Ch.

Genome- One unique set of DNA of an organism. We have two sets of 23 chromosomes
and one of those set is called genome.

Chromosome- is one long DNA molecule.

Gene- is functional region of a DNA molecule. Genes are simply the regions of
chromosomal DNA that are involved in the cell’s production of proteins.

Homologous- two chromosomes with the same gene array.

Nucleotides-consist of a phosphate group, a deoxyribose sugar molecule, and one of four

different nitrogenous bases: adenine, guanine, cytosine, or thymine.

Alleles- different forms of the same gene, located in the same position along the
chromosome in each individual.

Genotype- is the set of alleles that an individual possesses for a specific character.

Phenotype- visually observable expression of those messages.

DNA is the genetic material of all life form and viruses.

DNA Structure:

Most organism are diploid.

For human 2n = 46, n = 23
We have 23 different kinds of chromosomes and there are two of each kind.
Haploid set of chromosome is one set (n).

Prokaryotes- bacteria and cyanobacteria.

- no membrane bound nucleus and they have DNA.

Eukaryotes- animals, plant, fungi, and protists.

- have membrane bound nucleus. Important because the membrane create a
partition between two environments. Cytosol and nucleus.
- DNA is located inside the nucleus and in the mitochondria.
- the embryo get the mitochondria DNA from the mother and not from the
father.
Plant have DNA in the chloroplast which located in the cytosol.

Watson and Crick were the first to discover the model of DNA.

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DNA is a polymer. The monomer of DNA is nucleotide.

Nucleotide- is made up of phosphate group bonded to five carbon sugar, deoxyribose

sugar, then bonded to nitrogenous base.

In phosphate group, it has negative charge on the oxygen.

Deoxyribose sugar- the fifth carbon is not part of the ring.

- number the carbon by counting clockwise after the oxygen.

The phosphate group bonded to the fifth carbon and the nitrogenous base bonded
to the first carbon of the sugar.

Ribose sugar has a hydroxyl group on the second carbon.

Deoxyribose sugar only has hydrogen on the second carbon.

Nucleoside- is a nucleotide without the phosphate group.

Nucleotide is nucleoside monophosphate.

Nitrogenous bases:

Adenine (A), Guanine(G), Cytosine(C), Thymine(T).

Sugar- phosphate backbone : covalent bond that hold the backbone together.

The message of the DNA is the sequence of the nitrogenous base.

When reading the message of the DNA, you have to keep track of the polarity.

DNA consists of two sugar-phosphate backbones.

- The DNA strands are aligned antiparallel to each other.
- Adenine and guanine are double ring structure called purine.
- Thymine and cytosine are single ring structure called pyrimidine.
- Thymine always connected with adenine.
- Guanine always connected with cytosine.
- The two bases are connected with hydrogen bonds.
- Nitrogenous base is in a flat area stack on top of one another.
- Minor groove = less than 180 degree.
- Major groove = more than 180 degree.

The protein recognizes base pairs in the major groove because major groove have distinct
pattern. H- hydrogen, A- hydrogen acceptor, D- hydrogen donor, M- methylated.
Ex. G-C pattern is different form C-G pattern.

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 RNA substitute thymine with uracil.

CUT PY ---- cytosine, uracil, and thymine are pyrimidine.

Pyrimidine are single ring structure.

Purine are double ring structure, guanine and adenine.

DNA is a right- handed helix.

Amino acid always carbon bond it to hydrogen, carboxylic acid and NH2, and an R
group. What separates the different amino acid is the R group.

The R group fit into the protein receptor in the major groove.

The environment change the structure of DNA.

There are ten base pairs per spiral and equal to 34 angstroms. There are 3.4 A in between
two base pairs.

Structure of Genes:

DNA copied certain genes transcribe into RNA, and those genes carry the messages for
the amino acid sequence to make the necessary proteins.

Regulatory region- segment of DNA that regulate transcription by enabling transcription

of that genes to be turn on or off (initiation and terminal ).

Prokaryotic genes =>

Regulatory region, gene, regulatory region

RNA is polycistronic (one RNA stretch has messages for multiple

polypeptides to perform one related task).

Polycistronic- Pertaining to mRNA carrying information for the synthesis

of more than one protein.

Eukaryotic genes =>

Monocistronic - only one gene is transcribed onto RNA. Referring to
fully processed mRNA that codes for a single protein.
Remove segment ( intron) before it became mature RNA.
After the exons are connected, the mature RNA is much shorter than what
was transcribed from DNA.

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 Both prokaryotes and eukaryotes cell have regulatory region but the
regulatory region in the eukaryotes cell is more complex and larger.
Prokaryotes- have no nucleus membrane bound result in no intron, so no
splicing in the prokaryotes.
Extron have one more than intron.
Split gene- gene with intron.
Human have about 95 percent of out gene are split gene.
Spacer DNA- is the space between the adjacent gene.
The more complex the organism, the larger the space between the genes
and there are more intron but the MRA’s length is about same as other simpler
organisms.
Less than five percent have message for polypeptide.
More comlex the organism there are more split gene and longer gene
length.

Different type of genomes:

There are circular DNA and linear DNA. Circular DNA is where two ends are
attached.
Bacteria has main DNA (circular) and also have some accessory DNA called
plasmid.

1. Plasmid-
No membrane bound and circular DNA.
Plasmid can be transfer from one organism to another.
Most of the genes for antibiotic resistance in bacteria are in plasmid.
Bacteria have only one origin of replication, mean that they copy the whole DNA
at a time.
Eukaryote cell have multiply origin of replication, mean that replication can be
copy at many points on the DNA.

2. Organellar DNA-
Mitochondria and chloroplast also contain DNA that are circular.
Mitochondria was a bacteria long time ago and through evolution they
became part of our cell. Chloroplast used to be cyanobacteria.

3. Viruses- they are not alive but they have a genome that is surround by protein coat. It
can have either single or double stranded DNA or RNA.

Histone- they packed DNA into chromosome.

- only eukaryotes cell have histone.
- Eukaryotes cell’s nuclei has five different histone type proteins and 100
different non-histone proteins.

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DNA is copied onto RNA, and then RNA go through the processing by splicing the
intron. Then the RNA leaves the nucleus through the nucleopore.

Ribosome attached to the RNA molecule and move along the molecule. While the
ribosome move along the RNA, it make polypeptide.

Each species have a specific number of double stranded DNA molecules. Each DNA
molecule makes up a significant portion of a chromosome.

Since human have 46 chromosomes, so we have 46 double stranded DNA molecules in

each body cell.
- Chromosome is made up of a DNA molecule and associate
protein (some are histone type proteins and some are non-
histone proteins).
- Each chromosome are made out of two sister chromatids. Both
are identical to each other.
- (X) This chromosome contain two double stranded of DNA
molecule.
- The two sister chromatids are connected at the centromere.
- During cell division, the centromere is where the microtubules
attached and direct the chromosome movement in the cell.
- The centromere is structurally different from the rest of the
chromosome.

Recognition of the Chromosome:

1. We recognize the chromosome by how long is it.
2. Also by the location of the centromere.
- Each chromosome have a different location of its centromere.
- The centromere is divide the chromosome into a short and long
arm. P is for short arm and q is for long arm.
- Chromosomes are divided into group A – G and Longest to short
( chromosome one largest to chromosome 22 smallest).
Ex. G21 p site that mean is in the G group and it is the 21st
chromosome and it’s in the short arm.
- We actually pairs of homologous chromosomes ( same set of
genes at the same location). But for the male sex chromosome,
they have an X and Y chromosome that are not really
homologous but they pair up during meiosis as if they are
homologous. Since the X chromosome is larger than the Y
chromosome and so X chromosome have more genes that is not
in the Y chromosome.
- A special type in fruit fly is called polytene chromosome and it’s
in the malpigian tubules. When DNA replicated, the DNA stick
together.
- DNA have band and that distinguish between different
chromosome.

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Karyotype- a picture of arrange homologous chromosome in groups ( stain the
chromosome first so you can see the band then take a picture of it).

Certain band are rich in A-T base pairs or G-C base pairs that give morphology to the
chromosome ( help to distinguish between chromosome).

Chromomere- are beadlike, localize thickening found along the chromosome during the
early stages of nuclear division.

Nucleolus- where ribosomes are assembled.

Ribosomes are made out of protein and RNA.
Nucleolar Organizers Region (NOR)- Region of DNA that make ribosomal RNA
( rRNA).
We have five different chromosomes that have NOR, but since we have homologous
chromosomes so there are total of 10 NOR that coalesce to form the nucleolus.

DNA are exist as either heterochromatin or euchromatin configuration.

Heterochromatin- is where DNA remain condense. Ex. Centromere.
Euchromatin- region of the DNA that are not condense.

Telocentric- is where centromere is at one end of the chromosome.

Acrocentric- is where cetromere is close to one end of the chromosome.
Metacentric- is where centromere is in the middle of the chromosome.

Telomeres- the tip of linear chromosome and is not visably distinct from the other part of
the chromosome. Telomeres are always heterochromatic.

Facultative heterochromatin- sometimes condensed, and sometime not.

How DNA Packages:

First level of packaging-

There are five types of histone proteins: H1, H2A, H2B, H3, H4.

H2A, H2B, H3, H4- will form the core particle.

Dimer is made from two of H2A, two of H2B, two of H3, two of H4.

Nucleosome- is the beads of the necklace which made out of the core particle ( which is
8 histone proteins) and DNA wrapped around it.

The width of the nucleosome is 10 nm.

The DNA wrapped around the core particle (8 histones) twice.

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H1 histone protein bind to the linker DNA that just before and after the nucleosome.
Every 200 base pairs, you will get one nucleosome. There are 146 base pairs of DNA that
wrapped around the core particle and there are about 50 base pairs of linker DNA.

Second level of packaging:

H1 histone interact and they form a spiral ( solenoid). The width of solenoid is 30 nm.

One spiral in the solenoid contains six to seven nucleosomes.

If you digest the H1 histone protein then it will go back to level one a linear line of
nuclosome.

Scaffold is largely composed of the enzyme called topoisomerase II.

The loops of the solenoid attaches to the scaffold by special region along the DNA called
scaffold attachment region. Fig. 2-26

The width of the looped solenoid around the scaffold is 100 nm.

The width of the chromosome is 700 nm.

Histone H3 and H4 are highly conserved across all eukaryotes life forms.

Even though DNA is invisible and we cannot see the level of organization of the DNA
but there is still a level of organization. Fig. 2-13 when the chromosome is unwound
( chromatin) the DNA molecule of that chromosome still have their own territory in the
nucleus.

Ch. 3

Gene Function
Fig. 3-2. Within a normal DNA it contains gene and within the gene there is regulatory
region, exon, intron, and termination region.

When RNA is transcribed, it doesn’t include the regulatory region and may not include
portion of the termination sequence.

The RNA is process in the nucleus and the mature RNA come out to the cytosol.

The mature RNA contain the message to make polypeptide. The process of making
polypeptide in the cytosol is called Translation. The polypeptide then folded themselves

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up to form a three dimensional structure that have an active site that will bind to a
substrate and encourage the substrate to participate in the chemical reaction.

Mutation is occur in the DNA and it is on the exon. The mutation is still part of the
mature message RNA and translate onto the polypeptide. So when the polypeptide fold
up and it may not have the same form as the normal polypeptide (a different polypeptide )
and the substrate might not fit.

Structure of RNA:

There are different types of RNA molecules.

RNA polymers- are nucleotide hung together along the sugar phosphate backbone.

The main difference between RNA and DNA are the sugar and one of the base.
RNA have ribose sugar and DNA have deoxyribose sugar.
The main difference between ribose sugar and deoxyribose sugar- are the
hydroxyl group that attach to the second carbon on the ribose sugar. On deoxyribose
sugar, there is just a hydrogen on the second carbon.
RNA has uracil instead of thymine.
RNA has the same structure as DNA; with a sugar-phosphate backbone and
nitrogenous bases.

During transcription, the RNA polymerase moves along the DNA template strand from
the 3 prime to the 5 prime direction. The RNA polymerase starts making the RNA strand
in the 5 prime to the 3 prime direction. The nucleotides that are used in the RNA
molecule came from nucleoside tri-phosphates that originated from the cytosol. When
the nucleoside tri-phosphate comes into the nucleus, two phosphate molecules
(diphosphate) break off of it and give off energy, which is used to connect the remaining
nucleotide to the 3 prime end of the RNA strand. The phosphate coming in will connect
to the sugar of the previously added nucleotide making an ester bond between them.

The RNA that been transcribe look just like the non-template strand except for the uracil
and ribose sugar.

Fig. 3-7
A conventional way of reading and writing DNA strand is by reading and writing
the non-template strand only. From this we can easily know the RNA sequence and the
template strand.

Fig. 3-9

Nucleoside is made up of a base and a sugar.

Nucleoside tri-phosphate is a base, a sugar, and three phosphates.

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After the transcription of the RNA, the mature RNA comes out into the cytosol and then
a ribosome comes and starts reading the message along the RNA strand to make the
polypeptide.

Transfer RNA is an adapter molecule that brings the amino acids to the mRNA and
simultaneously reads the nucleotide sequence in the mRNA. tRNA is a very short RNA
molecule.

All the RNA’s that exist are created because of the instructions on the DNA.

Ribosomal RNA (rRNA) – ribosomal subunits are made up of a number of proteins and
some RNA molecules. The RNA molecules in a ribosome (rRNA) play both a structural
role and a functional role.

Spliceosome – splices out the introns in the RNA molecule. It’s located in the nucleus
and is as big as a ribosome. It’s made up of small proteins and small RNA molecules.
These small RNA molecules are only found in the nucleus and are called “small nuclear
RNAs” (snRNA). snRNA often combines with proteins to form “small nuclear
riboproteins” (snRNP). These snRNPs are often the vehicle that are used in the splicing
process.

Small cytoplasmic RNA molecules (scRNA) – RNA that starts in the nucleus and extends
out into the cytosol and ends up in the rough endoplasmic reticulum.

After the ribosome synthesizes the polypeptide in the cytosol, it attaches to the
endoplasmic reticulum to make up the rough E.R.

When the ribosome reads the mRNA and first synthesizes the polypeptide, the beginning
section of the polypeptide is the signal molecule. The amino acid in that signal molecule
is what directs the destination of the polypeptide.

When the polypeptide needs to be secret out the cell, it goes to the endoplasmic reticulum
so cytosol doesn’t go out of the cell and also the signal molecule needs to be linear
otherwise it won’t fit through the pore. For the signal molecule to remain linear, the small
cytoplasmic RNA (scRNA) will bind to the signal molecule, it’s complementary to the
RNA strand, and it will keep the signal molecule to remain straight and also so the signal
molecule can find it location on the endoplasmic reticulum.

Uracil is behave just like thymine in RNA.

The sequence of the nitrogenous bases in nucleic acid are coded to make other nucleic
acid and also are code for protein to recognize them.

RNA are easy for protein to recognize because it’s in a linear strand not like the DNA
double strand.

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Process of Transcription:

During transcription, it’s important to keep track of the polarity of the molecule. You
need to know if the molecule is transcribing 5 prime to 3 prime or vice versa.

Also keep track of whether you are transcribing prokaryote or eukaryotes.

Prokaryotes’ Transcription:

Initiation:

You can transcribe three place at the same time. A RNA polymerase start to transcribe
the template strand at a 3 prime to 5 prime. One component of the regulatory sequence is
going to identify which strand is going to be the template strand.

The promoter is the component in the regulatory sequence that said where the gene is
and which strand is the template strand.

The RNA polymerase is reading in a 3 prime to 5 prime direction and the RNA strand
that is produce is in a antiparallel direction in 5 prime to 3 prime direction.

Fig. 3-5
RNA polymerase attached to the promoter of the double strand DNA and start
pull the nitrogenous bases and move like a snowplow. The two sugar phosphate backbone
remain separate for only about 20 nucleotide.

Fig. 3-9
Dark blue vertical line is where transcription starts. Downstream is from plus one
to the right. Upstream is left of the blue line. Promoter region is those between the yellow
line. Consensus sequence is region in different genes where the nucleotide sequences are
the same and found more than fifty percent of the time.

Yellow region is the promoter region. That is where is RNA polymerase bind to the DNA
template strand. The promoter region is usually having A-T pairs because they only have
two hydrogen bonds and it’s easy to break.

Elongation:

You continue to make the RNA sequence.

Fig. 3-5b

Termination:
Fig. 3-10

Termination sequence in the template strand is consist of about 40 bp, ending in a G-C
rich stretch that is followed by a run of six or more A’s. Because the termination

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sequence is G-C rich, the RNA in this region is also G-C rich. This allows RNA to form
complementary bonds with itself result in a hairpin loop. And the loop is follow by a run
of U’s that correspond to A’s on the DNA template.

There are two types of termination in prokaryotes.

One is the hairpin loop.
The second is the Rho-dependent (ρ )- As the RNA polymerase synthesize the
RNA strand, a protein called rho came in and bind to the 5 prime end of the RNA strand
and read it 5 prime to 3 prime. As the RNA polymerase reached the termination
sequence, it’s slow down and that allows rho protein to catch up to the RNA polymerase,
and cause it to dissociated from the DNA. This is the end of transcription process.

There are only one type of RNA polymerase in prokaryotes cell.

Untranslated region or 5′ UTR- this is the region between the point where transcription
start and the point where protein-coding gene begin.

3′ UTR- transcription of a gene is terminate beyond the protein-coding of the gene. So

the region between the end of the protein-coding segment and the point of termination.

Eukaryote Cell:

Transcription in eukaryote cell have different RNA polymerase that make different type
of RNA.
RNA polymerase I- that polymerase that transcribe the ribosomal RNA (rRNA). It
transcribed all of them except the smallest one (5 s unit ). The smallest rRNA is
transcribe by RNA polymerase III.
Sedimentation coefficient ( sphedberg unit- s )- we used sedimentation unit to
differentiate between different molecule, the larger RNA molecule have a larger
sedimentation coefficient. The smallest rRNA is 5 s unit.

RNA polymerase II- will transcribe all the messenger RNA.

RNA polymerase III- will transcribe all transfer RNA (tRNA) and
also the 5 s rRNA.

The RNA polymerase doesn’t really recognize the promoter region at all, it need some
protein to bind to the promoter region then the RNA polymerase will attracted to that
protein complex. These protein are called transcription factor (written as TF).
Ex. TFII- is the transcription factor that is part of the regulatory region for RNA
polymerase II.

Fig. 3-11
Transcription using RNA polymerase II
The RNA polymerase II move along the DNA strand in a 3 prime to 5 prime direction
and synthesize mRNA in a 5 prime to 3 prime direction.

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After the mRNA reach thirty nucleotides long, you modify the 5 prime end of the mRNA
by putting a cap ( 5′ cap or 7-methylguanine cap ).
- The cap is like a nucleotide of guanine that has a methyl group on
the seven nitrogen atom. It has a guanine base ( methyl group
attached) attached to the sugar, and then attached to two
phosphate group and then it will attached to the phosphate of the
RNA strand.

As the RNA polymerase II move down and transcribe nucleotide sequence of AAUAAA
on the mRNA and that sequence recognize by an enzyme ( endonuclease ) which cut the
RNA strand twenty nucleotides down stream from AAUAAA.

Nuclease- an enzyme that digest a nucleic acid.

Endonuclease- digest nucleic acid from the middle.
Exonuclease- digest nucleic acid from the end.

An enzyme called poly (A) polymerase that add a sequence of about 150 to 200 Adenine
nucleotide to the 3 prime end of the RNA strand.
This sequence of Adenine is called Poly (A) tail. Poly (A) tail is important because it’s
determine how long the mRNA is going to live.

Intron’s Splicing:

Fig. 3-12

So far the mRNA is still in the nucleus with intron ( immature mRNA). Within an intron,
there is always (100%) a G U sequence on the 5 prime end (front) and there is always a
AG sequence on the 3 prime end (end ) and there is also within 50 base pairs upstream
from the 3 prime end there is always (100%) an Adenine, also called the branch point.

Spliceosome- is team of snRNP ( small nuclear ribonucleoprotein particle ) that constitute

a functional unit. snRNP is a complex of protein and snRNA ( small nuclear RNA).

Fig. 3-14 and Fig. 3-15

The splicesome attached to the mRNA, the snRNP cause the intron to fold up.

First, the 2 prime carbon of the Adenine branch point attacks the five prime end of
the intron and also attach to it, then the 3 prime end of the exon attacks the five prime of
the other exon that still connect to the intron.

During the splicing process, the mRNA is prevented from the leaving the nucleus
prematurely until all splicing occurred. This is because the spliceosome is attached to the
mRNA and mRNA can’t fit through the nucleus pores.

Ribozyme- is a RNA molecule that can act as an enzyme to performs a specific biological
reaction. Ex. splice their own intron without any protein.

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Polypeptide is amino acid connected by a peptide bond.

Amino acid is carbon that connected amino group, carboxylic acid group, hydrogen
group, and some kind of side chain (R).

Polypeptide are formed by condensation reaction when two amino acid joined. One
amino acid loses an H from the amino group and the other amino acid loses an OH from
the carboxylic acid. Condensation reaction is when you lose water.

The bond that form between the NH and CO is a peptide bond.

The backbone of the polypeptide is N-C-C-N-C-C…

An average length of a polypeptide is 600-700 amino acids long.

Dipeptide- two amino acids joined together
Tripeptide- three amino acids joined together

In polypeptide, there is amino end and a carboxyl end. We read the polypeptide from the
amino end to the carboxyl end. Ex. amino acid 20 amino acid 17 amino acid 1 , that is
how you would read the polypeptide.

Fig. 3-18
Primary Structure of Polypeptide:

The primary structure of polypeptide is one long chain of amino acid.

Secondary Structure of Polypeptide:

The partial positive on the amino hydrogen and partial negative on the oxygen are
attracted together to make a hydrogen bond. This caused the polypeptide to form an
alpha helix which is now in the secondary structure.

Something caused the primary structure to go in a opposite direction and you get
hydrogen bond that formed beta pleated sheet. The side chain is involved in the
bonding.

The protein that is embedded in the plasma membrane has a alpha helix structure. The
part of the protein that is inside the membrane is hydrophilic and the part that is outside
the membrane is hydrophobic. The inside of the plasma membrane is hydrophobic and
the exterior is hydrophilic.

Tertiary Structure of Polypeptide:

The tertiary structure is cause by the attraction between the side chain and folding of
secondary structure that created a complex three-dimensional shape.

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Quaternary Structure of Polypeptide:

Quaternary structure results when more than one polypeptide come together by
something other than hydrogen bond.

The protein that is most often interact with DNA are called Dimers.
Dimers are made of two polypeptides.

Homo-dimer- made of same two components.

Hetero-dimer- made of two different components.

There are two main categories of proteins:

Globular protein- they folded up to make a glob with an active site that accept a
specific substrate. When mutation occurred, they modified the protein structure but as
long as the active site doesn’t changed, the protein still do it job. If the mutation does not
affect the active site then it is not high deleterious (not a big changed).
Fibrous proteins- are linear shape protein such as hair and muscle.

Translation:

In prokaryotes, translation starts before the mRNA is fully transcribed.

Ribosome exists as two subunit- small subunit and large subunit. We distinguish between
small and large based on their sedimentation coefficient.
Prokaryotes- the two subunits’ sedimentation coefficient are 30s and 50s and
when we combine the two they make sedimentation coefficient 70s.
Eukaryotes- the two subunits are 40s and 60s, and when we combine the two they
make 80s.

Prokaryotes:
In the five prime end of the mRNA, there is a sequence in 5 prime UTR and this
sequence is called Shine-Dalgarno sequence. This sequence will bind to the small
ribosomal subunit. First some initiation factors have to bind to the small ribosomal
subunit. Then magnesium ions bind to the subunit, followed by GTP (guanasine
triphosphate) energy source. Then next thing is charge tRNA binds to. Then the small
ribosomal subunit binds to the shine-dalgarno sequence in the mRNA.

Prokaryotes have three initiation factors that are proteins.

There is a different tRNA molecule for every amino acid and some amino acid have more
than one tRNA molecule.

tRNA are small molecule with about 75-90 nucleotides long. They are made of sugar
phosphate backbone.

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Fig. 3-21
tRNA like all RNA have polarity in 5 prime to 3 prime and it in a clover shape leaf. The
3 prime end of tRNA is always end with CCA nucleotide sequence. tRNA folded up on
itself to form a secondary structure. There is also region with complementary bases that
bind together by hydrogen bond to created a stem shape. There is also region where the
base are not bind together that have a structure like loop. Within the loop there is a
modified base called psi (Ψ ), we basically take a uridine and modified it to
pseudouridine. The loop have a modified base that it doesn’t become part of the stem.

Within tRNA molecule, there is an anticodon loop that has a series of three bases that are
complementary to the codon on the mRNA molecule. They are connected by hydrogen
bond to mRNA in an antiparallel direction. The mRNA is read three bases at a time.
Codon is a triplet.

tRNA read the codon and position a specific amino acid at the 3 prime end using a
covalent bond.

Fig. 3-20
Genetic code is a triplet. Every amino acid have it own specific code but sometime there
is more than one codes that make the same amino acid. So they said the genetic code is
redundant but unambiguous.

Charged tRNA- is a tRNA that has an amino acid bound to it.

In the genetic code, we read three bases at a time. There is a start signal (AUG) and three
stop signal (UAA, UGA, and UAG).
The start signal for a eukaryotes has a codon (AUG) code for the amino acid
methianine.
The start signal for a prokaryotes has a codon (AUG) code for the amino acid that
has been modify to be formal methianine or Fmet.
Within a protein, the first amino acid is not methianine or Fmet because the first
few amino acid get cut off when the polypeptide got processed.

Fig. 3-21
The tRNA has an amino acid that is on the opposite side to the mRNA strand.
It has an anticodon seqence that is complementary and antiparallel to the codon of the
mRNA. The bases between the codon and anticodon are connected by hydrogen bond,
and the third bases of the codon is connected to the first base of the anti codon.

In the tRNA, base are modify so you can have a new base that can appear on the
anticodon for inosine (base).
One tRNA have a specific anticodon that can read more that one codon. So if one
tRNA is reading suite of codon then every member of that suites is code for the same
amino acid.
Every specific tRNA will be charge with one and only one amino acid.

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Wobble- is a phenomenon where an anticodon can read more than one codon.

Charge tRNA:
Amino acyl tRNA synthetases- are the enzyme that bring amino acid together and
covalently bind it to tRNA. There are 20 different amino acids so we need a different
amino acyl tRNA synthetases for every amino acid.
First the amino acid bind to the amino acyl tRNA synthetases, then ATP come
and lose two phosphate to make AMP and release energy. After that, amino acyl tRNA
synthetases used that energy to bind the amino acid to the CCA 3’ end of the tRNA.
Because of the wobble, we don’t need as many tRNA as we have codon. We have
61 different codon that messages for twenty amino acid. In human, we have 30 tRNA
because of the wobble. There are more than one tRNA for the same amino acid. All the
tRNA for a specific amino acid have to be recognize by the same enzyme.
All the RNA are transcribed from the DNA.

How Charge tRNA bind to mRNA:

Fig. 3-23

Ribosome are consist of large subunit and small subunit. The subunit are made up
of rRNA molecule bound with protein.
Initially, the large and small unit are not connect and floating in the cytosol. The
assemble of ribosome is part of the initiation stage of translation.
Initiation factor (IF)- are protein that small ribosomal subunit become associated
with.
There are there (IF) in prokaryotes and there are more in eukaryotes.
EIF- stand for eukaryotes initiation factor. When you just said IF is meant for the
initiation factor in prokaryotes.
After the IF bind the small ribosomal subunit, then a magnesium ion and an
energy source (GTP) also bind to it.

Then, the small ribosomal subunit binds to the mRNA. This is done by connecting the
rRNA in the small subunit to the Shine-Dalgarno sequence in the 5’ UTR of the mRNA.
The rRNA is complementary to the mRNA.

Approximately ten bases down stream from the shine-dalgarno sequences you have the
start codon (AUG). The small subunit get in position with the start codon (AUG). This is
all in prokaryotes.
In eukaryotes, there is no shine-dalgarno sequences , the five prime cap will bind
to the small ribosomal subunit. There is no specific distance between the five prime cap
and the start codon, so the small subunit attached to the five prime cap and starting search
for the start codon.

The tRNA is charged with Fmet (prokaryote example) and binds to the start
codon, then the large ribosomal subunit will assemble around the small subunit and you
get a body of ribosomes.

16
Functional Ribosome:

Fig 3-24
Ribosomes have 2 sites of activity: the “a” and “p” sites that are three dimensional
regiona within the ribosome.

The charged tRNA for FMet is in the P site, the A site is empty. Then another charged
tRNA enters the A site which is determined by the codon that’s in the A site.
Except for the very first charged tRNA (with FMet or Met), every other charged tRNA
enters in the A site.
The A site is on the 3’ side of the mRNA strand, and the P site is on the 5’ side.

The amino acid that enters the A site binds its amino group to the carboxyl group of the
amino acid that’s in the P site.
-The enzyme that regulates this reaction is not a protein, it’s the large rRNA
molecule (a ribozyme).

The tRNA in the P site gets discharged (loses its amino acid) and the tRNA in the A site
now has a dipeptide. This is called Elongation.

Now we have Translocation (where the ribosome moves down 3 bases on the mRNA)-
now the A site become the P site. Now the A site is moved to the next codon on the
mRNA strand then it will attract a charged tRNA to the A site. The amino group in the
amino acid on the new charged tRNA will bind to the carboxyl group on the previously
added peptide to make a new peptide bond. Then the tRNA that in the P site will be
discharged and forming a tripeptide in the A site. This process is continue until it reach a
stop codon.

Before leaving the ribosome, the discharge tRNA go into the E site just before it exit.

When the ribosome reach the stop codon, a protein called Release Factor goes into the A
site cleave the bond between the polypeptide and the tRNA in the P site. Then the
ribosome disassemble and now have an initial transcribed polypeptide.

Eukaryotes are monosystronic because the mRNA have message for only one
polypeptide.

Since prokaryotes are polysystronic that mean their mRNA have messages for more than
one kind of polypeptide. That means their mRNA is separately initiated for each of the
messages.

One DNA can make a lot of mRNA, each of these mRNA can be translated by a number
of ribosomes at the same time to make a lot of the same polypeptide, and each
polypeptide make a lot of the same chemical reaction.

17
We have a lot of redundant copies of DNA to make many copies of ribosomal RNA
(rRNA) so they can make enough ribosome for translation.

Fig. 3-28
We normally converted phenylalanine to tyrosine that is control by an enzyme. If
the enzyme is malfunction then you get a buildup phenylalanine which cause conversion
into phenylpyruvic acid which created nerve damage and lead to mental retardation.

Fig. 3-30

Fig. 3-27
Protein folded up to make three dimensional shape with an active site.
- Mutation in the promoter prevent the RNA polymerase from
recognizing the DNA ( so the polypeptide never forms in the first
place). A null mutation is when you don’t get a functional
enzyme.
- Mutation of the active site that is also null mutation.
- Mutation that is right on the boundary of the active site and rest
of the protein that result in a leaky mutation, does work partly.
- Mutation on the part of the exon that will not affect the active site
so it’s a silent mutation, there is mutation but the protein is still
functional.
- Mutation in the intron as a result in the null mutation, because if
there is a mutation on the intron’s branch point or the GU-AG
region then the intron will not be remove by the splicesome and
resulting polypeptide will not be the same.

Ch. 4

DNA Replication

When DNA replicate, the backbone have to be separated

Replication bubble- is a bubble where replication occur. Each replication bubble have
two replication forks.

Prokaryotes- have circular DNA so they only have one origin of replication in the
bacteria genome. The bubble enlarged to make the DNA look like Greek letter theta (θ )
configuration.

Eukaryotes- human have over 1000 origin of replication in their genome. That allow for
many bubble to form simultaneously. Then eventually all of the bubble will coalesce.

18
Prokaryotic DNA replication:
DNA polymerase- is the enzyme that they used to replicate DNA. There are three kind of
DNA polymerase ( I, II, III ).

Helicase- is the enzyme that unwind the DNA strand and separate it also.

