Mitochondrial genome of the colorless green alga Polytomella parva: two linear DNA molecules with homologous inverted repeat Termini

Most of the well-characterized mitochondrial genomes from diverse green algal lineages are circular mapping DNA molecules; however, Chlamydomonas reinhardtii has a linear 15.8 kb unit mitochondrial genome with 580 or 581 bp inverted repeat ends. In mitochondrial-enriched fractions prepared from Polytomella parva (=P. agilis), a colorless, naturally wall-less relative of C. reinhardtii, we have detected two linear mitochondrial DNA (mtDNA) components with sizes of 13.5 and 3.5 kb. Sequences spanning 97% and 86% of the 13.5- and 3.5-kb mtDNAs, respectively, reveal that these molecules contain long, at least 1.3 kb, homologous inverted repeat sequences at their termini. The 3.5-kb mtDNA has only one coding region (nad6), the functionality of which is supported by both the relative rate at which it has accumulated nonsynonymous nucleotide substitutions and its absence from the 13.5-kb mtDNA which encodes nine genes (i.e., large and small subunit rRNA [LSU and SSU rRNA] genes, one tRNA gene, and six protein-coding genes). On the basis of DNA sequence data, we propose that a variant start codon, GTG, is utilized by the P. parva 13.5-kb mtDNA-encoded gene, nad5. Using the relative rate test with Chlamydomonas moewusii (=C. eugametos) as the outgroup, we conclude that the nonsynonymous nucleotide substitution rate in the mitochondrial protein-coding genes of P. parva is on an average about 3.3 times that of the C. reinhardtii counterparts.     

Chloroplast DNA variation in Chlamydomonas and its potential application to the systematics of this genus

We have estimated the extent of chloroplast DNA (cpDNA) variation in three species of green algae belonging to the genus Chlamydomonas to determine if this variation could be used for taxonomic studies. The overall arrangement of sequences in the chloroplast genome of Chlamydomonas eugametos was compared with that of the closely related C. moewusii and that of the more distantly related C. reinhardtii. The results show that the chloroplast genomes of C. eugametos and C. moewusii are essentially co-linear and are highly homologous in sequence while those of C. eugametos and C. reinhardtii have been extensively rearranged and share a relatively low overall sequence homology. This wide range of chloroplast genome organization suggests that the analysis of cp-DNA variation will be useful for the classification of algae belonging to the Chlamydomonas genus.     

Group I introns interrupt the chloroplast psaB and psbC and the mitochondrial rrnL gene in Chlamydomonas.

The polymerase chain reaction was used to identify novel IAI subgroup introns in cpDNA-enriched preparations from the interfertile green algae Chlamydomonas eugametos and Chlamydomonas moewusii. These experiments along with sequence analysis disclosed the presence, in both green algae, of a single IA1 intron in the psaB gene and of two group I introns (IA2 and IA1) in the psbC gene. In addition, two group I introns (IA1 and IB4) were found in the peptidyltransferase region of the mitochondrial large subunit rRNA gene at the same positions as previously reported Chlamydomonas chloroplast introns. The 188 bp segment preceding the first mitochondrial intron revealed extensive sequence similarity to the distantly spaced rRNA-coding modules L7 and L8 in the Chlamydomonas reinhardtii mitochondrial DNA, indicating that these two modules have undergone rearrangements in Chlamydomonas. The IA1 introns in psaB and psbC were found to be related in sequence to the first intron in the C. moewusii chloroplast psbA gene. The similarity between the former introns extends to the immediate 5' flanking exon sequence, suggesting that group I intron transposition occurred from one of the two genes to the other through reverse splicing.     

