OBJECTIVETo investigate the expression of 14-3-3 sigma and heat shock protein 27 (HSP27) in colorectal carcinoma(CRC) tissue and its clinical significance.

METHODSThe expression of 14-3-3 sigma and HSP27 was detected by immunohistochemical staining in 50 pathologically verified CRC cases. The association of clinical data with 14-3-3 sigma and HSP27 expression was examined.

RESULTSThe positive expression rate of 14-3-3 sigma was 10% in normal control mucosa and 58% in CRC tissue (P<0.01). The positive expression rate of HSP27 was 16% in normal control mucosa and 54% in CRC tissue (P<0.01). No correlation between 14-3-3 sigma and HSP27 expression was found in CRC tissue (P>0.05). The expression of 14-3-3 sigma was associated with patient age, tumor diameter and lymph node metastasis (LNM) (P<0.05), but not with gender, tumor differentiation or serous membrane invasion (P>0.05). The expression of HSP27 was associated with LNM (P<0.05), but not with gender, age, differentiation, tumor diameter or serous membrane invasion (P>0.05).

CONCLUSIONThe abnormal expression of 14-3-3 sigma and HSP27 is significantly associated with LNM in CRC.

14-3-3 Proteins ; Adult ; Aged ; Biomarkers, Tumor ; metabolism ; Colorectal Neoplasms ; metabolism ; pathology ; Exonucleases ; metabolism ; Exoribonucleases ; Female ; HSP27 Heat-Shock Proteins ; metabolism ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Proteins ; metabolism ; Young Adult

Exonuclease Ⅷ (Exo Ⅷ), an ATP-independent dsDNA 5'-3' exonuclease, is a candidate protein with great application value for in vitro DNA recombination. However, the application of Exo Ⅷ in DNA recombination in vitro has not been reported. In this study, the recombinant expression vector of the truncated Exo Ⅷ (tExo Ⅷ) with the full exonuclease activity was built and used to achieve the overexpression of tExo Ⅷ in Escherichia coli. Based on the purified tExo Ⅷ protein with high-purity, the feasibility of tExo Ⅷ applied in vitro DNA recombination and effects of the reaction temperatures, reaction duration, and homology arm lengths were examined. The results showed that tExo Ⅷ was highly expressed in soluble form in E. coli. One liter of bacterial culture yielded 92.40 mg of purified tExo Ⅷ with the specific activity of 1.21×10⁵ U/mg. In a 10 μL recombination system containing 2.5 U tExo Ⅷ, the highest cloning efficiency was achieved in a reaction at 25 °C for 12.5 min and followed by incubation at 50 °C for 50 min. With addition of Pfu DNA polymerase, the homology arm extension strategy can effectively improve the recombination efficiency. Using competent E. coli Mach1 T1 with 2.2×10⁶ cfu/μg transformation efficiency as recipient cell, the recombination of a 1 kb fragment with a 21 bp homology arm and a 5.8 kb linearized vector can form about 1.1×10⁴ recombinant clones per μg vector, and the positive rates was over 80%. The recombination efficiency was increased with the increasing length of homology arm ranged from 8 to 21 bp. Under the optimal reaction condition, only 8 bp homology arm can still achieve valid DNA recombination. This novel in vitro DNA recombination system mediated by tExo Ⅷ was particularly characterized by its easy preparation, no limitation on restriction sites and high recombination cloning efficiency. All results revealed that the new efficient gene cloning system has potential application in the field of molecular biology.
Cloning, Molecular ; Escherichia coli ; genetics ; Exonucleases ; genetics ; Recombinant Proteins ; metabolism ; Recombination, Genetic

OBJECTIVETo determine whether 3'phosphorothioate-modified-2 terminal mismatched primers can turn off DNA polymerization mediated by Exo(+) polymerase.

METHODSTwo-directional primer extension was performed using polymerase with and without 3' exonuclease activity. The effects of unmodified primers and 3' phosphorothioate-modified primers on primer extension were evaluated.

