2000 Fiscal Year Final Research Report Summary
Development of DNA chips for ribosomal protein genes
| Project/Area Number |
11694300
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| Research Category |
Grant-in-Aid for Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General medical chemistry
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| Research Institution | University of the Ryukyus |
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Principal Investigator |
KENMOCHI Naoya University of the Ryukyus, Biochemistry, Instructor, 医学部, 助手 (00133124)
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| Co-Investigator(Kenkyū-buntansha) |
TANAKA Tatsuo University of the Ryukyus, Biochemistry, Professor, 医学部, 教授 (70018688)
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| Project Period (FY) |
1999 – 2000
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| Keywords | ribosome / DNA chip / human disease / ribosomal protein / microarray / gene expression / mutation screening |
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| Research Abstract |
In this study, we have developed DNA chips for human ribosomal protein (RP) genes, in collaboration with Dr.Metspalu at Tartu University in Estonia, to examine the possible involvement of RP gene defects in human disorders. We prepared following two types of DNA chips ; one for hybridization and the other for primer extension on the chip.
1. RPex (Ribosomal Protein gene expression) chip
PCR products (〜250 bp) of cDNAs for 40S ribosomal small subunit proteins were immobilized onto an epoxy-silanized glass surface. The gene expression at the mRNA level was measured by hybridization of fluorescently labeled DNA probes prepared from total mRNA of either wild-type HeLa cells or heat-shock HeLa cells. Although there were no significant differences of the RP gene expression in these two cells, we found the differences in RPS17, RPS18, RPS24, and RPS27 when the expression was compared between the HeLa and placental RNAs. This suggests that the changes of the RP gene expression level are involved in the carcinogenesis.
2. RPmu (Ribosomal Protein gene mutation) chip
Oligoncleotides (25 mer) specific to the small subunit protein genes were immobilized via 5'terminal amino group on an epoxy-silanized glass surface. After hybridization of target DNA to the array, target dependent oligonucleotide extension by a DNA polymerase is used to incorporate fluorescently labeled dideoxy terminators to the primers. More than 80% of the primers worked properly and precise single-base extensions were occurred on the array.
These two chips shotuld be useful for studying the RP gene defects or mutations in human disorders.
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