Biomedical subjects

J D Rochaix

Publications and source records attributed to J D Rochaix.

At least 73 records · Page 4Linked to original sources

PubMed1991

A small chloroplast RNA may be required for trans-splicing in Chlamydomonas reinhardtii.

In C. reinhardtii, the mature psaA mRNA is assembled by a process involving trans-splicing of three separate transcripts encoded at three widely scattered loci of the chloroplast genome. At least one additional chloroplast locus (tscA) is required for trans-splicing of exons 1 and 2. We have mapped this gene by transformation of a deletion mutant with a particle gun. The 0.7 kb region of the chloroplast genome that is sufficient to rescue tscA function has been subjected to insertion mutagenesis, showing that it does not contain significant open reading frames. We suggest from these experiments that the product of the tscA gene may be a small chloroplast RNA that acts in trans in the first trans-splicing reaction of psaA. A model for the mode of action of this RNA is presented, in which the characteristic structure of group II introns is assembled from three separate transcripts.

Amino Acid Sequence↗

PubMed1991

A transposon with an unusual LTR arrangement from Chlamydomonas reinhardtii contains an internal tandem array of 76 bp repeats.

TOC1, a transposable element from Chlamydomonas reinhardtii, is 5662 bases long. The 217 and 237 base long terminal repeat sequences of TOC1 are unusually arranged around the 4600 and 123 base unique regions: [217]-4600-[237] [217]-123-[237]. Although TOC1 contains long terminal repeats and most TOC1 elements are complete, features shared with virus-like retroposons, its unique 4600 base region is more similar to the structure of the L1 family of non-virus retroposons: first, 11 3/4 tandemly repeated copies of a 76 base repeat are found 813 bases from the left end of TOC1, and second using the universal genetic code large open reading frames were not found in TOC1. The relationship between TOC1, virus-like retroposons and the L1 family of non-virus retroposons is unclear and may be very distant since only poor similarity was found between the TOC1 encoded ORFs and retrovirus polypeptides. The length of the tandem array of 76 base repeat sequences was conserved in most TOC1 elements and solo 76 base repeat sequences were not found outside TOC1 elements in the C. reinhardtii genome. Nucleotide substitutions allow all copies of the 76 base repeat to be distinguished from one another.

Base Sequence↗

PubMed1991

Structure and inheritance of sense and anti-sense transcripts from a transposon in the green alga Chlamydomonas reinhardtii.

We have studied the transcription pattern of a 5700 base-pair transposon (TOC1) in Chlamydomonas reinhardtii. Northern blotting and nuclease S1 protection experiments define three classes of major TOC1 RNAs that accumulate to different levels in a number of strains and segregate independently in the progeny of crosses: class 1 RNAs are unstable near full-length sense transcripts whose 5' end maps to the left 217 base-pair repeat of TOC1, class 2 and class 3 RNAs are large, discrete chimaeric transcripts containing full-length sense (class 2) and anti-sense (class 3) copies of TOC1. Sequence motifs common to the 5' non-transcribed regions of C. reinhardtii genes were found upstream from the putative initiation site of class 1 transcripts. A functional polyadenylation site was located in the far-right 237 base-pair repeat of TOC1. Class 1 TOC1 transcripts are initiated, and probably terminated, within the terminal repeats of TOC1 and may represent retrotransposition intermediates. Class 2 and 3 TOC1 transcripts co-segregate with specific TOC1 elements identified on Southern blots. The loci that control the production of high levels of class 1 transcripts could correspond to specific TOC1 elements, i.e. only a few TOC1 elements are transcribed, or to a regulatory locus. The accumulation of an 11,500 to 12,000 base sense transcript (class 2) is reduced two- to fourfold by the presence of a 9500 to 9700 base antisense transcript (class 3). In contrast, the accumulation of the 5' ends of class 1 transcripts are unaffected by the anti-sense TOC1 transcript.

Base Sequence↗

PubMed1991

Chloroplast ribosomal intron of Chlamydomonas reinhardtii: in vitro self-splicing, DNA endonuclease activity and in vivo mobility.

All chloroplast 23S ribosomal RNA genes of the unicellular alga Chlamydomonas reinhardtii contain an 888 bp group I intron with an internal open reading frame (ORF). A precursor RNA encompassing the intron with its 5' and 3' flanking sequences was shown to self-splice both during in vitro transcription and upon incubation of the isolated pre-RNA under self-splicing conditions. Expression of the internal ORF in Escherichia coli in the presence of a plasmid containing a cDNA corresponding to the intronless form of the 23S rRNA gene resulted in specific cleavage of the cDNA at or close to the exon junction sequence. To test whether this ORF-encoded double-strand DNA endonuclease is involved in intron mobility in vivo, the same ribosomal cDNA was stably integrated into the C. reinhardtii chloroplast genome using particle gun mediated transformation. All the transformants with the cDNA integrated at the expected site in the chloroplast genome had the intron precisely inserted at the artificial exon junction site. These experiments demonstrate that the chloroplast ribosomal intron of C. reinhardtii behaves as a ribozyme in vitro and also as a mobile genetic element in vivo provided a target site is present.

Animals↗

PubMed1991

Directed chloroplast transformation in Chlamydomonas reinhardtii: insertional inactivation of the psaC gene encoding the iron sulfur protein destabilizes photosystem I.

The chloroplast gene psaC encoding the iron sulfur protein of photosystem I (PSI) from the green alga Chlamydomonas reinhardtii has been cloned and characterized. The deduced amino acid sequence is highly related to that of higher plants and cyanobacteria. Using a particle gun, wild type C. reinhardtii cells have been transformed with a plasmid carrying the psaC gene disrupted by an aadA gene cassette designed to express spectinomycin/streptomycin resistance in the chloroplast. Transformants selected on plates containing acetate as a reduced carbon source and spectinomycin are unable to grow on minimal medium lacking acetate and are deficient in PSI activity. Southern blot analysis of total cell DNA of the transformants shows that the wild type psaC gene has been replaced by the interrupted psaC gene through homologous recombination. While authentic transcripts of the psaC gene are no longer detected, aadA gives rise to a few transcripts in the transformants. Biochemical analysis indicates that neither PSI reaction center subunits nor the seven small subunits belonging to PSI accumulate stably in the thylakoid membranes of the transformants. Pulse-chase labeling of cell proteins shows that the PSI reaction center subunits are synthesized normally but turn over rapidly in the transformants. We conclude that the iron sulfur binding protein encoded by the psaC gene is an essential component, both for photochemical activity and for stable assembly of PSI. The present study suggests that any chloroplast gene encoding a component of the photosynthetic apparatus can be disrupted in C. reinhardtii using the strategy described.

Amino Acid Sequence↗

PubMed1990

Chloroplast transit peptides from the green alga Chlamydomonas reinhardtii share features with both mitochondrial and higher plant chloroplast presequences.

Chloroplast transit peptides from the green alga Chlamydomonas reinhardtii have been analyzed and compared with chloroplast transit peptides from higher plants and mitochondrial targeting peptides from yeast, Neurospora and higher eukaryotes. In terms of length and amino acid composition, chloroplast transit peptides from C. reinhardtii are more similar to mitochondrial targetting peptides than to chloroplast transit peptides from higher plants. They also contain the potential amphiphilic alpha-helix characteristic of mitochondrial presequences. However, in similarity with chloroplast transit peptides from higher plants, they contain a C-terminal region with the potential to form an amphiphilic beta-strand. As in higher plants, transit peptides that route proteins to the thylakoid lumen consist of an N-terminal domain similar to stroma-targeting transit peptides attached to a C-terminal apolar domain that share many characteristics with secretory signal peptides.

Amino Acid Sequence↗

PubMed1990

Trans-splicing mutants of Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii the three exons of the psaA gene are widely scattered on the chloroplast genome: exons 1 and 2 are in opposite orientations and distant from each other and from exon 3. The mature mRNA, encoding a core polypeptide of photosystem I, is thus probably assembled from separate precursors by splicing in trans. We have isolated and characterized a set of mutants that are deficient in the maturation of psaA mRNA. The mutants belong to 14 nuclear complementation groups and one chloroplast locus that are required for the assembly of psaA mRNA. The chloroplast locus, tscA, is remote from any of the exons and must encode a factor required in trans. The mutants all show one of only three phenotypes that correspond to defects in one or other or both of the joining reactions. These phenotypes, and those of double mutants, are consistent with the existence of two alternative splicing pathways.

