Department of Pathology, University Hospital, S-751 85 Uppsala, Sweden; and Department of Medical Genetics, University of Uppsala, Uppsala, Sweden.
Mol Cell Neurosci. 1993 Dec;4(6):549-61. doi: 10.1006/mcne.1993.1068.
Phorbol ester treatment of human SH-SY5Y neuroblastoma cells, which leads to mature neuron-like cells with a sympathetic phenotype, induces outgrowth of neurites which are terminated by growth cones. The neurite extension is parallelled by an increased expression of the growth-associated protein, GAP-43. At the mRNA level, two GAP-43 mRNA species of 1.4 and 1.6 kb, respectively, were detected in SH-SY5Y cells. Both the low- and high-molecular-weight GAP-43 transcripts cosedimented with a polysomal fraction, indicating translation of both types of transcripts. To structurally characterize these GAP-43 mRNAs, several cDNA clones were isolated. The only difference identified corresponded to various size extensions in the 5'-untranslated region. A human genomic DNA fragment extending 1145 bp 5' of the GAP-43 translation start site, including a putative promoter region of the GAP-43 gene, was also characterized. Comparison of human and rat GAP-43 genomic sequences revealed an 85% identity between the first 900 bp 5' of translation start site. By RNase protection analysis, two clusters of putative transcription start sites, located approximately 200 bp apart, were identified. DNaseI footprinting analyses, using nuclear extracts prepared from untreated and TPA-treated SH-SY5Y cells, revealed specific footprints primarily detected in extracts prepared from differentiating cells. These clustered at positions immediately 5' of the two putative transcription start site regions. GAP-43 mRNA expression was finally studied using a probe which specifically recognizes the high-molecular-weight GAP-43 transcripts. Five tested human neuroblastoma cell lines and human fetal brain tissue expressed these transcripts. Furthermore, during differentiation of SH-SY5Y and LA-N-5 cells, both sizes of GAP-43 transcripts were transiently induced with the larger slightly preceeding the smaller mRNA species.
佛波酯处理人 SH-SY5Y 神经母细胞瘤细胞，导致具有交感表型的成熟神经元样细胞，诱导轴突的生长，轴突的生长由生长锥终止。神经突的延伸伴随着生长相关蛋白 GAP-43 的表达增加。在 mRNA 水平上，在 SH-SY5Y 细胞中检测到分别为 1.4 和 1.6 kb 的两种 GAP-43 mRNA 种。低和高分子量的 GAP-43 转录本与多核糖体部分共沉淀，表明这两种类型的转录本都被翻译。为了对这些 GAP-43 mRNA 进行结构表征，分离了几个 cDNA 克隆。唯一的区别是在 5'非翻译区的不同大小延伸。一个延伸到 GAP-43 翻译起始位点上游 5'的 1145 bp 的人基因组 DNA 片段，包括 GAP-43 基因的一个推定启动子区，也被表征。人 GAP-43 基因组序列和大鼠 GAP-43 基因组序列的比较显示，翻译起始位点上游 5'的前 900 bp 之间有 85%的同一性。通过 RNase 保护分析，在大约 200 bp 分开的两个簇中鉴定出假定的转录起始位点。使用未处理和 TPA 处理的 SH-SY5Y 细胞制备的核提取物进行 DNaseI 足迹分析，显示出主要在分化细胞提取物中检测到的特异性足迹。这些足迹集中在两个假定的转录起始位点区域的 5'处。最后，使用专门识别高分子量 GAP-43 转录本的探针研究 GAP-43 mRNA 的表达。五种测试的人神经母细胞瘤细胞系和人胎脑组织表达这些转录本。此外，在 SH-SY5Y 和 LA-N-5 细胞的分化过程中，GAP-43 转录本的两种大小均被短暂诱导，较大的稍先于较小的 mRNA 种。
