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HUMAN CHROMOSOMES
Normal human somatic cells contain a diploid number of chromosomes (2n=46), so there are 23
pairs of chromosomes:
- 22 pairs are identical in man and women and are called autosomes;
- 1 pair of sex chromosomes: XY in men and XX women.
The maternally and paternally derived chromosomes present in a diploid cell that contain
equivalent genetic information, are similar in morphology, and pair during meiosis are called homologous
chromosomes.
The mature sexual sells – the gametes – contain a haploid number of
chromosomes (n=23); the sperm cells contain 22+X (23,X) or 22+Y (23,Y); the
egg cells contain 22+X (23,X).
A display of the metaphase chromosomes of a somatic cell of an
individual, based on their number, shape, size and other landmarks (secondary
constriction, satellites, bands) is called karyotype. The karyotype is normally
shown as a photomicrograph of the chromosomes arranged in a standard way.
Photographs of chromosome pairs are aligned to provide a visual representation of
the organism's chromosomal constitution. The process of preparing a
photomicrograph is known as karyotyping. Individual chromosomes are
identified by chromosome banding.
A typical metaphase chromosome consists of two sister chromatids
connected by centromere (primary constriction) - the point where the two
chromatids remain attached, but also containing the kinetochore, the point of
spindle attachment. Each chromatid contains two arms: short (p) and long (q),
separated by primary constriction.
The ends of chromatids are called telomeres, which contain repetitions of TTAGGG sequence.
They prevent the fusion of chromosomes, protect the DNA from the action of exonucleases and are
involved in full replication of DNA (through the activity of telomerase). Their biological role is thus
providing the stability and maintaining the integrity of chromosomes.
Some chromosomes contain less condensed and less stained fragments called secondary
constriction.
There are some morphological criteria based on chromosome’s size and configuration used for
identification of chromosomes:
- The chromosomal length. Usually the absolute length (in micrometers) or relative length is
used. The relative length is calculated using the formula:
The lenght of studied chromosome
X 100.
The total length of all chromosome s from a haploid cell
The chromosomes may be large, medium and small. Another classification criterion is based on
the position of centromere, which is characterized by centromere index (arm length ratio), calculated
p
through the formula: Ic 
X 100.
pq
Using this criterion, human chromosomes are divided in: metacentric, submetacentric, and
acrocentric.
Human Chromosomes
metacentric:
Ic = 46 – 49 %
submetacentric:
Ic = 31 – 45 %
acrocentric:
Ic = 17 – 30 %
Methods of Chromosome Analysis
Chromosomes could be analyzed through cytogenetic and molecular cytogenetic techniques.
Chromosomes can best be studied at metaphase, when chromosome condensation is maximal and
all chromosomes are in one plane. To visualize metaphase chromosomes, they must first be fixed on a
slide, and then stained, either uniformly with a dye such as orcein, or for more resolution and detail, with
banding techniques after gentle denaturing treatments. The chromosomes from actively dividing tissues
may be examined directly (marrow cells, tumors). The other cells should be cultured before studying.
There are two types of cell cultures, depending on examined tissues:
- Short time cultures – for blood cells;
- Long time culture – for skin, amniotic or solid tumor cells.
The steps of blood cell (lymphocytes) culture:
Collecting of blood in aseptic condition;
Culturing in sterile vials containing special media at 370C, during 72 hours. For mitosis induction a
special mitogen – phytohaemagglutinin – is added;
Blocking of mitosis at metaphase using colchicines or colcemid;
Adding of hypotonic solution, which disperses the chromosomes in the cells;
Adding of fixative (3:1, alcohol:acetic acid), which kills the cells and keeps the natural shape of
chromosomes;
Spreading of cells on a slide;
Staining, using an appropriate dye;
Examination, using a microscope;
Photographs of chromosome pairs are aligned to provide a visual representation of the chromosomal
constitution.
Uniform Staining
The chromosomes are stained without special treatment at the metaphase stage. Usually Giemsa
dye or orcein are used for staining. This method provides information only about the number and
morphology of chromosomes. The chromosomes could be grouped on the basis of their relative sizes
and the relative lengths of their two arms, i.e. the positions of their centromeres.
