Expressed sequence tags (ESTs) analysis of <italic>Acanthamoeba healyi</italic>

Hyun-Hee Kong; Mee-Yeul Hwang; Hyo-Kyung Kim; Dong-Il Chung

doi:10.3347/kjp.2001.39.2.151

Korean J Parasito > Volume 39(2):2001 > Article

Kong, Hwang, Kim, and Chung: Expressed sequence tags (ESTs) analysis of Acanthamoeba healyi

Original Article

The Korean Journal of Parasitology 2001; 39(2): 151-160.

Published online: June 30, 2001

DOI: https://doi.org/10.3347/kjp.2001.39.2.151

Expressed sequence tags (ESTs) analysis of Acanthamoeba healyi

Hyun-Hee Kong¹, Mee-Yeul Hwang¹, Hyo-Kyung Kim², Dong-Il Chung¹

¹Department of Parasitology, Kyungpook National University School of Medicine, Taegu 700-422, Korea.

²Department of Neurology, Ulsan University College of Medicine, Ulsan 682-060, Korea.

Corresponding author (dichung@knu.ac.kr)

Received March 12, 2001 Accepted May 22, 2001

Abstract

Randomly selected 435 clones from Acanthamoeba healyi cDNA library were sequenced and a total of 387 expressed sequence tags (ESTs) had been generated. Based on the results of BLAST search, 130 clones (34.4%) were identified as the genes enconding surface proteins, enzymes for DNA, energy production or other metabolism, kinases and phosphatases, protease, proteins for signal transduction, structural and cytoskeletal proteins, cell cycle related proteins, transcription factors, transcription and translational machineries, and transporter proteins. Most of the genes (88.5%) are newly identified in the genus Acanthamoeba. Although 15 clones matched the genes of Acanthamoeba located in the public databases, twelve clones were actin gene which was the most frequently expressed gene in this study. These ESTs of Acanthamoeba would give valuable information to study the organism as a model system for biological investigations such as cytoskeleton or cell movement, signal transduction, transcriptional and translational regulations. These results would also provide clues to elucidate factors for pathogenesis in human granulomatous amoebic encephalitis or keratitis by Acanthamoeba.

Key words: Acanthamoeba healyi, expressed sequence tags, novel genes

INTRODUCTION

The genus, Acanthamoeba, a human pathogen causing granulomatous amoebic encephalitis (GAE) and amebic keratitis (Sisson et al., 1994), has been known to be a vector for pathogenic microbes such as Mycobacterium spp., Listeria spp., and Legionella spp. (Jadin, 1973; Ly & Muller, 1990; Field, 1991). In addition to these medical importances, it is also well known that Acanthamoeba is a good model system to study eukaryotic cell biology due to its relatively large size, rapid growth in culture, active motility, and well developed cytoskeleton (Byers et al., 1990).

Although the ploidy and the total DNA content of the genus Acanthamoeba are unclear at the present, Byers et al. (1990) speculated that the genome size of the amoeba would be ~1×10⁸ bp of which the size could express more than 5,000 transcripts; however, only a few genes and proteins have been reported. Most of the genes identified are 18s rDNA sequences for the taxonomic purpose (Gast et al., 1996; Stothard et al., 1998), actin, myosins and actin binding proteins to study cytoskeleton (Nellen & Gallwitz, 1982; Cooper et al., 1986; Jung et al., 1989; Pollard & Rimm, 1991; Kelleher et al., 1995; Lee et al., 1999), and mitochondrial genome (Burger et al., 1995). Therefore most genes of Acanthamoeba have yet to be uncovered.

Characterization of the transcribed genes in a certain organism by expressed sequence tag (EST) analysis, single pass sequencing of randomly selected cDNA clones has been applied to many organisms including parasitic protozoa such as Plasmodium falciparum (Chakrabarti et al, 1994), Trypanosoma brucei rhodesiense (El-Sayed et al., 1995), Toxoplasma gondii (Wan et al., 1996; Manger et al., 1998), and Entamoeba histolytica (Azam et al., 1996; Tanaka et al., 1997). In addition to the candidate genes for pathogenic factors, many novel genes for stage specific, cell cycle regulatory, or related to cell signaling were identified in these protozoa by EST analysis.

