Mammalian sex-determining genes Peter Koopman The University of Queensland, Australia The recent cloning of the Y-linked sex-determining gene SRY has ended one of the most notorious gene hunts in mammalian molecular genetics. Attention has now been turned to characterizing this gene further and studying how it acts as a switch in the choice of male or female developmental pathways. Current Opinion in Biotechnology 1992, 3:603-606
Introduction The development of male or female characteristics in the embryo is the result of a combination of sex determination and subsequent sexual differentiation. Whilst the distinction between these two processes is nebulous it is generally agreed, based on Drosophila and nematode models, that sex determination in mammals involves a pathway of gene regulation. Understanding mammalian sex determination at the molecular level relies on isolating and studying all the genes in this pathway. The peculiar biology and genetics of sex determination have resulted in one of the genes from this pathw a y being singled out for special attention by molecular geneticists. The pioneering experiments of Jost [1] demonstrated that the testes are required for male development of rabbit fetuses; if the testes are removed, females develop. This observation suggested that in eutherian mammals, testis development is the key event that determines the sexual fate of the embryo. Cytogenetic analyses showed that sex in mammals is determined by the presence or absence of the Y chromosome, unlike the situation in Drosophila and Caenorhabditiseleganswhere the ratio of X chromosomes to autosomes provides the sex-determining switch. Combining these observations led to the theory that the Y chromosome contains a dominant locus which triggers maleness in mammals by directing testis formation from the bipotential embryonic gonad. This locus was said to encode 'testis-determining factor' and was dubbed TDF in humans and Tdy in mice. The search for TDF/Tdywas stimulated not only by its pivotal role in the sex determination pathway, but also by the amenability of this locus to genetic analysis in comparison with other loci involved in sex determination, the existence of which is assumed but not proven. First, the chromosomal location of TDF/Tdyhad been firmly established, mapping to the Y short arm in both man and mouse. Second, the proximity of TDF to the region of X-Y pairing and exchange means that TDFis occasionally transferred to the human X chromosome
by an accident of nature. This type of genetic accident results in sex reversal - maleness in XX individuals and is identified easily when these men seek treatment for sterility. Analysis of the Y-unique DNA on the X chromosomes of four such XX males allowed the positional cloning of the TDF candidate SRY[2], and its murine homologue Sty [3]. The properties of SRY/Srythat have allowed us to equate it genetically and functionally with TDF/Tdy are discussed elsewhere [4,5]. The purpose of this review is to summarize the progress of the past year in illuminating the function of SRY/Sryand its relationship to the genetic pathway of sex determination.
Transgenic mice Evidence equating SRY/Sry and TDF/Tdy culminated in the demonstration that S t y can bring about normal male development in chromosomally female (XX) transgenic mice [6"']. Sry was injected as a 14 kb fragment of Y-derived genomic DNA, named L741. Sequence analysis of L741 has shown that this fragment contains no genes other than Sry. These experiments provided long awaited proof that only one gene from the Y chromosome is required for male sex determination (although others are clearly associated with male-specific functions, such as spermatogenesis). Not suprisingly, some of the XX mice transgenic for L741 did not show sex reversal. Transgenes are generally susceptible to position effects which may suppress their expression depending on their site of integration into the genome. Founder mouse m32.10 had multiple copies of the Sty transgene and was a fertile female, allowing a transgenic line to be bred and expression of Sty to be examined amongst the offspring. Two findings have resulted from the analysis of this line (N Vivian, P Koopman and R Lovell-Badge, unpublished data). First, Sty was found to be expressed in embryos of this line, ruling out a lack of expression
Abbreviations bp--base pair; HMG--high mobility group.
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Mammaliangene studies due to position effects as an explanation for the lack of sex reversal. Second, some of the XX progeny of line 32.10 develop as males. These observations imply that a threshold of Sry activity is required for its function in male development, and that Sty expression in line 32.10 is close to this threshold; mice expressing Sty below the threshold level develop as females, whilst the few w h o s e expression exceeds the threshold level develop as males. An accurate assay for Sry expression is required to test this hypothesis and to define the threshold level. Efforts of several laboratories have failed to elicit sex reversal in mice w h e n human SRY is used as a transgene, even though the human gene is capable of being expressed in the genital ridge of transgenic embryos [6"]. It is not yet k n o w n whether this is due to a failure of the SRY protein to interact with mouse genes or proteins involved in male sex determination, or whether the regulation of the h u m a n gene in mice is subtly defective. Domain swap experiments will be important in clarifying these possibilities. A recently isolated yeast artificial c h r o m o s o m e containing human SRYwith 100 kb of 5' and 50 kb of 3' flanking sequence may be useful in the production of transgenic mice in which SRY is present in the context of its normal regulatory sequences [7].
