Nucleic Acids Convey Genetic Information

09:08

DNA: The molecule of heredity

1869 Friedrich Miesher discovers a weak acid abundant in cell
nuclei: deoxyribonucleic acid, DNA
1870s Chromosomes: thread-like objects in cell nuclei that
split and are passed on during cell division

09:08

DNA: The molecule of heredity

1869 Friedrich Miesher discovers a weak acid abundant in cell
nuclei: deoxyribonucleic acid, DNA
1870s Chromosomes: thread-like objects in cell nuclei that
split and are passed on during cell division
The number of chromosomes is constant within a species,
but may differ between species
Chromosomes are made up of DNA (simple structure) and
proteins (diverse structure)

09:08

DNA: The molecule of heredity

1869 Friedrich Miesher discovers a weak acid abundant in cell
nuclei: deoxyribonucleic acid, DNA
1870s Chromosomes: thread-like objects in cell nuclei that
split and are passed on during cell division
The number of chromosomes is constant within a species,
but may differ between species
Chromosomes are made up of DNA (simple structure) and
proteins (diverse structure)
The amount of DNA is constant between cells of an organism
while the amount and type of proteins differ
The complexity of protein made it the initial favorite candidate
for genetic material
09:08

Determining the genetic material:

The Griffith Experiment 1928

Rough
09:08

Smooth

Determining the genetic material:

The Avery, MacLeod and McCarty Experiment 1944
DNA extract from S cells

RNase or Protease

Transformation

09:08

DNase

No
Transformation

The Griffith Experiment 1928

The Avery, MacLeod and

McCarty Experiment 1944

09:08

Determining the genetic material:

The Hershey-Chase Experiment 1952
32

P DNA

S Protein

35

T2 Phage

DNA is the
genetic
material of all
cellular
organisms

Labeled
Progeny
09:08

No Labeled
Progeny

DNA Replication

2007-2008

Watson and Crick

1953 article in Nature

Double helix structure of DNA

It has not escaped our notice that the specific pairing we have postulated
immediately suggests a possible copying mechanism for the genetic
material.
Watson & Crick

Directionality of DNA
You need to
number the
carbons!

nucleotide

PO4

N base

it matters!
5 CH2
This will be
IMPORTANT!!

O
1

ribose
3
OH

The DNA backbone

Putting the DNA
backbone together
refer to the 3 and 5
ends of the DNA
the last trailing carbon
Sounds trivial, but
this will be
IMPORTANT!!

5
PO4
5 CH2
4

base
O
1

C
3

O P O
O
5 CH2

base
O

1
2

OH

Anti-parallel strands
Nucleotides in DNA
backbone are bonded from
phosphate to sugar
between 3 & 5 carbons

DNA molecule has direction

complementary strand runs in
opposite direction

Bonding in DNA
hydrogen
bonds

covalent
phosphodiester
bonds

.strong or weak bonds?

How do the bonds fit the mechanism for copying DNA?

Base pairing in DNA

Purines
adenine (A)
guanine (G)

Pyrimidines
thymine (T)
cytosine (C)

Pairing
A:T
2 bonds

C:G
3 bonds

Copying DNA
Replication of DNA
base pairing allows
each strand to serve as
a template for a new
strand
new strand is 1/2
parent template &
1/2 new DNA

Lets meet
the team

DNA Replication

Large team of enzymes coordinates replication

Replication: 1st step

Unwind DNA
helicase enzyme
unwinds part of DNA helix
stabilized by single-stranded binding proteins
helicase

single-stranded binding proteins

replication fork

Replication: 2nd step

Build daughter DNA
strand
add new
complementary bases
DNA polymerase III

DNA
Polymerase III

But
Wheres the
Were missing
ENERGY
something!
for the bonding!
What?

Energy of Replication
Where does energy for bonding usually come from?
We come
with our own
energy!

