Quirky Primase Bollixes Delicate Genetic Dance

Enzymatic Miscues Could Lead to DNA Mutations

By Robert Sheldon, Public Relations

The duplication of DNA is as precisely choreographed as a ballet. There are proteins that carefully unwind the two-stranded structure of the DNA molecule while a chorus of other proteins tiptoe in and out to keep the strands from dipping and twisting about each other until they are entwined once again, before each strand is copied and dances away with its new partner as the curtain falls on the creation of a brand new set of chromosomes.

It would appear to be a process with little tolerance for mistakes, no room for one of the dancers to trip and fall on stage, or miss the outstretched hand of a trusting partner as they swirl about one another in precarious leaps and bounds.

For most of us, this biochemical dance of life is a carefully choreographed sequence admired more for the beauty of its results than the practiced gyrations by which the final result is achieved. But others like NU Professor of Chemistry Mark Griep (shown above) seek to understand each and every step in a meticulously constructed performance in which no movement is, or should be, without meaning or significance.

When Griep discovered that one protein enzyme essential to DNA replication seemed to be stumbling around the stage, far more prone than most of its fellow cast members to making mistakes, he set out like a Ballanchine of the test tube to analyze its "curious" performance.

The stumbling ballerina, in scientific terms, is primase, a special kind of RNA polymerase; an enzyme that initiates, or primes, DNA synthesis by initiating the synthesis of nucleic acid polymers, the building blocks of the replicated strand.

"Other nucleic acid polymerases are far less error-prone," he said. "Most DNA and RNA polymerases make as few as one mistake in 100,000 or even a million. Primase makes as many as one mistake in 20."

In the DNA replication process, the two strands of the duplex DNA are separated into two single strands. Each of the single strands has to be copied by a DNA polymerase, a process accomplished in cooperation with primase. Of the two original strands, one is called the leading strand and is more easily copied than the other, called the lagging strand. The two strands are different because their orientation is antiparallel to one another-parallel but oriented in different directions-and the leading strand just happens to be properly oriented for the DNA polymerase to work efficiently.

On the lagging strand, however, certain chemical processes involving primase have to be done over and over. Griep said that as a result of this repetitive and more complicated replication procedure, every new piece of lagging strand DNA polymer ends up connected to a short RNA polymer.

"That means even more work for the cell because you now need other enzymes to remove the RNA polymer," Griep said. "If the RNA polymer remains attached, it will cause cells to die when they divide, or at least result in mutations in the new cells."

He says that the errors that primase makes could be an important factor in natural mutagenesis, the process by which mutant genes are formed. It could even be responsible for mutations that occur in response to external factors.

The concept of natural mutagenesis is related to the biological concepts of "survival of the fittest" and "population adaptability," Griep said. "The molecular biological definition of 'survival of the fittest' means that a successful, or 'fit,' organism leaves behind offspring who have the same sequence of DNA as they do. If the process of DNA replication creates so many errors that the offspring don't have the same DNA sequence as the parents, then the offspring won't be anything like their parents." To Griep, finding that nature uses such a magnificently error-prone enzyme like primase during DNA replication was a mystery worth solving.

It also might solve another mystery, offering evidence that could show some scientists that they have long been fingering the wrong suspect in the process by which mutant bacteria are produced.

Natural populations of bacteria do not all have the identical DNA sequence, Griep noted. Many scientists believe that this type of genetic diversity allows at least some of the bacteria in a population to survive and adapt in the face of assaults by anti-bacterial medicines.

"It's long been believed that natural mutations were the result of the rare mistakes made by DNA polymerases," Griep said. "With all the mistakes that primase makes, I see primase as a more likely cause."

If so, Griep may be on the way to discovering the basis for natural mutagenesis and possibly for the development of drugs that inhibit primase from working.

By learning how primase works, it may be possible to develop drugs that act against primase so that it will kill the bacterium within which it acts and prevent that bacterium from mutating into a drug-resistant strain. By studying primase, his lab will also determine which amino acids in this enzyme are responsible for its error-prone nature.

Griep speculates that if primase plays this role in natural mutagenesis, it may be "the thing that allows evolution to happen."

"I won't go so far as to say primase is responsible for evolution. But I think it's critical for making it happen."

Griep is seeking an extension of his three-year grant from the National Science Foundation to undertake such studies. He also hopes that the additional funding will enable him to extend his research into the study of human primase, which is also known to be very mistake-prone.

