- Email: [email protected]
Novel plastid gene minicircles in the dinoflagellate Amphidinium operculatum
Gene 331 (2004) 141 – 147 www.elsevier.com/locate/gene
Novel plastid gene minicircles in the dinoflagellate Amphidinium operculatum R. Ellen R. Nisbet, V. Lila Koumandou, Adrian C. Barbrook, Christopher J. Howe * Department of Biochemistry, Centre for Molecular Recognition, University of Cambridge, Downing Site, Tennis Court Road, Cambridge CB2 1QW, UK Received 15 September 2003; received in revised form 30 January 2004; accepted 4 February 2004 Received by W. Martin
Abstract Seven new minicircles, forming part of the fragmented plastid genome of the dinoflagellate Amphidinium operculatum, have been identified by PCR. Three minicircles are full-length, one of 2.6 kb encoding the 23S rRNA gene, one of 2.4 kb containing the psaB gene, and a third of 2.5 kb containing the psbD, psbE and psbI genes. This is the first report of a three-gene minicircle. All three genes are conventional in length, with the same codon bias found in other minicircle genes. The psbI gene sequence is very divergent. One empty minicircle, of 1.7 kb, and three ‘microcircles’, between 400 and 600 bp, have also been identified by PCR. They appear to be formed from full-length minicircles by homologous recombination and internal deletion. All three microcircles contain the core region common to minicircles, but are missing a coding region, providing further evidence that the core region is necessary for minicircle replication and maintenance. D 2004 Elsevier B.V. All rights reserved. Keywords: Chloroplast; Microcircle; Plasmid; Genome fragmentation; Episome; Integron
1. Introduction Plastid genomes of land plants and most algae usually contain about 120 genes on a 120– 150 kb circular DNA molecule. Many of the genes encode subunits of the complexes that carry out the light reactions of photosynthesis (photosystems I and II, the cytochrome b6f complex and the ATP synthase) as well as ribosomal proteins, rRNAs and tRNAs. Despite the broad range of plastids, and the complex evolutionary history they have followed, the plastid genome is remarkably well conserved, with many genes in a similar order across plant and algal groups (Martin et al., 1998; Stoebe and Kowallik, 1999). Despite a report in 1991 (Boczar et al., 1991) of a conventional plastid genome in dinoflagellate algae, recent papers have indicated that there is no full-length plastid genome in those species which have been studied in detail Abbreviations: bp, base pair; kb, kilobase pair; rRNA, ribosomal RNA; tRNA, transfer RNA. * Corresponding author. Tel.: +44-1223-333688; fax: +44-1223333345. E-mail address: [email protected] (C.J. Howe). 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.02.008
(Zhang et al., 1999; Barbrook and Howe, 2000; Barbrook et al., 2001; Hiller, 2001). Instead, genes for many plastid proteins are arranged on small plasmid-like minicircles of 2 –3 kb. Each minicircle contains one or two genes, as well as a highly conserved ‘core’ region of about 400 bp. All genes are in the same orientation with respect to the core region. The remainder of the minicircle is made up of non-coding DNA, and is not conserved. Core regions from Amphidinium operculatum contain a number of motifs that are seen in other species (Zhang et al., 1999; Barbrook and Howe, 2000; Barbrook et al., 2001; Hiller, 2001). Because the core region is highly conserved and contains possible secondary structural features, it seems likely that it is responsible for the maintenance and/or replication of the minicircle. How this fragmented genome arose remains unclear. However, it seems likely that the dinoflagellate plastid had a conventional genome at some stage. This genome fragmented into many minicircles, each with the ability to replicate. As there is as yet limited evidence for a full-length plastid genome in dinoflagellates, it seems most likely that the fragmentation event took place some time ago. The existence of subgenomic circles has been documented in some algae and plant mitochondria, but
142
R.E.R. Nisbet et al. / Gene 331 (2004) 141–147
these are in addition to a full-length genome (Lonsdale et al., 1984; Backert et al., 1997). It is also not clear whether the dinoflagellate minicircles are located in the plastid. There is indirect evidence that this is the case, including the demonstration of psbA mRNA in the dinoflagellate plastid (Takishita et al., 2003), but it remains to be tested directly. To date, 13 genes have been identified on minicircles in various dinoflagellate species, encoding polypeptides closely involved with the light reactions of photosynthesis (atpA, atpB, petB, petD, psaA, psaB, psbA, psbB, psbC, psbD, psbE) or rRNA species (16S, 23S). The atpA and petB genes and the psbD and psbE genes are located in pairs, respectively, on two minicircles, each containing two genes in both A. operculatum and Amphidinium carterae (Barbrook et al., 2001; Hiller, 2001). The members of each pair of genes are not located next to each other in conventional plastid genomes or on minicircles of the dinoflagellate Heterocapsa triquetra (Zhang et al., 1999). The atpA and petB genes have been shown to be represented on separate transcripts (Barbrook et al., 2001). In addition, we have reported the existence of ‘empty’ minicircles that do not contain any identifiable coding region (Barbrook et al., 2001). These minicircles are found across different dinoflagellate species (Hiller, 2001) and in different cultures of the same species (our unpublished observations). As the plastid genome should contain about 120 genes, it is clear that there are many genes that have not yet been located in dinoflagellates, and a number have been reported in only one species. We report here the identification of a number of additional minicircles from A. operculatum, containing the 23S rRNA gene, psaB, and psbD, psbE and psbI genes, all thought to be essential for plastid photosynthetic function. We show that the psbD, psbE and psbI genes are located on a single minicircle. This is the first report of a minicircle containing three genes. We also report the existence of much smaller molecules, which we term ‘microcircles’, derived by large-scale deletion and rearrangements of minicircles.
