[Novel complexes of gene expression and their role in the appearance and evolution of the genus Homo].

Using genetic (yeast two-hybrid system) and biochemical (co-precipitation of proteins from cellular lysates) approaches, we have performed a whole-genome wide search for interacting partners of the previously described by us variants of hRPB11 subunit of human RNA polymerase II - hRPB1 1balpha, hRPB11calpha and hRPB1 1bbeta, hRPB 11cbeta - in fetal brain and Jurkat cell line libraries. In consequence, the main spectrum of the protein partners of these human specific isoforms of the RNA polymerase II subunit hRPB 11 (POLR2J) has been established. Functional characteristics of the uncovered protein partners of hRPB 11balpha and hRPB 11calpha isoforms clearly indicate that these isoforms, similarly to the main (major) subunit hRPB11a, are components of the distinct transcription complexes participating not only in the transcription of the specific DNA matrices, but involving also in the later stages of mRNA biogenesis. The RNA polymerase I-III common subunit hRPB6 (POLR2F) and basal component of the exon-exon junction complex Y14 (RBM8A) have been found among the protein partners of the isoforms hRPB 11bbeta and hRPB 11cbeta together with a number of proteins involved in the biogenesis of microRNAs, including a novel, not previously described variant of the microRNA processing nuclease DROSHA, which indicates the existence of a special coordination between processes of transcription and RNA interference in the nuclei of human cells.

[PSM2 and POLR2J gene families as molecular markers of the higher primate evolution].

We have studied the molecular evolution of two gene families specific for primates: POLR2J of the transcription system and PMS2 of the MMR repair system. The appearance and improvement of the genetic structure in each of the families was shown to strongly correlate with the main stages of the higher primates biological evolution. Our results indicate that the PSM2 and POLR2J genes can serve as helpful and reliable molecular markers of anthropogenesis.

[Characteristics of steroid metabolism in transgenic Nicotiana tabacum plants bearing the CYP11A1 cDNA of cytochrome P450(SCC) from the bovine adrenal cortex].

In the mitochondria of animal steroidogenic tissues, cytochrome P450(SCC), encoded by the CYP11A1 gene, catalyzes the conversion of cholesterol into pregnenolone - the general precursor of all steroid hormones. In this work, we study the steroid metabolism in transgenic tobacco plants carrying the CYP11A1 cDNA cytochrome P450(SCC)from the bovine adrenal cortex. The transgenic plants under investigation markedly surpass the control wild-type plants by size and are characterized by a shortened period of vegetative growth (by rapid flowering); their leaves contain pregnenolone - the product of a reaction catalyzed by cytochrome P450(SCC). The level of progesterone in transgenic tobacco leaves is higher than in the control plants of the wild type. The seeds of the transgenic plants contain less (24R)-brassinosteroids than the wild-type tobacco plants. The results obtained indicate that the synthesis of an active P450(SCC) cytochrome in transgenic Nicotiana tabacum plants has a profound effect on steroid metabolism and is responsible for the specific phenotypic features of transgenic plants bearing CYP11A1 cDNA.

[The functional interaction of an RNA polymerase II Rpb11 subunit with the Med18 subunit (Srb5) of the Saccharomyces cerevisiae mediator complex].

The SRB5 gene encoding the Med18 (Srb5) subunit of the mediator complex of the Saccharomyces cerevisiae transcription apparatus was identified in the C-terminal region of the yeast RNA polymerase II Rpb11 subunit as a multicopy suppressor of the Leu111Ala (L111A) point mutation. Thus, the functional interaction between one of the mediator components and the core of the major transcription enzyme was first shown. It is also essential that the suppressed point mutation was located in the short C-terminal region of the Rpb11subunit, which plays an important role for the evolution of the eukaryotic transcription apparatus, as was demonstrated in our previous studies.

[Construction and characteristics of transgenic tobacco Nicotiana tabacum L. plants expressing CYP11A1 cDNA encoding cytochrome P450scc].

In steroidogenic animal tissues cytochrome P450scc catalizes the conversion of cholesterol into pregnenolone, a common metabolic precursor of all steroid hormones. To study the possibility of functioning of mammalian cytochrome P450scc in plants and the mechanism of its integration in the plant steroidogenic system, transgenic plants of tobacco Nicotiana tabacum L. were developed carrying cDNA of CYP11A1 encoding cytochrome P450scc of bovine adrenal cortex. Pregnenolone, a product of the reaction catalyzed by cytochrome P450scc, was discovered in the steroid-containing fraction of transgenic plants. Transgenic plants are characterized by a reduced period of vegetative development (early flowering and maturation of bolls) and increased productivity. The contents of soluble protein and carbohydrates in leaves and seeds of transgenic plants are essentially higher than the contents of these components in leaves and seeds of control plants.

