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).     

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.     

Characterization of chloroplast DNA in Chlamydomonas eugametos and C. moewusii and its inheritance in hybrid progeny

Summary The density, molecular weight, and cellular repetition of DNA molecules associated with the -DNA satellite of the interfertile algae Chlamydomonas eugametos and C. moewusii are reported. The similarities between these values and those for the chloroplast DNA (cpDNA) in the related alga Chlamydomonas reinhardtii indicate that these satellites represent cpDNA. The buoyant densities of C. eugametos and C. moewusii cpDNAs are indistinguishable from one another, as are those of their respective nuclear DNAs. These densities differ slightly from the densities of the homologous components of C. reinhardtii whole cell DNA. All three species differ with respect to additional minor satellite DNAs and low molecular weight DNAs of unknown cellular location.Differences in the Aval and Smal restriction endonuclease fragmentation patterns of C. eugametos and C. moewusii cpDNAs were employed to study the inheritance of cpDNA in an F1 hybrid which had inherited a non-Mendelian streptomycin resistance marker (sr-2) from the C. eugametos mating-type plus (mt ⁺) parent and in two homoplasmic mitotic segregants from a B 1 hybrid (F₁ × C. moewusii) which had been initially heteroplasmic for the resistance marker. Although the cpDNA patterns in the F1 hybrid were similar to those of the C. eugametos ml ¹ parent, important differences were noted which suggest that recombination between C. eugametos and C. moewusii cpDNA had occurred. Homoplasmic streptomycin resistant and sensitive mitotic segregants recovered from the B1 hybrid product reveal Aval restriction patterns similar to those of the respective resistant and sensitive parents. These data are consistent with the hypothesis that the sr-2 marker is located in cpDNA and that C. eugametos and C. moewusii cpDNA sequences can coexist in the same chloroplast and, at least sometimes, segregate without extensive recombination. The transmission of low molecular weight DNAs characteristic of C. moewusii but of unknown cellular origin shows no direct correlation with the transmission of the sr-2 marker.     

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.     

Mitochondrial DNA of Chlamydomonas reinhardtii: the ND4 gene encoding a subunit of NADH dehydrogenase

Summary The ND4 gene encoding a subunit of respiratory NADH dehydrogenase has been identified on the linear 15.8 kb mitochondrial DNA of Chlamydomonas reinhardtii. The gene maps downstream of ND5. The 1,332 bp nucleotide sequence presented is the first complete reported ND4 sequence from a photoautotrophic organism. The deduced protein of 443 amino acid residues shows 34%, 29% and 27% homology to the protein sequences of Aspergillus amstelodami, Drosophila yakuba and mouse, respectively. ND4 is the fifth and last mitochondrial gene of the NADH dehydrogenase complex on the 15.8 kb mitochondrial genome of C. reinhardtii.     

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.     

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.     

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.     

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.     

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     

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.     

Mitochondrial DNA of Chlamydomonas reinhardtii: the DNA sequence of a region showing homology with mammalian URF2

Summary A 1.27 kb DNA fragment of the 15 kb DNA of Chlamydomonas reinhardtii has been cloned and sequenced. A 906 bp long open reading frame was found showing homology with the URF2 genes of mammals and insects. This homology is functional evidence for Chlamydomonas reinhardtii 15 kb DNA representing indeed mitochondrial DNA. This is the first report of an URF2 gene in mitochondria of a photosynthetic organism. The absence of a TGA codon within the gene suggests that it is used as stop codon like in higher plants and not as tryptophan like in animal and fungal mitochondria.     

