Characterization of the gene and mRNA of the large subunit of ribulose-1,5-bisphosphate carboxylase in pea plants.

Abstract

mRNA coding for the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase was obtained by fractionating chloroplast polysomes on an affinity column, using anti-ribulose-1,5-bisphosphate carboxylase immunoglobulin G. Approximately 20%o of the polysomal RNA specifically bound to the affinity column. LS mRNA was also isolated by fractionating chloroplast polysomal RNA on sucrose gradients. The LS mRNA fraction was identified by translation in vitro followed by immunoprecipitation with anti-ribulose-1,5-bisphosphate carboxylase immunoglobulin G. Labeled LS mRNA was hybridized to a genomic digests of pea chloroplast DNA. The LS gene was localized on a 3.55-kilobase pair BamHI fragment in Sall-SmaI DNA fragment 4. The BamHI fragment containing the LS gene was cloned, and a restriction endonuclease map was constructed. The LS gene was localized on a 1.9-kbp KpnI-EcoRI fragment. The LS gene was analyzed by electron microscopy, using the R loop mapping technique. LS mRNA was colinear with the gene, and its size was 1.35 ± 0.2 kilobase pairs. When the LS mRNA was analyzed on methylmercury agarose gels, it comigrated with the 16S rRNA. The direction of transcription of the LS gene was in the same direction as that of the rRNA genes.

Sequence organization of a pea chloroplast DNA gene coding for a 34,500-dalton protein.

“…This gene is located at 67 kbp from the 3' end of the 23S rRNA gene and is transcribed in the same direction as the rRNA gene and the LS gene (23 (25). Similarly, at -102 nucleotides there is a sequence, TATACT, that is homologous to the sequence TATAAT of the E. coli promoters (25).…”

“…80 polypeptides. However, only a few proteins have phosphate carboxylase (LS) has been extensively studied (2,17,19,23,31). The genes for the three polypeptides a, 3, and e subunits of the coupling factor (CF1) (K. K. Oishi, Ph.D. thesis, University of California, Irvine, 1983) (15,28), and the cytochrome f gene (29) have also been mapped in ctDNA.…”

Highly purified pea chloroplast RNA polymerase transcribes both rRNA and mRNA genes

“…Lanes 1-5, same as A; pBR322 (HaeIII restriction) markers are indicated on the side (M). (D) Diagram of the PstI -XbaI restriction fragment from pCB1-3 recombinant containing upstream sequences of rbc-L gene from pea (Oishi and Tewari, 1983;Mullet et al, 1985). The restriction sites are indicated by downward arrows.…”

“…Lane 1, the control without antibodies; lane 2, tagetitoxin (2 units/assay); lane 3, the preimmune antibodies, lane 4, the native chloroplast RNA polymerase antibodies. pBR322 (HaeIII restriction) markers are indicated on the left side (M) and transcripts commencing at the -180 and -65 transcriptional start sites on the right side ing the pea rbc-L mRNA promoter regions from the XbaIPstI restriction fragment of pCBl-3 recombinant (Oishi and Tewari, 1983;Mullet et al, 1985; Fig. 5D).…”

“…5E, lane 2) genes. It may be noted that the tagetitoxin effect on the inhibition of transcription was not similar to that observed with the purified antibodies against native chloroplast RNA polymerase antibodies as tagetitoxin did not affect the promoter binding of the chloroplast RNA polyTo determine if the same chloroplast RNA polymerase was also active in transcribing an mRNA gene, the supercoiled pCB1-3 recombinant (Oishi and Tewari, 1983) was employed merase determined by the DNA mobility shift assay (data not shown).…”