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Chromosomes:
Structure and
Function
Changes in Chromatin Structure
Polytene chromosomes are giant chromosomes found
in certain tissues of Drosophila and some other organisms.
These large, unusual chromosomes arise when repeated
rounds of DNA replication take place without accompanying
cell divisions, producing thousands of copies of DNA that lie
side by side.
Chromosomal puffs—localized swellings of the chromosome. Each puff
is a region of the chromatin that has relaxed its structure, assuming a
more open state. If radioactively labeled uridine is added to a Drosophila
larva, radioactivity accumulates in chromosomal puffs, indicating that
they are regions of active transcription.
Appearance of puffs at particular locations on the chromosome can be
stimulated by exposure to hormones and other compounds that are
known to induce the transcription of genes at those locations. This
correlation between the occurrence of transcription and the relaxation
of chromatin at a puff site indicates that chromatin structure undergoes
dynamic change associated with gene activity.
Chromosomal puffs in condensed Drosophila chromosome show
states of de-condensing in expressed regions
A several experiments indicating that chromatin structure changes with gene activity is
sensitivity to DNase I, an enzyme that digests DNA.
The ability of this enzyme to digest DNA depends on chromatin structure: when DNA is
tightly bound to histone proteins, it is less sensitive to DNase I, whereas unbound DNA is
more sensitive to digestion by DNase I.
The results of experiments that examine the effect of DNase I on specific genes show that
DNase sensitivity is correlated with gene activity.
 For example, globin genes code for hemoglobin in the erythroblasts (precursors of
red blood cells) of chickens. The forms of hemoglobin produced in chick embryos and
chickens are different and are encoded by different genes (Fig.a).
 No hemoglobin is synthesized in chick embryos in the first 24 hours after
fertilization. If DNase I is applied to chromatin from chick erythroblasts in this first 24hour period, all the globin genes are insensitive to digestion (Fig.b).
Changes in Chromatin Structure
 From day 2 to day 6 after fertilization, after hemoglobin synthesis has
begun, the globin genes become sensitive to DNase I, and the genes
that code for embryonic hemoglobin are the most sensitive (Fig c).
 After 14 days of development, embryonic hemoglobin is replaced by
the adult forms of hemoglobin. The most sensitive regions now lie
near the genes that produce the adult hemoglobins (Fig.d).
DNA from brain cells, which produce no hemoglobin, remains
insensitive to DNase digestion throughout development (Fig.e).
Method: Sensitivity to DNase I was
tested on different tissues and at
different times in development
DNase I sensitivity is correlated with the
transcription of globin genes in
erythroblasts of chick embryos.
The U gene codes for embryonic
hemoglobin;
the D and A genes code for adult
hemoglobin.
Changes in Chromatin Structure
In summary, when genes become transcriptionally active, they also become sensitive to
DNase I, indicating that the chromatin structure is more exposed during transcription.
What is the nature of the change in chromatin structure that produces chromosome
puffs and DNase I sensitivity?
In both cases, the chromatin relaxes; histones loosen their grip on the DNA. One
process that appears to be implicated in changing chromatin structure is acetylation, a
reaction that adds chemical groups called acetyls to the histone proteins.
Enzymes called acetyltransferases attach acetyl groups to lysine amino acids at one end
(called a tail) of the histone protein.
This modification reduces the positive charges that normally exist on lysine and
destabilizes the nucleosome structure, and so the histones hold the DNA less
tightly. Proteins taking part in transcription can then bind more easily to the DNA
and carry out transcription.
Histones are subject to modification which
affect gene regulation
• Exposed regions of
the histone are
subject to
modification
• These modification
influence the
structure of
chromatin and it
regulator properties
• Some modification
– Acetylation
– Methylation
– Phosphorylation
How are chromosome organized in
a nucleus
Staining pattern
results in different
color specific for
each chromosome
Chromosomes
occupy discrete
territories in the
cell nucleus
Chicken nuclei
Features of human chromosome
territories
specific
location of p
and q arms
and specific
territories
Transparent
view
Three
dimensional
view of
chromsome
territories
Same specific territories for the
active and inactive X
chromosome
Where are gene-rich and gene-poor regions of
chromosome located
Gene-poor chromosome located at nuclear periphery
Gene-rich chromosome located in nuclear interior
So generally
silent regions
of
chromosomes
are located at
the nuclear
periphery
Essential Functional elements of
Chromosomes
 A centromere
 A pair of telomeres
 Origins of replication
Chromosomes as functioning
organelles Centromere
A constricted region of the
chromosome
where
spindle
fibers attach and is essential
for proper movement of the
chromosome in mitosis and
meiosis .
The first centromeres to be isolated
from Yeast (small, linear chromosomes).
Chromosome
fragments
that lack a
centromere
are lost in
mitosis.
