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A small spliceosomal-type intron occurs in a ribosomal protein gene of the microsporidian Encephalitozoon cuniculi1
Molecular and Biochemical Parasitology 94 (1998) 283 – 286
Short communication
A small spliceosomal-type intron occurs in a ribosomal protein gene of the microsporidian Encephalitozoon cuniculi 1 Corinne Biderre, Guy Me´te´nier, Christian P. Vivare`s * Laboratoire de Protistologie Mole´culaire et Cellulaire des Parasites Opportunistes, UPESA CNRS 6023, Uni6ersite´ Blaise Pascal, 63177, Aubie`re Cedex, France Received 30 March 1998; received in revised form 15 April 1998; accepted 27 April 1998
Keywords: Microsporidia; Encephalitozoon cuniculi; L27a ribosomal protein; Spliceosomal intron
Among unicellular eukaryotes, microsporidia are obligately intracellular amitochondrial parasites that are considered to be of very ancient origin as deduced from the prokaryotic features of their ribosomes [1– 3], and rRNA [4] and EF1a /EF2 phylogenies [5]. Microsporidia are also characterized by small nuclear genomes ranging from 19.6 Mb in Glugea atherinae [6] to only 2.9 Mb in Encephalitozoon cuniculi [7], and thus partially spanning the prokaryotic range. Sequencing data on the E. cuniculi genome have supplied information on noncoding regions but have not allowed identification of introns [8]. However, the relatively small sample of genes sequenced in miAbbre6iations: NLS, nuclear localization signal. * Corresponding author. Tel.: +33 473 407457; fax: + 33 473 407455; e-mail: [email protected] 1 Note: Nucleotide sequence data reported in this paper are available in the EMBL, GenBankTM and DDJB data bases under the accession number AF054829.
crosporidia may be insufficiently representative for detection of potential intervening sequences. During screening of a partial plasmid library from E. cuniculi genomic DNA, we isolated a clone (c141) coding for a homolog of a large subunit ribosomal protein (L27a or L29 in yeast) gene known to contain usually one or more introns. Blast alignments first revealed that a single reading frame accounts for the entire putative L27a coding region with a termination codon UAA. However, an AUG start codon could not be placed in the position expected on the basis of the conserved character of the L27a amino-terminal region. A thorough examination of this region identified an AT-rich (61%) intervening sequence, with a size of only 28 bp, that creates a frameshift and extends from just after an AUG codon (Fig. 1A). This intron harbors consensus spliceosomal boundaries (5%-GT...AG-3%) with a 5% region identical to the consensus of higher eukaryotes (GTAAGT). In the mould Neurospora crassa, one
0166-6851/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0166-6851(98)00064-4
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C. Biderre et al. / Molecular and Biochemical Parasitology 94 (1998) 283–286
intron also interrupts the extreme 5% region of L27a gene, but 6 additional introns are present [9]. Generally, the size of introns is at least 30 bp and can comprise over several thousands of nucleotides. However, very small introns have been identified in the processed somatic genome (macronuclear genome) of the ciliated protozoan Paramecium tetraurelia (20 – 33 bp) [10] and in the highly reduced nucleomorph genome (380 kbp) of a eukaryotic endosymbiont in chlorarachniophyte alga (18–20 bp) [11]. Such introns may be close to the minimum size. The occurrence of a spliceosomal-type intron in Encephalitozoon L27a agrees with recent lines of evidence for U2 and possibly also U6 RNAs in another microsporidian species, Vairimorpha necatrix [12]. Since no complementary sequence to the branch point GUAGUA conserved in the Vairimorpha U2 RNA was found in the Encephalitozoon intron, it may be assumed that the splicing process does not necessarily involve the complete set of the snRNAs. Either a branch point may be available for a simpler processing mechanism or only one or few adenosines may be required for the branch point. The intron frequency in nucleomorph and Paramecium genomes has been estimated to be 5.5 and 4 per kb, respectively. Considering the total length of Encephalitozoon hellem intronless b-tubulin gene [13] and seven other E. cuniculi gene sequences examined so far by us, the intron frequency is probably