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Technique offers new view of dynamic biological landscape

of epistatic genetic interactions at once."

The E-MAP technique consists of selectively "dialing down" the activity of a multitude of gene pairs and comparing the effects of those changes to those that result when each gene is dialed down individually. Many genes' activity could be reduced by eliminating them entirely, but for the subset of genes that are essential for yeast growth -- whose complete deletion would kill the cell -- the researchers invented a high throughput technique to manipulate the half-life of their messenger RNA (mRNA). Since mRNA is a genetic intermediate during the conversion of a gene to protein, reducing its lifespan by mutating the mRNA message lowers the amount of protein the cell can produce. The group called this approach "decreased abundance by mRNA perturbation" (DAmP).

"The DAmP technique gave us a way of lowering the abundance of a target gene's messenger RNA while maintaining its natural regulation," said Weissman. "Most of the mRNAs in yeast have half-lives of ten minutes or so, but our alterations destabilized them to have only a half-life of a couple of minutes. Consequently, they produce five- to ten-fold less protein," he said.

In developing E-MAP, the researchers faced a significant hurdle: Even yeast's relatively modest 6,000 genes would generate nearly 20 million possible gene pairs that would need to be tested. To narrow the number of possible interactions, they adopted a strategy called neighborhood clustering, which restricts analysis to genes that have related functions and that also cluster in one place in the cell. In the Cell paper, they applied the E-MAP technique to a "mini array" of 442 yeast genes that define a biological pathway called the early secretory pathway. This compartmentalized, interconnected pathway synthesizes and regulates lipids and secreted proteins in yeast.

Weissman and his colleagues also needed a way to quantify the epistatic effects of interacting mut
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Source:Howard Hughes Medical Institute


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