A family of selfish minicircular chromosomes with jumbled chloroplast gene fragments from a dinoflagellate

Chloroplast genes of several dinoflagellate species are located on unigenic DNA minicircular chromosomes. We have now completely sequenced five aberrant minicircular chromosomes from the dinoflagellate Heterocapsa triquetra. These probably nonfunctional DNA circles lack complete genes, with each being composed of several short fragments of two or three different chloroplast genes and a common conserved region with a tripartite 9G-9A-9G core like the putative replicon origin of functional single-gene circular chloroplast chromosomes. Their sequences imply that all five circles evolved by differential deletions and duplications from common ancestral circles bearing fragments of four genes: psbA, psbC, 16S rRNA, and 23S rRNA. It appears that recombination between separate unigenic chromosomes initially gave intermediate heterodimers, which were subsequently stabilized by deletions that included part or all of one putative replicon origin. We suggest that homologous recombination at the 9G-9A-9G core regions produced a psbA/psbC heterodimer which generated two distinct chimeric circles by differential deletions and duplications. A 23S/16S rRNA heterodimer more likely formed by illegitimate recombination between 16S and 23S rRNA genes. Homologous recombination between the 9G-9A-9G core regions of both heterodimers and additional differential deletions and duplications could then have yielded the other three circles. Near identity of the gene fragments and 9G-9A-9G cores, despite diverging adjacent regions, may be maintained by gene conversion. The conserved organization of the 9G-9A-9G cores alone favors the idea that they are replicon origins and suggests that they may enable the aberrant minicircles to parasitize the chloroplast's replication machinery as selfish circles.     

'Empty' minicircles and petB/atpA and psbD/psbE (cytb559 alpha) genes in tandem in Amphidinium carterae plastid DNA

Amphidinium carterae minicircle chloroplast DNA was separated from total DNA by centrifugation through a sucrose/NaCl gradient. Sequences of minicircles with psbA and 23S rRNA contained a common region of 67 bp. Primers designed from this generated numerous polymerase chain reaction products of 1.5-2.6 kb. These contained psaA and psaB as one gene/circle, and petB/atpA and psbD/psbE as two genes/circle. 'Empty' minicircles of 1.7-2.5 kb containing no identifiable genes or parts of genes were more abundant than gene-containing circles. From 15 minicircles a minimum common region of 48 bp was identified, with little identity to that from other dinoflagellate minicircles.     

Differential DNA rearrangements of plastid genes, psbA and psbD, in two species of the dinoflagellate Alexandrium

Plastomes of the peridinin-containing dinoflagellates are composed of a limited number of genes, which are carried individually on small circular molecules, termed 'minicircles'. Although the prevalent plastid chromosome of most algae and plants has only a single copy of each gene, our previous study showed that low copy numbers of multiple variants of the gene psbA co-exist with the 'ordinary' gene encoding the D1 protein in minicircles of Alexandrium tamarense. Although none of the psbA variants encoded the entire protein, they persisted in culture. In this study, we compared the distribution and structure of psbA and psbD variants in two species of Alexandrium to characterize DNA rearrangement within these genes. In addition to four previously reported psbA variants, three psbD variants were found in A. tamarense minicircles. The ordinary psbA and psbD genes also co-existed with variants in another species, A. catenella. The sequences of the ordinary genes were virtually identical in the two species. All the variants comprised insertion or deletion mutations, with no base substitutions being identified. Duplicated parts of the coding sequences were contained in most of the insertions. Short direct repeats (4-14?bp) and/or adenine?+?thymine-rich motifs were present in all mutation regions, although the position and/or the sequence of each DNA rearrangement was unique to each variant. The results indicated that replication-based repeat-mediated recombination was responsible for generation of the variants.     

