The Macromelanophore Locus and the Melanoma Oncogene Xmrk Are Separate Genetic Entities in the Genome of Xiphophorus

Corresponding author: Manfred Schartl, Physiologische Chemie I, Biozentrum der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany., schartl{at}biozentrum.uni-wuerzburg.de (E-mail).

Communicating editor: C. KOZAK

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Fish of the genus Xiphophorus are polymorphic for black pigmentation patterns. Certain intra- or interspecific hybrids exhibit enhanced expression of these patterns, leading in many cases to malignant melanoma. Because no recombination was ever observed between the pattern information and the genetic predisposition to develop melanoma after hybridization, a "tumor gene" (Tu) was postulated that encodes both phenotypes. A dominant oncogene, ONC-Xmrk, was then found to be necessary and sufficient for the transforming function of Tu. Here we present molecular evidence that ONC-Xmrk and the pigment pattern information are encoded by separate, although intimately linked loci. No ONC-Xmrk gene was present in the 15 Xiphophorus strains investigated which exhibit no black pigmentation pattern. Five different patterns from Xiphophorus maculatus, X. evelynae, X. milleri, X. cortezi, and X. montezumae were associated with ONC-Xmrk and were melanomagenic, while fish of X. helleri, X. variatus, X. nezahualcoyotl, and X. montezumae with five other patterns had no ONC-Xmrk and consequently did not produce hybrid melanoma. These data provide evidence that ONC-Xmrk is sufficient for tumorigenesis in Xiphophorus hybrids, and that a separate, pigment pattern-encoding locus is closely linked to it.

MOST species of the genus Xiphophorus are polymorphic for a variety of pigment patterns due to local high density of melanophores, xantho/erythrophores, and guanophores. For the melanin-containing pigment cell lineage two types are distinguished, micro- and macromelanophores. Micromelanophores have a diameter up to 100 µm. They constitute a reticular pattern on the body and are dispersed on the fins, making up the greyish wild-type pigmentation of all fish of this group. In addition they concentrate in certain areas of the skin or the underlying extracutaneous tissues where they give rise to certain spot patterns. These are most prominent in the peduncular and tail fin compartment and have been designated "tail spot patterns." They are encoded by an autosomal multiple allelic series of codominant genes (KALLMAN 1975 ; BOROWSKY 1984 ). Modifier genes that specifically interact with certain tail spot alleles have been described (GORDON 1956 ). Macromelanophores attain a size of 300–500 µm and even larger and have—in contrast to the evenly spaced micromelanophores—a tendency to overgrow each other at their margins. They compose certain bold markings, encoded by codominant alleles of the so-called macromelanophore locus (KOSSWIG 1929 ; GORDON 1931 , GORDON 1958 ; KALLMAN 1975 ; here referred to as Mdl, for macromelanophore determining locus), which in many species is located on the sex chromosomes. This locus has attracted special attention, because it has been shown for some of these patterns that upon certain crossings its expression is enhanced in the hybrids, giving rise to severe melanosis and even malignant melanoma. In the classical Gordon-Kosswig hybridization experiment (GORDON 1927 ; KOSSWIG 1928 ), the "spotted dorsal" pattern of the southern platyfish, Xiphophorus maculatus, is introduced via backcrossing into the genetic background of the green swordtail, X. helleri. F₁ hybrids have enlarged spots in the dorsal fin, and sometimes macromelanophores appear at ectopic sites, preferentially in the tail fin. The phenotype of backcross hybrids with X. helleri as the recurrent parent ranges from F₁-like fish to malignant melanoma. A genetic model to explain this and other observations from similar crossing experiments has been formulated by ANDERS and co-workers (AHUJA and ANDERS 1976 ; for review see ANDERS 1991 ). A simple two-locus system was proposed where the platyfish carries a sex-chromosomal "tumor gene" (Tu) whose activity is suppressed by an autosomal regulatory locus (R). The swordtail is supposed to have neither Tu nor R. By the crossing conditioned elimination of R-containing autosomes the Tu locus is released from its control. This leads to enhanced expression of Tu. R heterozygotes have enlarged spots. The absence of both copies of R in Tu containing backcross hybrids results in malignant melanoma. It was proposed as a consequence of this model that Tu itself specifies the aberrant phenotype of the macromelanophore even in the pure species and that therefore Tu is also the "macromelanophore" gene, synonymous to Mdl (ANDERS 1991 ). This was supported by the fact that no recombination was ever observed between the pattern information and the melanoma predisposing gene (KALLMAN 1975 ). Overexpression of Tu in hybrids was thus seen as simply leading to a stepwise overproduction of cells of the macromelanophore lineage depending on the dosage of R.

