| Budget Amount *help |
¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1992: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1991: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
1. Determination of the amount of mRNA for nucleotide pyrophosphatase(NPPase)----The amounts of the mRNAs in fibroblasts of normal individuals and patients with Lowe's syndrome were determined by Northern hybridization. The cells from most of the patients had a large amount of the mRNAs compatible with their elevated NPPase activities. However, the cells from several patients contained a small amount of the mRNA in spite of their elevated NPPase activities.
2. Chromosomal assignment of human NPPase gene----Chromosomal localization was accomplished by somatic cell hybrid analysis and in situ hybridization with a fragment from cDNA encoding human NPPase. The gene was mapped on 6q22-6q23. Lowe's syndrome is an X-linked inherited disease. Therefore, the result shows that the disease is not caused by mutation of NPPase gene.
3. Cloning of human NPPase gene----Genomic libraries were constructed by inserting fragments of genomic DNA to EMBL, pWE15 or SacB II. Genomic clones coverring most of the full-length cDNA sequence for the NPPase have been isolated with probes from cDNA for human NPPase. However, no genomic clones containing 5'-noncoding region and/or 5'-flanking region have been obtained from any of these libraries.
4. Expression of cloned gene for human NPPase in cultured CHO cells----Full-length cDNA for human NPPase was constructed from two truncated cDNAs and inserted into an expression vector pMSG, and then the recombinant vector was introduced into CHO cells. By cloning of the transfected cells, several clones expressing high NPPase activity were isolated. The activity was increased several times more in the transfected cells cultured in the presence of dexamethazone, suggesting that the activity is due to the NPPase expressed from human NPPase cDNA in CHO cell. The NPPase expressed in CHO cells had molecular weight of 130,000 and reacted with the antibodies raised against human NPPase.
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