Nucleotide analysis of Phaffia rhodozyma DNA fragment that functions as ARS in Saccharomyces cerevisiae

Abstract

The chromosomal DNA fragment from Phaffia rhodozyma CBS 6938 which is able to autonomously replicate in the yeast Saccharomyces cerevisiae was cloned on an integrative URA3 plasmid. Its minimal fragment exhibiting autonomously replicating activity in the S. cerevisiae gave a higher frequency transformation efficiency than that found for centromere-based plasmid, and enabled extrachromosomally stable transmission of the plasmids in one copy per yeast cell under non-selective culture condition. The 836-bp DNA element lacked an ORF and did not contain any acceptable match to an ARS core consensus. Sequence analysis, however, displayed a cluster of three hairpin-loop-sequences with individual ΔG25°C free energy value of - 10.0, -17.5, and -17.0 kcal · mol-1 as well as a 9-bp sequence with two base pair mismatches to the S. cerevisiae/E. coli gyrase-binding site. This 836-bp sequence also included one 7-bp sequence analogous to the core consensus of centromeric DNA element III (CD) of S. cerevisiae, but CD-like 7 bp sequence alone did not give a replicative function in this yeast.