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Measuring UV-induced Mutagenesis at the CAN1 Locus in Saccharomyces cerevisiae
紫外线诱导的酿酒酵母CAN1基因突变位点的检测   

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参见作者原研究论文

本实验方案简略版
PLOS Biology
Jan 2014

Abstract

There are several methods to measure the capacity of yeast cell to respond to environmental impacts on their genome by mutating it. One frequently used method involves the detection of forward mutations in the CAN1 gene. The CAN1 gene encodes for an arginine permease that is responsible for the uptake of arginine and it can also transport the toxic analog of arginine, canavanine (Whelan et al., 1979). When CAN1 cells are grown on a media containing canavanine but lacking arginine, the cells die because of the uptake of the toxic canavanine. However, if a mutation in the CAN1 gene inactivates the permease, that cell survives and forms a colony on the plate.

The following protocol describes the measurement of UV-induced mutagenesis at the CAN1 locus.

Keywords: Yeast (酵母), Mutagenesis (诱变), Forward mutations (正向突变), Canavanine (刀豆氨酸), UV light (UV光)

Materials and Reagents

  1. Strains that have the wild type CAN1 gene (e.g., BY4741, EMY747)
  2. Yeast nitrogen base [w/o amino acids and w (NH4)2SO4] (Difco)
  3. Adenine (Sigma-Aldrich)
  4. Arginine (Sigma-Aldrich)
  5. Canavanine (Sigma-Aldrich)
  6. Histidine (Sigma-Aldrich)
  7. Isoleucine (Sigma-Aldrich)
  8. Leucine (Sigma-Aldrich)
  9. Lysine (Sigma-Aldrich)
  10. Methionine (Sigma-Aldrich)
  11. Phenylalanine (Sigma-Aldrich)
  12. Tryptophan (Sigma-Aldrich)
  13. Tyrosine (Sigma-Aldrich)
  14. Uracil (Sigma-Aldrich)
  15. Valine (Sigma-Aldrich)
  16. Yeast extract
  17. Pepton
  18. D-glucose
  19. Bacto agar
  20. Yeast extract-pepton-D-glucose (YPD) media (see Recipes) (or other media needed for the strain in use)
  21. Synthetic complete media (SC) plates (see Recipes)
  22. SC-arginine plates containing canavanine (+can) (see Recipes)

Equipment

  1. 30 °C incubator-shaker (180-200 round per minute)
  2. 30 °C incubator
  3. Glass slide
  4. Centrifuge
  5. Microscope
  6. Vortex
  7. UV-irradiation machine
  8. Box
  9. Spreader
  10. Culture tube
  11. Waterbath sonicator
  12. Hemocytometer (Burker counting chamber)

