• Title/Summary/Keyword: DNA screening

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Characterization of a Phage Library Displaying Random 22mer Peptides

  • Lee, Seung-Joo;Lee, Jeong-Hwan;Kay, Brian K.;Dreyfuss, Gideon;Park, Yong-Keun;Kim, Jeong-Kook
    • Journal of Microbiology
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    • v.35 no.4
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    • pp.347-353
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    • 1997
  • We have characterized a phage library displaying random 22mer peptides which were produced as N-terminal fusions to the pIII coat protein of M13 filamentous phages. Among the sixty phages randomly picked from the library, 25 phages had the 22mer peptide inserts. The DNA sequence analysis of the 25 inserts showed the following results: first, each nucleotide was represented almost equally at each codon position except that there were some biases toward G bases at the first position of the codons. Secondly, the expected 47 sense codons were represented. The deduced amino acid sequences of the 25 inserts were analyzed to examine its diversity. Glycine and glutamate were the two most overrepresented residues above the expected value, whereas cysteine and threonine residues were underrepresented. The range of dicersity in dipeptide sequences showed that the amino acid residues were randomly distributed along the peptide insert. Acidic, basic, polar, and nonpolar amino acid residues were represented to the extent expected at most positions of the peptide inserts. The predicted isoelectric points and hydropathy indices of the 25 peptides showed that a variety of the peptide were represented in the library. These results indicate that this phage display library could be useful in fiuding ligands for a broad spectrum of receptors by affinity screening.

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Aburatubolactam C, a Novel Apoptosis-inducing Substance Produced by Marine Streptomyces sp. SCRC A-20

  • Bae, Myung-Ae;Yamada, Kaoru;Uemura, Daisuke;Seu, Jung-Hwan;Kim, Young-Ho
    • Journal of Microbiology and Biotechnology
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    • v.8 no.5
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    • pp.455-460
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    • 1998
  • In the course of screening for new antitumor substances, a novel cytotoxic agent inducing apoptotic cell death was isolated from the culture broth of marine bacterial strain SCRC A-20. Strain SCRC A-20 was separated from a mollusk and was chemotaxonomically identified as a Streptomyces sp. The cytotoxic substance was purified by organic solvent extraction followed by silica gel column chromatography and preparative TLC. HRFAB-MS determined its molecular formula to be $C_{30}H_{40}N_2O_5$ (MW 508). The 1D and 2D NMR spectral data demonstrated that the substance has a novel lactam structure of a 20-membered macrocycle coupled with a unique acyl tetramine and bicyclo[3.3.0] octane, which includes three methyl groups, six olefinic protons, five carbonyl groups, a conjugated diene and a dienone. The substance, named aburatubolactam C, appeared to be cytotoxic for various continuously proliferating tumor cells of human and murine origins. The $IC_{50}$ values determined by MIT assay were in the range of 0.3 to $5.8\mug/ml$. When Jurkat T cells were treated with $3\mug/ml$. of aburatubolactam C, the apoptotic DNA fragmentation was detectable within 3 h, indicating that the cytotoxic effect of aburatubolactam C on tumor cells is attributable to the induced apoptosis.

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Association Analyses with Carcass Traits in the Porcine KIAA1717 and HUMMLC2B Genes

  • Xu, D.Q.;Xiong, Y.Z.;Liu, M.;Lan, J.;Ling, X.F.;Deng, C.Y.;Jiang, S.W.
    • Asian-Australasian Journal of Animal Sciences
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    • v.18 no.11
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    • pp.1519-1523
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    • 2005
  • By screening a subtracted cDNA library constructed with mRNA obtained from the longissimus dorsi muscles of F1 hybrids Landrace${\times}$Yorkshire and their Yorkshire female parents, we isolated two partial sequences coding for the H3-K4-specific methyltransferase (KIAA1717) and skeletal muscle myosin regulatory light chain (HUMMLC2B) genes. In the present work we investigated two SNPs, one (C1354T) at the 3' untranslated region (UTR) of KIAA1717 and one (A345G) at the SINE (PRE-1) element of HUMMLC2B, in a resource population derived from crossing Chinese Meishan and Large White pig. The selected pigs were genotyped by means of a PCR-RFLP protocol. Significant associations were observed for the KIAA1717 C1354T polymorphic site with thorax-waist backfat thickness (p<0.05), buttock backfat thickness (p<0.05), average backfat thickness (p<0.05), loin eye height (p<0.05), loin eye area (p<0.05), carcass length to 1$^{st}$ spondyle (p<0.01) and carcass length to 1st rib (p<0.01). HUMMLC2B A345G were significantly associated with loin eye width (p<0.05), loin eye area (p<0.05). Further studies are needed to confirm these preliminary results.

