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Towards the minimal eukaryotic parasitic genome Christian P Vivarès* and Guy Méténier Microsporidia are well-known to infect immunocompromised patients and are also responsible for clinical syndromes in immunocompetent individuals. In recent years, evidence has been obtained in support of a very close relationship between Microsporidia and Fungi. In some species, the compaction of the genome and genes is remarkable. Thus, a systematic sequencing project has been initiated for the 2.9 Mbp genome of Encephalitozoon cuniculi, which will be useful for future comparative genomic studies. Addresses Laboratoire Parasitologie Moléculaire et Cellulaire, UMR CNRS 6023, Université Blaise Pascal, 63177 Aubière Cedex, France *e-mail: [email protected] Current Opinion in Microbiology 2000, 3:463–467 1369-5274/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations DHFR dihydrofolate reductase HSP heat-shock protein ITS internal transcribed spacer kbp kilobase pair Mbp megabase pair PTP polar tube protein rRNA ribosomal RNA TS thymidilate synthase UTR untranslated region

Introduction Microsporidia are amitochondrial unicellular eukaryotic and intracellular parasites. About 1000 species parasitize members of almost all animal phyla. They are opportunistic pathogens, infecting AIDS patients, and their prevalence in the European population is predicted to be about 8% [1]. They harbor some prokaryotic-like features: 70S ribosomes, 16S and 23S ribosomal RNA (rRNA), and fusion of the 5.8S sequence to the 23S rRNA 5′ end. Phylogenetical analysis of microsporidian small subunit ribosomal RNA (SSUrRNA) and translation elongation factors suggested a very early evolutionary origin of Microsporidia and lent credit to the hypothesis of a primitive amitochondrial state [2,3]. In contrast, the identification of separated coding regions for thymidilate synthase (TS) and dihydrofolate reductase (DHFR), as in fungi and metazoa, supported a late origin of Microsporidia [4]. The placement of these organisms among the fungi has been inferred from tubulin phylogenies [5]. The microsporidian life cycle occurs usually in a single host, starting after the ingestion of spores contaminating the environment or the diet. The invasion process is unique in the living world. The activated spore fires a very long tubular element (polar tube) into a target cell through which the sporoplasm including nucleus is then injected.

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Molecular karyotype variability in Encephalitozoon cuniculi. Three strains have been discriminated so far, on the basis of immunological and molecular criteria. A useful genetic marker is a tetranucleotide repeat (5′-GTTT-3′) within the first and unique internal transcribed spacer (ITS1) of the rDNA unit (three repeats for strain I, four repeats for strain II and two repeats for strain III). Hybridisation experiments have shown the conservation of 11 different chromosomes separated by pulsed-field gel electrophoresis (PFGE) in the strain I reference isolate. Isolates classified within different strains were found to be heterogeneous in respect of the PFGE karyotype [10]. Examples of profiles observed for these strains are illustrated here. The numbers down the side correspond to chromosomes in order of size. Each chromosome is identified by a specific gridded box. The left-most profile is characteristic of the strain I reference isolate. The boxes in white correspond to chromosomes that have not been clearly identified because of their small size range. Variations in banding pattern can be related to co-migration of some heterologous chromosomes, as well as to differential migration of some homologous ones. Chromosome size distributions in variants from strains II and III are shifted down (15–20 kbp) relative to strain I.

The intracellular development frequently takes place within a parasitophorous vacuole surrounded by numerous host mitochondria [6•]. The nuclear apparatus is represented by either single nucleus or paired nucleus (diplokaryon). The ploidy level is unknown. The intranuclear mitosis involves spindle pole bodies, and the cell divides by either binary or multiple fission. There is no evidence for meiosis in species parasitizing vertebrates.. The haploid genome size of Encephalitozoon cuniculi, a species parasitizing a wide range

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Figure 2 Simplified representation of a physical (restriction) map of the E. cuniculi genome. The map was deduced from the analysis of restriction patterns obtained with the enzymes BssHII and MluI, after two-dimensional pulsed-field gel electrophoresis separation of DNA fragments arising from either a single digestion of the molecular karyotype or a double digestion of individual chromosomes [14•]. BssHII and MluI sites are marked by black and open symbols, respectively. Each subtelomeric 16S-23S rDNA transcription unit is represented by a black arrow. The chromosome size estimated from the sum of different restriction fragments varies from 210 kbp for chromosome I to 304 kbp for chromosome XI, which is in agreement with the size range (215–315 kbp) deduced from the molecular karyotype. More than 60 chromosome-specific DNA probes were also localized and sequenced.