You
remember
ATP!
Are
Are there
there
other
other ways
energy
to
get energy
nucleotides?
out
You of
betit?
!

energy
energy

CTP
TTP
GTP
ATP

And we
leave behind a
nucleotide!

modified nucleotide

TMP
GMP
AMP
ADP
CMP

Energy of Replication
The nucleotides arrive as nucleosides
DNA bases with PPP
P-P-P = energy for bonding

DNA bases arrive with their own energy source for

bonding
bonded by enzyme: DNA polymerase III

ATP

GTP

TTP

CTP

Replication
energy
Adding bases
can only add
nucleotides to
3 end of a growing
DNA strand
need a starter
nucleotide to
bond to

strand only grows

53
B.Y.O. ENERGY!
The energy rules
the process

DNA
Polymerase III
energy
DNA
Polymerase III
energy
DNA
Polymerase III
energy
DNA
Polymerase III

need primer bases to add on to

energy
no energy
to bond

energy
energy

energy
energy

ligase
energy

energy

Okazaki

Leading & Lagging strands

Limits of DNA polymerase III

can only build onto 3 end of an

existing DNA strand
ents
m
g
a
r
f
ki
Okaza
3
5

ligase

growing
3
replication fork

Lagging strand

Leading strand
3

Lagging strand

Okazaki fragments
joined by ligase

spot welder enzyme

5
3

DNA polymerase III

Leading strand

continuous synthesis

Replication fork / Replication bubble

3

DNA polymerase III

leading strand
5

3
5

3
5

lagging strand

3
5

3
5
lagging strand

5
leading strand

growing
replication fork 5

growing
replication fork
3

leading strand

lagging strand
5 5

Starting DNA synthesis: RNA primers

Limits of DNA polymerase III

can only build onto 3 end of an

existing DNA strand

3
3

5
growing
3
replication fork

DNA polymerase III

primase
RNA 5

RNA primer

built by primase
serves as starter sequence for DNA
polymerase III

Replacing RNA primers with DNA

DNA polymerase I

removes sections of RNA primer and

DNA polymerase I
replaces with DNA nucleotides

5
3

ligase

growing
3
replication fork
RNA

5
3

But DNA polymerase I still

can only build onto 3 end of
an existing DNA strand

Chromosome erosion
All DNA polymerases can
only add to 3 end of an
existing DNA strand

Houston, we
have a problem!

DNA polymerase I
5
3

5
growing
3
replication fork

DNA polymerase III

RNA

Loss of bases at 5 ends

in every replication

chromosomes get shorter with each replication

limit to number of cell divisions?

5
3

Telomeres
Repeating, non-coding sequences at the end
of chromosomes = protective cap

limit to ~50 cell divisions

5
3

5
growing
3
replication fork

telomerase
5

Telomerase

enzyme extends telomeres

can add DNA bases at 5 end
different level of activity in different cells

high in stem cells & cancers -- Why?

TTAAGGG TTAAGGG TTAAGGG 3

Replication fork
DNA
polymerase I

5
3

DNA
polymerase III

ligase

lagging strand
primase

Okazaki
fragments

SSB

5
3

helicase

DNA
polymerase III

leading strand
direction of replication
SSB = single-stranded binding proteins

DNA polymerases
DNA polymerase III
1000 bases/second!
main DNA builder

Roger Kornberg
2006

DNA polymerase I
20 bases/second
editing, repair & primer removal
DNA polymerase III
enzyme

Arthur Kornberg
1959

Editing & proofreading DNA

1000 bases/second =
lots of typos!
DNA polymerase I
proofreads & corrects typos
repairs mismatched bases
removes abnormal bases
repairs damage
throughout life

reduces error rate from

1 in 10,000 to
1 in 100 million bases

Fast & accurate!

It takes E. coli <1 hour to copy
5 million base pairs in its single
chromosome
divide to form 2 identical daughter cells

Human cell copies its 6 billion bases &

divide into daughter cells in only few hours
remarkably accurate
only ~1 error per 100 million bases
~30 errors per cell cycle

What does it really look like?

3
4

Any Questions??

