Evolutionary Analyses of the Small Subunit of Glutamate Synthase: Gene Order Conservation, Gene Fusions, and Prokaryote-to- Eukaryote Lateral Gene Transfers

Lateral gene transfer has been identified as an important modeof genome evolution within prokaryotes. Except for the specialcase of gene transfer from organelle genomes to the eukaryoticnucleus, only a few cases of lateral gene transfer involvingeukaryotes have been described. Here we present phylogeneticand gene order analyses on the small subunit of glutamate synthase(encoded by gltD) and its homologues, including the large subunitof sulfide dehydrogenase (encoded by sudA). The scattered distributionof the sudA and sudB gene pair and the phylogenetic analysisstrongly suggest that lateral gene transfer was involved inthe propagation of the genes in the three domains of life. Oneof these transfers most likely occurred between a prokaryoteand an ancestor of diplomonad protists. Furthermore, phylogeneticanalyses indicate that the gene for the small subunit of glutamatesynthase was transferred from a low-GC gram-positive bacteriumto a common ancestor of animals, fungi, and plants. Interestingly,in both examples, the eukaryotes encode a single gene that correspondsto a conserved operon structure in prokaryotes. Our analyses,together with several recent publications, show that lateralgene transfers from prokaryotes to unicellular eukaryotes occurwith appreciable frequency. In the case of the genes for sulfidedehydrogenase, the transfer affected only a limited group ofeukaryotes—the diplomonads—while the transfer ofthe glutamate synthase gene probably happened earlier in evolutionand affected a wider range of eukaryotes.

INTRODUCTION

Top

Introduction

During the last 5 years it has become clear that lateral genetransfer (LGT) is a widespread and important evolutionary processamong prokaryotes (10, 22). Although multicellular eukaryotes,like humans, seem to be relatively immune to LGT (2, 23, 24),it is unclear if LGT is also an important mode of evolutionin unicellular eukaryotes (protists), due to the limited amountof genomic sequence data currently available for these organisms.Apart from the special case of prokaryote-to-eukaryote LGTs,which occur from the eukaryotic organelles with prokaryoticancestry (the mitochondrion and the chloroplast) to the nucleus,there are only a handful of well-documented cases of prokaryote-to-eukaryoteLGT events described in the literature (3, 8, 12, 16, 27). However,LGT appears to occur frequently between the two prokaryoticdomains of life, Bacteria and Archaea, indicating that domainboundaries do not prevent successful LGT (20, 21).

We have chosen diplomonads, a group of anaerobic protists, totest the hypothesis that LGT from prokaryotes to protists isoccurring at an appreciable rate. There is an ongoing whole-genomesequence project for Giardia lamblia, the most studied diplomonad,that releases newly generated sequences on a regular basis toGenBank (19). In our laboratory we have focused on another diplomonad,the Atlantic salmon parasite Spironucleus barkhanus (25), andwe are, together with our collaborators, performing a sequencesurvey project. Genes that were laterally transferred from prokaryotesto the diplomonad lineage are expected to show higher similarityto prokaryotic genes than to any eukaryotic gene. Therefore,candidate laterally transferred genes are easily identifiedin these projects with similarity searches against the publicsequence databases. Such genes have been further analyzed withphylogenetic methods that will give a much more reliable indicationof whether the gene originated via LGT. Furthermore, if thegene is present in both the Giardia and Spironucleus projects,the phylogeny will show whether the gene has a common sourcein both lineages or it was a recent acquisition in one of thetwo lineages. Here we present such an analysis of the sud gene,a diplomonad gene with greater similarities to prokaryotic thanto eukaryotic homologues. Sequences with the highest similaritiesto this gene corresponded to a conserved gene pair, sudA andsudB, present in Thermotoga maritima, Treponema pallidum, andPyrococcus furiosus, suggesting an LGT event from a prokaryoteto a eukaryotic ancestor of diplomonads. The gene pair encodesthe two subunits of sulfide dehydrogenase (15). However, theC-terminal part of the diplomonad protein also shows high similarityto the small subunit of eubacterial glutamate synthase (gltD).

