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MOLECULAR GENETICS
CLASS SESSIONS:
1. DNA, Genes, Chromatin
2. DNA Replication, Mutation, Repair
3. RNA Structure and Transcription
4. Eukaryotic Transcriptional Regulation
5. CLASS DISCUSSION GENETIC DISEASES
6. RNA Processing
7. Protein Synthesis and the Genetic Code
8. Protein Synthesis and Protein Processing
9. CLASS DISCUSSION GENETIC DISEASES
10. DNA Cloning and Isolating Genes
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THE FLOW OF GENETIC INFORMATION
DNA RNA PROTEIN
DNA
1
2 3
1. REPLICATION (DNA SYNTHESIS)2. TRANSCRIPTION (RNA SYNTHESIS)
3. TRANSLATION (PROTEIN SYNTHESIS)
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DNA Structure and Chemistry
a). Evidence that DNA is the genetic informationi). DNA transformation know this term
ii). Transgenic experiments know this process
iii). Mutation alters phenotype be able to define
genotype and phenotype
b). Structure of DNAi). Structure of the bases, nucleosides, and nucleotides
ii). Structure of the DNA double helix
iii). Complementarity of the DNA strands
c). Chemistry of DNAi). Forces contributing to the stability of the double helix
ii). Denaturation of DNA
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Thymine (T)
Guanine (G) Cytosine (C)
Adenine (A)
Structures of the bases
Purines Pyrimidines
5-Methylcytosine (5mC)
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[structure of deoxyadenosine]
Nucleoside
Nucleotide
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Nomenclature
Purines
adenine adenosineguanine guanosine
hypoxanthine inosine
Pyrimidinesthymine thymidine
cytosine cytidine
+ribose
uracil uridine
Nucleoside NucleotideBase +deoxyribose +phosphate
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polynucleotide chain
3,5-phosphodiester bond
ii). Structure of the
DNA double helixStructure of the DNA
polynucleotide chain
5
3
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A-T base pair
G-C base pair
Chargaffs rule: The content of A equals the content of T,
and the content of G equals the content of C
in double-stranded DNA from any species
Hydrogen bonding of the bases
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Double-stranded DNA
Major groove
Minor groove
5 3
5 33 5
B DNA
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Chemistry of DNA
Forces affecting the stability of the DNA double helix hydrophobic interactions - stabilize
- hydrophobic inside and hydrophilic outside stacking interactions - stabilize
- relatively weak but additive van der Waals forces hydrogen bonding - stabilize
- relatively weak but additive and facilitates stacking electrostatic interactions - destabilize
- contributed primarily by the (negative) phosphates
- affect intrastrand and interstrand interactions- repulsion can be neutralized with positive charges
(e.g., positively charged Na+ ions or proteins)
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Stacking interactions
Charge repulsion
Char g
erepul s
ion
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Model of double-stranded DNA showing three base pairs
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Denaturation of DNA
Double-stranded DNA
A-T rich regions
denature first
Cooperative unwinding
of the DNA strands
Extremes in pH or
high temperature
Strand separation
and formation of
single-strandedrandom coils
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Electron micrograph of partially melted DNA
A-T rich regions melt first, followed by G-C rich regions
Double-stranded, G-C rich
DNA has not yet melted
A-T rich region of DNA
has melted into a
single-stranded bubble
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Hyperchromicity
The absorbance at 260 nm of a DNA solution increases
when the double helix is melted into single strands.
260
Abs
orbance
Absorbance maximum
for single-stranded DNA
Absorbance
maximum for
double-stranded DNA
220 300
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100
50
0
7050 90
Temperature oC
Percen
thy
perchro
micity
DNA melting curve
Tm is the temperature at the midpoint of the transition
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Average base composition (G-C content) can be
determined from the melting temperature of DNA
50
7060 80
Temperature oC
Tm is dependent on the G-C content of the DNA
Percen t
hyperchro
mic
ity
E. coli DNA is
50% G-C
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Genomic DNA, Genes, Chromatin
a). Complexity of chromosomal DNAi). DNA reassociation
ii).Repetitive DNA and Alu sequences
iii). Genome size and complexity of genomic DNA
b). Gene structure
i). Introns and exonsii). Properties of the human genome
iii). Mutations caused by Alu sequences
c). Chromosome structure - packaging of genomic DNA
i). Nucleosomes
ii). Histones
iii). Nucleofilament structure
iv). Telomeres, aging, and cancer
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DNA reassociation (renaturation)
Double-stranded DNA
Denatured,
single-stranded
DNA
Slower, rate-limiting,
second-order process of
finding complementary
sequences to nucleate
base-pairing
k2
Faster,
zippering
reaction to
form long
moleculesof double-
stranded
DNA
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Cot1/2
DNA reassociation kinetics for human genomic DNA
Cot1/2 = 1 /k2 k2 = second-order rate constantCo = DNA concentration (initial)
t1/2 = time for half reaction of each
component or fraction
50
100
0
%
DNA
reass
ociated
I I I I I I I I I
log Cot
fast (repeated)
intermediate
(repeated)
slow (single-copy)
Kinetic fractions:fast
intermediate
slowCot1/2
Cot1/2
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high k2
106 copies per genome of
a low complexity sequence
of e.g. 300 base pairs
1 copy per genome of
a high complexity sequence
of e.g. 300 x 106 base pairs
low k2
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Type of DNA % of Genome Features
Single-copy (unique) ~75% Includes most genes 1
Repetitive
Interspersed ~15% Interspersed throughout genome betweenand within genes; includes Alu sequences 2
and VNTRs or mini (micro) satellites
Satellite (tandem) ~10% Highly repeated, low complexity sequences
usually located in centromeres
and telomeres
2Alu sequences are
about 300 bp in length
and are repeated about
300,000 times in the
genome. They can be
found adjacent to or
within genes in introns
or nontranslated regions.
