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Chromosome Structure

and DNA Sequence

Organization

John Donnie A. Ramos, Ph.D. College of Science University of Santo Tomas

España, Manila, Philippines

Fundamental Genetics Its all in the

“Packaging”

Each cell of Homo sapiens contains…

3 billion DNA bases

2 meters of DNA

25-30 thousand genes

46 chromosomes

Viral Chromosomes Single stranded or double stranded DNA or RNA

Circular or linear structures

Examples:

(Phi) X174 bacteriophage – single stranded circular DNA

Polyoma virus – double stranded circular DNA

Lambda phage – double stranded linear DNA (before infection) but forms a loop upon infection

T-even bacteriophages – double stranded linear DNA (before and after infection)

HIV – single stranded linear RNA

Phage lambda and its DNA

Packaging of Viral Chromosomes

Ability to package an exceedingly long nucleic acid into a relatively small volume.

Achieved by tremendous coiling and supercoiling mechanisms

Space available on viral head rarely exceeds the chromosome volume by a factor of 2

Genetic material is functionally inert once packaged inside the viral head T2 Phage DNA released by osmotic shock

Bacterial Chromosomes Double stranded DNA arranged into a nucleoid

Chromosomes are packaged using DNA-binding proteins

DNA-binding proteins contain high amounts of positively charged amino acids

Bacterial chromosomes are not functionally inert (can be replicated and transcribed)

E. coli DNA released by osmotic shock

Mitochondrial and Chloroplast DNA

Double stranded circular DNA

Highly conserved DNA sequences

Believed to have originated from prokaryotic symbionts

(Endosymbiotic Hypothesis)

First identified in mutant yeasts (Petites – abnormal

mitochondria thus defective in cellular metabolism)

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Endosymbiotic

Hypothesis

Circular DNA without chromosomal proteins

Double-membrane organelles

With own ribosomes

Ability to duplicate on its own (binary fission)

Undergo: Replication

Transcription

Translation

Endosymbiotic Hypothesis

Molecular Organization of

Mitochondrial DNA (mtDNA) Variable size in different organisms

Human - 16.6 Kb Mouse - 16.2 Kb Fruit Fly - 18.4 Kb Yeast - 75.0 Kb Pea - 110.0 Kb Mustard plant - 367.0 Kb

Variable sedimentation coefficients of mtRNA

Absence of introns

Lesser repetitive sequences

Molecular Organization of

Chloroplast DNA (cpDNA)

Larger in size compared to mtDNA (120-

200 Kb)

Double stranded circular DNA

Replicate semiconservatively

Free of associated chromosomal proteins

With genes coding for ribosomal RNAs (5S,

16S and 23S rRNA)

Example of cpDNA gene product: Large subunit of RuBP (ribulose-1-5-

biphosphate carboxylase) cpDNA from lettuce

Eukaryotic Chromosomes: Clues from YACs

Key Components of S. cerevisiae chromosomes: Centromere (CEN) – 100-1000 bp

Telomere (TEL)

Origin of Replication (ORI)

YACs with ORI but no CEN and TEL do not segregate properly

YACs with ORI and CEN but no TEL replicate and segregate fairly well when in circular form but undergoes fragmentation in linear form

Size of chromosome is important for proper function YACS with 11 kb insert = 50% segregation errors

YACS with 55 kb insert = 1.5% segregation errors

YACS with up to 100 kb insert = 0.3% segregation errors

Minimum length of YAC for normal function (100-150 kb)

Polytene Chromosomes

Giant chromosomes found in the salivary gland cells of the larvae of fruit flies

First identified by E.G. Balbiani in 1881

Can be identified in the nuclei of interphase cells using compound microscope

Linear series of alternating bands and interbands (chromomeres)

200-600 m long (each polytene chromosome)

They represent paired homologous chromosomes (in somatic cells)

Result of several rounds of replication without strand separation and cytokinesis

Presence of ‘puff” regions – indicating gene activity (transcription)

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Lampbrush Chromosomes

First discovered in1892 in shark oocytes

Meiotic chromosomes

Extended, uncoiled versions of normal meiotic chromosomes

Size ranging from 500-800 m

Contain condensed areas called chromomeres with lateral loops (lampbrush)

Lateral loops contain one DNA double helix while the main axis is composed of two DNA helices

Lateral loops are active in RNA synthesis

Chromatin Organization in Eukaryotes Each human chromosome ranges from 19,000-73,000 m in length

46 chromosomes in human extends 2 meters

Contained in a nucleus with 5-10 m diameter

Undergo supercoiling mechanism – folded-fiber model (10,000x contraction in length)

Chromatin fibers are composed of spherical particles called nucleosomes

The Human Genome

Haploid genome has 3.2 X 109 bp of DNA

Length of DNA: almost 2 m

25 X 106 nucleosomes per nucleus are complexed with 2 m of DNA

Average nucleus size: 5-10 m diameter

Packing ratio of 500:1 ( DNA length:length of structure containing it )

