The “Icing” on the DNA

XUEHUA ZHONG, an assistant professor of genetics, studies epigenetics, a growing area of research focused on how chemical tags on DNA can change the expression of genes. She and her team at the Zhong Lab of Epigenetic Regulation, located at the Wisconsin Institute for Discovery, are especially interested in the modification of genes involved in growth and development, and how epigenetics can be affected by the changing environment.

As evidence for a link between environmental factors and epigenetics grows, so does public interest in the topic as people consider the impact of their lifestyles and diets not only on themselves but also on the next generation. Zhong and her team hold talks for the public about their work and conduct a number of hands-on programs about epigenetics for undergraduate and K-12 students, including a summer science camp for local high school students, a field trip for middle-schoolers, a youth apprenticeship program in her lab and a “tabletop exploration station” about how lifestyle choices can affect gene expression. Zhong hopes opportunities such as these will raise interest in and encourage the next generation to study this rapidly growing field.

What is epigenetics?

It’s a very interesting question, I would say. The definition of epigenetics has been really challenging over the years because there are different concepts of epigenetics. Most people accept the definition that epigenetics is modifications on the genetic material, the DNA, that changes expression of the underlying genes. I like to say that epigenetics is like a Christmas decoration. You decorate the DNA in a different way, and then the expression of genes is different.

I would also use another comparison: If you think about a cake, the base of the cake is the DNA, the genes. Then the epigenetics is the frosting, the decoration on the cake. And the nice thing about that is if you don’t like the frosting, you can remove it. You can redecorate it differently, and it looks like a different cake.

Can epigenetics be passed on from one generation to the next?

This is another reason why epigenetics is so debatable—the question of inheritance. The modification on top of DNA has been well accepted, but whether it’s heritable is still being debated. Some modifications are very transient and unstable. But some of the modification, for example, methylation—the process of adding methyl groups to the DNA molecule—is fairly stable and can be inherited by the next generation. That is called transgenerational inheritance.

We talk a lot about how your diet, your exercise and your environment have a huge impact on you, obviously, but can also impact your children and even grandchildren through transgenerational inheritance. There are cases from World War II of women who lived through famine, and even 20 years later when they were leading a healthier life, those women tended to have children with more diseases and stress through- out life.

How is this inheritance being studied?

It’s very challenging to study transgenerational inheritance in humans. We’re talking about 60, 70 or 80 years for each generation. But in plants, it’s been very clear that certain epigenetic patterns can be transgenerationally inherited. For example, the Wisconsin cold can induce modifications of genes that can then be inherited. This is an area we are very interested in—environmentally induced epigenetic modifications and to what extent these modifications are transmitted to the next generation.

What plant do you use to study inherited epigenetics?

Currently we are primarily utilizing a flowering plant called Arabidopsis thaliana, or thale cress. It’s a model system that is widely used. We use it because it has a small genome, and because most of our studies are done at the whole genome scale, it’s cheaper than other model systems. Also, the generations are very short, only eight weeks. You can look at six generations in just a year. We’ve also started to extend our work to rice and maize through other collaborations on campus.

Can you explain what you’ve learned about plant aging in your work?

We have been finding that one epigenetic complex in particular is very important to make sure that a plant senesces, or ages, at the right time. Early senescence can reduce yields, so if we can find a way to delay senescence we can hopefully increase productivity. And that’s exactly what we see. If we get rid of the complex we’ve found, senescence is significantly delayed.

While we often talk about how delaying aging is good, the opposite can be true, too. Here in Wisconsin, we have relatively short windows for growing plants. If we can promote senescence, we can maybe shorten the plants’ growing season to better fit our weather patterns.

Now we are trying to understand the mechanism behind these changes because only when we know the mechanism can we really manipulate the system. Ideally, we will be able to manipulate things both ways by fine-tuning the epigenetics to different levels. It’s not all or nothing—it’s kind of an art.

How can your work help address concerns about climate change?

