This work was supported by NIH grant GM50936 to DKB.

注：以下一些步骤的协议要求在清洁通风橱工作。另外，该方法需要一个酵母四分体解剖显微镜配备一个10X长工作距离物镜并监控原生质球。显微镜应设置在提前罩。取出显微手臂和板夹从范围和放置这些物品在安全的地方，远离工作区。 1.准备原生质球<ol><li>生长所需条件下酵母。使用约2×10 8个细胞的每个样品，如4毫升培养的在OD 600 1.4 = 5×10 7个细胞/ ml。虽然样品的即时处理可以优选在某些情况下，对于大多数应用，细胞等份可以存储在冰上达8小时处理之前。 </li><li>转移细胞悬液至15ml离心管中，自旋在临床离心机中以设置图5（2345转/ 857.5 XG）处理3分钟，以沉淀细胞。 </l我&gt; <li>倒出培养基照顾不从粒料输细胞。轻轻重悬细胞于1ml ZK缓冲，然后添加40微升的1M二硫苏糖醇（DTT）。孵育2分钟，轻轻混合。如前颗粒细胞（见步骤1.2）。 </li><li>重悬粒料在1ml ZK缓冲器。添加酶解酶100T的新鲜制备的溶液已被充分混合5微升。孵育20-30分钟，在30℃以除去细胞壁和产生原生质球。 </li><li>测定在显微镜载玻片上的细胞悬浮液的10微升液滴，以确定是否原生质球是完整。细胞解剖显微镜下观察无盖玻片。细胞应该会出现浮肿和圆形而非略呈长方形，应该裂解以下加入20微升的水。如果细胞不能裂解，返回样品至30℃，并重新测定每10分钟，直到水诱导裂解容易观察到。如前颗粒细胞（见步骤1.2）。 </li><li>轻轻悬浮和洗涤细胞沉淀我ñ2.5毫升冷MES /山梨糖醇缓冲。如前颗粒细胞（见步骤1.2）。轻轻悬浮细胞沉淀在300-400微升冷MES /山梨糖醇缓冲。细胞可以保持在冰上，在此阶段进行数小时。 </li></ol> 2.染色体传播注：玻璃表面在其染色体将被传播，无论是幻灯片或盖玻片，应提前准备时间。每张幻灯片或盖玻片应浸入水中，然后乙醇，然后干燥，抛光用镜头纸。如果染色体将被摊在盖玻片，盖玻片应贴有一个透明胶带或橡皮泥滑。除非另有说明，否则该协议的其余部分将介绍在任幻灯片或传播盖玻片<ol><li>使用P20移液管，移液管20微升细胞悬浮液到一个干净的载玻片的表面上。 </li><li> 3％PFA /蔗糖溶液中使用的是P200的移液器，加入40微升，轻轻混合搜索解决方案N以“纷飞”用手滑动，直到纹影线消失。将滑动显微镜下，确认细胞是关注的焦点。煤灰溶液应该是新制备在使用的当天通风柜。 注意：如果目的蛋白质尚未审查的方法以前，最好是准备使用包含2和4％PFA解决方案，以确定是否保持该蛋白质是固定条件敏感附加幻灯片。 4％PFA /蔗糖溶液中使用的情况下，期望的可视化有关的微管蛋白的微管。较高固定液浓度的缺点是，染色体将是“underspread，”2％PFA中也往往产生“underspread”染色体。 </li><li>采用P200的移液器，加80微升1％LIPSOL的，如前漩涡混合解决方案，尽可能完整地，启动一个定时器。如果LIPSOL不可用，80微升1％NP40或80微升的dh 2 O可能取代（见代表结果，图2）。 </li><li>观看细胞仔细，轻轻地旋转幻灯片每15秒。当细胞大约80％的裂解（消失）停止计时，立即从显微镜取出载玻片上，加80微升的PFA /蔗糖溶液和漩涡混合。裂解应启动定时器之后发生从30至90秒。 注意：如果若干幻灯片显示裂解在大约相同的时间，可以最终省略微观监测用于幻灯片的其余部分，简单地仔细计时加成LIPSOL和另外的固定剂的最终等分试样之间的时期。然而，从同一样品准备多张幻灯片时，这个捷径是唯一可靠的。最好是监视各样品的裂解显微镜时不同载玻片从在不同时间采集的样品或来自不同文化的制备。 </li><li>放置幻灯片上清洁的平面上并扩散，液体在整个无结霜面传热e。