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">We thank Brian G. Coon and Claudia B. Hanna for helpful discussions and support.
This project was supported by start-up funds from the Dep. of Biological Sciences, Purdue University to R. Claudio Aguilar and an American Cancer Society Institutional Research Grant to R. Claudio Aguilar through the Purdue Cancer Center.</p>

<p class="jove_title">A. Cell Culture and Protein Induction</p>
<p class="jove_content">This section describes cell culture conditions, cell-cycle synchronization with Nocodazole, followed by induction of protein expression from a regulatable promoter in yeast cells.</p>
<ol>
  <li>The wild type yeast strain W303 (<em>leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15</em>) is transformed with high copy (2&mu;) plasmid DNA that contains our gene of interest (ENTH2) under the control of a methionine-repressible promoter <em>(MET25</em>). 2mM...

<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">This protocol describes how to monitor the development of a morphological phenotype (yeast cell unable to undergo proper cell division) upon protein overexpression. When doing this procedure it's important to remember to harvest yeast cells by pelleting at the recommended centrifugation speed as faster speeds may damage cells and obscure results. Methylene blue and Calcofluor white should be added to live cells just prior to imaging as they are toxic. This procedure can also be easily adapted for phenotypes observed unde...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Nocodazole</td>
<td>Reagent</td>
<td>Sigma (St. Louis, MO)</td>
<td>M1404</td>
<td>&nbsp;</td>
<tr>
<td>Methylene Blue 1% (w/v)</td>
<td>Reagent</td>
<td>VWR International</td>
<td>VW6276-0</td>
<td>&nbsp;</td>
<tr>
<td>Calcofluor White</td>
<td>Reagent</td>
<td>Sigma (St. Louis, MO)</td>
<td>F3543</td>
<td>&nbsp;</td>
<tr>
<td>Petroleum Jelly</td>
<td>Reagent</td>
<td>VWR International</td>
<td>VW3339-2</td>
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analysis of the development of a morphological phenotype as a function of protein concentration in budding yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in <em>Saccharomyces cerevisiae</em>. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved <u>E</u>psin <u>N</u>-<u>T</u>erminal <u>H</u>omology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

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">Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in <em>Saccharomyces cerevisiae</em>.</p>

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

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