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Translation of RNAs Synthesized In Vivo and In Vitro from Bacteriophage SP82 DNA 总被引:4,自引:2,他引:2
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The synthesis of 69 phage-specific polypeptides during the infection of Bacillus subtilis with bacteriophage SP82 was detected by pulse-labeling, one-dimensional electrophoresis, and autoradiography. SP82 virions were found to contain approximately 22 polypeptides, most of which were synthesized late in infection; evidence was obtained for the processing of the major virion protein. RNAs extracted at different times during infection were translated by using an Escherichia coli cell-free extract. Only smaller-molecular-weight peptides were produced efficiently in vitro; in the 9,000- to 60,000-molecular-weight range, 50 to 60% of the peptides synthesized in vivo were produced by translation of RNAs extracted from infected cells. Eight of the virion peptides were produced by in vitro translation of RNAs extracted from infected cells. RNAs were synthesized under defined conditions by RNA polymerase extracted from uninfected B. subtilis and by polymerases isolated from cells 8 and 20 min after infection with SP82. Translation of these RNAs yielded characteristic and different patterns of polypeptides. Nine of the 12 polypeptides produced by translation of RNAs synthesized by the host polymerase corresponded in mobility to peptides appearing in vivo in the 0 to 3 and 3 to 6 min intervals of pulse-labeling after infection; 12 of the 25 peptides synthesized from RNAs produced by polymerase extracted 8 min after infection corresponded in mobility to peptides detected in vivo 8 min after infection, and 15 of the 22 peptides directed by RNAs made by the polymerase isolated 20 min after infection corresponded to peptides present in vivo late in infection. Five of the peptides produced in vitro from the latter RNA corresponded to virion peptides. 相似文献
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Predominance of bacteriophage SP82 over bacteriophage SP01 in mixed infections of Bacillus subtilis
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In mixed infections with Bacillus subtilis phages SP82 and SP01, the SP82 genotype is predominant among the progeny. This predominance is determined by a specific region of the genome, the pos region, which apparently is located near genes 29 to 32 (by the SP01 numbering system). Recombination between SP82 and SP01 yields phage which have both the SP82 pos region and an SP01 mutation. This mutation then behaves in mixed infection as if it were part of an SP82 genome. 相似文献
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Heidrun Heilmann John N. Reeve Alfred Pühler 《Molecular & general genetics : MGG》1980,178(1):149-154
Summary P1 infected minicells synthesize approximately 50 phage-encoded polypeptides. Phage expression is temporally controlled, demonstrating phage polypeptides synthesized both early and late after infection. The P1 repressor, gpc1
1 (Mr=33,000), repressor bypass polypeptide, gprebA (Mr=27,500) and cistron 10 product, (gp10) (Mr=64,000), have been identified by infection of minicells with P1 amber mutants. The beta-lactamase gene product (gpbla) carried by the closely related phage P7 and the chloramphenicol acetyl-transferase gene product (gpcat) carried by P1 Cm (in Tn9) have been demonstrated. Infection of minicells by P1vir
s or P1c4 mutants results in increased synthesis of gprebA and a second polypeptide designated gprebB (Mr=40,000). The P1vir11 mutation leads to increased synthesis of a small polypeptide (Mr=3,500) but does not affect the amount of gpc1 synthesized. 相似文献
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Purification and properties of a new bacillus subtilis RNA processing enzyme. Cleavage of phage SP82 mRNA and Bacillus subtilis precursor rRNA 总被引:5,自引:0,他引:5
An RNA processing activity capable of cleaving Bacillus subtilis phage SP82 early mRNA has been purified to apparent homogeneity from crude extracts of uninfected B. subtilis. The enzyme, a functional monomer of Mr approximately 27,000, cleaves only at the 5' side of adenosine residues at processing sites and is competitively inhibited by double-stranded synthetic RNA polymers. Processed SP82 mRNAs were translated in an Escherichia coli cell-free system and no qualitative or quantitative effects of processing on the synthesis of polypeptides was observed. The processing enzyme does not cleave T7 mRNA, E. coli precursor rRNA, or double-stranded poly(AU). A recombinant plasmid containing portions of two B. subtilis rRNA gene sets was transcribed in vitro and the resulting RNA was cleaved in the spacer region between the 16 S and 23 S rRNA genes. The ability of the B. subtilis processing enzyme to cleave SP82 mRNA and B. subtilis precursor rRNA and the fact that the enzyme has high affinity for double-stranded RNA suggest that it is the functional analog of E. coli RNase III. 相似文献
