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
发布: 2014年10月20日第4卷第20期 DOI: 10.21769/BioProtoc.1272 浏览次数: 12264
评审: Kanika GeraEmilia Krypotou Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
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.
Mutagenesis (诱变)
Forward mutations (正向突变)
Canavanine (刀豆氨酸)
UV light (UV光)
Materials and Reagents
- Strains that have the wild type CAN1 gene (e.g., BY4741, EMY747)
- Yeast nitrogen base [w/o amino acids and w (NH4)2SO4] (Difco)
- Adenine (Sigma-Aldrich)
- Arginine (Sigma-Aldrich)
- Canavanine (Sigma-Aldrich)
- Histidine (Sigma-Aldrich)
- Isoleucine (Sigma-Aldrich)
- Leucine(Sigma-Aldrich)
- Lysine (Sigma-Aldrich)
- Methionine (Sigma-Aldrich)
- Phenylalanine (Sigma-Aldrich)
- Tryptophan (Sigma-Aldrich)
- Tyrosine (Sigma-Aldrich)
- Uracil (Sigma-Aldrich)
- Valine (Sigma-Aldrich)
- Yeast extract
- Pepton
- D-glucose
- Bacto agar
- Yeast extract-pepton-D-glucose (YPD) media (see Recipes) (or other media needed for the strain in use)
- Synthetic complete media (SC) plates (see Recipes)
- SC-arginine plates containing canavanine (+can) (see Recipes)
Equipment
- 30 °C incubator-shaker (180-200 round per minute)
- 30 °C incubator
- Glass slide
- Centrifuge
- Microscope
- Vortex
- UV-irradiation machine
- Box
- Spreader
- Culture tube
- Waterbath sonicator
- Hemocytometer (Burker counting chamber)
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
微生物学 > 微生物遗传学 > 诱/突变
微生物学 > 微生物遗传学 > DNA
分子生物学 > DNA > 诱/突变
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