Molecular Cloning and Identification of the Transcriptional Regulatory Domain of the Goat Neurokinin B Gene <i>TAC3</i>
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Neurokinin B (NKB), encoded by <i>TAC3</i>, is thought to be an important accelerator of pulsatile gonadotropin-releasing hormone release. This study aimed to clarify the transcriptional regulatory mechanism of goat <i>TAC3</i>. First, we determined the full-length mRNA sequence of goat <i>TAC3</i> from the hypothalamus to be 820 b, including a 381 b coding region, with the putative transcription start site located 143-b upstream of the start codon. The deduced amino acid sequence of NKB, which is produced from preproNKB, was completely conserved among goat, cattle, and human. Next, we cloned 5'-upstream region of goat <i>TAC3</i> up to 3400 b from the translation initiation site, and this region was highly homologous with cattle <i>TAC3</i> (89%). We used this goat <i>TAC3</i> 5'-upstream region to perform luciferase assays. We created a luciferase reporter vector containing DNA constructs from –2706, –1837, –834, –335, or –197 to +166 bp (the putative transcription start site was designated as +1) of goat <i>TAC3</i> and these were transiently transfected into mouse hypothalamus-derived N7 cells and human neuroblastoma-derived SK-N-AS cells. The luciferase activity gradually increased with the deletion of the 5'-upstream region, suggesting that the transcriptional suppressive region is located between –2706 and –336 bp and that the core promoter exists downstream of –197 bp. Estradiol treatment did not lead to significant suppression of luciferase activity of any constructs, suggesting the existence of other factor(s) that regulate goat <i>TAC3</i> transcription.
- Journal of Reproduction and Development
Journal of Reproduction and Development 59(5), 463-469, 2013
THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT