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`3110; Nucleic Acid Metabolism
Central Dogma – September 8, 11
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Molecular activity that govern biological properties of cells/virus
DNA is the brain of cell
Crick in 1958
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Fig 1.19 – info in nucleic acid can be perpetuated or transferred, but the
transfer of information into protein was irreversible
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DNA ----transcription-RNA----translation------Protein
Transcription “book in library example”
Translation – nucleic acid(nucleotide) -- converted into protein(amino
acid)
Darwin – natural selection of fit genes
Genotype forms phenotype, not the other way around ie)Giraffe neck
Irreversible
DNA ----transcription-RNA----translation------Protein
----reverse transcriptase(retrovirus AIDS)
RNA replication (Polio, Influenza)
AIDS; single stranded RNA has to go through double stranded intermediate
to replicate
Cricks statement of irreversibility was wrong because of reverse
transcriptase
Retrovirus only grow in eukaryotic cells
Fig 1.21 – mycoplasma – simplest
Plants – most complex
Origin of life
Protein is not the origin of life
Has to be DNA or RNA
RNA was beginning – Evidence
1) rRNA, tRNA, mRNA are central elements in translation apparatus
mRNA; template for protein synthesis; triplet codons
tRNA; carries amino acid to Ribosome(adaptor RNA) – crick postulated it
needed at least 1 nucleotide
rRNA – makes ribosome – various functions
2)catalyze certain rxns
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a) short RNA molecules can be synthesized in vitro on a RNA template
without protein but not in vivo
b)in vivo/vitro ; modify RNA molecules
1)ecoli RNase P ; RNA + protein, RNA had catalytic activity and protein used
for structure
Ribozyme catalyzes 5’ site specific cleavage of tRNA’s
Only specific for tRNA molecules but can be modified for other molecules
Ii)rRNA in tetrahymena(protozoan)
Splices out its own introns
Done by Tom Cech – won the nobel in chemistry
Iii) 23S rRNA in ecoli is peptidyl transferase activity (forms peptide bonds)
3) RNA is the genome of certain viruses
Therefore RNA was thought to be original molecule
Then over time DNA became molecule of choice
Because 2’OH is missing in DNA so it cant carry out catalytic rxns therefore
making it more stable
Protein are complex in structure and function so you generate more
structures and more specificity
This generates greater diversity in structure and function
20 amino acids used as building blocks versus 4 nucleotides gives more
things that can be formed
Dolittle and Darnell – Early stages of life
Postulate RNA was original molecule of life and had introns and exons
Introns can exist anywhere
5’ untranslated region,
3’ untranslated region
rRNA(tetrahemena)
tRNA(yeast)
Fig 3.12 – BERG and SINGER – genes and genomes
Condensation of nucleotides into short RNA
Catalyze – RNA synthesis, RNA cleavage, RNA splicing
Translation mechanism ; mRNA, rRNA, tRNA, genetic code developed, exon
shuffling (to have so many proteins exons have to be moved around)
RNA genomes – several genes/RNA
Reverese transcriptase lead to…
DNA genomes
Progenote (large DNA molecules, containing many genes, had many
chromosomes
Cells
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3 domains : Archae, eubacteria, eukarya
September 13, 11 - Cis acting elements/ Trans acting
factors
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2 guys – ??Jacobs, and francois??
fig 2.17 – genes contain control site to which protein bind to regulate gene
transcription
fig 2.18 – experiment was in e. coli to discover terms on lac operon
had to put 2 copies of same gene in ecoli
allele: alternate form of a gene; distinguished by mutation
analysis of expression of 2 genes
also have control protein made in excess; so it binds to both alleles
1st mutation in control site – control protein cannot bind to site : allele cannot
be expressed
2nd allele is wild type; so it is expressed
in cis acting mutation – affects only DNA molecule to which it is attached to
not diffusible
transacting mutations – affects both alleles – control protein is mutated??
Is diffusible – goes one place to another to regulate
In DNA replication : starts at OR, which is cis acting element, control proteins
are trans acting factors
Mutation in OR only DNA attached(cis acting)
Mutation in control protein(trans acting factor) affects everything
DNA Structure
- nucleotide/polynucleotide chains
- fig 1.2 Deoxynucleotides – structure of 4 nucleotides
- contain 5’ sugar – deoxy ribose bc lacks OH at 2’
- gives DNA more chemical stability than RNA
- 1’ attached nitrogenous base
- 2 purines (double ring) – adenine, guanine
- 2 pyrimindines (single ring) – thymine, cytosine
- adenine – adenosine
- guanine – guanosine – guanidine
- cytosine – cytidine
- thymine – thymidine
- uracil - uridine
- phosphate + sugar + base– nucleotide
- sugar + base = nucleoside – adenosine, thymidine, cytosine, guanosine
- dAMP – 5’
- dTMP
- dGMP
- dCMP
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fig 1.3 – linked covalently to 5’
called nucleotide residues because lost water molecule
linked together to have 5’ to 3’ polarity
phosphate attached to 3’
Double Helix
- DNA bonds
- Separating daughter from parental template
- to build daughter DNA they have to be separated – rules out covalent bonds
- weak bonds – have specificity
- 3 classes
- 1) ionic bonds/hydrogen bonds – attractive forces between oppositely
charged atoms
- 2)van der Waals forces – molecules of same shape come together due to
minor fluctuations of electrons in molecules
- 3)hydrophobic interactions – btwn non polar molecules
- water wants to associate together so it drives out excludes hydrophobic
molecules bc they have attraction to each other through hydrogen bonding
- no stoichiometry so called interactions not bonds
- complementarity – assume complementary shapes
- to have proper structure u need complementarity to provide surface area
btwn parental and daughter
- fig 1.9 – DNA measured and had width(diameter) of 20 A or 2 nm
- DNA was long, unbranched and had uniform width
- distance between atoms is Angstroms – easier to say than nm
- Rosalind Franklin – x ray crystallography
- DNA had uniform structure, and has 2 repeats (34 and 3.4 A)
- B –DNA – hydrated form 10 bases per repeat of 34 A, repeat at 3.4 A
- A – DNA (dehydrated) – more complicated to work with
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Chargaff rules – amount of A = T, G = C, G not equal to A, C not equal to T
Fig 1.12 – DNA double helix has a uniform width of 20 A
Fig 1.5 – 0.34 nm distance btwn adjacent base pairs - BDNA
10 base pairs – 360 degree turn – 3.4 nm - BDNA
right handed helix – twists in a clockwise direction
major groove/ minor groove – important for protein binding
Fig 1.13 – 20 A, 34 A
Strands are antiparallel
Problem for 5’ to 3’ polymerase replication, so 3’ to 5’ stand cannot be read
nor replicated – another strategy developed
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Fig 1.7 –
Hydrogen bonding btwn the bases
A –T – 2 hydrogen bonds : 1) amino(A) to keto(T)(2.85A)
2) 2 ring nitogens(2.90)
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G-C – 3 hydrogen bonds - bottom/top – amino/keto (2.83/2.84)
middle – 1 ring nitrogen (2.86)
Base stacking: maintains double helix of DNA; due to hydrophobic interactions
btwn bases on the same DNA strand
- Fig 3-10: exclusion of nonpolar bases from water
- Makes DNA a rigid single strand molecule
- Makes hydrogen bonding easier to occur to make double stranded rigid
- Hydrogen bonding promotes base stacking
- Synergistic/cooperative rxn btwn hydrogen bonding and base stacking
- Fig 4-9 : makes interior of DNA very stable
- Ends of DNA are frayed – single stranded:
Chargaff’s 2nd rule: Base composition in DNA is species specific and varies
amongst species – (G-C content)
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Fig 2.16 – amount of G-C in different species
G-C is harder to break than A-T bonds
September 15 – alternate forms of DNA
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Fig 1.8 - A DNA, B DNA, Z DNA
B DNA is 0.34nm
Space filling Van der Waals
B DNA – hydrated form
B DNA is right handed – twisting in clockwise
Most DNA is B DNA
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A DNA – dehydrated form – rare from
bases are twisted relative to helical axis
twisted 20 degrees from perpendicular of the plain of helix
reduces distance of base pairs 0.29nm
11-12 bases per complete turn
deeper major grove and shallower minor groove
deep major/shallow minor
form of double stranded RNA
form of DNA/RNA hybrids
proteins that bind recognize between A and B form
Z DNA
Discovered in the states
Twisting counter clockwise – left handed
Twists in opposite direction to B DNA
Fig 27-9 – sugar phosphate backbones are “zig-zag” – very close
together
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Factors that promote Z DNA formation…
1) nucleotide sequence in DNA – have alternating purines and pyrimidine’s
on the same strand
first strand found was CGCGCG – other strand is compliment
2) high salt conditions – in vivo; proteins mask negative charges of
phosphate groups and allow Z DNA to form
phosphate come closer together than B DNA – means more negative charges
one will have to mask negative charges,
high ion (Na+) shells mask negative charge
Z DNA in vivo – Fig 4-35: polytene chromosomes from salivary glands of
drosophila – amplified DNA
Obtain antibodies from Z DNA (don’t bind to B DNA)
Fluorescent tag covalently linked
Antibodies applied to polytene chromosome
Direct fluoresce would light up antibodies binding to Z DNA
Indicating there is Z DNA present in Drosophila DNA
Each chromosome has 1 single DNA molecule in it;
