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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Temperature-sensitive "leaky" mutants of phage MS2 having white dense ring around negative colonies are described. As these mutants are used for quantitative genetic studies, the white ring presents interest. Typical mutant 40 is used as a model for investigation. Light microscopy has shown, that cells from white ring zone have spore-like inclusions, which determine the characteristic structure of surrounding mutant negative colonies. Cytochemical reactions for the presence of glicogen, lipids, volutin, nuclear material and spores were negative. Electrone microscopy of negatively stained samples and ultrathin sections has revealed that cells from white ring zone, unlike phage-infected wild type cells, have two types of electron dense inclusions: 1) crystalline structures formed with great number of closely packed mature phage particles, and 2) large amorphic bodies. Electrone microscope-cytochemical data showed that inclusions remain intact under treatment of ultrathin sections of white zone ring with DNase and perchloric acid, while nuclear material was completely destroyed. Amorphic bodied were completely destructed after the treatment with RNase, while nuclear material and crystalline phage aggregated remained unchanged. Therefore, amorphic bodies consist of RNA, which has not been used to form virions. Single cycle of the development of mutant 40 at 37 degrees and 43 degrees C and under the temperature of incubation 37 degrees leads to 43 degrees C and 43 degrees leads to 37 degrees C in the course of intracellular reproduction is investigated. Influence of the phage on growth on infected culture is studied. The data obtained draw to a conclusion that the impaired function belongs to cystron protein of the phage membrane. As certain mutations in this cystrone of RNA-containing phage result in the depression of cystrone RNA polymerase, it is supposed that the formation of RNA containing bodies in infected cells, determining the formation of white rings in NA, together with cristalline aggregates of cells, is a result of mutation damage of cystrone protein of the phage MS2 membrane.
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PMID:[Effect of mutagens on RNA-containing phages and its infectious RNA. VII. Genetic nature of morphologic mutants of RNA-containing phage MS2]. 99 65

The recombinant hepatitis delta virus antigen was obtained as a chimaeric protein fused to the C-terminus of the phage MS2 RNA polymerase. Following induction of the temperature-sensitive promoter, two major polypeptides of about 34 kDa and 29 kDa, and two minor peptides about 21 kDa and 18 kDa, were obtained on PAGE. The 34-kDa protein was identified as the expected recombinant protein by confirming 82% of the primary structure using fast-atom-bombardment mass spectrometry. The most represented degradation product, i.e. the 29-kDa polypeptide, was also characterized by means of mass spectrometry and found to be produced by cleavage between amino acids 261 and 265. The presence of two main protein bands, with a similar difference in size, is also a typical feature of delta antigens, both extracted and recombinant, and it is considered to be derived either from heterogeneity of viral sequences, which can encode hepatitis delta antigen proteins of 195 and 214 amino acids, or from proteolysis of a single precursor. Since the data were obtained with a single viral sequence coding for 195 amino acids fused to 106 residues from MS2 polymerase, there is direct evidence that intrinsic structural properties of the protein sequence are able to cause a specific proteolysis resulting in the presence of two major forms, of which the smaller is 35-40 amino acids at the C-terminus. The recombinant protein can be used as an antigenic substitute of viral antigens both for immunoassays and for the preparation of anti-(hepatitis delta virus) antisera.
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PMID:Characterization by mass spectrometry of a recombinant hepatitis delta virus antigen and its proteolytic products. 154 Dec 67

Two immunogenic proteins, Sm31 and Sm32, originating from the gut of Schistosoma mansoni were evaluated for their potential as recombinant immunodiagnostic reagents. Sm31 and Sm32 cDNA fragments were cloned and expressed in Escherichia coli as polypeptides fused to RNA polymerase of bacteriophage MS2. The recombinant proteins were tested in enzyme-linked immunosorbent assays (ELISA) with paired sera of 182 persons from Mali with S. mansoni and S. haematobium infections collected before and one year after treatment with praziquantel. Pretreatment sera of the study population gave a strong antibody response to Sm31 and Sm32 in immunoblots of total worm extract with the sera. The sensitivities of both western blotting (86%) and ELISA (75%) using Sm31 and Sm32 fusion proteins compared well with a single egg count (84%). Chemotherapy resulted in an overall decline of egg counts. Posttreatment sera gave significantly lower reactivities than the pretreatment sera. Our results demonstrate the feasibility of detecting circulating antibodies with recombinant antigens in schistosomiasis.
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PMID:Immunological analysis of cloned Schistosoma mansoni antigens Sm31 and Sm32 with sera of schistosomiasis patients. 179 25

