Chromosomes structure and function

1. Chromosomes
: Structure
and Function
Dr.Kamlesh Shah

2. • Transmission
• Expression of genetic information.
• A centromere, which is most evident at metaphase, where it is
the narrowest part of the chromosome and the region at which
spindle fibers attach.
• Replication origins, certain DNA sequences along each
chromosome at which DNA replication can be initiated.
• Telomeres, the ends of linear chromosomes that have a
specialized structure to prevent internal DNA from being
degraded by nucleases.
Chromosomes

3. Appears as a distinct clump, the
nucleoid, which is confined to a
definite region of the cytoplasm.
If a bacterial cell is broken open
gently, its DNA spills out in a series
of twisted loops (Fig.a).
The ends of the loops are most
likely held in place by proteins
(Fig.b).
Many bacteria contain additional
DNA in the form of small circular
molecules called plasmids.
The Bacterial
Chromosome

4. The Eukaryotic Chromosome
Individual eukaryotic chromosomes contain enormous amounts of
DNA, and consists of a single, extremely long molecule of DNA.
The chromosomes are in an elongated, relatively uncondensed
state (less tightly packed than DNA in mitotic chromosomes)
during interphase of the cell cycle.

5. In the course of the cell cycle, the level of DNA packaging
changes— chromosomes progress from a highly packed state to
a state of extreme condensation.
DNA packaging also changes locally in replication and
transcription, when the two nucleotide strands must unwind so
that particular base sequences are exposed.
Thus, the packaging of eukaryotic DNA is not static but changes
regularly in response to cellular processes.
The Eukaryotic Chromosome

6. Packing DNA into Small Spaces
 Chromosome of the bacterium E. coli, a single molecule of DNA with
approximately 4.64 million base pairs. Stretched out straight, this DNA would be
about 1000 times as long as the cell within which it resides.
 Human cells contain 6 billion base pairs of DNA, which would measure some 1.8
meters stretched end to end.
 Even DNA in the smallest human chromosome would stretch 14,000 times the
length of the nucleus. DNA molecules must be tightly packed to fit into such
small spaces.

7. The structure of DNA can be considered at three hierarchical
levels:
the primary structure of DNA is its nucleotide sequence;
the secondary structure is the double stranded helix; and
the tertiary structure refers to higher order folding that
allows DNA to be packed into the confined space of a cell.
Chromosomal DNA is coiled
hierarchically

8. Most DNA found in cells is negatively supercoiled, which has two
advantages for the cell.
– First, supercoiling makes the separation of the two strands of DNA
easier during replication and transcription. Negatively supercoiled
DNA is underrotated; so separation of the two strands during
replication and transcription is more rapid and requires less energy.
– Second, supercoiled DNA can be packed into a smaller space
because it occupies less volume than relaxed DNA.
DNA Supercoils

9. Supercoiled DNA is
overwound or underwound,
causing it to twist on itself

10.  Supercoiling occurs
 only if the two polynucleotide strands of the DNA double helix are
unable to rotate about each other freely. If the chains can turn freely,
their ends will simply turn as extra rotations are added or removed, and the
molecule will spontaneously revert to the relaxed state.
 When the strain of over rotating or under rotating cannot be
compensated by the turning of the ends of the double helix, which is
the case if the DNA is circular— that is, there are no free ends. Some
viral chromosomes are in the form of simple circles and readily undergo
supercoiling.
 Eukaryotic DNA is normally linear but also tends to fold into loops stabilized by
proteins. In these chromosomes, the anchoring proteins prevent free rotation of
the ends of the DNA; so super coiling does take place.
Supercoiling; Tertiary Structures

11.  Topoisomerases, enzymes that add or remove rotations from the DNA
helix by temporarily breaking the nucleotide strands, rotating the ends
around each other, and then rejoining the broken ends.
 The two classes of topoisomerases are:
 Type I, which breaks only one of the nucleotide strands and reduces super
coiling by removing rotations.
 Type II, which adds or removes rotations by breaking both nucleotide
strands
DNA Supercoils: Enzymes