2. Materials and methods 2.1. A. Operculatum culture conditions A. operculatum (from the Culture Collection of Algae and Protozoa, Oban, UK, ref CCAP 1102/6) was cultured in Artificial Sea Water, as described by Barbrook and Howe (2000). 2.2. PCR amplification of minicircles containing genes A DNA fraction enriched in minicircles was isolated from A. operculatum essentially as described by Barbrook and Howe, 2000, except separation was carried out only
in the presence of ethidium bromide. PCR was carried out on this fraction using degenerate primers (Table 1) designed to conserved regions of other algal plastidencoded genes, taking into account the codon usage found in previously sequenced A. operculatum plastid genes (Barbrook and Howe, 2000). Further primers were designed based on the initial PCR products to amplify the remainder of the minicircles. PCR was carried out using Qiagen or Bioline Taq according to the manufacturer’s instructions, varying the annealing temperature and MgCl2 concentrations. 2.3. PCR analysis of other minicircles Four primers, CF1, CF2, CR1 and CR2 (Table 1), were designed to the conserved central core region. Primer pairs (CF1/CR1 and CF2/CR2) were used to amplify potential minicircles. The PCR products obtained were randomly ligated into pGEM-T-Easy (Promega) and sequenced. Further primers were designed from the sequences obtained in order to confirm that the potential minicircles were circular. To test if partially deleted minicircles could arise as PCR artefacts caused by repeated sequences, PCR was carried out using minicircles cloned into pGEM-T-Easy with primers CF1/CR1. 2.4. DNA sequencing and analysis PCR products were cloned into pGEM-T-Easy (Promega) using Escherichia coli TG1 as host (Gibson, 1984). Plasmids and PCR products were sequenced using an Applied Biosystems DNA sequencer. Genes were identified using BLAST analysis (www.ncbi.nlm.nih.gov/blast) and assemTable 1 Primer sequences
R.E.R. Nisbet et al. / Gene 331 (2004) 141–147
bled using the GCG Wisconsin package. Multiple alignments were generated using ClustalX (Thompson et al., 1997).
143
confirmed using Southern analysis on DNA from A. operculatum (data not shown). 3.2. PCR from core region to obtain non-coding minicircles
2.5. Southern analysis Minicircle enriched DNA was digested with restriction enzymes and subjected to electrophoretic analysis. Southern analysis was carried out using 5% w/v polyacrylamide gels as described by Sambrook et al., 1989. Fluorescently labelled probes were prepared using the appropriate PCR products and the Amersham random primer labelling system. Hybridization of the probes to the membranes was carried out overnight at 60 or 65 jC. Hybridization was visualized using the Gene Images CDP star detection system (Amersham).
3. Results 3.1. PCR amplification of minicircles containing 23S rRNA, psaB and psbD/psbE/psbI A DNA fraction previously shown to be enriched in minicircles was used as template for PCR. Primers 23SF1 and 23SR2 were designed to 23S rRNA gene sequences from related organisms (Guillardia theta, Odontella sinesis, Porphyra purpurea and Cyanophora paradoxa). PCR generated a product of 700 bp, which was sequenced and showed homology to algal 23S rRNA sequences. Based on the sequence of this product two further primers, 23SF3 and 23SR4, were designed, facing outwards from the sequenced region. PCR with these primers gave rise to a single product of 2500 bp confirming that the 23S rRNA gene was located on a minicircle. The product showed homology to algal 23S rRNA sequences and to the minicircle core region from A. operculatum. The sequences from the two PCR products were identical throughout the region of overlap and were therefore assembled, to generate a minicircle of 2655 bp containing the 23S rRNA gene. A similar strategy was used with primers psaBF1/psaBR2 and psaBF3/psaBR4 to identify a 2363 bp minicircle, containing the psaB gene. Degenerate primers (psbDF1 and psbDR2) for conserved regions of psbD (Table 1) gave a PCR product with sequence similarity to other psbD genes. Further PCR using primers psbDF1/psbER2 and psbEF1/psbDR2 showed that the psbD and psbE genes were located on a single minicircle of 2453 bp. In addition, sequence analysis indicated that the minicircle also contained the psbI gene. All three minicircles contain an approximately 400 bp core region found in previously sequenced minicircles. The genes are in the same orientation with respect to the core region as previously described (Barbrook et al., 2001). The sizes of all three minicircles obtained by sequencing were
Primers CF1/CR1 designed facing outwards from the central, highly conserved core region were used in PCR analysis of A. operculatum DNA enriched for minicircles. The products obtained were analysed using gel electrophoresis (Fig. 1). Two intense bands were seen, corresponding to 1700 and 2200 bp, together with a smear corresponding to shorter molecules, indicating that many additional products were formed. The entire reaction products were cloned into pGEM-T-Easy, and the ligation products introduced into E. coli by transformation. Plasmids were recovered from a number of E. coli colonies picked at random and subjected to sequence determination. Four plasmids were chosen for further study as they occurred most frequently. PCR was carried out with additional sets of primers to confirm that the initial products seen were from minicircles. In all, four new putative minicircles were found of 584, 514, 405 and 263 bp. All four share the same central core region, including the AAAAA motif. One minicircle (of 584 bp) is entirely comprised of regions of DNA homologous to sequences from the previously obtained psbB and psbC minicircles, as shown in Fig. 2. Another (of 514 bp) is entirely comprised of regions of DNA homologous to sequences from the psbB minicircle and empty circle 5 described below. The 405 and 263 bp minicircles were found to be homologous to part of the full-length 23S rRNA minicircle detailed in Section 3.1. However, both minicircles had substantial deletions, of 2250 and 2392 bp, corresponding to the majority of the 23S rRNA coding region. Similar experiments were carried out using primers CF2 and CR2 for the core region, which resulted in the recovery
Fig. 1. PCR products obtained with core primers CR1 and CF1. PCR was carried out on DNA enriched for minicircles. Lane 1, PCR products; lane 2, Hyperladder 1 (Bioline).
