Isolation And Characterization Mutants Defective In Cilia Regeneration In Chlamydomonas reindhartii

Abstract

Cilia and flagella are identical brush-like organelles found on the surfaces of eukaryotic cells and are involved in motility, sensing, and signaling. Defects in cilia assembly or function lead to polycystic kidney disease, congenital heart disease, Bardet Biedl syndrome, and other emerging ciliopathies. In Chlamydomonas reinhardtii, one of the most well-studied flagella model organisms, regeneration of flagella to normal length and normal function occurs within 90 minutes of acid-shock deflagellation. During this process hundreds of genes are induced. Much is left to be determined about how cells regulate expression of these genes: How do cells detect the presence or absence of cilia? How do cells send an ‘absence of cilia’ signal to the nucleus? How are the hundreds of genes encoding cilia proteins coordinated with each other? Previous experiments found that cells that failed to upregulate a reporter of flagella gene expression also had a delay in flagella regeneration. The goals of this experiment were to generate new C. reinhardtii insertional mutants defective in cilia regeneration, to identify the mutated genes in these strains, and ultimately, to identify transcription factors and signaling components needed for flagella assembly. 3000 hygromycin-resistant colonies were generated by the insertion of aph7” DNA fragment through electroporation. 42 of the 3000 colonies exhibited defective flagella structure, defective motility, or delayed flagella regeneration; 14 of these 42 mutants had delay in regenerating their flagella. To identify the mutated genes, genomic DNA for a subset of mutant strains was extracted, and Polymerase Chain Reaction (PCR) reactions were set up using the Restriction Enzyme Site Directed Amplification polymerase chain reaction (RESDA PCR) protocol with primers specific for the aph7” insert combined with four different degenerate primers. DNA fragments from successful PCR reactions were gel purified and sequenced. Using this method, mutated genes were identified and characterized to identify the proteins encoded by these genes. Flagella regeneration and reporter gene assays were conducted to further characterize the phenotypes of two of the mutants originally identified as having a delay.