• Tuberculosis and Respiratory Diseases
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• v.51 no.2
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• pp.108-121
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• 2001

Background : The $p16^{INK4a}$ (p16) twnor suppressor gene is frequently inactivated in hwnan non-small cell lung cancers (NSCLCs), predominantly through homozygous deletion or in association with aberrant promotor hypermethylation. Death-associated protein kinase (DAPK) gene influences interferon $\gamma$-induced apoptotic cell death and has important role in metastasis of lung cancer in animal model. Hypermethylation of promoter region of DAP kinase gene may suppress the expression of this gene. Methods : This study was performed to investigate the aberrant methylation of p16 or DAP kinase in 35 resected primary NSCLCs by methylation-specific PCR (MSP), and demonstrated frequency, diagnostic value and clinical implication of aberrant methylation of two genes. Results : Thirty-two cases were male patients, and 3 cases were female patients with an average age was 57. $8{\pm}10.5$ years. The histologic types of lung cancer were 22 of squamous cell carcinoma, 12 of adenocarcinoma, 1 of large cell carcinoma. Pathologic stages were 11 cases of stage I (1 IA, 10 IB), 13 cases of stage II (1 IIA, 12 IIB), and 11 cases of stage III (9 IIIA, 2 IIIB). Regarding for the cancer tissue, p16 aberrant methylation was noted in 13 case of 33 cases (39.4%), DAP kinase in 21 cases of 35 cases (60%). Age over 55 year was associated with p16 aberrant methylation significantly (p<0.05). Methylation status of two genes was not different by smoking history, histologic type, size of tumor, lymph node metastasis and disease progression of lung cancer. There was no correlation between p16 and DAP kinase hypermethylation. Conclusion: This investigation demonstrates that aberrant methylation of p16 tumor suppressor gene or DAP kinase showed relatively high frequency (74.3%) in NSCLCs, and that these genes could be a biologic marker for early detection of lung cancer.

• Tuberculosis and Respiratory Diseases
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• v.45 no.3
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• pp.587-595
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• 1998

Background: Recombinant adenovirus hold promise as vectors to carry therapeutic genes for several reasons: 1) they can infect both dividing and non-dividing cells; 2) they have the ability to directly transduce tissues in vivo; 3) they can easily be produced in high titer; and 4) they have an established record of safety as vaccination material. However, one of the major limitation in the use of adenoviruses is that transgene expression is quite short because adenovirusees insert their DNA genome episomally rather than by chromosomal integration, and an immune response against the virus destroys cells expressing the therapeutic gene. Since sodium butyrate has been reported to induce adenovirus-mediated gene expression, we hypothesized that treatment of tumor cells, transduced with herpes simples virus thymidine kinase(HSVtk) gene using adenoviral vector, with butyrate could augment the effect of gene therapy. Methods: We transduced HSVtk gene, driven by the cytomegalovirus promoter, into REN cell line(human mesothelioma cell line). Before proceeding with the comparison of HSVtk/ganciclovir mediated bystander killing, we evaluated the effect of butyrate on the growth of tumor cells in order to rule out a potential antitumor effect of butyrate alone, and also on expression of HSVtk gene by Western blot analysis. Then we determined the effects of butyrate on bystander-mediated cell killing in vitro. Results: There was no inhibition of growth of cells exposed to butyrate for 24 hours at a concentration of 1.5mM/L. Toxic effects were seen when the concentration of butyrate was greater than 2.0mM/L. Gene expression was more stable and bystander effect was augmented by butyrate treatment of a concentration of 1.5mM/L. Conclusion: These results provide evidence that butyrate can augment the efficiency of cell killing with HSVtk/GCV system by inducing transgene expression and may thus by a promising new approach to improve responses in gene therapy using adenoviral vectors.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.203-207
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• 2004

