Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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 expression of an Escherichia coli K12 flagellin gene, hagA48, is insensitive to the Salmonella H1 repressor (rh1+). By selecting merodiploid cells H2-rh1on-off/F'hag48 for motility in the presence of anti-H48 serum, mutants which had escaped from inhibition by the serum because of repression of their hag48 expression by rh1+ were isolated. Their nucleotide sequences were examined in the region containing the promoter, the position of which was confirmed by S1 nuclease analysis of the transcriptional initiation site. The two independently isolated mutants had the same heptamer insertion AGACGAT at a site overlapping with the promoter sequence, creating a putative operator sequence homologous to Salmonella H1, but not to H2. Other candidates for operator mutants had reduced flagellar synthesis because of mutations between the transcriptional and translational initiation sites or in the structural gene. The sequence analysis also revealed a repetitive extragenic palindrome (REP) consensus sequence and a transcriptional terminator of hag48 in a small, functionally unknown open reading frame (ORF).
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PMID:Isolation and characterization of Escherichia coli hag operator mutants whose hag48 expression has become repressible by a Salmonella H1 repressor. 265 72

The leading region of the Escherichia coli K12 F plasmid is the first segment of DNA to be transferred into the recipient cell during conjugal transfer. We report the nucleotide sequence of the 64.20-66.77F portion of the leading region immediately adjacent to the origin of transfer, oriT. The 2582 bp region encodes three open reading frames, ORF95, ORF169 and ORF273; the product of ORF273, is equivalent in size and map location to the 35 kDa protein, 6d, previously described (Cram et al. 1984). S1 nuclease analyses of mRNA transcripts have identified a potential promoter for ORF95 and ORF273 and indicated that these ORFs are transcribed as a single transcript; in contrast, ORF169 appears to be transcribed from two overlapping promoters on the complementary DNA strand. The products of ORF95 and ORF273 are mainly hydrophilic and are probably located in the cytoplasm. ORF273 shares some homology with DNA-binding proteins. There is a signal peptide sequence at the NH2-terminus of ORF169 and the mature form of ORF169 probably resides in the periplasm due to its hydrophilic nature. Both ORF273 and ORF169 are well conserved among conjugative F-like and a few non-F-like plasmids. On the other hand, ORF95 sequences are only present on some of these plasmids. Several primosome and integration host factor recognition sites are present implicating this region in DNA metabolism and/or replication functions.
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PMID:Nucleotide sequence of the leading region adjacent to the origin of transfer on plasmid F and its conservation among conjugative plasmids. 269 41

IMP dehydrogenase, the product of the guaB locus in Escherichia coli K12, catalyzes the synthesis of XMP by the NAD+ dependent oxidation of IMP. The guaB locus has been subcloned from the Clarke and Carbon plasmid pLC34-10. The sequence of the guaB structural gene and surrounding DNA was determined by the dideoxy chain termination method of Sanger. The 1.533 kb guaB gene encodes an IMP dehydrogenase subunit of molecular weight 54,512. S1 nuclease mapping placed the site of guaBA mRNA initiation approximately 188 bp from the start of the guaB structural gene. The -10 and -35 regions that define the guaBA promoter were located upstream of the start of the guaBA transcription initiation site. The control region of approximately 188 bp does not show any obvious potential for secondary structure. A secondary lambda att site has been identified 42 bp distal to the guaB start codon.
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PMID:Nucleotide sequence of the guaB locus encoding IMP dehydrogenase of Escherichia coli K12. 286 Jun 37

A cDNA library was efficiently synthesized from mouse neuroblastoma poly(A)+RNA. Several modifications of the oligo(dC)(dG) tailing procedure were used. After first strand synthesis, a dATP tail was added to the 3'-end of the cDNA. The second strand was primed for synthesis with oligo(dT). Blunt ends were produced on the cDNA by treatment with S1 nuclease. Size-enriched fractions of high molecular weight DNAs were obtained by passing the cDNA over a Sepharose CL-4B column. The optimal tailing time for each cDNA fraction was individually tested. Tailing reactions used terminal deoxynucleotidyl transferase and annealing reactions used a (G)-tailed Pst I cut pBR322. E. coli K12 RR1 cells were transformed and 2.5-5 X 10(6) transformants per microgram cDNA insert were obtained for each size fraction. The transformants had an average insert size of 1200 base pairs and were 98% ampicillin sensitive. Our modifications in the method for cDNA library synthesis had 3 advantages. (1) Homopolymer-primed cDNA treated with S1 nuclease allowed the blunt ends to be tailed synchronously. This allowed a higher transformation efficiency without loss of 5'-sequences. (2) Time tailing determined the most efficient tail length and optimized the transformation efficiency in each size fraction. (3) A Sephadex G-50 mini-column was used to desalt and dry nitrogen was used to concentrate the ds cDNA instead of the usual ethanol precipitation. This resulted in almost 100% recovery of synthesized products at each step of this procedure.
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PMID:High-efficiency cloning of DNA sequences complementary to mouse neuroblastoma polyadenylated RNA. 286 33

