Fundamental Genetics

Lecture 8

Linkage and
Chromosome Mapping
in Eukaryotes
John Donnie A. Ramos, Ph.D.
Dept. of Biological Sciences
College of Science
University of Santo Tomas

Linked Genes
‰ Genes located in the same
chromosomes
‰ Initiated by Thomas Morgan and
Alfred Sturtevant
‰ Will not segregate independently
‰ Affected by crossing over

1
Linkage vs Independent Assortment

Linked Genes in Drosophila

‰ Red eyes (bw+) dominant to
mutant brown eyes (bw)
‰ Thin wing veins (hv+) dominant to
mutant heavy wing veins (hv)

2
X-Linked Genes in Drosophila
Cross A
‰ Gray body (y+) dominant
to mutant yellow body (y)
‰ Red eyes (w+) dominant to
mutant white eyes (w)

Cross B
‰ Red eyes (w+) dominant to
mutant white eyes (w)
‰ Normal wings (m+)
dominant to mutant
miniature wings (m)

‰ Due to linkage and

crossing over that occurred
during meiosis
‰ Distance between linked
genes is related degree of
crossing over

Chromosome Mapping
‰ Determining the distances between genes and the order
of sequence in a chromosome
‰ Uses the frequency of crossing
‰ The shorter the distance between linked genes, the lower the
frequency of crossing-over.
‰ The longer the distance, the higher the frequency of crossing over
to occur.
‰ Frequencies of crossing over:
1. Yellow, white 00.5 %
2. White, Miniature 34.0 %
3. Yellow, miniature 35.4 %

‰ 1% of crossing over = 1 map unit (centimorgan, cM)

3
Distance Affects Crossover

Single Crossover

‰ 50% are recombinant gametes

‰ 50 % non-crossover gametes
‰ If distance between genes is more than 50 map units,
~100 % crossing over will occur.

4
Multiple Crossover

‰ Occurrence of more than one crossovers between non-sister

chromatids.
‰ Produces double crossover (DCO) gametes
‰ If the probability of crossover between A and B is 20% (0.20)
and the probility of crossover between B and C is 30% (0.30),
the frequency of DCO is 6 % (0.06)
‰ Product Law: (0.2)(0.3)=0.06

Three-Point Mapping
‰ The percentage of crossing over could be used
to map genes in a chromosome
‰ Three criteria needed for successful mapping:
‰Genotypes of organisms producing the
crossover gametes must be heterozygous
for all gene loci
‰Cross must be constructed so that the
genotypes of gametes could be determined
based on the phenotypes of the offspring.
‰Large number of offspring must be
produced

5
Traits considered:
1. Body color
Gray(y+)
dominant to
yellow (y)
2. Eye color
Red eyes (w+)
dominant to
white (w)
3. Eye shape
Normal (ec+)
dominant to
echinus (ec)

10,000

Determining Gene Sequence

Steps:
‰ Determine 3 possible orders
‰ w-y-ec (y at the middle)
‰ y-ec- w (ec at the middle)
‰ y-w-ec (w at the middle)
‰ Perform a theoretical double cross over
‰ Compare the theoretical DCO with actual DCO (least no.)
‰ Perform theoretical NCOI and NCOII and compare with data

White echinus eyes, gray body

Red normal eyes, yellow body

Yellow body, normal white eyes

Gray body, echinus red eyes

Yellow body, echinus red eyes

Gray body, white normal eyes

6
Unknown Gene Sequence

Total=1109

Unknown Gene Sequence

7
Not all crossovers can be detected

Degree of inaccuracy increases with

increasing distance between linked genes

Observed vs Expected DCO

‰ Observed DCO = double cross-over that actually

occurred
‰ Example: (44 + 42)/1109 = 0.078

‰ Expected DCO = theoretical double crossovers

‰ Product of all the SCOI and SCOII
‰ Example: (82+79+44+42) / 1109 = 0.223
(200+195+44+42) / 1109 = 0.434
DCO exp= (0.223)(0.434) = 0.097

8
 Coefficient of Coincidence
and Interference
‰ Coefficient of Coincidence (C)
‰The measure of actual DCOs that occurred
‰C = Observed DCO / Expected DCO
= 0.078/0.097
= 0.804 or 80.4%

