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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Porcine liver DNA polymerase gamma was shown previously to copurify with an associated 3' to 5' exonuclease activity (Kunkel, T. A., and Mosbaugh, D. W. (1989) Biochemistry 28, 988-995). The 3' to 5' exonuclease has now been characterized, and like the DNA polymerase activity, it has an absolute requirement for a divalent metal cation (Mg2+ or Mn2+), a relatively high NaCl and KCl optimum (150-200 mM), and an alkaline pH optimum between 7 and 10. The exonuclease has a 7.5-fold preference for single-stranded over double-stranded DNA, but it cannot excise 3'-terminal dideoxy-NMP residues from either substrate. Excision of 3'-terminally mismatched nucleotides was preferred approximately 5-fold over matched 3' termini, and the hydrolysis product from both was a deoxyribonucleoside 5'-monophosphate. The kinetics of 3'-terminal excision were measured at a single site on M13mp2 DNA for each of the 16 possible matched and mismatched primer.template combinations. As defined by the substrate specificity constant (Vmax/Km), each of the 12 mismatched substrates was preferred over the four matched substrates (A.T, T.A, C.G, G.C). Furthermore, the exonuclease could efficiently excise internally mismatched nucleotides up to 4 residues from the 3' end. DNA polymerase gamma was not found to possess detectable DNA primase, endonuclease, 5' to 3' exonuclease, RNase, or RNase H activities. The DNA polymerase and exonuclease activities exhibited dissimilar rates of heat inactivation and sensitivity to N-ethylmaleimide. After nondenaturing activity gel electrophoresis, the DNA polymerase and 3' to 5' exonuclease activities were partially resolved and detected in situ as separate species. A similar analysis on a denaturing activity gel identified catalytic polypeptides with molecular weights of 127,000, 60,000, and 32,000 which possessed only DNA polymerase gamma activity. Collectively, these results suggest that the polymerase and exonuclease activities reside in separate polypeptides, which could be derived from separate gene products or from proteolysis of a single gene product.
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PMID:Properties of the 3' to 5' exonuclease associated with porcine liver DNA polymerase gamma. Substrate specificity, product analysis, inhibition, and kinetics of terminal excision. 166 14

The human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) is a protein of 66 kDa, p66, which contains two domains, an amino-terminal DNA polymerase and an RNase H at the carboxy terminus of the molecule. In order to characterize the mode of action of the RNase H, two previously described mutant enzymes were used, with substitutions in the highly conserved histidine 539, which was mutated to the neutral amino acid asparagine and to the negatively charged aspartate. The purified wild-type (wt) and mutant (mt) enzyme activities are analyzed here using RNA-DNA hybrids consisting of in vitro transcribed RNA that harbors the polypurine tract (PPT) from HIV-1 and DNA oligonucleotides complementary to the PPT or to other regions of the RNA. Analysis of the radioactively labeled RNA of these model hybrids after RNase H treatment indicates that both, wt and mt enzymes, are capable of cleaving the RNA in an endonucleolytic manner. The mt enzymes exhibit a severely reduced exonuclease activity. They are more sensitive towards salt and competition with excess of unlabeled hybrid, suggesting a reduced substrate binding affinity. DNA elongation by the RT is coupled with RNA hydrolysis by the 3'-5' exonuclease of the wt RNase H. The RNase Hmt of the mt enzymes, however, does not exhibit such processive 3'-5' exonuclease activity during DNA synthesis but gives rise to sporadic endonucleolytic cuts, whereas the RT is not affected. The endonuclease activities of the RNase H mt enzymes exhibit cleavage preferences in the absence or presence of DNA synthesis different from those of the wt enzyme. They cannot recognize specific sequences required to generate a PPT-primer and therefore cannot initiate plus-strand DNA synthesis in vitro at the 3' end of the PPT, which is essential for viral replication.
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PMID:Mutations of a conserved residue within HIV-1 ribonuclease H affect its exo- and endonuclease activities. 171 5