Heat and drought will make the areas that can grow plants limited and challenging in the future. This is a big motivation for us. We want to know what kind of epigenetic modifications happen in response to heat and drought—how strongly, uniformly, stably and rapidly do these modifications happen? Also, is this inheritable? If we treat a plant with heat and collect its seeds, will the next generation “remember” that past experience? Can that memory help the plant?

Why is it difficult to study the influence of environmental factors on epigenetics?

In the lab, it’s simple because we can control each factor and use one kind of stress. But in the real world, you are going to have multiple factors, and how they crosstalk is very complicated. Heat is associated with drought, and there may be long, dark nights and short days as well. I am interested in finding the epigenetic complexes responding to all of these factors. Ideally I want to combine all this information to establish an environmental epigenetic regulatory network. And if there is one key complex responding to all kinds of factors, that can be our target.

Is there a way to do very targeted epigenetic work?

One area we are getting into is epigenome editing (also named epigenome engineering) using a modified CRISPR–dCas9 system that others are using for genomic editing. This lets us target the genes involved in aging, let’s say, and then change only those few genes we have identified to be important. We can put a modification only in that place or on those genes. It’s more efficient.

Using CRISPR–dCas9, the epigenetic changes hopefully will be stable. That’s a question right now because we haven’t gotten to that step yet, but I hope that’s true. Ideally once we have the modification on there, it should stay and do its job.

How are epigenetic studies being used beyond the lab?

I am most interested in how epigenetics can be applied to horticulture and agriculture, but many people are interested in epigenetics for drug discovery. In human medicine, there is already a drug used clinically called azacitidine, which is used to treat a bone marrow disorder called myelodysplastic syndrome and works by blocking the methylation of DNA. This is still a huge, growing area, and whether lab findings can be used in the field or in practice is a million-dollar question. We need efforts to take the discovery from the lab into the field. Making that connection is important and challenging work in all areas of research.

Xuehua Zhong uses plants to study epigenetics, an exciting new field that is broadening our understanding of how some traits might be passed down from one generation to the next. Photo credit: Sevie Kenyon BS’80 MS’06

Turning them on

CALS biochemistry professor Hazel Holden is excited about science. So when she witnessed science becoming “boring” in her daughter’s classroom—a feeling several classmates shared—she decided to take matters into her own hands.

Some five years ago she created Project CRYSTAL—Colleagues Researching with Young Scientists, Teaching and Learning—a program designed to challenge middle schoolers who show an aptitude for science. The program is funded by the National Science Foundation.

Each school year, Holden takes four eighthgrade students under her wing for weekly hands-on sessions. “We’re trying to de-stigmatize science by exposing kids to material they otherwise would never have been exposed to,” she says.

And it’s impressive stuff. The students start by extracting DNA from yeast cells they have grown themselves. They then use the extracted DNA to practice the art of polymerase chain reaction (PCR for short), the process by which a piece of DNA is replicated to produce thousands to millions of copies of a targeted DNA sequence.

Switching between 10-minute lecture and lab segments keeps the kids motivated, and with clever anecdotes sprinkled throughout the lecture material, the young students are never bored.

This class format is used to progress to more advanced skills such as protein purification—the isolation of proteins from, in this case, E. coli cells— and X-ray crystallography, a tool used by the students to identify the molecular structure of a crystalline protein. The year ends with a group poster presentation— a rite of passage that most students don’t experience until much later.

“I was able to work in a real lab and gain lab experience. I do not think many 12-year-olds are able to have an experience like that,” says Project CRYSTAL alumna Manpreet Kaur, now a high school senior. “Before the program I did not have any knowledge of X-ray crystallography, and now I am able to explain the process in science classes.”

The program inspired Kaur to take several AP Science classes and affirmed her plans to become a doctor.

Holden has published her curriculum as an 80-page book, and Project CRYSTAL was introduced at Indiana University–Purdue University Indianapolis during the current school year.

The program has benefited graduate students almost as much as the youngsters, giving them experience teaching complex science at the most basic level.

“This class puts our own graduate work in perspective. You get more excited about your own research by watching them get excited about the small things, like pipetting,” reflects biochemistry doctoral student Ari Salinger.

Looking ahead, Holden hopes that what she has created will inspire other universities to implement similar programs.

