• Korean Journal of Biological Psychiatry
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• v.6 no.1
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• pp.67-73
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• 1999

The cAMP-dependent protein kinase(PKA) is an intracellular enzyme with serine-threonine kinase activity that plays a key role in cell growth, differentiation, and apoptosis in eukaryotes. In order to understand the PKA signal transduction pathway regulating cell life cycle and identify its role, we focused on the characterization of up-/down-regulated genes by PKA using the differential display polymerase chain reaction. Seven differentially expressed sequence tags(DEST) have been obtained. Among these DESTs, 2 DESTs were homologous to the sequence of genes from BLAST search result. KC1-5 DEST that was up-regulated in A123.7 cells was highly corresponded to mouse apoptosis-related gene(MA-3) or mouse mRNA for topoisomerase inhibitor suppressed(TIS). MA-3 was induced in various types of apoptosis, specially in NGF-deprived apoptotic PC12 cells. TIS was down-regulated in the RVC lymphoma cells incubated with topoisomerase inhibitor that induces DNA strand breakages. PG1-1 DEST that was highly expressed in PC12 cells was corresponded to transposon Tn10 3'-end. Tnansposon Tn10 was up-regulated in differentiated myeloblastic ML-1 cells by 12-O-tetradecanoylphorbol-13-acetate. This study illuminates that MA-3/TIS was down-regulated by PKA activity, and transposon Tn10 was up-regulated by it.

• Development and Reproduction
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• v.8 no.1
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• pp.19-25
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• 2004

Pax6, a member of the highly conserved homeobox gene family, is known to be expressed in spatially and temporally restricted pattern during embryogenesis. To examine the spatial expression pattern of Pax6 in malaria vector mosquito Anopheles stephemi, in different molecular environment, the germ line transformation technique using piggyBac transposon combined with the use of Pax6 specific 3xp3-EGFP marker was utilized. Four transgenic lines with a transformation rate of 6.7% were established. Transgenes were stably expressed in subsequent several generations. The transgenic lines showed 3 different expression pattern with spatial specificity, possibly due to enhancing and/or silencing position effects. In two transgenic lines, noble expression pattern of Pax6 was observed in the region that has not been previously reported in any animal species. The results show that the tranposon piggyBac mediated germ line transformation system can be used as an efficient tool for the generation of diverse spatially restricted reporter gene expression.

• Journal of Microbiology and Biotechnology
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• v.15 no.6
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• pp.1337-1345
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• 2005

Vibrio vulnificus is the causative agent of foodborne diseases such as gastroenteritis and life-threatening septicemia. Microbial pathogenicity is a complex phenomenon in which expression of numerous virulence factors is frequently controlled by a common regulatory system. In the present study, a mutant exhibiting decreased cytotoxic activity toward intestinal epithelial cells was screened from a library of V. vulnificus mutants constructed by a random transposon mutagenesis. By a transposon-tagging method, an open reading frame, fexA, a homologue of Escherichia coli areA, was identified and cloned. The nucleotide and deduced amino acid sequences of the fexA were analyzed, and the amino acid sequence of FexA from V. vulnificus was $84\%\;to\;97\%$ similar to those of AreA, an aerobic respiration control global regulator, from other Enterobacteriaceae. Functions of the FexA were assessed by the construction of an isogenic mutant, whose fexA gene was inactivated by allelic exchanges, and by evaluating its phenotype changes in vitro and in mice. The disruption of fexA resulted in a significant alteration in growth rate under aerobic as well as anaerobic conditions. When compared to the wild-type, the fexA mutant exhibited a substantial decrease in motility and cytotoxicity toward intestinal epithelial cell lines in vitro. Furthermore, the intraperitoneal $LD_{50}$ of the fexA mutant was approximately $10^{1}-10^{2}$ times higher than that of parental wild-type. Therefore, it appears that FexA is a novel global regulator controlling numerous genes and contributing to the pathogenesis as well as growth of V. vulnificus.

• Journal of Microbiology and Biotechnology
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• v.4 no.3
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• pp.165-170
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• 1994

An efficient and convenient method of introducing transposable elements into acetic acid bacteria was developed by the method of conjugal transfer. The ampicillin-resistant strain, Acetobacter sp. HA, was selected to be conjugated with two E. coli strains, WA803 containing pGS9 and AC8001 harboring pJB4JI. The Tn5 containing suicide vector pGS9 or pJB4JI, was transferred from E. coli to Acetobacter sp. HA and kanamycin-ampicillin-resistant transconjugants obtained at high frequencies. The conjugal frequencies of pGS9 and pJB4JI were 6.20$\times$$l0^{-1} and 2.79$\times$l0{-1}$ per recipient, respectively. The transfer method was applied on four different strains of Acetobacter. The conjugal transfer frequencies ranged from 2.00$\times$$l0^{-2} to 4.45$\times$l0^{-8}$ per recipient in the three strains. Some transconjugants tested were found to contain Tn5 DNA in their genomes and this was confirmed by Southem blot analysis. This is the first study which shows that Tn5 mutagenesis can be applied to successfully isolate mutants of Acetobacter genus.