2007-2008

Transcription

Transcription
The synthesis of RNA molecules using
DNA strands as the templates so that
the genetic information can be
transferred from DNA to RNA.

Similarity between
replication and transcription
Both processes use DNA as the
template.
Phosphodiester bonds are formed in
both cases.
Both synthesis directions are from 5
to 3.

Differences between
replication and transcription
replication

transcription

template

double strands

single strand

substrate

dNTP

NTP

primer

yes

no

Enzyme

DNA polymerase

RNA polymerase

product

dsDNA

ssRNA

base pair

A-T, G-C

A-U, T-A, G-C

Section 1
Template and Enzymes

 The whole genome of DNA needs to

be replicated, but only small portion
of genome is transcribed in response
to the development requirement,
physiological need and
environmental changes.
DNA regions that can be transcribed
into RNA are called structural genes.

1.1 Template
The template strand is the strand
from which the RNA is actually
transcribed. It is also termed as
antisense strand.
The coding strand is the strand
whose base sequence specifies the
amino acid sequence of the encoded
protein. Therefore, it is also called as
sense strand.

5'

GCAGTACATGTC

3' coding

3'

CGTCATGTACAG

5'

strand
template
strand

transcription

5'

GCAGUACAUGUC

3'

RNA

Asymmetric transcription
Only the template strand is used for
the transcription, but the coding strand
is not.
Both strands can be used as the
templates.
The transcription direction on different
strands is opposite.
This feature is referred to as the
asymmetric transcription.

5'

3'

3'

5'

Organization of coding information in

the adenovirus genome

1.2 RNA Polymerase

The enzyme responsible for the RNA
synthesis is DNA-dependent RNA
polymerase.
The prokaryotic RNA polymerase is a
multiple-subunit protein of ~480kD.
Eukaryotic systems have three kinds of
RNA polymerases, each of which is a
multiple-subunit protein and responsible
for transcription of different RNAs.

Holoenzyme
The holoenzyme of RNA-pol in E.coli
consists of 5 different subunits: 2 .

holoenzyme
core enzyme

RNA-pol of E. Coli
subunit

MW

function

36512

Determine the DNA to be

transcribed

150618

Catalyze polymerization

155613

Bind & open DNA template

70263

Recognize the promoter

for synthesis initiation

 Rifampicin, a therapeutic drug for

tuberculosis treatment, can bind
specifically to the subunit of RNApol, and inhibit the RNA synthesis.
RNA-pol of other prokaryotic
systems is similar to that of E. coli in
structure and functions.

RNA-pol of eukaryotes
RNA-pol

II

III

products

45S rRNA

hnRNA

5S rRNA
tRNA
snRNA

Sensitivity
to Amanitin

No

high

moderate

Amanitin is a specific inhibitor of RNA-pol.

1.3 Recognition of Origins

Each transcriptable region is called
operon.
One operon includes several structural
genes and upstream regulatory
sequences (or regulatory regions).
The promoter is the DNA sequence that
RNA-pol can bind. It is the key point
for the transcription control.

Promoter

regulatory
sequences
5'
3'

promotor
RNA-pol

structural gene
3'
5'

Prokaryotic promoter
3'

5'
3'

-50

-40

-30

-20

-35
region
TTGACA
AACTGT

-10

-10
region
TATAAT
ATATTA
(Pribnow box)

Consensus sequence

10

start

5'

 The -35 region of TTGACA sequence

is the recognition site and the
binding site of RNA-pol.
The -10 region of TATAAT is the
region at which a stable complex of
DNA and RNA-pol is formed.

Section 2
Transcription Process

General concepts
Three phases: initiation, elongation,
and termination.
The prokaryotic RNA-pol can bind to
the DNA template directly in the
transcription process.
The eukaryotic RNA-pol requires cofactors to bind to the DNA template
together in the transcription process.

2.1 Transcription of Prokaryotes

Initiation phase: RNA-pol recognizes
the promoter and starts the
transcription.
Elongation phase: the RNA strand is
continuously growing.
Termination phase: the RNA-pol stops
synthesis and the nascent RNA is
separated from the DNA template.

a. Initiation
RNA-pol recognizes the TTGACA
region, and slides to the TATAAT
region, then opens the DNA duplex.
The unwound region is about 171 bp.

