• 미생물학회지
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• 제51권3호
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• pp.187-193
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• 2015

Candida (Pseudozyma로도 알려짐) antarctica lipase B(CAL-B)는 학문적으로 그리고 산업적으로 많이 활용되고 있다. CAL-B 자체에 대한 연구는 많이 진행되어온 반면, CAL-B 상동체에 관한 연구는 그리 알려진 바가 없다. 본 연구에서는 단백질 유사성 검색을 통해서 CAL-B의 상동체 탐색을 수행하였고, 6종의 단백질 서열을 찾았다. 해당하는 유전자들을 대장균에 대한 코돈 최적화를 수행하였고, 이를 바탕으로 유전자 합성을 진행하였다. 이들 유전자를 대장균 발현용 벡터에 클로닝한 후, 대장균 내에서 단백질 발현을 시도하여 이들 중 4종의 단백질이 성공적으로 발현되었다. 이들 단백질들이 가수분해 효소로서의 활성이 있는지 확인하기 위해서, 4-nitrophenyl acetate와 4-nitrophenyl butyrate를 반응기질로 하여 가수분해 반응성을 확인하였다. 이들 단백질들의 비활성(specific activity)값은 $(1.3-30){\times}10^{-2}{\mu}mol/min/mg$로 측정되었고, 이는 CAL-B의 비활성 수치보다는 다소 낮은 값에 해당하였다. (${\pm}$)-1-phenylethyl acetate의 가수분해 반응에 대한 입체선택성은 이들 상동체 효소들 중에서 Pseudozyma hubeiensis SY62에서 유래된 효소만이 CAL-B의 입체선택성과 유사함이 확인되었다.

• Asian-Australasian Journal of Animal Sciences
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• 제33권6호
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• pp.1023-1033
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• 2020

Objective: The efficiency of the knock-in process is very important to successful gene editing in domestic animals. Recently, it was reported that transient loosening of the nucleosomal folding of transcriptionally inactive chromatin might have the potential to enhance homologous recombination efficiency. The objective of this study was to determine whether histone deacetylases (HDAC) inhibitor and RAD51 recombinase (RAD51) expression were associated with increased knock-in efficiency on the β-casein (bCSN2) gene locus in mammary alveolar-large T antigen (MAC-T) cells using the transcription activator-like effector nucleases (TALEN) system. Methods: MAC-T cells were treated with HDAC inhibitors, valproic acid, trichostatin A, or sodium butyrate for 24 h, then transfected with a knock-in vector, RAD51 expression vector and TALEN to target the bCSN2 gene. After 3 days of transfection, the knock-in efficiency was confirmed by polymerase chain reaction and DNA sequencing of the target site. Results: The level of HDAC 2 protein in MAC-T cells was decreased by treatment with HDAC inhibitors. The knock-in efficiency in MAC-T cells treated with HDAC inhibitors was higher than in cells not treated with inhibitors. However, the length of the homologous arm of the knock-in vector made no difference in the knock-in efficiency. Furthermore, DNA sequencing confirmed that the precision of the knock-in was more efficient in MAC-T cells treated with sodium butyrate. Conclusion: These results indicate that chromatin modification by HDAC inhibition and RAD51 expression enhanced the homologous recombination efficiency on the bCSN2 gene locus in MAC-T cells.

• 한국동물생명공학회지
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• 제36권3호
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• pp.121-128
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• 2021

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 ㎛ 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.

