RAPID GENE DISCOVERY

描述：（申请人的摘要）：项目2（快速基因发现）具有 它的主要目标是发现被破坏的基因或在附近的基因， 与发育相关的个体平衡染色体畸变 异常我们将从项目1（高重复率FISH）接收BAC克隆 映射），其穿过平衡畸变的位点和来自 相应的患者。将对断点进行优先分析， 最大化断裂导致患者表型的可能性 或者断裂附近的基因可以被有效地评估为候选基因。使用 从人类基因组序列的PCR为基础的战略，我们将与 项目1：检测染色体断裂点的位置， BAC.改变的染色体序列将与 相应的正常序列以确定任何缺失的程度， 在断点处的重复或其它重排。我们将识别基因 在每个染色体上的断裂点附近， 数据库搜索，跨物种比较，计算基因预测， RT-PCR和基于PCR和杂交的cDNA文库筛选。基因 将通过cDNA FISH（项目1）和 用cDNA探测患者DNA的Southern印迹。我们将使用北方印迹法， RT-PCR分析以评估被破坏的基因是否产生新的转录物 并将延伸选定的cDNA以确定完整的编码序列， 这些蛋白质基序或同源性可能预测基因功能。哪里 可能的，被破坏的基因或位置效应失调的候选基因 将分析一组独立的非易位突变， 表现出相似表型的患者。我们的目标是初步分析8 每年的单个断点，从项目1 直接或通过本项目的帮助识别“交叉”BAC 用于GenBank或BAC文库中的基因组步移。项目2中发现的基因 将被转发到项目3进行发育表达的分析， 根据所有现有数据确定最具吸引力的选择的优先次序， 模型系统中的功能遗传分析。

DESCRIPTION: (Applicant's Abstract): Project 2 (Rapid Gene Discovery) has as its major goal the discovery of the gene(s) disrupted by or in the vicinity of individual balanced chromosome aberrations associated with developmental abnormalities. We will receive BAC clones from Project 1 (High Throughput FISH Mapping) that cross the sites of balanced aberrations and DNA from the corresponding patients. Breakpoints will be prioritized for analysis to maximize the likelihood that the break is responsible for the patient phenotype or that genes near the break can be effectively assessed as candidates. Using PCR-based strategies from the human genomic sequence, we will collaborate with Project 1 on detection of the site of the chromosomal breakpoint within the BAC. The sequence of altered chromosomes will be compared with the corresponding normal sequences to determine the extent of any deletion, duplication or other rearrangement at the breakpoints. We will identify genes in the vicinity of the breakpoint on each chromosome by a combination of database searching, cross-species comparison, computation gene prediction, RT-PCR and PCR-based and hybridization-based cDNA library screening. Genes disrupted by the breakpoint will be identified by cDNA FISH (Project 1) and by probing Southern blots of patient DNA with cDNAs. We will use Northern blot and RT-PCR analysis to assess whether the disrupted genes produce novel transcripts and will extend selected cDNAs to define the full coding sequence, screening these for protein motifs or homologies that might predict gene function. Where possible, disrupted genes or candidates for disregulation by position effects will be analyzed for mutations in an independent set of non-translocation patients displaying similar phenotypes. Our goal is to initially analyze 8 individual breakpoints per year, drawn from the chromosomes for which Project 1 identifies "crossing" BACs either directly, or through the help of this project for genomic walking in GenBank or a BAC library. Genes discovered in Project 2 will be forwarded to Project 3 for analysis of developmental expression and prioritization based on all available data of the most compelling choices for functional genetic analysis in model systems.