Long-range physical mapping around the human steroid sulfatase locus

GENOMICS6,528-539(1990)

Long-Range Physical Mapping around the Human Steroid Sulfatase Locus MARK T. ROSS,* ANDREA BALLmo,t *Genetics Laboratory, Department Medical Institute and Institute

AND IAN W. CRAIG*

of Biochemistry, South Parks Road, Oxford OX1 3QU, United Kingdom; and tHoward Hughes for Molecular Genetics, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030 Received September 1, 1989; revised November 27, 1989

The availability of STS cDNA clones provides an opportunity to analyze the locus in molecular detail, and there are several good reasons for wishing to do so. The STS gene is unique among well-studied Xlinked loci in that it escapesfrom the X-inactivation process that occurs in the somatic tissues of women. Classical genetic analysis in the mouse demonstrated the existence of an active allele on the Y chromosome which undergoes recombination with the X allele during male meiosis (i.e., is pseudoautosomal; Keitges et aZ., 1985). The human gene, however, exhibits X-linked behavior, although three principal lines of evidence indicate that the locus escapes, at least partially, from inactivation: STS levels are higher in women than in men in the tissues tested (Miiller et al., 1980; Lykkesfeldt et al., 1984); an inactive X chromosome isolated in a mouse-human hybrid cell line expressed STS activity (Mohandas et al., 1980); and fibroblast clones from women heterozygous for STS deficiency were found to produce the enzyme regardless of which X was inactive (Shapiro et al., 1979), although expression from the inactive X was lower (Migeon et al., 1982). This ability to evade the inactivation process may be intrinsic to the gene or may be conferred by regulatory elements close to it, since there is some evidence to suggest that inactivation can spread across the region without affecting STS activity (Mohandas et al., 1987). Further interest in the genetics of STS stems from the etiological association between STS deficiency and the syndrome of X-linked ichthyosis (XLI). The observation that an overwhelming number of cases involve deletions of the entire coding region-for example, in one recent survey 48 of 57 unrelated XL1 patients (84%) had deletions (Ballabio et al., 1989)is particularly fascinating. For no other human disease has such a high frequency of deletions been observed. A long-range physical map of the region could provide a starting point for the study of the molecular mechanisms underlying the intriguing nature of the

The region of the human X chromosome containing the steroid sulfatase locus was analyzed by pulsed-field gel electrophoresis. Restriction site maps were generated for the X chromosome in the blood of a normal male individual and that in the mouse-human hybrid cell line ThyB-X; these maps extend over approximately 4.3 Mb of DNA of the former, and 3.2 Mb of the latter. Physical linkage was defined between the STS locus and sequences detected by the probes GMGXS (DXS237), GMGXYlS (DYS74), CRIS232 (DXS278), and dic56 (DXS143), and the order telomere-(STS, DYS74)-DXS237-DXS278-DXSl43centromere was deduced. The pulsed-field maps were used to demonstrate a deletion of 180 kb of DNA from the X chromosome of an individual with X-linked ichthyosis. Also, possible locations for the Kallmann syndrome gene were revealed, and the distance between the steroid sulfatase locus and the pseudoautosomal region was estimated to be at least 4 Mb. OlBSOAcademicPmas,Inc.

INTRODUCTION

The human steroid sulfatase (STS) gene (EC 3.1.6.2) encodes a microsomal enzyme involved in the production of estrogens during pregnancy and is X-linked. Formerly, studies of X chromosomes from which material had been lost, owing to an X/autosome translocation event (Mohandas et al., 1979), to an X/Y translocation (Tiepolo et al., 1980), or to an apparently simple deletion (Curry et al., 1984), localized the STS locus in humans to the distal part of the X chromosome short arm and close, in cytogenetic terms, to the pseudoautosomal region. The isolation of complementary DNAs to the STS gene allowed confirmation of the localization to Xp22.3-Xpter and led to the detection of a pseudogene on the long arm of the Y chromosome (Ballabio et aZ., 1987; Fraser et al., 1987; Yen et al., 1987; Conary et al., 1987; Bonifas et al., 1987).

