Identification of DNA-binding protein(s) in the developing heart.
Identification of DNA-binding protein(s) in the developing heart.
复制标题
鉴定发育中心脏中的 DNA 结合蛋白。
DOI：
10.1006/dbio.1993.1088
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发表时间：
1993
影响因子：
2.7
通讯作者：
Bader,DM
中科院分区：
文献类型：
--
作者：
Litvin,J;Montgomery,MO;Goldhamer,DJ;EmersonJr,CP;Bader,DM
针对螺旋-环-螺旋（HLH）蛋白MyoD 1的第二螺旋的抗血清（抗H2）与禽类心肌细胞分化期间表达的蛋白反应。间接免疫组织化学整体包埋染色与抗H2检测到的蛋白质表达在第11阶段的心脏，但不是在心脏的老胚胎。在细胞水平，这种染色仅限于心肌细胞的细胞核，表明这些蛋白质可能具有DNA结合能力。几种蛋白质免疫沉淀抗H2从第11阶段的心脏组织。从类似阶段的心脏，蛋白质提取物与肌肉特异性增强子序列的肌肉肌酸激酶（MCK）孵育时，给出了一个阶段特异性的频带移动在电迁移率变动分析（EMSA），这些蛋白质-DNA复合物被识别和supershifted由抗H2。用含有突变E盒的MCK序列孵育不产生偏移。早在第6阶段就出现了特定的偏移，一直持续到第13阶段，并在第17阶段消失。这些数据表明，至少有一种蛋白质的存在下，是瞬时表达的分化心肌细胞，这是免疫化学反应的抗血清提出的第二个螺旋的MyoD 1，并结合到肌肉特异性DNA增强子序列。
An antiserum (anti-H2) directed at the second helix of the helix-loop-helix (HLH) protein MyoD1 reacts with a protein expressed during avian cardiac myocyte differentiation. Indirect immunohistochemical whole mount staining with anti-H2 detected a protein expressed in stage 11 hearts, but not in hearts of older embryos. At the cellular level, this staining is confined to the nucleus of cardiac cells suggesting that these proteins may have DNA-binding abilities. Several proteins were immunoprecipitated by anti-H2 from stage 11 heart tissue. Protein extracts from similarly staged hearts, when incubated with the muscle-specific enhancer sequence of muscle creatine kinase (MCK), gave a stage-specific band shift in electromobility shift assays (EMSA), and these protein-DNA complexes were recognized and supershifted by anti-H2. Incubation with a MCK sequence containing a mutated E box did not produce a shift. The specific shift was present as early as stage 6, remained through stage 13, and disappeared by stage 17. These data suggest the presence of at least one protein that is transiently expressed in the differentiating cardiac myocyte, that is immunochemically reactive with an antiserum raised against the second helix of MyoD1, and that binds to a muscle-specific DNA enhancer sequence.