Isolation, Restriction Mapping, and Promoter Sequence Analysis of an Isoperoxidase Gene from Korean-Radish, Raphanus sativus L.

Park, Jong-Hoon,Kim, Soung-Soo Korean Society for Biochemistry and Molecular Biol 1996 Journal of biochemistry and molecular biology Vol.29 No.1

A specific DNA fragment from Korean radish (Raphanus sativus L.) was amplified by performing PCR with oligonucleotide primers which correspond to the highly conserved regions of plant peroxidases. The size of the PCR product was ca. 400 bp, as expected from the known plant peroxidase genes. Comparison of the nucleotide and deduced amino acid sequences of the PCR product to those of other plant peroxidase-encoding genes revealed that the amplified fragment corresponded to the highly conserved region I and III of plant peroxidases. By screening a genomic library of Korean radish using the amplified fragment as a probe, two positive clones, named prxK1 and prxK2, were isolated. Restriction mapping studies indicated that the 5.2 kb Sail fragment of the prxK1 clone and the 4.0 kb EcoRI fragment of the prxK2 clone encode separate isoperoxidase genes. Analyses of the promoter region of the prxK1 clone shows that putative CAAT box, CMT box, and TGA1b binding sequence (5' TGACGT) are present 718 bp upstream from the start codon.

Hepatic Lipase C514T Polymorphism and its Relationship with Plasma HDL-C Levels and Coronary Artery Disease in Koreans

Park, Kyung-Woo,Choi, Jin-Ho,Chae, In-Ho,Cho, Hyun-Jai,Oh, Se-Il,Kim, Hyo-Soo,Lee, Myoung-Mook,Park, Young-Bae,Choi, Yun-Shik Korean Society for Biochemistry and Molecular Biol 2003 Journal of biochemistry and molecular biology Vol.36 No.2

Hepatic lipase is a key enzyme that is involved in HDL-C metabolism. The goal of this study was to find out the frequency of the hepatic lipase C514T polymorphism, and evaluate its relationship with plasma HDL-C levels and coronary artery disease (CAD) in Koreans. Two hundred and twenty four subjects with no previous history of lipid-lowering therapy, 118 patients with significant CAD, and 106 controls were examined with respect to their genotypes, lipid profiles, and other risk factors for CAD. The frequency of the -514T allele was 0.37 in men and 0.35 in women, which were higher than the frequency that was reported in Caucasians, but lower than the frequency that was reported in African-Americans. The -514T allele was associated with significantly higher HDL-C levels in women. After controlling for age, gender, BMI, DM, and smoking, the non-CC genotype was significantly associated with HDL-C levels, and explained 6% of the HDL-C variation in this study. When the genotypes-distribution was compared between the CAD and non-CAD patients, the hepatic lipase C-514T polymorphism was not associated with the presence of CAD. Koreans have a higher frequency of the hepatic lipase gene 514T allele than Caucasians, and the -514T allele is associated with higher plasma HDL-C levels in Korean women, and perhaps non-smoking men. However, our data does not suggest an association between the polymorphism and an increased risk of CAD.

Transforming Growth Factor-β3 Gene SfaN1 Polymorphism in Korean Nonsyndromic Cleft Lip and Palate Patients

Kim, Myung-Hee,Kim, Hyo-Jin,Choi, Je-Yong,Nahm, Dong-Seok Korean Society for Biochemistry and Molecular Biol 2003 Journal of biochemistry and molecular biology Vol.36 No.6

The nonsyndromic cleft lip and palate (NSCL/P) is a congenital deformity of multifactorial origin with a relatively high incidence in the oriental population. Various etiologic candidate genes have been reported with conflicting results, according to race and analysis methods. Recently, the ablation of the TGF-${\beta}3$ gene function induced cleft palates in experimental animals. Also, polymorphisms in the TGF-${\beta}3$ gene have been studied in different races; however, they have not been studied in Koreans. A novel A $\rightarrow$ G single nucleotide polymorphism (defined by the endonuclease SfaN1) was identified in intron 5 of TGF-${\beta}3$ (IVS5+104 A > G). It resulted in different genotypes, AA, AG, and GG. The objective of this study was to investigate the relationship between the SfaN1 polymorphism in TGF-${\beta}3$ and the risk of NSCL/P in the Korean population. The population of this study consisted of 28 NSCL/P patients and 41 healthy controls. The distribution of the SfaN1 genotypes was different between the cases and controls. The frequency of the G allele was significantly associated with the increased risk of NSCL/P [odds ratio (OR) = 15.92, 95% confidence interval (CI) = 6.3-41.0]. The risk for the disease increased as the G allele numbers increased (GA genotype: OR = 2.11, 95% CI = 0.38-11.68; GG genotype: OR = 110.2, 95% CI = 10.67 - 2783.29) in NSCL/P. A stratified study in patients revealed that the SfaN1 site IVS5+104A > G substitution was strongly associated with an increased risk of NSCL/P in males (p < 0.001), but not in females. In conclusion, the polymorphism of the SfaN1 site in TGF-${\beta}3$ was significantly different between the NSCL/P patients and the control. This may be a good screening marker for NSCL/P patients among Koreans.

cDNA Cloning and Overexpression of an Isoperoxidase Gene from Korean-Radish, Raphanus sativus L.

