| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2004: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
We aimed to identify and characterize nGTBP, a novel GT-mismatch binding protein that specifically binds to TRTRNB(R ; G or hypoxanthine mispaired with T) with a high affinity (Takata-Yahiro, M et al.(2003) : Tohoku J.Exp.Med.).
1. In order to determine a partial amino acid sequence of nGTBP by LC/MS/MS, the following procedure was tentatively established : 30Kg ultracentrifugation of nuclear extracts of HL60-c-15→Superose→heparin column ( 2 M KCl elution)→GC-match affinity column (flow through)→GT-mismatch affinity column. No protein bands specific to GT-mismatch probe were, however, found in SDS-PAGE. I, therefore, shifted my experiment to the molecular cloning of nGTBP.
2. A South-western screening was established using a 14-mer probe identical to bp-183〜-170 of CYBB with a GT-mismatch at-177. No definitively positive clones were, however, obtained from 3.6x10^7 clones, suggesting two possibilities ; one is poor quality of the library, and the other dull screening condition. The latter was overcome by using the high concentration of a probe of 3 x 20 mer identical to bp-187〜-168 with a GT-mismatch at-177) under the mild KCl concentration.
3. More than 6 x 10^7 clones were screened by using the method mentioned above, and were obtained 125 apparently positive clones. None of reproducible 75 clones was, however, specific to GT-mismatched probe.
4. All insert sequences of 15 reproducible clones had identical to the coding sequence for YB-1, a transcriptional protein, suggesting the screening procedure is sufficiently sensitive if non-specific binding of YB-1 is avoided. It may be done by using YB-1 specific DNA. Human cerebral library is now being screened.
5. Two-dimentional electrophoresis of the nuclear extract was successfully adapted to a South-western blotting, re-emerging the possibility of the partial amino acid sequence determination of nGTBP by LC/MS/MS.
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