Molecular characterization of the proteins involved in the regulation of cell polarity in budding yeast and fission yeast

本论文研究了芽殖酵母和裂殖酵母细胞极性的调控机制。1.芽殖酵母细胞类型决定了两种不同的空间芽殖模式。单倍体细胞表现出轴向模式，而二倍体细胞表现出双极模式。Axl 1是决定单倍体轴向模式的关键形态决定因子。我发现了一个基因，RAX 1，其功能的丧失抑制axi 1单倍体的缺陷双极模式到轴向模式，Rax 1是一个长寿的蛋白质，持续在分裂位点残留多代。Bud 8的本地化需要Rax 1。Rax 1是双极出芽标志的建立所必需的; 2.构建了裂殖酵母随机基因中断的基因文库，但由于一些克隆在裂殖酵母细胞中可以作为质粒复制，该文库不能用于随机基因中断; 3.利用cDNA过表达文库鉴定了裂殖酵母中参与细胞极性调控的几个基因。通过双杂交、基因破坏和显微镜分析对tpb 1、ckb 1和mid 2基因进行了鉴定。Tpb 1的PB 1结构域与Scd 2的PB 1结构域相互作用。mid 2突变株细胞不能出现新末端生长。ckb 1的过量表达导致mid 2突变体细胞出现新的末端生长。4.芽殖酵母二倍体细胞在氮限制条件下发生二态性转变，形成丝状体，称为假菌丝生长。我发现了一个酵母基因，SFG 1，当缺乏氮时，它的过度表达会增强表面假菌丝的生长。sfg 1与真菌发育的转录调节因子家族成员具有序列相似性。SFG 1基因突变株的细胞在假菌丝生长方面存在严重缺陷。Sfg 1可能独立于丝裂原活化蛋白激酶途径和cAMP依赖性蛋白激酶A途径发挥作用。

I studied about the regulatory mechanisms of cell polarity in budding yeast and fission yeast.1.In budding yeast, cell type determines two distinct spatial budding patterns. Haploid cells exhibit an axial pattern, whereas diploid cells exhibit a bipolar pattern. Axl1 is the key morphological determinant for the haploid axial pattern. I identified a gene, RAX1, whose loss of function suppresses the defective bipolar pattern of axi1 haploids to the axial pattern, Rax1 is a long-lived protein persisting at division site remnants for multiple generations. Rax1 is required for the localization of Bud8. Rax1 is necessary for the establishment of the bipolar budding landmark.2.I constructed a gene library for random gene disruptions in fission yeast However, this library could not be useful for random gene disruption because some clones could replicate as plasmids in cells of fission yeast.3.I identified several genes involved in the regulation of cell polarity in fission yeast using cDNA overexpression library. Genes, tpb1,ckb1 and mid2,were characterized by two-hybrid analysis, gene disruption and microscopic analysis. The PB1 domain of Tpb1 interacts with that of Scd2. Cells of mid2 mutant strain could not exhibit new-end growth. However, overexpression of ckb1 caused new-end growth in mid2 mutant cells.4.In response to nitrogen limitation, diploid cells of budding yeast undergo a dimorphic transition to a filamentous growth form known as pseudohyphal growth. I identified a yeast gene, SFG1,whose overexpression enhances superficial pseudohyphal growth when starved for nitrogen. Sfg1 has a sequence similarity to members of a family of transcriptional regulators of fungal development. Cells of SFG1-disrupted strain have a serious defect in pseudohyphal growth. Sfg1 may act separately from mitogen-activated protein kinase pathway and cAMP-dependent protein kinase A pathway.