• Journal of Life Science
• /
• v.21 no.10
• /
• pp.1370-1376
• /
• 2011

To study the functional genomic analysis of a crenachaeon Sulfolobus acidocaldarius, we have constructed an auxotrophic mutant based on pyrEF, which encodes the pyrimidine biosynthetic enzymes orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase. S. acidocaldarius was shown to be sensitive to 5-fluoroorotic acid (5-FOA), which can be selected for mutations in pyrEF genes within a pyrimidine biosynthesis cluster. Spontaneous 5-FOA-resistant mutants by ultraviolet, KH1U and KH2U, were found to contain two point mutations and a frame shift mutation in pyrE, respectively. Mutations at these sites from KH1U and KH2U decreased the activity of orotate phosphoribosyltransferase encoded by the pyrE gene and blocked the degradation of 5-FOA into toxic 5-FOMP and 5-FUMP that kill the cells. Therefore, KH1U and KH2U were uracil auxotrophs. Transformation of Sulfolobus-Escherichia coli shuttle vector pC bearing pyrEF genes from S. solfataricus P2 into S. acidocaldarius mutant KH2U restored 5-FOA sensitivity and overcame the uracil auxotrophy. This study establishes an efficient genetic strategy towards the systematic knockout of genes in S. acidocaldarius.

• Journal of Microbiology and Biotechnology
• /
• v.25 no.5
• /
• pp.606-611
• /
• 2015

As Salmonella enterica serovar Pullorum remains a major economic problem for the poultry industries of countries with no efficient control measures, we presented a multidrug resistance strain S06004 (isolated from a clinically sick chicken in China in 2006) for genome sequencing. The genome comparison showed that the strain contained two prophages, the ST104 and prophage-4 (Fels2) of E. coli LF82, which were not detected in the only published genomes of S. Pullorum RKS5078 and CDC1983-67. In addition, the GyrA Ser83 point mutation, drugresistant genes, and many antibiotic pump systems that are present in S06004 may be contributing to the multidrug resistance of this strain.

• KSBB Journal
• /
• v.23 no.5
• /
• pp.403-407
• /
• 2008

Recently, Candida antarctica lipase B (CalB) draws attention from industries for various applications for food, detergent, fine chemical, and biodiesel, because of its characteristics as an efficient biocatalyst. Since many industrial processes carry out in organic solvent and at high temperature, CalB, which is stable under harsh condition, is in demand from many industries. In order to reform CalB promptly, the expression system which has advantages of ease to use and low cost for gene libraries screening was developed using E. coli. The E. coli strains, Rosettagami with competence for enhanced disulfide bond formation, Novablue, and $DH5{\alpha}$, were exploited in this study. To obtain the soluble CalB, the pCold I vector expressing the cloned gene at $15^{\circ}C$ and the chaperone plasmids containing groES/groEL, groES/groEL/tig, tig, dnaK/dnaJ/grpE, and dnaK/dnaJ/grpE/groES/groEL were used for coexpression of CalB and chaperones. The colonies expressing functional lipase were selected by employing the halo plate containing 1% tributyrin, and the CalB expression was confirmed by SDS-PAGE. E. coli Rosettagami and $DH5{\alpha}$ harbouring groES/groEL chaperones were able to express soluble CalB effectively. From a facilitative point of view, E. coli $DH5{\alpha}$ is more suitable for further mutation study.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.39 no.9
• /
• pp.1313-1319
• /
• 2010

Rice cultivars of Goami2 (G2), Baegjinju (BJJ), and Sulgaeng (SG) with different amylose contents were developed by mutation breeding via N-methyl-N-nitrosourea (MNU) treatment to Ilpumbyeo (IP), high japonica rice. They were identified by different appearances such as grain size, color, and shape. In this experiment, the compositional and physical qualities of those cultivars were examined. The G2 rice classified as a high-amylose rice cultivar was significantly higher in its non-digestable carbohydrates contents. Linoleic and oleic acid were composed of 70~75% of all fatty acids composition regardless of milled and brown rice, except G2 rice in which palmitic acid was the major fatty acid followed by linoleic acid and oleic acid in order. Major amino acids were aspartic acid, glutamic acid, and hydroxy lysine. It was found that cysteine contents were higher in the cultivars of endosperm mutant rice. The DSC analysis revealed that enthalpy was the highest in BJJ followed by SG, IP, and G2 rice. The lowest enthalpy of G2 might be attributable to the higher amylose content. Ilpumbyeo in its cooked rice form showed the highest in Toyo value and less in hardness, but G2 was vise versa. Results of gelatinization and cooked rice properties suggest that G2 was less suitable for cooked rice, but has a potential for functional ingredients from nutritional point of view. The BJJ and SG could be used for traditional cooking as well as for processed foods.

