The immediate vicinity of mouse metallothionein-I gene contains two sites conferring glucocorticoid inducibility to the heterologous promoter.

Glucocorticoid responsive elements (GREs) located -252 to -209 by upstream and +1011 to +1054 bp downstream of the transcription initiation site of the mouse metallothionein-I (mMT-I) gene were identified in transient experiments. However, the promoter region of the mMT-I gene (-330 to +70 bp) was found to provide low, if any, glucocorticoid induction of the linked CAT gene, while showing strong cadmium regulation, comparable with the in vivo level.

Cyclic GMP phosphodiesterase from cattle retina. Amino acid sequence of the gamma-subunit and nucleotide sequence of the corresponding cDNA.

The primary structure of the gamma-subunit of cyclic GMP phosphodiesterase was determined by parallel analysis of the amino acid sequence of the protein and nucleotide sequence of the corresponding cDNA. The enzyme gamma-subunit contains 87 amino acid residues, its N-terminal amino group being acetylated.

The energetics of the B-Z transition in DNA.

The paper deals with the energetics of the transition to left-handed Z form in DNA with an arbitrary base sequence. There is a brief outline of the statistical-mechanical model of the B-Z transition allowing for three possible states of each base pair. The parameters of the model can be determined by comparing the theory with experimental data for the B-Z transition in inserts with given sequences in circular DNA. The model contains six energy parameters, most of which have been determined before. In order to find the remaining parameters of the model and test its adequacy, a number of oligonucleotide sequences were synthesized and inserted into the pUC 19 plasmid. Two-dimensional gel electrophoresis was used to determine the superhelical density at which the inserts adopt the Z form. A statistical-mechanical treatment of these data yielded a complete set of six energy parameters for the B-Z transition. The theoretical assumption that the free energy of Z-form pairs does not depend on the type of adjacent pairs proved to be in agreement with the experimental data.

[Sequences homologous to the rat ID element in mouse DNA]. / Posledovatel'nosti, podobnye élementu ID krysy, v DNK myshi.

Sequences homologous to the rat frain specific identifier sequences were found in the mouse genome. These sequences were defined by dot- and blotting-hybridisation in different DNA and RNA preparations. It was shown that there are (1-2) X 10(3) copies of these sequences per genome. They are not transcribed in mouse brain and possibly are not connected with mouse brain specific genes expression.

[Identification of the glucocorticoid receptor binding site at the 5'-flanking region of mouse metallothionein I gene: the effect of base substitutions on binding efficiency]. / Identifikatsiia uchastka sviazyvaniia gliukokortikoid-retseptornogo kompleksa v 5'-flankiruiushchem raione gena metallotioneina I myshi: vliianie nukleotidnykh zamen na éffektivnost' sviazyvaniia.

Interaction of highly purified glucocorticoid receptor complex (GIRC) with synthetic DNA-fragment of mouse metallotionein 1 gene promoter from -209 to -252 b.p. (MTwt) was investigated. By means of nitrocellulose filter binding assay this fragment was shown to contain specific GIRC-binding site. In order to analyse the fine structure of the site, two variants of this DNA-fragment were synthesized and used in gel retardation assay. GIRC specific binding was shown to retain throughout interaction with the fragment in which all base pairs in the surroundings of generally accepted GIRC-binding site consensus G--ACA---TGTTCT C--TGT---ACAAGA were substituted by means of transitions, but it was weaker than the GIRC-binding with MTwt, where the mentioned consensus was situated in the natural surroundings. Complete loss of the GIRC-binding ability was observed when five CG pairs were substituted by AT ones. Two of the CG pairs belonged to the mentioned consensus. Comparison of the data obtained with results of computer analysis allows to consider the consensus as a "core" of GIRC-binding site, flanked with additional elements, interacting with GIRC.

[Inclusion of biotinylated analogs of dUTP and dCTP in DNA by DNA-polymerases. Cloning DNA fragments, containing biotinylated deoxyribouridine in E. coli]. / Vkliuchenie v DNK biotinilirovannykh analogov dUTP i dCTP DNK- polimerazami. Klonirovanie v E. coli fragmenta DNA, soderzhashchego biotinilirovannyi dezoksiribouridin.

