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’Timeless’ gene found to play key role as timekeeper in mammals


In 1998, scientists found the mammalian version of a gene, known as timeless, which in flies is crucial for the biological clock. However, all but one of the research groups involved determined that timeless did not have such a role in mammals. Now that research group says timeless is indeed a key timekeeper in mammals.

In a new complex molecular study of rats, published in the Oct. 17 issue of Science, researchers at the University of Illinois at Urbana-Champaign blocked the functional ability of timeless, leaving the circadian clock in disarray.

The key difference between the previous studies and this new one was the identification of two timeless proteins -- one a full-length protein and the other a shorter, incomplete version.

"There has been a lot of dispute about the role of timeless, and timeless has been generally excluded in research done since 1998," said Martha U. Gillette, the head of the department of cell and structural biology at Illinois. In the initial studies, her lab had seen differences in timeless RNA expression. The other labs had not.

The research in Gillette’s lab, led by Jessica W. Barnes and Jeffrey A. Barnes, both doctoral students, and Shelley A. Tischkau, a professor of veterinary biosciences, continued with the goal to decipher the previously conflicting findings.

"This paper has substantial supportive data that provides definitive evidence that timeless needs to be back in the loop," Gillette said. Much of the supporting data, in fact, is presented online to complement the material appearing in the Science paper.

The "loop" is the 24-hour circadian rhythm in the brain and cells. It consists of an automatically regulated loop of transcription and translation of gene products important for many diverse physiological functions such as sleep, metabolism and reproduction.

The earlier findings had led to the conclusion that timeless was vital only to cellular development in mammals but not to the clock. "The other labs had targeted their reagents at the end of the gene where changes in only full-length timeless are difficult to isolate due to the over-abundance of the short isoform," Jessica Barnes said. "So their results were being confounded."

Working with the whole molecule, the interaction of timeless with the five other mammalian clock genes (three forms of mPER, mClk and bmal) became clear.

In normal and control-treated brain slices from the rats’ suprachiasmatic nucleus, the site of the circadian clock, normal activity occurred in the presence of timeless. When specially designed antisense molecules were added to block it, electrical rhythms were disrupted. "When you have really low levels of timeless, you also disrupt the other clock genes," Barnes said. "You get an uncoupling. The clock is very much in disarray."

Some clock genes send positive signals, triggering mRNA production. The Illinois team theorizes that timeless and another clock gene (mPER2) work in tandem as negative signals to shut down mRNA production during the 24-hour cycle. With timeless back in the mammalian equation, it means that the clock genes of Drosophila and mammals correspond and function similarly.

"This conservation of timeless is very important, that what is happening in Drosophila is holding true in the mammal," Gillette said. "Without timeless, you are missing a whole set of gears in an intricate mechanism."

Other contributors to the paper were postdoctoral researchers Jennifer W. Mitchell and Penny W. Burgoon, both in cell and structural biology, and Jason R. Hickok, a doctoral student in cell and structural biology.

The U.S. Department of Health and Human Services, the University of Illinois Scholars Program and the Illinois Governor’s Venture Technology Fund/Molecular & Endrocrine Pharmacology Program supported the research.

Jim Barlow | EurekAlert!
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