Noncoding sequences from the slowly evolving chloroplast inverted repeat in addition to rbcL data do not support gnetalean affinities of angiosperms

We developed PCR primers against highly conserved regions of the rRNA operon located within the inverted repeat of the chloroplast genome and used these to amplify the region spanning from the 3' terminus of the 23S rRNA gene to the 5' terminus of the 5S rRNA gene. The sequence of this roughly 500-bp region, which includes the 4.5S rRNA gene and two chloroplast intergenic transcribed spacer regions (cpITS2 and cpITS3), was determined from 20 angiosperms, 7 gymnosperms, and 16 ferns (21,700 bp). Sequences for the large subunit of ribulose bisphosphate carboxylase/oxygenase (rbcL) from the same or confamilial genera were analyzed in both separate and combined data sets. Due to the low substitution rate in the inverted repeat region, noncoding sequences in the cpITS region are not saturated with substitutions, in contrast to synonymous sites in rbcL, which are shown to evolve roughly six times faster than noncoding cpITS sequences. Several length polymorphisms with very clear phylogenetic distributions were detected in the data set. Results of phylogenetic analyses provide very strong bootstrap support for monophyly of both spermatophytes and angiosperms. No support for a sister group relationship between Gnetales and angiosperms in either cpITS or rbcL data was found. Rather, weak bootstrap support for monophyly of gymnosperms studied and for a basal position for the aquatic angiosperm Nymphaea among angiosperms studied was observed. Noncoding sequences from the inverted repeat region of chloroplast DNA appear suitable for study of land plant evolution.      

Photosystem II of rye. Cloning and determination of the nucleotide sequence of chloroplast DNA fragments, comprising the psbA gene coding for D1 proteins

EcoRI and BamHI fragments of rye chloroplast DNA comprising psbA gene were cloned and a 2729 bp region was sequenced. Cloning of EcoRI fragment into pTZ19R plasmid led to a single nucleotide deletion in the coding region of psbA gene. A scheme of full-length psbA gene cloning is proposed, allowing one to escape the damage effect of the psbA gene expression product on the host cell. The differences between monocot and dicot in nucleotide sequences of DNA downstream of psbA genes are discussed. Gene rps19 is located 131 bp downstream from psbA gene on the complementary strand. The amino acid sequences of D1 and S19 proteins of different species are compared.     

Nucleotide sequence of the single ribosomal RNA operon of pea chloroplast DNA

The nucleotide sequence of an 8 kbp region of pea ( Pisum sativum L.) chloroplast DNA containing the rRNA operon and putative promoter sites has been determined and compared to the corresponding sequences from maize, tobacco and the liverwort Marchantia polymorpha . The chloroplast DNA species of all vascular plants investigated, with the exception of a few legumes including pea, and of Marchantia contain an inverted repeat with an rRNA operon. The pea rRNA operon is the first sequenced rRNA operon from a plant with only one copy of the rRNA genes per molecule of chloroplast DNA. The organization of the operon is the same as for maize, tobacco and Marchantia . i.e. tRNA-Val gene/16S rRNA gene/spacer with intron-containing genes for tRNA-Ile and tRNA-Ala/23S rRNA gene/4.5S rRNA gene/5S rRNA gene. Current evidence suggests that the tRNA-Val gene may not be contranscribed with the other genes. For pea 16S, 23S, 4.5S and 5S rRNA have 1488, 2813, 105 and 121 nucleotides, respectively. The homologies of the entire operon (the tRNA-Val gene - 5S rRNA region) to those from tobacco, maize and Marchantia are 88, 82 and 79%, respectively. The corresponding homologies for tobacco/maize, tobacco/ Marchantia and maize/ Marchantia have similar values. The 16S and 23S rRNA genes from pea are more than 90% homologous to those from the 3 other species. We conclude that the fact that pea only has one set of rRNA genes per molecule of chloroplast DNA is apparently not correlated with any significant difference between the pea operon and the rRNA operons from tobacco, maize and Marchantia .     

Molecular organization of dinoflagellate ribosomal DNA: evolutionary implications of the deduced 5.8 S rRNA secondary structure