“The students want to learn more—and they are ready for it,” Holden says.

Will Dead Species Live Again?

Stanley A. Temple is the Beers-Bascom Professor Emeritus in Conservation in forest and wildlife ecology at CALS and a former chair of the conservation biology and sustainable development program at the Gaylord Nelson Institute for Environmental Studies. For 32 years Temple occupied the faculty position once held by Aldo Leopold, and while in that position he received every University of Wisconsin teaching award for which he was eligible. Since his retirement from academia in 2008 he has been a Senior Fellow of the nonprofit Aldo Leopold Foundation. He and his 75 graduate students have worked on conservation problems in 21 different countries and have helped save some of the world’s rarest and most endangered species. Last spring Temple gave a TED talk at a special event devoted to de-extinction, a concept that has captured the imagination of scientists and the general public alike.

What is “de-extinction”?
De-extinction is a recent term that involves bringing back an extinct species using DNA that’s been recovered from preserved material. There are two ways that it can be accomplished: one would be cloning to produce a copy of an extinct individual’s genome. The second way is through genetic engineering to re-create a close approximation of what the extinct species’ genome might have once been. The reality is that it’s no longer science fiction. We’re getting close to being able to revive extinct species from recovered DNA.

This must make for some unusual scientific partnerships.
It’s an interesting synthetic endeavor that matches the biotechnologists in the laboratory with conservationists in the field. The biotech crowd will be responsible for recovering DNA from an extinct species and through either cloning or engineering turning that DNA into individuals. But once they’ve done that, the next step involves people like myself who know how to recover endangered species by taking a small number of individuals and turning them into a viable population and getting them back into the wild.

What opportunities might this technology present to conservation efforts?
On the plus side, obviously, it would be exciting to bring back a species that human beings drove to extinction. But even if we weren’t able to do that, the technology presents an appealing opportunity to recover DNA from preserved specimens of an endangered species and use it to enhance the genetic diversity of the surviving population.

Can you please elaborate on that?
Conservationists have recovered many endangered species from very low population levels and saved them from extinction. The problem is, they’re often genetically depauperate, or lacking in genetic diversity. If we can recover some of the lost genes from preserved specimens collected before the population crashed, we might greatly improve the species’ prospects for long-term survival.

How would a conservation biologist go about actually applying this?
De-extinction is still an unproven concept, but it’s likely that sometime in the coming decades it will happen. Once they have revived individuals of an extinct species in the lab, then conservation biologists could try to recover the species by captive breeding and reintroducing the species to the wild. But conservation biologists get concerned about some of the details: Which species are going to be revived? Are they the right species? Are they the species that have the best chances for long-term survival in the world today? Are they species that might actually enhance the ecological health of the ecosystem that they were once part of, like the wolves reintroduced to the Yellowstone ecosystem? These are all questions of setting priorities for which species to actually revive.

How would you recommend setting priorities?
As a conservation biologist I would certainly look first at recently extinct species that were affected by a threat we’ve now overcome. Not only are those the ones for which we’re likely to have good quality DNA, but their ecological niche in the wild hasn’t been vacant for very long. And as a result, the ecological community that they were once part of has not readjusted itself to their absence, and might once again easily accommodate the species in its midst. On the other hand, if you’re dealing with a species that’s been extinct for a very long period of time—centuries or even millennia—the ecosystem that they were part of has moved on, and a species like that, once back in the system, could essentially be the equivalent of an invasive species. It might disrupt the system and threaten extant species.

How would you like to see this development proceed?
Considering the timeline that we probably have years or even decades to do this right—I and other individuals and groups that are thoughtful and somewhat skeptical about this would like to see a very broad discussion of the implications. We would like to see a lot of input in deciding the priorities about which species to bring back. We would not like to see this done in secret, which, unfortunately, is where this seems to be heading. This very expensive work is not receiving government funding and doesn’t have any sort of public oversight. Hence, privately funded biotech labs seem to be focusing on reviving spectacular extinct species, like mammoths and other Ice Age animals, rather than species that have a real chance of surviving in today’s world.

