A gene (PGII), which codes for a 34.5-kilodalton protein, has been isolated and cloned from pea chloroplast DNA. The production of its 1.2-kilobase mRNA is photodependent. The direction of transcription has been determined, the site of initiation of transcription has been found, and an in vitro protein product has been produced. The gene, including the 5' and 3'-flanking regions, has been sequenced. It shows ca. 95% homology to the photosystem II thylakoid membrane protein, photogene 32, from spinach and tobacco. There are no intervening sequences. The 5'-flanking region suggests similarities with Escherichia coli promoters. The 5'-flanking region is remarkably conserved among pea, spinach, and tobacco DNA.Chloroplasts from higher plants contain covalently closed circular DNAs which range in size from 119 to 150 kilobase pairs (kbp) (7,13,14). Physical mapping studies of chloroplast DNA (ctDNA) in higher plants have shown that ctDNA encodes the rRNA cistrons, 16S, 23S, and 5S genes (3. 6. 21). and ca. 30 to 40 genes for the tRNAs found in the chloroplast (8,20). The relative abundance and stability of these RNA species facilitated their localization in the higherplant ctDNA. The rRNA and tRNA genes account for only 3 to 5% of the coding sequences of ctDNA. Molecular hybridization studies between ctDNA and polysomal RNA show that RNA transcripts present in chloroplasts hybridize to ca.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.