Glutamate synthase is a complex iron-sulfur flavoprotein thatparticipates in ammonia assimilation processes. The eubacterialversion of the enzyme is composed of two subunits. The smallsubunit is a flavin adenine dinucleotide-dependent NADPH oxidoreductasewhich shows sequence similarity to several other protein domainsand enzyme subunits. Indeed, the small subunit of eubacterialglutamate synthase has been proposed to be a prototype of proteindomains or enzyme subunits used in many different contexts totransfer electrons from NAD(P)H to an acceptor protein or proteindomain (28). In the case of glutamate synthase, the small subunitprovides electrons to the large subunit, which binds L-glutamineand 2-oxoglutarate and forms L-glutamate (28).

Sulfide dehydrogenase is an iron-sulfur flavoprotein withoutglutamate synthase activity (15). The enzyme consists of twosubunits: the ${alpha}$ -subunit, which is clearly homologous to the smallsubunit of glutamate synthase, and the ß-subunit,which does not show any sequence similarity to the large subunitof glutamate synthase at all (15). Thus, the ${alpha}$ -subunit of sulfidedehydrogenase is an example of an enzymatic subunit that ishomologous (and very similar) to the small subunit of glutamatesynthase but that is used in a different context. The two subunitsof sulfide dehydrogenase are encoded by sudA and sudB, two genesfound next to each other on the chromosome in P. furiosus (15).Sulfide dehydrogenase not only catalyzes the reduction of polysulfideto H₂S in P. furiosus, but it has also been shown to functionas a reduced ferrodoxin-NADP oxidoreductase (15, 18). Thesedual functions of the protein complicate the interpretationof the physiological role for the enzyme. Indeed, the biologicalsignificance of the sulfide dehydrogenase activity in P. furiosushas recently been questioned, since the activity of the enzymedid not respond to changes in the availability of elementalsulfur (1).

To determine if the diplomonad gene shared a common ancestrywith other sulfide dehydrogenases that would be suggestive ofa shared function, and to elucidate its origin in the genomesof diplomonads, we retrieved all homologues of gltD and gltD-likegenes from the public databases, studied the gene organizationpatterns around the homologues, and performed phylogenetic analyses.We identified two cases of prokaryote-to-eukaryote LGT, showingthat interdomain LGT involving microbial eukaryotes may occurrelatively frequently.

MATERIALS AND METHODS

Top

Materials and Methods

Gene nomenclature. Unfortunately, the nomenclature of gltD and gltD-like genesis far from consistent in the public databases. To simplifythe presentation, the gene name gltD is only used for genesthat are linked to gltB (which encodes the large subunit ofglutamate synthase) on the chromosome. gltD-like genes thatare linked to sudB (see below) on the chromosome are annotatedas sudA. All other genes are simply referred to as gltD-like.

PCR assays and nucleotide sequencing. The gene for sulfide dehydrogenase--a fusion of sudA and sudB—wasamplified from S. barkhanus genomic DNA by degenerate PCR understandard conditions with the following amplification parameters:denaturation at 95°C for 30 s, annealing at 48°C for1 min, and extension at 72°C for 3 min. Forty cycles wereperformed and a final extension step of 72°C for 10 minwas used. The forward primer GLTDf1 (5'-GGTCGAGTHTGYCCHYARGA-3')and the reverse primer GLTDr1 (5'-CAATCCATDGCDACRTTDCC-3') wereused. The PCR product was purified and sequenced directly. UnevenPCR (7) was used to obtain the 5' end of the gene. Exact matchprimers from the obtained sequence were used together with arbitraryshort primers in a reaction that used two radically differentannealing temperatures (42 and 55°C) in alternate annealingsteps, as described previously (7). This procedure yielded aPCR product that covered the 5' end of the gene, which was cloned.Several independent clones were sequenced.