REVIEW—CONTRASTING MITOCHONDRIAL GENOME ORGANIZATIONS AND SEQUENCE AFFILIATIONS AMONG GREEN ALGAE: POTENTIAL FACTORS, MECHANISMS, AND EVOLUTIONARY SCENARIOS

The three green algal mitochondrial genomes completely sequenced to date — those of Chlamydomonas reinhardtii Dangeard, Chlamydomonas eugametos Gerloff, and Prototheca wickerhamii Soneda & Tubaki — revealed very different mitochondrial genome organizations and sequence affiliations. The Chlamydomonas genomes resemble the ciliate / fungal / animal counterparts, and the Prototheca genome resembles land plant homologues. This review points out that all the green algal mitochondrial genomes examined to date resemble either the Chlamydomonas or the Prototheca mitochondrial genome; the Chlamydomonas- like mitochondrial genomes are small and have a reduced gene content (no ribosomal protein or 5S rRNA genes and only a few protein-coding and tRNA genes) and fragmented and scrambled rRNA coding regions, whereas the Prototheca- like mitochondrial genomes are larger and have a larger set of protein-coding genes (including ribosomal protein genes), more tRNA genes, and 5S rRNA and conventional continuous small-subunit (SSU) and large-subunit (LSU) rRNA coding regions. It appears, therefore, that the differences previously observed between the mitochondrial genomes of C. reinhardtii and P. wickerhamii extend to the two green algal mitochondrial lineages to which they belong and are significant enough to raise questions about the causes and mechanisms responsible for such contrasting evolutionary strategies among green algae. This review suggests an integrative approach in explaining the occurrence of distinct evolutionary strategies and apparent phylogenetic affiliations among the known green algal mitochondrial lineages. The observed differences could be the result of distinct genetic potentials differentiated during the previous evolutionary history of the flagellate ancestors and / or of subsequent changes in habitat and life history of the more advanced green algal lineages.     

Genomewide analysis of box C/D and box H/ACA snoRNAs in Chlamydomonas reinhardtii reveals an extensive organization into intronic gene clusters

Chlamydomonas reinhardtii is a unicellular green alga, the lineage of which diverged from that of land plants >1 billion years ago. Using the powerful small nucleolar RNA (snoRNA) mining platform to screen the C. reinhardtii genome, we identified 322 snoRNA genes grouped into 118 families. The 74 box C/D families can potentially guide methylation at 96 sites of ribosomal RNAs (rRNAs) and snRNAs, and the 44 box H/ACA families can potentially guide pseudouridylation at 62 sites. Remarkably, 242 of the snoRNA genes are arranged into 76 clusters, of which 77% consist of homologous genes produced by small local tandem duplications. At least 70 snoRNA gene clusters are found within introns of protein-coding genes. Although not exhaustive, this analysis reveals that C. reinhardtii has the highest number of intronic snoRNA gene clusters among eukaryotes. The prevalence of intronic snoRNA gene clusters in C. reinhardtii is similar to that of rice but in contrast with the one-snoRNA-per-intron organization of vertebrates and fungi and with that of Arabidopsis thaliana in which only a few intronic snoRNA gene clusters were identified. This analysis of C. reinhardtii snoRNA gene organization shows the functional importance of introns in a single-celled organism and provides evolutionary insight into the origin of intron-encoded RNAs in the plant lineage.     

The plastid aldolase gene fromChlamydomonas reinhardtii: Intron/exon organization, evolution, and promoter structure

Genomic clones encoding the plastidic fructose- 1,6-bisphosphate aldolase ofChlamydomonas reinhardtii were isolated and sequenced. The gene contains three introns which are located within the coding sequence for the mature protein. No introns are located within or near the sequence encoding the transit-peptide, in contrast to the genes for plastidic aldolases of higher plants. Neither the number nor the positions of the three introns of theC. reinhardtii aldolase gene are conserved in the plastidic or cytosolic aldolase genes of higher plants and animals. The 5 border sequences of introns in the aldolase gene ofC. reinhardtii exhibit the conserved plant consensus sequence. The 3 acceptor splice sites for introns 1 and 3 show much less similarity to the eukaryotic consensus sequences than do those of intron 2. The plastidic aldolase gene has two tandemly repeated CAAT box motifs in the promoter region. Genomic Southern blots indicate that the gene is encoded by a single locus in theC. reinhardtii genome.     