Chromosomes as functioning
organelles Centromere
Centromeric sequences
are the binding sites for
proteins that function as the kinetochore, a complex
that assembles on the centromere and to which the
spindle fibers attach.
Kinetochores play a central role in this process, by
controlling
assembly
and
disassembly
of
the
attached microtubules and, through the presence
of
motor
molecules,
chromosome movement.
by
ultimately
driving
Chromosomes as functioning organelles
Centromere
•
In simple eukaryotes, the sequences that specify centromere function
are very short.
– Yeast Saccharomyces cerevisiae the centromere element (CEN) is
about 110 bp long, comprising two highly conserved flanking
elements of 9 bp and 11 bp and a central AT-rich segment of about
80–90 bp.
Centromeres consist of particular sequences repeated many times. This nucleotide
sequence is found in the point centromere of Saccharomyces cerevisiae.
•
•
The centromeres of such cells are interchangeable - a CEN fragment
derived from one yeast chromosome can replace the centromere of
another with no apparent consequence.
In mammals, centromeres comprise hundreds of kilobases of repetitive
DNA, some nonspecific and some chromosome-specific.
Chromosomes as functioning organelles
Centromere
Variations in centromeric sequences
Diffuse Centromeres, spindle fibers attach along the entire length of the
chromosome.
Localized centromeres; spindle fibers attach at a specific place on the
chromosome. Appear constricted, but there also can be secondary
constrictions at places that do not have centromeric functions.
Classes of localized centromeres
Point centromeres
Smaller and more compact. DNA sequences are both necessary and
sufficient to specify centromere identity and function in organisms with
point centromeres)
• Budding yeast (Saccharomyces cerevisiae) encompasses 125 bp of
DNA.
Regional centromeres (large amounts of DNA and are often packaged into
heterochromatin).
Most of the centromere is made up of short sequences of DNA that are
repeated thousands of times in tandem. Within these repeats are “islands”
of more complex sequence, primarily transposable element sequences.
Chromosomes as functioning organelles Centromere
•
Centromeric DNA shows remarkable sequences heterogeneity.
•
Universally marked by the presence of a centromere-specific variant of
histone H3, generically known as CenH3 (the human form of CenH3 is
named CENP-A).
•
At centromeres, CenH3/CENP-A replaces the normal histone H3 and is
essential for attachment to spindle microtubules.
•
Depending on centromere organization different numbers of spindle
microtubules can be attached.
Chromosomes as functioning organelles: origins of replication
Origins of replication are the sites where DNA synthesis begins;
Eukaryotic chromosomes have multiple origin of replication.
Eukaryotic origins of replication; yeast, where the presence of a putative
replication origin can be tested by a genetic assay.
Bacterial replication origin in the plasmid does not function in yeast,
therefore the few plasmids that transform at high efficiency must possess
a sequence within the inserted yeast fragment that confers the ability to
replicate extrachromosomally at high efficiency - that is an autonomously
replicating sequence (ARS) element.
Chromosomes as functioning organelles: origins of replication
•
•
ARS elements are thought to derive from authentic origins of replication and,
in some cases, this has been confirmed by mapping a specific ARS element to
a specific chromosomal location and demonstrating that DNA replication is
indeed initiated at this location.
ARS elements extend for only about 50 bp and consist of an AT-rich region
which contains a conserved core consensus and some imperfect copies of this
sequence.
– ARS elements contain a binding site for a transcription factor and a
multiprotein complex is known to bind to the origin.
Chromosomes as functioning organelles: origins of replication
•
Mammalian replication origins are less well defined because of the
absence of a genetic assay.
•
There are speculations that replication can be initiated at multiple sites
over regions tens of Kb long.
•
Computer analysis of regions encompassing several eukaryotic origins of
replication, including some human and other mammalian examples,
identified
a
consensus
DNA
sequence
WAWTTDDWWWDHWGWHMAWTT where W = A or T; D = A or G or
T; H = A or C or T; and M = A or C
Chromosomes as functioning organelles
Telomeres
Telomeres are specialized structures, comprising DNA and protein,
which cap the ends of eukaryotic chromosomes.
Functions
Maintaining the structural integrity of a chromosome.
Ensuring complete replication of the extreme ends of chromosomes.
Helping establish the three-dimensional architecture of the nucleus
and/or chromosome pairing.
The ability of telomerase to replicate a chromosome end depends on the
unique molecular structure of the telomere.
Chromosomes as functioning organelles
Telomeres
• Eukaryotic telomeres consist of a long array of tandem repeats.
One DNA strand contains TG-rich sequences and terminates in
the 3′ end; the complementary strand is CA-rich.
• Highly conserved in evolution - there is considerable similarity in
the simple sequence repeat,
• Example
– TTGGGG (Paramecium), TAGGG (Trypanosoma) TTTAGGG
(Arabidopsis) and TTAGGG (Homo sapiens)
Chromosomes as functioning organelles
Telomeres