Structure of the
Sry gene
Sequence analysis of genomic clones containing human and mouse SRY open reading frames s h o w e d that they contain a conserved, 237-base-pair (bp) region with h o m o l o g y to a motif that has come to be k n o w n as an HMG box. HMG boxes are found in a growing n u m b e r of eukaryotic genes encoding, amongst other things, the e p o n y m o u s high mobility group (HMG) proteins and a number of transcription factors (see below). As the HMG b o x is k n o w n to confer DNA-binding ability on several transcription factors, SRY/Sry is also assumed to be a transcription factor. Full length cDNA sequences have not yet been obtained for either mouse Sry or h u m a n SRY. These genes are k n o w n to e n c o d e mRNAs of approximately 1.2 kb in adult testes [2,3]. The HMG b o x of human SRY resides in a 670 bp open reading frame flanked by an 80 bp putative 3' untranslated region with a potential polyadenylation signal, suggesting that this is the final exon of the gene. There is some evidence that h u m a n SRY is encoded by a single exon (P Goodfellow and colleagues, personal communication). The coding sequence outside the HMG box region does not appear to contain motifs associated with transcriptional activation or repression, or show h o m o l o g y to any k n o w n genes. The structure of the mouse Sry gene is even less clear, although it may have more than one exon. Lack of h o m o l o g y between h u m a n SRY and mouse S ~ sequences outside the HMG box region casts some doubt
on the functional significance of these sequences. Curiously, the mouse Sty open reading frame is flanked in the genome by a large inverted repeat involving at least 15.5 kb on either side [8]. This duplication arose before the divergence of the Mus musculus musculus and M.m.domesticus subspecies 1-2 million years ago, yet the two arms of the repeat differ in only a few nucleotide positions and microsatellite polymorphisms. The significance of this repeat structure remains a mystery.
DNA binding studies The HMG box region of several transcription factors has b e e n shown to bind directly to specific DNA sequences. In particular, the T-cell transcription factor TCF-1 binds to the sequence AACAAAG [9]. As SRY is more closely related to this transcription factor than to any other HMG b o x protein, Harley and colleagues [10"'] tested the ability of SRY protein produced in vitro to bind to this sequence. Binding was confirmed by gel retardation assays, and mutations in positions 2,4,5 and 6 of the heptanucleotide sequence abolished binding, indicating that binding is sequence specific. In addition, mutant SRY protein, containing the amino acid substitutions found in five XY females, b o u n d little or no DNA, implying that DNA binding by SRY is required for sex determination. Similar results have been reported for SRYcontaining a frameshift mutation found in a human XY female [11]. SRY has been shown recently to bind to AACAAT, the target sequence of the yeast HMG box protein Rox-1, with six-fold higher affinity than to the TCF-1 binding site [12"q. SRY binding to the AACAAT motif induces a bend in the DNA helix; Giese and colleagues [12"] propose that this bending juxtaposes normally distant regulatory sequences, facilitating the assembly of higher order nucleoprotein structures involved in gene regulation. This possibility may explain the failure to detect funtional domains in SRY/Sry other than the HMG box involved in DNA binding.
Mutation studies As SRY/Sry is the pivotal gene determining the sexual fate of the embryo, one would expect to find mutations in this gene underlying at least some cases of XY sex reversal. Indeed, de novo mutations in the HMG box region in two XY w o m e n were found soon after the discovery of SRY[13,14]. Recently, three more de novo mutations have been described [15",16-], confirming bey o n d reasonable doubt that SRY is TDF. Of these five de novo mutations, three clearly inactivate the SRYprorein (one frameshift and two premature stop codons) whilst the other two change the protein more subtly (one Gly-Arg and one Met-Ile substitution). No mutations causing XY sex reversal have been described
Mammalian sex-determininggenes Koopman 605 outside the HMG b o x of SRY, adding further weight to the possibility that this is the only domain critical to the male-determining function of SRY. T w o further mutations in SRY have been found in XY females which are shared by their XY fathers (JR Hawkins and P Goodfellow, personal communication) [13]. It is thought that these variants cause conditional sex reversal depending on other genetic or environmental factors. The observation that one of these mutations reduces but does not abolish DNA-binding activity may explain its partial penetrance [10"']. Although a proportion of XY females carry mutations in the HMG box of SRY, the great majority clearly do not. These individuals are likely to have mutations in other parts of the SRY-coding sequence, in regulatory regions of the SRYgene, or in other genes in the maledetermining pathway. As more mutations in the SRYHMG box are found to be associated with XY sex reversal, a clearer picture will emerge of the amino acid positions that are critical to the male-determining ability of SRY. It is noteworthy that in the sequencing of SRY in over 100 normal XY males, not one polymorphism in the HMG box region has been detected.
