Proc. Nati. Acad. Sci. USA Vol. 83, pp. 135-139, January 1986 Genetics Cloning an expressed shared by the human sex (Xgtll/ceil-surface antigen/X and Y chromosomes/transfectants) S. M. DARLING*, G. S. BANTING*, B. PYM*, J. WOLFEt, AND P. N. GOODFELLOW* *Laboratory of Human Molecular Genetics, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom; and tDepartment of Genetics and Biometrics, University College London, Wolfson House, 4 Stephenson Way, London NW1 2HE, United Kingdom Communicated by G. Pontecorvo, August 15, 1985

ABSTRACT The existence of shared by mammalian reacts strongly with a 32-kDa cell-surface molecule ex- sex chromosomes has been predicted on both evolutionary and pressed on the surface of human but not mouse cells. The functional grounds. However, the only experimental evidence antibody also recognizes a 29-kDa cytoplasmic molecule for such genes in humans is the cell-surface antigen encoded by found in both mouse and human cells. The cell-surface loci on the X and Y chromosomes (MIC2X and MIC2Y, products of the human X and Y chromosomes cannot be respectively), which is recognized by the monoclonal antibody distinguished by either size or charge (13). These results 12E7. Using the bacteriophage Xgtll expression system in support the contention that the MIC2X and MIC2 Y loci are Escherichia coi and immunoscreening techniques, we have related, but the degree of homology between these genes isolated a cDNA clone whose primary product is recognized by cannot be gauged without knowing if the 12E7 antigen is the 12E7. Southern blot analysis using somatic cell hybrids con- primary polypeptide product of the MIC2 loci. A direct taining only the human X or Y chromosomes shows that the approach for resolving this question is to clone the DNA sequences reacting with the cDNA clone are localized to the sex sequences corresponding to the MIC2 loci. In this report, we chromosomes. In addition, the clone hybridizes to DNAs describe the isolation of a cDNA clone corresponding to an isolated from mouse cells that have been transfected with MIC2 locus. We have used this clone to investigate the human DNA and selected for 12E7 expression on the fluores- relationship between the 12E7 antigen and the MIC2 loci and cence-activated cell sorter. We conclude that the cDNA clone between the MIC2X and MIC2 Yloci. We encodes the 12E7 antigen, which is the primary product of the conclude that the 12E7 antigen is the primary product of the MIC2 loci. The clone was used to explore sequence homology MIC2 loci and that MIC2X and MIC2 Y are closely related, if between MIC2X andMIC2Y; these loci are closely related, ifnot not identical. identical. MATERIALS AND METHODS The human sex chromosomes have been shown to share a pair of related genes defined by the monoclonal antibody Cell Cultures. Cells were grown at 37°C in Dulbecco's 12E7. This antibody was raised against leukemic human cells modified Eagle's medium, or RPMI 1640 medium, supple- (1), reacts with a cell-surface antigen expressed on all human mented with 10% fetal bovine serum, penicillin, and cells, and fails to react with the surface of rodent cells (2). streptomycin. Where appropriate (HORL 9X and "12E7- Human-rodent somatic cell hybrids, which retain the human transfectants"), the medium was supplemented with HAT X , express the 12E7 cell-surface antigen. This medium (100 ,M hypoxanthine/10 ,uM methotrexate/16 ,M defines the MIC2X locus (3). Similarly, somatic cell hybrids thymidine). that retain the human Y chromosome also express the 12E7 Cell Lines. The following human cell lines were used: J6, a antigen (4) and this defines the MIC2 Y locus. No autosomal subclone of the male T-cell line Jurkat (14); Molt-4, a male locus is sufficient, or necessary, for 12E7 antigen expression T-cell line (15); GM1416B, a female B-cell line that contains in somatic cell hybrids. The MIC2X locus has been localized four X chromosomes (purchased from the Institute for to Xp22.32-Xpter (4, 5), the terminal region of the X Medical Research, Camden, NJ); OX, a B-cell line containing