• Journal of the korean academy of Pediatric Dentistry
• /
• v.27 no.2
• /
• pp.300-308
• /
• 2000

The Fibroblast growth factors(FGFs) plays an important role in the control of osteogenesis during skeletal development. Especially, FGF-2 is a potent mesodermal inducer during embryogenesis and FGF receptors (FGFRs) messages are strongly expressed in developing bones. In this study, we investigated the effect of bFGF on osteopontin(OPN) gene expression in ST-2 cells and tried to elucidate the mechanism of its stimulatory effects. The obtain results were as follows; The treatment of bFGF(1ng/ml) upregulates OPN, fibronectin mRNA levels and downregulates type I collagen mRNA levels. But, there was no remarkable difference in alkaline phosphatase mRNA levels between two groups. The OPN gene expression increased in a dose-dependent manner up to 10ng/ml and OPN gene began to occur at around 3h with continuous increase up to 24h then decreased to basal level at 48h. 30 minutues pretreatment with cycloheximide (500ng/ml), a protein synthesis inhibitor, prior to addition bFGF resulted in blocking bFGF induced OPN expression. These results suggest that bFGF increased the level of OPN mRNA in a dose and time-dependent manner via the synthesis of certain transcriptional regulatory proteins.

• BMB Reports
• /
• v.37 no.6
• /
• pp.709-714
• /
• 2004

The wild-type repressor CI of temperate mycobacteriophage L1 and the temperature-sensitive (ts) repressor CIts391 of a mutant L1 phage, L1cIts391, have been separately overexpressed in E. coli. Both these repressors were observed to specifically bind with the same cognate operator DNA. The operator-binding activity of CIts391 was shown to differ significantly than that of the CI at 32 to $42^{\circ}C$. While 40-95% operator-binding activity was shown to be retained at 35 to $42^{\circ}C$ in CI, more than 75% operator-binding activity was lost in CIts391 at 35 to $38^{\circ}C$, although the latter showed only 10% less binding compared to that of the former at $32^{\circ}C$. The CIts391 showed almost no binding at $42^{\circ}C$. An in vivo study showed that the CI repressor inhibited the growth of a clear plaque former mutant of the L1 phage more strongly than that of the CIts391 repressor at both 32 and $42^{\circ}C$. The half-life of the CIts391-operator complex was found to be about 8 times less than that of the CI-operator complex at $32^{\circ}C$. Interestingly, the repressor-operator complexes preformed at $0^{\circ}C$ have shown varying degrees of resistance to dissociation at the temperatures which inhibit the formation of these complexes are inhibited. The CI repressor, but not that of CIts391, regains most of the DNA-binding activity on cooling to $32^{\circ}C$ after preincubation at 42 to $52^{\circ}C$. All these data suggest that the 131st proline residue at the C-terminal half of CI, which changed to leucine in the CIts391, plays a crucial role in binding the L1 repressor to the cognate operator DNA, although the helix-turn-helix DNA-binding motif of the L1 repressor is located at its N-terminal end.

