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 solution hybridization/RNase protection assay with riboprobes was developed to quantitate apolipoprotein mRNA concentrations. Previously, radiolabeled DNA probes have been used in solution hybridization/S1 nuclease protection assays for this purpose. The new assay requires less time for probe preparation and hybridization compared to previous assays. In addition, the vector used for riboprobe preparation can also be used to conveniently produce cRNA required to generate the standard curve to quantitate absolute apolipoprotein mRNA levels. The solution hybridization RNase protection assay was used to quantitate apoB, A-I, and E mRNA levels in four human hepatoma cell lines, HepG2, Hep3B, WRL-68, SK-Hep2. HepG2 and Hep3B, but not WRL-68 and SK-Hep2 cells had concentrations of all three apolipoprotein mRNAs comparable to liver in vivo. These data suggest that HepG2 and Hep3B are suitable models to study liver specific apolipoprotein gene expression.
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PMID:A solution hybridization/RNase protection assay with riboprobes to determine absolute levels of apoB, A-I, and E mRNA in human hepatoma cell lines. 216 16

Degradation intermediates of the estrogen-regulated apolipoprotein (apo) II mRNA were identified by S1 nuclease mapping and primer extension analysis. S1 mapping of poly(A)-RNA detected a series of mRNAs truncated at specific sites in the 3'-noncoding region. Many of these sites were also detected by primer extension analysis indicating that truncated molecules resulted from endonucleolytic cleavage in the 3'-noncoding region. Identical cleavage sites were seen with RNA from estrogen-treated animals or from animals withdrawn from hormone under conditions where apoII mRNA degraded in the slow (t1/2 = 13 h) or rapid (t1/2 = 1.5 h) decay mode. No differences were seen in poly(A) tail length or heterogeneity among these conditions. These results indicate that the estrogen-induced alteration in apoII mRNA turnover does not involve a new pathway of degradation, but, more likely, involves an increased targeting of the mRNA for degradation by a preexisting pathway. These data are consistent with a mechanism in which the initial step in apoII mRNA degradation is an endonucleolytic cleavage in the 3'-noncoding region without prior removal of the poly(A) tail. The endonucleolytic cleavage sites occurred predominantly at 5'-AAU-3' or 5'-UAA-3' trinucleotides found in single-stranded domains in a secondary structure model of the naked mRNA (Hwang, S-P. L., Eisenberg, M., Binder, R., Shelness, G. S., and Williams, D. L. (1989) J. Biol. Chem. 264, 8410-8418). The structure of the 3'-noncoding region in polyribosomal messenger ribonucleoprotein was examined by titrations of liver homogenates with dimethyl sulfate and cobra venom RNase. The results suggest that the typical cleavage site is a 5'-AAU-3' or 5'-UAA-3' trinucleotide in an accessible single-stranded loop domain. Single-stranded domains alone or accessible domains alone are not sufficient for cleavage. Similarly, 5'-AAU-3' or 5'-UAA-3' trinucleotides alone are not sufficient for cleavage. Localization of these trinucleotides to accessible single-stranded domains in the polyribosomal messenger ribonucleoprotein may provide the specificity for cleavage during targeted degradation.
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PMID:Degradation of apolipoprotein II mRNA occurs via endonucleolytic cleavage at 5'-AAU-3'/5'-UAA-3' elements in single-stranded loop domains of the 3'-noncoding region. 255 Apr 65

The complete nucleotide sequence of the human apolipoprotein All gene together with 911 bases of 5' flanking sequence and 687 bases of 3' flanking sequence have been determined. The mRNA coding region is interrupted by three introns of 169, 293 and 395bp. The Intro-exon structure of the apo All gene is similar to that of the apo AI, apo CIII and apo E genes: three introns separate 4 coding sequences specifying the 5' untranslated region, pre-peptide, a short N-terminal domain and a C-terminal domain composed of a variable number of lipid-binding amphipathic helices. Intron II carries a 33bp dG-dT repetitive element adjacent to the 3' splice junction which has the potential to adopt the Z-DNA conformation. The 5' and 3' terminuses of the mRNA have been identified by primer extension and S1 nuclease mapping. A number of short direct repeats are found in the 5' flanking region and an inverted repeat occurs between the CAAT and TATA boxes. Downstream of the the gene is an Alu family repeat containing a polymorphic MspI site, the deletion of which is associated with increased circulating levels of apoAII. ApoAII gene expression was demonstrated in adult human liver and HepG2 cells but not in human small intestine. Of ten Rhesus monkey tissues examined apo All mRNA was detected only in liver.
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PMID:The human apolipoprotein AII gene: structural organization and sites of expression. 299 28

