Optional elements in the chloroplast DNAs of Chlamydomonas eugametos and C. moewusii: unidirectional gene conversion and co-conversion of adjacent markers in high-viability crossses

Unlike most polymorphic markers in the Chlamydomonas eugametos and Chlamydomonas moewusii chloroplast DNAs (cpDNAs), the C. moewusii 6- and 21-kb extra sequences and the C. eugametos-specific CeLSU ⋅ 5 intron are transmitted to all of the few viable progeny in reciprocal crosses between the two green algae. To determine whether this unidirectional transmission pattern is due to gene conversion or to selection for F1 hybrid survival, we followed the inheritance of the parental alleles at the loci featuring these three deletions/additions and at several other polymorphic cpDNA loci in zygospore clones derived from high-viability crosses. The great majority of the zygospore clones examined inherited exclusively the long alleles from the mt ^– parent at the loci containing the three optional cpDNA elements, but as expected, they preferentially inherited the markers from the mt ⁺ parent at most other loci. Our results therefore indicate that all three optional cpDNA sequences propagate themselves very efficiently by gene conversion in crosses between strains differing by the presence of these elements. The co-conversion tracts associated with these sequences are longer (>3 kb) than those previously reported for mobile elements spreading by gene conversion. Our results also revealed that less efficient gene conversion events occurred at two other cpDNA loci. Received: 12 February / 14 May 1996     

Extensive sequence rearrangements in the chloroplast genomes of the green algaeChlamydomonas eugametos andChlamydomonas reinhardtii

Summary We have compared the overall sequence organization of chloroplast DNA (cpDNA) from the unicellular green algaeChlamydomonas eugametos andChlamydomonas reinhardtii. Cloned restriction fragments whose locations are known on the chloroplast genome of one or the other alga were hybridized to Southern blots of cpDNA digests from both algae and the positions of hybridization signals were used to align the two algal cpDNA restriction maps. In agreement with the important biological differences reported betweenC. eugametos andC. reinhardtii, we found extensive sequence rearrangements and low overall sequence homology between the two cpDNAs. To explain the striking contrast between our results and the remarkable conservation reported for the sequence and the organization of angiosperm cpDNAs containing inverted repeats, we suggest that the divergence between theC. eugametos andC. reinhardtii chloroplast genomes simply reflects a longer time of separate evolution relative to the angiosperm lineages. However, we cannot exclude the possibility that the algal cpDNAs rearranged faster than angiosperm cpDNAs.     

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.     

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).     

Uniparental inheritance of chloroplast DNA sequences in interspecific hybrids of Chlamydomonas

Summary The meiotic transmission of chloroplast DNA (cpDNA) was studied in crosses between two species of Chlamydomonas (C. moewusii and C. eugametos) which have substantial differences in cpDNA restriction patterns. The results provide a direct demonstration that cpDNA can be inherited in a uniparental pattern, paralleling the transmission of a uniparentally inherited antibiotic resistance marker. Thus, cpDNA could carry the uniparental genes of these species, but other extrachromosomal DNAs are not excluded as possible carriers. For example, C. moewusii was found to contain a set of low molecular weight (LMW) DNA species which cannot be detected in C. eugametos. These LMW DNA species are also transmitted uniparentally in the tetrads studied. Uniparentai transmission may not be an exclusive property of cpDNA in Chlamydomonas species.     

Comparative analysis of the mitochondrial genomes of Chlamydomonas eugametos and Chlamydomonas moewusii

Summary We report the cloning and physical mapping of the mitochondrial genome of Chlamydomonas eugametos together with a comparison of the overall sequence structure of this DNA with the mitochondrial genome of Chlamydomonas moewusii, its closely related and interfertile relative. The C. eugametos mitochondrial DNA (mtDNA) has a 24 kb circular map and is thus 2 kb larger than the 22 kb circular mitochondrial genome of C. moewusii. Restriction mapping and heterologous fragment hybridization experiments indicate that the C. eugametos and C. moewusii mtDNAs are colinear. Nine cross-hybridizing restriction fragments common to the C. eugametos and C. moewusii mtDNAs, and spanning the entirety of these genomes, show length differences between homologous fragments which vary from 0.1 to 2.3 kb. A 600 bp subfragment of C. moewusii mtDNA, within one of these conserved fragments, showed no hybridization with the C. eugametos mtDNA. Of the 73 restriction sites identified in the C. eugametos and C. moewusii mtDNAs, five are specific to C. moewusii, eight are specific to C. eugametos and 30 are common to both species. Hybridization experiments with gene probes derived from protein-coding and ribosomal RNA-coding regions of wheat and Chlamydomonas reinhardtii mtDNAs support the view that the small and large subunit ribosomal RNA-coding regions of the C. eugametos and C. moewusii mtDNAs are interrupted and interspersed with each other and with protein-coding regions, as are the ribosomal RNA-coding regions of C. reinhardtii mtDNA; however, the specific arrangement of these coding elements in the C. eugametos and C. moewusii mtDNAs appears different from that of C. reinhardtii mtDNA.     

