Current retroviral replication models propose that during (+) strand synthesis, the initial (-) s... more Current retroviral replication models propose that during (+) strand synthesis, the initial (-) strand tRNA primer is partially replicated to reproduce the 18 nt primer-binding site (PBS). Subsequent removal of the tRNA primer from the (-) strand template exposes the PBS, which anneals to complementary sequences on a DNA acceptor template to enable (+) strand transfer. We used model templates composed of primed (-) strand DNA covalently linked with post-transcriptionally modified tRNA(3)(lys) along with natural sequence human immunodeficiency virus (HIV) acceptor DNA to study the generation of the (+) strand strong stop intermediate and the subsequent (+) strand transfer reaction. The rate of formation of the (+) strand transfer reaction products was modestly increased (threefold) by inclusion of nucleocapsid protein, suggesting an ancillary role for this protein in this stage of retroviral replication. In addition to the well-known stop site opposite G59 of the tRNA primer, we detected two additional stop sites opposite psi55 and at A38. Kinetic analysis showed that only the intermediates formed by stops opposite G59 and psi55 were active in the subsequent (+) strand transfer reaction. The surprising discovery of the longer, viable (+) strand interaction intermediate prompted us to survey retroviral sequences for a region complementary to the additional donor DNA nucleotides involved in this over-extension. Indeed, complementary sequences that could support this over-extension were found. A strong consensus sequence is immediately adjacent to and downstream of the PBS in lentiviruses and spumaviruses. This consensus sequence was not found in other genera of retroviruses. We have named this element the "primer over-extension sequence" (POS), and propose that it provides a complementary sequence for strand transfer reactions proceeding from intermediates that extend beyond the standard 18 nt complement of the PBS.
Typically, biochemical screens that employ pure macromolecular components focus on single targets... more Typically, biochemical screens that employ pure macromolecular components focus on single targets or a small number of interacting components. Researches rely on whole cell screens for more complex systems. Bacterial DNA replicases contain multiple subunits that change interactions with each stage of a complex reaction. Thus, the actual number of targets is a multiple of the proteins involved. It is estimated that the overall replication reaction includes up to 100 essential targets, many suitable for discovery of antibacterial inhibitors. We have developed an assay, using purified protein components, in which inhibitors of any of the essential targets can be detected through a common readout. Use of purified components allows each protein to be set within the linear range where the readout is proportional to the extent of inhibition of the target. By performing assays against replicases from model Gram-negative and Gram-positive bacteria in parallel, we show that it is possible to distinguish compounds that inhibit only a single bacterial replicase from those that exhibit broad spectrum potential.
... CIRJE-F-558 Suicide and Life Insurance Joe Chen Yun Jeong Choi Yasuyuki Sawada University of ... more ... CIRJE-F-558 Suicide and Life Insurance Joe Chen Yun Jeong Choi Yasuyuki Sawada University of Tokyo April 2008 ... by Joe Chen Faculty of Economics University of Tokyo joechen@eu-tokyo. ac.jp Yun Jeong Choi Faculty of Economics University of Tokyo yun@eu-tokyo.ac.jp ...
CIRJE Discussion Papers can be downloaded without charge from: ... Discussion Papers are a series... more CIRJE Discussion Papers can be downloaded without charge from: ... Discussion Papers are a series of manuscripts in their draft form. They are not intended for ... The Jump, Inertia, and Juvenization of Suicides in Japan ... Joe Chen National Chengchi University Yun Jeong ...
Current retroviral replication models propose that during (+) strand synthesis, the initial (-) s... more Current retroviral replication models propose that during (+) strand synthesis, the initial (-) strand tRNA primer is partially replicated to reproduce the 18 nt primer-binding site (PBS). Subsequent removal of the tRNA primer from the (-) strand template exposes the PBS, which anneals to complementary sequences on a DNA acceptor template to enable (+) strand transfer. We used model templates composed of primed (-) strand DNA covalently linked with post-transcriptionally modified tRNA(3)(lys) along with natural sequence human immunodeficiency virus (HIV) acceptor DNA to study the generation of the (+) strand strong stop intermediate and the subsequent (+) strand transfer reaction. The rate of formation of the (+) strand transfer reaction products was modestly increased (threefold) by inclusion of nucleocapsid protein, suggesting an ancillary role for this protein in this stage of retroviral replication. In addition to the well-known stop site opposite G59 of the tRNA primer, we detected two additional stop sites opposite psi55 and at A38. Kinetic analysis showed that only the intermediates formed by stops opposite G59 and psi55 were active in the subsequent (+) strand transfer reaction. The surprising discovery of the longer, viable (+) strand interaction intermediate prompted us to survey retroviral sequences for a region complementary to the additional donor DNA nucleotides involved in this over-extension. Indeed, complementary sequences that could support this over-extension were found. A strong consensus sequence is immediately adjacent to and downstream of the PBS in lentiviruses and spumaviruses. This consensus sequence was not found in other genera of retroviruses. We have named this element the "primer over-extension sequence" (POS), and propose that it provides a complementary sequence for strand transfer reactions proceeding from intermediates that extend beyond the standard 18 nt complement of the PBS.
Typically, biochemical screens that employ pure macromolecular components focus on single targets... more Typically, biochemical screens that employ pure macromolecular components focus on single targets or a small number of interacting components. Researches rely on whole cell screens for more complex systems. Bacterial DNA replicases contain multiple subunits that change interactions with each stage of a complex reaction. Thus, the actual number of targets is a multiple of the proteins involved. It is estimated that the overall replication reaction includes up to 100 essential targets, many suitable for discovery of antibacterial inhibitors. We have developed an assay, using purified protein components, in which inhibitors of any of the essential targets can be detected through a common readout. Use of purified components allows each protein to be set within the linear range where the readout is proportional to the extent of inhibition of the target. By performing assays against replicases from model Gram-negative and Gram-positive bacteria in parallel, we show that it is possible to distinguish compounds that inhibit only a single bacterial replicase from those that exhibit broad spectrum potential.
... CIRJE-F-558 Suicide and Life Insurance Joe Chen Yun Jeong Choi Yasuyuki Sawada University of ... more ... CIRJE-F-558 Suicide and Life Insurance Joe Chen Yun Jeong Choi Yasuyuki Sawada University of Tokyo April 2008 ... by Joe Chen Faculty of Economics University of Tokyo joechen@eu-tokyo. ac.jp Yun Jeong Choi Faculty of Economics University of Tokyo yun@eu-tokyo.ac.jp ...
CIRJE Discussion Papers can be downloaded without charge from: ... Discussion Papers are a series... more CIRJE Discussion Papers can be downloaded without charge from: ... Discussion Papers are a series of manuscripts in their draft form. They are not intended for ... The Jump, Inertia, and Juvenization of Suicides in Japan ... Joe Chen National Chengchi University Yun Jeong ...
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