• Journal of Microbiology
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• v.43 no.5
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• pp.417-423
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• 2005

In this study, the nematode-trapping fungus, Monacrosporium sphaeroides, was transformed with a plasmid harboring the hygromycin B phosphotransferase gene, via restriction enzyme-mediated integration (REMI). Frequencies of up to 94 transformants ${\mu}g^{-1}$ per linearized plasmid DNA were obtained by optimizing the PEG concentration, as well as the category and quantity of the added restriction enzyme. $90\%$ of the transformants were determined to be stable for drug resistance when 20 randomly selected transformants were tested. Southern analyses revealed that the transforming DNA was integrated into the M. sphaeroides genome either with or without rearrangement. Five mitotic stable mutant strains were obtained using this approach, all of which had been altered with regard to sporulation capacity and pathogenicity toward nematodes. Southern blot analyses of the five mutants revealed that foreign plasmid DNA had integrated into the genome. Three of the mutants, Tms2316, Tms3583 and Tms1536, exhibited integration at a single location, whereas the remaining two, Tms32 and Tms1913, manifested integration at double or multiple locations. Our results suggest that the transformation of M. sphaeroides via REMI will facilitate insertional mutagenesis, the functional analysis of a variety of genes, and the tagging or cloning of genes of interest.

• Korean Journal of Microbiology
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• v.53 no.4
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• pp.297-303
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• 2017

Leucine-responsive regulatory protein (LRP) is a regulatory protein of molecular weight 18.8 kDa and is widely known to regulate many metabolic and functional activities of operons in Escherichia coli. The gene for Lrp from Escherichia coli in pQE system of 6 ${\times}$ His-tagging was expressed and $^3H$-labeled protein, as well as the wild type Lrp, was purified. The crosslink experiments were performed to analyze the quaternary structure of Lrp at high of $5{\mu}M$ and at low concentrations below $0.3{\mu}M$ with cross linkers, such as glutaraldehyde, 1, 2, 3, 4-diepoxy-butane (DEB), and ethylene glycol bis (succinimidyl succinate) (EGS). In the experiments, we found that the Lrp protein can be formed higher conformation states of tetramer, hexamer, octamer, as well as dimeric state when incubated with the above cross linkers.

• Molecules and Cells
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• v.46 no.6
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• pp.351-359
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• 2023

Deamination of adenine or cytosine in RNA, called RNA editing, is a constitutively active and common modification. The primary role of RNA editing is tagging RNA right after its synthesis so that the endogenous RNA is recognized as self and distinguished from exogenous RNA, such as viral RNA. In addition to this primary function, the direct or indirect effects on gene expression can be utilized in cancer where a high level of RNA editing activity persists. This report identified actin-related protein 2/3 complex inhibitor (ARPIN) as a target of ADAR1 in breast cancer cells. Our comparative RNA sequencing analysis in MCF7 cells revealed that the expression of ARPIN was decreased upon ADAR1 depletion with altered editing on its 3'UTR. However, the expression changes of ARPIN were not dependent on 3'UTR editing but relied on three microRNAs acting on ARPIN. As a result, we found that the migration and invasion of cancer cells were profoundly increased by ADAR1 depletion, and this cellular phenotype was reversed by the exogenous ARPIN expression. Altogether, our data suggest that ADAR1 suppresses breast cancer cell mobility via the upregulation of ARPIN.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.51 no.3
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• pp.259-268
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• 2006

Miniature inverted transposable elements (MITEs) are abundant genomic components in plant including rice. MITE-transposon display (MITE-TD) is an Amplified Fragment Length Polymorphism (AFLP)-related technique based on MITE sequence. In this study, we used the MITE-AFLP for the analysis of diversity and relation-ship of the 114 japonica accessions. Of the several MITEs, the mPing family was applied to detect polymorphisms based on PCR amplification. The BfaI adaptor primer and the specific primer derived from mPing terminal inverted repeat (TIR) region were used to PCR amplification of 114 accessions. Nine primer pairs produced a total of 160 polymorphic bands. PIC values of the polymorphic bands generated by nine primer pairs ranged from 0.269 (BfaI + ACT) to 0.426 (BfaI + T). Each accession revealed a distinct fingerprint with two primer combinations, BfaI + G and BfaI + C. Cluster analysis using marker-based genetic similarity classified 114 accessions into five groups. MITE-AFLP markers were genetically mapped using a population of 80 BILs (BC1F7) derived from a cross between the rice accessions, Milyang 23 and Hapcheonaengmi 3. Eight of the markers produced with the primer pair BfaI + 0 were mapped on chromosomes 1, 2, 4, 5, 7, and 9. Considering that one MITE-AFLP marker on chromosome 7 was tightly linked to the Rc gene, the MITE-AFLP markers will be useful for gene tagging and molecular cloning.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.8
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• pp.3629-3633
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• 2014

