RESUMO
Multiple gene knockout systems developed in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius are powerful genetic tools. However, plasmid construction typically requires several steps. Alternatively, PCR tailing for high-throughput gene disruption was also developed in S. acidocaldarius, but repeated gene knockout based on PCR tailing has been limited due to lack of a genetic marker system. In this study, we demonstrated efficient homologous recombination frequency (2.8 × 104 ± 6.9 × 103 colonies/µg DNA) by optimizing the transformation conditions. This optimized protocol allowed to develop reliable gene knockout via double crossover using short homologous arms and to establish the multiple gene knockout system with one-step PCR (MONSTER). In the MONSTER, a multiple gene knockout cassette was simply and rapidly constructed by one-step PCR without plasmid construction, and the PCR product can be immediately used for target gene deletion. As an example of the applications of this strategy, we successfully made a DNA photolyase- (phr-) and arginine decarboxylase- (argD-) deficient strain of S. acidocaldarius. In addition, an agmatine selection system consisting of an agmatine-auxotrophic strain and argD marker was also established. The MONSTER provides an alternative strategy that enables the very simple construction of multiple gene knockout cassettes for genetic studies in S. acidocaldarius.
Assuntos
Técnicas de Inativação de Genes/métodos , Reação em Cadeia da Polimerase/métodos , Sulfolobus acidocaldarius/genética , Carboxiliases/deficiência , Desoxirribodipirimidina Fotoliase/deficiência , Recombinação Homóloga , Sulfolobus acidocaldarius/enzimologia , Transformação GenéticaRESUMO
Damage of DNA and Photosystem-II are among the most significant effects of UV-B irradiation in photosynthetic organisms. Both damaged DNA and Photosystem-II can be repaired, which represent important defense mechanisms against detrimental UV-B effects. Correlation of Photosystem-II damage and repair with the concurrent DNA damage and repair was investigated in the cyanobacterium Synechocystis PCC6803 using its wild type and a photolyase deficient mutant, which is unable to repair UV-B induced DNA damages. A significant amount of damaged DNA accumulated during UV-B exposure in the photolyase mutant concomitant with decreased Photosystem-II activity and D1 protein amount. The transcript level of psbA3, which is a UV-responsive copy of the psbA gene family encoding the D1 subunit of the Photosystem-II reaction center, is also decreased in the photolyase mutant. The wild-type cells, however, did not accumulate damaged DNA during UV-B exposure, suffered smaller losses of Photosystem-II activity and D1 protein, and maintained higher level of psbA3 transcripts than the photolyase mutant. It is concluded that the repair capacity of Photosystem-II depends on the ability of cells to repair UV-B-damaged DNA through maintaining the transcription of genes, which are essential for protein synthesis-dependent repair of the Photosystem-II reaction center.
Assuntos
Proteínas de Bactérias/genética , Reparo do DNA , DNA Bacteriano/química , Desoxirribodipirimidina Fotoliase/genética , Complexo de Proteína do Fotossistema II/genética , Synechocystis/efeitos da radiação , Proteínas de Bactérias/biossíntese , Dano ao DNA , DNA Bacteriano/metabolismo , Desoxirribodipirimidina Fotoliase/deficiência , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/biossíntese , Complexo de Proteína do Fotossistema II/metabolismo , Biossíntese de Proteínas/efeitos da radiação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Synechocystis/genética , Synechocystis/metabolismo , Transcrição Gênica/efeitos da radiação , Raios UltravioletaRESUMO
We report a remarkably high UV-radiation resistance in the extremely halophilic archaeon Halobacterium NRC-1 withstanding up to 110 J/m2 with no loss of viability. Gene knockout analysis in two putative photolyase-like genes (phr1 and phr2) implicated only phr2 in photoreactivation. The UV-response was further characterized by analyzing simultaneously, along with gene function and protein interactions inferred through comparative genomics approaches, mRNA changes for all 2400 genes during light and dark repair. In addition to photoreactivation, three other putative repair mechanisms were identified including d(CTAG) methylation-directed mismatch repair, four oxidative damage repair enzymes, and two proteases for eliminating damaged proteins. Moreover, a UV-induced down-regulation of many important metabolic functions was observed during light repair and seems to be a phenomenon shared by all three domains of life. The systems analysis has facilitated the assignment of putative functions to 26 of 33 key proteins in the UV response through sequence-based methods and/or similarities of their predicted three-dimensional structures to known structures in the PDB. Finally, the systems analysis has raised, through the integration of experimentally determined and computationally inferred data, many experimentally testable hypotheses that describe the metabolic and regulatory networks of Halobacterium NRC-1.
Assuntos
Halobacterium/genética , Halobacterium/efeitos da radiação , Animais , Proteínas Arqueais/fisiologia , Cricetinae , Reparo do DNA/genética , Desoxirribodipirimidina Fotoliase/deficiência , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica em Archaea/genética , Regulação da Expressão Gênica em Archaea/efeitos da radiação , Halobacterium/classificação , Halobacterium/enzimologia , Luz , Mesocricetus/genética , Camundongos , Mutação/genética , RNA Arqueal/genética , RNA Mensageiro/genética , Proteínas Repressoras/genética , Homologia de Sequência do Ácido Nucleico , Taxa de Sobrevida , Fatores de Tempo , Fatores de Transcrição/genética , Raios UltravioletaRESUMO
We produced a photolyase-deficient mutant by repeat induced point mutation using the Neurospora crassa photolyase gene cloned previously. This mutation identified a new gene, phr, which was mapped on the right arm of linkage group I by both RFLP mapping and conventional mapping. To investigate the relationship between photoreactivation and dark repair processes, especially excision repair, double mutants of phr with representative repair-defective mutants of different types were constructed and tested for UV sensitivity and photoreactivation. The results show that the phr mutation has no influence on dark repair. Tests with CPD and TC(6-4) photoproduct-specific antibodies demonstrated that the phr mutant is defective in CPD photolyase and confirmed that there is no TC(6-4) photolyase activity in N. crassa. Furthermore, N. crassa photolyase is not a blue light receptor in the signal transduction that induces carotenoid biosynthesis.
Assuntos
Desoxirribodipirimidina Fotoliase/genética , Neurospora crassa/genética , Mutação Puntual , Southern Blotting , Mapeamento Cromossômico , Reparo do DNA , DNA Fúngico/análise , DNA Fúngico/efeitos da radiação , Desoxirribodipirimidina Fotoliase/deficiência , Ensaio de Imunoadsorção Enzimática , Genes Fúngicos/genética , Genes Fúngicos/efeitos da radiação , Neurospora crassa/enzimologia , Neurospora crassa/efeitos da radiação , Polimorfismo de Fragmento de Restrição , Raios UltravioletaRESUMO
Damaged DNA can be repaired by three different mechanisms: photoreactivation, excision repair and postreplication repair. Each mechanism is regulated by a highly specific set of enzymes. Defects within these systems result in diseases which have one common feature: affected individuals are cancer prone. Recently, newly developed methods not only make it possible to diagnose affected patients but also to detect individuals at risk. Furthermore, the results obtained elucidate some mechanisms of carcinogenesis. Clinical applications are discussed.