Spring 1987 by Endeavors

Research and Graduate Education at The University

of North Carolina

at Chapel Hill/Spring l98i/Volume IV Number 3

Molecular

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BioIogT

Cooperation Between Federal and State Governments, the Priuate Sutor, and the University Communities Leads to Biotechnology Revolution

During the past decade the accumulated efforts of a particular area of basic science have found their way to the market place in a dramatic and unprecedented manner. The bio technology industry owes its origin rn toto to the results emanating from departments of biology, genetics, biochemistry microbiology, and molecular biology, to name but a few on the campuses of American research universities and research institutes. The availability of the new therapeutic and diagnostic reagents in medicine can be traced directly to the laboratory bench of the scientist. New protocols for plant protection or the enhancement of crop production have been derived from the greenhouse and laboratory of plant scientists. Moreover, the discoveries of the past ten to fifteen years are changing the science of genetics from a retrospective science into a prospective science, for in many cases we no longer need to await the appearance of offspring to confirm a specific genetic composition. Rather we can determine the genetics of the offspring beforehand using

of the recently developed techniques. The growth of biotechnology results from the close cooperation of the federal and state governments, the prirate sector, and the uni

the increased funding of basic research in academic units which are seminal for this industry. Clearly a beneficial harvest can be reaped from the biotechnology revolution.

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Dr. J. Dennis 0'Connor, the neutly appointed UNC-CH Vic*Chancellor for Research and Anduate Studia and Dean ol the Gnduate School. Dr. 1'Connor, who holds a joint appointment as professor in the Department of Biologr, comes to UNC fron llCLA, where he was Dean of the Division of Life Sciences and Professor of Biology.

several

versity communities. The strength of such a consortium is apparent not only in the rapid expression of the biotech industry but also in

However, concerns exist in several quarters. The close association of university scientists with the industrial sector can only continue under circumstances which protect the traditional values of academic research, including the free exchange of ideas and data in publications and conferences. Release of genetically altered organisms into the environment must be monitored so that to the best of our ability the delicate balance of a complex ecosystem is not disturbed. It is a pleasant coincidence that the first issue of Endeavors to be published since my appointment at UNC-CH is devoted to bio technology. I have been a participant in and witness to many of the exciting undertakings which have characterized the past twenty years in life science. It is with particular pleasure that I invite you to discover within this issue a sample of the significant research in bio technology occurring at Chapel Hill.

-J.

Dennis )'Connor

from the laboratory to industry agriculture, or medicine. Significantly, they also provide badly needed resources and support to stimulate creativity and help launch new proiects in this extremely costly area of scientific research.

Director,

)ffice of

-Ton K. Scott Research Services

-Suzanne Appelbaun Editor

The contents ol this Endeavors* represent a departure from our usual approach of covering a number of interest areas within each issue. Here, instead, we have focused on one category of research projects, advances in biology on the UNC-CH campus which have led or may lead to an exciting array of applications of biological technology, many of which could well prove beneficial to human health care and the quality of life. The research community is in the midst of

a revolution in its understanding of biological systems that has come about through mastely of powerful new techniques and tools. Many of the techniques involve manipulation of genetic

material and, with them, researchers are attempting to understand how genetic effects are regulated. Basic research on this campus has so far contributed to techniques for synthesizing proteins, manufacturing new molecules,

and unravelling the mystery of abnormal genetic expression, among a good many others. Possibilities for use of these new techniques that are explored in these pages include the development of new anticancer drugs; the development of more potent, yet less toxic,

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drugs of various types; vaccines against diseases

for which no effective means of immunization has yet been found; genetic manipulation to improve crop growth and yield; ways of mitigating the effects of environmental pollution; and creation of new man-made materials with improved properties of strength and durability over similar materials produced by nature-a list that is by no means exhaustive. Much of this work involves manipulating the structure, and consequently the function, of biological

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molecules. While the research is often of a highly technical nature, in the articles that follow an effort has been made to keep tech-

nical language to a minimum. This issue describes the work of investigators from several seemingly unrelated scientific disciplines on the campus under headings that tend to blur the traditional distinctions between departmental fields. Much of the research is interdisciplinary. Entities such as the Program

in Molecular Biology and Biotechnology on this campus and the North Carolina Biotechnology Center, located in the Research Ttiangle Park, assist in promoting such cooperative research as well as aid

in the transfer of biotechnology

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are indebted to Dr. Marshall Edgell, Director of in Molecular Biology and Biotechnology at UNCIH, and Dr. Laura Meagher and Mr. Stwen Burke, the Progran

of the North Carolina Biotechnology Center, for their help in the preparation of this edition of Endeavors.

could determine the sequence, the ability to change

it in a predetermined

manner became

an achievable objective.

Conholled Mutation: A Key to Genetic functlon

researcher can generate

all combinations in

one or two runs. Saturation mutagenesis relies on an automated machine that can synthesize single strands of DNA. The computer-operated machine has four bottles, each containing a

solution of one of the bases. When the

in a desired sequence, the machine takes a growing DNA chain and, automatically pumping in the appropriate base solution, adds nucleotides to the chain, researcher types

a team led by Dr. Clyde A. Hutchison III, professor of microbiology at UNC-CH, developed a method for creating specific mutations by changing a single base in a DNA 1977

sequence. This technique, called sitedirected mutagenesis, has since been applied in many it gave researchers the

ways. For instance,

ability to convert normal genes into cancercausing genes, 0r oncogenes, which greatly aided the study of the role of these genes.

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Molecular biologists employ a wide variety of techniques, ranging from genetic manipulations to organic chemistry. While some researchers apply these tools directly to advancing basic research and technology, others concentrate

More recently, Hutchison, Dr. Marshall H. Edgell, professor of microbiology, and Drs. Steven K. Nordeen and Kenneth Vogt, former postdoctoral researchers in Hutchison's laboratory have developed a related technique, called saturation mutagenesis, that allows researchers t0 construct quickly every possible mutation in

the nucleotide sequence of a gene segment. This method is much faster and cheaper than simple synthesis, in which an entire day is needed to generate each separate sequence and which involves costly reagents, according to Hutchison. Using saturation mutagenesis, a

one by one. The machine can build a good sequence up to about sixty bases long. To produce the mutations, each bottle is contaminated with each of the other three bases. "lf you synthesize with impure solutions, you

will

randomly generate some molecules

with the normal, or wild-type, sequence plus many variationsl' Hutchison says. By controlling

the degree of contamination, one can control the average number of mutations per molecule. "We set the solutions to aim for one to two mistakes per moleculel' he adds. Complementary strands needed to form

doublehelix segments are synthesized in a separate run. After the two strands are joined, the segments are reinserted into active DNA molecules and cloned to produce a collection, or library of mutants. The mutants are then identified by removing molecules at random and sequencing the synthesized segments.

on refining and inventing techniques that will open up new horizons for scientific inquiry. For instance,

1975 Dr. Frederick Sanger,

a British biochemist, invented a technique for sequencing deoxyribonucleic acid (DNA). The DNA molecule is basically a long chain, or

of linlis called nucleotides. These nucleotides are composed 0f a sugar (deoxyribose), a phosphate, and a nitrogenous base. There are four DNA bases: adenine (A), guanine (G), cytosine (C), and thymine The DNA molecule can be thousands of bases long, and sequence,

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the bases repeat in a sequence unique to the organism. The process of determining the

of bases is called sequencing. This relatively quick and easy technique led to further advances in DNA research. Once a researcher sequence

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With funding from the National Institutes of Health, Hutchison's team developed saturation mutagenesis using a DNA segment in mouse mammary tumor virus called the glucocorticoid response element (GRE). "Most people would consider the GRE an enhancer, something that can stimulate expression of genesl' Hutchison says. "lt's fairly mysterious at present how those things work, and one reason for trying

to make mutants of this particular segment

that they would be useful in figuring out which nucleotides in the sequence actually are important for the function of GREI' The target sequence was thirty nucleotides long, yielding ninety possible different mutations at a single base (thirty bases with three possible changes at each). 0f these, the team isolated 88, 74 as singlebase mutations. "Other researchers have started using these mutants to see what effects they havej' Hutchison notes. "Now one of the directions we're taking is trying to extend this technique to produce mutations in other sorts of genetic

ular hydrogen atom in the structure points

certain direction. Ericlson's laboratory however, can manufacture and use right-handed as well

as left-handed amino acids. Even though Ericlson is working with relatively short chains, the ability to synthesize amino acids increases by many orders of magnitude the number of possible proteins that can be made.

