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DNase I protection experiments have indicated that the cyclic AMP-catabolite gene activator protein complex binds to two regions preceding the chloramphenicol acetyl transferase (cat) gene in Escherichia coli. One of these lies adjacent to the RNA polymerase binding site, whereas the second lies approximately 130 base pairs upstream from the starting point of transcription. Additional DNase protection experiments and in vitro transcription experiments with modified templates indicate that the catabolite gene activator protein site proximal to the cat promoter functions independently of the distal site, indicating that in vitro the second of these sites is not required for transcriptional activation of the cat gene.In Escherichia .coli, the cyclic AMP (cAMP)catabolite gene activator protein (CAP) complex acts as a regulator of certain genes or operons, the most notable being the sugar utilization operons, although several other systems have been shown to be under a similar mode of regulation (20). Early studies with the lac operon suggested that the cAMP-CAP complex might recognize certain sequences in the vicinity of the RNA polymerase binding site (5), and Gilbert (7) has suggested that transcriptional activation could occur by (i) direct interaction between CAP and RNA polymerase or (ii) cAMP-CAPinduced destabilization of DNA. At present there is no direct evidence to suggest which of these mechanisms prevails, and in fact it may be that both are required. Ebright and Wong (6) have presented strong evidence that direct intercalation between the adenyl moeity of cAMP and a thymine residue within the CAP binding site may destabilize the DNA sufficiently to allow enhanced RNA polymerase interaction. Additionally, chemical and enzymatic probes have indicated that CAP and RNA polymerase occupy adjacent sites on the promoter, as in the lac promoter (26), or overlapping sites, as in the araC (10, 18), gal (27), pBR-P4 (21), and ompA (16) promoters, where contact between CAP and RNA polymerase could occur. The promoter for the araBAD operon has a CAP site displaced from the RNA polymerase site and contains an t Present address:

We have physically characterised a deletion mutant of the R plasmid R100 which has lost all of the antibiotic resistances, including chloramphenicol resistance (Cmr), coded by its IS1‐flanked r‐determinant. The deletion was mediated by one of the flanking IS1 elements and terminates within the carboxyl terminus of the Cmr gene. DNA sequence analysis showed that the mutated gene would produce a protein 20 amino acids longer than the wild‐type due to fusion with an open reading frame in the IS element. Surprisingly for a deletion mutation, rare, spontaneous Cmr revertants could be recovered. Two of the four revertants studied had frame shifts due to the insertion of a single AT base pair at the same position; the revertants would code for a protein five amino acids shorter than the wild‐type. The other two revertants had acquired duplications of the 34‐bp inverted terminal repeat sequences of the IS1 element and would direct the synthesis of a protein six amino acids longer than the wild‐type. The reverted Cmr markers were still capable of transposition. These observations suggest a role for point mutations and small DNA rearrangements in the formation of new gene organisations produced by mobile genetic elements.