have regions of Z and B DNA in same molecule
3) Cytosine methylation – plants more than ~20% methylated
regulation of gene expression
identifies genes – looking for GC
if cytosine is methylated – gene is inactive, not methylated – gene active
Molecular Weight and Length
DNA molecules are long and fragile
Average molecular weight of nucleotide is 330
Base pair (2 nucleotides) molecular weight = 660
1 Kb = 1000 bp
1 Kb = 660 000
B DNA – distance between base pairs is 0.34nm
Length = 1000 X 0.34nm = 340nm
E coli – MW = 3.6 X 10^9 - Circular double strand – 4 X 10^6bp, 1 mm long
E coli is haploid only
Mammalian DNA (diploid)– MW = 3 X 10^9 bp per haploid genome, length is
3 X10^9nm X 0.34nm - ~1m, MW =????
MW= 3 X10^9nm /0.34nm X 660 =
DNA fragments broken down are 50kb in length
If your careful you can get 70 kb in length
Pulse field gel electrophoresis – isolates different sizes of DNA molecules in
yeast
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DNA Bending
Caused by stretches of AAAAAAAA on one strand – gives greatest inclination
First discovered in trypanosomes – some DNA fragments did not migrate on
gels based on weight
Maybe involved in gene expression
Proteins also bend DNA
Enhancer – sequences in DNA that enhance gene expression – could be close
or far from gene of interest – specific sequence that regulatory proteins bind
to
Bending enhancer far away from gene of interest so they get close to the gene
of interest –
DNA can be linear or circular
In nuclei DNA is linear
Some bacteria/viruses are linear
Circular DNA in some bacteria/viruses, in mitochondria and chloroplasts,
plasmids
Lambda Phage – effects e coli – exists in circular and linear forms
4) Negative supercoiling – Fig 4-25
caused by under winding the DNA
putting a negative supercoil in relaxed circular double stranded DNA
first cut double strand
second anchor one side
move the other end 360 degrees counter clockwise
then seal ends
10 bp per 360 degree turn
region of locally disturbed bps ~ 10 bp
bp’s want to reform to relaxed form by breaking H+ bonds
then becomes tense negative supercoil
each unwinding 360 degrees creates a negative supercoil
ie)rotating 5 times by 360 degrees creates 5 supercoils
all naturally occurring DNA are negatively supercoiled
if it’s not then its because there is a “nick” in DNA
in linear DNA, both ends are anchored
DNA gyrase generates negative supercoils – example of type 2
topoisomerase
functions of negative supercoiling vary – breaking apart base pairs
1) DNA replication – separate strands at origin of replication
2) Transcription – occurs at promoter – generally AAAATTT rich
Easier to break AT(2 H+ bonds) than GC(3 H+ bonds)
3)DNA recombination
promoters are generally not open; proteins required to open promoter for
polymerase to get in, harder to open promoter in relaxed state – negative
supercoiling helps promoter or OR to open up
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In vitro: plasma DNA containing a gene: 2 forms
1)DNA is relaxed
2)DNA is negative supercoiled
measure amount of transcription
transcription of negative supercoiled DNA is much greater than relaxed DNA
which comes from frequency of initiation (~50 fold greater)
doesn’t matter where negative supercoiling occurs, as long as its affecting
some region allowing it to open up
Fig 2.17 – negative supercoiling promotes Z DNA formation
In vitro: high salt, in vivo: proteins would do this
B DNA is clockwise, Z DNA is counter clockwise can relieve stress in negative
supercoiled DNA
Supercoiled promotes Z DNA formation
Tense state In equilibrium with relaxed DNA with locally disturbed region
Shitty Drawn Picture
3 stages occur at equilibrium
Z DNA purpose is to inhibit or activate replication, transcription,
recombination
Drives these 3 processes
Proteins involved
September 20, 11 – DNA
denaturation/Renaturaction - RNA
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role of Z DNA – inhibit all the DNA processes
formation is relaxing entire region of DNA
not all organisms have double stranded DNA as genome ie)viruses have
single stranded DNA as genomes
ie) phi ΦX174 – affects e.coli - type os ssDNA
has to replicate to form double stranded DNA, called replicated form(rf)
from that you get single stranded DNA progeny
Denaturation
Important for ie)forming recombinant molecules
Fig 1.12 – H+ and base stacking are weak and can be disrupted by small
amounts of nrg
Take a double strand(native state) and break it apart to single
strands(denatured state)
Heat at 100 degrees for ~3-5 min
Measuring: look at UV absorbance of 260nm
Light at 260nm is absorbed by bases and amount of absorbance is due to
concentration of nucleic acids
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Ie)50micro g /ml of double stranded DNA A260=1.0, (absorbance of 2.0
then there would be 100micro g/ml of DNA)
50 micro g /ml of single stranded DNA, absorbance increases to 1.37, called
the hyperchromic shift – determines denaturation of DNA
with enzymes you could break down to free bases, A260 = 1.60
nucleotides are more exposed to UV light in single stranded DNA, in double
stranded nucleotides are in the interior of molecule
free bases have more absorbance because no base stacking, not covalently
attached to eachother – has maximum amount of exposure to UV light
fig 4.6 – temperature vs relative absorbance at 260nm – melting curve
at normal temp , DNA is double stranded
transition for native to denatured is sharp (6-8 degrees)
Tm = melting point, occurs when transition is half complete
Forces maintain double stranded structure are shown through melting
curves
H+ bonding and base stalking – act in a cooperative manner
Evidence for H+ bonds between bases –
Fig 1.13 – organisms vary in % of GC (Chargaff) – variation of GC content in
DNA and see what that does to Tm
1) Tm vs GC content – linear relationship – as GC increases, Tm increases
GC base pairs have 3 H+ bonds and AT base pairs have 2 H+
2) add urea and formamide compete with bases for base pairing
if you increase concentration, Tm decreases
Disrupting base pair formation of DNA
H+ causing base pairing NB for maintaining double stranded DNA
3) Fig – disrupt double stranded DNA with pH
Increase pH, Tm will decrease
pH greated than 11.3 – DNA is fully denatured – all bases fully
deprotonated
pka = amino protonated with H+, if you increase pka relative to that group
you will loose the H+, called deprotonation
Evidence for base Stacking
Comes from hydrophobic interactions – caused by water excluding non polar
substances from aqueous phase, bc it has an affinity for itslef
1) sodium-triflouroacetate – disrupts water shells – weakens hydrophobic
interactions(base stacking)
fig 4.8 – low conc to high conc – increase sodim –tri…., decrease Tm
2) alcohols or amines – (solubilize bases)increase solubility of hydrophobic
bases- as solubility increases they don’t stack as well to Tm decreases
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Tm decreases when you add these substances
Conclusion: H+ bonds and base stacking are NB for maintain double stranded
DNA
Force that destabilizes double stranded DNA;
Fig 4.8 – NaCl in solution and measure Tm
NaCl decreases, Tm decreases and vice versa [proportional]
NaCl repulses the negative P groups, in Z DNA we need to mask this
In B DNA, P groups are close together, and negatively charged so that
disrupts the structure
Forming Na+ clouds that mask negative P charge
At 0.2M NaCl all of the P groups are masked
Proteins that denature DNA
Gene 32 product of phage T4 – T even phages – infect e.coli
Discovered by Bruce Alberts in 1960– first author of Biochem book
In e.coli protein is called SSB(single stranded DNA binding protein)
Fig 4-11. SSB binds to single stranded DNA
Has a high affinity for itself – they line up beside each other along the DNA
Denatures bc DNA breathes and if they break then SSB bind
Significant role in DNA replication
DNA Renaturation/reassociation/annealing
Fig 1.14 – keep DNA denatured if you remove denaturing molecule quickly
Quickly = denatured
Slowly = renaturation
Ie) Put tube in hot water to denature then put into ice bucket – DNA remains
single stranded
Ie) High pH solution to denature DNA – then add neutralizing agent to keep
ssDNA
low salt – all DNA is single stranded,
high salt – single stranded DNA networks – inter/intra pairing bases
if you remove denaturing agent slowly – reanneals complementary strands
of DNA
generally done in high salt
H+ compete with single stranded DNA networks, eventually reanneal
DNA molecules together
Renaturation requires [salt] to mask P groups, usually 0.2M, 20 -25
degrees below Tm (most DNA its 65 degrees)
Temperature high enough to get rid of single stranded DNA networks –
competes with formation of double stranded DNA
Low enough temp to reform native DNA molecules
Fig 4.6 – range of 65 degrees – temp where you can get double stranded
DNA
Purpose of denaturation/renaturation experiements?
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Artificial reenactments of transcription, recombination, etc.…
Clues as to what’s present in genome of organism – in respect to highly,
moderate, or unique sequences of DNA
Recombinant DNA technology – identifying DNA molecules with probes
NB for determining structure of DNA
Probes – radioactive P32
RNA – types of RNA
Table 1.5 – RNA is more abundant than DNA in cells (~5-10X more)
RNA is NB for translation apparatus
rRNA – prokaryotes vs eukaryotes
1) Pro – 23S, 16S, 5S – S – how fast these molecules sediment through a
gradient, 23S is the fastest and largest
23S and 5S is in the large ribosomal subunit, 16S is in small ribosomal
subunit
2)Euk – 28S, 18S, 5.8S, 5S
large subunit – 28S, 5.8S, 5S, small subunit 18S
small RNA –
nucleus - SnRNA
cytolplasm – ScRNA
eukaryotes – heterogeneous RNA(in nucleus) – HnRNA – primary transcripts
of mRNA before introns are removed – large RNA molecules
mature RNA goes to cytoplasm from nucleus
September 22, 11 – RNA
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ecoli – rRNA – 80% of total RNA = stable
tRNA 15% - stable
mRNA 5% - unstable
fig 1.19(genes and genomes) – constituents of RNA
RNA contains 4 ribonucletoides – 3 same as DNA A, G, C
Urdine – sugar + phosphate + base
Lacks methyl group at 5 methyl group in base
Fig 1-20– nucleotides linked by 5’ to 3’ phosphodiester linkages
RNA - At 2’ position there is an OH rather than H+
2’OH – makes RNA unstable in presence of high pH will break down to free
nucleotides
hydrolyzes phosphodiester linkages
fig1.21(genes) ; once RNA synthesized the nucleotides can be chemically
modified – especially tRNA
modifications to RNA :
1) methylthiol + H+ substitutions in bases
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ie) uracil base – methyl group put on 5 position of base unlike thymine
intermediate is ribothymidine also called 5-methyluridine
5 thiouridine – sulfur on 4 position of base
H+ substitutions -> dihydrouridine
2) methylation may occur at 2’ C of sugar methyladenyic acid
3) altered linkage between the sugar and the base
ie)pseudouridine – ribose sugar linked to C group instead of Nitrogen
enzymes carry out these: prevalent in tRNA
RNA can range in length ; 70nucleotides  10 000 nucletotides
One gene involved in muscular dystrophy ~2X10^6 bp and produces
primary transcript of same length (2X10^g nucleotides)
Codes for dystrophan
About half the size of e.coli genome
Takes about a day to be made
Some phages and viruses have double stranded RNA as genome
Most have single stranded
Secondary structure of RNA – takes on A form structure
Brought about by internal bonding
16S rRNA in e coli 
depending on region – secondary structure sometimes more important than
primary structure
as long as you maintain secondary structure in certain regions even if you
change nucleotide sequence
Evidence 
1) 16S rRNA in e coli vs 18S rRNA in yeast (pro vs Eu)
very similar secondary structures
2) change sequence and see if it maintains secondary structure and function;
compensatory mutations
done by site generated mutagenesis
ie)mutate GC base pair to AU or CG or UA base pair
if you get restoration of function than secondary structure in NB, more
important than primary structure
ANDY WHITE Does these experiments at York
Important for studying regions in rRNA
NB in In tRNA – has a corelief secondary structure
All tRNA molecules have same secondary structure
3’OH end is amino acid accepting end
right TC loop
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left  DHU loop
bottom is anticodon
secondary structures of tRNA between e coli an yeast are exactly the same,
the nucleotide sequence varies
in single stranded RNA more flexible than double stranded
can form other kinds of base pairs
CG, AU – Watson crick base pairing (double stranded)
Can generate non standard base pairs – GU, GA, AC, AA, GG
non standard base contribute to secondary structure of tRNA
forms a tertiary structure
important for structures RNases have trouble destroying these molecules
in mRNA  template for protein synthesis, contains codons specify amino
acids and proteins
function, which is to convey info
lacks secondary structure so is unstable in cells, can be degraded easily by
RNases
Denaturation/Renaturation of RNA molecules
By heat,
unlike DNA cannot by denatured by high pH
denaturation
single stranded rRNA are easier to denature than double stranded RNA
bc double stranded regions in single stranded RNA are short, so don’t take as
much heat
single stranded RNA can be renatured to double stranded
easier to renature double stranded RNA which is linear as opposed to having
a secondary structure
RNA/DNA hybrids
If RNA is complimentary to DNA then they can form a hybrid
A form  form by Watson crick base pairs (dA – rU, dT – rA, dG – rC, dC
– rG)
More easily denatured than double stranded DNA at ie)lower temperatures –
less stable
If your carrying out a northern blot – run on RNA on gel through
nitrocellulose filter
Probe with P32(DNA probe)
Conditions that favour probe binding to RNA (hybrid) over double strand
DNA formation are – formamide + NaCl
Cant use pH to break down
Mapping genes;
Early way was genetic or linkage map; determine distance between
mutations in terms of recombination frequencies
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Ie) If RF is close then genes are close together
Map is limited bc mutations must affect phenotype
Also distances could be inaccurate bc RF’s can be distorted
DNA bound to protein in cells, can change RF
25% of genome have genes, 1% of total genome codes for protein(exons)
Physical maps: 2 ways
1) restriction enzymes
2) sequencing DNA; gives location of known genes
also gives protein coding potential of region of DNA
look for open reading frame (ORF’s) – does not have stop codons
reading frame is trinucleotide that codes for codon
