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1.
Cell Rep ; 20(7): 1553-1562, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28813668

RESUMO

Structure-specific endonucleases contribute to the maintenance of genome integrity by cleaving DNA intermediates that need to be resolved for faithful DNA repair, replication, or recombination. Despite advances in the understanding of their function and regulation, it is less clear how these proteins respond to genotoxic stress. Here, we show that the structure-specific endonuclease Mus81-Mms4/EME1 relocalizes to subnuclear foci following DNA damage and colocalizes with the endonucleases Rad1-Rad10 (XPF-ERCC1) and Slx1-Slx4. Recruitment takes place into a class of stress foci defined by Cmr1/WDR76, a protein involved in preserving genome stability, and depends on the E2-ubiquitin-conjugating enzyme Rad6 and the E3-ubiquitin ligase Bre1. Foci dynamics show that, in the presence of DNA intermediates that need resolution by Mus81-Mms4, Mus81 foci persist until this endonuclease is activated by Mms4 phosphorylation. Our data suggest that subnuclear relocalization is relevant for the function of Mus81-Mms4 and, probably, of the endonucleases that colocalize with it.


Assuntos
Reparo do DNA , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Endonucleases Flap/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Dano ao DNA , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Endonucleases Flap/metabolismo , Fosforilação , Ligação Proteica , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
2.
Appl Microbiol Biotechnol ; 99(3): 1145-53, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25472432

RESUMO

The efficiency of current methods for industrial production of the enzyme nuclease P1 is limited. In this study, we sought to improve fermentation methods for the production of nuclease P1. An immobilized fermentation system using an activated carbon filter sponge as a carrier was used for the production of nuclease P1. In an airlift internal loop reactor (ALR), the fermentation performance of three different fermentation modes, including free-cell fermentation, repeated-batch fermentation, and semi-continuous immobilized fermentation, were compared. The fermentation kinetics in the fermentation broth of the three fermentation modes, including dissolved oxygen (DO), pH value, cell concentration, residual sugar concentration, and enzyme activity, were tested. The productivity of semi-continuous immobilized fermentation reached 8.76 U/mL/h, which was 33.3 and 80.2% higher than that of repeated-batch fermentation and free-cell fermentation, respectively. The sugar consumption of free-cell, repeated-batch, and semi-continuous immobilized fermentations was 41.2, 30.8, and 25.9 g/L, respectively. These results showed that immobilized-cell fermentation by using Penicillium citrinum with activated carbon filter sponge in an ALR was advantageous for nuclease P1 production, especially in the semi-continuous immobilized fermentation mode. In spite of the significant improvement in nuclease P1 production in semi-continuous immobilized fermentation mode, the specific activity of nuclease P1 was almost equal among the three fermentation modes.


Assuntos
Proteínas Fúngicas/metabolismo , Penicillium/enzimologia , Penicillium/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Reatores Biológicos/microbiologia , Carboidratos/análise , Células Imobilizadas/enzimologia , Células Imobilizadas/metabolismo , Carvão Vegetal , Meios de Cultura/química , Fermentação , Proteínas Fúngicas/genética , Concentração de Íons de Hidrogênio , Oxigênio/análise , Penicillium/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética
3.
Appl Biochem Biotechnol ; 170(6): 1533-45, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23700147

RESUMO

Genome shuffling is a powerful approach for efficiently engineering industrial microbial strains with interested phenotypes. Here we reported a high producer of nuclease P1, Penicillium citrinum G-16, that was bred by the classical physics-mutagenesis and genome shuffling process. The starting populations were generated by (60)Co γ-irradiation mutagenesis. The derived two protoplast fractions were inactivated by heat-treatment and ultraviolet radiation respectively, then mixed together and subjected to recursive protoplast fusion. Three recombinants, E-16, F-71, and G-16, were roughly obtained from six cycles of genome shuffling. The activity of nuclease P1 by recombinant G-16 was improved up to 1,980.22 U4/ml in a 5-l fermentor, which was 4.7-fold higher than that of the starting strain. The sporulation of recombinant G-16 was distinguished from the starting strain. Random amplified polymorphic DNA assay revealed genotypic differences between the shuffled strains and the wild type strain. The close similarity among the high producers suggested that the genetic basis of high-yield strains was achieved by genome shuffling.


