Mutagenesis Research Articles (Page 1)

Synthetic gene construction is one of the components of synthetic biology. It can be used for various purposes such as to optimize gene expression. In this study, we proposed six predetermined criteria for designing oligos for the synthesis of the Beauveria bassiana protease gene. These criteria were set up to optimize the cost and to accommodate the oligos assembly. A total of 44 overlapping oligos were designed and synthesized 0.5 µM of oligos mixture was used in assembly PCR together with high fidelity DNA polymerase to produce 1.1 kbp fragment. The gene was visualized by agarose gel electrophoresis before subcloned into pCR™2.1-TOPO. The sequence of the gene was verified by DNA sequencing. Site-directed mutagenesis was performed to repair errors resulted from the gene synthesis. A sharp and distinguished band of the expected size of the protease gene was observed in agarose gel electrophoresis. Errors in the sequence which was detected by DNA sequencing were successfully repaired using our simplified site-directed mutagenesis protocol. The result indicated long DNA sequences (>1 kbp) can be synthesized with less error by using our method. Additionally, this method was easy to perform because it would require minimum optimization to synthesize other genes by following our guidelines.

Creating your own path: inducing novel traits using mutagenesis

Objective To explore the molecular and genetic mechanism of transcription factor GATA-6 in nonsyndromic conotruncal defect (CTD) in order to provide evidence for early prevention and inheritance consultation of CTD. Methods A total of 32 cases of patients with nonsyndromic CTD and 100 healthy individuals were enrolled in the study.A total of 7 exons and bilateral partial intron-exon boundaries of GATA-6 were amplified by means of polymerase chain reaction (PCR). The PCR products were purified and directly sequenced by using an ABI Genetic Analyzer 3100 Automatic DNA sequence equipment.The acquired GATA-6 gene sequence was compared with standard gene sequence published in National Center for Biotechnology Information database, as well as the healthy control group to observe the GATA-6 gene mutations.The mutations were introduced into pcDNA3.1(+ ) by site-directed mutagenesis PCR on the basis of pcDNA3.1(+ )-GATA-6 in order to generate the GATA6-G245R mutant constructs.Wild type GATA-6, GATA-6-G245R and atrial natriuretic factor-luciferase(ANF-luciferase) were cotransfected into HEK 293T cells in vitro, and the CMV-LacZ were cotransfected as internal reference.Luciferase and galactosidase activity were measured by using luminometer 24 h after transfection and detected in the downstream ANF-luciferase reporter gene. Results A heterozygous missense mutation in the GATA-6 gene was identified in a patient with double outlets of the right ventricle.The mutation was located in Gly245Arg(G245R) in exon 2 of GATA-6.The mutation of pcDNA3.1(+ )-GATA-6 expression vectors were successfully constructed.Through the detection of luciferase reporter gene activity, it was found that GATA-6-G245R and wild-type GATA-6 decreased by 41.3%, and the comparison between them was statistically significant (P<0.001). Conclusions Transcription factor GATA-6 gene mutation is associated with the occurrence of nonsyndromic CTD.Transcription factor GATA-6 gene may be susceptible gene in human nonsyndromic CTD. Key words: Nonsyndromic conotruncal defect; Transcription factor GATA-6; Mutation; Plasmid; Report gene

Multi-site Specific Mutagenesis by Multi-fragment Overlap Extension PCR

HeLa E-Box Binding Protein, HEB, Inhibits Promoter Activity of the Lysophosphatidic Acid Receptor Gene Lpar1 in Neocortical Neuroblast Cells.

Sodium azide (NaN3) is widely used to induce mutagenesis within in vitro plant systems. However, since this mutagenesis is undirected, its unintended effects demand characterization. This study investigated the mutagenic effects of sodium azide (0-0.45 mM) on selected growth (shoot multiplication rate and shoot cluster fresh weight) and biochemical (aldehydes, chlorophylls, carotenoids and phenolics) parameters in pineapple micropropagants within temporary immersion bioreactors (TIBs). The content of soluble phenolics in the culture medium was also evaluated. Irrespective of the concentration NaN3 decreased shoot multiplication rate (by 87% relative to the control at 0.45 mM) and fresh weight (by 66% relative to the control at 0.45 mM). Levels of chlorophyll a and b, and soluble phenolics in the culture medium were also negatively correlated with NaN3 concentration. Interestingly, NaN3 application increased shoot carotenoid and soluble phenolic levels but had no significant effect on a range of established plant stress biomarkers: cell wall-linked phenolic levels, malondialdehyde and other aldehydes. Given that 0.19 mM NaN3 decreased shoot multiplication rate by 50% and resulted in propagants that displayed no morphologically abnormalities, increased levels of photoprotective pigments (relative to the control) and no significant increase in lipid peroxidation products, the mutagen can be used at this concentration to induce pineapple mutagenesis in TIB based studies aimed at producing agriculturally-useful mutants.

