DNA-binding properties of the product of the testis-determining gene and a related protein

THE upstream region of the human glyceraldehyde-3-phosphate dehydrogenase gene contains an insulin-response element (IRE-A) responsible for insulin-dependent transcription of the gene. The open reading frame of a rat complementary DNA encoding a protein (IRE-ABP) that binds to this sequence contains an HMG box motif that is 67% identical to the mouse candidate gene for the testis-determining factor SRY, and 98% identical to the mouse SRY-like gene, a4. Here we report that IRE-ABP and SRY bind to IRE-A DNA with comparable specificity in a DNase-I footprinting assay. Two females with sex reversal were found to have a single amino-acid substitution in the HMG box domain of SRY at position 3 and 7, respectively. SRY derivatives containing corresponding mutations do not make contact with IRE-A DNA. These results are direct evidence that mouse SRY-like proteins are sequence-specific DNA-binding proteins and identify two amino acids critical to this interaction. Moreover, IRE-A is a candidate SRY-response element.