Name: determination of the mating efficiency of haploids in saccharomyces cerevisiae
Uploaded: 2022-12-02T14:00:00
Description: Watch this Scientific Journal Video about Determination of the Mating Efficiency of Haploids in Saccharomyces cerevisiae at JoVE.com

这项工作由DBT/威康信托基金会（印度联盟）赠款（IA/S/19/2/504632）资助，由DBT/威康信托基金会（印度联盟）资助（IA/S/19/2/504632）资助。A.M.作为高级研究员（09/087（0873）/2017-EMR-I）得到了印度政府科学与工业研究委员会（CSIR）的支持。作者感谢Paike Jayadeva Bhat的讨论。

注意：该协议广泛涉及以下步骤：（1）在YPD板上修补交配效率网格中的单倍体，（2）孵育24小时后将等数量的单倍体混合，并给混合单倍体几个小时的交配时间（本研究中为7小时），（3）将混合细胞接种在YPD上，以便在30°C下7小时后分离单个菌落， 最后，（4）确定使用营养不足标记物形成的二倍体的数量。下面将详细讨论这些步骤（另请参阅 图 2）。
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酿酒酵母交配效率的量化对于开展与交配途径相关基因相关的研究或研究外部环境对交配行为的影响至关重要。在过去的二十年中，酿酒酵母也已成为解决与物种形成14，36，37，38相关的问题的流行模型。两种交配类型的存在以及在实验室环境中易于进行遗传操作和维护，使其成为实时?...

酿酒酵母，通常称为出芽酵母，是一种单细胞真核生物。它有两种交配类型， a 和 α， 并表现出无性和有性生殖周期。 a 和 α 交配类型是单倍体，可以在周围环境中没有其他交配类型的情况下进行有丝分裂，这代表了酵母的无性循环。当两种交配类型非常接近时，它们停止有丝分裂并融合形成二倍体细胞。二倍体酵母可以在营养物质存在时有丝分裂，也可以在存在不可发酵的不良碳源（例如乙酸盐1）的情况下在氮饥饿的条件下进行减数分裂。这导致孢子的形成，孢子保持休眠状态，直到有有利的生长条件。当这些孢子萌发并且两种单倍体类型被释放回单倍体池2，3 时，生命周期就完成了（图1）。

酵母细胞的交配包括几个步骤，例如凝集，形成交配投影或“shmoo”，然后是细胞和核融合4，5。两种配接类型 a 和 α分别产生 a 因子和 α 因子以启动配接。这些因子是多肽信息素，与存在于相反交配类型5的细胞表面的受体（Ste2和Ste3）结合。信息素与受体的结合启动信息素反应途径，即丝裂原活化蛋白激酶（MAPK）信号转导途径6，7，8。这导致细胞周期在G1期停滞，导致代谢活跃的固定相9。然后细胞停止有丝分裂，合成交配所需的蛋白质。由于单倍体细胞不能相互移动，交配投影或“shmoo”指向交配伴侣。当细胞接触时，细胞壁降解，细胞质内容物融合，导致交配形成二倍体细胞10，11。单倍体之间的交配效率已被用作实验室进化菌株以及现存物种之间物种形成的量度12。

作为一种简单的真核生物，酵母是与复杂真核生物相关的大量研究问题的首选模型。其中一个问题与物种形成和生殖屏障的进化有关13，14。对于有性繁殖的生物，物种由Ernst Mayr15提出的生物物种概念（BSC）定义。根据这个概念，一个种群中的两个个体如果不能杂交并且繁殖隔离，则被称为属于两个不同的物种。有性生殖周期的破坏（包括配子融合形成受精卵，受精卵发育成后代，以及后代性成熟）导致生殖隔离。 如图1所示， 酿酒酵母 的生命周期与有性生殖周期相当：a）两种交配类型 a 和 α 的融合类似于有性繁殖生物中配子的融合;b）二倍体进行有丝分裂的能力相当于受精卵发育成后代;c）经历孢子形成的二倍体与配子发生过程相当14。

