Method quickly surveys yeast genome by gene function, not sequence

December 24, 2001

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 authored the paper.
-end-
On the Web: http://www.sciencemag.org

Media Contact: Joanna Downer (410) 614-5105
Email: jdowner1@jhmi.edu

Johns Hopkins Medical Institutions' news releases are available on an EMBARGOED basis on EurekAlert at http://www.eurekalert.org, Newswise at http://www.newswise.com and from the Office of Communications and Public Affairs' direct e-mail news release service. To enroll, call 410-955-4288 or send e-mail to bsimpkins@jhmi.edu.

On a POST-EMBARGOED basis find them at http://www.hopkinsmedicine.org and Quadnet at http://www.quad-net.com

Johns Hopkins Medicine

Related Immune System Articles from Brightsurf:

How the immune system remembers viruses
For a person to acquire immunity to a disease, T cells must develop into memory cells after contact with the pathogen.

How does the immune system develop in the first days of life?
Researchers highlight the anti-inflammatory response taking place after birth and designed to shield the newborn from infection.

Memory training for the immune system
The immune system will memorize the pathogen after an infection and can therefore react promptly after reinfection with the same pathogen.

Immune system may have another job -- combatting depression
An inflammatory autoimmune response within the central nervous system similar to one linked to neurodegenerative diseases such as multiple sclerosis (MS) has also been found in the spinal fluid of healthy people, according to a new Yale-led study comparing immune system cells in the spinal fluid of MS patients and healthy subjects.

COVID-19: Immune system derails
Contrary to what has been generally assumed so far, a severe course of COVID-19 does not solely result in a strong immune reaction - rather, the immune response is caught in a continuous loop of activation and inhibition.

Immune cell steroids help tumours suppress the immune system, offering new drug targets
Tumours found to evade the immune system by telling immune cells to produce immunosuppressive steroids.

Immune system -- Knocked off balance
Instead of protecting us, the immune system can sometimes go awry, as in the case of autoimmune diseases and allergies.

Too much salt weakens the immune system
A high-salt diet is not only bad for one's blood pressure, but also for the immune system.

Parkinson's and the immune system
Mutations in the Parkin gene are a common cause of hereditary forms of Parkinson's disease.

How an immune system regulator shifts the balance of immune cells
Researchers have provided new insight on the role of cyclic AMP (cAMP) in regulating the immune response.

Read More: Immune System News and Immune System Current Events
Brightsurf.com is a participant in the Amazon Services LLC Associates Program, an affiliate advertising program designed to provide a means for sites to earn advertising fees by advertising and linking to Amazon.com.