T-vectors are widely used for cloning the polymerase chain reaction (PCR) products. However, the low conversion efficiency of a plasmid into the linear T-vector usually results in non-recombinants. Here, we designed a new plasmid pNBQ-T to easily select the recombinant colonies harboring PCR products. pNBQ-T plasmid, which contains a DsRed indicator gene between two Nt.BspQI restriction cassettes, each of which contains palindromic sequences susceptible to Nt.BspQI nickase (5′-GCTCTTCT^GAAGAGC-3′) at each end. Thus, this plasmid can be easily converted into T-vectors by a nickase (quadruple nicking), which results in two double strand breaks with 3′-thymidine overhangs. DsRed indicator gene, which is inserted between the restriction sites, helps identifying the PCR recombinants. Using this pNBQ-T plasmid the insertion efficiency of a PCR product was examined. We successfully identified white colony of the recombinants with the inserted myostatin promoter gene: the cloning efficiency was 93%. Therefore, this simple method utilizing pNBQ-T plasmid will serve as a useful and efficient technique for preparation of home-made T-vectors.
Clone Cells ; Cloning, Organism ; Deoxyribonuclease I* ; Methods ; Myostatin ; Plasmids ; Polymerase Chain Reaction

OBJECTIVETo study the effect of DNase I on biofilm formation of Staphylococcus aureus.

METHODSThe growth curve of S. aureus was detected using a spectrophotometer. The adhesion of S. aureus was analyzed using flat colony counting method, and the biofilm formation was assayed using the 96-well crystal violet staining method.

RESULTSExposure to different concentrations of DNase I did not obviously affect the growth of S. aureus but significantly inhibit the formation of bacterial biofilms in a dose-dependent manner. DNase I inhibited the adhesion of S. aureus at different growth stages. When combined with antibiotics, DNase I resulted in a signi?cant decrease in the established bio?lm biomass compared to antibiotics or DNase I used alone.

CONCLUSIONDNase I can effectively inhibit biofilm formation of S. aureus and enhance the inhibitory effect of antibiotics against S. aureus biofilms.

Anti-Bacterial Agents ; Biofilms ; drug effects ; Deoxyribonuclease I ; chemistry ; Staphylococcus aureus ; growth & development

Animals ; Archaeal Proteins ; genetics ; metabolism ; Deoxyribonuclease I ; genetics ; metabolism ; Gene Editing ; methods ; Humans ; Natronobacterium ; enzymology ; genetics

Structural and functional changes in the major apurinic/apyrimidinic DNA endonuclease (APEX) gene in human colorectal cancers were investigated. DNAs were prepared from surgically removed 25 human colorectal tissues and direct sequencing of PCR-amplified APEX gene covering the entire protein coding region was performed. Point mutations in 3 and silent mutations in 3 out of 25 colorectal cancer patients were found. Base substitutions in intron II were also found in 2 patients. T<-> C or some A<-> G transitions were the most typical pattern of the mutations. AP DNA endonuclease (APE) activities in normal and tumor tissues were 65.7 EU/mg and 21.7 EU/mg, respectively. APEX protein was detected in both normal and tumor tissues and no remarkable difference in the amount of APEX protein between colorectal cancer tissues and their normal counterparts was observed. The incidence of APEX gene mutation in colorectal cancer was 12% which is relatively lower than that of other genes associated with colorectal tumor, but a significant reduction of APE enzyme activities in tumor tissues, especially in those with APEX mutations, was observed. These results indicate that the decreased APE enzyme activity might be closely related to the colorectal tumorigenesis, although no quantitative correlation between APE enzyme activity and APEX content exists.
Carcinogenesis ; Colorectal Neoplasms* ; Deoxyribonuclease I ; DNA Repair* ; DNA* ; Hominidae ; Humans* ; Incidence ; Introns ; Open Reading Frames ; Point Mutation

The rapid involution of the rat ventral prostate after castration is an active process initiated by removal of the inhibitory effects of androgen on prostatic cell death. The degradation of genomic DNA into nucleosomal-sized fragments if an early event in this process and is catalyzed by Ca2+Mg2+-dependent endonuclease activity which is dependent upon calcium ions. The morphologic correlation of the involution process involves a series of structural changes which are collectively referred to as apoptosis. Since the castration-induced endonuclease is dependent upon calcium ions for maximal activity, a potential involvement of a intracellular calcium in the castration-induced prostatic cell death was investigated. Acute disturbance in intracellular calcium homeostasis within the ventral prostate by means of a potent calcium channel blocker, nifedipine, simultaneous with castration resulted in a significant decrease in prostatic apoptosis. This result points to a potential role intracellular calcium levels in the mechanism of activation of castration-induced death of the androgen-dependent epithelial cells in the ventral prostate.
Animals ; Apoptosis* ; Calcium Channels* ; Calcium* ; Castration ; Cell Death ; Deoxyribonuclease I ; DNA ; Epithelial Cells ; Homeostasis ; Ions ; Nifedipine* ; Prostate* ; Rats*