below 0.2 per kb. Compilation of the nucleotide sequences flanking the translation initiation site has afforded several group-specific consensuses having in common the A-residue at position −3 [14]. In contrast, this position is occupied by a G-residue in the Encephalitozoon L27a. The current microsporidian consensus based on the comparison of 7 genes would be GAAATGACA. The mRNAs from several amitochondrial protozoans display short leader regions (1 – 18 nt), making a scanning model unlikely [15]. It should be noted that an interaction between a sequence downstream of the initiation codon (downstream box) and a zone of the 16S rRNA decoding region (anti-downstream box) may be involved in prokaryotic translation initiation independent of
the Shine-Dalgarno sequence [16]. When examining the beginning of the E. cuniculi L27a translated region, a 6-nt sequence (position + 19/+ 24) may be paired to a 16S rRNA sequence (position 1231/1241) with the same location as an anti-downstream box, and with a single noncanonical pair G.G. As illustrated in Fig. 1B, putative downstream boxes can also be identified in the coding regions of the partially sequenced E. cuniculi kinesin gene and E. hellem b-tubulin gene. Thus, an additional prokaryote-like feature of microsporidia may be that the translation initiation depends on an mRNA-16S rRNA interaction placing the start codon close to the decoding region. The chromosomal location of L27a in the E. cuniculi genome, which is divided into 11 chromo-
Fig. 1. (A) Part of the nucleotide and aminoacid sequences of the E. cuniculi homolog of ribosomal protein L27a including the intron (underlined) located just after the start codon and the NLS1 motif (in bold). The untranslated regions are represented in lower cases. (B) Sequences involved in translation initiation. The putative downstream boxes (DB) from L27a and kinesin genes in E. cuniculi and b-tubulin gene in E. hellem are underlined. The 16S rRNA regions complementary to the DB sequences (anti-downstream boxes: ADB) are indicated.
C. Biderre et al. / Molecular and Biochemical Parasitology 94 (1998) 283–286
somes [6], was determined after CHEF gel electrophoresis and Southern blotting. The c141 probe gave a hybridization signal with a single chromosomal DNA band (chromosome X). To estimate the number of gene copies, Encephalitozoon genomic DNA was digested with endonucleases EcoRI and P6uII that do not cut within the L27a coding region, and probed with c141 insert. The hybridization with a single fragment indicates there is only one copy of L27a per haploid genome, as in Neurospora crassa [9] and Drosophila melanogaster [17]. Assuming the first methionine is not removed, the mature protein should contain 147 residues (predicted molecular mass: 17015 Da), with a basic isoelectric point (10.51). The amino acid similarities only extend from 62.5 to 65.3% when compared to L27a/L29 proteins from either animals, plants, fungi or some other protozoans in the SwissProt data base [18]. A novel motif of 27 residues with an invariant glycine residue at position 11, named KOW (as an acronym for the authors’ surnames), is shared by the members of both the NusG family (a bacterial transcription elongation factor) and several large subunit ribosomal protein families, and has been viewed as the principal mediator for RNA-protein interactions [19]. A similar motif can be identified in the E. cuniculi L27a, as in other L27a/L29 homologs, referring to the invariant Gly-11 (Fig. 2). Its location close to the N-terminus is identical in the L27 and L24 families. It has been shown that the N-terminus of the yeast L29 protein promotes import into the nucleus by means of two nuclear localization sequences (NLSs) KTRKHRG and KHRKHPG, rich in basic amino acids, designated NLS1 and NLS2, at positions six and 23 respectively [20]. The E. cuniculi KOW-like motif spans two sequences (KTRKLRG and KHRKHSG at positions seven and 24) homologous to NLSs, with NLS2 characterized by a residue Ser in penultimate position. The yeast L29 can bind to 5.8S rRNA [21] but immunofluorescence and mutagenesis experiments failed to demonstrate a specific role of NLSs in such a binding. That microsporidia are devoid of free 5.8S rRNA [4] also indicates another involvement of these sequences in ribosomal assembly (possibly as ligands for a nuclear pore receptor).
