Abstract
In the heterothallic green alga, Chlamydomonas reinhardtii, the MID gene in the minus cell is required for expression of genes specific to the mating type. However, the detailed molecular mechanism of how MID regulates sex determination is unknown. To identify MID-interacting factor(s), MID-FLAG construct was introduced into the MID-deletion mutant, and two transformants showed a partially rescued phenotype. Soluble proteins prepared from the transformants were applied to anti-FLAG affinity gel to obtain the MID-FLAG complex. MID-interacting protein candidates in the fraction are under investigation. To identify novel factors involved in sex determination, a luciferase-reporter assay system was established. Approximately 2.0-kb promoter region of SAD1, which is under the control of MID and active only in mt(-) gametes, was fused with the luciferase gene (SAD1p:LucNC), and introduced into mt(-) strain. Transformants that induced Luc activity specifically during gametogenesis were obtained, and used for mutagenesis by tagging with a hygromycin-resistant gene. Three out of 992 transformants showed 1/10 Luc activity and 1/3 gamete formation efficiency compared with the host cells.
