III.I Introduction
The search for the testis determining factor TDF in humans, led to a 60kb region o f the short arm o f the Y chromosome proximal to the pseudoautosomal boundary (the region o f homology between the X and Y chromosome, PAR) (Palmer, Sinclair et al. 1989). This conclusion was reached based on the analysis o f XX sex-reversed individuals, that inherited Y sequences through abnormal XY interchange during meiosis. Subsequently, more detailed analysis o f these chromosomal rearrangements showed that the
breakpoints were clustered around a region that is approximately 35kb proximal to the PAR boundary, thus narrowing down the minimal portion o f the Y chromosome able to confer maleness. This 35 kb sequence was used to carry out a “chromosome walk” to identify genes within the region (Sinclair, Berta et al. 1990). Subclones from this region were used to probe Southern blots o f DNA extracted from human male and females, bovine and murine males and females. One particular subclone hybridised strongly to Y specific fragments in human, bovine and murine genomic DNA. The sequence o f this clone was found to be conserved across male eutherian species. When translated, this 0.9 kb H in d i fragment encoded a protein that shared some homology to a portion o f the
S. pom be Me protein, the product o f one o f the m ating-type genes, and had striking similarity to a conserved m otif in several non-histone proteins related to HM GI and HMG2. This clone was named SR Y (sex-determining region Y) and was shown to encode a testis specific transcript. The conserved m otif is now what we know to be the HMG box or DNA binding domain common to all known SRY proteins and SOX (SRY related HMG Box ) proteins. The gene was proposed to be a good candidate for the elusive testis determining gene.
Coincident with the identification o f SRY, the murine counterpart, Sry was also isolated and found to reside in a region on the Y-chromosome known to be involved in sex determination, and to be missing in a line o f XY female mice known to be mutant in Tdy
(Gubbay, Collignon et al. 1990). The most striking feature identified within the open reading frame was a conserved 237bp sequence, with an amino acid sequence homology o f 80% compared to the conserved m otif identified in human SRY. This region o f
similarity was later shown to encode the HMG box DNA binding domain. Sry was shown to be expressed in the XY genital ridge, between 10.5 dpc and 12.0 dpc.
Expression o f Sry was also detected in mutant mice that completely lack germ cells in the genital ridge. Hence, soon after the discovery o f Sry, it was confirmed to be expressed in somatic cells o f the developing testis. Sry was subsequently shown to be the definitive testis determining gene, when XX mice, carrying the Sry transgene and
14kb o f regulatory sequences, developed as phenotypic males (Koopman, Gubbay et al. 1991).
III.I.Ill Sry can induce testis development in XX female mice
XX mice carrying the Sry transgene (X XSry) develop as males, with no obvious visible differences compared to wild type males. X X S ry males were caged with normal female animals, and although copulation appeared normal, no pups were ever bom. Vaginal plugs were detected in females mated with XX Sry males, but the absence o f mature sperm in the plug explained their infertility. The presence o f two X chromosomes always results in sterility in male mice, as germ cell are arrested at the prospermatogonia stage. Moreover other genes from the Y chromosome are known to be required for spermatogenesis.
Sex reversed transgenic mice were examined internally, and no hermaphrodites were found. The vas deferens appeared normal, however the testes appeared smaller than normal males. Testis weights o f sex reversed animals were about one third that o f normal wild type controls. Adult testis sections revealed a recognisable testis tubule stmcture, but the lumen o f the testis tubules were devoid o f germ cells and were
vacuolated, most likely due to a disruption o f the cell contacts that would normally occur between germ cells and Sertoli cells.
III.LU Comparison between mouse and human SRY.
Comparison o f SR Y sequences from different mammalian species, including humans, rodents (M musculus ) and rabbits, all o f which are eutherian mammals, and finally a marsupial, the striped face dunnart {Sminthopsis macroura), indicates that the only region showing conservation is that encoding the HMG box, which binds and bends DNA (Whitfield, Lovell-Badge et al. 1993). Comparing the mouse and human SRY coding sequences, regions outside the HMG box show very poor conservation both at the nucleotide and amino acid level. Mouse SRY comprises 395 amino acids whereas the human counterpart is 204 amino acids (Fig 8). In mouse, the N-terminal domain is restricted to the first two amino acids followed directly by the 79 amino acid HMG box domain. In comparison, the human N-terminal domain is comprised o f 57 amino acids followed by the 79 amino acid HMG box. These figures demonstrate the considerable differences in size and therefore sequence between mouse and human N-terminal domains. In addition, the C-termini o f the two species have diverged completely. The mouse 314 amino acid C-terminal domain encodes a short linker domain followed by a long degenerative glutamine repeat domain. No similar glutamine repeat region is present in the human C-terminus, which is much shorter than that o f mouse, encoded by only 68 amino acids. The HMG box domains o f human and mouse SRY show 89% conservation in amino acid similarity based on automated sequence analysis and are 72% identical at the amino acid level (Koopman 2001). As the HMG box is highly conserved across mammalian species, and sequences outside o f the HMG box appear to be rapidly evolving, much emphasis has been placed on the functional relevance o f this domain.
Fig 8 Comparison o f Human and Mouse SRY sequences.
A. Human SRY is 204 amino acids, the HMG box domain is shaded in blue and a 7 amino acid PDZ protein interaction domain is located at the distal C-terminus. Mouse SRY is comprised o f 395 amino acids, with the HMG box shaded in blue, and the large 222 amino acid Q(glutamine) rich sequence is shaded in yellow.
B. Sequence comparison on the human and mouse HMG box domains, with the conserved amino acids in boxes. The two nuclear localisation signals NLS are
bracketed: these are well conserved between mouse and human. Two human mutations, defined by asterixes, perturb nuclear localisation o f human SRY, leading to sex reversal.