Taken together, the PFGE patterns (Fig. 1D) and Southern hybridization results (Fig. 3A and 3B) indicated that 76-9 and SA1-8 have the same chromosomal structure, and have undergone the same three rearrangement events. Since 76-9 is able to sporulate and to produce high-level avermectins, it can be concluded that the deleted central region within G1 is not responsible for the differentiation or avermectin production in S. avermitilis. Chromosomal circularization in SA1-6 The 1938-kb deletion region at both chromosomal PLX4032 price ends of SA1-6 was identified by walking PCR, including entire AseI-W, A, U, left part of AseI-P, and right part of AseI-D (Fig. 7A). No obvious retardation

of the AseI fragment of SA1-6 was observed in SDS-treated sample (data not shown), together with the intact chromosome remaining trapped in the gel well in PK-treated sample (Fig.

2A), indicating that the SA1-6 chromosome was circularized. The left and right deletion ends were located at 1611078 nt and 8698105 nt, respectively. Therefore, the size of the new AseI junction fragment NA4 was 489-kb and overlapped with AseI-G1 in the PFGE gel, which was confirmed by Southern hybridization using probe N4 spanning the fusion site (Additional file 1: Supplementary Fig. S3). Hybridization of probe N4 with the BglII-digested Torin 1 in vitro genomic DNA revealed that a 2.99-kb BglII fragment from the left AseI-P and a 13.0-kb BglII fragment from the right AseI-D in the wild-type strain were partially deleted and joined, generating a newly 8.7-kb BglII fragment in SA1-6 (Fig.

7B and 7C). No homology was found when the fusion sequence was compared with the corresponding left and right sequences from wild-type (Fig. 7D). Figure 7 Characterization of circular chromosome in SA1-6. (A) Schematic representation of the chromosomes of wild-type strain and mutant SA1-6, showing deletions at both ends. (B) Location of chromosomal deletion ends and fusion junction. Bg, BglII. (C) Southern analysis of fusion fragment with probe N4, which was prepared using primers 405 and 406. (D) Junction sequence, showing no obvious homology between the original sequences. Stability assay of chromosomal structure in Reverse transcriptase bald mutants Generational studies were performed to assess the chromosomal stability of bald mutants derived from the wild-type strain. Four bald strains were selected, and subjected to PFGE analysis following ten passages. The chromosomal structure of SA1-8 and SA1-6 was conserved, whereas that of SA1-7 and SA3-1 was changed (Additional file 1: Supplementary Fig. S4A). Both SA1-7 and SA3-1 lost their characteristic bands, and became indistinguishable from SA1-6. SA1-7 chromosome was further monitored in each passage, and found to change in the 4th passage (Additional file 1: Supplementary Fig. S4B). The corresponding fusion fragments of SA1-6 and SA1-8 were also detected in their progeny. These results indicate that chromosomal structure of SA1-6 and SA1-8 is stable.