Biologic resurfacing of the damaged joints is an area of great interest and clinical promise because of the limited potential ofdamaged articular cartilage healing. Several methods such as spongiolization. joint dehridement and ahrasion of suhchondral hone. perichondral grafts, and osteochondral grafts have heen used to repair cartilage defects, but the results were not satisfactory. Rccently autologous chondrocyle transplantation with a pcrioslcal patch was paid an altention for its advantage , the regeneration with hyalin cartilage. But it have many disadvantages such as too expensive cost. second staged operation, and technically difficult to isolatc chondrocytes from a small volume of donor site, so we performed that a definecl cartilaee delect in the ribbit patella was treated with transplanta1ion of in virto expanded allogenic chondrocvtes and then compared with an autologous chondrocytes transplantation. Adult rabbits were used to transplant autogenously and allogenously and allogenically harvested and in vitro cultured chondrocytes into patellar chondral lesions that had been made previously 3x 3mmin size , extending down to the calcified zone. Chondrocytes were isolated in the femoral condyle of the opposite knee or othe rabbit knee. And then enzymatic digestion ( collagenase A and DNase I ) was performed for 5 hours room temperature in a spinner bottle and cells were seeded in a 25cm2 culture flask in Dulheccos modified essential medium (DMEM), supplemented with l0% fetal hovine serum (FBS). The culture medium was changed twice weekly. After 14 days of culture, the cells were isolated hy irypsinization and transplanted into previously made chondral defects with an autogenous periosteal patch taken from the medial aspect of tibia. Healing ol' the defects was assessed by gross examination, immunohistochemical stain, and light microscope with hematoxylin-eosin stain at 8, 16, and 24 weeks. Allogenic and autologous chondrocytes transplantation significantly increased the amount of newly tormed repair tissue compared to that found in control knees in which the Jesion was solely covered hy a periosteal patch. The repair tissue, however, had a tendency of incomplete bonding to adjacent cartilage. This study shows that allogenic and autologous articular chondrocytes that have heen expanded for 2 weeks in vitro can stimulate the healing phase of chondral lesion. There is no signilicant diffcrence hetween allogenic and autologous chondrocytes transplantation.
Adult ; Cartilage ; Cartilage, Articular ; Chondrocytes* ; Collagenases ; Deoxyribonuclease I ; Digestion ; Humans ; Hyalin ; Joints ; Knee ; Patella* ; Rabbits ; Regeneration ; Tibia ; Tissue Donors ; Transplants

Genome editing is a useful research tool essentially applicable to gene therapy in the field of biotechnology, pharmaceutics and medicine. Scientists have developed three types of programmable nucleases for genome editing, and these include: Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) system particularly derived from bacterial adaptive immune system. Programmable nucleaseses occur double strand breaks (DSBs) on target strand, and a repair mechanism of DSBs introduces either non-homologous end joining (NHEJ) or homology directed repair (HDR), where the pathway is determined by presence of donor DNA template. In this sense, we can generate gene insertion, gene correction, point mutagenesis and chromosomal translocations via endogenous repair mechanism. However, these nucleases exhibit several discrepancies in the aspects of their compositions, targetable sites, efficiency and other characteristics. Here, we discuss on various characteristics of three programmable nucleases and potential outcomes of DSBs. Acknowledging the distinctions among these programmable nucleases will help scientists to select appropriate tools in genome engineering.
Biotechnology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Deoxyribonuclease I ; DNA ; Genetic Engineering ; Genetic Therapy ; Genome* ; Humans ; Immune System ; Mutagenesis ; Mutagenesis, Insertional ; Tissue Donors ; Translocation, Genetic

OBJECTIVETo investigate the change of gastric cancer cell proliferation and the expression of gastric cancer related gene 213 (GCRG213), a long interspersed nuclear element-1 (LINE-1) endonuclease variant, during hypoxia.

METHODSNormal gastric mucosa cell GES-1 and gastric cancer cell BGC-823 were cultured in 20% or 3% oxygen concentrations, respectively. MTT test was used to analyze the proliferation of the GES-1 and BGC-823 cells. The change of GCRG213 mRNA and protein expression in GES-1 and BGC-823 cells was detected by using RT-PCR and Western blot analysis. Blast was used at the NCBI Blast server to identify GCRG213 sequence to any alignment in the GeneBank databases.

RESULTSCompared with 20% oxygen condition, 3% oxygen concentration could promote cell growth. Mean-while, the expression of GCRG213 at mRNA and protein levels was increased. GCRG213 sequence shared high homology with LINE-1 endonuclease sequence.

CONCLUSIONGCRG213 is a variant of LINE-1 endonuclease. Hypoxia as in 3% oxygen condition can promote cell proliferation and lead to GCRG213 overexpression.

