|Budget Amount *help
¥3,300,000 (Direct Cost : ¥3,300,000)
Fiscal Year 1999 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Fiscal Year 1998 : ¥1,900,000 (Direct Cost : ¥1,900,000)
This study aimed at 1) cloning and sequencing of cDNAs for the maize proteins that cross-react with anti-Cdc42 antibody and 2) systematic isolation of Arabidopsis mutants defective in cortical microtubule function. Cdc42 is a small GTPase of the rho sub-family, found in many eukaryotes from yeast to human, and is known to be involved in the regulation of cytoskeleton and the activation of MAP kinase cascade. Previously we found that the antibody directed for C-terminal oligo-peptide of human Cdc42 recognized ca.50 kDa and ca.25 kDa proteins in cell extracts of maize and tobacco plants, and the antibody labeled microtubular structures as well as cytosol. Here, we report the cloning of cDNA clones by immunoscreening of λ expression library of maize cDNA and the DNA sequencing of the clones. The DNA sequence of one clone contained an ORF coding for a 57 kDa protein with a homology to members of the copper-binding oxidase family. It is further needed to confirm this protein's localization on microtubules. In the second project, we first tried to establish the efficient method for screening of temperature-sensitive mutants, because strong mutant alleles of microtubule-related genes are expected to be lethal. By cycling the culture temperature, 15-23-31-23℃, we could observe many candidates for mutant plants with various morphological abnormalities, as well as subsequent recovery of their seeds after the growth at 23℃, a Arabidopsis permissive temperature. Using this temperature-cycling method as the first step of screening, the second step screening by microscopic observation of microtubules will be carried out.