González C, Perdomo G, Tejera P, Brito N, Siverio J M
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.
Yeast. 1999 Sep 30;15(13):1323-9. doi: 10.1002/(SICI)1097-0061(19990930)15:13<1323::AID-YEA459>3.0.CO;2-1.
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.
我们实验室先前基于经验的证据表明，在多形汉逊酵母中进行一步法基因破坏并非易事。我们对几个可能影响基因破坏频率的因素进行了系统研究。我们发现，影响多形汉逊酵母一步法基因破坏的更关键因素是标记基因两侧目标基因区域的长度。标记基因两侧约1 kb的目标基因区域对于获得约50%的破坏频率是必要的。然而，基因标记，无论是同源的还是异源的，所检测的位点和菌株对破坏频率没有显著影响；使用酿酒酵母URA3基因作为标记，在菌株HMI39中获得的YNR1基因的最高破坏频率。由于基因标记两侧的长区域不允许使用为酿酒酵母开发的简单的PCR介导策略来获得用于破坏多形汉逊酵母中给定基因的构建体，因此开发了一种替代的PCR策略。
