JOURNAL OF BACTERIOLOGY,0021-9193/97/$04.0010

Nov. 1997, p. 6816–6823 Vol. 179, No. 21

Copyright © 1997, American Society for Microbiology

The tigA Gene Is a Transcriptional Fusion of Glycolytic GenesEncoding Triose-Phosphate Isomerase and Glyceraldehyde-3-

Phosphate Dehydrogenase in OomycotaSHIELA E. UNKLES,1* JOHN M. LOGSDON, JR.,2† KEITH ROBISON,3 JAMES R. KINGHORN,4

AND JAMES M. DUNCAN1

Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA,1 and School of Environmental and Evolutionary Biology,University of St. Andrews, Fife KY16 9TH,4 United Kingdom; Department of Biology, Indiana University, Bloomington,

Indiana 474052; and Harvard Biological Laboratories, Cambridge, Massachusetts 021383

Received 21 March 1997/Accepted 25 August 1997

Genes encoding triose-phosphate isomerase (TPI) and glyceraldehyde-3-phosphate dehydrogenase(GAPDH) are fused and form a single transcriptional unit (tigA) in Phytophthora species, members of the orderPythiales in the phylum Oomycota. This is the first demonstration of glycolytic gene fusion in eukaryotes andthe first case of a TPI-GAPDH fusion in any organism. The tigA gene from Phytophthora infestans has a typicalOomycota transcriptional start point consensus sequence and, in common with most Phytophthora genes, hasno introns. Furthermore, Southern and PCR analyses suggest that the same organization exists in other closelyrelated genera, such as Pythium, from the same order (Oomycota), as well as more distantly related genera,Saprolegnia and Achlya, in the order Saprolegniales. Evidence is provided that in P. infestans, there is at leastone other discrete copy of a GAPDH-encoding gene but not of a TPI-encoding gene. Finally, a phylogeneticanalysis of TPI does not place Phytophthora within the assemblage of crown eukaryotes and suggests TPI maynot be particularly useful for resolving relationships among major eukaryotic groups.

The enzymes triose-phosphate isomerase (TPI) and glycer-aldehyde-3-phosphate dehydrogenase (GAPDH) catalyze se-quential steps in the major pathways of carbohydrate metab-olism, including glycolysis and gluconeogenesis. GAPDHcatalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate following its isomerization from dihy-droxyacetone phosphate by TPI. In all organisms studied thusfar, GAPDH is a tetrameric protein, which is homomeric (EC1.2.1.12) in the prokaryotes, fungi, mammals, and plant cytosoland heteromeric (EC 1.2.1.13), composed of two types of sub-unit, in the plastid-localized enzymes of algae and plants. Thecrystal structure of this enzyme has recently been determinedfrom a bacterium (25) and a eukaryote (23). TPI is a ho-modimeric protein (EC 5.3.1.1), the crystal structure of whichhas been determined from a number of organisms (2, 11, 42).Comparative crystallographic studies have shown a high levelof structural conservation between TPIs of diverse eukaryoticorganisms (42).

Both enzymes from a wide range of organisms show highconservation of the protein sequence (26, 28, 29, 45), andindeed it was by using the Aspergillus nidulans gpdA gene as aheterologous probe that the Oomycota gene encodingGAPDH was isolated from Phytophthora infestans (30). At thattime, there was no indication of fusion of glycolytic genesoccurring in any organism, and in all other species studied, thegenes appeared to be discrete. The only known association ofTPI- and GAPDH-encoding genes is that in some eubacteria,where they are known to be present in the same operon, butalways with the pgk gene (encoding the glycolytic enzyme 3-

phosphoglycerate kinase) located between GAPDH and TPI(summarized in reference 6).

The Oomycota are a large and heterogeneous group (19),and although traditionally classed as lower fungi, it is nowaccepted from biochemical and DNA data that they are phy-logenetically distant from the true fungi (kingdom Fungi), suchas Ascomycota and Basidiomycota. Instead, Oomycota proba-bly belong to the protistan heterokont lineage, which includesdiverse species such as the chromophyte algae (e.g., brownalgae and diatoms), within the kingdom Chromista (3, 16, 19,40). The Oomycota include several saprophytic species, butmany are pathogenic to fish and plants, causing devastation toseveral commercially important crops. Despite their economicimpact, however, they are as a group poorly understood inmolecular terms.

