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Steroid sulphatase and the conservation of mammalian X chromosomes
TIG - - August 1986
reviews Steroid sulphataseand the conservation of mammal ian X chromosomes
The X and Y chromosomes of mammals are remarkably different from each other. Whereas the X chromosome appears to cant genetic informationin proportion to its size relative to that of other chromosomes, the Y chromosome, apart from its role lan W. Craig and Elinor Tolley in male determination, appears to be largely iner~ The evolution A g.eneforsteroidsulphatase is locatedcloseto the recentlyestablisbed'pseudoautosomal" of this heteromorphic chromoregion at tl~ @ of the human X chromosome. E..,_~me~,eb in f ~ exc~d those in some pair has been accommales, and tl~'e is evidence that this unusual locus escapesfrom the flormalpattern of panied, or possibly even preX-inactivagon. In mice, hou~er, enzyme levelsin the twosexesare similar; thisis t&mght ceded, by the development of a to be achieved by the expression of a Y-linked allele. mechanism resulting in the inactivation of one of the two X chromosomes in female esterol sulphate, oestrogen sulphate (OES) and desomatic cells. The consequent dosage compensation hydroepiandrosterone sulphate (DHES), as well as to ensures a similar level of message transcripts in male the synthetic substrates, p-nitrophenyl sulphate and 4and female cells and therefore maintains the same ratio methyl umbelliferone sulphate~'s. The literature has of autosomal to X-chromosomal gene products in both been unclear concerningthe relationship between these sexes. The range of X-linkedloci is constant in different enzymes and aryl sulphatase C, which is also micromammals; indeed, Olmot has argued that conservation somal; however, genetic evidence indicates that one is a necessary consequence of dosage compensation. enzyme has activity towards the majority of these Alterations to the distn'outionof gene loci between the X substrates. Clinical interest in the roles of these chromosome and autosomes are likely to be selectively microsonmlenzymes was aroused followingreports that disadvantageous since they would change the relative low levels, or absence, of steroid sulphatase activity in dosage of gene products. placentae was associated with late parturition (see Ref. Against this evolutionarybackground, the genetics of 5); monitoring the levels of the enzyme product steroid sulphatase are enigmatic. The locus for this (oestfioi) in later stages of pregnancy is a routine aspect otherwise unexceptional enzyme is X-linked in humans of prenatal care. It was subsequently recognizede that but, unlike the majority of other X-chromosomalgenes, the children at risk were male and that they frequently it appears to escape from inactivation and there is developed, in early childhood, the classical symptoms of evidence for a Y-linkedallele in mice. The close linkage the X-linked variety of a skin disease called ichthyosis of steroid sulphatase to other sex chromosome god, 0iteral!y, fish-like skin). The simplest interpretation of including those which may have a role in sex determi- the infounation available is that the lesion in ichthyosis nation, together with its atypical pattern of regulation, results from a defect at a single (X-chromoson@) locus. have singled it out as a target for investigation. Because affected males lack enzyme activity towards However, it was for different and more prosaic reasons several substrates includingOES, DHES and those used that the genetics of this enzyme first attracted attention. to define aryl sulphatase C, it is probable that a single gene product (hereafter referred to as steroid sulphatase, STS) is active toward the sulphate esters of Physiological role The aryl sulphatases are enzymes capable of hydro- several physiologically important steroids and to the lysinga ~ d e range of sulphate esters and several forms, artificial substrates. Alternatively, the expression of the designated A, B, and C have been described. They were X-linked locus may be necessary for the activity of a first distinguished on the basis of their substrate group of related enzymes sharing common control or specificity, pH optima and pattern of cellularlocalization. structural subonits. The former argument is the more Aryl sulphatases A and B are lysosomal and catalyse the straightforward and is also consistent with the genetic hydrolysis of a variety of natural and synthetic sulphate behaviour of the STS locus in somatic cell hybrids (see esters. The two enzymes have been assigned to below). Placental STS has been purified from microsomal chromosomes 22 and 5, respectively. Aryl sulphatase A has activity towards naturally occurring sulpholipids, fractions by treatment with detergentss and the apincludingcerebroside 3-sulphates found in brain, kidney parent molecular weight of the major polypeptide and testes. The presence of a galactose 3-sulphate observed was between 57000 and 62000. Recently, moiety appears to be a necessary feature of the antfeodies have been raised against the purified protein substrate structure recognized by this enzyme. Its and used to demonstrate the lack of immunologically deficiency is associated with metachromatic leucodyl- detectable enzyme protein in X-linkedichthyosis7. Such trophy, a neurological disorder characterized by the reagents may provide an approach to the identificationof accumulationof cerebroside sulphate in nervous tissues. the gene. Some reports have suggested that low levels of STS Aryl sulphatase B catalyses the hydrolysis of UDP Nacetyl galactosamine 4-sulphate, a constituent of con- may indicate a predisposition to testicular tumours8, but nective tissue. A deficiency of this enzyme (Maro- a recent survey of levels in affected patients and controls teaux-Lamy syndrome) causes gross skeletal deformi- failed to confirm this correlation9. ties and corneal opacity2. In addition to these well defined sulphatases, several Genetiebaekground In addition to the clinicalfeatures outlinedabove, two microsomal enzymes have been characterized which have activity toward steroid sulphates, including chol- further considerations have contn~uted to current 201 ~}1986, FAsevierScien:ePeblish~B.V., ~ 0168-9525/86/$0.200
reviews
~
TIG - - August 1986
MIC2
P
p 1.~ q 1 ~.
active human X chromosome can be achieved subsequently by back selection in a medium containing 6thioguanine. This has enabled the isolation of several hybrids which retain an inactivehumanX chromosome in_ the absence of an active counterpart. Such hybrids have MIC2 been found to express amounts of STS far in excess of TDF the very low amounts detected in the rodent parental lines and, although there is no unambiguous proof that the enzyme expressed is the human one, the circumstantial evidence for an active STS locus on the otherwise inactive human X chromosome is compelling.
