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214 (1~) 233-252 233 Overlapping direct repeats stimulate deletions in spedally designed derivatives of plasmid pBR325 in Escherichia cell Elias Balbinder, Cheryl Mac Vean and Robert E. Williams Department of Btochern~r~, Bioph),~c~ and Geneti~ and Colorado Cancer "Cev.ter, Universify of Colorado Health Sciences Center, 4200 F-,. 91h Ave., Box B-121, Denver, CO 80262, and Interaational Center for Cancer 2~esearch and Developmental Biology, Denver, CO (U.S.A.) (Received 19 December 1988) (Revision x~-ived 6 March 1989) (Accepted. 6 April 1989) Key~ord~: Deletions on plasmlds; Plasmids, deletions on; DNA, single-stranded, rrds~lignments; Chlormnphenicol acetyltransferase gene Summary Current misalignment mutagenesis models have identified certain sequences such as direct and inverted repeats, which can stab'flize transient misalignments on single-stranded DNA, as major straetural parame- ters for deletions. We have eomtmcted derivatives of the plasmid pBR325 to investigate fm~er the xelative roles of such sequences amder controlled conditions. The plasmid derivatives pOCE15, pRS1 and pRS4, were obtained by cloning fragments of the same approximate size (60-64 bp) but different sequence into the unique EcoR1 site of the ehloramphenieol aeetyl transferase (cat) gene of plasmid pBR325. The inse~t of pOCE15 is a perfeetiy palindromic lac operator fragment. Both pRS1 and pRS4 carry the same non-palindromic fragment but differ from each other in the sequence at the right (3') end of the insert. Plasmid pRS4 differs from pRS1 by a 9-bp duplication containing an additional EcoR1 site at the 3' end of the insert. This arrangement yields overlapping imperfect 17-18 bp and perfect 11 bp direct repeats at the deletion t~ and creates multiple opporttmities for the stab'dization of misaligned intermediates. The deletion.rate, meastwed from the reversion of chloramphenicol sensitivity (Cm~) to resistance (Cmr), was always highest in pRS4, intermediate in pOCE15 and lowest in pRS1, with an approximate ratio of 100:10:1. We have also obtained evidence for the participation of RecA in the genesis of deletions in these pBR325-derived plasmids. Deletions are an import, ant class of genetic rearrangements and have been fonnd to be at the basis of a number of cancers and other genetic diseases (Yunis, 1983; ~scot et ah, 1986; Monaco Correspondence: Dr. Elias Balb~der, D~ax~ment of Biochem- istry, Biophys~s aud Genetics, B-121, U~iv~s~ty of Colorado Health Seience~ Center, 4290 E. 9th Ave., Denver, CO 80262 et al., 1985). They have been extensively studied at the molecular level, mainly in prokaryotes, and most of. them are explained by misalignment mutagenesis models. These propose that deletions arise from the resolution of transient misalign- ments formed by slippage of single-stranded stretches of DNA during replication and stabi- lized by sequence homologies such as terminal direct repeats, inte~,ening inverted repeats (palin- dromes) or a eombinatioa of boih (Albertini et al., 602%5107/89/$03.50 ~ 1989 Ese~ier Sd~ce Publi~ers B.V. (Biomedical Div~on) 40000088