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Vol. 9, Issue 11, 1128-1134, November 1999

METHODS
A Genetic Strategy to Eliminate Self-Activator Baits Prior to High-Throughput Yeast Two-Hybrid Screens

Albertha J.M. Walhout, and Marc Vidal1

Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts 02129 USA

Large-scale sequencing projects have predicted high numbers of gene products for which no functional information is yet available. Hence, large-scale projects, such as gene knockouts, gene expression profiles, and protein-interaction mapping, are currently under way to initiate the understanding of the function of these gene products. The high-throughput strategies that are currently being developed to generate protein-interaction maps include automated versions of the yeast two-hybrid system. These strategies rely on the large-scale construction of DNA-binding domain/protein-of-interest hybrid constructs (DB-X baits). An inherent problem of large-scale two-hybrid systems is that a high percentage of cloned sequences encode polypeptides that, when fused to DB, can activate transcription in the absence of any two-hybrid-interacting partner protein. Here, we describe and validate a genetic strategy that efficiently eliminates such self-activator baits prior to screening procedures. The strategy is based on a negative-growth selection and is compatible with high-throughput settings.

1 Corresponding author.

9:1128-1134 ©1999 by Cold Spring Harbor Laboratory Press  ISSN 1088-9051/99 $5.00
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