Generation of antisera that discriminate among mammalian alpha-tubulins: introduction of specialized isotypes into cultured cells results in their coassembly without disruption of normal microtubule function.
Generation of antisera that discriminate among mammalian alpha-tubulins: introduction of specialized isotypes into cultured cells results in their coassembly without disruption of normal microtubule function.
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DOI：
10.1083/jcb.106.6.2011
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发表时间：
1988-06
期刊：
影响因子：
--
通讯作者：
Cowan NJ
中科院分区：
文献类型：
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作者：
Gu W;Lewis SA;Cowan NJ
为了测定在哺乳动物细胞中表达的多种但密切相关的α-微管蛋白多肽的功能意义，我们产生了三种特异性免疫血清，每种免疫血清独特地识别不同的α-微管蛋白同种型。所有三种同种型都以组织限制性方式表达：一种（M α 3/7）仅在成熟睾丸中表达，一种（M α 4）主要在肌肉和脑中表达，第三种（M α 6）在几种组织中表达水平很低。还产生了第四种特异性抗血清，其区分单个α-微管蛋白同种型的酪氨酸化和非酪氨酸化形式。由于个别微管蛋白同种型不能生化纯化，这些血清提出使用克隆的融合蛋白纯化宿主大肠杆菌细胞。为了抑制对共有表位的免疫应答，首先使动物耐受编码除一种以外的所有已知哺乳动物α-微管蛋白同种型的融合蛋白。随后用剩余的融合蛋白进行攻击，导致引发对独特表位的免疫应答。使用三个标准来确定所得血清的特异性：（a）它们区分代表所有已知哺乳动物α-微管蛋白同种型的克隆融合蛋白的能力;（B）它们独特地检测整个组织提取物中α-微管蛋白的能力;和（c）它们对用编码相应同种型的序列转染的细胞的固定制备物中微管染色的能力。转染实验用于证明（a）在HeLa细胞和NIH 3 T3细胞中M α 3/7、M α 4和M α 6共组装到间期和纺锤体微管中，和（B）M α 4同种型，其在哺乳动物α-微管蛋白中是独特的，因为它缺乏编码的羧基末端酪氨酸残基，就大多数培养细胞中发生的酪氨酸化/脱酪氨酸化周期而言，其表现类似于其它α-微管蛋白同种型。
To assay the functional significance of the multiple but closely related alpha-tubulin polypeptides that are expressed in mammalian cells, we generated three specific immune sera, each of which uniquely recognizes a distinct alpha-tubulin isotype. All three isotypes are expressed in a tissue-restricted manner: one (M alpha 3/7) only in mature testis, one (M alpha 4) mainly in muscle and brain, and the third (M alpha 6) in several tissues at a very low level. A fourth specific antiserum was also generated that distinguishes between the tyrosinated and nontyrosinated form of a single alpha-tubulin isotype. Because individual tubulin isotypes cannot be purified biochemically, these sera were raised using cloned fusion proteins purified from host Escherichia coli cells. To suppress the immune response to shared epitopes, animals were first rendered tolerant to fusion proteins encoding all but one of the known mammalian alpha-tubulin isotypes. Subsequent challenge with the remaining fusion protein then resulted in the elicitation of an immune response to unique epitopes. Three criteria were used to establish the specificity of the resulting sera: (a) their ability to discriminate among cloned fusion proteins representing all the known mammalian alpha-tubulin isotypes; (b) their ability to uniquely detect alpha-tubulin in whole extracts of tissues; and (c) their capacity to stain microtubules in fixed preparations of cells transfected with sequences encoding the corresponding isotype. The transfection experiments served to demonstrate (a) the coassembly of M alpha 3/7, M alpha 4, and M alpha 6 into both interphase and spindle microtubules in HeLa cells and NIH 3T3 cells, and (b) that the M alpha 4 isotype, which is unique among mammalian alpha-tubulins in that it lacks an encoded carboxy-terminal tyrosine residue, behaves like other alpha-tubulin isotypes with respect to the cycle of tyrosination/detyrosination that occurs in most cultured cells.