• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.483-489
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• 2004

Pseudomonas sp. S-47 is a bacterium capable of degrading benzoate as well as 4-chlorobenzoate (4CBA). Benzoate and 4CBA are known to be degraded via a meta-cleavage pathway characterized by a series of enzymes encoded by xyl genes. The meta-cleavage pathway operon in Pseudomonas sp. S-47 encodes a set of enzymes which transform benzoate and 4CBA into TCA cycle intermediates via the meta-cleavage of (4-chloro )catechol to produce pyruvate and acetyl-CoA. In the current study, the meta-pathway gene cluster was cloned from the chromosomal DNA of S-47 strain to obtain pCS1, which included the degradation activities for 4CBA and catechol. The genetic organization of the operon was then examined by cloning the meta-pathway genes into a pBluescript SKII(+) vector. As such, the meta-pathway operon from Pseudomonas sp. S-47 was found to contain 13 genes in the order of xylXYZLTEGFlQKIH. The two regulatory genes, xylS and xylR, that control the expression of the meta-pathway operon, were located adjacently downstream of the meta-pathway operon. The xyl genes from strain S-47 exhibited a high nucleoside sequence homology to those from Pseudomonas putida mt-2, except for the xylJQK genes, which were more homologous to the corresponding three genes from P. stutzeri AN10. One open reading frame was found between the xylH and xylS genes, which may playa role of a transposase. Accordingly, the current results suggest that the xyl gene cluster in Pseudomonas sp. S-47 responsible for the complete degradation of benzoate was recombined with the corresponding genes from P. putida mt-2 and P. stutzeri AN10.

• Proceedings of the Botanical Society of Korea Conference
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• 1999.07a
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• pp.11-15
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• 1999

In order to evlauate feasibility of the gene tagging by the maize transposable element Ac in heterologous plant systems, we have investigated physical distances and directions of transposition of the element in Arabidopsis thaliana and tobacco cultured cell line BY-2. We prepared a T-DNA construct that carried a non-autonomous derivative of Ac with a site for cleavage by endonuclease I-Scel (designated dAc-I-RS element). Another cleavage site was also introduced into the T-DNA region outside dAc-I-RS. A number of transgenic Arabidopsis plants were generated, each of which had a single copy of the T-DNA at a different chromosomal location. To examine the pattern of transposition, three out of these transgenic plants were crossed with the Arabidopsis plant that carried the gene for Ac transposase and progeny in which dAc-I-RS had been transposed were isolated. After digestion of the genomic DNA of these progeny with I-SceI, sizes of segment of DNA were determined byd pulse-field gel electrophoresis. We also performed linkage analysis for the transposed elements and sites of mutations near the elements. Our results with three transgenic lines showed that 50% of all transposition events had occurred within 1,700 kilo-base pairs (kb) on the same chromosome, with 35% within 200 kb, and that the elements transposed in both directions on the chromosome with roughly equal probability. The data thus indicate that the Ac-Ds system is most useful for tagging of genes that are present within 200 kb of the chromosomal site of Ac in Arabidopsis. In addition, determination of the precise localization of the transposed dAc-I-RS element should definitely assist in map-based cloning of genes around insertion sites. In the present paper, we report typical examples of such gene isolation studies.

• Proceedings of the Botanical Society of Korea Conference
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• 1985.08b
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• pp.51-57
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• 1985

In order to evlauate feasibility of the gene tagging by the maize transposable element Ac in heterologous plant systems, we have investigated physical distances and directions of transposition of the element in Arabidopsis thaliana and tobacco cultured cell line BY-2. We prepared a T-DNA construct that carried a non-autonomous derivative of Ac with a site for cleavage by endonuclease I-Scel (designated dAc-I-RS element). Another cleavage site was also introduced into the T-DNA region outside dAc-I-RS. A number of transgenic Arabidopsis plants were generated, each of which had a single copy of the T-DNA at a different chromosomal location. To examine the pattern of transposition, three out of these transgenic plants were crossed with the Arabidopsis plant that carried the gene for Ac transposase and progeny in which dAc-I-RS had been transposed were isolated. After digestion of the genomic DNA of these progeny with I-SceI, sizes of segment of DNA were determined byd pulse-field gel electrophoresis. We also performed linkage analysis for the transposed elements and sites of mutations near the elements. Our results with three transgenic lines showed that 50% of all transposition events had occurred within 1, 700 kilo-base pairs (kb) on the same chromosome, with 35% within 200 kb, and that the elements transposed in both directions on the chromosome with roughly equal probability. The data thus indicate that the Ac-Ds system is most useful for tagging of genes that are present within 200 kb of the chromosomal site of Ac in Arabidopsis. In addition, determination of the precise localization of the transposed dAc-I-RS element should definitely assist in map-based cloning of genes around insertion sites. In the present paper, we report typical examples of such gene isolation studies.

