Developmental Study on a Real-time Measurement of Gene Transcriptional Activity by Means of lon-channel Reporter Genes.

通过与基因编码区5‘上游连锁的各种报告基因来分析基因转录活性的调节。常用的报告基因有β-半乳糖基因、过氧化氢酶基因、荧光素酶基因，这些基因是通过酶活性检测或固定后的组织化学方法检测出来的。为了分析活细胞的转录活性，离子通道基因是非常合适的，因为离子通道的活性可以在活着的状态下通过电生理学来测量，有时还可以通过适当的荧光指示剂通过细胞内离子浓度的变化来光学检测。目的探讨离子通道基因在海鞘双细胞模型神经诱导系统中作为报告基因的可行性。获得的结果如下：(1)为了利用海鞘钠通道基因作为报告基因，我们从金枪鱼幼体的cDNA文库中克隆了金枪鱼I，并测定了其全核苷酸序列。对TTX的不敏感可能是由于SS2区的点突变所致。(2)与RSV启动子相连的金枪鱼I基因克隆获得了可变的表达效率，并将其合成了金枪鱼I基因的mRNA注入海鞘神经母细胞球，以阐明这种差异的原因。(3)当胸苷激酶基因编码区5‘端的一部分与金枪鱼I基因编码区的头部相连时，我们发现了高效率的表达，并得出结论，至少在海囊胚胎中，金枪鱼I对报告基因是有用的。(4)为了区分金枪鱼I的外源表达和内源表达，我们在SS2区域进行了TTX不敏感通道和TTX敏感通道的定点突变。

Regulation of transcriptional activities of a gene has been analyzed by means of various reporter genes linked to 5' upper stream of the coding region of the gene. The reporter genes generally used are beta-Gal, CAT,Luciferase genes, which are detected by the enzymatic activities in homogenates or by histochemical methods after fixation. In order to analyze transcriptional activities in living cells ion channel genes are extremely suitable because activities of ion channels can be measured electrophysiologically in living state and sometimes may be detected optically through changes in intracellular ion concentrations by appropriate fluorescent indicators. Aim in the present project is to test the possibility of using ion channel genes as reporter genes in the model neural induction system of the ascidian two-cell pair. Obtained results are : (1) In order to use an ascidian Na channel gene as a reporter we cloned TuNa I from larval cDNA library and whole nucleotide sequence was determined. The insensitivity to TTX was suggested to be due to a point mutation in SS2 region. (2) The cDNA clone of TuNa I linked to RSV promotor gave us variable expression efficiency and we synthesized TuNa I mRNA from cDNA to inject ascidian neural blastomeres to clarify causes of the variability. (3) We found high efficiency of expression, when a part of 5'end of the coding region of thymidine kinase mRNA was linked to the head of the coding region of TuNa I mRNA,and concluded that TuNa I was useful for a reporter gene at least in ascidian embryos. (4) In order to discriminate exogenous expression of TuNa I from the endogenous one we have tried to altemate the TTx insensitive channels to the TTX sensitive ones by site-directed mutagenesis in the region of SS2.