Background: Efficient gene editing technology is needed for successful knock-in. Homologous recombination (HR) is a major double-strand break repair pathway that can be utilized for accurately inserting foreign genes into the genome. HR occurs during the S/G2 phase, and the DNA mismatch repair (MMR) pathway is inextricably linked to HR to maintain HR fidelity. This study was conducted to investigate the effect of inhibiting MMR-related genes using CdCl2, an MMR-related gene inhibitor, on HR efficiency in HC11 cells. Methods: The mRNA and protein expression levels of MMR-related genes (Msh2, Msh3, Msh6, Mlh1, Pms2), the HR-related gene Rad51, and the NHEJ-related gene DNA Ligase IV were assessed in HC11 cells treated with 10 μM of CdCl2 for 48 hours. In addition, HC11 cells were transfected with a CRISPR/sgRNA expression vector and a knock-in vector targeting Exon3 of the mouse-beta casein locus, and treated with 10 μM cadmium for 48 hours. The knock-in efficiency was monitored through PCR. Results: The treatment of HC11 cells with a high-dose of CdCl2 decreased the mRNA expression of the HR-related gene Rad51 in HC11 cells. In addition, the inhibition of MMR-related genes through CdCl2 treatment did not lead to an increase in knock-in efficiency. Conclusions: The inhibition of MMR-related gene expression through high-dose CdCl2 treatment reduces the expression of the HR-related gene Rad51, which is active during recombination. Therefore, it was determined that CdCl2 is an inappropriate compound for improving HR efficiency.

Increasing the efficiency of HR (homologous recombination) is important for a successful knock-in. Rad51 is mainly involved in homologous recombination and is associated with strand invasion. The HR-related mismatch repair system maintains HR fidelity by heteroduplex rejection and repair. Therefore, the purpose of this study is to control Rad51, which plays a critical role in HR, through UV-induced DNA damage. It is also to confirm the effect on the expression of MMR related genes (Msh2, Msh3, Msh6, Mlh1, Pms2) and HR-related genes closely related to HR through treatment with the MMR inhibitor CdCl2. The mRNA expression of Rad51 gene was confirmed in both HC11 cells and mouse testes, but the mRNA expression of Dmc1 gene was confirmed only in mouse testes. The protein expression of Rad51 and Dmc1 gene increased in UV-irradiated HC11 cells. After 72 hours of treatment with 1 μm of CdCl2, the mRNA expression level of Msh3, Pms2, and Rad51 decreased, but the mRNA expression level of Msh6 and Mlh1 increased in HC11 cells. There was no significant difference in Msh2 mRNA expression between CdCl2 untreated-group and the 72 hours treated group. In conclusion, HR-related gene (Rad51) was increased by UV-induced DNA damage. Treatment of the MMR inhibitor CdCl2 in HC11 cells decreased the mRNA expression of Rad51.

A gram-positive bacterium was isolated from the spent substrate of Agaricus bisporus that showed a marked antagonistic activity against Pseudomonas agarici. It was identified as Bacillus safensis HC42 based on its cultural, biochemical, and physiological characteristics, and 16S rRNA sequence. B. safensis HC42 was saprophytic, but not parasitic or pathogenic, in cultivated mushrooms and showed strong inhibition of P. agarici in vitro. Moreover, it showed a control efficacy of 66 % against browning disease caused by P. agarici in Agaricus bisporus. Therefore, B. safensis HC42 may be useful in the future for the development of a biocontrol system.