The transgenic mice carrying human Interleukin-10 (hIL-10) gene in conjunction with bovine (3 -casein promoter express hIL-10 in milk during lactation. In this study, stability of germ line transmission and expression of hIL-10 transgene integrated into host chromosome were monitored up to generation F8 of transgenic mice. When male mouse of generation F8 was crossbred with normal females, approximately half of offspring (50.9±5.8%) were identified as transgenic mice. Generation F9 to F15 mice also showed similar transmission rates (66.0±20.1%, 61.5±16.7%, 41.1±8.4%, 40.7±20.3%, 61.3±10.8%, 49.2±18.8% and 43.8±25.9%, respectively), implying that hIL-10 transgene can be transmitted stably up to long term generation in the transgenic mice. Expression levels of human IL-10 from milk of generation F9 to F14 mice were 3.6± 1.2 mg/ml, 4.2±0.9 mg/ml, 5.7±1.5 mg/ml, 6.3±3.5 mg/ml, 6.8±4.5 mg/ml and 6.8±3.1 mg/ml, respectively, which was showed high-level expression compared with that of generation F1 (1.6 mg/ml) mice. In conclusion, our results suggest that transgenic mice can be continuously passed their transgenes to the progeny through the breeding program with the same productivity of human IL-10 protein in their milk.

• Korean Journal of Agricultural Science
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• v.28 no.2
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• pp.78-84
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• 2001

This study was analyzed by PCR technique with specific primer in order to investigate the characteristics of ${\beta}$-lactoglobulin (${\beta}$-LG) gene 5'flanking region in Korean native goat. This work confirmed amplified product of 1,077 bp fragments obtained from the amplification of ${\beta}$-LG promoter from genomic DNA using PCR in Korean native goat. The nucleotide sequence of ${\beta}$-LG gene 5'flanking region in Korean native goat as compared with that of sheep ${\beta}$-LG were different at 46 base of 897 nucleotides, and showed high homology as about 94.9% each breed. Especially we confirmed that the difference of nucleotide sequences between Korean native goat and sheep were consisted of $T{\rightarrow}C$ substitution and $C{\rightarrow}T$ substitution reversely. As a consequences, the sequences of ${\beta}$-LG gene 5'flanking region showed a high homology between Korean native goat and sheep. Furthermore we should be studied that relationships between the control of gene expression and nucleotide sequences of transcription factor in Korean native goat.

• Korean Journal of Microbiology
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• v.47 no.4
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• pp.281-288
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• 2011

To screen downstream genes of Aspergillus nidulans MsnA showing amino acid sequence similarity to the zinc finger region of Msn2/4 stress response transcription factors in Saccharomyces cerevisiae, differentially expressed genes (DEG) in MsnA overexpressed or msnA null mutant strains compared to wild type have been isolated. The cognate gene IDs were identified by DNA sequencing of the selected DEGs. Among those, DEG6 was known as mstB encoding a putative monosaccharide transporter. Expression level of mstB mRNA was increased in MsnA overproducing strains and MsnA bound directly to the promoter region of mstB in vitro. MstB containing twelve transmembrane domains exhibited 80% of amino acid sequence identities to A. niger MstA a high-affinity monosaccharide transporter. A null mutant of mstB was phenotypically undistinguishable to wild type. On the other hand, forced overexpression of MstB caused the increased formation of sexual structure cleistothecia in 0.1% glucose condition where wild type showed almost no cleistothecia. This result implies that mstB is involved in transport of monosaccharide required for sexual differentiation.

• BMB Reports
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• v.56 no.10
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• pp.563-568
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• 2023

DNA methylation regulates gene expression and contributes to tumorigenesis in the early stages of cancer. In colorectal cancer (CRC), CpG island methylator phenotype (CIMP) is recognized as a distinct subset that is associated with specific molecular and clinical features. In this study, we investigated the genome-wide DNA methylation patterns among patients with CRC. The methylation data of 1 unmatched normal, 142 adjacent normal, and 294 tumor samples were analyzed. We identified 40,003 differentially methylated positions with 6,933 (79.8%) hypermethylated and 16,145 (51.6%) hypomethylated probes in the genic region. Hypermethylated probes were predominantly found in promoter-like regions, CpG islands, and N shore sites; hypomethylated probes were enriched in open-sea regions. CRC tumors were categorized into three CIMP subgroups, with 90 (30.6%) in the CIMP-high (CIMP-H), 115 (39.1%) in the CIMP-low (CIMP-L), and 89 (30.3%) in the non-CIMP group. The CIMP-H group was associated with microsatellite instability-high tumors, hypermethylation of MLH1, older age, and right-sided tumors. Our results showed that genome-wide methylation analyses classified patients with CRC into three subgroups according to CIMP levels, with clinical and molecular features consistent with previous data.

