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1.
Sci Total Environ ; 703: 134977, 2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-31757553

RESUMO

The optimization of more sustainable fertilization practice to relieve phosphorus (P) resource scarcity and increase P fertilizer utilization, a better understanding of the regulatory roles of microbes in P mobilization is urgently required to reduce P input. The genes phoD and pqqC are responsible for regulating organic and inorganic P mobilization, respectively. Using high-throughput sequencing, the corresponding bacterial communities harbored by these genes were determined. We conducted a 4-year rice-rice-crop rotation to investigate the responses of phoD- and pqqC-harboring bacterial communities to the partial replacement of inorganic P fertilizer by organic manure with reduced P input. The results showed that a combination of organic and inorganic fertilization maintained high rice yield, and also produced a more complex and stable phosphate mobilizing bacterial community, which contributed to phosphatase activities more than their gene abundances in the model analysis. Compared with the conventional mineral fertilization, organic-inorganic fertilization with the reduced P input slightly increased pqqC gene abundance while significantly enhanced the abundance of phoD-harboring bacteria, especially the genera Bradyrhizobium and Methylobacterium known as potential organic P mineralizers which can maintain high rice production. Moreover, the increased pH was the most impactful factor for the phoD- and pqqC-harboring bacterial communities, by promoting microbial P turnover and greatly increasing bioavailable P pools (H2O-Pi and NaHCO3-Pi, NaOH-Pi) in this P-deficient paddy soil. Hence, our study demonstrated that the partial replacement of mineral P with organic manure could reshape the inorganic phosphate solubilizing and alkaline-phosphomonoesterase encoding bacterial communities towards more resilient and effective to the high P utilization and productivity over intense cultivation, providing insights into the potential of soil microbes in the efficient management of agricultural P fertilization.

2.
Sci Total Environ ; 666: 778-785, 2019 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-30812011

RESUMO

Livestock manure has gradually become an alternative fertilizer for maintaining soil fertility, whereas excessive application of manure leads to the release of phosphorus (P) and toxic metals that may cause complex environmental risks. To investigate the accumulation and migration of P within soil profiles, a mesocosm experiment was conducted to analyze the content and leaching of soil P, metals, and dissolved organic carbon after different fertilization treatments, including control (no fertilizer, CK), chemical fertilizer (CF), chemical fertilizer combined low (CF + LPM) and high (CF + HPM) rate of manure application. Results showed that a high rate of manure application significantly enhanced the accumulation of total soil P (by ~14%) and P availability (easily-available P, by ~24%; Olsen-P, by ~20%) in topsoil, and also increased the content of easily-available organic P (EA-Po) in both topsoil and subsoil compared to the CK treatment. The migration of dissolved inorganic and organic P (DIP and DOP) in leachate within soil profiles was strengthened by manure application. Moreover, significant positive correlations between P, metals, and dissolved organic carbon (DOC) in leachate indicated that downward co-migration occurred within the soil profiles, and also suggested that excessive manure application can intensify the risk of P loss by increasing the migration of manure-derived DOC. Overall, our findings provide insights into P accumulation and migration within soil profiles after excessive manure application, which is useful for predicting the potential risk of P and metal leaching from paddy soils.

3.
Int J Mol Sci ; 20(1)2018 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-30586940

RESUMO

Endonuclease IV (EndoIV) is a DNA damage-specific endonuclease that mainly hydrolyzes the phosphodiester bond located at 5' of an apurinic/apyrimidinic (AP) site in DNA. EndoIV also possesses 3'-exonuclease activity for removing 3'-blocking groups and normal nucleotides. Here, we report that Thermococcus eurythermalis EndoIV (TeuendoIV) shows AP endonuclease and 3'-exonuclease activities. The effect of AP site structures, positions and clustered patterns on the activity was characterized. The AP endonuclease activity of TeuendoIV can incise DNA 5' to various AP site analogues, including the alkane chain Spacer and polyethylene glycol Spacer. However, the short Spacer C2 strongly inhibits the AP endonuclease activity. The kinetic parameters also support its preference to various AP site analogues. In addition, the efficient cleavage at AP sites requires ≥2 normal nucleotides existing at the 5'-terminus. The 3'-exonuclease activity of TeuendoIV can remove one or more consecutive AP sites at the 3'-terminus. Mutations on the residues for substrate recognition show that binding AP site-containing or complementary strand plays a key role for the hydrolysis of phosphodiester bonds. Our results provide a comprehensive biochemical characterization of the cleavage/removal of AP site analogues and some insight for repairing AP sites in hyperthermophile cells.


