Inheritance of chloroplast DNA in Chlamydomonas reinhardtii

Two symmetrically located deletions of approximately 100 base pairs each have been identified in chloroplast DNA of Chlamydomonas reinhardtii. Although present in a mutant strain that requires acetate for growth, both deletions have been shown to be distinct from the nonphotosynthetic phenotype of this strain. These physical markers in the chloroplast genome and maternally inherited genetic markers showed strict cotransmission in reciprocal crosses. Thus, our results are consistent with the location of the well-characterized maternally inherited genetic markers in chloroplast DNA of C. reinhardtii.     

Perturbation of chloroplast DNA amounts and chloroplast gene transmission in Chlamydomonas reinhardtii by 5-fluorodeoxyuridine

5-Fluorodeoxyuridine selectively decreases the rate of chloroplast DNA replication in Chlamydomonas resulting after several generations of growth in equilibrium levels as low as one-seventh of normal. When the maternal parent is treated prior to mating, the decrease of chloroplast DNA appears to perturb the normal maternal transmission of chloroplast genes, dramatically increasing the proportion of exceptional zygotes transmitting chloroplast genes from the paternal parent.     

Structure and function of a chloroplast DNA replication origin of Chlamydomonas reinhardtii

Chloroplast DNA replication in Chlamydomonas reinhardtii is initiated by the formation of a displacement loop (D-loop) at a specific site. One D-loop site with its flanking sequence was cloned in recombinant plasmids SC3-1 and R-13. The sequence of the chloroplast DNA insert in SC3-1, which includes the 0.42-kilobase (kb) D-loop region, as well as 0.2 kb to the 5′ end and 0.43 kb to the 3′ end of the D-loop region, was determined. The sequence is A+T-rich and contains four large stem-loop stuctures. An open reading frame potentially coding for a polypeptide of 136 amino acids was detected in the D-loop region. One stem-loop structure and two back-to-back prokaryotic-type promoters were mapped within the open reading frame. The 5.5-kb EcoRI fragment cloned in R-13 contains the 1.05-kb SC3-1 insert and its flanking regions. A yeast autonomously replicating (ARS) sequence and an ARC sequence, which promotes autonomous replication in Chlamydomonas, have been mapped within the flanking regions [Vallet, J.-M. & Rochaix, J.-D. (1985) Curr. Genet. 9, 321-324]. Both R-13 and SC3-1 were active as templates in a crude algal preparation that supports DNA synthesis. In this in vitro system, chloroplast DNA synthesis initiated near the D-loop site.     

Active digestion of sperm mitochondrial DNA in single living sperm revealed by optical tweezers

In almost all eukaryotes, mitochondrial (mt) genes are transmitted to progeny mainly from the maternal parent. The most popular explanation for this phenomenon is simple dilution of paternal mtDNA, because the paternal gametes (sperm) are much smaller than maternal gametes (egg) and contribute a limited amount of mitochondria to the progeny. Recently, this simple explanation has been challenged in several reports that describe the active digestion of sperm mtDNA, down-regulation of mtDNA replication in sperm, and proteolysis of mitochondria triggered by ubiquitination. In this investigation, we visualized mt nucleoids in living sperm by using highly sensitive SYBR green I vital staining. The ability to visualize mt nucleoids allowed us to clarify that the elimination of sperm mtDNA upon fertilization is achieved through two steps: (i) gradual decrease of mt nucleoid numbers during spermatogenesis and (ii) rapid digestion of sperm mtDNA just after fertilization. One notable point is that the digestion of mtDNA is achieved before the complete destruction of mitochondrial structures, which may be necessary to avoid the diffusion and transmission of potentially deleterious sperm mtDNA to the progeny.     

Biogenic membranes of the chloroplast in Chlamydomonas reinhardtii

The polypeptide subunits of the photosynthetic electron transport complexes in plants and algae are encoded by two genomes. Nuclear genome-encoded subunits are synthesized in the cytoplasm by 80S ribosomes, imported across the chloroplast envelope, and assembled with the subunits that are encoded by the plastid genome. Plastid genome-encoded subunits are synthesized by 70S chloroplast ribosomes directly into membranes that are widely believed to belong to the photosynthetic thylakoid vesicles. However, in situ evidence suggested that subunits of photosystem II are synthesized in specific regions within the chloroplast and cytoplasm of Chlamydomonas. Our results provide biochemical and in situ evidence of biogenic membranes that are localized to these translation zones. A “chloroplast translation membrane” is bound by the translation machinery and appears to be privileged for the synthesis of polypeptides encoded by the plastid genome. Membrane domains of the chloroplast envelope are located adjacent to the cytoplasmic translation zone and enriched in the translocons of the outer and inner chloroplast envelope membranes protein import complexes, suggesting a coordination of protein synthesis and import. Our findings contribute to a current realization that biogenic processes are compartmentalized within organelles and bacteria.     

