THE VERY LARGE AMPLIFIABLE ELEMENT AUD2 FROM STREPTOMYCES-LIVIDANS-66HAS INSERTION SEQUENCE-LIKE REPEATS AT ITS ENDS

In a spontaneous, chloramphenicol-sensitive (Cm-s), arginine-auxotroph ic (Arg(-)) mutant of Streptomyces lividans 1326, two amplified DNA se quences were found. One of them was the well-characterized 5.7-kb ADS1 sequence, amplified to about 300 copies per chromosome. The second on e was a 92-kb sequence called ADS2. ADS2 encoding the previously isola ted mercury resistance genes of S. lividans was amplified to around 20 copies per chromosome. The complete ADS2 sequence was isolated from a genomic library of the mutant S. lividans 1326.32, constructed in the phage vector lambda EMBL4. In addition, the DNA sequences flanking th e corresponding amplifiable element called AUD2 in the wild-type strai n were isolated by using another genomic library prepared from S. livi dans 1326 DNA. Analysis of the ends of AUD2 revealed the presence of a n 846-bp sequence on both sides repeated in the same orientation. Each of the direct repeats ended with 18-bp inverted repeated sequences. T his insertion sequence-like structure was confirmed by the DNA sequenc e determined from the amplified copy of the direct repeats which demon strated a high degree of similarity of 65% identity in nucleic acid se quence to IS112 from Streptomyces albus. The recombination event leadi ng to the amplification of AUD2 occurred within these direct repeats, as shown by DNA sequence analysis. The amplification of AUD2 was corre lated with a deletion on one side of the flanking chromosomal region b eginning very near or in the amplified DNA. Strains of S. lividans lik e TK20 and TK21 which are mercury sensitive have completely lost AUD2 together with flanking chromosomal DNA on one or both sides.