1996 Fiscal Year Final Research Report Summary
Studies on DNA-binding, transcriptional stimulation and role in cell proliferation of HMG proteins.
| Project/Area Number |
07680660
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | Science University of Tokyo |
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Principal Investigator |
YOSHIDA Michiteru Science University of Tokyo, Department of Biological Science & Technology, Professor, 基礎工学部, 教授 (20005648)
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| Co-Investigator(Kenkyū-buntansha) |
SHIRAKAWA Hitoshi Science University of Tokyo, Department of Biological Science & Technology, Rese, 基礎工学部, 助手 (40206280)
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| Project Period (FY) |
1995 – 1996
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| Keywords | HMG1 protein / stimulation of transcription / DNA-binding protein / chromatin / nucleosome / surface plasmon resonance / chromosome / protein engineering |
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| Research Abstract |
DNA-binding abilities of the various peptides containing DNA-binding domain (s) in HMG1 and 2 proteins were analyzed using gel retardation assay and the measurement of surface plasmon resonance with a BIAcore instrument. The results showed the necessity of both the basic flanking regions of the second DNA-binding domain for strong binding and stabilization of DNA.The mutation of the hydrophobic and basic residues in second HMG1 DNA-binding domain indicated the involvements of the amino acid residues in the formation of hydrophobic core and of complex with DNA.The functional region for DNA-bending and unwinding activities of HMG was also identified by DNA ligation and relaxation assays. The results showed that the second DNA-binding domain and the flanking basic region are important to express the activities.
The tertiary structure of DNA-binding domains expressed in E.coli cells and purified in homogeneity is under investigation with NMR and X-ray diffraction.
HMG1 stimulates the transcription of the gene in a reporter plasmid. The relations between the transcriptional stimulation and chromatin structure of the reporter gene were analyzed. Minichromosomes derived from the reporter plasmid in HMG1 or HMG2 over-expression cells were digested by DNaseI and microccocal nuclease. Although the respective nucleosomal positions on minichromosomes were not different, the reporter gene in HMG1 over-expression cells was more sensitive to nucleases than that in HMG2 over-expression cells. These results suggested that the transcriptional stimulation by HMG1 may be due to the destabilization of the chromatin structure.
The cDNA coding for rat HMG2 was isolated and sequenced. One of anti-sense oligonucleotides designed on the basis of its sequence and transfected into rat 3Y1 cells reduced the cellular level of HMG2 protein. Apparent changes of expression of cellular proteins were observed according to the reduction of the HMG protein.
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