RESULTSExo(-) polymerase yielded products from matched and mismatched primers regardless of their modification. However, 3' phosphorothioate-modified primers with a single base mismatch at -2 position worked similarly to the terminal (-1) mismatched primers in triggering the novelly reported "off-switch" of Exo(+) polymerase.

CONCLUSIONThese data suggested that the "off-switch" can be of enormous application in the diagnosis of single gene diseases and in the association studies by single nucleotide polymorphism screening.

DNA Primers ; chemistry ; genetics ; Exonucleases ; metabolism ; Humans ; Phosphorothioate Oligonucleotides ; chemistry ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide

OBJECTIVE: To investigate the differentially expressed genes between gastric cancer and normal gastric mucosa by bioinformatics analysis, identify the important gene participating in the occurrence and progression of gastric cancer, and predict the functions of these genes. METHODS: The gene expression microarray data GSE100935 (including 18 gastric cancer samples and normal gastric mucosal tissues) downloaded from the GEO expression profile database were analyzed using Morpheus to obtain the differentially expressed genes in gastric cancer, and a cluster analysis heat map was constructed. The online database UALCAN was used to obtain the expression levels of these differentially expressed genes in gastric cancer and normal gastric mucosa. The prognostic value of the differentially expressed genes in gastric cancer was evaluated with Kaplan-Meier survival analysis. GO functional enrichment analysis was performed using Fun-Rich software, and the STRING database was exploited to establish a PPI network for the differentially expressed genes. RESULTS: A total of 45119 differentially expressed genes were identified from GSE100935 microarray data. Analysis with UALCAN showed an obvious high expression of EXD3 gene in gastric cancer, and survival analysis suggested that a high expression level of EXD3 was associated with a poorer prognosis of the patients with gastric cancer. GO functional enrichment analysis found that the differentially expressed genes in gastric cancer were involved mainly in the regulation of nucleotide metabolism and the activity of transcription factors in the cancer cells. CONCLUSIONS EXD3 may be a potential oncogene in gastric cancer possibly in relation to DNA damage repair. The up-regulation of EXD3 plays an important role in the development and prognosis of gastric cancer, and may serve as an important indicator for prognostic evaluation of the patients.
Computational Biology ; Databases, Genetic ; Exonucleases ; genetics ; Gastric Mucosa ; chemistry ; enzymology ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Neoplasm Proteins ; genetics ; Prognosis ; Stomach Neoplasms ; enzymology ; genetics ; mortality

The Rad1 gene is evolutionarily conserved from yeast to human. The fission yeast Schizosaccharomyces pombe Rad1 ortholog promotes cell survival against DNA damage and is required for G(2)/M checkpoint activation. In this study, mouse embryonic stem (ES) cells with a targeted deletion of Mrad1, the mouse ortholog of this gene, were created to evaluate its function in mammalian cells. Mrad1 (-/-) ES cells were highly sensitive to ultraviolet-light (UV light), hydroxyurea (HU) and gamma rays, and were defective in G(2)/M as well as S/M checkpoints. These data indicate that Mrad1 is required for repairing DNA lesions induced by UV-light, HU and gamma rays, and for mediating G(2)/M and S/M checkpoint controls. We further demonstrated that Mrad1 plays an important role in homologous recombination repair (HRR) in ES cells, but a minor HRR role in differentiated mouse cells.
Animals ; Cell Division ; Cell Proliferation ; DNA Damage ; DNA Repair ; Embryonic Stem Cells ; metabolism ; Exonucleases ; genetics ; metabolism ; physiology ; G2 Phase ; Gamma Rays ; Gene Deletion ; Hydroxyurea ; pharmacology ; Mice ; Ultraviolet Rays

OBJECTIVETo establish an exonuclease protection mediated polymerase chain reaction (PCR) assay for the non-radioactive, sensitive detection of the binding of protein and DNA.

