Gene (Rmmp1) of matrix metalloproteinase 1 (MMP1) of the bullfrog, Rana catesbeiana, has been shown previously to contain a thyroid hormone response element (TRE)-like sequence in its 5'-upstream region. The present study aimed to determine whether this TRE-like sequence is functional in vivo as a true TRE, and to characterize the sequences of the 5'-upstream region with respect to the regulation of the activity of the TRE when the TRE-like sequence was proved to be a true TRE. With this aim, various sequences of TRE-like sequence-containing 5'-upstream region were constructed and fused to the enhanced green fluorescent protein gene as a reporter gene. The fusion constructs were bombarded to the skin of bullfrog tadpoles and the activity of the TRE was quantitatively determined by measuring increased intensities of fluorescence when the animals were exposed to thyroid hormone. The present study clearly demonstrated that the sequence of Rmmp1 is a biologically active TRE in vivo. In addition, a unique 36 bp long sequence directly flanked to the 3'-end of the TRE was identified which worked co-operatively with TRE to regulate the transcriptional promoter activity. It should be emphasized that the presence of TRE in the Rmmp1 gene is unique, because its presence has not been reported in the known promoter region of vertebrate MMPs.

Growth hormone receptor (GHR) cDNA and gene of the Japanese flounder (Paralicthys olivaceus) were cloned and their molecular structures were characterized. The 641 amino acid sequence predicted from the cDNA sequence showed more than 75% overall sequence similarity with GHRs of other teleosts such as turbot and goldfish, and contained common structural features of vertebrate GHRs. The extracellular domain of flounder GHR had three pairs of cysteines and an FGEFS motif with a replacement E to D. The cytoplasmic domain contained two conserved motifs referred to as box 1 and box 2. The flounder GHR gene was cloned by PCR using primers designed from the sequence of the GHR cDNA. The GHR gene was composed of 10 exons. The sequence of exon 1 corresponded to the 5'-untranslated region of the cDNA, and exons 2-6 encoded most parts of the extracellular domain. The transmembrane domain was found in exon 7, and the intracellular domain was encoded in exons 8-10. Exon 10 also encoded the 3'-untranslated region. Comparison of the flounder GHR gene with the human GHR gene shows that the flounder gene contains no exons corresponding to exon 3 of the human GHR gene, and that the region corresponding to exon 10 in the human GHR gene is encoded by exons 9 and 10 in the flounder GHR gene. These findings indicate that the flounder GHR gene diverged from those of mammalian and avian GHR genes, especially in the organization of the exons encoding the cytoplasmic domain. In addition to the regular form of GHR mRNA, a 3'-truncated form lacking the region derived from exons 9 and 10 was detected as a minor species in the liver by RT-PCR and by RNase protection assay. RT-PCR analysis showed that both the regular and the 3'-truncated GHR mRNAs are expressed in a wide range of flounder tissues with the highest levels being found in the liver. The 5'-flanking region of the flounder GHR gene was cloned by inverse PCR, and three transcription start points were identified with similar frequency by RNase protection assay.

Insulin receptor substrate 1 (IRS-1) gene polymorphisms have been identified in type 2 diabetic patients; however, it is unclear how such polymorphisms contribute to the development of diabetes. Here we introduced obesity in heterozygous IRS-1 knockout (IRS-1(+/-)) mice by gold-thioglucose (GTG) injection and studied the impact of reduced IRS-1 expression on obesity-linked insulin resistance. GTG injection resulted in approximately 30% weight gain in IRS-1(+/-) and wild type (WT) mice, compared with saline-injected controls. There was no difference in insulin sensitivity between lean IRS-1(+/-) and lean WT. Elevated fasting insulin levels but no change in fasting glucose were noted in obese IRS-1(+/-) and WT compared with the respective lean controls. Importantly, fasting insulin in obese IRS-1(+/-) was 1.5-fold higher (P<0.05) than in obese WT, and an insulin tolerance test showed a profound insulin resistance in obese IRS-1(+/-) compared with obese WT. The islets of obese IRS-1(+/-) were 1.4-fold larger than those of obese WT. The expression of insulin receptor and IRS-1 and IRS-2 was decreased in obese IRS-1(+/-), which could in part explain the profound insulin resistance in these mice. Our results suggest that IRS-1 is the suspected gene for type 2 diabetes and its polymorphisms could worsen insulin resistance in the presence of other additional factors, such as obesity.