INT-1-RELATED GENES IN XENOPUS EMBRYOS

原癌基因的不适当表达可能与 各种癌症和出生缺陷。这导致了相当大的 对阐明正常的表达模式和功能的兴趣 脊椎动物发育过程中表达的原癌基因 胚胎。原癌基因Int-1有一个特别有趣的模式 表达--它在背侧中线以一种短暂的方式表达 在脊椎动物胚胎的神经管中，并可能参与信号传递 在细胞之间。脊椎动物胚胎表达额外基因的假说 与INT-1相关的基因尚未得到广泛研究。在建议的 项目中，我们将通过完成以下工作来检验这一假设 具体目的：1.用聚合酶链式反应方法克隆与INT-1相关的cDNA 从非洲爪哇胚胎mRNA中，使用互补于 几个物种的INT-1和IRP保守区。这些都是 技术是适当的，这从我们最近克隆的至少 用这种方法获得了4个与INT-1同源的非洲爪哇独特的cDNA。2.至 确定与int-1相关的cDNA互补的转录本是否 在发展过程中以一种临时的方式进行管理。以确定是否 与int-1相关的cDNA转录产物的表达为 限制在胚胎的特定区域，非洲爪哇胚胎将被 进行显微解剖、RNA提取和RNA印迹分析。3. INT-1相关蛋白的最低保守区将在 在表达载体中连接成框架的cDNA后，在大肠杆菌中 PRIT2T。将在兔体内制备多克隆抗血清。整架坐骑 将使用非洲爪哇胚胎的免疫细胞化学来确定 INT-1相关蛋白的空间分布。5.转录的mRNA 来自几乎全长的int-1相关cDNA将被显微注射到 使非洲爪哇受精卵解除对正常表达的调控。分析 由此产生的胚胎表型将提供对正常 INT-1相关蛋白的功能。6.消减cdna探针将 用于克隆因INT-1相关基因表达而改变的mRNAs 蛋白质。7.几条调查路线将追查手机 INT-1和INT-1相关蛋白的受体以及第二信使 参与信号转导的系统。总而言之，这些具体目标 将提供有关势的表达和功能的新信息 原癌基因在脊椎动物中的重要细胞信号和通讯 胚胎，以及人类癌症和出生缺陷。

Inappropriate expression of proto-oncogenes may be causally related to a variety of cancers and to birth defects. This has led to considerable interest in elucidating the normal pattern of expression and functions of proto-oncogenes which are expressed during development of vertebrate embryos. The proto-oncogene Int-1 has a particularly interesting pattern of expression-it is expressed in a transient manner in the dorsal midline of the neural tube of vertebrate embryos, and may be involved in signalling between cells. The hypothesis that vertebrate embryos express additional genes related to int-1 has not been widely studied. In the proposed project, we shall test this hypothesis by completion of the following specific aims: 1. cDNAs related to int-1 will be cloned by PCR approaches from Xenopus embryo mRNA, using oligonucleotide primers complementary to conserved regions of int-1 and IRP from several species. That these techniques are appropriate is evident from our recent cloning of at least four unique Xenopus cDNAs homologous to int-1 by this approach. 2. To determine whether transcripts complementary to the int-1-related cDNAs are regulated in a temporal manner during development. To determine whether the expression of the transcripts related to the int-1-related cDNAs is restricted to specific regions of embryos, Xenopus embryos will be microdissected, RNA extracted, and RNA blot analyses undertaken. 3. The least conserved region of the int-1-related proteins will be expressed in E. coli following ligation of the cDNAs in frame in the expression vector pRIT2T. Polyclonal antisera will be prepared in rabbits. Whole mount immunocytochemistry of Xenopus embryos will be used to determine the spatial distribution of the int-1-related proteins. 5. mRNA transcribed from nearly full-length int-1-related cDNAs will be microinjected into fertilized Xenopus eggs to deregulate normal expression. Analysis of resulting embryonic phenotypes will provide insight into the normal functions of the int-1-related proteins. 6. Subtractive cDNA probes will be used to clone mRNAs changing in response to expression of int-1-related proteins. 7. Several lines of investigation will pursue cellular receptors for int-1 and int-1-related proteins, as well as second messenger systems involved in signal transduction. Collectively these specific aims will provide new information on the expression and functions of potential proto-oncogenes important cell signalling and communication in vertebrate embryos, as well as human cancers and birth defects.