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Method for Identifying the Region of the Y-chromosome Containing the Testis Determining Factor in Humans

DNA specific for the Y chromosome can be isolated from recombinant DNA libraries made from male cells by screening DNA prepared from XX and XY cells. Y-specific sequences will bind only to DNA prepared from XY cells but not from XX cells (Figure 1). By screening such Y-specific sequences on DNA from individuals missing parts of the Y chromosome, one can determine the portion of the Y chromosome from which that particular sequence is derived (Page, 1986).

Figure 1
Figure 1   Protocol for screening Y-specific DNA clones. DNA from male cells is cloned into viruses and grown in E. coli. Each clone is separately cultured, its human DNA is isolated, and its strands are made radioactive. Each DNA fragment is denatured into single strands and hybridized with DNA from male or female cells that has been electrophoresed and Southern blotted. The Y-specific DNA fragments should bind specifically to the DNA from XY cells but not to that from XX cells.

In the human population, there exist both XX males (about 1 out of every 20,000 males) and XY females. The DNA hybridization results (Page et al., 1985; Vergnaud et al., 1986) demonstrated that XX males had Y-specific DNA from Y chromosome region 1 translocated onto one of their chromosomes. Moreover, all the XY females tested were missing this region of the Y chromosome (Figure 2). It seems most likely, then, that the gene for the testis-determining factor maps to region 1 of the Y chromosome, near the tip of the short arm. Similar evidence from mice also suggested that this region of DNA contains the testis-determining gene of the Y chromosome (Singh and Jones, 1982; Eicher et al., 1983).

Figure 2
Figure 2   Deletion mapping of the gene for the testis-determining factor(s) in humans. (A) The Y chromosome has been divided into nine intervals. Interval 4B is the kinetochore, and the stippled region is the "pseudoautosomal" portion, which contains DNA sequences in common with the X chromosome (and allows it to pair with the X during meiosis). XX males have DNA from region 1 whereas XY females have a deletion of this material from their Y chromosomes. As these regions are often too small to be seen microscopically, the presence or absence of the region was determined by DNA hybridization (as in Figure 21.5). Results of such a hybridization are shown in B, where the radioactive DNA was from region 1 of the Y chromosome. This Y-specific DNA is thought to contain the TDF, since the autoradiograph of the Southern blot demonstrates its presence in XY males and XX males, but not in XX females. (A after Page, 1985; B from Vergnaud et al., 1986, courtesy of D. Page.)

In 1990, Sinclair and colleagues narrowed the region to a 35,000 base-pair domain of the small arm of the Y chromosome. A history of this winnowing process is seen in Figure 3.

Figure 3
Figure 3   In 1959, the Y chromosome was shown to contain the testis determining factor in both humans and mice. By 1966, translocations and deletions of human chromosomes indicated that the TDF resides in the short arm of the Y chromosome (Yp). In the late 1980s, deletion mapping with male-specific DNA probes showed that the gene(s) resided in region 1 of the small arm of the Y chromosome. Studies of XY females, XX males, and transgenic mice have localized the TDF to a single region on the Y chromosome containing the SRY gene. (After McLaren, 1990.)

Literature Cited

Eicher, E. M., Phillips, S. J. and Washburn, L. L. 1983. The use of molecular probes and chromosomal rearrangements to partition the mouse Y chromosome into functional regions. In A. Messer and I. H. Porter (eds.), Recombinant DNA and Medical Genetics. Academic Press, New York, pp. 57-71.

McLaren, A. 1990. The making of male mice. Nature 351: 96.

Page, D. C. 1986. Sex-reversal: Deletion mapping of the male-determining function of the human Y chromosome. Cold Spring Harbor Symp. Quant. Biol. 51: 229-235.

Page, D. C., de la Chappelle, A. and Weissenbach, J. 1985. Chromosome Y-specific DNA in related human XX males. Nature 315: 224-226.

Singh, L. and Jones, K. W. 1982. Sex reversal in the mouse (Mus musculus ) is caused by a recurrent nonreciprocal crossover involving the X and an aberrant Y chromosome. Cell 28: 205-216.

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