Procedure

  1. By inoculating a single colony from a fresh plate, grow up 10 ml overnight cultures in YPD (one reference, or wild type strain, and the strains to be examined).
  2. Put the culture tube into a waterbath sonicator and sonicate the cells at room temperature for 3 min to disrupt clumps (40 Hz). Check for the presence of clumps under the microscope.
  3. Make 200x dilutions of each strain in water (5 µl cell culture into 995 µl water) and put 10 µl onto a Burker-chamber.
  4. Count the cells under the microscope in a big square of the Burker-chamber (bordered by three lines) (Figure 1). One cell in a big square means 1 x 104 cells/ml.
  5. Calculate the density of the original cultures (multiply the counted cell number by 200).
  6. Make 10x serial dilutions of each strain, starting from 108 to 103 cells/ml. If necessary concentrate cells to get 108 cells/ml. When calculating the volume of a given dilution step, take into consideration how many plates you will be plating from that dilution (see steps 8 and 9 below).
  7. Label the SC and the +can plates with the strain names and the UV doses you want to apply. For each UV dose, including zero, label 2 SC and 2 +can plates for each strain. The SC plates will be used to calculate survival at different UV doses, the +can plates will be used to calculate mutagenesis at different UV doses.
  8. Plate 200 µl on the control, 0 J/m2 SC plates from the 103 cells/ml dilution. For wild type, EMY747 or BY4741 cells that are quite resistant to UV showing 20% survival at 80 J/m2, for up to that dose the 103 cells/ml dilution should be used for plating. In case of more sensitive strains the expected survival rate should be taken into consideration when determining which dilution to use for plating for given UV doses.
  9. Plate 200 µl cells on the +can plates from the 108 cells/ml dilutions.
  10. Wait till plates absorb the moisture, then irradiate the plates without lids, with the required UV doses. Make sure the irradiated plates are not exposed to white light after irradiation (work with yellow light on), and they are placed right away in a box that shields them from light and put in the 30 °C incubator.
  11. Incubate the plates until colonies grow to 2-3 mm in diameter. For SC plates it takes usually 2-3 days, for +can plates it takes up to 5 days (check them under yellow light).
  12. Count the colonies on each plate.
  13. Calculate the percentage of survival on SC plates. Divide the average number of colonies of the two 0 J/m2 plates with the number of cells that were plated on one plate and multiply it by 100. That gives the percentage of cells that survived plating.
  14. Calculate the survival at each UV doses. Multiply the average colony number of the two parallel plates with the plating survival percentage calculated from 0 J/m2 plates (see step 13 above), and divide it by the number of cells that was plated. (Take into consideration the actual volumes you plated at different doses: 100 µl, or 200 µl!)
  15. Calculate mutagenesis from +can plates. Multiply with the survival percentage the number of cells plated on a plate at the given UV dose. Calculate the average number of colonies from the two parallel plates of the same UV dose. That gives you the number of mutants/plated cells. Based on that calculate how many mutants would be in 106 cells, because mutagenesis data usually corresponds to 106 cells. (Take into consideration the actual volumes you plated at different doses: 100 µl, or 200 µl!)


    Figure 1. Burker chamber. One cell in one blue area means 1 x 104 cells/ml. (Source: http://openwetware.org/wiki/IGEM:University_of_Debrecen:_transfection)

Representative data

  1. Since the result of this experiment depends on the number of inactivating mutations in the CAN1 gene inflicted upon by UV, the number of mutants can vary. Because of that average numbers should be calculated based on 3-5 experiments.

Recipes

  1. YPD liquid
    1% yeast extract
    2% pepton
    2% D-glucose
  2. SC plates
    Bacto-agar: 16.6 g/L
    D-glucose: 20 g/L
    12 media mix: 7.2 g/L
    12 media mix
    Yeast nitrogen base [w/o amino acids and w (NH4)2SO4]
    400 g
    Adenine
    1.8 g  
    Arginine
    1.2 g
    Histidine
    1.2 g
    Isoleucine
    1.8 g
    Leucine
    1.8 g
    Lysine
    1.8 g
    Methionine
    1.2 g
    Phenylalanine
    3.0 g
    Tryptophan
    1.2 g
    Tyrosine
    1.8 g
    Uracil
    1.2 g
    Valine
    9.0 g
  3. SC -arginine plates containing canavanine
    Same as the SC plates, but the media mix contains 3.6 g canavanine instead of arginine

Acknowledgments

We used this protocol in our work (Daraba et al., 2014). Funding support: Wellcome Trust, 070247/Z/03/A.

References

  1. 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.
  2. Whelan, W. L., Gocke, E. and Manney, T. R. (1979). The CAN1 locus of Saccharomyces cerevisiae: fine-structure analysis and forward mutation rates. Genetics 91(1): 35-51.

