Creation of targeted inversion mutations in plants using an RNA-guided endonuclease

Inversions are DNA rearrangements that are essential for plant gene evolution and adaptation to environmental changes. We demonstrate the creation of targeted inversions and previously reported targeted deletion mutations via delivery of a pair of RNA-guided endonucleases(RGENs) of CRISPR/Cas9. The efficiencies of the targeted inversions were2.6% and 2.2% in the Arabidopsis FLOWERING TIME(At FT) and TERMINAL FLOWER 1(At TFL1)loci, respectively. Thus, we successfully established an approach that can potentially be used to introduce targeted DNA inversions of interest for functional studies and crop improvement.

Relationship between apurinic endonuclease 1 Asp148Glu polymorphism and gastrointestinal cancer risk: An updated meta-analysis

AIM:To evaluate the relationship between apurinic endonuclease 1(APE1) Asp148 Glu polymorphism and the susceptibility to gastrointestinal(GI) cancers.METHODS:We searched Pub Med, ISI Web of Knowledge, and Chinese National Knowledge Infrastructure(CNKI) databases updated on July 15, 2014 for relevant studies.Only case-control studies comparing APE1 Asp148 Glu polymorphism and GI cancer risk were included.We excluded studies reporting only standardized incidence ratios without control groups and those without detailed genotyping data.Meta-analysis was performed on 17 studies involving 4856 cancer patients and 6136 cancer-free controls.Review Manager version 5.1 was used to perform the meta-analysis.The pooled odds ratios(ORs) and 95% confidence intervals(CIs) were estimated under the allele contrast, homozygous, heterozygous, dominant and recessive genetic models.We also conducted subgroup analyses stratified by ethnicity and cancer type.Publication bias was evaluated using Begg's test.RESULTS:The meta-analysis showed a significant association between APE1 Asp148Glu polymorphism and GI cancer risk in three genetic models in the overall population(G vs T:OR=1.18;95%CI:1.05-1.32;TG vs TT:OR=1.28;95%CI:1.08-1.52;TG+GG vs TT:OR=1.32;95%CI:1.10-1.57).Stratified analysis by ethnicity revealed a statistically increased GI cancer risk in Asians(G vs T:OR=1.27;95%CI:1.07-1.51;GG vs TT:OR=1.58;95%CI:1.05-2.38;TG vs TT:OR=1.30;95%CI,1.01-1.67;and TG+GG vs TT:OR=1.38;95%CI:1.07-1.78),but not in Caucasians.Furthersubgroup analysis by cancer type indicated that APE1Asp148Glu polymorphism may contribute to gastric cancer risk.However,Asp148Glu has no significant association with colorectal or esophageal cancer risk in any genetic model.CONCLUSION:This meta-analysis suggests that the APE1 Asp148Glu polymorphism G allele is associated with an increased GI cancer risk,especially in gastric cancer.

Differential expression of hepatic apurinic/apyrimidinic endonuclease 1,a DNA repair enzyme,in chronic hepatitis

AIM:To determine hepatic expression of apurinic/apyrimidinic endonuclease 1(APE-1)and 8-hydroxydeoxyguanosine(8-OHdG)in patients with chronic hepatitis B and C.METHODS:Liver biopsies were obtained from 27 patients with chronic hepatitis B virus(HBV),30 with chronic hepatitis C virus(HCV),6 with autoimmune hepatitis(AIH),and 6 with primary biliary cirrhosis(PBC).Normal liver tissue was obtained from surgical resection specimens of four patients.Hepatic APE-1 protein and mRNA expression were assayed by Western blot and by real-time polymerase chain reaction,respectively.Hepatocellular APE-1 and 8-OHdG expression were determined by immunohistochemistry.RESULTS:The staining intensity of hepatocellular nuclear APE-1 was lower in the HBV group than in the other groups(P < 0.05).Hepatic APE-1 protein levels were reduced in the HBV group relative to the other groups.Hepatic APE-1 mRNA levels were also lower in the HBV group.The proportion of hepatocytes with 8-OHdG-positive nuclei was increased in the HCV,AIH and PBC groups(P < 0.05),but not in the HBV group.Hepatocellular nuclear APE-1 levels were positively correlated with hepatocellular 8-OHdG levels in both the HBV and HCV groups(HBV,r = 0.34,P < 0.05;HCV,r = 0.54,P < 0.01).CONCLUSION:An imbalance between oxidative DNA damage and APE-1 expression may contribute to hepatocarcinogenesis in chronic viral hepatitis.

