Variants of a cloned synthetic lactose operator II. Chloramphenicol-resistant revertants retaining a lactose operator in the CAT gene of plasmid pBR325

The chloramphenicol transacetylase (CAT) gene of plasmid pBR325 is inactivated by insertion of a 40-bp lactose operator DNA fragment into the EcoRI site located 216 bp downstream of the initiation codon of the gene. Rare spontaneous Cm r revertants have been selected, some of which still contain a functional operator in the CAT gene. DNA sequencing in the operator region of two revertant plasmids revealed that both had suffered single base-pair deletions just upstream of the operator segment: ▪ In the revertant pOE223, the deletion converts the “upstream” EcoRI site to GAATC (a site for HinfI), while in pOE222 both EcoRI sites flanking the operator remain intact. Removal of the operator from pOE222 creates a new plasmid pJT7 in which CAT gene contains a (−1) frameshift at position 213. Reinsertion of the 40-bp operator, in the orientation shown above, recreates pOE222 and restores the Cm r phenotype. However, insertion of the operator in the reverse orientation into pJT7creates a new plasmid, pOE271, with a Cm s phenotype due to an in-phase TAA chain terminating (C.T.) codon in the operator as shown: ▪ Spontaneous, as well as mutT-induced, Cm r revertants of pOE271 have been recovered which still contain a 40-bp operator; ten have been sequenced and shown to contain single-base substitutions at positions 14 or 15 in the operator sequence in the C.T. codon. Three of these mutations significantly reduce repressor affinity for the operator, while the fourth does not.