Showing papers on "Transcription (biology) published in 1977"

Glucocorticoid-stimulated accumulation of mouse mammary tumor virus RNA: increased rate of synthesis of viral RNA

Abstract: Glucocorticoid hormones specifically increase the intracellular concentration of mouse mammary tumor virus (MMTV) RNA in a cultured cell line from a GR mouse mammary carcinoma (GR) and in an MMTV-infected rat hepatoma cell line (M1.19). In contrast, these steroids have no effect on the concentration of MMTV RNA in a lymphoma line, S49, from a Balb/c mouse. Using a molecular hybridization procedure to detect newly synthesized RNA, we have directly measured the effect of dexamethasone, a synthetic glucocorticoid, on the rate of MMTV RNA synthesis. In GR cells the hormone causes a 10-fold increase in the rate of synthesis of viral RNA without appreciably affecting the overall rate of cellular RNA synthesis. The transition from the basal to the maximally stimulated rate of MMTV RNA synthesis occurs within the earliest labeling period, 0-15 min after addition of the hormone. Thus, it appears that glucocorticoids regulate MMTV genes principally by this rapid and specific alteration of their rate of transcription. Similar results are obtained in M1.19 rat hepatoma cells. In contrast, dexamethasone does not affect the rate of viral RNA synthesis in S49 lymphoma cells.

Transcription termination at the trp operon attenuators of Escherichia coli and Salmonella typhimurium: RNA secondary structure and regulation of termination.

Abstract: Transcription termination at the attenuators of the trp operons of Escherichia coli and Salmonella typhimurium was studied in vitro using DNA restriction fragments as templates. Readthrough transcription beyond the terminators occurred with 5 and 30% efficiency, respectively, in E. coli and S. typhimurium. This difference is correlated with the stability of proposed secondary structures of the respective trp leader transcripts. Secondary structure analyses of the two leader transcripts revealed a well-conserved pattern of RNA base paring. This and the possibility that trp leader RNA is translated suggest a model for regulation of transcription termination that is based on ribosome movement along the RNA and a shift between alternative RNA base-pairing configuration.

Evidence that herpes simplex virus DNA is transcribed by cellular RNA polymerase B.

Abstract: In herpes simplex virus type 1 (HSV-1)-infected HEp-2 cells, amanitin added before or at various times after infection always reduced viral multiplication. Also, the three waves of transcription of HSV-1 DNA, which led to the synthesis of alpha, beta-, and gamma-polypeptides, were all sensitive to amanitin in HEp-2 cells, and the amanitin-sensitive RNA polymerase activities of isolated nuclei were equally sensitive to the inhibitor before and during the infection. On the contrary, HSV-1 DNA transcription was totally unaffected by amanitin in AR1/9-5B cells, a mutant subline of CHO cells that possesses an amanitin-resistant RNA polymerase B. Together, these results strongly suggest that HSV-1 DNA utilizes for its transcription a polymerase undistinguishable from host cell RNA polymerase B with respect to its sensitivity to amanitin.

Characterization of the autoregulation of simian virus 40 gene A.

Abstract: Cells infected by tsA mutants of simian virus 40 (SV40) overproduce early RNA. Overproduction results from failure of the temperature-sensitive A protein (T antigen) to inhibit early transcription. The amount of early RNA in the cytoplasm, determined quantitatively from the kinetics of hybridization to labeled complementary SV40 DNA, was elevated at both permissive (32°C) and nonpermissive (41°C) temperatures in all the early mutants tested (tsA7, -30, -58, and -209), but not in the late mutant tsB4. The amount of early RNA in a culture maintained at 32°C for 72 h and then shifted to 41°C was maximum when each cell was infected initially with at least one plaque-forming unit of tsA58. Azidocytidine (2′-deoxy-2′-azidocytidine), which inhibits initiation of DNA synthesis, did not cause overproduction of early RNA in cells infected with wild-type SV40, showing that the effect seen with tsA mutants is not due to interference with initiation of DNA synthesis per se. In parallel infections at 41°C, the amount of early RNA per copy of viral DNA was as much as 2,000 times greater with tsA58 than with wild-type SV40, even though there was no replication of the tsA58 DNA. Synthesis of late RNA could not be detected during the first 20 h of an infection by either virus at 32°C, indicating that late and early transcription are under different control. In three cell lines transformed by tsA mutants, the amount of early RNA increased moderately after a shift from 32 to 41°C, whereas with homologous cells transformed by wild-type virus, the amount of early RNA decreased, indicating that the A protein may be able to repress transcription of integrated SV40 DNA. All the observations are consistent with a simple model in which the binding of A protein at the origin of replication blocks either binding of RNA polymerase to the early promoter or its progress through the early gene(s).

