MOLECULAR CLONING AND CHARACTERIZATION OF METHANOL DEHYDROGENASE GENE FROM METHYLOBACILLUS SP. SK1

다국어 초록 (Multilingual Abstract)

This study was performed for identification and characterization of methanol dehydrogenase (mdh^(+)) gene from Methylobacillus sp. SK1. To use in immunoscreening of mdh^(+) gene, polyclonal anti-MDH antibodies were prepared by subcutaneous injection of a white rabbit with Methylobacillus MDH protein. The MDH antibody recognized specifically 60 kDa MDH protein from cell extract, as determined by immunoblot analysis. To clone the gene encoding the MDH protein, a genomic expression library of Methylobacillus sp. SK1 was constructed in λgt11 vector. From this library, an mdh^(+) gene was isolated by immunological screening method using anti-MDH antibody as a probe. From 10^(7) plaques screened, 9 putative clones were finally isolated. To examine whether these clones contain homologous inserts, cross-hybridization analysis was performed using 3.2 kb C-1 insert DNA as a probe. The DNA inserts of all clones, except clones C6 and C8, hybridized with the probe, indicating that the clones contained the same kinds of DNA inserts. In order to identify the transcript of mdh^(+) gene, Northern hybridization analysis was performed using the 3.2 kb insert DNA of clone C1 as a probe. The RNA transcript sizes of the cloned gene were 3.2 and 1.5 kb.