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)

A scheme is presented for cloning a double-stranded cDNA molecule that codes for a portion of chicken preproalbumin. This method, which does not require pure mRNA or cDNA, has widespread applicability. Chicken preproalbumin was identified as a Mr = 72,000 polypeptide by immunoprecipitation of proteins synehesized in a wheat germ cell-free translation system from total, guanidine.HCl-extracted, rooster liver RNA. After removal of the bulk of the ribosomal RNA by poly(U)-Sephadex G-10 chromatography, albumin mRNA was enriched approximately 2-fold by centrifugation through low salt, isokinetic sucrose gradients, until it represented about 30% of the mRNA sequences present. Double-stranded cDNA prepared from this mRNA was then inserted into the Pst 1 site of the plasmid PBR322 by the "G-C tailing" technique and the recombinant DNA was used to transform Echerichia coli stran X1776. Transformants containing putative albumin DNA sequences were identified by colony hybridization with a cDNA probe that was highly enriched for albumin cDNA sequences. This probe was isolated by hybridizing the partially purified RNA preparation to its cDNA, under conditions of RNA excess, to a R0t value such that only the most abundant cDNA sequences had hybridized. Unhybridized, less abundant, sequences were destroyed by subsequent S1 nuclease digestion. The identity of clones that hybridized to this abundant class cDNA was established by DNA-mRNA hybrid-arrested cell-free translation. Hybridization of nick-translated, albumin-containing, plasmid DNA to total liver poly(A)+ RNA, that had been separated on methyl mercury agarose gels and transferred to diazobenzyloxymethyl paper, established that avian albumin mRNA has a molecular weight of 850,000. This molecular weight corresponds to approximately 2,600 nucleotides, or 600 nucleotides longer than the size required to code for the preproalbumin polypeptide.
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PMID:Cloning of a double-stranded cDNA that codes for a portion of chicken preproalbumin. A general method for isolating a specific DNA sequence from partially purified mRNA. 71 69

We have isolated a 5-kilobase pair fragment of genomic DNA containing the entire coding region for the Chlamydomonas reinhardtii gene encoding the copper-repressible Cyt c6. A region comprising 2.6 kilobase pairs contains the entire transcribed region plus 852 nucleotides upstream of the Cyt c6 transcription start site and 495 nucleotides downstream of the conserved C. reinhardtii polyadenylation signal. Comparison of the genomic sequence with the cDNA sequence (Merchant, S., and Bogorad, L. (1987) J. Biol. Chem. 262, 9062-9067) revealed that the coding region is interrupted by two introns, each of which is flanked by C. reinhardtii consensus intron/exon boundaries. Primer extension and S1 nuclease protection analyses identified the 5' border of the Cyt c6 mRNA at approximately 79 base pairs upstream from the initiator methionine. Analysis of the 5' upstream region reveals no significant similarity to sequences found in upstream regions of other copper-regulated genes. Time-course studies indicate that 1) the mature Cyt c6 mRNA has a half-life of approximately 45-60 min and is completely lost within 4 h, and 2) the primary, unspliced transcript has a half-life of approximately 10 min and is completely lost within 30 min after the addition of copper ions to copper-depleted cells. These results indicate that the response to copper occurs very rapidly upon elevation of extracellular copper levels. Although this gene is unresponsive to silver ions in vivo, in contrast to the yeast copper-responsive CUP1 gene (Furst, P., Hu, S., Hackett, R., and Hamer, D. (1988) Cell 55, 705-717), it does respond to mercury ions, albeit with less sensitivity. Mercury ions cannot, however, substitute for copper in allowing the accumulation of plastocyanin in vivo.
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PMID:Isolation and structural characterization of the Chlamydomonas reinhardtii gene for cytochrome c6. Analysis of the kinetics and metal specificity of its copper-responsive expression. 171 51

