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)

Five major anfH-hybridizing mRNA species accumulated in Azobacter vinelandii cells derepressed for nitrogenase-3 (an alternative nitrogenase, which appears to lack Mo and V). Using anfH-, anfD-, anfG-, anfK-, and orflorf2-specific probes and mutant strains of A. vinelandii these mRNA species have been identified as encoding anfHDGKorflorf2 (6.0 kb), anfHDGK (4.3 kb), anfHD (2.6 kb), vnfHorfFd (1.3 kb), and vnfH and (or) anfH (1.0 kb). A 0.6-kb mRNA species, which hybridized only to the orflorf2-specific probe, and a 3.5-kb mRNA species, which hybridized to anfD or anfK, also accumulated under these conditions. Northern blot analysis and S1 nuclease mapping indicate that transcription of the anf structural gene cluster initiates at a unique nif consensus promoter situated 127 base pairs upstream from the anfH coding region. Observation of anfH-hybridizing mRNA species that accumulate in strains derepressed for nitrogen fixation demonstrates that transcription of the anfHDGKorflorf2 cluster is normally repressed by Mo, V, and NH4+, whereas transcription of the vnfHorfFd cluster does not require the presence of V and is repressed only by Mo, but not NH4+. Analysis of the accumulation of mRNAs in a tungsten-tolerant strain revealed that Mo and V repression of anf transcription must occur by different mechanisms.
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PMID:Characterization of transcripts expressed from nitrogenase-3 structural genes of Azotobacter vinelandii. 128 43

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

A 1.8 kb transcript corresponding to a region of the Anabaena 7120 chromosome 4 kb downstream of the nifHDK operon appears 12-18 h after heterocyst induction. The DNA corresponding to this transcript was sequenced and found to contain two open reading frames, designated ORF 1 and ORF 2. Two polypeptides, of 30 kDa and 13 kDa, encoded by these ORFs were expressed in Escherichia coli. An apparent start site for the transcript, detected by S1 nuclease protection, was located 42 bp upstream of the ATG start codon of ORF 1. ORF 2 shows strong sequence similarity to ORF 6 in the nif gene region of Azotobacter vinelandii. ORF 1 was interrupted using a 1.4 kb neomycin resistance cassette and the resulting mutant grew very slowly on medium lacking combined nitrogen. The mutant had 45% of wild-type acetylene reduction activity, which could be complemented by a 2.8 kb EcoRI fragment of wild-type Anabaena DNA containing only ORF 1 and ORF 2. Thus, one or both of these ORFs is required for efficient nitrogen fixation in Anabaena.
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PMID:Expression, nucleotide sequence and mutational analysis of two open reading frames in the nif gene region of Anabaena sp. strain PCC7120. 211 11

In Neurospora the expression of a set of unlinked structural genes, which allows utilization of various nitrogen-containing compounds, is controlled by the positive-acting nit-2 gene and the negative-acting nmr gene. The nucleotide sequence of the nmr gene has been determined and a long open reading frame which encodes a putative protein of 54854 daltons has been identified. A full-length cDNA clone was obtained and its the sequence revealed that the nmr gene contains no introns. The transcriptional start and stop sites have been mapped by S1 nuclease and primer extension. Site-directed mutagenesis was used to introduce stop codons at various locations in the nmr coding region. Transformation assays showed that the proteins lacking up to 16% of the carboxyl-terminus were still functional. Homology searches showed that the nmr protein is homologous to the yeast arginine regulatory gene AR-GRII.
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PMID:Nucleotide sequence and analysis of NMR, a negative-acting regulatory gene in the nitrogen circuit of Neurospora crassa. 214 84

A modified highly sensitive procedure for the evaluation of DNA damage in individual cells treated with alkylating agents is reported. The new methodology is based on the amplification of single-strandedness in alkylated DNA by heating in the presence of Mg2+. Human ovarian carcinoma cells A2780 were treated with nitrogen mustard (HN2), fixed in methanol, and stained with monoclonal antibody (MOAB) F7-26 generated against HN2-treated DNA. Binding of MOAB was measured by flow cytometry with indirect immunofluorescence. The maximal difference in fluorescence between untreated and HN2-treated cells was observed after heating at 100 degrees C for 5 min in PBS containing 1.25 mM MgCl2. Higher concentrations of MgCl2 inhibited MOAB binding to HN2-treated cells and heating at lower concentrations induced binding to control cells. Intensive binding of MOAB to control and drug-treated cells was observed after heating in Tris buffer supplemented with MgCl2. Thus, the presence of phosphates and MgCl2 during heating was necessary for the detection of HN2-induced changes in DNA stability. Fluorescence of HN2-treated cells decreased to background levels after treatment with single-strand-specific S1 nuclease. MOAB F7-26 interacted with single-stranded regions in DNA and did not bind to dsDNA or other cellular antigens. Specific reactivity of MOAB F7-26 with deoxycytidine was established by avidin-biotin ELISA. Single-stranded conformation was necessary for the binding of MOAB to deoxycytidine on the DNA molecule. It is suggested that alkylation of guanines decreased the stability of the DNA molecule and increased the access of MOAB F7-26 to deoxycytidines on the opposite DNA strand.
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PMID:Decreased stability of DNA in cells treated with alkylating agents. 225 76

