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Eukaryotic Cell, February 2004, p. 170-179, Vol. 3, No. 1
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.1.170-179.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

An Entamoeba histolytica LINE/SINE Pair Inserts at Common Target Sites Cleaved by the Restriction Enzyme-Like LINE-Encoded Endonuclease

Prabhat K. Mandal,1 Anindya Bagchi,2,{dagger} Alok Bhattacharya,1 and Sudha Bhattacharya2*

School of Environmental Sciences,2 School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India1

Received 4 September 2003/ Accepted 6 November 2003

The non-long-terminal-repeat (non-LTR) retrotransposons (also called long interspersed repetitive elements [LINEs]) are among the oldest retroelements. Here we describe the properties of such an element from a primitive protozoan parasite, Entamoeba histolytica, that infects the human gut. This 4.8-kb element, called EhLINE1, is present in about 140 copies dispersed throughout the genome. The element belongs to the R4 clade of non-LTR elements. It has a centrally located reverse transcriptase domain and a restriction enzyme-like endonuclease (EN) domain at the carboxy terminus. We have cloned and expressed a 794-bp fragment containing the EN domain in Escherichia coli. The purified protein could nick supercoiled pBluescript DNA to yield open circular and linear DNAs. The conserved PDX12-14D motif was required for activity. Genomic sequences flanking the sites of insertion of EhLINE1 and the putative partner short interspersed repetitive element (SINE), EhSINE1, were analyzed. Both elements resulted in short target site duplications (TSD) upon insertion. A common feature was the presence of a short T-rich stretch just upstream of the TSD in most insertion sites. By sequence analysis an empty target site in the E. histolytica genome, known to be occupied by EhSINE1, was identified. When a 176-bp fragment containing the empty site was used as a substrate for EN, it was prominently nicked on the bottom strand at the precise point of insertion of EhSINE1, showing that this SINE could use the LINE-encoded endonuclease for its insertion. The nick on the bottom strand was toward the right of the TSD, which is uncommon. The lack of strict target site-specificity of the restriction enzyme-like EN encoded by EhLINE1 is also exceptional. A model for retrotransposition of EhLINE1/SINE1 is presented.

* Corresponding author. Mailing address: School of Environmental Sciences, Jawaharlal Nehru University, New Mehrauli Road, New Delhi 110067, India. Phone: 91-11-26704308. Fax: 91-11-26172438. E-mail: sb{at}mail.jnu.ac.in.

{dagger} Present address: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Eukaryotic Cell, February 2004, p. 170-179, Vol. 3, No. 1
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.1.170-179.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Mandal, P. K., Rawal, K., Ramaswamy, R., Bhattacharya, A., Bhattacharya, S. (2006). Identification of insertion hot spots for non-LTR retrotransposons: computational and biochemical application to Entamoeba histolytica. Nucleic Acids Res 34: 5752-5763 [Abstract] [Full Text]  
  • MacFarlane, R. C., Singh, U. (2006). Identification of Differentially Expressed Genes in Virulent and Nonvirulent Entamoeba Species: Potential Implications for Amebic Pathogenesis. Infect. Immun. 74: 340-351 [Abstract] [Full Text]  
  • Anbar, M., Bracha, R., Nuchamowitz, Y., Li, Y., Florentin, A., Mirelman, D. (2005). Involvement of a Short Interspersed Element in Epigenetic Transcriptional Silencing of the Amoebapore Gene in Entamoeba histolytica. Eukaryot Cell 4: 1775-1784 [Abstract] [Full Text]  
  • Pritham, E. J., Feschotte, C., Wessler, S. R. (2005). Unexpected Diversity and Differential Success of DNA Transposons in Four Species of Entamoeba Protozoans. Mol Biol Evol 22: 1751-1763 [Abstract] [Full Text]  
  • Kojima, K. K., Fujiwara, H. (2005). An extraordinary retrotransposon family encoding dual endonucleases. Genome Res 15: 1106-1117 [Abstract] [Full Text]  


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