 The first nucleotide on RNA transcript

is always purine triphosphate. GTP is
more often than ATP.
The pppGpN-OH structure remains on
the RNA transcript until the RNA
synthesis is completed.
The three molecules form a
transcription initiation complex.
RNA-pol ( 2) - DNA - pppGpN- OH 3

 No primer is needed for RNA

synthesis.
The subunit falls off from the RNApol once the first 3 ,5 phosphodiester
bond is formed.
The core enzyme moves along the
DNA template to enter the elongation
phase.

b. Elongation
The release of the subunit causes
the conformational change of the
core enzyme. The core enzyme
slides on the DNA template toward
the 3 end.
Free NTPs are added sequentially to
the 3 -OH of the nascent RNA strand.

 RNA-pol, DNA segment of ~40nt and

the nascent RNA form a complex
called the transcription bubble.
The 3 segment of the nascent RNA
hybridizes with the DNA template,
and its 5 end extends out the
transcription bubble as the synthesis
is processing.

Transcription bubble

RNA-pol of E. Coli

RNA-pol of E. Coli

Simultaneous
transcriptions and
translation

c. Termination

The RNA-pol stops moving on the

DNA template. The RNA transcript
falls off from the transcription
complex.
The termination occurs in either
-dependent or -independent manner.

The termination function of factor

The factor, a hexamer, is a ATPase

and a helicase.

-independent termination
The termination signal is a stretch of
30-40 nucleotides on the RNA
transcript, consisting of many GC
followed by a series of U.
The sequence specificity of this
nascent RNA transcript will form
particular stem-loop structures to
terminate the transcription.

DNA

rplL protein

5TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGGCACCAGCCTTTTT... 3
5TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGTCACCAGCCTTTTT... 3

RNA
UUUU...
UUUU...

Stem-loop disruption
The stem-loop structure alters the
conformation of RNA-pol, leading to
the pause of the RNA-pol moving.
Then the competition of the RNARNA hybrid and the DNA-DNA hybrid
reduces the DNA-RNA hybrid
stability, and causes the
transcription complex dissociated.
Among all the base pairings, the
most unstable one is rU:dA.

2.2 Transcription of Eukaryotes

a. Initiation
Transcription initiation needs
promoter and upstream regulatory
regions.
The cis-acting elements are the
specific sequences on the DNA
template that regulate the
transcription of one or more genes.

Cis-acting element
cis-acting element
GCGC

CAAT

TATA

structural gene
exon

intron exon

start
TATA box

enhancer
GC box

CAAT box

(Hogness box)

TATA box

Transcription factors
RNA-pol does not bind the promoter
directly.
RNA-pol II associates with six
transcription factors, TFII A - TFII H.
The trans-acting factors are the
proteins that recognize and bind
directly or indirectly cis-acting
elements and regulate its activity.

TF for eukaryotic transcription

Pre-initiation complex (PIC)

TBP of TFII D binds TATA
TFII A and TFII B bind TFII D
TFII F-RNA-pol complex binds TFII B
TFII F and TFII E open the dsDNA
(helicase and ATPase)
TFII H: completion of PIC

Pre-initiation complex (PIC)

RNA pol II
TF II F
TF II
A

TBP TAF

TATA

TF II
B

TF II E

TF II H

DNA

Phosphorylation of RNA-pol
TF II H is of protein kinase activity to
phosphorylate CTD of RNA-pol. (CTD
is the C-terminal domain of RNA-pol)
Only the p-RNA-pol can move toward
the downstream, starting the
elongation phase.
Most of the TFs fall off from PIC
during the elongation phase.

b. Elongation
The elongation is similar to that of
prokaryotes.
The transcription and translation do
not take place simultaneously since
they are separated by nuclear
membrane.

nucleosome
RNA-Pol

moving
direction

RNA-Pol

RNA-Pol

c. Termination
The termination sequence is
AATAAA followed by GT repeats.
The termination is closely related to
the post-transcriptional modification.