To develop the baboon model for molecular genetic studies of atherosclerosis, we have cloned and sequenced the baboon apolipoprotein E (apo E) gene. The baboon apo E gene encodes the E4 isoform with respect to specific amino acid positions, suggesting that the common epsilon 3 allele is not the primal human allele. Rather than accumulating predominantly synonymous nucleotide changes, 50% of substitutions in human and baboon apo E gene coding regions cause amino acid substitutions. However, comparisons of these apo E proteins show conservation of amphipathic helices required for apo E--lipid interactions. The human and baboon apo E genes have diverged less extensively than those from rat and mouse, providing further evidence for a slowing of molecular evolution in primate species. The baboon and rhesus monkey apo E genes (intron 2) contain two Alu repeats that are absent in the human gene, indicating insertion after the divergence of human and cercopithecine lineages, but before the baboon/rhesus divergence. S1 nuclease studies show that transcription of the baboon apo E gene starts at two different positions, one of which corresponds to the human gene start site. To examine linkage of apolipoprotein genes in the baboon genome, we have used a human cDNA probe to detect apo C-I gene sequences approximately 4 kb from the 3' end of the baboon apo E gene.
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PMID:The baboon apolipoprotein E gene: structure, expression, and linkage with the gene for apolipoprotein C-1. 322 Apr 72

We have investigated RNAs originating from the 5'-flanking region of the chicken very-low-density apolipoprotein II (apoVLDLII) gene. S1 nuclease mapping and primer extension experiments revealed two minor upstream transcription start points located 1105 and 1530 nucleotides in front of the apoVLDLII gene. Transcription starting at these points is dependent upon estradiol as is transcription from the major start points. The transcripts are polyadenylated, but are not detectable in polysomes. Run-on assays indicated that the low concentration of the upstream initiated transcripts is due both to low transcription levels and to low transcript stability. The sequence around the upstream start points does not show strong homologies with consensus sequences of promoters for eukaryotic protein encoding genes. Nevertheless, the upstream sequences are transcribed in vivo by RNA polymerase II.
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PMID:Estradiol-dependent transcription initiation upstream from the chicken apoVLDLII gene coding for the very-low-density apolipoprotein II. 378 Dec 48

We have isolated and sequenced the 5' proximal exons and flanking regions of the chicken very low density apolipoprotein II (apo-VLDLII) and serum albumin genes. These genes specify the most abundant mRNA species present in livers of hens or estrogen-treated roosters. Sequencing revealed that the promoter region of the estrogen-dependent apo-VLDLII gene contained at least two potential transcriptional initiation sites, preceded by appropriately positioned "ATA" sequences. One is homologous with the cap site of the ovalbumin gene, and the other exhibits a match of 10 out of 12 nucleotides with the cap site of the serum albumin gene. S1 nuclease mapping indicates that both sites are active in vivo, although the "ovalbumin"-like site is by far the most efficient at all stages of the estrogenic response. The relative efficiencies of these two sites are maintained during in vitro transcription of truncated templates. A third site, that was not anticipated from sequencing data, is active in vivo but inactive in vitro. A conserved 5' flanking element, initially identified during studies on egg white protein genes, is also present upstream from the apo-VLDLII and serum albumin genes. Its removal does not affect initiation site selection in vitro. Regions of the apo-VLDLII and ovalbumin genes extending 100 nucleotides downstream from the "TATA box" contain several striking homologies that suggest a common mode of evolution.
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PMID:The 5' noncoding and flanking regions of the avian very low density apolipoprotein II and serum albumin genes. Homologies with the egg white protein genes. 618 37

We have precisely determined the positions of the first three exons for the major chicken vitellogenin gene (VTG II) by a combination of S1 nuclease protection, primer extension and DNA sequencing experiments. In addition, we have determined the nucleotide sequences of the 5' flanking nuclease hypersensitive sites that we have previously shown are induced during the estrogen mediated activation of the VTG II gene in liver (1). One of these sites is found to be nearly identical to the enhancer core sequence of SV40. A computer assisted analysis of the DNA sequences upstream from the VTG II gene has revealed four short (7 to 9 base pair) sequence elements that are present in similar positions flanking the other major estrogen inducible gene for liver, very low density apolipoprotein II (apoVLDL II). For VTG II, these sequences are located between two of the induced nuclease hypersensitive sites that are liver specific. Sequences homologous to one element, located approximately 100 base pairs upstream from the mRNA cap sites of the VTG II and apoVLDL II genes, are also observed for three estrogen inducible genes that are expressed in the oviduct, although for each of these genes the sequence falls further upstream, between -220 and -200. We suggest that these conserved sequences may be important in mediating the tissue specific responses of these genes to estrogen.
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PMID:Identification and sequence analysis of the 5' end of the major chicken vitellogenin gene. 669 8