• BMB Reports
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• v.38 no.4
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• pp.414-419
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• 2005

The production of recombinant antibodies has been generally recognized as time-consuming and labor-intensive. The aim of our study is to construct mammalian expression vectors containing the cDNA encoding the human constant regions and murine variable regions to massively and cost-effectively produce full-length chimeric antibodies. Unique restriction sites flanking the Ig variable region were designed to allow for the replacement of variable regions generated by PCR. Western blot analysis of the chimeric antibodies revealed that the expressed products were of the predicted size, structure and specificity. The usefulness of the vectors was confirmed by construction of human-mouse chimeric antibody-HCAb which secretes murine antibody against the human colorectal cancer. Selected in medium containing gradually increasing methotrexate (MTX), clones with increased expression of the product gene can be efficiently generated. The secretion of recombinant chimeric antibody-HCAb yielded $30\;pg\;cell^{-1}\;day^{-1}$ at $10^{-6}\;M$ MTX. With this high-level expression from pools, the convenient and rapid production of over 100 milligram amounts per liter of recombinant antibodies may be achieved, which indicates the significant roles of pYR-GCEVH and pYR-GCEVL in the production of chimeric antibodies.

• Korean Journal of Microbiology
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• v.38 no.4
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• pp.230-230
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• 2002

Class Ⅲ chitin synthases in filamentous fungi are important for hyphal growth and differentiation of several filamentous fungi. A genomic clone containing the full gene encoding Chs4, a class Ⅲ chitin synthase in Penicillium chrysogenum, was cloned by PCR screening and colony hybridization from the genomic library. Nucleotide sequence analysis and transcript mapping of chs4 revealed an open reading frame (ORF) that consisted of 5 exons and 4 introns and encoded a putative protein of 915 amino acids. Nucleotide sequence analysis of the 5′flanking region of the ORF revealed a potential TATA box and several binding sites for transcription activators. The putative transcription initiation site at -716 position was identified by primer extension and the expression of the chs4 during the vegetative growth was confirmed by Northern blot analysis. Amino acid sequence analysis of the Chs4 revealed at least 5 transmembrane helices and several sites for past-transnational modifications. Comparison of the amino acid sequence of Chs4 with those of other fungi showed a close relationship between P chrysogenum and genus Aspergillus.

• Proceedings of the Korea Society of Environmental Toocicology Conference
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• 2002.10a
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• pp.170-170
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• 2002

College of Pharmacy, Ewha womans University, Seoul, 120-750, Korea 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent halogenated aromatic hydrocarbon congener that induces expression of several genes including CYP1A1. Exposure to TCDD results in many toxic actions such as carcinogenesis, hepatotoxicity, immune suppression, and reproductive and developmental toxicity. Dramatic differences in dioxin toxicity have been observed between the sexes of some animal species, suggesting hormonal modulation of dioxin action. Many studies have been reported and propose several mechanisms of anti-estrogenic effects of TCDD. In contrast, the effect of estrogen on the regulation of CYP1A1 are not clear at present. There are several reports showing conflicting results. It seems that induction/inhibition of CYP1A1 may be dependent on cell-type and concentration. The purpose of this study was to investigate the regulation of TCDD-induced CYP1A1 gene expression by estradiol and its metabolites. We examined whether estradiol and its metabolites altered TCDD-mediated induction of CYP1A1 enzyme activity. 17 ${\beta}$ estradiol and 16 ${\alpha}$ estriol at non cytotoxic concentrations caused a significant concentration dependent decline of TCDD-induced EROD activity To determine whether reduced EROD activity reflected altered CYP1A1 mRNA expression, we measured CYP1A1 mRNA level by RT-PCR. And to examine whether estradiol and its metabolites have effects on TCDD-induced CYP1A1 gene expression at the transcription level, we also peformed transient transfection with an AhR responsive reporter plasmid containing the 5' flanking region of the human CYP1A1 gene to examine whether estradiol and its metabolites have effects on TCDD-induced CYP1A1 gene expression at the transcription level.