• Journal of Plant Biotechnology
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• v.50
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• pp.56-62
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• 2023

Transgenic lilies have been obtained using Agrobacterium tumefaciens (AGL1) with the plant scale explants, followed by DL-phosphinothricin (PPT) selection. In this study, scales of lily plants cv. "red flame" were transformed with the pCAMBIA3301 vector containing the gus gene as a reporter and the blpR gene as a selectable marker, as well as a gene of interest showing herbicide tolerance, both driven by the CaMV 35S promoter. Using a 20-minute infection time and a 5-day cultivation period, factors that optimized and demonstrated a high transformation efficiency were achieved. With these conditions, approximately 22-27% efficiency was observed for Agrobacterium-mediated transformation in lilies. After transformation with Agrobacterium, scales of lilies were transferred to MS medium without selective agents for 2 weeks. They were then placed on selection MS medium containing 5 mg/L PPT for a month of further selection and then cultured for another 4-8 weeks with a 4-week subculture regime on the same selection medium. PPT-resistant scales with shoots were successfully rooted and regenerated into plantlets after transferring into hormone-free MS medium. Also, most survived putatively transformed plantlets indicated the presence of the blpR gene by PCR analysis and showed a blue color indicating expression of the gus gene. In conclusion, when 100 scales of lily cv. "red flame" are transformed with Agrobacterium, approximately 22-27 transgenic plantlets can be produced following an optimized protocol. Therefore, this protocol can contribute to the lily breeding program in the future.

• Korean Journal of Plant Tissue Culture
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• v.24 no.1
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• pp.1-35
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• 1997

Fertilized egg, by successive cell divisions, differentiates into different tissues and organs with various structures and functions. Different cells and tissues contain different proteins, products of selective gene expression. Not all the genes in any genomes are equally active, temporal and spatial gene expression being the general rule. Present paper attempts to review the tanscriptional mechanisms or the initiations of transcription from several angles. In some of the organisms the genes in the process of transcription or the genes in the inactive state can be seen under the light microscope. Some bands of Drosophila polytene chromosomes may exhibit a swollen or puff appearance under certain conditions. A puff, unfolded or decondensed form of chromomere, represents sets of intense transcriptional activity or RNA synthesis. The heterochromatic X chromosome whose genes remain inactive in the female mammals can be visualized as a dark staining structure called Barr body, Configuration of chromatin differs between transcribed and nontranscribed chromatin. Modification to the chromatin facilitates RNA synthesis. The movement of large polymerase molecule along the DNA would probably be facilitated if some modifications of the chromatin configuration is effected. Methylation of cytosines in CG sequences is associated with inactive genes. Methylation can play a role in determination of mammalian cells during embryogenesis. Demethylation is necessary for the gene to be expressed during development A histone modification that is also known to be correlated with transcriptional capacity of chromatin is acetylation of the lysine residues of the core histones. Chromatin containing a high level of histone acetylation is very sensitive to DNase 1. For the transcription to occur TBP must first bind to the TATA box. Another TF, TF IIB, then binds to the promoter-TBP complex, facilitating the access of RNA polymerase to the transcription initiation site. As recently as eight years ago researchers assumed that histones were irrelevant to the regulation of gene expression. Histones combine with the DNA to form nucleosome of the chromatin. Histones are vital participant in gene regulation. Histone and basal factors compete for access to TATA box. When DNA is exposed to basal factors before histones are introduced, the basal factors assemble on TATA boxes preventing the access of histones, allowing transcription to occur, for transcription to begin, activator protein at the upstream activation sequence or enhancer must interact with the tail of histone H4 at TATA box and cause the histone role particle to dissociate from the TATA box leading to partial breakup of the histone core particle and allowing the basal factors to bind to the TATA box. New concept of genomic flux in contrast to the old concept of static genome has been developed based on the powerful new molecular techniques. Genomic changes such as repetitive DNAs and transposable elements, it is assumed but not yet proved, may affect some of the developmental patterns that characterize particular cells, tissues, organs, and organisms. In the last decade or so remarkable achievement have been made in the researches of the structures and functions of TFs and the specific target sequences located in promoters or enhancers where these TFs bind. TFs have independent domains that bind DNA and that activate transcription. DNA binding domain of TFs serves to bring the protein into the right location. There are many types of DNA binding domains. Common types of motifs can be found that are responsible for binding to DNA. The motifs are usually quite short and comprise only a small part of the protein structure. Steroid receptors have domains for hormone binding, DNA binding, and activating transcription. The zinc finger motif comprises a DNA binding domain. Leucine zipper consist of a stretch of amino acids with a leucine residue in every seventh position Two proteins form a dimer because they interact by means of leucine zippers on similar α-helical domain. This positions their DNA binding basic domains for interaction with the two halves of a DNA sequence with dyad symmetry of TGACTCA, ACTGAGT.