Stable nuclear transformation of Chlamydomonas reinhardtii by using a C. reinhardtii gene as the selectable marker.

We have developed a stable nuclear transformation system for the unicellular green alga Chlamydomonas reinhardtii. Transformation was accomplished by introducing the cloned C. reinhardtii oxygen-evolving enhancer protein 1 (OEE1) gene into C. reinhardtii cells by bombardment with DNA-coated tungsten particles. The recipient strain was an OEE1-deficient, nonphotosynthetic, acetate-requiring mutant, which recovered photosynthetic competence after transformation, and was therefore able to grow in the absence of acetate. Analysis of several transformants indicates that transformation has proceeded via second-site integration of the cloned gene, leaving the endogenous mutant gene intact. In genetic crosses of transformants with wild type, both mutant and wild-type phenotypes were recovered, showing that the photosynthetic competence of transformants was due not to reversion of the original locus but rather to expression of the introduced gene. We suggest that the success of the present system is largely due to using a homologous C. reinhardtii gene, leading to stable maintenance and expression of the gene. Transformation with heterologous genes may be problematic because of poor expression due to an unusual codon bias in C. reinhardtii.     

Transmission of chloroplast genes in triploid and tetraploid zygospores of Chlamydomonas reinhardtii: Roles of mating-type gene dosage and gametic chloroplast DNA content

Diploid clones homozygous (mt⁺/mt⁺ or mt^-/mt^-) or heterozygous (mt⁺/mt^-; phenotypically mt^-) for the mating-type locus and homoplasmic for a chloroplast marker conferring resistance to an antibiotic were isolated by artificially induced cell fusion or sexual mating. These diploids were crossed with haploid or diploid strains of opposite mating type and carrying another chloroplast marker. The transmission of the chloroplast genes was analyzed in the triploid and tetraploid zygospores in comparison with diploid zygospores used as controls. The transmission was almost exclusively maternal (mt⁺) (>94%) in the crosses mt⁺ × mt^-, mt⁺/mt⁺ × mt^-, and mt⁺/mt⁺ × mt^-/mt^-. The transmission was preferentially maternal (>76%) in the crosses mt⁺ × mt^-/mt^- whereas in the crosses mt⁺ × mt⁺/mt^-, <50% of the zygospores transmitted the chloroplast allele of maternal (mt⁺) origin. The zygospores produced in crosses mt⁺/mt⁺ × mt⁺/mt^- transmitted the alleles from both parents in >60% of cases. The results show that (i) the presence of one mt⁺ allele in the mt⁺/mt^- (phenotypically mt^-) diploid gametes and (ii) the higher amount of chloroplast DNA molecules (input) present in the diploid gametes versus the haploid ones favor the transmission of the chloroplast allele contributed by these gametes. Moreover, because the zygospores issued from crosses mt⁺/mt⁺ × mt^- and mt⁺ × mt⁺/mt^- were genotypically identical mt⁺/mt⁺/mt^-) but behaved very differently in their chloroplast gene transmission, it was concluded that the molecular events leading to preferential elimination of paternal DNA copies must occur before the fusion of nuclei or chloroplasts in the newly formed zygotes.     

Extensive sequence homology in the DNA coding for elongation factor Tu from Escherichia coli and the Chlamydomonas reinhardtii chloroplast.

Considerable DNA sequence homology can be detected between the Escherichia coli genes coding for translational components and Chlamydomonas reinhardtii chloroplast DNA. Labeled chloroplast DNA was found to hybridize to restriction fragments of the transducing phage lambda fus3 that code for elongation factor Tu. The chloroplast probe also reacts with fragments coding for ribosomal proteins carried by this phage. The region homologous to the elongation factor genes was located on the physical map of the chloroplast genome by probing restriction fragments of chloroplast DNA with cloned fragments, labeled in vitro, carrying the E. coli elongation factor Tu genes.     

The persistence of maternal inheritance in Chlamydomonas despite hypomethylation of chloroplast DNA induced by inhibitors.

We have used inhibitors of methylation to evaluate the proposal that the extent of methylation of chloroplast DNA ( cpDNA ) of the mating type-plus (mt+) parent occurring during gametogenesis in wild-type Chlamydomonas renhardtii is directly correlated with the uniparental transmission of chloroplast genes by this parent [ Sager , R., Grabowy , C. & Sano , H. (1981) Cell 24, 41-47]. As detected by high-pressure liquid chromatography, the methylation of cpDNA was at its lowest level in the vegetative stage; the mt+ cells had a deoxycytidine methylation index (the percentage of deoxycytidine methylated) of 0.5, while the mating type-minus (mt-) index was lower by at least a factor of 3. This basal level of cpDNA methylation increased more than 20-fold after gametogenesis to give a methylation index of 12.1 and 4.3 for mt+ and mt- gametes, respectively. Another striking increase was detected at the 7-hr-zygote stage, resulting in the methylation of nearly half of the total deoxycytidine residues. The extent of zygotic cpDNA methylation was shown to be dependent on the preexisting methylation level of both parental gametic cpDNAs . L-Ethionine and 5-azacytidine effectively inhibited cpDNA methylation during gametogenesis and ensuing zygotic development as shown by both Hpa II/Msp I digestion patterns and HPLC. The transmission of chloroplast genes was analyzed concomitantly with the inhibitor studies. The two inhibitors produced different patterns of inhibition of methylation in mt- and mt+ cells at a given developmental stage. Our overall results demonstrate that the extent of mating type-specific and gamete-specific methylation during gametogenesis is not correlated with the frequency of maternal transmission of chloroplast genes.     

