Reconstruction of Appropriate Tubulin and Actin Gene Regulation After Transient Transfection of Cloned β-Tubulin and β-Actin Genes

Abstract

Most animal cells rapidly depress the synthesis of new α- and β-tubulin polypeptides in response to microtubule inhibitors that increase the pool of depolymerized subunits. This apparent autoregulatory control of tubulin synthesis is achieved through the modulation of tubulin mRNA levels. To begin to analyze the molecular mechanism responsible for such regulation, we have introduced exogenous β-tubulin gene sequences into cultured mouse cells by DEAE-dextran-mediated DNA transfection. We find that the heterologous tubulin genes are expressed and that their RNA transcripts are accurately processed to mature mRNAs. Moreover, after drug-induced microtubule depolymerization, the expression of unintegrated tubulin gene sequences is regulated coordinately with the endogenous mouse α- and β-tubulin RNA transcripts. Such regulation appears to be specific for transfected tubulin genes, since similar down-regulation is not observed in a contransfected β-actin gene. Curiously, in response to microtubule depolymerization, the amount of RNA transcripts from a transfected β-actin gene increases twofold, which qualitatively and quantitatively parallels that seen by the RNAs encoded by the endogenous actin genes. Thus, the transient DNA transfection approach may permit the unambiguous elucidation of regulatory sequences involved in establishing the proper level of expression of these two important cytoskeletal gene families.