Here we report the first demonstration of a fusion betweenthe tpi and gpd genes encoding TPI and GAPDH, a genearrangement which appears to be prevalent throughout thephylum Oomycota and perhaps unique to this eukaryoticgroup.

MATERIALS AND METHODS

Strains and growth conditions. Escherichia coli DH5a was used for all cloningand propagation of plasmids. P. infestans ATCC 48720 and ATCC 36609, P.infestans I117 (from the collection at the Scottish Crop Research Institute) and89/AF1 (a gift from D. Shaw, University of Bangor, Wales, United Kingdom),Pythium ultimum, P. violae, and P. sylvaticum were grown in 20 ml of pea brothextract in 100-ml Erlenmeyer flasks (7) for 3 days at 20°C. P. cactorum CAC23,P. nicotianae NIC1, P. citricola CIT2, P. fragariae var. rubi FVR11, and P.cinnamomi CIN8, from the collection at the Scottish Crop Research Institute,were cultivated in a modified GYP medium (43) for 2 to 7 days (depending onthe strain) at 20°C. Mycelial mats were harvested, washed with sterile distilledwater, pressed dry, and stored at 280°C. Frozen mycelia of Achlya radiosa andSaprolegnia turfosa were kindly provided by M. Dick, University of Reading,Reading England, United Kingdom.

Recombinant DNA procedures. Genomic DNA was extracted from myceliumby using a Nucleon II kit (Scotlab) adapted as previously described (41). TotalRNA was prepared as described by MacCabe et al. (27). PCRs were performedin a Techne Thermal Cycler. For reverse transcription-PCR (RT-PCR), 5 mg of

* Corresponding author. Present address: Department of Microbi-ology, Monash University, Clayton, Victoria 3168, Australia. Phone: 613 9905 4323. Fax: 61 3 9905 4811. E-mail: shiela.unkles@med.monash.edu.au.

† Present address: Department of Biochemistry, Dalhousie Univer-sity, Halifax, Nova Scotia B3H 4H7, Canada.

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total RNA was reverse transcribed in the presence of 0.5 mg of oligo(dT)15 and1 mM deoxynucleoside triphosphates (dNTPs), using 200 U of Moloney murineleukemia virus reverse transcriptase, followed by 30 cycles of 95°C for 20 s, 52°Cfor 20 s, and 72°C for 30 s, in the presence of 100 mM dNTPs, 1 mM each primersT1 and G1, and 2.5 U of Taq polymerase (Life Technologies). Sequences ofprimers T1 and G1 were 59-GCTACCGTCAAGGCTTC (position 1151) and59-TAGCACTGGCACGCAGC (position 1832), respectively. Degenerateprimer sequences were designed to match highly conserved regions of TPI andGAPDH and used at concentrations of 5 3 N mM (where N is the primerdegeneracy) in reactions with 50 ng of DNA template (HindIII digested orunrestricted), 100 mM dNTPs, and 2.5 U of Taq polymerase. Primers used wereTPI5 (59-TGGGCCATCGGNACNGG; position 1505), TPI7 (59-GGCTTCCTGGTNGGNGG; position 1685), GAP1R (59-GCCAATACGACCRAANCC;position 1807), and GAP2R (59-CAGACARTTNGTNGTRCA; position11233). Cycling conditions for 35 cycles were 95°C for 30 s, annealing for 10 s attemperatures between 50 and 58°C (depending on the primer combination), and72°C for 2 min. PCR fragments were isolated from 2% agarose gels by usingGeneclean (Bio 101) and either (i) cloned into pCR-SCRIPT vector followingpolishing with Pfu polymerase as recommended by the manufacturer (Strat-agene) and sequenced by using a Sequenase version 2 kit (Amersham) or (ii)sequenced directly, using the appropriate degenerate primer and the protocolrecommended by Amersham for PCR-derived sequencing templates. The nucle-otide sequence of each PCR product was determined with only one strand.Northern and Southern blotting and hybridization conditions have been de-scribed previously (27).

Phylogenetic analyses. Amino acid sequence alignments were initially pre-pared by using ClustalW (37) and then edited by visual inspection. The aminoacid alignment used for phylogenetic reconstruction includes all nearly full-length eukaryotic TPI-encoding genes available as of December 1996 from Gen-Bank, with the exception that TPI sequences .98% identical at the amino acidlevel were represented by a single arbitrarily chosen sequence. Regions of am-biguous alignment at the protein termini were excluded from the final alignmentand subsequent analyses. The complete alignment is available by request toJ.M.L. Distance phylogenetic analyses were performed with the PHYLIP version3.57c package (15) on a Sun SparcStation 20. Distance matrices were calculatedwith PROTDIST, using the Dayhoff PAM matrix, and neighbor-joining treeswere calculated with NEIGHBOR. One thousand bootstrap resamplings werecarried out by using SEQBOOT, with the bootstrap consensus tree calculated byCONSENSE. Maximum parsimony analyses were performed with PAUP version3.1.1 (36).

