STABLE, SELECTABLE, INTEGRATIVE DNA TRANSFORMATION IN PHYSARUM

The Physarum polycephalum actin promoter, PardC, can drive transient e xpression of heterologous genes in Physarum amoebae. The hph gene, enc oding hygromycin (Hy) phosphotransferase, can confer resistance to Hy on a broad spectrum of organisms. When PardC is translationally fused to hph and transformed into yeasts on high-copy-number vectors, the ye asts become Hy resistant (Hy(R)), showing that PardC-hph is a function al, selectable genetic element. To establish a stable transformation s ystem for Physarum, we electroporated plasmids bearing PardC-hph into Physarum amoebae and then selected for Hy(R) transformants. We show th at Hy(R) amoebae arise upon the stable integration of PardC-hph into t he nuclear genome in single copy. These results establish a. transform ation system that can be used to add plasmid-borne genetic information to Physarum.