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An enzyme, 5'-hydroxyl polyribonucleotide kinase, which catalyzes the phosphorylation of 5'-hydroxyl ends of RNA in the presence of ATP, has been isolated from extracts of HeLa cell nuclei. The kinase requires a divalent cation (Mg2+ or Mn2+) for activity, has an alkaline pH optimum, and is sensitive to the sulfhydryl antagonist N-ethylmaleimide. 5'-hydroxyl terminated polydeoxyribonucleotides are phosphorylated much less efficiently than the 5'-hydroxyl terminated polyribonucleotides, and the kinase preparation is inactive on ribonucleoside 3'-monophosphates. Enzyme activity is inhibited by ADP and by pyrophosphate. The sedimentation coefficient of the kinase is estimated to be 5.6 S from glycerol gradient centrifugation. 相似文献
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The interferon-induced protein kinase PK-i from mouse L cells. 总被引:8,自引:0,他引:8
A Kimchi A Zilberstein A Schmidt L Shulman M Revel 《The Journal of biological chemistry》1979,254(19):9846-9853
Interferon-treated L cells are characterized by an increased protein kinase activity that can selectively phosphorylate the small subunit of eukaryotic initiation factor 2. This protein kinase, PK-i, has been extensively purified and shown to be a potent inhibitor of mRNA translation. The purified PK-i contains the endogenously phosphorylated 67,000 Mr protein characteristic of interferon-treated cell extracts. PK-i can also phosphorylate arginine-rich histones. Purified PK-i can be activated by preincubation with ATP (but not adenylyl imidodiphosphate) and low concentrations of double-stranded RNA. The activation results in an increase in the first rate of eIF-2 phosphorylation. Activated PK-i becomes resistant to high concentrations of double-stranded RNA and more thermostable. A stimulator of PK-i activity, factor A, was isolated, as well as a specific phosphoprotein phosphatase that dephosphorylates the 67,000 Mr protein and eIF-2. These two factors, which are present in untreated L cells, may regulate the translation inhibitory activity of the interferon-induced and double-stranded RNA-activated protein kinase PK-i. 相似文献
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Messenger RNA turnover in mouse L cells 总被引:36,自引:0,他引:36
The turnover of polyadenylic acid-containing messenger RNA and histone messenger RNA, which lacks poly(A), was studied in exponentially growing mouse L cells by measuring the kinetics of approach to steady-state uridine labeling. Constant specific activity of precursor pools was verified by showing that the data for stable RNA components, like ribosomal RNA and transfer RNA, follow theoretically predictable curves. In agreement with a previous report by Greenberg (1972), the data for poly(A)-containing mRNA (poly(A)(+)mRNA) follow theoretical curves for a class of molecules turning over with first-order (stochastic) kinetics. Cells growing with doubling times of 13·5 hours at 37 °C and 41 hours at 30 °C exhibited mean lifetimes for their poly(A)(+)mRNA of 15 hours and 42 hours, respectively, suggesting a parallelism between growth and turnover rates. The kinetic data for histone mRNA are not indicative of a stochastic process. Rather, they suggest an age-dependent decay or a zero-order (ordered) turnover with a mean lifetime of about six hours. One model, which gave a good fit to the data, considers that the histone messages persist for a fixed duration of the cell cycle, e.g. the DNA synthetic phase, and are then destroyed in a “sensitive period” after this phase. These results are discussed with regard to the possible implications of the poly(A) sequences in messenger RNA aging. 相似文献
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Comparison of methylated sequences in messenger RNA and heterogeneous nuclear RNA from mouse L cells 总被引:9,自引:0,他引:9
A variety of methylated oligonucleotides were derived from mouse L cell messenger RNA and heterogeneous nuclear RNA by digestion with specific ribonucleases, and the cap-containing oligonucleotides separated from those containing internal m6A by chromatography on diborylaminoethyl-cellulose. Cap-containing sequences of the type m7GpppXmpG, m7GpppXmpY(m)pG, m7GpppXmpY(m) pNpG and m7GpppXmpY(m)p(Np)> 1G have distinctive non-random compositions of the 2′-O-methylated constituent Xm; yet sequences of a particular type and composition occur with a remarkably similar frequency in mRNA and hnRNA2. For example, approximately 20% of the cap sequences in both hnRNA and mRNA are m7Gppp(m6)AmG, whereas less than 1% are m7GpppUmpG. The high degree of similarity in cap sequences is consistent with the previously postulated precursor-product relationship between hnRNA caps and mRNA caps.The composition of the Y position in capped hnRNA molecules was determined to be (29% G, 20% A, 51% Py), which differs considerably from the composition of Ym in the cap II forms of mRNA (8% Gm, 11% Am, 81% Py). Given the precursor-product relationship between hnRNA caps and mRNA caps, this result provides strong evidence that only a restricted subclass of mRNA molecules receive the secondary methylation at position Y.In both hnRNA and mRNA the internal m6A occurs in well-defined sequences of the type: , the 5′ nearest-neighbor of m6A being G in about three-quarters of the molecules and A in about one-quarter of the molecules. The nucleotide N1 is a purine about 90% of the time and the nucleotide N2 is rarely a G. These same sequences are present in large (> 50 S), as well as small (14 S to 50 S) hnRNA. These results raise the possibility that the internal m6A, like caps, may be conserved during the processing of large hnRNA into mRNA. Two models based on this idea are discussed. 相似文献