Ralstonia solanacearum strain PRS-84 used in this study was isolated from diseased Pogostemon cablin plants in our previous study.The competent cells of R.solanacearum strain PRS-84 were transformed by electroporation with Tn5 transposon and then were plated on TTC agar plates containing kanamycin to select for kanamycin-resistant colonies.The detection of kanamycin-resistant gene in kanamycin-resistant colonies was performed by PCR.Further,the flanking fragments of Tn5 transposon insertion site in the mutants were amplified by inverse PCR,and the flanking fragments were sequenced and analyzed.The results indicated that the kanamycin-resistant colonies were obtained in the transformation experiment of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon.A specific band of approximately 700 bp was amplified by PCR from kanamycin-resistant colonies.The flanking sequences of Tn5 transposon insertion site in the transformants were obtained by inverse PCR.After sequencing and sequence analysis of Tn5 transposon insertion site in mutants,we preliminarily speculated that the Tn5 transposon inserted in the typ A gene,rec O gene and gid A gene in three mutants,respectively.A random mutagenesis system of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon has been established,and the Tn5 insertion mutants have been obtained.This study might facilitate the creation of mutant library and the discovery of the virulence gene of R.solanacearum isolated from P.cablin.

Introduction: Interactions of free radicals from ionizing radiation with DNA can induce DNA damage and lead to mutagenesis and carsinogenesis. With respect to radiation damage to human, it is important to protect humans from side effects induced by ionizing radiation. In the present study,the effects of arbutin were investigated by using the micronucleus test for anti- clastogenic activity, to calculate the ratio of polychromatic erythrocyte to polychromatic erythrocyte plus normochromatic erythrocyte (PCE/PCE+NCE) in order to show cell proliferation activity. Materials and Methods: Arbutin (50, 100, and 200 mg/kg) was intraperitoneally (ip)administered to NMRI mice two hours before gamma radiation at 2 and 4 gray (Gy). The frequency of micronuclei in 1000 PCEs (MnPCEs) and the ratio of PCE/PCE+NCE were calculated for each sample. Data were statistically evaluated using one-way ANOVA,Tukey HSD test, and t-test. Results: The findings indicated that gamma radiation at 2 and 4 Gy extremely increased the frequencies of MnPCE (P<0.001) while reducing PCE/PCE+NCE (P<0.001) compared to the control group. All three doses of arbutin before irradiation significantly reduced the frequencies of MnPCEs and increased the ratio of PCE/PCE+NCE in mice bone marrow compared to the non-drug-treated irradiated control (P<0.001). All three doses of arbutin had no toxicity effect on bone marrow cells. The calculated dose reduction factor (DRF) showed DRF=1.93 for 2Gy and DRF=2.22 for 4 Gy. Conclusion: Our results demonstrated that arbutin gives significant protection to rat bone against the clastogenic and cytotoxic effects of gamma irradiation.

CRISPR/Cas9 pooled screening permits parallel evaluation of comprehensive guide RNA libraries to systematically perturb protein coding sequences in situ and correlate with functional readouts. For the analysis and visualization of the resulting datasets, we develop CRISPRO, a computational pipeline that maps functional scores associated with guide RNAs to genomes, transcripts, and protein coordinates and structures. No currently available tool has similar functionality. The ensuing genotype-phenotype linear and three-dimensional maps raise hypotheses about structure-function relationships at discrete protein regions. Machine learning based on CRISPRO features improves prediction of guide RNA efficacy. The CRISPRO tool is freely available at gitlab.com/bauerlab/crispro.

