• 제목/요약/키워드: amino acid substitution

검색결과 170건 처리시간 0.027초

DNA shuffling을 이용한 Alcaligenes faecalis T1의 PHB depolymerase 활성 증진 (Enhancement of PHB depolymerase Activity from Alcaligenes faecalis T1 by DNA Shuffling)

  • 신동성;이영하;남진식
    • 미생물학회지
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    • 제39권2호
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    • pp.76-82
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    • 2003
  • Alcaligenes faecalis T1의 Poly(3-hydroxybutyrate)(PHB) depolymerase활성 증진을 위해 DNA shuffling방법을 이용하였다. 제조된 A. faecalis T1의 PHB depolymerase 돌연변이 유전자의 library를 Pseudomonas syringae의 icenucleation protein유전자를 포함하는 발현벡터 pJHCll에 클로닝하여 약 7,000개의 형질전환체를 얻었다. 탄소원으로 PHB또는 poly(3-hydroxybutyrate-co-3-hydroxyvalerate)를 포함하는 M9최소배지를 이용하여 형질전환체들로부터 활성이 서로 다른 돌연변이주들을 선별하였다. 이들의 PHB depolymease 활성은 평판배지에서의 halo형성 및 배양 상등액을 이용한 탁도 감소 실험으로 확인하였으며,형질전환체들 중에서 shuffling전의 대조군에 비하여 사용된 기질에 따라 효소활성이 1.8-3.2배 증진된 II-4 돌연변이주를 얻었다. DNA 염기서열의 분석을 통하여 II-4의 PHB depolymease에는 3개의 아미노산 치환(A1a209Va1, Leu258Phe, Asp263Thr)이 이루어졌음을 확인하였다. 여러 가지 돌연변이주의 아미노산 서열의 변화를 분석한 결과, PHB depolymerase의 catalytictriad주위에 기존 아미노산에 비하여 보다 소수성인 아미노산으로의 치환이 소수성 기질인 PHB에 대한분해 활성 중진에 기여하는 것으로 추정되었다.

Variability in the Viral Protein Linked to the Genome of Turnip Mosaic Virus Influences Interactions with eIF(iso)4Es in Brassica rapa

  • Li, Guoliang;Zhang, Shifan;Li, Fei;Zhang, Hui;Zhang, Shujiang;Zhao, Jianjun;Sun, Rifei
    • The Plant Pathology Journal
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    • 제37권1호
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    • pp.47-56
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    • 2021
  • Plants protect against viruses through passive and active resistance mechanisms, and in most cases characterized thus far, natural recessive resistance to potyviruses has been mapped to mutations in the eukaryotic initiation factor eIF4E or eIF(iso)4E genes. Five eIF4E copies and three eIF(iso)4E copies were detected in Brassica rapa. The eIF4E and eIF(iso)4E genes could interact with turnip mosaic virus (TuMV) viral protein linked to the genome (VPg) to initiate virus translation. From the yeast two-hybrid system (Y2H) and bimolecular fluorescence complementation (BiFC) assays, the TuMV-CHN2/CHN3 VPgs could not interact with BraA.eIF4E.a/c or BraA.eIF(iso)4E.c, but they could interact with BraA.eIF(iso)4E.a in B. rapa. Further analysis indicated that the amino acid substitution L186F (nt T556C) in TuMV-UK1 VPg was important for the interaction networks between the TuMV VPg and eIF(iso)4E proteins. An interaction model of the BraA. eIF(iso)4E protein with TuMV VPg was constructed to infer the effect of the significant amino acids on the interaction of TuMV VPgs-eIF(iso)4Es, particularly whether the L186F in TuMV-UK1 VPg could change the structure of the TuMV-UK1 VPg protein, which may terminate the interaction of the BraA.eIF(iso)4E and TuMV VPg protein. This study provides new insights into the interactions between plant viruses and translation initiation factors to reveal the working of key amino acids.

