RESUMO
In Escherichia coli, RecA-single-stranded DNA (RecA-ssDNA) filaments catalyze DNA repair, recombination, and induction of the SOS response. It has been shown that, while many (15 to 25%) log-phase cells have RecA filaments, few (about 1%) are induced for SOS. It is hypothesized that RecA's ability to induce SOS expression in log-phase cells is repressed because of the potentially detrimental effects of SOS mutagenesis. To test this, mutations were sought to produce a population where the number of cells with SOS expression more closely equaled the number of RecA filaments. Here, it is shown that deleting radA (important for resolution of recombination structures) and increasing recA transcription 2- to 3-fold with a recAo1403 operator mutation act independently to minimally satisfy this condition. This allows 24% of mutant cells to have elevated levels of SOS expression, a percentage similar to that of cells with RecA-green fluorescent protein (RecA-GFP) foci. In an xthA (exonuclease III gene) mutant where there are 3-fold more RecA loading events, recX (a destabilizer of RecA filaments) must be additionally deleted to achieve a population of cells where the percentage having elevated SOS expression (91%) nearly equals the percentage with at least one RecA-GFP focus (83%). It is proposed that, in the xthA mutant, there are three independent mechanisms that repress SOS expression in log-phase cells. These are the rapid processing of RecA filaments by RadA, maintaining the concentration of RecA below a critical level, and the destabilizing of RecA filaments by RecX. Only the first two mechanisms operate independently in a wild-type cell.
Assuntos
Escherichia coli/metabolismo , Resposta SOS em Genética/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Divisão Celular , Quebras de DNA de Cadeia Simples , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/citologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Ligação ProteicaRESUMO
Based on reflective optics at 13.5 nm, extreme-UV lithography is the ultimate top-down technique to define structures below 22 nm but faces several challenges arising from the discrete nature of light and matter. Owing to the short wavelength, mask surface roughness plays a fundamental role in the increase of speckle pattern contrast, compromising the uniformity of the printed features. Herein, we have used a mask with engineered gradient surface roughness to illustrate the impact that speckle has on the resulting photoresist pattern. The speckle increases the photoresist roughness, but surprisingly, only when the mask surface roughness is well above existing manufacturing capabilities.
RESUMO
A series of high-performance polycarbonates have been prepared with glass-transition temperatures and decomposition temperatures that are tunable by varying the repeat-unit chemical structure. Patterning of the polymers with extreme UV lithography has been achieved by taking advantage of direct photoinduced chain scission of the polymer chains, which results in a molecular-weight based solubility switch. After selective development of the irradiated regions of the polymers, feature sizes as small as 28.6 nm have been printed and the importance of resist-developer interactions for maximizing image quality has been demonstrated.
RESUMO
Genomic integrity is critical for an organism's survival and ability to reproduce. In Escherichia coli, the UvrD helicase has roles in nucleotide excision repair and methyl-directed mismatch repair and can limit reactions by RecA under certain circumstances. UvrD303 (D403A D404A) is a hyperhelicase mutant, and when expressed from a multicopy plasmid, it results in UV sensitivity (UV(s)), recombination deficiency, and antimutability. In order to understand the molecular mechanism underlying the UV(s) phenotype of uvrD303 cells, this mutation was transferred to the E. coli chromosome and studied in single copy. It is shown here that uvrD303 mutants are UV sensitive, recombination deficient, and antimutable and additionally have a moderate defect in inducing the SOS response after UV treatment. The UV-sensitive phenotype is epistatic with recA and additive with uvrA and is partially suppressed by removing the LexA repressor. Furthermore, uvrD303 is able to inhibit constitutive SOS expression caused by the recA730 mutation. The ability of UvrD303 to antagonize SOS expression was dependent on its 40 C-terminal amino acids. It is proposed that UvrD303, via its C terminus, can decrease the levels of RecA activity in the cell.
Assuntos
DNA Helicases/química , DNA Helicases/farmacologia , Escherichia coli K12/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/farmacologia , Regulação Bacteriana da Expressão Gênica , Mutação , Recombinases Rec A/metabolismo , Resposta SOS em Genética/efeitos dos fármacos , DNA Helicases/genética , DNA Helicases/efeitos da radiação , DNA Bacteriano/genética , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/efeitos da radiação , Recombinases Rec A/genética , Recombinação Genética , Raios UltravioletaRESUMO
There are few cohorts of individuals who have survived infection with HIV-1 for more than 20 years, reported and followed in the literature, and even fewer from Africa. Here we present data on a cohort of subtype C-infected individuals from rural northern Malawi. By sequencing multiple clones from long-term survivors at different time points, and using multiple genotyping approaches, we show that 5 of the 11 individuals are predicted as CXCR4 using (by ≥3/5 predictors) but only one individual is predicted as CXCR4 using by all five algorithms. Using any one genotyping approach overestimates the number of predicted CXCR4 sequences. Patterns of diversity and divergence were variable between the HIV-1 long-term survivors with some individuals showing very small amounts of variation and change, and others showing a greater amount; both patterns are consistent with what has been described in the literature.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/metabolismo , HIV-1/isolamento & purificação , Receptores CXCR4/metabolismo , Sequência de Bases , Primers do DNA , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/classificação , Humanos , Funções Verossimilhança , Malaui , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
Here we present new sequence data from HIV-1 subtype C-infected long-term survivors (LTS) from Karonga District, Malawi. Gag and env sequence data were produced from nine individuals each of whom has been HIV-1 positive for more than 20 years. We show that the three amino acid deletion in gag p17 previously described from these LTS is not real and was a result of an alignment error. We find that the use of dried blood spots for DNA-based studies is limited after storage for 20 years. We also show some unlikely amino acid changes in env C2-V3 in LTS over time and different patterns of genetic divergence among LTS. Although no clear association between mutations and survival could be shown, amino acid changes that are present in more than one LTS may, in the future, be shown to be important.
Assuntos
Genes env/genética , Antígenos HIV/genética , Sobreviventes de Longo Prazo ao HIV/estatística & dados numéricos , Soropositividade para HIV/genética , HIV-1/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Adulto , Sequência de Aminoácidos , Feminino , Soropositividade para HIV/epidemiologia , HIV-1/isolamento & purificação , Humanos , Malaui/epidemiologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Filogenia , Reação em Cadeia da Polimerase , Deleção de Sequência/genéticaRESUMO
Line edge roughness (LER) of patterned features in chemically amplified (CA) resists is formed in the acid generation stage and expected to be moderated by the acid diffusion and development process. It is essential to obtain information on the limit of LER in order to realize next-generation lithographies such as electron beam or extreme ultraviolet. Here, we report for the first time a process simulator based on physical and chemical reaction mechanisms. The LER of a positive-tone CA resist after development is investigated by Monte Carlo simulation and Mack's dissolution model. We found that the LER (high frequency) of less than 1.2 nm is achievable, although the process conditions and material design for achieving such a small LER are strict.