• Journal of Aquaculture
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• v.6 no.3
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• pp.221-233
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• 1993

Cell size, DNA content, chromosome and PCR-based mitochondrial 12S rRNA gene analyses were conducted to obtain basic informations for genetic stock identification of spotted flounder (Verasper variegatus) from Yeocheun, Korea. The mean erythrocytic and nuclear volumes of spotted flounder were $211.10{\mu}m^3$ and $23.03{\mu}m^3$, respectively. The haploid DNA content of this species was 0.79 pg/cell which correspond to $46.5\%$ of carp and to $22.6\%$ of mammals. Spotted flounder had the 2n = 46 acrocentric chromosomes but no heteromorphic sex chromosomes was found. Mitochondrial DNA gene for 12S ribosomal RNA was amplified by polymerase chain reaction (PCR) and the PCR products were subjected to digestion with 15 restriction endonucleases. Restriction enzyme analyses revealed that Ava I, Mae II, Sma I and Xba I had one restriction site in the mitochondrial 12S rRNA gene segment of spotted flounder, while Mae I had two. Segments of 12S rRNA gene from mitochondria in spotted flounder were sequenced and compared with channel catfish and human as controls. The 12S rRNA gene of this species was more similar to that of channel catfish than to human's.

• Reproductive and Developmental Biology
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• v.33 no.2
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• pp.107-111
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• 2009

The transfection efficiency of a transgene into pig and goat fetal fibroblast cells (PFF and GFF, respectively) was tested using co-transfection of a human tissue-type plasminogen activator (tPA) transgene and neomycin-resistant ($Neo^r$) gene, followed by G418 selection. To initially test G418 resistance, GFF and PFF were incubated in culture medium containing different concentration of G418 for 2 weeks, and cell survival was monitored over time. Based on the obtained results, the concentrations chosen for G418 selection were 800 ug/ml and 200 ug/ml for GFF and PFF, respectively. For co-transfection experiments, the pBC1/tPA and $Neo^r$ vectors were co-transfected into GFF and PFF ($1{\times}10^6$ cells in each case) using the FuGENE6 transfection reagent, and resistant colonies were obtained following 14 days of G418 selection. We obtained 96 and 93 drug-resistant colonies of GFF and PFF, respectively, only 54 and 39 of which, respectively, continued proliferating after drug selection. PCR-based screening revealed that 23 out of 54 analyzed GFF colonies and 5 out of 39 analyzed PFF colonies contained insertion of the tPA gene. Thus, the experimentally determined transfection efficiencies for tPA gene co-transfection with the $Neo^r$ gene were 42.6% for GFF and 12.8% for PFF. These findings suggest that co-transfection of a transgene with the $Neo^r$ gene can aid in the successful integration of the transgene into fetal fibroblast cells.

• Microbiology and Biotechnology Letters
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• v.31 no.2
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• pp.117-123
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• 2003

For screening of autoregulator receptor gene from Saccharopolyspora erythraea, PCR was performed with primers of receptor gene designed on the basis of amino acid sequences of autoregulator receptor proteins with known function. PCR products were subcloned into the BamHI site of pUC19 and transformed into the E. coli DH5$\alpha$. The isolated plasmid from transformant contained the fragment of 120 bp, which was detected on 2% gel after BamHI treatment. The insert, 120 bp PCR product, was confirmed as the expected internal segment of gene encoding autoregulator receptor protein by sequencing. Southern and colony hybridization using Saccha. erythraea chromosomal DNA were performed with the insert as probe. The plasmid (pEsg) having 3.2 kbp SacI DNA fragment from Saccha. erythraea is obtained. The 3.2 kbp SacI DNA fragment was sequenced by the dye terminator sequencing. The nucleotide sequence data was analyzed with GENETYX-WIN (ver 3.2) computer program and DNA database. frame analyses of the nucleotide sequence revealed a gene encoding autoregulator receptor protein which is a region including KpnI and SalI sites on 3.2 kbp SacI DNA fragment. The autoregulator receptor protein consisting of 205 amino acid was named EsgR by author. In comparison with known autoregulator receptor proteins, homology of EsgR showed above 30%.

