Changes in chloroplast genome composition and recombination during the maturation of zygospores of Chlamydomonas reinhardtii

Summary In crosses of the unicellular green alga Chlamydomonas reinhardtii, the chloroplast genes are normally transmitted exclusively by the maternal parent to zygospore progeny. However, transmission of the paternal chloroplast alleles can be increased markedly by certain pretreatments of the maternal parent prior to mating. As zygospores age prior to induction of meiosis, they display decreased biparental transmission of chloroplast alleles and increased transmission of chloroplast alleles from only the maternal or paternal parent. In this report, chloroplast genome composition of biparental zygospores is shown to change in several ways during zygospore maturation. Allelic ratios of chloroplast genes within biparental zygospore clones become maternally or paternally skewed as the zygospores age, cotransmission of chloroplast alleles is reduced, and recombination increases, resulting in an expansion of genetic map distances between chloroplast markers used in this cross. The recovery of unequal frequencies of zygospore progeny expressing reciprocal recombinant genotypes confirms and extends other reports of the predominance of nonreciprocal recombination in organelle genetic systems.     

About biased and non-biased transmission of chloroplast genes following artificial fusion of gametes in Chlamydomonas reinhardtii

Summary Artificial polyethyleneglycol induced fusions of gametes of opposite mating-types carrying chloroplast markers give rise to fusion products transmitting either both markers or the marker from the mt ⁺ or from the mt ^– parent exclusively. The frequencies of the three classes of products were approximately equal in our experiments (Matagne 1981). Similar experiments performed by Matsuda et al. (1983) gave different results, namely a preferential transmission of chloroplast gene from the mt ⁺ parent, very similar to that observed in vegetative zygotes obtained in sexual crosses. Results described here show that in experimental conditions used by Matsuda et al., sexual copulation does occur, leading to formation of zygotes which were misinterpreted as artificial fusion products and gave a biased transmission of chloroplast genes.     

Cytological demonstration of chloroplast DNA behavior during gametogenesis and zygote formation in Chlamydomonas reinhardtii

Summary We used the flourescent dye DAPI to visualize nucleoids of chloroplast DNA and follow their behavior through sexual reproduction by counting nucleoids in fixed cells at various stages. Nucleoid number varied greatly among cells at each stage. The mean number of nucleoids per cell was similar in mt ⁺ and mt ^– vegetative cells, and declined similarly during gametogenesis. Longer periods of nitrogen starvation reduced the mean nucleoid number further. Mean nucleoid number declined again in mating pairs, and continued to drop in zygotes up to the latest stage that can be examined (24-h zygotes). The oldest zygotes had means of about 2 to 3 nucleoids in different experiments, significantly fewer than in the mt ⁺ gametes (usually 4 to 5). The quantitative data on nucleoid number, mating efficiency, and germination efficiency allowed us to show that the decrease in nucleoid number is not limited to gametes that do not mate, or to zygotes that do not germinate. These data are consistant with earlier biochemical studies showing loss of chloroplast DNA during gametogenesis in both mating types, and with the degradation of paternal chloroplast DNA detected biochemically and (in non-quantitative studies) by DAPI staining. There may also be some fusion of nucleoids, although if it occurs it is not complete by 24 h of zygote maturation.     

Behavior of chloroplast genes during the early zygotic divisions of Chlamydomonas reinhardtii

Summary Chloroplast mutations in the green alga Chlamydomonas reinhardtii exhibit a predominantly maternal pattern of inheritance and this pattern can be perturbed by UV irradiation of the maternal gametes prior to mating. In a series of crosses over a range of UV doses, the transmission, segregation, and recombination of mutations at three closely linked chloroplast loci have been examined by pedigree analysis of products arising from the first three post-zygotic divisions. Stocks used in these crosses were constructed to permit identification of the nuclear products of each of the two meiotic divisions and the first post-meiotic mitotic division.A bias toward maternal alleles at all three chloroplast loci was observed in all pedigrees and in zygote clones analyzed from the same crosses many generations after meiosis. This bias decreased with increasing UV dose and with each subsequent division. Segregation of chloroplast genes was rapid during the first three post-zygotic divisions. The type of segregation event from which a given heteroplasmic cell arose had a significant effect on its most likely segregation. pattern in the subsequent division. The results presented here have been discussed in terms of published models of chloroplast gene segregation.     

