• Clinical and Experimental Pediatrics
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• v.45 no.10
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• pp.1263-1272
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• 2002

Purpose : Rett syndrome(RTT) is an X-linked dominant neurodevelopmental disorder affecting 1 per 10,000-15,000 female births worldwide. It was initially described by Andreas Rett in 1966. RTT involves developmental regression characterized stereotypic hand movements, tremors, gait apraxia, seizures, deceleration of head growth after the age of 6-18 months. The disease-causing gene was identified as MECP2 on chromosome Xq28. We carried out mutational analysis of MECP2 genes in RTT patients. Methods : Whole blood(5 cc) of 34 sporadic RTT patients was collected in EDTA-anticoagulated tubes. Genomic DNA was extracted from peripheral blood using the E.Z.N.A. blood DNA kit. Four exons of the MECP2 gene were amplified by PCR in 34 Korean with RTT. We carried out PCR divided the exon three into two parts and the exon four into five parts. Primer sequences designed by Amir et al. in 1999 were almost used(AF030876). Sequencing primers used were the same as PCR. DNA sequencing reactions were performed using an ABI 377 DNA sequencer and ABI PRISM dye terminator cycle sequencing reaction kit(Perkin-elmer). The results were compared with the normal DNA sequence(X99686). To confirm the change of sequence on novel mutations, RFLP analysis was performed. Results : The MECP2 mutations were detected in 23(67.6%) of the 34 patients. The mutations consisted of 12 different types including nine missense and three nonsense mutations. Of these, three (L100V, G161E and T311M) mutations were newly identified. Most of the mutations discovered are located within MBD(39.1%) and TRD(39.1%). In this study, three(T158M, R270X, R306C) mutations were identified high frequency. Conclusion : MECP2 gene was also an important cause of Korean RTT patients. MECP2 gene study is an important tool for diagnosis of Korean RTT patients.

• Journal of the Korean Chemical Society
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• v.53 no.6
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• pp.742-748
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• 2009

In the meat industry, correct breed information in food labeling is required to assure meat quality. Genetic markers provide corroborating evidence to identify breed. We described the development of DNA markers to discriminate between Korean beef cattle (Hanwoo), Holstein, and mixed cow beefs. As most breeds are standardized for coat colour, the melanocortin 1 receptor (MC1R) gene, involved in the regulation of eu/pheomelanins synthesis, has been suggested as marker for breed traceability of products of animal origin. We also designed sex-determining region Y (SRY) gene specific primers for Y chromosome detection. In this study, fragments of MC1R gene and SRY gene were amplified by multiplex-PCR and subjected to digestion by MspA1I restriction endonuclease. Reaction products were analysised by denaturing high performance liquid chromatography (DHPLC). As a result, we identified 6 DHPLC peak types from MC1R gene and SRY gene analysis. DHPLC method showed more sensitive than RFLP method for DNA fragments analysis. Therefore, DHPLC method can apply to identify for Hanwoo, Holstein and mixed beef.

• Biomedical Science Letters
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• v.4 no.1
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• pp.11-25
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• 1998

The HLA genes located in the short arm of chromosome 6 specify heterodimeric glycoproteins involved in the regulation of the immune response. Recently, in the elucidation of HLA polymorphism, serological and cellular typing methods have been replaced by DNA typing using polymerase chain reaction (PCR). The purpose of this study was to establish the HLA DNA typing methods and determine gene frequencies of HLA molecules in Koreans. PCR-SSP (sequence specific primers) and PCR-RFLP (restriction fragment length polymorphism) techniques were used for the analysis of HLA-A, -B, -C, DRBl genes and HLA-DQAl, DQBl, DPBl genes, respectively. The results of B-lymphoblastoid cells used for control experiment were consistent with the previous data identified in the 11th International Histocompatibility Workshop. Seventeen, 23, 16, 8, 16, 13 and 37 types of HLA-A, B, C, DQAl, DQBl, DPBl and DRBl alleles were found, respectively, in a total of unrelated 120 Korean individuals. The most frequent HLA alleles were $A^*$02 (27.0%), B$^*$40 (17.6%), Cw$^*$01 (19.2%), DQAl$^*$0301 (32.1%), DQBl$^*$0303 (12.9%), DPBl$^*$0501 (31.3%) and DRBl$^*$1501 (9.2%) among Koreans. This study shows that DNA typing method using PCR technique is a relatively simple, fast and practical tool for the determination of the HLA-class I and II genes. Moreover, the data of HLA gene frequencies could be useful for the Korean database before clinical applications, including organ and unrelated bone marrow transplantation, anthropological study, disease association and individual identification.

