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.     

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.     

A microsatellite genetic linkage map for Xiphophorus

Interspecies hybrids between distinct species of the genus Xiphophorus are often used in varied research investigations to identify genomic regions associated with the inheritance of complex traits. There are 24 described Xiphophorus species and a greater number of pedigreed strains; thus, the number of potential interspecies hybrid cross combinations is quite large. Previously, select Xiphophorus experimental crosses have been shown to exhibit differing characteristics between parental species and among the hybrid fishes derived from crossing them, such as widely differing susceptibilities to chemical or physical agents. For instance, genomic regions harboring tumor suppressor and oncogenes have been identified via linkage association of these loci with a small set of established genetic markers. The power of this experimental strategy is related to the number of genetic markers available in the Xiphophorus interspecies cross of interest. Thus, we have undertaken the task of expanding the suite of easily scored markers by characterization of Xiphophorus microsatellite sequences. Using a cross between Xiphophorus maculatus and X. andersi, we report a linkage map predominantly composed of microsatellite markers. All 24 acrocentric chromosome sets of Xiphophorus are represented in the assembled linkage map with an average intergenomic distance of 7.5 cM. Since both male and female F1 hybrids were used to produce backcross progeny, these recombination rates were compared between "male" and "female" maps. Although several genomic regions exhibit differences in map length, male- and female-derived maps are similar. Thus Xiphophorus, in contrast to zebrafish, Danio rerio, and several other vertebrate species, does not show sex-specific differences in recombination. The microsatellite markers we report can be easily adapted to any Xiphophorus interspecies and some intraspecies crosses, and thus provide a means to directly compare results derived from independent experiments.     

Xiphophorus interspecies hybrids as genetic models of induced neoplasia

Fishes of the genus Xiphophorus (platyfishes and swordtails) are small, internally fertilizing, livebearing, and derived from freshwater habitats in Mexico, Guatemala, Belize, and Honduras. Scientists have used these fishes in cancer research studies for more than 70 yr. The genus is presently composed of 22 species that are quite divergent in their external morphology. Most cancer studies using Xiphophorus use hybrids, which can be easily produced by artificial insemination. Phenotypic traits, such as macromelanophore pigment patterns, are often drastically altered as a result of lack of gene regulation within hybrid fishes. These fish can develop large exophytic melanomas as a result of upregulated expression of these pigment patterns. Because backcross hybrid fish are susceptible to the development of melanoma and other neoplasms, they can be subjected to potentially deleterious chemical and physical agents. It is thus possible to use gene mapping and cloning methodologies to identify and characterize oncogenes and tumor suppressors implicated in spontaneous or induced neoplasia. This article reviews the history of cancer research using Xiphophorus and recent developments regarding DNA repair capabilities, mapping, and cloning of candidate genes involved in neoplastic phenotypes. The particular genetic complexity of melanoma in these fishes is analyzed and reviewed.     

Proteomic analyses of the Xiphophorus Gordon-Kosswig melanoma model

Interspecies hybridization between the platyfish X. maculatus Jp 163 A, and the swordtail X. helleri (Sarabia), generates F(1) hybrids with pronounced melanin pigmentation. Backcrossing of F(1) hybrids with the X. helleri parent results in 25% of progeny that will spontaneously develop melanoma. We have applied proteomic methods to this Gordon-Kosswig (G-K) melanoma model to identify candidate proteins that exhibit modulated expression in fin tissue due to interspecies hybridization and progression of hybrid tissues to spontaneous melanoma. Difference Gel Electrophoresis (DIGE) was used to minimize the variability commonly observed in quantitative analyses of comparative protein samples. Following identification of up- or down-regulated protein expression by DIGE, candidate protein spots were identified by mass spectrometric sequencing. Several protein expression differences displayed in interspecies hybrids were identified and compared to distinct differences that occur upon backcrossing and progression to melanoma. These studies are important for the identification of distinct biochemical pathways involved in the variety of Xiphophorus interspecies hybrid tumor models.     

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.     

