• Journal of Animal Science and Technology
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• 제45권6호
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• pp.933-938
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• 2003

본 연구는 한우 mt DNA D-loop 영역의 염기변이 다형성과 경제형질간의 관련성을 분석하기 위하여 수행하였다. 한우의 mtDNA D-loop 영역에서 단일염기의 치환 의해 총 25개의 polymorphic site가 확인되었다. 그중 주요 Polymorphic site의 염기변이 빈도는 169, 16042, 16093, 16119, 16255 및 16302번째 위치에서 0.891, 0.117, 0.109, 0.182, 0.197 및 0.117로 검출되었다. 169 및 16119번째 위치에서의 염기치환에 의한 MS의 효과는 -1.08(p〈0.05), 1.29(p〈0.01)로 나타났으며, 169 및 16042번째 위치에서의 염기치환에 의한 BF의 효과는 -0.31(p〈0.01)과 0.34(p〈0.01)로 나타났다. 본 연구에서 검출한 한우 mtDNA내 D-loop 영역의 염기서열 변이 빈도 등은 한우집단의 유전적 변이성 추정과 좀 더 다양한 경제형질과의 관련성 분석은 물론 모계유전 양상 분석을 통한 한우의 형성과정과 타 품종과의 계통분류적 상호 관계 등의 분석에 유용하게 활용할 수 있을 것으로 기대된다.

• 미생물학회지
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• 제41권2호
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• pp.140-145
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• 2005

상업적으로 중요한 macrolide계 항진균 학생물질인 natamycin을 생산하는 Streptomyces natalensis ATCC27448의 환자 유전학적인 연구를 위해 대장균으로부터 S. natalensis로 plasmid DNA를 직접 도입하는 형질전환법을 확립하였다. 이러한 S. natalensis의 형질전환은 oriT와 attP 단편을 가지고 있는, ${\Phi}C31$ 유래의 integration 벡터인 pSET152를 이용하여 Escherichia coli ET12567/pUZ28002을 DNA 공여체(donor)로 이용한 접합전달법(conjugal transfer)을 사용하여 확립하였다. 접합전달의 가장 높은 효율은 10 mM의 $MgCl_2$를 포함한 MS 배지에서, $6.25\times10^8$의 E. coli 공여체와 열처리를 하지 않은 S. natalensis의 포자를 사용하여 얻어졌다. 또 얻어진 접합전달체 (exconjugant)에 대하여 southern blot hybridization과 벡터가 삽입된 염색체부분의 염기서열분석을 통해 attB site와 pseudo-attB site를 확인하다. attB site의 경우에는 다른 방선균들처럼 S. natalensis 염색체의 pirin 상동체를 코드하는 ORF내에 존재하였으나 pseudo-attB site는 염색체내 다른 site (GenBank accession no. $YP\_117731$)에 존재하였고 그 염기서열은 attB 염기서열과 차이를 나타내었다.

• Journal of Microbiology and Biotechnology
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• 제14권3호
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• pp.620-627
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• 2004

On the foundation of a database of genome sequences and protein analyses, the ability to clone a gene based on a peptide analysis is becoming more feasible and effective for identifying a specific gene and its protein product of interest. As such, the current study conducted a protein analysis using 2-D PAGE followed by MALDI- TOF and ESI-MS to identify a highly expressed gene product of C. parasitica. A distinctive and highly expressed protein spot with a molecular size of 47.2 kDa was randomly selected and MALDI-TOF MS analysis was conducted. A homology search indicated that the protein appeared to be a fungal enolase (enol). Meanwhile, multiple alignments of fungal enolases revealed a conserved amino acid sequence, from which degenerated primers were designed. A screening of the genomic $\lambda$ library of C. parasitica, using the PCR amplicon as a probe, was conducted to obtain the full-length gene, while RT-PCR was performed for the cDNA. The E. coli-expressed eno 1 exhibited enolase enzymatic activity, indicating that the cloned gene encoded the C. parasitica enolase. Moreover, ESI-MS of two of the separated peptides resolved from the protein spot on 2-D PAGE revealed sequences identical to the deduced sequences, suggesting that the cloned gene indeed encoded the resolved protein spot. Northern blot analysis indicated a consistent accumulation of an eno1 transcript during the cultivation.

