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)

Plasmid pRO1957 contains a 26.5-kb BamHI restriction endonuclease-cleaved DNA fragment cloned from the chromosome of Pseudomonas pickettii PKO1 that allows P. aeruginosa PAO1c to grow on toluene, benzene, phenol, or m-cresol as the sole carbon source. The genes encoding enzymes for meta cleavage of catechol or 3-methylcatechol, derived from catabolism of these substrates, were subcloned from pRO1957 and were shown to be organized into a single operon with the promoter proximal to tbuE. Deletion and analysis of subclones demonstrated that the order of genes in the meta cleavage operon was tbuEFGKIHJ, which encoded catechol 2,3-dioxygenase, 2-hydroxymuconate semialdehyde hydrolase, 2-hydroxymuconate semialdehyde dehydrogenase, 4-hydroxy-2-oxovalerate aldolase, 4-oxalocrotonate decarboxylase, 4-oxalocrotonate isomerase, and 2-hydroxypent-2,4-dienoate hydratase, respectively. The regulatory gene for the tbuEFGKIHJ operon, designated tbuS, was subcloned into vector plasmid pRO2317 from pRO1957 as a 1.3-kb PstI fragment, designated pRO2345. When tbuS was not present, meta pathway enzyme expression was partially derepressed, but these activity levels could not be fully induced. However, when tbuS was present in trans with tbuEFGKIHJ, meta pathway enzymes were repressed in the absence of an effector and were fully induced when an effector was present. This behavior suggests that the gene product of tbuS acts as both a repressor and an activator. Phenol and m-cresol were inducers of meta pathway enzymatic activity. Catechol, 3-methylcatechol, 4-methylcatechol, o-cresol, and p-cresol were not inducers but could be metabolized by cells previously induced by phenol or m-cresol.
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PMID:Genetic organization and regulation of a meta cleavage pathway for catechols produced from catabolism of toluene, benzene, phenol, and cresols by Pseudomonas pickettii PKO1. 185 61

The alkaline plasmid DNA extraction method of Birnboim and Doly was applied for the isolation of human papillomavirus (HPV) from warts. Tissue from common and plantar warts was digested with proteinase K, and the extrachromosomal circular covalently-closed form of HPV-DNA was rapidly extracted by alkaline sodium dodecyl sulphate and phenol-chloroform treatment. Recovery of HPV-DNA from the tissue was sufficient for determination of endonuclease restriction patterns by agarose gel electrophoresis.
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PMID:Human papillomavirus DNA from warts for typing by endonuclease restriction patterns: purification by alkaline plasmid methods. 196 33

Although the last decade has seen much activity devoted to the phylogeny of the Platyhelminthes, such activity has relied in the main on traditional anatomical, ultrastructural, and developmental data. Extension of these studies to the molecular level has been impeded by the lack of a reliable method for the isolation of DNA from these organisms, especially marine triclads whose DNA is particularly difficult to isolate using methods currently used with other eukaryotic or prokaryotic organisms. We report here the details of a method that results in the rapid isolation of relatively pure DNA in good yields from small amounts of material. The method involves the treatment of the animals with high concentrations of NaDodSO4 followed by phenol extraction, resulting in a product that is an excellent substrate for restriction endonuclease digestion and Southern blotting.
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PMID:A method for the preparation of high-molecular-weight DNA from marine and freshwater triclads (Platyhelminthes, Turbellaria). 196 73

A 26-kilobase BamHI restriction endonuclease DNA fragment was cloned from Pseudomonas pickettii PKO1, a strain isolated from a soil microcosm that had been amended with benzene, toluene, and xylene. This DNA fragment, cloned into vector plasmid pRO1727 and designated pRO1957, allowed Pseudomonas aeruginosa PAO1c to grow on phenol as the sole source of carbon. Physical and functional restriction endonuclease maps have been derived for the cloned DNA fragment. Two DNA fragments carried in trans and derived from subclones of pRO1957 show phenol hydroxylase activity in cell extracts of P. aeruginosa. Deletion and subcloning analyses of these fragments indicated that the gene encoding phenol hydroxylase is positively regulated. Phenol and m-cresol were shown to be inducers of the enzyme. o-Cresol and p-cresol did not induce enzymatic activity but could be metabolized by cells that had been previously exposed to phenol or m-cresol; moreover, the enzyme exhibited a rather broad substrate specificity and was sensitive to thiol-inhibiting reagents. A novel polypeptide with an estimated molecular mass of 80,000 daltons was detected in extracts of phenol-induced cells of P. aeruginosa carrying plasmid pRO1959.
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PMID:Molecular cloning, characterization, and regulation of a Pseudomonas pickettii PKO1 gene encoding phenol hydroxylase and expression of the gene in Pseudomonas aeruginosa PAO1c. 211 72