The 5.8 S rRNA gene of Prorocentrum micans, a primitive dinoflagellate, has been cloned and its 159 base pairs (bp) have been sequenced along with the two flanking internal transcribed spacers (ITS 1 and 2), respectively, 212 and 195 bp long. Nucleotide sequence homologies between several previously published 5.8 S rRNA gene sequences including those from another dinoflagellate, an ascomycetous yeast, protozoans, a higher plant and a mammal have been determined by sequence alignment. Two prokaryotic 5'-ends of the 23 S rRNA gene have been compared owing to their probable common origin with eucaryotic 5.8 S rRNA genes. Several nucleotides are distinctive for dinoflagellates when compared with either typical eucaryotes or procaryotes. This is consistent with an early divergence of the dinoflagellate lineage from the typical eucaryotes. The secondary structure of dinoflagellate 5.8 S rRNA molecules fits the model of Walker et al. (1983). Conserved nucleotides which distinguish dinoflagellate 5.8 S rRNA from that of other eucaryotes are located in specific loops which are assumed to play a structural role in the ribosome. A 5.8 S rRNA phylogenetic tree which is proposed, based on sequence data, supports our initial assumption of the dinoflagellates.     

Identification and transcription of transfer RNA genes in dinoflagellate plastid minicircles

The dinoflagellate chloroplast genome is unique in that the genes are found on small circular DNA molecules carrying from one to three genes. In addition, only 14 of the typical chloroplast-located genes have so far been discovered on minicircles, while a number have been transferred to the nucleus. We have sequenced four new minicircles from the dinoflagellate Heterocapsa triquetra, three of which carry a single protein-coding gene (psbD, psbE, petD) and one that appears to be an "empty" circle. Using the tRNA prediction programs ARAGORN and tRNAscan-SE, tRNA-Met was found in the petD circle immediately downstream of the end of petD, while tRNA-Trp and tRNA-Pro were detected in the psbE and petD circles as well as in several chimeric circles of H. triquetra and the psbA minicircles of Heterocapsa pygmaea. RT-PCR showed that the tRNAs were co-transcribed with the protein-coding genes that preceded them, and cleaved from the precursor before a poly(U) tail was added to the mRNA.     

Nonuniform concerted evolution and chloroplast capture: heterogeneity of observed introgression patterns in three molecular data partition phylogenies of Asian Mitella (saxifragaceae)

Interspecific hybridization is one of the major factors leading to phylogenetic incongruence among loci, but the knowledge is still limited about the potential of each locus to introgress between species. By directly sequencing three DNA regions: chloroplast DNAs (matK gene and trnL-F noncoding region), the nuclear ribosomal external transcribed spacer (ETS) region, and internal transcribed spacer (ITS) regions, we construct three phylogenetic trees of Asian species of Mitella (Saxifragaceae), a genus of perennials in which natural hybrids are commonly observed. Within this genus, there is a significant topological conflict between chloroplast and nuclear phylogenies and also between the ETS and the ITS, which can be attributed to frequent hybridization within the lineage. Chloroplast DNAs show the most extensive introgression pattern, ITS regions show a moderate pattern, and the ETS region shows no evidence of introgression. Nonuniform concerted evolution best explains the difference in the introgression patterns between the ETS region and ITS regions, as the sequence heterogeneity of the ITS region within an individual genome is estimated to be twice that of an ETS in this lineage. Significant gene conversion patterns between two hybridizing taxa were observed in contiguous arrays of cloned ETS-ITS sequences, further confirming that only ITS regions have introgressed bidirectionally. The relatively slow concerted evolution in the ITS regions probably allows the coexistence of multiple alleles within a genome, whereas the strong concerted evolution in the ETS region rapidly eliminates heterogeneous alleles derived from other species, resulting in species delimitations highly concordant with those based on morphology. This finding indicates that the use of multiple molecular tools has the potential to reveal detailed organismal evolution processes involving interspecific hybridization, as an individual locus varies greatly in its potential to introgress between species.     

Molecular diversity of dinoflagellate symbionts of Cnidaria: the psbA minicircle of Symbiodinium

Dinoflagellate algae of the genus Symbiodinium are important symbionts within corals and other benthic marine animals. The molecular diversity of Symbiodinium has been described mainly by use of ribosomal DNA sequence data. We tested whether minicircle sequences, which appear to form the chloroplast genome in many dinoflagellates, could be used as a marker for molecular diversity among symbionts found in corals and sea anemones. Partial and full-length sequences for psbA were obtained from environmental samples of coral and sea anemones of wide-ranging geographical distribution. Phylogenetic trees constructed with partial psbA sequences were consistent with the known phylotypes of the isolates. Further sequencing suggested that the psbA gene is present on a minicircle in all Symbiodinium phylotypes. The length and DNA sequence of the non-coding portion of the minicircles varied considerably among Symbiodinium phylotypes. In two Symbiodinium isolates from different phylotypes an elaborate pattern of repeat sequences of unknown function was found in the non-coding region. Phylogenetic analysis of the non-coding region of the psbA minicircle indicates that minicircle sequences could be a useful chloroplast-derived marker for differentiating both closely related and distantly related Symbiodinium isolates.     

