[Unique steroid 21-hydroxylase gene CYP21A2 polymorphism in patients with hyperandrogenism signs].

Hyperandrogenism is a medical condition characterized by excessive production of male sex hormones (androgens) in woman organism. One of the major causes of hyperandrogenism is the autosomal-recessive disorder--congenital adrenal hyperplasia (CAH). The mutational defects in the steroid 21-hydroxylase CYP21A2 gene causing steroid 21-hydroxylase deficiency account for over 90% of CAH cases. Our paper describes the sequencing results of entire CYP21A2 gene from 15 patients with hyperandrogenism signs, which had not nine most prevalent mutations associated with nonclassic CAH as it was previously established. 26 polymorphisms were found by sequencing among which 25 were known previously and 23 of them are referred to "normal" gene variants which do not associated with CAH. At the same time the gene of every patient had unique its own distinctive combination of polymorphisms. New SNP represents synonymous substitution C --> T in 3' part of exon 8. All detected SNPs are not regularly distributed but are clustered along the gene. Notably, they were found in the neighborhood of initiation and termination codons and near the intron-exon boundaries of introns 2, 6 and 8. We hypothesize that "normal" clinically insignificant per se SNPs in unique combinations may influence spatial structure of CYP21A2 mRNA or its pre-mRNA splicing efficiency and decrease gene expression level. This assumption may explain the mechanism of pathological phenotype development in our patients.

[A new DNA diagnostic system for the detection of human CYP21 gene mutations associated with adrenal cortex hyperplasia].

Congenital Adrenal Hyperplasia (CAH) is one of the most widespread severe autosomal recessive hereditary diseases. CAH is caused by the impaired biosynthesis of the key human hormones cortisol and aldosterone and is accompanied by the excess synthesis of androgens. Over 90% of CAH cases are caused by a deficiency of the steroid 21-hydrohylase (P450c21). The degree of damage in this enzyme is responsible for the severity of the clinical manifestation of CAH from potentially lethal to mild symptoms. Various mutations of the gene encoding this enzyme are the main source of the reduced activity of the 21-hydrolase. The location of the highly homological pseudogene CYP21P in close proximity to the functional gene impedes the DNA diagnostics of CAH. To detect the eight most frequent CYP21 gene mutations associated with CAH, we developed a new real-time PCR-based system of DNA diagnostics using new allele-specific primers and TaqMan probes for the analyzed mutations. The method was primarily tested on artificial DNA templates, where the analyzed mutations were introduced by site-directed mutagenesis. Then, it was tested on DNA samples from 43 patients with clinical and biochemical manifestations of CAH; seven patients were used as a control. Two mutant alleles were detected in two different individuals: the nonsense Q318X and the missense V281L mutations.

[Some practical notes about performing polymerase chain reaction and electrophoretic fractionation of amplificated products].

Practical aspects concerning standardization of molecular-genetic expertise performing with the use of the method of DNA are considered. Examples of difficulties, which can occur at nonobservance of requirements of polymerase chain reaction and electrophoresis performing, are described; practical recommendations of their elimination are given.

Prevalence of genes encoding exfoliative toxins among Staphylococcus hyicus isolated in Russia and Germany.

In the present study, previously characterized Staphylococcus hyicus isolated in Russia (n=23) and Germany (n=17) were investigated for the prevalence of the exfoliative toxin encoding genes exhA, exhB, exhC and exhD by multiplex PCR resulting in the detection of exhD positive strains among the S. hyicus isolated from pigs with exudative epidermitis in Russia and the detection of exhC and exhD for one and two strains isolated from exudative epidermitis in Germany respectively. The toxin gene negative strains were generally isolated from apparently healthy pigs, from other animals and from specimens where the relation between the isolation of S. hyicus and the clinical symptoms remained unclear. Partial sequencing of the toxin genes of selected exhC and exhD positive strains and comparing the sequencing results with sequences of exhC and exhD reference strains revealed an almost complete identity. The results of the present study were in agreement with the findings of Andresen and Ahrens (J. Appl. Microbiol., 96, 2004, 1265) and Andresen (J. Vet. Rec., 157, 2005, 376) that the presented multiplex PCR could be used to investigate S. hyicus for toxinogenic potential and that there is an association between the presence of toxin genes in S. hyicus strains from exudative epidermitis. However, comparable with the S. hyicus strains isolated in Germany which were investigated previously by Andresen (J. Vet. Rec., 157, 2005, 376), exhD seems to predominate in S. hyicus strains from Russia.

