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 plasmid pO61 that was isolated from an E. coli genomic DNA library and codes for O6-alkylguanine (O6AG) DNA alkyltransferase (ATase) activity (1) has been further characterised. Subclones of the 9 Kb insert of pO61 showed that the ATase activity was encoded in a 2Kb Pst1 fragment but a partial restriction endonuclease map of this was different to that of the E. coli ada gene that codes for O6-AG and alkylphosphotriester dual ATase protein. Fluorographic analyses confirmed that the molecular weight of the pO61-encoded ATase was 19KDa i.e. similar to that of the O6AG ATase function that is cleaved from the 39KDa ada protein but rabbit polyclonal antibodies to the latter reacted only very weakly with the pO61-encoded protein. A different set of hybridisation signals was produced when E. coli DNA, which had been digested with a variety of restriction endonucleases was probed with 2Kb Pst 1 fragment or the ada gene. These results provided evidence for the existence of a second ATase gene in E. coli. The 2Kb Pst-1 fragment of pO61 was therefore sequenced and an open reading frame (ORF) that would give rise to a 19KDa protein was identified. The derived amino acid sequence of this showed a 93 residue region with 49% homology with the O6AG ATase region of the ada protein and had a pentamer and a heptamer of identical sequence separated by 34 amino acids in both proteins. The pentamer included the alkyl accepting cysteine residue of the ada O6AG ATase. The hydrophobic domains were similarly distributed in both proteins. Shine-Dalgarno, -10 and -35 sequences were identified and the origin of transcription was located by primer extension and S1 nuclease mapping. The amino-terminal amino acid sequence of the protein was as predicted from the ORF.
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PMID:Characterisation and nucleotide sequence of ogt, the O6-alkylguanine-DNA-alkyltransferase gene of E. coli. 282 31

We have used enzymic and chemical probes to search for altered DNA conformations in the 5' flanking region of the gene for a high mobility group protein (HMG-T) from trout. This search was conducted in order to identify potential genetic elements that might be involved in the transcriptional control of the HMG-T gene. We identified, in supercoiled plasmid DNA molecules containing a 900 base pair insert of the 5' region of the gene, an S1-sensitive site situated within an (AT)12 sequence approximately 120 base pairs upstream from the start of the HMG-T gene. Chemical modification of supercoiled DNA with the single-strand-selective reagent bromoacetaldehyde was limited to a region coincident with the S1 nuclease site. T7 endonuclease I, a probe highly specific for four-way helical junctions, cleaved predominantly at the boundaries of the (AT)12 stretch. These data are most consistent with the interpretation that the (AT)12 sequence adopts a cruciform structure when torsionally stressed by negative supercoiling. DNase I footprinting analyses demonstrated that HMG-T protects two regions almost equidistant from the center of the (AT)12 sequence, indicating that HMG-T is a sequence-specific DNA binding protein.
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PMID:Induction by torsional stress of an altered DNA conformation 5' upstream of the gene for a high mobility group protein from trout and specific binding to flanking sequences by the gene product HMG-T. 283 70

The capacities of four synthetic sequences containing runs of perfectly alternating purine-pyrimidine base pairs (bp) to adopt left-handed structures were evaluated in a homologous family of recombinant plasmids. All the sequences had the same G+C content (50%) and consisted of simple tetranucleotide repeat units but differed in the relative orientations of these units. For some of the sequences, several alternate secondary structures were theoretically possible; a variety of probes (S1 nuclease, bromoacetaldehyde, OsO4, T7 gene 3 endonuclease, supercoil-induced gel relaxation studies) under a wide range of reaction conditions was used to determine which structures were adopted as a function of superhelical stress. The precise positions at the bp level of reactions with these chemical and enzymatic probes were determined. We conclude that for short (20-24 bp) sequences containing runs of alternating (T-G) and (C-A), the cruciform state is preferred over the similarly allowable left-handed form provided that symmetry constraints allow. However, these sequences can be induced to form a left-handed helix under appropriate conditions. This is the first demonstration of plasmid inserts which will adopt more than one unusual DNA structure in response to negative superhelical stress. The structural properties of a molecule containing a Z-Z junction were studied, and we conclude that the disruption caused by this feature extends over only a few bp although it requires a high energetic penalty.
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PMID:The role of DNA sequence in the formation of Z-DNA versus cruciforms in plasmids. 283 74

