11/30/2015

Fundamental Genetics

Chromosome Structure
and DNA Sequence
Organization

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

John Donnie A. Ramos, Ph.D.

College of Science
University of Santo Tomas
Espaa, Manila, Philippines

Viral Chromosomes

Packaging of 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

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)

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

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)

E. coli DNA released by osmotic shock

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Endosymbiotic
Hypothesis

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

Molecular Organization of
Mitochondrial DNA (mtDNA)

Molecular Organization of
Chloroplast DNA (cpDNA)

Variable size in different organisms

Human
Mouse
Fruit Fly
Yeast
Pea
Mustard plant

16.6 Kb
16.2 Kb
18.4 Kb
75.0 Kb
110.0 Kb
367.0 Kb

Variable sedimentation coefficients of mtRNA

Absence of introns
Lesser repetitive sequences

Larger in size compared to mtDNA (120200 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-1-5biphosphate carboxylase)

Polytene Chromosomes

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

cpDNA from lettuce

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)

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)

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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

Chromatin Structure
DNA is associated with histones
(+charged proteins) and nonhistones (less +charged
proteins)
Packing of chromatins

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 )

Association of DNA and Histones

Nucleosome level (160 bp)

Solenoid level
Chromatin fiber loops level
Chromatid supercoiling level

Histones in Calf Thymus

Histone
H1

Basic / Acidic
Amino Acids
5.4

Molecular
Weight (D)
23,000

Total amino
acid residues
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)

Histone Dimers
8 histone molecules comprise a
nucleosome core
Organized into 4 heterodimers

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)

Two H2A-H2B dimers

Two H3-H4 dimers

Dimerization mediated by Cterminal 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

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 chromosomes
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:

CENP-A (H3A variant)

kinetochore assembly
H3.3 Transcription

Histone Code
State of activity of a gene is
under the influence of histone
proteins
Modifications of histones (Nterminal tail) affects gene activity
DNA properties affected by
histones:
Degree of compaction
Likelihood of transcription

Modifications:

Methylation (Arg and Lys)

Acetylation (Lys)
Phosphorylation (Ser)

Mammalian Y chromosome
Barr body

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

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

Variable Number
Tandem Repeats

Short
Interspersed
Elements

Long
Interspersed
Elements

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