ESTs analysis of Acanthamoeba could characterize the expression pattern of the genes, providing invaluable information to understand the genetics and the identification of many novel genes in this genus.

In the present study, we report the results of ESTs analysis of A. healyi OC-3A strain isolated from the brain of a GAE patient.

MATERIALS AND METHODS

Amoeba culture

An isolate of Acanthamoeba from the brain of a GAE patient, A. healyi OC-3A, was obtained from ATCC and cultured in Proteose peptone-Yeast extract-Glucose medium at 25℃.

RNA preparation

Trophozoites of A. healyi, washed with phosphate-buffered saline (PBS), were homogenized with β-mercaptoethanol and RNA denaturation solution (Stratagene, San Diego, CA, USA). One milliliter of 3 M sodium acetate (pH 4.0) was added to the solution and mixed by inversion. Water saturated phenol was added and mixed well with the solution. The mixture was shaken vigorously after adding chloroform/isoamyl alcohol (24:1), and then was incubated on ice for 15 min. The supernatant of the mixture after centrifugation was moved to a new tube and incubated with 1 volume of isopropanol at -20℃ for 1 hr for precipitation of the RNA. The RNA pellet was dissolved with the RNA denaturation solution and reprecipitated with equal volume of isopropanol. DEPC treated Q-water was added to the ethanol washed and vacuum dried RNA pellet. mRNA was purified from total RNA sample using Poly (A) Quick mRNA isolation kit (Stratagene, San Diego, CA, USA).

Construction of cDNA library

A unidirectional oligo (dT)-primed EcoRI/Xho I cDNA library was constructed in UNI-ZAP™ (Stratagene, San Diego, CA, USA). Briefly, first strands, synthesized by reverse transcription of mRNA of A. healyi, were used to make second strands by the action of RNase H and DNA polymerase I. cDNA with blunted termini were ligated with Eco RI adapters and digested with Xho I. After size fractionation, cDNA over the size 400 bp were packaged into UNI-ZAPTM XR vector arms by ligation. Packaged cDNAs were incubated with the host cell of XL1-blue MRF strain on agarose LB medium. More than 5 millions clones of this library were amplified and the aliquots of the library were stored at 4℃ until use.

Sequencing of randomly selected cDNA clones

cDNA library mixture were incubated with XL1-Blue MRF cell to allow in vivo excision using ExAssist helper phage, transfected into Escherichia coli SOLR strain, and plated on LB-ampicillin agar plates. Each randomly selected colony from the plates was inoculated into LB-ampicillin broth and incubated at 37℃ overnight. Plasmid with the cDNA insert was extracted with plasmid DNA purification system (Wizard® Plus Minipreps, Promega, USA). The size of cDNA inserts obtained by digestion of the plasmid DNA with EcoR I and Xho I were estimated by electrophoresis on agarose gel with Hind III digested λ phage DNA, a DNA size standard. Alkaline denaturation of the plasmid DNA and the dideoxy chain termination method using DNA sequencing kit (T7 Sequenase version 2.0, Amersham, USA) and ³⁵S dATP were applied to elucidate the sequence data of the randomly selected cDNA clones. After electrophoresis of the reaction samples for sequencing, the vacuum dried acrylamide gel was exposed on X-ray film. Sequence data were edited to remove vector and ambiguous sequences, and less than 100 bases were also rejected.

Basic Local Alignment Search Tool (BLAST) search

The sequence data of cDNA clones by random partial sequencing were subjected to examine similarities in the nucleic acid and protein databases using the BLAST on the National Center for Biotechnology Information (NCBI). The cDNA sequences were compared against nucleotide data by the program BLASTN, and the conceptual translation products of query sequences against translated nucleic acid and protein databases by BLASTX. Matches were considered to be significant only when the probability (P) was less than 0.0001 and scores were >160 for BLASTN and >80 for BLASTX.

