PubMed Health⌕ Search

Biomedical subjects

J D Rochaix

Publications and source records attributed to J D Rochaix.

At least 55 records · Page 3Linked to original sources
PubMed1995

Isolation of a psaF-deficient mutant of Chlamydomonas reinhardtii: efficient interaction of plastocyanin with the photosystem I reaction center is mediated by the PsaF subunit.

The PsaF polypeptide of photosystem I (PSI) is located on the lumen side of the thylakoid membrane and its precise role is not yet fully understood. Here we describe the isolation of a psaF-deficient mutant of the green alga Chlamydomonas reinhardtii generated by co-transforming the nuclear genome of the cw15-arg7A strain with two plasmids: one harboring a mutated version of the psaF gene and the other containing the argininosuccinate lyase gene conferring arginine prototrophy. This psaF mutant still assembles a functional PSI complex and is capable of photoautotrophic growth. However, electron transfer from plastocyanin to P700+, the oxidized reaction center chlorophyll dimer, is dramatically reduced in the mutant, indicating that the PsaF subunit plays an important role in docking plastocyanin to the PSI complex. These results contrast with those obtained previously with a cyanobacterial psaF-, psaJ- double mutant where no phenotype was apparent.

Animals↗
PubMed1995

A mutant strain of Chlamydomonas reinhardtii lacking the chloroplast photosystem II psbI gene grows photoautotrophically.

The product of the chloroplast psbI gene is associated with the photosystem II reaction center. To gain insights into the function of this polypeptide, we have disrupted its gene in Chlamydomonas reinhardtii with an aadA expression cassette that confers resistance to spectinomycin through biolistic transformation. The transformants are still able to grow photoautotrophically in dim light, but not in high light, and they remain photosensitive when grown on acetate containing medium. The amounts of photosystem II complex and oxygen evolving activity are both reduced to 10-20% of wild-type levels in these psbI-deficient mutants. It appears that the PsbI polypeptide plays a role in the stability of photosystem II and possibly also in modulating electron transport or energy transfer in this complex.

Amino Acid Sequence↗
PubMed1995

The trans-spliced intron 1 in the psaA gene of the Chlamydomonas chloroplast: a comparative analysis.

In the secondary structure model that has been proposed for the trans-spliced intron 1 in the Chlamydomonas reinhardtii psaA gene, a third RNA species (tscA RNA) interacts with the 5' and 3' intron parts flanking the exons to reconstitute a composite structure with several features of group-II introns. To test the validity of this model, we undertook the sequencing and modelling of equivalent introns in the psaA gene from other unicellular green algae belonging to the highly diversified genus Chlamydomonas. Our comparative analysis supports the model reported for the C. reinhardtii psaA intron 1, and also indicates that the 5' end of the tscA RNA and the region downstream from the psaA exon 1 cannot be folded into a structure typical of domain I as described for most group-II introns. It is possible that a fourth RNA species, yet to be discovered, provides the parts of domain I which are apparently missing.

Animals↗
PubMed1995

Chlamydomonas reinhardtii as the photosynthetic yeast.

The green unicellular alga Chlamydomonas reinhardtii has long been used as a model system for studying photosynthesis, chloroplast biogenesis, and flagellar function and assembly because of its well-defined genetics. The value of this organism has been greatly increased recently by the development of efficient methods for nuclear and chloroplast transformation. While homologous recombination appears to occur at a low frequency in the nuclear genome, random integrations can be exploited to tag genes of interest by insertional mutagenesis. Cloning of the nuclear genes by genomic complementation is also possible. Chloroplast genetic engineering has provided new and important insights into the molecular mechanisms of chloroplast gene expression and into the function of chloroplast proteins. C. reinhardtii is probably the best organism in which to perform chloroplast DNA surgery because of the ease of chloroplast transformation in this alga. C. reinhardtii might be described as the photosynthetic yeast. However, it offers additional new promising areas of research, such as chloroplast-mitochondrial interactions, phototransduction, and the behavioral response to light.

Animals↗
PubMed1994

Determinants for stability of the chloroplast psbD RNA are located within its short leader region in Chlamydomonas reinhardtii.

Stability of the chloroplast psbD mRNA encoding the D2 protein of the photosystem II reaction center is drastically decreased in the nuclear photosynthetic mutant nac2-26 of Chlamydomonas reinhardtii. Using biolistic transformation and genetic crosses we have introduced chimeric genes consisting of the psbD leader fused to a reporter gene into the chloroplast in both wild-type and mutant nuclear backgrounds. The chimeric message is destabilized in the latter, but not in the former case, indicating that the 74 nt psbD leader includes one of the target sites for psbD RNA degradation in the absence of wild-type NAC2 function. Increased instability of the psbD leader in mutant versus wild-type chloroplast lysates is also demonstrated in vitro and the primary cleavage sites have been mapped. The instability of the psbD RNA in the mutant correlates with the loss of binding of a 47 kDa protein to the psbD leader RNA, suggesting that this factor acts as message stabilizer in wild-type.

