CLONING PALLID A MUTANT AFFECTING INNER EAR AND BLEEDING

这个项目的目标是克隆一个基因， 苍白的变种人 这个突变体是11个基因突变体之一， 不同的突变体影响毛色和出血时间。 这些影响是 通过黑素体和血小板致密颗粒的改变介导， 这两种基因都存在，但在突变小鼠中有缺陷。 一些 突变体，包括pallid，也对a 第三个细胞器，溶酶体。 Pallid是三种突变体之一 内耳，而突变体表现出共济失调，因为耳石， 平衡所需要的，都不见了。 克隆正常基因将允许 分析基因在其复合体所有部分的正常功能 表型，在分子和细胞水平，和分析的 突变表型 克隆这种基因的方法包括鉴定 最接近小鼠2号染色体上苍白突变的分子标记。 这将使用种间回交（C57 BL/6 J-pa a1/pa a1 X M. spretus-+ A/+ A）F1 X C57 BL/6 J-pa a1/pa a1，已经生产了123辆 后代 初步数据表明，β 2-微球蛋白是一个非常接近 标记。 将获得含有所选标记的YAC克隆， 开始走向苍白 将克隆YAC的末端， 测序以鉴定重叠的YAC。 回交动物含有 将识别重组断点并首先用于确定 步行的方向，然后监视 起点和目标。 YAC克隆重叠之间的间隔 位于苍白突变侧翼的重组断点将是 所需克隆的候选人。 为了确定YAC上的任何基因是否在细胞中表达， 巨核细胞（血小板前体）或皮肤中，来自皮肤的cDNA文库 骨髓细胞将在一个“片段化载体”中产生， YAC中不存在的选择性酵母标记和酵母端粒。 的 将文库转化到含有候选YAC的酵母细胞中 YAC上的单个cDNA和基因之间的重组体将被 选定. 这将允许鉴定一个或多个候选基因， 因为候选基因和端粒之间的区域将被删除， 重组YAC被载体序列取代。 实验来 检测该基因座的功能涉及DNA从 将候选YAC或其粘粒亚克隆导入骨髓细胞， 将这些细胞重新引入小鼠体内，以确定 充满致密颗粒的血小板数量增加。 以确定是否 可以校正色素的稀释，转基因小鼠将通过 将相同的构建体注射到对于所述抗体纯合的受精卵中， 变种人

The goal of this project is to clone a gene that complements the mouse pallid mutant. This mutant is one of a group of eleven genetically distinct mutants affecting coat color and bleeding time. These effects are mediated through alterations in melanosomes and platelet dense granules, both of which are present, but defective in the mutant mice. Some of the mutants, including pallid, also have some effects on the function of a third organelle, the lysosome. Pallid is one of three mutants that affect the inner ear, and the mutants display ataxia because the otoliths, required for balance, are missing. Cloning the normal gene will allow analysis of the normal function of the gene in all parts of its complex phenotype, both at the molecular and cellular level, and analysis of the mutant phenotype. The proposed approach to cloning this gene involves identifying the molecular marker closest to the pallid mutation on mouse chromosome 2. This will be done using an interspecific backcross (C57BL/6J-pa a1/pa a1 X M. spretus-+ A/+ A)F1 X C57BL/6J-pa a1/pa a1, that has already produced 123 progeny. Preliminary data suggest that beta2-microglobulin is a very close marker. A YAC clone containing the selected marker will be obtained and used to start the walk to pallid. The ends of the YAC will be cloned and sequenced to identify overlapping YACs. Backcross animals containing recombination breakpoints will be identified and used first to determine the direction of the walk and later to monitor progress between the starting point and the target. YAC clones overlapping the interval between the recombination breakpoints flanking the pallid mutation will be candidates for the desired clones. To determine whether any of the genes on the YAC are expressed in megakaryocytes (platelet precursors) or in skin, cDNA libraries from skin and bone marrow cells will be made in a 'fragmenting vector' which contains a selectable yeast marker not present in the YAC and a yeast telomere. The libraries will be transformed into yeast cells containing the candidate YAC and recombinants between individual cDNAs and genes on the YAC will be selected. This will allow identification of one or more candidate genes, as regions between the candidate gene and the telomere will be deleted in the recombinant YACs, being replaced by vector sequences. Experiments to detect the function of the locus involve transformation of DNA from the candidate YAC or its cosmid subclones into bone marrow cells and reintroduction of these cells into mice to determine whether the fraction of platelets with filled dense granules is increased. To determine whether the pigment dilution can be corrected, transgenic mice will be made by injecting the same constructs into fertilized eggs homozygous for the mutant.