Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.1 (S1 nuclease)
 3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The two 23S rRNA-5S rRNA-tRNAGly operons from the extreme thermophilic eubacterium Thermus thermophilus HB8 were used to characterized the in vivo processing and termination of 23S rRNA-5S rRNA-tRNAGly primary transcripts in this organism by nuclease S1 mapping. A processing site in the pre-23S rRNA 3'-flanking region is located approximately 25 nucleotides upstream of 5S rRNA and precedes a putative 23S-5S rRNA spacer antitermination box A. Cleavage at this site and 5S rRNA 5' end formation were shown to be inseparable events. Termination of transcription at the uridine cluster following the termination-associated hairpin was shown to be efficient but leaky. Subsequent to the operon, a functional promoter was detected whose -35 box coincided with the uridine-rich termination region. The promoter directed synthesis of a beta-galactosidase fusion protein in Escherichia coli.
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PMID:Processing and termination of 23S rRNA-5S rRNA-tRNA(Gly) primary transcripts in Thermus thermophilus HB8. 201 80

The effects of inhibitors of protein synthesis upon transcription have been re-examined. Cycloheximide (1 microgram/ml) inhibits incorporation of uridine into RNA of P1798.S20 lymphosarcoma cells. Filter hybridization studies indicate that labeling of pre-rRNA is inhibited 60-80% after 1 h and quantitative S1 nuclease mapping reveals a corresponding decrease in the amount of cellular pre-rRNA. Cycloheximide also inhibits labeling of 5 S RNA and tRNA, but incorporation of uridine into poly(A+) RNA is unaffected. Transcription experiments carried out in nuclei from cycloheximide-treated cells indicate that the inhibitor causes a selective decrease in the activity of RNA polymerases I and III. Cell-free extracts from P1798.S20 were used to transcribe the cloned mouse rRNA gene, Syrian hamster 5 S RNA gene, and the Drosophila tRNAArg gene. Extracts from cycloheximide-treated cells were inhibited in this respect. Transcription of rRNA and 5 S RNA genes was inhibited by 90% after 2 h and 50% inhibition occurred within 20-30 min. Transcription of the tRNA gene was inhibited 75% after 2 h with a half-time of approximately 1 h. Inhibition was due neither to a direct effect of cycloheximide nor to the presence of nucleases or diffusible inhibitors of transcription. Moreover, transcription of rDNA in extracts from cycloheximide-treated cells could be restored by the addition of a partially purified initiation factor preparation. The data indicate that inhibition of protein synthesis results in rapid depletion of transcription factors that are required for initiation by RNA polymerases I and III. Among these is the glucocorticoid-regulated rDNA initiation factor designated TFIC.
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PMID:The effects of cycloheximide upon transcription of rRNA, 5 S RNA, and tRNA genes. 241 18

To determine whether RNA polymerase pauses during transcription in vivo, we have examined transcripts of the trp operon leader regions of Serratia marcescens and Escherichia coli. Labeled RNAs synthesized in E. coli strains containing plasmids bearing wild-type or mutant trp leader regions of S. marcescens or E. coli were isolated by hybridization and analyzed by polyacrylamide gel electrophoresis. The labeled RNAs synthesized in vivo on the S. marcescens wild-type and deletion mutant plasmids were the same size as the in vitro pause and leader transcripts. Hybridization of the presumed in vivo pause RNAs, and control in vitro pause RNAs, to M13 phage DNA containing a trp leader region deletion followed by treatment with S1 nuclease produced identical protected RNA species, proving that the in vitro and in vivo RNAs were identical. The amount of labeled pause RNAs relative to leader RNAs decreased following a chase with unlabeled uridine. E. coli RNAs identical to the previously characterized in vitro pause and leader transcripts were demonstrated by electrophoretic band position and fingerprint analysis. The finding that transcription pausing occurs in vivo is consistent with the view that transcription pausing and ribosome release of paused transcription complexes are responsible for the coupling of translation with transcription that is crucial to attenuation.
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PMID:Detection of transcription-pausing in vivo in the trp operon leader region. 243 19