144
R.E.R. Nisbet et al. / Gene 331 (2004) 141–147
Fig. 2. Alignments showing how the four microcircles could have arisen. All circles are shown linearised, and the core region is shaded. Regions of sequence identity or very high similarity (represented by boxes) are aligned. Minicircle sequences are numbered according to the NCBI sequence. Regions of no homology are shown as lines. Panel (i) shows the 584 bp minicircle; (ii) the 514 bp minicircle; (iii) the 405 bp minicircle; (iv) the 263 bp minicircle. Numbers at the end denote the points at which circles have been notionally linearized. Numbers within the molecules indicate the start and end of regions of sequence identity or very high similarity.
of a minicircle of 1.7 kb that did not contain any open reading frame or potential RNA coding region of significant length. This was designated ‘empty circle 5’, following the numbering of Barbrook et al. (2001). A section of the 514 bp minicircle described above was identical to part of this minicircle. 3.3. Verification of minicircles Southern analysis was carried out to confirm the existence of the minicircles. DNA enriched for minicircles was cut with XmnI, run on a 5% acrylamide gel and blotted onto a nitrocellulose membrane. In addition, cloned minicircles were used as templates in PCR to generate products of similar size and sequence to the equivalent minicircles linearized with XmnI. These PCR products were used as positive controls in the Southern analyses. The blots were
analysed with probes to (i) the region of the 584 bp minicircle showing homology to psbC, (ii) the region of the 514 bp minicircle showing homology to empty circle 5, and (iii) a region common to the 405 bp, 263 bp and 23Scoding minicircles. Hybridization was seen to bands corresponding to the 584, 514 and 405 bp minicircles (Fig. 3). (The minor differences in mobility between the controls can be ascribed to slight differences in the actual sizes of the molecules due to the primers used and the effects of loading different amounts of DNA in different lanes.) The signals corresponding to the sub-minicircles were less intense than those corresponding to the ‘full-length’ minicircles, and bands of other sizes were also seen. Little or no hybridization was seen corresponding to the 263 bp minicircle. Given the low level of hybridisation to A. operculatum DNA corresponding to the 263 bp minicircle, it seemed possible that this was an artefact derived from the 23S
R.E.R. Nisbet et al. / Gene 331 (2004) 141–147
145
Fig. 3. Verification of minicircles. A. operculatum DNA enriched for minicircles was cut with the restriction enzyme XmnI and probed for the minicircles in Southern analyses. Samples were run alongside PCR products from cloned minicircles, with Hyperladder 1 as a marker. Size indicators are to the right of each figure. Panel (i) was probed with the region of the 584 bp minicircle showing homology to psbC: Lane 1, minicircle DNA, arrow A indicates the psbC minicircle, arrow B the linearised 584 bp minicircle; lane 2, PCR products from cloned 584 bp minicircle; lane 3, Hyperladder 1. Panel (ii) was probed with the region of the 514 bp minicircle showing homology to empty circle 5: Lane 1, minicircle DNA, arrow C indicates a fragment of empty circle 5, arrow D indicates the linearised 514 bp minicircle; lane 2 Hyperladder 1; lane 3, PCR products from the cloned 514 bp minicircle. Panel (iii) was probed with a region common to the 405 bp, 263 bp and 23S-coding minicircles. Lane 1, minicircle DNA, arrow E indicates a fragment of the full-length 23S minicircle, arrow F indicates the linearised 405 bp minicircle; lane 2, Hyperladder 1; lane 3 PCR products from the cloned 263 bp minicircle; lane 4 PCR products from the cloned 405 bp minicircle.
minicircle by ‘jumping PCR’ across the repeated sequences described below. PCR was therefore carried out using the full-length 23S minicircle cloned into pGEM-T-Easy as a template and the CF1/CR1 original primers. This reaction gave rise to a product of approximately 250 bp (data not shown). This was cloned and found to be identical in sequence to the 263 bp minicircle. No band corresponding to the 405 bp minicircle was seen in the PCR products.
4. Discussion 4.1. Coding minicircles The results described here extend the number of minicircles characterized from A. operculatum that contain fulllength potential coding sequences. They are all of a similar size to the minicircles reported before from this species, and have the coding region in the same orientation with respect to the core region. The 23S rRNA coding region has been localized to a minicircle of 2655 bp. Sequence analysis of the core region of the full-length 23S minicircle shows that it is most similar to the core region found in the psbB, psbD/ psbE/psbI, psbC and petD minicircles. The psaB minicircle is 2363 bp in length, and contains a full-length psaB coding region of 1965 bp, and a core. The psbD, psbE and psbI genes are located on a single minicircle of 2453 bp. All three genes are in the same orientation with respect to the core region, as are atpA and petB on the two-gene minicircle (Barbrook et al., 2001). The genes are in the order psbD, psbE, psbI. The psbD and psbE genes are separated by a region of 119 bp. The psbI gene (Fig. 4) is 259 bp further
downstream from psbE. This is the first report of a threegene minicircle in the dinoflagellates. A. carterae, a closely related dinoflagellate, also carries psbD and psbE on a single minicircle (Hiller, 2001). Although the latter study did not identify the psbI gene, inspection of the reported sequence shows that psbI is in fact present, but was not obvious because the gene is short and poorly conserved. It is interesting that the psbI gene appears to be most similar to the Chlamydomonas reinhardtii one, although the significance of this is not clear. The psbD, psbE and psbI genes are not located near each other in previously sequenced plastid genomes or in cyanobacterial genomes (Reith and Munholland, 1995). This suggests the group was not generated by fragmentation of a larger molecule, unless it had a different organization from anything reported to date. It is possible that the three-gene minicircle was derived by fusion of single-gene circles, perhaps to facilitate the control of expression. Since the genes are all in the same orientation, it is possible that they are co-transcribed, although our preliminary Northern analysis suggests this is not the case in A. operculatum (unpublished data). Essentially nothing is known about ribosome binding sites on dinoflagellate minicircle genes, so it is not possible to say if there is a separate ribosome binding site for each open reading frame. 4.2. Non-coding minicircles In addition to the coding minicircles and empty circle 5, we isolated a number of small minicircles. One of these, of 584 bp, could be derived by recombination between psbB and psbC minicircles across sequences common to the two minicircles. The psbB and psbC minicircles have two
146
R.E.R. Nisbet et al. / Gene 331 (2004) 141–147
Fig. 4. ClustalX alignment of PsbI protein sequences from a variety of plants and algae. Stars (*) indicate identities, dots (.) and colons (:) similarities as defined by ClustalX. Identity and similarity designations above the alignment are shared in all sequences. Identity and similarity designations below the alignments are those shared between C. reinhardtii and A. operculatum.
