Copyright  1999 by the Society of America

Intragenic -Chromosomal Crossovers of Xmrk Oncogene Affect Pigment Pattern Formation and the Severity of Melanoma in Xiphophorus

Heidrun Gutbrod and Manfred Schartl Physiological Chemistry I, Biocenter, University of Wu¨rzburg, D-97074 Wu¨rzburg, Germany Manuscript received July 23, 1998 Accepted for publication October 21, 1998

ABSTRACT The X and Y of the platyfish (Xiphophorus maculatus) contain a region that encodes several important traits, including the determination of sex, pigment pattern formation, and predisposition to develop malignant melanoma. Several sex-chromosomal crossovers were identified in this region. As the melanoma-inducing oncogene Xmrk is the only molecularly identified constituent, its genomic organization on both sex chromosomes was analyzed in detail. Using X and Y -specific sequence differences a high proportion of the crossovers was found to be intragenic in the oncogene Xmrk, concentrating in the extracellular domain-encoding region. The genetic and molecular data allowed establishment of an order cM. It further revealed a sequence located within several kilobases of the extracellular 0.6ف of loci over domain-encoding region of Xmrk that regulates overexpression of the oncogene.

N the platyfish Xiphophorus maculatus, three sex chro- Two pigment pattern loci of X. maculatus also locate to I mosomes coexist. They have been characterized as the SD and P-containing region. The red-yellow pattern X, Y, and W. Over a large geographic range the individ- (RY) [this locus has been referred to by other ual populations are polymorphic for these three chro- research groups as XANT (see Morizot et al. 1993), or mosomes. A balanced genetic system gives rise to WX, Ptr (see Anders 1992)] is responsible for different red, WY, and XX females as well as XY and YY males (Orzack brown, orange, and yellow patterns (Kallman 1975) et al. 1980; Kallman 1984). that are due to local high concentrations of pigment The X and Y appear to be very similar. Meiotic recom- cells of the xanthophore/erythrophore lineage in the bination has been observed over the entire linkage iris, on the body, and the fins. This locus appears to group, suggesting that the pseudo-autosomal region is consist of a series of closely linked (Kallman still very large (Kallman 1975, 1984; Morizot et al. 1975). The macromelanophore-determining locus, Mdl, 1991). Both X and Y carry a set of genetically well- contains the genetic information for a specific type of characterized, closely linked loci. melanin-containing pigment cells, which are much The locus that defines the identity of the gonosome larger than the normal melanophores, the micromela- is the sex-determining locus, SD (in Xiphophorus earlier nophores. Macromelanophores compose certain bold also referred to as SEX; see Morizot 1990). Nothing is black markings. known about the biological nature of SD and how it is The macromelanophore locus has attracted special related to the mammalian testes-determining factor, attention, because it has been shown for some of these SRY. The genetic analysis of sex determination in the patterns that upon certain crossings their expression is platyfish led to the hypothesis that a male-determining enhanced in the hybrids, giving rise to severe melanosis (M) is present on X, W, and Y, but only the Y-chro- and even malignant melanoma (Kallman 1975; Anders mosomal allele is active due to suppression of MX and 1991; Schartl 1995). The capacity to develop mela- MW by autosomal repressors. The W is thought to contain noma is contributed by a dominant oncogene, ONC- another suppressor specific for MY (Kallman 1984). Xmrk, which is very closely linked to Mdl (Weis and The second gene in this region is the so-called pituitary Schartl 1998). ONC-Xmrk codes for a growth factor (P) gene that determines the onset of sexual matura- receptor from the epidermal growth factor receptor tion. At present, nine alleles of P have been found. subclass of receptor tyrosine kinases (RTKs; Wittbrodt Again, the platyfish populations are polymorphic for P et al. 1989). Its oncogenic action is suppressed in the alleles, leading to a wide variety of phenotypes that wild platyfish by an autosomal tumor suppressor locus, range from very early to very late maturing animals R. Hybridization experiments set up so that the R-con- (Kallman 1989; Schreibman et al. 1994). taining autosomes are substituted through introgressive breeding by R-free chromosomes from Xiphophorus fish from other populations or species release ONC- Corresponding author: Manfred Schartl, Physiological Chemistry I, Biocenter, University of Wu¨rzburg, Am Hubland, D-97074 Wu¨rzburg, Xmrk from its control, and neoplastic growth of macro- Germany. E-mail: [email protected] melanophores occurs.