problems with analyzing ORF’s
humans have 25000 genes c.elegans have 18000 genes
September 27, 11 – Recombinant DNA technology(RDT)
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test next Tuesday oct 4
calculators permitted
16 MC questions
office hours: Monday 244B Farq 11am-1pm
DNA was discover in 1953 by Watson and crick
Central dogma proposed in 1958
mRNA found in 1959 in bacteria and bacteriophage
genetic code in mid 1960’s
prokaryotic is more known than eukaryotes
bc its easier to study and little known about euk(more complex)
ie) e.coli use genetic exchange with conjugation
biochem + molec(genetics) = understanding of things at end of 1960’s
single stranded DNA virus phiX174 – in vitro, enzymatically synthesize a
virus and make it fully infectious
viruses are not alive – they require cells
genes isolated in e.coli using bacteriophage – lac operon/trp operon
isolated in late 1960’s before RDT
euk  yeast, neurospora, drosphila
mid 1970’s – development of RDT
work in done on bacteria and viruses
mimic bacterial exchange in vitro and mimic it in vivo
for 6 months scientists were not allowed to carry out RDT before tests were
done
e.coli grows in rich nutrient medium in vitro so they can’t survive in
environment
methodology
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involves 2 steps
1)formation of RD molecule
mammalian DNA + vector cut with restriction endonuclease ie)EcoR1
EcoR1 leasves AATT(leading) and TTAA(lagging) sticky ends
AATT 5’ overhangs
Sticky ends annealed with DNA ligase
Ie)EcoR1 – leaves 5’ AATT overhangs
2)transformation and cloning
Problem: length of DNA(3X10^9bp); so long that you have to make a lot of
your gene to make sure it gets inserted
An e.coli cell will take up 1 DNA molecule
Mammalian needs 10^6 clones in genomic DNA library – that why you have
to make so many genes of interest
Strain of e.coli must be recombination deficient
After plating on nutrient + agar plate overnight at 37 °C
Select colony that is Amp resistance
Ampicilin disrupts bacterial cell wall synthesis
AmpR – means that the gene that cuts in the ringed ampicillin is present
3) screening genomic DNA library
fig 6.21 – singer and berg:
1) transfer colonies with a tooth pick from agar+Amp plate to agar +
nitrocellulose
2) lyse cells
3)denature DNA
4) neutralize; with high pH then to pH 7
5)remove NaOH quickly
6) add a radioactive probe (labeled with 32P DNA or RNA)
7)anneal
8)put on x-ray film; dark spots on film have DNA of interest
lambda (λ) DNA vectors fig 11.3
viruses like phage lambda used as vectors
viral DNA vector is 49kb in length
can cut out middle portion and insert DNA
virus has 2 infection cycles; lysogenic(middle) and lytic(left/right arms)
affects ecoli in 2 ways
first organism used to view development
1)lytic cycle
replicates on its own – no integration in host DNA
at end of cycle it lyses e.coli and you get progeny phage(~100 phage
produced)
2)lysogenic cycle –
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integrates into e.coli genome – site specific recombination
called a prophage when it integrates
it remains silent and replicates with host DNA
there is a flip flop between 2 pathways (1/10^4 phage change)
fig 1.7 : lambda phage grown on e.coli lawn on agar plate
1 phage will infect e.coli cell and plate overnight at 37 degrees C
as phage grow and lyse you get a clearing (plaque)
you screen plaques
fig 6.22 – put nitrocellulose on top of plate for 30 seconds
some phage will be picked up
denature/neutralize
add probe to disk
wash and expose to xray film
about 10^4 plaques per plate
duplicate to make sure dark spot is not an artifact
Restriction Endonuclease
Discovered by host restriction and modification
Done with phage lambda in lytic cycle
Phage Grown prevoiously in e.coli B
Phage grown previously in e.coli A
After both have successful infection and have 100% yield
When you grow phage B on ecoli.A you get poor infection (0.001% yield)
Called restriction; ecoli A is restricting growth of lambda B
Progeny that get through cycle are called lambda A
If you infect ecoli A then you get 100% yield because they have been
modified
If you infect e.coli B, they grow with poor efficeny (0.001% yield – restricted
by E.coli B
If you infect e.coli B with the ones that made it you get 100% yield(modified
by e.coli B)
TERM TEST #2
September 29, 11 – lambda B/K - RE
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Test on discovery of enzymes not type 2 RE
Plating efficiency
Host restriction modification: how restriction enzymes were
discovered
Nuclease(digestion) vs methylase(modification)
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E.coli B – lambda B
E.coli K – lambda K
Infection K 100% B 0.001% B 100% K 0.001% 100%
In E.coli B : Eco B nuclease makes a cognate enzyme so it doesn’t digest its
own DNA: methylase
It methylates the DNA and so it can’t bind to its own DNA
Lamba K made it through an infection on E.coli B: Fig 2.17:
Usually nuclease will get to lamba and chop it up to pieces: defense
mechanism
In rare occasions: methylase will get to DNA and stop the binding of nuclease
so it won’t be chopped up
Very rare 1 in 10^5
Modification: modified by methylation at specific sites so that nuclease will
not degrade viral DNA
Lamba with 100% efficiency on Ecoli B is lambda B
If put back on e.coli K then there is poor efficiency
After processing lambda K can’t grow on K if it intermediates between B
1) E.coli K has a different modification system than B – it has Eco K nuclease
that digests lamba B: so it binds to different sites
Eco K and Eco B bind to different sites on DNA
Cognate: if makes nuclease then makes methylase: so gene is not degraded
2) no longer methylated at Eco K site: ecoli B doesn’t have Eco K methylase it
has Eco B methylase – when replicating it loses its methylation
rare ones get through become modified by Eco K methylase
start with lamba K: 100% on Eco K, methylated at Eco K
when infects ecoli B, they get digested, but rare ones that get through are
methylated by Eco B methylase
rare ones 0.001% that get through can then grow on ecoli B at 100%
then when they infect K again only 0.001%, rare ones that get through ger
modified by Eco K methylase
then they can infect Ecoli K at 100%
Q: if you mix ecoli K and B together and then infect it with lambda and
you get poor infection?  they work together
Q: Both lambda B and K can grow on ecoli C – doesn’t not have a host
restriction modification system
Before restriction endonuclease, used DNases, which cut randomly
Fig. pg 239 – DNA fragments electrophorese based on size: small on bottom,
large on top
Sizes based on where restriction enzyme cuts
Mapping with restriction enzymes: restriction maps
Restriction enzymes generate staggered ends that make single stranded DNA
overhangs
Can make RDT with overrhands
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Pg 240 11.19 : blunt ends, sticky ends
Type 1, 2, 3 RE
Type 1: bind to DNA at specificity but don’t cut at specific site
Cut at a long distance from binding and cut randomly
Don’t cut DNA reproducibly
Type 1 & 3: cuts reproducibly but cognate methylase are physically
associated with them
You don’t want to use that in RDT because you could methylate DNA you
want to cut and then it wouldn’t cut
RE
Only Type 2 is used for RDT
cut DNA reproducibly: yield reproducible set of fragments(same size)
DNA cutting requires magnesium (Mg2+) ; hydrolyzing phosphodiester bond
Anything that cuts phosphate needs Mg2+
Leaves 5’PO4 and 3’OH DNA ligase will recognize this, if it was the other
way around ligase couldn’t do it (5’OH, 3’PO4)
Don’t have methylase physically associated with them  purify from cognate
methylase
EcoR1 is a type 2 RE
Cuts at 5’GAATTC 3’ – leading , 3’CTTAAG 5’ – lagging
Cuts between G and A – leaves 5’ AATTC 3’
Binding site is a palindrome: same sequence both ways
Forms 5’ protruding tails
RDT: anneal 2 molecules that have been cut by same RE with ligase
~400-500 type 2 RE
~100 Restriction sites: some cut at the same site: isoschizomers
ie) MBO1 and Sau3A – recognize same site and cut in same way
get there names from bacterial cells that they were isolated from
BamH1 – bacillus
3 classes of RE
1)recognizes palindromic sequences
differ: length and sequence of the restriction site
6 cutters: EcoR1, BamH1, Hind3, Pst1
5 cutters: Hinf1
4 cutters Mbo1, Sau3A
8 cutters: Not1 – cuts least frequently
2) differ with respect to the nature of the ends produced
EcoR1, BamH1, Hind3 – produce protruding 5’end – cut top left, bottom
right
Pst1 produces protruding 3’ ends – top right, left bottom
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Flush ends: Sma1 – cuts in the middle
BamH1 and Mbo1 – 6 and 4 cutter – cut at same sequence, Mbo1 cuts more
frequenty
3) don’t recognize palindromic sequences
ie)Mbo2 – leave a 1 nucleotide tail
October 6, 11 – Restriction Enzymes
-
ETBR, lamba V + light
Mobility of the DNA fragment is inversely proportional to the loagarithm of
the size
1)single and double RE digests
2) parital digets
FIG 2.4
use probe on one side (p32)
use RE, cuts only once
smallest fragement will be at bottom
lablel right to left
Fig 4.17 – DNA Ligase
Can get from e.coli, mammalian cells, usually phage T4
Need 3’OH and 5’PO4 at nick, will join these together to make
phosphodiester bond
Difference btwn nick and gap =
Nick = A break in phosphodiester bond, all base pairs are still there
Could be 3’OH or 5’PO4, or 3’PO4 and 5’OH
Gap = lost one or more nucleotides lost
T4 DNA ligase can join sticky ends generated by RE
If you have nick it will seal that nick
Unique to T4 can join blunt end(flush ends) molecules together = low
efficient, requires different solutions
Fig 4.18; E.coli DNA Polymerase 1
First DNA polymerase discovered in e.coli in 1950’s
Synthesizes DNA, requires a template DNA strand
Also requires primer
Synthesize at 3’ end of primer
From 5’ to 3’ direction on dna
Also requires 4 dNTPs as substrates
DNA primer = fidelity of putting correct nucleotide into newly synthesized
DNA
Terminal deoxynucleotidyl transferase(TdT)
20
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Comes from calf thymus
Requires primer, but doesn’t require template
Doesn’t use template, cant make single strand tails of any one of the bases
Ie) DNA + dATP (TdT) DNA-AAAAA
Will add single stranded tail base
DNA + dTTP (TdT)DNA-TTTTTT
DNA + dGTP (TdT)DNA-GGGGGG
DNA + dCTP (TdT)DNA-CCCCC
Fig 4.23: when joining 2 blunt ended DNA molecules, can do this by T4 DNA
Ligase
To make more efficient add complimentary tails to end
2 tubes add A in one and T in the other will molecule of interest and TdT
then anneal the ends of A and T together
add Pol 1 to seal gaps
use DNA ligase and
first restriction technology ever used
used in immune system to generate variability in antibodies bc doesn’t use
template
Techniques uses in RT: 3 types
Southern Blot
Used to identify restriction fragement from digestion or DNA inserted into
vector
Fig 6.1: run mammalian DNA on gel – use 6 cutter
Will see a smear of DNA on gel
Smear = Re will cut DNA at specific site, but will be random in DNA
Ie) 6 cutter frequency = 1/ 4^6 = 1/4096
Number of fragments of mammalian DNA = 3x10^6 / 4096 = ~7x10^5 DNA
fragements
4 cutter is more frequent than 6 cutter
Fig 6.2: made by Ed Southern
Digest DNA with RE, then run on gel
Take gel and put in solution of high pH to denature DNA in gel to make single
stranded ~30min
Then put into pH 7 to neutralize and stay single stranded
Then transfer DNA to nitrocellulose filter
Place filter and DNA goes up
Process like screening: put filter in bag
In bag put p32 DNA single stranded
Put in solution that allows for certain temperature, high salt to anneal DNA
fragements to complementary strand
Wash it then do audoradiography
Identify fragements that hybridize to probe
northern blot = for RNA
ie)mRNA to identify length
21
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probe is gene or DNA fragements
probe is cDNA
probe = synthetic oligonucleotides – chemically synthesized
Western blot = run protein on gel and transfer to nitrocelluslose filter
Probes are usually antibodies
Protein binds to DNA sequence – could use DNA as probe
Protein binds to other protein – can use protein as probe
cDNA probes
fig 4.20: when don’t have cloned DNA fragement to use as probe
you have RNA that you have isolated and make into DNA
cDNA = copy DNA – copy of mRNA
eukaryotic cell – mRNA have poly A tails at 3’ end
poly A – added when mRNA is needed for translation initiated – forms circle
allow synthetic oligionucleotide to bind to it, called oligo dT
anneal oligo dT to 3’ tail of mRNA molecule
then add reverse transcriptase + 4 dNTPs
enzyme that synthesizes DNA using RNA as a template
first strand DNA synthesis
adds DNA 5’ to 3’ direction – makes hybrid
to make double stranded cDNa copy, first you have to remove RNA
use alkali solution or use RNase H(enzyme that is an endo/exonuclease that
digests RNA in a RNA/DNA hybrind)
single standed cDNA will fold over on 3’ end due to base pairing
second strand DNA synthesis: use reverse transcriptase or e.coli DNA
polymerase 1 + 4 dNTPs
reverse transcriptase: can work on DNA or RNA template
will make a loop
S1 nuclease – digests single stranded DNA or single stranded RNA to get rid
of loops
Ds cDNA will be blunt ended and can tail it with TdT and put it into a vector
First method used to make cDNA
2 problems with method–
never got full length cDNA, don’t cover entire sequence of mRNA, short at
ends
1)use of S1 nuclease - dsDNA linear DNA are frayed(single stranded)(bc lack
of cooperatively between base stacking and H+ bonding)
in vivo proteins do this, but not in vitro
so S1 nuclease digests those ends
2)secondary structure in mRNA – reverse transcriptase stops copying at
secondary structure
now possible to make full length cDNA
synthetic oligonucleotide probes
made with gene machines – chemically synthesized
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1) can be used to make small genes
first gene synthesized was human gene: somatostatin
synthesized 8 over lapping fragements that were overlapping so they would
hyberdize
sealed nicks with DNA ligase
2nd was for human insulin: humulin
bc insulin was used from pig or cow
synthesized A and B chains
got to be expressed in e.coli using expression vectors
then put A and B together
active insulin = C portion is removed
October 18, 11 – chemically synthesized
oligonucleotides - Probes
-
1)making small genes
making large genes is a problem with chemical synthesis
2)can make probes for isolating large genes
problem with probe: degeneracy of code
many amino acids coded for by more than one codon
3)primers – DNA sequencing and PCR
4)Restriction site linkers
ie)can chemically synthesize restriction sites and insert into vector to be able
to clone DNA fragment into it
?oligoDT  makes cDNA?
