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Genomewide trapping of genes that encode secreted and transmembrane proteins repressed by oncogenic signaling.

ABSTRACT: A retroviral gene trap containing a human CD2 cell surface antigen/neomycin-phosphotransferase fusion gene in the U3 region of its LTR (U3Ceo) was used to screen the mammalian genome for genes encoding secreted and/or transmembrane proteins that are repressed by oncogenic transformation. From an integration library consisting of cells transformable by the insulin-like growth factor 1 (IGF-1), a collection of neomycin resistant (Neo(R)) clones was obtained; 86% also expressed the CD2 cell surface antigen. Molecular analysis of a random sample of Neo(R) clones revealed that the U3Ceo gene trap preferentially disrupted genes coding for secreted and transmembrane proteins. In each case, the signal sequence of the endogenous gene was fused in-frame to the CD2/neomycin-phosphotransferase reporter gene due to a cryptic splice acceptor site embedded in the coding region of the CD2 cDNA. When the library was transformed by IGF-1 and selected against CD2 expression, integrations were obtained in genes that are repressed by transformation. Molecular analysis of six randomly chosen integrations revealed that, in each case, U3Ceo captured a signal sequence from proteins involved in oncogenic transformation and metastatic spread.

SUBMITTER: Gebauer M

PROVIDER: S-EPMC311167 | biostudies-literature | 2001 Nov

REPOSITORIES: biostudies-literature

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Genomewide trapping of genes that encode secreted and transmembrane proteins repressed by oncogenic signaling.

Gebauer M M von Melchner H H Beckers T T

Genome research 20011101 11

A retroviral gene trap containing a human CD2 cell surface antigen/neomycin-phosphotransferase fusion gene in the U3 region of its LTR (U3Ceo) was used to screen the mammalian genome for genes encoding secreted and/or transmembrane proteins that are repressed by oncogenic transformation. From an integration library consisting of cells transformable by the insulin-like growth factor 1 (IGF-1), a collection of neomycin resistant (Neo(R)) clones was obtained; 86% also expressed the CD2 cell surface ...[more]

PMID: 11691852

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Project description:BackgroundMorphogenetic events that shape the Drosophila melanogaster embryo are tightly controlled by a genetic program in which specific sets of genes are up-regulated. We used a suppressive subtractive hybridization procedure to identify a group of developmentally regulated genes during early stages of D. melanogaster embryogenesis. We studied the spatiotemporal activity of these genes in five different intervals covering 12 stages of embryogenesis.ResultsMicroarrays were constructed to confirm induction of expression and to determine the temporal profile of isolated subtracted cDNAs during embryo development. We identified a set of 118 genes whose expression levels increased significantly in at least one developmental interval compared with a reference interval. Of these genes, 53% had a phenotype and/or molecular function reported in the literature, whereas 47% were essentially uncharacterized. Clustering analysis revealed demarcated transcript groups with maximum gene activity at distinct developmental intervals. In situ hybridization assays were carried out on 23 uncharacterized genes, 15 of which proved to have spatiotemporally restricted expression patterns. Among these 15 uncharacterized genes, 13 were found to encode putative secreted and transmembrane proteins. For three of them we validated our protein sequence predictions by expressing their cDNAs in Drosophila S2R+ cells and analyzed the subcellular distribution of recombinant proteins. We then focused on the functional characterization of the gene CG6234. Inhibition of CG6234 by RNA interference resulted in morphological defects in embryos, suggesting the involvement of this gene in germ band retraction.ConclusionOur data have yielded a list of developmentally regulated D. melanogaster genes and their expression profiles during embryogenesis and provide new information on the spatiotemporal expression patterns of several uncharacterized genes. In particular, we recovered a substantial number of unknown genes encoding putative secreted and transmembrane proteins, suggesting new components of signaling pathways that might be incorporated within the existing regulatory networks controlling D. melanogaster embryogenesis. These genes are also good candidates for additional targeted functional analyses similar to those we conducted for CG6234.See related minireview by Vichas and Zallen: http://www.jbiol.com/content/8/8/76.

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Genomewide trapping of genes that encode secreted and transmembrane proteins repressed by oncogenic signaling.

Publications

Genomewide trapping of genes that encode secreted and transmembrane proteins repressed by oncogenic signaling.

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