Biomaterial Database

Isolation and restriction endonuclease analysis of mycobacterial DNA. 1986

R Patel, and J T Kvach, and P Mounts

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.

UI	MeSH Term	Description	Entries
D008745	Methylation	Addition of methyl groups. In histo-chemistry methylation is used to esterify carboxyl groups and remove sulfate groups by treating tissue sections with hot methanol in the presence of hydrochloric acid. (From Stedman, 25th ed)	Methylations
D009161	Mycobacterium	A genus of gram-positive, aerobic bacteria. Most species are free-living in soil and water, but the major habitat for some is the diseased tissue of warm-blooded hosts.	Mycobacteria
D009162	Mycobacterium avium	A bacterium causing tuberculosis in domestic fowl and other birds. In pigs, it may cause localized and sometimes disseminated disease. The organism occurs occasionally in sheep and cattle. It should be distinguished from the M. avium complex, which infects primarily humans.
D009167	Mycobacterium lepraemurium	The etiologic agent of rat leprosy, also known as murine leprosy.
D009168	Mycobacterium phlei	A saprophytic bacterium widely distributed in soil and dust and on plants.
D009169	Mycobacterium tuberculosis	A species of gram-positive, aerobic bacteria that produces TUBERCULOSIS in humans, other primates, CATTLE; DOGS; and some other animals which have contact with humans. Growth tends to be in serpentine, cordlike masses in which the bacilli show a parallel orientation.	Mycobacterium tuberculosis H37Rv
D009170	Nontuberculous Mycobacteria	So-called atypical species of the genus MYCOBACTERIUM that do not cause tuberculosis. They are also called tuberculoid bacilli, i.e.: M. abscessus, M. buruli, M. chelonae, M. duvalii, M. flavescens, M. fortuitum, M. gilvum, M. gordonae, M. intracellulare (see MYCOBACTERIUM AVIUM COMPLEX;), M. kansasii, M. marinum, M. obuense, M. scrofulaceum, M. szulgai, M. terrae, M. ulcerans, M. xenopi.	Atypical Mycobacteria,Mycobacteria, Atypical,Mycobacterium duvalii,Mycobacterium flavescens,Mycobacterium gilvum,Mycobacterium gordonae,Mycobacterium obuense,Mycobacterium szulgai,Mycobacterium terrae,Mycolicibacter terrae,Mycolicibacterium duvalii,Mycolicibacterium flavescens,Mycolicibacterium gilvum,Mycolicibacterium obuense,Tuberculoid Bacillus,Atypical Mycobacterium,Mycobacterium, Atypical,Non-Tuberculous Mycobacteria,Nontuberculous Mycobacterium
D004262	DNA Restriction Enzymes	Enzymes that are part of the restriction-modification systems. They catalyze the endonucleolytic cleavage of DNA sequences which lack the species-specific methylation pattern in the host cell's DNA. Cleavage yields random or specific double-stranded fragments with terminal 5'-phosphates. The function of restriction enzymes is to destroy any foreign DNA that invades the host cell. Most have been studied in bacterial systems, but a few have been found in eukaryotic organisms. They are also used as tools for the systematic dissection and mapping of chromosomes, in the determination of base sequences of DNAs, and have made it possible to splice and recombine genes from one organism into the genome of another. EC 3.21.1.	Restriction Endonucleases,DNA Restriction Enzyme,Restriction Endonuclease,Endonuclease, Restriction,Endonucleases, Restriction,Enzymes, DNA Restriction,Restriction Enzyme, DNA,Restriction Enzymes, DNA
D004269	DNA, Bacterial	Deoxyribonucleic acid that makes up the genetic material of bacteria.	Bacterial DNA
D004587	Electrophoresis, Agar Gel	Electrophoresis in which agar or agarose gel is used as the diffusion medium.	Electrophoresis, Agarose Gel,Agar Gel Electrophoresis,Agarose Gel Electrophoresis,Gel Electrophoresis, Agar,Gel Electrophoresis, Agarose