Transmission of chloroplast genes in crosses between Chlamydomonas reinhardtii diploids: Correlation with chloroplast nucleoid behavior in young zygotes

Summary In crosses between diploid strains of Chlamydomonas reinhardtii, the mode of chloroplast gene transmission was studied and correlated with the behavior of DAPI-stained chloroplast nucleoids in young tetraploid zygotes. Phenotypically plus (female) diploids with mating-type genotypes of mt ⁺/mt ⁺ or mt ⁺/0 (hemizygote) and phenotypically minus (male) diploids with genotypes of mt ⁺/mt ^– or mt ^–/mt ^– or 0/mt ^– were obtained by either crosses or polyethylene glycol induced somatic fusions between haploid strains. When crosses were made between mt ⁺/mt ⁺ or mt ⁺/0 and mt ^–/mt ^– or 0/mt ^–, the transmission of chloroplast genes occurred mostly from the female parent, indicating a typical maternal inheritance. In these cases the chloroplast nucleoids of one of the two parents disappeared within 2.5 h after mating. In contrast, when female diploids were crossed to mt ⁺/mt ^– male diploids, the chloroplast genes from both parents were transmitted to the progeny at equal frequency. The chloroplast nucleoids derived from the two parents were conserved for at least 2.5 h after mating. It is concluded that the chloroplast genes derived from the heterozygous male (mt ⁺/mt ^–) are protected from the degradation that takes place soon after zygote formation, by virtue of the presence of mt ⁺ gene although this allele is recessive with respect to the phenotypic expression of sexuality in diploids.     

Chloroplast gene inheritance studied by somatic fusion in Chlamydomonas reinhardtii

Summary Somatic fusion between strains of Chlamydomonas containing complementing cell-wall and auxotrophic mutations, having the same mating-type (mt) and bearing chloroplast markers, have been performed to study the mode of chloroplast gene inheritance in the fusion products. About one third of the fusion products (mt ⁺/mt ⁺ or mt ^–/mt ^–) transmitted chloroplast markers from both parents (= biparental fusion products). The rest of the population was equally distributed between fusion products transmitting the chloroplast marker of one parent or the other (uniparental fusion products) exclusively. Incubation of the fusion products in the dark for 48 hours, immediately after the fusion, decreases the frequency of biparental fusion products. The results indicate that the general process of elimination of chloroplast alleles is independent of the presence of both mt ⁺ and mt ^– alleles in the cell. In contrast, directional elimination (i.e. preferential elimination of paternal chloroplast alleles) does appear to depend upon heterozygosity at the mt locus. These results are discussed in relation to the models which have been proposed to explain the maternal inheritance of chloroplast genes in Chlamydomonas.     

Detection of chloroplast DNA by using fluorescent monoclonal anti-bromodeoxyuridine antibody and analysis of its fate during zygote formation in Chlamydomonas reinhardtii

Summary A monoclonal anti-bromodeoxyuridine antibody conjugated to fluorescein was used to detect the chloroplast nucleoids after specific incorporation of bromodeoxyuridine (BUdR) into the chloroplast DNA of Chlamydomonas reinhardtii. The incorporation of BUdR was enhanced by simultaneous treatment with fluorodeoxyuridine (FUdR). The method was applied to analyze the fate of chloroplast DNA in zygotes resulting from mating between BUdR-treated gametes (mt ⁺ or mt ^-) and untreated gametes of opposite mating-type. In crosses between wild-type strains, the nucleoids of mt ⁺ origin remained in the large majority of zygotes whereas those of mt ^- origin most often disappeared within the first hours following copulation. In crosses of the type mat-3 mt ⁺xwild-type mt ^- (the mat-3 mutation permits a high transmission of chloroplast genes from the mt ^- parent), the nucleoids of mt ^- origin were generally not eliminated which indicates that the mat-3 mutation prevents the selective destruction of paternal chloroplast DNA in the zygote.     

