期刊
GENETICS
卷 172, 期 4, 页码 2113-2122出版社
GENETICS SOCIETY AMERICA
DOI: 10.1534/genetics.105.052688
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Genomewide 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 cerevisine, 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 for a new sporulation-specific regulatory site that we identified and several neutral spacer elements. This selective system achieved tip to 1:10(4) 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 Late 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 similar to 100 examples of active and inactive promoters could not be distinguished by simple binary rules; instead, the best model for the data was it linear regression fit of a quantitative measure of gene activity to multiple features of the regulatory sequence.
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