Section 3
Post-Transcriptional
Modification

 The nascent RNA, also known as

primary transcript, needs to be
modified to become functional
tRNAs, rRNAs, and mRNAs.
The modification is critical to
eukaryotic systems.

3.1 Modification of hnRNA

Primary transcripts of mRNA are called as
heteronuclear RNA (hnRNA).
hnRNA are larger than matured mRNA by
many folds.
Modification includes

Capping at the 5 - end

Tailing at the 3 - end
mRNA splicing
RNA edition

a. Capping at the 5 - end

OH

OH

O
N
H2N

H2C O P

5'

HN

5'

O P O P O CH2
O

NH

NH2

N
O

CH3

Pi

O
O

P O
O

m7GpppGp----

OH
AAAAA-OH 3'

 The 5 - cap structure is found on

hnRNA too. The capping process
occurs in nuclei.
The cap structure of mRNA will be
recognized by the cap-binding protein
required for translation.
The capping occurs prior to the
splicing.

b. Poly-A tailing at 3 - end

There is no poly(dT) sequence on the
DNA template. The tailing process
dose not depend on the template.
The tailing process occurs prior to
the splicing.
The tailing process takes place in the
nuclei.

c. mRNA splicing
mRNA

DNA

The matured mRNAs are much shorter than

the DNA templates.

Split gene
The structural genes are composed of
coding and non-coding regions that
are alternatively separated.

B C D

7 700 bp

A~G no-coding region 1~7 coding region

Exon and intron

Exons are the coding sequences that
appear on split genes and primary
transcripts, and will be expressed to
matured mRNA.
Introns are the non-coding sequences
that are transcripted into primary
mRNAs, and will be cleaved out in the
later splicing process.

mRNA splicing

Splicing mechanism

lariat

Twice transesterification
intron

5'

5'exon

U pA

G pU

3'exon

3'

first transesterification

pG-OH
pGpA
5'

UOH

3'

G pU

second transesterification
5' pGpA
5'

U pU

3'
GOH 3'

d. mRNA editing
Taking place at the transcription
level
One gene responsible for more than
one proteins
Significance: gene sequences, after
post-transcriptional modification,
can be multiple purpose
differentiation.

Different pathway of apo B

Human apo B
gene
hnRNA (14 500 base)
CAA to UAA
At 6666

liver
apo B100
500 kD

intestine
apo B48
240 kD

3.2 Modification of tRNA

Precursor transcription
DNA
TGGCNNAGTGC

GGTTCGANNCC

RNA-pol III

tRNA precursor

Cleavage

RNAase P
endonuclease

ligase

Addition of CCA-OH

tRNA nucleotidyl
transferase
ATP

ADP

Base modification

1
1

1. Methylation
AmA, GmG
2. Reduction
UDHU
3. Transversion
U

3
4

4. Deamination
AI

3.3 Modification of rRNA

45S transcript in nucleus is the
precursor of 3 kinds of rRNAs.
The matured rRNA will be assembled
with ribosomal proteins to form
ribosomes that are exported to
cytosolic space.

rRNA
18S

28S

5.8S

45S-rRNA

transcription

splicing
18S-rRNA

5.8S and 28S-rRNA

3.4 Ribozyme
The rRNA precursor of tetrahymena
has the activity of self-splicing
(1982).
The catalytic RNA is called ribozyme.
Self-splicing happened often for
intron I and intron II.

 Both the catalytic domain and the

substrate locate on the same
molecule, and form a hammer-head
structure.
At least 13 nucleotides are conserved.