12. Chromatin Structure
Eukaryotic DNA is closely associated with proteins, creating chromatin. Two
basic types of chromatin are:
o Euchromatin, which undergoes the normal process of condensation and
decondensation in the cell cycle
o Heterochromatin, which remains in a highly condensed state throughout the
cell cycle, even during interphase.
Proteins in chromatin are the histones, which are relatively small, positively
charged proteins of five major types: H1, H2A, H2B, H3, and H4.
All histones have a high percentage of arginine and lysine, positively charged
amino acids that give them a net positive charge.
The positive charges attract the
negative charges on the phosphates
of DNA and holds the DNA in contact
with the histones.

13. Euchromatin
 Exist in extended state, dispersed through the nucleus and staining diffusely.
 Early-replicating and GC rich region.
 In prokaryotes, euchromatin is the only form of chromatin present.
 Genes may oy may not expressed
Heterochromatin
darkly stained two types
1. Constitutive ; always inactive and condensed.
Consist of repetitive DNA
Late replicating and AT rich region
Present at identical positions on all chromosomes in all cell types of an organism.
 Genes poorly expressed.
 Human chromosomes 1, 9, 16, and the Y chromosome contain large regions of
constitutive heterochromatin.
Occurs around the centromere and near telomeres.
2. Facultative;
Genetically active(decondensed) and inactive (condensed)
Variable in its expression. It varies with the cell type and may be manifested as
condensed, or heavily stained.

14. Non-histone chromosomal proteins make up about half of the protein mass of the
chromosome.
Non-histone chromosomal proteins serves structural roles and those that take
part in genetic processes such as transcription and replication.
Chromosomal scaffold proteins are revealed when chromatin is treated with a
concentrated salt solution, which removes histones and most other chromosomal
proteins, leaving a chromosomal protein “skeleton” to which the DNA is attached.
These scaffold proteins may play a role in the folding and
packing of the chromosome.
Other structural proteins make up the kinetochore, cap the
chromosome ends by attaching to telomeres, and constitute the
molecular motors that move chromosomes in mitosis and meiosis.
Chromatin Structure

15. • Other types of nonhistone chromosomal proteins play a role in genetic
processes.
– components of the replication machinery (DNA polymerases, helicases,
primases and proteins that carry out and regulate transcription (RNA
polymerases, transcription factors, acetylases).
• High-mobility-group proteins are small, highly charged proteins that vary
in amount and composition, depending on tissue type and stage of the cell
cycle.
– these proteins may play an important role in altering the packing of
chromatin during transcription.
Chromatin Structure

16. Nucleosome
Chromatin has a highly complex structure with several levels of organization.
The simplest level is the double helical structure of DNA . At a more complex level, the
DNA molecule is associated with proteins and is highly folded to produce a
chromosome.
When chromatin is isolated from the nucleus of a cell and viewed with an electron
microscope, it frequently looks like beads on a string (Fig.a), If a small amount of
nuclease is added to this structure, the enzyme cleaves the string between the beads,
leaving individual beads attached to about 200 bp of DNA (Fig.b).
If more nuclease is added, the enzyme chews up all of the DNA between the beads and
leaves a core of proteins attached to a fragment of DNA (Fig.c).
The repeating core of protein and DNA produced by digestion with nuclease enzymes is
the simplest level of chromatin structure, the Nucleosome.
The nucleosome is a core particle consisting of DNA wrapped about two times around
an octamer of eight histone proteins (two copies each of H2A, H2B, H3, and H4) (Fig.
d).
The DNA in direct contact with the histone octamer is between 145 and 147 bp in
length, coils around the histones in a left-handed direction, and is supercoiled. It does
not wrap around the octamer smoothly; there are four bends in its helical structure as
it winds around the histones.