0888-7543/90$3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form

528 Inc. reserved.

PHYSICAL

MAPPING

AROUND

STS gene. In the context of control, it would be of interest to establish whether there is a CpG island associated with the STS locus, as is the case with other housekeeping genes (Bird, 1986; Gardiner-Garden and Frommer, 1987). Theoretical considerations based on the occurrence of the dinucleotide CpG and its degree of methylation, and on the proportion of C + G, in bulk DNA and in CpG islands, as well as empirical evidence (Brown and Bird, 1986), indicate that a cluster of accessible sites for methylation-sensitive restriction enzymes is diagnostic of an island. Site maps for such enzymes should, therefore, pinpoint CpG islands. The reasons for the particular susceptibility of this region to deletion events, over other types of mutation, could also be approached within the framework of a long-range map. Insight might follow directly from knowledge of the sizes and positions of deletions in cases of ichthyosis-for example, are the same deletions found in unrelated individuals?-or, more likely, indirectly, if these data should suggest appropriate strategies for the cloning of deletion endpoints, We report here the construction of such a map based on data obtained with pulsed-field gel electrophoresis (PFGE). MATERIALS

Preparation DNA

and Digestion

AND

METHODS

of High-Molecular-

Weight

DNA plugs were made from the somatic cell hybrid ThyB-X (Lund et al., 1983), containing the X chromosome as its only human contribution; from the peripheral blood leukocytes of a normal male; and from the cultured fibroblasts of a male with X-linked ichthyosis. Cells were encapsulated in low-gelling-temperature agarose (0.5% in PBS; Sea Plaque, FMC Bioproducts) at a concentration of 1.5 X 10’ cells/ml for the short-run-time gels (32-36 h) or at 0.75 X lo7 cells/ ml for longer run times. Plugs were formed by cooling this mixture in Perspex molds (approximately 100 ~1 per plug), and a third of a plug was used per digestion under conditions described previously (Anand, 1986). For double digestions, all traces of the first enzyme were washed away with 10 n&f Tris-HCl, 1 mM EDTA, pH 8.0 (TE), before reequilibration in the second restriction buffer. SfiI, NotI, EagI, NarI, and BssHII were from New England Biolabs, Inc., and SstII, SaZI, SmaI, NruI, and C&I were from Gibco BRL.

Pulsed-Field

Gel Electrophoresis

Electrophoresis was carried out in a rotating gel, or “waltzer,” apparatus (Southern et al., 1987) under 0.5X TAE buffer (1X TAE is 0.04 M Tris-acetate, 0.001 M EDTA). For resolution of fragments from 50 to 1000

THE

HUMAN

STS

529

LOCUS

kb the running parameters were 1.5% agarose (Sigma Type-II); 18°C; pulse time, 65-73 s; run time, 32-36 h; field strength, 6 V/cm. Increasing the resolution to 3.5 Mb required the following changes: 0.6% agarose; 5°C; pulse time, 40 min; run time, 5-10 days; field strength, 1 V/cm. Identical parameters were set to resolve above 5.7 Mb, except that the pulse time was increased to 60 min. Size markers were chromosomes of Saccharomyces cereuisiue strain X2180-lB, oligomers of bacteriophage X (cI857Sam7, monomer size 48.5 kb), and, for the sizing of larger fragments, chromosomes of Schizosacchuromyces pombe (Fan et al., 1988).

Blotting

and Hybridization

Following electrophoresis, DNA was transferred onto a nylon membrane (GeneScreen Plus, New England Nuclear) in 0.4 M sodium hydroxide. Filters were neutralized in 0.2 M Tris-HCl, pH 7.5/2X SSC, rinsed in 2X SSC, and then dried at room temperature. Hybridization was carried out at 65°C in 7% sodium dodecyl sulfate (SDS), 0.5 M sodium phosphate, 1% bovine serum albumin, 1 mA4 EDTA, pH 7.2, using DNA probes labeled by the random-primer technique with 32P (Feinberg and Vogelstein, 1983). Unbound probe was removed by washing at 65°C down to 40 mh4 sodium phosphate, pH 7.2/l% SDS. Filters were exposed to Fuji-RX film for l-10 days and were stripped for reuse by washing in 2 m&f Tris-HCl, pH 7.5, 1 mM EDTA at 65°C for 15-30 min. Where necessary, stripping was verified by exposure to X-ray film for an appropriate period.