HtrA2 Interacts with Aβ Peptide but Does Not Directly Alter Its Production or Degradation

  • Liu, Meng-Lu;Liu, Ming-Jie;Kim, Jin-Man;Kim, Hyeon-Jin;Kim, Jeong-Hak;Hong, Seong-Tshool
    • Molecules and Cells
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    • v.20 no.1
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    • pp.83-89
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    • 2005
  • HtrA2/Omi is a mammalian mitochondrial serine protease homologous to the E. coli HtrA/DegP gene products. Recently, HtrA2/Omi was found to have a dual role in mammalian cells, acting as an apoptosis-inducing protein and being involved in maintenance of mitochondrial homeostasis. By screening a human brain cDNA library with $A{\beta}$ peptide as bait in a yeast two-hybrid system, we identified HtrA2/Omi as a binding partner of $A{\beta}$ peptide. The interaction between $A{\beta}$ peptide and HtrA2/Omi was confirmed by an immunoblot binding assay. The possible involvement of HtrA2/Omi in $A{\beta}$ peptide metabolism was investigated. In vitro peptide cleavage assays showed that HtrA2/Omi did not directly promote the production of $A{\beta}$ peptide at the ${\beta}/{\gamma}$-secretase level, or the degradation of $A{\beta}$ peptide. However, overexpression of HtrA2/Omi in K269 cells decreased the production of $A{\beta}40$ and $A{\beta}42$ by up to 30%. These results rule out the involvement of HtrA2/Omi in the etiology of Alzheimer's disease. However, the fact that overexpression of HtrA2/Omi reduces the generation of $A{\beta}40$ and $A{\beta}42$ suggests that it may play some positive role in mammalian cells.

The DeveloDment of PaDain which is Extremely Stable to Negative Ionic Environment by Directed Molecular Evolution (방향성 분자진화에 의한 음이온에 안정한 Papain 개발)

  • Kang, Whan-Koo;Hwang, Sun-Duk;Kim, Hyoung-Sik;Jeung, Jong-Sik;Lee, Bheong-Uk
    • KSBB Journal
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    • v.21 no.5
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    • pp.394-400
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    • 2006
  • When the papain, which is a sort of Cystein protease, is applied to the outer skin, it decomposes the protein which forms the peeled outer skin and speeds up metabolism. Therefore, it is one of the most important cosmetics compositic which keeps the function of skin normal. When the papain is used in cosmetics with surfactant, the activity of papain is reduced rapidly. In this study, the modified papain with extreme stability negative ionic environment was developed by directed evolution

Toll-like Receptor 4 Polymorphism and Periodontitis in Korean Population

  • Park, Ok-Jin;Shin, Seung-Yun;Chung, Chong-Pyoung;Ku, Young;Choi, Young-Nim;Kim, Kack-Kyun
    • International Journal of Oral Biology
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    • v.31 no.1
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    • pp.1-6
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    • 2006
  • The primary cause of periodontitis is plaque-associated anaerobic gram-negative bacteria. As shown in the patients with defects in the number or function of neutrophils, innate immunity plays an important role in resistance to bacterial infection and periodontitis. Toll-like receptor 4(TLR4) is one of the key receptors that recognize the molecular patterns of microbes and initiate innate immune response. To understand the role of TLR4 in the pathogenesis of periodontitis, we investigated whether Asp299Gly of TLR4 mutation is associated with periodontitis in Korean population. Subjects for this study included 90 healthy subjects and 98 periodontitis patients. The Asp299Gly mutation was screened by PCR-Restriction Fragment Length Polymorphism(RFLP) of genomic DNA from blood cells using a primer that creates a NcoI restriction site only in the mutant allele. The Asp299Gly mutation was not found in all subjects tested. Our results suggest that the Asp299Gly mutation of TLR4 is very rare in a Korean population. Further mutation screening may be required to determine the role of TLR4 in the pathogenesis of periodontitis.

Arterial Tortuosity Syndrome in a Neonate

  • Kim, Kyung Min;Hur, Sun-Mi;Yoon, Ji Hong;Lee, Eun-Jung;Lee, Jae Young
    • Neonatal Medicine
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    • v.25 no.1
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    • pp.49-52
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    • 2018
  • Arterial tortuosity syndrome (ATS) is a very rare autosomal recessive connective tissue disease characterized by generalized elongation and tortuosity of the medium- to large-sized arteries, and other systemic connective tissue manifestations. To date, this disease entity has not been reported in Korea. We report a case of ATS diagnosed in a neonate who presented with severe elongation and tortuosity of the aorta and its major branches, as well as the intracranial arteries. Additionally, the patient presented with a tortuous dilatation of the inferior vena cava, an aneurysmal dilatation of the extra-hepatic bile ducts, and an inguinal and sliding hiatal hernia. The diagnosis was confirmed using DNA sequencing analysis, and the patient demonstrated a compound heterozygosity for two novel mutations (c.738delG [p.Gln247Serfs*33] and c.362T>C [p.Ile121Thr]) in exon 2 of the SLC2A10 gene. Genetic analysis also confirmed that both parents were heterozygous carriers of the responsible mutations. Owing to such clinical manifestations, ATS is often misdiagnosed as other connective tissue diseases including Loeys-Dietz syndrome, Marfan syndrome, and Ehlers-Danlos syndrome. In patients presenting with a high index of suspicion, thorough clinical evaluation and screening for ATS including computed tomography or magnetic resonance angiography and target gene analysis are necessary for early diagnosis and management.

