by Carol L. Spence

The lab in the Plant Sciences Building was unusually empty, as Bob Houtz and Roberta Magnani awaited results from a radioactive assay she was running on the protein calmodulin.

Roberta Magnani and Robert Houtz made the discovery others had been seeking for decades.

The easy rapport between professor and post-doc highlights their respect for each other, built over a seven-year professional relationship. Houtz, chair of the College of Agriculture’s Horticulture Department, had been so impressed with Magnani when she was a visiting graduate student, he held a research position open for her while she finished her doctorate at the University of Bologna in her home country of Italy.

So they chat about everyday things, as they wait for the test results.

Swing east 119 longitudinal degrees to Beersheba, Israel. Ruth Parvari is studying a gene in humans known as C2orf34, meaning it’s the 34th gene on the second chromosome. Orf means open reading frame, scientific terminology indicating that no one knows what it is or what it does. Parvari knows the deletion of the gene contributes to mental disabilities and muscle weakness in people, but why remains a mystery.

Franca Cambi is exploring the effects of methylation on autism spectrum disorders.

Back at UK, neuro-geneticist Dr. Franca Cambi studies human brain development. Her research does not focus specifically on C2orf34. That’s about to change.

Some of Reddy Palli’s studies in UK’s Department of Entomology have focused on the red flour beetle—a pantry pest that feeds off processed grains. It, too, carries C2orf34, though it’s not called that in insects, because the gene resides in a different spot on a different chromosome. But it’s the same gene, and it seems to be as important as it is in humans.

Across Cooper Drive in the Kentucky Tobacco Research and Development Center Indu Maiti conducts research using tobacco and Arabidopsis plants. Plants, too, carry that ubiquitous gene, and Maiti will soon discover that without it, plants respond in distinctive ways.

It is believed that every major life form on this planet carries this gene. In laboratories across the country and around the world, scientists in wide-ranging research fields sought its function.

On Jan. 14, 2009 at 2:07 p.m., in Houtz’ laboratory, Magnani’s test results emerged. She’d found the answer.

Green Light to Explore

Magnani’s search started two months earlier. She went to Houtz and said she wanted to identify the enzyme that enables the methylation of the protein calmodulin. In order to perform certain tasks, calmodulin needs to bond with three methyl groups, each composed of one carbon and three hydrogen atoms.

Picture yourself as a cell. You have all this genetic material in your nucleus, and you are bounded by a membrane so your inner workings don’t spill out. But you have to detect events that happen outside your walls, and you have to be able to react when you get a signal from outside. That signal could be from a hormone, a protein, even a pathogen or organism. How do you react? How do you protect yourself? By activating certain genes in the nucleus and deactivating others. And calmodulin is part of that communication system.

When a flux of calcium comes into the cell, it binds to calmodulin, which dramatically changes its entire shape, enabling it to bind to and activate any of 300 other proteins. Some of these proteins recognize whether or not calmodulin is methylated. If a gene doesn’t produce that enzymatic catalyst to methylate calmodulin, calmodulin can’t bond to those proteins, and the entire chain of action is shut down before it starts. But no one knew what gene was responsible for this.

“Everybody who knows anything about this area of research, protein methyltransferases, knew that this particular enzyme was out there,” said Houtz, whose laboratory has been recognized nationally and internationally for its work in this field. “I think a lot of people thought with the sequencing of genomes and comparing different genes in a number of organisms, that this one would show itself, but it never did.”

So Houtz gave Magnani the green light.

Roberta Magnani and Ag Biotechnology senior James Jasis.

Magnani began by isolating an enzyme she felt was a good candidate from some calmodulin-rich tissue. She sequenced the chain of amino acids in the chosen protein and compared that sequence to the human genome database. There she found C2orf34, the gene responsible for producing that protein.