Chromosomal Banding
If chromosomes are treated briefly with protease before staining then each chromosome has a
characteristic-banding pattern. Different dyes provide different patterns of banding. The two main
banding techniques used are Giemsa banding (G banding) and reverse banding (R banding), which
are grossly complementary and enable the detection of some 150200 bands generally agreed upon
and officially recognized by the ISCN (International System for Human Cytogenetic Nomenclature).
A still higher resolution can be obtained by synchronizing cell division and blocking the
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Human Chromosomes
chromosomes in a prometaphase stage, so that the chromosomes are more elongated and may show
up to 600 and even 1000 bands.
Type
G-banding
Q-banding
R-banding
(revers)
Different Approaches of Chromosome Banding
Dye
Band Origin
Practical Role
Giemsa
The positive bands heterochromatin
The negative bands –
euchromatin
Quinacrin
(fluorescent)
The positive bands heterochromatin
The negative bands –
euchromatin
Giemsa or
fluorescent dyes
The positive bands euchromatin
The negative bands –
heterochromatin
Exact identification of chromosomes,
identification structural abnormalities
Exact identification of chromosomes,
identification structural
abnormalities
Exact identification of chromosomes,
identification structural abnormalities
Exact identification of chromosomes
C-banding
(centromere)
Giemsa or
fluorescent dyes
The positive bands –
heterochromatin which
surround centromere
Exact identification of chromosomes
T-banding
(telomere)
Giemsa or
fluorescent dyes
The positive bands –
heterochromatin in
telomeres
FISH (Fluorescent in situ Hybridization)
This method is based on complementary binding of single-stranded DNA labeled with fluorescent
dye (hybridization probe) to denatured
chromosomes.
Hybridization
allows
visualization of a DNA fragment of as little
as 1 - 2 kb (but more usually 40 - 50 kb) at
an efficiency approaching 100%. It is useful
for identifying the position of a gene in the
chromosome, as well as chromosomal
aberration.
Chromosomal Painting
This technique represents a variety
of FISH, when complex probes made from
entire chromosomes are used. Different
parts of the chromosome are colored
specifically. In this way the presence of a
chromosome can be ascertained, as well as
different rearrangements.
The principle of FISH
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Human Chromosomes
Alternatively, each chromosome may be painted in a specific color: Spectral Karyotyping
(SKY) and Multiplex Fluorescence in situ Hybridization (M-FISH). SKY and M-FISH are
molecular cytogenetic techniques that permit the simultaneous visualization of all human
chromosomes in different colors, considerably facilitating karyotype analysis. SKY/M-FISH is
particularly useful in: mapping of chromosomal breakpoints; detection of subtle translocations,
characterization of complex rearrangements.
Comparative Genomic Hybridization (CGH)
Comparative genomic hybridization (CGH) is a fluorescent molecular cytogenetic technique
that identifies DNA gains, losses, and amplifications, mapping these variations to normal metaphase
chromosomes. It is a powerful tool for screening chromosomal copy number changes in tumor
genomes and has the advantage of analyzing entire genomes within a single experiment. It is
particularly applicable to the study of tumors which do not yield sufficient metaphases for
cytogenetic analysis.
Autoradiography. Is based on introducing of radioactive labeled nucleotides (dTTP, treated
with tritium) during replication in vivo. It assures identification of newly synthesized strands of
DNA. So it is possible to identify which chromosome or part of chromosome replicates early or
lately during S-phase (e.g. chromosomes 8, 13, 18, 21 and one of X in women replicate last).
Classification of Human Chromosomes
Based on different quantity criteria (length and centromere index) and quality criteria all
human chromosomes are divided in 7 groups, marked A, B, C, D, E, F, G.
Chromosomes Landmarks
Notes
1–3
1qh+
the largest chr.
4, 5
6 – 12 and X
9qh+
16 chr. in ♀ and 15 in ♂
13 – 15
ps+; ph+ h contain nucleolar organizing genes
16
16qh+
17, 18
19, 20.