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of mammalian hosts, is only 2.9 megabase pairs (Mbp) [7].. Here we review current advances in our understanding of the nuclear genome of Microsporidia, with emphasis on the E. cuniculi genome, which may be assumed to be close to a minimal genome for an eukaryotic cell. In order to justify a reduction in gene diversity and/or gene copy number in this organism, a combination of various factors can be considered: limite biosynthetic capacities in relation with a strict parasitism, absence of some protein rich organelles (i.e. mitochondria, peroxisomes), ribosomes of prokaryotic size, asexual cycle and very small cell size.

Comparisons of several isolates from the same microsporidian species indicate that a polymorphism in chromosome number and/or size may exist. This has been recently documented in E. cuniculi and E. hellem [10–12]. The study of 15 E. cuniculi isolates from different geographic areas and hosts, representative of three strains, has revealed six karyotype variants [10] (Figure 1).

Genome size and karyotype variability

In the reference isolate of E. cuniculi, several repetitive DNA probes derived from a partial genomic library hybridize with all chromosomal bands. These dispersed repeated sequences correspond to rDNA genes and micro- or minisatellites [13].

The molecular karyotypes of three mammal-infecting Encephalitozoon species, all forming very small spores (~2 µm), are characterized by 10–12 chromosomes with extremely small sizes of between 175 and 315 kilobase pairs (kbp). The haploid genome size was estimated to be less than 3 Mbp (ranging from 2.3 Mbp in E. intestinalis to 2.9 Mbp in E. cuniculi), which is within the range of prokaryotic genomes [8••]. However, larger variations in genome size (up to 19.5 Mbp in Glugea atherinae) and in chromosome number (from 8 in Vairimorpha sp. to 18 in Nosema bombycis) also exist in the phylum Microspora. The individual chromosome size can be as small as 130 kbp in Vavraia oncoperae and as large as 2.7 Mbp in Glugea atherinae [9].

A physical map was constructed using the restriction enzymes BssHII and MluI and two-dimensional pulsedfield gel electrophoresis (Figure 2) [14•]. The terminal regions of the chromosomes show a common pattern covering roughly 15 kbp and each containing one 16S-23S rDNA unit. Telomeric DNA is represented by heterogeneous repeats and may be up to 1.2 kbp in length (Katinka M, et al. Abstract 8, Genomes 2000, ASM-Institut Pasteur, Paris, April 2000). Results of hybridization and molecular combing experiments indicate a palindromic-like orientation of the two subtelomeric rDNA copies. This organization is reminiscent of the mini-chromosomes,

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which exclusively contain rDNA genes and the vestigial nucleomorph genome in Chlorarachniophytes (Figure 3). The only difference is their opposite orientation relative to E. cuniculi [14•]. The rDNA genes are located only on one chromosome and probably tandemly arranged in Nosema genus [15]. A 3 kbp deletion/insertion event accounts for the size difference between two homologous chromosome III in the reference isolate. The comparison of two karyotype variants from the strain I provided evidence for size polymorphism of subtelomeric regions from five chromosomes. The sites of deletion/insertion are upstream of rDNA units, within repetitive DNA regions marking the transition with the gene-rich core (JF Brugère, E Cornillot, G Méténier, CP Vivarès, unpublished data). The gene density is high and the intergenic regions are often very short (as small as 29 bp) [16,17] (Figure 4).

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Gene structure

Comparison of chromosomes from different eukaryotic organisms known to contain two subtelomeric rDNA copies (black boxes). (a) Palindromic rDNA dimers are found in slime molds (Dictyostelium, Physarum) or in the macronucleus of some ciliates (Tetrahymena). (b) In Encephalitozoon, each chromosome also exhibits two transcriptionally divergent rDNA copies, but contains a central domain rich in protein-coding genes (grey boxes). (c) A similar organization exists for very small chromosomes of the nucleomorph, a vestigial nucleus of a green alga endosymbiont in chlorarachniophytes, except that rDNA units have centripetal orientation.