Hammer-head

Significance of ribozyme
Be a supplement to the central
dogma
Redefine the enzymology
Provide a new insights for the origin
of life
Be useful in designing the artificial
ribozymes as the therapeutical
agents

Artificial
ribozyme
Thick lines:
artificial ribozyme
Thin lines:
natural ribozyme
X: consensus
sequence
Arrow: cleavage
point

05/09/15

Translation

Central Dogma of Molecular

Biology

The flow of information in the

cell starts at DNA, which
replicates to form more DNA.
Information is then transcribed
into RNA, and then it is
translated into protein. The
proteins do most of the work in
the cell.
Information does not flow in the
other direction. This is a
molecular version of the
incorrectness of inheritance of
acquired characteristics.
Changes in proteins do not
affect the DNA in a systematic
manner (although they can
cause random changes in DNA.

Translation
Translation of mRNA into protein is accomplished by the
ribosome, an RNA/protein hybrid. Ribosomes are
composed of 2 subunits, large and small.
Ribosomes bind to the translation initiation sequence on
the mRNA, then move down the RNA in a 5 to 3
direction, creating a new polypeptide. The first amino
acid on the polypeptide has a free amino group, so it is
called the N-terminal. The last amino acid in a
polypeptide has a free acid group, so it is called the Cterminal.
Each group of 3 nucleotides in the mRNA is a codon,
which codes for 1 amino acids. Transfer RNA is the
adapter between the 3 bases of the codon and the
corresponding amino acid.

Transfer RNA

Transfer RNA molecules are short RNAs

that fold into a characteristic cloverleaf
pattern. Some of the nucleotides are
modified to become things like
pseudouridine and ribothymidine.
Each tRNA has 3 bases that make up the
anticodon. These bases pair with the 3
bases of the codon on mRNA during
translation.
Each tRNA has its corresponding amino
acid attached to the 3 end. A set of
enzymes, the aminoacyl tRNA
synthetases, are used to charge the
tRNA with the proper amino acid.
Some tRNAs can pair with more than one
codon. The third base of the anticodon is
called the wobble position, and it can form
base pairs with several different
nucleotides.

Initiation of Translation
In prokaryotes, ribosomes bind to specific translation
initiation sites. There can be several different initiation
sites on a messenger RNA: a prokaryotic mRNA can
code for several different proteins. Translation begins at
an AUG codon, or sometimes a GUG. The modified
amino acid N-formyl methionine is always the first amino
acid of the new polypeptide.
In eukaryotes, ribosomes bind to the 5 cap, then move
down the mRNA until they reach the first AUG, the codon
for methionine. Translation starts from this point.
Eukaryotic mRNAs code for only a single gene.
(Although there are a few exceptions, mainly among the
eukaryotic viruses).
Note that translation does not start at the first base of the
mRNA. There is an untranslated region at the beginning
of the mRNA, the 5 untranslated region (5 UTR).

More Initiation
The initiation process
involves first joining the
mRNA, the initiator
methionine-tRNA, and the
small ribosomal subunit.
Several initiation
factors--additional
proteins--are also
involved. The large
ribosomal subunit then
joins the complex.

Elongation

The ribosome has 2 sites for tRNAs, called P and A. The initial
tRNA with attached amino acid is in the P site. A new tRNA,
corresponding to the next codon on the mRNA, binds to the A site.
The ribosome catalyzes a transfer of the amino acid from the P site
onto the amino acid at the A site, forming a new peptide bond.
The ribosome then moves down one codon. The now-empty tRNA
at the P site is displaced off the ribosome, and the tRNA that has the
growing peptide chain on it is moved from the A site to the P site.
The process is then repeated:
the tRNA at the P site holds the peptide chain, and a new tRNA binds to
the A site.
the peptide chain is transferred onto the amino acid attached to the A
site tRNA.
the ribosome moves down one codon, displacing the empty P site tRNA
and moving the tRNA with the peptide chain from the A site to the P site.

Elongation

Termination

Three codons are called stop

codons. They code for no amino
acid, and all protein-coding
regions end in a stop codon.
When the ribosome reaches a
stop codon, there is no tRNA that
binds to it. Instead, proteins
called release factors bind, and
cause the ribosome, the mRNA,
and the new polypeptide to
separate. The new polypeptide is
completed.
Note that the mRNA continues on
past the stop codon. The
remaining portion is not translated:
it is the 3 untranslated region (3
UTR).