The synthesis of a biotinylated derivative of dCTP, viz. N4-[(N-biotinyl)-4-amino-butoxyl]-2'-deoxycytidine 5'-triphosphate (I), is described. DNA polymerase I (Klenow fragment) incorporates (I) in DNA chains instead of thymidine, although with a lower efficiency than previously described biotinylated dUTP derivative (II), whereas highly purified DNA polymerase alpha from human placenta uses as substrate derivative (II) but not (I). A DNA fragment bearing biotin residues in one of strands was synthesized with the use of DNA polymerase alpha and dUTP derivative (II); its cloning in the plasmid vector pBR322 revealed that the DNA nucleotide sequence remained intact.

[Localization of a lysine residue near the site of initiating substrate binding of T7 bacteriophage RNA polymerase]. / Lokaliatsiia ostatka lizina v oblasti sviazyvaniia initsiruiushchego substrata RNK-polimerazy bakteriofaga T7.

A highly selective affinity label was introduced into the T7 phage RNA polymerase by means of GMP ortho-formylphenyl ester and [alpha-32P]UTP nearby the enzyme's active site, which was located using limited cleavage technique. Hydroxylamine, bromine, N-chlorosuccinimide, and cyanogen bromide were employed as the reagents. Analysis of gel-electrophoretic patterns of the cleavage products led to a conclusion that Lys631 is the target of labelling. The region nearby this residue has a high degree of sequence homology with regions of RNA polymerases from T3 and SP6 phages and yeast mitochondria.

[An improved chemico-enzymatic method of synthesizing long DNA segments]. / Usovershenstvovannyi khimiko-fermentativnyi metod sinteza protiazhennykh fragmentov DNK.

A simple and economy method of the biochemical assembling of long double-stranded DNA segments is described. A single-stranded polydeoxynucleotide 122 bases long representing a fragment of synthetic gene of human beta-interferon was assembled from three synthetic fragments 36 (two) and 50 bases long on four complementary 12-mers as templates. This single-stranded polynucleotide was converted, in the presence of DNA polymerase 1 and a 12-meric primer, in to the full-length double-stranded DNA (the beta-interferon gene segment). It was cloned into an E. coli plasmid vector pBR322 and its sequence confirmed.

[Rapid automated synthesis of polydeoxynucleotides]. / Bystryi avtomaticheskii sintez polidezoksinukleotidov.

A rapid automatic method of synthesis of deoxypolynucleotides from 5'-O-dimethoxytritylnucleoside-3'-H-phosphonates is described. An improved construction of synthesizer "Gene-2" adapted for this method has been developed. The modified scheme of synthesis included detritylation with trifluoroacetic acids in dichloromethane, washing with acetonitrile instead of pyridine--acetonitrile mixture and one-step oxidation with iodine solution in acetic acid and pyridine instead of two-step oxidation in the presence of amines. By means of this method, more then 160 polynucleotides containing 8 to 83 monomers were prepared for various biochemical goals including synthesis of promotor 9(260 bp) of the mouse metallothionein-I gene and of promotor and leader sequence (120 bp) of gene of the E. coli alkaline phosphatase.

[Cloning of single-stranded synthetic DNA]. / Klonirovanie odnotsepochechnykh sinteticheskikh DNK.

The method for cloning a single-stranded synthetic DNA with the short complementary oligonucleotides, that form corresponding restriction sites, is proposed. The potency of the method is demonstrated by cloning a single-stranded polynucleotide A (93 nucleotide residues (n. r.] in plasmid vector pBR327. The polynucleotide A includes a leader structure of the human fibroblast interferon gene. Oligonucleotides (IV) (20 n. r.) and (VI) (16 n. r.) were taken as strengthening complements and to create the sticky ends for the restrictases HindIII and EcoRI. 72% of the obtained clones appeared to be hybrid. Four hybrid clones were analyzed, and three of them carried the desirable insertion. The primary structures of these insertions are confirmed by sequencing.

