Nonrandom Distribution of Chloroplast Recombination Events in Chlamydomonas Reinhardtii: Evidence for a Hotspot and an Adjacent Cold Region

Intermolecular recombination of Chlamydomonas chloroplast genes has been analyzed in sexual crosses and following biolistic transformation. The pattern and position of specific exchange events within 15 kb of the 22-kb inverted repeat have been mapped with respect to known restriction fragment length polymorphism markers that distinguish the chloroplast genomes of the interfertile species Chlamydomonas reinhardtii and Chlamydomonas smithii. Recombinant progeny were selected from two- and three-factor crosses involving point mutations conferring herbicide (dr) and antibiotic resistance (er and spr) in the psbA, 23S and 16S ribosomal RNA genes, respectively. Exchange events were not randomly distributed over the 15-kb region, but were found to occur preferentially in a 0.7-kb sequence spanning the 3' end of the psbA gene and were much less common in an adjacent region of ca. 2.0 kb. These findings are corroborated by data showing that the dr mutation is unlinked genetically (3% recombination/kb) to the er and spr rRNA mutations, which are themselves linked and show ca. 1% recombination/kb. This discrepancy is significant since the dr-er and er-spr intervals are about the same length (ca. 7 kb). During chloroplast transformation, the 0.7-kb recombination hotspot also functions as a preferential site for exchange events leading to the integration of donor psbA gene sequences. The 0.7-kb hotspot region contains four classes of 18-37-bp direct repeats also found in other intergenic regions, but no open reading frame. Using deletion constructs in a chloroplast transformation assay, the hotspot was localized to a 500-bp region that lacks most of these repeats, which suggests that the repeats themselves are not responsible for the increased recombination frequency. Within this region, a 400-bp sequence is highly conserved between the chloroplast genomes of C. reinhardtii and C. smithii and includes several structural motifs characteristic of recombination hotspots in other systems.     

Generation and maintenance of tandemly repeated extrachromosomal plasmid DNA in Chlamydomonas chloroplasts

Unusual chloroplast transformants of Chlamydomonas reinhardtii that contain 2000 copies of a mutant version of the chloroplast atpB gene, maintained as an extrachromosomal tandem repeat, have recently been described. In this paper studies have been undertaken to (i) address possible mechanisms for generating and maintaining the amplified DNA and (ii) determine whether it is possible to use chloroplast gene amplification to overexpress chloroplast or foreign genes. Data presented here indicate that high copy number transformants harbor characteristic rearrangements in both copies of the chloroplast genome large inverted repeat. These rearrangements appear to be a consequence of, or required for, maintenance of the amplified DNA. In an attempt to mimic the apparently autonomous replication of extrachromosomal DNA in the chloroplast, transformation was carried out with a plasmid that lacked homology with the chloroplast genome or with the same plasmid carrying a putative chloroplast DNA replication origin ( oriA ). Transformants were recovered only with the plasmid containing oriA , and all transformants contained an integrated plasmid copy at oriA , suggesting that establishment or maintenance of the extrachromosomal tandem repeat requires conditions that were not replicated in this experiment. To determine whether other genes could be maintained at high copy number in the chloroplast, plasmids carrying the wild-type atpB gene or the bacterial aadA gene were introduced into a high copy number transformant. Surprisingly, the copy number of the plasmid tandem repeat declined rapidly after the secondary transformation events, even when strong selective pressure for the introduced gene was applied. Thus, chloroplast transformation can either create or destabilize high copy number tandem repeats.     

Long regions of homologous DNA are incorporated into the tobacco plastid genome by transformation.

We investigated the size of flanking DNA incorporated into the tobacco plastid genome alongside a selectable antibiotic resistance mutation. The results showed that integration of a long uninterrupted region of homologous DNA, rather than of small fragments as previously thought, is the more likely event in plastid transformation of land plants. Transforming plasmid pJS75 contains a 6.2-kb DNA fragment from the inverted repeat region of the tobacco plastid genome. A spectinomycin resistance mutation is encoded in the gene of the 16S rRNA and, 3.2 kb away, a streptomycin resistance mutation is encoded in exon II of the ribosomal protein gene rps12. Transplastomic lines were obtained after introduction of pJS75 DNA into leaf cells by the biolistic process and selection for the spectinomycin resistance marker. Homologous replacement of resident wild-type sequences resulted in integration of all, or almost all, of the 6.2-kb plastid DNA sequence from pJS75. Plasmid pJS75, which contains engineered cloning sites between two selectable markers, can be used as a plastid insertion vector.     

