extrachromosomal ribosomal DNA circle of Entamoeba ... School of Life Sciences and 'School of Environmental Sciences, Jawaharlal Nehru University,.
.::/ 1991 Oxford University Press
Nucleic Acids Research, Vol. 19, No. 10 2777
analysis of a DNA fragment with yeast autonomously replicating sequence activity from the extrachromosomal ribosomal DNA circle of Entamoeba histolytica Sequence
V.Mittal, S.Ramachandran', D.Sehgal, A.Bhattacharya and S.Bhattacharya1 School of Life Sciences and 'School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India EMBL accession no. X58630
Submitted April 15, 1991 The 4.4 kb EcoRI fragment of the 25 kb extrachromosomal ribosomal DNA circle of Entamoeba histolytica (1, 2) contains a stretch of tandem DraI repeats (2). We have demonstrated autonomously replicating sequence (ARS) activity in this 4.4 kb EcoRI fragment by cloning it in the yeast shuttle vector, YIP5, which lacks an ARS. The resulting construct (pYHM-d) could transform ura3 hisS Saccharomyces cerevisiae at high frequency (596 transformants/,Lg DNA and 104 transformants/yg DNA for two independent clones with the 4.4 kb insert) as against the negligible frequency (0 transformant/tg DNA) with YIP5 plasmid alone and 474 transformants/,ug DNA with p175B7, a plasmid containing yeast ARSI and CEN4. The Ura+ yeast transformants arising from pYHM-d were not due to homologous recombination since the Ura+ phenotype was lost upon repeated subculturing (five cycles) in nonselective medium. The sequence of one complete DraI repeat unit is presented (Fig. 1). It is 170 bp long and contains three, 12-nucleotide sequences which are completely homologous with the 1 1-nucleotide yeast ARS consensus sequence (A/TTTTATA/GTTTA/T) except for the presence of an extra nucleotide (Fig. 2) in each. The extra nucleotide is located at a position shown to be less critical for ARS activity in yeast (3). It remains to be determined whether the DraI repeat unit does indeed serve as ARS in the extrachromosomal rDNA of E. histolytica.
REFERENCES 1. Bhattacharya,S., Bhattacharya,A., Diamond,L.S. and Soldo,A.T. (1989) J.
Protozool. 36, 455-458. 2. Huber,M., Koller,B., Gitler,C., Mirelman,D., Revel, M., Rozenblatt,S. and Garfmkel,L. (1989) Mol. Biochem. Parasitol. 32, 285-296. 3. Kipling,D. and Kearsey,S.E. (1990) Mol. Cell. Biol. 10, 265-272.
1 CAATCAATTA ATCAATCATT AAAGAATATT AAAATAATAA ATGTTTAGAA TTTAATTATG
61 AACAACATTT ACCTTATGAA TATTTATACT TTAGTTGATA TTTTTTGAGA TATTAAATAT ------------>
121 TATTTATTTA AATGATAAAA AAGAAAGACA ATCAAATAAA AAAAGAAAGA
Figure 1. A single Dral repeat unit. The yeast consensus ARS sequences are underlined, with arrows to denote orientation.
Yeast consensus ARS
E.histolYtica sequences
5'-A T T T A T A T T T A-3' T G T :
T 5'-A T T T A T A T T T T-3' C 5'-A T T T A T A T T T A-3'
ACKNOWLEDGEMENTS This work was funded by grants from Department of Science and Technology and Council of Scientific and Industrial Research, and research fellowships from U.G.C. (V.M. and S.R.) and C.S.I.R. (D.S.).
C 5'-T T T T A T A T T T A-3'
Figure 2. The three ARS consensus sequences in the Dral repeat. For comparison with the yeast sequence, the 'extra' nucleotide in the E. histolytica sequence is shown above.
