• 한국미생물·생명공학회지
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• 제19권1호
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• pp.45-51
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• 1991

$cI_{857}$ repressor 단백질을 대량으로 얻기위해 tac promoter 하류에 $cI_{857}$ 구조 유전자를 삽입하는 것을 검토하였다. $cI_{857}$ 유전자를 포함하는 DNA 단편을 plasmid PUC12를 이용하여 대량생산후, HphI으로 부분 분해하여 $cI_{857}$ 구조 유전자만을 취하고, tac promoter 하류에 삽입시켰다. 그리고 $\lambda$ phage $cI_{90}$에 의해 $30^{\circ}C$에서는 용원성을, $42^{\circ}C$에서는 용균성을 보이는 균주를 선택함으로 tac promoter 하류에 cI857 구조 유전자가 삽입된 pDR540-$cI_{857}$을 선택할 수가 있었다. 이 plasmid는 $lacI^q$ JM103 뿐만 아니라 각종 E.coli에서 $cI_{857}$-repressor 단백질을 균체 단백질당 약 17까지 생산하였다.

• Journal of Microbiology and Biotechnology
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• 제10권3호
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• pp.287-292
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• 2000

Recombinant Aspergillus niger was constructed to express and secrete a biologically active murine granulaocyte macrophage-colony stimulating factor (mGM-CSF). A 500 bp fragment encoding the signal peptide and terminator of glyceraldehyde-3-phosphate dehydrogenase (gpd). The hygromycin phosphotrasferase gene (hph) was used as a selection marker for the fungal transformants. An expression vector was introduced into A. niger ATCC 9642, and a Northern blot analysis indicated the presence of a considerable amount of transcripts from the introduced mGM-CSF. The biological activity of recombinant mGM-CSF (rmGM-CSF) isolated from the culture filtrate was confirmend by measuring the proliferationof the GM-CSF dependent FDC-P1 cell line. It appeared that rmGM-CSF was amenable to the proteolytic activity produced by A. niger, since biological actibity was only observed when the transformants were grown in a protease-repressing medium, and the activity of rmGM-CSF dramatically decreased with an increase of age of the culture. The yield of rmGM-CSF, as determined by ELISA. was 640 ng/l of culture filtrate. Accordingly, its specific activity is estimated to be approximately two-and-a-half times higher than that of a commercial preparation from E. coli.

• 한방재활의학과학회지
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• 제30권1호
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• pp.1-12
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• 2020

Objectives Depletion of ovarian function after menopause in women induces estrogen deficiency leading to increased fat and decreased muscle mass. In this study, we examined the effect of herbal medicines by measuring hormone expression in muscle tissue of estrogen-deficient rats induced by ovariectomy. Methods Ovariectomy was performed to induce estrogen deficiency, and mice were given herbal prescription (HP) for 6 weeks. Estrogen-deficient rats were divided into two groups: one group (HPH) which were orally administered HP 200 mg/kg and the other group (HPL) administered HP 40 mg/kg. Weight changes in both groups were measured using polymerase chain reaction (PCR). After extraction of the femoral muscles in mice, the expression of the leptin, lipoprotein lipase (LPL), diacyl glycerol acyltransferase (DGAT)1, peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, NADH dehydrogenase (NDH), farnesyl diphosphate farnesyltransferase (FDFT)1, lanosterol synthase (LSS), phosphatidylethanolamine N-methyltransferase (PEMT), and peroxiredoxin (Prdx6) were measured using PCR. Results HP increased the expression of leptin, LPL, DGAT1, PGC-1α, NDH, FDFT1, LSS, PEMT, and Prdx6. HP affects body fat metabolism and is effective in improving menopausal obesity and obesity complications caused by estrogen deficiency. However, HP does not affect the expression of tumor necrosis factor-α and 3-hydroxy-3-methylglutaryl-CoA reductase, and thus will not be effective in obesity-related metabolic diseases. Conclusions HP is thought to inhibit weight gain by regulating hormone expression related to glucose metabolism and lipid metabolism in muscle tissue of estrogen-deficient rats.

