• BMB Reports
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• 제46권5호
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• pp.270-275
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• 2013

It is essential to analyze rare mutations in many fields of biomedical research. However, the detection of rare mutations is usually failed due to the interference of predominant wild-type DNA surrounded. Herein we describe a sensitive and facile method of detecting rare point mutation on the basis of allele-specific amplification in emulsion PCR. The identification and selective amplification of rare mutation are accomplished in one-pot reaction. The allele-specific primers coupled on magnetic beads allow the exclusive amplification and enrichment of the mutant amplicons. The productive beads bearing mutant amplicons are subsequently stained with the fluorescent dyes. Thus, the rare point mutations with a percentage as low as 0.1%, can be detected by fluorescent analysis. The relative percentages of mutation among different samples can be roughly accessed by counting the fraction of fluorescent positive beads through flow cytometry.

• Korean Journal of Veterinary Research
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• 제41권2호
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• pp.189-195
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• 2001

Single base substitution and deletion mutation have been introducted into the verotoxin 2 (VT2)A subunit gene from O157:H7 isolates to reduce cytotoxicity of VT2 and the cytotoxicity between wild type toxin and mutant toxoid were compared. A M13-derived recombinant plasmid pEP19RF containing a 940bp EcoRI-PstI fragment of VT2A gene was constructed for oligonucleotide-directed mutagenesis. The duoble mutant pDOEX was constructed by point and deletion mutation of two different highly conserved regions of VT2A encoding active site cleft of enzymatic domain. The key residue, Glu 167(GAA) and the pentamer(WGRIS) consisting of the enzymatic domain were replaced by ASP(GAC) and completely deleted in nucleotide sequence analysis of mutant, respectively. In the comparision of vero cell cytotoxicity between wide type toxin and toxoid from mutant, the wild type toxin expressed cytotoxicity in dilution of $10^{-6}$, but the toxid from mutant did not show cytotoxicity to vero cells.

• Microbiology and Biotechnology Letters
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• 제19권5호
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• pp.491-496
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• 1991

To maximize the production of aminoglycoside-3'-phosphotransferase of E. coli ATCC 21990 carrying R factor which encodes aminoglycoside-3'-phosphotransferase (APH(3')) phosphorylating the 3'-hydroxyl group of aminoglycoside, mutants M1 and M2, media composition and several factors affecting the enzyme production during fermentation were studied. Although the specific activity of APH(3') produced by a mutant M1 was increased as much as four times than that of E. coii ATCC 21990, the growth rate was decreased. The increase of the enzyme production was obtained by increased biomass during fermentation. A mutant M2 was obtained to increase the cell growth rate. Mutant M2 cells were cultivated with optimal media and pure oxygen gas in a fed-batch mode of fermentor operation. The specific activity of APH(3') was decreased, but total enzyme activity of APH(3') was increased as much as two point five times than that of mutant MI.

• Biomedical Science Letters
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• 제12권4호
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• pp.413-418
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• 2006

Charcot-Marie-Tooth disease (CMT) is blown as one of the inherited disorder of peripheral nervous system. Recently, it was found that point mutations in the neurofilament light subunit (NF-L) gene cause CMT. Neurofilaments (NFs) are heteropolymers consist of NF-L, NF-M and NF-H. To assess the relationship between CMT and NF-L mutation in cellular level, we performed phenotypic analysis of the mutant NF-L (E397K) using cultured cell lines. Vimentin-deficient human adrenal carcinoma SW13 (Vim-) cells have a potential to form the intermediate filaments when the cells are expressing both NF-L and NF-M. Our results show that co-expression of wild type NF-L with NF-M showed intermediate filament formation in SW13 (Vim-) cells, while E397K with NF-M did not. This result means that E397K mutant lost its ability to form the intermediate filament in vivo, and further suggests that the E397K mutation is closely related to CMT.

