*Department of Biochemistry and Molecular Biology, Dalhousie University;
Bigelow Laboratory for Ocean Sciences
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Abstract |
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Introduction |
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The sequencing of jakobid mitochondrial genomes has provided startling insight into mitochondrial evolution and has further suggested a pivotal role for the jakobids in our understanding of the evolution of eukaryotes. These organisms possess the most bacterial-like mitochondrial genomes characterized thus far (Lang et al. 1997
; Gray et al. 1998
; Gray, Burger, and Lang 1999
). Most striking is the mitochondrial DNA (mtDNA) of Reclinomonas americana, which encodes 97 genes, more than are present in any other mitochondrial genome. Several of these have never before been found encoded in mtDNA, including a gene for a bacterial translation factor (tufA), a putative cytochrome oxidase assembly protein (cox11), a secretion pathway protein (secY), and genes for four subunits of a bacterial-type RNA polymerase (rpoA-D; single subunit phage-type RNA polymerases are thought to function in all other known mitochondria) (Lang et al. 1997
; Gray et al. 1998
). Operon-like ribosomal protein gene clusters similar to those found in bacteria are also present.
More recently, the jakobids have been considered members of a much larger assemblage of protists, the excavate taxa. The excavates are a diverse group of amitochondriate, mitochondriate, and hydrogenosomal lineages that share as their uniting feature the presence of a ventral feeding groove (Patterson, Simpson, and Weerakoon 1999
; Simpson and Patterson 1999
). In addition to the jakobids (Jakoba, Reclinomonas, and Histiona) and the jakobid-like nanoflagellate Malawimonas (O'Kelly and Nerad 1999
), the excavates include the heteroloboseans, diplomonads, retortamonads, Trimastix, and Carpediemonas (Simpson and Patterson 1999
). Whereas ultrastructural data suggest that these organisms share a common excavate (i.e., feeding groove bearing) ancestor, there is no consensus view on the relationships amongst the various excavate taxa or their relationship to other mitochondriate, amitochondriate, and hydorogenosome-containing groups. Indeed, current views on the origin and evolution of eukaryotes are in a state of flux. Many of the putatively deep-branching and primitively amitochondriate protist lineages (including some of the excavate taxa) are now thought to be derived from mitochondrion-bearing ancestors (see Roger 1999
for recent review). Further, the ability of current phylogenetic methods to accurately reconstruct the deepest branches of phylogenetic trees has come into question (Hirt et al. 1999
; Stiller and Hall 1999
; Philippe and Germot 2000
, and references therein). Philippe and Adoutte (1998)
suggested that a big bang occurred at the base of eukaryotes and that the major cladogenetic events in eukaryotic evolution occurred in quick succession. Thus, there is at present no clear picture as to which protist groupsif anyactually represent early diverging lineages.
We are studying the evolution of chaperonins in diverse amitochondriate and mitochondriate protists. Group II chaperonins, a class of molecular chaperone found in Archaea and the eukaryotic cytosol, are double-ring protein complexes that mediate the proper folding of nascent or denatured proteins (for review see Willison and Horwich 1996
). The eukaryotic cytosolic chaperonin complex (CCT or TriC) is best known for its role in mediating the proper folding of the cytoskeletal proteins actin and tubulin (Willison and Kubota 1994
; Kubota, Hynes, and Willison 1995
). Recent experiments suggest that a large number of newly translated proteins may also interact with CCT (Melki et al. 1997
; Thulasiraman, Yang, and Frydman 1999
). Unlike bacterial and organellar chaperonins, which are usually homo-oligomeric, the eukaryotic CCT complex contains eight different homologous subunits, alpha, beta, gamma, delta, epsilon, zeta, eta, and theta (Willison and Horwich 1996
). The duplications producing the different CCT genes are known to have occurred prior to the divergence of the parabasalids and diplomonads from other eukaryotes (Archibald, Logsdon, and Doolittle 2000
). Individual CCT subunits are over 500 amino acids in length and are highly conserved, both desirable properties for a phylogenetic marker. The presence of eight distinct CCT paralogs makes it possible to perform multiple reciprocal rootings of the phylogenetic tree of eukaryotes. Unlike SSUrRNA, EF-1
, actin, and tubulin, the CCTs are relatively poorly sampled. Focusing on the alpha subunit of CCT, we therefore sought to broaden the diversity of taxonomic representation and add to the Trichomonas vaginalis and Giardia lamblia CCTalpha's sequenced in a recent study (Archibald, Logsdon, and Doolittle 2000
).
