NERVE-SPECIFIC EXPRESSION OF NONMUSCLE MYOSIN HEAVY CHAINS

先前的工作表明，编码肌球蛋白重链-B的mRNA (MHC-B)脑和脊髓的亚型包含30个插入片段 25-50kD和50-20kD结构域附近的核苷酸和63个核苷酸 分别位于头部区域的边界。在这个项目中，我们 重点介绍了这些插页的功能。我们的研究结果如下。 1)我们筛选了6种不同的人类癌细胞系，使用 逆转录-竞争性聚合酶链式反应 每个插入物外的引物组。只有神经母细胞瘤细胞系(SK- Nsh和SK-NMC)在其mRNAs中显示了25-50kD的连接插入， 未见细胞系出现50-20kD连接插入。2)PC12细胞(大鼠 嗜铬细胞瘤细胞株)，在NGF刺激前，显示 都不是插入物。经50 ng/mlNGF刺激后，这些细胞 显示含有25-50kD连接的mRNA的比例增加 插入到总的NMMHC-BmRNA(1wk-12%，2wks-38%)。 在形态上，它们经历了大量的轴突生长(1周)， 最终，形成了一个丰富的流程网络(3周)。3)之后 停止NGF刺激后，插入物的数量减少 神经突起在一周内消失了。4)我亚克隆了一条 与大鼠NMMHC-B和-A头部区域对应的产物和 已排序。与插入相对应的推导氨基酸序列 在不同物种(鼠、人、鸟)和所有 含有一个可作为底物的推定序列(-S-P-K-) 对于细胞周期蛋白-p34-cdc2(Cdc2)和cdc2样激酶。5)50fm的反义 相应的寡核苷酸插入序列抑制形态 NGF和相同浓度的NGF刺激PC12细胞后的变化 正义寡核苷酸不能抑制细胞形态的改变。然而， 插入形式的信使核糖核酸水平无明显变化。我们是 目前正在努力量化MHC的数量。一个 PC12细胞向神经元分化的相关性研究 NMMHC-B中插入10个氨基酸的表型和表达 同源异构体是假定的。

Previous work has shown that the MRNA encoding the myosin heavy chain-B (MHC-B) isoform of brain and spinal cord contains insertions of 30 nucleotides and 63 nucleotides near the 25-50 Kd and 50-20 Kd domain boundaries, respectively, in the head region. In this project, we focused on the function of these insertions. Our results are as follows. 1) We screened 6 different kinds of human cancer cell lines, using the reverse transcription-competitive polymerase chain reaction, with a set of primers outside of each insertion. Only neuroblastoma cell lines (SK- NSH and SK-NMC) showed the 25-50 Kd junction insertion in their MRNAS and no cell line showed the 50-20 Kd junction insertion. 2) PC12 cells (rat pheochromocytoma cell line), prior to stimulation with NGF, showed neither insertion. After stimulation with 50 ng/ml NGF, these cells showed an increase in the ratio of MRNA containing the 25-50 Kd junction insertion to the total NMMHC-B MRNA (1 wk- 12%, 2 wks- 38%). Morphologically, they underwent massive neurite outgrowth (1 wk) and, ultimately, a rich network of processes was formed (3 wks). 3) After cessation of NGF stimulation, the amount of the insert decreased and disappeared as did the neurites within one week. 4) I subcloned a PCR product corresponding to the head region of rat NMMHC-B and -A and sequenced. Deduced amino acid sequences corresponding to the insertion were conservative among different species (rat, human, avian) and all contained a putative sequence (-S-P-K-) which could serve as a substrate for cyclin-p34-cdc2 (cdc2) and cdc2-like kinases. 5) 50 fM of antisense oligonucleotide corresponding inserted sequence inhibit the morphological change of PC12 cells stimulated by NGF and the same concentration of sense oligonucleotide did not inhibit morphological change. However, the inserted form of MRNA level did not change significantly. We are presently making an effort to quantitate the amount of MHC. A correlation between the differentiation of PC12 cells to a neuronal phenotype and expression of the 10 amino acid insertion in the NMMHC-B isoform is postulated.