| Project/Area Number |
06670761
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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 |
Pediatrics
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| Research Institution | TOHOKU UNIVERSIT,SCHOOL OF MEDICINE |
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Principal Investigator |
SUZUKI Yoichi TOHOKU UNIV., School of Medical, Departerment of BIOCHEMICAL GENETICS.ASSISTANT, 医学部, 助手 (80216457)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUBARA Yoichi TOHOKU UNIV., School of Medical, Departerment of BIOCHEMICAL GENETICS,ASSOCIATE, 医学部, 助教授 (00209602)
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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: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1994: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | HOLOCARBOXYLASE SYNTHETASE / BIOTIN / MAPPING / cDNA CLONING / MULTIPLE CARBOXYLASE SYNTHETASE |
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| Research Abstract |
Using human HCScDNA as a probe, we isolated a clone coding for human holocarboxylase synthetase (HCS) gene from pWEX15 cosmid library. Fluorescence in situ hybridization analysis with this clone as a probe showed that the human HCS gene was located at band q22.1 of chromosome 21. This result was consistent with PCR anaysis of DNA panel of human-hamster hybrid cells. In addition, Southern blot analysis of genomic DNA digested with each of four restriction enzymes gave single band on hybridization with 111 bp fragment of HCScDNA.Taken these together, we concluded that the human genome contains one HCS gene per haploid. When we digested the isolated clone with Not I,the insert was divided into 12.5,9.4,4.5 kb fragments. Mapping by a partial digestion methed was carried out for Bgl II,Ecor I,Hind III,and Spe I restriction sites. Analysis of exon/intron boundary is under way.
The first mutations found in human HCS gene were a missense mutation and a one base deletion. We further analyzed the HCScDNA in 4 unrelated Japanese families and found that these two mutations accounted for 7 of 8 mutant alleles.
We developed two methods for the diagnosis of HCS deficiency. One is a method for DNA diagnosis based on restriction-site generation PCR which detects the two identified mutations in small amount of blood samples. The other is a method for detection of HCS activity in cultured cells using apo-carboxy1 carrier protein of E.coli as a substrate. The sensitivity of this assay was so high that we were able to characterize a mutant enzyme whose activity had not been previously detected. These new methods will enable us to diagnose HCS deficiency faster and more accurately than we did with previous methods.
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