NOVEL HOMEOBOX GENES IN DEVELOPING GENITOURINARY TRACT

泌尿生殖道发育中的新型同源盒基因

• 批准号: 2016580
• 负责人: Peter Igarashi
• 金额: $ 26.25万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 1998-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2016580
• 关键词: antisense nucleic acid; antiserum; developmental genetics; embryo /fetus tissue /cell culture; gene expression; genetic library; genetically modified animals; homeobox genes; in situ hybridization; laboratory mouse; laboratory rabbit; mammalian embryology; molecular cloning; nucleic acid sequence; polymerase chain reaction; reproductive development; tissue /cell culture; urinary tract

The causes of many developmental disorders affecting the kidney and genitourinary tract (e.g., renal agenesis, hypoplasia, and cystic dysplasia) remain unknown, largely because little is known about the molecular mechanisms underlying development of the normal kidney. Homeobox-containing genes are responsible for organ differentiation in many species, and disruption of one such gene (Hox-1.5) produces congenital abnormalities in the mouse. Recently, we cloned a novel class of homeobox-containing genes (named cux) from mouse. Cux genes are related to the cut gene of Drosophila melanogaster which is required for normal development of Malpighian tubules, the insect excretory organ that serves as a primitive kidney. The inferred structures of the cux gene products indicate that they also are likely to encode nuclear transcription factors involved in gene regulation. Moreover, we found that at least one of the genes (cux-1) is expressed in kidney, testis, and renal epithelial cells suggesting that it may have a role in the normal development of the mammalian kidney and genitourinary tract. Abnormalities of cux-1 expression, then, might be involved in the pathogenesis of urogenital abnormalities or neoplasms. To further test these hypotheses, full-length cDNAs encoding cux-1, cux-2, and related murine cut homologs will be cloned by library screening and PCR. The temporal and spatial patterns of expression of cux-1 and cux-2 in the developing genitourinary tract of mouse will be further investigated using in situ hybridization and semi-quantitative PCR. Specific antisera raised to cux fusion proteins will be used to identify and immunolocalize the cux gene products in the mature and developing kidney and testis. The promoter/regulatory regions of the cux-1 and cux-2 genes will be cloned, sequenced, and expressed. Whether the cux-1 gene product binds to its own promoter/regulatory region will be evaluated as a first step towards identifying its DNA-binding specificity. The functional roles of the cux-1 gene will be assessed by specific inhibition in renal epithelial cells using antisense oligonucleotides. From the effects on cell proliferation, morphology, polarity, epithelial organization, and gene expression, functional roles may be ascribed to the cux-1 gene. Whether the cux-1 gene is required for development of the kidney and genitourinary tract in mouse will be tested by disruption using homologous recombination and expression in transgenic mice. Our long-term aims will be to address the role of cux genes in a cascade of genetic regulatory mechanisms responsible for nephrogenesis. In these future studies, steps in pathways that are more proximal to cux (regulation of cux genes and gene products) and steps that are distal (target genes) will be examined in greater detail. Such studies should contribute to our fundamental understanding of genitourinary tract development. Moreover, further characterization of this novel class of homeobox-containing genes would be relevant to the overall study of eukaryotic gene regulation.

许多影响肾脏的发育障碍的原因， 泌尿生殖道（例如，肾发育不全、发育不全和囊性 发育不良）仍然未知，主要是因为对 正常肾脏发育的分子机制。 含有同源框的基因负责器官分化， 许多物种，其中一个这样的基因（Hox-1.5）的破坏产生 小鼠的先天性异常。 最近，我们克隆了一本小说 一类来自小鼠的含同源框基因（命名为cux）。 Cux基因 与果蝇的cut基因有关， 昆虫排泄器官马氏管的正常发育 作为原始肾脏。 Cux的推断结构 基因产物表明，它们也可能编码核 参与基因调控的转录因子。 而且，我们发现 至少一种基因（Cux-1）在肾脏，睾丸， 和肾上皮细胞，这表明它可能在 哺乳动物肾脏和泌尿生殖道的正常发育。 因此，cux-1表达的缺失可能与 泌尿生殖系统异常或肿瘤的发病机制。 为了进一步测试 这些假设，全长cDNA编码cux-1，cux-2，和相关的， 小鼠切割同源物将通过文库筛选和PCR进行克隆。 的 Cux-1和Cux-2在乳腺癌组织中表达的时间和空间模式 将进一步研究小鼠泌尿生殖道的发育 采用原位杂交和半定量PCR。 具体 针对Cux融合蛋白产生的抗血清将用于鉴定 将cux基因产物免疫定位在成熟和发育的肾脏中 和睾丸 Cux-1和Cux-2的启动子/调节区 基因将被克隆、测序和表达。 cux-1基因是否 产品与其自身启动子/调控区的结合将被评价为 这是鉴定其DNA结合特异性的第一步。 的 cux-1基因的功能作用将通过特异性的 使用反义寡核苷酸在肾上皮细胞中的抑制。 从对细胞增殖、形态学、极性、上皮细胞 组织和基因表达，功能作用可能归因于 cux-1基因 cux-1基因是否是发育所必需的 小鼠肾脏和泌尿生殖道将通过破坏试验 使用同源重组和在转基因小鼠中表达。 我们 长期的目标将是解决cux基因在级联反应中的作用。 负责肾发生的遗传调节机制。 在这些 未来的研究，更接近Cux的途径的步骤 （调节cux基因和基因产物）和步骤， （靶基因）将被更详细地检查。 这种研究 有助于我们对泌尿生殖道 发展 此外，进一步表征这类新的 含有同源框的基因将与整体研究有关， 真核基因调控