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We have developed a generic procedure to purify proteins expressed at their natural level under native conditions using a novel tandem affinity purification (TAP) tag. The TAP tag allows the rapid purification of complexes from a relatively small number of cells without prior knowledge of the complex composition, activity, or function. Combined with mass(More)
Identification of components present in biological complexes requires their purification to near homogeneity. Methods of purification vary from protein to protein, making it impossible to design a general purification strategy valid for all cases. We have developed the tandem affinity purification (TAP) method as a tool that allows rapid purification under(More)
The MSL5 gene, which codes for the splicing factor BBP/ScSF1, is essential in Saccharomyces cerevisiae, yet previous analyses failed to reveal a defect in assembly of (pre)-spliceosomes or in vitro splicing associated with its depletion. We generated 11 temperature-sensitive (ts) mutants and one cold-sensitive (cs) mutant in the corresponding gene and(More)
Removal of introns from pre-mRNA is an essential step of gene expression. The splicing reaction is catalyzed in a large complex termed the spliceosome. Introns are recognized during the early steps of spliceosome assembly with the formation of commitment complexes. Intron recognition is mediated by the interaction of splicing factors with conserved(More)
Gene disruption and tagging can be achieved by homologous recombination in the yeast genome. Several PCR-based methods have been described towards this end. However these strategies are often limited in their applications and/or their efficiencies and may be technically demanding. Here we describe two plasmids for C-terminal tagging of proteins with the IgG(More)
We are investigating the importance of histone acetylation to gene regulation using two model regulators, the Tuplp repressor and the Gcn5p histone acetyltransferase. Tuplp interacts with the amino-terminal 'tails' of histones H3 and H4 and concomitant mutation of these domains limits repression. Acetylation of either histone inhibits Tuplp binding,(More)
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