The proteins are also unnatural in the way they are built. When proteins are made in nature

through ribosomal synthesis, the chains are built link by link, or stepwise, from the amino end of the chain. In the laboratory using

way of linking versus iust adding unnatural amino acids to create new proteinsl' The technique also provides even greater versatility

in design, he adds. The model for this new technique is a protein called betabellin (see back corcr), which Ericlson has been developing over the last three years in collaboration with iane S. and David C. Richardson,

Ericlson says. Ericlson is collaborating with Dr. David Eisenberg of the Molecular Biology Institute at the University of California at Los Angeles in designing the molecule. "This work is on the cutting edge of synthetic protein productionl' he adds. Betabellin's unusual doublechain design has required a new kind of protein molecular structure. Singlechain protein molecules have structures that fold in patterns of alpha helices

Dr. Bruce W. Ericlaon, professor of chemistry

and beta sheets. Because betabellin has two chains, the first goal of the project has been to get each chain to fold in the zigzag pattern of a beta sheet and to lie backtoback with the other sheet. The question for the moment is which combination of acids, in what order, will give this desired folding pattern. Erickon's

is developing techniques for synthesizing small, unnatural proteins by organic chemistry rather than genetic means. His team is one of only three groups in the world working on this type of protein engineering. "We're inventing new proteins that nature can't makel' he says. Although "unnatural" in this sense, these proteins

are not significantly different from natural ones. "lf it's pure, there's no distinction between a synthetic and a natural proteinl' Ericlson

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points out. The major advantage of unnatural proteins, he adds, is the ability to create many more variations than exist in nature, which is

a technique called solid-phase peptide synthe

are fully developed, clients will be able to order proteins tailored to their needs. The proteins being synthesized in Ericlaon's laboratory are unnatural in several respects. All proteins are chains of amino acids at least fifty link long. While natural proteins are made of only twenty amino acids, many more amino acids exist and can be made in the laboratory-all arailable to the protein synthe sizer. Also, amino acids in natural proteins are "left handedl' which means that a partic-

has discovered a way to branch one chain into two identical chains that can then both be extended simultaneously. "No one has ever tried to do thisl' Erickson says. "lt involves a new

Professors

Conctructing New Kinds of Protelnc

When methods for creating these molecules

Another difference in these proteins lies in their structure. Nature can only make chains in a single, continuous line. Erickon, however,

biochemists at Duke University. The Office of Naval Research and the Army Research 0ffice have jointly funded the work. A second protein that linls three chains, called betacylindrin, is "on the drawing boardsl'

elements. I'm also interested in developing techniques for doing DNA sequencing as fast as possiblel'

especially useful in designing drugs and vaccines or for medical diagnoses and treatment.

purity and yield of [he'end productl' Ericlson explains. decrease the

Eri&son

sis, Ericlson's team builds the molecules stepwise but backwards, from the acid end of the chain. They build them stepwise because, as Ericlson notes, it worls best. "Nature has developed a perfect systeml' Ericlaon says. "lt has essentially come up with one answer for how to make proteins and has used it for millions of yearsl' The team builds the molecules backwards for the same reasonthe method worls best. More specifically, it prevents certain adverse reactions during the process. "Normally, if you add amino acids in the way the ribosomes add them, you run into the problem of some of the left-handed amino acids being converted into right-handed amino acids, a serious problem that will

team has synthesized eight versions of betabellin so far, learning from the mistakes each time and improving the design in the process. For stability, the two sheets have been designed to fit together and to repell water, and thus be mutually athacting, on the interior surfaces. The layers are held together by a bond between two sulfur atoms, a design feature

borrowed from an antibody molecule. An increased surface charge also helps maintain the

integrity of the structure. The betabellin research team includes research associates Scott Daniels, Anantha Reddy, and Raju Mohan plus graduate student Elisabeth

Albrecht. After the team has found a structure that will fold properly and maintain its integrity, they will focus on the end of the molecule that forms a pocket for interaction with other molecules. The nert phase of the protein engineering proiect

will be to

develop

a strat-

likely to produce a binding pocket for a given ligand. Various possible changes can be entered

into a computer, which will generate educated guesses about the resulting molecules. The

best possibilities are then tried. For instance, one might try three different amino acids at each of six places (three on each chain), producing 36, or 729, different molecules. These variations are then combined with a ligand that is similar to the desired ligand. The molecules are next combined with the desired ligand to see which 0f the 729 variations are more attracted to it than to the

first ligand. This process, called competition affinity chromatography, yields the yariations that bind best with the desired ligand. Finally, the amino acid sequences of these mriations are determined to learn which changes produced

The first goal of the betabellin proiect is to be able tct fold a protein in this predetermined pattern, such that it becones a new structural supporl for various t'Ltnctional sites. The second and third soals of lhe proiect are kt be able t0 crcate t'unctional sites for binding and calalysis by subtlv altering the protein and tailorinq it lo specific bindinq Nents and desired reactjons egy for tailoring the structure such that a ligand, a molecule that binds to another, will bind to the pocket. "We'll be designing a strategy that can be used with any ligandl'

Erichon notes. The threefourths of the betabellin molecule that determines folding will be held constant while the remaining fourth, which forms the binding pocket, will be subtly altered until its structure is the best match

for the desired ligand. Chemists normally work in the opposite direction, designing the ligand to fit a given binding pocket. But Ericlson wants to offer researchers the ability to work in either direction. The methodology for developing this strategy involves changing the amino acids within the binding pocket section of the chains. The first step is t0 figure out which changes are most

the best binding. "The advantage of this process is that you can use educated guesses and test a whole repertoire of possibilities at oncel' Ericlson says. "ln the process you have bought information. You've eliminated 99 percent of the 729 possibilities to get six 0r seven that workl' This cycle, which should take two to four weeh, is followed by another. Holding the first round of changes constant, the researcher tries changing other amino acids. "Our guess is that if you do this two or three times, you will get new engineered proteins that bind their ligands as tightly as do natural proteinsi' The third phase of the project involves the same methodology but aims at designing a catalytic site. The process of converting one compound into another requires a tremendous amount of energy. However, using a ligand that has structural properties of both compounds and that represents an intermediate stage bs tween the two could reduce the amount of energy needed and speed up the chemical reaction. Erickson hopes to develop a strategy whereby the binding pocket can be altered to

bind with such a ligand as this better than with either the initial or the final compound. The resulting engineered protein may actually be a new catalyst for the conversion reaction. To carry out these experiments, Ericlson has set up a protein chemistry laboratory in collaboration with the National Institute of Environmental Health Sciences. The Protein Dr. Anantha Reddy $tanding), who constructed the amino acid that branches to forn the double chain in betabellin. looks on as Dr. Scott Daniels assembles a betabellin molecule with the aid of a solid phase svnthesizer, designed and built bv Erickson and Eldex Laboratories of California and now under development at t-iNC-CH. With this new machine, a betabellin-sized nolecule can be synthesized in sixty hours. Manually, the same iob would take about one month. The nolecule is formed in a glass cclumn around polystvrene beads that are snaller than fine grains of sand. Under the control ol a nicroprocessor, reagents and solvents are punped into the column as needed. llhen the desired peptide is fully assembled. it is renoved

from the beads and purified.

Chemistry Laboratory is available to every researcher on campus. "We now have the modern automatic equipment to make and purify a small protein and to determine its amino acid composition and sequencej' Erickson notes.

-Diantha J. Pinner

Scientists conducting basic research in the area of molecular biology are asking very fundamental questions: What happens at a

microtubules that attach to a central part of the chromosome, called the centromere, to help pull it to a pole. It is not known, however,

certain stage of cell division? What regulates precisely when a gene goes into action? and What principles underlie interaction between protein molecules? Understanding the answers to these questions wil[, of course, broaden

what the centromere's function is in this process or how the microtubules attach. "lt's a 'black box' area of sciencel' Bloom says. 'All we know is that tubulin, the protein that makes up the spindle fibers, doesn't bind with the centromere

our knowledge of life processes and help form

DNA. The solution is much more complex

new questions. Eventually, it may also help solve specific problems that humans face by giving us tools to avert Down's syndrome or to develop a more potent, yet less toxic, drug. But these applications are many miles down

than thatl' To work toward an understanding of segregation in human cells, Bloom is currently looking at the centromere and microtubules in yeast. He is using yeast as a model because the cells are strikingly similar to human cells

the investigative road.

The Role of the Centromere in Cell Division One UNC-CH researcher, Dr. Kerry S. Bloom, of biology, is studying

associate professor

chromosome segregation, the stage during cell division when the duplicated chromosome splits apart and the sister strands migrate to opposite poles within the cell. lf all goes well at this stage, the proper equal number of chromosomes

is apportioned to each of the two new cells that are formed. But all too frequently in many organisms including humans, mistakes occur during segregation, and some cells wind up with the wrong number of chromosomes. This condition, called aneuploidy, results in a variety of problems, including Down's syndrome

and many forms of cancer, and is usually Iethal.

Although scientists have learned much about

other cellular processes, such as DNA replication, they still do not know exactly how chromosome segregation works. They do know that the cell generates a group of fibers, called the spindle. The spindle is comprised of

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in structure, reproduction, and chromosomal behavior. Yeast also has many advantages over other kinds of organisms. Experiments can be completed quickly because the cells grow well in a culture dish and divide

about every two

hours-much more frequently than mouse or human cells. Alsq yeast is easy to manipulate genetically and has less DNA than humans107 base

pairs compared to l0e-which makes

it simpler to work with. 'Anything you

can

dream of, anything you can build by molecular biology, you can put back into a yeast cell to see how it worlsl' Bloom says. With funding from the National Institutes of Health and the National Institute of Environmental Health Sciences, Bloom's laboratory is applying two approaches to this study: genetics and biochemistry. Using genetics, Bloom is studying the function of the centromere through conditional mutants, mutations in a DNA sequence that produces a protein. "Conditional mutants are a powerful tool for studying func-

tionl' Bloom says. In the classical

analyses of

conditional mutants, the researcher causes the protein produced by a sequence to fall apart by changing the temperature of the environment.

