• Journal of Microbiology and Biotechnology
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• 제30권7호
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• pp.982-995
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• 2020

A putative multidrug efflux gene, yddA, was cloned from the Escherichia coli K-12 strain. A drug-sensitive strain of E. coli missing the main multidrug efflux pump AcrB was constructed as a host and the yddA gene was knocked out in wild-type (WT) and drug-sensitive E. coliΔacrB to study the yddA function. Sensitivity to different substrates of WT E.coli, E. coliΔyddA, E. coliΔacrB and E. coliΔacrBΔyddA strains was compared with minimal inhibitory concentration (MIC) assays and fluorescence tests. MIC assay and fluorescence test results showed that YddA protein was a multidrug efflux pump that exported multiple substrates. Three inhibitors, ortho-vanadate, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and reserpine, were used in fluorescence tests. Ortho-vanadate and reserpine significantly inhibited the efflux and increased accumulation of ethidium bromide and norfloxacin, while CCCP had no significant effect on YddA-regulated efflux. The results indicated that YddA relies on energy released from ATP hydrolysis to transfer the substrates and YddA is an ABC-type multidrug exporter. Functional study of unknown ATP-binding cassette (ABC) superfamily transporters in the model organism E. coli is conducive to discovering new multidrug resistance-reversal targets and providing references for studying other ABC proteins of unknown function.

• Asian-Australasian Journal of Animal Sciences
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• 제28권1호
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• pp.20-24
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• 2015

Insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding protein-1 (IGFBP-1) play a pivotal role in regulating cellular hypoxic response. In this study, we cloned and characterized the genes encoding IGF-1 and IGFBP-1 to improve the current knowledge on their roles in highland Bos grunniens (Yak). We also compared their expression levels in the liver and kidney tissues between yaks and lowland cattle. We obtained full-length 465 bp IGF-1 and 792 bp IGFBP-1, encoding 154 amino acids (AA) IGF-1, and 263 AA IGFBP-1 protein, respectively using reverse transcriptase-polyerase chain reaction (RT-PCR) technology. Analysis of their corresponding amino acid sequences showed a high identity between B. grunniens and lowland mammals. Moreover, the two genes were proved to be widely distributed in the examined tissues through expression pattern analysis. Real-time PCR results revealed that IGF-1 expression was higher in the liver and kidney tissues in B. grunniens than in Bos taurus (p<0.05). The IGFBP-1 gene was expressed at a higher level in the liver (p<0.05) of B. taurus than B. grunniens, but it has a similar expression level in the kidneys of the two species. These results indicated that upregulated IGF-1 and downregulated IGFBP-1 are associated with hypoxia adaptive response in B. grunniens.

• Asian-Australasian Journal of Animal Sciences
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• 제29권9호
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• pp.1363-1370
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• 2016

Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene recombinant Pichia pastoris strain could serve as a novel source for rennet production. Yaks total RNA was extracted from the abomasum of an unweaned yak. The yak preprochymosin, prochymosin, and chymosin genes from total RNA were isolated using gene specific primers based on cattle chymosin gene sequence respectively and analyzed their expression pattern byreal time-polymerase chain reaction. The result showed that the chymosin gene expression level of the sucking yaks was 11.45 times higher than one of adult yaks and yak chymosin belongs to Bovidae family in phylogenetic analysis. To express each, the preprochymosin, prochymosin, and chymosin genes were ligated into the expression vector $pPICZ{\alpha}A$, respectively, and were expressed in Pichia pastoris X33. The results showed that all the recombinant clones of P. pastoris containing the preprochymosin, prochymosin or chymosin genes could produce the active form of recombinant chymosin into the culture supernatant. Heterologous expressed prochymosin (14.55 Soxhlet unit/mL) had the highest enzyme activity of the three expressed chymosin enzymes. Therefore, we suggest that the yak chymosin gene recombinant Pichia pastoris strain could provide an alternative source of rennet production.

