Showing papers by "Charles R. Hauser published in 1998"

Isolation and characterization of a mutant protoporphyrinogen oxidase gene from Chlamydomonas reinhardtii conferring resistance to porphyric herbicides

Abstract: In plant and algal cells, inhibition of the enzyme protoporphyrinogen oxidase (Protox) by the N-phenyl heterocyclic herbicide S-23142 causes massive protoporphyrin IX accumulation, resulting in membrane deterioration and cell lethality in the light. We have identified a 40.4 kb genomic fragment encoding S-23142 resistance by using transformation to screen an indexed cosmid library made from nuclear DNA of the dominant rs-3 mutant of Chlamydomonas reinhardtii. A 10.0 kb HindIII subclone (Hind10) of this insert yields a high frequency of herbicide-resistant transformants, consistent with frequent non-homologous integration of the complete RS-3 gene. A 3.4 kb XhoI subfragment (Xho3.4) yields rare herbicide-resistant transformants, suggestive of homologous integration of a portion of the coding sequence containing the mutation. Molecular and genetic analysis of the transformants localized the rs-3 mutation conferring S-23142 resistance to the Xho3.4 fragment, which was found to contain five putative exons encoding a protein with identity to the C-terminus of the Arabidopsis Protox enzyme. A cDNA clone containing a 1698 bp ORF that encodes a 563 amino acid peptide with 51% and 53% identity to Arabidopsis and tobacco Protox I, respectively, was isolated from a wild-type C. reinhardtii library. Comparison of the wild-type cDNA sequence with the putative exon sequences present in the mutant Xho3.4 fragment revealed a G→A change at 291 in the first putative exon, resulting in a Val→Met substitution at a conserved position equivalent to Val-389 of the wild-type C. reinhardtii cDNA. A sequence comparison of genomic Hind10 fragments from C. reinhardtii rs-3 and its wild-type progenitor CC-407 showed this G→A change at the equivalent position (5751) within exon 10.

Regulation of Chloroplast Translation

Abstract: During the evolution of chloroplasts from cyanobacterial endosymbionts, many genes encoding components necessary for protein synthesis and photosynthesis have been transferred to the nucleus. Assembly of the machinery for both processes now relies on the concerted expression of genes in the nuclear and plastid genomes. Evidence accumulating in C. reinhardtii indicates that the expression and regulation of chloroplast genes probably differs in certain respects from the prokaryotic (E. coli) paradigm. Both mutational analysis of putative Shine-Dalgarno (SD) sequences, and creation of canonical SD sequences (-9 to -5) reveals that initiation is not mediated by SD-16S rRNA interactions for the majority of chloroplast-encoded mRNAs. Recent evidence in cyanobacteria, the most likely ancestors of the Chlamydomonas chloroplast, indicates that most genes lack SD sequences at the classical position. Interactions between cis-acting sequences in the 5′ untranslated regions (UTRs) and specific trans-acting nuclear gene products appear to mediate translation of chloroplast-encoded mRNAs for specific genes. Both UV crosslinking and gel mobility shift assays with several chloroplast leaders show that multiple proteins interact with these sequences. Some of these appear to be involved in gene-specific regulation, whereas others may be core proteins of a general ribonucleoprotein (RNP) complex. Inverted repeat sequences present in most chloroplast leaders predict higher order structures within the 5′ UTR of mRNAs that could serve as scaffolding for the formation of the RNP complex. One model predicts that the RNP complex directly enhances ribosome binding and translation initiation. A second model predicts that the RNP complex plays a role in making topological distinctions in the sites of synthesis of chloroplast mRNAs. A challenge in the coming years will be to determine which, if any, of these models are correct.