酵母细胞周期调控的研究进展

酵母是一种研究细胞周期调控的好材料,在细胞周期的调控研究中具有重要作用。现在通常以芽殖酵母和裂殖酵母为代表进行研究。这两种酵母的细胞周期进程与高等真核生物相比各有其特点。 酵母细胞周期运行中存在有三个不同的控制点,即start点、S期启动点、G2/M转换处。在这三个不同的控制点起作用的CDK的组成是不同的。芽殖酵母分别是Cdc28-Clns;Cdc28-Clb5和Cdc28-Clb6;Cdc28-Clb1、Cdc28-Clb2、Cdc28-Clb3和Cdc28-Clb4。裂殖酵母中分别是Cdc2-Cig2;Cdc2-Cig2;Cdc2-Cdc13和Cdc2-Cig1,其中芽殖酵母中的Cdc28和裂殖酵母中的cdc2是等效基因。不同的控制点存在着不同 的调控机制,它们涉及到大量的基因,其中以G2/M转换处的调控机制研究得最早也最透彻。另外,APC途径在M中期/后期转化中起着重要作用。     

酵母细胞周期及其调控

酵母菌是一类多形的、不运动的、单细胞的真核微生物的统称。多数酵母菌都以出芽方式进行繁殖,称为芽殖酵母,也有少数种类的酵母以二分裂方式进行繁殖,称为裂殖酵母,例如粟酒裂殖酵母（＆hizosaccha－roapcesPombe）．芽殖酵母是研究细胞周期GI向S期过渡调控机制的好材料;而裂殖酵母为研究GZ向M期过渡调节的典型材料．下面简要介绍酵母细胞周期及其调控机制．1＄母细胞周期酵母细胞周期是由四个连续的时期组成,即：M期（有丝分裂期,包括核分裂和胞质分裂）、GI期（介于有丝分裂期与DNA合成之间的间歇期）、S期（DNA合成期）和…     

PHO80、PHO85基因和芽殖酵母金属离子耐受

PHO8 5基因是芽殖酵母中的一个多功能基因。它参与了无机磷酸的代谢、碳源利用、糖原积累、特定蛋白质的降解和细胞周期调控。研究了酵母株YPH499及其衍生的pho85缺失株、pho80缺失株、pap1(pcl7)缺失株在不同浓度的不同金属离子中的存活情况 ,结果表明和芽殖酵母YPH499相比 ,pho85缺失株和pho80缺失株表现出对K 、Mg2 、Zn2 、Ca2 和Mn2 的耐受下降 ,而PAP1基因的缺失则不会导致芽殖酵母对上述金属离子的敏感性的变化 ;而对Cu2 ,3株突变株都表现出和YPH499相同的耐受性。同时测定了各缺失株和YPH499对上述金属离子的半致死浓度以及pho85缺失株、pho80缺失株和YPH499的细胞内总钙量。这些结果显示 ,PHO85蛋白激酶通过和它的PCLPHO80而不是PAP1结合 ,参与了芽殖酵母K 、Mg2 、Zn2 、Ca2 和Mn2 离子平衡的调控。PHO85和PHO80基因的缺失损害了芽殖酵母钙的储存。     

芽殖酵母ASH1 mRNA的定位机制

mRNA定位是一种基因转录后水平的重要调控机制,对细胞的生理活动和分化发育都有着极其重要的作用。在芽殖酵母有丝分裂中,ASH1 mRNA在子细胞芽尖因不对称定位表达抑制了子细胞交配类型的转换。本综述介绍了芽殖酵母ASH1 mRNA定位的分子机制。     

芽殖酵母和裂殖酵母中异染色质形成机制

芽殖酵母(Saccharomyces cerevisiae)和裂殖酵母(Schizosaccharomyces pombe)是用来研究异染色质形成、细胞周期、DNA复制等重要细胞功能的理想单细胞真核生物.本文主要介绍这2种酵母中异染色质形成的机制.异染色质是一种抑制基因转录和DNA重组的特殊染色质结构.尽管在芽殖酵母和裂殖酵母中异染色质形成都需要组蛋白修饰,但异染色质建立的机制不同.在芽殖酵母中参与异染色质形成的主要蛋白是Sir1-4蛋白（其中Sir2为组蛋白H3去乙酰化酶）,而组蛋白H3赖氨酸9甲基化酶Clr4和异染色质蛋白Swi6在裂殖酵母异染色质形成中起关键的作用.在这两个酵母中,参与异染色质形成的组蛋白修饰蛋白由DNA结合蛋白招募到异染色质.此外,裂殖酵母也利用RNA干扰系统招募组蛋白修饰蛋白.     

