Identification of the fla4 Mutation in Chlamydomonas reinhardtii

Abstract

The unicellular green alga Chlamydomonas reinhardtii is commonly used as a model organism to study flagella and their associated proteins. The fla4 mutant exhibits normal flagellar function at a permissive temperature of 21C, but sheds its flagella at a restrictive temperature of 32C. Transformation with large insert BAC clones had previously identified a candidate FLA4 gene. I confirmed the identity of the FLA4 gene by transformation with a smaller subclone to rescue the mutant phenotype. The flagella of two transformed strains functioned normally at the restrictive temperature. Using reverse transcriptase-polymerase chain reactions (RT-PCR), gel electrophoresis, and sequence comparisons between wild-type and fla4 DNA, I determined the complementary DNA (cDNA) sequence of the wild-type FLA4 gene and identified the specific mutation in fla4. The predicted amino acid sequence corresponds to a conserved TPR repeat protein. This protein shows significant similarity to proteins found in many higher organisms, such as human and mouse. In animals, this protein has been associated with defects in nervous system development, but its cellular function is unknown. Further study of its function in C. reinhardtii may provide insight into its role in higher organisms.