Single stranded binding proteins (ssbp)- these are specialize protein that are inserted in
each strand to stabilize it and to keep the bubble open.

When the bubble gets bigger, that caused strain on the rest of the double helix strand on
the two end of the bubble. One way of releasing the tension is cut one or both end of the
backbone so the strand can twist around and reconnect to relieve the tension.
Topoisomerase I- is an enzyme that cut one side of the bubble.
Topoisomerase II- is an enzyme that cut on both sides of the bubble.

Prokaryotes have a special type of topoisomerase called gyrase that cut the circular DNA
and give them a figure 8 configuration. It called super coiling and it cause relaxation of
the tension cause by this untwisting. Gyrase is not significant in eukaryotes because we
don’t have circular DNA.

DNA polymerase always read the message in a 3’ to 5’ direction and synthesize the new
DNA in an antiparallel direction.

The problem is DNA polymerase need to attach at a vacant 3’ end of the strand. The
DNA polymerase come in and attaches to the 3’ end of the leading strand and start
making the DNA complementary to the leading strand. But with the lagging strand there
is no 3’ end for the DNA polymerase to attach to. So an enzyme called primase which is
an RNA polymerase lay down a small RNA primer that is complementary to the lagging
strand that allow the DNA polymerase to attach to the 3’ end of the RNA primer and
adding DNA nucleotide to it that are complementary to the lagging strand.
As the bubble expand, new RNA primer have to add to the lagging strand.

The leading strand synthesizes complement that is continuous and for lagging strand it’s
discontinuous.

Okazaki fragment- is the fragment of DNA that is between RNA primer and
complementary to the DNA in the lagging strand.

DNA polymerase III- is what makes the complement of the leading strand and okazaki
fragment in the lagging strand.

All DNA polymerases in prokaryotes have exonuclease activities. DNA polymerase III
constantly proof read the new complement. As it make a new nucleotide in a 5’ to 3’
direction, it recheck to make sure it the right nucleotide added. If a wrong nucleotide is
added then it will remove it and put a new one in. It makes a 5’ to 3’ complement so it

19
have to remove in a 3’ to 5’ direction. DNA polymerase III have a 3’ to 5’ exonuclease
activity.
DNA polymerase I have a 5’ to 3’ exonuclease activities (according to the
complement of the leading strand). Also it digest the RNA primer.

DNA ligase- make the two okazaki fragment continuous through a covalent bond.

DNA polymerase II- function in DNA repair. It cut out the bad fragment and try to make
a good one again. The mutation observe in individual are the ones that escape the repair
process.

A nucleoside triphosphate have two phosphates cleaved off before the nucleotide is add
to the DNA. The energy that release after cleavage is called pyrophosphate. That energy
is used to connected the new nucleotide to the 3’ end of the new strand. The two
nucleotides are binds by a covalent bond.

Fig. 4-6; 4-4

Fig.4-7
The DNA polymerase is anchored in nucleus matrix (skeleton) and the position of
the forks stay the same. The bubble of the DNA is expanding perpendicularly to the
forks.

Eukaryotes:
There are a lot of DNA polymerase. Only two function in synthesize DNA in the
nucleus, DNA polymerase alpha (α ) and DNA polymerase delta (∆ ).
DNA polymerase gamma (χ ) functions in replicating DNA in the mitochondria.

DNA polymerase beta (β ) and DNA polymerase epsilon (ε ) are function in

DNA repair.

Unlike DNA polymerase I in prokaryotes, none of the eukaryotes DNA

polymerase have 5’ to 3’ exonuclease activity.
To get rid of the primer, an endonuclease called H1 recognize the junction
between the RNA primer and the DNA and break the sugar phosphate backbone
connecting them. Then an RNA exonuclease called FEN1 digest the primer and replace it
with DNA.

Cell Division:
Cell cycle contains- G1, S, G2, and M phases. M phase is either mitosis or
meiosis. In the interphase ( G1, S, G2) are when DNA are not condense so we can’t see
it. Only in the M phase do we see the DNA in its condense form.

G1- gap 1; G2- gap 2; S- synthesis.

When DNA is condensed, it’s not transcribe.

20
DNA replication is occur in the S phase.

All of the histone are produce in the S phase.

The gene for the histone are transcribe at the end of the G1 phase.

Most of RNA transcription occurs in G1 and G2 phase.

There are conditions that have to be met to go from one phase to another.
Restriction point regulate whether the cells dividing or not. Located between G1 and S
phase. If a cell stalls, it in the G0 stage that mean it doesn’t divide or anything.
Example- scientists are trying to bring neuron cell from G0 to G1 phase so these
nerve cells can divide.

In the S phase, DNA replicate to make duplicate of itself and identical double stranded
DNA molecule. These two double stranded DNA are still connected together by a
complex of proteins. This region where they are connected is called centromere.

A chromosome is making up of two sister chromatids connected by a centromere.

A sister chromatid is make up of a double strand DNA.

Chromosomes are invisible until they reach the m phase.

Each chromosome have one centromere.

Each centromere have two kinetochores. And each kinetochores is associated with each
chromatid. The microtubules interacted with the kinetochores. The microtubules from
one pole attach to one kinetochore and same for the other pole. The microtubules guide
the movement of the chromosome.

What tell you that you about the leave G2 phase and enter the M phase is that you have a
region in the cell called centrosome which is invisible. Within the centrosome there are a
pair of centrioles. When you about the enter the M phase the centrosome duplicates so we
have four centrioles. Microtubules start to extent outward from each of centrosome. As
the microtubules extent from both of the centrosome, they start interacting and start
pushing the centrosome away from each other. DNA is now condense and you can see
the chromosome.
Prophase of mitosis- The DNA condensed, and the chromosomes are visible. The
centrosomes are moved to the opposite end of the cell and having the microtubules
extending between them. Nuclear envelop disassembles and become part of the
endoplasmic reticulum. Mitotic spindle- created by the microtubules extending between
the two poles.
Metaphase- centromeres lined up along the equatorial plane ( metaphase plate) so
that one member of each sister chromatid pair go to one pole and other sister chromatid
go to the other pole.

21
An enzyme called protease is release and dissolve the protein complex which make up
the centromere.

Anaphase- the sister chromatids are pull apart because of the microtubules and
migrate to opposite poles. Now each chromosome have only one chromatid. When these
chromatid reach the S phase, it will duplicate and then one chromosome will have two
sister chromatid.
Now the cell has two nuclei. Mitosis is just a nuclear division, it not a cell
division unless the cytoplasm is divided.
Cytokinesis- is the division of the cytoplasm. In animal cells, cytokinesis
involves the formation of cleavage furrow. In plant cells, you have formation of cell
plate. In plant, the golgi apparatus secrets vesicle that coalesce in the center and the
vesicle contain a glue like material called middle lamella and it keep grow out and make
a cell plate. Algae have centromere and most plant don’t have centromere
Cytokinesis is occur in telophase of mitosis- The two nuclear envelope
reassemble and DNA decondenses.

Somatic cell is the body cell and they divide using mitosis. Each human cell have 46
chromosomes and when it in the S phase it duplicate to have 92 and when it get to m
phase it reduces to 46 chromosomes in each cells.

Meiosis:

The gametes cells are haploid and those are eggs and sperm cells. We produce gametes
cell through meiosis.
Fig. 4-22
Meiosis is series of two sequential nuclear divisions.

Prophase 1- nuclear envelope disappear. chromosomes condense, homologous

chromosomes pair in synapsis. When they pair up, the base position of the homologs are
next to one another. Chiasmata- region of the overlapping chromatids. After the
chromatids overlap, the chromosomes move apart. Then crossing over occurs when the
chiasma migrates to the tip of the chromosome. This is called terminalization.

For every meiotic division, there is at least one crossing over per homologous
chromosome. The two homologous chromosomes move to the opposite poles. In meiosis
1, each homolog consists of two sister chromatids. At the end of meiosis 1, you have half
number of chromosomes. Meiosis one is also called reduction division. The probability
of transmitting all of one parent’s chromosomes to the same nucleus is (1/2) 23 . It’s really
hard to get all of chromosomes of one parents to one cell.

Metaphase 1- homologous chromosomes line up on opposite side of the equatorial plane.

The arrangement of each homologous pair is independent of the other nonhomologous
pairs.

Anaphase 1- homologous chromosomes segregate and move to different poles.

22
Telophase 1- Each pole now have a haploid chromosome set, but each chromosome still
has two sister chromatids. Cytokinesis is occur simultaneously with telophase 1.

Prophase II to telophase II is the same as in mitosis.

Meiosis gives rise to gametes cell in animals.

The second meiotic division is called the equational division.

According to Mendel’s Law of Segregation- each individual have two copies of every
gene and only one copy get transmitted to that individual’s offspring.

Law of independence absorptment- if you look at two different genes, each gene is
transferred independent to one another.

Prokaryotes do not under mitosis or meiosis cell division.

Fig. 4-9
In prokaryotes, cell go through binary fisson the divide cell.

Fig. 4-10
In virus undergo rolling circle model is how DNA replicate in virus. The nuclease cut
the outer strand of the circular DNA, then pull the 5’ end out. As it being pull, a
complement is added to 3’ end using the inner strand as a template. The horizontal single
strand doesn’t have a 3’ end so the RNA polymerase come in and add the primer to it.
The DNA polymerase III created okazaki fragment.
The strand contain multiple genomes. An endonuclease come in and separate
those genome out.
Concatamer- is the double strand DNA that contain multiple genomes in virus.

Fig. 4-24

Fig. 4-11
For linear DNA, H1 cuts the primers from the DNA and FEN1 digests the primers
away. Once that happens, it makes the lagging strand shorter than the leading strand
(because of the primer on the far right being digested in the figure).
Telomerase – an enzyme that’s made up of a complex of protein and RNA. It
extends the 3’ end of the lagging strand by adding multiple copies of a simple noncoding
sequence (that’s 6 base pairs long). Then you add an RNA primer to the template strand
and extend it with DNA, after which you clip off the primer. Now you have a double-
stranded DNA strand that’s longer than the original DNA. The DNA is synthesized
under the instructions of RNA (called reverse transcription).

An enzyme that directs reverse transcription is called reverse transcriptase.

23
Telomerase is reverse transcriptase.
It is a ribozyme because the RNA part of telomerase that have the enzymatic capacity.

Fig. 4-16
Animals
The gametes is the only stage in our life cycle that is haploid. We don’t have a
multicellular haploid stage.
The gametes go through a process of syngamy where they give rise to cell that are
2N (this is zygote). That 2N cell go to mitotic division to give rise to an adult individual.

Fungus
The two haploid multicellular organism that contribute cell to form a zygote.
The diploid stage is fungus is only a single cell. The diploid cell will go through meiosis
to form haploid cell which then go through mitosis to form a multicellular organism.
They have a multicellular haploid stage and the diploid stage is unicellular.
The haploid stage contains spores.
Spore – is a haploid cell that goes thru mitotic divisions producing multicellular haploid
individuals.

Plant
Meiosis occurs in the flower of the plant to make haploid cells which then go thru
mitotic divisions to produce multicellular haploid individuals (7 in the female and 3 in the
male). These go thru mitosis to make sperm and egg which fuse together to make the
zygote which then goes thru mitosis. Unlike animals, plants’ gametes are immediately
preceded by mitosis.

Fig. 4-14

Top picture – a bacterium undergoes binary fission to make identical daughter

cells (this is not mitosis or meiosis).

Fig. 4-20

In most mammal, telomerase is not actively transcribe. Telomerase become inactive after
fifty cell division (turn off). Because of this, the cell DNA shorten with every division
until the cell die.
In tumor, stem cell, and sperm cell- telomerase is constantly produce.

Ch. 5
The Inheritance of Single-Gene Differences

Product rule- the probability that two independent events simultaneously occur is equal
to the product of their individual probabilities.

24
 Probability- relative frequency of an event occurring is equal to the number of
observation divided by the total number of trial.
Theoretical probability- is what we would expect.

Independent event- when one event occurs and it doesn’t affect the probability
of the other event to occur.

Ex. to get a snake eyes on the dice, the probability of getting 1 on one dice is 1/6 and the
probability of getting 1 on the other dice is 1/6 so the product rule of independent event
says getting two dices with 1 is (1/6 times 1/6 = 1/36).

Summation Rule- the probability of obtaining any of a set of mutually exclusive events
is equal to the sum of their individual probability.
Mutually exclusive events- is when if one event occurs the other event could not
have occurred.
Ex. the probability of getting an odd number on a dice is (1, 3, 5) equal to the sum
of their individual probability (1/6 + 1/6 + 1/6 = ½ ).

Patterns of Inheritance:
Take two individuals know phenotype or genotype and cross them and see the
result. Then cross their offspring also.

Parental generation (P)- is two individuals we start out with.

P1 x P2  F1

F1 generation- stand for the first filial generation. Filial means family.

F1 x F1  F2 generation …

Phenotype - the visual or measurable expression regarding some type of characteristic.

Genotype- is the underlying genetic blueprint of the phenotype.

Most individual have more than one genotype that can give rise to same phenotype.

For every gene, there can be more than one form. Because we’re diploid, we have two
forms for a gene.

AA = homozygous dominant
aa = homozygous recessive
Aa = heterozygous (usually the phenotype is one of the homozygous types)

The heterozygous phenotype is that of the homozygous dominant.

25
Fig. 5-5
Mendel performs a monohybrid cross. In a monohybrid cross, you use two
parental units that have different phenotypes for the same character and you know that
they are true breeding.

true breeding – the descendants all have the same phenotype. True breeding is
code for homozygous.

If you have diploid parents that are true breeding and they have different phenotypes,
then one parent has to be homozygous for one allele and the other parent has to be
homozygous for the other allele (one is homozygous dominant and the other is
homozygous recessive).

Homozygous dominant plants make dominant gametes and homozygous recessive plants
make recessive gametes. When you cross the two parents, you all of the F1 generation
will be heterozygous.

By looking at the phenotype of the F1 generation, you can tell which parent has dominant
phenotype and which parent has recessive phenotype.
Then we self the F1 generation and half of the male’s gametes is dominant and half is
recessive and same for female.

The F2 genotypic ratio of a monohybrid cross is 1:2:1 (homozygous dominant:

heterozygous: homozygous recessive)

The F2 phenotypic ratio of a monohybrid cross is 3:1 (three have dominant characteristic
and one have recessive characteristic). This is because all the probabilities of events are
mutually exclusive.

When we do an experiment, we set Mendel’s phenotypic ratio as the null hypothesis and
if it didn’t turn out like that, that doesn’t mean it didn’t go the way Mendel said, it means
that some of his assumptions have been violated.

Ex. White flowers x Red flowers make pink flowers (the F1 generation’s
genotype is heterozygous but the phenotype doesn’t take on the form of the dominant
allele).

When Mendel did his experiment, he didn’t know about chromosomes or meiosis. What
he said was that all individuals have two copies of a message that are controlled in each
character. When an individual reproduces, it only transmits only one copy of that
message to the offspring. What Mendel was describing was really the reduction division
in meiosis.

Mendel was studying autosomal chromosomes, not the sex chromosomes (X is the big
one and Y is the small one). If Mendel was working with a character that was on a sex
chromosome, he would have gotten different results.

26
In mammals, if an individual has two X chromosomes, it’s a female; if an individual has
one X chromosome and one Y chromosome, it’s a male. If a female produces gametes,
the sex chromosome in the gamete will only be an X. If a male produces gametes, it will
produce a sperm that has either an X chromosome or a Y chromosome.

Because the female only produces gametes like her own, we call her homogametic.
Because the male can produce different types of gametes, we call him heterogametic.

-in mammals, males are the heteogametic sex and females are the homogametic
sex. In birds and butterflies, it is opposite.

Fig. 5-8 a
Sex (X)-linked inheritance – when you’re studying traits that are on the X chromosome.

Examples of sex-linked inheritance phenotypes:

-eye color in fruit flies

-hemophilia in man
-Duchenne muscular dystrophy in man
-red/green color blindness in man

The wild type phenotype is the phenotype that is most normal (the most common
phenotype in a population for that character would be referred to as the wild type
phenotype). Alleles that give rise to the wild type are given a plus (+) as superscript.

If it doesn’t have a plus (+) superscript, then it’s the opposite of the wild type, also called
the mutant phenotype

In the picture, we have a female with homozygous wild type alleles crossed with a
hemizygous male.
-the male is not homozygous or heterozygous because he has only one allele for
this gene (they do not apply here).
The female is a true breeding form and is homogametic; in contrast, the male can produce
a gamete that has an X chromosome or a Y chromosome, which will be produced with ½
probability each. These are the fertilization events that will occur. We see that red is
dominant over white in the F1 generation. Then we mate the two F1 individuals together.
The female is still homogametic when she produces gametes, but she’s just heterozygous.
In the F2 generation, it just looks like a monohybrid cross with a 3:1 phenotypic ratio.
But it’s different because all the females are red eyed, and half of the grandsons are red
eyed and half are white eyed. We had nothing like this with autosomal monohybrid
crosses, we never had to worry about the gender of the F2 (both ¾ths of males and ¾ths
of females had the dominant phenotype; that’s very different from what we’ve seen here).
We have a different ratio so it has to have a different explanation, which has to do with
sex-linked inheritance.

27
Sex linked inheritance skip one generation. If there is a mutant in the grandfather then it
will occur in the grandson.

The way to determine if it a sex linked inheritance or not is by performing a reciprocal

cross. You reverse the phenotype of the male and female. For example when you cross
the female wild type with the male mutant you will have one result and when you cross
the female mutant with the male wild type then you have another result then you know it
sex linked inheritance.

W+ the plus is that allele is the most common form. Common form can be recessive or
dominant but most of the time is dominant.

Family tree is also called pedigree.

Fig. 5-9
A square is to symbolize a male and a circle is to symbolize a female. A shade square or
circle is mean that individual have a mutant phenotype.

Fig. 5-10
Propositus- is male with the mutant phenotype.
Proposita- is a female with the mutant phenotype.

We number each generation by Roman numeral and we number each individual in the
each generation from left to right. There is usually a lot recessive allele that is rarely
express in a population so they rarely cause problem. If related individual mate then there
is a greater chance that the parents are heterozygous for the same so there is a greater
chance the offspring will be homozygous recessive (phenotypic abnormal).

The recessive allele is harmful (deleterious) but they are recessive and most of the time
they are mask by a dominant form.

The mode of inheritance is to determine if the mutant allele is recessive or dominant.

And also is the mutant allele is a sex chromosome or an autosomal chromosome.

If a trait that is not express in the parents but is express in the offspring then it is
recessive trait. If it dominant trait then it will express in the parents.

If there is a recessive trait in the male then it will not be pass to the next generation
(offspring). It skip one generation.

Sex linked traits are appear more frequently in male than in female.

If the trait is recessive then it will appear in only one sex either male or female.

Ex. If both parents are heterozygous that the offspring does not have the mutant
phenotype then the probability that that individual heterozygous, carrying the mutant

28
allele, is 2/3. This is conditional probability- mean were calculating the probability using
an factor that we normally wouldn’t know (the trait is not homozygous recessive).

Fig. 5-12
Each person have two copies of their chromsome number 11 and so there are two genes
to make the enzyme for your skin color. If both genes are functional then the reaction will
occur to make melanin then you will have skin color. If only one functional gene on
chromosome number 11 then you still make melanin. But if both genes are nonfunctional
then you cannot make melanin so you are an albano.

Fig. 5-14
This is a dominant autosomal inheritance pattern because the parents have it that is why it
is dominant and it autosomal because it occur every generation.

Fig. 5-16
The black dot in the middle of the circle mean that the individual is a carrier but not
express the trait.

P. 137 Calculating Risks in Pedigree Analysis:

The parents of both sides are heterozygous so the probility of the son to be heterozygous
is 2/3 and the ratio of the grandson being heterozygous is ½ and the ratio of the great
grandson to be homozygous recessive is 1/36.

Charles Darwin- is a naturalist and try to explain the origin of species. He thoughts that
there is a perfect form for every species but is always a mistakes within the species. He
try to explain how organisms can look very different even though they in the same
species.

Darwin believe in the blending theory and try to explain how organisms in the same
species can maintained their individualism.

Mendel said that inheritance is particular in nature. When two individual mate together,
they maintained their integrity within the offspring.

Ch. 6

Genetic Recombination in Eukaryotes

Fig. 6-4

Nonhomologous chromosomes are independent of each other.

If you take a homozygous dominant form of two genes and cross with a homozygous
recessive of two genes. The result is dominant dominant, recessive recessive, dominant
recessive, and recessive dominant.

29
Fig. 6-5

Recombination Frequency – number of recombinant gametes divided by number of

parental type gametes plus number of recombinant gametes.
Parental-type gametes are the gametes that are the same as the gametes which that
individual’s parents contributed.
The best way to determine the genotypes of the gametes of a dihybrid diploid is to
perform a testcross. By crossing that individual’s gametes with a tester (which has
homozygous recessive alleles for its genes).
After the testcross, the offspring’s phenotype will not be affected by the tester’s
phenotype. It’s only affected by the phenotype of that individual’s gametes.

Ex. If an individual has a dominant form for a gene, then the dominant form will
appear in the offspring, and if the individual has the recessive form of the gene, then the
recessive form will be shown in the offspring.

The tester allows you to know the ratio of gametes that the individual is producing.
1:1:1:1 ratio (pg. 152). If the genes are on different chromosomes, independent
assortment will produce 1:1:1:1 ratio of gametes.

If the recombination frequency is not 50 percent then the genes are on the same
chromosome.

Fig. 6-7

Inheritance of two genes:

Round is dominant over wrinkled and yellow is dominant over green.

Mendel used peas that are true-breeding parents. He used peas with homozygous alleles.

Ex. In Parental, he cross RR / yy * rr / YY  Rr / Yy (F1 generation).

When you cross two heterozygous genes, there are four possible phenotype combination:
A_ B_ (dominant dominant)
A_bb (dominant recessive)
aaB_ (recessive dominant)
aabb (recessive recessive)

When you cross the two heterozygous genes you will get 9 that is dominant dominant, 3
that is dominant recessive, and three that is recessive dominant, and one that is recessive
recessive.

The F2 phenotypic ratio for a dihybrid cross is 9:3:3:1.

30
1 AABB 2 AABb 1 AAbb
2 AaBB 4 AaBb 2 Aabb
1 aaBB 2 aaBb 1 aabb

This is the genotypic ratio for a dihybrid F2.

If you violate one of Mendel’s assumption, then you would get different ratio.

Punnett Square:
The number of column and row is 2^n.
(n) is the number of heterozygous genes that you dealing with. (type of gene ).

Use the product rule of independent event

Genotype:
AaBbCCDdee x aaBbCcDdEe  AaBbCcDdEe

Aa x aa the probability of getting Aa is ½.

Bb x Bb the probability of getting Bb is ½.
P (Cc) = ½
P (Dd) = ½
P (Ee) = ½
Because all of these probability are occurring independently (one probability does not
affect the other one) so we can use the product rule for independent event.
½ of the time you will get big A multiply with 100% of little a on the other, then in the
end you will get ½ Aa.

We multiply when we have independent event (product rule).

We add when we have mutually exclusive event (summation rule). When one event is
occur that mean the other event could not have occur.

Phenotype:
AaBbCCDdee x aaBbCcDdEe  A_B_C_D_ee
½ ¾ 1 ¾ ½ = 9/64

When you cross with a homozygous recessive then you ignore the homozygous recessive
and just look at the probability of the other one.

When you cross with a homozygous dominant then you will always get the dominant trait
and never the recessive trait.

When you cross two heterozygous then you will get ¾ dominant and ¼ recessive.

31
Pg. 146 problem 25

If the parents do not express the trait and their offspring does then it is a recessive trait.
It not a sex link trait because both male and female can have it then it is an autosomal
recessive trait. If both parents don’t express the trait then we know they are heterozygous.
We usually consider individual marrying in to be homozygous wild type.

Test cross:
That the offspring that are produce in the test cross are produced in same ratio as
the individual that is being test crossed is produced gametes.

When you cross a heterozygous dihybrid with the tester then you will get gametes with
four different phenotype:
AaBb x aabb

A_B_
A_bb
aaB_
aabb

These are 1:1:1:1 ratio

We want to determine if the genes are assorting independently or not?

We have 15000 different genes and only 23 chromosomes and it’s possible that two
genes can be on the same chromosome.

Cis configuration- where the dominant forms of the genes are on the same chromosome
and recessive forms are on the same but different chromosome from the first one.

Trans configuration- where the dominant form of one gene and the recessive form of
another are on the same chromosome, and the recessive form of the first gene and the
dominant form of the second gene are on the same chromosome that is different form the
first one.

Cis ---A----B---
---a-----b---

Trans ---a-----B---
---A----b---

32
An individual with the cis configuration like above will go through meiosis after
duplicating it chromosomes it forms four gametes with two gametes types.

Two of these ---A----B---

and
Two of these ---a-----b---

Crossing over may or may not change the linkage arrangements between genes. If there
crossing over then there will be four types of gametes produced.

If the crossing over is at middle between the two loci ( two genes) the you would get four
different type of gametes. It depends on how far apart they are.

There is at least one crossing over for each homologous pair.

If the two are on the opposite end of the chromosome then crossing over will almost
always occur between them. If the two genes are really close together then there is a low
probability of crossing over occur between them.

Linked genes- are those that are on the same chromosome.

AABB x aabb
↓
AaBb
↓
AB This is parental form (greater than ¼)
aB This is recombinant form (less than ¼)
Ab This is recombinant form (less than ¼)
ab This is parental form (more than ¼)

The recombinant frequency of the recombinant is never more than 50% percent.

The number of recombinant is always less than the number of parental.

When the genes are sorting independently then the recombination frequency is equal to
50%.

You use the recombination frequency to determine if the genes are linked or if they are
assorting independently. Recombination frequency is less than 50% if the genes are on
the same chromosome (linked).

Fig. 6-9, 6-10, 6-11.

The recombination frequency tell us how many recombinants are produced relative to the
number of parentals. If the RF has a high value then the loci are far apart. If the RF is low
then the loci is close together. We refer to this as the map distance. The unit for map unit
is centimorgan (cM). If RF is 1% then the map unit is 1 cM. The higher the map unit the

33
more crossing over. There is more crossing over at euchromatic region than at
heterochromatic region. The map unit between any two loci is never more than 50%.

If you have double crossing over between the same two chromatids then it like
you have zero crossing over.
If you have double crossing over between three chromatids then it like have 50%
recombinant or recombination frequency.
If you have double crossing over between four chromatids then you have 100%
recombinant.

The average of 0%, 50%, and 100% is 50% that is why you will never get more than
50%.

First we were violating Mendel ratio because of sex linked genes. Now we are going to
violate Mendel ratio by linkage between the genes.

If there are two different recombinants and the recombination frequency is 20% then the
recombination frequency of each recombinant is 10%.

Pg. 162 – Three Point Cross (trihybrid test cross)

First you take two parents that are true-breeding and differ phenotypically between 3
genes. The trihybrid (F1) will then be heterozygous for all three genes. Then, you take
the trihybrid and cross it with a tester (homozygous recessive for the 3 genes). The
trihybrid will make eight different types of gametes which should all occur with the same
frequency. If the gametes do not occur w/ the same frequency, then there is evidence that
the genes are linked.

PAGE 162
A gene with a plus superscript is the wild type. A gene with no plus is the mutant form.

Notation:
v b+ p+ = v++ = v

a true breeding parents (with either homozygous dominant or homozygous recessive

alleles) when they cross together, each gamete from the parents combine to make the F1
individual.

P1xP2 OR P1xP2
Z| |Z x z| |z z| |z x Z| |Z
Y| |Y y| |y Y| |Y y| |y
X| |X x| |x x| |x X| |X

↓ ↓
F1: F1:

Z| |z z| |Z

34
 Y| |y Y| |y
X| |x x| |X
If there is crossing over b/w the y and z alleles, then
the recombinant gametes will be x-Y-Z and X-y-z

The F1 makes parental gametes in a higher frequency than the recombinant gametes
(frequency of the recombinant gametes add up to less than 50%).

If there’s a crossing over b/w the x and y alleles and another crossing over b/w the y and
z alleles, that’s called double crossing over.
The probability of crossing over b/w y and z is less than 1; and the probability of crossing
over b/w x and y is also less than 1. So the probability of them both simultaneously
occurring is the product of each individual crossing over (so you get a double crossing
over).
Of the eight phenotypes of the trihybrid gametes, the 2 with the highest frequency are the
parentals. If there are two gametes w/ very low frequency compared to the parentals then
they must be because of double crossing over.

Linkage arrangement of the genes is the first question to answer on TEST 1 (order of the
genes along the chromatid). In this example, the parental linkage arrangement is z-Y-x
and Z-y-X. The two double crossing overs in this example are z-y-x and Z-Y-X. First
you pick one of the two double crossing overs and compare it with the two parental
gamete types to see which parental form shares two of the alleles in the double-crossing
over recombinant. The allele that they do not share is always the centrally located locus.
So by looking at the results of the F1 testcross, you’re able to know which alleles are
linked together and which of those three genes are located in the middle.

The four F1 gametes in the middle with frequencies that are not the most or the least are
due to single-crossing overs.

v|| ||v+
||x||
p+|| ||p
||x||
b+|| ||b

To calculate the map distance between v and p, we add up all of the frequency in which
crossing over occur between v and p that include single crossing over and double
crossing over. Then you divide the sum by the total frequencies (gametes).
Single crossing:
v p b frequency is 89
v+ p+ b+ frequency is 94

Double crossing:
v+ p+ b frequency is 5
v p b+ frequency is 3.

35
The map distance between v and p = (89+94+5+3)/1448 x 1000 = 13.2

The map distance between p and b is the same thing.

(45+40+5+3) /1448 x100 =6.4

To get the map distance between v and b, you just add up the two map distance between
v-p and p-b. So 13.2 + 6.4 = 19.6

Crossing over does not have to occur independently and these are control by an enzyme.
So when we analyze the three point cross we assume they are occur independently to one
another.

One way that crossing over do not occur independently is when one enzyme that attached
to the DNA to start crossing over at one point will tend to inhibit the crossing at the other
point nearby.

Another way is that the euchromatic region is where actively transcribe and the
heterochromatic region of the DNA that are dense up. So crossing over occur more
frequently in euchromatic region than in the heterochromatic region.

Coefficient of coincidence = observe double crossing over divide by expected double

crossing over. Assuming independent.
First we change the map distance between v-p and p-b from percentage to decimal
by dividing by 100. So 13.2.132 and 6.4.064
Expected probability of double crossing over = .132 x .064

Expected number of double crossing over = expected probability x total frequency

of offspring. = (.132 x .064 ) x 1448 = 12.2

Total observe double crossing over = 5+ 3 = 8.

Coefficient of coincidence (COC) = 8 / 12.2 = .65

Coefficient of interference = 1 - coefficient of coincidence. 1 - .65 = .35

If the coefficient of interference is positive then the coefficient of coincidence is
saying that crossing over at one location will inhibit the crossing over nearby.
If the coefficient of interference is negative then the observe double crossing over
is larger than expected double crossing over then we will have negative coefficient of
interference so that mean that there is a greater chance that the other crossing over will
also occur.

Fig. 6-12
The two point cross will give us the distance between two genes a and b or a and c but
you don’t know the arrangement of the three genes. So you need to use the three point
cross to figure it out.

36
Scientific method:
Scientific methods require that you formulate a hypothesis in such a way that the
hypothesis can be falsified. When you falsified a hypothesis, then you can make at least
one strong statement (null hypothesis is wrong).

When you reject the null hypothesis then you generate a alternative hypothesis and use it
as our new working hypothesis.

If your alternative hypothesis is not disprove after many experiments then you start to
believe it might be true.

We use statistic to determine if our empirical observation are consistent or inconsistent

with Mendel’s ratio. The ratio of our experiment will never be exactly the same as
Mendel’s ratio but how different do they have to be before we reject the hypothesis.

When we utilize scientific method, we going to create indices and if the index is
sufficiently high or sufficiently low that mean we have extreme result and we need to
reject the hypothesis. Our hypothesis could be true with these extremes differences when
comparing the result but the chance of it being true is very low.

We going to set criterion- set the probability of making type I error. We accept a five
percent chance of making mistake by rejecting the null hypothesis even those it’s true.