This study was conducted to investigate optimum conditions for mass production of ntagonistic microbes Alcaligenes sp. HC12. Alcaligenes sp. HC12 had a potent biological control agent to control browning disease caused by Pseudomonas agarici. Alcaligenes sp. HC12 markedly showed the antagonistic activity against Pseudomonas agarici, the most destructive pathogen of cultivated mushrooms. To define the optimum conditions for the mass production of the Alcaligenes sp. HC12, we have investigated optimum culture conditions and effects of various nutrient source on the bacterial growth. The optimum initial pH and temperature were determined as pH 9.0 and 30o, respectively. The optimal concentration of medium elements for the growth of pathogen inhibitor bacterium(Alcaligenes sp. HC12) was determined as follows: 0.5% dextrine, 1.5% yest extract, 1.0% NaNO3, 0.5% KH2PO4, and 1.5% asparagine.

The button mushroom, Agaricus bisporus, is one of the major economical crops cultivated in Korea. This mushroom showed the 5th production to 11,493 M/T in 2014. Several fungus are known as the causal agents of diseases of the cultivated button mushroom (Agaricus bisporus) and oyster mushroom (Pleurotus ostreatus). Cladobotryum mycophilum is the causal agent of cobweb disease of commercial mushrooms. Early symptoms were noticed as round, fleshy, yellowish brown lesions on mushroom caps. Late symptoms progressed when the parasitic fungus formed white cobweb circular colonies on dead or damaged pinheads, spread on the surface of the casing, and covered entirely fruiting bodies. A Gram-positive bacterium was isolated from mushroom media that markedly showed the antagonistic activity against Cladobotryum mycophilum, the most destructive pathogen of cultivated mushrooms. The HC57 strain was selected as antagonistic bacterium by inhibition zone method and it was identified as Bacillus subtilis by the cultural, morphological and physiological characteristics, and analysis of the 16S rDNA. The isolated bacterium is saprophytic but not parasitic nor pathogenic to cultivation mushroom. The isolated bacterium for Cladobotryum mycophilum cell, was sufficient for inhibition in vitro. Inoculation of the isolated bacterium prevents the development of bacterial disease in Cladobotryum mycophilum. Control efficacy of browning disease of strain HC57 treatment was 71% on Agaricus bisporus. The optimal culture medium for the antagonistic bacteria growth was determined as follows: 1.5% Xylose, 2% Soytone, 1% NH4H2PO4, 7 mmol CaCl2, and 0.5% Histidine at pH 6.0 at 25℃. The suppressive bacterium may be useful in future for the development of biocontrol system and the construction of genetically modified edible fungi resistant to the disease caused by Cladobotryum mycophilum.

버섯 세균갈성색무늬병원균인 Pseudomonas tolaasii에 대한 길항미생물로 보고된 Pseudomonas azotoformans HC5 균주의 배양적 특성과 대량 배양을 위한 최적배양조건을 설정하였다. HC5 균주의 생육온도는 10~20oC, pH는 6.0~9.0의 범위에서 왕성한 생육을 보였다. 대량배양을 위한 효율적인 영양원 선발을 위하여 기본배지에 탄소원 fructose 등 18종, 무기질소원 NH4Cl 등 6종, 유기질소원 peptone 등 6종 그리고 아미노산 asparagine 등 11종을 각각 1%씩 첨가하였고, 무기염류 13종을 1 mM 농도로 첨가하여 각각에 대한 생육에 미치는 영향을 조사하였다. 또한 선발된 각각의 영양성분들에 대한 최적 농도를 조사하기 위하여 각각의 성분을 최소 0.1%에서 최대 4.0%까지 배지에 첨가하여 배양 후 생육정도를 조사하였다. 그 결과, 대량배양을 위한 생육최적조건은 온도 15oC, pH 6, 탄소원 0.6% adonitol, 유기질소원 1.5% yeast extract, 무기질소원 0.8% NH4H2PO4,아미노산 0.2% asparagine 그리고 무기염류는 5 mM MgSO4에서 왕성한 생육을 보였다.