Assuntos
Embaralhamento de DNA/métodos , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Melhoramento Genético/métodos , Genoma Fúngico/genética , Mutagênese/genética , Penicillium/fisiologia , Endonucleases Específicas para DNA e RNA de Cadeia Simples/biossíntese , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética , Proteínas Fúngicas/isolamento & purificação , Endonucleases Específicas para DNA e RNA de Cadeia Simples/isolamento & purificação
4.
Nat Struct Mol Biol ; 19(9): 964-71, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22885325

RESUMO

Holliday junctions can be formed during homology-dependent repair of DNA double-strand breaks, and their resolution is essential for chromosome segregation and generation of crossover products. The Mus81-Mms4 and Yen1 nucleases are required for mitotic crossovers between chromosome homologs in Saccharomyces cerevisiae; however, crossovers between dispersed repeats are still detected in their absence. Here we show that the Rad1-Rad10 nuclease promotes formation of crossover and noncrossover recombinants between ectopic sequences. Crossover products were not recovered from the mus81Δ rad1Δ yen1Δ triple mutant, indicating that all three nucleases participate in processing recombination intermediates that form between dispersed repeats. We suggest a new mechanism for crossovers that involves Rad1-Rad10 clipping and resolution of a single Holliday junction-containing intermediate by Mus81-Mms4 or Yen1 cleavage or by replication. Consistent with the model, we show accumulation of Rad1-dependent joint molecules in the mus81Δ yen1Δ mutant.


Assuntos
Cromossomos Fúngicos/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Endonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Cromossomos Fúngicos/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Resolvases de Junção Holliday/genética , Resolvases de Junção Holliday/metabolismo , Mutação , Plasmídeos/genética , Plasmídeos/metabolismo , Recombinação Genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética
5.
Mar Biotechnol (NY) ; 14(1): 87-95, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21647618

RESUMO

An extracellular nuclease was purified 165-fold with a specific activity of 41,250 U/mg poly(U) by chromatography with modified chitosan from the culture of marine fungus Penicillium melinii isolated from colonial ascidium collected near Shikotan Island, Sea of Okhotsk, at a depth of 123 m. The purified nuclease is a monomer with the molecular weight of 35 kDa. The enzyme exhibits maximum activity at pH 3.7 for DNA and RNA. The enzyme is stable until 75°C and in the pH range of 2.5-8.0. The enzyme endonucleolytically degrades ssDNA and RNA by 3'-5' mode to produce 5'-oligonucleotides and 5'-mononucleotides; however, it preferentially degrades poly(U). The enzyme can digest dsDNA in the presence of pregnancy-specific beta-1-glycoprotein-1. The nuclease acts on closed circular double-stranded DNA to produce opened circular DNA and then the linear form DNA by single-strand scission. DNA sequence encoding the marine fungus P. melinii endonuclease revealed homology to S1-type nucleases. The tight correlation found between the extracellular endonuclease activity and the rate of H³-thymidine uptake by actively growing P. melinii cells suggests that this nuclease is required for fulfilling the nucleotide pool of precursors of DNA biosynthesis during the transformation of hyphae into the aerial mycelium and conidia in stressful environmental conditions.


Assuntos
Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , Penicillium/enzimologia , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Sequência de Aminoácidos , Proteínas Fúngicas/genética , Regulação Enzimológica da Expressão Gênica , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética , Especificidade por Substrato , Temperatura Ambiente
6.
Sheng Wu Gong Cheng Xue Bao ; 28(11): 1388-97, 2012 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-23457791