In the filamentous cyanobacterium Anabaena variabilis, specialized cells called heterocysts occur in a regular pattern along the filament and are the sites of nitrogen fixation. We used two different types of DNA-excess RNA hybridization techniques to estimate the number of genes expressed in recently differentiated, mature heterocysts. In the first, RNA and DNA were incubated in a phosphate buffer at 60 degrees C, and the hybrids were separated from the unhybridized material by hydroxylapatite chromatography. In the second, the nucleic acids were incubated at 50 degrees C in a buffer containing 50% formamide, and the fraction of DNA in duplexes was assayed by S1 nuclease digestion. Both techniques revealed that approximately 65% of the A. variabilis genome was expressed in vegetative cells and 45% of the genome was expressed in heterocysts. Two experiments were conducted to estimate the number of heterocyst-specific mRNA transcripts. In one, hybridization of heterocyst RNA to a null DNA probe (DNA not transcribed in vegetative cells) revealed that heterocyst-specific transcripts were encoded by 25% of the DNA sense strand, representing approximately 1,000 genes (assuming each to be 1,500 nucleotides in length). The second approach, in which total cell DNA was hybridized to a mixture of heterocyst and vegetative cell RNA, indicated that 14.7% of the DNA sense strand, or about 600 genes, was transcribed exclusively in the heterocyst. The remaining 900 to 1,300 transcripts present in the heterocyst appeared to be constitutively produced in both vegetative cells and heterocysts. The heterocyst-specific transcripts were present in abundant copies in the cell, while transcripts that occurred in both cell types were present at much lower frequency.
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PMID:Estimation of gene expression in heterocysts of Anabaena variabilis by using DNA-RNA hybridization. 242

A mutation in the gene IRA1 (formerly called PPD1) was originally characterized as a deficiency of a phosphoprotein phosphatase. The IRA1 gene has been cloned and sequenced. A large open reading frame (8,817 base pairs) which can encode a protein of 2,938 amino acids was found. Northern (RNA) blot analysis detected a message of about 10 kilobases, and nuclease S1 protection demonstrated mRNA start points at 97 and 98 base pairs upstream from the putative initiator ATG codon. Disruption of the IRA1 gene resulted in sensitivity to nitrogen starvation and heat shock. Diploids homozygous for the disrupted IRA1 gene were deficient in sporulation. Disruption of the IRA1 gene suppressed the lethality of the cdc25 mutation but did not suppress the lethality of either the ras1 ras2 or the cyr1 mutations. Deficiency of the phosphoprotein phosphatase was not reproducible in the disruption mutant of the IRA1 gene. Moreover, the ira1 mutant showed an increased level of cyclic AMP. Our results suggest that the IRA1 protein inhibits the function of the RAS proteins in a fashion antagonistic to the function of the CDC25 protein in the RAS-cyclic AMP pathway in Saccharomyces cerevisiae.
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PMID:IRA1, an inhibitory regulator of the RAS-cyclic AMP pathway in Saccharomyces cerevisiae. 254 Apr 26

Transcription of the structural genes for nitrogenase (nifHDK) in Azospirillum brasilense Sp7 was analysed using Northern blots of total RNA extracted from cultures grown under nitrogen-fixing conditions. Hybridization with an internal nifH probe revealed two transcripts, a major one (by concentration) of 1.1 kb corresponding to nifH and a minor one of 5.6 kb corresponding to nifHDK. Hybridization with nifD or nifK probes revealed the minor transcript of 5.6 kb. This confirms that the nifHDK genes are organized as a single transcription unit and suggests regulation at the level of termination of transcription. The complete nucleotide sequence of nifH was established and the DNA region upstream of the initiation codon was analysed for transcription and translation signals. The nifH open reading frame (ORF) is preceded by an NtrA-dependent promoter and two elements homologous to upstream activator sequences (UAS) required for NifA-mediated activation in other diazotrophs. Promoter mapping with S1 nuclease revealed two start sites located 10 bp and 40 bp downstream of the NtrA-dependent promoter.
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PMID:Regulation of transcription and promoter mapping of the structural genes for nitrogenase (nifHDK) of Azospirillum brasilense Sp7. 260 30

The production of the bacterial DNA replication inhibitor Microcin B17 is induced as cultures enter stationary phase. Using S1 nuclease protection assays we have shown that this induction is the result of increased levels of transcription initiation from a promoter located upstream from mcbA, the structural gene for Microcin B17. Upstream from the start site of transcription there is a rather typical -35 region. However, there is no good homology to the consensus -10 region. While most of the cell's transcription is shut off as a result of the cessation of growth, transcription from the mcbA promoter continues for several hours in stationary phase. A single-copy gene fusion between mcbA and lacZ was used to monitor the response of the promoter to different nutritional conditions and in different host backgrounds altered in metabolic regulatory loci. Starvation for nitrogen, phosphate or carbon sources all induced transcription from the promoter. Levels of transcription were reduced in ompR backgrounds. In contrast, mutations in other global regulatory loci, fnr, relA and cya had little or no effect.
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PMID:An E. coli promoter induced by the cessation of growth. 283 80

We have been investigating the structure and regulation of the lignin peroxidase genes from Phanerochaete chrysosporium. The gene for lignin peroxidase isozyme H8 was isolated and sequenced. The gene is split into nine exons and eight introns. The introns have consensus sequences both at the splice junctions and at an internal element which may be involved in the splicing mechanism. The promoter contains the eukaryotic consensus elements: a TATA box at -78 and a ACAAT box at -106. Transcription initiates downstream from these sequences as verified by S1 nuclease mapping. Induction of the genes for the lignin peroxidase isozymes during growth under nitrogen and under carbon limitation were compared by analyses of RNA and protein. The results indicate that the lignin peroxidase isozymes are differentially regulated in response to environmental stress.
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PMID:Structure and regulation of a lignin peroxidase gene from Phanerochaete chrysosporium. 284 76


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