Detection of Low-abundance Point Mutations by Competitive Strand Assisted Endonuclease Ⅳ Signal Amplification System

Genetic mutations are important molecular biomarkers for cancer diagnosis and surveillance. Therefore, the development of methods for mutation detection characterized with straightforward, highly specific and sensitive to low-level mutations within various sequence contexts is extremely needed. Although some of the currently available methods have shown very encouraging results, their discrimination efficiency is still very low. Herein, we demonstrate a fluorescent probe coupled with blocker and property of melting temperature discrimination, which is able to identify the presence of known or unknown single-base variations at abundances down to 0.1% within 20 min. The discrimination factors between the perfect-match target and single-base mismatched target are determined to be 10.15–38.48. The method is sequence independent, which assures a wide range of application. The new method would be an ideal choice for high-throughput in vitro diagnosis and precise clinical treatment.

Kinetic Studies on the Irreversible Inhibition of Restriction Endonuclease Pst I by Site-Specific Inhibitors

The irreversible modifying effects onPst I of several inhibitors have been studied with the irreversible inhibition kinetic theory of single substrate reaction provided by Tsou, C. L. Pyridoxal phosphate (PLP), p-chloromercuribenzoic acid (PCMB), diisopropyl fluorophosphate (DEP), 2,3-diacetyl (DAC) and N-ethyl-5-phenylisoxazoliun-3′-sulfonate (woodward's reagent K, WRK) modify the lysine, cysine, serteine, arginine and carboxyl groups of the protein molecule respectively. These five inhibitors have been found to inhibit both the prime activity and star activity ofPst I. Used with the irreversible inhibition theory, the apparent inhibition rate constant,A and the microcosmic inhibition rate constants,k +0 andk′ +0 of every inhibitor were calculated. We also found that their inhibition effects belong to the noncompetitive irreversible inhibition. Results show that among the groups to be modified, some have nothing to do with the combination with the substrate, and some may have, but any of them isn't the only factor involved in the specific binding. Despite all this, they may take part in the catalysis of enzyme or have important effects on maintaining the active structure of enzyme molecules. Furthermore, serine and arginine residues are related to the alteration ofPst I conformation and then influence the ability ofPst I recognizing and incising DNA specifically.

Alteration of the Specificity of PstⅠ Restriction Endonuclease

The influence of factors on the subst rate-specificity of Pst Ⅰ restric tion endonuclease has been studied with the method of electrophoresis. The resu lts show that, the specificity of Pst Ⅰ almost can not be influenced by th e sin gle alteration of the concentration of Tris·HCl, Mg 2+ or Na+ in the rea ction s ystem, but it can be altered by the reduction of any two of them. The specificit y can not be altered by the single alteration of pH or the replacement of Mg 2+ w ith Mn 2+. The addition of glycerol or dimethylsulphoxide (DMSO) to the rea ction system results in the relaxation of the substrate-specificity of Pst Ⅰ , b ut dim ethylmethylformide, glycol and ethyl alcohol can not bring about the alteration of Pst Ⅰ specificity. Through the method of cloning and sequencing, the nuc leot ides of No.1 and 6 in the recognition sequence of Pst Ⅰ have changed (1C→A or 6 G→T). Used with the enzyme analysis of an artificially synthetic DNA segment co ntaining a special sequence, the nucleotides of No.1 and 6 have both changed (1C →A and 6G→T). The recognition sequence of Pst Ⅰ is speculated to be chan ged from CTGCA↓G to TGCA↓.

