[The development and approbation of methodology on the basis of multiplex polymerase chain reaction in real-time to determine clinically significant micro-deletion in Y-chromosome.]

One of the prevalent genetic causes of idiopathic male sterility is related to micro-deletions in AZF locus located in Y-chromosome. In total population, rate of such micro-deletions makes up to 1:4000. however, in infertile males their rate varies from 2% to 10%. In AZF locus three subregions are distinguished: AZFa, AZFb and AZFc. The loss of one or several subregions can result in disorder of spermatogenesis of various degree - from decreasing of its activity to Sertoli-cell syndrome manifested by azoospermia or oligospermia of severe degree. Therefore, implementation of genetic testing for presence of micro-deletions in AZF locus is a necessary test in case of prognosis of male sterility and its treatment. The purpose of study is to develop and test a diagnostic system of detection of micro-deletions in subregions of AZF locus using multiplex polymerase chain reaction in real-time. As a reference method a technique was implemented described in guidelines of the European Academy of Andrology conjointly with European Molecular Genetics Quality Network. The technique testing specified analysis of 33 samples of DNA separated from blood of males with azoospermia and oligospermia of severe degree. No discordant results were received as compared with reference method. In 27 DNA samples the deletions were detected in AZF locus: 4 AZFa deletions (15%), 2 AZFb deletions (7%), 17 AZFc deletions (63%) and 6 combined deletions of AZFb+candи AZFa+b+с (22%). The proposed technique permits detect micro-deletions of subregions of AZF locus.

Enzymatic activity of protein kinase LOSK: possible regulatory role of the structural domain.

LOSK (LOng Ste20-like Kinase) protein kinases of mammals belong to a recently identified family of GCK kinases which are involved in the induction of apoptosis. LOSK have an N-terminal acidic catalytic domain and a long C-terminal basic structural domain which is cleaved off in cells by caspases during apoptosis. To study the LOSK enzymatic activity and its dependence on the structural domain, two preparations of this protein kinase were prepared: a natural full-length protein immunoprecipitated from CHO-K1 cultured cells and a recombinant N-terminal catalytic fragment synthesized in E. coli. Both preparations displayed the ability for autophosphorylation and the ability for phosphorylation of MBP and of H1 histone, and their activities were comparable. H1 histone was a better substrate for LOSK than casein and ATP was a better substrate than other nucleotides. The pH dependence of the activity of the immunoprecipitated protein was more pronounced than the pH dependence of its recombinant fragment deprived of the C-terminal domain. The catalytic and the structural domains of LOSK can interact through electrostatic forces; therefore, effects were studied of various polyions at the concentration of 0.1 mg/ml on the activity. Heparin, protamine sulfate, and poly(L-Lys) decreased tenfold the ability of the full-length kinase to phosphorylate H1 histone. Heparin did not affect the activity of the recombinant fragment, whereas protamine sulfate and poly(L-Lys) had a slight effect. Moreover, protamine increased fourfold the autophosphorylation of the immunoprecipitated protein kinase. These data suggest that the structural C-terminal domain of LOSK should be involved in the regulation of its protein kinase activity: the LOSK protein kinase with C-terminal domain cleaved off could significantly less depend on conditions in the cell than the full-size enzyme.

[The translation regulation of the synthesis of proteins responsible for dorsoventral differentiation of clawed toad embryos]. / Transliatsionnaia reguliatsiia sinteza belkov, otvetstvennykh za dorsoventral'nuiu differentsirovku zarodyshei shportsevoi liagushki.

We studied the distribution of mRNAs involved in dorsoventral differentiation of Xenopus laevis embryos (Xbra, chordin, Xnot, Xvent1, Xvent2, and Xwnt11) among polyribosomes (the translated form of mRNA) and informosomes (the nontranslated form of mRNA). It was shown using molecular hybridization that all of the studied templates are in the informosomes until the midgastrula stage, thus suggesting regulation at the level of translation. At the midgastrula stage (stage 11), translation begins on mRNAs of chordin, Xnot2, Xvent1, and Xbra, although mRNAs of Xvent1 and Xbra are partially located in the informosomes. The matrices of Xwnt11 are localized predominantly in the informosomes at the midgastrula stage, while those of Xvent2 are not seen in the polyribosomes until the end of gastrulation. We propose that the fate of different mRNAs is determined by different mechanisms of r-translational regulation in different cell lineages. It cannot be excluded that the Xvent2 transcript is not involved in translation and fulfills its morphogenetic functions in the form of RNA or RNP.