Ex. A monohybrid cross to give a phenotypic ratio of observation = 80 and 20 but our
expect ratio = 75 and 25 (3:1).

The index value tells us how close our observation is to our expectation.
Chi square index (χ 2)= (observe – expected)2 / expected
Ex. [(80-75)^2 / 75 + (20-25)^2 /25 ] =
So we do chi square for each phenotype and add them up.

For the coin example, you want to figure a value (63 head out 100) that you willing to bet
with him. So add the probability of getting 100 head + p(99) + p(98)… you continue to
add all the probability until the get a value of .05. The chance of getting 63 or more is
only five percent. If we get 63 or more heads then we reject the null hypothesis and we
get under 63 heads then we accept the null hypothesis.

Type I error- is when we reject the null hypothesis even though it is true. We willing to
take a 5% chance of incorrectly rejecting the null hypothesis.

Table 6-1:
Each row represent is a different set of theoretical chi square distribution. Degree
of freedom is the number of phenotype minus one.
A monohybrid cross have two potential phenotype and a dihybrid have four
potential phenotypes. So a monohybrid have 1 degree of freedom.

37
 If the chi square value is greater than 3.841 then we reject the null hypothesis and
if it lower than 3.841 then we accept the null hypothesis.

Type I error is when you reject the null hypothesis even though it really true.
Type II error when you accept the null hypothesis even though it’s really wrong.

If we used .01, then we have a greater chance of making type II error.

If you reduce the chance of making type I error then you will increase the chance of
making type two error.

Pg. 167 and 168

AbBb With this heterozygous genes, we want to see if these genes assorting
independently or not?
1. First we test cross it and look at the phenotype of the progeny and if they all
have the same ratio then we know that they are assort independently.
AaBb x aabb  112 AaBb: 105 aabb: 96 Aabb: 97 aaBb
↓ ↓
AB x ab  AaBb
Ab x ab  Aabb
aB x ab  aaBb
ab x ab  aabb

If the genes are assorting independently then the offspring phenotype

should be 1:1:1:1
The total frequency of the offspring is 400.
If they are not assorting independently then the number of recombinant is
less than the total number of parental.

χ 2 = (112 –100)2 / 100 + (105 –100)2 / 100 + (96 –100)2 / 100 + (97 –100)2 / 100 =
1.94

We take that 1.94 with three degree of freedom and compare with the critical chi square
of 7.815 with five percent error. As a result we accept the null hypothesis that they are
assort independently.

End of Test One:

If we see that it follow Mendel ratio and we see that one genotype occur less frequently
that could be because it has a higher mortality rate and it doesn’t mean they are not
assorting independently.

38
Problem 30 on page 182:

a. When we first look at the problem we see that there are four phenotype so we
know that there are two genes that are heterozygous and one must be homozygous of
something. Then we look at the phenotype of the offspring and we see that all of them are
wide so we know that they are homozygous dominant.
We know they are linked genes because they do not occur 1:1:1:1 ratio.
By looking at the highest frequencies, we know which are the parentals. From the
parentals we can tell if they are arrange in cis or trans configuration.
T| | t
r| | R

From the parentals, they are in trans configuration because on one parent we have
tall and white and another parent is short and red.
We cannot find the map distance for the width gene because they occur in every
offspring so we cannot use crossing over frequency to tell how far apart it is from another
gene.
Next we can calculate the map distance between height and color by using
offspring frequency and it come out to be 4 map unit (in percentage).
b. Now we self the parent TtRr x TtRr.
We want to know the probability of getting short, white, and wide individual.
From the map distance, we can see that the chance of crossing over occurring and we end
up with short white gametes is (.02). .04 is the total frequency for all crossing overs so
the frequency of crossing over between t and r is (.02) so .02 is the probability of one
parent making short and white gametes. The probability of the other parent of making the
same gamete is also .02
We use the product rule because the gametes are assorting independently.
.02 x.02 = .0004

Page 146 problem 24

10/06/03
Chapter 8 – Recombinant DNA

Recombinant DNA – combine DNA from one organism with DNA from a different
organism

One organism is a donor. Recipient organism is the vector.

Fig. 8-2 a
Melting the DNA – when you separate the two sugar-phosphate backbones by heating the
DNA up so the H bonds will break before the strong covalent bonds.

If you cool the DNA down again, then the two single strands will find their complements
and will re-anneal (combine).

39
You can also add RNA strands to the DNA solution and then some DNA will bind with
the RNA; one DNA strand combines with an RNA strand.
-when you cool it down really slowly, then the DNA will only combine with an
exact complement (only with another DNA strand). This is called a high stringency
condition.
-in constrast, if you melt the DNA and then cool it down a lot, then 2 sugar-
phosphate backbones that are almost complementary can still come back and bind
together. This is called a low stringency condition.

Pg. 219, Fig 8-6

An endonuclease is an enzyme that digests DNA (or a nucleic acid) right in the center; it
doesn’t need to start at an end
Restriction Endonuclease – an enzyme produced in bacteria
-it’s a defense mechanism of bacteria to avoid being
attacked by viruses.
Phage – a virus that attacks bacteria
A virus injects its nucleic acid into the bacterium. That nucleic acid makes many
copies of itself. And then it makes many proteins that assemble the viruses. The
bacterium dies and then the viruses are released.
If a restriction endonuclease finds nucleic acids inside the cell that have recognition sites,
then it cuts up the virus nucleic acid so it won’t damage the bacterium.

Pg 219 - The identifying site that we’re going to cut is called a palindrome. Palidrome-
is means that both strands have the same nucleotide sequence but in antiparallel
orientation. GAATTC on one strand and CTTAAG on the other strand. After you cut,
you create sticky ends on the end of the DNA. Why? Because if you give them a chance,
then they’ll come back together. You need a ligase to fuse the backbones together.
When the restriction endonuclease cut, it doesn’t cut straight down but instead it make
staggered cuts.

We name each restriction endonuclease by the name of the organism in which it was
isolated from.
Example: EcoR1, EcoE. coli, Rrough strain of E. coli, 1first restriction
endonuclease that was isolated from E. coli

EcoR1 always searches for the same recognition sequence in any DNA. There
can be more that one sequence in the same strand.

The DNA strand is cut by a restriction endonuclease and inserted into a plasmid.
The sticky ends of the plasmid join with the sticky ends of the DNA so now you
create a DNA that didn’t exist before. You can then make many copies of the
new DNA.

Pg. 218 Fig. 8-5, pg. 220 Fig. 8-7

40
 Ex. If you isolate a gene that makes insulin and insert it into a plasmid vector,
then the plasmid will start making insulin. The problem with using prokaryotes to
make eukaryote protein is that eukaryotes have split genes (introns). So the
prokaryotes can’t remove the introns and that means the protein will not be the
same because it also includes intron codes.

What type of DNA do we want to introduce into the recombinant DNA; is the
DNA from the genome of the organism? If it is, then it will include the introns
from the DNA. We want to remove all of the introns from the donor eukaryote
DNA.

What type of DNA do we want to use?

Pg. 217 Fig. 8-4

Using an enzyme called reverse transcriptase. It uses a mature RNA
molecule as a template to synthesize a complementary DNA strand.
Synthesize a nucleic acid with a poly-T as a primer. The poly-T is
complementary to the poly-A on the mRNA. With the poly-T, you now have a 3’
end to extend from and make DNA.

Solution:
The poly-T is complement to the Poly (A) tail on the mRNA. The Poly-T serve as
the primer for reverse transcriptase to elongate the 3’ end of the DNA strand. It also make
a hair pin loop and then DNA polymerase extend the 3’ end of the hair pin loop. Then
you digest away the hair pin loop with endonuclease. Now we have a double-stranded
DNA without any introns. This represents the DNA form of the gene of a polypeptide
without any introns. It’s called complementary DNA or cDNA.
You have to know when you want to use genomic DNA or cDNA.

10/08/03

Fig 8-9
-circular DNA acts as a vector.
-If you have two different segment that have the same sticky end in the donor
DNA then by chance, sometimes segment 1 or segment 2 will be incorporated
into the vector.

Transformation – process where DNA goes into the bacterium. The DNA is
naked DNA (DNA that’s not associated with histones). Both the bacterial
genome and the plasmids replicate themselves once the plasmid is in the
bacterium. Then the bacterium replicates thru binary fission so you wind up with
millions of copies of that bacterium that all have the plasmid with the
recombinant DNA. All of the bacteria are in one big soup. Then you take a Petri

41
 dish with growth agar and streak the bacteria onto the surface so you create
colonies of the bacteria. Each colony originated from a single bacterium. Some
colonies have the recombinant DNA and some don’t.

Page 224, Fig 8-10

Two systems that show how to we use plasmids as vectors to make clones of
recombinant DNA.

One system is called pBR322 and the other is called pUC18. p stands for
plasmid. Plasmids often are where the drug resistance of bacteria reside. When a
bacterium is exposed to antibiotics for a long period of time, it becomes resistant
to it due to certain gene products that are produced by genes that reside on
plasmids.

pBR322
In pBR322, we’re using plasmids that have resistance against 2 types of
antibiotics: tetracycline and ampicillin. Purple region is the gene that has
resistance against tetracycline. Dark green is the region for ampicillin resistance.
For each gene, there are recognition sites where the restriction endonuclease cuts.
There is also an origin of replication for the plasmid  ori. We have an
endonuclease that cuts the donor DNA and also cuts in the purple region for
tetracycline resistance.
We split the message of tetracycline resistance and add some other message so it
will be sensitive to tetracycline. Now we know that the plasmid with the donor
DNA is tetracycline sensitive. If it’s not sensitive, then we know that the plasmid
vector DNA just rejoined itself after being cut by endonuclease. Now we put all
the plasmids in bacteria and expose the bacteria to ampicillin. If the bacteria are
transformed, it has a plasmid in it with ampicillin resistance. But we don’t know if
the plasmids are sensitive to tetracycline or not. If we expose the bacteria to
tetracycline and if the bacteria die, then we know that they have the recombinant
DNA.

But we don’t want them to die, so what we have to do is something called

replica plating. We take a nitrocellulose membrane and place it on top of the
Petri dish and then peel it off. Now we have a mirror image of the growth plate
on the membrane (some of the bacteria are moved to the membrane). Then we
expose the membrane to tetracycline and any colonies that die are the ones with
the recombinant DNA. Now we know which colonies on the Petri plate to isolate.

pUC18
pUC18 is a faster method to use than pBR322. We use a different gene
called β-galactosidase which breaks down galactose into glucose and another
sugar. If we have β-galactosidase cut a mimic of galactose, then it produces a
product that is blue in color. If we took this vector (the plasmid), add it in a
bacterium and expose it to ampicillin, then the bacteria without the plasmids will

42
 die, leaving us only with the bacteria that have the plasmids. If we provide the
bacteria with a galactose mimic molecule, then the bacteria with the plasmids that
don’t have the donor DNA would digest galactose and release a blue coloration.
Therefore any colony that was digesting galactose would be blue. We don’t want
the blue colonies because the plasmid just rejoined after being cut. But if we have
an endonuclease cut the region for β-galactosidase and insert a donor DNA
(before we add ampicillin), then the β-galactosidase will be inactivated and the
colony will be white instead of blue (after we streak it on a growth plate). This is
how we know that we’ve got recombinant DNA.

Plasmids are usually good vectors to use for stretches of DNA up to ~20,000
kilobases.
Other vectors we can use for recombinant DNA are phages. Phages are viruses
that attack bacteria.

Page 225, Fig 8-11

We’re going to cut up the genomic DNA and what we have are a region called
phage lambda. The yellow region of the phage DNA is not necessary for
function of the phage. We’re going to open up the phage and get 2 sticky ends
and use another endonuclease to cut the phage on either side of the yellow region.
We cut this phage into 3 segments, and then what we can do is eliminate by bases
of its density the yellow region, remove it from a mixture of lots of these phages
and then we have a mixture of phages with only the green and purple regions. We
then mix the green and purple regions of the phage and hope that the blue donor
DNA will fit in place of the excluded yellow region. We use restriction
endonuclease that is similar to the one that cut the yellow region to cut the blue
donor DNA (they both have the same sticky end). Then we will have a
concatamer (rolling circle model) of the recombinant DNA. We can then pack up
the concatamer to make the nucleic acid of the phage. It will load itself into a
capsule (protein coat) that is the outer portion of the virus. The phage will then
inject its DNA into a bacterium and the bacterium will now have the recombinant
DNA. Then the bacterium replicates to make many copies of the recombinant
DNA. You will be able to detect which bacterial colonies have the recombinant
DNA and the phages because after a while, they will cause the bacteria to burst.
You can then remove some the growth surface with the phages and the ones that
attacked this particular colony are going to contain one region of the donor DNA
in it. Phages can contain donor DNA up to 20 kilobases.

If we have a long length of DNA to clone then we have to copy many segments of it to
make clones of the long DNA strand. You can make clone a lot of small segment or you
can clone fewer long segments of the donor DNA.
There are a variety of other cloning vectors.
-a cosmid is a combination of a plasmid and a phage. You can take the minimum
amount of DNA that’s necessary to lower the nucleic acid of the protein coat. That’s the
only part of the phage with the only DNA structures of phage (viral DNA). Then you

43
bind that with a plasmid so that you get the origin of replication from a plasmid and that
is called a cosmid. A cosmid can accept large stretches of donor DNA (~35-45 kb).
They can accept up to 2-3 times of DNA that a phage can hold. So cosmids allow you to
do only half or a third of the work.

Other cloning vectors are called BACs, PACs, and YACs.

BAC – bacterial artificial chromosome

PAC – plasmid artificial chromosome
YAC – yeast artificial chromosome.

BACs and PACs are very similar but BACs use a plasmid called an F-Factor and PACs
use something other than an F-Factor. They all have to have origins of replications and
they all have to be able to get in and out of cells. BACs are used with the greatest
frequency; they can take donor DNA up to ~300 kb.

YACs – yeast are eukaryotes. You’re going to create an extra pair of homologous
chromosomes. So you need centromeres, telomeres, and origins of replications for a
functional chromosome. If you incorporate some donor DNA into that then it will
replicate just like any other chromosome. YACs can take large stretches of donor DNA.

10/10/03

The polymerase chain reaction called PCR, it lets you make a large number of copies of
short lengths of DNA in vitro (outside life).

Fig. 8-8 The polymerase chain reaction called PCR

a) You heat up the DNA at 90 degree Celsius to separate the

backbone, then you cooled to 50-60 degree Celsius.
b) You have two primers, one for each strand. The DNA between
the two primer is what we’re making copies of. The faster you
cool the less stringent it will be (RNA primer will bind to the
DNA.
c) Use Taq polymerase (which is a DNA polymerase don’t
denature because their peak function is at 72 degree Celsius) to
extend the primer with DNA that is complementary to the other
strand.
d) Then you heat again to isolate the new copies and cool it to add
the primers again.
e) When Taq extends the primers, the new strands are what we
want many copies of.
f) Then heat again and cool again to add primers again. (There is a
sequence on the first strand that have a complement the bottom

44
 primer. On the bottom strand, there is a sequence that the top
primer can attach to.)
g) Now every time you repeat the process, you will get copies of
the same length of DNA that you want.

Taq stand for (thermus aquaticus).

Taq polymerase read the DNA in 3’ to 5’ and synthesize in 5’ to 3’.
You use a thermocycle for the PCR to heat the DNA.
Now how do you get the primers for the PCR reaction?
In humans, primers are developed in equal length and are published so you can
get primers that will replicate certain regions in the genome. Or you could make and use
primers that are conserved between very different organisms. You could also take donor
DNA, chop it up randomly with restriction endonucleases, insert that donor DNA into a
plasmid. You don’t know what donor DNA is inserted but you know the plasmid, so you
cut the plasmid around the inserted DNA. You know the sequence of the plasmid, so
then you use the plasmid to generate primers and make many copies of the little plasmid
DNA.

Gel Electrophoresis (it’s a technique)

Refer to picture in notes.

The gel used is named from the different types of material such as agarose or ?trilamide?.
You apply electrical potential to the gel. You put a positive charge on the bottom and
negative charge on the top. Then you put a solution material in the wells at the top of the
plate. If you put nucleic acids in the well, you know that nucleic acids are made up of
nucleotides that have a phosphate which is negatively charged, so the nucleic acids are
attracted and begin to move toward the positive charge of the voltage source.

If you magnify the gel, you see that it’s made up of solid particles with spaces between
them. The nucleic acid has to snake its way between the particles, so the smaller
molecules move faster thru the gel and migrate further than the larger molecules. You
can’t see the nucleic acids, so you could stain them with ethidium bromide. If you run it
for an hour, then the cluster of nucleic acids will separate and you see the individual
bands. The positions of the bands in the same lane are a function of their size and how
long you’ve exposed them to the electrical charge.

One way to separate the 2 strands is by heating them or changing the chemical
environment around them. If you put the DNA in a hydrophobic environment, then
the 2 strands will separate.

Pg. 233, Fig 8-18

What we’ve done so far is use PCR, phages and plasmids to make many copies some
double-stranded DNA to be processed. We don’t know if the DNA we’ve made copies of
is the DNA we’re interested in. We’re randomly cutting up the DNA and by chance,

45
they’re incorporated into the vector. We don’t know if it’s the DNA we’re interested in.
So we develop probes to identify it is the DNA we’re looking for.
We label the probes (short stretches of nucleic acids) with radioactivity and the
probes are complementary to a region of DNA we’re interested in. We create a region of
nucleic acids that we know is complementary to the region we’re looking for in the DNA.
We radioactively label that probe and then we melt the DNA and allow that probe to bind
to the DNA. If it binds to the DNA, then we know that the DNA has the region we’re
looking for.

Southern Blotting Fig. 8-18

We chop up DNA, put it in an electrophoretic apparatus and expose it to an

electrical charge and allow their migration. But the DNA is invisible so we know the
bands are there but we don’t see them. Then, we remove the electrical charge and put the
gel in a tank with a (membrane) filter on top of the gel and paper towels on top of the
membrane.
The filter is a nitrocellulose filter. You put a solvent in the Petri dish. The
solvent migrates up the paper towels thru a process called imbibation. As the solvent
moves thru the gel, it dissolves some of the DNA and the DNA is pulled up as well and
some of the DNA is caught into the nitrocellulose filter. The DNA has the same pattern
as the underlying electrophoretic gel. You take the filter off of the gel and put it in a
plastic bag filled with radioactive probes, short sections of nucleic acids complementary
to the DNA we’re looking for. The probes will bind to the DNA complements on the
filter and you have radioactive regions on the filter. Now you take the filter out of the
bag, wash it and only certain bands will be radioactively labeled. You take a
photographic plate used for X-rays and the X-rays will expose the photographic plate.
So you get an autoradiogram that exposes where the probe is bound to on the
filter; and the filter is a duplicate image of the electrophoretic gel. So now you can circle
where the DNA is on the electrophoretic gel, and then you can cut out the piece of gel
with DNA in it (isolate the DNA you want).

Southern Blotting is named after a man called Southern. Then someone

discovered you could do it with RNA also, and that’s called Northern Blotting.
Western Blotting – where you do it for proteins.

Where do get the probes from ?

The sequence of gene in one organism sometime can be similar to sequence of
gene in another organism. You can get a stretch of nucleic acid synthesize for you that
have that particular sequence. You radioactive that sequence and then use that as a probes
to find out where that gene is in human. You can use that probe to identify the DNA that
is coding for same polypeptide for human. The polypeptide are not identical but very
similar between different organisms.

Sequencing DNA
Pg. 237 fig. 8-22

46
The molecule we see is called dideoxynucleoside triphosphate (dideoxynucleotide) that
have no oxygen on the 2’ carbon and 3’ carbon on the sugar. When we synthesize DNA,
take 3’ end we add new nucleotide to it. If the last nucleotide added is dideoxynucleoside
triphosphate then you can’t extend the DNA anymore with the polymerase. This is the
basis of the Sanger method for sequencing DNA.

What is the sequence of the DNA that we make a lot of copy of ?

We make a lot of copies of the DNA and added in a solution and we melt the
DNA chemically to make single strand. We used an RNA primer that is radioactively
labeled. We used the single strand as the template and extend the radioactive primer with
DNA polymerase. Then we added deoxynucleoside triphosphate for A, T, C, and G
(ATP, TTP, CTP,GTP). These are what the polymerase needed to extend the chain. In
addition to that, we take dideoxynucleoside triphosphate for A ( called ddATP). Now you
have two types of ATP, the normal kind and the ddATP. If the DNA template sequence is
CTGTAAC, then there is a chance that the ddATP could bind with either the first T or the
second T in the sequence. If the ddATP does bind, then the strand synthesizing will be
stop there.
You have alooooot of DNA template strands then if the ddATP add to the first T
then you would have a shorter strand than if it add to the second T. So everytime we have
a T on the template then we will have some strand that is will end there.
Now these new strand (with ddATP at the end) are put in well of the
electrophoretic gel and run it. The electrophoresis showed us where the T are on the
original template strand. If you do the same for dideoxy A, dideoxy T, dideoxy C, and
dideoxy G then you will figure out the sequence of the template strand.

Fig. 8-22 b

This shows where the newly synthesize strand ends. The shorter length of the newly
synthesize strand is the 5’ end because we read the DNA strand from 3’ to 5’ and we
make a new strand that is 5’ to 3’.
The newly synthesize strand is 5’ TGCGGG…..3’
The sequence of the template strand is anti-parallel complement is 3’ACGCCC….5’.

The strand that is in the figure is the template strand.

10/13/03

Fig. 8-24
This is automatic sequencer printout. The machine put fluorescence dye on the
nitrogenous bases that how you distinguish the different bases. A computer will search
for the start codon (AUG). But the computer actually looks for TAC on the template
strand of the DNA, b/c TAC is the complement of AUG on the mRNA. You have to run
each strand three times because the other two bases in the first codon could be base
position 1. In a double-stranded DNA molecule, sometimes one strand has the message
and sometimes the other strand has the message served as the template. The promoter
tells us which of those strands are meaningful. So you have to run both strands 3 times.

47
When there is AUG on the strand, then you find a potential open reading frame,
basically mean you have found something that has the potential to be a gene. There are 3
stop signals and if you find a start codon right next to a stop codon, that means the
polypeptide will be very short; open reading frame is unlikely to have a message for a
polypeptide, it could just be random noise. A good open reading frame has the start
signal separated from the stop signal by several hundred bases. Some genes are very long
b/c of the introns (gene for dystrophin ,that has a mutation in it, is 2 million bases long).
That’s why it’s very difficult to find a whole gene in eukaryotes because of all the
introns.

Pg. 235; Figure 8-20

Restriction Fragment Length Polymorphisms
We want to map fragments by locating where cutting sites for restriction enzymes
occur.
If you have a long double-stranded DNA molecule and expose it to a restriction
endonuclease, the endonuclease was isolated from E. coli. This is our DNA, but
by chance we have the same sequences as E. coli (that are recognition sites) that
tells it where to cut. If it cuts in four locations, then we get 5 DNA fragments.
First you mix the DNA with the isolated restriction endonuclease from a
bacterium. We have no idea what’s going to happen. So we run the contents thru
an electrophoretic gel and stain them with ethidium bromide. Then we will find
the 5 segments shown as 5 binds.
In the left column, you see 3 dark bands that occur from one restriction
endonuclease. Now you take the donor DNA and expose it to a different
restriction endonuclease; you can tell the enzyme cuts only one time b/c you see 2
bands (middle column). Finally, we do the same all over again but add both
enzymes simultaneously so the DNA is cut by enzymes 1 and 2 at the same time.
We want to make a map of the DNA. We would like to take the DNA molecule
and put recognition sites where they would be located along that DNA. We
would have to be able to figure out where one site is relative to another site.

We have this 8 kb length that was produced by the enzyme 1 cut. Enzyme 2
didn’t anywhere in there, so we get that entire length in the double cut. This 8kb
band existed in a single cut and it also existed in a double cut, what does that
mean? It means that the enzyme (enzyme 2?) that did not produce the single cut
could not have cut within that region. So in other words, the cutting location for
enzyme 2 could not have been in here, within this region. So lets look at this one
(6 kb); this length exists in a single cut and it exists in a double cut, so enzyme 2
could not have been cutting in this region. So lets look at this one (3 kb); this
exists in a single cut and it disappears in a double cut so that means enzyme 2
must be cutting in this region (it’s cutting site must be in this region). Now let’s
look over here, if we look at the single cuts for enzyme 2, this band is for enzyme
2 but it disappears in the double cut; that means enzyme one must be cutting in
this (10 kb) region. It’s the same for the 7 kb region (enzyme 1 must be cutting in

48
 that region b/c the 7 kb region disappears in the double cut). So those are your
clues; now you have to come up with the map which shows where the cutting
sites are for the enzymes in their relative positions. So what you could do to start
is assume perhaps the largest section here that’s not cut is at one end. Then let’s
try alternative arrangements. What you have to do is see if your pattern is
consistent with your information by trial and error. If in the double cut, the band
disappears, you know the 2nd enzyme has to be cutting in that region. If in the
double cut, the band is maintained, then you know the 2nd enzyme can’t be cutting
in that region. This allows us to see that this restriction endonuclease cuts DNA
right here. Now that could be useful b/c we might find that there might be
something else we could follow that was linked closely to the cutting site of
DNA. Suppose you have a mutation for a gene and you have no idea where that
gene occurred. But you know you can get an individual that expresses some
genetic disease. If we see that the gene is linked closely to one of these cutting
sites, then we could use that cutting site as a proxy for whether or not an
individual is going to have that disease.???

10/15/03
CHAPTER 9

Fig 9-3 (a)

We want to develop markers along the length of the chromosome and we do this by
creating molecular markers. We get lengths of DNA; we want to get different lengths
of DNA that cover the whole length of the DNA (they overlap and this is called a
physical map). We want to see which molecular markers reside in which clones. If
we find a gene that is of interest to us and we find that it is closely linked with some
molecular marker, then we can say that the molecular marker is also on this segment
here so possibly if we want to study the gene in detail and know the sequence, we
don’t have to sequence the entire length of DNA, we can just work with this subunit.
The 1st part of the chapter talks about how we get this
The 2nd part of the chapter talks about how we’re going to interpret that database and how
we’re going to go about searching for genes. How we’re going to figure out which
region of that sequence codes for polypeptides because it’s the polypeptides that regulate
the chemical reactions.
This is a very confusing chapter. Now we’re going to talk about molecular markers and
first we’re going to talk about restriction fragment length polymorphisms.

(picture in notes)
This is a DNA and we have a restriction endonuclease that cuts in four cutting sites,
so we get 5 different lengths here. The DNA has to have a specific sequence, usually
a palindrome, that the endonuclease will recognize the cutting site. Since we have
homologous chromosomes, the 2 DNA molecules are very similar because of the
same genes but since they have different messages, so the DNA can also have some
differences. Now if these recognition sites are such that this site here has mutated and
does not exist in this DNA strand, we will get only 4 lengths when it’s cut with a

49
 restriction endonuclease. And this length is different than any other length we’ve
seen before. So we can an organism like this as if it was heterozygous for this locus.
But we can identify this DNA molecule b/c if we ran an electrophoretic gel, it would
generate 5 bands. We could run this, and it would generate 4 bands. And we get a
band corresponding to this and 2 bands corresponding to this, and we could treat this
as if this was a heterozygous individual. The allelic form is not something we can
recognize visually, but it’s something we could recognize using electrophoresis to try
to separate these bands.

Fig. 9-5
So to see what we would do here, what we’re going to do is take an organism and this
organism has a DNA molecule with 3 recognition sites that cut here, here, and here.
This is the same DNA where this central recognition site is missing. For us to
visualize the DNA (remember the DNA is invisible to us when we run an
electrophoretic gel unless we label it somehow) sometimes we label it with
radioactivity but we always need a probe. So we get a probe that is a stretch of
nucleic acids that’s complementary to a stretch of nucleic acids in the DNA we’re
searching for. [picture] let’s say this is the bottom one and let’s say this is the
recognition site that the endonuclease attaches to. We put the DNA in our
electrophoretic gel, we label a probe that will be complementary to this region but
this will be radioactively labeled (the phosphorus are radioactive). The probe will
bind to this region here, so we cut this up and the probe binds. So what will happen is
this segment from here to here is going to migrate a certain distance in the gel and it’s
going to display radioactivity so we can visualize that in an autoradiogram. [Back
to figure] So look at what’s happened here; here’s that single band here, we’ve
recognized it. The probe is bound to a region and is going to be on either side of
where the cut actually is. The point is there was no cut here so we get single bands.
When we use that same one on this DNA strand, this strand is going to be cut so that
is opens right over here and the probe can attach either here or here. So we will get
some probes that will bind with each segment of DNA. So where it’s cut, we wind up
getting 2 bands. So when you have the recognition site existing, we’re going to get 2
bands; when the recognition site is this here, you’ll only get 1 band. Now what that
does is create a genetic locus that we can identify and look for things that potentially
might be linked to it. So suppose we’re studying a homozygous recessive genetic
disease. And we find that individuals that are homozygous recessive are tightly linked
to this recognition site, so what we can do is if we cross an individual that’s
heterozygous with a homozygous recessive (a testcross), we’re going to get offspring
like this.

The heterozygous individual produces this single band down here because it’s a
diploid individual, one band b/c it doesn’t have the recognition site and 2 addition
bands b/c each of these are cut; so we 3 bands for the heterozygous individual but
with the homozygous recessive individual we only get 1 band. So you do that and the

50
 offspring are either going to have 1 band or 3 bands; and you look at that
phenotypically and you see that they express the trait or they don’t express the trait.
But look at individual #8 here, it is heterozygous so it should express 3 bands but it is
only expressing one band. Why, b/c it indicates a recombinant. So by that how far
away this gene that’s causing a genetic disease is from the site that you’re studying,
you take the # of recombinants over the total # of individuals and it gives you the map
distance away; and we get approximately how far away this genetic disease gene is
from one of your molecular markers. So if there’s very little crossing over, it’s very
close to your marker. So you may not know anything about the structure of this gene
that’s causing a genetic disease, but you might be able to say you do know that it is
near this particular maker that you’re studying. And with that information, that
marker may allow you to then get to a segment of DNA that is cloned which then you
can engage in more thorough investigation that smaller segment of DNA more about
the gene that is associated with this disease. This is called restriction fragment
length polymorphisms. What do you have to have to make this work? You have to
have for restriction sites, you have to have existing within the population two forms
of it. One that will be recognized by the endonuclease and one that won’t be
recognized by the endonuclease and you have to have a probe that will extend to
either side of the actual cutting site. The problem with this is it’s not easy to get
these. And what happens is not only is it not necessarily easy to get these, but
individuals tend not to be heterozygous for them. In other words, one of these forms
tends to be overwhelmingly more common, so you don’t get a lot of heterozygosity,
but it’s still surprisingly useful b/c now we’re able to put markers along the DNA that
something other than markers for blue eyes vs. brown. The phenotypic markers that
we have, these genes that we recognize by their phenotypes are really few relative to
the total amount of DNA that we have. So we struggle to add additional phenotypes;
that’s what this basically is, is adding additional phenotypes so we can recognize even
though it’s an elaborate way to recognize.

