LOCALIZATION OF THE WILMS TUMOR SUSCEPTIBILITY GENE(S)

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，肿瘤抑制基因存在于 在11p13和11p15.5。这些地区的缺失导致 肾母细胞瘤的起源。要了解的基本缺陷 这种癌症，位于11p13和11p15.5的Wilms肿瘤基因(WTG)必须 被隔离并对其产品进行分析。为此，一个 人类11号染色体重叠文库将在酵母中制作 利用啮齿类动物的DNA构建人工染色体载体 杂交细胞系。插入的DNA将被部分消化并调整大小 精选&gt；400kb。YAC文库将与已知的 来自11p13和11p15.5区域的探针。全面 11p13的1500kb和11p13的20mb的重叠文库 11p15.5将被创建。YAC刀片将与 这些地区的现有地图。将使用YAC染色体行走 以填补已知探测器之间的空白。任何无法实现的差距 要克隆到YAC中的基因将被克隆到粘粒或lambda载体中。 这些区域将使用脉冲场凝胶进行物理映射 能够分辨50-5000kb DNA片段的电泳法。 来自Wilm肿瘤的DNA面板将被用于更精确地绘制地图 WTG基因座位于11p15.5。11p13和11p13的转录区域 11p15.5文库将通过几种方法进行分离：1) 带有可选择标记的插入突变；2)杂交 3)未甲基化CpG的鉴定 岛屿；4)跨物种杂交。探测到的探测器 表达的基因将被杂交到来自中国的 多发性肾母细胞瘤。检测可变大小或 一些肾母细胞瘤的mRNA水平下降将是 候选WTG外显子探针。此外，青年交流会将接受测试 通过将球体融合到Wilm‘s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 极大地促进了风力发电机组的国产化。

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.