Rapd Technique Cloning Macromolecular Antitumor Antibiotic C1027 Biosynthesis Genes

C1027, produced by S. globisporus C1027, is a new macromolecular antitumor antibiotic composed of an enediyne chromophore and an apoprotein through non-covalent bond binding. The chromophore represents most of the biological activity attributable to the holoantibiotic in contrast to the chromophore-stabilizing activity of the apoprotein. DNA encoding apoprotein of macromomycin and C1027 have been cloned and sequenced. Hower little is known about the chromophore genes of this chromoprotein family of antitumor antibiotics. In our previous research, we amplified part of the C1027 apoprotein gene by PCR. The amplified fragment was used to probe the biosynthetic gene cluster of C1027 chromophore. For unknown reason, we failed.In this study a new method was used to clone C1027 pathway genes. The method is based on random-amplified polymorphic DNA (RAPD) in which strain-specific arrays of DNA fragment are generated by PCR with single arbitrary oligonucleotides to prime DNA synthesis from genomic sites they completely or partially match.Five C1027 nonproducing mutants were isolated by treatment of C1027 producer with acriflavine. A set of seven random sequence primers that were 10 bases long were synthesized, with average GC content of 60%-70%. Three relatively long (more than 17-nt) primers designed for other purposes were also chosen for this experiment. We established optimal reaction conditions under which amplification are both efficient and consistent.Gonomic DNAs from C1027 wild-type strain as well as five C1027 blocked mutants were amplified with different oligonucleotide primers. The procedure generated RAPD profiles containing between 0-5 amplified DNA...