Stable expression of antibiotic-resistant gene ble from Streptoalloteichus hindustanus in the mitochondria of Chlamydomonas reinhardtii

The mitochondrial expression of exogenous antibiotic resistance genes has not been demonstrated successfully to date, which has limited the development of antibiotic resistance genes as selectable markers for mitochondrial site-directed transformation in Chlamydomonas reinhardtii. In this work, the plasmid pBSLPNCB was constructed by inserting the gene ble of Streptoalloteichus hindustanus (Sh ble), encoding a small (14-kilodalton) protective protein into the site between TERMINVREP-Left repeats and the cob gene in a fragment of mitochondrial DNA (mtDNA) of C. reinhardtii. The fusion DNA-construct, which contained TERMINVREP-Left, Sh ble, cob, and partial nd4 sequence, were introduced into the mitochondria of the respiratory deficient dum-1 mutant CC-2654 of C. reinhardtii by biolistic particle delivery system. A large number of transformants were obtained after eight weeks in the dark. Subsequent subculture of the transformants on the selection TAP media containing 3 ìg/mL Zeomycin for 12 months resulted in genetically modified transgenic algae MT-Bs. Sequencing and Southern analyses on the mitochondrial genome of the different MT-B lines revealed that Sh ble gene had been integrated into the mitochondrial genome of C. reinhardtii. Both Western blot, using the anti-BLE monoclonal antibody, and Zeomycin tolerance analysis confirmed the presence of BLE protein in the transgenic algal cells. It indicates that the Sh ble gene can be stably expressed in the mitochondria of C. reinhardtii.     

Structure,organization and expression of the genes encoding mitochondrial cytochrome c(1) and the Rieske iron-sulfur protein in Chlamydomonas reinhardtii

The sequence and organization of the Chlamydomonas reinhardtii genes encoding cytochrome c(1) ( Cyc1) and the Rieske-type iron-sulfur protein ( Isp), two key nucleus-encoded subunits of the mitochondrial cytochrome bc(1) complex, are presented. Southern hybridization analysis indicates that both Cyc1 and Isp are present as single-copy genes in C. reinhardtii. The Cyc1 gene spans 6404 bp and contains six introns, ranging from 178 to 1134 bp in size. The Isp gene spans 1238 bp and contains four smaller introns, ranging in length from 83 to 167 bp. In both genes, the intron/exon junctions follow the GT/AG rule. Internal conserved sequences were identified in only some of the introns in the Cyc1 gene. The levels of expression of Isp and Cyc1 genes are comparable in wild-type C. reinhardtii cells and in a mutant strain carrying a deletion in the mitochondrial gene for cytochrome b (dum-1). Nevertheless, no accumulation of the nucleus-encoded cytochrome c(1) or of core proteins I and II was observed in the membranes of the respiratory mutant. These data show that, in the green alga C. reinhardtii, the subunits of the cytochrome bc(1) complex fail to assemble properly in the absence of cytochrome b.     

The complete mitochondrial DNA sequence of the green alga Pseudendoclonium akinetum (Ulvophyceae) highlights distinctive evolutionary trends in the chlorophyta and suggests a sister-group relationship between the Ulvophyceae and Chlorophyceae

