One-step, PCR-mediated, gene disruption in the yeast Hansenula polymorpha.
Previous evidence based on the experience of our laboratory showed that one-step gene disruption in the yeast Hansenula polymorpha is not straightforward. A systematic study of several factors which could affect gene disruption frequency was carried out. We found that the more critical factor affecting one-step gene disruption in H. polymorpha is the length of the target gene region flanking the marker gene. Target gene regions of about 1 kb flanking the marker gene were necessary to obtain a disruption frequency of about 50%. However, the gene marker, either homologous or heterologous, the locus and the strain examined did not significantly affect the frequency of disruption; the highest disruption frequency obtained for the YNR1 gene was in the strain HMI39, using the Saccharomyces cerevisiae URA3 gene as a marker. Since long regions flanking the gene marker do not allow the easy PCR-mediated strategies, developed for S. cerevisiae, to obtain constructs to disrupt a given gene in H. polymorpha, an alternative PCR strategy was developed.
González
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C
Departamento de Bioquímica y Biología Molecular, Grupo del Metabolismo del Nitrógeno-Consejo Superior de Investigaciones Científicas, Universidad de La Laguna, E-38206 La Laguna, Tenerife, Canarias, Spain.
Perdomo
G
G
Tejera
P
P
Brito
N
N
Siverio
J M
JM
D015139:Blotting, Southern; D009154:Mutation; D010843:Pichia / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D014170:Transformation, Genetic