Cloning of macromomycin apoprotein gene from Streptomyces macromomyceticus by use of 50-mer deoxynucleotide probes.

A mixed probe consisting of two synthetic deoxynucleotides (52 and 54 mers referred to as 50-mer) with arbitrarily chosen C or G for the third letters was prepared based on the amino acid sequences No. 31-48 and No. 72-90 of macromomycin (MCM) apoprotein and successfully used to clone the MCM apoprotein gene. Digestion with Sph I of total DNA of MCM-producing Streptomyces macromomyceticus M480-M1 yielded a 2.6-kb fragment that hybridized strongly to the probes. The hybridized probe was stable to washing with 3 x SSC at 75 degrees C. Radioactivity derived from the hybridized probe was comparable to that expected theoretically from hybridization between the probe and the true target sequence. The 2.6-kb fragment was cloned into Escherichia coli RR1 with pBR322 and subsequently subcloned into Streptomyces lividans TK21 with pIJ702. Nucleotide sequence analysis of the cloned fragment verified the existence of the sequence corresponding to the amino acid sequence of MCM apoprotein and about 90% homologies with the probes. Thus, the use of relatively long deoxynucleotide probes with arbitrarily chosen C or G for the third letters will be advantageous in cloning Streptomyces protein genes where more than 90% of the third letters have been known to be C or G. In addition, theoretical diagnosis of hybridization should be a great help to distinguish true positives from false ones.