• Asian-Australasian Journal of Animal Sciences
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• 제20권5호
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• pp.638-644
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• 2007

Differences of meat qualities between Japanese Black and Holstein have been known in Japan, however, the causative proteins and/or the genetic background have been unclear. The aim of this study was to identify candidate proteins causing differences of the meat qualities between the two breeds. Using technique of two-dimensional gel electrophoresis, protein profiling was compared from samples of the longissimus dorsi muscle and subcutaneous adipose tissue. Five protein spots were observed with different expression levels between breeds. By using LC-MS/MS analysis and Mascot program, three of them were identified as ankyrin repeat protein 2, phosphoylated myosin light chain 2 and mimecan protein. Subsequently, we compared the DNA coding sequences of three proteins between breeds to find any nucleotide substitution. However, there was no notable mutation which could affect pI or molecular mass of the proteins. The identified proteins may be responsible for different characteristics of the meat qualities between Japanese Black and Holstein cattle.

• Asian Pacific Journal of Cancer Prevention
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• 제17권2호
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• pp.581-590
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• 2016

Resistance to anoikis, a cell-detachment induced apoptosis, is one of the malignant phenotypes which support tumor metastasis. Molecular mechanisms underlying the establishment of this phenotype require further investigation. This study aimed at exploring protein expression profiles associated with anoikis resistance of a metastatic breast cancer cell. Cell survival of suspension cultures of non-metastatic MCF-7 and metastatic MDA-MB-231 cells were compared with their adherent cultures. Trypan blue exclusion assays demonstrated a significantly higher percentage of viable cells in MDA-MB-231 than MCF-7 cell cultures, consistent with analysis of annexin V-7-AAD stained cells indicating that MDA-MB-231 possess anti-apoptotic ability 1.7 fold higher than MCF-7 cells. GeLC-MS/MS analysis of protein lysates of MDA-MB-231 and MCF-7 cells grown under both culture conditions identified 925 proteins which are differentially expressed, 54 of which were expressed only in suspended and adherent MDA-MB-231 but not in MCF-7 cells. These proteins have been implicated in various cellular processes, including DNA replication and repair, transcription, translation, protein modification, cytoskeleton, transport and cell signaling. Analysis based on the STITCH database predicted the interaction of phospholipases, PLC and PLD, and 14-3-3 beta/alpha, YWHAB, with the intrinsic and extrinsic apoptotic signaling network, suggesting putative roles in controlling anti-anoikis ability. MDA-MB-231 cells grown in the presence of inhibitors of phospholipase C, U73122, and phospholipase D, FIPI, demonstrated reduced ability to survive in suspension culture, indicating functional roles of PLC and PLD in the process of anti-anoikis. Our study identified intracellular mediators potentially associated with establishment of anoikis resistance of metastatic cells. These proteins require further clarification as prognostic and therapeutic targets for advanced breast cancer.

• Molecules and Cells
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• 제40권9호
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• pp.621-631
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• 2017

Vaccinia-related kinase 1 (VRK1) and VRK3 are members of the VRK family of serine/threonine kinases and are principally localized in the nucleus. Despite the crucial roles of VRK1/VRK3 in physiology and disease, the molecular and functional interactions of VRK1/VRK3 are poorly understood. Here, we identified over 200 unreported VRK1/VRK3-interacting candidate proteins by affinity purification and LC-MS/MS. The networks of VRK1 and VRK3 interactomes were found to be associated with important biological processes such as the cell cycle, DNA repair, chromatin assembly, and RNA processing. Interactions of interacting proteins with VRK1/VRK3 were confirmed by biochemical assays. We also found that phosphorylations of XRCC5 were regulated by both VRK1/VRK3, and that of CCNB1 was regulated by VRK3. In liver cancer cells and tissues, VRK1/VRK3 were highly upregulated and its depletion affected cell cycle progression in the different phases. VRK3 seemed to affect S phase progression and G2 or M phase entry and exit, whereas VRK1 affects G1/S transition in the liver cancer, which could be explained by different interacting candidate proteins. Thus, this study not only provides a resource for investigating the unidentified functions of VRK1/VRK3, but also an insight into the regulatory roles of VRK1/VRK3 in biological processes.

• 농업과학연구
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• 제41권3호
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• pp.187-191
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• 2014

The cultivation area and use of genetically modified (GM) crops have been increased continuously over the world. Concerns about the potential risks of GM crops are also increasing. Safe management for the development and production of GM crops is required according to Living Modified Organism Act in Korea. Planning about the methods, duration, and frequency of environmental monitoring is also required for commercial use of GM crops. GM Zoysia japonica Steud. (event name: JG21) expressing resistance to glufosinate-ammonium has been generated previously. By using gamma ray treatment to JG21 we also developed male sterility and dwarf Z. japonica (event name: JG21-MS). The objective of this study was to establish the monitoring system for environment release of JG21-MS. In this study we extracted RNA from JG21 and JG21-MS and conducted RAPD (random amplified polymorphic DNA) method to distinguish JG21 and JG21-MS.

