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)

Shiga-like toxin type II (SLT-II) and Shiga-like toxin type II variant (SLT-IIv) are cytotoxins produced by certain strains of Escherichia coli. Nucleotide sequence analyses had revealed that the structural genes for the A subunit and B subunit of SLT-II or SLT-IIv are arranged in an operon. Primer extension and S1 nuclease protection analyses identified a promoter for the slt-II operon 118 bases upstream of the slt-IIA gene. The slt-IIv promoter was demonstrated to be identical to the slt-II promoter. The slt-II and slt-IIv promoters differed significantly from the previously characterized Shiga toxin (stx) and Shiga-like toxin type 1 (slt-I) promoters. The transcriptional efficiencies of the stx and slt-II promoters were compared in fusions to the chloramphenicol acetyltransferase gene, and constitutive expression of the slt-II promoter was found to be equivalent to derepressed expression of the stx promoter. In contrast to the stx and slt-I promoters, the slt-II and slt-IIv promoters did not contain sequences for binding of the Fur repressor protein, and SLT-II production was not determined by iron levels in the media in various E. coli strains with wild-type or mutant ferric uptake regulation (fur) alleles. Northern (RNA) blot analysis demonstrated a single mRNA transcript for the slt-II operon, and further analysis of the slt-II operon by primer extension did not reveal an independent promoter for the B subunit gene. A putative rho-independent transcription terminator was identified 274 bases downstream of slt-IIB. These data indicated that the slt-II and slt-IIv operons differ from the stx/slt-I operon in regulation of their transcription by iron. Whether these regulatory differences enable the type I and type II groups of Shiga-like toxins to perform different roles in the pathogenesis of infectious diseases remains to be established.
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PMID:Transcription of the Shiga-like toxin type II and Shiga-like toxin type II variant operons of Escherichia coli. 222 65

The nucleotide sequence of the promoter region of the gene for cholesterol oxidase (choA) from Streptomyces sp. strain SA-COO was determined. We found an open reading frame (choP) that is located between a potential promoter sequence and the structural gene for the ChoA protein. Deletion analysis showed that the promoter region for choP is essential for expression of the choA gene. Mappings of S1 nuclease and primer extension of transcripts generated in vivo suggested that the synthesis of mRNA starts at a site 41 bases upstream from the ATG initiation codon of the choP gene. By Northern (RNA) blot analysis of the transcripts, we found a 2.9-kilobase transcript that is identical in size to the total sequence of the choP and choA genes. These results suggest that the two genes, choP and choA, are transcribed polycistronically under the control of the promoter that is upstream from the structural gene for choP. The choP gene encodes a protein of 381 amino acids with a calculated Mr of 41,668. The nucleotide sequence of the choP gene has a high degree of similarity to the sequence of the genes for cytochrome P-450s from humans and Pseudomonas species. A region of homology with the cytochrome P-450s from various organisms was identified in the choP protein and may represent a region associated with a binding site for heme iron. Analysis of the CO difference spectrum of an extract of Streptomyces lividans cells that carry a plasmid which includes the choP gene revealed a unique peak, characteristic of cytochrome P-450, which is identical to that obtained with the parent strain.
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PMID:An operon containing the genes for cholesterol oxidase and a cytochrome P-450-like protein from a Streptomyces sp. 236 41

Transcription of the tox gene in lysogenic Corynebacterium diphtheriae strains C7(beta tox+), C7 (gamma tox) and the hypertoxigenic PW8 (omega tox+) was analyzed and compared with transcription of the C. diphtheriae tox gene in the recombinant strain Escherichia coli (pDT201). In all cases S1 nuclease mapping localized the 5' terminus of the tox mRNA to a site 8 or 9 base pairs (bp) downstream of a region similar to the -10 consensus sequence of E. coli promoters. In C. diphtheriae the tox transcript was observed only in strains that were grown under iron-limiting conditions; in the presence of excess iron, transcription beyond bp 38 of the tox coding region was not observed. In contrast, in E. coli(pDT201) tox was expressed at equivalent levels in both iron-depleted and iron-supplemented media. The DNA insertion in the tox gene of the nontoxigenic corynephage gamma was found to occur at bp 54 of the tox coding region. The insertion event resulted in the duplication of a 7-bp target sequence, and the ends of the insert were found to constitute an imperfect inverted repeat of approximately 26 bp. Transcription from the tox promoter in C7(gamma tox) was found to initiate at the same nucleotides as in C7(beta tox+), PW8, and E. coli(pDT201) and remained sensitive to iron inhibition. These observations are discussed in relation to the mechanism of iron-mediated regulation of the tox gene.
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PMID:Characterization of the diphtheria tox transcript in Corynebacterium diphtheriae and Escherichia coli. 241 14