• Radiation Oncology Journal
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• v.21 no.4
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• pp.306-314
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• 2003

Purpose : In our Previous study, we have shown the main cel1 death pattern Induced by irradiation or protein tyrosine kinase (PTK) inhibitors in K562 human myeiogenous leukemic cell line. Death of the cells treated with irradiation alone was characterized by mitotic catastrophe and typical radiation-induced apoptosis was accelerated by herblmycin A (HMA). Both types of cell death were inhibited by genistein. In this study, we investigated the effects of HMA and genistein on cell cycle regulation and its correlation with the alterations of radiation-induced cell death. Materials and Methods: K562 cells In exponential growth phase were used for this study. The cells were Irradiated with 10 Gy using 6 MeV Linac (200-300 cGy/min). Immediately after irradiation, cells were treated with 250 nM of HMA or 25 $\mu$N of genistein. The distributions of cell cycle, the expressions of cell cycle-related protein, the activities of cyclin-dependent kinase, and the yield of senescence and differentiation were analyzed. Results: X-irradiated cells were arrested In the G2 phase of the cell cycle but unlike the p53-positive cells, they were not able to sustain the cell cycle arrest. An accumulation of cells in G2 phase of first ceil-cycle post-treatment and an increase of cyclin Bl were correlated with spontaneous, premature, chromosome condensation and mitotic catastrophe. HMA induced rapid G2 checkpoint abrogation and concomitant p53-independent Gl accumulation. HMA-induced cell cycle modifications correlated with the increase of CDK2 kinase activity, the decrease of the expressions of cyclins I and A and of CDK2 kinase activity, and the enhancement of radiation-induced apoptosis. Genistein maintained cells that were arrested in the G2-phase, decreased the expressions of cyclin Bl and cdc25c and cdc25C kinase activity, increased the expression of pl6, and sustained senescence and megakaryocytic differentiation. Conclusion: The effects of HMA and genistein on the radiation-induced cell death of KS62 cells were closely related to the cell cycle regulatory activities. In this study, we present a unique and reproducible model in which for investigating the mechanisms of various, radiation-induced, cancer cell death patterns. Further evaluation by using this model will provide a potent target for a new strategy of radiotherapy.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.227-233
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• 2017

Pig has been known to be one of the most feasible animals as a bioreactor to produce pharmaceuticals in milk and as a mediator in xenotransplantation research. Previously, we generated transgenic pigs for both purposes, which were expressing Factor 8, vWF, hTPA, and hEPO in milk, along with expression of MCP at GalT gene locus ($GalT^{-MCP/-MCP}$) as well as expressing MCP at GalT gene loci with CD73 expression ($GalT^{-MCP/+}/CD73$). In this study, we performed comparative analyses of sperm parameters between wild type male (WT) pig and those transgenic males to examine the effects of transgenes integrated into the pigs on motility, morphology, viability, and acrosome integrity of the spermatozoa. Our results showed that the rates of actively motile spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 85.0%, 83.3%, 82.5%, 83.3%, 82.5%, 77.5%, and 78.7%, respectively. Whereas, the rates of morphologically normal spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 90.0%, 80.0%, 80.0%, 83.3%, 85.0%, 91.8%, and 80.8%, respectively. In addition, the viability in spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 93.9%, 82.4%, 89.9%, 83.9%, 87.4%, 92.8%, and 83.6%, respectively. The rates of spermatozoa with normal acrosome integrity in WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 98.1%, 98.6%, 98.6%, 98.7%, 98.1%, 99.5%, and 95.1%, respectively. There were no significant differences in motility, morphology, viability, and acrosome integrity of the spermatozoa among WT, Factor 8, vWF, hTPA, and hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs. These mean that neither random integration nor targeted integration of the transgene into chromosome of pig effect on characteristics of spermatozoa. Ultimately, the transgenic male pigs subjected in this study could apply to propagate their progenies for production of human therapeutic proteins and advancing the xenotransplantation research.