Back in the lab, she cloned the gene, injected it into E. coli bacteria and forced it to produce its enzyme—an enzyme that, indeed, showed the methylation of calmodulin. It took Magnani a mere two months to solve the mystery other scientists had been trying to crack for years. Her lab journal #5 records her reaction on that January day. Scrawled in the left-hand margin is, “We have it, we have it, we have it!”

Mystery Solved, Work Progresses

Ruti Parvari has identified certain human disorders with symptoms of mental retardation and severe muscle weakness as being the result of the deletion or mutation of C2orf34. A researcher and educator on the health faculty of Ben Gurion University in Israel, she has spent years studying genetic diseases.

When Houtz became aware of Parvari’s research, he contacted her, offering to collaborate.

Parvari offered to send cell cultures from affected patients, so they could biochemically demonstrate that calmodulin was not methylated in patients without the gene. Houtz, however, does not have the necessary licenses to handle human cells. So Magnani contacted her friend, medical doctor and fellow Italian, Franca Cambi, who did have permission.

“Sure enough, the evidence was pretty dramatic,” Houtz said. “The calmodulin was not methylated in those patients.”

The repercussions of Magnani’s discovery began to spread. In Israel, Parvari has developed a mouse model to study the deleted gene’s effects. So far, mice show the same symptoms as humans.

In UK HealthCare’s Neurology Department, Cambi is beginning a study on whether methylation affects patients with autism spectrum disorders.

Autistic features—which are not autism, she is quick to point out—are often seen in patients with mental retardation. Houtz and Magnani are collaborating with Cambi on the study.

A Twist

“But now there’s an additional little twist to the story,” Houtz said. “Anybody who has studied calmodulin—and loads of people have—also knows that calmodulin is never methylated in insects. Never.”

But the gene is there—active and completely capable of the task.
“We also know that if we mess that gene up in insects, we see specific and serious consequences,” Houtz said.

In his research pursuing the purpose of methylation in insects, Reddy Palli concentrates on fruit flies, the lab rat of insects.

So one day Reddy Palli took a call from Houtz, who asked him to investigate. Palli knocked out the gene in the red flour beetle and watched for a change.

“It didn’t do much. The larvae became pupae, and then the pupae became adults. Then the adults mated, and they laid eggs,” he said. “They laid eggs, but the larvae did not hatch. We found that the missing gene affected embryogenesis, the formation of the embryo within the egg.”

But if calmodulin were never methylated in insects, why would knocking out that gene make any difference at all?

“We are thinking that at certain stages in the life cycle, insect calmodulin does get methylated, but not all the time,” Palli said.

“So when people looked at it, they may have looked at the times when it was not methylated. They did not look in the embryos where it may get methylated.”

Palli is now studying the gene’s effects in fruit flies, where he can isolate the exact place where the methylation happens. His study could not only lead to applied research in pest control, it also could eventually help in understanding human diseases.

“There is a lot of work on human diseases that is done with an insect model,” he said. “In my lab, I work on both angles (human health and controlling insects in agricultural environs). I get funding from the National Institutes of Health to improve our knowledge of diseases, and I get funding from the U.S. Department of Agriculture to control insect pests.”

New Growth, New Ideas

Using tobacco and Arabidopsis, Indu Maiti is investigating the gene’s expression in plants, where a different scenario plays out.

Indu Maiti suspects the methylation process has something to do with controlling disease in plants.

“We’re finding that in plants, the protein seems to be expressed in young tissue that is regenerating: at the top of the plant, which has lots of small cells or young leaves or sometimes in the tips of the roots. We also see it in flower buds,” said Maiti, a researcher in plant genetics. “Something about the regeneration process requires the methylation of calmodulin.”

He also suspects the process has something to do with controlling disease. When he infects a plant with a pathogen, the gene seems to activate at the site of the infection. It also seems to activate when the plant is under environmental stress, but whether either has to do with regeneration or signaling has yet to be ascertained.

Research in this field, too, can have ramifications in human health. Maiti sees a time when plants will regularly be used to make proteins that would be useful for medicine.

Just the Beginning

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