简介

有几种方法来测量酵母细胞通过突变对其基因组的环境影响作出反应的能力。 一种常用的方法包括检测CAN1基因中的正向突变。 CAN1基因编码负责精氨酸摄取的精氨酸通透酶,并且它还可以运输精氨酸,刀豆氨酸的有毒类似物(Whelan等人,1979) 。 当在含有刀豆氨酸但缺乏精氨酸的培养基上生长CAN1 细胞时,细胞死亡,因为吸收了有毒的刀豆蛋白。 然而,如果在CAN1基因中的突变使通透酶失活,该细胞存活并在平板上形成集落。
以下协议描述了在CAN1 位点处UV诱导诱变的测量。

关键字:酵母, 诱变, 正向突变, 刀豆氨酸, UV光

材料和试剂

  1. 具有野生型 CAN1 基因(例如,BY4741,EMY747)的菌株
  2. 酵母氮碱[w/o氨基酸和w(NH 4)2 SO 4](Difco)
  3. 腺嘌呤(Sigma-Aldrich)
  4. 精氨酸(Sigma-Aldrich)
  5. 刀豆蛋白(Sigma-Aldrich)
  6. 组氨酸(Sigma-Aldrich)
  7. 异亮氨酸(Sigma-Aldrich)
  8. 亮氨酸(Sigma-Aldrich)
  9. 赖氨酸(Sigma-Aldrich)
  10. 甲硫氨酸(Sigma-Aldrich)
  11. 苯丙氨酸(Sigma-Aldrich)
  12. 色氨酸(Sigma-Aldrich)
  13. 酪氨酸(Sigma-Aldrich)
  14. 尿嘧啶(Sigma-Aldrich)
  15. 缬氨酸(Sigma-Aldrich)
  16. 酵母提取物
  17. Pepton
  18. D-葡萄糖
  19. 细菌琼脂
  20. 酵母提取物 - 蛋白胨D-葡萄糖(YPD)培养基(参见Recipes)(或使用菌株所需的其他培养基)
  21. 合成完全培养基(SC)培养基(参见配方)
  22. 含有刀豆氨酸(+ can)的SC-精氨酸平板(见配方)

设备

  1. 30℃恒温振荡器(每分钟180-200转)
  2. 30℃培养箱
  3. 玻璃片
  4. 离心机
  5. 显微镜
  6. 涡流
  7. 紫外线照射机

  8. 撒布机
  9. 文化管
  10. 水浴声波器
  11. 血细胞计数器(Burker计数室)

程序

  1. 通过接种来自新鲜平板的单个菌落,在YPD(一种参考或野生型菌株,和待检测的菌株)中培养10ml过夜培养物。
  2. 将培养管放入水浴超声仪,在室温下超声处理细胞3分钟以破坏团块(40 Hz)。在显微镜下检查团块的存在。
  3. 使每个菌株在水中的200x稀释(5微升细胞培养物,995微升水),并将10微升到伯克室。
  4. 在显微镜下的一个大广场的伯克室(由三条线边界)计数细胞(图1)。大方块中的一个细胞是指1×10 4细胞/ml。
  5. 计算原始培养物的密度(将计数细胞数乘以200)。
  6. 制备每个菌株的10×系列稀释液,从10 8个细胞/ml开始至10 3个细胞/ml。如果必要,浓缩细胞以获得10 8个细胞/ml。当计算给定稀释步骤的体积时,取 考虑从稀释液中电镀多少板(见下面的步骤8和9)
  7. 用应变名称和要应用的紫外线剂量标记SC和+ can板。对于每个UV剂量,包括零,每个菌株的标记物2SC和2 +罐平板。 SC板将用于计算不同UV剂量的存活率,+罐板将用于计算在不同UV剂量下的诱变。
  8. 将200μl在对照,0μg/ml的SC板上从10 3个细胞/ml稀释液中取出。对于野生型,对于在80J/m 2下显示20%存活的UV相当耐受的EMY747或BY4741细胞,对于高达该剂量的10 3个细胞/ml稀释应用于电镀。在更敏感的菌株的情况下,当确定用于给定UV剂量的电镀的稀释时,应该考虑预期的存活率。
  9. 在+罐平板上,从10 8个细胞/ml稀释液上铺板200μl细胞
  10. 等待板吸收水分,然后照射所需的紫外线剂量的无盖板。确保辐射板在照射后不暴露于白光(用黄色光打开),并立即放置在一个盒子中,将其遮住光线,并放入30℃的培养箱中。
  11. 孵育平板,直到菌落生长到直径2-3mm。对于SC板,通常需要2-3天,+罐板需要5天(在黄灯下检查)。
  12. 计算每个平板上的菌落。
  13. 计算SC板上的存活百分比。将两个0J/m 2平板的平均菌落数与铺板在一个平板上的细胞数目相乘,并将其乘以100.这给出了存活的细胞的百分比。 br />
  14. 计算每个UV剂量的存活率。将两个平行板的平均菌落数乘以从0J/m 2培养板计算的接种存活百分比(参见上述步骤13),并除以接种的细胞数。 (考虑到不同剂量下的实际体积:100μl或200μl!)
  15. 计算从+罐板的诱变。将存活百分比乘以在给定UV剂量下平板上铺板的细胞数。计算来自相同UV剂量的两个平行板的平均菌落数。这给你突变体/镀细胞的数量。基于计算在10 6个细胞中有多少突变体,因为诱变数据通常对应于10 6个细胞。 (考虑到不同剂量下的实际体积:100μl或200μl!)


