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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