ORF 10 of the K2 plasmid from Kluyveromyces lactis encodes a small basic protein (22.3% lysine). The function of its product has been investigated. Western blot analysis, using an antibody against MS2 RNA polymerase/ORF 10 fusion protein, reveals a protein band with an apparent molecular weight of 14 kDa. The protein can bind a DNA-Sepharose column, and is eluted by 350 mM-salt. Immunoprecipitation experiments show that the ORF 10 protein coprecipitates with the linear genomic DNAs of the two killer plasmids (K1 and K2). From Western/Southern blot data, it is possible to conclude that the interaction between protein and DNA occurs directly, rather than via other protein(s). ORF 10 is easily detected by Western blot and its transcript is one of the most abundant of the K2 plasmid, suggesting that this protein may have a structural rather than a regulatory function. This possibility is also suggested by the observed sequence homology between the ORF 10 protein and the family of histone-like proteins.
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PMID:Killer system of Kluyveromyces lactis: the open reading frame 10 of the pGK12 plasmid encodes a putative DNA binding protein. 188 49

We have expressed two cDNA sequences encoding 121 and 230 amino acids of the C-terminus of the Schistosoma mansoni Hsp 70 in Escherichia coli. The products were synthesized as polypeptides fused to the RNA polymerase of bacteriophage MS2, and their reactivities were tested in ELISAs, using sera from human and murine infections. Anti-Hsp70 antibodies were detected in a significant number of individuals suffering from chronic schistosomiasis mansoni, but not in patients with known recent infections. This, together with the finding that antibodies directed at S. mansoni-specific Hsp70 determinants during the course of infection of experimental mice were not detectable until 5-6 weeks post-infection, suggests that the protein may be a useful marker for distinguishing late and early infections. The diagnostic specificity of Hsp70 was evaluated with sera from humans infected with different schistosome species and other parasitic diseases. While some subjects infected with S. haematobium produced antibodies which recognized the S. mansoni Hsp70, no such antibodies were generated in S. japonicum infected individuals. However, cross-reactive antibodies were elicited in donors with other parasitic diseases such as filariasis and malaria. The absence of antibodies in early infection and the observed cross-reactivities led us to conclude that Hsp70 will be of limited value in the diagnosis of schistosomiasis.
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PMID:The humoral response to heat shock protein 70 in human and murine Schistosomiasis mansoni. 211 91

Various agents were tested for their effects on microbial proteases, which activity was monitored by the analysis of cleaved peptide bands in SDS-polyacrylamide gel electrophoresis. Using casein as a substrate, fungal protease (type XIX) was inhibited by the phenyl methyl sulphonyl fluoride, chymostatin, antipain and leupeptin, while bacterial protease (type XXVI) was inhibited by phosphatidyl glycerol, phosphatidyl inositol and sphingosine. MS2 RNA exerted minor inhibition on the bacterial proteolysis of regulatory subunits of cyclic AMP-dependent protein kinase (A-PK). The cleavage of DNA binding protein by both proteases was inhibited, in the presence of MS2 RNA and lambda DNA. In comparison, phosphatidyl serine slightly stimulated the fungal protease on the cleavage of ribonuclease T1. RNA polymerase is a good substrate of the bacterial protease as indicated by the generation of multiple cleaved peptide fragments, whereas alkaline phosphatase is not susceptible to proteolysis.
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PMID:A further study on the regulation of microbial proteases. 222 36

