Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:6.5.1.2 (DNA ligase)
 2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bacillus circulans NRRL B-3312, a nonpathogenic bacterium that produces the aminoglycoside antibiotic butirosin, is known to contain an aminoglycoside phosphotransferase that is similar to the neomycin phosphotransferases of clinically isolated antibiotic-resistant bacteria. Purified DNAs from B. circulans and the plasmid ColE1-ApR were digested with EcoRI endonuclease and the resulting fragments covalently joined with polynucleotide ligase. The recombined DNA was used to transform E. coli and ampicillin-neomycin resistant colonies were selected. Analysis of several clones indicated that neomycin resistance in the E. coli transformants was due to the presence of the B. circulans phosphotransferase gene. This observation is consistent with the notion that anitbiotic-modifying enzymes from antibiotic-producing organisms may be the sources of antibiotic resistance in plasmid-containing bacteria.
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PMID:Aminoglycoside-modifying enzyme of an antibiotic-producing bacterium acts as a determinant of antibiotic resistance in Escherichia coli. 32 54

Plasmids carrying various portions of colicin E1 plasmid (ColE1) DNA have been isolated in an attempt to determine the regions of ColE1 DNA which are required for maintenance of the plasmid in bacteria. To construct the plasmids, the DNA of a ColE1 derivative that contains a gene which controls ampicillin resistance was cleaved by the restriction endonuclease HaeII. The digestion products were joined by T4 DNA ligase and then used to transform bacteria to ampicillin resistance. The plasmid derivatives obtained in this way were always composed of certain HaeII segments. These contain approximately 10% of the ColE1 genome and include the origin of replication of ColE1. We presume that the region of ColE1 which is common to all these derivatives is required for maintenance of the plasmid. After a description of these results, the nucleotide sequence of this region is presented, and possible roles of the region in plasmid replication and maintenance are discussed.
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PMID:Nucleotide sequence of the region required for maintenance of colicin E1 plasmid. 39 52

A composite plasmid (pAT2010) has been constructed in vitro from RSF2124 and Bacillus subtilis IFO3022 plasmid (pAT1060) by covalent joining of the two DNA molecules by means of Escherichia coli DNA ligase through the cohesive ends generated by restriction endonuclease RI (EcoRI) cleavage. The composite plasmid was selected by transformation of E. coliC600r-m- with the ligated mixture after enrichment for composite plasmid by preparative agarose gel electrophoresis, and plating of the transformants on a medium containing ampicillin and colicin E1. Treatment of the composite plasmid with EcoRI yielded two fragments corresponding to the linear forms of the parental plasmids. The composite plasmids replicated as biologically functionally units in E. coli, and expressed genetic information carried by RSF2124. In the presence of chloramphenicol, the composite plasmids continued to replicate and the copy number gradually increased. Such nature of replication in the presence of chloramphenicol is characteristic to RSF2124 derived from colicin E1 factor, and so it is suggested that the replicator of RSF2124 is functional in the composite plasmid. The composite plasmid was found to synthesize mRNA of B. subtilis plasmid in cell-free extracts of E. Coli, by hybridization of the mRNA to the original plasmid DNA of pAT1060.
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PMID:Molecular cloning and in vitro transcription of Bacillus subtilis plasmid in Escherichia coli. 41 72

The highly specific restriction endonucleases Sa1I and BamI produce DNA fragments with complementary, cohesive termini that can be covalently joined by DNA ligase. The Escherichia coli kanamycin resistance factor pML21 has one SalI site, at which DNA can be inserted without interfering with the expression of drug resistance or replication of the plasmid. A more convenient cloning vehicle can be made with the tetracycline resistance factor pSC101, since insertion of DNA either at its single site for Sa1I or at that for BamI inactivates plasmid-specified drug resistance but not replication. To take advantage of this insertional inactivation, pSC101 was joined to a Co1E1-ampicillin resistance plasmid having no Sa1I site, and to a Co1E1-kanamycin resistance plasmid having no BamI site. Chimeras formed with the resulting hybrid vehicles can be identified simply by replica plating. These three vehicles, which all replicate under relaxed control, have been used to clone and amplify Drosophila melanogaster DNA fragments.
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PMID:Molecular cloning of DNA fragments produced by restriction endonucleases Sa1I and BamI. 81 38

The essentiality of the vaccinia virus DNA ligase gene, SalF 15R, for virus growth was tested by insertional mutagenesis. A plasmid containing E. coli gpt inserted within a large deletion in the DNA ligase gene was transfected into vaccinia virus-infected cells and recombinant viruses selected by three cycles of plaque purification in the presence of mycophenolic acid (MPA). Surprisingly, in some isolates, which replicated in a manner indistinguishable from wild type (WT) virus, the WT gene was replaced by the gpt allele, demonstrating that the DNA ligase gene is nonessential for growth in cultured cells. In other isolates the entire plasmid was integrated into the virus genome by a single crossover event and a functional copy of the DNA ligase was retained. Southern blot analyses of the latter, drug-resistant viruses indicated extra DNA fragments, of sizes inconsistent with predicted viral structures, which represent the plasmid products of homologous recombination. Hirt extracts from cells infected with such multiply plaque purified virus isolates yielded plasmids that produced ampicillin-resistant colonies after transformation of E. coli. These plasmids were of two structures, representing either the original plasmid used for transfection, or a plasmid containing the WT ligase gene rescued by recombination with the virus genome. Similarly, insertional mutagenesis of the vaccinia virus thymidine kinase (TK) gene with gpt yielded plasmids containing mutant or wild type TK alleles when recombinant viruses were selected in MPA. Such plasmids were not isolated when TK minus viruses were selected in 5-bromodeoxyuridine (BUdR).
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PMID:Vaccinia virus DNA ligase is nonessential for virus replication: recovery of plasmids from virus-infected cells. 198 87