‰ Interference (I)
phenomenon when a crossover event in one region of a
chromosome inhibits a second event to occur in a nearby
region)
I = 1-C
= 1-0.804 = 0.196 or 19.6%

Interpretation: 19.6% of expected DCO did not occur or only

80.4% of expected DCO was observed

Problem 1
A stock of corn homozygous for the recessive linked genes colorless (c), shrunken
(sh), and waxy (wx) was crossed to a stock of homozygous for the dominant wild
type alleles of the genes (+ + +). The F1 plants were then backcrossed to the
homozygous recessive stock. The F2 results were as follows:

Phenotype Number Phenotype Number

+++ 17,959 + + wx 4,455
c sh wx 17,699 c sh + 4,654
+ sh wx 509 + sh + 20
c++ 524 c + wx 12

a. Determine the distance between the c and sh

b. Determine the distance between the sh and wx
c. Determine the distance between c and wx
d. Give the coefficient of coincidence
e. Compute for the interference

9
Problem 1: Solution
c sh wx + + +
c sh wx
x + + +

c sh wx c sh wx
+ + + x c sh wx

NCO + + + = 17,959
35,658 = 77.80 %
c sh wx = 17,699
SCOI + sh wx = 509
c + + = 524 1,033 = 02.25 %
SCOII + + wx = 4,455
c sh + = 4,654 9,109 = 19.87 %
DCO + sh + = 20
32 = 00.07 %
c + wx = 12
Total = 45,832

Problem 1: Solution

Distance between c and sh = (509 + 524 + 20 +12) / 45,832

= 0.0232 or 2.32 %

Distance between sh and wx = (4466 + 4654 + 20 + 12) / 45832

= 0.1994 or 19.94 %

Distance between c and wx = 2.32 + 19.94

= 22.26

10
Problem 1: Solution

c sh wx

2.32 mu 19.94 mu
22.26 mu

C = (0.0007) / (0.0232)(0.1984)
= 0.1521 or 15.21%

I = 1-C
= 1-0.1521
= 0.8479 or 84.79 %

Problem 2
In a variety of tomato plant, the mutant genes o (oblate fruit), h (hairy fruit), and c
(compound inflorescence) are all located in chromosome 2. These genes are recessive
to their wild type alleles round fruit, hairless and single inflorescence, respectively. A
testcross mating of an F1 heterozygote for all three genes resulted in the following
phenotypes:

Phenotypes Number Phenotypes Number

+++ 73 ++c 348
+h+ 2 +hc 96
o++ 110 o+c 2
oh+ 306 ohc 63

a. Determine the sequence of the 3 genes in chromosome 2

b. Give genotypes of the homozygous parents (P1) used in making the F1 heterozygote.
c. Compute for the map distances between the genes
d. Give the coefficient of coincidence and interference

11
Problem 2: Solution
+++ ohc
x
ohc ohc Inference from given data:
‰ Sequence of genes is not correct
NCO: o h + = 306
‰ One chromosome contains 2 wild
+ + c = 348 type alleles while the homolog
DCO: +h+ = 2 contains the 3rd wild type allele

o+c = 2
Three possible orders of the genes involved:
oh+
‰o h c Find a sequence Satisfies NCO but not DCO
++c
‰o c h that will satisfy +ch
both NCO and Satisfies DCO but not NCO
o++
‰h o c DCO
+oh Satisfies both NCO and DCO
c++

Problem 2: Solution
+oh ohc Try if the sequence can
x
c++ ohc satisfy the SCOs

NCO: oh+ (same as + o h) = 306 654 = 0.654 or 65.4%

++c (same as c + +) = 348
DCO: +h+ (same as + + h) = 2
4 = 0.004 or 0.4%
o+c (same as c o +) = 2
SCO I: +++ = 73 136 = 0.136 or 13.6%
o h c (same as c o h) = 63
SCOII: o + + (same as + o +) = 110
+ h c (same as c + h) = 96 206 = 0.206 or 20.6%
Total = 1,000

