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Title 

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

Authors 

Kang Hyun AhWon Kyuong HongJung Hoon SohnEui Sung ChoiSang Ki Rhee

Publisher 

Springer Verlag (Germany)

Issue Date 

2001

Citation 

Applied Microbiology and Biotechnology, vol. 55, no. 6, pp. 734-741

Keywords 

actinDNAmessenger RNArecombinant proteinyeastantibiotic resistanceDNA sequencegenetic manipulationhansenula polymorphamolecular cloning

Abstract 

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

ISSN 

0175-7598

Link 

http://dx.doi.org/10.1007/s002530100605

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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