• BMB Reports
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• v.46 no.1
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• pp.53-58
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• 2013

We constructed deletion mutants and seven point mutants by polymerase chain reaction to investigate the specificity of feline foamy virus integrase functional domains. Complementation reactions were performed for three enzymatic activities such as 3'-end processing, strand transfer, and disintegration. The complementation reactions with deletion mutants showed several activities for 3'-end processing and strand transfer. The conserved central domain and the combination of the N-terminal or C-terminal domains increased disintegration activity significantly. In the complementation reactions between deletion and point mutants, the combination between D107V and deletion mutants revealed 3'-end processing activities, but the combination with others did not have any activity, including strand transfer activities. Disintegration activity increased evenly, except the combination with glutamic acid 200. These results suggest that an intact central domain mediates enzymatic activities but fails to show these activities in the absence of the N-terminal or C-terminal domains.

• Journal of Life Science
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• v.5 no.4
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• pp.162-169
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• 1995

The glpD genes encoding gly-3-p dehydrogenase is essential for the aerobic growth of E. coli on glycerol or gly-3-p. The glpE gene, the function of which is unknownm is transcribed divergently with respect to glpD gene. Expression of the adjacent but divergently transcribed glpD the glpE genes is positively regulated by the cAMP-CRP complex. In this study, for a precise investigation of the functional elements in the regulatory region for transcription activation by cAMP-CRP, deletion mutation have been introducted into the regulatory region. The effect of the deletion mutant on transcriptional regulation was tested in vivo by $\beta$-galctosidase activity. Deletion mutants in the regulatory region of glpD demonstrated that the presence of the CRP-binding site resulted in an sixfold increase in promoter activity. And also deletion mutants of glpE gene demonstrated that the presence of the CRP-binding site resulted in an eightfold increase in promoter activity. Insertion of 22 bp oligomer in the deletion mutants has shown that the CRP binding site is need for maximal expression of glpD and glpE genes. glpD and glpE gene, cAMP-CRP complex, deletion mutant, transcriptional regulation.

• YAKHAK HOEJI
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• v.49 no.3
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• pp.198-204
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• 2005

Human foamy virus (HFV) integrase mediates integration of viral c-DNA into cellular DNA. In this process, HFV integrase recognizes its own viral DNA specifically and catalyzes insertion of viral c-DNA. In order to study catalytic domains and residues, three deletion mutants and two point mutants of HFV integrase were constructed and analyzed with respect to enzymatic activities. The C-terminal deletion mutant showed decreased enzymatic activities while the N-terminal deletion mutant lost the activities completely, indicating that the N-terminal domain is more important than the C-terminal domain in enzymatic reaction. The point mutants, in which an aspartic acid at the 164th position or a glutamic acid at the 200th position of the HFV integrase protein was changed to an alanine, lost the enzymatic activities completely. However, they were well complemented with other defective deletion mutants to recover enzymatic activities partially. Therefore, these results suggest that the aspartic acid and glutamic acid at the respective 164th and 200th positions are catalytic residues for enzymatic reaction.

• Korean Journal of Microbiology
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• v.49 no.4
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• pp.419-423
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• 2013

Three genes, nsdD, nsdC, and veA are known to be necessary for positive regulating sexual development of Aspergillus nidulans. Since the mutants of those genes hardly form fruiting bodies in heterokaryons constructed by cross between two of them, it is difficult to isolate double mutants. In this work, double mutants of ${\Delta}nsdD$ ${\Delta}veA$ and ${\Delta}nsdD$ ${\Delta}nsdC$ were isolated using the characteristic of the nsdD deletion mutant that it could develop mature cleistothecia in hypoxic and low temperature culture condition. According to the phenotypes of double mutants, the nsdD gene controls the apical growth independently with veA or nsdC. Deletion of veA or nsdC was epistatic to nsdD deletion for pigment production. Conidia formation in submerged culture with lactose as sole carbon source was observed in ${\Delta}nsdD$ ${\Delta}nsdC$ double mutant implicating it to be unique phenotype of nsdC deletion.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.727-730
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• 2001

Various recombinant deletion mutants were constructed from cycloinulo - oligosaccharide fructanotransferase(CFTase) gene of Xanthomonas oryzae #5 . The mutants were expressed in Escherichia coli DH5${\alpha}$. We were able to obtain three recombinant proteins were purified, and examine their CFTase and hydrolyzing activity. N-terminal deletion mutant had both CFTase activity and hydrolyzing activity. however, in C-terminal and N,C-terminal deletion mutant disappeared CFTase activity, but hydrolyzing activity remained. From there results, it seems that the C-terminal region(amino acid $1173{\sim}1333$) is important for cyclization.

• Animal cells and systems
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• v.5 no.1
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• pp.65-69
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• 2001

Obtaining mutant animals is important for studying the function of a particular gene. A chemical mutagenesis was first carried out to generate mutations in C. elegans. In this study, we used ultraviolet-activated 4,5',8-trimethylpsoralen to induce small deletion mutations. A library of mutagenized worms was prepared for recovery of candidate animals and stored at $15^{\circ}C$ during screening instead of being made into a frozen stock library. In order to isolate deletion mutations in target genes, a polymerase chain reaction (PCR)-based screening method was used. As a result, two independent mutants with deletions of approximately 1.0 kb and 1.3 kb were isolated. This modified and improved reverse genetic approach was proven to be effective and practical for isolating mutant animals to study gene function at the organismal level.