The ada gene of Escherichia coli K12, the regulatory gene for the adaptive response of bacteria to alkylating agents, was cloned and placed under the control of the lac regulatory region on a multicopy runaway plasmid, thereby yielding a hybrid plasmid pYN3059. Ada protein with a molecular weight of about 38,000 was overproduced when cells harboring pYN3059 were incubated at 42 degrees C in the presence of a lac inducer, isopropyl-beta-D-thiogalactoside. Taking advantage of overproduction of Ada protein, we purified the protein to apparent physical homogeneity. The purified 38,000-dalton Ada protein transferred the methyl group from the O6-methylguanine residue of alkylated DNA to the Ada protein, per se. Although the Ada protein was degraded to smaller polypeptides when crude extracts or partially purified preparations were incubated in a high ionic-strength buffer at neutral pH, the purified Ada protein remained stable under the same conditions, indicating that the Ada protein may not undergo autodegradation. An amino-terminal sequence and total amino acid composition of the purified Ada protein were in accord with nucleotide sequence of the ada gene, determined by the dideoxy method using M13 phage. It was deduced that Ada protein comprises 354 amino acids and its molecular weight is 39,385. The promoter for the ada gene was determined by S1 nuclease mapping.
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PMID:Purification and structure of the intact Ada regulatory protein of Escherichia coli K12, O6-methylguanine-DNA methyltransferase. 298 51

The ilvC gene of Escherichia coli K12 encodes acetohydroxy acid isomeroreductase, the second enzyme in the parallel isoleucine-valine biosynthetic pathway. Previous data have shown that transcription of the ilvC gene is induced by the acetohydroxy acid isomeroreductase substrates, acetohydroxybutyrate or acetolactate, and that this substrate induction of ilvC expression is mediated by a positive activator encoded by the ilvY gene. We report here the isolation and complete nucleotide sequence of the ilvY and ilvC genes. The ilvY and ilvC genes encode polypeptides of Mr 33,200 and 54,000, respectively. In vitro transcription-translation of these gene templates results in the synthesis of gene products of these identical molecular weights. The ilvC gene is transcribed in the same direction as the genes of the adjacent ilvGMEDA operon. The ilvY gene is transcribed in a direction opposite to the ilvC and ilvGMEDA genes. The in vivo transcriptional initiation sites of the ilvY and ilvC genes have been determined by S1 nuclease protection experiments. These transcriptional initiation sites are 45 nucleotides apart, and transcription of the ilvY and ilvC genes is initiated via divergent overlapping promoters. The nucleotide sequence of the ilvY and ilvC promoters and 5'-coding regions of Salmonella typhimurium LT2 have been determined. A comparison of these sequences with E. coli K12 suggests regions important in the promotion, regulation, and translation of the ilvY and ilvC genes. A model is presented in which the ilvY-encoded activator binds to an operator site in the overlapping promoter region and reciprocally regulates the transcription of the ilvY and ilvC genes. The carboxyl-terminal amino acid sequence of threonine deaminase encoded by the ilvA gene of the ilv-GMEDA operon of E. coli K12 has been identified by homology with the previously deduced threonine deaminase amino acid sequence encoded by the ilv1 gene of Saccharomyces cerevisiae. Based on the deduced amino acid sequences of the ilvA and ilvY genes, the translational termination codons for both genes are shown to be separated by 52 nucleotides. The proximity of the ilvA and ilvY genes suggests that the 3'-ends of these transcripts overlap.
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PMID:Nucleotide sequence and in vivo expression of the ilvY and ilvC genes in Escherichia coli K12. Transcription from divergent overlapping promoters. 300 15