regions of homology, one across the core region and the other across 18 bp of the psbC and psbB minicircles (corresponding to residues 1668– 1685 of the psbB minicircle (AJ250263) and residues 1827 –1844 of the psbC minicircle (AF426172), Fig. 2). A double recombination event across these two regions could give rise to the 584 bp minicircle. Similarly, the 514 bp minicircle could be derived by a double recombination event between the psbB minicircle and the newly reported non-coding minicircle, designated ‘empty circle 5’ (Fig. 2). The 263 and 405 bp minicircles could be derived by deletion within the 23S rRNA minicircle. The 263 bp minicircle could be derived by recombination and the consequent deletion of the internal region across a repeated sequence of GAACGATTAGGTAA found at residues 69 – 82 and 2461 –2474 of the 23S minicircle (Fig. 2). However, it may be a PCR artefact caused by the repeated sequence as no hybridization was seen in Southern anaysis. The deletion giving rise to the 405 bp minicircle starts close to this but ends at a site with no sequence similarity to it, so it cannot be explained by simple recombination (Fig. 2). Zhang et al. (1999) reported the existence of three forms of 23S rRNA minicircles. One minicircle was full length, while two contained deletions in the core region of 23 and 35 base pairs. However, neither contained deletions in the 23S coding region, nor were the deletions of the magnitude of 2 kbp. Santos et al. (2002) also found deletions of 6, 23, 208, 250 and 298 bp in the coding region of the 23S rRNA minicircle in the dinoflagellate genus Symbiodinium, with genes having one or more of these deletions present. We have not observed such small scale deletions in A. operculatum. The generation of novel minicircles by fusion of different circles and subsequent deletion has been reported before (Barbrook et al., 2001; Zhang et al., 2002). Recombination between circles could account for the generation of the 584 and 514 bp minicircles. However, the existence of such
small minicircles has not been reported before. We term these very small minicircles ‘microcircles’. This is also the first report of the generation of small minicircles by simple deletion within one minicircle species, as with the 405 bp microcircle. The Southern hybridization showed that all these molecules, with the possible exception of the 263 bp, exist in vivo, although probably at lower abundance than the conventional coding minircles. The 584, 514 and 405 bp microcircles have been found several times in different cultures of A. operculatum over a period of 18 months, suggesting that the microcircles are stable. The generation of microcircles may in some cases be associated with repeated sequences, suggesting a recombination-mediated process, but this is not invariably the case. The existence of these microcircles is reminiscent of the situation with plant mitochondria, where there are varying levels of sub-genomic molecules generated by recombination across repeated sequences (Lonsdale et al., 1984; Backert et al., 1997) and very low levels of ‘sublimons’ generated by aberrant recombination events (Small et al., 1987). It remains to be seen whether the dinoflagellate microcircles have any function, and indeed whether they are located in the same cellular compartment as the more conventional minicircles.
5. DNA sequences DNA sequences for the four minicircles and three microcircles have accession numbers AJ582639 – AJ582644 and AJ620761.
Acknowledgements We thank Roger Hiller for helpful discussions, and the Biotechnology and Biological Sciences Research Council,
R.E.R. Nisbet et al. / Gene 331 (2004) 141–147
the Cambridge European Trust and Corpus Christi College, Cambridge for financial support. References Backert, S., Nielsen, B.L., Bo¨rner, T., 1997. The mystery of the rings: structure and replication of mitochondrial genomes from higher plants. Trends Plant Sci. 2, 477 – 483. Barbrook, A.C., Howe, C.J., 2000. Minicircular plastid DNA in the dinoflagellate Amphidinium operculatum. Mol. Gen. Genet. 263, 152 – 158. Barbrook, A.C., Symington, H.A., Nisbet, R.E.R., Larkum, A., Howe, C.J., 2001. Organisation and expression of the plastid genome of the dinoflagellate Amphidinium operculatum. Mol. Gen. Genomics 266, 632 – 638. Boczar, B.A., Liston, J., Cattolico, R.A., 1991. Characterization of satellite DNA from three marine dinoflagellates (dinophyceae): Glenodinium sp. and two members of the toxic genus, Protogonyaulax. Plant Physiol. 97, 613 – 618. Gibson, T.J., 1984. Studies on the Epstein – Barr virus genome. PhD thesis, Cambridge University, UK. Hiller, R.G., 2001. ‘Empty’ minicircles and petB/atpA and psbD/psbE (cytb559 alpha) genes in tandem in Amphidinium carterae plastid DNA. FEBS Lett. 505, 449 – 452. Lonsdale, D.M., Hodge, T.P., Fauron, C.M., 1984. The physical map and organisation of the mitochondrial genome from the fertile cytoplasm of maize. Nucleic Acids Res. 12, 9249 – 9261. Martin, W., Stoebe, B., Goremykin, V., Hansmann, S., Hasegawa, M., Kowallik, K., 1998. Gene transfer to the nucleus and the evolution of chloroplasts. Nature 393, 162 – 165.
147
Reith, M., Munholland, J., 1995. Complete nucleotide sequence of the Porphyra purpurea chloroplast genome. Plant Mol. Biol. Rep. 13, 333 – 335. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York. Santos, S.R., Taylor, D.J., Kinizie III, R.A., Sakai, K., Coffroth, M.A., 2002. Molecular phylogeny of symbiotic dinoflagellates inferred from partial chloroplast large subunit (23S)-rDNA sequences. Mol. Phylogenet. Evol. 23, 97 – 111. Small, I., Suffolk, R., Leaver, C.J., 1987. Stoichiometric differences in DNA-molecules containing the atpA gene suggest mechanisms for the generation of mitochondrial genome diversity in maize. EMBO J. 6, 865 – 869. Stoebe, B., Kowallik, K.V., 1999. Gene-cluster analysis in chloroplast genomics. Trends Genet. 15, 344 – 347. Takishita, K., Ishikura, M., Koike, K., Maruyama, T., 2003. Comparison of phylogenies based on nuclear-encoded SSU rDNA and plastid-encoded psbA in the symbiotic dinoflagellate genus Symbiodinium. Phycologia 42, 285 – 291. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876 – 4882. Zhang, Z., Green, B.R., Cavalier-Smith, T., 1999. Single gene circles in dinoflagellate chloroplast genomes. Nature 400, 155 – 159. Zhang, Z., Cavalier-Smith, T., Green, B.R., 2002. Evolution of dinoflagellate unigenic minicircles and the partially concerted divergence of their putative replicon origins. Mol. Biol. Evol. 19, 489 – 500.