Escherichia coli K-12 strains isolates carrying plasmid pBR322 were grown in the presence of subinhibitory concentrations of lincomycin, which stimulated beta-lactamase synthesis about 2.5-fold, and the effects of the drug on the synthesis and degradation of bla mRNA were studied. The bla mRNA levels determined by 1-min pulse-labeling with [3H]uridine were significantly higher in a lincomycin-containing culture than in the control culture, indicating that stimulation of beta-lactamase synthesis is caused by an increase in the amount of bla mRNA. The enhancing effect of lincomycin was observed in strains harboring pBR322 delta P1 and pBR322 delta P3, which lacked the P1 or P3 promoter, respectively, as well as in the strain harboring pBR322. S1 nuclease analysis showed that the half-life of bla mRNA increased about 2.7-fold when lincomycin was present. These results indicate that the increase in beta-lactamase synthesis caused by lincomycin is due to an increase in the stability of bla mRNA rather than activation of its synthesis.
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PMID:Lincomycin increases the half-life of beta-lactamase mRNA. 266 25

Hereditary orotic aciduria is an autosomal recessive disease in which there is a severe deficiency in the activity of the de novo pyrimidine pathway enzyme uridine 5'-monophosphate (UMP) synthase. UMP synthase is a bifunctional enzyme containing the two activities orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase, which catalyze the two-step conversion of orotic acid to UMP. Cell lines from three orotic aciduria patients have been characterized for UMP synthase gene and mRNA content. Restriction-enzyme analysis of DNA from the deficient cells revealed no changes in the gene structure compared with normal cell DNA structure. The amount of UMP synthase mRNA was not decreased, nor was there a detectable difference in the size of the UMP synthase mRNA in the deficient cells. Analysis of the mRNA by hybridization with a nearly full-length UMP synthase cDNA followed by S1 nuclease digestion showed no alteration in the mRNA structure. The UMP synthase activity of the deficient cells ranges from 2% to 7% of the normal cell level. The activity can be significantly increased by growing the deficient cells in barbituric acid. Our data indicate that UMP synthase gene transcription in the orotic aciduria cells produces the expected amount of a stable, correctly processed mRNA. The mRNA appears to code for a mutant enzyme that has reduced stability or altered kinetic properties.
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PMID:Analysis of UMP synthase gene and mRNA structure in hereditary orotic aciduria fibroblasts. 283 86

The fumarate reductase enzyme complex allows Escherichia coli to grow anaerobically with fumarate as a terminal electron acceptor for oxidative phosphorylation when the preferred compounds oxygen and nitrate are not available. We used the pKO promoter test vectors to identify a single promoter for the frdABCD genes which encode fumarate reductase. Expression of galactokinase from the frd promoter-galK operon fusion plasmid was repressed by oxygen and by nitrate and was induced by fumarate, indicating that frd gene expression is regulated at the transcriptional level by these terminal electron acceptors. S1 nuclease analysis, using a single-stranded DNA probe from the frd promoter region and mRNA isolated from a fumarate reductase-induced culture, revealed that the frd mRNA transcript initiates with an adenine residue 93 bases prior to the start of frdA translation. No promoters internal to the frd genes were revealed with the plasmid promoter screening system. S1 nuclease analysis revealed that the frd mRNA terminates in a uridine-rich region centered at 46 bases after the last codon of frdD. A stem and loop structure previously described as the growth rate-dependent attenuator for the linked ampC gene precedes the frd mRNA terminus. This result confirms the proposal that the stem and loop structure serves the dual role of a frd terminator anaerobically and an ampC attenuator aerobically. The four frd genes encoding the subunits of the fumarate reductase complex thus comprise an operon which is regulated at the transcriptional level in response to the cellular availability of the alternate electron acceptors oxygen, nitrate, and fumarate.
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PMID:Transcription of the Escherichia coli fumarate reductase genes (frdABCD) and their coordinate regulation by oxygen, nitrate, and fumarate. 299 70

The main early and late transcription termination sites in vivo in bacteriophage phi 29 DNA were determined by nuclease S1 mapping. Transcription of the phi 29 early genes located at the left end of the viral genome terminated at the very end of the DNA molecule and within the HindIII G fragment of the viral DNA. Transcription termination of the early genes located at the right end of the genome and that of the late viral genes overlapped in a specific region of the phi 29 DNA within the EcoRI D fragment. Stem-loop structures followed by uridine-rich tails could be derived close to the 3' ends of early and late mRNAs, suggesting Rho-independent transcription termination in phi 29 DNA.
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PMID:In vivo transcription of bacteriophage phi 29 DNA: transcription termination. 303 5