First isolated from the protozoan Tetrahymena thermophila and
possess multiple copies of the sequence:
Human Telomeres
• The (TTAGGG)" array of a human telomere spans about 10-15
kb.
• A very large protein complex shelterin, or the telosome
contains several components that recognize and bind to
telomeric DNA.
• Two telomere repeat binding factors (TRFl and TRF2) bind to
double-stranded TTAGGG sequences.
• G-rich strand has a Single-stranded overhang at its 3' end that
is typically 150-200 nucleotides long.
• This can fold back and form base pairs with the other, C-rich,
strand to form a telomeric loop known as theT-loop.
Protect the telomere DNA from natural
cellular mechanisms that repair doublestranded DNA breaks.
Chromosomes as functioning organelles
Telomeres
• Telomeres have now been
isolated from protozoans,
plants, humans, and other
organisms; most are
similar in structure.
Chromosomes as functioning organelles
Telomere Structure
The G-rich strand often protrudes beyond the complementary C-rich strand
at the end of the chromosome.
The length of the telomeric sequence varies from chromosome to
chromosome and from cell to cell, suggesting that each telomere is a dynamic
structure that actively grows and shrinks.
The telomeres of Drosophila chromosomes are different in structure.
They consist of multiple copies of the two different
retrotransposons , Het-A and Tart, arranged in tandem repeats.
Apparently, in Drosophila, loss of telomere sequences during
replication is balanced by transposition of additional copies of the
Het-A and Tart elements.
Cytogenetics