Genetics 151: 773–783 (February 1999) 774 H. Gutbrod and M. Schartl

The unit of ONC-Xmrk and certain alleles of Mdl is MATERIALS AND METHODS equivalent to the earlier defined Tu-locus sensu Anders Fish: All fish used in this study (see Table 1) were bred (Anders 1991; see Weis and Schartl 1998). Many of and maintained in the aquarium facilities of the Biozentrum these Mdl-ONC-Xmrk combinations exist and each gives (Wu¨rzburg, Germany) under standard conditions. F1 hybrids a peculiar phenotype of the pigment pattern and the of X. maculatus with X. helleri were produced by artificial insemi- melanoma with respect to the onset, compartment, in- nation. The melanoma phenotype was analyzed in F1 and backcross hybrids with X. helleri (strain 17), while for RFLP tensity, and the extension of the pattern on the one linkage analysis F1 and backcross hybrids with X. gordoni and hand and the onset, location, and severity of the cancer- X. couchianus (strains 13 and 14, respectively) were used. For ous disease on the other hand (Schartl and Well- mapping INV-Xmrk, backcross hybrids of X. variatus with X. brock 1998). It is unknown whether these features helleri were used. The and recombinant sex chromo- attribute either to Mdl or to the region controlling somes arose either spontaneously or following X-ray mutagen- esis (Anders et al. 1973) and were maintained in the genetic of ONC-Xmrk, or to structural differences background of the original population from which the wild- in the transcribed part that alter the biochemical prop- type originated. All were generously made avail- erties of the oncogenic growth factor receptor. able by A. and F. Anders (Gießen, Germany), except for the The platyfish populations are highly polymorphic Sr crossover 3084B (strain 10) and the wild platyfish strain 4, for the multiple alleles of Mdl-ONC-Xmrk, which are which were kindly supplied by K. D. Kallman, and platyfish grouped in five classes: the spotted dorsal (Sd), the strain 3, which was obtained from the Xiphophorus Genetic Stock Center (San Marcos, TX). striped (Sr), spotted (Sp), nigra (Ni), and spotted belly DNA extraction and PCR amplifications: Genomic DNA (Sb) patterns (Gordon 1948). The percentage of mac- from pooled organs of individual fish was extracted as pre- romelanophore-pattern-carrying fish can range from viously described (Schartl et al. 1995). From X. maculatus Ͻ1% to more than half of the individuals of a popula- (Rio Jamapa, ONCϪ, strain 3), genomic DNA for PCR was tion (Gordon and Gordon 1957). ONC-Xmrk has so prepared from fin clips according to the protocol of Altsch- mied et al. (1997). As template for amplification of the introns far been found only in conjunction with Mdl. However, of Xmrk, either phage Sac1-3-1 isolated from a subgenomic it should be noted that in other Xiphophorus species library of X. maculatus (Rio Jamapa, Sd, strain 1; see Table 1; Mdl alleles that are unlinked to ONC-Xmrk exist. These Adam et al. 1991), which contains exon 2 to 22 of ONC-Xmrk, are not melanomagenic (Weis and Schartl 1998). or genomic DNA of female X. maculatus (strain 1) was used. ONC-Xmrk has arisen through a PCR amplifications were performed for 35 cycles with a dena- turing step at 94Њ for 30 sec, annealing at 2Њ below Tm of the event from its corresponding proto-oncogene, INV- respective primer for 30 sec, and an extension at 72Њ for 30 Xmrk (Adam et al. 1993). This gene is also located in to 330 sec according to the size of the expected product. In the same region where ONC-Xmrk, Mdl, RY, P, and SD the first cycle, denaturation was for 180 sec and in the last are residing (Schartl 1990). Although some sugges- cycle extension time was 180 sec. Allele-specific primers for tions about a gene order have been made (Anders et the X and Y copies of ONC-Xmrk had the following sequences: Ј Ј Ј al. 1973; Kallman 1975; Schartl 1990; Sohn 1991), Xfor 5 -CTTACGTTGAAAGCACGTGA-3 , Xrev 5 -AAAGGA GGCTTCATGGAGGG-3Ј, Yfor 5Ј-TTTGGTGTCTTACTTCT so far a fine mapping has been impossible to achieve GTG-3Ј, and Yrev 5Ј-TTCCTCCTACTTGGCTAAAC-3Ј (Cough- due to the extremely low frequency of recombination lan et al. 1998). Primers flanking a 1.4-kb in the car- between these loci. This information, however, is boxyterminal domain of the X allele are Ins4 (5Ј-GCCTC needed for positional cloning approaches employing CTGGGAGGACAGCGAC-3Ј) and Ins5 (5Ј-AGCGAGCCCTGC chromosome walking out of the single cloned sites from ATCCCGCCG-3Ј). Products of different size for the X- and Y-ONC-Xmrk allele (X, 5.3 kb; Y, 3.4 kb) from the first intron this region, the Xmrk genes. were generated with primers Hg93 (5Ј-CTGCAGTCGTCAT The W chromosome is very different, despite being GGAAACC-3Ј) and Hg96 (5Ј-CCTCCTGCCGAATCGTTCAG- involved in sex determination. No P factor was identi- 3Ј) using 1 ␮l of a mixture of 84:1 units of Taq polymerase fied on the W chromosomes (Kallman 1989). The wild- (Gibco BRL, Eggenstein, Germany) and Pwo DNA polymer- type W is also devoid of RY and Mdl alleles (Kallman ase (AGS, Heidelberg, Germany) in a Taq Extender buffer (Stratagene, Heidelberg, Germany). Differentiation between 1975) and, as it does not predispose to melanoma, obvi- X- and Y-ONC in the promoter region is possible by using the ously also of ONC-Xmrk. Whether INV-Xmrk is present oligonucleotides Prom2 (5Ј-CCGCTCCTCCGCGCAGAAAC-3Ј) is unknown at present. Only six cases of W/Y crossovers and Prom3 (5Ј-AATGACTGGGCAGTGCTAAGG-3Ј). Sequence have been documented with the remaining product al- information on the 96 primers for amplification of Xmrk in- ways beingaW(Kallman 1984). trons and exons is available at the Xiphophorus internet web In the work described in this article, several , site (http://sprd1.mdacc.tmc.edu/skazianis/mainpage.html/). PCR products were subsequently analyzed on 0.8–1.6% agarose including X/Y crossovers that involved the Mdl/ONC- gels. Xmrk region, were analyzed for structural differences Single-strand conformation polymorphism (SSCP): For from the wild-type chromosome to understand the re- SSCP analysis PCR products were labeled by incorporation of sulting differences in the phenotype of the macromela- [32P]dCTP in 10-␮l reactions containing 1 ␮l10ϫ PCR buffer nophore pattern and, most importantly, the melanoma. (100 mm Tris pH 8.85, 500 mm KCl, 15 mm MgCl2, 1% Triton X-100, 2 mg/ml BSA), 50 ng genomic DNA, 5 pmol of each The structural data were then also used to establish a primer, 20 ␮m dATP, dGTP, dTTP, 2 ␮m dCTP, 1 ␮Ci gene order of this important region of the sex chromo- [32P]dCTP (3000 Ci/mm), and 0.75 units Taq DNA polymer- some. ase. Reactions were overlaid with mineral oil and amplifica- Intragenic Crossovers of Xmrk Oncogene 775