• Journal of Microbiology and Biotechnology
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• v.11 no.5
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• pp.763-771
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• 2001

Biphenyl dioxygenase (BPDO), which catalyzes the first step in the bacterial degradation of biphenyl and polychlorinated biphenyls, was characterized in Pseudomonas sp. B4. The bphA locus containing the four structural genes encoding BPDO were cloned and sequenced. A regulatory gene as well as a putative regulatory sequence were identified upstream of this locus. A transposase-like gene was found within a 1-kb region further upstream, thereby suggesting that the bphA locus may be carried on a transposable element. The three components of the BPDO enzyme have been separately overexpressed and purified from E. coli. The ferredoxin and terminal dioxygenase components showed biochemical properties comparable to those of two previously characterized BPDOs, whereas the ferredoxin reductase exhibited an unusually high lability. The substrate selectivity of BPDO was examined in vivo using resting cell assays performed with mixtures of selected polychlorinated biphenyls. The results indicated that para-substituted congeners were the preferred substrates. In vitro studies were carried out on a BPDO complex where the reductase from strain B4 we replaced by the more stable isoform from Comamonas testosteroni B-356. The BPDO enzyme had a specific activity of $0.26{\pm}0.02 {\mu}mol {min^-1}{mg^-1}\;of\;ISP_{BPH}$ with biphenyl as the substrate. The 2,3-, 4,4'-, and 2,4,4'-chlorobiphenyls were converted to single dihydrodiols, while 2,4'-dichlorobiphenyl gave rise to two dihydrodiols. The current data also indicated that 2,4,4'-trichlorobiphenyl was a better substrate than the 4,4'-dichlorinated congener.

• Korean Journal of Poultry Science
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• v.42 no.3
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• pp.239-245
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• 2015

Transgenic animals have been widely used for developmental biology studies, as disease models, and even in industry such as transgenic bioreactor animals. For transgenic birds, quail has the great advantages of small body size, short generation time, and frequent egg production. To date, retroviral or lentiviral transduction has been used to generate transgenic quail for various purposes. However, the efficiency of transgenic offspring production with these methods is relatively low and viral vector usage has safety issues. Unfortunately, non-viral transgenesis has not been established in quail due to a deficiency of stem cell and germ cell culture systems. In this study, we established a direct in ovo lipofection method that could be used to create transgenic quail without germline-competent cells or viruses. To optimize the injection stage during embryo development, the liposome complex (containing piggyBacCMV-GFP and transposase plasmids) was introduced into an embryonic blood vessel at 50 hr, 55 hr or 60 hr. GFP expression was detected in various tissues (heart, kidney, liver and stomach) on day 12 of incubation under a fluorescence microscope. Additionally, GFP-positive cells were detected in the recipient embryonic gonads. In conclusion, the direct in ovo lipofection method with the piggyBac transposon could be an efficient and useful tool for generating transgenic quail.

• Molecules and Cells
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• v.27 no.4
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• pp.459-465
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• 2009

MUG1 is a MULE transposon-related domesticated gene in plants. We assessed the sequence diversity, neutrality, expression, and phylogenetics of the MUG1 gene among Oryza ssp. We found MUG1 expression in all tissues analyzed, with different levels in O. sativa. There were 408 variation sites in the 3886 bp of MUG1 locus. The nucleotide diversity of the MUG1 was higher than functionally known genes in rice. The nucleotide diversity (${\pi}$) in the domains was lower than the average nucleotide diversity in whole coding region. The ${\pi}$ values in nonsynonymous sites were lower than those of synonymous sites. Tajima D and Fu and Li $D^*$ values were mostly negative values, suggesting purifying selection in MUG1 sequences of Oryza ssp. Genome-specific variation and phylogenetic analyses show a general grouping of MUG1 sequences congruent with Oryza ssp. biogeography; however, our MUG1 phylogenetic results, in combination with separate B and D genome studies, might suggest an early divergence of the Oryza ssp. by continental drift of Gondwanaland. O. long-istaminata MUG1 divergence from other AA diploids suggests that it might not be a direct ancestor of the African rice species.

• Molecules and Cells
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• v.43 no.12
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• pp.1011-1022
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• 2020

Cell type specification is a delicate biological event in which every step is under tight regulation. From a molecular point of view, cell fate commitment begins with chromatin alteration, which kickstarts lineage-determining factors to initiate a series of genes required for cell specification. Several important neuronal differentiation factors have been identified from ectopic over-expression studies. However, there is scarce information on which DNA regions are modified during induced pluripotent stem cell (iPSC) to neuronal progenitor cell (NPC) differentiation, the cis regulatory factors that attach to these accessible regions, or the genes that are initially expressed. In this study, we identified the DNA accessible regions of iPSCs and NPCs via the Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq). We identified which chromatin regions were modified after neuronal differentiation and found that the enhancer regions had more active histone modification changes than the promoters. Through motif enrichment analysis, we found that NEUROD1 controls iPSC differentiation to NPC by binding to the accessible regions of enhancers in cooperation with other factors such as the Hox proteins. Finally, by using Hi-C data, we categorized the genes that directly interacted with the enhancers under the control of NEUROD1 during iPSC to NPC differentiation.

