Genomic organization of Trypanosoma brucei kinetoplast DNA minicircles

The sequences of seven new Trypanosoma brucei kinetoplast DNA minicircles were obtained. A detailed comparative analysis of these sequences and those of the 18 complete kDNA minicircle sequences from T. brucei available in the database was performed. These 25 different minicircles contain 86 putative gRNA genes. The number of gRNA genes per minicircle varies from 2 to 5. In most cases, the genes are located between short imperfect inverted repeats, but in several minicircles there are inverted repeat cassettes that did not contain identifiable gRNA genes. Five minicircles contain single gRNA genes not surrounded by identifiable repeats. Two pairs of closely related minicircles may have recently evolved from common ancestors: KTMH1 and KTMH3 contained the same gRNA genes in the same order, whereas KTCSGRA and KTCSGRB contained two gRNA genes in the same order and one gRNA gene specific to each. All minicircles could be classified into two classes on the basis of a short substitution within the highly conserved region, but the minicircles in these two classes did not appear to differ in terms of gRNA content or gene organization. A number of redundant gRNAs containing identical editing information but different sequences were present. The alignments of the predicted gRNAs with the edited mRNA sequences varied from a perfect alignment without gaps to alignments with multiple mismatches. Multiple gRNAs overlapped with upstream gRNAs, but in no case was a complete set of overlapping gRNAs covering an entire editing domain obtained. We estimate that a minimum set of approximately 65 additional gRNAs would be required for complete overlapping sets. This analysis should provide a basis for detailed studies of the evolution and role in RNA editing of kDNA minicircles in this species.     

'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.     

Minicircle-encoded guide RNAs from Crithidia fasciculata.

Although the mitochondrial uridine insertion/deletion, guide RNA (gRNA)-mediated type of RNA editing has been described in Crithidia fasciculata, no evidence for the encoding of gRNAs in the kinetoplast minicircle DNA has been presented. There has also been a question as to the capacity of the minicircle DNA in this species to encode the required variety of gRNAs, because the kinetoplast DNA from the C1 strain has been reported as essentially containing a single minicircle sequence class. To address this problem, the genomic and mature edited sequences of the MURF4 and RPS12 cryptogenes were determined and a gRNA library was constructed from mitochondrial RNA. Five specific gRNAs were identified, two of which edit blocks within the MURF4 mRNA, and three of which edit blocks within the RPS12 mRNA. The genes for these gRNAs are all localized with identical polarity within one of the two variable regions of specific minicircle molecules, approximately 60 bp from the "bend" region. These minicircles were found to represent minor sequence classes representing approximately 2% of the minicircle DNA population in the network. The major minicircle sequence class also encodes a gRNA at the same relative genomic location, but the editing role of this gRNA was not determined. These results confirm that kinetoplast minicircle DNA molecules in this species encode gRNAs, as is the case in other trypanosomatids, and suggest that the copy number of specific minicircle sequence classes can vary dramatically without an overall effect on the RNA editing system.     

Evidence for horizontal gene transfer from bacteroidetes bacteria to dinoflagellate minicircles

Dinoflagellate protists harbor a characteristic peridinin-containing plastid that evolved from a red or haptophyte alga. In contrast to typical plastids that have ～100-200 kb circular genomes, the dinoflagellate plastid genome is composed of minicircles that each encode 0-5 genes. It is commonly assumed that dinoflagellate minicircles are derived from a standard plastid genome through drastic reduction and fragmentation. However, we demonstrate that the ycf16 and ycf24 genes (encoded on the Ceratium AF490364 minicircle), as well as rpl28 and rpl33 (encoded on the Pyrocystis AF490367 minicircle), are related to sequences from Algoriphagus and/or Cytophaga bacteria belonging to the Bacteroidetes clade. Moreover, we identified a new open reading frame on the Pyrocystis minicircle encoding a SRP54 N domain, which is typical of FtsY proteins. Because neither of these minicircles share sequence similarity with any other dinoflagellate minicircles, and their genes resemble bacterial operons, we propose that these Ceratium and Pyrocystis minicircles resulted from a horizontal gene transfer (HGT) from a Bacteroidetes donor. Our findings are the first indication of HGT to dinoflagellate minicircles, highlighting yet another peculiar aspect of this plastid genome.     

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.     

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.     

Mitochondrial genes are found on minicircle DNA molecules in the mesozoan animal Dicyema

Animal mitochondrial DNA genomes are generally single circular molecules, 14-20 kb in size, containing a number of functional RNAs and 13 protein-coding genes. Among these, the COI, COII and COIII genes encode three subunits of cytochrome c oxidase. We have isolated and characterized these three mitochondrial genes from the mesozoan Dicyema, a primitive multicellular animal. Surprisingly, the COI, COII and COIII genes are encoded on three small, separate circular DNA molecules (minicircles) of length 1700, 1599 and 1697 bp, respectively. We estimated the copy number of each minicircle at 100 to 1000 per cell, and have shown a mitochondrial localization of the minicircles by in situ hybridization. Furthermore, we could not detect a putative "maxicircle" DNA molecule containing any combination of the COI, COII and COIII genes using either PCR or genomic Southern hybridization. Thus, our results show a novel mitochondrial genome organization in the mesozoan animal Dicyema.     

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.     

Evolution of dinoflagellate unigenic minicircles and the partially concerted divergence of their putative replicon origins

Dinoflagellate chloroplast genes are unique in that each gene is on a separate minicircular chromosome. To understand the origin and evolution of this exceptional genomic organization we completely sequenced chloroplast psbA and 23S rRNA gene minicircles from four dinoflagellates: three closely related Heterocapsa species (H. pygmaea, H. rotundata, and H. niei) and the very distantly related Amphidinium carterae. We also completely sequenced a Protoceratium reticulatum minicircle with a 23S rRNA gene of novel structure. Comparison of these minicircles with those previously sequenced from H. triquetra and A. operculatum shows that in addition to the single gene all have noncoding regions of approximately a kilobase, which are likely to include a replication origin, promoter, and perhaps segregation sequences. The noncoding regions always have a high potential for folding into hairpins and loops. In all six dinoflagellate strains for which multiple minicircles are fully sequenced, parts of the noncoding regions, designated cores, are almost identical between the psbA and 23S rRNA minicircles, but the remainder is very different. There are two, three, or four cores per circle, sometimes highly related in sequence, but no sequence identity is detectable between cores of different species, even within one genus. This contrast between very high core conservation within a species, but none among species, indicates that cores are diverging relatively rapidly in a concerted manner. This is the first well-established case of concerted evolution of noncoding regions on numerous separate chromosomes. It differs from concerted evolution among tandemly repeated spacers between rRNA genes, and that of inverted repeats in plant chloroplast genomes, in involving only the noncoding DNA cores. We present two models for the origin of chloroplast gene minicircles in dinoflagellates from a typical ancestral multigenic chloroplast genome. Both involve substantial genomic reduction and gene transfer to the nucleus. One assumes differential gene deletion within a multicopy population of the resulting oligogenic circles. The other postulates active transposition of putative replicon origins and formation of minicircles by homologous recombination between them.