TABLE 1 Fish strains and genotypes

Strain Geographical Macromelanophore Strain number Species origin pattern RY pattern name Genotype 1 X. maculatus Rio Jamapa Spotted dorsal (Sd) Dorsal red (Dr) Sd Males: Y ArSr/X DrSd Striped (Sr) Anal red (Ar) Females: X DrSd/X DrSd 2 X. maculatus Rio Jamapa Spotted (Sp) Anal red (Ar) Sp Males: Y ArSr/X Sp Striped (Sr) Females: X Sp/X Sp 3 X. maculatus Rio Jamapa — — ONCϪ Males: X ϩ/Y ϩ Females: X ϩ/X ϩ 4 X. maculatus Rio Usumacinta Spotted4 (Sp4) — Sp4 Males: Y Sp4/Y Sp4 Females: W/Y Sp4 5 X. maculatus Rio Usumacinta — — Males: Y ϩ/Y ϩ Females: W ϩ/Y ϩ 6 X. maculatus Belize River Nigra (Ni1) Brown (Br) N1 Males: Y BrNi1/Y BrNi1 Females: W ϩ/Y BrNi1 7 X. maculatus Laboratory stock Nigra-extended (Ni2) Brown (Br) N2 Males: Y BrNi2/Y BrNi2 Females: W ϩ/Y BrNi2 8 X. maculatus Laboratory stocka Spotted belly (Sb) Ruby throat (Rt) Sb Males: Y RtSb/Y RtSb Females: W ϩ/Y RtSb 9 X. maculatus DrSr-strain Striped (Sr) Dorsal red (Dr) DrSr Females: X DrSr/X DrSr Anal red (Ar) Males: X DrSr/Y ArSr 10 X. maculatus 3084B-strain Striped (Sr) Dorsal red (Dr) 3084B Females: X DrSr/X DrSr Anal red (Ar) Males: X DrSr/Y ArSr 11 X. maculatus Laboratory stock Lineatus (Li) Dorsal red (Dr) DrLi Females: X DrLi/X DrLi Striped″ (Sr″) Anal red (Ar) Males: X DrLi/Y ArSr ″ 12 X. maculatus/ Backcross hybrid Lineatus Mut (LiMut) Dorsal red DrLi (mut) X DrLi-Mut/—b X. helleri to X. helleri, more than 10ϫ 13 X. gordoni Laguna St. Tecla — — 14 X. couchianus La Huasteca — — 15 X. meyeri Muzquiz — — 16 X. variatus Rio Panuco Lineatus (Li) Orange (Or) Li Males: X Li/Y OrPu1 Punctatus-1 (Pu1) Females: X Li/X Li 17 X. helleri Rio Lancetilla — — a The Y RtSb chromosome is most probably derived from wild fish from the Rio Tonto (Kallman 1975) and was introduced from the ornamental stock “Bleeding heart” into the genetic background of X. maculatus from Rio Usumacinta (stock 5). b The mutant X. maculatus sex chromosome is present in the genetic background of X. helleri, which has a polyfactorial sex determination. Usually, the X. maculatus X-SD overrides the X. helleri factors. Thus Ͼ95% of the hybrids are females. tions carried out as described above. Before loading, labeled gel purified with QIAEX (QIAGEN, Hilden, Germany) and PCR products were diluted 15- to 20-fold with formamide- sequenced using the cycle sequencing kit (Pharmacia, Frei- loading dye (95% formamide, 0.05% bromophenyl blue, burg, Germany). Those fragments that could not be reliably 0.05% xylene cyanol), denatured for 5 min at 95Њ, and chilled sequenced directly were cloned into pUC18 using the Sure- on ice. Electrophoresis was carried out under two different Clone Ligation kit (Pharmacia), and at least four independent conditions: samples were either electrophoresed on 6% non- clones were sequenced by Sanger dideoxy sequencing with denaturing polyacrylamide gels containing 10% glycerol, 0.5ϫ the Sequenase kit (United States Biochemical, Cleveland). TBE at 1.8 W constant power overnight at room temperature Sequences are deposited in GenBank under accession nos. or on 6% nondenaturing polyacrylamide gels without glycerol AF091399 and AF092692–AF092694. at 20 W constant power for 2.5 hr at 4Њ. Gels were dried on Southern analysis: A total of 4.5 ␮g of genomic DNA was Whatman paper and autoradiographed. Exons larger than digested with EcoRI or BglII, separated on a 0.8% agarose gel, 200 bp were digested with an appropriate restriction enzyme and blotted onto nylon membrane (Hybond Nϩ; Amersham after amplification taking 1 ␮l of PCR product and 1 unit of Buchler, Braunschweig, Germany). A 0.7-kb BamHI fragment enzyme in a total volume of 10 ␮l. SSCP of exon 1 was carried of the first intron of Xmrk (probe b, J. Altschmied, unpub- out using the primer Hg91 (5Ј-GTGCTCAGCATCAGCC lished results) and the PCR fragment amplified with the prim- GCTG-3Ј), which is located 3Ј adjacent to the variable length ers Hg93/Hg96 (probe c) were used as hybridization probes. CTG repeat in exon 1 and primer Hg92 (5Ј-CCTGAACTCA The localization of INV-Xmrk was determined using a PCR GTGAAACTGCAG-3Ј). To discriminate PCR products that are product as hybridization probe, which was amplified from amplified from the proto-oncogenic version of Xmrk, male genomic DNA of a female from strain 1 with the primers and female X. maculatus Rio Jamapa fish without ONC-Xmrk Ex1/Jd9 (Ex1, 5Ј-ATGGAGTTTCTGCGCGGAGG-3Ј; Jd9, 5Ј- were analyzed in parallel. CAAATTTCTCCTGAACTCACAGC-3Ј) and digested with Sequencing: For direct sequencing, PCR fragments were AvaII. The 900-bp fragment was gel purified and used as probe 776 H. Gutbrod and M. Schartl