DNA Fingerprinting

So what you have to recognize is a very common where you have a sequence like this
[notes] where it’s repeated. We have ATCT 3 times, so where you have repeat sequences
in DNA, there’s a tendency during replication for these to have duplications or deletions.
This is showing you 3 copies of four nucleotides, if you have a situation like that and
you’re looking at the homologous chromosome now, instead of having 3 three copies of
it, the homologous chromosome may have 5 copies. And that’s b/c during replication,
these repeated segments are either duplicated or they lose both their copies. What this is
called is an sslp (simple-sequence length polymorphism). The thing about this is could
treat this as one allele and treat this as a separate allele and what happens for these sslp’s,
instead of it being just one allele of 2 alleles, it may be within a population of 15 alleles,
and what happens is b/c there’s so many different forms, each of these forms are
relatively common so there’s a high probability that an individual’s heterozygous for one
of these forms. Now, what happens is this sequence may appear at one genetic locus in
the DNA and this would be at the homologous locus on the homologous chromosome.
This is a pretty simple sequence so it could occur somewhere else by chance in the DNA

51
as well at another locus and the same phenomenon would happen at a separate locus
independent of this one.
So look at page 274 Fig. 9-6
Look at the very left hand column of light brown, dark brown, and purple (part b).
That’s a single individual and it has 3 separate loci where this phenomenon has
occurred with the same sequence. So this sequence, this pair here, might represent
their chromosome that you’re seeing in light brown. Somewhere else in the
genome, there’s a pair that would represent with the brown (the same sequences
but different numbers of copies). What we have to do when we want to do this
remember DNA is invisible. So to visualize it on an electrophoretic gel is we have
to label it with a probe so it becomes visible. So when we’re dealing with these
things, we find a probe and the probe should be something like this
(complementary to sequence in figure). So this probe would recognize all of the
different stretches because they’re all made up of repeats of this basic set of 4
nucleotides. That probe will bind to every one of these segments [notes picture]
where we have these repeats. Now, what we can do is subject this DNA to a
restriction endonuclease and it will cut up the DNA in a random fashion. Now we
take that DNA, run them thru an electrophoretic gel, and expose them to a probe.
The only place the probe will attach is here, here, and here. So we’re going to get
3 bands from this DNA that are going to be visible and those bands will be of
different lengths for the top DNA strand. But let’s say the restriction sites are the
same on the homologous chromosome but the number of repeats are different. So
if instead of 2 copies over here I have 4 copies over here, this length (band) is
going to be longer so it has a lower mobility than this band. Because there’s a
higher degree of heterozygosity, we going to get 2 bands for each of these loci,
and each one will have a different mobility. The recognition sites remain constant,
but the amount of DNA between those sites vary because of these sslp’s. We take
the DNA, cut it up with restriction endonucleases, add a probe, run it thru the gel,
and we get [fig. 9-6]: for individual A, we’ve got 6 bands for that probe and that
repeat segment. For individual B we’ve got 6 bands, but the mobility pattern is
different from A. Same for individual C. Now we’ve developed a DNA
fingerprint if we use this one probe we’re going to get a certain pattern (we can
also use another probe). So we can characterize an individual based on this unique
mobility pattern we see in the DNA. If we want to study genetic systems with
this, look at
Fig. 9-7
Here, we use this to generate a marker for a female and a marker for a male. The male
expresses some phenotypic trait (look at pedigree). We can tell whether the offspring
express the phenotype or don’t, but then we can look at the banding pattern that the
offspring display. If this is an individual that has 2 different alleles for this particular
band, this individual when it reproduces because of segregation will only transmit one
of these bands to its offspring. You identify the markers (A, B, C…). Marker A
occurs in the female and she doesn’t express the trait, it also occurs in offspring 1, 3,
and 5. Band B occurs in the female, and it’s not in 1, not in 3, and not in 5. So the
idea is that they don’t have to be adjacent to each other in real life, but it looks like A
and B are the 2 alleles that the female has and she’s going to transmit only one of

52
 them to each of her offspring (either A or B but not both). Because of segregation,
you can think that maybe those 2 are at the same genetic locus. You also see that F
and H are always inherited together, so that means those may be linked on the same
chromosome or it could be close. So for this, we need a probe that will recognize the
sslp and you have to have restriction enzymes that will cut up the DNA.
Minisatellites – repeats of DNA around 15-100 nucleotides long.
Usually when we’re doing this technique, we’re looking for repeats that are around 25
nucleotides long.
Microsatellites – a couplet of nucleotides (ACACACACAC – we repeat a dinucleotide a
number of times). If you have a situation like that, you’re going to have the same problem
where they slipped during replication and you get different numbers of them. So when
you’re dealing with microsatellites, we typically analyze them with PCRs. If these are
microsatellites, what we want to get right here and right here are primers that will allow
us to make many copies of this stretch of DNA thru PCR. So we need a primer that will
recognize the DNA over here and a primer that will recognize the DNA over here. If we
have those 2 primers, it’s very easy to get a probe that will recognize ACACACAC
(because the probe will have to have TGTGTGTG to recognize).

10/22/03

pg 273 Fig. 9-5

One way to generate markers.
You have to have a probe for each restriction site you’re setting. So you want to
examine lots of markers using these RFLPs. You have to have a probe for every
marker that you’re considering setting and that can be bonded to those markers like
that. So that’s a big problem.

Pg. 274 Fig 9-6 shows how you do DNA fingerprinting

DNA fingerprinting utilizes these VNTRs. These are minisatellites that are 15-100
nucleotides long that they repeat most of the time.
You have repeated sequences in tandem (right next to each other) so often times there
is slippage during replication. So we’ll wind up with regions that have 5 copies,
regions that have 3 copies, regions that have 7 copies. And all of these regions are
connected to alleles and when we recognize those alternative alleles, if we cut up the
DNA using restriction enzymes randomly, that creates segments that will include
these VNTRs. We need a probe that will recognize something in that minisatellite
region so then we can detect it on an electrophoretic gel. And then we run the
electrophoretic gel and see the mobility of the bands; the greater the mobility, the
smaller the fragment created by cutting up with the restriction enzymes. So we wind
up getting these DNA fingerprints that we described in Fig. 9-6. Usually if we find
one of these VNTRs, it will be indicated at a specific locus and also on the
homologous chromosome of that locus; so quite often, occurs in other locations along
the DNA as well. [picture] If we had an individual that is heterozygous for a VNTR at
3 separate loci so we get 6 bands when we run an individual like that, so the only
thing you need for DNA fingerprinting is a probe that will recognize some component
on the minisatellite.

53
Now the 3rd technique we use are using microsatellites. Microsatellites are repeats of
dinucleotide pairs and you have the same slippage properties that the minisatellites have.
So now you’re talking about something like ACACAC… It’s very easy if you’re going to
have to locate something that short that repetitive; you create a probe that’s
complementary to it, all you need to find the AC sequence is the corresponding TG
sequence multiple times. When we have microsatellites, instead of using these random
restriction sites to create the fragments, we take 2 PCR primers that occur that will be
just outside of where these microsatellites occur and we create many copies of the PCR
product and then separate them in an electrophoretic gel and probe them. Then we get
what we see in Fig. 9-8 where for each microsatellite locus, we get 2 bands because
individuals are heterozygous for those 2 bands for that locus. And we would get
separation like you see there; and you can potentially find a microsatellite that will be
linked to a genetic disorder that you’re trying to study.
What we’ve done now is created molecular morphology within the genome. Now we
want to discuss how we can utilize our knowledge of the chromosomes to try to identify
where certain genes are and that’s what’s called cytogenetic maps.
Pg. 278 One type of cytogenetic map is called fluorescent in situ hybridization (FISH).
In FISH, you take chromosomes you process them so they will stick to a filter, spread
the chromosomes out and then denature them so that the double-stranded DNA
separates. Then you add a radioactive probe (in this case a fluorescent probe) that will
find the portion of chromosomes that’s complementary to that probe and bind to it.
Then you get what’s in Fig. 9-11. On the lower left, you see two homologous
chromosomes that have been identified by the probe. This gives you an approximate
location of where you’re looking to study in that genome.

Another technique: if you are growing cells in culture, you can get a single cell and with
the right techniques, you can make it go thru mitotic divisions and cytokinesis, producing
a number of cells. This is called cell culturing.
If you take a cell culture from a human cell and mix it with a cell culture for a rodent
(mouse) cell, then the cells will fuse together. You incorporate a virus called a
sintheye? virus and it embeds itself in the cell membranes of the mouse and human
cells, so then the 2 cells are very close and they will coalesce to form one big cell.
That one big cell will have the complete complement of mouse chromosomes and the
complete complement of human chromosomes. That fused cell will go thru mitotic
division and as this happens, then the chromosomes will randomly incorporated with
the human chromosomes. After a number of divisions, you’ll have a hybrid cell that
will have all of the mouse chromosomes and a subset of human chromosomes. the
subset of human chromosomes that you wind up with is some random subset. Once
you get down to a reasonable subset of human chromosomes, it seems to stabilize and
that hybrid cell will….in the lab. So one cell might get chromosomes 1, 14, 18, 21;
and another cell might get 5, 7, and 9. Those genes will position onto the You can
take the hybrid cell and see what biochemical is being produced by them and you’d
know that if it wasn’t produced by that, then the gene for it wasn’t on this
chromosome. Thru a series of experiments, that’s how we identify which

54
 chromosomes contain which genes in humans. But they do something even easier
now:
Fig. 9-14
We take a human cell and before we fuse it with a mouse, we expose it to high
voltages X-rays; X-rays are high energy radiation and one thing they can do is break
the sugar-phosphate backbones. The higher the dosage, the more breaks that will
occur. So you take a human cell with half chromosomes, expose it to high energy
radiation, break the chromosomes down into segments (randomly), and then you fuse
it with a mouse cell. So now we get broken human chromosomes together with mouse
chromosomes and expose that hybrid to another dose of radiation, so you break the
mouse chromosomes less severely than we broke the human chromosomes, and then
sometimes a section of human chromosomes will be incorporated into mouse
chromosomes as the mouse cell tries to repair its chromosomes. So overall, human
chromosomes will be randomly incorporated into mouse chromosomes. Now if you
have markers that exist in the human chromosome, then we find that those markers
exist together in the new hybrid in the figure, then we can persist with high frequency
that we know that it’s not just random, they must be linked together. If they are not
linked and they are, we would expect them to only to be transmitted into this hydrid
cell with a probability that would be equal to the product of their individual
probabilities (product rule of independent independents). Ex: if 10% of the time
marker 1 is passed on and 5% of the time marker 2 is passed on; if they were not
linked together, the amount the time they both should be passed on would be
(.1*.05=.005). We would expect them to be passed on by random if they’re not linked
as if they were independent events. If they get passed on with a higher frequency than
that, then we conclude that those 2 loci were linked together. That’s how we construct
a map of the linkage groups within humans in that way.

Here’s what we really want to do. We’ve got these markers installed and we’ve
learned how to find DNA and create a genomic library. So what we have to do is put
all of these sections of donor DNA, that we’ve cloned, together somehow.
On Pg. 281 we see a problem here. We’ve created those 4 segments at the top of that
figure by randomly recombinant DNA but we really want to do is all of those
segments represent segments that aren’t some long continuous DNA strand. So we
want to make sure we’ve got the entire length of DNA included in all of our
fragments. Basically, if have markers we can identify (in segment 1, we have markers
Q, R, U, and V) we can separate them and see that 2 of them share the same pair
(markers J and K), so we get overlap in the DNA. In those 2 donor segments, they
might be the same region of DNA; so we have a continuous region from AB to MO
and we try to piece them all together and what we’re doing is once you’ve created a
synthetic DNA molecule on a computer that is synthesized by learning that these 2
segments really overlap like this, that’s called a contig. So we try to assemble contigs
that will encompass the entire DNA length of DNA that we’re studying. If we do this
for the entire genome, then we have constructed a physical map of the genome. It’s
important because if we can identify that some gene exists in one these segments,
trying to learn the molecular nature of that gene is much easier by studying that

55
 cloned insert than studying the entire genome. We’ll study how we assemble these
contigs.

10/24/03

STS- sequence tag sites- where you trying to get certain sequences within a clone
DNA.

Now you already have a donor DNA that is place in the plasmid and now you want to
find out the sequence of the donor DNA. First you have to make a primer that is
complementary to the donor DNA and extending it.

Pg. 281
These short length of probes can be recognized by these sequence tag sites. Then you
want to see which clones share the same STS. The AB-JK share JK tag with the JK-
MO. If they share the same sequence tag then they must be overlapping at that region
and that how you assemble the contigs. Then now you have a long continuous
sequences from AB to YZ. Now you have a physical map of genome. Now we have a
large genome and we cut it up with individual pieces which individually clone.

There are two ways in which we assemble the contigs.

1. Pg. 282 Fig. 9-16 One way is using the STS.
2. Fig. 9-15 Another way is by using DNA fingerprints. We take each individual
clone of DNA and we do a fingerprint of that clone. The we stain all the band
with ethidium bromide. Then we see the fingerprint for clone A, B, C, and D
when we run it through the electrophoretic gel. We use the mobility pattern to
finger out. We look for the percentage of shared bands between each of the
clones. For the clone A and B they both share the same top band. Then the
next band is not share by them and the next pair share. Then you will get the
proportion of A band that are shared with B. Then you get the proportion
between A and C and between B and C. The clone that share the most band
are said overlap the most. After that you can see that A is overlap with B and
B is overlap with C and A is overlap a little bit with C and D does not overlap
at all. Then you will have to do more clone to fill in the region between C and
D. When you doing DNA fingerprinting you are not trying to give a lot of
emphasis to the fact that they a share band but just the proportion of bands
they share. Now we get the physical map of the genome and we can take
every clone we have in the physical map and we have robots that can insert
the DNA of those clones in a specific position on a membrane.
Fig. 9-17 page 283
That is a membrane and each of those dots is the DNA from a separate clone; but
they’re not just randomly positioned there. Let’s say if we looked at FIG. 9-16, we
would put E on the top left and C to the right of that, A to the right of that, and so on.
So each position on this membrane represents a particular clone with the physical

56
 map of the genome. Now we’ve got them attached to this membrane, we denature
them which causes the DNA to separate but they still are bound to the membrane.
And then if we have any DNA that we’re interested in that we’ve obtained from some
species (maybe it’s cDNA, something to do with what we’re interested in studying),
all we have to do is radioactively label that and use it as a probe, bathe this filter with
it, and the probe will bind to wherever it’s complementary to. DNA that they were
studying here was complementary to DNA that was in clone 332 and 333, so now any
time we want to study any DNA from that taxonomic group, we get the DNA and
label it and bath a filter with it and you know within that genome where the DNA is
going to be found.
With this, you can identify certain things that are going on based upon these
microarrays.
Page 288
A phenomenon that we see in eukaryotic organisms is that a lot of the DNA is very
repetitive in nature and we want to know where this repetitive DNA came from and
what it’s doing. So we first need to classify the different types of repetitive DNA.
Less than 5% of our DNA contains messages for polypeptides. So why is that DNA
there and can we understand anything about that. A large amount of that DNA is
repetitive in nature. There are 2 ways you can have DNA repeat itself: it can be in
tandem repeats and in non-tandem repeats. A tandem repeat means we have a
sequence here and it’s immediately adjacent to another sequence that’s identical. A
non-tandem repeat would have some sequence over here and that sequence may exist
somewhere else in the genome, perhaps on a different chromosome.
Now we talk about tandem repeats that are arrays of DNA that don’t code for genetic
material. And non-coding tandem repeats exist in the telomeres and also occur in the
regions that form the centromeres. The regions that include the centromeres are usually
large heterochromatic regions that in some organisms contain a lot of DNA. We take
DNA and chop it up with an endonuclease into smaller fragments, then we can measure
the density of each smaller fragment. The frequency of those density we would expect it
to be one big hill (bell shape curve and that mean we have a normal distribution) but it
turn out in eukaryotes that it has one small hill follow by a big hill.
The satellite DNA is what form the small mode. It doesn’t appear to be the same
composition of the DNA. The density of the DNA is going to be is going to be a function
of the relative amount of CG pairs vs. AT pairs. CG pairs are more dense than AT pairs.
When you cut up the DNA fragment, you would normally have the same proportion of
CG as AT. In satellite DNA, it has a different relative constitution of AT vs. CG that is
why we get that small mode. Satellite DNA are repeats of stretches high in AT
concentration that surrounding the centromere and are non-coding repeat because they
don’t code for polypeptide.
Coding repeat- fig 9-22 on page 288.
Think about the problem you’re facing. What proteins do you need that cost a lot of –
histones, they synthesize during S phase of the cell cycle. So you have to double the
amount of all of those histones in that short phase. One way to make all of the
histones is to have a lot of copies of those genes that make the same mRNA that can
translate for histones simultaneously. So tandemly repeated genes (like for these
histones) are important because when we need them, we need a lot of them.

57
 [notes] We’re amplifying the effect of one gene. One gene make many mRNA, make
many polypeptide, make many reactions. That is effective when the end product is a
polypeptide. But some genes make tRNAs and rRNAs as their end product, there’s no
amplification for those genes. So if you want a lot of rRNA or tRNA, you need to
have multiple copies of those genes that make them, so they are often repeated in the
genome many times. But they are not always found tandemly.

Fig. 9-25
45% of the human genome is made up of those type of repeats. These repeats
represent foreign DNA that has invaded our genome and used our cells to make
copies of themselves. They invaded the cells of our ancestors and used our own
replication of DNA to make copies of themselves. They are called transposable
elements.

3 types of transposition, transposable elements (transposons):

1. Conservative form of transposition – DNA from this region can leave the
genomic DNA and insert itself in another location in the DNA. If we have multiple
chromosomes, it can insert in another chromosome. If it inserts in the middle of a
coding sequence, that could lead to mutation. This is one type of transposon and it
needs an endonuclease; it has to be autonomous (needs endonuclease to cut it out and
open another section up). It has to contain a transposase (type of endonuclease) in this
region.

2. Replicative form of transposition - Another way where this region of DNA can
be replicated on its own to make another copy of DNA. This other copy then gets
inserted somewhere else.
3. Retrotransposon - the DNA is going to make mRNA. That mRNA contains a
message for a reverse transcriptase which makes complementary DNA from this
mRNA copy. Then the cDNA gets inserted somewhere else in the genome. If we
go this way then the mRNA gets processed and introns are removed. Then what
gets inserted is not exactly the same as the original copy, but it’s just the coding
portion of the original copy.

These are the DNA transposons that invaded our genome one time. It replicated
and made many copies of itself. And thru time, mutations accumulate there and it
loses its functionality. But we still have the remnants of what it produced when it
was replicative.
This would be a transposon that is functional to do its job (it’s the endonuclease).
If the endonuclease mutates and is nonfunctional, then we still have its remnants
but it can no longer transpose itself. So these are the DNA transposons.
These are transposons that originated from retroviruses (a virus that attacks an
animal cell and inserts its RNA; the RNA has message for reverse transcriptase
and it makes the DNA copy of its RNA, the DNA copy inserts itself into the host
genome). Now everytime you replicate your genome, you also replicate the DNA
from the retrovirus. A retrovirus has little regions for insertions, but it has several

58
 genes. Some of the genes are associated with the virus, some are the reverse
transcriptase, and some are the endonuclease. So if we can have some retrovirus
DNA that repeated many times in our genome, but that can mutate and no longer
be functional but the remnants of this remain. It will be a nonautonomous
retrovirus.
We have 2 parts of our genome. This is called a LINE (a long interspersed
element) and they have 2 open reading frames, one codes for the reverse
transcriptase and one codes for the endonuclease. But it originated from a
retrovirus but part of the retrovirus message was lost, the part that was for the
natural viral part so we make RNA that then makes cDNA that’s inserted using
the endonuclease. That’s what a LINE is, 21% our genome is made up of this. If
this gets truncated even more, where it’s no longer capable of autonomous
transposition, then you get a SINE (a short interspersed element). The most
common sequence in our DNA is a sequence called Alu which is a repetitive
SINE. Alu makes up 10% of our genome, it’s one short sequence that’s repeated
many times. It’s called Alu because it’s recognized by a restriction endonuclease.
Alu occurs every 1000 bases in our DNA, so we use a restriction endonuclease
that recognizes this short stretch and cuts the DNA up into very small segments
about 1000 nucleotides long.

This make up 45% of our DNA. It doesn’t code for anything and it’s called
parasitic (selfish) DNA. It somehow got into our DNA and it used our DNA to
make many copies of itself, and it’s no advantage to us.

Fig. 9-27 page 291

That top thing is a gene, the light green part of the gene is the introns. The dark green
are the exons. Very little of a eukaryotic gene has message for polypeptides. The next
row shows the Alus in that gene, those blue bands are just these highly repetitive
sequences. You find lots of them in the introns. Other SINEs are in purple in that
gene in the next row. You also have LINEs in the gene (yellow) and you also have
LTRs (long term repeats) and SSRs (short segment (sequence) repeats). LTRs are
like minisatellites and SSRs are like microsatellites. So you see most of what’s
making up the gene is introns and within the introns are these repetitive DNA.

Fig. 9-28
Now you see what a chromosome looks like. We have this region around the
centromere that’s heterochromatic and doesn’t code for anything; the only genes are
green cylinders. We have lots of SINEs, most of the gene is made up of introns rather
than exons. Then the nucleolar organizer (blue region) is where we have the tandem
repeats for rRNA.

10/27/03

We want to know how to interpret that database (genome). DNA (a large nucleic acid)
that has docking sites for other nucleic acids or other proteins to bind to the DNA. We
have regulatory proteins binding sites in the DNA. Binding sites for RNA polymerase.

59
The ribosome has binding site for mRNA. Translation has stop signal. Signal to cleave
mRNA 20 nucleotides downstream from AAUAAA sequence (binding site for the
endonuclease to cut it).
How do you know what’s a gene? You need to figure out where the genes are. Less than
5% of the DNA has message for polypeptide, how do you know where that 5% is? We
still don’t know how many genes there are in humans.

We have computer search programs that search for genes. They try to identify potential
genes (potential genes are things that are open reading frames), so they have to have a
start signal for translation and a stop signal. So the computer search for open reading
frames, but not all open reading frames are genes b/c the AUG could be in the middle of
the gene coding for methianine or in an intron (it could be between genes and have
nothing to do with them). So one of the first levels of investigating the genome is to
search for open reading frames and try to find a lot of genes that way. But then if you’re
looking for open reading frames, what’s the size limit you’re going to look for for a
potential gene. We know there’s always some minimal length that functioning
polypeptides will typically not be below, but how about a maximum length?
Ex. Dystrophin gene is 2 million bases long (when it mutates, it leads to muscular
dystrophy). That a lot longer than we think we have to look for a specific gene.

So once we identify potential genes, we then have to see if it actually is a gene or not.
One way to do this is use mRNAs to make cDNA. If we create cDNA, we know that has
to be produced from mRNA, so we know the cDNA’s existence is the result of a gene
after it was sliced and transcribed. So we see if the cDNA is complementary to certain
regions in the genomic DNA. If it is complementary to a region we think a potential gene
resides in, then it’s nice evidence that that might be real gene. One thing we do with
cDNA is remember what we do when we tried to make a physical map of the genome, we
put donor DNA into a vector like this and then use the DNA that we know would be at
the end of the vector as primers in sequencing reactions, that the sequence of about 300
bases on either side and you would break STSs (sequence tag sites) and the nice thing
about those STSs is then we could make primers for PCR; and any region of DNA that
would be bound by those 2 PCR primers you would expect it would have overlap over
here. So you take mRNA, we make cDNA out of it, and then we create STSs on the
cDNA (on this region and this region). And from that we make probes and we use those
probes on the genome DNA. Is type of STS is called an ETS (express tag sequence).
ETSs bind to the beginning and end of what we think is a potential gene. Then it’s
probably because that’s the gene that codes for mRNA that we use to develop the cDNA
from.

Other things we look for is if we have a gene that exists in another species, that same
gene is likely to be similar in all species to a certain degree. If we’re closely related to a
species, the more similar the sequence is. What we’re often able to do is if somebody
studies fruit flies or nematodes and find a gene similar in humans, the sequence will not
be identical to the sequence in humans but it will have enough similarity that we can use
computer algorithms that will find non-exact matches.

60
So far all we talk about is the DNA. What we really want to know is what proteins exist
in a cell b/c it’s the proteins that control the biochemistry. And that is really defining
what life is. So we really have to understand that this genome is the blueprint for making
a set of proteins. But the proteins that are expressed at any time in a specific cell may be
different than the proteins that are expressed in a different time in that cell. Or they may
be different than the proteins that are expressed in another cell at the same time. And the
most common way we study this is using DNA chips.

Page 304
This is part of what we call functional genomics. What you can do with robots is take
sets of DNA, perhaps an entire set of DNA thats developed from all the different
cDNA that we could establish from a particular species. We take them and precisely
anneal them to a chip about the size of a microscope cover-slip. And we put them in
very precise locations and what happens are these are cDNA; then we denature them
to become single-stranded and now what we do is collect RNAs from tissue at
different times in an organisms life or different types of tissue at the same time. When
you do that and bathe this chip on them, the RNA molecules will bind to the cDNA
that they’re complementary to; now we’ve labeled this mRNA, then the (this is done
with fluorescence in this case) they create a grid where one color indicates that the
mRNA exists in the cell at that point in time and another color means that it’s
under… we’re not finding it. So if you get green, you’re expressing it; you get a red,
it’s not being expressed.
And look at what happens as a for instance in Fig. 9-38. What this is doing is looking
at a particular cell type thru time. Time is going from left to right. Each row within a
column represents a different gene and what you can see is early on the genes near the
top are being expressed later on the middle portion starts becoming expressed.
Previously we had a photograph of the complete set of messages that exist. Now this
is telling us how we’re using those messages thru time. You can use these DNA chips
to get a profile of a person to see if they have this type of cancer or some other type of
cancer. Problem: a lot of our mRNAs are transcribed and sit in out cytosol and is not
translated immediately; so what this is doing right now is looking at the profile of
mRNAs effectively, that exist in cells at any different time or stage. That’s what this
microchip thing is doing.

But now people are starting to look to see how different this profile for the mRNAs is
different from the protein profiles that exist in the cell. Because if the mRNAs are not
actively translated even though they exist in the cytosol that doesn’t mean they’re
producing proteins. And lots of research is being done on this, especially cancer research.

CHAPTER 10 – Gene Mutations

Usually we characterize mistakes that are made if they are visible to us (thru a
microscope) or if they’re invisible to us. The invisible ones are categorized as
point (or gene) mutations. You want to distinguish those that have occurred and
changed the inheritance pattern from are invisible to us to those that we could examine a
cell with a microscope and then we might be able to recognize that something big has

61
happened. Those are called chromosomal mutations (mutations we can see). So the 2
major categories of mutations are gene mutations and chromosomal mutations.

When we talk about gene mutations, a gene mutation always may involve a single
nucleotide or a few nearby nucleotides. But if something like that happens, the nucleotide
is too small for us to visualize. Those are the ones we’re going to talk about first.
Now, one type of a gene mutation is simply a base substitution. If what we do is we
replace a nitrogenous base with an alternative nitrogenous base that is different, that’s
called a base substitution. A lot of the mutations are base substitutions. This chapter has
a lot of detail, so you have to see the tree thru the forest. When we talk about base
substitutions, we have these four bases in the DNA [notes]. If we replace an A with a G
or a G with an A, G and A are both purines; so if we replace a purine with a purine (or a
pyrimidine with a pyrimidine), that’s called a transition.
Transition – when you replace a base with a similar base.
In contrast, if we replace a T with a G or a G with a T, an A with a T or a T with an A, an
A with a C or a C with an A, a C with a G or a G with a C, then we’re replacing a purine
with a pyrimidine or a pyrimidine with a purine. That type of base substitution is called a
transversion. There are 8 possible transversions and four possible transitions. You
should expect more transversions than transitions, but that’s not what happens. Base
substitutions are twice as frequenly transitions as they are transversions. So transitions
are the most common base susbstitutions. In this chapter, most of the mutations that occur
lead to a transition. So you memorize the ones that are transversions to make it easier to
remember which mutations lead to transitions.

Page 317, Table 10-2

Right now bombarded with radiation from the sun, and this causes mutations. The shorter
the wavelengths of radiation, the more energy that’s contained and the more mutagenic
they are. So in any point in time, lots of mutations are occurring. Those are background
mutations and we refer to them as spontaneous mutations.
Spontaneous mutations – mutations that we have no control for and they would occur
even if we didn’t do anything to you.
So we take organisms and expose them different environmental conditions and what
we’re expecting are some of the environmental conditions we expose them to are
mutagenic in nature. So if mutations that arise in an organisms lifetime that have been
exposed to what we think is a mutagenic agent, what we do is we assign all of those
mutations as being due to mutagenic agents, even though some of them might have
spontaneously occurred anyway. But what we’re hoping is the agent that we’re studying
is sufficiently a greater magnitude in causing mutations that it dominates the background.
Induced mutations – all of those mutations that we try to create by exposing an
organism to specific environments.

Fig. 10-3
This is showing you X-ray dosage vs. mutation rates in fruit flies. You get a straight
line and that means if you double the X-ray dosage, you double the amount of

62
 mutations. If you expose it to two dosages at 500 units of X-rays, then you get the
percentage of mutations as if it was exposed to one dosage at 1000 units of X-rays.
For this linear relationship, we don’t know if it’s for humans or not.
[notes] But for mice, if you expose it to low levels, then there’s no increase in
mutation rate b/c they have repair systems to repair the low levels of radiation
damage. When we see a mutation, what that is is a net mutation; mutations occur all
the time in our DNA and the ones we see are those that have escaped the repair
system. Now, we don’t know if we’re closer to mice or fruit flies but we think we’re
closer to mice.
X-rays cause damage in 2 ways. One thing they can do is break the sugar-phosphate
backbone if they hit the DNA. But if they miss the DNA, they can hit nearby
molecules, knocking electrons out of orbitals. So what that does is create ions, and
those ions then become reactive where they weren’t expected to be reactive, and it
cause secondary chemical reactions to occur because of this ionization.
Mutations that occur in the soma may be serious to you but they’re not that of a big
deal because they don’t occur in the gonads.

10/29/03

page 319 – Different forms of a mutation

3 types of point mutations

a) you can change the base
b) you can damage the base
c) you can modify the base

you can also have additions (insertions) or deletions

Fig. 10-4
Frameshift mutations – arise from an addition or deletion of a nucleotide
-you modify the messages for all of the amino acids downstream from the
mutation.
-then you get a nonsense (nonsensical) protein
-if you have a frameshift of 3x bases, then you have a polypeptide that may be
functional.
-if it’s not in multiples of 3, then it’s nonfunctional.
Nonsense mutations
-cause nonfunctional polypeptides, highly deleterious (lethal)
in contrast to the nonsense mutation, if this were the wild type message and we had a
base substitution like this, in this example it’s the codon for the third amino acid in
the third base position of that codon, we replace a T with a C. But both CGT and
CGC code for the same amino acid argenine. So with that, we have a mutation b/c
something modified the DNA but despite that modification, the polypeptide is going

63
 to be translated from the modified DNA exactly the same as the original wild type
message. That would be generally characterized as a missense mutation, but it’s a
special type of missense mutation. It’ll be called a synonymous mutation or
synonymous substitution b/c what you’re doing is changing one codon for another
codon that codes for the same amino acid.

In contrast, we could have a missense mutation where we replace one nucleotide for
another nucleotide and what happens here is all we’re doing is changing one codon
for one amino acid, so if the polypeptide is 1000 amino acids long and we change one
amino acid; that can be highly deleterious. Mutations like that lead to things like
sickle cell anemia and cystic fibrosis. That could be deleterious, but most of the time
it may not be important b/c the polypeptide folds up and it’s the active site that’s
really important; so if the mutation is not in the active site, then might have very little
effect on the polypeptide. So a missense mutation is unpredictable whether it’s highly
deleterious or not b/c it depends on where they occur.

Now, sometimes we could have a position in the polypeptide that’s very …to the
polypeptide. The only thing that’s important about that position is the characteristics
of the amino acids. So perhaps what we need is an amino acid that has an electrical
charge, where if that’s what we need, any amino acid that has the appropriate
electrical charges do well. What we need is amino acid that has a basic side chain. So
if we replace one amino acid with another amino acid (a different amino acid) but
they both are chemically similar, that might have very little effect on the polypeptide.
That is called a conservative substitution. Where we replace one amino acid with
something that’s chemically quite different, that would be a nonconservative
substitution.
If we have conservative substitutions, the functioning of the polypeptide may be
virtually identical in the mutant one to the wild type. In that case, we call that a
neutral mutation.
As we talked about previously, we could have a mutation in the regulatory sequence
that’s not in the coding region. And that could prevent even the transcription of the
message. Or we could have a mutation in the introns at a portion of the intron that has
to be recognized by the splicosome; if you don’t splice out the introns, then it’s like
you have a large insertion or it could be a frameshift mutation. When we have these
frameshift mutations, we could have a frameshift by an insertion or by a deletion so
mutations like this are indel.
Indels – mutations that combine the concept of insertions/deletions.

