• Title/Summary/Keyword: Genetic screening

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Directed Evolution in Protein Functionality Improvement (단백질 기능 향상을 위한 방향적 진화)

  • 강환구;김학성
    • KSBB Journal
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    • v.16 no.2
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    • pp.107-114
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    • 2001
  • The dynamic evolution process has resulted in the myriad shapes, functions, and systems evident in every living organism. For centuries, people have been harnessing the power of evolution to produce new varieties of plants and animals, such as producing tomatoes from berries and Chihuahuas from wolves. Now scientists are using it to produce better molecules, ranging from drugs to industrial chemicals, and doing it in days or weeks rather than eons. The ingenious process, which creates genetic diversity and selects those with desired features in the laboratory, is called directed evolution or test tube evolution. In this paper, concepts of directed molecular evolution and some examples will be discussed.

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Clinical and Laboratory Characteristics of Galactokinase Hyperactivity (GALK Hyperactivity로 인한 갈락토스혈증의 임상적 특성에 관한 연구)

  • Yang, Seung Do;Lee, Jungho;Shin, Young Lim;Lee, Dong Hwan;Hong, Yong Hee
    • Journal of The Korean Society of Inherited Metabolic disease
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    • v.16 no.3
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    • pp.135-140
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    • 2016
  • Purpose: Galactose is metabolized to galactose-1-phosphate by galactokinase (GALK), galactose-1-phosphate uridyltransferase (GALT) and UDP-galactose-4-epimerase (GALE), and galactosemia occurs when each enzyme is deficient. In Korea, unlike foreign countries, classic galactosemia is rare and transient galactosemia due to GALK hyperactivity is reported, but studies on frequency, clinical significance, and genetic variation are lacking. In this study, we analyzed the clinical characteristics of patients with galactosemia due to GALK hyperactivity. Methods: We investigated 85 patients who had an elevated galactose level in the neonatal screening test without deficiency of enzymes at Department of Pediatrics, Seoul & Bucheon Soonchunhyang University Hospital from January 2008 to June 2016. We investigated the level of galactose, galactose-1-phosphate, GALK and duration of galactose normalization, and analyzed the correlation between GALK elevation and galactose, galactose-1-phosphate and duration of galactose normalization. And the levels of galactose, galactose-1-phosphate, and duration of galactose normalization were compared between the galactose-free formula feeding group and non-feeding group. Results: Mean age of visit was $26.7{\pm}16.1days$. Duration of galactose normalization was $35.3{\pm}20.5days$. Mean galactose level was $18.5{\pm}7.3mg/dL$ in the neonatal screening and follow-up galactose level in serum was $2.3{\pm}5.4mg/dL$. The mean value of galactose-1-phosphate was $6.0{\pm}4.7mg/dL$ and the mean GALK level was $3.84{\pm}1.28{\mu}mol/Hr/gHb$. There was no significant correlation between GALK levels and galactose levels in the neonatal screening test (P=0.351), and we analyzed the correlation between GALK levels and follow-up galactose levels in serum, there was no significant correlation (P=0.101). There was a significant correlation between GALK levels and galactose-1-phosphate (P=0.015), and the correlation between GALK levels and duration of galactose normalization was not statistically significant (P=0.176). 49% of the patients were fed galactose-free formula, and 45% were not. Galactose and galactose-1-phosphate levels in the neonatal screening test were statistically significantly higher (P=0.004, 0.034) in using galactose-free formula group. Duration of galactose normalization was not related to the use of galactose-free formula (P=0.266, 0.249). Conclusion: Galactosemia due to GALK hyperactivity seems to be a temporary phenomenon and may not require galactose restriction. More research is needed on the role of the nuclear protein, racial traits and genetic variations in Korean patients.

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Development of cell models for high-throughput screening system of Charcot-Marie-Tooth disease type 1