The Tu locus was found to encode an activated version of the growth factor receptor tyrosine kinase gene Xmrk (WITTBRODT et al. 1989 ; ADAM et al. 1993 ; for review see SCHARTL 1995 ). This oncogene has all features of a dominant-acting transforming gene (WITTBRODT et al. 1992 ; WINKLER et al. 1994 ; DIMITRIJEVIC et al. 1998 ). It was shown that in X. maculatus the oncogenic version of Xmrk (ONC-Xmrk) is present as an additional copy besides the corresponding proto-oncogene (INV-Xmrk) which is a normal constituent of the genome of all fish of the genus Xiphophorus and serves a so far unknown physiological function. The oncogene arose through a nonhomologous recombination event leading to a gene duplication of INV-Xmrk on one of the sex chromosomes. During this process the duplicated copy was fused in its 5' region to another sequence. This generated a new promoter which is responsible for the transcriptional activation of this additional Xmrk copy (ONC-Xmrk) in the macromelanophore lineage of the hybrids (ADAM et al. 1993 ).

From all data available to date, ONC-Xmrk appears to be sufficient for bringing about the Tu phenotype in hybrids, namely an increase in cells of the macromelanophore lineage. In accordance with earlier hypothesis it was proposed that ONC-Xmrk also functions in specifying the macromelanophore phenotype in the nontransformed state, e.g., in parental wild platyfish (WOOLCOCK et al. 1994 ). With molecular probes now available from the Tu-encoded melanoma-inducing Xmrk gene we have investigated if the tumor gene and the macromelanophore-determining locus (Mdl) are indeed the same or if they may be encoded by separate loci. Here we show that several macromelanophore-spotting loci exist in some Xiphophorus species that do not contain an Xmrk oncogene and, consequently, produce neither enhanced pigmentation, melanosis, nor melanoma in hybridization experiments, thus defining Mdl and Tu as separate genetic loci.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Fish:
All fish used in this study (see Table 1) were bred and maintained in the aquarium facilities of the Biocenter under standard conditions (KALLMAN 1975 ). F₁ hybrids of X. maculatus, X. evelynae, and X. variatus with X. helleri were produced by artificial insemination (ZANDER 1969 ). All stocks are derived from wild fish and are kept as closed colonies. Most were established after a few generations of selective breeding in the laboratory from single pairs of fish homozygous or hemizygous for a certain pigmentation pattern.

For description of the pattern phenotype in wild fish see ATZ 1962 , ZANDER 1969 , KALLMAN 1975 , and RAUCHENBERGER et al. 1990 . So far undescribed patterns are briefly characterized in Table 1. Some patterns in different species which look more or less similar had earlier been given the identical genetic term by some authors. To avoid confusion the macromelanophore pattern of X. milleri (earlier "striped"), X. birchmanni (earlier "spotted-caudal"), and X. nezahualcoyotl (earlier "spotted") have been renamed (see Table 1).