Mutations occur all the time in our cells and some of those mutations are due to things
like cosmic rays and X-rays that are emitted from the sun. There are other spontaneous
forms of mutations as well. They occur at a rate of 10^-5 to 10^-8 or 1 in a hundred
thousand to one in a hundred million per locus per cell generation.

How to induce mutations by some artificial mechanism

We have to understand a few chemical concepts about tautomerization.

64
Fig. 10-5
What happens is normally this is what we think of C, G, T, and A. We normally that
C always bond with G (by 3 hydrogen bonds) and T always bond with A (by 2
hydrogen bonds). But it turns out that each of these 4 molecules tend to assume that
configuration the majority of their time. What happens is they are able to exist in
alternative forms relatively stable and what they would do is spontaneously convert
from one form to another form and back. These forms you see here are
overwhelmingly the most common arrangement of the atoms for these 4 molecules.
But what happens is when they assume the alternative (or rare) form, we call that
phenomenon tautomerization. They are in the rare tautomeric form 1% of the time.
Let’s see what happens when this occurs.

Fig. 10-6
When cytosine is in its rare tautomeric form, it takes the imino form. Normally it has
3 positions for hydrogen bonds in the common form. When cytosine enters the imino
form, it has 2 positions and now instead of base pairing like we assumed it would
with Guanine, what it does is base pair with Adenine. So in the rare tautomeric form,
C will pair with A instead of G.

The rare tautomer for Thymine is called the enol form. Thymine usually has 2
positions for hydrogen bonds to pair with Adenine; in its rare form, it has 3 positions
for hydrogen bonds and pairs with Guanine.

Adenine in its common form has 2 positions for hydrogen bonds to pair with
Thymine; in its rare form, it has also 2 positions, but now it pairs with Cytosine.

The rare form for Guanine has 3 positions but those 3 positions pair with Thymine
instead of Cytosine normally.

Normally you have CG pair but if it goes in to the rare tautomer, you have the rare
tautomer A pair. So you’ve replaced a G with an A and that’s replacing a purine with
a purine, so that’s a transition.
Normally you have a TA pair, in the rare tautomer it’s the rare tautomer T with a
normal G so you’ve replaced an A with a G. That’s replacing a purine with purine,
also a transition.
Same for the other two. You replace a T with a C and then a C with a T and both are
transitions.
So the effect of tautomerization leads to a transition mutation.
So if you are told there’s going to be tautomerization of A, you know that the rare
tautomer of A must pair with another pyrimidine and the only other pyrimidine is a C.
So it has to pair with C. It will be a lot easier to figure out if it’s a transition or a
transversion if we know what’s going on.
2 ways to get tautomerization: [notes] What can happen is during replication each one
of the existing sugar-phosphate backbones act as template; so if there’s a tautomer

65
 there, it would serve as a template with the incorrect nucleotide to be inserted
opposite in the growing DNA strand.
Another way you could do it is the incoming nucleoside triphosphate could be a
tautomer (the rare form) instead of the common form. Both of those ways would wind
up leading to transitions.

Fig. 10-8
This is happening spontaneously in our cells all the time. We can use things that are
called base analogs.
Base analog – a molecule that is incorporated in a nucleoside triphosphate and it can
behave as one of our common nucleosides. A very common base analog is called 5-
bromouracil (5-BU). That’s incorporated instead of by normal nitrogenous bases in
the nucleotide; 5-BU behaves just like a Thymine. It’s got one ring, so it’s a
pyrimidine and it serves as the template for Adenine. So if we’ve somehow created
nucleoside triphosphates that had 5-BU in it, sometimes that would get incorporated
in the DNA and behaves like a Thymine. 5-BU will tautomerize and the ratio of the
rarer form of 5-BU is not like 99:1, it’s a lot higher possibility that the 5-BU is in the
uncommon form. If this happens, then the uncommon form will bond with Guanine
instead of Adenine.
So we take advantage by introducing these chemicals (5-BU) they get incorporated
into the DNA, spontaneously convert in their rarer form at a much higher frequency
than our normal nitrogenous bases and therefore, that leads to a replacement of an A
with a G. If we replace an A with a G, that type of base substitution is a purine with a
purine, so it’s another transition.

Fig. 10-9
Another base analog is called 2-aminopurine (2-AP). It behaves the same way; it
acts as an adenine normally which would pair with a Thymine. But when it takes its
rare form, instead of pairing with a Thymine it pairs with a Cytosine. So we
effectively replace a T with a C (a pyrimidine with a pyrimidine) which is another
transition.
So what we’ve done in this case is replace one base with a base analog. We take
advantage of them because of their frequency with which they assume their rare
tautomeric state.

Page 321
Now we’re going to talk about base alterations. And there are a variety of mutagenic
agents called alkylating agents. If you look at the top of Pg. 321, you see a green
colored group off of the O-6-Ethylguanine. That’s an alkyl group. If we have an
alkylating agent like EMS, EMS will transfer an alkyl group to Guanine and convert
it into O-6-Ethylguanine which has different bonding patterns than Guanine, it will
pair with T instead of C. So we would replace a GC pair with an alkylated T so we’re
replacing a C with a T and that’s pyrimidine with a pyrimidine (transition). So if EMS
is operating on a G, it will modify G to pair with t instead of C causing a transition
mutation. If EMS is working on Thymine, it alkylates it and the alkylated form will

66
 now pair with Guanine instead of Adenine. So we’re replacing a purine with a purine
which is another transition.
So those two base alterations are base modifications.

Page 322 Fig. 10-11

Now we have different mutagenic agents called intercalating agents which stick
something in the middle. There are a variety of them like proflavin, acridine orange,
and ICR compounds). They are the same size as a nitrogenous base and if we expose
cells to these intercalating agents, what they do is wedge themselves into the DNA
molecule. When they do this, it leads to an indel mutation or a frameshift mutation.
So what you can do is generate frameshift mutations by exposing organisms to these
intercalating agents.

Fig. 10-12
We mess around with the DNA, but the cell’s always trying to repair the DNA. So we
wind up modifying the DNA and if the cell repairs it, then it’s like nothing happens.
The only mutations that we can monitor are the ones that escape DNA repair.

There are certain situations that are referred to as mutational hotspots where under
certain circumstances, mutations have a higher rate of occurrence than other
circumstances. Before we were talking about VNTRs; there a lot of sequences where
we have microsatellites and minisatellites where we have the same sequence as
varying numbers of tandem repeats. If that happens in the coding region for a cell,
that would lead to something like a frameshift mutation (it would be like an indel
mutation). But what turns out are indel mutations occur with much higher frequency
when there are these tandem repeats. This explains why the indels occur. There are 2
columns, look at the left one. Each double-stranded DNA here is in the process of
being replicated, the light blue strand is the template and the dark blue is what’s being
synthesized. If this is the template and this is the DNA strand that’s being replicated,
what happens is they come in contact like this but it does something like breathing
during replication where the hydrogen bonds are forming and breaking over and over.
In (a), we’ve got four T’s; now a little breathing occurs and because there’s a lot of
A’s in a row, the 2nd T binds with the 1st A mistakenly instead of the 2nd A. So we
have the T’s 2-4 bind with the A’s 1-3 and the first T is looped out (it doesn’t bond
with anything). But now we add another two T’s at the end of those three T’s b/c
there’s two more A’s. So at the end, the dark blue copy has an insertion in it b/c of
this phenomenon that occurs b/c of the repetitive DNA (it could also be with ATAT
or ACAC, etc.). In the right hand column, you get a deletion. The repetitive DNA is
CTCTCT; we have breathing and when they come back, part of the template has
looped out and that does not bind with part of the newly synthesized DNA, so the end
result is the newly synthesized DNA is missing one of the GA’s it should have. That’s
a deletion. What we see here is how insertions and deletions occur and they occur in a
high frequency where we have this repetitive DNA. So repetitive DNA is a potential
hotspot for mutation; an insertion of 1 nucleotide or a deletion of 2 nucleotides would
lead to a frameshift mutation.

67
There are a number of genetic mutations in humans that are caused by insertions or
deletions; insertions of trinucleotide repeats. We wind up getting genetic loci where we
have these repeats. And if we get above a certain number of these copies of trinucleotide
repeats, that leads to a wide variety of genetic diseases. This model is one of the
explanations for these trinucleotide repeats.

Fig. 10-14 (a)

This is a DNA molecule and there’s a variety of types of damage that can occur,
usually caused by UV radiation. When we have 2 pyrimidines adjacent to each other
on the same sugar-phosphate backbone, the UV radiation can cause covalent bonds to
occur b/w them. This is called a pyrimidine dimer.
(b) this is another way you can get a pyrimidine dimer. Two thymines can bind
together like this. And if that occurs, the pyrimidines will no longer pair with their
complements on the other strand; and we’re going to get a distorted DNA molecule.
If this happens, the DNA polymerase gets to the dimer during replication and unless
that dimer has been repaired, it will not be able to read past it. So if you’re a
bacterium and you don’t replicate your DNA, it’s over. So what happens is there’s a
system called the SOS system where it invokes a different DNA polymerase where it
lays down in a 5’ to 3’ direction but doesn’t proofread in the 3’ to 5’ direction. What
the SOS repair does is invoke that polymerase that reads past the dimer and put
nucleotides there and not check to see whether they were good nucleotides. But no
matter what, they have to get past the dimer b/c if they don’t, it’s all over for them.
So you take the chance of putting down the incorrect nucleotide in the newly
synthesized strand, so you have a big chance for error (mutation) in the SOS repair.
Most of the time, this leads to transition mutations (it can occasionally lead to
transversions). Where the transversions occur is in the dimer (pg. 323 5’CC3’…
mutation).

10/31/03

Pg. 325 Fig. 10-15

Aflatoxin B1- is derive from a fungus attaches to guanine in the DNA and it break
guanine from the DNA resulting in apurinic site (in the DNA). So in the DNA, there
is just a phosphate and the sugar without the base (guanine). A short form of apurinic
site is called AP site. Apurinic site is more common than apyrimidic sites. Getting the
AP site is not spontaneous.

The SOS sytem gets invoked and puts an Adenine opposite the apurinic site. It was
guanine before so you replace a C with an A. This is a transversion. Aflatoxin B1 is a
mutagen that lead to transversion.

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 Spontaneous Mutation
- Two common mutations are Depurinations and Deaminations.
o Depurinations- is where you lose lot of purines making AP sites. So you
need a mechanism for DNA repairs.
o Deaminations- spontaneously occurred. Pg. 326 fig. 10-17. Cytosine
normally it has an amino group. Cytosine will spontaneously undergo
deamination so the amino group is removed and replaced with a double
bonded oxygen. When this happen, it modified cytosine into uracil. Now
you have an DNA molecule with uracil in it. Through evolution, we
created enzyme that search for unusual bases. This is called DNA
glycosylase. A special type of this enzyme is called uracil DNA
glycosylase and what is does is break the glycosidic linkage between the
uracil and the sugar. So it converted nucleotide into AP site. This is
transitional mutation because a G is replace with an A and this happen if
the DNA glycosylase doesn’t break the linkage.
o One of the problem with DNA is you methylate some of the nitrogenous
bases. The bacterium methylate the bases to prevent the restriction
endonuclease from recognizing where to cut so the bacterium is
protecting itself. Methylation typically shut down gene if it near them.
Methylation is very common. In vertebrate, the most common methylate
bases is the one where we have 5’ CpG3’. This is called CpG sequences.
When this occurred, most of the time we methylate the cytosine.
o Fig. 10-17 If we have C in CpG sequence then it will be methylate like in
this figure. If deamination spontaneous occurred then we removed the
amino group and replaced it with a double bond oxygen. But now the
molecule is thymine instead of uracil because of the methyl group. Now
with thymine there instead of uracil, we can take advantage of this
protection system because thymine is the normal part of the DNA unlike
uracil. When it got converted to T it not corrected so the CpG sequences
lead to transition. So a methylate C is a mutational hot spot. We do not
find many CpG in human because they all mutated to T.

Fig. 10-18 We look at DNA within a gene of E coli. The higher the bar, the greater the
number of spontaneous mutation that occurred. All the highest mutation are the one that
has an methylated C in them. So whenever you methylated a C, it represents a mutational
hot spot.

Two major types of mutational hot spot are Tandom repeat in the DNA where you get
slippage during replication. And second type are CpG site where we have methylated
cytosine.

Fig. 10-19 Pg. 328

- Another way bases can get damaged is when we have oxygen that highly
reactive. That lead to oxidation where it not supposed to occurred. The
superoxide radical are all highly reactive species of oxygen. They damaged DNA
and modified it. The figured show two damaged nitrogenous bases that resulted

69
 from oxidation (guanine get converted to 8-oxo dG). What this does is instead of
pairing with C will pair with A. So C is replaced with an A and that is
transversion.

Fig. 10-20 – It showed you that there are hot spot of mutation where we have tandom
repeat.

Trinucleotide repeat- they caused serious human diseases. Trinucleotide repeat are indel
mutation where the sequence that being repeated is a trinucleotide (is a size of a codon).
If you have frameshift mutation in multiple of three then you don’t have a frameshift. So
when we get this slippage during replication and it in multiple of three, it does not lead to
frameshift mutation.
- We categorize these trinucleotide repeat by seeing if they are repeat of CAG
(glutamine) or not because CAG repeat are associated with neurodegenerative diseases.
That polypeptide with a lot of glutamine will interact with other protein. These repeat are
in coding region of the gene.

A trinucleotide repeat of CGG occurred in the untranslated region. What happen is you
modified the length of the UTR that have some influence the amount of polypeptide that
will be translated from the mRNA. This lead to fragile –X syndrome or minefeld
syndrome (they are the leading caused of mental retardation). The trinucleotide repeat
caused a constrict region at the telomeric end to be broken off.

Pg. 330 Fig. 10-22

A normal individual has 50 or less CGG repeat coding for the UTR. If the female and the
male have the normal number then the offspring will be likely to have the normal number
of CGG repeat. But sometime due to slippage you can get an excess number so you can
have from 50-200 copies. This individual is normal but that called premutation. If you
have a number that greater 50 then the DNA become very unstable and when it replicated
and the children of this individual are going to have a very large number of repeat. When
the number of repeat get over 200 then the disease is occurred and they are mentally
retarded. So you can see if the child will be affected if you know how many repeats the
parents have.

11-3-03

Problem that could be on the test; sequencing problem, restriction mapping problem,
probes, southern blotting, microsatillate DNA, DNA fingerprinting.

They all generate heterozygous locus on an individual. You have to know fingerprints of
different mobilities are usually 2 alleles of the same gene.

From last time- when you see the sequence 5’CG 3’ then the C has high frequency for
being methylate so that a mutational hot spot.

Another case of mutation can arise through transposition-

70
 - Transposition- creates new DNA that is a copy of old DNA. And that new DNA
get inserted somewhere else in the genome. If it inserted in a coding region of a
gene then it a mutation.
- When we talk about transposons we categorize them as retrotransposon or just
transposon. The retrotransposon first copy to a messenger RNA and then the
mRNA get converted to cDNA through reverse transcriptase. That cDNA is what
get inserted into a new location. If it inserted in a gene then it lead to a mutation.
- The other types of transposon is called conservative or replicative transposons. It
what they are is the initial DNA is either removed from it position and inserted
somewhere else (and in that case it been conserved and you don’t get additional
copies). So where it left it change and where it inserted is change and if those two
places are the parts of a gene then that determine if it functional or not. The
replicative transposon are where the DNA is left intact where is was initially but
a DNA copy of that was produced which get inserted somewhere else. In a
replicated transposon, you get an additional copy of that DNA in a new position.
That will lead to mutation.

DNA Repair System

-Mutation that occur are those that has pass the DNA repair system. The most
important repair system we have is proofreading. If the DNA polymerase has no proof
reading then we would make a mistake one out of 100,000 times and that very high. After
all the repair system we have a mistake one out of 10^10 times. How we get from 10^5 to
10^10 because of this proofreading. We correct 99% of the wrong bases through
proofreading. DNA polymerase have 3’ to 5’ exonuclease activity to check the bases that
it lay down to prevent mistake. That just bring us to one to 10^7 time and we need to get
to one out of 10 billion times we get a mistake.
- An enzyme called superoxide dismutase- this minimize the superoxide radical.
Some reactive oxygen species can cause damage so the superoxide dismutase neutralize
those effect.
- Pyrimidine Dimer- they lead to two adjacent pyrimidine to bound covalently.
When we have this problem and the DNA polymerase can’t read past that so you can
invoke the SOS system. There are other systems to fix this.
1. In bacteria, they have an enzyme called photolyase. If it attach
to the dimer and have blue light (the blue component of normal sun light) to energize the
photolyase enzyme break the covalent bond between the adjacent pyrimidine to correct
the dimer.
2. there also enzyme called alkyltransferase that remove the alkyl
group that cause the problem previously.

General excision repair- is a type of repair system. Located distortion in the DNA
which result of a dimer. It give two endonucleases. One endonuclease which cut
upstream from the Dimer and downstream from the dimer to remove that region. The gap
they removed is about 12 nucleotide long in prokaryotes and 27 nucleotide long in
eukaryotes. Then we can get a polymerase to used the other strand as the template to

71
replace the gap which was cut out and replace it with new nucleotide and the ligase which
will bind the 3’ end of the new segment to the 5’ end from the old strand.

Specific excision repair- pg. 341- the distortion of the DNA is easily bound by enzyme
if it involve something as large as pyrimidine primer. This is a big distortion that would
occur. But whenever the DNA is modified in an unsual fashion then it distort the DNA.
This specific excison repair. Fig. 10-38- we have an unmethylated cytosine and it get
deaminated so it become uracil. So the red nucleotide is uracil and we would have an
enzyme called uracil DNA glycosylase that search the DNA for uracil nucleotide. When
it find uracil nucleotide, it break the glycosidic linkage so the uracil get cleave off and we
have AP site. This is the first part of repair of these deamination.

Fig. 10-39

You see the AP site, that AP site will cause distortion but the distortion is not as
significant as the pyrimidine dimer. An AP endonuclease makes one cut at the AP site. In
general excision repair we have two cuts. Now the exonuclease open the place where it
cut. We use the 3’ end of the open DNA and extend it and use ligase to seal the backbone
of the DNA. This is specific excision repair.

Mismatched Repair
- So have a replicate DNA that has the wrong base pair like A-C which is not
complementary. So the problem is you don’t know which base is the wrong base.
Like should the A be a G or the C be T. As DNA being replicate what happen is
the old DNA has specific site that are going to be replicate. So when the new
DNA is being synthesize, it does not get synthesize with these methylated
nucleotide incorporate immediately. What happen first is we replicate the DNA
and then after replication, a short period of time later the new sugar phosphate
backbone get methylate as well. What happen now we inspect these two double
strand DNA molecule and we find a mismatch over here then we can know
which one is the template strand and which is the new strand by examining the
strands for methylation. The methylation may be 5000 nucleotides away from the
site where we have a mismatch. An enzyme with attach to and find the methylate
strand. The methylated strand is the old strand. If there is a mismatch between the
new and the old strand, the new strand nucleotide will cleave by specific excision
repair and replace with the appropriate base. In prokaryotes, the mismatch repair
recognition enzyme searches for 5’ GATC 3’ sequence. In eukaryotes we don’t
know the exact sequence but it similar with prokaryotes.

Recombination repair- Fig. 10-41

- If there is an error here like a dimer then the new strand cannot be replicate pass
it. So you going to cleave off the dimer with an exonuclease. Then we cleave out
the same sequence from the other new strand and add it to the other old strand
where you have the dimer error. The template in the leading strand will make the
complement to make the up the part that is have been cut. In the lagging strand,

72
 now the have a right template strand and you can use that to make the right
lagging complement.

Fig. 10-42
An individual with skin cancer. The individual with xeroderma pigmentosum cannot live
in sun light. If they go to sun light, then the UV light cause cancer. The reason it affect
them because have mutation in their DNA repair system. They cannot fix it. They are
more prone for skin cancer. This figure showed that survival ship curve. There are
different form of this disease. Each of the brown band represent different mutation
category. On the X axis it is time and on the Y axis it is survival ship. The more the steep
the curve, the more detriminal for that form of disease.

Ch. 11

Chromosome Mutation

Chromosome mutation have two major categories but all of them can be visibly observe
under microscope.
The two categories are either the wrong number of chromosome or structural
modification of the chromosome.

First we’re going to talk about mutation with the wrong number of chromosomes.
1. By having wrong number multiple of the haploid genome. Example human
being 3n = 69 instead of 2n = 46. This individual with the wrong number of
haploid genome is called euploid. Euploidy- mean there is a true differences
in the number of haploid genome you have. There are two major categories in
euploid. They involve whether or not the haploid genomes we have came
from the same species or from more than one species.
a. The individual that have their haploid genomes that came from the
same species is called autopolyploid. Example is an individual that
has three copies of the haploid human genome. It turn out
autopolyploid are not common in animals but they are very common in
plant. The problem is: most species have to undergo sexual
reproduction and if they go sexual reproduction and they have an odd
number of haploid genome then you get unbalance gametes. 3n = 9.
Then it will not produce a viable offspring. Watermelon are
autopolyploid so that are seedless because they have odd number of
haploid genome so they can’t make offspring. Also banana.
b. Allopolyploids- the haploid genomes arose from more than one
species. 2n1 x 2n2  (n1 + n2). This is rarely happen in animals. The n1
chromosome is very different from the n2 chromosomes so you don’t
get an normal metaphase 1 in meiosis because they are not homolog. If
they are closely related species then sometime they can synapse. And
sometime they have somewhat normal metaphase 1. Plant are capable
of asexual reproduction. In the branch if something happen where you

73
 do not get segregation during mitotic division, then you can create a
cell that has two copies of it genome [before it was n1+n2; now it’s
2(n1+n2) or 2n1+2n2] so now it’s called an amphidiploid. This
amphidiploid can now go thru normal meiosis; this will produce a
gamete that’s n1+n2 and this will produce a gamete that’s n1+n2. This is
why euploidy is common in plants.
11/05/03
if the tree is a hybrid, it is n1+n2. It can’t go thru meiosis b/c you can’t
have homologs here. If the mitotic spindle breaks down and the cell
goes thru replication, you double the number of chromosomes and you
don’t go thru mitosis. Now you get (2n1+2n2). Now you have
homologs for n1 and n2. Every cell further out on the limb is 2(n1+n2),
including the flower. The eggs have (n1+n2) and it can reproduce
sexually. What we can do is add a chemical called a cultureseen which
breaks down the microtubules so you double the # of chromosomes in
each cell; this is how agriculturists make new species.
2. Aneuploidy- are not true ploidy. They have a wrong number of chromosome
but not in multiples of the haploid genome. Like 45 or 47 chromosomes in
humans.
Monosomic – if the diploid individual is missing one chromosome. It only
has one chromosome where it normally has a pair of homologous
chromosomes.
Trisomic – if the human has 47 chromosomes; it has 3 of one of those
chromosomes instead of 2.
Fig. 11-3
This happens during meiosis. If this is metaphase I of meiosis, when the two
chromosomes pair up they move to the equatorial plane and what normally
happens is the 2 centromeres go to opposite poles. But if they both migrate to
the same pole, then you have a primary nondisjunction b/c this gamete has
24 chromosomes and this gamete has 22 chromosomes. Then we undergo the
2nd meiotic division (equational division) and you wind up with 2 gametes that
have an extra chromosome and 2 gametes that are missing one chromosome.
This is what happens in primary nondisjunction. This structure where you
have 4 chromotids that have synapsed together is called a tetrad. So the tetrad
has to maintain its integrity; the 2 chromosomes or 4 chromatids have to move
as a unit to the equatorial plane. What helps the tetrad maintain its integrity is
the crossing over that occurs, and b/c the chismata form that holds the
homologous together. When chiasma don’t form, there’s a larger chance of
such nondisjunction.
You can also get secondary nondisjunction. In secondary nondisjunction, the
messup occurs in the secondary meiotic division rather than the first
nondisjunction. So here we have a normal reductional division (reduced the
chromosomes in half), but now during metaphase II and anaphase II the
chromatids on this one move apart from each other normally but in this
nucleus they don’t (they migrate to the same pole). So in this case when you

74
 have secondary nondisjunction, half of the gametes are normal and half of the
gametes are not.

Now what you want to do is see the consequences of aneuploids in humans. Now we’re
going to look at individuals with chromosomal abnormalities. The first syndrome is
Turner ’s syndrome.

Fig. 11-14 – Turner syndrome is symbolized like that. You would describe the
chromosomal complement of a human as 45, X. What that means is when you see 45,
you know it should be 46 so this individual is missing a chromosome. And this tells you
that the individual is missing one of the sex chromosomes. So this individual has an X
chromosome but we know it should either have 2 X chromosomes or an X and a Y
chromosome. We don’t really know which will be lost in this process but this indicates
that the chromosomal abnormality is associated with the sex chromosomes. Now
individuals that have Turner syndrome are mentally retarded, are very short, and have
webbed necks. Phenotypically, individuals with Turner syndrome are female.

Fig. 11-16
Klinefelter syndrome: humans that have Klinefelter syndrome are 47, XXY. They
are sterile. Phenotypically, they are male.
In mammals if it you have a Y chromosome, then you are phenotypically male.
Ex:
Something like this 47, XXX. That would be called triplo X, and individuals that
are triplo X are female b/c they don’t have a Y chromosome.
Individuals that are 47, XYY are males; they have a Y chromosome. Except
individuals like this, that have extra Y chromosomes have an interesting history b/c they
think it makes an individual super aggressive.

A couple things to notice:

When we have aneuploids in humans, they are often for our sex chromosomes.
Aneuploids are much less frequent in indivuals if they’re for autosomal (somatic)
chromosomes. Why? What’s the problem with being 45, X or 47, XXY? Well, females
only have X chromosomes, So you’ve got all the genes here but now with the X
chromosome, the amount of copying that the genetic being produced is half of the
amount of gene product for any of the autosomal chromosomes. so in this situation, there
can be an unbalance between what’s expected and what you have with a relative
concentration of enzymes produced with the X chromosomes. Yet, an individual like this
(46, XY) is totally normal, it’s a normal male. So why is this abnormal and why is this
normal; well what happens is:
This is the normal chromosomal complement for us whether it’s male or female
(46 XY; 46 XX). Somehow, both of these phenotypes have to come out normal. What is
required there is somehow you balance the genes. The way that’s done in mammals is if
this is the fertilized egg, it divides over and over again. Around the 64 cell stage (where
we have about 64 cells), if this is a female what will happen is one of these X

75
chromosomes is going to be randomly inactivated in a cell. The X chromosome that’s
inactivated in this cell is independent of the X chromosome that’s in another cell. So once
it’s inactivated, then every cell that descends from this cell is going to have the same X
chromosome inactivated. So if you look at one of these cells in a microscope, what you
would see is something like that [notes]. A large, darkly stained body would be near the
inside of the nuclear membrane; that’s the inactivated X chromosome, it condenses up
and becomes heterochromatin and is passed on that way in future generations. So this
structure is called a Barr body. The Barr body is the adaptation mammals have adopted
to make sure that the phenotype of a male and the phenotype of a female can be
somewhat normal even though they both have different numbers of X chromosomes.

A female that’s triplo-X (47 XXX), what she will do is inactivate two of her X
chromosomes in every cell. So she would have two Barr bodies. And all of that is a way
to make gains in genetic balance so we could say if you’re a human individual that makes
sure you’re going to behave as if you have normal X chromosomes.
This is what they first started doing as gender determiners in international athletic
competitions.

There are lots of complications with the genotype or the phenotype of a gender, b/c what
happens which actually determines sex in mammals is the Y chromosome produces one
gene product called the testes determining factor which if it’s not present, then the cells
develop into ovaries. If it is present, then the cells differentiate into testes which release
testosterone. Testosterone then diffuses and moves thru circulatory system where the
male external genitalia develop, and those hormones interact with a hormone receptor
inside the cell. So if you have a Y chromosome, you produce testosterone but to be
phenotypically male you have to have a functional hormone receptor in your cells; there
can be mutations which cause a nonfunctional hormone receptor then even though you
would be producing testosterone, there’s nothing for it to bind with and then you would
not produce the external male genitalia. The default in humans and mammals is to be
female.
Cells that are derived from the barr body cell are typically clumped together in an
individual’s adult body. What you find are females that are heterozygous for this allele
will have some regions on their body that express one allele and other regions that
express the other allele. This is called the Lyon Hypothesis.
The point is aneuploids of the sex chromosomes can survive; very few aneuploids for
autosomal chromosomes survive. We have none that are monosomic for autosomes.

We have three trisomies that survive more than a few weeks. One survives a few weeks,
one a few months, and one is Down syndrome.
There are 2 causes of Down syndrome. The most common cause is called trisomy 21
which causes 95% of the cases of Down syndrome. Individuals with Down syndrome die
in their teens; males are sterile but females are capable of reproducing but the significant
thing is one extra copy of one small chromosome can lead to profound phenotypic
effects.
Pg. 361 Fig. 11-18

76
 Having a baby with Down syndrome increases with maternal age. One
explanation for this is: all females when they are embryos, after a few months, all of the
cells begin meiosis. So within the embryo, all of the cells that will become eggs start
meiotically dividing but they get to a stage in prophase I and then they arrest there. They
remain in the arrested state until the female ovulates around 13 yrs. All of this is caused
by nondisjunction. What’s keeping the homologous chromosomes paired up is the
tenuous crossing over connection by chiasmata; so that has to survive from the time the
female is infant to the time she ovulates those eggs. If the pairing is no longer effective
(by UV radiation or something), then they pairing breaks and you have a good chance for
nondisjunction. That is probably the cause of this maternal effect.

There are 2 other trisomies:

a) trisomy 13 (Patau syndrome) – die after 4 months
b) trisomy 18 (Edward syndrome) – die after a few weeks
For both of those, individuals are born but they die as infants.

We have no monosomies for autosomal chromosomes; the only one is Turner syndrome.
Down syndrome is not an inherited condition; a female with gives birth to a child with
trisomy 21 they could give birth to another child with trisomy 21 simply because they’re
old. 5%of the cases of Down syndrome are caused by a chromosomal structural
abnormality called a translocation; that type of Down syndrome is inherited (if you have
one child like that, then there’s a higher chance you have another child like that).

11/07/03

Southern blotting
- Cut up DNA we’re looking for with restriction endonuclease you. You need a
probe for it. They found for a different organism and you hope it will be similar
to your species. You alternative species’s gene as a probe.

We know trisomy 21 is a very small chromosome so maybe we can by with it because it

has very little affect.

Structural Modification of Chromosome

Fig. 11-19 Part A pg. 364
- They occur during random breakage and problems due to crossing over.
- In the first structural modification, we lose loci two and three and that is a
deletion. So if there’s deletion in one chromosome then there will duplication
(insertion) in homologous chromosome.
- The next one down is an inversion where you do not lose or gain any genetic
material. We take one section out and invert it and then put it back. It become
1324.
- Translocation- is taking a region of one chromosome and put it in a non-
homologous chromosome. When we talking about translocation, we’re often
talking about reciprocal translocation. Reciprocal translocation- is where you

77
 take a part of one chromosome and put on a non-homologous chromosome and
vice versa where you take a non-homologous chromosome and put it in the first
chromosome.

Inversion- We have the common form is called the wild type chromosomal region.
Anything that have an inversion is called an inverted region.

There are two types of inversion.

- And we recognize them by seeing if the region that being inverted contain the
centromere or it does not contain a centromere.
- Pericentric inversion- is where the part that being inverted contains the
centromere.
- Paracentric inversion- the part that being inverted does not contains the
centromere.

Fig. 11-22
Paracentric inversion- The top strand is the normal strand (ABCDE) and the bottom
strand is the inverted one (ADCBE). It normally BCD but it has been inverted to be DCB
and the region does not contain a centromere.
What important about these inversion is how they behave during prophase one of
meiosis because synapse must occur so an inversion loop must occur. Crossing over that
occur in the inverted region is inviable that mean it never get pass it on to the offspring.
Let say there is a crossing over between B and C. you start at a centromere and begin
tracing.
The two parentals, make a dot and ABCDE and another one is dot adcbe.
So one from crossing over is dot ABcda dot this is dicentric chromatid. And what happen
in anaphase 1 of meiosis is the two centromere go to opposite pole then the dicentric
chromatid is going to break somewhere in the middle at random.
The gene products produced be the dicentric chromatid are always inviable so they
can’t make offspring and they also called deletion product.
Another one is ebCDE and this section has no centromere so there is nothing to guide it
to the poles so it just get loss so we called that the accentric product.

