Gallouzi 454 Sept 09

Gallouzi 454 Sept 09 - Mutational analysis to identify an...

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Mutational analysis to identify an NLS sequence - SV40 is a member of the Poliomaviridae family of small DNA tumor viruses. - We will use the large T antigen of SV40 that contains an NLS domain (it s a 90kDa protein required for viral DNA replication)
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Gene Fusion to identify an NLS sequence
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- Pyruvate Kinase (PK) is a cytosolic protein. - When PK is fused to the NLS domain of the SV40 protein and expressed in HeLa cells it, translocates to the nucleus.
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There are other methods to study protein import from the cytoplasm to the nucleus, such as the import assay performed in mammalian cells.
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Import : The movement of proteins [with or without their cargo] and/or other cellular components (RNAs…) from the cytoplasm to the nucleus. Export : The movement of proteins [with or without their cargo] and/or other cellular components (RNAs…) from the nucleus to the cytoplasm.
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These proteins can shuttle between the nucleus and the cytoplasm either to affect a specific process, such as translation or mRNA decay, and/or to transport their cargo (RNA or protein) to a specific area in the cell. There are proteins that harbour both and NLS and NES
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Shuttling : The back and forward movements [Return back to the starting point] of proteins [with or without their cargo] and/or other cellular components (RNAs…).
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Method used to define a shuttling protein The Heterokaryon Fusion Experiment: detecting nuclear-cytoplasmic shuttling
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