• 생명과학회지
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• 제33권10호
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• pp.828-834
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• 2023

쌍별귀뚜라미(Gryllus bimaculatus)에서 분리한 막전단백질 258(transmembrane protein 258, Tmem258)을 코딩하는 cDNA를 GbTmem258로 이름 붙였다. 이 단백질은 80개의 아미노산으로 구성되어 있으며, N-glycosylation site가 없고, 각각 2개의 serine과 threonine, 1개의 tyrosine 잔기로 구성된 5개의 잠재적 인산화 부위를 가지고있다. GbTmem258 단백질은 분자량은 9.06 kDa이며 이론적 등전점은 5.5으로 계산되었으며, alpha-helix (52.5%), random coils (22.5%), extended strands (16.25%), beta turns (8.75%)의 2차 구조 정보를 기반으로 GbTmem258의 3차 구조가 작성되었다. 그리고, GbTmem258은 다른 종의 Tmem258와 아미노산 수준에서 높은 상동성을 보였다. 이 연구에서는 기아와 먹이 공급에 의해 GbTmem258 발현 조절이 어떻게 영향을 받는지 조사하였다. 기아가 지속되는 동안 hindgut에서 GbTmem258 발현이 점진적으로 증가하여 기아 6일 후 대조군보다 1.5배 높은 수준이 되었다. 그러나 6일간의 기아상태가 끝난 후 하루 또는 이틀 동안 다시 먹이를 주면 GbTmem258 발현이 대조군 수준으로 회복되었다. 지방에서는 기아 동안 대조군에 비해서 GbTmem258 발현이 최대 3배까지 증가했지만, 6일 기아 후 하루 또는 이틀 동안 다시 먹인 후에는 발현이 약 2.5배 증가로 감소되었다. 굶기고 다시 먹이는 실험 내내 각각의 조직에서 주목할만한 GbTmem258 발현은 관찰되지 않았다.

• 한국어병학회지
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• 제9권1호
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• pp.33-40
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• 1996

자연산 보리새우(Penaeus japonicus) 모하의 병리검사 결과 사상체의 mollicute와 유사한 미생물의 감염을 확인하였다. 검사에 사용된 보리새우 모하는 일본산이며, 한국으로 수입되었다. 일반 조직검사 결과 병리학적인 변화는 관찰되지 않았다. 투과전자현미경적 관찰 결과 간췌장 상피세포에서 세포내 다형태성 사상체 세균의 광범위한 감염을 발견하였다. 사상체 세균은 직경이 약 60nm이며, 길이가 300nm에서 $1{\mu}m$ 이상의 크기를 나타내었다. 이 세균의 구조는 사상체이며, 끝마디를 가진 가지로 분지 되는 특징을 보였다. 세포벽은 없으며, 원형질막으로만 둘러싸여 있었으며, 전형적인 원핵세포성 리보소음과 선형 DNA와 유사한 가닥을 가지고 있었다. 그 외 다른 세포내 기관은 관찰되지 않았다. 이러한 형태적인 특징들과 세포내 감염부위로 볼 때 이 세균은 mollicutes로 판단된다.

• Parasites, Hosts and Diseases
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• 제36권1호
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• pp.47-54
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• 1998

소의 주혈원충인 Reileria sp.의 small subunit ribosomal RMh (SSU rRNA) 유전자의 염 기서 열분 석을 위해 한국의 전북 장수로부터 분리하여 계대보관 중인 실험실 보관주 (KLS)와 김제 분리주 (KCB), 그리고 일본의 Shintoku 분리주 (JHS)를 실험에 사용하였다. 이들 분리주로 부터 원충을 회수 한 후 유전자를 추출하여 중합효소연쇄반응에 의 해 1.8 kb의 SSU rRNA 유전자를 증폭시 킬 수 있었으며, 증폭된 유전자를 이용하여 클론을 제작하고. 이들 클론으로 부터 플라스미드를 추출하여 유전자 염기서열분석을 실시하였다. 각 ReTheileria 분리주의 SSU rRNA유전자의 염기서열분석은 forma터와 reverse양쪽 다중복하여 실시하였으며 연속적인 primer를 이용하였다. 그 결과 한국의 소로부터 분리 한 Theue4n sp. (KLS. KCB)의 SSU rRNA유전자 염 기서 열 (Type A로 명명하였슴)은 일본 분리주와 동 일하였으며, 이 Type A를 GenBank로부터 유전자 검색을 해본 결과 Kenya의 Marula 분리주인 Reixerin buffeli의 SSU rRNA유전자 염기서열과 일치 하였다.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제22권2호
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• pp.164-173
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• 2000

