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

---

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

A chimeric plasmid has been constructed in vitro from colicin E1 factor (Col E1), nontransmissible R-factor RSF-1010, and Drosophila melanogaster DNAs by the sequential action of Escherichia coli endonuclease RI(Eco RI) and T4 phage DNA ligase. The chimeric plasmid was assembled in two stages--first, a composite plasmid consisting of Col E1 and RSF 1010 was constructed, followed by partial digestion of the composite with Eco RI (in order to open one of the susceptible cleavage sites) and ligation with an Eco RI-digested D. melanogaster DNA preparation. The chimeric plasmid was selected and amplified in vivo by sequential transformation of E. COLI C with the ligated mixture, selection of transformants in medium containing streptomycin plus colicin E1, followed by amplification in the presence of chloramphenicol and purification of the extracted plasmid by dye-buoyant density gradient centrifugation in ethidium bromide-CsCl solution. Treatment of the chimeric plasmid with Eco RI yields three fragments with mobilities corresponding to the linear forms of the constituents--COL E1, mol wt 4.2 times 106, RSF 1010, mol wt 5.5 times 106 and D. melanogaster DNA, mol wt 4.0 times 106. The buoyant densities of the three constituents are respectively 1.706, 1.719, and 1.697 g/cm3, while the buoyant density of the composite factor is 1.712 and that of the chimeric plasmid is 1.705. Serratia marscesens endonuclease R (Sma) which introduces a single cut in Col E1, but not in RSF 1010, converts the chimeric plasmid to a single linear molecule (mol wt 13.7 times 106) and sequential digestion with both Sma and Hin III yields two distinct fragments, mol wt 3.7 and 10.0 times 10.6, respectively; this implies that the two sites are unique and occur at distinctly different positions. Sequential digestion with both Hin III and Eco RI reveals that the Hin III cut is in the D. melanogaster segment; neither Col E1 nor RSF 1010 contain sites susceptible to digestion with Hin III. In the presence of chloramphenicol, the chimeric plasmid continues toreplicate for 9 hr while bacterial chromosomal DNA replicates at a much slower rate. As in the case of the composite plasmid, continued synthesis is the presence of chloramphenicol suggests that the replicator of Col E1 is functional in the chimeric plasmid as well. Examination of the chimeric plasmid by partial denaturation mapping permits identification of its constituents, each of which presents a characteristic profile. The D. melanogaster segment reveals a wealth of detail at the molecular level pertaining to the distribution of AT-rich regions.
...
PMID:Construction and characterization of a chimeric plasmid composed of DNA Pfrom Escherichia coli and Drosophila melanogaster. 80 34

The joining of duplex DNA at base-paired ends by bacteriophage T4 DNA ligase was confirmed using either a synthetic duplex decamer or restriction endonuclease fragments of ColE1 DNA as substrates. The reaction was not linearly dependent on enzyme concentration but increased markedly at high enzyme concentrations. Although T4 RNA ligase did not catalyze this blunt end joining, it makedly stimulated the DNA ligase reaction particularly at low DNA ligase concentrations. The apparent Km for the decamer was 50 micronM in the presence or absence of RNA ligase. In the presence of RNA ligase, T4 DNA ligase had about the same turnover number for blunt end and cohesive end joining. The joining of duplex DNA at base-paired ends was proven by several techniques including restriction endonuclease cleavage of the products. The products of the ligation reaction using restriction enzyme fragments were mostly linear oligomers but included some circular duplexes. Escherichia coli DNA ligase in the presence or absence of RNA ligase did not catalyze blunt end joining. RNA ligase only moderately affected the joining of cohesive ends by T4 DNA ligase or E. coli DNA ligase and did not itself catalyze this reaction.
...
PMID:Interaction of bacteriophage T4 RNA and DNA ligases in joining of duplex DNA at base-paired ends. 86 10

An endonuclease (AP-endonuclease II) that specifically attacks double stranded or single stranded depurinated DNA, resulting in single-strand nicks, has been purified 320-fold from Micrococcus luteus. The enzyme is not stimulated by 0.002 M MgCl2, it induces 3'OH-5'PO4 breaks on the 5' side of apurinic sites, it has no activity towards UV-irradiated DNA and has a molecular weight of about 30 000. In cooperation with DNA-polymerase from M. luteus and T4 DNA ligase, AP-endonuclease II has been shown capable of carrying out complete excision repair of depurinated DNA in vitro.
...
PMID:[Isolation and properties of Micrococcus luteus endonucleas acting in DNA depurinization but inactive with pyrimidine dimers]. 88 77