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Materials and Methods |
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Cloning and Sequencing of CCT Genes
Degenerate PCR primers were used to amplify CCT genes from gDNA. A combination of universal chaperonin primers (Archibald, Logsdon, and Doolittle 2000
) and CCTalpha-specific primers were used to amplify the CCTalpha gene (forward primers: CCT-2-for [5'-AACGACGGTGCNACNATHYT-3'], CCT-9-for [5'-CCAGTCGGTCTNGAYAARATG-3']; reverse primers: CCT-4-rev [5'-CTCTACAGCNCCNSCNCC-3'], CCT-10-rev [5'-TGATCAGRTCRTCDATNC-3'], CCT-11-rev [5'-AGGTCGTCGATGCKNARDAT-3'], TF-9-rev [GCAGCTATCARRTCRTCDAT-3']). With most primer combinations, 90%95% of the gene was amplified. The CCTdelta gene was amplified with CCT-9-for (above) and CCT-7-rev [5'-ACGATGCACATNGHRTCRTG-3'].
PCR reactions were carried out on an MJ Research Inc. PTC-100 thermal cycler using GIBCO-BRL Taq polymerase, buffer, and dNTP. The PCR reactions performed with R. americana and M. jakobiformis DNAs contained 5% acetamide (final concentration). Following an initial denaturation of 3 min at 92°C, reactions were performed with 4045 cycles of 92°C for 15 s, 5054°C for 30 s, and 72°C for 3 min. PCR products of the expected size were cloned directly from low-melt agarose with the TOPO-TA cloning kit (Invitrogen). Clones were screened for the presence of inserts by EcoRI restriction of isolated plasmid DNAs or by PCR-screening directly from E. coli cells with the M13 universal forward and reverse primers. Sequencing was performed manually (T7 sequencing kit, Pharmacia) and with LiCor and ABI automated sequencers.
CCT sequences from Plasmodium falciparum were identified by BLAST (Altschul et al. 1990
) at the PlasmoDB website (http://www.plasmodb.org/). CCTalpha was obtained from chromosome 11 sequence, whereas CCTdelta was found in genomic sequence from chromosome 13. Preliminary sequence data for P. falciparum chromosomes 10 and 11 was obtained from The Institute for Genomic Research website (www.tigr.org). Sequencing of chromosomes 10 and 11 was part of the International Malaria Genome Sequencing Project and was supported by award from the National Institute of Allergy and Infectious Diseases, National Institutes of Health. Sequence data for P. falciparum chromosome 13 was obtained from The Sanger Centre website at http://www.sanger.ac.uk/Projects/P_falciparum/. Sequencing of P. falciparum chromosome 13 was accomplished as part of the Malaria Genome Project with support by The Wellcome Trust.
For T. brucei, a small fragment of the CCTalpha gene was found by searching genomic data from unfinished microbial genomes. Preliminary sequence data was obtained from The Institute for Genomic Research website at http://www.tigr.org. Based on this sequence an exact-match reverse primer (Tbru.CCTa.R-1 [5'-GTTAAGGCGAACACAGTCAT-3']) was designed and used in combination with degenerate forward primers (CCT-2-for, CCT-9-for; above) to amplify most of the CCTalpha coding sequence from T. brucei gDNA. From the sequence of five independent clones, two different CCTalpha clones were apparent, one of which matched the original fragment present in the T. brucei genome data. The ambiguities between the two clone types were almost all synonymous substitutions, suggesting the presence of multiple copies of the CCTalpha gene in T. brucei (see Results). The sequences presented in this study have been deposited in GenBank under the following accession numbers: AF322043AF322050.
Southern Hybridization
To confirm the source and copy number of the CCT genes, PCR products obtained from R. americana (ATCC number 50394) and M. jakobiformis (ATCC number 50310) were isolated (BIORAD, Prep-a-gene), labeled with 32P (Prime-It II random primer labeling kit, Stratagene), and hybridized to restricted gDNA.