Background: Glioblastoma (GBM) is an immunosuppressive tumor whose median survival time is only 12-15 months, and patients with GBM have a uniformly poor prognosis. It is known that heredity contributes to formation of glioma, but there are few genetic studies concerning GBM. Materials and Methods: We genotyped six tagging SNPs (tSNP) in Han Chinese GBM and control patients. We used Microsoft Excel and SPSS 16.0 statistical package for statistical analysis and SNP Stats to test for associations between certain tSNPs and risk of GBM in five different models. ORs and 95%CIs were calculated for unconditional logistic-regression analysis with adjustment for age and gender. The SHEsis software platform was applied for analysis of linkage disequilibrium, haplotype construction, and genetic associations at polymorphism loci. Results: We found rs891835 in CCDC26 to be associated with GBM susceptibility at a level of p=0.009. The following genotypes of rs891835 were found to be associated with GBM risk in four different models of gene action: i) genotype GT (OR=2.26; 95%CI, 1.29-3.97; p=0.019) or GG (OR=1.33; 95%CI, 0.23-7.81; p=0.019) in the codominant model; ii) genotypes GT and GG (OR=2.18; 95%CI, 1.26-3.78; p=0.0061) in the dominant model; iii) GT (OR=2.24; 95%CI, 1.28-3.92; p=0.0053) in the overdominant model; iv) the allele G of rs891835 (OR=1.85; 95%CI, 1.14-3.00; p=0.015) in the additive model. In addition, "CG" and "CGGAG" were found by haplotype analysis to be associated with increased GBM risk. In contrast, genotype GG of CCDC26 rs6470745 was associated with decreased GBM risk (OR=0.34; 95%CI, 0.12-1.01; p=0.029) in the recessive model. Conclusions: Our results, combined with those from previous studies, suggest a potential genetic contribution of CCDC26 to GBM progression among Han Chinese.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.302-311
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• 2004

Comamonas testosteroni (formerly Pseudomonas sp.) NCIMB 10643 can grow on biphenyl and alkylbenzenes $(C_2-C_7)$ via 3-substituted catechols. Thus, to identify the genes encoding the degradation, transposon-mutagenesis was carried out using pAG408, a promoter-probe mini-transposon with a green fluorescent protein (GFP), as a reporter. A mutant, NT-1, which was unable to grow on alkylbenzenes and biphenyl, accumulated catechols and exhibited an enhanced expression of GFP upon exposure to these substrates, indicating that the gfp had been inserted in a gene encoding a broad substrate range catechol 2,3-dioxygenase. The genes (2,826 bp) flanking the gfp cloned from an SphI-digested fragment contained three complete open reading frames that were designated bphCDorfl. The deduced amino acid sequences of bphCDorfl were identical to 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC), 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (BphD), and OrfI, respectively, that are all involved in the degradation of biphenyl/4-chlorobiphenyl (bph) by Ralstonia oxalatica A5. The deduced amino acid sequence of the orfl revealed a similarity to those of outer membrane proteins belonging to the OmpW family. The introduction of the bphCDorfl genes enabled the NT-l mutant to grow on aromatic hydrocarbons. In addition, PCR analysis indicated that the DNA sequence and gene organization of the bph operon were closely related to those in the bph operon from Tn4371 identified in strain A5. Furthermore, strain A5 was also able to grow on a similar set of alkylbenzenes as strain NCIMB 10643, demonstrating that, among the identified aromatic hydrocarbon degradation pathways, the bph degradation pathway related to Tn4371 was the most versatile in catabolizing a variety of aromatic hydrocarbons of mono- and bicyclic benzenes.

• Genomics & Informatics
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• v.6 no.3
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• pp.99-109
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• 2008

Protein phosphorylation at tyrosine residues is a key regulatory event that modulates insulin signal transduction. We studied the PTPN1 gene with regard to susceptibility to Korean type 2 diabetes mellitus (T2DM) and its related quantitative traits. A total of seven SNPs [g.36171G>A (rs941798), g.58166G>A (rs3787343), g.58208A>G (rs2909270), g.64840C>T (rs754118), g.69560C>G (rs6020612), g.69866G>A (rs718050), and g.69934T>G (rs3787343)] were selected based on frequency (>0.05), linkage disequilibrium (LD) status, and haplotype tagging status. We studied the seven SNPs in 483 unrelated patients with type 2 diabetes (age: $64{\pm}2.8$ years, onset age: $56{\pm}8.1$ years; 206 men, 277 women) and 1138 nondiabetic control subjects (age: $64{\pm}2.9$; 516 men, 622 women). The SNP rs941798 had protective effects against T2DM with an odds ratio of 0.726 (C.I. $0.541{\sim}0.975$) and p-value=0.034, but none of the remaining six SNPs was associated with T2DM. Also, rs941798 was associated with blood pressure, HDL cholesterol, insulin sensitivity. rs941798 also has been associated with T2DM in previous reports of Caucasian-American and Hispanic-American populations. This is the first report that shows an association between PTPN1 and T2DM in the Korean as well as Asian population.

