Molecular characterization of the actin-encoding gene and the use of its promoter for a dominant selection system in the methylotrophic yeast Hansenula polymorpha

The actin gene (ACT) from the methylotrophic yeast Hansenula polymorpha was cloned and its structural feature was characterized. In contrast to the ac tin genes of other ascomycetous yeasts, which have only one large intron, t he H. polymorpha ACT gene was found to be split by two introns. The H. poly morpha ACT introns were correctly processed in the heterologous host Saccha romyces cerevisiae despite appreciable differences in the splice site seque nces. The promoter region of H. polymorpha ACT displayed two CCAAT motifs a nd two TATA-like sequences in a configuration similar to that observed in t he S. cerevisiae actin promoter. A set of deleted H. polymorpha ACT promote rs was exploited to direct expression of the bacterial hygromycin B resista nce (hph) gene as a dominant selectable marker in the transformation of H. polymorpha. The resistance level of H. polymorpha transformants to the anti biotic was shown to be dependent on the integration copy number of the hph cassette. The selectivity of the hygromycin S resistance marker for transfo rmants of higher copy number was remarkably increased with the deletion of the upstream TATA-like sequence, but not with the removal of either CCAAT m otif. from the H. polymorpha promoter. The dosage-dependent selection syste m developed in this study should be useful for genetic manipulation of H. p olymorpha as an industrial strain to produce recombinant proteins.