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A vector was selected for homologous recombination to generate mouse embryonic stem cell clones with disrupted platelet-derived growth factor A (PDGF-A) chain gene. A chimeric mouse with targeted PDGF-A gene has been created.

A highly specific procedure for quantitative assay of the homoserine kinase activity in an optimized enzymatic reaction using 14C-labelled homoserine or [gamma 33P]-ATP as substrates, and paper or thin-layer chromatography for separation of the formed o-phosphohomoserine, is described. The procedure is simple, sensitive and allows the assay for the activity of both purified and non-purified homoserine kinases.

Two human genomic genes for the hair high-sulphur keratins were for the first time cloned in a 15 kb fragment. The primary structures of the coding regions of the genes and their 5'- and 3'-flanks were determined. In the 5'-flanking region, TATA boxes, initiating codons and a 18 nucleotide sequence, previously described in sheep keratin genes and designated as "the matrix-specific" sequence was revealed. Basing on the nucleotide sequences, the encoded amino acid sequences of the high-sulphur keratins were determined for the first time. The suggested functional role of the structural elements (regions) revealed in the proteins primary structure and problems concerning their evolution tendencies are discussed.

A transgene rabbit with the human growth hormone releasing factor gene was produced by a method of microinjections into fertilized oocytes; a mouse metallothionein I gene was used as a promoter. Gene expression was accompanied by a phenotypical effect, expressed in increasing the rates of development. The maximum difference among the transformant, transplants and control was revealed on the 30-45th day of postnatal development. Analysis of the hormonal status of the transgene animal has shown change in the levels of the majority of hormones: a 6-10-fold increase in insulin; a 2-3-fold increase in the level of triiodothyronine, thyroxin; a reduced somatostatin concentration, a two-fold decrease in the level of progesterone, and a four-fold decrease in the level of testosterone. Activation of the promoter zone with Zn++ salts for 5 weeks resulted in a further increase in the transformant body mass by 10%. However blood hormone levels in the transgene rabbit returned to normal. Proceeding from the above it can be assumed that exogenous gene expression probably increased somatotropin secretion which determined dysfunction of most of the endocrine glands; the effect of somatotropin was probably insulin-mediated.

Transgenic mice were obtained inheriting the human erythropoietin gene under the control of viral regulatory elements. The reliable difference in haematocrit, the content of haemoglobin and percentage of reticulocytes in peripheral blood were not revealed. The level of serum erythropoietin in transgenic mice is several fold higher than in control mice. The increased pool of erythroid cells was observed in the bone marrow of transgenic mice, especially of normoblasts (3-fold) and reticulocytes (4,5-fold).

Human and mice nuclear extracts from livers and mice spleen extract were analysed in an attempt to find any proteins capable of binding to the human alpha 1-antitrypsin gene promoter. The nuclei of all studied tissues contain such proteins. The proteins were partially purified on DEAE-trisacryl, heparin sepharose and phosphocellulose columns. The multiple sites for liver nuclear proteins binding to the human alpha 1-antitrypsin gene promoter were found by the DNAse I footprinting technique.

We have demonstrated that the ability to induce benign neoplasms We have dominant mode of inheritance in Drosophila melanogaster is the specific feature of oncoviral DNAs. It is supposed that development of this type of neoplasms in Drosophila is connected with the changes in expression of protooncogenes in mutant genome: firstly, the genetic factors directing the development of neoplasms and Drosophila protooncogenes which shared the homology with v-src are localised in the same regions; secondly, there are structural rearrangements in c-src/fps (29A) protooncogene in mutant stocks which display the ability for neoplastic growth.

We have demonstrated that mutations induced in Drosophila melanogaster by the microinjections of adenovirus Sa7 DNA in early embryos are of insertional nature. The role of insertional elements is played by the Drosophila transposons, but not by the virus DNA. The ability of oncoviral DNA to induce transpositions of mobile elements in recipient genome is the molecular basis of this system of genetic instability.

