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)

The gene for cytosolic phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) from the chicken was isolated from a recombinant library containing the chicken genome in phage lambda Charon 4A. The isolated clone, lambda PCK1cc, contains the complete gene for the enzyme as well as both 5' and 3' flanking sequences. The gene is approximately 8 kilobases in length divided into 8 exons, as demonstrated by restriction endonuclease mapping and DNA-RNA heteroduplex analysis. Southern blotting of chicken chromosomal DNA digested with various restriction enzymes shows a pattern predicted from the restriction map of lambda PCK1cc. The phosphoenolpyruvate carboxykinase gene is present as a single copy in the haploid chicken genome. The 5' region of the gene was defined by S1 nuclease mapping and by sequencing. Two mRNA species with discrete 5' ends were observed using S1 nuclease mapping. The ratio between the amounts of these multiple forms of mRNA is the same in chicken kidney and liver and is not affected by induction of the enzyme mRNA by cAMP. Examination of sequence homologies with the gene for rat cytosolic phosphoenolpyruvate carboxykinase indicates a putative control region contained in flanking sequences at the 5' end of the gene.
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PMID:The gene encoding the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the chicken. 609 81

The 5' terminus of Saccharomyces cereviasiae 35S pre rRNA was mapped on the rDNA using two methods: 1) Suitable restriction endonuclease fragments were hybridized to total high molecular weight RNA and extended with reverse transcriptase to the 5' end of the RNA template. 2) Other restriction fragments spanning the 5' terminus of 35S pre rRNA and radioactively labeled at their ends were hybridized to high molecular weight RNA and the non hybridized nucleic acids were digested with S1 nuclease. On the basis of these experiments, the 5' terminus of 35S pre rRNA was placed approximately 670 nucleotides upstream from the 17S rRNA coding region. The exact position was determined by reverse transcription as above, but in the presence of dideoxyribonucleoside triphosphates, which served as a way of sequencing the 5' terminal region. 35S pre rRNA synthesis is initiated at a site in EcoRI restriction fragment B which is 48 nucleotides upstream from the EcoRI cleavage site in the coding strand.
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PMID:The 5' terminus of the precursor ribosomal RNA of Saccharomyces cerevisiae. 615 79

Human thyroglobulin mRNA was isolated from Graves' goitres by size selection of total poly(A)-rich RNA in a sucrose gradient. It sedimented at 33 S, as in other mammalian species, and showed a single component of approximately 8500 bases by gel electrophoresis. cDNA was synthesized from the 33-S RNA by using reverse transcriptase in the presence of human placenta ribonuclease inhibitor and in conditions allowing the formation of long transcripts. The latter was made double-stranded using reverse transcriptase and blunt-ended with nuclease S1. After tailing with dCTP and terminal transferase, the double-stranded cDNA was annealed to pBR322 DNA that had been cleaved at the endonuclease PstI site and tailed with dGTP. The resulting plasmids were used to transform Escherichia coli C600 cells and four cloned recombinants were selected. Each plasmid DNA was shown to contain a sequence complementary to human thyroglobulin mRNA by hybridization with a labeled 33-S mRNA, visualization of cDNA . mRNA hybrids by electron microscopy and filter hybridization selection of mRNA directing the synthesis of immunologically related thyroglobulin peptides in the reticulocyte lysate. The four inserted DNA sequences were 1400 - 1800 base pairs long, two of them showing an homologous sequence of 1100 base pairs. Together, the four cloned DNA fragments represented 63% of the 8500 bases of human thyroglobulin mRNA.
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PMID:Cloning of four DNA fragments complementary to human thyroglobulin messenger RNA. 617 25