Phylogenetic analysis. All published gltD, gltD-like, and sudA sequences and all availableunpublished sudA sequences were retrieved from the NationalCenter for Biotechnology Information. The Dictyostelium discoideumGenome Project database (http://dicty.sdsc.edu) was also searchedfor homologues, with a negative result. Although the N-terminalpart of eukaryotic dihydropyrimidine dehydrogenase has beenshown to be homologous to the small subunit of glutamate synthase(28), we did not include it in our data set since the gene isonly distantly related to gltD. This resulted in a data setwith 399 unambiguously aligned positions. Sequences with >85%amino acid sequence identities were excluded from the data setto reduce the computational time. At the initial phylogeneticanalysis, five sequences failed the ${chi}$ ² tests for deviation ofamino acid frequencies implemented in TREE-PUZZLE, version 4.02(26) (a partial Entamoeba histolytica gltD-like sequence, aRhodobacter capsulatus gltD-like sequence, a Plasmodium falciparumgltS sequence, and gltD sequences from Pseudomonas aeruginosaand Campylobacter jejuni). These sequences were excluded sincethe currently available phylogenetic methods cannot deal withstrong amino acid compositional biases in the data (13). Thisprocedure resulted in a 53-taxon data set with 399 amino acids(aa). Protein maximum likelihood (ML) phylogenies were inferredusing PROML within the PHYLIP package, version 3.6 (11), witha Dayhoff (PAM 001) substitution model (the only supported modelin version 3.6a of PROML) and a mixed four-category discrete-gammamodel of among-site rate variation plus invariable sites (PAMplus ${Gamma}$ plus Inv). Ten random additions with global rearrangementswere used to find the optimal tree (shown below in Fig. 1).The ${Gamma}$ shape parameter, ${alpha}$ , was estimated to be 1.07, and the fractionof invariable sites, P_inv, was 0.01 using TREE-PUZZLE, version4.02 (26). Protein ML distance bootstrap values for bipartitionswere calculated by analysis of 500 resampled data sets usingPUZZLEBOOT (http://www.tree-puzzle.de) with a Jones-Taylor-Thornton(JTT) substitution model and a mixed eight-category discrete-gammamodel ( ${alpha}$ = 1.25) of among-site rate variation plus invariablesites (P_inv = 0.01) (JTT plus ${Gamma}$ plus Inv). Due to the computationalintensity, full ML bootstrapping was not performed on the 53-taxondata set.

View larger version (74K):
[in this window]
[in a new window]
FIG. 1. The ML tree of the inferred amino acid sequence of gltD, sudA, and gltD-like genes and their gene arrangements. See Materials and Methods for gene nomenclature. The different typefaces of the species names indicate whether the species belongs to the eukaryotes (all capitals), Archaea (lowercase, normal), or Bacteria (lowercase italic). Three main parts of the tree are indicated with large boxes, as follows: a clade with mostly gltD genes (A); a clade with only sudA genes (B); and a paraphyletic group with different gltD-like genes (C). The tree was arbitrarily rooted between the gltD clade and the paraphyletic group of gltD-like genes. Black boxes represent the homologous part of the gltD, gltD-like, and sudA sequences (used in analysis), open boxes represent gltB genes, gray boxes represent sudB genes, and boxes with dotted borders represent unique N- or C-terminal extensions of the gltD-like genes. Note that there is no detectable sequence homology between the extensions represented by dotted boxes. A thin line between two boxes indicates neighboring genes on the chromosome. Two boxes attached to each other indicate fusion of the genes. Protein ML-distance bootstrap values >50% for bipartitions are shown.

In a separate phylogenetic analysis, the sudA and sudB geneproducts were combined, yielding 671 aa that could be unambiguouslyaligned. Aquifex aeolicus, Geobacter sulfurreducens, Desulfovibriovulgaris, and T. pallidum failed the ${chi}$ ² amino acid compositiontest implemented in TREE-PUZZLE, version 4.02 (26), and wereremoved from further analysis. Protein ML phylogenies were inferredas described above except that an eight-category discrete-gammamodel was used ( ${alpha}$ = 0.88 with no invariable sites detected).Protein ML bootstrap values for bipartitions were calculatedby analysis of 500 resampled data sets using PROML within thePHYLIP package, version 3.6a (11), with one random additionwith global rearrangements.

Nucleotide sequence accession numbers. The G. lamblia sudA sequence was assembled using the followingsingle-pass reads deposited in GenBank by the Josephine BayPaul Center for Comparative Molecular Biology and Evolution(19) with the following accession numbers: AC030366, AC031039,AC031040, AC035392, AC043148, AC048816, AC051395, AC062548,AC084971, AC084972, AC086120, and AC082857. The S. barkhanussequence reported here was deposited in GenBank under the accessionnumber AF455034.

