GENETIC ENGINEERING (synonym genetic engineering) — the direction of researches in molecular biology and genetics which ultimate goal is receiving by means of laboratory receptions of organisms with new, including and not meeting in the nature, combinations of hereditary properties. At the heart of G. and. the possibility of a purposeful manipulation caused by the last achievements of molecular biology and genetics with fragments nucleinic to - t lies. It is necessary to refer establishment of universality to these achievements genetic code (see), i.e. the fact that at all live organisms inclusion of the same amino acids in a proteinaceous molecule is coded by the same sequences of nucleotides in a chain of DNA; achievements of the genetic enzymology which made available to the researcher a set of the enzymes allowing to receive in the isolated look separate genes or fragments nucleinic to - you, to carry out in vitro synthesis of fragments nucleinic to - t, to combine the received fragments in a whole. Thus, change of hereditary properties of an organism with G. and. comes down to designing from various fragments of new genetic material, to administration of this material in a retsipiyentny organism, to creation of conditions for its functioning and stable inheritance.
One of ways of receiving genes — chemical synthesis. After Halle (A. Holli) in the USA, A. A. Bayeva in the USSR and to other researchers was succeeded to decipher structure of various transport RBGK (TRNK), X. The Koran with soavt, carried out chemical synthesis of DNA coding alanine TRNK of pressed yeasts.
But the most effective method of artificial synthesis of genes is connected with use of the RNA-dependent enzyme of a DNA polymerase (the return transcriptase) found by Baltimore (D. Baltimore) and H. Temin in oncogenous viruses (see). This enzyme is allocated and purified from the cells infected with some RNA-containing oncogenous viruses, including a virus of a bird's myeloblastosis, Raus's sarcoma, mouse leukemia. The return transcriptase provides synthesis of DNA on a matrix of information RNA (IRNK). Use of molecules IRNK as matrixes for synthesis of DNA substantially facilitates artificial synthesis of separate structural genes of the higher organisms as the sequence of nitrogen bases in molecule IRNK is the exact copy of the sequence of nitrogen bases of the corresponding structural genes, and the technique of allocation of various molecules IRNK is rather well developed. Progress in allocation of IRNK of protein of the globin which is a part of hemoglobin of the person, animals and birds IRNK of protein of lens, IRNK of an immunoglobin, IRNK of specific protein of a malignant tumor (myeloma) allowed to carry out synthesis of a structural part of the genes coding some of these proteins by means of the return transcriptase.
However in an organism structural genes function together with regulatory which nucleotide sequence is not reproduced molecule IRNK. Therefore any of the specified ways does not allow to carry out synthesis of set of a structural and regulatory gene. The solution of this problem became possible after development of methods of allocation of separate genes. For allocation of bacterial genes use the small DNA-containing cytoplasmatic structures capable to be replicated (see. Replication ) irrespective of a bacterial chromosome. These structures form uniform group of extra chromosomal genetic elements of bacteria — plasmids (see. Plasmids ). Some of them can be implemented into a bacterial chromosome, and then is spontaneous or under the influence of the inducing agents, e.g. UF-radiations to pass from a chromosome into cytoplasm, taking with itself and adjacent chromosomal genes cells of the owner. The extra chromosomal genetic elements of bacteria having such properties are called episomes [F. Jacob, E. Wollman]. To to episomes (see) carry moderated a phage (see. Bacteriophage ), sexual factor of bacteria, factors medicinal stability of microorganisms (see), bakteriotsinogenny factors (see). In cytoplasm the genes taken by episomes are replicated in their structure and often form a set of copies. Development of an effective method of allocation of plasmids, in particular the moderate phages bearing genetic material of a bacterial chromosome, and allocation of the bacteriophage of a fragment of a chromosome of a bacterial cell included in a genome allowed in 1969 to Bekvitu (j. Beckwith) with soavt, to allocate a lactose operon — group of the genes controlling synthesis of the enzymes necessary for assimilation by colibacillus of lactose. The similar equipment was used for allocation and cleaning of the gene controlling synthesis of tirozinovy acceptor RNA colibacillus (see. RNA ).
Use of plasmids gives the chance to receive in the isolated look practically any bacterial genes and consequently, and an opportunity to design molecules DNA from various sources. Such hybrid structures can be saved up in cells in significant amounts as many plasmids in certain conditions are intensively replicated in cytoplasm of bacteria, forming tens, hundreds and even thousands of copies.