RESULTS

Among 435 cDNA clones sequenced, the sequence data of 378 clones were submitted for blast search (Table 1). It was 130 clones (34.4%) identified by high homology with the DNA sequence of Acanthamoeba or other organisms in the public data base. Although 15 clones were matched with Acanthamoeba genes already studied, they were just 3 kinds of genes and twelve of them were the actin gene, the most commonly found gene in this study. The other 115 clones (88.5%, 94 different genes) are reported in the present paper for the first time in this genus.

Based on the results of BLAST search, ESTs with predicted or known functions were classified into putative cellular roles (Table 2). They were 4 clones for surface protein, 2 clones for DNA metabolism, 12 clones for energy metabolism, 10 clones for kinase and phosphatase, 17 clones for other metabolism, 1 clone for protease, 10 clones for signal transduction, 18 clones for structure and cytoskeleton, 3 clones for cell cycle related proteins, 5 clones for transcription factor, 32 clones for transcription and translational machinery, 4 clones for transporters, and 12 clones for not classified.

DISCUSSION

It is the first time to analyze ESTs of Acanthamoeba known as an human pathogen and a good model system for biological studies. The most frequently presented gene was actin gene which appeared 12 times. This was as expected because Acanthamoeba has well developed cytoskeleton and move very actively (Pollard, 1982). The biggest class among identified clones was genes for protein synthesis with the number of 32 from 130 clones. Similar results were reported in the EST analysis of Entamoeba histolytica, Typanosoma cruzi, and E. dispar (Tanaka et al., 1997; Verdun et al., 1998; Sharma et al., 1999).

Among four clones of genes for surface protein, two were identified as non-integrin type laminin binding protein. Adhesion would be the very first and important step to infect host by tissue invading parasites. Laminin binding proteins has been reported from several parasitic organisms including Trichomonas vaginalis (Silva Filho et al., 1988), Trypanosoma brucei rhodesiense (gene bank, W99296), Leishmania donovani (Ghosh et al., 1996) and Echinococcus granulosus (Zhang et al., 1997). The ability to recognize extracellular matrix proteins such as laminin or fibronectin has been known to correlate with invasiveness (Silva Filho et al., 1988; Ghosh et al., 1999). Further characterization of laminin binding protein in A. healyi would help to discover the mechanisms of the invasion by the amoeba.

A lot of genes for proteins involved in various metabolism were found in Acanthamoeba for the first time in this study except for the ubiquitin (Ub) gene (Hu & Henney, 1997). Six clones were identified to be associated with Ub-proteasome protein destruction system. Ub-proteasome pathway of intracellular proteolysis has been shown to be involved in various biologically important processes, such as the cell cycle, cellular metabolism, apoptosis, signal transduction, immune response, and protein quality control (Hilt and Wolf, 1996; Ciechanover, 1998; Tanaka, 1998).

Little information has been reported for signal transduction in genus Acanthamoeba. In this study, many kinds of cell signaling molecules, including Rabs, 14-3-3 protein and rac, were identified. Rabs regulate the flux through individual steps of the intracellular membrane trafficking pathway. The small GTPase Rab2 is a resident of pre-Golgi intermediates and required for protein transport from the endoplasmic reticulum to the Golgi complex (Tisdale et al., 1992). The GTP binding motif, GDTGVGKS, was conserved in the sequence of the clone Ahc040 identified as Rab2. A Rab protein isolated by EST analysis had been characterized in Trypanosoma brucei by Field et al. (1999).

In addition, gene for prohibitin known to negatively regulate cell proliferation in mammals was identified in Acanthamoeba. Further studies on this gene would give information in the regulation of Acanthamoeba proliferation and development. Three clones were identified as gene for vault protein showing high homology with that of Dictyostelium discoideum. Although vault proteins are found in nearly all eukaryotic cells, the function of the protein has yet to be elucidated.