Probes
How to make them radioactiver – in libraires, for southern blot
1)use of polynucleotide kinase
add radioactive p32 create 5’PO4 ends
Fig 4.16: 5’OH ends – can use PNK – comes from phage t4
Presence of ATP a P end will be put onto end and product is ADP
2 ATP, and 2 ATP released
have ATP A-P-P-P alpha, beta, gamma
label at gamma phosphate
DNA fragements made by restriction enzymes have a PO4 there, so it must be
removed
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Fig 4.15: Remove 5’PO4 by alkaline phosphatase(from e.coli) also called
phosomonoesterase
Then you have 5’OH and can be labeled by radioactive ATP
2)nick translation
done with E.coli DNA Pol 1
has 3 different activites, from 3 different active sites
1)polymerizing: works in presence of 4 dNTPs
works in 5’ to 3’ direction
2)3’ to 5’ Exonuclease Activity
only works on single stranded DNA(ssDNA)
used as an editing function
3) 5’ to 3’ Exonuclease Activity
works on doulble stranded DNA(dsDNA)
or RNA in a RNA/DNA hybrid
function: remove RNA primers
coppenhagen did an experiment:
mild digestion with trypsin: protein bridge between 2 active sites
trypsin bridge was cleaved
Klenov fragment  activity 1 and 2
Other fragment had 5’ to 3’ activity
5’ to 3’ Activity usually on another protein in other organisms, called a RNase
H
over evolution the klenov fragment came together with 5’ to 3’ activity to
make a more efficient enzyme
nick translation (1+3)
inserting new DNA
a weird reaction with Pol 1: when activit 1 works with activity 3
Fig 14.8: when Pol works with 5’ to 3’ activity
Start with nick (3’OH and 5’PO4)
3’OH can be primer for Pol
remove base with 5’ to 3’ and add 4 dNTPs with Pol
labeling DNA with p32,
make a nick with pancreatic DNase 1
add DNA Pol 1 + 4 dNTPS  one is radioactive
labeled PO4 is at alpha position
pyrophosphate will be released(2 PO4 released)
if region was RNA, it would be replaced with dNTPs
Random Priming DNA
Method of choice nowadays
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3rd method of Making a DNA probe radioactive
denature probe by heating and quick cooling
add chemically synthesized oligoprimers
have a random sequence
aneal to probe
add Pol 1 and 4 dNTPs
one dNTP is labeled at alpha PO4
DNA Sequencing
first macromolecule to be sequenced was protein
first protein was done in 1950s bc proteases had been found that can cut
peptides at specific amino acids
Sanger sequenced insulin first
Showed L-isomer was used instead of D-isomer
Only 1 protein was a product of a gene
Then RNA was sequenced in 1960s
First tRNA was sequenced
Easy to sequence bc have 3D structure
RNases cut at specific nucleotides
End of 1960s ssRNA genome of MS-2 virus sequenced
Compared RNA codons to amino acids in proteins
Showed in vivo and vitro were the same
1970s DNA could be sequenced
needed RE to cut DNA into reproducible fragments (discovered in early
1970s)
2 techniques for DNA sequencing
1)chemical sequencing:
2)enzymatic sequencing  dideoxy chain termination method
SANGER SEQUENCING
developed by Fred Sanger
uses DNA Pol from e.coli
requires a primer and template strand
Fig 2.10: covalently attaches alpha PO4 to end of primer
Fig 2.11: template strand provides specificity
Fig 7.12: template DNA + primer
DNA synthesis goes 5’ to 3’ starts at 3’ end
Need dNTPs
Designed to stop synthesis when you reach a certain point
Add dideoxy nucleoside triphosphate, which lacks OH at 2’ and 3’OH
Need 3’OH to continue DNA synthesis  stops DNA synthesis
4 test tubes
each test tube add one dNTPs
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?ie) add dATP and ddATP
?dATP>ddATP  evens out stops
you get a nested set of sequence fragments
you will have fragments on different sizes in same rxn tube
read sequence by putting on polyacridimide gel
fragments will separate by 1 nucleotide difference
make on dNTPs radioactive
add 8M urea in gel
urea competes with base pairs for H+ bonding
helps to denature DNA
sequence fragements should be single stranded  no secondary structure
put gel on xray film and read up from 5’ to 3’
Fig 7.4:gives u sequence of synthesized fragment
October 20, 11 – Sequencing
-
sequencing – hve to make template DNA ss
clone into M13 (ssphage of e.coli)
M13 doesn’t lyse e.coli
also need a primer DNA for DNA synthesis
buy a universal primer: oligonucleotide that is complimentary to M13 DNA
that is next to template DNA
can also make a synthetic primer
automated DNA sequencing
recognizes the newly sequenced strand
so template is complimentary to that
instead of using autoradiography
tag each ddNTPs with flouescent molecule
will emit different colours based on ddNTPs
4 different colours and computer will tell u what base they are
as they run down polyacrilimide gel a laser light will excite a fluorescent tag
detector sends info to computer
Phage phiX174 – ssDNA
Codons in vitro used in vivo
Has overlapping genes: more protein than amount of DNA – how can it do
this?
Overlapping genes are read in different reading frames
One region – shared region – can give more than one protein
Easier to sequence DNA than protein, so sequence DNA than use rules to
discover protein
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PCR
Amplify DNA
Karry mullis
Fig 7.49:
First make chemically synthesized oligonucleotide primers
have them excess so u don’t have to keep putting them back in
at ends of region
1) denature DNA; by heat ~94 degrees for 5 minutes
2)anneal primers to ssDNA ~50-68 degrees for ~30 sec
3)primer extension ~70 degrees for 2-5min
need polymerase and 4 dNTPs
polymerase from thermos aquaticus, called Taq polymerase
the longer DNA, the more likely Taq will make mutations
first cycle have 2 dsDNA
cycle
1
2
3
4
60
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dsDNA at end of cycle
2
4
8
18
2^60 = 1X10^18
can also use PCR to amplify RNA, called RTPCR
1)have to make into cDNA by adding reverse transcriptase
applications of PCR
confirm mutations
take wild type DNA and mutated, amplify both and compare both
can use this to diagnose genetic diseases
population genetics
presence of infective agents and how much ie) AIDS(ssRNA)
bacterial food poisoning
forensic medicine: DNA fingerprinting
DNA fingerprinting an animal: red tail deer was going extinct, wanted to
know if red tail deer came from one or more than one
Restriction fragment length polymorphisms (RFLPs)
in individuals
genetic polymorphism: multiple alleles in region of DNA, came about by
mutation
might lead 2 different phenotypes
polymorphism doesn’t have to give rise to altered phenotypes could still be
wild type
majority are wild type(don’t change phenotype)
looking at variation in restriction maps
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Fig 5.1: DNA has 3 Restriction sites and the other has 1 restriction site
Targeting genes
RFLPs are tightly linked to genes of interest
Fig 5.4: RFLPs can be associated with disease genes
comparing patients with a disease and normal individuals
Make DNA probes for different regions of genome
Do southern blot with DNA probe
find what band is common to patients and what is common to normal people
can give probability of having a genetic disease by LOV stats
GENE STRUCTURE
Isolated gene from recombinant DNA library
First gene analyzed was chicken ovalbumin gene
Cloned gene into lambda vector
FIG 7.1:
1) size insert by Eco R1 and run fragements on agarose gel
2) Restriction Mapping: order the internal Eco R1 fragments by RM
3)locate gene of intrest
a)southern blot with cDNA probe
b)heteroduplex mapping: can also look at intron/exon arrangement
2.35kb DNA fra  dentaure  anneal to mature mRNA of cDNA
Fig 7.3: ovalalbumin annealed to mRNA
Heteroduplex is formed
some RNA that doesn’t anneal: have not isolated entire gene
ssDNA loops - introns
c) sequence genes
October 25, 11 – introns/exons, chromosome
walking, zooblots, exon trapping
-
interrupted genes: genes that contain introns
can be found everywhere
found in…
present in bacteria & bacteriophage(extremely rare)
found in yeast  only a few genes contain introns
higher eukaryotes  most genes contain introns
introns are larger than exons – gives rise to long genes in higher eukaryotes
introns in mRNA, rRNA, tRNA
mitochondria, chloroplasts
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related genes usually have similar intron/exon arrangements
ie)hemoglobin gene family  encode proteins alpha and beta globin
tetramer
FIG 7.5/7.6  alpha/beta globin cluster of genes
Represent gene family that encode embryonic, fetal, and adult hemoglobin’s
Beta globin  arranged in order of when they are transcribed, embryonic is
transcribed early on in humans (8 weeks)
Then fetal (3-9 months)
Then adult (from birth)
Need to change affinity for oxygen throughout life stages
There are pseudogenes present in gene clusters
Beta has 1, alpha has 3
No longer active and non functional  slowly decaying over evolution bc of
mutations
Once expressed, but no longer expressed
Humans have ~700 pseudogenes of the 25 000 genes, which is 3%
50% of pseudo genes are olfactory – humans no longer need it bc we have a
good sense of vision
in favour of darwins natural selection
FIG 4.4: all alpha and beta have similar arrangements have 3 exons
separated by 2 introns
Introns positions are homologous between exons
Introns vary in length
Single ancestral globin gene that had this exon/intron arrangement and
duplicated over time to give a family
Conservation of exons have been maintained
Exons are conserved
Introns are not conserved
FIG 4.5: DHFR genes: structure of genes across mammalian species
Exon are conserved  protein coding exons
Introns are not conserved
Exon conservation allows the identification and isolation of genes
EXONS
2 distinctive properties of exons:
1)must have ORF (not containing stop codons for translation)
2)since conserved can find related sequence in other species
allowed for the development of chromosome walking: finding a diseased
gene
FIG: Chrmosome walking
Found an RFLP with a probe and found little recombination with a disease
gene, which means its very close to disease gene
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Theoretical: use sub clones as probes(radioactive), can go right or left,
rescreen DNA library, make sure genomic library has overlapping ends
Make sure sub clones have unique sequence of DNA and not repetitive, by
using southern blot
About 100kb/month can be sequenced
Problem: how do you identify gene?
Take pieces of DNA from walk and make them radioactive to see if there
exons in the region on DNA zooblot
FIG: zooblot: southern blot of DNA from closely related animals
Digest with RE
Run it on gel
Transfer to nitrocellulose
Human is positive control
Isolate DNA regions and use as a probe to see what’s happening on gel
DNA probe hybridized and was complimentary to DNA present in
organisms: shows conserved region in DNA probe
Therefore the probe contains an exon
If there were introns then there would be no hybridization
Once you think there is an ORF: then sequence probe to see if there are no
stop codons
Can use many probes on nylon filter
FIG 5.6: How muscular dystrophy was isolated by chromosome walk
Located in band 21 of short arm(p) of X chromosome
Cloned DNA from Xp21
Made a library of DNA from Xp21
Then did chromosome walk to 70kb on right hand side
Noted deletions occurring in DNA
to the right, left, and internal deletions of patients genes
Fig 5.7: then made probes for zooblots
Sequenced DNA clones and found ORF
Used fragments to isolate cDNA(mRNA) and found it to be 14kb in length
Gene is 2000kbp, takes about a day for gene to be transcribed
Gene is so large and deletions occur randomly to cause muscular dystrophy
Protein is 1500kDa  seen by western blot
Dystrophan cross anchors interior of muscle to outside of muscle cell
filaments
Anchorage causes breakdown of muscle membrane when contraction
Breathing muscles goes first bc contract all the time
Cystic fibrosis also found by chromosome walking
FIG 5.8: exon trapping: technique to identify exons:
A special vector is used for exon trapping
Has strong promoter
Vector has 2 exons that are spliced together
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Then splice out
Works with splice junctions
Will recognize exons present in DNA fragments
Can do by RTPCR  amplifying DNA  make cDNA  amplify to compare
with mRNA
Similar to exon shuffling in vivo
GRAPHS
Fig 4.8: number of exons vs % of number of genes
Comparison of yeast, fly, and mammals
Yeast: most genes don’t have introns
Yeast: Most genes don’t have more than 4 exons
insects and mammals have more introns
Mammals  6% do not contain introns
Fig: Size of gene vs % size of genes
Yeast have the smallest
Mammals have largest
Bc more introns and exons in higher organisms
FIG 4.10: Exon length vs % exons
Exon length is small
In flies and humans  an exon codes for ~50 amino acids
Yeast  exons are larger than flies and humans bc lack of introns
Fig: intron length vs % introns
Worms, flies and humans
Worms and flies intron length not much larger than exon length
Humans greater variation in intron length
bc introns are so large compared to exons….
