Scientists from DNA Plant Technology Corp. (DNAP) have used agenetic technique known as heterologous transposon tagging toisolate an acidity-controlling gene from petunias.
The results, which appear in the current issue of the journalThe Plant Cell, are the first published demonstration ofisolating a plant gene using a transposon from another plantspecies as a probe, the Cinnaminson, N.J., company(NASDAQ:DNAP) announced Wednesday.
Plant molecular biologists are often stuck with the problem oftrying to isolate genes that have only been identified by thechange they produce in a plant's phenotype. Even if geneticistshave mapped the putative gene of interest to a particular plantchromosome, the exact location of that gene remains elusive.
One method developed to tackle this problem has beenheterologous transposon tagging. Transposons are discretesegments of DNA that can excise from one chromosomallocation and move to another (or "jump"). If they insert withina gene, they can inactivate it, producing a mutant phenotype.Thus transposons can be used as molecular probes and geneticmarkers. And transposons tagged to isolate genes containselectable and/or screenable markers for monitoring theirprogress.
The particular transposon the DNAP scientists used is the Acelement from maize, which "most of the time tends to jumpinto genes on the same chromosome," explained John Bedbrook,DNAP's executive vice president and director of science.
The gene for acidity in petunia -- which affects flower colorbecause anthocyanin pigments are pH-sensitive -- has beendefined and mapped genetically, so its approximatechromosomal location was already known, Bedbrook explained.
"We placed the transposable element on the same chromosomeas the acidity gene," Bedbrook said, "and then selected apopulation of individuals that had the Ac element close to thegene. ... From then on it was relatively simple. We screened forplants whose flower color had changed from red to blue."
Bedbrook added that since the Ac element is unstable (it jumpsin and out of genes), the scientists were looking for flowers thathad "sectors of changed color," indicating that the transposableelement had landed in the acidity gene, however briefly.
"When we engineer the transposon into the plant, we have itembedded in the gene for resistance to the antibioticspectinomycin (or streptomycin)," Bedbrook said. "Theseedlings come up white if they are incubated in this antibiotic;if they're resistant to it, they come up green.
"Thus," he said, "we can select those seedlings on the premisethat if the transposon has jumped out of the antibioticresistance gene, it's jumped into another gene. Then we plantthose and look for changes in flower color."
Once the researchers correlated the location of the transposonwith the phenotype, they used the transposable element as aDNA probe to obtain the gene sequences. After the gene isisolated, it becomes possible to introduce that back into themutant to see whether it will complement the phenotype,Bedbrook explained. This is a way of confirming that the clonedDNA fragment corresponds to the gene.
A tremendous amount of effort by many different researchgroups has gone into developing the Ac transposon system,according to Nina Fedoroff, who heads the lab that originallyisolated the Ac transposon, and was the first to show that itworks in a heterologous system.
And although the DNAP scientists are the first to publish thesuccessful isolation of a gene via transposon tagging, thereshould be a "spate of papers in the next year or two" reportingsuccessful gene isolations by this technique, Fedoroff toldBioWorld.
"Somebody has to be the first to publish, but it's far moreimportant that there are so many researchers" working in thisarea, said Fedoroff, who is a staff scientist at the CarnegieInstitution of Washington's department of embryology inBaltimore.
Meanwhile, DNAP has an issued patent on its heterologoustransposon tagging system, and has filed a patent applicationon the particular acidity-controlling gene, Bedbrook toldBioWorld. DNAP plans to use the gene in tomatoes and otherfruits and vegetables to control the acidity component of flavor.
-- Jennifer Van Brunt Senior Editor
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