[Promoters of genes MTHFR from patients with hyperhomocysteinemia and PTEN from patients with malignant and benign endometrial and ovarian tumors].

Mutational changes in the promoter regions of MTHFR genes from patients with hyperhomocysteinemia and PTEN genes from patients with endometrial and ovarian tumors were studied. An increased level of homocysteine was found in a part of the patients with a heterozygous C677T mutation in the MTHFR gene, although a moderate hyperhomocysteinemia is usually associated with homozygous mutation. We hypothesized that, in this case, the allele lacking the C677T mutation may be inactivated by the promoter mutation. The sequencing of both DNA strands of the minimal promoter region of the MTHFR gene in ten patients did not reveal any mutation, which implied another mechanism of the development of hyperhomocysteinemia in these patients. A PCR analysis of the minimal promoter region of the tumor suppressor PTEN in the presence of 2-pyrrolidone in 101 patients from Moscow clinics revealed changes in it in patients with endometrial (56%) or ovarian (29%) cancer, as well as in patients with endometrial hyperplasia and benign ovarian tumors (34.6 and 29%, respectively). It was presumed that the found PTEN gene promoters may arise from epigenetic alterations (erroneous methylation) or may (more rarely) be induced by mutations. As a result of the studies, new molecular markers associated with endometrial and ovarian tumors were revealed and a simple and effective method of detection of these markers was developed.

[The need of regulation of production of components for forensic-medical molecular-genetic technologies and improvement of legal basis of forensic-medical molecular-genetic expert examinations in the Russian Federation].

Of late, Ministry of Health of Russian Federation has developed instructions concerning forensic-medical molecular genetic methods of analysis promoting creation of standardized forensic-medical genetic service. However, some legal uncertainty exists in respect to design and production of the materials for forensic-medical molecular-genetic technologies, unification and standardization of molecular-genetic kits and methods. It is thought necessary to regulate legally forensic medical molecular-genetic technologies from foreign countries and production and use of domestic components for forensic medical molecular-genetic expert examinations.

[Possible unfavourable effects of using in-house components for forensic expert molecular-genetic technologies].

So-called in-house production of reagents, components and kits for DNA analysis which are made without strict quality control and standards are now widely practiced in forensic molecular-genetic examinations. Markers of molecular mass were studied to illustrate problems which may arise in use of such in-house components. Other difficulties and negative sequelae of in-house products are also demonstrated.

[Study on the polymorphism of the coagulase gene by the method of sequencing in methicillin-resistant Staphylococcus aureus strains, isolated in hospitals in different regions of Russia and Belarus].

This method was used for typing of 31 Staphylococcus aureus methicillin-resistant (MRSA) strains; of these, 27 were clinical isolates obtained in hospitals of different cities of Russia and Belarus and 4 were international epidemic strains EMRSA-1, -2, -3, -12. The sequencing of the variable area, located in the middle part of the coagulase gene between nucleotides 979-1355 and detected with the use of information technologies, was carried out. The results of this sequencing were compared with those of the earlier study on the polymorphism of the area of the same gene between nucleotides 1513-2188, carried out by the method of PCR-restrictive fragment length polymorphism. The sequencing of the part of the coagulase gene made it possible to confirm the presence of essential differences in the nucleotide sequences of the coagulase gene in international strains EMRSA-1, -3, -12, grounds for classifying clinical isolates of MRSA strains with two groups (4 and 5), as well as the genetic relationship of different phage types, isolated in different clinics. The study revealed considerable similarity in the nucleotide composition of strains EMRSA-2 and EMRSA-12 despite the fact that, according to the results of Cfol restriction of the 3'-end, they were classified with different groups; the study also revealed the identity of the nucleotide sequences of the coagulase gene in the cultures of group 5, isolated in hospitals of Moscow, St. Petersburg, Orenburg, and strain EMRSA-2, as well as methicillin-sensitive S. aureus strain 8325-4; in addition, in clinical isolates of group 4 and strain EMRSA-1 a considerable degree of homology was revealed. The study of two different loci made it possible to find out the strain with the recombinant form of the coagulase gene. The approach used in this study permitted the differentiation of the international epidemic strains EMRSA-1, -2, -3 and -12 into individual groups, which coincided with the results of Enright et al. (2002) who used multilocus sequencing.