"If human primase causes mutations in humans, then it may contribute to the initiation of cancer. That's a good reason to be concerned about it," he said.

Griep, vice chair of the Chemistry Department, has created a zoetrope movie on his web page to show the repetitive process by which DNA is duplicated. He says that his web page is visited by several people a day and that he has received many e-mail comments from students from all around the world who appreciate being able to "see" the whole process in action. The web address for the movie is http://chemmgriep2. unl.edu/replic/forkmov.html.

David Shelton, Stephen Baenziger, Bob Graybosch developed Nuplains Wheat.

Nebraska's First Hard White Wheat Soon Ready to Roll

By Cheryl Alberts, IANR News Service

Nuplains, Nebraska's first hard white wheat variety, is scheduled to be available to certified seed growers this fall and farmers for planting in fall 2000.

Nebraska-grown white wheat has the potential to join a growing market for tortillas, pita breads and Asian noodles, said Robert Graybosch, a research geneticist with the U.S. Department of Agriculture's Agricultural Research Service at the University of Nebraska. ARS scientists teamed with NU wheat breeders and the Nebraska Wheat Board to develop white wheat for Nebraska.

Nuplains is just the first of several Nebraska white wheat varieties, predicts Steve Baenziger, NU wheat breeder who will assume responsibility for Nebraska's white wheat development in the next three to four years.

"You can't have just one wheat and stop," Baenziger said, because no one variety is ideal for every farm.

Graybosch agreed. "White wheats are being developed because customers want them. And if Nebraska is not in a position to provide them, customers will shop elsewhere." He predicts two or three other white wheat varieties will be forthcoming in the next couple of years.

In 1998 Nebraska produced 84.6 million bushels of wheat, half of which are exported, Baenziger said. Asia imports 400 million bushels of white wheat, from Australia and other countries. Nebraska wheat promoters hope to tap this growing market.

Nuplains is a cross between Abilene, a hard red winter wheat, and a Kansas experimental hard wheat. Although other states have white wheat programs, Nebraska needed its own, Graybosch said. Wheat varieties are bred to grow in a particular climate and to resist that area's insects and diseases. Kansas cultivars, for example, aren't winter-hardy enough for Nebraska.

Right now few if any premiums exist for white wheat. However, Nebraska wheat growers may need to adapt if they wish to remain competitive, Graybosch added.

A new education committee is spreading the word about white wheat's potential. Hard White Wheat 2000 will target Nebraska wheat growers, seed suppliers, grain purchasers, elevators, flour millers and consumers.

Committee member Drew Lyon, NU dryland crops specialist at NU's Panhandle Research and Extension Center at Scottsbluff, describes white wheat's advantages.

The differences between white and red wheats are in the genes, Lyon said. White wheat has no major genes for color. The gene that gives red wheat its color also contains tannins, which cause bitterness. The absence of bitter tannins in white wheats mean millers can mill the grain closer to the hull, ultimately getting more flour and using less sugar in the product.

The U.S. milling and baking industry also is interested in using more white wheat. For example, NU's Institute of Agriculture and Natural Resources Nebraska Wheat Quality Laboratory evaluates new wheat lines for their end uses, including wet Asian noodles.

Red and white wheats have many similarities -- seeding dates and rates, fertilization, harvest, yields and test weights. They must not be mixed, however. Mixed wheats are worth much less. Until commercial facilities are dedicated to white wheat storage, Lyon noted, some producers may need to store white wheat on the farm.

Keeping the two wheats separate is so important that planning needs to begin this year if white wheat is to be planted in fall 2000, Baenziger said. To ensure a pure grade of white wheat, a completely different crop, such as alfalfa, must be planted where the first crop of white wheat will be. That eliminates any possibility of volunteer red wheat mixing with white wheat.

Hard white wheat has the potential to grow especially well in the Panhandle because of western Nebraska's dry climate. A drier climate helps prevent seeds sprouting in the wheat head if harvest is delayed. Sprouting also occasionally occurs in red wheat, Baenziger noted.

Sprouting devalues wheat, Baenziger said, because enzyme levels increase, and test weight and flour quality decrease. All cause the wheat's flour to lose its ability to make good bread and noodles.

White wheat, mostly from Australia, has the edge on the wet Asian noodle market now. In another decade, Lyon said white wheat could be fairly common and could even surpass the number of red wheat acres in Nebraska.