An examination of hybridization between the cattail species typha latifolia and typha angustifolia using random amplified polymorphic DNA and chloroplast DNA markers

Typha glauca represents a significant portion of the biomass of the wetlands surrounding the Great Lakes, USA. It is generally accepted to be a form of hybrid between T. latifolia and T. angustifolia, which itself appears to be an exotic introduction from Europe. Based on morphological and isozyme data, conflicting theories have been proposed for the hybrid nature of T. glauca: it has been described as a hybrid swarm, a distinct hybrid species and an F1 hybrid. Therefore, we developed random amplified polymorphic DNA (RAPD) and chloroplast DNA markers, specific to the parental species, to assess hybrids. Ten RAPD primers gave 17 fragments specific to T. angustifolia and 13 fragments specific to T. latifolia. All of the interspecific hybrids contained each of the species-specific markers, indicating an F1 hybrid status. Furthermore, all hybrids tested contained the T. angustifolia chloroplast haplotype, which is consistent with differential interspecific crossing success found previously. Additional confirmation of an F1 hybrid status was gained by examining seedlings from T. glauca. These progeny were expected to be advanced-generation hybrids, as opposed to the F1 hybrid parent. Analysis of the seedlings revealed segregating marker patterns consistent with patterns observed in experimental advanced-generation hybrids, although these advanced hybrids do not appear to be a significant part of mature stands. Our data do not provide support for extensive gene flow between T. latifolia and T. angustifolia. However, our results suggest that hybridization between the native and introduced Typha species has impacted the native population through the spread of the F1 hybrid, T. glauca.     

The use of inter-specific hybrids in aquaculture and fisheries

Inter-specific hybrid fishes have been produced for aquaculture and stocking programmes to increase growth rate, transfer desirable traits between species, combine desirable traits of two species into a single group of fishes, reduce unwanted reproduction through production of sterile fish or mono-sex offspring, take advantage of sexual dimorphism, increase harvestability, increase environmental tolerances, and to increase overall hardiness in culture conditions. Hybrids constitute a significant proportion of some countries' production for certain taxa; for example, hybrid striped bass in the USA, hybrid clarid catfish in Thailand, hybrid characids in Venezuela, and hybrid tilapia in Israel. Despite its widespread use, there is a general impression that inter-specific hybridization is not a very useful tool for aquaculture. We believe this impression stems from inaccurate reporting of some useful hybrids, limited testing of strains used for hybrids, and from early work on salmonids that did not result in hybrids of commercial advantage.Experimentation with new hybrid fishes is ongoing, especially in marine culture systems where sterile fish may be preferred because of the concern that fish may escape into the marine and coastal environment.Hybridization has been used in tandem with polyploidization to improve developmental stability in hybrid progeny. The results of inter-specific hybridization can be variable and depend on the genetic structure (including the sex) of the parent fish. Inadvertent hybridization and backcrossing can lead to unexpected and undesirable results in hybrid progeny, such as failure to produce sterile fish, loss of color pattern, and reduced viability.Hybridization is only one tool to improve aquaculture production and will require knowledge of the genetic structure of the broodstock, good broodstock management and monitoring of the viability and fertility of the progeny. Hybridization does represent a genetic modification wherein genes are moved between different species; implications for biodiversity conservation and regulation of this type of modification are discussed.     

Replicated hybrid zones of Xiphophorus swordtails along an elevational gradient

Natural hybrid zones provide opportunities to study a range of evolutionary phenomena from speciation to the genetic basis of fitness-related traits. We show that widespread hybridization has occurred between two neo-tropical stream fishes with partial reproductive isolation. Phylogenetic analyses of mitochondrial sequence data showed that the swordtail fish Xiphophorus birchmanni is monophyletic and that X. malinche is part of an independent monophyletic clade with other species. Using informative single nucleotide polymorphisms in one mitochondrial and three nuclear intron loci, we genotyped 776 specimens collected from twenty-three sites along seven separate stream reaches. Hybrid zones occurred in replicated fashion in all stream reaches along a gradient from high to low elevation. Genotyping revealed substantial variation in parental and hybrid frequencies among localities. Tests of F(IS) and linkage disequilibrium (LD) revealed generally low F(IS) and LD except in five populations where both parental species and hybrids were found suggesting incomplete reproductive isolation. In these locations, heterozygote deficiency and LD were high, which suggests either selection against early generation hybrids or assortative mating. These data lay the foundation to study the adaptive basis of the replicated hybrid zone structure and for future integration of behaviour and genetics to determine the processes that lead to the population genetic patterns observed in these hybrid zones.     

