GEN01

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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
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3
DNA
RNA
PROTEIN
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DNA
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
information i). 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 DNA i). Structure of the bases,
nucleosides, and nucleotides ii). Structure of
the DNA double helix iii). Complementarity of
the DNA strands c). Chemistry of DNA i).
Forces contributing to the stability of the
double helix ii). Denaturation of DNA
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Structures of the bases
Purines
Pyrimidines
Thymine (T)
Adenine (A)
5-Methylcytosine (5mC)
Guanine (G)
Cytosine (C)
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Nucleoside
structure of deoxyadenosine
Nucleotide
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Nomenclature
Nucleoside Nucleotide Base
deoxyribose phosphate
Purines adenine adenosine guanine guanosine
hypoxanthine inosine Pyrimidines thymine thy
midine cytosine cytidine ribose
uracil uridine
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ii). Structure of the DNA double helix
Structure of the DNA polynucleotide chain
5
3

• polynucleotide chain
• 3,5-phosphodiester bond

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A-T base pair
Hydrogen bonding of the bases
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
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Double-stranded DNA
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3
Major groove
Minor groove
B DNA
5
3
3
5
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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
Charge repulsion
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Model of double-stranded DNA showing three base
pairs
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Denaturation of DNA
Strand separation and formation
of single-stranded random coils
Double-stranded DNA
A-T rich regions denature first
Extremes in pH or high temperature
Cooperative unwinding of the DNA
strands
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Electron micrograph of partially melted DNA
Double-stranded, G-C rich DNA has not yet
melted
A-T rich region of DNA has melted into
a single-stranded bubble

• A-T rich regions melt first, followed by G-C
  rich regions

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Hyperchromicity
Absorbance maximum for single-stranded DNA
Absorbance maximum for double-stranded DNA
Absorbance
260
220
300
The absorbance at 260 nm of a DNA solution
increases when the double helix is melted
into single strands.
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Tm is dependent on the G-C content of the DNA
E. coli DNA is 50 G-C
Percent hyperchromicity
50
70
60
80
Temperature oC
Average base composition (G-C content) can
be determined from the melting temperature of DNA
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Genomic DNA, Genes, Chromatin
a). Complexity of chromosomal DNA i). DNA
reassociation ii). Repetitive DNA and Alu
sequences iii). Genome size and complexity of
genomic DNA b). Gene structure i). Introns and
exons ii). 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
Faster, zippering reaction to form
long molecules of double- stranded DNA
k2
Slower, rate-limiting, second-order process
of finding complementary sequences to
nucleate base-pairing
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DNA reassociation kinetics for human genomic DNA
Cot1/2 1 / k2 k2 second-order rate
constant Co DNA concentration
(initial) t1/2 time for half reaction
of each component or fraction
Kinetic fractions fast intermediate slow
0
fast (repeated)
intermediate (repeated)
Cot1/2
DNA reassociated
50
Cot1/2
slow (single-copy)
Cot1/2
100
I I I I I I I
I I
log Cot
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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
high k2
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 between
and 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 2 Alu 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 in the
repetitive DNA fraction
0
fast 10
intermediate 15
50
slow (single-copy) 75
100
I I I I I I I
I I
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Classes of repetitive DNA
Interspersed (dispersed) repeats (e.g., Alu
sequences)
GCTGAGG
GCTGAGG
GCTGAGG
Tandem repeats (e.g., microsatellites)
TTAGGGTTAGGGTTAGGGTTAGGG
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Genome sizes in nucleotide pairs (base-pairs)
plasmids
viruses
bacteria
fungi
plants
algae
insects
mollusks
bony fish
The size of the human genome is 3 X 109
bp almost all of its complexity is in
single-copy DNA. The human genome is thought to
contain 30,000 to 40,000 genes.
amphibians
reptiles
birds
mammals
104
108
105
106
107
1011
1010
109
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Gene structure
promoter region
exons (filled and unfilled boxed regions)
1
introns (between exons)
transcribed region
mRNA structure
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3
translated region
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The (exon-intron-exon)n structure of various genes
histone
total 400 bp exon 400 bp
b-globin
total 1,660 bp exons 990 bp
HGPRT (HPRT)
total 42,830 bp exons 1263 bp
factor VIII
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 gt75 exons
• genes vary in length from lt100 to gt2,300,000 bp
• Alu sequences are present throughout the genome
• Mitochondrial genome
• circular genome of 17,000 bp
• contains lt40 genes

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Alu sequences can be mutagenic

• Familial hypercholesterolemia
• autosomal dominant
• LDL receptor deficiency

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
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5
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Alu repeats in exons
unequal crossing over
4
5
6
Alu
Alu
X
Alu
Alu
4
5
6
one product has a deleted exon
5 (the other product is not shown)
Alu
4
6
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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 charged
• Phosphorylation
• Poly(ADP) ribosylation
• Methylation
• Acetylation
• Hypoacetylation
• by histone deacetylase (facilitated by Rb)
• tight nucleosomes
• assoc with transcriptional repression
• Hyperacetylation
• by histone acetylase (facilitated by TFs)
• loose nucleosomes
• assoc with transcriptional activation

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Nucleofilament structure
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Condensation and decondensation of a chromosome
in the cell cycle
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Telomeres are protective caps on
chromosome ends consisting of short 5-8 bp
tandemly repeated GC-rich DNA sequences, that
prevent chromosomes from fusing and
causing karyotypic rearrangements.
Telomeres and aging
Metaphase chromosome
lt1 to gt12 kb
telomere structure
(TTAGGG)many
young
(TTAGGG)few
senescent

• 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

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Class Assignment (for discussion on Sept
9th) Botchkina GI, et al. Noninvasive detection
of prostate cancer by quantitative analysis of
telomerase activity. Clin Cancer Res. May
111(9)3243-3249, 2005 PDF of article is
accessible on the website