当观察到配种交配时，就会发生合子前分离。如果有机会与两种遗传上不同的 A 型交配， α 型优先与一种交配，反之亦然14。在单倍体在不同环境中进化的进化实验中，可以通过进行交配测定来确定交配前屏障的存在。与祖先相比，交配效率的降低表明交配前屏障的进化。由于二倍体无法进行有效的有丝分裂和/或孢子形成单倍体孢子，因此可能会出现合子分离后14。这些可以通过分别测量二倍体的生长速率和计算孢子形成效率来量化。因此，为了研究生殖屏障的进化，需要强大的方法来量化（a）交配效率，（b）二倍体的有丝分裂生长和（c）二倍体的孢子形成效率。在这项工作中，报告了一种量化酵母菌株交配效率的稳健方法。

在实验室实验中，检测交配发生的方法之一是使用补充营养需求的营养标志物。当两种交配类型对两种不同的氨基酸具有营养性时，只有两种交配类型融合形成的二倍体细胞才能在缺乏两种氨基酸的培养基上生长。因此，营养型标记物可用于定性和定量检测交配。定性测试足以确定减数分裂16 后菌株的交配类型。当一个人有兴趣在研究交配途径中涉及的基因时确定交配减少时，定量测试是必不可少的17，18.此外，随着酵母越来越多地用于物种形成研究，一种方便且可重复的交配测定是必要的，因为交配效率的量化是合子前屏障的衡量标准。

两种酵母交配类型之间的交配效率先前已量化为16，19，20。大多数以前使用的方法在设计上都是相似的，只有一些变化16，21，22，23，24，25。其中一些使用早期对数期培养物，而其他一些使用单倍体菌株的对数期中期培养物。两种交配类型混合的比例存在差异。几乎所有的方案都使用硝酸纤维素膜。从先前生长的培养物中取出的两种交配类型的悬浮液混合并过滤到放置在YPD板上的硝酸纤维素膜上。在协议的变体之一中，单倍体悬浮液直接贴在YPD板21上。在处理参与两种交配类型信息素产生的基因的实验中，在使两种交配类型的悬浮液24的同时向外部添加信息素。

混合单倍体后孵育几个小时（通常约5小时）后，将细胞从膜上洗掉，稀释并铺在选择性培养基上。在1973年报道的早期方法之一中，受精卵形成或交配的效率是通过使用血细胞计数器在显微镜下计数芽细胞，未出芽细胞和交配对的数量来计算的。然而，后来报道的大多数方法使用营养标记来区分单倍体和二倍体。交配效率计算为二倍体细胞相对于细胞池中二倍体和单倍体细胞数量的百分比16，21，23。

然而，尽管有许多报道使用酵母作为模型生物来研究物种形成，但到目前为止，文献中还没有报告用于计算交配效率的标准化方案。对数期的细胞可能不是量化交配效率的理想选择。在交配过程中，两个单倍体的细胞周期被阻止，因此，交配过程中的细胞没有分裂9。由于已知细胞周期在固定相27的细胞中被类似地阻滞，因此使用这种细胞可以使方案更具可重复性。固定相细胞可以混合并放置在YPD板上（即营养丰富的环境）上进行交配。传统程序还需要硝酸纤维素膜并冲洗掉细胞，使该过程繁琐且容易出现处理错误。此外，迄今为止使用的协议以一个单倍体来量化交配效率。然而，在测量生殖隔离时，对特定单倍体组合而不是单个单倍体的交配效率进行量化。

为了解决这些问题，我们在这里报告了一种用于定量酵母交配效率的稳健方法，该方法具有高度可重复性和易于使用性。此外，这种方法和这里采用的酵母菌株也可用于研究基因流对交配屏障进化的影响。