Novel plastid gene minicircles in the dinoflagellate Amphidinium operculatum R. Ellen R. Nisbet, V. Lila Koumandou, Adrian C. Barbrook, Christopher J. Howe * Department of Biochemistry, Centre for Molecular Recognition, University of Cambridge, Downing Site, Tennis Court Road, Cambridge CB2 1QW, UK Received 15 September 2003; received in revised form 30 January 2004; accepted 4 February 2004 Received by W. Martin
Abstract Seven new minicircles, forming part of the fragmented plastid genome of the dinoflagellate Amphidinium operculatum, have been identified by PCR. Three minicircles are full-length, one of 2.6 kb encoding the 23S rRNA gene, one of 2.4 kb containing the psaB gene, and a third of 2.5 kb containing the psbD, psbE and psbI genes. This is the first report of a three-gene minicircle. All three genes are conventional in length, with the same codon bias found in other minicircle genes. The psbI gene sequence is very divergent. One empty minicircle, of 1.7 kb, and three ‘microcircles’, between 400 and 600 bp, have also been identified by PCR. They appear to be formed from full-length minicircles by homologous recombination and internal deletion. All three microcircles contain the core region common to minicircles, but are missing a coding region, providing further evidence that the core region is necessary for minicircle replication and maintenance. D 2004 Elsevier B.V. All rights reserved. Keywords: Chloroplast; Microcircle; Plasmid; Genome fragmentation; Episome; Integron
1. Introduction Plastid genomes of land plants and most algae usually contain about 120 genes on a 120– 150 kb circular DNA molecule. Many of the genes encode subunits of the complexes that carry out the light reactions of photosynthesis (photosystems I and II, the cytochrome b6f complex and the ATP synthase) as well as ribosomal proteins, rRNAs and tRNAs. Despite the broad range of plastids, and the complex evolutionary history they have followed, the plastid genome is remarkably well conserved, with many genes in a similar order across plant and algal groups (Martin et al., 1998; Stoebe and Kowallik, 1999). Despite a report in 1991 (Boczar et al., 1991) of a conventional plastid genome in dinoflagellate algae, recent papers have indicated that there is no full-length plastid genome in those species which have been studied in detail Abbreviations: bp, base pair; kb, kilobase pair; rRNA, ribosomal RNA; tRNA, transfer RNA. * Corresponding author. Tel.: +44-1223-333688; fax: +44-1223333345. E-mail address: [email protected] (C.J. Howe). 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.02.008
(Zhang et al., 1999; Barbrook and Howe, 2000; Barbrook et al., 2001; Hiller, 2001). Instead, genes for many plastid proteins are arranged on small plasmid-like minicircles of 2 –3 kb. Each minicircle contains one or two genes, as well as a highly conserved ‘core’ region of about 400 bp. All genes are in the same orientation with respect to the core region. The remainder of the minicircle is made up of non-coding DNA, and is not conserved. Core regions from Amphidinium operculatum contain a number of motifs that are seen in other species (Zhang et al., 1999; Barbrook and Howe, 2000; Barbrook et al., 2001; Hiller, 2001). Because the core region is highly conserved and contains possible secondary structural features, it seems likely that it is responsible for the maintenance and/or replication of the minicircle. How this fragmented genome arose remains unclear. However, it seems likely that the dinoflagellate plastid had a conventional genome at some stage. This genome fragmented into many minicircles, each with the ability to replicate. As there is as yet limited evidence for a full-length plastid genome in dinoflagellates, it seems most likely that the fragmentation event took place some time ago. The existence of subgenomic circles has been documented in some algae and plant mitochondria, but
142
R.E.R. Nisbet et al. / Gene 331 (2004) 141–147
these are in addition to a full-length genome (Lonsdale et al., 1984; Backert et al., 1997). It is also not clear whether the dinoflagellate minicircles are located in the plastid. There is indirect evidence that this is the case, including the demonstration of psbA mRNA in the dinoflagellate plastid (Takishita et al., 2003), but it remains to be tested directly. To date, 13 genes have been identified on minicircles in various dinoflagellate species, encoding polypeptides closely involved with the light reactions of photosynthesis (atpA, atpB, petB, petD, psaA, psaB, psbA, psbB, psbC, psbD, psbE) or rRNA species (16S, 23S). The atpA and petB genes and the psbD and psbE genes are located in pairs, respectively, on two minicircles, each containing two genes in both A. operculatum and Amphidinium carterae (Barbrook et al., 2001; Hiller, 2001). The members of each pair of genes are not located next to each other in conventional plastid genomes or on minicircles of the dinoflagellate Heterocapsa triquetra (Zhang et al., 1999). The atpA and petB genes have been shown to be represented on separate transcripts (Barbrook et al., 2001). In addition, we have reported the existence of ‘empty’ minicircles that do not contain any identifiable coding region (Barbrook et al., 2001). These minicircles are found across different dinoflagellate species (Hiller, 2001) and in different cultures of the same species (our unpublished observations). As the plastid genome should contain about 120 genes, it is clear that there are many genes that have not yet been located in dinoflagellates, and a number have been reported in only one species. We report here the identification of a number of additional minicircles from A. operculatum, containing the 23S rRNA gene, psaB, and psbD, psbE and psbI genes, all thought to be essential for plastid photosynthetic function. We show that the psbD, psbE and psbI genes are located on a single minicircle. This is the first report of a minicircle containing three genes. We also report the existence of much smaller molecules, which we term ‘microcircles’, derived by large-scale deletion and rearrangements of minicircles.
