Characterization of new DNA sequences of Chlamydomonas reinhardtii that replicate autonomously in Saccharomyces cerevisiae

Abstract

After cloning in pUC9 ura3, a newly constructed 3.84 kb yeast integrating vector, two Sau3A DNA fragments of 0.4 kb (ARS09) and 1.5 kb (ARS10) promoting plasmid autonomous replication in yeast have been isolated from a Chlamydomonas purified nuclear DNA fraction slightly contaminated with chloroplast DNA. Plasmids carrying these fragments were unstable in yeast but transformed it at high frequency. ARS09 and ARS10 were shown to hybridize to the chloroplast EcoRI fragments R19 and R21 for ARS09 and to the fragment R26 for ARS10. Since the amount of chloroplast DNA in the DNA fraction used was minor it can be concluded that the frequency of ARSs in nuclear DNA is low. The sequence analysis of ARS09 and ARS10 shows a high AT content, numerous direct and inverted repeats and 11 bp elements closely related to the yeast ARS consensus sequence. ARS09 and ARS10 contain in addition an 11 bp sequence differing by one or two nucleotides only from the 3′ conserved element present in many yeast ARSs and which might play a role in promoting ARS activity.