• Bulletin of the Korean Chemical Society
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• v.34 no.12
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• pp.3665-3670
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• 2013

The R3V6 peptide includes a hydrophilic arginine stretch and a hydrophobic valine stretch. In previous studies, the R3V6 peptide was evaluated as a gene carrier and was found to have low cytotoxicity. However, the transfection efficiency of R3V6 was lower than that of poly-L-lysine (PLL) in N2A neuroblastoma cells. In this study, the transfection efficiency of R3V6 was improved in combination with high mobility group box 1A domain (HMGA). HMGA is originated from the nuclear protein and has many positively-charged amino acids. Therefore, HMGA binds to DNA via charge interaction. In addition, HMGA has a nuclear localization signal peptide and may increase the delivery efficiency of DNA into the nucleus. The ternary complex with HMGA, R3V6, and DNA was prepared and evaluated as a gene carrier. First, the HMGA/DNA complex was prepared with a negative surface charge. Then, R3V6 was added to the complex to coat the negative charges of the HMGA/DNA complex, forming the ternary complex of HMGA, R3V6, and DNA. A physical characterization study showed that the ternary complex was more stable than the PLL/DNA complex. The HMGA/R3V6/DNA complex had a higher transfection efficiency than the PLL/DNA, HMGA/DNA, or R3V6/DNA complexes in N2A cells. Furthermore, the HMGA/R3V6/DNA complex was not toxic to cells. Therefore, the HMGA/R3V6/DNA complex may be a useful gene delivery carrier.

• Microbiology and Biotechnology Letters
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• v.19 no.3
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• pp.222-227
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• 1991

Rhizobium fredii USDA193 is one of the causal organism for root nodule formation in soybean (peking). Previously we cloned the pectate lyase gene (SY1) of R. fredii USDA193. The $pel^-$ mutants (SY1$\Omega$ and SY1$\Omega$1) of SY1 were obtained using the in vitro insertional omega mutagenesis of RpelB (of Rhizobium pel) and fill-in reaction of RpelE (of Rhizobium pel) gene respectively, and we constructed two mutants (R, fredii USDA193$\Omega$ and R. fredii USDA193$\Omega$1) in pectate lyase function by marker-exchange with pe1B::$\Omega$ and R. fredii USDA193 strain (rif). The pectate lyase activity of two pel- mutant of R. fredii USDA193 was determined by spectrophotometric method. However, all pectate lyase activity of these mutants was not lost upon the mutagenesis by marker-exchange. This suggests that other pectate lyase genes may be present on the plasmid or the chromosome of R. fredii. As yet we do not have evidence linking RpelB and RpelE genes of R. fredii directly to the early nodulation process.

• International Journal of Stem Cells
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• v.16 no.2
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• pp.145-155
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• 2023

Background and Objectives: Embryologically, mesodermal development is closely related to the development of various organs such as muscles, blood vessels, and hearts, which are the main organs that make up the body. However, treatment for mesoderm developmental disorders caused by congenital or acquired factors has so far relied on surgery and drug treatment for symptom relief, and more fundamentally, treatment for mesoderm developmental disorders is needed. Methods and Results: In our study, microRNA (miRNA), which plays an important role in the mesoderm development process, was identified and the developmental function was evaluated. miRNAs consist of small nucleotides, which act as transcription factors that bind to the 3' untranslated region and suppressed target gene expression. We constructed the human embryonic stem cell (hESC) knockout cell line and analyzed the function and characteristics of miR-5739, which plays an important role in mesoderm lineage. miR-5739 acts as a transcription factor targeting SMA, Brachyury T, Hand1, which controls muscle proliferation and differentiation, and KDR gene, which regulates vessel formation in vitro. In vivo results suggest a role in regulating muscle proliferation and differentiation. Gene ontology analysis confirmed that the miR-5739 is closely related to genes that regulate muscle and vessel proliferation and differentiation. Importantly, abnormal expression of miR-5739 was detected in somatic cells derived from patients with congenital muscle disease. Conclusions: Our study demonstrate that miR-5739 gene function significantly affects transcriptional circuits that regulate muscle and vascular differentiation during embryonic development.