• Journal of Life Science
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• v.28 no.12
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• pp.1416-1423
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• 2018

With strong biotin binding affinity ($K_D=10^{-14}M$), the tetrameric feature of streptavidin could be used to increase the antigen binding activity of a camel heavy chain (VHH) antibody through their fusion, here stained with biotinylated horseradish peroxidase and subsequent immunoassays ELISA and Western blot analysis. For this application, we cloned the streptavidin gene amplified from the Streptomyces avidinii chromosome by PCR, and this was fused to the gene of the 8B9 VHH antibody which is specific to green fluorescent protein (GFP) antigens. To express a soluble fusion protein in Escherichia coli, we used the pUC119 plasmid-based expression system which uses the lacZ promoter for induction by IPTG, the pelB leader sequence at the N-terminus for secretion into the periplasmic space, and six polyhistidine tags at the C-terminus for purification of the expressed proteins using an $Ni^+$-NTA-agarose column. Although streptavidin is toxic to E. coli because of its strong biotin binding property, this soluble fusion protein was expressed successfully. In SDS-PAGE, the size of the purified fusion protein was 122.4 kDa in its native condition and 30.6 kDa once denatured by boiling, suggesting the tetramerization of the monomeric subunit by non-covalent association through the streptavidin moiety fusing to the 8B9 VHH antibody. In addition, this fusion protein showed biotin binding activity similar to streptavidin as well as GFP antigen binding activity through both ELISA and Western blot analysis. In conclusion, the protein resulting from the fusion of an 8B9 VHH antibody with streptavidin was successfully expressed and purified as a soluble tetramer in E. coli; it showed both biotin and GFP antigen binding activity suggesting the possible production of a tetrameric and bifunctional VHH antibody.

• Tuberculosis and Respiratory Diseases
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• v.61 no.3
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• pp.248-255
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• 2006

Background: LOH11A is a region with frequent allele loss (>75%) in lung cancer that is located on the centromeric part of chromosome 11p15.5. Clinical and cell biological studies suggest that this region contains a gene associated with metastatic tumor spread. RRM1 encoding the M1 subunit of ribonucleotide reductase, which is an enzyme that catalyses the rate-limiting step in deoxyribonucleotide synthesis, is located in the LOH11A region. Methods: Polymorphisms were found at nucleotide position (-)37 (C/A) and (-)524 (C/T) from the beginning of exon 1 of the RRM1 gene that might regulate the expression of RRM1. We studied the polymorphisms in 127 Korean individuals (66 lung cancer and 61 normal controls) and compared with those of 140 American patients with lung cancer. Results: CC, AC and AA were found at the (-)37 position in 64(50.4%), 55(43.3%), and 8(6.3%) out of 127 Korean individuals (66 cancer, 61 non-cancer patients), respectively. There was a similar frequency of allele A at (-)37 in the American(27.9%) and Korean population(28.0%). CC, CT and TT was found at the (-)524 position in 24(18.9%), 44(34.6%), and 59(46.5%) out of the 127 Korean individuals, respectively. There was a similar frequency of allele C at (-)524 in the American(34.6%) and Korean population(36.2%). There was no difference in the frequency of the (-)37 and (-)524 genotypes between the cancer and non-cancer group. However there was a significant correlation of the genotypes between (-)37 and (-)524 (p<0.001), which suggests the possible coordination of these polymorphisms in the regulation of the promoter activity of the RRM1 gene. Conclusion: RRM1 promoter polymorphisms were not found to be significant risk factors for lung cancer. However, a further study of the promoter activity and expression of the RRM1 gene according to the pattern of the polymorphism will be needed.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.175-180
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• 2005

