RESUMO
Chemically synthesized small interfering RNAs (siRNAs) are tools used for silencing the expression of a single gene. They are mainly employed in basic research applications, but may also have great potential in therapeutic applications. Longer double-stranded RNAs, such as Dicer-substrate 27mers, trigger gene silencing through the intrinsic RNAi pathway. The design of these Dicer-substrate 27mers has been optimized so they can be oriented by Dicer to consistently select the antisense (guide) strand after cleavage to shorter siRNAs, leading to predictable mRNA cleavage. In this paper we describe evidence that these Dicer-substrate 27mers produce more potent and sustained gene silencing for four genes when compared with synthetic 21mers that have the same guide-strand sequence. Furthermore, improved silencing by these 27mers is often more pronounced at lower concentrations.
Assuntos
Inativação Gênica , Ribonuclease III/metabolismo , Sequência de Bases , Biotecnologia , Quinase 2 Dependente de Ciclina/genética , Primers do DNA/genética , Genes p53 , Células HeLa , Humanos , Modelos Genéticos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-raf/genética , Interferência de RNA , RNA Interferente Pequeno/síntese química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade por Substrato , Fatores de Tempo , TransfecçãoRESUMO
Cockayne syndrome (CS) is a progressive childhood neurodegenerative disorder associated with a DNA repair defect caused by mutations in either of two genes, CSA and CSB. These genes are involved in nucleotide excision repair (NER) of DNA damage from ultraviolet (UV) light, other bulky chemical adducts and reactive oxygen in transcriptionally active genes (transcription-coupled repair, TCR). For a long period it has been assumed that the symptoms of CS patients are all due to reduced TCR of endogenous DNA damage in the brain, together with unexplained unique sensitivity of specific neural cells in the cerebellum. Not all the symptoms of CS patients are however easily related to repair deficiencies, so we hypothesize that there are additional pathways relevant to the disease, particularly those that are downstream consequences of a common defect in the E3 ubiquitin ligase associated with the CSA and CSB gene products. We have found that the CSB defect results in altered expression of anti-angiogenic and cell cycle genes and proteins at the level of both gene expression and protein lifetime. We find an over-abundance of p21 due to reduced protein turnover, possibly due to the loss of activity of the CSA/CSB E3 ubiquitylation pathway. Increased levels of p21 can result in growth inhibition, reduced repair from the p21-PCNA interaction, and increased generation of reactive oxygen. Consistent with increased reactive oxygen levels we find that CS-A and -B cells grown under ambient oxygen show increased DNA breakage, as compared with xeroderma pigmentosum cells. Thus the complex symptoms of CS may be due to multiple, independent downstream targets of the E3 ubiquitylation system that results in increased DNA damage, reduced transcription coupled repair, and inhibition of cell cycle progression and growth.
Assuntos
Síndrome de Cockayne/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA/genética , Reparo do DNA/genética , Regulação da Expressão Gênica/genética , Transcrição Gênica/genética , Ciclo Celular/genética , Linhagem Celular , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/fisiopatologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Dano ao DNA/efeitos da radiação , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Humanos , Estresse Oxidativo/fisiologia , Proteínas de Ligação a Poli-ADP-Ribose , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Raios UltravioletaRESUMO
Mutants sensitive to ionizing radiation in yeast and mammals include an assortment of DNA repair genes. The majority of these DNA repair genes are involved in the repair of DNA double-strand breaks. In this study a forward genetic screen is used to identify gamma-sensitive mutants of Arabidopsis thaliana. The gamma-plantlet screen used here also reveals two general mutant classes based on size of cotyledons and hypocotyls. One of the mutants discovered is a homologue of the mammalian nucleotide excision repair gene ERCC1.
Assuntos
Arabidopsis/efeitos da radiação , Reparo do DNA/genética , Proteínas de Ligação a DNA , Endonucleases , Proteínas/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis , Ciclo Celular/efeitos da radiação , Cotilédone/crescimento & desenvolvimento , Cotilédone/efeitos da radiação , Reparo do DNA/efeitos da radiação , DNA de Plantas/química , DNA de Plantas/genética , Relação Dose-Resposta à Radiação , Raios gama , Teste de Complementação Genética , Humanos , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/efeitos da radiação , Meristema/crescimento & desenvolvimento , Meristema/efeitos da radiação , Mutação , Fenótipo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/efeitos da radiação , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/efeitos da radiação , Proteínas/metabolismo , Análise de Sequência de DNA , Fatores de Tempo , Raios UltravioletaRESUMO
Extracts from several different photocopies were mutagenic in the Ames Salmonella assay. The mutagenic behavior was similar for extracts from copies and corresponding toners indicating that toners are directly responsible for the mutagenicity. The mutagenicity is caused by at least two classes of compounds which may be present either alone or in combination in any toner.
