A screen for Chlamydomonas insertional mutants unable to normally induce gene expression following deflagellation
Jason M. Brown, Danjuma X. Quarless, Karl F. Lechtreck and George B. Witman
Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA
Deflagellation of Chlamydomonas results in rapid and coordinate induction of hundreds of genes. The resulting new protein synthesis is required for regeneration of full-length flagella. Although Chlamydomonas is the preeminent model organism for the study of flagellar structure and function, the proteins required for deflagellation-stimulated gene induction are unknown. We have developed a reporter strain, LC8GLuc4, in which expression of a codon-optimized Gaussia princeps luciferase (Shao and Bock, 2008, Curr Genet) is driven by the promoter for dynein light chain LC8, an abundant flagellar protein upregulated by deflagellation. Deflagellation of LC8GLuc4 results in an increase in luciferase activity similar to increases in expression of genes encoding flagellar proteins. Induction is detectable using a standard luciferase assay within 15 minutes after deflagellation and peaks at ~10- to 20-fold by 60 minutes. The ease of the luciferase assay makes feasible high-throughput screening for insertional mutants in the deflagellation-stimulated gene-induction pathway. To this end, we developed a method for reproducible deflagellation and flagellar regeneration in a 96-well plate format. We mutagenized LC8GLuc4 by transformation with a fragment of pHyg3 (Berthold et. al., 2002, Protist). Two percent of hygromycin-resistant transformants showed a reproducible induction of only 2-fold or less. Since the assay depends on an active luciferase enzyme, some of these strains may be defective in proper luciferase folding or secretion. However, in some strains, decreased GLuc induction mirrored a decreased induction of LC8 mRNA, indicating a probable defect in normal deflagellation-stimulated signaling or transcription. Further characterization of these mutants is underway.
e-mail address of presenting author: Jason.Brown2@umassmed.edu