TABLE 2 Segregation analysis of Mdl, ONC-Xmrk, INV-Xmrk, and SD

Cosegregating loci EcoRI fragments (kb)a Parental Recombinant Mdl Sr-ONC-Xmrk/INV-Xmrkmac 6.5/7.0/10.0 13 0 Mdl Sr-ONC-Xmrk/INV-Xmrk couch 10.0 12 0 Mdl Sp-ONC-Xmrk/INV-Xmrkmac 5.0/7.0/10.0 45 0 Mdl Sp-ONC-Xmrk/INV-Xmrk gord 10.0 41 0 Mdl Sd-ONC-Xmrk/INV-Xmrkmac 5.0/7.0/10.0 27b 1 Mdl Sd-ONC-Xmrk/INV-Xmrk gord 10.0 20b 0 Mdl-ONC-Xmrk/INV-Xmrk/SD 45 0 a Detected with the Xmrk p17-2 probe; the 10.0-kb INV-Xmrkcouch or gord fragment is present in all backcross hybrids, because X. couchianus or X. gordoni was used as the recurrent parent. b Data from Schartl (1990). a. For linkage analysis of INV-Xmrk with ONC-Xmrk, the cDNA in our study between any of the Xmrk genes and either probe p17-2 (Wittbrodt et al. 1989) encompassing the tyro- Mdl or SD. sine kinase and carboxyterminal domains of the Y-chromo- somal ONC-Xmrk was used. The fragments were radiolabeled To analyze for the presence of INV-Xmrk on W, WY- by random priming according to Feinberg and Vogelstein females (strain 5) that do not have an ONC-Xmrk gene (1983). Hybridization was done at 42Њ in buffer containing were analyzed. Primers that flank a variable length trip- 50% formamide, 5ϫ SSC. Filters were washed at 68Њ with 0.1ϫ let repeat in exon 1 of both Xmrk genes were designed SSC, 1% SDS, and autoradiographed. (Schartl et al. 1998). Two PCR-products were obtained from 125 females. This indicated two alleles of INV- Xmrk and thus heterozygosity. Males (n ϭ 116) of this RESULTS strain gave only one PCR product. For RFLP analysis, W/Y females from strains 4 and 7 were crossed with X. Linkage of ONC- and INV-Xmrk: In a previous study couchianus. In 16 F hybrids cosegregation of W and the linkage of both ONC- and INV-Xmrk to Mdl Sd on the X 1 7.0-kb EcoRI INV-Xmrk fragment from X. maculatus was chromosome has been shown (Schartl 1990). In X. found, while 7 F hybrids showed cosegregation of Y, maculatus an EcoRI RFLP for the kinase domain encod- 1 the corresponding Mdl-ONC-Xmrk complex, and the ing the genomic region of Xmrk allows differentiation 7.0-kb EcoRI INV-Xmrk fragment. This also confirmed of the X- as well as the Y-chromosomal copy of ONC- the presence of an INV-Xmrk allele on W. Xmrk from INV-Xmrk (5, 6.5, and 7 kb, respectively). Structure of the ONC-Xmrk alleles of X. maculatus: Cosegregation of ONC- and INV-Xmrk was analyzed in Thus far, information about the genomic structure of backcross hybrids of X. maculatus (strains 1 and 2) with ONC-Xmrk was available only for the very 5Ј-end, the either X. couchianus or X. gordoni as the recurrent parent. region coding for the tyrosine kinase domain and the In these two species no ONC-Xmrk is present, and INV- carboxyterminus of the receptor (Adam et al. 1991), Xmrk is represented by a 10-kb EcoRI fragment in the while for its extracellular and transmembrane part the RFLP analysis. Hence, the X. maculatus INV-Xmrk can sequence is known only on the cDNA level (Wittbrodt be easily distinguished from the X. couchianus or gordoni et al. 1989). To establish the full genomic organization, INV-Xmrk. The segregation analysis revealed the pres- first the exon/intron structure of the missing large ge- ence of INV-Xmrk also on the Y chromosome and cose- nomic region was analyzed (Figure 1). Primers were Sr gregation of the Y allele of ONC-Xmrk and Mdl without designed from the ends of each exon and amplification recombination (in n ϭ 25 backcross segregants; Table of the intervening sequences was done. This was possible 2). The linkage of the X-chromosomal ONC- and INV- from all missing introns with the exception of the large Sp Xmrk loci to each other and of both to Mdl was estab- first intron. The amplified introns were sequenced com- lished in n ϭ 86 backcross segregants without recombi- pletely or at least from both ends. nation, which confirms our earlier data (Table 2). If all Comparison of exon/intron arrangement and exon data of this study are combined, the overall recombina- borders with that of the closely related chicken epider- tion frequency between INV- and ONC-Xmrk would be mal growth factor-receptor (EGF-R) gene revealed high 0.6% (parental, 158; recombinant, 1), which roughly conservation with a few exceptions. First, intron 12, equals 180 kb according to Morizot et al. (1991). For which divides subdomains III and IV of the extracellular those segregants that were analyzed after they had ma- domain of the chicken EGF-R (Callaghan et al. 1993), tured (and thus information on the sex was available), is absent in ONC-Xmrk and INV-Xmrk as detected by linkage of INV-Xmrk, ONC-Xmrk, and Mdl to SD was sequencing across the deduced exon 12/13 border of established (Table 2). No recombination was observed phage ␭Sac1-3-1, cosmids containing different alleles Figure 1.—(A) Schematic maps of the X- and Y-chromosomal ONC-Xmrk loci. Numbers give the size of the EcoRI fragments in kilobases. E, EcoRI sites. Homologous sites are connected by lines. (B) Intron/exon arrangement and location of allele-specific landmarks. Exons are indicated by black bars, open symbols above arrows point to structural characters diagnostic for the X locus, while solid symbols are for Y-chromosomal markers. Triangles represent insertions/deletions, rectangles are identified sequence differences, diamonds indicate positions for allele-specific PCR differences, and circles are SSCP markers. Primers (arrows) and probes (hatched boxes) used for detecting allele-specific differences between X and Y are as follows: 1a,1b/2, Prom3,4/Prom2; 3/4, Hg91/Hg92; 5/6, Hg93/Hg96; 7/8, Hg65/Hg66; 9/10, Hg69/Hg70; 11/12, Hg73/Hg74; 13a/14a, Xfor/Xrev; 13b/14b, Yfor/Yrev; 15/16, Hg81/Hg82; 17/18, Hg85/Hg86; 19/20, Ins4/Ins5; a, Ex1/Jd9 PCR product, digested with AvaII; b, 0.7-kb BamHI fragment; c, Hg93/Hg96 PCR product. 778 H. Gutbrod and M. Schartl

