• Title/Summary/Keyword: Restriction sites

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Association of the Interleukin-1$\beta$ and Interleukin-1 Receptor Antagonist Genetic Polymorphism and Korean Gastric Cancer (Interleukin-1$\beta$ 및 Interleukin-1 Receptor Antagonist의 유전적 다형성과 한국인 위암과의 연관 관계)

  • Park Jik Young;Cho Young Gu;Kim Chang Jae;Park Yong Kyu;Kim Young Sil;Park Cho Hyun;Lee Sug Hyung;Yoo Nam Jin;Lee Jung Young;Park Won Sang
    • Journal of Gastric Cancer
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    • v.2 no.3
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    • pp.163-167
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    • 2002
  • Purpose: Interleukin 1$\beta$ (IL-1$\beta$) polymorphisms are associated with hypochlorhydria, atrophic gastritis, and increased risk of gastric cancer in Caucasians. We tried to determine whether the IL-1.. and IL-1 receptor antagonist (IL-1 RN) genetic polymorphisms contribute to the development of gastric cancer and the specific type of gastritis in Korean. Materials and Methods: The study population was comprised of 128 gastric cancer patients with histologically proven carcinoma and 63 normal healthy individuals. Sixty-eight carcinomas were of intestinal-type and sixty tumors were of diffuse-type. No patient had a familial gastric cancer history. The 511 bp and 31 bp polymorphisms in the IL-1.. were genotyped by polymerase chain reaction (PCR)-restriction fragment length polymorphism. The polymorphism of the IL-1 RN was analyzed with variable number tandem repeat after PCR. Results: The genotype of 511C/-31T of IL-1$\beta$ and allele 1 of IL-1 RN was dominant in the present subjects. The allelic frequencies of the C allele IL-1$\beta$, which is a high risk genotype for gastric cancer, were 0.551 and 0.429 in gastric cancer and normal controls, respectively. Statistically, significant difference in allelic frequencies of three polymorphic sites between gastric cancer patients and normal controls, and between intestinal-type and diffuse-type was not observed. Conclusions: These results suggest that the polymorphisms of IL-1$\beta$ and IL-1 RN may not contribute to the development of Korean gastric caner and that other endogenous or exogenous factors will be important for gastric carcinogenesis.

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Characterization of Barley ${\alpha}$-Amylase Chimeric Enzymes Expressed in Pichia pastoris (Pichia pastoris에서 발현된 보리 알파아밀라제 Chimera 효소들의 특성)

  • Kim, Tae-Jip;Yuk, Jeong-Bin;Choi, Seung-Ho;Jang, Myoung-Uoon;Svensson, Birte
    • Korean Journal of Microbiology
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    • v.46 no.1
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    • pp.80-85
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    • 2010
  • Two different ${\alpha}$-amylase isozymes (AMY1 and AMY2) found in barley malt share up to 80% of amino acid sequence identity with each other, but their enzymatic properties differ remarkably. AMY1 shows the highest activity at low concentration of calcium ion, while AMY2 is highly active at high calcium concentration. Meanwhile, BASI (Barley ${\alpha}$-Amylase/Subtilisin Inhibitor) protein specifically inhibits only AMY2. In the present study, three separate regions in AMY genes (I, II, and III) were assigned on the basis of restriction enzyme sites and four kinds of chimeric amylases have been obtained by swapping a part of regions with each other. Each chimera gene was successfully over-expressed in Pichia pastoris. From the results of enzymatic characterization, both AMY211 and AMY122 showed the mixed or intermediate type of calcium-dependent activity between AMY1 and 2. Meanwhile, only AMY221 chimera could be significantly inhibited by BASI protein. As a result, it can be proposed that some amino acid residues in the region I and II, except region III, of barley ${\alpha}$-amylases play very important roles in calcium-dependency and interaction with BASI.

A Study on a Coping Method of the Family Caregivers of Demented Patients (치매노인 가족부양자의 대처방법에 관한 연구)

