• Title/Summary/Keyword: Point mutation

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Isolation and Characterization of Engineered Nucleoside Deoxyribosyltransferase with Enhanced Activity Toward 2'-Fluoro-2'-Deoxynucleoside

  • Yoo, Yeon-Jin;Choi, Kang-Hyun;Kim, Byoung-Kyun;Choi, Si-Sun;Kim, Eung-Soo
    • Journal of Microbiology and Biotechnology
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    • v.32 no.8
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    • pp.1041-1046
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    • 2022
  • Nucleoside deoxyribosyltransferase (NDT) is an enzyme that replaces the purine or pyrimidine base of 2'-deoxyribonucleoside. This enzyme is generally used in the nucleotide salvage pathway in vivo and synthesizes many nucleoside analogs in vitro for various biotechnological purposes. Since NDT is known to exhibit relatively low reactivity toward nucleoside analogs such as 2'-fluoro-2'-deoxynucleoside, it is necessary to develop an enhanced NDT mutant enzyme suitable for nucleoside analogs. In this study, molecular evolution strategy via error-prone PCR was performed with ndt gene derived from Lactobacillus leichmannii as a template to obtain an engineered NDT with higher substrate specificity to 2FDU (2'-fluoro-2'-deoxyuridine). A mutant library of 214 ndt genes with different sequences was obtained and performed for the conversion of 2FDU to 2FDA (2'-fluoro-2'-deoxyadenosine). The E. coli containing a mutant NDT, named NDTL59Q, showed 1.7-fold (at 40℃) and 4.4-fold (at 50℃) higher 2FDU-to-2FDA conversions compared to the NDTWT, respectively. Subsequently, both NDTWT and NDTL59Q enzymes were over-expressed and purified using a His-tag system in E. coli. Characterization and enzyme kinetics revealed that the NDTL59Q mutant enzyme containing a single point mutation of leucine to glutamine at the 59th position exhibited superior thermal stability with enhanced substrate specificity to 2FDU.

Generation of a transgenic mouse model to study cranial suture development; Apert syndrome (두개봉합 발육 연구를 위한 형질변환 쥐의 개발 : 어퍼트 신드롬)

  • Lee, Kee-Joon;Ratisoontorn, Chootima;Baik, Hyoung-Seon;Park, Young-Chel;Park, Kwang-Kyun;Nah, Hyun-Duck
    • The korean journal of orthodontics
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    • v.33 no.6 s.101
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    • pp.485-497
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    • 2003
  • The form and function of the craniofacial structure critically depend on genetic information. With recent advances in the molecular technology, genes that are important for normal growth and morphogenesis of the craniofacial skeleton are being rapidly uncovered, shaping up modem craniofacial biology. One of them is fibroblast growth factor receptor 2 (FGFR2). Specific point mutations in the. FGFR2 gene have been linked to Apert syndrome, which is characterized by premature closure of cranial sutures and craniofacial anomalies as well as limb deformities. To study pathogenic mechanisms underlying craniosynostosis phenotype of Apert syndrome, we used a transgenic approach; an FGFR2 minigene construct containing an Apert mutation (a point mutation that substitute proline at the position 253 to arginine; P253R) was introduced into fertilized mouse germ cells by DNA microinjection. The injected cells were then allowed to develop into transgenic mice. We used a bone-specific promoter (a DNA fragment from the type I collagen gene) to confine the expression of mutant FGFR2 gene to the bone tissue, and asked whether expression of mutant FGFR2 in bone is sufficient to cause the craniosynostosis phenotype in mice. Initial characterization of these mice shows prematurely closed cranial sutures with facial deformities expected from Apert patients. We also demonstrate that the transgene produces mutant FGFR2 protein with increased functional activities. Having this useful mouse model, we now can ask questions regarding the role of FGFR2 in normal and abnormal development of cranial bones and sutures.