Figure 2.—Informative SSCP for exon 23, amplified with the primers Hg81/ Hg82 and digested with HpaI. (1) X. maculatus Sd, X/ X female; (2) X. maculatus SdSr, X/Y male; (3) X. macu- latus Sr crossover 3084B, X/X female; (4) X. maculatus DrLi, X/X female; (5) X. macula- tus/X. helleri DrLi (mut) F1 hy- brid female; (6) X. macula- tus Sb, Y/Y male; (7, 8) X. maculatus without ONC-Xmrk, X/X female, X/Y male; (9) X. maculatus N1, Y/Y male; (10) X. maculatus Sp4, Y/Y male; (11) X. maculatus N2, Y/Y male; (12) X. maculatus Sd, X/X female.

(ONC, INV) of Xmrk, as well as PCR products from type phenotype of any of the sex chromosomal loci. genomic DNA of several X. maculatus genotypes (strains The Sr macromelanophore pattern is unchanged in

8, 11, and 12). Thus the Xmrk gene is composed of 27 purebred platyfish and in F1 as well as in backcross (Y-ONC-Xmrk, INV-Xmrk) or 26 exons (X-ONC-Xmrk) hybrids with X. helleri. The RY-locus allele Dr remained instead of the 28 of the chicken EGF-R gene (Figure linked to the X-chromosomal SD in both cases. In the 1). To facilitate structural comparison to other RTKs, DrSr strain the structure of ONC-Xmrk is the same as the fused exon in Xmrk is denominated 12/13. For ex- that for the Y-chromosomal wild-type allele, indicating ons 9 and 10, 11 and 12, and 16 and 17 the correspond- that the crossover took place outside ONC-Xmrk. In the ing exon/intron borders are shifted by 1 or 3 bp. Sr crossover 3084B strain, analysis of ONC-Xmrk for the The design of primers matching to intron ends made allele-specific characters revealed that the 5Ј markers it possible to analyze for sequence differences between up to intron 1 are diagnostic for the Y allele and that the corresponding exons of the X and the Y allele of all further downstream markers give the pattern for ONC-Xmrk by SSCP analysis. The ONC-Xmrk SSCP the X allele. This indicates that obviously an intragenic bands could be differentiated from the corresponding crossover of the X- and Y-chromosomal allele took place INV-Xmrk exons by comparison to the pattern obtained in a region between the 3Ј-end of intron 1 and exon from fish that possess only INV-Xmrk but not ONC-Xmrk. 15 (Figure 3b). For exons 1, 15, 19, 23, and 25 informative SSCP patterns The Mdl Sb-carrying Y chromosome (strain 8; see Table were obtained (Figure 2), recognizable by an additional 1) originates most probably from fish taken from the Rio band appearing only in the hemizygous male fish. Be- Tonto (Kallman 1975). Its linked ONC-Xmrk produces sides SSCP, strategic sequencing revealed informative severe malignant melanoma originating from the spot- base changes in exons 1 and 17. Further allele-specific ted belly pattern, even in F1 hybrids with X. helleri. In landmarks for each of the ONC-Xmrk alleles, namely the case of the Sb chromosome the 3Ј portion of the large size differences due to insertions/deletions, are Xmrk oncogene was exchanged against X-ONC se- detectable by PCR. They were found in the 5Ј promoter quences by a crossover event that must have taken place region, the first intron, and the 3Ј carboxyterminal do- somewhere between the 3Ј-end of intron 1 and exon main. A multiplex PCR detects polymorphic base pairs 15 (Figure 3b). It is assumed that this happened in the between X-ONC and Y-ONC in intron 22 and sequences wild before the strain was collected. unique for Y-ONC in intron 25. In summary, 20 struc- The Y chromosome with the MdlSp4 allele (strain 4; tural characters that are specific either for the X or the see Table 1) leads to malignant melanoma, comparable Y allele were identified (Figure 1). to the well-studied Sp1 tumors. However, it shows a very Mutant analysis and structure of mutant ONC-Xmrk pronounced gene-dosage effect. Whereas heterozygous alleles: With the availability of X- and Y-chromosomal nonhybrid platyfish have only scattered small spots, in ONC-Xmrk-specific molecular characters (see Figure 1) the homozygotes the entire peduncle is bold black. Anal- an analysis of sex chromosomal crossover mutants was ysis of ONC-Xmrk indicates a combination of 5Ј up- possible. Both mutant X chromosomes (strains 9 and stream sequences of the X allele and the 3Ј-part of 10; see Table 1) that carry the Mdl Sr from Y are the Y-ONC (Figure 3c). The gene shares a polymorphic result of an X/Y crossover that did not affect the wild- nucleotide in exon 1 with Y-ONC. However, it shows an Figure 3.—Schematic map of the ONC-Xmrk loci of the sex chromosomal crossover mutants. (a) DrLi; (b) DrLi (mut), Sr crossover 3084B, Sb; (c) Sp4, N1; and (d) N2. For symbol explanations see Figure 1. Hatched symbols represent X. variatus-specific landmarks. n.d., strategic sequencing of exon 17 was not performed for Sp4 and N2. 