GENOMICS

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Isolation of the Human Sex Determining Region from a Y-Enriched Yeast Artificial Chromosome Library ANTHONY

P. MONACO,’

ULRICH MijLLER,*‘t

ZOIA LARIN, SEBASTIAN MEIER-EWERT,

AND HANS LEHRACH

Genome Analysis Laboratory, imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2a 3PX, United Kingdom; *Genetics Division, The Children‘s Hospital, Boston, Massachusetts; and tDepartment of Pediatrics, Harvard Medical School, Boston, Massachusetts 02 17 5 Received

March

27, 1991;

revised

July 19, 1991

We isolated and characterized four overlapping yeast artificial chromosome (YAC, Burke et uZ., 1987) clones using probes from this interval to establish the continuity and stability of the region in yeast and for future functional studies of the male sex determining region in transgenic mice. The analysis of these and other YAC clones isolated from the YAC library enriched for human Y sequences suggested that partial degradation of DNA occurred during YAC library construction. The YAC insert sizes were much smaller than the size of fragments selected by PFGE and only contained the right arm of the cloning vector pYAC4 (Burke et al., 1987). Partial degradation of high-molecular-weight DNA isolated from agarose after PFGE has been shown to occur when agarose-containing DNA was melted (Larin et al., 1991). This problem was eliminated by protecting the DNA with polyamines before melting the agarose and has resulted in human (48,XxXx) and mouse (C3H, male) YAC libraries with much larger average insert sizes (>600 kb, Larin et aZ., 1991). We used the same protocols with polyamines to construct a second Y-enriched YAC library of 3000 clones with an average insert size of 627 kb.

We describe the isolation and characterization of yeast artificial chromosome(YAC) clonesspanning the male sex determining region on the short arm of the human Y chromosome.The cloneswere isolated by hybridizing probesin the interval between the genesMIC2 and ZFY to a Y chromosome-enrichedYAC library. The YAC cloneswere consistent with the order of probesestablishedfor this interval and may be useful for functional studies of the region in malesex determination. However, many of the YAC clones from this library carried only one arm of the vector (“halfYACs”), deleted sequencesfrom one end, and contained much smaller inserts (148 kb average) than the size of ligated fragments selectedby pulsed-field gel electrophoresis (>440 kb). These problems were overcome by protecting DNA with polyamines during YAC library construction and a secondY-enriched YAC library was constructed with an average insert size of 627 kb. Q ml Academic press, IUC. INTRODUCTION

The male sex determining region on the short arm of the human Y chromosome has been localized between the genes for MIC2 (Darling et al., 1986) and ZFY (Page et al., 1987) in a 35-kb region proximal to the pseudoautosomal boundary (PAB) (Palmer et aZ., 1989). A candidate gene for male sex determination has been isolated in both mouse and man using conserved sequences from this 35-kb area (Sinclair et aZ., 1990; Gubbay et al., 1990). The region between MIC2 and ZFY has been well characterized by chromosome walks from the 3’ end of MIC2 toward ZFY (Ellis et al., 1989) and from the probe Y286 (Muller, 1987) and has been estimated to be 260-290 kb by pulsed-field gel electrophoresis (PFGE; Pritchard et al., 1987; Verga and Erikson, 1989; Muller and LaLande, 1990).

MATERIALS

AND METHODS

YAC Library Construction Both YAC libraries were constructed from DNA isolated from the cell line OXEN (49,XYYYY; Sirota et al, 1981) in low-melting-point agarose blocks containing 1 X 10’ cells (Herrmann et al., 1987). DNA in blocks was partially digested to produce fragments ranging from 100 to 2000 kb using a mixture of EcoRI (1 p) and EcoRI methylase (200 p) for 4 h at 37°C as described (Larin et al., 1991). The digests were terminated by adding EDTA and proteinase K to 10 mM and 200 pg/ml, respectively, and incubated at 37°C for 30 min.