The rDNA unit that lacks the internal transcribed spacer (ITS) 2 has been fully sequenced in two microsporidia: E. cuniculi [8••] and Nosema apis [18]. It seems likely that the putative expressed tag sequences (ETSs) are short, as it is for the coding regions and ITS1. Unlike in prokaryotes or some protozoans and fungi, no neighboring 5S rRNA or tRNA gene was detected. The length of the sequence between the start of the 16S rRNA gene and the predicted end of the 23S rRNA gene is close to 3.75 kbp, with an ITS1 of only 38 bp in E. cuniculi (strain I) and 33 bp in N. apis. As judged from models of rRNA secondary structure, the highly reduced size (below Escherichia coli counterparts) can be related to the deletion of some universal and eukaryote-specific helices for 16S rRNA as the truncation or loss of most divergent domains and variable stems of the conserved core for 23S rRNA [8••,18,19•] With respect to protein-coding genes, the first report on the systematic sequencing of the E. cuniculi chromosome I described a cluster of three genes (dhfr, ts, shmt [serine hydroxymethyl transferase]) encoding enzymes involved in nucleotide metabolism, facing an aminopeptidase gene [16]. A gene pair for two different structural proteins of the polar tube (PTPs) needed for invasion has been shown to be conserved in the three Encephalitozoon species, providing a first example of synteny in Microsporidia (Delbac F, Peuvel I, Méténier G, Vivarès CP, unpublished data) (Figure 4)..

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Transcription start sites have been determined by 5′ rapid amplification of cDNA ends for a spore wall protein (SWP1) gene in E. cuniculi, revealing a 5′ untranslated region (UTR) of only 3–8 nucleotides [20]. We predict that most 5′ UTRs from densely packed genes will be very reduced in length; this is supported by alignments of the 5′ flanking regions of several protein-coding genes that show that a putative TATA box generally extends less than 20 nucleotides upstream of the start codon [16,20]. A putative signal for translation initiation control would be equivalent to the prokaryotic ‘downstream box’ — a proximal segment of the translated region pairing with the most distal 16S rRNA hairpin and acting independently of the Shine–Dalgarno sequence located in the 5′ UTR [16,21]. A typical polyadenylation signal (AATAAA) was generally identifiable in the 3′ flanking regions, and cDNA sequencing data provided evidence for 3′ UTRs of variable length, which are associated with poly(A) tails [20,22].

Figure 4 Gene clustering in E. cuniculi. Examples of regions showing packed protein-coding genes on three chromosomes, involved either in related functions (e.g. common metabolism for DHFR, TS and SHMT [serine hydroxymethyl transferase], transport of molecules mediated by ATP-binding cassette [ABC] transporters) or in the genesis of a same cellular structure (PTPs for the polar tube). HypP, hypothetical protein. Intergenic distances are given in base pairs.

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The translated regions of some microsporidial protein-coding genes (DHFR, TS, kinesin-related protein, heat-shock protein [HSP]70) are shorter than their known eukaryotic homologues [16,17,23], indicating that genome reduction may have also involved various deletions in coding regions. That the microsporidian genome contains a gene encoding a HSP70 protein with signatures sequences shared by mitochondrial and α-proteobacterial HSP70s does not support the ‘Microsporidia-early’ hypothesis. A parsimonious interpretation is that Microsporidia once harbored mitochondria or their endosymbiotic progenitors [23]. As with tubulin phylogenies, a late divergence of Microsporidia has been deduced from phylogenetic trees constructed from gene sequences encoding other conserved proteins (valyl-tRNA synthetase, RNA polymerase II, TATA-box-binding protein) [24–26] or large subunit (LSU) rRNA [8••,19•]. The deep branching in trees obtained with either SSU rRNA or translational elongation factors is considered as an artefact, taking into consideration the among-site rate variation [19•]. Thus, the current view on the phylogenetical position of the Microsporidia consists in a close relationship with fungi.