In heterocysts of the filamentous cyanobacterium Anabaena 7120 a specific [2Fe-2S] ferredoxin is synthesized, serving as immediate electron donor to nitrogenase. The structural gene for this heterocyst ferredoxin, fdxH, was isolated from a recombinant lambda library, using an oligonucleotide probe derived from a unique segment of the N-terminal amino acid sequence of the purified protein. The sequence of the entire fdxH coding region was determined including 3' and 5' flanking sequences. Assuming proteolytic cleavage of the first methionine residue, the molecular weight of Anabaena 7120 heterocyst ferredoxin is 10,806. Compared with the ferredoxin from vegetative cells, 47 out of 98 amino acid residues are different, including conversions within a highly conserved region responsible for binding of the iron-sulfur cluster. Northern hybridization with a 0.64 kb EcoRI DNA fragment containing the entire fdxH gene indicated two major transcripts of 0.59 and 1.85 kb, which are expressed at a late stage of heterocyst differentiation. By S1 nuclease digestion and primer extension a possible start site of transcription was mapped, 132 bp upstream of fdxH; however, neither a typical Escherichia coli nor nif-type promoter sequence was apparent. Southern hybridization detected only one copy of the fdxH gene in the Anabaena 7120 genome. FdxH is located approximately 7 kb downstream from the nifHDK gene cluster.
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PMID:Molecular cloning and nucleotide sequence analysis of the gene coding for heterocyst ferredoxin from the cyanobacterium Anabaena sp. strain PCC 7120. 246 84

The promoter of the high-affinity iron assimilation system coded in an approximately 8-kilobase-pair segment of the large Escherichia coli plasmid ColV-K30 was localized to a 0.7-kilobase HindIII-SalI fragment by in vitro runoff transcription. By an S1 nuclease protection assay, with in vitro-transcribed RNA and total in vivo-synthesized RNA, the major start site for transcription was mapped within this fragment and found to be identical in vitro and in vivo. A minor initiation site was located about 50 base pairs upstream from the major site. DNA sequencing of the HindIII-SalI fragment revealed the presence of two promoter-like structures within an extremely AT-rich region with transcriptional initiation sites at 30 and about 80 base pairs upstream from the initiation codon for the first structural gene. Numerous potential secondary structures were found in the DNA sequence around the major promoter. The major transcriptional start site was determined precisely by sequencing the 5' end of in vitro-transcribed RNA. The effect of iron on both the level of specific RNA, as determined by a quantitative S1 nuclease mapping assay, and on beta-galactosidase activity in a iucA'-'lacZ protein fusion, showed that the aerobactin operon is regulated at the transcriptional level. The iron-regulatory sequences are contained within a 152-base-pair Sau3A fragment of the promoter region.
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PMID:Promoter mapping and transcriptional regulation of the iron assimilation system of plasmid ColV-K30 in Escherichia coli K-12. 258 32

Transferrin is an iron-binding protein that is expressed as a major product in liver and secreted into the plasma. To study the tissue-specific regulatory regions of this gene, the genomic mouse transferrin (mTf) gene was cloned and characterized by partial sequence analysis and S1 nuclease mapping of the transcriptional start site. Fusion genes containing the transferrin gene promoter and 5'-flanking sequences were ligated to the human growth hormone (hGH) gene and used to produce transgenic mice. A deletion construct containing the -581 to +50 region of the transferrin gene was sufficient to direct a high level of liver-specific expression resembling endogenous transferrin gene expression. Deletion to -139 base pairs of 5'-flanking sequence gave a construct which retained liver specificity, but the magnitude of expression decreased severalfold. These results demonstrate the presence of a liver-specific transcriptional element between -139 and +50 and suggest the presence of a distal element between -581 and -139 that can further increase expression. Surprisingly, fusion constructs containing -3 kilobase pairs (kb) of 5'-flanking sequence gave higher levels of mRNA in nonhepatic tissues than did either the -581 or -139 construct. Further studies indicated that the high levels of circulating hGH in these transgenic mice specifically induced the endogenous transferrin and albumin genes in liver and also stimulated the normally low levels of expression of the endogenous transferrin gene in brain, heart, kidney, and muscle. A mutated hGH gene that does not produce active growth hormone was fused to the -3- to +50-kb transferrin sequences to produce the -3-kb mTf-hGX construct. A liver-specific pattern of expression was observed in transgenic mice harboring the -3-kb mTf-hGX construct, and this mutated transgene was shown to be induced four- to sevenfold by either bovine or human growth hormone. These results demonstrate the presence of a growth hormone-responsive element between -3 and +50 kb in the 5'-flanking region of the mTf gene promoter.
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PMID:Expression from the transferrin gene promoter in transgenic mice. 260 14