12
Problem 1: Solution

Distance between c and o = (73 + 63 + 2 + 2) / 1,000

= 0.140 or 14 % / cM

Distance between o and h = (110 + 96 + 20 + 12) / 1000

= 0.210 or 21 % / cM

Distance between c and wx = 14 + 21

= 35 cM

Problem 1: Solution

c o h

14 cM 21.0 cM
35 cM

C = (0.004) / (0.14)(0.21)
= 0.1361 or 13.61%

I = 1-C
= 1-0.1361
= 0.8639 or 86.39 %

13
 Fundamental Genetics
Lecture 10

DNA Replication
and Synthesis
John Donnie A. Ramos, Ph.D.
Dept. of Biological Sciences
College of Science
University of Santo Tomas

The Flow of Biological Information

Replication

DNA

Transcription

RNA

Translation

Protein

1
 Modes of DNA Replication

Semiconservative Replication

2
Semiconservative Replication in Prokaryotes
‰ Mathew Messelson and Franklin Stahl (1958)
‰ 15N– heavy isotope of N (contains 1 more
neutron) compared to 14N
‰ 15N has high sedimentation rate in cesium
chloride compared to 14N

Semiconservative Replication in Prokaryotes

‰ Expected results of the Messelson-Stahl experiment

3
 Semiconservative Replication in Eukaryotes
‰ J. Herbert Taylor, Philip
Woods, and Walter Hughes
(1957)
‰ Used root tip cells from Vicia
faba (broad bean)
‰ Monitored replication using
3H-Thymidine to label DNA

‰ Used autoradiography to
determine the incorporation
of 3H-Thymidine
‰ Arrested cells at metaphase
using colchicine

Replication of E. coli Plasmid

‰ Shown by John Cairns (1981) using
radioisotopes and radiography
‰ Replication starts in a single OriC –
origin of replication (245 bp)
‰ Replication is bidirectional
‰ Replication fork – unwound DNA helix
‰ Replicon – replicated DNA
‰ Ter region – region of replication
termination

4
 DNA Synthesis in Microorganisms
‰ DNA polymerase I (928 aa) – catalyses the synthesis of DNA in vitro
(A. Kornberg, 1957)
‰ Requirements:
‰ Deoxyribonucleoside triphosphates, dNTPs (dATP, dCTP, dGTP, dTTP)
‰ DNA template
‰ Primer

Chain Elongation
‰ 5’ to 3’ direction of DNA synthesis (requires 3’ end of the DNA template)
‰ Each step incorporates free 3’ OH group for further elongation

‰ DNA replication using DNA polymerase is of high fidelity (highly

accurate)
‰ With exonuclease activity (proofreading ability)

5
 DNA Polymerases
‰ All 3 types requires a primer
‰ Complex proteins (100,000 Da)
Functions of DNA polymerases
in vivo
‰ DNA Pol I – proofreading;
removes primers and fills gaps
‰ DNA Pol II - mainly involved in
DNA repair from external
damage
‰ DNA Pol III – main enzyme
involved in DNA synthesis
‰ a holoenzyme (>600,000 Da)
– forms replisome when
attached to a replication fork.

Replication in Prokaryotes
1. Unwinding of DNA helix
2. Initiation of DNA synthesis
3. DNA synthesis proper (elongation)
4. Sealing gaps
5. Proofreading and error correction

6
 Unwinding of DNA Helix
‰ Takes place in oriC (245 bp) –
repeating 9mers and 13mers
‰ Function of helicases (Dna A, B, C)
– requires ATP hydrolysis to break
hydrogen bonds
‰ Initiated by Dna A – binds to 9mers
‰ Binding of Dna B and Dna C to
unwound helix
‰ Single-stranded binding proteins
(SSBPs) – prevents reannealing of
replication bubble.
‰ DNA gyrase (a DNA topoisomerase)
– relaxes the supercoiling of DNA
helix

Initiation of DNA Synthesis

‰ Synthesis of RNA primer – 5 to 15 RNA bases complementary to

the DNA template
‰ Catalysed by primase (an RNA polymerase)
‰ Pimase does not require free 3’ end to initiate synthesis (not unlike
DNA polymerase III)
‰ Function of primase will be continued by DNA polymerase III.

