THE PLANT CELL, Vol 2, Issue 11 1051-1057, Copyright © 1990 by American Society of Plant Biologists
Missense Mutations at Lysine 350 in [beta]2-Tubulin Confer Altered Sensitivity to Microtubule Inhibitors in Chlamydomonas
V. D. Lee and B. Huang
Department of Molecular Biology, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, California 92037
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.
