lecture 2 genetic information.ppt; protein synthesis.ppt; genetic engineering.ppt; genetic...
TRANSCRIPT
Nucleic Acids Convey Genetic Information
09:37
• 1869 Friedrich Miesher discovers a weak acid abundant in cell nuclei: deoxyribonucleic acid, DNA
• 1870’s Chromosomes: thread-like objects in cell nuclei that split and are passed on during cell division
DNA: The molecule of heredity
09:37
• 1869 Friedrich Miesher discovers a weak acid abundant in cell nuclei: deoxyribonucleic acid, DNA
• 1870’s 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)
DNA: The molecule of heredity
09:37
• 1869 Friedrich Miesher discovers a weak acid abundant in cell nuclei: deoxyribonucleic acid, DNA
• 1870’s 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
DNA: The molecule of heredity
09:37
Rough Smooth
Determining the genetic material:The Griffith Experiment 1928
09:37
DNA extract from S cells
RNase or Protease
Transformation
DNase
NoTransformation
Determining the genetic material:The Avery, MacLeod and McCarty Experiment 1944
09:37
The Avery, MacLeod andMcCarty Experiment 1944
The Griffith Experiment 1928
09:37
DNA is the genetic
material of all cellular
organisms
32P DNA 35S Protein
LabeledProgeny
No LabeledProgeny
T2 Phage
Determining the genetic material:The Hershey-Chase Experiment 1952
09:37
2007-2008
DNA Replication
Watson and Crick1953 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!– it matters!
OH
CH2
O
4
5
3 2
1
PO4
N base
ribose
nucleotide
This will beIMPORTANT!!
The DNA backbone
• Putting the DNA backbone together– refer to the 3 and 5
ends of the DNA• the last trailing carbon
OH
O
3
PO4
base
CH2
O
base
OPO
C
O–O
CH2
1
2
4
5
1
2
3
3
4
5
5
Sounds trivial, but…this will beIMPORTANT!!
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
3
5
5
3
Bonding in DNA
….strong or weak bonds?How do the bonds fit the mechanism for copying DNA?
3
5 3
5
covalentphosphodiesterbonds
hydrogenbonds
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
DNA Replication • Large team of enzymes coordinates replication
Let’s meetthe team…
Replication: 1st step• Unwind DNA
– helicase enzyme• unwinds part of DNA helix• stabilized by single-stranded binding proteins
single-stranded binding proteins replication fork
helicase
DNAPolymerase III
Replication: 2nd step
But…We’re missing something!What?
Where’s theENERGYfor the bonding!
Build daughter DNA strand add new
complementary bases DNA polymerase III
energy
ATPGTPTTPCTP
Energy of ReplicationWhere does energy for bonding usually come from?
ADPAMPGMPTMPCMPmodified nucleotide
energy
We comewith our ownenergy!
And weleave behind anucleotide!
YourememberATP!Are there other waysto get energyout of it?
Are thereother energynucleotides?You bet!
Energy of Replication• The nucleotides arrive as nucleosides
– DNA bases with P–P–P• 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
• 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
DNAPolymerase III
DNAPolymerase III
DNAPolymerase III
DNAPolymerase III
energy
energy
energy
Replicationenergy
3
3
5B.Y.O. ENERGY!The energy rulesthe process
5
energy
35
5
5
3
need “primer” bases to add on to
energy
energy
energy
3
no energy to bond
energy
energy
energy
ligase
3 5
Limits of DNA polymerase III can only build onto 3 end of an
existing DNA strand
Leading & Lagging strands
5
5
5
5
3
3
3
53
53 3
Leading strand
Lagging strand
Okazaki fragments
ligase
Okazaki
Leading strand continuous synthesis
Lagging strand Okazaki fragments joined by ligase
“spot welder” enzyme
DNA polymerase III
3
5
growing replication fork
DNA polymerase III
Replication fork / Replication bubble
5
3 5
3
leading strand
lagging strand
leading strand
lagging strandleading strand
5
3
3
5
5
3
5
3
5
3 5
3
growing replication fork
growing replication fork
5
5
5
5
53
3
5
5lagging strand
5 3
DNA polymerase III
RNA primer built by primase serves as starter sequence for DNA
polymerase III
Limits of DNA polymerase III can only build onto 3 end of an
existing DNA strand
Starting DNA synthesis: RNA primers
5
5
5
3
3
3
5
3 53 5 3
growing replication fork
primase
RNA
DNA polymerase I removes sections of RNA primer and
replaces with DNA nucleotides
But DNA polymerase I still can only build onto 3 end of an existing DNA strand
Replacing RNA primers with DNA
5
5
5
5
3
3
3
3
growing replication fork
DNA polymerase I
RNA
ligase
Loss of bases at 5 ends in every replication
chromosomes get shorter with each replication limit to number of cell divisions?
