Biomedical subjects
Publications and source records attributed to V I Evtushenko.
Low molecular weight nuclear RNAs (LMWN RNAs) from rat liver, Zajdela hepatoma and rat hepatoma induced by DENA have been studied. The changes in the relative content of nucleoplasmic U1 and U2 RNAs, nucleolus U3 RNA and chromatine 5S RNA have been discovered in both hepatomas. Whereas 8S1 and U3 RNA were found in polysomes from rat liver but not in those from Zajdela hepatoma.
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Some fractions of low molecular weight (LMW) nuclear RNAs were shown to be present in the cytoplasm of rat liver cells. In addition to known 4S tRNA, 5S and 5,8S rRNAs U3 and 8S1 LMW nuclear RNAs, 8SII and 8SIII LMW RNAs have been detected in RNA preparations of free total and membrane-bound polysomes. The U3 and 8SI polysoma I RNAs seem to be associated with high molecular weight polysomal RNA. Using thermal phenol fractionation, that some LMW RNAs were shown to be slightly bound to the cellular structures whereas some others are bound more tightly. Considerable amounts of LMW RNAs are tightly bound to the chromosome-nucleolar apparatus. They can be extracted only at 85 degrees C. The data presented are discussed with regard to LMW nuclear and polysomal RNAs functions.
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Embryonic stem cells (ESC) are widely employed as an experimental model owing to the similarity of molecular events occurring in their in vitro differentiation and in early embryo development. Subtraction hybridization was used to clone genes that display enhanced transcription in simple embryoid bodies (EB), which are formed on ESC culturing in suspension and are characterized by the presence of endodermal cells. The cloned sequences proved to include three new genes lacking homologs in databases. Northern analysis of the transcript tissue distribution in adult mice confirmed higher expression of these genes in differentiated cells compared with ESC.
Hybridization with cDNA arrays was used to obtain expression profiles of 214 protein-tyrosine kinase, protein-tyrosine phosphatase, dual-specific phosphatase, and other genes for kidney carcinomas (KC) and normal kidney tissues of 34 patients and for seven carcinoma cell lines. Computer analysis revealed three clusters of genes coexpressed in KC. A proliferating-cell gene cluster included MET, VIM, MYC, TOP2A, PCNA, etc. A neoangiogenesis and blood-cell gene cluster included LCK, HCK, FGR, MMP9, CSFR1, VEGF, FLT1, and KDR. A cluster corresponding to normal, differentiated kidney cells included ERBB2 (HER2) for receptor protein-tyrosine kinase, several phosphatase genes (PTPRE, PTPRB, DUSP9), and EGF. The results suggested that MET, DUSP9, PCNA, TOP2A, and VIM may serve as diagnostic and prognostic markers in KC. Tubulin and topoisomerase II were assumed to be promising targets for cell proliferation inhibitors in KC.
Hybridization with cDNA arrays was used to obtain expression profiles of 263 protein-tyrosine kinase (PTK), protein-tyrosine phosphatase (PTP), dual-specific phosphatase (DuSP), and other genes for the normal prostate tissue, primary prostate carcinomas (PC) of 84 patients, 7 xenografts, and 5 carcinoma cell lines. Analysis of 96 profiles revealed eight clusters of genes coexpressed in PC (coefficient of correlation r > 0.7). According to the known functions of their genes, the clusters were designated as proliferating-cell (CDC42, TOP2A, FGFR3, MYC, etc.), neoangiogenesis and blood-cell (LCK, VAV1, KDR, VEGF, MMP9, SYK, PTPRS, and FLT4), invasion-1 and invasion-2 (ADAM17, TRPM2, DUSP6, VIM, CAV1, CAV2, JAK1, PTPNS1, FYN, and PDGFB), HER2, and PSA/PSM/HER3. Basing on expression profiles of 66 genes, a molecular classification of PC was constructed and allowed discrimination between PC and cell lines or xenografts at 98.9% probability. The results suggested that, along with PSA, PSM (FOLH1), kallikrein-2, and a-2-macroglobulin, cell signaling genes EGFR, HER2, HER3, TOP2, KRT8, KRT18, VEGF, CD44, VIM, CAV1, and CAV2 may serve as diagnostic and prognostic markers in PC. The HER2, VEGF, and CD44 genes and the MMP and ADAM families were assumed to be promising targets for inhibitors of PC cell proliferation and metastasis.