G6PD
2E ~
2
StS
Crm
4
SiS X
Y
o/interest. interest in the enzyme. Followingthe realization that the enzyme defect was not confined to placental tissue, assays have been carried out on a wide variety of cell types. An investigation of more than I00 tibroblast clones from a female heterozygous for X-linked ichthyosis found them all to have substantial STS activityl°; a finding in contrast to the expected pattern for Xinactivation, in which about equal numbers of positive and negative clones should result. This observation provided evidence that a second X-linked genetic marker escaped from inactivation. The X-linked blood group Xg had previously been reported to act in this way but, because its expression is confined to red cells, unambiguouscbaracte~ation of its behaviour had been difficult. Data that have accumulated on the levels of enzyme in norn~ male and female fibroblasts and leucocytes entirely support the concept that the STS locus is not dosage compensated (see Refs 11 and 12). Females have substantially higher levels of STS activity than males, although not necessarily twice as much (see below). C o n v i n ~ evidence of its escape from inactivationhas come from studies on somatic cell hybrids. Humun-rodent cell hybrids are often selected on the basis of complementation, by the human parent, of an Xlinked enzyme defect (HPRT deficiency)in the mouse, or hamster, parent. Eliminationof hybrids retmalng an 2O?.
Partial or complete escape from inactivation? A survey of the information available for the female: male ratios for STS levels has revealed an average value of 1.6 for libmblasts and placentae, substantially less than that expected for a direct dosage relationship. Ratios observed for peripheral white cells are even lower. Furthermore, individualswith multiple X chromosomes do not exhibit the proportionately high levels of enzyme anticipated (see Refs 11 and 12). Escape from inactivation may therefore not be complete. An elegant study has pro'sued this poss~llity further by eyaaniningSTS levels in fibroblast clones from ~ndividnalsheterozygous for deficienciesin both STS and for the X-linked marker glucose 6-phosphate dehydrogenase 11. This enabled the identification of dunes in which one or other of the two X chromosomes was active and the demonstration that the STS+ allele was expressed at about half the level from the inactive chromosome compared with its expression from the active homologue. Part~ ina~'vation is the most satisfactory explanatiou for this observation and would also account for the deviation from a strict dosage relationship observed for the female:male ratio. X0 individuals and STS-deficiency heterozygotes have enzyme levels below the normal male range, the heterozygotes signific.~tly so; this observation could result from the partial random inactivation of the normal allele. Although a Y-linked STS locus has been postulated for mice (see below), there is no substantial evidence supporting tles suggestion for humans. Studies on cells carcying an X-autosomal translocation in which the breakpoint is above the STS locus (demonstrated by the retention of high levels of enzyme in human-mouse hybrids retaining the 'derived-X' chromosome) indicated that the normal X was preferentially inactivated in vivo. This suggests that the translocation deletes a chromosomal feature which norreally confers local resistance to inactivation. Random inactivation of the derived X lacking this postulated feature could therefore be transmitted through the S'I~ region into the autosonml material attached. This would result in a decreased output of autosomal gene products and would explain the observed preferential advantage to cells with the normal X chromosome inactivatedIs. Chromosomal localization Two approaches have independently assigned the STS locus to the tip of the human X short ann (see Fig. 1). Early studies (see Ref. 5) of somatic cell hybrids retaining different X-autosomal rearrangements enabled the assignment of STS and, by virtue of its close linkage, Xg to the distal portion of the short arm (Xp22ter) or (Xp11-ter). Further refinement of the map po~itioa ~oXp223-ter was achieved later by studies on a
reviews
TIG - - August 1986
hybrid carrying a resn-anged X chromosome which lacked only this tenninal segment (See Ref. 14). A similar location has also been suggested by observations on ichthyotic males who lacked STS and who were karyotypically abnormal. Such patients are typically e~racterized by ap~.ently unbalanced X-Y trans. locations resulting from an interdmnge which deleted the terminal segment of the X chromosome, Xp223-ter 0tefs 15 and 16). There is little doubt that both STS and Xg are located at the short ann terminus of the X chromosome and we are therefore presented with an intriguing pattern of observations which distinguish this potlion from the remainder of the X chromosome. Firstly, studies on rnei,otic preparations indicate that this region is preferenEallyinvolved in pairing with the Y chromoson~ 17and that this ~ g may be accompanied by an obligate exchangeTM. Secondly, the region contains loci which escape from inactivation and thirdly, it has been suggested that it contains a regulator gene for sex detennimtion (see Ret. 19). The consensus view is that the distal short arms of the X and Y chromosomes represent a conserved remnant of an original pair of homologous chromosomes. Very recently, pseudoautosomal sequences have been d ~ which are regularly exchanged in male meiosis