Mutations Lobe induced by the microinjections of RSV cDNA into Drosophila melanogaster early embryos are characterized by permanent genetic instability; the level of this instability is being changed in time. Based on the results of genetic analyses of Lobe mutations and molecular analysis of white and ADH mutations induced at high frequency in this system of gene instability, we supposed that unstable mutations which arose under the influence of retroviral cDNA are of the insertional nature.

The functioning of Escherichia coli threonine operon isolated genes thrB and thrC was studied by using the genetic complementation and enzymatic activity determination techniques. A new gene thrBC was obtained by the genes merging. The genes thrB and thrC were shown to function in Escherichia coli cells independent of the operon and the polipeptide encoded by the thrBC gene combined the functions to express the products of both genes in bacterial cell. At the same time the enzyme coded for by the merged genes demonstrates the level of activity compared with the ones of the isolated genes.

Human cDNAs coding for angiogenin were isolated from Li7 hepatoma. Analysis of the nucleotide sequences of isolated cDNA clones and its comparison with recently published sequences of cDNA and human angiogenin gene permitted to suggest that an intron is present in the 5' region of the gene, dividing the coding and 5' untranslating regions. The size of the intron exceeds 1700 b.p. Up to now it has been known that the angiogenin gene is a single copy gene. However our results of blot-hybridization with genomic DNA of some mammals showed that there are 2-3 copies of the gene in their genomes. According to our results the angiogenin gene is conserved in mammalian genomes.

Previously cloned PstI-repeats located in 5'- and 3'-flanking sequences of the bovine growth hormone gene were studied in details. The size of the repeats (1,200-2,000 b.p.), the degree of homology, the orientation and direction of their transcription were determined. They were shown to be transcribed in thymus, liver, kidneys, pancreas, adenohypophysis, spleen, although with different efficiency. The transcripts are polyadenylated and heterogeneous in size. Partial asymmetry of PstI-repeat family transcription was shown.

A comparative study has been made of erythroid cell development pathways in the peripheral blood of pigeons during severe, moderate and weak forms of anaemia. Three modes of erythrocyte formation from bone marrow precursor are described: 1. A reserve erythropoiesis--the principal process during severe anaemia; the bone marrow precursors are basophylic erythroblasts which are reversibly blocked in phase G2 of the cell cycle; in results the rapid, increase of erythrocyte population above the normal level, although the cells have 25-30 per cent deficiency in haemoglobin content. 2) A mode of erythropoiesis, whose precursors are proliferating polychromatophylic erythroblasts; this is the principal mode of erythropoiesis at the moderate anaemia, leading to restoration of the normal quantity of erythrocytes with a normal haemoglobin content. 3) A mode of erythropoiesis with proliferating orthochromatic erythroblasts being precursors (which do not divide normally); this is the principal mode during the weak anaemia to result in a slow restoration of the number of erythrocytes with an excess in haemoglobin content. It is shown that regulation of the restoration processes during anaemia are characterized by a specific combination of cell proliferation and differentiation.

The hybridization kinetics of nuclear RNAs of loach embryos labelled with [3H]uridine for 1 hour with DNA excess shows that during embryogenesis (from the blastula to the gastrula stage) the number of newly formed RNA molecules transcribed from repeating DNA sequences in considerably reduced. This occurs both in the RNA fraction extracted from the nuclei with phenol pH 7.7 and having a low sedimentation coefficient and a low degree of polyadenylation, and in the RNA fraction extracted with phenol pH 9.0 having a higher sedimentation coefficient and a higher degree of polyadenylation.

Using cytophotometric and autoradiographic methods, it was shown that on days 2-3 of embryogenesis primary erythroid cells (PEC) divided actively. The distribution of erythroblasts (EB) according to their DNA content is not, however, typical of a proliferating population: it contains an unusually large number of 4c cells resulting from the cell cycle arrest at the G2 phase. It is established that reticulocytes (RC) do not divide and are arrested at G1 or G2 phases, since they do not incorporate 3H-thymidine after their formation is complete and their DNA contents are strictly confined to either 2c or 4c. All types of PEC include a large number of cells containing H2c DNA which is due either to the cell cycle arrest at the S phase, or to the formation of accessory nuclei. All PECs have much higher contents of hemoglobin and total protein than do adult hen erythrocytes (EC). Hemoglobin and total protein contents of H2c and accessory nuclei containing cells are much higher than those in 2c-cells. We have calculated that adult birds and embryos contain the same amount of hemoglobin per gram of weight, but the quantity of red blood cells in the former is ten times higher. A conclusion is drawn that proliferation and cytodifferentiation regulation mechanisms are directed, in primary erythropoiesis, to intense hemoglobinization of the cells, and, in adult erythropoiesis, to increasing their number. In both the cases homeostatic regulation of erythropoiesis works.(ABSTRACT TRUNCATED AT 250 WORDS)