[C-terminal domain (CTD) of the subunit Rpb1 of nuclear RNA polymerase II and its role in the transcription cycle].

The review deals with analysis of the structure, functions and molecular evolution of the CTD (C-terminal domain) of the largest subunit Rpb1 of nuclear RNA polymerase II. All the discovered ways of CTD modifications are summarized. Its interactions with various partners are described in the context of the CTD central role in coupling of the major transcription steps (initiation, elongation and termination) with the main stages of pre-mRNA processing (capping, splicing and polyadenylation) in eukaryotes during different stages of the cell cycle.

[New genes on human chromosome 7: bioinformatic analysis of a gene cluster from the POLR2J family]. / Novye geny na khromosome 7 cheloveka: bioinformatsionnyi analiz klastera genov iz semeistva POLR2J.

Four independent genes encoding various variants of the hRPB11 subunit of Homo sapiens RNA polymerase II were revealed in human chromosome 7. Three genes (POLR2J1, POLR2J2, and POLR2J3) form a cluster of total length 214530 bp in the genetic locus 7q22.1 on the long arm of chromosome 7 (contig NT_007933). The fourth gene (POLR2J4, 31040 bp) was localized in the cytogenetic locus 7p13 of the short arm of chromosome 7 (contig NT_007819). An analysis enabled us to refine dissimilar experimental data on the mapping of the hRPB11 subunit gene on chromosome 7. In particular, the presence of three sites of its localization according to data on hybridization with fluorescent-labeled probes (the FISH method) was explained. It was established that, upon the expression of the four human POLR2J genes, at least 14 types of mature mRNAs encoding somewhat differing hRPB11 isoforms can be synthesized. Eleven of these mRNAs were revealed (as full-length copies or clearly identifiable fragments) in the available databases of expressed sequence tags and cDNAs. The most probable scheme of origination of the multiple genes of the POLR2J family, as a result of three consecutive segmented duplications increasing in size, was proposed and substantiated. On the basis of the scheme, some assumptions on the pathways of evolution of separate human genes and the mechanisms of generation of protein diversity in higher eukaryotes were made. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.

[Structure and function of eukaryotic nuclear DNA-dependent RNA polymerase I]. / Structura i funktsii iadernoi DNK-zavisimoi RNK-polimerazy I éukariot.

In the eukaryotic cell, normal protein biosynthesis is sustained by several million ribosomes, which contain rRNA as an essential component. The high-molecular-weight precursor of large and 5.8S rRNAs is synthesized by DNA-dependent RNA polymerase I (Pol I) in the nucleolus. Data on DNA regulatory elements, protein factors involved in rDNA transcription by Pol I, subunit composition of Pol I, and on the interactions and possible functions of individual subunits are summarized.

[Chromosomal localization of rpb9+ and tfa1+ genes, coding for components of the mRNA synthesis apparatus of Schizosaccharomyces pombe]. / Khromosomnaia lokalizatsiia genov rbp9+ i tfa1+, kodiruiushchikh komponenty apparata sinteza mRNK Schizosaccharomyces pombe.

Using DNA hybridization on cosmid filters of high density, we established chromosomal localization of the rpb9+ gene encoding one of the specific subunits of RNA polymerase II of Schizosaccharomyces pombe and thus filled in the last gap in the mapping of the genes encoding components of RNA polymerase II of the fission yeast. The primary structure of three extended regions of the Sz. pombe chromosome I was elucidated and, as a result, genes neighboring on rpb9+ were identified. One of them proved to be the tfa1+ gene, encoding the large (alpha) subunit of the general factor of transcription initiation TFIIE.

[Molecular cloning of some components of the translation apparatus of fission yeast Schizosaccharomyces pombe and a list of its cytoplasm ic proteins genes]. / Molekuliarnoe klonirovanie nekotorykh komponentov apparata transliatsii deliashchikhsia drozhzhei Schizosaccharomyces pombe i pervichnyi unifitsirovannyi spisok genov tsitoplasmaticheskikh ribosomnykh belkov etogo mikroorganizma.