Nucleotide sequence accession number. The sequence shown in Fig. 1 hasbeen assigned EMBL accession number X64537.

RESULTS

Sequence of the tigA gene. The gpdA gene, encodingGAPDH, of P. infestans was cloned previously by our group asa 3.5-kb EcoRI fragment in pUC19 (pSTA33 [Fig. 1A]), and itsDNA sequence was determined (30). The gene showed highsimilarity at the deduced protein level to the equivalent gene ofA. nidulans, but in place of the expected initiation codon ATGwas the sequence GTG. Although unusual, the use of GTG toencode methionine at the initiation position is not unique inthe filamentous fungi (17), and in the absence of obvious in-tron consensus sequences, this was assumed to be the transla-tional start of the protein. Later database searches, however,showed DNA sequence homology to cloned tpi genes (TPIencoding) in the sequences upstream from the proposed gpdAinitiation codon. The entire DNA sequence upstream of gpdAin pSTA33 was therefore determined and shown to encodeTPI. The sequence of the complete gene is shown in Fig. 1B.The whole gene, which incorporates the gpdA sequence, hasbeen designated tigA. The initiation codon at the position ex-pected by homology to other tpi genes is ATG. In addition,there is a motif, GCTCATTCTCGGATTT, 97 nucleotides up-stream from this ATG which is conserved at the transcriptionalstart site of most Oomycota genes studied so far (31). Com-parison to published sequences suggests that there are no in-trons within the tigA gene. The lack of introns is not unex-pected, as introns are present in only 3 of 22 Oomycota genessurveyed (26a). The coding regions of tpi and gpd are separatedby a 21-bp sequence without stop codons, indicating that theprotein is translated as a TPI-GAPDH fusion. We have noevidence of other glycolytic genes in close association distal to

tigA. Probes from the region of pSTA33 downstream from tigAhybridize to highly repetitive genomic sequences, and DNAsequence at the distal end of pSTA33 shows no significantsimilarity to database sequences (data not shown).

tigA is a single transcriptional unit. To demonstrate that theapparent gene fusion was not a cloning artifact and that thegene was transcribed as a single unit, we carried out RT-PCRwith total RNA from the strain (ATCC 48720) used originallyto prepare the gene library and from another strain in ourcollection (89/AF1). The positions of the primers T1 and G1,which straddle the fusion, are shown in Fig. 1A. RT-PCRproducts of the expected size (680 bp) were obtained fromboth strains, but only in reactions containing reverse transcrip-tase (Fig. 2A; compare lanes 3 and 5 to lanes 2 and 4). Inaddition, we performed Northern blotting experiments usingas probes a 0.58-kb SacI fragment specific to the tpi region ora 0.78-kb XhoI-HindIII fragment specific to the gpd region(Fig. 1A). Using total RNA from strains ATCC 48720 (lane 1)and 89/AF1 (lane 2), a transcript of the same size (2 kb)hybridized to both the tpi and gpd probes (Fig. 2B). A secondtranscript of around 1 kb hybridized to the gpd-specific probeonly.

A discrete gpd, but not tpi, gene is present in Phytophthora.The Northern blots (Fig. 2B) showed two gpd-specific signalssuggesting the presence of the fusion transcript and one otherof a size which would be compatible with transcription of gpdalone. This may have been the result of some sort of posttran-scriptional processing of the fusion transcript or the presenceof another expressed copy of gpd. Genomic DNA of fourstrains of P. infestans was digested, blotted, and hybridized tothe same tpi- or gpd-specific probes as above (Fig. 2B). Thetpi-specific probe hybridized to bands of the size expected fromthe restriction map of pSTA33 (Fig. 1A). Although the ploidylevel of our strains is unknown, P. infestans is at least diploid,and it is likely that in these strains, the ploidy is higher. It istherefore not unexpected to observe extra bands which prob-ably represent restriction fragment length polymorphisms indifferent alleles, for example, in the HindIII digest of strainsATCC 48720 and ATCC 36609 (Fig. 3). This phenomenon isquite different from that observed with the gpd-specific probe,where there are additional hybridizing bands with each restric-tion digest and in each strain as well as those of the sizesexpected from the pSTA33 map. Restriction fragment lengthpolymorphism bands can also be seen, for example, in theSacI-digested 89/AF1 DNA. The hybridizations were per-formed under conditions of high stringency and indicate thatthere must be at least one copy of gpd, other than that which ispart of the fusion. In contrast, we find no evidence for anotherdiscrete gene copy of tpi.