Sequence Characterization and Phylogeny
A combination of BLAST and DNA Strider (Douglas 1995
) was used to determine the positions of introns in the new CCT genes and in CCTs retrieved from the public databases. Inferred amino acid sequences from the Reclinomonas, Malawimonas, heterolobosean, Trypanosoma and Monocercomonas CCTs were added manually, based on globally conserved regions, to an alignment constructed previously (Archibald, Logsdon, and Doolittle 2000
). From this master data set, smaller alignments containing subsets of the data were constructed. Each alignment was assessed individually and regions of ambiguity were removed to ensure that only confidently aligned amino acid positions were used for phylogenetic reconstruction. Gaps for missing data (e.g., the extreme N- and C-termini missing from PCR-generated sequences) were also removed. The complete CCTalpha-only alignment contained 26 taxa and 424 unambiguously aligned amino acid positions. The data sets for rooted analyses contained 23 CCTalpha sequences (three highly similar mammalian CCTs were removed) and between five and eight outgroup sequences chosen for maximal taxonomic diversity. These data sets contained the following number of sites: CCTalpha-beta381 sites, CCTalpha-gamma360 sites, CCTalpha-delta378 sites, CCTalpha-eta369 sites, CCTalpha-epsilon351 sites, CCTalpha-theta334 sites, and CCTalpha-zeta357 sites. All alignments are available from J.M.A. upon request (jarch@interchange.ubc.ca).
Phylogenetic trees were inferred from amino acid sequences using maximum likelihood (ML) and ML-distance methods of tree reconstruction. ML analyses were performed with the following programs: proML in PHYLIP 3.6 (http://evolution.genetics.washington.edu/phylip.html), using the Dayhoff amino acid substitution matrix, the global rearrangements option, a randomized sequence input order (10 jumbles), and an among-site rate variation (ASRV) model with eight rate categories plus an invariable rate category (the relative rates for each category were estimated in PUZZLE 4.02 [Strimmer and von Haeseler 1997
]); protML (using the JTT-F amino acid substitution matrix) in MOLPHY (Adachi and Hasegawa 1996
); quartet puzzling in PUZZLE 4.02, accounting for ASRV with an eight rate-category discrete approximation to the
distribution plus an invariable rate category. ML-distance trees were inferred from
-corrected distance matrices calculated in PUZZLE 4.02 using FITCH (with global rearrangements) in PHYLIP, version 3.57 (Felsenstein 1993
). Support for proML trees was obtained by bootstrapping (100 replicates). For protML trees, RELL values (obtained from quick-add searches of the best 1,000 or 2,000 trees in protML options -q -n 1,000, 2,000; Adachi and Hasegawa 1996
) were used as measures of statistical support. Support values for ML-distance trees were obtained by bootstrapping (500 replicates) with PUZZLEBOOT 1.02 (A. Roger and M. Holder; http://members.tripod.de/korbi/puzzle/). PUZZLE was used to statistically assess the significance of different tree topologies using the Kishino-Hasegawa test (Kishino and Hasegawa 1989
).
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Results |
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The jakobid-malawimonad CCT genes presented here are among the first nuclear protein-coding genes to be characterized from these protists. The most striking feature of the R. americana and M. jakobiformis CCTs is the presence of multiple spliceosomal introns. The CCTalpha gene from R. americana possessed five introns (ranging from 67 to 145 nt in length, with some size heterogeneity between homologous introns in the two strains), whereas the M. jakobiformis CCTalpha contained seven introns (58127 nt long). The M. jakobiformis CCTdelta gene possessed seven introns between 61 and 73 nt in length, despite being a shorter fragment of coding sequence than that obtained for CCTalpha (1 kb of ORF). When the putative introns were removed, the inferred protein sequences were readily alignable with orthologs from a wide range of other eukaryotes, with no size heterogeneity at the intron-exon boundaries. All the introns possessed standard 5'-GT...AG-3' intron boundaries, with the exception of a single intron in one of the two CCTdelta clones from M. jakobiformis, which possessed a CT at the 5' intron-exon boundary. It is unclear whether this represents a PCR-generated artifact or a legitimate noncanonical 5' intron boundary. A summary of the sizes and positions of the introns found in the R. americana and M. jakobiformis CCT genes is shown in figure 1
. With the exception of a single intron in the A. rosea CCTalpha, none of the nonjakobid-nonmalawimonad CCTalpha genes sequenced in this study contained introns.