The button mushroom, Agaricus bisporus, is one of the major economical crops cultivated in Korea. This mushroom showed the 5th production to 10,996 M/T in 2012. Several fungus are known as the causal agents of diseases of the cultivated button mushroom (Agaricus bisporus) and oyster mushroom (Pleurotus ostreatus). Cladobotryum mycophilum is the causal agent of cobweb disease of commercial mushrooms. Early symptoms were noticed as round, fleshy, yellowish brown lesions on mushroom caps. Late symptoms progressed when the parasitic fungus formed white cobweb circular colonies on dead or damaged pinheads, spread on the surface of the casing, and covered entirely fruiting bodies. A Gram-positive bacterium was isolated from mushroom media that markedly showed the antagonistic activity against Cladobotryum mycophilum, the most destructive pathogen of cultivated mushrooms. The HC7 strain was selected as antagonistic bacterium by inhibition zone method and it was identified as Bacillus altitudinis. by the cultural, morphological and physiological characteristics, and analysis of the 16S rDNA. The isolated bacterium is saprophytic but not parasitic nor pathogenic to cultivation mushroom. The isolated bacterium for Cladobotryum mycophilum cell, was sufficient for inhibition in vitro. Inoculation of the isolated bacterium prevents the development of bacterial disease in Cladobotryum mycophilum. Control efficacy of browning disease of strain HC7 treatment was 78% on Agaricus bisporus. The optimal culture medium for the antagonistic bacteria growth was determined as follows: 3% Soluble startch, 10% Soytone, 1% (NH4)2HPO4, 1 mmol KCl, and 0.5% L-asparagin at pH 6.0 at 30°C. The suppressive bacterium may be useful in future for the development of biocontrol system and the construction of genetically modified edible fungi resistant to the disease caused by Cladobotryum mycophilum.

The button mushroom, Agaricus bisporus, is one of the major economical crops cultivated in Korea. This mushroom showed the 5th production to 10,996 M/T in 2012. Several bacteria are known as the causal agents of diseases of the cultivated button mushroom (Agaricus bisporus) and oyster mushroom (Pleurotus ostreatus). Pseudomonas agarici is the causal agent of browning disease of commercial mushrooms. Colonization of mushroom caps by the bacterium results in development of browning lesions on pileus. These lesions are superficial brown spots and can be round or spreading. But P. agarici never caused sunken lesions and rotting of the mushroom tissues. A Gram-positive bacterium was isolated from mushroom media that markedly showed the antagonistic activity against Pseudomonas agarici, the most destructive pathogen of cultivated mushrooms. The HC42 strain was selected as antagonistic bacterium by inhibition zone method and it was identified as Bacillus safensis. by the cultural, morphological and physiological characteristics, and analysis of the 16S rDNA.. The isolated bacterium is saprophytic but not parasitic nor pathogenic to cultivation mushroom. The isolated bacterium for P. agarici cell, was sufficient for inhibition in vitro. Inoculation of the isolated bacterium prevents the development of bacterial disease in Agaricus bisporus. Control efficacy of browning disease of strain HC42 treatment was 66% on Agaricus bisporus. The optimal culture medium for the antagonistic bacteria growth was determined as follows: 1.5% D-galactose, 1.5% yest extract, 1% NH4Cl, 1.5% KCl, and 1.0% L-asparagin at pH 6.0 at 25℃. The suppressive bacterium may be useful in future for the development of biocontrol system and the construction of genetically modified edible fungi resistant to the disease caused by P. agarici.

버섯 세균갈성색무늬병원균인 Pseudomonas tolaasii에대한 독소저해균으로 보고된 Pseudomonas sp. HC1 균주의 배양적 특성과 최적 배양을 위한 대량배지를 선발하였다. CH1균주의 생육온도는 20~40oC, pH는 5.0~11.0까지넓은 범위에서 왕성한 생육을 보였다. 대량배양을 위한효율적인 영양원 선발을 위하여 기본배지에 탄소원fructose 등 18종, 무기질소원 NH4Cl 등 6종, 유기질소원peptone 등 6종 그리고 아미노산 asparagine 등 11종을 각각 1%씩 첨가하였고, 무기염류 13종을 1mM 농도로 첨가하여 각각에 대한 생육에 미치는 영향을 조사하였다.또한 선발된 각각의 영양성분들에 대한 최적 농도를 조사하기 위하여 각각의 성분을 0.1%에서 4.0%까지 배지에첨가하여 배양후 생육정도를 조사하였다. 그 결과, 대량배양을 위한 생육최적조건은 온도 20oC, pH5, 탄소원 0.9%dextrine, 유기질소원 1.5% yeast extract, 무기질소원0.5% (NH4)2HPO4, 아미노산 3.0% cysteine, 무기염류4mM FeCl3에서 왕성한 생육을 보였다.