    图1.打嗝室。一个蓝色区域中的一个细胞是指1×10 4个细胞/ml。 (资料来源:http://openwetware.org/wiki/IGEM:University_of_Debrecen:_transfection

代表数据

  1. 由于该实验的结果取决于由UV施加的CAN1 基因中的失活突变的数目,突变体的数目可以变化。 因为平均数应该根据3-5个实验计算。

食谱

  1. YPD液体
    1%酵母提取物
    2%pepton
    2%D-葡萄糖
  2. SC板
    细菌琼脂:16.6g/L
    D-葡萄糖:20g/L
    12介质混合物:7.2g/L
    12培养基混合物
    酵母氮碱[w/o氨基酸和w(NH 4)2 SO 4子]
    400克
    腺嘌呤
    1.8 g  
    精氨酸
    1.2克
    组氨酸
    1.2克
    异亮氨酸
    1.8 g
    亮氨酸
    1.8 g
    赖氨酸
    1.8 g
    甲硫氨酸
    1.2克
    苯丙氨酸
    3.0克
    色氨酸
    1.2克
    酪氨酸
    1.8 g
    尿嘧啶
    1.2克
    缬氨酸
    9.0克
  3. 含有刀豆蛋白的SC-精氨酸平板
    与SC板相同,但培养基混合物含有3.6g刀豆蛋白,而不是精氨酸

致谢

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. 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。
  2. Whelan,W.L.,Gocke,E。和Manney,T.R。(1979)。 酿酒酵母的CAN1基因座:精细结构分析和正向突变 。 91(1):35-51。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Unk, I. and Daraba, A. (2014). Measuring UV-induced Mutagenesis at the CAN1 Locus in Saccharomyces cerevisiae. Bio-protocol 4(20): e1272. DOI: 10.21769/BioProtoc.1272.
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Ying Wu
University of Maryland College Park
Hi,

Right now I am trying to doUV mutagenesis for powdery mildew (a biotroph fungi). However, I am not sure what machine and dose I should use. Could you tell me the UV machine you used? I have a UV-crosslinker. The power is 10,000uJ/cm2. Do you think if I can use it for mutagenesis?

Thanks!

Best,
Ying

5/29/2017 4:53:44 AM Reply
Ildiko Unk
The Institute of Genetics, Biological Research Center of The Hungarian Academy of Sciences, Hungary

Hi,
The machine was made here at the institute according to my request.
I have never worked with powdery mildew, so you have to search the literature, or try different doses.
In my experience, UV cross linkers do not work well for irradiating fungi on plate, because they can only irradiate with one single dose, but you usually have to apply a broad range of doses to perform an experiment, e.g. a killing curve.
My advice is to look for a lab in/around your institute that already has a machine you need.
Best,
Ildiko

8/24/2017 7:07:37 AM