Instability of SD repeats shown by restriction enzyme analyses.

Abstract

<p>(A) pNZ3004-ClfA.SAR.rRWM.C9 plasmid map showed locations of the restriction enzymes and sizes of different segments. (B) After 3 rounds of propagation, instability of constructs pNZ3004-ClfA.SAR.rRWM.C9.C1 was detected by restriction enzyme analyses. Lane 1: Uncut plasmid; Lane 2: Digested by <i>Bam</i>HI; Lane 3: Digested by <i>Bam</i>HI/<i>Sal</i>I; Lane 4: Digested by <i>Pst</i>I; Lane 5: Digested by <i>Sal</i>I; Lane 6: Digested by <i>Eco</i>RI; Lane 7: <i>Bam</i>HI restriction enzyme activity control. Plasmid PF01 digested by <i>Bam</i>HI; Lane 8: ultraRanger 1 kb DNA ladder (Kb). Arrows A and B indicated the incomplete digestion by <i>Bam</i>HI and the failed double digestion by <i>Bam</i>HI/<i>Sal</i>I, respectively. Arrow C showed the mixture of the shorter plasmid DNA. Arrow D revealed the rearrangement in the region between two <i>Pst</i>I sites. (C) The rearranged variants from the transformants of pNZ3004-ClfA.SAR.rRWM.C9.C7 digested by <i>Pst</i>I. Lane 1: pNZ3004-ClfA.SAR.rRWM.C9.C7.C1 (deletion in SD repeats between SAR and rRWM). Lane 2: pNZ3004-ClfA.SAR.rRWM.C9.C7.C2 (deletion in SD repeats between SAR and rRWM). Lane 3: pNZ3004-ClfA.SAR.rRWM.C9.C7.C3 (deletion in SD repeats between SAR and rRWM). Lane 4: pNZ3004-ClfA.SAR.rRWM.C9.C7.C4 (deletion in SD repeats between SAR and rRWM). Lane 5: pNZ3004-ClfA.SAR.rRWM.C9.C7.C5 (deletion in SD repeats between SAR and rRWM). Lane 6: pNZ3004-ClfA.SAR.rRWM.C9.C7.C6 (deletion in SD repeats between SAR and rRWM). Lane 7: pNZ3004-ClfA.SAR.rRWM.C9.C7.C7 (expansion in SD repeats between SAR and rRWM). Lane 8: pNZ3004-ClfA.SAR.rRWM.C9.C7.C8 (deletion in SD repeats between SAR and rRWM). Lane 9: pNZ3004-ClfA.SAR.rRWM.C9.C7.C9 (no rearrangement). Lane10: pNZ3004-ClfA.SAR.rRWM.C9.C7.C10 (deletion in SD repeats between SAR and rRWM). (D) The rearranged variants from the transformants of pNZ3004-ClfA.SAR.rRWM.C9.C7 double-digested by <i>Bam</i>HI and <i>Sal</i>I. Lane 1: pNZ3004-ClfA.SAR.rRWM.C9.C7.C1 (rearrangement occurred between SAR and rRWM, lost <i>Bam</i>HI site). Lane 2: pNZ3004-ClfA.SAR.rRWM.C9.C7.C2 (rearrangement occurred between SAR and rRWM, lost <i>Bam</i>HI site). Lane 3: pNZ3004-ClfA.SAR.rRWM.C9.C7.C3 (rearrangement occurred between SAR and rRWM, lost <i>Bam</i>HI site). Lane 4: pNZ3004-ClfA.SAR.rRWM.C9.C7.C4 (rearrangement occurred between SAR and rRWM, lost <i>Bam</i>HI site). Lane 5: pNZ3004-ClfA.SAR.rRWM.C9.C7.C5 (rearrangement occurred between SAR and rRWM, lost <i>Bam</i>HI site). Lane 6: pNZ3004-ClfA.SAR.rRWM.C9.C7.C6 (rearrangement occurred between SAR and rRWM, lost <i>Bam</i>HI site). Lane 7: pNZ3004-ClfA.SAR.rRWM.C9.C7.C7 (SAR segment increase, may have expansion in SD repeats of SAR). Lane 8: pNZ3004-ClfA.SAR.rRWM.C9.C7.C8 (deletion in SD repeats of SAR). Lane 9: pNZ3004-ClfA.SAR.rRWM.C9.C7.C9 (no rearrangement). Lane10: pNZ3004-ClfA.SAR.rRWM.C9.C7.C10 (rearrangement occurred between SAR and rRWM, lost <i>Bam</i>HI site). Lane 11: ultraRanger 1 kb DNA ladder (Kb).</p

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