  • You, Kwang-Soo
    • Research in Community and Public Health Nursing
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    • v.13 no.4
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    • pp.648-667
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    • 2002
  • This was a descriptive study designed to identify the level of coping method and its influencing factors on the family caregivers of demented patients, and resolve the family caregivers' level of stress. The data were collected from September 10 to October 10, 2001. Subjects for this study were recruited from four clinics, which were chosen from 15 clinics located in Chunbuk-Do as the study sites because of their cooperation for the study. They were similar in terms of size, the characteristics of the local community. and the population and registration status of the demented patients. The instruments used for the study were as follows: 1. Problematic behaviors of demented patients are measured by the Memory and Behavior Problem Checklist (Zarit, 1980), and the Linguistic Communication Symptoms Questionnaire (Bayles and Tomoeda, 1991) 2. The ability to carry out daily activities was measured using the Barthel Index (1965) and Katz Index (1963), which as well-known ADL assessment methods. 3. Burden was measured using Cost of Care Index by the Kosberg and Cairl (1986). 4. Coping strategy was measured Bell's 18 methods (1977). The data were analyzed using SPSS/PC. The study results were as follows: 1. The total stress score was 2.90 out of a maximum score of 5. The highest score reported was 3.09 on the dimension of restriction of individual and social activities, and the lowest region reported was 2.58 on the dimension of mental and physical health. 2. The total score of the coping method was 2.65 out of a maximum score of 5. The highest score reported was 4.01 on the dimension of thinking that includes an ideation such that it is better than any possible worst case, and the lowest score reported was 1.45 on the dimension of the self-image as a scapegoat. 3. There were significant differences in coping method among the subjects by age (F=2.752 p=0.04), caregiver (F=4.33 p=0.003), care-giving period (F=2.68 p=0.049), and dementia stage (F=2.87 p=0.034). 4. There were highly negative correlations ($\gamma$=-0.301 p=0.000) between problematic behaviors of demented patients and the coping method of their family caregivers. The highest correlation coefficient ($\gamma$=-0.339 p=0.000) was found between aggressive behaviors of the demented patients and the coping method of their family caregivers. 5. There was a low negative correlation ($\gamma$=-0.201 p=0.019) between the ADL of the demented patients and the coping method of their family caregivers. 6. There were highly negative correlations ($\gamma$=-0.213 p=0.005) between stress and the coping method of the family caregivers. The highest correlation was found between financial burden ($\gamma$=-.327 P=.000) and the coping method of the family caregivers. There was no significant correlation among unpleasant aspects of the demented patients, willingness to the demented patients, and the coping method of the family caregivers.

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Molecular Cloning of Human Genomic DNA for Epinephrine Synthesizing Enzyme, Phenylethanolamine N-Methyltransferase (Epinephrine 합성효소인 phenylethanolamine N-methyltransferase의 인간 genomic DNA의 유전자 크로닝)

  • Suh, Yoo-Hun;Huh, Sung-Oh;Chun, Yang-Sook;Kim, Hun-Sik;Lim, Jung-Kyoo;Park, Chan-Woong
    • The Korean Journal of Pharmacology
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    • v.24 no.1
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    • pp.1-10
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    • 1988
  • To obtain information about the structure of the human phenylethanolamin N-methyltransferase (PNMT) and to further define the extent of the evolutionary relationships among PNMT molecules of several spesies, a full length cDNA clone for bovine adrenal PNMT was used to screen a charon 4A genomic library. One phage was isolated and identified, which included the entire PNMT gene. The length of inserted genomic DNA was 13.1-Kilobase (Kb) containing two internal EcoRI sites. Construction of a restriction map and subsequent Southern and dot blot analysis with 5'-and3'-specific cDNA probes allowed the identification of exon-containing fragments. This is the first report of the cloning of gene for human epinephrine synthesizing enzyme.

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Pre-Packing, Early Fixation, and Multi-Layer Density Analysis in Analytic Placement for FPGAs (FPGA를 위한 분석적 배치에서 사전 패킹, 조기 배치 고정 및 밀도 분석 다층화)

  • Kim, Kyosun
    • Journal of the Institute of Electronics and Information Engineers
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    • v.51 no.10
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    • pp.96-106
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    • 2014
  • Previous academic research on FPGA tools has relied on simple imaginary models for the targeting architecture. As the first step to overcome such restriction, the issues on analytic placement and legalization which are applied to commercial FPGAs have been brought up, and several techniques to remedy them are presented, and evaluated. First of all, the center of gravity of the placed cells may be far displaced from the center of the chip during analytic placement. A function is proposed to be added to the objective function for minimizing this displacement. And then, the density map is expanded into multiple layers to accurately calculate the density distribution for each of the cell types. Early fixation is also proposed for the memory blocks which can be placed at limited sites in small numbers. Since two flip-flops share control pins in a slice, a compatibility constraint is introduced during legalization. Pre-packing compatible flip-flops is proposed as a proactive step. The proposed techniques are implemented on the K-FPGA fabric evaluation framework in which commercial architectures can be precisely modeled, and modified for enhancement, and validated on twelve industrial strength examples. The placement results show that the proposed techniques have reduced the wire length by 22%, and the slice usage by 5% on average. This research is expected to be a development basis of the optimization CAD tools for new as well as the state-of-the-art FPGA architectures.