• Journal of Microbiology and Biotechnology
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• v.21 no.4
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• pp.374-378
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• 2011

To find alternative genetic resources for D-serine dehydratase (E.C. 4.3.1.18, dsdA) mediating the deamination of D-serine into pyruvate, metagenomic libraries were screened. The chromosomal dsdA gene of a wild-type Escherichia coli W3110 strain was disrupted by inserting the tetracycline resistance gene (tet), using double-crossover, for use as a screening host. The W3110 dsdA::tet strain was not able to grow in a medium containing D-serine as a sole carbon source, whereas wild-type W3110 and the complement W3110 dsdA::tet strain containing a dsdA-expression plasmid were able to grow. After introducing metagenome libraries into the screening host, a strain containing a 40-kb DNA fragment obtained from the metagenomic souce derived from a compost was selected based on its capability to grow on the agar plate containing D-serine as a sole carbon source. For identification of the genetic resource responsible for the D-serine degrading capability, transposon-${\mu}$ was randomly inserted into the 40-kb metagenome. Two strains that had lost their D-serine degrading ability were negatively selected, and the two 6-kb contigs responsible for the D-serine degrading capability were sequenced and deposited (GenBank code: HQ829474.1 and HQ829475.1). Therefore, new alternative genetic resources for D-serine dehydratase was found from the metagenomic resource, and the corresponding ORFs are discussed.

• Korean Journal of Veterinary Research
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• v.42 no.4
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• pp.505-512
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• 2002

대장균 K99 섬모의 생합성은 8개로 구성된 K99의 특이 유전자의 발현과 숙주유래 인자에 의해 조절되는 다른 유전자들의 발현에 의존된다. 본 연구에서는 K99섬모 유전자군중 제 3지역 발현에 유전조절자의 관련성 여부를 연구하였다. Gel retardation 분석 방법올 통하여 제3지역의 발현에 관련된 유전조절단위를 함유한 fanF 지역의 단백질 인자가 부착됨을 암시하였다. 이 분석방법을 이용한 결과는 또한 이 단백질 인자가 K99 유전자에서 유래되지 않고 대장균 염색체에서 유래됨을 지적하였다. 이를 보다 더 조사하기 위하여 대장균 염색체에 Tn10 transposon 유전자 변이 실험을 수행하였다. K99 유전자군으로부터 제 1지역과 제2지역의 유전자를 제거시키고, 제 3지역의 유전자인 fanG에 transposon TnlacZ를 삽입한 pTL65-1 plasmid을 제작하였다. 이 pTL65-1는 다시 Tn10으로 염색체가 변이된 대장균에 주입하였다. 3개의 pTL65-1 주입된 Tn10 대장균 변이체 내에서 fanG의 발현이 증가되었다. 이들 변이대장균으로부터 Tn10이 어떤 염색체 유전자 부위를 변이 시켰는지 확인하기 위해서 변이부위 유전자를 cloning하여 염기서열을 분석하였다. 이중 2개의 clone이 동일하였으며 지금까지 알려지지 않은 유전자였다. 이들 2개의 변이체 내에서 fanG의 발현은 대조군과 비교해 약 4.2배 증가 되였다. 결론적으로 이들 2개의 clone으로부터 유래된 인자는 지금까지 알려지지 않은 제 3지역의 억제 조절자임을 나타내었다.

• Molecules and Cells
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• v.26 no.6
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• pp.611-615
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• 2008

DNA methylation is an epigenetic mechanism for gene silencing. In Arabidopsis, MET1 is the primary DNA methyltransferase that maintains CG DNA methylation. Plants having an overall reduction of MET1 activity, caused by a met1 mutation or a constitutively expressed MET1 antisense gene, display genome hypomethylation, inappropriate gene and transposon transcription, and developmental abnormalities. However, the effect of a transient reduction in MET1 activity caused by inhibiting MET1 expression in a restricted set of cells is not known. For this reason, we generated transgenic plants with a MET1 antisense gene fused to the DEMETER (DME) promoter (DME:MET1 a/s). Here we show that DME is expressed in leaf primordia, lateral root primoridia, in the region distal to the primary root apical meristem, which are regions that include proliferating cells. Endogenous MET1 expression was normal in organs where the DME:MET1 a/s was not expressed. Although DME promoter is active only in a small set of cells, these plants displayed global developmental abnormalities. Moreover, centromeric repeats were hypomethylated. The developmental defects were accumulated by the generations. Thus, not maintaining CG methylation in a small population of proliferating cells flanking the meristems causes global developmental and epigenetic abnormalities that cannot be rescued by restoring MET1 activity. These results suggest that during plant development there is little or no short-term molecular memory for reestablishing certain patterns of CG methylation that are maintained by MET1. Thus, continuous MET1 activity in dividing cells is essential for proper patterns of CG DNA methylation and development.