7
 DNA Synthesis (Elongation)
‰ Function of DNA polymerase III
‰ Requires free 3’ end
‰ Direction of elongation: 5’ to 3’
‰ DNA synthesis is continuous in 3’ to 5’
DNA strand (leading strand) and
discontinuous in the 5’ to 3’ DNA strand
(lagging strand).
‰ Okazaki fragments – short DNA
fragments produced in the lagging
strand
‰ Concurrent synthesis of leading and
lagging strands occur by using DNA pol
dimer and by a looping mechanism for
the lagging strand

Sealing of Gaps, Proofreading

and Error Correction
‰ DNA polymerase I removes all RNA bases produced
by primase (creates gaps in the lagging strand) and
replaces it with DNA bases (U to T).
‰ DNA ligase seals the gaps by forming
phosphodiester bonds
‰ Exonuclease proofreading (identification of
mismatched bases) is a function of both DNA
polemerase I and III (both with 3’-5’ exonuclease
activity)
‰ ε subunit of DNA polymerase III is involved in
proofreading.
‰ Assures high fidelity of DNA replication

8
 Mutations Affect Replication

Replication in Eukaryotes
‰ Presence of multiple replication origin
(faster replication, guarantees replication
of a big genome) – 25K replicons in
mammalian cells
‰ Autonomously replicating sequences
(ARSs) – origin of replication in yeasts
(11 bp)
‰ Origin site is AT rich region
‰ Helicase unwinds double stranded DNA
and removes histone proteins from DNA
‰ Histones reassociates while DNA
synthesis occurs.

9
 Eukaryotic DNA Polymerases
‰ Pol α - initiates nuclear DNA synthesis
‰ 4 subunits (2 acts primase – produces RNA primers)
‰ Acts on both leading and lagging strands
‰ 2 other subunits continue elongation step (DNA synthesis)
‰ Low processivity (short length of synthesized DNA prior to dissociation)
‰ Pol δ - replaces Pol α (called polymerase switching)
‰ High processivity (during elongation)
‰ With 3’-5’ exonuclease activity (proofreading)
‰ Pol ε - nuclear DNA synthesis
‰ Pol β - DNA repair (the only eukaryotic DNA polymerase with single
subunit)
‰ Pol ξ - DNA repair
‰ Pol γ - mitochondrial DNA synthesis (encoded by nuclear gene)

‰ Eukaryotes has a high copy number of DNA polymerases (ex. Pol α

may be up to 50K copies)

Eukaryotic DNA Replication

‰ Telomeres – linear ends of
eukaryotic chromosomes
‰ Problem with lagging
strand: no 3’ needed by
DNA polymerase I (after
removal of RNA primers)
‰ Possible result:
chromosome with shorter
lagging strand every
replication step

10
 Telomerase
‰ Enzyme that adds TTGGGG
repeats on the telomeres (first
identified in Tetrahymena)
‰ Prevents shortening of
chromosomes
‰ Forms a “hairpin loop” on
chromosome ends using G-G
bonds
‰ Creates a free 3’ on lagging
strand that can be used by
DNA polymerase I to replaced
the removed RNA primer
‰ Telomerase is a
ribonucleoprotein and contains
RNA sequence (5’ AACCCC 3”-
serving as template) – reverse
transcriptase
‰ Cleavage of loop after DNA
synthesis

DNA
Recombination
‰ Exchange of genetic
material
‰ Homologous
recombination
‰ Ex. Rec A protein
(produces recB, recC
and recD genes)

11
 Gene Conversion
‰ Exchange of genetic information between non-homologous
chromosomes
‰ Type of chromosome mutation (recombination)
‰ First identified in Neurospora (by Mary Mitchell)
‰ Can be repaired but forms recombined genetic material

12
Fundamental Genetics
Lecture 9

The Genetic Code

and Transcription

John Donnie A. Ramos, Ph.D.