Phylogeny of Ultra-Rapidly Evolving Dinoflagellate Chloroplast Genes: A Possible Common Origin for Sporozoan and Dinoflagellate Plastids

Complete chloroplast 23S rRNA and psbA genes from five peridinin-containing dinoflagellates (Heterocapsa pygmaea, Heterocapsa niei, Heterocapsa rotun-data, Amphidinium carterae, and Protoceratium reticulatum) were amplified by PCR and sequenced; partial sequences were obtained from Thoracosphaera heimii and Scrippsiella trochoidea. Comparison with chloroplast 23S rRNA and psbA genes of other organisms shows that dinoflagellate chloroplast genes are the most divergent and rapidly evolving of all. Quartet puzzling, maximum likelihood, maximum parsimony, neighbor joining, and LogDet trees were constructed. Intersite rate variation and invariant sites were allowed for with quartet puzzling and neighbor joining. All psbA and 23S rRNA trees showed peridinin-containing dinoflagellate chloroplasts as monophyletic. In psbA trees they are related to those of chromists and red algae. In 23S rRNA trees, dinoflagellates are always the sisters of Sporozoa (apicomplexans); maximum likelihood analysis of Heterocapsa triquetra 16S rRNA also groups the dinoflagellate and sporozoan sequences, but the other methods were inconsistent. Thus, dinoflagellate chloroplasts may actually be related to sporozoan plastids, but the possibility of reproducible long-branch artifacts cannot be strongly ruled out. The results for all three genes fit the idea that dinoflagellate chloroplasts originated from red algae by a secondary endosymbiosis, possibly the same one as for chromists and Sporozoa. The marked disagreement between 16S rRNA trees using different phylogenetic algorithms indicates that this is a rather poor molecule for elucidating overall chloroplast phylogeny. We discuss possible reasons why both plastid and mitochondrial genomes of alveolates (Dinozoa, Sporozoa and Ciliophora) have ultra-rapid substitution rates and a proneness to unique genomic rearrangements. Received: 27 December 1999 / Accepted: 24 March 2000     

Sequence,proposed secondary structure,and phylogenetic analysis of the chloroplast 5S rRNA gene of the brown alga Pylaiella littoralis (L.) Kjellm

Summary The chloroplast 5S rRNA gene of the brown alga Pylaiella littoralis (L.) Kjellm has been cloned and sequenced. The gene is located 23 bp downstream from the 3 end of the 23S rRNA gene. The sequence of the gene is as follows: GGTCTTG GTGTTTAAAGGATAGTGGAACCACATTGAT CCATATCGAACTCAATGGTGAAACATTATT ACAGTAACAATACTTAAGGAGGAGTCCTTT GGGAAGATAGCTTATGCCTAAGAC. A secondary structure model is proposed, and compared to those for the chloroplast 5S rRNAs of spinach and the red alga Porphyra umbilicalis. Cladograms based on chloroplast and bacterial 5S rRNA and rRNA gene sequences were constructed using the MacClade program with a user-defined character transformation in which transitions and transversions were assigned unequal step values. The topology of the resulting cladogram indicates a polyphyletic origin for photosynthetic organelles.Offprint requests to: S. Loiseaux-de Goër     

Nucleotide sequence of a 'truncated rRNA operon' of the Euglena gracilis chloroplast genome.