• The Plant Pathology Journal
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• v.36 no.4
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• pp.355-363
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• 2020

Bacterial traits for virulence of Ralstonia solanacearum causing lethal wilt in plants were extensively studied but are not yet fully understood. Other than the known virulence factors of Ralstonia solanacearum, this study aimed to identify the novel gene(s) contributing to bacterial virulence of R. solanacearum. Among the transposon-inserted mutants that were previously generated, we selected mutant SL341F12 strain produced exopolysaccharide equivalent to wild type strain but showed reduced virulence compared to wild type. In this mutant, a transposon was found to disrupt the murI gene encoding glutamate racemase which converts L-glutamate to D-glutamate. SL341F12 lost its motility, and its virulence in the tomato plant was markedly diminished compared to that of the wild type. The altered phenotypes of SL341F12 were restored by introducing a full-length murI gene. The expression of genes required for flagella assembly was significantly reduced in SL341F12 compared to that of the wild type or complemented strain, indicating that the loss of bacterial motility in the mutant was due to reduced flagella assembly. A dramatic reduction of the mutant population compared to its wild type was apparent in planta (i.e., root) than its wild type but not in soil and rhizosphere. This may contribute to the impaired virulence in the mutant strain. Accordingly, we concluded that murI in R. solanacearum may be involved in controlling flagella assembly and consequently, the mutation affects bacterial motility and virulence.

• The Plant Pathology Journal
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• v.28 no.4
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• pp.439-445
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• 2012

The chitinase producing Lysobacter enzymogenes C-3 has previously been shown to suppress plant pathogens in vitro and in the field, but little is known of the regulation of chitinase production, or its role in antimicrobial activity and biocontrol. In this study, we isolated and characterized chitinase-defective mutants by screening the transposon mutants of L. enzymogenes C-3. These mutations disrupted genes involved in diverse functions: glucose-galactose transpoter (gluP), disulfide bond formation protein B (dsbB), Clp protease (clp), and polyamine synthase (speD). The chitinase production of the SpeD mutant was restored by the addition of exogenous spermidine or spermine to the bacterial cultures. The speD and clp mutants lost in vitro antifungal activities against plant fungal pathogens. However, the gluP and dsbB mutants showed similar antifungal activities to that of the wild-type. The growth of the mutants in nutrient rich conditions containing chitin was similar with that of the wild-type. However, growth of the speD and gluP mutants was defective in chitin minimal medium, but was observed no growth retardation in the clp and dsbB mutant on chitin minimal medium. In this study, we identified the four genes might be involved and play different role in the production of extracellular chitinase and antifungal activity in L. enzymogenes C-3.

• Korean Journal of Veterinary Research
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• v.39 no.2
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• pp.327-337
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• 1999

The transposon TnphoA was used to generate avirulent mutants from a type A Pasteurella multocida. A suicide vector plasmid pRT733 carrying TnphoA, having the kanamycin resistant gene and harbored in Escherichia coli K-12 strain SM10(${\lambda}pir$), was mated with streptomycin resistant P. multocida P-1059 strain as recipient. This resulted in the generation of two TnphoA insertion mutants (transconjugants, tc95-a and tc95-b) which were resistant both to kanamycin ($Km^{R}$) and streptomycin ($Sm^{R}$), secreted alkaline phosphatase, and were avirulent to turkeys. Southern blot hybridization using two probes derived from internal fragments of TnphoA, confirmed the insertion of TnphoA into 12.9kb or 13.7kb DNA fragment from the EcoRV digested genomic fragments of transconjugants. The two transconjugants, tc95-a and tc95-b, were distinguishable from their parent strains by differences in ribotypes, and outer membrane protein profiles. TnphoA insertion in both transconjugants also resulted in constitutive expression of a 33Kd iron regulated outer membrane protein (IROMP). The gene encoding $Sm^{R}$ was also located within the same 12.9kb EcoRV genomic fragment from both transconjugants. Furthermore, our finding that the recipient P. multocida P-1059 $Sm^{R}$ strain and both transconjugants were avirulent to turkeys suggest that the either 12.9kb or 13.7kb genomic DNA contains the virulence gene and speculate that the presence of $Sm^{R}$ gene or TnphoA insertion may be responsible for regulating and inactivating the gene(s) encoding virulence in P. multocida.