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Several species of the genus Xiphophorus are polymorphic for specific pigment patterns. Same of these give rise to malignant melanoma following the appropriate crossings. For one of these pattern Iod from the platyfish Xiphophorus maculatus the melanoma-inducing gene has been doned 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 melanomapredisposing 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 geneduplication event, thereby acquiring the oncogenic potential. The homology of the melanomainducing Iod points to a similar mechanism of tumor suppression in all feral fish populations of the different species of the genus Xiphophorus.
Malignant melanomas (MM) in the fish Xiphophorus and in humans were studied both by transmission electron microscopy (TEM) and freeze-etching (FE). In both fish and human melanomas the cells show interdigitations of the,plasma membranes. The nuclei are large and lobulated and have many nuclear pores. Melanosomes are abundant and melanosome complexes ("compound melanosomes") occur regularly. Pinocytotic vesicles could be demonstrated in fish and human melanomas showing iocal differences in frequency and distribution patterns in the tumor. lntercellular junctions are lacking in MM cells from fish and humans. The FE technique showed considerable advantages in demonstrating membrane-surface peculiarities such as nuclear pores or pinocytotic vesicles. The FE replicas of fish melanomas are like those of humans. These findings may support the hypothesis that melanoma in fish and humans reflect the same biological phenomenon.
The observation of a slower migrating form of pp6oc-src in neural tissue of chicken and mouse has recently been shown to be due to an alternative transcript form of tbe c-src gene (Martinez et al.: Science 237:411-415, 1987; Levy et al.: Mol Cell Bio17:4142- 4145, 1987). An insertion of 18 basepairs between exons 3 and 4, presumed to be due to alternative splicing of a mini-exon, gives rise to six amino acid residues not found in the non-neuronal (termed flbroblastic) form of pp60\(^{c-src}\). Wehave addressed the question of the evolutionary origin of the c-src neuronal insert · and its functional signiflcance regarding neural-speciflc expression of the c-src gene. To this end we have investigated whether the c-src gene of a lower verlebrate (the teleost fish Xiphophorus) gives rise to a neural-specific transcript in an analogous manner. We could show that the fish c-src gene does encode for a "fibroblastic" and a "neuronal" form of transcript and that the neuronal transcript does indeed arise by way of alternative splicing of a mini-exon. The miniexon is also 18 basepairs long and we could demoostrate directly that this exon lies within the intron separating exons 3 and 4. For comparative purposes we have examined whether the fish c-yes gene, the member of the src gene family most closely related to c-src, also encodes a neural tissue-specific transcript. No evidence for a second transcript form in brain was obtained. This result suggests that the mini-exon arose within the c-src gene lineage sometime between the srclyes gene duplication event and the divergence of the evolutionary lineage giving rise to the teleost fish. Published genomic sequence of src-related genes in Drosophila and our own results with Hydra demoostrate no intron in these species at the analogous location, consistent with first appearance of this mini-exon sometime between 550 and 400 million years ago.
Animal sex chromosome evolution has started on different occasions with a homologous pair of autosomes leading to morphologically differentiated gonosomes. In contrast to other vertebrate classes, among fishes cytologically dernonstrahle sex chromosomes are rare. In reptiles, certain motifs of simple tandemly repeated DNA sequences like (gata)\(_n\)/(gaca)\(_m\) are associated with the constitutive heterochromatin of sex chromosomes. In this study a panel of simple repetitive sequence probes was hybridized to restriction enzyme digested genomic DNA of poeciliid fishes. Apparent male heterogamety previously established by genetic experiments in Poecilia reticulata (guppy) was correlated with male-specific hybridization using the (GACA)\(_4\) probe. The (GATA)\(_4\) oligonucleotide identifies certain male guppies by a Y chromosomal polymorphism in the outbred population. In cantrast none of the genetically defined heterogametic situations in Xiphophorus could be verified consistently using the collection of simple repetitive sequence probes. Only individuals from particular populations produced sex-specific patterns of hybridization with (GATA)\(_4\). Additional poeciliid species (P. sphenops, P. velifera) harbour different sex-specifically organized simple repeat motifs. The observed sex-specific hybridization patterns were substantiated by banding analyses of the karyotypes and by in situ hybridization using the (GACA)\(_4\) probe.
The demonstration ofthe chromosomal mode ofsex determinationvia genetic experiments as well as the absence of heteromorphic sex chromosomes affirm poeciliid fishes as a unique group among vertebrates that are endowed with the mostprimitive form of sex chromosornes. In many different taxa the evolutionary process involved in the differentiation ofadvanced sex chromosomes is outlined through sex specifically organized repetitive sequences. In this investigation hydridization of synthetic probes specific to genomic simple repeat motifs uncovers a sex-specific hybridization pattern in certain viviparaus fishes ofthe family Poeciliidae. The hybridization pattern together with specific staining ofthe constitutive heterochromatin by C-banding reveals heterogamety in males (Poecilia reticulata) as weil as in females (P. sphenops). In P. velifera, however, C-banding alone fails to unravel the heterogametic status. The female specific W-chromosome can be detected by simple repetitive sequence probes. Therefore, the principal significance of heterochromatization as a means of generating differentiated sex chromosomes is evident.