Conclusion Following the discovery of SRY/Sry in 1990, evidence from expression studies in mice and genetic studies of h u m a n XY females was soon obtained to support its proposed role as the primary testis-determining gene in mammals. The causal link between Sry and male development provided by mice transgenic for Sty formally ended the search for TDF/Tdy. Recent progress has b e e n made in elucidating the role of SRY/Sry as a transcription factor in the pathway of gene regulation involved in sex determination in various species. These studies have also led to the characterization of a n e w family of transcription factors, the Sox genes, which are highly conserved in evolution suggesting that they have important but as yet u n k n o w n roles in the developing embryo. At the same time the search is continuing for genes which control or are controlled by SRY/Sry in the sex-determining cascade. The isolation of these genes and the study of their interaction with each other and with SRY/Sry will allow the genetic jigsaw of sex determination to be pieced together.
Acknowledgements I am grateful to Robin Lovell-Badge and Peter Goodfellow for sharing unpublished data.
Sry in other species Southern blots hybridized with a probe derived from the HMG box of SRYshow a male-specific band in all mammalian species tested, including human, mouse, tiger, pig, rabbit, cow, horse and chimp [2,3,17]. These observations have recently been extended to include four marsupial species [18",19"]. It appears that SRY took on its testis-determining function prior to the divergence of marsupials and eutherian mammals 80-180 million years ago. In all mammalian species a large number of bands can be seen in both sexes in addition tO the male-specific (Sry) band. These bands correspond to a conserved family of non-Y-linked genes, referred to as Sox genes (Sry-type HMG box genes), which are thought to be involved in aspects of embryo development other than sex determination (J Collignon and R Lovell-Badge, personal communication) [3]. Tiersch and colleagues [17] tested the phylogenetic conservation of SRY/Sry using Southern blot analysis of DNA from various non-mammalian species. These included birds (which have a ZZ/ZW sex-determining system where the females are heterogametic), reptilian species having temperature-dependent, genetic or ZZ/ZW sex-determining mechanisms, and lower vertebrates. In no case was any difference seen between male and female hybridization patterns. The emerging picture is that detection of a sex-specific SRY/Sry homotogue is complicated by the existence of structural and functional homologues of Sox genes, and there is no evidence to date that a functional homologue of SRY/Sry exists outside of the class Mammalia.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest •. of oustanding interest 1.
JOST A: R e c h e r c h e s s u r la D i f f e r e n t i a t i o n S e x u e l l e de l ' E m b r y o n de Lapin. Arch A n a t Microsc Morphol Exp 1947, 36:271-315.
2.
SINCLAIRAH, BERTA P, PALMER MS, HAWKINSJR, GRIFFITHS BL, SMITH MJ, FOSTER J'W, FRISCIIAUF A-M, LOVELL-BADGE R, GOODFELLOW PN: A G e n e f r o m t h e H u m a n Sexd e t e r m i n i n g R e g i o n E n c o d e s a P r o t e i n w i t h I-Iomolo g y to a C o n s e r v e d DNA-binding Motif. Nature 1990, 346:240-244.
3.
GUBBAYJ, COLLIGNONJ, KOOPMAN P, CAPEL B, ECONOMOU A, MUNSTERBERG A I VIVIANN, GOODFELLOWF, LOVELL-BADGER: A Gene Mapping to the Sex-determining Region of the M o u s e Y C h r o m o s o m e is a M e m b e r o f a N o v e l Fami l y o f E m b r y o n i c a l l y E x p r e s s e d Genes. Nature 1990, 346:245-250.
4.
HAWKINSJR, KOOPMANF, BERTA P: T e s t i s - d e t e r m i n i n g Fact o r a n d Y - l i n k e d Sex Reversal. Curt Opin Genet Dev 1991, 1:30-33.
5.
CAPEL B, LOVELL-BADGER: T h e S t y G e n e a n d S e x Determ i n a t i o n i n M a m m a l s . Adv Develop Bio 1992, in press.
KOOPMANP, GUBBAY J, VIVIAN N, GOODFELLOW P, LOVELLBADGER: Male D e v e l o p m e n t o f C h r o m o s o m a U y F e m a l e Mice T r a n s g e n i c f o r Sty. Nature 1991, 351:117-121. Demonstrates that a 14kb Y-derived genomic DNA fragment containing only Sry is sufficient to induce male development in XX transgenic mice, proving that Sty and Tdy are one and the same. 6. •.