285
Fig. 2. Comparison of the characteristic KOW motif found in all members of the ribosomal protein families L24 (and K34), L26 and L27. Sequence alignments of multiple L27a genes show that such a motif exists in this family with an invariant glycine residue at position 11 (in bold). Numbering denotes the position of the motif in the protein. Accession numbers are indicated. L27: P. s.: Pyrobotris stellata; L27a: E. c.: Euplotes crassus; T. b.: Trypanosoma brucei brucei; S. c.: Saccharomyces cere6isiae; D. m: Drosophila melanogaster; L24: E. coli: Escherichia coli; B. s.: Bacillus subtilis; K24: P. s.: Pisum sati6um; N. t.: Nicotiana tabacum.
Finally, the evidence reported here that a microsporidian gene may be interrupted by a spliceosomal-type intron, combined with data supporting a secondary loss of mitochondria during evolution of microsporidia [22,23], is consistent with the theory that spliceosomal introns originated from fragmentation of group II selfsplicing ones [24]. A subsequent loss of many nuclear introns may be a result of a general evolutionary trend to genome and cell miniaturization in Microsporidia.
References [1] Ishihara R, Hayashi Y. Some properties of ribosomes from sporoplasm of Nosema bombycis. J Invertebr Pathol 1968;11:377 – 85. [2] Curgy JJ, Vavra J, Vivare`s CP. Presence of ribosomal RNAs with prokaryotic properties in Microsporida, eukaryotic organisms. Biol Cell 1980;38:49 – 52. [3] Vossbrinck CR, Woese CR. Eukaryotic ribosomes that lack a 5.8S RNA. Nature 1986;320:287 – 8. [4] Vossbrinck CR, Maddox JV, Friedman S, DebrunnerVossbrinck BA, Woese CR. Ribosomal RNA sequence suggests microsporidia are extremely ancient eukaryotes. Nature 1987;326:411 – 4.
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C. Biderre et al. / Molecular and Biochemical Parasitology 94 (1998) 283–286
[5] Kamaishi T, Hashimoto T, Nakamura Y, Masuda Y, Nakamura F, Okamoto KI, Shimizu M, Hasegawa M. Complete nucleotide sequences of the genes encoding translation elongation factors 1a and 2 from a microsporidian parasite, Glugea plecoglossi: implications for the deepest branching of eukaryotes. J Biochem 1996;120:1095– 103. [6] Biderre C, Page`s M, Me´te´nier G, David D, Bata J, Prensier G, Vivare`s CP. On small genomes in eukaryotic organisms: molecular karyotypes of two microsporidian species (Protozoa) parasites of vertebrates. CR Acad Sci Paris 1994;317:399 – 404. [7] Biderre C, Page`s M, Me´te´nier G, Canning EU, Vivare`s CP. Evidence for the smallest nuclear genome (2.9 Mb) in the microsporidium Encephalitozoon cuniculi. Mol Biochem Parasitol 1995;74:229–31. [8] Biderre C, Duffieux F, Peyretaillade E, Glaser P, Peyret P, Danchin A, Page`s M, Me´te´nier G, Vivare`s CP. Mapping of repetitive and non-repetitive DNA probes to chromosomes of the microsporidian Encephalitozoon cuniculi. Gene 1997;191:39–45. [9] Kreader CA, Heckman JE. Isolation and characterization of a Neurospora crassa ribosomal protein gene homologous to CYH2 of yeast. Nucleic Acids Res 1987;1015:9027– 42. [10] Russell CB, Fraga D, Hinrichsen RD. Extremely short 20 – 33 nucleotide introns are the standard length in Paramecium tetraurelia. Nucleic Acids Res 1994;22:1221– 5. [11] Gilson PR, McFadden GI. The miniaturized nuclear genome of a eukaryotic endosymbiont contains genes that overlap, genes that are cotranscribed, and the smallest known spliceosomal introns. Proc Natl Acad Sci USA 1996;93:7737 – 42. [12] DiMaria P, Palic B, Debrunner-Vossbrinck BA, Lapp J, Vossbrinck CR. Characterization of the highly divergent U2 RNA homolog in the microsporidian Vairimorpha necatrix. Nucleic Acids Res 1996;24:515–22.
.