Lots of the genetic information of Acanthamoeba obtained in this study would be very helpful to figure out pathogenetic mechanisms of GAE or keratitis by Acanthamoeba and to develop therapeutic reagents specific to the amoeba.

Notes

This study was in part supported by a grant from Kyungpook National University.

Note: EST sequence data reported in this paper is available in GenBankTM database under the accession number from AT001240 to AT001616.

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Table 1.

Acanthamoeba healyi cDNA library sequencing

EST category	No. of clones
Total clones sequenced	435
ESTs submitted for BLAST search	378
ESTs identified by homology	130
Unique ESTs identified	94
ESTs with homology to Acanthamoeba genes	15
Redundant ESTs	20
Common ESTs
actin	12
60S acidic ribosomal protein PO	4
elongation factor 1 alpha	3
major vault protein	3

Table 2.

Significant matches of Acanthamoeba healyi ESTs with database sequences from other organisms

Clone No.	Length	Identification	Organism	Data base	Accession No.	Score	Probability
Surface protein
Ah161	208	coatomer delta subunit	Oryza sativa	sp^a)	P49661	182	3.80E-17
Ah217	275	laminin receptor like protein (P40)	Daucus carota	dbj^b)	AB012702	214	3.10E-21
Ah477	256	laminin receptor like protein (P40)	D. carota	dbj	AB012702	202	1.20E-19
Ah489	202	70 kD peroxisomal membrane protein	Mus musculus	sp	P55096	145	4.30E-12
DNA metabolism
Ah468	206	methyl transferase	Saccharomyces cerevisiae	sp	P25087	226	2.60E-23
Ah534	308	double-strand break repair protein	Caenorhabditis elegans	gb^c)	U40029	236	3.50E-24
Energy metabolism
Ah015	153	acyl-CoA dehydrogenase	C. elegans	emb^d)	AL032621	102	1.40E-06
Ah029	227	ATP synthase gamma chain	Arabidopsis thaliana	sp	Q01908	89	0.00034
Ah035	180	3-hydroxyisobutyrate dehydrogenase	Rabbit	sp	P32185	107	5.50E-07
Ah089	262	adenylosuccinate synthetase precursor	A. thaliana	sp	Q96529	217	1.00E-21
Ah110	261	NADP-isocitrate dehydrogenase	Glycine max	gb	AF095445	286	3.20E-31
Ah335	256	NADP-isocitrate dehydrogenase	Pig	gb	M86719	523	1.80E-33
Ah452	193	6-phosphogluconate dehydrogenase	Cunninghamella elegans	emb	Y17297	140	1.90E-11
Ah475	181	fatty acid synthase, subunit alpha	Candida albicans	sp	P43098	206	1.20E-20
Ah532	364	NADH-ubiquinone oxidoreductase	Homo sapiens	sp	Q16795	277	1.00E-29
Ah542	281	enolase	Spongilla sp.	gb	U85829	198	5.00E-19
Ah577	254	fatty acid synthase, subunit alpha	C. albicans	sp	P43098	135	5.40E-23
Ah595	137	transketolase	Spinacia oleracea	gb	L76554	127	5.50E-10
Immunology
Ah342	228	immunoglobulin heavy chain V-D-J region	Oryctolagus cuniculus	gb	AF058603	77	0.015
Ah464	244	Leukotriene A-4 hydrolase	Cavia porcellus	sp	P19602	110	4.90E-07
Kinase and phosphatase
Ah042	225	cytohesin 2	H. sapiens	emb	Z94160	100	1.00E-05