In mammals, insects, birds, average gene is 5X larger in length of mRNA
And in mRNA there are 5’ and 3’ untranslated regions so protein is even
smaller
October 27, 11 – alternative splicing,
evolution, packaging
-
some genes encode more than one proten
single gene  more than one protein starting or terminating
starting or terminating at different sites
1)FIG 4.12  same gene, one protein is smaller than the whole protein
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same reading frame
2) Fig 4.13 – overlapping proteins
mRNA is read in 2 or more reading frames
different reading frames
Fig 13.28  phage phiX174  overlapping genes and are read in different
reading frames
3)Fig 4.15: making multiple proteins from single DNA region
alternative splicing of the primary transcript
optional exons; either there or not there in mature mRNA
splicing and recombining exons  gives rise to shorter proteins
ie)exon 2 is optional in splicing pathway
exons that are mutually exclusive  both exons are not their, one or the
other
Fig 4.14 : ie) alpha and beta variants of ` Troponin T
Muscle specific protein; regulates speed of contraction of muscle
Primary transcript has alpha and beta
Alternative splicing  alpha + W + Z, beta removed
Beta + W + Z, alpha removed
Could be regulated: one splicing pathway in a cell and a different pathway in
another cell
Pathways could be regulated by environmental cues
We have 25 000 genes in our genomes
About 60% of genes in humans are alternatively spliced to yield multiple
proteins
Major reason why we have introns: so we can carry out alternative splicing
Nrg cost for large introns 
Regulation of alternative splicing is faster than initiation of transcription
First gene to have alternative splicing was a muscle gene: has 44 different
ways to alternatively splice  44 different variants of one gene
By duplicating exons can have alternative splicing
Reduces amount of DNA we need
Cuts down nrg cost of DNA replication
How did interrupted genes evolve?
Evidences…
Amount of protein is explained by mutations, exon duplication and shuffling
In vivo; FIG; similar to exon trapping
Exon trapping done in vitro, insert into vector
Random translocations
Exons are small relative to introns
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Random recombination; piece of DNA is more likely to be inserted into intron
Exon if they are inserted into old gene, the exon is flanked by intervening
sequences
Fig 3.18: exon flanked by partial introns
By recombination it may be inserted into intron in preexisting genes
Introns of old and new become one new intron
New exon has to be read in same reading frame as old exons  1/6 chance
that it works  then will have insertion of new protein domain
To maintain exon duplication and shuffling there must be a benefit
Do exons encode protein domains with different functions?
Ie)humeral immunoglobulin (Fig 4.16)
Consist of a light and heavy protein chains
Light protein chain  has variable portion and constant portion
Variable portion there to bind antigen
Constant portion there to provide pathways to destroy pathogens
V and C are coded by different exons
exon 3 encodes for quaternary structure  which is variable
do distinct genes share exons
fig 4.17: LDL receptor
region in the middle of it that shares exons with EGF precursor
also shares with C9 complement factor (punches holes in bacterial cells that
are infecting it)
LDL shares exons with 2 genes
As proteins increase in size the genes have a greater number of exons
Increase in number of exons over evolution
Advantageous for: proteins can bind to speed up reactions
When you sequence DNA, you can see positions of introns and exons
The sites in a protein represented by the exon/intron boundaries in a
gene are located on the surface of the protein
Cannot put new exon into protein bc will destroy function of protein
- DNA Packaging
- DNA has to be highly packaged to exist in a small cell
- FIG 7.9: DNA of e.coli vs length of DNA of e.coli
- The width of DNA is very small, so can be packaged
- DNA makes 1/3 of volume of e.coli cell
Compartment
Shape
Dimensions(micometers) DNA length
e.coli cell
Cylinder
1.7 X 0.65
1.3 micrometers
length X diamter
Human nucleus
Sphere
6 (diameter)
2 meters
33
(diploid)
-
has to carry out stuff while compact
DNA concentration of DNA = 100mg/ml
DNA is very viscous and not broken down
Can’t be done in vitro
Don’t know how TF find target sites in viscous solution
DNA packaging must be very flexible for…
1)gene expression + replication
2) eukaryotic cell cycle
packing ratio = length of DNA/length of unit that contains it
ie) chromosome ;
DNA length is 4.6 X 10^7 bp = 15 640 micrometers
Chromosome length = 2 micrometers
= 15 640/2 = 7820
harder to find ratio in interphase chromatin (about 5-10X less packing ratio)
November 1, 11 – Bacterial,Eukaryotic DNA
Packaging, MARs, Centomeres, CDE
-
test from RE to centromeres
OFFICE hours, Mon 11am-1pm
Bacterial
Nucleoids
Bacteria don’t have histones
Fig 7.9: DNA packaged as a nucleoid body
Can use flurosecent tag bound to DNA, in middle of cell  makes up 1/3 of
cell
If you lyse e.coli cell  DNA will spill out
DNA is present in loops, attached to pertinacious scaffold
Disrupt nucleoid body with RNase and Protease  shows nucleoid body is
maintained by RNA and protein
Loops/negative supercoils
Has a Proteinaceous scaffold
Has multiple DNA loops
Loops are negatively supercoiled
Negative supercoiling involved in promoting DNA melting to promote DNA
replication
Loops are 10-40kb in length
Has 1 negative supercoil/turn/100 bp
Fig 9.7 loops secured by unknown mechanism
Proteins in nucleoid  histone like proteins: protein Hu and protein H1
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Positively charged proteins
No repeating nucleoid structures in bacteria cells
Eukaryotic Packaging
have histones
Loops and scaffolds (seen after histone removal)
IPMAT(mitosis figure
Packaging must be flexible  for transcription/replication to occur and for
cell cycle
Interphase  DNA diffuse, but highly packaged (chromatin)
Prior to cell divisonn DNA packaged into chromosomes (highly packaged)
Metaphase are 10X more condensensed than interphased DNA
Lyse interphase nucleoid and remove histones
DNA contains loops(nucleoid bodies)
~85kb in length
1 negative supercoil/200bp
Interphase has euchromatin &
Fig 9.9: Metaphase chromosomes; can see DNA loops
These loops are 30-90kb
Are attached to proteinaceous scaffold
Scaffold between sister chromatids
Nuclear matrix is a spehere that is inside nuclear membrane  is
filamentous
If you remove hitones, loops will radiate out and you can see attached to
membrane
Q: DNA regions that attach to supports in interphase to matrix/metaphase to
scaffold do they have specific sequencing?
Do these DNA regions are they the same or different in interphase vs
methaphase?
In interphase regions are called matrix attachment regions(MARs)  DNA
sequences that bind to matrix
How can we look at these regions?
2 experiements
1) in vivo approach: cleave with RE  remove DNA that’s been cleaved 
analyze DNA present on nuclear matrix
2)in vitro approach: partial degradation with DNase(pancreatic DNase
(doesn’t bind to specific site, can cut at MARs, and RE)  will still have
fragments  add back fragments and see what fragments bind
remove fragments than add back, analyze by sequence analysis
MARs sequences are AT rich, but no consensus sequence that is present in all
MARs regions
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Have cis-acting sites for transcription regulation
Have topoisomerase 2 binding sites (how negative supercoiling is generated)
DNA gyrase in e.coli is a topoisomerase 2 to generate negative supercoiling
Sequences of all experiments are the same
interphase attached to matrix
mitosis attached to scaffold
topoisomerase 2 is common in both
DNA regions that attach to scaffolds are similar, but proteins differ
Histones
Chromosomes + chromatin structure with histones
Fig 9.11: picture of metaphase chromosomes;
Consists of a pair of sister chromatids
Are derived from previous DNA replication event
Joined together in metaphase by centromere
Fig 10.34: highly coiled fiber in sister chromatids: 30nm Fiber(being the
diameter of the fiber)
30nm fiber exists in interphase, but its less coiled
Interphase chromatin
2 states seen cytologically
1) Euchromatin: diffuse state: most regions of DNA genome, less densly
packed
DNA can be transcribed, 10X less packed than metaphase
2)Heterochromatin: regions of DNA that are very densely packed, as dense
as DNA in metaphase chromosomes
replicated late in S-phase
this DNA is not transcirptively active
there are active genes in it
exist at centromeres and other areas
length of DNA molecule can have hetero and Eu on same molecule
Fig 9.12: Heterchromatin in nucleous: located near nucleolus and nuclear
membrane
Q: do particular DNA sequences occur in the same place? Do genes folded
into euchromatin exist in the same place? In nuclei or same place in
metaphase chromosome?
Difficult to map genes in nucleus
Depends on cell type:
In muscle near nuclei  different than ie) liver cells
Metaphase chromosomes always located in same position
Fig 9.13: G-banding: shows reproducibility of metaphase folding
Treat chromosomes
Stain with dye (Geinsa dye)
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Human karyotype 23 chromosomes
Can be used to diagnose genetic disorders: ie) dystrophy
Can locate genes
Banding pairs are the same in each chromosome shows DNA folds the same
way
Centromere
Fig 9.22: metaphase sister chromatids attached by centromeres
Anaphase  centromere duplicates and are pulled apart
Contains kinetochores: DNA in centromere,
Kinetochore binds microtubules at centromere
Microtubules are part of the spindle fiber
Sister chromatids are pulled to the poles
Centromeres can be visualized by C banding
Fig 9.23: proteins at centromere that you can get antibodies(have tag on
antibodies) and can visualize centromere
Dark region is where the centromere are located
Attachment of microtubule to kinetochore
Centromere is more resistant to nuclease bc its surrounded by proteins
Fig 9.24: Proteins that bind to centromere: microtubule binding proteins
How can we isolate centromeres?
Centromeres are necessary for proper segregation of chromosomes to poles
Isolation of centromere elements in yeast
Yeast have plasmid like vectors: capable of independent replication (have
own ORF) but don’t have centromere
Unstable plasmid and cannot segregate
Lost over time in yeast population
Yeast plasmid (minus CEN) = unstable
Yeast plasmid (plus CEN) = stable
Yeast plasmid (minus CEN) = high copy number (~30 plasmids per cell)
Yeast plasmid (plus CEN) = low copy number (~1-2 plasmids per cell)
Centromere regulates replication of chromosome
Start with plasmids minus CEN, unstable, high copy number, erratic
segregations
Put DNA into plasmid
If DNA made plasmid stable with low copy number means DNA has CEN
region in it
Fig 9.49: 1st centromere was isolated from CEN 11 in yeast
Close to MET 14 gene (product is enzyme that synthesizes methionine)
If you could isolate MET14 than you can isolate CEN11 with it
Using plasmid minus CEN as a vector:
Make recombinant DNA molecules by putting in yeast DNA into vector
Yeast DNA had wild type MET14 gene (MET14+)
Yeast genomic library was made that MET14+
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Then put them into yeast
Transform into yeast that had no MET14 gene (MET14-)
To select grow transformed yeast on media not containing methionine
Survivors had MET14+ and maybe centromere located close to it was
isolated
By looking at low copy number and if it segregates properly
Once centromere was isolated, deletion analysis was done to see what limit
of CEN is
CEN is ~120bp long
Fig 9.5: Contain 3 elements: CDE 1, 2,3
Sequence comparison of CEN in diff yeast
CDE 1,3: highly conserved, short DNA sequences, 1=9bp, 3 =11bp
CDE 2: longer (~80-90bp length), not conserved in sequence but is greater
than 90% AT rich
If you make mutations in 1,2  reduction but not elimination of CEN
function in mitosis
Element 3 mutations will stop CEN function, required for mitosis
In meiosis all three elements are essential for proper segregation
Proteins that bind to CDE have been isolated
Yeast Artifical Chromosomes
Can put more DNA in these than in lambda phage vectors
After centromeres were isolated and telemoeres were discovered YAKs were
synthesizied
Need yeast centromere
Need yest ORF
And telomeres at the end
You can clone lots of DNA in these
November 10, 11 – Histone Modifications,
semiconservative, replication
Test EADCEAEEDABBAACE
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Histones modification are transient (there or not there)
Acetylation/phosphorylation can change charge of protein
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Modification associated with replication and transcription
Phosphorylation of specific amino acids occur during specific processes
Ie) Serine 10 of Histone H3 is phosphorylated chromosomes condense
at mitosis
Replication/transcription is a local event occurs at the promotor
Modification can also be long term with above example
Fig 28.26: Acetylation of H3 and H4 with active chromatin
Methylation of H3 and H4 with inactive chromatin and heterochromatin
Modification at one site will activate/inhibt modification at another amino
site
Histone code: Combinations of modifications generate different chromatin
states for inactivation/activation
This is an epigenetic code, above DNA
Controls gene expression
Histone acetylation
Ie) methylation/acetylation of certain histones leads to activation 
chromatin remodeling/histone modifiers work together
Fig 10.18: occurs at 2 different circumstances inactive gene without
acetylation, active gene with acetylation
1)histone acetylation during replication, before they are incorporated into
nucleosomes
during replication DNA doubles so amount of histones doubles in S phase in
eukaryotic cells
Histone acetylation allow them to be incorporated in nucleosomes better
After incorporated the acetyl goup removed (specific for DNA replication)
2)when genes are transcribed: nucleosomes are already on the DNA
acetylation is reversible: ezymes that add and enzymes that remove
acetylation
Histone acetyltransferase (HATs): add acetyl  coactivator of transcription
Fig 28.22: PCAF + CBP/p300(HATs) (activation of gene transcription)
Ie) androgen receptor goes out of wack and causes prostate cancer, receptor
will bind to proteins and will acetylate DNA
2 things happen at promoters
1)histone modification
2) proteins that contact DNA pol 2: will stabilize complex
TF react with basal apparatus of DNA pol 2 to stabilze transcription
HATs open them up or bump them off to allow for transcription
Aceylation removes +ve charge on lysine residues  becomes –ve, might
loosen nucleosome to be displaced by promoter
Histone deacetylases (HDACs): Enzymes that deacetylate(repression of
promoters) histones (repression of gene transcription)
Fig 28.24: repressor complex might contain 3 different kinds of protein
1) DNA binding subunit: binds to specific region on DNA
2) corepressor: inhibit basal apparatus, to inhibt initiation of transcription
3) Histone deactylase: remoes acetyl groups from region of promoter
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Deactylation is also a feature of heterochromatin
Promoter Activation:
TF binding to DNA; TF recruits histone modifiers
Histone modifiers are methyl/acetyl/phosphor
Histone modications can occur at promoter
Combinations can happen at promoter (histone code)
Leads to recruitment of remodeling complex
The remodeling complex recruited based on what modifications are present
Ie) lysine can be trimethylated  3 methyl groups, histone has a pocket that
can fit around lysine.