DNA Probes Two partial STS cDNA probes were used in this study: 3’P2A7 contains part of the most 3’ exon of the STS gene and is a P&I fragment of the almost fulllength cDNA P2A7; ST12 is derived from the 5’ end of the locus and contains sequences homologous to the Y-STS pseudogene. References relating to these probes and the others are P2A7, Fraser et al. (1987); ST12, A. Ballabio (unpublished observation); GMGXS (DXS237), Gillard et al. (1987); GMGXY19 (DYS74), Affara et al. (1987); CRI-S232 (DXS278), Knowlton et al. (1989); dic56 (DXS143), Middlesworth et al. (1985); MlA (DXS31), Koenig et al. (1984). RESULTS Data for mapping were obtained from the X chromosome in the peripheral blood leukocytes of a normal male and from that in the hybrid cell line ThyB-X, whose sole human chromosomal representative is the X (Lund et al., 1983). DNA from these cell types, protected from shearing forces by encapsulation within an

530

ROSS,

BALLABIO,

agarose matrix, was subjected to digestion with a range of infrequently cutting restriction enzymes both singly and in pairs, and the products were separated by PFGE on a waltzer apparatus (Southern et al., 1987). The sizes of fragments detected by the probes detailed under Materials and Methods are given in Tables 1 and 2. On the basis of these observations, restriction site maps for the two X chromosomes were constructed

Hybridization

AND

and are illustrated in Fig. 1. Approximately 4.3 Mb of DNA was mapped in male blood and over 3 Mb in ThyB-X. It is important to note that the estimation of the sizes of very large fragments (>1650 kb) is beset by certain difficulties, these being the lack of a sufficient range of size markers and the very great effect of initial DNA concentration on running characteristics. The

TABLE

1

Results

(Single Probe

Male 3’P2A7” (STS)

GMGXS (DXS237)

800

800

840 750

840 750

Sal1

LM

NM?1

800

100 130 LM 800

EagI

800

Enzyme SstII

Digests)

(locus)

blood

ThyB-X CR1 S232 800 830 940 320b 840 750 280 390 710 670b LM

SmI

800

800

800 830 LM 800 1650 2400 830 940 ND

NarI

ND

1650

1650

BssHII

ND

2400 1650

Not1 NruI

ND ND

1650 3000

2400 1650 780 940 1650 3000

MluI cl01

ND 650 710 250

3400 ND

3400 ND

800 1650 2400

CRAIG

dic56 (DXS143)

3’P2A7” (STS)

GMGXS (DXS237)

CR1 S232

dic56 (DXS143)

750 710

800

800

800 830 940

200

750 840

750 840

750 840 280 390 710

ND

650 750

180 90

ND

ND

800

800

650 750 2400

800

800

500 550 100 800 650 LM 800 1650 2400

100 175

ND

ND

650 750 2400 750

1300 750 1650 3400 ND

2400

360 430 280 470 590 ND

175 200 220 290 590 1650

ND

2400 2400

ND ND ND >lOOO

2400 2400 2700 >5700 ND

Note. Sizes are given in kb, and faint bands are indicated in italics. ND, not done. LM refers to hybridization of the appropriate figure. a 3’P2A7 and GMGXY19 detected common X fragments with the enzymes tested (SstII, S/i I, SalI, NaeI, b Faint fragments detected by CRI-S232 (designated DXS278) in DNA from blood, but not from ThyB-X,

ND

200

ND

1650 2400

ND

ND

2400
2400

ND 2400 2700 ND ND

2400
at the limiting

25700 ND

mobility

region

EagI, and SmaI). may be from the Y chromosome.

PHYSICAL

MAPPING

AROUND

Hybridization Male

sfi 1+sst11 +NocI +BssHII +EogI +NotI +NarI

+saw

sst11+soW

+NruI

3P2Al (SW 800 710 800 710 800 710 800 710 800 710 800 840 750 520 550 640 320* 480 510 600 800 710

HUMAN

TABLE

2

Results

(Double

STS

531

LOCUS

Digests)

blood

ThyB-X Probe

Enzymes

THE

GMGXS (DXS237)

(locus)

Probe CR1 S232

dic56 (DXS143)

-

-

-

-

-

-

-

-

-

-

-

-

3P2A7 W’S)

Enzymes

710 800

-

SalI+NoeI

480 590 480

-

+NarI +clu1

650 590

-

510 700 520 620

-

+NotI

100 130 600 340

-

-

-

+sfi1

SstII+SalI +clo1

-

750 710

BssHII+NruI +MluI

-

1650

1650 1650 1

750 750

MluI+NruI

-

2400

2400

750

Note. Sizes are given in kb, and faint bands are indicated in italics. of clarity, were consistent with the site positions of Fig. 1. a The STS cDNA used for these hybridizations was ST12. * This fragment is derived from the Y chromosome.

values assigned to such fragments should therefore be regarded as approximate, although, in most cases, estimates were based on more than one gel, under different running conditions. In the description that follows, the SstII site immediately to the left of the STS locus, in the given map orientation, is designated S&II (O), and other sites are assigned values relative to this; positive positions are to the right of this site on the maps (i.e., proximal) and are measured in kilobase pairs (kb). Linkage of the STS Locus with DXS237 and DYS74 Physical linkage between the STS gene and sequences detected by probes GMGXS (DXS237) and GMGXY19 (DYS74) was demonstrated with several enzymes on gels resolving to a maximum size of around