2. Materials and methods 2.1. A. Operculatum culture conditions A. operculatum (from the Culture Collection of Algae and Protozoa, Oban, UK, ref CCAP 1102/6) was cultured in Artificial Sea Water, as described by Barbrook and Howe (2000). 2.2. PCR amplification of minicircles containing genes A DNA fraction enriched in minicircles was isolated from A. operculatum essentially as described by Barbrook and Howe, 2000, except separation was carried out only
in the presence of ethidium bromide. PCR was carried out on this fraction using degenerate primers (Table 1) designed to conserved regions of other algal plastidencoded genes, taking into account the codon usage found in previously sequenced A. operculatum plastid genes (Barbrook and Howe, 2000). Further primers were designed based on the initial PCR products to amplify the remainder of the minicircles. PCR was carried out using Qiagen or Bioline Taq according to the manufacturer’s instructions, varying the annealing temperature and MgCl2 concentrations. 2.3. PCR analysis of other minicircles Four primers, CF1, CF2, CR1 and CR2 (Table 1), were designed to the conserved central core region. Primer pairs (CF1/CR1 and CF2/CR2) were used to amplify potential minicircles. The PCR products obtained were randomly ligated into pGEM-T-Easy (Promega) and sequenced. Further primers were designed from the sequences obtained in order to confirm that the potential minicircles were circular. To test if partially deleted minicircles could arise as PCR artefacts caused by repeated sequences, PCR was carried out using minicircles cloned into pGEM-T-Easy with primers CF1/CR1. 2.4. DNA sequencing and analysis PCR products were cloned into pGEM-T-Easy (Promega) using Escherichia coli TG1 as host (Gibson, 1984). Plasmids and PCR products were sequenced using an Applied Biosystems DNA sequencer. Genes were identified using BLAST analysis (www.ncbi.nlm.nih.gov/blast) and assemTable 1 Primer sequences
R.E.R. Nisbet et al. / Gene 331 (2004) 141–147
bled using the GCG Wisconsin package. Multiple alignments were generated using ClustalX (Thompson et al., 1997).
143
confirmed using Southern analysis on DNA from A. operculatum (data not shown). 3.2. PCR from core region to obtain non-coding minicircles
2.5. Southern analysis Minicircle enriched DNA was digested with restriction enzymes and subjected to electrophoretic analysis. Southern analysis was carried out using 5% w/v polyacrylamide gels as described by Sambrook et al., 1989. Fluorescently labelled probes were prepared using the appropriate PCR products and the Amersham random primer labelling system. Hybridization of the probes to the membranes was carried out overnight at 60 or 65 jC. Hybridization was visualized using the Gene Images CDP star detection system (Amersham).
3. Results 3.1. PCR amplification of minicircles containing 23S rRNA, psaB and psbD/psbE/psbI A DNA fraction previously shown to be enriched in minicircles was used as template for PCR. Primers 23SF1 and 23SR2 were designed to 23S rRNA gene sequences from related organisms (Guillardia theta, Odontella sinesis, Porphyra purpurea and Cyanophora paradoxa). PCR generated a product of 700 bp, which was sequenced and showed homology to algal 23S rRNA sequences. Based on the sequence of this product two further primers, 23SF3 and 23SR4, were designed, facing outwards from the sequenced region. PCR with these primers gave rise to a single product of 2500 bp confirming that the 23S rRNA gene was located on a minicircle. The product showed homology to algal 23S rRNA sequences and to the minicircle core region from A. operculatum. The sequences from the two PCR products were identical throughout the region of overlap and were therefore assembled, to generate a minicircle of 2655 bp containing the 23S rRNA gene. A similar strategy was used with primers psaBF1/psaBR2 and psaBF3/psaBR4 to identify a 2363 bp minicircle, containing the psaB gene. Degenerate primers (psbDF1 and psbDR2) for conserved regions of psbD (Table 1) gave a PCR product with sequence similarity to other psbD genes. Further PCR using primers psbDF1/psbER2 and psbEF1/psbDR2 showed that the psbD and psbE genes were located on a single minicircle of 2453 bp. In addition, sequence analysis indicated that the minicircle also contained the psbI gene. All three minicircles contain an approximately 400 bp core region found in previously sequenced minicircles. The genes are in the same orientation with respect to the core region as previously described (Barbrook et al., 2001). The sizes of all three minicircles obtained by sequencing were
Primers CF1/CR1 designed facing outwards from the central, highly conserved core region were used in PCR analysis of A. operculatum DNA enriched for minicircles. The products obtained were analysed using gel electrophoresis (Fig. 1). Two intense bands were seen, corresponding to 1700 and 2200 bp, together with a smear corresponding to shorter molecules, indicating that many additional products were formed. The entire reaction products were cloned into pGEM-T-Easy, and the ligation products introduced into E. coli by transformation. Plasmids were recovered from a number of E. coli colonies picked at random and subjected to sequence determination. Four plasmids were chosen for further study as they occurred most frequently. PCR was carried out with additional sets of primers to confirm that the initial products seen were from minicircles. In all, four new putative minicircles were found of 584, 514, 405 and 263 bp. All four share the same central core region, including the AAAAA motif. One minicircle (of 584 bp) is entirely comprised of regions of DNA homologous to sequences from the previously obtained psbB and psbC minicircles, as shown in Fig. 2. Another (of 514 bp) is entirely comprised of regions of DNA homologous to sequences from the psbB minicircle and empty circle 5 described below. The 405 and 263 bp minicircles were found to be homologous to part of the full-length 23S rRNA minicircle detailed in Section 3.1. However, both minicircles had substantial deletions, of 2250 and 2392 bp, corresponding to the majority of the 23S rRNA coding region. Similar experiments were carried out using primers CF2 and CR2 for the core region, which resulted in the recovery
Fig. 1. PCR products obtained with core primers CR1 and CF1. PCR was carried out on DNA enriched for minicircles. Lane 1, PCR products; lane 2, Hyperladder 1 (Bioline).
