BIOCHEMISTRY OF GENE EXPRESSION IN HIGHER EUKARYOTES

我们使用的是早期果蝇胚胎的巨型单细胞 (500微米x 150微米)作为模型系统 选定的细胞质组织方面的生化基础。 对这种不寻常现象中高度组织化的细胞质的理解 需要细胞来理解空间图案化的细胞 决定过程是它发展成为一个 复杂的多细胞生物。我们的方法是以一项研究为中心 组成合胞体细胞质的细胞骨架蛋白。 细胞骨架非常复杂，我们的主要目标是 分析几种主要的多蛋白复合体的功能 涉及分子细节的。我们也希望开发出方法来 鉴定和分离更多的次要细胞骨架蛋白 疑似定位选定的mRNAs(如双核糖核酸)或 卵子皮质的特定区域的蛋白质。 我们的大多数研究计划都是从利用两个相似性开始的 层析程序花了我们几年的时间来 发展到令人满意的状态：肌动蛋白细丝亲和力 层析和微管亲和层析。这些 这些方法已经通过对已知蛋白质的实验进行了验证 与肌动蛋白细丝和微管结合。当习惯于 果蝇早期(原肠胚前期)胚胎的分级提取物， 它们支持40多个(肌动蛋白细丝柱)和60个(微型 小管柱)要选择和纯化的不同蛋白质。 到目前为止产生的针对这些蛋白质的抗体 并用来给胚胎染色表明大多数蛋白质 通过亲和层析分离的与其相关的 各自的细胞骨架网络(肌动蛋白细丝或微管) 在牢房里。现在的主要问题是做出明智的选择 单项蛋白质的纯化至均一及其研究 细节。这一选择是在考虑结果后做出的 细胞学定位，将抗体注射到胚胎中 阻断功能，以及识别蛋白质的生化分级 复合体和活动。到目前为止，我们已经选择了详细的 一种释放的205千道尔顿微管结合蛋白的研究 从带有三磷酸腺苷的微管和定位到中心体，以及230 Kilodalton肌动蛋白结合蛋白，其单抗 果蝇体内注射干扰核间距的研究 胚胎。此外，我们一直在净化到同质化 存在于体内的几种中间丝状蛋白 合胞体期胚胎中大量存在。

We are using the giant single cell of the early Drosophila embryo (500 um x 150 um) as a model system in which to examine the biochemical basis for selected aspects of cytoplasmic organization. An understanding of the highly organized cytoplasm in this unusual cell will be required to understand the spatially patterned cell determination processes that underlie its development into a complex multicellular organism. Our approach centers on a study of the cytoskeletal proteins that organize the syncytial cytoplasm. The cytoskeleton is very complicated, and our major goal is to analyze the function of a few of the major multiprotein complexes involved in molecular detail. We also wish to develop methods for identifying and isolating more minor cytoskeletal proteins that are suspected to localize selected mRNAs (such as bicoid mRNA) or proteins to specific regions of the egg cortex. Most of our research plans begin by exploiting two affinity chromatography procedures that have taken us several years to develop to a satisfactory state: actin filament-affinity chromatography and microtubule-affinity chromatography. These procedures have been validated by experiments with known proteins that bind to actin filaments and microtubules. When used to fractionate extracts of early (pre-gastrula) Drosophila embryos, they enable more than 40 (actin filament columns) and 60 (micro- tubule columns) different proteins to be selected and purified. The antibodies that have thus far been produced to these proteins and used to stain embryos suggest that most of the proteins isolated by affinity chromatography are associated with their respective cytoskeletal network (actin filament or microtubule) inside the cell. The main problem now is to make a wise selection of individual proteins to purify to homogeneity and study in detail. This selection is being made after considering the results of cytological localizations, antibody injections into embryos to block function, and biochemical fractionation that identify protein complexes and activities. We have thus far selected for detailed study a 205 kilodalton microtubule-binding protein that is released from microtubules with ATP and localizes to centrosomes, and a 230 kilodalton actin-binding protein whose monoclonal antibody interferes with nuclear spacing when injected into Drosophila embryos. In addition, we have been purifying to homogeneity several intermediate filament-type proteins that are present in large amounts in the syncytial stage embryo.