The results

+NruI BssHII+NruI +NarI

of other

double

-

590 480 590 480 650 4230

+EagI

-

GMGXS (DXS237)

sfi 1+sst11

+BssHII

100 130 640 340

(locus)

digestions,

which

-

480 590 360 430 800 710

-

-

1650 1650 2400

are not shown

-

for the sake

1 Mb. The STS cDNA probe 3’P2A7 and GMGXS detected common fragments in most instances (Fig. 2). Identical patterns of hybridization were observed with the enzymes S&II, SfiI, NaeI, and EagI, and the only difference noted between male blood and ThyB-X in these caseswas the reduced accessibility in the former of the site SfiI (+710). Differential hybridization patterns of 3’P2A7 and GMGXS were observed only with SmaI and SalI. Actually, these probes were found to lie on a common SmuI fragment of blood DNA, but with ThyB-X their major sites of hybridization differed, although a common fragment that had arisen from partial cleavage was evident (see Fig. 2). Digestion with the enzyme Sal1 allowed the relative localization of the STS locus and DXS237 on the maps. The results of hybridizing an STS cDNA probe and GMGXS to male and female

532

ROSS,

NO Ea as N.2 SfSs

tdWtlOl0 +

Nr

MI

t

t

:

BALLABIO,

Ea St Nr He Sf R

sasasa

AND

CRAIG

NO SS Nr Na Ea es

SS Nr m Ea as

MI

No

t

t

t

T

zixs

STS GMGXVlS

centromem

dic5S Tb2SZ

Thy&X

Nr

.R

t

tn

No Es BS m Sfss

clclR

En Sf m Ss

SaSf

ttt T TT

T

T

Nr

Na

.sTs 1

dies

iZX9

GMGxVfS

I

m Nr

b NO Na R

CRI-SZ32

I

i

0 400 kb

FIG. 1. Long-range restriction maps around the STS locus in DNA from the peripheral blood leukocytes of a normal male and the mousehuman hybrid cell line ThyB-X. Two types of site are defined: ones at which cleavage is good, though not necessarily complete (0); and others at which cleavage is very poor (0). It should be noted that the maps do not necessarily contain all the accessible sites for some of the enzymes. Positions of loci recognized by the probes shown are indicated by bars. The location of %I1 (0) is indicated on the scale. Key to enzymes: Ss, Sⅈ Ne, NueI; Bs, BssHII; Ea, EC& No, NotI; Na, NarI; Nr, NruI; MI, MluI; Sf, Sfi I; Cl, CU.

blood DNA digested with Sal1 alone, with Sal1 and S&II, or with Sal1 and SfiI are shown in Fig. 3. With blood, the STS probe ST12 hybridized primarily to a SalI fragment beyond the resolution of this gel (>940 kb) but also, weakly, to several products of partial digestion. Double digestion with Sal1 and SstII produced fragments of 480,510, and 600 kb recognized by ST12, while with Sal1 and SflI together, the bands measured 520, 550, and 640 kb; other evidence (see tables) indicated the existence of an SfiI site at -40 kb; therefore, it was concluded that there were SalI sites at +480, +510, and +600 kb. GMGXS, too, hybridized with the 600-kb SalI/SstII and 640-kb SalI/ SfiI partial-digestion products, as well as to a Sal1 fragment of around 100 kb. These results placed the STS locus between S&II (0) and Sal1 (+480) and DXS237 between Sal1 (+510) and Sal1 (+600). The maximum distance between the end of the 146-kb STS gene (Yen et al., 1986) and DXS237 is, therefore, 450 kb. Differences were apparent between the Sal1 cleav-

age patterns of DNA from blood and ThyB-X (see Tables 1 and Z), although these could be accounted for by differential methylation. The probe GMGXY19 (DYS74) detected the same X-chromosomal fragments as 3’PZA7 in all digests. Additionally, GMGXY 19 hybridized with fragments from the long arm of the Y chromosome in DNA from male blood, the same Y-chromosomal fragments were seen with the probe ST12, which contains sequences homologous to the STS pseudogene on Yq (M. A. Jobling, personal communication). The Orientation

of the Map

For a number of enzymes, it proved necessary to raise the level of resolution to 3.5 Mb in order to visualize the fragments recognized by the various probes; this was achieved by applying the conditions given under Materials and Methods. Figure 4 shows the outcome of such an experiment involving GMGXS, CRIS232 (DXS278), and dic56 (DXSl43).