Structure and properties of chromosomes,

Chromosomal behaviour during mitosis and meiosis,

Chromosomal influence on the phenotype and the factors
that cause chromosomal changes.
Related to disease status caused by abnormal chromosome
number and/or structure.
Methods for chromosomal analysis:
Karyotyping and Banding
The collection of all the chromosomes is referred to as a
Karyotype.
The method used to analyze the chromosome constitution of an
individual, known as chromosome banding.
Chromosomes are displayed as a karyogram.
Obtaining and preparing cells for
chromosome analysis

Cell source:
–
Blood cells
–
Skin fibroblasts
–
Amniotic cells / chorionic villi
 Increasing the mitotic index
- proportion of cells in mitosis using colcemid

Synchronizing cells to analyze prometaphase
chromosomes
Key Procedure
In the case of peripheral (venous) blood

A sample is added to a small volume of nutrient medium containing
phytoheamagglutinin, which stimulates T lymphocytes to divide.

The cells are cultured under sterile conditions at 37C for about 3
days, during which they divide, and colchicine is then added to each
culture.

This drug has the extremely useful property of preventing formation
of the spindle, thereby arresting cell division during metaphase, the
time when the chromosomes are maximally condensed and therefore
most visible.

Hypotonic saline is then added, which causes the red blood cells to
lyze and results in spreading of the chromosomes, which are then
fixed , mounted on a slide and stained ready for analysis
PREPARATION OF CHROMOSOMES
Karyotype Analysis
Following Steps are involved;
 Counting the number
metaphase spread
of
cells,
sometimes
referred
as
 Analysis of the banding pattern of each individual chromosome
in selected cells.
 Total chr. Count is determined in 10-15 cells, but if mosaicism
is suspected then 30 or more cell count will be undertaken.
 Detailed analysis of the banding pattern of the individual
chromosomes is carried out in approx. 3-5 metaphase spread,
which shows high quality banding.
 The banding pattern of each chromosome is specific and shown
in the form of Idiogram.
MITOTIC CHROMOSOMAL SPREAD
Chromosome Banding
 Chromosome banding is developed based on the
presence of heterochromatin and euchromatin.
 Heterochromatin is darkly stained whereas
euchromatin is lightly stained during chromosome
staining.
Types of chromosome banding
 G-banding
 C-banding
 Q-banding
 R-banding
 T-banding
G-Banding
۩-
G-banding is obtained with Giemsa stain following digestion of
chromosomes with enzyme trypsin.
۩-۩-
It is a mixture of methylene blue and eosin.
It is specific for the phosphate groups of DNA and attaches
itself to regions of DNA where there are high amounts of
adenine-thymine bonding.
۩-
Yields a series of lightly and darkly stained bands – the dark
regions tend to be heterochromatic, late-replicating and AT
rich.
۩-
The light regions tend to be euchromatic, early-replicating
and GC rich .
G-Banding
G-banding of human female metaphase chromosomes
Q-Banding
-
Q-banding is a fluorescent pattern obtained using quinacrine for
staining. The pattern of bands is very similar to that seen in Gbanding.
- Chromosomes are stained with a fluorescent dye which binds
preferentially to AT-rich DNA, such as Quinacrine.
-
Quinacrine banding (Q-banding) was the first staining method
used to produce specific banding patterns for mammalian
chromosomes.
-
It is especially useful for distinguishing the Y chromosome.
Q-Banding
Q-banding of human male metaphase chromosomes
R-Banding

R-banding is the reverse of G-banding (the R stands for
"reverse").
The chromosomes are heat-denatured in saline before being
stained with Giemsa. The heat treatment denatures AT-rich DNA.

Dark regions are euchromatic (guanine-cytosine rich regions) and
the bright regions are heterochromatic (thymine-adenine rich
regions).

Telomeres are stained well by this procedure.
R-Banding
R-banding of human female metaphase chromosomes
T-Banding
Identifies a subset of the R bands which are especially
concentrated at the telomeres.
The T bands are the most intensely staining of the R bands and are
visualized by employing either a particularly severe heat treatment
of the chromosomes prior to staining with Giemsa, or a combination
of dyes and fluorochromes.
C-Banding
- C-banding stains the constitutive heterochromatin, which usually lies near
the centromere. The chromosomes are typically exposed to denaturation with a
saturated solution of barium hydroxide, prior to Giemsa staining.
Chromosomes of mouse
Chromosomes of human female
Molecular Cytogenetics
Molecular cytogenetics locates specific DNA sequences on
chromosomes
• Analysis of the gross structural organization of chromosomes
• Higher resolution analyses
• Target DNA Sequence
• Probe
– probes are often 15-50 nucleotides long and are chemically
synthesized.
High Resolution Karyotype
• Advantages
– “Whole genome scan”
– Relative low cost
• Disadvantages
– Labor intensive
– Detection above 5 Mb
Molecular Methods for chromosomal
analysis
Molecular Cytogenetics