DNA analysis:
DNA was extracted from individual fish as described by SCHARTL et al. 1995 , digested with different restriction enzymes, blotted onto positively charged nylon membranes (Hybond N⁺), and hybridized to probes from the Xmrk gene under conditions of high stringencies [hybridization at 42° in a buffer containing 50% formamide, 0.1% Na-pyrophosphate, 50 mM Tris-Cl, pH 7.5, 5x SSC, 1% sodium dodecyl sulfate (SDS), 5x Denhardt's, 100 µg/ml calf thymus; washing generally at 68° with 0.1x SSC, 1% SDS]. Hybridization probes were generated by PCR amplification of fragments spanning nt 75 to 396 (nt numbering according to ADAM et al. 1991 ; K-probe) and nt 4297–4589 (C-probe) of Xmrk and labeled by random priming (FEINBERG and VOGELSTEIN 1983 ). The following primers were used: Ins2 (5' AGG GAA TGA ACT ACC TGG AAG AGC 3') and Mey3 (5' CGT AGC TCC ACA CGT CGC TCT G 3') for the K-probe and SW1 (5' TGA GAA TCC AGT TTC AAC C 3') and SW2 (5' GCT GAC GGG ATG AAC GC 3') for the C-probe. The PCR was done under the following conditions: 95° for 30 sec, 67° (K-probe) or 50° (C-Probe) for 30 sec, 72° for 60 sec (for 35 cycles). For the linkage analysis of the Cs pattern of X. birchmanni with an additional copy of Xmrk, the 6.5-kb EcoRI insert of pY-Xmrk (ADAM et al. 1991 ) was used as probe. For cloning the homologous genomic breakpoint of ONC-Xmrk copies associated with macromelanophore pattern from various species primers were derived from the corresponding region of the X. maculatus ONC-Xmrk. PCR was done with primers Prom2 (5' CCG CTC CTC CGC GCA GAA AC 3') located 3' of the breakpoint, and Prom4 (5' CCT CTA CCT TAT ATA ATG AGA GCG) at 35 cycles of 95°, 65°, 72° for 30 sec each for X. birchmanni with Cs or Prom3 (5' AAT GAC TGG GCA GTG CTA AGG 3') for X. evelynae with Se and X. milleri with Sl at 35 cycles of 95°, 30 sec, 64°, 30 sec, and 72°, 60 sec, both located 5' of the breakpoint. The products were cloned into the SmaI site of pUC 18 using the Sure Clone Kit (Pharmacia, Freiburg, Germany). Four independent clones each were sequenced. The sequences are deposited under accession nos. AF047372, AF056306, and AF056307 at GenBank.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Although in the past extensive crossing studies in the genus Xiphophorus have been performed, our data base concerning the inheritance of macromelanophore (M) pattern under the aspect of melanoma formation in hybrids was still far from complete. Especially for some only recently described species and newly detected M patterns such information was totally lacking. We therefore hybridized M-pattern-exhibiting fish with M-pattern-free X. helleri, X. gordoni, or X. couchianus. These species have generally been found to give strong enhancement of various M patterns. For studying in hybrids the expression of the X. helleri Db2-pattern crossings were done with M-pattern-free X. helleri and X. andersi. We found that the M-pattern loci Sd, several alleles of Sp, Ni1, Ni2, and Sb of X. maculatus, Li of X. variatus, Sl of X. milleri, Ma of X. montezumae, and Se of X. evelynae are strongly enhanced in expression and give rise to severe melanosis and melanoma. Expression of Sr of X. maculatus was moderately enhanced, even after repeated backcrossing to X. helleri. Enhancement of Sc of X. cortezi and Cs of X. birchmanni occurred only in higher backcrosses. No enhancement or even reduction of the pattern was observed in hybrids with the Db2 pattern from X. helleri, the Ss pattern from X. nezahualcoyotl, and the Sf pattern of X. montezumae (Table 2). These data group the M patterns into three categories: those which can give rise to malignant melanoma in certain hybrids, those which are enhanced to a certain degree, but not to melanosis or melanoma, and those which are either unaffected, or even reduced. From the literature, Pu2 of X. variatus (BOROWSKY 1973 ) and Fl of X. xiphidium (ATZ 1962 ; ANDERS and KLINKE 1965 ; ZANDER 1969 ; SCHARTL 1990 ) can be added to class I, At of X. cortezi (ATZ 1962 ; ZANDER 1969 ) to class II, and Pu1 of X. variatus (ATZ 1962 ; ZANDER 1969 ; ANDERS et al. 1973 ) to class III.

Fish from the species X. meyeri have dark spots on the body side that can merge to large patches. This pattern originates in the underlying extracutaneous compartment, mostly in the myoseptae, but associates to the dermis. The cells are intermediate in size between typical micromelanophores that make up the extracutaneous spot patterns of X. couchianus and X. gordoni, and the macromelanophores found so far in 10 of the remaining 19 species of the genus (SCHARTL and SCHRODER 1987 ). Crossing X. meyeri to X. maculatus and X. andersi resulted always in reduction of this pattern (data not shown).

