CLONING CHROMOSOME 11 GENES INVOLVED IN NEOPLASIA

建议进行研究，以识别、克隆和表征潜在的 肿瘤或生长抑制基因，通过失活， 会导致多种癌症的发生， 包括那些在贝克维斯中表现出患病率增加的人- Wiedemann综合征（BWS）。染色体11p15.5区间 匿名DNA标记D11 S12和胰岛素样生长因子 2（IGF 2）基因与肿瘤抑制和BWS有关。我们有 映射到这个区域的两个染色体重排（一个来自一个 横纹肌样瘤和其他与BWS相关的），可能会破坏 并因此精确定位生长或肿瘤抑制基因。长- 范围限制映射表明， D11 S12和IGF 2之间有2200 kb的基因组长度， 适合于在重叠的YAC中克隆。使用37号电池 探针（每60 kb一个探针）同源YAC将从 4-折叠染色体11 YAC文库和通过探针组装的重叠群 内容分析和步移使用YAC衍生的RT-PCR产物。一 使用此YAC库进行遍历的显著优点 方法是人-人嵌合克隆的罕见发生率 （<1%）。发现YAC克隆在染色体重排中作图 断裂点区域将用于分析中期染色体 通过荧光原位杂交检测含有重排的 （FISH）。那些被发现“越过”断点的YAC将被用于 分离相应粘粒克隆，然后通过 基因编码序列存在的外显子扩增。这些 然后将外显子用于分离cDNA。或者， 跨度断点或相应的余弦将用于标识 通过cDNA选择技术获得编码基因。突变试验 候选基因将用于分析肿瘤和其他BWS 患者克隆和表征假定的生长或 肿瘤抑制基因在这个染色体区域将有助于解决 与参与的基因的数量和类型有关的问题 广泛的肿瘤表现出杂合性缺失（洛）， 这个基因片段此外，两者的分子测定 诊断和预后价值可能会发展，一旦基因 有助于肿瘤发生的特征。

Research is proposed to identify, clone and characterize potential tumor or growth suppressor genes that, through inactivation, contribute to the development of a large variety of cancers, including those exhibiting increased prevalence in Beckwith- Wiedemann Syndrome (BWS). The chromosome 11p15.5 interval between the anonymous DNA marker D11S12 and the insulin-like growth factor 2 (IGF2) gene is implicated in tumor suppression and BWS. We have mapped into this region two chromosome rearrangements (one from a rhabdoid tumor and the other associated with BWS) that may disrupt and therefore pinpoint a growth or tumor suppressor gene(s). Long- range restriction mapping indicates that there is little more than 2200 kb separating D11S12 and IGF2, a genomic length easily amenable to cloning in overlapping YACs. Using a battery of 37 probes (one probe per 60 kb) cognate YACs will be isolated from a 4-fold chromosome 11 YAC library and contigs assembled by probe content analysis and walking using YAC-derived Alu-PCR products. A significant advantage of using this YAC library for a walking approach is the rare incidence of human-human chimeric clones (<1%). YAC clones found to map in the chromosome rearrangement breakpoint region will be used to analyze metaphase chromosomes containing the rearrangements by fluorescence in situ hybridization (FISH). Those YACs found to "cross" the breakpoints will be used to isolate corresponding cosmid clones which will then be screened by exon amplification for the presence of gene coding sequences. These exons will then be used to isolate cDNAs. Alternatively, YACs that span breakpoints or corresponding cosmids will be used to identify encoded genes by a cDNA selection technique. Mutation assays for candidate genes will be used to analyze tumors and additional BWS patients. The cloning and characterization of putative growth or tumor suppressor genes in this chromosome region will help address questions pertaining to the number and type of genes involved in the wide spectrum of tumors showing loss of heterozygosity (LOH) in this genomic segment. Furthermore, molecular assays of both diagnostic and prognostic value may be developed once genes contributing to tumorigenesis are characterized.