Browsing by Subject "cloning"

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    Characterizing Novel Fluorescent Protein Pairings for Förster Resonance Energy Transfer (FRET)
    (Office of the Vice Chancellor for Research, 2011-04-08) Ariss, Corey R.; Hays, Nicole; Day, Richard N.
    Since it’s cloning, the sequence encoding the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has been engineered to produce fluorescent proteins (FPs) with emission in the blue to yellowish-green range of the visible spectrum. Furthermore, many FPs with homology to the Aequorea GFP have been cloned from marine organisms, providing new proteins that fluoresce into deep red spectrum. These new FPs expanded the repertoire of applications to include multi-color imaging of protein co-localization and behavior inside living cells. However, it is their use as donor and acceptor pairs for Förster Resonance Energy Transfer (FRET) microscopy that has generated the greatest interest. The most precise methods for measuring FRET detect the quenching of the donor by the acceptor, and Fluorescence Lifetime Imaging Microscopy (FLIM) can accurately measure the shorter donor lifetimes that result from FRET. Currently, the Aequorea GFP variants known as Cerulean (cyan) and Venus (yellow) are the most popular FRET pair. However, Venus has poor photostability, and the emission near 500 nm limits its utility as an acceptor. The objective of this study was to use FLIM to test the utility of different FP pairings for FRET studies with the goal to identify potentially improved FRET pairs.
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    The yeast 8-oxoguanine DNA glycosylase (Ogg1) contains a DNA deoxyribophosphodiesterase (dRpase) activity
    (1997-10) Sandigursky, Margarita; Yacoub, Adly; Kelley, Mark R.; Xu, Yi; Franklin, William A.; Deutsch, Walter A.
    The yeast OGG1 gene was recently cloned and shown to encode a protein that possesses N-glycosylase/AP lyase activities for the repair of oxidatively damaged DNA at sites of 7,8-dihydro-8-oxoguanine (8-oxoguanine). Similar activities have been identified for Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and Drosophila ribosomal protein S3. Both Fpg and S3 also contain a deoxyribophosphodiesterase (dRpase) activity that removes 2-deoxyribose-5-phosphate at an incised 5′ apurinic/apyrimidinic (AP) sites via a β-elimination reaction. Drosophila S3 also has an additional activity that removes trans-4-hydroxy-2-pentenal-5-phosphate at a 3′ incised AP site by a Mg2+-dependent hydrolytic mechanism. In view of the substrate similarities between Ogg1, Fpg and S3 at the level of base excision repair, we examined whether Ogg1 also contains dRpase activities. A glutathione S-transferase fusion protein of Ogg1 was purified and subsequently found to efficiently remove sugar-phosphate residues at incised 5′ AP sites. Activity was also detected for the Mg2+-dependent removal of trans-4-hydroxy-2-pentenal-5-phosphate at 3′ incised AP sites and from intact AP sites. Previous studies have shown that DNA repair proteins that possess AP lyase activity leave an inefficient DNA terminus for subsequent DNA synthesis steps associated with base excision repair. However, the results presented here suggest that in the presence of MgCl2, Ogg1 can efficiently process 8-oxoguanine so as to leave a one nucleotide gap that can be readily filled in by a DNA polymerase, and importantly, does not therefore require additional enzymes to process trans-4-hydroxy-2-pentenal-5-phosphate left at a 3′ terminus created by a β-elimination catalyst.