Two types of biosynthetic processes associated with cytodifferentiation underlie the "reserve" erythropoiesis of pigeons during anemia: 1) cells rapidly increase their nuclear DNA amount up to the level of 4c, whereas the total protein remains at the initial level; furtheron, cells double also their protein amount, and then enter into the phase of differentiation and specialization; after having hemoglobin accumulation they divide; 2) a limited increase of DNA amount takes place up to the level not exceeding 130% of the initial level; in this case a synchronous increase of the hemoglobin amount is detected in the cell. The first type processes provide the accumulation of the specific product and the accelerated increase of the number of erythrocytes in the population. The second way is aimed only to the accumulation of the specific product in a greater amount.

We have demonstrated that larval tissues of mutant stocks induced by injections of oncogene virus DNA (Sa7 and RSV) show the neoplastic mode of growth after transplantation into the body cavity of wild-type flies. Neoplasms tested were shown to be ordinary benign insect neoplasms, and at the same time they show the dominant mode of heredity typical of the neoplasms of vertebrates. Genetic factors responsible for the neoplastic growth are localised in the 3rd chromosome in the stocks obtained after injections of the adenoviral Sa7 DNA, and in the 2nd and 3rd chromosomes in the mutant stocks induced by the retroviral DNA.

The steady-state content of globin-coding sequences in nuclear and cytoplasmic RNA of pigeon erythroid cells was estimated by hybridization in the excess of nuclear 28S RNA and cytoplasmic poly(A) + RNA with [3H]DNA, synthesized on globin mRNA. Sequences of 9S globin mRNA are found in 0.06% of molecules of non-ribosomal 28S nuclear RNA (pre-mRNA) of erythroblasts and in 0.5% of molecules of non-ribosomal 28S nuclear RNA of reticulocytes. The content of globin mRNA in erythroblast cytoplasm is, respectively lower than in that of reticulocytes.

A constant fine regulation of gene expression is needed for the normal development to proceed and for the physiological homeostasis to be maintained. In many cases such a regulation in eukaryotes is realized at the level of transcription, involving various cis- and trans-regulatory genomic elements. The review provides the data on the structure of elements determining the level of gene transcription in response to the action of various environmental factors and effectors, responsible for coordinated expression of the genes which provide for tissue-specific transcription and self-regulation of gene transcription. The data were considered on regulation of gene activity by mobile genetic elements and a relationship between the mechanisms of regulation of gene expression and evolution has been formulated.

The phage clones containing a gene coding for bovine growth hormone were isolated from a bovine genomic library. Comparison of the 5' and 3' regions flanking the bovine growth hormone gene by Southern blot hybridization revealed that they share homology. Screening the bovine genomic library by nick-translated DNA fragment from 5' flanking region leads to conclusion that this sequence is present in 0.1% of clones. Each analysed clone carrying the sequence contains some copies of it.

Reassociation kinetics of the fragments of DNA consisting of interspersed repetitive and non-repetitive nucleotide sequences is considered in this paper. Based on the model, suggested by Gavrilov and Mazo (Mol. biol., 11, 101 1977), which takes into account the random DNA shearing, both reassociation kinetics of the total DNA in the region corresponding to interspersed repeat reassociation and that of the isolated preparation of interspersed repetitive sequences are calculated. In both cases influence of the repeat length on the reassociation rate is demonstrated. The estimation of the repetition frequency of rare repeats from pigeon genome is specified using calculations performed.

