[Influence of individual domains of the translation termination factor eRF1 on induction of the GTPase activity of the translation termination factor eRF3].

Translation termination in eukaryotes is governed by two proteins, belonging to the class-1 (eRF1) and class-2 (eRF3) polypeptide release factors. eRF3 catalyzes hydrolysis of GTP to GDP and inorganic phosphate in the ribosome in the absence of mRNA, tRNA, aminoacyl-tRNA and peptidyl-tRNA but needs the presence of eRF1. It's known that eRF1 and eRF3 interact with each other in vitro and in vivo via their C-terminal regions. eRF1 consists of three domains - N, M, and C. In this study we examined the influence of individual domains of the human eRF1 on induction of the human eRF3 GTPase activity in the ribosome in vitro. It was shown that none of the N-, M-, C- and NM-domains induces eRF3 GTPase activity in presence of the ribosomes. MC-domain does induce GTPase activity of eRF3 but four times less efficient than full-length eRF1, therefore, MC-domain (and very likely M-domain) binds to the ribosome in the presence of eRF3. Based on these data and taking into account the data available in literature, a conclusion was drawn that the N domain of eRF1 is not essential for eRF1-dependent induction of the eRF3 GTPase activity. A working hypothesis is formulated, postulating that GTPase activity eRF3 during the translation termination is associated with the intermolecular interactions of GTP/GDP, GTPase center of the large ribosomal subunit (60S), MC-domain of eRF1, C-terminal region and GTP-binding domains of eRF3, but without participation of the N-terminal region of eRF3.

[Transcription TIMP3, DAPk1 and AKR1B10 genes in squamous cell lung cancer].

Lung cancer is one of the most frequent neoplasia in the Russia, the United States and Europe. This cancer is associated with functional activity changes of many genes. In the present study TIMP3, DAPK1 and AKR1B10 genes transcription analysis of squamous cell lung cancer specimens was carried out using reverse transcription-PCR. Substantial increasing of AKR1B10 transcription level is revealed in 80% tumor samples. TIMP3 and DAPK1 transcription level is considerably decreased in 76 and 72% tumor specimens, accordingly. These results may point out that all three genes are important for squamous cell lung cancer tumorogenesis while AKR1B10 is potential oncogene whereas TIMP3 and DAPK1 are potential tumor suppressor genes. We suggest that revealed substantial transcription level-changes of investigated genes may be used for oncodiagnostics.

[Analysis of expression of a series of lymphoma-specific genes in human fibroblasts immortalized by SV40 virus]. / Analiz ékspressii riada limfomospetsificheskikh genov v immortalizovannykh virusom SV40 fibroblastakh chelovaka.

Subtraction hybridization was earlier used to obtain cDNA clones corresponding to human genes upregulated in HIV-associated centroblast lymphoma (CL) as compared with HIV-associated immunoblast lymphoma (IL). With inverse subtraction hybridization, clones were isolated that correspond to genes upregulated in IL compared with CL. In addition to cDNAs characterized earlier, the resulting clones contained several (seven CL-specific and three IL-specific) sequences with unknown functions. To identify the lymphoma-specific genes that are overexpressed in early carcinogenesis, Northern blotting was used to assess the level of gene transcription in two human fibroblast lines and in their derivatives immortalized with either a temperature-sensitive mutant of SV-40 or with pSV3neo carrying the SV-40 A gene, considering the latter as a model of early cell malignant transformation. Increased expression in at least one immortalized line compared with normal fibroblasts was observed for set, a-myb, ND1, ND2, ND4 (NADH dehydrogenase subunits 1, 2, and 4), COX2, COX3 (cytochrome-c-oxidase subunits 2 and 3), KIAA0129, and the gene corresponding to cDNA hss2-1-7-10. High expression of these genes was assumed to be associated not only with lymphomogenesis, but also with early transformation (immortalization) of other, nonlymphoid cells. Expression of the calpain gene and the gene corresponding to cDNA hss2-2-9-5 proved to be lower in immortalized than in normal fibroblasts. This was considered indicative of an alternative mechanism of fibroblast transformation or of different processes regulating the expression of these genes in early and late carcinogenesis.

[Identification of genes induced to chondrocytes by nitric oxide]. / Poisk i identifikatsiia genov, indutsiruemykh v khondrotsitakh pod deistviem okisi azota.

Nitric oxide (NO) acts as a short-lived paracrine factor and selectively activates transcription of certain genes. The spectrum of inducible genes was studied in primary chondrocytes. A cDNA library was obtained by subtraction hybridization with RNAs isolated from rabbit chondrocytes before and after treatment with nitrosoglutathione, an NO-generating agent. Some of the cloned cDNAs were homologous to known mammalian genes and human EST. NO-dependent transcriptional activation was demonstrated for the stromelysin 1 and cyclooxygenase 2 genes and, for the first time, for mcl1 coding for an apoptosis suppressor.

[Amplification of transcription of separate mitochondrial genes in human and monkey B-cell non-Hodgkin's lymphoma]. / Usilenie transkriptsii otdel'nykh mitokhondrial'nykh genov v B-kletochnykh nekhodzhkinskikh limfomakh cheloveka i obez'iany.

