Cloning of a human thyroid hormone responsive gene

3，3‘，5-三碘甲腺原氨酸(T3)是甲状腺激素的一种活性形式，在人类胎儿发育和整个生命过程中发挥着重要作用。T3通过与其核受体结合，调节靶基因的表达而发挥作用。因此，识别不同组织中的T3反应基因对于在分子水平上阐明T3在人体中的作用具有重要意义。利用“聚合酶链式反应差异显示mRNAs”的方法，从培养的人皮肤成纤维细胞中克隆了一个名为ZAKI-4的T3反应基因的cDNA片段。Northern印迹分析表明，Zaki-4mRNAs有两个片段(3.4和1.4kb)，生理浓度的T3在12hr内均能使这两个片段的mRNA量增加。这种增加是由于Zaki-4基因转录的增加，而不是由于T3增加了Zaki-4mRNA的稳定性。放线菌酮可消除T3对ZAKI-4表达的影响，提示…的调节作用可能T_3诱导的Zaki-4 m RNA中更多的n是间接的，通过T_3诱导的中间产物。Zaki-4基因在心、脑、肝和骨骼肌中表达，而在胎盘、肺、肾和胰腺中不表达。在皮肤成纤维细胞和骨骼肌中，3.4kb的信使核糖核酸是主要物种，而在心脏、脑和肝脏中，1.4kb的信使核糖核酸占主导地位。利用快速扩增技术(RACE)克隆了该基因的全长c DNA。序列数据证实了存在两种可能由交替多聚腺苷作用产生的mRNAs，并表明这些mRNAs编码一个192个氨基酸的单一蛋白质。转录耦合体外翻译结果显示，这两种基因编码的蛋白质的相对分子质量均为26 kDa，与Zaki-4基因的序列分析结果基本一致。在1例因T3受体基因β亚型缺失而导致甲状腺激素抵抗患者的成纤维细胞中，T3能够诱导正常成纤维细胞中的Zaki-4基因表达。因此，提示另一种异构体--trα可介导Zaki-4基因表达的上调。较少

3,3', 5-Triodothyronine (T3), an active form of the thyroid hormone, plays an important role in fetal development and throughout life in humans. T3 exerts its effect through binding to its nuclear receptor and regulating expression of target genes. Thus, identification of T3-responsive genes in various tissue is important to elucidate T3 action at a molecular level in human. Utilizing a method called "differential display of mRNAs by means of polymerase chain reaction", the cDNA fragment of a T3-responsive gene named ZAKI-4 was cloned from cultured human skin fibroblasts. Northern blot analysis revealed that there were tow ZAKI-4mRNA species (3.4 and 1.4kb) and both mRNAs were increased by a physiological concentration of T3 within 12hr. This increase was due to an increased transcription of ZAKI-4 gene rather than increased stability of ZAKI-4 mRNA by T3. The T3 effect on ZAKI-4 expression was abolished by the treatment with cycloheximide, indicating the possibility that the regulatio … More n of ZAKI-4 mRNA by T3 is indirect, through an intermediate product induced by T3. ZAKI-4 mRNA was detected in heart, brain, liver, and skeletal muscle but not in placenta, lung, kidney and pancreas. In skin fibroblasts and skeletal muscle, 3.4-kb mRNA was the major species, whereas 1.4-kb mRNA was dominant in heart, brain, and liver. By using Rapid Amplification of cDNA Ends (RACE), the full-length cDNA was cloned. The sequence data confirmed the presence of two mRNA species which are possibly generated by an altermative polyadenylation and showed that these mRNAs code a single protein of 192 amino acids. Transcription coupled in vitro translation of the two mRNA species revealed both the two mRNA species code a protein with a molecular mass of 26 kDa, that roughly corresponds to the data of sequence analysis od ZAKI-4 cDNA.In fibroblasts from a patient with thyroid hormone resistance due to deletion of beta isoform of T3 receptor (TR) gene, T3 was able to induce ZAKI-4 mRNA like in normal fibroblasts. It is thus suggested that the other isoform, TR alpha can mediate the up-regulation of ZAKI-4 mRNA expression. Less