1 To whom correspondence should be addressed at Human Genetics Laboratory-ICRF, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK. 1049

All

Copyright Q 1991 rights of reproduction

OSSS-7543/91$3.00 by Academic Press, Inc. in any form reserved

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MONACO ET AL.

For the first YAC library construction, blocks were washed (twice for 20 min each) at 50°C in 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA (1X TE) followed by 1X TE with 0.04 mg/ml phenylmethylsulfonyl fluoride (PMSF) and 1X TE with 30 mM NaCl. Blocks were melted at 68°C for 10 min and cooled to 37°C. The vector pYAC4, which had been digested to completion with BamHI and EcoRI and dephosphorylated with calf intestinal phosphatase, was added in an equal weight ratio to genomic DNA. Ligation reactions were carried out in 50 mM Tris-HCl, pH 7.5,lO mM MgCl,, 10 mM DTT, 0.5 mM ATP, and 10 u/p1 T4 DNA ligase at 16°C for 16 h. Ligation reactions were terminated by adding EDTA to 20 mM, heated at 68°C for 10 min and cooled to 37°C. The molten mix was loaded into a trough of a 1% low-meltingpoint agarose gel (Beckman) in 0.25X TBE. The gel was run on a CHEF apparatus (EMBL, Heidelberg) for 16 h at 170 V at a switch time of 30 s. The outside lanes containing some of the ligation mix and yeast chromosomes as size markers were stained with ethidium bromide, and the leading edge of the limiting mobility (>440 kb) was notched under uv light. The marker lanes were then placed adjacent to the preparative unstained gel and the limiting mobility up to and including the gel slots was removed. The gel slice (l-2 ml) was washed four times (30 min each) at 4°C in 1.0 M sorbitol, 10 mM Tris-HCl, pH 7.5, 30 mM NaCl, and 2 mM EDTA. The solution was removed and the gel slice was melted at 68°C for 10 min and then cooled to 37°C. Agarase (CalBiochem) was added slowly at 100 u/ml agarose, incubated for 16 h at 37”C, and then stored at 4°C. The second YAC library was constructed from 49,XYYYY DNA using protocols described in Larin et a2. (1991). Briefly, EcoRI partially digested DNA in blocks was size selected to ~440 kb by PFGE. The limiting mobility was excised, washed in ligase buffer and polyamines (0.75 mM spermidine/0.30 mM spermine), melted, ligated to pYAC4, and size-selected on a second pulsed-field gel to >440 kb. The limiting mobility was excised and washed in 10 mM Tris-HCL, pH 7.5, 1 mM EDTA, 30 n-&f NaCl, and polyamines prior to melting and agarase treatment as described above.

Yeast Transformation, Transformation

Replication,

and Screening

of yeast cells from the SaccharoAB1380 (Burke et al., 1987) was carried out according to the protocol of Burgers and Percival (1987). Yeast cells (150 ~1) were transformed at a final concentration of 4 X lO’/ml with 500 ng of ligated DNA (50-100 ~1 of DNA in digested agarose solution), and 5 pg of sheared salmon sperm DNA (in the first YAC library only). Ten tubes con-

myces cerevisae strain

taining 200-250 ~1 each of transformed ceils were pooled and mixed with 50 ml of 2.5% top agar (Rothstein, 1985) kept at 48°C and poured onto the surface of a 2% agar plate (22 X 22 cm). Plates were incubated at 30°C for 4 days, Recombinant YAC clones embedded in the top agar of regeneration plates were replicated to the surface of another plate (22 X 22 cm) lacking sorbitol and uracil by a metal grid transfer device as described previously (Larin and Lehrach, 1990; Larin et at., 1991). Hybond N+ filters (Amersham) were used to make colony filter replicas and hybridized with radioactively labeled probes (Larin and Lehrach, 1990). Potential positive YAC clones were picked from the primary library plates and rescreened by colony hybridization.

