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Expressed sequence tags (ESTs) analysis of Acanthamoeba healyi
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Original Article

Expressed sequence tags (ESTs) analysis of Acanthamoeba healyi

Hyun-Hee Kong1, Mee-Yeul Hwang1, Hyo-Kyung Kim2, Dong-Il Chung1
The Korean Journal of Parasitology 2001;39(2):151-160.
Published online: June 30, 2001

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

Copyright © 2001 by The Korean Society for Parasitology

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  • 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
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.
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.
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.
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.

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
Table 1.
 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
Table 2.
Clone No. Length Identification Organism Data base Accession No. Score Probability
Surface protein
 Ah161 208 coatomer delta subunit Oryza sativa spa) P49661 182 3.80E-17
 Ah217 275 laminin receptor like protein (P40) Daucus carota dbjb) 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 gbc) U40029 236 3.50E-24
Energy metabolism
 Ah015 153 acyl-CoA dehydrogenase C. elegans embd) 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 pire) 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.

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Expressed sequence tags (ESTs) analysis of Acanthamoeba healyi
Korean J Parasitol. 2001;39(2):151-160.   Published online June 30, 2001
DOI: https://doi.org/10.3347/kjp.2001.39.2.151
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Expressed sequence tags (ESTs) analysis of Acanthamoeba healyi
Korean J Parasitol. 2001;39(2):151-160.   Published online June 30, 2001
DOI: https://doi.org/10.3347/kjp.2001.39.2.151
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Expressed sequence tags (ESTs) analysis of Acanthamoeba healyi
Expressed sequence tags (ESTs) analysis of Acanthamoeba healyi
 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
Clone No. Length Identification Organism Data base Accession No. Score Probability
Surface protein
 Ah161 208 coatomer delta subunit Oryza sativa spa) P49661 182 3.80E-17
 Ah217 275 laminin receptor like protein (P40) Daucus carota dbjb) 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 gbc) U40029 236 3.50E-24
Energy metabolism
 Ah015 153 acyl-CoA dehydrogenase C. elegans embd) 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 pire) 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
Table 1. Acanthamoeba healyi cDNA library sequencing
Table 2. Significant matches of Acanthamoeba healyi ESTs with database sequences from other organisms

Database abbreviation:

sp, swissprot;

dbj, Data Base of Japan;

gb, GenBank;

emb, EMBL;

pir, Protein Informatio Resource.

Table 1.

Table 2.

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