Molecular cloning of a novel thyroid hormone-responsive gene, ZAKI-4, in human skin fibroblasts

Utilizing a method called ''differential display of mRNAs by means of polymerase chain reaction'', the cDNA fragment of a thyroid hormone-responsive gene ZAKI-4 was cloned from cultured human skin fibroblasts, Northern blot analysis revealed that there were two ZAKI-4 mRNA species (3.4 and 1.4 kilobases (kb)), and they were up-regulated by a physiological concentration of triiodothyronine (T-3). This T-3 effect was abolished by the treatment with cycloheximide, indicating the possibility that gene ZAKI-4 is regulated by T-3 in an indirect fashion, through an intermediate product of T-3, rather directly by T-3 itself, No effect of T-3 on ZAKI-4 mRNA stability suggested that T-3 induces the mRNA at the transcriptional level. Rapid amplification of cDNA ends confirmed the presence of two mRNA species. 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. The sequence analysis suggested that the two mRNA species arise from alternative polyadenylation and code a single protein of 192 amino acids. No homologous protein sequence was found in a data base. Elucidation of the function of ZAKI-4 gene product will provide new insights into an important role of T-3 in various organs.