The rates of formation and processing of rRNA and transcription of rDNA were examined during the differentiation of MM14DZ mouse myoblasts into muscle fibers. Analyses of the incorporation of [3H]uridine into 18 and 28 S rRNAs and the UTP precursor pool indicate that the rate of rRNA formation is 2.3-fold higher in myoblasts as compared to muscle fibers. To determine if the rate of rRNA formation is regulated at the level of pre-rRNA processing, the labeling kinetics and steady state levels of pre-rRNAs were measured in myoblasts and fibers. These experiments suggest that the time required for and the efficiency of pre-rRNA processing are the same in myoblasts and fibers, but that the steady state levels of all pre-rRNAs detected (32, 34, 37, 41, and three 45 S subspecies) are reduced 2.5- to 3.0-fold in fibers. This suggests that the reduced rate of rRNA formation in fibers is not regulated at the level of pre-rRNA processing but is due to a decrease in the rate of rDNA transcription. The transcriptional rates for rRNA in myoblasts and fibers were calculated from measurements of the total rate of transcription and from hybridization analyses of radioactive rRNA formed in isolated nuclei or in short pulses of cultured cells. These experiments indicate that the rates of 18 and 28 S rDNA and 5 S rDNA transcription are reduced 2.5 to 3.0-fold in fibers. Thus, a coordinate change in the rate of rDNA transcription is the primary mechanism regulating the formation of rRNA during the differentiation of this myoblast line. This decreased transcription in fibers appears to be due to modulation of the activity of the known mouse rDNA promoter, as S1 nuclease mapping experiments indicate that rDNA transcription initiates at the same location in both myoblasts and fibers.
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PMID:rDNA transcription and pre-rRNA processing during the differentiation of a mouse myoblast cell line. 364 62

Although antibodies directed against bromodeoxyuridine (BrdU) are being used in both clinical and basic research laboratories as tools to study and monitor DNA synthesis, little is known about the epitopes with which they react. Four monoclonal antibodies directed against BrdU were produced and were characterized to learn more about the epitopes on BrdU which are important for antibody recognition, to identify compounds other than BrdU which react with the antibodies and which might interfere with immunologic assays for BrdU, and to characterize the reaction of these antibodies with BrdU-containing DNA. By radioimmunoassays, the antibodies generally reacted well with 5-iododeoxyuridine, 5-fluorodeoxyuridine, and 5-nitrouracil. However, none of the antibodies reacted well with uridine--indicating that a substituent on uridine C5 was essential for antibody reactivity--or with 5-bromo- or iodo-cytosine, indicating that the region around pyrimidine C4 is important for antibody recognition. Although the antibodies reacted with 5-halogen-substituted uracil bases, the antibodies reacted much better with the corresponding halogenated nucleosides, indicating that the sugar moiety was important for recognition. The presence of a triphosphate group on C'5 of BrdU (i.e., BrdUTP) did not detectably alter antibody recognition. Three of the antibodies reacted only with purified DNA containing BrdU, whereas one antibody, which exhibited a weak interaction with thymidine, also reacted with BrdU-free DNA. S1 nuclease treatment of purified DNA suggested that all four monoclonal antibodies reacted exclusively with single-stranded regions of BrdU-containing DNA. Comparison of detecting DNA synthesis by [3H]TdR incorporation followed by autoradiography with that by BrdU incorporation followed by indirect immunofluorescence indicated that the latter technique was both an accurate and a sensitive measure of DNA synthesis.
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PMID:Interaction of monoclonal antibodies directed against bromodeoxyuridine with pyrimidine bases, nucleosides, and DNA. 395 Apr 2

We have been able to isolate several species of 5-S ribosomal RNA from Escherichia coli A19. These molecules were separated on the basis of their differing stabilities during electrophoresis on 12% polyacrylamide gels in 7 M urea. This differing stability is shown, in one case, to be due to a different primary sequence. We have determined the sequence of the least stable of these molecules and have found only one difference to the published sequence of E. coli A19 5-S RNA, namely a uridine in place of a cytidine at position 92. The consequent G x U base pair, formed in a normally highly stable G x C-rich region, is responsible for a drastic reduction in the stability of the molecule. This instability leads to a less constrained, more compact molecule which thus migrates faster in electrophoresis under denaturing conditions. This species of 5-S RNA is shown to make up 30% of the total 5-S RNA in the 50-S ribosomal subunits in this organism. Further structural studies were carried out using S1 nuclease digestion, sodium bisulphite modification and thermal melting analysis. All these methods indicate a 5-S RNA drastically destabilized in parts of its secondary and tertiary structure. Finally, the ability of the variant 5-S RNA to recognize and form a complex with its 50-S subunit binding proteins was examined and found to be impaired.
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PMID:The effect of a cytidine-to-uridine transition on the stability of Escherichia coli A19 5-S RNA. 618 24


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