Restriction endonucleases digesting DNA in PCR buffer

Six commonly used restriction endonucleases (REs) (Acc I, Ban II, EcoR I, Hind III, Sac I, Sca I) were tested for their ability to directly digest DNA completely in the Polymerase Chain Reaction (PCR) buffers. The results showed that: with the requirement for addi- tional magnesium supplemented as activator, REs, except EcoR I appeared star activity, completely digested unmethylated lambda DNA af- ter overnight incubation in PCR buffer and functioned as equally well as in recommended Restriction Enzyme Buffer provided with each enzyme; all REs tested completely digested PCR products in PCR buffer, it implied digestion of PCR products may often be performed di- rectly in the PCR tube without the requirement for any precipitation or purification steps; and the concentration of MgCl2 from 2.5 mmol·L-1 to 10 mmol·L-1 did not significantly affect activity of REs in PCR buffer. This simplified method for RE digestion of PCR prod- ucts could have applications in restriction fragment length polymorphism (RFLP) analysis and single-stranded conformational polymor- phism (SSCP) analysis of large PCR products. However, usage of this procedure for cloning applications needs further data.

Sensitive detection of alkaline phosphatase based on terminal deoxynucleotidyl transferase and endonuclease Ⅳ-assisted exponential signal amplification

Alkaline phosphatase(ALP)is widely expressed in human tissues.ALP plays an important role in the dephosphorylation of proteins and nucleic acids.Therefore,quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods.Terminal deoxynucleotidyl transferase(TdT)catalyzes continuous polymerization of deoxynucleotide triphosphates at the 30-OH end of single-stranded DNA in the absence of a template.In this study,we developed a highly sensitive and selective method based on TdT and endonuclease Ⅳ(Endo Ⅳ)to quantify ALP activity.After ALP hydrolyzes the 30-PO_(4) end of the substrate and generates 30-OH,TdT can effectively elongate the 30-OH end with deoxynucleotide adenine triphosphate(dATP)and produce a poly A tail,which can be detected by the poly T probes.Endo Ⅳ digests the AP site in poly T probes to generate a fluorescent signal and a new 30-OH end,leading to the generation of exponential fluorescence signal amplification.The substrate for TdT elongation was optimized,and a limit of detection of 4.3×10^(-3) U/L was achieved for ALP by the optimized substrate structure.This method can also detect ALP in the cell lysate of a single cell.This work has potential applications in disease diagnosis and biomedical detection.

EXPRESSION AND DELETION ANALYSIS OF EcoRII ENDONUCLEASE AND METHYLASE GENE

Objective. To clone complete EcoRII restriction endonuclease gene (ecoRIIR) and methyltransferase gene (ecoRIIM) in one vector and to analyze the coordinating expression of this whole R M system.Methods. Unidirectional deletion subclones were constructed with ExoIII.ecoRIIR/M genes were preliminarily located in the cloned fragment according to the enzyme activities of subclones. Exact deletion sites were determined by sequencing, and transcriptional start sites were determined by S1 mapping. Results. The DNA fragment which was cloned into pBluescript SK+contained intact ecoRIIR gene andecoRIIM gene, and two transcriptional start sites of ecoRIIR gene were determined. 132bp to 458bp from 3’end of ecoRIIR gene are indispensable to enzyme activities and deletion of 202bp from 3’end of ecoRIIM gene made enzyme lose the capability in DNA protection to resist specific cut with EcoRII endonuclease (EcoRII.R). Deletion of the coding and flanking sequences of one gene did not affect the expression of the other gene, and the recombinants only containing ecoRIIR gene appeared to be lethal to dcm+host. Conclusion. ecoRIIM gene linking closely to ecoRIIR gene is very important for the existence of the R M system in process of evolution, but the key tocontrol EcoRII R M order may not exist in transcriptional level .

