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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)

We report here the transcriptional analysis of the fixABCXORF1 region of Azorhizobium caulinodans. This led to the identification of a 0.9 kb transcript covering fixX and ORF1, which was synthesized only under conditions of nitrogen fixation. The 5' end of this transcript was mapped by primer extension and S1 nuclease protection analyses and shown to be located 70 +/- 1 nucleotides upstream of the fixX start codon. By means of transcriptional fixX- and ORF1-lacZ fusions, it was shown that fixX and ORF1 were most probably transcribed from the fixA promoter and that expression of fixX and ORF1 was dependent on NifA activation. This suggests that the 0.9 kb mRNA results from post-transcriptional processing of a large mRNA covering fixA,B,C,X and ORF1. In addition, ORF1 mutants were constructed and were shown not to be impaired in nitrogenase activity.
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PMID:Transcriptional analysis of the fix ABCXORF1 region of Azorhizobium caulinodans suggests post-transcriptional processing of the fix ABCXORF1 mRNA. 128 16

The bean rust fungus, Uromyces appendiculatus, undergoes thigmotropic differentiation to produce infection structures. Six differentiation-specific genes have been isolated and one, INF24, has been characterized [Bhairi et al., Gene 81 (1989) 237-243]. Here, we report the structure of a second gene, INF56, which was subcloned on a 2.6-kb fragment and sequenced. The location of the 1.0-kb INF56 transcript was determined by S1 nuclease protection and primer extension. A TATA box was found 38 bp upstream and a CAAT box 130 bp upstream from the major transcription start point (tsp). The gene contains two open reading frames: ORF2 is nested within ORF1; they share a 67-bp intron. ORF1 encodes a 14.1-kDa polypeptide which has an amino acid sequence rich in Gly, Pro and Ser. It has sequence similarity to a functional domain (V2) of mammalian cytokeratin type II. ORF2 encodes a 10.1-kDa polypeptide which is rich in Pro. It shares similarity with the cell-surface recognition region of chicken fibronectin. Hybrid selection and in vitro translation of the INF56 mRNA yielded two polypeptides of 15.5 and 23 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. INF56 is constitutively expressed at a low level, but the abundance of its steady-state transcript is upshifted 4.5 h after spore hydration during the period that infection structures are formed.
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PMID:Characterization of INF56, a gene expressed during infection structure development of Uromyces appendiculatus. 154 77

The 4.4-kb BamHI-E fragment of the orf virus (OV) genome contains three discrete open reading frames designated ORF-pp, ORF-1, and ORF-3, all of which are flanked by vaccinia virus-like early transcriptional control sequences. To determine whether the vaccinia transcriptional machinery would recognize these promoters and faithfully transcribe OV genes in vivo the BamHI-E fragment was inserted into the thymidine kinase (TK) locus of vaccinia virus and the recombinant used in transcription studies. Northern blotting analysis of early RNA isolated from 143B-TK- cells infected with the recombinant virus showed that OV genes were transcribed and that the three transcripts of 0.70-(ORF-pp), 0.48- (ORF1), and 0.75-kb (ORF-3) were the same size as their counterparts in OV-infected cells. Analysis of the 5' end of transcripts by S1 nuclease and primer extension showed that the transcriptional start points (tsp) of ORF-pp, ORF-1, and ORF-3 in the recombinant were identical or within four nucleotides of the tsps of the same ORFs in OV. However, there were quantitative differences. ORF-1 was transcribed more efficiently in recombinant virus-infected cells than in those infected with OV and analysis of the putative promoter, 5'-AAAATTGTAAATGTA, showed that it was similar to the 7.5-kDa early promoter of vaccinia virus. This demonstrates that the transcriptional control sequences of OV genes are recognized by vaccinia virus transcriptional factors but that quantitative differences exist suggesting that the generically different transcriptional factors have different promoter sequence requirements for maximal transcription.
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PMID:In vivo recognition of orf virus early transcriptional promoters in a vaccinia virus recombinant. 154 49

