Bazinet C, Katzen A L, Morgan M, Mahowald A P, Lemmon S K
Department of Genetics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Genetics. 1993 Aug;134(4):1119-34. doi: 10.1093/genetics/134.4.1119.
The clathrin heavy chain (HC) is the major structural polypeptide of the cytoplasmic surface lattice of clathrin-coated pits and vesicles. As a genetic approach to understanding the role of clathrin in cellular morphogenesis and developmental signal transduction, a clathrin heavy chain (Chc) gene of Drosophila melanogaster has been identified by a combination of molecular and classical genetic approaches. Using degenerate primers based on mammalian and yeast clathrin HC sequences, a small fragment of the HC gene was amplified from genomic Drosophila DNA by the polymerase chain reaction. Genomic and cDNA clones from phage libraries were isolated and analyzed using this fragment as a probe. The amino acid sequence of the Drosophila clathrin HC deduced from cDNA sequences is 80%, 57% and 49% identical, respectively, with the mammalian, Dictyostelium and yeast HCs. Hybridization in situ to larval polytene chromosomes revealed a single Chc locus at position 13F2 on the X chromosome. A 13-kb genomic Drosophila fragment including the Chc transcription unit was reintroduced into the Drosophila genome via P element-mediated germline transformation. This DNA complemented a group of EMS-induced lethal mutations mapping to the same region of the X chromosome, thus identifying the Chc complementation group. Mutant individuals homozygous or hemizygous for the Chc1, Chc2 or Chc3 alleles developed to a late stage of embryogenesis, but failed to hatch to the first larval stage. A fourth allele, Chc4, exhibited polyphasic lethality, with a significant number of homozygous and hemizygous offspring surviving to adulthood. Germline clonal analysis of Chc mutant alleles indicated that the three tight lethal alleles were autonomous cell-lethal mutations in the female germline. In contrast, Chc4 germline clones were viable at a rate comparable to wild type, giving rise to viable adult progeny. However, hemizygous Chc4 males were invariably sterile. The sterility was efficiently rescued by an autosomal copy of the wild-type Chc gene reintroduced on a P element. These findings suggest a specialized role for clathrin in spermatogenesis.
网格蛋白重链（HC）是网格蛋白包被小窝和小泡细胞质表面晶格的主要结构多肽。作为一种理解网格蛋白在细胞形态发生和发育信号转导中作用的遗传学方法，通过分子遗传学和经典遗传学方法相结合，鉴定出了黑腹果蝇的网格蛋白重链（Chc）基因。基于哺乳动物和酵母网格蛋白HC序列设计简并引物，通过聚合酶链式反应从果蝇基因组DNA中扩增出HC基因的一个小片段。以该片段为探针，从噬菌体文库中分离并分析基因组和cDNA克隆。从cDNA序列推导的果蝇网格蛋白HC的氨基酸序列与哺乳动物、盘基网柄菌和酵母的HC分别有80%、57%和49%的同源性。原位杂交到幼虫多线染色体显示，在X染色体的13F2位置有一个单一的Chc基因座。一个包含Chc转录单元的13kb果蝇基因组片段通过P因子介导的种系转化重新导入果蝇基因组。该DNA补充了一组映射到X染色体同一区域的EMS诱导致死突变，从而确定了Chc互补群。Chc1、Chc2或Chc3等位基因的纯合或半合突变个体发育到胚胎发生后期，但未能孵化到第一幼虫期。第四个等位基因Chc4表现出多相致死性，大量纯合和半合后代存活至成年。Chc突变等位基因的种系克隆分析表明，这三个紧密致死等位基因是雌性种系中的自主细胞致死突变。相比之下，Chc4种系克隆以与野生型相当的比率存活，产生可存活的成年后代。然而，半合的Chc4雄性总是不育的。野生型Chc基因的常染色体拷贝通过P因子重新导入可有效挽救不育。这些发现表明网格蛋白在精子发生中具有特殊作用。