使用保持低于“水坑”的弯月面，但在滑动本身， 即表面上方的清洁，一次性牧场吸管的侧滑动的。不“耙”与吸管滑动的表面上。不要重复使用吸管在不同的幻灯片，以避免样品的污染。 </li><li>离开幻灯片在通风柜干燥过夜。不溶性的核部件将落户到滑动面和绑定到它。对于大型实验，规划利用空间的干燥步骤是重要的。 </li><li>一旦干燥，最佳结果是通过发展到免疫染色阶段在同一天获得。然而，干燥的蔗糖溶液中嵌入扩频染色体在一个“蜂蜜”，允许样品的冷冻：幻灯片可以被转移到一个塑料载玻片盒，并存储在-20℃多年。 </li></ol> 3.免疫染色<ol><li>浸载玻片在0.2％光弗洛30秒。除去蜂蜜。精益幻灯片上的边缘，以在纸巾边缘休息，以去除残留的光弗洛。 </li><li>浸载玻片在1×TBS中5分钟以洗涤。通过在边缘滑靠在去除多余的液体，但不要让他们干出来的。 </li><li>铺设幻灯片磨砂朝上，吸管300微升TBS / BSA上横跨滑板的无结霜部滑动。 </li><li>孵育在潮湿室中在室温持续15分钟。 （下穿孔金属板或塑料滑梯盒内衬水的纸巾用饱和纸巾大型塑料容器）。 </li><li>由搁在纸巾上滑动的一个短边和靠在一个适当的支持所述滑动件（如一个试管架）漏的幻灯片。不要让表面干燥。 </li><li>立即应用80微升含有适当稀释的第一抗体的TBS / BSA缓冲液。如果抗体限制，使用40微升和22×22毫米盖玻片。对于粗兔血清，这是一个典型的1/50和1/500稀释之间。 注意：执行滴定新抗体制剂。最稀制剂能够产生高信号上述背景选择。为了控制的背景染色的水平，核应从适当缺失突变株和/或重复的载玻片制备应与预免疫血清被染色。 </li><li>将在幻灯片上避免气泡抛光22×50毫米盖玻片。握住拇指和食指沿长边之间的盖玻片在靠近一短边缘位置做到这一点。保持盖玻片，在30°角相对于滑动，从手指的短边最远被降低，直到其位于所述滑动只是“水坑”最接近载片的磨砂区域的边缘内侧。然后慢慢放下盖玻片，在平滑的运动，直到拿着幻灯片触摸板凳手指，然后松开。不要试图一次它的土地调整盖玻片的位置。 注：如果利差染色体上制备贴盖玻片（而不是直接在载玻片的表面上），该样品盖玻片将保持固定到整个染色和洗涤步骤的幻灯片。染色和随后丢弃期间额外盖玻片将被放置在样品上盖玻片均匀分布抗体溶液的顶部。 </li><li>在一个密封的腔潮湿的地方幻灯片和孵化一夜之间在4℃。 </li><li>保持磨砂边缘转移的幻灯片到载玻片染色齿条和淹没在含有TBS一个染色缸。 </li><li>轻轻蘸上下滑动，除去盖玻片。尽量不要用力过猛做到这一点。通过浸没在TB​​S洗载玻片2×10分钟。从机架上卸下幻灯片和通过触摸边纸巾排出多余液体。不要让表面干燥。 </li><li>在柔和的灯光工作，立即加入含1/1000倍稀释荧光标记的二次抗体（或40微升用22×22毫米盖玻片）的80微升TBS / BSA。广告ð盖玻片作为前孵育在4℃下2小时，在湿室中在黑暗中。 </li><li>除去盖玻片和以前一样，漏的幻灯片，并允许表面风干1至2小时在黑暗中。如前纸巾精益幻灯片。 注：如果在安装盖玻片进行传播，干燥分离前从幻灯片盖玻片。如在先前步骤中描述的幻灯片盖玻片，然后干燥。 </li><li>在柔光的工作，加入约30微升含安装DAPI（3个10微升滴）中，然后小心地放下盖玻片。如果利差盖玻片上，将安装介质的液滴在干净的玻片，降低盖玻片，染色体端下来，到幻灯片。 </li><li>仍在柔和的光，等待2分钟的盖玻片定居和密封盖玻片用透明指甲油的边缘。将幻灯片平坦滑动箱。 注：对于STED显微镜，用安装延长黄金。使用30微升延长黄金的没有DAPI并允许治愈过夜。用指甲油密封是不必要的。 </li><li>查看幻灯片由宽视场荧光显微镜用40或100X目的使用适当的设置进行DAPI集中的过滤器。可选择存放在4℃的幻灯片在黑暗中了好几个星期。不要冷冻。 </li></ol>