• 한국가축번식학회지
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• 제25권3호
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• pp.277-286
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• 2001

기존의 미세주입 및 정자 electroporation에 의한 보다 효율적인 유전자 도입방법을 개선하여 간단하고 고효율성의 유전자 변환기술을 위한 유전자 도입장치의 시제품 개발로 다수의 난자를 전기적으로 단순화할 수 있는 상업화의 가능성을 보여주었다. 도입된 유전자는 모든 초기배에서 발현됨을 보여 주었다. 특히 난황내의 합포체세포(syncytium)에서의 transient성의 강한 발현은 전기자극에 의해 많은 수의 난자에 유전자를 도입하고 100% 발현되는 체계를 이용하여 transient 시기에서 인간 유용단백질 생산의 가능성을 타진할 수 있는 결과를 보여 주었다. 어류유전자 발현의 작동되는가를 검색하기 위해 신경세로조직특이 tubulin promoter 를 이용한 결과 gfp의 발현이 뇌주변과 척추를 중심으로 체내 전반의 신경세포내에 발현이 강하게 나타남을 보여 주었다. 한편 reporter 유전자 이외에 간세포로부터 전체 RNA를 분리시켜 vitellogenin의 분해산물인 phosvitin cDNA의 길이와 promoter지역인 1.6 kb에 대한 primer쌍들을 선정한 상태에서 PCR에 의해 각각 cDNA와 gDNA로부터 cloning 중에 있으며 human factor Ⅶ과 epidermal growth factor, vitellogenin의 3종의 target 단백 질유전자를 구축 및 검정 화인 중에 있다.

• Reproductive and Developmental Biology
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• 제32권1호
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• pp.65-70
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• 2008

Members of the Vps (Vacuolar protein sorting) protein family involved in the formation of the retromer complex have been discovered in a variety of species such as yeast, mouse, and human. A mammalian retromer complex is composed of Vps26, Vps29, and Vps35 proteins and plays and important role in cation-independent mannose-6-phosphate receptor retrieval from the endosome to the trans-Golgi network. In this study, we have identified the full-length sequences of the retromer components of Vps26, Vps29, and Vps35 in micro pigs. The cDNA sequences of these retromer components have been determined and the result showed there is 99% homology among the component counterparts from mouse, micro pigs, and humans. In addition, the retromer complexes formed with hetero-components were found in the brain of micro pigs. Based on above results, we suggest mammalian Vps components are well conserved in micro pigs.

• Animal Bioscience
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• 제35권9호
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• pp.1360-1366
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• 2022

Objective: The present study analyzed the influence of co-transferring embryos with high and low cloning efficiencies produced via somatic cell nuclear transfer (SCNT) on pregnancy outcomes in dogs. Methods: Cloned dogs were produced by SCNT using donor cells derived from a Tibetan Mastiff (TM) and Toy Poodle (TP). The in vivo developmental capacity of cloned embryos was evaluated. The pregnancy and parturition rates were determined following single transfer of 284 fused oocytes into 21 surrogates and co-transfer of 47 fused oocytes into four surrogates. Results: When cloned embryos produced using a single type of donor cell were transferred into surrogates, the pregnancy and live birth rates were significantly higher following transfer of embryos produced using TP donor cells than following transfer of embryos produced using TM donor cells. Next, pregnancy and live birth rates were compared following single and co-transfer of these cloned embryos. The pregnancy and live birth rates were similar upon co-transfer of embryos and single transfer of embryos produced using TP donor cells but were significantly lower upon single transfer of embryos produced using TM donor cells. Furthermore, the parturition rate for TM dogs and the percentage of these dogs that remained alive until weaning was significantly higher upon co-transfer than upon single transfer of embryos. However, there was no difference between the two embryo transfer methods for TP dogs. The mean birth weight of cloned TM dogs was significantly higher upon single transfer than upon co-transfer of embryos. However, the body weight of TM dogs did not significantly differ between the two embryo transfer methods after day 5. Conclusion: For cloned embryos with a lower developmental competence, the parturition rate and percentage of dogs that remain alive until weaning are increased when they are co-transferred with cloned embryos with a greater developmental competence.

• 농업과학연구
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• 제25권1호
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• pp.79-88
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• 1998