YAC Clone Analysis and DNA Probes Single positive colonies were grown in liquid culture and yeast chromosomes were isolated in agarose blocks (Larin and Lehrach, 1990). Undigested YAC clones were analyzed on 0.75% agarose gels in 0.25X TBE on a CHEF apparatus at 170 V for 36 h at a switch time of 70 s. For restriction enzyme digests, blocks were prepared by washing in 1X TE twice (20 min each) at 5O”C, 1X TE with 0.04 mg/ml PMSF twice at 5O”C, and then 1X TE twice at room temperature. After restriction enzyme digests, YAC DNA was loaded adjacent to human genomic DNA samples and fractionated on 0.75% agarose gels in circulating 1X TAE buffer for 16 h at 2.5 V/cm. All gels were vacuum blotted for 1.5 h in 0.5 M NaOH and 1.5 M NaCl onto Hybond Nf membranes (Amersham), vacuum baked at 80°C for 10 min, and uv-crosslinked for 2 min. DNA probes used in this study were 3’ RSA, a genomic probe from the 3’ end of MIC2 (Ellis et al, 1989); HF0.2, a genomic probe that detects sequences from the PAB (Ellis et al., 1989); and PO.9 and 27a, Y-specific probes that are proximal to the PAB (Pritchard et al., 1987; Ellis et al., 1989). More proximally, three Y-specific genomic fragments, Y286-1, Y286-la5, and Y286-lOBE, were isolated from a phage walk from Y-286 (Muller, 1987), and cDNA 5.8 was used as a ZFY probe (Palmer et al., 1990). One additional probe, Y286-6a4, was generated by isolating YAC Y286-la5 No. 1 from a low-melting-point agarose FIGE gel and digesting it to completion with EcoRI. The agarose was melted at 68°C and extracted with phenol and chloroform, and the DNA was precipitated twice with ethanol and then ligated to the plasmid vector Bluescript (Stratagene). Recombinant plasmids were amplified, inserts were isolated and tested by hybridization against Hind111 and EcoRI digests of YAC DNA and human genomic DNA for Y chromosome specificity. Y286-6a4 is the smallest 0.6-

YACs

FOR

HUMAN

SEX

DETERMINING

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REGION

3’ RSA-

PO.9-

,234

1234 A

1 B

234 c

1234 0

FIG. 1. PFGE of undigested YACs isolated with Y286la5. Lanes 1,2, and 3 are three DNA preparations from single colonies of YAC Y286-la5 No. 1. Lane 2 has the original and deleted YAC. Lane 4 is a single colony preparation of YAC Y286la5 No. 2 containing both the original and the deleted YAC. Hybridizations are done with (A) 3’ RSA, (B) P0.9, (C) 27a, and (D) Y286-la5.

kb fragment released by EcoRI the recombinant plasmid.

and PstI digestion

of

RESULTS

Isolation of the Sex Determining Clones

12

1234567

3

A

4

5

67

B

of three DNA probes to human genoFIG. 2. Blot hybridization mic DNA samples (lane 1, 4Y cell line; lane 2, normal male) and YAC DNA digests (lane 3, PO.9 YAC No. 2; lane 4, Y286-la5 YAC No. 1; lane 5, YAC No. 1 with original and deleted YAC; lane 6, YAC No. 2; lane 7, YAC No. 5). (A) DNA samples digested with EcoRI and probed simultaneously with 3’ RSA and P0.9. (B) DNA samples digested with Hi&II and probed with Y2Wla5. HindIIIdigested EDNA was used as size markers as indicated.