• Journal of Life Science
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• v.21 no.8
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• pp.1134-1141
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• 2011

Transmembrane protein 21 (TMP21) is a member of the p24 cargo protein family and has been shown to modulate ${\alpha}$-secretase-mediated A${\beta}$ production which was specifically observed in the brains of subjects with Alzheimer's disease (AD). In order to investigate whether TMP21 could affect nerve growth factor (NGF) receptor signaling pathway, the alteration of NGF receptors and their downstream proteins were detected in TMP21 over-expressed cells. CMV/hTMP21 vector used in this study was successfully expressed into TMP21 proteins in B35 cells after lipofectamin transfection. Expressed TMP21 proteins induced the down-regulation of ${\gamma}$-secretase complex components including Presenlin-1 (PS-1), PS-2, Nicastrin (NST), Pen-2 and APH-1. Also, the expression level of NGF receptor $p75^{NTR}$ and RhoA were significantly higher in CMV/hTMP21 transfectants than vehicle transfectants, while their levels returned to vehicle levels after NGF treatment. However, the phosphorylation of NGF receptor TrkA was dramtically decreased in NGF No-treated CMV/hTMP21 transfectants compared with vehicle transfectants, and increased in NGF treated CMV/hTMP21 transfectants. In TrkA downstream signaling pathway, the phosphorylation level of ERK was also decreased in CMV/hTMP21 transfectants, while the phosphorylation of Akt was increased in the same transfectants. Furthermore, NGF treatment induced the increase of phosphorylation level of Akt and ERK in CMV/hTMP21 transfectants. Therefore, these results suggested that over-expression of TMP21may simultaneously induce the up-regulation of $p75^{NTR}$/RhoA expression and the down-regulation of TrkA/ERK phosphorylation through the inhibition of ${\gamma}$-secretase activity.

• Journal of Life Science
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• v.23 no.11
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• pp.1317-1324
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• 2013

High-molecular weight glutenin subunits (HMW-GS) have been shown to play a crucial role in determining the processing properties of the wheat grain. We have produced marker-free transgenic rice plants containing a wheat Glu-1Bx7 gene encoding the HMG-GS from the Korean wheat cultivar 'Jokyeong' using the Agrobacterium-mediated co-transformation method. The Glu-1Bx7-own promoter was inserted into a binary vector for seed-specific expression of the Glu-1Bx7 gene. Two expression cassettes comprised of separate DNA fragments containing only Glu-1Bx7 and hygromycin phosphotransferase II (HPTII) resistance genes were introduced separately to the Agrobacterium tumefaciens EHA105 strain for co-infection. Each EHA105 strain harboring Glu-1Bx7 or HPTII was infected to rice calli at a 3:1 ratio of Glu-1Bx7 and HPTII, respectively. Then, among 216 hygromycin-resistant $T_0$ plants, we obtained 24 transgenic lines with both Glu-1Bx7 and HPTII genes inserted into the rice genome. We reconfirmed integration of the Glu-1Bx7 gene into the rice genome by Southern blot analysis. Transcripts and proteins of the wheat Glu-1Bx7 were stably expressed in the rice $T_1$ seeds. Finally, the marker-free plants harboring only the Glu-1Bx7 gene were successfully screened at the $T_1$ generation.