GENETIC MAPPING AND CLONING OF THE HD GENE USING YACS

亨廷顿病基因(HD)位于端粒染色体带上 4p16.3，位于遗传标记D4S125两侧的2.5兆碱基区域内 和D4S168(Whaley等人，1991；Bates等人，1991)。巨大的…… 这一地区表明有必要进一步完善候选人。 区域通过连锁不平衡研究，例如 在囊性纤维化基因的克隆中具有无可估量的价值。它是 预期某些标记等位基因与 HD突变可能提供必要的遗传信息 HD基因的精确定位。该提案的第一部分将 寻求鉴定高度多态的(dc-da).(dg-dt)二核苷酸重复序列 在候选区域中。至少已经有四次这样的重复 从已经被鉴定的噬菌体和宇宙线虫中进行亚克隆 在这一地区绘制了地图。进一步提出了基于聚合酶链式反应的检测方法。 促进从酵母中直接鉴定这些(CA)n标记 为候选区域分离的人工染色体；(CA)n 含有Alu间聚合酶链式反应扩增产物的 Southern杂交到(CA)n探针及其两侧的序列 (Ca)n重复区段将通过对凝胶进行测序直接确定- 用Alu引物或(CA)n和(GT)n寡聚体纯化的PCR产物。这个 提案的第二部分将寻求确定和描述 HD的候选基因。这将需要采取几种方法：a) 以凝胶纯化的YAC为探针直接筛选cDNA文库， B)将凝胶纯化的YAC固定在膜过滤器上，并应用PCR- 扩增的cDNAs插入作为探针。

The Huntington's disease gene (HD) lies in the telomeric chromosome band 4p16.3, within a 2.5 megabase region flanked by genetic markers D4S125 and D4S168 (Whaley et al., 1991; Bates et al., 1991). The enormity of this region suggests the need for further refinement of the candidate region through linkage disequilibrium studies, which for example have been invaluable in the cloning of the cystic fibrosis gene. It is expected that nonrandom association of certain marker alleles with the HD mutation may provide the genetic information necessary for more precise placement of the HD gene. The first part of this proposal will seek to identify highly polymorphic (dC-dA).(dG-dT) dinucleotide repeats in the candidate region. At least four such repeats have already been identified and are being subcloned from phages and cosmids that have been mapped in this region. A PCR-based assay is further proposed to facilitate the identification of these (CA)n markers directly from yeast artificial chromosomes (YACs) isolated for the candidate region; (CA)n containing inter-Alu PCR amplification products will be identified by Southern hybridization to a (CA)n probe and the sequences flanking the (CA)n repeat block will be directly determined by sequencing the gel- purified PCR product using Alu primers or (CA)n and (GT)n oligomers. The second part of the proposal will seek to identify and characterize candidate genes for HD. This will entail several approaches: a) utilizing gel-purified YACs as probes to directly screen cDNA libraries, b) immobilizing gel-purified YACs onto membrane filters and applying PCR- amplified cDNA inserts as probes.