Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada
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Abstract |
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Introduction |
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Here we show that in addition to normal functional minicircles, Heterocapsa triquetra contains a family of related, probably nonfunctional minicircular chromosomes carrying fragments of chloroplast genes, and we present complete sequences of five such nongenic minicircles. Each minicircle consists of jumbled chloroplast gene fragments derived from four separate unigenic circles and the very conserved tripartite 9G-9A-9G region. Plasmid-like DNA has previously been found in algal chloroplasts but has not been well characterized: in the diatom Cylindrotheca fusiformis (Hildebrand et al. 1992
; Jacobs et al. 1992
) and the green alga Acetabularia (Ebert, Tymms, and Schweiger 1985
), homology between parts of the plasmid sequence and chloroplast DNA has been shown by hybridization. The linear plasmids of the green alga Ernodesmis verticillata are localized around the chloroplast pyrenoid (La Claire and Wang 2000a
) and have a unique hairpin-like structure (La Claire and Wang 2000b
) but appear to contain only fragments of chloroplast genes (La Claire et el. 1998
).
In the case of the aberrant H. triquetra minicircles we report here, it is unlikely that the fragments of chloroplast genes encode functional polypeptides, so the aberrant minicircles are probably nonfunctional selfish DNA like that widespread in nuclear genomes (Doolittle and Sapienza 1980
; Orgel and Crick 1980
). The very conserved tripartite 9G-9A-9G region of these minicircles may predispose them to form heterodimers by homologous recombination at these sites. We propose a model for the origin of all five circles from ancestral heterodimeric circles containing fragments of four genespsbA, psbC, and 16S rRNA and 23S rRNA genesfollowed by numerous deletions and some duplications.
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Materials and Methods |
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DNA Sequencing and Sequence Assembly
Plasmid DNA preparation and the sequencing were as described (Zhang, Green, and Cavalier-Smith 1999
). Sequencing reactions used the Perkin-Elmer GeneAmp 9600 with the ABI cycle sequencing protocol: 94°C for 5 s, 50°C for 5 s, and 60°C for 4 min for 25 cycles. Each reaction contained 23 µl FS-Taq or Bigdye, 2030 ng DNA (purified PCR product) or 100150 ng DNA (plasmids), 35 pmol primers, and distilled water to 10 µl. The sequencing samples were precipitated by adding 1/10 volume 3 M sodium acetate (pH 5.2) and 2 volumes 95% ethanol, quenched on ice for 10 min, centrifuged for 20 min, air-dried, and analyzed by an ABI 373 or 377 automatic sequencer. Sequences were imported into Gap 4 of Staden and edited using Trev; contigs were generated from overlapped clone sequences using the options "shotgun assembly" and "find internal joins" of Gap 4 (http://www.mrc-lmb.cam.ac.uk/pubseq).
PCR Reactions
Specific primers to verify sequences and circularity were based on the sequences of normal H. triquetra psbA, psbC, 16S rRNA, and 23S rRNA genes. PCR reactions were for 35 cycles of 94°C for 30 s and 55°C for 30 s followed by 2 min at 72°C in a GeneAmp PCR system 9600 (Perkin-Elmer). Each reaction (50 µl) contained 0.2 mM dNTP, 1 x PCR buffer, 0.11.0 µg template DNA, 50200 pmol primers, 2.0 or 2.5 mM MgCl2, and 1.52.5 U Taq polymerase (Sigma or Rose). PCR products were purified from 1% agarose gels by a purification kit (Amersham-Pharmacia Biotech) and sequenced directly. Sequences of PCR products were integrated into appropriate contigs as above.
BLAST Searches
NCBI's sequence similarity search tool BLAST (http://www.ncbi.nlm.nih.gov/BLAST) was used to analyze DNA sequences. Each sequence was compared against the entire GenBank database, which included the nine unigenic circles; homologs of these sequences were further characterized by carefully examining multiple alignments of related regions using the Staden package (http://www.mrc-lmb.cam.ac.uk/pubseq).
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Results |
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Each gene fragment on an aberrant circle has the same orientation with respect to the 9G-9A-9G region as its homologous segment on the normal unigenic circle (fig. 1 and table 1 ). This regularity would not have been found had the fragments been generated artificially and randomly ligated during cloning, but it is exactly what would be expected if they evolved in vivo from naturally chimeric molecules through many deletions. The patterns of sequence identity shared between the aberrant and normal circles are complex and are shown in figure 1b. Comparison of the gene fragments with normal circles showed that all originate from only five regions: A (720 bp) and B (399 bp) of the 23S rRNA circle, C (181 bp) of 16S rRNA, D (322 bp) of psbA, and E (361 bp) of psbC circles. Fragments homologous to each region are present in at least three circles, often with identical boundaries indicative of a common origin (table 1 ); e.g., fragments from the A, B, and C regions are on circles 1, 2, and 3, fragments from the E region are on circles 3, 4, and 5, and fragments from the D region are on circles 1, 2, 4, and 5.