RESUMO

To establish a prokaryotic expression and purification protocol for nuclease P1 (NP1), we first obtained a synthetic NP1 by splicing 22 oligonucleotides with overlapping PCR. We constructed and transformed a secretory expression vector pMAL-p4X-NP1 into Escherichia coli host strains T7 Express and Origami B (DE3) separately. Then, the recombinant NP1 was purified by amylose affinity chromatography, and its activity, thermo-stability and metal-ion dependence were investigated systematically. The results indicated that the expressed fusion proteins MBP-NP1 (Maltose binding protein-NP1) existed mainly in soluble form both in host strains T7 Express and Origami B (DE3), but the specific activity of recombinant protein from Origami B(DE3) strain was higher than T7 Express strain (75.48 U/mg : 51.50 U/mg). When the MBP-tag was cleaved by protease Factor Xa, the specific activity both increased up to 258.1 U/mg and 139.2 U/mg. The thermal inactivation experiments demonstrated that the recombinant NP1 was quite stable, and it retained more than 90% of original activity after incubated for 30 min at 80 degrees C. Zn2+ (2.0 mmol/L) could increase enzyme activity (to 119.1%), on the contrary, the enzyme activity was reduced by 2.0 mmol/L Cu2+ (to 63.12%). This research realized the functional expression of NP1 in the prokaryotic system for the first time, and provided an alternative pathway for NP1 preparation.


Assuntos
Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/biossíntese , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Genes Sintéticos , Vetores Genéticos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética
7.
Extremophiles ; 15(5): 619-24, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21667093

RESUMO

The hyperthermophilic Sulfolobus islandicus rod-shaped virus 2 (SIRV2) encodes a 25-kDa protein (SIRV2gp19) annotated as a hypothetical protein with sequence homology to the RecB nuclease superfamily. Even though SIRV2gp19 homologs are conserved throughout the rudivirus family and presumably play a role in the viral life cycle, SIRV2gp19 has not been functionally characterized. To define the minimal requirements for activity, SIRV2gp19 was purified and tested under varying conditions. SIRV2gp19 is a single-strand specific endonuclease that requires Mg(2+) for activity and is inactive on double-stranded DNA. A conserved aspartic acid in RecB nuclease superfamily Motif II (D89) is also essential for SIRV2gp19 activity and mutation to alanine (D89A) abolishes activity. Therefore, the SIRV2gp19 cleavage mechanism is similar to previously described RecB nucleases. Finally, SIRV2gp19 single-stranded DNA endonuclease activity could play a role in host chromosome degradation during SIRV2 lytic infection.


Assuntos
Rudiviridae/enzimologia , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Proteínas Virais/metabolismo , DNA Arqueal/genética , DNA Arqueal/metabolismo , Rudiviridae/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/química , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/isolamento & purificação , Sulfolobus/enzimologia , Sulfolobus/genética , Sulfolobus/virologia , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação
8.
Acta Histochem ; 113(4): 409-15, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20546858

RESUMO

Rad14 is a DNA damage recognition protein in yeast Nucleotide Excision Repair (NER) and believed to function early in the cascade of events. The function of Rad14 presumably precedes that of the Rad1-Rad10 endonuclease complex, which functions in a downstream step incising DNA 5' to the site of DNA damage. We investigated whether recruitment of Rad10 to UV-induced DNA damage sites in live cells is dependent on Rad14 using fluorescence microscopy. Experiments were carried out using Saccharomyces cerevisiae strains in which the gene for Rad14 was fused to Cyan Fluorescent Protein (Rad14-CFP) and that of Rad10 was fused to Yellow Fluorescent Protein (Rad10-YFP). Rad14-CFP forms nuclear localized CFP fluorescent foci in response to UV irradiation with the peak induction occurring 15min post-irradiation. In contrast, Rad10-YFP foci form in response to UV with the peak induction occurring 2h post-irradiation. Recruitment of Rad14-CFP is not dependent on the RAD10 gene but Rad10-YFP is recruited to UV-induced YFP foci in a RAD14-dependent fashion. Time-lapse experiments indicate that Rad14-CFP foci are transient, typically persisting less than 6min. Together these data support the model that yeast NER protein assembly is step-wise whereas Rad14 required to recruit Rad10 and Rad14 involvement is transient.