참담치(Mytilus coruscus) 유래 항균 펩타이드 분리 및 정제 (Isolation and Purification of Antimicrobial Peptide from Hard-shelled Mussel, Mytilus coruscus)

  • 오륜경;이민정;김영옥;남보혜;공희정;김주원;안철민;김동균
    • 생명과학회지
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    • 제26권11호
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    • pp.1259-1268
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    • 2016
  • 항생제 남용의 문제점 및 사용제한에 따른 천연물 유래 항생제 대체제 개발을 위하여 우리나라와 중국, 일본에서만 서식하는 고유종인 참담치의 족부 근육 추출물로부터 항균 펩타이드를 분리 및 정제하였다. $C_{18}$ 역상 컬럼을 사용하여 항균 펩타이드를 정제하였으며, MALDI-TOF/MS로 분석 결과 분자량은 6,701 Da에 해당하였다. edman degradation 방법으로 N-말단 아미노산 서열 분석을 통하여 20개의 아미노산 서열을 밝혔으며 이는 캘리포니아 홍합(Mytilus californianus)의 sperm-specific protein 또는 protamine-like PL-II/PL-IV precursor와 100% 상동성을 지님을 밝혀냈다. 이러한 정보를 바탕으로 ORF를 분석한 결과 60개의 아미노산을 코딩하는 183 bp로 캘리포니아 홍합의 protamine-like PL-II/PL-IV precursor와 아미노산 서열이 100% 일치하였고 유전자 염기서열은 97.2%의 상동성을 나타냈다. 합성된 항균 펩타이드는 그람 양성 균주 Bacillus cereus (MEC, $20.8{\mu}g/ml$), Bacillus subtilis (MEC, $0.2{\mu}g/ml$), Streptococcus mutans (MEC, $0.2{\mu}g/ml$), 그람 음성 균주 Pseudomonas aeruginosa (MEC, $5.7{\mu}g/ml$), Escherichia coli (MEC, $2.6{\mu}g/ml$) 그리고 진균류 Candida albicans (MEC, $56.3{\mu}g/ml$)의 항균활성을 나타냈다. 또한 높은 열안정성을 지녔으며, C. albicans를 제외하고 염안정성을 지닌 항균 펩타이드로 확인되었다. 이를 통해 참담치 유래의 항균 펩타이드는 항생제 대체제의 후보소재로 높은 가능성을 지녔다고 할 수 있다. 따라서 본 연구에서 분리한 참담치 족부 근육 유래 항균 펩타이드는 향후 추가적인 연구를 통하여 항생제를 대체할 수 있는 물질의 개발이 가능할 것으로 사료된다. 그리고 해양무척추동물의 선천성 면역 기작에 대한 정보를 제공할 것으로 기대한다.

국민식생활(國民食生活) 향상(向上)을 위(爲)한 곡류제품(穀類製品)의 경제적( 經濟的) 영향강화(營養强化)에 관(關)한 연구(硏究) (A Study on the Economical Nutrition Supplement of Cereal Food for Improvement in our National Eating Habits)

