Altered Sensitivity to Microtubule lnhibitors in Chlam ydomonas

Two 0-tubulin mutants of Chlamydomonas reinhardtii, coP4 and coP75, were previously isolated in our laboratory. Each mutant expressed an acidic 8-tubulin variant as a result of an alteration in the coding sequence of one of the two p-tubulin genes in C. reinhardtii, which in the wild type encode identical proteins. In this report, we describe the identity of the specific 0-tubulin altered in the cop mutants and the precise nature of the genetic lesions. Hybrid selection of mutant poly(A)+ RNA with cDNA probes specific for the two p-tubulins in C. reinhardtii indicated that both mutations resided in the B2-tubulin gene. cDNA libraries were constructed with mutant poly(A)+ RNA, and 82tubulin cDNA clones were isolated. Results of in vitro transcription of cloned 02-tubulin cDNAs confirmed the identity of the altered genes. Sequencing of the entire coding regions of the B2-tubulin cDNA clones revealed that the mutants carried different single-base substitutions in the same codon for the amino acid at position 350 in the 82tubulin sequence, effecting a change from a lysine to a glutamic acid in the coP4 variant and to a methionine in the coP75 variant. These changes in amino acids are consistent with the difference in the charge of the two variant polypeptides obsewed in isoelectric focusing. Because both the coP4 and coP15 mutations confer an altered sensitivity to a number of different microtubule inhibitors and herbicides, lysine 350 appears to be of functional importance in the structure of the tubulin molecule.