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首页> 外文学位 >Identification of novel Ras p21-interacting proteins using yeast two-hybrid analysis.
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Identification of novel Ras p21-interacting proteins using yeast two-hybrid analysis.

机译：使用酵母双杂交分析鉴定与Ras p21相互作用的新型蛋白。

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摘要

Ras proteins are encoded by a closely related set of genes, initially identified in acutely transforming sarcoma viruses and subsequently in human tumor cell lines. Ras genes are GTPases (guanosine triphosphatase hydrolases), which play a major role in signal transduction, cellular proliferation and transformation. Ras protein function is controlled by a guanosine triphosphate-guanosine diphosphate (GTP-GDP) cycle. The transforming Ras oncogenes are activated by point mutation, so that they encode proteins that are locked in active GTP-bound state. The immediate signaling targets of Ras proteins include not only the well-characterized Raf/mitogen activated protein (MAP) kinase cascade but also non-Raf involved pathways.;In the current study, the Ras DNA sequences (500bp) were utilized in a yeast two-hybrid system to identify unknown proteins interacting with Ras. The Yeastmaker(TM) yeast transformation system 2 (Clontech) allowed screening of a complete human cDNA library derived from normal pancreatic cells and cloned in the AD (activation domain) vector, for protein-protein interaction between the library proteins and Ras protein. First, the Ras DNA sequences were amplified from original pNdr-1r vector with cDNA specific primers and then the amplified Ras sequences were cloned within the pGBKT7 vector (BD). The Ras clones (Ras sequences in the BD vector) were transformed with yeast strain via EZ Transformation kit. The transformed yeast cell was subsequently grown and co-transformed with the cDNA library in AD vector via the Yeastmaker(TM) yeast transformation system 2 (Clontech). Future work involves the screening for protein-protein interactions and extraction of purified proteins from those clones for comparison with DNA and protein databases.;Further characterization of isolated Ras interacting proteins will certainly give new clues to determine the role of aberrant/normal Ras function in human tumorigenesis and will help define the mechanism by which Ras mediates its actions in normal and tumor cells. These novel proteins may also help develop antagonists of Ras as novel approaches for cancer treatment.
机译：Ras蛋白由一组紧密相关的基因编码，最初在急性转化的肉瘤病毒中发现，随后在人类肿瘤细胞系中发现。 Ras基因是GTPases（鸟苷三磷酸酶水解酶），在信号转导，细胞增殖和转化中起主要作用。 Ras蛋白功能受鸟苷三磷酸-鸟苷二磷酸（GTP-GDP）周期控制。转化的Ras癌基因通过点突变激活，因此它们编码的蛋白质被锁定在活跃的GTP结合状态。 Ras蛋白的直接信号转导靶点不仅包括特征明确的Raf /丝裂原活化蛋白（MAP）激酶级联反应，还包括非Raf参与的途径。；在当前的研究中，酵母中利用了Ras DNA序列（500bp）双杂交系统，以鉴定与Ras相互作用的未知蛋白质。 Yeastmaker TM酵母转化系统2（Clontech）允许筛选完整的人cDNA文库，该文库来自正常胰腺细胞并克隆到AD（激活域）载体中，用于文库蛋白质和Ras蛋白质之间的蛋白质相互作用。首先，使用cDNA特异性引物从原始pNdr-1r载体扩增Ras DNA序列，然后将扩增的Ras序列克隆到pGBKT7载体（BD）中。通过酵母转化菌株，通过EZ转化试剂盒转化Ras克隆（BD载体中的Ras序列）。随后使转化的酵母细胞生长，并通过YeastmakerTM酵母转化系统2（Clontech）与AD载体中的cDNA文库共转化。未来的工作包括筛选蛋白质与蛋白质之间的相互作用，并从这些克隆中提取纯化的蛋白质，以便与DNA和蛋白质数据库进行比较。对分离的Ras相互作用蛋白质的进一步表征无疑将为确定异常/正常Ras功能在体内的作用提供新的线索。人类肿瘤发生，将有助于确定Ras在正常细胞和肿瘤细胞中介导其作用的机制。这些新蛋白还可能有助于开发Ras拮抗剂，作为癌症治疗的新方法。

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  • 作者

    Sarfraz, Yasmeen.;

  • 作者单位

    Long Island University, The Brooklyn Center.;

  • 授予单位 Long Island University, The Brooklyn Center.;
  • 学科 Biology Molecular.
  • 学位 M.S.
  • 年度 2009
  • 页码 94 p.
  • 总页数 94
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分子遗传学;
  • 关键词

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