What would be an important takeaway point for the general public?
De-extinction doesn’t mean we can ignore the significance of extinction—to think, “Oh well, we can let species go extinct because we can always save some DNA and bring them back later.” This would just be an open door for activities that have been constrained by concerns for biodiversity and basically give the green light to go ahead and precipitate extinctions of species that are already with us.

Making It Personal

It was one of the strangest homework assignments Erin Syverson had ever had. The senior genetics major was asked to open a small vial and start spitting.

“I would much rather have gotten my blood drawn, but it’s a simple, effective way to collect DNA at home without a medical professional,” notes Syverson, who submitted her saliva to 23andMe, a private company that analyzes a person’s DNA—all 23 pairs of chromosomes, hence the name—for $99.

Syverson underwent the analysis as part of Genetics 677, Genomic and Proteomic Analysis. While DNA testing is not required for the course, professor Ahna Skop encourages her students to undergo it. Students may use their own results as the basis of their individual semester-long class project, which requires doing in-depth research about a particular genetic disease or disorder and presenting findings in class and on a website the student creates.

“Because they have a vested interest in their project, they are emotionally engaged and seek out answers from me, their classmates and beyond the classroom—for example, from doctors and their families,” says Skop. “The payoff I see in my course is deeper, longer-lasting learning due to this emotional investment.”

Those benefits are being cited all around the nation as more and more college genetics courses encourage students to get tested. They were confirmed by a recent study in the journal PLOS One showing that 70 percent of students who underwent personal genome testing self-reported a better understanding of human genetics on the basis of having undergone testing. They also demonstrated an average 31 percent increase in pre- to post-course scores on knowledge questions, which was significantly higher than students who did not undergo testing.

Syverson didn’t end up basing her research project on her own results, but she still found the testing worthwhile. “Through learning to interpret my own results and scrutinize them, I have learned a lot about not only the diseases they tested me for, but also how to think critically about genetic results,” she says. “I’ve also learned a lot about the state of the field and how to explain it to others, which will be very helpful for my future career as a genetic counselor.”

The course will be offered again next spring. Student presentations are posted at

Getting to the heart of a problem

When Marion Greaser set out to study titin, the largest natural protein known to man, his goal was to answer some basic questions about its role in the body. A major protein of skeletal muscle that’s also found in heart tissue, titin gives muscle its elasticity and is known for its massive size, which ranges from around 27,000 to 33,000 amino acid residues in length.

“Initially we were just going to look at whether titin was related to muscle growth in animals,” says Greaser, a CALS professor of animal sciences.

Working in rats, his team looked at changes in the size of the titin protein over the course of animal development—and immediately came across something strange. In most cases the titin protein shifted from a larger form to a smaller form during development due to natural changes in protein processing known as alternative splicing. But in some rats the titin didn’t change. It stayed big.

The team wondered if they’d mixed up the samples. “But we’d kept good track of things and, in fact, all of the weird samples were from the same litter of rats,” says Greaser. “Then the light bulb went off: There must be some genetic reason why these samples are different. These rats had a genetic mutation affecting the alternative splicing of the titin.”

But where was the mutation? They first checked the titin gene itself, but it was fine. With hard work, they were able to pinpoint the mutation to a little-studied gene called RBM20, which had been previously linked to dilated cardiomyopathy and sudden death in humans.

Dilated cardiomyopathy affects approximately one in 2,500 people. Sufferers have enlarged hearts, with thin walls, that don’t pump blood very well. People with the RBM20 mutation need heart transplants and, without them, tend to die quite early: between ages 25 and 30.

Scientists first linked RBM20 to hereditary dilated cardiomyopathy in 2009, but they hadn’t yet figured out how a faulty RBM20 gene worked—or didn’t work—to cause disease inside the body.

Greaser’s accidental discovery, as described in Nature Medicine, filled in the blank. In healthy individuals, the RBM20 protein is involved in the alternative splicing that helps trim titin down to its smaller, adult form. Without it, titin doesn’t get processed correctly, and the presence of extra-large titin in heart tissue leads to disease.