chromosome short arm; the MIC2 Y locus has been assigned one X chromosome and four Y chromosomes (see ref. 16; a to Ypter-Yqll, the euchromatic region ofthe Y chromosome gift from M. Fellous, Institut Pasteur, Paris). (4). As might be predicted for an expressed X-linked gene * The following mouse cell lines were used: the L cells IR with a Y-linked homologue, the MIC2X locus escapes X- (17) and LMTK- (18); the adenocarcinoma cell line RAG inactivation, thus maintaining functional gene dosage for the (19). The human-mouse somatic cell hybrids were used: MIC2 loci in males and females (6). Two other genes, STS and HORL 9X (mouse parent IR) retains the X chromosome as its Xg, map to the tip of the X chromosome short arm (7, 8) and only human genetic contribution (20); 3E7 (mouse parent escape X-inactivation (9, 10); however, Y-linked homologues RAG), a hybrid retaining the human Y chromosome only of these genes have not been described in humans. (21). Full details of the 12E7 antigen-positive transfectant The level of expression of the 12E7 antigen on erythro- lines will be presented elsewhere (unpublished data). Briefly, cytes, but not on nucleated cells, is polymorphic (11). In LMTK- cells were transfected with DNA from Molt-4 and females, the polymorphism is controlled by the Xg blood the selectable plasmid pTK1 (22) by using the calcium group locus, and in males it is controlled byXg and a Y-linked phosphate precipitation method derived from Wigler et al. locus, Yg (11, 12). The precise genetic and biochemical (23). The transfectants were sorted for 12E7 antigen expres- relationship between the MIC2 loci and the Xg and Yg loci sion on the fluorescence-activated cell sorter (FACS) and the has not been resolved. primary positive transfectant TKM1EP5 was isolated. This The 12E7 antibody is a poor reagent for immunoprecip- mass positive population was sorted further for increased itation experiments; however, in immunoblot analysis it 12E7 antigen expression. TKM1EP7, TKM1EP9, and TKMlEP13 are mass cultures that express increasing The publication costs of this article were defrayed in part by page charge amounts of 12E7 antigen (primary amplified transfectants). payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: FACS, fluorescence-activated cell sorter. 135 Downloaded by guest on September 28, 2021 136 Genetics: Darling et al. Proc. Natl. Acad. Sci. USA 83 (1986) DNA from TKM1EP7 was transfected into LMTK- cells as down to 15 mM NaCl/1.5 mM sodium citrate at 650C and described above, and two independent secondary positive exposed to XAR-5 film (Kodak) at -700C with an intensifying transfectant populations, EP5A and EP5C, were isolated by screen. FACS selection. Genomic DNA was isolated from these cultered cell lines by standard techniques (24). RESULTS Construction of cDNA Library. The library, a gift from H. Analysis of the Products Encoded by the MIC2 Loci. The Kataoka and M. Collins (Imperial Cancer Research Fund, monoclonal antibody 12E7 reacts in a species-specific man- London), was constructed as follows: cDNA was synthe- ner with a of 32.5-kDa that is expressed on human sized from J6 poly(A)+ RNA (25), and the RNADNA hybrid cells and on human-rodent somatic cell hybrids when the was converted to double-stranded cDNA by treatment with human X and/or Y chromosome is present. A smaller band RNAse H, DNA polymerase I, and Escherichia coli DNA (29-kDa) is also recognized by the 12E7 antibody and is ligase (26). The cDNA was methylated with EcoRI methylase present in both human and mouse cell extracts (ref. 13; Fig. and ligated to synthetic EcoRI linkers and oligomeric termi- 1). Both polypeptides were detected in immunoblot analysis nal linkers cleaved with EcoRI. The excess linkers were of primary, primary amplified, and secondary DNA removed by gel filtration through Bio-Gel A-50 and the cDNA transfectant cell lines (Fig. 1) that had been selected for 12E7 was ligated to Xgtll arms (27) previously digested with EcoRI surface-antigen expression. In the amplified primary and dephosphorylated with calf intestinal alkaline phospha- transfectant, the 32.5-kDa species-specific protein is ampli- tase. After in vitro packaging, the library of 2.5 x 106 fied in expression, whereas the common band of 29-kDa plaque-forming units (pfu) was amplified in E. coli Y1088, a protein remains at approximately the same level. laclq strain that represses transcription of the /3-galactosid- Screening of the cDNA Library. The poor reaction of the ase-cDNA fusion protein (28). 