Region in YAC

The first 4Y YAC library consisted of 15,000 clones plated on three primary library filters (22 X 22 cm). Two DNA probes (Y286-la5 and P0.9) were used to isolate a set of four overlapping YAC clones that included the 3’ end of MICP but not the 5’ end of ZFY. All four YAC clones were checked by probes along this interval on PFGE blots of undigested chromosomes (Fig. 1) and on blots of DNA digested with HindIII and EcoRI (Fig. 2). Two site-specific deletions were identified in YAC clones Y286-la5 Nos. 1 and 2 (Fig. 1). The deletion in Y286-la5 No. 1 included the 3’ end of MICB, the probe HF0.2 (PAB), and the probe PO.9. The deletion in Y286-la5 No. 2 included the probe 27a and the subclone Y286-6a4. Deleted YACs were seen alone or in combination with the original YAC when grown from single colonies and always at the same size and deleted for the same probes on hybridization of HindIII and EcoRI digests (data not shown). A schematic diagram summarizing the YACs isolated from this region and the extent of their deletions is shown in Fig. 3. The distances between probes and between the genes MIC2 and ZFY have been determined from chromosome walks in this region (Ellis et aZ., 1989; Palmer et al., 1990, Muller, 1987) and longrange PFGE maps (Pritchard et al., 1987; Verga and Erikson, 1989; Muller and LaLande, 1990). The probe Y286-1 identified an altered Hind111 and EcoRI fragment in PO.9 YAC No. 2 showing it to be at one end since this YAC was negative for probes Y286-la5 and

Y286-10BE. The YAC subclone Y286-6a4 recognized an end junction fragment in YAC Y286-la5 No. 5, indicating that the YAC did not extend as far distally as YAC Y286-la5 No. 2.

Problems

with the First YAC Library

The first Y-enriched YAC library of 15,000 clones had an average insert size of 148 kb from the PFGE analysis of 20 randomly selected clones and 17 positive YACs isolated by hybridization with specific probes (range, 85-600 kb; Fig. 4A). Although the ligated genomic DNA was size-selected by PFGE to

I ID0

I o

Kb

I

I 200

300

TEL

CEN

5

3

PAB PO.9

278

“286

5

3

1 1 (de,,

2(&l, 5

diagram of genes, DNA probes, and YAC FIG. 3. Schematic clones in the male sex determining region of the Y chromosome (SRY). YACs Nos. 1, 2, and 5 were isolated with Y286-la5 and YACs No. 1 and PO.9-2 were isolated with P0.9. The telomere of Yp is indicated toward the left and the centromere toward the right.

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MONACO

AL.

picked clones and 3 clones positive for specific probes the average insert size was 627 kb (range, 280-1800 kb, Fig. 4B). Most of the YAC clones in this second library were larger than 440 kb, the size selected by PFGE and all clones grew on double selection of (-)Ura, (--)Trp. The library (approximately 0.5 genome equivalents) was screened by hybridization and three positive clones have been isolated. This second YAC library has been picked into microtiter dishes for permanent storage.

24 22 20 18 16 14 NO. YACs

ET

12 10 8

DISCUSSION

6 4 2

kb

B 16 14 12 10 NO. YACs

8 6

LIL average size

627kb

n

600

700

800800

>looo

kb

FIG. PFGE probe. (B) 34 structed

The phenomenon of “half-YACs” explained why many YAC clones from the first library would not grow on double selection, and perhaps was responsible for the deletion of sequences from one end. There would be no telomere, centromere or autonomous replicating sequences present from the left arm of the vector and one or more of these functions may be required from the human genomic DNA insert for YAC stability. Without the yeast telomere sequences seeded from the left arm of pYAC4, the naked DNA ends would be degraded during replication but may stop at specific human sequences that may function to seed the addition of yeast telomeres. The halfYACs may also explain why the average insert size of the library (148 kb) was so much lower than the size of ligated DNA fractionated by PFGE (>440 kb). If there was degradation of the ligated material after PFGE size selection, then few molecules would contain both arms of the vector. We have recently investigated the degradation of size-selected DNA in agarose and have found it to occur at the melting step at 68°C (Larin et aZ., 1991). This degradation could be inhibited by protecting the DNA with polyamines before any melting step of DNA in agarose and has resulted in mouse and human (48,xXxX) YAC libraries with 700- and 620-kb average insert sizes, respectively (Larin et al., 1991). We describe here a second Y-enriched YAC library of 3000 clones with an average insert size of 627 kb. The consistency of large inserts in the second library shows the reliability of including polyamines when isolating DNA from agarose during YAC library construction. We have isolated overlapping YAC clones for the region between MICP and ZFY, one of which contains the gene for male sex determination. If the human equivalent of the male sex determining gene is functional in mice, transgenics could be constructed after microinjection of isolated YAC DNA and sex-reversed progeny identified. Recently, mice transgenic for a 14-kb genomic fragment containing the mouse Sry gene exhibited sex reversal although 25 kb of the human genomic region failed to reverse sex in female transgenic mice even though it was expressed at the

