Rate Variation as a Function of Gene Origin in Plastid-Derived Genes of Peridinin-Containing Dinoflagellates

Peridinin-pigmented dinoflagellates contain secondary plastids that seem to have undergone more nearly complete plastid genome reduction than other eukaryotes. Many typically plastid-encoded genes appear to have been transferred to the nucleus, with a few remaining genes found on minicircles. To understand better the evolution of the dinoflagellate plastid, four categories of plastid-associated genes in dinoflagellates were defined based on their history of transfer and evaluated for rate of sequence evolution, including minicircle genes (presumably plastid-encoded), genes probably transferred from the plastid to the nucleus (plastid-transferred), and genes that were likely acquired directly from the nucleus of the previous plastid host (nuclear-transferred). The fourth category, lateral-transferred genes, are plastid-associated genes that do not appear to have a cyanobacterial origin. The evolutionary rates of these gene categories were compared using relative rate tests and likelihood ratio tests. For comparison with other secondary plastid-containing organisms, rates were calculated for the homologous sequences from the haptophyte Emiliania huxleyi. The evolutionary rate of minicircle and plastid-transferred genes in the dinoflagellate was strikingly higher than that of nuclear-transferred and lateral-transferred genes and, also, substantially higher than that of all plastid-associated genes in the haptophyte. Plastid-transferred genes in the dinoflagellate had an accelerated rate of evolution that was variable but, in most cases, not as extreme as the minicircle genes. Furthermore, the nuclear-transferred and lateral-transferred genes showed rates of evolution that are similar to those of other taxa. Thus, nucleus-to-nucleus transferred genes have a more typical rate of sequence evolution, while those whose history was wholly or partially within the dinoflagellate plastid genome have a markedly accelerated rate of evolution. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Debashish Battacharya]     

The plastid-encoded psbA gene in the dinoflagellate Gonyaulax is not encoded on a minicircle

In all dinoflagellate species studied to date, the plastid genome is highly reduced, with many genes normally found in the plastid genome found instead encoded by the nucleus. Furthermore, those genes still remaining in the plastid are found as primarily single gene minicircles whose size is typically only 2-3 kb. We show here that the plastid genome architecture in the dinoflagellate Gonyaulax polyedra is unusual for this class of organism. In particular, the psbA gene is associated with DNA of roughly 50-150 kb and appears to have an unusually high complexity.     

Plastid-derived single gene minicircles of the dinoflagellate Ceratium horridum are localized in the nucleus

Recent reports show that numerous chloroplast-specific proteins of peridinin-containing dinoflagellates are encoded on minicircles-small plasmidlike molecules containing one or two polypeptide genes each. The genes for these polypeptides are chloroplast specific because their homologs from other photosynthetic eukaryotes are exclusively encoded in the chloroplast genome. Here, we report the isolation, sequencing, and subcellular localization of minicircles from the peridinin-containing dinoflagellate Ceratium horridum. The C. horridum minicircles are organized in the same manner as in other peridinin-containing dinoflagellates and encode the same kinds of plastid-specific proteins, as previous studies reported. However, intact plastids isolated from C. horridum do not contain minicircles, nor do they contain DNA that hybridizes to minicircle-specific probes. Rather, C. horridum minicircles are localized in the nucleus as shown by cell fractionation, Southern hybridization, and in situ hybridization with minicircle-specific probes. A high-molecular-weight DNA was detected in purified C. horridum plastids, but it is apparently not minicircular in organization, as hybridization with a cloned probe from the plastid-localized DNA suggests. The distinction between C. horridum and other peridinin-containing dinoflagellates at the level of their minicircle localization is paralleled by C. horridum thylakoid organization, which also differs from that of other peridinin-containing dinoflagellates, indicating that a hitherto underestimated diversity of minicircle DNA localization and thylakoid organization exists across various dinoflagellate groups.     

