From Big Medical Encyclopedia

GENETICS OF SOMATIC CELLS (grech, genetikos relating to an origin; soma, somatos a body) — the field of genetics studying patterns of heredity and variability elementarnyuy a base and functional unit of a metaphyte — a somatic cell. G.'s allocation by the village to. there were in connection with development in the mid-fifties 20 century of methods of cultivation out of an organism of the isolated zooblasts. Of page to. developed under the influence of genetics of unicells. Its development allowed to bypass such difficulty of the genetic analysis of the person as impossibility of the directed crossing, gave the chance to carry out studying of the genetic events happening to low frequency. The methods of a research applied in G. by page to., have great practical value. They promote studying of a pathogeny at cellular molecular level and are used during the development on this basis of new diagnostic methods, treatment and prevention of diseases which developing is directly connected with pathology of a cell (hereditary diseases of a metabolism, malignant tumors, a cellular immunopathology).

The cells supported in culture by passages in the breeding state (cellular lines) can or change slightly in comparison with initial (diploid lines), or to undergo more serious changes (heteroploid lines). Diploid cultures of cells genetically are also biochemical stabler, but is still long it is possible to cultivate only diploid cells of a connective tissue origin. Heteroploid lines are genetically heterogeneous that is shown not only in a variety of chromosomal complements of the cells making them, but also in variability of nutritious requirements, activity of enzymes, medicinal stability, etc. It is connected with continuous emergence in such cell populations of options on different signs.

Genetic analysis (see) it is impossible without studying of posterity of individual cells. In G. of page to. this problem is solved by cloning of cellular cultures — cultivation on solid substrate of a clone (genetically homogeneous posterity of a separate cell) in the form of cellular colony. Success in cultivation of the isolated cells of the person, especially in cloning, depends on creation of adequate conditions for their reproduction, first of all on satisfaction of specific nutritious requirements of cells. This problem is finally not solved; searches are conducted on the way of creation of completely synthetic mediums considering specific requirements of the differentiated cells of different fabrics. Creation of such environments at the same time is a necessary condition for stabilization gene and a phenotype of the cultivated cellular lines.

A necessary condition of carrying out the genetic analysis on somatic cells — marking of cells on the signs controlled by individual genes. Development of this problem is still far from end. It is in a complete dependence from progress in biochemistry of somatic cells and creation of methods of selection of mutants. There are two methods of receiving genetically marked cells of the person. The first — explantation of cells from patients with hereditary enzymopathies. Several tens of hereditary defects of exchange at the person which cornerstone the mutations which are accurately shown at the cellular level in the form of disturbance of normal synthesis of specific enzyme are are described. An example of such markers in the cultivated connective tissue cells is lack of activity galaktozo-1-phosphate-uridiltransferazy — G1FUT or гипоксантин-гуанин-фосфорибозилтрансферазы — GGFRT (Lesh's disease — Naykhana), insufficiency of glyukozo-6-fosfatde-hydrogenase — G6FD (at some hemolitic anemias). As markers often serve also normal biochemical signs: enzymes of group of dehydrogenases (G6FD, a lactate dehydrogenase — LDG, a 6-fosfoglyukonatdegidrogenaza — 6FGD, a malate dehydrogenase — MDG, etc.), kinases (a thymidinekinase — shopping mall, phosphoglycerate kinase — FGK, etc.), transferases (GGFRT, G1FUT, etc.). Use antigenic markers less often: antigens of blood groups and antigens of histocompatibility.

Fig. 1. The scheme of allocation of auxotroph mutants (auxotrophs) by means of the BDU minimum environment and uviolizing (UF): auxotrophs — the cells breeding in the environment in the presence of additional substances; the BDU minimum environment (5-bromdezoksiuridin) sensibilizes prototrophs to UF; prototrophs — the cells capable to independently synthesize substances, necessary for growth