Park, Jong-Hoon,Kim, Soung-Soo Korean Society for Biochemistry and Molecular Biol 1996 Journal of biochemistry and molecular biology Vol.29 No.2

A partial cDNA encoding a Korean radish isoperoxidase was obtained from a cDNA library prepared from 9 day old radish root. In order to obtain Korean radish isoperoxidase cDNA, 5' RACE (rapid amplification cDNA end) PCR was performed and a cDNA (prxK1) encoding a complete structural protein was obtained by RT (reverse transcription)-PCR. Sequence analysis revealed that the length of the cDNA was 945 base pairs, and that of the mRNA transcript was ca. 1.6 kb. The deduced amino acid of the protein were composed of 315 amino acid residues and the protein was 92% homologous to turnip peroxidase, and 46% to 50% homologous to other known peroxidases. The 945 bp cDNA encoding Korean radish isoperoxidase was overexpressed in Escherichia coli up to approximately 9% of total cellular protein. The recombinant fusion protein exhibited 43 kDa on SDS-PAGE analysis and the activity level of the recombinant nonglycosylated protein was two fold higher in IPTG induced cell extracts than that of uninduced ones.

Korean BAC Library Construction and Characterization of HLA-DRA, HLA-DRB3

Park, Mi-Hyun,Lee, Hye-Ja,Bok, Jeong,Kim, Cheol-Hwan,Hong, Seong-Tshool,Park, Chan,Kimm, Ku-Chan,Oh, Berm-Seok,Lee, Jong-Young Korean Society for Biochemistry and Molecular Biol 2006 Journal of biochemistry and molecular biology Vol.39 No.4

A human bacterial artificial chromosome (BAC) library was constructed with high molecular weight DNA extracted from the blood of a male Korean. This Korean BAC library contains 100,224 clones of insert size ranging from 70 to 150 kb, with an average size of 86 kb, corresponding to a 2.9-fold redundancy of the genome. The average insert size was determined from 288 randomly selected BAC clones that were well distributed among all the chromosomes. We developed a pooling system and three-step PCR screen for the Korean BAC library to isolate desired BAC clones, and we confirmed its utility using primer pairs designed for one of the clones. The Korean BAC library and screening pools will allow PCR-based screening of the Korean genome for any gene of interest. We also determined the allele types of HLA-DRA and HLA-DRB3 of clone KB55453, located in the HLA class II region on chromosome 6p21.3. The HLA-DRA and DRB3 genes in this clone were identified as the DRA*010202 and DRB3*01010201 types, respectively. The haplotype found in this library will provide useful information in future human disease studies.

The Association between the T102C Polymorphism of the HTR2A Serotonin Receptor Gene and HDL Cholesterol Level in Koreans

Choi, Jin-Ho,Zhang, Shu-Ying,Park, Kyung-Woo,Cho, Young-Seok,Oh, Byung-Hee,Lee, Myoung-Mook,Park, Young-Bae,Kim, Hyo-Soo Korean Society for Biochemistry and Molecular Biol 2005 Journal of biochemistry and molecular biology Vol.38 No.2

5-HT2A is one of major serotonin receptor that is involved in the action of serotonin-targeting drugs. Previous clinical studies have shown an unexpected association between lower cholesterol level and psychiatric diseases, in which T102C polymorphism of HTR2A, gene of 5-HT2A serotonin receptor, might be involved. Therefore, we hypothesized a potential association between lower cholesterol level and T102C polymorphism. The effect of the T102C polymorphism on the serum lipid profiles of 646 subjects without specific psychiatric disease was investigated. Genotype was determined by polymerase chain reaction and restriction fragment length polymorphism analysis. There were significantly lower levels of total cholesterol ($193.6{\pm}35.0$ versus $202.1{\pm}45.5\;mg/dl$, p = 0.016) and HDL-cholesterol ($42.7{\pm}11.6$ versus $46.3{\pm}12.7\;mg/dl$, p = 0.004) in CC genotype than non-CC genotypes. Moreover, multivariate analysis showed that the CC genotype is a strong predictor of a lower HDL-cholesterol level (p < 0.001). In conclusion, this study shows that the CC genotype of the HTR2A gene is related to lower HDL-cholesterol level in Koreans. This is the first demonstration showing the potential genetic relationship between the serotonin receptor gene polymorphism and the HDL-cholesterol level.