The copy number of chloroplast gene minicircles changes dramatically with growth phase in the dinoflagellate Amphidinium operculatum

The chloroplast genome of algae and plants typically comprises a circular DNA molecule of 100-200kb, which harbours approximately 120 genes, and is present in 50-100 copies per chloroplast. However, in peridinin dinoflagellates, an ecologically important group of unicellular algae, the chloroplast genome is fragmented into plasmid-like 'minicircles', each of 2-3kb. Furthermore, the chloroplast gene content of dinoflagellates is dramatically reduced. Only 14 genes have been found on dinoflagellate minicircles, and recent evidence from EST studies suggests that most of the genes typically located in the chloroplast in other algae and plants are located in the nucleus. In this study, Southern blot analysis was used to estimate the copy number per cell of a variety of minicircles during different growth stages in the dinoflagellate Amphidinium operculatum. It was found that minicircle copy number is low during the exponential growth stage but increases during the later growth phase to resemble the situation seen in other plants and algae. The control of minicircle replication is discussed in the light of these findings.     

Subcellular localization of minicircle DNA in the dinoflagellate Amphidinium massartii

Peridinin‐containing dinoflagellates have small circular DNA molecules called minicircle DNAs, each of which encodes one, or occasionally a few, plastid proteins or ribosomal RNA. Dinoflagellate minicircle DNA is composed of two parts: a gene‐coding sequence and a non‐coding sequence that consists of several variable and core regions. The core regions are identical among the minicircle DNAs with different genes within a species or strain. Because such structure is very different from those of well known plastid DNAs, many functional and evolutionary questions have been raised for the minicircle DNAs, and several studies that focus on answering those questions are underway. However, the localization of minicircle DNA is still controversial: several lines of indirect evidence have implied plastid localization, whereas the nuclear localization of minicircle DNA has also been suggested in a species. In order to understand the evolution and function of minicircle DNA, it is important to know its precise localization. In this study, we sequenced two typical minicircle DNAs, one encodes psbA and the other encodes 23S rRNA genes, from an Amphidinium massartii strain (TM16). To determine the subcellular localization of these minicircle DNAs, we performed DNA‐targeted whole cell fluorescence in situ hybridization with A. massartii minicircle DNA‐specific probes and demonstrated that minicircle DNAs were present in plastids. This study provides the first direct evidence for the plastid localization of dinoflagellate minicircle DNAs.     

Migration of the plastid genome to the nucleus in a peridinin dinoflagellate

Dinoflagellate algae are important primary producers and of significant ecological and economic impact because of their ability to form "red tides". They are also models for evolutionary research because of an unparalleled ability to capture photosynthetic organelles (plastids) through endosymbiosis. The nature and extent of the plastid genome in the dominant perdinin-containing dinoflagellates remain, however, two of the most intriguing issues in plastid evolution. The plastid genome in these taxa is reduced to single-gene minicircles encoding an incomplete (until now 15) set of plastid proteins. The location of the remaining photosynthetic genes is unknown. We generated a data set of 6,480 unique expressed sequence tags (ESTs) from the toxic dinoflagellate Alexandrium tamarense (for details, see the Experimental Procedures in the Supplemental Data) to find the missing plastid genes and to understand the impact of endosymbiosis on genome evolution. Here we identify 48 of the non-minicircle-encoded photosynthetic genes in the nuclear genome of A. tamarense, accounting for the majority of the photosystem. Fifteen genes that are always found on the plastid genome of other algae and plants have been transferred to the nucleus in A. tamarense. The plastid-targeted genes have red and green algal origins. These results highlight the unique position of dinoflagellates as the champions of plastid gene transfer to the nucleus among photosynthetic eukaryotes.     

Genome fragmentation is not confined to the peridinin plastid in dinoflagellates

When plastids are transferred between eukaryote lineages through series of endosymbiosis, their environment changes dramatically. Comparison of dinoflagellate plastids that originated from different algal groups has revealed convergent evolution, suggesting that the host environment mainly influences the evolution of the newly acquired organelle. Recently the genome from the anomalously pigmented dinoflagellate Karlodinium veneficum plastid was uncovered as a conventional chromosome. To determine if this haptophyte-derived plastid contains additional chromosomal fragments that resemble the mini-circles of the peridin-containing plastids, we have investigated its genome by in-depth sequencing using 454 pyrosequencing technology, PCR and clone library analysis. Sequence analyses show several genes with significantly higher copy numbers than present in the chromosome. These genes are most likely extrachromosomal fragments, and the ones with highest copy numbers include genes encoding the chaperone DnaK(Hsp70), the rubisco large subunit (rbcL), and two tRNAs (trnE and trnM). In addition, some photosystem genes such as psaB, psaA, psbB and psbD are overrepresented. Most of the dnaK and rbcL sequences are found as shortened or fragmented gene sequences, typically missing the 3'-terminal portion. Both dnaK and rbcL are associated with a common sequence element consisting of about 120 bp of highly conserved AT-rich sequence followed by a trnE gene, possibly serving as a control region. Decatenation assays and Southern blot analysis indicate that the extrachromosomal plastid sequences do not have the same organization or lengths as the minicircles of the peridinin dinoflagellates. The fragmentation of the haptophyte-derived plastid genome K. veneficum suggests that it is likely a sign of a host-driven process shaping the plastid genomes of dinoflagellates.     