PHYSICAL

ss

----e-

Sf

Sa

FIG. 2. Physical linkage of the (b). Size markers were S. cereuisiae 1, and Sm, SmaI. The faint 590-kb kb SmaI fragment of blood DNA is

Ne

MAPPING Ea

AROUND

THE

Sm W

HUMAN

STS

so ------

Sf

533

LOCUS

Sa

Ne

Ee

Sm

cTCYTOTCfTc7TdTdc

STS locus with DXS237. DNA from ThyB-X (T) or male blood (6) was probed with 3’P2A7 (a) or GMGX9 chromosomes (C; sizes given in kb) and X phage DNA oligomers (not shown). Key to enzymes as in Fig. Snal fragment of ThyB-X DNA recognized by both probes is indicated by an arrow. The common 900very faint owing to poor digestion.

GMGXS exposed several differences between the two cell lines in this experiment and in others: for example, the BssHII fragment measured only 1650 kb in blood DNA compared with 2400 kb in ThyB-X; further differences were noted following digestion with NruI, Not1 (data not shown for ThyB-X/NotI), and EugI, the last revealing a difference in the pattern of partial digestion products. The pattern of GMGXS hybridization for the two cell lines was identical in the case of NarI digestion (data for ThyB-X not shown). When taken together to produce the restriction maps of the region (Fig. l), the data obtained with the various probes and digestions indicated that the differences between the two cell lines were due to polymorphism or methylation rather than to a large-scale rearrangement of the DNA in this region of the ThyB-X chromosome. At the level of resolution of genetic data, the probe CRI-S232 recognizes a single locus or a group of closely linked loci on the X chromosome (Knowlton et al., 1989). The pulsed-field data on this probe were more consistent with the existence of multiple loci, at least one of which could be linked with the STS locus. CRIS232 recognized all those fragments seen with GMGXS in the experiment of Fig. 4, and several additional fragments in both cell lines. Subsequent hybridization to the filter of Fig. 2 showed conclusively that one of the loci detected by CRI-S232 lies between S&II (0) and

SfiI (+710) (data not shown); results with the enzyme Sal1 suggested its location as proximal to DXS237. The locus DXS143, detected by the probe dic56, also proved to lie within the boundaries of the map. A 2400kb BssHII fragment of ThyB-X DNA recognized by dic56 and GMGXS is visible in Fig. 4; the partial fragments of 2400 kb detected by GMGXS with Nor1 (blood DNA only) and EugI (Fig. 4) could also be seen with dic56 following prolonged exposure. Furthermore, it was clear that the two loci lay upon the same Not1 fragment of ThyB-X DNA and the same MluI fragment of blood DNA (data not shown). The probes, however, failed to recognize common NruI fragments; this placed DXS143 between NruI (+1650) and BssHII (+2400). Deletion mapping has located DXS143 proximal to the STS locus (Ballabio et at., in press; Yates et aZ., 1987; Mondello et al., 1987), and thus the map was oriented telomere-(STS, DYS74)-DXS237-DXS278-DXS143centromere. The data shown in Fig. 4 hinted that the EugI fragment of 1650 kb recognized by dic56 in ThyB-X DNA also contained sequences homologous to CRIS232. A tentative conclusion is that a second locus detected by this probe lies proximal to EagI (+800) and distal to EugI (+2400). If this is the case, then there must be at least three loci homologous to CRI-S232, since the BssHII fragments of less than 1000 kb seen in male

534

ROSS,

BALLABIO,

AND

CRAIG

c 2 @Q -al

Sa L

c

sa

5-s

sa

Sa

Sa Sa

s;

s’r

s’r

940 830 790 750 680 590

360 280 245

FIG. 3. Detection by ST12 (a) and GMGXS (b) of common partial digestion products of blood DNA. Tracks contain DNA from male (6) or female (0) blood. Results obtained with the control female indicate that the Sal1 fragment of 320 kb detected by ST12 is Y-chromosomal in origin. Migration differences between identical digests of male and female DNA indicate the effect of DNA concentration. Size markers were S. cereuisioe chromosomes (C, sizes given) and X phage DNA oligomers (L). Key to enzymes as in Fig. 1.

