Characterization of a chloroplast mutation in the psaA2 gene of Chlamydomonas reinhardtii

Summary The synthesis of polypeptides related to the CPI chlorophyll-protein complex of photosystem I has been studied by pulse-labeling experiments in twenty chloroplast mutants of Chlamydomonas reinhardtii. Three mutations of the same locus (Girard-Bascou 1987) result in the absence of these CPI-related polypeptides. Among these mutations one, (FUD26) leads to the synthesis of a new polypeptide presumed to be a truncated CPI apoprotein. The molecular characterization of this mutation in the psaA2 gene has been achieved by DNA sequencing the 3 end of this gene. The FUD26 mutation is a 4 base pair deletion resulting in frameshift and premature termination of the protein.     

Studies on mutants deficient in the photosystem I reaction centers in Chlamydomonas reinhardtii

Summary Genetic analysis of 25 nuclear mutants defective in the chlorophyll-protein complex CP1 was undertaken. The mutants belong to 13 complementation groups scattered throughout the nuclear genome. All these mutants lack the apoprotein of CP1 and, in addition, a specific set of six low molecular weight thylakoid polypeptides. System I particles obtained by treating WT thylakoid membranes with detergent specifically contain those polypeptides which the mutants lack. These observations suggest that a particular sub-structure of the thylakoid membrane associated with the photosystem I activity is missing from all 25 mutants studied, and that this general phenotype can result from mutation at any one of several unlinked Mendelian loci.     

Prokaryotic promoters in the chloroplast DNA replication origin of Chlamydomonas reinhardtii

Summary In Chlamydomonas reinhardtii, one displacement loop region which initiates the replication of chloroplast DNA was located on a 1.05 kb restriction fragment. This fragment was cloned and sequenced. In this report, the galK expression plasmid, pK01 was used to screen for the presence of any prokaryotic promoter within the cloned fragment. The insertion of 2 AluI fragments yielded galK⁺ colonies. Sequence analyses of these Alul inserts revealed prokaryotic promoter consensus regions. Cloning into pKOTWI and subsequent DNA sequencing were used to determine the promoter-active orientation of each insert. Two back-to-back prokaryotic promoters were mapped on a 79 by Alul fragment located within the displacement loop region.     

Isolation and characterization of Chlamydomonas temperature-sensitive mutants affecting gametic differentiation under nitrogen-starved conditions

Summary Two conditional mutants of Chlamydomonas reinhardtii, dif-1 and dif-2, affecting gametic differentiation under conditions of nitrogen (N)-starvation, have been isolated. These mutant cells remain vegetative at the restrictive temperature (35°C) in — N medium, as defined by assays of cell body-agglutinin and cell wall lytic enzyme activities in the soluble fractions of cell homogenates. Moreover, the mutants fail to form mating structures at the restrictive temperature, but do so at the permissive temperature (25°C). Temperature-shift experiments show that mutant cells which have differentiated into gametes at 25°C dedifferentiate into vegetative cells under N-starvation conditions after transfer to 35°C, but differentiate again into gametes at 25°C. Genetic analyses indicate that the dif-1 and dif-2 genes are recessive and unlinked to each other or to the matingtype locus; the dif-1 phenotype cosegregates with a conditional flagellales phenotype expressed in both +N and-N medium at the restrictive temperature.     

Characterization of a cDNA encoding the 20-kDa photosystem I light-harvesting polypeptide of Chlamydomonas reinhardtii

A cDNA encoding the precursor to a major 20-kDa thylakoid polypeptide of Chlamydomonas reinhardtii (P22), previously localized to the photosystem I light-harvesting complex (LHCI), was characterized. N-terminal sequencing of P22 identified the precursor cleavage site. Genomic Southern blots and polymerase chain reaction analyses show that the gene for P22 (Lhca1^*1) is single-copy and contains at least one intron. Northernblot analyses show that Lhca1^*1 mRNA is highly regulated in light-dark synchronized cells. The primary sequence and predicted topology of P22 has features characteristic of light-harvesting chlorophyll a/b-binding proteins from higher plants. Sequence comparisons indicate that P22 has significantly greater identity with the Type-I LHCI protein of tomato, compared to other LHC proteins. This result suggests that the divergence of LHCI proteins into the classes found in higher plants may have occurred early in evolution, prior to the separation of green algae and land plants.     