780 H. Gutbrod and M. Schartl additional base exchange different from all other ONC 3084B and of Sb, the breakpoint region is localized be- alleles, except N1. This possibly represents a sequence tween the 3Ј-end of the first intron and exon 15 (Figure polymorphism specific to these strains. Southern experi- 3b). ments revealed that the first intron is neither identical Sr″ is an X-ray-induced of the wild-type to Y-ONC as would have been expected nor to the Mdl Sr-ONC-Xmrk carrying Y (Anders et al. 1973). The X-ONC copy. The markers for Y-ONC were found for Sr pattern in these platyfish is considerably enhanced the 3Ј-end from exon 23 onward. Thus the composite through an increase in numbers of macromelanophores structure of this ONC-Xmrk allele could not be resolved that spread all over the body but are arranged following exactly. the basic reticular pattern preformed by the margins of The Mdl N1-carrying Y chromosome (strain 6; see Table the scale pouches. While the wild-type Sr pattern is only 1) was isolated from the Belize River. Its associated ONC- slightly enhanced in hybrids and does not give rise to Xmrk is very similar to Xmrk of Sp4 (Figure 3c); it also tumors, Sr″ leads to melanosis and melanoma already has the nucleotide in exon 1, which is different from in F1 hybrids with X. helleri. The SSCP analysis of all all other ONC alleles. exons, as well as sequencing, PCR, and Southern blot The Mdl N2 chromosome (strain 7; see Table 1) is a analysis for the size-specific landmark positions of the spontaneous mutation of the N1 chromosome. In pa- Xmrk oncogene of Sr″, did not reveal differences in the rental platyfish the large blotches encoded by Mdl N1 are wild-type Y-ONC. However, the breakpoint for the DrLi extended in N2 to a coverage of the whole body side mut chromosome where the 5Ј region of Sr″ was trans- by macromelanophores, and in the hybrids with X. helleri ferred to the X-ONC shows that the mutation responsi- a more severe melanoma phenotype is observed. The ble for the Sr″ phenotype must lie 3Ј to that breakpoint, 3Ј-part of the Xmrk oncogene showed all markers of the because the transferred 5Ј region restored the normal Y allele (Figure 3d). Exon 1 again shows the unusual adult wild-type phenotype of the Sr pattern. nucleotide exchange like the parental chromosome. Localization of INV-Xmrk: The structure of the DrLi The 5Ј-flanking promoter region upstream (nt Ϫ44) of X chromosome that combines complementary portions the TATA box, however, is totally different compared of the X chromosomes of two different Xiphophorus with all other known X and Y sequences. species provided the chance to determine the position The DrLi X chromosome (strain 11; see Table 1) of the Xmrk proto-oncogene relative to ONC-Xmrk. An arose obviously by a crossover of the X. maculatus X RFLP that differentiates between INV-Xmrk and ONC- chromosome harboring Mdl Sd-ONC-Xmrk and the X. var- Xmrk of X. maculatus and X. variatus, respectively, was iatus X chromosome carrying Mdl Li-ONC-Xmrk. The RY found in BglII digestions of genomic DNA using the allele Dr linked to Mdl Sd was retained (Anders et al. 0.7-kb BamHI fragment from intron 1 as a probe and 1973). The macromelanophore pattern of these fish, rehybridization of the filter with a PCR probe (Ex1/ however, does not resemble either Sd or Li. The pig- Jd9) that spans a region 5Ј of the 0.7-kb BamHI frag- ment cells are dispersed over the whole body at high ment. This region harbors a 4.5-kb transposon-like ele- density. The pattern is most similar to Sr″ (see below). ment in INV-Xmrk of X. maculatus, which is absent in Unlike Li, which only leads to melanoma in hybrids ONC-Xmrk and in INV-Xmrk of the other species ( J. with X. helleri that are homozygous for Mdl Li-ONC-Xmrk, Altschmied and J. N. Volff, unpublished results). In fish with a DrLi X chromosome develop melanoma even fish that are homozygous for the DrLi X chromosome, in F1 and readily in heterozygous backcross hybrids with only the INV-Xmrk band specific for X. maculatus was X. helleri. In the molecular analysis the breakpoint region detected (Figure 4). This clearly indicated that INV- could be narrowed down to the 5Ј-part of the first intron Xmrk maps to the part of the DrLi X chromosome that of the Xmrk oncogene (Figure 3a). is derived from X. maculatus and thus is downstream of A second mutation (DrLi mut) occurred when a male ONC-Xmrk. platyfish (XDrLi/Y Sr ″ ) was crossed to a X. helleri female. One of the offspring did not exhibit the strongly en- DISCUSSION hanced macromelanophore expression typical for ei- ″ ther Sr or DrLi hybrids, but a phenotype like F1 of wild- Intragenic crossovers have as yet rarely been de- type Sr. Adults of the pedigree with this new mutant scribed (March et al. 1993; Kim et al. 1994) because of chromosome are indistinguishable from wild-type Sr. the difficulty in detecting their products when allele- Interestingly, a variable fraction of neonates of this specific landmarks are scarce and the crossover does strain develops severe melanosis and even invasive mela- not result in an altered phenotype. Determination of noma over the course of the first two months. Then all the genomic structure of both X- and Y-chromosomal lesions gradually regress over a period of two to three alleles of ONC-Xmrk from the Rio Jamapa platyfish made months until the normal Sr phenotype is reached. What it possible to identify characteristic landmarks for distin- happened to the recombined Xmrk gene was a substitu- guishing between the two alleles. These could then be tion of all 5Ј-X. variatus sequences by those of YSr ″ by used to analyze several mutants of the Mdl-Xmrk com- another crossover. As in the case of the Sr crossover plex locus. Regarding the relatively low number of cross- Intragenic Crossovers of Xmrk Oncogene 781

