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Proc Natl Acad Sci U S A. 2002 May 14;99(10):7033-8. Epub 2002 May 7.

Genome-wide screening for trait conferring genes using DNA microarrays.

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  • 1Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.

Abstract

We report a DNA microarray-based method for genome-wide monitoring of competitively grown transformants to identify genes whose overexpression confers a specific cellular phenotype. Whereas transcriptional profiling identifies differentially expressed genes that are correlated with particular aspects of the cellular phenotype, this functional genomics approach determines genes that result in a specific physiology. This parallel gene-trait mapping method consists of transforming a strain with a genomic library, enriching the cell population in transformants containing the trait conferring gene(s), and finally using DNA microarrays to simultaneously isolate and identify the enriched gene inserts. Various methods of enrichment can be used; here, genes conferring low-level antibiotic resistance were identified by growth in selective media. We demonstrated the method by transforming Escherichia coli cells with a genomic E. coli library and selecting for transformants exhibiting a growth advantage in the presence of the anti-microbial agent Pine-Sol. Genes conferring Pine-Sol tolerance (19 genes) or sensitivity (27 genes) were identified by hybridizing, on DNA microarrays containing 1,160 E. coli gene probes, extra-chromosomal DNA isolated from transformed cells grown in the presence of various levels of Pine-Sol. Results were further validated by plating and sequencing of individual colonies, and also by assessing the Pine-Sol resistance of cells transformed with enriched plasmid library or individual resistance genes identified by the microarrays. Applications of this method beyond antibiotic resistance include identification of genes resulting in resistance to chemotherapeutic agents, genes yielding resistance to toxic products (recombinant proteins, chemical feedstocks) in industrial fermentations, genes providing enhanced growth in cell culture or high cell density fermentations, genes facilitating growth on unconventional substrates, and others.

PMID:
11997466
[PubMed - indexed for MEDLINE]
PMCID:
PMC124523
Free PMC Article
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