Abstract
A simple method for inserting foreign genes into the T-region of Agrobacterium Ti-plasmids is described. A modified cosmid (pHC 79) was introduced into a predetermined site of the T-region of pTi C58. An Agrobacterium strain harboring this modified Ti-plasmid was used as an acceptor strain into which genes, cloned in pBR322, can be introduced by mobilization from Escherichia coli. pBR322-derived plasmids cannot replicate in Agrobacterium, but can be maintained by integration into the T-region of the modified Ti-plasmids by homologous recombination. This method was used to introduce the genes for ovalbumin and alpha-actin from chicken into tobacco tumors. Southern blotting and re-isolation of the inserted genes by reverse cloning showed that the animal DNA was transferred and integrated into the plant genome without rearrangements. The alpha-actin gene is not transcribed in plant tumors, whereas transcription of the ovalbumin gene was observed, however the initiation point of transcription was different from the one used in the chicken oviduct. The RNA transcribed from the chicken ovalbumin gene is polyadenylated and ranges in size between 2 and 7 kb.