Correlation Between Primary Tuberculous Pleurisy and NRAMP1 Genetic Polymorphism (결핵성 흉막염 환자에서 NRAMP1 유전자 다형성에 대한 연구)

  • Kim, Je-Hyeong;Kim, Byung-Gyu;Jung, Ki-Hwan;Park, Sang-Myun;Lee, Sang-Youb;Lee, Sin-Hyung;Sin, Cheol;Cho, Jae-Youn;Shim, Jae-Jeong;In, Kwang-Ho;Yoo, Se-Hwa;Kang, Kyung-Ho
    • Tuberculosis and Respiratory Diseases
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    • v.48 no.2
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    • pp.155-165
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    • 2000
  • Background: The phagolysosomal function of alveolar macrophage against M. tuberculosis infection is influenced by Nramp1, which is encoded by the NRAMP1 gene. There are several genetic polymorphisms in NRAMP1, and these polymorphisms affect the innate host resistance through the defect in production and function of Nramp1. To investigate this relationship, the NRAMP1 genetic polymorphism in patients with primary tuberculous pleurisy was determined. Methods: Fifty-six primary tuberculous pleurisy patient, who were diagnosed by pleural biopsy, were designated to the pleurisy group and 45 healthy adults were designated to the healthy control group. Three genetic polymorphisms of NRAMP1, such as a single point mutation in intron 4(469+14G/C, INT4), a nonconservative single-base substitution at codon 543 that changes aspartic acid to asparagine(D543N) and a TGTG deletion in the 3' untranslated region(1729+55delI4, 3'UTR), were determined. Polymerase chain reaction(PCR) and polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) were used. Results: The frequencies of mutant genotypes of INT4 and 3'UTR were significantly high in pleurisy group(p=0.001, p=0.023). But the frequencies of D543N were not significantly different between the two groups(p=0.079). The odds ratios, which are a comparison with wild genotype for determining mutant genotypes, were 8. 022(95% confidence interval=2.422-26.572) for INT4 and 5.733(95% confidence interval = 1.137~28.916) for 3'UTR ; these were statistically significant But the ratio for D543N was not significant In the combined analysis of the INT4 and 3'UTR polymorphisms, the odds ratios were 6.000(95% confidence interval = 1.461~24.640) for GC/++ genotype and 14.000(95% confidence interval=1.610~121.754) for GC/+del when compared with GG/++ homozygotes ; these were statistically significant. Conclusion: Among the NRAMP1 genetic polymorphisms, a single point mutation in intron 4(469+14G/C, INT4) and a TGTG deletion in the 3' untranslated region(1729+55del4, 3'UTR) were closely related to the primary tuberculous pleurisy.

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Digital painting: Image transfonnation, simulation, heterologie and transfonnation (현대회화에서의 형태와 물질 -Digital Transfiguration에 관한 연구-)

