Expression of Nicotiana Chloroplast Genes in vivo

Expression of Nicotiana Chloroplast Genes in vivo

The plasmid-directed protein synthesis system was employedto test whether the cloned ct-DNA can be expressed in vivo in maxicells CSR 603.Since UV light inhibits chromosomal DNA replication，all the proteins synthesized in the UV irradiated maxicells are plasmiddirected.Thus，the expression of any ct-DNA inserts on the plasmid can be detected.［35 S］methionine was incorporated into almost every protein synthesized by E.coli（CSR 603）without UV treatment［see Figure 5（a）］as compared to Figure 5（b）.However，the polypeptide synthesis directed by the plasmid after UV irradiation is quite evident as revealed by autoradiography［see Figure 5（c）］.Figure 5（c）illustrates the synthesis of a major protein by plasmid pBR322 alone.In this case a 31 kD protein［see Figure 5（c）］was made which is most likely theβ-lactamase of plasmid pBR322 origin（Sancar et al.，1979）.When many cloned fragments of ct-DNA were tested for in vivo expression in this manner plasmid pBR322 carrying ct-DNA inserts of BamHI 7，12，and 16（in clone#17）produced three additional products of approximately 43，29，and 13.5 kD［see Figure 5（c）］.Thus，the in vivo expression of chloroplast genes in E.coliis clearly demonstrated.Furthermore，in seven clones where different inserts were ligated to pBR322，a 2-3 fold enrichment of theβlactamase synthesis was also observed［see Figure 5（c）］.