Browsing by Subject "Chloroplasts"

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Distinct roles for the 5' and 3' untranslated regions in the degradation and stability of chloroplast tufA mRNA
(2005) Zicker, Alicia A.; Herrin, David L.
The tufA gene encodes the chloroplast elongation factor Tu and is found in the chloroplast genome in Chlamydomonas reinhardtii, whereas it is in the nucleus in land plants. When C. reinhardtii is treated with chloramphenicol (CAP), an inhibitor of ribosome elongation, the stability of the 1.7-kb tufA mRNA is increased, and a RNA of ~1.5 kb over-accumulates. When CAP and the transcription inhibitor rifampicin are added together, tufA mRNA can be chased into the 1.5-kb RNA (which is subsequently degraded), suggesting that it is an intermediate in the degradation pathway. Mapping of the 5' and 3' ends of the 1.5-kb RNA showed that it is truncated at the 5' end, and is missing 177-181 nucleotides; there was no evidence of truncation at the 3' end. The 5' end of this intermediate lies 76-80 nucleotides upstream of the tufA start codon, and within a vii small ORF that has predicted secondary structure. Interestingly, the degradation intermediate did not over-accumulate in a chloroplast ribosome-deficient mutant, suggesting that its stabilization requires elongation-arrested ribosomes. Finally, evidence was obtained that the intermediate accumulates in the absence of CAP, especially in older cultures. These results indicate that a major pathway for degradation of tufA mRNA in vivo begins at the 5' end of the RNA, probably with an endonucleolytic cleavage, but 5' to 3' exonuclease activity cannot be ruled out. To determine whether the 5' or 3' UTR of tufA is destabilizing, the corresponding regions of an atpA-aadA-rbcL gene (which gives high mRNA levels) were replaced with one or both tufA UTRs, and transformed into the chloroplast. Northern blot analysis of the transformants indicated, quite surprisingly, that the 3' UTR of tufA is strongly destabilizing. The results suggest that the UTRs of tufA have distinct, but also interacting roles in mediating decay of tufA mRNA. To determine if the 3' UTR of tufA can regulate protein expression, constructs were made using Renilla luciferase as reporter, and transformed into chloroplasts. Luminescence measurements of the transformants indicated that the 3' UTR of tufA can regulate expression at the protein level, but no more so than the 5' UTR
Molecular systematics of the Loasaceae
(1995-12) Hempel, Alice Louise; Not available
Mutagenesis and suppression of a light-regulated group I intron in the chloroplast psbA gene of Chlamydomonas reinhardtii
(2003) Lee, Jaesung; Herrin, David L.
Four chloroplast psbA introns in the green alga Chlamydomonas reinhardtii (Cr.psbA1-Cr.psbA4) are self-splicing group I introns and their splicing is lightpromoted. Because evidence indicates that some chloroplast mRNAs are in great excess over what is needed to sustain translation rates, the physiological significance of light-promoted psbA splicing has not been clear. Also, splicing factor(s) for these introns have not been identified. I have generated several point mutations in the core helices of the Cr.psbA4 intron, and tested their effects on self-splicing in vitro. I also replaced the wild-type intron in vivo with each mutant, and compared the in vitro self-splicing data to the effects of these mutations in vivo. The results indicate that destabilizing mutations in the intron core have less effect in vivo than in vitro, suggesting that there is considerable stabilization of the intron’s active structure in vivo. Interestingly, however, there were approximate correlations between in vivo Cr.psbA4 splicing efficiency, synthesis of full-length D1, and autotrophic growth rates. For example, the P4′-3,4 mutant, which showed a 45 % reduction in psbA mRNA, had a 28 % decline in synthesis of full–length D1, and a 18 % decline in photoautotrophic growth rate. These results indicate that psbA mRNA is not in great excess, and that efficient splicing of the psbA introns, which is light-dependent, is essential for maintaining robust autotrophic growth. I have also found that three nuclear suppressors of a mutated 23S rRNA intron (7120, 7151, and 71N1) also suppressed the P4′-3,4 mutation in the Cr.psbA4 intron. This was accomplished by replacing the wild-type Cr.psbA4 intron in the suppressors with the P4′-3,4 mutant; higher steady-state levels of spliced psbA mRNA and less precursor was observed in the suppressors. This result indicates that these genes play a role in splicing of multiple group I introns in the chloroplast. In addition, further genetic analysis was performed with the 7120 suppressor, which had been only partially characterized previously. Tetrad analysis of a cross of 7120 (mt+) with wild-type (mt-) indicated that it is a single nuclear gene mutation. Complementation analysis in diploids demonstrated that the suppressor mutation is dominant. We propose to call this gene css2 (for chloroplast splicing suppressor).
Nuclear genes that promote splicing of the chloroplast group-I 23S rRNA intron and an organelle intron database, FUGOID
(2002-08) Li, Fei; Herrin, David L.
Cr. LSU is a self-splicing Group I intron in the chloroplast 23S rRNA gene of the green alga Chlamydomonas reinhardtii. Trans-acting factors are likely required to promote the splicing of this intron in vivo; however, nothing is known of the trans requirements for Group I intron splicing in chloroplasts. I have used a genetic approach in this study of possible splicing factors for Cr. LSU. Single nucleotide substitutions were made in the core helices P4, P6, and P7, and in the metal-binding GAAA motif in the J4/5 region of the intron. In vitro assays showed that these substitutions had very strong effects on Cr.LSU self-splicing; however, splicing of all but the P6 mutations could be partially recovered by vi increasing the Mg2+ concentration, indicative of structural effects. The mutant constructs were transformed into chloroplasts to replace the wild-type intron. Surprisingly, only the P4 mutants became homoplasmic, indicating that the other mutations were lethal. The splicing-deficient P4 125A mutant, which exhibited slow growth and light sensitivity, was used to isolate suppressor strains that showed a substantial restoration of Cr.LSU splicing. Genetic analysis of the 7151, 7120 and 71N1 suppressors indicated that these mutations are in at least 2 nuclear genes. The 7151 suppressor, which defines the css1 (chloroplast splicing suppressor) gene, was shown to have no discernible phenotype with the wild-type intron, and to be dominant in vegetative diploids containing the mutant intron. These results indicate that the Cr.LSU intron is unusually sensitive to single base changes in the core, and, moreover, provide the first identification of plant genes that promote splicing of a group I intron in vivo. Toward the eventual cloning of the css1 gene, a library was constructed in a novel cosmid vector, SCBN, using genomic DNA from the 7151 suppressor mutant of C. reinhardtii. Part of the library was used to generate an indexed collection of 10,340 individual clones in 110 96-well microtiter plates. The library can be easily replicated (for DNA preparation) and has an average insert size of ~37 kb. Thus, it covers almost 4 genome equivalents. The library may allow the cloning of css1 by complementation of the P4 125A mutant. Finally, I have developed a web-based relational database, FUGOID (Functional Genomics of Organellar Introns Database) that collects and integrates vii viii various functional (and some structural) data on organellar (mitochondrial and chloroplast)introns. The main information provided by FUGOID includes intron sequence, subclass, resident ORF, self-splicing capability, host gene, protein factor(s) involved in splicing, mobility, insertion site, twintron, seminal references and taxonomic position of host organism. Users can access the database with any common web browser on a variety of operating systems. The main page of the database is available at http://wnt.cc.utexas.edu/~ifmr530/introndata/main.htm.
Starch deposition in Cosmarium subtumidum
(1933) Cuyler, Esther Arsinoé Solcher; McAllister, F. (Frederick), 1876-1949