Complete chloroplast genome sequences from Korean ginseng (Panax schinseng Nees) and comparative analysis of sequence evolution among 17 vascular plants.

The nucleotide sequence of Korean ginseng (Panax schinseng Nees) chloroplast genome has been completed (AY582139). The circular double-stranded DNA, which consists of 156,318 bp, contains a pair of inverted repeat regions (IRa and IRb) with 26,071 bp each, which are separated by small and large single copy regions of 86,106 bp and 18,070 bp, respectively. The inverted repeat region is further extended into a large single copy region which includes the 5' parts of the rpsl9 gene. Four short inversions associated with short palindromic sequences that form stem-loop structures were also observed in the chloroplast genome of P. schinseng compared to that of Nicotiana tabacum. The genome content and the relative positions of 114 genes (75 peptide-encoding genes, 30 tRNA genes, 4 rRNA genes, and 5 conserved open reading frames [ycfs]), however, are identical with the chloroplast DNA of N. tabacum. Sixteen genes contain one intron while two genes have two introns. Of these introns, only one (trnL-UAA) belongs to the self-splicing group I; all remaining introns have the characteristics of six domains belonging to group II. Eighteen simple sequence repeats have been identified from the chloroplast genome of Korean ginseng. Several of these SSR loci show infra-specific variations. A detailed comparison of 17 known completed chloroplast genomes from the vascular plants allowed the identification of evolutionary modes of coding segments and intron sequences, as well as the evaluation of the phylogenetic utilities of chloroplast genes. Furthermore, through the detailed comparisons of several chloroplast genomes, evolutionary hotspots predominated by the inversion end points, indel mutation events, and high frequencies of base substitutions were identified. Large-sized indels were often associated with direct repeats at the end of the sequences facilitating intra-molecular recombination.     

Sequence and spacing requirements of a retrovirus integration site

Following infection, retroviruses insert a DNA copy of their RNA genome into the host cell genome. This integrative recombination reaction occurs at specific sites on the viral DNA: inverted repeat sequences near the termini of the linear DNA form of the viral genome. We have described elsewhere the generation and analysis of deletion mutations at one of the inverted repeat sequences in Moloney murine leukemia virus. We describe here the effects of insertion mutations made at this locus. Our results show that substantial sequence changes at the site of recombination can be tolerated, and that the spacing between the cleavage sites on the viral DNA can be expanded as well as contracted while still allowing efficient viral integration. After several rounds of virus replication, each of the insertion mutants gave rise to pseudorevertants with new alterations at the integration site.     

Complete structure of the chloroplast genome of Arabidopsis thaliana.

The complete nucleotide sequence of the chloroplast genome of Arabidopsis thaliana has been determined. The genome as a circular DNA composed of 154,478 bp containing a pair of inverted repeats of 26,264 bp, which are separated by small and large single copy regions of 17,780 bp and 84,170 bp, respectively. A total of 87 potential protein-coding genes including 8 genes duplicated in the inverted repeat regions, 4 ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acid species were assigned to the genome on the basis of similarity to the chloroplast genes previously reported for other species. The translated amino acid sequences from respective potential protein-coding genes showed 63.9% to 100% sequence similarity to those of the corresponding genes in the chloroplast genome of Nicotiana tabacum, indicating the occurrence of significant diversity in the chloroplast genes between two dicot plants. The sequence data and gene information are available on the World Wide Web database KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/arabi/.     

Sequence organization of repetitive elements in the flanking regions of the chloroplast ribosomal unit of Chlamydomonas reinhardii.

The flanking regions and the end of the chloroplast ribosomal unit of Chlamydomonas reinhardii have been sequenced. The upstream region of the ribosomal unit contains three open reading frames coding for 111, 117 and 124 amino acids, respectively. The latter polypeptide is partially related to the ribosomal protein L16 of E. coli. Two of the open reading frames overlap each other and are oriented in opposite direction. The region between these open reading frames and the 5' end of the 16S rRNA gene contains numerous short direct and inverted repeats which can be folded into large stem-loop structures. Sequence elements that resemble prokaryotic promoters are found in the same region. Several of the repeated elements are distributed throughout the non-coding regions of the chloroplast inverted repeat. Sequence comparison between the 5S rRNA and its gene does not reveal any significant sequence heterogeneity between the chloroplast 5S rRNA genes.     