RESULTS AND DISCUSSION

Top

Results and Discussion

A sulfide dehydrogenase gene in diplomonads. Among the sequences released from the ongoing genome projectof the intestinal human parasite G. lamblia (19), we found agene with greater similarities to prokaryotic genes encodingsulfide dehydrogenase than to any eukaryotic homologues, suggestingan LGT event from prokaryotes to an ancestor of Giardia. Furthermore,the gene appeared to represent a gene fusion, since it correspondedto two distinct genes in prokaryotes (sudA and sudB). No othersequences with significant homology to the small and/or largesubunit of glutamate synthase were found among the availableG. lamblia sequences. In order to determine if the sulfide dehydrogenasegene was a recent acquisition in the lineage leading to Giardiaor if it was present in the last common ancestor of diplomonads,we amplified and sequenced the gene from the distantly relateddiplomonad S. barkhanus. The amplified gene did indeed showthe highest similarity to the Giardia gene and appeared to bethe result of the same gene fusion. Some diplomonads, includingS. barkhanus, use an alternative genetic code where TAG andTAA encode glutamine rather than termination (17). Seven in-frameTAG codons and five in-frame TAA codons were found in the S.barkhanus sulfide dehydrogenase gene, which show that it hasameliorated to the genetic code used in the organism. To clarifythe evolutionary origin of the gene in diplomonads, we retrievedhomologues of the gene from the public databases and performedphylogenetic and gene order analyses.

Phylogenetic analysis of gltD and its homologues. The protein ML tree of the gene sequences of gltD and its homologuesis shown in Fig. 1. The phylogenetic tree can be divided intothree distinct parts: one well-supported clade (Fig. 1A) thatincludes mostly gltD sequences; one clade with sudA sequences(Fig. 1B); and a paraphyletic group (Fig. 1C) with six gltD-likesequences. However, the root of the tree is unknown and couldbe within one of these groupings. Within clade A, three distinctgroups are found, two well-supported clades consisting of ${alpha}$ -and ${gamma}$ -proteobacteria, respectively, and a large clade with amonophyletic eukaryotic clade nested within low-GC gram-positivebacteria, indicating an LGT from this eubacterial group to anearly eukaryote (see discussion below) (Fig. 1A). With a fewexceptions (see below), all eubacterial sequences within thegltD clade form a gene pair with gltB, which encodes the largesubunit of glutamate synthase (Fig. 1A). This indicates thatthe glutamate synthase operon structure initially identifiedin Escherichia coli (5) is conserved within eubacteria, withthe exception that the gene order between gltB and gltD is reversedin ${alpha}$ -proteobacteria. In eukaryotes, glutamate synthase is encodedby a single gene, gltS, which arose by gene fusion of the eubacterialgltB and gltD genes (Fig. 1A).

Interestingly, there are also three eubacterial gltD-like sequences—twoaegA genes from E. coli (6) and the dsrL gene from Allochromatiumvinosum (GenBank accession number U84760)—nested amongthe gltD sequences (Fig. 1A). These genes probably encode functionsdistinct from glutamate synthase; they have N- or C-terminalextensions unrelated to the large subunit of glutamate synthase(Fig. 1) and they are not linked to gltB. It is likely thatthese enzymes have originated from gene duplications of gltD,followed by fusions with different domains, and they have acquirednew functions in these ${gamma}$ -proteobacteria.

A clade consisting of sulfide dehydrogenase genes. A second large set of gene order conservation, including theP. furiosus sulfide dehydrogenase genes, was found for cladeB (Fig. 1B). The sudB genes are found upstream of, or fusedto, the sudA genes in all species in the clade except A. aeolicus.Gene order conservation is rare in distantly related prokaryotesand is very unlikely to indicate ancient organization that wasretained solely by chance (29). Therefore, the gene order conservationbetween the sudA and sudB homologues (Fig. 1) strongly suggeststhat the gene products functionally, and probably physically,associate (interact) in vivo. Three iron-cluster-binding motifshave been proposed for the P. furiosus sulfide dehydrogenase(15). These show a high degree of conservation within the clade;all positions are conserved in all species containing the sudgene pair except in G. lamblia, which has substitutions in 2of the 11 proposed iron-binding positions (Fig. 2). The monophylyof sudA (Fig. 1), the gene order conservation of sudA and sudB(Fig. 1), and the conservation of the iron-cluster-binding motifsstrongly suggest that the gene pair (or a single gene in thediplomonad case) encodes sulfide dehydrogenases in these organisms.The most likely origin for the gene in the two diplomonads isby LGT, since the gene sequences are nested among prokaryotesand the gene is only distantly related to other eukaryotic sequences(see discussion below).

