The Preliminary Study Of A Novel Thyroid Hormone Receptor β Isoform TRβ△

Thyroid hormone (T3) activates nuclear receptor transcription factors, encoded by the TRa (NR1A1) and TRb (NR1A2) genes, to regulate target gene expression in the positive or negative manner. Thyroid hormone plays critical roles in differentiation, growth, and metabolism. Several TR isoforms exist, TR α gene encodes five isoforms, and TR ft gene encodes four isoforms. Only four of them are confirmed as functional receptors, they are α 1, β 1, β 2 and β 3,bioroles of others are still unclear. These nine isoforms have arisen by different splicing or transcription from separated promoters.This is part work of National Nature Science Foundation. In this experiment, RNAs was extracted from rat's liver and it served as the template for producing the first strand of the cDNAs. Then amplified the TR B 1 with the cDNAs . TR ft I gene was inserted into cloning vector pGEM-T easy . Then had the cloned TR B 1 gene sequenced, then compared it with the sequence which has been published on the net. The result shew that the newly cloned sequence has an additional sequence of 108bp, blasted the additional sequence on NCBI, the sequence met with the part of the 6224bp 3-4 intron of the thyroid hormone receptor. The change take place before the changing point, the sequence located in what previously considered as highly conserved sequence in vertebrates. From our result, A novel TR B isoform was found in our experiment which is longer than TR ft 1. The sequence has been published on Gene Bank of NCBI, the register number is DO191165. it is named TR B A. It is considered that there is a changing point in TR β mRNA. The invariant splice site between the divergent N termini and the first of the common exons is known as the changing point.Upstream of the changing point the splicing manner is highly variable, but downstream of the changing point the splicing manner is highly conserved in vertebrates.The difference between TR {3 1 and TR 0 A is that the later has an additional fragment of 108bp appeared between exon3 and exon4.We think this 108bp DNA fragment is maybe a newly found exon behind the changing point and it break the conserved splicing manner. The result is a challenge to the previous theory of the changing point, whether this changing point exists should be doubted .The TR 3 A gene segment was inserted into fusion expressing vector pQE-30Xa. Furthermore, recombinant vector pQE-30Xa/ TR P A was transformed into E.coli Ml5 [pREP4] . The target proteins containing 6XHis affinity tag facilitates binding to Ni-NTA resin in metal chelating affinity chromatography. After the proteins binding non-specifically to resin were removed by washing solutions with gradient increasing concentration of imidazole, the target proteins were eluted from resin with apparent higher concentration of imidazole and concentrated by centrifuge ultrafiltration. Store the protein at — 80 °C for the further research.