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Korean J Parasitol > Volume 35(3):1997 > Article

Original Article
Korean J Parasitol. 1997 Sep;35(3):203-210. English.
Published online Sep 20, 1997.  http://dx.doi.org/10.3347/kjp.1997.35.3.203
Copyright © 1997 by The Korean Society for Parasitology
cDNAs encoding the antigenic proteins in pathogenic strain of Entamoeba histolytica
K I Im,*J T Choi,Y P Hong,T E Kim and M S La
Department of Parasitology and Institute of Tropical Medicine, Yonsei University College of Medicine, Seoul 120-752, Korea.
Received June 20, 1997; Accepted September 01, 1997.

Abstract

The differential display reverse transcription polymerase chain reaction (DDRT-PCR) analysis was performed to identify the pathogenic strain specific amplicons. mRNAs were purified from the trophozoites of the pathogenic strain YS-27 and the non-pathogenic strain S 16, respectively. Three kinds of first stranded cDNAs were reverse transcribed from the mRNAs by one base anchored oligo-dT11M (M: A, C, or G) primers. Each cDNA template was used for DDRT-PCR analysis. A total of 144 pathogenic strain specific amplicons was observed in DDRT-PCR analysis using primer combinations of the 11 arbitrary primers and the 3 one base anchored oligo-dT11M primers. Of these, 31 amplicons were verified as the amplicons amplified only from the mRNAs of the pathogenic strain by DNA slot blot hybridization. Further characterization of the 31 pathogenic strain specific amplicons by DNA slot blot hybridization analysis using biotin labeled probes of the PCR amplified DNA of cysteine proteinase genes revealed that 21 of them were amplified from the mRNAs of the cysteine proteinase genes. Four randomly selected amplicons out of the rest 10 amplicons were used for screening of cDNA library followed by immunoscreening and all of them were turned out to be amplified from the mRNA.

Figures


Fig. 1
An example of differential display using one base anchored oligo-dT primers and one arbitrary 10-mer primer (#228). P denotes pathogenic Entamoeba histolytica YS-27 and NP denotes non-pathogenic E. dispar S 16. Arrows denote the bands specific for pathogenic E. histolytica YS-27. S lane denotes standard size marker of 100 bp DNA ladder.


Fig. 2
Identification of DDRT-PCR amplicons of differentially expressed genes by DNA slot blot analysis using the pooled DDRT-PCR products as hybridization probes. Duplicated sets of the reamplified PCR products of the selected 31 YS-27 specific amplicons were immobilized onto nylon membranes. Each membrane was hybridized with the pooled DDRT-PCR products from Entamoeba histolytica YS-27 (left) and from E. dispar S 16 (right), respectively. Amplicons representing differentially expressed mRNA are indicated by arrow-heads.


Fig. 3
Identification of DDRT-PCR amplicons of Entamoeba histolytica cysteine proteinase genes by DNA slot blot analysis. PCR reamplified products of the selected 31 unique amplicons for the YS-27 were immobilized onto nylon membrane and hybridized with the PCR amplified sequences of the E. histolytica cysteine proteinase genes. Twenty-one amplicons representing differentially expressed cysteine proteinase mRNA are indicated by arrows.


Fig. 4
Result of the immunoscreening. Dots on each blot denote the positive clones detected by anti-Entamoeba histolytica human serum. (A) clone #232-G, (B)clone #250-G, (C)clone #282-G, (D)clone #282-C.

Tables


Table 1
Number of differential amplified fragment of the combination of 11 arbitrary 10-mer and one base anchored oligo-dT11 primers


Table 2
DDRT-PCR products hybridized only with the probe synthesized from the pooled DDRT-PCR products fo Entamoeba histolytica YS-27

References
1. Chang JK, Im K, Soh CT. Axenization of Entamoeba histolytica, a Korean strain YS-27. Korean J Parasitol 1995;33(4):387–390.
  
2. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162(1):156–159.
  
3. Diamond LS, Harlow DR, Cunnick CC. A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans R Soc Trop Med Hyg 1978;72(4):431–432.
  
4. Espelund M, Stacy RA, Jakobsen KS. A simple method for generating single-stranded DNA probes labeled to high activities. Nucleic Acids Res 1990;18(20):6157–6158.
  
5. Garfinkel LI, Giladi M, Huber M, Gitler C, Mirelman D, Revel M, Rozenblatt S. DNA probes specific for Entamoeba histolytica possessing pathogenic and nonpathogenic zymodemes. Infect Immun 1989;57(3):926–931.
 
6. Gitler C, Calef E, Rosenberg I. Cytopathogenicity of Entamoeba histolytica. Philos Trans R Soc Lond B Biol Sci 1984;307(1131):73–85.
  
7. Huber M, Garfinkel L, Gitler C, Mirelman D, Revel M, Rozenblatt S. Entamoeba histolytica: cloning and characterization of actin cDNA. Mol Biochem Parasitol 1987;24(3):227–235.
  
8. Hubank M, Schatz DG. Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res 1994;22(25):5640–5648.
  
9. Keene WE, Petitt MG, Allen S, McKerrow JH. The major neutral proteinase of Entamoeba histolytica. J Exp Med 1986;163(3):536–549.
  
10. Lee M, Hong ST. Differentiation of Entamoeba histolytica and Entamoeba dispar in cyst-passers by immunoblot. Korean J Parasitol 1996;34(4):247–254.
  
11. Leippe M, Ebel S, Schoenberger OL, Horstmann RD, Muller-Eberhard HJ. Pore-forming peptide of pathogenic Entamoeba histolytica. Proc Natl Acad Sci U S A 1991;88(17):7659–7663.
  
12. Liang P, Averboukh L, Zhu W, Pardee AB. Ras activation of genes: Mob-1 as a model. Proc Natl Acad Sci U S A 1994;91(26):12515–12519.
  
13. Liang P, Pardee AB. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 1992;257(5072):967–971.
  
14. Lushbaugh WB, Hofbauer AF, Pittman FE. Entamoeba histolytica: purification of cathepsin B. Exp Parasitol 1985;59(3):328–336.
  
15. Nishio Y, Aiello LP, King GL. Glucose induced genes in bovine aortic smooth muscle cells identified by mRNA differential display. FASEB J 1994;8(1):103–106.
 
16. Orozco E, Lazard D, Sanchez T, Sanchez MA, Hernandez R, Silva EF. A variable DNA region of Entamoeba histolytica is expressed in several transcripts which differ in genetically related clones. Mol Gen Genet 1993;241(3-4):271–279.
  
17. Ravdin JI. Pathogenesis of disease caused by Entamoeba histolytica: studies of adherence, secreted toxins, and contact-dependent cytolysis. Rev Infect Dis 1986;8(2):247–260.
  
18. Reeves RE, Bischoff JM. Classification of Entamoeba species by means of electrophoretic properties of amebal enzymes. J Parasitol 1968;54(3):594–600.
  
19. Robinson GL. The laboratory diagnosis of human parasitic amoebae. Trans R Soc Trop Med Hyg 1968;62(2):285–294.
  
20. Sun Y, Hegamyer G, Colburn NH. Molecular cloning of five messenger RNAs differentially expressed in preneoplastic or neoplastic JB6 mouse epidermal cells: one is homologous to human tissue inhibitor of metalloproteinases-3. Cancer Res 1994;54(5):1139–1144.
 
21. Tannich E, Horstmann RD, Knobloch J, Arnold HH. Genomic DNA differences between pathogenic and nonpathogenic Entamoeba histolytica. Proc Natl Acad Sci U S A 1989;86(13):5118–5122.
  
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