View larger version (45K):
[in this window]
[in a new window]
FIG. 2. Amino acid alignment of the iron-cluster binding motifs in sulfide dehydrogenase, proposed by Hagen et al. (15). The numbers above the alignments refer to the amino acid position in the P. furiosus SudB (cluster I) and SudA (cluster II and III) proteins. The proposed ligands are indicated by shadowed boxes. Note that cluster I has been proposed to have a novel Asp(Cys)₃ binding motif (15).

gltD-like sequences. Finally, there is a paraphyletic group of six gltD-like sequencesthat are linked neither to the large subunit glutamate synthasenor to sudB (Fig. 1C). Four of these sequences do have N- and/orC-terminal extensions that indicate that the gltD-like partof the polypeptide is likely functioning in a different contextin these proteins. Possibly, the two gltD-like sequences thatlack significant N- or C-terminal extensions are functioningtogether with other, not-yet-identified genes. Preliminary phylogeneticanalyses indicated that the partial E. histolytica sequencethat was excluded from the large phylogenetic analysis due toa strong amino acid composition bias probably belongs to thisparaphyletic group (data not shown).

Several cases of LGT within the sulfide dehydrogenase clade. Preliminary individual phylogenetic reconstructions showed congruitybetween the sudA and sudB gene topologies (data not shown),suggesting that they share a common history. We therefore concatenatedthe two proteins in a combined phylogeny to increase the resolutionwithin the sulfide dehydrogenase clade. Five nodes in the proteinML tree of the sud genes show strong bootstrap support (Fig.3). The Pyrococcus and the diplomonad sequences form two stronglysupported monophyletic clusters, as expected (Fig. 3). The Fibrobactersuccinogenes sequence forms a clade with the Treponema denticolasequence, with the Porphyromonas gingivalis sequence as an immediateoutgroup. Finally, the low-GC gram-positive Enterococcus faecalisforms a strongly supported monophyletic grouping with T. maritima(Fig. 3), which is inconsistent with accepted views of eubacterialphylogeny (4) and which strongly suggests an LGT event betweenthe two lineages. This is unlikely to be a rare case of LGTin the history of the sud genes. The scattered distributionof the sud genes among microbes from the three domains of lifeis unlikely to be solely due to differential gene loss; clearly,that would require a vast number of independent gene lossesin all three domains of life. Thus, propagation of the sud genesvia multiple LGT events combined with vertical transmissionis the most parsimonious explanation of the distribution ofsud genes among microbes. Since the gene is only found in diplomonadsamong the studied eukaryotes and the diplomonads are nestedwithin a prokaryotic clade (Fig. 1 and 3), an ancestor of diplomonadsalmost certainly acquired the gene from a prokaryotic sourcevia LGT. Unfortunately, the donor lineage cannot be determineddue to the limited resolution in the phylogenetic reconstructions(Fig. 1B and 3).

View larger version (27K):
[in this window]
[in a new window]
FIG. 3. Phylogeny of concatenated sudA and sudB protein sequences. Protein ML bootstrap and ML distance bootstrap support values are shown above and below the branches, respectively. Only support values >50% for bipartitions are shown.

It would be imprudent to speculate on what might have driventhe propagation of the gene, since the biological activity ofsulfide dehydrogenase is not very well understood (1, 15). Atany rate, all organisms in the sud clade are anaerobes (Fig.1B), which hints at a biological role related to anaerobiosisfor the protein. However, only biochemical studies can proveor disprove if the gene products have sulfide dehydrogenaseactivity in the members of the clade. Such experiments in diplomonads,combined with genome data from the ongoing G. lamblia genomeproject (19), will deepen our understanding of sulfur metabolismin these organisms, which currently is very limited. It mayturn out that diplomonads metabolize elemental sulfur to modulateredox potential or as an alternative energy source. If so, itwould be another example where anaerobic eukaryotes metabolizeinorganic sulfur compounds; some animals use sulfide as an inorganicenergy source during mitochondrial sulfide oxidation (14).

Eukaryotic gltS may have a eubacterial origin via LGT. The LGT of the sud genes from a prokaryote to the diplomonadlineage may not be the only case of prokaryote-to-eukaryotetransfer in the evolution of the gltD and gltD-like genes. Interestingly,eukaryotic gltS is clearly nested (bootstrap support value of96) within a bacterial clade consisting of mostly gram-positivebacteria (Fig. 1A). This is unlikely the result of an endosymbioticgene replacement from the mitochondria or chloroplast, sincethe eukaryotic sequences fail to show affinity to proteobacteriaor cyanobacteria, the ancestors of mitochondria and chloroplasts,respectively. Therefore, an origin via LGT independent of theeukaryotic organelles, perhaps from a gram-positive eubacteriumin the common ancestor of animals, plants, and fungi, is themost likely scenario.