The detailed organization of the RNAs transcribed from an early gene cluster encoded by vaccinia virus has been determined from the information derived from several complementary techniques. These include hybrid selection coupled with cell-free translation to locate DNA sequences complementary to mRNAs encoding specific polypeptides; RNA filter hybridization to size and locate on the DNA mature RNAs as well as higher-molecular-weight RNAs; S1 nuclease mapping to precisely locate the 5' and 3' ends of the RNAs; S1 nuclease mapping to precisely locate the 5' and 3' ends of the RNAs; and fractionation of hybrid-selected mRNAs in an agarose gel containing methyl mercury hydroxide followed by the cell-free translation of these mRNAs to definitively ascertain the size of the mRNA encoding each polypeptide. The early gene cluster is located between 21 and 26 kilobases from the left end of the vaccinia virus genome and is encoded by a 5.0-kilobase EcoRI fragment which spans the HindIII-N, -M, and -K fragments. Transcribed towards the left terminus are four mature mRNAs, 1,450, 950, 780, and 400 nucleotides in size, encoding polypeptides of 55, 30, 20, and 10 kilodaltons, respectively. These mRNAs are colinear with the DNA template and are closely spaced such that the 5' terminus of one mRNA is within 50 base pairs of the 3' terminus of the adjacent RNA. In addition to the mature size mRNAs, there are higher-molecular-weight RNAs, 5,000, 3,300, 2,350, 2,300, 1,800, 1,700, and 1,350 nucleotides in size. The 5' and 3' termini of the high-molecular-weight RNAs are coterminal with the 5' and 3' termini of the mature size mRNA. The implications of this arrangement and the biogenesis of these early mRNAs are discussed.
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PMID:Organization of RNA transcripts from a vaccinia virus early gene cluster. 608 46

Metallothioneins are small cysteine-rich proteins that bind heavy metals such as zinc, cadmium, copper and mercury. Recent interest in these proteins has focused on the part they play in zinc metabolism and heavy metal detoxification. Our interest in metallothionein genes stems largely from the observations that these proteins are inducible by both heavy metals and glucocorticoid hormones. To explore the regulation of these genes, we have isolated cDNA and genomic clones corresponding to mouse metallothionein-I (MT-I), and have used them to show that both inducers act at the transcriptional level in vivo and in a wide variety of cell lines. We have also shown that the MT-I gene is amplified during selection for cadmium resistance. To investigate the mechanisms of gene regulation, knowledge of the primary DNA sequence is necessary. Here we present the entire sequence of mouse MT-I gene along with approximately 300 bases of 5' flanking region that presumably includes promoter and regulatory sites. The 5' mRNA sequence, defined by S1 nuclease mapping, was combined with sequences of the coding and 3' untranslated regions obtained previously to allow a computer prediction of the most stable secondary structure of MT-I mRNA.
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PMID:Structure of mouse metallothionein-I gene and its mRNA. 725 20

The broad-spectrum mercury resistance of Streptomyces lividans 1326 is mediated by six open reading frames (orf). These are arranged in two divergently transcribed operons. The orfs mer A (mercuric reductase) and mer B (organolyase) form one of the two operons. These genes and their regulation were further studied by deletion analysis and transcriptional fusion to the reporter gene xylE in the plasmid pXE4. An increase in XylE activity in response to the presence of mercuric ions was observed. The function of ORF2 (MerT) and ORF3 (MerP) as mercury-specific transport proteins, previously postulated based on the structural features of the predicted proteins, was confirmed. Transcription of the mer genes starts within the intercistronic region and two divergent promoters were identified by S1 nuclease mapping. Expression of the genes was negatively regulated by the product of orf1, now called merR. The repressor function was confirmed by gel retardation assays. MerR, produced in Escherichia coli, bound to two sites (operators) in the fragment containing the promoter region between merA and merR. Addition of mercuric ions and phenylmercuric acetate prevented the binding of MerR.
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PMID:Regulation of the operon responsible for broad-spectrum mercury resistance in Streptomyces lividans 1326. 867 73