Molecular and Biochemical Parasitology, 50 (1992) 185 188

185

:~'~ 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00 MOLBIO 01665

Short Communication

Isolation of an Entamoeba histolytica c D N A clone encoding a protein with a putative zinc finger domain S a m u e l L. Stanley, Jr. 1"2 a n d Ellen Li 1~3 Departments ~f l Medicine, 2Moleeular Microbiology and 3Biochemistry and Molecular Biophysics, Washington Universi O, School o[" Medicine, Saint Louis, MO, U.S.A. (Received 3 September 1991; accepted 12 September 1991)

Key words: Entamoeba histolytiea; Zinc finger

Despite the medical importance of the protozoan parasite Entamoeba histolytica, little is known regarding the genetics and control of gene expression in this pathogen. Recently, we used differential screening of a c D N A library derived from the pathogenic E. histolytica strain H M I : I M S S to isolate E. histolytica specific c D N A clones [1,2]. In this report we show that one of these clones, designated c3, encodes an E. histolytica peptide of 84 amino acids which contains a putative zinc finger region. The c3 clone was isolated from the H M I : I M S S c D N A library as described in ref. 1. R N A blot hybridization (conditions as described in ref. 2) revealed that the c3 clone hybridized with a 0.3-kb species in axenic strains of E. histolytica but did not hybridize with R N A from the E. histolytica-like Laredo strain, Entamoeba moshkovskii (which may be identical to Laredo; ref. 3), or Entamoeba invadens (data not shown). Correspondence address: Samuel L. Stanley, Jr., Department of Medicine, Washington University School of Medicine, 660 S. Euclid Avenue, Campus Box 8051, St. Louis, MO 63110, U.S.A. Tel.: 314-362-1070: Fax: 314-362-9230. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number M77240.

Southern blot analysis (conditions as described in ref. 2) of Sau3a-digested D N A from 3 E. histolytica strains and the E. histolyticalike Laredo strain showed that the c3 clone hybridized to species of 4.0, 2.7, 2.2 and 1.8 kb in all 3 E. histolytica strains, but did not hybridize with any species in Laredo (Fig. IA). With longer autoradiograph exposures (72 h) a low intensity band could be seen at 2.8 kb in Laredo (data not shown), suggesting that Laredo may have a homologous gene. Southern blot analysis of H M I : I M S S D N A digested with HincII (1 internal site), HindIII (1 internal site), EcoRI (no internal sites), and Sau3a demonstrated 4 fragments for HincII, EcoRI, and Sau3a (Fig. I B). The 4 E. histolytica genes sequenced to date [4-7], as well as the gene encoding the serine-rich E. histolytica protein (Li, E. et al., submitted for publication), contain no introns. If no introns are present in the c3 gene, the Southern blot analysis suggests that the c3 gene is part of a multigene family. Exact determination of the c3 gene copy number will require isolation and sequence analysis of c3 genomic DNA. The c3 c D N A clone was sequenced by the technique of Maxam and Gilbert [8]. The c3 c D N A clone contained 251 nucleotides with one open reading frame. Because the size of the c D N A clone was slightly smaller than expected

186

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Fig. 1. Southern blot analysis of amebic D N A probed with the c3 clone. (A) Hybridization of the c3 clone with Sau3a digestcd D N A from: lane 1, E. histolylica H M I:IM SS; lane 2, E. histolytica H K9; lane 3, E. hislol)'lica 200:NI H; lane 4, E. h istol),ticalike Laredo species. (B) Hybridization of the c3 clone with E. hixto@tica HMI:IMSS D N A digested with: lane 1, Hincll; lane 2, Hindlll: lane 3. EcoRl; lane 4, Sau3a. The numbers on the leli represent sizes in kb.

based on the transcript seen on Northern blotting, the c3 c D N A sequence was completed by primer extension as described [9] using the antisense oligonucleotide A G C T T C A G A A G C A G C A G derived from the sequence of c3. The completed c3 c D N A sequence

contained 290 nucleotides with a continuous open reading frame beginning with a putative initiator methionine at nucleotide 15 to a TAA termination at nucleotide 267 (Fig. 2A). The agreement between the size of the completed c3 c D N A clone and the transcript identified by

A.

cagcttccaaag

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GTT GAC TTG TTG D L L N P

ACT GCT GCT TCT - - T A A S

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Fig. 2. (A) Nucleotide and derived amino acid sequence oF the c3 cDNA. The region of overlap bctwcen the primer cxtcnsion transcript and the sequence derived from the c3 c D N A clone is underlined. The amino acids comprising the putative zinc finger region are shown in bold face, with the component cysteine residues underlined. (B) Comparison of the putative zinc finger region from the derived amino acid sequence of c3 (EHZc3) with one of the zinc finger domains from the derived amino acid sequence of the c-erh-A gene.

