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Synchronization of Saccharomyces cerevisiae Cells in G1 Phase of the Cell Cycle
酿酒酵母细胞在细胞周期G1阶段的同步化   

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PLOS Biology
Jan 2014

Abstract

The baker’s yeast, Saccharomyces cerevisiae is a widely used model organism in molecular biology because of the high homology it shares with human cells in many basic cellular processes such as DNA replication, repair, recombination, transcription, and because of the ease its genome can be manipulated. Other advantages of working with yeast are its fast production rate which is comparable to bacteria’s, and its cheap maintenance.

To examine certain phenomena, for example whether a mutation affects the passage through a cell cycle phase, it can be necessary to work with a yeast culture, in which all the cells are in the same phase of the cell cycle. Yeasts can be arrested and kept in different phases of the cell cycle. Here we describe the method that allows synchronizing and keeping yeast cells in the G1 phase of the cell cycle with the mating pheromone, α-factor. Only MATa cells can be synchronized with α-factor which is produced by MATα cells. It is highly recommended to use a MATa bar1 deletion strain. The BAR1 gene encodes for an extracellular protease that inactivates α-factor by cleaving it (MacKay et al., 1988). To counteract the Bar1 protease activity when using BAR1 cells, 100-1000 times more α-factor is needed as compared to bar1 deletion cells (α-factor is quite expensive!), and still the synchrony will be transient. In contrast, bar1 deletion cells can be kept in G1 phase with α-factor for several hours, and the degree of synchronization is usually higher than using a BAR1 strain. Moreover, bar1 deletion cells can be synchronized even at high cell density, whereas BAR1 cells, due to the activity of the secreted Bar1 protease, only at low cell density.

Keywords: Alpha mating factor (α交配因), G1 phase (G1期), Yeast (酵母)

Materials and Reagents

  1. MATa bar1 deletion strain (can be engineered in the lab, or purchased from stock repositories like EUROSCARF)
  2. Yeast extract
  3. Peptone
  4. D-glucose
  5. 95% ethanol
  6. Yeast extract-peptone-dextrose (YPD) media (see Recipes) (or other media needed for the strain in use)
  7. α-factor (α-mating factor acetate salt) (Sigma-Aldrich, catalog number: T6901-5MG ) (see Recipes)
  8. Pronase (protease, from streptomyces griseus) (Sigma-Aldrich, catalog number: P6911-5G ) (see Recipes)

Equipment

  1. 30 °C incubator-shaker (180-200 rounds per minute)
  2. Microscope
  3. Glass slide
  4. Coverslips
  5. Centrifuge
  6. Spectrophotometer
  7. Vortex
  8. Culture tube
  9. Culture flask

Procedure

  1. Grow up a small overnight starter culture inoculated from one single colony from a YPD plate in YPD in a culture tube (180 rpm, 30 °C). Usually 1/10th of the final working culture is enough for the starter.
  2. From the overnight culture inoculate into fresh media to get OD600:0.2 in a culture flask or tube (this is your working culture with its working volume).
  3. Grow the culture to get the required amount of cells, but do not go higher than OD600:0.8 because at very high density even bar1 deletion cells do not synchronize. Keep in mind that during synchronization the density of the culture will increase since cells that already passed G1 will complete the cycle until they reach G1 of the next cycle.
  4. Spin down the cells and discard the media (3 min with 3,000 rpm at room temperature).
  5. Wash the cell pellet with at least 10 cell pellet volumes of water.
  6. Spin down the cells and discard the water (3 min, 3,000 rpm RT).
  7. Resuspend the cells in working volume of fresh YPD containing 50 ng/ml α-factor.
  8. Grow the cells.
  9. After 60, 90, 120 min check the cells under the microscope with 40x magnification. Before checking vortex the samples thoroughly to disturb clamps. G1 arrested cells have typical pear shape. Usually between 90 and 120 min ~100% synchrony can be observed.
  10. To release the cells from the arrest, spin down the cells and wash them with water.
  11. Repeat washing.
  12. Resuspend the cells in the working volume of fresh YPD containing 50 µg/ml pronase. It is important to add pronase because even a remaining small amount of α-factor can inhibit release.
  13. After adding pronase cells will enter the cell cycle. Depending on your goal, you can let them cycle, treat them with different agents, or collect them at any desired time points. Pronase does not affect cell growth.
    This experiment is easy to reproduce once you can recognize arrested cells (Figure 1). If your strain has a selectable marker, you can carry out the experiment using the appropriate media instead of YPD.