Microsatellite analysis as a tool for discriminating an interfamily hybrid between olive flounder and starry flounder

An interspecific artificial hybrid was produced between two economically important aquaculture flatfish: olive flounder (Paralichthys olivaceus) and starry flounder (P. stellatus). This hybrid displays the rapid growth characteristic of the former and tolerance to low temperatures and low salinity of the latter, but the genetics of inheritance in this hybrid have not been elucidated. Polymorphic microsatellite markers developed for P. olivaceus and P. stellatus were tested to determine if these markers can be used for analysis of parentage and genetic inheritance. Multiplex PCR using two primer sets that were specific to each species produced PCR products of different sizes; these could be used for the identification of interspecific hybrids. Among the 192 primers derived from olive flounder, 25.5% of the primer sets successfully amplified genomic DNA from starry flounder, and 23% of the 56 primer sets originating from starry flounder amplified DNA from olive flounder. Analysis of genetic inheritance in the hybrid using seven of the 62 microsatellite markers common to both species demonstrated classic Mendelian inheritance of these markers in the hybrid progeny, with the exception of one locus identified as a null allele in the hybrid. These results demonstrate that cross-specific microsatellite markers can be used tools for parentage analysis of hybrid flatfish, for mapping quantitative trait loci, for marker-assisted selective breeding, and for studies of the evolution of fish.     

Factors enforcing the species boundary between the human pathogens Cryptococcus neoformans and Cryptococcus deneoformans

Hybridization has resulted in the origin and variation in extant species, and hybrids continue to arise despite pre- and post-zygotic barriers that limit their formation and evolutionary success. One important system that maintains species boundaries in prokaryotes and eukaryotes is the mismatch repair pathway, which blocks recombination between divergent DNA sequences. Previous studies illuminated the role of the mismatch repair component Msh2 in blocking genetic recombination between divergent DNA during meiosis. Loss of Msh2 results in increased interspecific genetic recombination in bacterial and yeast models, and increased viability of progeny derived from yeast hybrid crosses. Hybrid isolates of two pathogenic fungal Cryptococcus species, Cryptococcus neoformans and Cryptococcus deneoformans, are isolated regularly from both clinical and environmental sources. In the present study, we sought to determine if loss of Msh2 would relax the species boundary between C. neoformans and C. deneoformans. We found that crosses between these two species in which both parents lack Msh2 produced hybrid progeny with increased viability and high levels of aneuploidy. Whole-genome sequencing revealed few instances of recombination among hybrid progeny and did not identify increased levels of recombination in progeny derived from parents lacking Msh2. Several hybrid progeny produced structures associated with sexual reproduction when incubated alone on nutrient-rich medium in light, a novel phenotype in Cryptococcus. These findings represent a unique, unexpected case where rendering the mismatch repair system defective did not result in increased meiotic recombination across a species boundary. This suggests that alternative pathways or other mismatch repair components limit meiotic recombination between homeologous DNA and enforce species boundaries in the basidiomycete Cryptococcus species.     

Etiology of MNU-induced melanomas in Xiphophorus hybrids

Genetic hybrids of the genus Xiphophorus have historically been useful models for study of the genetic aspects of tumor formation. In the most studied Xiphophorus tumor model, two-gene loci, XMRK and DIFF, are implicated as critical both to UV-induced and spontaneous melanoma formation in BC(1) hybrids of crosses between X. maculatus and X. helleri, with X. helleri as the recurrent backcross parent. In addition to UV, the direct-acting carcinogen N-methyl-N-nitrosourea (MNU) has been used to induce tumors in Xiphophorus BC(1) hybrids from several cross types. In the present study, we address the hypothesis that excess melanomas in MNU-treated BC(1) hybrids may have been generated by direct mutation of CDKN2AB, a candidate gene for DIFF. MNU treatment of F(1) and BC(1) hybrid fish significantly increased tumor incidence at 6 months; however, no association was found between MNU-induced tumor formation and zygosity of the candidate tumor tumor-suppressor CDKN2AB in BC(1) hybrids, consistent with previously reported results. Sequence analysis of the X. maculatus CDKN2AB locus of heterozygous individuals (both BC(1) and F(1) hybrids) did not reveal any mutations caused by MNU, suggesting that the mechanism of MNU-induced melanoma formation in this Xiphophorus model does not involve direct mutation of CDKN2AB but may result from mutation of other critical genes.     