To analyze fish from different species and populations either with or without an M pattern for the presence of ONC-Xmrk, an approach utilizing restriction length polymorphisms detected by Southern analysis was chosen. It was reasoned that the presence of ONC-Xmrk besides INV-Xmrk in the genome should be recognized as additional hybridizing fragments due to restriction site polymorphisms between both copies. Two independent hybridization probes were employed, one from the kinase domain encoding region (K-probe), the other from the carboxyterminal part (C-probe) of Xmrk. Both probes were kept as short as possible (K = 322 bp, C = 303 bp) to minimize the number of restriction fragments detected per copy. The assay was first tested on X. maculatus females which are homozygous for the X-chromosomal Xmrk oncogene of the Sd locus. Using 15 different restriction enzymes, in 6 cases with the K-probe and 10 with the C-probe, an additional band was detected. For comparison an X. maculatus strain was analyzed which has no macromelanophore locus and, consequently, has no melanoma oncogene. Here only the fragments of the proto-oncogene were seen. Another X. maculatus strain which carries the M-pattern locus Ni1 that leads to highly malignant melanoma in hybrids with X. helleri gave similar results with a total of three restriction enzymes for K and nine for C being informative by generating additional fragments (Table 3). In general, for those restriction fragments for which information from the cloned genomic loci of the proto-oncogene and the oncogene is available from X. maculatus, the predicted size was noted.

Comparing M-pattern-bearing fish with the corresponding animals that have no macromelanophores, additional Xmrk fragments were obtained for X. cortezi Sc, X. montezumae Ma (see Figure 1), and X. milleri Sl. For X. evelynae Se, no corresponding strain without M pattern was available, however, with two enzymes for the K-probe and nine for the C-probe, two hybridizing fragments indicate the presence of an additional Xmrk copy. On the contrary, for the M patterns Db1 and Db2 of X. helleri (Rio Sontecomapan and Rio Lancetilla strains), Ss of X. nezahualcoyotl, Pu1 of X. variatus, and Sf of X. montezumae no additional bands were found if compared to the restriction fragment pattern of the corresponding M-pattern-free fish (Table 3 and Table 4).

View larger version (65K): In this window In a new window Download PPT slide   Figure 1. Southern blot analysis of X. montezumae with Ma or Sf and without any M pattern. Hybridized (top) with the C-probe, and (bottom) with the K-probe.

The Cs pattern of X. birchmanni was tested for cosegregation with an additional 6.5-kb HindIII fragment that is absent in macromelanophore-free individuals. 34 Cs-expressing backcross hybrids with X. helleri (strain no. 29, BC₁ and BC₂) had also inherited this diagnostic fragment, while 35 siblings without Cs had no additional Xmrk band. No recombinants were seen in this analysis.

To obtain further evidence for the homology of the Cs-pattern-associated additional Xmrk copy of X. birchmanni with the ONC-Xmrk of X. maculatus, a PCR analysis was done with primers derived from the 5' (promoter) and 3' (exon 1) flanking region of the chromosomal breakpoint in ONC-Xmrk. Such primers give rise to an amplification product only from ONC-Xmrk but not from INV-Xmrk. Cloning and sequencing confirmed the homology of the Cs-associated Xmrk copy of X. birchmanni with ONC-Xmrk from X. maculatus. Similarly, breakpoint-spanning homologous products were obtained from X. evelynae with Se and X. milleri with Sl (Figure 2).

View larger version (43K): In this window In a new window Download PPT slide   Figure 2. Sequences of homologous ONC-Xmrk fragments from several Xiphophorus species. The fragments were obtained after PCR with primers flanking the chromosomal breakpoint of ONC-Xmrk linked to MdlSd in X. maculatus, from DNA of X. evelynae with MdlSe, X. milleri with MdlSl, and X. birchmanni with MdlCs. For X. maculatus the complete sequence is given, while for the other species only divergent sites are recorded. A dash indicates a missing nucleotide. The oncogene-specific breakpoint is indicated by an arrow.