Cell Line, Tumor ; Cell Proliferation ; Cells, Cultured ; Deoxyribonuclease I ; genetics ; Gastric Mucosa ; cytology ; Gene Expression ; Humans ; Hypoxia ; Peptide Hormones ; genetics ; Stomach Neoplasms ; genetics ; pathology

OBJECTIVETo study the effect of extracellular DNA(eDNA) on the formation of Streptococcus mutans(Sm) biofilms during different growth periods in sucrose environment.

METHODSSm biofilms were established on smooth glass surfaces under the environment of 1% sucrose and cultured in the condition of 37 ℃, 5% O2, 85% N2 and 10% CO2. Samples were randomly divided into four groups based on fourculture time(6,12, 24 and 48 h), respectively. Each group was further divided into two subgroups: control group(without deoxyribonuclease Ⅰ[DNaseⅠ] treatment) and test group(with DNaseⅠtreatment). DNaseⅠ was added 1 h advance in the treatment group to a final concentration of 100 U/ml. Each sample was stained with mixed SYTO-9/PI fluorescent dye. Confocal laser scanning microscopy was used for biofilm observation and scanning. The total biomass, the thickness and the volume of red fluorescence of each biofilm sample were measured following three-dimensional reconstruction using the softwear of Imaris 7.2.3.

RESULTSUnder the environment of 1% sucrose, the Sm bacterial adhesion and distribution density increased over time, the quantity of eDNA and membrane-damaged bacteria which were indicated by red fluorescence also increased within 24 h but dropped later. The biofilm biomasses of Sm biofilm in 6, 12, 24 and 48 h DNaseⅠ treatment group reduced significantly(P<0.05) compared to those in the corresponding control groups by 81.3%, 85.0%, 90.1% and 12.4%, respectively. The biofilm thicknesses in each DNase Ⅰ treatment group (except 6 h group) also reduced significantly(P<0.05) compared to those in the corresponding control group by 34.4%, 45.6% and 23.6%, respectively. The quantities of eDNA and membrane-damaged bacteria reduced in each treatment group except 48 h group compared to that in the corresponding control group.

CONCLUSIONSUnder the environment of 1% sucrose, eDNA plays an important role in promoting the formation of Sm biofilm.

Bacterial Adhesion ; drug effects ; Biofilms ; growth & development ; DNA ; physiology ; Deoxyribonuclease I ; pharmacology ; Microscopy, Confocal ; Streptococcus mutans ; physiology ; Sucrose ; Sweetening Agents ; Temperature

BACKGROUNDPrevious studies have suggested that interrupted clearance of nuclear DNA-protein complexes after cell death might initiate and propagate systemic lupus erythematosus (SLE). Deoxyribonuclease I (DNaseI) may be responsible for the removal of DNA from nuclear antigens at sites of high cell turnover, thus preventing the onset of SLE. The purpose of this study was to genotype the single nucleotide polymorphisms (SNPs) of DNase1 and characterize its gene expression and alternatively spliced transcripts in Chinese patients with SLE in order to understand the pathogenic role of DNase1 in human SLE.

METHODSFour SNPs located at the 3' end of the DNase1 gene, as listed on the SNP website, were selected for analysis. Those SNPs with relatively high heterozygosity were chosen for genotyping in 312 Chinese SLE families using the Taqman minor groove binder (MGB) allelic discrimination method. Haplotypes were constructed and linkage disequilibrium tests were performed using GeneHunter. DNase1 mRNA expression was detected using real-time polymerase chain reaction (PCR), and alternatively spliced transcripts were isolated using capillary electrophoresis. Any effects the specific SNP haplotypes had on DNase1 gene expression and the alternatively spliced transcripts were also assessed.

RESULTSrs179982 and rs1053874 had high heterozygosity, about 0.5 in this Chinese cohort, while rs1059857 was also found to be heterozygous. Analysis of the haplotype combining rs179982-rs1030874 (C-G) and rs179982-rs1030874-rs1059857 (C-G-G) revealed a skewed transmission in favor of affected offspring. DNase1 gene expression was higher in SLE patients than in normal controls (P < 0.001), but this was not related to disease activity or SNP haplotype. Capillary electrophoresis revealed that the pattern of alternatively spliced transcripts in patients differed from that of normal controls. Furthermore, different SNP haplotype combinations generated different transcript patterns in SLE patients.

CONCLUSIONSThe SNP haplotypes are in linkage disequilibrium in Chinese SLE patients and may induce the disease through a modification of DNase1 mRNA splicing rather than at the level of mRNA expression. There is a relatively unique transcript band in SLE patients independent of special haplotype, which suggests that other unknown factors might be involved in adjusting gene expression.

Adolescent ; Adult ; Alternative Splicing ; Deoxyribonuclease I ; genetics ; Female ; Haplotypes ; Humans ; Linkage Disequilibrium ; Lupus Erythematosus, Systemic ; genetics ; Male ; Middle Aged ; Polymorphism, Single Nucleotide