MOLECULAR BIOLOGY OF THE MENKES SYNDROME GENE

门克斯综合征基因的分子生物学

• 批准号: 2143535
• 负责人: THOMAS W GLOVER
• 金额: $ 11.73万
• 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/2143535
• 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.

我们建议克隆和表征基因负责门克斯 综合征 Menkes综合征是一种X连锁隐性铜障碍 以早期生长迟缓和严重的 神经损伤 这种病没有有效的治疗方法 而其在生化层面上的确切原因尚不清楚。 近日一 患有这种疾病的女性和从头X-常染色体易位， 鉴定 Xql 3处的易位断裂点与先前的易位断裂点一致。 门克斯基因座的连锁分配和可能的位置， 小鼠中的同源斑驳基因座。 因此，几乎可以肯定， 破坏门克斯综合征基因的正常表达， 我们的克隆策略是基于 鉴定和克隆该易位处DNA序列 断点。 易位断裂点相对于多个 Xq 13探针已通过利用体细胞杂交确定 含有易位染色体。 发现易位 在PGK-1位点附近破坏X染色体。 与此 知识，该地区的长期物理地图开始尝试， 检测易位断点。 发现断点位于 在Sfil消化的DNA上的300 kb的PGK-1基因座。 已经开始努力 以获得跨越该区域的酵母人工染色体（YACS）。 在 这个修订后的应用程序，我们建议确定YAC克隆，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或与门克斯综合征基因紧密相连的序列。 一 将构建该区域的物理图并鉴定CpG岛。 总YAC和λ亚克隆都将用于筛选cDNA文库 用于鉴定候选基因。 候选克隆将用于 分析门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，并可能通过在 体外门克斯易位提供了一个宝贵的资源， 是该基因的“路标”，也是少数X连锁和常染色体遗传的基因之一。 在人类疾病基因位点的易位可用于研究这一点， kind. 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，但也有新的发现， 铜的代谢。