Enhancing the Activity of LkTADH by Site-Directed Mutagenesis to Prepare Key Chiral Block of Statins

Objective To evaluate the performance of MTHFR 677 genotyping external quality assessment (EQA) program using plasmid DNA constructed in vitro as quality control samples and discuss the problems in clinical laboratories enrolled in the program. Methods Recombinant plasmid carrying MTHFR 677C locus sequence was constructed as wild type sample and plasmid with MTHFR 677T mutation was generated with site-directed mutagenesis as mutant type sample. Heterozygous mutant samples were obtained after equal proportion of the two plasmids. EQA scheme were held twice a year in 2016 and 2017, and sample panels contained 5 different samples using recombinant plasmid DNA containing all types of MTHFR 677 locus genotypes. Participating laboratories were asked to test samples using their routine methods and report the results before deadlines. 26, 28, 52 and 56 effective reports were received respectively in the four EQA schemes. The scores of each lab were calculated based on their results and the overall compliance of different samples as well as the sensitivity and specificity of different methods were calculated using Microsoft Excel. Results MTHFR 677 locus genotypes of the constructed plasmid were verified by Sanger sequencing and there was no failure of sample detection in the four EQA schemes, which suggest that the plasmid has good clinical applicability. About 96.15%(25/26), 100%(28/28), 96.15%(50/52)and 98.21%(55/56)of the laboratories submitted correct results for all samples in the four EQA schemes. The overall compliance rate were 99.23% (129/130), 100%(140/140), 96.92% (252/260) and 98.93% (277/280) respectively. All laboratories using digital FISH and microarrays got full marks in four EQA schemes. The compliance rates for fluorescent PCR were 97.5% (39/40), 100% (45/45), 94.29% (66/70) and 100% (95/95) respectively, while the rates were 100% (20/20), 100% (15/15), 90% (36/40) and 92.5% (37/40) for Sanger sequencing. Conclusions The recombinant plasmid DNA constructed in this study can effectively detect the performance of reagents with good clinical applicability. The results of EQA programs suggested that the overall accuracy rate of laboratories enrolled was high enough, while some laboratories′ performance still needs to be improved. Quality controls in clinical laboratories were essential to assure the accuracy of results.(Chin J Lab Med, 2018, 41: 749-754) Key words: Methylenetetrahy drofolate reductase(NADPH); Plasmids; Genetic testing; Quality control

EvolvR-ing to targeted mutagenesis.

Structural Biology Maintaining the correct balance of calcium concentrations between the cytosol and the mitochondria is essential for cellular physiology. A calcium-selective channel called the mitochondrial calcium uniporter (MCU) mediates calcium entry into mitochondria. Yoo et al. report the high-resolution structure of MCU from Neurospora crassa. The channel is formed by four MCU protomers with differing symmetry between the soluble and membrane domains. The structure, together with mutagenesis, suggests that two acidic rings inside the channel provide the selectivity for calcium. Science , this issue p. [506][1] [1]: /lookup/doi/10.1126/science.aar4056

Objective To study how CD73 is shed from the retinal pigment epithelium (RPE) surface. Methods CD73 shedding was induced by treating RPE with lipopolysaccharides (LPS) and TNF-α. After Phospholipase C (PLC) or pan matrix metalloproteinase (MMP) inhibitors were added, surface amount of CD73 was evaluated by flow cytometry (FACS). Then selective inhibitors or their corresponding siRNAs of MMP-2 and MMP-9 were applied to the treatments of RPE; and their effects on induced CD73 shedding were evaluated by FACS. By site directed mutagenesis, mutations were introduced to Lys547-Phe548 coding sites of CD73 cDNA, which was cloned in a pcDNA mammalian expression vector. Both wt-CD73 and mutated-CD73 were over expressed in CD73-/- RPE and their induced shedding was compared. Results LPS and TNF-α induced CD73 shedding from RPE was completely blocked by the addition of pan MMP inhibitor but not PLC inhibitor. Selective MPP-9, but not MMP-2, inhibitor or its siRNA blocked CD73 shedding. In CD73-/- RPE induced CD73 shedding was happened to overexpressed wt-CD73 but not Lys547- Phe548 sites mutant CD73. Conclusion MMP-9 is responsible for shedding CD73 from RPE through hydrolyzing its Lys547-Phe548 sites. Key words: Matrix metalloproteinase 9/antagonists i 5′-Nucleotidase; Retinal pigment epithelium

Improving Production of Tacrolimus In Streptomyces Tacrolimicus (ATCC 55098) Through Development of Novel Mutant by Dual Mutagenesis

Optimization of EMS Mutagenesis Condition and Screening of Mutants in Gossypium arboretum L.

Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) is a family of evolutionarily conserved cytidine deaminases, encoded by eleven genes located in the human genome. APOBECs play key roles in innate immunity through their ability to mutagenize viral DNA and restrict rival replication. Recent cancer genomics revealed APOBEC3 subtype-mediated APOBEC-signature mutations are common in a broad spectrum of human cancers. The pervasive APOBEC3 activation in the host genome which converts cytosine to uracile during RNA editing has been suggested to depend on ATR/chk1 pathways. In this review, we highlight how microRNAs interact with the APOBEC gene family and post-transcriptionally regulate APOBEC gene expression, and we speculate how targeting specific microRNAs may reduce host genome mutagenesis via inactivation of APOBEC deaminases.

Recent advances in genome editing technologies have opened the possibility for treating genetic diseases, such as Duchenne muscular dystrophy(DMD), by correcting the causing gene mutations in dystrophin gene. In fact, there are several reports that demonstrated the restoration of the mutated dystrophin gene in DMD patient-derived iPS cell or functional recovery of forelimb grip strength in DMD model mice. For future clinical applications, there are several aspects that need to be taken into consideration:efficient delivery of the genome editing components, risk of off-target mutagenesis and immunogenicity against genome editing enzyme. In this review, we summarize the current status and future prospective of the research in applying genome editing technologies to DMD.

Mutagenic agents had been used to enhance production of enzymes in microorganisms, but the effects of the agents on the kinetic parameters and catalytic efficiency of the produced enzymes had not been critically evaluated. A study was conducted to assess the effects of ultraviolet (UV) mutagenesis on the kinetic parameters and catalytic efficiency of endo-1,4-β-D-glucanase enzyme obtained from wild and mutated strains of Aspergillus niger and Penicillium citrinum obtained from decaying wood wastes modified by ultraviolet radiation. The endo-1, 4-β-Dglucanase from both wild and mutant strains of the fungi were purified by centrifugation, ammonium sulfa-te precipitation and anion exchange chromatography using Sephadex A25-120 and Whatman DE-52 resins. The kinetic parameters determined from Lineweaver-Burk plot, using the velocity or reaction rates at varying concentrations of carboxymethyl cellulose substrate ranging from 0 - 20 mg/mL were validated by Hanes-Woolf plots for convergence, as the Michaelis-Menten kinetic equation is non-linear. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) for the wild A. niger were 15.31 g/L and 4.67 g/L respectively, while values for corresponding mutated strain were 7.9 g/L and 3.39 g/L respectively. The Km and Vmax for wild P. citrinum were 7.94g/L and 6.81g/L respectively, while the values for the mutated strain were 6.60 g/L and 6.84 g/L respectively. Catalytic efficiency of endoglucanase of wild and mutated strains of A. niger were 0.305 and 0.42 M-1s-1 respectively, while the values for endoglucanase of wild and mutated strains of P. citrinum were 0.858 and 1.036 M-1s-1 respectively. Mutagenesis using ultraviolet radiation therefore enhanced the catalytic efficiency of endoglucanase enzyme.Keywords: Endo-1,4-β-D-glucanase, Aspergillus niger, Penicillium citrinum, UV Mutagenesis, Michaelis-Menten Kinetic Equation, Lineweaver-Burk Plot, Hanes Woolf Plot, Catalytic Efficiency

We obtained a glabrous leaf and hull mutant from a population of radiation mutagenesis of an indica rice cultivar R401. The mutant produced smooth leaves and hairless glumes under normal growth conditions. An F2 population was developed from a cross between a japonica cultivar Nipponbare and the glabrous leaf and hull mutant. By investigating the performance of the F2 population, we found that the mutant phenotype was controlled by a single recessive gene, temporarily designated GLR3. Bulked segregant analysis (BSA) based on the F2 mapping population revealed that GLR3 is located on chromosome 6. By analyzing 417 typical glabrous leaf F2 plants using molecular markers, GLR3 was mapped to a 0.2 cM interval between InDel markers ID27101 and ID27199, and the physical distance between the two markers is 98 kb. Thus we have mapped the gene GLR3, and our work will provide basis for future mechanistic analysis of GLR3 function.