Regulation of Early and Late Simian Virus 40 Transcription: Overproduction of Early Viral RNA in the Absence of a Functional T-Antigen

Abstract: Virus-specific RNA synthesized in monkey cells after infection by both wild-type simian virus 40 (SV40) and the early SV40 temperature-sensitive mutant tsA58 has been analyzed. The fraction of SV40-specific RNA increased throughout infection with either wild-type SV40 or with tsA58 in direct proportion to the accumulation of progeny DNA molecules, suggesting their role in the late transcriptional process. Cytoplasmic fractions from cells infected at various temperatures (31.5 to 41°C) by wild-type virus and harvested 48 h later contained 4 to 8% virus-specific RNA, of which 5 to 10% was early SV40 RNA. In contrast, though 5 to 8% of the cytoplasmic RNA from tsA 58-infected cells incubated at 31.5 to 37°C for 48 h was virus specific, the percentage of early virus-specific RNA ranged from 25 to 80% as the incubation temperature increased. In tsA58-infected cultures incubated for 48 h at 41°C (a temperature at which essentially no tsA 58 DNA synthesis occurred), only 0.4% of the cytoplasmic RNA was virus specific, but at least 90% of this RNA was early. In experiments where cells were inoculated at 32°C and shifted at 48 h postinfection to 40°C for various times, the percentage of virus-specific pulse-labeled RNA varied from 3.5 to 10.0%. Of the virus-specific RNA, early SV40 RNA ranged from 14 to 65% in tsA 58-infected cultures. Analogous studies with Sarkosyl-extracted viral transcription complexes to incorporate label into nascent (unprocessed) viral RNA yielded essentially identical results. This finding strongly suggests that the overproduction of early SV40 RNA occurs at the level of synthesis. While cytosine arabinoside effectively terminated most viral DNA replication in wild-type-infected cells, the ratio of early to late viral RNA remained less than 1:9. These results demonstrate that: (1) the amount of virus-specific RNA synthesized depends directly on the amount of viral DNA available for use as templates; once viral DNA replication has occurred, presumably providing progeny SV40 DNA molecules for templates, the level of transcription remains high; (ii) termination of viral DNA replication does not terminate late SV40 transcription; (iii) early SV40 RNA is overproduced by tsA 58 at all temperatures, but especially at higher temperatures; and (iv) overproduction of early SV40 RNA appears to be correlated with defectiveness of the tsA mutant T-antigen. These results suggest that T-antigen may regulate its own production either by repressing the synthesis of early viral RNA or by stimulating the synthesis of late SV40 RNA or both.

Cell-free translation of simian virus 40 early messenger RNA coding for viral T-antigen.