An extra 16S rRNA gene (s-16S rDNA) from the Euglena gracilis chloroplast genome and several hundred positions of its flanking regions have been sequenced. The structural part has 1486 positions and is to 98% homologous in its sequence with the 16S rRNA gene in functional chloroplast rRNA operons. Sequences of about 200 positions upstream and 15 positions downstream of the structural part of the s-16S rRNA gene region are highly homologous with corresponding parts in the functional operon. Neither tRNA genes (A1a, I1e) nor parts of the 23S and 5S rRNA genes are found within 557 positions after the 3' end of the s-16S rRNA gene, i.e., the 330 bp homology, observed in electron microscopic studies of heteroduplexes (4), between the s-16S rDNA downstream region and the 6.2 kb repeated segment containing the functional rRNA operon, must be due to a DNA stretch in the interoperon spacer. A structural model of the "truncated rRNA operon" is presented. Results from S-1 endonuclease analysis suggest that the s-16S rDNA region is probably not transcribed into stable s-16S rRNA.     

基于核及叶绿体基因序列探讨中国绿水螅共生单细胞绿藻系统发生地位

研究对中国绿水螅共生绿藻的核18S rRNA基因全长序列及其叶绿体9个基因(atpA、chlB、chlN、petA、psaB、psbA、psbC、psbD及rbcL)片段序列进行了克隆和测序, 并基于18S rRNA基因序列及叶绿体9个基因序列的整合数据分别通过最大似然法(Maximum-likelihood)和贝叶斯分析(Bayesian inference)对中国绿水螅(Hydra sinensis)共生单细胞绿藻的系统发生地位进行了探讨。系统发生表明: (1)中国绿水螅共生绿藻属于共球藻纲(Trebouxiophyceae)小球藻目(Chlorellales), 但不属于其中的小球藻属(Chlorella); (2)来源于草履虫、水螅、地衣及银杏的共生绿藻均在共球藻纲支系, 而来源于蛙类和蝾螈的共生绿藻属于绿藻纲(Chlorophyceae)支系。无论在共球藻纲支系还是在绿藻纲支系, 不同来源的共生藻并没有排他性地聚为单系群而在系统树中与其他自由生活的绿藻混杂排列, 来自不同宿主的共生绿藻没有共同起源。     

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.     

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.     

Genetic diversity of Gambierdiscus spp. (Gonyaulacales, Dinophyceae) in Japanese coastal areas

The genetic diversity of the ciguatera fish poisoning-related dinoflagellate distributed in Japanese coastal areas was investigated. The entire sequence of the 5.8S rRNA gene and two internal transcribed (ITS) regions were determined, which included putative pseudogenes, from 19 strains of dinoflagellates assigned to the genus Gambierdiscus Adachi et Fukuyo collected from Japanese subtropical and temperate coastal areas. The sequences obtained from the 19 strains were divided into two types based on sequence similarity. Here we designate the two types as type 1 and type 2. For the relationship between the genotypes and origins of the strains used, the strains collected from subtropical areas possessed the type 1 sequence; whereas those from temperate areas possessed the type 2. This observation led us to question former reputations that Gambierdiscus cells observed in Japanese temperate areas are immigrants from Japanese subtropical areas. Subsequently, we sequenced a part of the 18S rRNA gene from two strains from subtropical areas and two from temperate areas. Unfortunately, phylogenetic analysis including the sequences obtained from various gonyaulacales dinoflagellates failed to determine the species phylogenetically closely related to and possible origin(s) of the Gambierdiscus sp. from the Japanese coastal areas.     

Streptomycin-resistance of Euglena gracilis chloroplasts: identification of a point mutation in the 16S rRNA gene in an invariant position.

We sequenced the chloroplast 16S rRNA gene of two Euglena gracilis mutants which contain streptomycin-resistant chloroplasts (Smr 139.12/4 and Smr 139.20/2). These mutants are known to contain a single intact rrn operon per circular chloroplast genome. Nucleotide sequence comparison between a 16S rRNA gene of wild type Euglena gracilis, strain Z, with streptomycin-sensitive chloroplasts, and the 16S rRNA gene of both Smr-strains reveals a single base change (C to T) at position 876. This position is equivalent to the invariant position 912 of the E. coli 16S rRNA gene. The analogous position is also conserved in all chloroplast small subunit RNA genes from lower and higher plants sequenced so far. Light dependent protein synthesis with purified chloroplasts from streptomycin-resistant cells is not inhibited by streptomycin. Based on the results reported here we postulate linkage between the observed point mutation on the 16S rRNA gene and streptomycin-resistance of chloroplast 70S ribosomes.