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Not only the gene fragments but also their arrangement are strikingly shared between the five circles, apart from many insertions and deletions, suggesting that similar chimeric regions from the separate circles had a common evolutionary origin. The observed pattern makes it highly improbable that the circles were assembled directly from the gene fragments; instead, almost all regions outside of the 9G-9A-9G region can be derived from just five ancestral segments, each with three, four, or five gene fragments. Those segments shown by the same Roman numerals in figure 1a are essentially identical between different circles: segment I is shared by circles 1 and 2 (part of it also occurs in circles 35), segment II is shared by circles 2 and 3, and segment IV is shared by circles 35. This is true not only of genic regions shared with the unigenic circles, but also of the intervening "nongenic" regions (e.g., S2; see below).
Deletions and Duplications Within Fragments
All five sets of homologous gene fragments have suffered deletions and duplications relative to the putatively ancestral single-gene circles (fig. 1
). For example, region A or segment 23S(5), starting from *9G to 9GL of circle 1 (fig. 1a
), has three gaps of 10, 5, and 23 bp compared with the 23S rRNA gene (fig. 1b
), and there are also two 116-bp repeats, with a deletion of 24 bp in the second ( in figs. 1b and 2a
). Segment 23S(5) is 98% identical to the corresponding segment of the complete 23S rRNA gene apart from gaps and duplications (table 1
). Fragment 23S(6) (337 bp) on circle 2 is 99% identical to the B region of the 23S rRNA gene and has three 29-bp repeats, the second with an internal repeat of 6 bp (ß,
in figs. 1b and 2b
). Similarly, fragments homologous to region B on circle 3 have a gap of 26 bp, two 39-bp repeats (
in fig. 1b
), and two repeats of 25 bp (
in fig. 1b
).
Segments IV, IV', and IV'' (fig. 1a
) are each essentially a chimera of the long nongenic region (S2; see below) and region E of the psbC minicircle that encodes part of the fifth transmembrane helix and the following loop region of the PsbC protein (fig. 2c
). It seems that this region, with the central part of psbC juxtaposed to a noncoding region of unknown origin, had already evolved prior to the divergence of circles 35 from a common ancestor. Sequences of psbC fragments on circles 35 are identical to the E region of the normal psbC gene apart from indels (figs. 1 and 2c
). The psbC fragment on circle 3 has a 165-bp deletion; psbC fragments on circles 4 and 5 have two and three 33-bp repeats respectively, which encode 11 amino acids of PsbC protein ( in figs. 1b and 2c
). Sequence comparison showed an insertion of 11 bp at the 40th bp from the 5' end of the psbC fragments on the three circles (arrow in fig. 1b
). The translation products of the psbC segments on the three circles gave two significant BLAST hits on PsbC protein: a small peptide of 13 amino acids and, in a different reading frame, a large peptide of 51, 117, and 128 amino acids on circles 3, 4, and 5, respectively (fig. 2c
); all peptides have a potential ATG or ATA start and TAA stop.
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Nongenic Regions
We have previously shown (Zhang, Green, and Cavalier-Smith 1999
) that the 9G-9A-9G noncoding region on the nine unigenic circles consists of three very conserved regions, the 9GL (135 bp), 9A (188 bp), and 9GR (135 bp) cores (named after their central mononucleotide tracts), with less conserved D2 and D3 intervening regions (fig. 1
). The cores are almost identical among all normal gene circles, and the 9GL and 9GR sequences are highly related to each other. The 9G-9A-9G regions on the aberrant circles are very closely related to those of the normal single-gene circles. The 9G cores share 108 identical base pairs with those of the normal circles, while the 9A cores differ by very few substitutions. However, the 9GL sequences of circles 24 have several mutations and are the most divergent of all 9GL sequences. The presumed 9GR of circle 3 is a core with a run of 8G's instead of 9G's; sequence alignment indicated that the 8G core was highly related to a conventional 9GR core except for a few base substitutions.