Assuntos
Enzimas Reparadoras do DNA/genética , Reparo do DNA , DNA Fúngico/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética , Proteínas de Bactérias , Dano ao DNA/efeitos da radiação , Enzimas Reparadoras do DNA/metabolismo , DNA Fúngico/efeitos da radiação , Proteínas de Fluorescência Verde , Proteínas Luminescentes , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Raios Ultravioleta
9.
Radiat Res ; 172(2): 141-51, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19630519

RESUMO

Abstract Illegitimate recombination can repair DNA double-strand breaks in one of two ways, either without sequence homology or by using a few base pairs of homology at the junctions. The second process is known as microhomology-mediated recombination. Previous studies showed that ionizing radiation and restriction enzymes increase the frequency of microhomology-mediated recombination in trans during rejoining of unirradiated plasmids or during integration of plasmids into the genome. Here we show that radiation-induced microhomology-mediated recombination is reduced by deletion of RAD52, RAD1 and RAD10 but is not affected by deletion of RAD51 and RAD2. The rad52 mutant did not change the frequency of radiation-induced microhomology-mediated recombination but rather reduced the length of microhomology required to undergo repair during radiation-induced recombination. The rad1 and rad10 mutants exhibited a smaller increase in the frequency of radiation-induced microhomology-mediated recombination, and the radiation-induced integration junctions from these mutants did not show more than 4 bp of microhomology. These results suggest that Rad52 facilitates annealing of short homologous sequences during integration and that Rad1/Rad10 endonuclease mediates removal of the displaced 3' single-stranded DNA ends after base-pairing of microhomology sequences, when more than 4 bp of microhomology are used. Taken together, these results suggest that radiation-induced microhomology-mediated recombination is under the same genetic control as the single-strand annealing apparatus that requires the RAD52, RAD1 and RAD10 genes.


Assuntos
Enzimas Reparadoras do DNA/genética , Reparo do DNA/genética , DNA Bacteriano/genética , Endonucleases/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , DNA Bacteriano/efeitos da radiação , Mutação/genética , Recombinação Genética/efeitos da radiação , Saccharomyces cerevisiae/efeitos da radiação , Homologia de Sequência , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética
10.
J Med Chem ; 52(9): 2863-74, 2009 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-19385599

RESUMO

The importance of DNA supercoiling in transcriptional regulation has been known for many years, and more recently, transcription itself has been shown to be a source of this superhelicity. To mimic the effect of transcriptionally induced negative superhelicity, the G-quadruplex/i-motif-forming region in the c-Myc promoter was incorporated into a supercoiled plasmid. We show, using enzymatic and chemical footprinting, that negative superhelicity facilitates the formation of secondary DNA structures under physiological conditions. Significantly, these structures are not the same as those formed in single-stranded DNA templates. Together with the recently demonstrated role of transcriptionally induced superhelicity in maintaining a mechanosensor mechanism for controlling the firing rate of the c-Myc promoter, we provide a more complete picture of how c-Myc transcription is likely controlled. Last, these physiologically relevant G-quadruplex and i-motif structures, along with the mechanosensor mechanism for control of gene expression, are proposed as novel mechanisms for small molecule targeting of transcriptional control of c-Myc.


Assuntos
DNA Super-Helicoidal/química , Desenho de Drogas , Quadruplex G/efeitos dos fármacos , Regulação da Expressão Gênica , Genes myc/genética , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , Composição de Bases , Sequência de Bases , Bromo/farmacologia , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , DNA Super-Helicoidal/metabolismo , Desoxirribonuclease I/metabolismo , Humanos , Mutação/efeitos dos fármacos , Desnaturação de Ácido Nucleico , Cloreto de Potássio/farmacologia , Permanganato de Potássio/farmacologia , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Transcrição Genética
11.
Chembiochem ; 8(6): 649-56, 2007 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-17394189