We have studied excision-repair of UV-irradiated phiX174 RFI DNA in vitro with UV-specific endonuclease from Micrococcus luteus (UV-endo), DNA polymerase I from Escherichia coli and DNA ligase from phage T4 infected E. coli. Excision-repair was measured a) by physico-chemical methods, i.e. by determination of the conversion of RF I DNA into RF II DNA by UV-endo and by the subsequent conversion of RF II DNA ligase, b) by biological methods i. e. by measuring the ability of the reaction product to form phages upon incubation with spheroplasts from the appropriate strains of E. coli. Using the first method, we have shown, that more than 90% of the pyrimidine dimers can be repaired in vitro; with the latter method we have shown, that the molecules which are repaired as defined by method a) have regained full biological activity. Exonuclease III was found to be not essential for excision-repair in vitro and also did not stimulate repair. From this result we conclude that UV-endo generates 3'OH endgroups, in agreement with results obtained by Hamilton et al. (1974). The usefulness of the method presented in this paper with regard to the study of excision-repair is discussed.
...
PMID:Physico-chemical and biological study of excision-repair of UV--irradiated phiX174 RF DNA in vitro. 105 35

A composite plasmid has been constructed in vitro from colicin E1 factor (mass of 4.2 megadaltons [Md]) and nontransmissible resistance factor RSF 1010 (mass, 5.5. Md) deoxyribonucleic acids (DNAs) by the sequential action of Escherichia coli endonuclease (RI (Eco RI) and T4 phage DNA ligase on the covalently closed circular forms of the constituents. The composite plasmid was selected and amplified in vivo by sequential transformation of E. coli C600 with the ligated mixture and selection of transformants in medium containing streptomycin plus colicin E1, followed by amplification in the presence of chloramphenicol and purification of the extracted plasmid by dye-buoyant density gradient centrifugation in ethidium bromide-cesium chloride solution. Treatment of the composite plasmid with Eco RI yielded two fragments with mobilities corresponding to the linear forms of the parental plasmids, whereas Serratia marscesens endonuclease R (SmaR), which introduces a single scission in the colicin E1 factor but not in RSF 1010, convErted the composite plasmid to a single linear molecule (mass, 9.7 Md). Sequential degradation of colicin E1 factor with Sma R and Eco RI produced two fragments with masses of 3.5 and 0.7 Md; sequential degradation of RSF 1010 produced only one fragment (due to the cleavage with Eco RI), and sequential degradation of the composite plasmid produced the expected three fragments--an RSF 1010 Eco RI linear and the two expected products from the colicin E1 factor moiety. The composite plasmid conferred on the host cell resistance to streptomycin, sulfonamides, and colicin E1, but colicin E1 itself was not synthesized. In contrast, colicin E1 was synthesized by cells containing simultaneously both colicin E1 factor and RSF 1010 as separate entities. In the presence of chloramphenicol, the composite plasmid continued to replicate for 6 h. whereas replication of RSF 1010 and chromosomal DNA stopped within 2 h. Continued replication in the presence of chloramphenicol suggests that the replicator of the colicin E1 factor is functional in the composite plasmid.
...
PMID:Construction of a colicin E1-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid. 109 May 74

Plasmid lambdadv1, which is in a dimeric form, was converted to a linear monomer duplex by the action of EcoRI restriction endonuclease that incises at a unique site in this plasmid genome. The resulting products were then joined by Escherichia coli DNA ligase to produce molecules with various oligomeric forms, and from these monomeric, dimeric, or trimeric circular molecules were purified. By transformation of cells with these DNAs, clones were obtained that carried lambdadv1 in a monomeric or dimeric form. The former type of clones have not been generated in vivo, except for one in a different host strain, and carriers of timeric or tetrameric lambdadv1's have not been obtained so far. It was observed that a considerable fraction of these oligomeric circular DNAs were converted to lower oligomers (e.g., from trimer to dimer) during transformation. The characteristics of the monomeric lambdadv1 carriers obtained were compared with those of dimeric lambdadv1 carriers. The stabilities of the plasmids of the two forms were the same. However, the monomeric plasmid carriers were less tolerant to lambdavir phage infection and perpetuated about 30% less plasmid genomes in monomer units. Furthermore, dimeric plasmid carriers appeared spontaneously and accumulated in cultures of the monomeric lambdadv1 carriers.
...
PMID:In vitro construction of different oligomeric forms of lambdadv DNA and studies of their transforming activities. 109 30

DNA from lambdagt-lambdaB bacteriophage was cleaved with EcoRI endonuclease and fragments from EcoRI-digested E. coli DNA were inserted. This DNA was used to infect E. coli, and phages containing the gene for DNA ligase were isolated by genetic selection. Two different hybrids were found with the same E. coli segment inserted in opposite orientations. Both hybrids produced similar levels of ligase as measured in crude extracts of infected cells.
...
PMID:In vitro construction of bacteriophage lambda carrying segments of the Escherichia coli chromosome: selection of hybrids containing the gene for DNA ligase. 110 46

