The Tenth Annual NC State University
Undergraduate Research Symposium

Student Author(s):	DiGangi, Brian A.
Department(s):	Animal Science
Research Mentor(s):	Brenda Alston-Mills/Animal Science
Title of Presentation:	A Comparison of Beta-lactoglobulin Profiles of Mastitic and Healthy Milk

Beta-lactoglobulin (B-LG) is a protein present in the whey fraction of the milk of many species.  Its chemical properties have been well characterized but relatively little is known of its biological functions.  The purpose of the study is to examine the concentration and estimate the molecular mass of B-LG in the milks from healthy and mastitic cows.  Genetic mutations of the protein (known as A and B variants) were visualized by polyacrylamide gel electrophoresis and were investigated.  Milk samples originating from cows with both clinical and sub-clinical mastitis and those from healthy cows were compared.  Analyses included percent fat and total whey proteins, concentrations of  B-LG as well as protein profiles.  Ten milk samples (10 milliliters each) from cows diagnosed with mastitis and five samples from healthy cows were tested.  A bicinchoninic acid protein assay measured total whey protein. B-LG was assayed by immunoassay. Polyacrylamide gel electrophoreses determined the whey protein profile.  A standard of B-LG (A and B variants) was applied to the gel.  The electrophoretic zones for B-LG in milks of normal and mastitic cows were compared.  Additionally, an aliquot of milk was used to determine fat composition using a modified Werner-Schmidt protocol.  The study attempted to demonstrate whether or not B-LG was altered by the development of mastitis or whether there was any indication of polymorphism.

Student Author(s):	Doutova, Anastassia V.
Department(s):	Biochemistry
Research Mentor(s):	William L. Miller/Biochemistry
Title of Presentation:	In Vitro Testing of a Gene Switch for Use in Farm Animals: Turning On and Off Reproductive Hormones

In this research we tested the tetracycline controlled transcriptional switch for the production of the b subunit of Follicle-Stimulating Hormone (FSH) in vitro.  Two separate constructs were used, one with FSHa and one with FSHb promoter driving tetracycline receptor production.  Preliminary studies showed that one FSHa construct was capable of 7600 fold induction of FSH after stimulation with doxycycline (a tetracycline analog). The FSHb driven switch was induced 2-18 fold. Final data from one established FSHa line showed an 11-fold stimulation with an ED50 of 5x10-7 M doxycycline.  Future work with this gene switch will establish its effectiveness in transgenic animals and at the same time study the effects of hyper FSH secretion on reproduction.  This gene switch may ultimately be used to give farmers better control of their livestock physiology.

Student Author(s):	Dow, Anna E.
Department(s):	Biological Life Sciences
Research Mentor(s):	Alicia Moore/National Institute of Environmental Health Sciences Darlene Dixon/National Institute of Environmental Health Sciences
Title of Presentation:	Tissue Microarray Analysis of Inter-tumor and Intra-tumor Proliferative Activity in Uterine Leiomyomas in Women During the Menstrual Cycle

Uterine leiomyomas ("fibroids" or "myomas") clinically affect at least 1 out of every 4 women in the United States. They  are benign tumors that consist of smooth muscle and fibrous connective tissue. Uterine leiomyomas are usually multiple and may be located within the submucosal, intramural or subserosal regions of the uterine wall. Low mitotic activity (<4 mitotic figures/10 high-power fields [HPF]) is a common feature of uterine leiomyomas and is used as criterion for diagnosing these tumors.  Although uterine leiomyomas have low mitotic activity, they may range in size from 1mm to greater than 20 cm  in diameter. Immunohistochemical detection of endogenous markers of cell replication such as Ki-67 and Proliferating Cell Nuclear Antigen (PCNA) is commonly used to assess cell proliferation. We have shown there is increased proliferation in uterine leiomyomas compared to the myometrium of the same patient, and that Ki-67 seems to be a better indicator than PCNA of this proliferative activity. We have also found there is tumor to tumor variation of labeling indices for Ki-67 and PCNA in an individual woman. We are now interested in using tissue microarray technology to confirm our earlier observations of inter-tumor variation in proliferation within a woman, and to expand our studies to assess whether regional differences exist. The recently developed tissue microarray technology will allow for much more accurate analysis between tumors while conserving tissue samples.  We plan to evaluate 20 women, 10 in the secretory phase and 10 in the proliferative stage of the menstrual cycle, all within the same age range.

Student Author(s):	DuBose, Melinda J.
Department(s):	Microbiology Biochemistry
Research Mentor(s):	Leo W. Parks/ Microbiology W. David Dotson/ Microbiology
Title of Presentation:	Assessment of Regulatory and Phenotypic Effects Resulting from Mutations in UPC2

Sterols serve multiple growth dependent functions in yeast, in addition to mediating the physical properties of eukaryotic cellular membranes. The brewer's yeast, Saccharomyces cerevisiae, has often been used as a model organism for genetic analysis. Its genome has been completely sequenced and a wide variety of mutants have been characterized.  In this study, we have focused on the gene UPC2, which encodes a protein involved in the regulation of sterol uptake. Wild type yeast strains do not accumulate sterols from the growth medium under aerobic conditions. However, when oxygen is not available, sterols cannot be synthesized and uptake from the growth medium is required for survival. Strains with the upc2-1 allele take up high levels of sterol under aerobic conditions, while at the same time they exhibit a markedly increased level of sterol synthesis.  We have analyzed the effect of different mutations in UPC2 on some oxygen related growth characteristics, in otherwise isogenic strains.  Additionally, we have used Northern analyses to determine the effects of mutations in UPC2 on the expression of certain ergosterol biosynthetic, and oxygen regulated genes.  Our results are consistent with the predicted role of Upc2p, as a transcriptional regulator mediating sterol homeostasis in response to the availability of oxygen.