“Now doctors can analyze people showing symptoms of dilated cardiomyopathy, see if they’re carrying this mutation and factor this information into their treatments,” says Greaser. That treatment would probably start with careful monitoring to catch any further deterioration of the heart condition, Greaser notes.

Protecting our Pollinators

People and bees have a long shared history. Honeybees, natives of Europe, were carried to the United States by early settlers to provide honey and wax for candles. As agriculture spread, bees became increasingly important to farmers as pollinators, inadvertently fertilizing plants by moving pollen from male to female plant parts as they collected nectar and pollen for food. Today, more than two-thirds of the world’s crop plants—including many nuts, fruits and vegetables—depend on animal pollination, with bees carrying the bulk of that load.

It’s no surprise that beekeeping has become a big business in the farm-rich Midwest. Wisconsin is one of the top honey-producing states in the country, with more than 60,000 commercial hives. The 2012 state honey crop was valued at $8.87 million, a 31 percent increase over the previous year, likely due in part to the mild winter of 2011–2012.

But other numbers are more troubling. Nationwide, honeybee populations have dropped precipitously in the past decade even as demand for pollination-dependent crops has risen. The unexplained deaths have been attributed to colony collapse disorder (CCD), a mysterious condition in which bees abandon their hives and simply disappear, leaving behind queens, broods and untouched stores of honey and pollen. Annual overwintering losses now average around 30 percent of managed colonies, hitting 31.1 percent this past winter; a decade ago losses were around 15 percent. Native bee species are more challenging to document, but there is some evidence that they are declining as well.

Despite extensive research, CCD has not been linked to any specific trigger. Parasitic mites, fungal infections and other diseases, poor nutrition, pesticide exposure and even climate change all have been implicated, but attempts to elucidate the roles of individual factors have failed to yield conclusive or satisfying answers. Even less is known about native bees and the factors that influence their health.

Poised at the interface of ecology and economy, bees highlight the complexity of human interactions with natural systems. As reports of disappearing pollinators fill the news, researchers at CALS are investigating the many factors at play—biological, environmental, social—to figure out what is happening to our bees, the impacts of our choices as farmers and consumers, and where we can go from here.

Seeding an Organic Future

As a wicker basket containing old, faded seed packets made its way around the room, Tom Stearns asked each person to grab a packet and pour a few seeds into their hands. Some of the seeds were green and shriveled, others were tiny, shiny and black.

“Check them out,” encouraged Stearns, founder and president of Vermont-based High Mowing Organic Seeds, the only seed company in the nation to sell 100 percent organically produced seeds.

Addressing participants and speakers attending the Student Organic Seed Symposium at the Lakeview Inn in tiny Greensboro, Vermont, Stearns asked the group to consider what they could—and couldn’t—tell about the seeds just by looking at them. For many, all it took was a quick glance to know what plants they’d grow into.

But seeds hide an important part of their story beneath their coats. Just looking at a handful, it’s impossible to know who developed them and to what end. These details, however, have a lot to do with a farmer’s success.

Plant breeders have enormous influence over the varieties they develop, making key decisions about how, when and where they’ll grow best. Plants bred with high-input, conventional systems in mind (which generally employ chemical fertilizers and pesticides) tend to thrive in those systems. Likewise, those bred for organic systems tend to flourish in organic systems. Yet relatively little of this latter type of breeding work has been done over the past 50 years, mostly due to meager financial support. Today’s organic growers have difficulty finding organic-adapted seeds, and they are often forced to choose among conventional varieties.

To Stearns, this situation is ludicrous, on par with giving a beef cow to a dairy farmer. “You will get milk out of a beef cow, but not a lot—they haven’t been selected to produce milk. Beef cattle don’t have the right genetics for what dairy farmers are trying to do,” he explained to the group. “That’s what I think organic growers are dealing with. We don’t even know what we’re missing. The seeds we’re using aren’t genetically adapted to the kind of systems that we have.”

The most obvious solution is to have more plant breeders doing organic work. And, as Stearns looked around the room that day at the Lakeview Inn, he had reason to hope.

