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Genetic relatedness of the elongationfactor-1 alpha in blastocystis isolates
Poster Sessions I Parasitology International 47 (Suppl.) (1998) 283-389
P-0802 GENETIC RELATEDNESS FACTOR-l
OF THE ELONGAnON ALPHA IN BLASTOCYSTIS ISOLATES
Ho*, Singh M*, Jeyaseelan K**, and Yap EH Departments of * Microbiology and ** Biochemistry, Faculty of Medicine, National University of Singapore, Singapore 119260 Blastocystis is an intestinal protozoan parasite found in a wide variety of animal hosts e.g. mammals, reptiles, birds and insects. This study examines nucleotide sequences of the elongation factor1 alpha (EF-la) in isolates of Blastocystis from humans, reptiles and rats. A pair of sense and anti-sense primers comprising of 21 and 18 nucleotides respectively, for the EF- I a wassynthesized and used in PCR with DNA from 13 Bkzstocystis isolates. A 900bp product consistently amplified in all the isolates was used in the evaluation of their genetic relatedness.
p-o804
337
FASCIOLAHEPATZCACATHEPSIN L : GENE STRUCTURE, EXPRESSIONIN BACTERIA, AND IN VITRO PROCESSING
Yamasaki l-l and Aoki T. Department of Parasitology, Unwersity School of Medicine, Tokyo113-0@33, Japan
Juntendo
Cathepsin L in Fusciolahepatica is thought to be involved in important functions including host protein digestion, tissue penetration, and so on. We have reported that the enzyme is synthesized as an inactive preprocathepsin L, targeted to the secretory granules in intestinal epithehal cells, and secreted into the intestinal lumen. In the present paper, the genomic structure of the F. h+ib cathepsin L gene (fhCATL) is clarified. The processing mechanism of procathepsin L to mature cathepsin L and the possible function of the pro-region of procathepsin L were also analyzed using recombinant procathepsin L and cathepsin L. [Materiala and Methods] fhCATLwas cloned from a hEMBL3/genomic DNA library using digoxigenin-labeled Fuscdn cathepsin L cDNA as a probe, and sequenced. For fhCATL expression in Escberichti coli, two cDNAs encoding procathepsin L and cathepsin L were amplified by PCR, and inserted into IPTG-inducible pGEX-4T-1. Recombinant proteins fused with glutathione-S-transferase (GST) were solubilized in 8M urea and then renatured in the presence of reduced and oxidized glutathiones. GST was removed by thrombin digestion. The enzyme activity was assayed using Z-Phe-Arg-MCA as a substrate. [Rasalta] A fhCATL (822-2). localized on the cloned 16kb-genomic DNA fragment, consists of 4 e~ons and 3 introns. Exon 1 encodes the signal sequence, pro-peptide, and N-terminal 21 amino acid residues of the mature form. The active site cysteine is contained in exon 2, and the active site histidine and asparagine are in exon 4. The insertion positions of 3 introns are highly conserved among CATLsfrom other enkaryotes. Southern blot analysis indicates that fhCATLs exist as multiple copies that are sparsely organized in the genome. Inactive recombinant procathepsin L (36kDa) was autocatalytically processed to active 26kDa-cathepsin L at pH 4.555. However, the Nterminal sequence of the processed form differs from that of native cathepsin L. No enzyme activity was detected for recombinant cathepsin L lacking the pro-peptide. [Coachdon] The fhCATLis more compact compared with mammalian CATLs. It was found that the processing of procathepsin L takes place by pH-dependent autolysis in vitro, and the pro-region of procathepsin L is essential for the folding of functional cathepsin L.
P-0803
PHYLOGENETIC POSITION OF BUSTOCYSTIS HOMINIS.