The second method — use as markers of the mutations arising is spontaneous or induced already in the cultivated cells. Can be such markers: lack of growth on the Wednesday without separate amino acids (see. Auksotrofnost ), resistance to chemical substances (analogs of nitrogen bases or nucleosides, antibiotics), physical. to factors (ultraviolet or ionizing radiation, temperature), viruses. Allocation of clones of somatic cells — auxotrophs which cannot breed on the Wednesday, free of these or those nutrients is especially convenient. One of the offered ways of their allocation is as follows. Cells grow up on the full-fledged medium providing survival of rare auxotroph mutants; then the mixed cellular culture is transferred to the minimum environment with 5-bromdezoksiuridiny (BDU) where only prototrophs, substances, capable to synthesize necessary for growth, breed. BDU is an analog of nitrogen base of thymine and therefore actively joins in DNA of the growing cells. In DNA of cells auxotrophs (their growth because of absence in the environment of necessary amino acid is inhibited) inclusion of BDU does not happen. Thus, DNA of the cells growing on the minimum environment contains BDU, and DNA of the cells which are not growing on this Wednesday is deprived it. In turn presence of BDU at cells increases their sensitivity to lethal influence of UV rays. The subsequent uv radiation destroys sensibilized BDU prototrophs. At crops of the irradiated cellular material only colonies of an auxotroph mutant (fig. 1) adequate to the environment grow by the full-fledged selection environment. By this way in cultures of cells of the Chinese hamster the stable auxotrophs incapable of synthesis of glycine (gli-), hypoxanthine (type) and other metabolites are received. Studying of the spontaneous and induced mutational process on the cultivated cells makes the independent section G. of page to. also it is carried out on gene and it is especially wide on chromosomal levels.

Fig. 2. The scheme of intergene complementation (mutual addition) on which selection of the hybrid cells (HC) is based: 1 and, 2 — parent cells; in the first there is no synthesis of гипоксантин-гуанин-фосфорибозилтрансферазы (GGFRT-) enzyme, and in the second there is no synthesis of enzyme of a thymidinekinase (shopping mall-); in the selection environment due to intergene complementation only hybrid cells breed; in the hybrid cell (HC) there is a synthesis of гипоксантин-гуанин-фосфорибозилтрансферазы enzyme (GGFRT+) and enzyme of a thymidinekinase (TK+).

Necessary condition of carrying out the hybridological analysis of somatic cells — exchange of hereditary factors and their recombination at descendants. Nek-rym an equivalent of these processes in G. of page to. the phenomenon of hybridization of somatic cells with the subsequent segregation of chromosomes of one of parents serves. The essence of hybridization of somatic cells consists in education of two cells of one hybrid cell (HC) by merge. Such cell has two independent kernels (heterokaryon) in the beginning. In case of their association premises for the subsequent reproduction of hybrid cells are created [Lordly, Efrussi (G. Barski, V. of Ephrussi)]. Frequency of spontaneous formation of group of companies is extremely low (one group of companies on tens of thousands of parent), and chances of the selection replacement of parent forms by them from the mixed cell population are absolutely insignificant. By means of the inactivated parainfluenza virus Sendai is possible to increase the frequency of cell fusion to 5 — 10%. Release of cultures of group of companies from parent forms is carried out by means of the selection mediums. Offered is widely used. for this purpose J. W. Littlefield the AT environment G including hypoxanthine, Amethopterinum and thymidine. For hybridization take cells, one of which in the environment with Amethopterinum are not formative due to the lack of enzyme of a thymidinekinase (shopping mall-), others — гипоксантин-гуанин-фосфорибозилтрансферазы (GGFRT-) enzyme, providing digestion of the thymidine which is present at the environment and hypoxanthine respectively. Amethopterinum is necessary for blocking of synthesis of derivative these substances. Hybrid cells like (TK+GGFRT-) X (TK-GGFRT+) thanks to mutual addition (complementation) with normal alleles of the mutating genes will grow on such environment (fig. 2). Use of the semi-selection environments when the mutation is born by cells of only one of parents, and cells of the second parent is possible or are sowed in small number, or slowly breed, or do not grow in a monolayer (culture of lymphocytes of blood). By means of the specified methodical receptions it is possible to receive hybrids of cells of various fabrics of one species of animals (intraspecific) or different types, including the types relating to taxonomical groups far remote from each other (trans-species), napr, hybrids of cells of people — a mouse, the person — the Chinese hamster, the person — a mosquito. In hybrid cells parent genomes combine in a uniform integral genome. At the same time separate signs of stem cells can disappear, there can also be new signs that connect with change of activity of the corresponding genes. At the same time the majority of parent genes remains functioning without changes. In interspecies hybrids thanks to distinctions in physical. - chemical properties (e.g., electrophoretic mobility) manages to divide homologous biochemical, markers of parents (enzymes). It is one of the reasons of use of interspecies hybrids for genetic mapping of chromosomes. The possibility of education in group of companies of heteropolymeric molecules of enzymes when subunits of a molecule are coded by genes of both parent forms is established. The segregation of genes involving their recombination is provided in group of companies with spontaneous and gradual elimination of chromosomes of one of parent forms. So, in hybrid systems of people — the mouse or the person — the Chinese hamster are lost chromosomes of the person. It is the second reason of use of interspecies hybrids for genetic mapping of chromosomes.