Student Author(s):	Dukes, B.A. Wolford, S.C.
Department(s):	Animal Science
Research Mentor(s):	T.A.T.G. van Kempen/Animal Science
Title of Presentation:	Caffeine Increases Heat Production in Newborn Pigs and May Offer a Means for Reducing Piglet Mortality Under Production Conditions

A farrowing house is designed to provide temperature levels that are comfortable to the sow.    However, newborn pigs need a higher temperature to prevent hypothermia, a condition that often leads to death in production environments.  The present techniques for improving the environment for newborn pigs are geared to heat the newborn pig by localized external heating.  These techniques are poor at increasing core temperature and place weaker smaller pigs at a disadvantage because of difficulty finding these zones and also because larger stronger pigs block access to localized heating zones.  If the pigs were internally producing the heat required to maintain their core temperature, the competition for heating zones would be eliminated.  Administering caffeine, a compound with thermogenic properties, is one option allowing for the newborn pigs to create their own heat.  In experiment 1, 46 pigs were given a one-time dose of 0, 5, 10, 20, 40, 80, and 120 mg of caffeine mixed in 20 ml of condensed milk with a control of 20 ml of water while the piglets remained with their mother. Based on regression analysis, 60 mg of caffeine was the most effective in raising the radiation temperature with 1.4±0.6°C above the water control two hours after administration.  In experiment 2, 55 pigs were given a one-time dose of 0 mg, 30 mg, 60 mg, 90 mg, or 120 mg of caffeine per 20 ml of condensed milk or a control of 20ml of water per kg of body weight. In experiment 2, variability was reduced by placing the piglets in a controlled environment and by adjusting the dose rate to the body weight of the animals, but the results were similar to experiment 1 with 60 mg of caffeine being the most effective at raising the temperature with 2.0±0.5°C. These data prove that caffeine can substantially increase heat production in newborn pigs, and as a result may well reduce mortality resulting from hypothermia under production conditions.

Student Author(s):	Ferguson, Jamie L.
Department(s):	Animal Science
Research Mentor(s):	Robert M. Petters/Animal Science  Jeffrey R. Sommer/Animal Science
Title of Presentation:	Detecting the Expression of Cre Mediated Recombination from a VP22/Cre Recombinase Chimeric Protein

VP22 is a protein translated from the genome of the herpes simplex virus type 1 (HSV-1). This protein is capable of leaving the cell it is produced in and entering the nucleus of cells that surround it in order to spread the virus.  Cre recombinase is a protein capable of performing specific recombination reactions at specific DNA sequences called Lox sites.  By fusing VP22 and Cre recombinase and transfecting fibroblast cells with this VPCre chimera, we hope to manipulate fibroblast cells into producing the complex and exporting the Cre recombinase activity to its neighboring cells.  Colonies that successfully produced the VPCre complex were incubated with other fibroblast cells to see if they could transfer the VPCre to the nucleus of surrounding cells.  By using a green fluorescent protein (GFP) reporter gene incorporated into the genome of reporter cells, we were able to detect expression of the Cre activity with a fluorescence microscope.  Antibodies specific to either Cre recombinase, VP22 or GFP were used to confirm the presence of the chimeric VPCre protein and its functional recombinase activity.  Western blot analysis was used to detect the expression of the fusion protein in transfected cells.

Student Author(s):	Geisenhoffer, Kristen M.
Department(s):	Genetics
Research Mentor(s):	Rebecca M. Riley/Genetics Gregory C. Gibson/Genetics
Title of Presentation:	Microarray Analysis of Variation in the Genomic-Wide Transcriptional Response to Tyramine in Drosophila melanogaster

After nearly a century of genetic research, fruit fly geneticists are now in a position to array thousands of fly genes to probe their function in physiological processes such as response of the nervous system to drugs.  Neurotransmission in Drosophila, as in all other metazoa, depends on the transmission of signals between neurons that is mediated by neurotransmitters including tyramine and its derivatives. Tyramine receptors are primarily located in the head, and belong to the G-protein coupled receptors, activation of which is thought to lead to changes in gene expression.  To characterize genetic variation for the response to tyramine, we started with two lines of fruit flies (A14 and A19, both derived from single females trapped in Ann Arbor, Michigan) that have divergent survival times when exposed to dietary tyramine.  Both lines become disoriented within a day of drug exposure, but A14 survives for a mean of 75 hours, while A19 flies die after just 30 hours.
        In order to analyze whether and how gene expression changes differently after exposure to tyramine, high-density cDNA microarrays containing five thousand Drosophila melanogaster expressed sequenced tags were constructed.  For each line, replicate RNA pools from treated and untreated controls of each sex were isolated and reverse transcribed in the presence of fluorescent dies, and then hybridized to the arrays.  Analysis of variance was used to obtain statistical measures of the fraction of the genome that responds to tyramine, providing the first estimate of the genome-wide transcriptional response to drug exposure.