We have used an in vitro system to characterize ribonuclease activities present in spinach chloroplasts. We show that 3' end maturation of petD mRNA, which encodes subunit IV of the cytochrome b6/f complex, is affected by a 33-kDa protein that binds to a hairpin structure at the 3' end of the mature mRNA. Binding of the 33-kDa protein to the petD hairpin structure decreases the efficiency of 3' end maturation, probably by impeding the progress of the processive 3'-5' exonuclease activity involved in chloroplast mRNA processing. A two-base mutation in the stem of the petD hairpin structure creates a novel recognition site for a ribonuclease which competes with the normal processing exonuclease activity. This mutation results in a very low 3' end processing efficiency for mutant petD transcripts, and instead generates a second processing product that lacks a complete hairpin structure. An endonuclease activity which is biochemically distinct from the previously characterized exonuclease activities has also been identified. This endonuclease activity is EDTA-insensitive, and cleaves petD RNA both at the termination codon and at the mature RNA 3' end. Cleavage of petD mRNA at the termination codon leads to rapid degradation of upstream RNA. The possible roles of these ribonuclease activities in chloroplast mRNA decay in vivo are discussed.
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PMID:Specific ribonuclease activities in spinach chloroplasts promote mRNA maturation and degradation. 172 Oct 67

Enkephalins are opiate peptides found in a variety of tissues including brain and pituitary. In brain, they function as neurotransmitters, neuromodulators and neurohormones. Recent studies show that proenkephalin mRNA is expressed early in development both in mammals and the amphibian, suggesting that enkephalins may play a unique role in embryogenesis. In order to characterize factors which regulate the onset and patterning of expression of this gene in adult and developing frog embryos, the proenkephalin A gene was cloned from Xenopus laevis. The clones have been characterized by DNA sequencing and restriction endonuclease mapping. The gene is made up of three exons which span approximately 12 kb. Exon I encodes the 5' untranslated region of the mRNA. Exon II contains the signal peptide and the N terminus of the mature protein. Biologically active opioid peptides are generated from exon III. Comparison to mammalian proenkephalin genomic sequence indicated that nucleotide sequences of the 5' flanking region, noncoding exon I and exon II were not well conserved but exon III was highly conserved. Primer extension and RNase protection assay analyses of the RNA transcripts revealed two major 5' ends. The putative TATA box, CAAT box, CRE and Pit 1 elements have been identified on this gene by sequence homology to published consensus sequences. To assay for sequences that could potentially regulate Xenopus proenkephalin expression, we transfected constructs that contained upstream genomic sequences linked to the CAT reporter gene into various eukaryotic cell lines. The expression of the fusion gene constructs were detected and could be induced 10- to 30-fold upon treatment with forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of Xenopus laevis proenkephalin gene. 172 92

Estrogen administration to male Xenopus causes the cytoplasmic destabilization of the hepatic serum protein coding mRNAs, most notably, albumin, yet has little effect on mRNAs encoding intracellular proteins such as ferritin. This report describes an estrogen-inducible ribonuclease activity found in liver polysomes that degrades albumin mRNA 4 times faster in vitro than it degrades ferritin mRNA. This differential rate of degradation was observed upon incubation of polysome extract with free liver RNA, isolated liver mRNPs, or transcripts from plasmid vectors. A cleavage fragment consisting of a doublet of approximately 194 nucleotides in length was consistently observed upon digestion of transcripts for the full length or 5' half of albumin mRNA. The generation of this cleavage fragment was used as an assay to study properties of the polysome nuclease activity. The 194 doublet is produced by the action of a Mg(2+)-independent endonuclease. This distinguishes the Xenopus liver enzyme from the enzymes that degrade histone or c-myc mRNA in vitro. It is inactivated by 400 mM NaCl or heating at 90 degrees C, but not by placental ribonuclease inhibitor or N-ethylmaleimide. Finally, the polysomal nuclease activity does not degrade double-stranded RNA. We believe the estrogen-induced nuclease activity contains an enzyme(s) that may mediate hormone-regulated changes in mRNA stability in this tissue.
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PMID:Estrogen-induced ribonuclease activity in Xenopus liver. 193 72

Two derivatives of pancreatic ribonuclease and endonuclease of Staphylococcus aureus, insolubilized on corn cob, have been used to reduce the percentage of nucleic acids in single cell protein (SCP) concentrates from yeasts. These derivatives are thermostable and active at 45 degrees C. At these temperatures the contamination by bacteria is negligible. The thermostability is remarkable, since the native nuclease is deactivated at above 39 degrees C. The hydrolysis of the nucleic acids in SCP is carried out first with the ribonuclease derivative followed by the endonuclease derivative. The catalytic activity of the insolubilized derivatives is similar to that of the native enzymes in the hydrolysis of RNA but not of DNA. The percentage of nucleic acids is reduced from 5-15 to 0.5%, with a loss of protein of 6%. These percentages are lower than those previously reported.
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PMID:New insolubilized derivatives of ribonuclease and endonuclease for elimination of nucleic acids in single cell protein concentrates. 196 85