G.'s progress and. are connected with development of the technology of merging of genetic structures from various istoch-i nicknames in one molecule DNA. In designing of the hybrid molecules in vitro use of endonucleases of restriction — the special enzymes capable to cut molecules DNA in strictly certain sites was decisive. Such enzymes are found in cells of Escherichia coli bearing the plasmids like R causing stability of bacteria to a nek-eye to medicines in cells of Haemophilus influenzae, Serratia marcescens and other microorganisms. One of the most often used enzymes of this type — endonuclease of restriction EcoRI synthesized by a plasmid of RI in cells of E. coli. Enzyme distinguishes the site of DNA with the unique sequence from six couples of nucleotides and cuts two-filamentous structure of DNA on this site thus that the single-stranded ends on both sides are formed of four nucleotides (the so-called sticky ends). As enzyme cuts molecules DNA irrespective of their origin strictly definitely, all fragments of DNA formed as a result of effect of enzyme will have the same sticky ends. The Komplementarny sticky ends of any fragments of DNA combine hydrogen bindings, forming hybrid ring DNA (fig). For stabilization of hybrid molecule DNA use other enzyme — the polinukleotidligaza reestablishing the covalent bonds terminated by enzyme of restriction. The sequence, it is specific recognizable EcoRI, meets in DNA not more often than through 4000 — 16 000 couples of nucleotides. Therefore, the fragment of DNA formed under the influence of EcoRI can include at least one the gene unimpaired with enzyme (one gene on average contains 1000 — 1500 couples of nucleotides).
Use of endonucleases of restriction and some other enzymes gives the chance to receive complex recombinant DNA. The group of researchers under the leadership of P. Berg managed to combine in the USA as a part of one molecule DNA genetic information from three sources: full genome (see) oncogenous virus of monkeys of SV40, part of a genome of a temperate backteriophage λ and group of the genes of colibacillus responsible for assimilation of a galactose. The designed recombinant molecule was not investigated on functional activity as authors of this work stopped before potential danger of spread of oncogenous viruses of animals to populations of the bacteria living in intestines of the person. It is known that the purified DNA of viruses can get into various cells of mammals and it is stable be inherited by them.
For the first time S. Cohen managed to design functionally active molecules of hybrid DNA in the USA et al. S. Cohen's group consistently solved a problem of association and cloning (selective accumulation) of the molecules DNA allocated from the types more and more remote from each other in the phylogenetic relation. The procedure of cloning usually is in what DNA from various sources fragment by means of endonucleases of restriction, then these fragments combine in vitro in the general structure and enter into a retsipiyentny organism, the Crimea serves in Coen's experiences colibacillus. It is established that cells of several species of bacteria (including Escherichia coli, Salmonella typhimurium, Staphylococcus aureus) can be transformed (see. Transformation ) by means of recombinant molecules DNA. At the same time a plasmid part of a hybrid molecule (or one of plasmids if as a part of a hybrid molecule two plasmids from various sources are integrated) serves as a vector, i.e. provides transfer in retsipiyentny cells phylogenetic of alien genetic material and its reproduction in them. The plasmid of pSC101 received by it in vitro controlling tetracycline resistance of bacteria was the first plasmid used by Coen with soavt as a vector. This small plasmid consists of only 8000 couples of nucleotides. It is attacked by EcoRI enzyme only in one site, and enzyme does not damage ability of a plasmid to the subsequent replication in cells of E. coli and to control tetracycline resistance. These features allowed to use it for designing of in vitro of hybrid molecules DNA. At the first stages attached the plasmid DNA emitted from different types of bacteria, and then and from the higher organisms to pSC101. The «himerny» plasmids (i.e. not capable to arise in nature) which combined in the structure the genetic material of colibacillus, the site of DNA from oocytes of a shportsevy frog of Xenopus laevis controlling synthesis of ribosomal RNA, and the site of DNA of a sea hedgehog controlling synthesis of proteins - histones, or DNA of mitochondrions of a mouse were so created. In cells of colibacillus into which entered such hybrid «himerny», plasmids, work of genes of the higher organisms was registered.