Student Author(s):	Gupton, Stephanie L.
Department(s):	Botany
Research Mentor(s):	Nina Stromgren Allen/Botany
Title of Presentation:	Confocal Imaging Demonstrates that the Nuclear Invaginations and Channels Found in Tobacco NT-1 Cells Originate During Cell Division

The nuclei of plant cells can have deep grooves, invaginations and channels that are stable, contain cytoplasmic cores and contain actin bundles that support cytoplasmic streaming (Collings et al, 2000).  To determine the possible origins of nuclear invaginations and channels during and after mitosis, tobacco NT-1 suspension cultured cells stably expressing endoplasmic reticulum-targeted GFP (GFP-ER) were used as this indicator faithfully reports any fold of the nuclear envelope.  Cells were imaged during interphase and mitosis with confocal laser scanning and confocal spinning disk microscopy.  Interphase cells contained dynamic arrays of subcortical ER, with approximately 20% of nuclei demonstrating invaginations and channels.  During prophase, the nuclear envelope breaks down, transvacuolar strands vastly decrease in number, subcortical ER remains in a lattice network, and ER accumulates in the former nuclear area. During metaphase and anaphase, the spindle contains expansive amounts of tubular ER aligned in the direction of the spindle microtubules and interdigitating the condensed chromosomes.  Puffy aggregates of ER were also observed beyond the spindle poles. Nuclear channels were visualized during late anaphase and early telophase immediately after nuclear envelope reformation and were aligned with the microtubules of the spindle.  During anaphase and telophase the phragmoplast and cell plate contained extensive amounts of ER.  Spinning disk confocal microscopy caused less light damage than traditional laser scanning confocal microscopy and high-resolution dynamic images could be rapidly recorded.

Student Author(s):	Guthrie, Amanda L.
Department(s):	Animal Science
Research Mentor(s):	C. Scott Whisnant/Animal Science
Title of Presentation:	Alternate and Improved Methods of Estrus Synchronization in Cattle

Estrus synchronization in cattle allows producers to make better use of techniques for genetic improvement such as artificial insemination and to increase efficiency of beef production and improve marketability of calves.  Several methods of estrus synchronization exist in cattle with advantages and disadvantages to all.  The objective of the current project is to develop a single injection method.  This would offer producers a simple, easy-to-use method.  In the preliminary esperiments reported here, several formulations containing progesterone alone or estradiol  and progesterone were used.  In these formulations, the steroids were mixed with microspheres, which allow for a sustained release of the hormone.  This results in a single injection providing  elevated levels of the hormone for up to one week.  Two trials were conducted.  In trial one, 24 heifers were injected with one  of six different formulations containing estradiol and progesterone. Blood samples were taken on day -1, 0, 1, 2, 4, 6, 8, 10, and 12 of hormone treatment for determination of serum progesterone concentrations. Estrus detection was also performed on these animals by visual observation.  In trial 2, 20 heifers were given one of five different formulations containing progesterone alone and blood samples were taken and estrus detection performed as described above.  Measurement of progesterone in samples from trial 1 revealed that some formulations produced serum progesterone concentrations of the desired level. However, many of the heifers displayd estrus shortly after injection  perhaps due to the estradiol in these formulations.  Samples from trial 2 are currently being assayed and will be presented in the poster. Identification of the proper formulation will lead to further trials with larger numbers of cattle in which fertility of the synchronized estrus will be tested.

Student Author(s):	Haltom, Mary I.
Department(s):	Botany/Center for Applied Aquatic Ecology
Research Mentor(s):	Brant W. Touchette/Botany  JoAnn M. Burkholder/Botany
Title of Presentation:	Environmental Factors that Influence Phosphatase Activity in Surface Waters of the Lower Neuse River

Phosphatases (PAs; also referred to as phosphomonoesterases) are exoenzymes that hydrolyze organic phosphorus compounds into orthophosphate (Pi) and alcohol.  These enzymes play an important role in phosphorus acquisition/remineralization in aquatic organisms, especially during periods of increased phosphorus demand.  PAs tend to be substrate non-specific, allowing the release of Pi from a broad range of phosphorus containing compounds. Following enzyme-mediated hydrolysis, PO4-3 can be assimilated by the cell and used for a variety of metabolic processes. The ecological role of PAs, especially alkaline phosphatase, has been linked to phosphorus deprivation in both phytoplankton and bacteria. As Pi becomes less available, many planktonic microbes respond by enhancing PA activity, thereby increasing the potential for Pi utilization from environmentally derived organic compounds. Because phosphatases are induced during periods of low inorganic phosphorus levels, the measurement of alkaline phosphatase activity in lakes and rivers can be used as an indicator for P-limited growth. Increased eutrophication has been known to adversely affect aquatic habitats by increasing primary production, increasing bottom-water hypoxia, and promoting the development of  harmful algal blooms (including outbreaks of certain toxic dinoflagellate species such as Pfiesteria piscicida).  Planktonic species play a major role in the phosphorus cycle, through the mineralization of phosphorus from organic compounds via phosphatases. These enzymes allow phytoplankton and bacterioplankton to grow and persist under conditions of low dissolved inorganic phosphorus. In this study we evaluated alkaline PA activity in the lower Neuse River to determine what factors (e.g., physical, chemical, biological) influence surface water PA activity. By understanding what environmental factors affect PA activity, we may develop a better understanding of the components that regulate plankton dynamics (from a physiological point of view) in estuarine waters.