[Plasmid vector pSSC1 for cloning synthetic polynucleotides and their regeneration with a unique nucleotide sequence]. / Plazmidnyi vektor pSSC1 dlia klonirovaniia sinteticheskikh polinukleotidov i ikh regeneratsii s unikal'noi nukleotidnoi posledovatel'nost'iu.

For subcloning separate synthetic gene fragments, a plasmid vector pSSC1 was constructed by inserting a synthetic polylinker into plasmid pBR 327 at the EcoRI-PstI sites. There are two FokI and HgaI sites at the ends of this polylinker in the opposite orientation, with the EcoRI and PstI sites between them. DNA fragments cloned at the EcoRI and PstI sites can be regenerated by either FokI or HgaI, the EcoRI and PstI sites being deleted from the cloned sequences. Such fragments have unique cohesive ends that allows their directed ligation into longer DNA (genes).

[Determination of the primary structure of oligodeoxyribonucleotides containing P-S-C(5') bonds]. / Opredelenie pervichnoi struktury analogov oligodezoksiribonukleotidovs P-S-C(5')-sviaziami.

Analogues of oligodeoxynucleotides with P-S-C(5') bonds, which, due to their unusual substrate properties, may find interesting applications in molecular biology, can not be structurally analysed by the Maxam-Gilbert or Sanger (fingerprinting) methods. We, therefore, devised a modification of the fingerprinting technique making possible the sequence determination of these analogues.

[Current approaches to the synthesis and reconstruction of genetic material]. / Sovremennye podkhody k sintezu i rekonstruktsii geneticheskogo materiala.

Advanced approaches to the synthesis and reconstruction of genetic material developed in the Institutes of Molecular Biology and Genetics during the past years are summarized. The evolution of methods for oligonucleotide synthesis and scopes for their use in gene production are discussed. The principles of localised mutagenesis methods developed in the Institute are described, such as: a) mutagenesis directed to the regulatory gene regions; b) segment-localized mutagenesis; c) mutagenesis directed by phosphotriester analogues of oligonucleotides. Examples of employing these methods for induction of regulatory mutants of phage lambda, production of fused genes, mutant interferon genes, construction of new DNA vectors, construction of hybrid H1-H3 subtype haemagglutinine gene of influenza virus etc. are presented. The approach to in vivo site-directed mutagenesis is experimentally substantiated.

[An experimental infection of the seal Phoca sibirica with the morbilli virus]. / Eksperimental'noe infitsirovanie nerpy Phoca sibirica morbillivirusom.

A Baikal seal (Phoca sibirica) was experimentally infected with Baikal seal morbillivirus (BSMV) isolated from Baikal seals during an epizootic in 1987-1988. The seal was infected with BSMV with an infectious titer of 10(7.0) TCD50/ml, and daily observations of the animal clinical condition were made. The virus-specific antibodies in the seal serum were determined by ELISA and virus neutralization test. The clinical picture showed a mild infection. The ELISA-active antibodies were detected 10 days postinfection and reached the maximum in 20 days. Virus-neutralizing antibodies were detected in 16 days after infection, reached the maximum titer of 1:640 in 20 days and remained at this level for 39 days (the observation period). These data indicate that BSMV can induce a disease in the natural host with production of virus-neutralizing antibodies. The results of this work and the earlier reports show that the epizootic in Baikal seals was induced by BSMV.

[Antibodies to morbilli virus of the Baikal seal in its natural host]. / Antitela k morbillivirusu baikal'skoi nerpy u prirodnogo khoziaina.

Morbillivirus of Baikal seal (BSM) was isolated from organs of a dead animal during 1987-1988 epizootic of Baikal seal (Phoca sibirica). A method of cellular enzyme immunoassay for testing for virus-specific antibodies was developed using BSM. The method was used for antibody detection in sera of 115 apparently normal seals collected in the spring of 1989. Antibody to BSM were found in sera from 75 animals. Examinations of seropositive animals of different age and sex were carried out. The results obtained indicate a possible role of BSM in the epizootic.