HUMAN CHROMOSOMES. - The position of the centromere, which is characterized by centromere index, calculated p using formula: Ic X100.

Transcription

1 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 consists of different chromosomes in men (XY) and women (XX) sex chromosomes. The maternally and paternally derived chromosomes present in a diploid cell that bear 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 or 22+Y; the egg cells contain 22+X. A display of the metaphase chromosomes of a somatic cell of an individual to show 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. The process of preparing such a photomicrograph is known as karyotyping. Individual chromosomes are identified by chromosome banding and in a formal karyotype; photographs of chromosome pairs are aligned to provide a visual representation of the organism's chromosomal constitution. A typical metaphase chromosome consists of two sister chromatids connected by centromere (located in primary constriction). Each chromatid contains two arms: short (p) and long (q), separated by primary constriction. The ends of chromatids are called telomeres. 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 is used absolute length (in microns) or relative length, calculated using formula: The lenght of studied chromosome X100. The total length of all chromosomes from a haploid cell The chromosomes may be large, medium and small. - The position of the centromere, which is characterized by centromere index, calculated p using formula: Ic X100. p q Using this criteria human chromosomes are divided in: metacentric: Ic = submetacentric: Ic = acrocentric: Ic = 17-30

2 - Presence of satellites. 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). - Presence of secondary constrictions. The secondary constrictions represent less condensed and less stained fragments of chromosomes. They may be present in all chromosomes, but are usual near the centromere in long arms of chromosomes 1, 9, 16, 19. Methods of chromosome analysis 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 used 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 37 0 C, during 72 hours. For inducing of mitosis 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 organism's chromosomal constitution. Uniformly 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. Chromosome 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 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 chromosomes in a prometaphase stage, so that the chromosomes are more elongated and may show up to 600 and even 1000 bands. 2

3 Different methods of chromosome banding Type Used dye Band s origin Practical role G-banding Giemsa The positive bands - heterochromatin The negative bands euchromatin Exact identification of chromosomes, identification structural abnormalities Q-banding Quinacrin (fluorescent) The positive bands - heterochromatin The negative bands euchromatin Exact identification of chromosomes, identification structural abnormalities R-banding (revers) Giemsa or fluorescent dyes The positive bands - euchromatin The negative bands heterochromatin 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 G-banding of human chromosomes (46,XX) FISH (Fluorescent In Situ Hybridisation). This method is based on complementary binding of single-stranded DNA labeled with fluorescent dye (hybridisation probe) to denatured 3

4 chromosomes. It is useful for identifying the position of a gene in the chromosome, as so as chromosomal aberration. Chromosome 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 that chromosome can be ascertained and also whether it has been subject to any rearrangements. This picture shows a probe made from chromosome 22, which has been used to "paint" a cell line, which has a small mysterious extra chromosome. The probe hybridizes to this as well as to the two normal chromosome 22s showing that the small "marker" chromosome is derived from chromosome 22. Alternatively, each chromosome may be painted in a specific color (SKY method). The principle of FISH 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 quantitative criteria (length and centromere index) and qualitative criteria all human chromosomes are divided in 7 groups, marked A, B, C, D, E, F, G. - Group A (large, metacentric) 1 3, the largest chromosomes. Chromosome 1 may contain a secondary constriction (1qh+). - Group B (large, submetacentric) 4, 5. - Group C (medium, submetacentric) 6 12 and X. There are 16 chromosomes in women and 15 in men. Chromosome 9 contains a secondary constriction (9qh+). - Group D (medium, acrocentric) May contain satellites on short arms. 4

5 Length Human chromosomes - Group E (medium metacentric) 16 and (small submetacentric) 17, 18. Chromosome 16 may contain a secondary constriction (16qh+). - Group F (small metacentric) 19, Group G (small acrocentric) 21, 22, Y. Chromosome Y may contain a secondary constriction (Yqh+). There are 4 chromosomes in women (2x x 22) and 5 chromosomes in men (2x x 22 + Y). Position of centromere Metacentric Submetacentric Acrocentric - Large A 1, 2, 3 B 4, 5 Medium E 16 С 6, 7, 8, 9, 10, 11, 12, X D 13, 14, 15 Small F 19, 20 E 17, 18 G 21, 22, Y An idealized human karyotype after G-banding (46,XY) Chromosomal formulas In each chromosome may be distinguished some landmarks: - Short arm p - Long arm q - 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). 5

6 - In each region consists of bands, which are marked with numbers in direction from centromere to telomere. The metaphase chromosomes contain 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 subbands. This stage is used for high resolution karyotyping. 46,XX for women 46,XY for men The normal karyotype formulas: the number of chromosomes, coma, sex chromosomes The formulas of karyotypes containing numeric errors: Errors of sex chromosomes - the number of chromosomes, coma, 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 of an X chromosome Errors of autosomes - the number of chromosomes, coma, sex chromosomes, coma, 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: Simbol Name Characterization del deletion Loss of a segment of the genetic material from a chromosome dup duplication A chromosome segment occurs more then once inv inversion The order of several genes is reversed from the normal order i (iso) isochromosome A chromosome with two identical arms r ring Ring chromosome rob Robertsonian translocation The long arms of two acrocentric chromosomes become joined to a common centromere t translocation 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 1-st 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 6

7 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 loose one of chromosome X and become 45,X In men after 70 years old ~ 2% of cells may loose the chromosome Y and become 45,X Some chromosomes (1, 9, 16, Y) contain a very long secondary constriction. Some times satellites may be observed in chromosomes 17, 18. The bands width (Q, G, C) may differ in chromosomes from different origin. These peculiarities offer possibility to identify the origin of chromosomes (maternal or paternal) Main terms: centromere, telomere, chromatid, primary constriction, secondary constriction, satellite, centromere index, autosome, sex chromosome, karyotype, karyotyping, band, subband, chromosomal formula, chromosomal landmark, 7