18. The fifth type of histone, H1, is not a part of the core particle but plays an
important role in the nucleosome structure. The precise location of H1 with
respect to the core particle is still uncertain.
H1 sits outside the octamer and binds to the DNA where the DNA joins and
leaves the octamer. However, the results of recent experiments suggest that
the H1 histone sits inside the coils of the nucleosome.
H1 helps to lock the DNA into place, acting as a clamp around the nucleosome
octamer.
Together, the core particle and its associated H1 histone are called the
chromatosome, the next level of chromatin organization.
The H1 protein is attached to between 20 and 22 bp of DNA, and the
nucleosome encompasses an additional 145 to 147 bp of DNA; so about 167 bp
of DNA are held within the chromatosome.
Chromatosomes are located at regular intervals along the DNA molecule and
are separated from one another by linker DNA, which varies in size among cell
types—most cells have from about 30 bp to 40 bp of linker DNA.
Non-histone chromosomal proteins may be associated with this linker DNA, and
a few also appear to bind directly to the core particle.
Nucleosome

19. Higher-order chromatin
structure
In chromosomes, adjacent nucleosomes are not separated by space equal to the
length of the linker DNA; rather, nucleosomes fold on themselves to form a
dense, tightly packed structure.
This structure is revealed when nuclei are gently broken, open and their contents
are examined with the use of an electron microscope; much of the chromatin
that spills out appears as a fiber with a diameter of about 30 nm (Fig a).
The next-higher level of chromatin structure is a series of loops of 30-nm
fibers, each anchored at its base by proteins in the nuclear scaffold.
On average, each loop encompasses some 20,000 to 100,000 bp of DNA and is
about 300 nm in length, but the individual loops vary considerably.
The 300-nm fibers are packed and folded to produce a 250-nm-wide fiber. Tight
helical coiling of the 250-nm fiber, in turn, produces the structure that appears
in metaphase: an individual chromatid approximately 700 nm in width.

20. Adjacent nucleosomes pack together to form a 30-nm fiber.

21.  Nucleosomes associate with each other to form a more compact
structure termed the 30 nm fiber
 Histone H1 plays a role in this compaction
 At moderate salt concentrations, H1 is removed
 The result is the classic beads-on-a-string morphology
 At low salt concentrations, H1 remains bound
 Beads associate together into a more compact morphology
Nucleosomes Join to Form a 30 nm Fiber

22.  The 30 nm fiber shortens the total length of
DNA another seven-fold
 Its structure has proven difficult to determine
 The DNA conformation may be substantially altered
when extracted from living cells
 Two models :
 Solenoid model
 Three-dimensional zigzag model

23. Regular, spiral
configuration
containing six
nucleosomes per turn
Irregular
configuration where
nucleosomes have
little face-to-face
contact

24.  The two events we have discussed so far have shortened the DNA
about 50-fold
 A third level of compaction involves interaction between the 30 nm
fiber and the nuclear matrix
 The nuclear matrix is composed of two parts
 Nuclear lamina
 Internal matrix proteins
 10 nm fiber and associated proteins
Further Compaction of the
Chromosome

26.  The third mechanism of DNA compaction involves the
formation of radial loop domains
Matrix-attachment
regions
Scaffold-attachment
regions (SARs)
or
MARs are anchored
to the nuclear matrix,
thus creating radial
loops
25,000 to
200,000 bp

27.  The attachment of radial loops to the nuclear matrix is important in
two ways
1. It plays a role in gene regulation
2. It serves to organize the chromosomes within the nucleus
 Each chromosome in the nucleus is located in a discrete
and nonoverlapping chromosome territory
Further Compaction

29. Compaction level
in euchromatin
Compaction level
in heterochromatin
During interphase
most chromosomal
regions are
euchromatic

31. Chromatin has a highly complex structure with several levels of organization

32. Changes in Chromatin Structure
Eukaryotic DNA must be tightly packed to fit into the cell nucleus, it must also periodically
unwind to undergo replication and transcription. Evidence of the changing nature of
chromatin structure is seen in the puffs of polytene chromosomes and in the sensitivity of
genes to digestion by DNase I.
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.
When polytene chromosomes are stained with dyes, numerous bands are revealed.
Under certain conditions, the bands may exhibit 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.

33. 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 24-hour period, all the globin genes are insensitive to digestion (Fig.b).