The mitochondrial genome has undergone radical changes in both the Chlorophyta and Streptophyta, yet little is known about the dynamics of mtDNA evolution in either of these lineages. In the Chlorophyta, which comprises four of the five recognized classes of green algae (Prasinophyceae, Trebouxiophyceae, Ulvophyceae, and Chlorophyceae), the mitochondrial genome varies from 16 to 55 kb. This genome has retained a compact gene organization and a relatively complex gene repertoire ("ancestral" pattern) in the basal lineages represented by the Trebouxiophyceae and Prasinophyceae, whereas it has been reduced in size and gene complement and tends to evolve much more rapidly at the sequence level ("reduced-derived" pattern of evolution) in the Chlorophyceae and the lineage leading to the enigmatic chlorophyte Pedinomonas. To gain information about the evolutionary trends of mtDNA in the Ulvophyceae and also to gain insights into the phylogenetic relationships between ulvophytes and other chlorophytes, we have determined the mtDNA sequence of Pseudendoclonium akinetum. At 95,880 bp, Pseudendoclonium mtDNA is the largest green-algal mitochondrial genome sequenced to date and has the lowest gene density. These derived features are reminiscent of the "expanded" pattern exhibited by embryophyte mtDNAs, indicating that convergent evolution towards genome expansion has occurred independently in the Chlorophyta and Streptophyta. With 57 conserved genes, the gene repertoire of Pseudendoclonium mtDNA is slightly smaller than those of the prasinophyte Nephroselmis olivacea and the trebouxiophyte Prototheca wickerhamii. This ulvophyte mtDNA contains seven group I introns, four of which have homologs in green-algal mtDNAs displaying an "ancestral" or a "reduced-derived" pattern of evolution. Like its counterpart in the chlorophycean green alga Scenedesmus obliquus, it features numerous small, dispersed repeats in intergenic regions and introns. Its overall rate of sequence evolution appears to be accelerated to an intermediary level as compared with the rates observed in "ancestral" and "reduced-derived" mtDNAs. In agreement with the finding that Pseudendoclonium mtDNA exhibits features typical of both the "ancestral" and "reduced-derived" patterns of evolution, phylogenetic analyses of seven mtDNA-encoded proteins revealed a sister-group relationship between this ulvophyte and chlorophytes displaying "reduced-derived" mtDNAs.     

Isolation and characterization of a new transposable element in Chlamydomonas reinhardtii

A new transposable element, Tcr3, was identified in the unicellular green alga Chlamydomonas reinhardtii. The Tcr3 element contained imperfect terminal inverted repeat sequences of 56 bp and created a 2 bp target site duplication upon insertion. Insertion of Tcr3 into the 3-untranslated region of the NIT8 gene, which is essential for nitrate assimilation, prevented expression of the gene. Excision of the Tcr3 element correlated with reversion of the mutant phenotype and left behind a 3 bp footprint. Tcr3 was found in all Chlamydomonas isolates tested and should prove to be useful for transposon-tagging experiments in Chlamydomonas.     

Nuclear-mitochondrial interactions in cytoplasmic male-sterile sorghum

Summary Variation in mitochondrial genome organization and expression between male fertile and sterile nuclear-cytoplasmic combinations of sorghum has been examined. Cytoplasmic genotypes were classified into eleven groups on the basis of restriction endonuclease digestion of mitochondrial DNA (mtDNA) and five groups on the basis of mitochondrial translation products. These cytoplasms were further characterized by hybridization of specific gene probes to Southern blots of EcoRI digested mtDNA, and identification of the fragment location of four mitochondrial genes. Variation was observed in the genomic location and copy number of the F₁ ATPase -subunit gene, as well as the genomic location and gene product of the cytochrome c oxidase subunit I gene. The effect of nuclear genotype on mitochondrial genome organization, expression and the presence of two linear plasmid-like mtDNA molecules was examined. Our results indicate that nuclear-mitochondrial interactions are required for regulation of mitochondrial gene expression. When a cytoplasm is transferred from its natural to a foreign nuclear background some changes in the products of in organello mitochondrial protein synthesis occur. In a number of cytoplasmic genotypes these changes correlate with the expression of cytoplasmic male sterile phenotype, suggesting a possible molecular basis for this mutation.     