Use of the Synthetic Gene Encoding the Truncated Human Rotavirus VP8* Protein in Escherichia coli for Production of Vaccine Candidates or Development of Diagnostic Antibodies (합성 유전자를 이용하여 Escherichia coli에서 백신 후보의 생산 혹은 진단용 항체의 개발을 위한 인간 rotavirus VP8* 부분 단백질의 발현)

  • Kim, Sang-Rae;Lee, Bheong-Uk
    • Journal of Life Science
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    • v.28 no.4
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    • pp.478-482
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    • 2018
  • Human rotavirus is a causative agent of acute diarrhea among children. The artificial gene encoding the truncated $VP8^*$ protein of human rotavirus A (serotype 1 strain WA) was synthesized according to the Escherichia coli codon preference. The synthetic $VP8^*$ gene also possessed the NdeI and HindIII restriction sites for the convenient in-frame cloning for translation and a 6-histidine tag at C-terminus for Ni+ affinity purification. Molecular weight of the truncated $VP8^*$ protein deduced from the nucleotide sequences of the artificial gene was a 19.7-kDa. This synthetic $VP8^*$ DNA fragment was inserted into the pT7-7 expression vector and transformed into E. coli BL21 (DE3). Transformants harboring the synthetic gene encoding the $VP8^*$ protein was induced by supplement of a final concentration of 0.05 mM ITPG at $20^{\circ}C$. Protein crude extract from the E. coli transformants was subjected to Western blotting with the mouse anti-rotavirus capsid antibody, showing ~20-kDa $VP8^*$ protein band. The truncated $VP8^*$ protein band was also observed by Western blotting using the rabbit polyclonal antibody serum made against the truncated $VP8^*$ protein. This study suggested that the synthetic gene could be used as an easy way to produce the antigenic vaccine candidate for control of virus-associated diseases or to develop antibodies for diagnostic purpose.

Influence of Genotype and Haplotype of MDR1 (C3435T, G2677A/T, C1236T) on the Incidence of Breast Cancer - a Case-Control Study in Jordan

  • Abuhaliema, Ali M;Yousef, Al-Motassem F;El-Madany, Nirmeen N;Bulatova, Nailya R;Awwad, Nemah M;Yousef, Muhammad A;Al Majdalawi, Khalil Z
    • Asian Pacific Journal of Cancer Prevention
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    • v.17 no.1
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    • pp.261-266
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    • 2016
  • Background: Breast cancer is the leading cause of cancer death among women and the second in humans worldwide. Many published studies have suggested an association between MDR1 polymorphisms and breast cancer risk. Our aim was to study the association between genetic polymorphism of MDR1 at three sites (C3435T, G2677A/T, and C1236T) and their haplotype and the risk of breast cancer in Jordanian females. Materials and Methods: A case-control study involving 150 breast cancer cases and 150 controls was conducted. Controls were age-matched to cases. The polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP) technique and sequencing were performed to analyse genotypes. Results: The distribution of MDR1 C3435T genotypes differed between cases and controls [cases, CC 45.3%, CT 41.3%, and TT 13.3%; controls, CC 13.4%, CT 43.3%, and TT 30.2%, p < 0.001]. Similarly, the distribution of G2677A/T significantly differed [cases, GG 43.1 %, GT+GA 50.9% and AA+TT 6%; controls, GG 29.6 %, GT+GA 50.9%, and AA+TT 19.4%, p = 0.004]. On the other hand, genotype and allelotype distribution of C1236T was not statistically different between cases and controls (p=0.56 and 0.26, respectively). The CGC haplotype increased the risk to breast cancer by 2.5-fold compared to others, while TGC and TTC haplotypes carried 2.5- and 5-fold lower risk of breast cancer, respectively. Conclusions: Genetic polymorphisms of MDR1 C3435T and G2677A/T, but not C1236T, are associated with increased risk of breast cancer. In addition, CGC, TGC and TTC haplotypes have different impacts on the risk of breast cancer. Future, larger studies are needed to validate these findings.

Relationship between Genetic Polymorphisms in MTHFR (C677T, A1298C and their Haplotypes) and the Incidence Of Breast Cancer among Jordanian Females - Case-Control Study

