Retroelement

1. proteins (gag, env) budding cDNA integration REVERSE TRANSCRIPTION translation pseudogene mRNA infection transcription REVERSE LTR LTR LTR LTR LTR LTR LTR LTR LTR LTR spliced- mRNA TRANSCRIPTION transcription integration Cellular gene provirus cDNA splicing mRNA mRNA transcription transcription A A A A A A A A A A A A retrotransposon Enodgenous retrovirus translation translation REVERSE TRANSCRIPTION proteins (gag) cDNA Particle formation proteins (gag, env) no re-infection re - integration REVERSE TRANSCRIPTION re - integration cDNA Particle formation(budding) Retroelement RNA intermediate - LTR element + LTR element Retroposon - env + env Retrotransposon - RT + RT LTR LTR SINE Yeast Ty1/copia/truncated HERVs P ORF1 LTR LTR ORF2 Poly(A) Human Alu Human THE1 Retrovirus LINE gag pol env LTR LTR P ORF1 ORF2 Full-length HERVs/exogenous retrovirus Poly(A) L1 Exogenous Retroviruses Pseudogenes Retrotransposons Endogenous Retroviruses Twenty Different Human tissue cDNAs HERVs (Human endogenous retroviruses) and LTR (long terminal repeat) - like elements are dispersed over 8% of the whole human genome. There are at least 22 independent HERV families within the human genome, which originated from germ-cell infection by the exogenous retrovirus during primate evolution. Elucidation of expression pattern in HERV elements should provide information about fundamental cellular activities and the pathogenesis of multifactorial diseases such as cancer and autoimmune disease. HERV-W env gene is related to multiple sclerosis, and has potential roles for normal differentiation of human villous cytotrophoblast into syncytiotrophoblast. HERV-W env gene was expressed differentially in human tissues. Especially, it was highly expressed in human placenta. This phenomenon indicates HERV-W env gene have the different roles in each tissues. Here, we applied realtime RT-PCR for detection of its expression in various human tissues. We also analysed such amplification using cancer cells and monkey tissues, and discussed in relation to physiological function. ____________________ 2. W-env standard 1. Gapdh standard 3. Meting graph 4. Tissue expression graph Other region SINE 13% 16% LINE 20% HERV element 5. Tissue melting graph 8% Gene-related Sequence DNA element Pseudogene 3% 1% 36% Coding sequence 3% 6. Relative quantitative analysis of w-env expression Experimental Procedure Skeletal muscle Adrenal gland Salivary gland Bone marrow Spinal cord Cerebellum Fetal brain Fetal liver Placenta Prostate Trachea Thymus Thyroid Kidney Uterus Testis Heart Brain Lung Liver Primer Design Quantitative Analysis 7. Relative quantitative analysis of w-env expression except for placenta Skeletal muscle Adrenal gland Salivary gland Bone marrow Spinal cord Cerebellum Fetal brain Fetal liver Prostate Trachea Thymus Thyroid Kidney Uterus Testis Heart Brain Lung Realtime RT-PCR using SYBR Green I Liver Extension(I) phase Annealing phase relative quantification normalisation external calibration curve without any reference gene via one reference gene via reference gene index >3HKG SYBR Green I Extension(II) phase End of PCR cycle without real-time PCR efficiency correction with real-time PCR efficiency correction Yi JM, Osamu T and Kim HS 2003. Molecular characterization and phylogenetic relationship of HERV-W family in the Macaca fuscata. Arch. Virol. 148(8) 1613-1622. 2(-△△CP) REST, qGene LC software & etc... Yi JM and Kim HM and Kim HS 2004. Expression of the human endogenous retrovirus HERV-W family in various human tissues and cancer cells. J. Gen. Virol. 85: 1203-1210. Detection of fluorescence