Maintenance and expression of heterologous genes in chloroplast ofChlamydomonas reinhardtii

The chloroplast genome ofChlamydomonas reinhardtii has been transformed with a chimeric gene consisting of the chloroplastatpA promoter and the bacterial gene for aminoglycoside adenine transferase (aadA). TheatpA-aadA cassette has been placed within the chloroplast DNAEcoRI restriction enzyme fragment 14, or within the chloroplastBamH1 fragment 10. The chimeric constructs were introduced into the chloroplast by particle bombardment. Integration of the cassette into chloroplast DNA then occurred via homologous recombination of sequences flanking the cassette with their corresponding chloroplast sequences. We demonstrate that the chloroplastatpA promoter inatpA-aadA routinely recombines with its endogenous counterpart, resulting in heteroplasmic chloroplast DNA populations that may persist for many generations. The heterologous gene does not require a 3 inverted repeat sequence for its expression. TheatpA-aadA gene copy number, which is dictated here by its position in the chloroplast genome, is proportional to the steady state level ofatpA-aadA mRNA. However, neither genomic position, gene copy number, or mRNA level have a significant effect on cellular resistance to spectinomycin, nor activity of theaadA gene productin vitro. These results suggest that, in the case ofaadA, the limiting step for expression of this gene is at the translational or post-translational level. TheatpA-aadA cassette should prove a useful model for future studies on the maintenance and expression of heterologous genes inC. reinhardtii chloroplasts.     

Targeted disruption of chloroplast genes in Chlamydomonas reinhardtii.

Summary We have developed an efficient procedure for the disruption of Chlamydomonas chloroplast genes. Wild-type C. reinhardtii cells were bombarded with microprojectiles coated with a mixture of two plasmids, one encoding selectable, antibiotic-resistance mutations in the 16S ribosomal RNA gene and the other containing either the atpB or rbcL photosynthetic gene inactivated by an insertion of 0.48 kb of yeast DNA in the coding sequence. Antibiotic-resistant transformants were selected under conditions permissive for growth of nonphotosynthetic mutants. Approximately half of these transformants were initially heteroplasmic for copies of the disrupted atpB or rbcL genes integrated into the recipient chloroplast genome but still retained photosynthetic competence. A small fraction of the transformants (1.1% for atpB; 4.3% for rbcL) were nonphotosynthetic and homoplasmic for the disrupted gene at the time they were isolated. Single cell cloning of the initially heteroplasmic transformants also yielded nonphotosynthetic segregants that were homoplasmic for the disrupted gene. Polypeptide products of the disrupted atpB and rbcL genes could not be detected using immunoblotting techniques. We believe that any nonessential Chlamydomonas chloroplast gene, such as those involved in photosynthesis, should be amenable to gene disruption by cotransformation. The method should prove useful for the introduction of site-specific mutations into chloroplast genes and flanking regulatory sequences with a view to elucidating their function.     

The gene for the large subunit of ribulose-1,5-bisphosphate carboxylase in Euglena gracilis chloroplast DNA: location, polarity, cloning, and evidence for an intervening sequence

The gene for the large subunit (LS) of ribulose-1,5,-bisphosphate carboxylase of Euglena gracilis Z chloroplast DNA has been mapped by heterologous hybridization with DNA restriction fragments containing internal sequences from the Zea mays and Chlamydomonas reinhardii LS genes. The Euglena LS gene which has the same polarity as the Euglena rRNA genes has been located with respect to Pst I, Pvu I, and HindIII sites within the Eco RI fragment Eco A. The region of Euglena chloroplast DNA complementary to an 887 bp internal fragment from the Chlamydomonas chloroplast LS gene is interrupted by a 0.5-1.1 kbp non-complementary sequence. This is the first chloroplast protein gene located on the Euglena genome, and the first evidence for an intervening sequence within any chloroplast protein gene.     

Physical and gene mapping of chloroplast DNA from Atriplex triangularis and Cucumis sativa.