The Ile repressor protein negatively controls expression from the ilv and thr promoters of Escherichia coli K12. Its existence was inferred from an analysis of the phenotypes of the ileR mutant avr-16 (Johnson, D. I., and Somerville, R. L. (1983) J. Bacteriol. 155, 49-55). The nucleotide sequence of ileR, the structural gene for Ile repressor, has been determined. A DNA segment of 300 base pairs constitutes the ileR gene. The predicted gene product, a protein of 100 amino acids (molecular weight 11,823) has primary structural features reminiscent of other double-stranded DNA-binding regulatory proteins. S1 nuclease mapping of the 5' terminus of ileR mRNA revealed two discrete species whose startpoints differed by approximately 47 nucleotides. The ileR gene, like other repressors for amino acid biosynthetic systems, is autogenously regulated at the transcriptional level. Within the ileR promoter region lie two 18-base pair segments of DNA bearing significant homology to putative operator targets also found within the thr and ilv promoters. A second open reading frame capable of specifying a protein of 83 amino acids, designated orf83, is transcribed divergently from the ileR gene. There are 202 base pairs separating the first codons of the two genes. S1 nuclease mapping of the 5' terminus of orf83 mRNA revealed two discrete species whose startpoints differed by approximately 27 nucleotides. The upstream promoters for ileR and orf83 overlap in their -35 regions.
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PMID:Structural analysis of the ileR locus of Escherichia coli K12. 352 38

We have determined the DNA sequence surrounding the transcription terminator following rpoC, the gene that codes for the beta' subunit of RNA polymerase in E. coli K12. The 2044 bp sequence obtained contains the distal 335 codons of rpoC followed by a 212 bp non-coding region and a second open reading frame (ORFa) of 179 codons. The final 181 nucleotides of the sequence form the 5' end of a third open reading frame (ORFb). The in vivo 3' end of the rpoC mRNA was located by analysis of RNA/DNA hybrids cleaved with nuclease S1 (S1 mapping). These results indicated that the major transcription termination of the rplJL-rpoBC transcription unit occurs a short distance past the translation stop codon for rpoC. Four regions of symmetry, suggesting secondary structure in the mRNA, were found in the DNA sequence near the rpoC translation termination codon. The last of these hairpin structures is similar to other rho-independent transcription terminators and its 3' end coincides with the end of the rpoC mRNA as predicted by S1-mapping. Inspection of the open reading frames indicates that rpoC uses a high percentage of codons that are recognized by the major tRNA species of E. coli while ORFa and ORFb contain many codons recognized by minor tRNA species. ORFa specifies a very basic peptide.
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PMID:Nucleotide sequence at the end of the gene for the RNA polymerase beta' subunit (rpoC). 627 50

The nucleotide sequence has been determined of a 900 bp segment of chromosomal DNA located between 2.6 and 3.5 kb left of the origin of replication, oriC. This segment, which overlaps with the known sequence of the atp operon coding for the eight subunits of the Escherichia coli K12 ATP synthase, contains two coding sequences with the same polarity (counterclockwise) as the atp genes: One of these, designated atpI, which codes for the N-terminal part of a 14 kD polypeptide, is located in front (upstream) of the atpB gene (the first structural gene in the atp operon), the other one codes for the C-terminal part of the gidB gene. The 606 bp segment located between the gidB and the atpI genes contains no coding sequences. By employing the nuclease S1 mapping technique, we have determined a promoter, designated atpIp, for the atp operon located in front of the atpI gene; two additional, weak transcription starts were located within the atpI gene. No transcription start sites were detected up to 1,000 bp upstream of the atpIp promoter, neither were any transcription start sites detected within the cluster of the eight structural atp genes. The atp operon transcription terminates at a site approximately 50 bp downstream from the atpC gene.
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PMID:The promoters of the atp operon of Escherichia coli K12. 631 52

Resistant strains for trimethoprim, a potent inhibitor of dihydrofolate reductase, were obtained by transforming the ligated products of Escherichia coli K12 DNA and plasmid pBR322 BamH I fragments. The strains carry a 13.6 kbp plasmid, pTP1, which contains the trimethoprim- and ampicillin-resistance determinant genes. The trimethoprim-resistance determinant gene was estimated to consist of more than 500 nucleotides and less than 1,500 nucleotides and was restricted by EcoR I and Sal I. Trimethoprim-, ampicillin-, and tetracycline-resistant plasmids were made in the following way, and the resultant plasmids contained a unique EcoR I "insertional inactivation" site for trimethoprim resistance: the DNA sequences extraneous to the determinant gene of the trimethoprim resistance on BamH I fragment of pTP 1 were eliminated by digestion with a double-strand-specific exonuclease BAL 31, and the resultant fragments were ligated with pBR 322 which had been digested by EcoR I and a single-strand-specific nuclease S1. The strains carrying pTP 1 or trimethoprim-resistant plasmids produced about 10 times more dihydrofolate reductase than control strains. The enhancement of the enzyme production, which is due to an increase in the copy number of the enzyme gene, seems to be responsible for the trimethoprim resistance of the transformed cells.
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PMID:Cloning of dihydrofolate reductase gene of Escherichia coli K12. 704 10


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