Mushroom is cultivated as one of the major economical crops in many areas in Korea. The total production has steadily increased from approximately 186,400 M/T in 2007 to 190,111 M/T in 2011. The button mushroom, Agaricus bisporus, showed the 5th production to 13,052 M/T in 2011. Several bacteria are known as the causal agents of diseases of the cultivated button mushroom (Agaricus bisporus) and oyster mushroom (Pleurotus ostreatus). Pseudomonas agarici is the causal agent of browning disease of commercial mushrooms. Colonization of mushroom caps by the bacterium results in development of browning lesions on pileus. These lesions are superficial brown spots and can be round or spreading. But P. agarici never caused sunken lesions and rotting of the mushroom tissues. Antagonists against P. tolaasii, HC42 were selected and their control efficacy of browning disease was investigated in this study. Antagonists against P. agarici, HC42 were selected and their control efficacy of browning disease was investigated in this study. After proceeding antagonistic test, HC42 was selected as a strong antagonist against P. agarici and the HC42 strain was identified as P. safensis with the cultural, physiological and biochemical properties and analysis of the 16S rRNA. The optimal culture medium for the antagonistic bacteria growth was determined as follows: 1.5% D-galactose, 1.5% yest extract, 1% NH4Cl, 1.5% KCl, and 1.0% L-asparagin at pH 6.0 at 25℃. Control efficacy of browning disease by HC42 treatment was 66% on Agaricus bisporus..

The total production has steadily increased from approximately 186,400 M/T in 2007 to 198,563 M/T in 2009. Winter mushroom, Flammulina velutipes, with 61,057913 M/T in 2009, showed the highest production. Several bacteria are known as the causal agents of diseases of the cultivated button mushroom (Agaricus bisporus) and oyster mushroom (Pleurotus ostreatus). Pseudomonas tolaasii is the causal agent of brown blotch disease of commercial mushrooms. Antagonists against P. tolaasii, HC5 were selected and their control efficacy of brown blotch disease was investigated in this study. After proceeding antagonistic test, HC5 was selected as a strong antagonist against P. tolaasii and the HC5 strain was identified as P. azotoformans with the cultural, physiological and biochemical properties and analysis of the 16S rRNA. Control efficacy of brown blotch disease by HC5 treatment was 73% on Agaricus bisporus, 78% on Flammulina velutipes and 71% on pleurotus ostreatus respectively.

