Physical mapping, nucleotide sequencing and expression in E. coli minicells of the gene for the large subunit of ribulose bisphosphate carboxylase from Petunia hybrida

Abstract

The Petunia hybrida rbcL gene was identified and located on the physical map within the Sall S9 fragment of the Petunia hybrida cpDNA by heterologous hybridization with the cloned rbcL gene of spinach (pSoc3BE148). In E. coli minicells harbouring the S9 fragment inserted into pBR322, the rbcL polypeptide is synthesized as was shown by molecular weight determination, immunoprecipitation and proteolytic digestion. However, the size of the rbcL polypeptide synthesized in minicells appeared to be dependent on the orientation of the S9 fragment in pBR322. In minicells harbouring the S9 fragment inserted into pBR322 in the clockwise orientation the molecular weight of the rbcL polypeptide is approximately 53 kD, whereas in minicells harbouring the S9 fragment in the opposite orientation, the rbcL polypeptide synthesized has a molecular weight of 52 kD. The difference in molecular weight of the two rbcL polypeptides is the result of transcription and translation into the flanking pBR322 sequences. This is due to the absence of the terminal part (6 codons), including the translation stop codon, of the rbcL gene on the cloned S9 fragment as was determined by nucleotide sequencing. The observed expression of the cloned part of the rbcL gene of Petunia hybrida indicates that the E. coli minicell system can be used as a suitable and convenient system for the identification and physical mapping of chloroplast genes.Comparison of the sequence of the untranslated 3'-end of the rbcL gene of Petunia hybrida with that of Nicotiana tabacum revealed a striking similarity of the region in which stem and loop structures can be formed that are most likely involved in termination of transcription of the rbcL gene. This region appears to be highly conserved in the rbcL genes of P. hybrida, N. tabacum, S. oleracea and Z. mays.