MOLECULAR BIOLOGY OF THE MENKES SYNDROME GENE

门克斯综合征基因的分子生物学

• 批准号: 3245630
• 负责人: THOMAS W GLOVER
• 金额: $ 16.09万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-30 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3245630
• 关键词: DNA replication; Menkes' syndrome; RNA biosynthesis; SDS polyacrylamide gel electrophoresis; artificial chromosomes; autosomal recessive trait; biopsy; chromosome translocation; complementary DNA; copper; cytogenetics; gene expression; genetic library; human tissue; in situ hybridization; inborn metal metabolism disorder; laboratory mouse; linkage mapping; mental retardation; molecular biology; molecular cloning; nucleic acid sequence; phosphoglycerate kinase deficiency; point mutation; polymerase chain reaction; postnatal growth disorder; restriction mapping; southern blotting; transfection /expression vector

We propose to clone and characterize the gene responsible for Menkes syndrome. Menkes syndrome is an X-linked recessive disorder of copper metabolism characterized by early growth retardation and severe neurological impairment. There is no effective treatment for the disease and its exact cause at the biochemical level is unknown. Recently, a female with the disease and a de novo X-autosome translocation was identified. The translocation breakpoint at Xql3 coincides with a previous linkage assignment of the Menkes locus and with the probable location of the homologous mottled locus in the mouse. It therefore almost certainly disrupts proper expression of the Menkes syndrome gene and may very likely directly interrupt it. Our cloning strategy is based on the physical identification and cloning of DNA sequences at this translocation breakpoint. The location of the translocation breakpoint with respect to a number of Xql3 probes has been determined by utilizing somatic cell hybrids containing the translocation chromosome. The translocation was found to break the X chromosome just proximal to the PGK-1 locus. With this knowledge, a long range physical map of the region was begun in attempt to detect the translocation breakpoint. The breakpoint was found to be within 300kb of the PGK-1 locus on Sfil digested DNA. Efforts have been initiated to obtain yeast artificial chromosomes (YACS) that span this region. In this revised application, we propose to identify YAC clones that cross the Menkes syndrome translocation breakpoint and will very likely contain sequences from within or closely linked to the Menkes syndrome gene. A physical map of the region will be constructed and CpG islands identified. Both total YACs and lambda subclones will be used to screen cDNA libraries for identification of candidate genes. Candidate clones will be used to analyze DNA and RNA from Menkes syndrome patients and from mottled mice for mutations. The gene will be characterized by sequence analysis, expression in different tissues and possibly by expression studies in vitro. The Menkes translocation provides an invaluable resource as a "signpost" for the gene and is one of but a few X-linked and autosomal translocations at a human disease gene locus available for studies of this kind. The proposed studies should not only lead to better understanding of the basic defect in Menkes syndrome, but also to new findings concerning copper metabolism in general.

我们建议克隆并鉴定与门克斯有关的基因 综合症。门克斯综合征是一种X连锁的铜隐性遗传病 新陈代谢表现为早期生长迟缓和严重的 神经损伤。这种疾病没有有效的治疗方法。 而它在生化水平上的确切原因尚不清楚。最近，一位 患有这种疾病并有新的X常染色体易位的女性是 确认身份。Xql3的易位断点与之前的 孟克斯基因座的连锁定位及其可能的位置 小鼠体内的同源斑驳基因。因此，几乎可以肯定的是 干扰了门克斯综合征基因的正常表达，很可能 直接打断它。我们的克隆策略是基于物理 该易位DNA序列的鉴定和克隆 断点。 易位断点相对于多个 利用体细胞杂交种确定了Xql3探针 含有易位染色体的。易位被发现是 将X染色体打断在PGK-1基因座的近端。有了这个 在认识到这一点之后，开始绘制该地区的长期实物地图，试图 检测易位断点。断点被发现在 SFIL上300kb的PGK-1基因座消化DNA。已经开始努力 获得跨越该区域的酵母人工染色体(YAC)。在……里面 在这份修订后的申请中，我们建议识别跨越 门克斯综合征易位断点，很可能包含 来自门克斯综合征基因内部或与之紧密连锁的序列。一个 将绘制该区域的实物地图，并确定CPG岛屿。 将使用总YAC和lambda亚克隆来筛选cDNA文库 用于识别候选基因。候选克隆将用于 分析孟克斯综合征患者和斑点小鼠的DNA和RNA 突变。该基因将通过序列分析来表征， 在不同组织中的表达，并可能通过对 体外培养。门克斯易位提供了一种非常宝贵的资源 是该基因的“路标”，是为数不多的X连锁常染色体 人类疾病基因座上的易位可用于这方面的研究 善良。拟议的研究不仅应有助于更好地了解 门克斯综合征的基本缺陷，但也是关于 铜的代谢一般。