Mutations at three different nuclear loci of Chlamydomonas suppress a defect in chloroplast psbD mRNA accumulation

In the photosynthetic chloroplast mutant PRB2A of Chlamydomonas reinhardtii the psbD mRNA is unstable. Three strains were isolated, in which the underlying site-directed mutation within the psbD 5′ UTR (untranslated region) is suppressed. In all three suppressors, psbD RNA levels and RNA 5′ maturation are restored to a varying extent, suggesting a tight coupling of RNA stabilization and 5′ processing. Expression of the psbA gene is not compromised in these strains. Genetic crosses revealed that the suppressor mutations affect three unlinked nuclear loci, which may encode new factors involved in psbD gene expression. Received: 30 August / 26 October 1999     

Heteroplasmic suppression of an amber mutation in the Chlamydomonas chloroplast gene that encodes the large subunit of ribulosebisphosphate carboxylase/oxygenase

Summary The 18-5B and 18-7G mutants of Chlamydomonas reinhardtii lack ribulosebisphosphate carboxylase/oxygenase holoenzyme and contain nonsense mutations in the chloroplast gene that encodes the protein's large subunit. Spontaneous revertants of the 18-5B opal (UGA) mutant were found to be heteroplasmic in a previous study (Spreitzer et al. 1984). They appeared to contain both mutant and wild-type alleles of a suppressor gene within the chloroplast. However, revertants of the 18-7G amber (UAG) mutant could not be recovered spontaneously. In the present investigation, revertants of the opal and amber mutants were recovered after a mutagen treatment. Heteroplasmic suppression of the 18-7G amber mutant was observed, suggesting that heteroplasmic suppression may be a common genetic mechanism of polyploid genomes. Although a diversity of other revertant types was also observed, no significant alteration occurred in the oxygen sensitivity of ribulose-bisphosphate carboxylase acitivity.This research was supported in part by USDA Grant No. 85-CRCR-1-1563, and is published as Paper No. 8185, Journal Series, Nebraska Agricultural Research Division     

Chloroplast gene transmission in Chlamydomonas reinhardtii. A model for its control by the mating-type locus

Summary In sexual crosses of Chlamydomonas reinhardtii, genes residing in the chloroplast (cp) are most often transmitted from the mating-type plus (mt ⁺) parent only. Galloway and Goodenough (1985) proposed a model in which the mt locus (linkage group VI) is a complex region containing several genes involved in the control of both gametic differentiation and cp inheritance. The mt ⁺ locus contains: the sfu locus necessary for sexual fusion between gametes; the upp locus (uniparental plus) which controls cp gene inheritance and also perhaps zygote maturation; and the sad locus which functions in sexual adhesion. The mt ^– locus also contains a sad locus as well as a regulatory element (mid) necessary for the minus dominance in mt ⁺/mt ^– diploid gametes. This model has been extended to include new genetic functions linked to the mt ⁺ or mt ^– locus. In this new system, there is a group of genes (maps for mating-type plus structure), present in both plus and minus strains, controlling some mt ⁺ phenotypical traits as well as the synthesis of an activator of the cp DNA nuclease; two genes contained in the mt ^– locus — one (cge for cp genomic elimination) coding for a nuclease (in an inactive form) located in the chloroplast and another (mid) coding for a repressor of maps; one gene (upp) contained in the mt ⁺ locus, coding for a substance preventing the synthesis or the activity of the nuclease and perhaps also acting on the maps product. The model can be applied to explain the complex dominance/ recessivity relationships observed between the mt ⁺ and mt ^– alleles in heterozygous diploid gametes, the basis for mitotic vs meiotic zygote formation and the differences in transmission observed for these two physiologically distinct zygote classes. Application of the model for prediction of mutant phenotypes and the design of future experiments is also described.     

Evidence for informational suppression within the chloroplast of Chlamydomonas reinhardtii

Summary A balanced polymorphism within the single chloroplast of the green alga Chlamydomonas reinhardtii has been postulated to account for stable heteroplasmicity. Heteroplasmicity of mutant and wild-type alleles of a suppressor gene appears to be required for the suppression of a nonsense mutation in the chloroplast large-subunit gene (rbcL) of ribulosebisphosphate carboxylase/oxygenase. Whereas nonsense suppression has been demonstrated, its molecular basis remains to be determined. In the present study, the suppressed strain was found to synthesize both truncated and full-length large-subunit proteins. Since the rbcL nonsense mutation created a new restriction enzyme site, restriction-enzyme analysis and DNA hybridization were employed to show that only the mutant gene was present in the suppressed strain. It is thus likely that informational suppression is involved in the heteroplasmic suppression mechanism: the original nonsense mutation is present, but normal enzyme is produced comprised of full-length large subunits.     