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.     

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.     

Transmission of chloroplast alleles in somatic fusion products obtained from vegetative cells and/or ‘Gametes’ of Chlamydomonas reinhardi

Summary Somatic fusion between cells of Chlamydomonas containing complementing cell wall and auxotrophic mutations and bearing chloroplast markers for resistance to antibiotics (streptomycin or spectinomycin) have been performed to analyze the mode of chloroplast gene transmission in the fusion products. Prototrophic colonies developed from mitotic divisions of diploid fusion products were isolated on minimal medium and analyzed for their resistance to antibiotics. Fusion was performed between vegetative or nitrogen-starved cells (non-flagellated gametes) of the same or of opposite mating type. In all cases, about one third of the fusion products (mt ⁺/mt ⁺, mt ^–/mt ^–, or mt ⁺/mt ^–) transmitted chloroplast markers from both parents (= biparental fusion products). The rest of the population was equally distributed between fusion products transmitting the chloroplast marker of one parent or the other exclusively (uniparental, or UP fusion products). The results indicate that the preferential elimination of paternal chloroplast alleles (i.e. maternal inheritance) observed after sexual fusion does not occur following artificially induced cell fusion, and that heterozygosity at the mt locus is not sufficient to ensure a directionality in uniparental chloroplast gene transmission.When somatic fusions were made between vegetative cells and nitrogen-starved cells, preferential transmission of the chloroplast alleles of the vegetative parent was observed, independently of the mating type of the parent. The data can be interpreted in terms of differences in the input frequencies of parental chloroplast genomes at the time of cell fusion. The possible importance of flagellar contact between opposite mating types in determining patterns of chloroplast gene transmission is also discussed.     

A model for the rapid vegetative segregation of multiple chloroplast genomes in Chlamydomonas: Assumptions and predictions of the model

Summary Physical evidence indicates that the chloroplast DNA of Chlamydomonas reinhardtii is composed of approximately 75 copies of a small unique sequence. Genetic analysis of zygotes biparental for chloroplast genes shows rapid vegetative segregation of parental chloroplast alleles. Zygote clones composed entirely of homoplasmic progeny cells predominate within 10–20 post-mating generations. A model is proposed here which reconciles the high multiplicity of chloroplast genes with their rapid vegetative segregation rates. Clustering of genomes into a small number of discrete areas (nucleoids) within the chloroplast reduces the effective number of segregating units. A non-random distribution of nucleoids to daughter cells, dictated solely by the spatial arrangement of parental nucleoids with respect to the plane of chloroplast division, further increases the rate of segregation from heteroplasmic cells. Recombination between parental chloroplast genomes is viewed as an indication of nucleoid fusion, and can account for differences in the patterns and rates of segregation at different gene loci. Within such fused nucleoids, clustering of parental genomes and a non-random distribution, again based solely on physical positioning of the genomes, to daughter nucleoids, could act to promote rapid genetic purification of heteroplasmic nucleoids. The effects of biased parental nucleoid ratios, and of potentially unequal nucleoid distributions to daughter chloroplasts are also discussed with respect to observed rates and patterns of chloroplast gene segregation.     