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CCTalpha Phylogeny
In an attempt to determine the relationships between the jakobid and malawimonad flagellates and their relationship to other eukaryotes, we performed phylogenetic analyses on the CCTalpha data set. We also performed multiple rooted analyses using the seven other CCT paralogs to root the CCTalpha tree. Figure 2
shows a ML-distance phylogeny inferred from CCTalpha protein sequences. Interestingly, the jakobid R. americana and the jakobid-like M. jakobiformis showed no affinity for one another in the CCTalpha tree. Whereas the phylogenetic position of M. jakobiformis with respect to the other protists was quite unstable, R. americana consistently (but weakly) branched with the two heteroloboseans (as in fig. 2
) and only occasionally with T. brucei (in proML analyses; data not shown). In many ways, the CCTalpha topology is similar to those obtained with more widely used phylogenetic markers, such as SSUrRNA, actin, EF-1, and alpha- and beta-tubulin. For example, the sisterhood of animals and fungi, strongly supported by an ever-increasing wealth of molecular data (see Baldauf 1999
for recent review) is moderately well supported with all phylogenetic methods. Also, the single mycetozoan representative in the CCTalpha data set, Dictyostelium discoideum, branches weakly but consistently at the base of animals and fungi, in line with suggestions that the Mycetozoa are an outgroup to the animal-fungal clade (Baldauf 1999
). The alveolates, represented in our data set by the ciliate Tetrahymena pyriformis and the apicomplexan parasite P. falciparum, receive reasonable support as a monophyletic grouping, and as expected, the two heterolobosean sequences (A. rosea and Nagleria gruberi) branch together with strong support and with all phylogenetic methods. The parabasalid CCTalphas (Monocercomonas sp., sequenced here and T. vaginalis, sequenced previously; Archibald, Logsdon, and Doolittle 2000
) are characterized by extremely long branches, similar to phylogenies constructed with other molecules.
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Given the general lack of resolution among the protists in the CCTalpha phylogeny, we examined the significance of alternate topologies using the Kishino-Hasegawa test (Kishino and Hasegawa 1989
), in particular, the relative positions of the R. americana and M. jakobiformis sequences. Interestingly, trees in which R. americana and M. jakobiformis were specific sister groups were found to be worse than the topology shown in figure 2
at a 5% level of significance, as were trees in which M. jakobiformis was placed as an outgroup to the R. americana-Heterolobosea-Giardia-Trypanosoma clade. We also tested the effect of removing various long-branch taxa on the support for the topology obtained with the full CCTalpha data set. When the parabasalids were removed, the overall topology was the same, except for the placement of M. jakobiformis, which moved to a position adjacent to the alveolates (data not shown). Interestingly, the support for the R. americana-Heterolobosea grouping increased with most phylogenetic methods, as did the support for Giardia branching with T. brucei (data not shown).
When CCTalpha was rooted with each of the seven other CCT paralogs, different topologies were often obtained from different data sets and from the same data set analyzed with different phylogenetic methods. However, the parabasalids were usually the deepest branch in the CCTalpha tree, and the alveolates were often the next deepest branch (sometimes as a paraphyletic group). Figure 3A shows the CCTalpha-theta tree, which represents the most commonly obtained topology with the various methods and data sets. Significantly, G. lamblia was not attached to the long branch of the outgroup in most analyses but grouped with T. brucei. Overall, the internal topology was very similar to that obtained in the unrooted analyses (fig. 2 ). Figure 3B shows the CCTalpha-beta tree, where the G. lamblia sequence branches near the base of CCTalpha, and R. americana branches with T. brucei and not with the heteroloboseans.
