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.     

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.     

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.     

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.     

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.     

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.     

A group-I intron in the mitochondrial small subunit ribosomal RNA gene of Sclerotinia sclerotiorum

A 1 380-bp intervening sequence within the mitochondrial small subunit ribosomal RNA (mt SSU rRNA) gene of the fungus Sclerotinia sclerotiorum has been sequenced and identified as a group-I intron. This is the first report of an intron in the mt SSU rRNA gene. The intron shows close similarity in secondary structure to the subgroup-IC2 introns from Podospora (ND3i1, ND5i2, and COIi5) and Neurospora (ND5i1). The intron has an open reading frame (ORF) that encodes a putative protein of 420 amino acids which contains two copies of the LAGLI-DADG motif. The ORF belongs to a family of ORFs identified in Podospora (ND3i1, ND4Li1, ND4Li2, ND5i2, and COIi5) and Neurospora (ND5i1). The putative 420-aa polypeptide is also similar to a site-specific endonuclease in the chloroplast large subunit ribosomal RNA (LSU rRNA) gene of the green alga Chlamydomonas eugametos. In each clone of S. sclerotiorum examined, including several clones which were sampled over a 3-year period from geographically separated sites, all isolates either had the intron or lacked the intron within the mt SSU rRNA gene. Screening by means of Southern hybridization and PCR amplification detected the intron in the mt SSU rRNA genes of S. minor, S. trifoliorum and Sclerotium cepivorum, but not in other members of the Sclerotiniaceae, such as Botrytis anamorphs of Botryotinia spp., or in other ascomycetous and basidiomycetous fungi.     

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.     

Intragenic suppressors that restore the splicing and homing activities of the protein encoded by the second intron of the Saccharomyces capensis cyt b gene

The second (bi2) intron of the mitochondrial cyt b gene from Saccharomyces capensis encodes a bifunctional protein which acts both as a maturase, promoting intron splicing, and as a homing-endonuclease, I-ScaI, promoting intron mobility. In this work we isolated and characterized revertants from a respiratory-deficient mutant in which both functions of the protein have been lost. Intragenic revertants resulted mainly from monosubstitutions in the mutated codon and in one case from a distant second site mutation. All novel variants of the S. capensis bi2 intron-encoded protein are competent for the maturase activity but only two of them can partially complement the homing function. Received: 7 January 2000 / Accepted: 12 August 2000     

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     

Two co-existing mechanisms account for the large-scale deletions of mitochondrial DNA in Podospora anserina that involve the 5′ border of a group-II intron

A degenerative syndrome associated with the accumulation of site-specific deletions within mitochondrial chromosomes occurs in strains of Podospora anserina carrying the AS1-4 nuclear mutation. The site-specific deletion event has been assumed to result from the transposition of a group-II intron (intron α) behind an IBS motif, followed by recombination between the two intron repeats. We show here that a number of distinct deletions can accumulate in AS1-4 strains. Most of them are present in low amounts in wild-type cells where they are only detectable in PCR experiments. The deletions can be divided into two classes. In class I, intron α is joined to an IBS motif. In class II, the intron is not joined to an IBS site, it can be truncated or contain a few upstream exonic nucleotides; some junctions carry non-templated nucleotides. These results indicate that at least two mechanisms are involved in the generation of large-scale mitochondrial deletions in Podospora. One of them seems to be based on the transposition properties of the group-II α intron, the other one on illegitimate recombination. We propose that these two mechanisms use DNA double-strand breaks occurring within the 5′ region of intron α. Received: 15 May / 18 August 1998     

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.     

Mystery of intron gain

For nearly 15 years, it has been widely believed that many introns were recently acquired by the genes of multicellular organisms. However, the mechanism of acquisition has yet to be described for a single animal intron. Here, we report a large-scale computational analysis of the human, Drosophila melanogaster, Caenorhabditis elegans, and Arabidopsis thaliana genomes. We divided 147,796 human intron sequences into batches of similar lengths and aligned them with each other. Different types of homologies between introns were found, but none showed evidence of simple intron transposition. Also, 106,902 plant, 39,624 Drosophila, and 6021 C. elegans introns were examined. No single case of homologous introns in nonhomologous genes was detected. Thus, we found no example of transposition of introns in the last 50 million years in humans, in 3 million years in Drosophila and C. elegans, or in 5 million years in Arabidopsis. Either new introns do not arise via transposition of other introns or intron transposition must have occurred so early in evolution that all traces of homology have been lost.     

The trans-spliced intron 1 in the psaA gene of the Chlamydomonas chloroplast: a comparative analysis

In the secondary structure model that has been proposed for the trans-spliced intron 1 in the Chlamydomonas reinhardtii psaA gene, a third RNA species (tscA RNA) interacts with the 5 and 3 intron parts flanking the exons to reconstitute a composite structure with several features of group-II introns. To test the validity of this model, we undertook the sequencing and modelling of equivalent introns in the psaA gene from other unicellular green algae belonging to the highly diversified genus Chlamydomonas. Our comparative analysis supports the model reported for the C. reinhardtii psaA intron 1, and also indicates that the 5 end of the tscA RNA and the region downstream from the psaA exon 1 cannot be folded into a structure typical of domain I as described for most group-II introns. It is possible that a fourth RNA species, yet to be discovered, provides the parts of domain I which are apparently missing.     