<ol>
	<li>Loidl, J., Nairz, K., Klein, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiotic+chromosome+synapsis+in+a+haploid+yeast.">Meiotic chromosome synapsis in a haploid yeast.</a> Chromosoma. 100 (4), 221-228 (1991).</li><li>Loidl, J., Klein, F., Engebrecht, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+morphological+approaches+for+the+analysis+of+meiotic+chromosomes+in+yeast.">Genetic and morphological approaches for the analysis of meiotic chromosomes in yeast.</a> Methods in cell biology. 53, 257-285 (1998).</li><li>Lydall, D., Nikolsky, Y., Bishop, D. K., Weinert, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+meiotic+recombination+checkpoint+controlled+by+mitotic+checkpoint+genes.">A meiotic recombination checkpoint controlled by mitotic checkpoint genes.</a> Nature. 383 (6603), 840-843 (1996).</li><li>Bishop, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RecA+homologs+Dmc1+and+Rad51+interact+to+form+multiple+nuclear+complexes+prior+to+meiotic+chromosome+synapsis.">RecA homologs Dmc1 and Rad51 interact to form multiple nuclear complexes prior to meiotic chromosome synapsis.</a> Cell. 79, 1081-1092 (1994).</li><li>Rabitsch, K. P., Galova, M., Schleiffer, A., Buonomo, S. B., Nasmyth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomics+identifies+Monopolin:+a+kinetochore+protein+required+for+segregation+of+homologs+during+meiosis+I.">Functional genomics identifies Monopolin: a kinetochore protein required for segregation of homologs during meiosis I.</a> Cell. 103 (7), 1155-1168 (2000).</li><li>Gasior, S. L., Olivares, H., Ear, U., Hari, D. M., Weichselbaum, R., Bishop, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+RecA-like+recombinases:+Distinct+roles+for+mediator+proteins+in+mitosis+and+meiosis.">Assembly of RecA-like recombinases: Distinct roles for mediator proteins in mitosis and meiosis.</a> PNAS. 98 (15), 8411-8418 (2001).</li><li>Biggins, S., Murray, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+budding+yeast+protein+kinase+Ipl1/Aurora+allows+the+absence+of+tension+to+activate+the+spindle+checkpoint.">The budding yeast protein kinase Ipl1/Aurora allows the absence of tension to activate the spindle checkpoint.</a> Genes &amp; Development. 15 (23), 3118-3129 (2001).</li><li>Dresser, M. E., Giroux, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiotic+chromosome+behavior+in+spread+preparations+of+yeast.">Meiotic chromosome behavior in spread preparations of yeast.</a> The Journal of Cell Biology. , (1988).</li><li>Lao, J. P., Cloud, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiotic+crossover+control+by+concerted+action+of+Rad51-Dmc1+in+homolog+template+bias+and+robust+homeostatic+regulation.">Meiotic crossover control by concerted action of Rad51-Dmc1 in homolog template bias and robust homeostatic regulation.</a> PLOS Genetics. 9 (12), e1003978-e1003978 (2013).</li><li>Vonesch, C., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fast+thresholded+landweber+algorithm+for+wavelet-regularized+multidimensional+deconvolution.+IEEE+transactions+on+image+processing+:+a+publication+of+the.">A fast thresholded landweber algorithm for wavelet-regularized multidimensional deconvolution. IEEE transactions on image processing : a publication of the.</a> IEEE Signal Processing Society. 17 (4), 539-549 (2008).</li></ol>

The authors declare no competing financial interests.