Spotted X. meyeri were not distinguishable from unspotted fish by additional fragments, excluding a second copy of Xmrk in these fish. X. helleri (Laguna Catemaco), X. variatus (Rio Vinazco), X. gordoni, and X. couchianus, all of which have no macromelanophores, showed a simple hybridization pattern with generally a single band per enzyme. This, as expected, indicates only presence of INV-Xmrk. In some cases an interindividual population-specific polymorphism for fragment length was seen with certain enzymes leading in heterozygotes to two hybridizing bands. However, single bands in the homozygotes easily distinguished this from the true additional band situation of fish which have the extra oncogenic Xmrk copy. Such polymorphisms were especially evident in the X. helleri stocks from the Rio Sontecomapan and in X. nezahualcoyotl where up to five alleles could be detected. Sex-chromosome-specific restriction fragment length polymorphisms (RFLPs) for INV-Xmrk were detected in X. variatus (Rio Vinazco strain) with AccI (K- and C-probe) and BamHI (C-probe), in X. helleri (Rio Sontecomapan strain) with XbaI (K-probe), X. andersi with DraI (K-probe), and X. evelynae (SstI, C-probe). Here double bands were restricted to the heterogametic sex, the males (Table 3).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In this study additional restriction fragments have been used to score for the presence of ONC-Xmrk as an additional gene copy in the genome of the fish. This assay was tested for fidelity in X. maculatus, where through extended molecular analysis including the cloning of the entire proto-oncogene and oncogene genomic loci the organization of the different Xmrk copies is known in detail and complete restriction maps are available (WITTBRODT et al. 1989 ; ADAM et al. 1991 ; DIMITRIJEVIC et al. 1998 ; S. WEIS and M. SCHARTL, unpublished results). The occurrence of the expected fragment sizes proved the usefulness of this approach. For further analyses of fish where no data on the genomic organization of Xmrk are available, by taking additional fragments as indicators of a duplicated Xmrk two complications had to be considered: (1) A restriction site may be located in the region that is spanned by the probe in INV-Xmrk of the M-pattern-carrying fish. This should, however, equally likely occur in the proto-oncogene of the M-pattern-free stocks, thus the frequency can be calculated from the M-pattern-free stock fish. From our analysis this rate is 8% (15 restriction enzymes, K- and C-probe, 14 stocks; Table 3). We therefore scored only those genotypes positive for the presence of the oncogene that had a frequency of more than 30% of additional bands. (2) A restriction site polymorphism may exist between both proto-oncogene copies. For sex-chromosomal location this will generate two fragments in the heterogametic sex. Such RFLPs were found in X. variatus, X. helleri, and X. evelynae indicating that the Xmrk proto-oncogene in these species is also sex-chromosomally located like in X. maculatus. Other interindividual RFLPs which generate two bands in heterozygotes were distinguished by detection of the corresponding homozygous individuals.

All patterns that readily produce melanoma in hybrids (class I) were found to be associated with an additional, oncogenic Xmrk, while those that are unaltered or reduced (class III) have no extra Xmrk copy. For the intermediate class II (patterns which are enhanced, but not melanomagenic) from the Sr-pattern-encoding Y chromosome of X. maculatus an oncogenic copy of Xmrk has been cloned (ADAM et al. 1991 ), Sc of X. cortezi was identified in the RFLP assay to be associated with an additional copy of Xmrk, and the Cs pattern of X. birchmanni cosegregated with an additional Xmrk without recombination. These M patterns were found to be enhanced after hybridization, but formation of malignant melanoma was not observed in our crossing experiments. The findings for Sr are consistent with reports on similar crossings and those using other species for introduction of the M pattern (ATZ 1962 ; ZANDER 1969 ; ANDERS et al. 1973 ). However, in one certain hybridization experiment backcross hybrids with X. cortezi exhibited even "small melanoma" on the body sides (KOSSWIG 1961 ). For Sc of X. cortezi similar results to ours (ANDERS and KLINKE 1965 ; ZANDER 1969 ) were obtained, while in other crossing experiments severe melanosis and melanoma were obtained (ATZ 1962 ). One explanation is that a different allele of Sc has been used. On the other hand, it is known that the ability to enhance M pattern is different, not only between species but also between different populations (KALLMAN 1970 , KALLMAN 1975 ). Thus also a different partner for the crossing experiment might explain this discrepancy. Clearly Sc was enhanced to melanoma even in F₁ animals when X. nezahualcoyotl (montezumae) was used for hybridization (ATZ 1962 ; ZANDER 1969 ). We interpret these findings that the Xmrk alleles linked to Sr and Sc are weak alleles with respect to their tumorigenicity; for Sc even strong and weak alleles might coexist in a population.