• Journal of Crop Science and Biotechnology
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• 제10권4호
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• pp.231-236
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• 2007

Differentially expressed genes(DEG) were identified in a rice variety, Sathi, an indica type showing high allelopathic potential against barnyardgrass(Echinochloa crus-galli(L.) Beauv. var. frumentaceae). Rice plants were grown with and without barnyardgrass and total RNA was extracted from rice leaves at 45 days after seeding. DEG full-screening was performed by $GeneFishing^{TM}$ method. The differentially expressed bands were re-amplified and sequenced, then analyzed by Basic Local Alignment Search Tool(BLAST) searching for homology sequence identification. Gel electrophoresis showed nine possible genes associated with allelopathic potential in Sathi, six genes(namely DEG-1, 4, 5, 7, 8, and 9) showed higher expression, and three genes(DEG-2, 3 and 6) showed lower expression as compared to the control. cDNA sequence analysis showed that DEG-7 and DEG-9 had the same sequence. From RT PCR results, DEG-6 and DEG-7 were considered as true DEG, whereas DEG-1, 2, 3, 4, 5, and 8 were considered as putative DEG. Results from blast-n and blast-x search suggested that DEG-1 is homologous to a gene for S-adenosylmethionine synthetase, DEG-2 is homologous to a chloroplast gene for ribulose 1,5-bisphosphate carboxylase large subunit, DEG-8 is homologous to oxysterol-binding protein with an 85.7% sequence similarity, DEG-5 is homologous to histone 2B protein with a 47.9% sequence similarity, DEG-6 is homologous to nicotineamine aminotransferase with a 33.1% sequence similarity, DEG-3 has 98.8% similarity with nucleotides sequence that has 33.1% similarity with oxygen evolving complex protein in photosystem II, DEG-7 is homologous to nucleotides sequence that may relate with putative serin/threonine protein kinase and putative transposable element, and DEG-4 has 98.8% similarity with nucleotides sequence for an unknown protein.

• 한국동물생명공학회지
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• 제38권3호
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• pp.99-108
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• 2023

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.

• Journal of Microbiology and Biotechnology
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• 제32권2호
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• pp.248-255
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• 2022

Phlebia sp. MG-60 is the salt-tolerant, white-rot fungus which was isolated from a mangrove forest. This fungus expresses three kinds of manganese peroxidase (MGMnP) isozymes, MGMnP1, MGMnP2 and MGMnP3 in low nitrogen medium (LNM) or LNM containing NaCl. To date, there have been no reports on the biochemical salt-tolerance of these MnP isozymes due to the difficulty of purification. In present study, we established forced expression transformants of these three types of MnP isozymes. In addition, the fact that this fungus hardly produces native MnP in a high-nitrogen medium (HNM) was used to perform isozyme-selective expression and simple purification in HNM. The resulting MGMnPs showed high tolerance for NaCl compared with the MnP of Phanerochaete chrysosporium. It was worth noting that high concentration of NaCl (over 200 mM to 1200 mM) can enhance the activity of MGMnP1. Additionally, MGMnP1 showed relatively high thermo tolerance compared with other isozymes. MGMnPs may have evolved to adapt to chloride-rich environments, mangrove forest.

• Journal of Photoscience
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• 제4권3호
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• pp.141-145
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• 1997

Polymerase chain reaction(PCR) was conducted with a pea cDNA library using two primers synthesized from homology analysis of amino acid sequences for animal and plant cytosolic FBPases. A PCR product with 650 bp long was cloned into pGEM-T vector and sequenced. The deduced amino acid sequence of the cDNA fragment was 98, 91, and 85% homologous with those of cytosolic FBPases from spinach, sugarbeet, and sugarcane, respectively. It was 51% homologous with amino acid sequence of FBPase from pea chloroplasts. Northern blot analysis was proceeded with the cDNA clone resulting that 1.2 kb transcript was highly expressed in light-grown pea leaves but almost not expressed in dark-grown etiolated pea seedlings. When peas grown in the light for 10 days were transferred to darkness, the transcript was gradually decreased with dark treatment, indicating that the expression of the enzyme was induced by continuous white light but suppressed by dark treatment. Pea cytosolic FBPase was highly expressed in leaves with trace amounts in stems. but almost not expressed in roots.