• Pediatric Infection and Vaccine
• /
• v.16 no.2
• /
• pp.199-204
• /
• 2009

Purpose : Various proteins encoded in the early region 3 (E3) of adenoviruses protect cells from being killed by cytotoxic T cells and death-inducing cytokines. We sought to find out whether the genetic heterogeneity of the E3 gene might contribute to the molecular diversity of adenoviruses. Methods : Sequences in the E3 region were analyzed for 14 adenovirus type 3 (Ad3) strains that were isolated from children with lower respiratory tract infections in the Seoul National University Children's Hospital during the period 1991-2000. Full-length adenoviral DNA was purified from the infected A549 cell lysates using a modified Hirt procedure. Results : There was 98% homology between 14 Korean Ad3 strains with a reference strain (M15952). Homology within the Korean Ad3 strains was 98.7%. Variation was found in the region of transcripts 20.1 kDa, 20.6 kDa, truncated 7.7 kDa, 10.3 kDa, 14.9 kDa, and 15.3 kDa. In particular, all 14 Korean strains showed a missense single point mutation at the start codon of the truncated 7.7 kDa. In addition, a deletion was found in the truncated 7.7 kDa region by 58 base pairs in 10 strains and 94 base pairs in 4 strains. Variations in amino acids were observed in the receptor internalization and degradation complex (10.3 kDa/14.9 kDa) which stimulates the clearance from the cell surface and subsequent degradation of the receptors for the Fas ligand and TRAIL, while no variations were observed in another immunoregulatory transcript, 19 kDa. Conclusion : Sequence analysis of the immunoregulatory region of adenovirus E3 shows that genetic heterogeneities are related to genome type patterns.

• Proceedings of the Korean Society for Noise and Vibration Engineering Conference
• /
• 2006.05a
• /
• pp.164-170
• /
• 2006

This paper proposes a RSM-based hybrid evolutionary algorithm (RHEA) which combines the merits of the popular programs such as genetic algorithm (GA), tabu search method, response surface methodology (RSM). This algorithm, for improving the convergent speed that is thought to be the demerit of genetic algorithm, uses response surface methodology and simplex method. The mutation of GA offers random variety to finding the optimum solution. In this study, however, systematic variety can be secured through the use of tabu list. Efficiency of this method has been proven by applying traditional test functions and comparing the results to GA. And it was also proved that the newly suggested algorithm is very effective to find the global optimum solution to minimize the weight for avoiding the resonance of fresh water tank that is placed in the rear of ship. According to the study, GA's convergent speed in initial stages is improved by using RSM method. An optimized solution is calculated without the evaluation of additional actual objective function. In a summary, it is concluded that RHEA is a very powerful global optimization algorithm from the view point of convergent speed and global search ability.

• Korean Journal of Microbiology
• /
• v.30 no.5
• /
• pp.360-365
• /
• 1992

Clinical isolates of 8 ofloxacin resistant Staphylococcus auresu (ORSA) were subjected to MIC test, Southern analysis on gyrA locus and nucleotide sequence analysis of 290 bp of gyrA gene (gyrA-290) spanning amino acid 26 to 121 in order to understand the mechanism of quinolone resistance in Staphylococcus aureus. ORSAs showed highlevel resistance against quinolones (8-250 fold increase of MICs) and also significant resistance agianst ${\beta}-lactams$ (2-32 fold increase of MICs). However, ORSs did not show any change in sensitivity agianst vancomycin. Southern analysis of ORSAs with HindIII, PstI and AluI revealed RFLPs on gyrA locus. In order to further analyze the gyrA gene, gyrA-290 was amplified by PCR and cloned to pTZ vector. Subsequent nucleic acid sequence analysis of gyrA-290 demonstrated a point mutation of C to T resulting amino acid change of Ser-84 to Leu-84 in all 8 ORSA strains. The substitution at 84th amino acid of tyrase A might confer one mechanism of high level quinolone resistance in Staphylococcus aureus.

• Korean Journal of Plant Taxonomy
• /
• v.35 no.4
• /
• pp.273-285
• /
• 2005

Genetic variation of notorious invasive plants, common ragweed (Ambrosia artemisiifolia L.) and giant ragweed (Ambrosia trifida L.) were examined using the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA. A total of 18 different ITS types were detected from 156 individuals of common ragweed sampled mainly from the southern part of Korean peninsula whereas four types were identified from 46 individuals of giant ragweed. High sequence diversity observed from common ragweed in Korean populations was interpreted as multiple introduction. Genetic recombination was suggested as possible method for the production of some of the ITS types while point mutation was mainly responsible for the origin of the sequence diversity. This study provided some of basic genetic information needed for understanding of the evolutionary process in ragweed during invasion.