[Identification of changes in gene loci potentially associated with cervical cancer using NotI microarrays].

The investigation of the cancer-associated structural and epigenetic changes in cell genome is a major approach for understanding mechanisms of cancerogenesis. To investigate these genome changes, novel technique of microarrays comprising NotI-linking genome clones was developed. Twenty eight samples from patients with cervical cancer were analyzed using NotI microarrays of human chromosome 3. Deletions, amplifications and methylation were detected for 109 out of 182 NotI clones with different frequency. Notably, 17 NotI-linking clones showed genomic changes in more than 35% of tumor samples investigated, which suggests involvement of genes associated with these clones in development of cervical cancer.

[New forms of gene transcripts in human and mouse intersectin]. / Novye formy transkriptov gena intersektina cheloveka i myshi.

New human and mouse cDNAs which are homologous to human intersectin gene (ITSN) mapped on a q22.1-22.2 region of human chromosome 21 have been obtained. ITSN gene structure has been determined using nucleotide sequences of human chromosome 21 presented in nucleotide's data bases. The analysis of human and mouse ITSN gene transcripts revealed that their pre-mRNA splicing could occur in different ways. New form of ITSN gene transcripts with alternatively spliced SH3C domain (exon 25 and 26) was detected in different human and mouse tissues. The other splice form with absence of exons 6-14 that results in reading frame shift and stop-codon formation was identified in the mouse adult lung and kidney. In addition, we showed alternative splicing of exons 20, 25 and part of exon 6.

[Complete nucleotide sequence of Rous sarcoma virus variants adapted to duck cells]. / Polnaia nukleotidnaia posledovatel'nost' adaptirovannogo k kletkam utok varianta virusa sarkomy rausa.

Subgroup C avian sarcoma viruses efficiently infect and transform but poorly replicate in duck cells. Nucleotide sequence analysis of Prague strain of Rous sarcoma virus adapted by numerous passages on duck embryonic fibroblasts (daPr-RSV-C) showed that adaptation of originally chicken virus to duck cells correlated with changes in viral genome, first of all in gp85-coding domain of env-gene. Besides, changes in LTR and src-gene sequences could play a role in widening of host range for this virus. The major changes of daPr-RSV-C in comparison with original Pr-RSV-C appeared to be the result of homologous recombinations with corresponding regions of chicken endogenous retroviruses.

[Molecular basis of retrovirus adaptation: nucleotide sequence of Rous sarcoma virus adapted to duck cells]. / Molekuliarnye osnovy adaptatsii retrovirusov: nukleotidnaia posledovatel'nost' virusa sarkomy Rausa, adaptirovannogo k utinym kletkam.

The host range of retroviruses is rather complex and specific. It is controlled by the products of viral structural genes that interact with the determinants both on the surface and within the cell. The possibility to infect and transform duck embryo fibroblasts is shown for the Prague strain of chicken Rous sarcoma virus (subgroup C), though virus production in these cells is restricted. However, after the 6th passage the "adapted" virus gave the titre practically the same as it was for chicken embryo fibroblasts. Provirus of RSV adapted to the duck embryo fibroblasts and integrated into host DNA was isolated from the library of nucleotide sequences of duck embryo fibroblasts transformed by this virus. The nucleotide sequence of such provirus was determined. The alterations in gp85 coding region of the env gene which proved to be the result of recombination with endogeneous RAV-0 sequences were shown. The formation of viral particles with rather high titre was induced by the proviral transfection on both chicken and duck embryo fibroblasts. The contribution of the revealed alterations in the genome of transformation active virus and possible participation of its td mutant in the adaptation to the new host are discussed.

[The family of env genes of avian retroviruses: molecular analysis of Rous sarcoma virus adapted to duck cells]. / Semeistvo genov env ptich'ikh retrovirusov: molekuliarnyi analiz virusa sarkomy Rausa, adaptirovannogo k utinym kletkam.

For the elucidation of the molecular basis of RSV adaptation to conditionally permissive host from the genome library of duck embryo fibroblasts, transformed by Rous sarcoma virus in 30 passages on these cells, recombinant bacteriophages that include provirus sequences, were obtained. Complete and transformation-defective proviruses were characterized, nucleotide sequences of their env-genes were compared with their counterparts the original RSV (Pr-RSV-C) and with viruses of other subgroups (A, B, D and E). The possible relation of the revealed changes in domains coding gp85 and gp37, with the changes of chicken RSV characteristics during adaptation to duck cells is discussed.

[Isolation of reverse transcriptase from the avian myeloblastosis virus in preparative quantities]. / Vydelenie obratnoi transkriptazy virusa mieloblastoza ptits v preparativnykh kolichestvakh.

Inverse transcriptase of bird myeloblastosis virus is a unique instrument for artificial synthesis of structural genes of viruses, plants, animals. Methods for the virus production in preparative amounts are developed due to selection of the corresponding line of chickens, conditions of their maintenance, diet infection methods and myeloblastosis diagnostics. Main demands to the inverse transcriptase preparations (their high activity, absence of nuclease impurities, high concentration of the enzyme preparation solutions and their stability in storage) are ensured by zonal centrifugation purification of the virus in a sucrose density gradient, described methods of inverse transcriptase isolation and purification as well as conditions of its storage.