and are presumed to be located in a region of active recombination near the short ann termini. A decreasing probability of exchange is observed for sequences further from the telomeres (see Ref. 18 and Fig. 2). The gene (MIC2) coding for the cell surface,antigen 12E7 has been cloned and assigned, by in situ hybridization, to the tips of both the X and Y chromosomesm,9-t. The STS locus in humans must lie below the region of X-Y exchange, as no convindng evidence for Y localization has been reported. Furthermore, the dMerence in enzyme levels observed for males and females is inconsistent with the existence of a Y-linked locus (that is, one expressed in the normal range of tissues examined). The location of the STS locus in this region and the relatively simple assay for its gene product have led to its use as a marker for investigations into the aetiology of XX males and other dysgenic sexual types. A combination of studies has suggested that at least some XX males result from an aberrant exchange between the X and Y chromosomes at paternal meiosis- resulting in the transmission to the affected offspring of an X chromosome which has exchanged its distal short ann sequences for Y short ann material including the putative testis-detennining factor (TDF)2z.m. Whereas presence of Y sequences can be detected using male-specific probes, loss (or inactivation) of distal X material may be inferred from the STS levels observed for some individuals, which are in the male range (see Ref. 5). Steroid sulphatase in rodents The rationale explaining the conservation of the X chromosome does not extend to genes which escape from inactivation. The behaviour of the STS locus in this context in other mammals is therefore of interest. Apart from a preliminary observation sugges"_tij]g.the absence of X-linkedSTS in AustrAliAnmarsupials~, most studies have concentrated on wood-lemmings and mice. Woodlemmings have an unusual sex deter "n.ingmechanism in which two different types of X chromosome participate. STS levels in various classes of woed-lemming were found to correlate with the number of X
chromosomes2s. As in humans, the gene appears to be X-linked, but not subj__e_ectto inactivation. Mice also have an X-linked STS locus~, but do not show sex differences in dosage and there is no evidence for loci which escape from inactivation on the mouse X chromosome~ This observation, remarkable in fight of the evidence from wnod-lemmings, could be explained in two ways. If X linkage is accepted, then either the locus must be subjected to the normal pattern of inactivation, or there must be, additionally, a Y-linkedallele bringing the level of enzyme of the male into a range similar to that of the female. The second suggestion is favoured by Keitgss et al.m, who were able to demonstrate, in different types of crosses, that STS deficiency could behave either as an X-linked or as an autosonml recessive trait. These observations would be explained if both the X and Y chromosomes possess functional STS alleles which undergo obfigstory recombination during meiosis. The TDF in mice is located proxit~Jy on the Y chromosome and therefore not at risk of being included in the recombination event. In this context, it is interesting to note that in the progeny of crosses with male mice carrying the sex r e v e r s ~ trait (Sxr), the levels of
Xpter
Ypter r"
m
!
Xg
TDYgl=
STS
p
II II II
II II II
X
Y
Fig. 2. $ ~ dia~am of s~rt arm terminal ~ of hummz X and Y chfomosome.~indicating the pseudmutosomal which has ~ e s ~ to amtain 2000-6000 kb (see Refs lg ami30). Thm is ~ that MIC2 may be ~ at ~ ~ CP. C,oo~eaow, pen. an,ram.). Yg is t ~ p~uz~tiw ~ t o ~ b ~ Snup ~ Xe. $ ~ is placed below TDF because the pfem~gve X - Y e~w2ua~ r ~ g n g in XX w l ~ is o~y ~ a ~ o d ~ u~thto~ of S'l'S a c ~ . 203
reviews
TIG - - August 1986
steroid sulphatase are similar in XX males, normal males, males carrying Sxr and normal females 27, suggesting that the duplication of the male-determining region on the Y chromosome of the Sxr male does not include the STS locus. Recent studiesz9 suggest that Sts is indeed linked to the most distally located, conventional marker on the X chromosome (Cream; see Fig. 1). This linkage, which was demonstrated through female meiosis, is not incompatible with the pseudoautosomal behaviour of the postulated Y-linked locus in males, given the differences in recombination observed for the region in human males and femalesa°. Conclusion The intriguing behaviour of the steroid sulphatase locus and its significance in terms of understanding fundamental biological problems, including that of the control of inactivation processes and the evolutionary relationship between the mammalian X and Y chromosomes, make it an attractive target for direct molecular studies. Difficultiesin the purificationof the protein have so far prevented the isolation of coding sequences through oligonucleotideor expression vector strategies; nevertheless, one can be confident that the cloning of the structural gene will generate great interest among the afidonados of mammalian sex chromosome genetics. References