At a professional gathering about a year earlier, Stearns had met Claire Luby and Adrienne Shelton, graduate students in the Plant Breeding and Plant Genetics program at CALS, along with Alex Lyon MS’08, a CALS agroecology graduate now working on a doctorate at the Nelson Institute. During a dinner reception at the 2011 meeting of the Vegetable Breeding Institute—a Cornell University-based public-private partnership that fosters interaction between vegetable breeders and seed and food companies—the trio had shared with Stearns some of their experiences doing organic-focused work. While the students were excited about the work, they also felt unsure about their career paths and somewhat isolated and discouraged. Graduate students working in organic plant breeding, like their faculty advisors, are few and far between, and they lack the support network enjoyed by their conventional-focused peers.

“There are a lot of activities and events geared toward graduate students who are going to work at the bigger plant breeding companies,” explains Shelton. “But it’s really hard to connect with other students doing organic plant breeding because the organic seed industry is so small in comparison, and there are just a few of us—at best—at each land-grant university.”
Before dinner was over, a plan had sprouted to put on a symposium, dubbed the Student Organic Seed Symposium (SOSS), to give this scattered group of students a much-needed opportunity to come together and feel like part of something bigger—part of the new and growing agricultural movement that they comprise. Luby, Lyon and Shelton would organize it, with support from their advisors. Stearns would help host it in Vermont. There would be talks by experts, farm tours and a visit to High Mowing Organic Seeds. There would also be time to just hang out and get to know each other.

“The whole idea was to try to build these connections, to create a scientific community that could support us throughout our careers,” says Shelton.

It all came together in early August 2012, with 20 graduate students cupping seeds in their hands, eager to develop new plant varieties to meet the needs of organic growers.

Humans have been breeding plants since antiquity. Simply by selecting which seeds to save and plant the following spring, people make decisions that alter the overall genetic makeup of their crops. It’s a powerful technique, known as selection, that plant breeders still use to this day.

Modern plant breeders have many more tools at their disposal and bring a scientific approach to the whole process. A significant portion of the work involves making crosses. To do so, breeders pick two varieties with desirable traits, transferring the pollen from one to the pistil of the other, purposefully mixing together the good genes of both. The new plants created this way then go through years and years of re-crossing and selection until the breeder is satisfied with the final product. Only then is it released as a new variety. It’s a time-consuming process, taking up to a decade and sometimes more.

Crossing and selecting are classical plant-breeding techniques that look pretty much the same whether they’re used to breed plants for organic or conventional systems, so context is key.

“One of the underlying paradigms of plant breeding is you should breed for the conditions under which the crops are going to be grown,” says Bill Tracy, chair of the agronomy department at CALS.

And organic farms have a special set of conditions. Without chemical options to control weeds, insects and microbial diseases, organic farmers need varieties with a unique set of traits. For instance, they need varieties that are fast-growing and preferably dense-growing to out-compete and shade out weeds. They also need varieties with natural pest and disease resistance. At the same time, these plants need to produce a large, beautiful bounty.

“But to date there’s been very little breeding for organic conditions, so there are opportunities and needs out there that aren’t being met,” says Tracy, whose breeding program encompasses both conventional and organic sweet corn.

Safer Snacking?

What do Americans love more than french fries and potato chips? Not much—but perhaps we love them more than we ought to. Fat and calories aside, both foods contain high levels of a compound called acrylamide, a potential carcinogen.

First discovered in foods in 2002, acrylamide is produced whenever starchy foods are fried, roasted or baked, meaning that it’s found in everything from doughnuts to coffee beans. But fries and chips are relatively high in acrylamide compared to most starch-based snacks, and potato processors are eager to change that.

CALS plant geneticist Jiming Jiang, a professor of horticulture, has a solution. His lab has developed a promising new kind of potato with reduced levels of acrylamide, an innovation he created with support from USDA-ARS plant physiologist Paul Bethke, a professor of horticulture. As a bonus, these potatoes also could help producers significantly reduce food waste.