• Journal of Microbiology and Biotechnology
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• v.12 no.5
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• pp.773-779
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• 2002

A gene (hap) encoding aminopeptidase from the chromosomal DNA of Bacillus licheniformis was cloned. The gene is 1,347 bp long and encodes a 449 amino acid preproprotein with a major mature region of 401 amino acids (calculated molecular mass 43,241 Da). N-Terminal sequence of the purified protein revealed a potential presence of N-terminal propeptide. The deduced primary amino acid sequence and the mass analysis of the purified protein suggested that a C-terminal peptide YSSVAQ was also cleaved off by a possible endogeneous protease. Tho amino acid sequence displayed 58% identity with that of the aminopeptidase from alkaliphilic Bacillus halodurans. This bacterial enzyme was overexpressed in recombinant Escherichia coli and Bacillus subtilis cells. Clones containing the intact hap gene, including its own promoter and signal sequence, gave rise to the synthesis of extracellular and thrmostable enzyme by B. subtilis transformants. The secreted protein exhibited the same biochemical properties and the similar apparent molecular mass as the B. lichenzyormis original enzyme.

• Plant Biotechnology Reports
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• v.2 no.4
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• pp.233-238
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• 2008

Kadainou R-1, an interspecific hybrid grape derived from red (Vitis ficifolia var. ganebu) and white (V. vinifera cv. Muscat of Alexandria) grapes, accumulates high concentrations of anthocyanin in the berry skin. Hence, the expression of uridine 50 -diphosphate (UDP)-glucose:flavonoid 3-O-glucosyltransferase (UFGT), the key enzyme of the anthocyanin pathway, was examined in the berry skin of Kadainou R-1. As information on gene sequences of V. ficifolia var. ganebu and other wild grape species was unavailable, we performed GeneChip hybridization using biotin-labeled genomic deoxyribonucleic acid (DNA) to investigate how the genomic sequences of V. vinifera varieties and that of V. ficifolia var. ganebu differ. The study showed a lower correlation coefficient between V. vinifera cultivars and V. ficifolia var. ganebu than that among V. vinifera cultivars. The sequences of the UFGT gene derived from both parents of the red and white cultivars were sequenced in Kadainou R1 and revealed that both were expressed irrespective of the fact that it was not expressed in the white grape (male parent).

• Journal of fish pathology
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• v.34 no.2
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• pp.177-184
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• 2021

Vaccines based on single-cycle viruses that are replication-incompetent due to knockout of replication-related structural gene(s) are more immunogenic than inactivated or subunit vaccines and can be used as delivery vehicles for foreign antigens without concerns on the reverting to virulent forms. The aim of this study was to develop a delivery vehicle for nervous necrosis virus (NNV)-like particles (VLPs) using G gene deleted single-cycle VHSV (rVHSV-𝚫G). Recombinant single-cycle VHSVs carrying NNV capsid protein gene between N and P gene of rVHSV-𝚫G genome (rVHSV-𝚫G-NNV_Cap) were rescued by reverse genetic technology. The successful expression of NNV capsid protein in cells infected with rVHSV-𝚫G-NNV_Cap was demonstrated by Western blot analysis, and the production of NNV VLPs in infected cells was confirmed using an electron microscopy. The results suggest that single-cycle VHSVs can be used as a safe delivery vehicle for NNV VLPs, and can be extended to other pathogens for the development of prophylactic vaccines.

• Animal cells and systems
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• v.3 no.3
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• pp.295-301
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• 1999

The nucleotide sequence of a 447 base pair fragment in the mitochondrial cytochrome b gene and the complete sequence of the mitochondrial 12S ribosomal RNA gene, 938 bp, were analyzed to infer inter- and intraspecific genetic relationships of Hyla japonica and H. suweonensis from Korea and H, japonica from Japan. In the mitochondrial cytochrome b gene, genetic differentiation among H. japonica populations were 9.62% and 15.66% between H. japonica and H. suweonensis. Based on the Tamura-Nei distance, the level of sequence divergence ranged from 0.45% to 2.75% within Korean H. japonica, while 8.31%-8.87% between Korean and Japanese H. japonica and 11.51%-12.46% between H. japonica and H. suweonensis. In the neigh-bor-joining tree, Korean populations of H. japonica were clustered first at 2.22% and followed by Japanese H. japonica and H. suweonensis at 8.51% and 12.29%, respectively. In mitochondrial 12S rRNA gene, genetic differentiation between H. japonica and H. suweonensis nras 7.17% (68 bp) including 7 gaps. Based on Tamura-Nei distance, the level of sequence divergence ranged 3.53% between Korean and Japanese H. japonica and from 4.93% to 5.41% between H. japonica and H. suweonensis. Phenogram pattern of the 12S rRNA gene sequence corresponded with that of the mitochondrial cytochrome b gene.