The tigA gene fusion occurs throughout the genus Phytoph-thora. Members of the genus Phytophthora have been charac-terized by biochemical and growth characteristics into six ma-jor groups (35). One representative member of each group waschosen for analysis by restriction digestion and Southern blot-ting of genomic DNA followed by hybridization using theabove tpi- and gpd-specific probes. Hybridizations were per-formed under conditions of low stringency (Fig. 4) and clearlyshow that each probe hybridizes to similar-sized or overlappingfragments in each digest. Therefore, the hybridization patternsobtained with the tpi and gpd probes are evidently superim-posable. Although it is possible that the tpi and gpd genes aresimply adjacent and not actually fused, the fact that distantlyrelated genera clearly have the tigA fusion (see below) stronglysuggests that the same tigA fusion arrangement exists in allthose Phytophthora species examined in this study.

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The tigA gene fusion is present in other oomycetous genera.Genomic DNA was prepared from three species (P. sylvaticum,P. violae, and P. ultimum) of the genus Pythium, closely relatedto Phytophthora within the order Pythiales. DNA was digested,Southern blotted, and hybridized at low stringency, using thesame tpi- and gpd-specific probes as above. The pattern ofhybridizing bands in the DNA of each species was similar foreach probe (data not shown), suggesting once again that thetigA fusion occurs also in this genus.

Attempts to repeat these hybridizations with genomic DNAfrom species of other Oomycota genera, however, produced

equivocal results, and so a PCR approach was adopted. De-generate primers were designed on the basis of sequence con-servation between species, and their relative positions areshown in Fig. 1A. A range of annealing temperatures andextension times was used with each primer combination andeach template DNA, resulting in a number of PCR productswith all the conditions used. Bands around the sizes expected(assuming no introns) were excised from agarose gels, and theDNA sequence was determined directly or following cloninginto pCR-SCRIPT. Positive identity of the fragments was ob-tained by sequence comparison with the P. infestans tigA gene.

FIG. 1. Organization (A) and nucleotide sequence (B) of the tigA gene. (A) The restriction map of the original gpdA clone, pSTA33, is shown, with the positionsof the TPI and GAPDH coding regions represented by hatched bars. The linking sequence is a solid bar. Arrows indicate the positions of the primers used in PCRsrelative to the restriction map. E, EcoRI; S, SacI; X, XhoI; H, HindIII. (B) Nucleotide sequence of tigA. Numbers to the left refer to nucleotides. The sequence reportedpreviously (30) starts at nucleotide position 1631 relative to the translational start (11). The deduced amino acid sequence is given in one-letter code and numberedto the right. The conserved motif at position 297 found around Oomycota transcriptional start points is underlined. The sequence linking the tpi and gpd coding regionsis in bold.

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Such positively identified fragments were amplified from P.ultimum by using primer combination TPI5 and GAP1R, con-firming the Southern analysis results, as well as from S. turfosaand A. radiosa, using primer combination TPI7 and GAP2R.

These latter species belong to another order, Saprolegniales,within the Oomycota. Alignment of the deduced amino acidsequences of the S. turfosa and A. radiosa PCR fragments withthe corresponding regions in the P. infestans gene are shown inFig. 5. These alignments serve to confirm identity of the PCRproduct and show a not unexpected, high degree of amino acidsequence conservation between the species within these shortstretches.

PCR amplification was also attempted with more distantlyrelated chromophyte species, using genomic DNA kindly pro-vided by G. W. Saunders, University of New Brunswick, Fre-dericton, New Brunswick, Canada. No specific PCR productswere obtained from these species under the conditions used,suggesting but not demonstrating that the gene fusion may notextend beyond the Oomycota into related groups of theChromista.

