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.
A highly purified RNA polymerase preparation from pea chloroplasts has been shown to specifically transcribe the 16S rRNA gene in vitro using the recombinant pCB2-8 DNA as a template. The RNA polymerase has been found to show maximum activity and specificity with pea supercoiled rDNA as a template. At low concentrations of ribonucleoside triphosphates, the RNA polymerase selectively initiates transcription on the 16S rRNA gene. A part of the 16S rRNA gene has been sequenced. The mature 16S rRNA has been found by S1 nuclease analysis to contain sequences starting from GAAGCT. The in vitro synthesized RNA has been found to protect the same nucleotides GAAGCT. In addition, the in vitro synthesized RNA was also found to strongly protect bases starting with TATG located at about 260 bases away from the start site of the mature 16S rRNA.
Eight transfer RNA (tRNA) genes which were previously mapped to five regions of the Pisum sativum (pea) chloroplast DNA (ctDNA) have been sequenced. They have been identified as tRNA(Val)(GAC), tRNA(Asn)(GUU), tRNA(Arg)(ACG), tRNA(Leu)(CAA), tRNA(Tyr)(GUA), tRNA(Glu)(UUC), tRNA(His)(GUG), and tRNA(Arg)(UCU) by their anticodons and by their similarity to other previously identified tRNA genes from the chloroplast DNAs of higher plants or from E. gracilis. In addition,two other tRNA genes, tRNA(Gly) (UCC) and tRNA(Ile)(GAU), have been partially sequenced. The tRNA genes are compared to other known chloroplast tRNA genes from higher plants and are found to be 90-100% homologous. In addition there are similarities in the overall arrangement of the individual genes between different plants. The 5' flanking regions and the internal sequences of tRNA genes have been studied for conserved regions and consensus sequences. Two unusual features have been found: there is an apparent intron in the D-loop of the tRNA(Gly)(UCC), and the tRNA(Glu)(UUC) contains GATTC in its T-loop.
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.
Silica gel G is mixed with an aqueous solution of ferric chloride, and the resulting slurry spread onto glass plates. After spotting, development, and drying, the plates are sprayed with sulfuric acid. Cholesterol, ergosterol, and Δ5-pregnen-3β-ol-20-one produce colored spots (pink-violet, greenish black, and pink-violet, respectively) without further treatment, but some other steroids require heating at 70 °C for full color development. Other colors generated are: estrone, orange; Δ4-androsten-3,17-dione, blue-green; androsterone, light brown; and 5α-androstan-17β-ol-3-one, tan. After prolonged heating at 70 °C, 6-keto-cholestanol produces an orange-brown spot. Color reactions are more reproducible than they are when the ferric chloride is sprayed on the plate.
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