Student Author(s):	Hansman, Roberta L.
Department(s):	Biochemistry  Marine, Earth, & Atmospheric Sciences
Research Mentor(s):	Donna Wolcott/Marine, Earth, & Atmospheric Sciences Jack DiTullio/Grice Marine Laboratory Alan Lewitus/South Carolina Department of Natural Resources
Title of Presentation:	Effects of Light and Nutrients on Taxon-Specific Variability in Pigment Ratios of Coastal Phytoplankton Isolates

Ratios of pigments in photosynthetic pathways are routinely used to identify species of phytoplankton in natural samples using a software program, CHEMTAX.  To identify whether environmental factors might alter pigment ratios and interfere with species identification, batch cultures of estuarine phytoplankton species were grown under various nutrient and light conditions, and cells were harvested for analysis at different stages of growth and during varying periods of light and dark.  Pigments were separated and analyzed using high-performance liquid chromatography, and pigment ratios determined for each set of conditions.  Differences in the ratios of pigments involved in the xanthophyll cycle were found amongst samples taken in light and dark in cells grown under high light intensity.  Pigment ratios for phytoplankton in log growth phase appear to be higher than those in lag or stationary phases.  Care should be taken in order to determine initial pigment ratio matr ices for use in the program CHEMTAX due to the potential for ratio differences under various light intensities and growth stages.

Student Author(s):	Harris, Brittany A.
Department(s):	Plant Pathology
Research Mentor(s):	Jim Moyer/Plant Pathology  Elizabeth Parks/Plant Pathology
Title of Presentation:	Development of Retrotransposon-Based Marker System in Floral Crops

Retrotransposons are mobile genetic elements that play an important role in plant genome evolution.  The nuclear DNA content of plants can be comprised of over 50% retrotransposons that propagate through the reverse transcription of an RNA intermediate. The excision of transposable elements has been associated with variable phenotypes in many crops including corn and tobacco. Understanding how retrotransposons affect traits, such as leaf color, morphology, and disease resistance, will be highly valuable to breeders in developing new cultivars, and will be an available source of genetic markers in these crops. Although retrotransposons provide fundamental insight into the nature and development of floral crop genomes, only few retrotransposons have been identified. The objective of this project is to identify long terminal repeat (LTR) retrotransposons in floral crops such as poinsettia, petunia, geranium, and others, in order to develop a new marker system, thus expanding upon the availability of genetic markers for commercially useful phenotypes.  Retrotransposons exist in two forms, those with LTRs, and those without LTRs.  The conserved nature of the LTR sequence, coupled with insertional polymorphisms, make retrotransposons useful as a source of genetic makers.  LTR retrotransposons are usually comprised of Gag and Pol regions, with identical long terminal repeats on either end.  The reverse transcriptase gene, located in the Pol region, has highly conserved regions across plant species. Primers designed from these conserved regions were utilized to amplify and sequence this gene.  The sequence of this gene was used to design additional primers, which were used to obtain the remaining retrotransposon sequence, including the LTRs, by primer extension.  Subsequently, primers complementary to the LTR region can be used to amplify into the region surrounding the retrotransposon insertion point, as a source of potential molecular markers for commercially important traits.

Student Author(s):	Hefley, Catherine M.  Wagner, Victoria A.
Department(s):	Animal Science
Research Mentor(s):	Charlotte E. Farin/Animal Science
Title of Presentation:	Effect of In Vitro Production of Bovine Embryos on Levels of Fetal IGF Binding Protein-2

Large Offspring Syndrome (LOS) is a condition that has been associated with production of bovine and ovine embryos using in vitro fertilization and embryo culture techniques.  A wide range of symptoms accompany this condition and can include increased birth weight, increased mortality and morbidity, increased gestation length, and placental abnormalities.  Embryo and fetal growth is regulated in part by the Insulin-Like Growth Factor (IGF) family.  This family consists of two ligands, (IGF-I,-II), their receptors, and their binding proteins (IGFBPs).  It has been suggested that LOS may result from altered expression of the IGF system.  For example, bovine fetuses from embryos produced in vitro had higher levels of IGF-II mRNA in liver compared to in vivo produced controls.
        The objective of this study was to compare the relative levels of IGFBP-2, another component of the IGF family, in the serum of bovine fetuses from embryos produced in vitro versus in vivo. Serum samples from 7 month-old fetuses (n=12 In Vitro, n=10 In Vivo) were denatured for 10 min at 60 C and electrophoresed on 12% SDS-PAGE gels. Proteins were transferred to nitrocellulose membranes and immunoblotted with IGFBP-2 antibody (1:2000).  The IGFBP-2 levels were detected using an enhanced chemiluminescence detection system (ImmunoStar, Biorad) according to the manufacturer's recommendations.  The signal intensity of the IGFBP-2 bands were compared using video imaging analysis.  Results indicate that there is no significant difference in levels of IGFBP-2 in the serum of  fetuses from embryos produced in vitro compared to in vivo produced controls.