Post-Translational Modification
New polypeptides usually fold themselves spontaneously
into their active conformation. However, some proteins
are helped and guided in the folding process by
chaperone proteins
Many proteins have sugars, phosphate groups, fatty
acids, and other molecules covalently attached to certain
amino acids. Most of this is done in the endoplasmic
reticulum.
Many proteins are targeted to specific organelles within
the cell. Targeting is accomplished through signal
sequences on the polypeptide. In the case of proteins
that go into the endoplasmic reticulum, the signal
seqeunce is a group of amino acids at the N terminal of
the polypeptide, which are removed from the final protein
after translation.

The Genetic Code

Each group of 3 nucleotides on the mRNA is a codon. Since there

are 4 bases, there are 43 = 64 possible codons, which must code
for 20 different amino acids.
More than one codon is used for most amino acids: the genetic
code is degenerate. This means that it is not possible to take a
protein sequence and deduce exactly the base sequence of the
gene it came from.
In most cases, the third base of the codon (the wobble base) can
be altered without changing the amino acid.
AUG is used as the start codon. All proteins are initially translated
with methionine in the first position, although it is often removed
after translation. There are also internal methionines in most
proteins, coded by the same AUG codon.
There are 3 stop codons, also called nonsense codons.
Proteins end in a stop codon, which codes for no amino acid.

More Genetic Code

The genetic code is almost universal. It is used
in both prokaryotes and eukaryotes.
However, some variants exist, mostly in
mitochondria which have very few genes.
For instance, CUA codes for leucine in the
universal code, but in yeast mitochondria it
codes for threonine. Similarly, AGA codes for
arginine in the universal code, but in human and
Drosophila mitochondria it is a stop codon.
There are also a few known variants in the code
used in nuclei, mostly among the protists.

Protein synthesis
1.
2.
3.
4.

DNA unwinds
mRNA copy is made of one of the DNA strands.
mRNA copy moves out of nucleus into cytoplasm.
tRNA molecules are activated as their complementary amino acids are
attached to them.
5. mRNA copy attaches to the small subunit of the ribosomes in cytoplasm. 6
of the bases in the mRNA are exposed in the ribosome.
6. A tRNA bonds complementarily with the mRNA via its anticodon.
7. A second tRNA bonds with the next three bases of the mRNA, the amino
acid joins onto the amino acid of the first tRNA via a peptide bond.
8. The ribosome moves along. The first tRNA leaves the ribosome.
9. A third tRNA brings a third amino acid
10. Eventually a stop codon is reached on the mRNA. The newly synthesised
polypeptide leaves the ribosome.

Summary

Genetic Engineering
First, the nucleus of human cells are burst

Nucleus

Human cell

Genetic Engineering
The chromosomes are cut up into small fragments and the required
gene identified.

Fragment containing
required gene

Chromosome
fragments

Genetic Engineering
Next the fragments are spread out and the required one isolated.

Segment with required gene

Genetic Engineering
Cytoplasm

Bacterial cell wall

Plasmid

Bacterial chromosome

Structure of a typical bacterium

Genetic Engineering
Plasmid

Plasmids are loops of DNA separate from the main chromosome. They carr
genes for things like antibiotic resistance. This makes them very useful to th
Genetic engineer.

Genetic Engineering
P

In the above plasmid, the YELLOW gene is one that gives the bacterium
resistance to one antibiotic (eg Penicillin).
The GREEN gene gives
resistance to a different antibiotic (eg
Tetracycline)

Genetic Engineering
P

Cut
here T
By using special enzymes, we can make a cut in the midst of ONE of these
antibiotic resistance genes.In this example, we will cut open the T gene

Genetic Engineering

Prepared
human gene

Next, we introduce the prepared HUMAN gene to the mixture.

If all goes according to plan, the human gene will fit into the cut in the plasm
so that the green T gene will no longer work correctly.