• Microbiology and Biotechnology Letters
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• v.50 no.4
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• pp.563-573
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• 2022

Xanthomonas arboricola pv. juglandis (Xaj) is a globally important bacterial pathogen of walnut trees that causes substantial economic losses in commercial walnut production. Although prophages are common in bacterial plant pathogens and play important roles in bacterial diversity and pathogenicity, there has been limited investigation into the distribution and function of prophages in Xaj. In this study, we identified and characterized 13 predicted prophages from the genomes of 12 Xaj isolates from around the globe. These prophages ranged in length from 11.8 kb to 51.9 kb, with between 11-75 genes and 57.82-64.15% GC content. The closest relatives of these prophages belong to the Myoviridae and Siphoviridae families of the Caudovirales order. The phylogenetic analysis allowed the classification of the prophages into five groups. The gene constitution of these predicted prophages was revealed via Roary analysis. Amongst 126 total protein groups, the most prevalent group was only present in nine prophages, and 22 protein groups were present in only one prophage (singletons). Also, bioinformatic analysis of the 13 identified prophages revealed the presence of 431 genes with an average length of 389.7 bp. Prokka annotation of these prophages identified 466 hypothetical proteins, 24 proteins with known function, and six tRNA genes. The proteins with known function mainly comprised prophage integrase IntA, replicative DNA helicase, tyrosine recombinase XerC, and IS3 family transposase. There was no detectable insertion site specificity for these prophages in the Xaj genomes. The identified Xaj prophage genes, particularly those of unknown function, merit future investigation.

• Genomics & Informatics
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• v.21 no.2
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• pp.19.1-19.13
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• 2023

Liver cancer, particularly hepatocellular carcinoma (HCC), poses a significant global threat to human lives. To advance the development of innovative diagnostic and treatment approaches, it is essential to examine the hidden features of HCC, particularly its 3D genome architecture, which is not well understood. In this study, we investigated the 3D genome organization of four HCC cell lines-Hep3B, Huh1, Huh7, and SNU449-using in situ Hi-C and assay for transposase-accessible chromatin sequencing. Our findings revealed that HCC cell lines had more long-range interactions, both intra-and interchromosomal, compared to human mammary epithelial cells (HMECs). Unexpectedly, HCC cell lines displayed cell line-specific compartmental modifications at the megabase (Mb) scale, which could potentially be leveraged in determining HCC subtypes. At the sub-Mb scale, we observed decreases in intra-TAD (topologically associated domain) interactions and chromatin loops in HCC cell lines compared to HMECs. Lastly, we discovered a correlation between gene expression and the 3D chromatin architecture of SLC8A1, which encodes a sodium-calcium antiporter whose modulation is known to induce apoptosis by comparison between HCC cell lines and HMECs. Our findings suggest that HCC cell lines have a distinct 3D genome organization that is different from those of normal and other cancer cells based on the analysis of compartments, TADs, and chromatin loops. Overall, we take this as evidence that genome organization plays a crucial role in cancer phenotype determination. Further exploration of epigenetics in HCC will help us to better understand specific gene regulation mechanisms and uncover novel targets for cancer treatment.

• Journal of Life Science
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• v.33 no.11
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• pp.897-904
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• 2023

This study was done to develope genetic markers with the unique characteristics of genes according to the genomic information of Bacillus velezensis K10. B. velezensis K10 maintained a total of 4,159,835 bps, which was found to encode 5,136 open reading frames (orfs). B. velezensis K10 was found to have much more gene migration due to external factors overall compared to standard strain B. velezensis JS25R. In order to discover genetic selection markers, orfs on the genome to be easily induced to gene mutation were surveyed such as recombinase, integrase, transposase, and phage-related genes. As a result of the investigation, 9 candidate markers were isolated with high possibility as genetic selection markers. Although a part in the various origin's areas showed specificities in comparison with homology, the selected markers were all existed in phage-related areas because they were relatively lower homologies in phage-related genes. PCR analysis was done on B. licheniformis K12, B. velezensis K10, B. subtilis, and B. cereus to establish them as inter-species candidate selection markers. As a result, it was confirmed that B. velezensis K10-specific PCR products were formed in a total of 6 primer sets such as BV3 and BV5 to 9. On the other hand, analysis at the subspecies level observed the formation of B. velezensis K10-specific PCR products in 4 primer sets such as BV3, 5, 8, and 9. Among them, since BV5 and BV8 were detected by very specific results, we suggest that BV5 and 8 can be used as B. velezensis K10 gene selection markers at the species and sub-species level.