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DELETIONS ON SPECIALLY DESIGNED DERIVATIVES OF PLASMID pBR325 IN ESCHERICHIA COLI by Elias Balbinder, Cheryl Mac Vean and Robert E. Williams Dept. of Biochemistry, Biophysics and Genetics University of Colorado Health Sciences Center 4200 E. 9th Ave. - B-121 Denver, CO 80262 40000205

ABSTP, ACZ DNA fragments cloned into the EcoRl site of the chloramphen- icol acetyl transferase (CAT) gene of plasmid pBR325 are deleted precisely between the two EcoRl restriction sites generated as a result of the insertion. Plasmids pOCEI5, pRSl and pRS4 carry 66- 68 bp fragments inserted at this site. The insert in pOCEI5 is a perfectly palindromic lac operator fragment .pRSI and pRS4 both contain the same non-palindromic insert, but while pRSl has a single EcoRl site at each end pRS4 is asymmetric having one EcoRl site at the 5' end and two tandem EcoRl sites at the 3' end of the insert. Deletion frequency is measured as the reversion from Cms (chloramphenicol sensitivity) to Cmr (chloramphenicol resistance). On these plasmids, the potential to form cruciforms favored deletion between the same terminal repeats by at least a factor of i0 (pOCEI5 vs pRSI) but, surprisingly, the presence of an extra EcoRl site on the 3' side of the non-palindromic insert (pRS4) increased its frequency of deletion 5-10 fold over that of a palindrome (pOCE15). Thus the number and arrangement of direct end repeats can play a major role in determining deletion prone- ness. We observed a slight but consistent increase in deletion rate in rec_~A+ over reck- cells and a somewhat higher increase in the presence of recA 730, a mutant allele of recA+ which makes the SOS response constitutive and also increases homologous recombination frequency. These results show that recA+ and some of its mutant alleles can stimulate the occurrence of deletions on pBR~25 derived plasmids, but do not differentiate between mechanisms involving homologous recombination or SOS processing. 40000208