IANR scientists have worked with the Nebraska Wheat Board to plant about six acres of hard white wheat near Sidney, Neb. Barring unforeseen circumstances, grain from these plots will be harvested this summer and sold to certified seed producers this fall. They will plant and harvest it for sale to producers in fall 2000.

Hard White Wheat 2000 committee members include representatives from NU Cooperative Extension educators and specialists, Nebraska Wheat Board, Nebraska Department of Agriculture, Nebraska Grain and Feed Association and the Nebraska Wheat Growers Association.

The Nebraska Wheat Board and the USDA help fund NU's wheat breeding and development program in cooperation with IANR's Agricultural Research Division.

Arts Are Basic Grant Now Includes Three West Nebraska Counties

By Kathe Andersen, College of Fine and Performing Arts

Cherry County Rural Schools, in partnership with the College of Fine and Performing Arts' Arts Are Basic aesthetic education program, has received a $91,199 Excellence in Education Council (lottery) grant to continue a cross-curricular arts project in Cherry County and expand it to Scotts Bluff and Banner counties.

Two Cherry County teachers, Fay Ann Blaylock of Mother Lake #190 in Ashby and Katy O'Kief of Evergreen Dist. #53 in Valentine, will be mentors for the newly trained teachers in Scottsbluff and Banner. They have been trained in aesthetic education and have incorporated its principles and methods into their lesson plans.

Blaylock and O'Kief hope to attend the Arts Are Basic Summer Workshop June 7-18 at the Johnny Carson Theater . New and returning teachers to the Arts Are Basic aesthetic education program participate in this summer workshop. Week one (June 7-11) focuses on visual art and theater, and week two (June 14-18) focuses on music and dance.

Wendy Weiss, a professor in the Department of Textiles, Clothing and Design, will conduct a workshop in August in Valentine for Cherry County's 39 rural school teachers. She will teach design techniques to create quilts and banners, the medium which school children will use during the year to create a school quilt.

Two extension educators from the Institute of Agriculture and Natural Resources will also train at the workshop.

Last year, Cherry County Rural Schools and Arts Are Basic successfully collaborated on the pilot program "Storytelling Through Music," which will be replicated this year in Scotts Bluff and Banner counties. Cherry County will pilot a new aesthetic education program with Arts Are Basic and Extension Educators, "Storytelling Through Visual Art." Both programs incorporate arts education into core subject areas to increase student learning, social skills and exploration of student talent.

"We hope by the end of the school year 1999-2000, the schools in Cherry County will have quilts and banners that reflect the history of their school or ranch," said Kit Voorhees, director of Arts Are Basic.

Students will study aerial photos of their school's location, develop patterns from this study and then create designs that will provide inspiration for their school quilt or banner. Students and teachers will gather family, school and area stories, write them down, tell them to fellow students and parents, and then create designs that will reflect some facet of each stories. These designs will be incorporated into a cloth quilt or banner. Student work will then be exhibited in each school next April and will be installed for permanent display in the schools. Quilts from the university's extensive collections and nearby Indian reservations will also be studied.

Banner and Scotts Bluff rural schools will replicate this year's "Storytelling Through Music." Michael Fitzsimmons, an Arts Are Basic master teaching artist and composer, will teach the musical component. Students will make their own instruments, create accompaniment to prose and poetry writings, and write and tell their original stories. These stories, gathered from family and friends, will reflect school and area history, and may be made into books. Students will create orchestral accompaniment for their stories, select instruments, and oversee classmates' performances of their musical stories. Student compositions will be performed in April.

This project brings the arts to rural students who might otherwise not experience it.

"Many of these students will never have the chance to meet a composer or artist," Voorhees said. "But they need to expand their universe in this ever-shrinking world. Art is a celebration and a non-threatening way of giving them a window into the world at large."

In Cherry County, the 20 rural schools vary in size from one to 22 students.

Voorhees said the intellectual growth of students in Cherry County last year was evident in the assessment statements from the students. At the beginning of the program last fall, and then again at the end this spring, students were asked to finish the following statement: "A composer is...."

Last fall, a typical response was, "A composer is an actor who sings." By spring, one student wrote "A composer is anyone who believes in writing music."

"That's a big concept for them to grasp," Voorhees said.

Master teaching artist Fitzsimmons said the students in Cherry County were very creative and receptive.

"The kids seemed more receptive and excited about an artist coming in," he said. "And they see the importance of valuing your own story and area where you live."

Voorhees agrees.