4. Histogram of the distribution of YAC insert sizes by analysis and hybridization with a human-specific repeat (A) 37 YAC clones from the first 49, XYYYY YAC library. YAC clones from the second 49, XYYYY YAC library conusing polyamines.

>440 kb, most of the YAC clones were between 100 and 200 kb (Fig. 4A). The second problem with YAC clones isolated from this library was that very few clones grew on double selection of (-)Ura, (-)Trp. To test for pYAC4 arm-specific fragments, the four YACs isolated by Y286-la5 and PO.9 were digested with Hi&II or EcoRI and hybridized with pBR322 (data not shown). This revealed that all four clones were half-YACs containing only one copy of the right arm of the pYAC4 vector (Ura 3) and no sequences from the left vector arm (pBR322 ori and Ampicillin resistance gene).

The Second YAC Library The second YAC library enriched for Y sequences was constructed using polyamines and consisted of 3000 clones. From the PFGE analysis of 31 randomly

YACs FOR HUMAN

SEX DETERMINING

appropriate stage in the genital ridge (Koopman et al., 1991). An alternative approach may be to fuse spheroplasted yeast cells containing YACs to embryonic stem cells as was recently described with mouse Lcells and embryonal carcinoma cells (Pachnis et 4, 1990; Pavan et aZ., 1990). Embryonic stem cells containing the intact YAC stably integrated could then be injected into fertilized embryos to produce transgenie mice. ACKNOWLEDGMENTS We thank Matthijs Smith, Nathan Ellis, Mark Palmer, Andrew Sinclair, and John Robbins for DNA probes used to isolate and characterize YACs and Peter Goodfellow for discussions on the manuscript. U.M. is supported by NIH Grant HD24381-02. A.P.M. was supported in part by the Muscular Dystrophy Association of America.

REFERENCES 1. BURGERS, P. M. J., AND PERCIVAL, K. J. (1987). Transformation of yeast spheroplasts without cell fusion. Anal. Biochem. 163: 391-397. 2. BURKE, D. T., CARLE, G. F., AND OLSON, M. V. (1987). Cloning of large DNA segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236: 806812. 3. DARLING, S. M., BANTING, G. S., PYM, B., WOLFF, J., AND GOODFELLOW, P. N. (1986). Cloning an expressed gene shared by the human sex chromosomes. Proc. Natl. Acad. Sci. USA 83: 135-139. 4. ELLIS, N. A., GOODFELMW, P. J., PYM, B., SMITH, M., PALMER, M., FRISCHAUF, A.-M., AND GOODFELLOW, P. N. (1989). The pseudoautosomal boundary in man is defined by an Alu repeat sequence inserted on the Y chromosome. Nature (London) 337: 81-84. 5. GUBBAY, J., COLLIGNON, J., KOOPMAN, P., CAPEL,B., ECONOMOU, A., MUNSTERBERG, A., VIVIAN, N., GOODFELLOW, P., AND LOVELL-BADGE, R. (1990). A gene mapping to the sex determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes. Nature (London) 346: 245-250. 6. HERRMANN, B. G., BARLOW, D. P., AND LEHRACH, H. (1987). An inverted duplication of more than 650 kbp in mouse chromosome 17 mediates unequal but homologous recombination between chromosomes heterosygous for a large inversion. CeU 48: 813-825. 7. LARIN, Z., AND LEHRACH, H. (1990). Yeast artificial chromosomes: An alternative approach to the molecular analysis of mouse developmental mutations. Genet. Res. 56: 203-208.

