[The study of preimplantation development of mice embryos labelled with green fluorescent protein gene (EGFp)].

One of the crucial problems of developmental biology is the study of mechanisms of regulation of gene expression in early embryogenesis. Here we studied dynamics of mosaic appearance of a marker fluorescent protein in in vitro developing mice embryo derived from zygotes after microinjections to male pronuclei of cloned DNA fragment carrying EGFP under control of different promoters. Main attention was paid to initial stages of development, when structural rearrangements and reprogramming of both parental genomes, activation of zygotic genes, and control of development by embryo genome take place.

[Primary structure of cDNA for bovine beta-casein]. / Pervichnaia struktura kDNK beta-kazeina korovy.

In this work the complete sequence of 1097 nucleotide bovine beta-casein cDNA has been determined. Sequencing of end labeled fragments was performed using the method of Maxam and Gilbert. Bovine beta-casein cDNA consist of a 672 nucleotide coding region, flanked by 60 nucleotide 5' and 358 nucleotide 3'-noncoding region. The restriction map of beta-casein cDNA was constructed. The computer analysis was done and the comparisons of the complete sequence of bovine beta-casein cDNA with other sequences, coding caseins in bovine, rat and guinea-pig was performed. It was determined, that cloned cDNA is related to genetic variant A1.

[Activation of a trans-activating factor of NF1 transcription in a lactating mammary gland]. / Aktivatsiia trans-deistvuiushchego faktora transkriptsii NF1 v laktiruiushchei molochnoi zheleze.

Functional differentiation of a bovine mammary gland in the course of lactation is characterized by significant increase of tissue-specific expression of genes encoding milk proteins (caseins, lactoglobulin etc.). The NF1 is known as a ubiquitous transcription factor which modulates a tissue-specific transcription of different genes (including beta-casein) cooperating with tissue-specific and other ubiquitous transcription factors. We have observed a dramatic increase of NF1-binding activity in nuclear extracts of bovine mammary gland during lactation. The NF1 transcription factor appears to have a cytoplasmic precursor pool. This cytoplasmic precursor as well as NF-kappa B cytoplasmic precursor could be activated in vitro by deoxycholate (DOC) treatment which caused possibly dissociation of a complex of NF1 and its cytoplasmic inhibitor. There was an inverse proportion between concentrations of active nuclear NF1 factor and its cytoplasmic precursor. We have observed an increase of nuclear factor binding and a simultaneous decrease of the cytoplasmic precursor pool in the course of lactation. We have determined the NF1 protein subunit composition using UV-cross-linking 32P labeled NF1-oligonucleotide with nuclear and cytoplasmic proteins of mammary gland. The main subunits of NF1 factor were p50 and p20. The drastic increase of nuclear NF1 binding activity was correlated with significant increase of the p20 subunit concentration in nuclear protein during lactation.

[Complexity and diversity of poly(A)-containing mRNAs from different tissues of Bos taurus]. / Slozhnost' i geterogennost' poli(A)-soderzhaschikh informatsionnykh RNK iz raznykh organov Bos taurus.

The kinetic complexity of poly(A)+-mRNA isolated from Bos taurus liver, kidney and spleen polysomes has been studied using the mathematical analysis of kinetic curves of hybridisation between mRNA and complementary DNA (cDNA) synthesized by means of reverse transcriptase. Liver, kidney and spleen polysomal mRNA have been found to be represented by 9300, 10 200 and 7200 mRNA types respectively. These mRNA are transcribed from 0.4--0.6% of genome. The sequences of the studied mRNAs are grouped in four discrete classes differentiated by the complexity of polynucleotide sequences and by the frequency per cell. The comparative study of homology of nucleotide sequences of liver, kidney and spleen mRNA has been undertaken using cross hybridization of cDNA with heterologous mRNA. About 40--67% of molecules of the total poly(A)+-mRNA are common for all the tissues under study.

[Specific interaction of a nuclear protein factor from the CTF/NF-I family with 2 different promoter regions of the rat tyrosine aminotransferase gene]. / Spetsificheskoe vzaimodeistvie iadernogo belkovogo faktora semeistva CTF/NF-I s dvumia raznymi uchastkami promotora gena tirozinaminotransferazy krysy.