606
Mammalian gene studies MONACO AP, MULLER U, LAR1NZ, MEIER-EWERT S, LEHRACHH: I s o l a t i o n o f t h e H u m a n Sex D e t e r m i n i n g R e g i o n f r o m a Y - e n r i c h e d Yeast Artificial C h r o m o s o m e Library. Gen o m i c s 1991, 11:1049-1053. GUBBAYJ, VIVIANN, ECONOMOU A, JACKSON D, GOODFELLOW P, LOVELL-BADGE R: I n v e r t e d R e p e a t S t r u c t u r e o f t h e S r y Locus i n Mice. P r o c N a t l A e a d Sci U S A 1992, 89:7953-7957. VAN DE WETERING M, OOSTERWEGEL M, DOOIJES D, CLEVERS H: I d e n t i f i c a t i o n a n d C l o n i n g o f TCF-1, a T L y m p h o c y t e - s p e c i f i c T r a n s c r i p t i o n F a c t o r Containi n g a S e q u e n c e - s p e c i f i c HMG Box. E M B O J 1991, 10:123-132. 10.
HARLEY MR, JACKSON DI, I~IEXTALLPJ, HAWKINSJR, BERKOVITZ GD, SOCKANATHAN S, LOVELL-BADGE R, GOODFELLOW PN: DNA B i n d i n g Activity o f R e c o m b i n a n t SRY f r o m N o r m a l Males a n d XY Females. S c i e n c e 1992, 255:453-456. Human SRY protein generated in vitro is s h o w n to bind to the motif AACAAAG in a sequence-dependent manner. SRY protein containing point mutations present in five XY females binds poorly to this sequence, implicating DNA binding in SRY function in vivo. °*
11.
NASRINN, BUGGS C, FU KONG X, CARNAZZAJ, GOEBL M, ALEXANDER-BRIDGES M: DNA-binding P r o p e r t i e s o f t h e P r o d u c t o f t h e T e s t i s - d e t e r m i n i n g G e n e a n d a Related P r o t e i n . N a t u r e 1991, 354:317-320.
12.
GIESEK, COX J, GROSSCHEDL R: T h e HMG Domain_ o f L y m p h o i d E n h a n c e r F a c t o r 1 B e n d s D N A a n d Facilitates A s s e m b l y o f F u n c t i o n a l N u c l e o p r o t e i n Structures. Cell 1992, 69:185-195. Provides evidence that AACAAT is a high affinity binding sequence for SRY protein, and that SRY acts by bending DNA, bringing normally distant regulatory sequences into close proximity. •.
13.
BERTAP, HAWKINSJR, SINCLAIRAt-t, TAYLORA, GRIEFITHSBL, GOODFELLOW PN, FELLOUS M: Genetic E v i d e n c e E q u a t i n g S R Y a n d t h e Male Sex D e t e r m i n i n g Gene. N a t u r e 1990, 348:448~450.
14.
JAGER RJ, ANVRET M, HALL K, SCHERER G: A H u m a n XY F e m a l e w i t h a F r a m e Shift Mutation i n t h e C a n d i d a t e T e s t i s - d e t e r m i n i n g G e n e S R Y . N a t u r e 1990, 348:452-454.
15.
HAWKINSJR, TAYLOR A, BERTA P, LEVILLIERS J, VAN DER AUW~ERA B, GOODFELLOWPN: Mutational A n a l y s i s o f SRY: N o n s e n s e a n d M i s s e n s e M u t a t i o n s i n X Y Sex Reversal. H u m a n Genel 1992, 88:471-474. Describes two mutations in the HMG box region of S R Y in XY females, and confirms that the frequency of XY sex reversal attributable to de n o v o mutations in S R Y i s about 10-15%, 16.
MCELREAVEY KD, VILAIN E, BOUCEKKINE C, VIDAUD M, JAUBERT F, RICHAUD F, FELLOUS M: XY S e x R e v e r s a l Associated with a N o n s e n s e Mutation i n SRY. Genomics 1992, 13:838-4340. A further mutation giving rise to XY sex reversal is described. 17.
TIERSCH TR, MITCHELLMJ, WACHTELSS: Studies o n t h e Phylogenetic Conservation of the SRY Gene. Human Genet 1991, 87:571-573.
18.
FOSTER JW, BRENNAN FE, HAMPIKIAN GK, GOODFELLOW PN, SINCLAIR AH, LOVELL-BADGE R, SELWOOD L, RENFREE MB, COOPER DW, MARSHALL GRAVES JA: The H u m a n Sex D e t e r m i n i n g G e n e S R Y Detects H o m o l o g o u s Seq u e n c e s o n t h e M a r s u p i a l Y C h r o m o s o m e . N a t u r e 1992, 359:531-533. Provides a 'missing link' in the evidence that S R Y / S r y is the primary testis-determining gene by showing that marsupials have an homologous gene mapping to the Y chromosome. 19.
