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 兴趣阐明正常模式的表达和功能， 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 在背中线以短暂的方式表达 脊椎动物胚胎的神经管，并可能参与信号 细胞之间。 脊椎动物胚胎表达额外的 与int-1相关的基因尚未被广泛研究。 拟议 项目，我们将通过完成以下内容来测试这一假设 具体目标：1. 通过PCR方法克隆与int-1相关的cDNA 从非洲爪蟾胚胎mRNA，使用互补的寡核苷酸引物， 几个物种的int-1和IRP保守区。 这些 从我们最近克隆的至少 四个独特的非洲爪蟾cDNA同源的int-1的这种方法。 2.到 确定与int-1相关cDNA互补的转录物是否 在发育过程中以时间的方式调节。 以确定是否 与int-1相关的cDNA相关的转录物的表达是 仅限于胚胎的特定区域，非洲爪蟾胚胎将被 显微解剖，提取RNA，并进行RNA印迹分析。 3. 的 int-1相关蛋白的最小保守区域将在 E.将cDNA连接到表达载体的读框内， pRIT 2T。 将在家兔中制备多克隆抗血清。 整装 非洲爪蟾胚胎的免疫细胞化学将用于确定 int-1相关蛋白的空间分布。 5. 转录的mRNA 将来自几乎全长int-1相关cDNA的DNA显微注射到 非洲爪蟾受精卵去调节正常表达。 分析 由此产生的胚胎表型将提供对正常的 int-1相关蛋白的功能。 6. 消减cDNA探针将 用于克隆响应int-1相关基因表达而改变的mRNA。 proteins. 7. 几条调查线将追踪细胞 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。 这些具体目标 将提供新的信息的表达和功能的潜力 原癌基因在脊椎动物中重要的细胞信号和通讯 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.