Name: analysis of the development of a morphological phenotype as a function of protein concentration in budding yeast
Uploaded: 2010-03-24T16:05:00
Description: Watch this Scientific Journal Video about Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast at JoVE.com

<p class="jove_content">我们感谢布莱恩G.库恩和克劳迪亚B。汉纳有益的讨论和支持。这个项目是从DEP的启动资金支持。生物科学，普渡大学的R.克劳己立和美国癌症协会的机构研究资助通过普渡大学癌症中心的R.克劳阿吉拉尔。 </p>

<p class="jove_title"> A.细胞培养和蛋白质感应</p><p class="jove_content">本节介绍了细胞培养条件下，从regulatable启动子在酵母细胞中蛋白表达诱导细胞周期同步，与噻氨酯哒唑。 </p><ol><li>在野生型酵母株<em>W303（LEU2 - 3，112 TRP1 - 1 CAN1 - 100 URA3 - 1 ADE2 - 1 HIS3 - 11，15）</em>是转变高复制（2μ）的质粒DNA，包含的控制下我们感兴趣的基因（ENTH2）蛋氨酸repressible子<em>（MET25）。</em> 2MM蛋氨酸足以抑制启动子活性，而缺乏蛋氨酸的?...

<ol>
	<li>R&#246;nicke, V., Graulich, W., Mumberg, D., M&#252;ller, R., Funk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+conditional+promoters+for+expression+of+heterologous+proteins+in+Saccharomyces+cerevisiae.">Use of conditional promoters for expression of heterologous proteins in Saccharomyces cerevisiae.</a> <em style='font-style: italic'>Methods Enzymol</em>. <b>283</b>, 313-322 (1997).</li><li>Gietz, R. D., Woods, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>High efficiency transformation with lithium acetate in Molecular Genetics of Yeast: A Practical Approach</em>. , 121-134 (1994).</li><li>Mukherjee, D., Coon, B. G., Edwards, D. F., Hanna, C. B., Longhi, S. A., McCaffery, J. M., Wendland, B., Retegui, L. A., Bi, E., Aguilar, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yeast+endocytic+protein+Epsin+2+functions+in+a+cell-division+signaling+pathway.">The yeast endocytic protein Epsin 2 functions in a cell-division signaling pathway.</a> <em style='font-style: italic'>J Cell Sci</em>. <b>122</b>, 2453-2463 (2009).</li><li>Sherman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Getting started with yeast in Guide to Yeats genetics and Molecular Biology</em>. , 3-21 (1991).</li></ol>

<p class="jove_content">本协议描述了如何监督的形态学表型蛋白表达（酵母细胞无法进行正常的细胞分裂）后的发展。执行此过程时，重要的是要记住造粒建议离心速度更快的速度可能会损伤细胞和晦涩的结果收获的酵母细胞。应增加活细胞成像前，因为它们是有毒的亚甲基蓝和Calcofluor白色。此过程也可以很容易地适应蛋白质镇压条件下观察到表型的目标是从一个可控的发起人表示。 </p>

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analysis of the development of a morphological phenotype as a function of protein concentration in budding yeast

<p class="jove_content">基因缺失和蛋白表达研究蛋白质功能的常用方法。在这篇文章中，我们描述了作为一个人口功能的蛋白浓度和单细胞水平分析表型的发展协议<em&gt;酿酒酵母</em&gt;。虽然此协议基础上的一种蛋白质的过度表达，它可以很容易地适应依赖于抑制蛋白表达的形态学表型。我们的实验室是在兴趣学习吞适配器蛋白epsin的信号。为了这个目的，我们使用了一个占主导地位的消极做法，在我们过度表达的保守<u&gt;电子</u&gt; psin<u&gt; N</u&gt; -<u&gt; T</u&gt; erminal<u&gt; H</u&gt; omology（ENTH）域，以干扰内源性epsin - 2（Ent2或YLR206W）的功能。我们观察到，在野生型细胞，一个细胞分裂的缺陷，是依赖于一个家庭的骨架蛋白，septins mislocalization Ent2（ENTH2）ENTH域的过度表达。</p>

Watch this Scientific Journal Video about Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast at JoVE.com

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

<p class="jove_content">基因缺失和蛋白表达研究蛋白质功能的常用方法。在这篇文章中，我们描述了作为一个人口功能的蛋白浓度和单细胞水平分析表型的发展协议<em&gt;酿酒酵母</em&gt;。</p>

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