Two mRNA species differing by 258 nucleotides at the 5′ end are formed from the barley chloroplast rbcL gene

Abstract

The region of the barley chloroplast genome encoding the 5′ third of the rbcL gene has been subcloned in M13 vectors and sequenced by the dideoxy chain termination method. Some of the recombinant M13 clones have been used as a template for the synthesis of highly radioactive single stranded DNA probes. These are specifically hybridizing to short regions at the 5′ end of the two transcripts, which were previously found to be encoded by the rbcL region in barley cpDNA. The two transcripts differ in size by more than 200 nucleotides, are encoded by the same strand and contain the full coding capacity for the large subunit of ribulose bisphosphate carboxylase-oxygenase. The short transcript is predominant in dark grown seedlings. Of two probes employed in this study one hybridized only to the long and the other to both rbcL mRNA species. Thereby it was possible to quantitate the rbcL mRNAs by hybridization with the probes and subsequent S1-nuclease digestion of the single stranded molecules. I found this mRNA to be aproximately 1.38% of total plastid RNA (excluding the small rRNAs and the tRNAs) in dark grown seedlings and 2.20% in greened seedlings. The two probes were also used in primer extension experiments using the two transcripts as template. After hybridizing the probe to the mRNA reverse transcription was performed in the presence of cold deoxynucleotides. The reverse transcribed strands were separated on sequencing gels. The probe which acted as a specific primer for the short transcript was extended by 29 nucleotides, and the probe specific for the longer transcript was extended by 91 nucleotides. The end point of the latter transcript was only found with template RNA from plastids of greened barley seedlings. Thus it was shown that the 5′ ends of the two transcripts begin at position-58 and-316 from the ATG triplet of the rbcL reading frame. A comparison of the sequences upstream from the ATG codon of the rbcL gene of barley with those of maize, spinach and tobacco revealed completely conserved prokaryote type transcription promotors in front of the 5′ end of the long transcript, while no such promotors are evident close to the 5′ end point of the short transcript. The two monocot species differ from the dicot species by an insertion of about 140 nucleotides in the mRNA leader sequence.