Student Author(s):	Herbert, Farah R.
Department(s):	Biochemistry
Research Mentor(s):	Dennis T. Brown/Biochemistry
Title of Presentation:	Protein-Protein Interactions in Sindbis Virus

Sindbis virus, the prototype alpavirus, contains two structural glycoproteins, E1 and E2, which interact as trimers of heterodimers to form the icosehedral shell of the virus. Past experiments using chemical crosslinkers led to the determination of the structure of the surface spikes to have E1 maintaining the basal structure and E2 at the top of the spike. Using two different protein crosslinking methods, I am also examining the protein-protein interactions of the surface proteins of Sindbis virus and visualizing the results through polyacrylamide gel electrophoresis. The two techniques used to perform the crosslinks were irradiation with ultraviolet light and the introduction of an iodine solution. Both techniques formed crosslinks, but at this time the precise nature of those interactions is still being investigated. Those results are expected to justify the arrangement of E1 and E2 projected earlier. The advantages to these techniques as opposed to using chemical crosslinkers is that neither requires that the reactive sites of the proteins maintain a specified distance, however they also cannot be separated for analysis once the interactions are formed. Further analysis performed this week will indicate the success of these methods in confirming the spatial arrangement of the E1 and E2 glycoproteins.

Student Author(s):	Holbrook, Jennifer A.
Department(s):	Biological Sciences
Research Mentor(s):	John G. Scandalios/Genetics
Title of Presentation:	The Effects of Copper and Zinc on SOD and CAT-2 Null Maize Lines

An excess of copper sulfate (CuSo₄) ions may cause alterations in the efficiency of essential enzymes such as catalase (CAT) or superoxide dismutase (SOD) in maize.  The SOD and CAT enzymes work together to convert free oxygen radicals to water and molecular oxygen. A change in the function of either the SOD or CAT enzyme could result in damage to the plant.  Three maize lines were treated with varying concentrations of CuSO₄> and zinc sulfate (ZnSO₄) to analyze the effects of the metals on the CAT and SOD enzymes.  The control line, W64A is normal for both CAT and SOD activity, while the other two lines are low or null in either CAT or SOD.  Line A350 has low Cu/Zn SOD activity and normal CAT activity and the third line, A351, is CAT-2 null and has low Cu/Zn SOD activity.  Varying concentrations of CuSO₄ and ZnSO₄ were used as treatments ranging from 1mM to 100mM.  The effects of Cu/Zn at the given concentrations were also analyzed.  Treatment with Zn resulted in decreased enzymatic activity, in both SOD and CAT, in all tissue types within the three lines, while the addition of Cu or Cu/Zn caused slight increases in the SOD and CAT activity in the maize lines.

Student Author(s):	Kapfer, Katharine L.
Department(s):	Biochemistry
Research Mentor(s):	Dennis Brown/Biochemistry
Title of Presentation:	Investigation into Protein-Protein Interactions in the Sindbis Virus Membrane Glycoprotein to Block Virus Assembly

Sindbis Virus, a highly structured capsid enveloped alphavirus, serves as a model for the study of viral structure assembly and development of alphaviruses.  Various experiments have been conducted in which the structure of the virus has been altered to preclude the assembly of the virus structure to stop the transmission of the virus from its host, mosquitoes, to mammals.  Two mutations were conducted to the E2 glycoprotein tail of the capsid structure of the virus.  In the first mutation, a second lysine was added just after the lysine that is already present at the plasma membrane and cytosol junction pushing the rest of the amino acids behind it one position back.  The lysine that is already present was also changed to a phenylalanine with a second phenylalanine inserted, as was the second lysine.  The mutations were examined using Polymerase Chain Reaction (PCR), transfection of vertebrate and insect cells, and harvesting of the mutated viruses from insect and vertebrate cells.  These mutated viruses were used to infect into both insect and vertebrate cells to determine the viruses' infectivity through plaque assay.  It was found that both mutants produced a plaque assay titer, the number of plaques, that was lower than that produced by the wild type mutant.  While several of these experiments have altered the structure of the virus as to decrease the infectivity or to prevent the assembly of Sindbis, there has been no means of modifying the virus structure such that it would be feasible transmission to a wild population.

Student Author(s):	Keeling, E. Blaire
Department(s):	Zoology
Research Mentor(s):	Craig V. Sullivan/Zoology
Title of Presentation:	Utilization of Microsatellite DNA Loci to Enhance Selective Breeding of Striped Bass, Morone saxatilis

Striped bass and their hybrids support one of the fastest growing forms of fish farming in North Carolina and the nation.  The productivity of breeding programs for striped bass, Morone saxatilis, can be enhanced through the use of genetic markers.  Current breeding programs are inefficient and expensive because selection methods are based solely on fish phenotypes.  Alternatively, identifying microsatellite loci or short tandem repeats in the species' genome can reduce the capricious nature of breeding programs by selectively breeding striped bass that have such markers linked to genes for desirable traits, including disease resistance, stress tolerance and growth rate.  Utilizing multiple microsatellite loci for accurate determination of parentage also will permit rearing of offspring for performance testing in a "common garden" environment, removing confounding effects of genotype-environment interactions.  We genotyped 166 captive striped bass broodstock held at The North Carolina State University Pamlico Aquaculture Field Laboratory in Aurora, North Carolina.  DNA was amplified using polymerase chain reaction and genotypes were determined using an ABI Prism sequencer and software.  To date, genotypes at three loci have been successfully determined for all fish, and the genotyping at three additional loci is nearing completion.  Genotypic information will be used during the upcoming 2001 spawning season to simplify design of experimental crosses and facilitate selective breeding efforts.