  • Jeong, Suk-Yeong
    • Journal of Science of Art and Design
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    • v.10
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    • pp.161-181
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    • 2006
  • The words which appeared in my theoretical study and work are image transformation to digital painting, simulation, heterologie and transfiguration, etc. Firstly, let's look into 'digital era' or 'new media era'. Nowadays, the image world including painting within the rapid social and cultural change, which is called as digital era, is having the dramatic change. Together with the development of scientific technology, large number of events which was deemed to be impossible is happening as real in image world Moreover, these changes in image world is greatly influencing to our life. The word which compresses this change of image world and shows is 'digital'. Digit, which means fingers in Latin, indicates separately changing signal, and to be more narrow, it indicates the continual signal of '0' and ' 1' in computer. The opposite word is 'analogue'. As analogue is the word meaning 'infer' or 'similarity', it indicates the signal or form which continuously changes along the series of time when it is compared to digital. Instead of analogue, digital is embossed as a major ruler along the whole area of our current culture. In whole culture and art area, and in whole generalscience, digital is appearing as it has the modernism and importance. The prefix, 'digital', e.g. digital media, digital culture, digital design, digital philosophy, etc, is treated as the synonym of modernism and something new. This advent of digital results the innovative change to the image world, creates the new beauty experience which we could not experience before, and forecasts the formation of advanced art and expansion of creative area. Various intellectual activities using computer is developing the whole world with making the infrastructure. Computer in painting work immediately accomplishes the idea of painters, takes part in simulation work, contingency such as abrupt reversal, extraction, twisting, shaking, obscureness, overlapping, etc, and timing to stimulate the creativity of painters, and provides digital formative language which enables new visual experience to the audience. When the change of digital era, the image appeared in my work is shown in 'transfiguration' like drawing. The word, 'transfiguration' does not indicate the completed and fixed real substance but indicate endlessly moving and floating shape. Thus, this concept is opposite to the substantial consideration, so that various concepts which is able to replace this in accordance with the similar cases are also exist such as change, deterioration, mutation, deformity of appearance and morphing which is frequently used in computer as a technical word. These concepts are not clearly classified, and variably and complicatedly related. Transfiguration basically means the denial of "objectivity' and '(continual) stagnation' or deviation from those. This phenomenon is appeared through the all art schools of art ever since the realism is denied in the 19th century. It is called as 'deformation' in case of expressionism, futurism, cubism, etc, in the beginning of the century, which its former indication is mostly preserved within the process of structural deviation and which has the realistic limit which should be preserved. On the contrary, dramatic transfiguration which has been showing in the modern era through surrealism is different in the point that dramatic transfiguration tends to show the deterioration and deviation rather than the preservation of indicated object. From this point, transfiguration coming out from morphing using computer deteriorates and hides the reality and furthermore, it replaces the 'reality'. Moreover, transfiguration is closely approached to the world of fake or 'imaginary' simulation world of Baudrillard. According to Baudrillard, the image hides and deteriorates the reality, and furthermore, expresses 'not existing' to 'imaginary' under the name of transfiguration. Certain reality, that is, image which is absent from the reality is created and overflowed, so that it finally replaces the reality. This is simulation as it is said by Baudrillard. In turn, Georges Bataille discusses about the image which is produced by digital technology in terms of heterologie. Image of heterologie is the visual signal which is established with the media. Image of media is to have the continuous characteristics of produce, extinction, and transformation, and its clear boundary between images becomes meaningless. The meaning of composition, excess, violation, etc of digital image is explained to heterological study or heteologie suggested as important meaning of Georges Bataille who is a heretic philosopher. As the form and image of mutation shows the shape in accordance with mechanical production, heterologie is introduced as very low materialism (or bas materialisme), in this theory. Heterologie as low materialism which is gradually changing is developing as a different concept and analysis because of the change of time in the late 20s century beside high or low meaning. Including my image, all images non-standardizes and transforms the code. However, reappearance and non-standardization of this code does not seem to be simple. The problem of transformation caused by transfiguration which appears in my digital drawing painting, simulation, heterologie, etc, are the continual problems. Moreover, the subject such as existence of human being, distance from the real life, politics and social problems are being extended to actual research and various expressing work. Especially, individual image world is established by digital painting transfiguration technique, and its change and review start to have the durability. The consciousness of observers who look at the image is changing the subject. Together with theoretical research, researchers are to establish the first step to approach to various image change of digital era painting through transfiguration technique using our realistic and historical image.

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Detection of Point Mutations in the rpoB Gene Related to Drug Susceptibility in Mycobacterium Tuberculosis using an Oligonucleotide Chip (올리고뉴클레오티드 칩(Oligonucleotide Chip)을 이용한 항결핵제 감수성과 관련된 Mycobacterium tuberculosis rpoB 유전자의 점돌연변이 판별 방법)

  • Kim, Hyun-Jung;Kim, Seong-Keun;Shim, Tae-Sun;Park, Yong-Doo;Park, Mi-Sun
    • Tuberculosis and Respiratory Diseases
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    • v.50 no.1
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    • pp.29-41
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    • 2001
  • Background : The appearance of multiple-drug-resistant Mycobacterium tuberculosis strains has been seriously compromising successful control of tuberculosis. Rifampin-resistance, caused by mutations in the rpoB gene, can be indicative of multiple-drug-resistance, and its detection is of great importance. The present study aimed to develop an oligonucleotide chip for accurate and convenient screening of drug-resistance. Methods : In order to detect point mutations in the rpoB gene, an oligonucleotide chip was prepared by immobilizing specific probe DNA to a microscopic slide glass by a chemical reaction. The probe DNA that was selected from the 81 bp core region of the rpoB gene was designed to have mutation sites at the center. A total of 17 mutant probes related to rifampin-resistance including 8 rifabutin-sensitive mutant probes were used in this study. For accurate determination, wild type probes were prepared for each mutation position with an equal length, which enabled a direct comparison of the hybridization intensities between the mutant and wild type. Results : Mycobacterial genomic DNA from clinical samples was tested with the oligonucleotide chip and the results were compared with those of the drug-susceptibility test in addition to sequencing and INNO-LiPA Rif. TB kit test in some cases. Out of 15 samples, the oligonucleotide chip results of 13 samples showed good agreement with the rifabutin-sensitivity results. The two samples with conflicting result also showed a discrepancy between the other tests, suggesting such possibilities as existence of mixed strains and difference in drug-sensitivity. Further verification of these samples in addition to more case studies are required before the final evaluation of the oligonucleotide chip can be made. Conlcusion : An oligonucleotide chip was developed for the detection of rpoB gene mutations related to drugsusceptibility. The results to date show the potential for using the oligonucleotide chip for accurate and convenient screening of drug-resistance to provide useful information in antituberculosis drug therapy.