METHODSThe 1 pmol/L-10 nmol/L 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) dissolved in dimethyl sulphoxide (DMSO), was added into 100 microl SD rat hepatic cytosol in vitro, which contained different amount of aromatic hydrocarbon receptors (AhR) and relative proteins, and ligand-AhR-DRE complex were formed in addition to 1 fmol/L-100 nmol/L DNAs probes containing the sequence of DRE. With the digestion of Exonuclease III and S1 nuclease, free DNAs were digested to oligonucleotide and binding DNA remained due to protein (AhR) protection and be amplified by PCR. The results of PCRs were shown by loading on 2% agarose electrophoresis. DMSO was used as negative control and blank control was set up.

RESULTSTarget DNA (285 bp) could be observed in the ligand groups, but not in the control group. The minimal amount of receptor was 2.5 fmol/L and the minimal amount of DNA probes was 2 fmol.

CONCLUSIONSExonuclease protection mediated PCR assay should be a good non-radioactive tool to quantify the interaction of protein and DNA with high sensitivity and simplicity.

Animals ; Aryl Hydrocarbon Receptor Nuclear Translocator ; genetics ; metabolism ; Binding, Competitive ; DNA Probes ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Exonucleases ; metabolism ; Male ; Polymerase Chain Reaction ; methods ; Rats ; Rats, Sprague-Dawley ; Receptors, Aryl Hydrocarbon ; genetics ; metabolism

Crystallography, X-Ray ; DNA Repair ; Exodeoxyribonucleases ; chemistry ; Humans ; Protein Conformation

Chilblain lupus erythematosus (LE) is a rare, chronic form of cutaneous LE (CLE), which presents mostly in women as erythematous to violaceous plaques on the acral areas and face, precipitated by cold and damp climates. It may be accompanied by discoid LE (DLE) lesions or other forms of CLE. Up to 20% of patients develop systemic LE (SLE). Although two missense mutations in TREX1, encoding the 3'-5' repair exonuclease 1, were described in familial chilblain LE, the pathogenesis of sporadic chilblain LE remains unknown. To our knowledge, there are a few reports of chilblain LE in the Korean dermatologic literature. Herein, we present a rare and interesting case of sporadic chilblain LE in 71-year-old man and review the Korean literatures.
Aged ; Chilblains ; Climate ; Cold Temperature ; Exodeoxyribonucleases ; Female ; Humans ; Lupus Erythematosus, Cutaneous ; Mutation, Missense

Gene cloning is one of the most important and widely used technologies in molecular biology research. Generally, DNA fragment is cut with restriction enzyme, and then the product is ligated to a linearized vector with complementary sticky end or blunt end by DNA-ligase. This traditional DNA cloning method requires compatible enzyme recognition sites existing in both PCR fragment and targeted vector. Several ligase-free methods have been established to avoid the using of restriction enzyme. However, those methods are time-consuming, labor-intensive and expensive. To overcome these shortcomings, we developed an Exonuclease III based DNA cloning method that takes only 30 minutes with high cloning efficiency and significant economic advantage. Therefore, this method is suitable for large-scale gene cloning.
Cloning, Molecular ; methods ; DNA ; chemistry ; DNA Restriction Enzymes ; Exodeoxyribonucleases ; chemistry ; Genetic Vectors ; Polymerase Chain Reaction

DNA binding compounds were previously shown to bind to the right-handed DNA forms and hybrid B-Z forms in a highly cooperative manner and indicate that structural specificity plays a key role in a ligand binding to DNA. In this study, the modes of binding and structural specificity of agents to unusual DNA are examined by a variety of fluorescence techniques (intensity, polarization and quenching, etc.) to explore a reliable method to detect the association environment of ligands to deoxyoligonucleotides initially containing a B-Z junction between the left-handed Z-DNA and right-handed B-DNA. The results of fluorescence energy transfer measurement demonstrated that the ligand molecules bind to the allosterically converted DNA structures by intercalation. In the absence of high-resolution structural data, this fluorescence energy transfer measurement allowed reliable measures and infer the binding environment of ligands to the allosteric DNA structures.
Allosteric Regulation ; Circular Dichroism ; DNA/*chemistry/*metabolism ; Energy Transfer ; Ethidium/*metabolism ; Exodeoxyribonucleases/metabolism ; Ligands ; Motion ; Nucleic Acid Conformation ; Oligodeoxyribonucleotides/*metabolism