Genome annotation: some preliminary data The systematic sequencing of the E. cuniculi genome has been recently completed and the annotation is in progress (Katinka M, et al. Abstract 8, Genomes 2000, ASM-Institut Pasteur, Paris, April 2000). Here we briefly describe some of the principal findings. The G+C content of the whole genome is close to 47%. Less than 2,000 protein-coding genes are predicted; of these, about 48% have been assigned a putative function through computer searching of similarities with sequences from databases. Duplicated genes are rare. Not surprisingly, a large fraction (46%) is devoted to the basic machinery for DNA replication, repair, transcription and protein synthesis. In spite of the low frequency of introns, the presence of different small nuclear RNAs supports a functional spliceosome [27•,28•]. The set of ribosomal proteins is predicted to be reduced, relative to other eukaryotes, and can be correlated with 70S ribosomes and small-sized rRNAs. In regard to the central metabolism, E. cuniculi has potential genes for intact glycolytic and pentose-phosphate pathways, which agrees with biochemical data [29]. Possible use of trehalose as a chief sugar reserve can be inferred from the finding of genes involved in synthesis and degradation of this disaccharide. No gene for the tricarboxylic acid cycle, aerobic respiratory chain or F-type ATP synthase has been found so far. This is therefore consistent with the absence of oxygen-utilizing organelles. We assume that energy production occurs only through substrate-level phosphorylations. The ability to import the ATP from the host cell is supported by the presence of ADP/ATP translocase genes. As expected for intracellular parasites, the E. cuniculi genome contains numerous genes encoding ABC transporters and very few genes for biosynthesis of amino acids. About 52% of the predicted gene sequences have weak or no similarity to proteins of other organisms, and should include a set of

Microsporidia-specific genes such as those required for the construction of the typical cell wall and invasion apparatus during sporogony. Accordingly, the recently identified genes encoding SWP1 [20] and two PTPs ([30]; Delbac F, Peuvel I, Méténier G, Poyrotaillade E, Vivarès CP, unpublished data) were ‘re-found’ in this category.

Conclusions A striking feature of the E. cuniculi genome is the interchromosomal conservation of the symmetrical organization of subtelomeric regions, each marked by a single rDNA transcription unit. The intraspecific karyotype variability is suggestive of deletions/insertions whose sites are restricted to a region extending upstream of the rDNA. We speculate that reciprocal translocations may have contributed to the dispersion of rDNA units in the evolutionary history of Encephalitozoon species, assuming an ancestral situation with tandemly arranged units. Translocations among the genomes of closely related species in the Saccharomyces complex have been considered to depend on ectopic recombination between repeated sequences including Ty elements, yeast transposons preferentially localised upstream of tRNA genes [31]. Future work should aim to map precisely the translocation breakpoints and evaluate the role of repeated sequences in the chromosomal rearrangements of Microsporidia. The prokaryotic-like features of Microsporidia appear as derived characters owing to a reductive evolution of small parasitic organisms clearly marked by a trend to genome compaction. We propose that, as in obligate intracellular parasitic bacteria (Rickettsia, Chlamydia, Buchnera), genome degradation might have occurred in response to the host environment through the accumulation of mutations leading to the inactivation and possible loss of genes for the synthesis of metabolites that could be obtained from the host cell [32••]. The absence of nuclear genes encoding compartmentalized enzymes for aerobic pyruvate degradation and ATP production supports this hypothesis, in the context of a secondary loss of mitochondria during Microsporidian evolution. Transcriptome analysis will be of principal importance to define functional genes. We hope that potential drug targets and vaccine antigens against Microsporidia infection will be identified, and that the way will be open for exploring the proteome in a rather simplified eukaryotic model system. We expect that the E. cuniculi genome sequence will be useful for comparative genomics in order to identify Microsporidia-specific genes, to facilitate phylogenetical approaches and to test the hypothesis that there are genes that are common to all intracellular eukaryotic parasites.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest •• of outstanding interest 1.

Van Gool T, Vetter JCM, Weinmayr B, Van Dam A, Derouin F, Dankert J: High seroprevalence of Encephalitozoon species in immunocompetent subjects. J Infect Dis 1997, 175:1020-1024.

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Vossbrinck CR, Maddox JV, Friedman S, Debrunner-Vossbrinck BA, Woese CR: Ribosomal RNA sequence suggests Microsporidia are extremely ancient eukaryotes. Nature 1987, 326:411-414.

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Kamaishi T, Hashimoto T, Nakamura Y, Nakamura F, Murata S, Okada N, Okamoto K, Shimizu M, Hasegawa M: Protein phylogeny of translation elongation factors EF-1a suggests Microsporidians are extremely ancient eukaryotes. J Mol Evol 1996, 42:257-263.

