해양성 메탄올 산화세균 Methylophaga sp. MP에서 메탄올 산화에 관여하는 mxaF 유전자의 클로닝

다국어 초록 (Multilingual Abstract)

The mxaF gene encoding the large subunit of methanol dehydrogenase(MDH) was cloned from a marine methylotroph Methylophaga sp. MP. The gene was smplified from the Methylophaga chromosomal DNA by polymerase chain reaction (PCR) using a pair of conserved sequences in MDH as primers. A 537 bp PCR product in size was cloned into TOPO-cloning vector and the nucleotide sequence was determined. The calculated GC content was 43.99%. However, 14 kinds of codons including AGA and AGG were not found within the open reading frame of the sequenced mxaF gene. It was revealed by the predicted amino acid sequence analysis that there was about 49% homology, comparing to those from 16 methanotrophs and methylotrophs. The allgned amino acid sequences showed that several key amino acids (asparagine 287, aspartate 327, arginine 357, and asparagine 420) required for the enzyme activity were located at the active site of MDH, as commonly found in soll methylotrophic bacteria. The tryptophan docking motlfs corresponding to W4 and W5 were also found in the residues 282 to 292 and 337 to 347, respectively. It was confirmed by Southern blot analysis that the cloned mxaF gene was located in chromosomal DNA of the marine methylotroph Methylophaga sp-MP in a single copy. In addition, the mxaF gene could hybridize to 14 kb of EcoRⅠ, 9.4 kb and 5.6 kb of HindⅢ, 7.0 kb and 3.8 kb of PstⅠ, and 7.0 kb of ClaⅠ fragments, respectively. These results suggest that MDH gene cluster might be located in chromosomal DNA of Methylophaga sp. MP as a gene cluster. These results suggest that mxaF gene is also highly conserved in marine methylotrophic bacterium.

목차 (Table of Contents)

• CONTENTS = ⅰ
• LIST OF TABLES = ⅲ
• LIST OF FIGURES = ⅳ
• ABSTRACT = ⅴ
• INTRODUCTION = 1
• MATERIALS AND METHODS = 9
• Ⅰ. Bacterial strains and plasmids = 9
• Ⅱ. Culture of E. coli and Methylophaga strains = 9
• Ⅲ. Enzymes and reagents = 9
• Ⅳ. Isolation of Methylophaga sp. MP chromosomal DNA = 12
• Ⅴ. PCR amplification = 12
• Ⅵ. Electrophoresis and elution of PCR products = 13
• Ⅶ. Cloning and transformation of PCR products = 13
• Ⅷ. Preparation of plasmid DNA = 14
• Ⅸ. Sequencing analysis of mxaF gene = 14
• Ⅹ. Southern blot analysis = 14
• RESULTS AND DISCUSSION = 16
• Ⅰ. PCR amplification of mxaF gene fragments = 16
• Ⅱ. Cloning of the PCR products into T-tailed plasmid vector = 16
• Ⅲ. Sequence analysis of the cloned mxaF gene and analysis of codon usage = 16
• Ⅳ. Amino Acid Sequence Alignment of the cloned mxaF Gene = 21
• Ⅴ. Southern Blot Analysis = 22
• 적요 = 28
• REFERENCES = 30
• 감사의 글 = 38