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Analysis of Mitochondrial Gene Sequence in Etoxazole Resistant Two-Spotted Spider Mite, Tetranychus urticae (Etoxazole 저항성 점박이응애의 미토콘드리아 유전자 서열 분석)

  • Park, Sang-Eun;Koo, Hyun-Na;Yoon, Chang-Mann;Choi, Jang-Jeon;Kim, Gil-Hah
    • The Korean Journal of Pesticide Science
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    • v.16 no.1
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    • pp.54-61
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    • 2012
  • The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is one of the most important pest species devastating many horticultural and ornamental crops and fruit trees. Difficulty in managing this mite is largely attributed to its ability to develop resistance to many important acaricides. Development of 3,700-folds resistance to etoxazole was found in the population of T. urticae collected from rose greenhouses in Buyeo, Chungnam Province in August 2000. This population has been selected for eleven years with etoxazole (over 500 times), and increased over 5,000,000-folds in resistance as compared with susceptible strain. Also, etoxazole-resistant strain was shown to be maternally inherited. The objective of this study was to determine whether resistance of T. urticae to etoxazole was linked with point mutations in the mitochondrial gene. DNA sequencing of cytochrome c oxidase subunit I (COX1), COX2, COX3, cytochrome b (CYTB), NADH dehydrogenase subunit 1 (ND1), ND2, ND3, ND4, ND5, and ND6 were analyzed by comparing two etoxazole-susceptible and etoxazole-resistant strains. As a result, differences were not detected between the nucleotide sequences of two strains within a mitochondrial gene.

Land Use Optimization using Genetic Algorithms - Focused on Yangpyeong-eup - (유전 알고리즘을 적용한 토지이용 최적화 배분 연구 - 양평군 양평읍 일대를 대상으로 -)

  • Park, Yoonsun;Lee, Dongkun;Yoon, Eunjoo;Mo, Yongwon;Leem, Jihun
    • Journal of Environmental Impact Assessment
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    • v.26 no.1
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    • pp.44-56
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    • 2017
  • Sustainable development is important because the ultimate objective is efficient development combining the economic, social, and environmental aspects of urban conservation. Despite Korea's rapid urbanization and economic development, the distribution of resources is inefficient, and land-use is not an exception. Land use distribution is difficult, as it requires considering a variety of purposes, whose solutions lie in a multipurpose optimization process. In this study, Yangpyeong-eup, Yangpyeong, Gyeonggi-do, is selected, as the site has ecological balance, is well-preserved, and has the potential to support population increases. Further, we have used the genetic algorithm method, as it helps to evolve solutions for complex spatial problems such as planning and distribution of land use. This study applies change to the way of mutation. With four goals and restrictions of area, spatial objectives, minimizing land use conversion, ecological conservation, maximizing economic profit, restricting area to a specific land use, and setting a fixed area, we developed an optimal planning map. No urban areas at the site needed preservation and the high urban area growth rate coincided with the optimization of purpose and maximization of economic profit. When the minimum point of the fitness score is the convergence point, we found optimization occurred approximately at 1500 generations. The results of this study can support planning at Yangpyeong-eup.ausative relationship between the perception of improving odor regulation and odor acceptance.

P53 Expression in Squamous Cell Carcinomas of Tongue and Tonsil (설과 편도 편평 상피세포암에서 P53의 표현양상에 관한 연구)