9 Tolley, E., Craig, I. W., Jomsson, J., CartwrighL It/L and Jones, W. G. (1985) Lcmcet i, 563 10 Shapiro, L. J., Mohandas, T., Weiss, R. and Romeo, G. (1979) Science 204, 1224 11 Migeon, B. R,, Shapiro, L.J., Norm, R. A., Mohandas, T, Axelman, J. and Dabora, R. L (1982) Nature 229, 838-840 12 Lykkesfeld,G,, Lykkesfeld,A. E. andShakkabaeck,N. E. (1984) Hum. Genet 65, 355-357 13 Crocker, M., Jonasson, H. and Patei, C. (1985)Hum. Gcnet. 28, 556-560 14 Miller, O. J, and Siniscalco, M. (1982) Cytogenct Cell Greet. 32, 179-190 15 Tiepelo, L., Zuffar~, 0., Fraccaro, M., di Natale, D.o Garganflni, L., Muller, C. P,,. and Ropera, H. H. (1980)Hum. Greet. 54, 205--206 16 Ferguson-Smith, M. A., Sanger, It, TippeR, P., Aitken, D. A. and Boyd, E. (1982)Cytogcnet. Cell Greet. 32, 273-274 17 Pearson, P. L. and Bobrow, M. (1970) Nature 226, 959-961 18 Burgoyne, P. S. (1986) Nature 319, 258-259 19 Polani, P. E. (1982)Hum. Genet. 60, 207-211 20 Darling,S. M., Banting, G. S., Pym, B., Wolfe,J. andGoodfellow, P. N. (1986) Proc. Natl Acad. Sd. USA 83, 135-139 2/ Buckle, V., Mondello, C., Darling, S.M., Craig, I.W. and Goodfellow, P. N. (1985) Nature 317, 739-740 22 Page, D. C., de la Chapelle, A. and Weissenhach, J. Nature 315, 224-227 23 Guellaen, G., Cassanova, M., Bishop, C. E., Geldwerth, D., Andre, G., FeHous, M. and Weissenhach, J. (1984) Nature 307, 172-173 24 Cooper, D. W., McAIlen, B. M., Donald, J. A., Dawson, G., Dobrovic~ A. and Graves, A. M. (1984)Cytogenct. CeliGmet 37, 439 25 Ropers, K H. and Wiberg, U. (1982) Nature 296, 766-768 26 Gm'tler, S. M. and Rivest, M. (1983) Gencgcs 103, 137-141 27 Crocker, N. and Craig, I. W. (1983) Nature 303, 721-722 28 Keitges, E., Rivest, M., Siniscalco, M. and Gartler, S. M. (1985) Nature 315, 226-227 29 Cattenach, B. and Crocker, M. (1986) Mouse News Letter 74, 94-95 30 Rouyer, F., Simmler, M-C., Jolmsson, C., Vergnand, G., Cooke, H. J. and Weissenbach, J. (1986)Nature 319, 291-295
I Olmo,S. (1967)SexCbro~o~a~ISezLinkedGu~s, Springer Verlag 2 Farooqui,A. A. andMandel, P. (1977)Int. ]. Biochem. 8, 685-691 3 lwamori, M., Moser, H. W. and Kishimoto, Y. (1976) Arch. Biod~n. Biop~s. 174, 199-208 4 Dolly,J. O., Dodgson, K. S. and Rose, F. A. (1972)B~chem. ]. 128, 337-345 5 Shapiro, L. J. (1985)Adv. Hum. Genet. 14, 331--381 6 Shapiro, L. J., Weiss, R., Btvamn, M. M., Vidgoff,J., Dimond, R. L., Roller, J. A. and Wells, R. S. (1978) Lancet fi, 756-757 7 Epstein, E. H. and Bonifas,J. M. (1985)Hum. Genct. 71, 201-210 L IV. Craig and E. Tolley are at the Gozetics Laboratory, 8 Lykkesfeldt, G., Hoyer, H., Lykkesfeldt, A. E. and Shakkabaek, University of Oxford, South Parks Road, Oxford OXI 3QU, L. E. (1983) Lancet ii, 1456 UK.
Suppression is defined as the reversal of a mutant phenotype due to a mutation at a site distinct from that giving rise to the mutant phenotype. The result is a revertant produced by Eric Kubli the interaction of either allelic (intragemc suppression) or nonallefic (intergenic suppression) Mobile elements contain a vasty of r@ulato~ sigaals which can profoundly affect the mutant genes. Intergenic supexpressionofgenes near the sites at which the elementsare insertedin the gonome. These effectscan be compensatedbysuppressormutalfims. Thus, geneticand molecularsb~iies pression can be exerted at the of suppressionphenomena can yield important insights into the mechanism of eukaryotic transcriptional, translational or gene r@ulagon. post-translational level. This review concentrates on recent advances in our understanding of molecular aspects of intergenic suppression involving part of it belonging to three distinct classes of MEs: mobile elements in Drosophila melano&aster. The copia-like-, foldback- and P-elements. In Drosophila, all mutations and suppressors that willbe described act on mobile elements involved in suppression phenomena the affected genes at the level of transcription. that have been studied at the molecular level belong to the copia family~ (e.g. copla, gypsy, 412, etc.). They all Mobile elements and gene expression share common structural features (Fig. 1) similar to The existence of mobile or transposable elements those of the yeast Ty element and to integrated (ME) in Drosophila mela~wgaster has been well provirases of RNA tumour viruses. The suggestion that documented. About 12% of the Drosophila genome these elements are retroviral in character is strongly consists of moderately repetitive DNA, a considerable supported by sequence analysis, and by the demonstra204 ~) 1986,ElsevierSciencePubl/shemB.V.,Armterdam 0168-9525/86~0.200
Molecular mechanisms of suppression in Drosophila
reviews Steroid sulphataseand the conservation of mammal ian X chromosomes
The X and Y chromosomes of mammals are remarkably different from each other. Whereas the X chromosome appears to cant genetic informationin proportion to its size relative to that of other chromosomes, the Y chromosome, apart from its role lan W. Craig and Elinor Tolley in male determination, appears to be largely iner~ The evolution A g.eneforsteroidsulphatase is locatedcloseto the recentlyestablisbed'pseudoautosomal" of this heteromorphic chromoregion at tl~ @ of the human X chromosome. E..,_~me~,eb in f ~ exc~d those in some pair has been accommales, and tl~'e is evidence that this unusual locus escapesfrom the flormalpattern of panied, or possibly even preX-inactivagon. In mice, hou~er, enzyme levelsin the twosexesare similar; thisis t&mght ceded, by the development of a to be achieved by the expression of a Y-linked allele. mechanism resulting in the inactivation of one of the two X chromosomes in female esterol sulphate, oestrogen sulphate (OES) and desomatic cells. The consequent dosage compensation hydroepiandrosterone sulphate (DHES), as well as to ensures a similar level of message transcripts in male the synthetic substrates, p-nitrophenyl sulphate and 4and female