Assuntos
DNA de Cadeia Simples/química , DNA/química , Desoxirribonuclease IV (Fago T4-Induzido)/metabolismo , Thermococcus/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , DNA/metabolismo , Clivagem do DNA , Reparo do DNA , DNA de Cadeia Simples/metabolismo , Desoxirribonuclease IV (Fago T4-Induzido)/classificação , Desoxirribonuclease IV (Fago T4-Induzido)/genética , Cinética , Filogenia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Especificidade por Substrato
4.
Nucleic Acids Res ; 46(17): 9027-9043, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30102394

RESUMO

Nucleases play important roles in nucleic acid metabolism. Some archaea encode a conserved protein known as Hef-associated nuclease (HAN). In addition to its C-terminal DHH nuclease domain, HAN also has three N-terminal domains, including a DnaJ-Zinc-finger, ribosomal protein S1-like, and oligonucleotide/oligosaccharide-binding fold. To further understand HAN's function, we biochemically characterized the enzymatic properties of HAN from Pyrococcus furiosus (PfuHAN), solved the crystal structure of its DHH nuclease domain, and examined its role in DNA repair. Our results show that PfuHAN is a Mn2+-dependent 3'-exonuclease specific to ssDNA and ssRNA with no activity on blunt and 3'-recessive double-stranded DNA. Domain truncation confirmed that the intrinsic nuclease activity is dependent on the C-terminal DHH nuclease domain. The crystal structure of the DHH nuclease domain adopts a trimeric topology, with each subunit adopting a classical DHH phosphoesterase fold. Yeast two hybrid assay confirmed that the DHH domain interacts with the IDR peptide of Hef nuclease. Knockout of the han gene or its C-terminal DHH nuclease domain in Haloferax volcanii resulted in increased sensitivity to the DNA damage reagent MMS. Our results imply that HAN nuclease might be involved in repairing stalled replication forks in archaea.


Assuntos
Proteínas Arqueais/química , Reparo do DNA , DNA de Cadeia Simples/química , Exonucleases/química , Pyrococcus furiosus/enzimologia , RNA Arqueal/química , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Cátions Bivalentes , Clonagem Molecular , Cristalografia por Raios X , Quebras de DNA de Cadeia Simples , Dano ao DNA , Replicação do DNA , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exonucleases/genética , Exonucleases/metabolismo , Expressão Gênica , Haloferax volcanii/química , Haloferax volcanii/efeitos dos fármacos , Haloferax volcanii/enzimologia , Haloferax volcanii/genética , Cinética , Manganês/química , Manganês/metabolismo , Metanossulfonato de Metila/farmacologia , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Pyrococcus furiosus/química , Pyrococcus furiosus/efeitos dos fármacos , Pyrococcus furiosus/genética , RNA Arqueal/genética , RNA Arqueal/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
5.
DNA Repair (Amst) ; 65: 54-63, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29609115

RESUMO

In cells, degrading DNA and RNA by various nucleases is very important. These processes are strictly controlled and regulated to maintain DNA integrity and to mature or recycle various RNAs. NanoRNase (Nrn) is a 3'-exonuclease that specifically degrades nanoRNAs shorter than 5 nucleotides. Several Nrns have been identified and characterized in bacteria, mainly in Firmicutes. Archaea often grow in extreme environments and might be subjected to more damage to DNA/RNA, so DNA repair and recycling of damaged RNA are very important in archaea. There is no report on the identification and characterization of Nrn in archaea. Aeropyrum pernix encodes three potential Nrns: NrnA (Ape1437), NrnB (Ape0124), and an Nrn-like protein Ape2190. Biochemical characterization showed that only Ape0124 could degrade ssDNA and ssRNA from the 3'-end in the presence of Mn2+. Interestingly, unlike bacterial Nrns, Ape0124 prefers ssDNA, including short nanoDNA, and degrades nanoRNA with lower efficiency. The 3'-DNA backbone was found to be required for efficiently hydrolyzing the phosphodiester bonds. In addition, Ape0124 also degrads the 3'-overhang of double-stranded DNA. Interestingly, Ape0124 could hydrolyze pAp into AMP, which is a feature of bacterial NrnA, not NrnB. Our results indicate that Ape0124 is a novel Nrn with a combined substrate profile of bacterial NrnA and NrnB.