• KSBB Journal
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• 제13권2호
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• pp.214-219
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• 1998

Polymerase chain reaction(PCR) was conducted to obtain the gene encoding glucose oxidase(GOD) from Aspergillus niger(ATCC 2110) and the DNA sequence determined was coincided with published GOD sequence from A. niger. Recombinant transforming vector containing GOD and hygromycin B(hyg.B) resistant gene(hph) was constructed and used for further transformation of A. niger ATCC 2110. Selectivity of hyg.B against A. niger differed depending on which media were used i.e., nutrient-rich media such as potato dextrose agar(PDA) and complete medium(CM) showed only 50% growth inhibition at 400 $\mu$m ml$^-1$ of hyg.B while the minimal media inhibited mycelial growth completely at 200 $\mu$m ml$^-1$ of hyg.B. Twenty to sixty putative transformants were isolated from the hyg.B-containing minimal top agar, transferred successively onto alternating selective and nonselective media for a mitotic stability of hyg.B resistance and, then, single-spored. Among the stable transformants, the transformant(GOD1-6) grown by flask culture showed the considerable increase of extracellular GOD activity, which was estimated to the degree of 50% - 100% comparing to that of wild type. Transformation of tGOD1-6 was resulted from integration of the vectors into heterologous as well as homologous regions of the A. niger genome. Southern blot analysis revealed that there were two independent integrations of vector into fungal genome and one into the GOD gene due to homologous recombination. In addition, GOD activity and sodium gluconate production when tGOD1-6 was fed-batch fermented were enhanced 11 fold and 2.25 fold, respectively, compared to that of the wild type.

• 한국균학회지
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• 제38권1호
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• pp.48-53
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• 2010

팽이버섯의 다양한 유전자의 기능을 연구하기 위한 방법으로 팽이 자실체의 주름조직을 사용하여 A. tumefaciens을 매개로 한 형질전환을 수행하였다. Hygromycin 항생제 저항성 유전자를 포함하고 있는 AGL-1 pBGgHg를 팽이버섯의 자실체 조직에 도입시킴으로서 형질전환체들을 얻을 수 있었다. 형질전환체 선발은 첫 번째로 hygromycin 선발 배지에서 선발한 뒤 gpd-FH, hph-R primer를 이용하여 PCR로 확인 할 수 있었다. hygromycin은 30 ug/ml에서 선발 효율이 가장 높았다. 형질전환체 중 일부는 갈색 자실체를 갖는 wild type과는 달리 백색 자실체를 나타내었다. 형질전환체의 southern blot결과 외래유전자가 팽이버섯 내부에 하나 또는 그 이상의 다양한 copy 수로 삽입되어 있음을 알 수 있었다. 또한 삽입된 유전자의 주변 염기서열에 대한 정보는 Inverse PCR을 통해 알 수 있었다.

• 식물조직배양학회지
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• 제26권3호
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• pp.219-222
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• 1999

부정아를 유도하기 위하여 발아 7∼10일된 일본 결구상추 2품종과 국내 잎상추 (Lactuca sativa L.)4품종의 하배축 및 자엽절편으로부터 식물체 재분화시스템을 확립하였다. 하배축 및 자엽절편을 BA와 NAA가 조합 처리된 MS 및 SH배지에 치상하여 5주 동안 명배양하였다. 일반적으로 하배축보다는 자엽절편에서, SH 배지보다는 MS 배지에서 배양하였을 때에 부정아 형성률이 높았다. MS배지에 BA는 0.5 ㎎/L, NAA는 0.1∼l ㎎/L로 처리하였을 때에 최고의 부정아 형성률을 보였으며, 품종에 따라 30% ( '만추대청치마')에서 86% 까지의 부정아 형성률을 나타내었다. 형성된 shoot의 95% 이상이 MS 기본배지에서 발근하여 소식물체로 발달하였다.

• Journal of Plant Biotechnology
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• 제5권3호
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• pp.163-168
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• 2003

A routine system based on particle bombardment of embryogenic callus for recovery of fertile transgenic rice (Oryza sativa L.) plants was developed. Embryogenic callus was established within 2-3 months from calli derived from mature seeds of Korean rice cultivar, Nagdongbyeo. The callus was bombarded with the plasmid pRQ6 containing the $\beta$-glucuronidase gene (gusA) and hygromycin phosphotransferase gene (hph, conferring resistance to hygromycin B), both driven by CaMV 35S promoter. Placement of cells on an osmoticum-containing medium (0.2 M sorbitol and 0.2 M mannitol) 4 hrs prior to and 16 hrs after bombardment resulted in a statistically significant increase with 3.2-fold in transient expression frequency gusA. In five independent experiments, the average frequency of transformation showing GUS activities was $8.86\%$. A large number of morphologically normal, fertile transgenic rice plants were obtained. Integration of foreign gene into the genome of $R_0$ transgenic plants was confirmed by Southern blot analysis. GUS and HPT were detected in $R_1$ progeny and Mendelian segregation of these genes was observed in $R_1$ progeny.

