|Budget Amount *help
¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1998: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1997: ¥1,900,000 (Direct Cost: ¥1,900,000)
In this study, we try to isolate a novel gene for a sugar-transferase by differential display between a mutant HG794 defective in mucin-type carbohydrates and a wild type AX2. Differential display was carried out by using total RNA from the mutant strain HG794 and the wild type AX2 whose cells were allowed to develop for 3 h. Twenty kinds of primers in 5' end and four kinds of primers in 3' end were purchased as a RNAmapTM kit from GenHunter Co. Differential bands between the mutant HG794 and AX2 were identified to be negative in the mutant HG794 and to be positive in the wild type AX2.
Those bands were cut, extracted, amplified by PCR, and ligated into apBluescript II vector. As aresult, in combination of primers in 3' and 5' ends, two novel genes, band 1 and band 2, in T_<12>MT/AP1, one novel gene, band3, in T_<12>MG/AP11 and one novel gene, band7, in T_<12>MG/AP17, the gene for actin-bundling protein in T_<12>MA/AP5 and the gene for a phosphodiesterase inhibitor in T_<12>MN/AP5 and T_<12>MC/AP5 were identified and isolated. Four novel genes were thought to be a good candidate for asugar-trasferase gene. Therefore, each novel gene, bandi, 2, 3, 7 was used as a probe to isolate full lengh cDNAs, using cDNA library prepared newly from mRNAs of cells developed for 3 h.
A full length cDNA of 2,169 bp including a start codon and a stop codon, band3-3, was isolated by using probe 3. Also, a full length cDNA of 910 bp, band7-1, was isolated by using probe 7. In the case of probes 1 and 2, isolation of full length cDNAs is now in progress. The functional analysis for each gene will be done by isolating knockout mutants.