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.     

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.     

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     

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     

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 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.     

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).     

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.     

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.     

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.     

Conservation of chloroplast genome structure among vascular plants

Summary We have constructed the first physical map of a gymnosperm chloroplast genome and compared its organization with those of a fern and several angiosperms by heterologous filter hybridization. The chloroplast genome of the gymnosperm Ginkgo biloba consists of a 158 kb circular chromosome that contains a ribosomal RNA-encoding inverted repeat approximately 17 kb in size. Gene mapping experiments demonstrate a remarkable similarity in the linear order and absolute positions of the ribosomal RNA genes and of 17 protein genes in the cpDNAs of Ginkgo biloba, the fern Osmunda cinnamomea and the angiosperm Spinacia oleracea. Moreover, filter hybridizations using as probes cloned fragments that cover the entirety of the angiosperm chloroplast genome reveal a virtually colinear arrangement of homologous sequence elements in these genomes representing three divisions of vascular plants that diverged some 200–400 million years ago. The only major difference in chloroplast genome structure among these vascular plants involves the size of the rRNA-encoding inverted repeat, which is only 10 kb in Osmunda, 17 kb in Ginkgo, and about 25 kb in most angiosperms. This size variation appears to be the result of spreading of the repeat through previously single copy sequences, or the reverse process of shrinkage, unaccompanied by any overall change in genome complexity.     

Restriction site and genetic map of Cucurbita pepo chloroplast DNA

Summary A detailed restriction map of squash chloroplast DNA (cpDNA) was constructed with five restriction endonuclease, SalI, PvuII, BglI, SacII, and PstI. The cleavage sites were mapped by sequential digestion of cpDNA using low-gelling temperature agarose. The restriction map shows that squash cpDNA is an approximately 153 kilobase (kb) circle with a large inverted repeat sequence of 23.3 kb, separated by a large (83.7 kb) and a small (22.7 kb) single copy region. Genes for a number of chloroplast polypeptides were localized on the map by hybridizing the cpDNA restriction fragments to heterologous gene-specific probes from tobacco, pea, tomato, maize, and spinach chloroplasts. The gene locations and organization of squash cpDNA are highly conserved and similar to chloroplast genomes of tomato, pepper, and Ginkgo.Abbreviations cpDNA chloroplast DNA - kb kilobases - IR inverted repeat. Gene names follow the nomenclature recommendation of Hallick and Bottomley (1983)     

Localization of two novel chloroplast genome functions: trans-splicing of RNA and protochlorophyllide reduction

Summary Chloroplast DNA deletions in the unicellular green alga Chlamydomonas reinhardtii localize two novel chloroplast gene functions. One of these, tscA, is required in trans for splicing, in trans, of the first and second, but not the second and third, exons of the RNA of the chloroplast gene psaA. Previously, no chloroplast genes were known to be required in trans for the splicing of chloroplast RNA. The other chloroplast gene function is required for light-independent reduction of protochlorophyllide, a key step in the algal pathway of chlorophyll biosynthesis. Both functions reside in the same 4 kbp region of chloroplast DNA.     

Chloroplast DNA from Adiantum capillus-veneris L., a fern species (Adiantaceae); clone bank,physical map and unusual gene localization in comparison with angiosperm chloroplast DNA

Summary We cloned all of Adiantum capillus-veneris chloroplast DNA PstI fragments longer than 1.0 kb, which cover 98% of the genome. These cloned fragments were used to construct a physical map for five restriction enzymes. The genome of A. capillus-veneris is approximately 153 kb long and contains a 24 kb inverted repeat. Mapping of 12 chloroplast DNA genes and heterologous hybridization, involving A. capillus-veneris chloroplast DNA and angiosperm chloroplast DNA probes, demonstrated that chloroplast DNA of A. capillus-veneris has a different gene order from typical angiosperm cpDNA (e.g., tobacco) in the inverted repeat region and the flanking segment of the large single copy region.     

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.     

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 nuclear mutant of Chlamydomonas that exhibits increased sensitivity to UV irradiation,reduced recombination of nuclear genes,and altered transmission of chloroplast genes

Summary Meiotic progeny of Chlamydomonas reinhardtii normally receive chloroplast genomes only from the mt ⁺ parent. However, exceptional zygotes, which transmit the chloroplast genomes of both parents or, more rarely, only those of the mt ^- parent, arise at a low frequency. Mutations at the mt ⁺-linked mat-3 locus were found previously to elevate the transmission of chloroplast genomes from the mt-parent, resulting in a much higher than normal frequency of exceptional zygotes. In this paper we demonstrate that an ultraviolet-sensitive nuclear mutation mapping at the uvsE1 locus, which is unlinked to mating type, also promotes chloroplast genome transmission from the mt ^- parent. This mutant, which was previously shown to reduce recombination of nuclear genes in meiosis, acts synergistically which the mat3-3 mutation to produce an extremely high frequency of exceptional zygotes. Through the use of restriction fragment length polymorphisms existing in the chloroplast genomes of C. reinhardtii and the interfertile strain C. smithii, we show that chloroplast DNA fragments from the mt ^- parent normally begin to disappear shortly after zygote formation. However, this process appears to be blocked totally in the absence of wild-type uvsE1 and mat-3 gene products. Our findings are consistent with the hypothesis that both gene products contribute to the mechanism responsible for uniparental inheritance of the chloroplast genome from the mt ⁺ parent.     

Chloroplast origins of DNA replication are distinct from chloroplast ARS sequences in two green algae

Summary A 5.3 kb chloroplast restriction fragment of Chlamydomonas reinhardii containing an origin of DNA replication and a sequence capable of promoting autonomous replication in C. reinhardii (ARC sequence) also carries an ARS sequence (autonomous replication in yeast). The ARC and ARS elements have been physically mapped and shown to be distinct from the origin of DNA replication. Similarly, restriction fragments containing the origin of chloroplast DNA replication from Euglena gracilis are unable to promote autonomous replication in yeast.     

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.