The P. falciparum gltS sequence (excluded from the large phylogeneticanalysis due to its strong amino acid composition bias) showedaffinity to the large bacterial clade in preliminary phylogeneticanalyses but failed to branch with the other eukaryotic gltSsequences (data not shown). However, the P. falciparum sequenceappears to have experienced the same fusion event as the othereukaryotic gltS sequences, indicating that the failure of thissequence to group with other eukaryotic gltS sequences is likelyan artifact caused by its extreme amino acid composition bias.

Two examples of gene fusion events of prokaryotic operons in eukaryotes. The gene arrangements of glutamate synthase and sulfide dehydrogenasein prokaryotes versus eukaryotes are strikingly similar; inboth cases a conserved gene pair occurs in prokaryotes thatcorresponds to a single fused gene in eukaryotes. Furthermore,in both cases, the eukaryote homologues probably originatedvia LGT from a prokaryotic source. The probability of successfultransfers of the two subunits simultaneously was certainly increasedby the conserved gene arrangements within prokaryotes. The genefusions in eukaryotes likely reflect the differences in generegulation between eukaryotes and prokaryotes. Fusions of genesthat code for different subunits of the same enzyme are probablystrongly selectively advantageous in eukaryotes, which lackoperon structures, because they provide an easy solution forcoregulation of the expression of the different subunits.

The gene fusions reported in this study probably resulted fromtwo distinct fusion events: one that created the eukaryoticglutamate synthase gene and one that yielded the diplomonadsulfide dehydrogenase gene. Both appear to have occurred relativelysoon after the LGT events. However, the evolutionary signalin gene fusions should not be overstated. The presence of similarfusions in distantly related eukaryotes does not necessarilymean that the genes are the result of a single transfer followedby a fusion. Two independent transfers of the same prokaryoticoperon to two different eukaryotic lineages can easily leadto two independent gene fusions, since selection for coordinateregulation probably strongly favors such fusion events. Therefore,analyses of gene fusions should be interpreted in conjunctionwith phylogenetic analyses based on gene sequences.

LGT might play an important role in protistan genome evolution. Although it is always difficult to discern mechanisms for ancientevolutionary events, it has been suggested that a gene transferratchet mechanism may exist for eukaryotes whereby genes fromprokaryotes taken up as food are incorporated into the eukaryoticnucleus (9). Such a mechanism would explain the transfer ofsulfide dehydrogenase from a prokaryote to the common ancestorof diplomonads, which likely engulfed prokaryotes as food, aswell as the putative transfer of glutamate synthase from a gram-positiveeubacterium to a common ancestor of fungi, animals, and plants—anancestor that most certainly was a single-celled organism.

Our analysis of sulfide dehydrogenase, together with severalrecently reported cases of prokaryote-to-eukaryote gene transfers(3, 8, 12, 16, 27), indicates that LGT may play an importantrole in protistan genome evolution. In addition, the putativetransfer of glutamate synthase suggests that LGT also shapedthe genomes of the protist ancestors of major multicellulareukaryote lineages.

ACKNOWLEDGMENTS

We thank T. Martin Embley and David S. Horner (Natural HistoryMuseum, London, United Kingdom) for a generous gift of genomicS. barkhanus DNA. We also thank Yuji Inagaki, Camilla Nesbø,and Åsa Sjögren for discussions and critical readingof the manuscript, and we thank Lesley Davis for help with sequencing.We utilized the preliminary data generated by The Institutefor Genomic Research (www.tigr.org), the Utah Genome Center(www.genome.utah.edu), and the Josephine Bay Paul Center forComparative Molecular Biology and Evolution (www.mbl.edu/Giardia).

J.O.A. is supported by a Postdoctoral Fellowship from the Wenner-GrenFoundations. A.J.R. is supported by the CIAR Program in EvolutionaryBiology. This work was supported by a Natural Sciences and EngineeringResearch Council Genomics Project, grant 228263-99, awardedto A.J.R.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada. Phone: (902) 494-2881. Fax: (902) 494-1355. E-mail: joanders{at}is.dal.ca.

REFERENCES

Top