Chlamydomonas↗

PubMed1990

Instability of a yellow mutation in Chlamydomonas reinhardtii is not due to TOC1 elements.

yc4 is an unstable yellow-in-the dark mutation found in the unicellular green alga Chlamydomonas reinhardtii. yc4 reverts to the wild-type green-in-the-dark phenotype at a frequency of 1 X 10(-4) revertants per cell division. We have tested whether mutation and reversion of yc4 is due to the insertion and excision of the C. reinhardtii transposon TOC1. Southern blots showed differences in the distribution of TOC1 elements when the original mutant yc4 strain, which contained over 30 TOC1 elements, was compared with green-in-the-dark-revertants. However, analysis of the yc4 mutation in a different genetic background containing half the number of TOC1 elements showed that reversion of yc4 is not associated with loss of TOC1 elements.

Blotting, Southern↗

PubMed1989

Mutation at the Chlamydomonas nuclear NAC2 locus specifically affects stability of the chloroplast psbD transcript encoding polypeptide D2 of PS II.

The nuclear NAC2 locus of C. reinhardtii encodes a trans-acting factor that confers stability on the chloroplast message for the D2 protein of photosystem II (PS II). A mutant that carries a lesion in the NAC2 gene is unable to accumulate the psbD transcript encoding D2, but synthesizes all other PS II proteins normally. However, the PS II complex is completely unstable in the nac2-26 mutant, and all major PS II polypeptides, including the three oxygen-evolving enhancing proteins, are absent or greatly reduced in this strain because of posttranslational degradation. In C. reinhardtii the second exon of the trans-spliced psaA mRNA is cotranscribed with psbD (Choquet et al., 1988), but the nac2-26 mutation normally has no effect on psaA message maturation or stability. However, in double mutants carrying the nac2-26 mutation, as well as a mutation that prevents psaA splicing, splicing intermediates carrying psbD sequences are degraded. These results suggest that the NAC2 gene product acts in a very specific manner to control the half-life of psbD transcripts.

Chlamydomonas↗

PubMed1989

Nuclear and chloroplast mutations affect the synthesis or stability of the chloroplast psbC gene product in Chlamydomonas reinhardtii.

The psbC gene of Chlamydomonas reinhardtii encodes P6, the 43 kd photosystem II core polypeptide. The sequence of P6 is highly homologous to the corresponding protein in higher plants with the exception of the N-terminal region where the first 12 amino acids are missing. Translation of P6 is initiated at GUG in C. reinhardtii. The chloroplast mutant MA16 produces a highly unstable P6 protein. The mutation in this strain maps near the middle of the psbC gene and consists of a 6 bp duplication that creates a Ser-Leu repeat at the end of one transmembrane domain. Two nuclear mutants, F34 and F64, and one chloroplast mutant, FuD34, are unable to synthesize P6. All of these mutants accumulate wild-type levels of psbC mRNA. The FuD34 mutation has been localized near the middle of the 550 bp 5' untranslated region of psbC where the RNA can be folded into a stem-loop structure. A chloroplast suppressor of F34 has been isolated that partially restores synthesis of the 43 kd protein. The mutation of this suppressor is near that of FuD34, in the same stem-loop region. These chloroplast mutations appear to define the target site of a nuclear factor that is involved in P6 translation.

Amino Acid Sequence↗

PubMed1989

The argininosuccinate lyase gene of Chlamydomonas reinhardtii: an important tool for nuclear transformation and for correlating the genetic and molecular maps of the ARG7 locus.