Mitochondrial genes that are overexpressed in human and monkey B-cell non-Hodgkin lymphomas (B-NHLs) were sought via subtraction hybridization, cloning, and differential screening of the resulting cDNA libraries. The cDNAs of mitochondrial genes made an appreciable proportion of all lymphoma-specific cDNAs. Lymphomogenesis was associated with overexpression of a mitochondrial gene set which varied with lymphoma type and always included NADHIV. A possible association between overexpression of certain mitochondrial genes and cell malignant transformation is discussed.

[Effects of the nef and tat genes of the human immunodeficiency virus type I on rodent cells in vivo and in vitro]. / Vliianie genov nef i tat virusa immunodefitsita cheloveka tipa 1 na kletki gryzunov in vivo i in vitro.

The tat and nef regulatory genes of the human immunodeficiency virus type I (HIV-1) under the control of eukaryotic promoters were transferred in vivo into mice and in vitro into rat cell cultures. The development was disturbed and adenocarcinomas of the lacrimal glands and pancreas appeared in transgenic mice carrying the HIV-1 tat gene. Transfection with the tat gene altered morphology and increased proliferative activity of Rat-2 pseudonormal cells. The tat gene also induced the formation of neoplastic foci in a primary rat embryo fibroblast culture. The results obtained showed that the HIV-1 tat gene can act as an oncogene and activate the proliferation of cultured cells. Cell proportions in peripheral blood and bone marrow were altered and mitogen-induced lymphocyte proliferation was decreased in transgenic mice carrying the HIV-1 nef gene. This gene also significantly suppressed proliferation but had no effect on morphology of Rat-2 cells. Thus, the HIV-1 nef gene appeared to suppress proliferation of various animal cells.

[Physical and genetic organization of the plasmid R15]. / Fizicheskaia i geneticheskaia organizatsiia plazmidy R15.

The conjugative IncN plasmid R15 (SmrSurHgr, 62.3 kb) is cleaved by the hexanucleotide-specific endonucleases BglII, HindIII, EcoRI, BamHI, SmaI, SalI, PstI and XhoI into 9, 9, 6, 5, 4, 4, 4 and 2 fragments, respectively. The restriction sites were located on the physical map of the R15 genome. Distribution of the cleavage sites is strongly asymmetric. 28 of 32 sites for BamHI, EcoRI, HindIII, SalI, SmaI and PstI were located close to or within the sequences of transposable elements Tn2353 and Tn2354. According to the results of analysis of R15::Tn1756 deletion derivatives and recombinant plasmids harboring fragments of R15, the genetic determinants for resistance to Sm, Su and Hg were mapped, as well as the regions necessary for EcoRII restriction--modification and for plasmid replication and conjugation. The features of physical and genetic structures of R15 and other IncN plasmids are discussed.

[Enzymatic synthesis and molecular cloning of the pigeon alpha-globin structural gene]. / Fermentativnyi sintez i molekuliarnoe klonirovanie strukturnogo gena al'fa-globina golubia.

Double-stranded DNA synthesized from the pigeon globin mRNA by the subsequent actions of avian myeloblastosis virus reverse transcriptase and E. coli DNA polymerase I was split with nuclease S1 and inserted into PstI site in the plasmid pBR322 by poly(dG) times poly(dC) homopolymer extension technique using terminal deoxynucleotidyl transferase. E. coli transformants have been shown to contain pigeon globin sequences by colony hybridization with pigeon globin [32P]cDNA. The inserted DNA fragment deleted from recombinant DNA by PstI treatment hybridizes with globin cDNA. The maximal length of the inserted fragment measured in agarose gel was found to be 550--560 base pairs. Inserted sequences subjected to analysis by hybridization with alpha- and beta-[32P]cDNA have been ascribed to the pigeon alpha globin chain. EcoRI, HindIII, BglII, SalI, BamHI, PstI restriction enzymes did not cleave the inserted DNA fragment. Pigeon DNA coding alpha-globin chain contains recognition sites for AluI, HindII and HaeIII restriction enzymes. Part of the recombinant clones remains resistant to ampicillin and therefore in some of these clones the globin gene could be expressed.

[Globin-coding sequences in the nuclear 28S pre-mRNA and in the cytoplasmic RNA of pigeon erythroid cells]. / Posledovatel'nosti, kodiruiushchie globin, v 28S pre-mRNK i v tsitoplazmaticheskoi RNK éritroidnykh kletok golubia.

The steady-state content of globin-coding sequences in nuclear and cytoplasmic RNA of pigeon erythroid cells was estimated by hybridization in the excess of nuclear 28S RNA and cytoplasmic poly(A) + RNA with [3H]DNA, synthesized on globin mRNA. Sequences of 9S globin mRNA are found in 0.06% of molecules of non-ribosomal 28S nuclear RNA (pre-mRNA) of erythroblasts and in 0.5% of molecules of non-ribosomal 28S nuclear RNA of reticulocytes. The content of globin mRNA in erythroblast cytoplasm is, respectively lower than in that of reticulocytes.