Inheritance of mitochondrial and chloroplast genome markers in backcrosses of Chlamydomonas eugametos x Chlamydomonas moewusii hybrids

Summary Southern blot analysis of AvaI-digested total cellular DNA from the interfertile species Chlamydomonas eugametos and Chlamydomonas moewusii with a coxI mitochondrial gene probe from Chlamydomonas reinhardtii revealed single hybridizing fragments of 5.0 and 3.5 kb, respectively. The transmission of these mitochondrial DNA physical markers along with that of chloroplast genetic markers for resistance to streptomycin and resistance to erythromycin was studied in the fourth backcrosses of F₁ hybrids to one or the other parent. Viability in these backcrosses is high in contrast to the cross C. eugametos x C. moewusii and its reciprocal which are associated with considerable meiotic product lethality. The resulting zygospores were found to transmit the mitochondrial and chloroplast genome markers uniparentally or preferentially from the mating-type-plus parent. Thus the species pair C. eugametos and C. moewusii differs from the pair Chlamydomonas reinhardtii and Chlamydomonas smithii in which mitochondrial genome markers are transmitted uniparentally by the mating-type minus parent, while the chloroplast genome markers are transmitted uniparentally by the opposite parental mating-type (Boynton et al. 1987).     

Localization of two novel chloroplast genome functions: trans-splicing of RNA and protochlorophyllide reduction

Summary Chloroplast DNA deletions in the unicellular green alga Chlamydomonas reinhardtii localize two novel chloroplast gene functions. One of these, tscA, is required in trans for splicing, in trans, of the first and second, but not the second and third, exons of the RNA of the chloroplast gene psaA. Previously, no chloroplast genes were known to be required in trans for the splicing of chloroplast RNA. The other chloroplast gene function is required for light-independent reduction of protochlorophyllide, a key step in the algal pathway of chlorophyll biosynthesis. Both functions reside in the same 4 kbp region of chloroplast DNA.     

True reversion of a mutation in the chloroplast gene encoding the large subunit of ribulosebisphosphate carboxylase/oxygenase in Chlamydomonas

The ribulosebisphosphate carboxylase/oxygenase-defective Chlamydomonas mutant, 10-6C, was the first mutant to be physically defined in chloroplast DNA. In this report, a photosynthesis-competent revertant of the 10-6C mutant has been found to result from true reversion within the chloroplast large-subunit gene. This result supports the original assignment of the 10-6C mutation within the large-subunit gene.     

Mutations in a nuclear gene of Chlamydomonas cause the loss of two chloroplast ribosomal proteins,one synthesized in the chloroplast and the other in the cytoplasm

Summary The allelic nuclear mutations of Chlamydomonas reinhardtii, cr-6 and cr-7, result in the loss of two proteins from the large subunit of the chloroplast ribosome. One of these proteins, L-13, is synthesized in the chloroplast and the other, L29, is made in the cytoplasm. The loss of these two proteins is correlated with the inability of the large subunits of the chloroplast ribosomes to form monomers which incorporate labeled phenylalanine at normal rates in response to a polyuridylic acid template. Using antisera raised against L13 and L29, we found that protein L-13 was synthesized in appreciable amounts in pulse labeled cells of cr-6 and cr-7, but protein L-29 was not. We conclude that the inability to synthesize protein L29 is a primary defect in both cr-6 and cr-7 and that this protein is required for the stable assembly of protein L-13 into chloroplast ribosomes. The absence of one or both of these proteins from the large subunit of chloroplast ribosomes of the mutants interferes with the ability of the small and large subunits to associate properly into normal 70S monomers.     