Tea polyphenols increase X-ray repair cross-complementing protein 1 and apurinic/apyrimidinic endonuclease/redox factor-1 expression in the hippocampus of rats during cerebral ischemia/reperfusion injury

Recent studies have shown that tea polyphenols can cross the blood-brain barrier, inhibit apoptosis and play a neuroprotective role against cerebral ischemia. Furthermore, tea polyphenols can decrease DNA damage caused by free radicals. We hypothesized that tea polyphenols repair DNA damage and inhibit neuronal apoptosis during global cerebral ischemia/reperfusion. To test this hypothesis, we employed a rat model of global cerebral ischemia/reperfusion. We demonstrated that intraperitoneal injection of tea polyphenols immediately after reperfusion significantly reduced apoptosis in the hippocampal CA1 region; this effect started 6 hours following reperfusion. Immunohistochemical staining showed that tea polyphenols could reverse the ischemia/reperfusion-induced reduction in the expression of DNA repair proteins, X-ray repair cross-complementing protein 1 and apudnic/apyrimidinic endonuclease/redox factor-1 starting at 2 hours. Both effects lasted at least 72 hours. These experimental findings suggest that tea polyphenols promote DNA damage repair and protect against apoptosis in the brain.

Phylogeny derived from homodimeric endonuclease correlates with its pre-RNA substrates

Amongst endonuclease, the homodimeric variety is found in many prokaryotes for processing of the introns out from pre-RNAs. But as the variety and the complexity of introns rise with evolution, do the homodimeric endonuclease adapt to the changes? The correlations between evolving pre-RNAs and adapting homodimeric endonuclease in lower prokaryotes is investigated in this paper. First, we construct and observe the appearance of a long branch in the phylogeny based on homodimeric endonuclease. To appreciate the finer aspects of accelerating evolution near this long branch, we delve deeper into the pre-RNA substrates of the endonuclease. Computational evidence of an as-yet-unreported noncoding RNA gene then emerges from this study. The capabilities of homodimeric endonuclease and the complexities of its pre-RNA substrates appear to evolve in steps together.

Staurosporine-Induced Cell Death in Trypanosoma brucei and the Role of Endonuclease G during Apoptosis

Apoptosis in single-cell organisms like Trypanosoma or Leishmania was characterized in several studies in the last few years [1]-[4]. Cell death in these caspase lacking protozoa is still poorly understood and a conclusive apoptotic pathway has not been identified so far. In the work presented here, we studied the effects of prostaglandin D2 and staurosporine induced cell death in blood-forms of Trypanosoma brucei in a time dependent manner and focused on the role of a nuclease similar to endonuclease G of higher eukaryotes. We found that these parasites undergo apoptotic cell death as demonstrated by the appearance of several canonical hallmarks of apoptosis in higher eukaryotes, but that different stimuli induce remarkable differences in the way these cells die. We compared the effects of prostaglandin D2 and staurosporine in trypanosomes with and without endonuclease G overexpression by flow cytometric and electron microscopic methods with the result that endonuclease G overexpression led to a significant modification of intracellular organelles and accelerated apoptotic cell death in prostaglandin D2 or staurosporine treated cells. Our results demonstrate that different stimuli induce apoptosis even in these ancient organisms in different caspase-independent ways. Whereas central processes of apoptosis like ROS formation, loss of mitochondrial membrane potential, endonuclease G release, phosphatidylserine exposure and DNA fragmentation appeared in the same chronology during treatment with either one of both drugs, other effects like cell cycle arrest or change of cell shape occurred only in the case of prostaglandin D2 or staurosporine treatment. We conclude from these results that trypanosomes react to stimuli of apoptosis with the concerted action of cellular responses but cannot control the final outcome if additional stress, as in the case of staurosporine, is superimposed.

Polymorphism in Mitochondrial DNA of Six Fowl Breeds Revealed by Restriction Endonuclease

The patterns of cleavage of mtDNA by restriction endonucleases was analysed for six fowl breeds and of. Hyline White, Hyline Brown, ISA Brown, Hisex Brown, Lohmann White, Abor Acres and mtDNA polymorphisms were detected in the restriction patterns with the following eight enzymes, Ava Ⅰ, Ava Ⅱ, EcoR Ⅰ, Hind Ⅲ, Bam HI, Pvu Ⅱ, Pst Ⅰ, Hinc Ⅱ. The restriction cleavage patterns were identical among these breeds. Hyline White, Hyline Brown, ISA Brown, Hisex Brown, Lohmann White—A type. The patterns of Abor Acres were B type. Based on their mtDNA restriction types, all the breeds were classified into two groups. Genetic distances among these groups were calculated in order to define their phylogenetic relationships. The relationship among five egg line breeds is close, while Abor Acres (Broiler fowl) is relatively far from them. The results suggest that the difference of mtDNA could result from the different origins. The polymorphic sites in mtDNA of Hyline White has been located on a restriction map.

