GENETIC APPROACH STUDY OF PIGMENT GRANULE TRANSPORT

分子遗传学的发展部分的成员正在使用经典的小鼠毛色突变[稀释（d），灰（ash），铅（ln）]，以及反向遗传学，以分子解剖的生化途径（s）重要的色素颗粒运输。d、ash和ln突变由于色素颗粒运输缺陷而导致毛色变浅，而d也由于小脑浦肯野细胞中的内质网（ER）运输缺陷而导致致命的神经系统疾病。 以前，我们发现这三个突变被另一个突变[稀释抑制因子（dsu）]抑制，并提供证据表明这三个突变在一个共同的生化途径中起作用。 我们还发现dsu可以抑制眼睛颜色，但不能抑制与另外两个突变相关的被毛颜色变淡[ruby-eye（ru）和ruby-eye-2（ru 2）]。 此外，我们发现，d编码非常规肌球蛋白VA（MyoVA），一个主要的细胞囊泡运输马达，而其他人表明，人类MYOVA突变产生Griscelli病，一种罕见的常染色体隐性遗传疾病，其特征是色素稀释，可变的细胞免疫缺陷，神经系统疾病，以及急性期不受控制的淋巴细胞和巨噬细胞活化。 在最近的研究中，我们使用基于RT-PCR的测序来鉴定17个可行的稀释等位基因的突变，并使用酵母双杂交试验来鉴定与MyoVA相互作用的蛋白质。 这些研究确定了蛋白质的重要功能结构域，并为选择性剪接产生的不同MyoVA同种型编码重要的细胞类型特异性功能的概念提供了支持。 这些研究还表明，MyoVA可以结合一个主要的细胞微管囊泡运输马达，重链中的遍在因子（KhcU），并首次表明，囊泡运输可以通过不同马达分子的直接相互作用在细胞中协调。 未来的研究旨在利用定位克隆来鉴定由其他毛色突变编码的基因产物，然后确定它们在细胞中的功能。

Members of the Molecular Genetics of Development Section are using classical mouse coat-color mutations [dilute (d), ashen (ash), leaden (ln)], as well as reverse genetics, to molecularly dissect the biochemical pathway(s) important for pigment granule transport. The d, ash, and ln mutations cause a lightening of coat color due to defects in pigment granule transport, while d also causes a fatal neurological disease due to endoplasmic reticulum (ER) transport defects in cerebellar Purkinje cells. Previously, we showed that these three mutations are suppressed by another mutation [dilute suppressor (dsu)] and provided evidence suggesting that all three mutations function in a common biochemical pathway. We also showed that dsu can suppress the eye color but not the diluted coat color associated with two other mutations [ruby-eye (ru) and ruby-eye-2 (ru2)]. In addition, we showed that d encodes unconventional myosin VA (MyoVA), a major cellular vesicle transport motor, while others showed that human MYOVA mutations produce Griscelli disease, a rare autosomal recessive disorder characterized by pigment dilution, variable cellular immunodeficiency, neurological disorders, and acute phases of uncontrolled lymphocyte and macrophage activation. In more recent studies, we used RT-PCR-based sequencing to identify the mutations responsible for 17 viable dilute alleles and yeast two-hybrid assays to identify proteins that interact with MyoVA. These studies identified important functional domains of the protein and provided support for the notion that the different MyoVA isoforms produced by alternative splicing encode important cell-type-specific functions. These studies also showed that MyoVA can bind a major cellular microtubule vesicle transport motor, ubiquitous kines in heavy chain (KhcU), and suggested for the first time that vesicle transport can be coordinated in the cell via the direct interaction of the different motor molecules. Future studies are aimed at using positional cloning to identify the gene products encoded by the other coat-color mutations and then to determine how they function in the cell.