The mitochondrial tRNAs of Trypanosoma brucei are nuclear encoded

The mitochondrial DNA of Trypanosoma brucei is organized as a catenated network of maxicircles and minicircles. The maxicircles are equivalent to the typical mitochondrial genome except that the genes for the mitochondrial tRNAs have not been identified by sequence analysis of the maxicircle DNA. The apparent absence of tRNA genes in the maxicircle DNA suggests that the mitochondrial tRNAs are encoded by either the minicircle or the nuclear DNA. In order to determine their genomic origin, we isolated and identified the mitochondrial tRNAs of T. brucei. We show that these mitochondrial tRNAs are truly mitochondrially located in vivo and that they are free from detectable contamination by cytosolic RNAs. By hybridization analysis, using mitochondrial tRNAs as the probe, we determined that the mitochondrial tRNAs are encoded by nuclear DNA. This implies that RNAs, like proteins, are imported into the mitochondria. We investigated the relationship between the cytosolic and the mitochondrial tRNA genes and show that there are unique cytosolic tRNA genes, unique mitochondrial tRNA genes, and tRNA genes which appear to be shared and whose products are therefore targeted to both the cytosol and the mitochondrion.     

Parasite plastids: maintenance and functions

Malaria and related parasites retain a vestigial, but biosynthetically active, plastid organelle acquired far back in evolution from a red algal cell. The organelle appears to be essential for parasite transmission from cell to cell and carries the smallest known plastid genome. Why has this genome been retained? The genes it carries seem to be dedicated to the expression of just two "housekeeping" genes. We speculate that one of these, called ycf24 in plants and sufB in bacteria, is tied to an essential "dark" reaction of the organelle--fatty acid biosynthesis. "Ball-park" clues to the function of bacterial suf genes have emerged only recently and point to the areas of iron homeostasis, [Fe-S] cluster formation and oxidative stress. We present experimental evidence for a physical interaction between SufB and its putative partner SufC (ycf16). In both malaria and plants, SufC is encoded in the nucleus and specifies an ATPase that is imported into the plastid.     

Mitochondrial minicircles in the free-living bodonid Bodo saltans contain two gRNA gene cassettes and are not found in large networks

In trypanosomatids, the majority of the guide (g) RNAs that provide the information for U-insertion/deletion RNA editing are encoded by minicircles that are catenated into large networks. In contrast, in the distantly related cryptobiid Trypanoplasma borreli, gRNA genes appear to reside in large 180-kb noncatenated DNA circles. To shed light on the evolutionary history and function of the minicircle network, we have analyzed minicircle organization in the free-living bodonid Bodo saltans, which is more closely related to trypanosomatids than T. borreli. We identified 1.4-kb circular DNAs as the B. saltans equivalent of minicircles via sequence analysis of 4 complete minicircles, 14 minicircle fragments, and 14 gRNAs. We show that each minicircle harbors two gRNA gene cassettes of opposite polarity residing in variable regions of about 200 nt in otherwise highly conserved molecules. In the conserved region, B. saltans minicircles contain a putative bent helix sequence and a degenerate dodecamer motif (CSB-3). Electron microscopy, sedimentation, and gel electrophoresis analyses showed no evidence for the existence of large minicircle networks in B. saltans, the large majority of the minicircles being present as circular and linear monomers (85-90%) with small amounts of catenated dimers and trimers. Our results provide the first example of a kinetoplastid species with noncatenated, gRNA gene-containing minicircles, which implies that the creation of minicircles and minicircle networks are separate evolutionary events.     

'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.