RNase MRP is a site-specific endoribonuclease that processes primer RNA from the leading-strand origin of mammalian mitochondrial DNA replication. It is present in active form as isolated from the nucleus, suggesting a bipartite cellular location and function. The relatively high abundance of nucleus-localized RNase MRP has permitted its purification to near homogeneity and, in turn, has led to the identification of protein components of this ribonucleoprotein. Analysis of the mode of RNA cleavage by nuclear RNase MRP revealed the surprising and unprecedented ability of the endonuclease to process RNA at multiple discrete locations. Substrate cleavage is dependent on the presence of a previously described G-rich sequence element adjacent to the primary site of RNA processing. Downstream cleavage occur in a distance- and sequence-specific manner.
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PMID:Nuclear RNase MRP processes RNA at multiple discrete sites: interaction with an upstream G box is required for subsequent downstream cleavages. 206 76

A method is described for the rapid isolation of chromosomal deoxyribonucleic acid from species of the genus Mycoplasma. The method involves incubation of washed cells at elevated temperature in the presence of an ionic detergent, chelating agents, and proteinase K prior to the removal of residual protein and ribonucleic acid with ribonuclease and chloroform. It results in a good yield of high molecular weight material that is shown to be free of endogenous nuclease and substantially free of protein or ribonucleic acid contamination without the use of phenol. The isolated DNA is shown to be an excellent substrate for restriction endonuclease digestion and ligation with T4 DNA ligase.
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PMID:An improved method for the rapid isolation of chromosomal DNA from Mycoplasma spp. 218 71

Adrenodoxin reductase (ferrodoxin:NADP+ oxidoreductase, EC 1.18.1.2) is a flavoprotein mediating electron transport to all mitochondrial forms of cytochrome P450. We cloned the human adrenodoxin reductase gene and characterized it by restriction endonuclease mapping and DNA sequencing. The entire gene is approximately 12 kilobases long and consists of 12 exons. The first exon encodes the first 26 of the 32 amino acids of the signal peptide, and the second exon encodes the remainder of signal peptide and the apparent FAD binding site. The remaining 10 exons are clustered in a region of only 4.3 kilobases, separated from the first two exons by a large intron of about 5.6 kilobases. Two forms of human adrenodoxin reductase mRNA, differing by the presence or absence of 18 bases in the middle of the sequence, arise from alternate splicing at the 5' end of exon 7. This alternately spliced region is directly adjacent to the NADPH binding site, which is entirely contained in exon 6. The immediate 5' flanking region lacks TATA and CAAT boxes; however, this region is rich in G + C and contains six copies of the sequence GGGCGGG, resembling promoter sequences of "housekeeping" genes. RNase protection experiments show that transcription is initiated from multiple sites in the 5' flanking region, located about 21-91 base pairs upstream from the AUG translational initiation codon.
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PMID:Cloning and sequence of the human adrenodoxin reductase gene. 223 61

We present in this paper the structural analysis of two members of a small cellulase gene family, designated cel1 and cel2, from avocado. These genes were isolated by screening a lambda EMBL3 genomic library with a ripening-induced cellulase cDNA. Restriction endonuclease and Southern blot analyses showed that the cel1 gene is highly homologous to the cellulase cDNA and thus represents a ripening-related cellulase gene. The other cellulase gene, cel2, is closely related to cel1, but is divergent at its 5' end. The nucleotide sequence of a 5 kb region encompassing the cel1 gene was determined. Four previously characterized cellulase cDNAs from ripe fruit are identical to the eight exons of the cel1 gene. RNase protection and primer extension analyses were used to define the transcription start site of cel1 and to quantitate cel1 transcripts in ripening fruit. The cel1 mRNA was present at a low level in unripe fruit and increased 37-fold during ripening. Partial DNA sequence analysis of cel2 and comparison to the cel1 sequence revealed a high degree of similarity both at the DNA and deduced amino acid sequence levels. No characterized cellulase cDNAs derived from ripe fruit represent cel2 transcripts. These data suggest that the cel1 gene is responsible for a major portion, if not all, of the cellulase transcripts in ripe fruit. The DNA sequence of 1.4 kb of 5' flanking DNA of the cel1 gene was compared to the upstream sequence of other ethylene-regulated genes. Several interesting upstream sequence motifs were identified and are discussed.
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PMID:Isolation and characterization of a cellulase gene family member expressed during avocado fruit ripening. 225 45


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