Construction and initial analysis of bacterial artificial chromosome (BAC) contigs from the sex-determining region of the platyfish Xiphophorus maculatus

Despite the major importance of sex determination in aquaculture, no master sex-determining gene has been identified so far in teleost fish. In the platyfish Xiphophorus maculatus, this master gene is flanked by two receptor tyrosine kinase genes, the Xmrk oncogene responsible for melanoma formation in some Xiphophorus interspecific hybrids, and its proto-oncogenic counterpart. Both Xmrk genes, which have already been characterised at the molecular level, delimit a region of about 1 Mb that contains other gene loci involved in sexual maturity, pigmentation and melanoma formation. We have constructed a genomic bacterial artificial chromosome (BAC) library of X. maculatus with a tenfold coverage of the haploid genome and walked on both X and Y sex chromosomes starting from both Xmrk genes. This led to the assembly of BAC contigs from the sex-determining region covering approximately 950 kb of the X and 750 kb of the Y chromosome. To our knowledge, these are the largest contigs reported so far for sex chromosomes in fish. Molecular analysis suggests that the sex-determining region of X. maculatus frequently undergoes retrotranspositions and other kinds of rearrangements. This genomic plasticity might be related to the high genetic variability observed in Xiphophorus for sex determination, sexual maturity, pigmentation and melanoma formation, which are encoded by gene loci located in the sex-determining region.     

Pharmacological properties of angiotensin II receptors in cultured rabbit gingival fibroblasts

Certain interspecific hybrids of the fish Xiphophorus spontaneously develop melanoma induced by the derepression of the Xmrk oncogene. Xmrk is a recent duplicate of an orthologue of the mammalian epidermal growth factor receptor gene Egfr. In addition to a specific overexpression in melanoma, amino-acid substitutions in the extracellular domain leading to ligand-independent dimerisation and constitutive autophosphorylation are responsible for the tumorigenic potential of Xmrk. The Xmrk receptor induces several signal transduction pathways mediating cell proliferation and resistance to apoptosis and initiating dedifferentiation. Moreover, Xmrk upregulates the expression of the secreted protein osteopontin, inducing an autocrine loop possibly allowing invasion and survival in the dermis as a first step in malignancy. Hence, Xmrk is able to induce pathways essential for a transformed phenotype. Some of these events are equivalent to those found downstream of the mammalian Egfr, but others have clearly evolved differently or are specific for pigment cells. Xmrk is potentially hazardous, nonessential and located in a very unstable genomic region. Nevertheless, Xmrk has been maintained under purifying selection in divergent Xiphophorus species. Hence, Xmrk has probably a beneficial function under certain conditions. The analysis of this function is a major challenge for future research in the Xiphophorus model.     

The Xmrk receptor tyrosine kinase is activated in Xiphophorus malignant melanoma.

Xmrk encodes a putative transmembrane glycoprotein of the tyrosine kinase family and is a melanoma-inducing gene in Xiphophorus. We attempted to investigate the biological function of the putative Xmrk receptor by characterizing its signalling properties. Since a potential ligand for Xmrk has not yet been identified, it has been difficult to analyse the biochemical properties and biological function of this cell surface protein. In an approach towards such analyses, the Xmrk extracellular domain was replaced by the closely related ligand-binding domain sequences of the human epidermal growth factor receptor (HER) and the ligand-induced activity of the chimeric HER-Xmrk protein was examined. We show that the Xmrk protein is a functional receptor tyrosine kinase, is highly active in malignant melanoma and displays a constitutive autophosphorylation activity possibly due to an activating mutation in its extracellular or transmembrane domain. In the focus formation assay the HER-Xmrk chimera is a potent transforming protein equivalent to other tyrosine kinase oncoproteins.     

PI3-Kinase Is Involved in Mitogenic Signaling by the Oncogenic Receptor Tyrosine Kinase Xiphophorus Melanoma Receptor Kinase in Fish Melanoma

