genomic dna 2

8/13/2019 Genomic DNA 2

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2. 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 repetitive sequences

c). Chromosome structure - packaging of genomic DNA

i). Nucleosomes

ii). Histones

iii). Nucleofilament structure and chromosome

condensation

iv). Telomeres and aging


2/23

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

molecules

of double-

stranded

DNA

a). Complexity of chromosomal DNA


3/23

DNA reassociation kinetics (for a single DNA species)

Cot1/2= 1/k2 k2 = second-order rate constant

Co= DNA concentrationt1/2= time for half reaction

Cot1/2

50

100

0

%D

NAreassociated

log Cot

Ideal second-order DNA reassociation curve (Cot curve)


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10-5 10010-4 10-3 10-1 10410310210-6 10-2 101

101 106102 103 105 1010109108100 104 107Complexity expressed as base-pairs (bp)

Cot

1 2 3 4 5

1 = poly(dT)-poly(dA)

2 = purified human satellite DNA

3 = T4 bacteriophage DNA

4 = E. coli genomic DNA

5 = purified human single-copy DNA

Cot1/2

There is a direct

relationship between

Cot1/2and complexity


5/23

Cot1/2

DNA reassociation kinetics for a mixture of DNA species

Cot1/2= 1/k2 k2 = second-order rate constantCo= DNA concentrationt1/2= time for half reaction

50

100

0

%D

NAreassociated

I I I I I I I I I

log Cot

fast (repeated)

intermediate

(repeated)

slow (single-copy)

human genomic DNA

Kinetic fractions:

fastintermediate

slowCot1/2

Cot1/2


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k2>>>>>>>>>> k2

110,000


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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 centromeresand telomeres

2Alu sequences are

about 300 bp in length

and are repeated about

300,000 times in thegenome. 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%


8/23

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 109bp;

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


10/23

5 3

promoterregion

exons (filled and unfilled boxed regions)

introns (between exons)

transcribed region

translated region

mRNA structure

+1

b). Gene structure


11/23

(exon-intron-exon)nstructure 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


12/23

Properties of the human genome

Nuclear

the haploid human genome has ~3 X 109bp 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 genomegenes 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

circular genome of ~17,000 bp

contains


13/23

Familial hypercholesterolemia

autosomal dominant

LDL receptor deficiency


14/23

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)


15/23

EM of protein-depleted human metaphase chromosome

There are 23 pairs of chromosomes in diploid human cells.

The average metaphase chromosome has ~1.3 X 108bp DNA.


16/23


17/23

Nucleosome structure

Nucleosome core (left)

146 bp DNA; 1 3/4 turns of DNA

DNA is negatively supercoiledtwo each: H2A, H2B, H3, H4 (histone octomer)

Nucleosome (right)

~200 bp DNA; 2 turns of DNA plus spacer

also includes H1 histone


18/23


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Hi t (H1 H2A H2B H3 H4)


20/23

Histones (H1, H2A, H2B, H3, H4)

small proteins

arginine or lysine rich: positively charged

interact with negatively charged DNAcan be extensively modified - modifications in

general make them less positively chargedphosphorylation

poly(ADP) ribosylation

methylationacetylation

hypoacetylation

by histone deacetylase (facilitated by Rb)

tight nucleosomesassoc with transcriptional repression

hyperacetylation

by histone acetylase (facilitated by TFs)

loose nucleosomes

assoc with transcriptional activation


21/23

Nucleofilament structure

C d i d d d i


22/23

Condensation and decondensation

of a chromosome in the cell cycle

T l t ti


23/23

Telomeres and aging

Metaphase chromosome

centromere telomeretelomere

telomere structureyoung

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

genomic dna 2

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