• 한국환경보건학회지
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• 제42권6호
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• pp.385-395
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• 2016

Objectives: The aim of this study was to assess exposure to VOCs and PAHs and the health effects on volunteers who participated in an oil spill cleanup in Yeosu. Methods: Atmospheric VOCs were evaluated in the vicinity of the accident site and questionnaire surveys were conducted to identify personal characteristics and acute health symptoms of clean-up workers seven days after the accident. The levels of metabolites of VOCs (t,t-MA, HA, PGA, MA, MHA) and PAHs (2-NAP, 1-OHP, 2-HF, 1-HPH), oxidative stress markers (TABARS, 8-OHdG) in the urine of workers were analyzed. Their correlation was determined by multiple regression analysis with SAS ver. 9.4. Results: Although the concentration of atmospheric VOCs in the residential areas were low at the time of survey, the levels of VOCs and PAHs metabolites in clean-up workers were higher than those in the control group after clean-up activities. The levels of urinary VOC and PAH metabolites were significantly increased after clean-up compared to those measured before participation. The thiobarbituric acid reactive substance (TBARS) concentrations were also increased and showed significant correlations with those of metabolites of benzene. Conclusion: This study shows that oil spill clean-up activities affect exposure to VOCs and PAHs and the health of clean-up workers. The results suggest the need for check-ups of participants in oil spill cleaning work.

• Biomolecules & Therapeutics
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• 제24권1호
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• pp.85-93
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• 2016

We already reported that genetically engineered resveratrol-enriched rice (RR) showed to down-regulate skin melanogenesis. To be developed to increase the bioactivity of RR using calli from plants, RR was adopted for mass production using plant tissue culture technologies. In addition, high-pressure homogenization (HPH) was used to increase the biocompatibility and penetration of the calli from RR into the skin. We aimed to develop anti-melanogenic agents incorporating calli of RR (cRR) and nanoparticles by high-pressure homogenization, examining the synergistic effects on the inhibition of UVB-induced hyperpigmentation. Depigmentation was observed following topical application of micro-cRR, nano-calli of normal rice (cNR), and nano-cRR to ultraviolet B (UVB)-stimulated hyperpigmented guinea pig dorsal skin. Colorimetric analysis, tyrosinase immunostaining, and Fontana-Masson staining for UVB-promoted melanin were performed. Nano-cRR inhibited changes in the melanin color index caused by UVB-promoted hyperpigmentation, and demonstrated stronger anti-melanogenic potential than micro-cRR. In epidermal skin, nano-cRR repressed UVB-promoted melanin granules, thereby suppressing hyperpigmentation. The UVB-enhanced, highly expressed tyrosinase in the basal layer of the epidermis was inhibited by nano-cRR more prominently than by micro-cRR and nano-cNR. The anti-melanogenic potency of nano-cRR also depended on pH and particle size. Nano-cRR shows promising potential to regulate skin pigmentation following UVB exposure.

• Mycobiology
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• 제49권5호
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• pp.491-497
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• 2021

An endolichenic fungus Xylaria grammica EL000614 produces grammicin, a potent nematicidal pyrone derivative that can serve as a new control option for root-knot nematodes. We optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for X. grammica to support genetic studies. Transformants were successfully generated after co-cultivation of homogenized young mycelia of X. grammica with A. tumefaciens strain AGL-1 carrying a binary vector that contains the bacterial hygromycin B phosphotransferase (hph) gene and the eGFP gene in T-DNA. The resulting transformants were mitotically stable, and PCR analysis showed the integratin of both genes in the genome of transformants. Expression of eGFP was confirmed via fluorescence microscopy. Southern analysis showed that 131 (78.9%) out of 166 transformants contained a single T-DNA insertion. Crucial factors for producing predominantly single T-DNA transformants include 48 h of co-cultivation, pretreatment of A. tumefaciens cells with acetosyringone before co-cultivation, and using freshly prepared mycelia. The established ATMT protocol offers an efficient tool for random insertional mutagenesis and gene transfer in studying the biology and ecology of X. grammica.