Identification of nuclear receptor mRNAs by RT-PCR amplification of conserved zinc-finger motif sequences

Abstract

Highly degenerate PCR primers were designed based on the amino acid sequence of the zinc finger motifs of known nuclear receptor members. Reverse transcription (RT)-PCR was performed using these degenerate primers starting with total RNA isolated from human umbilical vein endothelial cells (HUVEC). We identified 13 nuclear receptors of which three were novel. Of the novel orphans two-RZR alpha and PHR-1-were further characterised. Cloning of their cDNAs and sequence comparisons revealed that RZR alpha is related to Drosophila DHR3, RAR and RXR. PHR-1 turned out to be the human homologue of the recently published murine LRH-1. By Northern blot analyses we found RZR alpha mRNA expressed in a variety of organs, whereas a high amount of PHR-1 mRNA was found in pancreas and less in liver. This technique not only aides in the identification of additional members of the nuclear receptor superfamily but also measures relative changes in the expression of nuclear receptors between two different biological states, for example, normal and disease states.