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.

Molecular cloning and sequencing of omul prolactin cDNA.

We constructed a complementary DNA (cDNA) library from Baikal omul (Coregonus autumnalis migratorius Georgi) pituitary glands. Synthetic oligonucleotide probes corresponding to chum salmon prolactin (PRL) cDNA were used to select the recombinant plasmids carrying the omul PRL gene. The larger insert was sequenced and found to encode a polypeptide of 210 amino acid residues, including a putative signal sequence of 23 amino acids. Nucleotide and predicted amino acid sequence of omul PRL showed high homology with other salmonid PRLs.

[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.

[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.

Comparison of two morbilliviruses isolated from seals during outbreaks of distemper in north west Europe and Siberia.

Recently morbilliviruses were isolated from harbour seals (Phoca vitulina) in North West Europe (phocid distemper virus-1: PDV-1) and from Baikal seals (Phoca sibirica) in Siberia (phocid distemper virus-2: PDV-2) during outbreaks of severe disease which resembled distemper in dogs. PDV-1 and PDV-2 were passaged in SPF dogs, in which they caused distemper-like disease symptoms, and were subsequently passaged in Vero cells in which they caused cytopathic changes. PDV-1, PDV-2, and canine distemper virus (CDV) were compared with respect to their biological, morphological, physical, protein chemical, and antigenic properties. It was concluded that PDV-1 should be considered a newly recognized member of the genus Morbillivirus, whereas PDV-2 proved to be quite similar if not identical to CDV.

Energetics of the B-H transition in supercoiled DNA carrying d(CT)x.d(AG)x and d(C)n.d(G)n inserts.

We have studied the B-H transition in the d(AG)x inserts of varying length under superhelical stress. The new data and previously published results for the d(G)31 insert are treated within a phenomenological model of the B-H transition, making it possible to obtain, for the first time, the energy parameters of the B-H transition in the d(AG)x and d(G)n sequences.

[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.

[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.

[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.

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.