[Recognition of the potential SF-1 binding sites by SiteGA method, their experimental verification and search for new SF-1 target genes].

The SF-1 (Steroidogenic Factor-1) is a transcription factor known as a key regulator of the steroidogenic gene expression. SF-1 is required for the development and functioning at all levels of the hypothalamic-pituitary-gonadal and adrenal axis. Also it plays an essential role in sex determination. SF-1 is a member of the nuclear receptor superfamily and it activates gene expression by binding to DNA in a monomeric form. Here, we report the results of potential SF-1 binding sites identification by using the SiteGA recognition method. The SiteGA method was implemented using a genetic algorithm (GA) involving a iterative discriminant analyses of local dinucleotide context characteristics. These characteristics were compiled not only over the core binding sites region but over its flanks as well. Developed SiteGA method is characterized by considerably better recognition accuracy when compared to that for the weight matrix method. The experimental tests demonstrated that 83% of the sites recognized by the SiteGA method in the regulatory regions of steroidogenic genes, indeed, interact with the SF-1 factor. We also estimated the density of predicted sites in regulatory region of genes, the members of different functional groups and developed the criterion to search for new SF-1 target genes in genome sequences.

Binding sites for transcription factor SF-1 in promoter regions of genes encoding mouse steroidogenesis enzymes 3betaHSDI and P450c17.

Using gel retardation of DNA samples and specific antibodies, binding sites for the transcription factor SF-1 were found in positions -53/-44 and -285/-270 in the promoter region of the mouse Cyp17 gene and in position -117/-108 of the promoter region of the mouse 3betaHSDI gene.

Consensus sequence of transcription factor SF-1 binding site and putative binding site in the 5' flanking regions of genes encoding mouse steroidogenic enzymes 3betaHSDI and Cyp17.

Using 42 nucleotide sequences extracted from the Transcription Regulatory Regions Database (TRRD) containing SF-1 transcription factor binding site, we have determined the decanucleotide (GTCAAGGTCA) consensus sequence for SF-1 binding. In the frequency matrix of this sequence nucleotides between the 3rd and the 7th position had the highest frequency and guanine nucleotides at the 6th and the 7th positions were recognized in all nucleotide sequences. The latter suggests a crucial role of these guanines for the interaction of DNA with SF-1 protein. The determined consensus and frequency matrix were used for search of putative SF-1 binding sites in regulatory regions of two genes, encoding mouse Cyp17 (17alpha-hydroxylase/17-20-lyase) and 3betaHSDI (3beta-hydroxysteroid dehydrogenase/4delta-5delta-isomerase I), the microsomal enzymes involved in steroidogenesis. 5;-Flanking regions of genes encoding Cyp17 and 3betaHSDI were shown to contain six and five such binding sites, respectively. The presence of the putative SF-1 binding sites in the regulatory regions of mouse Cyp17 and 3betaHSDI suggests that gene SF-1 could represent one of the putative genes which (as we predicted earlier) determine coordinated inheritable variability of hormonal activity in mouse Leydig cells.

[The role of genotype, social stress, and season of the year in regulation of hormonal function of murine testis in vitro]. / Rol' genotipa, sotsial'nogo stressa i sezona goda v reguliatsii gormonal'noi funktsii semennikov in vitro u myshei

Micropopulations consisting of six male mice of different genotypes were studied (each of lines A/He, CBA/Lac, C57BL/6J, DD, YT, and PT was represented by one male). Interlinear differences in the level of social dominance and the effects of genotype, social hierarchy, and season on in vitro testosterone production by testes were examined under different incubation conditions. The testosterone production was estimated under control conditions and under stimulation with human chorionic gonadotropin (CG). Significant genetic differences in the initial and CG-stimulated testosterone production by testes incubated in vitro were found. By the control production, the genotypes fell into two groups: lines C57BL/6J, A/He, and CBA/Lac had low production of the hormone; lines YT, PT, and DD, high production. By responsiveness of gonads to CG, the genotypes fell into three groups: line CBA/Lac had low testosterone production by testes; lines C57BL/6J, A/He, YT, and DD, line PT, intermediate production; and line PT, high production. The obtained data indicate stability of genetic polymorphism for the responsiveness of testes to gonadotropins, because neither season nor the formation of social hierarchy could significantly change the interlinear differences. In line PT characterized by high hormonal activity of gonads in the control and under stimulation with gonadotropins, males became dominant in a significantly greater number of cases studied during the formation of hierarchy in micropopulations. The dynamics of both control production of a male sex hormone and responsiveness of testes to CG was established in vitro during the formation of social hierarchy; the effects of season on this dynamics were revealed. Specific characteristics of secretory activity of testes were detected in the control and under stimulation with gonadotropins, depending on incubation conditions. Seasonal and genotypic characteristics of the responsiveness of testes to CG were revealed under different incubation conditions. Genotypic characteristics indicate interlinear differences in the degree of inertia of testosterone biosynthesis on exposure to gonadotropins.

Transcription regulatory regions database (TRRD): its status in 2000.

Transcription Regulatory Regions Database (TRRD) has been developed for accumulation of experimental information on the structure-function features of regulatory regions of eukaryotic genes. Each entry in TRRD corresponds to a particular gene and contains a description of structure-function features of its regulatory regions (transcription factor binding sites, promoters, enhancers, silencers, etc.) and gene expression regulation patterns. The current release, TRRD 4.2.5, comprises the description of 760 genes, 3403 expression patterns, and >4600 regulatory elements including 3604 transcription factor binding sites, 600 promoters and 152 enhancers. This information was obtained through annotation of 2537 scientific publications. TRRD 4.2.5 is available through the WWW at http://wwwmgs.bionet.nsc.ru/mgs/dbases/trrd4/

Transcription Regulatory Regions Database (TRRD):its status in 1999.

The Transcription Regulatory Regions Database (TRRD) is a curated database designed for accumulation of experimental data on extended regulatory regions of eukaryotic genes, the regulatory elements they contain, i.e., transcription factor binding sites, promoters, enhancers, silencers, etc., and expression patterns of the genes. Release 4.1 of TRRD offers a number of significant improvements, in particular, a more detailed description of transcription factor binding sites, transcription factors per se, and gene expression patterns in a computer-readable format. In addition, the new TRRD release provides considerably more references to other molecular biological databases. TRRD 4.1 is installed under SRS and is available through the WWW at http://www.bionet.nsc.ru/trrd/

[Determination of metabolism of steroid hormones and activity of their synthetic enzymes in murine Leydig cells by high performance liquid chromatography]. / Opredelenie metabolizma steroidnykh gormonov i aktivnosti fermentov ikh sinteza v kletkakh Leidiga myshei metodom vysokoéffektivnoi zhidkostnoi khromatografii.

A very effective procedure was developed for separation of sex hormones of the delta 4 pathway using the microcolumn technique of high performance liquid chromatography (HPLC). A five-step gradient containing methanol, acetonitrile and water was used as a mobile phase, while Lychrosorb RP-18 with particles of 5 microns was used as a sorbent. Metabolism of steroid hormones and activity of 3 beta-steroid dehydrogenase, 17 alpha-hydroxylase, C17-20-lyase and 17-ketosteroid reductase were studied in Leydig cells of two mouse strains PT and A/He. The higher rate of steroid metabolism and of the enzymatic activity was detected in the mouse cells of PT strain as compared with those of A/He strain.