Student Author(s):	Lahre, Kirsten
Department(s):	Genetics
Research Mentor(s):	Stephanie Curtis/Genetics
Title of Presentation:	Characterization of Genes Up-regulated During Heterocyst Development in the Cyanobacterium Anabaena

Anabaena  sp. strain PCC 7120 is a photosynthetic cyanobacterium capable of fixing atmospheric nitrogen.  Nitrogen fixation is inhibited by the presence of O2, a byproduct of photosynthesis.  When starved for fixed nitrogen, Anabaena differentiates some of its cells into heterocysts which are specialized for nitrogen fixation.  Heterocysts lose their photosynthetic capabilities which keeps O2 physically separated from the nitrogen fixation process.  A number of clones containing genomic sequences that are up-regulated during differentiation have been identified.  My project is to characterize the clone AD206.2 which was shown to contain portions of two genes.  The sequence of one open reading frame (orf1) was completed and shown to encode one part
of a two-component response regulatory system.  Sequencing upstream of this gene revealed that the second component of the system does not lie adjacent to orf1.  AD206.2 contains a second gene (orf2) for which partial sequence was obtained.  Orf2 appears to encode an ATP-binding cassette component of an ABC transporter.  Analysis of orf1 and orf2 transcripts across a developmental timecourse revealed that neither gene is expressed during growth on fixed nitrogen, and that both are strongly induced at the transcript level after nitrogen starvation.

Student Author(s):	Lambert, Cynthia F.
Department(s):	Horticultural Science
Research Mentor(s):	Dennis J. Werner/Horticultural Science
Title of Presentation:	Examination of Various Morphological and Pollen Characteristics for Inference of Ploidy Level in Styrax japonicus ‘Emerald Pagoda’

Japanese snowbell, Styrax japonicus Sieb. and Zucc. is a graceful, small ornamental tree that flowers in May in USDA hardiness zone 7. S. japonicus is diploid. In 1986, the late Dr. J. C. Raulston introduced to the United States a unique clone of S. japonicus from Sohukson Island off the coast of Korea. This clone, now named ‘Emerald Pagoda’, has larger leaves and flowers than the typical species, and is highly regarded for its ornamental value. The increased leaf and flower size of ‘Emerald Pagoda’ suggest that ‘Emerald Pagoda’ may be a polyploid. Comparison of stomate size, pollen viability, and pollen size of ‘Emerald Pagoda’ and ‘Crystal’, a typical diploid cultivar, revealed no significant differences in any of these parameters that often differ in magnitude as a consequence of change in ploidy level. Our results suggest that the distinct increase in leaf and flower size in ‘Emerald Pagoda’ is unrelated to a change in ploidy, but rather to other genetic factors.

Student Author(s):	Marsil, GiGi
Department(s):	Animal Science
Research Mentor(s):	Theo Van Kempen/Animal Science Adam Moeser/Animal Science
Title of Presentation:	The Effects of Degermed, Dehulled Corn on Animal Performance of  Nursery Pigs

Excessive manure production resulting from intensive swine production has increased public concerns regarding the potential negative impact on the environment. A developing method to reduce these negative impacts of swine waste is processing corn to remove indigestible dietary fiber thereby increasing its digestibility as compared to normal corn. This product is degermed, dehulled corn (DGDH).
        In this context, an experiment was designed to assess the effects of feeding DGDH on growth performance in nursery pigs. The design consisted of 96 nursery pigs (48 barrows and 48 gilts) that were blocked by sex and weight and were housed in nursery pens with 4 pigs per pen. Two starter diets were formulated to contain either DGDH (56%) or regular corn (58%) as the major grain source. Diets were formulated to contain similar levels of metabolizable energy and ileal digestible lysine. All pigs were weighed weekly and feed disappearance was recorded.
         Degermed, dehulled corn had no effect on ADG. There was a trend for lower ADFI in pigs fed DGDH corn (P = .09) which resulted in a 4% increase in feed conversion (P <.05). Results from this study imply that DGDH corn was more digestible than regular corn. A highly digestible corn product could have major implications in reducing waste production and potentially odor and volatile gas emissions.

Student Author(s):	Matson, Cynthia P.
Department(s):	Biochemistry
Research Mentor(s):	Dennis T. Brown/Biochemistry Traci M.T. Hall/The National Institute of Environmental Health Sciences
Title of Presentation:	Characterization and Crystallization of the STAR domain protein, QK1

A mutation of the quaking gene in mice, which encodes the QK1 protein, causes mice to shake uncontrollably, thus the name "quaking."  This quaking is due to a severe lack of myelin throughout the nervous system.  Another mutation in the quaking gene has been shown to be embryonic lethal at nine days of gestation.  The protein, QK1, has been shown to repress translation in vitro, and belongs to a family of proteins that all contain the STAR domain, which stands for Signal Transduction and Activator of RNA.  The study of this protein and others like it could lead to a better understanding of the regulation of embryonic development and of its problems and disorders.  In my research, I have subcloned a construct encoding the QK1 STAR domain and have recombined the gene into an expression vector for growth in bacterial cells.  I have refined the protocol for growth, expression and purification of this protein from a protocol for another STAR protein, improving the final yield of pure QK1 from approximately 2-3 mg per liter of culture to approximately 7-10 mg per liter, with the protein staying reasonably soluble and stable in solution.  I have created constructs of the gene containing the embryonic lethal mutation of QK1 and a variant of it for examination as well.  I have examined the RNA binding properties and have done some preliminary crystallization work for X-ray crystallography, which will be used to determine the structure of the protein in the future.  The goal of these continuing studies is to understand the relationship between structure and function in this protein, which will help to clarify its role in translational repression.