The synergistic effect of X-rays and deficiencies in DNA repair in P-M hybrid dysgenesis in Drosophila melanogaster.

X-rays and deficiencies in DNA repair had a synergistic effect on genetic damage associated with P-element mobility in Drosophila melanogaster. These interactions, using sterility and fecundity as endpoints, were tested in dysgenic males deficient in either excision or post-replication DNA repair. Three sublines of the Harwich P strain were used for the construction of hybrid males. These sublines differ in P-induction ability based on gonadal dysgenesis sterility (GD) and snw mutability tests, in P-element insertion site pattern, and in the types of defective P-elements, such as KP elements, they possess. A lower degree of gonadal dysgenesis was correlated with the presence of KP elements. GD sterility and snw mutability were not always correlated. Dysgenic hybrids originating from the standard reference subline, Harwich(white), were much more sensitive to the post-replication repair than the excision repair defect. In contrast, sterility of hybrids derived from the weak subline was least affected by, and that of hybrids of the strongest subline was most affected by either DNA repair deficiency. The exacerbation by X-rays of the effects of DNA repair deficiencies on genetic damage indicates that both repair mechanisms are required for processing DNA lesions induced by the combined effect of P activity and ionizing radiation.     

Linkage group VI of fish of the genus Xiphophorus (Poeciliidae): Assignment of genes coding for glutamine synthetase,uridine monophosphate kinase,and transferrin

Electrophoretic variation ascribable to three protein-coding loci, coding for glutamine synthetase (GS), uridine monophosphate kinase (UMPK), and transferrin (Tf), was observed in three species of fish of the genus Xiphophorus. Electrophoretic patterns in interspecific F1 hybrid heterozygotes suggested monomeric subunit structures of UMPK and Tf and a multimeric structure of undetermined subunit number of GS. Linkage analyses in backcross hybrids indicated a recombination map of GS-0%-Tf-10.8%-UMPK. This group (designated Xiphophorus linkage group VI) was shown to assort independently from the 14 enzyme loci assigned to linkage groups I-V and from 19 other informative markers within the limits of the data.     

Analysis of an esterase linked to a locus involved in the regulation of the melanoma oncogene and isolation of polymorphic marker sequences inXiphophorus

Melanoma formation in Xiphophorus hybrids is mediated by a growth factor receptor tyrosine kinase oncogene encoded by the Tu locus. In the wild-type parental fish no tumors occur due to the activity of a locus that regulates the activity of the melanoma oncogene. Molecular identification of this regulatory locus (R) requires a precise physical map of the chromosomal region. Therefore we studied esterase isozymes in Xiphophorus, two of which have been previously reported to be linked to locus R. We confirm that ES1 is a distant marker for R (approx. 30cM), and contrary to earlier studies, we show that this isozyme is present in all species of the genus and at similar activity levels in all organs tested. ES4, which has also been reported to be linked to R, was found to be a misclassification of liver ES1. In an attempt to identify markers that bridge the large distance between ES1 and R, we have generated DNA probes which are highly polymorphic. They will be useful in finding landmarks on a physical map of the R-containing chromosomal region.     

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.      

Amaranthus hybridus can be pollinated frequently by A. tuberculatus under field conditions

Recent studies have confirmed that weedy Amaranthus species are capable of interspecific hybridization, and such hybridization may foster the evolution of herbicide resistance. However, the extent to which hybridization among these species occurs in nature is unknown. The purpose of this study was to determine the frequency under field conditions at which A. hybridus, a monoecious and predominantly self-pollinated species, would be pollinated by A. tuberculatus, a dioecious species. To do this, parents carrying different alleles at the ALS locus, which encodes a herbicide target site, were used. Male A. tuberculatus parents were homozygous for a dominant herbicide-insensitive allele, while A. hybridus parents were homozygous for a sensitive form. Hybrid progeny therefore could be detected via herbicide selection. Mean hybridization frequencies between 0.4 and 2.3% were obtained, depending on the proximity between parents (P=0.02). The robustness of the hybrid selection assay was verified using a molecular marker and DNA content analyses. Using these techniques, more than 99% of the progeny that survived the herbicide were confirmed to be hybrids. Frequencies obtained in this study were many times higher than the generally expected rate of mutation. Therefore, even minimal fertility in hybrid progeny would support the view that hybridization could play a role in adaptive evolution of weedy Amaranthus species.