• Journal of Microbiology and Biotechnology
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• v.22 no.4
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• pp.510-515
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• 2012

A recombinant putative dextransucrase (DexT) was produced from Leuconostoc citreum KM20 as a 160 kDa protein, but its productivity was very low (264 U/l). For optimization, we examined enzyme activity in 7 Escherichia coli strains with inducer molecules such as lactose or IPTG. E. coli BL21-CodonPlus(DE3)-RIL exhibited the highest enzyme activity with lactose. Finally, DexT activity was remarkably increased by 12-fold under the optimized culture conditions of a cell density to start induction ($OD_{600}$) of 0.95, a lactose concentration of 7.5 mM, and an induction temperature of $17^{\circ}C$. These results may effectively apply to the heterologous expression of other large DexT genes.

• Journal of Microbiology and Biotechnology
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• v.19 no.11
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• pp.1456-1463
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• 2009

Vibrio parahaemolyticus possessing urease-positive property is relatively rare, but such strains consistently exhibit the TDH-related hemolysin (TRH) gene. In this study, we examined the effects of incubation temperature on urease activity expression, using the TH3996 and AQ4673 strains where the enzyme activity is known to be temperature-dependent and -independent, respectively. In the TH3996 strain, $\beta$-galactosidase activity was 4.4-fold lower after $30^{\circ}C$ cultivation than after $37^{\circ}C$ in a ureR-lacZ fusion strain, but temperature dependency was not found in ureD- or nikA-lacZ fusion strains. However, ureR-, ureD-, and nikA-lacZ fusions of the AQ4673 strain was not influenced by incubation temperature. We compared the promoter sequences of ureR between the above two strains. Intriguingly, we detected mismatches of two nucleotides between the two strains located at positions -66 and -108 upstream of the methionine initiation codon for UreR. Additionally, urease activity was not affected by culture temperature at either $30^{\circ}C$ or $37^{\circ}C$ by allelic introduction of the AQ4673 ureR gene into the TH3996 ureR deletion mutant. Taken together, our study demonstrates that the transcriptional factor UreR is involved in the temperature dependency of urease activity, and two nucleotides within the ureR promoter region are of particular importance for the urease activity dependency of V. parahaemolyticus.

• Journal of Animal Science and Technology
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• v.47 no.2
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• pp.167-176
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• 2005

PIGK(phosphatidylinositol glycan, class K) is a subunit of GPI transamidase that cleaves the signal peptide in proproteins and replaces it with GPI. In addition, the structure and synthesis of GPI are critically involved in some of the cellular actions of insulin. Therefore, PIGK would be essential for mammalian development and many specific cellular functions as well as for metabolic activity of insulin associated with GPI. Two types of" full-length cDNAs of porcine PIGK were cloned through RT-PCR and RACE experiments. One is thought to be a normal form(consist of 395 amino acids) and the other is considered as an alternative spliced form(consist of 371 amino acids) which contains additional 63 bps in intron 7. Since a stop codon was contained within the insertion, the spliced form has a shorter coding sequence than that of normal form. A missense mutation (T314I) in exon 6 was detected and used for genotyping to estimate association with the growth and fat deposition traits for 545 $F_2$ animals(Korean native boars ${\times}$ Landrace). From the PCR-RFLP analysis using HpyCH4III, CT genotype showed highly significant relationship(P< 0.01) with carcass fat contents.

• Microbiology and Biotechnology Letters
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• v.18 no.3
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• pp.309-316
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• 1990

The sequence of a 1795 bp restriction fragment containing the B. circulans F-2 gene for NaC1- dependent $\alpha$-amylase (CI-amylase) is reported. The probable coding region of the gene is 1005 base pairs (335 amino acida) long. The NaC1-dependent $\alpha$-amylase (el-amy) sequence shows an open reading frame (ORF) with the translated molecular weight of about 38, 006, which correspond to a molecular weight of about 35, 000 (Mi). The gene is preceded by the sequence resembling promoter for the vegetative B, subtitis RNA polymerases. These are followed by the sequences resembling a B. subtilis ribosome binding site 5 nucleotides before the first codon of the gene. Homologous regions with other amylases were found. The N-terminal sequences of the mature proteins expressed in E. eoli were identical to the N-terminal sequences which are anaIysed.

