Sequence analysis of a DNA fragment with yeast autonomously replicating sequence activity from the extrachromosomal ribosomal DNA circle of Entamoeba histolytica

The 4.4 kb EcoRI fragment of the 25 kb extrachromosomal ribosomal DNA circle of Entamoeba histolytica (1,2) contains a stretch of tandem Dral 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 his5 Saccharomyces cerevisiae at high frequency (5% transformants//ig DNA and 104 transformants//ig DNA for two independent clones with the 4.4 kb insert) as against the negligible frequency (0 transformant//ig DNA) with YIP5 plasmid alone and 474 transformants//ig DNA with pl75B7, a plasmid containing yeast ARS1 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.