Gene, 10 (1980) 329-338 329 © Elsevier/North-Holland Biomedical Press IS-like element IS8 in RP4 plasmid and its involvement in cointegration (Fusion of RP4 and Ti replicons;Agrobacterium tumefaciens; direct repeats) A. Depicker, M. De Block, D. Inz6, M. Van Montagu and J. Schell Laboratorium voor Genetica en Histologie, Ri/ksuniversiteit Gent, B-9000 Gent (Belgium) (Received February 6th, 1980) (Accepted in revised form March 31st, 1980) SUMMARY The structure of the cointegrate plasmids formed by fusion of RP4 and the tumour-inducing plasmid (pTi) of Agrobacterium tumefaciens was analyzed. In all of the nine independently isolated pTi: :RP4 cointegrates, the integration occurred at the same site on the RP4 genome. Moreover, a 1.2 Md (1750 bp) RP4 sequence (IS8) was directly repeated at both junction sites of the two replicons. The insertion of RP4 generated deletions, starting from the IS8 sequence and extending into the Ti part of the cointegrate. Dissociation of the cointegrates resulted in wild-type RP4 and Ti-plasrnids with the IS8 sequence inserted at the original RP4 insertion site. The processes of integration and dissociation and the genetic properties of the cointegrates indicate that the IS8 sequence has unique characteristics defining a new insertion sequence. INTRODUCTION RP4, a broad host-range plasmid of the P incom- patibility group, specifies resistance to ampicillin, tetracycline and kanamycin (Datta et al., 1971). The functional map of RP4 was derived by insertion of transposon Tn7 at different sites within RP4 (Barth and Grinter, 1977; Barth et al., 1978). Com- parison of the restriction map of RP4 (Depicker et al., 1977) with that of the plasmid RK2 (Meyer et al., 1978), RP1 (Grinsted et al., 1977) and R68 (Leemans et al., 1980) indicates that all four are dif- ferent isolates Of an identical plasmid. RP4 or related plasmids have been frequently used to extend- by mobilisation-the host-range of other plasmids Abbreviations: bp, base pairs; IS, insertion sequence; Md, megadaltons;pTi, tumour-inducing plasmid. (Hedges and Jacob, 1976); e.g. the pTi of Agrobacteri- um tumefaciens can be transferred by mobilisation with RP4 (Levin et al., 1976; Chilton et al., 1976; Van Larebeke et al., 1977; Hooykaas et al., 1977). Holsters et al. (1978a) reported the isolation of cointegrate pTi::RP4 plasmids, which were sub- sequently used to study the expression of the Ti- plasmid phenotypes in Escherichia coli (Hoisters et al., 1978b), a host in which the Ti-plasmids nor- mally cannot replicate. Hemalsteens et al. (1978) have used the pTi::RP4 cointegrates to isolate Ti- plasmids which carry the Tn7 transposon. pTiB6S3 and pTiC58, respectively, are octopine and nopaline tumour-inducing plasrnids of Agrobac- terium. In the present paper we describe the physical study of a pTiB6S3::RP4 cointegrate and eight independently isolated pTiC58::RP4 cointegrates. Several deletion derivatives and dissociation products