Studies on DNA-binding, transcriptional stimulation and role in cell proliferation of HMG proteins.

HMG 蛋白的 DNA 结合、转录刺激及其在细胞增殖中的作用的研究。

基本信息

  • 批准号:
    07680660
  • 负责人:
  • 金额:
    $ 1.47万
  • 依托单位:
  • 依托单位国家:
    日本
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
  • 财政年份:
    1995
  • 资助国家:
    日本
  • 起止时间:
    1995 至 1996
  • 项目状态:
    已结题

项目摘要

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.
采用凝胶阻滞法和BIAcore仪器测量表面等离子体共振,分析了HMG1和2蛋白中含有dna结合结构域的各种肽的dna结合能力。结果表明,第二DNA结合域的两个基本侧翼区域对于DNA的强结合和稳定都是必要的。HMG1第二DNA结合域的疏水残基和碱性残基的突变表明,这些氨基酸残基参与了疏水核的形成以及与DNA的复合物的形成。HMG的DNA弯曲和解绕功能区域也通过DNA连接和松弛试验确定。结果表明,第二dna结合域和侧翼碱基区是表达活性的重要区域。用核磁共振和x射线衍射研究了大肠杆菌细胞中表达和纯化的dna结合域的三级结构。HMG1刺激报告质粒中该基因的转录。分析了报告基因的转录刺激与染色质结构的关系。在HMG1或HMG2过表达细胞中,由报告质粒衍生的小染色体被DNaseI和微球菌核酸酶消化。虽然小染色体上核小体的位置没有差异,但HMG1过表达细胞的报告基因对核酸酶的敏感性高于HMG2过表达细胞。这些结果表明,HMG1的转录刺激可能是由于染色质结构的不稳定。分离并测序了大鼠HMG2基因的cDNA编码。其中一个根据其序列设计的反义寡核苷酸转染大鼠3Y1细胞后,可降低HMG2蛋白的细胞水平。根据HMG蛋白的减少,观察到细胞蛋白表达的明显变化。

项目成果

期刊论文数量(20)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Sobajima, J.: "Novel autoantigens of perinuclear anti-neutrophil cytoplasmic antibodies (P-ANCA) in ulcerative colitis : non-histone chromosomal proteins, HMG1 and HMG2." Clin.Exp.Immunol.107. 135-140 (1997)
Sobajima, J.:“溃疡性结肠炎中核周抗中性粒细胞胞浆抗体 (P-ANCA) 的新型自身抗原:非组蛋白染色体蛋白、HMG1 和 HMG2。”
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    0
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Ogawa, Y.: "Stimulation of transcription accompanying with relaxation of chromatin structure of cells over-expressing HMG1 protein." J.Biol.Chem.270. 9272-9280 (1995)
Okawa, Y.:“转录刺激伴随着过度表达 HMG1 蛋白的细胞染色质结构的松弛。”
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    0
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Shirakawa,H.: "Nuclear accumulation of HMG2 protein is mediated by basic regions interspaced with a long DNA-binding sequence,and retention within the nucleus requires the acidic carboxyl-terminus." Biochemistry. 36(in press). (1997)
Shirakawa, H.:“HMG2 蛋白的核积累是由长 DNA 结合序列间隔的碱性区域介导的,并且保留在细胞核内需要酸性羧基末端。”
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    0
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Shirakawa,H.: "Existence of a transcription factor for the human HMG2 gene positively related to the level of HMG2 mRNA in the cells." Biochemistry. 34. 2521-2527 (1995)
Shirakawa, H.:“人类 HMG2 基因转录因子的存在与细胞中 HMG2 mRNA 的水平呈正相关。”
  • DOI:
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    0
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  • 通讯作者:
Shirakawa, H.: "Existence of a transcription factor for human HMG2 gene." Biochemistry. 34. 2521-2527 (1995)
Shirakawa, H.:“人类 HMG2 基因转录因子的存在。”
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YOSHIDA Michiteru其他文献

YOSHIDA Michiteru的其他文献

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{{ truncateString('YOSHIDA Michiteru', 18)}}的其他基金

The functions and their mechanisms of DNA-binding proteins, HMG1 and HMG2
DNA结合蛋白HMG1和HMG2的功能及其机制
  • 批准号:
    09680601
  • 财政年份:
    1997
  • 资助金额:
    $ 1.47万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
STUDIES ON TRANSCRIPTION PROGRESSION FACTORS AND THE APPLICATION
转录进展因子的研究及应用
  • 批准号:
    03660096
  • 财政年份:
    1991
  • 资助金额:
    $ 1.47万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (C)
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