Student Author(s):	McCall, Erin E.
Department(s):	Biological Sciences
Research Mentor(s):	Michael D. Schulman/Sociology and Anthropology
Title of Presentation:	Occupational Hazards and Exposures among Teenage Construction Workers

Construction workers are at higher risk compared to other trades of experiencing serious occupational injury or death.  Not limited to adults, construction also ranks high as a source of teen occupational injury and fatality.  It is believed that one cause of the high risk of injury and death is teenagers handling machinery and working in environments that are in violation of Federal and State Child Labor Laws.  As an attempt to gain some understanding of the causes for the increased risk of injury to teens in construction, a statewide telephone survey of teens working in construction in North Carolina during the summer of 2000 was conducted.  In this survey of 123 teenage construction workers, responses to questions asking about specific types of work conducted and equipment used reveal that teens in construction work in hazardous situations that often violate Federal and State Child Labor Laws.  Results from this and from further surveys will be used to increase public awareness of the risks to youth in construction and to develop recommendations for injury prevention.

Student Author(s):	Murray, G. Chris
Department(s):	Soil Science
Research Mentor(s):	Daniel W. Israel/Soil Science
Title of Presentation:	Influence of Crop Management Systems on Mineralizable Soil Nitrogen in Swine Lagoon Effluent Spray Fields

Effluent from anaerobic swine lagoons is commonly applied to spray fields cropped with coastal bermuda grass for hay or for grazing in Eastern North Carolina.  Hay production removes large quantities of this applied nitrogen, which minimizes potential for nitrate movement to nearby streams after leaching to the shallow groundwater.  In grazed pastures much of the nitrogen is recycled into the soil system by the excrement of the grazing animals, which could lead to an elevated pool of mineralizable organic nitrogen in the soil.  The purpose of this experiment was to test the hypothesis that spray fields managed in grazed pastures have larger pools of mineralizable organic nitrogen than spray fields managed for hay production.
        A study was conducted to quantify the mineralizablenitrogen in soils from hay and grazed pasture spray fields.  Four replicates of soil samples werecollected at 0-6" and 6-12" depths from each field. After incubation in the laboratory, ammonium and nitrate mineralized from the organic nitrogen  pool were extracted with 1M KCl and quantified with a Lachat autoanalyzer system. After six weeks incubation at 28 C, similar amounts of nitrogen was mineralized from hay and grazed pasture spray fields (12 micrograms/g dry soil).  This indicated that crop management did not have a large impact in the size of the organic nitrogen pool in the soil.  Furthermore, there is likely some alternate pathway for the escapeof the nitrogen into the environment such as ammonia volatilization.

Student Author(s):	New, Stephen L.
Department(s):	Biological Sciences
Research Mentor(s):	James W. Moyer/Plant Pathology Jorge Abad/Plant Pathology Jan Speck/Plant Pathology
Title of Presentation:	Novel Strategy for Detection and Identification of Unrecognized Viruses Infecting Sweetpotatoes

Sweetpotato viruses are known to infect nearly all sweetpotatoes (Ipomea batatas), interfering with the exchange of germplasm and resulting in numerous symptoms reducing the yield, quality, and thus, commercial value of the sweetpotato crop.  Unfortunately, not enough information is known about viruses infecting sweetpotatoes to make diagnostic techniques reliable.  For instance, sweetpotato viruses often occur in viral complexes impeding viral isolation in sweetpotatoes.  Progress is further hindered due to compounds inhibitory to virus transmission in sweetpotato extracts, very low titer, and narrow host range.  Thus, in response to the need to develop diagnostic tools for the prevention of outbreaks from viruses involved in international exchange of germplasm, strategies for viral isolation and diagnosis were developed.  First, sweetpotato plants displaying viral symptoms were grafted into indicator plants.  Following the development of symptoms in the indicator plants, plant tissue was assayed for known viruses via membrane immuno-binding assay.  In this technique, crude extract from the tissue was transferred to nitrocellulose membranes and exposed to specific antibodies corresponding to known viruses.  For samples with negative results to sweet potato feathery mottle virus, the most common virus among sweetpotatoes, double stranded (ds)RNA purification was completed to verify the presence of a virus.  The dsRNA is a relatively stable intermediate in viral replication.  It is diagnostic to the genus level and provides substrate for the synthesis of virus specific diagnostic tools.  At present, our results obtained with current diagnostic techniques suggest the presence of novel viruses and/or novel strains of known viruses that can be used to generate clones for hybridization assays and sequencing for the synthesis of PCR primers that will facilitate rapid detection of exotic plant viruses.