.5 Gy prenatal neutron irradiation at the 17 day of gestation resulted in activation of gene expression in brain. Elevated level of neuronal (GAP-43, NCAM, protein kinase C, calmodulin) and protooncogene (c-foc, c-jun, c-mas) gene transcripts in brain of 2-4 weeks old rats well correlated both with brain maturation in normal animals and development of brain weight deficiency in the irradiated rats. Activation of gene expression appears to be a compensatory response of surviving brain cells to irradiation. Suppression of the gene activity in brain of 18 months old rats correlated with impairment of brain functions at later periods after prenatal irradiation.
An attempt was made to elucidate possible participation of low molecular weight nuclear RNA's (LMWN RNA's) in the transcription process. For this purpose, we studied the effect of individual fractions of LMWN RNA's, isolated by polyacrylamide gel electrophoresis, on the endogenous RNA synthesis in isolated nuclei. We have found no influence of LMWN RNA's on the incorporation of labeled precursors in the acid-insoluble material under the conditions when RNA polymerase I is predominantly active. The results obtained thus indicate that LMWN RNA's do not participate in the regulation of 45S pre-rRNA synthesis and they do not belong to limiting factors in pre-rRNA synthesis.
Small RNAs were studied in the cell-free system containing cytosol and nucleoside triphosphates. The specific incorporation of NMPs into small RNAs was shown. While AMP and CMP were incorporated predominantly into tRNA, UMP was incorporated into 4.5S, 5S, and U1 RNAs. GMP was not incorporated into small RNAs in this system. The data suggest that maturation of 4.5S, 5S, and U1 RNAs involves post-transcriptional addition of uridylate residues at their 3'-ends.
Nuclear RNA was isolated from the majority of adult rat tissues, and from the whole embryos at three different stages of gestation. 13 different mRNA preparations were mixed and the sequence complexity of this total RNA (sigma RNA) was measured by RNA-driven hybridization to [125I]DNA under conditions of maximal stringency. With unique DNA as a probe, the complexity of sigma RNA was equivalent (by assuming asymmetric transcription) to 55.4% of the single-copy genome, corresponding to enough information for 2.2 . 10(5) different 4.5 k.b. genes. With DNA probe containing both unique and middle-repetitive sequences (C0t greater than or equal to 0.02), the complexity of sigma RNA was equivalent to 67.6% of the genome. Nearly all middle-repetitive DNA is represented (by assuming asymmetric transcription) in embryos nuclear RNA. The value of genetic complexity of sigma RNA (55.4 and 67.6%) represents the highest published value of RNA complexity and approach the maximal theoretical capacity of the mammalian genome. We conclude that despite the dramatic increase of the genome size in higher animals, the relative values of their genetic information expression (at the level of nuclear RNA) is comparable with that in lower eukaryotes and bacteria.
Present evidence on the use of genetic engineering methods in studying the molecular mechanism of radiation damage and repair of DNA, as well as radiation mutagenesis and carcinogenesis has been summarized. The new approach to radiobiological research has proved to be extremely fruitful. Some previously unknown types of structural disorders in DNA molecule have been discovered, some repair genes isolated and their primary structure established, some aspects of radiation mutagenesis elucidated, and research into deciphering the molecular bases of neoplastic transformations of exposed cells are being successfully investigated. The authors discuss the perspectives of using genetic engineering methods in radiobiology.
Possible involvement of small nuclear RNAs in transcription was explored. The fraction of small (4-8S) nuclear or cytoplasmic RNAs was isolated by Sepharose 4B gel chromatography and its influence on the RNA synthesis in isolated nuclei was studied. Small nuclear or cytoplasmic RNAs stimulated RNA synthesis in isolated nuclei both in the presence and in the absence of cytosol. On the contrary, the influence of E. coli tRNA or high molecular weight cytoplasmic RNA varied depending on the presence of cytosol. The data indicate that small nuclear RNAs may participate in transcription although the mechanisms of their involvement demand further investigation.
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