In paracentric inversion, two of the products are parentals and are viable. The other two
are produce by dicentric chromatid and are inviable. You will never get accentric
product.

Fig. 11-23
Pericentric inversion-
One of the parental has inverted B and C.
One parental is AB . CD and the other one is ac . bd
One product is AB centromere CD and the other one is ac centromere bd
The other product are AB centromere ca and this is not dicentric because it has only one
centromere and it has two copy of A and no copy of D. This is duplication and deletion
where you duplicate A and delete D.

78
The other product is DC centromere bc and this is duplication of D and deletion of A.
The one that you have duplication and deletion are inviable.

When you have pericentric inversion this is the long arm and one short arm and that
called the acrocentric chromosome. For the other chromosome the ratio of arms length is
the same.
In pericentric inversion after one chromosome has an inversion then the ratio of arm
length for the two chromome are now different. You can recognize pericentric inversion
by the position of the centromeres because of this.
For paracentric inversion after one chromosome get inverted then there is no change in
the arm length ratio between the chromosome. Having a paracentric inversion doesn’t
change the arm length.

If there is an inversion between loci and one of them go from euchromatic region to a
heterochromatic region then that gene or locus is turn off. The amount of gene that
transcribe is influence by the location of the gene in the genome this is called the position
effect. So if you change the position of a gene to a new location then that has potential
for a position effect and that can change the phenotype of the organism.

Fig. 11-24
Reciprocal Translocation-
N is the normal chromosome and T is the translocate chromosome.
The N1 and T1 centromere synapse and the N2 and T2 centromere synapse. In meiosis
you get this cross like configuration if you have a reciprocal translocation. In anaphase 1
you have segregation occur. Let’s look at how segregation could occur, it could occur
like this if T1 and N2 go to the same pole. What would happen? Well we would be
missing this whole large yellow arm; there would be a major deletion there would be two
copies of these purple arms which is duplication. These lead to inviable products. Same
thing would happen over here if N1 and T2 segregate together. This is called adjacent
segregation where the adjacent chromatids segregated to the same pole, and then you get
inviable gametes.
You can get viable gametes if N1 follow with N2 and T1 follows with T2; so if the
translocated moves with the translocated and the normal moves with the normal. This
would produce a gamete that was the parental type (had no translocation in it); these
would produce a gamete that had the translocation in it.
So half the time you’ll have alternate segregation and half the time you’ll have adjacent
segregation and that means when you have a reciprocal translocation, half of the time
you’re going to produce inviable gametes. This is important for female because they
have a limited number of eggs. This is called semisterility where half the gametes are
nonfunctional. If there’s a drop in fecundity, then you would expect that individuals are
producing half the number of viable gametes and that’s a tip-off for reciprocal
translocation.

The other type of structural modifications you can have are deletions. When you have
deletions, the homologs have to synapse as well. So for that to happen, there has to be a

79
deletion loop and the organism is hemizygous for that region. High deletions are highly
deleterious. We only have one really large deletion in humans and on the short arm of
chromosome 15, and it’s called cri du chat. It you have recessive alleles in the deletion
loop, then they will be expressed because you hemizygous in that region. That is called
pseudodominance.

11/10/03
-key things to remember about deletions:
cytologically, we have to be able to visualize each of the chromosomal
mutations essentially through a microscope
-during synapsis, certain characteristic pairings of homologous chromosomes that are
heterozygous for one of these chromosomal aborations
-in an inversion-loop de loop that formed
-reciprocal tranlslocation that a cross forms
-in deletion
if one chromosome has a region deleted, it then pairs with a wild type
chromosome
:the deleted region that existed in the wild type chromosome will have no
region to pair with-fall out and forms deletion loop
:any genes that are contained in the deletion loop are going to be expressed
whether they are dominant or recessive
:recessive allele may become heterozygous with a chromosome that has a
deletion on it-it will become expressed/appear to be dominant with respect to
the phenotype-pseudodominance
-mapping chromosomes-pg. 370
:recessive alleles of a-g on it
:heterozygous with a chromosome that is wild type on those positions
:wild type chromosome has had a deletion on it and it’s the region that
contains loci b and c deleted on the wild type chromosome
:you will see phenotypically, an individual that is heterozygous for these
two chromosomes will express the b and c mutants which it previously
did not
-pt. 371-fig. 11-21
:series of deletions perhaps in a laboratory stock, you can position the
deletions along the chromosomes and know where the deletions are
due to the banding patterns (phenotypically speaking)-that can identify
where certain genetic loci are-deletion mapping
-small deletions may be handled; large deletions are deleterious
:small deletion in the frameshift can cause a nonsense mutation to form
-duplications
:trinucleotide repeats
:number of genes that represent tandem repeats-example rRNA gene-have
many copies of those; those apparently evolved for situations where we
need more gene product than can be produced than simply having a
single gene-one thing that happens that is functionally useful for the
organism

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 :gene duplications are important evolutionarily because what happens is
if we go from one copy of a gene to two copies, one copy can make
copies of that gene, then what will happen, is because of that redundancy,
some genes can essentially act as evolutionary experiments
:have enough copies of a functional gene that can produce the gene
product we need, but these other loci that began as identical copies of that
original gene can go through mutations and will have little effect on the
organism’s phenotype-because that organism will always have the
wild-type existing as well
-pg. 373-fig. 11-31
-pg. 375-fig. 11-33
:human chromosome has 22-autosomal chromosomes and an XY-23
pairs of chromosomes
:mice have 40-pairs of chromosomes
:both are mammals
:color-code chromosomes; look for linkage groups in mice; continuous
blocks of DNA in different mouse chromosomes that are actually
consist of regions of specific chromosome
:species that are evolutionarily related appear to have their genomes
evolved by regions of the DNA through translocations, inversions, etc.
rearranging themselves/there still appears to be remnant of linkage
groups that existed in one species that you can visualize in another
species
-pg. 376-fig. 11-35
:5% of Downs syndrome cases due to reciprocal translocation
:punnet square-left are gametes from normal parent/top-
translocation occurring in an invidiual-top right-pairing
of chromosomes during metaphase I of meiosis
:4 gamete types-1 type leads to aneuploidy-causes death
:3 individuals that are produced-1 out of 3 will have
Downs syndrome, 1 out of 3 will be normal, 1 out of 3 will
be a translocation carrier
:child that has Downs syndrome due to this translocation, that is an
inherited form
:chances of having another child with Downs syndrome is approx. 1-in-3
:trisomy 21 Down syndrome is an independent event; requires an
independent nondisjunction to occur each time it happens

11/14/03

Ch. 13
Gene Regulation

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When we have an DNA sometime the gene turn on and sometime turn off in a cell.
Sometime in two different fission, a gene turn on in one fission and turn off in another
fission. There is a lot of regulation in the product of the gene produce.

Gene regulation is involve in when we either have positive or negative gene control.
We going to talk about protein interact with DNA. A protein is going to bind to the DNA
and the individual gene is shown in purple here and is going to bind the region between
the promoter and where the transcription are going to begin. When this protein bind the
DNA so we going to transcribe the DNA and this is refer to be positive control.
Positive control- first the protein bind to the DNA and that turn the gene on. And
that same gene with the protein removed and we don’t transcribe the DNA.

Negative control- is when the protein bind to the DNA between the promoter and
where the transcription initiation site and that serve as a blockage and prevent
transcription. And when the protein are remove the gene would be transcribe.

What integrate each either type of regulation we have a system called inducible system
that repress these system. In the inducible system, the thing that determine the
functioning of the control of the gene is the function of the concentration of the substrate.
When the substrate get high we turn some gene on or off. By having a high concentration
of substrate we turn the gene on. We can turn on a gene by either a through a positive
control or negative control.

A repressible system, when the concentration of the substrate get high we turn of the
gene. It can turn of a gene either through positive control or negative control.

We can have an inducible system with positive control or negative control.

We can have an repressible system with positive control or negative control.

Fig. 13-4
These regulatory proteins is for positive control it is an activator. If it for negative control
is called repressor. These generically drawn regulatory protein have allosteric site which
another protein can bind to. The protein that bind to these regulatory protein is called an
effector protein.
In an activator, if there is no effector protein bound to the activator then the activator will
not bound to the DNA. If the effector is present at the allosteric site of the regulatory
protein that will change to conformation of the activator then the regulatory protein can
bound to the DNA.

In a repressor, without the effector present. The repressor would normally bound to the
DNA and the only way to get the repressor off is to have an effector bound to an
allosteric bound to the repressor and that will remove the repressor from the DNA.

The major different between gene regulation in prokaryote and eukaryote. We have to
appreciate the way biochemical pathway regulate in prokaryotes vs. eukaryotes. Bacteria
have circular DNA and they need a biochemical pathway like this and each step is control

82
by different enzyme. For this biochemical pathway to occur they need the enzyme alpha,
beta, and gamma. They are going to be encode for by machine in the DNA of this
prokaryote and these genes would be adjacent to each other in the DNA of this bacteria.
And the bacteria would have promoter over here and this promoter will be recognize by
an RNA polymerase and the RNA polymerase will attach to it and begin reading the
DNA and it would transcribe that entire region of the DNA before the transcription would
be terminated. We would wind up with an messenger RNA that has the gene alpha, beta,
and gamma on the same RNA strand. This is a polycistronic messenger RNA mean
several genes on the same RNA strand. So the trick is to get this biochemical pathway in
prokaryotes is somehow have messenger RNA recognize this specific promoter.

But in eukaryotes, each gene have it own messenger RNA. So the messenger RNA is
monocistronic.

Gene regulation in prokaryotes

- The operon is a set of structural genes like alpha, beta, and gamma and we
also have a promoter and the region between the structural gene and the
promoter we have this region called operator. So an operon have a set of
structural gene, promoter region, and the operator.
- Transcription occur when RNA polymerase look for promoter region and then
bind to the promoter and start moving down to transcribe the DNA. Now we
have think about the process of when the RNA polymerase recognize the
promoter and whether they can move pass the operator or not to get to the
structural gene. We have the repressor bound to the operator this RNA bound
bound the promoter and there is a repressor there and it can’t pass the
operator.
- The lac operon- is the operon that use to break down lactose. It include three
genes which are beta galactosidase break lactose down to glucose and
permease is an enzyme that become part of the membrane system and allow
the transfer of lactose from the outer of the cell into the cell. Tran acytalase
is an another enzyme.

The bacteria that live in you will reproduce very fast. The bacteria live in your gut when
you were fetus and they have spend their entire life in your gut and they gain all the
nutrients that come in you. The E coli get nutrients from your intestinal tract. These E
coli that live in you when you were young. And the sugar in the breast milk is lactose and
the only source of energy you get is lactose sugar. You have to have to be able to
breakdown lactose and after a few years you stop drinking milk. And so there is no
lactose present in the baby after he/ she stop drinking breast milk. There is a region on the
DNA that is near the operon or distance from the operon. The regulator gene is symbolize
‘I’ that normally transcribe to producing a repressor protein. This repressor protein then
no effector attach to it so the repressor simply bind to the operator and negative control
because it block the RNA polymerase to get to the structural gene. Normally the lac
operon is turn off. After you drink a glass of milk and these lactose out here and these
bacteria want to get lactose. Some lactose can pass through the plasma membrane but

83
very inefficiently. And if you have to permease activate then the permease will speed up
the rate of reaction of the lactose be transport to the inside of the cell. Lactose will
behave as an effector and it will bind to the repressor that change shape of the
repressor and it will no longer bind to the DNA and this is repressor bound by
lactose. Then now the polymerase can transcribe the gene and when it transcribe the beta
galactosipermease and beta galactosidase. We will keep the gene turn on if lactose is
there. And galactosidase will break down lactose to glucose and galactose and after all
the lactose is break down then there will be no more lactose to bind to the repressor and
that repressor will bind to the DNA again. When concentration of lactose is drop we go
negative control, we shut the operon down. We call this inducible system with negative
control.

Lac operon is the first operon that discover and common role of regulation of
transcription in prokaryote.
- The prokaryotes side is what determine a gene in prokaryote is going to turn
on or off.

In eukaryotes, we are a multicellular cell and each cell have the same DNA blueprint but
we have to turn on and off genes in different tissues at different time. This is going to
involve a complex set of communication and complex set of signal that have to be made
within an organism. It amazing because all of these cell in different tissues have to
originate from one single cell which is the mother fertilize egg. This cell have to
somehow differengase so that the descendents are able to know which gene to turn on
and off despite that they all have identical gene.

11/17/03

Regulator gene = I

Normally the repressor prevents RNA polymerase from transrcribing the gene.
Lactose cause gene to be turned on.
Permease is a transporter gene.

If lactose is available then they want to utilize lactose. But if lactose is not available then
they don’t want to maintance the expense biochemical machinery is continue the
production of beta galactosidase and permease if lactose is not present. We want to
understand how does this establish genetically.
Table 13-1, 13-2, 13-3 on page 426

Table 13-1 Let look at the operon which is the structural gene and the operator. We don’t
want to worry about the third structural gene which is tran acetylase. We only worry
about the Z and Y where Z is the beta galactosidase and Y is permease. Normally we
have the operon which is the operator cause the wild type Z and the wild type Y. On the

84
first row we can see that beta galactosidase and permease is both inducible. So if lactose
is present then the enzymes are produce and if lactose is not present then the enzymes
will not be produce. This is a bacteria genome and normally it going to have the operon-
the promoter, the operator, and the structural gene and it also going to have regulatory
gene on it. There are variability different types of plasmid present in the bacteria. One
type of plasmid is called fertility factor (F). Plasmid have some DNA that form circular
DNA that is different from the bacteria genome. We can inserted in the F factor certain
DNA that was obtain from a bacteria. We can inserted part of the operon into the F
factor. We can inserted the operator and the structural gene in the plasmid. We called this
bacteria an F prime that mean it has an F factor that has some gene on it from the
bacteria. In this bacteria it has two copies of this gene so this bacterium is a partial
diploid for the gene we talking about. And the allele here and here can be the same or
different. If they are the same they are homozygous and if they are different then they are
heterozygous. If we create heterozygous gene then one of them is dominant over the
other.

As we can see from the table 13-1 that they symbolize O+Z+Y+ that mean the operon on
the bacteria genome. Then you have slash and anything after the F’ is everything that
on the plasmid.

So we can see that on the plasmid we have a wild type O and a mutant Z and a wild type
Y. And we can see that the Z gene is induce just like previously just as the permease. So
that tell you that the wild type Z is dominant over the mutant Z.

Also on the third row we have a mutation on the operator region. We symbolize a
mutation on the O by having Oc. We can compare the third row with the first row and we
can see that no longer inducible because the gene never got turn on. So whether we have
lactose present or lactose absence we got beta galactosidase and permease produce and
this type of mutation is called constitutive mutation. Constitutive mean that it always
turn on. If this is DNA over and normally the regulator recognize the operator and bind to
it and turn it off when there is no lactose and if the operator mutate such that the regulator
can no longer recognize the binding site so it can’t serve as a blockage. So the RNA
polymerase can gain excess and move pass the operator and transcribe the gene.

Look at row four, in our genomic operon we have a mutant Z and a wild type O and a
wild type Y. And on the plasmid we have a mutant operator and a wild type Z and a
mutant Y. This tell us that the Z is turn on whether there is a present or not. But the Y. the
only effect of the operator is for the controlling DNA that is on the same DNA strand as
the operator is. The constitutive mutation on the plasmid keep the Z gene always on
therefore it never got turn off. The wild type operator on the genomic DNA regulate the y
gene in the appropriate fashion. What we looking for is whether or not the regulatory
system work at the cis functioning or at the trans functioning unit. If it on the Cis
functioning then it has to be on the same DNA to be able to control the gene. Tran
functioning regulatory unit may be on the same DNA or it may on the different DNA
strand. So cis mean the regulator on the same strand as the structural gene. The trans
requirement simply mean it does not exist because it can’t be on the same or different.

85
Table 13-2
- This is mutation on the regulator and we assume we have wild type operator.
- On row one we have wild type I, wild type Z, and wild type Y and it showed
work appropriate.
- On row two we have mutation on the regulator and we never turn off the
structural gene. We now create a nonfunctional regulator and even though we
have a working operator and waiting for the regulator but the regulator is not
functional so the polymerase continue to move pass and read the structural
gene whether there is lactose or not. And mutation on the regulator as a
constitutive mutation.
- Now we have create an F’ and this partial diploid will act operator that is wild
type regulator on the genome and a mutant type Z and a wild type Y and on
the plasmid we have a mutant regulator, wild type Z and a wild type Y. We
going back to normal functioning inducible system. If lactose is not present
then we don’t synthesize either beta galactosidase and permease. As long as
we have a functioning regulator somewhere and it doesn’t matter if the
regulator is in cis configuration or trans configuration and we can control the
operon just as well.
- Look at row four, look at the genomic DNA we have a regulator produce a
repressor protein that will bind to the operator. So if there is mutant operator
over here then it used the operator on the plasmid. This tell you that the
operon is not include the operator, the operator is come far from the operon.
Strand four tell us that the operator is act as a trans configuration.

Table 13-3
- Strand one tell us the normal functioning system.
- Strand two we mutate the regulator gene, we indicate this mutation with a
superscript S. this is called a super repressor. We never synthesize gene
products from structural gene. So when there is a mutation on the regulator
and it going to produce a protein and the protein recognize the operator and
the repressor must be remove. We have lactose bind allosterically to the
repressor and change it shape and remove the suppressor. But with a super
repressor, it bind to the operator and mutation modified the allosteric site so
the lactose can no longer recognize the repressor. If lactose don’t bind to the
repressor then nothing it going to remove the repressor. So you keep repressor
on even if there are lactose or not.

Pg. 431 fig. 13-15 and fig. 13-16

The lac operon is not the primary control mechanism for lactose utilization in bacteria.
- Positive control, the promoter sequence have a sequence that can be recognize
by RNA polymerase. If all we have is promoter by itself then the RNA
polymerase can’t recognize it very well. The promoter by itself is not very
attractive to the RNA polymerase. So we have to modified the promoter

86
 region to make it more attractive. There is a site on the promoter that is
recognize by a another protein CAP protein. There is a CAP protein that can
bind to the promoter then the promoter become very attractive to the
polymerase. The polymerase will attach there and whether they get pass the
operator or not depend on the regulator mechanism previous discuss.
- If you drink a glass of milk then there will be a lot of lactose so bacteria can
used it as an energy source. The bacteria can use beta galactosidase to break
down lactose to galactose and glucose. In the cell membrane there is an
enzyme called adenylate cyclase and this enzyme will convert ATP to cyclic
AMP. This enzyme can be turn on and off. If there is a lot of glucose in the
cell then it will break down glucose to carbon dioxide and the intermediate of
this process will activate adenylate cyclase. If there is glucose is available
then this enzyme will be turn off. And if this enzyme turn off then we don’t
produce cyclic AMP. If glucose if not available then this enzyme will be turn
on then we con convert ATP to cyclic AMP. What determine if we going to
have cyclic AMP or not is whether we have glucose.
- What it turn out that the CAP protein will not bind to the promoter by itself. It
only bind to the promoter if CAP protein form complex with cyclic AMP. If
cyclic AMP is available then it will form complex with CAP protein and those
together will bound to the promoter. And the only time you get cyclic AMP is
when you don’t have glucose. Now we got the CAP-cyclic AMP bound here
that attractive to the RNA polymerase and RNA polymerase can bind to the
promoter. And whether or not if the RNA polymerase pass through the
operator is depend if there is a present of lactose or not.
- If there is a lot of lactose and there is not a repressor here but without cyclic
AMP the CAP doesn’t bind and so the RNA polymerase is not attractive to the
promoter and so you will never transcribe a structural gene. This is an
example of positive control. The only way we can transcribe the structural
gene if is the CAP protein bound to DNA.

In eukaryotes gene regulation:

The regulation to what we looking for is by how much functional enzyme is going to
produce. When the functional enzyme produce and how much is it going to produce
because this is what control the biochemical reaction that going to occur. We can control
this by how often we create messenger RNA from DNA. That can be control by
transcriptional level. 40% of human gene are slice in multiple way.The length of the poly
A tail will effect on how long the messenger RNA is going to survive. And some of the
message in the UTR can effect the stability and the rate at which they attract ribosome.
How fast you process that pre mRNA and get it out the nucleus can effect. We can
produce a lot of RNA that remain inerted status until they get activated this is happen
when woman begin producing milk. How long lived this RNA is and how attractive it is
to ribosome is going to effect how many enzyme we going to produce. How long that
polypeptide. All of these stages are being regulate.

Transcriptional regulation

87
The promoter will have TATA box. The TATA box is prokaryote occur ten nucleotide
upstream from where transcription initiation. But in eukaryotes this TATA box occur 30
nucleotide upstream from the transcription initiation site. In the promoter region there is
also a region called promoter proximo region. It has a CAT box 100 bases upstream and a
GC rich region about 200 base pairs upstream.

Fig. 13-22
This show us the frequency in which transcription occur. That we have base substitution
at various point upstream from the point of transcription initiation site. At a level of 1.0 is
a normal transcription for the wild type. If you look at the TATA box, GC rich, and the
CAT box mutation have dramatically reduce the rate of transcription for those gene. The
actual transcription process is going to be affected by region of DNA as well. None of the
region of that DNA that affected transcription are going to have this specific spatial
orientation. These are particular region that is important toward transcription have to
occur spatially in those specificity.

Enhancer and silencer- these are region of DNA that can occur within 20000 base pairs
or so upstream, downstream, or within intron. There are other region that going to
affected the rate of transcription but they are not limited to these specific location.

What occur at every gene? This is called a basal level of transcription. This promoter
and these promoter proximal regions will not attract to RNA polymerase at all. These
region are attach to other protein. So other protein will bind to these region and now it
become attractive to RNA polymerase.

The protein that bind the DNA is called transcription factor (TF). We have different set of
transcription factor for different RNA polymerase. In eukaryotes we have RNA
polymerase I, II, III. So we going to have transcription factor to work with each of the
RNA polymerase. RNA polymerase II is what synthesize the messenger RNA.

TFII- is transcription factor for RNA polymerase II.

TFIIA- mean that is the first one that was isolated.

Each of these transcription factor are holoenzymes. They are made up of multiple
polypeptide.

Trancription factor II is what recognize the DNA. One of the polypeptide that is a part of
TFIID is called the TATA binding protein (TBP). This is what recognize the TATA box.
The TBP will recognize the TATA box and recognize it at the minor groove and it going
to fit in the minor groove. And the rest of the holoenzyme for TFIID is assemble there
and then you have the whole TFIID enzyme assemble around the TATA box.

Fig. 13-25b
TBP bind to the TATA box and it going to fold the DNA and that change the architecture
of the DNA at that region.

88
If you have the transcription factor assemble and you get RNA polymerase II recognizing
this complex. After you assemble the protein, the RNA polymerase will attract to it. for
transcription to occur, the RNA polymerase has to leave the complex and begin moving
along the DNA. You have to get the RNA polymerase in and have to get the RNA
polymerase out. You have to attract the RNA polymerase and initiate transcription. One
way to increase the rate of transcription is by making the complex more attractive or
increase the rate at which the RNA polymerase leave it and begin transcription. To get
the RNA polymerase to leave we have to phosphorylate RNA polymerase.

11/19/03

TATA box is part of the core promoter and the other two parts of the promoter proximal
regulatory region.

TBP- is the TATA box binding protein. TBP is part of the TFIID.

All of these proteins are called transcription factor and they are holoenzyme which mean
that are made up of multiple polypeptide. One of the protein within the TFIID called
TBP. The TBP protein is what recognize the TATA box. The protein bind to the minor
groove of the DNA and recognize a sequence within the DNA. After it bind it cause the
DNA to fold in a 180 degree folding. And now it make that region within the DNA more
attractive for an additional transcription factor.

Fig. 13-25
First we bind to TFIID to the promoter, then we have transcription factor B and A, then
we have RNA polymerase II come in with transcription factor II F. this is happen to
every gene and we have not done any regulation yet.
Part of it is by assembling this complex and part of it by getting the RNA polymerase to
leave the complex and initiate transcription. The rate at which this complex assemble and
attract RNA polymerase II will influence how many transcription we made. In addition,
how fast we can process the RNA polymerase and so it can leave and begin transcription
this will going to effect how fast we transcribe these thing.

The histone, with the octomer wrap the DNA around. All protein have an amino end and
a carboxyl end. All of these system have an amino end hanging and if we add an acetyl
group these end here that change the mediate structure and we said that we remodeling
the chromatin. Because now we make the DNA more available for protein to recognize.
If we can add acetyl group or acetylate the histone and that mean it loosely bound the
histone to the DNA and now that DNA can now be recognize by thing like transcription
factor. We turn on a lot of gene by just adding a acetyl group to them. The enzyme that
do this are histone acetyl transferase (HAT). This enzyme will add an acetyl group to
the histone and that make that region be available to be recognize by proteins.

89
We can inactivate a gene by removing the acetyl and now the histone bind tightly and
other protein can’t recognize the DNA. The enzyme that remove an acetyl group from the
histone are called Histone Deacetyl Transferase enzyme (HDAT).

The nitrogenous base itself got methylated. We have these methyl transferase that
methylate certain nucleotide. The region that is highly methylated region also have
activity of HDAT that cause the acetyl group to be remove which turn the gene off.

Mechanism that control these thing:

Fig. 13-24
If we eliminate the activator. There are region on the DNA that are not location specific.
These enhancer is a sequence of DNA that attract to a different type of transcription
factor. And these transcription factor will recognize these enhancer and these
transcription factor that recognize the enhancer are called activator. This activator will
have a domain that’s complementary to some domain in our basal transcription factor
complex. So what it does is bind over here and what that does is bend this DNA back.
That’s why the location of the enhancers is entirely important because if this enhancer is
5000 bases away, the DNA can still fold over here. So what we’re doing is creating a
larger complex here that has some different characteristics than just the basal complex
has. This could be more attractive for an RNA polymerase if the activator’s in here, or it
could cause the RNA polymerase to leave faster therefore the transcribing more copies.
Now this is what an activator would be; so here’s another activator but maybe this
activator can’t recognize this complex. So there are things called coactivators which will
bind there. The whole idea is to create a much more attractive complex to RNA
polymerases or to create a complex that’s going to cause the RNA polymerase to be more
efficient-making. In addition to enhancers, there are also things called silencers. A
silencer can attract proteins also, but the proteins it attracts are called repressors. So if
we attract a repressor here, this repressor now can bind to the complex to make it less
attractive to RNA polymerase; or it may prevent the RNA polymerase from leaving. So
we’re regulating the rate of transcription in a cell not by changing the basal transcription
complex itself. What we’re doing is each tissue has cells in it that are producing
activators or repressors and the combination of activators and repressors you have, that’s
going to recognize a gene, are going to determine the rate of transcription of that gene.
Another way a silencer can work is if it’s right next to an enhancer, then look at what
happens. If a repressor binds to the silencer, then the enhancer is unavailable for binding
because it blocks any protein from getting access to that enhancer. Now the way a lot of
these activators or enhancers work is they have HAT activity. In other words, it this
comes over here what it can do is acetylate the DNA and by modifying the structure of
the chromatin, that may make it much easier to begin the transcription process. So we
want to know how to turn on genes in some places at certain times when we want to. The
way we turn on a gene at a particular time is by that cell producing the appropriate
activator or combination of activators/silencers, now we’re regulating things. So we
know at the location of the gene how we’re going to do it. We still don’t know when to
produce these activators or these silencers because remember ultimately these proteins all
have to be synthesized by this cell itself. So somewhere else in the genome there has to

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be genes that code for these activators and silencers. What we now have to try to
understand is why some cells turn on these activators and other cells don’t turn them on.
You have 210 different cell types in the body. We know some cells produce some gene
products and other cells produce different gene products (proteins); we know one thing
that’s happening is that they’re producing different activators and repressors.

Skip to page 440, 441

Transcription factors - these are proteins that can recognize DNA. Any protein that can
recognize DNA has to have a domain that can read the DNA, so it has to have some way
to interact with the DNA. There are certain motifs that proteins have in their domains that
are regularly affected for reading DNA.

Page 441, Fig. 13-34

That’s showing you a motif that’s called a helix-turn-helix. Each of those purple
cylinders represent alpha helixes that’s part of the proteins structure, and that turn is a
very specific turn. The rest of the protein may be around here but part of the protein looks
like this. And what happens is that this alpha helix over here can fit right into the major
groove of DNA, and it will fit in if it has sequences in its side chain that will be
complementary to specific sequences that can occur along the DNA. So what it does is
this causes this whole protein to position itself along the DNA. If this is positioned in the
major groove, then it’s positioning the whole protein and that protein may have another
domain that’s attractive to another set of proteins and so on. So as long as you have a
DNA recognition site, I can put a marker along the DNA that can be then used as an
assembly point for out protein. This could be an activator protein here or it could be part
of the basal transcription complex or it could be a silencer, etc.
What happens is sometimes you produce lots of copies of one transcription factor and it
will have a recognition site at multiple locations in the genome. So a transcription factor
might recognize the DNA here, or it might recognize it on a different chromosome. So if
we produce transcription factors like that, then we can simultaneously turn on different
sets of genes at points.
So remember how prokaryotes do it. Prokaryotes do it because genes are organized in
operons. They have polysystronic mRNA’s. What we can do is create a transcription
factor, maintain copies of this same TF but what this TF can recognize is multiple regions
of DNA that have the same sequence.

Fig. 13-35
Another motif that recognizes DNA is called a zinc finger. What you see is the element
Zn will actually form a complex with a polypeptide and will cause usually cause systine
amino acids to bind with zinc in some combination here. If you see 2 systines and 2
histadines covalently binding with zinc but when that happens, if this is where the Zn
molecule is going to be and I bind like this, then I have this extension out here. And what
we can do is create a protein that looks something like this. Each of these is a zinc finger,
and now you have a protein that has a certain pattern of zinc fingers that will fit into the
major groove of DNA also. If we have the right zinc fingers then we can fit into some of
the major grooves of the DNA and if one of the zinc fingers is wrong then maybe it

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doesn’t fit in there. So that’s another type of recognition site. So we’re searching the
DNA of the genome and if we know the basic patterns that exist that will be recognition
motifs of DNA, then we can find these genes that code for these transcription factors by
searching out their DNA using computer algorithms that are looking for something that
will give you a helix-turn-helix or a zinc finger.

There’s another motif that’s not in the book that’s called a leucine zipper. When
you have polypeptides like this that has a number of leucines along it and you get another
polypeptide that also has a number of leucines, what happens is the leucines attract each
other and bind. So look at this, this almost looks like a pair of scissors. So what happens
is pair will go over to a DNA and this one will fit into one major axis and this one will fit
into another major axis. So the functioning protein is a dimer, it’s got 2 polypeptides
bound together. But this is a really tricky one b/c one way we control regulation is
sometimes if we have 2 identical polypeptides then that would be called a homodimer. If
what happens is we have one protein like this and a different protein like that, they could
come together too. So the homodimer may act as a recognition site for DNA but the
heterodimer may not. So this may not be able to bind where this one could. So one way
that we can regulate whether or not the proteins are going to recognize the DNA is by
whether we’re creating hetero- or homodimers.

Page 444

Calico cat – fur color in cats is controlled by a gene on the X chromosome and what
happens is that’s a mammal, so at 64 cell stage of embryonic development females
inactivate one of their X chromosomes and make Barr bodies of it. So what happens is a
mature female is a mosaic and you can see the different colors you get if you have that
way.