Animals↗
PubMed1994

The chloroplast ycf8 open reading frame encodes a photosystem II polypeptide which maintains photosynthetic activity under adverse growth conditions.

We have engineered and analyzed a chloroplast mutant of Chlamydomonas reinhardtii that lacks ycf8, the chloroplast open reading frame 8, which is highly conserved in location and predicted amino acid sequence in land plants and C.reinhardtii. The ycf8 sequence was replaced with the aadA cassette which confers resistance to spectinomycin when expressed in the chloroplast. Although the mutant is able to grow phototrophically, photosystem II function and cell growth are impaired under stress conditions such as high light intensity and diminished chloroplast protein synthesis induced by spectinomycin. Use of an antibody generated against the ycf8 product has revealed that this hydrophobic polypeptide is associated with photosystem II, based on its severely reduced levels in various photosystem II-deficient mutants and on its copurification with photosystem II. This protein, therefore, appears to be (i) a novel photosystem II subunit and (ii) required for maintaining optimal photosystem II activity under adverse growth conditions.

Animals↗
PubMed1994

Complementation of a Chlamydomonas reinhardtii mutant using a genomic cosmid library.

We report the rescue of an arginine-requiring mutant (arg7-8) of Chlamydomonas reinhardtii by complementation using total DNA from a genomic cosmid library. Using the glass-bead transformation method of Kindle [8] four putative transformants able to grow in the absence of exogenous arginine were obtained from 3 x 10(9) treated cells. Southern blot analysis reveals that at least three of the clones have acquired an additional copy of the gene (ARG7) encoding argininosuccinate lyase (ASL). The arginine-independent phenotype is stable in the absence of selective pressure and high levels of ASL activity are detected in all four clones. We conclude that these represent true transformants and that any stable nuclear mutant of Chlamydomonas could be rescued using this approach.

Animals↗
PubMed1994

Directed disruption of the Chlamydomonas chloroplast psbK gene destabilizes the photosystem II reaction center complex.

Using particle gun-mediated chloroplast transformation we have disrupted the psbK gene of Chlamydomonas reinhardtii with an aadA expression cassette that confers resistance to spectinomycin. The transformants are unable to grow photoautotrophically, but they grow normally in acetate-containing medium. They are deficient in photosystem II activity as measured by fluorescence transients and O2 evolution and they accumulate less than 10% of wild-type levels of photosystem II as measured by immunochemical means. Pulse-labeling experiments indicate that the photosystem II complex is synthesized normally in the transformants. These results differ from those obtained previously with similar cyanobacterial psbK mutants that were still capable of photoautotrophic growth (Ikeuchi et al., J. Biol. Chem. 266 (1991) 1111-1115). In C. reinhardtii the psbK product is required for the stable assembly and/or stability of the photosystem II complex and essential for photoautotrophic growth. The data also suggest that the stability requirements of the photosynthetic complexes differ considerably between C. reinhardtii and cyanobacteria.

Algal Proteins↗
PubMed1994

The 5' leader of a chloroplast mRNA mediates the translational requirements for two nucleus-encoded functions in Chlamydomonas reinhardtii.

In the green alga Chlamydomonas reinhardtii, the nuclear mutations F34 and F64 have been previously shown to abolish the synthesis of the photosystem II core polypeptide subunit P6, which is encoded by the chloroplast psbC gene. In this report the functions encoded by F34 and F64 are shown to be required for translation of the psbC mRNA, on the basis of the finding that the expression of a heterologous reporter gene fused to the psbC 5' nontranslated leader sequence requires wild-type F34 and F64 alleles in vivo. Moreover, a point mutation in the psbC 5' nontranslated leader sequence suppresses this requirement for wild-type F34 function. In vitro RNA-protein cross-linking studies reveal that chloroplast protein extracts from strains carrying the F64 mutation contain an approximately 46-kDa RNA-binding protein. The absence of the RNA-binding activity of this protein in chloroplast extracts of wild-type strains suggests that it is related to the role of the F64-encoded function for psbC mRNA translation. The binding specificity of this protein appears to be for an AU-rich RNA sequence motif.

Animals↗
PubMed1993

Characterization of the cleavage site and the recognition sequence of the I-CreI DNA endonuclease encoded by the chloroplast ribosomal intron of Chlamydomonas reinhardtii.

The chloroplast ribosomal intron of Chlamydomonas reinhardtii encodes a sequence-specific DNA endonuclease (I-CreI), which is most probably involved in the mobility of this intron. Here we show that I-CreI generates a 4 bp staggered cleavage just downstream of the intron insertion site. The I-CreI recognition sequence is 19-24 bp in size and is located asymmetrically around the intron insertion site. Screening of natural variants of the I-CreI recognition sequence indicates that the I-CreI endonuclease tolerates single and even multiple base changes within its recognition sequence.

