| Project/Area Number |
59480137
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Institute of Medical Science, University of Tokyo, |
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Principal Investigator |
NAGATA Shigekazu Research Associate, Institute of Medical Science, University of Tokyo, 医科学研究所, 助手 (70114428)
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| Co-Investigator(Kenkyū-buntansha) |
KANEGASAKI Shiro Professor, Institute of Medical Science, University of Tokyo,, 医科学研究所, 教授 (10012767)
ASANO Shigetaka Assistant Professor, Institute of Medical Science, University of Tokyo,, 医科学研究所, 助教授 (50134614)
KAZIRO Yoshito Professor, Institute of Medical Science, University of Tokyo, 医科学研究所, 教授 (90012690)
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| Project Period (FY) |
1984 – 1986
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| Project Status |
Completed (Fiscal Year 1986)
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| Budget Amount *help |
¥6,300,000 (Direct Cost: ¥6,300,000)
Fiscal Year 1986: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1985: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1984: ¥3,900,000 (Direct Cost: ¥3,900,000)
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| Keywords | hemopoiesis / granulocyte colony stimulating factor / interferon- <gamma> / cDNA chromosomal gene / gene engineering / bovine papilloma virus / ミエロペルオキシダーゼ / 遺伝子工学 |
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| Research Abstract |
Proliferation, differentiation and activation of hematopoietic cells are regulated by several protein factors like interferons (IFNs) and colony stimulating factors (CSFs). Granulocyte colony stimulating factor (G-CSF) is a regulator for neutrophilic granulocytes, and during differentiation of granulocytes, some enzymes such as myeloperoxidase (MPO), are specifically induced. In this project, we have isolated cDNAs for human G-CSF and MPO. Human G-CSF was purified from the medium conditioned with human squamous carcinoma CHU-2 cells producing G-CSF constitutively, and the partial amino acid sequence of the G-CSF was determined. By using an oligonucleotide as probe, two different cDNAs for human G-CSF were isolated, each encoding 207 or 204 amino acids. Then, by using the human G-CSF cDNA as probe, cDNA for murine G-CSF and chromosomal genes for G-CSF were isolated. There is a single gene (-2.5 kb long) for G-CSF in human and mouse genome. Only in human gene, two splice donor sites are
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arranged in tandem at the 5' end of the 2nd intron, which suggests that two different mRNAs for human G-CSF are generated by an alternative splicing. Human and murine G-CSF have homologies of 69.3 % and 72.6 % on the nucleotide and amino acid sequence level, respectively.
The cDNA for human MPO was isolated from the cDNA library of HL-60 cells. The protein structure elucidated from the cDNA indicated that the MPO protein is synthesized as a precursor of Mr.84,000, and subsequent modification and cleavage result in the heavy chain (Mr. 55-60,000) and the light chain (Mr. 15,000).
The cDNAs for human IFN- <gamma> and G-CSF were placed under the control of SV40 early promoter, and introduced into mouse C127I cells using bovine papilloma virus as a vector. Some of the transformants could produce IFN- or G-CSF very efficiently ( 1-20 mg / 1 ) in a low serum medium, and each proteins was purified to homogeneity. They are indististiguishable physicochemically from the native proteins. When the recombinant G-CSF was subcutaneously administrated into mice, a remarkable granulopoiesis and splenomegaly were observed. Less
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