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Bordetella bacteriophages generate diversity in a gene that specifies host tropism. This microevolutionary adaptation is produced by a genetic element that combines the basic retroelement life cycle of transcription, reverse transcription and integration with site-directed, adenine-specific mutagenesis. Central to this process is a reverse transcriptase-mediated exchange between two repeats; one serving as a donor template (TR) and the other as a recipient of variable sequence information (VR). Here we describe the genetic basis for diversity generation. The directionality of information transfer is determined by a 21-base-pair sequence present at the 3' end of VR. On the basis of patterns of marker transfer in response to variant selective pressures, we propose that a TR reverse transcript is mutagenized, integrated into VR as a single non-coding strand, and then partially converted to the parental VR sequence. This allows the diversity-generating system to minimize variability to the subset of bases under selection. Using the Bordetella phage cassette as a signature, we have identified numerous related elements in diverse bacteria. These elements constitute a new family of retroelements with the potential to confer selective advantages to their host genomes.

Transposase (tnp) expression from insertion sequence IS10 is controlled, in part, by an antisense RNA, RNA-OUT, which pairs to the translation initiation region of the tnp mRNA, RNA-IN. Genetic experiments suggest that control occurs post-transcriptionally. Here, we present evidence that bears on the control mechanism. Specific ribosome binding at the tnp translation initiation site is demonstrated in vitro. Two mutations that alter tnp translation in vivo are shown to have corresponding effects in vitro. Most importantly, RNA-OUT/RNA-IN pairing is shown to block ribosome binding. In conjunction with the work described in the accompanying paper, we propose that inhibition of ribosome binding also occurs in vivo, and that it is sufficient to account for control. Implications for translational control in analogous systems are discussed.

RNA stability is an important component of gene expression, and antisense RNAs have been proposed to alter target RNA stability. We show here that the IS10 transposase mRNA, RNA-IN, is rendered unstable during control by the IS10 antisense RNA, RNA-OUT. Destabilization requires RNA-OUT/RNA-IN pairing and ribonuclease III cleavage. Independent of such cleavage, RNA-OUT is rendered unstable through disruption of its secondary structure. Pairing has no other obvious effects on RNA-IN transcription or stability. Nevertheless, RNA-IN destabilization is not required for antisense control in vivo. In the accompanying paper [Ma,C. and Simons, R.W. (1990) EMBO J., 9, 1267-1274 we show that pairing blocks ribosome binding to RNA-IN. Were it not for control at this level, destabilization would play a more prominent role.

IS10 transposition is regulated by an approximately 70 nt anti-sense RNA, RNA-OUT. RNA-OUT folds into a duplex 'stem-domain' topped by a loosely paired 'loop-domain'. The loop-domain is critical for RNA-RNA pairing per se; pairing initiates by interaction of the RNA-OUT loop with the 5' end of the target mRNA. We show here that RNA-OUT is unusually stable in vivo (half-life 60 min) and that this stability is conferred by specific features of the RNA-OUT stem-domain. One critical feature is stable base-pairing: mutations that disrupt stem pairing destabilize RNA-OUT in vivo and abolish anti-sense control; combinations of mutations that restore pairing also restore both stability and control. We propose that the stem renders RNA-OUT resistant to 3' exoribonucleases. Other features of the stem-domain prevent this essential duplex from being an effective substrate for double-strand nucleases: two single base mutations disrupt antisense control by making RNA-OUT susceptible to RNase III. Mutations in the loop region have little effect on RNA-OUT stability. Implications for IS10 biology and the design of efficient anti-sense RNAs are discussed.