|Budget Amount *help
¥7,500,000 (Direct Cost : ¥7,500,000)
Fiscal Year 1996 : ¥1,900,000 (Direct Cost : ¥1,900,000)
Fiscal Year 1995 : ¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1994 : ¥3,600,000 (Direct Cost : ¥3,600,000)
We have studied about functions and modulation of expression of auxin-regulated genes, arcA,parB and parC to gain insight into the action mechanism of auxins at the molecular level. The initiation of expression of arcA,isolated from tobacco BY-2 cells, by the application of auxin precedes the induction of cell division. Sequence analysis revealed that arcA belongs to an expanding gene family, including the genes for beta subunits of G proteins. These proteins all have a series of internal repeats of 40 amino acids called the WD-40 repeat. Members of the WD-40 repeat family are involved in various cellular functions. Most known WD-40 proteins form multiprotein complexes, sometimes interacting with other proteins through the WD-40 repeat region. So we isolated several cDNA clones of arcA-interacting proteins using two-hybrid screen in yeasts and examined their direct binding capabilities with arcA products by the in vitro system. One of these cDNA clones had the extensive homology to a b
eta subunit of voltage-dependent K^+ channel well characterized in mammalian cells. The antibodies against arcA products revealed that arcA products accumulated at the maximum level 2 days after the subculture of BY-2, at which time the cells were most actively dividing.
We investigated auxin-responsive cis elements of parA,parB and parC from tobacco mesophyll protoplasts in transgenic tobaccos using GUS as a reporter gene. Detailed analysis including point mutation experiments showed that the interaction of separately located sequences was required for the auxin-responsiveness. Furthermore, different nuclear proteins bound to each of auxin-responsive elements of these genes. Although parA,parB and parC are all isolated from tobacco mesophyll protoplasts, auxin mediated activation of transcription depends on different cis and trans factors. These results suggest that various physiological effects of auxins are underlain by the composite molecular mechanisms of trascriptional regulation.