Zhang S P, Natsukari N, Bai G, Nichols R A, Weiss B
Department of Pharmacology, Medical College of Pennsylvania, Philadelphia 19129.
Neuroscience. 1993 Jul;55(2):571-82. doi: 10.1016/0306-4522(93)90525-k.
Calmodulin, a ubiquitous calcium-binding protein which is involved in many biological processes, including cell proliferation and differentiation, has been shown to be encoded by three genes from which five calmodulin messenger RNAs are transcribed. In our previous studies, using the PC12 pheochromocytoma cell line as a model system for neuronal differentiation, all five calmodulin messenger RNAs were found to be present, and treatment with both nerve growth factor and dibutyryl cyclic AMP, which induce neurite outgrowth in these cells, increased the level of calmodulin and differentially increased the levels of the various calmodulin messenger RNAs. In an attempt to uncover the nature of the differential increase in the calmodulin messenger RNAs during neuronal differentiation, we examined here the subcellular distribution of the individual calmodulin messenger RNAs in PC12 cells treated with nerve growth factor and dibutyryl cyclic AMP by in situ hybridization cytochemistry, using radiolabeled oligodeoxynucleotide probes. Using an oligodeoxynucleotide probe which detects all of the calmodulin transcripts, the calmodulin messenger RNAs were found to be distributed throughout the cell bodies of differentiated PC12 cells; significant amounts of calmodulin messenger RNAs were also found in most neurites (approximately 70% of the total number). Using specific probes for the calmodulin messenger RNAs derived from each calmodulin gene, distinct patterns of localization of the different calmodulin messenger RNAs were revealed. The messenger RNAs from calmodulin genes I and II were readily detected in all cell bodies and in about one-half of the neurites. In contrast, a weak signal for the messenger RNAs from calmodulin gene III was associated with cell bodies, while no significant signal was found in neurites. A population distribution analysis of the labeling of individual PC12 cell bodies, as determined by counting autoradiographic grains, revealed differences in the relative abundance of each group of messenger RNAs derived from each of the three calmodulin genes. The order of relative abundance of the messenger RNAs in cell bodies was found to be: calmodulin gene II messenger RNA > calmodulin gene I messenger RNAs >> calmodulin gene III messenger RNAs. An analysis of the labeling density along neurites indicated a similar density of neuritic messenger RNAs from calmodulin gene I and calmodulin gene II, whereas there was no significant signal for the messenger RNAs from calmodulin gene III.(ABSTRACT TRUNCATED AT 400 WORDS)
钙调蛋白是一种普遍存在的钙结合蛋白，参与包括细胞增殖和分化在内的许多生物过程，已被证明由三个基因编码，转录出五种钙调蛋白信使核糖核酸。在我们之前的研究中，以PC12嗜铬细胞瘤细胞系作为神经元分化的模型系统，发现所有五种钙调蛋白信使核糖核酸均存在，用神经生长因子和二丁酰环磷腺苷（二者均可诱导这些细胞的神经突生长）处理后，钙调蛋白水平升高，且不同程度地提高了各种钙调蛋白信使核糖核酸的水平。为了揭示神经元分化过程中钙调蛋白信使核糖核酸差异增加的本质，我们在此通过原位杂交细胞化学，使用放射性标记的寡脱氧核苷酸探针，研究了经神经生长因子和二丁酰环磷腺苷处理的PC12细胞中各个钙调蛋白信使核糖核酸的亚细胞分布。使用能检测所有钙调蛋白转录本的寡脱氧核苷酸探针，发现钙调蛋白信使核糖核酸分布于分化的PC12细胞的整个细胞体中；在大多数神经突中也发现了大量的钙调蛋白信使核糖核酸（约占总数的70%）。使用针对每个钙调蛋白基因衍生的钙调蛋白信使核糖核酸的特异性探针，揭示了不同钙调蛋白信使核糖核酸的独特定位模式。钙调蛋白基因I和II的信使核糖核酸在所有细胞体和大约一半的神经突中易于检测到。相比之下，钙调蛋白基因III的信使核糖核酸信号较弱，与细胞体相关，而在神经突中未发现明显信号。通过对单个PC12细胞体标记的群体分布分析（通过计数放射自显影片上的银粒确定），揭示了源自三个钙调蛋白基因中每个基因的每组信使核糖核酸相对丰度的差异。发现细胞体中信使核糖核酸相对丰度的顺序为：钙调蛋白基因II信使核糖核酸>钙调蛋白基因I信使核糖核酸>>钙调蛋白基因III信使核糖核酸。对神经突上标记密度的分析表明，钙调蛋白基因I和钙调蛋白基因II的神经突信使核糖核酸密度相似，而钙调蛋白基因III的信使核糖核酸没有明显信号。（摘要截短于400字）