A novel plasmid vector pSELECT-1 is described which can be used for highly efficient site-directed in vitro mutagenesis. The mutagenesis method is based on the use of single-stranded DNA and two primers, one mutagenic primer and a second correction primer which corrects a defect in the ampicillin resistance gene on the vector and reverts the vector to ampicillin resistance. Using T4 DNA polymerase and T4 DNA ligase the two primers are physically linked on the template. The non-mutant DNA strand is selected against by growth in the presence of ampicillin. In tests of the vector, highly efficient (60-90%) mutagenesis was obtained.
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PMID:Efficient site directed in vitro mutagenesis using ampicillin selection. 219 59

Casein messenger RNAs (mRNAcsn) were purified from lactating mammary glands of BALB/c mice and used as a starting material for cloning of casein gene sequences. Double-stranded casein cDNA (ds-cDNAcsn) was prepared and blunt-end ligated to HindIII-specific DNA linker molecules. After digestion with HindIII, the dsDNAcsn was inserted into the HindIII site of plasmid pBR322, using T4 DNA ligase. Escherichia coli strain RH202 was transformed with the hybrid plasmids, and transformants were selected for resistance to ampicillin. Electrophoresis of HindIII-digested hybrid plasmid DNAs, followed by Southern transfer and hybridization to [32P]cDNAcsn, revealed that one of the hybrid-plasmid-containing colonies, designated pCas51, contained a 400-bp insert which hybridized to the [32P]cDNAcsn. Purification of the individual casein mRNAs (mRNAcsn alpha, beta, and gamma) and solution hybridization of nick-translated insert DNA to each of these revealed that pCas51 contained sequences complementary primarily to mRNAcsn beta.
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PMID:Cloning of mouse beta-casein gene sequences. 729 56

Here, we describe assembly PCR as a method for the synthesis of long DNA sequences from large numbers of oligodeoxyribonucleotides (oligos). The method, which is derived from DNA shuffling [Stemmer, Nature 370 (1994a) 389-391], does not rely on DNA ligase but instead relies on DNA polymerase to build increasingly longer DNA fragments during the assembly process. A 1.1-kb fragment containing the TEM-1 beta-lactamase-encoding gene (bla) was assembled in a single reaction from a total of 56 oligos, each 40 nucleotides (nt) in length. The synthetic gene was PCR amplified and cloned in a vector containing the tetracycline-resistance gene (TcR) as the sole selectable marker. Without relying on ampicillin (Ap) selection, 76% of the TcR colonies were ApR, making this approach a general method for the rapid and cost-effective synthesis of any gene. We tested the range of assembly PCR by synthesizing, in a single reaction vessel containing 134 oligos, a high-molecular-mass multimeric form of a 2.7-kb plasmid containing the bla gene, the alpha-fragment of the lacZ gene and the pUC origin of replication. Digestion with a unique restriction enzyme, followed by ligation and transformation in Escherichia coli, yielded the correct plasmid. Assembly PCR is well suited for several in vitro mutagenesis strategies.
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PMID:Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. 759 Mar 20

The aim of study was to investigate the construction, identification and expression of human B7.1 (CD80) eukaryotic expressing vector on HL-60 cells. B7.1 gene was subcloned from the cloning vector using PCR. The PCR products and eukaryotic expressing vector (pHook) both were separately digested with ApaI, SalI and were ligated using T4 DNA ligase. The ligases products were transduced into DH-5alpha. B7.1 gene containing clones was selected by digestion with ApaI and SalI, and were further confirmed by sequencing of DNA. HL-60 cells were transfected with B7.1 by using lipofectamine and detected by immunofluorescence, SABC and FACS methods. The results showed that the size of PCR products was about 620 bp. Five clones were ampicillin-resistant and all could be digested by ApaI and SalI to produce 620 bp gene fragment that had the same size of B7.1, which means that the B7.1 recombinant vector has been constructed successfully. Further sequencing confirmed the validity of the construction. No nucleotide mutation was found, B7.1 effectively expressed on HL-60 cells with 70%, 65% and 92.7% by immunofluorescence, SABC and FACS respectively. It is concluded that the human B7.1 (CD80) eukaryotic expressing vector can be successfully constructed by molecular cloned methods and can stably effectively express on the membrane of B7.1-negative acute myelocytic leukemia (AML) cell line HL-60. It is inferred that the vaccine prepared by using this method may have immunotherapeutic and immuno-protective effects.
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PMID:[Construction, identification of human B7.1 eukaryotic expressing vector and its expression on leukemic cells]. 1937 86