What happens to the cell when the protein is disrupted indicates what the protein is required for and, consequently, the sequence's function. The difficulty in using this method to study the centromere, however, is that the centromere does not produce a protein. To solve this difficulty, Bloom found a way to control the function of the centromere by manipulating the environment in a different way. He and graduate student Alison Hill spliced in a DNA section containing the promoter for galactose

chromosome with two centromeres. Each of the centromeres will then be attracted to an opposite pole, breaking the chromosome im-

properly again, unless one of the centromeres can be deactiyated, In the case of trisomy, where a cell has received an extra chromosome, the third chromosome will cause severe prob lems unless it can be shut down by turning off its centromere. fuo other graduate students working in Bloom's laboratory are following related questions. Michael Saunders is looking al the structure of centromeres with mutant nucleotide sequences, trying to correlate centromeric structure and function. Elaine Yeh is investigating the presence of RNA in a region flanking the

centromere. Yeh has shown that one of the

this RNA is required for meiosis, one of the forms of cell division. "We suspect that its location near the centro mere is not just fortuitous, that it is related to centromeric functionl' Bloom notes. processes carried out by

Using the biochemistry approach, Bloom is isolating the proteins at the centromere to discover what they are and how they work. Assisted by graduate students Margaret Kenna and Enrique Amaya, Bloom has developed a new method. While other researchers are isolating a strand of DNA without its proteins and then seeing which proteins will bind to it, Bloom

is letting the strand bind its proteins naturally and then clipping out the centromere particle to analyze and identify the proteins attached to

it. This method has the advantage of isolating only those proteins related to the centromere, whereas the other method might yield proteins that will bind but do not naturally occur in that region. "This way you know you have the right proteinsl' Bloom explains. Several pro teins have been identified, and the team is starting to look at how they function.

Gene Regulation In another laboratory across campus, Dr. Kenneth F. Bott, director of the Curriculum in Genetics and professor of microbiology, is working toward understanding how genes are regulated by studying a common soil bacterium called Bacl/us subfrTis ln higher forms of life, every cell in an individual organism contains the same set of genes. Yet only the genes appropriate to a given cell's functions are activated. For instance, while certain genes in liver cells stimulate liver functions, other genes in heart cells control heart functions, and the liver functions are not expressed in the heart cells. What accounts for this differentiation? What regulates which genes will be activated

in a specific type of cell? Understanding this mechanism may be an important step in unraveling one of the mysteries 0f cancer, a disease in which the cell's genetic information fails to be regulated correctly.

metabolizing enzymes (i.e., GAL l-10 divergent promoter).

They discovered that when the cell is bathed galactose the GAL promoter's function is

initiated and the centromeric function is turned off. When the cell is bathed in glucose, the switches are reversed-the GAL promoter is turned off and the centromere is turned on. Thus, the GAL promoter/centromere section can

be made to work like a conditional mutant, causing the centromeric process t0 shut down when galactose is introduced into the environment.

This technique will allow Bloom and Hill to study the function of the centromere throughout the cell cycle by simply turning it off at successive points and seeing what is inhibited. However, it also provides a clue to a mechanism for correcting an imbalance in the number

of centromeres or chromosomes. Sometimes chromosomes break in the wrong place, and resulting DNA repair process may produce a

Dr. Kenneth F. Bott lohn

Moomaw.

re,,'iet^is

a diagramned sectton ol the qenetic map of B. subtilis wrli Catherine lves and

mental process is triggered The siructure ana ii"te cycte of B. subtilis are fairty simple. The bacterium is a singlecelled organism, each cell containing a single, circular, doublestranded molecule of DNA, or "chrom0s0me." When nutrients are available, the cells proliferate rapidly by generating copies of the DNA and then dividing into pairs of cells, each containing a copy of the DNA. The copying

at a point called the origin of replication and proceeds in both directions around the circle until the new pieces meet at the opposite pole. During this growing phase, the genes conprocess begins

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trolling reproduction are active. But eventually the bacteria outgrow the arrailable nutrients. At that point, Bott says, a molecular "switch" near the origin of replication is activated, and the genes controlling spore development are turned on. The cells stop replicating and, instead, use the energy available from the remaining nutrients to synthesize new proteins that will generate spore production. The spores will then lie dormant until conditions are again suitable for growth. To understand the mechanisms of this cycle, Bott's first task is to isolate the origin of replication. He knows that it lies in a certain region of the chromosome, but the specific spot has never been isolated. Catherine lves, a graduate student in microbiology, and John Moomaw, a graduate student in genetics, are

aiding Bott's search for the origin by cloning

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The genetic nap of B. subtilis. Iile origin of replication is at the twelve o'clock position, and the region under investigation by Bottb tean is the set of genes on either side of it. Diagran fron P. l. Piggot and l. A. Hoch, Microbiological Reviews 49J58 (I'985), reprinted by pernission of the authors.

and deciphering the function of several different genes in this region. Steven Colman, another graduate student in genetics, is using a similar approach to characterize an even simpler bacterium, Mycoplasma pneumoniae, in hopes of associating selected gene functions with specific DNA fragments

in the

organism.

Once the researchers can prove that they have

isolated the specific "switch" region and

it takes to stimulate it in the controlled environment of the test tube, they can begin to catalog the precise changes required to initiate sporulation. "We expect to use the clones of the origin of replication segment to characterize the tipes of proteins that bind to it and regulate the initiation of sporulationj' Bott explains. "This will then be a first step toward artificially controlling the switch from know what

Bott has chosen B. subtr'/rs because it can be genetically manipulated and because it is typical of one of the two major types of bacteria. Escherichia coli, lhe bacterium that we

set of genes that trigger spore production when the environment does not contain enough nutrients t0 support active replication. This means that those genes can be artificially ac-

know most about, is typical of one type, called Gram negative, while B. suDlrTq a species that undergoes a form of differentiation, is typical of the other, Gram positive. "8. subtilis is used as a prototype Gram positive bacterium for many molecular genetics studies because so much is known about its physiology, metabolism, and genetic malleability," Bott

tivated

says.

The bacterium is also particularly useful for studying gene regulation because

carries

in the laboratory by simply limiting the nutrients, thus providing a synchronized way of studying the mechanism that initiates spore production. "We're using a simple bacterium that we know a lot about geneticallyl' Bott explains. "We're not trying to explain the whole thing; we're just trying to isolate and uplain the triggerl' Achieving this much, he adds, can lead to understanding how normal DNA synthesis is regulated and how that particular function is stopped when the develop-

active replication to coordination of spore specific gene regulation and a first step toward controlling the coordinated expression of a series of genes required for more complex forms

of differentiationl' The research is funded by the National Science Foundation and by the National Institutes of Health, which provides training grants for graduate students.

Predicting lllolecular Interactions Dr. Jan Hermans, professor of biochemistry is developing theories about how protein molecules behave, based

on information about their

structure and dynamics. Each protein has its own unique, complex molecular architecture, consisting of many hundreds of atoms in precise locations. "lf you can understand in detail the forces within that hold the structure together and understand the internal and external motion

of the molecule, you have taken the first step in understanding the function of the proteinl' Hermans notes. How these molecules interact

with other molecules is an area of particular interest to Hermans, for this is where the most exciting applications

will be found. For

instance, the ability to predict interactions would be very useful in developing more potent, yet less toxic, drugs. Potency depends on how well a drug interacts with its targeted protein. If one knows enough about how that protein behaves, one can design a drug that will produce a better interaction and thus be a more effec-

tive treatment. Assisted by graduate student Amil Anderson, Hermans has been working on several small proteins. The largest of these is myoglobin, a small muscle protein containing about 1,000 atoms and 153 residues. Hermans' work begins with the structure of the molecule, which has been determined by protein crystallographers using X-ray diffraction. The structure is so complex that Hermans uses a sophisticated computer graphics system for visualization and interpretation. The computer system, which was funded by grants from the National Science Foundation and the National Institutes of Health, holds a threedimensional representation of the molecule. It displays the molecule as colored lines, indicating the chemical bonds, and dots, indicating the molecular surface, on a nineteen-inch graphics screen, which is basically a very high-performance television monitor. A computer

program written by Hermans and Michael Carson, now at the University of Alabama, allows the user to shift, rotate, tilt, enter, and change the size of the molecule by turning dials.

It can also simulate the motion of

the

molecule and trace that motion through time. Because these simulations take

an immense

amount of time to calculate, they are first computed on a Cray-2 supercomputer at the University of Minnesota. The results are then analyzed on Hermans' system. But even with

this machinery the molecules are too large and complu to be comprehended easily.

Dr. Jan Hernans and Amil Anderson manipulate a computer-sinulated representation of a nyoglobin molecule.