Assuntos
Aeropyrum/enzimologia , DNA de Cadeia Simples/metabolismo , Desoxirribonucleases/metabolismo , RNA/metabolismo , Ribonucleases/metabolismo , Proteínas Arqueais/metabolismo , Especificidade por Substrato
6.
Genes (Basel) ; 8(9)2017 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-28837073

RESUMO

Bacterial nuclease RecJ, which exists in almost all bacterial species, specifically degrades single-stranded (ss) DNA in the 5' to 3' direction. Some archaeal phyla, except Crenarchaea, also encode RecJ homologs. Compared with bacterial RecJ, archaeal RecJ exhibits a largely different amino acid sequence and domain organization. Archaeal RecJs from Thermococcus kodakarensis and Pyrococcus furiosus show 5'→3' exonuclease activity on ssDNA. Interestingly, more than one RecJ exists in some Euryarchaeota classes, such as Methanomicrobia, Methanococci, Methanomicrobia, Methanobacteria, and Archaeoglobi. Here we report the biochemical characterization of two RecJs from Methanocaldococcus jannaschii, the long RecJ1 (MJ0977) and short RecJ2 (MJ0831) to understand their enzymatic properties. RecJ1 is a 5'→3' exonuclease with a preference to ssDNA; however, RecJ2 is a 3'→5' exonuclease with a preference to ssRNA. The 5' terminal phosphate promotes RecJ1 activity, but the 3' terminal phosphate inhibits RecJ2 nuclease. Go-Ichi-Ni-San (GINS) complex does not interact with two RecJs and does not promote their nuclease activities. Finally, we discuss the diversity, function, and molecular evolution of RecJ in archaeal taxonomy. Our analyses provide insight into the function and evolution of conserved archaeal RecJ/eukaryotic Cdc45 protein.

7.
Genes (Basel) ; 8(1)2017 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-28106786

RESUMO

Sulfolobus acidocaldarius encodes family 4 and 5 uracil-DNA glycosylase (UDG). Two recombinant S. acidocaldarius UDGs (SacUDG) were prepared and biochemically characterized using oligonucleotides carrying a deaminated base. Both SacUDGs can remove deoxyuracil (dU) base from both double-stranded DNA and single-stranded DNA. Interestingly, they can remove U linked with deoxyribose from single-stranded RNA backbone, suggesting that the riboses on the backbone have less effect on the recognition of dU and hydrolysis of the C-N glycosidic bond. However, the removal of rU from DNA backbone is inefficient, suggesting strong steric hindrance comes from the 2' hydroxyl of ribose linked to uracil. Both SacUDGs cannot remove 2,2'-anhydro uridine, hypoxanthine, and 7-deazaxanthine from single-stranded DNA and single-stranded DNA. Compared with the family 2 MUG, other family UDGs have an extra N-terminal structure consisting of about 50 residues. Removal of the 46 N-terminal residues of family 5 SacUDG resulted in only a 40% decrease in activity, indicating that the [4Fe-4S] cluster and truncated secondary structure are not the key elements in hydrolyzing the glycosidic bond. Combining our biochemical and structural results with those of other groups, we discussed the UDGs' catalytic mechanism and the possible repair reactions of deaminated bases in prokaryotes.