A notable feature of circles 13 is that each has an extra 9G core segment lying between other gene fragments instead of within a tripartite core region (fig. 1a ). Sequence comparison showed that the extra 9G of circle 1 is identical to the 9GR of the 23S rRNA circle, and the extra 9G of circle 2 is identical to the 9GR of the 16S rRNA and psbC circles except for two base substitutions. On circle 1, apart from the two small indels, the A region of 791 bp (23S(5)) is 98% identical to the 9GR region and the following region of the normal 23S rRNA gene circle, supporting the idea that this whole segment originated from a 23S rRNA circle. However, the extra 9G region of circle 3 (#9G in fig. 1a ) is identical to the 9GL of circles 1 and 5, as well as to that of the four normal chloroplast gene circles, except for three base substitutions. Therefore, the extra 9G regions on circles 13 may have originated independently from the 9GR or 9GL regions of different minicircles. Overall, none of the 9G-9A-9G regions of the chimeric circles are identical to each other or to any of the putatively ancestral single gene circles, although they are all very closely related to each other.
Shared variants indicative of gene conversion were found in the D2 and D3 regions of normal gene minicircles (Zhang, Green, and Cavalier-Smith 1999
), and similar shared sequences were found in the five chimeric minicircles (fig. 3
). The D2 region of circles 3 and 4 share 148 bp containing two 41-bp repeats with a few substitutions, making it longer than that of other minicircles; 65 of the 148 bp shared by circles 3 and 4 are present in circle 2. Gene conversions were also found between circle 1 and the 23S rRNA circles (20 bp) and between circle 5 and the 16S rRNA circle (30 bp) (fig. 3
). The D3 and D4 regions (downstream of 9GR) are more conserved than the D2 region among both chimeric and unigenic circles.
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The S2 nongenic region present on circles 25 is 200400 nt long (236, 307, 422, and 499 bp on circles 25). Sequence analysis showed that S2 on each circle consisted of four motifs (fig. 4 ), one (the 19-bp motif 3) present in variable numbers, accounting for the length difference. Motifs 1 and 2 lack homologs in the databases. Motif 1 is almost identical on circles 35 but is totally different from motif 1 on circle 2 (fig. 4a ). The sequences of motifs 24 are almost identical among the four circles except for several deletions (3, 1, and 6 bp) and substitutions on circle 2 (fig. 4 ). Motif 4 is immediately upstream of 9GL; its sequence is homologous to the sequence upstream of 9GL on circle 1 and the 16S and 23S rRNA and psbA unigenic circles (fig. 4b ). It is also related to the sequence upstream of the extra 9G of circles 13 (fig. 4b ). The similarities of nongenic regions among the aberrant circles show that they did not originate independently. Both chloroplast gene fragments and nongenic regions on the aberrant circles must be evolutionarily related, as is particularly clearly indicated by the sharing of segment IV (psbC plus S2) by circles 3, 4, and 5.
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Discussion |
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It is most improbable that any of the gene fragments on these circles are functional. Most of the protein gene fragments lack start codons or have internal stop codons. It would be difficult to detect transcription of these fragments against the background of transcription from normal single-gene circles, which give products of the length expected for monocistronic transcripts (Zhang, Green, and Cavalier-Smith 1999
), but it is hard to imagine that fragmentary genes would be functional in the presence of the corresponding complete genes on the unigenic circles. The coexistence of normal and fragmentary genes in H. triquetra chloroplasts is not comparable to the fragmented mitochondrial rRNA genes in the green alga Chlamydomonas (Nedelcu and Lee 1998
) and in apicomplexans (Wilson and Williamson 1997
), where the normal full-length genes no longer exist in the mitochondrial genomes.
The great conservation of the 9G-9A-9G regions, in marked contrast to the fragmented and jumbled gene fragments of these chimeric circles, strongly suggests that such conservation is essential for their replication and persistence. This conservation is probably a sufficient explanation for their replication and transmission from generation to generation, even if the jumbled "coding" regions are entirely devoid of function. If so, then these tiny circles are genetic parasites of the chloroplast replication machinery, and it is reasonable to regard them as selfish DNA (Doolittle and Sapienza 1980
; Orgel and Crick 1980
). The small size of both the functional unigenic circles and the aberrant circles, along with the high frequency of deletions in the latter, suggests that both kinds of circles are under strong selection for small size and may imply that smaller molecules have a replicative advantage over larger ones in dinoflagellate chloroplasts.
This conservation of the noncoding 9G-9A-9G regions of the five selfish circles provides a very strong argument that they must be located in the same cell compartment as the functional minicircles. We know that the noncoding regions diverge exceedingly rapidly and are totally different between different species of the same genus (Zhang, Green, Cavalier-Smith 1999
). This means that the 9G-9A-9G regions of both the normal circles and the chimeric selfish circles must be being kept similar in both length and sequence by a combination of gene conversion and positive selection. Positive selection for retaining the 9G-9A-9G regions in both kinds of molecules is likely to arise because of their interactions with the replication initiation machinery and/or the DNA segregation machinery, with which they must coevolve. Both kinds of machinery must be very different between chloroplasts and other cell compartments, such as the nucleus or the mitochondria. Because of this, and because gene conversion between normal and selfish circles could occur only if they were in the same compartment, we conclude that the five selfish circles must have been present in the same compartment as the normal circles for most, and probably all, of their evolutionary history during which they diverged so radically from each other and from the normal circles. As explained in the introduction, the arguments for the chloroplast location of the normal minicircles are very strong. Therefore, although direct evidence is lacking for either, it is almost certain that both kinds of minicircles are actually located within the chloroplasts.