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The synthesis of adenovirus type 2 (Ad2)-induced early polypeptides was examined in vivo and in vitro by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis alone and specific immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of total [35S]methionine-labeled polypeptides synthesized in vivo at 3 h postinfection allowed us to detect in infected cells at lease 13 distinct polypeptides that are either absent or less conspicuous in extracts from mock-infected cells. These Ad2-induced early polypeptides have molecular weights ranging from 72 x 10(3) to 10.5 x 10(3) and have accordingly been designated as E72K to E10.5K. Nine of the in vivo synthesized early polypeptides can be precipitated specifically from infected cell extracts by antisera with specificity against early adenovirus proteins. In vitro translation of mRNA extracted from mock-infected cells and from Ad2-infected cells was carried out in preincubated Ehrlich ascites cell extracts. All the early Ad2-induced polypeptides identified in the extracts from infected cells labeled in vivo were also detected among the polypeptides immunoprecipitated specifically from the in vitro reaction mixtures programmed by RNA extracted at 4 h postinfection from Ad2-infected cells. 相似文献
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A wheat germ cell-free translation system has been used to analyze populations of abundant messenger RNA from sea urchin eggs and embryos and from amphibian oocytes and ovaries. We show directly that sea urchin eggs and embryos contain translatable mRNA of three general classes: poly(A)+ mRNA, poly(A)? histone mRNA, and poly(A)? nonhistone mRNA. Additionally, some histone synthesis appears to be promoted by poly(A)+ RNA. Sea urchin eggs seem to contain a higher proportion of prevalent poly(A)? nonhistone mRNAS than do embryos. Some differences in the proteins encoded by poly(A)+ and poly(A)? RNAs are detectable. Many coding sequences in the egg appear to be represented in both poly(A)+ and poly(A)? RNAs, since the translation products of the two RNA classes exhibit many common bands when run on one-dimensional polyacrylamide gels. However, some of this overlap is probably due to fortuitous comigration of nonidentical proteins. Distinct stage-specific changes in the spectra of prevalent translatable mRNAs of all three classes occur, although many mRNAs are detectable throughout early development. Particularly striking is the presence of an egg poly(A)? mRNA, encoding a 70,000–80,000 molecular weight protein, which is not detected in morula or later-stage embryos. In amphibian (Xenopus laevis and Triturus viridescens) ovary RNA, the translation assay detects the following three mRNA classes: poly(A)+ nonhistone mRNA, poly(A)? histone mRNA, and poly(A)+ histone mRNA. Amphibian ovary RNA appearently lacks an abundant poly(A)? nonhistone mRNA component of the magnitude detectable in sea urchin eggs. mRNA encoding histone-like proteins is found in the very earliest (small stage 1) oocytes of Xenopus as well as in later stage oocytes. During oogenesis there appear to be no striking qualitative changes in the spectra of prevalent translatable mRNAs which are detected by the cell-free translation assay. 相似文献
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Characterization of Ehrlich ascites tumor cell messenger RNA specifying ribosomal proteins by translation in vitro 总被引:2,自引:0,他引:2
We have examined the messenger RNA which codes for the ribosomal proteins in Ehrlich ascites tumor cells. Poly(A)-containing mRNA was isolated from polysomes and fractionated into 11 size classes whose average molecular weights were between 1.8 × 105 and 24 × 105. These mRNAs were used to direct protein synthesis in a fractionated translational system that was derived completely from Ehrlich ascites tumor cells. More than 90% of the ribosomal proteins which we could identify were coded for by mRNAs averaging in size between Mr = 180 × 103 and 320 × 103. The small size of these mRNAs indicates that the cytoplasmic mRNAs which specify the ribosomal proteins are monocistronic. We could detect the synthesis of 36 of 48 ribosomal reference proteins as well as 20 additional polypeptides which had characteristics similar to ribosomal protein. The ribosomal proteins were identified on the basis of their positive charge, small size, electrophoretic properties on two-dimensional polyacrylamide gels and chromatographic characteristics on carboxymethyl-cellulose. 相似文献
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Micheline Giphart-Gassler Carel Wijffelman John Reeve 《Journal of molecular biology》1981,145(1):139-163
This paper describes the identification and functional role of late gene products of bacteriophage Mu, including an analysis of the structural proteins of the Mu virion.In vitro reconstitution of infectious phage particles has shown that four genes (E, D, I, J) control the formation of phage heads and that a cluster of eight genes (K, L, M, N, P, Q, R, S) controls the formation of phage tails.Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of Mu polypeptides synthesized in Escherichia coli minicells infected by Mu phages carrying amber mutations in various late genes has resulted in the identification of the products of gene C (15.5 × 103Mr); H (64 × 103Mr); F (54 × 103Mr); G (16 × 103Mr); L (55 × 103Mr); N (60 × 103Mr); P (43 × 103Mr) and S (56 × 103Mr). Minicells infected with λpMu hybrid phages and deletion mutants of Mu were used to identify polypeptides encoded by the V-β region of the Mu genome. These are the products of genes V, W or R (41.5 × 103Mr, and 45 × 103Mr); U (20.5 × 103Mr) and of genes located in the β region (24 × 103Mr (gpgin) and 37 × 103Mr (possibly gpmom)).Analytical separation of the proteins of the Mu virion revealed that it consists of a major head polypeptide with a molecular weight of 33 × 103, a second head polypeptide of 54 × 103 (gpF) and two major tail polypeptides with molecular weights of 55 × 103 and 12.5 × 103 (gpL and gpY, respectively). In addition, there are five minor components of the tail (including gpN, gpS and gpU) and approximately seven minor components of the head structure of the virion (including gpH). 相似文献