• Korean Journal of Microbiology
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• 제54권4호
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• pp.343-353
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• 2018

Our previous research demonstrated the essential role of the xenB gene in stress response to RDX by using Pseudomonas sp. HK-6 xenB knockout. We have extended this work to examine the cellular responses and altered proteomic profiles of the HK-6 xenA knockout mutant under RDX stress. The xenA mutant degraded RDX about 2-fold more slowly and its growth and survival rates were several-fold lower than the wild-type HK-6 strain. SEM revealed more severe morphological damages on the surface of the xenA mutant cells under RDX stress. The wild-type cells expressed proportionally-increased two stress shock proteins, DnaK and GroEL from the initial incubation time point or the relatively low RDX concentrations, but slightly less expressed at prolonged incubation period or higher RDX. However the xenA mutant did not produced DnaK and GroEL as RDX concentrations were gradually increased. The wild-type cells well maintained transcription levels of dnaA and groEL under increased RDX stress while those in the xenA mutant were decreased and eventually disappeared. The altered proteome profiles of xenA mutant cells under RDX stress also observed so that the 27 down-regulated plus the 3 up-regulated expression proteins were detected in 2-DE PAGE. These all results indicated that the intact xenA gene is necessary for maintaining cell integrity under the xenobiotic stress as well as performing an efficient RDX degradation process.

• Interdisciplinary Bio Central
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• 제4권3호
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• pp.8.1-8.7
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• 2012

The motor neuron degeneration 2 (mnd2) mice carry a point mutation of A to T nucleotide transversion at the serine 276 residue of high temperature requirement A2 (HtrA2), resulting in losses of an AluI restriction enzyme site (5'AGCT3') and the HtrA2 serine protease activity. Moreover, dysfunctions of HtrA2 are known to be intimately associated with the pathogenesis of neurodegenerative diseases, including Parkinson's disease. Thus, this mnd2 mouse is an invaluable model for understanding the physiological role of HtrA2 and its pathological role in neurodegenerative diseases. Nevertheless, many molecular and cellular biologists in this field have limited experience in working with mutant mouse models due to the necessity of acquired years of the special techniques and knowledges. Herein, using the mnd2 mouse model as an example, we describe easy-to-use standard protocols for web-based analyses of target genes, such as HtrA2, and a novel approach for simple and accurate PCR-AluI-RFLP-based genotype analysis of mnd2 mice. In addition, band resolution of AluI-RFLP fragments was improved in 12% polyacrylamide gel running in 1X Tris-Glycine SDS buffer. Our study indicates that this PCR-AluI-RFLP genotype analysis method can be easily applied by the molecular and cellular biologist to conduct biomedical science studies using the other mutant mouse models.

• Journal of Microbiology and Biotechnology
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• 제32권8호
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• pp.1041-1046
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• 2022

Nucleoside deoxyribosyltransferase (NDT) is an enzyme that replaces the purine or pyrimidine base of 2'-deoxyribonucleoside. This enzyme is generally used in the nucleotide salvage pathway in vivo and synthesizes many nucleoside analogs in vitro for various biotechnological purposes. Since NDT is known to exhibit relatively low reactivity toward nucleoside analogs such as 2'-fluoro-2'-deoxynucleoside, it is necessary to develop an enhanced NDT mutant enzyme suitable for nucleoside analogs. In this study, molecular evolution strategy via error-prone PCR was performed with ndt gene derived from Lactobacillus leichmannii as a template to obtain an engineered NDT with higher substrate specificity to 2FDU (2'-fluoro-2'-deoxyuridine). A mutant library of 214 ndt genes with different sequences was obtained and performed for the conversion of 2FDU to 2FDA (2'-fluoro-2'-deoxyadenosine). The E. coli containing a mutant NDT, named NDT^L59Q, showed 1.7-fold (at 40℃) and 4.4-fold (at 50℃) higher 2FDU-to-2FDA conversions compared to the NDT^WT, respectively. Subsequently, both NDT^WT and NDT^L59Q enzymes were over-expressed and purified using a His-tag system in E. coli. Characterization and enzyme kinetics revealed that the NDT^L59Q mutant enzyme containing a single point mutation of leucine to glutamine at the 59^th position exhibited superior thermal stability with enhanced substrate specificity to 2FDU.