BIANCHINO, BLANCH] MS: Male-specific S t y P a t t e r n s i n M a r s u p i a l s . J M a m m o l 1992, in press. Two marsupial species are s h o w n to have a male-specific homologue of SRY/Sry.
P Koopman, Centre for Molecular Biology and Biotechnology, The University of Queensland, Brisbane 4072, Australia.
Introduction The development of male or female characteristics in the embryo is the result of a combination of sex determination and subsequent sexual differentiation. Whilst the distinction between these two processes is nebulous it is generally agreed, based on Drosophila and nematode models, that sex determination in mammals involves a pathway of gene regulation. Understanding mammalian sex determination at the molecular level relies on isolating and studying all the genes in this pathway. The peculiar biology and genetics of sex determination have resulted in one of the genes from this pathw a y being singled out for special attention by molecular geneticists. The pioneering experiments of Jost [1] demonstrated that the testes are required for male development of rabbit fetuses; if the testes are removed, females develop. This observation suggested that in eutherian mammals, testis development is the key event that determines the sexual fate of the embryo. Cytogenetic analyses showed that sex in mammals is determined by the presence or absence of the Y chromosome, unlike the situation in Drosophila and Caenorhabditiseleganswhere the ratio of X chromosomes to autosomes provides the sex-determining switch. Combining these observations led to the theory that the Y chromosome contains a dominant locus which triggers maleness in mammals by directing testis formation from the bipotential embryonic gonad. This locus was said to encode 'testis-determining factor' and was dubbed TDF in humans and Tdy in mice. The search for TDF/Tdywas stimulated not only by its pivotal role in the sex determination pathway, but also by the amenability of this locus to genetic analysis in comparison with other loci involved in sex determination, the existence of which is assumed but not proven. First, the chromosomal location of TDF/Tdyhad been firmly established, mapping to the Y short arm in both man and mouse. Second, the proximity of TDF to the region of X-Y pairing and exchange means that TDFis occasionally transferred to the human X chromosome
by an accident of nature. This type of genetic accident results in sex reversal - maleness in XX individuals and is identified easily when these men seek treatment for sterility. Analysis of the Y-unique DNA on the X chromosomes of four such XX males allowed the positional cloning of the TDF candidate SRY[2], and its murine homologue Sty [3]. The properties of SRY/Srythat have allowed us to equate it genetically and functionally with TDF/Tdy are discussed elsewhere [4,5]. The purpose of this review is to summarize the progress of the past year in illuminating the function of SRY/Sryand its relationship to the genetic pathway of sex determination.
Transgenic mice Evidence equating SRY/Sry and TDF/Tdy culminated in the demonstration that S t y can bring about normal male development in chromosomally female (XX) transgenic mice [6"']. Sry was injected as a 14 kb fragment of Y-derived genomic DNA, named L741. Sequence analysis of L741 has shown that this fragment contains no genes other than Sry. These experiments provided long awaited proof that only one gene from the Y chromosome is required for male sex determination (although others are clearly associated with male-specific functions, such as spermatogenesis). Not suprisingly, some of the XX mice transgenic for L741 did not show sex reversal. Transgenes are generally susceptible to position effects which may suppress their expression depending on their site of integration into the genome. Founder mouse m32.10 had multiple copies of the Sty transgene and was a fertile female, allowing a transgenic line to be bred and expression of Sty to be examined amongst the offspring. Two findings have resulted from the analysis of this line (N Vivian, P Koopman and R Lovell-Badge, unpublished data). First, Sty was found to be expressed in embryos of this line, ruling out a lack of expression
Abbreviations bp--base pair; HMG--high mobility group.
© Current Biology Ltd ISSN 0958-1669
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604
Mammaliangene studies due to position effects as an explanation for the lack of sex reversal. Second, some of the XX progeny of line 32.10 develop as males. These observations imply that a threshold of Sry activity is required for its function in male development, and that Sty expression in line 32.10 is close to this threshold; mice expressing Sty below the threshold level develop as females, whilst the few w h o s e expression exceeds the threshold level develop as males. An accurate assay for Sry expression is required to test this hypothesis and to define the threshold level. Efforts of several laboratories have failed to elicit sex reversal in mice w h e n human SRY is used as a transgene, even though the human gene is capable of being expressed in the genital ridge of transgenic embryos [6"]. It is not yet k n o w n whether this is due to a failure of the SRY protein to interact with mouse genes or proteins involved in male sex determination, or whether the regulation of the h u m a n gene in mice is subtly defective. Domain swap experiments will be important in clarifying these possibilities. A recently isolated yeast artificial c h r o m o s o m e containing human SRYwith 100 kb of 5' and 50 kb of 3' flanking sequence may be useful in the production of transgenic mice in which SRY is present in the context of its normal regulatory sequences [7].