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The genomic RNA of a member of the “Nudaurelia β virus” group functioned as a mRNA in vitro. The translation products included a protein, which comigrated with the single virus capsid protein, and a stable 100 × 103 MW protein, which was synthesized by cleavage of a precursor protein. No precursor proteins were involved in synthesis of the putative capsid protein. Attempts to inhibit proteolytic cleavage did not result in the appearance of a product corresponding to the entire coding capacity of the genome. 相似文献
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Greene FC 《Plant physiology》1981,68(3):778-783
Free and membrane-associated polysomes were isolated in approximately equal amounts from endosperm of wheat kernels harvested 20 days after anthesis. The presence of heparin in the homogenizing buffer minimized polysome degradation. Ribonucleic acid from the isolated polysomes, when translated in vitro in a wheat germ system, yielded products ranging in size from about 12,000 to about 80,000 daltons, including at least two polypeptides that co-migrated with seed extract proteins in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The nature of the translation products of free and membrane-associated RNA are distinctly different, with membrane-associated RNA yielding a higher proportion of polypeptides in the size range of 30,000 to 37,000 daltons. Analysis of membrane-associated 3′-terminal polyadenylyl-containing RNA in vitro translation products, by solubility in 70% ethanol and by immunoprecipitation, indicates that the 33,000- to 37,000-dalton polypeptides contain gliadins, and the analysis provides evidence that these proteins are synthesized in association with membranous cell organelles. Gliadin polypeptides synthesized in vitro are larger than authentic gliadins and probably are precursors which, in vivo, undergo modification to yield the smaller final products. 相似文献
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Polyadenylated RNA was isolated from total RNA extracted from cotyledons of ungerminated or 18-hour-germinated chick-pea seeds by affinity chromatography on oligo(dT)-cellulose. Both poly(A)-containing RNA fractions exhibited a template activity when assayed in two cell-free translation systems, wheat germ extracts, and nuclease-treated reticulocyte lysates. Translation of preformed mRNA from cotyledons of dry seeds was completely abolished in the presence of several inhibitors of polypeptide chain initiation and also in the presence of the two “cap” analogues m7 GTP and m7 GMP. The patterns of polypeptides synthesized by translation of poly(A)-containing RNAs from cotyledons of ungerminated or 18-hour-germinated seeds, in the wheat germ system, analyzed by electrophoresis and autoradiography, were similar but not identical. It is concluded that cotyledons of dry Cicer arietinum L. seeds contain preformed mRNA. 相似文献
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Identification of Messenger Ribonucleic Acids and Proteins Synthesized by the Bacteriocinogenic Factor Clo DF13 in Purified Minicells of Escherichia coli 总被引:22,自引:12,他引:10
It has previously been shown that the cloacinogenic factor Clo DF13 (Clo DF13) segregates into minicells of strain Escherichia coli P678-54 that harbors Clo DF13 and that this Clo DF13 factor is the only deoxyribonucleic acid (DNA) present in these otherwise chromosomeless minicells. The study reported here shows that minicells prepared from P678-54(Clo DF13) are able to incorporate radioactive precursors into ribonucleic acid (RNA) and protein. The RNA synthesized in these purified minicells is Clo DF13 specific, as shown by RNA-DNA hybridization experiments. The results indicate that all the de novo synthesized gene products in Clo DF13 minicells are Clo DF13 specific. Polyacrylamide gel electrophoretic patterns show that in these minicells at least three polypeptides (molecular weight about 70,000, 20,000, and 11,000) and one major species of messenger RNA (mRNA) (S value about 21.3) are synthesized. To investigate the factor in its induced state, we isolated a Clo DF13 mutant with an enhanced level of cloacin production. Minicells harboring this Clo DF13 mutant produce five additional polypeptides (molecular weight about 58,000, 44,000, 28,000, 16,000, and 14,000). Three additional mRNA species (S value about 19.5, 14, and 12) could be distinguished. The total molecular weight of the eight polypeptides corresponds to 85% of the total coding capacity of the mRNAs (303,000). The total molecular weight of the four mRNAs is 2.55 x 10(6), which covers 85% of the Clo DF13 DNA (molecular weight 6 x 10(6)). 相似文献
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