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Treatment of mouse L cells with mouse IFN gamma induced a cytoplasmic Ca-dependent protein kinase, which highly phosphorylated cellular enzymes such as phosphodiesterase and RNase in vitro. The kinase partially purified from IFN gamma-treated cells (100 units/ml, 12 h at 37 degrees C) was different from IFN-induced dsRNA-dependent protein kinase since it was dsRNA independent. The kinase may have played an important role in mediating IFN-induced biological effects, since cellular enzymes were found to alter enzyme activity after phosphorylation by the kinase in vitro. 相似文献
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Nucleotide sequence of nuclear 5S RNA of mouse cells 总被引:5,自引:0,他引:5
The nucleotide sequence of nuclear 5S RNA of mouse cells was determined. The 5S RNA is 117 nucleotides long with one mole each of m32,2,7G, Gm, Am and Cm, two moles of Um, and three moles of ψ as modified nucleosides, and it is rich in uridylate residues (about 36 %). The 5′-terminal hexanucleotide-containing cap structure, m32,2,7GpppAm-Um-A-C-U-, is identical with that of U1 RNA. This RNA contains sequences complementary to the terminal sequences of the introns of heterogeneous nuclear RNAs. 相似文献
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5-methylnicotinamide is an inhibitor of poly(ADP-ribose) synthetase, and it enhances the cytotoxicity of alkylating agents and of radiation in mouse lymphoma L1210 cells. We have isolated a spontaneous variant L1210 cell which shows increased resistance to 5-methylnicotinamide and has a reduced potentiation of cell-killing by methylnitrosourea and by γ-radiation. This observation is further evidence in support of the participation of (ADP-ribose)n in DNA excision repair and in cell survival. This variant may be of use in the molecular analysis of this component of DNA repair. 相似文献
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TFT is an effective selective agent for TK-deficient mutants of L5178Y TK+/- -3.7.2C mouse lymphoma cells. Mutants can be classified by colony size into small colonies (many of which show readily observable chromosome abnormalities associated with chromosome 11--the location of the TK gene) and large colonies (which may represent events affecting only the expression of the TK gene). The precise nature of the induced damage causing the loss of the TK-enzyme activity for both mutant type is not known and is currently under investigation. The hypomethylating agent 5-azacytidine can be utilized to investigate the possibility that mutants might be the result of a suppressed rather than an altered TK gene. Mutant cell lines are treated with 5-azacytidine and then evaluated for re-expression of the TK enzyme as measured by resistance to THMG. In these studies, 11 mutants have been evaluated. None of the 11, including 10 small-colony mutants (6 with chromosome 11 translocations) and 1 large-colony mutant, show a high conversion to TK competency following 5-azacytidine treatment. 相似文献
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Ribonuclease resistance of RNA from mammalian cells was studied as a possible measure of the extent of base pairing. Ribonuclease-sensitive and resistant components of the RNA were discerned from the biphasic kinetics of the ribonuclease reaction. The amount of resistant component increased progressively with increasing salt concentration until it comprised 20–40% of the total RNA. Comparisons between different RNA fractions from L1210 cells revealed differences in the content of resistant component, in the order: nucleolar ribosomal-precursor RNA > mature ribosomal RNA > nucleoplasmic RNA. In each RNA fraction, the amount of resistant component was greater when measured by a cytidine label than by a uridine label. Comparison with known double-stranded RNA indicated that the bulk of the resistant component consists of lessstable structures, such as short or imperfect helices. 相似文献
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The recombinant locus ORAgp5 containing the regions homologous to protooncogene mos was previously cloned from the human genome. In order to monitor the expression of this human genome region, we have transfected the recombinant phage gp5 into mouse LMtk cells. One of these transfectants contained several copies of gp5. ORAgp5 sequences were found to be expressed in 1.5 kb RNA from this clone. 相似文献
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We describe some fundamental properties of the cytidine 5'-diphosphate (CDP) and guanosine 5'-diphosphate (GDP) reductase activity from mouse L cells. Both activities increased in a nonlinear fashion at low protein concentrations; this may be due to dissociation of two protein subunits of the enzyme at very low concentrations. CDP reductase activity was greatly stimulated in the presence of ATP and required magnesium and iron for maximum activity. GDP reductase required 2'-deoxythymidine 5'-triphosphate for maximum activity. Also apparent Km values of 0.14 mmol/l for CDP and 0.05 mmol/l for GDP were determined from double reciprocal plots of velocity against substrate concentrations. Activity in extracts of logarithmically growing mouse L cells was very high indicating that attempts to purify the enzyme from this source should be rewarding. 相似文献
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D Kessel 《The Journal of biological chemistry》1968,243(18):4739-4744
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Regulation of thymidine kinase (TK) activity was examined in L(O)c133 and L(H3) cells carrying varicella-zoster virus-TK gene. TK activity of L(O)c133 cells was similarly high in either medium but that of L(H3) cells was high in HAT medium and low in non-HAT medium. Cell growth was well correlated with TK activities of L(O)c133 and L(H3) cells in medium conditions. Regulation of the TK gene in L cells carrying the VZV-TK gene is discussed. 相似文献