• Environmental Analysis Health and Toxicology
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• v.20 no.3 s.50
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• pp.215-221
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• 2005

Environmental polycyclic aromatic hydrocarbones (PAHs), which are formed during incomplete combustion of fossil fuels, are widely distributed in our environment. Human exposure to PAHs may occur through smoking, polluted air, food consumption and occupational contact. Urinary naphthols, 1-and 2-naphthol, have been suggested as route -specific biomarkers for exposure to airborne PAHs. Cytochrome p450 2E1 (CYP2E1) is known to be a great importance for the metabolism of organic solvents, which is a precacinogens with small molecular weight. This study describes the metabolic differences between PstI and RsaI polymorphisms (c1 allele: PstI-. RsaI+ ; c2 allele: PstI+, RsaI-) of CYP2E1 5-flanking region by genetically modified HepG2 cells, which overexpress the polymorphic regions. The results of CAT assay and western blot in the c2 allele overexpressed cells have higher activities than the cl allele over-expressing cells. However, the metabolism of naphthalene to 2-naphthol has no difference due to the two genotypes. In this study, we established the CYP2E1 polymorphic allele transduced HepG2 cells to screen susceptibility -differences in PAH exposure. In conclusion, the CYP2E1 polymorphism may hardly induce susceptibility differences in PAH exposure monitoring with urinary naphthols.

• Molecules and Cells
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• v.26 no.3
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• pp.250-257
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• 2008

Microsatellites or simple sequence repeats (SSR) are widely distributed in eukaryotic genomes and are informative genetic markers. Despite many advantages of SSR markers such as a high degree of allelic polymorphisms, co-dominant inheritance, multi-allelism, and genome-wide coverage in various plant species, they also have shortcomings such as low polymorphic rates between genetically close lines, especially in Capsicum annuum. We developed an alternative technique to SSR by normalizing and alternating anchored primers in random amplified microsatellite polymorphisms (RAMP). This technique, designated reverse random amplified microsatellite polymorphism (rRAMP), allows the detection of nucleotide variation in the 3' region flanking an SSR using normalized anchored and random primer combinations. The reproducibility and frequency of polymorphic loci in rRAMP was vigorously enhanced by translocation of the 5' anchor of repeat sequences to the 3' end position and selective use of moderate arbitrary primers. In our study, the PCR banding pattern of rRAMP was highly dependent on the frequency of repeat motifs and primer combinations with random primers. Linkage analysis showed that rRAMP markers were well scattered on an intra-specific pepper map. Based on these results, we suggest that this technique is useful for studying genetic diversity, molecular fingerprinting, and rapidly constructing molecular maps for diverse plant species.

• Korean Journal of Microbiology
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• v.40 no.3
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• pp.221-225
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• 2004

Novel cytochrome P450 hydroxylase (CYP) genes were isolated and characterized from P. autotrophica cosmid DNA library using an actinomycete CYP-specific PCR product as a screening probe. The cosmids containing four unique CYP genes (pESK601, 602, 603, 604, 605) were identified, and the four CYP genes were completely sequenced to be homologous to other known Actinomycetes CYP genes involved in various secondary metabolic pathways. Among all novel actinomycete CYP genes found in P. autotrophica, the CYP genes present in pESK601 were revealed to be highly homologous to the CYP genes involved in polyene-type amphotericin and nystatin antibiotic biosynthesis. The nucleotide sequences of the CYP flanking region in pESK601 also revealed the polyene-type biosynthetic genes, implying the presence of a cryptic polyene-type antifungal biosynthetic gene cluster in P. autotrophica.

• Journal of Food Hygiene and Safety
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• v.15 no.1
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• pp.51-54
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• 2000

In the 5'-flanking region of the D-AAT, AspAT and AlaDH gene, I found three or two pairs of sequences(designated as Pl, P2, P3) which show significant similarity to the E.coli consensus sequences of -35 and -10 for promoters. The spacing between -35 and -10 is 16 to 18bp in all the three putative promoters Pl, P2 and P3 which is in good agreement with the preferred spacer length in E.coli and in B.subtilis. Therefore, the putative promoters may also function to increase the efficiency of transcriptional initiation. The most stable, double-helical“Shine-Dalgarno”pairing is formed with a free energy change(ΔG) of -13.0 kcal/mol, -9.6 kcal/mol, -15.8 kcal/mol.

