RESUMO
We used a genetic screening methodology, a human cell line bearing a retinoic-acid-responsive enhanced GFP reporter, and a flow sorter to recover dominant modulators of reporter expression. Four inducers and three suppressors that were fused to the C terminus of a protein scaffold for stability were isolated and their mechanisms of action studied. Mutagenesis experiments indicated that six of these dominant agents exerted their effects at the protein level. The single cDNA coding fragment that was isolated comprised the central 64-amino-acid section of human cyclophilin B, which contained its peptidyl-prolyl isomerase domain; this cyclophilin fragment repressed expression of the retinoic-acid-responsive reporter. The remaining clones encoded peptides shorter than 30 amino acids unrelated to known gene open reading frames. Genetic epistasis studies between the strongest inducer, R3, and a dominant-negative mutant of RARalpha suggest that the two factors function in the same pathway. Transcript microarray analyses suggest that R3 induced a subset of the retinoid-responsive genes in melanoma cells. Finally, yeast two-hybrid assays and co-immunoprecipitation studies of human cell extracts identified PAT1 as a protein that interacts with R3.
Assuntos
Testes Genéticos/métodos , Receptores do Ácido Retinoico/genética , Seleção Genética , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Ciclofilinas/química , Ciclofilinas/genética , DNA Complementar/genética , DNA Mitocondrial/genética , Biblioteca Gênica , Genes Reporter , Humanos , Dados de Sequência Molecular , Peptidilprolil Isomerase , Mapeamento por Restrição , TransfecçãoRESUMO
Transdominant genetic selections can yield protein fragment and peptide modulators of specific biochemical pathways. In yeast, such screens have been highly successful in targeting the MAP (mitogen-activated protein) kinase growth-control pathway. We performed a similar type of selection aimed at recovery of modulators of the mammalian MAP kinase cascade. Two pathway activators were identified, fragments of the TrkB and Raf-1 kinases. In a second selection directed at the beta-catenin growth-control pathway, three different clones encoding cadherin fragments were recovered. In neither selection were peptide inhibitors observed. We conclude that some transdominant selections in mammalian cells can readily yield high-penetrance protein fragments, but may be less amenable to isolation of peptide inhibitors.
Assuntos
Proteínas do Citoesqueleto/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Seleção Genética , Transdução de Sinais/genética , Transativadores/genética , Células 3T3 , Animais , Bioensaio , Proteínas do Citoesqueleto/fisiologia , Técnicas In Vitro , Camundongos , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Transdução de Sinais/fisiologia , Transativadores/fisiologia , beta CateninaRESUMO
Retroviruses are useful for genetics studies to deliver genes that express proteins, peptides, and RNAs. Several steps, including DNA preparation, transfection, packaging, transduction, and assay, are required to execute screens using retroviral constructs. Unlike screens with purified components, whole-cell assays using retroviral constructs need a large number of steps with microplate manipulations. The nature of these steps, especially the involvement of cultured mammalian cells, limits the throughput of such screens. To improve the efficiency of genetic experiments with retroviral expression vectors, an automated system for retroviral screening in microplates was devised and tested. The system, called Somata, provides high throughputs and robust, reproducible performance.