• Journal of Life Science
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• 제33권1호
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• pp.8-14
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• 2023

Peroxidasin (PXDN), a multidomain heme peroxidase containing extracellular matrix (ECM) motifs, as well as a catalytic domain, catalyzes the sulfilimine crosslink of collagen IV (Col IV) to reinforce Col IV scaffolds. We previously reported that PXDN is required for endothelial cell (EC) survival and growth signaling through sulfilimine crosslink-dependent matrix assembly. In this study, we examined whether peroxidase activity is required for PXDN function in ECs. First, we constructed a mutant PXDN by point mutation of two highly conserved amino acids, Q823 and D826, which are present in the active site of the peroxidase domain. After isolation of HEK293 clones highly expressing the mutant protein, conditioned medium (CM) was obtained after incubating the cells in serum-free medium for 24 hours and then analyzed by Western blot analysis under nonreducing conditions. The results revealed that the mutant PXDN formed a trimer and that it was cleaved by proprotein convertase-like wild-type (WT) PXDN. However, peroxidase activity was not detected in the CM containing the mutant PXDN, in contrast to that of WT PXDN. In addition, the sulfilimine crosslink ability of the mutant PXDN was lost. Moreover, the CM containing the mutant PXDN failed to promote the growth of PXDN-depleted ECs, unlike the CM containing WT PXDN. These results suggest that the peroxidase activity of PXDN affects EC growth by forming a sulfilimine crosslink.

• Asian Pacific Journal of Cancer Prevention
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• 제16권18호
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• pp.8281-8285
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• 2016

Background: Helicobacter pylori is a cause of chronic gastritis, peptic ulcer disease, and gastric malignancy, infection being a serious health problem in Thailand. Recently, clarithromycin resistant H. pylori strains represent the main cause of treatment failure. Therefore this study aimed to determine the prevalence and pattern of H. pylori resistance to clarithromycin in Suranaree University of Technology Hospital, Suranree University of Technology, Nakhon Ratchasima, Northeastern Thailand, Nakhon Ratchasima province, northeast of Thailand. Materials and Methods: This hospital-based cross-sectional study was carried out between June 2014 and February 2015 with 300 infected patients interviewed and from whom gastric mucosa specimens were collected and proven positive by histology. The gastric mucosa specimens were tested for H. pylori and clarithromycin resistance by 23S ribosomal RNA point mutations analysis using real-time polymerase chain reactions. Correlation of eradication rates with patterns of mutation were analyzed by chi-square test. Results: Of 300 infected patients, the majority were aged between 47-61 years (31.6%), female (52.3%), with monthly income between 10,000-15,000 Baht (57%), and had a history of alcohol drinking (59.3%). Patient symptoms were abdominal pain (48.6%), followed by iron deficiency anemia (35.3%). Papaya salad consumption (40.3%) was a possible risk factor for H. pylori infection. The prevalence of H. pylori strains resistant to clarithromycin was 76.2%. Among clarithromycin-resistant strains tested, all were due to the A2144G point mutation in the 23S rRNA gene. Among mutations group, wild type genotype, mutant strain mixed wild type and mutant genotype were 23.8%, 35.7% and 40.5% respectively. With the clarithromycin-based triple therapy regimen, the efficacy decreased by 70% for H. pylori eradication (P<0.01). Conclusions: Recent results indicate a high rate of H. pylori resistance to clarithromycin. Mixed of wild type and mutant genotype is the most common mutant genotype in Nakhon Ratchasima province, therefore the use of clarithromycin-based triple therapy an not advisable as an empiric first-line regimen for H. pylori eradication in northeast region of Thailand.

• Korean Journal of Microbiology
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• 제41권4호
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• pp.246-254
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• 2005

Analysis of the complete genome of Neurospora crassa reveals that at least 19 proteins contain tetratricopeptide repeat (TPR) motifs. One of them shows over $60\%$ homology to Ssn6 of Saccharomyces cerevisiae, a universal repressor that mediates repression of genes involved in various cellular processes. Mutant strains generated by RIP (repeat-induced point mutation) process showed four distinctive vegetative growth patterns and slow growth in various rates. Firstly, a mutant showed denser mycelial growth, yellow, csp, and looked like ropy mutant. Secondly, slower growth, dense mycelial, and conidial phenotype. Thirdly, extremely slower growth and aconidial. And finally, flat, tittle aerial hyphae, acon, and similar with a rco-1 RIP mutant. They are all male-fertile, yet female-sterile and produced little or no perithecium. It seems that various phenotypes were occurred depending upon mostly likely, the degree of RIP. These results indicate that this gene may be involved in several cellular possess during vegetative growth, and asexual and sexual development. Therefore it is pleiotropic. Sequence analysis of cDNA shows that it encodes a putative 102 kDa protein composed of 917 amino acids, and has six introns. It is designated rcm-1 (regulation of conidiation and morphology).