RESUMO

Herein, we describe our initial steps towards identifying the RNA secondary structure motifs that are recognized by small molecules. We selected members of an RNA 3x3 internal loop motif library that bind kanamycin A, an RNA-binding aminoglycoside antibiotic, by using only one round of selection. A small internal-loop library was chosen because members are likely to be present in other larger, biologically relevant RNAs. We have identified several internal loops of various size and base composition that kanamycin A prefers to bind. The highest affinity structures are two 5'-UU/3'-CU 2x2 internal loops closed by AU pairs. Binding is specific for the selected internal loops with the highest affinities, since binding to the RNA cassette used to display the library or to DNA is >150-fold weaker. Enzymatic mapping experiments also confirm binding of kanamycin A to the internal loops. This method lays the foundation for finding RNA secondary structure elements that bind small molecules and for interrogating factors affecting RNA-ligand interactions. Information from these and subsequent studies will: 1) facilitate the rational and modular design of drugs or probes that bind target RNAs with high affinity, provided the secondary structure of the target is known and 2) give insight into the potential bystander RNAs that aminoglycosides bind.


Assuntos
Canamicina/metabolismo , RNA/análise , Azidas/química , Sequência de Carboidratos , Hidrólise , Canamicina/química , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos/síntese química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonuclease T1/química , Ribonucleases/genética , Ribonucleases/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo
12.
J Control Release ; 107(3): 537-46, 2005 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-16087268

RESUMO

Polyethylenimine (PEI) is one of the most potent non-viral vectors. We have developed a lactosylated PEI (Lac-PEI) to enhance cell-specific transfection and have shown that Lac-PEI is more efficient than unsubstituted PEI for gene transfer into immortalized cystic fibrosis airway epithelial SigmaCFTE29o-cells. As both intact PEI/plasmid and Lac-PEI/plasmid complexes are found in the cell nucleus, we have investigated the transcription efficiency of the plasmid complexed with PEI or Lac-PEI, according to the polymer nitrogen/DNA phosphate (N/P) ratio (from 0 to 20). The initiation of transgene transcription was analyzed in an acellular nuclease S1 transcription assay. For both PEI and Lac-PEI complexes, transcription efficiency varied with the N/P ratio of the complexes. Transcription inhibition was observed when plasmid DNA was either loosely (N/P<5) or tightly condensed (N/P>15). For an N/P ratio of 5 and up to 15, transcription of the complexed plasmid was as efficient as that of the free plasmid. Similar results were observed when gene expression was studied after nuclear microinjection of the complexes into SigmaCFTE29o-cells. Our study shows that condensation of DNA influences the accessibility of the plasmid to the transcription machinery. Interestingly, the charge ratios that allow the most efficient transcription are those usually known to be the most efficient for gene transfer in vitro and in vivo.


Assuntos
DNA/genética , Plasmídeos/genética , Polietilenoimina/química , Transcrição Genética , Fenômenos Químicos , Química Física , Eletroquímica , Eletroforese em Gel de Ágar , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Lactose/química , Microinjeções , Microscopia Eletrônica , Endonucleases Específicas para DNA e RNA de Cadeia Simples/biossíntese , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética
13.
Leg Med (Tokyo) ; 5(4): 233-7, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14602167

RESUMO

The allele-specific inverse polymerase chain reaction (PCR) technique, which has been explored to detect two linked polymorphic regions simultaneously, was applied to genotype the Se system. The major alleles of the Se system in Japanese are Se, sej defined by a single nucleotide substitution in the Se allele, and se(fus) generated by recombination between the Sec1 and FUT2 genes. The first PCR products using gene-specific primers were self-ligated, and each allele was detected by the second inverse-PCR using allele-specific primers. The 340, 331 and 353-bp products were finally amplified from Se, sej and se(fus) templates, respectively. The first PCR products without mung bean nuclease treatment were not self-ligated and non-specific fragments were amplified in the second PCR, suggesting that non-templated adenylation occurred at the termini during the first PCR. Nuclease digestion of the first PCR products that blunts their termini was found to reduce interference of non-templated adenylation with the intramolecular ligation and to improve the genotyping markedly. This modified allele-specific inverse-PCR method is applicable to analyze haplotypes consisting of separated single nucleotide polymorphisms and recombinant genes.