1
Some genes are repeated a few times to thousands-fold and thus would be inthe repetitive DNA fraction
50
100
0
I I I I I I I I I
fast ~10%
intermediate
~15%
slow (single-copy)
~75%
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Classes of repetitive DNA
Interspersed (dispersed) repeats (e.g., Alu sequences)
TTAGGGTTAGGGTTAGGGTTAGGG
Tandem repeats (e.g., microsatellites)
GCTGAGG GCTGAGGGCTGAGG
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viruses
plasmids
bacteria
fungi
plants
algae
insects
mollusks
reptiles
birds
mammals
Genome sizes in nucleotide pairs (base-pairs)
104 108105 106 107 10111010109
The size of the human
genome is ~ 3 X 109 bp;
almost all of its complexityis in single-copy DNA.
The human genome is thought
to contain ~30,000 to 40,000 genes.
bony fish
amphibians
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5 3
promoterregion
exons (filled and unfilled boxed regions)
introns (between exons)
transcribed region
translated region
mRNA structure
+1
Gene structure
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The (exon-intron-exon)n structure of various genes
-globin
HGPRT
(HPRT)
total = 1,660 bp; exons = 990 bp
histone
factor VIII
total = 400 bp; exon = 400 bp
total = 42,830 bp; exons = 1263 bp
total = ~186,000 bp; exons = ~9,000 bp
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Properties of the human genome
Nuclear genome
the haploid human genome has ~3 X 109 bp of DNA single-copy DNA comprises ~75% of the human genome the human genome contains ~30,000 to 40,000 genes
most genes are single-copy in the haploid genome genes are composed of from 1 to >75 exons genes vary in length from 2,300,000 bp Alu sequences are present throughout the genome
Mitochondrial genome
circular genome of ~17,000 bp contains
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Familial hypercholesterolemia autosomal dominant LDL receptor deficiency
Alu sequences can be mutagenic
From Nussbaum, R.L. et al. "Thompson & Thompson Genetics in Medicine," 6th edition (Revised Reprint), Saunders, 2004.
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LDL receptor gene
Alu repeats present within introns
Alu repeats in exons
4
4
4
5
5
5 6
6
6
Alu Alu
AluAlu
X
4 6
Alu
unequal
crossing over
one product has a
deleted exon 5(the other product is not shown)
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Chromatin structure
EM of chromatin shows presence of
nucleosomes as beads on a string
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Nucleosome structure
Nucleosome core (left) 146 bp DNA; 1 3/4 turns of DNA
DNA is negatively supercoiled two each: H2A, H2B, H3, H4 (histone octomer)
Nucleosome (right) ~200 bp DNA; 2 turns of DNA plus spacer
also includes H1 histone
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Histones (H1, H2A, H2B, H3, H4) small proteins arginine or lysine rich: positively charged
interact with negatively charged DNA can be extensively modified - modifications in
general make them less positively chargedPhosphorylation
Poly(ADP) ribosylation
MethylationAcetylation
Hypoacetylation
by histone deacetylase (facilitated by Rb)
tight nucleosomes
assoc with transcriptional repressionHyperacetylation
by histone acetylase (facilitated by TFs)
loose nucleosomes
assoc with transcriptional activation
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Nucleofilament structure
Condensation and decondensation
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Condensation and decondensation
of a chromosome in the cell cycle
Telomeres are protective
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Telomeres and aging
Metaphase chromosome
centromer
e
telomeretelomere
telomere structure
young
senescent
Telomeres are protective
caps on chromosome
ends consisting of short
5-8 bp tandemly repeated
GC-rich DNA sequences,
that prevent chromosomesfrom fusing and causing
karyotypic rearrangements.
(TTAGGG)many
(TTAGGG)few
telomerase (an enzyme) is required to maintain telomere length in
germline cells
most differentiated somatic cells have decreased levels of telomerase
and therefore their chromosomes shorten with each cell division
12 kb
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Class Assignment (for discussion on Sept 9th)
Botchkina GI, et al.
Noninvasive detection of prostate cancer byquantitative analysis of telomerase activity.
Clin Cancer Res. May 1;11(9):3243-3249, 2005
PDF of article is accessible on the website