Figure 4.—Southern hybridization of BglII-digested genomic DNA of wild-type and mutant Xiphophorus fish. (A) Filter probed with the 0.7-kb BamHI fragment (probe b) common to ONC and INV copies of Xmrk. (B) Filter reprobed with the Ex1/ Jd9 PCR product (probe a), digested with AvaII. Different fragments for INV-Xmrk were detected in X. maculatus with both probes because of the presence of an inserted transposon-like element in the region covered by probe a (J. Altschmied and J. Duschl, unpublished results), which is absent in the INV copies of the other Xiphophorus species. Arrows point to the allele- specific fragments; * indicates a band that is most probably due to a partial restriction digest; arrowheads point to the X. maculatus INV-Xmrk-specific fragments. Filters were hybridized under stringent conditions (50% formamide) and washed with 0.1ϫ SSC, 1% SDS at 68Њ. overs affecting the whole chromosomal region, the pro- ments of different alleles from both the Mdl and the portion of intragenic crossovers in the ONC-Xmrk gene Xmrk genes. was surprisingly high. The breakpoints all lie within the Combining the molecular data from the crossover 5Ј portion of the gene somewhere between the end of mutants with the phenotype of the mutants and pheno- exon 1 and the beginning of the kinase domain-encod- genetic data from X/Y crossovers outside ONC-Xmrk ing part. Although it was impossible to find the exact allowed us to establish a fine map of this region of the crossover points on the single nucleotide level for each sex chromosomes. The pattern of macromelanophores mutant, it appears that despite this obvious concentra- encoded by the Mdl locus in all intragenic crossover tion in a certain defined region at least the DrLi mutants is that of the parental chromosome that con- breakpoint is different from the DrLi (mut), 3084B, and tributed the 5Ј portion of the respective ONC-Xmrk lo- Sb group as well as from the Sp4, N1, and N2 group. cus; e.g., a wild-type Sr pattern is seen in the Sr crossover The finding that crossovers are concentrating in a 3084B mutant. This places the sequences responsible for defined region in the 5Ј-part of the Xmrk oncogene may the Mdl phenotype 5Ј of ONC-Xmrk. Similarly, the RY point to a recombination hotspot. The analysis of the and SD locus can be placed 3Ј of ONC-Xmrk. Crossover available sequence of this region, however, has not yet chromosomes with the 3Ј part of the X-chromosomal revealed obvious GT-rich sequences that have been pro- allele have the RY pattern of the wild-type X chromo- posed to be a prerequisite in DNA strand exchange some. The 3Ј-part of ONC-Xmrk is also diagnostic for protein-mediated DNA recombination (Tracy et al. the linked female or male SD locus, indicating location 1997). A similar unexpectedly high recombination fre- of SD also downstream of ONC-Xmrk. A possible excep- quency has been described for the Duchenne muscular tion is the Sb chromosome, which is a Y, but has an dystrophy gene (DMD; Grimm et al. 1989). Contrary to X-chromosomal 3Ј-end of ONC-Xmrk. As this chromo- the DMD gene, crossovers of ONC-Xmrk exhibit pheno- some was isolated from a wild population, its precursors typic effects leading to new macromelanophore patterns are unknown and this exceptional structure might be probably due to the new combination of regulatory ele- reasonably explained by a double crossover. 782 H. Gutbrod and M. Schartl