12E7 antibody in immunoprecipitation (13) precluded classi- Antibody Screening of cDNA Library and Immunoblot cal cDNA cloning approaches. However, its reactivity in Analysis. The amplified library was propagated in the E. coli immunoblotting suggested that direct recognition of fusion strain Y1090 as described by Young and Davis (29). After in a prokaryotic expression system might be suc- induction of the B3-galactosidase-cDNA fusion protein for 3.5 cessful. In bacteriophage Xgtll, eukaryotic peptides can be hr at 42TC, the nitrocellulose filters were oriented, and then expressed as part of a /3-galactosidase fusion protein. Previ- blocked by incubation in phosphate-buffered saline (PBS) ous experiments had suggested that 12E7 and RFB1 recog- (pH 7.5) supplemented with 0.5% Tween 20 and 50 kallikrein nize the same antigens (unpublished observations). Since it units of aprotinin per ml (PBS/Tween) for 15 min at room had been proposed that polyclonal antisera might be more temperature with gentle agitation (30). The filters were efficient for screening Xgtll expression libraries (27), we used incubated at room temperature overnight with antibody in a mixture of 12E7 and RFB-1 in an attempt to increase the heat-sealed plastic bags. sensitivity of the detection procedure. We screened 2.5 x 106 A mixture of two monoclonal antibodies, 12E7 (1) and pfu; 30 bacteriophage RFB-1 (31), was used for the screening; both antibodies have plaques produced positive signals. been shown to recognize the products of the MIC2 loci by XSG1 was isolated after two subsequent rounds of plaque immunoblot analysis (ref. 13; unpublished results). The purification. DNA from XSG1 was prepared, digested with antibody source was hybridoma tissue culture supernatant EcoRI, and found to contain a 1-kilobase insert. that was concentrated 10-fold and then diluted 1:20 with Analysis of Fusion Proteins. Initially, we made lysogens of PBS/Tween. After incubation, free antibody was removed E. coli strain Y1089 by infection with XSG1 and analyzed by a 15-min wash in PBS/Tween. Bound antibody was isopropyl ,B-D-thiogalactopyranoside (IPTG)-induced lysates detected by using horseradish peroxidase-conjugated rabbit for the presence of a ,B-galactosidase-cDNA fusion protein. anti-mouse IgG (purchased from Dako, Denmark) diluted Since the ,B-galactosidase-cDNA fusion protein is a much 1:200 with PBS/Tween and incubated for 1 hr with the filters. larger protein than the majority of the other E. coli proteins, The filters were washed 2 x 20 min in PBS/Tween and 1 x detection of the product in Coomassie blue-stained gels is 10 min in PBS before color detection using 4-chloronaphthol (13, 30). Immunoreactive bacteriophage were picked and purified through two additional rounds of screening. Subse- k>a kDa quently, bacteriophage inserts were recloned into the EcoRI 93- sites of either pEX2 (32) or pUC8 (33). E. coli pop 2136 (a gift from K. Stanley, European Molecular Biology Laboratory, 69- -93 Heidelberg) was used for transformation of the pEX2 con- structs. - 69 For immunoblot analysis (34), proteins from either cell line 46- lysates or E. coli lysates were separated by electrophoresis through NaDodSO4/polyacrylamide gels and transferred on- to nitrocellulose filters as described (13). The filters were -46 treated as described above for antibody screening. A mono- clonal anti-p-galactosidase antibody was a gift from D. Lane 30 - _ (Imperial College, London). RFB1, a gift from M. Bodger (New Zealand), is a monoclonal antibody that recognizes the ' ~ ~~~~~30 same antigens as 12E7. Southern Analysis. Genomic DNAs (20 ug) were cleaved with restriction enzymes following the manufacturer's rec- 1 2 3 4 5 6 7 8 ommended conditions and fractionated through 0.8% agarose gels. The gels were denatured, neutralized, and FIG. 1. Immunoblot analysis of proteins. Cell lysates were transferred to separated on NaDodSO4/polyacrylamide gel and transferred to nitrocellulose filters as described (24). DNA as probe was nitrocellulose. The filter was incubated with the 12E7 antibody and labeled with [a-32P]dCTP by the method of random priming reacting antigenic determinants were detected as described. Lanes: (35) and hybridized to the filters at 420C in 40% forma- 1, RAG; 2, 3E7; 3, HORL9X; 4, 1R; 5, LMTK-; 6, TKM1EP7; 7, mide/6% polyethylene glycol 6000. Filters were washed TKM1EP13; 8, EP5C. Downloaded by guest on September 28, 2021 Genetics: Darling et al. Proc. Natl. Acad. Sci. USA 83 (1986) 137 possible (27). However, we could not detect a fusion protein 1 2 3 4 5 6 M in Coomassie blue- or silver-stained gels. After immunoblot - 23 analysis of these gels and probing with the 12E7 antibody, a single small band of 27-kDa was seen but no large fusion -66 protein was detected. This polypeptide was not constitutively expressed and its production was IPTG inducible. Therefore, g v t -~~~~~4.4 we concluded that there was breakdown of the fusion proteins; similar observations have been reported elsewhere (36). This problem was circumvented by using another prokary- otic expression system-the pEX vectors (32). These vectors contain a cro-lacZ gene fusion, which is under the control of the PR promoter of bacteriophage X. A polylinker sequence -2.3 has been inserted into the 3' end of the lacZ gene in all three -2.0 translational reading frames so any open reading frame DNA may be expressed as a fusion protein. Insert from XSG1 was cloned into the EcoRI site ofpEX2. The recombinant plasmid was introduced into E. coli pop 2136, amplified at 30'C, and then transient expression was induced at 42TC. A 130-kDa cro-o-galactosidase-cDNA fusion protein was detected after Coomassie staining of the gel. In immunoblot analysis the fusion protein reacted with both 12E7 and a monoclonal anti-p-galactosidase antibody. These results strongly suggest that the 12E7 antigenic determinant is entirely defined by a FIG. 3. Chromosomal localization of sequences that react with specific sequence of amino acids and is therefore part of a XSG1. Southern analysis ofEcoRI-cleaved genomic DNA. Lanes: 1, primary gene product (Fig. 2C). 3E7; 2, mouse control; 3, HORL9X; 4, GM1416B; 5, OX; 6, J6; M, Chromosomal Localization of Sequences Reacting with the molecular size markers in kilobase pairs. cDNA Probe. The insert from XSG1 was subcloned into the vector pUC8 (33) for easier manipulation and was called to DNA from the 12E7 expressing genomic DNA transfect- pSG1. This probe was hybridized to a panel of DNAs ants. Primary transfectants and independent secondary consisting of human-mouse hybrids containing the human X transfectants both react with pSG1 (Fig. 4). DNA from the or Y chromosomes and human cell lines with various num- secondary transfectants fails to react with Alu repeat or total bers of sex chromosomes (Fig. 3). pSG1 reacts with DNA human DNA (data not shown), and thus the total amount of from the human X and Y chromosomes. No autosomal human DNA in these cells must be limited. In addition, sequences react with pSG1 (data not shown). amplification of 12E7 antigen expression in the transfectants The observations that the pSG1 insert in pEX2 encodes a is associated with amplification of the sequences defined by fusion polypeptide that reacts with the 12E7 antibody and pSG1 (Fig. 4). that pSG1-related sequences are present on the X and Y Sequence Homology Between MIC2X and MIC2Y. The pSG1 indicates that pSG1 is a cDNA clone homologous to MIC2 clone has been used to explore the relationship between sequences. This was MIC2X and MIC2 Y. The human-rodent hybrids HORL9X suggestion confirmed by hybridization and 3E7 containing only the X and Y chromosomes as their A B C human component, respectively, were cleaved with 12 dif- ferent restriction enzymes and Southern analysis was per- kDa formed with pSG1. No difference in hybridization pattern 200 - 1 2 3 4 5 6 7 M "., OP W. -23 w- -9.4 -6.6 40 - 93 -4.4