Full-length cDNAs of four new genes encoding cytoplasmic ribosomal proteins L14 and L20 (large ribosomal subunit) and S1 and S27 (small ribosomal subunit) were isolated and sequenced during the analysis of the fission yeast Schizosaccharomyces pombe genome. One of the Sz. pombe genes encoding translation elongation factor EF-2 was also cloned and its precise position on chromosome I established. A unified nomenclature was proposed, and the list of all known genetic determinants encoding cytoplasmic ribosomal proteins of Sz. pombe was compiled. By now, 76 genes/cDNAs encoding different ribosomal proteins have been identified in the fission yeast genome. Among them, 35 genes are duplicated and three homologous genes are identified for each of the ribosomal proteins L2, L16, P1, and P2.

[Characteristics of the cDNA of the Schizosaccharomyces pombe rpa43+ gene: structural similarity of the Rpa43 subunit of RNA-polymerase I with the Rpc25 subunit of RNA-polymerase III]. / Kharakteristika kDNK gena rpa43+ Schizosaccharomyces pombe: strukturnoeskhodstvo sub''edinitsy Rpa43 RNK-polimerazy I s sub''edinitsei Rpc25 RNK-polimerazy III.

We isolated and characterized full-length cDNA of the rpa43+ gene encoding one of subunits of the nuclear RNA polymerase I of Schizosaccharomyces pombe. The gene contains two introns and is located on chromosome II. Comparison of the primary structure of the subunit Rpa43 of Sz. pombe (173 aa; M 19,385 Da; pl 5.36), deduced from the cDNA obtained, with the amino acid sequences of subunits A43 from Saccharomyces cerevisiae and Drosophila melanogaster demonstrates a high divergence of this protein in evolution. A comparison of the Rpa43 with other proteins from the SwissProt database revealed a similarity of this subunit to subunit Rpc25 of RNA polymerase III, which, as was shown previously, is structurally similar to subunit Rpb7 of RNA polymerase II. Thus, including the Rpa43<-->Rpc25<-->Rpb7 family, nuclear RNA polymerases I-III contain at least 11 identical and/or similar subunits. This fact illustrates a pronounced resemblance of the organization of all three enzymes of the eukaryotic transcription apparatus. Moreover, at least ten out of these eleven families of eukaryotic RNA polymerase subunits have homologues in the 13-subunit archaeal RNA polymerase.

[Molecular evolution and structure of eukaryotic nuclear RNA polymerase subunits in light of the exon-intron organization of their genes]. / Molekuliarnaia évoliutsiia i struktura sub''edinits iadernykh RNK-polimeraz éukariot v svete ékzon-intronnoi organizatsii ikh genov.

Analysis of literary data (for Saccharomyces cerevisiae, Caenorhabditis elegans, Arabidopsis thaliana, Homo sapiens, and some other Eucarya) and our data (for Schizosaccharomyces pombe) on the exon-intron organization of the genes encoding subunits of nuclear RNA polymerases showed that introns in the orthologous genes from different organisms are arranged nonrandomly, namely, their positions, if projected on the map of the comparison of the amino acid sequences of the orthologous subunits, not infrequently coincide in evolutionarily distant species. As a rule, intron positions correspond to the boundaries of the structurally conserved regions (domains) or to the sites of possible turns of the polypeptide chain. For example, introns flank the secondary structure elements in the Rpb8 subunit with the known three-dimensional structure or the structure-function modules in subunits Rpb10 and Rpc10. These facts are in agreement with the idea of the ancient origin of introns, and with the notion of evolution of ancient protein sequences through the assembly of their genes from short protoexons selected by the nature as far back as the RNA world times. Comparative analysis of the primary structures of the subunits of eukaryotic RNA polymerases allowed us to reveal a nuclear localization signal in subunit Rpb10 and some hypothetical archaeal homologues of subunit Rpc10.

[Exon-intron structure of the fet5+ gene of Schizosaccharomyces pombe and physical mapping of genome encompassing regions]. / Ekzon-intronnaia struktura gena fet5+ Schizosaccharomyces pombe i fizicheskoe kartirovanie okaimliaiushchikh ego uchastkov genoma.