P-@O5
CYSTElNE Paragonirus
INFERRED FROM MULTIPLE MOLECULAR SEQUENCE DATA
Arisue N’, Hashimoto T’, Nakamora Y**, Yoshikawa H***, Hasegawa M’ *The lnstitote of Statistical Mathematics, Tokyo, Japan, **Laboratory of Gene Manipulation, Showa University, Tokyo, Japan and ***Department of Biology, Nara Women’s University, Nara, Japan Blartocystis horn& is an enigmatic human parasite that has cytochrome&ee mitoohondria. Anaerobic mitoohondria are always present and are considered to be timetional, but no activity of many mitochondrial enzymes has been detected. Because of the unique features of its mitoehoodria, B. hominis is an organism of great interest in eukaryotic evolutionary biology. However, the phylogenetic position of B. hominisstill remains unclear because of the lack of sequence information. Racent molecular phylogenetic study based on the comparison of small subunit ritmsomai RNA &RNA) sequences suggested that 5. hominis is placed withii the slramenopiles inclnding goldan-brown algae, chrysophytes, oomycetes, and others. In contrast, onr previous phylogeny based on the amino acid sequences of elongation factor (EF) -1 D positioned B. homhiqon the far earlier branch in the eukaryotic tree, in disagreement with the SrRNA phylogeny. To make sure the phylogenetic position of E. hominis with more sequence information, we isolated and sequenced cDNA clones that putatively encode EF-2, non-catalytic ‘8’ subunit of vacwlar ATPase, and cytowlic-type 70kDa beat shock protein. Deduced amino acid sequence of each protein was aligned with all the cormsponding seqnences available in the database, and phylogenetic reconstrnction analysis was performed by the use of maximum likelihood method of protein phylogeny. Although the results derived tiom the individual proteins were inoonsistent with each other, total evidence of these results (including the EF-I 0:) suggested that the divergence of B. hominis is later than that of the dlplomonad, Gwdio 1nmb[io, but is earlier than that of kinetoplastids, still in disagreement with the S~RNA phylogeny. Because there is great discrepancy between the protein and the SrRNA phylogenies, we cannot simply conclude the phyiogenetic position of E. hominis based only on a single moiecuie. Fnrther pbylogenetic analyses using more sequence data With enOt4$ species sampling will be necesmry to more precisely settle this issue.
Cornelo_Medina *Department **Institute Lima, Peru The releases
of of
PROTEINASES xexlcanus
IN EXTRACTS OF METACERCARIAE
j@, RoJas-MorBn N** Medical Microbiology Pathology, San MarCOS
metacercariae proteolytlc
Paragonixus
and UniverSitY
mexicanus
enzymes which are thought to be important for penetration of the host tissue as well as for nutrition. In this report, homogenates of these organisms were prepared and their proteolytic enzymes examined. Gelatin-substrate sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed seven bands of activity at pH 7.0, in the apparent molecular size range 20-70 ham. Most of them, ware Cysteine Proteinases since they were stimulated by 5 nM dithiothreitol, and were sensitive to 1euPePtin and L-transepoxysuccinyl-leucylamido(4-guanldino)butane_
P-0802 GENETIC RELATEDNESS FACTOR-l
OF THE ELONGAnON ALPHA IN BLASTOCYSTIS ISOLATES
Ho*, Singh M*, Jeyaseelan K**, and Yap EH Departments of * Microbiology and ** Biochemistry, Faculty of Medicine, National University of Singapore, Singapore 119260 Blastocystis is an intestinal protozoan parasite found in a wide variety of animal hosts e.g. mammals, reptiles, birds and insects. This study examines nucleotide sequences of the elongation factor1 alpha (EF-la) in isolates of Blastocystis from humans, reptiles and rats. A pair of sense and anti-sense primers comprising of 21 and 18 nucleotides respectively, for the EF- I a wassynthesized and used in PCR with DNA from 13 Bkzstocystis isolates. A 900bp product consistently amplified in all the isolates was used in the evaluation of their genetic relatedness.
p-o804
337
FASCIOLAHEPATZCACATHEPSIN L : GENE STRUCTURE, EXPRESSIONIN BACTERIA, AND IN VITRO PROCESSING
Yamasaki l-l and Aoki T. Department of Parasitology, Unwersity School of Medicine, Tokyo113-0@33, Japan
Juntendo
Cathepsin L in Fusciolahepatica is thought to be involved in important functions including host protein digestion, tissue penetration, and so on. We have reported that the enzyme is synthesized as an inactive preprocathepsin L, targeted to the secretory granules in intestinal epithehal cells, and secreted into the intestinal lumen. In the present paper, the genomic structure of the F. h+ib cathepsin L gene (fhCATL) is clarified. The processing mechanism of procathepsin L to mature cathepsin L and the possible function of the pro-region of procathepsin L were also analyzed using recombinant procathepsin L and cathepsin L. [Materiala and Methods] fhCATLwas cloned from a hEMBL3/genomic DNA library using digoxigenin-labeled Fuscdn cathepsin L cDNA as a probe, and sequenced. For fhCATL expression in Escberichti coli, two cDNAs encoding procathepsin L and cathepsin L were amplified by PCR, and inserted into IPTG-inducible pGEX-4T-1. Recombinant proteins fused with glutathione-S-transferase (GST) were solubilized in 8M urea and then renatured in the presence of reduced and oxidized glutathiones. GST was removed by thrombin digestion. The enzyme activity was assayed using Z-Phe-Arg-MCA as a substrate. [Rasalta] A fhCATL (822-2). localized on the cloned 16kb-genomic DNA fragment, consists of 4 e~ons and 3 introns. Exon 1 encodes the signal sequence, pro-peptide, and N-terminal 21 amino acid residues of the mature form. The active site cysteine is contained in exon 2, and the active site histidine and asparagine are in exon 4. The insertion positions of 3 introns are highly conserved among CATLsfrom other enkaryotes. Southern blot analysis indicates that fhCATLs exist as multiple copies that are sparsely organized in the genome. Inactive recombinant procathepsin L (36kDa) was autocatalytically processed to active 26kDa-cathepsin L at pH 4.555. However, the Nterminal sequence of the processed form differs from that of native cathepsin L. No enzyme activity was detected for recombinant cathepsin L lacking the pro-peptide. [Coachdon] The fhCATLis more compact compared with mammalian CATLs. It was found that the processing of procathepsin L takes place by pH-dependent autolysis in vitro, and the pro-region of procathepsin L is essential for the folding of functional cathepsin L.