버섯에서 가장 문제가 되고 있는 세균성갈반병은 주로 Pseudomonas tolaasii에 의해 발생한다. 병징은 초기에 버섯 갓의 표면에 황갈색의 점무늬가 생기고 점차 진갈색의 불규칙한 큰 병반으로 확대된다. 발병이 심하면 버섯 표면은 점액성을 띄고 부패한다. 어린 버섯에 감염되면 뚜렷한 갈색 점무늬가 생기고 아주 어린 자실체는 전체가 갈변 부패한다. 이 병원균은 톨라신(tolaasin)이라는 독소를 분비하여 버섯의 세포막을 파괴하며, 세포막이 파괴되면서 분비되는 물질이 산화되어 갈색으로 변색되고, 세포의 파괴가 심하면 그 부분이 움푹 파이는 증상을 보이게 된다. 따라서 P. tolaasii에 의해 생성되는 톨라신을 분해하는 균을 선발하여 방제에 이용하고자 실험을 하였다. P. tolaasii가 생성하는 독소를 효과적으로 분해하는 세균들을 버섯배지로부터 분리하여 16s rDNA로 동정한 결과 Pseudomonas otitidis, Ochrobactrum anthropi, Bacillus safensis, Bacillus altitudinis 등으로 동정되었다. 이들 중 가장 효과가 좋은 Pseudomonas otitidis을 선택하여 독소분해 정도 및 배양특성을 조사하였다. 균의 최적 배양온도는 30℃였고, pH5에서 생육이 가장 좋았다. 그리고 탄소원Dextrine을 가장 잘 이용하였고, 질소원은 Yeast extract, 아미노산은 Arginine을 가장 잘 이용하였으며, 톨라신 분해효과도 높았다.

우리나라의 버섯 생산량은 매년 조금씩 증가하고 있으며, 버섯 생산의 안전성을 저해하는 여러 가지 요인에 의해 농가의 피해는 증가되고 있는 추세이다. 그 중에서도 세균성 병원균에 의한 갈반병은 수확량 감소 및 품질 저하로 커다란 손실을 초래하고 있다. 양송이버섯에 발생하는 세균성병해 중 Pseudomonas tolaasii는 세균성갈반병을 일으키고, Psudomonas agarici는 세균성갈색무늬병을 일으키는 것으로 알려져 있다. 양송이의 세균성갈색무늬병은 Pseudomonas agarici에 의해 일어나며, 갓 표면의 갈변증상에 의해 상품가치의 하락을 초래하여 재배농가에 많은 피해를 주고 있다. 따라서 P. agarici에 대한 길항균을 선발하여 균의 동정 및 배양특성을 조사한 결과를 보고하고자 한다. 길항미생물은 버섯배지로부터 분리한 1800균주 중 디스크 확산 법을 통하여 길항성이 높은 8개의 균주를 선발하여 16s rDNA로 동정한 결과 Alcaligenes faecalis, Enterococcus mundtii, Bacillus safensis 등으로 동정되었다. 이들 중 길항성이 가장 높은 Alcaligenes faecalis을 선택하여 배양특성을 조사한 결과 pH9와 30℃에서 생육이 가장 좋았으며, 탄소원은 Dextrose에서 가장 생육이 좋았고, 질소원은 Yeast extract에서 아미노산은 Proline에서 생육이 가장 좋았으며, 길항성도 높은 것으로 나타났다.

우리나라 버섯재배 농가에 심각한 피해를 주는 세균성갈반병은 Pseudomonas tolaasii에 의해 발생되며 초기에는 버섯 갓의 표면에 황갈색의 점무늬가 생기고 점차 진갈색의 불규칙 한 큰 병반으로 확대되어 심하면 표면이 점액성을 띄고 부패한다. 본 연구에서는 Pseudomonas tolaasii에 대하여 길항미생물을 선발하고 길항미생물의 대량배양 조건을 설정하기위하여 최적 pH, 배양온도, 탄소원, 질소원 및 아미노산의 이용성에 대한 실험을 수행하였다. P. tolaasii에 대한 길항균은 버섯 배지로부터 분리한 1800균주 중 길항성이 좋은 3균주를 선발하여 16s rDNA로 동정결과 Pseudomonas azotoformans, Stenotrophomonas maltophilia, Pseudomonas otitidis로 동정되었다. 이들 선발길항균 중 길항성이 가장 좋은 Pseudomonas azotoformans균주를 선택하여 최적배양조건을 조사한 결과 pH6과 20℃에서 가장 생육이 좋았고, 병원균에 대해 강한 억제력을 나타내었다. 탄소원의 선발에서는 Dextrine, Adonitol에서 균생육이 가장 좋았고, 질소원에서는 Soytone, Tryptone, Yeast extract에서 생육과 길항성이 좋았으며 아미노산은 Cysteine에서 가장 생육이 좋았다.

