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research-article

DNA amplification in Streptomyces achromogenes subsp. rubradiris is accompanied by a deletion, and the amplified sequences are conditionally stable and can be eliminated by two pathways.

School of Pharmacy, University of Wisconsin Madison 53706.

ABSTRACT

Southern blot analysis of BglII-digested DNA isolated from wild-type Streptomyces achromogenes, which harbors the 8.8-kilobase amplifiable unit of DNA, AUD-Sar 1, and of similarly digested DNA from 12 strains carrying an array of 200 to 300 tandem copies of a specific AUD-Sar 1-derived 8.0-kilobase DNA sequence, ADS-Sar 1, revealed the absence of the 12.4-kilobase BglII AUD-Sar 1-chromosome right junction band in the latter strains, whereas the corresponding 26.0-kilobase left junction band remained unaltered. Further Southern analyses indicated in all of the seven amplified strains tested the occurrence of a deletion of at least 10 kilobases of the DNA adjacent to the right side of the AUD. The deletion has one endpoint in the vicinity of the ADS array. Corroborating and expanding upon previously reported results, we found that the amplified DNA of strain C010 was stably maintained for at least 20 transfers when the transfers involved mycelia propagated in spectinomycin-free liquid medium. In contrast, when strain C010 was subjected separately to one cycle of protoplast formation and regeneration or to three cycles of spore germination, aerial mycelium formation, and sporulation on spectinomycin-free media, only approximately 20% of the protoplast regenerants and spores retained the reiterated DNA sequences and the ability subsequently to form colonies on media containing high levels of spectinomycin. Approximately 80% of these units completely deleted the reiterated DNA and left adjacent sequences and exhibited sensitivity to 25 micrograms of spectinomycin per ml. One among 24 protoplast-derived deletants apparently retained the left portion of the AUD-ADS left direct repeat plus left adjacent sequences.