본 연구는 축협중앙회 한우개량부에서 사육중인 한우의 혈액으로부터 genomic DNA를 추출하여 한우의 면역체계에 중요한 역할을 담당하고 있는 BoLA DRB3 exon2 유전자를 PCR기법을 이용하여 증폭하고 친자확인을 위한 이들 대립유전자들의 염기서열을 분석하여 한우의 효율적인 육종에 분자유전수준에서의 접목을 위한 기초자료를 얻고자 실시하였던바, 얻어진 결과를 요약하면 다음과 같다. 1 한우의 혈액에서 추출된 genomic DNA를 1.5% agarose gel에서 전기영동한 결과, 12.2kb이상의 단일밴드로 나타나, genomic DNA는 아주 잘 분리된 것으로 판단되었으며, 한편 genomic DNA로부터 PCR기법을 이용하여 BoLA DRB3 exon2 유전자를 증폭한 결과 284kb의 단편이 증폭 되었음을 확인하였다. 2. PCR 증폭산물을 pCR 2.1 vector를 사용하여 cloning 하고, 균주에 형질전환을 시켜 재조합 plasmid를 추출한 후, EcoR 1으로 처리한 결과 300bp 단편이 확인되어 증폭되어진 BoLA DRB3 exon2 유전자가 vector 내에 잘 삽입되었음을 확인하였다. 3. 부와 모의 BoLA DRB3 exon2 대립유전자의 염기서열을 분석한 결과 염기서열의 상동성은 부의 대립유전자간에는 82.0% 이었고, 모의 대립유전자간에는 90.1%이었다. 4. 친자를 확인하기 위해 부와 모 그리고 자손간의 가계에 대한 BoLA DRB3 exon2 대립유전자의 염기서열을 분석한 결과 Mendel 의 법칙에 따라 유전된다는 사실을 확인 할 수 있었다. 5. 이상의 결과를 종합하여보면 한우의 BoLA DRB3 exon2 유전자의 부와 모, 그리고 자손의 대립유전자에 대한 염기서열 수준에서의 친자확인이 가능하였으며, 이들 연구결과는 축우의 유전적 능력개량에 중요한 분자유전학적 기초 자료가 될 것으로 판단되었다.

• Animal cells and systems
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• 제13권3호
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• pp.297-304
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• 2009

A complete mRNA sequence of transferrin from the wax moth, Galleria mellonella, was obtained, and compared with those of other species. We previously reported that the sequence was most similar to those of Manduca sexta and Bombyx mori. As in other moths, G. mellonella transferrin had only one iron-binding site at its N-terminal region. Semi-qRT PCR was conducted to investigate tissue-specific distribution and transcriptional regulation of the wax moth transferrin mRNA. Larval muscle and fat body contained larger quantity of mRNA than other tested tissues. In this study, it was observed that iron and cadmium regulated transferrin transcription, and this regulation pattern was tissue specific. Iron up-regulated transferrin mRNA level in fat body, while suppressed it in the Malpighian tubules and silk glands. Cadmium decreased the mRNA level in fat body, muscle, and Malpighian tubules, but significantly increased the mRNA level in silk glands. In addition, the mRNA expression was induced by all tested pathogen-associated molecular patterns (PAMPs) including LPS, lipoteichoic acid (LTA), glucan, and even chitin.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.85.2-86
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• 2003

Fusarium graminearum is an important pathogen of cereal crops in many areas of the world causing head blight and ear rot of small grains. In addition to serious economic losses, this fungus produces mycotoxins, such as trichothecenes and zearalenone on diseased crops and has been a potential threat to human and animal health. To massively identify pathogenesis-related genes from F. graminearum, two representative strains (SCKO4 from rice and Z03643 from wheat) were mutagenized using restriction enzyme-mediated integration (REMI). In total, 20,DOD REMI transformants have been collected from the two strains. So far, 63 mutants for several traits involved in disease development such as virulence, mycotoxin production, and sporulation have been selected from 3,000 REMI transformants. Now, selected mutants of interest have being genetically analyzed using a newly developed outcross method (See Jungkwan Lee et al poster). In addition, cloning and characterization of genomic DNA regions flanking the insertional site in the genome of the mutants are in progress.

• International Journal of Industrial Entomology and Biomaterials
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• 제2권1호
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• pp.87-89
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• 2001

The nucleotide sequence of the domestic silkworm (Bombyx mori) mitochondrial (mt) control region and its flanking genes was determined from PCR clones. The control region of the silkworm mt genome was located between the small ribosomal RNA gene and transfer RN $A^{Met}$. This 499 bp control region hale 95.4% A+T content. Extensive comparative analysis studies performed with similar control region of other insect genomes could not reveal a highly conserved region containing conserved motifs of animal mito-chondrial genome. The remarkable feature that found in this control region was the presence of tandem motifs containing nine repetitive sequences. The potential usefulness of this motif sequences for Bombyx species or their taxonomically related species is enhanced by its unique localization in the maternally inheritance mitochondrial molecule.e.