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8. LARIN, Z., MONACO, A. P., AND LEHRACH, H. (1991). Yeast artificial chromosome libraries containing large inserts from mouse and human DNA. Proc. Natl. Acad. Sci. USA 88: 41234127. 9. KOOPMAN, P., GUBBAY, J., VIVIAN, N., GOODFELLOW, P., AND NOVELL-BADGE, R. (1991). Male development of chromosomally female mice transgenic for Sry. Nature (London) 351: 117-121. 10. MULLER, U. (1987). Mapping of the testis-determining locus on Yp by the molecular genetic analysis of XX males and XY females. Development 101: 51-58. 11. MULLER, U., AND LALANDE, M. (1990). A physical map of the human Y-chromosome short arm. Genomics 7: 517-523. 12. PAGE, D. C., MOSHER, R., SIMPSON, E. M., FISHER, E. M. C., MARDON, G., POLLACK, J., MCGILLIVRAY, B., DE LA CHAPELLE, A., AND BROWN, L. G. (1987). The sex-determining region of the human Y chromosome encodes a finger protein. Cell 51: 1091-1104. 13. PACHNIS, V., PEVNY, L., ROTHSTEIN, R., AND CONSTANTINI, F. (1990). Transfer of a yeast artificial chromosome carrying human DNA from Saccharomyces cerevisiae into mammalian cells. Proc. Natl. Acad. Sci. USA 87: 5109-5113. 14. PALMER, M. S., SINCLAIR, A. H., BERTA, P., ELLIS, N. A., GOODFELLOW, P. N., ABBAS, N. E., AND FELLOUS, M. (1989). Genetic evidence that ZFY is not the testis-determining factor. Nature (London) 342: 937-939. 15. PALMER, M. S., BERTA, P., SINCWR, A. H., PYM, B., AND GOODFELLOW, P. N. (1990). Comparison of human ZN and ZFX transcripts. Proc. Natl. Ad. Sci. USA 87: 1681-1685. 16. PAVAN, W. J., HEmR, P., AND REEVES, R. H. (1990). Modification and transfer into an embryonal carcinoma cell line of a 360-kilobase human-derived yeast artificial chromosome. Mol. Cell. Bid. 10: 4163-4169. 17. PRITCHARD, C. A., GOODFELLOW, P. J., AND GOODFELLOW, P. N. (1987). Mapping the limits of the human pseudoautosoma1 region and a candidate sequence for the male-determining gene. Nature (London) 328: 273-275. 18. ROTHSTEIN, R. (1985). Cloning in Yeast. In “DNA Cloning” (D. M. Glover, Ed.), Vol. 2, pp. 45-65. IRL Press, Oxford. 19. SIROTA, L., ZLOTOGORA, Y., SHABATAI, F., HALBRECHT, I., AND ELIAN, E. (1981). 49, XYYYY: A case report. Clin, Genet. 19:87-93. 20. SINCLAIR, A. H., BERTA, P., PALMER, M. S., HAWKINS, J. R., GRIFFITHS, B. L., SMITH, M. J., FOSTER, J. W., FRISCHAIJF, A.-M., LOVELL-BADGE, R., AND GOOD-W, P. N. (1990). A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature (London) 346: 240-244. 21. VERGA, V., AND ERICKSON, R. P. (1989). An extended longrange restriction map of the human sex-determining region on Yp, including ZFY, finds marked homology on Xp and no detectible Y sequences in an XX male. Am. J. Hum. Genet. 44: 756-765.