The argininosuccinate lyase (ASL) gene of Chlamydomonas reinhardtii has been cloned using four oligonucleotide probes corresponding to highly conserved regions of the ASL polypeptide sequence. The identity of the gene was confirmed by partial sequencing. It is unique, contains several introns and spans a region less than 7.8 kb that includes highly repetitive sequences. Using a particle gun, a reliable nuclear transformation system has been established by complementing three mutants deficient in ASL activity with the wild-type ASL gene. Analysis of the transformants reveals variable patterns of integration of the transforming DNA into the nuclear genome. Previous work has mapped the mutations in the mutants arg2 and arg7 to either end of the ARG7 locus 1.0 to 1.6 recombination map units apart. Our transformation results show that these two mutations are located within a region of 7.8 kb. This allows for the first correlation of the recombination map and the molecular map at the ARG7 locus and indicates a high recombination frequency in this region of the nuclear genome.

Amino Acid Sequence↗

PubMed1989

Isolation and characterization of cDNA clones encoding photosystem I subunits with molecular masses 11.0, 10.0 and 8.4 kDa from Chlamydomonas reinhardtii.

cDNA clones encoding three photosystem I subunits of Chlamydomonas reinhardtii with apparent molecular masses 13, 5 and 3 kDa (thylakoid polypeptides 28, 35 and 37; P28, P35 and P37, respectively) were isolated using gene specific oligonucleotides as probes. The sequences of these oligonucleotides were deduced from the N-terminal amino acid sequences of the proteins. The cDNAs were sequenced and used to probe Southern and Northern blots. The Southern blot analysis indicates that the proteins are encoded by single-copy genes. The mRNA sizes of the three components are 960 (P28), 1120 (P35) and 790 (P37) nucleotides. Comparison between the open reading frames of the cDNAs and the N-terminal amino acid sequences of the proteins indicates that the nascent polypeptides possess N-terminal transit sequences that are removed to give mature proteins of 11.0 (P28), 10.0 (P35) and 8.4 (P37) kDa. Analysis of the deduced protein sequences suggests that P28 and P35 are extrinsic membrane proteins and that P37 spans the thylakoid membrane. All three proteins have short transit peptides that probably route them to the stromal side of the thylakoid membrane.

Amino Acid Sequence↗

PubMed1989

Characterization of transcribed dispersed repetitive DNAs in the nuclear genome of the green alga Chlamydomonas reinhardtii.

Four cDNAs (cDNAs 1-4), 162, 338, 321 and 167 bp in size, that contain repetitive DNA sequences, were isolated from C. reinhardtii. cDNAs 1, 2 and 3 hybridized to multiple transcripts in poly A+ RNA. Each of the four repeat families is comprised of an extremely heterogeneous population of interspersed nuclear DNA sequences most of which are less than 0.5 kbp in size. A large number of restriction fragment length polymorphisms were uncovered by using cDNAs 1 and 2 as hybridization probes. cDNA2 was compared to two different genomic DNA sequences: the first sequence was complementary to a central 136 bases of cDNA2, which is bordered by a 15-bp imperfect direct repeat; the second sequence lacks a poly-dA tail, but is otherwise colinear along its entire length with cDNA2. This suggests that some members of the cDNA2 repeat family contain signals for polyadenylation. The majority of accumulated transcripts that hybridize to cDNA2 have the same 5'-3' orientation as cDNA2.

Base Sequence↗

PubMed1989

Preparation of chlamydomonas chloroplasts for the in vitro import of polypeptide precursors.

To study the import of polypeptide precursors we have adapted and compared two procedures for the isolation of competent chloroplasts from the green unicellular alga, Chlamydomonas reinhardtii: silicasol gradient centrifugation and elutriation. The chloroplasts actively import the precursor of the small subunit of ribulose bisphosphate carboxylase-oxygenase in vitro.

Journal Article↗

PubMed1989

Molecular and biophysical analysis of herbicide-resistant mutants of Chlamydomonas reinhardtii: structure-function relationship of the photosystem II D1 polypeptide.