In conclusion, our data show that all macromelanophore pattern loci that can be enhanced in expression after hybridization, frequently even up to the malignant state, are associated with ONC-Xmrk, while those that remain unaltered or are reduced in expression after hybridization are not (Table 4). This is supported by the comparative Xmrk RFLP analysis of an X. helleri strain polymorphic for Db2 (SCHARTL 1990 ) and results from a study using a EcoRI RFLP for Xmrk in several X. variatus strains polymorphic for the Pu1 and Pu2 pattern (KAZIANIS and BOROWSKY 1995 ).

The fact that several macromelanophore loci (Db1, Db2, Ss, Pu1, Sf) exist, which are not associated with an additional oncogenic copy of Xmrk (Table 4), can most likely be interpreted by the reasoning that the Xmrk oncogene is not Mdl or, in the classical genetic terms, that Tu is not the macromelanophore gene. The fact that all M-pattern loci that are enhanced or even tumorigenic after the appropriate crossings are indeed associated with a second Xmrk copy lends further support to the extrapolation of the unambiguous data from X. maculatus that the Xmrk oncogene is the melanoma oncogene of the whole genus Xiphophorus and that the different alleles from the various species are homologous (SCHARTL 1990 ).

In the current absence of further molecular data it cannot be excluded as an alternative hypothesis that the macromelanophore patterns are not being coded for by alleles at a single locus, Mdl, but that perhaps there are two distinct loci, one for the tumorigenic patterns (class I and II), which is associated with the extra Xmrk copy ("Mdl-1"), and the other for the class III pattern, not containing an extra sequence ("Mdl-2"). The perfect similarity of some class I, II, and III pattern phenotypes in the nonhybrid wild fish would argue against structurally very divergent "Mdl-1" and "Mdl-2" loci; however, in the absence of recombinants among patterns (which might be too rare to observe or due to the lack of any way to predict recombinant phenotypes), this issue cannot be clarified at present.

It appears appropriate to adjust the genetic nomenclature for the pigment pattern and melanoma genes on the basis of the findings that the tumor gene is not equivalent to the macromelanophore gene. The Tu locus sensu Anders consists of two different loci, namely of one allele of Mdl and one closely linked allele of ONC-Xmrk. The different phenotypes of the macromelanophore pattern are ascribed to Mdl as different alleles, because also in those cases where an Mdl is not linked to an ONC-Xmrk gene different patterns can be clearly recognized (e.g., Db1 and Db2). Consequently Tu-Sd is now designated Mdl^Sd-Xmrk. The locus encoding the pattern Dabbed-2 of X. helleri that neither contains an ONC-Xmrk gene nor gives rise to crossing conditioned pattern enhancement, melanosis, or melanoma, is encoded by Mdl^Db2. Here the earlier used denomination Tu-Db2 would be especially misleading. We reserve the acronym Tu as a designation for the Mdl-Xmrk compound locus.

Although the argument that ONC-Xmrk oncogene is not Mdl is supported by our data as presented in Table 3, this makes it difficult to explain the phenotype of a loss-of-function mutant (lof-1) of the spotted dorsal locus. Fish with the wild-type Mdl^Sd-Xmrk locus give rise to hybrid offspring, e.g., with X. helleri as the recurrent parent for backcrossing, which develop malignant melanoma originating in the Sd compartment. The Mdl^Sd-Xmrk loss-of-function mutant lof-1 carries an insertion in ONC-Xmrk which disrupts the reading frame (WITTBRODT et al. 1989 ) and leads to a nonfunctional, prematurely terminated protein (M. SCHARTL, U. HORNUNG and C. WELLBROCK, unpublished results). The consequence of this gene disruption is a loss of the ability to develop hybrid melanoma from this locus. Unexpectedly, these mutant fish do not show an M pattern like parental fish. This, however, would have been predicted only if Mdl is identical to the Xmrk oncogene. Obviously, the Xmrk oncogene and Mdl are closely linked, because so far no recombinants have been found. It is possible that the large, 7-kb insertion in the Xmrk oncogene also has a remote effect on a closely located Mdl-locus, for instance by altering chromatin structure.

