CLONING CHROMOSOME 11 GENES INVOLVED IN NEOPLASIA

建议进行研究以识别、克隆和表征潜力 肿瘤或生长抑制基因，通过失活， 促进了多种癌症的发展， 包括那些在贝克韦斯表现出更高流行率的人- 维德曼综合征(BWS)。染色体11p15.5之间的间隔 匿名DNA标记D11S12与胰岛素样生长因子 2(IGF2)基因与肿瘤抑制和BWS有关。我们有 将两个染色体重排映射到该区域(一个来自 横纹肌样瘤和其他与BWS相关的肿瘤)，可能会破坏 并因此精确定位生长或肿瘤抑制基因(S)。长- 范围限制映射表明，除了 分离D11S12和IGF2的2200kb，基因组长度容易 可以在重叠的YAC中进行克隆。使用37英寸的电池 探针(每60kb一个探针)同源YAC将从 4倍体11号染色体YAC文库和探针组装的重叠群 使用YAC衍生的Alu-PCR产物进行含量分析和步行。一个 使用这个YAC图书馆散步的显著优势 方法是罕见的人-人嵌合克隆 (&lt；1%)在染色体重排中发现定位的YAC克隆 断裂点区域将用于分析中期染色体 用荧光原位杂交法检测重排 (鱼)。那些被发现“越过”断点的YAC将被用来 分离相应的粘粒克隆，然后通过 外显子扩增用于基因编码序列的存在。这些 然后将使用外显子来分离cDNA。或者，YAC认为 SPAN断点或相应的COSMID将用于识别 通过cDNA选择技术对基因进行编码。基因突变检测 候选基因将用于分析肿瘤和其他BW 病人。推定生长的Or基因的克隆与鉴定 该染色体区域的肿瘤抑制基因将有助于解决 关于涉及的基因数量和类型的问题 表现为杂合性缺失(LOH)的广谱肿瘤 这个基因组片段。此外，这两种物质的分子检测 一旦发现基因，就可能产生诊断和预后价值 对肿瘤发生的贡献是有特征的。

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.