Nakazawa T, Motoyama T, Horiuchi H, Ohta A, Takagi M
Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Japan.
J Bacteriol. 1997 Aug;179(16):5030-6. doi: 10.1128/jb.179.16.5030-5036.1997.
We observed that a YCp-type vector having the centromeric DNA (CEN) sequence previously isolated from the genome, but not a YRp-type vector lacking the CEN sequence, induced pseudohyphal growth in a dimorphic fungi, Candida maltosa, which had been shown to be closely related to Candida albicans by phylogenetic analysis. Deletion analysis of the CEN sequence revealed that the intact CEN sequence was not required for the induction, but part of it, having partial centromeric activity, was enough for the induction. By screening the gene library of this yeast for the sequences which induced pseudohyphal growth, we isolated three different DNA fragments which also had part of the centromere-like sequence. Partial centromeric activity of these fragments was confirmed by three criteria: low copy number and high stability of the plasmids carrying these fragments and rearrangement at high frequency of the plasmid DNA with one of these fragments plus the CEN sequence. Furthermore, when the GGTAGCG sequence commonly found in one copy in each of these four sequences was mutated in the CEN sequence by site-directed mutagenesis, both partial centromeric activity and pseudohyphal growth-inducing activity of the CEN sequence were lost. These results indicated that part of CEN region with partial centromeric activity induces pseudohyphal growth in C. maltosa. It is suggested that some cellular components which interact with the sequence containing GGTAGCG required for centromeric activity are involved in the regulation of the transition between yeast forms and pseudohyphal forms of the cells.
我们观察到,一种带有先前从基因组中分离出的着丝粒DNA(CEN)序列的YCp型载体,而非缺乏CEN序列的YRp型载体,能在一种二态真菌——麦芽糖假丝酵母中诱导假菌丝生长,系统发育分析表明该真菌与白色念珠菌密切相关。对CEN序列的缺失分析显示,诱导作用并不需要完整的CEN序列,但其部分具有部分着丝粒活性的序列就足以引发诱导。通过筛选该酵母的基因文库以寻找能诱导假菌丝生长的序列,我们分离出了三个不同的DNA片段,它们也含有部分着丝粒样序列。这些片段的部分着丝粒活性通过三个标准得以证实:携带这些片段的质粒拷贝数低且稳定性高,以及携带其中一个片段与CEN序列的质粒DNA高频重排。此外,当通过定点诱变使这四个序列中每个序列都共有的一个拷贝中的GGTAGCG序列在CEN序列中发生突变时,CEN序列的部分着丝粒活性和假菌丝生长诱导活性均丧失。这些结果表明,具有部分着丝粒活性的CEN区域的一部分在麦芽糖假丝酵母中诱导假菌丝生长。这表明一些与着丝粒活性所需的包含GGTAGCG的序列相互作用的细胞成分参与了细胞酵母形态和假菌丝形态之间转变的调控。