ABSTRACT

1. uterus breast stomach liver colon liver cancer breast cancer colon cancer stomach cancer uterus cancer Hep G2 Caki-1 NCI-H250 C-33A Jurkat RT4 OVCAR-3 U-937 MIA-PaCa-2 COLO205 CLASSⅠ SINE HERV5 Other region HERV4 13% HERVP71A 16% HERV12 HERV5_6 HERV5_5 HERV5_3 HERV5_4 HERV5_2 HERV5_1 HERV4_2 HERV4_1 HERV12_1 HERV4_7 HERV4_4 HERV4_6 HERV12_2 HERVIP10F HERV11 HERVI HERV4_3 HERV17 HERV11_1 HERV4_5 HERV9 HERV11_2 HERV30 HERV10_1 HERV2 LINE HERV10 HERV10_2 HERV2 HERV7_1 PABL B 20% HERV7_2 GALV HERV7 PERV HERV6 BaEV HERV6 HERVS71 HERVE Harlequin HERV15 HERV3 HERVFH19 HERVH48 HERV1_2 HERVH HERV1_4 PRIMA4 HERV1_1 HERV-P HERV1 HERV1_3 testis placenta trachea lung prostate kidney uterus fetal brain thymus Brain fetal liver liver heart thyroid Gene-related Sequence spinal cord cerebellum adrenal gland bone marrow HERVK4 salivary gland HERVG25 HERV element HERVK14C DNA element HERV18 skeletal muscle HFV HERVK10 HERVL HERVK14 FeFV 36% 8% HERVK9 MMTV RERV HIV HERVK11D Pseudogene HERVK3 HERVK13 HERVK11 HERVK22 3% Coding sequence CLASSⅢ HERV16 CLASSⅡ 1% 0.1 3% gag pol env U3 R U5 U5 R U3 LTR LTR Heart Kidney Colon Ovary Lung Liver Trachea Uterus Spleen Stomach Cerebellum Ccerebrum Kidney Colon SK-muscle Lung Adrenal gland Liver Testis Trachea Spleen Salivary gland Stomach Cerebellum Heart Ccerebrum Adrenal gland SK-muscle Salivary gland HERVs R K E D H Y C T Q N S G I F W M V A P L Lysine (lys) Threonine (thr) Methionine (met) Phenylalanine (phe) Asparagine (asn) Tryptophan (trp) Tryosine (tyr) Glutamine (gln) Aspatic acid (asp) Histidine (his) Arginine (arg) Cysteine (cys) Glycine (gly) Isoleucine (ile) Proline (pro) Leucine (leu) Serine (ser) Valine (val) Alanine (ala) Glutamic acid (glu) Establishment of quantitative RT-PCR method for the detection of HERV-K family using SYBR green Sang-Je Park1, Kung Ahn1, Jae-Won Huh2, Dae-Soo Kim3, and Heui-Soo Kim1 1Division of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea 2National Primate Research Center (NPRC), KRIBB, Ochang, Chungbuk 363-883, Republic of Korea 3Korean BioInformation Center, KRIBB, Daejeon 305-806, Republic of Korea http://www.primate.or.kr INTRODUCTION ABSTRACT Human endogenous retroviruses (HERVs) have been distributed in human genome. HERVs have been considered as etiological cofactors in chronic disease such as cancer, auto immunity, and neurological disease. Among the various HERV families, only HML(human mouse mammary tumor virus-like) group harbor the complete provirus which could amplify their descendant by retrotransposition mechanism. High rate of relationship with cancer development and polymorphic character has been highlighted in biology field. To investigate the expression profiles of HML-4 families, conserved RT(reverse transcriptase) and IN(integrase) domains of pol region were used as target for quantitative real-time RT-PCR analysis. Using the various human and rhesus monkey tissues, HML-4 family was amplified by SYBR green based quantitative real-time RT-PCR. Although, the pol region of HML-4 family was expressed ubiquitously in human and rhesus monkey tissues, SYBR green based quantitative real-time RT-PCR method could be effective technique for the investigation of HERV families. Structure of HERV-K MA (matrix) protein CA (capsid) protein NC (nucleotide-binding) protein SU (surface) protein TM (transmembrane) protein RT (reverse transcriptase) IN (integrase) protein MATEIRALS & METHODS Real-time RT-PCR Bioinformatics Primer design Sample Preparation Measured fluorescence Data analysis RESULTS & DISCUSSION HML-4 cancer cell-line HML-4human normal tissues 20 panel HML-4 human normal tissues vs cancer tissues HML4 Rhesus monkey Female HML4 Rhesus monkey male SYBR green based quantitative real-time RT-PCR method could be effective technique for the investigation of HERV families. REFERENCES • Rafael Contreras-Galindo, Marta Gonzalez, Sharilyn Almodovar-Camachob, Sandra Gonzalez-Ramirez , Eric Lorenzo , Yasuhiro Yamamura. (2006) A new Real-Time-RT-PCR for quantitation • of human endogenous retroviruses type K (HERV-K) RNA load in plasma samples: Increased HERV-K RNA titers in HIV-1 patients with HAART non-suppressive regimens. J Virol Methods. • 136(1-2):51-7. 2. Polavarapu N, Bowen NJ, McDonald JF.(2006) Newly identified families of human endogenous retroviruses. J Virol. 80(9):4640-2. 3. Muradrasoli S, Forsman A, Hu L, Blikstad V, Blomberg J.(2006) Development of real-time PCRs for detection and quantitation of human MMTV-like (HML) sequences HML expression in humantissues. J Virol Methods. 136(1-2):83-92. -GENOME INFORMATION LABORATARY-