美科学家发现25个“长寿基因”

美国科学家日前通过对芽殖酵母和线虫的基因分析,鉴别出2种生物共有的25个负责调控寿命长短的基因。     

丝/苏氨酸蛋白激酶Plk1研究进展

Plk1是一类从酵母到人类都高度保守的丝氨酸/苏氨酸蛋白激酶。Plk1与不同的细胞周期检查点的精密调控有关,从而确保了细胞周期事件按照严格的时间和顺序正常进行。Plk1在增殖活跃的细胞中呈高水平表达,Plk1的高度表达和肿瘤患者的低存活率之间具有显著的统计相关性。Plk1可能是非常有效的抗癌药物设计的靶点。     

人乳头瘤病毒E7蛋白对细胞周期G1/S检查点的影响

高危型人乳头瘤病毒(HPV)感染与宫颈癌的发生关系已经明确,HPV编码的早期蛋白E7是HPV致宫颈癌的主要相关蛋白之一。G_1/S检查点是细胞周期中不可逆的关键点,决定了细胞进入细胞周期与否,与肿瘤的发生发展关系密切。pRb蛋白是调控细胞周期G_1/S检查点中的限速底物,是起调控作用的主要因子之一。E7通过影响pRb蛋白、E2F家族、Cyclin/CDK2复合物、p27蛋白、Dyrk1B等细胞周期相关蛋白来影响G_1/S检查点的正常工作,形成失控的细胞增殖。高危型HPV E7蛋白对细胞周期检查点的影响,既是HPV致癌机制的重要组成部分,也可能成为攻克此类癌症的突破口。本文综述了高危型HPV E7蛋白对细胞周期G_1/S检查点的影响。     

裂殖酵母复制检查点调控

细胞在敏殖时可能遇到各种各样的干扰,因此细胞需要用检查点时时监控基因组,当复制干扰发生时,复制检查点被激活并保证M期在S期完成后进行。检查点通过感受器,信号转导蛋白,效应器之间的一系列信号传导级联反应,作用于其最终调控对象,并排除“故障”,由于裂殖酵母同哺乳动物的检查点结构最为相似,文章将详细论述裂殖酵母复制检查点调控过程及其与人类检查点的相似之处。     

蛋白质的磷酸化作用和泛肽化降解作用与芽殖酵母细胞周期的调控

在芽殖酵母（Ｓａｃｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅ）细胞中,Ｇ１期的三种ｃｙｃｌｉｎｓ和Ｓ、Ｍ期的五种ｃｙｃｌｉｎｓ之周期性的合成和分解调节着Ｃｄｃ２８的活性,驱动细胞周期的正常运转。除了ＣＤＫ的磷酸化作用外,蛋白质的泛肽化降解作用间接或直接调控细胞周期：ＣＤＣ３４泛肽化途径通过降解Ｃｄｃ２８的专一抑制子而起始ＤＮＡ复制;ＡＰＣ泛肽化途径通过降解Ｍ期后期的抑制子和Ｍ期ｃｙｃｌｉｎｓ,使姐妹染色体分离和Ｍ期终止。     

Morphogenesis and cell cycle progression in Candida albicans

Candida albicans, an opportunistic human pathogen, displays three modes of growth: yeast, pseudohyphae and true hyphae, all of which differ both in morphology and in aspects of cell cycle progression. In particular, in hyphal cells, polarized growth becomes uncoupled from other cell cycle events. Yeast or pseudohyphae that undergo a cell cycle delay also exhibit polarized growth, independent of cell cycle progression. The Spitzenk?rper, an organelle composed of vesicles associated with hyphal tips, directs continuous hyphal elongation in filamentous fungal species and also in C. albicans hyphae. A polarisome mediates cell cycle dependent growth in yeast and pseudohyphae. Regulation of morphogenesis and cell cycle progression is dependent upon specific cyclins, all of which affect morphogenesis and some of which function specifically in yeast or hyphal cells. Future work will probably focus on the cell cycle checkpoints involved in connecting morphogenesis to cell cycle progression.     

