Michelle Miller

MAPPING THE MIA1 GENE IN CHLAMYDOMONAS REINHARDTII. Michelle Miller¹, Susan K. Dutcher², Biology Department, Washington University, St. Louis, MO¹; Genetics Department, Washington University Medical School, St. Louis, MO²

Chlamydomonas reinhardtii is a unicellular green alga that is biflagellate and can live under a wide range of conditions. With the entire genome sequenced, it is easy to study genetic factors underlying observed phenotypes, and it is important in medical research, especially for studying cellular motility, as many cells in the human body have cilia. Mutations affecting non-motile cilia in human cells have profound effects that can lead to polycystic kidney disease and vision loss. Mutations that affect motile cilia cause the syndrome primary cilia dyskinesia, and patients can have sterility, increased respiratory infections, and reversal of the asymmetrical organs like the heart and stomach. These patients often have defects in the dynein arms of their cilia. Chlamydomonas has ten dynein arm complexes and one of the nine inner dynein arms known as I1 or f is required for responding to directional light. Chlamydomonas regulates phototaxis, or reaction to light, both through response to environmental factors and genetic expression.

A screen for phototactic mutants conducted by King and Dutcher (1997) identified a strain with a modified inner dynein arm (mia1) in its flagella, which caused it to move much slower than wild-type cells, and they found that isolated flagella from mia1 lack both a 35 kD and a 74 kD protein. One of the subunits of the I1 inner dynein arm also showed hyperphosphorylation. These observations led to the hypothesis that the gene may encode a phosphatase or a negative regulator of a kinase. King mapped MIA1 to the second largest assembled contig, close to the ACT1 gene. Mutations in the ACT1 gene confer cycloheximide resistance, and I sequenced it and then used it as a marker. Different CAPS markers were designed around the MIA1 gene. To facilitate scoring the phenotype, the mia1 mutant was first crossed with oda7. ODA7 is a gene that affects the outer dynein arm of the flagella, and the double mutants are aflagellate. The mia1 oda7 mutants were then crossed with the polymorphic lab strain, S1-D2, so that there would be polymorphisms to score where recombination events may have occurred. Progeny exhibiting the mia1 oda7 phenotype (aflagellates) were selected from the screen and then scored with the approximately 25 CAPS markers. An additional cross of mia1 oda7 was performed with act1 to verify previously published data, which had indicated MIA1 was within 4 cM of ACT1. With the more than 175 tetrads analyzed this time, that distance appears closer to 10 cM. Recombinants flanking mia1 were identified. I have narrowed the region from 3 Mb to just 70 kb, which contains 11 possible gene candidates and is covered by two BACs.

I am currently performing transformations to see if the mutants can be rescued with one of these BACs. In addition, I will determine if any of the candidate genes are transcriptionally up regulated, which is evidence of a flagellar-localized protein. The goal is to identify a single gene and then sequence it to look for mutations. Then it can be further studied to aid in understanding the role of MIA1 in the control of the phosphorylation of the inner dynein arm and the processes of gene regulation, flagellar motility, and phototaxis.