A group I intron in the chloroplast large subunit rRNA gene of Chlamydomonas eugametos encodes a double-strand endonuclease that cleaves the homing site of this intron

Summary During interspecific crosses between Chlamydomonas eugametos and Chlamydomonas moewusii, an optional group I intron of 955 base pairs (CeLSU· 5) in the C. eugametos chloroplast large subunit rRNA gene undergoes a duplicative transposition event which is associated with frequent co-conversion of flanking cpDNA sequences. In the present study, we show that the basic protein of 218 amino acids encoded by CeLSU· 5 could mediate the phenomenon of intron transposition, also called intron homing. We overexpressed the ORF specifying this protein in E. coli using expression vectors that contain a C. moewusii cpDNA sequence encompassing the intron homing site. The expression product was found to exhibit a double-strand DNA endonuclease activity that is specific for the homing site. This activity was detected in vivo by self-linearization of the expression plasmids.     

Physical mapping of differences between the chloroplast DNAs of the interfertile algae Chlamydomonas eugametos and Chlamydomonas moewusii

Summary The chloroplast genomes from the interfertile green algae Chlamydomonas eugametos and C. moewusii have been compared in their overall sequence organization. Physical mapping of Aval, BstEII and EcoRI restriction sites on the C. moewusii chloroplast genome revealed that this 292 kilobase-pair (kbp) genome is 49 kbp larger than the C. eugametos genome. Heterologous fragment hybridizations indicated the same order of common sequence elements on the two algal genomes. Almost all of the 49 kbp size difference is accounted for by the presence of two large extra sequences in C. moewusii: a 21 kbp sequence in the inverted repeat and a 5.8 kbp sequence in the single copy-region bordering the 16S ribosomal RNA (rRNA) genes. In addition to these two major deletion/addition differences, 42 restriction site and fragment length differences (ranging from 100 to 500 base pairs) were mapped on the two algal genomes. Surprisingly, the greatest density of these differences was found to be confined within the inverted repeat, one of the most conserved regions of land plant chloroplast genomes.     

An optional group I intron between the chloroplast small subunit rRNA genes of Chlamydomonas moewusii and C. eugametos

Summary We report the presence of a 402 by group I intron in the chloroplast small subunit (SSU) rRNA gene of Chlamydomonas moewusii. The intron is inserted within the highly conserved 530 loop, at a site corresponding to positions 531–532 of the E. coli 16rRNA. Residues surrounding the insertion site almost certainly play an important role in ribosomal proofreading function as they proved to be protected by tRNAs in E. coli 16S rRNA (Moazed and Noller 1986; Stern et al. 1986). The C. moewusii intron revealed a secondary structure model which differs substantially from those of the typical subgroup IA and IB introns. This model, however, shows striking similarities with the structures of the C. reinhardtii chloroplast 23S rRNA gene intron (Rochaix et al. 1985), the S. cerevisiae mitochondrial COB3 intron (Holl et al. 1985) and the three introns of phage T4 in the nrdB, td and sunY genes (Shub et al. 1988). The SSU rRNA gene intron is absent from C. eugametos, an alga that is interfertile with C. moewusii. The presence/absence of the intron account for a 390 by restriction fragment length polymorphism between the two algal SSU rRNA genes, a polymorphic locus that is strictly co-inherited with a tightly linked streptomycin resistance mutation (sr-2) in interspecific hybrids between the two algae.     