A 12.5-kilobase segment of Bacillus subtilis chromosomal DNA containing the entire pyrimidine biosynthetic (pyr) gene cluster has been cloned and sequenced. The sequenced DNA has seven cistrons encoding the six enzymes of de novo pyrimidine nucleotide biosynthesis and two open reading frames of unknown function. Based on the sequence and mapping of transcripts, the genes in this cluster appear to be transcribed on one large polycistronic message in the order ORF1, pyrB, pyrC, pyrAA, pyrAB, ORF2, pyrD, pyrF, pyrE. The deduced amino acid sequences for six pyrimidine biosynthetic enzymes from B. subtilis and comparisons with the corresponding sequences from numerous other species are presented. The 3' ends of the reading frames overlap the 5' ends of the downstream open reading frames for all cistrons in the cluster except ORF1 and pyrB, which are separated by a 145-base pair intercistronic region. The start of transcription was mapped by primer extension to a G residue 158 nucleotides upstream from the translation initiation codon of ORF1. This site is preceded by a typical B. subtilis sigma A-dependent promoter. A promoter indicator plasmid was used to show that this region carried a promoter from which transcription was regulated by pyrimidines. Transcription from this promoter was not detected in primer extension experiments using mRNA prepared from B. subtilis cells grown in the presence of excess uracil. No transcripts initiating from the intercistronic space between ORF1 and pyrB were detected with S1 nuclease mapping; however, a transcription terminator was detected in this region that reduced but did not fully block transcriptional readthrough. This terminator was not regulated by pyrimidines in the growth medium under the conditions tested. The region immediately following the promoter and 5' to ORF1 has a potential transcription terminator sequence. The roles, if any, of these transcription terminators in the regulation of pyr operon expression are yet to be determined. However, deletion of the terminator immediately following the promoter abolished pyrimidine regulation of transcription in the indicator plasmid.
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PMID:Functional organization and nucleotide sequence of the Bacillus subtilis pyrimidine biosynthetic operon. 170 62

The nucleotide sequence of the RNA genome of foxtail mosaic virus (FMV), a member of the potexvirus family, is 6151 nucleotides long, exclusive of a poly(A) tail. The RNA contains five principal open reading frames (ORFs), designated from the 5' terminus as encoding proteins with Mr values of 152.3K (ORF1), 26.4K (ORF2) which overlaps an 11.3K (ORF3) product, 5.8K (ORF4) which overlaps a 28.8K readthrough protein (ORF5A) which leads into the coat protein cistron of 23.7K (ORF5). The sizes and composition of the proteins encoded by the ORFs are generally similar to those found in other potexviruses; the least similar is the coat protein which nonetheless retains apparently critical consensus regions. The 5' terminus of the previously reported 0.9 kb subgenomic (sg) RNA was determined by S1 nuclease mapping and shown to begin with the sequence GAAGA, 43 nucleotides upstream from the first nucleotide of the coat protein initiation codon. The positions of the 5' end of this sgRNA and of that deduced from the nucleotide sequence for a 1.9 kb sgRNA are entirely consistent with the previously published sizes of these sgRNAs.
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PMID:The entire nucleotide sequence of foxtail mosaic virus RNA. 184 Jun 10

picA is an Agrobacterium tumefaciens chromosomal locus, identified by Mu d11681 mutagenesis, that is inducible by certain acidic polysaccharides found in carrot root extract. Cloning and genetic analysis of a picA::lacZ fusion defined a region of the picA promoter that is responsible for the induction of this locus. Furthermore, we identified a possible negative regulator of picA expression upstream of the picA locus. This sequence, denoted pgl, has extensive homology to polygalacturonase genes from several organisms and inhibited the induction of the picA promoter when present in multiple copies in A. tumefaciens. DNA sequence analysis indicated at least two long open reading frames (ORFs) in the picA region. S1 nuclease mapping was used to identify the transcription initiation site of picA. Mutation of ORF1, but not ORF2, of the picA locus was responsible for an increased aggregation of A. tumefaciens, forming "ropes" in the presence of pea root cap cells. In addition, a potato tuber disk virulence assay indicated that a preinduced picA mutant was more virulent than was the wild-type control, a further indication that the picA locus regulates the surface properties of the bacterium in the presence of plant cells or plant cell extracts.
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PMID:Genetic and molecular analyses of picA, a plant-inducible locus on the Agrobacterium tumefaciens chromosome. 186 Aug 22

By a chromosome walking strategy the DNA region from Methanococcus vannielii flanking the genes for protein synthesis elongation factor (EF) 1 alpha and EF-2 was cloned and sequenced. A gene organization of 5' - beta' - open reading frame (ORF) 1 - ORF2 - S12 - S7 - EF-2 - EF-1 alpha - S10 - ORF3 - ORF4 - 3' was found where beta', S12, S7, S10, EF-2, and EF-1 alpha represent gene products with sequences similar to the beta' subunit of RNA polymerase, ribosomal proteins S12, S7, and S10, and EF-G and EF-Tu from Escherichia coli, respectively. ORF1-4 represent gene products with no known eubacterial counterparts. Northern blot analysis of transcripts and nuclease S1 mapping showed that transcription initiates between beta' and ORF1 and terminates at the 3' side of the S10 gene and that the genes from ORF1 to S10 are cotranscribed. Apart from the presence of two additional ORFs, ORF1 and ORF2, and of the gene for S10, this organization is identical to that of the eubacterial "streptomycin operon." ORF1 displays sequence similarity to rat liver ribosomal protein L30 and may represent one of the "additional" ribosomal proteins of Methanococcus. The sequenced part of the beta' gene and the EF-2 and EF-1 alpha gene products from Methanococcus are more similar to their eukaryotic than to their eubacterial counterparts. It appears, therefore, that the genetic organization of the translational components resembles the situation in eubacteria, whereas their primary structures are more eukaryotic in nature.
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PMID:Organization and nucleotide sequence of a transcriptional unit of Methanococcus vannielii comprising genes for protein synthesis elongation factors and ribosomal proteins. 247 40