    Figure 1. Yeast cells treated with α-factor. (Source: Arkowitz, 2009)

Recipes

  1. YPD liquid
    1% yeast extract
    2% peptone
    2% D-glucose
  2. α-factor
    1 mg/ml stock solution in EtOH
    Stored at -20 °C
  3. Pronase
    40 mg/ml stock solution in sterile distillated water
    Stored at -20 °C

Acknowledgments

This protocol is a modified form of the one published by Amberg et al. (2005). We used this protocol in our work (Daraba et al., 2014). Funding support: Wellcome Trust, 070247/Z/03/A.

References

  1. Amberg, D. C., Burke, D. J. and Strathern, J. N. (2005). Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, 2005 Edition. ISBN  978-087969728-0.
  2. Arkowitz, R. A. (2009). Chemical gradients and chemotropism in yeast. Cold Spring Harb Perspect Biol 1(2): a001958.   
  3. Daraba, A., Gali, V. K., Halmai, M., Haracska, L. and Unk, I. (2014). Def1 promotes the degradation of Pol3 for polymerase exchange to occur during DNA-damage--induced mutagenesis in Saccharomyces cerevisiae. PLoS Biol 12(1): e1001771.
  4. MacKay, V. L., Welch, S. K., Insley, M. Y., Manney, T. R., Holly, J., Saari, G. C. and Parker, M. L. (1988). The Saccharomyces cerevisiae BAR1 gene encodes an exported protein with homology to pepsin. Proc Natl Acad Sci U S A 85(1): 55-59.

简介

面包酵母,酿酒酵母(Saccharomyces cerevisiae)是分子生物学中广泛使用的模式生物体,因为它在许多基本细胞过程如DNA复制,修复,重组,转录中与人细胞具有高同源性,并且因为的易于其基因组可以操纵。使用酵母的其他优点是其快速的生产速率与细菌相当,并且其廉价的维护。
为了检查某些现象,例如突变是否影响通过细胞周期阶段,可能有必要与酵母培养物一起工作,其中所有细胞处于细胞周期的相同阶段。酵母可以被捕获并保持在细胞周期的不同阶段。在这里我们描述了允许同步和保持酵母细胞在细胞周期的G1期与交配信息素,α因子的方法。只有MATa细胞可以与由MATα细胞产生的α因子同步。强烈建议使用MATa bar1 缺失菌株。 BAR1 基因编码胞外蛋白酶,通过切割它使α因子失活(MacKay等人,1988)。为了在使用BAR1细胞时抵消蛋白酶活性,相比于bar1 缺失细胞,需要100-1000倍的α因子(α-因子是相当昂贵的!) ,同步仍然是瞬态的。相反,bar1 缺失细胞可以在具有α因子的G1期保持几小时,并且同步程度通常高于使用BAR1 株。此外,由于分泌的Bar1蛋白酶的活性,仅在低细胞密度下,即使在高细胞密度下, bar1 缺失细胞也可以同步,而BAR1 细胞。

关键字:α交配因, G1期, 酵母

材料和试剂

  1. MATa bar1 缺失菌株(可在实验室中设计,或从EUROSCARF等库存库购买)
  2. 酵母提取物
  3. 蛋白胨
  4. D-葡萄糖
  5. 95%乙醇
  6. 酵母提取物 - 蛋白胨 - 葡萄糖(YPD)培养基(见Recipes)(或使用菌株所需的其他培养基)
  7. α-因子(α-交配因子乙酸盐)(Sigma-Aldrich,目录号:T6901-5MG)(参见配方)
  8. 链霉素(蛋白酶,来自灰色链霉菌)(Sigma-Aldrich,目录号:P6911-5G)(参见Recipes)