8.
Nucleic Acids Res ; 45(21): 12551-12564, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30053256

RESUMO

RecJ nucleases specifically degrade single-stranded (ss) DNA in the 5' to 3' direction. Archaeal RecJ is different from bacterial RecJ in sequence, domain organization, and substrate specificity. The RecJ from archaea Pyrococcus furiosus (PfuRecJ) also hydrolyzes RNA strands in the 3' to 5' direction. Like eukaryotic Cdc45 protein, archaeal RecJ forms a complex with MCM helicase and GINS. Here, we report the crystal structures of PfuRecJ and the complex of PfuRecJ and two CMPs. PfuRecJ bind one or two divalent metal ions in its crystal structure. A channel consisting of several positively charged residues is identified in the complex structure, and might be responsible for binding substrate ssDNA and/or releasing single nucleotide products. The deletion of the complex interaction domain (CID) increases the values of kcat/Km of 5' exonuclease activity on ssDNA and 3' exonuclease activity on ssRNA by 5- and 4-fold, respectively, indicating that the CID functions as a regulator of enzymatic activity. The DHH domain of PfuRecJ interacts with the C-terminal beta-sheet domain of the GINS51 subunit in the tetrameric GINS complex. The relationship of archaeal and bacterial RecJs, as well as eukaryotic Cdc45, is discussed based on biochemical and structural results.


Assuntos
Proteínas de Bactérias/química , Exodesoxirribonucleases/química , Pyrococcus furiosus/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/fisiologia , Cátions , Proteínas de Ciclo Celular , Sequência Conservada , Cristalografia por Raios X , Reparo do DNA , Replicação do DNA , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Exodesoxirribonucleases/fisiologia , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Fosfodiesterase I/metabolismo , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
9.
Front Microbiol ; 6: 943, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26441878

RESUMO

Hyperthermophile Pyrococcus furiosus grows optimally near 100°C and is an important resource of many industrial and molecular biological enzymes. To study the structure and function of P. furiosus proteins at whole genome level, we constructed expression plasmids of each P. furiosus gene using a ligase-independent cloning method, which was based on amplifying target gene and vector by PCR using phosphorothioate-modified primers and digesting PCR products by λ exonuclease. Our cloning method had a positive clone percentage of ≥ 80% in 96-well plate cloning format. Small-scale expression experiment showed that 55 out of 80 genes were efficiently expressed in Escherichia coli Strain Rosetta 2(DE3)pLysS. In summary, this recombinant expression library of P. furiosus provides a platform for functional and structural studies, as well as developing novel industrial enzymes. Our cloning scheme is adaptable to constructing recombinant expression library of other sequenced organisms.

10.
Nucleic Acids Res ; 41(11): 5817-26, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23605041

RESUMO

Replicative DNA polymerases require an RNA primer for leading and lagging strand DNA synthesis, and primase is responsible for the de novo synthesis of this RNA primer. However, the archaeal primase from Pyrococcus furiosus (Pfu) frequently incorporates mismatched nucleoside monophosphate, which stops RNA synthesis. Pfu DNA polymerase (PolB) cannot elongate the resulting 3'-mismatched RNA primer because it cannot remove the 3'-mismatched ribonucleotide. This study demonstrates the potential role of a RecJ-like protein from P. furiosus (PfRecJ) in proofreading 3'-mismatched ribonucleotides. PfRecJ hydrolyzes single-stranded RNA and the RNA strand of RNA/DNA hybrids in the 3'-5' direction, and the kinetic parameters (Km and Kcat) of PfRecJ during RNA strand digestion are consistent with a role in proofreading 3'-mismatched RNA primers. Replication protein A, the single-stranded DNA-binding protein, stimulates the removal of 3'-mismatched ribonucleotides of the RNA strand in RNA/DNA hybrids, and Pfu DNA polymerase can extend the 3'-mismatched RNA primer after the 3'-mismatched ribonucleotide is removed by PfRecJ. Finally, we reconstituted the primer-proofreading reaction of a 3'-mismatched ribonucleotide RNA/DNA hybrid using PfRecJ, replication protein A, Proliferating cell nuclear antigen (PCNA) and PolB. Given that PfRecJ is associated with the GINS complex, a central nexus in archaeal DNA replication fork, we speculate that PfRecJ proofreads the RNA primer in vivo.