The appearance of spread nuclei critically depends upon the balance between fixation and lysis/detergent treatment. As discussed above, the preservation of varying cellular structures requires the use of different PFA concentrations. For most proteins, 3% PFA is optimal. However, preservation of spindles requires use of 4% PFA. Even with a single set of reagents, the timing of lysis relative to fixation can also affect the quality of spread nuclei. For the most consistent results, the time of lysis should be normalize...

在芽殖酵母提供了许多优势，为生物过程的分子机制，包括控制染色体功能的蛋白质的研究研究。虽然有出芽酵母基因，分子和生物化学研究的公知的优点，蛋白质在细胞核分布的细胞学研究是由它的小尺寸复杂。典型的酵母核的直径小于一微米，这是可见光的只有约5倍的分辨率极限。因此，对核蛋白质，可以从常规的免疫染色或通过使用荧光蛋白标记，如绿色荧光蛋白（GFP）来获得的分配信息的数量是有限的。一个有用的方法来表征蛋白质的亚核的分布是染色体的表面扩散。该方法包括去除细胞壁，扰乱细胞和核膜，和一个llowing核的不溶性内容沉降到显微镜载玻片的表面上。这些不溶性组分包括核基质和染色体。除了允许去除可溶性核内容，从而增强检测的染色质结合的蛋白质的能力，所述染色体扩频方法导致染色体大幅度减压，使得传播核具有的直径约3至5微米的（对于二倍体减数分裂核）和2至3微米为二倍体有丝分裂核。这种减压使检测核亚结构是相对困难或不可能解决在完整的细胞核。 一个明显的缺点，以染色体扩频这样的可能性，即在扩展过程可以部分或完全破坏感兴趣的结构。特别值得关注的是，一个特定的染色体结合蛋白可能会丢失作为扩展过程的结果。这种潜在的并发症寿LD解释数据时必须牢记这一点。的蛋白质的一个例子是于扩展过程敏感的β-微管蛋白。在某些情况下，主轴，其由主要的微管蛋白，被扩频3中保存。主轴的可视化往往是有益的舞台感兴趣的原子核。然而，可视化微管蛋白需要有高浓度的固定剂处理;主轴下述的标准条件下丢失。这个实施例说明，在分析以前未表征的蛋白质的分布时，它改变固定液以确定蛋白质如何敏感是这种变化的浓度是重要的。尽管关于扩频条件对染色体结构的影响的关注，扩展方法的实用性和功率已被证明在许多情况下，具有在有丝分裂的表征，特别是减数分裂细胞4-7广泛的用途。 TWø扩频方法已被广泛使用。第一这些方法，通过修整和吉鲁8研制，避免了使用洗涤剂的，并且可以得到显示时染色的DNA特异性染料DAPI具有相对保存良好染色体形态传播的制剂。然而，这种方法是比较困难的完善和传播核的质量显着地发生变化，当一个滑动件的一个区域被与其他区域相比。这个问题变得更复杂，涉及很多成像细胞核未选定从一个幻灯片，以避免数据采集偏见定量方法。第二种染色体传播方法，通过Loidl和克莱因1发达，要平衡固定由多聚甲醛，用裂解和染色质解由洗涤剂溶液提升。当适当地进行，该方法提供了用更少的区域对区域的变化非常可重复的结果相比，德莱和吉鲁方法。本演讲将重点在modified版，因为它的可靠性和简易Loidl和克莱因的方法，。 染色体扩展并不复杂或费时;高达100幻灯片可以用于免疫染色在一天制备。此外，传播制剂可贮存在冷冻多年之前免疫染色，因此实验室可以开发冷冻染色体差，可以当新的生物问题出现的或新的染色试剂变得可用可使用的存储库。 染色体扩频方法是最常用的与免疫染色和宽场荧光显微镜组合，但是也可能制备载玻片超分辨率光学显微镜方法如受激发射损耗（STED）显微镜。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Zymolyase</td>
			<td>US Biological&#160;</td>
			<td>Z1004</td>
			<td>Prepare 20 mg/mL solution in 50 mM Tris pH 7.5 supplemented with 2% glucose. Prepare fresh each experiment and store at 4&#176;C until ready for use.</td>
		</tr>
		<tr>
			<td>Lipsol&#160;</td>
			<td>L.I.P. Ltd&#160;</td>
			<td>no longer commercially available</td>
			<td>Prepare 1% (v/v) solution in water. Store on ice.</td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>USB</td>
			<td>19628</td>
			<td>Prepare 1% (v/v) solution in water. Store on ice.</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P2287</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>Corning</td>
			<td>2948-75x25</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard coverslip</td>
			<td>Fisher</td>
			<td>12-544-E or 12-540-B</td>
			<td></td>
		</tr>
		<tr>
			<td>High resolution coverslips</td>
			<td>Fisher</td>
			<td>12-542-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Photo-Flo 200 solution</td>
			<td>Kodak</td>
			<td>P-7417</td>
			<td>Prepare 0.2% (v/v) solution in water.&#160;</td>
		</tr>
		<tr>
			<td>TBS</td>
			<td></td>
			<td></td>
			<td>137 mM NaCl, 2.7 mM KCl, 24.7 mM Tris, pH 8</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td>Prepare a 1% (w/v) solution in TBS. Store at 4&#176;C for up to a month.</td>
		</tr>
		<tr>
			<td>Primary antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Donkey Anti-Rabbit</td>
			<td>Invitrogen</td>
			<td>A-21206</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG (H+L) Antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield mounting media with DAPI</td>
			<td>Vector 
			Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic slide box</td>
			<td>Fisher</td>
			<td>03-448-1</td>
			<td>Store slides containing dried spreads in slots at -20&#176;C. Also, use as a wet chamber.</td>
		</tr>
		<tr>
			<td>Cardboard slide box</td>
			<td>Fisher</td>
			<td>12-587-10</td>
			<td>Use to conveniently transport stained/sealed slides or store at 4&#176;C.</td>
		</tr>
		<tr>
			<td>Coplin jar</td>
			<td>Fisher</td>
			<td>08-816</td>
			<td>Use as a wash basin for slides.&#160;</td>
		</tr>
	</tbody>
</table>