Isolation and characterization of Pioneer1, a novel Chlamydomonas transposable element

During the course of this study a novel family of Chlamydomonas mobile elements has been identified in natural isolate strain 224. The first member of this class to be characterized, a 2.8-kb element named Pioneer1, was trapped in an intron of the nitrate reductase structural gene, NIT1. This element has been cloned and completely sequenced and found to be unusual in structure. Pioneer elements are present in a very low-copy number of three per genome in strain 224. The copy number increased by one upon transposition of Pioneer1. Hybridization of Pioneer1 to a variety of Chlamydomonas strains confirmed that this element differed from previously described Chlamydomonas transposons. It also indicated that related elements are present in low-copy number in natural isolate strains 356 and S1D2, but not in the most commonly used laboratory strains 137c and 21 gr. For these reasons, members of the Pioneer family might prove useful as insertional mutagens.     

Transfer RNA genes and the genetic code in Chlamydomonas reinhardtii mitochondria

Summary Only three tRNA genes are present within a sequenced 12.35 kbp region of the 15.8 kbp mtDNA of Chlamydomonas reinhardtii, a unicellular green alga. The corresponding tRNAs, whose anticodons are specific for TGG (Trp), CAA/G (Gln) and ATG (Met) codons, all display conventional secondary structures. The tRNA^Met gene encodes an elongator rather than initiator species. The standard genetic code is used in C. reinhardtii mitochondria, but codon distribution is highly biased: in a collection of six identified protein coding genes, nine codons (including TGA) are not used at all, while four other sense codons occur very infrequently. In spite of the absence of certain codons, a minimum of 23 tRNAs (assuming separate initiator and elongator tRNAs^Met are used) is needed to translate the C. reinhardtii mitochondrial genetic code. It appears unlikely that this minimal tRNA set is encoded by C. reinhardtii mtDNA.     

The transcriptional program of synchronous gametogenesis in Chlamydomonas reinhardtii

Cells of Chlamydomonas reinhardtii undergo gametogenesis to produce sexually competent gametes under nitrogen-starved conditions. By using a synchronized system for gametogenesis of early G1 cells, several previously identified marker genes and 18 novel nitrogen-starved gametogenesis (NSG) genes isolated by macroarray analysis were placed into at least three temporal classes of expression. Early genes are induced transiently in the first 2 h after transfer to nitrogen-free medium. Middle genes are strongly induced between 3 h and 4 h after nitrogen removal, a time corresponding to the acquisition of mating competency, suggesting their involvement in the gamete program. Late genes are induced between 5 h and 8 h after nitrogen removal, a time after the completion of gametic differentiation, suggesting that they are not directly involved in the formation of sexually competent gametes. All of the 18 NSG genes examined are induced in both mating-type plus and minus gametes and about two-thirds of the genes are also expressed in the mitotic cell cycle, especially at S/M phases.     

Translational accuracy and sexual differentiation in Chlamydomonas reinhardtii

Summary A renewal of ribosomes has been previously reported to occur during gametogenesis in C. reinhardtii. In order to further characterize these new ribosomes, we performed pulse-labelling experiments on whole cells of C. reinhardtii, during gametogenesis and in the presence of various aminoglycosides known to alter translational accuracy: Hygromycin and Paromomycin are assumed to increase the rate of translational errors at the level of 80S and 70S ribosomes whereas Kasugamycin is assumed to induce the opposite effect. Three lines of evidence support an increased inaccuracy in protein translation during gametogenesis: (1) gamete cells displayed a higher sensitivity than vegetative cells to Hygromycin and Paromomycin; 4 g/ml Hygromycin cancelled cytoplasmic protein synthesis in gametes but not in vegetative cells; Paromomycin induced the synthesis of new polypeptides of high molecular weight and of nuclear origin in gametes but not in vegetative cells. In addition, chloroplast protein synthesis was more sensitive to Hygromycin and Paromomycin in gametes than in vegetative cells. (2) Kasugamycin-sensitive alterations of thylakoid membranes were detected during gametogenesis. (3) ³⁵S-misincorporation in the OEE3 polypeptide, of nuclear origin and normally devoid of sulphur containing amino acids, was more than three times higher in gametes than in vegetative cells. This increase was prevented by Kasugamycin, suggesting that 80S translation in gametes was more inaccurate than in vegetative cells. The possible significance of these changes occurring during gametogenic differentiation is discussed in light of the importance of a modulation of translational accuracy at particular stages of the life cycle in other lower eukaryotes.Abbreviations Hm Hygromycin B - Pm Paromomycin - Ks Kasugamycin - CAP Chloramphenicol - OEE Oxygen evolving enhancer