A series of Tn917lac insertions define the comG region of the Bacillus subtilis chromosome. comG mutants are deficient in competence and specifically in the binding of exogenous DNA. The genes included in the comG region are first expressed during the transition from the exponential to the stationary growth phase. From nucleotide sequence information, it was concluded that the comG locus contains seven open reading frames (ORFs), several of which overlap at their termini. High-resolution S1 nuclease mapping and primer extension were used to identify the 5' terminus of the comG mRNA. The sequence upstream from the comG start site closely resembled the consensus recognition sequence for the major B. subtilis vegetative RNA polymerase holoenzyme. Complementation analysis confirmed that the comG ORF1 protein is required for the ability of competent cultures to resolve into two populations with different cell densities on Renografin (E. R. Squibb & Sons, Princeton, N.J.) gradients, as well as for full expression of comE, another late competence locus. The predicted comG ORF1 protein showed significant similarity to the virB ORF11 protein from Agrobacterium tumefaciens, which is probably involved in T-DNA transfer. The N-terminal sequences of comG ORF3 and, to a lesser extent, the comG ORF4 and ORF5 proteins were similar to a class of pilin proteins from members of the genera Bacteroides, Pseudomonas, Neisseria, and Moraxella. All of the comG proteins except comG ORF1 possessed hydrophobic domains that were potentially capable of spanning the bacterial membrane. It is likely that these proteins are membrane associated, and they may comprise part of the DNA transport machinery. When present in multiple copies, a DNA fragment carrying the comG promoter was capable of inhibiting the development of competence as well as the expression of several late com genes, suggesting a role for a transcriptional activator in the expression of those genes.
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PMID:Nucleotide sequence and genetic organization of the Bacillus subtilis comG operon. 250 24

The DNA sequence of the Salmonella typhimurium metC gene and its flanking regions was determined. The metC gene contains an open reading frame of 1185 nucleotides encoding a polypeptide of 395 amino acids with a predicted molecular weight of 42,874 daltons. S1 nuclease mapping experiments located the transcription start site of the metC gene. The nucleotide sequence and the deduced amino acid sequence for the metC genes of S. typhimurium and Escherichia coli were compared. Although there are 279 nucleotide replacements, most do not change the amino acid sequence. Nucleotide sequence analysis of the flanking regions of the S. typhimurium metC gene shows that there is an open reading frame upstream and an open reading frame downstream of the gene. The existence of the divergently transcribed upstream open reading frame (designated ORF1) was confirmed by the construction of an ORF1-lacZ fusion. The transcription start site of ORF1 was determined by S1 nuclease mapping.
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PMID:DNA sequence of the metC gene and its flanking regions from Salmonella typhimurium LT2 and homology with the corresponding sequence of Escherichia coli. 265 70

The newly identified sin gene affects late growth processes in Bacillus subtilis when it is overexpressed or inactivated in the chromosome. S1 nuclease mapping of the sin gene transcripts in vivo reveals the existence of three transcripts (RNAI, RNAII, and RNAIII). By correlating 5' ends of sin gene transcripts with DNA sequence, we have identified three different promoterlike sequences (P1, P2, and P3) for these transcripts. 3'-End mapping of these transcripts identified three prominent termination sites at the end of the sin gene. These termination sites are localized on two hairpin structures previously identified from the DNA sequence. The most abundant transcript, RNAIII, coded only for the sin gene, while the polycistronic transcripts RNAII and RNAI coded for the sin gene and ORF1 that precedes the sin gene. S1 mapping and translational lacZ fusion studies indicated that ORF1 and the sin gene are regulated differently. ORF1 expression is under developmental control, increasing at the end of vegetative growth, and requires functional spo0A and spo0H gene products. The sin gene is expressed at an almost constant and relatively low level throughout growth and remains largely unaffected by spo0A and spo0H mutations.
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PMID:Structure and expression of the Bacillus subtilis sin operon. 312 49


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