Perturbation of DNA repair gene expression due to interspecies hybridization.

The effect of interspecies hybridization on gene regulation was examined using real-time polymerase chain reaction (RT-PCR) to measure the expression of five base-excision repair genes in brain, eye, gill, liver, and tailfin tissues from Xiphophorus parental species and F(1) hybrids. Relative mRNA levels of uracil N-glycosylase (Ung), Apurinic/apyrimidinic endonuclease (Ape1), polymerase-beta (Polb), flap endonuclease (Fen1), and DNA ligase (Lig1) were measured in three parental Xiphophorus species (X. maculatus Jp 163 B, X. helleri Sarabia, and X. andersi andC) and in two interspecies F(1) hybrids, the Sp-helleri hybrid (X. maculatus Jp 163 BxX. helleri Sarabia) and the Sp-andersi hybrid (X. maculatus Jp 163 BxX. andersi) to identify genes that undergo changes in expression levels upon interspecies hybridization. Significant differences in gene expression were observed between parental animals and their respective F(1) hybrids in both interspecies crosses. Generally, marked increases in DNA repair gene mRNA levels were observed across all tissues in F(1) hybrid animals from the Sp-helleri cross compared to either X. maculatus or X. helleri parents. In contrast, the Sp-andersi F(1) hybrid animals generally exhibited decreased base-excision repair gene expression, although this trend was more specific to individual tissues than observed for Sp-helleri hybrids.

Cloning of JunA and JunB and comparison of mRNA expression levels in two Xiphophorus melanoma models.

The cloning and mRNA expression analysis of Xiphophorus maculatus JunA and JunB proto-oncogenes (designated X-JunA and X-JunB, respectively) is described. In mammals, JunA and JunB proteins make up the activator protein-1 (AP-1) transcription factor with related Fos proteins. The deduced amino acid sequences of X-JunA and X-JunB exhibit moderate degrees of similarity when compared to their human homologues, while the regions considered functionally critical, namely, the transactivation domains, DNA-binding domain, and the leucine zipper, are highly conserved. X-JunA and X-JunB mRNA expression levels in six X. maculatus Jp 163 A tissues were assayed by real-time RT-PCR. In addition, X-JunA and X-JunB mRNA levels are compared in skin and tumor tissues derived from two distinct Xiphophorus backcross hybrid tumor models, one of which develops melanoma spontaneously, whereas the other requires induction via UVB exposure for melanoma development. X-JunB mRNA expression was higher than X-JunA expression in tissues from X. maculatus parental animals. X-JunB was also more highly expressed than X-JunA in both spontaneous and induced melanoma tissue and nonmelanotic skin tissue. However, X-JunA mRNA levels were significantly higher in the spontaneous melanomas compared to melanomas induced by UVB exposure. The authors speculate that these findings may indicate that JunA regulation is affected by regulatory differences between the two melanoma model systems.

DNA polymerase beta mRNA and protein expression in Xiphophorus fish.

Herein we report Xiphophorus DNA polymerase beta (XiphPolbeta) mRNA and protein expression levels in brain, liver, gill, and testes tissues from Xiphophorus maculatus, Xiphophorus helleri, and Xiphophorus couchianus parental line fish and two different tumor-bearing Xiphophorus interspecies hybrids. Polymerase beta protein levels in the Xiphophorus tissues were measured by Western blot, and mRNA was measured with a quantitative real time RT-PCR method which employed cRNA construction to produce accurate calibration curves. We found significant differences in both mRNA and protein levels between the tumor-bearing hybrid animals and the three parental species. However, there were no significant differences in either mRNA levels or protein expression observed between the parental species. Thus, interspecies hybridization results in dysregulation of Polbeta expression and this may manifest a modulation in DNA repair capability and susceptibility to latent tumorigenesis.

Gene structure, purification and characterization of DNA polymerase beta from Xiphophorus maculatus.

