GENETIC APPROACH STUDY OF PIGMENT GRANULE TRANSPORT

分子遗传学发育组的成员正在使用经典的小鼠毛色突变[稀释(d)，灰白（ash），铅色（ln）]以及反向遗传学，从分子上剖析色素颗粒运输的重要生化途径。d、ash和ln突变由于色素颗粒运输缺陷导致毛色变浅，而d也由于小脑浦肯野细胞内质网（ER）运输缺陷导致致命的神经系统疾病。先前，我们发现这三种突变被另一种突变[稀释抑制因子（dsu）]抑制，并提供证据表明这三种突变在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，但不能抑制与其他两个突变[红宝石眼（ru）和红宝石眼2 (ru2)]相关的稀释毛色。此外，我们发现d编码非常规的肌球蛋白VA (MyoVA)，这是一种主要的细胞囊泡运输马达，而其他人则发现人类MyoVA突变会产生Griscelli病，这是一种罕见的常染色体隐性遗传病，其特征是色素稀释、可变细胞免疫缺陷、神经系统疾病以及淋巴细胞和巨噬细胞激活失控的急性期。在最近的研究中，我们使用基于rt - pcr的测序方法鉴定了17个活性稀释等位基因的突变，并使用酵母双杂交测定法鉴定了与MyoVA相互作用的蛋白质。这些研究确定了该蛋白的重要功能域，并支持了由不同剪接产生的不同MyoVA亚型编码重要的细胞类型特异性功能的观点。这些研究还表明，MyoVA可以结合细胞微管囊泡运输马达——重链泛在激酶（ubiquitous kines in heavy chain, KhcU），并首次表明细胞内的囊泡运输可以通过不同马达分子的直接相互作用来协调。未来的研究目标是利用定位克隆来鉴定其他毛色突变所编码的基因产物，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.