The 330 residue-long N-terminal domains (NTDs) of beta and beta' subunits of the Escherichia coli RNA polymerase (RPase) core enzyme were found to be significantly homologous to the entire length of its alpha subunit. The C-terminal domains (CTDs) of the RPase beta subunit and DNA primase (dnaG protein) were not only strongly homologous to each other but also considerably homologous to the RPase alpha, suggesting that an alpha subunit-like enzyme must have been commonly ancestral to core enzyme subunits and primase. The N-terminal region (NTR) of RPase alpha was also found to show significant homologies with NTRs of the E. coli EF-Tu and F1-ATPase alpha subunit, and a possible weak homology with ribosomal protein L3. A most important finding was that the C-terminal regions (CTRs) of DNA polymerase (DPase) I, T7 phage DPase and MS2 phage RNA replicase beta subunit are closely homologous with one another. These CTRs showed considerable homologies to RPase alpha NTD and RPase beta CTD. These conclusions are based on statistical evaluations of homologies in base and/or amino acid sequence alignments.
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PMID:Amino acid and nucleotide sequence homologies among E. coli RNA polymerase core enzyme subunits, DNA primase, elongation factor Tu, F1-ATPase alpha, ribosomal protein L3, DNA polymerase I, T7 phage DNA polymerase, and MS2 phage RNA replicase beta subunit. 286 46

The release of the ribonucleic acid (RNA)-containing phage MS2 from Escherichia coli is accompanied by cellular lysis at 37 C, whereas at 30 C phage are released from intact cells. Chloramphenicol or rifampin prevents the release of progeny phage particles at both temperatures. Neither drug causes an immediate cessation of phage release and after inhibition of protein synthesis by chloramphenicol phage release proceeds for about 17 min at 37 C and about 35 min at 30 C. Rifampin does not inhibit phage release from mutant cells possessing a rifampin-resistant deoxyribonucleic acid-dependent RNA polymerase. The results indicate that a short-lived host-controlled protein(s) is essential for the release of RNA phage particles at both temperatures.
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PMID:Ribonucleic acid bacteriophage release: requirement for host-controlled protein synthesis. 410 40

The effects of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), both produced by E. coli, were measured on the activities of several genes in a cell-free system. Gene activity is measured as gene-directed synthesis of biochemically competent protein or transfer RNA. Both ppGpp and pppGpp stimulated the activities of the ara, lac, and trp operons and inhibited the arg operon. Production of transfer-RNA(Tyr) was unaffected by moderate levels of either ppGpp or pppGpp and only slightly inhibited at higher levels of ppGpp. Since the cell-free reaction mixtures hydrolyze pppGpp to ppGpp, we performed similar studies with a hydrolysis-resistant analog of pppGpp, the beta-gamma methylenyl derivative (pcppGpp). In general, pcppGpp shows the same inhibitory potency as pppGpp for the arg operon, but lacks the stimulatory effects on the ara, lac, and trp operons. This result suggests that the stimulation of these gene activities is specific for ppGpp.Under similar conditions, pppGpp and ppGpp show a slight inhibitory effect on the messenger-directed synthesis of beta-galactosidase and no effect on the messenger-directed synthesis of MS2 viral-coat protein. These observations, together with the fact that in the same system these nucleotides affect coupled transcription and translation, lead us to surmise that the activities of pppGpp and ppGpp are exerted at the level of RNA polymerase activity.
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PMID:Effects of guanosine tetraphosphate, guanosine pentaphosphate, and beta-gamma methylenyl-guanosine pentaphosphate on gene expression of Escherichia coli in vitro. 435 31

In an MS2 phage ribonucleic acid (RNA)-directed in vitro protein-synthesizing system, the coat protein cistron and the adjacent RNA polymerase cistron are translated non-continuously. The ribosomes which have completed the synthesis of coat protein dissociate from the MS2 RNA and do not read through the intercistronic gap. Translation of the adjacent RNA polymerase cistron requires ribosomes other than those translating the coat protein cistron.
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PMID:Non-continuous translation of coat protein and ribonucleic acid polymerase cistrons in MS2 bacteriophage ribonucleic acid. 458 4


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