et al. 1989; Adam et al. 1991; Ma¨ueler et al. 1993). Therefore, the regulatory elements controlling the al- lele-specific overexpression of ONC-Xmrk in hybrid mel- anoma have to be searched in the region 5Ј of exon 15. This is also confirmed by the DrLi (mut) phenotype, Figure 5.—Putative gene order on the sex chromosome of which is—at least in adult fish—like the wild-type Sr X. maculatus. SD, sex-determining locus; RY, locus for red- Sr yellow pattern; Mdl, macromelanophore-determining locus; (from Mdl -Xmrk on the Y chromosome of the Rio Ja- CEN, ; TEL, telomere. The localization of INV- mapa platyfish, strain 1) and is, as judged from all land- Xmrk is 3Ј of ONC-Xmrk, but the exact position cannot yet be marks, structurally like the Sr crossover 3084B. It also determined. has its crossover point somewhere between intron 1 and exon 15. Interestingly, the 5Ј portion of the DrLi (mut) locus stems from the Sr″ chromosome. Sr″ fish are char- The gene order of RY and SD with respect to ONC- acterized by an extremely malignant melanoma in hy- Xmrk can be determined from crossovers where the brids and by an overabundance of macromelanophores whole pigmentary gene-containing part was transferred in nonhybrid X. maculatus. SSCP analysis of all exons Dr Sd to the other sex chromosome, e.g., the RY -Mdl -Xmrk revealed no mutation in Sr″. Thus a mutation in the from the X to the Y chromosome (Kallman 1975). nontranslated portion of ONC-Xmrk that leads to an Thus the gene order is Mdl-5Ј ONC-Xmrk 3Ј-RY-SD (see enhanced expression of Xmrk appears as the most likely Figure 5). cause for the Sr″ phenotype, although such a mutation INV-Xmrk is also 3Ј of ONC-Xmrk, but exact placement was not detected with the tools used in this study. The in this linkage group was not possible with the currently structure of the DrLi (mut) ONC-Xmrk allele would available material. The that can be cal- place such a mutation 3Ј of its crossover point, re- -cM; however, this stricting the localization of the putative regulatory ele 0.6ف culated from our data would be is based on the one recombinant in all 159 fish analyzed. ment to a region downstream of this breakpoint up to Analysis of other crosses will reveal more data, which exon 15. finally should allow precise fine mapping of INV-Xmrk. The Mdl DrLi-Xmrk chromosome gives its carrier a mac- Referring to Anders et al. (1973), it appears reason- romelanophore phenotype that is neither characteristic able to suggest that the whole linkage group is oriented for the paternal Mdl Sd-Xmrk (that contributed the 3Ј in a way that SD is toward the centromere and Mdl portion) nor for the Mdl Li-Xmrk from which the 5Ј re- toward the telomere. The X/Y crossover rate over the gion and exon 1 is derived. The novel phenotype might whole segment between SD and Mdl is very low, usually then be due to the inappropriate interaction of the in the range of Յ1% (Gordon 1937; Kallman 1975; 5Ј regulatory region from X. variatus and a regulatory K. D. Kallman, personal communication, and our own element located somewhere in intron 1 or in the follow- unpublished data). As this whole complex is located ing introns up to exon 15. Alternatively, the crossover in the subtelomeric region of the sex chromosome (I. process may have occurred within such an element and Nanda, M. Schmidt and M. Schartl, unpublished re- has compromised its structure and function. This infor- sults), where recombination frequency and physical map distance are not expected to be considerably bi- mation should be useful for molecularly identifying the .kb (according to Morizot et regulatory elements of ONC-Xmrk in the future 300ف ased, it might cover al. 1991) or less. This should allow cloning of the whole We thank Jean-Nicolas Volff for critically reading the manuscript set of genes by a chromosome walk starting from the and for discussions, Ute Hornung for technical assistance, Georg available ONC- and INV-Xmrk genomic clones. Schneider, Hugo Schwind, and Petra Weber for breeding of the fish, Christine Moeller for help in preparing the manuscript, and Steven So far nothing is known regarding what determines Kazianis (Austin, TX) and an anonymous reviewer for many useful the profound differences in the pathophysiological phe- suggestions. Founder fish for some of our stocks were obtained from notype of the melanoma arising from the different Mdl- Annerose and Fritz Anders, Gießen, Klaus D. Kallman, New York, and Xmrk alleles. From the fact that the Sr crossover 3084B the Xiphophorus Genetic Stock Center at the Southwest Texas State has the identical macromelanophore pattern pheno- University, San Marcos, TX. This work was supported through grants to M.S. supplied by the Deutsche Forschungsgemeinschaft through type in the X. maculatus genetic background and in Sonderforschungsbereich 465 and 165, the European Commission hybrids as the wild-type Sr, it can be deduced that all (CI1* CT94-0021, FAIR PL97-3796), and the Fonds der Chemischen the genetic elements determining the time of onset, Industrie. the body compartment of melanoma appearance, and malignancy of this Mdl-Xmrk complex are located 5Ј of exon 15 of the oncogene. This is in agreement with LITERATURE CITED earlier data suggesting that pattern information is con- tained within Mdl (Weis and Schartl 1998). Adam, D., W. Ma¨ueler and M. Schartl, 1991 Transcriptional acti- vation of the melanoma inducing Xmrk oncogene in Xiphopho- The malignancy of the melanoma is highly correlated rus. Oncogene 6: 73–80. with the amount of ONC-Xmrk transcripts (Wittbrodt Adam, D., N. Dimitrijevic and M. 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