MOLECULAR GENETICS OF SEGMENT IDENTITY

片段同一性的分子遗传学

• 批准号: 3329608
• 负责人: RICHARD S MANN
• 金额: $ 15.14万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3329608
• 关键词: DNA binding protein; Drosophilidae; antibody; centrifugation; developmental genetics; gene expression; gene mutation; genetic regulation; genetic transcription; histogenesis; homeobox genes; intermolecular interaction; invertebrate embryology; laboratory mouse; lethal genes; molecular genetics; mutant; nucleic acid hybridization; protein sequence; protein structure function; tissue mosaicism

The morphogenesis of a complex organism depends upon the execution of an intricate genetic hierarchy that provides individual cells with positional and cell fate information. In Drosophila the homeotic selector genes play a central role in this hierarchy because they interpret positional information and determine cell fate. These genes have highly conserved homologs in many animals, including humans, suggesting that their analysis in Drosophila may be a key to understanding vertebrate development as well. The molecular dissection of vertebrate development will, in turn, provide insights into human diseases, including birth defects and various cancers, that appear to result from mistakes in the execution of this genetic hierarchy. Drosophila is an especially well suited organism in which to study this hierarchy because of its experimental versatility, particularly with regard to its molecular biology and genetics. The homeotic selector genes provide an attractive entry into these studies because of the large amount of genetic and molecular information already available. The products of these genes all contain the DNA-binding homeodomain and act, at least in part, by regulating the transcription of downstream target genes. The experiments proposed here have two complementary goals: to identify and characterize target genes that are regulated by the homeotic selector gene, Ultrabithorax, and to understand in detail how homeotic selector proteins regulate target gene expression. The first goal will be approached in two ways. A subtractive hybridization approach has succeeded in isolating 20 genes that are induced by Ultrabithorax gene products. Three of these genes appear to be bona fide downstream target genes and will be studied in detail. A genetic approach involves isolating dominant mutations that enhance or suppress Ultrabithorax-induced homeotic transformations. Genes identified in this screen will be examined to determine if they are indeed Ultrabithorax target genes. If they appear to be true target genes they will be further characterized by DNA sequencing, determining their in vivo spatial and temporal expression patterns, and examining their genetic interactions with other homeotic mutations. The second goal of this proposal is to better understand how homeotic selector proteins regulate target gene expression. Ibis will be accomplished by making mutant Ultrabithorax proteins and studying them using in vivo assays that measure homeotic protein functions. Assays are already available for studying functions required during embryonic development. In addition to these, new in vivo assays will be developed to measure Ultrabithorax functions required for adult morphogenesis. These in vivo studies will be paralleled by experiments utilizing new assays that measure the in vitro DNA binding properties of mutant Ultrabithorax proteins. These experiments will determine if particular mutations affect Ultrabithorax functions by affecting their ability to bind DNA or by affecting some other property, such as specific protein-protein interactions.

一个复杂有机体的形态发生依赖于一个 复杂的遗传层次，为单个细胞提供 位置和细胞命运信息。 在果蝇中， 选择基因在这个层次中起着核心作用，因为它们 解释位置信息并确定细胞命运。这些基因 在许多动物中有高度保守的同源物，包括人类， 这表明在果蝇中对它们的分析可能是 了解脊椎动物的发育。 分子解剖 脊椎动物发育的研究将反过来提供对人类 包括出生缺陷和各种癌症在内的疾病， 都是由于遗传等级制度的错误执行造成的。 果蝇是一个特别适合研究这个问题的生物体 层次结构，因为它的实验多功能性，特别是 关于它的分子生物学和遗传学。 同源异型选择子 基因为这些研究提供了一个有吸引力的切入点，因为大的 大量的基因和分子信息已经可用。的 这些基因的产物都含有DNA结合同源结构域和ACT， 至少部分通过调节下游靶基因的转录， 基因. 这里提出的实验有两个互补的目标： 鉴定和表征由同源异型调节的靶基因 选择基因，超双胸，并详细了解如何同源异型 选择蛋白调节靶基因表达。 第一个目标将 可以通过两种方式接近。 一种消减杂交方法 成功分离了20个由超双胸基因诱导的基因， 产品. 其中三个基因似乎是真正的下游目标， 基因，并将进行详细研究。 遗传学方法包括 分离显性突变， Ultrabithorax诱导的同源异型转化。 在此过程中发现的基因 将检查屏幕以确定它们是否确实是超双胸 靶基因 如果它们看起来是真正的靶基因， 通过DNA测序进一步表征， 空间和时间表达模式，并检查其遗传 与其他同源异型突变的相互作用。 第二个目标是 一项提议是更好地了解同源异型选择蛋白如何调节 靶基因表达。 朱鹭将通过使突变 Ultrabithorax蛋白质，并使用体内测定法研究它们， 测量同源异型蛋白质功能。 检测试剂盒已可用于 研究胚胎发育所需的功能。 除了 这些，新的体内试验将开发测量超双胸 成年形态发生所需的功能。 这些体内研究将 利用新的测定方法进行实验， 突变体超双胸蛋白的体外DNA结合特性。 这些 实验将确定特定的突变是否会影响超双胸 通过影响它们结合DNA的能力或通过影响一些 其他性质，如特定的蛋白质-蛋白质相互作用。