"When the kids write stories out of their life, it seems mundane to them," she said. "But when the school supports them and their classmates join in as the accompaniment, what is theirs acquires a luster of value. That is a rare thing."

Voorhees hopes to expand the program to other interested areas throughout rural Nebraska with additional grant support.

"Cherry County is the resident reference for other counties to see how this program can become sustained," she said.

Roxie Bullock shoots the breeze with professor David Beukelman before addressing his speech disorders class. Roxie uses a laptop computer called a liberator to speak to large groups.

Professor's Help Gives Prom Queen Freedom of Speech

By Curtis Bright, Public Relations

Roxie Bullock was speechless as she accepted the crown for Lincoln East High School's prom queen. This was unusual for Roxie. The bubbly 18-year-old is a seasoned public speaker, but when the MC announced her name, a smiling Roxie accepted the honor silently from her remote-controlled wheelchair.

Roxie has cerebral palsy, and she speaks through a talking laptop computer. That day it was out for repairs.

Roxie learned how to use her computer with the help of David Beukelman, professor of special education and communication disorders at the University of Nebraska-Lincoln's Barkley Memorial Center. Beukelman specializes in augmentative and alternative communication, or to put it more simply, he uses technology to find voices for people who can't speak clearly on their own.

"There's quite a variety of people who have difficulty speaking well enough so they're understood." Beukelman said. "Examples include someone with multiple sclerosis or Lou Gehrig's disease, or someone who's had a stroke later in life, or severe Parkinson's disease. A child with cerebral palsy or a teen-ager with traumatic brain injury, might be an example of someone who's unable to speak for physical reasons."

Because every person is different, Beukelman has developed methods to assess each individual's needs and find the best communication strategy available. This often involves a combination of physical rehabilitation and computer technology . In October of last year Beukelman won an award for his research from the Rehabilitation Engineering Center on Communication Enhancement, which is a consortium of Duke, Penn State, New York-Buffalo, the University of North Carolina and Temple universities. He is also a 1996 recipient of NU's Outstanding Research and Creative Activity Award.

"To help someone talk who can't speak is a pretty remarkable experience," Beukelman said. "You see the power that it gives individuals. Even though with the equipment they don't speak as rapidly or as efficiently as before, it is a really rewarding experience."

Beukelman has worked with Roxie since she started preschool.

"I remember her as a young child having this very infectious laugh before she really had all of the equipment that made her seem like part of the group and become accepted by other students," Beukelman said.

Over the next decade computer technology improved dramatically, and today Roxie uses a portable computer that is much faster and easier for people to understand.

"The system is a liberator," Beukelman said. "It speaks for her and has a printing system so she can print memos, messages and assignments. She controls it by physically touching areas on a keypad. Instead of letter by letter spelling, which is very slow, she has a system of storing words and sentences which she can use to enhance her communication speed."

A day before her 18th birthday Roxie used her liberator to speak to Beukelman's augmentative communication class.

"My favorite class is P.E.," Roxie explained through her computer voice. "Sometimes I play volleyball. Other times I get out of my chair to go swimming or ride scooters. I like to talk to boys and listen to country music."

A bit nervous at first, Roxie quickly relaxed and soon had the class laughing as she poked fun at Beukelman.

"She's kind of returning the favor in that she comes here to the university.," Beukelman said. "She speaks to my students in class, and that gives her some practice in formal public speaking."

It also gives Roxie a chance to talk about her post-high school plans.

"Just like anyone else, I have goals for my future," said Roxie. "Some day I would like to have a job doing music. I could work in a music store, or a radio station or even write music. My liberator helps me reach goals by being my typewriter and voice."

Grant Winners Bernard Doudin, above, and Diandra Leslie-Pelecky, below.

Two Win NSF Career Development Grants

By Tom Simons, Public Relations

Two University of Nebraska-Lincoln physicists have won prestigious four-year National Science Foundation Faculty Early Career Development grants.

Bernard Doudin was awarded a $300,000 grant and Diandra Leslie-Pelecky a $320,000 grant in a program that is designed to emphasize the importance NSF places on integrating research and education in the beginning stages of a faculty member's career. Applicants for NSF Career grants are required to submit long-term plans that include both research and teaching components.

"The Career program is even more competitive that the regular NSF grants program," said Roger Kirby, professor and chair of the department of physics and astronomy at NU. "Only about 15 percent of applications are approved and it's rare for one department to get two awards, so we are especially pleased that both Diandra Leslie-Pelecky and Bernard Doudin were chosen."