Hormone Receptors

This is our cell here and what happens is if we have steroid hormones, hormones don’t
have to be steroids. They can be steroids or they can be proteins. But if they’re steroids
they have an interesting feature; steroids can cross cell membranes because they are small
nonpolar molecules. So what happens is we have things called hormone receptors, which
are proteins that exist in our cytosol and sometimes they also exist within our nucleus.
And what happens is they will have a domain that will bind to a hormone, let’s say
testosterone. If we produce testosterone, it can move across our cell membrane and bind
to this receptor. When it does, it allosterically changes the shape of this receptor,
activating a portion of that receptor that then will be a DNA recognition site. So the way
hormones work is they bind to one region of the receptor, that receptor changes shape,
gets activated, and then goes and binds to a DNA region which is called a hormone
receptor element in the DNA but this is just like a transcription factor binding to the
DNA. That’s how hormones turn on genes. So a hormone receptor has to have one site
that will recognize the hormone and one site that will recognize DNA.

End of Chapter 13

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11/21/03

CHAPTER 14

Pleiotropy – one gene affects more than one phenotype.

For one phenotype it can be dominant and for another phenotype it can be
recessive.

In addition to pleiotropy, there are situations where one phenotypic trait can be controlled
by more than one gene.

Pg. 464
For instance in an example like this, for us to get blue pigment what has to happen is
some precursor molecule has to be converted to an intermediate molecule and that
intermediate molecule then has to be converted into blue pigment. Each of these steps
requires chemical reactions that’s controlled by an enzyme. So therefore if either enzyme
1 or enzyme 2 were missing you would not get blue flower color. So the phenotype we
would be looking at in this case would be flower color but flower color is going to be
controlled by 2 separate genes. Now we’re going to talk about that today and that
essentially involves a phenomenon called epistasis.

But before we get into any of the complications that I just referred to, I want you to
understand how geneticists make certain decisions because let’s say you’re looking at a
blue flowered plant and a mutant arose in your lab that resulted in the plant having white
flowers. Now I’ve already showed you the example there, in that case what could happen
is either of those two genes one controlling enzyme 1, the other controlling enzyme 2 as
you just discussed. Okay, so you know you’ve got a mutation and we’re going to assume
for the time being that that mutation is a recessive mutation. Some guy in Chicago is
studying the same species as you are and he gets all of a sudden a white flowered plant.
So he’s got a mutation causing instead of blue (the wild type) to have white flowers. Now
the question that I asked you is are the two mutations you’ve each isolated for the same
gene or are they for different genes? So we realize what we could have is you could have
isolated one form of mutation for a gene and he could have isolated another form of a
mutation for that same gene therefore there would be multiple alleles for that gene. In
contrast, you could have isolated a mutation for gene 1 and he could have isolated a
mutation for gene 2 and that could be what’s going on. So the first thing you have to do
when you have 2 recessive mutant mutations is to decide whether or not these mutations
are alleles for the same gene or they represent different genes, b/c different genes can
affect the same phenotype. So in order to do that we use what’s called a
complementation test.
Pg. 458 Fig. 14-3
Here’s the wild-type blue flowered individual. You don’t really know the
genotype they’re showing you. So right now you’re studying this species and it has blue
flowers. Here are 3 mutations that have been isolated. We’re going to call them the dollar
mutation, the pound mutation, and the yen mutation. Now what happens is what you have

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to do is get an individual that’s homozygous for each mutant form. So this individual is
homozygous dollor and what it turns out is phenotypically the offspring have white
flowers. This individual is homozygous pound and the individuals have white flowers.
This individual is homozygous yen and the individuals have the white phenotype. Now if
we want to study whether these are genes or not genes, you take 2 individuals that are
each homozygous for a mutant mutation that we’ve uncovered and we cross them. If we
cross a dollar individual with a pound individual, we do not have any functional genes for
the gene on the left. We have a functional gene, a homozygous wild type, for the gene on
the right. So look at what would happen in this biochemical pathway, it would stop right
at the colorless precursor because we do not have the enzyme necessary to convert that to
the intermediate. So we would have a white flowered individual. In contrast, look at what
happens if we cross pound with yen. If we cross pound with yen, the F1 individual is
going to be heterozygous wild type for each of these 2 chromosomes. So there will be at
least a functional copy of the gene producing this first enzyme 1 as well as the gene
producing enzyme 2. So what happens is we cross 2 white individuals and they lead to a
blue individual. What happens is the mutations complement each other. So a wild type
from one gene can mask the expression of the mutation and the same thing happens to the
other gene. If two mutations complement each other, what that means is you’re dealing
with 2 separate genes. If two mutations do not complement each other, you’re dealing
with one gene but 2 different alleles. So if you’re trying to figure out what you’ve got,
you have to know whether you’re dealing with the same gene or multiple genes. So that’s
what the complementation is.

Now we want to look at how different alleles from one gene can interact. What we’ve
done so far is assume that if we had individuals from these genotypes:
AA
Aa
Aa
that a homozygous dominant and a heterozygous individual are phenotypically
indistinguishable from one another. Both of these can be distinguished from the
homozygous recessive. That’s not always the case. Let’s say if you look at snap dragons.
If you look at snap dragons and you get a white flowered snap dragon and cross it with a
red flowered snap dragon, what you know is that all of the F1’s are going to be
heterozygous individuals. This F1 individual in this case can be distinguished from either
parent because the F1 tends to have pink flowers. If you then bring that down to F2, what
you wind up with is a 1:2:1 ratio. You get this type of punnet square from making this
cross. This individual would have a red phenotype, these 2 would have pink phenotypes,
and this would have a white phenotype. So in the 1:2:1 ratio, the phenotypes have the
same ratio as the genotypes. Now in this case, why this happens is very simple. We have
some precursor molecules that get converted to red pigment and it’s converted as the
result of the presence of a certain enzyme. This reaction is enzyme limited, so if you have
more of the enzyme you produce more red pigment. So if you don’t have a lot of the red
pigment, instead of the flower looking red it tends to look pink. Now what happens is we
have 2 chromosomes since it’s a diploid individual and therefore if it is diploid we
assume the amount of gene product produced is related to the number of gene copies that
you have. So if the process is enzyme limited, only having one copy of the allele for

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producing red pigment would produce less enzyme and therefore less pigment. If we
were homozygous dominant, in this case we would produce more mRNA’s and therefore
more gene product (enzyme for red pigment) in our example. This mode of inheritance is
called incomplete dominance.

There’s another form of gene interaction that can give you the same 1:2:1 ratio in the F2
generation. This is referred to as codominance. There’s a slight intellectual distinction
b/w codominance and incomplete dominance. The best example of this are the antigens
that exist on your red blood cells. If this is a RBC, you have proteins embedded in the cell
membrane of the RBC. These proteins are all ultimately going to be synthesized by the
cell so there has to be a gene for each category of protein that’s causing the production of
that protein. Well we have a specific locus that codes for one of these membrane bound
proteins, and it’s one that’s of importance to us. It’s referred to as the ABO system. What
happens is there are 3 different alleles that can exist. Now each individual can only have
2 different alleles because you only have 2 copies of each gene. Those 2 copies could be
identical to each other or they could be different, but none of you can have all 3 of them.
But all 3 alleles for this gene exist within this room. These proteins get embedded in the
membrane and when they’re embedded, they can act as antigens. An antigen is a protein
that can be recognized by your immune system (which is in charge of checking your
internal components to make sure there are no foreign bodies inside you). Your immune
system has to be able to distinguish proteins that exist in your bodies and are supposed to
exist there versus ones that are foreign to your body. So we say that our immune system
recognizes self from nonself. Let’s say this represents a type of protein that we’ll call an
A antigen. There’s another type of protein that we could call the B antigen and there’s
another type of protein that actually doesn’t elicit an immune response which we’ll refer
to as O. Any of these 3 alleles can exist at this gene. The way we describe them
symbolically is like this: IA, IB, o. These 2 are in capital letters and this is in lower case
letter. So what happens is if I were a heterozygous BO, you would have type B blood. If
you’re heterozygous AO, you would have type A blood because B and A are both
dominant over the O antigen. However if you had A and B, you would look like this
where you would be presenting both antigens. What happens is in response to foreign
antigens, you immune system produces things called antibodies. One way antibodies
work is if these were a bunch of antigens and antibody would stick the antigens together
forming clumps. Then you’ve got a big phagocytic cell that will engulf it and dispose of
the foreign body. What happens if you’ve got antigens that exist in the RBC? Well
you’ve got a big old cell here, a big old cell here, and a big old cell here. So you’ve got a
big clump and this is happening in your circulatory system. So all of a sudden you’ve got
clumping and you clog up the circulatory system which isn’t good. So that’s what
happens if you get a transfusion from somebody and the transfusion puts blood in you
that’s different with regard to the antigens it presents versus what you normally have in
your own body.

Let’s look at transfusions. For transfusions we can have a donor and a recipient. If an
individual looks like this: IAIA or IAi, that’s a type A individual. This is a type B
individual: IBIB or IBi, either of those two genotypes. This is a type AB individual: IAIB.

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And this is a type o individual: ii. So we recognize 4 phenotypes depending upon the
genotype, so it’s one gene with 3 alleles.

Donor
Recipient A B AB O
(down)
A √ X X √
B X √ X √
AB √ √ √ √
O X X X √

If we donate type A blood to an A recipient, that’s good. But it we donate type B blood to
an A recipient, that would be putting foreign antigens in, the immune system of the
recipient will attack them, clog the blood up, and that would be a bad transfusion. If you
put AB blood in recipient, the recipient won’t attack the A antigen but it will attack the B
antigen. If you put blood from a type O individual into a type A individual, that’s good
b/c there’s no antigens in that donor blood.
Type O individuals are universal donors. Type AB individuals are universal recipients.
If you look at this from a genetic point of view, I can easily talk to you about let’s say the
dispute about the paternity of a child. If we know the mothers ABO system and we have a
potential father, we can see if whether the child’s ABO genotype is consistent with that
male having fathered the child or not. The fact that it’s consistent with that male doesn’t
prove that the male doesn’t prove the male was the father, but if it was inconsistent it
could prove the male was not the father. This is how we used to do paternity tests because
there’s about 90 different loci that produce membrane proteins that can act as antigens.
Now it’s all done with DNA. With the product rule of independent events, we can figure
out the likelihood of some offspring having a genotype that’s consistent with that father.
So I would give you anything more complicated than just simply working with the ABO
system.
There’s another interesting part to this story, there’s a separate genetic locus
called the secretor locus. What it does is it hangs a hydroxyl group on these antigens.
This makes them water soluble. So what happens is if you’re a secretor, if you have A
antigens or B antigens, they’re water soluble so they’re going to come out in your saliva,
in you sweat, in your urine, in you semen. So what happens is in cases of rape if there’s
bodily fluids left on females and if the male was a secretor, not only do you know that he
was a secretor but you can determine his ABO blood type. Now that’s 70% of north
american males are secretors. So what that means is if a male has the dominant allele for
the secretor locus, then you would be able to determine the ABO blood type from things
like saliva or something like that. That’s one way this is useful.

Now what can happen is you can have one gene that has more than one
phenotypic effect. And with this is manifest in a very interesting fashion.
Page 462 Fig. 14-10
What you see are mice and some of the mice are yellow. Now what happens is
that if you cross a yellow mouse with a yellow mouse, you’re going to find that 2 out of

96
every 3 of the offspring are yellow; 1 out of every 3 is the wild type. Now what genetic
system does that remind you of? None, you haven’t learned any genetic system like that.
So that’s a very puzzling pattern of transmission genetics, let’s look at what’s happening.
What happens is color is controlled by a gene and this gene has pleiotropic effects. In
terms of how it affects color if you have the allele causing yellow, that allele is dominant
over the allele causing wild-type. So for instance, if you were like this: AYAY, this is the
yellow allele of gene A. If you were like this, you would be yellow. If you were like this:
AYa+, you would be wild-type. And if you were like this: a+a+ you would be wild-type.
Here’s what happens though, so with regard to fur color the allele acts as a dominant. But
with regard to lethality the allele acts as a recessive. So what happens is this allele if you
this genotype with regard to it’s effect on lethality this would cause it to die (AYAY).
These individuals are never born. These individuals over here (AYa+, a+a+) survive
because they have at least one dominant allele for survivability. So this allele (AY) acts as
the dominant for fur color and also acts as the recessive for survivorship.
So if we took a yellow individual and selfed it, we know from our mendelian
ratios to expect a 1:2:1 genotypic ratio. We expect:
AYa+ X AYa+  1 AYAY, 2 AYa+,1 a+a+

But these (AYAY) die and are never born so we get a ratio of offspring that’s 2:1.
That’s a tipoff of a system where we have lethal alleles.

Page 462 Fig. 14-11

Manx cat that has no tail; that’s controlled by a gene and when you’re homozygous for
that, what happens is it causes a distortion of the spinal cord. If you’re heterozygous, the
spinal cord gets distorted and you can see that cat has a humped back. But also no tail
develops. If you’re homozygous for that allele, the distortion of your spinal cord is so bad
that you’re never born. So Manx cats would have the same phenotypic ratio in a genetic
cross of a Manx crossed with a Manx, you get 2 Manx’s for every wild (normal one).
Because if you’re homozygous for the Manx allele, you die and you’re not even born.

11/24/03

epistasis  where more than 1 gene contribute to the phenotype.

Fig. 14-14
Normally we do AaBb x AaBb and get 9:3:3:1 for 2 genes controlling 2 different
traits. Here we’re looking at 2 genes controlling 1 trait.
9 A_B_, 3 A_bb, 3 aaB_, 1 aabb. The aaB_ and aabb give the same phenotype.

B_ = black pigment
bb = brown pigment
E_ = pigment is deposited
ee = pigment is not deposited in the hair even though it’s produced

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 Individuals like this: eeB_ eebb don’t have pigment in the hair, it’s like the same
situation above where aaB_ and aabb give same phenotype. Individuals that do
not deposit pigment in their hair are going to be yellow labs. If they have a
dominant E, then they will be black if they have a dominant B. If they have a
dominant E and are homozygous recessive B they will be brown labs. So what
you do here is you look at the phenotypic ratio of fur color in Labrador retrievers
and the ratio is 9:3:4. That’s different from any ratio you’ve seen now for a single
phenotype. So if you were just looking at fur color in Labrador retrievers, you
would get a ratio that on the surface you could not explain by Mendelian
inheritance because Mendel assumed each of the genes was controlling a single
phenotype. So Mendel considered the situation where 2 genes controlled 2
phenotypes; now we have 2 genes controlling 1 genotype. This is not an
uncommon phenomenon and the trick to appreciate that this is happening is
you’re looking at one phenotype but the phenotypic ratios, if you add them up,
add up to 16. This is the punnet square, 4 gametes by one parent, 4 gametes by the
other, 16 possible fertilization events. So we get a 9:3:3:1 ratio but if we add them
up, 9+3+3+1 add up to 16. But that punnet square was assuming we were dealing
with 2 phenotypic traits. The pattern of fertilization is going to be the same
independent of the phenotype. So a lot of those retrievers, if it was doubly
heterozygous it has 4 potential gametes it could produce and if it mated with
another individual that was doubly heterozygous it would also have 4 different
potential gametes it could produce. So there would be 16 different fertilization
events and the ratios of the offspring would occur in multiples of 1/16. This is one
form of epistasis. Any grouping of these 9:3:3:1 is possible. So you could have
epistasis where it’s 9:7; this phenotype is distinguishable from the other three yet
they are indistinguishable amongst themselves. You could also have a 9:6:1 ratio
or a 15:1 ratio or a 13:3 ratio. When you see offspring when you’re studying a
single trait (phenotype) and yet we’re getting phenotypic categories that are in
multiples of a sixteenth, then you’re dealing with an epistatic situation.

Page 476, problem 7

These are squash here and look at the ratios of the F2. There’s 270 that have the
disk shape phenotype, 178 have the sphere type, and 32 have the long phenotype. You
should read those numbers as being 270, 180, and 30 because there’s sampling error
when you just have a small number. If you say 30 is 1/16, then 180 is 6/16 and 270 is
9/16. So the long squash is doubly recessive and the disk shaped ones are doubly
dominant and the spheres have one dominant. There are going to be a few problems like
this on the final. It comes down to recognizing that this is not Mendel’s typical ratio and
from that, you’re going to figure out what’s going on.

One other thing on how more than one gene can interact is referred to as suppression.
There are different ways you get suppression genetically.

One way is in Fig. 14-16 on page 467

Let’s say we have to create a multiple polypeptide protein; for the protein to
function appropriately, it has to have 2 polypeptides come together to form a

98
 quaternary structure. So the functional protein is the dark purple and the light
purple docked together. So to get the functional phenotype you need that docking,
but is if you have a mutation to that one gene that gives rise to the deep purple
functional polypeptide. Look what it does, it changes the male part form
rectangular to triangular. If that happens, the mutant form can no longer dock with
the normal wild-type form of the light purple. So you do not get the protein that is
necessary for us to recognize the phenotype, that they can’t come together. So
that’s the mutation. Now what can happen is there can be another mutation that
reverses the effect of that first mutation. In this case they’re showing you a
mutation in the gene that gives rise to the light people gene product. So instead of
the female docking site being rectangular, now the female docking site is
triangular. So what we have are 2 mutations and if they both occur, the 2 gene
products can dock and form a functional complex. That would be referred to as
suppression. This is suppression at the protein level.
What happens, a common type of mutation that occurs is a nonsense mutation
which is a mutation that occurs in the DNA and the DNA is supposed to be transcribed
into this mature mRNA. And what it does is we have a mutation here and a premature
translation stop signal arises out of context. So rather than the protein being synthesized
according to the instructions on this entire mRNA molecule, what it does is it ends right
here; so instead of having a protein that’s a thousand amino acids long, we have a protein
that’s translated that’s a hundred amino acids long. Almost always, those are highly
deleterious. But what can happen, remember the mRNA is normally read by a charged
tRNA that has an anticodon down here. If this were a nonsense mutation that became a
stop signal, it would be read by a release factor that would cause the release of the
polypeptide but lets think about how it was supposed to occur. It’s supposed to have a
particular amino acid positioned here along the growing polypeptide. What it turns out is
there are mutations to the transfer RNA gene. Remember tRNAs are repetitive, for each
tRNA we make many copies of that same gene in the DNA. So this normally codes for
amino acid 1 lets say; this tRNA binds to AA1 and this would read the codon for AA1.
Now the message here is no longer for AA1, it’s for a stop signal. Sometimes the main
thing is just to get any amino acid in here so that instead of being 100 AA long, it can
keep reading and get one that’s 1000 AA long. Remember, the active site is the important
part of the protein; if it doesn’t affect the active site, that’s ok. That’s why nonsense the
mutations are so serious, we mess up so much of the polypeptide that the tertiary
structure is meaningless. So what we could do is as long as we get any AA here, it’s fine.
So what happens is there can be a mutation to the anticodon region of one copy of the
tRNA gene; there could be 40 copies of that. We mutate one copy of it, there’s still 39
good copies of it. But if we mutate one copy of it, what can happen then is this could be
complementary to a stop codon and we position something random (AA8) into the
growing polypeptide. That would be like a base substitution mutation where sometimes
the polypeptide might be functional. So sometimes by having a mutation to the anticodon
region of the tRNA, that could read a stop signal and read past these mutations that were
nonsense mutations. Therefore, reverse the serious effect of a nonsense mutation. That’s
a nonsense suppressor mutation.

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Pg. 470 and 471 Fig. 14-19
Temperature Sensitive mutation
Sometime the phenotype is dependent not only on the genotype but also on the
environment it in. The phenotype can be dependent on the external environment of the
organism or the other polypeptide that is synthesized. The genotype give us the potential
expression of the phenotype but if the environment doesn’t allow the expression of that
phenotype then we not going to see that phenotype. It could be other polypeptide in the
cell or it could also be the external environment of the organism. There are some
mutation that express themselves in some environment condition but not in some other.
Both the cat and the mouse have a mutation to a gene normally produces melanin which
is a dark pigment. But the mutation normally express itself if the temperature is above
threshold level. The cat is endothermic and the bulk of it heat is in the center of the torso.
As you move away from the torso there is not as much body heat so the mutation express
are around the torso of the cat but not on the extremities. The gene are normal for the
extremities so it produce melanin there.

In other cases, a mutation may be lethal at one temperature but not at another
temperature. So you can maintain the mutation that are lethal by keeping it in the
temperature where it not lethal so the mutation can be express.

Pg. 471 fig. 14-22

The two concept of penetrance and expressivity. Sometime if the A gene have
mutation and you have homozygous recessive A then don’t always get the mutation
phenotypically because of these environment factors. The environment doesn’t have to be
temperature it can the rest of the genotype of the organism making other gene product.
- Penetrance is 100% if you express the mutant phenotype everytime you get
the mutant genotype.
- Often time you can have the mutant genotype but express it phenotype
because of the environmental factors so in that case penetrance is less than
100%.

In the figure 14-22, the top row of eggs all have the mutant genotype to express the
mutant purple egg color but only five of them express the mutant phenotype so the
penetrance is 5/9.
So if you doing cross and individual like this then things that have low penetrance are
hard to study through control mating. The mutation we want to study are those that has
high penetrance.

Expressivity- is the range of phenotype that the mutant genotype expressed. In row two
there is a wide range of expressivity so it very hard to assign eggs to different phenotypic
categories if you have a wide range.

You can also have variable penetrance and also variable expressivity like in the bottom
row.

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 Ch. 15
Cell cycle in Cancer genetic

Before we thought by understanding the cell cycle we can understand the cancer but it
opposite.

The most common thing we do in cell to control what going on in term of activating thing
or not is we phosphorylate thing. If something is phosphorylated then it activity changed.
It can be more active or less active. We can activate thing when we want them to be
activated and deactivated thing when we want them to be deactivated and we do this by
adding or removing a phosphate. An enzyme that causes another enzyme to be
phosphorylated is called a kinase. The enzyme that has a new phosphate is activated or
deactivated. A phosphotase is an enzyme that removes a phosphate from another
enzyme; the other enzyme is activated or deactivated.

When we go through cell cycle and were going from G1 to S, somehow we have to know
that we fulfill all the necessary things to get to S. There are various things that are called
check points that regulate moving from one phase to another. There is an important check
point in G1, at the end of G2, and in M when we going from metaphase to anaphase. The
check point in G1 is called the restriction point. If the cell goes through the restriction
point then it will go through the cell cycle. A lot of mature cells stop going through the
cell cycle because they can’t get pass the restriction point. So things like neurons, when
they are mature they never divide again.

Fig. 15-3
What happens when you make sure that those things get done before the check point; it’s
all run by kinases. But these kinases can’t dock with other proteins themselves. So there
are other enzymes that are produced in the cell called cyclins. When cyclins are in high
concentration they bind to these kinases and the kinases they bind with are called cyclin-
dependent kinases (cdk) and now you have this complex. And this complex now has the
capacity to recognize a target protein. So the cyclin recognizes the target protein and it
brings the CDK along with it. When this complex occurs, now the kinase can
phosphorylate the target protein and the complex now leaves so you phosphorylated the
target. The shape of the target protein is now changed and an active site is produced
which was not there before. The trick is this may be the protein that has to be in this form
before the cell can go pass the restriction point. The kinases remain in relatively constant
concentration in the cell cycle. But the cyclin concentration does change.

Pg. 487 Fig. 15-4

The gold circle is the cell cycle and the thing around it is the complex and the thickness
of it is the relative concentration of these complex. And the concentration of these
complex is control by the concentration of the cycle. As we can see that the concentration
of the cycle increase and drop dramatically. For this to happen, the messenger RNA that
are activate to produces the message to be translate they have to be very unstable which
mean they have a short half life so you produce a lot of them and you stop so they

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disappear fast. The big dark purple on top, you can see that there is a lot of concentration
but it drop and disappear.

Dispite this complex forming, these CDK complex is not ready to phosphorylated the
final product they need a message from outside the cell to come in and tell the complex to
phosphorylate the product. The complex will not do anything until they get the message
from the outside. There are certain criteria that must be fill before the cell move past the
restriction point. When the cell keep dividing, then you will have a tumor and it originate
from a single cell and it going to use up all the resource in your body and keep the rest of
your body from working.

12/01/03

One ways cells communicate is by chemically, paracrine system, and endocrine system.

Signals are called ligands (can’t move through membrane). Hormones can move right
thru the cell membrane.

You have to have receptors: RTK and G-protein receptor regions. The signal attaches to
the receptor in the cell membrane. The cytosolic end of the receptor changes shape and
you go thru a transduction pathway in the cytosol. The end product can be a transcription
factor that is now turned on (activated) and goes in the nucleus to transcribe DNA.

Different signals can turn on different genes. Different cells respond differently to these
external signals; if they have different receptor molecules or if in these transduction
pathways they have different enzymes available and if we don’t have a receptor molecule
here then nothing will turn on the DNA. If we have these external signals and some cell
will response and some cells won’t, that is going to depend on the selection of gene
products that these cells have toward the external signal pathway. Now we can see maybe
how one cell behaves differently from another cell.

When a nearby cell releases a signal molecule that’s going to affect its neighbor and
usually cause the neighbor to divide. So the way the paracrine system works is that it
releases ligands that bind to a nearby neighbor and what the nearby neighbor does in
response to these ligands is it mitotically divides from one cell into two cells. Ligands
like that are called growth factor.

When we talking about cancer genetics they often talk about TGF’s or EGF’s. EGF is just
an epidermal growth factor. Those things are ligands, so then you have to have a growth
factor receptor in the membrane.

Pg. 495 Fig. 15-13

One important mechanism. This is a very typical pathway that can stimulate mitosis in
the recipient cell. So a ligand is produced by a signaling cell and that stimulates an RTK.
The RTK then starts a series of signal transduction pathways. One of the important

102
protein that got mutate in 30% of human cancer and that protein called Ras. And Ras
behave like a G-protein and G-protein are made up of three polypeptide bond together
and when stimulated that break out to one monomer and one dimer and each can then
signal something. Ras is like that but doesn’t make up of three polypeptides but instead it
make up of one polypeptide.
- When you stimulate a G-protein what happen is that G-protein will replace
GDP with GTP. And when GTP bind with the G-protein and that will activate
the G-protein. And now G-protein leaves the receptor and migrate and act as
kinase for signal transduction pathway. Once you understand how this causes
mitotic division then it help you understand how most cancer are occur. If
anything that causes these transcription factors to continuously produce and
that have the cell continuously divide and one cell keeps dividing uncontrolled
and proliferates and cause a tumor.

A good communication signal is when you can turn on the process and also turn the
process off. What Ras does like all G-proteins do, is they get stimulated to exchange their
GDP’s for GTP’s, it turns on but the protein itself has a GTPase activity so it gets turned
on and it breaks the GTP down to GDP it turns itself off. What it does is it first starts the
pathway and then turns itself off. That is a good communication molecule.

When Ras mutates and causes cancer, one of the reasons it does this is the most common
thing it does is it loses this GTPase activity. When it loses the GTPase activity, then once
you turn it on, you can’t turn it off and it keeps signaling this transduction pathway and
therefore the cell keeps dividing. That’s one way a cancer can exist.

Another way cancers exist is if the receptor protein mutates so the cytosolic side is
always in an activated stage so it continuously signals Ras to divide over and over again.
So that’s another way you can have a cancer.

Another way you can have a cancer is where did this ligand come from, this cell over
here. And you know that this cell has the same DNA as this cell. So that means the other
cell can also produce that ligand. So what happens is if you incorrectly turn on the
production of this signal molecule then you’re signaling yourself. And you divide
continuously b/c you’re incorrectly signaling yourself.

All of these are cancers that are caused by stepping on the accelerator (encouraging cell
proliferation). The gene causing these mutations are a normal part of the cell cycle
control system. So when they’re functioning normally, they’re called proto-oncogenes.
When they mutate and they no longer are functioning appropriately for the cell cycle
control, then they’re simple called oncogenes. The oncogenes can arisen from a cell or if
a virus has inserted some nucleic acid that contains some of these proto-oncogenes in a
mutated fashion, which can do the same thing.

Pg. 501 Fig. 15-20

The most successful medical cure for a cancer. There are normally 2 genes on 2
different chromosomes, chromosome 22 and chromosome 9.

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Both of these genes are kinases, but they each are controlled by their own regulatory
system. If translocation occurs such that the abl gene attaches to the bcr gene, now the abl
gene is being controlled by the regulatory region of the bcr gene. These are not
uncommon b/c you have cells that produce antibodies. When antibodies are produced,
you remove parts of the genes that make them and stick the ends together, that’s how you
get variation in the antibodies you’re producing. If you’re breaking up the DNA and
hoping to rejoin it, what can happen is something else can come over and join easily. So
you get a number of translocations. In this case, the new chromosome that arises from
that is called a Philadelphia chromosome and what it leads to is a type of cancer called
chronic myelogenous leukemia (CML). CML was virtually 100% fatal and this is what
caused it. But with individuals that are like this, now you have a unique protein that’s a
combination of the bcr and the abl protein that never existing before. What that has is a
region that binds with ATP, so a molecular biologist got a molecule that recognizes this
unique protein and binds to its ATP binding site. So that ATP binding site is necessary
for the kinase activity, so now they add this drug and it binds to this ATP binding site of
the Philadelphia chromosome which prevents the kinase activity from that chromosome.
So this cures 90% of the cases of CML.
Once we can find something that’s unique to a type of cancer, then we can try to
create a magic bullet that can attack that. So this was the first magic bullet that was
produced.
Let’s look at the other ways you can have cancer. Instead of messing around with
the accelerator, you can mess around with the brake where you inhibit cell division.
When we inhibit cell division, genes that do that are called tumor suppressor genes. Of
you remember that RB gene, it was a tumor suppressor b/c it normally bound to E2F and
as long as it was bound, E2F could not act as a transcription factor allowing you to get
into the S phase of the cell cycle. If that tumor suppressor gene mutates (the suppression
is removed) and can no longer bind with E2F, then you can’t turn off the mitotic division.
Pg. 503
That individual has a form of cancer called retinoblastoma and that is a common
childhood cancer that occur. Some of these cancers are inherited with out a hundred
percent penetrance. If you’re heterozygous for one wild type RG gene and one mutated
RB gene, the one wild type gene is enough to produce the tumor suppressor that you need
to turn off the E2F. As you’re going thru your life, all of your cells are mutating with
every mitotic division. Now you only have one copy that has to mutate for this to be
expressed. So if you’re a carrier for this, your offspring may look fine for a while but
there’s a higher chance that the wild type RB gene will mutate somewhere along the line.
That’s a way where you can have an inherited form of cancer that way.

p53 is called the guardian of the genome. Remember last time, one way p53 can
influence this thing is thru its interaction with p21. If you mutate p53, you can’t produce
p21 and that can lead to cell proliferation. Another thing p53 does is it checks the DNA
making sure there are no gross mutations. If you check your DNA and the p53 is doing
that it will prevent you from going thru the restriction point b/w G1 and S if there’s bad
DNA, that’s a safeguard. You’re preventing the mutations from being passed on prior to
them being corrected by our DNA repair system. If p53 mutates and you lose that
checkpoint, then you’re prone to more mutations and what is cancer. Cancer is a series of

104
usually several mutations that have to occur in the same cell before you go haywire.
Having a defective p53 not only modifies the control mechanism with p21, but it leads to
greater mutations.
The other way p53 works is if it gets in high enough concentration, that’s telling you that
we’ve been trying to repair the DNA and p53 is getting in higher and higher
concentration, we can’t seem to repair the DNA, so we commit suicide. If p53 gets in
high enough concentration, it stimulates apoptosis in the cell. p53 is a key molecule b/c it
decides whether or not the cell is going to undergo apoptosis as a result of damage,
whether or not it’s going to stall for DNA repair, or whether its going to influence the RB
E2F via the p21system.

