Chlamydomonas reinhardtii tubulin-gene disruptants for efficient isolation of strains bearing tubulin mutations Article Swipe
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· 2020
· Open Access
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· DOI: https://doi.org/10.1371/journal.pone.0242694
· OA: W3109817910
The single-cell green alga Chlamydomonas reinhardtii possesses two α-tubulin genes ( tua1 and tua2 ) and two β-tubulin genes ( tub1 and tub2 ), with the two genes in each pair encoding identical amino acid sequences. Here, we screened an insertional library to establish eight disruptants with defective tua2 , tub1 , or tub2 expression. Most of the disruptants did not exhibit major defects in cell growth, flagellar length, or flagellar regeneration after amputation. Because few tubulin mutants of C . reinhardtii have been reported to date, we then used our disruptants, together with a tua1 disruptant obtained from the Chlamydomonas Library Project (CLiP), to isolate tubulin-mutants resistant to the anti-tubulin agents propyzamide (pronamide) or oryzalin. As a result of several trials, we obtained 8 strains bearing 7 different α-tubulin mutations and 12 strains bearing 7 different β-tubulin mutations. One of the mutations is at a residue similar to that of a mutation site known to confer drug resistance in human cancer cells. Some strains had the same amino acid substitutions as those reported previously in C . reinhardtii ; however, the mutants with single tubulin genes showed slightly stronger drug-resistance than the previous mutants that express the mutated tubulin in addition to the wild-type tubulin. Such increased drug-resistance may have facilitated sensitive detection of tubulin mutation. Single-tubulin-gene disruptants are thus an efficient background of generating tubulin mutants for the study of the structure–function relationship of tubulin.
