| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1999: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1998: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
Gene 1 is one of the dna genes of Bacteriophage φ29, and its product (gp1) may regulate the expression of other dna genes ofφ29 through specific binding to mRNA. In this project, we tried to elucidate the structure and function of gp1.
For the structural analysis of proteins, the use of X-ray refraction of the crystals is a very powerful tool. Therefore, we started to purify a large amount of gp1 from E. coli cells after modifications including the addition of His-tag region, and trials of crystallization have been carried out. Though extensive conditions have been tested, the formation of crystal was not observed. Two possibilities which affected the formation of crystals were considered. One is the presence of His-tag. Therefore, we changed the DNA sequence to remove the His-tag region by protease. The other was the dispersion of gp1. By scattering experiments, addition of 2% CHAPS was found to be necessary for the single dispersion of gp1 at 20℃. By employing these improvements, more trials to crystallize gp1 will be done.
We have also tried to isolate mutants of gp1. The gene 1 region was randomly mutagenized by PCR in the presence of MgィイD1++ィエD1 ions After DNA sequencing, we have obtained 31 mutants of single amino acid substitution. All the sites of mutations dispersed among the coding region. These mutants will be useful for the structural and functional analysis.
For the localization of gp1 binding region on the polycistronic mRNA (target RNA of gp1), the mRNA was segmented into 18 regions. By using these segmented mRNAs, gp1 was shown to have a significantly high affinity to two overlapping segments, suggesting specific binding to the target. Since the binding regions located far down stream from the repressed genes, the repression mechanism may be different from simple operator-repressor binding.
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