• Microbiology and Biotechnology Letters
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• v.26 no.1
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• pp.34-39
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• 1998

We have already prepared a human lysozyme (HLY) structural gene from chemically synthesized 38 oligomers with high codon usage in Saccharomyces cerevisiae. For directing the synthesis and secretion of HLY in S. cerevisiae, two types of expression vectors, a YCp centromere-based vector, pHK101 and a YEp 2-$\mu\textrm{m}$ circle-based vector, pHK501 were constructed. With the resulting plasmids, we have confirmed that yeast transformant harboring pHK501 has more secreted HLY than pHK101-transformant by using a lysoplate and a turbidimetric assay. In flask cultivation, pHK501-transformant produced active HLY about 8 times (55 units/$m\ell$) higher than pHK101-transformant. From batch cultivation, the HLY productivity was obtained with 1.12 units/$m\ell$/h, corresponding to a 1.8-fold increase compared with flask fermentation. These results indicate that yeast transformant with pHK501 vector overexpressed and secreted HLY than that of YCp type vector.

• The Journal of Korean Society of Virology
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• v.29 no.2
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• pp.107-118
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• 1999

To characterize the molecular nature of human immunodeficiency virus (HIV)-1, we determined the full-length HIV-1 sequences from cultured peripheral blood mononuclear cells (PBMC) of a Korean long-term nonprogressor (LTNP). Without antiretroviral therapy, the individual has maintained CD4+ T counts over $500/{\mu}l$ from 1989 to 1999. Plasma viral RNA copy was 992 U/ml in 1998. Culture supernatant showed positive from culture days 9. A series of 9 overlapping PCR products were amplified from cultured PBMC and cloned About 9.2 kb from R of 5' LTR to R of 3' LTR was determined by automated sequencing. The G-to-A hypermutations were shown throughout the entire region. As a result of G to A hypermutations, premature stop codon was found in integrase coding region. Though there was no recombination between subtypes over all genomes, TATA box in both LTRs was TAAAA which is detected in subtype E instead of TATAA in subtype B. And, there were nucleotide GC insertion between $NF-{\kappa}B$ I and Sp1 III, and duplication of $TCF-1{\alpha}$ in LTR. We could not find any deletion of amino acid in Nef, Gag, Pol and Env gene. This study is the first report on molecular nature of full genomes of HIV-1 isolated in Korea.

• Journal of Genetic Medicine
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• v.13 no.1
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• pp.41-45
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• 2016

Marfan syndrome (MFS) is an inherited connective tissue disorder with a mutation in the fibrillin-1 (FBN1) gene. Fibrillin is a major building block of microfibrils, which constitute the structural component of the connective tissues. A 10-year-old girl visited our hospital with the chief complaint of precocious puberty. According to her medical history, she had a pulmonary wedge resection for a pneumothorax at 9 years of age. There was no family history of MFS. Mid parental height was 161.5 cm. The patient's height was 162 cm (>97th percentile), and her weight was 40 kg (75th-90th percentile). At the time of initial presentation, her bone age was approximately 11 years. From the ophthalmologic examination, there were no abnormal findings except myopia. There was no wrist sign. At the age of 14 years, she revisited the hospital with the chief complaint of scoliosis. Her height and weight were 170 cm and 50 kg, respectively, and she had arachnodactyly and wrist sign. We performed an echocardiograph and a test for the FBN1 gene mutation with direct sequencing of 65 coding exons, suspecting MFS. There were no cardiac abnormalities including mitral valve prolapse. A cytosine residue deletion in exon 7 (c.660delC) was detected. This is a novel mutation causing a frameshift in protein synthesis and predicted to create a premature stop codon. We report the case of a patient with MFS with a novel FBN1 gene missense mutation and a history of pneumothorax at a young age without cardiac abnormalities during her teenage years.

