LOCALIZATION OF PROTEIN CHIMERAS THAT INCLUDE GREEN FLUORESCENT PROTEIN (GFP)

绿色荧光蛋白(GFP)已被证明是巨大的 在为细胞生物学家提供一种标记方面很有价值 体内有强大的信号，但对蛋白质基本上是无害的 它与之融合在一起。在酵母和其他遗传有机体中，它是 通常可以将内源基因替换为融合的版本 GFP，并展示了野生型功能是通过这个 “标记的”等位基因。当在基因的正常启动子下表达时， 也避免了过度表达的伪像。我们发现，一个 亲和纯化的兔抗鼠疫多克隆抗体 卡哈纳的绿色荧光蛋白识别其抗原的S/N为~200，甚至 在我们的快速冷冻/冷冻替代固定的严酷之后 (参见子项目T12)。采用这些制备方法，Morphew 现已将类动蛋白Kar3p和Kip2p定位于 萌芽酵母。她还定位了一种新的蛋白质，它的突变 用Kar3-Delete，Sms1p合成致命性。结果是 研究酵母细胞骨架的学生对此非常感兴趣 和有丝分裂，因为我们的结果已经让人怀疑 关于活体运动位置的假说。我们也在分享这一点 马克·维尼、特丽莎·戴维斯和卡尔·埃克沃尔的实验室的技术， 他们正在用它来制造几种额外的酵母蛋白 本地化。 C11

The green fluorescent protein (GFP) has proven to be tremendously valuable in providing cell biologists with a marker that gives a robust signal in vivo but is largely non-destructive to the proteins with which it is fused. In yeasts and other genetic organisms it is often possible to replace an endogenous gene with a version, fused with the GFP, and show that wild type function is fulfilled by this "tagged" allele. When expressed under the gene's normal promoter, artifacts of over-expression are also avoided. We have found that an affinity purified, polyclonal antibody raised in rabbits against the GFP by Kahana will recognize its antigen with a S/N of ~200, even after the rigors of our fast freezing/freeze-substitution fixation (see Subproject T12). Employing these preparation methods, Morphew has now localized the kinesin-like proteins, Kar3p and Kip2p in budding yeast. She has also localized a novel protein whose mutation is synthetically lethal with kar3-delete, Sms1p. The results are proving extremely interesting to students of the yeast cytoskeleton and mitosis, because our results have already cast doubt on several hypotheses about motor position in vivo. We are also sharing this technology with Mark Winey's, Trisha Davis', and Karl Ekwall's labs, and they are using it for several additional yeast protein localizations. C11