[Synthesis and cloning of DNA, complementary to rabbit globin pre-mRNA]. / Sintez i klonirovani DNA, komplementarnoi pre-mRNK globina krolika.

cDNA synthesized on rabbit bone marrow erythroid cells pre-mRNA was cloned in bacterial plasmids. Cold phenol extracted pre-mRNA was a several times more effective template in the reaction of reverse transcription without oligo(dT) 10-primer than hot phenol extracted pre-mRNA. There was no yield increase of DNA-product on hot phenol extracted pre-mRNA in the reaction of reverse transcription with the oligo(dT)10-primer addition. The "hot phenol" poly (A)+-pre-mRNA was used to obtain the representative, full-sized cDNA. The double-stranded form of this cDNA, obtained with the help of DNA-polymerase I, was inserted into the PstI-site of pBR322 plasmid. About 25% E. coli JC5183 clones, transformed with this hybrid plasmid, were found to contain the globin sequences.

[Electron microscopic study of the DNA products of reverse transcription]. / Elektronno-mikroskopicheskoe issledovanie DNK-produktov obratnoi transkriptsii.

DNA-products synthesized on pre-mRNA's from rat liver and rabbit erythroidal bone marrow cells, on rabbit globin mRNA and the RNA-templates themselves have been examined by electron microscopy. In spreading conditions providing extension of molecules the sizes of DNA-products corresponded to sizes of RNA templates. Globin mRNA, pre-mRNA as well as cDNA synthesized on these templates in the presence of actinomycine D were seen as single-stranded molecules by electron microscopy. The preparations of cDNA synthesized in the absence of actinomycine D on the pre-mRNA template were represented by double-stranded molecules along side with sigle-sranded. Up to 10% of double-stranded DNA-product appeared to be branched; all branches of such molecules had the thickness and rigidity of double-stranded structures. The possible ways of formation of branched structures are discussed.

[mRNA of mouse plasmacytoma. Reverse transcription and translation in cell-free systems]. / Matrichnye RNK plazmotsitony myshei. Obratnaia transkriptsiia i transliatsiia v beskletochnoi sisteme sinteza.

Total poly(A)-mRNA from polyribosomes of MOPC 21 mouse myeloma were investigated. Poly(A)-mRNA was released by two successive chromatography on oligo (dT)-cellulose. A 14S fraction of total poly(A)-mRNA was obtained and partially purified by sucrose gradient centrifigation followed by acrylamide gel electrophoresis. As estimated from the electrophoretic analysis, the 14S mRNA has three components, one of which appears to be 18S rRNA and two others--mRNAs with molecular weight of 5.2.10(5) and 3.8.10(5), respectively. Total poly(A)-mRNA and partially purified 14S mRNA were active when employed as a template in a reverse transcription and cell-free system from wheat germ. DNA complementary to the 14S mRNA was prepared with avian myeloblastosis virus RNA-dependent DNA polymerase. This cDNA was heterogeneous in size with the average size of about 800 nucleotides when analyzed by gel electrophoresis in 98% formamide. The maximal length was about 1100 nucleotides that consistent with full template length. About half of the translation product directed by the 14S mRNA migrated as mature L-chain Ig (upon polyacrylamide gel electrophoresis in sodium dodecylsulfate). The presented data suggested that 14S mRNA species contain mRNA L-chain Ig.

[DNA synthesis on the heterogeneous nuclear RNA template catalysed by DNA polymerase of avian myeloblastosis virus]. / Sintex DNK na matritse geterogennoi iadernoi RNK DNK-polimerazoi virusa mieloblastoza ptits

Template activity of nuclear pre-mRNA has been investigated in DNA-polymerase reaction. Active synthesis of DNA was demonstrated on pre-mRNA as a template in the absence of primer. A part of synthetic activity may be attributed to the traces of DNA present in the pre-mRNA preparation. Addition of oligo(dT)10 to the template stimulated the synthesis of DNA product due to transcription of heteropolymeric regions near the poly(A). The rate of DNA synthesis was different depending on the fraction of template used: the RNA extracted by hot phenol at 85 degrees showed higher template activity without adding of primer than the 65 degrees C fraction. On the contrary 65 degrees C pre-mRNA which is known to contain greater quantity of molecules with poly(A) at the 3'-end is more strongly stimulated by addition of oligo(dT). The nuclear RNA corresponding to the precursors of rRNAs extracted at 40 degrees C were not transcribed by the reverse transcriptase. The size of the DNA-product (about 7-8S in alkaline sucrose gradient) did not depend on the size of the template neither on the presence of oligo(dT)10 primer. The inhibition of the second DNA strand synthesis with actinomycin D had also no influence on the size of DNA-product.