Assuntos
Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética , Alelos , Genótipo , Humanos , Reação em Cadeia da Polimerase
14.
Sheng Wu Gong Cheng Xue Bao ; 19(2): 240-3, 2003 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-15966330

RESUMO

S1 nuclease (from Aspergillus oryzae) is a specific enzyme to degrade single stranded DNA or RNA molecules. It has been reported to be able to convert superhelical circular DNA molecules into open circle or linear forms under certain conditions, but this function has not been well explored. In order to use the action of S1 nuclease to linearize circular DNA and develop a novel way of cloning microcircular DNAs, the pUC19 was used to investigate the relationship between the linearization efficiency of S1 nuclease and the amount of enzyme used. By this way the optimal conditions for linearization of circular DNAs by S1 nuclease would be determined. 0.3u to 17u S1 nuclease per 100ng pUC19 DNA was added into a 25 microL system, respectively, to perform the reaction. The effectiveness of enzyme digestion was realized by electrophoresis in a 1.2% agarose gel. The results showed that along with the increase in enzyme amount from 0.3u to 17u a gradual decrease in the superhelical form, a gradual increase in the linear form and then in the circular form was obvious. The conversion from superhelical form to linear and circular form was directly related to the enzyme amount used. A higher proportion of linear DNA molecules was achieved by using 5 to 17u S1 nuclease per 100ng DNA. Besides, electrophoretic mobility of the S1 nuclease-linearized pUC19 was the same as that of the linear form produced by restriction enzyme digestion. According to the result of phiX174 digested by S1 nuclease it has been proposed that the enzyme cleaves first randomly on one site of one strand, thus converting the superhelical molecules into open circle form, and then on the same site of the complementary strand to produce the linear form. Therefore, the S1 nuclease-linearized DNA molecules are intact in the sense of their length and can be used for cloning. The plasmid-like DNA pC3 from cucumber mitochondria is a double stranded circular DNA molecule with about 550bp and the smallest known plasmid-like DNA in eukaryotic mitochondria. Many attempts have been made to linearize the molecule by using restriction enzymes but failed. Therefore, S1 nuclease was used to linearize pC3 based on the results obtained with pUC19. The linearized pC3 DNA molecules formed a very sharp band in a 2.5% agarose gel after electrophoresis. They were then recovered from the gel, added an "A" tail and ligated with T-vector. After transformation into E. coli JM109 cells, the positive clones were, screened by the blue-white selection. The insert was then cut using restriction enzymes EcoRI and Pst I. The result of electrophoresis shows that the electrophoretic mobility of the insert is just the same as that predicted. A 32 P-labled probe was synthesized using pC3 as the template and Southern blot analysis was carried out. The result shows that the inserted DNA is hybridized to the probe, which indicates that the cloned DNA fragment is from pC3. The sequence information of the insert shows that the plasmid-like DNA pC3 was 537bp in length. The nucleotide sequence was deposited in the GenBank (the accession number is AF522195).


Assuntos
Clonagem Molecular/métodos , DNA Circular/metabolismo , Proteínas Fúngicas/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Southern Blotting , DNA Circular/genética , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética
15.
Mol Carcinog ; 34(3): 140-50, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12112308

RESUMO

We have systematically varied microsatellite sequence composition to determine the effects of repeat unit size, G+C content, and DNA secondary structure on microsatellite stability in human cells. The microsatellites were inserted in frame within the 5' region of the herpes simplex virus thymidine kinase (HSV-tk) gene. The polypyrimidine/polypurine microsatellites displayed enhanced S1 nuclease sensitivity in vitro, consistent with the formation of non-B-form DNA structures. Microsatellite mutagenesis studies were performed with a shuttle vector system in which inactivating HSV-tk mutations are measured after replication in a nontumorigenic cell line. A significant increase in the HSV-tk mutation frequency per cell generation was observed after insertion of [TTCC/AAGG]9, [TTTC/AAAG]9, or [TCTA/AGAT]9 sequences (P

Assuntos
Repetições de Microssatélites , Mutação , Alelos , Células Cultivadas , Análise Mutacional de DNA/métodos , Feminino , Vetores Genéticos/metabolismo , Humanos , Mutagênese , Simplexvirus/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Timidina Quinase/genética
16.
Mol Microbiol ; 40(4): 804-14, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11401688