We describe a subtractive hybridization protocol which is designed to permit subtractions between cDNA libraries. The method uses single-stranded phagemids with directional inserts as both the driver and the target. We modified the M13 phagemid vector pBluescript for the directional cDNA cloning and subtractive hybridization. Two simplified methods for efficient construction of directional cDNA libraries are also described. Using a model system, we found that one round of subtractive hybridization results in a 5,000-fold specific subtraction of abundant molecules. We used two methods to quantify the efficiency and verify the specificity of the subtraction. In order to obtain these subtraction efficiencies, it was necessary to develop a method to purify the single-stranded DNA to homogeneity. The single-stranded purification involved using potassium iodide (KI) density centrifugation, restriction endonuclease digestion and phenol extraction in the presence of magnesium. We describe the several advantages of using directional inserts for the subtraction procedure.
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PMID:Subtractive hybridization system using single-stranded phagemids with directional inserts. 216 39

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

We have developed a new, rapid method for the extraction of human genomic DNA from whole blood samples. Traditionally, genomic DNA has been extracted from blood by overnight proteinase K digestion of lysed peripheral lymphocytes followed by phenol/chloroform extraction. In addition to being time consuming, the use of phenol involves inherent risks due to the toxic nature of the reagent. Our method for the extraction of DNA from whole blood uses sodium perchlorate and chloroform instead of phenol with a significant time savings realized as well as fewer hazards to the technician. Furthermore, DNA prepared by this new method is an excellent substrate for restriction endonuclease digestion and Southern hybridization analysis.
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PMID:Purification of human genomic DNA from whole blood using sodium perchlorate in place of phenol. 255 54

A simple and efficient method of purifying linear plasmid DNA from contaminating DNA fragments is described. Both vector and insert containing plasmids may be used without extensive purification, in particular without cesium chloride centrifugation. Careful deproteinization with phenol-chloroform allows efficient restriction enzyme digestion. Fragment separation can be performed immediately after restriction endonuclease digestion in a single 6% polyacrylamide gel. Extraction of DNA fragments from the gel is easy and gives a good yield. The DNA may be used for ligation and transformation without further purification.
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PMID:A protocol for DNA fragment extraction from polyacrylamide gels. 285 97

A method for the isolation of DNA from mycobacteria propagated in vitro is described that utilizes organic solvents to extract lipoidal components from the outer membrane, and digestion with a protease (nagarse) and lysozyme to penetrate the cell wall. The mycobacterial cells were lysed by the addition of detergent and the DNA was purified by digestion with pronase, sequential phenol and chloroform extractions, and digestion with RNAase A. The isolated DNA, which was obtained in good yields, was of a relatively high Mr and could be readily digested by restriction endonucleases. By this method, the genomes of Mycobacterium avium, M. intracellulare, M. lepraemurium, 'M. lufu', M. marinum, M. phlei, M. scrofulaceum, M. smegmatis and M. tuberculosis were isolated and the restriction endonuclease digestion patterns analysed. Each species could be distinguished by the digestion patterns, indicating that this approach can be used for identifying mycobacterial species. This approach is also sufficiently sensitive to differentiate strains since we were able to distinguish two independently isolated strains of M. tuberculosis, H37 and H4. In addition, no evidence was obtained for the presence of methylcytosine residues in the sequences 5'.CCGG.3',5'.CCCGGG.3',5'.CC(A/T) GG.3' or for methyladenine at 5'.GATC.3' in the DNA of the nine mycobacterial species examined using pairs of restriction enzymes that recognize and cleave at the same nucleotide sequence but differ in their sensitivity to 5-methylcytosine or 6N-methyladenine.
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PMID:Isolation and restriction endonuclease analysis of mycobacterial DNA. 301 65

A simple and reliable method for the recovery of specific fragments of DNA from agarose gels is presented. The electroelution of the DNA onto the NENSORB cartridge matrix with the subsequent elution of the bound DNA by a methanol (50% v/v) wash has been shown to result in the quantitative recovery of the restriction fragment. Of importance is the fact that the DNA purified by this procedure is a viable substrate for further digestion by a second restriction endonuclease. The method does not require either phenol extraction or extensive desalting of the sample.
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PMID:Affinity chromatographic procedure for the quantitative recovery of DNA fragments from agarose gels. 301 50


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