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Discussion |
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The weak but consistent affinity of R. americana for the heteroloboseans and euglenozoa in CCTalpha phylogenies (figs. 2, 3A, and 3B
) is interesting for two reasons. First, it is consistent with beta-tubulin and combined alpha-betatubulin trees (Edgcomb et al. 2001
). Second, it is consistent with mitochondrial gene-content data. The mtDNA of the heterolobosean N. gruberi contains (in addition to many genes shared with plant and protist mtDNAs) two genes thus far only identified in jakobid mitochondrial genomes (atp3 and cox11; M. W. Gray, personal communication; http://megasun.bch.umontreal.ca/ca.ogmp/projects/ngrub/gen.html). Whereas the grouping of R. americana with the Heterolobosea is consistent with beta-tubulin and combined alpha-betatubulin phylogenies (Edgcomb et al. 2001
) and mitochondrial gene-content data, it is at odds with mitochondrial cristal morphology. Like N. gruberi and the euglenozoan T. brucei, M. jakobiformis possesses discoidal cristae, yet it is R. americana, which possesses tubular mitochondrial cristae, that shows the most affinity for the Heterolobosea and T. brucei in the CCTalpha and tubulin trees. Whereas cristal morphology has often been taken to be an evolutionarily stable character, these data suggest that it may not always be reliable for tracking the deepest divisions in eukaryotic evolution. More generally, the significance of the retention of ancestral features in jakobid and malawimonad mtDNAs, in terms of their relationship to other eukaryotes, is as yet unclear. The M. jakobiformis mtDNA possesses three genes (rpl18, 19, 31) that have thus far not been observed in nonjakobid protist mtDNAs (http://megasun.bch.umontreal.ca/ogmp/projects/mjako/gen.html), yet all phylogenetic analyses performed thus far on both mitochondrial and nuclear genes suggest that M. jakobiformis and the jakobids do not form a monophyletic group.
On the whole, CCTalpha phylogenies show a great deal of congruence with more commonly used phylogenetic markers: animals and fungi appear to be each other's closest relatives, the mycetozoan Dictyostelium appears as an immediate outgroup to the animal-fungal clade, and the alveolates and Heterolobosea are monopyletic groups. Most interesting was the placement of the diplomonad G. lamblia. Almost without exception, rooted SSUrRNA and protein phylogenies place diplomonads at or near the base of the eukaryotic tree (Roger 1999
). In most rooted analyses of CCTalpha, Giardia was not positioned at the base of the eukaryotic tree but was nested within mitochondrion-containing groups. This is consistent with the suggestion that diplomonads have lost their mitochondria secondarily (Roger et al. 1998
; Roger 1999
). The analyses presented here illustrate the problem with deep phylogeny, long-branch attraction (e.g., Germot and Philippe 1999
; Stiller and Hall 1999
; Philippe and Germot 2000
). It is clear that the two parabasalids in the CCTalpha data set have the longest branches of all the taxathey also emerge consistently at the base of the CCT tree in rooted analyses. For this reason, the deepest branches of the rooted CCTalpha phylogenies presented here should be viewed with caution.
The high density of introns in the jakobid and malawimonad CCT genes raises the question of their origin. Were they recently acquired, or do they represent the retention of old introns? Separate from the issue of the origin of introns themselves (i.e., introns-early vs. introns-late; Logsdon et al. 1995
; de Souza et al. 1998
; Logsdon 1998
) is the question of the diversity and antiquity of spliceosomal introns within eukaryotes. For the most part, protist genes are intron sparse, and many of the lineages that have figured prominently in hypotheses of early events in eukaryotic evolution (e.g., diplomonads and parabasalids) seem to lack introns entirely (Logsdon 1998
). Remarkably few intron positions are known to be conserved between animals, fungi, and plants (Fast, Logsdon, and Doolittle 1999
; Palmer and Logsdon 1991
) and fewer still between animals or fungi (or both), plants, and protists. Interestingly, the CCTalpha data set contains two introns of the latter sort (positions 2 and 21), and CCTdelta contains an intron shared between M. jakobiformis, one of three fungi, and animals (position 13). Unfortunately, the CCTalpha data set is somewhat biased against the presence of old introns because of the fact that the sole fungal representatives, Saccharomyces cerevisiae and Schizosaccharomyces pombe, are known to be relatively intron sparse compared to other fungi (the intron density in S. cerevisiae is only 0.1 per kb; Logsdon 1998
). In the CCTdelta data set, three introns were found in the N. crassa CCTalpha (N. crassa has a higher intron density), and two of these were shared with animals. The CCTdelta data set is similarly biased. No introns were found in the S. cerevisiae or S. pombe CCTdelta genes, and despite the fact that the intron density in plant protein-coding genes is generally quite high (Logsdon 1998
), neither of the plant CCTdeltas (Arabidopsis or Oryza) contained a single intron. With few protist introns available for comparison, it is difficult to make strong inferences about whether the potentially old jakobid-malawimonad introns are a result of the retention of ancestral intron positions or because of independent parallel insertions. More CCTalpha sequences from each of the major eukaryotic groups may resolve this issue.