The p16 protein is a cyclin dependent kinase inhibitor that inhibits cell cycle progression from $G_1$ phase to S phase in cell cycle. Many p16 gene mutations have been noted in many cancer-cell lines and in some primary cancers, and alterations of p16 gene function by DNA methylation have been noticed in various kinds of cancer tissues and cell-lines. There have been a large body of literature has accumulated indicating that abnormal patterns of DNA methylation (both hypomethylation and hypermethylation) occur in a wide variety of human neoplasma and that these aberrations of DNA methylation may play an important epigenetic role in the development and progression of neoplasia. DNA methylation is a part of the inheritable epigenetic system that influences expression or silencing of genes necessary for normal differentiation and proliferation. Gene activity may be silenced by methylation of up steream regulatory regions. Reactivation is associated with demethylation. Although evidence or a high incidence of p16 alterations in a variety of cell lines and primary tumors has been reported, that has been contested by other investigators. The precise mechanisms by which abnormal methylation might contribute to carcinogenesis are still not fully elucidated, but conceivably could involve the modulation of oncogene and other important regulatory gene expression, in addition to creating areas of genetic instability, thus predisposing to mutational events causing neoplasia. There have been many variable results of studies of head and neck squamous cell carcinoma(HNSCC). This investigation was studied on 13 primary HNSCC for p16 gene status by protein expression in immunohistochemistry, and DNA genetic/epigenetic analyzed to determine the incidence, the mechanisms, and the potential biological significance of its Inactivation. As methylation detection method of p16 gene, the methylation specific PCR(MSP) is sensitive and specific for methylation of any block of CpG sites in a CpG islands using bisulfite-modified DNA. The genomic DNA is modified by treatment with sodium bisulfate, which converts all unmethylated cytosines to uracil(thymidine). The primers designed for MSP were chosen for regions containing frequent cytosines (to distinguish unmodified from modified DNA), and CpG pairs near the 5' end of the primers (to provide maximal discrimination in the PCR between methylated and unmethylated DNA). The two strands of DNA are no longer complementary after bisulfite treatment, primers can be designed for either modified strand. In this study, 13 paraffin embedded block tissues were used, so the fragment of DNA to be amplified was intentionally small, to allow the assessment of methylation pattern in a limited region and to facilitate the application of this technique to samlples. In this 13 primary HNSCC tissues, there was no methylation of p16 promoter gene (detected by MSP and automatic sequencing). The p16 protein-specific immunohistochemical staining was performed on 13 paraffin embedded primary HNSCC tissue samples. Twelve cases among the 13 showed altered expression of p16 proteins (negative expression). In this study, The author suggested that low expression of p16 protein may play an important role in human HNSCC, and this study suggested that many kinds of genetic mechanisms including DNA methylation may play the role in carcinogenesis.

• 생명과학회지
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• 제30권3호
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• pp.313-319
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• 2020

최근, 국내에서 발생하는 대가축의 질병은 바이러스 혹은 세균 등과 같은 병원체가 사료 섭취, 가축 간의 신체접촉, 호흡 등 다양한 경로를 통해 전파되어 발병되는 전염성 질병이다. 전염성 질병은 가축의 건강을 위협하고 생산성을 감소시키기 때문에 현장에서 조기 진단하여 개체 격리와 같은 통제 관리가 필수적이다. 기존 사용되고 있는 진단 키트들은 현장에서 사용하기에 용이하지 않으며 극소량의 감도에서 진단이 제한적인 단점을 가지고 있다. 그러므로, 현장에서 극소량의 감도와 진단의 편이성을 고려하여 DNA와 RNA 수준에서 진단할 수 있는 CRISPR/Cas 시스템은 최적의 시스템이라 할 수 있다. 본 연구논문에서는 대가축의 전염성 질병들을 현장에서 조기 진단함에 있어 CRISPR/Cas 시스템의 활용전략에 대해 소개하고자 한다. 최근 발견된 CRISPR/Cas 효소들은 2개의 클래스와 6가지 하위유형으로 분류되었다. 이 중에서 클래스 2에 포함되는 Cas 효소들은 대표적으로 제 2형에 Cas9, 제 5형에 Cas12a와 Cas12b, 제 6형에 Cas13a와 Cas13b가 있다. 현재까지 개발된 CRISPR/Cas 시스템들은 간단한 시각 신호를 통해 표적에 대한 정량 및 다중 감지가 가능하고 특히, 극소량 수준의 초고감도에서도 표적만을 진단할 수 있으며 단시간 이내에 진단 결과를 얻을 수 있다. 하지만 초고감도 DNA 혹은 RNA를 진단하기 위해 최적의 신호 증폭 방법과 결합되어야 하고 표적 DNA 혹은 RNA를 진단에 적합하도록 DNA를 RNA로, RNA를 DNA로 전변해야 하는 단점이 있다. 따라서, 현장에서 대가축의 전염성 질병을 조기에 진단할 수 있는 CRISPR/Cas 바이오센서를 개발하는데 있어 가축의 전염 매개체로부터 추출되는 병원체 유형(DNA 혹은 RNA)을 고려하여 최적의 Cas 효소를 선정하여야 하고 이에 따른 적절한 신호 증폭 방법이 결합되어야 한다. 따라서, CRISPR/Cas 시스템은 유전자 편집 방법을 사용하는 빠르고 효율적인 진단 도구이며 이 시스템은 소의 전염병을 조기에 진단하고 감염 확산방지에 도움될 수 있을 것으로 판단 되어진다.