surface spreading and immunostaining of yeast chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

传播核的外观需要依靠染色体固定和脱压实之间的平衡。即使当试剂被适当平衡，变异在染色体脱压实度可发生在相同的幻灯片之间和/或不同的幻灯片的不同区域。因此，图像被解释之前以幻灯片的给定区域差的质量进行评估。 可使用抗减数分裂重组蛋白来说明的“笼压”和“underspreading”的影响。在图1中 ，减数分裂细胞核双重免疫染色两种真核生物链交换的?...

Watch this Scientific Journal Video about Surface Spreading and Immunostaining of Yeast Chromosomes at JoVE.com

Surface Spreading and Immunostaining of Yeast Chromosomes

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

表面扩散和酵母染色体免疫染色

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...

surface-spreading-and-immunostaining-of-yeast-chromosomes

Research

JoVE Journal

Biology

9.8K 次观看.  University of Chicago. A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

视频: Surface Spreading and Immunostaining of Yeast Chromosomes

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

科研

生物学

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

高活性酵母核糖体的色谱分离纯化

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Immunostaining of Dissected Zebrafish Embryonic Heart

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

免疫染色的解剖斑马鱼胚胎心脏

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

基于亲和力，外的相互作用筛选（AVEXIS）外低亲和力受体 - 配体相互作用的可扩展检测

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

高通量使用自动染色体的物理图谱<em&gt;原位</em&gt;杂交

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

解剖和免疫染色成虫盘的<em&gt;果蝇</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

对于荧光DNA的简易方法<em&gt;原位</em&gt;杂交来压扁染色体

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

裂殖酵母性生命周期的显微镜

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

在裂殖酵母中Cytokinetic活动的时空分析

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...