Using gel-retardation and DNase I footprinting assays, we have analysed sequence-specific DNA-protein interactions within proximal promoter fragment (from -2 to -210 bp relative to the transcription start) of the rat tyrosine aminotransferase (TAT) gene. Two distinct DNase I protection regions flanked at either boundary by sites of DNase I hypersensitivity were observed with the rat-liver nuclear extracts. The internal sequence of the region I non-coding strand, (-155)TGGGCCACCTTCCAAT(-170), is highly homologous to the NF-I consensus sequence TGG(N)6-7TGCCAA and also shares a CCAAT-box; the region II noncoding strand sequence includes asymmetrically positioned (-37)AGCCAAT(-43) recognition motif. Since there have been a number of reports about multiple DNA-binding factors that recognize CCAAT homologies, both regions were likely to interact with either a single or distinct factors. Here we show that both region I and II of the TAT gene promoter are binding to the same factor related to the human CTF/NF-I. The evidence for that is based on competition experiments using the DNA fragment containing a synthetic consensus sequence for the NF-I recognition site and on the indistinguishable chromatographic properties of the activity specifically binding to each of three DNA fragments containing NF-I consensus, region I and region II sequences.

[Nuclear factor A5 from the rat liver that requires a metal for specific interaction in vitro with distal element of the tyrosine aminotransferase gene promoter]. / Iadernyi belkovyi faktor A5 iz pecheni krysy, kotoromu neobkhodim metall dlia spetsificheskogo vzaimodeistviia in vitro s distal'nym élementom promotora gena tirozinaminotransferazy.

To detect nuclear protein factors which might account for a tissue-specific and inducible expression of the rat tyrosine aminotransferase (TAT) gene promoter, extracts from rat liver and spleen nuclei have been fractionated by heparin-sepharose chromatography and the fractions assayed for sequence-specific binding to the distal TAT gene promoter element (sequence between -313 and -210). Gel retardation experiments carried out in the presence or absence of Mg2+, Ca2+, or Zn2+ ions showed that there are at least two nuclear factors (A3 and A4) binding to the distal promoter element only in the presence of the chelator (20 mM EDTA). Incubation of the protein fractions with Zn2+ or Ca2+ instead of commonly used Mg2+allowed: (i) to avoid 3 2P-DNA-probe degradation by "contaminating" endogenous nucleases; and (ii) to detect another sequence-specific nuclear factor, A5. No other specific binding activities were found in the rat-liver nuclear fractions tested under these conditions. As the metal ions became inaccessible to chelation in excess of EDTA and EGTA when protein factor A5 was complexed to DNA we assumed that factor A5 is metalloprotein which requires Zn or Ca to maintain a structure of its DNA-binding domain. To identify the polypeptide possessing this domain, a protein gel blotting procedure was employed. By incubating gel blots with the 3 2P-DNA-probe in the buffer containing Zn2+, specific binding to the only polypeptide with approximate Mr 30 kDa was clearly revealed. Both gel retardation and gel blotting assays consistently showed that nuclear factor A5 is present in the liver, but not in the spleen extracts.

Isolation of a hybrid between rat ribosomal RNA and DNA.

A procedure for the isolation and purification of a specific hybrid between rat 28S and 18S ribosomal RNA's and nucleolar DNA is described. The method employed includes the following steps: 1) isolation of the nucleolar DNA, 2) hybridization of [14C]rRNA with the nucleolar DNA, and 3) isolation and purification of the rRNA-DNA hybrid complex by chromatography on hydroxylapatite and centrifugation in a CsCl density gradient. In the isolated hybrid complex the RNA:DNA ratio is close to 1:1, and the degree of enrichment of the DNA by the rRNA cistrons is about 1500 times. The hybrid obtained has a sedimentation constant on the order of 20S, is resistant to the action of pancreatic RNase and RNase T1 and sheep brain DNase, and is characterized by high thermostability. Acording to the physicochemical tests used, the rRNA-DNA hybrid complex is a double-stranded poly-nucleotide with an ordered secondary structure.

[Primary structure of cDNA of Bos taurus kappa-casein macropeptide]. / Pervichnaia struktura kDNK makropeptida kappa-kazeina Bos taurus.

By means of nucleotide sequencing in a library of clones, containing cDNA of Bos taurus mammary gland, a clone corresponding to kappa-casein has been identified. In addition, the region encoding for the complete nucleotide sequence of cDNA of kappa-casein B macropeptide has been detected. The nucleotide changes in the DNA sequence have been identified which mean that the isolated clone corresponds to the genetic variant B of kappa-casein.

