Multiple Sry genes in the Okinawa spiny rat encode proteins with an A-to-S substitution in the HMG domain that retain DNA-binding ability.

The mammalian sex-determining gene SRY is highly conserved across species, with only a few exceptions. The Japanese rodent genus Tokudaia is known for its unique sex chromosome evolution. The Okinawa spiny rat T. muenninki (TMU) acquired neo-sex chromosomes with multiple Sry copies by sex chromosome-autosome fusions. All SRY copies in TMU have a substitution from alanine to serine at position 21 in the high-mobility group (HMG) box, a critical DNA-binding domain, suggesting that they are nonfunctional. However, the sex determination system in TMU remains unclear, in part because the species is endangered and it is therefore extremely difficult to obtain experimental samples. In this study, we performed in silico and in vitro analyses to investigate the molecular properties and function of SRY using recently obtained whole-genome sequence and RNA-seq data. A comparison of SRY sequences from 225 species showed that TMU is the only species with a substitution at the 21st position. This result highlights the rarity and specificity of this substitution. Structural predictions, DNA docking simulations, electrophoretic mobility shift assays and fluorescence anisotropy showed that although the affinity was slightly lower than that of the mouse homolog, DNA-binding ability was retained. However, Sry expression was not detected in the testis, liver or brain in adult TMU. The complete absence of Sry expression in the adult tissues, despite an intact sequence, strongly indicates a loss of regulatory function. These findings provide insight into the unique evolution of the Sry gene in this species.