Irradiation of DNA in situ i.e. in phage particles or in the cell leads to alterations of single DNA nucleotides as well as to clustered lesions such as double strand breaks or unpaired DNA regions the latter being sensitive to digestion by S1 nuclease. A contribution will be made to the configuration of such S1-nuclease-sensitive sites (S1 sites). DNA from irradiated lambda phage containing S1 sites was treated with gamma endonuclease from M. luteus which is known to split the nucleotide strand at the position of oxidized pyrimidine base. It was found that the gamma endonuclease induces double-strand breaks at some of the S1 sites indicating double base damage within this site. However, half of the S1 sites are not converted into a double-strand break by the gamma endonuclease, indicating base damage only on one strand within the unpaired region.
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PMID:Action of gamma endonuclease on clustered lesions in irradiated DNA. 283 62

DNA sequences of the X-chromosome-linked hypoxanthine phosphoribosyltransferase (HPRT) and glucose 6-phosphate dehydrogenase (G6PD) genes have revealed the presence of clusters of CpG dinucleotides, raising the possibility that such clusters are involved in the control of expression of these genes, which are expressed in all tissues. Although CpG clusters are not exclusive features of the X chromosome, the analysis of X-linked genes provides the means to determine whether CpG clusters are control elements; one of the two homologous X loci in female mammals is not expressed, so that active and inactive versions of the gene can be compared. In fact, it has been shown that these CpG clusters are undermethylated when the gene is active and extensively methylated when the gene is inactive. In addition to hypomethylation, chromatin hypersensitivity to endonuclease digestion is a known hallmark of regulatory sequences in eukaryotic genes. We report here that the CpG clusters of the active hprt and g6pd genes are not only undermethylated, but also hypersensitive to MspI, DNase I and S1 nuclease, further supporting the suggestion that they are involved in the control of expression of these genes.
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PMID:Clusters of CpG dinucleotides implicated by nuclease hypersensitivity as control elements of housekeeping genes. 298 78

DNA coding for ribosomal RNA in Podospora anserina has been cloned and was found as a tandemly repeated 8.3 kb sequence. The cloned rDNA was characterized by restriction endonuclease mapping. The location of 5.8S, 18S and 28S rRNA coding regions was established by DNA-RNA hybridization and S1 nuclease mapping. The organization of P. anserina rRNA genes is similar to that of Neurospora crassa and Aspergillus nidulans. The rDNA unit does not contain the sequence coding for 5S RNA.
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PMID:Cloning and characterization of the rDNA repeat unit of Podospora anserina. 298 47

We have examined a cDNA displacement synthesis procedure in which the extent of precursor incorporation and the unusual kinetics of displacement synthesis suggest a unique replicative form of DNA and the occurrence of multiple rounds of displacement synthesis, leading to amplification of mRNA sequences. Globin double-stranded DNA containing a hairpin loop was extended by the addition of a homopolymer to the 3' end. This was followed by displacement synthesis with the Klenow fragment of DNA polymerase I that was primed by an oligonucleotide hybridized to the homopolymer. Thus, the hairpin cDNA was copied to form an open duplex with an inverted repetition of globin sequences. These molecules can then serve as templates for additional synthesis which would be primed from oligomers bound the homopolymer. Globin cDNA sequences appear to be amplified 10-fold or more by this procedure. Globin cDNA obtained by displacement synthesis was similar in size to the original template. However, displaced molecules associate to the extent that they are not readily resolved by electrophoresis or sedimentation under nondenaturing conditions. Restriction endonuclease digests of 32P-labeled displaced strands gave fragment patterns similar to rabbit globin cDNA hairpin molecules. S1 nuclease studies demonstrated that displaced complexes and replication intermediates are partially single stranded, which might account for their aggregation properties.
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PMID:Displacement synthesis of globin complementary DNA: evidence for sequence amplification. 298 27

