Cloning of a human thyroid hormone responsive gene

人甲状腺激素反应基因的克隆

• 批准号: 07671125
• 负责人: MURATA Yoshiharu
• 金额: $ 1.6万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07671125/
• 关键词: thyroid hormone; fibroblast; human; cloning; gene

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

3,3', 5-三碘甲状腺原氨酸 (T3) 是甲状腺激素的一种活性形式，在胎儿发育和人类一生中发挥着重要作用。 T3通过与其核受体结合并调节靶基因的表达来发挥作用。因此，鉴定不同组织中的 T3 反应基因对于在分子水平上阐明人类 T3 作用非常重要。利用一种称为“通过聚合酶链式反应差异显示mRNA”的方法，从培养的人皮肤成纤维细胞中克隆了名为ZAKI-4的T3反应基因的cDNA片段。 Northern印迹分析显示存在两种ZAKI-4mRNA种类（3.4和1.4kb）并且两种mRNA在12小时内均增加了T3的生理浓度。这种增加是由于 T3 导致 ZAKI-4 基因转录增加，而不是 ZAKI-4 mRNA 稳定性增加。放线菌酮处理消除了 T3 对 ZAKI-4 表达的影响，表明 T3 对 ZAKI-4 mRNA 的调节可能是通过 T3 诱导的中间产物间接调节的。在心脏、脑、肝脏和骨骼肌中检测到 ZAKI-4 mRNA，但在胎盘、肺、肾和胰腺中未检测到。在皮肤成纤维细胞和骨骼肌中，3.4-kb mRNA 是主要种类，而1.4-kb mRNA 在心脏、脑和肝脏中占主导地位。通过使用 cDNA 末端快速扩增 (RACE)，克隆了全长 cDNA。序列数据证实了两种 mRNA 的存在，这两种 mRNA 可能是由选择性多腺苷酸化产生的，并表明这些 mRNA 编码 192 个氨基酸的单一蛋白质。两种 mRNA 的转录耦合体外翻译显示，这两种 mRNA 均编码分子量为 26 kDa 的蛋白质，该蛋白质大致对应于 ZAKI-4 cDNA 的序列分析数据。 在因 T3 受体 (TR) 基因的 β 亚型缺失而导致甲状腺激素抵抗的患者的成纤维细胞中，T3 能够诱导 ZAKI-4 mRNA，如 正常成纤维细胞。因此表明另一种异构体 TR α 可以介导 ZAKI-4 mRNA 表达的上调。较少的

项目成果

S.Yamaguchi: "Hepatocytes cultured in monolayers have a lower responsiveness to thyroid hormone and retinoic acid than do hepatocytes cultured in spheroids : Possible role of retinoid X receptor." Environmental Medicine. 40(1). 47-50 (1996)

S.Yamaguchi：“单层培养的肝细胞对甲状腺激素和视黄酸的反应性比球状体培养的肝细胞低：类视黄醇 X 受体的可能作用。”

RE.Weiss, Y.Hayashi, T.Nagaya, KJ.Petty, Y.Murata, H.Tunka H.Seo, S.Refetoff: "Dominant inheritance of resistance to thyroid hormone not linked to defects in the thyroid hormone receptors alpha or beta genes may be due to a defective co-factor." J.Clin.En

RE.Weiss、Y.Hayashi、T.Nagaya、KJ.Petty、Y.Murata、H.Tunka H.Seo、S.Refetoff：“甲状腺激素抵抗的显性遗传与甲状腺激素受体 α 或 α 或 α 的缺陷无关。

T. Miyazaki: "Molecular cloning of a novel thyroid hormone-responsive gene, ZAKI-4, in human skin fibroblasts." Journal of Biological Chemistry. 271. 14567-14571 (1996)

T. Miyazaki：“在人类皮肤成纤维细胞中分子克隆一种新型甲状腺激素反应基因 ZAKI-4。”

RE. Weiss: "Dominant inheritance of resistance to thyroid hormone not linked to defects in the thyroid hormone receptors a or β genes maby be due to a defective co-factor." Journal of Clinical Endocrinology and Metabolism. 81. 1496-4203 (1996)

RE. Weiss：“甲状腺激素抵抗的显性遗传与甲状腺激素受体 a 或 β 基因的缺陷无关，可能是由于辅助因子缺陷所致。” 81. 1496-4203 (1996) ）

S. Yamaguchi: "Hepatocytes cultured in monolayers have a lower responsiveness to thyroid hormone and retinoic acid than do hepatocytes cultured in spheroids : Possible role of retinoid X receptor." Environmental Medicine. 40. 47-50 (1996)

S. Yamaguchi：“单层培养的肝细胞对甲状腺激素和视黄酸的反应性比球状体培养的肝细胞低：类视黄醇 X 受体的可能作用。”