  • 주진순;유종열;김숙희;이기열;한인규
    • Journal of Nutrition and Health
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    • 제6권1호
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    • pp.1-9
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    • 1973
  • I. Subject of the Study: Studies on the economical nutrition supplement of cereal foods for the improvement in our notional eating habits. II. Purpose and Importance of the Study: 1. Our nation is confronted with the situation that the rice, a principal food, short of some essential amino acids, lysine and threonine, leads to imbalanced meals insufficient in the nutrient of protein, to bring many difficulties in the elevation of our national physique. 2. The shortage of even the rice imperfect in the nutrient of protein makes the import of lots of foreign rice inevitable. It is considered that the protein supplement and decrease in the consumption amount, of rice, is a serious key to the solution of our food difficulty, and then a way of the proetin supplement of rice through the addition of essential amino acid is to be rarely applied in the view of the our present finance and situation. 3. In the present experiment, therefore, it aims to the suggestion of an aspect of the improvement in our national eating habits guiding in the nutrition elevation which our nation can afford economically through the development of first, a way of the protein supplement by the mixture of cereals producted plentifully in our country, and second, a way of the decrease in the consumption amount and the improvement in the nutrition of rice through the substitution of the other cereals for rice. III. Contents of Scope of the Study: 1. Objects of the study: Objects of the study are the following three items; a) The nutrition supplement of rice through the mixture of cereals. Our nation makes mainly rice as a principal food, but practically many kinds of cereal are produced in our country. They contain different levels and qualities of each nutrient and they are different from one another in the kinds of essential amino acid consisting protein. For that reason, the mutual complement efficeincy of insufficient nutrients is observed through the mixture of cereals. b) The nutrition supplement of rice through the addition of superior protein sources to rice, a principal food. The development of rice as superior foods in the sense of nutrition is conducted through the risement in protein quality by the addition of protein sources in good quality, for example, fish flour (anchovy flour), egg powder, milk powder, and so on, and through the supplement of vitamins and minerals. c) The decrease in the consumption amount of rice through the substitution of the other cereals for rice, as a principal food. The compensation for the short amount of rice is made by the reduction in the consumption of rice through the discovery of a way of substitution of the other cereals for rice, as a principal food and of the settlement of problems in nutrition and finance subsequent to this. 2. Contents of the study: a) An ideal mixture-ratio of cereals is established for rats by feeding mixed foods(rice-barely or rice-wheat) containing 5%, 15%, 25%, 35% and 45% level of either barely or wheat. b) The nutritive value is determined in the whole subsititution of other foods for rice, and then, a way of the complement of over and under nutrients is devised. c) The ideal combination is investigated for rats through feeding mixed foods of main food, rice and supplement foods of protein sources, soy bean, fish flour, egg powder and milk Powder. d) According to results from the above three experiments, the concise functional test for men and the examination of economical property are made. 3. Scope of the study: a) The observation of the effect of each diet on the growth rate for rats. The growth rate of rats was observed for 15 groups of mixed foods of a main food, rice, and wheat flour, barley powder or soy bean powder, respectively, and 12 groups of wheat flour diets supplemented with $1{\sim}3%$ milk powder, and rice or wheat flour diets supplemented with 5% of milk powder, egg powder, fish flour or soy bean powder, respectively. b) The determination of food consumption. The food consumption was determined at weekly intervals for 27 kinds of diet described in a) item. c) The determination of food efficiency rate. The food efficiency rate for each diet was determined by calculation from the gained body weight and the food consumption amount at the same intervals described in b) item. d) The determination of protein efficiency rate. The protein efficiency rate for each diet was determined by calculation form gained body weight and the protein amount of the food consumption amount at the same intervals described c) item. e) The determination of the body component. The hematocrite and hemoglobin levels in the blood, total nitrogen in the serum, blood sugar, and lipids and glycogen in the liver were determined. f) The observation of nitrogen balance. As a means of the observation of nitrogen balance, the total nitrogen in the urine was determined. g) The analysis of economical property. The economical property was analyzed as the gained body weight to the amount equivalent to one won through the conversion of the food consumption amount into money. h) The functional test for men. The concise functional test for men was made in order to establish if the best diet for experimental animals can be applied to men. IV. Results of the Study: The national food product plan, nationwide nutritive enlightment and the improvement activities in our country eating habits, especially, mixed and powder food problems are to be significantly referred, and the following results must be applied. a) In the mixed foods of cereals, the mixed food of the rice-barley containing $5{\sim}15%$ level of barley is best in terms of nutrition. b) The addition of superior protein sources, egg, Bilk, soy bean, or fish, respectively to either rice or wheat flour makes a great risement in the nutritive value. c) The animal protein is more effective in the elevation of nutritive value of cereals. d) Rice takes the most nutritive operation and has the highest preference, among rice, wheat flour and barley. e) Wheat flour is more economical than rice in evaluation of the gained body weight to the regular money, and the addition of fish or soy bean is more economical than that of any other supplement food. But the above results are true of the range of nutrition and economical property. f) The study on the nutrition composition and barley will lead to the improvement in our national eating habits as mixed food of the rice-barley containing $5{\sim}15%$ level of barley is more nutritive. g) This study on the nutrition only for the growing animal can not be considered as a perfect and entire evaluation. Consequently, the perfect data for our national nutrition can be obtained from the experiment similar to this for the much longer period examining, in details, the growth rate, change of physical strength, mental and bodily change, average life span, and resistance ability to infectious diseases.

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O-Methylation of Flavonoids Using DnrK Based on Molecular Docking

  • Kim, Na-Yeon;Kim, Jeong-Ho;Lee, Youn-Ho;Lee, Eun-Jung;Kim, Jin-Young;Lim, Yoong-Ho;Chong, You-Hoon;Ahn, Joong-Hoon
    • Journal of Microbiology and Biotechnology
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    • 제17권12호
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    • pp.1991-1995
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    • 2007
  • O-Methylation is a common substitution reaction found in microbes as well as in mammalians. Some of the O-methyltransferases (OMTs) have broad substrate specificity and could be used to methylate various compounds. DnrK from Streptomyces peucetius encodes an anthracycline 4-O-methyltransferase, which uses carminomycin as a substrate, and its crystal structure has been determined. Molecular docking experiments with DnrK using various flavonoids were successfully conducted, and some of the flavonoids such as apigenin and genistein were predicted to serve as substrates. Based on these results, O-methylations of various flavonoids with the DnrK were successfully carried out. The methylation position was determined to be at the hydroxyl group of C7. Important amino acid residues for the enzymatic reaction of DnrK with apigenin could be identified using site-directed mutagenesis. Molecular docking could be useful to predict the substrate specificity ranges of other OMTs.