Differential Expression Patterns of Crystallin Genes during Ocular Development of Olive Flounder (Paralichthys olivaceus)

  • Yang, Hyun;Lee, Young Mee;Noh, Jae Koo;Kim, Hyun Chul;Park, Choul Ji;Park, Jong Won;Hwang, In Joon;Kim, Sung Yeon;Lee, Jeong Ho
    • Development and Reproduction
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    • v.16 no.4
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    • pp.301-307
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    • 2012
  • Olive flounder Paralichthys olivaceus is one of the most widely cultured fish species in Korea. Although olive flounder receive attention from aquaculture and fisheries and extensive research has been conducted eye morphological change in metamorphosis, but little information was known to molecular mechanism and gene expression of eye development- related genes during the early part of eye formation period. For the reason of eyesight is the most important sense in flounder larvae to search prey, the screening and identification of expressed genes in the eye will provide useful insight into the molecular regulation mechanism of eye development in olive flounder. Through the search of an olive flounder DNA database of expressed sequence tags (EST), we found a partial sequence that was similar to crystallin beta A1 and gamma S. Microscopic observation of retinal formation correspond with the time of expression of the crystallin beta A1 and gamma S gene in the developmental stage, these result suggesting that beta A1 and gamma S play a vital role in the remodeling of the retina during eye development. The expression of crystallin beta A1 and gamma S were obviously strong in eye at all tested developing stage, it is also hypothesized that crystallin acts as a molecular chaperone to prevent protein aggregation during maturation and aging in the eye.

Screening Differential Expressions of Defense-related Responses in Cold-treated 'Kyoho' and 'Campbell Early' Grapevines

  • Ahn, Soon Young;Kim, Seon Ae;Han, Jae Hyun;Kim, Seung Heui;Yun, Hae Keun
    • Horticultural Science & Technology
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    • v.31 no.3
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    • pp.275-281
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    • 2013
  • Low temperature is one of the major environmental factors that affect productivity including reduced growth and budding of vines, and changes of metabolic processes in grape (Vitis spp.). To screen the specific expression of abiotic stress-related genes against cold treatment in 'Kyoho' and 'Campbell Early' grapevines, expression of various defense-related genes was investigated by RT-PCR and real-time PCR. Among the 67 genes analyzed by RT-PCR and real-time PCR, 17 and 16 types of cDNA were up-regulated, while 5 and 6 types were down-regulated in cold-treated 'Kyoho' and 'Campbell Early' grapevines, respectively. Genes encoding carotene (Cart3564 and Cart4472), chalcone isomerase (CHI), cytochrome P450 (CYP), flavonol synthase (FLS), endo-${\beta}$-glucanase precursor (Glu), glutathione peroxidase (GPX), glutathione-S-transferase (GST), leucine-rich repeats (LRR), manganese superoxide dismutase (Mn-SOD), phenylalanine ammonia lyase (PAL), polygalacturonase-inhibiting protein (PGIP), proline rich protein 2 (PRP2), small heat shock protein (sHSP), temperature induced lipocalin (TIL), and thaumatin-like protein (TLP) were up-regulated, while those encoding CBF like transcription factor (CBF1), chitinase-like protein (CLP), cold induced protein (CIP), glycerol-3-phosphate acyltransferase (GPAT), and mitogen-activated protein kinase (MAPK) were down-regulated by low temperature treatment in both in 'Kyoho' and 'Campbell Early'.

Screening of Differentially Expressed Genes between PC12 Cells and A123.7 Cells (PC12 세포와 A123.7 세포에서 차별적으로 발현되는 유전자의 검색)

  • Baik, Seung-Youn;Yang, Byung-Hwan;Chai, Young-Gyu
    • Korean Journal of Biological Psychiatry
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    • v.6 no.1
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    • pp.67-73
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    • 1999
  • The cAMP-dependent protein kinase(PKA) is an intracellular enzyme with serine-threonine kinase activity that plays a key role in cell growth, differentiation, and apoptosis in eukaryotes. In order to understand the PKA signal transduction pathway regulating cell life cycle and identify its role, we focused on the characterization of up-/down-regulated genes by PKA using the differential display polymerase chain reaction. Seven differentially expressed sequence tags(DEST) have been obtained. Among these DESTs, 2 DESTs were homologous to the sequence of genes from BLAST search result. KC1-5 DEST that was up-regulated in A123.7 cells was highly corresponded to mouse apoptosis-related gene(MA-3) or mouse mRNA for topoisomerase inhibitor suppressed(TIS). MA-3 was induced in various types of apoptosis, specially in NGF-deprived apoptotic PC12 cells. TIS was down-regulated in the RVC lymphoma cells incubated with topoisomerase inhibitor that induces DNA strand breakages. PG1-1 DEST that was highly expressed in PC12 cells was corresponded to transposon Tn10 3'-end. Tnansposon Tn10 was up-regulated in differentiated myeloblastic ML-1 cells by 12-O-tetradecanoylphorbol-13-acetate. This study illuminates that MA-3/TIS was down-regulated by PKA activity, and transposon Tn10 was up-regulated by it.

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