Expression and function of beta-tubulin isotypes in Chinese hamster ovary cells.
The availability of isotype-specific antisera for beta-tubulin, coupled with genetic and biochemical analysis, has allowed the determination of beta-tubulin isotype expression and distribution in Chinese hamster ovary (CHO) cells. Using genetic manipulations involving selection for colcemid resistance followed by reversion and reselection for drug resistance, we have succeeded in isolating cell lines that exhibit three major and one minor beta-tubulin spots by two-dimensional gel electrophoresis. In concert with isotype-specific antibodies, analysis of these mutants demonstrates that CHO cells express two copies of isotype I, at least one copy of isotype IV, and very small amounts of isotype V. All three isotypes assemble into both cytoplasmic and spindle microtubules and are similar in their responses to cold, colcemid, and calcium-induced depolymerization. They have comparable turnover rates and are equally sensitive to depression of synthesis upon colchicine treatment. These results suggest that beta-tubulin isotypes are used interchangeably to assemble microtubule structures in CHO cells. However, of 18 colcemid-resistant mutants with a demonstrable alteration in beta-tubulin, all were found to have the alteration in isotype I, thus leaving open the possibility that subtle differences in isotype properties may exist.
Sawada
T
T
Department of Pharmacology, University of Texas Medical School, Houston 77225.
Cabral
F
F
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D008870:Microtubules / Q000648:ultrastructure; D009154:Mutation; D010053:Ovary; D008941:Spindle Apparatus / Q000648:ultrastructure; D014404:Tubulin / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology
D014404:Tubulin