Molecular phylogeny of TPI-encoding genes. The TPI-en-coding portion of the tigA gene represents the first reportedheterokont TPI sequence. Thus, to address the phylogeneticplacement among other eukaryotes, and potentially provideinsight into the timing of the tigA fusion, phylogenetic analysesof 31 eukaryotic TPI sequences were carried out. Figure 6shows the neighbor-joining (distance) tree. When outgrouprooted on the probable deep-branching protist genus Giardia,the tree shows that the Oomycota TPI (Phytophthora) branchesprior to the divergence of all other eukaryotic TPIs, an ar-rangement supported by a moderate bootstrap value of 86%.Nonetheless, the relationships between major eukaryoticgroups are not resolved on this tree, as they are not supportedby bootstrap analyses. Although the Phytophthora TPIbranches in the same position in maximum parsimony analysis(data not shown), the support is very weak (bootstrap value,,50%). Similar to the distance analysis, parsimony did notstrongly support any specific relationships among major eu-karyotic groups, nor did it contradict any of the strongly sup-ported groups.

FIG. 2. Analysis of P. infestans total RNA. (A) Amplification products fromtotal RNA of P. infestans ATCC 48720 (lanes 2 and 3) and 89/AF1 (lanes 4 and5), using primers T1 and G1. For lanes 2 and 4, a reverse transcriptase reactionprior to amplification was not included, while for lanes 3 and 5, amplification wascarried out following reverse transcription. Lane 1, control without templateRNA; lane 6, positive control using pSTA33 as template; lane L, molecular sizemarkers (100-bp ladder). (B) Northern analysis of total RNA isolated from P.infestans ATCC 48720 (lane 1) and 89/AF1 (lane 2) hybridized with the 0.58-kbSacI-SacI tpi-specific fragment (tpi) or the 0.78-kb XhoI-HindIII gpd-specificfragment (gpd) of pSTA33 indicated in Fig. 1B. Hybridization was performed athigh stringency (42°C with 50% formamide and washed with 13 SSC [0.15 MNaCl plus 0.015 M sodium citrate]). The positions of molecular size markers areindicated in kilobases.

FIG. 3. Southern blot analysis of P. infestans strains. DNA from strains ATCC 48720 (lane 1), 89/AF1 (lane 2), I117 (lane 3), and ATCC 36609 (lane 4) was digested withEcoRI, HindIII, SacI, or KpnI, electrophoresed in 0.8% agarose, and Southern blotted in duplicate. Hybridizations were carried out at high stringency (65°C, washed with 0.23SSC), using the tpi-specific (A) and gpd-specific (B) probes described in the legend to Fig. 2. The positions of molecular size markers are indicated in kilobases.

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DISCUSSION

The gene fusion tigA found in the Oomycota is unprece-dented among the glycolytic genes in any other organism, pro-karyotic or eukaryotic. In the prokaryotes, genes encodingGAPDH are commonly found in an operon arrangement alongwith other glycolytic genes, typically those encoding phospho-glycerate kinase (PGK) and TPI (14, 33). However, althoughcoordinately transcribed, they are translated as individualgenes. One exception to this has recently been found in thehyperthermophilic bacterial genus Thermotoga. In T. maritima(34) and T. neopolitana (44), the genes encoding PGK and TPIare fused but in different reading frames with respect to eachother. Translation of the fus gene results in a monofunctionalPGK or, following a programmed translational frameshift, adifunctional PGK-TPI protein with TPI C terminal to PGK. Inthe Oomycota, the tpi region is 59 to the gpd region and a truetranscriptional fusion is present, resulting in a mRNA with

both tpi and gpd in the same reading frame. Also, the secondgpd transcript probably derives not from the gene fusion but, asshown by Southern analysis, from another locus in the genomicDNA. In all other eukaryotes studied, including fungi, algae,higher plants, and animals, tpi and gpd specify monofunctionalproteins. In those eukaryotic organisms for which rigorousclassical genetic systems as well as karyotyping are available,these genes have been found to be unlinked to each other.

Although the Oomycota have been traditionally regarded asfilamentous fungi, they are now considered, on the basis ofsmall subunit rRNA sequence comparisons, to be more closelyrelated to certain diverse protists, chromophytes, together withwhich they form the heterokonts (5, 8–10, 16, 38, 40), part ofthe late-arising assemblage of crown eukaryotes (5, 20). Thecrown group includes fungi, plants, animals, and some protists,representing the rapid evolutionary diversification of eukary-otic species. However, other analyses based on large subunit

FIG. 4. Southern blot analysis of Phytophthora species. DNA from P. infestans ATCC 48720 (lane 1) and 89/AF1 (lane 2), P. cactorum CAC23 (lane 3), P. nicotianaeNIC1 (lane 4), P. citricola CIT2 (lane 5), P. fragariae FVR11 (lane 6), and P. cinnamomi CIN8 (lane 7) was digested with EcoRI, HindIII, or SacI, electrophoresed in0.8% agarose, and Southern blotted in duplicate. Hybridizations were carried out at low stringency (53°C, washed with 33 SSC), using the tpi-specific (A) and thegpd-specific (B) probes described in the legend to Fig. 2. The positions of molecular size markers are indicated in kilobases.