Dept. of Biological Sciences
College of Science
University of Santo Tomas

Flow of Genetic Information

1
 The Genetic Code
‰ Linear form (mRNA derived from DNA)
‰ Triplet codons (triplets of ribonucleotides coding for 1
amino acid)
‰ Unambiguous (1 codon = 1 amino acid only)
‰ Degenerate ( 1 amino acid can be specified by several
codons)
‰ Contains specific start and stop codons
‰ Commaless (no breaks once translation starts until the
stop codon is reached)
‰ Non-overlapping (single reading frame)
‰ Universal (same ribonucleotide used by all organisms)

The Discovery of the Genetic Code

‰ Francois Jacob and Jacques Monod
(1961) – messenger RNA (mRNA)
‰ Sydney Brenner (1960s) – codon in
triplets (minimal use of the 4 mRNA
bases to specificy 20 aa) (43=64)
‰ Francis Crick – frameshift mutations
alters the codons
‰ Mariane Manago and Severo Ochoa -
polynucleotide phosphorylase
(synthesis of RNA without template)-
paved the way to the production of
RNA polymeres in cell free-systems

2
 The Discovery of the Genetic Code
‰ Marshall Nirenberg and J. Heinrich Matthaei (1661) – codons
‰ used cell-free protein synthesizing system and polynucleotide
phosphorylase
‰ RNA Homopolymers (UUUUUU…, AAAAAAA…, CCCCCC…, GGGGG…)
‰ UUU (Phenylalanine)
‰ AAA (Lysine)
‰ CCC (Proline)
‰ RNA Mixed Copolymers

1A:5C (1/6 A: 5/6C)

The Triplet Binding Assay

‰ Developed by M. Nirenberg and P. Leder (1964)
‰ Mimics the in vivo translation of proteins where a mRNA-tRNA-
ribosome complex is formed when all three macromolecules are
allowed to interact.

3
 Repeating Copolymers
‰ Developed by Gobind Khorana (1960s)
‰ Synthetic long RNAs with repeating sequences

Results of Repeating Copolymers

4
 The Universal Genetic Code

‰ Degeneracy
‰ Wobble
Hypothesis
‰ Start codon
(N-formylmethionine)

‰ Termination
codons
‰ Universal
Viruses
Bacteria
Archaea
Eukaryotes

Exceptions to the Universal Code

5
 Transcription
‰ Uses DNA as a template
‰ Catalyzed by RNA polymerase
(holoenzyme of 500 kD)
‰ αββ’σ subunits
‰ Sense strand / template strand –
DNA strand used as a template
for transcription
‰ Promoter region – DNA sequence
recognized by σ factor to initiate
transcription (60 bases).
(upstream of a gene)
‰ TATA box (Pribnow box) –
TATAAT sequence
‰ Sigma factor (σ70, σ28, σ32, σ54)

Transcription
‰ RNA polymerase don’t need
primers
‰ Elongation in 5’ to 3’ direction
‰ Rate in E coli: 50 bases/sec at
37°C
‰ Termination is a function of rho
(ρ) factor – hexameric protein
interacting with the end of a
gene
‰ Polycistronic mRNA – bacterial
mRNA containing information for
the synthesis of proteins of
related function
‰ Monocystronic mRNA –
eukaryotic mRNA containing
information for a single protein.

6
 Eukaryotic Transcription
‰ Features of eukaryotic transcription different from prokaryotic
transcription:
‰ Transcription inside the nucleus under the direction of 3 different
RNA polymerases

‰ Presence of protein factors (promoters, enhancers, etc.) binding to

the upstream portion of a gene (cis-acting elements) during
initiation step.
‰ Presence of post-transcriptional regulation.

Cis -acting Elements

‰ TATA Box (Goldberg-Hogness Box)
‰ Located 30 bases upstream from the start of
transcription (-30)
‰ Consensus sequence: TATAAAA
‰ Facilitates denaturation of helix because it is AT-
rich region

‰ CAAT Box
‰ Located 80 bases upstream from the start of
transcription (-80)
‰ Consensus sequence: GGCCAATCT
‰ Influence the efficiency of the promoter

7
 Trans -acting Factors
‰ Transcription factors – facilitates template
binding during the initiation of transcription
‰ Example:
‰ TFIID (TATA-binding protein or TBP) – binds to
TATA-box