Fluorescent in situ Hybridization (FISH)

Chromosome painting

Comparative Genomic Hybridization (CGH)

Molecular karyotyping and Multiplex
FISH(M-FISH)

Spectral Karyotyping

Array CGH
In situ hybridization
In situ hybridization, DNA probes can be used to determine the
chromosomal location of a gene or the cellular location of an
mRNA in a process called in situ hybridization.
The name is derived from the fact that DNA or RNA is visualized
while it is in the cell (in situ).
The maximum resolution of conventional FISH on metaphase
chromosomes is several megabases.
Prometaphase chromosomes can permit 1 Mb resolution.
Fluorescent IN SITU Hybridization (FISH)
•
A technology in which labeled nucleic acid sequence/ probes are used
for the visualization of specific DNA or RNA sequences on mitotic
chromosome preparations or in interphase cells.
 Fluorescently labeled DNA probes to detect or confirm gene or
chromosome abnormalities that are generally beyond the resolution of
routine Cytogenetics.
 The sample DNA (metaphase chromosomes or interphase nuclei) is first
denatured, a process that separates the complimentary strands within
the DNA double helix structure.
 The fluorescently labeled probe of interest is then added to the
denatured sample mixture and hybridizes with the sample DNA at the
target site as it reanneals (or reforms itself) back into a double helix.
 The probe signal can then be seen through a fluorescent microscope and
the sample DNA scored for the presence or absence of the signal.
Concept: A simple procedure for mapping genes and other DNA
sequences is to hybridize a suitable labeled DNA probe against
chromosomal DNA that has been denatured in situ.
Fluorescent in situ Hybridization
Originally, probes were radioactively labeled and detected with autoradiography, but
now many probes carry attached fluorescent dyes that can be seen directly with the
microscope (Fig.a).
Fluorescent probes are used to mark the locations
of specific gene sequences on chromosomes.
Different types of FISH
Probes
•
•
•
•
•
•
Centromeric Probes; consist of repetitive DNA sequences found in
and arround the centromere of a specific chromosomes.
Used for rapid diagnosis of trisomies 13, 18, 21.
Chomosomes specific unique sequence probes; specific for a
particular single locus. Locus specific probes for chromosomes
13q14 and the critical region for down syndrome on
chr.21(21q22.13-21q22.2), X and Y chromosomal abnormalities.
Telomeric probes; Complete set of telomeric probes for all 24
chromosomes, used for subtelomeric abnormalities (deletions,
translocations).
Whole chromosome paint probes; consist of a cocktail of probes
obtained from different parts of a particular chromosome, used for
ring chromosomes and translocations.
Different types of Fish Probes
•
Probes derived from flow- sorted chromosomes; Because of their
size and DNA composition, chromosomes bind different amount of
fluorescent dyes, some of which bind specifically to GC sequences
and others to AT sequences.
– This property of differential binding allows chromosomes to be separated
by the process of flow cytometry or fluorescent activated cell sorting
(FACS). This will stained the metaphase chromosomes with a florescent
DNA- binding dye and then projecting them across a laser beam which
excites the chromosome to fluoresce. This fluorescence intensity is
measured and analyzed by a computer that draws up a distribution
histogram of chromosomes size called as a flow karyotype.
Green signals indicate positive hybridization of a YAC from human 3q26.3 to metaphase chromosomes from a patient with
Cornelia syndrome with a balanced translocation (breakpoints at 3q26.3 and 17q23.1). Red signals indicate simultaneous
hybridization with a chromosome 17 centromere probe. The YAC spans the 3q26.3 breakpoint (green signals on one
normal chromosome 3 + the two translocation chromosomes). One translocation chromosome is small and carries a
chromosome 17 centromere (red signal); the other has a chromosome 3 centromere and is about the same size as the
normal chromosome 3.