2. uterus breast stomach liver colon liver cancer breast cancer colon cancer stomach cancer uterus cancer Caki-1 Hep G2 NCI-H250 C-33A Jurkat RT4 OVCAR-3 U-937 MIA-PaCa-2 COLO205 testis placenta lung prostate trachea kidney fetal brain Brain uterus fetal liver thymus liver heart thyroid spinal cord cerebellum adrenal gland bone marrow salivary gland skeletal muscle CLASSⅠ HERV5 HERV4 HERVP71A HERV12 HERV5_6 HERV5_5 HERV5_3 HERV5_4 HERV5_2 HERV5_1 HERV4_2 HERV4_1 HERV12_1 HERV4_7 HERV4_4 HERV4_6 HERV12_2 HERVIP10F HERV11 HERVI HERV4_3 HERV17 HERV11_1 HERV4_5 HERV9 HERV11_2 HERV30 HERV10_1 HERV2 HERV10 HERV10_2 HERV2 HERV7_1 gag pol env PABL B U3 R U5 U5 R U3 HERV7_2 GALV HERV7 PERV HERV6 BaEV HERV6 HERVS71 HERVE LTR LTR Harlequin HERV15 HERV3 HERVFH19 HERVH48 HERV1_2 HERVH HERV1_4 HERV1_1 PRIMA4 HERV-P HERV1 HERV1_3 HERVK4 HERVG25 HERVK14C HERV18 HFV HERVK10 HERVL HERVK14 FeFV HERVK9 MMTV RERV HIV HERVK11D HERVK13 HERVK3 HERVK11 HERVK22 CLASSⅢ HERV16 Heart Kidney CLASSⅡ Colon Ovary Lung Liver Trachea Uterus Spleen 0.1 Stomach Cerebellum Ccerebrum Kidney Colon Adrenal gland SK-muscle Lung Testis Liver Trachea Spleen Salivary gland Stomach Cerebellum Heart Cerebrum Adrenal gland SK-muscle Salivary gland Establishment of real-time RT-PCR method for the detection of HML-4 family using SYBR green Sang-Je Park1, Kung Ahn1, Jae-Won Huh1, Dae-Soo Kim2, and Heui-Soo Kim1, 2 1Division of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea 2PBBRC, Interdisciplinary Research Program of Bioinformatics, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea http://www.primate.or.kr ABSTRACT RESULTS & DISCUSSION Human endogenous retroviruses (HERVs) have been distributed in human genome. HERVs have been considered as etiological cofactors in chronic disease such as cancer, auto immunity, and neurological disease. Among the various HERV families, only HML(human mouse mammary tumor virus-like) group harbor the complete provirus which could amplify their descendant by retrotransposition mechanism. High rate of relationship with cancer development and polymorphic character has been highlighted in biology field. To investigate the expression profiles of HML-4 families, conserved RT(reverse transcriptase) and IN(integrase) domains of pol region were used as target for quantitative real-time RT-PCR analysis. Using the various human and rhesus monkey tissues, HML-4 family was amplified by SYBR green based quantitative real-time RT-PCR. Although, the pol region of HML-4 family was expressed ubiquitously in human and rhesus monkey tissues, SYBR green based quantitative real-time RT-PCR method could be effective technique for the investigation of HERV families. HML-4human normal tissues 20 panel HML-4 cancer cell-line HML-4 human normal tissues vs cancer tissues INTRODUCTION SINE Other region 13% 16% LINE 20% HML4 Rhesus monkey male HML4 Rhesus monkey Female Gene-related Sequence HERV element DNA element 8% 36% Pseudogene 3% Coding sequence 1% 3% Structure of HERV-K MA (matrix) protein CA (capsid) protein NC (nucleotide-binding) protein SU (surface) protein TM (transmembrane) protein RT (reverse transcriptase) IN (integrase) protein SYBR green based quantitative real-time RT-PCR method could be effective technique for the investigation of HERV families. MATEIRALS & METHODS Bioinformatics Primer design Sample Preparation HERVs R K E D H Y C T Q N S G I F W M V A P L Real-time RT-PCR Lysine (lys) Threonine (thr) Methionine (met) Phenylalanine (phe) Tryosine (tyr) Asparagine (asn) Tryptophan (trp) Glutamine (gln) Aspatic acid (asp) Arginine (arg) Histidine (his) Cysteine (cys) Serine (ser) Glycine (gly) Isoleucine (ile) Valine (val) Alanine (ala) Proline (pro) Leucine (leu) Glutamic acid (glu) REFERENCES • Rafael Contreras-Galindo, Marta Gonzalez, Sharilyn Almodovar-Camachob, Sandra Gonzalez-Ramirez , Eric Lorenzo , Yasuhiro Yamamura. (2006) A new Real-Time-RT-PCR for quantitation of human endogenous retroviruses type K (HERV-K) RNA load in plasma samples: Increased HERV-K RNA titers in HIV-1 patients with HAART non-suppressive regimens. J Virol Methods. 136(1-2):51-7. 2. Polavarapu N, Bowen NJ, McDonald JF.(2006) Newly identified families of human endogenous retroviruses. J Virol. 80(9):4640-2. 3. Muradrasoli S, Forsman A, Hu L, Blikstad V, Blomberg J.(2006) Development of real-time PCRs for detection and quantitation of human MMTV-like (HML) sequences HML expression in humantissues.J Virol Methods. 136(1-2):83-92. Measured fluorescence Data analysis GENOME INFORMATION LAB.