The problem starts with storage. Because fry and chip processors need potatoes year-round, most of the fall harvest goes into storage, where low temperatures can cause simple sugars to accumulate in the tubers, a phenomenon known as “cold-induced sweetening.” During cooking, those sugars react with free amino acids to produce acrylamide. The same reaction also causes fries and chips to turn dark brown during processing, making them unsalable.

Jiang’s solution is to insert a small segment of a potato’s own DNA back into its genome. The extra DNA helps block the gene that converts sucrose into glucose and fructose, the sugar culprits that cause both acrylamide formation and browning. Through this process Jiang has created a numberof potato lines that produce very little acrylamide when cooked.

“Regular potato chips can have acrylamide levels up around 1,000 parts per billion,” says Jiang. “Ours are down around 150.” Jiang’s process, potentially of enormous use to the food industry, is now being patented by the Wisconsin Alumni Research Foundation.

But because they are genetically modified (GM), Jiang’s potatoes can’t be grown for consumption in the United States, where only a handful of GM crops have been approved and widely cultivated.

Jiang hopes that will change, and notes that GM versions of corn and soybeans, which are now added to many processed food items, contain DNA from other species. The extra DNA in his low-acrylamide potatoes, on the other hand, comes from the potato genome itself.

Down the line, especially if scientists confirm acrylamide’s link to human cancer, consumers may have to make a choice: accept a new GM crop or cut back on fries and chips.

Berry Good Science

It was a yummy connection to a science badge. Nearly 50 Girl Scouts from southern Wisconsin extracted DNA from strawberries during two “Biochemistry in the Kitchen” workshops led by graduate students and postdoctoral fellows with the Integrated Program in Biochemistry’s Student–Faculty Liaison Committee. The scouts used salt water and dishwashing detergent to extract DNA from mashed-up strawberries, and then added rubbing alcohol to make its spaghetti-like strands visible. As a keepsake, the girls got to bring their results home in test tubes.

How DNA Profiling Works

EVEN THOUGH 99.9 PERCENT OF HUMAN DNA is exactly the same in all people, a single droplet of blood or stray eyelash collected at a crime scene still carries all the genetic information needed to convict a criminal. Back at the lab, forensic scientists simply probe the remaining 0.1 percent of the genome—3 million nucleotide bases—for telltale variations. This process, known as DNA profiling or genetic fingerprinting, reveals a suite of variations in the genetic code that, taken together, constitute an individual’s unique DNA profile. Here’s how it works:

1. Collect a sample and extract its DNA. Scientists only need a tiny amount of DNA—around 100 micrograms—to construct a DNA profile from a crime scene sample. That’s so little, a few cells from saliva on a straw will do.

2. Amplify the telltale regions. Scientists use a powerful technique called Polymerase Chain Reaction (PCR) to make millions of copies of the sample’s telltale DNA regions. In particular, they home in on regions known as Short Tandem Repeats, or STRs, which are composed of short units of DNA—just four or five bases long—that are repeated numerous times in a row. What makes these regions telltale is that the number of repeats they contain varies widely from person to person. In criminal investigations, 13 such STR regions, all located in the non-coding DNA between our genes, are analyzed for the number of repeated units they contain.

3. Count the repeats. During PCR, fluorescent dyes are attached to all the STR copies that get made—one type of dye for each STR region—so that all of the DNA copies from a given region can be distinguished from the others in the mix. Scientists run the mixture through a capillary electophoresis machine, which separates the various DNA fragments by size. From there, it’s a fairly easy thing to calculate the length of each STR region, and, therefore, the number of repetitive units at each site.

4. Look for a match. To convict a suspect, his or her STR repeats must match those in the crime scene sample—at all 13 STR regions. According to the FBI, when all 13 STR sites match perfectly, it’s virtually guaranteed you’ve got your culprit; the odds of fingering the wrong person are about one in 1 billion. A single STR mismatch, however, is enough to exonerate a suspect and spur investigators to search CODIS, the nation’s database of DNA profiles, in hopes of solving the crime.