DNA polymerase III
All DNA polymerases can only add to 3 end of an existing DNA strand
Chromosome erosion
5
5
5
5
3
3
3
3
growing replication fork
DNA polymerase I
RNA
Houston, we have a problem!
Repeating, non-coding sequences at the end of chromosomes = protective cap
limit to ~50 cell divisions
Telomerase enzyme extends telomeres can add DNA bases at 5 end different level of activity in different cells
high in stem cells & cancers -- Why?
telomerase
Telomeres
5
5
5
5
3
3
3
3
growing replication fork
TTAAGGGTTAAGGGTTAAGGG
Replication fork
3’
5’
3’
5’
5’
3’
3’ 5’
helicase
direction of replication
SSB = single-stranded binding proteins
primase
DNA polymerase III
DNA polymerase III
DNA polymerase I
ligase
Okazaki fragments
leading strand
lagging strand
SSB
DNA polymerases
• DNA polymerase III– 1000 bases/second!– main DNA builder
• DNA polymerase I– 20 bases/second– editing, repair & primer removal
DNA polymerase III enzyme
Arthur Kornberg1959
Roger Kornberg2006
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
1
2
3
4
What does it really look like?
2007-2008
Any Questions??
Transcription
The synthesis of RNA molecules using DNA strands as the templates so that the genetic information can be transferred from DNA to RNA.
Transcription
• Both processes use DNA as the template.
• Phosphodiester bonds are formed in both cases.
• Both synthesis directions are from 5´ to 3´.
Similarity 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
Differences between replication and transcription
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.
G C A G T A C A T G T C5' 3'
3' C G T C A T G T A C A G 5' template strand
coding strand
transcription
RNAG C A G U A C A U G U C5' 3'
• 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.
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.
core enzymeholoenzyme
Holoenzyme
The holoenzyme of RNA-pol in E.coli consists of 5 different subunits: 2 .
subunit MW function
36512Determine the DNA to be transcribed
150618 Catalyze polymerization
155613 Bind & open DNA template
70263Recognize the promoter
for synthesis initiation
RNA-pol of E. Coli
• Rifampicin, a therapeutic drug for tuberculosis treatment, can bind specifically to the subunit of RNA-pol, and inhibit the RNA synthesis.
• RNA-pol of other prokaryotic systems is similar to that of E. coli in structure and functions.
RNA-pol I II III
products 45S rRNA hnRNA
5S rRNA
tRNA
snRNA
Sensitivity to Amanitin
No high moderate
RNA-pol of eukaryotes
Amanitin is a specific inhibitor of RNA-pol.
• 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.
§1.3 Recognition of Origins
5'
3'
3'
5'
regulatory sequences structural gene
promotorRNA-pol
Promoter
5'
3'
3'
5'-50 -40 -30 -20 -10 1 10
start -10 region
T A T A A T A T A T T A
(Pribnow box)
-35 region
T T G A C A A A C T G T
Prokaryotic promoter
Consensus sequence
• 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 co-factors 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 RNA-pol 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.
RNA
5TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGGCACCAGCCTTTTT... 3 DNA
UUUU...…
rplL protein
UUUU...…
5TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGTCACCAGCCTTTTT... 3
• The stem-loop structure alters the
conformation of RNA-pol, leading to the pause of the RNA-pol moving.
• Then the competition of the RNA-RNA 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.
Stem-loop disruption
§2.2 Transcription of Eukaryotes
• 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.
a. Initiation
structural geneGCGC CAAT TATA
intronexon exon
start
CAAT box
GC box
enhancer
cis-acting element
TATA box (Hogness box)
Cis-acting element
TATA box
• 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.
Transcription factors
TF for eukaryotic transcription
• 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)
Pre-initiation complex (PIC)
RNA pol II
TF II F
TBP TAFTATA
DNATF II A
TF II B
TF II E
TF II H
• 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.
Phosphorylation of RNA-pol
• The elongation is similar to that of prokaryotes.
• The transcription and translation do not take place simultaneously since they are separated by nuclear membrane.
b. Elongation
RNA-Pol
RNA-Pol
RNA-Pol
nucleosome
moving direction
• The termination sequence is AATAAA followed by GT repeats.
• The termination is closely related to the post-transcriptional modification.
c. Termination
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.
• 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
§3.1 Modification of hnRNA
CH3
O
O OH
CH2
PO
O
O
N
NHN
N
O
NH2
AAAAA-OH
O
Pi
5'
3'
O
OHOH
H2CN
HNN
N
O
H2N O P
O
O
O P
O
O
O P
O
O
5'
a. Capping at the 5- end
m7GpppGp----
ppp5'NpNp
pp5'NpNp
GTP
PPi
G5'ppp5'NpNp
methylating at G7
methylating at C2' of the first and second nucleotides after G
forming 5'-5' triphosphate group
removing phosphate group
m7GpppNpNp
m7Gpppm
2'Npm2'Np
Pi
• 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.