cells and therefore maintains the same ratio methyl umbelliferone sulphate~'s. The literature has of autosomal to X-chromosomal gene products in both been unclear concerningthe relationship between these sexes. The range of X-linkedloci is constant in different enzymes and aryl sulphatase C, which is also micromammals; indeed, Olmot has argued that conservation somal; however, genetic evidence indicates that one is a necessary consequence of dosage compensation. enzyme has activity towards the majority of these Alterations to the distn'outionof gene loci between the X substrates. Clinical interest in the roles of these chromosome and autosomes are likely to be selectively microsonmlenzymes was aroused followingreports that disadvantageous since they would change the relative low levels, or absence, of steroid sulphatase activity in dosage of gene products. placentae was associated with late parturition (see Ref. Against this evolutionarybackground, the genetics of 5); monitoring the levels of the enzyme product steroid sulphatase are enigmatic. The locus for this (oestfioi) in later stages of pregnancy is a routine aspect otherwise unexceptional enzyme is X-linked in humans of prenatal care. It was subsequently recognizede that but, unlike the majority of other X-chromosomalgenes, the children at risk were male and that they frequently it appears to escape from inactivation and there is developed, in early childhood, the classical symptoms of evidence for a Y-linkedallele in mice. The close linkage the X-linked variety of a skin disease called ichthyosis of steroid sulphatase to other sex chromosome god, 0iteral!y, fish-like skin). The simplest interpretation of including those which may have a role in sex determi- the infounation available is that the lesion in ichthyosis nation, together with its atypical pattern of regulation, results from a defect at a single (X-chromoson@) locus. have singled it out as a target for investigation. Because affected males lack enzyme activity towards However, it was for different and more prosaic reasons several substrates includingOES, DHES and those used that the genetics of this enzyme first attracted attention. to define aryl sulphatase C, it is probable that a single gene product (hereafter referred to as steroid sulphatase, STS) is active toward the sulphate esters of Physiological role The aryl sulphatases are enzymes capable of hydro- several physiologically important steroids and to the lysinga ~ d e range of sulphate esters and several forms, artificial substrates. Alternatively, the expression of the designated A, B, and C have been described. They were X-linked locus may be necessary for the activity of a first distinguished on the basis of their substrate group of related enzymes sharing common control or specificity, pH optima and pattern of cellularlocalization. structural subonits. The former argument is the more Aryl sulphatases A and B are lysosomal and catalyse the straightforward and is also consistent with the genetic hydrolysis of a variety of natural and synthetic sulphate behaviour of the STS locus in somatic cell hybrids (see esters. The two enzymes have been assigned to below). Placental STS has been purified from microsomal chromosomes 22 and 5, respectively. Aryl sulphatase A has activity towards naturally occurring sulpholipids, fractions by treatment with detergentss and the apincludingcerebroside 3-sulphates found in brain, kidney parent molecular weight of the major polypeptide and testes. The presence of a galactose 3-sulphate observed was between 57000 and 62000. Recently, moiety appears to be a necessary feature of the antfeodies have been raised against the purified protein substrate structure recognized by this enzyme. Its and used to demonstrate the lack of immunologically deficiency is associated with metachromatic leucodyl- detectable enzyme protein in X-linkedichthyosis7. Such trophy, a neurological disorder characterized by the reagents may provide an approach to the identificationof accumulationof cerebroside sulphate in nervous tissues. the gene. Some reports have suggested that low levels of STS Aryl sulphatase B catalyses the hydrolysis of UDP Nacetyl galactosamine 4-sulphate, a constituent of con- may indicate a predisposition to testicular tumours8, but nective tissue. A deficiency of this enzyme (Maro- a recent survey of levels in affected patients and controls teaux-Lamy syndrome) causes gross skeletal deformi- failed to confirm this correlation9. ties and corneal opacity2. In addition to these well defined sulphatases, several Genetiebaekground In addition to the clinicalfeatures outlinedabove, two microsomal enzymes have been characterized which have activity toward steroid sulphates, including chol- further considerations have contn~uted to current 201 ~}1986, FAsevierScien:ePeblish~B.V., ~ 0168-9525/86/$0.200
reviews
~
TIG - - August 1986
MIC2
P
p 1.~ q 1 ~.
active human X chromosome can be achieved subsequently by back selection in a medium containing 6thioguanine. This has enabled the isolation of several hybrids which retain an inactivehumanX chromosome in_ the absence of an active counterpart. Such hybrids have MIC2 been found to express amounts of STS far in excess of TDF the very low amounts detected in the rodent parental lines and, although there is no unambiguous proof that the enzyme expressed is the human one, the circumstantial evidence for an active STS locus on the otherwise inactive human X chromosome is compelling.