Missing Piece

Horticulture professor Jiming Jiang studies centromeres, large regions of DNA that help match up and then separate pairs of chromosomes during cell division. Long ignored by most genome scientists, centromeres now appear to be key in creating artificial chromosomes—complete, self-replicating packages of genetic material that could revolutionize crop improvement in plants and gene therapy in humans.

What is a centromere?

Humans have about 30,000 genes carried by our 46 chromosomes. Each chromosome has one centromere, a stretch of DNA that ensures the accurate transmission of the chromosomes—our genetic material—into daughter cells during cell division.

You can actually see the centromere under the microscope—it looks like a constriction on the chromosome. It’s an extremely complex structure. There’s a lot of protein involved, and the centromere’s DNA—how to describe it? It’s junk DNA, basically. It doesn’t have genes, just a lot of repetitive junk DNA.

When did scientists discover the centromere is full of junk DNA? When they sequenced the human genome?

Understanding the structure, function and evolution of centromeres in plants will definitely help on the human side.

Scientists say that the human genome has been sequenced, that the mouse genome has been sequenced, but people don’t realize that none of the centromeres have been sequenced. They just don’t count it. And most scientists don’t care because there are no genes [in those regions]. Plus, it’s almost impossible to sequence centromeres with current technology—they are too long and contain too much repetitive DNA.

But rice is a different story. The centromere on rice chromosome 8 is not particularly repetitive, so my team was able to sequence it back in 2004. We were the first team to sequence a centromere from a multicellular species, and, surprisingly, we found genes in it!

How did this rice centromere end up with genes in it?

Let me try to explain what we think is going on in this strange case. In the scientific community, people are starting to believe that centromeres originate somewhere. They don’t just exist, right? And when a new centromere emerges—a neo-centromere—it may look like a regular piece of DNA, with genes in it. Over time, however, as it evolves, the centromere accumulates junk DNA for whatever reason.

So, the rice centromere that we sequenced, we believe, is somewhere in the middle of this evolutionary process. It’s like a caveman. It is starting to accumulate some repetitive, junk DNA, but it still has some genes in it.

It’s interesting to consider that centromeres can evolve.

With funding from the NSF, we are now trying to understand the evolution of this rice centromere over the past 10 million years. To get at this question, we’re sequen-cing this centromere in five different species of wild rice, which diverged from cultivated rice between 1 million and 10 million years ago. We’ll be able to see what kinds of changes happened over that time—how the genes moved away, how the junk DNA accumulated.

This work will help us figure out the minimum requirements needed to make a centromere. There are a lot of things we don’t know right now, but if we can figure out the answers, this work will ultimately help us design artificial chromosomes. That’s the long-term goal.

How to See a Gene Work

RESEARHERS OFTEN WANT TO KNOW when and where a particular gene is “turned on” inside an organism. How do they accomplish such a feat? When genes are on, they produce telltale proteins. Unfortunately, it’s no good looking for these directly; proteins are much too small to see, even with the most powerful microscopes. Over the years, scientists have come up with a number of innovative workarounds. Here’s a particularly bright (green) one:

  • Tag the gene. Scientists splice the gene for Green Fluorescent Protein (GFP) to the end of their gene of interest. This way, when the gene is expressed, it will produce a protein with a GFP “tail” attached to its end.
  • Transfer the tagged gene. The methods used to deliver this extra DNA vary from organism to organism. For the common fruit fly, by way of example, a tiny needle is used to inject the DNA into early fly embryos.
  • Switch on the black light. GFP, originally discovered in a naturally fluorescing jellyfish, emits green light after absorbing certain wavelengths of UV light. This makes the cells expressing tagged genes glow bright green, while cells with no gene activity remain black. With special microscopes, scientists can see exactly which cells inside an organism are producing these glowing proteins, and then monitor changes in those protein levels over time.
  • Location, location, location. For researchers trying to develop safe genetic therapies, it’s vitally important that they be able to control where therapeutic genes are expressed inside the body. Using GFP-tagged proteins, scientists can quickly determine whether a new gene therapy approach targets the correct organs or tissues. To make this kind of analysis possible in larger organisms, scientists introduced GFP into the core DNA of several lab animals, including mice, cats, pigs and fish.