| Project/Area Number |
06807176
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Human genetics
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| Research Institution | Nagoya City University |
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Principal Investigator |
SUGIYAMA Kohachiro Nagoya City University, Medical School Assistant Professor, 医学部, 講師 (60117827)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1995: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1994: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | Pyrimidine metabolism / UMP synthase / Hereditary orotic aciduria / Bifunctional enzyme |
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| Research Abstract |
1. A lambdaEMBL-3 genomic DNA library was obtained from the Japanese Cancer Research Resources Bank. Approximately 6x10^6 phage clones were screened with the human UMP synthase cDNA.Six positive clones were digested with EcoRI or hindIII and mapped. Three clones overlapping one another and forming the longest continuous sequence describe a single-copy gene which spans approximately 15 kb. Ncleotide sequence data of the exon/intron boundaries show that all introns begin with a 5'GT and conclude with a 3'AG terminus. The 5' flanking region of human UMP synthase was also sequenced about 1.0kb upstream of the putative transcription initiation site. There were no consensus TATA or CAAT boxes. However, the region around and immediately 5'of the promotor region of house keeping genes.
2. The UMP synthase cDNA was cloned into pVL1392 baculovirus expression vector. Expression of the cDNA in Sf21 insect cells yielded a protein of approximately 52 kD and a significant rise in UMP synthase activity. The recombinant protein was purified to homogeneity using FPLC system. Approximately 4x10^8 cells were disrupted by sonication and the supernatant was applied to a hydroxylapatite column followed by an ion exchange chromatography on a MonoQ column. SDS polyacrylamide gel electrophoresis of the final product demonstrated a single band of 52 kD.
3. The T to G transversion causing a valine to glycine change at codon 109 was introduced to the UMP synthase cDNA.This mutation had been described in a hereditary orotic aciduria patient diagnosed at our institute. The expressed mutated UMP synthase by the baculovirus system demonstrated a marked reduction in UMP synthase activity.
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