• Molecules and Cells
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• v.20 no.3
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• pp.416-422
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• 2005

We previously used Southern blot analysis to detect restriction-length polymorphisms between male fertile and cytoplasmic male sterile (CMS) cytoplasms at the coxII and atp6 loci of the mtDNA of Capsicum annuum L. Two copies of atp6 were found in each male fertile and CMS pepper lines. Interestingly, one of the copies of atp6 in CMS pepper was a 3'-truncated pseudogene. The open reading frame of the coxII gene was the same in the fertile (N-) and CMS (S-) lines. However, the nucleotide sequence in the S-cytoplasm diverged from that in the N-cytoplasm 41 bp downstream of the stop codon. To develop CMS-specific sequence-characterized amplified region (SCAR) markers, inverse PCR was performed to characterize the nucleotide sequences of the 5' and 3' flanking regions of mitochondrial atp6 and coxII from the cytoplasms of male fertile (N-) and CMS (S-) pepper plants. Based on these data, two CMS-specific SCAR markers, 607 and 708 bp long, were developed to distinguish N-cytoplasm from S-cytoplasm by PCR. The CMS-specific PCR bands were verified for 20 cultivars containing either N- or S-cytoplasm. PCR amplification of CMS-specific mitochondrial nucleotide sequences will allow quick and reliable identification of the cytoplasmic types of individual plants at the seedling stage, and assessment of the purity of $F_1$ seed lots. The strategy used in this report for identifying CMS-specific markers could be adopted for many other crops where CMS is used for F1 seed production.

• BMB Reports
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• v.42 no.8
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• pp.529-534
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• 2009

Marbling score (MS) is the major trait that affects carcass quality in beef cattle. In this study, we investigated the association between genetic polymorphisms of the adipose differentiation-related protein gene (ADFP) and carcass traits in Korean cattle (also known as Hanwoo). Using direct DNA sequencing in 24 unrelated Korean cattle, 25 novel polymorphisms were identified within all exons and their flanking regions of ADFP, including the promoter region (1.5 kb). Among them, 21 polymorphic sites were selected for genotyping in the beef cattle (n = 425). Statistical analyses revealed that one promoter polymorphism (c.-56-18A > G) was associated with MS (P = 0.009). The 'A' allele of c.-56-18A > G exerted a lowering effect on MS, e.g., the lowest MS was found in 'A/A' (MS = 2.09 ${\pm}$ 1.23), intermediate in 'A/G' (MS = 2.11 ${\pm}$ 1.31), and the highest in 'G/G' (MS = 2.47 ${\pm}$ 1.47). Our findings suggest that these polymorphisms in ADFP might be important genetic factors involved in carcass quality in beef cattle.

• Animal cells and systems
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• v.6 no.4
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• pp.317-322
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• 2002

To improve conventional yeast one-hybrid screening, we have developed an efficient mammalian one-hybrid system that allows rapid isolation of com-plementary DNAs which are able to induce human p14$^{ARF}$. tumor suppressor gene. A 1.5 kb promoter region of p14$^{ARF}$ was fused to EGFP to generate ARF promoter-EGFP reporter vector. This reporter plasmid was stably trans-fected into NIH3T3 cells for generation of reporter cell line. When the reporter cell line was infected with E2F-1 together with excess amounts of empty vector, the cells that received the positive modulator were readily identifiable by green fluorescence using FACS. The GFP-positive cells were cloned directly from the cultured cells and expanded in bulk culture. The genomic DNAs from GFP-positive cells were prepared and the CDNA insert in integrated retroviral genome was recovered by PCR using primers annealing to the retroviral vector sequences flanking the insert-cloning site. This system should be useful for efficient screening of expression CDNA libraries in mammalian cells to identify novel upstream regulators for spe-cific genes by one-hybrid interaction.ion.