• Journal of Plant Biology
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• 제38권4호
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• pp.381-388
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• 1995

해녀콩(Canavalia lineata) 뿌리혹으로부터 만들어진 cDNA library를 뿌리의 총 RNA, leghemoglobin 및 uricasell cDNA를 competitor로 사용하여 차등 혼성화반응으로 후기 뿌리혹에서 발현되며 Cno이, Cne2, Cne3, Clb2로 명명된 4개의 cDNA 클론을 얻었다. Cnod1은 벼의 cysteine proteinase를 암호화하는 유전자와 유사한데 1450nt의 전사체외 혼성화 반응을 보였으며 뿌리혹에서만 발현되고 뿌리혹 발달 후기에서 가장 높은 발현을 보였다. Cne2는 벼의 Expressed Sequence Tag의 한 종류와 유사하고 900 nt의 전사체와 혼성화 반응을 보였으며 뿌리혹에서 주로 발현되었다. Cne2는 접종 후 13일째 발현이 증폭된 후 일정하게 유지되었다. Cne2는 완두의 tonoplast 막 내재 단백질 TRG31을 암호화하는 유전자와 유사하며 뿌리에서 가장 많이 발현되었다. Cne3는 1700 nt와 1400 nt의 전사체와 혼성화반응을 보였으며 뿌리혹의 성장, 발달과 일치하는 발현 양상을 보였다. Clb2는 800 nt의 전사체와 혼성화반응을 보였으며 접종 후 8일째부터 발현이 시작되어 13일째 증폭되고 그 이후 일정한 수준을 유지하였다.

• 한국발생생물학회지:발생과생식
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• 제20권2호
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• pp.141-147
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• 2016

Rad51 is a key component of homologous recombination (HR) to repair DNA double-strand breaks and it forms Rad51 recombinase filaments of broken single-stranded DNA to promote HR. In addition to its role in DNA repair and cell cycle progression, Rad51 contributes to the reprogramming process during the generation of induced pluripotent stem cells. In light of this, we performed reprogramming experiments to examine the effect of co-expression of Rad51 and four reprogramming factors, Oct4, Sox2, Klf4, and c-Myc, on the reprogramming efficiency. Co-expression of Rad51 significantly increased the numbers of alkaline phosphatase-positive colonies and embryonic stem cell-like colonies during the process of reprogramming. Co-expression ofRad51 significantly increased the expression of epithelial markers at an early stage of reprogramming compared with control cells. Phosphorylated histone H2AX (${\gamma}H2AX$), which initiates the DNA double-strand break repair system, was highly accumulated in reprogramming intermediates upon co-expression of Rad51. This study identified a novel role of Rad51 in enhancing the reprogramming efficiency, possibly by facilitating mesenchymal-to-epithelial transition and by regulating a DNA damage repair pathway during the early phase of the reprogramming process.

• Journal of Microbiology and Biotechnology
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• 제21권7호
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• pp.679-685
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• 2011

Xanthomonas oryzae pv. oryzae (Xoo) produces a putative effector, XoAvrBs2. We expressed XoAvrBs2 homologously in Xoo with a TAP-tag at the C-terminus to enable quantitative analysis of protein expression and secretion. Addition of rice leaf extracts from both Xoo-sensitive and Xoo-resistant rice cultivars to the Xoo cells induced expression of the XoAvrBs2 gene at the transcriptional and translational levels, and also stimulated a remarkable amount of XoAvrBs2 secretion into the medium. In a T3SS-defective Xoo mutant strain, secretion of the TAPtagged XoAvrBs2 was blocked. Thus, we elucidated the transcriptional and translational expressions of the XoAvrBs2 gene in Xoo was induced in vitro by the interaction with rice and the induced secretion of XoAvrBs2 was T3SSdependent. It is the first report to measure the homologous expression and secretion of XoAvrBs2 in vitro by rice leaf extract. Our system for the quantitative analysis of effector protein expression and secretion could be generally used for the study of host-pathogen interactions.