Herbicide resistance in Chlamydomonas reinhardtii results from a mutation in the chloroplast gene for the 32-kilodalton protein of photosystem II

We report the isolation and characterization of a uniparental mutant of Chlamydomonas reinhardtii that is resistant to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2-chloro-4-ethylamino-6-isopropylamino-s-triazine (atrazine). Such herbicides inhibit photosynthesis by preventing transfer of electrons in photosystem II from the primary stable electron acceptor Q to the secondary stable electron acceptor complex B, which is thought to contain a protein of 32 kDa and a bound quinone. It has been proposed that herbicide binding to the 32-kDa protein alters the B complex so that electron transfer from Q is prohibited. Both whole and broken-cell preparations of the mutant alga show a resistance to the effects of herbicide on electron transfer from Q to B, as measured by fluorescence-induction kinetics. In the absence of herbicide, mutant cells exhibit a slower rate of Q to B electron transfer than do wild-type cells. The 32-kDa protein from wild-type cells, but not mutant cells, binds azido[¹⁴C]atrazine at 0.1 μM. We have isolated psbA, the chloroplast gene for the 32-kDa protein, from both wild-type and herbicide-resistant algae and sequenced the coding regions of the gene that are contained in five exons. The only difference between the exon nucleotide sequences of the wild-type and mutant psbA is a single T-A to G-C transversion. This mutation results in a predicted amino acid change of serine in the wild-type protein to alanine in the mutant. We suggest that this alteration in the 32-kDa protein is the molecular basis for herbicide resistance in the C. reinhardtii mutant.     

Differential methylation of chloroplast DNA regulates maternal inheritance in a methylated mutant of Chlamydomonas

In Chlamydomonas, the maternal inheritance of chloroplast genes correlates with the differential methylation of chloroplast DNA (chlDNA) in females (mt⁺) but not in males (mt^-). Our previous studies have supported our methylation-restriction model in which the maternal transmission is accounted for by the differential methylation in gametes which protects female but not male chlDNA from degradation during zygote formation. In the mutant me-1 [Bolen, P. L., Grant, D. M., Swinton, D., Boynton, J. E. & Gillham, N. W. (1982) Cell 28, 335-343], chlDNA of vegetative cells of both mating types is heavily methylated even before gametogenesis; nonetheless, maternal inheritance occurs in mutants as in wild type. To investigate the mechanism of maternal inheritance in the me-1 mutant, we have compared restriction fragment patterns after agarose gel electrophoresis of chlDNAs from mutant vegetative cells and gametes with those from wild type, by using a set of 32 restriction enzymes of which 17 were methylation-sensitive in this system. We find that additional methylation occurs during gametogenesis in the mutant female (mt⁺) but not in the corresponding male (mt^-). Thus, gamete-specific, mating-type-specific methylation occurs in the me-1 mutant as in the wild type, consistent with our methylation-restriction model. In the me-1 mutant, gametic methylation occurs on a background of vegetative cell methylation not present in wild-type cells and irrelevant to the regulation of chloroplast inheritance. Comparison of the me-1 mutation with the mat-1 mutation [Sager, R., Grabowy, C. & Sano, H. (1981) Cell 24, 41-47] provides evidence for the existence of two different chlDNA methylation control systems: mat-1, linked to the mating type locus and regulating the mating-type-specific methylation that correlates with maternal inheritance, and me-1, unlinked to the mating type locus and unrelated to the regulation of maternal inheritance.     

Role of methylation in the modification and restriction of chloroplast DNA in Chlamydomonas.

The different metabolic paths followed by homologous chloroplast DNAs of maternal and paternal origins in zygotes of Chlamydomonas were examined by prelabeling parental cells, before mating them, with [3H]adenine, [3H]thymidine, and [3H]deoxycytidine. Within 6 hr after mating, maternal chloroplast DNA was extensively methylated to 5-methylcytosine and its bouyant density decreased. Paternal chloroplast DNA was largely degraded. Some radioactivity from deoxycytidine of maternal origin reappeared in thymine, and residual paternal DNA contained radioactivity in a base tentatively identified as uracil. These results confirm and extend our previous findings and support our hypothesis that modification (methylation) and restriction enzymes determine maternal inheritance of chloroplast DNA and that the two parental DNAs have different metabolic fates within the zygote.