A MICROTUBULE-ASSOCIATED PROTEIN IN DROSOPHILA-MELANOGASTER - IDENTIFICATION, CHARACTERIZATION, AND ISOLATION OF CODING SEQUENCES
A MICROTUBULE-ASSOCIATED PROTEIN IN DROSOPHILA-MELANOGASTER - IDENTIFICATION, CHARACTERIZATION, AND ISOLATION OF CODING SEQUENCES
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DOI：
10.1083/jcb.102.6.2076
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发表时间：
1986-06-01
影响因子：
7.8
通讯作者：
MCINTOSH, JR
中科院分区：
文献类型：
--
作者：
GOLDSTEIN, LSB;LAYMON, RA;MCINTOSH, JR
通过紫杉醇依赖性聚合程序，从黑腹果蝇的培养细胞中分离出微管和微管相关蛋白（MAP）。主要的 MAP 是一组具有相似电泳迁移率的四种多肽，对应于 .apprx。 205,000 先生（205K MAP）。这些蛋白质能够抵抗煮沸沉淀。制备了一种针对 Mr 205,000 MAP 的小鼠单克隆抗体和一种多克隆兔抗血清，并通过免疫印迹和间接免疫荧光进行了表征。两种抗体制剂均对果蝇细胞匀浆免疫印迹中的 Mr 205,000 分子和 Mr 255,000 分子进行染色；兔子抗血清也对 150,000 先生的三胞胎染色。两种制剂均对有丝分裂纺锤体的微管进行染色，兔抗血清也对细胞质微管进行染色。使用亲和纯化的兔抗血清进行的实验表明，Mr 205,000 物种位于有丝分裂器和细胞质微管上。在表达载体 lambda gt11 中产生随机剪切基因组文库，并用兔抗血清筛选以分离编码这些多肽的 DNA 序列。回收了几个交叉杂交克隆，显示其编码 Mr 205,000 MAP 中的抗原决定簇，并通过与 mRNA 的 Northern 印迹和基因组 DNA 的 Southern 印迹杂交来表征。原位杂交分析表明，编码205K MAP的基因位于多线区100EF。
Microtubules and microtubule-associated proteins (MAPs) have been isolated from cultured cells of Drosophila melanogaster by a taxol-dependent polymerization procedure. The principal MAPs are a group of four polypeptides with similar electrophoretic mobilities corresponding to .apprx. Mr 205,000 (the 205K MAP). These proteins are resistant to precipitation by boiling. One mouse monoclonal antibody and one polyclonal rabbit antiserum specific for the Mr 205,000 MAP were produced and characterized by immunoblotting and indirect immunofluorescence. Both antibody preparations stain the Mr 205,000 molecules and an Mr 255,000 molecule in immunoblots of Drosophila cell homogenates; the rabbit antiserum also stains an Mr 150,000 triplet. Both preparations stain the microtubules of the mitotic spindle, and the rabbit antiserum stains the cytoplasmic microtubules as well. Experiments using affinity-purified rabbit antiserum demonstrate that it is the Mr 205,000 species that is located in the mitotic apparatus and on cytoplasmic microtubules. A random shear genomic library was produced in the expressing vector lambda gt11 and screened with the rabbit antiserum to isolate the DNA sequences encoding these polypeptides. Several cross-hybridizing clones were recovered, shown to encode antigenic determinants in the Mr 205,000 MAP, and characterized by hybridization to Northern blots of mRNA and Southern blots of genomic DNA. Analysis by in situ hybridization reveals that the gene encoding the 205K MAP is located in polytene region 100EF.