title: Insertion sequence-based cassette PCR: cultivation-independent isolation of gamma-hexachlorocyclohexane-degrading genes from soil DNA.
abstract: gamma-Hexachlorocyclohexane (gamma-HCH) is a highly chlorinated pesticide that has caused serious environmental problems. Based on the frequently observed association of insertion sequence IS6100 with lin genes for gamma-HCH degradation in several gamma-HCH-degrading bacterial strains isolated to date, DNA fragments flanked by two copies of IS6100 were amplified by nested polymerase chain reaction (PCR) technique using a DNA sample extracted from soil contaminated with HCH. Four distinct DNA fragments with sizes of 6.6, 2.6, 1.6, and 1.3 kb were obtained, three of which carried lin genes: the 6.6-kb fragment carried linD and linE as well as linR; the 2.6-kb fragment showed a truncated form of linF; and the 1.6-kb fragment carried linB. Our approach, named as insertion sequence (IS)-based cassette PCR, was successful in the isolation of the lin genes from HCH-contaminated soil without cultivation of host cells and is applicable for the culture-independent isolation of other functional genes bordered by other IS elements.
authors.affiliation: Department of Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Japan.
authors.forename: Genki
authors.initials: G
authors.lastname: Fuchu
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Ohtsubo
authors.forename: Michihiro
authors.initials: M
authors.lastname: Ito
authors.forename: Ryo
authors.initials: R
authors.lastname: Miyazaki
authors.forename: Akira
authors.initials: A
authors.lastname: Ono
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nagata
authors.forename: Masataka
authors.initials: M
authors.lastname: Tsuda
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001673:Biodegradation, Environmental; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D001556:Hexachlorocyclohexane / Q000378:metabolism*; D008969:Molecular Sequence Data; D010575:Pesticides / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D012987:Soil / Q000032:analysis; D012988:Soil Microbiology; D042301:Sphingomonadaceae / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D010575:Pesticides; D012987:Soil; D001556:Hexachlorocyclohexane