设备

  1. 30℃恒温摇床(180-200转/分钟)
  2. 显微镜
  3. 玻璃片
  4. 盖舌
  5. 离心机
  6. 分光光度计
  7. 涡流
  8. 文化管
  9. 培养瓶

程序

  1. 在培养管(180rpm,30℃)中培养从YPD板中的一个单菌落接种到YPD中的小的隔夜起始培养物。 通常,最终工作文化的1/10 th 就足够了
  2. 从过夜培养物接种到新鲜培养基中以在培养瓶或管中获得OD <600>:0.2(这是具有其工作体积的工作文化)。
  3. 生长培养物以获得所需量的细胞,但不高于OD 600:0.8,因为在非常高的密度下,即使bar1 缺失细胞不同步。 记住,在同步期间,培养物的密度将增加,因为已经通过G1的细胞将完成循环,直到它们达到下一个循环的G1。
  4. 旋下细胞并丢弃培养基(室温下3000转/分钟,3分钟)
  5. 用至少10个细胞团块的水洗涤细胞沉淀
  6. 旋转细胞并丢弃水(3分钟,3,000rpm转)。
  7. 在含有50 ng/mlα因子的新鲜YPD工作体积中重悬细胞
  8. 生长细胞。
  9. 在60,90,120分钟后,在显微镜下以40x放大检查细胞。 在彻底涡旋样品以干扰夹具之前。 G1逮捕的细胞具有典型的梨形状。 通常在90和120分钟之间〜100%同步可以观察到
  10. 要释放细胞从逮捕,旋下细胞和用水洗它们
  11. 重复清洗。
  12. 重悬细胞在工作体积的新鲜YPD含50μg/ml链霉蛋白酶。重要的是添加链霉蛋白酶,因为即使剩余少量的α-因子也能抑制释放
  13. 加入链霉蛋白酶后,细胞会进入细胞周期。根据您的目标,您可以让他们循环,用不同的代理处理他们,或收集他们在任何所需的时间点。链霉蛋白酶不影响细胞生长。
    这个实验很容易重现一旦你可以识别被拘捕的细胞(图1)。如果你的菌株有一个可选择的标记,你可以使用适当的媒介,而不是YPD进行实验

    图1.用α-因子处理的酵母细胞(来源:Arkowitz,2009)

食谱

  1. YPD液体
    1%酵母提取物
    2%蛋白胨
    2%D-葡萄糖
  2. α因子
    1mg/ml储备溶液,在EtOH中
    储存于-20°C
  3. pronase
    40 mg/ml无菌蒸馏水中的储备溶液
    储存于-20°C

致谢

该协议是由Amberg等人(2005)发表的修改形式。 我们在我们的工作中使用了这个协议(Daraba ,2014)。 资金支持:Wellcome Trust,070247/Z/03/A。

参考文献

  1. Amberg,D.C.,Burke,D.J。和Strathern,J.N。(2005)。 Methods in Yeast Genetics:A Cold Spring Harbor Laboratory Course Manual,2005 Edition。 ISBN  978-087969728-0。
  2. Arkowitz,R.A。(2009)。 酵母中的化学梯度和趋化性。 Cold Spring Harb Perspect Biol em> 1(2):a001958。   
  3. Daraba,A.,Gali,V.K.,Halmai,M.,Haracska,L.and Unk,I.(2014)。 Def1促进聚合酶交换的Pol3的降解发生在DNA损伤诱导的诱变过程中, em> Saccharomyces cerevisiae 。 12(1):e1001771。
  4. MacKay,V.L.,Welch,S.K.,Insley,M.Y.,Manney,T.R.,Holly,J.,Saari,G.C.and Parker,M.L。(1988)。 酿酒酵母 BAR1基因编码与胃蛋白酶同源的输出蛋白。 Proc Natl Acad Sci USA 85(1):55-59。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Unk, I. and Daraba, A. (2014). Synchronization of Saccharomyces cerevisiae Cells in G1 Phase of the Cell Cycle. Bio-protocol 4(20): e1273. DOI: 10.21769/BioProtoc.1273.
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