RESUMO

Morphological changes leading to aerial mycelium formation and sporulation in the mycelial bacterium Streptomyces coelicolor rely on establishing distinct patterns of gene expression in separate regions of the colony. sigmaH was identified previously as one of three paralogous sigma factors associated with stress responses in S. coelicolor. Here, we show that sigH and the upstream gene prsH (encoding a putative antisigma factor of sigmaH) form an operon transcribed from two developmentally regulated promoters, sigHp1 and sigHp2. While sigHp1 activity is confined to the early phase of growth, transcription of sigHp2 is dramatically induced at the time of aerial hyphae formation. Localization of sigHp2 activity using a transcriptional fusion to the green fluorescent protein reporter gene (sigHp2-egfp) showed that sigHp2 transcription is spatially restricted to sporulating aerial hyphae in wild-type S. coelicolor. However, analysis of mutants unable to form aerial hyphae (bld mutants) showed that sigHp2 transcription and sigmaH protein levels are dramatically upregulated in a bldD mutant, and that the sigHp2-egfp fusion was expressed ectopically in the substrate mycelium in the bldD background. Finally, a protein possessing sigHp2 promoter-binding activity was purified to homogeneity from crude mycelial extracts of S. coelicolor and shown to be BldD. The BldD binding site in the sigHp2 promoter was defined by DNase I footprinting. These data show that expression of sigmaH is subject to temporal and spatial regulation during colony development, that this tissue-specific regulation is mediated directly by the developmental transcription factor BldD and suggest that stress and developmental programmes may be intimately connected in Streptomyces morphogenesis.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA , Fator sigma/genética , Streptomyces/fisiologia , Fatores de Transcrição , Proteínas de Bactérias/metabolismo , Sequência de Bases , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Etanol/farmacologia , Regulação Bacteriana da Expressão Gênica , Proteínas de Fluorescência Verde , Resposta ao Choque Térmico , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Mutação , Óperon , Regiões Promotoras Genéticas , Fator sigma/efeitos dos fármacos , Fator sigma/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Esporos Bacterianos , Streptomyces/efeitos dos fármacos , Streptomyces/crescimento & desenvolvimento , Transcrição Genética
17.
DNA Seq ; 12(5-6): 355-9, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11913781

RESUMO

Obtaining the complete DNA sequence of a genome is often not straightforward. After standard shotgun sequencing strategies have been employed there are often gaps remaining and these can be the most intractable regions, frequently containing repeat sequences, "uncloneable" sequences and/or regions of potential secondary structure or differential base composition. In genomes with a high A/T content, such as Plasmodium falciparum and Dictyostelium discoideum, solving these gaps is a particularly difficult problem as the sequences concerned are "fragile" and easily denatured, commonly uncloneable and have a paucity of good oligonucleotide priming sites. Reported here is a simple, yet reliable method for determining the sequence of A/T-rich gap-spanning PCR products. This method relies on the slippage of the specificity of mung bean nuclease so that it digests A/T-rich double-stranded DNA into a set of deletion fragments that can then be cloned into M13, sequenced and the original sequence assembled therefrom.


Assuntos
Sequência Rica em At/genética , Genoma , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética , Animais , Bacteriófago M13 , Clonagem Molecular , Vetores Genéticos , Plasmodium falciparum , Análise de Sequência de DNA/métodos
18.
Med Dosw Mikrobiol ; 52(1): 67-74, 2000.
Artigo em Polonês | MEDLINE | ID: mdl-11107780

RESUMO

The aim of this study was standardization of PCR for the detection of gene encoding the P1 protein, 16S rRNA and elongation factor Tu of M. pneumoniae. A total of 13 strains of M. pneumoniae, 28 strains of other mycoplasmas and 14 strains of different bacteria causing respiratory tract infections were tested. In all of tested M. pneumoniae strains the presence of the sought genes was confirmed. The specificity of DNA was confirmed by the restriction endonuclease analysis with enzymes Hind III, Alu I and Hha I. With none of primers specific for the M. pneumoniae genes amplification of DNA from other bacteria was noted. The PCR method with the selected primers allowed to detect from 10(2) to 10(4) cfu M. pneumoniae/ml suspended in broth. The obtained results indicate that the PCR method can be used for detection of M. pneumoniae genes. A very good sensitivity and specificity predestine++ PCR as a potential quick diagnostic method for identification of M. pneumoniae in clinical specimens.


