[w~OMMENT

The continuing search for the mammalian sex-determining gene HOWARD COOKE

MRC HUMAN GENETICS UNIT, WESTERN GENERAL HOSPITAL,

CREWE ROAD, EDINBURGH EH4 2XU, UK.

Sex determination repre::ents a choice between two developmental pathways and as such provides an attractive model system for studying the regulation of suck switches. Mechanisms of sex deter- mination are diverse; in Drosophila and Caenorhabditis elegans the switch is controlled by the ratio of X chromosomes to autosomes, in crocodiles by the incubation temperature of the eggs and in mammals by the presence of a single Y chromosome. In mammals, a Y chromosome is suffi- cient to change gonadal development from the de- fault female pathway to that of the male, regardless of the number of auto- somes or X chromosomes. The location on the Y chromosome of the pre- sumed gene (or genes) responsible for male sex determination (TDF in humans, Tdy in mice) has been possible because of the relatively frequent occurrence of humans with sex reversal - males with two X chromosomes and females with an XY karyotype. These chro- mosomes arise because of aberrant exchanges between the X and Y chromosomes outside the pseudoautosomal region (the region that normally recombines between X and Y chromosomes). The product of such an abnor- mal exchange is an X chromosome with Y chro- mosome DNA on the distal short arm.

By assembling data on these and other structurally abnormal Y chromosomes, Page and co-workers were able to construct a map of the human Y chromosome and to define a relatively

small region which was sufficient for testis determination. Walking from the centromere-proximal part of this region towards the pseudo- autosomal region and the telomere, and checking for DNA sequence conservation and sex specificity of hybridization patterns, they were able to find a gene with an open reading frame that codes for a zinc finger protein (ZFY) 1. This gene

Pseudoautosomal region

× ¥ i i

SRY + - - - ~ X chromosome

X Y

Pseudoautosomal .., boundary

-" ~L t 5 k b SRY

30 kb I1.,1t1_ Break points in II 4XX males

-200 kb

ZFY

~ Abnormal XY interchange

× Y

FIGil Localization of TDF. The 35 kb region of the Y chromosome adjacent to the pseudoautosomal boundary, which does not include ZFL is sufficient to confer male sex determination properties on an X chromosome. Within this region, the SRYopen reading frame is a good candidate for TDF.

was present in all the XX inter- change males available to them.

Clearly, this was a good candi- date for the human sex- determining gene but further work cast increasing doubt on this hypothesis. An X chromosome homologue was found that escapes X-inactivation, making a switch mechanism based on the dosage of this gene unlikely. Marsupials,

with a sex determination mechanism similar to mammals, were found to have autosomal rather than sex-linked copies of ZFY- related genes. Two lines of evidence finally excluded ZFY as the primary deter- minant of maleness in mammals: the discovery of human XX males lacking the ZFY gene 2 and the observation that We/W e mice, which form testes without germ cells, do not express Y-linked homologs of ZFY in their testes (the mouse genes are called Zf¥- I and Zfv-2) 3. Zfv- 1 in mice and ZFY in humans may be involved in spermatogenesis. Nonethe- less, the basic strategy used to define the interval in which the Tdy gene might lie, and the approach used to isolate ZFY, remain valid; the new XX males may move a goal- post but they do not alter the rules of the game. This is especially true since the reason for the apparently conflicting results has been made clear by further study of the Y chromo- some used to define one end of the region that con- tains ZFY and which was previously thought to con- tain the sex-determining gene; rather than having a simple deletion, this chro- mosome turns out to have

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at least two - one that deletes ZFY and another more distal one 4.

Sinclair et al. report in a recent issue of Nature5 the results of a search in the 35 kb of DNA that is present between the pseudoauto- somal boundary and the break point region in the ZFY- XX males (see Fig. 1). Again using the 'Noah's ark blot' approach (a zoo blot with male and female of each species) they found a genomic fragment which contained two open reading frames and which detected related sequences on the Y chromosomes of the mammals tested. The larger open reading frame has been given the acrooym SRY and is deleted in the XY female used by Page et al. Given the relatively small region of the Y chromosome remaining between the goal-posts, this must be a strong candidate for the TDF gene but the strength of its candidacy is increased by the accompanying paper from Gubbay et aL 6, which analyses mouse homologs of SRY.

