Fragments D and W correspond to the right and left ends of the chromosome, respectively, which covalently bind terminal proteins. In comparison to AseI patterns of wild-type chromosome, all the bald mutants derived from wild-type (designated SA) displayed chromosomal rearrangements. Some of the mutants shared BI 10773 datasheet similar PFGE profile representatively shown in Fig. 1B and 1C, although the chromosomal structures among these mutants might be different. Fragments AseI-W

(63-kb) and A (1422-kb) on the left chromosomal arm were involved in nearly all deletion events, most of which extended to fragment U (85-kb). Considering that the overlapping band D/E became fainter and thinner, it is most likely that the right terminal fragment D was missing, although the possibility that centrally located fragment E could also be missing can not be excluded. Meanwhile, some new AseI bands appeared in the SA mutants. In contrast, the spontaneous bald mutants derived from 76-9 showed High Content Screening no apparent chromosomal rearrangements in comparison to the AseI pattern of 76-9 (Additional file 1: Supplementary Fig.

S1). Figure 1 Gross chromosomal rearrangements in spontaneous bald mutants from S. avermitilis wild-type (WT) strain ATCC31267. (A) AseI restriction map of wild-type chromosome. (B and C) AseI restriction patterns of genomic DNA of bald mutants (SA). (D) Similar AseI profiles of 76-9 and SA1-8. PFGE conditions for separating large fragments were: (B and D) 1.2% agarose, 4.5 V/cm, 20-130 s pulses, 36 h; 4.5 V/cm, 60-90 s pulses, 2 h; 4.5 V/cm, 5-10 s pulses, 8 h; conditions for separating small fragments were: (C) 1.5% agarose, 6 V/cm, 5-10 s pulses, 24 h. Fragments D and E overlapped because of their extremely similar migration; overlap was

also found for fragments G1/G2, O/P/N, and S/T. SAP1: 94.3-kb linear plasmid. Solid arrows: missing fragments; Open arrows: potential missing fragments; Triangles: new bands. Among the rearrangement types of SA mutants, the AseI profile of SA1-6 showed no novel bands apart from the deleted fragments (Fig. 1B and 1C). On the other hand, the AseI profile of SA1-8 revealed two new fragments, and was quite similar to that of 76-9 (Fig. 1D), suggesting that SA1-8 and 76-9 may share Calpain the same chromosomal structure. Therefore, SA1-6 and SA1-8 were selected for further study of chromosomal architecture. Both the linear chromosome and plasmid maintain a circular conformation in vivo because of the interaction of two terminal proteins. When intact DNA samples are MAPK inhibitor treated with Proteinase K (PK), the covalently bound terminal proteins are removed and the DNA acquires a linear conformation. Whereas the intact DNA in the SDS-treated sample is trapped in the slot, since just noncovalently bound proteins are removed and the linear DAN keeps a circular form [3]. It has been reported that the wild-type strain ATCC31267 has a linear chromosome and a linear plasmid SAP1 of 94.3-kb [4].