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     

Recombination within the inverted repeat sequences of the Chlamydomonas reinhardii chloroplast genome produces two orientation isomers

Summary Two orientations of the Chlamydomonas reinhardii chloroplast (ct) genome are shown to be produced by recombination within the inverted repeat (IR) sequences that separate the two single copy (SC) regions. SC region 1 is bounded on its two ends by EeoRI restriction endonuclease fragments of 3.2 and 4.7 kilobase pairs (kb) (Rochaix 1978). The 3.2 kb EeoRI fragment overlaps a 51.3 kb BglII fragment spanning one of the 19.7 kb IR sequences, and the 4.7 kb EcoRI fragment overlaps a 42.1 kb BglII fragment spanning the other 19.7 kb IR sequence. We have shown by hybridization analysis that the 3.2 kb fragment also overlaps a BgIII fragment with a predicted size of 52.3 kb, and that the 4.7 kb fragment also overlaps a BglII fragment of a predicted size of 41.1 kb. The second set of BglII fragments are isomers produced by recombination localized to the IR region. The two isomers are present in approximately equimolar ratio. Knowledge of the isomeric composition of the C. reinhardii ctDNA is essential for establishing a correlation between genetic and physical maps of the ct genome.     

Restriction map of the mitochondrial DNA of the true slime mould,Physarum polycephalum: linear form and long tandem duplication

Summary The mitochondrial DNA (mtDNA) of the true slime mould, Physarum polycephalum strain CH934xCH938, was isolated and characterized by restriction mapping. Cloned fragments of the mtDNA were assembled and used to construct the restriction map. This map showed that the mtDNA was a linear molecule of 86.0 kb with a tandem duplication of 19.6 kb. The terminal fragments were identified by sensitivity to Bal31 exonuclease. One of the duplications was located at the right end and the other was located 5 kb from the left end. Each duplicated segment contained 26 restriction sites for ten enzymes and these restriction sites were completely conserved in each duplication. Genes for the large and small rRNAs were mapped to positions about 30 kb from the right end of the mtDNA by hybridization with its own rRNAs. With the exception of a probe for the gene for the large rRNA in Tetrahymena pyriformis mtDNA, various probes from the mtDNAs of Saccharomyces cerevisiae and T. pyriformis showed no significant hybridization to any of the restriction fragments of the mtDNA from P. polycephalum.     

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.     

Autonomously replicating sequences in young and senescent mitochondrial DNA from Podospora anserina

Summary Senescence in the filamentous fungus Podospora anserina is characterized by the accumulation of multimeric circular mitochondrial DNA molecules, known as senDNAs. These tandemly repeated DNA sequences, which originate from broadly dispersed regions of the young mitochondrial genome, behave as independently replicating molecules. In this study, the yeast transformation system was used to assay senDNAs and their young mtDNA counterparts for the presence of autonomously replicating sequences. P. anserina mtDNA fragments were cloned into the yeast vector YIp5 and the hybrid YPM plasmids were used to transform yeast. All of the senDNAs and their homologous young mtDNAs promoted high frequency transformation and extrachromosomal maintenance of YPM plasmids. The putative origin of replication for the P. anserina mitochondrial genome was also cloned into YIp5 and shown to confer autonomously replicating properties.     

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.     

The atpF group-II intron-containing gene from spinach chloroplasts is not spliced in transgenic Chlamydomonas chloroplasts

In order to determine whether the group-II trans-splicing machinery of the chloroplast of Chlamydomonas reinhardtii can splice a heterologous group-II cis intron, the atpF gene of spinach was transferred into the chloroplast genome of C. reinhardtii using the atpX expression vector. The atpF gene contains a group-II intron which, like other higher plant chloroplast introns, does not self-splice in vitro. The chimeric transgene was expressed at high levels, based on the accumulation of the precursor; however, spliced products could not be detected by Northern blotting, or by RT-PCR coupled with Southern-blot hybridization of the amplified products with an exon-junction probe. These results indicate that the spinach atpF intron is not spliced in transgenic C. reinhardtii chloroplasts. Thus, splicing of chloroplast introns mediated by cellular factors may be species-specific; alternately, the group-II splicing machinery of C. reinhardtii is specific for trans-spliced introns.