F
21, 22
ph+; ps+ ph form NOR (contain rRNA)
G
Y
Yqh+
4 chr. in ♀ and 5 chr. in ♂
The satellites represent short fragments of heterochromatin at the end of chromosome, separated
by secondary constriction. They are present in chromosomes 13, 14, 15, 21, 22 (all acrocentric
chromosomes, except Y). In the short arms of acrocentric chromosomes, secondary constrictions are
associated with the nucleolar organizing regions (NOR).
The secondary constrictions represent less condensed and less stained fragments of chromosomes.
In addition to acrocentric chromosomes, they are usual for long arms of chromosomes 1, 9, 16, 19. These
constrictions do not contain NOR, but consist of repetitive DNA sequences. These regions show
considerable variations in length (ex: shorter than the average, 1qh-, or longer than the average, 16qh+),
which are considered clinically insignificant and represent normal polymorphism (heteromorphism).
Group
A
B
C
D
E
Size, Shape
large, metacentric
large, submetacentric
medium, submetacentric
medium, acrocentric
medium, metacentric
small, submetacentric
small, metacentric
small, acrocentric
Length
Large
Medium
Small
Metacentric
A 1, 2, 3
E 16
F 19, 20
Position of centromere
Submetacentric
B 4, 5
С 6, 7, 8, 9, 10, 11, 12, X
E 17, 18
Acrocentric
D 13, 14, 15
G 21, 22, Y
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Human Chromosomes
An idealized human karyotype after G-banding (46,XY)
Karyotype: Chromosomal Formulas

Some landmarks may be distinguished in each chromosome:
The arms may contain one or some regions separated by secondary constriction or prominent,
large bands. The regions are marked with numbers, beginning from the centromere. Different
chromosomes contain diverse number of regions (e.g. chromosome 1 contains 3 regions in p-arm and
4 regions in q-arm).

Each region consists of bands,
which are marked with numbers in
direction from centromere to telomere.
The metaphase chromosomes contain
400 – 500 bands.

At the prometaphase stage (short
stage
between
prophase
and
metaphase), when the chromosomes
are less condensed, the bands may be
divided in some subbands. The
prometaphase chromosomes contain
1800 – 2000 subbands. This stage is
used for high resolution karyotyping.
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Human Chromosomes
The normal karyotype formulas: the number of chromosomes, coma, sex chromosomes
46,XX – for women (♀)
46,XY – for men (♂)
The formulas of karyotypes containing numeric errors:
 Errors of sex chromosomes - the number of chromosomes, comma, sex chromosomes
47,XXX – a woman with an extra X chromosome
47,XXY – a man with an extra X chromosome
47,XYY – a man with an extra Y chromosome
45,X – a woman missing a sex chromosome
 Errors of autosomes - the number of chromosomes, comma, sex chromosomes, comma, plus
(minus) chromosome
47,XX,+13 – a woman with an extra 13 chromosome
47,XY,+21 – a man with an extra 21 chromosome
The formulas of karyotypes containing structural abnormalities:
The structural aberrations are marked with special symbols.
Symbol
del
dup
inv
i (iso)
r
rob
t
Name
deletion
duplication
inversion
isochromosome
ring
Robertsonian
translocation
translocation
Characterization
Loss of a segment of the genetic material from a chromosome
A chromosome segment occurs more than once
The order of several genes is reversed from the normal order
A chromosome with two identical arms
Ring chromosome
The long arms of two acrocentric chromosomes become joined to a
common centromere
Interchange of parts between nonhomologous chromosomes
46,XX,del(1)(q11q13) – a woman, 46 chromosomes, deletion of a segment between band 1 and band 3
of region 1 of short arm of 1st chromosome.
46,XY,inv(2)(p21q31) – a man, 46 chromosomes, inversion of the segment between region 2, band 1 in
short arm and region 3 band 1 in long arm of chromosome 2.
46,XX,dup(1)(q21.1q21.3) – a woman, 46 chromosomes, duplication of a segment in long arm of
chromosome 1 between region 2, band 1, subband 1 and region 2, band 1, subband 3
46,X,i(Xq) [or 46,X,iso(Xq)] – a women, 46 chromosomes, one of chromosomes X contains only long
arms.
