The mouse androgen receptor. Functional analysis of the protein and characterization of the gene

Abstract

Screening a mouse genomic DNA library with human androgen-receptor (hAR) cDNA probes resulted in the isolation and characterization of eight genomic fragments that contain the eight exons of the mouse androgen-receptor (mAR) gene. On the basis of similarity to the hAR gene, the nucleotide sequences of the protein-coding parts of the exons as well as the sequences of the intron/exon boundaries were determined. An open reading frame (ORF) of 2697 nucleotides, which can encode an 899-amino-acid protein, could be predicted. The structure of the mAR ORF was confirmed by sequence analysis of mAR cDNA fragments, which were obtained by PCR amplification of mouse testis cDNA, using mAR specific primers. A eukaryotic mAR expression vector was constructed and mAR was transiently expressed in COS-1 cells. The expressed protein was shown by Western blotting to be identical in size with the native mAR. Co-transfection of HeLa cells with the mAR expression plasmid and an androgen-responsive chloramphenicol acetyltransferase (CAT) reporter-gene construct showed mAR to be able to trans-activate the androgen-responsive promoter in a ligand-dependent manner. Transcription-initiation sites of the mAR gene were identified by S1-nuclease protection experiments, and the functional activity of the promoter region was determined by transient expression of mAR promoter-CAT-reporter-gene constructs in HeLa cells. Structural analysis revealed the promoter of the mAR gene to be devoid of TATA/CCAAT elements. In addition, the promoter region is not remarkably (G + C)-rich. Potential promoter elements consist of a consensus Sp1 binding sequence and a homopurine stretch. The polyadenylation sites of mAR mRNA were identified by sequence similarity to the corresponding sites in the hAR mRNA.