• Asian-Australasian Journal of Animal Sciences
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• 제29권5호
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• pp.631-639
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• 2016

China has a long history of sheep (Ovis aries [O. aries]) breeding and an abundance of sheep genetic resources. Knowledge of the complete O. aries mitogenome should facilitate the study of the evolutionary history of the species. Therefore, the complete mitogenome of O. aries was sequenced and annotated. In order to characterize the mitogenomes of 3 Chinese sheep breeds (Altay sheep [AL], Shandong large-tailed sheep [SD], and small-tailed Hulun Buir sheep [sHL]), 19 sets of primers were employed to amplify contiguous, overlapping segments of the complete mitochondrial DNA (mtDNA) sequence of each breed. The sizes of the complete mitochondrial genomes of the sHL, AL, and SD breeds were 16,617 bp, 16,613 bp, and 16,613 bp, respectively. The mitochondrial genomes were deposited in the GenBank database with accession numbers KP702285 (AL sheep), KP981378 (SD sheep), and KP981380 (sHL sheep) respectively. The organization of the 3 analyzed sheep mitochondrial genomes was similar, with each consisting of 22 tRNA genes, 2 rRNA genes (12S rRNA and 16S rRNA), 13 protein-coding genes, and 1 control region (D-loop). The NADH dehydrogenase subunit 6 (ND6) and 8 tRNA genes were encoded on the light strand, whereas the rest of the mitochondrial genes were encoded on the heavy strand. The nucleotide skewness of the coding strands of the 3 analyzed mitogenomes was biased toward A and T. We constructed a phylogenetic tree using the complete mitogenomes of each type of sheep to allow us to understand the genetic relationships between Chinese breeds of O. aries and those developed and utilized in other countries. Our findings provide important information regarding the O. aries mitogenome and the evolutionary history of O. aries inside and outside China. In addition, our results provide a foundation for further exploration of the taxonomic status of O. aries.

• Journal of Life Science
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• 제9권2호
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• pp.9-14
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• 1999

Pseudomonas iodinum IFO 3558 adenosine deaminase(ADA) gene was cloned by the polymerase chain reaction and deduced the amino acid sequence of the enzyme. DNA sequence homology of Pseudomonas iodinum IFO 3558 ADA gene was compared to those of E. coli, human and mouse ADA genes. Unambiguous sequence from both strands of pM21 was obtained for the region believed to encode ADA. The sequence included a 804-nucleotide open reading frame, bounded on one end by sense primer and on the other end by two antisense primer. This open reading frame encodes a protein of 268 amino acids having a molecular weight of 29,448. The deduced amino acid sequence shows considerable similarity to those of E. coli, mouse and human ADA. Pseudomonas iodinum IFO 3558 nucleotide sequence shows 98.5% homology with that of the E. coli ADA sequence and 51.7% homology with that of the mouse ADA sequence and 52.5% homology with that of the human ADA sequence. The ADA protein sequence of Pseudomonas iodinum IFO 3558 shows 96.9% homology with that of the E. coli and 40.7% homology with that of the mouse and 41.8% homology with that of the human. The distance between two of the conserved elements, TVHAGE and SL(1)NTDDP has veen exactly conserved at 76 amino acids for all four ADAs. Two of the four conserved sequence elements shared among the four ADAs are also present in the yeast, rat, human (M), and Human(L) AMP deaminase. The SLSTDDP sequence differs only in the conservative substitution of a serine for an asparagine. A conserved cysteine with conserved spacing between these two regions is also found. Thus, sequence analysis of four ADAs and four AMP deaminases revealed the presence of a highly conserved sequence motif, SLN(S)TDDP, a conserved dipeptide, HA, and a conserved cysteine residue.