Plants and green algae can develop resistance to herbicides that block photosynthesis by competing with quinones in binding to the chloroplast photosystem II (PSII) D1 polypeptide. Because numerous herbicide-resistant mutants of Chlamydomonas reinhardtii with different patterns of resistance to such herbicides are readily isolated, this system provides a powerful tool for examining the interactions of herbicides and endogenous quinones with the photosynthetic membrane, and for studying the structure-function relationship of the D1 protein with respect to PSII electron transfer. Here we report the results of DNA sequence analysis of the D1 gene from four mutants not previously characterized at the molecular level, the correlation of changes in specific amino acid residues of the D1 protein with levels of resistance to the herbicides atrizine, diuron, and bromacil, and the kinetics of fluorescence decay for each mutant, which show that changes at two different amino acid residues dramatically slow PSII electron transfer. Our analyses, which identify a region of 57 amino acids of the D1 polypeptide involved in herbicide binding and which define a D1 binding niche for the second quinone acceptor, QB of PSII, provide a strong basis of support for structural and functional models of the PSII reaction center.

Amino Acid Sequence↗

PubMed1988

Mutant phenotypes support a trans-splicing mechanism for the expression of the tripartite psaA gene in the C. reinhardtii chloroplast.

The chloroplast psaA gene of the green unicellular alga Chlamydomonas reinhardtii consists of three exons that are transcribed from different strands. Analysis of numerous nuclear and chloroplast mutants that are deficient in photosystem I activity reveals that roughly one-quarter of them are specifically affected in psaA mRNA maturation. These mutants can be grouped into three phenotypic classes, based on their inability to perform either one or both splicing reactions. The data indicate that the three exons are transcribed independently as precursors which are normally assembled in trans and that the splicing reactions can occur in either order. While some chloroplast mutations could act in cis, the nuclear mutations that fall into several complementation groups probably affect factors specifically required for assembling psaA mRNA.

Base Sequence↗

PubMed1988

Nuclear mutations specifically affect the synthesis and/or degradation of the chloroplast-encoded D2 polypeptide of photosystem II in Chlamydomonas reinhardtii.

To study the interaction of the nuclear and chloroplast genomes in the biogenesis of the photosynthetic apparatus, nuclear mutants of Chlamydomonas reinhardtii deficient in photosystem II (PSII) activity were analyzed. Two independently-isolated, allelic nuclear mutants show a pleiotropic reduction in a set of functionally related PSII polypeptides. Immunoblot analysis reveals that the two mutants, nac-1-18 and nac-1-11, accumulate reduced amounts of the chloroplast-encoded polypeptides P5 and P6 and are completely deficient in polypeptides D1 and D2. Polypeptides of the oxygen-evolving and light-harvesting complexes associated with PSII, however, are present at wild-type levels. Analysis of mRNAs encoding PSII polypeptides from these mutants indicates that all messages are present, although some species, including the D2 message, are significantly elevated. When mutant cells are pulse-labeled for 10 min with [C]acetate, a greatly reduced amount of labeled D2 protein is observed, while all other PSII polypeptides are synthesized normally. These data indicate that the mutations present in nac-1-18 and nac-1-11 affect a nuclear gene whose product specifically controls the translation and/or degradation of the chloroplast-encoded D2 polypeptide.

Journal Article↗

PubMed1988

A transposon with an unusual arrangement of long terminal repeats in the green alga Chlamydomonas reinhardtii.

We have isolated a 5.7-kbp dispersed moderately repeated DNA sequence (TOC1) from the mutant OEE1 gene of the Chlamydomonas reinhardtii strain FUD44. The copy number (2 to over 30) and genomic locations of TOC1 elements vary widely in different C. reinhardtii strains. Our standard laboratory photosynthetic strain exhibits a high degree of TOC1 instability during short periods of mitotic growth. TOC1 appears to be a retrotransposon: it contains LTRs and an oligonucleotide stretch that corresponds to a conserved pentapeptide of reverse transcriptase. TOC1 is an unusual retrotransposon: it is not flanked by a target site duplication in the OEE1 gene, the left end of TOC1 only contains a fraction of the LTR the remainder of which is present at its right end and TOC1 does not start with a 5' TG and end with a 3' CA. In most cases, TOC1 excision leaves behind a complete solo LTR sequence (577 bp) and in one case a deleted solo LTR sequence (191 bp). Solo LTR sequences form a separate family of repeated sequences in most of the strains tested.

Base Sequence↗