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)

Two hundred thirty-nine base pairs upstream from acoXABC, which encodes the Alcaligenes eutrophus H16 structural genes essential for cleavage of acetoin, the 2,004-bp acoR gene was identified. acoR encodes a protein of 668 amino acids with a molecular mass of 72.9 kDa. The amino acid sequence deduced from acoR exhibited homologies to the primary structures of transcriptional activators such as NifA of Azotobacter vinelandii, NtrC of Klebsiella pneumoniae, and HoxA of A. eutrophus. Striking similarities to the central domain of these proteins and the presence of a typical nucleotide-binding site (GETGSGK) as well as of a C-terminal helix-turn-helix motif as a DNA-binding site were revealed. Between acoR and acoXABC, two different types of sequences with dual rotational symmetry [CAC-(N11 to N18)-GTG and TGT-(N10 to N14)-ACA] were found; these sequences are similar to NtrC and NifA upstream activator sequences, respectively. Determination of the N-terminal amino acid sequence of an acoR'-'lacZ gene fusion identified the translational start of acoR. S1 nuclease protection assay identified the transcriptional start site 109 bp upstream of acoR. The promoter region (TTGCGC-N18-TACATT) resembled the sigma 70 consensus sequence of Escherichia coli. Analysis of an acoR'-'lacZ fusion and primer extension studies revealed that acoR was expressed at a low level under all culture conditions, whereas acoXABC was expressed only in acetoin-grown cells. The insertions of Tn5 in six transposon-induced acetoin-negative mutants of A. eutrophus were mapped within acoR. On the basis of these studies, it is probable that AcoR represents a regulatory protein which is required for sigma 54-dependent transcription of acoXABC.
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PMID:Identification of acoR, a regulatory gene for the expression of genes essential for acetoin catabolism in Alcaligenes eutrophus H16. 137 52

In a previous publication (Wen, L.-P., Ruettinger, R. T., and Fulco, A.J. (1989) J. Biol. Chem. 264, 10996-11003), we reported that about 1 kilobase of 5' flanking sequence was required for barbiturate-inducible expression of the cytochrome P450BM-3 gene in Bacillus megaterium. We have now found, by analysis of various deletion and frameshift derivatives of this region, that an open reading frame immediately upstream of the B. megaterium cytochrome P450BM-3 structural gene encodes a protein, designated Bm3R1, which negatively controls the expression of the P450BM-3 gene at the transcriptional level. A helix-turn-helix DNA binding motif was found near the N-terminal portion of Bm3R1 protein. The 5' terminus of the bm3R1 transcript generated in vivo was determined by nuclease S1 mapping and primer extension analysis to be 44 base pairs upstream of the translation initiation sequence GTG of bm3R1. A putative promoter sequence with a high degree of similarity to the -35 and -10 consensus sequence recognized by the Bacillus subtilis sigma-43 factor was located at an appropriate distance from the transcription start site. A B. megaterium mutant which highly constitutively produced P450BM-3 protein was isolated and complementation of this cytochrome P450BM-3-constitutive mutant by a DNA fragment containing the wild-type bm3R1 gene indicated that the mutation in this locus was trans-dominant. Sequence analysis of the bm3R1 gene and its upstream region from this mutant, after amplification by the polymerase chain reaction, identified a single base change that resulted in a glycine to glutamate substitution in the beta-turn region of the DNA binding motif. By placing the bm3R1 gene under the control of a tac promoter and changing the translation initiation sequence from GTG to ATG, we succeeded in overproducing the Bm3R1 protein in Escherichia coli. A 20-bp perfect palindromic putative operator site, located between the presumed promoter sequences and the bm3R1 structural gene, was defined both by in vivo titration of Bm3R1 repressor and by gel mobility shift assays using the cell-free extracts containing the overproduced wild-type or mutant Bm3R1 protein. The barbiturate effect in mediating the induction of cytochrome P450BM-3 appears to be indirect but probably involves, in part, the release of inhibition by Bm3R1 repressor protein by interfering with its binding to the palindromic putative operator sequence and perhaps to other sites on the regulatory region of the gene encoding cytochrome P450BM-3.
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PMID:Barbiturate-mediated regulation of expression of the cytochrome P450BM-3 gene of Bacillus megaterium by Bm3R1 protein. 154 26