PA-E18G substitution in influenza A virus confers resistance to ZX-7101,a cap-dependent endonuclease inhibitor

Cap-dependent endonuclease(CEN)in the polymerase acidic protein(PA)of influenza A virus(IAV)represents a promising drug target due to its critical role in viral gene transcription.The CEN inhibitor,baloxavir marboxil(BXM),was approved in Japan and the US in 2018 and several other countries subsequently.Along with the clinical use of BXM,the emergence and spread of IAV variants with reduced susceptibility to BXM have aroused serious concern.Herein,we comprehensively characterized the in vitro and in vivo antiviral activities of ZX-7101A,an analogue of BXM.The active form of prodrug ZX-7101 showed broad-spectrum antiviral potency against various IAV subtypes,including pH1N1,H3N2,H7N9 and H9N2,in MDCK cells,and the 50%effective concentration(EC_(50))was calculated to nanomole level and comparable to that of baloxavir acid(BXA),the active form of BXM.Furthermore,in vivo assays showed that administration of ZX-7101A conferred significant protection against lethal pH1N1 challenge in mice,with reduced viral RNA loads and alleviated pulmonary damage.Importantly,serial passaging of H1N1 virus in MDCK cells under selection pressure of ZX-7101 led to a resistant variant at the 15th passage.Reverse genetic and sequencing analysis demonstrated that a single E18G substitution in the PA subunit contributed to the reduced susceptibility to both ZX-7101 and BXA.Taken together,our results not only characterized a new CEN inhibitor of IAV but also identified a novel amino acid substitution responsible for CEN inhibitor resistance,which provides critical clues for future drug development and drug resistance surveillance.

Ginsenoside Rd Attenuates DNA Damage by Increasing Expression of DNA Glycosylase Endonuclease VIII-like Proteins after Focal Cerebral Ischemia

Background： Ginsenoside Rd （GSRd）, one of the main active ingredients in traditional Chinese herbal Panax ginseng, has been found to have therapeutic effects on ischemic stroke. However, the molecular mechanisms of GSRd＇s neuroprotective function remain unclear. Ischemic stroke-induced oxidative stress results in DNA damage, which triggers cell death and contributes to poor prognosis. Oxidative DNA damage is primarily processed by the base excision repair （BER） pathway. Three of the five major DNA glycosylases that initiate the BER pathway in the event of DNA damage from oxidation are the endonuclease VIII-like （NELL） proteins. This study aimed to investigate the effect of GSRd on the expression ofDNA glycosylases NEILs in a rat model of focal cerebral ischemia. Methods： NEIL expression patterns were evaluated by quantitative real-time polymerase chain reaction in both normal and middle cerebral artery occlusion （MCAO） rat models. Survival rate and Zea-Longa neurological scores were used to assess the effect of GSRd administration on MCAO rats. Mitochondrial DNA （mtDNA） and nuclear DNA （nDNA） damages were evaluated by the way of real-time analysis of mutation frequency. NEIL expressions were measured in both messenger RNA （mRNA） and protein levels by quantitative polymerase chain reaction and Western blotting analysis. Apoptosis level was quantitated by the expression of cleaved caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling assay. Results： We found that GSRd administration reduced mtDNA and nDNA damages, which contributed to an improvement in survival rate and neurological function; significantly up-regulated NEIL1 and NEIL3 expressions in both mRNA and protein levels of MCAO rats; and reduced cell apoptosis and the expression of cleaved caspase-3 in rats at 7 days after MCAO. Conclusions： Our results indicated that the neuroprotective function of GSRd for acute ischemic stroke might be partially explained by the up-regulation of NEILI and NEIL3 expressions.

