BIOCHEMISTRY OF GENE EXPRESSION IN HIGHER EUKARYOTES

我们用的是果蝇早期胚胎的巨大单细胞 (500 um x 150 um）作为模型系统， 细胞质组织的某些方面的生化基础。 对这个不寻常的细胞中高度组织化的细胞质的了解 细胞将需要理解空间图案化的细胞 决定的过程，其发展成为一个 复杂的多细胞生物 我们的方法集中在一项研究上 构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物的功能 涉及分子的细节。 我们还希望开发方法， 鉴定和分离更多的次要细胞骨架蛋白， 怀疑定位选定的mRNA（如bicoid mRNA），或 卵皮质的特定区域。 我们的大多数研究计划开始都是利用两种亲和力 我们已经花了几年的时间， 发展到令人满意的状态：肌动蛋白亲和性 层析和微管亲和层析。 这些 已经通过用已知蛋白质的实验验证了这些方法 与肌动蛋白丝和微管结合。 当用于 早期（原肠胚前）果蝇胚胎的碎片提取物， 它们使超过40（肌动蛋白丝柱）和60（微- 小管柱）选择和纯化不同的蛋白质。 迄今为止针对这些蛋白质的抗体 并用于染色胚胎表明大多数蛋白质 通过亲和层析分离的与它们的 各自的细胞骨架网络（肌动蛋白丝或微管） 在细胞内。 现在的主要问题是作出明智的选择 将单个蛋白质纯化至均匀，并在 详细 这一选择是在考虑结果后作出的 细胞学定位，抗体注射到胚胎中， 阻断功能和生化分离来鉴定蛋白质 综合体和活动。 到目前为止，我们已经选择了详细的 研究一种205千道尔顿的微管结合蛋白， 从微管与ATP和定位到中心体，和230 千道尔顿肌动蛋白结合蛋白 当注射到果蝇体内时， 胚胎 此外，我们一直在净化同质化 几种存在于 在合胞期胚胎中大量存在。

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.