Increasing tubC beta-tubulin synthesis by placing it under the control of a benA beta-tubulin upstream sequence causes a reduction in benA beta-tubulin level but has no effect on microtubule function.

Abstract

We have constructed a chimeric beta-tubulin gene that places the structural gene for the tubC beta-tubulin of Aspergillus nidulans under the control of the benA beta-tubulin promoter. Introduction of either this chimeric gene or a second wild-type benA gene into a benomyl-resistant benA22 strain causes it to become benomyl sensitive, indicating that the introduced genes are functional. Analysis of the tubulin proteins synthesized in benA22 strains into which a second wild-type benA beta-tubulin gene was transformed showed that the total amount of beta-tubulin protein was the same as in the parental strain with a single benA gene. Thus the level of beta-tubulin must be regulated. This was also true of transformants carrying an extra copy of the chimeric beta-tubulin gene. The total amount of beta-tubulin was the same as in the parental strain. Two-dimensional gel analysis showed that the endogenous benA22 and the introduced chimeric tubC gene contributed equally to the total beta-tubulin pool. The fact that one-half of the benA beta-tubulin could be replaced by tubC beta-tubulin with no effect on the growth of the cells suggests that the benA and tubC beta-tubulins are functionally interchangeable.