The reactivation of the nuclei of erythrocytes, reticulocytes and bone marrow cells has been studied by means of hybridization of the pigeon erythroid cells with the human embryonic cells A1. The process of reactivation of the erythroid nucleus was shown to depend on the stage of erythroid cell differentiation. The nuclei of cells at the earlier stages of differentiation give a higher percentage of heterocaryons (40%, 70%) than those of more mature cells (9%). The activated nuclei of immatur cells formed nucleoli already 24 hrs, whereas those of mature erythrocytes only 3-5 days after the cell fusion.

Organization of sequences in pigeon genome and the spectrum of their repetition frequencies were studied by means of DNA/DNA reassociation. Reassociation of 125I-labelled DNA fractions isolated from pigeon total DNA attested the presence of rare repetitions with an average of 50 copies within a gaploid genome. The disposition of repetitive and unique sequences was studied by reassociation of the labelled fragments of different length with an essential excess of short fragments of an unlabelled DNA. Additional evidence was provided by estimation of hyperchromicity and resistance to nuclease S1 of long DNA fragments, reassociated to the given C0t values. It was demonstrated that approximately one fourth of the pigeon genome consists of intermittent repetitive and unique sequences with individual elements of average length of 2 and 37 kb, respectively (1 kb = = 1000 nucleotide base pairs). A hypothetical organization of palindromic sequences in pigeon genome is discussed in terms of the dependence of the value of zero binding to hydroxyapatite on the fragment length.

The existence and extent of homology between the 28S fraction of messenger-like nuclear RNA on one hand, and giant (greater than 45S) molecules of heterogeneous nuclear RNA and various fractions of cytoplasmic RNA on the other hand were studied by the method of competitive hybridization. The results obtained lead to the conclusion that the poly(A)-subfraction of hnRNA molecules with the size of greater than 15 000 nucleotides contains molecules subjected to rapid but incomplete processing with the formation of chains approximately 5000 nucleotides long (approximately 28S), which are then stored in the nucleus for up to 30 hours. Later polyadenylated and non-polyadenylated molecules of cytoplasmic RNA with the size of less than or equal to 2000 nucleotides are formed from the stored precursors.

Anemia was induced in rats by phenylhydrazine. There were found in the blood basophil erythroblasts, macrocytes, both ortochromatic and polychromatophil and basophil reticulocytes (the number of which could attain 60%). The role of macrocytosis in the reaction of the organism is discussed. The mitotic activity of erythroid cells increased in the bone marrow. On the basis of comparison of hemopoiesis in mammals (rats) and birds, by the previous authors' publication, a suggestion is put forward to the effect that during evolution "reserve" erythropoiesis is preserved only in the forms with nucleate erythrocytes (birds) or in the cells which have not lost the nucleus (in the bone marrow of rats).

RNA-containing Raus sarcoma virus, recombinant plasmid pBR322 inserted by kDNA RSV (pPrC11) and Sa7 adenovirus DNA were injected into the polar region of Drosophila melanogaster early embryons. The exogenic genetic material injected was shown to induce mutations, many of them unstable. In a number of cases, virus-specific sequences were found in DNA isolated from mutant flies. It is hypothesized that mutations induced by DNA of oncogenic viruses are of the insertion type.

The recent data on the structural organization and expression of genome in eukaryotes are reviewed. Different types of nucleotide sequences (repetitions, palindromes, homopolymers) represented in the regions of genome transcription, as well as in mRNA's of the cytoplasm and nuclear messenger-like RNA's are analyzed. Probable functions of different types of sequences are discussed. The problem of nuclear precursors of cytoplasmic mRNA's and in particular heterogeneous nuclear RNA is considered. It is noted that this population of molecules is heterogeneous not only by the molecular weight, but also by some other parameters. Stable messenger-like 28S and 18S RNA's found in the nuclei are described. In addition to the already known scheme of rapid posttranscriptional processing of pre-mRNA, a hypothesis is put forward to the effect that the selective transport of mRNA molecules stored in the nucleus from the nucleus into the cytoplasm is realized at the expense of extended processing.