Thus, in constructing a theory Hermans starts

with a computerized map of the molecule. He then applies the theory to simulate the molecule's dynamics and to extract relevant information about the predicted interaction. Finally, he compares the results with existing experimental data as a critical test of the validity of the theoretical method. "0ur work has passed through several critical testsl' he notes, "and the results are very encouragingi' An example of this process is his work with myoglobin. The crystallographers found that myoglobin can interact with xenon, a simple non-reactive molecule. When the xenon and myoglobin are brought together at a certain pressure, one molecule of xenon binds at a particular site within the myoglobin molecule and remains there. The interaction is particularly

interesting because

occurs inside the

myoglobin rather than on its surface. Using a theory under development, Hermans was able to calculate the amount of pressure necessary for binding and then tested his prediction with existing data, with very good matching

mental data so that the methodology can be checked and developed. The next step will be to study the interaction of a molecule which has some importance for biochemistry and for which there is no experimental data. Eventually, once Hermans has worked out the method, he will start predicting affinity of unknown structures that have not been studied with a particular protein. "That's when the applications

will comel' he says. The primary applications will probably be in drug development. Drug companies typically have many ideas about what molecule might make a good drug or how to improve an existing drug, Hermans says. But it is very expensive to make and test the molecules in the laboratory. "lt is, in principle, much cheaper to subject each molecule to a prediction of the kind that will be possible as a result of this workl' Hermans notes. "Some

will look much better than

others based on

the prediction, and these are the ones that should first be made in the laboratory and tested for efficacyi'

results.

For the present, Hermans is working only on problems for which there is existing experi-

-Diantha J. Pinner

0ncogenes Investiqations into Genes that Cause Cancer Are Providing New Infornation

About the Normal Development ol Cells

The riddle of the cancer cell has absorbed medical researchers for a very long time, producing numerous strategies aimed at a solution as well as a wealth of accumulated knowledge. One approach to this problem began in 1910, and led eventually to the discovery of oncogenes, genes within the normal vertebrate cell that

during reproduction, thus transmitting the "blueprint" for the replication of the organism.

with RNA, and use DNA as the "messenger" that carries the genetic blueprint 0f the organism. A retrovirus invades a cell and integrates its DNA Retroviruses, however, start

message into the DNA of the cell. Thus, when the cell reproduces it also replicates the genetic information of the retrovirus. Often, such a union is benign to both the virus and the cellular host. Sometimes, however, the virus may carry a gene that can cause the host cell t0 convert to cancerous growth. Such a gene is called an oncogene. The method by which oncogenes instigate malignancies in cells is being mapped out by the analysis of the Rous sarcoma virus. The oncogene in that virus, called a src (pronounced "sark") gene because it induces sarcoma

somehow regulate growth and that may, if abnormally expressed, cause cancer. Laboratories throughout the world are now looking closely at oncogenes in an effort to understand their function both when they behave normally as well as when they produce malignancies.

The research being conducted by Dr. Patricia Maness, associate professor

of biochemistry

UNC-CH, is contributing to our knowledge about

the role of oncogenes in normal cellular development.

The Rous Sarcoma Virus ln 1910, Peyton Rous of the Rockefeller Institute for Medical Research withdrew tissue fluid from a chicken tumor called a sarcoma. He filtered out all then-known infectious agents from the tissue fluid, and injected this cell-free filtrate into healthy chicken tissue.

l-tr Pllti, i.t 11,ti,r r' ,tiit)l,rk,|11rt9i.r1r1 1y' 1111r itetitisltt. L ttnler:; *itl; l).,(lrfrl'llrr,l iiii,rrl [']ll /ril,[lli,rnt. .!r't]Ie{i ,ri iftr' r:r' r,.,,,,;'

tumors, produces a protein kinase, an enzyme that attaches a phosphate group onto a target

use of the electron microscope confirmed Rous' discoveries. In 1966, at the age of 85, he was awarded a tardy but well-deserved

cancerous growth.

Nobel Prize.

The Rous sarcoma virus, which is only one

New sarcomas were produced; Rous postulated a viral agent t0 account for them. In essence, he was suggesting that viruses cause cancer. His report met with widespread skepticism and

of a number of tumor viruses that have been

he abandoned this aspect of his research. Decades later, though, the introduction of new techniques for purifying filtrates and the

duction. In the ordinary course of events, the DNA in an organism is used as a template for

discovered since

the 1960s, belongs to

group known as retroviruses. Retroviruses reverse the normal process of genetic repro

the RNA, which duplicates the genetic sequences

protein within the cell. This process, known as phosphorylation, changes the activity of the genetic material of the cell, leading to Phosphorylation, however, also plays a role in regulation of normal cellular growth. Why phosphorylation appears t0 control regular

cellular development but at times leads to cancer is still unknown. In an effort to answer this question, researchers are looking

for the "targets" for the protein kinases, that is, the specific proteins in the cell upon which the src kinase acts. If they learn precisely which proteins are affected by phosphorylation,

embryonic development. What Maness found was that the src protein is expressed

two

phases: early on while the nervous tissue cells are dividing, as well as later when these cells are differentiating (reaching their maturity). Maness concludes that the src may play a pivotal role in regulating the development and

laboratory. fuo graduate students, Carol Shores and Michael Cox, are working with the electric eels, while Wayne Matten is carrying out phosphopeptide mapping studies on the expression of the src in neural tissue. Postdoctoral

maturation of nervous tissue. By extension, this

fellow Muriel Aubrey and research technician Pam Kitchen are working on src target sites, while Chris Ingraham, another postdoctoral fellow, is investigating the role of other onco genes in nervous system dwelopment. The lab also boasts a fourth-year medical student with a Ph.D. in organic chemistry David Ward. The National Institutes of Health is a major

suggests that other cellular oncogenes may be essential for the normal growth of other healthy

cell tissue; proteins which allow an embryo to develop into an adult organism may be the same proteins that in certain circumstances give rise to cancer. Currently, Maness is extending her research in an effort to develop new technologies to characterize the oncogene and its gene products

funder of Maness' research and recently awarded her a Research Career Development Award to

further her work.

order to define more fully the role these products play in the development of normal /le,iearch leiliiriciari 1';rrrr Aitchen skt rint neu rcn al ,r,il rr/tuiri..s u.qerl the slud.v tti the sr. pratein.

itr

neural tissue. She thinks the electric eel will provide an appropriate animal model for her

work. Its electric organ is the source for a relatively abundant supply of src protein kinase, the enzyme that provides the basis they may gain clues to what goes wrong in oncogenesis-the creation 0f tumors. Research 0n retroviruses has led to a startling

discovery: the viral src gene has a close relative in normal cellular DNA. Indeed, there are a number of retrovirus oncogenes that have cellular "cousinsl' Researchers have concluded that these relatives are proto-oncogenes,

the originals for which viral oncogenes are the copies. [n other words, somewhere in the recent past, speaking from an evolutionary perspective, retroviruses picked up cellular oncogenes from normal cellular DNA. The implication 0f this is that the cancer-causing potential of the oncogene is present in the normal cell. Significantly, then, cells do not

The amount of work the research involves a number of people busy in Maness'

keeps

Ultimately, Maness hopes to find out more about the nervous system through the study

of its embryonic development. "The brain is so complex that its development is difficult to understand by examining the mature organismj'

for phosphorylation. Maness is engaged in the search for targets that may lead to a picture of the mechanism of oncogenic activity and wants t0 extend the research beyond the confines of the src gene. "We're taking a general

in looking at the process of neural differentiationl' she says. "There are thirty t0 fifty known oncogenes, all of them as important as the src gene. We are beginning to examine their expression in nervous system development and hope to be able t0 put together a sequence of events in which they may come togetheri' approach

she says. "We think we can learn something new if we dissect the mechanism of oncogene action in the embryol' Furthermore, by understanding such basic processes of human biology, Maness hopes to gain clues leading to the solution of problems like cancer. "Thanla to molecular biology,' she says, "the strides made in understanding the cancer process so far are enormous. What we can look forward to will be a greater understanding of related aspects

of embryonic developmentl'

-Tim

Jenkins

need viruses to become malignant. While

scuttling the idea of viral agents as a primary cause

of all cancers, this discovery has pro

vided scientists with a new focal point for

their

research:

the cellular

oncogene which

both regulates growth and can, under conditions still unknown, lead to oncogenesis.

Oncogenes and Normal Cellular Development Dr. Maness is interested in the role the cellular oncogene plays in normal cell growth and development. Recently, her work has focused

on the activity of the cellular src gene (the prototype for the viral src gene discussed above) in the nervous tissue of the chick during

Medical student David Ward. Chris Ingrahant. ltlaness, and graduale student Wayne Matten are examirttng an

auloradilgran showing phosphopeptides of the

src protein.

Mapping Out the Brain Investiqations into the Structure and FLrnction

Pharmacological

of Nerve Cells and the Neurobioiogy of Nlodifiable Brain Processes

The laboratory of Dr. Raymond Dingledine,

ularly, seems to rely on synaptic transmission mediated by NMDA receptors, and behavioral studies have indicated that the area of the brain involved in this process is the hippo campus, which, interestingly enough, possesses one of the highest densities of NMDA receptors of any brain region.

associate professor of pharmacology, is providing

some of the pieces to a complex puzzle involving the physiological basis of human learning and memory. This research may lead to better drug-based treatments for neurological disorders such as epilepsy. Dingledine is studying excitatory amino acid (EAA) receptors in the central nervous system. Although not well understood, these molecules clearly play a pivotal role in the relaying of messages within the brain. Dingledine wants to unravel some of the molecular properties of these

What neuronal changes underlie learning and memory? For almost a decade, electrophysiologists have used thin slices of'hippocampus to study a phenomenon called "long-term potentiation" (LTP). nP, which can be produced by activating certain excitatory synapses repeatedly over a brief period of time, results in much stronger and more effective communication among the participating neurons. In this way transmission through certain nerve circuits in the brain can be selectively strengthened. The realization that UIP in the

receptors and understand their role in the transmission of signals between neurons.