Synthesis of polyoma DNA in nuclei isolated from virus-infected 3T6 mouse fibroblasts leads to the selective labeling of replicative intermediates. Digestion of these replicative intermediates with the restriction endonuclease HpaII resulted in three highly labeled heterogeneous species in addition to the expected full-length fragments. These three species migrated more slowly in agarose than did any of the full-length restriction fragments and were shown to represent families of replication forks by criteria of sensitivity to S1 nuclease, kinetics of labeling both in vitro and in vivo, electron microscopy, and migration behavior during agarose gel electrophoresis. Subsequent digestion with other restriction enzymes showed that the two largest of the three fork bands originated from HpaII fragments 1 and 2. These fragments flank the putative terminus located 180 degrees relative to the origin. The third fork-containing band was less labeled and was derived from fragment 3, which is juxtaposed to the replication origin on the side corresponding to late transcription. A two-dimensional gel system revealed the presence of a fourth fork band, derived from fragment 4, that was obscured by full-length fragments 1 and 2 in the single-dimension electrophoresis. Resolution of the fork families revealed multiple discrete species within the major bands, implying the existence of stops or hesitations during replication of a given region of the genome. This conclusion is consistent with the presence of multiple species upon electrophoresis of the fork bands under denaturing conditions.
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PMID:Isolation and characterization of replication forks from discrete regions of the polyoma genome. 618 49

An assay was developed to investigate the fate of specific segments of beta-lactamase (bla) and ompA gene transcripts in Escherichia coli. DNA probes cloned in bacteriophage M13 were treated with an endonuclease capable of cleaving single-stranded DNA, the fragments produced were annealed with total cellular RNA, and the resulting RNA . DNA hybrids were subjected to S1 nuclease treatment and gel fractionation. By using this assay, direct evidence was obtained for 3'-to-5' directionality in the decay of the long-lived mRNA encoded by the ompA gene, and no preferential stability was observed for translated versus untranslated mRNA segments. In the case of bla mRNA, initial cleavage of the full-length transcript was rate limiting, and no decay intermediates were detected. No difference in degradation rate was seen for bla transcripts having variant 3' or 5' termini.
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PMID:Decay of mRNA in Escherichia coli: investigation of the fate of specific segments of transcripts. 618 1

Cultivation of E. coli cells in the presence of 5-bromodeoxyuridine (BUdR) leads to formation of lesions in the cellular DNA which affect its secondary structure, as reflected by changes in temperature profiles. Such DNA contains single-stranded regions susceptible to endonuclease S1. One of the major sources of the BU-induced lesions appears to be dehalogenation of incorporated 5-bromouracil (BU) residues, with accompanying formation of uracil. The presence of uracil residues in such DNA was demonstrated directly by chromatography of hydrolyzates, and by the susceptibility of such residues to uracil-DNA glycosylase. The number of uracil residues was dependent on the extent of damage in the DNA, and decreased during the DNA repair that accompanied reactivation of bromouracil-inactivated cells. Dehalogenation of incorporated BU presumably results in formation of apyrimidinic sites by uracil-DNA glycosylase, and then single-strand nicks either by AP-endonuclease and/or dehalogenation. The findings are relevant to the mechanism of BU-induced mutagenesis.
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PMID:Identification of uracil as a major lesion in E. coli DNA following the incorporation of 5-bromouracil, and some of the accompanying effects. 618 38

The K562 human leukemia cell is an erythroid-like cell that may serve as a model for the study of globin gene expression in transcriptionally active human erythroid cells. K562 cells express all globin genes with the exception of that for beta-globin; failure to produce beta-globin could result from an acquired mutation in each of the beta-globin genes or from an alteration in the regulatory factor environment of the beta-globin gene. To uncover a possible acquired mutation, restriction endonuclease analysis of genomic K562 DNA and expression studies of a cloned K562 beta-globin gene were carried out. Restriction endonuclease analysis revealed no structural alteration of the K562 beta-globin genes. Analysis of the polymorphic Ava II site in intervening sequence 2 of the beta-globin gene showed that K562 cells contain two different beta-globin alleles, both of which are inactive. A K562 beta-globin gene was cloned, ligated into the expression vector pLTN3B, and introduced into COS cells. Transcripts were analyzed by RNA blot, dot blot, S1 nuclease mapping, and primer extension assay. The cloned K562 beta-globin gene was transcribed in COS cells as efficiently as a normal beta-globin gene introduced into COS cells; the mRNA was 10 S and polyadenylylated; the 5' and 3' termini and the processing of transcripts were identical to that of mRNA transcribed from a normal gene. Based on these data we suggest that the absence of beta-globin gene expression results not from an alteration in the beta-globin gene, but from a quantitative or qualitative alteration in a trans-acting factor important in beta-globin gene expression.
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PMID:A beta-globin gene, inactive in the K562 leukemic cell, functions normally in a heterologous expression system. 620 98