P-0803
PHYLOGENETIC POSITION OF BUSTOCYSTIS HOMINIS.
P-@O5
CYSTElNE Paragonirus
INFERRED FROM MULTIPLE MOLECULAR SEQUENCE DATA
Arisue N’, Hashimoto T’, Nakamora Y**, Yoshikawa H***, Hasegawa M’ *The lnstitote of Statistical Mathematics, Tokyo, Japan, **Laboratory of Gene Manipulation, Showa University, Tokyo, Japan and ***Department of Biology, Nara Women’s University, Nara, Japan Blartocystis horn& is an enigmatic human parasite that has cytochrome&ee mitoohondria. Anaerobic mitoohondria are always present and are considered to be timetional, but no activity of many mitochondrial enzymes has been detected. Because of the unique features of its mitoehoodria, B. hominis is an organism of great interest in eukaryotic evolutionary biology. However, the phylogenetic position of B. hominisstill remains unclear because of the lack of sequence information. Racent molecular phylogenetic study based on the comparison of small subunit ritmsomai RNA &RNA) sequences suggested that 5. hominis is placed withii the slramenopiles inclnding goldan-brown algae, chrysophytes, oomycetes, and others. In contrast, onr previous phylogeny based on the amino acid sequences of elongation factor (EF) -1 D positioned B. homhiqon the far earlier branch in the eukaryotic tree, in disagreement with the SrRNA phylogeny. To make sure the phylogenetic position of E. hominis with more sequence information, we isolated and sequenced cDNA clones that putatively encode EF-2, non-catalytic ‘8’ subunit of vacwlar ATPase, and cytowlic-type 70kDa beat shock protein. Deduced amino acid sequence of each protein was aligned with all the cormsponding seqnences available in the database, and phylogenetic reconstrnction analysis was performed by the use of maximum likelihood method of protein phylogeny. Although the results derived tiom the individual proteins were inoonsistent with each other, total evidence of these results (including the EF-I 0:) suggested that the divergence of B. hominis is later than that of the dlplomonad, Gwdio 1nmb[io, but is earlier than that of kinetoplastids, still in disagreement with the S~RNA phylogeny. Because there is great discrepancy between the protein and the SrRNA phylogenies, we cannot simply conclude the phyiogenetic position of E. hominis based only on a single moiecuie. Fnrther pbylogenetic analyses using more sequence data With enOt4$ species sampling will be necesmry to more precisely settle this issue.
Cornelo_Medina *Department **Institute Lima, Peru The releases
of of
PROTEINASES xexlcanus
IN EXTRACTS OF METACERCARIAE
j@, RoJas-MorBn N** Medical Microbiology Pathology, San MarCOS
metacercariae proteolytlc
Paragonixus
and UniverSitY
mexicanus
enzymes which are thought to be important for penetration of the host tissue as well as for nutrition. In this report, homogenates of these organisms were prepared and their proteolytic enzymes examined. Gelatin-substrate sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed seven bands of activity at pH 7.0, in the apparent molecular size range 20-70 ham. Most of them, ware Cysteine Proteinases since they were stimulated by 5 nM dithiothreitol, and were sensitive to 1euPePtin and L-transepoxysuccinyl-leucylamido(4-guanldino)butane_