Excitatory Amino Acid Receptors

hippocampus is blocked by NMDA antagonists has contributed to the growing consensus that

Excitatory amino acid receptors, one type

of neurotransmitter receptors, are protein recognition molecules used by a large fraction of lhe neurons in the brain to relay messages through synaptic transmission.

"ln

NMDA receptors are involved I

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response which signals the completion

of the

transmission of the chemical message from the firsi neuron to the second. This process continues, involving more and more neurons. While the overall function of the EAA receptors is recognized, there are questions concerning details of the structure and function

of these molecules. One of the most important is: Precisely what processes of the brain are EAAs responsible for? To answer this question, researchers are employing receptor antagonists, synthetic drugs that block EAA functions. Scientists observe the processes that are impaired when the receptor is blocked and can

processes

learning.

As stated above, NMDA receptors seem to be involved in the normal functioning of the

synaptic

transmission the nerve cells in this class communicate with one another by releasing a small amino acid moleculel' Dingledine explains. The molecule diffuses to a nearby neuron where it encounters an EAA receptor. The subsequent interaction triggers an electrical

in the

of spatial memory and in some form of

thereby deduce the function of that particular

protein in the brain. At this point, however, there are very few EAA receptor antagonist drugs available with the required selectivity, and so the business of assigning functicns to the different receptors is proceeding slowly. In fact, according t0 Dingledine, there is only one EAA receptor that is well characterized, the NMDA receptor, named after a synthetic drug (N-methyl-D-aspartate) that strongly activates

it. Thanks largely to the work of Dr. Jeff Watkins, a biological chemist in Bristol, England, a number of good antagonists have been developed against the NMDA receptor. As a result, several varied and interesting functions have been discovered for this amino acid that seem to deal primarily with "plasticl' or modifiable, brain processess including

learning and memory. Spatial memory, partic-

brain. But what happens when things go wrong? Mounting evidence indicates that overstimulation of these receptors can lead to seizures and the death of neurons. As it turns out, NMDA antagonists are good anti-convulsants

in animal models of epilepsy, and Dingledine has shown that they can reduce the severity of epileptic neuronal discharges in the hippocampus in these animals. NMDA receptor antagonists also prevent or reduce neuronal death following brain ischemia (tissue damage) induced in animals by blocking the carotid artery. None of these antagonists is in human clinical trials yet, but the research now being conducted is promising. Dingledine and his coworkers hope that their work will help t0 target a new generation of drugs to combat seizure disorders and the consequences of ischemia,

and will help improve understanding ol the neurophysiology of these problems.

it possible to examine these receptors more easily. These techniques employ frog oocl.tes (eggs before they are laid), which are have made

tricked into synthesizing receptors normally found only in the mammalian brain. In 1972, J.B. Gurdon of 0xford University reasoned that oocytes could be viewed as large bags of protein synthesis machinery. He iniected oocytes with messenger RNA (mRNA)

that he had isolated from rabbit red blood cells; the oocytes immediately began making globin, a rabbit protein that frog oocytes would normally never make. Messenger RNA is the nucleic acid template that participates in the synthesis of proteins; most mRNAs relay the "blueprint" in this synthesis for only a single protein. Frog cells can read the code of rabbit mRNA because the protein synthesis machinery of all higher animals is essentially the same. A new technique was born in Gur-

0rariuate .studtrtts

*arkinq uith llre

'ilxld

don's laboratory for manufacturing specific proteins from almost any species in the large, It'rdrtrtrn

arr17

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Frog Eggs and Voltage Clamps While work concerning the function of NMDA receptors is proceeding, efforts are being made to characterize the molecular properties of these and other EAA receptors. Major questions remain about these proteins, according

to Dingledine, including those concerning

the

regulation of receptor synthesis-how do neurons

know how much of the receptor to make, and is this part of a long-term "plan" suggesting the production of finite amounts due to genetic coding, or a shortterm "planl' lndicating a complex response to environmental changes? The answers to these questions

hinge partly on whether or not the receptors are very simple molecules that mainly pass on signals, or much more complex structures that are involved in a number of interrelated processess controlling transmission of neuronal messages. Dingledine believes the second hypothesis to be the case. The above questions clearly involve the structure of the receptor molecule, about which almost nothing is known. "The traditional approaches to studying the molecular properties

of receptors, raising antibodies to the receptor

or purifying it

away from other brain proteins,

in the case of EAA receptorsl' says Dingledine. New techniques

of tlte lntq ottc.vtes. \bnloorn and Kleckner prepare llir:,sr e{{s lor tnjedton rilh ntf,\.1.

hardy frog oocyte.

Clo.seup

Dingledine has adapted the oocyte technology to his research problem. This part of the work is carried out by graduate students Todd Verdoorn, who brought a knowledge of the key molecular biology techniques to the lab, and Nancy Kleckner, the newest member of the team. They isolate mRNA from rat brain,

v,ill

which contains the code for the synthesis of the receptor proteins. A minute quantity of the mRNA is then injected into several oocytes. The protein synthesis machinery of the oocytes reads the mRNA and is able to produce the foreign receptors. Verdoorn and Kleckner then assay for the presence of the EAA receptors in the oocytes by using a voltage clamp. EAA receptors are coupled in the oocyte outer membrane to ion channels, aqueous pores that allow the passage of charged ions, this flow of ions across the membrane results

in a measurable electrical

current. In the voltage clamp technique the ionic currents are measured between two microelectrodes, one inserted into the oocyte, the other placed in a solution just outside the cell. EAA receptor agonists, drugs that stimulate the receptors, are then introduced into the solution and if the receptors are present, an increase in current flow can be measured.

With these techniques Kleckner hopes to isolate specific pieces of mRNA and characterize

them for various receptors. Eventually, the frog oocyte could be used to help clone this purified mRNA for different receptors, thereby

have proved daunting tasks

creating larger quantities of the receptor proteins

from the field of molecular biology, however,

to study in a durable form. Slowly, Dingledine and his associates are beginning to unravel

the structural mysteries of this class of neuro transmitter receptor. Dingledine credits the spirit of cooperation among his colleagues for much of the initial success in his laboratory. Dr. Kay Lund of the Department of Physiology was especially important in helping him with the technical information he needed to get his project off the ground. Seed money from the North Carolina

Biotechnology Center and grants from the National Science Foundation and the National Institutes of Health have also helped to make

his work possible. Dingledine is both excited and cautious about the potential ramifications of his research. "The maximum payoff will depend on how complicated these receptors turn out to be on the molecular scalel' he says. "l suspect and hope that they will be complicated enough to serve as targets for a whole new generation of drugs which wilt modify the receptor functions, rather than block or stimulate them. [f analysis of previously studied receptors is any hint, this witl be the direction future research will take, and the consequences may be significant for improving our understanding of brain function. The receptors must be studied

in isolation,

however,

and this is where the oocyte technology comes in. The basic research has to be done first; applications that result will depend on what is

foundl'

-Tin

Jenkins

AYO

t,)

What Makes a Better Pathogen? Microbiology is Uncovering Clues to the Mystery of Bacterial Defense Systems

Gonorrhea is the most common reportable venereal disease in America today, infecting more than two million people annually. While it is treatable with antibiotics, an effective vaccine has yet to be developed due to the gonococcus' peculiar characteristic of varying

protein are produced. What, in effect, makes

Neixeria a better pathogen, and how can the process by which

Meningococcal meningitis is a far less prevalent disease, affecting about four thousand people

To answer these questions, Cannon's laboratory is employing the techniques of molecular biology in order to study the structure of the antigens, and the genes responsible for

conse-

quences can be devastating, sometimes killing

its victims within eight hours of the first appearance of symptoms. Vaccines do exist for

it, but their efficacy

leaves something

their expression and variation. The first of these techniques involves the production and employment of monoclonal antibodies. Monoclonal antibodies are made by immortalized antibody-producing cells called hybridomas. To make such cells, a mouse is

desired and the search continues for better methods of immunization against the disease. Gonorrhea and meningococcal meningitis, then, represent significant public health hazards, Both belong to the same genus of pathogenic bacteria, called flersseria. Both are under scrutiny in the laboratory of Dr. Janne Cannon, assistant professor of microbiology at UNC.CH.

Neisseria: Nice and IUot So Nice The genus Neisseria includes a number of different species which have a variety of properties and biochemical activities in common. Among these species, however, two broad categories of bacteria can be distinguished: commensal and pathogenic. Commensal bacteria live

within a host without adversly affecting it or being so affected by it: pathogenic organisms, on the other hand, produce diseases in their hosts. fleriseri2 gonorrhoeae and N. neningi-

tidis, the bacteria which cause gonorrhea and meningococcal meningitis, belong to this second group. Both have developed effective ways of escaping the antibody defenses of their hosts by producing antigenic variants. Antigens are

evades host defenses be

Monoclonal Antibodies and Gene Sequencing

the chemical makeup of its outer surface.

in the United States every year, but its

circumvented?

