Rapid selection against truncation mutants in yeast reverse two-hybrid screens.
The yeast reverse two-hybrid system is a powerful technique for isolating mutations in a protein that abolish its interaction with a known partner. Selection is based on abrogation of growth suppression imposed when wild-type interactions confer 5-fluoroorotic acid (5-FOA) sensitivity to yeast cells. A laborious component of this system is to eliminate those mutations that cause protein truncation. By fusing the green fluorescent protein (GFP) to the C-terminus of a protein of interest, dynein light chain (LC8), we were able to rapidly isolate mutations that did not result in protein truncation.
Puthalakath
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Walter and Eliza Hall Institute, Melbourne, Australia.
Strasser
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Huang
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DC
D051017:Apoptosis Regulatory Proteins; D000072224:Bcl-2-Like Protein 11; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D029721:Drosophila Proteins; D004398:Dyneins; D005434:Flow Cytometry; D005472:Fluorouracil / Q000378:metabolism / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008565:Membrane Proteins; D016296:Mutagenesis; D020125:Mutation, Missense; D009963:Orotic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics*; D014162:Transfection; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D051017:Apoptosis Regulatory Proteins; D000072224:Bcl-2-Like Protein 11; D002352:Carrier Proteins; D029721:Drosophila Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D009963:Orotic Acid; C001242:5-fluoroorotic acid; D001616:beta-Galactosidase; D004398:Dyneins; D005472:Fluorouracil