In order to study the divergence of teleost sex chromosomes, subtractive cloning was carried out between genomic DNA ofmales and females ofthe rainbow trout (XX/XY) and of Leporinus elongatus (ZW /ZZ). Inserts cloned in a plasmid vector were individually tested on Southern blots of DNA of males and females for sex specificity. No sex-specific insert was obtained from trout, but two out of ten inserts cloned from L. elongatus showed sex-specific patterns in this species: one corresponds to a sequence present on both Z and W chromosomes, while the other is W specific. Sequences of these two inserts show neither clear homology with other known sequences, nor an open reading frame. They cross-hybridize with the genomic DNA of Leporinusfriderici, but without sex-specific patterns. Twenty-four L. elongatus adults were sexed by gonadal observation, chromosomed examination and Southern hybridization with one or the other insert. Ten males and 11 females had chromosomes and hybridization patterns typical of their sex. One ZW female was recognized as a male with the W-specific probe. This was also the case for two unusual ZW males, one having a male hybridization pattern with the other probe. These three atypical individuals may result from single genetic exchanges between four regions of the Z and the W, giving rise to three atypical W chromosomes. Finding males with such atypical heterochromosomes in a female heterogametic species may indicate that a gradual transition occurs between the heterogametic systems.
Hereditary melanoma in Xiphophorus hybrids canying the melanoma·induclng Tu-Sd locus is caused by transcriptional activation of the Xmrk gene that resides at the Tu·Sd locus and encodes a novel member of receptor tyrosine kinases (RTK). ln this study, a total of 17 hereditary melanomas from various hybrid genotypes harbouring 7 different Tu alleles were also found to aver-express the correspondlng Xmrlc alleles. The Ievei of over-expression correlated with the degree of malignancy of the melanoma. ln addition, Xsrc expression was high ln many malignant melanomas. Expression pattems and Ieveis of the Xiphophorus EGF-receptor gene (Xerb B), the c-myc (Xmyc), and the PDGF (Xsls) gene(s) were not intriguing. Transcription of the ras gene(s) may be correlated to secondary events of melanoma progression. Expression pattems of Xfms, the Xiphophorus CSF-1 receptor homologue, can be explained by different contents of infiltrating macrophages in the tumors. ln carcinogen-induced tumors includlng one melanoma no significant expression of the Xmrk oncogene could be detected. Xsrc expression, however, was strikingly high. This indicates that activation of oncogenes other than Xmrk ls instrumental in tumorigenesls of neoplasia of non-hereditary origin.
DARWIN\(^1\) believed that sexual selection accounts for the evolution of exaggerated male ornaments, such as the sword-like caudal fin extensions of male fishes of the genus Xiphophorus, that appear detrimental to survival. Swordtails continue to feature prominently in empirical work and theories of sexual selection; the pre-existing bias hypothesis has been offered as an explanation for the evolution of swords in these fishes\(^{2,3}\). Based upon a largely morphological phylogeny, this hypothesis suggests that female preference to mate with sworded males arose in ancestrally swordless species, thus pre-dating the origin of the sword itself and directly driving its evolution. Here we present a molecular phylogeny (based on mitochondrial and nuclear DNA sequences) of Xiphophorus which differs from the traditional one: it indicates that the sword originated and was lost repeatedly. Our phylogeny suggests that the ancestor of the genus is more likely to have possessed a sword than not, thus questioning the applicability of the pre-existing bias hypothesis as an explanation for the cvolution of this sexually selected trait.
The melanoma·inducing gene of Xiphophorus fish encodes the Xmrk receptor tyrosine kinase. U sing a highly specific antiserum p~oduced against the recombinant receptor expressed with a baculovirus, it is shown that Xmrk is the most abundant phosphotyrosine protein in fish melanoma and thus highly activated in the tumors. Studies on a melanoma cellline revealed that these cells produce an activity that considerably stimulates receptor autophosphorylation. The stimulating activity induces receptor down-regulation and can be depleted from the melanoma cellsupernatant by the immobilized recombinant receptor protein. The fish melanoma cells can thus be considered autocrine tumor cells providing a source for future purification and characterization of the Xmrk ligand.
The promoter of the rainbow trout metallothionein B gene ( tMTb) was isolated from genomic DNA by the polymerase chain reaction (PCR ), fused to the bacterial chloramphenicol acetyltransferase (CAT) genein an expression vector, and functionally analyzed in one human cellline and four fish celllines. This promoter exhibited an extremely low basal expression in all celllines and was zincand cadmium-inducible except in the fish melanoma cell line where the promoter was completely inactive. The metal-induced expression patterns were cellline-specific. In general the fish promoter was more species- and cell type-specific than its human counterpart. In a transient assay it was functional in developing embryos of the medaka ( Oryzias /atipes). These properties make this promoter suitable for inducible, tissue-specific expression of transgenes and for in vivo studies of gene function and regulation.
The silver carp (Hypophthalmichthys molitrix) growth hormone (GH) genewas isolated and sequenced following amplification from genomic DNA by the polymerase chain reaction. The gene spans a region of approx. 2.5 kb nucleotides (nt) and consists of five exons. The sequence predicts a polypeptide of 210 amino acids (aa) including a putative signal peptide of 22 hydrophobic aa residues. The arrangement of exons and introns is identical to the GH genes of common carp, grass carp, and very similar to mammals and birds, but quite different from that for the GH genes of tilapia and salmonids. The silver carp GH gene shares a high homology at the nt and aa Ievels with those of grass carp (95.3% nt, 99.5% aa) and of common carp (81% nt, 95.7% aa).
To investigate the regulation of metallothionein-encoding genes in fish, we have isolated and sequenced the rainbow trout metallothionein-A-encoding gene (tMT-A) by polymerase chain reaction. This gene spans about 1.1 kb, consists of three exons and two introns, and has an A+ T-rieb 5' -region which contains a TATAAA signal, and two metal responsive elements (MREs). The transcription start point is centered around an A residue 81 nt upstream of the ATG codon.