Dr. Janne Cannon. assistant professor of nicrobiology

proteins on the surface of the bacteria which are recognized by the immune system of the host as a foreign element to be eliminated. The pathogenic Neisseria constantly vary the proteins that make up their surface, thus effectively baffling antibody defenses. They are among the chameleons of the bacterial world and the chemical quick changes they instigate on their surfaces make it difficult to develop a vaccine, since the strategy of immunization is to cause the host to produce antibodies that attack specific antigens. In the case of the Neisseria bacteria, as s00n as the antigens vary, the immunization is rendered ineffective. Cannon and her staff are working to understand the basis of antigenic variability in Neisseria. She wants to know what the structural difference is in the pathogenic group, as well as how the changes in the surface

injected with the organism that the researcher wants t0 study-in this case, with the surface membrane of the pathogenic tYerssera. The mouse's immune system produces antibodies

in response to the bacteria. The mouse is then destroyed and his spleen cells, which contain the antibody-producing cells, are harvested and fused to immortal cancer cells. What results is a hybrid having the characteristics of an antibody-producing cell and the immortality of a cancer cell. "The monoclonal antibody techniquel' says Cannon, "provides a powerful and precise molecular tool for researchers, in contrast to the previously available method of studying serum taken from an immunized animal. It provides a single standard material that can be used again and again in experiments without introducing unnecessary variablesl' In her efforts to understand the mechanism of antigenic variation, Cannon looked for clues in the structure of the surface proteins that could help distinguish pathogenic from commensal bacteria. She tested a variety of monoclonal antibodies for reactions with several

Eil

expression was DNA sequencing. The technology

in this process has developed rapidly in the last few years. The rationale behind involved

the technique is that while the study of the structure of a protein is extremely difficult when that protein is observed directly, the same information about the protein's nature

adenine, guanine, cytosine, and thymine, or A, G, C, and T. The arrangement of these bases in a given gene, that is, the sequence

of the beads on the strand, constitutes the code that leads to the expression of a given amino acid; GCC, for instance, will produce

Electron micrograph of gonococci, showing the

variation in upression ol a surface protein. A monoclonal antibody specific to one of the surface proteins was attached to microscopic gold beads. llhen the antibody binds to the protein on the surface of an organisn, the gold beads are attached to the organisn, and are visualized in the electron micrograph as black dots. In this photograph, there are

four pairs of organisns; two of the pairs bind

the monoclonal antibody, and are covered with the gold beads. However, two of the pairs were not upressing the particular surtace protein, and did

nol bind the monoclonal

antibody.

cultures of different species of flersserua. In essence, Cannon had a number of keys, the hybridomas, and a number of locks, the Nelssena cultures, and she was searching for a fit. The power of the monoclonal antibody technique was that she could keep track of her keys, so t0 speak, so that if she should find her fit, she could readily identify the key that matched the lock. What she discovered was that a specific monoclonal, named H.8, constantly recognized (reacted with) some antigen common to the pathogenic species of

with the commensal species. Clearly, some component of the surface membrane of the bacteria was common to the Neisseria, but not

pathogenic species alone. What the role of this component was in pathogenesis, and what

its ability was for stimulating antibody production, was still unclear, but Cannon now had an identifiable structural detail of disease producing Neisseria to study, which might lead to insights into the causes of surface variability and pathogenesis. Another technique from the repertoire of molecular biology employed by Cannon to study

the structure 0f the H.8-binding antigenic compound and the gene responsible for its

< ^*"-

(ou) \_-/

aesnrdoi Eraue

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different amino acid than GAC, The rules for these codes are well understood. By isolating the gene responsible for the antigenic protein and determining its sequencing, Cannon could begin the task of understanding the structure

of her component. To be able to sequence the gene, Cannon had

first to isolate it, or "purify" it for $udy

**"**"

,^. ,//

blueprint (or "codes") for the expression of the protein. DNA is composed of a series of chemical bases strung out like beads on a strand. There are only four of these bases,

iryT\6u

and synthesis can be obtained much more easily by examining the gene that provides the

J) -\

,/\

X\' Y

(see

diagram). What she discovered after doing so was that her antigenic component was a "conserved"

protein, a protein that remained stable on the surface of the bacteria and did not vary along with the other antigens present. This information was enormously exciting, because it was precisely that variability of the surface antigens that made it so difficult to develop vaccines against pathogenic bacteria. The discovery of a stable antigen suggested that an immunological strategy against the disease-bearing Neisseria was a distinct and viable possibility

in the not too distant

future.

Cannon's research is now moving in two different directions. Working with graduate students Terry Connell and Ellen Aho, postdoctoral fellow George Murphy, and lab tech-

nician JoAnne Dempsey, she continues to explore the regulation of the process of antigenic

variability in pathogenic,{elsseria. Current research, funded by the National Institutes of Health, involves comparing the characteristics of the genetic structure of the bacteria before and after variation takes place. The group's findings suggest that there is actually a change in the DNA sequence of certain genes when variation occurs. Until now it was generally thought that the sequence of DNA bases in the gene remained constant whether or not the gene was actually expressing. Another approach Cannon is taking involves further study of the H.8-binding conserved antigen she has discovered. Working along

Purification of the gene responsible for the antigenic protein. DNA is extracted from the glnococcus. Restriction enzymes are added to the gonococcal DNA; these enzymes "clip" the DNA into snall pieces which are inserted into vectot plasnids. The plasnids are introduced rnlo Escherichia coli, a rod-shaped bacteriun. The particular E. coli containing the gene that will produce the antigenic protein are identified with the help of monoclonal antibodies and allowed to replicate. ln the process, large quantities of the gene of interest will be pro duced within the vector plasnid. The plasnid containing the gene is then ertracted hon the E. coli, and restriction enzymes are again added to isolate the gene hon the plasnids. The result is a "pure" gene that can be studied for the deternination of its sequence and the prediction ol the resultant structurc of the protein.

with her in this effort are graduate students Tom Kawula and Jon Woods, postdoctoral fellow

Stan Spinola, and lab technician Diana Barritt.

"The H.8 antigen is a tough protein to Continued on inside back cover

The Evolutionary Response to Pollution Investigatinq the Process bv Which Orqanisms Develop Resistance

Currently, one of the biological community's major concerns is the effect of toxic industrial

Role of Molecular Biology

biology at UNC-CH, is focusing on the process by which the lruit fly, Drosophila melanogaster,

to metals whose

Molecular biology makes it possible to study the organization and control of the MT gene and also to test whether certain changes (mutations) are selected under particular envi-

concen-

tration in the environment is affected by human activities. Funded by a grant from the National Institute of Environmental Health Sciences, Maroni is conducting experiments on a gene

ronmental conditions, according to Maroni. Previously, it would have been necessary to

that carries information for the protein metallothionein (MT). MT is found in the cells of all organisms from yeast to humans; it binds highly toxic metals, such as cadmium and copper, thus preventing them from damaging

breed thousands of flies and examine several generations of their progeny to determine if

flies from a specific locality are resistant to a specific metal. Now it is possible to examine a small number of flies directly to test for the presence of those mutations.

vital processes. "0ne of the reasons that we are working with this particular protein;' says Maroni, "is that although it has the property of being able to protect organisms from toxic metals, we do not yet know whether that is its only functionl' It must have some role other than t0 protect organisms from toxic metals, Maroni explains, since in nature, in the absence of human activity, there are only very small amounts of these toxic metals available to the organism. "We now find it important to understand all its functions, since humans are putting s0 much toxic metal into the environmentl' One clue as to whether MT has some function other than binding toxic metals is that it also

Wastes

MT to transfer zinc, when needed in the cellular metabolism, while at the same time not releasing the toxic metalsl'

waste products on evolutionary development. Dr. Gustavo Maroni, associate professor of

develops resistance

to 'Ioxic

Maroni and his coworkers, research associates

1)r (iititaii,

1/,t",.,

binds certain beneficial metals, such as zinc, which are not toxic except in very high concentrations. "One possibilityl' says Maroni, "is that MT could be a reservoir or storage site for zinc, but it may also bind other metals of the same type, such as cadmium and copper. If this is the case, there must be a mechanism for

Donna Lastowski-Perry and Janet Wise, graduate student Edward 0tto, and technician Julie Young, have kept fruit flies on a diet containing a high concentration of cadmium. The activity 0f the MT gene is induced by the presence of cadmium, which causes it to make the metallothionein protein in an amount proportional to the amount of cadmium. Too much cadmium will be toxic, since the gene cannot make enough protein to bind it. Maroni's group used a concentration of cadmium that allowed only a few flies to survive until the next generation. After this procedure had

been repeated for about fifteen generations the flies began to build up a tolerance for the cadmium and to reproduce at almost normal

survival rates. They had therefore become cadmium resistant. The researchers then looked at the gene for MT to determine if any changes had occurred. They discovered that instead of one gene, there were now two. The presence of this mutation meant that the gene could now pro duce twice as much of the protein and could therefore bind twice as much cadmium. Scientists had already known that cells exist with multiple copies of genes in order to produce enough of a particular product. "However, it is much rarer to be able to study this phenomenon in whole organismsl' explains Maroni. "ln this case, every cell carries the mutation and the characteristic is transmitted through the gametes (reproductive cells) to the next generation. Mutations of

this particular kind are called duplicationsl' In an attempt to answer the question of how duplications occur, Maroni and his group have isolated several of them and are now sequencing the DNA near the site of each. One observation that has been noted so far is that different-sized portions of DNA are duplicated in different strains of fruit flies, but more data are needed before any sort of pattern can become apparent.