Comparative Evolutionary Analysis of Cell Cycle Proteins Networks in Fission and Budding Yeast

Fission yeast and budding yeast are the two distantly related species with common ancestors. Various studies have shown significant differences in metabolic networks and regulatory networks. Cell cycle regulatory proteins in both species have differences in structural as well as in functional organization. Orthologous proteins in cell cycle regulatory protein networks seem to play contemporary role in both species during the evolution but little is known about non-orthologous proteins. Here, we used system biology approach to compare topological parameters of orthologous and non-orthologous proteins to find their contributions during the evolution to make an efficient cell cycle regulation. Observed results have shown a significant role of non-orthologous proteins in fission yeast in maintaining the efficiency of cell cycle regulation with less number of proteins as compared to budding yeast.     

The decisive role of the Saccharomyces cerevisiae cell cycle behaviour for dynamic growth characterization.

The dynamic behaviour of the cell cycle and the physiology of Saccharomyces cerevisiae was monitored in transient experiments. Frequent flow cytometric analyses of the DNA (nuclear phase state) and the cell size enabled us to characterize the proliferation properties of yeast cells under well controlled and undisturbed cultivation conditions. Preliminarily, the correlation between flow cytometric light scattering measurements and the cell size was attested for yeasts. These flow cytometric results are compared with the physiological behaviour of the culture that was detected by high resolution on-line analyses and off-line measurements. The presented results focus on the importance of the yeast cell cycle behaviour for the dynamic growth characterization. Any kind of transients in yeast cultures induced partial synchronization. The characteristics and the time course of the yeast cell cycle were found to be strongly dependent on the physiological environment.     

Extracellular calmodulin regulates growth and cAMP-mediated chemotaxis in Dictyostelium discoideum

Zymocin and PaT are killer toxins that induce cell cycle arrest of sensitive yeast cells in G1 and S phase, respectively. Recent studies have revealed that these two toxins cleave specific tRNAs, indicating that the cell growth impairment is due to the tRNA cleavage. Additionally, we have previously shown that the active domain of colicin D (D-CRD), which also cleaves specific Escherichia coli tRNAs, statically impairs growth when expressed in yeast cells. To verify that phase-specific cell cycle arrest is also induced by the expression of D-CRD, D-CRD and the subunits of zymocin and PaT that have tRNA cleaving activity were expressed in yeast cells and cell cycle status was analyzed. Our results indicate that phase-specific arrest does not commonly occur by tRNA cleavage, and it saves the cell viability. Furthermore, the extent of protein synthesis impairment may determine the phase specificity of cell cycle arrest.     

Synchronization of cell cycle of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by using a cell chip platform

Cell synchrony is a critical requirement for the study of eukaryotic cells. Although several chemical and genetic methods of cell cycle synchronization are currently available, they have certain limitations, such as unnecessary perturbations to cells. We developed a novel cell cycle synchronization method that is based on a cell chip platform. The budding yeast, Saccharomyces cerevisiae, is a simple but useful model system to study cell biology and shares many similar features with higher eukaryotic cells. Single yeast cells were individually captured in the wells of a specially designed cell chip platform. When released from the cell chip, the yeast cells were synchronized, with all cells in the G1 phase. This method is non-invasive and causes minimal chemical and biological damage to cells. The capture and release of cells using cells chips with microwells of specific dimensions allows for the isolation of cells of a particular size and shape; this enables the isolation of cells of a given phase, because the size and shape of yeast cells vary with the phase of the cell cycle. To test the viability of synchronized cells, the yeast cells captured in the cell chip platform were assessed for response to mating pheromone (α-factor). The synchronized cells isolated using the cell chip were capable of mediating the mating signaling response and exhibited a dynamic and robust response behavior. By changing the dimensions of the well of the cell chip, cells of other cell cycle phases can also be isolated.     

Carbon source-dependent regulation of cell growth by murine protein kinase C epsilon expression in Saccharomyces cerevisiae