Rules for DNA target-site recognition by a lactococcal group II intron enable retargeting of the intron to specific DNA sequences

Group II intron homing occurs primarily by a mechanism in which the intron RNA reverse splices into a DNA target site and is then reverse transcribed by the intron-encoded protein. The DNA target site is recognized by an RNP complex containing the intron-encoded protein and the excised intron RNA. Here, we analyzed DNA target-site requirements for the Lactococcus lactis Ll.LtrB group II intron in vitro and in vivo. Our results suggest a model similar to yeast mtDNA introns, in which the intron-encoded protein first recognizes a small number of nucleotide residues in double-stranded DNA and causes DNA unwinding, enabling the intron RNA to base-pair with the DNA for reverse splicing. Antisense-strand cleavage requires additional interactions between the protein and 3' exon. Key nucleotide residues are recognized directly by the intron-encoded protein independent of sequence context, and there is a stringent requirement for fixed spacing between target site elements recognized by the protein and RNA components of the endonuclease. Experiments with DNA substrates containing GC-clamps or "bubbles" indicate a requirement for DNA unwinding in the 3' exon but not the distal 5' exon region. Finally, by applying the target-site recognition rules, we show that the L1.LtrB intron can be modified to insert at new sites in a plasmid-borne thyA gene in Escherichia coli. This strategy should be generally applicable to retargeting group II introns and to delivering foreign sequences to specific sites in heterologous genomes.     

The catalytic group-I introns of the psbA gene of Chlamydomonas reinhardtii : core structures, ORFs and evolutionary implications

The sequences and predicted secondary structures of the four catalytic group-I introns in the psbA gene of Chlamydomonas reinhardtii, Cr.psbA-1–Cr.psbA-4, have been determined. Cr.psbA-1 and Cr.psbA-4 are subgroup-IA1 introns and have similar secondary structures, except at the 3′ end where Cr.psbA-1 contains a large inverted-repeat domain. Cr.psbA-4 is closely related to intron 1 of the Chlamydomonas moewusii psbA gene, with which it shares the same location, high nucleotide identity in the core, and an identically placed ORF that shows 58% amino-acid identity. Cr.psbA-2 is a subgroup-IA3 intron, and shows similarities to the Chlamydomonas eugametos rRNA intron, Ce.LSU-1. Cr.psbA-3 is a subgroup-IA2 intron, and is remarkably similar to the T4 phage intron, sunY. Interestingly, a degenerate version of Cr.psbA-3 is located in the intergenic region between the chloroplast petA and petD genes. All four introns contain ORFs, which potentially code for basic proteins of 11–38 kDa. The ORFs in introns 2 and 3 contain variants of the GIY-YIG motif; however, the Cr.psbA-2 ORF is free-standing, whereas the Cr.psbA-3 ORF is contiguous and in-frame with the upstream exon. The Cr.psbA-4 ORF contains an H-N-H motif, and possibly a GIY-YIG motif. These data indicate that the C. reinhardtiipsbA introns have multiple origins, and illustrate some of the evolutionary DNA dynamics associated with group-I introns in Chlamydomonas. Received: 24 November 1998 / 23 March 1999     

Splicing of the mitochondrial group-II intron rl1: conserved intron-exon interactions diminish splicing efficiency

The mitochondrial intron rI1 is a self-splicing group-II intron of algal mitochondria that can be transferred into chloroplasts from the green alga Chlamydomonas reinhardtii for in vivo investigations (Herdenberger et al. 1994). Thus, rI1 is a suitable system to compare in vitro and in vivo RNA processing. Interestingly, rI1 shows correct RNA splicing, although typical cis-acting exon-sequences (IBS2, δ) of group-II introns are lacking. In order to examine the effect of these exon-intron interactions on splicing, we introduced the endogenous mitochondrial IBS2 sequence in order to produce optimal IBS2-EBS2 base pairing. In addition, the first nucleotide of the 3′exon (δ′) was substituted to create an optimal δ-δ′ interaction. Neither of the two mutations, nor a combination of both, had any effect on the precision of the splice-site selection. Unexpectedly, introduction of IBS2 led to a reduction in the efficiency of the second splicing step in vitro but not in vivo. These findings lead us to conclude that trans-acting factors are present in vivo to optimize splicing efficiency. The possibility is discussed that these factors may, for example, stabilize tertiary intron structures that are a prerequisite for correct RNA processing. Furthermore, our data indicate that similar trans-acting factors promote correct intron splicing in chloroplasts and mitochondria. Received: 18 October / 4 December 1997