[13] Edlind TD, Li J, Visvesvara GS, Vodkin MH, McLaughlin GL, Katiyar SK. Phylogenetic analysis of b-tubulin sequences from amitochondrial protozoa. Mol Phylogenet Evol 1996;5:359 – 67. [14] Yamauchi K. The sequence flanking translational initiation site in protozoa. Nucleic Acids Res 1991;19:2715 – 20. [15] Katiyar SK, Visvesvara GS, Edlind TD. Comparisons of ribosomal RNA sequences from amitochondrial protozoa: implications for processing, mRNA binding and paromomycin susceptibility. Gene 1995;152:27 – 33. [16] Sprengart ML, Fuchs E, Porter AG. The downstream box: an efficient and independent translation initiation signal in Escherichia coli. EMBO J 1996;15:665 – 74. [17] Garwood J, Lepesant JA. The Drosophila melanogaster homolog of ribosomal protein L27a. Biochem Biophys Res Com 1994;198:748 – 54. [18] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215:403 – 10. [19] Kyrpides NC, Woese CR, Ouzounis CA. KOW: a novel motif linking a bacterial transcription factor with ribosomal proteins. Trends Biochem Sci 1996;21:425 – 6. [20] Underwood MR, Fried HM. Characterization of nuclear localizing sequences derived from yeast ribosomal protein L29. EMBO J 1990;9:91 – 9. [21] Lee JC, Henry B, Yeh Y. Binding of proteins from the large subunits to 5.8S rRNA of Saccharomyces cere6isiae. J Biol Chem 1983;258:854 – 8. [22] Germot A, Philippe H, Le Guyader H. Evidence for the loss of mitochondria from a mitochondrial-type HSP70 in Nosema locustae. Mol Biochem Parasitol 1997;87:159 – 68. [23] Peyretaillade E, Broussolle V, Peyret P, Me´te´nier G, Gouy M, Vivare`s, CP. Microsporidia, amitochondrial protists, possess a 70-kDa heat-shock protein gene of mitochondrial evolutionary origin. Mol Biol Evol (in press). [24] Roger A, Doolittle WF. Why introns-in-pieces? Nature 1993;364:289 – 90.
Short communication
A small spliceosomal-type intron occurs in a ribosomal protein gene of the microsporidian Encephalitozoon cuniculi 1 Corinne Biderre, Guy Me´te´nier, Christian P. Vivare`s * Laboratoire de Protistologie Mole´culaire et Cellulaire des Parasites Opportunistes, UPESA CNRS 6023, Uni6ersite´ Blaise Pascal, 63177, Aubie`re Cedex, France Received 30 March 1998; received in revised form 15 April 1998; accepted 27 April 1998
Keywords: Microsporidia; Encephalitozoon cuniculi; L27a ribosomal protein; Spliceosomal intron
Among unicellular eukaryotes, microsporidia are obligately intracellular amitochondrial parasites that are considered to be of very ancient origin as deduced from the prokaryotic features of their ribosomes [1– 3], and rRNA [4] and EF1a /EF2 phylogenies [5]. Microsporidia are also characterized by small nuclear genomes ranging from 19.6 Mb in Glugea atherinae [6] to only 2.9 Mb in Encephalitozoon cuniculi [7], and thus partially spanning the prokaryotic range. Sequencing data on the E. cuniculi genome have supplied information on noncoding regions but have not allowed identification of introns [8]. However, the relatively small sample of genes sequenced in miAbbre6iations: NLS, nuclear localization signal. * Corresponding author. Tel.: +33 473 407457; fax: + 33 473 407455; e-mail: [email protected] 1 Note: Nucleotide sequence data reported in this paper are available in the EMBL, GenBankTM and DDJB data bases under the accession number AF054829.
