Mamma Mia!: Mapping the MIA1 gene in Chlamydomonas reinhardtii

Michelle Miller, Jessica M. Esparza, Susan K. Dutcher, Department of Genetics, Washington University School of Medicine

Chlamydomonas reinhardtii is a unicellular, biflagellate green alga that is extremely useful for studying a variety of functions, which include motility and phototaxis.  Chlamydomonas cells can sense light; they swim away from intense light and swim towards a moderate intensity light source.  There are two classes of mutants that affect phototaxis.  The first class responds to the intermediate intensity light signal aberrantly and swims away rather than towards light.  The DRM1 mutant shows this phenotype.  It encodes a quinone reductase.  I tested whether chemicals known to bind to quinone reductase affected the ability of wild-type cells to swim towards light.    The other class of mutants fails to orient towards light.  The mia1 mutant, which has a modified inner-arm, has two phenotypes.  It moves more slowly than wild-type cells and it cannot orient to light.  The MIA1 gene maps to linkage group II, close to the ACT1 gene (<5cM).  Mutations in the ACT1 gene confer cycloheximide resistance.  Cycloheximide resistance is conferred by mutations in ribosomal protein L27 in other microbial organisms.  To begin to positionally clone MIA1, I sequenced the R27a gene from the mutant act1 strain as an entry point.  There was a single base pair change after the stop codon in the sequence, which is likely to be just a polymorphism.  Chlamydomonas may acquire cycloheximide resistance by another means.  Four molecular markers were designed on linkage group II and the mia1 mutant was crossed with the polymorphic lab strain, S1D2.  Progeny exhibiting the Mia1 phenotype were selected and then thirty-two were scored with the markers to look for recombination to define a minimum region of interest.  Initial data suggests that the MIA1 gene is approximately six mu from the molecular marker 20341, which is about 250 kb from the ACT1 gene. Eventually, the location of the MIA1 gene will be more precisely determined by finding markers on both sides and moving in closer until there is a region with no recombination is found. By studying both the drm1 and the mia1 mutants, it will perhaps give rise to a better understanding of responses to light in Chlamydomonas.