Protein kinase C is known to play a role in cell cycle regulation in both lower and higher eucaryotic cells. Since mutations in yeast proteins involved in cell cycle regulation can often be rescued by the mammalian homolog and since significant conservation exists between PKC-signalling pathways in yeast and mammalian cells, cell cycle regulation by mammalian PKC isoforms may be effectively studied in a simpler genetically-accessible model system such as Saccharomyces cerevisiae. With this objective in mind, we transfected S. cerevisiae cells with a plasmid (pYECepsilon) coding for the expression of murine protein kinase C epsilon (PKCepsilon) under the control of a galactose-inducible promoter. Unlike mock-transfected cells, yeast cells transformed with pYECepsilon expressed, in a galactose-dependent manner, an 89 kDa protein that was recognized by a human PKCepsilon antibody. Extracts from these pYECepsilon-transfected cells could phosphorylate a PKCepsilon substrate peptide in a phospholipid/phorbol ester-dependent manner. Moreover, this catalytic activity could be inhibited by a fusion protein in which the regulatory domain of murine PKCepsilon was fused in frame with GST (GST-Repsilon), further confirming the successful expression of murine PKCepsilon. Induction of PKCepsilon expression by galactose in cells transformed with pYECepsilon increased Ca++ uptake by the cells approximately 5-fold and resulted in a dramatic inhibition of cell growth in glycerol. However, when glucose was used as the carbon source, PKCepsilon expression had no effect on cell growth. This was in contrast to what was observed upon bovine PKCalpha or PKCbeta-I expression in yeast, where expression of these PKC isoforms strongly and moderately inhibited growth in glucose, respectively. Visualization of the cells by phase contrast microscopy indicated that murine PKCepsilon expression in the presence of glycerol resulted in a significant increase in the number of yeast cells exhibiting very small buds. Since overall growth of the cells was dramatically decreased, the data suggests that PKCepsilon expression potently inhibits the progression of yeast cells through the cell cycle after the initiation of budding. In addition, a small amount of the PKCepsilon-expressing yeast cells (1-2%) exhibited gross alterations in cell morphology and defects in both chromosome segregation and septum formation. This suggests that for those cells which do complete DNA synthesis, murine PKCepsilon expression may nevertheless inhibit yeast cell growth by retarding and/or imparing cell division. Taken together, the data suggests murine PKCepsilon expression potently reduces the growth of yeast cells in a carbon source-dependent fashion by affecting progression through multiple points within the cell cycle. This murine PKCepsilon-expressing yeast strain may serve as a very useful tool in the elucidation of mechanism(s) by which external environmental signals (possibly through specific PKC isoforms) regulate cell cycle progression in both yeast and mammalian cells.     

Effects of 50 Hz magnetic field on cell cycle kinetics and the colony forming ability of budding yeast exposed to ultraviolet radiation.

To investigate the effects of extremely low frequency magnetic fields on ultraviolet radiation (UV) exposed budding yeast, haploid yeast (Saccharomyces cerevisiae) cells of the strain SEy2101a were exposed to 50 Hz sine wave magnetic field (MF) of 120 microT with simultaneous exposure to UV radiation. Most of the UV energy was in the UVB range (280-320 nm). The biologically weighted (CIE action spectrum) dose level for the UV radiation was 175 J/m2. We examined whether 50 Hz MF affected the ability of UV irradiated yeast cells to form colonies (Colony Forming Units, CFUs). In addition, the effect of coexposure on cell cycle kinetics was investigated. Although the significant effect of MF on the cell cycle phases of UV exposed yeast cells was seen only at one time point, the overall results showed that MF exposure may influence the cell cycle kinetics at the first cycle after UV irradiation. The effect of our particular MF exposure on the colony forming ability of the UV irradiated yeast cells was statistically significant 420 min after UV irradiation. Moreover, at 240, 360, and 420 min after UV irradiation, there were fewer CFUs in every experiment in (UV+MF) exposed populations than in only UV exposed yeast populations. These results could indicate that MF exposure in conjunction with UV may have some effects on yeast cell survival or growth.     

Improved Flow Cytometric Analysis of the Budding Yeast Cell Cycle

The budding yeast, Saccharomyces cerevisiae has been a remarkably useful model system for the study of eukaryotic cell cycle regulation. Flow cytometric analysis of DNA content in budding yeast has become a standard tool for the analysis of cell cycle progression. However, popular protocols utilizing the DNA binding dye, propidium iodide, suffer from a number of drawbacks that confound accurate analysis by flow cytometry. Here we show the utility of the DNA binding dye, SYTOX Green, in the cell cycle analysis of yeast. Samples analyzed using SYTOX Green exhibited better coefficients of variation, improved linearity between DNA content and fluorescence, and decreased peak drift associated with changes in dye concentration, growth conditions or cell size.

Key Words:

Flow cytometry, Cell cycle, Saccharomyces cerevisiae, SYTOX Green, Propidium iodide