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生科四甲 報告學生 : 李殿強

Sequence, Distance, and Accessibility are Determinants of 5' End-Directed Cleavages by Retroviral RNase Sharon J. Schultz, et al. ( 2005 ). J. Biol. Chem. M510504200. 生科四甲 報告學生 : 李殿強. Introduction:. Retroviral replication. RNA virus:. Minus-strand DNA( - ) Plus-strand DNA(+). 專有名詞介紹 :.

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生科四甲 報告學生 : 李殿強

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  1. Sequence, Distance, and Accessibility are Determinants of 5' End-DirectedCleavages by Retroviral RNaseSharon J. Schultz, et al. (2005). J. Biol. Chem.M510504200 生科四甲 報告學生:李殿強

  2. Introduction: • Retroviral replication

  3. RNA virus: Minus-strand DNA( - ) Plus-strand DNA(+)

  4. 專有名詞介紹: • Reverse transcriptase • Moloney murine leukemia virus(M-MuLV) • The reverse transcriptase of HIV-1 • The reverse transcriptase of M-MuLV • polypurine tract (PPT)

  5. RNase H contributes to reversetranscription in three distinct ways: • First, RNase H carries out degradation of the RNA genome both during and after minus-strand DNA synthesis to facilitate plus-strand DNA synthesis. • Second, RNase H creates the polypurine tract (PPT) primer from the viral genome. • third, RNase H removes the PPT and tRNA primers used to prime plus-strand and minus-stand DNA synthesis.

  6. Asking three fundamental questions concerning retroviral RNases H? • First, does sequence influence 5' end- directed cleavages? • Second, what distances from the RNA 5' end are acceptable for 5'end-directed cleavage sites? • Third, how large of a gap is required between the 3' terminus of an upstream RNA and the 5' end of a downstream RNA?

  7. EXPERIMENT:Enzymes and reagents– • HIV-1 reverse transcriptase • M-MuLV reverse transcriptase • T7 DNA polymerase • T4 polynucleotide kinase, T4 DNA ligase, T4 DNA polymerase • restriction enzymes • Gel-purified in denaturing polyacrylamide gels(跑RNA用)

  8. Preparation ofRNAs:pGEM- Vector SystemsMd1 – Md10, PPT20, PPT62, and NPPTPreparation of hybrid substrates:RNAs were annealed to the appropriate template DNAs T7 RNA polymerase Template-dependent

  9. RNAs:

  10. Result:HIV-1 5´ end-labeling I +27/+28 H +22/+23 G +19/+20 F +16/+17 E +13/+14

  11. HIV-1 3´ end-labeling

  12. M-MuLV 5´ end-labeling

  13. M-MuLV 3´ end-labeling

  14. Distance:

  15. HIV-1

  16. M-MuLV

  17. Prefer Base:

  18. EXPERIMENT:GAP SIZE: M-MuLV

  19. HIV-1

  20. Conclusion: 1.An alignment of sequences reveals that both enzymes strongly prefer A or U at the +1 position and C or G at the –2 position, and additionally for HIV-1, A is disfavored at the –4 position. 2.For both enzymes, 5' end-directed cleavages occurred when sites were positioned between the 13th and 20th nucleotides from the RNA 5' end, a distance termed the cleavage window.

  21. 3.The importance of accessibility to the RNA 5' end, it was found that the extent of 5' end-directed cleavages observed in substrates was most comparable to substrates with a gap of 2 or 3 bases between the upstream and downstream RNAs.

  22. The End

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