Mitochondrial genome sequence evolution in Chlamydomonas

The mitochondrial genomes of the Chlorophyta exhibit significant diversity with respect to gene content and genome compactness; however, quantitative data on the rates of nucleotide substitution in mitochondrial DNA, which might help explain the origin of this diversity, are lacking. To gain insight into the evolutionary forces responsible for mitochondrial genome diversification, we sequenced to near completion the mitochondrial genome of the chlorophyte Chlamydomonas incerta, estimated the evolutionary divergence between Chlamydomonas reinhardtii and C. incerta mitochondrial protein-coding genes and rRNA-coding regions, and compared the relative evolutionary rates in mitochondrial and nuclear genes. Synonymous and nonsynonymous substitution rates do not differ significantly between the mitochondrial and nuclear protein-coding genes. The mitochondrial rRNA-coding regions, however, are evolving much faster than their nuclear counterparts, and this difference might be explained by relaxed functional constraints on the mitochondrial translational apparatus due to the small number of proteins synthesized in Chlamydomonas mitochondria. Substitution rates at synonymous sites in a nonstandard mitochondrial gene (rtl) and at intronic and synonymous sites in nuclear genes expressed at low levels suggest that the mutation rate is similar in these two genetic compartments. Potential evolutionary forces shaping mitochondrial genome evolution in Chlamydomonas are discussed.     

Organization of heterogeneous mitochondrial DNA molecules in mitochondrial nuclei of cultured tobacco cells

Summary The molecular size of mitochondrial DNA (mtDNA) molecules and the number of copies of mtDNA per mitochondrion were evaluated from cultured cells of the tobacco BY-2 line derived fromNicotiana tabacum L. cv. Bright Yellow-2. To determine the DNA content per mitochondrion, protoplasts of cultured cells were stained with 4,6-diamidino-2-phenylindole (DAPI), and the intensity of the fluorescence emitted from the mitochondrial nuclei (mt-nuclei) was measured with a video-intensified photon counting microscope system (VIM system). Each mitochondrion except for those undergoing a division contained one mt-nucleus. The most frequently measured size of the DNA in the mitochondria was between 120 and 200 kilobase pairs (kbp) throughout the course of culture of the tobacco cells. Mitochondria containing more than 200 kbp of DNA increased significantly in number 24 h after transfer of the cells into fresh medium but their number fell as the culture continued. Because division of mitochondria began soon after transfer of the cells into fresh medium and continued for 3 days, the change of the DNA content per mitochondrion during the culture must correspond to DNA synthesis of mitochondria in the course of mitochondrial division. By contrast, the analyses of products of digestion by restriction endonucleases indicated that the genome size of the mtDNA was at least 270 kbp. Electron microscopy revealed that mtDNAs were circular molecules and their length ranged from 1 to 35 m, and 60% of them ranged from 7 to 11 rn. These results indicate that the mitochondrial genome in tobacco cells consists of multiple species of mtDNA molecules, and mitochondria do not contain all the mtDNA species. Therefore, mitochondria are heterogeneous in mtDNA composition.Abbreviations DAPI 4, 6-diamidino-2-phenylindole - mtDNA mitochondrial DNA - mt-genome mitochondrial genome - mt-nucleus mitochondrial nucleus - ptDNA proplastid DNA - pt-nucleus proplastid nucleus - VIM system video-intensified photon counting microscope system     