Identification of a novel mutation in a patient with pseudohypoparathyroidism type Ia

  • Lee, Ye Seung;Kim, Hui Kwon;Kim, Hye Rim;Lee, Jong Yoon;Choi, Joong Wan;Bae, Eun Ju;Oh, Phil Soo;Park, Won Il;Ki, Chang Seok;Lee, Hong Jin
    • Clinical and Experimental Pediatrics
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    • 제57권5호
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    • pp.240-244
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    • 2014
  • Pseudohypoparathyroidism type Ia (PHP Ia) is a disorder characterized by multiform hormonal resistance including parathyroid hormone (PTH) resistance and Albright hereditary osteodystrophy (AHO). It is caused by heterozygous inactivating mutations within the Gs alpha-encoding GNAS exons. A 9-year-old boy presented with clinical and laboratory abnormalities including hypocalcemia, hyperphosphatemia, PTH resistance, multihormone resistance and AHO (round face, short stature, obesity, brachydactyly and osteoma cutis) which were typical of PHP Ia. He had a history of repeated convulsive episodes that started from the age of 2 months. A cranial computed tomography scan showed bilateral calcifications in the basal ganglia and his intelligence quotient testing indicated mild mental retardation. Family history revealed that the patient's maternal relatives, including his grandmother and 2 of his mother's siblings, had features suggestive of AHO. Sequencing of the GNAS gene of the patient identified a heterozygous nonsense mutation within exon 11 (c.637 C>T). The C>T transversion results in an amino acid substitution from Gln to stop codon at codon 213 ($p.Gln213^*$). To our knowledge, this is a novel mutation in GNAS.

Associations of Polymorphisms in the Mx1 Gene with Immunity Traits in Large WhitexMeishan F2 Offspring

  • Li, X.L.;He, W.L.;Deng, C.Y.;Xiong, Y.Z.
    • Asian-Australasian Journal of Animal Sciences
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    • 제20권11호
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    • pp.1651-1654
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    • 2007
  • The mouse myxovirus resistance protein 1 (Mx1) is known to be sufficient to confer resistance to influenza viruses, and the gene encoding Mx1 is, therefore, an interesting candidate gene for disease resistance in farm animals. The porcine Mx1 gene has already been identified and characterized based on its homology with mouse Mx1; the full-length coding region of the pig Mx1 gene spans 2,545 bp (M65087) and is organized into 17 exons compared with the human ortholog mRNA. In this study, the exons 9, 10 and 11 and introns 6 and 9 of the porcine Mx1 gene were cloned and sequenced. Two SNPs were identified in exons 9, 10 and 11 but none of the SNPs led to an amino acid exchange, and the other eleven variants were detected in introns 6 and 9, respectively. Differences in allele frequency between Meishan and other pig breeds were observed within intron 6, of which an $A{\rightarrow}G$ substitution at position 371 was detected as an SnaBI PCR-RFLP. The association analysis using the Large White${\times}$Meishan $F_2$ offspring suggested that the Mx1 genotype was associated with variation in several immunity traits that are of interest in pig breeding. However, further investigations in more populations are needed to confirm the above result.

Molecular Characterization and Expression Analysis of Equine Vascular Endothelial Growth Factor Alpha (VEGFα) Gene in Horse (Equus caballus)