Variations in chromosome number and structure in persons with normal phenotype
In women after 60 years old ~ 7% of cells may lose one of chromosome X and become 45,X.
In men after 70 years old ~ 2% of cells may lose the chromosome Y and become 45,X.
Some chromosomes (1, 9, 16, Y) contain a very long secondary constriction. Sometimes satellites
may be observed in chromosomes 17, 18. The bands width (Q, G, C) may differ in chromosomes of
different origin. These peculiarities offer possibility to identify the origin of chromosomes (maternal or
paternal)
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Human Chromosomes
HUMAN SEX CHROMOSOMES
In humans, the sex chromosomes are labeled X and Y. Females have two X
chromosomes and males have one X and one Y chromosome. All the eggs produced
during meiosis have an X chromosome (23,X). Half of the sperm produced by a male
contain an X chromosome (23,X) and the other half have a Y chromosome (23,Y).
Thus, sperm determine the sex of the offspring. If the egg is fertilized by a sperm with
an X chromosome, the zygote develops into a female. If the sperm contains a Y
chromosome, the offspring is a male. There is a difference between the sex
chromosomes. While the X chromosome is relatively large (approximately 6% of
nuclear DNA), the Y chromosome is quite small, and only a few genes have been
assigned to it.
X-chromosome inactivation
In 1961, the British
geneticist Mary Lyon proposed
that the X chromosomes in
somatic cells of mature females are
of two types. One is active and
expresses its full complement of
genetic information; the other is
inactivated in some manner and
does not serve as a source of
genetic information. The biological
meaning of suppression of the
functional activity of one of two X
chromosomes
is
the
dose
compensation, as in male karyotypes there is only one X chromosome present, and in female - two.
Thus the genotypic possibilities of male and female karyotype are equalized. It is important that this
inactivation occurs randomly, so that in early embryonic life (after 16 days) different cells may have
alternative X chromosomes inactivated. So, in some cells, the X-linked genes inherited from the
mother are expressed, whereas in other cells, the X-linked genes inherited from the father are active.
The somatic tissues of females are thus said to be mosaic because they represent the contribution
of genes from different X chromosomes. In each somatic cell the genes in only one X chromosome
will be expressed, but the X chromosome that is genetically active will differ from cell to cell. The
mosaicism has been observed in women who are heterozygous for an X-linked recessive mutation
resulting in the absence of sweat glands; these women exhibit areas of skin in which sweat glands are
present (these parts derived from embryonic cells with normal X chromosome active and the mutant
inactive), and other areas of skin in which sweat glands are absent.
The molecular basis for X chromosome inactivation is not completely understood. The
process begins with activation of a gene called X-inactivation-specific transcript (XIST) on the long
arm of the X chromosome (Xq13). XIST is expressed only in the inactive X chromosome and
produces an RNA molecule (not translated into protein) that transmits the inactivation signal
throughout the chromosome. The process involves physical reorganization of the DNA within the
chromatin and also the addition of methyl groups to the DNA bases.
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Human Chromosomes
Barr Body
One of the two X chromosomes
in female cells is condensed in form of
facultative
heterochromatin
during
interphase and forms the Barr body. A
Barr body is about 1 micrometer in
diameter and is located at the periphery
of the nuclear membrane. The number of
Barr bodies is one less than the number
of X chromosomes. Cells of normal
females have one Barr body; cells of
normal males have none.
Individuals with two or more X
chromosome have a number of Barr
bodies equal to the number of X
chromosomes minus one (that is, equal to the number of inactivated X chromosomes). For example,
XXX individuals have two Barr bodies, XXXX individuals have three, and XXXXX individuals have
four. Thus, an XYY male has no Barr bodies, and XXY or XXYY males have one Barr body.
Determination of X-chromatin in mucosa cells
Barr bodies can be determined most easily in buccal mucosa, hair roots and fibroblast cells.
The normal positive range for sex chromatin bodies is 20-60 percent.
Determination of sex chromatin in mucosa cells includes the following stages:
1) making the preparation; 2) microscope analysis of the preparation; 3) conclusions making.
Epithelium cells of the mucosa from the internal part of a cheek serve as material.
 Before taking the scrape of the cells, the mouth should be rinsed with clean water.