crosporidia may be insufficiently representative for detection of potential intervening sequences. During screening of a partial plasmid library from E. cuniculi genomic DNA, we isolated a clone (c141) coding for a homolog of a large subunit ribosomal protein (L27a or L29 in yeast) gene known to contain usually one or more introns. Blast alignments first revealed that a single reading frame accounts for the entire putative L27a coding region with a termination codon UAA. However, an AUG start codon could not be placed in the position expected on the basis of the conserved character of the L27a amino-terminal region. A thorough examination of this region identified an AT-rich (61%) intervening sequence, with a size of only 28 bp, that creates a frameshift and extends from just after an AUG codon (Fig. 1A). This intron harbors consensus spliceosomal boundaries (5%-GT...AG-3%) with a 5% region identical to the consensus of higher eukaryotes (GTAAGT). In the mould Neurospora crassa, one
0166-6851/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0166-6851(98)00064-4
284
C. Biderre et al. / Molecular and Biochemical Parasitology 94 (1998) 283–286
intron also interrupts the extreme 5% region of L27a gene, but 6 additional introns are present [9]. Generally, the size of introns is at least 30 bp and can comprise over several thousands of nucleotides. However, very small introns have been identified in the processed somatic genome (macronuclear genome) of the ciliated protozoan Paramecium tetraurelia (20 – 33 bp) [10] and in the highly reduced nucleomorph genome (380 kbp) of a eukaryotic endosymbiont in chlorarachniophyte alga (18–20 bp) [11]. Such introns may be close to the minimum size. The occurrence of a spliceosomal-type intron in Encephalitozoon L27a agrees with recent lines of evidence for U2 and possibly also U6 RNAs in another microsporidian species, Vairimorpha necatrix [12]. Since no complementary sequence to the branch point GUAGUA conserved in the Vairimorpha U2 RNA was found in the Encephalitozoon intron, it may be assumed that the splicing process does not necessarily involve the complete set of the snRNAs. Either a branch point may be available for a simpler processing mechanism or only one or few adenosines may be required for the branch point. The intron frequency in nucleomorph and Paramecium genomes has been estimated to be 5.5 and 4 per kb, respectively. Considering the total length of Encephalitozoon hellem intronless b-tubulin gene [13] and seven other E. cuniculi gene sequences examined so far by us, the intron frequency is probably below 0.2 per kb. Compilation of the nucleotide sequences flanking the translation initiation site has afforded several group-specific consensuses having in common the A-residue at position −3 [14]. In contrast, this position is occupied by a G-residue in the Encephalitozoon L27a. The current microsporidian consensus based on the comparison of 7 genes would be GAAATGACA. The mRNAs from several amitochondrial protozoans display short leader regions (1 – 18 nt), making a scanning model unlikely [15]. It should be noted that an interaction between a sequence downstream of the initiation codon (downstream box) and a zone of the 16S rRNA decoding region (anti-downstream box) may be involved in prokaryotic translation initiation independent of
the Shine-Dalgarno sequence [16]. When examining the beginning of the E. cuniculi L27a translated region, a 6-nt sequence (position + 19/+ 24) may be paired to a 16S rRNA sequence (position 1231/1241) with the same location as an anti-downstream box, and with a single noncanonical pair G.G. As illustrated in Fig. 1B, putative downstream boxes can also be identified in the coding regions of the partially sequenced E. cuniculi kinesin gene and E. hellem b-tubulin gene. Thus, an additional prokaryote-like feature of microsporidia may be that the translation initiation depends on an mRNA-16S rRNA interaction placing the start codon close to the decoding region. The chromosomal location of L27a in the E. cuniculi genome, which is divided into 11 chromo-
Fig. 1. (A) Part of the nucleotide and aminoacid sequences of the E. cuniculi homolog of ribosomal protein L27a including the intron (underlined) located just after the start codon and the NLS1 motif (in bold). The untranslated regions are represented in lower cases. (B) Sequences involved in translation initiation. The putative downstream boxes (DB) from L27a and kinesin genes in E. cuniculi and b-tubulin gene in E. hellem are underlined. The 16S rRNA regions complementary to the DB sequences (anti-downstream boxes: ADB) are indicated.