A rapid and simple method for constructing restriction maps of large DNAs (100-200 kb) is presented. The utility of this method is illustrated by mapping the Sal I, Sac I, and Hpa I sites of the 152 kb Atriplex triangularis chloroplast genome, and the Sal I and Pvu II sites of the 155 kb Cucumis sativa chloroplast genome. These two chloroplast DNAs are very similar in organization; both feature the near-universal chloroplast DNA inverted repeat sequence of 22-25 kb. The positions of four different genes have been localized on these chloroplast DNAs. In both genomes the 16S and 23S ribosomal RNAs are encoded by duplicate genes situated at one end of the inverted repeat, while genes for the large subunit of ribulose-1,5-bisphosphate carboxylase and a 32 kilodalton photosystem II polypeptide are separated by 55 kb of DNA within the large single copy region. The physical and genetic organization of these DNAs is compared to that of spinach chloroplast DNA.     

Recombination of Chlamydomonas chloroplast DNA occurs more frequently in the large inverted repeat sequence than in the single-copy regions

Summary It is well documented that chloroplast DNA (cpDNA) recombination occurs at a relatively high frequency during sexual reproduction of unicellular green algae from the Chlamydomonas genus. Like the cpDNAs of most land plants, those of Chlamydomonas species are divided into two single-copy regions by a large inverted repeat sequence, part of which encodes the chloroplast rRNA genes. In the present study, we scored the inheritance of polymorphic loci spanning the entire chloroplast genome in hybrids recovered from reciprocal interspecific and F₁ crosses between Chlamydomonas eugametes and C. moewusii, and from these data, estimated the density of recombination junctions within each region of recombinant cpDNAs. Our results indicate that recombination junctions occur at highly variable frequencies across the three main domains of the chloroplast genome. The large inverted repeat sequence was found to exhibit at least a five-fold higher density of recombination junctions compared to one of the singlecopy regions, whereas junctions in the latter region were five-fold more abundant relative to those in the other single-copy region. This marked difference in the densities of recombination junctions implies that the extent of genetic linkage between two given chloroplast loci will depend not only on their physical distance, but also on their locations within the genome.     

Variable copy number DNA sequences in rice

Summary We have cloned two types of variable copy number DNA sequences from the rice embryo genome. One of these sequences, which was cloned in pRB301, was amplified about 50-fold during callus formation and diminished in copy number to the embryonic level during regeneration. The other clone, named pRB401, showed the reciprocal pattern. The copy numbers of both sequences were changed even in the early developmental stage and eliminated from nuclear DNA along with growth of the plant. Sequencing analysis of the pRB301 insert revealed some open reading frames and direct repeat structures, but corresponding sequences were not identified in the EMBL and LASL DNA databases. Sequencing of the nuclear genomic fragment cloned in pRB401 revealed the presence of the 3rps12-rps7 region of rice chloroplast DNA. Our observations suggest that during callus formation (dedifferentiation), regeneration and the growth process the copy numbers of some DNA sequences are variable and that nuclear integrated chloroplast DNA acts as a variable copy number sequence in the rice genome. Based on data showing a common sequence in mitochondria and chloroplast DNA of maize (Stern and Lonsdale 1982) and that the rps12 gene of tobacco chloroplast DNA is a divided gene (Torazawa et al. 1986), it is suggested that the sequence on the inverted repeat structure of chloroplast DNA may have the character of a movable genetic element.     

Mutants of Chlamydomonas reinhardtii with physical alterations in their chloroplast DNA

We have isolated nonphotosynthetic (acetate-requiring) mutants with physical alterations in chloroplast DNA following growth of haploid cells in the chloroplast specific mutagen 5-fluorodeoxyuridine (FdUrd) or treatment of FdUrd-grown diploid cells with X rays. About one-third of the nonphotosynthetic mutations resulting from FdUrd treatment alone show simple deletions. All eight of the mutants examined so far which were obtained with FdUrd plus X rays have deletions that are accompanied by rearrangements, including inversions or duplications. All the alterations extend into one of the two inverted repeat regions of the chloroplast genome which contain the ribosomal RNA cistrons. However, Southern hybridization experiments reveal that the rRNA cistrons are not deleted but instead are contained in new fragments. The relocated rRNA cistrons appear to be functional, since the mutants have normal levels of chloroplast ribosomes. In most cases the deletions and rearrangements are symmetrical and affect both inverted repeats in a similar fashion. An exception is the mutant ac-u-c-2–43, which lacks one inverted repeat region almost completely, including an entire set of rRNA genes. Three additional mutants, which fail to recombine with ac-u-c-2–43 to give photosynthetically competent cells, have smaller deletions in the same region of the genome. These physical mapping studies have allowed us to place the ac-u-c locus itself in a region of unique sequence DNA in a fragment, Ba10, which also includes the right-hand end of one inverted repeat.