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rRNA (12, 39, 40), actin (1, 3, 4, 13), elongation factor 1-a (1,18), and b-tubulin (1, 22) show inconsistencies in placementfor the Oomycota, possibly due to sparse taxon sampling. Inparticular, a recent phylogenetic analysis of GAPDH could notunambiguously position Phytophthora GAPDH (encoded bytigA) among eukaryotic groups (32). Our phylogenetic analysisof TPI indicates a phylogenetic position for Phytophthora asbranching before the crown, but taking into account the lack ofavailable TPI sequences for comparison from relevant, i.e.,other heterokont, groups, and possibly the relatively shortlength of the gene itself, we cannot unequivocally exclude theOomycota from the crown.

The inherent uncertainties of both the current GAPDH (32)and TPI phylogenies do not at this point allow interpretation

based on sequence comparisons into the origin or evolution ofthe tigA fusion. However, the tigA gene fusion in the Oomycotadoes serve to illustrate further the divergent evolution of thisgroup of organisms. As this arrangement of glycolytic genesappears to be unique to this group, the fused gene may providea useful diagnostic tool for the identification of this phylum or,potentially, a unique target for antimetabolic agents activeagainst the pathogenic members of the group.

Finally, this study poses the question of whether the proteinis posttranslationally processed to give separate TPI andGAPDH products. If so, the subunits would be free to assumetheir conventional arrangements as homodimers and homotet-ramers, respectively. However, if processing does not occur, itis unclear whether the protein functions as a dimer or as a

FIG. 5. Comparison of deduced amino acid sequences of tigA genes from three Oomycota species. PCR products from S. turfosa and A. radiosa were amplified byusing primers TPI7 and GAP2R and were partially sequenced from each end. Residues identical among all three species are indicated below the alignment by asterisks,and gaps introduced for alignment are shown by dashes. Numbers to the left and right of the P. infestans sequence indicate positions relative to Fig. 1B. The aminoacid sequence corresponding to the primer is in bold, and the linking sequence between the TPI and GAPDH domains is underlined. The left portion shows thesequence obtained from primer TPI7; the sequence to the right was obtained from primer GAP2R.

FIG. 6. Phylogenetic analysis of eukaryotic TPI sequences. The neighbor-joining tree is shown; branches supported by bootstrap percentages of $50 are labeled,based on 1,000 replicates. Major eukaryotic groups are labeled to the right. Taxa are given by genus names only, except where more than one species is shown; in thelatter case, the species name is abbreviated by a single letter as follows: Schistosoma mansoni (m.) and japonicum (j.), Trypanosoma brucei (b.) and cruzi (c.), and Giardialamblia (l.) and intestinalis (i). Plant TPI genes which are nuclear encoded yet function in chloroplasts are denoted by cp.

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tetramer. Recent evidence from the prokaryotes might indi-cate that the latter is at least a possibility and that TPI canfunction as a tetramer. Two instances of tetrameric TPI, bothfrom hyperthermophilic organisms, have been reported andsuggested to represent an adaptation for stability at highgrowth temperatures (21, 24). The TPIs from T. maritima andthe related bacterial species T. neopolitana are expressed astetrameric fusion proteins with PGK (34, 44), while the TPI ofthe hyperthermophilic archaea Methanothermus fervidus andPyrococcus woesei are tetramers derived from an independenttpi gene (24). However, the Oomycota are a mesophilic orpsychrophilic group, and so the structural relevance of a di-functional tetrameric protein is uncertain.

ACKNOWLEDGMENTS

We gratefully acknowledge time and effort given by colleagues whogenerously provided mycelium or DNA samples from the organismsused in this study. In particular, we thank Michael Dick (University ofReading, Reading, United Kingdom) and Gary Saunders (Universityof New Brunswick, Fredericton, New Brunswick, Canada). Addition-ally, we thank S. Baldauf for helpful comments on the manuscript.

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