  • Choi, Yu-Ri;Jung, Sung-Chul;Shin, Jinhee;Yoo, So Young;Lee, Ji-Su;Joo, Jaesoon;Lee, Jinho;Hong, Young Bin;Choi, Byung-Ok
    • Journal of Genetic Medicine
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    • v.12 no.1
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    • pp.25-30
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    • 2015
  • Purpose: Charcot-Marie-Tooth disease (CMT) is a peripheral neuropathy mainly divided into CMT type 1 (CMT1) and CMT2 according to the phenotype and genotype. Although molecular pathologies for each genetic causative have not been revealed in CMT2, the correlation between cell death and accumulation of misfolded proteins in the endoplasmic reticulum (ER) of Schwann cells is well documented in CMT1. Establishment of in vitro models of ER stress-mediated Schwann cell death might be useful in developing drug-screening systems for the treatment of CMT1. Materials and Methods: To develop high-throughput screening (HTS) systems for CMT1, we generated cell models using transient expression of mutant proteins and chemical induction. Results: Overexpression of wild type and mutant peripheral myelin protein 22 (PMP22) induced ER stress. Similar results were obtained from mutant myelin protein zero (MPZ) proteins. Protein localization revealed that expressed mutant PMP22 and MPZ proteins accumulated in the ER of Schwann cells. Overexpression of wild type and L16P mutant PMP22 also reduced cell viability, implying protein accumulation-mediated ER stress causes cell death. To develop more stable screening systems, we mimicked the ER stress-mediated cell death in Schwann cells using ER stress inducing chemicals. Thapsigargin treatment caused cell death via ER stress in a dose dependent manner, which was measured by expression of ER stress markers. Conclusion: We have developed genetically and chemically induced ER stress models using Schwann cells. Application of these models to HTS systems might facilitate the elucidation of molecular pathology and development of therapeutic options for CMT1.

Screening of Humectant to Improve Conservatation of Bacterial Culture Agar Plate (미생물 배양 배지의 보관성을 개선하기 위한 보습성분의 탐색)

  • Hong, Su-Young;Seo, Yu-Mi;Kang, Chang-Youl;Lee, Byung-Woo;Oh, Sung-Kwan;Kwon, Moo-Sik;Lee, Jin-Sung
    • Proceedings of the KAIS Fall Conference
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    • 2009.12a
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    • pp.937-941
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    • 2009
  • 현재 가장 일반적으로 사용되는 미생물 배양 용기인 petri dish를 통해 제조된 미생물 배양 배지의 보관은 냉장과 실온 보관 시 수분 증발에 대한 보호 효과가 극히 낮아 한 달 이상으로 배지 품질을 유지하기 어려운 단점이 있다. 따라서 본 연구는 petri dish를 이용하여 미생물 배양 배지를 제조할 때 glycerol 등 7종의 보습성분을 첨가하여 이에 따른 미생물 배양 배지의 수분 증발 억제 효과와 표준균주별 증식반응을 비교, 분석한 것으로 결과적으로 sodium lactate를 제외한 6종의 보습성분이 배지의 수분 증발을 억제하는 효과가 있으며 이들에 대한 표준균주별 증식반응에서도 양성의 결과를 보여주었다. 앞으로 본 연구의 결과는 즉시 사용 배지(PPM, Pre-Plated Media)의 유통기한 연장 효과 및 품질향상에 유용한 자료로 활용될 수 있을 것으로 기대된다.

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Hesperidin Suppresses Melanosome Transport by Blocking the Interaction of Rab27A-Melanophilin

  • Kim, Bora;Lee, Jee-Young;Lee, Ha-Yeon;Nam, Ky-Youb;Park, JongIl;Lee, Su Min;Kim, Jin Eun;Lee, Joo Dong;Hwang, Jae Sung
    • Biomolecules & Therapeutics
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    • v.21 no.5
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    • pp.343-348
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    • 2013
  • We investigated the inhibitory effects of hesperidin on melanogenesis. To find melanosome transport inhibitor from natural products, we collected the structural information of natural products from Korea Food and Drug Administration (KFDA) and performed pharmacophore-based in silico screening for Rab27A and melanophilin (MLPH). Hesperidin did not inhibit melanin production in B16F10 murine melanoma cells stimulated with ${\alpha}$-melanocyte stimulating hormone (${\alpha}$-MSH), and also did not affect the catalytic activity of tyrosinase. But, hesperidin inhibited melanosome transport in melanocyte and showed skin lightening effect in pigmented reconstructed epidermis model. Therefore, we suggest that hesperidin is a useful inhibitor of melanosome transport and it might be applied to whitening agent.