Nuclear ribosomal RNA genes and algal phylogeny--the Chlamydomonas example

The nuclear ribosomal RNA genes (rDNA) of Chlamydomonas reinhardtii, C. moewusii and C. eugametos were examined with restriction endonuclease fragment and direct rRNA sequencing analyses. These comparative molecular data confirm similarity between C. moewusii and C. eugametos, and dissimilarity between the strains and C. reinhardtii. For C. moewusii and C. eugametos, the fragment analysis of digests with 16 (six base pair recognition site) restriction endonucleases revealed either no or minor differences. These minor differences appear to be confined to length and site variation in the rapidly evolving intergenic spacer region of the algal rDNA repeat unit. In contrast, patterns of digests for C. reinhardtii were completely different from those of C. moewusii and C. eugametos for all enzymes tested. Over two regions of the 18S ribosomal RNA (spanning approx. 300 bases) in C. moewusii and C. eugametos, we observed three possible base substitutions and no insertion/deletion events. The same comparison between C. reinhardtii and C. moewusii (or C. eugametos) revealed 31 base substitutions and eight insertion/deletion events. Overall, the rDNA comparisons support the proposed conspecificity of C. moewusii and C. eugametos, as well as the hypothesis that intraspecific variation in the algal ribosomal RNA coding region is minimal and that comparisons of rDNA sequences at higher taxonomic levels can be useful indicators of algal phylogeny. The degree of difference in the sequences of the 18S coding region between C. reinhardtii and C. moewusii or C. eugametos is comparable to that between an angiosperm and Equisetum and may reflect an ancient divergence between two species in one algal genus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure and expression of the overlapping ND4L and ND5 genes of Neurospora crassa mitochondria

Summary Genes homologous to the mammalian mitochondrial NADH dehydrogenase subunit genes ND4L and ND5 were identified in the mitochondrial genome of the filamentous fungus Neurospora crassa, and the structure and expression of these genes was examined. The ND4L gene (interrupted by one intervening sequence) potentially encodes an 89 residue long hydrophobic protein that shares about 26% homology (or 41% homology if conservative amino acid substitutions are allowed) with the analogous human mitochondrial protein. The ND5 gene (which contains two introns) encodes a 715 residue polypeptide that shares 23% homology with the human analogue; a 300 amino acid long region is highly conserved (50% homology) in the two ND5 proteins. The stop codon of the ND4L gene overlaps the initiation codon of the downstream ND5 gene, and the two genes are contranscribed and probably cotranslated. A presumed mature dicistronic (ND4L plus ND5) RNA was detected. The postulated mRNA (about 3.2 kb) contains 5 and 3 non-coding regions of about 86 and 730 nucleotides, respectively; this species is generated from very large precursor RNAs by a complex processing pathway. The ND4L and ND5 introns are all stable after their excision from the precursor species.Abbreviations bp base pairs - rRNA ribosomal RNA - ND NADH dehydrogenase - URF unidentified reading frame - kDal kilodaltons; a.a., amino acid     

Nucleated assembly of Chlamydomonas and Volvox cell walls

The Chlamydomonas reinhardtii cell wall is made up of hydroxyproline-rich glycoproteins, arranged in five distinct layers. The W6 (crystalline) layer contains three major glycoproteins (GP1, GP2, GP3), selectively extractable with chaotropic agents, that self-assemble into crystals in vitro. A system to study W6 assembly in a quantitative fashion was developed that employs perchlorate-extracted Chlamydomonas cells as nucleating agents. Wall reconstitution by biotinylated W6 monomers was monitored by FITC-streptavidin fluorescence and quick-freeze/deep-etch electron microscopy. Optimal reconstitution was obtained at monomer concentrations (0.2-0.3 mg/ml) well below those required for nonnucleated assembly. Assembly occurred from multiple nucleation sites, and faithfully reflected the structure of the intact W6 layer. Specificity of nucleated assembly was demonstrated using two cell-wall mutants (cw-2 and cw-15); neither served as a substrate for assembly of wild-type monomers. In addition, W6 sublayers were assembled from purified components: GP2 and GP3 coassembled to form the inner (W6A) sublayer; this then served as a substrate for self-assembly of GP1 into the outer (W6B) sublayer. Finally, evolutionary relationships between C. reinhardtii and two additional members of the Volvocales (Chlamydomonas eugametos and Volvox carteri) were explored by performing interspecific reconstitutions. Hybrid walls were obtained between C. reinhardtii and Volvox but not with C. eugametos, confirming taxonomic assignments based on structural criteria.