• Asian-Australasian Journal of Animal Sciences
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• 제29권11호
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• pp.1576-1584
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• 2016

Acetyl xylan esterase (AXE), which hydrolyzes the ester linkages of the naturally acetylated xylan and thus known to have an important role for hemicellulose degradation, was isolated from the anaerobic rumen fungus Neocallimastix frontatlis PMA02, heterologously expressed in Escherichi coli (E.coli) and characterized. The full-length cDNA encoding NfAXE1 was 1,494 bp, of which 978 bp constituted an open reading frame. The estimated molecular weight of NfAXE1 was 36.5 kDa with 326 amino acid residues, and the calculated isoelectric point was 4.54. The secondary protein structure was predicted to consist of nine ${\alpha}$-helixes and 12 ${\beta}$-strands. The enzyme expressed in E.coli had the highest activity at $40^{\circ}C$ and pH 8. The purified recombinant NfAXE1 had a specific activity of 100.1 U/mg when p-nitrophenyl acetate (p-NA) was used as a substrate at $40^{\circ}C$, optimum temperature. The amount of liberated acetic acids were the highest and the lowest when p-NA and acetylated birchwood xylan were used as substrates, respectively. The amount of xylose released from acetylated birchwod xylan was increased by 1.4 fold when NfAXE1 was mixed with xylanase in a reaction cocktail, implying a synergistic effect of NfAXE1 with xylanase on hemicellulose degradation.

• Reproductive and Developmental Biology
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• 제32권3호
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• pp.183-191
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• 2008

This study was conducted to investigate the development and gene expression in miniature pig nuclear transfer (mNT) embryos produced under different osmolarity culture conditions. Control group of mNT embryos was cultured in PZM-3 for 6 days. Treatment group of mNT embryos was cultured in modified PZM-3 with NaCl (mPZM-3, 320 mOsmol) for 2 days, and then cultured in PZM-3 (270 mOsmol) for 4 days. Blastocyst formation rate of the treatment group was significantly higher than the control and the apoptosis rate was significantly lower in treatment group. Bax-$\alpha$ and caspase-3 mRNA expression were significantly higher in the control than the treatment group. Also, the majority of imprinting genes were expressed aberrantly in in vitro produced mNT blastocysts compared to in vivo derived blastocyst H19 and Xist mRNA expression were significantly lower in the control than the treatment group or in vivo. IGF2 mRNA expression was significantly higher in the control than the treatment group or in vivo. IGF2r mRNA expression was significantly lower in the control. Methylation profiles of individual DNA strands in H19 upstream T-DMR sequences showed a similar methylation status between treatment group and in vivo. These results indicate that the modification of osmolarity in culture medium at early culture stage could provide more beneficial culture environments for mNT embryos.

• Imaging Science in Dentistry
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• 제33권1호
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• pp.51-57
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• 2003

Purpose: To investigate the effects of irradiation on the phenotypic expression of the MC3T3-El osteoblastic cell line, particularly an the expression of type I collagen and alkaline phosphatase mRNA. Materials and Methods: Cells were irradiated with a single dose of 0.5, 1, 2, 4, and 8 Gy at a dose rate of 5.38 Gy/min using a cesium 137 irradiator. The specimens were then harvested and RNA extraction was carried out at 1 and 3 days after irradiation. The extracted RNA strands were reverse-transcribed and the resulting cDNA fragments were amplified by PCR. Results: The irradiated cells demonstrated a dose-dependent increase in type I collagen mRNA expression relative to the control group, with a maximum level of type I collagen mRNA expression occurring at 8 Gy. The degree of type I collagen mRNA expression increased significantly at 1 day after irradiation, but little differences were found between the control group and at the 3rd day. The amount of alkaline phosphatase mRNA expression increased significantly at land 3 days after irradiation in the 1 Gy exposed group compared with the control group. Conclusion: The amount of type I collagen and alkaline phosphatase mRNA expression increased significantly 1 day after irradiation when compared with the control group.