11:10-12:00, Rm 103

RNA biologyT-18-14Rapid molecular diagnosis using real-time nucleic acid sequence basedamplification (NASBA) for detection of Influenza A virus subtypesJaewon Lim¹, Yoonjung Cho¹, In Soo Lee², Hyunwoo Jin¹, Hyeeun Bang¹,Hyeyoung Lee¹and Tae Ue Kim¹¹Department of Biomedical Laboratory Science, College of Health Science, Yonsei University,Wonju 220-710, ²Department of Clinical Laboratory Science, Hyejeon College, Hongseong 350-702, KoreaInfluenza A virus of the Orthomyxoviridae family is a contagious respiratory pathogen thatcontinues to evolve and burden in the human public health. Influenza A viruses areclassified into different subtypes by Antigenicity base on their hemagglutinin (HA) andneuraminidase (NA) proteins. The sudden emergence of influenza A virus subtypes andaccess for epidemiological analysis of this subtypes demanded a rapid development ofspecific diagnostic tools. In this study, our aim is to detection of influenza A virus subtypesby using real-time NASBA which has high sensitivity and specificity through molecularbeacon. We selected major pandemic influenza A virus subtypes, H1N1, H2N2, H3N2and H5N1. Then, three influenza A virus gene fragments were targeted, heamgglutinin(HA) gene, neuraminidase (NA) gene and Matrix protein (M) gene, respectively. Wedesigned specific primers and molecular beacons for each targeted RNA segments. Theresults show that the specificity and the sensitivity of the method using real-time NASBAwere higher then that of real-time PCR. This study suggests that rapid detection of neoappearancepandemic influenza A virus using real-time NASBA has the potential todetermine the subtypes of respiratory pathogen.T-18-15Estrogen receptor alpha expression with miR-206 trans-acting regulationroles in Breast cancer cell linesYoung Eun Choi, Junkyu Song, Kyung-Sik YoonDepartment of Biochemistry and Molecular Biology, Kyung Hee university, Seoul 130-701, KoreaEstrogen receptor-alpha (ER-α) is present in 65% of human breast tumors.Tamoxifen(Tam) was only effective in ER-αexpressed breast cancer cell lines. Also, itwas reported that miR-206 decreases endogenous ER-αmRNA and protein levels inhuman MCF-7 breast cancer cells. We expect that effect of invasion and apoptosis inbreast cancer cells by regulating expression of miR-206. Hence, we demonstrated thattrans-acting regulation roles by ER-αand miR-206 in breast cancer cell line. MCF-7(ERhyper-expressed), MDA-MB231(ER hypo-expressed), MCF-10A(ER hypo-expressednormal breast cell line) cells were targeted experiments. Cell viability was analyzed bydose-dependent Tamoxifen with MTT assay. Whole-cell extracts were observed forprotein levels of ER-α, ERK, p38, p-ATM, ATM, p-chk2, chk2, p-p53, p53, cyclinD1 withwestern blotting. By using, ER-α, p38, miR206 activator/inhibitor and real-time PCR.Wesuggest that these roles of miR-206 will take the strategy for valuable breast cancertherapy.T-18-17Cooperative interaction in 23S rRNA methylation by Erm proteinHak Jin Lee, Sung Keun Kim, Jea Woong Lim and Hyung Jong JinDepartment of Bioscience and Biotechnology, College of Natural Science, the University of Suwon,Hwaseong 445-743, KoreaErm family of proteins catalyze S-adenosyl-L-methionine dependent modification of aspecific adenine residue (A2058, E. coli numbering) in bacterial 23S rRNA, therebyconferring resistance to clinically important macrolide, lincosamide and streptogramin B(MLS) antibiotics. ErmSF from Streptomyces frudiae is one of proteins in this family. Allthe structural elements required for recognition and methylation by Erm reside in domainV (stem 73 to 92) of 23S rRNA. 72nt RNA composed of only the stems 73 and 74,despite it’s severe truncation, showed very high methyl group accepting activity. Weused chemical and enzymatic footprinting to identify the interactions between ErmSF and72nt. ErmSF protected the almost all part of single stranded region containing A2058 andthe proximal part of both stems adjacent to it. We choose protected 3 consecutive GCbase pairs which were found to be strongly protected by ErmSF in footprinting andinversion mutation at those sites was introduced into domain V, complete substrate.While other GC base pairs does not affect substrate activity considerably, G2069C-C2442G mutation