144
R.E.R. Nisbet et al. / Gene 331 (2004) 141–147
Fig. 2. Alignments showing how the four microcircles could have arisen. All circles are shown linearised, and the core region is shaded. Regions of sequence identity or very high similarity (represented by boxes) are aligned. Minicircle sequences are numbered according to the NCBI sequence. Regions of no homology are shown as lines. Panel (i) shows the 584 bp minicircle; (ii) the 514 bp minicircle; (iii) the 405 bp minicircle; (iv) the 263 bp minicircle. Numbers at the end denote the points at which circles have been notionally linearized. Numbers within the molecules indicate the start and end of regions of sequence identity or very high similarity.
of a minicircle of 1.7 kb that did not contain any open reading frame or potential RNA coding region of significant length. This was designated ‘empty circle 5’, following the numbering of Barbrook et al. (2001). A section of the 514 bp minicircle described above was identical to part of this minicircle. 3.3. Verification of minicircles Southern analysis was carried out to confirm the existence of the minicircles. DNA enriched for minicircles was cut with XmnI, run on a 5% acrylamide gel and blotted onto a nitrocellulose membrane. In addition, cloned minicircles were used as templates in PCR to generate products of similar size and sequence to the equivalent minicircles linearized with XmnI. These PCR products were used as positive controls in the Southern analyses. The blots were
analysed with probes to (i) the region of the 584 bp minicircle showing homology to psbC, (ii) the region of the 514 bp minicircle showing homology to empty circle 5, and (iii) a region common to the 405 bp, 263 bp and 23Scoding minicircles. Hybridization was seen to bands corresponding to the 584, 514 and 405 bp minicircles (Fig. 3). (The minor differences in mobility between the controls can be ascribed to slight differences in the actual sizes of the molecules due to the primers used and the effects of loading different amounts of DNA in different lanes.) The signals corresponding to the sub-minicircles were less intense than those corresponding to the ‘full-length’ minicircles, and bands of other sizes were also seen. Little or no hybridization was seen corresponding to the 263 bp minicircle. Given the low level of hybridisation to A. operculatum DNA corresponding to the 263 bp minicircle, it seemed possible that this was an artefact derived from the 23S
R.E.R. Nisbet et al. / Gene 331 (2004) 141–147
145
Fig. 3. Verification of minicircles. A. operculatum DNA enriched for minicircles was cut with the restriction enzyme XmnI and probed for the minicircles in Southern analyses. Samples were run alongside PCR products from cloned minicircles, with Hyperladder 1 as a marker. Size indicators are to the right of each figure. Panel (i) was probed with the region of the 584 bp minicircle showing homology to psbC: Lane 1, minicircle DNA, arrow A indicates the psbC minicircle, arrow B the linearised 584 bp minicircle; lane 2, PCR products from cloned 584 bp minicircle; lane 3, Hyperladder 1. Panel (ii) was probed with the region of the 514 bp minicircle showing homology to empty circle 5: Lane 1, minicircle DNA, arrow C indicates a fragment of empty circle 5, arrow D indicates the linearised 514 bp minicircle; lane 2 Hyperladder 1; lane 3, PCR products from the cloned 514 bp minicircle. Panel (iii) was probed with a region common to the 405 bp, 263 bp and 23S-coding minicircles. Lane 1, minicircle DNA, arrow E indicates a fragment of the full-length 23S minicircle, arrow F indicates the linearised 405 bp minicircle; lane 2, Hyperladder 1; lane 3 PCR products from the cloned 263 bp minicircle; lane 4 PCR products from the cloned 405 bp minicircle.
minicircle by ‘jumping PCR’ across the repeated sequences described below. PCR was therefore carried out using the full-length 23S minicircle cloned into pGEM-T-Easy as a template and the CF1/CR1 original primers. This reaction gave rise to a product of approximately 250 bp (data not shown). This was cloned and found to be identical in sequence to the 263 bp minicircle. No band corresponding to the 405 bp minicircle was seen in the PCR products.
4. Discussion 4.1. Coding minicircles The results described here extend the number of minicircles characterized from A. operculatum that contain fulllength potential coding sequences. They are all of a similar size to the minicircles reported before from this species, and have the coding region in the same orientation with respect to the core region. The 23S rRNA coding region has been localized to a minicircle of 2655 bp. Sequence analysis of the core region of the full-length 23S minicircle shows that it is most similar to the core region found in the psbB, psbD/ psbE/psbI, psbC and petD minicircles. The psaB minicircle is 2363 bp in length, and contains a full-length psaB coding region of 1965 bp, and a core. The psbD, psbE and psbI genes are located on a single minicircle of 2453 bp. All three genes are in the same orientation with respect to the core region, as are atpA and petB on the two-gene minicircle (Barbrook et al., 2001). The genes are in the order psbD, psbE, psbI. The psbD and psbE genes are separated by a region of 119 bp. The psbI gene (Fig. 4) is 259 bp further
downstream from psbE. This is the first report of a threegene minicircle in the dinoflagellates. A. carterae, a closely related dinoflagellate, also carries psbD and psbE on a single minicircle (Hiller, 2001). Although the latter study did not identify the psbI gene, inspection of the reported sequence shows that psbI is in fact present, but was not obvious because the gene is short and poorly conserved. It is interesting that the psbI gene appears to be most similar to the Chlamydomonas reinhardtii one, although the significance of this is not clear. The psbD, psbE and psbI genes are not located near each other in previously sequenced plastid genomes or in cyanobacterial genomes (Reith and Munholland, 1995). This suggests the group was not generated by fragmentation of a larger molecule, unless it had a different organization from anything reported to date. It is possible that the three-gene minicircle was derived by fusion of single-gene circles, perhaps to facilitate the control of expression. Since the genes are all in the same orientation, it is possible that they are co-transcribed, although our preliminary Northern analysis suggests this is not the case in A. operculatum (unpublished data). Essentially nothing is known about ribosome binding sites on dinoflagellate minicircle genes, so it is not possible to say if there is a separate ribosome binding site for each open reading frame. 4.2. Non-coding minicircles In addition to the coding minicircles and empty circle 5, we isolated a number of small minicircles. One of these, of 584 bp, could be derived by recombination between psbB and psbC minicircles across sequences common to the two minicircles. The psbB and psbC minicircles have two
146
R.E.R. Nisbet et al. / Gene 331 (2004) 141–147
Fig. 4. ClustalX alignment of PsbI protein sequences from a variety of plants and algae. Stars (*) indicate identities, dots (.) and colons (:) similarities as defined by ClustalX. Identity and similarity designations above the alignment are shared in all sequences. Identity and similarity designations below the alignments are those shared between C. reinhardtii and A. operculatum.