34.  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).
Changes in Chromatin Structure

35. 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.
Method: Sensitivity to DNase I was
tested on different tissues and at
different times in development

36. 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.
Changes in Chromatin Structure

37. 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

38. How are chromosome organized in
a nucleus
Chromosomes
occupy discrete
territories in the
cell nucleus
Chicken nuclei
Staining pattern
results in different
color specific for
each chromosome

39. 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

40. Where are actively expressing
genes located
• The interchromatin
compartment (IC)
contains various types
of non-chromatin
domains with factors
for transcription,
splicing, DNA replication
and repair.
• This suggest active gene
expression in these
compartments
non-snRNP splicing factors
Overlay

41. Chromosome Morphology
Under the microscope chromosomes appear as thin, thread-like structures.
• They all have a short arm and long arm separated by a primary constriction called the
centromere. The short arm is designated as p and the long arm as q.
• The centromere is the location of spindle attachment and is an integral part of the
chromosome. It is essential for the normal movement and segregation of chromosomes during
cell division.
Human metaphase chromosomes can be categorized according to the length of the short and long
arms and also the centromere location.
• Metacentric chromosomes have short and long arms of roughly equal length with the
centromere in the middle.
• Submetacentric chromosomes have short and long arms of unequal length with the centromere
more towards one end.
• Acrocentric chromosomes have a centromere very near to one end and have very small short
arms. They frequently have secondary constrictions on the short arms that connect very small
pieces of DNA, called stalks and satellites, to the centromere.
• The stalks contain genes which code for ribosomal RNA.
• The diagrams showing region on chromosomes, called ideograms.

42. Metacentric
(Chromosome 1)
Submetacentric
(Chromosome 9)
Acrocentric
(Chromosome 14)

43. •The ideogram is basically a "chromosome map" showing the relationship between the
short and long arms, centromere (cen), and in the case of acrocentric chromosomes the
stalks (st) and satellites (sa). Each band is numbered to aid in describing
rearrangements.

44. Chromosome Nomenclature
 The International System for Human Cytogenetic Nomenclature (ISCN) is fixed by the Standing
Committee on Human Cytogenetic Nomenclature (Mitelman, 1995).
 Basic terminology for banded chromosomes was decided at a meeting in Paris in 1971, and is often
referred to as the Paris nomenclature.
 Short arm locations are labeled p (petit)
 Long arms are labelled q (queue).
 Each chromosome arm is divided into regions labeled p1, p2, p3 etc., and q1, q2, q3, etc., counting
outwards from the centromere.
 Regions are delimited by specific landmarks, which are consistent and distinct morphological features,
such as the ends of the chromosome arms, the centromere and certain bands.
 Regions are divided into bands labeled p11 (one-one, not eleven), p12, p13, etc.,
 sub-bands labeled p11.1, p11.2, etc., and
 sub-sub-bands e.g. p11.21, p11.22, etc., in each case counting outwards from the centromere.

45. Chromosome nomenclature
Figure. Different chromosome banding resolutions can resolve bands, sub-bands and sub-
sub-bands.
• (A) G-banded chromosome 1 at different banding resolutions.
• (B) Numbering of bands, sub-bands, and sub-sub-bands.
 The centromere is designated ‘cen' and the telomere ‘ter'.
 Proximal Xq means the segment of the long arm of the X that is closest to the
centromere, while distal 2p means the portion of the short arm of chromosome 2
that is most distant from the centromere, and therefore closest to the telomere.

46. Chromosomes are identified by their size, centromere position and
banding pattern
Autosomes are numbered from largest to smallest, except that chromosome 21 is smaller
than chromosome 22.
Group Chromosomes Description
A 1–3 Largest; 1 and 3 are metacentric but 2 is submetacentric
B 4,5 Large; submetacentric with two arms very different in size
C 6–12,X Medium size; submetacentric
D 13–15 Medium size; acrocentric with satellites
E 16–18 Small; 16 is metacentric but 17 and 18 are submetacentric
F 19,20 Small; metacentric
G 21,22,Y Small; acrocentric, with satellites on 21 and 22 but not on
the Y