• Molecules and Cells
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• v.21 no.2
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• pp.284-293
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• 2006

Even though Ac/Ds gene-tagging systems have been established in many higher plants, maize is the only major plant in which short-distance transposition of Ac/Ds has been utilized to probe gene function. This study was performed to evaluate the efficiency of obtaining new alleles and functional revertants from Ds insertion loci in rice. By analyzing 1,580 plants and the progeny of selected lines, the insertion sites and orientations of Ds elements within 16 new heritable alleles of three rice loci were identified and characterized. Intragenic transposition was detected in both directions from the original insertion sites. The closest interval was 35 bp. Three of the alleles had two Ds elements in cis configuration in the same transcription units. We also analyzed the excision footprints of intragenic and extragenic transpositions in Ds-inserted alleles at 5 loci. The 134 footprints obtained from different plants revealed predominant patterns. Ds excision at each locus left a predominant footprint at frequencies of 30-75%. Overall, 66% of the footprints were 7-bp additions. In addition, 16% of the excisions left 0-, 3-, 6-, and 9-bp additions with the potential of conserving reading frame.

• Journal of Korean Society of Forest Science
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• v.87 no.2
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• pp.164-172
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• 1998

This study was conducted to find the optimum transformation condition using Agrobacterium harboring promoterless GUS gene. The optimal medium for shoot induction from leaves of Populus nigra${\times}$P. maximowiczii was MS medium supplemented with $0.1mg/{\ell}$ NAA, $0.5mg/{\ell}$ BAP(94% regeneration frequency and 11.5 average number of shoot) According to the test using pBI121, the concentration of antibiotics for selection marker gene was $100mg/{\ell}$ kanamycin or $60mg/{\ell}$ geneticin in the SIM(shoot inducing medium) 3. Two weeks later, callus was induced in the SIM 3 and this callus grew up to 0.5-1cm shoots after 6 weeks in the new SIM 3. And the treatment with methylation inhibitor(5-azacytidine) led to a dramatic increase in foreign gene expression rate from 5.7% to 26.7%. The vector systems showed. different transformation efficiencies based on the fluorometric and histochemical GUS assay. In this study the vector systems used for transformation seemed to affect transformation frequency, in which pEHA101 yielded more transformants(35.9%) than LBA4404/pBI121 did(5.7%). This result indicated that pEHA101 was effective to insert the promoterless foreign gene into a poplar genome.

• Journal of Plant Biotechnology
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• v.35 no.4
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• pp.307-316
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• 2008

Transposon-mediated insertional mutagenesis is one of powerful strategy for assessing functions of genes in higher plants. In this report, we have selected highly susceptible and tolerance plant by screening about high salt (3% NaCl) and cold stresses ($4^{\circ}C$) from F2 seeds of 30,000 Ac/Ds insertional mutagenesis lines in rice (Oryza sativa L. cv. Dongjin). In order to identify the gene tagging, insertion of Ds element was analyzed by Southern blot and these results revealed that 19 lines were matched genotype of selected lines with phenotype from the first selected 212 lines, and 13 lines have one copy of Ds elements. The Franking Sequence Tags (FSTs) of selected mutant lines showed high similarities with the following known function genes: signal transduction and regulation of gene expression (transpoter, protease family protein and apical meristem family protein), osmotic stress response (heat shock protein, O-methyltransferase, glyceraldehyde-3-phosphate dehydrogenase and drought stress induce protein), vesicle trafficking (SYP 5 family protein) and senescence associated protein. The expression pattern of 19 genes were analyzed using RT-PCR under the abiotic stresses of 9 class; 250mM NaCl, osmotic, drought, 3% $H_2O_2$, $100{\mu}M$ ABA, $100{\mu}M$ IAA, 0.1 ppm 2,4-D, $4^{\circ}C$ cold and $38^{\circ}C$ high temperature. Isolated knock-out genes showed the positive response about 250 mM NaCl, drought, $H_2O_2$, PEG, IAA, 2,4-D, ABA treatment and low ($4^{\circ}C$) and high temperature ($38^{\circ}C$). The results from this study indicate that function of selected knock-out genes could be useful in improving of tolerance to abiotic stresses as an important transcriptional activators in rice.