• Journal of The Korean Society of Inherited Metabolic disease
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• v.1 no.1
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• pp.23-27
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• 2001

Fanconi-Bickel Syndrome (FBS) is a rare autosomal recessive disorder of carbohydrate metabolism recently demonstrated to be caused by mutations in the GLUT 2 gene for the glucose transporter protein 2 expressed in liver, pancreas, intestine, and kidney. This disease is characterized by hepatorenal glycogen accumulation, both fasting hypoglycemia as well as postprandial hyperglycemia and hyperglactosemia, and generalized proximal renal tubular dysfunctions. We report the first Korean patient with FBS diagnosed based on clinical manifestations and identification of a novel mutation in the GLUT 2 gene. She was initially diagnosed having a neonatal diabetes mellitus due to hyperglycemia and glycosuria at 3 days after birth. In addition, newborn screening for galactosemia revealed hypergalactosemia. Thereafter, she has been managed with lactose free milk, insulin therapy. However, she failed to grow and her liver has been progressively enlarging. Her liver functions were progressively deteriorated with increased prothrombin time. Liver biopsy done at age 9 months indicated micronodular cirrhosis with marked fatty changes. She succubmed to hepatic failiure with pneumonia at 10 months of age. Laboratory tests indicated she had generalized proximal renal tubular dysfuctions; renal tubular acidosis, hypophosphatemic rickets, and generalized aminoaciduria. Given aforementioned findings, the diagnosis of FBS was appreciated at age of 2 months. The DNA sequencing analysis of the GLUT 2 gene using her genomic DNA showed a novel mutation at 5th codon; Lysine5 Stop (K5X).

• Molecular & Cellular Toxicology
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• v.1 no.4
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• pp.217-223
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• 2005

To assess that the XRCC1 399Gln variant contributes to sensitivity to ionizing radiation treatment and is associated with progression-free and overall survival, one hundred and ninety-five lung cancer patients were recruited at the Asan Medical Center from 2000 to 2003. We determined the genotypes of the XRCC1 genes by PCR-RFLP. Kaplan-Meier survival curves and the log-rank test were used to analyze the effects of genotypes on survival. Hazard ratios, adjusted for age, sex, and other potential confounders, were calculated using the Cox-proportional hazard model. Patients carrying the 399Gln variant allele under radiotherapy only had a shorter progression-free and overall survival than those with the 399Arg homozygote. However, when we analyzed for the effect of the XRCC1 Arg399Gln polymorphism in the combined treatment of surgical resection and radiotherapy, we found that patients with the 399Gln variant allele had a longer progression-free and overall survival. This study shows different associations between the XRCC1 Arg399Gln polymorphism and progression-free or overall survival depending on treatment protocol in patients with NSCLC.

• Journal of Microbiology
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• v.34 no.1
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• pp.68-73
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• 1996

An nprX gene of Bacillus subtilis NS15-4 encoding a neutral protease was cloned and its molecular characteristics were analyzed. The complete nucleotide sequence indicated that there is an open reading frame (0RF) possibly encoding 521 amino acid polypeptide. The ORF used all codons expected two cysteine and a proline having a codon bias index (CBI) of 0.09 in Escherichia coli. There were homologous sequences to the consensus sequence of -35 and -10 regions of E. coli promoters and to a Shine-Dalgarno (SD) sequence located 25 bp downstream of a mojor transcription initiation site. Moreover, there were also five minor transcription initiation sites at 6. 7. 8. 14 and 15 nt downstream of the major site. Northern blot analysis revealed the presence of about 1.8 kb mRNA transcript in E. coli having the nprX gene. The nucleotide sequence was identified in GenBank to be a gene for a neutral protease of B. sutilis with six nucleotide difference in the ORF region. The flanking regions of the NprX ORF showed much more differences form those of other neutral protease genes except the nprE gene of B. subtilis, which has the most homology to the nprX gene, and of which the flanking regions were identical to those of the nprX gene.