The nucleotide sequence of a cloned 2.8-kilobase-pair BamHI-PstI fragment containing dcmA, the dichloromethane dehalogenase structural gene from Methylobacterium sp. strain DM4, was determined. An open reading frame with a coding capacity of 287 amino acids (molecular weight, 37,430) was identified as dcmA by its agreement with the N-terminal amino acid sequence, the total amino acid composition, and the subunit size of the purified enzyme. Alignment of the deduced dichloromethane dehalogenase amino acid sequence with amino acid sequences of the functionally related eucaryotic glutathione S-transferases revealed three regions containing highly conserved amino acid residues and indicated that dcmA is a member of the glutathione S-transferase supergene family. The 5' terminus of in vivo dcmA transcripts was determined by nuclease S1 mapping to be 82 base pairs upstream of the GTG initiation codon of dcmA. Despite a putative promoter sequence with high resemblance to the Escherichia coli -10 and -35 consensus sequences, located at an appropriate distance from the transcription start point, dcmA was only marginally expressed in E. coli. The strong induction of dichloromethane dehalogenase in Methylobacterium sp. by dichloromethane was abolished by deleting the 1.3-kilobase-pair upstream region of dcmA. Plasmid constructs devoid of this region directed expression of dichloromethane dehalogenase at a constitutively induced level.
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PMID:Sequence analysis and expression of the bacterial dichloromethane dehalogenase structural gene, a member of the glutathione S-transferase supergene family. 210 2

Vibrio ordalii is a major cause of vibriosis in wild and cultured marine salmonids and carries pMJ101, a 30-kb cryptic plasmid that replicates in the absence of DNA polymerase I without producing single-stranded intermediates. A recombinant derivative harboring the pMJ101 replication region proved to be compatible with pJM1, a plasmid containing the iron acquisition system required for the virulence of V. anguillarum 775, another important pathogen that causes vibriosis. Sequence analysis of a 1.56-kb fragment harboring the pMJ101 replication region revealed the presence of typical features found in DNA origins including an AT-rich region, 11 dam-methylation sites of which 5 are within the putative ori region, and five copies of the 9-bp consensus sequence for DnaA binding. Gel retardation assays demonstrated that the latter replication element indeed binds DnaA purified from Escherichia coli. A potential open reading frame encoding a hydrophilic protein with a predicted pI of 10.3 and an M(r) of 33,826 was found adjacent to the ori region. Although these properties are typical of DNA-binding proteins, no significant homology was found between this predicted protein, named RepM, and other previously characterized proteins. Reverse transcriptase-polymerase chain reaction analysis of total RNA demonstrated the presence of repM mRNA in V. ordalii. The major initiation site of this mRNA was located 187 nucleotides upstream of the GTG initiation codon as determined by nuclease S1 protection assays. This transcription initiation site is preceded by putative -10 and -35 promoter sequences that control the expression of the repM replication gene. These results demonstrate that the replication region of pMJ101 shares some structural and sequence similarities with other DNA replication regions, which include DnaA binding and methylation sites and an open reading frame encoding a distinct protein required for its replication.
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PMID:Analysis of the replication elements of the pMJ101 plasmid from the fish pathogen Vibrio ordalii. 1041 62

CcaR is a positive-acting transcriptional regulator involved in cephamycin C and clavulanic acid biosynthesis in Streptomyces clavuligerus. Previous sequence analyses of the ccaR gene revealed two possible start codons, an ATG, and a GTG located in-frame 18 bp downstream of the ATG. To determine the true start codon, ccaR was expressed, either from the GTG or ATG codon, in Escherichia coli. A protein product was only obtained from the ATG construct. Similarly, ccaR constructs originating from ATG or GTG and designed for expression from a glycerol-regulated promoter in Streptomyces species were prepared and used to complement a S. clavuligerus ccaR mutant. Bioassays showed that only the ATG construct could complement the ccaR mutant to restore cephamycin C production, and Western analysis confirmed the presence of CcaR in the mutant complemented with the ATG construct only. To ensure that expression of ccaR from its native promoter also initiated at the ATG rather than GTG, a conservative point mutation was introduced into ccaR, converting the GTG to GTC. The GTC construct still fully complemented a ccaR mutant, confirming that ATG is the true start codon. Inspection of the region upstream of ccaR by S1 nuclease protection and primer extension analyses indicated the presence of two transcript start points that mapped to residues located 74 and 173 bp upstream of the ATG codon.
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PMID:Transcriptional and translational analysis of the ccaR gene from Streptomyces clavuligerus. 1558 66