• Proceedings of the Korean Society of Plant Pathology Conference
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• 1995.06b
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• pp.1-19
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• 1995

The plasmid pJEL 101 contains a highly repetitive element from the genome of Xanthomonas oryae pv. oryzae that has properties of an insertional element. The insertional nature of the element, hereto referred to as IS203, was confirmed by molecular analyses of the element and three related elements that were isolated from X. oryzae. The related sequences were isolated on the basis of transposition to the transposon-trapping vector pL3SAC and hybridization with pJEL101. The trapped elements (IS203a, IS203b, and IS203c) were each composed of 1,055 base pairs with 25 base terminal inverted repeats. The elements caused a three base pair target site duplication at the site of insertion in the sacRB gene. The sequence of pJEL 101 has 96% base pair identity with IS203a and 99% identity with IS203a and IS203c but lacks three nucleotides of the consensus left terminal repeat. IS203b has the same DNA sequences as IS203c but is inserted ito the sacRB gene in the opposite orientation. The longest open reading frame of IS203a could code for a protein of 318 amino acids and molecular weight of 37, 151. A search of the Genbank database revealed that IS203 has 51% identity with 909 nucleotides of IS4551 from Escherichia coli. The predicted protein of ORF1 has 40% and 30% amino acid identity to the ORF1 of Tn4551 and the transposase of IS30, respectively.

• Journal of Life Science
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• v.10 no.4
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• pp.333-339
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• 2000

To identify genes involved in the decolorization of crystal violet, we isolated random mutants generated by transponson insertion in crystal violet-declorizing bacterium, Citrobacter sp. The resulting mutant bank yielded mutants with six distinct phenotypes, and Southern hybridization with a Tn5 fragment as a probe showed a single hybridized with six distinct phenotypes, and Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in the mutants Ctg 2, 5 an 6, whereas two and three bands were detected in Ctg1, 4 and 3, respectively. Tn5-inserted genes were isolated and the DNA sequence flanking Tn5 was determined. From comparison with a sequence database, putative protein product encoded by ctg 5 was identified as E. coli maltose transproter(Mal G) homolog, whereas the deduced amino acid sequence of the other ctg genes did not show any significant similarity with any DNA or protein sequency. Therefore, these results indicate that the other ctg genes except ctg 5 encode new proteins responsible for decolorization of crystal violet.

• Korean Journal of Microbiology
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• v.27 no.4
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• pp.323-333
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• 1989

Plasmid DNA molecules containing strong promoter upstream from IS50L or IS50R, the two insertion sequences that flank Tn5, were constructed to amplify the synthesis of Tn5-encoded polypeptides. When proteins made by cells that contain these plasmids were analyzed on polyacrylamide gels, enhanced synthesis of IS50R polypeptides could be detected. Synthesis of this polypeptide apparently is initiated within the large open reading frame of this element. In addition, the stability of IS50L-and IS50R-encoded polypeptides was analyzed. It was found that IS50L polypeptides are relatively unstable in vivo. This instability could account for the observed inability of this element to promote transposition.

• Korean Journal Plant Pathology
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• v.10 no.1
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• pp.39-46
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• 1994

Pseudomonas fluorescens Biovar III strains S-2 antagonistic to Rhizoctonia solani was subjected to Tn5 mutagenesis by the transposon vector pGS9. Ampicillin and kanamycin resistant (Ampr, Kmr) transconjugants were recovered at a frequency of 1.3$\times$10-7 per initial recipient cell, when recipient cells were washed twice in TE buffer before conjugation. Of the ca. 3000 transconjugants, a frequency of noninhibitory (Inh-), nonfluorescent (Flu-) and auxotorphic (Pro-) mutants were 0.27%, 0.47% and 0.40%, respectively. In these mutants, all Inh- mutants showed the same colony morphology as wild type, whereas all Flu- and Pro- mutants inhibited the growth of R. solani. These mutants were also susceptible to chloramphenicol, indicating only the Tn5 element, except for parts of pGS9, was integrated into the recipient genome. In a Southern blot analysis, the Tn5 element inserted into one site on the chromosome for each of the chosen mutants. However, Tn5 insertion sites of Inh-, and Pro- mutants were differed in each other. These indicate that the genes essential for R. solani inhibition, fluorescent production and auxotrophic are chromosomally located, but not linked to each other.

• Journal of Microbiology and Biotechnology
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• v.29 no.9
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• pp.1488-1493
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• 2019

The rising cases of multidrug-resistant Acinetobacter baumannii (Ab) and the lack of effective drugs call for quick attention. Here, based on a Tn7 transposon and Xer/dif system, we constructed a stable, selectable marker-free autoluminescent Ab capable of producing visible light without extra substrates. Utilization of this autoluminescent reporter strain has the potential to reduce the time, effort and costs required for the evaluation of activities of anti-Ab drug candidates in vitro.

• The Plant Pathology Journal
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• v.39 no.1
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• pp.62-74
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• 2023

Plant pathogenic Pectobacterium species cause severe soft rot/blackleg diseases in many economically important crops worldwide. Pectobacterium utilizes plant cell wall degrading enzymes (PCWDEs) as the main virulence determinants for its pathogenicity. In this study, we screened a random mutant, M29 is a transposon insertion mutation in the metC gene encoding cystathionine β-lyase that catalyzes cystathionine to homocysteine at the penultimate step in methionine biosynthesis. M29 became a methionine auxotroph and resulted in growth defects in methionine-limited conditions. Impaired growth was restored with exogenous methionine or homocysteine rather than cystathionine. The mutant exhibited reduced soft rot symptoms in Chinese cabbages and potato tubers, maintaining activities of PCWDEs and swimming motility. The mutant was unable to proliferate in both Chinese cabbages and potato tubers. The reduced virulence was partially restored by a complemented strain or 100 µM of methionine, whereas it was fully restored by the extremely high concentration (1 mM). Our transcriptomic analysis showed that genes involved in methionine biosynthesis or transporter were downregulated in the mutant. Our results demonstrate that MetC is important for methionine biosynthesis and transporter and influences its virulence through Pcc21 multiplication in plant hosts.