Genetic Engineering
Intact P gene
and defective T
gene

P and T
Genes intact

No P or T
gene

As plasmids are extremely small, we cannot tell by looking which ones have g
the human gene in the right place. We need to use a shotgun approach and
incubate thousands of plasmids with hundreds of bacterial cells

Genetic Engineering

Required cell

Cell with P and T intact

Cell with neither P or

T
Some cells will take up the recombinant plasmid, some will take up original
plasmids, others will take up no plasmds at all or ones without antibiotic
resistance genes.

Genetic Engineering
Agar containing
penicillin

Colonies growing
from single cells that
are resistant to
penicillin

An agar plate containing Penicillin is used to allow only those cells which have
taken up a suitable plasmid to survive and divide. These cells must have resis
to Penicillin

Genetic Engineering

Next, these colonies are sub-cultured onto agar containing

tetracycline.
Only cells resistant to BOTH antibiotics will be able to grow.
We are interested in those cells which WONT grow in the presence of
Tetracycline

Genetic Engineering
These cells must
have intact T
genes

These cells must

have intact P
genes and
defective T genes

Next, these colonies are sub-cultured onto agar containing tetracycline.

Genetic Engineering

This colony will probably have the correct plasmid to produce the product
from the
human gene. Cells from this colony will be grown on a large scale and the
medium
analysed for the presence of the product from the human gene, eg growth
hormone

Genetic Counseling

 Genetic Counseling
Definition
History: Models of Genetic Counseling
Process
Profession

Prenatal Genetic Counseling

Process
Indications
Prenatal Testing
Psychosocial Issues
Ethical Implications

Genetic Counseling

How would you define genetic

counseling?
What experiences (if any) have
you had with genetic
counseling?

Genetic Counseling: Definition

The genetic counselor is a health professional
who is academically and clinically prepared to
provide genetic services to individuals and
families seeking information about the
occurrence, of risk of occurrence, of a genetic
condition or birth defect. The genetic counselor
communicates genetic, medical, and technical
information in a comprehensive,
understandable, non-directive manner with
knowledge of an insight into the psychosocial
and ethno cultural experiences important to
each client and family. The counselor provides
client-centered, supportive counseling
regarding the issues, concerns, and experiences
meaningful to the clients circumstances.
American Board of Genetic

History: Models of Genetic Counseling

Eugene Model (well born)
Sheldon Reed (1947) coined term
Genetic Counseling
Bateson (1906) Study of hereditary
Advising people about inherited traits

Eugenics Records Office at Cold Spring

Harbor
Collected data and provided information to
affected families

Mandatory Sterilization of mentally

defective (1926)
23 out of 48 United States

Models
Medical/Preventive Model
1940s
Retreat from advisement with a focus on
prevention by offering risk information

Decision-Making Model
1950s Discovery of cytogenetics of
several chromosomal conditions
Emphasis on providing information in an
interactive process

Models

Psychotherapeutic Model
Provision of information alone is not enough
Focus on response and experiences related
to genetic conditions
Framework
Client-centered therapy Carl Rogers
Non-directiveness

Genetic Counseling Profession

Masters Training Programs
1971 Sarah Lawrence College
Currently 28 training Programs (USA)

National Society of Genetic

Counselors
1979

American Board of Genetic

Counseling
Certification process - 1981

Philosophy of Genetic Services

Voluntary utilization
Equal Access
Client Education
Complete disclosure
of Information

Nondirective
counseling
Attention to
Psychosocial and
Affective Dimensions
in counseling
Confidentiality

Process of Genetic Counseling

Information Gathering
Family and Medical History

Risk Assessment
Actual risk vs. perceived risk

Information Giving
Educators

Psychosocial Counseling

Genetic Counseling Contexts

Reproductive Issues**
Preconception counseling
Prenatal
Infertility

Pediatrics
Newborn Screening
Specialty Clinics

Adult-Onset conditions
Specialty Clinics
Pre-symptomatic testing: Breast and Colon Cancer,
Huntingtons disease