Gene Expression from Random Libraries of Yeast Promoters

Abstract. Genome wide techniques to assay gene expression and transcription factor binding are in widespread use, but are far from providing predictive rules for the function of regulatory DNA. To investigate more intensively the grammar rules for active regulatory sequence we made libraries from random ligations of a very restricted set of sequences. Working with the yeast Saccharomyces cerevisiae we developed a novel screen based on the sensitivity of ascospores lacking dityrosine to treatment with lytic enzymes. We tested two separate libraries built by random ligation of a single type of activator site either for a well characterized sporulation factor, Ndt80, or a new sporulation-specific regulatory site we identified, and several neutral spacer elements. This selective system achieved up to 1:104 enrichment of the artificial sequences that were active during sporulation, allowing a high throughput analysis of large libraries of synthetic promoters. This is not practical with methods involving direct screening for expression, such as those based on fluorescent reporters. There were very few false positives, since active promoters always passed the screen when retested. . The survival rate of our libraries containing roughly equal numbers of spacers and activators was a few percent that of libraries made from activators alone. The sequences of approximately 100 examples of active and inactive promoters could not be distinguished by simple binary rules, instead the best model for the data was a linear regression fit of a quantitative measure of gene activity to multiple features of the regulatory sequence.

Key Words: S.cerevisiae, selection screen, sporulation, transcriptional regulation

Online ISS 1091-6490
Copyright 2008 by the Genetics Society of America
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