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Northern blotting, suggest the full coding region is contained within the c3 c D N A sequence shown in Fig. 2A. The derived amino acid sequence for the peptide encoded by the c3 c D N A (designated EHZc3) contains 84 amino acids and has an estimated molecular weight of 9248. A search of the on-line GenBank and National Biomedical Research Foundation (NBRF) data banks (February 1991) revealed no sequences with widespread homology to the nucleotide sequence of c3. However analysis of the EHZc3 sequence demonstrated a region of 29 amino acids which showed 28% identity with a portion of the gag polyprotein from Visna virus [10]. Inspection of the homologous sequence from EHZc3 revealed the presence of a single Cys-X2-Cys-X,-Cys-X2-Cys domain (bold in Fig. 2A), consistent with a zinc finger region [I 1 13]. This region was identical in 9 of 23 amino acids (40%) with the zinc finger domain from the thyroid hormone receptor protein c-erb-A (Fig. 2B) [14]. While the sequence of the putative zinc finger domain from EHZc3 resembles that of the steroid hormone receptor, the entire EHZc3 sequence clearly differs from those proteins in its small size and in the presence of only 1 zinc finger domain, as opposed to the paired domains that characterize the steroid hormone receptors [14]. In this respect EHZc3 resembles the 289 amino acid protein encoded by the 13S product of the adenovirus E lA gene [15], a factor that may be involved in protein-protein interactions [15]. An additional similarity between the two gene products is the presence of multiple basic residues at the C-terminus of the EHZc3 peptide; an analogous region in the E1A gene is associated with nuclear localization of the protein product [16].

Acknowledgements We thank Lynne Foster and Cindy KunzJenkins for technical assistance, and Drs. J. Gordon, J. Milbrandt, and P. Cieslak for critical reading of the manuscript. This work was supported in part by the Lucille P. Markey Charitable Trust Foundation.

References 1 Stanley Jr., S.L., Becker, A., Kunz-Jenkins, C., Foster, L. and Li, E. (1990) Cloning and expression of a membrane antigen of Entamoeba histolytica possessing multiple tandem repeats. Proc. Natl. Acad. Sci. USA 87, 4976 4980. 2 Burch, D.J., Li, E., Reed, S., Jackson, T.F.H.G. and Stanley Jr., S.L., (1991) Isolation of a strain-specific Entamoeba histoO, tica cDNA clone. J. Clin. Microbiol. 29, 696 701. 3 Clark, C.G. and Diamond, L.S. (1991) The Laredo strain and other "Entamoeba histolytica-like' amoebae are Entamoeba moshkovskii. Mol. Biochem. Parasitol. 46,11 18. 4 Edman, U., Meza, I. and Agabian, N.M. (1987) Genomic and eDNA actin sequences from a virulent strain of Entamoeba histolytica. Proc. Natl. Acad. Sci. USA 84, 3024 3028. 5 Huber, M., Garfinkel, L., Gitler, C., Mirelman, D., Revel, M. and Rozenblatt, S. (1987) Entamoeba histolytica: cloning and characterization of actin eDNA. Mol. Biochem. Parasitol. 24, 277 235. 6 Huber, M., Garfinkel, L., Gitler, C., Mirelman, D., Revel, M. and Rozenblatt, S. (1988) Nucleotide sequence analysis of an Entamoeba histolytica ferredoxin gene. Mol. Biochem. Parasitol. 31, 27 34. 7 [~dman, U., Meraz, M.A., Rausser, S., Agabian, N. and Meza, 1. (1990) Characterization of an immunodominant variable surface antigen from pathogenic and nonpathogenic Entamoeba histolvtica. J. Exp. Med. 172, 879 888. 8 Maxam, A.M. and Gilbert, W. (1991) Sequencing endlabeled DNA with base-specific chemical cleavages. Methods Enzymol. 65, 499 559. 9 Heuckeroth, R.O.. Birkenmeier, E.H., Levin, M.S. and Gordon, J.I. (1987) Analysis of the tissue-specific expression, developmental regulation, and linkage relationships of a rodent gene encoding heart fatty acid binding protein. J. Biol. Chem. 262, 9709 9717. 10 Sonigo, P., Alizon, M., Staskus, K., Klatzman, D., Cole, S., Danos, O., Retzel, E., Tiollais, P., Haase, A. and Wain-Hobson, S. (1985) Nucleotide sequence of the visna lentivirus: relationship to the AIDS virus. Cell 42, 369 382. 11 Berg, J.M. (1986) Potential metal-binding domains in nucleic acid binding proteins. Science 232, 485~487. 12 Klug, A. and Rhodes, D. (1987) Zinc fingers: a novel protein motif for nucleic acid recognition. Trends Biochem. Sci. 12, 39%402. 13 Berg, J.M. (1990) Zinc fingers and other metal binding domains. J. Biol. Chem. 265, 6513 6516. 14 Weinberger, C., Thompson, C.C., Ong, E.S., Lebo, R., Gruol, D.J. and Evans, R.M. (1986) The c-erb-A gene encodes a thyroid hormone receptor. Nature 324, 641 646. 15 Moran, E. and Mathews, M.B. (1987) Multiple functional domains in the adenovirus EIA gene. Cell 48, 177 178. 16 Krippt, B., Ferguson, B., Jones, N., Rosenberg, M. and Westphal, H. (1985) Mapping of functional domains in adenovirus EIA proteins. Proc. Natl. Acad. Sci. USA 82, 7480 7494.

Isolation of an Entamoeba histolytica cDNA clone encoding a protein with a putative zinc finger domain.

Molecular and Biochemical Parasitology, 50 (1992) 185 188 185 :~'~ 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00...
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