C. Biderre et al. / Molecular and Biochemical Parasitology 94 (1998) 283–286
somes [6], was determined after CHEF gel electrophoresis and Southern blotting. The c141 probe gave a hybridization signal with a single chromosomal DNA band (chromosome X). To estimate the number of gene copies, Encephalitozoon genomic DNA was digested with endonucleases EcoRI and P6uII that do not cut within the L27a coding region, and probed with c141 insert. The hybridization with a single fragment indicates there is only one copy of L27a per haploid genome, as in Neurospora crassa [9] and Drosophila melanogaster [17]. Assuming the first methionine is not removed, the mature protein should contain 147 residues (predicted molecular mass: 17015 Da), with a basic isoelectric point (10.51). The amino acid similarities only extend from 62.5 to 65.3% when compared to L27a/L29 proteins from either animals, plants, fungi or some other protozoans in the SwissProt data base [18]. A novel motif of 27 residues with an invariant glycine residue at position 11, named KOW (as an acronym for the authors’ surnames), is shared by the members of both the NusG family (a bacterial transcription elongation factor) and several large subunit ribosomal protein families, and has been viewed as the principal mediator for RNA-protein interactions [19]. A similar motif can be identified in the E. cuniculi L27a, as in other L27a/L29 homologs, referring to the invariant Gly-11 (Fig. 2). Its location close to the N-terminus is identical in the L27 and L24 families. It has been shown that the N-terminus of the yeast L29 protein promotes import into the nucleus by means of two nuclear localization sequences (NLSs) KTRKHRG and KHRKHPG, rich in basic amino acids, designated NLS1 and NLS2, at positions six and 23 respectively [20]. The E. cuniculi KOW-like motif spans two sequences (KTRKLRG and KHRKHSG at positions seven and 24) homologous to NLSs, with NLS2 characterized by a residue Ser in penultimate position. The yeast L29 can bind to 5.8S rRNA [21] but immunofluorescence and mutagenesis experiments failed to demonstrate a specific role of NLSs in such a binding. That microsporidia are devoid of free 5.8S rRNA [4] also indicates another involvement of these sequences in ribosomal assembly (possibly as ligands for a nuclear pore receptor).
285
Fig. 2. Comparison of the characteristic KOW motif found in all members of the ribosomal protein families L24 (and K34), L26 and L27. Sequence alignments of multiple L27a genes show that such a motif exists in this family with an invariant glycine residue at position 11 (in bold). Numbering denotes the position of the motif in the protein. Accession numbers are indicated. L27: P. s.: Pyrobotris stellata; L27a: E. c.: Euplotes crassus; T. b.: Trypanosoma brucei brucei; S. c.: Saccharomyces cere6isiae; D. m: Drosophila melanogaster; L24: E. coli: Escherichia coli; B. s.: Bacillus subtilis; K24: P. s.: Pisum sati6um; N. t.: Nicotiana tabacum.
Finally, the evidence reported here that a microsporidian gene may be interrupted by a spliceosomal-type intron, combined with data supporting a secondary loss of mitochondria during evolution of microsporidia [22,23], is consistent with the theory that spliceosomal introns originated from fragmentation of group II selfsplicing ones [24]. A subsequent loss of many nuclear introns may be a result of a general evolutionary trend to genome and cell miniaturization in Microsporidia.
References [1] Ishihara R, Hayashi Y. Some properties of ribosomes from sporoplasm of Nosema bombycis. J Invertebr Pathol 1968;11:377 – 85. [2] Curgy JJ, Vavra J, Vivare`s CP. Presence of ribosomal RNAs with prokaryotic properties in Microsporida, eukaryotic organisms. Biol Cell 1980;38:49 – 52. [3] Vossbrinck CR, Woese CR. Eukaryotic ribosomes that lack a 5.8S RNA. Nature 1986;320:287 – 8. [4] Vossbrinck CR, Maddox JV, Friedman S, DebrunnerVossbrinck BA, Woese CR. Ribosomal RNA sequence suggests microsporidia are extremely ancient eukaryotes. Nature 1987;326:411 – 4.
