ISOLATING THE GENE FOR CHOROIDEREMIA

脉络膜血症(McKusick#30310)是一种未知的X连锁视网膜营养不良 导致进行性视力丧失和眼球萎缩的发病机制 半合子男性的光感受器和视网膜色素上皮(RPE) 根据家系和家系的连锁分析，疾病基因定位于xq21.2 关于患者染色体缺失和易位的研究。一位女性 脉络膜血症在XQ和13P之间有平衡易位 显然会干扰脉络膜血症基因并通过 正常X的失活。分离到的匿名探针，pH 165.1 这个实验室绘制了易位的50千碱基的近端 这只雌性的断裂点。Lambda or中重叠的基因组序列 粘粒向量将被隔离，直到整个区域在PH165.1和 断点，以及断点远端的序列已经被克隆。 来自这一区域的所有DNA都将接受测试，以确定它是否包含一个 基于(I)DNA同源性保守性的候选基因 人类和其他哺乳动物，(Ii)该序列在视网膜和RPE中的表达， (Iii)这些推定的突变、缺失或其他突变的证明 脉络膜病先证者的外显子序列。当候选基因是 确认身份， (1)对其完整的mRNA序列和基因组结构进行了表征。 (2)导致这种疾病的突变将通过以下方式确定 猪瘟病毒DNA中该基因的外显子和内含子-外显子边界的测序 病人 (3)将产生针对基因产物的抗体并用于检测 视网膜中表达该基因的组织(S)和细胞 以及基因产物的亚细胞定位。 (4)负责基因调控的启动子和增强子序列 表达将被识别。 (5)分离同源小鼠基因，用于评价 在小鼠胚胎中表达的发育时序以及 开始研究通过同源重组发展小鼠模型 胚胎干细胞系统中突变基因序列的研究。 长期目标是确定人类眼病的基因。 通过它的位置而不是它的功能来描述 基因及其表达以及导致这种疾病的突变 人类的疾病。最终目标是了解基因产物是什么 以及基因缺陷是如何导致疾病的。

Choroideremia (McKusick #30310) is an X-linked retinal dystrophy of unknown pathogenesis that causes progressive vision loss and atrophy of the photoreceptors and retinal pigmented epithelium (RPE) in hemizygous males. The disease locus maps to xq21.2 based on linkage analysis in families and on chromosome deletion and translocation in affected patients. A female with choroideremia has a balanced translocation between Xq and 13p that apparently interrupts the choroideremia gene and causes disease through inactivation of the normal X. An anonymous probe, pH165.1, isolated in this laboratory maps proximal to and <50 kilobases from the translocation breakpoint in this female. Overlapping genomic sequences in lambda or cosmid vectors will be isolated until the entire region between pH165.1 and the breakpoint, and sequences distal to the breakpoint have been cloned. All DNA from this region will be tested for whether it contains exons of a candidate gene based on (i) conservation of nucleic acid homology between man and other mammals, (ii) expression of the sequence in retina and RPE, (iii) demonstration of mutation, deletional or otherwise, of these putative exonic sequences in choroideremia probands. When a candidate gene is identified, (1) its entire mRNA sequence and genomic structure will be characterized. (2) the mutations responsible for the disease will be determining by sequencing the exons and intron-exon boundaries of the gene in the DNA of patients (3) antibodies to the gene product will be raised and used to determine the tissue(s) in the retina in which the gene is expressed and the cellular and subcellular localization of the gene product. (4) the promoter and enhancer sequences responsible for control of gene expression will be identified. (5) the homologous murine gene will be isolated to use for assessing the developmental timing of expression in mouse embryos as well as for beginning studies to develop a mouse model through homologous recombination of mutant gene sequences in the embryonal stem cell system. The long-term objective is to identify the gene for a human eye disease through its location rather than its function and then to characterize the gene and its expression as well as the mutations responsible for the disease in man. The ultimate goal is to understand what the gene product is and how defects in the gene produce disease.