G6PD
2E ~
2
StS
Crm
4
SiS X
Y
o/interest. interest in the enzyme. Followingthe realization that the enzyme defect was not confined to placental tissue, assays have been carried out on a wide variety of cell types. An investigation of more than I00 tibroblast clones from a female heterozygous for X-linked ichthyosis found them all to have substantial STS activityl°; a finding in contrast to the expected pattern for Xinactivation, in which about equal numbers of positive and negative clones should result. This observation provided evidence that a second X-linked genetic marker escaped from inactivation. The X-linked blood group Xg had previously been reported to act in this way but, because its expression is confined to red cells, unambiguouscbaracte~ation of its behaviour had been difficult. Data that have accumulated on the levels of enzyme in norn~ male and female fibroblasts and leucocytes entirely support the concept that the STS locus is not dosage compensated (see Refs 11 and 12). Females have substantially higher levels of STS activity than males, although not necessarily twice as much (see below). C o n v i n ~ evidence of its escape from inactivationhas come from studies on somatic cell hybrids. Humun-rodent cell hybrids are often selected on the basis of complementation, by the human parent, of an Xlinked enzyme defect (HPRT deficiency)in the mouse, or hamster, parent. Eliminationof hybrids retmalng an 2O?.
Partial or complete escape from inactivation? A survey of the information available for the female: male ratios for STS levels has revealed an average value of 1.6 for libmblasts and placentae, substantially less than that expected for a direct dosage relationship. Ratios observed for peripheral white cells are even lower. Furthermore, individualswith multiple X chromosomes do not exhibit the proportionately high levels of enzyme anticipated (see Refs 11 and 12). Escape from inactivation may therefore not be complete. An elegant study has pro'sued this poss~llity further by eyaaniningSTS levels in fibroblast clones from ~ndividnalsheterozygous for deficienciesin both STS and for the X-linked marker glucose 6-phosphate dehydrogenase 11. This enabled the identification of dunes in which one or other of the two X chromosomes was active and the demonstration that the STS+ allele was expressed at about half the level from the inactive chromosome compared with its expression from the active homologue. Part~ ina~'vation is the most satisfactory explanatiou for this observation and would also account for the deviation from a strict dosage relationship observed for the female:male ratio. X0 individuals and STS-deficiency heterozygotes have enzyme levels below the normal male range, the heterozygotes signific.~tly so; this observation could result from the partial random inactivation of the normal allele. Although a Y-linked STS locus has been postulated for mice (see below), there is no substantial evidence supporting tles suggestion for humans. Studies on cells carcying an X-autosomal translocation in which the breakpoint is above the STS locus (demonstrated by the retention of high levels of enzyme in human-mouse hybrids retaining the 'derived-X' chromosome) indicated that the normal X was preferentially inactivated in vivo. This suggests that the translocation deletes a chromosomal feature which norreally confers local resistance to inactivation. Random inactivation of the derived X lacking this postulated feature could therefore be transmitted through the S'I~ region into the autosonml material attached. This would result in a decreased output of autosomal gene products and would explain the observed preferential advantage to cells with the normal X chromosome inactivatedIs. Chromosomal localization Two approaches have independently assigned the STS locus to the tip of the human X short ann (see Fig. 1). Early studies (see Ref. 5) of somatic cell hybrids retaining different X-autosomal rearrangements enabled the assignment of STS and, by virtue of its close linkage, Xg to the distal portion of the short arm (Xp22ter) or (Xp11-ter). Further refinement of the map po~itioa ~oXp223-ter was achieved later by studies on a
reviews
TIG - - August 1986
hybrid carrying a resn-anged X chromosome which lacked only this tenninal segment (See Ref. 14). A similar location has also been suggested by observations on ichthyotic males who lacked STS and who were karyotypically abnormal. Such patients are typically e~racterized by ap~.ently unbalanced X-Y trans. locations resulting from an interdmnge which deleted the terminal segment of the X chromosome, Xp223-ter 0tefs 15 and 16). There is little doubt that both STS and Xg are located at the short ann terminus of the X chromosome and we are therefore presented with an intriguing pattern of observations which distinguish this potlion from the remainder of the X chromosome. Firstly, studies on rnei,otic preparations indicate that this region is preferenEallyinvolved in pairing with the Y chromoson~ 17and that this ~ g may be accompanied by an obligate exchangeTM. Secondly, the