We determined the nucleotide sequence of the Shiga-like toxin-1 (SLT-1) genes carried by the toxin-converting bacteriophage H-19B. Two open reading frames were identified; these were separated by 12 base pairs and encoded proteins of 315 (A subunit) and 89 (B subunit) amino acids. The predicted protein subunits had N-terminal hydrophobic signal sequences of 22 and 20 amino acids, respectively. The predicted amino acid sequence of the B subunit was identical to that of the B subunit of Shiga toxin. The A chain of ricin was found to be significantly related to the predicted A1 fragment of the SLT-1 A subunit. S1 nuclease protection experiments showed that the two cistrons formed a single transcriptional unit, with the A subunit being proximal to the promoter. A probable promoter was identified by primer extension, and transcription was found to increase dramatically under conditions of iron starvation. A 21-base-pair sequence with dyad symmetry was found in the region of the SLT-1 -10 sequence, which was found to be 68% homologous to a region of dyad symmetry found in the -35 region of the promoter of the iucA gene on plasmid ColV-K30, which specifies the 74,000-dalton ferric-aerobactin receptor protein. Betley et al. (M. Betley, V. Miller, and J. Mekalanos, Annu. Rev. Microbiol. 40:577-605, 1986) have recently summarized evidence suggesting that the slt operon is under the control of the fur regulatory system. The area of dyad symmetry found in both promoters may represent a regulatory site. A rho-independent terminator sequence was found 230 base pairs downstream from the B cistron stop codon.
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PMID:Nucleotide sequence and promoter mapping of the Escherichia coli Shiga-like toxin operon of bacteriophage H-19B. 304 Jun 89

Adrenodoxin is an iron-sulfur protein that serves as an electron transport intermediate for all mitochondrial forms of cytochrome P450. To facilitate studying the regulation of adrenodoxin, we have cloned and determined the structure of the human adrenodoxin gene. It spans more than 20 kb, containing four exons and three introns. The first exon encodes the 60-amino-acid signal peptide, directing transport of the protein into the inner mitochondrial matrix. The mature peptide of 124 amino acids is encoded by the other three exons. The third exon encodes the portion of the protein containing the iron-sulfur center and a domain which binds other components of the electron transport chain. The transcriptional start sites were determined by primer extension and S1 nuclease mapping. The 5'-flanking region of this gene contains canonical promoters including a TATA box at nucleotide position -30 and two GC boxes at nucleotide positions -60 and -100. The sequence at nucleotides -234 to -252 is also highly homologous to the glucocorticoid-responsive element and the estrogen-responsive element.
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PMID:Cloning and structure of the human adrenodoxin gene. 322 85

Methidiumpropyl-EDTA . iron(II) [MPE . Fe (II)] cleaves double-helical DNA with considerably lower sequence specificity than micrococcal nuclease. Moreover, digestions with MPE . Fe(II) can be performed in the presence of certain metal chelators, which will minimize the action of many endogenous nucleases. Because of these properties MPE . Fe(II) would appear to be a superior tool for probing chromatin structure. We have compared the patterns generated from the 1.688 g/cm3 complex satellite, 5S ribosomal RNA, and histone gene sequences of Drosophila melanogaster chromatin and protein-free DNA by MPE . Fe(II) and micrococcal nuclease cleavage. MPE . Fe(II) at low concentrations recognizes the nucleosome array, efficiently introducing a regular series of single-stranded (and some double-stranded) cleavages in chromatin DNA. Subsequent S1 nuclease digestion of the purified DNA produces a typical extended oligonucleosome pattern, with a repeating unit of ca. 190 base pairs. Under suitable conditions, relatively little other nicking is observed. Unlike micrococcal nuclease, which has a noticeable sequence preference in introducing cleavages, MPE . Fe(II) cleaves protein-free tandemly repetitive satellite and 5S DNA sequences in a near-random fashion. The spacing of cleavage sites in chromatin, however, bears a direct relationship to the length of the respective sequence repeats. In the case of the histone gene sequences a faint, but detectable, MPE . Fe(II) cleavage pattern is observed on DNA, in some regions similar to and in some regions different from the strong chromatin-specified pattern. The results indicate that MPE . Fe(II) will be very useful in the analysis of chromatin structure.
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PMID:Cleavage of chromatin with methidiumpropyl-EDTA . iron(II). 640 8

Many bacteria respond to a lack of iron in the environment by synthesizing siderophores, which act as iron-scavenging compounds. Fluorescent pseudomonads synthesize strain-specific but chemically related siderophores called pyoverdines or pseudobactins. We have investigated the mechanisms by which iron controls expression of genes involved in pyoverdine metabolism in Pseudomonas aeruginosa. Transcription of these genes is repressed by the presence of iron in the growth medium. Three promoters from these genes were cloned and the activities of the promoters were dependent on the amounts of iron in the growth media. Two of the promoters were sequenced and the transcriptional start site were identified by S1 nuclease analysis. Sequences similar to the consensus binding site for the Fur repressor protein, which controls expression of iron-repressible genes in several gram-negative species, were not present in the promoters, suggesting that they are unlikely to have a high affinity for Fur. However, comparison of the promoter sequences with those of iron-regulated genes from other Pseudomonas species and also the iron-regulated exotoxin gene of P. aeruginosa allowed identification of a shared sequence element, with the consensus sequence (G/C)CTAAAT-CCC, which is likely to act as a binding site for a transcriptional activator protein. Mutations in this sequence greatly reduced the activities of the promoters characterized here as well as those of other iron-regulated promoters. The requirement for this motif in the promoters of iron-regulated genes of different Pseudomonas species indicates that similar mechanisms are likely to be involved in controlling expression of a range of iron-regulated genes in pseudomonads.
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PMID:Identification of a DNA sequence motif required for expression of iron-regulated genes in pseudomonads. 789 66


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