Assuntos
Proteínas Arqueais/metabolismo , Replicação do DNA , Exorribonucleases/metabolismo , Pyrococcus furiosus/enzimologia , RNA/metabolismo , Pareamento Incorreto de Bases , DNA/química , DNA/metabolismo , DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Pyrococcus furiosus/genética , RNA/química
11.
DNA Repair (Amst) ; 12(2): 140-7, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23291401

RESUMO

DNA polymerase I (DNApolI) catalyzes DNA synthesis during Okazaki fragment maturation, base excision repair, and nucleotide excision repair. Some bacterial DNApolIs are deficient in 3'-5' exonuclease, which is required for removing an incorrectly incorporated 3'-terminal nucleotide during DNA elongation by DNA polymerase activity. The key amino acid residues in the exonuclease center of Chlamydophila pneumoniae DNApolI (CpDNApolI) are naturally mutated, resulting in the loss of 3'-5' exonuclease. Hence, the manner by which CpDNApolI proofreads the incorrectly incorporated nucleotide during DNA synthesis warrants clarification. C. pneumoniae encodes three 3'-5' exonuclease activities: one endonuclease IV and two homologs of the epsilon subunit of replicative DNA polymerase III. The three proteins were biochemically characterized using single- and double-stranded DNA substrate. Among them, C. pneumoniae endonuclease IV (CpendoIV) possesses 3'-5' exonuclease activity that prefers to remove mismatched 3'-terminal nucleotides in the nick, gap, and 3' recess of a double-stranded DNA (dsDNA). Finally, we reconstituted the proofreading reaction of the mismatched 3'-terminal nucleotide using the dsDNA with a nick or 3' recess as substrate. Upon proofreading of the mismatched 3'-terminal nucleotide by CpendoIV, CpDNApolI can correctly reincorporate the matched nucleotide and the nick is further sealed by DNA ligase. Based on our biochemical results, we proposed that CpendoIV was responsible for proofreading the replication errors of CpDNApolI.


Assuntos
Proteínas de Bactérias/metabolismo , Chlamydophila pneumoniae/enzimologia , Reparo de Erro de Pareamento de DNA , DNA de Cadeia Simples/metabolismo , Desoxirribonuclease IV (Fago T4-Induzido)/metabolismo , Ribonucleotídeos/metabolismo , Pareamento Incorreto de Bases , Quebras de DNA de Cadeia Simples , DNA Bacteriano/biossíntese
12.
Protein Expr Purif ; 87(2): 79-86, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23147204

RESUMO

We describe the biochemical characterization of Methanocaldococcus jannaschii (M. jannaschii) DNA ligase and its potential application in single nucleotide polymorphism (SNP) genotyping. The recombinant M. jannaschii DNA ligase is an ATP-dependent ligase. The ligase activity was dependent on metal ions of Mg(2+) and Mn(2+). The optimal concentrations of ATP cofactor and Mg(2+) ion were 0.01-2 and 10 mM, respectively. The optimal pH value for DNA ligation was 8.5. High concentrations of NaCl inhibited DNA ligation. The effects of mismatches on joining short oligonucleotides by M. jannaschii DNA ligase were fully characterized. The mismatches at the first position 5' to the nick inhibited ligation more than those at the first position 3' to the nick. The mismatches at other positions 5' to the nick (3rd to 7th sites) exhibited less inhibition on ligation. However, the introduction of a C/C mismatch at the third position 5' to the nick could completely inhibit the ligation of the terminal-mismatched nick of an oligonucleotide duplex by M. jannaschii DNA ligase. Therefore, introducing an additional mismatch at the third position 5' to the SNP site is a more effective approach in genotyping by M. jannaschii DNA ligase.


