A NOVEL AUTONOMOUSLY REPLICATING SEQUENCE (ARS) FOR MULTIPLE INTEGRATION IN THE YEAST HANSENULA-POLYMORPHA DL-1

Several autonomously replicating sequences of Hansenula polymorpha DL- 1 (HARSs) with the characteristics of tandem integration were cloned b y an enrichment procedure and analyzed for their functional elements t o elucidate the mechanism of multiple integration in tandem repeats. A ll plasmids harboring newly cloned HARSs showed a high frequency of tr ansformation and were maintained episomally before stabilization, Afte r stabilization, the transforming DNA was stably integrated into the c hromosome, HARS36 was selected for its high efficiency of transformati on and tendency for integration, Several tandemly repeated copies of t he transforming plasmid containing HARS36 (pCE36) integrated into the vicinity of the chromosomal end, Bal31 digestion of the total DNA from the integrants followed hy Southern blotting generated progressive sh ortening of the hybridization signal, indicating the telomeric localiz ation of the transforming plasmids on the chromosome. The minimum regi on of HARS36 required for its HARS activity was analyzed by deletion a nalyses. Three important regions, A, B, and C, for episomal replicatio n and integration were detected, Analysis of the DNA sequences of regi ons A and B required for the episomal replication revealed that region A contained several AT-rich sequences that showed sequence homology w ith the ARS core consensus sequence of Saccharomyces cerevisiae, Regio n B contained two directly repeated sequences which were predicted to form a bent DNA structure, Deletion of the AT-rich core in region A re sulted in a complete loss of ARS activity, and deletion of the repeate d sequences in region B greatly reduced the stability of the transform ing plasmid and resulted in retarded cell growth, Region C was require d for the facilitated chromosomal integration of transforming plasmids .