Animals↗
PubMed1992

Characterization of chlorophyll a/b proteins of photosystem I from Chlamydomonas reinhardtii.

In this study we have isolated the chlorophyll a/b-binding proteins from a photosystem I preparation of the green alga Chlamydomonas reinhardtii and characterized them by N-terminal sequencing, fluorescence, and absorption spectroscopy and by immunochemical means. The results indicate that in this organism, the light-harvesting complex of photosystem I (LHCI) is composed of at least seven distinct polypeptides of which a minimum number of three are shown to bind chlorophyll a and b. Both sequence homology and immunological cross-reactivity with other chlorophyll-binding proteins suggest that all of the LHCI polypeptides bind pigments. Fractionation of LHCI by mildly denaturing methods showed that, in contrast to higher plants, the long wavelength fluorescence emission typical of LHCI (705 nm in C. reinhardtii) cannot be correlated with the presence of specific polypeptides, but rather with changes in the aggregation state of the LHCI components. Reconstitution of both high aggregation state and long wavelength fluorescence emission from components that do not show these characteristics confirm this hypothesis.

Amino Acid Sequence↗
PubMed1992

Tentoxin sensitivity of chloroplasts determined by codon 83 of beta subunit of proton-ATPase.

Tentoxin is a naturally occurring phytotoxic peptide that causes seedling chlorosis and arrests growth in sensitive plants and algae. In vitro, it inhibits activity of the beta subunit of the plastid proton-adenosine triphosphatase (ATPase) from sensitive species. Plastid atpB genes from six closely related, tentoxin-sensitive or -resistant Nicotiana species differ at codon 83, according to their response to the toxin: glutamate correlated with resistance and aspartate correlated with sensitivity. The genetic relevance of this site was confirmed in Chlamydomonas reinhardtii by chloroplast transformation. The alga, normally tentoxin-resistant, was rendered tentoxin-sensitive by mutagenesis of its plastid atpB gene at codon 83. Codon 83 may represent a critical site on the beta subunit that does not compete with nucleotide binding or other catalytic activities.

Adenosine Triphosphate↗
PubMed1992

A chloroplast gene is required for the light-independent accumulation of chlorophyll in Chlamydomonas reinhardtii.

The light-independent pathway of chlorophyll synthesis which occurs in some lower plants and algae is still largely unknown. We have characterized a chloroplast mutant, H13, of Chlamydomonas reinhardtii which is unable to synthesize chlorophyll in the dark and is also photosystem I deficient. The mutant has a 2.8 kb deletion as well as other rearrangements of its chloroplast genome. By performing particle gun mediated chloroplast transformation of H13 with defined wild-type chloroplast DNA fragments, we have identified a new chloroplast gene, chlN, coding for a 545 amino acid protein which is involved in the light-independent accumulation of chlorophyll, probably at the step of reduction of protochlorophyllide to chlorophyllide. The chlN gene is also found in the chloroplast genomes of liverwort and pine, but is absent from the chloroplast genomes of tobacco and rice.

Algal Proteins↗
PubMed1992

Accumulation of chloroplast psbB RNA requires a nuclear factor in Chlamydomonas reinhardtii.

We have isolated and characterized a nuclear mutant, 222E, in Chlamydomonas reinhardtii, which is defective in photosystem II (PSII). Polypeptide P5, the product of psbB, is not produced in this mutant, leading to a destabilization of other PSII components. The mutant specifically fails to accumulate psbB transcripts and displays an altered transcription pattern downstream of psbB. Pulse-labelling experiments suggest that mRNA stability and/or processing are affected by the alteration of a nuclear gene product in this mutant. We show that the C. reinhardtii psbB gene is co-transcribed with a small open reading frame that is highly conserved in location and amino acid sequence in land plants. The 5' and 3' termini of the psbB transcript have been mapped to 35 bases upstream of the initiation codon and approximately 600 bases downstream of the stop codon. The 3' flanking region contains two potential stem-loops, of which the larger (with an estimated free energy of -46 kcal) is near the 3' terminus of the transcript.

Amino Acid Sequence↗
PubMed1991

Conservation in structure of TOC1 transposons from Chlamydomonas reinhardtii.

TOC1 transposons from Chlamydomonas reinhardtii have an unusual arrangement of long terminal repeats. Polymorphic regions between TOC1 transposons were identified by restriction mapping on Southern blots. The variation in size of an internal MluI fragment defines two subclasses of TOC1 elements. Full-length cloned members of each subclass of TOC1 element were compared by electron microscope heteroduplex analysis. The cloned elements were co-linear over their entire length with no large sequence discontinuities. Base substitutions and small insertion/deletion events of less than 50 bp are responsible for forming the two subclasses of TOC1 elements.

Animals↗