ABSTRACT
Small aquarium fishes become increasingly important in the study of normal vertebrate development and disease. Differential DNA methylation might play a role in these processes. In the teleost Xiphophorus, a well-established animal model for melanoma formation, tumour-specific hypomethylation of the melanoma-inducing gene ONC-Xmrk has been observed. We have isolated a cDNA for the DNA-(cytosine-5)-methyltransferase XDNMT-1 from this organism, which encodes the first full-length protein from a fish species. Linkage analysis showed that Xdnmt-1 is different from the Xiphophorus tumour suppressor R, which is involved in the transcriptional repression of the ONC-Xmrk melanoma oncogene in healthy fish. As methylation has been implicated in the regulation of ONC-Xmrk expression, XDNMT-1 might play a role by acting up- or downstream of R. Expression analysis demonstrated that the Xdnmt-1 transcript is present in all adult tissues and cell lines tested. However, developing embryos show a spatially and temporally regulated expression pattern suggesting that the enzyme might play a role during development in fish.
Subject(s)
Cyprinodontiformes/genetics , DNA (Cytosine-5-)-Methyltransferases/genetics , Fish Proteins , Animals , Cell Line , Cyprinodontiformes/embryology , DNA/genetics , DNA, Complementary/chemistry , DNA, Complementary/genetics , Embryo, Nonmammalian/metabolism , Embryonic Development , Female , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Genetic Linkage , In Situ Hybridization , Male , Melanoma/genetics , Molecular Sequence Data , Polymorphism, Restriction Fragment Length , Receptor Protein-Tyrosine Kinases/genetics , Sequence Analysis, DNA , Tumor Cells, CulturedABSTRACT
Enhanced chemiluminescence was applied to detect the binding of monoclonal antibodies to surface antigens on intact cells. The fast and simple assay is performed in the microtiter scale and thus allows for the simultaneous processing of a large number of samples with a sensitivity comparable to conventionally used techniques such as cytometry or Western blot analysis. In two model experiments, we demonstrate (a) the detection of a heterologously expressed cytokine receptor subunit on the surface of suspension cells and (b) the screening of hybridoma clones for the production of antibodies specifically recognizing surface antigens on a tumor cell line. Moreover, the assay is shown to be suitable for the determination of antibody affinities and of antibody binding sites per cell.
