An Introduction

Abstract

A prerequisite to the development of gene cloning and expression of homologous and heterologous genes in any organism is the identification of a suitable vector for the introduction and stable maintenance of cloned DNA in the host. During progressive application of rDNA technology in Streptomyces, the problems of efficient introduction of vectors by transformation of protoplasts were solved. However, the problems of stable maintenance of the cloned DNA in the host still persist. Streptomyces strains are often genetically unstable (Schrempf et al., 1987) and their mycelial growth habit hampers the study of plasmid instability. In general, two types of instability are observed and studied in gram-positive bacteria: one corresponding to the loss of the entire vector from the cell, the other to rearrangements, most often deletions of plasmid sequences. The first was named segregational, the second structural plasmid instability (Ehrlich et al., 1987). Recently attempts to study the problems of plasmid segregational instability in Streptomyces were reported. Because of their mycelial growth habit and irregular cell division, plasmids do not segregate into individual cells after replication. This fact makes investigation of plasmid inheritance difficult in Streptomyces. Many vectors currently used in Streptomyces are derivatives of pIJ101 (Kieser et al., 1982). It is interesting to note that no evidence exists for a par locus on such plasmids which replicate via rolling circle, i.e. there are no membrane attachment sites that physically aid segregation of sufficient numbers of plasmids to be established in each daughter cell (Cesareni et al., 1987). In these plasmids, stability seems to be coupled with replication and not with a discrete par-like function (Gruss and Ehrlich, 1989).