 A scrape is made using a sterile scalpel.
 The scrape is spread over the object-plate, which is dipped into methyl alcohol for fixation.
 10-15 minutes later the preparation is taken out of the alcohol, air-dried.
 Then one or two drops of acetoorceine are added on the preparation, covered with a cover-glass
and the excess of dye is removed by blot.
 Microscopic examination of preparation follows.
For microscope analysis, only the nuclei with chromatin appeared as oval or kidney-shaped
bodies are taken into account. The bodies are usually localized by the inner surface of nuclear
envelope. About 100-150 cells are analyzed; those containing Barr bodies are counted. The
frequency of cells containing Barr bodies is calculated in %.
Examination of Barr bodies is a rapid and convenient test. It is useful to:
 determine the sex in prenatal stage;
 determine the sex of a person with ambiguous genitalia,
 detect abnormal karyotypes such as Turner syndrome (45,X) and Klinefelter syndrome (polysomy
X in male).
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Human Chromosomes
Y Chromosome
The Y chromosome likely contains approximately 400 genes. Because only
males have the Y chromosome, the genes on this chromosome tend to be involved
in male sex determination and development. Sex is determined by the SRY (Sexdetermining Region Y) gene, located on the short arm, which is responsible for the
development of a fetus into a male. SRY is thus a Y-linked gene, because it is
found only on the Y chromosome. Other genes on the Y chromosome are
important for male fertility.
In case of a translocation of SRY gene to X chromosome a 46,XX
testicular disorder of sex development occurs. This means that a fetus with two X
chromosomes, one of which carrying the SRY gene, will develop as a male despite
not having a Y chromosome.
Deletions of genetic material in regions of the Y
chromosome called azoospermia factor (AZF) usually
cause Y chromosome infertility. Genes in these regions are
believed to provide instructions for making proteins
involved in sperm cell development, although the specific
functions of these proteins are unknown.
Many genes are unique to the Y chromosome, but
genes in areas known as pseudoautosomal regions (PAR)
are present on both sex chromosomes. As a result, men and
women each have two functional copies of these genes.
Many genes in the pseudoautosomal regions are essential
for normal development.
Structure of Y chromosome
Y-chromatin
Y-chromatin or F body is formed by 2/3 of q arm of the Y chromosome and can be observed
microscopically as an intensive fluorescent staining body in the nucleus of interphase cells. Its size is
about 0.25 µm and it is situated apart or is attached to nuclear membrane. The frequency of cells
containing F bodies varies in different tissues of male organism. For example, it is 70-85% in
fibroblasts and it is about 45% in sperm cells.
The number of Y-chromatin bodies is equal with the number of Y-chromosomes.
Thus, cells of normal females have no F bodies, cells of normal males have one F body, XYY
males have two F bodies. Cells with is isochromosome Y containing two long arms will present one
F-body twice bigger than normal (0.5 µm).
F-body test is used for identification of the changes in the number of copies of Y
chromosome. Such changes might include:
48,XXYY syndrome. Extra genetic material from the X chromosome interferes with male
sexual development, preventing the testes from functioning normally and reducing the levels of
testosterone (male hormone). A shortage of testosterone during puberty can lead to reduced facial
and body hair, poor muscle development, low energy levels, and an increased risk for breast
enlargement (gynecomastia). Dental problems are frequently seen with this condition; they include
delayed appearance of the primary or secondary teeth, thin tooth enamel, crowded and/or misaligned
teeth, and multiple cavities. Extra copies of genes from the pseudoautosomal region of the extra X
and Y chromosome contribute to the signs and symptoms of 48,XXYY syndrome; however, the
specific genes have not been identified.
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Human Chromosomes
47,XYY syndrome, also called Jacob's syndrome or YY syndrome. Although males with this
condition may be taller than average, this chromosomal change typically causes no unusual physical
features. Most males with YY syndrome have normal sexual development and are able to father
children. It is unclear why an extra copy of the Y chromosome is associated with tall stature, learning
problems, and other features in some men. A small percentage of males with Jacob's syndrome are
diagnosed with autistic spectrum disorders, which are developmental conditions that affect
communication and social interaction.
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