HERVs (Human endogenous retroviruses) and LTR (long terminal repeat) - like elements are dispersed...

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. ____________________

Retroelement

Retroposon

SINE

Retrotransposon

RetrovirusLINE

RNA intermediate- LTR element + LTR element

- env + env

- RT + RT

gag pol envLTR LTR

Human Alu

ORF1 ORF2P

Poly(A)L1Full-length HERVs/exogenous retrovirus

P

LTR LTR

LTR LTRORF2ORF1Poly(A)

Yeast Ty1/copia/truncated HERVs

Human THE1

Pseudogenes

LTR LTR

provirus

LTR LTR

Enodgenous retrovirus

Exogenous Retroviruses

RetrotransposonsEndogenous Retroviruses

buddingproteins(gag, env)

translation

infection

REVERSE

TRANSCRIPTION

transcription

integrationcDNA

AAA

mRNA

AAA

mRNAtranscription

LTR LTR

re - integration

translation

proteins(gag, env)

REVERSETRANSCRIPTION

cDNA

Particle formation(budding)

LTR LTR

re - integration

Particle formationno

re-infection

LTR LTR

retrotransposonAAAtranscription

mRNA

cDNA

REVERSETRANSCRIPTION

Cellular gene

pseudogene

splicing

AAA

REVERSETRANSCRIPTION

cDNAintegration

transcriptionspliced- mRNA

translation

proteins(gag)

Other region16%

Gene-related Sequence

36%

LINE

20%

SINE13%

Coding sequence3%

Pseudogene1%

HERV element8%

DNA element3%

Primer Design

Twenty Different Human tissue cDNAs

QuantitativeAnalysis

Realtime RT-PCR using SYBR Green I

Extension(II) phase

Extension(I) phase

End of PCR cycle

Annealing phase

SYBR Green I

Detection of fluorescence

Experimental Procedure

relative quantification

normalisation

via onereference

gene

via referencegene index

>3HKG

externalcalibration

curve withoutany reference

gene

without real-time PCRefficiency correction

2(- CP)△△

with real-time PCRefficiency correction

REST, qGeneLC software & etc...

1. Gapdh standard 2. W-env standard

3. Meting graph

5. Tissue melting graph

4. Tissue expression graph

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0

0.002

0.004

0.006

0.008

0.01

0.012

0.014

0.016

6. Relative quantitative analysis of w-env expression

7. Relative quantitative analysis of w-env expression except for placenta

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.

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.

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