We have used single strand specific nucleases to map DNA distortion in the adult chicken beta A-globin gene. We have detected two structures of that kind and have mapped nuclease-cutting sites at one base resolution. One prominent site is centered at -190 relative to the RNA capping site and is positioned at the center of a stretch of contiguous C residues. The second site is near the first intron/exon junction (+620) and appears as a series of discrete 1-base-long enzyme-cutting sites. Based upon the pattern of nuclease cutting and the kinetics of nuclease cutting we conclude that the "poly(C)" stretch may assume a looped geometry in supertwisted DNA molecules which is similar to that proposed by Felsenfeld (Nickol, J. M., and Felsenfeld, G. (1983) Cell 35, 467-477). We show that S1 nuclease cuts within the intron occur mainly at the end points of polypurine segments and suggest that such end points may assume a distorted transitional geometry. We find that Neurospora crassa endonuclease cuts both the promotor and intron sites in linear DNA molecules but that in linear DNA the cutting process is limited by a first order conformation change of the DNA substrate. Based upon those kinetics we propose that in unstressed DNA, each of the two sites can convert between a distorted and undistorted geometry. In the enzyme assay buffer at 37 degrees C, the time constant for the equilibrium is nearly 10 h for the promotor site and 7 h for the intron.
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PMID:An equilibrium between distorted and undistorted DNA in the adult chicken beta A-globin gene. 298 80

A nuclease S1 mapping procedure was used to identify sites accessible to nucleases in the 3'-noncoding region of the rabbit globin mRNAs. A complex structure was evident in the alpha-globin species, with one highly accessible single-stranded site, large portions in an accessible double-stranded configuration, and a portion not accessible to any of the nucleases. In the beta-globin mRNA, the region was more uniformly accessible to RNase T1 and to a cobra venom enzyme specific for double-stranded RNA, but it had only a single site highly accessible to a bulkier Neurospora endonuclease. The patterns of cleavage were nearly identical in the deproteinized mRNAs and in the mRNAs associated with polyribosomes in reticulocyte extracts. In both species, a zone of secondary structure occurred around the poly(A) junction. In each species, virtually all the molecules had a poly(A) sequence of at least 20-25 AMP residues. A periodicity in poly(A) size distribution was observed. These results indicate that the beginning of this sequence is well protected against degradation inside the cell and that zones of partial protection occur at measured intervals. In crude extracts, where the poly(A) is covered with proteins, this sequence was protected against nuclease digestion.
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PMID:Structural features in the 3'-terminal region of polyribosome-bound rabbit globin messenger RNAs. 300 Oct 55

Accessible sites in the 5' noncoding region of the rabbit alpha- and beta-globin mRNAs were identified and compared in deproteinized RNA and in the mRNAs engaged in translation in the reticulocyte lysate. Preparations of RNA and lysate were subjected to limited nuclease digestion by RNase T1 and Neurospora endonuclease, and the cleavage sites were analyzed by a nuclease S1 mapping procedure. The free alpha-globin mRNA contained few nuclease-sensitive sites and its initiation codon AUG was masked. The free beta-globin mRNA contained a larger number of accessible sites and its AUG was highly exposed. The distribution of sensitive sites differed considerably in the lysate. In both mRNA species, a site near the 5' terminus became the one most accessible to Neurospora endonuclease. Also the accessibility of the AUG in beta-globin mRNA decreased considerably. The distribution of accessible sites in the lysate was the same when the mRNAs were undergoing rapid initiation and when initiation became limited after prolonged incubation. Inhibition of initiation by the cap analogue 7-methylguanosine 5'-triphosphate was accompanied by increased sensitivity of some of the sites in both mRNA species. One of the accessible sites in each mRNA species had a sequence complementary to the 3'-terminal portion of the 18S ribosomal RNA.
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PMID:Structural features of the 5' noncoding region of the rabbit globin messenger RNAs engaged in translation. 300 32


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