Akiyama T, Whitaker B, Federspiel M, Hughes S H, Yamamoto H, Takeuchi T, Brumbaugh J
Department of Biology, Keio University, Yokohama, Japan.
Exp Cell Res. 1994 Sep;214(1):154-62. doi: 10.1006/excr.1994.1244.
A mouse tyrosinase cDNA has been combined with different promoters and inserted into several replication-competent avian leukosis proviruses and the viruses were transferred into cultured albino chick cells by viral infection. Expression of the tyrosinase gene depended on one of four promoter sequences: the resident constitutive promoter (Rous sarcoma virus long-terminal repeat; RSV-LTR), 471 bp from the mouse tyrosinase gene-associated promoter, 519 bp from the Japanese quail tyrosinase gene associated promoter, or 369 bp from the quail tyrosinase promoter. The infected cells expressed tyrosinase and produced pigment which could be seen with the light microscope. Immunofluorescence microscopy, using an anti mouse tyrosinase T1-specific antibody, also showed the presence of mouse tyrosinase. When infected with the same viral titer, gene expression was highest with the constitutive LTR promoter. The quail tyrosinase promoter, while less efficient than the LTR, was more efficient than the other tyrosinase promoter. Fibroblasts and hepatocytes infected with the construct carrying the constitutive promoter or the truncated quail promoter expressed tyrosinase. The mouse and quail promoters appeared to show tissue-specific expression since fibroblasts and hepatocytes infected with viruses carrying these promoters did not express mouse tyrosinase. Toxicity is associated with constitutive expression of tyrosinase in nonmelanocytes. Therefore the viruses that carry the tissue specific promoters should be useful for in vivo studies.
小鼠酪氨酸酶cDNA已与不同的启动子结合，并插入到几种具有复制能力的禽白血病前病毒中，然后通过病毒感染将这些病毒转移到培养的白化鸡细胞中。酪氨酸酶基因的表达取决于四个启动子序列之一：常驻组成型启动子（劳氏肉瘤病毒长末端重复序列；RSV-LTR）、来自小鼠酪氨酸酶基因相关启动子的471 bp、来自日本鹌鹑酪氨酸酶基因相关启动子的519 bp或来自鹌鹑酪氨酸酶启动子的369 bp。被感染的细胞表达酪氨酸酶并产生色素，在光学显微镜下可以看到。使用抗小鼠酪氨酸酶T1特异性抗体的免疫荧光显微镜检查也显示了小鼠酪氨酸酶的存在。当感染相同病毒滴度时，组成型LTR启动子的基因表达最高。鹌鹑酪氨酸酶启动子虽然比LTR效率低，但比其他酪氨酸酶启动子效率高。用携带组成型启动子或截短的鹌鹑启动子的构建体感染的成纤维细胞和肝细胞表达酪氨酸酶。小鼠和鹌鹑启动子似乎表现出组织特异性表达，因为用携带这些启动子的病毒感染的成纤维细胞和肝细胞不表达小鼠酪氨酸酶。毒性与酪氨酸酶在非黑素细胞中的组成型表达有关。因此，携带组织特异性启动子的病毒应该对体内研究有用。