[Genetic analysis of wild measles virus strains isolated in the european part of the Russian Federation]. / Geneticheskii analiz dikikh shtammov virusa kori, izolirovannykh v evropeiskoi chasti RF.

Eleven wild measles virus isolated, in 1988 and in 1999-2001 in the European territory of the Russian Federation, were investigated. On the basis of an analysis of N-gene region sequences, encoding the COOH terminal end of nucleoprotein, the isolates were divided into 2 subgroups. According to the WHO classification, subgroup 1 was in line with genotype A and subgroup 2--with genotype D. Subgroup 2 was close to genotype D4 but differed from it according to its composition of nucleotides on the average by 2.8%, and according to its amino-acid composition--by 2.6%. with respect to the WHO criteria, the latter can be referred to preliminarily as an independent genotype. Finally, the measles viruses' strains of genetic groups A and D circulated in the Russian Federation in 1988, and in 1999-2001.

[Influence of tumor necrosis factor on the growth of vegetative and nonculturable forms of Salmonella]. / Vliianie factora nekroza opukholi na razmnozhenie vegetativnykh i nekul'tiviruemykh form sal'monell.

The growth rate of the vegetative forms and the recultivation rate of the uncultivable forms of Salmonella isogenous strains, one of these strains carrying mutation in gene pqi, were studied. The multiplication rate of the vegetative and uncultivable forms of Salmonella control strain in the spleen of infected animals at the initial stages of the infectious process was shown (in vivo) to be considerably accelerated after the preliminary incubation of the culture with cytokine (tumor necrosis factor). The multiplication rate, in vivo and in vitro, of Salmonella vegetative and uncultivable forms with mutation in gene pqi did not change after the incubation of the cells with cytokine, which is indicative of an important role played by the product of this gene in the process of the interaction of bacteria with cytokines. The full nucleotide sequence Salmonella gene pqi was determined.

A new approach for point mutation detection based on a ligase chain reaction.

A new method for the identification of point mutations is proposed. The method is based on ligase chain reaction (LCR) and it includes a procedure for correction of ligation by Cleavase. Reaction products are detected by a colorimetric method after adsorption of the resulting DNA duplexes to the solid phase. One strand of LCR products carries biotin to be bound on a streptavidin-coated microwell. Another strand contains a single-stranded region that is to be coupled with an oligonucleotide carrying a substrate for colorimetric detection. The suggested method has two advantages: (i) use of Cleavase increases the accuracy of ligation and (ii) a template independent ligation does not occur in LCR due to a special design of primers.

[A model prokaryotic promotor. III. Cloning and study of functional activity in vivo]. / Model'nyi prokarioticheskii promotor. III. Klonirovanie i issledovanie funktsional'noi aktivnosti in vivo.

The efficiency of two schemes of oligonucleotide-directed insertion mutagenesis was studied in comparison with standard cloning of DNA duplexes based on blunt-end ligation. Using new approaches, the 30-bp consensus-like prokaryotic promoter was inserted in proper orientation in a promoter-testing plasmid at an almost 100% frequency. Data on the marker gal operon expression and S1-nuclease mapping of the transcription initiation points indicate formation of an active promoter in the region of the insertion.

[Features of connecting short oligonucleotides with phage T4 DNA ligase]. / Osobennosti soedineniia korotkikh oligonukleotidov DNK-ligazoi faga T4.

To understand the influence of different factors on the efficiency of T4 DNA ligase-catalyzed connection of oligonucleotides, a comparative study of polycondensation of AT-containing concatemer octanucleotides (some of them potentially can form "parallel" duplexes) has been undertaken. It has been shown that the size of substrate duplex is the most significant factor, while the sequence of oligonucleotides influences the enzymatic reaction indirectly, by determining the thermal stability of double-stranded complexes. In practice, none of the octanucleotides under study does form "parallel" duplexes.

[An effective method for site-directed mutagenesis in plasmids and cloning single-stranded DNA fragments]. / Effektivnyi metod napravlennogo vvedeniia mutatsii v plazmidy i klonirovaniia odnotiazhevykh fragmentov DNA.

A three primer variant of the earlier devised oligonucleotide-directed mutagenesis in plasmids is described, useful also for the fast cloning of single-stranded DNA products of the asymmetric polymerase chain reaction (PCR). Using this method for plasmid pHD-001-14-11, a 59 b. p. deletion and a 7 b. p. insertion were simultaneously introduced at 81% frequency, and the PCR-copied phage fd transcription terminator (26 b. p.) was inserted with the yield of 67%.