Ah066	271	mevalonate kinase	Rat	sp	P17256	100	2.10E-06
Ah119	243	c2 domain	H. sapiens	gb	AC005278	177	2.90E-16
Ah172	269	3-phosphoglycerate kinase	Thermotoga maritime	emb	X75437	254	8.90E-27
Ah177	160	myosin heavy chain kinase B	Dictyostelium discoideum	sp	P90648	91	1.30E-07
Ah233	232	nucleoside diphosphate kinase 3	A. thaliana	gb	AF044265	227	9.30E-23
Ah401	119	S-phase kinase associated protein like	D. discoideum	gb	U73686	90	9.50E-05
Ah436	207	Skb1Hs	H. sapiens	gb	AF015913	167	4.40E-15
Ah463	223	phosphatases pleiotropic regulator	A. thaliana	sp	Q39190	280	1.40E-30
Ah482	242	phosphoenolpyruvate carboxykinase	Chlorobium limicola	sp	Q08262	116	7.30E-08
Ah584	272	protein kinase	Schizosaccharomyces pombe	emb	AL022245	80	8.90E-03
Other metabolism
Ah030	208	ADP-ribosylglycohydrolase	Methanococcus jannaschii	pir^e)	C64448	83	0.0017
Ah039	237	phosphomannomutase	C. elegans	emb	AL021481	84	2.00E-03
Ah043	190	ubiquitin	Acanthamoeba castellnii	pir	S45304	252	6.30E-27
Ah090	217	thioredoxin peroxidase	Trypanosoma brucei rhodesiense	sp	Q26695	213	2.30E-21
Ah111	293	protein disulfide isomerase	D. discoideum	gb	AF019112	245	1.80E-25
Ah162	214	dihydrolipoamide dehydrogenase	garden pea	pir	A42494	99	1.10E-05
Ah171	268	thioredoxin peroxidase	Pig	sp	P52552	117	4.00E-09
Ah173	224	ubiquitin fusion degradation protein 1	M. musculus	sp	P70362	112	5.70E-09
Ah174	185	protein disulfide isomerase	Drosophila melanogaster	sp	P54399	168	3.30E-15
Ah179	275	uricase	Sus scrofa	sp	P16164	61	1.20E-06
Ah346	236	proteosome subunit DD5	D. discoideum	sp	P34120	555	3.50E-36
Ah381	260	20S proteosome subunit PAC1	A. thaliana	gb	AF043521	335	5.50E-38
Ah421	151	glutamate decarboxylase	D. melanogaster	sp	P20228	117	1.20E-08
Ah458	150	ubiquitin	A. castellnii	sp	P49634	148	6.60E-13
Ah461	229	proteosome subunit DD5	D. discoideum	sp	P34120	310	1.10E-34
Ah581	228	ELAV protein (RNA metabolism)	D. melanogaster	sp	P16914	102	5.50E-06
Ah582	190	thiazole mono	Escherichia coli	sp	Q46948	97	2.30E-07
Protease
Ah068	195	aminopeptidase-like protein	A. thaliana	emb	Z99708	104	1.80E-06
Signal transduction
Ah040	260	GTP binding protein (rab2)	Chlamydomonas reinhardtii	sp	Q39570	187	1.40E-17
Ah155	250	ras related protein RAB4	D. discoideum	sp	P36410	386	4.60E-45
Ah184	284	visinin like protein 3 (VILIP-3)	H. sapiens	sp	P37235	67	2.00E-05
Ah348	182	Guanine nucleotide binding protein	Hydra vulgaris	sp	Q25189	143	5.90E-12
Ah487	185	14-3-3 protein	D. melanogaster	sp	P92177	198	8.30E-19
Ah501	170	14-3-3 protein	D. melanogaster	sp	P92177	183	1.20E-16
Ah503	283	rac GTP binding protein Arac10	A. thaliana	gb	AF079485	275	1.20E-29
Ah578	246	small GTP binding protein Rab-7	H. sapiens	gb	U44104	188	8.90E-18
Ah603	307	GTP binding nuclear protein SPI1	S. pombe	sp	P28748	386	7.00E-45
