GENE MAPPING/ISOLATION BY CHROMOSOME TRANSFER AND POSITIONAL CLONING

通过染色体转移和定位克隆进行基因定位/分离

• 批准号: 6242184
• 负责人: Markus Grompe
• 金额: $ 25.92万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6242184
• 关键词: DNA repair; animal genetic material tag; autosomal recessive trait; cell fusion; chemical cleavage; chromosome deletion; clone cells; complementary DNA; congenital aplastic anemia; family genetics; gene complementation; genetic disorder diagnosis; genetic mapping; human genetic material tag; linkage mapping; molecular cloning; molecular genetics; polymerase chain reaction; subtraction hybridization

Several possible strategies for the cloning of new FA genes exist. cDNA complementation of the DNA cross-linking hypersensitivity has already resulted in the cloning of one FA gene. This method, however, is limited by 2 factors: a) the cDNA has to be relatively small and b) properly regulated expression of the gene must not he essential for cell survival. An alternative method for isolating FA genes is positional cloning. Due to the fact, that multiple complementation groups exist and that the number of families is relatively limited, the mapping of the FA genes by genetic linkage studies alone appears difficult We here propose to use chromosome transfer to map the location of FA genes at a gross level, followed by genetic linkage and physical mapping to further refine the chromosomal position and to ultimately clone the genes. Human chromosomes marked with the 0418 resistance gene will be transferred into transformed fibroblasts from FA patients, which are known to not represent complementation group C. Selection with DNA crosslinking agents will identify complemented clones and the chromosome conferring the resistance will be identified. The size of the correcting cDNA is not relevant in this method and proper regulation of the gene in question is also given. Once a complementing chromosome has been identified, the more precise localization of the FA gene will be identified by deletion mapping in somatic cell hybrids and genetic linkage. Existing markers as well as new markers generated by chromosome microdissection and PCR will be employed for genetic mapping. This step will be followed by isolation of YACs from the critical region, physical mapping and the cloning of expressed sequences. Candidate cDNAs will be screened by chemical mismatch cleavage for possible mutations. An additional approach to be used is the identification of mouse chromosomes bearing FA genes as described above. FA cells complemented by mouse DNA could then be used to prepare subtractive cDNA libraries and direct cloning of FA candidate cDNAs.

存在用于克隆新FA基因的几种可能的策略。cDNA DNA交联超敏反应的互补已经 结果克隆了一个FA基因。然而，这种方法是有限的。 通过2个因素：a）cDNA必须相对较小，以及B）适当地 基因的调节表达对于细胞存活一定不是必需的。 分离FA基因的另一种方法是定位克隆。由于 事实上，存在多个互补组， 家庭数量相对有限，FA基因的定位， 单靠遗传连锁研究似乎很困难 我们在这里建议使用染色体转移来定位FA 基因在一个粗略的水平，其次是遗传连锁和物理映射 为了进一步细化染色体位置并最终克隆 基因. 标记有0418抗性基因的人类染色体将被 转移到来自FA患者的转化成纤维细胞中， 已知不代表互补群C。DNA选择 交联剂将识别互补的克隆和染色体 将识别赋予抗性的物质。纠正的大小 cDNA在该方法中是不相关的，并且基因的适当调节在 也提出了问题。一旦互补染色体 鉴定，FA基因的更精确定位将是 通过体细胞杂种中的缺失作图和遗传学鉴定， 联动现有标记以及染色体产生的新标记 将采用显微切割和PCR进行遗传作图。该步骤 随后将YAC与关键区域隔离，物理 表达序列的定位和克隆。候选cDNA将是 通过化学错配切割筛选可能的突变。 另一种使用的方法是识别小鼠 如上所述携带FA基因的染色体。FA细胞补充 然后可以用小鼠DNA制备消减cDNA文库， FA候选cDNA的直接克隆。