Doudin and Leslie-Pelecky, both assistant professors, were funded for separate studies that involve magnetic properties of extremely small structures.

"My research plan is to make very small electronic devices and have magnetic control of the electronics," said Doudin, who earned his doctorate at the University of Lausanne in Switzerland (1991) and joined the Nebraska faculty in 1997.

"The advantage of having a magnetic control is you have one more possibility to tune the electrical characteristics of the device. One application is the new generation of hard disks. You can gain a factor of nearly 10 by using a new type of reading heads based on a magnetic electronic device."

Theoretically, he said it is possible to get a magnetic device that can work based on the detection of single electrons.

"If you have one magnetic orientation, you can accept one electron, if you have the other, you cannot," he said." If you control the magnetic orientation, you control the device. The main drawback is you must be small, about 10,000 atoms, and you must cool down the device a lot."

The educational part of Doudin's plan involves the creation of an interdisciplinary upper-level electronics course that will be accessible to non-physics science majors.

Leslie-Pelecky, who earned her doctorate at Michigan State (1991) and joined the NU faculty in 1996, received her grant for "Cluster-Assembled Magnetic Nanostructures." In lay terms, her project will involve assembling tiny clusters of magnetic atoms and compacting the clusters under extremely high pressure into disks about one-third of an inch in diameter and a few microns thick. The disks can then be used to measure the material's magnetic properties.

"My particular area is magnetism and I'm interested in looking at ferromagnets-nickel, iron, cobalt-and how their magnetic properties change as you decrease their size down to a few nanometers (billionths of an inch), which is a fraction of a human hair's width," she said.

"Obviously, if you're going to use these materials for applications, you can't use a 50-nanometer piece of material. You're going to have to have an ensemble, a giant group of these."

Leslie-Pelecky said the technique she uses gives her control not only over the size of the clusters, but also over the distribution of their sizes and therefore great breadth of research material.

"How we make the material, the size of the particles, the size distribution of the particles, how we put them together-all these things go into making a material that hopefully has unique properties and we hopefully also understand why those properties arise," she said.

"These materials have special importance because they have applications for magnetic recording and for permanent magnets, which are mainstays in things like motors."

For the last three years, Leslie-Pelecky has run the physics department's Science Works program, which takes demonstrations of current university research to schools and youth groups, and she said she wants to build on that for the education-outreach portion of her grant.

"It's kind of hard to assess the impact of the Science Works visits, so I wanted to take an alternative model," she said. "I'm going to take one class and work with that class intensively on a long-term project."

The class Leslie-Pelecky "adopted" is taught by Sue Kirby and Peg Honeycutt at Lincoln's Clinton Elementary School, 1520 N. 29th St. The students were fourth-graders last year and will be in the fifth grade in 1999-2000.

LuAnn Wandsnider, center, and Peter Bleed, dark shirt, along with three students examine a find from the 17A site.

Class Privy to Early Lincoln Secrets

Archeological Dig in Lot 17A Precedes Kauffman Construction

By Tom Simons, Public Relations

The closing of a parking lot for the construction of the Esther L. Kauffman Academic Residential Center to house the J.D. Edwards Honors Program in Computer Science and Management, will give university anthropologists an opportunity to research a bit of Lincoln's history.

Work crews are removed pavement from part of Lot 17A, southeast of 14th and U streets, for a dig that will continue until approximately June 14, when construction of the Kauffman building will begin.

The area along 14th Street was a residential area from the late 1880s until the university purchased the land in the 1950s. Peter Bleed, professor of anthropology, said the area was not served by city sewer lines until the early 20th century and he and his crew will search for intact septic tanks, cisterns and privy trenches. "People threw a lot of things in privies and once they were there, they stayed there, so privies tend to be documents of earlier times," Bleed said. "They will give us a measure of the affluence of folks who lived on the margins of the university in the late 19th and early 20th century."

Bleed said another potentially interesting site is on the 15th Street side of the lot, where Hattie Painter, a Civil War doctor, operated an infirmary from 1879 until her death in 1889. After her death, the infirmary was turned into a hospital operated by the Tabitha Home, "so it was the first Tabitha Hospital," Bleed said.

Sixteen parking stalls will be lost during the archeological dig, but an expanded Lot 17C will be available in the mall east of Memorial Stadium. The mall will be temporarily paved for lots that will be used pending construction of additional parking garages on campus.