Differential effects of Ca^(2+) and Mg^(2+) on endonuclease activation in isolated promyelocytic HL-60 cell nuclei

In autodigestion assays, endonuclease activity in non apoptotic HL 60 promyelocytic leukemia cell nuclei cleaved the chromatin of the autologous cells to an oligonucleosomal length pattern. Both EGTA and EDTA inhibited the activation of endonuclease activity in isolated HL 60 cell nuclei. The inhibition by EDTA could be reversed by exogenous Ca 2+ , but not by exogenous Mg 2+ . In Ca 2+ /Mg 2+ free nuclei digestion buffer, addition of Ca 2+ （1\10 mmol/L） induced endonuclease activity in the isolated nuclei, while addition of Mg 2+ had no effect. In the presence of Ca 2+ （0.1 mmol/L）, endonuclease activity was enhanced by exogenous Mg 2+ （0.1\10 mmol/L）. These results suggest that the endonuclease responsible for internucleosomal DNA fragmentation in HL 60 cells during apoptosis is activated by Ca 2+ and further modulated by Mg 2+ in the presence of Ca 2+ .

Elimination of inter-domain interactions increases the cleavage fidelity of the restriction endonuclease Dralll

Dralll is a type liP restriction endonucleases （REases） that recognizes and creates a double strand break within the gapped palindromic sequence CACTNNN^GTG of double-stranded DNA indicates nicking on the bottom strand; indicates nicking on the top strand）. However, wild type Dralll shows significant star activity. In this study, it was found that the prominent star site is CATSGTT;GTG, consisting of a star 5＇ half （CAT） and a canonical 3＇ half （GTG）. Dralll nicks the 3＇ canonical half site at a faster rate than the 5＇ star half site, in contrast to the similar rate with the canonical full site. The crystal structure of the Dralll protein was solved. It indicated, as supported by mutagenesis, that Dralll possesses a ~13a- metal HNH active site. The structure revealed extensive intra-molecular interactions between the N-terminal domain and the C-terminal domain containing the HNH active site. Disruptions of these interactions through site- directed mutagenesis drastically increased cleavage fidelity. The understanding of fidelity mechanisms will enable generation of high fidelity REases.

Human DNA contains sequences homologous to the 5'-non-coding region of hepatitis C virus: characterization with restriction endonucleases reveals individual varieties

Objective To investigate a 272 base pair section of the 5' non coding region of genomic DNA from the peripheral blood monounuclear cells of healthy hepatitis virus C (HCV) negative human subjects (not patients) Mothods This sequence section bears interest because ① it harbors several potential methylation (Cp rich) sites, and ② it represents the largest part of its internal ribosomal entry site A pre PCR digestion protocol was established making consistent use of four restriction endonucleases selected for certain features: SmaI, XmaCI, MspI, and HpaII are inhibited if methylation(s) are present at certain cytosines within their cutting sequences Results The suspected HCV specific sequence was found in the DNA of each subject tested The pre PCR digestion assay reveals individual differences in their pattern of methylation, which may be due to possible epigenetic phenomena Conclusions The results provide formal proof that these HCV specific sequences are contained in the genomic or extra chromosomal target DNA, and probably belong to a new class of endogenous sequences

A NEW TYPE Ⅱ RESTRICTION ENDONUCLEASE, BsaO Ⅰ, FROM Bacillus stearothermophilus

A new type Ⅱ restriction endonuclease, BsaO Ⅰ, has been isolated from the thermophilic bacillus Bacillus stearothermophilus O-122. This enzyme cleaves pBR322 DNA at 7 sites, pUC19 DNA at 5 sites and φ X 174 DNA at one site. The recognition sequence of BsaO Ⅰ was determined by mapping its cleavage sites on φ X 174 DNA to position 4600 and

STUDIES ON POLYMORPHISM IN β-GLOBIN GENE CLUSTER IN CHINESE——THE POLYMORPHIC RESTRICTION ENDONUCLEASE HIND Ⅲ SITES WITHIN γ-GLOBIN GENE (Ⅲ)

The polymorphism of human globin genes as a genetic marker has been widely used in studies related to anthropology, genetics and prenatal diagnosis of genetic diseases (e.g. prenatal diagnosis of β-thalassemia and sickle cell anemia). Since 1978, several polymorphic restriction endonuclease sites in the human in β-globin gene cluster