Unlike pSC101 which is present at a cell only in 4 — the 6th copies, some other plasmids used as vectors in certain conditions can repeatedly be replicated, forming several thousands of copies in one cell. The plasmid of ColEI controlling synthesis of colicine has such properties, e.g., (see. Bakteriotsinogeniya ). pSC101 is similar, ColEI is cut by EcoRl enzyme only in one site, and the alien DNA which is also processed by EcoRI easily joins the formed linear molecule with the sticky ends. Thus, it was succeeded «to hem» genes of a triptofanovy operon of colibacillus to ColEI. In the cells bearing a set of copies of the designed hybrid plasmid products of the proteins-enzymes controlled by genes of biosynthesis of tryptophane sharply increased. In in the vitro system it was succeeded to attach a plasmid of ColEI to a nek-eye of R-fakto-frames and to a moderate phage. Similar works are for the first time performed in the USSR under the leadership of the academician A. A. Bayev and professor S. I. Alikhanyan. The combined vector plasmids formed by ColEI and R-factors are capable to breed intensively in bacterial cells, ColEI is similar, and at the same time cause resistance of cells to antibiotics that considerably simplifies selection of bacteria — carriers of hybrid plasmids.
As vectors use also moderated a phage. In in the vitro system the hybrid particles of a bacteriophage which included bacterial genes, DNA of other phages or the higher organisms in the structure are designed (e.g., DNA of a fruit front sight drosophila).
Functional activity of hybrid DNA is determined by a possibility of their transfer in cells of retsipiyentny organisms and the subsequent multiplication (amplification) in these cells. As recipients already now effectively use not only bacteria about what it was mentioned above, but also cells of the higher organisms, so far, however, only in the form of culture of the fabric cultivated out of an organism. There are instructions on a possibility of penetration of DNA of the phages bearing bacterial genes in connective tissue cells (fibroblasts) of the person, in protoplasts or in undifferentiated culture (kallus) of cells of plants. In 1971 an amer. researcher Merrill (Page R. Merril) with soavt, reported about experiments on correction of hereditary defect — galactosemia (see) by introduction to «sick» cells of galaktozny genes of the bacteria included in composition of DNA of a transdutsiruyushchy phage. As a result of a cell of the patient with a galactosemia, defective on бета-D-галактозо-1-фосфатуридилтрансферазе enzyme, not capable to acquire a galactose, recovered normal growth potential in the presence of a galactose, and in their extracts earlier being absent enzymatic activity was registered. The similar result was received by J. Horst with soavt, at introduction of the bacterial gene controlling synthesis of a beta galactosidase in fibroblasts of the patient with the generalized gangliozidoz which is characterized by sharp insufficiency of this enzyme. W. Munyon and it sotr. by means of a virus of herpes transferred the gene controlling synthesis of a thymidinekinase from cells of the person to cells of a mouse, having recovered ability of defective mouse fibroblasts to synthesize this enzyme.
One of ways of transfer of genetic information in culture of cells of the person, animals and plants is the hybridization of somatic cells developed by Efrussi (V. of Ephrussi) and G. Barski. Efficiency of this method considerably increased after it was revealed that particles of the inactivated virus of a parainfluenza like Sendai increase the frequency of cell fusion from the most various sources. The possibility of transfer of separate genes from the isolated chromosomes of the Chinese hamster in connective tissue cells of a mouse is shown. Hybrids of cells of the person and a mouse in which a part of chromosomes of the person is removed are described, and a part remains functionally active. Development of methods of microsurgery of cells allowed to replace cellular kernels from somatic cells in oospores and to receive as a result absolutely identical organisms. Hybridization of cells gave the chance to induce synthesis of a globin of the person in formative cells of a frog. All these examples show potentialitys of G. and.
Practical value G. and. for medicine it is connected with perspectives of correction of hereditary defects of exchange at the person (see. Gene therapy ), creations of the microorganisms which lost the pathogenicity, but kept ability to formation of the immunity, synthesis of antibiotics, amino acids, hormones, vitamins, enzymes, immunoglobulins etc. based on use of the microorganisms which included the corresponding genes. Exclusive results can be received G. in the nearest future and. plants. By means of methods G. and. try to create the plants capable to acquire atmospheric nitrogen and to improve proteinaceous structure of vegetable food. The successful solution of these tasks will allow to increase sharply productivity of plants, to reduce production and consumption of mineral nitrogen, and thereby considerably to revitalize environment (see). The possibility of creation of brand new forms of animals and plants due to overcoming trans-species barriers of combining ability is studied. However at G.'s assessment and. as new form of development of wildlife it is necessary to consider not only its possible revolutionizing role in biology, medicine and agriculture, but also the possibilities of emergence of new forms of pathogenic microorganisms arising in connection with its development, danger of distribution in populations of the bacteria living at the person, the hybrid DNA bearing Oncogenous viruses, etc. Of course, deliberate use of achievements of science, and including G. and., in the inhuman, misanthropic purposes it is possible only in society, in Krom the benefit of the person is sacrificed to a profit and aggression.
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L. S. Chernin.