Ah622	251	G protein beta subuit like	M. musculus	pir	I49700	288	3.80E-31
Ah627	255	G protein beta subuit like	M. musculus	pir	I49700	299	5.40E-33
Structural and cytoskeletal
Ah016	225	F-actin capping protein bets subunit	D. discoideum	sp	P13021	220	2.80E-22
Ah031	195	actin	A. castellanii	sp	P02578	280	9.90E-31
Ah072	245	actin-bundling protein	Physarum polycephalum	pir	S32566	93	2.50E-12
Ah145	217	actin	A. castellanii	sp	P02578	337	1.80E-38
Ah215	286	actin	A. castellanii	sp	P02578	425	2.60E-50
Ah219	173	actin-related protein 2 (Arp2)	A. castellanii	sp	P53487	197	1.10E-19
Ah220	192	gelation factor	D. discoideum	sp	P13466	162	3.30E-14
Ah221	268	actin	A. castellanii	sp	P02578	384	1.40E-44
Ah231	271	tubulin alpha 1	O. sativa	sp	P28752	297	1.00E-32
Ah341	275	actin	A. castellanii	sp	P02578	296	4.50E-38
Ah344	197	actin	A. castellanii	sp	P02578	227	2.90E-30
Ah354	159	actin	A. castellanii	sp	P02578	233	1.20E-24
Ah361	287	actin	A. castellanii	sp	P02578	461	2.80E-55
Ah388	270	actin	A. castellanii	sp	P02578	469	2.00E-56
Ah402	167	actin	A. castellanii	sp	P02578	277	1.00E-30
Ah499	295	actin	A. castellanii	sp	P02578	498	2.30E-60
Ah599	164	actin	A. castellanii	sp	P02578	260	2.30E-28
Ah614	211	coronin (actin binding protein)	D. discoideum	sp	P27133	164	1.20E-14
Cell cycle related
Ah114	193	prohibitin	Toxocara canis	gb	U97204	100	5.90E-06
Ah151	234	diaphanous 1	H. sapiens	gb	AF051782	131	6.00E-10
Transcription factors
Ah147	263	myocyte-enhancer-binding factor 2	D. melanogaster	emb	X83527	114	4.40E-13
Ah163	230	myocyte-enhancer-binding factor 2	Cyprinus carpio	dbj	AB012884	85	1.10E-10
Ah235	271	SNF4-like protein (Pv42p)	Phaseolus vulgaris	gb	U40713	90	3.60E-04
Ah356	150	dribble protein	D. melanogaster	emb	Z96931	151	2.50E-13
Ah396	205	dribble protein	D. melanogaster	emb	Z96931	280	1.10E-30
Transcription and translational machinery
Ah006	289	ribosomal protein L38	rat	emb	X57007	215	8.90E-08
Ah082	249	elongation factor 2	C. elegans	sp	P29691	106	1.80E-14
Ah085	266	elongation factor 1 gamma	C. elegans	sp	P54412	108	4.00E-07
Ah116	228	ribosomal protein L7	H. sapiens	pir	S30212	114	2.60E-07
Ah146	237	40S ribosomal protein S16	Fritillaria agrestis	sp	O22647	215	1.50E-21
Ah148	258	RNAse L inhibitor	M. musculus	gb	U90446	181	8.90E-17
Ah156	264	elongation factor 1-alpha	Blastocystis hominis	sp	P54959	335	5.50E-38
Ah181	288	ATP-dependent RNA helicase	H. sapiens	emb	Y14768	198	1.10E-20
Ah187	270	elongation factor 1B gamma	O. sativa	dbj	D89802	151	1.40E-12
Ah193	247	60S acidic ribosomal protein PO	Bos taurus	sp	Q95140	185	2.30E-17
Ah208	275	RNA binding protein (multiple splicing)	H. sapiens	dbj	D84107	114	1.90E-07
Ah232	167	40S ribosomal protein S14	Maize	sp	P19951	149	4.70E-13
Ah339	255	elongation factor 2	Trypanosoma cruzi	dbj	D50806	161	5.20E-14