[Nucleotide sequence of the cDNA of kappa casein in cows]. / Analiz nukleotidnoi posledovatel'nosti kDNK kappa-kazeina korovy.

Nucleotide sequence of the cloned DNA complementary to cow kappa-casein mRNA was determined. The complete sequence is composed of 854 bases and includes 60 bases of 5'-noncoding region, 570 bases of the coding region and 209 bases of 3'-noncoding region without poly(A). The cloned sequence codes for kappa-casein, the genetic variant B2. Several restriction sites were defined that permitted the identification of genetic variants and size polymorphism of restriction fragments in the structural region of the gene. Upon the comparative analysis of kappa-casein mRNAs of cow and rat, it was shown that, apart from functionally important regions in the coding sequence, high homology is characteristic of the 5'- and 3'-noncoding regions.

[The casein genes of Bos taurus. II. Isolation and characteristics of the beta-casein gene]. / Geny kazeinov Bos taurus. Soobshchenie II. Vydelenie i kharakteristika gena beta-kazeina.

The expression of the casein genes in mammary gland cells is regulated by peptide and steroid hormones. To study underlying regulatory mechanisms, the bovine beta-casein gene was isolated and characterized from lambda bacteriophage bovine DNA library. The beta-casein gene is 8.6 kb long and is 7.8 times longer than the mature casein mRNA coded for by 9 exons. The genomic clones incorporate additional 8.5 and 4.5 kb of the 5'- and 3'-flanking regions. The nucleotide sequences of 5' and 3' ends of the beta-casein gene are determined. Conserved sequences identical or homologous to potential sites of binding with the nuclear factor CTF/NF-1, glucocorticoid and progesterone receptors were identified. The regulatory region of the casein gene contains two different TATA signals flanking the duplication site in the promoter region.

[Restriction analysis of cDNA for cow alpha s1-, beta- and kappa-caseins]. / Restriktsionnyi analiz kDNK alpha s1-, beta- i kappa-kazeinov korovy.

By means of in situ hybridization to cloned cDNA fragments coding for cow alpha s1-, beta- and kappa-caseins, screening of the library of clones containing the cDNA complementary to mRNA of lactating cow mammary gland was carried out. The clones containing the sequences of alpha s1-, beta- and kappa-casein cDNAs were shown to constitute 4.0, 3.2 and 0.7% of all the colonies, respectively. The analysis of the data on cross-hybridization points to the absence of extensive regions of homology between the above-mentioned cDNAs. The restriction analysis of cDNAs of the selected clones was carried out and the restriction maps of cDNAs of these three caseins were constructed. The restriction analysis data and determination of the nucleotide sequence of 5'-termini of the studied cDNAs indicated that the cloned sequences were the full-length mRNA copies of alpha s1-, beta- and kappa-caseins. The data obtained on restriction analysis are utilized in mapping the corresponding natural genes of cow caseins.

[Synthesis and cloning of DNA complementary to mRNA of the bovine mammary gland]. / Sintez i klonirovanie DNK, komplementarnykh mRNK molochnoi zhelezy korovy.

Poly(A+)mRNA from bovine mammary glands was used to synthesize double-stranded cDNAs that were subsequently inserted into the plasmid vector pBR322 at the Pst1 site by means of oligo(dG)-oligo(dC) tailing. After transfection of Escherichia coli JC5183, recombinant plasmid library containing 5400 clones was screened by serial rounds of colony hybridization in situ to total [23P] poly(A+)mRNA and electrophoretically homogenious [32P]16SmRNA of mammary glands. Then hybrid selection of mRNA and subsequent in vitro translation of selected mRNAs were performed. In this manner, recombinant clones coding for alpha S1- beta-, kappa-casein were identified. cDNA clones range in size from 35% for beta-casein, 65% for alpha S1-casein to about 95% for kappa-casein, in comparison with their respective mRNAs.

[Loss and stabilization of aminopterin resistance in murine cell lines]. / Poteria i stabilizatsiia ustoichivosti k aminopterinu v kletochnykh liniiakh myshi.

