LOCALIZATION OF THE WILMS TUMOR SUSCEPTIBILITY GENE(S)

威尔姆斯肿瘤易感基因的定位

• 批准号: 3087067
• 负责人: STANLEY F. NELSON
• 金额: $ 8.04万
• 依托单位: STANFORD UNIVERSITY
• 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/3087067
• 关键词: chromosome walking; gene expression; genetic library; genetic manipulation; genetic mapping; genetic transcription; human genetic material tag; molecular cloning; neoplasm /cancer immunology; neoplastic cell; nephroblastoma; northern blottings; nucleic acid probes; nucleic acid sequence; pulsed field gel electrophoresis; tumor suppressor genes; yeasts

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 在11 p13和11p15.5。 这些区域的删除有助于 威尔姆瘤的起源 要理解的基本缺陷， 在这种癌症中，位于11 p13和11p15.5的Wilms肿瘤基因（WTG）必须 分离并分析其产物。 为此， 人类11号染色体的重叠文库将在酵母中制备 使用来自人类：啮齿动物的DNA的人工染色体（YAC）载体 杂交细胞系 插入的DNA将被部分消化， 选择> 400 kb。 YAC文库将用已知的 来自11 p13和11p15.5区域的探针。 全面 11 p13的1500 kb和20 mb的 11p15.5将被创建。 将YAC衬垫与 现有的地图。 将使用YAC染色体步移 以填充已知探针之间的间隙。 任何不能 将克隆到粘粒或λ载体中。 这些区域将用脉冲场凝胶进行物理映射 能够分辨50- 5000 kb DNA片段的电泳。 来自Wilm肿瘤的DNA组将用于更精确地绘制 11p15.5的WTG位点。 11 p13的转录区域， 11p15.5文库将通过几种方法分离：1） 用选择标记插入诱变; 2）杂交 3）未甲基化CpG的鉴定 岛屿; 4）跨物种杂交。 探测器探测 表达的基因将与来自以下的mRNA的北方印迹杂交： 多发性肾母细胞瘤 检测可变大小或 一些肾母细胞瘤的mRNA水平降低， 候选WTG外显子探针。 此外，将对YAC进行测试 通过原生质球融合到Wilm's细胞中来实现肿瘤抑制功能 肿瘤细胞系G401。 如果YAC克隆体能够抑制 致瘤性、缺失分析和插入突变将 大大方便了风力发电机国产化。