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Eukaryotic Cell, January 2009, p. 69-76, Vol. 8, No. 1
1535-9778/09/$08.00+0     doi:10.1128/EC.00254-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Promoter Analysis of Palindromic Transcription Units in the Ribosomal DNA Circle of Entamoeba histolytica{triangledown}

Sunil K. Panigrahi,1 Gagan Deep Jhingan,1 Indrani Som,2 Alok Bhattacharya,3 William A. Petri Jr.,4 and Sudha Bhattacharya1*

School of Environmental Sciences, Jawaharlal Nehru University, New Mehrauli Road, New Delhi 110 067, India,1 LCDB/NIDDK, National Institutes of Health, Bethesda, Maryland 20892,2 School of Life Sciences, Jawaharlal Nehru University, New Mehrauli Road, New Delhi 110 067, India,3 Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia 22908-13404

Received 29 July 2008/ Accepted 23 October 2008

rRNA genes of Entamoeba histolytica are organized as palindromic ribosomal DNA (rDNA) units (I and II) in a 24.5-kb circle. Although the two rDNAs are identical in sequence, their upstream spacers are completely different. Since the intergenic sequences (IGS) of all rDNA copies in other organisms are conserved and contain transcription regulatory sequences, the lack of sequence conservation in the IGS prompted the question of whether both rDNAs are indeed transcriptionally active. We mapped the transcriptional start points (tsp's) and promoters of the two rDNAs. A 51-bp sequence immediately upstream of the tsp's was highly conserved in both units. In addition, both units had an A+T-rich stretch upstream of the 51-bp core. Analysis of reporter gene transcription showed promoter activity to reside in the regions from positions –86 to +123 (rDNA I) and positions –101 to +140 (rDNA II). The promoter-containing fragments from both units could bind and compete with each other for protein(s) from nuclear extracts. Protein binding was especially dependent on the A+T-rich region upstream of the 51-bp core (positions –53 to –68). The requirement of >80 bp downstream of the tsp was striking. Although this sequence was not conserved in the two units, it could potentially fold into very long stem-loops. Both rDNAs transcribed with comparable efficiency, as measured by nuclear runon. Thus, both rDNAs share very similar organization of promoter sequences, and in exponential culture both rDNAs are transcribed. It remains to be seen whether the different IGS affect the regulation of the two units under adverse conditions.

* Corresponding author. Mailing address: School of Environmental Sciences, Jawaharlal Nehru University, New Mehrauli Road, New Delhi 110 067, India. Phone: 91-11-26704308. Fax: 91-11-26172438. E-mail: sb{at}mail.jnu.ac.in

{triangledown} Published ahead of print on 31 October 2008.

Eukaryotic Cell, January 2009, p. 69-76, Vol. 8, No. 1
1535-9778/09/$08.00+0     doi:10.1128/EC.00254-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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