Mouse L-cell lines (B-82, tk-) were obtained using the stepwise selection method, their aminopterin (AP) resistance being 10(3)-5 X 10(4) times higher than that of parental cells. This resistance increase results from dihydrofolate reductase (DHFR) gene amplification which was determined from the 15-120-fold rise of the enzyme activity and with the cytogenetical techniques. The development and loss of AP resistance have been studied and karyological analysis of the lines obtained carried out. Two types of karyological changes were found in stable DM and HSR cells which correspond to extrachromosomal and intrachromosomal forms of the amplified material organization. Localization of the DHFR gene in HSR was proved using the in situ hybridization technique. Extrachromosomal localization of the amplified genes in DM providing unstable AP resistance is dominant at the early stages of the development of resistance and for a long time. It was demonstrated that DM and HSR can exist in one cell during the prolonged period. DHFR gene copy number in such cells is regulated by a change in the DM number, whereas the HSR size and localization are highly stable. HSR covers 1.7-1.9% of the genome length and 38-40% of the marker chromosome length. The genes localized in HSR provide stable AP resistance. Evidence on some intermediate, relative stabilization of the resistance has been obtained. This stabilization is mediated by temporary integration of DHFR copies into other chromosomal sites, in addition to HSR.

[The Bos taurus casein genes. Isolation and characterization of the kappa-casein gene]. / Geny kazeinov Bos taurus. Vydelenie i kharakteristika gena kappa-kazeina.

A region spanning 25 kb of genomic DNA containing the kappa-casein gene, has been isolated from two genomic libraries in EMBL3 and EMBL4 phage vectors. Five phage clones containing kappa-casein gene have been found. Gene organisation has been determined using restriction mapping and a partial sequencing the 5' and 3' flanking regions. The kappa-casein gene includes 5 exons, the first of them coding for 64 nucleotides from the 5' untranslated mRNA zone. The gene is 12.5 kb long, which is almost 16 times longer than the corresponding mRNA. The first intron spans 2.5 kb, the second is the largest one and spans 5.5 kb. The 5' flanking region sequence has been analysed; it contains a TATA box from -30 to -25 bp, somewhat different from the canonic sequence, and a CAAT box at -80 bp.

[Genetic activity of para-aminobenzoic acid. The intensification of DNA polymerase I-dependent repair induced by chemical mutagens in toluene-treated Escherichia coli cells]. / Geneticheskaia aktivnost' para-aminobenzoinoi kisloty. Usilenie DNK-polimeraza-I-zavisimoi reparatsii, indutsirovannoi khimicheskimi mutagenami v toluolizirovannykh kletkakh Escherichia coli.

Alkylatio of Escherichia coli DNA that have been made permeable to nucleotides by toluene treatment results in the expression of DNA polymerase I-directed repair synthesis. The system only permits measurement of DNA polymerase I-directed repair synthesis. The latter is not observed in mutant cells deficient in this polymerase. DNA ligation is intentionally prevented by the addition of the inhibitor, nicotinamide mononucleotide. MNU, ENU and MMS elicit DNA polymerase I-directed repair synthesis. MNU and MMS are especially potent in this regard, while EMS is a poor inducer of DNA polymerase I activity in permeabilized cells. The natural compound para-aminobenzoic acid itself (0,0002 mM - 20 mM) doesn't induce DNA polymerase I-directed repair synthesis. However, when PABA is used in complex with alkylating agents as the inducers, the repair synthesis increased 2,0, 1,2 and 2,8 times for MNU, ENU and EMS, respectively, as compared to that elicited by "pure" mutagens. The increasing of DNA repair synthesis in permeabilized bacteria in the experiments with PABA may serve as the foundation for its reparagenic activity. The latter was discovered previously by the authors in experiments on mutagenesis of bacterial cells.

[Cloning and nucleotide sequence analysis of the cDNA of the immunoglobulin lambda light chain from the bovine udder]. / Klonirovanie i analiz nukleotidnoi posledovatel'nosti kDNK legkoi lambda-tsepi immunoglobulina iz molochnoi zhelezy korovy.

A cDNA library was constructed from the mRNA of bovine mammary gland which contained Ig lambda producing plasmacytes. Overlapping clones encompassing the major portion of the coding sequence of the Ig lambda mRNA were isolated and sequenced. Predicted amino acid sequence shows a mature polypeptide of 217 residues in which V, J and C regions can be distinguished. The constant region has greatest homology with human Ig lambda mRNA constant region. The area of the V region between CDR3 and CDR2 has also great homology with the same area of human lambda chain.