Post-transcriptional Processing
‰ 7-methylguanosine cap (7mG)
‰ Protection from nucleases
‰ Role mRNA transport across the
nuclear membrane
‰ 3’ cleavage site:
‰ AAUAAA
‰ Failure of 3’ cleavage results to
absence of poly A tail
‰ Split genes – contains intervening
sequences

8
 RNA Splicing

‰ Ribozyme – RNA with

catalytic activity
‰ Self-excision process –
process of RNA splicing or
intron removal.
‰ Transesterification –
interaction between
guanosine and the
transcript.
‰ 2 successive
transesterification processes

The Spliceosome

‰ Alternative splicing
‰ Small nuclear ribonucleoproteins
(snRNP or snurps) – bonds to GU
or AG sites of introns
‰ 2 transesterification processes
‰ Snurps form a loop (lariat) in the
branch point region
‰ Produces isoforms of proteins

9
 RNA Editing

‰ Substitution editing
‰ changes in the nucleotide bases of a given mRNA
‰ Common in mitochondrial RNA and chloroplast RNA
‰ Example: Apoliprotein B (Apo B) – C to U change CAA
to UAA
‰ Insertion / deletion editing
‰ addition or removal of nucleotide sequences
‰ Common in mitochondrial RNA or guide RNA (gRNA)

10
Fundamental Genetics
Lecture 12

Translation and
Proteins

John Donnie A. Ramos, Ph.D.

Department of Biological Sciences
College of Science
University of Santo Tomas

The Products of Transcription

‰ Messenger RNA (mRNA)
‰ primary structure
‰ linear sequence of RNA bases
‰ carries the genetic information in
the form of codons
Codons
‰ Ribosomal RNA (rRNA)
‰ assumes a 3D structure
(complexed with proteins)
‰ site of protein synthesis
‰ Transfer RNA (tRNA)
‰ assumes a cloverleaf structure
‰ carries amino acids from
cytoplasm to ribosomes

1
 Ribosome
‰ Encoded by rDNA
(ribosomal gene)
‰ Synthesized by RNA
polymerase I in the
nucleolus
‰ Complex of RNA and
proteins (monosome)
‰ Prokaryotes: 10K/cell

Transfer RNA
‰ 75-90 nucleotides
‰ Nucleotides are post-transcriptionally
modified
‰ 2D cloverleaf structure (Rodbert Holley)
due to base pairing

‰ Amino acid binding site - ends

in CCA3’ and 5’G
‰ Anticodon loop – contains
RNA bases complementary to
3D Structure the codons
‰ Other loops serves as
recognition sites for enzymes
2D Structure during translation

2
 Steps in Translation
1. Charging of tRNA
2. Initiation of translation
3. Elongation of polypeptide chain
4. Termination of translation
Charging of tRNA
‰ Loading of specific amino acid to its own tRNA
‰ Catalyzed by aminoacyl tRNA synthetase
‰ 32 different tRNA (despite the presence of 61
codons (bec. Of wobbling mechanisms)
‰ 20 different aminoacyl tRNA synthetases
‰ Isoaccepting tRNA – tRNA that binds to aa
‰ End product: aminoacyl-tRNA complex

Initiation of Translation

‰ Shine-Dalgarmo sequence (5’AGGAGG3’)

– sequence that precedes the first codon
in prokaryote m RNA
‰ Formylmethionine (fmet) – the first amino
acid of most polypeptides

3
 Elongation of Polypeptide Chain

‰ Peptidyl site (P site) – contains the elongating

peptide
‰ Aminoacyl site (A site) – contains the amino acid to
be added
‰ Exit site (E site) – exit of uncharged tRNA
‰ Peptidyl transferase – catalyzes the formation of
peptide bond
‰ High efficiency (error rate 10-4)
‰ Rate of elongation: 15 aa/sec at 37°C (E. coli)

Protein Factors Involved in Translation

4
 Termination of Translation
‰ Signaled by stop codons (UAG, UAA,
UGA)
‰ Release Factor 1 (RF1) – recognizes stop
codon UAA and UAG
‰ Release Factor 2 (RF2) – recognizes stop
codons UGA and UAA
‰ Release factors are GTP dependent
‰ Post-translational modification starts
after release from ribosome