Mice have a number of mutant Y (strictly X and Y) chromosomes that have different phenotypic effects. Sxr is a mutation that has placed the testis-determining locus Tdy in the pseudoautosomal region. This is probably the result of a translocation of the Y chromo- some short arm, which in mouse is the location of Tdy, to the pseudo- autosomal region (in mouse the distal long arm of the X and Y chromosomes). Genes present in this region are a minor histo- compatibility antigen gene (Hya, a previous candidate for Tdy), a gene involved in spermatogenesis and Zfy-1 and Zfy-2. Sxr' is a reduced version of the same chromosome that retains only Zfy-1 and Tdy. A further mutant Y chromosome has been derived from retroviraUy infected embryonal stem cells and is no longer capable of determining male sex. At the cytological and molecular level this is a normal Y chromosome; the mutation in sex determination could be com- plemented by the minimal amount of Y chromosome present in Sxr' and on this basis it was presumed to be a mutation in Tdy itself (Tdyml). No alterations in the structure or expression of Zfy-1 or Zfy-2 genes have been detected on this Y chromosome.

This set of mutants can be used to follow the cosegregation of Tdy and a candidate gene. By isolating the murine homologue of the human SRYgene and probing these mutants, Gubbay et al. have found that both Sxr and Sxr' XX male mice have a sequence homologous to SRE Importantly, this gene is absent from the retroviraUy mutated, non-male-determining Y chromosome. In fact, probes adjoin- ing the SRY open reading frame detect one end of a deletion start- ing about 2 kb from the coding sequences. So far so good: this gene fulfils the criterion of cosegre- gating with Tdy/TDF in both mice and humans. Other immediately testable criteria are the pattern and timing of transcription of the gene. Sinclair et al. show that a 1.1 kb message is expressed in adult testis, and Gubbay et al. that a message detectable by polymerase chain reaction (PCR) amplification of cDNA is present in adult testes and 11.5 day male but not female gonadal ridges. This is consistent with the expression pattern ex- pected for a testis-determining gene, the action of which is postulated to be the induction of Sertoli cell formation in cells that would other- wise become follicle cells in the ovary.

Can we get any clues about the possible mechanism of action of the SRYgene? Sequence analysis of the human genomic DNA shows two open reading frames of 99 and 223 amino acids that overlap and are oriented 5' to 3' away from the centromere. Only the longer reading frame matches sequences in the databases. This is a con- served motif, spanning 80 amino acids, found in the human upstream binding factor (a tran- scription factor for ribosomal RNA), the chromatin-associated non- histone proteins HMG1 and HMG2, and the M c protein involved in mating type switching in the fission yeast Scbizosaccbaromyces pombe. A DNA segment from the rabbit Y chromosome has been cloned on the basis of cross-hybridization to the human SRY probe. Conser- vation between rabbit and human amino acid sequences within the motif region is 80% (90% if con- servative chat,g,~s are scored as identity). Outside this region the

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homology drops to 54%. PCR amplification from the RNA in humans, using a primer based on a poly(A) site and another derived from the genomic DNA sequence, suggests that the DNA cloned rep- resents the last exon of the gene.

Neither the human nor the mouse Y-encoded cDNAs have been cloned, although the mRNAs have been detected. In the mouse, Gubbay et al. have been able to obtain a number of cDNA clones but these are derived from genes that are probably autosomal, tiiough possibly X-linked. Se- quencing these cDNAs reveals the 80 amino acid HMG motif. Of these amino acids, 36 are identical in all the cloned SRY-related genes of humans and mice and a number of additional residues are common to the Y-linked genes of human, mouse and rabbit. The authors point out that the conserved motif has been suggested to be a DNA-binding domain and speculate that the product of the SRY gene may be a transcription factor - always an attractive idea when thinking in terms of a regulatory protein.

SRY is a gene that so far fulfils the criteria for the mammalian sex-determining gene. It remains possible that other candidate genes could exist. They would have to be within the 35 kb of DNA between the pseudoautosomal boundary and the break points in the XX males or the deletion responsible for the Tdy ml mutation in mouse. If thev had the same expression pattern as SRY they would be equally strong candidates for the TDF gene. A number of direct tests of the role of SRY or any other candidate gene are possible. First, introduction of the complete gene and nothing else (perhaps by using a cDNA construct with an added intron) into the female germ line would be predicted to give rise to XX males. A careful choice of mouse strains would have to be made, as some mouse Y chro- mosomes do not work in the wrong genetic background. A second test would be to show complementation of the Tdymt mutation by such a construct. However, position effects could render a negative result meaning- less. The third possibility would be to mutate the conserved HMG