Missense mutations at lysine 350 in beta 2-tubulin confer altered sensitivity to microtubule inhibitors in Chlamydomonas.
Two beta-tubulin mutants of Chlamydomonas reinhardtii, colR4 and colR15, were previously isolated in our laboratory. Each mutant expressed an acidic beta-tubulin variant as a result of an alteration in the coding sequence of one of the two beta-tubulin genes in C. reinhardtii, which in the wild type encode identical proteins. In this report, we describe the identity of the specific beta-tubulin altered in the colR mutants and the precise nature of the genetic lesions. Hybrid selection of mutant poly(A)+ RNA with cDNA probes specific for the two beta-tubulins in C. reinhardtii indicated that both mutations resided in the beta 2-tubulin gene. cDNA libraries were constructed with mutant poly(A)+ RNA, and beta 2-tubulin cDNA clones were isolated. Results of in vitro transcription of cloned beta 2-tubulin cDNAs confirmed the identity of the altered genes. Sequencing of the entire coding regions of the beta 2-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 beta 2-tubulin sequence, effecting a change from a lysine to a glutamic acid in the colR4 variant and to a methionine in the colR15 variant. These changes in amino acids are consistent with the difference in the charge of the two variant polypeptides observed in isoelectric focusing. Because both the colR4 and colR15 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.
Lee
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VD
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Huang
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000187:drug effects* / Q000235:genetics; D003001:Cloning, Molecular; D003078:Colchicine / Q000494:pharmacology*; D004351:Drug Resistance / Q000235:genetics*; D015723:Gene Library; D008239:Lysine / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D014404:Tubulin / Q000235:genetics*
D014404:Tubulin; D008239:Lysine; D003078:Colchicine