Physical evidence for recombination of chloroplast dna in hybrid progeny of Chlamydomonas eugametos and C. moewusii

Summary Differences in the restriction endonuclease fragmentation patterns of chloroplast DNA (cpDNA) from C. eugametos and C. moewusii have been used to study the inheritance of these DNAs in interspecific hybrids. Analysis of the cpDNAs from ten randomly selected F₁ hybrids, in each case revealed cpDNA to be recombinant for AvaI and BstEII restriction sites, although fragments characteristic of C. eugametos, the mt+ parent, were typically found in excess of those for C. moewusii, the mt– parent. In backcrosses between an F ₁ mt+ hybrid and C. moewusii mt–, seven randomly selected B₁ hybrids showed cpDNA restriction patterns either identical to or highly similar to that of the mt+ parent. We propose that cpDNA molecules are predominantly transmitted by the mt+ parent in both F₁ and B₁ generations but that selection favors survival of F₁ progeny with recombinant chloroplast genomes which avoid interspecific incompatibilities. On the surface, the inheritance of recombinant cpDNA contrasts with the simultaneous uniparental inheritance of two putative chloroplast markers (sr-2 and er-nM1 ⁺). However, it may be that these two markers are by chance associated with cpDNA sequences of the mt+ parent which were selected in all F₁ hybrids.     

Dispersive labelling of Chlamydomonas chloroplast DNA in 15N-14N density transfer experiments

Summary ¹⁵N-¹⁴N density transfer experiments with synchronized vegetative cultures of Chlamydomonas reinhardtii revealed a dispersive labelling of chloroplast DNA (cpDNA) while the labelling of nuclear DNA was consistent with semiconservative replication. The dispersive labelling of cpDNA was progressive and extensive as after less than two net doublings of this DNA in ¹⁴N-medium no significant amount of fully heavy, ¹⁵N-strands could be detected in denatured cpDNA preparations; the average size of DNA in these preparations corresponded to 6% of the intact chloroplast genome or about 12 kbp. The density shifts of native cpDNA samples were found to be consistent with the net amounts of cpDNA synthesized. This observation indicates that essentially all ¹⁵N atoms incorporated prior to the transfer were conserved and that metabolic turnover of cpDNA was probably absent. Our results are best explained by the exchange of homologous single-stranded segments between cpDNA molecules to form heteroduplex regions and by each DNA molecule undergoing several rounds of heteroduplex formation.     

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.     

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).     

A uniparental mutant of Chlamydomonas reinhardtii resistant to chloramphenicol

Summary A uniparental mutant of Chlamydomonas resistant to chloramphenicol was selected following treatment of wild-type cells with 5-fluorodeoxyuridine. Under heterotrophic conditions, growth and chloroplast protein synthesis of this mutant (CAP1) are resistant to chloramphenicol. Under phototrophic conditions, CAP1 is sensitive to chloramphenicol. In addition CAP1 displays thermosensitivity when grown phototrophically in the absence of antibiotics: at the restrictive temperature, a specific reduction of those thylakoid membrane polypeptides which are synthesized inside the chloroplast is observed. Alternative explanations for the pleiotropic phenotype of CAP1 are discussed.     

Studies on homologous recombination in the green alga Chlamydomonas reinhardtii

The introduction of exogenous DNA into the nuclear genome of Chlamydomonas reinhardtii occurs predominantly via non-homologous (illegitimate) recombination and results in integration at apparently-random loci. Using truncated and modified versions of the C. reinhardtii ARG7 gene in a series of transformation experiments, we demonstrate that homologous recombination between introduced DNA molecules occurs readily in C. reinhardtii, requires a region of homology of no more than 230 bp, and gives rise to intact copies of ARG7 in the nuclear genome. Evidence is presented for homologous recombination between introduced ARG7 DNA and the resident copy of the gene, and for the de-novo synthesis of the ARG7 sequence during transformation.     

Net synthesis of chloroplast DNA throughout the synchronized vegetative cell-cycle of Chlamydomonas

Summary The accumulation of chloroplast and nuclear DNAs during the 12 h light and 12 h dark synchronized vegetative cell-cycle of Chlamydomonas reinhardtii was monitored by the direct optical quantification of these DNAs in the analytical ultracentrifuge. Net synthesis of nuclear DNA was sharply discontinuous and this synthesis occurred during the first 6 h of the dark period. In contrast, the net synthesis of chloroplast DNA appeared continuous throughout the cell-cycle. The rate of this accumulation, however, was greatest in the dark period.