Histone octamers might move along DNA and is displaced from DNA
TF can bind to those sites at basal apparatus for initiation of transcription
May also recruit HATs  acetylation of Histone: results in stabilization of
complex for initiation to occur
Nucleic Acid Replication
Purpose is to duplicate nucleic acid material
Once duplicated, nucleic acid can be passed to progeny cells or viruses
Replication relies on specificity of base pairing
Organisms can have dscells viruses can have all dsDNA, ssRNA, ssDNA,
dsRNA
DNA Replication
Best known in bacterial cells (dsDNA in e.coli)
Replication of ssDNA viruses
So complicated bc if improper replication progeny can have mutations
Minimize replication errors
Replicate DNA with high fidelity
Proof reading functions built into system
Ie) 1 in 10^5 mutation of RNA  doesn’t matter
But mutation in DNA becomes fixed will be passed onto future generations
RNA viruses don’t have proof reading ie)AIDS, influenze  problem is
proteins are always changing
Separate DNA strands: into ssDNA so they act as templates
Other proteins to stabilize ssDNA so they don’t hybridize together
DNA pol needs a primer(RNA primer)
Can only go 5’ to 3’  bc template are antiparallel, lagging needs
discontinuos synthesize
As DNA unwinds as replication fork, u get overwinding: positive supercoils
Mehlson and Stahl  DNA replication is semiconservative
dsDNA being replicated
when Watson and Crick wrote a paper “base pairing, 1 chain is the
complement of the other”
also suggested means of replication is semiconservative
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means each daughter DNA strand is composed of 1 conserved parental DNA
and 1 newly synthesized strand
1 problem with this mode: DNA is very long, for this to work u have to
unwind all DNA molecule  problem for linear/circular DNA molecules
Fig 2.23: as you unwind DNA at replication fork, DNA ahead of fork gets
tigher and overwound and becomes +ve supercoil
DNA is initially –ve supercoil, once used up becomes very wound up
Fig 2.2: conservative vs. semiconservative vs. Dispersive
Conservative  like transcription, opening up DNA and closing it up
dsRNA virus is conservative
dispersive  recombination btwn 2 different strand
each single strand has conserved parental DNA and newly synthesized DNA
in 1957 solved this problem for cells using e.coli
SEE NOTEBOOK
A year before Meselson/Stahl, Taylor ruled out conservative, but couldn’t
rule out dispersive in plants
Taylors experiment
Labeled DNA in cells (eukaryotic cells)  grown for many generations in
radioactive nucleotide (3H(tritiated) thymidine, (3H-Tdr))  thymidine is
unique to DNA not RNA
At time 0 switched growth medium and grew in non-radiactive thymidine
(cold, not radioactive)
Looked at metaphase chromosomes 1 gen after switch into cold thymidine
Put metaphase chromosomes on slides and put emolgen on slide to see
where thymidine is in 2 sister chromatids
Would see dots in one sister chromatid and the sister chromatid also had
tridium
That means each sister chromatid is daughter DNA from previous replication
DNA
Tridium specific for template DNA
Meant replication was semi-consrevative
If conservative: 1 sister would be radioactive, they other not
Couldn’t rule out dispersive
Both sister chromatids become radioactive with dispersive model
What happens during DNA replication?
Different modes of DNA replication
Replicons, origins, termini
Replicons: unit of DNA which an individual act of replication occurs
Need an origin of replication(Ori), where DNA replication is initiated
There are cis-acting elements that mutations effect
Replicons also have termini  cis acting sites in DNA
Eukaryotic cells don’t have termini
Bacterial cells entire genome is one replicon
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Bacterial viruses and plasmids are single replicons, able to replicate many
times before DNA replications
In eukaryotic cells the nuclear DNA has multiple replicons
In a single DNA molecule can have many Oris
Acticated at different times
Not activated simulataneosuly
Need to be one firing event (single firing event)
Theta mode
November 14, 11 – replication, Ori, ARS,
tertrap, theta,
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Unidirectional vs birectional replication
cells low amount of 3H TDR
pulse label seconds
high amount of 3H Tdr (nascent DNA (new DNA))
if unidirectional 1 fork is heavley labeled
EM autoradiography
bidirectional both forks are heavely labeled
with tdr can see origin
start with low then add high for a very short period of time
low to high shows newly synthesized DNA
circular DNA  we see replicating theta structure when we look at eyes
occurs when u initiate replication with RNA primers
FIG: theta mode of replication
FIG: linear DNA genome: DNA replication you get a y structure
Again with RNA primers  so called theta mode
When use RNA primers its theta mode
AT rich opens up origin
Proteins, and negative supercoiling
E.coli DNA replication
Fig 11.9:
Circular DNA genome
2 replication forks (birectional)
single origin of replication (OriC)
allow initiation of replication
can do deletion analysis to see how large it is (245bp long)
13mer + 9mer repeats
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replication forks moving in opposite directions around genome
move at ~50 000bp/min or 1000bp/sec
faster than eukaryotes (2000bp/min)
above is elongation
termination replication forks meet at top of DNA genome
there are tertraps (termination sites):
2 termination sites  left: terE,DA, right: terC,B
if one replication fork is faster than the other then it will be trapped by a
termination site to slow it down and let the other catch up
each tersite is 23bp long  Tus binds to it (preotein)
stops DNA replication fork: stops the DNA from being unwound
Tus binds asymmetrically
Can only stop 1 fork at a time
Ie) rep fork 2 delayed, rep 1 will bypass terCB and be trapped by terEDA
bc terCB is opposite direction
If 2 is going too fast then will bypass terEDA and get trapped by terCB
100kb of DNA between tertraps
if not delay: rep forks will meet
why does e.coli have tertraps?
If replication fork meets up with RNA polymerase synethesizing RNA,
replication fork cant get by
If its in same direction, rep fork can proceed
If transcription and replication go in opposite direction can’t proceed
If didn’t have tertrap would replicate right around and hit RNA polymerase
and can be lethal
Tertraps prevent collision between RNA polymerase and rep fork
If rep forks are going in same direction of tersites then they will be delayed
Rep forks first pass tertrap going in opposite direction and bump off Tus,
then get caught at next tersite going in same direction
Eukaryotic DNA replication
Fig 2.9: have Ori’s
Linear DNA molecule
Replication starts at a number of Oris on same DNA molecule
Will proceed until individual replicons fuse
Bidirectional
Individual replicons are small
Yeast & drosophila replicons 40kb
Higher eukaryotes 100kb
Fig 11.14: origin 1 active first, then origin 2 becomes active: then 1 and 2
become fused replicon
During S phase: (DNA replication phase) not all origins are active at same
time
In a somatic cell 15% of origins are active at any one time
Many replicons = faster replication
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To change rate of replication, cells do it by frequency of initiation of
replication
E.coli will speed up by initiating more often at ori (only 1 origin in E.coli)
Eukaryotes speed up by firing more often at same time ie)drosophila embryo
development, rapid cell division at beigining, does this by firing origins at
same time
Euchromatin and heterochromatin: Euchromatin first gets replicated then
heterochromatin
Yeast Origins
Yeast Origin of Replication: ARS Elements (Autonomously replicating
sequence)
When put into DNA without Ori, it will initiate replication in yeast
Isolated  when put into any DNA molecule
Different from e.coli origin
Cant put yeat origin in e.coli and get initiation
Are AT rich
Have sites when mutated can = loss or reduction in origin function
Smaller than E.coli Ori
50bp long (yeast Ori)
called Ori in bacteria
Fig 11.16: mutational analysis, with single base mutations along origin
A domain (14 bp core): AT rich
Inside 14bp core there is 11 bp consensus sequence
If you mutate A domain = no origin function
B1, B2, B3 domains if mutated = reduce origin function but don’t abolish it
ORC: protein that binds to origin (400kDa); origin recognition complex
Required for initiation in yeast
Binds to 400 ARS to start initiation in yeast
Mitochondrial origins
Unidirectional
Fig 2.5: There are 2 origins of replication
1) Ori R: synthesis of L strand
2) Ori D: synthesis of H strand
Fig 11.20: initiation at Ori occurs first
RNA primer made by mitochondrial RNA polymerase
Will make a long RNA molecule
The RNA primer is cleaved, which will eventually be replaced by DNA
After cleaved generates 3OH priming for DNA
Creates a D loop: displacing parental strand
Dloop: ssDNA on one strand and dsDNA on other strand
When D loop gets to Ori D then initiation starts
Ori D goes the opposite direction
At Ori D Primase synthesizes short RNA so doesn’t have to be cleaved
Completion: L strand first, sealed by ligases
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Release of partially replicated mitochondrial genome started at Ori D
Mitochondrial polymerase then has to replicate that
Mitochondria have no lagging strand synthesis
DNA synthesized in continuous fashion
Doesn’t need discontinuous replication
1) Theta mode of replication: has priming with RNA
Bacteria, eukarotes, mitochondira, chloroplast, some viruses
Some virsues use different modes of replication, viruses use something else
Other modes of DNA replication…
2) Alternate strand mode
carried out by adenovirus (used for gene therapy: patients over time will
develop antibodies against proteins adenovirus makes, so have to add more
adenovirus)
Fig 12.2: Replication will start at end of DNA molecule in 1 direction
1 strand will be replicated and displaces other strand
displaced strand hyberdizes to itself at end
then it gets replicated
1 strand gets replicated first, then other gets replicated later
how does initiation start at end?
Fig: adenovirus DNA genome first isolated, showed protein attached to 5’
ends (viroteral protein?)
Initiator protein starts replication at end (80Kda)
Initiator protein attached to dCMP
dCMP hyperdizes at end bc there is a G at end of template end
dCMP provides 3OH primer so polymerase can synthesize DNA
80kDa becomes 55kDA when starts initiation
50Kda called terminal protein
top strand circularizes
then dCMP binds to G
3) Rolling circle mode
November 17, 11 – ASS, rolling, virus
replication, lagging, Pol
alternate strand synthesis (ASS)
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Fig:?
Fig: ?