Overexpression of the mutationally activated receptor tyrosine kinase Xiphophorus melanoma receptor kinase (Xmrk) initiates formation of hereditary malignant melanoma in the fish Xiphophorus. In melanoma as well as in a melanoma-derived cell line (PSM) this receptor is highly activated resulting in constitutive Xmrk-mediated mitogenic signaling. In order to analyze mitogenic signaling triggered by Xmrk a possible involvement of phosphatidylinositol 3 (PI3)-kinase in Xmrk signal transduction was examined. Constitutive binding of the p85 adapter subunit of PI3-kinase to the Xmrk receptor was detected in PSM melanoma cells. Further analyses in BHK cells expressing a Xmrk chimera (HER-mrk) showed that p85 association with the intracellular part of Xmrk was dependent on autophosphorylation of the receptor. In vitro binding studies revealed that the interaction is mediated mainly through the N-terminal SH2 domain of p85 which directly binds to a sequence motif around phosphorylated Tyr-983 in the Xmrk carboxy-terminus. In accordance with recruitment of p85 by Xmrk in PSM cells, the PI3-kinase downstream target Akt was found to be highly phosphorylated on Ser-473, indicating efficient PI3-kinase signaling in melanoma cells. PI3-kinase activation was also detected in Xiphophorus melanoma. Moreover, malignant melanomas exhibited an increased level of PI3-kinase activity which was about three times higher than that in benign pigmented lesions. Inhibition of PI3-kinase activity in PSM melanoma cells by both Wortmannin and LY294002 blocked entry into S-phase. Together these data demonstrate that PI3-kinase is a substrate of the oncogenic Xmrk receptor and plays a significant role in mitogenic signaling of melanoma cells and the formation of malignant melanoma in Xiphophorus.     

Homology of Melanoma-Inducing Loci in the Genus Xiphophorus

Several species of the genus Xiphophorus are polymorphic for specific pigment patterns. Some of these give rise to malignant melanoma following the appropriate crossings. For one of these pattern loci from the playfish Xiphophorus maculatus the melanoma-inducing gene has been cloned and found to encode a novel receptor tyrosine kinase, designated Xmrk. Using molecular probes from this gene in Southern blot analyses on single fish DNA preparations from 600 specimens of different populations of various species of the genus Xiphophorus and their hybrids, either with or without melanoma-predisposing pattern, it was shown that all individuals contain the Xmrk gene as a proto-oncogene. It is located on the sex chromosome. All fish that carry a melanoma-predisposing locus which has been identified by Mendelian genetics contain an additional copy of Xmrk, closely linked to a specific melanophore pattern locus on the sex chromosome. The melanoma-inducing loci of the different species and populations are homologous. The additional copy of Xmrk obviously arose by a gene-duplication event, thereby acquiring the oncogenic potential. The homology of the melanoma-inducing loci points to a similar mechanism of tumor suppression in all feral fish populations of the different species of the genus Xiphophorus.     

Activation of phosphatidylinositol 3-kinase by a complex of p59fyn and the receptor tyrosine kinase Xmrk is involved in malignant transformation of pigment cells.

Malignant melanoma in the fish Xiphophorus is induced by overexpression of the Xmrk-oncogene, encoding a subclass I receptor tyrosine kinase. The mutationally activated Xmrk protein triggers constitutive mitogenic signalling in fish melanoma cells. In recent studies we showed that in melanoma cells phosphatidylinositol (PtdIns) 3-kinase, as well as p59fyn, has elevated levels of kinase activity. Both bind directly to different phosphotyrosine residues in the Xmrk receptor C-terminus through their SH2 domains. To analyse the mechanism of regulation of these Xmrk-associated kinases in melanoma we characterized the protein-protein interactions between PtdIns 3-kinase, p59fyn and the Xmrk receptor in detail. A ternary complex in which the p85 subunit of PtdIns 3-kinase is associated with p59fyn as well as with Xmrk was identified. Contrary to complexes described for other receptors, the adaptor protein p120Cbl was not involved in these interactions. Thus, we describe here a new mechanism of activation of PtdIns 3-kinase by a receptor of the epidermal growth factor receptor family in which p59fyn acts as an adaptor as well as an activator of PtdIns 3-kinase. Activation of PtdIns 3-kinase activity by fyn was also found in vivo. The fact that this was only detectable in highly transformed Xmrk overexpressing melanomas but not in benign lesions points to the essential role of the Xmrk receptor in this mechanism of regulation.     

Evolutionary Origin and Molecular Biology of the Melanoma-Inducing Oncogene of Xiphophorus

Melanoma formation in platyfish/swordtail hybrids of genus Xiphophorus is due to overexpression of the receptor tyrosine kinase oncogene Xmrk. This gene is the molecular equivalent to the Tu-locus of platyfish, formerly identified by Mendelian genetics. The supposed evolutionary origin of the Xmrk oncogene is a nonhomologous recombination event in the 5’region of the corresponding Xmrk protooncogene with an anonymous sequence, D. This event led to a gene duplication of Xmrk, whereby the new copy obtained a novel promoter derived from D. Inactivity of this promoter in parental fish warrants lack of tumorigenicity of the Xmrk oncogene in wild playfish. In hybrids, however, the promoter is active. This leads to the pigment cell transforming overexpression of Xmrk.     

