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December 24, 2001
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Method Quickly Surveys Yeast Genome By Gene Function, Not Sequence

Combining a decade of research advances, scientists have implemented a new method that essentially searches the entire yeast genome in an instant, looking for what the genes do rather than what they look like, say the researchers from Johns Hopkins and Rosetta Inpharmatics, Inc.

The scientists mixed more than 4,600 yeast mutants, each lacking a different gene, and put the pooled mutants in an environment that tested their ability to repair DNA. They were then able to sort out how each mutant performed by using microarray technology, according to the report in the Dec. 21 issue of the journal Science.

Amounting to a "functional" microarray, these experimental steps marry classical genetics, which was used initially to identify many of the genes, with high-tech genomics, whose goal is determining the function of genes, say the researchers.

"The sequence of the yeast genome is available and the human genome is in draft form, so now there's a big push to figure out what the genes do," says Jef Boeke, Ph.D., D.Sc., professor of molecular biology and genetics at Johns Hopkins. "We tested DNA repairing ability, but we can use this method to identify genes involved in many other cellular processes. It should dramatically speed our efforts to understand genes' functions."

Some 6,000 yeast genes are known, and a mutant strain for each one was made thanks to an international effort, including major contributions from Boeke's lab (pronounced BU-ka). It isn't difficult to see whether a mutant can last in a given environment, which is how scientists evaluate the missing gene's function, but until now, each mutant had to be tested separately.

Testing all 4,600-plus existing mutants at the same time depends on a "barcode" system developed by Dan Shoemaker of Rosetta Inpharmatics, Inc., that identifies the mutants as easily as the varying stripes of a UPC symbol distinguish brands and sizes of packaged foods. (Mutations in the remaining genes are fatal regardless of environment, so those are not considered.)

The genetic barcodes, first reported in 1996, are unique, 20-block-long pieces of DNA inserted into the genetic material of each mutant. Shoemaker now has barcodes in all of the existing yeast mutants. He's also developed a microarray that acts as a "checkout scanner" to read these barcodes.

The microarray is a grid of thousands of tiny spots on a piece of glass roughly one-fourth the size of a dollar bill. Each spot holds a unique "sensor," a strand of DNA that precisely matches one of the barcodes. Machines then read the microarray "chip" to determine which of the sensors found matching barcodes.

"While regular checkout scanners at the grocery store can only read one barcode at a time, the chip can read all the barcodes at once," says Boeke, co-director of a new microarray facility that will soon open to serve researchers at the Johns Hopkins School of Medicine. Initial funding for the facility was provided by the school's Institute for Cell Engineering. The facility is co-directed by Forrest Spencer, Ph.D., of the school's McKusick-Nathans Institute of Genetic Medicine.

To prove they could identify genes' functions by pooling the mutants and using the barcodes, Boeke turned to his lab's study of how cells fix DNA, a process that is crucial in the immune system and in preventing cancer cells from forming, he says. For their experiments with yeast, the process also was crucial for the organism's survival.

"We're interested in how cells break strands of DNA and put them back together again, so we used the technique to search all the yeast mutants to find ones that couldn't 'fix' a test piece of DNA," says Boeke, whose studies were funded by the National Institutes of Health.

Siew Loon Ooi, a graduate student in Boeke's lab, tested a repair process that fixes DNA that has breaks in both strands. Normally a matching set of two strands, DNA may be irreparably damaged if both strands break in the same area. By adding what would seem to be "broken" DNA to the yeast, the scientists tested the mutants' abilities to fix these breaks.

In the experiments, strains died if their missing gene was required for repairing these breaks, and their barcodes then were absent from the analyzed microarray. In addition to identifying genes already tied to this type of DNA repair, Ooi found that a new gene, called NEJ1, also is crucial in the process. Two other groups have also reported finding NEJ1's function by using other methods.

The method identifies genes crucial in a given process, but detailed examination of those genes and their mutants is needed to figure out exactly how the genes work, notes Boeke. He, Shoemaker and Ooi are co-authors on the paper.

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