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C. Biderre et al. / Molecular and Biochemical Parasitology 94 (1998) 283–286
[5] Kamaishi T, Hashimoto T, Nakamura Y, Masuda Y, Nakamura F, Okamoto KI, Shimizu M, Hasegawa M. Complete nucleotide sequences of the genes encoding translation elongation factors 1a and 2 from a microsporidian parasite, Glugea plecoglossi: implications for the deepest branching of eukaryotes. J Biochem 1996;120:1095– 103. [6] Biderre C, Page`s M, Me´te´nier G, David D, Bata J, Prensier G, Vivare`s CP. On small genomes in eukaryotic organisms: molecular karyotypes of two microsporidian species (Protozoa) parasites of vertebrates. CR Acad Sci Paris 1994;317:399 – 404. [7] Biderre C, Page`s M, Me´te´nier G, Canning EU, Vivare`s CP. Evidence for the smallest nuclear genome (2.9 Mb) in the microsporidium Encephalitozoon cuniculi. Mol Biochem Parasitol 1995;74:229–31. [8] Biderre C, Duffieux F, Peyretaillade E, Glaser P, Peyret P, Danchin A, Page`s M, Me´te´nier G, Vivare`s CP. Mapping of repetitive and non-repetitive DNA probes to chromosomes of the microsporidian Encephalitozoon cuniculi. Gene 1997;191:39–45. [9] Kreader CA, Heckman JE. Isolation and characterization of a Neurospora crassa ribosomal protein gene homologous to CYH2 of yeast. Nucleic Acids Res 1987;1015:9027– 42. [10] Russell CB, Fraga D, Hinrichsen RD. Extremely short 20 – 33 nucleotide introns are the standard length in Paramecium tetraurelia. Nucleic Acids Res 1994;22:1221– 5. [11] Gilson PR, McFadden GI. The miniaturized nuclear genome of a eukaryotic endosymbiont contains genes that overlap, genes that are cotranscribed, and the smallest known spliceosomal introns. Proc Natl Acad Sci USA 1996;93:7737 – 42. [12] DiMaria P, Palic B, Debrunner-Vossbrinck BA, Lapp J, Vossbrinck CR. Characterization of the highly divergent U2 RNA homolog in the microsporidian Vairimorpha necatrix. Nucleic Acids Res 1996;24:515–22.
.
[13] Edlind TD, Li J, Visvesvara GS, Vodkin MH, McLaughlin GL, Katiyar SK. Phylogenetic analysis of b-tubulin sequences from amitochondrial protozoa. Mol Phylogenet Evol 1996;5:359 – 67. [14] Yamauchi K. The sequence flanking translational initiation site in protozoa. Nucleic Acids Res 1991;19:2715 – 20. [15] Katiyar SK, Visvesvara GS, Edlind TD. Comparisons of ribosomal RNA sequences from amitochondrial protozoa: implications for processing, mRNA binding and paromomycin susceptibility. Gene 1995;152:27 – 33. [16] Sprengart ML, Fuchs E, Porter AG. The downstream box: an efficient and independent translation initiation signal in Escherichia coli. EMBO J 1996;15:665 – 74. [17] Garwood J, Lepesant JA. The Drosophila melanogaster homolog of ribosomal protein L27a. Biochem Biophys Res Com 1994;198:748 – 54. [18] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215:403 – 10. [19] Kyrpides NC, Woese CR, Ouzounis CA. KOW: a novel motif linking a bacterial transcription factor with ribosomal proteins. Trends Biochem Sci 1996;21:425 – 6. [20] Underwood MR, Fried HM. Characterization of nuclear localizing sequences derived from yeast ribosomal protein L29. EMBO J 1990;9:91 – 9. [21] Lee JC, Henry B, Yeh Y. Binding of proteins from the large subunits to 5.8S rRNA of Saccharomyces cere6isiae. J Biol Chem 1983;258:854 – 8. [22] Germot A, Philippe H, Le Guyader H. Evidence for the loss of mitochondria from a mitochondrial-type HSP70 in Nosema locustae. Mol Biochem Parasitol 1997;87:159 – 68. [23] Peyretaillade E, Broussolle V, Peyret P, Me´te´nier G, Gouy M, Vivare`s, CP. Microsporidia, amitochondrial protists, possess a 70-kDa heat-shock protein gene of mitochondrial evolutionary origin. Mol Biol Evol (in press). [24] Roger A, Doolittle WF. Why introns-in-pieces? Nature 1993;364:289 – 90.