The endpoints of an inversion in wheat chloroplast DNA are associated with short repeated sequences containing homology toatt-lambda

Summary The physical mapping of Aval, BstEII and EcoR1 restriction sites on the chloroplast genome of the green alga Chlamydomonas eugametos is presented. The circular map, with a size of 243 kilobase pairs, is the largest yet reported for a chloroplast genome. It features a large inverted repeat sequence, part of which encodes the 16S and 23S ribosomal RNAs (rRNAs), the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcL) and the 32-kdodalton thylakoid membrane protein (psbA). Such an rRNA-encoding inverted repeat sequence is also found in the chloroplast genomes of Chlamydomonas reinhardtii and most land plants. These genomes, however, differ from that of C. eugametos by the absence of the rbcL gene from the inverted repeat sequence of C. reinhardtii and by the absence of both the rbcL and psbA genes from the inverted repeat sequence of land plants. Possible evolutionary implications of these differences are discussed.Abbrevations cpDNA chloroplast DNA - kbp kilobase pairs - psbA 32 kilodalton thylakoid membrane protein gene - rbcL ribulose-1,5-bisphosphate carboxylase-oxygenase large subunit gene - rRNA ribosomal RNA     

The chloroplast gene cluster containing psbF,psbL, petG and rps3 is conserved in Chlamydomonas

We have sequenced a 6.8-kb segment of the Chlamydomonas eugametos chloroplast DNA which contains the psbF, psbL, petG and rps3 genes. As in the distantly related green alga Chlamydomonas reinhardtii, these genes reside in this order (53) on the same DNA strand, suggesting that such a chloroplast gene cluster was present in the most recent common ancestor of all Chlamydomonas species. For each of the four genes, with the exception of rps3, the C. eugametos and C. reinhardtii coding regions were found to be identical, or very similar, in length, whereas each of the intergenic spacers is substantially longer in C. eugametos than in C. reinhardtii. The central portion of both Chlamydomonas rps3 genes features a long extra coding region relative to other rps3 sequences. We have shown that the insertion sequence in the C. eugametos rps3 is not excised at the RNA level.     

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.     

Chloroplastic genomes of Ginkgo biloba and Chlamydomonas moewusii contain a chlB gene encoding one subunit of a light-independent protochlorophyllide reductase

We have cloned and sequenced a Chlamydomonas moewusii chloroplastic DNA fragment that includes a 563 amino-acid open reading frame (ORF563, chlB) presenting 89% amino-acid homology with ORF513 from Marchantia polymorpha. It is also homologous to ORF510 from Pinus thumbergii but includes two insertions absent in both M. polymorpha and P. thunbergii. The derived polypeptide is 54% similar to the product of bchB from Rhodobacter capsulatus, identified as one subunit of a light-independent NADH-protochlorophyllide reductase. We also isolated and sequenced an homologous chloroplastic gene from the gymnosperm Ginkgo biloba. Northern hybridizations performed on RNA isolated from synchronized Chlamydomonas eugametos cells showed higher expression between the tenth hour of light and the eighth hour of darkness, peaking during the first 2 h of darkness.     

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.     

Cell type determines plastid transmission in tomato intergeneric somatic hybrids

Summary Mesophyll (M)- and suspension culture (S)-derived protoplasts of both Lycopersicon esculentum, tomato, and its wild relative Solanum lycopersicoides were fused as S+M, M+M and S+S combinations, respectively, to resolve the role of parental cell types in determining cpDNA transmission to intergeneric somatic hybrid plants. The mesophyll cpDNA was preferentially transmitted to 96% of the plants, each regenerated from a separate callus, in M+S and S+M fusion combinations. In contrast, for the M+M combination there was an equable distribution of either tomato cpDNA or that of S. lycopersicoides among the 34 hybrid plants. The number of plastids or proplastids in mesophyll or suspension protoplasts was not a factor regulating cpDNA transmission. Mesophyll or suspension protoplasts of both fusion partners had comparable frequencies of either plastid type with a mean of 23. The biased transmission of plastids from the mesophyll parent in somatic hybrid plants of S+M and M+S combinations appears to be due to differential multiplication of plastids, possibly conditioned by an unequal input of the nucleoids found in plastids versus proplastids. In the M+M fusion, plastid and nucleotid input and subsequent plastid multiplication are apparently equal, and when combined with random sorting out leads to an equal distribution of parental cpDNAs in the regenerated somatic hybrid plants. For the S+S combination, 22 somatic hybrid plants have exclusively tomato cpDNA, an outcome that is not readily explained by donor cell input.     