Origin of Chimeric Circles by Recombination and Multiple Deletions
It is clear from our data that all five circles are related and carry gene fragments of four nonhomologous genes that originated from four separate unigenic circles. We suggest that the sharing of the 9G-9A-9G regions by all of the unigenic circles in H. triquetra, kept similar by gene conversion, predisposes normal circles to form dimers by homologous recombination between their almost-identical 9G-9A-9G regions. Normally, such dimers (both homodimers and heterodimers) would be resolved into separate unigenic circles by a DNA topoisomerase II, but if by chance one 9G-9A-9G region were accidentally deleted before this happened, the dimer could persist for a while. Given the apparent selection for smaller DNA circles in dinoflagellate chloroplasts, variants with partial deletions would remain longer, perhaps indefinitely if they were smaller than unigenic circles. Such chimeric circles could also undergo recombination with other circles to form higher-order chimeras bearing more gene fragments.
We propose a model (fig. 5b
) where a heterodimer of psbA and psbC minicircles was formed as just suggested and stabilized and compressed by one or more large deletions including a 9G-9A-9G region (e.g., double-headed arrow in fig.5b
). This compressed psbA/psbC dimer could have given rise directly to circles 4 and 5 with more small deletions and small duplications of the D4 region of psbA (fig. 1b
) and a duplication of 33 bp within the psbC fragments (). Circle 5 would have had a further duplication of 63 bp near the N terminus of the psbA gene (
). A recombinant 23S/16S rRNA minicircle might have been formed in a similar fashion. However, this mechanism would not have placed a 16S gene fragment between two 23S rRNA fragments, as is observed in circle 3. Therefore, it is more likely that a 16S/23S rRNA heterodimer was generated by illegitimate recombination (fig. 5a
) at the TTC sites found in both molecules at the exact point of the present 16S/23S chimeric boundary. The 3' end of the 16S fragment (TTC) could have formed a heteroduplex with the TTC of the 23S gene at the integration site (table 1
). Following deletion of most of the 16S circle, this chimeric 16S/23S rRNA circle (fig. 5a
) could then have recombined homologously with the compressed psbA/psbC dimer at the 9G-9A-9G region to yield an intermediate circle with all five gene regions (AE). Further deletions and duplications of this multiply chimeric circle could have generated circles 13. Deletions and duplications in other regions, such as D2 and S2, possibly stabilized these circles at an appropriate size (
2 kb).
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Gene Conversion Between Aberrant and Normal Minicircles
Interpretation of the phylogenetic origins of the extra 9G region on the aberrant minicircles is complicated by the prevalence of gene conversion among dinoflagellate minicircles. The 9G-9A-9G regions are strongly conserved among the genic minicircles and provide compelling evidence for extensive gene conversion between 9G-9A-9G regions of different gene circles (Zhang, Green, and Cavalier-Smith 1999
). All the patterns of identity or near identity between different parts of the 9G-9A-9G regions of the chimeric circles can also be explained as the result of mutation and gene conversion, making gene conversion equally important in the evolution of the chimeric circles.
Functional Implications
The 9G-9A-9G region was proposed to be the replication origin based on its high degree of conservation across all single gene circles and its propensity to fold into complicated hairpin structures (Zhang, Green, and Cavalier-Smith 1999
). The fact that only this region is also well conserved in the selfish circles strongly supports this proposal. We argued above that a circle with two 9G-9A-9G regions would be quickly resolved into two separate ones. It is interesting that three of the five selfish circles have an extra 9G core, indicating that partial deletion of the 9G-9A-9G region is sufficient to stabilize a dimer. It is tempting to suggest that the 9A region, which would more easily undergo strand separation, is at the heart of the replicon origin and that the extra 9G core fragment of circles 13 does not function in replication.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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1 Present address: Department of Plant Biology, Carnegie Institution,
Stanford University.
2 Present address: Department of Zoology, University of Oxford,
Oxford, England.
1 Keywords: dinoflagellate
Heterocapsa triquetra,
chloroplast DNA minicircles
gene fragments
selfish DNA
2 Address for correspondence and reprints: Beverley R. Green, Department
of Botany, University of British Columbia, #3529-6270 University
Boulevard, Vancouver, British Columbia, Canada V6T 1Z4.
brgreen{at}interchange.ubc.ca
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References |
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