Ah345	257	60S acidic ribosomal protein PO	B. taurus	sp	Q95140	185	2.60E-17
Ah347	209	elongation factor 1-alpha	Bombyx mori	sp	P29520	241	2.90E-25
Ah365	236	ribosomal protein S16	Mouse	sp	P14131	160	5.90E-14
Ah390	287	elongation factor 1 alpha	B. mori	sp	P29520	415	6.20E-49
Ah399	219	elongation factor 1 beta	D. melanogaster	emb	AL031863	86	7.80E-04
Ah420	134	ribosomal protein L41	K. fragilis	gb	M62394	154	7.90E-07
Ah448	200	tRNA splicing protein SPL1	C. maltosa	sp	P87187	146	3.10E-12
Ah454	222	60S ribosomal protein L14EB	S. cerevisiae	sp	P38754	136	3.30E-11
Ah460	160	60S acidic ribosomal protein PO	Plasmodium falciparum	sp	Q94660	91	4.70E-05
Ah493	301	40S ribosomal protein RS16	S. cerevisiae	sp	P40213	294	3.30E-32
Ah500	218	40S ribosomal protein S3A	H. sapiens	sp	P48154	135	1.40E-10
Ah511	204	40S ribosomal protein S7	Avicennia marina	gb	AF098519	149	1.20E-12
Ah514	207	60S acidic ribosomal protein PO	P. falciparum	sp	Q94660	145	4.80E-12
Ah575	219	elongation factor 1 beta	D. melanogaster	emb	AL031863	86	7.90E-04
Ah579	134	phenylalanyl-tRNA synthetase	C. albicans	emb	AL033503	120	4.90E-09
Ah615	211	60S ribosomal protein L14 (CAG-ISL 7)	H. sapiens	sp	P50914	163	1.60E-14
Ah616	240	ribosomal protein L32	Kluveromyces lactis	gb	L05772	174	1.80E-04
Ah621	255	splicing factor	H. sapiens	sp	Q08170	95	6.50E-05
Ah623	223	elongation factor 1 alpha	B. mori	sp	P29520	315	2.10E-35
Transporters
Ah037	171	mitochondrial carrier protein DIF-1	C. elegans	pir	S55056	119	8.50E-10
Ah074	177	protein transport protein SEC13	H. sapiens	sp	P55735	87	4.10E-05
Ah528	241	adenine nucleotide translocator	Lupinus albus	emb	AJ003197	266	1.40E-28
Ah572	181	protein transport protein SEC23	M. musculus	pir	I60247	141	1.10E-11
Not classified
Ah008	215	hypothetical 69.8 kD protein	S. cerevisiae	sp	Q06053	142	1.40E-11
Ah013	311	yeast UTR3 protein	S. cerevisiae	emb	Z72510	119	4.90E-08
Ah087	222	hypothetical 36.8 kD protein	S. pombe	sp	Q10169	93	8.70E-05
Ah335	214	Major vault protein alpha	D. discoideum	sp	P34118	198	3.30E-25
Ah384	196	hypothetical protein YER007c-a	S. cerevisiae	pir	S53543	99	2.50E-09
Ah446	169	hypothetical protein YER007c-a	S. cerevisiae	pir	S53543	101	3.40E-06
Ah457	240	SGT protein	Rattus norvegicus	emb	AJ222724	90	3.10E-05
Ah478	185	Nbr1	M. musculus	gb	U73039	102	2.90E-06
Ah486	291	Down syndrome critical region protein A	H. sapiens	sp	O14972	215	2.40E-21
Ah492	248	EST yk400g3.3	C. elegans	emb	Z81592	86	1.10E-03
Ah543	218	Major vault protein alpha	D. discoideum	sp	P34118	165	1.00E-14
Ah574	175	Major vault protein beta	D. discoideum	sp	P54659	137	2.10E-11
rRNA
Ah197	256	large subunit ribosomal RNA	Plumbago auriculata	gb	AF036492	746	5.60E-71

Database abbreviation:

^a) sp, swissprot;

^b) dbj, Data Base of Japan;

^c) gb, GenBank;

^d) emb, EMBL;

^e) pir, Protein Informatio Resource.