In order to clone the entire SOD gene, inverse PCR method was adopted. Genomic DNA, which had been previously digested with SphI (for subcloning of 5′-end) or AccIII (for subcloning of 3′-end), was self-ligated and used for template DNA.
For the analysis of DNA fragments, agarose gel electrophoresis was performed under standard condition . GeneClean kit (Bio 101, La Jolla, CA) was used to recover DNA fragment from agarose gel slices. The PCR amplified gene fragment was ligated independently into the cloning vector pCR2.1 (Invitrogen Corp.), with TA cloning kit (Invitrogen Corp.), and used for transformation of E. coli DH5α. Nucleotide sequence of the gene GANT61 molecular weight was determined by using ABI PRISM 310 genetic analyzer (Applied Biosystems Japan). The nucleotide
and amino acid sequence of P24, Mn-SOD of strain B23, has been deposited in the EMBL/GeneBank/DDBJ under accession number BAA95631. Cloning of genes encoding P21 and P16 In order to clone the genes encoding P21 and P16, their internal amino acid sequences were determined as follows. Target proteins were prepared by slicing the SDS-PAGE gel and eluting out by vortex with 20 mM Tris-HCl (pH 8.0) containing 1% SDS overnight. After digestion of the protein with lysyl endopeptidase (LEP) under standard condition , each peptide fragment was fractionated by reverse phase HPLC (column: AQUAPORE RP300, 4.6 × 250 mm, Applied Biosystems Japan) and its N-terminal amino acid sequences was determined. Based on these amino acid sequences, PCR primers were constructed to amplify the target ATPase inhibitor gene loci. A part of the gene encoding P21 was amplified by PCR with primers designed for N-terminal amino acid sequence, AFPLSGVGGFTISADLI (P21-N), and one of the internal amino acid sequences, PSLNTHYMSAGSITIPSMK (P21-37). B23 genome library was screened to obtain a phage clone containing the entire gene encoding P21. The nucleotide
sequence of this gene and its flanking region has been submitted to EMBL/GenBank/DDBJ under accession number AB047106. A part of the gene encoding P16 was amplified by, what we call, armed-PCR method using lambda EMBL3-B23 genomic DNA library as template DNA. The PCR amplification primers were designed for second right arm of EMBL3 vector (5′-CGTCCGAGAATAACGAGTGGATC-3′) and one of the internal amino acid sequences, AAQEFQTGADNITIDNGN (P16-16). The PCR amplified DNA fragments (1.8 kb) were ligated into the cloning vector pCR2.1. The complete nucleotide sequence was determined and found that the DNA fragment encodes a part of the P16 gene, including 5′-end. Utilizing this gene fragment as a probe, B23 genome library was screened to obtain a phage clone containing the entire gene encoding P16. The nucleotide sequence of this gene fragment has been submitted to EMBL/GenBank/DDBJ under accession number AB049820. Northern hybridization and RT-PCR Cultures were taken from a bottle after 0, 4, and 10 days cultivation in the presence of alkanes.