blood and ThyB-X DNA (Fig. 4) cannot be accommodated within the region of the maps containing the other two putative loci. Genetic mapping data have shown that the CRI-S232 loci lie distal to DXS143 (Knowlton et aZ., 1989). If this is the case, a scrutiny of the pulsed-field maps indicates that the BssHII fragments just described, containing sequences homologous to CRI-S232, would have to be located distal to the STS locus. A CpG Island

in the Region of the STS Gene

The presence of a CpG island distal to the STS locus was revealed in the course of mapping. Double digestions with SalI, for ThyB-X, or SfiI, for blood (Fig. 5), and probing with 3’P2A7 showed the island to contain sites for S&II, NueI, BssHII, NotI, NurI, and EagI. The data were insufficient to confirm or exclude the possibility of this island’s association with the gene; neither the orientation of the gene nor its proximity to the island could be defined using the available range of probes and enzymes. Further study of the island is required, although preliminary observations on individuals with two X chromosomes indicate that the site SstII (0) is unmethylated on the inactive X chromosome.

Analysis

of DNA

from an Individual

with XLI

A study was made by PFGE of an individual with X-linked ichthyosis. Conventional electrophoretic analysis had revealed the deletion of the 3’ end of the STS locus and of the X homolog of DYS74, although not of 5’STS or DXS237, from the DNA of this individual (Ballabio et al., 1989, and in press). This deletion is of interest for two reasons: first, it should be relatively simple to isolate the deletion endpoints for detailed analysis; and second, from the point of view of the work here, it held the prospect of providing the orientation of the gene on the pulsed-field map. Probing fibroblast DNA from a normal male and from the ichthyotic male with ST12 (5’STS cDNA) revealed the expected 800-kb SstII fragment in the case of the former but an altered fragment of 620 kb in the latter (Fig. 6a). An altered fragment of precisely the same size was seen in an NueI digest of DNA from the ichthyotic male. The likely explanation for these data is that the deletion does not encompass the CpG island distal to the STS locus. However, another possibility is that the deletion does remove the distal island but stops short of a second, more distal, island also with sites for SstII and NueI. The knowledge that DYS74 (GMGXY19) is within the deletion provided no clar-

PHYSICAL a)

--

Nil

Ea

CdTd’T

b)

Bs ---

MAPPING No

AROUND

Nr

P~T~T@TP

Bs ---

?!%k cdTdT

No

Nr

P@TCfT@TP

THE

HUMAN

STS

535

LOCUS

weight to the idea that the deletion does not remove the sites S&II (0) and?+& (0). ST12 recognized a Sal1 fragment of 520 kb and a partial product of 590 kb in the deletion case (Fig. 6a). These were reduced to 350 and 420 kb, respectively, on double digestion with SstII (Fig. 6b), indicating the presence of a Sal1 site 170 kb distal to the distal SstII site in this individual. Such an arrangement of sites was also found on the normal X chromosome present in ThyB-X (Sal1 (-170) and SstII (0); Fig. 1). Furthermore, the pattern of Sal1 cleavage detected in the DNA of this hybrid cell line would appear to be identical to that in fibroblast DNA, since ST12 recognized a Sal1 fragment of 650 kb in both the normal male fibroblast control (Fig. 6a) and ThyB-X. This similarity between deleted and normal X chromosomes in both the order and the separation of sites distal to the STS locus is consistent with a deletion of 180 kb of DNA lying between SstII (0) and DXS237. GMGXS, like ST12, recognized Sal1 fragments of 520 and 590 kb in the DNA from the ichthyotic male (data not shown). The detection of identical fragments by the two probes would be consistent with the deletion

Sf L

-- Na

EL!

0% -w-

No

C

Sf

Ss

Ne

Bs

No

Na

Ea MI

Nr

=I

CdTdT

PdTdTdTP

FIG. 4. Physical linkage on large DNA fragments of loci detected by GMGXS (a), CRI-S232 (b), and dic56 (cl. Tracks contain DNA from male blood (8) or ThyB-X (T). Size markers were chromosomes of S. cereuisiae (C) and S. pornbe (P). Sizes of second largest cereuisiae and smallest pornbe chromosome are given. NurI and Not1 failed to digest the ThyB-X DNA in this particular experiment. Key to enzymes as in Fig. 1.

ification of the situation, since the pulsed-field data on GMGXY19 showed the locus to lie in very close proximity to the STS gene, and possibly within a 3’ intron. However, results obtained with Sal1 lend considerable

FIG. 5. Experiment to show the existence of a CpG island close to the STS locus. The Sfi I fragments of male blood DNA detected by 3’P2A7 are reduced in size in an identical fashion by double digestion with S&I, NacI, BssHII, EagI, and NotI. Prolonged exposure indicated that NarI cuts very partially in the island, but that there is no site for it4luI. Size markers were chromosomes of S. cereuisiae (C, sizes given) and oligomers of X phage DNA (L). Key to enzymes as in Fig. 1.