It is certainly clear from both data sets that recent intron gain has occurred. The Arabidopsis thaliana CCTalpha gene alone contained 12 introns in positions not found in any other taxa, and the CCTalpha from the ciliate T. pyriformis contained another three (fig. 4A
, see Results). For the jakobid-malawimonad CCTalpha's, one of the R. americana introns and two of the M. jakobiformis introns were located in unique positions, as were four of seven M. jakobiformis CCTdelta introns. Data from the jakobid tubulin genes also support this notion. As predicted from ultrastructure and mitochondrial genome content data, R. americana and J. libera branched strongly together in beta- and alpha-betatubulin phylogenies. Yet despite this apparent close relationship, their respective beta-tubulins each possessed a single intron located in different positions, neither of which is present in known beta-tubulin genes (Edgcomb et al. 2001
).
As for intron loss, the data are more ambiguous. In general, this uncertainty stems from the lack of a robust phylogeny of the major protist groups upon which intron gain-loss scenarios can be evaluated. Nevertheless, a case for intron loss can be inferred for the Heterolobosea. Consistent with the low intron density observed thus far for heterolobosean protein-coding genes (Logsdon 1998
), only one intron was found in the CCTalpha gene from A. rosea, and none were present in the N. gruberi sequence. However, the intron in the A. rosea CCTalpha is shared with the two R. americana strains, M. jakobiformis and Arabidopsis (intron position 10; fig. 4A
), suggesting that it may predate the divergence of plants and these protists.
It seems significant that in cases where only one or a few introns are present in the CCT genes (e.g., Acrasis, Plasmodium, Schizosaccharomyces; fig. 4A and B
) they are located near the 5' end (in CCTalpha, intron positions 110 are located in the first 20% of the coding sequence, whereas introns 15 in CCTdelta are present in the first 11% of the gene). Such a bias is also observed for the vast majority of introns in yeast (Fink 1987
; Spingola et al. 1999
), and for the few remaining introns in the highly reduced nucleomorph genome of the cryptomonad alga Guillardia theta (Douglas et al. 2001
). In yeast, a process of incomplete reverse transcriptasemediated intron loss has been suggested to account for such a nonrandom distribution (Fink 1987
). However, it is known that at least some of these 5'-end introns play regulatory roles in gene expression (Spingola et al. 1999
), suggesting that they may persist not because of incomplete reverse transcription but because of selection against intron loss. Either way, the clustering of introns near the 5'-ends of genes is consistent with loss from a more intron-rich ancestral state. On the other hand, it is also possible that such a pattern simply reflects a bias in the process of intron insertion.
Although no introns have been described in genes from the amitochondriate diplomonads and parabasalids, indirect evidence for their existence in the parabasalid T. vaginalis has come from the discovery of a homolog of PRP8, a highly conserved protein component of the spliceosome (Fast and Doolittle 1999
). A gene whose product has significant similarity to PRP8 is also present in the near complete genome of the diplomonad G. lamblia (Smith et al. 1998
) (identified by searching the high-throughput genome sequence database at NCBI). Taken as a whole, the data are consistent with the possibility that the intron-sparse or intron-free nature of many protist genes is a derived feature.
How intron-rich will jakobid and malawimonad nuclear genes turn out to be? At present, such a small sample size makes it impossible to predict. Nevertheless, the data presented here suggest a different picture of spliceosomal intron evolution than is often assumed, one in which intron loss has been a significant factor in shaping eukaryotic nuclear genomes.
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Acknowledgements |
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Footnotes |
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1 Present address: Department of Botany, University of British Columbia, Vancouver, Canada
Keywords: jakobids
eukaryotic evolution
introns
chaperonins
CCT
Address for correspondence and reprints: John M. Archibald, Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4. jarch{at}interchange.ubc.ca
.
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