Transfer and amplification of a mutant beta-tubulin gene results in colcemid dependence: use of the transformant to demonstrate regulation of beta-tubulin subunit levels by protein degradation.
Total genomic DNA from a temperature-sensitive, colcemid-resistant Chinese hamster ovary (CHO) cell mutant expressing an electrophoretic variant beta-tubulin was used to transform wild-type CHO cells to colcemid-resistant cells at 37 degrees C. Southern blot analysis of the transformant demonstrated the three- to fivefold amplification of one of many beta-tubulin sequences compared with that of the wild type or mutant, thereby identifying a functional tubulin gene in CHO cells. This amplification of one tubulin-coding sequence resulted in a threefold increase in two beta-tubulin mRNA species, suggesting that both species may be encoded by a single gene. Pulse-chase experiments showed that in the transformant, total beta-tubulin was synthesized and degraded faster than in the revertant or wild-type cells, so that the steady-state levels of beta-tubulin and alpha-tubulin were unchanged in the transformant compared with those of wild-type, mutant, or revertant cells. Increased ratios of mutant to wild-type beta-tubulin made the transformant dependent on microtubule-depolymerizing drugs for growth at 37 but not 34 degrees C and supersensitive to the microtubule-stabilizing drug taxol at 34 degrees C.
D000818:Animals; D002455:Cell Division; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D003703:Demecolcine / Q000494:pharmacology*; D005260:Female; D005784:Gene Amplification; D005796:Genes / Q000187:drug effects; D005809:Genes, Regulator / Q000187:drug effects; D046911:Macromolecular Substances; D009154:Mutation; D010053:Ovary; D012333:RNA, Messenger / Q000235:genetics; D014404:Tubulin / Q000235:genetics* / Q000302:isolation & purification
D046911:Macromolecular Substances; D012333:RNA, Messenger; D014404:Tubulin; D003703:Demecolcine