Structural alterations of the chloroplast genome found in grasses are not common in monocots

The distribution of structural rearrangements of the chloroplast genome found in grass cpDNA in comparison to that of tobacco was systematically checked in the cpDNAs of representative monocots. The physical map of lily cpDNA, which shares a key position in the diversity of monocotyledonous plants, was constructed to assess whether three inversions found in grass cpDNA are common in monocots. Specific probes for the detection of (1) intron loss in the rpoC1 gene, (2) insertional sequence gain in rpoC2, (3) deletion of ORF2280 in the inverted repeats, (4) non-reciprocal translocation of rpl23, and (5) rearrangements of ORF512, were hybridized to cpDNAs of lily, onion, spiderwort, two turf grasses, and wheat. The existence of intervening sequences in the rpoC1 and rpoC2 genes was also confirmed by PCR analysis. All markers used in the study revealed that structural rearrangements of the chloroplast genome were restricted to grasses, indicating that drastic structural alterations of the chloroplast genome had occurred in the ancestor(s) of grasses. These results also suggest that structural analysis of the chloroplast genome is applicable to the phylogenetic reconstruction of related plants.     

Evidence for persistence of chloroplast markers in the heteroplasmic state in Chlamydomonas reinhardtii

Summary In the green alga Chlamydomonas reinhardtii, reciprocal crosses between strains carrying non-allelic chloroplast mutations to streptomycin dependence (sd-u) produce streptomycin sensitive (sd-u ⁺) recombinant progeny. Transfer of these sd-u ⁺progeny to streptomycin-containing medium results in a much higher frequency of recovery of streptomycin dependent isolates than expected by mutation. Failure to recover the more commonly encountered class of streptomycin resistant mutants also suggests that mutation is not responsible for appearance of the new dependent isolates. Backcrosses of these new sd-u isolates to strains carrying the original sd-u mutations demonstrate their allelism with the sd-u mutation contributed by the mt ⁺parent. Earlier work by Schimmer and Arnold (1969, 1970a-d) indicated that newly isolated sensitive revertants of the streptomycin dependent mutant sd-u-3-18 also yielded high frequencies of sd-u cells but these were never analyzed genetically. We have now obtained new sd-u. isolates from streptomycin sensitive revertants of sd-u-318 and shown them all to be allelic with the original sd-u3-18 mutation. Thus hidden sd-u alleles can coexist with sd-u ⁺alleles in heteroplasmic cells. These heteroplasmic cells are streptomycin sensitive in phenotype and may arise in crosses or from new mutation.     

An automated GCxGC‐TOF‐MS protocol for batch‐wise extraction and alignment of mass isotopomer matrixes from differential 13C‐labelling experiments: a case study for photoautotrophic‐mixotrophic grown Chlamydomonas reinhardtii cells

Two dimensional gas chromatography coupled to time‐of‐flight mass spectrometry (GCxGC‐TOF‐MS) is a promising technique to overcome limits of complex metabolome analysis using one dimensional GC‐TOF‐MS. Especially at the stage of data export and data mining, however, convenient procedures to cope with the complexity of GCxGC‐TOF‐MS data are still in development. Here, we present a high sample throughput protocol exploiting first and second retention index for spectral library search and subsequent construction of a high dimensional data matrix useful for statistical analysis. The method was applied to the analysis of ¹³C‐labelling experiments in the unicellular green alga Chlamydomonas reinhardtii. We developed a rapid sampling and extraction procedure for Chlamydomonas reinhardtii laboratory strain (CC503), a cell wall deficient mutant. By testing all published quenching protocols we observed dramatic metabolite leakage rates for certain metabolites. To circumvent metabolite leakage, samples were directly quenched and analyzed without separation of the medium. The growth medium was adapted to this rapid sampling protocol to avoid interference with GCxGC‐TOF‐MS analysis. To analyse batches of samples a new software tool, MetMax, was implemented which extracts the isotopomer matrix from stable isotope labelling experiments together with the first and second retention index (RI1 and RI2). To exploit RI1 and RI2 for metabolite identification we used the Golm metabolome database (GMD [1] with RI1/RI2‐reference spectra and new search algorithms. Using those techniques we analysed the dynamics of ¹³CO₂ and ¹³C‐acetate uptake in Chlamydomonas reinhardtii cells in two different steady states namely photoautotroph and mixotroph growth conditions. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Extensive restriction fragment length polymorphisms in a new isolate ofChlamydomonas reinhardtii

Summary A new field isolate of the unicellular green algaChlamydomonas reinhardtii with useful properties for restriction fragment length polymorphism mapping is described in this report. The isolate, S1-D2 (mating type-), was the only strain found among 24Chlamydomonas isolates taken from many locations which was interfertile with laboratory strains ofC. reinhardtii. It mates at high efficiency, giving tetrads with excellent viability. Using cloned probes for both nuclear and chloroplast genes, we have found numerous restriction fragment length polymorphisms between Sl-D2 and laboratory strains ofC. reinhardtii.     

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.