Screen for Photoreceptor and Other Eyespot Defective Mutants in Chlamydomonas

Abstract

Chlamydomonas are single celled photosynthetic organisms that rely on their single eyespot to help them navigate towards light for use as an energy source. Their eyespot is crucial for their survival and as a result, the genetics behind the formation of the eyespot is regularly studied. To study and analyze several specific genes thought to be involved in eyespot function and formation, I collected phototaxis-defective mutants after mutagenic transformation of the arg7-2 auxotrophic strain with the ARG7 gene (arginine biosynthesis). Transformation of the Chlamydomonas genome was done by electroporation. The genes targeted in this study code for COP4 (photoreceptor), SOUL3 (affects eyespot size and placement), the MORN Repeat (attaches proteins to the membrane), MEC17 and MAP65 (affects tubulin dynamics), and the (PAP)-fibrillin domain (stabilizes pigment granule arrays) proteins. The ARG7 gene inserted into the Chlamydomonas genome randomly, and I intended to screen ultimately for strains with specific disruptions in these genes. The electroporation transformants were collected and analyzed using phototaxis techniques in order to find strains containing mutations that affect the functionality of the eyespot. Phototaxis-deficient mutants will be screened by PCR to identify those with disruptions in any of the targeted genes. Fluorescence and light microscopy techniques will be used to identify the mutant phenotypes and sequencing will determine the precise location of the gene insertion.