536

ROSS,

BALLABIO,

Ic.

b3

a)

c

sa SaSs

940

660

FIG. 6. Detecting altered fragments in the DNA of an individual with XLI. (a) Tracks show digests of ichthyotic male (1~) or control male DNA (8) probed with ST12. Sal1 fragments at about 320 kb are from the STS pseudogene on the Y chromosome. (b) DNA from ichthyotic male digested with Sal1 alone or with Sal1 and SstII together and probed with ST12. The unaltered fragment of 320 kb is from the Y chromosome. Size markers were chromosomes of S. cereuisiae (C, sizes given). Key to enzymes as in Fig. 1.

having removed all sites for Sal1 between the two loci. However, closer inspection of the result with ST12 revealed a faint fragment of 480 kb not recognized by GMGXS (Fig. 6a), presumably the product of weak digestion at a Sal1 site between the two loci. The deletion, therefore, would appear to cross neither the CpG island at position (0) on the maps nor the Sal1 site at (+480), and so provided no information on the orientation of the STS gene on the maps. Distance

to the Pseudoautosomal

Region

Physical linkage with markers proximal to the STS locus was not possible, although some useful information was obtained with these. The probe MlA (DXS31) detected an NruI fragment of about 2700 kb in blood DNA. Previous work has shown that the first X-chromosome-specific NruI site in blood lies close, and proximal, to the pseudoautosomal boundary (Brown, 1988). There must, therefore, be at least 4 Mb of DNA between the STS locus and the pseudoautosomal region. DISCUSSION

The distal part of the short arm of the human X chromosome contains much that is of interest to the

AND

CRAIG

molecular biologist. The 2.5 Mb of DNA from the telomere has been defined as pseudoautosomal (Burgoyne, 1982), that is, a region of ancestral X/Y homology maintained by an obligatory recombination event during each male meiosis, which serves to ensure the orderly segregation of the sex chromosomes into the gametes. Both within the pseudoautosomal region of the X chromosome and proximal to its boundary are examples of genes that evade the process of inactivation, the consequence of which is dosagecompensation. The best studied of the latter class of loci is the gene that encodes steroid sulfatase. Interest in the genetics of STS stemmed initially from a clinical source: it was noted several years ago that difficulties in pregnancy, specifically delayed parturition and failure of cervical dilatation, were often associated with low levels of the enzyme in the placenta and that the offspring of such pregnancies, invariably male, went on to develop ichthyosis, characterized by brown, scaly skin (reviewed in Shapiro, 1985). These boys lacked STS activity and immunologically crossreacting material (Epstein and Bonifas, 1985), and there now seemslittle doubt that this X-linked ichthyosis is due directly to the deficiency of the enzyme. Further attention was focused on the locus by the elucidation of its unusual pattern of expression, namely that, despite its X linkage, the gene remains active on the inactive X chromosome, and, moreover, that this escape from inactivation is only partial (Migeon et aZ., 1982). An analysis of the molecular basis of this phenomenon required the cloning of the gene, which was achieved in several laboratories (Ballabio et al., 1987; Yen et al., 1987; Conary et al., 1987; Bonifas et al., 1987). One of the cloned STS cDNAs has been employed here to construct a long-range restriction map of the X chromosome in the region of the STS locus. Physical linkage was defined between the STS locus and loci detected by the probes GMGXS (DXS237), GMGXY19 (DYS74), CRI-S232 (DXS278), and dic56 (DXS143). Close linkage between DXS237 and the STS locus had been established previously by family studies (Yates et al., 1987); their proximity was confirmed here, with DXS237 localized to within 450 kb of the STS gene. The X homolog of DYS74 is closer still to the STS locus: the probe GMGXY19 detected an array of Xchromosomal fragments identical to that recognized by the STS cDNA probe 3’P2A7, and additionally is contained within a deletion that was shown here to have removed only 180 kb of DNA including the 3’ part of the STS locus; a minimum of 20 kb of DNA from the 3’ end of the gene is lost in this case (A. Ballabio, unpublished observation), which places DYS74 either within the gene or a maximum of 160 kb downstream. CRI-S232 detects multiple X-chromosomal loci, but at