Hybridization of somatic cells is used for different types of genetic researches. Widely actually genetic analysis (studying of linkage groups of genes and genetic mapping of chromosomes of the person) develops what apply interspecies hybrids to. Mapping of chromosomes is carried out by comparison of manifestation to group of companies biochemical, markers and presence of chromosomes of the person. As in process of reproduction of group of companies chromosomes of the person eliminirut, the simultaneous and reproduced in repeated experiences preservation or loss of two or more markers in a genome of group of companies will indicate their coupling. If such manifestation of markers is correlated with behavior of a certain chromosome, the possibility of «binding» of genes to this chromosome appears. The task is facilitated by the fact that the problem of identification of chromosomes of the person and the laboratory animals used for hybridization is solved now. On the basis of a method of hybridization of somatic cells and some other methods genetic mapping of chromosomes of the person is begun. Data on localization of these or those genes in all chromosomes of one person, in particular about localization more than 20 genes in an autosome of one person among which the genes controlling synthesis of enzymes 6FGD, FGM (phosphoglucomutase), peptidase C about coupling of the genes controlling synthesis of G6FD, GGFRT, FGK enzymes and an alpha galactosidase and their localization in X-chromosome are received.

The method of intraspecific hybridization of auxotroph mutants allows to carry out the analysis of intergene and intragenic (interallelic) complementation (see. Mutational analysis ) at the person. For this purpose use cells from different donors with an identical mutant phenotype. If the mutation mentions the same loci, there is no complementation, groups of companies on the selection environment do not proliferate. Reproduction of hybrids demonstrates to existence of interallelic complementation, i.e. that control of this sign is exercised more than one allele. By means of such analysis existence of interallelic complementation at a galactosemia which is characterized by lack of activity of G1FUT was shown. It speaks about a poliallelnost of the gene coding this enzyme.

On system of hybrids between genetically repressed cellular genome (a chicken erythrocyte) and actively functioning genome (a cell of the Hela line of the person and the L mouse) reversibility of an inactivation of genes in the high-differentiated cells is proved and the chain of events at derepression of a genome is investigated [Harris (N. of Harris)]. Hybridization of normal and tumor cells allows to estimate a role of mutational changes of the genetic device and the changes connected with a condition of genetic activity in a malignancy of a cell.

G.'s achievements of page to. begin to be implemented into medicogenetic practice. Diagnosis of hereditary defects of a metabolism and studying of their pathogeny are transferred more and more to the cellular level since such diagnosis provides the analysis not simply patol, metabolites, and the defective enzymes which are the cornerstone of their emergence. Such approach allows to solve more profoundly a number of practical problems of diagnosis, prevention and treatment of hereditary enzymopathies (expansion of a circle of the diagnosed diseases, accuracy of differential diagnosis, a possibility of prenatal diagnosis, etc.). By means of complementation analysis studying of genetic heterogeneity of a number of hereditary diseases is possible; in particular, genetic heterogeneity of some mukopolisakharidoz and Lesh's diseases — Naykhana is already shown. Use of cellular lines from patients with hereditary defects of exchange allows to pass to check of methods of specific therapy. So, from urine and blood of healthy people specific factors, apparently the enzymes correcting abnormal biochemical, a phenotype of the cultivated cells sick with nek-ry mukopolisakharidoza are allocated.

Researches on G. of page to. so far are carried out only in the few institutions of the USSR, the USA, England, Sweden, France, etc. Specialized independent institutions on this branch of science do not exist yet. The specialized periodic and reference media, the managements and monographs are single.

See also Genome , Genotype , Conjugation of chromosomes , Mosaicism , Mutation , Chromosomes .

Bibliography: Vakhtin Yu. B. Genetics of somatic cells, L., 1974; Zakharov A. F. Methodical problems of development of genetics of somatic cells of mammals and person, Vestn. USSR Academy of Medical Sciences, No. 9, page 78, 1966; Siniskal-k about M. Hybrids of somatic cells, Ontogenesis, t. 2, No. 2, page 115, 1971, bibliogr.; Shapiro N. I. Urgent problems of genetics of somatic cells, Geneticist, t. 11, No. 6, page 159, 1975; E f-r at with with and B., Hybridization of somatic cells, the lane with English, M., 1976; Harris H. Cell fusion, Dunham lectures, Oxford, 1970; Harris M. Cell culture and somatic variation, N. Y., 1969; Human gene mapping 2, Rotterdam conference (1974), Cytogenet. cell Genet., v. 14, JVs 3—6, 1975, bibliogr.; Krooth R. S. a. S e 1 1 E. K. The action of Mendelian genes in human diploid cell strains, J. cell. Physiol., v. 76, p. 311, 1970; P u with k T. T. Some considerations on genetic analysis at the single gene level in man, Ann. N.Y., Acad. Sci., v. 171, p. 406, 1970.

A. F. Zakharov.