This study was conducted to examine the effects of demecolcine-assisted enucleation and recipient cell cycle stage on the development of bovine somatic cell nuclear transfer (NT) embryos. In vitro cultured oocytes for $16\~20$ h were classified by first polar body (1st PB) extrusion and cell cycle stage (MI and MII) and treated $0.4\;{\mu}L/mL$ demecolcine for 40 min before enucleation. Enucleated oocytes were fused electrically with bovine ear skin cells, activated by Ca-ionophore+DMAP, and cultured in vitro. Most of eggs ($86.2\%$) treated with demecolcine protruded a chromosome mass and enucleated efficiently ($98.8\%$, (P<0.05). Demecolcine did not have a deteriorative effect on the development of NT embryos. Developmental rate of NT embryos reconstituted with oocytes extruded 1st PB significantly higher than that of NT embryos produced by oocytes without 1st PB ($18.2\%\;vs.\;4.6\%\cdot$, P<0.05). Cleavage and blastocyst formation rate of embryos reconstituted with MI oocytes ($69.4\%\;and\;5.9\%$, respectively) were significantly lower than those of embryos reconstituted with MII oocytes ($96.7\%\;and\;23.9\%$, respectively, P<0.05). From the present result, it is suggested that domecolcine is useful for the enucleation of recipient oocytes in bovine NT procedures, and MII oocytes rather than MI oocytes are more appropriate for recipient cytoplasm. Although, the potential to develop into blastocysts of NT embryos produced by 1st PB-nonextruded and MI oocytes was very low, these oorytes could be used for NT.

• Clinical and Experimental Pediatrics
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• v.51 no.3
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• pp.315-322
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• 2008

Purpose : Prader-Willi syndrome (PWS) is a complex genetic disorder, caused by the deletion of the paternally derived 15q11-13 region or the maternal uniparental disomy of chromosome 15 (mUPD(15)). In this study, we compared phenotypic differences between those patients whose disease was caused by microdeletion and those caused by mUPD(15). In addition, a comparison of the efficacy of growth hormone (GH) therapy between these two PWS genotypes was analyzed. Methods : Fifty-three patients were diagnosed as having PWS based on molecular and cytogenetic analyses and clinical features. Data that included maternal age, birth weight, a feeding problem in the neonatal period, cryptorchidism, developmental delay or mental retardation, short stature, hypopigmentation, changes in height, weight, and body mass indexes (BMI) before and after GH treatment were obtained by a retrospective review of medical records. The data from the patients with microdeletion were compared with those from the patients with mUPD(15). Results : Of the 53 patients with genetically confirmed PWS, 39 cases had microdeletion and 14 mUPD(15). Maternal ages were significantly higher in the mUPD(15) group, and hypopigmentation and a feeding problem in the neonatal period were more frequent in the microdeletion group. Growth hormone was administered to 20 patients [14 with microdeletion, 6 with mUPD(15)]. There were no differences between the two groups in height velocity, weight and height SDS, and BMI after GH therapy. Conclusion : Phenotype and genotype correlations were observed in Korean PWS patients, such as more advanced maternal ages in the mUPD(15) group and more feeding problems and hypopigmentations in the microdeletion group. Further long-term prospective studies are needed to correlate other aspects of the phenotypes.

• Korean Journal of Microbiology
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• v.52 no.4
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• pp.406-414
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• 2016

Among 6 leu codons, CUG is the most frequently used codon in E. coli. It is recognized by leu-tRNA(CAG) encoded by four genes scattered on two chromosomal loci (leuT and leuPQV ). In the process of constructing a strain with no functional leu-tRNA (CAG) gene on chromosome, we made two mutant strains separately, one on leuPQV locus (${\Delta}leuPQV$), and the other on leuT locus [$leuT^*$(GAG)], where the anticodon of leuT was changed from CAG to GAG, thereby altering its recognition codon from CUG to CUC. We attempted to combine these two mutations by transduction using $leuT^*$(GAG) strain as a donor and ${\Delta}leuPQV$ strain as a recipient. Large and small colonies appeared from this transduction. From PCR and DNA sequencing, large colony was confirmed to be the reciprocal recombinant as expected, but the small colonies contained both mutant $leuT^*$(GAG) and wild type leuT (CAG) genes in the cell. This heterozygous diploid strain did not show any unusual morphology under microscopic observation, but, interestingly, it showed a linear growth curve in rich medium with much slower growth rate than wild type cell. It always formed homogenous small colonies in the selection medium, but, when there was no selection, it readily segregated into $leuT^*$(GAG) and leuT (CAG). From these observations, we suggested that the strain with both $leuT^*$(GAG) and leuT (CAG) genes was not a partial diploid (merodiploid), but a full diploid cell having two different chromosomes. We proposed a model explaining how such a heterozygous diploid cell was formed and how and why its growth showed a linear growth curve.