CHARACTERIZATION OF HOMEOBOX GENES IN THE LEECH

高级后生动物中的许多细胞，特别是神经元，获得了独特的， 在开发过程中特定于职位的身份。与果蝇一起工作， 果蝇，已经导致了许多基因的鉴定 参与了这一基本过程。其中大约30个已经被证明是 在一个叫Homeo box的区域有直接的DNA序列同源性，该区域有 编码60个氨基酸的DNA结合的保守序列。 不同物种基因组DNA与其他物种的杂交 果蝇探测器表明，同源盒在进化中是保守的 并导致在青蛙、老鼠和人类中分离出同源基因。 因为大多数果蝇的同源框基因都会影响发育 过程，特别是分段和分段区分，它具有 有人提出，同源基因框也可能是这些基因的一个标记 其他物种。最近的研究还表明，在 神经系统，并提示这些基因在决定 神经元的命运和分化中。一群动物，在其中进行一项测试 这些想法特别合适的是水蛭。水蚤，和其他的一样 环节动物和节肢动物，表现出明显的节段性组织 身体。目前的进化论有节肢动物和环节动物 从一个共同的、未分割的祖先进化而来，暗示了一种可能性 这两组人可能有相似或相关的同源基因 功能。我们之前的观察已经证明了 水蛭同源盒基因及其表达，特别是在中央 神经系统，在时空模式中，就像在Homeo Box中发现的那样 脊椎动物的基因和苍蝇的同源异型基因。核苷酸序列 相似性表明这些可能是苍蝇同源异型的水蚤同源物。 基因，因此，是很好的候选基因之一 在水蚤中指定分段标识。这个项目的中心目标是 鉴定这一类水蚤的基因并探索它们的功能 干扰他们的表达。特别是，我们将利用 能够与已识别的水蛭神经元一起工作，以确定哪些表达 并寻找与这些基因相关的表型差异 差异表达模式。在此过程中将采用的技术 工作包括标准的分子克隆、原位杂交、 融合蛋白的构建及抗体、反义RNA的提高 注射、免疫组织化学、细胞内染料注射和 电生理记录和计算机辅助结构分析。这个 这次调查的结果应该对我们的 对控制早期发育和基因进化的理解 这些基因如何控制神经元的表型。

Many cells in advanced metazoans, particularly neurons, acquire unique, position-specific identities during development. Work with the fruit fly, Drosophila melanogaster, has led to the identification of many of the genes involved in this fundamental process. About 30 of these have been shown to have direct DNA sequence homology in a region called the homeo box that has been shown to code for a DNA-binding, conserved sequence of 60 amino acids. Cross-hybridization of genomic DNA from various other species with Drosophila probes has shown that the homeo box is conserved in evolution and has led to the isolation of homologous genes in frogs, mice and humans. Because most of the Drosophila homeo box genes affect developmental processes, in particular segmentation and segmental differentiation, it has been proposed that the homeo box could also be a marker of such genes in other species. Recent work also shows high levels of expression in the nervous system and suggests a role for these genes in the determination of neuronal fate and in differentiation. A group of animals in which a test of these ideas is particularly appropriate is the leeches. Leeches, like other annelids and arthropods, display an obvious segmental organization of their bodies. Current evolutionary theory has the arthropods and the annelids descending from a common, unsegmented ancestor, suggesting the possibility that the two groups may have cognate genes with similar or related functions. Our previous observations have demonstrated the existence of leech homeo box genes and their expression, particularly in the central nervous system, in spatiotemporal patterns like those found for homeo box genes in vertebrates and for homeotic genes in the fly. Nucleotide sequence similarities indicate that these may be leech cognates of fly homeotic genes and are, therefore, excellent candidates to be among the genes that specify segmental identity in the leech. The central aim of this project is to characterize leech genes of this class and to explore their functions by interfering with their expression. In particular, we will exploit the ability to work with identified leech neurons to determine which express these genes and to search for phenotypic differences that correlate with differential expression patterns. The techniques to be employed in this work include standard molecular cloning, in situ hybridization, construction of fusion proteins and raising antibodies, antisense RNA injection, immunohistochemistry, intracellular dye injection and electrophysiological recording, and computer-aided structural analysis. The results of this investigation should have a significant bearing on our understanding of the evolution of genes that control early development and how these genes may control neuronal phenotype.