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Vivarès CP, Biderre C, Duffieux F, Peyretaillade E, Peyret P, Méténier G, Pages M: Chromosomal localization of five genes in Encephalitozoon cuniculi (Microsporidia). J Euk Microbiol 1996, 43:97S.

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Edlind T, Li J, Visvesvara G, Vodkin MH, McLaughlin GL, Katiyar SK: Phylogenetic analysis of β-tubulin sequences from amitochondrial protozoa. Mol Phylogen Evol 1996, 5:359-367.

6. Wittner M (Ed): The Microsporidia and Microsporidiosis. Washington: • ASM Press; 1999. A useful book providing a good view on biology of Microsporidia and pathology and diagnosis of microsporidiosis with emphasis on vertebrates. 7.

Biderre C, Pagès M, Méténier G, Canning EU, Vivarès CP: Evidence for the smallest nuclear genome (2,9 Mb) in the microsporidium Encephalitozoon cuniculi. Mol Biochem Parasitol 1995, 74:229-231.

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Peyretaillade E, Biderre C, Peyret P, Duffieux F, Méténier G, Gouy M, Michot B, Vivarès CP: Microsporidian Encephalitozoon cuniculi, a unicellular eukaryote with a unusual chromosomal dispersion of ribosomal genes and a LSU rRNA reduced to the universal corE. Nucleic Acids Res 1998, 26:3513-3520. A report on highly reduced Encephalitozoon genomes and distribution of rDNA genes on all chromosomes. The full sequencing of 23S rRNA enables the construction of a model for the secondary structure, showing the dramatic shortening of variable domains. Through the comparison of the 23S rRNA tree by two phylogenetics methods, the placement of Microsporidia is discussed; finally, Microsporidia appear in the crown of life trees. 9.

Biderre C, Pagès M, Méténier G, David D, Bata J, Prensier G, Vivarès CP: On small genomes in eukaryotic organisms: molecular karyotypes of two Microsporidian species (Protozoa) parasites of vertebrates. Crit Rev Acad Sci Paris 1994, 317:399-404.

10. Biderre C, Mathis A, Deplazes P, Weber R, Metenier G, Vivarès CP: Molecular karyotype diversity in the Microsporidian Encephalitozoon cuniculi. Parasitology 1999, 118:439-445. 11. Biderre C, Méténier G, Canning EU, Vivarès CP: Comparison of isolates of Encephalitozoon hellem and E. intestinalis by pulse field gel electrophoresis. Eur J Protistol 1999, 35:194-196. 12. Sobottka I, Albrecht H, Visvesvara GS, Pienazek NJ, Deplazes P, Schwartz DA, Laufs R, Elsner HA: Inter- and intra-species karyotype variations among Microsporidia of the genus Encephalitozoon as determined by pulsed-field gel electrophoresis. Scand J Infect Dis 1999, 31:555-558. 13. Biderre C, Duffieux F, Peyretaillade E, Glaser P, Peyret P, Danchin A, Pages M, Méténier G, Vivarès CP: Mapping of repetitive and non repetitive DNA probes to chromosome of the Microsporidian Encephalitozoon cuniculi. Gene 1997, 191:39-45 14. Brugere JF, Cornillot E, Méténier G, Bensimon A, Vivarès CP: • Encephalitozoon cuniculi (Microspora) genome: physical mapping and evidence for telomere-associated rDNA units on all chromosomes. Nucleic Acids Res 2000, 26:2026-2033. The first mapping of a Microsporidian genome by the procedure of two-dimensional pulsed-field gel electrophoresis. 15. Gatehouse HS, Malone LA: The ribosomal RNA gene region of Nosema apis (Microspora): DNA sequence for small and large subunit rRNA genes and evidence of large tandem repeat unit sizE. J Invertebr Pathol 1998, 71:97-105. 16. Duffieux F, Peyret P, Roe BE, Vivarès CP: First report on the systematic sequencing of the small genome of Encephalitozoon cuniculi (Protozoa, Microsporidia): gene organization of a 4.3 kbp region on chromosome I. Microb Comp Genomics 1998, 3:1-11. 17.

Biderre C, Méténier G, Vivarès CP: Sequencing of several proteincoding genes of the chromosome X from the Microsporidian Encephalitozoon cuniculi. J Euk Microbiol 1999, 35:194-196.

18. DeRijk P, Gatehouse HS, De Wachter R: The secondary structure of Nosema apis large subunit ribosomal RNA. Biochim Biophysic Acta 1998, 1442:326-328.