• International Journal of Industrial Entomology and Biomaterials
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• v.1 no.2
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• pp.171-175
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• 2000

Determined sequences of 384 randomly selected clones in a PCR-based cDNA library of Antheraea yamamai could identify expressed sequence tags (ESTs) of highly expressed gene. One EST (fibroin) appeared 15 times, one EST (40S ribosomal protein S18) twelve times, one EST (ribosomal protein S24a) eleven times, ten times (ribosomal protein S8), nine times (60S ribosomal protein L10A), seven times (60S ribosomal protein S15A, S17, S17 and seroin), six times (ribosomal protein S8), five times (ribosomal protein S24, mariner transposase and P8 protein), four times (serpin 2), three times (heat shock protein 70 and poly A binding protein), and the remaining 6 ESTs twice (amylase, KIAA1006, elongation factor-1, transposon mag, translation initiation factor 4C, QM protein, transposase). Therefore, the 94 EST make it possible to identify 24 redundant clones that are candidates for highly expressed genes in posterior silk gland of this insect. The 24 redundant EST clones were identified in GenBank, but none of them was related to A. yamamai, suggesting that there are many unidentified genes which are highly expressed in the A. yamamai genome.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.6
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• pp.886-892
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• 2017

Objective: Transgenic technology is widely used for industrial applications and basic research. Systems that allow for genetic modification play a crucial role in biotechnology for a number of purposes, including the functional analysis of specific genes and the production of exogenous proteins. In this study, we examined and verified the cumate-inducible transgene expression system in chicken DF1 and quail QM7 cells, as well as loxP element-mediated transgene recombination using Cre recombinase in DF1 cells. Methods: After stable transfer of the transgene with piggyBac transposon and transposase, transgene expression was induced by an appropriate concentration of cumate. Additionally, we showed that the transgene can be replaced with additional transgenes by co-transfection with the Cre recombinase expression vector. Results: In the cumate-GFP DF1 and QM7 cells, green fluorescent protein (GFP) expression was repressed in the off state in the absence of cumate, and the GFP transgene expression was successfully induced in the presence of cumate. In the cumate-MyoD DF1 cells, MyoD transgene expression was induced by cumate, and the genes controlled by MyoD were upregulated according to the number of days in culture. Additionally, for the translocation experiments, a stable enhanced green fluorescent protein (eGFP)-expressing DF1 cell line transfected with the loxP66-eGFP-loxP71 vector was established, and DsRed-positive and eGFP-negative cells were observed after 14 days of co-transfection with the DsRed transgene and Cre recombinase indicating that the eGFP transgene was excised, and the DsRed transgene was replaced by Cre recombination. Conclusion: Transgene induction or replacement cassette systems in avian cells can be applied in functional genomics studies of specific genes and adapted further for efficient generation of transgenic poultry to modulate target gene expression.

• Molecules and Cells
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• v.40 no.4
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• pp.262-270
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• 2017

MicroRNAs (miRNAs) are single-stranded, small RNAs (21-23 nucleotides) that function in gene silencing and translational inhibition via the RNA interference mechanism. Most miRNAs originate from host genomic regions, such as intergenic regions, introns, exons, and transposable elements (TEs). Here, we focused on the palindromic structure of medium reiteration frequencies (MERs), which are similar to precursor miRNAs. Five MER consensus sequences (MER5A1, MER53, MER81, MER91C, and MER117) were matched with paralogous transcripts predicted to be precursor miRNAs in the horse genome (equCab2) and located in either intergenic regions or introns. The MER5A1, MER53, and MER91C sequences obtained from RepeatMasker were matched with the eca-miR-544b, eca-miR-1302, and eca-miR-652 precursor sequences derived from Ensembl transcript database, respectively. Each precursor form was anticipated to yield two mature forms, and we confirmed miRNA expression in six different tissues (cerebrum, cerebellum, lung, spleen, adrenal gland, and duodenum) of one thoroughbred horse. MER5A1-derived miRNAs generally showed significantly higher expression in the lung than in other tissues. MER91C-derived miRNA-5p also showed significantly higher expression in the duodenum than in other tissues (cerebellum, lung, spleen, and adrenal gland). The MER117-overlapped expressed sequence tag generated polycistronic miRNAs, which showed higher expression in the duodenum than other tissues. These data indicate that horse MER transposons encode miRNAs that are expressed in several tissues and are thought to have biological functions.

• The Plant Pathology Journal
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• v.25 no.1
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• pp.77-85
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• 2009

Pseudomonas fluorescens strain Gpf01, isolated from ginseng rhizosphere showed antiviral activity against Cucumber mosaic virus, when tested in a local host of CMV, Chenopodium amaranticolor. Transposon mutant library of Gpf01 was prepared using pGS9::Tn5 and the mutant Gpf01-RS19 was found to loose antiviral production. We developed primers from the flanking region of Tn5 and found a cosmid clone pAV1123, harboring 1.2 kb antiviral compound producing (avcf01) locus. When a sub-clone pPH9, which carried 9.3 kb region of pAV1123, was introduced into antivirus deficient P. fluorescens wild type strain B16, it exhibited antiviral activity. Using Tn3-gus mutagenesis and complementation analysis, it was found that the genes related to antiviral activity production resided in a 9.3 kb HindIII-HindIII fragment of pAV1123, indicating that the plasmid carries an essential genes promoting antiviral activity.