Melanoma loss-of-function mutants in Xiphophorus caused by Xmrk-oncogene deletion and gene disruption by a transposable element.

The overexpression of the Xmrk oncogene (ONC-Xmrk) in pigment cells of certain Xiphophorus hybrids has been found to be the primary change that results in the formation of malignant melanoma. Spontaneous mutant stocks have been isolated that have lost the ability to induce tumor formation when crossed with Xiphophorus helleri. Two of these loss-of-function mutants were analyzed for genetic defects in ONC-Xmrk's. In the lof-1 mutant a novel transposable element, TX-1, has jumped into ONC-Xmrk, leading to a disruption of the gene and a truncated protein product lacking the carboxyterminal domain of the receptor tyrosine kinase. TX-1 is obviously an active LTR-containing retrotransposon in Xiphophorus that was not found in other fish species outside the family Poeciliidae. Surprisingly, it does not encode any protein, suggesting the existence of a helper function for this retroelement. In the lof-2 mutant the entire ONC-Xmrk gene was found to be deleted. These data show that ONC-Xmrk is indeed the tumor-inducing gene of Xiphophorus and thus the critical constituent of the tumor (Tu) locus.     

Interaction of Xiphophorus and murine Fyn with focal adhesion kinase

The Src family kinase/Focal Adhesion Kinase (FAK) complex is a signaling platform playing a crucial role in transformation downstream of oncogenic growth factor receptors. In the case of melanoma in Xiphophorus fish, the oncogenic EGF receptor orthologue Xiphophorus melanoma receptor kinase (Xmrk) effects continuous activation of the Src family kinase Fyn, but not of the other family members Src or Yes. Here, Fyn is strongly involved in promoting many tumorigenic events. Although Fyn is expressed in most mammalian tissues, there are only few reports of its involvement in the development of solid tumors. To find out whether the prominent role of Xiphophorus Fyn is based on an altered binding to its important binding partner FAK when compared to its mammalian Fyn counterparts, we performed yeast-two-hybrid analyses. We compared Xiphophorus and murine Fyn with respect to their binding to full-length and truncated FAK constructs. We found that interaction with FAK occurs similarly for Xiphophorus and mouse Fyn. Both phosphorylated FAK residue Y397 and FAK proline-rich domain are involved in Fyn binding. We also found interaction of FAK and Fyn in human melanoma cell lines. These data suggest a possible, yet unrecognized role of Fyn in the tumorigenesis of human melanoma, too.     

Identification of a second egfr gene in Xiphophorus uncovers an expansion of the epidermal growth factor receptor family in fish

The epidermal growth factor receptor (EGFR) gives name to a family of receptors formed by four members in mammals (EGFR, ErbB2, ErbB3, and ErbB4). Members of this family can be activated to become potent oncogenes, and many human and animal tumors express qualitatively or quantitatively altered receptors from this group. We have isolated and characterized a second egfr gene in the melanoma model fish Xiphophorus. Both Xiphophorus egfra and egfrb duplicates are co-orthologs of the mammalian egfr gene. Database analysis showed that not only egfr but also erbB3 and erbB4 are present as duplicates in some fish species. They originated from ancient duplication events that might be consistent with the hypothesis of a fish-specific genome duplication. In Xiphophorus, the egfrb gene underwent a second duplication that generated the melanoma-inducing oncogene Xmrk. The study and comparison of some of the functional characteristics of both Xiphophorus EGF receptors, including expression profile, ligand-binding abilities, and intracellular signal transduction revealed that Xiphophorus Egfra not only shares common features with Egfrb and the human EGFR but also shows significant differences in its functional characteristics. The mechanism of maintenance of these duplicates remains to be clarified.     