DNA pol and adenovirus DNA, done at origin at adenovirus
Other proteins stimulate this 200X
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Initiator protein has serine residue and dcmp is attached to serine
Continuous synthesis (not lagging)
Initiates at end of adenovirus DNA no problems with termination like other
linear DNA that initiate with RNA primers
Rolling circle/toilet paper mode of DNA replication
dsDNA circular DNA
viruses
can be genome or dsRF(replicated form), derived from ssDNA virus
initiator protein makes a nick in the duplex circular DNA
3oh and 5po4
3OH acts as primer for Pol
displace strand with 5PO4
1 revolution = 1 unit length genomic DNA
many revolutions can contain multiple unit lengths genomic DNA
(concatamers)
Fig on board (looks likes a snail): template strand
Direction of replication fork is 5’ to 3’
Can make ssDNA viruses (phiX174)
Template  dsRF  progeny single strands by rolling mode
Can also make dsDNA circular genomes
Lagging strand has to be primed by RNA primers
Okazaki fragments are covalently joined
Fig: lambda DNA replication; dsDNA genome from rolling circle
Examples
Replication of phiX174 making ssDNA
Ssphage virus phiX174; ssDNA virus
2 modes of replication
1)ssDNA (plus + strand)  dsRF ( has + and – (minus) strand) (theats mode
of replication, RNA primers) using - (minus) strand as template it makes +
strand progeny virus
thetha mode early in infection
rolling circle late in infection
Fig: A protein makes nick on the + strand DNA
A protein made by virus not by e.coli
Covalently attached to 5’ end of nick
3OH acts as primer for DNA pol and u get DNA synthesis around circle
until origin becomes exposed
once ORI is exposed, A protein cleaves
the + strand is closed to make circular DNA by A protein
A protein 2 functions: 1)nick DNA at origin 2)2nd nick of DNA at Ori, then join
5PO4 and 3OH of displaced strand (+)
DNA element is acting as a cis acting factor
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Only affects DNA molecule which it I attached
+ strand can act to make more dsRF or it can be packaged into protein to
make virus
Lambda as dsDNA genome
Undergoes rolling circle replication
Phage uses both theta and roliing circle mode of replication when infecting
e.coli
When packaged into phage protein the DNA is linear
Can use linear DNA as cloning vector
When it enters e.coli is circularizes (froms dsDNA circular)
Has stick ends that form cohesive ends (Cos-sites)
Cos sites hyperdize together and DNA ligase will seal nicks to make a dsDNA
circular molecule
In e.coli cell can go through 2 modes
1)lysoegenic mode: integrates in e.coli genome as prophage
2) lytic cycle: replicates apart from e.coli genomic DNA
early on replicates by theta mode to make a few dsDNA
then replicates by rolling circle mode (get multiple unit length genomes)
one unit length genome has cos sites at end
when it gets packaged into phage head the DNA gets cleaved at subsequent
cos stie)
gives a virus that has linear dsDNA, which has cos sites
packaging enzymes cleave DNA at cos sites
when u use lambda DNA as vector do this last part in vitro
Fig: molecular mechanism to replication in e.coli
Quick stop vs slow stop came from restrictive temperature
Mutants in DNA replication
Quick stop: If raised the temperature proteins would work at elongation
Slow stop would work at initiation or termination
In vitro complementation: mutants have done is made it able to isolate
proteins
Add wild type to mutant extract and if it works then isolate wild type
Mutations made it possible to isolate proteins in DNA replication
Leading and Lagging Strand Synthesis
Fig 14.12: DNA replication has a number of problems
DNA/RNA pol can only synthesize in 5’ to 3’ but DNA is antiparallel
Continuous fashion on leading
Discontinues fashion on lagging
Lagging has to be synthesized in opposite direction in okazaki fragments
Okazaki fragments are 1000-2000 nucleotides long in prokaryotes
In eukaryotes are 100-200 nucleotides long
Leading strand u need one priming event
Lagging u need multiple priming events
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Fig: 2.12: okazaki fragments are covalently joined together after RNA primer
is removed and replaced with DNA
Fig 9.3: circular DNA molecule in lambda (theta mode)
Fig 14.15: RNA primers are made by DNA G primase
Primers are 10 nucleotides long
Primers are made 1 for each okazaki fragments
Then replace primers with DNA
Fig 2.14: standard base pairs AT, GC
Unstable forms AC(imino), GT(enol) (rare) leads to mispairing
Fig 2.15 what happens in nucleotides are misincorporated
DNA Pol have 3’ to 5’ exonuclease (only degrade ssDNA not dsDNA)
misincorporation at very low rate  not 100%
Increases the fidelity of DNA replication by 100X
DNA Polymerases
Catalyze reactions
dNMP(primer) + dNTP(substrate) <------polymerase, template specificty--------->(dNMP)n+1 + pp(inorganic pyrohphosphate)
use dNTP for nrg
equilibrium is going to left
how can u make the rxn go to the right
inorganic pyrophosphotase breaks down pyrophosphotase so shifts rxn to
the right
this explains why diphosphates are not used for substrates
triphosphate : dN-P-P-P  dN-P + PPi  2Pi
diphosphate: d-N-P-P  dN-P + Pi
Fig: strucuture of klenow fragment of Pol1 (contains activity 1 and 3)
Fingers(ssDNA + substrate), Palm (active site), Thumb (primer template
helix)
Pol active site and Klenow fragment (3’ to 5’ exo) is 30 Angstroms away
November 22, 11 – Pol 1, Pol 3, helicase,
primers, okazaki, primosome
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DNA pol: finger, palm, thumb motif
E.coli DNA pol 1 in DNA replicaction
3’ to 5’ work exo works on ssDNA
5’ to 3’ exonuclease activity  hydrolysis of nucletodide
5’ to 3’ only works on dsDNA not ssDNA
nucleotide must be base paired
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this activity can also digest RNA in RNA/DNA hybrid, called RNase H
main function of e.coli Pol 1 is to remove RNA primers at OR and okazaki
fragments
okazaki made in lagging
DNA pol 1 removes RNA primer and replaces it with DNA
Prereq for joing of okazaki fragements with ligase
Uses nick translation: combination of Pol activity
Nick translation: binds to nick and uses 5’ to 3’ exonuclease an pol activity
Works behind replication fork not at replication fork
Fills in gaps between okazaki fragements, then replaces RNA primers
Then ligase will be able to seal the nick
E.coli DNA pol 1 first pol to be discovered in e.coli
Mutations in pol A gene (codes pol 1), reduced pol 1 activity was still able to
replicate DNA
Lead to discovery of E.coli DNA pol 3
There are 5 DNA pol in e.coli
The function of the other 3 is repair not replication
DNA pol 3 is main enzyme in DNA replication
Pol 3 Works at replication fork
Lacks 5’ to 3’ exonuclease, so requires pol 1
Consists of 10 protein subunits
The enzymatic activity Is called core enzyme
3 subunits
alpha subunit is polymerizing activity
epsilon subunit has 3’ to 5’ exonuclease activity
theta subunit required for assembly of the core, also stimulates 3’ to 5’
exonuclease activity
problem with core is low processivity (abilty to stick on DNA when
synthesizing DNA)
can synethsize 11 nucletotides before it falls off
1 core enzyme synthesizes leading, 1 core enzyme synthesizes lagging
beta sliding clamp increases the processivity
clamp has to be loaded onto DNA by gamma complex(clamp
loader/unloader)
gamma complex consists of 5 protein subunits
tau subunits dimerize core enzymes
coordinates leading and lagging strand synthesis
When everything put together called the pol 3 holoenzyme
Holozyme is assymetric, has 2 of everything except gamma complex
1 Beta sliding clamp is needed for each okazaki fragment
therefore During elongation only need 1 gamma complex
Beta sliding clamp
2 protein subunits: homodimer
forms a protein ring around DNA
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there are 1 to 2 water molecules on DNA to facilitate movement
closed circular proteins
to lad onto DNA requires nrg
journal of cell, 92: 295-305, 1998
load beta sliding clamp onto DNA
1) ATP binds to gamma complex and exposes its delta subunit
(conformational change)
2) Delta subunit then binds beta clamp and opens beta ring
3) gamma complex recognizes the primer-template junction and brings beta
clamp to DNA
4) ATP hydrolysis to ADP + Pi = delta subunit buried in gamma complex is
released and then beta clamp and then snaps shut around DNA
lagging strand alpha subunit has to dissociate and reassociates from DNA,
doesn’t happen on leading strand
DNA unwinding
at replication fork dsDNA must come apart and has to be unwound
done by DNA B Helicase (product of gene B)
unwinds DNA at replication fork
is a hexamer, consisting of 6 subunits
circles DNA
requires nrg in form of ATP to ADP + pi to break apart H+ bonds at
replication fork
1 ATP hydrolyzed for each base pair unwound
closely associated with Pol 3 holoezymes
DNA B helicase alone, DNA unwinds at a speed of 35nucleotides/sec
DNA B helicase + tau subunit in Pol 3 that contacts DNA B helicase and
speeds up rate of unwinding to 1000nucleotides/sec
2 functions of tau 1)dimerize core to coordinate leading and lagging strand
synthesis 2)contact DNA B helicase to speed up unwinding at replication fork
SSB (single stranded binding protein)
Maintenace of ssDNA template
Can denature linear dsDNA
Has affinity for itself to other SSB will bind to it and line up next to eachother
some proteins keeps DNA single stranded
prevents 2 strands from reannealing together again
eliminates secondary structure in ssDNA template
might get deletion of DNA if has secondary structure because Pol 3 will go
past it
Formation of RNA primers
How RNA primers were discovered
ssDNA phage and looked at when ssDNA was making a dsRF (theta mode)
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then make ss progeny by rolling circle mode
viruses that infect e.coli: m13, G4, phiX174
M13 (ssDNA for template DNA)  RNA primer is synthesized by RNA
polymerase
G4 (ssDNA bacteriophage) uses DNA G primase (made by e.coli DNA G gene)
to synthesize RNA primer
Both cases need formation of hairpin loop in DNA
phiX174, instead of hairpin loop u need primosome (protein complex with 6
subunits)(1 subunit is DNA B helicase)
primosome activates DNA G primase to synthesize RNA primer
in e.coli primosome is simpler in structure (1 protein = DNA B helicase)
helicase acts as unwinding and primosome
RNA primers are short, 10 nucleotides long
Primase will activate DNA G primase when sees 3’—GTC—5’
Primase will put in 5’---pppAG-----3’
DNA G primase is in transient association with DNA B helicase
Replisome: Pol 3 holoenzyme + DNA B helicase (helicase and primosome) +
DNA G primase (RNA primers) + SSB
Pol 3 alpha subunit on leading strand moves continuously (has beta sliding
clamp attached to it)
On lagging strand DNA is pulled through DNA G primase and polymeraze =
formation of loop
DNA B helicase comes in contact with tau subunit of pol 3 to increase rate or
unwinding
DNA B helicase also contacts DNA G primase to initate okazaki fragment
DNA G primase is off/on (not a stable component of event)
DNA G primase also interacts with tau subunit, which leads to limiting the
length of RNA primers to 10 nucleotides
Tau has 3 functions 1)dimerizes core 2)contacts DNA B helicase 3)contacts
DNA G primase to limit length of primers
Tau is a central scaffolding element
Movement is 1000nucleotdies/sec
Okazaki fragment is 1000 to 2000 nucleotides long
Okazaki fragments
1) recruitment of DNA G primase by DNA B
2) synthesis of RNA primer
3)loading beta sliding clamp on DNA/RNA  done by gamma complex
4) transfer of lagging strand Pol 3 to new beta clamp
5)synthesis of okazaki fragment
6) repeat
Beta sliding clamps are in excess
2 proteins that are in transient with replisome, beta sliding clamp, and DNA g
primase
E.coli replication is bidirectional so there are 2 replisomes
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Replisome is only in one part of e.coli cell
DNA is moving not replisome
November 24, 11 – ligase, topoisomerase,
DNA A, telomeres
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DNA Ligase
blume syndrome  low amount of DNA ligase
bacteriophage + mammalian ligases – ATP
AMP-denyl group
Bacterial ligases
Fig. 14.25 genes 10
Bacterial ligases ; NAD
1)adenylation; AMP –PO4-LYS-enzyme
2)transadenylation AMP-PO4-PO4-DNA
3)ligation
stuff happening ahead of replication fork
Supercoiling
DNA is +ve supercoiled
needs to relieve +ve supercoiling
not enough –ve supercoiling to replicate entire genome
introduce ss nick to relieve +ve supercoiling
if replication fork catches up to nick before its sealed then one of the
branches will be lost
class of enzymes that create ss nicks
topoisomerase 1; make nick in the DNA and attached covalently through Tyr
to PO4 at nick
enzyme then rotates once around nick and then enzyme reseals nick and falls
off DNA
one supercoil is relieved per rxn cycle
relieves stress of supercoiling
in bacteria type 1 topoisomerase relieves –ve supercoiled DNA, can’t relieve
+ve supercoiling
in e.coli top 1 fcn is to relieve –ve supercoils
type 2 top (gyrase) makes –ve supercoiling
balance supercoiling
in eukaryotes top 1 relives both +ve and –ve supercoils
gyrase
DNA gyrase (type 2 topoisomerase) relieves +ve supercoiling in bacteria
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Gyrase makes a double strand cut and passes one circle to the other
Then releases ds cut
Resolves Intertwined circles (catenanes)
Process is called decatenation
DNA gyrase rxn cycle is called sign inversion model
1) gyrase binds to DNA and twists it to make a +ve node
a –ve node forms spontaneously elsewhere in DNA
-ve node relieves tension of +ve node
2) converts +ve node to a –ve node
to convert +ve no –ve makes a ds cut in the backstrand and pulls backstrand
to the front
have 2 negative nodes produces
experiment to see if gyrase is involved in DNA replication
in vitro there is nad (naladixic acid) inhibits DNA gyrase
if you grow e.coli cells in nad they stop relication
problem with experiment is do we know if nad is specific for DNA gyrase or
other enzymes in cell?
How do we show specificity for DNA gyrase?