  • Choi, Geon;Kim, Man-Su;Choi, Jong-Ouck;Hwang, Soon-Jae;Yoo, Hong-Kyun
    • Proceedings of the KOR-BRONCHOESO Conference
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    • 1993.05a
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    • pp.83-83
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    • 1993
  • The nuclear phosphoprotein p53 is expressed in all normal cells and appears to function in cell cycle regulation. Abnormally high levels of the protein are found in many different types of cancer. In human cancer overexpression of p53 is associated with point mutations within highly conserved regions of p53 gene. These altered genes encode stable p53 proteins that can detected by standard immunocytochemical techniques unable to detect rapidly degraded wild-type protein. Using of a monoclonal antibody to p53 antigen, immunocytochemical analysis of 29 squamous cell carcinomas of tongue(n= 19) and tonsil(n= 10) was performed. Non-tumor nuclei showed all negative reactivity. Positive reactivity was found in 4/29(13.8%)of SCCs of tongue and tonsil. In sizes of primary tumor, the cases over 4cm showed more positive reactivity than the cases under 4cm(p < 0.05). There was no stastical correlation between the reactivity and histopathologic grades, the primary sites of tumor or the presence of cervical metastasis.

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Chromosomal Localization and Mutation Detection of the Porcine APM1 Gene Encoding Adiponectin (Adiponectin을 암호화하는 돼지 APM1 유전자의 염색체상 위치파악과 돌연변이 탐색)

  • Park, E.W.;Kim, J.H.;Seo, B.Y.;Jung, K.C.;Yu, S.L.;Cho, I.C.;Lee, J.G.;Oh, S.J.;Jeon, J.T.;Lee, J.H.
    • Journal of Animal Science and Technology
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    • v.46 no.4
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    • pp.537-546
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    • 2004
  • Adiponectin is adipocyte complement-related protein which is highly specialized to play important roles in metabolic and honnonal processes. This protein, called GBP-28, AdipoQ, and Acrp30, is encoded by the adipose most abundant gene transcript 1 (APM1) which locates on human chromosome 3q27 and mouse chromosome 16. In order to determine chromosomal localization of the porcine APM1, we carried out PCR analysis using somatic cell hybrid panel as well as porcine whole genome radiation hybrid (RH) panel. The result showed that the porcine APM1 located on chromosome 13q41 or 13q46-49. These locations were further investigated with the two point analysis of RH panel, revealed the most significant linked marker (LOD score 20.29) being SIAT1 (8 cRs away), where the fat-related QTL located. From the SSCP analysis of APM1 using 8 pig breeds, two distinct SSCP types were detected from K~ native and Korean wild pigs. The determined sequences in Korean native and Korean wild pigs showed that two nucleotide positions (T672C and C705G) were substituted. The primary sequence of the porcine APM1 has 79 to 87% identity with those of human, mouse, and bovine APM1. The domain structures of the porcine APM1 such as signal sequence, hypervariable region, collagenous region. and globular domain are also similar to those of mammalian genes.

Diagnosis of Pigs Producing PSE Meat using DNA Analysis (DNA검사기법을 이용한 PSE 돈육 생산 돼지 진단)

  • Chung Eui-Ryong;Chung Ku-Young
    • Food Science of Animal Resources
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    • v.24 no.4
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    • pp.349-354
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    • 2004
  • Stress-susceptible pigs have been known as the porcine stress syndrome (PSS), swine PSS, also known as malignant hyperthermia (MH), is characterized as sudden death and production of poor meat quality such as PSE (pale, soft and exudative) meat after slaughtering. PSS and PSE meat cause major economic losses in the pig industry. A point mutation in the gene coding for the ryanodine receptor (RYR1) in porcine skeletal muscle, also known calcium (Ca$^{2+}$) release channel, has been associated with swine PSS and halothane sensitivity. We used the PCR-RFLP(restriction fragment length polymorphism) and PCR-SSCP (single strand conformation polymorphism) methods to detect the PSS gene mutation (C1843T) in the RYR1 gene and to estimate genotype frequencies of PSS gene in Korean pig breed populations. In PCR-RFLP and SSCP analyses, three genotypes of homozygous normal (N/M), heterozygous carrier (N/n) and homozygous recessive mutant (n/n) were detected using agarose or polyacrylamide gel electrophoresis, respectively. The proportions of normal, carrier and PSS pigs were 57.1, 35.7 and 7.1% for Landrace, 82.5, 15.8 and 1.7% far L. Yorkshire, 95.2, 4.8 and 0.0% for Duroc and 72.0, 22.7 and 5.3% for Crossbreed. Consequently, DNA-based diagnosis for the identification of stress-susceptible pigs of PSS and pigs producing PSE meat is a powerful technique. Especially, PCR-SSCP method may be useful as a rapid, sensitive and inexpensive test for the large-scale screening of PSS genotypes and pigs with PSE meat in the pork industry.y.