The wild-type strain Streptomyces ambofaciens DSM 40697 exhibits a high degree of genetic instability. Pigment-defective (26). Genomic instabilities are associated with these phenotypic instabilities. Thus, molecular analysis of mutant progeny often reveals genomic rearrangements such as large deletions including genes directly involved in the following phenomena: chloramphenicol sensitivity in S. coelicolor A3(2) and S. lividans 66 (1, 7), streptomycin sensitivity in S. glaucescens (11), melanin formation in S. reticuli (25), S. glaucescens (12), and several Streptomyces species (26), and A-factor biosynthesis in S. bikiniensis (14). However, in some cases of reversible phenotypes, it was suggested that the genes involved were affected by transposable elements (8,9).Highly amplified DNA sequences (ADS) have been detected within the DNA isolated from variants of many Streptomyces species arising either spontaneously (1,3,25) or during vegetative growth in the presence of ethidium bromide (10,19,24,25), formation and regeneration of protoplasts (6), or interspecific protoplast fusion (23). In some cases, ADS were associated with particular phenotypes, such as sequential loss of resistance to chloramphenicol and arginine synthesis (2) and loss of resistance to tetracyclihe and nitrogen assimilation (5). Some others were selected on the basis of high-level antibiotic resistance (7,15,17,21) Table 1. Most of the media used in the culture conditions were described previously (13). S. ambofaciens strains were grown at 37°C on plates of HickeyTresner (HT) medium (22) for maintenance, sporulation, and mutant isolation. Auxotrophic mutants were detected by replica-plating the WT cultures on minimal medium and further characterized on minimal medium supplemented with combinations of growth requirements. For large-scale isolations of genomic DNA, the S. ambofaciens cultures were grown aerobically at 37°C for 48 h in YEME liquid medium supplemented with glycine (0.25%). Small-scale isolations of genomic DNA were performed with mycelium grown at 37°C in HT liquid medium for 24 h.DNA extraction and restriction endonuclease analyses. The myceliumn was harvested by centrifugation, resuspended in TE buffer (10 mM Tris hydrochloride, 1 mM EDTA, pH 8.0) and mixed with lysozyme (Boehringer Mannheim) (2 mg/ml). Protoplasts were formed by incubation of the mixture at 30'C for 30 min and lysed with sodium dodecyl sulfate (1%) in the presence of proteinase K (50 ,ug/ml). DNA was purified by two phenol-chloroform extractions followed by one chloroform extraction. The aqueous phase was treated with RNase A (Sigma) (5 mg/ml) for 1 h at 37°C. DNA was precipitated with sodium acetate (0.3 M, pH 5.2) and isopropanol at -20°C. After centrifugation, the pellet was vacuum dried and dissolved in TE buffer. For the small-scale isolations, DNA was purified by the Geneclean process (Bio 101 Inc.). For the large-scale isolations, the DNA was purified by equilibrium density centrifugation in CsCl-ethidium bromide gradients, using a vTi 65.2 rotor ...