Structure of the
Sry gene
Sequence analysis of genomic clones containing human and mouse SRY open reading frames s h o w e d that they contain a conserved, 237-base-pair (bp) region with h o m o l o g y to a motif that has come to be k n o w n as an HMG box. HMG boxes are found in a growing n u m b e r of eukaryotic genes encoding, amongst other things, the e p o n y m o u s high mobility group (HMG) proteins and a number of transcription factors (see below). As the HMG b o x is k n o w n to confer DNA-binding ability on several transcription factors, SRY/Sry is also assumed to be a transcription factor. Full length cDNA sequences have not yet been obtained for either mouse Sry or h u m a n SRY. These genes are k n o w n to e n c o d e mRNAs of approximately 1.2 kb in adult testes [2,3]. The HMG b o x of human SRY resides in a 670 bp open reading frame flanked by an 80 bp putative 3' untranslated region with a potential polyadenylation signal, suggesting that this is the final exon of the gene. There is some evidence that h u m a n SRY is encoded by a single exon (P Goodfellow and colleagues, personal communication). The coding sequence outside the HMG box region does not appear to contain motifs associated with transcriptional activation or repression, or show h o m o l o g y to any k n o w n genes. The structure of the mouse Sry gene is even less clear, although it may have more than one exon. Lack of h o m o l o g y between h u m a n SRY and mouse S ~ sequences outside the HMG box region casts some doubt
on the functional significance of these sequences. Curiously, the mouse Sty open reading frame is flanked in the genome by a large inverted repeat involving at least 15.5 kb on either side [8]. This duplication arose before the divergence of the Mus musculus musculus and M.m.domesticus subspecies 1-2 million years ago, yet the two arms of the repeat differ in only a few nucleotide positions and microsatellite polymorphisms. The significance of this repeat structure remains a mystery.
DNA binding studies The HMG box region of several transcription factors has b e e n shown to bind directly to specific DNA sequences. In particular, the T-cell transcription factor TCF-1 binds to the sequence AACAAAG [9]. As SRY is more closely related to this transcription factor than to any other HMG b o x protein, Harley and colleagues [10"'] tested the ability of SRY protein produced in vitro to bind to this sequence. Binding was confirmed by gel retardation assays, and mutations in positions 2,4,5 and 6 of the heptanucleotide sequence abolished binding, indicating that binding is sequence specific. In addition, mutant SRY protein, containing the amino acid substitutions found in five XY females, b o u n d little or no DNA, implying that DNA binding by SRY is required for sex determination. Similar results have been reported for SRYcontaining a frameshift mutation found in a human XY female [11]. SRY has been shown recently to bind to AACAAT, the target sequence of the yeast HMG box protein Rox-1, with six-fold higher affinity than to the TCF-1 binding site [12"q. SRY binding to the AACAAT motif induces a bend in the DNA helix; Giese and colleagues [12"] propose that this bending juxtaposes normally distant regulatory sequences, facilitating the assembly of higher order nucleoprotein structures involved in gene regulation. This possibility may explain the failure to detect funtional domains in SRY/Sry other than the HMG box involved in DNA binding.
Mutation studies As SRY/Sry is the pivotal gene determining the sexual fate of the embryo, one would expect to find mutations in this gene underlying at least some cases of XY sex reversal. Indeed, de novo mutations in the HMG box region in two XY w o m e n were found soon after the discovery of SRY[13,14]. Recently, three more de novo mutations have been described [15",16-], confirming bey o n d reasonable doubt that SRY is TDF. Of these five de novo mutations, three clearly inactivate the SRYprorein (one frameshift and two premature stop codons) whilst the other two change the protein more subtly (one Gly-Arg and one Met-Ile substitution). No mutations causing XY sex reversal have been described
Mammalian sex-determininggenes Koopman 605 outside the HMG b o x of SRY, adding further weight to the possibility that this is the only domain critical to the male-determining function of SRY. T w o further mutations in SRY have been found in XY females which are shared by their XY fathers (JR Hawkins and P Goodfellow, personal communication) [13]. It is thought that these variants cause conditional sex reversal depending on other genetic or environmental factors. The observation that one of these mutations reduces but does not abolish DNA-binding activity may explain its partial penetrance [10"']. Although a proportion of XY females carry mutations in the HMG box of SRY, the great majority clearly do not. These individuals are likely to have mutations in other parts of the SRY-coding sequence, in regulatory regions of the SRYgene, or in other genes in the maledetermining pathway. As more mutations in the SRYHMG box are found to be associated with XY sex reversal, a clearer picture will emerge of the amino acid positions that are critical to the male-determining ability of SRY. It is noteworthy that in the sequencing of SRY in over 100 normal XY males, not one polymorphism in the HMG box region has been detected.