Chromatin Structure DNA is associated with histones

(+charged proteins) and non-histones (less +charged proteins)

Packing of chromatins Nucleosome level (160 bp)

Solenoid level

Chromatin fiber loops level

Chromatid supercoiling level

Association of DNA and Histones Histones in Calf Thymus

Histone Basic / Acidic

Amino Acids

Molecular

Weight (D)

Total amino

acid residues

H1 5.4 23,000 224

H2A 1.4 13,960 129

H2B 1.7 13,774 125

H3 1.8 15,273 135

H4 2.5 11,236 102

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Histones High content of basic amino

acids (Arginine and Lysine)

Highly conserved

pea plant and cow cells

histones = 102 amino acids

with only 2 amino acid

difference

Reasons:

interaction with similar DNA

backbones (in all organisms)

interaction with DNA or other

histones (conserved

structural function)

Histone Dimers 8 histone molecules comprise a

nucleosome core

Organized into 4 heterodimers

Two H2A-H2B dimers

Two H3-H4 dimers

Dimerization mediated by C-

terminal domains (alpha helices)

N-terminal segment of each

histone forms a long flexible tail

that extends pass the DNA

Histone modifications:

H2AX – DNA repair

H2AZ – transcription

macroH2A – X-chromosome

inactivation

CENP-A (H3A variant) –

kinetochore assembly

H3.3 Transcription

Heterochromatin Parts of chromosomes that remain condensed (in

contrast to euchromatin=uncoiled)

Found in centromeres, telomeres, and sometimes the whole chromosomes

Genetically inactive

Either lack genes or contain repressed genes

Replicates later during the S phase

Involvement in maintenance of chromosome’s structural integrity (ex. chromosome movement during cell division)

Position effect: the position of a gene or group of genes relative to all other genetic material may affect their expression (ex. translocation of heterochromatin)

Examples:

Mammalian Y chromosome

Barr body

Histone Code State of activity of a gene is

under the influence of histone proteins

Modifications of histones (N-terminal tail) affects gene activity

DNA properties affected by histones:

Degree of compaction

Likelihood of transcription

Modifications: Methylation (Arg and Lys)

Acetylation (Lys)

Phosphorylation (Ser)

Heterochromatin Formation

Methylation of Lysine 9 (K9) on H3

histone results to heterochromatin

formation

Catalyzed by histone

methyltransferase (SUV39H1)

Binding of methylated histones to

heterochromatic protein 1 (HP1)

Promotes the formation of

interconnected network of

methylated nucleosomes.

Leads to heterochromatin

formation

Non-Histone Proteins

Heterogenous group of proteins involved in wide a range of

functions

Uneven distribution (different amounts and different proportions)

Functions:

Structural role (chromosome scaffolds)

chromosome movement (CENP-E in kinetochore)

seprartion of sister chromatids

Replication (polymerase and other replication factors)

Transciption (transcription factors)

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Eukaryotic DNA is repetitive in nature

Variable Number

Tandem Repeats

Short

Interspersed

Elements

Long

Interspersed

Elements

Highly Repetitive DNA Centromeric and telomeric DNA sequences

5% of the human genome (10% in mouse genome)

Called CEN gene (250 bp) in C. elegans Region I (8 bp) – highly conserved

Region II (80-85 bp) – extremely AT rich

Region III (26 bp) – highly conserved (mutations critical to function)

Binding region of kinetocore

Repetitive Genes in Humans

Alphoid gene family

Highly repetitive satellite DNA sequences

Located in centromere regions

About 170 bp each in length

Repeated in tandem arrays up to 1 million base pairs

Telomeric DNA Sequences

short tandem repeats

contributes to integrity and stability of chromosomes

GGGATT (highly conserved)

Telomere-associated sequences

adjacent to and within telomeres

Vary in different organisms

Middle Repetitive DNA Variable Number Tandem Repeats (VNTRs)

15-100 bp long of no known function

Minisatellites (1-5 Kb in length) – molecular markers for DNA fingerprinting

Microsatellites (5-50 repeats of CA sequence)

Short Interspersed Elements (SINEs) Less than 500 bp

Present 500,000x in the human genome

Example: Alu family (5% of the human genome) – presence of AluI RE site

Produced via reverse transcription

Long Interspersed Elements (LINEs) 6400 bp long

Present 100,000x in human genome

Produced via reverse transcription (retro-transposons) – encoded by L1 gene

What proportion of the genome

encodes proteins?

Human genome = ~5%

Sea urchin genome = less than 10%

Drosophila genome = 5-10%

Why?

Non-coding Repetitive sequences

Non-coding single copy sequences

Psuedogenes

Mutated genes