Abstract: Simian virus 40 (SV40) mRNA was isolated by hybridization of cytoplasmic RNA, from SV40-infected BS-C-1 monkey cells early in lytic infection, to SV40 DNA immobilized on Sepharose. The early viral mRNA, when added to a wheat-germ translation system, directed the synthesis of a unique class of products including a 90,000 molecular weight (Mr) polypeptide. It was found that this 90,000 Mr product as well as a prominent 17,000 Mr polypeptide could be specifically immunoprecipitated with hamster antiserum to SV40 T-antigen, but not with hamster control serum. Similar immunoprecipitation of extracts of SV40-infected cells with hamster anti-T serum yielded 90,000 Mr and 17,000 Mr polypeptides; these polypeptides were not found in immunoprecipitates of uninfected cell extracts. SV40 cRNA, prepared by asymmetric transcription of plaque-purified SV40 DNA, directed the cell-free synthesis of several products, including a 70,000 Mr polypeptide that could be specifically immunoprecipitated with anti-T serum. However, no T-antigen-related polypeptide was found in infected cells that corresponded in size to the major immunoprecipitated cRNA product.

Vesicular stomatitis virus: mode of transcription.

Abstract: Recent studies on the mechanism by which the virion-associated RNA polymerase of vesicular stomatitis virus transcribes RNA have revealed several new biological features of general interest. The mode of synthesis of the 5'-terminal cap structure of the mRNAs, the sequential transcription of the genes and the presence of a transcribed "leader" RNA segment are properties which are either not shown by other viruses, or have not yet been described. These features are probably inter-related with the primary transcription process, which itself may be a useful model for future studies on mRNA biosynthesis in eukaryotic systems.

Purified DNAs are transcribed after microinjection into Xenopus oocytes.

Abstract: The possibility of using DNA-injected Xenopus laevis oocytes and eggs for studying the control of transcription in eukaryotes has been investigated. When purified DNA of simian virus 40 (SV40) is injected into Xenopus laevis oocytes, tritiated RNA precursors are incorporated into DNase-I-resistant, RNase-A- and alkali-sensitive material that hybridizes specifically to SV40 DNA. This viral transcription continues for at least 5 days and occurs only when the injected DNA is directed to the nucleus of the oocyte. The quantity of SV40-specific RNA produced is roughly proportional to the amount of DNA injected; above 1 ng per oocyte, most of the nonribosomal RNA made in successfully injected oocytes is virus-specific. Transcription also occurs, although at a lower efficiency, after injection of the DNA into unfertilized eggs. The DNAs of adenovirus 5, cloned Drosophila melanogaster histone genes, and even bacteriophage phiX174 replicative form, bacteriophage phi80plac, and the ColE1 plasmid are also transcribed after injection into oocytes or eggs.

Genes transcribed at diverse rates have a similar conformation in chromatin

Abstract: We have analyzed the DNA generated upon treatment of oviduct nuclei with pancreatic DNase I (deoxyribonucleate 3′-oligonucleotidohydrolase; EC 3146), with cDNA copies of specific mRNA sequences to study the structure and organization of transcriptionally active genes in chromatin In this report we examine the kinetics of digestion of three classes of genes in the oviduct which are transcribed at significantly different rates Our results indicate that the ovalbumin genes appear to be organized by chromatin proteins in such a way that they are rendered exceedingly sensitive to digestion by DNase I This sensitivity is not observed in the liver, a tissue in which these genes are transcriptionally inert Furthermore, the transcriptionally inactive globin genes in the oviduct are not selectively sensitive to nuclease attack and are digested 5 times more slowly in the ovalbumin genes in this tissue In addition, we have examined the accessibility of a complex subset of genes that are rarely represented in the mRNA and are likely to be transcribed at a frequency orders of magnitude below that of the ovalbumin gene Comparison of the accessibility of these sequences with that of the ovalbumin gene indicates that these two subsets of genes are recognized and cleaved by DNase I at similar rates These results suggest that the maintenance of an active conformation about specific genes does not reflect the polymerase distribution about these genes This active conformation is therefore not confined to sequences actively engaged in the transcription process and may reflect the structure about a subpopulation of the genome which represents the transcriptional potential of a given cell type

Steroid induction of mouse mammary tumor virus: effect upon synthesis and degradation of viral RNA.