Conclusion Following the discovery of SRY/Sry in 1990, evidence from expression studies in mice and genetic studies of h u m a n XY females was soon obtained to support its proposed role as the primary testis-determining gene in mammals. The causal link between Sry and male development provided by mice transgenic for Sty formally ended the search for TDF/Tdy. Recent progress has b e e n made in elucidating the role of SRY/Sry as a transcription factor in the pathway of gene regulation involved in sex determination in various species. These studies have also led to the characterization of a n e w family of transcription factors, the Sox genes, which are highly conserved in evolution suggesting that they have important but as yet u n k n o w n roles in the developing embryo. At the same time the search is continuing for genes which control or are controlled by SRY/Sry in the sex-determining cascade. The isolation of these genes and the study of their interaction with each other and with SRY/Sry will allow the genetic jigsaw of sex determination to be pieced together.
Acknowledgements I am grateful to Robin Lovell-Badge and Peter Goodfellow for sharing unpublished data.
Sry in other species Southern blots hybridized with a probe derived from the HMG box of SRYshow a male-specific band in all mammalian species tested, including human, mouse, tiger, pig, rabbit, cow, horse and chimp [2,3,17]. These observations have recently been extended to include four marsupial species [18",19"]. It appears that SRY took on its testis-determining function prior to the divergence of marsupials and eutherian mammals 80-180 million years ago. In all mammalian species a large number of bands can be seen in both sexes in addition tO the male-specific (Sry) band. These bands correspond to a conserved family of non-Y-linked genes, referred to as Sox genes (Sry-type HMG box genes), which are thought to be involved in aspects of embryo development other than sex determination (J Collignon and R Lovell-Badge, personal communication) [3]. Tiersch and colleagues [17] tested the phylogenetic conservation of SRY/Sry using Southern blot analysis of DNA from various non-mammalian species. These included birds (which have a ZZ/ZW sex-determining system where the females are heterogametic), reptilian species having temperature-dependent, genetic or ZZ/ZW sex-determining mechanisms, and lower vertebrates. In no case was any difference seen between male and female hybridization patterns. The emerging picture is that detection of a sex-specific SRY/Sry homotogue is complicated by the existence of structural and functional homologues of Sox genes, and there is no evidence to date that a functional homologue of SRY/Sry exists outside of the class Mammalia.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest •. of oustanding interest 1.
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SINCLAIRAH, BERTA P, PALMER MS, HAWKINSJR, GRIFFITHS BL, SMITH MJ, FOSTER J'W, FRISCIIAUF A-M, LOVELL-BADGE R, GOODFELLOW PN: A G e n e f r o m t h e H u m a n Sexd e t e r m i n i n g R e g i o n E n c o d e s a P r o t e i n w i t h I-Iomolo g y to a C o n s e r v e d DNA-binding Motif. Nature 1990, 346:240-244.
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GUBBAYJ, COLLIGNONJ, KOOPMAN P, CAPEL B, ECONOMOU A, MUNSTERBERG A I VIVIANN, GOODFELLOWF, LOVELL-BADGER: A Gene Mapping to the Sex-determining Region of the M o u s e Y C h r o m o s o m e is a M e m b e r o f a N o v e l Fami l y o f E m b r y o n i c a l l y E x p r e s s e d Genes. Nature 1990, 346:245-250.
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HAWKINSJR, KOOPMANF, BERTA P: T e s t i s - d e t e r m i n i n g Fact o r a n d Y - l i n k e d Sex Reversal. Curt Opin Genet Dev 1991, 1:30-33.
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Mammalian gene studies MONACO AP, MULLER U, LAR1NZ, MEIER-EWERT S, LEHRACHH: I s o l a t i o n o f t h e H u m a n Sex D e t e r m i n i n g R e g i o n f r o m a Y - e n r i c h e d Yeast Artificial C h r o m o s o m e Library. Gen o m i c s 1991, 11:1049-1053. GUBBAYJ, VIVIANN, ECONOMOU A, JACKSON D, GOODFELLOW P, LOVELL-BADGE R: I n v e r t e d R e p e a t S t r u c t u r e o f t h e S r y Locus i n Mice. P r o c N a t l A e a d Sci U S A 1992, 89:7953-7957. VAN DE WETERING M, OOSTERWEGEL M, DOOIJES D, CLEVERS H: I d e n t i f i c a t i o n a n d C l o n i n g o f TCF-1, a T L y m p h o c y t e - s p e c i f i c T r a n s c r i p t i o n F a c t o r Containi n g a S e q u e n c e - s p e c i f i c HMG Box. E M B O J 1991, 10:123-132. 10.