Plasmid pYUK3 bearing the fet5+ gene of Schizosaccharomyces pombe was isolated from a genomic library of the fission yeast, and a detailed physical map of the whole genomic insert (ca. 9.6 Kbp) was constructed. The primary structure of the fet5+ gene and its flanking regions is established. The gene contains a single 45-bp intron in its distal part. A typical TATA-box (TATAAG) was found in the 5'-noncoding region ca. 50 bp upstream of the putative start of transcription, and the 3'-noncoding region contains AT-rich palindromes, which are probably involved in termination of the fet5+ transcription. A previously unidentified gene of Sz. pombe encoding a protein with some similarity to one of the transcriptional activators from the TBP (TATA-binding protein) group of SPT factors of transcription was found in the vicinity of the fet5+ gene. Taking into account that cDNA of the fet5(+)-gene was isolated as a suppressor of the genetic-defect of nuclear RNA polymerases I-III (Bioorg. Khim., 1997, vol. 23, No 3, pp. 234-237), this vicinity may be the first evidence of possible clustering, in the genome of the fission yeast, of genes participating in transcription regulation.

[Molecular cloning and characteristics of rpc19+ and rpc40+ Schizosaccharomyces pombe genes, coding for common subunits of nuclear RNA polymerase I and III]. / Molekuliarnoe klonirovanie i kharakteristika genov rpc19+ i rpc40+ Schizosaccharomyces pombe, kodiruiushchikh obshchie sub''edinitsy iadernykh RNK-polimeraz I i III.

Full-length copies of cDNAs of the rpc19+ and rpc40+ genes encoding the common subunits of nuclear RNA polymerases I and III and the corresponding fragments of chromosomes were isolated from genomic and cDNA libraries of Schizosaccharomyces pombe and characterized. It was established that the cloned genes are located on chromosomes III and II of the fission yeast, respectively. The rpc40+ gene lacks introns, and the rpc19+ gene contains two intervening sequences. The comparison of subunits Rpc19 (125 aa; M 13 722 Da; pI 4.51) and Rpc40 (348 aa; M 39 141 Da; pI 5.40) of Sz. pombe, whose characteristics were deduced from the sequences of their cDNAs, with the orthologous components of other eukaryotes allowed the most conserved structure-functional domains of these proteins to be identified.

[Structural-functional characteristics of the Schizosaccharomyces pombe rpb8+ gene, coding the subunit of RNA polymerase I-III, specific only for eukaryotes]. / Strukturno-funktsional'naia kharakteristika gena rpb8+ Schizosaccharomyces pombe, kodiruiushchego sub''edinitsu RNK-polimeraz I-III, specifichnuiu tol'ko dlia éukariot.

A full-length cDNA of the rpb8+ gene encoding a common subunit Rpb8 of nuclear RNA polymerases I-III only specific for Eucarya was isolated from an expression library of the fission yeast Schizosaccharomyces pombe. The primary structure of the corresponding fragment of the Sz. pombe genome was also established. The rpb8+ gene contains two short introns, 59 and 48 bp long. Only short segments of homology were found upon comparing the Rpb8 subunit homologs from various eukaryotic species, and substantial differences exist between the corresponding proteins of unicellular and multicellular organisms. Subunit Rpb8 of Sz. pombe proved to be the smallest one among the known related proteins: it lacks the 21-aa fragment corresponding to amino acids residues 68-88 of the central part of the homologous subunit ABC14.5 of Saccharomyces cerevisiae. Accordingly, subunit Rpb8 of the fission yeast was not capable of substituting in vivo subunit ABC14.5 in nuclear RNA polymerases of the baker's yeast.

[Molecular cloning and characterization of cDNA of the rpc10+ gene encoding the smallest subunit of nuclear RNA polymerases of Schizosaccharomyces pombe]. / Molekuliarnoe klonirovanie i kharakteristika kDNK gena rpc10+, kodirue shchego samuiu maluiu cyb''edinitsu iadernykh rnk-polimeraz Schizosaccharomyces pombe.

The full-length cDNA of the rpc10+ gene encoding mini-subunit Rpc10, which is common for all three nuclear RNA polymerases of the fission yeast Schizosaccharomyces pombe, was cloned and sequenced. The Rpc10 subunit of Sz. pombe and its homologs from S. cerevisiae and H. sapiens are positively charged proteins with a highly conserved C-terminal region and an invariant zinc-binding domain (Zn-finger) of a typical amino acid composition: YxCx2Cx12RCx2CGxR. Functional tests of heterospecific complementation, using tetrad analysis or plasmid shuffling, showed that the Rpc10 subunit of Sz. pombe can successfully replace the homologous ABC10 alpha subunit in nuclear RNA polymerases I-III of S. cerevisiae.