The DNA-binding activity of heat shocktranscription factor (HSF) was induced by heat shock (HS) of a whole cell extract. Addition of antiserum, specific toCaM, to a whole cell extract reduced bind of the HSF to the he...The DNA-binding activity of heat shocktranscription factor (HSF) was induced by heat shock (HS) of a whole cell extract. Addition of antiserum, specific toCaM, to a whole cell extract reduced bind of the HSF to the heat shock element (HSE) with maize, and the re-addition of CaM to the sample restored the activity of the HSF forbinding to HSE. In addition, DNA-binding activity of the HSF was also induced by directly adding CaM to a whole cell extract at non-HS temperature with maize. Similar resultswere obtained with wheat and tomato. Our observationprovide the first example of the involvement of CaM inregulation of the DNA-binding activity of the HSF.展开更多
Dear Editor, The LATERAL ORGAN BOUNDARIES DOMAIN （LBD）/ ASYMMETRICLEAVES2-LIKE （ASL） genes （hereafter referred to as LBD） encode proteins containing a conserved plant-specific LOB domain and play roles in later...Dear Editor, The LATERAL ORGAN BOUNDARIES DOMAIN （LBD）/ ASYMMETRICLEAVES2-LIKE （ASL） genes （hereafter referred to as LBD） encode proteins containing a conserved plant-specific LOB domain and play roles in lateral organ development （Iwakawa et al., 2002; Shuai et al., 2002; Majer and Hochholdinger, 2011）. The LOB domain is approximately 100 amino acids in length and contains a conserved four-Cys motif （CX2CX6CX3C）, the Gly-Ala-Ser block （GAS）, and the leucine-zipper-like coiled-coil motif （LX6LX3LX6L） （Shuai et al., 2002）. The leucine-zipper-like coiled-coil motif in the LOB domain is predicted to function in protein dimerization. LOB, AS2, and LBD4 preferentially bind unique DNA sequences in electrophoretic mobility shift assays （EMSAs） （Husbands et al., 2007）. The LOB domain of AS2 cannot be functionally replaced by those of other members of the LOB family,展开更多
猪肺炎支原体的持续性感染问题频发,同源重组介导的抗原变异扮演重要角色。解螺旋酶RuvA调节霍利迪连接体变构是参与同源重组的关键步骤。鉴于此,本研究旨在原核表达猪肺炎支原体解螺旋酶RuvA重组蛋白,鉴定其DNA结合活性并制备多克隆抗...猪肺炎支原体的持续性感染问题频发,同源重组介导的抗原变异扮演重要角色。解螺旋酶RuvA调节霍利迪连接体变构是参与同源重组的关键步骤。鉴于此,本研究旨在原核表达猪肺炎支原体解螺旋酶RuvA重组蛋白,鉴定其DNA结合活性并制备多克隆抗体。试验通过分子克隆构建原核表达质粒pET21a-RuvA,经诱导表达和纯化获得RuvA M hp重组蛋白;免疫家兔制备抗RuvA M hp多克隆抗体,再利用间接ELISA和Western blot试验进行效价测定和特异性验证;利用凝胶迁移试验结合表面等离子共振技术分析RuvA M hp的DNA结合活性;利用凝胶迁移试验结合Western blot试验鉴定RuvA M hp的寡聚特性。结果显示:原核表达的RuvA M hp重组蛋白约为26 ku;制备的抗RuvA M hp多克隆抗体效价为1∶256000,且具有良好的特异性;RuvA M hp具有较强的DNA结合活性,对霍利迪连接体的亲和力高达624.4 pmol·L^(-1)(K D),并且主要以八聚体形式与其形成稳定复合物。通过RuvA M hp的原核表达、多克隆抗体制备及活性鉴定,为后续探索RuvA调解同源重组介导猪肺炎支原体抗原变异的分子机制奠定了基础。展开更多
基金supported by the National Key Basic Research Special Funds of China(Grant No.G1999011700)Hebei Provincial Natural Science Foundation(Grant No.301447).
文摘The DNA-binding activity of heat shocktranscription factor (HSF) was induced by heat shock (HS) of a whole cell extract. Addition of antiserum, specific toCaM, to a whole cell extract reduced bind of the HSF to the heat shock element (HSE) with maize, and the re-addition of CaM to the sample restored the activity of the HSF forbinding to HSE. In addition, DNA-binding activity of the HSF was also induced by directly adding CaM to a whole cell extract at non-HS temperature with maize. Similar resultswere obtained with wheat and tomato. Our observationprovide the first example of the involvement of CaM inregulation of the DNA-binding activity of the HSF.
文摘Dear Editor, The LATERAL ORGAN BOUNDARIES DOMAIN （LBD）/ ASYMMETRICLEAVES2-LIKE （ASL） genes （hereafter referred to as LBD） encode proteins containing a conserved plant-specific LOB domain and play roles in lateral organ development （Iwakawa et al., 2002; Shuai et al., 2002; Majer and Hochholdinger, 2011）. The LOB domain is approximately 100 amino acids in length and contains a conserved four-Cys motif （CX2CX6CX3C）, the Gly-Ala-Ser block （GAS）, and the leucine-zipper-like coiled-coil motif （LX6LX3LX6L） （Shuai et al., 2002）. The leucine-zipper-like coiled-coil motif in the LOB domain is predicted to function in protein dimerization. LOB, AS2, and LBD4 preferentially bind unique DNA sequences in electrophoretic mobility shift assays （EMSAs） （Husbands et al., 2007）. The LOB domain of AS2 cannot be functionally replaced by those of other members of the LOB family,
文摘猪肺炎支原体的持续性感染问题频发,同源重组介导的抗原变异扮演重要角色。解螺旋酶RuvA调节霍利迪连接体变构是参与同源重组的关键步骤。鉴于此,本研究旨在原核表达猪肺炎支原体解螺旋酶RuvA重组蛋白,鉴定其DNA结合活性并制备多克隆抗体。试验通过分子克隆构建原核表达质粒pET21a-RuvA,经诱导表达和纯化获得RuvA M hp重组蛋白;免疫家兔制备抗RuvA M hp多克隆抗体,再利用间接ELISA和Western blot试验进行效价测定和特异性验证;利用凝胶迁移试验结合表面等离子共振技术分析RuvA M hp的DNA结合活性;利用凝胶迁移试验结合Western blot试验鉴定RuvA M hp的寡聚特性。结果显示:原核表达的RuvA M hp重组蛋白约为26 ku;制备的抗RuvA M hp多克隆抗体效价为1∶256000,且具有良好的特异性;RuvA M hp具有较强的DNA结合活性,对霍利迪连接体的亲和力高达624.4 pmol·L^(-1)(K D),并且主要以八聚体形式与其形成稳定复合物。通过RuvA M hp的原核表达、多克隆抗体制备及活性鉴定,为后续探索RuvA调解同源重组介导猪肺炎支原体抗原变异的分子机制奠定了基础。