Page 505 Fig. 15-24

You take a biopsy and send to a pathologist, he tells you if you have cancer or not
and which type of cancer you have. Then he gives you a suite of potential
treatments.
There’s a type of cancer called diffuse large B-cell lymphoma (DLBCL). What
you see is a microarray profile for individuals. A single vertical column represents
a single individual; each row is a different cDNA. It’s telling you the type of
mRNA’s that are being produced. All of those individuals (the whole width of
that) were diagnosed with DLBCL histologically. The green areas show which
mRNA exist and the red areas show which mRNA’s are underexpressed.
Histologically, all of them have DBLCL, but there are 2 forms when you look at
the molecular biology. All the individuals of the left have one microarray profile
and the ones on the left have a different microarray profile. So these individuals
are treated with this strategy, this approach to curing. It seems that half the people
that got this treatment were okay and half the people were not. Now look at part b.
This is a survivorship curve and of the 21 patients that have one form, 16 of them
died receiving this treatment. For the other profile, of the 19 patients that received
that, only 6 of them died. So the treatment was very affective if we can identify
what the disease profile is. Instead of doing things histologically, we’re going to
run microarrays. This is a winner b/c now you can not expose the people that are
part of the blue profile to that treatment b/c it doesn’t do them much good. Any
person that has the red profile are in really good shape b/c the treatment works
good.
You’re taking mRNA from the cell and using that to make cDNA and from that,
they’re assuming that the cDNA is being transcribed and translated. But some
mRNA’s in your cell are not actively translated. This is telling you what mRNA’s
exist in your cell. You’re not sure which ones are being translated. So the next
step is looking at the protein profile associated with cDNA to see which of these
are actually producing mRNA that at the moment is being translated.

CHAPTER 16 – Developmental Biology

105
Now we know how we can control some events in here by some external signals by
controlling any of these key proteins along the pathway. But how does one cell get
different proteins here than another cell? So now we want to know how the fertilized egg
which has just divided, and divided, and divided, how are some of those cells going to
have different membrane proteins or different signal transduction components than
others. That’s going to involve how the fertilized egg is going to differentiate into the
different cell types. The zygote that’s going to produce you is going to produce every
kind of cell that’s necessary for your life, 210 different kinds of cells. What happens is as
the divisions occur, they start to lose their potential to produce every kind of cell. A cell
that can produce every other kind of cell is called totipotent. That is what people are
looking for in these stem cells, b/c they are totipotent. You have stem cells that utilize
each of your tissue types in your mature body. What you don’t know is how much of the
overall potential those mature stem cells have lost. So if we have a stem cell that’s going
to produce blood cells, could that produce other types of cells as well? Well maybe, that’s
where the controversy is, but we don’t know. You don’t know if the stem cell has lost its
totipotency or the procedure you’re using is not effective for doing what you’re trying to
do. Why scientists are trying to use embryonic stem cells is b/c then you won’t be
strained by using inappropriate cells. The descendent cells of the zygote have to
differentiate to produce cell types of a fewer set than the totipotent cells do. We want to
understand how cells can become different. There are a couple of ways this happens.

One thing you should recognize is, this is your mother’s fertilized egg. The egg
has half of the genetic material from the mother and half from the father. That DNA
there, 50% is from the mother and 50% is from the father. But all the stuff in the cytosol
came from the mother. So early on during the development, before any of the nuclear
DNA is transcribed, all of the mRNA that’s controlling the development of that organism
is from the mother. Things that are controlled by this are controlled by maternal effect
genes. So early on during embryonic development, a lot of the gene products (the
proteins that are produced) are not produced from transcribing and translating this DNA
but they’re the result of the mRNA that the mother included in the egg of that organism.
The other thing is if I have a bunch of cells here and this cell starts releasing
something over here, the nearby cells are going to get higher concentration of the ligand
than this cell. So a spatial pattern can develop that’s due to the relative concentration of
these ligands that are released by some cell. All the cells in your body as they divide are
getting positional information as to where they are within the developing body plan of the
organism based upon these external ligands that are being produced.

Fig. 16-2
That’s the fertilized egg of a fruit fly. There are very basic things you have to
know if you’re going to be a developing organism. You have to know anterior from
posterior, dorsal from ventral. So we have to create positional information that’s going to
allow some gradient from the anterior to posterior end of the developing organism. We’ll
see as that gets refined from some gross partitioning from anterior to posterior to a very
finely repetitive partitioning. And we’re also going to have to learn up from down, dorsal

106
to ventral. When we talk about the developmental genetics, we’re going to talk about the
anterior to posterior axis and how that axis is produced along with the dorsal to
ventral axis. We’re going to do this in a fruit fly.

The other thing we have to do is that there are certain things that are going to
happen that are very critical. We have things that are called binary decisions. A binary
decision is: is the cell going to be part of your soma or part of your germ cell? Cells in
your soma have no influence on your offspring, it’s only cells that are part of your germ
line that are going to be passed on to your offspring. So cells of your germ line are
qualitatively different from the cells that are part of your body, your body from an
evolutionary perspective is not important. So a binary decision is whether cells are going
to be part of your germ line or part of your soma. Another binary decision is if you’re
going to be a male or if you’re going to be a female (gender differentiation, how you’re
going to become male or female).

In mammals, if you have a Y chromosome then you’re a male; if not, then you’re
a female. That’s not how it occurs in fruit flies. Now you have this ratio:
(XX)/(AA) = 1.0
This is the number of X chromosomes for the number of sets of autosomes. Fruit flies
have four different chromosomes, this would be one haploid set of autosomes (A). So this
is a normal diploid female. A female in drosophila will have as many X chromosomes as
it does sets of autosomal chromosomes. So this ratio would be 1.0 and it would be a
female. If it looks like this:
(X)/(AA) = 0.5
this individual would have a male phenotype. The ratio is 0.5, this would be a sterile
male. If you have a mutation where you’re triplo X (XXX/AA) then you’re called a
metafemale. If you’re (X/AAA), then you’re a metamale. If the ratio is in between, then
you would be intersex. But is normal is 0.5 = male and 1.0 = female. What happens is sex
is determined on an individual cell basis; every cell in the fruit fly’s body decides what
sex it’s going to be. Usually every cell in the fruit fly’s body will have the same ratio and
the decision will be unanimous. Let’s see how that’s done next time.

12/3/03

Pg. 514 Fig. 16-4

This shows where gene regulation can occur. We were looking at how it can occur at the
transcription level, but it can also occur during the splicing process and later too.

Last time we started talking about how gender is determined in fruit flies.
For this protein, that has a helix-turn-helix motif, act as a transcription factor we have to
have two copies of it and they have to be identical. So when we have 2 identical
monomers come together like this, what that forms is a homodimer. In contrast what can
happen is that there can be a monomer like this and a different monomer like this and
they can come together to form a heterodimer, but if they come together they will not
form a functional transcription factor. So to determine whether it’s going to be a

107
functional transcription factor or not is if you have a homodimer; if you have a
heterodimer like this then it’s not going to be functional.

Pg. 517 Fig. 16-6

What we do is refer to the genes that are contributing to this on the X
chromosome as numerator genes (NUM), and that are contributing to this on the
autosomes are denominator genes (DEM). In a normal fly, you’re always going to have
the same number of denominator genes producing one type of monomer. However if you
have two X chromosomes, you’re to produce twice as many numerator genes as it would
if you had only one X chromosome. So what’s going to determine if a functional
transcription factor arises is if a numerator monomer binds to another numerator
monomer creating this homodimer. So if we want to produce transcription factor, we
would have to get gene product from the numerator coming together with gene product
from the numerator. But what happens is numerator gene products will bind just as easily
with monomers from the denominator. If we have an equal number of numerator and
denominator, then we’ll often get this heterodimer; but if we have more numerator then
we’ll get homodimers spontaneously arising with some frequency.
In the figure if we want the transcription factor, we want a pair of red dots to
come together (they come together randomly). If we have a lot of numerator genes then
we have a greater chance of 2 red dots coming together, the red dots are produced from
the numerator gene and the green dots are produced by the denominator gene. If there’s a
greater chance of 2 red dots coming together, then there’s a greater chance of producing a
functional transcription factor.
Of you look at the left hand part of (b), there’s a couple of homodimers (red-reds)
along with some green-greens and some green-reds. But you’re getting an appreciable of
red-reds (or homodimers) that act as transcription factor. Now what happens over here is
that we only have one X chromosome, we’re going to have many more green dots than
red dots so what you see is the pattern on the right there. There is one red-red there, but
not as many red-red homodimers as we have on the left. This is occurring normally in
every cell in the fruit fly. So what’s happening with gender determination in the fruit fly
is that it’s occurring on a cellular to cellular basis. Basically, each cell decides what sex
it’s going to be, but because of the phenomenon of this random aggregation of the
monomer what’s happening is if you have 2 X chromosomes (which is the same number
of sets of autosomes you have) you’ll have a significant chance of producing these red-
red dimers that can act as transcription factors. Generally what happens is you get the
transcription factors in the females and you don’t get them in the male. All of this action
occurs in the first two hours after the fertilization of the egg, so it’s really early during
embryonic development.
What happens are these transcription factors are going to stimulate the
transcription of a gene called sex lethal (sxl). If we get the functional homodimer (the
female gender controller) it stimulates this promoter and what that does is cause the
transcription of the sxl gene. And the sxl gene therefore produces a protein called sex
lethal (SXL). When you’re talking about genes, they’ll be in lowercase and when you’re
talking about gene products (proteins) they’ll be in uppercase. This sxl gene product
(SXL) is only produced by the female and what the sxl gene product will do is it will
have several functions. But the primary function is it acts as the RNA and what it will do

108
is control how that RNA is spliced, remember in eukaryotes these introns are going to be
removed. So what the SXL protein does is it binds to the RNA molecules that are the
initial pre-mRNA and it controls the splicing pattern of them. Perhaps what it does is it
will bind to the pre-mRNA for the sxl gene itself and it will splice that in such a way that
you’ll produce more sxl gene products (SXL’s), but without that you’ll not produce sex
lethal gene products. Here’s where it gets a little bit trickier. This gene is first encoded for
by this promoter during this early 2 hour period. So later these numerator denominator
genes have no influence so what happens is: that is the early promoter (PE) and what
happens is that there’s a late promoter (PL) and the late promoter is going to be
recognized by transcription factors that have not been …with the sex lethal. So what they
do is they will begin transcribing a larger mRNA transcript that will be spliced one way if
sex lethal gene product (SXL) exists but an alternative way if sex lethal gene product
(SXL) doesn’t exist. So look at what happens: if we turn sxl on early, it ensures that sxl
will always be on; we don’t turn sxl on early, we’ll never have a sxl gene to correctly
splice this mRNA and what will happen is a stop codon will arise and we’ll have a very
truncated protein that’s not a functional SXL protein. So this little feedback mechanism is
how we’re turning on the gender identity very early on and it’s maintained throughout the
organisms life. This concept of taking a mRNA and splicing it one way and then
potentially splicing that same mRNA a different way is really common. About 60% of
our genes are spliced like that. Now we’ve learned how we can keep sxl on, these cells
will always produce SXL if they’re female. They’ll never produce SXL if they’re male.
SXL will not only bind to pre-mRNA’s for SXL, but it will also bind to pre-mRNA’s for
another gene called transformer (tra). So the tra gene is going to be transcribed in both
males and females but it will be spliced such that you get a functional tra gene product
(TRA) only in females because the sxl gene product binds to the mRNA, you get that
mRNA spliced appropriately and it will produce a functional gene product. So we’ve got
TRA produced in females but not in males because males don’t have SXL, and what that
does is the TRA binds to the gene product (pre-mRNA) from another gene which is
called double sex (dsx). So there’s another gene called dsx, this transformer gene product
binds to the mRNA of dsx and what it does is cause alternative splicing in the pre-mRNA
for double sex and it produces two forms of the DSX. One is called double sex male
(DSX-M) and the other is called double sex female (DSX-F). So if we have transformer
that’s going to become a female, so it will cause splicing of DSX such that you produce a
gene product called DSX-F. If you don’t have transformer, that double sex mRNA is
spliced so that you produce DSX-M. What DSX-M does is it shuts down (it acts as a
repressor) for all the genes that would give female characters. What DSX-F does is it acts
as a repressor for all the genes that give male character. So that’s how sex is determined
in fruit flies. It’s determined on a cell by cell basis. If things don’t work, then you’re
going to become a male; if something happens here where you don’t produce SXL or you
don’t produce TRA, in either of those cases what happens is you become a male. So the
default gender is male in fruit flies. Now let’s look at how sex is determined in
mammals.

Pg. 520,
If you have a Y chromosome, you’re male; if you don’t have a Y
chromosome, you’re female. What’s actually occurring is there’s a gene on the Y

109
chromosome that produces a gene product called testis-determining factor (TDF). What
TDF does is for the first 2 months of your embryonic life your gonads are forming and
your gonads form from tissue that sits on your developing kidneys. And what happens is
the gene that produces the TDF is on the Y chromosome. So if you have that gene, then
you’ll make TDF which will diffuse over to this region of the embryo near the kidney and
it will cause that tissue to converge and produce testis from those specialized cells which
are called Leydig cells. If TDF is not present then those Leydig cells develop into
ovaries. So the same cells are going to be either ovaries or testes depending whether TDF
is produced by genes on the Y chromosome. If TDF is produced, then these Leydig cell
produce a sex hormone called testosterone which is released from the testes and diffused
to all the cells within the organism’s body. Testosterone is a steroid so it can move right
across the cell membrane and we have steroid receptors inside these cells. So if
testosterone binds to a receptor, then it allosterically changes the receptor to make it a
transcription factor and what this does then is binds to specific genes within the DNA
turning on the male characteristics. So this is the process where a mammal becomes a
male. So the default for human is if you don’t have a Y chromosome, you’re going to
become a female. So there’s lots of ways to become default: you could have the Y
chromosome missing (have two X chromosomes) or you could also have a deletion in the
Y chromosome for this region that produces TDF. You could look at the cells and it
would appear to be XY, but if there was a deletion in that region then phenotypically you
would be female. Also, there could be a translocation and if an X chromosome picked up
a small part of a Y chromosome and that part of the Y chromosome is now attached to the
X chromosome, if that had the gene to make TDF then an individual would look like
they’re XX and yet produce TDF so they would then be phenotypically a male. Before
what could happen, I told you about jamy lee curtis, she has this mutation where she
doesn’t produce this hormone receptor. If you don’t produce that hormone receptor, it
doesn’t matter that testosterone was being produced by these Leydig cells, they get to the
cell and there’s nothing for them to stimulate and convert to a TF so you don’t turn on the
genes that are necessary for the male appearance. Notice how different these things are,
not only is the default gender different but what happens is mammals have a central
region that’s telling us what is going to be controlled; our gonads are what releases
testosterone or doesn’t release it and that’s the key. Releasing that hormone signals all of
our cells so they all know to do the male function or the female function. Where for the
drosophila, each cell is essentially independent; it’s making the decision on its own but
the decision is usually the same decision made by all the cells. What we’re really talking
about at this point are binary decisions, early on you have to decide if you’re going to be
a male or a female. Once that decision is made, you don’t to revert you want it to be
maintained. Another decision you have to make is: is the cell going to be part of your
germ line or part of your soma.

The cytoskeleton that makes up your cell is really important for this. There are
three main components of it: microfilaments, microtubules, and intermediate
filaments. Microfilaments and microtubules act as monorails which is really important.
We have molecules called motor molecules which come in two forms: dyenin and
kinesin.

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Pg. 524
This is showing you a kinesin motor molecule in green there. So we’re looking at
a yellow microtubule there and remember they look something like this. They’re hollow
and they’re made up of dimers of tubulin molecules, but the dimer there’s two types
there’s an alpha and a beta. So we always put them like this so they’re organized and then
there’s another dimer down here with its alpha and beta. So this microtubule always has
one end that has the alpha side and one end that has the beta side so that gives a polarity
to microtubules.
Page 521 fig. 16-10
Just like microtubules have polarity, microfilaments are made up of actin
molecules and they look like this oooooo (like pearls on a string) and each one is an
individual actin protein. They’ve coated the microfilament with a protein and the protein
binds very specifically, it binds like this >>>>>>>. So there’s a polarity here also.
Page 524
These motor molecules have two attachments that can effectively walk along the
cytoskeleton, but they only walk in one direction. What you see is kinesin and kinesin
will always move from negative to positive along the microtubule; dyenin will go in the
opposite direction. This part of the motor molecule that’s called the head (the circle in
figure) will bind to other things like mRNA or organelles. If it binds to mRNA, then it
walks and you concentrate the mRNA on this positive side of the cell. So we can create
polarity within the cell this way.

One of the most widely studied organisms in biology called C.elegans because at
adult maturity it has a little over 1000 cells in its body. It’s also transparent so you can
follow where every one the cells descended from. So you start with one cell, it makes two
cells, etc. Some of the cells die b/c of apoptosis also. We will talk about with regard
which cells become the germ line and which become the soma.

Fig. 16-13
You see the fertilized egg of C.elegans and in it you have things that are called
polar granules (P granules) which are shown as red dots. They get to only side of the
zygote b/c they’re attached to motor molecules, they follow a microfilament and when
they were distributed randomly before, now they’re concentrated at one end of the cell.
now when that cell undergoes mitosis, one descendant is called an AB cell and the other
descendant is called a P1 cell and the P1 cell contains all the granules. Then that cell goes
thru a mitotic division and once again the P granules are only distributed to only one of
its descendants. This continues until you get down to P4 which give rise to all the germ-
line cells. Those other cells that had no P granules in them are going to be the soma of the
organism. That’s essentially how whether the cell is going to be part of the soma or part
of the germ-line is determined.
All of next time is going to be about fruit flies so need to give information on the
embryonic development of fruit flies b/c it’s unusual.

What happens in fruit flies is we start out with one cell with one nucleus, that’s
the zygote. When we have our first mitotic division, remember mitosis is nuclear
division, so after the first nuclear division you get two nuclei but you don’t get

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cytokinesis. This happens again and again and after the first 13 divisions, you get all
these nuclei that are contained in one cell and they’re arranged around the perimeter here.
So this is called a syncitial arrangement (syncitium) where we have more than one
nucleus within the same cell.

Pg. 525, Fig. 16-14

After the ninth nuclear division, you see that some of the cells pinch out like this
and they close off; so the nuclei (of the pole cells) there are going to part of cells and not
part of the syncitium. Those cells generate the germ cells in fruit flies and that
identification is going to be done by polar granules attaching to microtubules and moving
toward that pole, that’s why that pole is a place for invagination or evagination.

We have this mass of nuclei here and what we have to do is identify is this the
dorsal side or is this where the legs are, b/c you have cells that do different things up near
your end than do towards your posterior end. So somehow you have to identify, those
cells have to be given information so they know where they are in the scheme of things
and how they should potentially differentiate. There’s only two ways that can happen.
They have to get some positional information and the positional information can come
from within or it can come from without. So you’re going to get two examples: when
you’re developing the AP axis, that’s established all within the cell (the information came
from within the cell); and when going to develop the DV axis, that’s going to be an
example of how you get positional information from outside the cell that cleave this in a
slightly different way.

Developing the anterior-posterior axis or AP axis

We have a zygote now and this zygote has the information that’s going to start us
identifying the anterior from the posterior end of this axis. The way that happens is the
only proteins that occur in the cytoplasm of this zygote have been contributed by the
mother. So even though this nucleus has DNA from the mother and the father, we haven’t
had transcription and translation of that yet. But all of the proteins in the cytosol were
contributed from the mother, so these gene products are called maternal effect gene
products and they’re not produced by the DNA of the actual individual. What happens is
within this cell we have microtubules that have polarity (negative and positive end). The
mRNA’s that the mother contributed to this egg are going to attach to the microtubules,
but they’re going to attach to motor molecules which bring them to one end. The negative
side is going to be the anterior and the positive side is going to be the posterior end.
Remember, the mRNA’s for a polypeptide have a 5’ and a 3’ UTR. This UTR binds to
the motor molecules that get concentrated to the negative end of the cell.

Fig. 16-15
These are the proteins that are concentrated. The first one is the bicoid (BCD) and
what it does is its 3’ UTR attaches to a motor molecule that then gets concentrated at the
negative end here. So the mother’s mRNA is concentrated here and when that’s translated
in the organism, all of the protein is in that area also but it’s also diffusing; so there’s a
little bit of the protein on the positive side but there’s higher concentration of it on the

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negative side b/c that’s where the mRNA that’s being translated is concentrated. That’s
what gives us the high [BCD] on the anterior end and the low [BCD] on the posterior
end, b/c of this phenomenon.

12/5/03

You have bcd mRNA, hb-m mRNA, and nos mRNA that make BCD, HB-M,
and NOS. Those mRNA in the cytosol are from the mother and the protein they make
(BCD, HB-M, and NOS) are transcription factors. These TF’s occur in gradients, towards
the anterior side of this syncitium, we’re going to have high concentration of both BCD
(bicoid) and HB-M (hunchback). Towards the extreme anterior side, HB-M is in lower
concentration than BCD. But as we proceed posteriorly, the ratio is reducing between the
two and finally HB-M reaches a state where it’s in higher concentration than BCD. What
the DNA is able to do is these TF’s are going to act as enhancers and stimulate the
transcription of certain genes but the genes they are going to control are very sensitive to
the relative concentrations of these two transcription factors. So when there’s an
extraordinary bias of HB-M over BCD, one thing will happen; when they’re at equal
concentrations, different genes will be turned on and turned off and so on. So it’s the
relative concentration of those two things that affect this. Now this concentration is all
occurring in the syncitium, there is still one cell. Now how do you create these gradients?
From last time, the key is the microtubules of the cytoskeleton. The 3’ UTR of the bcd
mRNA becomes tethered to the negative side of the microtubules. That’s going to
become the anterior side of the organism. The 3’ UTR plays a major role in regulation
where it’s determining how many copies of polypeptides will be synthesized from a
mature mRNA in two ways. (One way is establishing how long lived the mRNA is and
the other way is it influences how attractive the mRNA is to the initiation complex for
translation.) So you’ve got the mRNA here and that’s where translation of that mRNA
occurs. All of the BCD protein is generated from a messenger RNA that’s concentrated at
the negative end of the microtubule. So the BCD protein is concentrated at that end also
and the BCD is anywhere else if it diffuses away from that negative end. That’s where we
get the high BCD, but the HB-M is very different.

The hb-m mRNA (produced by the mother) is evenly distributed throughout the
cytoplasm. But that doesn’t seem to be what we’re seeing in Fig. 16-15 graph b/c the
[HB-M] has a gradient, higher at the anterior and lower at the posterior end. What
happens is there’s another protein called NOS (which is produced by the nanose gene)
which will prevent translation of the hb-m mRNA. So the nos mRNA that’s produced by
the mother has its 3’ UTR attached to the positive end of the microtubule. So you
concentrate the nos mRNA over here and when the NOS protein is produced, it’s also in
high concentration here. What NOS does is bind to the hb-m mRNA and prevents it from
being translated so you don’t get that much HB-M at the positive end of the microtubule.
So you’ve got a different form of regulation here; you make a protein that prevents hb-m
translation and when it’s not translated you’re going to have low concentration of HB-M
where there’s high concentration of NOS. Over here there’s lower [NOS] so more of the
hb-m mRNA’s will be translated. So in Fig. 16-4 (pg. 514) that’s all the different ways
eukaryotes can regulate their cells. We’ve already talked about regulation at the

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transcription level and we’ve already talked about regulation at the splicing level (sxl,
gender establishment in drosophila). This is an example of Subcellular localization and
post-transcriptional regulation. The localization of the mRNA (bcd’s and nos’s) and the
post-transcriptional regulation is what we’re talking about for hb-m.

So what this does is take a very symmetric cell in beginning and making that
gradient you see on page 526. And since these are all TF’s, what that does is turn on
some genes at one relative concentration and different genes at different relative
concentrations. Some genes will be turned on in these nuclei and different genes will be
turned on in these nuclei in the syncitium. That’s one way we can get positional
information. All of that positional information was established right within the cell itself.
The other way we can do it is how we establish the DV gradient or the dorsal-ventral
axis.
Developing the dorsal-ventral axis or DV axis

You see the syncitium on page 529, and going to describe the DV differentiation
on page 532.
Fig. 16-20
What you see in part (a) is in purple there is the nuclei arranged around the
perimeter of the egg. So where yellow things are the, that’s the membrane that includes
that egg. The mother is releasing ligands (signal molecules) which look like red dots and
they’re called spaetzle ligands (SPZ) and what they do is bind to transmembrane
receptors that are yellow color. Those are called TOLL receptors. If the egg is
positioned in the mother (as shown in figure) and she’s producing these SPZ ligands from
one direction, the [SPZ] is higher on side of the egg than the other. Whether these SPZ
ligands will bind to the TOLL receptors is strictly concentration dependent. If there’s a
high concentration of SPZ, then they’ll bind to the TOLL receptors.
Part (b). When the SPZ binds to the TOLL receptor it starts a signal transduction
pathway within that receptor cell so now you’re getting membrane around the nuclei at
one end of the syncitium and they’re becoming cells. There are two proteins that exist
within the recipient cell (the cells that exist at this point) and they’re called cactus
(CACT) and dorsal (DL). What happens is normally CACT and DL bind to each other
and remain in an inactive state. If SPZ stimulates this signal transduction pathway, it
causes a series of kinases to be stimulated and eventually CACT and DL become
phosphorylated. When they get phosphorylated, they separate from each other. The key
one there is DL; b/c DL is not sequestered by CACT, it can now enter the nucleus and act
as a transcription factor. But what it does is a high concentration of the DL transcription
factor gives rise to the ventral side of the organism.
So at the bottom of part (a), you’re going to have activated DL. The DNA in all of
those nuclei are then going to be turned on by the DL TF and that will give rise to the
ventral side of the organism.
The AP gradient was established by positional information that was established
within the zygote itself, it didn’t communicate with anything outside of the zygote to
identify what the anterior end was and what the posterior end was. But for the DV axis,
these SPZ ligand chemicals that are released from somewhere outside of the cell are

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identifying to the cell its position; you’re effectively forming the control system from
outside of the cell.

Refining the AP gradient

There’s a much finer gradient in organisms than just front and back, like if you’re
looking at drosophila you’re going to have segments that become the head, segments that
become the thorax, and segments that become the abdomen. You can’t do that with just a
course beginning like this b/c this beginning all came from the mother. During the early
positioning, we have not transcribed a single gene of the zygote yet and we’ve developed
this polarity within the cell. But now, these things will turn on different genes depending
upon their relative gradients.

The first genes that are targeted by AP and DV TF’s and are turned on in a
developing embryo are called cardinal genes; the more common name for them (AP
cardinal genes) are gap genes. So the gap genes are now the first of the newly created
organism’s genes that are going to produce gene products.

Pg. 535 Fig. 16-24 (left column)

They show you kruppel and knirps; these are two gap genes. You see what a
normal larva would look like on the top left. If you had a mutation to one of the gap
genes that’s a kruppel, the zygote will develop as shown to the right of that. The
organism develops and if there’s a mutation to that gene, then a whole gap of that larva is
eliminated. Different gap genes cause different regions of the organism to be missing
during the developmental process. Gap genes are the first genes to be transcribed from
the developing organism.

Pg. 539, Fig. 16-28

We have the initial gradient in BCD and HB-M; what that does is lead to a more
fine-scaled partitioning of the organism from anterior to posterior based upon what gap
genes get turned on and you wind up then creating a region that’s dominated by the
zygotic hunchback (HB-M), the kruppel (KR), the knirps (KNI), and the zygotic HB-M
again on the right. So what we’ve done now is we’ve subdivided the organism into 4 part
based upon the gap proteins that are produced; and what gap proteins are produced where
is dependent upon these maternal effect proteins (BCD, HB-M, NOS).

Now once we’ve expressed the gap proteins, they do two different things. One
thing they do is further refine these subdivisions. Now when we further subdivide the
organism from anterior to posterior in a more fine scaled fashion, the gap proteins are
going to act as transcription factors as well. So the gap proteins regulate the pair-rule
genes and the segment-polarity genes. The pair-rule genes tell each segment which is
the anterior and which is the posterior side of that segment. What this is really doing is
finely partitioning the organism in an anterior to posterior direction and each time along
the way you’re more finely partitioning.
These same gap genes not only subdivide the organism into fine scale segments
but now they tell the organism what those segments should become. On the left, it

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determines how many segments there are and on the right, it determines what each of
those segments are. So it’s the gap genes that are turning on genes that are called
homeotic genes.

Pg. 509
On the top, you see a normal fruit fly’s head with normal antennae where the first
segment of the fruit fly is. On fruit fly immediately below that, it’s antennae is replaced
with legs. This called a homeotic mutation which is a mutation to the genes on the
bottom right of fig. 16-28 (the homeotic genes). These genes are establishing what each
of these segments should be, so they’re establishing things called segment identity.
Pg. 537
The center picture is what a normal fruit fly looks like. Fruit flys have 2 wings,
where almost all other insects have 4 wings. You can get a mutation to one of these genes
here and then you get the fly on the bottom which has 4 wings just like all other insects.
So the homeotic genes are essentially what’s determining the major phenotypic character
of an organism.

What’s interesting is this is how we develop also. We have this system of

developmental genes that’s very analogous to fruit flies. There are two clusters of genes
in fruit flies. The genes that caused these wrong identities being placed on segments (like
legs on fly head) are called homeotic genes and they occur in fruit flies in two clusters.
There are several homeotic genes in one cluster and several in another cluster and they
are tandem to each other, but the clusters are in two separate locations within the fly
genome. On chromosome 3 they’re separated with these two separate regions. Now what
happens when you look at other insects, they have the same genes but the clusters are
adjacent to each other. So it’s thought that primitively in insects, these two clusters of
homeotic genes actually had once been one cluster and the chromosome had some type of
rearrangement like an inversion or something like that that would separate parts of them.

Fig. 16-28 bottom right

So let’s pretend this was the set of homeotic genes. If we’re looking at the
organism like this and these are the different homeotic genes. This is the DNA [note] and
this is the organism [figure]. If we start looking at the gene from 5’ to 3’ here, the first
gene is expressed in the most anterior part of the organism, the next gene is expressed in
the next most anterior part, and so on. So these are co-linear, we have sequences of sets
of genes over here and the first gene we come to establishes the phenotype of the most
anterior portion of the organisms body. The second gene in the pathway does the second
one; there’s no need for that but we get this linear pattern that occurs and we don’t know
why.
What these homeotic genes do is if they’re transcribed and translated, they
produce a TF that has different domains. One characteristic domain is a homeodomain
which is made up of 60 amino acids and what it will do is: if we create this polypeptide
here [note] that has a homeodomain here, this will recognize certain enhancers for a
whole set of other genes. So one protein that’s produced by this gene will act as a TF and
perhaps turn on 15 different genes simultaneously. As long as you produce this, then you
simultaneously turn on whole sets of genes. What that does then as you’re developing,

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during your development process, at some point you’re going to need a certain collection
of gene products. So if you can turn this gene on at the right time, this gene produces a
product that will turn on a set of perhaps a dozen widely separated genes b/c it has this
homeodomain part in it. So we have things like that too.

Fig. 16-33
These are the two homeotic clusters of genes in fruit flies. This one is called
antennapedia (ANT-C) and this one is called Bithorax (BX-C); there’s sets of genes in
there. Now what they’ve shown here is they’ve taken those two clusters that are separated
from each other in drosophila and they’ve shown them adjacent to each other b/c this is
how they look like in other insects.
What’s interesting to us is that what happened in vertebrates, during early
vertebrate evolution, is there was two consecutive duplications of the genome. So instead
of having one set of our genes, there’s two sets. And that double set of genes duplicated
again, now we wound up having four sets. So we have, to a large extent, 4 possible
redundant sets of all of our genes (we being pretty high up on the evolutionary pathway).
Because we’ve got this redundancy in genes, we can go thru these evolutionary mutations
where if we mutate and one becomes nonfunctional then we still have another one that
can allow us to survive. But look, once we’ve got the DNA from these homeotic genes
from insects, and now we have the DNA from mammals, we can find these same genes
exist in mammals and they exist in four separate clusters (from Hox A to Hox D). So we
have four of these homeotic clusters and these are the genes which we also have. So in
some clusters we’re missing a few genes, but look, the amazing thing is that the sequence
of the gene in an insect is analogous to the sequence here (in humans). So you can tell
that these and this have a common ancestor. So now we studied what’s going on in fruit
flies; we can do experiments in fruit flies but not in humans. But we learn that there must
have been a common from which these disparate life forms arose from at one time. And
by studying one, we can learn a lot about how others are organized. So what this says is
whatever gave rise to this developmental pathway that leads to the segment identification,
it must have been pretty good b/c all animal life forms are using it.

Pg. 641 Fig. 19-21

On the left is the pathway for establishing the DV orientation in drosophila. We
use the system on the right for our DV development…similar.

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