• 식물조직배양학회지
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• 제26권1호
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• pp.21-26
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• 1999

쌍떡잎식물체 형질전환에 널리 쓰이는 Agrobacterium의 binary vector를 이용하여 왜화성을 나타내는 pGA643-rolC gene을 엽절편 transformation방법으로 petunia에 도입하였다. Petunia hybrida의 재분화에 있어 엽조직으로부터 식물체 재분화에 미치는 생장조절물질의 효과는 0.1mg/L NAA와 1.0mg/L BA의 조합에서 높았고 재분화된 식물체도 양호하였다. 식물체 형질전환 선발배지에 에틸렌 억제제인 AgNO$_3$와 KMnO$_4$의 첨가시 형질전환체 재분화율이 높게 나타났으며, AgNO$_3$에 비해 KMnO$_4$처리구에서 보다 많은 식물체 재분화율을 나타내었으나 AgNO$_3$와 KMnO$_4$의 고농도 첨가시 다소의 유리화 현상이 발생하였으므로 3mg/L KMnO$_4$ 첨가가 식물체 재분화에 적합한 것으로 생각된다. 항생제 200mg/L kanamycin, 500mg/L carbenicylin 과 1.0 mg/L BA, 0.1mg/L NAA가 첨가된 형질전환 선발배지에서 엽절편으로 부터 형질전환 된 것으로 추정되는 식물체들을 일차 선발하였고 기외로 이식한 후 genomic DNA를 분리하여 Southern blot analysis법으로 분석한 결과 외래 유전자가 식물체의 genomic DNA내로 삽입된 것을 확인할 수 있었다.

• Asian-Australasian Journal of Animal Sciences
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• 제20권3호
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• pp.328-333
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• 2007

Carboxypeptidase E (CPE) plays an important role in the regulation of the body fat content. Therefore, it has been suggested as candidate gene for traits related to meat quality in beef cattle. This study was conducted to identify single nucleotide polymorphisms (SNPs) in the CPE gene and to investigate association of SNP marker with carcass and meat quality traits in Korean cattle. Three SNPs were identified in the intron 4 (A309G SNP and C445T SNP) and exon 5 (C601T SNP) of the CPE gene by sequence analyses of CPE cDNA and genomic DNA samples. The sequences have been deposited in GenBank database with accession numbers AY970664 and AY970663. Genotyping of the gene-specific SNP marker was carried out using the PCR-RFLP with restriction enzymes DdeI for C445T SNP and NlaIII for C601T SNP. The frequencies of C and T alleles were 0.43 and 0.57 for C445T SNP and 0.42 and 0.58 for C601T SNP, respectively. Statistical analysis indicated that the C445T SNP showed a significant effect (p<0.05) on marbling score (MS) and breeding value of backfat thickness (BF-EBV), respectively. Animals with the CT genotype showed higher marbling score and backfat thickness than those with the TT genotype. This marker also showed a significant dominance effect for the MS and BF-EBV (p<0.05). However, no significant associations were observed between C601T SNP genotypes and all traits examined. The results suggest that the CPE gene may be used as a marker for carcass traits in Korean cattle.

• Parasites, Hosts and Diseases
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• 제51권3호
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• pp.327-334
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• 2013

Vaccination is considered a promising alternative for controlling tick infestations. Haemaphysalis longicornis midgut proteins separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane were screened for protective value against bites. The western blot demonstrated the immunogenicity of 92 kDa protein (P92). The analysis of the P92 amino acid sequence by LC-MS/MS indicated that it was a H. longicornis paramyosin (Hl-Pmy). The full lenghth cDNA of Hl-Pmy was obtained by rapid amplification of cDNA ends (RACE) which consisted of 2,783 bp with a 161 bp 3' untranslated region. Sequence alignment of tick paramyosin (Pmy) showed that Hl-Pmy shared a high level of conservation among ticks. Comparison with the protective epitope sequence of other invertebrate Pmy, it was calculated that the protective epitope of Hl-Pmy was a peptide (LEEAEGSSETVVEMNKKRDTE) named LEE, which was close to the N-terminal of Hl-Pmy protein. The secondary structure analysis suggested that LEE had non-helical segments within an ${\alpha}$-helical structure. These results provide the basis for developing a vaccine against biting H. longicornis ticks.