PHYSICAL

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least one of these was mapped to between S&II (0) and SfiI (+710), its most likely position being proximal to DXS237. A second locus with homology to CRI-S232 might lie between EagI (+800) and EagI (+2400), and a third is probably distal to the STS gene. The locus DXS143 detected by the probe dic56 was placed on the restriction maps between NruI (+1650) and BssHII (+2400). This sequence had previously been localized proximal to the STS gene (Mondello et al., 1987; Yates et al., 1987), thereby orienting the pulsed-field maps. The primary reason behind constructing these maps was to provide a framework for the physical analysis of the genetic lesions occurring in cases of XLI. The cloning of STS cDNAs made it possible to study such cases using conventional electrophoretic techniques; what emerged was that by far the most common type of genetic lesion was the deletion of the entire STS coding region (Ballabio et al., 1989). The location of the STS gene close to the pseudoautosomal region prompted speculation that illegitimate recombination during male meiosis was to blame for the high deletion frequency; after all, there is a precedent for this in the etiology of XX maleness. However, data obtained here indicate a minimum distance of 4 Mb between the STS locus and the proximal boundary of the pseudoautosomal region, this compared with the estimate of only about 200 kb between the Y pseudoautosomal boundary and the putative testis determinant(s) (Page et al., 1987). Furthermore, the retention of distal X-linked markers and the absence of detectable Y sequences, in most cases of deletions of the STS gene (Gillard et al., 1987; Yen et al., 1988; Ballabio et al., 1989), are inconsistent with this simple mechanism of stray recombination. Information on the sizes and locations of deletions in unrelated XL1 cases may throw light on the mechanisms at work in their generation and can be obtained by PFGE. As an example, an ichthyotic individual was studied here and shown to lack approximately 180 kb of DNA on the X chromosome. The type of lesion in this individual, leaving the 5’ end of the STS gene intact, is exceptional among cases of ichthyosis, the entire gene generally being deleted. In the majority of cases DXS237 is also removed (Gillard et al., 1987), and so the minimum deletion size in most cases may be as high as 600 kb. Many genes are associated with CpG islands, which contain most of the accessible sites for methylationsensitive restriction enzymes; and many CpG islands are associated with transcribed regions of the genome (Bird, 1986; Lindsay and Bird, 1987). Using the defining criterion of a cluster of two or more rare-cutter sites, four CpG islands appear on the pulsed-field maps at positions (0), (+800), (+1650), and (+2400); most of the sites in the island at (+1650) would appear to

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be methylated in ThyB-X but available for cleavage in blood. From the point of view of its expression, it would be of interest to establish whether there is an association between the STS locus and the distal CpG island at position (0), and, if such an association were demonstrated, to ask whether this island is methylated on the inactive X chromosome. Cases that involve XL1 in combination with other specific disorders have been described, these are presumed to be due to the deletion of contiguous genes (see Curry et al., 1984; Ballabio et al., 1989). One gene believed to lie close to the STS locus on this basis is that for Kallmann syndrome. This locus has been mapped proximal to STS but distal to DXS143 (Ballabio et al., in press), and so must lie upon the pulsedfield map. The CpG islands at (+800) and (+1650) should therefore be considered as defining candidate positions for the Kallmann gene. The loci DNF22Sl and DNF22S2 (Bardoni et al., 1988) flank the Kallmann gene (Ballabio et al., in press); placing these loci on the pulsed-field map should allow the exclusion of one-or possibly both-of the islands and might indicate the best strategy for approaching the Kallmann locus. Terminal deletions of the X short arm that remove the Kallmann locus and DXS143 are known; these deletions involve a loss of at least 10 Mb of DNA with relatively mild phenotypic consequences. If there are 30,000 CpG islands in the genome, then only 4 islands in 4 Mb of DNA is a dearth, and it is tempting to speculate that the region between the pseudoautosomal boundary and the Kallmann syndrome locus, and beyond, is relatively genetically inert. This may reflect the region’s escape from inactivation and the inability to tolerate dosage differences for most genes. ACKNOWLEDGMENTS We thank Mark Jobling and Thomas Meitinger for invaluable assistance at all stages of the work; Thomas Meitinger also built the excellent pulsed-field box. We are also indebted to William Brown, Chris Tyler-Smith, and Rakesh Anand for expert advice; Professor M. Ferguson-Smith and Drs. N. Affara and E. Gillard for the probes GMGXS and GMGXYlS; Dr. L. Kunkel for the probe dic56; Dr. J.-L. Mandel for the probe MlA; Collaborative Research, Inc., for the probe CRI-S232; and Rebecca Oakey for the gift of plugs of male fibroblast DNA.

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