Assuntos
Proteínas de Bactérias/biossíntese , DNA Ligases/biossíntese , Técnicas de Genotipagem/métodos , Methanococcales/enzimologia , Proteínas Recombinantes/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Pareamento Incorreto de Bases , DNA Ligases/química , DNA Ligases/genética , DNA Ligases/isolamento & purificação , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Methanococcales/genética , Polimorfismo de Nucleotídeo Único , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Cloreto de Sódio/química
13.
PLoS One ; 6(11): e27248, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22087273

RESUMO

Recombinant uracil-DNA glycosylase (UDG) from Aeropyrum pernix (A. pernix) was expressed in E. coli. The biochemical characteristics of A. pernix UDG (ApeUDG) were studied using oligonucleotides carrying a deoxyuracil (dU) base. The optimal temperature range and pH value for dU removal by ApeUDG were 55-65°C and pH 9.0, respectively. The removal of dU was inhibited by the divalent ions of Zn, Cu, Co, Ni, and Mn, as well as a high concentration of NaCl. The opposite base in the complementary strand affected the dU removal by ApeUDG as follows: U/C≈U/G>U/T≈U/AP≈U/->U/U≈U/I>U/A. The phosphorothioate around dU strongly inhibited dU removal by ApeUDG. Based on the above biochemical characteristics and the conservation of amino acid residues, ApeUDG was determined to belong to the IV UDG family. ApeUDG increased the yield of PCR by Pfu DNA polymerase via the removal of dU in amplified DNA. Using the dU-carrying oligonucleotide as an inhibitor and ApeUDG as an activator of Pfu DNA polymerase, the yield of undesired DNA fragments, such as primer-dimer, was significantly decreased, and the yield of the PCR target fragment was increased. This strategy, which aims to amplify the target gene with high specificity and yield, can be applied to all family B DNA polymerases.


Assuntos
Aeropyrum/enzimologia , DNA Polimerase Dirigida por DNA/metabolismo , Reação em Cadeia da Polimerase/métodos , Uracila-DNA Glicosidase/metabolismo , Cátions Bivalentes , Fragmentação do DNA , Desoxiuridina , Concentração de Íons de Hidrogênio , Cloreto de Sódio , Temperatura , Uracila-DNA Glicosidase/genética
14.
Protein Sci ; 19(5): 967-73, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20217896

RESUMO

Function studies of many proteins are waited to develop after genome sequencing. High-throughout technology of gene cloning will strongly promote proteins' function studies. Here we describe a ligation-independent cloning (LIC) method, which is based on the amplification of target gene and linear vector by PCR using phosphorothioate-modified primers and the digestion of PCR products by lambda exonuclease. The phosphorothioate inhibits the digestion and results in the generation of 3' overhangs, which are designed to form complementary double-stranded DNA between target gene and linear vector. We compared our phosphorothioate primer cloning methods with several LIC methods, including dU primer cloning, hybridization cloning, T4 DNA polymerase cloning, and in vivo recombination cloning. The cloning efficiency of these LIC methods are as follows: phosphorothioate primer cloning > dU primer cloning > hybridization cloning > T4 DNA polymerase cloning >> in vivo recombination cloning. Our result shows that the 3' overhangs is a better cohesive end for LIC than 5' overhang and the existence of 5'phosphate promotes DNA repair in Escherichia coli, resulting in the improvement of cloning efficiency of LIC. We succeeded in constructing 156 expression plasmids of Aeropyrum pernix genes within a week using our method.


Assuntos
Aeropyrum/genética , Clonagem Molecular/métodos , Oligonucleotídeos Fosforotioatos/metabolismo , Aeropyrum/metabolismo , Exonucleases/metabolismo , Genes Bacterianos , Oligonucleotídeos Fosforotioatos/química
15.
Anal Biochem ; 398(1): 83-92, 2010 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-19891952

RESUMO

Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation. SNPs are important markers that link sequence variations to phenotypic changes. Because of the importance of SNPs in the life and medical sciences, a great deal of effort has been devoted to developing accurate, rapid, and cost-effective technologies for SNP analysis. In this article, we describe a novel method for SNP genotyping based on differential fluorescence emission due to cleavage by Thermus thermophilus RNase HII (TthRNase HII) of DNA heteroduplexes containing an SNP site-specific chimeric DNA-rN(1)-DNA molecular beacon (cMB). We constructed a loop sequence for a cMB that contains a single SNP-specific ribonucleotide at the central site. When the cMB probe is hybridized to a target double-stranded DNA (dsDNA), a perfect match of the cMB/DNA duplex permits efficient cleavage with TthRNase HII, whereas a mismatch in the duplex due to an SNP greatly reduces efficiency. Cleavage efficiency is measured by the incremental difference of fluorescence emission of the beacon. We show that the genotypes of 10 individuals at 12 SNP sites across a series of human leukocyte antigen (HLA) can be determined correctly with respect to conventional DNA sequencing. This novel TthRNase HII-based method offers a platform for easy and accurate SNP analysis.