HARLEY MR, JACKSON DI, I~IEXTALLPJ, HAWKINSJR, BERKOVITZ GD, SOCKANATHAN S, LOVELL-BADGE R, GOODFELLOW PN: DNA B i n d i n g Activity o f R e c o m b i n a n t SRY f r o m N o r m a l Males a n d XY Females. S c i e n c e 1992, 255:453-456. Human SRY protein generated in vitro is s h o w n to bind to the motif AACAAAG in a sequence-dependent manner. SRY protein containing point mutations present in five XY females binds poorly to this sequence, implicating DNA binding in SRY function in vivo. °*
11.
NASRINN, BUGGS C, FU KONG X, CARNAZZAJ, GOEBL M, ALEXANDER-BRIDGES M: DNA-binding P r o p e r t i e s o f t h e P r o d u c t o f t h e T e s t i s - d e t e r m i n i n g G e n e a n d a Related P r o t e i n . N a t u r e 1991, 354:317-320.
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GIESEK, COX J, GROSSCHEDL R: T h e HMG Domain_ o f L y m p h o i d E n h a n c e r F a c t o r 1 B e n d s D N A a n d Facilitates A s s e m b l y o f F u n c t i o n a l N u c l e o p r o t e i n Structures. Cell 1992, 69:185-195. Provides evidence that AACAAT is a high affinity binding sequence for SRY protein, and that SRY acts by bending DNA, bringing normally distant regulatory sequences into close proximity. •.
13.
BERTAP, HAWKINSJR, SINCLAIRAt-t, TAYLORA, GRIEFITHSBL, GOODFELLOW PN, FELLOUS M: Genetic E v i d e n c e E q u a t i n g S R Y a n d t h e Male Sex D e t e r m i n i n g Gene. N a t u r e 1990, 348:448~450.
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JAGER RJ, ANVRET M, HALL K, SCHERER G: A H u m a n XY F e m a l e w i t h a F r a m e Shift Mutation i n t h e C a n d i d a t e T e s t i s - d e t e r m i n i n g G e n e S R Y . N a t u r e 1990, 348:452-454.
15.
HAWKINSJR, TAYLOR A, BERTA P, LEVILLIERS J, VAN DER AUW~ERA B, GOODFELLOWPN: Mutational A n a l y s i s o f SRY: N o n s e n s e a n d M i s s e n s e M u t a t i o n s i n X Y Sex Reversal. H u m a n Genel 1992, 88:471-474. Describes two mutations in the HMG box region of S R Y in XY females, and confirms that the frequency of XY sex reversal attributable to de n o v o mutations in S R Y i s about 10-15%, 16.
MCELREAVEY KD, VILAIN E, BOUCEKKINE C, VIDAUD M, JAUBERT F, RICHAUD F, FELLOUS M: XY S e x R e v e r s a l Associated with a N o n s e n s e Mutation i n SRY. Genomics 1992, 13:838-4340. A further mutation giving rise to XY sex reversal is described. 17.
TIERSCH TR, MITCHELLMJ, WACHTELSS: Studies o n t h e Phylogenetic Conservation of the SRY Gene. Human Genet 1991, 87:571-573.
18.
FOSTER JW, BRENNAN FE, HAMPIKIAN GK, GOODFELLOW PN, SINCLAIR AH, LOVELL-BADGE R, SELWOOD L, RENFREE MB, COOPER DW, MARSHALL GRAVES JA: The H u m a n Sex D e t e r m i n i n g G e n e S R Y Detects H o m o l o g o u s Seq u e n c e s o n t h e M a r s u p i a l Y C h r o m o s o m e . N a t u r e 1992, 359:531-533. Provides a 'missing link' in the evidence that S R Y / S r y is the primary testis-determining gene by showing that marsupials have an homologous gene mapping to the Y chromosome. 19.
BIANCHINO, BLANCH] MS: Male-specific S t y P a t t e r n s i n M a r s u p i a l s . J M a m m o l 1992, in press. Two marsupial species are s h o w n to have a male-specific homologue of SRY/Sry.
P Koopman, Centre for Molecular Biology and Biotechnology, The University of Queensland, Brisbane 4072, Australia.