Polyribosomes
‰ Single mRNA being used by different ribosomes for the process of
translation
‰ Also called polysomes
‰ A mechanism to produce more polypeptide (protein) copies

Translation of hemoglobin Translation in giant salivary

mRNA in rabbit reticulocyte gland cells of midgefly

5
 Translation in Eukaryotes
‰ mRNAs stays in the cytoplasm for longer periods before degradation by
RNAses (hours)
‰ Ribosomes are much bigger
‰ mRNA is capped with 7-methyguanosine (7MG)
‰ mRNA contains an initiation sequence called Kozak sequence (ACCAUGG)
discovered by Marilyn Kozak
‰ Formylmethionine (fMet) is not required for initiation but met is often used
as a start codon
‰ More complex protein factors involved in different steps
‰ Elongating polypeptide enters the ER immediately as translation occurs

Proteins Form Phenotypes

Phenyketonuria
‰ Mental retardation
‰ Autosomal
recessive
‰ Inability of Phe to
converted to Tyr
‰ Accumulation of
Phe and its
derivatives in
cerobrospinal fluid

Alkaptonuria
‰ Autosomal recessive
‰ Darkening ears and nose
‰ Benign arthritic conditions

6
 Genes and Proteins
‰ One gene: one enzyme
hypothesis
‰ Proposed by George Beadle
and Edward Tatum (1940s)
‰ Experiments in Neurospora
mutants

Genes and Proteins

‰ One gene: one protein (polypeptide chain)

‰ Not all protein are enzymes
‰ Example: Sickle Cell Anemia (mutant hemoglobin)

7
 Amino Acids

Protein Structure

Primary Structure Secondary Structure

Tertiary Structure
Quaternary Structure

8
 Post-translational Modifications
‰ Cleavage of formylmethionine
‰ Cleavage of signal peptides
‰ Acetylation of amino group
‰ Phosphorylation of certain amino acids
‰ Glycosylation
‰ Trimmining of polypetides
‰ Addition of metallic groups

‰ Molecular Chaperons – help proteins undergo correct protein folding

to become functional molecules.

Protein Function
‰ Structural Function ‰ Defense Function
‰ Collagen ‰ Antibodies
‰ Keratin ‰ Complement proteins
‰ Actin ‰ Catalytic Function
‰ Myosin ‰ Enzymes
‰ Regulatory Function ‰ Ribozymes
‰ Hormones ‰ Others
‰ Hemoglobin ‰ Histones
‰ Myoglobin ‰ Receptors

Enzyme Activity

9
Protein Domains and Exon Shuffling

Structural domains of a fibronnectin molecule

DNA organization of an LDL receptor gene

10
Fundamental Genetics
Lecture 9

DNA Structure
and Analysis
John Donnie A. Ramos, Ph.D.
Dept. of Biological Sciences
College of Science
University of Santo Tomas

DNA: The String of Life

James Watson Francis Crick

1
 Characteristics of the
Genetic Material
‰ Replication
‰ Storage of
information
‰ Expression of
information
‰ Variation by
mutation

Central Dogma of Molecular Genetics

Early Studies on the

Genetic Material
‰ Friedrick Miescher (1868) – acid substance from nuclei called nuclein
‰ Phoebus Levene (1910) – tetranucleotide hypothesis (equal amount
of nucleotides)
‰ Frederick Griffith (1927) – In vivo transformation experiment
‰ Oswald Avery, Colin MacLeod, Maclyn McCarty (1944) – In vitro
transformation experiment (bacteriophage)
‰ Alfred Hershey, Martha Chase (1952) – Bacteriophage
transformation
‰ William Astbury (1938) – X-ray diffraction analysis of DNA
‰ Rosalind Franklin (1950) – improved X-ray diffraction analysis of
DNA
‰ James Watson and Francis Crick (1953) – DNA double helix
structure

2
In Vivo Transformation Experiment

“Transformation
might be due to the
polysaccharide
capsule or some
compound required
for capsule
synthesis”

In Vitro Transformation Experiment

DNA is responsible for the transformation of
avirulent strain to a virulent type!

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 Hershey-Chase Experiment
DNA (and not protein) is the genetic
material in phage T2.