elicited the significant change in substrate activity, lowering Vmax andincreasing Km. Footprinting study showed that this mutation caused the disruption ofinteractions with protein at several sites concomitantly, suggesting that this site shouldbind first to protein for other site to interact with protein, that is hierarchical cooperativebinding. [Supported by the Research Program for New Drug Target Discovery (2010-0020407) and the Basic Science Research Program (2010-0011442) through theNational Research Foundation of Korea (NRF) funded by the Ministry of Education,Science and Technology]T-18-18microRNA-185 regulates time-dependent expression of mouseCRYPTOCHROME1Kyung-Ha Lee¹, Kyung-Chul Woo¹, Do-Yeon Kim¹, Sung-Hoon Kim², Hwa-Rim Lee²and Kyong-Tai Kim¹ , ²¹Department of Life Science, POSTECH(Pohang University of Science and Technology), Pohang790-784, Korea, ²School of Interdisciplinary Bioscience and Bioengineering, POSTECH(PohangUniversity of Science and Technology), Pohang 790-784, KoreaThe mammalian circadian rhythm is observed not only at the suprachiasmatic nucleus, amaster pacemaker, but also throughout the peripheral tissues. Until now, researches inclock genes’expression and working have mainly focused on transcription and posttranslationalmodification. However, little is known about post-transcriptional regulation ofthese genes. In the present study, we investigated the role of the 3’-untranslated region(UTR) of the mouse Cryptochrome 1 (mCry1) gene at the post-transcriptional level,especially microRNA (miRNA) mediated regulation. Knockdown of Drosha, Dicer orArgonaute2 showed increased level of mCry1-3′UTR reporter. miRNA recognitionelement (MRE) of mCry1 which is important for miR-185 binding led to a decrease inprotein level but not in mRNA quantity. Indeed, mutation of miR-185 binding regionincreased reporter protein level. Overexpression of miR-185 led to decrease in reporteractivity. Our results suggest that miR-185 plays a role as a fine regulator, contributing tothe mCry1 mRNA translation rate.T-18-16Essential RNA structure to be recognized and methylated by Ermprotein, 23S rRNA methyltransferaseHak Jin Lee, Sung Keun Kim, Jea Woong Lim and Hyung Jong JinDepartment of Bioscience and Biotechnology, College of Natural Science, the University of Suwon,Hwaseong 445-743, KoreaErm proteins recognize specific adenine residue (A2058, E. coli coordinate) and methylate at itsexocyclic amino group to confer resistance to MLS antibiotics on a variety of microorganisms rangingfrom antibiotic producers to pathogens. Domain V of 23S rRNA has been known as a completesubstrate containing all the structural elements to be recognized by Erm proteins and variousstructural features of it contribute to methylation reaction in different ways such as recognition andbinding to substrate, induced fit, binding for methylation, methyl group transfer and release fromenzyme. However, they are too complicated to deduce function of each amino acid-nucleotideinteraction or group of interactions in methylation with complete substrate, domain V. Defining theminimalist substrate structure which contains all the least but essential structural elements to berecognized and methylated by Erm protein could be a start point to define all the structural andinteraction characteristics in each step of methylation. As domain V was truncated, stem 73 wasfound to be essential as noted before, while sequences in stem 74 are not essential but helpful inmethyl group accepting activity. Structures away from the methylatable adenine could be removedwithout abolishing the substrate activity to get 20nt stem-loop structure which is the smallest of all thepublished minimal substrate to date. Long N-terminal end domain of ErmSF may allow defining theminimalist substrate of 20nt. The importance of each nucleotide in substrate activity was pursued byin vitro site directed mutagenesis and activity test and those will be discussed in detail. [Supported bythe Research Program for New Drug Target Discovery (2010-0020407) and the Basic ScienceResearch Program (2010-0011442) through the National Research Foundation of Korea (NRF)funded by the Ministry of Education, Science and Technology]338 Korean Society for Biochemistry and Molecular Biology