The matured mRNAs are much shorter than the DNA templates.
DNA
mRNA
c. mRNA splicing
A~G no-coding region 1~7 coding region
L 1 2 3 4 5 6 77 700 bp
The structural genes are composed of coding and non-coding regions that are alternatively separated.
Split gene
EA B C D F G
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
U pA G pU5' 3'5'exon 3'exon
intron
pG-OH
pGpA
G pU 3'U5' OH
first transesterification
Twice transesterification
second transesterification
U5' pU 3'
pGpA
GOH
5'
3'
• 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.
d. mRNA editing
Different pathway of apo B
Human apo B gene
hnRNA (14 500 base)
liverapo B100
( 500 kD) intestineapo B48
( 240 kD)
CAA to UAAAt 6666
§3.2 Modification of tRNA
tRNA precursor
RNA-pol III
TGGCNNAGTGC GGTTCGANNCC
DNA
Precursor transcription
RNAase Pendonuclease
Cleavage
ligase
tRNA nucleotidyl transferase
ATP ADP
Addition of CCA-OH
Base modification
( 1 )( 1 )
( 3 )
( 2 )
( 4 )
1. Methylation A→mA, G→mG
2. Reduction U→DHU
3. Transversion U→ψ
4. DeaminationA→I
§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
transcription
splicing
45S-rRNA
18S-rRNA5.8S and 28S-rRNA
28S5.8S18S
• 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.
§3.4 Ribozyme
• 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
• 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
Significance of ribozyme
Artificial ribozyme
• Thick lines: artificial ribozyme
• Thin lines: natural ribozyme
• X: consensus sequence
• Arrow: cleavage point
04/21/23
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 “C-terminal”.
• 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 synthesis1. DNA unwinds
2. mRNA copy is made of one of the DNA strands.
3. mRNA copy moves out of nucleus into cytoplasm.
4. 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 EngineeringGenetic Engineering
First, the nucleus of human cells are burst
Human cellNucleus
Genetic EngineeringGenetic Engineering
The chromosomes are cut up into small fragments and the required gene identified.
Chromosome fragments
Fragment containing required gene
Genetic EngineeringGenetic Engineering
Next the fragments are spread out and the required one isolated.
Segment with required gene
Genetic EngineeringGenetic Engineering
Cytoplasm
Bacterial chromosomeBacterial cell wall
Plasmid
Structure of a typical bacterium
Genetic EngineeringGenetic Engineering
Plasmid
Plasmids are loops of DNA separate from the main chromosome. They carry genes for things like antibiotic resistance. This makes them very useful to theGenetic engineer.
Genetic EngineeringGenetic Engineering
In the above plasmid, the YELLOW gene is one that gives the bacterium resistance to one antibiotic (eg Penicillin).
PP
TT
The GREEN gene gives resistance to a different antibiotic (eg Tetracycline)
Genetic EngineeringGenetic Engineering
By using special enzymes, we can make a cut in the midst of ONE of theseantibiotic resistance genes.In this example, we will cut open the ‘T’ gene
PP
TT
Cut here
Genetic EngineeringGenetic Engineering
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 plasmidso that the green ‘T’ gene will no longer work correctly.
Prepared human gene
Genetic EngineeringGenetic Engineering
As plasmids are extremely small, we cannot tell by looking which ones have gotthe human gene in the right place. We need to use a ‘shotgun’ approach andincubate thousands of plasmids with hundreds of bacterial cells
No P or T gene
Intact P gene Intact P gene and ‘defective’ T and ‘defective’ T genegene
P and T Genes intact
Genetic EngineeringGenetic Engineering
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 antibioticresistance genes.
Required cellRequired cell Cell with P and T intactCell with P and T intact Cell with neither P or Cell with neither P or TT
Genetic EngineeringGenetic Engineering
An agar plate containing Penicillin is used to allow only those cells which havetaken up a suitable plasmid to survive and divide. These cells must have resistanceto Penicillin
Agar containingpenicillin
Colonies growing from single cells that are resistant to penicillin
Genetic EngineeringGenetic 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 WON’T grow in the presence of Tetracycline
Genetic EngineeringGenetic Engineering
Next, these colonies are sub-cultured onto agar containing tetracycline.
These cells must have intact T genes
These cells must have intact P genes and defective T genes
Genetic EngineeringGenetic Engineering
This colony will probably have the correct plasmid to produce the product from thehuman gene. Cells from this colony will be grown on a large scale and the mediumanalysed 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 client’s circumstances.”
American Board of Genetic Counseling
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– 1940’s– Retreat from “advisement” with a focus on
prevention by offering risk information
• Decision-Making Model– 1950’s – 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