A soluble extract prepared from T7-infected E. coli is able to initiate DNA synthesis on an exogenous T7 DNA template. We have developed a fractionation procedure to resolve and identify the proteins required for T7 DNA synthesis. By this method we have purified the following T7 replication-related proteins (each greater than 50% pure as judged by sodium dodecyl sulfate gel electrophoresis): T7 DNA-binding protein (27,000 daltons), T7 RNA polymerase (105,000 daltons), T7 DNA polymerase (gene 5-protein, 85,000 daltons, plus host-factor), T7 DNA ligase (40,000 daltons), and T7 DNA-priming protein (65,000 daltons). The T7 DNA-priming protein, synthesized between 7.5 and 15 min following infection, was not detectable if the infecting phage carried an amber mutation in gene 4. Using an in vitro complementation assay which specifically measures the stimulation of DNA synthesis in an extract prepared from T7 gene 4-mutant infected cells, we have purified the DNA-priming protein about 2,000-fold. The purified priming protein preparations are essentially free of endonuclease, exonuclease, DNA ligase and DNA polymerase activity, but they do contain measurable DNA-dependent RNA synthetic acitvity. The enzyme is rapidly inactivated by heating to 46 degrees C and by treatment with N-ethylmalemide. In the presence of T7 DNA-binding protein and all four ribonucleoside triphosphates, the DNA-priming protein enables T7 DNA polymerase to initiate DNA synthesis on intact duplex T7 DNA. Closer studies of its enzymatic function as well as of the possible roles of the other proteins in the T7 replication system will be presented in the accompanying paper.
...
PMID:Studies on bacteriophage T7 DNA synthesis in vitro. I. Resolution of the T7 replication system into its components. 110 17

In Saccharomyces cerevisiae, the genes encoding the HO endonuclease, G1-specific cyclins CLN1 and CLN2, as well as most proteins involved in DNA synthesis, are periodically transcribed with maximal levels reached in late G1. For HO and the DNA replication genes, cell cycle stage-specific expression has been shown to be dependent on the Cdc28 kinase and passage through START. Here, we show that cells released from cdc28ts arrest in the presence of cycloheximide show wild-type levels of induction for HO, CLN1, and CDC9 (DNA ligase). Induction is gradual with a significant lag not seen in untreated cells where transcript levels fluctuate coordinately with the cell cycle. This lag may be due, at least in part, to association of the Cdc28 peptide with G1 cyclins to form an active kinase complex because overexpression of CLN2 prior to release in cycloheximide increases the rate of induction for CDC9 and HO. Consistent with this, release from pheromone arrest (where CLN1 and CLN2 are not expressed) in cycloheximide shows no induction at all. Transcriptional activation of CDC9 is likely to be mediated through a conserved promoter element also present in genes for other DNA synthesis enzymes similarly cell cycle regulated. The element contains an intact MluI restriction enzyme recognition site (consensus approximately 5'-A/TPuACGCGTNA/T-3'). Insertion of a 20-bp fragment from the CDC9 promoter (containing a MluI element) upstream of LacZ confers both periodic expression and transcriptional induction in cycloheximide following release from cdc28ts arrest. High levels of induction depended on both the MluI element and CDC28. These results suggest that the activity of trans-acting factors that operate through the MluI element may be governed by phosphorylation by the Cdc28 kinase.
...
PMID:Direct induction of G1-specific transcripts following reactivation of the Cdc28 kinase in the absence of de novo protein synthesis. 131 70

The size of the repair patch produced by E. coli DNA polymerase (Pol I) following the removal of a pyrimidine dimer from DNA in response to the nicking activity of T4 endonuclease (T4 endo V) was determined. A 48-bp DNA containing a pyrimidine dimer at a defined location was labelled in the damaged strand and incubated with T4 endo V and E. coli endonuclease IV. Subsequently, DNA synthesis by DNA Pol I was carried out in the presence of four dNTPs, ATP and DNA ligase. Analysis of the reaction products on a sequencing gel revealed a ladder of only 4-oligonucleotides, 1-4 nucleotides greater in length than the fragment generated by the combined nicking activities of T4 endo V and E. coli endonuclease IV. Thus we conclude that the in vitro repair patch size of T4 endo V is 4 nucleotides and that in some cases the repaired DNA is not ligated.
...
PMID:In vitro characterization of repair synthesis initiated by T4 endonuclease V on a synthetic DNA substrate. 151 8


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---