Cloning and gene map assignment of the Xiphophorus DNA ligase 1 gene

Fishes represent the stem vertebrate condition and have maintained several gene arrangements common to mammalian genomes throughout the 450 Myr of divergence from a common ancestor. One such syntenic arrangement includes the GPI-PEPD enzyme association on Xiphophorus linkage group IV and human chromosome 19. Previously we assigned the Xiphophorus homologue of the human ERCC2 gene to linkage group U5 in tight association with the CKM locus. CKM is also tightly linked to the ERCC2 locus on human chromosome 19, leading to speculation that human chromosome 19 may have arisen by fusion of two ancestral linkage groups which have been maintained in fishes. To investigate this hypothesis further, we isolated and sequenced Xiphophorus fish genomic regions exhibiting considerable sequence similarity to the human DNA ligase 1 amino acid sequence. Comparison of the fish DNA ligase sequence with those of other species suggests several modes of amino acid conservation in this gene. A 2.2-kb restriction fragment containing part of an X. maculatus DNA ligase 1 exon was used in backcross hybrid mapping with 12 enzyme or RFLP loci. Significant linkage was observed between the nucleoside phosphorylase (NP2) and the DNA ligase (LIG1) loci on Xiphophorus linkage group VI. This assignment suggests that the association of four DNA repair-related genes on human chromosome 19 may be the result of chance chromosomal rearrangements.      

Reorganization of the X chromosome in voles of the genus Microtus

Comparative chromosomal analysis is a powerful tool in the investigation of the mechanisms of chromosomal evolution. The accuracy of the analysis depends on the availability of region-specific markers to follow the fate of the particular chromosomal region through the evolution of species. We have assigned 12 unique sequences to the euchromatic part of the vole X chromosome, which serve as reliable markers of chromosomal segments. Together with region-specific libraries and GTG banding, these markers allow us to delineate the homologous regions of the X chromosomes in five species of the genus Microtus. We found that X chromosomes of these species differ by numerous rearrangements and all rearrangements are clustered at specific breakpoints. Moreover, these breakpoints were found to colocalise with repetitive and/or duplicated DNA sequences. We suggest that clusters of repeated and/or duplicated DNA sequences have played a crucial role in the formation of rearrangement hot spots during evolution of the X chromosome in the subgenus Microtus.     

Chromosome Rearrangement by Ectopic Recombination in Drosophila Melanogaster: Genome Structure and Evolution

Ectopic recombination between interspersed repeat sequences generates chromosomal rearrangements that have a major impact on genome structure. A survey of ectopic recombination in the region flanking the white locus of Drosophila melanogaster identified 25 transposon-mediated rearrangements from four parallel experiments. Eighteen of the 25 were generated from females carrying X chromosomes heterozygous for interspersed repeat sequences. The cytogenetic and molecular analyses of the rearrangements and the parental chromosomes show: (1) interchromosomal and intrachromosomal recombinants are generated in about equal numbers; (2) ectopic recombination appears to be a meiotic process that is stimulated by the interchromosomal effect to about the same degree as regular crossing over; (3) copies of the retrotransposon roo were involved in all of the interchromosomal exchanges; some copies were involved much more frequently than others in the target region; (4) homozygosis for interspersed repeat sequences and other sequence variations significantly reduced ectopic recombination.     

A Sex Chromosomal Restriction-Fragment-Length Marker Linked to Melanoma-Determining Tu Loci in Xiphophorus

In Xiphophorus, the causative genetic information for melanoma formation has been assigned by classical genetics to chromosomal loci, which are located on the sex chromosomes. In our attempts to molecularly clone these melanoma-determining loci, named Tu, we have looked for restriction-fragment-length markers (RFLMs) linked to the Tu loci. These RFLMs should be useful in obtaining a physical map of a Tu locus, which will aid in the cloning of the corresponding sequences. DNA samples from various Xiphophorus strains and hybrids including those bearing different Tu wild-type, deletion and translocation chromosomes, were screened for the presence of random RFLMs using homologous or heterologous sequences as hybridization probes. We find an EcoRI restriction fragment which shows limited crosshybridization to the v-erb B gene--but not representing the authentic c-erb B gene of Xiphophorus--to be polymorphic with respect to different sex chromosomes. Linkage analysis revealed that a 5-kb fragment is linked to the Tu-Sd locus on the X chromosome, a 7-kb fragment is linked to the Tu-Sr locus on the Y chromosome, both of Xiphophorus maculatus, and that a 12-kb fragment is linked to the Tu-Li locus on the X chromosome of Xiphophorus variatus. Using different chromosomal mutants this RFLM has been mapped to a frequent deletion/translocation breakpoint of the X chromosome, less than 0.3 cM apart from the Tu locus.