Molecular and Biochemical Studies on the DNA Replication of Bacteriophage T7: Functional Analysis of Amino-terminal Region of Gene 2.5 Protein

Kim, Young-Tae,Lee, Sung-Gu,Kim, Hak-Jun Korean Society for Biochemistry and Molecular Biol 1995 Journal of biochemistry and molecular biology Vol.28 No.6

The product of bacteriophage T7 gene 2.5 is a single-stranded DNA binding protein and plays an important role in T7 DNA replication, recombination, and repair. Genetic analysis of T7 phage defective in gene 2.5 shows that the gene 2.5 protein is essential for T7 DNA replication and growth (Kim and Richardson, 1993). The C-terminal truncated gene 2.5 protein ($GP2.5-{\Delta}21C$) cannot substitute for wild-type gene 2.5 protein in vivo; suggesting that the C-terminal domain of gene 2.5 protein is essential for protein-protein interactions (Kim and Richardson, 1994; J. Biol. Chem. 269, 5070-5078). Truncated gene 2.5 proteins lacking 19 residues ($GP2.5-{\Delta}19N$) and 39 residues ($GP2.5-{\Delta}39N$) from the amino-terminal domain were constructed by in vitro mutagenesis. $GP2.5-{\Delta}19N$ can support the growth of T7 phage lacking gene 2.5 while $GP2.5-{\Delta}39N$ cannot substitute for wild-type gene 2.5 protein in vivo; however, its ability to bind to single-stranded DNA is not affected. These results clearly demonstrate that the 20~39 amino-terminal region of gene 2.5 protein is required for T7 growth in vivo but may not be involved in DNA binding activity.

Purification of a Pore-forming Peptide Toxin, Tolaasin, Produced by Pseudomonas tolaasii 6264

Cho, Kwang-Hyun,Kim, Sung-Tae,Kim, Young-Kee Korean Society for Biochemistry and Molecular Biol 2007 Journal of biochemistry and molecular biology Vol.40 No.1

Tolaasin, a pore-forming peptide toxin, is produced by Pseudomonas tolaasii and causes brown blotch disease of the cultivated mushrooms. P. tolaasii 6264 was isolated from the oyster mushroom damaged by the disease in Korean. In order to isolate tolaasin molecules, the supernatant of bacterial culture was harvested at the stationary phase of growth. Tolaasin was prepared by ammonium sulfate precipitation and three steps of chromatograpies, including a gel permeation and two ion exchange chromatographies. Specific hemolytic activity of tolaasin was increased from 1.7 to 162.0 HU $mg^{-1}$ protein, a 98-fold increase, and the purification yield was 16.3%. Tolaasin preparation obtained at each purification step was analyzed by HPLC and SDS-PAGE. Two major peptides were detected from all chromatographic preparations. Their molecular masses were analyzed by MALDI-TOF mass spectrometry and they were identified as tolaasin I and tolaasin II. These results demonstrate that the method used in this study is simple, time-saving, and successful for the preparation of tolaasin.

Quantitation of Hepatitis C Viral RNA Using Direct CRT-PCR

Park, Young-Suk,Lee, Kyung-Ok,Oh, Moon-Ju,Chai, Young-Gyu Korean Society for Biochemistry and Molecular Biol 1997 Journal of biochemistry and molecular biology Vol.30 No.3

Chronic hepatitis C virus (HCV) infection is associated with the rapid development of cirrhosis and hepatocellular carcinoma. It has been reported that the amount of HCV RNA may be correlated with the progression of hepatitis and may be a prognostic marker for treatment of HCV patients. The direct detection of HCV RNA by reverse transcription-polymerase chain reaction (RT-PCR) is widely used to determine the presence of circulating virions. The most relevant limit of this approach is the lack of quantitative information about the viral titer. In the present study, we developed the method for HCV quantitation using competitive reverse transcription (CRT)-PCR using the deleted HCV standard. The serially diluted standard was added in titrated amounts to the target HCV RNA. The mixture was then reverse transcribed and amplified in the same reaction tube. The methods were evaluated using over 110 HCV-PCR positive samples in Koreans. About 59% of the samples were judged to contain $10^{5}-10^{6}$ copies of HCV RNA in 1 ml of serum.

Mapping of the Interaction Domain of DNA Topoisomerase $II{\alpha}$ and $II{\beta}$ with Extracellular Signal-Regulated Kinase 2

Park, Gye-Hwa,Bae, Young-Seuk Korean Society for Biochemistry and Molecular Biol 2001 Journal of biochemistry and molecular biology Vol.34 No.1

Both topoisomerase $II{\alpha}$ and $II{\beta}$ east as phosphoproteins in the cells. Recently it was reported that DNA topoisomerase $II{\alpha}$ associates with and is phosphorylated by the extracellular signal-regulated kinase 2 (ERK2). Also, ERK2 stimulates the activity of topoisomerase II by a phosphorylation-independent manner [Shapiro et al., (1999) Mol. Cell. Biol. 19, 3551-3560]. In this study, a yeast two-hybrid system was used to investigate the binding site between topoisomerase $II{\alpha}$ or $II{\beta}$ and ERK2. The two-hybrid test clearly showed that topoisomerase $II{\beta}$ residues 1099-1263, and topoisomerase $II{\alpha}$ residues 1078-1182, mediate the interaction with ERK2, and that the leucine zipper motifs of topoisomerase $II{\alpha}$ and $II{\beta}$ are not required for its physical binding to ERK2. Our results suggest that topoisomerase $II{\beta}$ residues 1099-1263, and topoisomerase $II{\alpha}$ residues 1078-1182, may be common binding sites for activator proteins.