regions of homology, one across the core region and the other across 18 bp of the psbC and psbB minicircles (corresponding to residues 1668– 1685 of the psbB minicircle (AJ250263) and residues 1827 –1844 of the psbC minicircle (AF426172), Fig. 2). A double recombination event across these two regions could give rise to the 584 bp minicircle. Similarly, the 514 bp minicircle could be derived by a double recombination event between the psbB minicircle and the newly reported non-coding minicircle, designated ‘empty circle 5’ (Fig. 2). The 263 and 405 bp minicircles could be derived by deletion within the 23S rRNA minicircle. The 263 bp minicircle could be derived by recombination and the consequent deletion of the internal region across a repeated sequence of GAACGATTAGGTAA found at residues 69 – 82 and 2461 –2474 of the 23S minicircle (Fig. 2). However, it may be a PCR artefact caused by the repeated sequence as no hybridization was seen in Southern anaysis. The deletion giving rise to the 405 bp minicircle starts close to this but ends at a site with no sequence similarity to it, so it cannot be explained by simple recombination (Fig. 2). Zhang et al. (1999) reported the existence of three forms of 23S rRNA minicircles. One minicircle was full length, while two contained deletions in the core region of 23 and 35 base pairs. However, neither contained deletions in the 23S coding region, nor were the deletions of the magnitude of 2 kbp. Santos et al. (2002) also found deletions of 6, 23, 208, 250 and 298 bp in the coding region of the 23S rRNA minicircle in the dinoflagellate genus Symbiodinium, with genes having one or more of these deletions present. We have not observed such small scale deletions in A. operculatum. The generation of novel minicircles by fusion of different circles and subsequent deletion has been reported before (Barbrook et al., 2001; Zhang et al., 2002). Recombination between circles could account for the generation of the 584 and 514 bp minicircles. However, the existence of such
small minicircles has not been reported before. We term these very small minicircles ‘microcircles’. This is also the first report of the generation of small minicircles by simple deletion within one minicircle species, as with the 405 bp microcircle. The Southern hybridization showed that all these molecules, with the possible exception of the 263 bp, exist in vivo, although probably at lower abundance than the conventional coding minircles. The 584, 514 and 405 bp microcircles have been found several times in different cultures of A. operculatum over a period of 18 months, suggesting that the microcircles are stable. The generation of microcircles may in some cases be associated with repeated sequences, suggesting a recombination-mediated process, but this is not invariably the case. The existence of these microcircles is reminiscent of the situation with plant mitochondria, where there are varying levels of sub-genomic molecules generated by recombination across repeated sequences (Lonsdale et al., 1984; Backert et al., 1997) and very low levels of ‘sublimons’ generated by aberrant recombination events (Small et al., 1987). It remains to be seen whether the dinoflagellate microcircles have any function, and indeed whether they are located in the same cellular compartment as the more conventional minicircles.
5. DNA sequences DNA sequences for the four minicircles and three microcircles have accession numbers AJ582639 – AJ582644 and AJ620761.
Acknowledgements We thank Roger Hiller for helpful discussions, and the Biotechnology and Biological Sciences Research Council,
R.E.R. Nisbet et al. / Gene 331 (2004) 141–147
the Cambridge European Trust and Corpus Christi College, Cambridge for financial support. References Backert, S., Nielsen, B.L., Bo¨rner, T., 1997. The mystery of the rings: structure and replication of mitochondrial genomes from higher plants. Trends Plant Sci. 2, 477 – 483. Barbrook, A.C., Howe, C.J., 2000. Minicircular plastid DNA in the dinoflagellate Amphidinium operculatum. Mol. Gen. Genet. 263, 152 – 158. Barbrook, A.C., Symington, H.A., Nisbet, R.E.R., Larkum, A., Howe, C.J., 2001. Organisation and expression of the plastid genome of the dinoflagellate Amphidinium operculatum. Mol. Gen. Genomics 266, 632 – 638. Boczar, B.A., Liston, J., Cattolico, R.A., 1991. Characterization of satellite DNA from three marine dinoflagellates (dinophyceae): Glenodinium sp. and two members of the toxic genus, Protogonyaulax. Plant Physiol. 97, 613 – 618. Gibson, T.J., 1984. Studies on the Epstein – Barr virus genome. PhD thesis, Cambridge University, UK. Hiller, R.G., 2001. ‘Empty’ minicircles and petB/atpA and psbD/psbE (cytb559 alpha) genes in tandem in Amphidinium carterae plastid DNA. FEBS Lett. 505, 449 – 452. Lonsdale, D.M., Hodge, T.P., Fauron, C.M., 1984. The physical map and organisation of the mitochondrial genome from the fertile cytoplasm of maize. Nucleic Acids Res. 12, 9249 – 9261. Martin, W., Stoebe, B., Goremykin, V., Hansmann, S., Hasegawa, M., Kowallik, K., 1998. Gene transfer to the nucleus and the evolution of chloroplasts. Nature 393, 162 – 165.
147
Reith, M., Munholland, J., 1995. Complete nucleotide sequence of the Porphyra purpurea chloroplast genome. Plant Mol. Biol. Rep. 13, 333 – 335. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York. Santos, S.R., Taylor, D.J., Kinizie III, R.A., Sakai, K., Coffroth, M.A., 2002. Molecular phylogeny of symbiotic dinoflagellates inferred from partial chloroplast large subunit (23S)-rDNA sequences. Mol. Phylogenet. Evol. 23, 97 – 111. Small, I., Suffolk, R., Leaver, C.J., 1987. Stoichiometric differences in DNA-molecules containing the atpA gene suggest mechanisms for the generation of mitochondrial genome diversity in maize. EMBO J. 6, 865 – 869. Stoebe, B., Kowallik, K.V., 1999. Gene-cluster analysis in chloroplast genomics. Trends Genet. 15, 344 – 347. Takishita, K., Ishikura, M., Koike, K., Maruyama, T., 2003. Comparison of phylogenies based on nuclear-encoded SSU rDNA and plastid-encoded psbA in the symbiotic dinoflagellate genus Symbiodinium. Phycologia 42, 285 – 291. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876 – 4882. Zhang, Z., Green, B.R., Cavalier-Smith, T., 1999. Single gene circles in dinoflagellate chloroplast genomes. Nature 400, 155 – 159. Zhang, Z., Cavalier-Smith, T., Green, B.R., 2002. Evolution of dinoflagellate unigenic minicircles and the partially concerted divergence of their putative replicon origins. Mol. Biol. Evol. 19, 489 – 500.