PLASMID REORGANIZATION DURING INTEGRATIVE TRANSFORMATION IN HANSENULA-POLYMORPHA

During studies of integrative transformation in Hansenula polymorpha, it was found that transformants with plasmids possessing the LEU2 gene of H. polymorpha were frequently unstable and lost plasmids while growing on non-selective medium. These transformants possessed reorganized plasmids capable of replication in H. polymorpha. Two such plasmids were isolated and characterized. It was shown that they contain additional DNA segments which were not present in the original plasmid used for transformation. Southern hybridization analysis carried out with labeled DNA probes derived from these segments showed that they consisted of H. polymorpha DNA. The hybridization patterns indicated that corresponding sequences were homologous to several chromosomal regions. These chromosomal DNA segments apparently carried H. polymorpha autonomous replicating sequences (HARS), since plasmids bearing them could transform H. polymorpha with high efficiency and were maintained in transformants in an autonomous state. Sequence analysis of one such captured chromosomal fragment revealed several eight- to ten-base AT-rich blocks similar to the presumed HARS sequence defined by Roggenkamp et al. (1986). Analogous reorganization was also observed with respect to integrative plasmids carrying the TRP3 and HIS3 genes of H. polymorpha and the ADE2 gene of Saccharomyces cerevisiae as selectable markers.