region contains loci which escape from inactivation and thirdly, it has been suggested that it contains a regulator gene for sex detennimtion (see Ret. 19). The consensus view is that the distal short arms of the X and Y chromosomes represent a conserved remnant of an original pair of homologous chromosomes. Very recently, pseudoautosomal sequences have been d ~ which are regularly exchanged in male meiosis and are presumed to be located in a region of active recombination near the short ann termini. A decreasing probability of exchange is observed for sequences further from the telomeres (see Ref. 18 and Fig. 2). The gene (MIC2) coding for the cell surface,antigen 12E7 has been cloned and assigned, by in situ hybridization, to the tips of both the X and Y chromosomesm,9-t. The STS locus in humans must lie below the region of X-Y exchange, as no convindng evidence for Y localization has been reported. Furthermore, the dMerence in enzyme levels observed for males and females is inconsistent with the existence of a Y-linked locus (that is, one expressed in the normal range of tissues examined). The location of the STS locus in this region and the relatively simple assay for its gene product have led to its use as a marker for investigations into the aetiology of XX males and other dysgenic sexual types. A combination of studies has suggested that at least some XX males result from an aberrant exchange between the X and Y chromosomes at paternal meiosis- resulting in the transmission to the affected offspring of an X chromosome which has exchanged its distal short ann sequences for Y short ann material including the putative testis-detennining factor (TDF)2z.m. Whereas presence of Y sequences can be detected using male-specific probes, loss (or inactivation) of distal X material may be inferred from the STS levels observed for some individuals, which are in the male range (see Ref. 5). Steroid sulphatase in rodents The rationale explaining the conservation of the X chromosome does not extend to genes which escape from inactivation. The behaviour of the STS locus in this context in other mammals is therefore of interest. Apart from a preliminary observation sugges"_tij]g.the absence of X-linkedSTS in AustrAliAnmarsupials~, most studies have concentrated on wood-lemmings and mice. Woodlemmings have an unusual sex deter "n.ingmechanism in which two different types of X chromosome participate. STS levels in various classes of woed-lemming were found to correlate with the number of X
chromosomes2s. As in humans, the gene appears to be X-linked, but not subj__e_ectto inactivation. Mice also have an X-linked STS locus~, but do not show sex differences in dosage and there is no evidence for loci which escape from inactivation on the mouse X chromosome~ This observation, remarkable in fight of the evidence from wnod-lemmings, could be explained in two ways. If X linkage is accepted, then either the locus must be subjected to the normal pattern of inactivation, or there must be, additionally, a Y-linkedallele bringing the level of enzyme of the male into a range similar to that of the female. The second suggestion is favoured by Keitgss et al.m, who were able to demonstrate, in different types of crosses, that STS deficiency could behave either as an X-linked or as an autosonml recessive trait. These observations would be explained if both the X and Y chromosomes possess functional STS alleles which undergo obfigstory recombination during meiosis. The TDF in mice is located proxit~Jy on the Y chromosome and therefore not at risk of being included in the recombination event. In this context, it is interesting to note that in the progeny of crosses with male mice carrying the sex r e v e r s ~ trait (Sxr), the levels of
Xpter
Ypter r"
m
!
Xg
TDYgl=
STS
p
II II II
II II II
X
Y
Fig. 2. $ ~ dia~am of s~rt arm terminal ~ of hummz X and Y chfomosome.~indicating the pseudmutosomal which has ~ e s ~ to amtain 2000-6000 kb (see Refs lg ami30). Thm is ~ that MIC2 may be ~ at ~ ~ CP. C,oo~eaow, pen. an,ram.). Yg is t ~ p~uz~tiw ~ t o ~ b ~ Snup ~ Xe. $ ~ is placed below TDF because the pfem~gve X - Y e~w2ua~ r ~ g n g in XX w l ~ is o~y ~ a ~ o d ~ u~thto~ of S'l'S a c ~ . 203
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TIG - - August 1986
steroid sulphatase are similar in XX males, normal males, males carrying Sxr and normal females 27, suggesting that the duplication of the male-determining region on the Y chromosome of the Sxr male does not include the STS locus. Recent studiesz9 suggest that Sts is indeed linked to the most distally located, conventional marker on the X chromosome (Cream; see Fig. 1). This linkage, which was demonstrated through female meiosis, is not incompatible with the pseudoautosomal behaviour of the postulated Y-linked locus in males, given the differences in recombination observed for the region in human males and femalesa°. Conclusion The intriguing behaviour of the steroid sulphatase locus and its significance in terms of understanding fundamental biological problems, including that of the control of inactivation processes and the evolutionary relationship between the mammalian X and Y chromosomes, make it an attractive target for direct molecular studies. Difficultiesin the purificationof the protein have so far prevented the isolation of coding sequences through oligonucleotideor expression vector strategies; nevertheless, one can be confident that the cloning of the structural gene will generate great interest among the afidonados of mammalian sex chromosome genetics. References