Exictence of the lllutation in Nature

.lUlie Younq ;tncj Erlward Otta exilrrliile at) \-ra\, l.iln n etal I ot h i itnei n t)ity.tl i t at t tt ;

to lelerntint,

lhe sequence of nucleotitles

one

the

Having succeeded

isolating laboratory

strains of flies resistant to toxic metals, Maroni's group has sought to determine if these mutations exist in nature. They have collected flies from a rrariety of locales and have found duplications in some of them. "Rather than testing many hundreds of offspring from each trapped fly to determine whether they are more tolerant to cadmium we only need ten flies to test whether they have a duplication for the MT genel' Maroni explains.

The studies have shown a higher rate of duplications in some places than in others. The data collected so far suggest a rough correlation between the number of duplications and the degree of industrialization in the area studied. The question naturally arises as to whether there is also a correlation with the amount of metal in the environment. One location from which specimens have been collected is a French vineyard. Various derivatives of copper are sprayed in vineyards to control mold. Since copper is one of the

toxic metals bound by MT, Maroni's group wants

is a higher-thannormal concentration of copper in the soil of measure whether there

vineyards. Specifically, they are interested in

whether the decaying fruit that nourishes the flies has a high concentration of metal. Maroni feels that studies of this nature may also prove useful in areas where there are copper and zinc mines and in the neighborhood of industrial plants using cadmium. Tiaditional studies to answer these sorts of questions were extremely laborious and often inconclusive. "We hope to use the more efficient techniques of molecular biology to establish whether a correlation existsl' Maroni says. He points out that while gene duplications have been recognized to exist for a long time, it has never before been possible to investigate

the process by which these mutations occur and thus perhaps be able to draw some con-

clusions as to how evolution occurs as response to the environment.

future

Goalc

"We want to produce a mutant that does not have any MT," Maroni says. The group is using X+ays and other mutagens to try to obtain this mutation. "We would expect it to be very sensitive to metals, for one thing. But, what other effects would such a deficiency have?'You don't know what you have until you've lost itl What is true for human affairs is true for genes, in this case. The best way to find out what MT is for is to make flies without it and see what happens to theml' As far as we know, explains Maroni,

it would be the

only

case of a multicellular organism without MT.

-Suzanne Appelbaun

ilD

A Moveable Feast Exploiting Nature's Defenses Against Ground Water Pollution

Until as recently as six or seven years ago,

was not known that microorganisms are present in aquifers (the ground water in the subsurface of the earth). These microbes have been shown to be capable of breaking down chemical pollutants in the water. However, the normal action of the microbes on the pollutants is very slow. Since the water itself

typically moves at a rate of only one to two meters per year, the time available for microbial action on the pollutant is very long. Therefore, even this very slow rate of activity is significant, and since it involves the process of metabo-

lism, has the potential to be manipulated to some extent. In the laboratory of Dr. Frederic Pfaender of the UNC-CH Department of Environmental Sciences and Engineering, work is underway to try t0 understand the physiology of these subsurface microorganisms in order to influence

the rate at which they act in the environment. "What we are dealing with herel' says Pfaender, "is the phenomenon of adaptation-the adjustment of the microbial community to the chemical pollutant. Some adaptation will take place naturally, enabling the organisms already present in the ground water t0 grow and metabolize more of the chemical. The other kinds of adaptive changes that can occur naturally are either genetic or molecular, and usually occur very quickly, or require a very long time. However, there appear to be ways to manipulate the existing physiology to accomplish the same goalsl' Commercially, there are some dozen or so companies in the business of providing ground

water remediation. They have traditionally relied on one or more methods of adding organic nutrients or oxygen to speed up the metabolic process. "lf it's done right, it worksl' explains Pfaender. "We are now experimenting by adding new things to the environment to make the pollutant break down faster. Our aim is to help companies that are

Drs. Frederic Pfaender and Cass Miller perforn a transfer step as part of the biodegradation measurenent procedure.

doing this work understand what they are doing and how to do it betterl'

this project and laboratory studies of the action of microbes are Environmental Sciences

ground water. Previous models have not included

and Engineering graduate students C. Margerie Aelion, Durell Dobbins, and 0u Jiang, along with postdoctoral associate C. Michael Swindoll. Pfaender's group, which has previously relied on samples of contaminated subsurface soil/ water imported from sites in Oklahoma or Michigan, now has access to a contaminated ground water site in North Carolina and is launching a field-scale study of the site with funding from the Environmental Protection

microbiology-in this case the attempt is to

Agency. They

include the indigenous community of micro organisms. Assisting Pfaender and Miller with

the entire plume, or range of movement, of the pollutant.

Modelling the Flow Pfaender, in collaboration with Dr. Cass Miller, a ground water hydrologi$ in the Department of Environmental Sciences and Engineering, is seeking to devise a model of the transport and fate of pollutant in the

will be able to define and sample

Examinlng the Effects of Pollutrntr on Uncontamlnated Soil In the laboratory

Pfaender's group

will

employ a technique they have developed in their earlier studies of unpolluted ground water, only in this instance they will take a clean

soil and contaminate it to see how it will respond. (Pfaender orplains that getting uncontaminated samples out of the subsurface

is very difficult, requiring an expensive drilling rig and a special attachment to bring up the core samples. The team will have access to a rig belonging to the state of North Carolina, and will use an attachment which they have designed themselves-see back cover.) The experimental procedure involves first taking

each sample and making a slurry with water. Dividing the sample into several subsamples, they add radiolabeled chemicals (would-be pollutants) in various concentrations. They

then let the bottles incubate for varying lenEhs of time, at which point they measure the amount of radiolabel metabolized in each bottle. fuo measurements are taken, one of the carbon dioxide produced and one of the pollutant that is taken up into the cells of the microbes. The relative amount of radioactive carbon converted to these two products indicates the amount of metabolism or breakdown of chemical or pollutant. A recent

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tffii,im *:!IijiJ.'iiii: WATEB FLOW

CONFININ'JG LAYER

Diagran of ground water flow and sources of pollution. As water slowlv approaches site use, nicrobial activity will metabolize some or all of pollutant.

access

for human

refinement of this technique employs a chemical called polyvinylpyrollodone, which does a good job of releasing the cells from the soil

of the sample. The cells are then transferred to the liquid in the bottle, filtered, and the radioisotopes counted. Data analpis is based on

kinetics-how long

takes to metabolize the chemicals relative to their concentration. "We are trying to put

time scales on how long these things takel' explains Pfaender. If a pollutant is neutralized by natural processes before it reaches a location where it will become a problem, then no intervention will be necessary. For the other cases, Pfaender

is hoping to

dervise ways to

speed up the process by which indigenous microbes metabolize the pollutant.

-Suanne Appelbaun

Dr. C. Michael Swindoll, Durell Dobbins, and 0u liang add soil sanples to containe$ that will be used lor measuring the persistence ol chenical in ground water.

Phytochroffio, the Key to Controlling Plant Growth Molecular studies

in plants represent a

new

for

UNC-CH, but one that is growing, according to Dr. Alan M. Jones, commitment

assistant professor of biology. Jones is analyzing

the molecular structure of phytochrome, a light-sensitive protein found in plants, and studying how it carries out its functions in crop plants, especially corn. Jones projects that eventual applications of his work might include improving crop yield by altering the sensitivity of leaves to light and, consequently, altering the plant's structure. Although scientists do not understand precisely

how phytochrome works, they know that it measures three qualities of the light environment: the amount of light, its color, and even length of darkness. Stimulated by the tight environment, phytochrome triggers various developmental responses, such as greening, flowering, and altered growth rates. For instance, the spindly appearance of a plant in a window providing insufficient light is due to the elevated growth rate of the stem, a response

that is under the control of phytochrome. Similarly, the yellow color of grass that has been covered lor a few days the phytochrome molecule.

is induced

Apart from the amount of light, the color of light can also determine a response. Vines in the forest, where green foliage filters out less and less red light as a plant approaches the sky, will grow up rapidly until they sense sufficient red light. "lndependent of whether it's a

cloudy day or not, the growing plant tip determines relatively how high it is in the lorest canopy by utilizing the dependable redlight gradientl'Jones explains. And, through the mediation of phytochrome, the length of darkness signals many plants to drop their leaves when the nights grow longer in autumn. Phytochrome has two photoconvertible forms:

one absorbs light in the red region of the spectrum (660 nanometer$ while the other absorbs in the far-red region (730 nanometers). These forms are referred to as Pr, the redabsorbing form, and Pfr, the far-red-absorbing

f, ffi Dr..|lan !fi..lones

measures the nesocotyl of corn plants grown in complete darkness, using a very sensitive instruntenl thal he built. As the stem pushes upward. it tilts a balance arm that triggers an electrical impulse and sends it to a recording device. The instrunent measures growth on a minut*b.v-ninLtte basis to a resolution

ttf .0lmm.

form. They act much like a light switch with on and off positions, but control developmental events in plants rather than an electric bulb. Red light, such as a plant might receive from daylight, causes the phytochrome molecule to switch to its Pfr form. Then, in the absence of red light-during the night or on the forest floor, for instance-some 0f the Pfr molecules revert back to Pr, the amount depending on the length of darkness or on the amount of

the mechanism by which light and phytochrome control the response, Growth is regulated by the hormone auxin and also by its receptor, a molecule that allows the plant to perceive the presence of auxin. Jones has discovered that red

light, mediated by phytochrome, decreases the level of auxin and of its receptor, thus inhibiting growth. "Taken together, the decreases would

explain why a structure called the mesocotyl, specialized underground stem, stops growing

far-red light.

when

This switching lunction is reversible, a feature that is unique to phytochrome. The molecule can be flipped back and forth, and, depending on the way the switch is flipped, the phytochrome causes the plant to respond in different

Jones says. His goal over the next ten years is to confirm and understand this process. The major task of his work will be to isolate the auxin receptor, which misht take three to six years, "Sometimes the hardest part in

ways. "The presence of Pfr induces many biological responses; the lack of Pfr generally

biology is extracting the extraneous part of the organism away from the molecule to be studiedl' he says. To isolate and purify the receptor, he will use the techniques of affinity labeling and monoclonal antibodies. Both

cancels theml' Jones explains.