69-

46- - -2.3

FIG. 4. Southern analysis of 12E7 transfectant DNAs cleaved 1 2 1 2 1 2 with EcoRI. The insert from pSG1 was hybridized to DNAs isolated from mouse cells that had been transfected with human DNA and FIG. 2. Detection of fusion polypeptides. The insert from XSG1 selected for 12E7 expression on the FACS. Lanes: 1, J6; 2, EP5A (a was cloned into the pEX2 vector. The plasmid was amplified at 30°C secondary 12E7 positive transfectant); 3, EP5C (a second indepen- and transient expression was induced at 42°C. Cell lysates were dent secondary 12E7 positive transfectant); 4, TKMlEP13 (a pri- electrophoresed through NaDodSO4/polyacrylamide gels and the mary transfectant showing greater expression of 12E7 antigen than fusion polypeptides were detected by either staining with Coomassie TKM1EP9); 5, TKM1EP9 (a primary transfectant showing increased blue (A) or immunoblot analysis and probing either with 12E7 (B) or expression of 12E7 antigen); 6, TKM1EP7 (a primary 12E7 positive an anti-3-galactosidase monoclonal antibody (C). Lanes: 1, pEX2; 2, transfectant); 7, LMTK-; M, molecular size markers in kilobase pEX2SG1. pairs. Downloaded by guest on September 28, 2021 138 Genetics: Darling et al. Proc. Natl. Acad. Sci. USA 83 (1986) Table 1. Restriction enzyme analysis of sequence homology Evidence from restriction analysis leads us to conclude that between MIC2X and MIC2Y the MIC2X and MIC2Y loci are closely related, if not No. of identical. Restriction analysis of the genomic clones for fragments Minimum MIC2X and MIC2Y will allow us to investigate their relat- obs. to no. of No. of edness over a greater distance. Restriction hybridize* sites bp This is the first described probe for a Y-linked expressed enzyme Recognition site to XSG1 defined identical gene and the first probe for an X-linked gene that escapes X inactivation. The existence of structural genes on both sex Alu I AGCT 3 4 16 chromosomes makes a relationship between MIC2 and the Bgl I GCCNNNNNGGC 3 4 24 minor histocompatibility antigen H-Y unlikely. Pvu II CAGCTG 6 7 42 Although the human sex chromosomes differ extensively HindIII AAGCTT 3 4 24 in morphology and genetic content (39), they are thought to Pst I CTGCAG 6 7 42 have evolved from a homologous pair of chromosomes (40). Sau3A GATC 3 4 16 The sharing of genes by the sex chromosomes might reflect Xba I TCTAGA 3 4 24 this common origin or might reflect a recent exchange of Bgl II AGATCT 2 3 18 genetic material. It has been proposed that sex chromosomes Dde I CTNAG 1 2 8 share sequences to facilitate meiotic chromosomal pairing EcoRI GAATTC 4 5 30 and that such sequences might be exchanged between the sex HincII GTPyPuAC 4 5 30 chromosomes by normal recombination (41, 42). Such se- Hinfl GANTC 3 4 16 quences have been termed pseudoautosomal, as they would Total 53 290 not be expected to show sex linkage. The existence of bp, Base pairs. pseudoautosomal genes has been supported by the finding *Hybridization patterns for HORL9X and 3E7 were identical. that the Sts and abnormal Sxr regions are exchanged between Hybridizing fragments were counted after 1-week exposure of the the X and Y chromosomes in the mouse (43-45). filters at -70'C; longer exposure results in the appearance of more Early studies suggested that the pairing, and consequently bands. the homology, involved the distal third ofthe short arm ofthe human X chromosome and almost the entire Y chromosome was found between MIC2X and MIC2Y (Table 1). Two short arm (46, 47). More recently, experiments have shown restriction enzymes, Taq I and Msp I, have been excluded that pairing varies extensively between spreads (48). Homol- from this analysis because they recognize extensive X- and ogy of single-copy DNA sequences between the X and Y Y-linked polymorphisms. The 12 restriction enzymes recog- chromosomes was