The structure of human adenosine deaminase mRNA from normal and mutant lymphoblasts was examined by sequence analysis of a cDNA for normal mRNA and electrophoretic analyses of DNA fragments generated by S1 endonuclease cleavage of mRNA-cDNA hybrids. The 1,533-base sequence of the cloned cDNA represents the complete mRNA sequence with the possible exception of some of the 5' untranslated region. S1 nuclease analyses of hybrids between cloned cDNA and normal adenosine deaminase mRNA confirmed that a 76-base sequence in a previously examined adenosine deaminase cDNA is an intron. S1 nuclease analyses of mRNAs from seven mutant cell lines demonstrated that four of the mutants, those in the GM-2471, GM-2756, GM-4258, and GM-2606 cells, contain small defects, such as single-base changes, that are not detectable by the S1 nuclease technique. Three of the mRNAs, those in GM-3043, GM-2294, and GM-2825A cells, do contain defects detectable with S1 nuclease. These defects differ from each other and have been mapped to specific regions of the mRNA. Some or all of these defective mRNAs are postulated to result from anomalous RNA processing.
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PMID:Structure of adenosine deaminase mRNAs from normal and adenosine deaminase-deficient human cell lines. 620 79

Bacteriophage T7 DNA reacts uniformly with trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene(anti-BPDE). The reaction product retains the native configuration so that only one site sensitive to S1 nuclease is produced for every 70 anti-BPDE adducts. DNA treated with anti-BPDE is retained on benzoylated naphthoylated DEAE-cellulose even after washing with 1.0 M salt solutions. About 100 adducts per T7 molecule are required for adherence which is not due to breaks or single-stranded regions since adherence is not affected by S1 nuclease treatment. The binding of anti-BPDE reacted DNA to benzoylated naphthoylated DEAE-cellulose is cooperative and requires many residues per bound fragment. Treatment of T7 DNA treated with anti-BPDE with restriction endonuclease yields smaller molecules, still containing adducts, which do not adhere. We interpret these results to mean that reaction with BPDE does not involve deformation of the DNA structure and that the adducts lie in a position which they are readily accessible for interaction with aromatic groups on the column resin.
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PMID:Reaction of T7 DNA with a polycyclic aromatic hydrocarbon. Lack of structural perturbation. 624 97

Cleavage of rat liver nuclear DNA with EcolR1 restriction endonuclease yields 14 discrete fragments ranging from 2300 to 93 base pairs in length, representing approx. 10.5% of the rat genome. Fragments of 1500, 180, and 93 base pairs are reiterated over 100 000 times; fragments of 2300, 880, 290, and 200 base pairs are reiterated over 20 000 times; the remaining fragments are present in over 1000 copies per genome. When compared to whole rate DNA, 11 were 1-5% richer in A . T base pairs and five were 1.5-2.5 times more methylated. From the criteria of the banding patterns in complete and incomplete digests, base composition and extent of methylation, none of these fragments appeared to be generated as oligomers of a basic shorter repeat. The reassociation of EcoR1 fragments was monitored on hydroxyapatite and by S1 nuclease treatment in order to assess band reiteration frequency and the possibility of interpersion or short internal repeats. The renaturation of the four smallest EcoR1 fragments gave no indication of short internal repeats from hyperpolymer formation nor interpersion with lower frequency sequences by size reduction after S1 nuclease treatment. Anomalous renaturation of several large fragments was observed, possibly due to internal repeats.
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PMID:Analysis of highly repeated DNA sequences of rat with EcoR1 endonuclease. 624 98


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