  • Song, Ki-Duk;Cho, Hyun-Woo;Lee, Hak-Kyo;Cho, Byung Wook
    • Asian-Australasian Journal of Animal Sciences
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    • 제27권5호
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    • pp.743-748
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    • 2014
  • The objective of this study was to determine the molecular characteristics of the horse vascular endothelial growth factor alpha gene ($VEGF{\alpha}$) by constructing a phylogenetic tree, and to investigate gene expression profiles in tissues and blood leukocytes after exercise for development of suitable biomarkers. Using published amino acid sequences of other vertebrate species (human, chimpanzee, mouse, rat, cow, pig, chicken and dog), we constructed a phylogenetic tree which showed that equine $VEGF{\alpha}$ belonged to the same clade of the pig $VEGF{\alpha}$. Analysis for synonymous (Ks) and non-synonymous substitution ratios (Ka) revealed that the horse $VEGF{\alpha}$ underwent positive selection. RNA was extracted from blood samples before and after exercise and different tissue samples of three horses. Expression analyses using reverse transcription-polymerase chain reaction (RT-PCR) and quantitative-polymerase chain reaction (qPCR) showed ubiquitous expression of $VEGF{\alpha}$ mRNA in skeletal muscle, kidney, thyroid, lung, appendix, colon, spinal cord, and heart tissues. Analysis of differential expression of $VEGF{\alpha}$ gene in blood leukocytes after exercise indicated a unimodal pattern. These results will be useful in developing biomarkers that can predict the recovery capacity of racing horses.

Polymorphism of Exon 2 of BMP15 Gene and Its Relationship with Litter Size of Two Chinese Goats

  • Wang, Yuqin;Yuanxiao, Li;Nana, Zhang;Zhanbin, Wang;Junyan, Bai
    • Asian-Australasian Journal of Animal Sciences
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    • 제24권7호
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    • pp.905-911
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    • 2011
  • Polymorphisms of BMP15 gene exon 2 and its relationship with prolificacy of goats were detected by PCR-SSCP and DNA sequencing methods in Chinese two local goat breeds. The results showed that the product amplified by the primers displayed polymorphisms. Three genotypes (AA, BB and AB) were detected in Funiu white goats, and their frequency was 0.071, 0.715, 0.214, respectively. Two genotypes (AB and BB) were detected in Taihang black goats, and their frequency was 0.342 and 0.658, respectively. Sequencing revealed that four mutations (456T${\rightarrow}$G, 466C${\rightarrow}$G, 510C${\rightarrow}$T, 511T${\rightarrow}$C) occurred in genotype BB of Funiu white goat, which resulted in amino acid substitution of V155G and S171P. No mutation was detected in Taihang black goat. The Funiu white goat with genotype BB had 0.91 or 0.82 kids, more than those with AB or AA, respectively. The difference of the least squares means for litter size between BB and AB was not significant (p>0.05) in Taihang black goat. It is concluded that the BMP15 gene may be a major gene which affects the prolificacy in Funiu white goats. This study could provide basic molecular data on the reproductive characteristics of local breeds of Henan province in China, and a scientific basis for the conservation and utilization of those two goat breeds.

Importance of Leu-5 and Pro-6 in the Inhibitory Activity of the Serratia marcescens Metalloprotease Inhibitor (SmaPI)

  • Bae, Kwang-Hee;Kim, Dong-Min;Kim, Sun-Taek;Kim, Tae-Hoon;Shin, Yong-Chul;Byun, Si-Myung
    • BMB Reports
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    • 제34권2호
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    • pp.109-113
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    • 2001
  • The Serratia marcescens metalloprotease inhibitor (SmaPI) is a proteinase inhibitor toward Serratia marcescens metalloprotease (SMP). The three-dimensional structure of SmaPI was calculated by computer modeling using the structure complex between SMP and the Erwinia chrysanthemi inhibitor as a template. Based on this model structure, the substitution of the amino acid residues, Ala4, Leu-5, Pro-6, and Thr-7, were located at the hinge region of the N-terminal segment by site-directed mutagenesis. Although the A4R and T7A mutant SmaPIs showed a nearly full inhibitory activity, the inhibitory activity of SmaPI decreased significantly when the Leu-5 was converted to Ala, Gly, Ile, or Val. Surprisingly, the L5I and L5V mutant SmaPIs showed less inhibitory activities than the L5A mutant. From these results, we suggested that the orientations and positions of respective aliphatic groups in the side-chain of position 5 mainly affected the inhibitory activity of SmaPI. The overall side-chain hydrophobicity was only slightly affected. The side-chain of the Leu-5 residue contributed approximately 0.79 kcal/mol out of 8.44 kcal/mol to the binding of SmaPI with SMP The inhibitory activities of P6A and F6G were also severely decreased. The Pro-6 may have a critical role in maintaining the strict conformation of the N-terminal portion that may be important in the inhibitory activity of SmaPI. In conclusion, Leu-5 and Pro-6 have crucial roles in the inhibitory function of SmaPI toward SMP.

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