first reported by Page et al. (49) for the nize a minimum total of53 sites and have sampled a minimum markerDXYSI . However, in situ hybridization revealed that total of 290 base pairs. We conclude that the MIC2X and DXYSJ maps to the long arm ofthe X chromosome and to the MIC2 Y loci are closely related in sequence and may be short arm ofthe Y (50), outside the postulated pairing region. identical. Because of this close degree of homology and the Other single-copy probes have been mapped to the human X fact that the RNA used to construct the cDNA library was and Y chromosomes, although again none has been shown to derived from J6, a male T-cell line, we do not know whether reside within the pairing region (51, 52). The MIC2Xlocus has we have isolated a cDNA clone corresponding to MIC2X or been mapped to Xp22.32-Xpter (4, 5), and the MIC2Y locus MIC2 Y. has been mapped to Yqll-Ypter (4), locations that include the postulated pairing regions. Using the cloned cDNA to DISCUSSION isolate genomic clones we will be able to investigate the extent of homology surrounding the MIC2 loci and whether We have isolated a cDNA clone that corresponds to a MIC2 these loci are exchanged between the sex chromosomes by locus and conclude that the 12E7 antigen is the primary normal recombination. product ofthe MIC2 loci. Several experiments lead us to this conclusion. First, a recombinant bacteriophage, XSG1, ex- pressing an antigenic determinant that is recognized by the We would like to thank H. Kataoka and M. Collins for the Xgtll 12E7 antibody has been isolated. The insert from this library; K. Stanley for the pEX vectors and E. coli pop 2136; D. Lane a for the monoclonal anti-3-galactosidase antibody; G. Lam and L. bacteriophage, after subcloning into plasmid expression Davis for expert assistance on the FACS; P. Lamb, C. Mondello, C. system, has been shown to express a cro-3galactosid- Pritchard, M. V. Wiles, and B. Williams for helpful suggestions; and ase-cDNA fusion protein that is recognized by both the 12E7 C. Middlemiss for typing the manuscript. antibody and a p-galactosidase antibody. Second, hybridiza- tion analysis of the insert shows localization to the human X and Y chromosomes. Third, sequences that share homology 1. Levy, R., Dilley, J., Fox, R. I. & Warnke, R. (1979) Proc. with an insert from XSG1 are present in DNAs isolated from Natl. Acad. Sci. USA 76, 6552-6556. mouse cells that have been transfected with human DNA and 2. Goodfellow, P. (1983) Differentiation 23, S35-S39. selected for 12E7 antigen expression on the FACS. It has 3. Goodfellow, P., Banting, G., Levy, R., Povey, S. & transfectants McMichael, A. (1980) Somatic Cell Genet. 6, 777-787. been estimated that L-cell obtained by using 4. Goodfellow, P., Banting, G., Sheer, D., Ropers, H.-H., Caine, cotransfection with the thymidine kinase gene incorporate A., Ferguson-Smith, M. A., Povey, S. & Voss, R. (1983) -0.1% of the (37, 38). Thus, the probability Nature (London) 302, 346-349. of finding by chance a sequence of interest in two indepen- 5. Curry, C. J. R., Magenis, E. R., Brown, M., Lonman, J. T., dent secondary transfectants isolated by transfection from Tsai, J., O'Lague, P., Goodfellow, P., Mohandas, T., Bergner, DNA of a primary transfectant line is 1 in 106. Finally, E. A. & Shapiro, L. J. (1984) N. Engl. J. Med. 311, 1010-1015. increased antigen expression in the primary transfectants is 6. Goodfellow, P., Pym, B., Mohandas, T. & Shapiro, L. J. associated with an increased amount of the 32.5-kDa poly- (1984) Am. J. Hum. Genet. 36, 777-782. peptide and increased amounts of the sequences defined by 7. Mohandas, T., Shapiro, L. J., Sparkes, R. S. & Sparkes, the insert from bacteriophage XSG1. M. C. (1979) Proc. Natl. Acad. Sci. USA 76, 5779-5783. Previous biochemical analysis had not detected a differ- 8. Ferguson-Smith, M. A., Sanger, R., Tippett, P., Aitken, D. A. ence between the MIC2X and MIC2Y gene products (13). & Boyd, E. (1982) Cytogenet. Cell Genet. 