Amino acid transporters play an important role in supplying organic nutrient to cells. The expression profile of L-type amino acid transporter 1 (LAT1) and its subunit 4F2 heavy chain (4F2hc) on different differentiation stages in 4-NQO induced rat tongue carcinogenesis was examined using immunohistochemical analysis. The gradually increasing LAT1 and 4F2hc expression detected during the multistep progressive change shows that the protein may have an important role i n the multistep tongue c arcinogenesis. Conclusively, LAT1 and 4F2 hc c an b e a useful b iomarker f or a better understanding of multistep tongue carcinogenesis, while the specific inhibition o f LAT1 and 4F2 hc would be a new rationale for suppressing tumor cell growth in tongue cancer.

Amino acid transporters play an important role in supplying organic nutrient to cells. The expression of L-type arnino acid transporter 1 (LATl) and its subunit 4F2 heavy chain (4F2hc) was evaluated to deterrnine the alterations to these transporters in oral norrnal mucosa (ONM) , oral precancerous lesion (OPL) and oral squamous cell carcinoma (OSCC). Sections from formalin-ftxed, paraffm-embedded S따nples of ONM, OPL or OSCC were exarnined using immunohistochernical staining to detect LATl and 4F2hc proteins. 까le LATl and 4F강lC expression increased progressively from ONM to hypeφ，Iastic and to dysplastic lesions and OSCC. In partiαlar， LATl rnay be a more S야dftc indicator of tumor prog~않sion than 4F2hc. 까le gradually increasing LA Tl and 4F2hc expression detected during the multistep progressive change shows that the protein rnay have an important role in the early stages of multistep oral carcinogenesis. In addition, the specific inhibition of LA Tl and 4F2hc rnight be a new rationale to suppress oral cancer progression.

We have observed the 10-9 transitions of HC3N and its 13C substitutes (H13CCCN,HC13CCN, and HCC13CN), and the vibration ally excited 12-11 (vr=1) HC3N transition toward the Sgr B2 molecular cloud. The observed HC3N emission shows an elongated shape around the Principal Cloud (~4.5 pc in R.A. × 7.4 pc in Decl.). The optically thin H13CCCN line peaks around the (N) core and we derive the total column density N(H13CCCN) = 4 ×10 13 cm-2 at this position. Toward the 2' N cloud which shows the peculiar chemistry, the HC3N lines show enhancements compared to the extended envelope. The shocks of the 2' N may have resulted in the enhancement of HC3N. The hot component of HC3N is strongly concentrated around the (N) core and its HPW is ~0.9 pc in diameter. We derive the lower limit of the abundance ratio N(HC3N)/N(H13CCCN) to be larger than 40 in most regions except the (M) and (N) cores. The fractionation processes of 13C at this region may not be as effective as previously reported.

We have observed the emission of HC3N J=4-3, 5-4,10-9 and 12-11 transitions toward the Sgr B2 central region in an area of $150 with resolutions of 16'-48'. The intensities and central velocities of line profiles show significant variations with positions. In contrast to the intensities of the low J-level transitions which gradually increase from the central source toward the outside region, the HC3N emission of the high J-level transition become stronger toward 'the central radio continuum source MD5. Systematic change in the radial velocity of each line profile occurs along north-south direction. There are a few peaks in most line profiles, and these indicate that there are multiple velocity components along the line of sight. Distributions of excitation temperature and column density which were estimated from the excitation calculations show the existence of a small (1 X 2pc), hot (Tex>50K) core which contains two temperature peaks at-15' east and north of MD5. The column density of HC3N is (1-3)X1014cm-2 Column density at distant position from MD5 is larger than that in the central region. We have deduced that this 'hot-core' has a mass of 105M⊙, which is about an order of magnitude larger than those obtained by previous studies.