The use of pNJ5000 as an intermediate vector for the genetic manipulation of Agrobacterium Ti-plasmids

Abstract

The use of broad-host-range plasmids derived from RP4 as intermediate vectors for the transfer of narrow-host-range recombinant plasmids from Escherichia coli to Agrobacterium tumefaciens as a preliminary to marker exchange is described. Recombinant plasmids having a ColE1 type origin were linked to the RP4 derivative. Cointegrate formation appeared to take place by RecA-independent, homologous recombination within a short piece of DNA derived from the beta-lactamase gene of Tn1/Tn3 carried by both vector components, so that it never disrupted the recombinant portion of the construction. pNJ5000 provides an unstable intermediate vector for use in marker exchange experiments, while its stable relative pNJ1020 provides a carrier for use in binary vector systems.