DNA of simian adenovirus Sa7 injected into polar plasma of early Drosophila melanogaster y1snw*; bw; st stock embryos induced one to three unstable visible X-linked mutations in the absence of intact P-element. Numerous mutational events (reversions, new mutations) occur only in four precisely destabilized by the Sa7 DNA loci of X-chromosome and take place during 4-5 generations; in the next generations the level of instability decreased. At the same time, Sa7 DNA induced reversions and new allelic mutations in the snw mutant locus, without exogenous intact P-element.

DNAs of seven transgenic mice and one transgenic rabbit was divided into fractions according to reassociation kinetics and GC-content. Moderate and/or frequent (reverse) repeated sequences of the genome were revealed in all cases next to different transgenes. DNA fractions containing foreign sequences differed by the GC-content in different transgenic animals.

Isolated from pigeon genome short (approximately 450 nucleotides) and long (approximately 8000 nucleotides) single-stranded DNA fragments containing prolonged (greater than or equal to 300 nucleotides) polydeoxyandenylic sequences have been studied. Based on the analysis carried out we come to the conclusion that for the pigeon genome the prolonged poly(dA)-sequences are located as clusters where a large amount of short palindromic sequences occur.

The bone marrow of three intact male mice of C57Bl/6 line, fixed by perfusion of isotonic fixative of Karnovsky, has been studied by means of the scanning electron microscopy method. The surface of erythroid cells, that are immediately connected with macrophages of the erythroblasts islets, is analysed. According to the surface form, the erythroid cells are devided into 5 types. Every maturation stage of the erythroid cells is characterized by a certain type of surface. For identification of basophilic and polychromatophilic proerythrocytes the combined method of light and electron scanning microscopy of the cells in suspension is used. The bone marrow cells, obtained from the two male mice of C57Bl/6 line are fixed with the same fixative on special glasses with grids traced on them, stained after Romanovsky-Giemsa method and in moist preparations are examined in the light microscope. After further treatment the surface of the same cells in studied in the scanning electron microscope.

A review is given of the principal achievements in studying the structure of mRNA in eukaryotic cells. The data are provided on the size and life time, complexity and distribution of different kinds of mRNA by the frequency of repetitions; composition and structure of mRNP. The structures of individual mRNA's and general pattern of the structure of eukaryotic mRNA and mRNP are considered.

An antiserum with the antibody titer of 1 : 4096 was obtained by immunization of rabbits with the tRNA-histone H5 complex from pigeon erythrocytes. The specificity of the antiserum was studied quantitatively from the reaction of the complement binding to a homologous antigen (histone H5) and its modifications (I, II, III), differing in the degree of phosphorylation. It was shown that phosphorylation of histone H5 increases the ability of the antigen to bind to antibodies, which is especially well-pronounced at the antiserum dilutions as high as 20480. The comparison of the antigenic properties of histones H5 from pigeon and chicken erythrocytes revealed beside structural differences of the proteins the presence of common antigenic determinants. A similar observation was made when histones H5 and H1 from pigeon erythrocytes were compared. Histone H1 from chicken erythrocytes and histone H1 from calf thymus did not produce criss-cross reactions with antiserum H5.

A method for direct registration of the first derivative of DNA reassociation kinetics in spectrophotometric cuvettes is proposed. Using this method, the rat liver DNA obtained by chromatography on hydroxyapatite was studied. The differential curves of reassociation and changes in hyperchromic spectra revealed the presence in the rat genome of 4 types of repeating sequences differing in their GC-content. A scheme of preparative separation of DNA with respect to the reassociation rate was developed. Three types of sequences repeating 3.10(5), 600 and 1 times in the genomes with the GC-content of 50, 42, and 33%, respectively, were obtained in a homogeneous state. The advantages of the differential method of registration of the kinetic curves for the study of DNA structure and calculation of fractionation schemes are discussed.