Cloning of the Xiphophorus maculatus Polbeta gene and overexpression of the recombinant Polbeta protein has been performed. The organization of the XiphPolbeta introns and exons, including intron-exon boundaries, have been assigned and were found to be similar to that for human Polbeta with identical exon sizes except for exon XII coding for an additional two amino acid residues in Xiphophorus. The cDNA sequence encoding the 337-amino acid X. maculatus DNA polymerase beta (Polbeta) protein was subcloned into the Escherichia coli expression plasmid pET. Induction of transformed E. coli cells resulted in the high-level expression of soluble recombinant Polbeta, which catalyzed DNA synthesis on template-primer substrates. The steady-state Michaelis constants (Km) and catalytic efficiencies (kcat/Km) of the recombinant XiphPolbeta for nucleotide insertion opposite single-nucleotide gap DNA substrates were measured and compared with previously published values for recombinant human Polbeta. Steady-state in vitro Km and kcat/Km values for correct nucleotide insertion by XiphPolbeta and human Polbeta were similar, although the recombinant Xiphophorus protein exhibited 2.5-7-fold higher catalytic efficiencies for dGTP and dCTP insertion versus human Polbeta. In contrast, the recombinant XiphPolbeta displayed significantly lower fidelities than human Polbeta for dNTP insertion opposite a single-nucleotide gap at 37 degrees C.

Characterization and purification of flap endonuclease-1 (xiFEN-1) from Xiphophorus maculatus.

The cloning, gene structure, and expression of flap endonuclease-1 (xiFEN1) from Xiphophorus maculates are presented. The xiFEN1 gene structure was found to include 8 exons and 7 introns. The Xiphophorus FEN1 cDNA sequence contained an open reading frame that encoded a 380 amino acid protein with a predicted mass of 43 kDa. The intact FEN1 cDNA was subcloned into a bacterial expression vector (pET101-xiFEN1ct) and recombinant xiFEN1 enzyme purified from E. colicell extracts. The pET101-xiFEN1ct translation product was a 3' fusion protein with a ~3 kDa vector-encoded carboxy terminal extension designed to facilitate protein recognition and purification. The xiFEN1 fusion protein was purified and its amino acid sequence verified by Western blot analysis and tryptic peptide mass fingerprinting. The purified recombinant protein was assessed for enzyme specificity using several different oligonucleotide substrates having select flap overhangs. Also reported are Michaelis steady state kinetic values of enzymatic activity for the xiFEN1 directly compared with human FEN1 activity. xiFEN1 displayed a five-fold greater Km and six-fold lower catalytic efficiency (kcat/Km) than observed for the hFEN1.

Structural organization, mapping, characterization and evolutionary relationships of CDKN2 gene family members in Xiphophorus fishes.

Xiphophorus fishes and their hybrids are used as models for the study of melanoma and other diseases. The cyclin-dependent kinase inhibitor gene family in humans is comprised of four members, including CDKN2A (P16), and dysregulation of this gene is implicated in numerous neoplasms including melanomas. We have investigated the status of the gene family in the southern platyfish X. maculatus. Xiphophorus harbors at least two such loci, which we now term CDKN2A/B and CDKN2D. Both loci map to Xiphophorus linkage group 5, a genomic area that has long been known to harbor the DIFF tumor suppressor locus. Within this report, we report on the complete cloning, genomic exon/intron boundary delineation, linkage mapping and expressional characteristics of Xiphophorus CDKN2D. We also compare and contrast this expression to that of the previously isolated CDKN2AB locus in normal and neoplastic tissues derived from non-hybrid and hybrid fishes. The hypothetical evolutionary relationships of gene family members and their involvement in melanoma is evaluated. In comparison to CDKN2A/B, the RNA expression of Xiphophorus CDKN2D differs in normal tissues and is not associated with melanotic/pathologic tissues, confirming functional divergence between obvious homologues.

The genetic map of Xiphophorus fishes represented by 24 multipoint linkage groups.

Hybrids between distinct Xiphophorus species have been utilized for over 70 years to study melanoma and other neoplasms that can develop spontaneously in hybrid offspring. Genetic linkage mapping has proven to be important in delineating genomic areas that harbor oncogenes and tumor suppressors. Within this report, two parallel backcrosses have been utilized to generate a genetic linkage map for Xiphophorus fishes. Isozyme/allozyme, RFLP and PCR-based mapping techniques, including AP-PCR/RAPDs and microsatellite loci were utilized. The derived linkage map provides a total of 403 mapped polymorphisms distributed among 24 linkage groups, representative of 24 acro- and telocentric chromosome pairs. Genomic coverage is approximately one marker per 5.8 cM. Detailed genotypic analysis of the utilized hybrids revealed two areas of the genome that show significant segregation distortion. Loci within the linkage group harboring the sex determining locus (LG 24) and an autosomal linkage group (LG 21) show highly significant deviations from Mendelian expectations. This phenomenon is not present in a hybrid cross that utilizes a different backcross hybrid progenitor species. The derived map with sequence-tagged markers provides a framework for physical map generation, large-scale genomic sequencing and will further enable cross-genome comparisons of vertebrate genomes.