9 Tolley, E., Craig, I. W., Jomsson, J., CartwrighL It/L and Jones, W. G. (1985) Lcmcet i, 563 10 Shapiro, L. J., Mohandas, T., Weiss, R. and Romeo, G. (1979) Science 204, 1224 11 Migeon, B. R,, Shapiro, L.J., Norm, R. A., Mohandas, T, Axelman, J. and Dabora, R. L (1982) Nature 229, 838-840 12 Lykkesfeld,G,, Lykkesfeld,A. E. andShakkabaeck,N. E. (1984) Hum. Genet 65, 355-357 13 Crocker, M., Jonasson, H. and Patei, C. (1985)Hum. Gcnet. 28, 556-560 14 Miller, O. J, and Siniscalco, M. (1982) Cytogenct Cell Greet. 32, 179-190 15 Tiepelo, L., Zuffar~, 0., Fraccaro, M., di Natale, D.o Garganflni, L., Muller, C. P,,. and Ropera, H. H. (1980)Hum. Greet. 54, 205--206 16 Ferguson-Smith, M. A., Sanger, It, TippeR, P., Aitken, D. A. and Boyd, E. (1982)Cytogcnet. Cell Greet. 32, 273-274 17 Pearson, P. L. and Bobrow, M. (1970) Nature 226, 959-961 18 Burgoyne, P. S. (1986) Nature 319, 258-259 19 Polani, P. E. (1982)Hum. Genet. 60, 207-211 20 Darling,S. M., Banting, G. S., Pym, B., Wolfe,J. andGoodfellow, P. N. (1986) Proc. Natl Acad. Sd. USA 83, 135-139 2/ Buckle, V., Mondello, C., Darling, S.M., Craig, I.W. and Goodfellow, P. N. (1985) Nature 317, 739-740 22 Page, D. C., de la Chapelle, A. and Weissenhach, J. Nature 315, 224-227 23 Guellaen, G., Cassanova, M., Bishop, C. E., Geldwerth, D., Andre, G., FeHous, M. and Weissenhach, J. (1984) Nature 307, 172-173 24 Cooper, D. W., McAIlen, B. M., Donald, J. A., Dawson, G., Dobrovic~ A. and Graves, A. M. (1984)Cytogenct. CeliGmet 37, 439 25 Ropers, K H. and Wiberg, U. (1982) Nature 296, 766-768 26 Gm'tler, S. M. and Rivest, M. (1983) Gencgcs 103, 137-141 27 Crocker, N. and Craig, I. W. (1983) Nature 303, 721-722 28 Keitges, E., Rivest, M., Siniscalco, M. and Gartler, S. M. (1985) Nature 315, 226-227 29 Cattenach, B. and Crocker, M. (1986) Mouse News Letter 74, 94-95 30 Rouyer, F., Simmler, M-C., Jolmsson, C., Vergnand, G., Cooke, H. J. and Weissenbach, J. (1986)Nature 319, 291-295
I Olmo,S. (1967)SexCbro~o~a~ISezLinkedGu~s, Springer Verlag 2 Farooqui,A. A. andMandel, P. (1977)Int. ]. Biochem. 8, 685-691 3 lwamori, M., Moser, H. W. and Kishimoto, Y. (1976) Arch. Biod~n. Biop~s. 174, 199-208 4 Dolly,J. O., Dodgson, K. S. and Rose, F. A. (1972)B~chem. ]. 128, 337-345 5 Shapiro, L. J. (1985)Adv. Hum. Genet. 14, 331--381 6 Shapiro, L. J., Weiss, R., Btvamn, M. M., Vidgoff,J., Dimond, R. L., Roller, J. A. and Wells, R. S. (1978) Lancet fi, 756-757 7 Epstein, E. H. and Bonifas,J. M. (1985)Hum. Genct. 71, 201-210 L IV. Craig and E. Tolley are at the Gozetics Laboratory, 8 Lykkesfeldt, G., Hoyer, H., Lykkesfeldt, A. E. and Shakkabaek, University of Oxford, South Parks Road, Oxford OXI 3QU, L. E. (1983) Lancet ii, 1456 UK.
Suppression is defined as the reversal of a mutant phenotype due to a mutation at a site distinct from that giving rise to the mutant phenotype. The result is a revertant produced by Eric Kubli the interaction of either allelic (intragemc suppression) or nonallefic (intergenic suppression) Mobile elements contain a vasty of r@ulato~ sigaals which can profoundly affect the mutant genes. Intergenic supexpressionofgenes near the sites at which the elementsare insertedin the gonome. These effectscan be compensatedbysuppressormutalfims. Thus, geneticand molecularsb~iies pression can be exerted at the of suppressionphenomena can yield important insights into the mechanism of eukaryotic transcriptional, translational or gene r@ulagon. post-translational level. This review concentrates on recent advances in our understanding of molecular aspects of intergenic suppression involving part of it belonging to three distinct classes of MEs: mobile elements in Drosophila melano&aster. The copia-like-, foldback- and P-elements. In Drosophila, all mutations and suppressors that willbe described act on mobile elements involved in suppression phenomena the affected genes at the level of transcription. that have been studied at the molecular level belong to the copia family~ (e.g. copla, gypsy, 412, etc.). They all Mobile elements and gene expression share common structural features (Fig. 1) similar to The existence of mobile or transposable elements those of the yeast Ty element and to integrated (ME) in Drosophila mela~wgaster has been well provirases of RNA tumour viruses. The suggestion that documented. About 12% of the Drosophila genome these elements are retroviral in character is strongly consists of moderately repetitive DNA, a considerable supported by sequence analysis, and by the demonstra204 ~) 1986,ElsevierSciencePubl/shemB.V.,Armterdam 0168-9525/86~0.200
Molecular mechanisms of suppression in Drosophila