本研究使用了两种不同的酿酒酵母菌株。其中一个菌株来源于SK1背景;在我们的实验室中，通过在MAT位点附近添加营养不良标志物对此进行了修改。所得的单倍体基因型见表1 28、29、30。在SK1菌株中，单倍体在MAT位点附近插入TRP1基因，α单倍体在MAT位点附近插入LEU2基因。在ScAM菌株中，TRP1和URA3基因分别插入a和α单倍体中。插入的位置位于III号染色体的ARS区域（Chr III：197378..197609）。对于这里报告的协议，基因组上任何地方的营养标记就足够了。然而，在MAT位点附近具有营养性标记意味着这些菌株也可用于研究基因流对物种形成的影响31，32。标记物被添加到MAT位点附近，以防止由于重组而导致标记物的重新洗牌。因此，该协议可用于量化涉及物种形成的研究中的交配效率，以及在研究参与交配途径的蛋白质时确定交配效率的改变。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adenine</td>
			<td>Sigma Life Science</td>
			<td>A8626</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar Powder regular grade for bacteriology</td>
			<td>SRL</td>
			<td>19661 (0140186)</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Sulphate, Hi-AR</td>
			<td>HiMedia</td>
			<td>GRM1273</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-glucose</td>
			<td>Sigma Life Science</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Petri plates</td>
			<td>HiMedia</td>
			<td>PW008</td>
			<td>&#160;90 mm x 15 mm dimension</td>
		</tr>
		<tr>
			<td>L-Arginine</td>
			<td>Sigma Life Science</td>
			<td>A8094</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Aspartic acid</td>
			<td>Sigma Life Science</td>
			<td>A7219</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Histidine monochloride monohydrate</td>
			<td>Sigma Life Science</td>
			<td>H5659</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Isoleucine</td>
			<td>Sigma Aldrich</td>
			<td>I2752</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Leucine</td>
			<td>Sigma Life Science</td>
			<td>L8912</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Lysine</td>
			<td>Aldrich</td>
			<td>62840</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Methionine</td>
			<td>Sigma Life Science</td>
			<td>M5308</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Phenylalanine</td>
			<td>Sigma Life Science</td>
			<td>P5482</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Threonine</td>
			<td>Sigma Aldrich</td>
			<td>T8625</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tyrosine</td>
			<td>Sigma Life Science</td>
			<td>T8566</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Valine</td>
			<td>Sigma Life Science</td>
			<td>V0513</td>
			<td></td>
		</tr>
		<tr>
			<td>Mating efficiency grid</td>
			<td></td>
			<td></td>
			<td>1 cm x 1.5 cm rectangular grid drawn on the Petri plate</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>Tarsons</td>
			<td>500010</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>HiMedia</td>
			<td>RM001</td>
			<td></td>
		</tr>
		<tr>
			<td>Uracil</td>
			<td>Sigma Life Science</td>
			<td>U0750</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract Powder</td>
			<td>HiMedia</td>
			<td>RM027</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Nitrogen Base w/o Amino acids and Ammonium Sulphate</td>
			<td>BD Difco</td>
			<td>233520</td>
			<td></td>
		</tr>
	</tbody>
</table>

determination of the mating efficiency of haploids in saccharomyces cerevisiae

酿酒酵母 是遗传学、进化和分子生物学中广泛使用的模式生物。近年来，它也成为研究物种形成相关问题的流行模式生物。酵母的生命周期涉及无性和有性生殖阶段。易于进行进化实验和生物体的短生成时间允许研究生殖屏障的进化。两种配接类型（a 和 α）配合形成 a/α 二倍体的效率称为配接效率。单倍体之间交配效率的任何降低都表明存在合子前屏障。因此，为了量化两个单倍体之间的生殖隔离程度，需要一种可靠的方法来量化交配效率。为此，这里提出了一个简单且高度可重复的协议。该方案涉及四个主要步骤，包括修补YPD板上的单倍体，将等数量的单倍体混合，稀释和接种单个菌落，最后，根据脱落板上的菌落数量计算效率。使用营养标记来清楚地区分单倍体和二倍体。

两种插拔类型配接效率的量化 此处描述的方案用于量化两种酵母菌株之间的交配效率 - SK1AM a 和 SK1AMα 之间以及 ScAMa 和 ScAMα 之间的交配效率（图 3A）。在这些实验中，两个单倍体之间的交配重复至少12次。在实验的每个重复中，至少有100个菌落在双脱落培养基上划线。该方案的稳健性使得SK1AM和ScAM两种菌株?...

Watch this Scientific Journal Video about Determination of the Mating Efficiency of Haploids in Saccharomyces cerevisiae at JoVE.com

Determination of the Mating Efficiency of Haploids in Saccharomyces cerevisiae

在这项工作中，描述了一种定量酿 酒 酵母交配效率的稳健方法。该方法对于物种形成研究中合子前屏障的定量特别有用。

酿酒酵母中单倍体交配效率的测定

Saccharomyces cerevisiae is a widely used model organism in genetics, evolution, and molecular biology. In recent years, it has also become a popular ...