Abstract: Steroid hormones have been demonstrated to induce in tissue culture the production of mouse mammary tumor viral (MMTV) RNA, proteins, and particles 10-fold compared with constitutive levels. However, previous data of increased viral RNA levels did not distinguish between an increased rate of viral-specific RNA synthesis and a slower rate of viral RNA degradation. According to the recently developed assay of Coffin et al. (1974) for measuring rates of viral RNA synthesis, short-term labeling experiments of a mouse mammary tumor cell line indicate that the glucocorticoid hormone dexamethasone stimulates a 3-fold increase in the synthesis of MMTV-specific RNA within 10 min after the addition of hormone and that stimulation of RNA synthesis reaches 5- to 10-fold within 30 to 60 min, while the synthesis of Moloney leukemia virus-specific RNA in the same cell is unaffected by steroids. The decay rates of pulse-labeled and accumulated MMTV RNA in the presence or absence of dexamethasone show this RNA to have a half-life of greater than 8 h. In addition, hormone-stimulated MMTV RNA appears to have an increased rate of decay compared to basal MMTV RNA, thus ruling out an increased stability of MMTV RNA in the presence of steroid hormones as the basis for increased RNA levels. Thus, the magnitude, rapidity, and specificity of hormone action on MMTV RNA synthesis indicate a primary effect upon transcription.

High-fidelity transcription of 5S DNA injected into Xenopus oocytes.

Abstract: Purified DNA, from Xenopus erythrocytes, containing genes for 5S ribosomal RNA (58 DNA) is transcribed faithfully when it is injected into the nucleus of a Xenopus oocyte. Full length 5S RNA of the correct sequence is transcribed from the injected DNA, and the newly synthesized transcripts hybridize predominantly with the coding strand of 5S DNA. The majority of transcripts hybridize with the gene region as opposed to the spacer region of the coding strand. Accurate transcription starts within the first 3 hr and continues for up to 4 days after injection of 5S DNA. Radioactive 5S RNA is detected in as short a labeling time as 1 hr.

RNA Methylation and Control of Eukaryotic RNA Biosynthesis

Abstract: The role of RNA methylation in the control of ribosome production in mammalian cells has been reinvestigated through a study of the effects in vivo of cycloleucine, a specific and reversible inhibitor of nucleic acid methylations. No close coupling is observed between methylation and transcription. During an extensive blocking of methylation, the synthesis of preribosomal 45-S RNA continues, although at a slightly reduced rate. Transcription and methylation can be temporarily uncoupled in vivo without impairing significantly the efficiency of the subsequent maturation of the transcript which takes place when the methylation is resumed. At the post-transcriptional level, two main observations are made. First, the cleavage pattern of ribosomal RNA is not qualitatively modified by the drug treatment. Preribosomal 45-S RNA which is synthesized during an extensive blocking of methylations (95%) is cleaved in vivo in a stepwise fashion into molecules resembling the species of the normal processing. Undermethylation does not result in the appearance of new size products and no extensive or rapid degredation of rRNA precursors can be detected. Second, the global efficiency of the process of ribosomal RNA maturation is severely affected, in quantitative terms; the undermethylation partially inhibits several stages in the maturation pathway and does not block selectively at a definite step of processing, as previously reported by others. Two main modifications are observed. (a) In the nucleus, the life times of the various undermethylated intermediates of ribosomal RNA maturation are markedly increased and a significant accumulation of these forms is detected by long-term labelling studies. (b) The rate of appearance of ribosomal 28-S RNA into the cytoplasm is severely affected (85–90%) inhibition), in much higher proportions than the formation of its immediate precursor, nuclear 28-S RNA, thus suggesting a particular sensitivity to a lack of methylation of the last stage of preribosome maturation (possibly the release to cytoplasm). Finally, if a normal level of methylation does not appear to be stringently required for the completion of ribosomal RNA maturation, our results indicate that the degree of RNA methylation can modulate the general efficiency of the maturation processess.