The repeated sequences situated in 5'- and 3'-flanking regions of the bovine growth hormone gene were cloned and characterized. It was shown that they are related to a described before PstI-family of bovine repeats with a frequency of repetition about 10(5). PstI-repeats are found only in genomes of sheep and goats subfamily (Carpinae) and not found in DNA of other analysed vertebrate species. Repeats of this family are situated in the genome as a number of dispersed clusters, they have conservative structure and are alike by the frequency of repetition in the DNA of all organisms that contain them. The presence of sequences homologous to PstI-repeats are shown in bovine pituitary poly(A)+ cytoplasmic RNA by means of North-hybridization.

From pigeon DNA two families of repeats have been isolated: one (frequency of repetitions 30--40 times per haploid genome) actively transcribing and the second (frequency of repetitions 2000--2800 times) weakly transcribing in erythroid cells. The reassociation kinetics, GC-content and size of isolated repeats were investigated. Actively transcribing repeats (average length of 350 nucleotides) contain about 55% GC-nucleotides and alternate in the genome with AT-rich sequences of the unique type. Comparison of isolated families of repeats revealed differences in the structure and posttranscriptional fate of RNA transcribed from them.

The 17-mer oligonucleotide probe homologous to the fragment of the gene for human erythrocyte differentiation factor erythropoietin was used to screen the human genomic library for this gene. Restriction analysis and partial sequencing of one of the identified clones have confirmed that the clone does contain the human erythropoietin gene. We are planning to use the cloned human erythropoietin gene for developing a stably transfected mammalian cell line that should secrete erythropoietin.

The erythropoiesis during anemia is characterized by the reduction of the bone marrow period of cell development. Two immature forms, absent in the normal state, were shown to be present under anemia: basophilic erythroblasts and reticulocytes with basophilic cytoplasm. These forms are interposed between the bone marrow basophilic erythroblasts and erythrocytes. Some basophilic erythroblasts turn out in the peripheral blood and transform into basophilic reticulocytes there. The level of transcription in anemic animals during the transformation of basophilic erythroblasts into basophilic reticulocytes does not decrease and is higher than that in polychromatophilic erythroblasts. The total chromatin condensation does not reflect the decrease of the transcription level.

Two discreet in size molecular classes of metabolically stable messenger-like RNA molecules with sedimentation coefficientes about 28S and 18S have been revealed in the nuclei of pigeon bone marrow cells. The structural pecularities of 28S RNA class were investigated more carefully. It was shown to constitute the largest fraction of nuclear messenger-like RNA and is characterized by a GC/AU ratio 1.13. As pulse-labeled nuclear RNA, 28S stable nuclear RNA hybridizes with the unique and rare repeated DNA, it does not contain poly(A)-sequences and is found in a form of RNP particles with density of 1.41 g/sm3, tightly bound to chromatin.

Proliferation and differentiation processes of chick embryo primary erythroid cells (PEC) were studied. A novel differentiation pathway was discovered by which cells of proerythroblastic and erythroblastic stages are blocked in G1 or G2 phases, to develop then directly into reticulocytes, i.e. terminally differentiated non-dividing cells with high hemoglobin contents differing in shape from erythrocytes. These cells appear in blood two days earlier than erythrocytes, then they co-exist with the latter and are eliminated in parallel with them. This pathway leads to a rapid enrichment of PEC with hemoglobin. A fraction of PEC forms accessory nuclei, which, as it is shown here, contain an extra quantity of DNA. Compared to the diploid ones, such cells reveal increased hemoglobin contents which enabled us to assume that they may have amplified the globin genes. The above-mentioned pecularities of cytodifferentiation may be presumably an adaptation to oxygen supply of growing embryos which are known to stay in hypoxia. A comparison of these results with results of our earlier study on experimental anemia makes it possible to suppose that pecularities of these two types of cytodifferentiation may be based on similar or, perhaps, analogous mechanisms of regulation.

Heterogenic population of the follicular oocytes of bovine ovaries has been divided into 5 groups on the basis of morphological characteristics. Chromatin structure, frequency of degenerative changes and the ability to maturation in vitro of different oocyte groups have been studied. The correlation of morphological characteristics of native oocytes and their ability to mature in vitro have been shown. The bovine follicular oocyte classification allows to pick out more homogeneous oocyte groups in comparison with the population as a whole and the oocyte group which is the best for maturation in vitro.