RESUMO
We have used a multiplex selection approach to construct a library of DNA-protein interaction sites recognized by many of the DNA-binding proteins present in a cell type. An estimated minimum of two-thirds of the binding sites present in a library prepared from activated Jurkat T cells represent authentic transcription factor binding sites. We used the library for isolation of "optimal" binding site probes that facilitated cloning of a factor and to identify binding activities induced within 2 hr of activation of Jurkat cells. Since a large fraction of the oligonucleotides obtained appear to represent "optimal" binding sites for sequence-specific DNA-binding proteins, it is feasible to construct a catalog of consensus binding sites for DNA-binding proteins in a given cell type. Qualitative and quantitative comparisons of the catalogs of binding site sequences from various cell types could provide valuable insights into the process of differentiation acting at the level of transcriptional control.
Assuntos
Clonagem Molecular/métodos , Oligodesoxirribonucleotídeos , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Sequência Consenso , DNA Complementar , Bases de Dados Factuais , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Fito-Hemaglutininas/farmacologia , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ets , Homologia de Sequência do Ácido Nucleico , Relação Estrutura-Atividade , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
The Contingent replication assay (CRA) is a rapid assay for the screening and isolation of cDNAs by protein-protein or protein-DNA interactions in mammalian cells. The method has been shown to enrich a plasmid containing a cDNA encoding the bacterial replication-related protein, R6K, from a mixture of two plasmids. In this report we present data illustrating the sensitivity and selectivity of the method. Using the small subunit of TFIIF (Rap30) as a target, we demonstrate the enrichment of a clone encoding the large subunit, Rap74, from a cDNA library. Additional cDNA clones including human Rap30 and an anonymous cDNA clone homologous to members of the human cdc2 kinase family were enriched and isolated by a modified screening approach. The structure of these additional clones suggest that the CRA enriches for products that interact not only directly with the target protein but also through bridging by endogenous proteins.
