LOCALIZATION OF THE WILMS TUMOR SUSCEPTIBILITY GENE(S)

威尔姆斯肿瘤易感基因的定位

• 批准号: 3087070
• 负责人: STANLEY F. NELSON
• 金额: $ 5.87万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3087070
• 关键词: Wilms' tumor; artificial chromosomes; chromosome walking; gene expression; genetic library; genetic manipulation; genetic mapping; genetic transcription; human genetic material tag; molecular cloning; neoplasm /cancer immunology; neoplastic cell; northern blottings; nucleic acid probes; nucleic acid sequence; pulsed field gel electrophoresis; tumor suppressor genes

Wilm's tumor is one of the most common malignancies of children. Strong evidence indicates that tumor suppressor genes are located at 11p13 and 11p15.5. Deletions in these regions contribute to the genesis of Wilm's tumor. To understand the basic defect in this cancer, the Wilms tumor genes (WTG) at 11p13 and 11p15.5 must be isolated and their products analyzed. To this end, an overlapping library of human chromosome 11 will be made in yeast artificial chromosome (YAC) vectors using DNA from a human:rodent hybrid cell line. Insert DNA will be partially digested and size selected >400kb. The YAC library will be screened with known probes from the 11p13 and 11p15.5 regions. Comprehensive overlapping libraries of the 1500kb of 11p13 and the 20mb of 11p15.5 will be created. The YAC inserts will be compared to existing maps of the regions. YAC chromosome walking will be used to fill in gaps between known probes. Any gaps that are not able to be cloned in YACs will be cloned in cosmid or lambda vectors. The regions will be physically mapped with pulsed field gel electrophoresis capable of resolving 50-5000kb fragments of DNA. Panels of DNA from Wilm's tumors will be used to more precisely map the WTG locus in 11p15.5. Transcribed regions of the 11p13 and 11p15.5 libraries will be isolated by several methods: 1) insertional mutagenesis with a selectable marker; 2) hybridization with cDNA from human kidney; 3) identification of unmethylated CpG islands; and 4) cross-species hybridization. Probes that detect expressed genes will be hybridized to Northern blots of mRNA from multiple Wilm's tumors. Probes that detect variable sizes or decreased levels mRNA from some of the Wilm's tumors will be candidate WTG exon probes. Additionally, the YACs will be tested for tumor suppression function by spheroplast fusion into a Wilm's tumor cell line, G401. If a YAC clone is able to suppress tumorigenicity, deletion analysis and insertional mutagenesis would greatly facilitate localization of a WTG.

肾母细胞瘤是儿童最常见的恶性肿瘤之一。 强有力的证据表明肿瘤抑制基因位于 11p13 和 11p15.5。 这些区域的删除有助于 肾母细胞瘤的起源。 了解基本缺陷 对于这种癌症，位于 11p13 和 11p15.5 的 Wilms 肿瘤基因 (WTG) 必须 进行分离并分析其产品。 为此，一个 人类11号染色体重叠文库将在酵母中构建 使用人类 DNA 的人工染色体 (YAC) 载体：啮齿动物 杂交细胞系。 插入 DNA 将被部分消化并调整大小 选择 >400kb。 YAC文库将用已知的筛选 来自 11p13 和 11p15.5 区域的探针。 综合的 11p13 的 1500kb 和 11p13 的 20mb 的重叠文库 11p15.5 将被创建。 YAC 插入物将与 现有的地区地图。 将使用YAC染色体行走 填补已知探针之间的空白。 任何无法做到的间隙 要克隆到 YAC 中的将被克隆到粘粒或 lambda 载体中。 这些区域将用脉冲场凝胶进行物理绘制 电泳能够解析 50-5000kb 的 DNA 片段。 来自维尔姆氏肿瘤的 DNA 组将用于更精确地绘制地图 11p15.5 的 WTG 基因座。 11p13 和 11p13 的转录区域 11p15.5 文库将通过以下几种方法进行分离：1) 使用选择标记的插入诱变； 2) 杂交 含有来自人类肾脏的 cDNA； 3) 未甲基化CpG的鉴定 岛屿; 4）跨物种杂交。 检测的探针 表达的基因将与 mRNA 的 Northern 印迹杂交 多发性肾母细胞瘤。 检测可变尺寸或 一些肾母细胞瘤的 mRNA 水平下降 候选WTG外显子探针。 此外，YAC 还将接受测试 通过将原生质球融合到肾母细胞中来实现肿瘤抑制功能 肿瘤细胞系，G401。 如果 YAC 克隆能够抑制 致瘤性、缺失分析和插入突变将 极大地方便了风力发电机组的国产化。