该协议是一种简单的方法来量化不同菌株和酵母种类相互交配的效率。该方法简单、稳健、高效。该方法可以扩展到任何酵母菌株。本研究中描述的方法可用于了解酵母中物种形成是如何发生的。此外，我们描述的菌株对于设计基因流实验特别有用。该方法的优势在于它非常简单。根据所使用的特定菌株，可能需要进行一些细微的调整，例如孵育时间。通过将酵母提取物蛋白胨葡萄糖或YPD琼脂平板上的酵母接种物条纹，开始从冷冻原液中恢复单倍体A和Alpha。让它们在 30 摄氏度下生长 48 小时以获得单个菌落。孵育后，从YPD板和5毫升YPD培养基中接种单个菌落，并在30摄氏度下以250RPM振荡孵育48小时。在此孵育期之后，细胞处于生长的固定阶段。在一个新的YPD板上，将配接效率网格绘制为1 x 1.5厘米的矩形，分为三个盒子，每个盒子的尺寸为1 x 0.5厘米。在最左侧和最右侧的矩形上接种两种交配类型的 5 微升 YPD 培养物。该体积大约相当于每个部分中第五个单倍体细胞的5乘以10。将板在 30 摄氏度下孵育 24 小时。为了混合相同数量的酵母细胞，使用无菌牙签去除外盒中产生的约1/3的酵母生长，并将去除的细胞重悬于无菌1.5毫升小瓶中的20微升水中。对每个单倍体都遵循此操作。然后，在两毫升水中稀释五微升该悬浮液，并使用600纳米的分光光度计测量光密度。基于光密度值和一种光密度下的每毫升细胞数，在新鲜无菌的1.5毫升小瓶中加入所需体积的两个单倍体。用移液管充分混合。这种悬浮液的最终体积通常在六到八微升左右。接下来，在中心网格中接种此悬浮液。将板在 30 摄氏度下孵育七个小时，让单倍体交配。孵育七小时后，使用牙签或移液器吸头从中心矩形刮下细胞，并用两毫升无菌水稀释。进行进一步稀释，然后将细胞悬液散布在YPD琼脂上以获得单个菌落。铺板后，将YPD板在30摄氏度下孵育36至48小时以形成单个菌落。确保从每个交配实验中获得几百个单独的菌落进行筛选，以确保数据的统计意义。为了鉴定平板上的二倍体菌落，通过将单个菌落单独划线到双脱落板上来转移单个菌落，缺乏菌株具有营养性的氨基酸。将板在30摄氏度下孵育48小时并计算交配效率nu。该方案的稳健性使得SK1AM和ScAM两种菌株之间的交配效率变得容易。虽然SK1菌株的配效率非常高，但ScAM菌株的配效率相对较低。当环境中不含葡萄糖作为主碳源时，ScAM菌株的交配效率显著下降，表明交配是一个能量昂贵的过程，交替碳源的生长会定性地降低交配效率。当允许交配不同时间段时，前四到五个小时没有观察到交配。第一个二倍体仅在四到五个小时后出现，表明这是在给定环境中完成交配过程所需的时间。之后，插拔效率迅速提高。超过七个小时，交配效率计算受到有丝分裂生长在板上开始的事实的影响。确保混合两个单倍体的数量相等。偏离这一点将导致协议的独立重复的变化。基因流如何影响适应和物种形成是一个悬而未决的问题。由于膨养标记物插入我们菌株中的MAT位点旁边，这有助于解决这一重要问题。

Research

JoVE Journal

Biology

Determination of the Mating Efficiency of Haploids in Saccharomyces cerevisiae

Dissection of Saccharomyces Cerevisiae Asci

Dissection of Saccharomyces Cerevisiae Asci

剖析酿酒酵母 ASCI

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

科研

生物学

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

脂肪酸定量磷酸化刺激酿酒酵母</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Microarray Analysis for Saccharomyces cerevisiae

Microarray Analysis for Saccharomyces cerevisiae

微阵列分析FOR酿酒酵母</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

囊性纤维化跨膜传导调节蛋白表达和纯化酿酒酵母

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

囊性纤维化跨膜电导调节蛋白表达纯化<em&gt;酿酒酵母</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

EPR监控的辅助因子氧化还原滴定<em&gt;酿酒酵母</em&gt; Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA的衰变率的测量<em&gt;酿酒酵母</em&gt;使用<em&gt; rpb1-1</em&gt;株

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...