Evidences Favoring DNA as the

Genetic Material
‰ DNA is found only where genetic function is known to occur
but protein is ubiquitous.
‰ DNA content of cells is directly correlated with the number of
sets of chromosomes present but not for proteins

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 Evidences Favoring DNA as the
Genetic Material
‰ DNA absorbs UV at the
same wavelength where
mutation occurs (action
spectrum) but proteins
absorbs at different
wavelength
‰ Recombinant DNA
Technology (transgenic
organisms) – direct
evidence

RNA: Genetic Material in

Some Viruses

‰ First identified in 1956 in

tobacco mosaic virus (TMV)
‰ Uses RNA replicase to duplicate
genetic material
‰ Retroviruses – undergo reverse
transcription (RNA to cDNA)
using reverse transcriptase

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 DNA Structure
‰ Proposed by Watson
and Crick in 1953
based on:
‰ Base composition
analysis of
hydrolyzed samples
of DNA
‰ X-ray diffraction
studies of DNA
‰ Sequence of
nucleotides codes for
the genetic
information (4n where
n refers to the no. of
nucleotides)

DNA Structure

6
 DNA Structure

‰ Precursor molecule in nucleic acid synthesis

‰ Source of energy (ATP)

Nucleotide Linkage

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Base Composition Studies
‰ First studied by Erwin Chargaff (1949-1953)
‰ Agrees with Watson and Crick DNA model

Chargaff Rule

‰ Amount of A is proportional to T
while C is proportional to G
‰ Sum of purines (A+G) equal to
sum of pyrimidines (C + T)
‰ Percentage of G + C does not
necessarily equal to percentage of
A+T

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 The Watson-Crick DNA Model
‰ Right-handed double helix
‰ Antiparallel chains
‰ Nitrogenous bases as flat
structures inside the helix
‰ Bases are 3.4 A apart
‰ Base complementarity (A-T
and G-C)
‰ 10 bases every 360° turn
‰ 34 A every complete turn
‰ Double helix diameter is 20 A
‰ Semiconservative mode of
replication

Types of DNA
Criteria B DNA A DNA Z DNA
Bases / 360° turn 10 bp 11 bp 12 bp
Length / 360° turn 34 A 37.4 A 40.8
Diameter of helix 20 A 23 A 18 A
Direction of turn Right-handed Right-handed Left-handed
Major groove Present Modified Absent

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 RNA Structure
‰ Ribose sugar
‰ Same nitrogenous bases as DNA except that T replaced by U
‰ Single stranded (but can form double strands)
‰ Forms:
‰ Ribosomal RNA (rRNA) ‰ Small Nuclear RNA (snRNA)
‰ Messenger RNA (mRNA) ‰ Telomerase RNA
‰ Transfer RNA (tRNA) ‰ Antisense RNA

‰ Differs by sedimentation rate (Svedverg Coefficient)

Nucleic Acid Unique Characteristics

‰ Hydrogen bonds breaks at high temperature (denaturation or
unwinding)
‰ Hydrogen bonds reform at lower temperature (annealing)
‰ Melting Temperature (Tm)= temperature at which 50 % of H bonds are
broken (DNA with higher GC content has higher Tm)
‰ Can be measured using spectrophotometer (absorbance at 260 nm)
‰ With increasing temperature, the viscosity of DNA decreases and UV
absorption increase

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 Molecular Hybridization

‰ Annealing of nucleic acid

(DNA or RNA) strands
sharing nucleotide
sequence similarity
‰ Used to identify
homologous genes in
different species
‰ Example: In situ
hybridization or
Fluorescence in situ
hybridization (FISH)

Reassociation Kinetics
‰ Measures the rate of annealing between
complementary strands
‰ Measures half reaction time (point when
½ of the reaction are double stranded)
‰ Half Reaction is lower in smaller genomes
‰ Used to measure repetitive DNA
sequences (characteristic of eukaryotes)

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 Electrophoresis
‰ Agarose gel
electrophoresis
‰ Polyacrylaminde gel
electrophoresis
‰ Separates nucleic acids
by size under an
electrical field
‰ DNA is negatively
charged (travels to +
charge)
‰ Southern Blot –
detection of DNA
‰ Northern Blot –
detection of RNA

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