Figure 1.
Nucleotide substitutions in the SRY and SF1 binding sites of Tokudaia TESCO.
Identical residues are indicated by boldface. Tokudaia species-specific nucleotides are indicated by gray boxes. The number of each binding site is in accordance with a previous study [12].
Table 1.
The identity of TESCO sequences.
Figure 2.
Contrasting enhancer activities in mouse and Tokudaia TESCO.
(A) Enhancer activities of mouse TESCO performed by reporter gene assays with mSRY or mSOX9 in COS7 cells. The 18–20-fold activation was observed when co-transfected with a combination of SF1 and mSRY or mSOX9. This synergistic regulation of Sox9 expression corresponded with a previous study [12]. (B) Tokudaia TESCO had no enhancer activity. For all Tokudaia species and all combinations of transfections, TESCO did not show significant activities. Means and standard deviations from at least three independent experiments are shown.
Figure 3.
Mutations in the binding sites depress the enhancer activities of Tokudaia TESCO.
Mutated TESCO, in which all binding sites were replaced by mouse sequence, showed recovery of activities by co-transfection of TMU_SOX9 and SF1. However, no significant activity was observed by TMU_SRY. Means and standard deviations from at least three independent experiments are shown. *P<0.05.
Figure 4.
Impaired transcription factor function of T. muenninki SRY.
(A) Comparison of the enhancer activity of mouse TESCO by co-transfecting mSRY with SF1 and TMU_SRY with SF1. T. muenninki SRY did not increase the activity of mouse TESCO. (B) One amino acid substitution at the 21st position within HMG-box had no effect on the function of T. muenninki SRY. The TMU_SRY_StoA also did not increase the activity of mouse TESCO. Means and standard deviations from at least three independent experiments are shown.
Figure 5.
Evolution of the Y chromosome and sex-determining mechanism in the genus Tokudaia.
Nucleotide substitutions accumulated in SRY and SF1 binding sites in TESCO of the common ancestor after emergence of a new sex-determining gene, leading to inactivation of TESCO, and SRY could not function as a transcription factor. In the common ancestor or in a lineage of T. muenninki, SRY itself lost function caused by mutations in the SRY sequence. Neo-X and neo-Y chromosomes were acquired by fusion of a pair of autosomes with the X and Y chromosomes. Many pseudo SRY copies amplified and were distributed throughout the heterochromatic region in the neo-Y chromosome. In a common ancestor of T. osimensis and T. tokunosimensis, a part of the Y region translocated to the distal region of X chromosome and the remaining Y chromosome region containing SRY (and other genes) was completely lost.