32, 273-274. Downloaded by guest on September 28, 2021 Genetics: Darling et al. Proc. Natl. Acad. Sci. USA 83 (1986) 139 9. Shapiro, L. J., Mohandas, T., Weiss, R. & Romeo, G. (1979) 30. Batteiger, B., Newhall, V. W. J. & Jones, R. B. (1982) J. Science 204, 1224-1226. Immunol. Methods 55, 297-307. 10. Race, R. R. & Sanger, R. (1975) Blood Groups in Man 31. Bodger, M. P., Francis, G. E., Delia, D., Granger, S. M. & (Blackwell, Oxford). Janossy, G. (1981) J. Immunol. 127, 2269-2271. 11. Goodfellow, P. & Tippett, P. (1981) Nature (London) 289, 32. Stanley, K. K. & Luzio, J. P. (1984) EMBO J. 3, 1429-1434. 404-405. 33. Vieira, J. & Messing, J. (1982) Gene 19, 259-268. 12. Tippett, P., Shaw, M. A., Daniels, G. L. & Green, C. A. 34. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl. (1984) Cytogenet. Cell Genet. 37, 595. Acad. Sci. USA 76, 4350-4354. 13. Banting, G. S., Pym, B. & Goodfellow, P. (1985) EMBO J. 4, 35. Feinberg, A. P. & Vogelstein, B. (1984) Anal. Biochem. 137, 1967-1972. 266-267. 14. Schneider, S., Schwerk, H. V. & Bornkamm, G. (1977) Int. J. 36. Young, J. F., Hockmeyer, W. T., Gross, M., Ballou, W. R., Cancer 19, 621-626. Wirtz, R. A., Trosper, J. H., Beaudoin, R. K., Hollingdale, 15. Minowada, J., Ohnuma, T. & Moore, G. E. (1972) J. Natl. M. R., Miller, L. H., Diggs, C. L. & Rosenberg, M. (1985) Cancer Inst. 49, 891-895. Science 228, 958-962. 16. Bishop, C. E., Guellaen, G., Geldwerth, D., Voss, R., Fell- 37. Kavathas, P., Sukhatme, V. P., Herzenberg, L. A. & Parnes, ous, M. & Weissenbach, J. (1983) Nature (London) 303, J. R. (1984) Proc. Natl. Acad. Sci. USA 81, 7688-7692. 831-832. 38. Perucho, M., Hanahan, D. & Wigler, M. (1980) Cell 22, 17. Nabholz, M., Miggiano, V. & Bodmer, W. F. (1969) Nature 309-317. (London) 223, 358-363. 39. Miller, 0. J., Drayna, D. & Goodfellow, P. (1984) Cytogenet. 18. Kit, S., Dubbs, D. R., Piekawshi, K. L. J. & Hsu, T. C. (1963) Cell Genet. 37, 176-204. Exp. Cell Res. 31, 297-312. 40. Ohno, S. (1967) Sex Chromosomes and Sex-Linked Genes 19. Klebe, R. J., Chen, T. R. & Ruddle, F. H. (1970) J. Cell Biol. (Springer, New York). 45, 74-82. 41. Koller, P. C. & Darlington, C. D. (1934) J. Genet. 29, 159-173. 20. Goodfellow, P. N. (1975) Dissertation (Oxford University). 42. Burgoyne, P. S. (1982) Hum. Genet. 61, 85-90. 21. Marcus, M., Trantravali, R., Der, V. G., Miller, D. A. & 43. Singh, L. & Jones, K. (1982) Cell 28, 205-216. Miller, 0. J. (1976) Nature (London) 262, 65-67. 44. Evans, E. P., Burtenshaw, M. & Cattanach, B. M. (1982) 22. Wilkie, N. M., Clements, J. B., Boll, W., Montei, N., Nature (London) 300, 443-445. Lonsdale, D. & Weissman, C. (1979) Nucleic Acids Res. 7, 45. Keitges, E., Rivest, M., Siniscalco, M. & Gartler, S. M. (1985) 859-877. Nature (London) 315, 226-227. 23. Wigler, M., Silverstein, S., Lee, L. S., Pellicer, A., Cheng, 46. Moses, M. J., Counce, S. J. & Paulson, D. F. (1975) Science Y.-C. & Axel, R. (1977) Cell 11, 223-232. 187, 363-365. 24. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular 47. Solari, A. J. (1980) Chromosoma (Berlin) 81, 315-337. Cloning: A Laboratory Manual (Cold Spring Harbor Labora- 48. Chandley, A. C., Goetz, P., Hargreave, T. B., Joseph, A. M. tory, Cold Spring Harbor, NY). & Speed, R. M. (1984) Cytogenet. Cell Genet. 38, 241-247. 25. Wickens, M. P., Buell, G. N. & Schimke, R. T. (1978) J. Biol. 49. Page, D., de Martinville, B., Barker, D., Wyman, A., White, Chem. 253, 2483-2495. R., Francke, U. & Botstein, D. (1982) Proc. Natl. Acad. Sci. 26. Okayama, H. & Berg, P. (1982) Mol. Cell. Biol. 2, 161-170. USA 79, 5352-5356. 27. Young, R. A. & Davis, R. W. (1983) Proc. Natl. Acad. Sci. 50. Page, D. C., Harper, M. E., Love, J. & Botstein, D. (1984) USA 80, 1194-1198. Nature (London) 311, 119-123. 28. Young, R. A. & Davis, R. W. (1983) Science 222, 778-782. 51. Cooke, H. J., Brown, W. R. A. & Rappold, G. A. (1984) 29. Young, R. A. & Davis, R. W. (1985) in Genetic Engineering, Nature (London) 311, 259-261. eds. Setlow, J. K. & Hollaender, A. (Plenum, New York), 52. Koenig, M., Camerino, G., Heilig, R. & Mandel, J.-L. (1984) Vol. 7, in press. Nucleic Acids Res. 12, 4097-4109. Downloaded by guest on September 28, 2021