Isolated e.coli mutants that are resistant to nad
Grow wild type in nad and look for mutant that grows in nad
Then take cells and break them apart and look at DNA gyrase
DNA gyrase was now resistant to nad
Mutation occurring in cells bc of mutation in DNA gyrase that made resistant
to nad
Molecular events that happens at OR
At start separate 2 strands to get ssDNA at replication eye
Then assemble machinery at origin (primase, helicase, polymerase (e.coli pol
3)
Then have something to remodel that so machinery can move away
4 steps
1) DNA A; initiator protein (e.coli makes), different than A protein of phiX174
product of DNA A gene
ori C (origin in e.coli) is AT rich (easy to open up),
ori c has 2 groups of repeating sequences (4 X 9 bp repeats and 3 X 13 bp
repeats)
245 bp long origin
first step is initiator binding to ori C and DNA melting
DNA A binds to 9bp repeats, have affinity for each other so they will align
together and contact 13bp repeats, then melt DNA at 13bp repeats
2) When DNA A protein is bound to the origin it recruits DNA helicase as a
complex with a loading factor DNA C, which is bound to ATP
This step is called recruitment of DNA B Helicase
In prokaryotes  initator binding  recruitment
DNA C has to be bound to ATP molecules
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3) remodeling; recruitment complex is stable and cant move away from
origin
ATP hydrolysis  ADP + Pi
Initially DNA C is bound to ATP and allowed to recruit complex
Hydrolysis allows DNA C to leave complex
4) then you get primase & polymerase loading
DNA B helicase recruits DNA G primase to make RNA primer at origin
DNA B helicase also recruits Pol 3 holoenzyme
Need initiator protein, loading/remodeling factors, DNA helicase
In yeast initiator protein is ORC
Termination of DNA replication
1) circular DNA
replication of circular DNA what happens at end you get catenation
catenation has to be resolved
the ezyme that does this is type 2 topo (e.coli DNA gyrase)
2)linear DNA
linear DNA molecules replicated by theta mode (RNA primers) have a
problem
adenovirus has no problem bc starts right at end
when you remove RNA primer you have a gap formed
can’t be filled in by Pol bc its in the wrong direction
something has to be done to maintain lagging strand
T7 bacteriophae  RNA removed at end but can’t add DNA, therefore DNA
molecules will get shorter
Experiment to see whats at the ends of chromosomes
telomeres
In tetrahymena there are 20-70 repeats of 5’-CCCCAA-3’ C-rich strand and
other strand 5’-TTGGGG-‘3’ G rich strand
These are called telomeres
Ends fold over to make G-G interactions to protect ends from DNA
exonucleases
Telomerase makes teleomere
Ends unfold and G rich strand acts as a primer for telomerase
C rich strand  primase makes RNA primer and Pol
Telomerase consists of protein and RNA
RNA had same sequence of C rich strand
Protein worked like reverse transcriptase
RNA acts as a template
RNA binds to G rich strand so uses RNA template for DNA synthesis
G rich  Elongation, Translocation, Elongation
C rich  Primer synthesis(primase)(pol), DNA replication, Primer removal
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function of telomeres are….
1) to maintain chromosome length following replication
2) protect chromosome ends from degradation
cells without telomerase after each replication chromosome ends get shorter
after about 60 cell cycles
problem with cancer cells  make a lot of telomerase
November 29, 2011 – retrovirus, gag, pol,
env, integrase, RNA virus
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Replication of retrovirus
ssRNA  dsDNA  integration in host genome ssRNA genomes
reverse transcriptase  integrase  provirus  RNA pol 2 
reverese transcriptase can use RNA/DNA as template
RNA pol 2 makes mRNA, structure at ends of retrovirus have 5’Cap (7-methyl
guanyic acid), 3’ Poly A tail
Necessary for translation of eukaryotic mRNA
Gag, po, and env  Each gene is translated made into polyprotein and
translated by retrovirus protease into individual protein
5’CAP-------start-----gag------stop------pol------stop------env----stop---3’AAAA
In eukarotic translation ribosome binds to 5’cap then goes to first AUG
(translation start)
End of gag, pol, and env there are stop codons
Have only 1 AUG
Problem retovirus have, they have stop after gag
When you stop at gag you get gag protein
How do we get pol protein? Pol protein makes reverse transcriptase and
integrase
Pol translation 2 ways depending on retrovirus
1) use a suppressor of the stop codon; glutanyl tRNA
suppressor tRNA is inserted at stop so read through to pol
done when gag and pol have the same reading frame
2)ribosomal frameshift
ribsome reading along gag, right before it reaches stop it changes its reading
frame  bypasses stop codon
pol and gag are read in different reading frames
both are 5% efficient, means that gag protein is 20X more abundant than pol
how is env translated?
There is a splicing mechanism that splices out from start (AUG) to beginning
of env
This gives rise to subgenomic RNA
Has start AUG and env next to that and then its stop codon
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Once these proteins are translated a protease cuts them up into individual
prtoins
Gag  ???????
Env  envelope protein (outer surface of retrovirus verion)
When researching AIDS  first went for reverse transcriptase
AIDS
Cant go for env proteins and can make vaccines for you to make antibodies
Retrovirus have low proofreading ability so during infection it mutates and
vaccine wont work
Then drug companies targeted protease
Protease needs to be dimeric to function properly  drugs that inhibit
dimerization
Other things that are required in RNA genome to ssDNA for dsDNA
intermediate
5’CAP-r-u5-p-------gag----------pol------------env----pu-u3-r-3’AAAA
a section at 5’ and 3’ called repeats (r)
next to r is 5’ untranslated region (u5)
next to 3’ r is u3 (3’ untranslated region)
next to u5 is p (primer to bind reverse transcriptase), primer similar to tRNA
for host strand synthesis
next to u3 there is pu (RNA primer used for second strand synthesis)
lengths vary depending on retrovirus
linear dsDNA intermediate is different from ssRNA
ends have reapeats called LTRs(long terminal repeats)
LTRs are u3/R/u5
u3/R/u5------------------------------------------- u3/R/u5  dsDNA
to make a promoter enchancer for pol 2 want to generate ssDNA
u3 has promoter and enhancer for RNA Pol 2
integration is random
has to make its own promoter/enhancer for pol 2
dsDNA products  linear dsDNA products with LTRs an circular DNA
products
the linear dsDNA integrates into host genome
circular are dead end
how retrovirus replicates
Fig 2.41
integration
Site specific integration after synthesis of dsDNA with integrase
Loss of a few bp at end of host virus and repeats at host cell DNA at both ends
Lose a few bp at both 5’ and 3’ ends of dsDNA when integrating into host
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Ie) loss of 2 bp at the ends of the virus and a repeat of 4 bp in host cell DNA
following integration
Integrase (made by retrovirus) chops off 2 nucleotides at 3’ end of viral DNA
Now it has recessed ends
In host cell integrase makes staggered cut of 4 bp (get 4 bp repeats)
Then 5’ of staggered cuts are joined to 3’ ends of viral DNA
Then 2 more nucleotides are lost and gaps are filled in
How to get 4 bp repeat
Gaps are filled in 5’ to 3’
Enzymes that do this integrate DNA into host DNA have repeats
Integrase can make 6bp staggered cut and repeat would be 6bp
enzymes can make 10bp staggered cut and repeat would be 10bp
once integrated RNA pol 2 binds to u3 and transcribes RNA
LTRs function…..
1) promter/enchancer for pol 2 and u
2)sed for integration (provide sequence for integration event to make
provirus)
RNA intermediates/ RNA viruses
Can occur in prokaryotic and eukaryotic
Can be ssDNA and dsDNA
They have an enzyme that synthesizes RNA on RNA template called RNA
dependant Replicase
RNA and DNA polymerization work the same way
Growing RNA chain - covalent linakage between 3’ and 5’ alpha phosphate
DNA dependant DNA polymerase (e.coli pol 1 and 3)
RNA dependant DNA polymerase (reverse transcriptase)
DNA dependant RNA polymerase (pol 2)
RNA dependant RNA polymerase (replicase)
ssRNA viruses
MS2, QB, R17  discovered in Switzerland in 1960s
plus strand RNA viruses, bc genome has correct sequence to be translated
into protein
minus strand is complement of plus strand and cannot make protein before
making intermediate RNA
December 1, 2011 – +/- virus, MS2,
reovirus, A, CP, REP, RBS, BOB, POP
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Plus strand RNA viruses  infect bacteria
Phage R17, MS2, QBeta
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Bacteriophage MS2
Can be uses as template and mRNA
5’---RBS----AUG----A----STOP-----RBS---AUG----CP---STOP---RBS—AUG—
REP---STOP----3’
Example how a phage regulates protein products both amounts and
temporally
RBS – ribosomal binding site;
10 bp upstream of the initiator AUG
purine rich
binds to 16S rRNA and 30S ribosomal subunit (rSU)
bacterial translation can get internal RBS in prokaryotes unlike eukaryotes
RBS are complimentary to 16S rRNA
A – attachment protein ; attaches to 1 RNA progeny genome and gets it
incorporated into phage head
Works at end of infection cycle (late in cycle)
1:1 ratio 1 A protein for each progeny genome
made late in infection bc binds to progeny plus strand and brings it into
empty phage head
plus stand and A protein goes into phage head
1 A protein attaches to 1 plus strand
10 000 A proteins made bc 10 000 genomes made at end of infectious cycle
full length RNA genome A RBS is blocked so made late
region upstream hybridizes to A RBS and blocks it
when synthesizing plus strand on the minus strand, the A protein is made
first and before a secondary structure forms an A protein is made
therefore every plus strand makes A protein
CP – coat protein
Makes up phage head
200 CP per phage head
makes lots of CP
RBS is always open for CP so 30S and 50S can bind
About 10 000 phage made at end of infection and need about 2X10^6 CP
made
Translation of CP gene disrupts secondary structure blocking REP RBS
Allows REP gene product to be made early in infection
Late in infection don’t want REP
Late in infection: CP has a weak affinity for binding to REP RBS
When REP increases in concentration will bind to REP RBS and block it and
stop translation of REP (at 500 molecules of REP) 30S won’t bind to it
REP – replicase (RNA dependent); synthesizes RNA
Start off replicating right away
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Made early in infection
Reusable enzyme so don’t have to make too much
Saves energy in making correct amounts
RBS of REP Initially closed bc of secondary structure in RNA genome
500 REP molecules made per infectious cycle
QBeta Replicase Complex
Rep protein- viral encoded
Below encoded by e.coli cell
EF-TS protein – elongation factor for translation (put tRNA on)
EF-TU protein - elongation factor for translation (put tRNA on)
S1 protein – protein in small ribosomal subunit (L1 means protein in large
ribosomal subunit)
Proteins keeps replicase on RNA template (processivity factors like Beta
sliding clamp for Pol 3)
Minus strand synthesis & plus strand synthesis
3’-------------+-------------5’ – can be replicated to make minus strand
does not require a primer
plus and minus strand joined together at REP (only a few nucleotides)  like
RNA transcription
this allows phage to make more minus strands before first minus strand is
completed
once minus strand made then plus strand can be made using minus strand as
a template
plus stand can be packaged or make more minus strand
Plus strand RNA virus that infects eukaryotes
Polio virus  RNA primer for REP protein
Minus strand viruses;
influenza virus; has a lot of RNA molecules that encode distinct genes
when infection template RNA cannot act as mRNA  must be made into plus
strand
plus strand made by REP, which is in the virion
Reovirus  dsRNA virus
Infects respiratory and GI of humans  harmless
Requires REP in virion
Plus strand is not available for translation
Virion dsRNA is not released from the virion as free RNA
10 RNA molecules per virion
each RNA molecule codes its own gene
infectious cycle
1)start with nucleoprotein core with 10 dsRNA molecules
encapsulates by protein shell
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2) once it gets into cell, shell is removed
REP is activated
3) plus strand synthesis coming out of core into cytoplasm
4) plus strand translated in cytoplasm to make core(transcription) & shell
proteins(translation)
5) core proteins bind to ssplus strands to make a precore(ss)
6) minus strand synthesis in precore
7) core is made and shell proteins are added to make mature virion
replication is conservative bc parental dsRNA is conserved in initial core
lambda phage recombination
in e.coli genome
lambda makes prophage in lysogenic cycle
in lytic cycle lambda comes out of e.coli DNA
site specific DNA recombination
circular lambda phage DNA gets inserted into linear bacteria DNA
recombination site; attB (attachment site bacteria)  BOB’ sequence
in lambda; attP (attachment site phage)  POP’ sequence
O is common between phage and bacteria
O is 15 nucleotides long
located btween galactose and Biotin
recombination through O site
then change attachment site
-----------BOP’ (attL)------Prophage---------POR’(attR)------attL = left
attR – right
change in sequence makes integration stable
to get back to circular phage need to make enzymes so it doen’t happen
immediately
Enzymes in lambda
integration
int – integrase – lambda int gene; makes cut at O site
2 ssnicks 7 bp apart
has endonuclease activity
has topoisomerase 1 activity  takes care of topological problems
seals nicks at end
IHF; integration host factor – made by e.coli gene
Required to make integrase bind to attB and attP
Excision
Int, IHF, and Excisionase –
Integration is stable because excisionase isn’t made
Excisionase make integrase recognize attL and attR