RESUMO
We have measured the induction and removal of UV-induced cyclobutane pyrimidine dimers from defined, DNA sequences in brains isolated from wild-type Drosophila melanogaster third instar larvae. Brains were exposed to a single dose of 500 J/m2 UVB and kept in the dark for up to 48 h. Within 48 h after irradiation, 50% of the dimers are removed from the actively transcribed genes Gart and Notch. Moreover, these kinetics are similar to the time course of dimer removal measured in the transcriptionally inactive white gene. It is further demonstrated that the genome overall is repaired at a similar rate. The results are discussed with respect to the in vivo irradiation of brains and to the data found for gene-specific repair in other eukaryotes.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Ciclobutanos/farmacologia , Reparo do DNA , Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas do Olho , Genes de Insetos/efeitos dos fármacos , Genes de Insetos/efeitos da radiação , Proteínas de Insetos/genética , Proteínas de Membrana/genética , Raios Ultravioleta/efeitos adversos , Animais , Northern Blotting , Southern Blotting , Encéfalo/metabolismo , DNA/isolamento & purificação , Sondas de DNA/genética , Endonucleases/metabolismo , Cinética , Dímeros de Pirimidina/metabolismo , Receptores Notch , Transcrição GênicaRESUMO
Nucleotide excision repair (NER) of ultraviolet (UV) light induced cyclobutane pyrimidine dimers (CPDs) was assayed in a Drosophila melanogaster Kc subline that responds to treatment with the steroid hormone 20-hydroxyecdysone (20-OH-E; beta-ecdysone, ecdysterone). In this cell line the hormone induces transcription of the beta 3-tubulin gene which is not expressed under standard culture conditions. Cells were exposed to either 10 or 15 J/m2 UV (predominantly 254-nm) and removal of CPDs from several genes, including beta 3-tubulin, and total cellular DNA was assayed. We show that upon induction of transcription of the beta 3-tubulin gene, its repair is not enhanced. In non-treated as well as 20-OH-E treated cells, repair kinetics in beta 3-tubulin resemble those in the active genes Gart and Notch, the inactive locus white and total cellular DNA. Moreover, in the presence as well as in the absence of transcription, the separate strands of the beta 3-tubulin gene are repaired with the same rate and to the same extent: about 90% after 24 h. It can be concluded from these observations that transcription is not a prerequisite for the efficient repair of CPDs in the Drosophila embryonic Kc cell line.
Assuntos
Reparo do DNA , Dímeros de Pirimidina , Transcrição Gênica , Tubulina (Proteína)/genética , Animais , Autorradiografia , Northern Blotting , Linhagem Celular , Sondas de DNA , Drosophila melanogaster , PlasmídeosRESUMO
Strand-specific excision repair of UV-induced cyclobutane pyrimidine dimers was investigated in three genes: Gart, Notch and white in the permanent Kc cell line derived from wild-type Drosophila melanogaster embryonic cells. In this cell line Gart and Notch are transcriptionally active, whereas white is not expressed. Cells were irradiated with 10 or 15 J/m2 ultraviolet (UV) light (predominantly 254 nm). In all three genes, cyclobutane pyrimidine dimers were removed from the non-transcribed strand at the same rate and to the same extent as from the transcribed strand, indicating the absence of strand-specific repair in permanent Drosophila embryonic cell lines.
Assuntos
Reparo do DNA , Drosophila melanogaster/genética , Dímeros de Pirimidina/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Expressão Gênica , Genes , Dados de Sequência Molecular , Mapeamento por Restrição , Transcrição Gênica , Raios UltravioletaRESUMO
The excision repair of UV-induced pyrimidine dimers was investigated in three genes: Gart, Notch and white in a permanent Drosophila cell line Kc, derived from wild type Drosophila melanogaster embryonic cells. In this cell line Gart and Notch are actively transcribed, whereas white is not expressed. In all three genes UV-induced pyrimidine dimers were removed with the same rate and to the same extent: 60% removal within 16 hours, up to 80-100% in 24 hours after irradiation with 10 or 15 J/m2 UV. These kinetics are similar to the time course of dimer removal measured in the genome overall. No difference in repair of the inactive white locus compared to the active Gart and Notch genes was found. Similar results were obtained using a different wild type cell line, SL2, although repair appeared to be somewhat slower in this cell line. The results are discussed with respect to the data found for gene specific repair in other eukaryotic systems.