Cloning and Expression of Bovine Polyadenylate Binding Protein 1 cDNA in Mammary Tissues

  • Kim, J.H.;Jeon, D.H.;Choi, Y.J.;Baik, M.G.
    • Asian-Australasian Journal of Animal Sciences
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    • v.14 no.6
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    • pp.771-776
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    • 2001
  • A pregnant-induced clone was identified by differential screening from a cDNA library of bovine mammary gland. The clone was identified as a cDNA encoding a polyadenylate binding protein 1 (PABP). The cDNA clone had a total length of 1,911 nucleotides coding for 636 amino acids. The nucleotide sequence of the bovine PABP was 95% and 94% identical to those of human and mouse species, respectively. Comparison of the deduced amino acid sequences of bovine PABP with those of human species showed 100% identity. Induction of the PABP mRNA was observed in bovine mammary tissues at pregnant 7 and 8 months compared to virgin, lactating and involuted states. Expression of the PABP gene was examined in mammary epithelial HC11 cells at proliferating, differentiated and apoptotic conditions. The mRNA levels of PABP gene were similar between proliferating and differentiated cells, but expression levels were very low in apoptotic cells compared to other conditions. Results demonstrate that the PABP gene is induced during pregnancy at which stage mammary epithelial cells are actively proliferating.

Identification of New Microsatellite Markers in Panax ginseng

  • Kim, Joonki;Jo, Beom Ho;Lee, Kyoung Lyong;Yoon, Eui-Soo;Ryu, Gi Hyung;Chung, Ki Wha
    • Molecules and Cells
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    • v.24 no.1
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    • pp.60-68
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    • 2007
  • Microsatellites, also called simple sequence repeats (SSR), are very useful molecular genetic markers commonly used in crop breeding, species identification and linkage analysis. In the present study, we constructed a microsatellite-enriched genomic library of Panax ginseng, and identified 251 novel microsatellite sequences. Tri-nt repeat units were the most abundant (46.6%), followed by di-nt repeats (35.5%). The $(AG)_n$ motif was most common (23.1%), followed by the $(AAC)_n$ motif (22.3%). From the genotyping of 94 microsatellites using marker-specific primer sets, we identified 11 intraspecific polymorphic markers as well as 14 possible interspecific polymorphic markers differing between P. ginseng and P. quinquefolius. The exact allele structures of the polymorphic markers were determined and the alleles were named. This study represents the first report of the bulk isolation of microsatellites by screening a microsatellite-enriched genomic library in P. ginseng. The microsatellite markers could be useful for linkage analysis, genetic breeding and authentication of Panax species.

Haplotype Analysis of BRCA1 Gene D17S855 and D17S1322 Markers in Iranian Familial Breast Cancer Patients

  • Miresmaeili, Sayed Mohsen;Tamandani, Dor Mohammad Kordi;Kalantar, Seyed Mehdi;Moshtaghiun, Seyed Mohammad
    • Asian Pacific Journal of Cancer Prevention
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    • v.17 no.7
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    • pp.3615-3617
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    • 2016
  • Background: Breast cancer molecular analysis by linkage analysis has the advantage of facilitating early diagnosis in asymptomatic genetic carriers, with a view to the preventive follow-up of these subjects and genetic counseling. The aim of this study was to evaluate BRCA1 gene D17S855 and D17S1322 markers in breast cancer patients. Materials and Methods: A series of 85 BC patients and 85 unrelated healthy women were recruited for haplotype analysis performed using two short tandem repeat markers located within the BRCA1 gene locus. Each marker was amplified with PCR genomic DNA from each individual and fluorescently end-labeled primers. Results: Both D17S855 and D17S1322 markers included 12 kinds of alleles. Results indicate that most of the BC patients shared two common 121-150 (11.2%, RR=1.56 and p=0.02) and 121-146 (5.6%, RR=1.9 and p=0.02) haplotypes. Conclusions: Our results should be helpful to understand the haplotype phase in the BRCA1 gene and establish a genetic screening strategy in the Iranian population.

Screening of a New Fibrinolytic Substances-Producing Yeast (혈전용해활성이 우수한 효모의 탐색)

  • Jang, In-Taek;Kim, Young-Hun;Yi, Sung-Hun;Lim, Sung-Il;Lee, Jong-Soo
    • The Korean Journal of Mycology
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    • v.39 no.3
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    • pp.227-228
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    • 2011
  • Fibrinolytic activities of culture concentrates of various yeasts were investigated. The concentrates of the culture broth of Saccharomyces cerevisiae Y99-7 showed the strongest fibrinolytic activity of 25 mm (clear zone). The fibrinolytic activity of Saccharomyces cerevisiae Y99-7 was more high in the culture concentrates from PD broth rather than that of yeast extract-peptone dextrose cultures (clear zone : 22.7 mm).