  • Awwad, Nemah;Yousef, Al-Motassem;Abuhaliema, Ali;Abdalla, Ihab;Yousef, Muhammad
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.12
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    • pp.5007-5011
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    • 2015
  • Background: Breast cancer is a major cause of morbidity and mortality in Jordan and worldwide. Abnormality of DNA methylation is a possible mechanism for the development of cancer. Methylenetetrahydrofolate reductase (MTHFR) is involved in DNA methylation. Our aim was to study the association between genetic polymorphisms of MTHFR at two sites (C677T and A1298C) and their haplotypes and the risk of breast cancer among Jordanian females. Materials and Methods: A case-control study involving 150 breast cancer cases and 150 controls was conducted. Controls were age-matched to cases. Polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP) technique and sequencing were conducted to determine the genotypes. Results: There was a significant difference in genotype frequency of C677T in the 41-60 year age category [cases: CC (37.4%), CT (49.5%) and TT (13.2%); controls: CC (56.3%), CT (35.6%) and TT (8%), p= 0.04; $OR_{TT\;vs.\;CC}$: 2.5, 95% CI: (0.9-6.9); $OR_{at\;least\;on\;T$: 2.1, 95%CI: (1.2-3.9)]. There was no significant difference in genotype frequency of A1298C between cases and controls [cases: AA (46.6%), AC (41.8%) and CC (11.6%); controls: AA (43%), AC (47.4%) and CC (9.6%); p= 0.6]. There was a significant difference of MTHFR genetic polymorphism haplotypes among breast cancer cases and controls [cases/control: CA: 38.3/45.4%; CC: 28.9/25.2%; TA: 29.2/21; TC: 3.6/8.3; p value= 0.01; $OR_{TA\;vs.\;CA}=1.6$; 95% CI (1.1-2.5); p= 0.02]. Conclusions: Genetic polymorphism of MTHFR C677T may modulate the risk of breast cancer especially in the 41-60 year age group. Additionally, TA haplotype amends the risk of breast cancer. Future studies with a larger sample size are needed to validate the role of MTHFR genetic polymorphisms in breast cancer.

PCR-based Determination of the Correct Orientation of Sub cloned DNA Fragments, and its Application in the Rapid Cloning and Recombinant Expression of Rat Urocortin in Eukaryotic Cells (중합효소 연쇄반응에 근거한 벡터 클로닝된 DNA조각의 방향성 결정 및 이를 이용한 랫트 Urocortin의 진핵 세포주상에서의 발현과 클로닝의 수행)

  • Jung-Hyun Park;Yun-Jung Lee;Shin-Young Na;Kil Lyong Kim
    • Biomedical Science Letters
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    • v.6 no.1
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    • pp.73-82
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    • 2000
  • Blunt-end DNA fragments can be inserted in two different orientations. Conventionally, their directions are determined by restriction enzyme digestion or by DNA sequencing, however, these methods are often limited in their use due to the lack of appropriate enzyme sites or large sample numbers, respectively. In the present study, a novel strategy and the corresponding protocol for the simple determination of insert orientation is introduced. Using conventional sequencing primers and PCR primers that have been used for amplification of the insert, single clones, which have inserted the fragment in the desired orientation, were easily identified by this PCR-based method. The fidelity of this system was confirmed by cloning of a tar urocortin cDNA, which is a recently discovered neuropeptide. Recombinant clones identified by this method were further shown to be fully functional, and using these, for the first time, urocortin was recombinantly expressed in eukaryotic cells.

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Cloning and Nucleotide Sequence Analysis of the Virulence Gene Cassette from Vibrio cholerae KNIH002 Isolated in Korea (국내 분리주인 Vibrio cholerae KNIH002로부터 독성 유전자 카세트의 클로닝 및 염기서열 분석)

  • 신희정;박용춘;김영창
    • Korean Journal of Microbiology
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    • v.35 no.3
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    • pp.205-210
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    • 1999
  • 16brio cholerne is an important pathogenic organism that causes dimhea in human beings. V ciaoleroe KNIH002 was isolated from patients suffering with dian.heal disease in Korea. From Southern hybridization using the amplified PCR product of 307 bp as a probe. which was obtained from PCR reaction using primer detecting cholera toxin gene, we have found that the c b gene located in 4.5-kb fragmenl double digested with Pstl and BgllI of the chromosome. Therefore, we made mini-libraries of the isolate using PstI and Bgm restriction endonuclease and pBluescript SKU(+) vector. As a result. we cloned 4.5-kb PstI-BglII fragment containing the c a gene encoding a cholera toxin from the constructed mini-libraries of V olzolerae KNlH002 by colony hybridization using the same probes. This recombinant plasmid was named pCTX75. E. coii XL1- Blue harboring pCTX75 showed the cytotoxicity on Chinese Hamster Ovary cells. From the sequencing of he cloned recombinant plasmid, we confinned that it has virulence gene cassette consisting of ace, zot, ctx.4 and cf"~B gene. The ace and zot genes were composed of 291 hp and 1.200 bp with ATG initiation codon and TGA lennination codon, respectively. Nucleotide sequence of the ace gene exhibited 100% identity with that of V cholera E7946 El Tor Ogawa strains. But, nucleolide and amino acid sequence comparison of the zot gene exhibited 99% and 98.8% identity with that of V cholerae 395 Classical Ogawa stram, respectively. Specially. the Ala-100, Ala-272 and Ala-281 sites of Zoi polypeptide presented in V choleme 395 Classical Ogawa strain are replaced by Val in V cholerae KNIH002.

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