• Journal of Microbiology and Biotechnology
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• v.31 no.1
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• pp.115-122
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• 2021

Phenol-soluble modulins (PSMs) are responsible for regulating biofilm formation, persister cell formation, pmtR expression, host cell lysis, and anti-bacterial effects. To determine the effect of psm deletion on methicillin-resistant Staphylococcus aureus, we investigated psm deletion mutants including Δpsmα, Δpsmβ, and Δpsmαβ. These mutants exhibited increased β-lactam antibiotic resistance to ampicillin and oxacillin that was shown to be caused by increased N-acetylmannosamine kinase (nanK) mRNA expression, which regulates persister cell formation, leading to changes in the pattern of phospholipid fatty acids resulting in increased anteiso-C15:0, and increased membrane hydrophobicity with the deletion of PSMs. When synthetic PSMs were applied to Δpsmα and Δpsmβ mutants, treatment of Δpsmα with PSMα1-4 and Δpsmβ with PSMβ1-2 restored the sensitivity to oxacillin and slightly reduced the biofilm formation. Addition of a single fragment showed that α1, α2, α3, and β2 had an inhibiting effect on biofilms in Δpsmα; however, β1 showed an enhancing effect on biofilms in Δpsmβ. This study demonstrates a possible reason for the increased antibiotic resistance in psm mutants and the effect of PSMs on biofilm formation.

• Journal of Marine Bioscience and Biotechnology
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• v.1 no.2
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• pp.84-90
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• 2006

Various ${\Delta}opp$ mutants of Vibrio fluvialis were constructed by allelic exchange method. The mutants occurred in target genes were confirmed by PCR and Southern hybridization analyses. After the exact mutants were identified, cell growth and biofilm production were examined using the respective mutants. The growth of wild strain was more rapid than mutants within 4hr incubation. Thereafter, the growth of wild strain and mutants reached to same level. When the productivities of wild strain and mutants were examined, ${\Delta}oppA$ mutant showed the highest productivity. Though ${\Delta}oppC,D$ and F mutants produced the lower production than that of ${\Delta}oppA$ mutant, the productivities of those mutants were much higher than that of wild strain.

• Journal of Microbiology and Biotechnology
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• v.23 no.12
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• pp.1683-1690
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• 2013

The cyclic AMP receptor protein (CRP) homolog, GlxR, controls the expression of several genes involved in the regulation of diverse physiological processes in Corynebacterium glutamicum. In silico analysis has revealed the presence of glxR binding sites upstream of genes ptsG, adhA, and ald, encoding glucose-specific phosphotransferase system protein, alcohol dehydrogenase (ADH), and acetaldehyde dehydrogenase (ALDH), respectively. However, the involvement of the GlxR-cAMP complex on the expression of these genes has been explored only in vitro. In this study, the expressions of ptsG, adhA, and ald were analyzed in detail using an adenylate cyclase gene (cyaB) deletion mutant and glxR deletion mutant. The specific activities of ADH and ALDH were increased in both the mutants in glucose and glucose plus ethanol media, in contrast to the wild type. In accordance, the promoter activities of adhA and ald were derepressed in the cyaB mutant, indicating that glxR acts as a repressor of adhA. Similarly, both the mutants exhibited derepression of ptsG regardless of the carbon source. These results confirm the involvement of GlxR on the expression of important carbon metabolic genes; adhA, ald, and ptsG.

• Journal of Veterinary Science
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• v.21 no.2
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• pp.20.1-20.13
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• 2020

Actinobacillus pleuropneumoniae (APP) causes a form of porcine pleuropneumonia that leads to significant economic losses in the swine industry worldwide. The apxIBD gene is responsible for the secretion of the ApxI and ApxII toxins and the pnp gene is responsible for the adaptation of bacteria to cold temperature and a virulence factor. The apxIBD and pnp genes were deleted successfully from APP serotype 1 and 5 by transconjugation and sucrose counter-selection. The APP1ΔapxIBDΔpnp and APP5ΔapxIBDΔpnp mutants lost hemolytic activity and could not secrete ApxI and ApxII toxins outside the bacteria because both mutants lost the ApxI- and ApxII-secreting proteins by deletion of the apxIBD gene. Besides, the growth of these mutants was defective at low temperatures resulting from the deletion of pnp. The APP1ΔapxIBDΔpnp and APP5ΔapxIBDΔpnp mutants were significantly attenuated compared with wild-type ones. However, mice vaccinated intraperitoneally with APP5ΔapxIBDΔpnp did not provide any protection when challenged with a 10-times 50% lethal dose of virulent homologous (APP5) and heterologous (APP1) bacterial strains, while mice vaccinated with APP1ΔapxIBDΔpnp offered 75% protection against a homologous challenge. The ΔapxIBDΔpnp mutants were significantly attenuated and gave different protection rate against homologous virulent wild-type APP challenging.