With funding from a Junior Faculty Development Award, Jones is studying one of these responses, that of growth, trying to elucidate

reaches

light at the top of the soill'

conlinued on inside back cwer

Synthetic Ceramics and the Mollusc Shell Investigations at the UNC-CH Dental Research Center Employ an Interdisciplinary Approach

to the Area of Materials

Development

in the development of composite materials that can be used to restore teeth. He is helping Crenshaw create the technology needed to understand the processess behind shell mineralization, technologies he hopes will lead to a method for making synthetic polymer interested

material that possesses

all the

a ceramic and none of the

advantages of

disadvantages.

The nacre, the lovely mother-of-pearl surface of molluscan shells, is the obiect of Crenshaw and Ttrner's research. "lt has a simple, elegant, and subtle microstructurel' says TLrner, "and that helps to make it interesting to

studyi' It is also extremely resilient, with the combined qualities of strength, firmness, and

flexibility. The reason for this is that nature has provided the nacre with what Thrner calls Dr. lliies

('renshav+'. prrl-e.s.sor

the Curriculunt lor

,&/arlne Jcrenr:es anrl the Dental Re.search Cenler,

and

Derek Turner. prufessor

the l)ental

"good packing characteristics resulting in a high mineral contentl' The rigid mineralized sections are surrounded by a network of polymer

protein molecules greatly swollen with wdter. lf a crack appears in a microsection of the shell, it only ertends until it reaches the polymer and then stops. The polymer prevents the crack from propagating throughout the shell and offsets susceptibility to catastrophic fracture-the main deficienry limiting wider applications of ceramic material. Crenshaw and'lbrner want to borrow the protective design that nature has evolved for

the mollusc and apply it to the manufacture of synthetic materials. The end result, they hope, will be a composite material much like a super ceramic. It will have the mechanical properties of a ceramic, and will be as rigid; however, it will not have a ceramic's brittleness. Flexible, durable, light, and nearly unbreakable, the new material would have a wide applicability in dentistry orthopedics, "anpvhere continued on inside back cover

Research ('enter

Marine biology, polymer science, dental ,'research, at first

and biotechnology seem, glance, an unlikely group of disciplines to bring together in any experimental effort, especially when that effort is focused on answering such questions as: Can science make a better ceramic? Drs. Miles Crenshaw and Derek lhrner are doing just that, however,

and their investigations, while still at an early stage, promise significant payoffs

materials

development for crack-resistant ceramics,

well as a better understanding of the mechanisms that allow molluscs to produce their shells. Crenshaw, a professor with a joint

in the Dental Research Center and the Curriculum for Marine Sciences, has developed a hypothesis about the formation of molluscan shells which he is testing in his laboratory. Tirrner, also a professor in the Dental Research Center, is a polymer chemist

= o

appointment

Fracture surfaL:e

the nacre. the mother-ct'-pearl lining

of the ntolluscan

shell

nov approach based on Crenshaw's hypothesis of shell formation. He belierrs that a mollusc creates its covering by smreting a protein molecule which induces mineralization

government, includes assaying the polymers for their mineral induction capacities. Edward Greenfield, a graduate student in the Marine Sciences Program, is working on a molluscan model sptem in an attempt to induce mineral formation in a test tube. Doug Wilson, a graduate student in chemistry is working on analytical aspects of the synthetic ceramic, especially the relatirc dispersion of the biological polymer in it. Tom Wilson, a postdoctoral felloly from North Carolina State University, is working on a sophisticated

of calcium carbonate in the surrounding sea water by a process involving coordinated ionic

characterization of the nov materials by dynamic chemical analysis. The laboratories

attraction. Each mineral crystal so formed is then encapsulated by protein, and constitutes

are funded largely by the 0ffice of Naval Research and the North Carolina Biotechnology

continud fron page 20

and errerywhere ceramics are used toda/'

sap Crenshaw. Current attempts to create such a material involve a crude mixing of organic materials with inorganic ceramic substances, but the ruults are unsatisfactory. Crenshaw and lhrner are trying a

Erdcrvon Research and Graduate Education at

the University

of North Carolina at Chapel Hill Spring 1987 Volume IV Number 3 Endeavors is a magazine published three times a

Center.

year by the 0ffice of Research Services, a division of the Graduate School of the University of North Carolina at Chapel Hill. Each issue of Endeavors describes only a few of the many research projects undertaken by faculty and students of the University.

in the distinctive microstructure of the shell.

Perhaps the most unique aspect of Crenshaw and TLrner's research is their method for

Requests

Crenshaw has ortracted this protein hom

attempting to create a super ceramic. Instead

mollusa and bound it to synthetic molecules with a hydrogel, a crosslinked polymer network swollen with water. ("lt's like jellol' sap ILrner, 'bnly strongerl') Within this artificial environment the two ruearchers have successfully induced mineralization on a small scale. They are nolv attempting to create synthetic macromolecules to replace the organic ones extracted from the mollusa. Sarcral people are working along with Crenshaw and Tbmer in their laboratories, and their contributions are 'trucial" according to Crenshaw. Dr. Mrian Lussi, from Bernq

of crudely mixing materials at hand, they

microsection of the shell. The mineralization produces specific crystalline patterns that

ruult

trying to reproduce, synthetically, the natural process by which the molluscan shell is created. "This is not an improvement on old techniquesj'

explains Crenshaw, "but a radically different

approachl' "We think we can figure out how to do itl' sap lhrner. "The organisms we study did it after all, and we know we're smarter than they are. But thenl' he adds with a wry smile, "they'rc had millions of years

to perfect their techniques. We're only

funded for threei'

should be sent to Editor, Enduvon,0ffice of Research Services, 300 Bynum Hall 008A, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27514 (telephone 919/966-5625). Chancellor: Christopher C. Fordham, III Vice Chancellor for Research and Craduate Studies and Dean ofthe Graduate School: J. Dennis 0'Connor Director: 0ffice of Research Services: Tom K. Scott Editor: Suzanne Appelbaum Assistant Editors: Tim Jenkins

Diantha J. Pinner

Switzerland, is a chemical engineer with a dental degree; his work, funded by the Swiss

continued from page

are

for permission to reprint material, readers' comments, and requests for extra copies

-Tim

Jenkins

Photographer: Will Owens Lynn M. Kenney Donna S. Slade

Designer: Diagrams:

continued hom page 14

understanding crop growth and yield, which, of course, is very important to the farmerj'

studyl' says Cannon. "The bacteria don't make very much of it, and so it's difficult to get it in quantity. Alsq it behaves strangely if you try t0 purify it, suggesting that it has some unique features to its structure. It's clearly an unusual proteinl' The majority of Cannon's work in this area, also funded by the National Institutes of Health, is basic research, so she can make no promises about potential applications. However, she hopes that it may lead someday to a viable vaccine for gonorrhea and more effective vaccinations

Jones says. Through this knowledge, scientists

against meningococcal meningitis.

techniques operate by binding something to the receptor-either a special molecule or an antibody-that marks it and allows it to be extracted from the plant cells. Jones will also use a type of chemical measurement called gas chromatography-mass spectrometry to plot levels

of the hormone through the growth

cycle.

0nce he has isolated the receptor, Jones will trace the gene that specifies the code for its production. "Having the gene is important in

could engineer a plant to respond better to given conditions. "For examplel' he adds, "you could change the amount of growth that goes into stem elongation and divert that energy into producing more or larger ears of corn insteadl'

-Diantha J. Pinner

1987 by

Chapel

The University of North Carolina

Hill in the United States. All rights

reserved.

No part of this publication may be reproduced without the consent of The University of North Carolina at Chapel Hill. Cover: Researchers at UNC-CH are making signifi-

cant contributions to many areas of molecular biology and biotechnology. Their studies employ diverse systems, from the fruit fly to crystalline mineral deposits, but all are aimed at addressing human needs. Drawing by Donna S. Slade.

-Tin Jenkins The staff of Endeawrs would like to acknowledge the contribution of Professor G. Philip Manire as a founder of the modern or "new" biologr on the UNC{H campus, and his help in perpetuating these efforts during his tenure as Dean of the Gnduate School.

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