RESUMO
Mice defective in the mismatch repair (MMR) gene Msh2 manifest an enhanced predisposition to skin cancer associated with exposure to UVB radiation. This predisposition is further heightened if the mice are additionally defective for the nucleotide excision repair gene Xpc. To test the hypothesis that the predisposition of Msh2 mutant mice to skin cancer reflects a mutator phenotype associated with increased proliferation of skin cells following exposure to UV radiation, Msh2 mutant mice were exposed to the tumor promoter TPA. Such mice showed a robust proliferative response in the skin, but did not manifest evidence of dysplasia or neoplasia. We conclude that the predisposition of Msh2 mice to UVB radiation-induced skin cancer reflects an interaction between the processes of mismatch repair and some other excision repair mode, the exact nature of which remains to be established.
Assuntos
Pareamento Incorreto de Bases/genética , Reparo do DNA/genética , Predisposição Genética para Doença/genética , Neoplasias Induzidas por Radiação/genética , Proteínas Proto-Oncogênicas/genética , Neoplasias Cutâneas/genética , Pele/efeitos da radiação , Animais , Carcinógenos/farmacologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Heterozigoto , Homozigoto , Camundongos , Camundongos Knockout , Proteína 2 Homóloga a MutS , Neoplasias Induzidas por Radiação/patologia , Neoplasias Cutâneas/patologia , Acetato de Tetradecanoilforbol/farmacologia , Raios UltravioletaAssuntos
2-Acetilaminofluoreno/toxicidade , Carcinógenos/toxicidade , Reparo do DNA , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/genética , Proteína Supressora de Tumor p53/genética , Animais , Cocarcinogênese , Cruzamentos Genéticos , Primers do DNA/química , Proteínas de Ligação a DNA/deficiência , Feminino , Predisposição Genética para Doença , Genótipo , Heterozigoto , Homozigoto , Humanos , Hidroxiacetilaminofluoreno/toxicidade , Neoplasias Pulmonares/induzido quimicamente , Masculino , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase , Proteína Supressora de Tumor p53/deficiênciaRESUMO
The understanding of gene function increasingly requires the characterization of DNA segments containing promoters and their associated regulatory sequences. We describe a novel approach for linking multiple DNA segments, here applied to the generation of promoter::reporter fusions. Promoters from Caenorhabditis elegans genes were cloned using the MultiSite Gateway cloning technology. The capacity for using this system for efficient construction of chimeric genes was explored by constructing promoter::reporter gene fusions with a gfp reporter. The promoters were found to provide appropriate expression of GFP upon introduction into C. elegans, demonstrating that the short Gateway recombination site between the promoter and the reporter did not interfere with transcription or translation. The recombinational cloning involved in the Gateway system, which permits the highly efficient and precise transfer of DNA segments between plasmid vectors, makes this technology ideal for genomics research programs.
Assuntos
Caenorhabditis elegans/fisiologia , Expressão Gênica , Genes Reporter/fisiologia , Técnicas Genéticas , Genoma , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/fisiologia , Animais , Fusão Gênica Artificial , Clonagem Molecular , DNA Recombinante/biossíntese , DNA Recombinante/genética , Estudos de Viabilidade , Técnicas de Transferência de Genes , Plasmídeos/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Recombinação GenéticaRESUMO
The ability to clone and manipulate DNA segments is central to molecular methods that enable expression, screening, and functional characterization of genes, proteins, and regulatory elements. We previously described the development of a novel technology that utilizes in vitro site-specific recombination to provide a robust and flexible platform for high-throughput cloning and transfer of DNA segments. By using an expanded repertoire of recombination sites with unique specificities, we have extended the technology to enable the high-efficiency in vitro assembly and concerted cloning of multiple DNA segments into a vector backbone in a predefined order, orientation, and reading frame. The efficiency and flexibility of this approach enables collections of functional elements to be generated and mixed in a combinatorial fashion for the parallel assembly of numerous multi-segment constructs. The assembled constructs can be further manipulated by directing exchange of defined segments with alternate DNA segments. In this report, we demonstrate feasibility of the technology and application to the generation of fusion proteins, the linkage of promoters to genes, and the assembly of multiple protein domains. The technology has broad implications for cell and protein engineering, the expression of multidomain proteins, and gene function analysis.