If the presence of either Mdl or ONC-Xmrk are plotted as characters on the DNA sequence based phylogenetic tree of the genus Xiphophorus (MEYER et al. 1994 ), it becomes apparent that neither one is concordant with a single cluster of species (Figure 3). Moreover, both are distributed unevenly with the oncogenic Xmrk always linked to melanomagenic Mdl. Without exception, all species in which Mdls and Xmrk oncogenes are found are polymorphic for this character. Because the different alleles of Mdl of all species appear to be homologous and the Xmrk oncogene alleles are also homologous (KALLMAN and ATZ 1966 ; SCHARTL 1990 ), it is reasonable to explain this situation by assuming that the Mdl as well as the gene duplication leading to the Xmrk oncogene originated prior to the divergence of the extant species of the genus, and that both have been frequently lost in the different branches. The same conclusion is reached if the allozyme and morphology based phylogeny (RAUCHENBERGER et al. 1990 ) or the data from BOROWSKY et al. 1995 based on nuclear RAPD characters are used. These differ from the sequence-based tree only by the clustering of X. birchmanni and malinche with cortezi, of pygmaeus and multilineatus with nigrensis, and by supporting the traditional placement of clemenciae in the helleri group, making an even more consistent superposition of ONC-Xmrk and Mdl states on the tree possible. But again the most parsimonious hypothesis remains to place the origin of Mdl and the Xmrk gene duplication at the base of the tree. Using a maximally conservative estimate for the timing of the first presence of Mdl and Xmrk [employing a mitochondrial molecular clock of 1% mutations/10⁶ years and the data set from MEYER et al. 1994 ], both genes should be older than approximately 5–6 million years.

View larger version (33K): In this window In a new window Download PPT slide   Figure 3. Phylogenetic tree of the genus Xiphophorus with character tracing for the presence of Mdl (A) and the Xmrk oncogene (B) using Mac Clade [ MADDISON and MADDISON 1992 ]. , presence of the character; , absence of the character. In B for several taxa the information is incomplete (no square). It is assumed that all those species which do not have macromelanophore pattern also lack the ONC-Xmrk, as an extrapolation of the existing data set (see text).

It has been shown that a broad spectrum of pigmentation phenotypes is obtained for one and the same Mdl and Xmrk allele depending on the host species or even population. M-pattern-carrying chromosomes may cause malignant melanoma even in F₁ with one species, but might be totally suppressed after hybridization with another species or population (ATZ 1962 ; ANDERS and KLINKE 1965 ; ZANDER 1969 ; KALLMAN 1970 , KALLMAN 1975 ). Also, another M pattern from one species may behave differently in the same genetic background. This can be explained by postulating that a variety of factors like the R-locus of the Rio Jamapa population of X. maculatus exist which control ONC-Xmrk expression to a different extent. However, it has now also to be envisaged that another set of population and species-specific genes influence the expression of the Mdl from a foreign genome. If, for instance, in a hybrid the development of macromelanophores is suppressed or if they are kept in a differentiation stage not competent for neoplastic transformation (e.g., through stage-specific absence of the appropriate signal transduction machinery), the oncogene Xmrk will lack its targets, and proliferation will not occur. Similarly, the ONC-Xmrk effect can be augmented by such macromelanophore differentiation genes interacting with Mdl.

	ACKNOWLEDGMENTS

We thank U. HORNUNG and B. WILDE for technical assistance, G. SCHNEIDER, H. SCHWIND, and P. WEBER for breeding and maintaining of the fish, and C. MÖLLER for help in preparing the manuscript. This work was supported by grants to M.S. supplied by the Deutsche Forschungsgemeinschaft through Sonderforschungsbereiche 465, 172, and 165, the European Commission (contract no. CI1*-CT94-0021), and the Fonds der Chemischen Industrie.

Manuscript received January 28, 1998; Accepted for publication May 1, 1998.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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ATZ, J. W., 1962  Effects of hybridization on pigmentation in fishes of the genus Xiphophorus. Zoologica 47:153-181.

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