Assuntos
Sondas de Oligonucleotídeos/química , Polimorfismo de Nucleotídeo Único , Ribonuclease H/metabolismo , Ribonucleotídeos/química , Espectrometria de Fluorescência , Pareamento Incorreto de Bases , Sequência de Bases , Clivagem do DNA , Corantes Fluorescentes/química , Genótipo , Antígenos HLA/genética , Humanos , Ácidos Nucleicos Heteroduplexes/química , Análise de Sequência de DNA
16.
Protein Expr Purif ; 70(1): 1-6, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19825417

RESUMO

The recombinant thymine-DNA glycosylase (TDG) from Aeropyrum pernix (A. pernix) was expressed in Escherichia coli. The enzymatic activity of recombinant A. pernix TDG (ApeTDG) was characterized using oligonucleotides containing a thymine/uracil base as substrate. ApeTDG had distinct glycosylase activity on T/G mismatch. The optimal temperature and pH for thymine removal were 65-70 degrees C and pH 7.0-8.5, respectively. High concentration of NaCl inhibited the thymine removal. Divalent ions had different influence on the thymine removal by ApeTDG. Ca(2+) and Mg(2+) had no inhibition on the enzymic activity, but Ni(2+), Co(2+), Cu(2+), Mn(2+), and Zn(2+) completely inhibited the excision reaction. As derived from a hyperthermophilic archaea, ApeTDG protein was heat-resistant at 75 degrees C. ApeTDG also had a relatively weak DNA glycosylase activity on uracil base, with the following order: U/C>U/G approximately U/T>U/U approximately U/I approximately U/AP approximately U/->U/A. Additional mismatch located at 3' of T/G had less inhibition on the thymine removal than that located at 5' of T/G, and two additional mismatches located at each side of T/G completely inhibited the excision of thymine. Together, these data suggest that ApeTDG is a TDG protein with weak UDG activity.


Assuntos
Aeropyrum/enzimologia , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Timina DNA Glicosilase/química , Timina DNA Glicosilase/metabolismo , Aeropyrum/metabolismo , Proteínas Arqueais/isolamento & purificação , Sequência de Bases , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Temperatura , Timina/química , Timina/metabolismo , Timina DNA Glicosilase/isolamento & purificação
17.
Wei Sheng Wu Xue Bao ; 47(4): 721-4, 2007 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-17944381

RESUMO

Attempts to transform Klebsiella pneumoniae resulted in very low efficiencies because of capsule polysaccharide (CPS). It was reported that some chelating agents could reduce CPS production and improve transformation efficiency. These methods mentioned above could not improve transformation efficiency apparently by incorporating such agents to liquid medium. However, this method introduces a simple way for efficient transformation of K. pneumoniae. In this method, K. pneumoniae strains NTUH-K2044 and magA(-) mutant are envolved as recipients. The plasmids used in this way are composed of pIP843T, pIP843TdhaB, pIP843TdhaT with different sizes. The sole critical step is to harvest bacteria on LB plates to prepare competent cells. 150 +/- 10, 1.3 x 10(3) +/- 100, 2 x 10(5) +/- 300, and 3.4 x 10(7) +/- 500 transformants were obtained per microgram plasmid DNA with NTUH-K2044 liquid cells, magA(-) liquid cells, NTUH-K2044 solid cells, and magA(-) solid cells, respectively. The number of transformants per microg DNA obtained by electroplating solid cells is at least 10(3) fold higher than that of transformants with liquid-cultured bacteria. This method will benefit gene manipulation and genetic study in K. pneumoniae.


Assuntos
Eletroporação/métodos , Klebsiella pneumoniae/genética , Plasmídeos/genética , Transformação Bacteriana
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