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19. Van de Peer, Ben Ali A, Meyer A: Microsporidia; accumulating • molecular evidence that a group of amitochondriate and suspectedly primitive eukaryote are just curious fungi. Gene 2000, 246:1-8. A critical phylogenetic analysi of Microsporidia supporting the divergence within fungal group. 20. Bohne W, Ferguson DJ, Kohler K, Gross U: Developmental expression of tandemly repeated, glycin- and serine-rich spore wall protein in the Microsporidian pathogen Encephalitozoon cuniculi. Infect Immun 2000, 68:2268-2275. 21. Sprengart ML, Fuchs E, Porter AG: The downstream box: an efficient independent translation initiation signal in Escherichia coli. EMBO J 1996, 15:665-674. 22. Broussolle V, Fumel S, Peyret P, Vivarès CP: First DDRT-PCR analysis of gene expression in a host-parasite system involving the Microsporidian Encephalitozoon cuniculi. J Euk Microbiol 1999, 46:25-26S. 23. Peyretaillade E, Broussolle V, Peyret P, Méténier G, Gouy M, Vivarès CP: Microsporidia, amitochondrial protist, possess a 70-kDa heat-shock protein gene of mitochondrial evolutionary origin. Mol Biol Evol 1998, 15:683-689. 24. Brown JR, Dolittle WF: Root of the universal tree of life based on ancient aminoacyl-tRNA synthetase gene duplications. Proc Natl Acad Sci USA 1995, 92:2441-2445. 25. Hirt RP, Logsdon JM Jr, Healy B, Dorey MW, Doolittle WF, Embley TM: Microsporidia are related to fungi: evidence from the largest subunit of RNA Polymerase II and other proteins. Proc Nat Acad Sci USA 1999, 96:580-585. 26. Fast NM, Logsdon JM Jr, Doolittle WF: Phylogenetic analysis of the TATA box binding protein (TBP) gene from Nosema locustae: evidence for a Microsporidia–fungi relationship and spliceosomal intron loss. Mol Biol Evol 1999, 16:1415-1419. 27. •

Biderre C, Méténier G, Vivarès CP: A small spliceosomal-type intron occurs in a ribosomal protein gene of the Microsporidian Encephalitozoon cuniculi. Mol Biochem Parasitol 1998, 94:283-286. See annotation for [28•]. 28. Fast NM, Roger AJ, Richardson CA, Doolittle WF: U2 and U6 • snRNA genes in the Microsporidian Nosema locustae: evidence for a functional spliceosome. Nucleic Acids Res 1998, 26:3202-3207. Two complementary papers [27•,28•] revealing the presence of a small intron and components of the spliceosome in two Microsporidia from different genera. 29. Weidner E, Findlay AM, Dolgikh V, Sokolova J: Microsporidian biochemistry and physiology. In The Microsporidia and Microsporidiosis. Edited by Wittner M. Washington: ASM Press; 1999:172-195. 30. Delbac F, Peyret P, Méténier G, David D, Danchin A, Vivarès CP: On proteins of the Microsporidian invasive apparatus: complete sequence of a polar tube protein of Encephalitozoon cuniculi. Mol Microbiol 1998, 29:825-834. 31. Fischer G, James SA, Roberts IN, Oliver SG, Louis EJ: Chromosomal evolution in Saccharomyces. Nature 2000, 405:451-454. 32. Andersson JO, Andersson SGE: Insights into the evolutionary •• process of genome degradation. Curr Opin Genet Dev 1999, 9:664-671. An interesting review on reductive evolutionary processes acting on the genome of obligate intracellular bacteria. The presence of pseudogenes and a large fraction of non-coding DNA in the rickettsial genome well suggest high rate of gene inactivation. Whether similar processes also occur in eukaryotic parasite, particularly in Microsporidia, deserves future studies.

Now in press The work referred to in the text as (JF Brugere, E Cornillot, G Metenier, CP Vivares, unpublished data) is now in press. 33. Brugere JF, Cornillot E, Metenier G, Vivares CP: Occurence of subtelomeric rearrangements in the genome of the Microsporidia parasite Encephatozoon cuniculu, as revealed by a new finger-printing procedure based on two-dimensional pulsed-field gel electrophoresis. Electrophoresis 2000, 21:2576-2581.