Assuntos
DNA Bacteriano/isolamento & purificação , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/isolamento & purificação , Reação em Cadeia da Polimerase/normas , Mycoplasma pneumoniae/classificação , Mycoplasma pneumoniae/patogenicidade , Fator Tu de Elongação de Peptídeos/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 16S/isolamento & purificação , Sensibilidade e Especificidade , Sorotipagem/métodos , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética , Especificidade da Espécie
19.
Biochem Biophys Res Commun ; 240(1): 203-7, 1997 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-9367910

RESUMO

Anti-gene is a potent inhibitor of transcriptional promoter activity and subsequent gene expression. This property has been exploited to suppress the expression of a variety of oncogenes for regulating tumor proliferation or viral activities. In this paper, we describe a novel retroviral vector designed to express human c-erbB anti-gene RNA and to reduce the promoter activity in the cells. Mouse fibroblast NIH3T3 cells were stably transfected with an expression construct containing a truncated human c-erbB gene promoter fused to the firefly luciferase reporter gene. Infection into these cells of the c-erbB anti-gene retroviral vector targeted to the 26 bp pyrimidine-rich element in the human c-erbB gene promoter resulted in a dose-dependent decrease in the luciferase activity of the cells. Retroviral vector expressing anti-gene RNA may be useful as an alternative program of gene regulation in the cells.


Assuntos
Regulação da Expressão Gênica , Genes erbB , Vetores Genéticos/metabolismo , Regiões Promotoras Genéticas , Retroviridae/genética , Células 3T3 , Animais , Sequência de Bases , Citomegalovirus/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Genes erbB/efeitos dos fármacos , Vetores Genéticos/farmacologia , Humanos , Luciferases/genética , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA/biossíntese , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética
20.
J Neurochem ; 67(1): 26-36, 1996 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-8666999

RESUMO

The cis elements mediating activation of the tyrosine hydroxylase gene by angiotensin II were examined by transfecting tyrosine hydroxylase promoter-luciferase constructs into cultured bone adrenal medullary cells. Angiotensin II-responsive elements are located within -54/+25-bp and -269/-55-bp promoter regions and were identified, respectively, as cyclic AMP (CRE)- and 12-O-tetradecanoylphorbol 13-acetate responsive element (TRE)-like sequences. Unlike CRE, TRE also supports basal promoter activity. Mutations of TRE or CRE that reduced angiotensin II stimulation abolished in vitro binding of nuclear proteins to those elements, suggesting that proteins forming CRE- and TRE-inducible complexes may mediate angiotensin II stimulation. The TRE is adjacent to a dyad symmetry element. Those two sites form a common regulatory unit in which the dyad symmetry element acts as a repressor of the TRE site. Isolated dyad symmetry element did not bind nuclear proteins in vitro. In supercoiled DNA it exhibited S1 nuclease sensitivity and was recognized by a DNA cruciform-specific antibody consistent with the extrusion of a cruciform structure that overlaps with the TRE. A mutation that abolished formation of the cruciform correlated with a loss of repressor activity. We propose a novel model of tyrosine hydroxylase gene regulation in which functions of the TRE are modulated via structural transition in the adjacent DNA.


Assuntos
Angiotensina II/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Endopeptidases , Proteínas de Fusão Oncogênica/genética , Proteínas Oncogênicas , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética , Tirosina 3-Mono-Oxigenase/genética , Animais , Sequência de Bases , Bovinos , Células Cultivadas/enzimologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , DNA/análise , DNA/química , DNA/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Teste de Complementação Genética , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Proteínas de Fusão Oncogênica/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas , Endonucleases Específicas para DNA e RNA de Cadeia Simples/química , Ubiquitina Tiolesterase
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