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

包含绿色荧光蛋白 (GFP) 的蛋白质嵌合体的定位

• 批准号: 6117497
• 负责人: MARY MORPHEW
• 金额: $ 3.13万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6117497
• 关键词: Chordata; biomedical resource; endocrine gland /system; growth /development

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

绿色荧光蛋白（GFP）已被证明是非常重要的 为细胞生物学家提供了一种标记物， 在体内的鲁棒的信号，但在很大程度上是非破坏性的蛋白质 与之融合。 在酵母和其他遗传生物中， 通常可以用融合的形式替换内源基因， 与GFP，并显示野生型功能是由这个 “标记”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假象。 我们发现， 亲和纯化的多克隆抗体，在兔中产生的抗 Kahana的GFP将以~200的S/N识别其抗原，甚至 在我们的快速冷冻/冷冻替代固定的严酷之后， (see次级项目T12）。 采用这些制备方法，Morphew 现在已经将驱动蛋白样蛋白Kar 3 p和Kip 2 p定位于 芽殖酵母 她还定位了一种新的蛋白质， 与kar 3-缺失Sms 1 p具有合成致死性。 结果 对研究酵母细胞骨架的学生来说非常有趣 和有丝分裂，因为我们的研究结果已经对一些 关于体内运动位置的假设。 我们也在分享这个 Mark Winey，Trisha Davis和Karl Ekwall实验室的技术， 他们用它来制造其他的酵母蛋白 本地化 C11