Student Author(s):	Newcomb, Dawn C.
Department(s):	Chemistry
Research Mentor(s):	Linda D. Martin/Anatomy, Physiological Sciences, and Radiology
Title of Presentation:	Effects of Glucose and Interleukin-13 on Mucus Secretion in Human Airway Epithelial Cells

Chronic inflammation in the asthmatic airway leads to epithelial cell injury.  Repair involves proliferation of epithelial cells, with a final increase in mucus-producing cells and airway mucus.  Recently, we have shown the proinflammatory protein interleukin-13 (IL-13) to induce proliferation and an increase in mucus cells and secreted mucus from differentiated, normal human bronchial epithelial (NHBE) cells in vitro.  These cells mimic the morphology and function of the in vivo airway epithelium.  Using this model system, we have begun to examine potential interplay between IL-13 and glucose in the development of this mucus phenotype.  By immunohistochemistry, we have observed a decrease in glucose transporter 1 (GLUT1) expression in cultures exposed chronically to IL-13 (10 days).  Expression of insulin receptor substrate 2 (IRS-2), a protein likely involved in the signal transduction pathway governing GLUT1 expression, increases with exposure to IL-13.  Interestingly, its phosphorylation is increased by day 3 of IL-13 treatment, and decreased as the cells become more differentiated (day 9, day 12).   ELISAs were used to measure secreted mucus in supernatants from cells grown on plastic (undifferentiated) and continuously exposed (10 days) to media with normal (100mg/dL) or high glucose (225mg/dL), with or without IL-13.  Secreted mucus was collected on days 3, 7, 9, and 12 post-confluence from all cells.  The presence of glucose in the undifferentiated cultures, or IL-13 in the differentiating cultures, correlated with an increase in secreted mucus compared to control cultures.  From these data, we conclude that undifferentiated NHBE cells respond to high glucose (225mg/dL) by mimicking the IL-13-induced mucus phenotype observed in differentiating NHBE cell cultures.  Further investigation will determine whether IRS-2 phosphorylation and GLUT1 expression play a role in glucose-induced mucus cell hyperplasia.  These results have implications for potential production of excess airway mucus in mildly hyperglycemic patients with inflammatory airway disease.

Student Author(s):	Oshimura, Tomohiro
Department(s):	Biological Sciences
Research Mentor(s):	Edward K. Lobenhofer/National Institute of Environmental Health Sciences Scott Chilton/Botany
Title of Presentation:	Identification of Estrogen Responsive Genes in MCF7-C7 Breast Tumor Cell Line Using cDNA Microarrays

The binding of estrogen to the ligand-binding site of the estrogen nuclear receptor induces a vast cascade of signaling within estrogen responsive cells.  With the use of the cDNA mircoarray, it becomes possible to monitor the gene expression changes of thousands of genes with a single experiment.  Of key interest are the identification of cell cycle genes and the changes in their expression.  Genes that are implicated in cell proliferation provide potential targets for pharmacological inhibitors.   We exposed cells from the hormone responsive ER+ tumor cell line, MCF7-C7, to 1 x 10-8M estradiol over several time periods.  From the mRNA isolated from these cells we were able to create cDNA, which was hybridized to a cDNA gene chip and scanned in order to obtain a qualitative analysis of gene expression changes.  The resulting outlier list identified that several genes such as c-Fos, c-Myc, c-Jun, and Cyclin A as well as other genes related to cell cycle progression were regulated by estrogen.  After identification of these genes, several candidate genes were selected for verification by northern blot.  These blots are ongoing and will be used to strengthen the data and quantify expression differences.  This study provided a key list of genes who’s pharmacological inhibition may stop cell proliferation in estrogen responsive tumors.

Student Author(s):	Pike, David A.
Department(s):	Zoology
Research Mentor(s):	Nick M. Haddad/Zoology
Title of Presentation:	A Novel Approach for Assessing Open-Habitat Corridor Use by a Variety of Insect Orders

Habitat corridors are often proposed in conservation efforts to help preserve species diversity, maintain population sizes, and increase genetic diversity among populations. Using malaise traps, a passive capture technique, I studied the effects of open-habitat corridors on movements of abundant insect orders out of experimental patches in eight large-scale experimental units at the Savannah River Site, S.C.  A pine forest matrix surrounded these systems, providing an "edge" that differed greatly in habitat type, mimicking potential areas of actual corridor implementation. The only insect order to show higher abundances in corridor traps was Homoptera, which showed a significantly greater abundance at corridor areas than at matrix edges (p=0.0549); though Lepidopterans showed a significantly greater preference for the matrix area (p=0.0352).  No other order was found to be significant at either edge.  These results are consistent with selected habitats in that Homoptera are open area species and Lepidopterans, in this study mainly moths, may prefer forest edges.  Overall abundances varied by experimental unit and time, as herbaceous ground cover, soil composition, and matrix habitat differed because of geographical isolation from one another. Weather and insect life history may have played a role in abundance differences during the experimental period.  Although my technique may not have been effective at determining corridor use at the insect order level because of variable responses of species within specific orders, it may be effective in assessing insects with consistent responses to habitat edges.
 

Student Author(s):	Powell, Erin K.
Department(s):	Microbiology
Research Mentor(s):	James W. Brown/Microbiology
Title of Presentation:	RNase New P Sequences from Unknown Organisms

Since the early 1980 it has been known that RNase P is essential to all living cells; it is required in processing precursor tRNAs needed for protein synthesis.  Currently, this molecule is being studied for its potential use in developing new antimicrobials.  A large part of what makes RNase P a potential target for antimicrobials is its evolutionary variability.  As gene sequencing becomes easier and more common, the RNase P database continues to grow.  One particular group of organisms that has yet to be fully explored is the green non-sulfur bacteria such as Chloroflexus and Herpetosiphon.  In attempting to sequence the RNase P RNA gene from PCR products of relatives of these two bacteria, new primers specific for green non-sulfur bacteria (based on the known sequence from close relative Thermomicrobium roseum) were designed.  These new primers were used with isolated environmental DNA samples from sludge and ruminal fluid.  In sequencing PCR products from these samples, four different RNase P RNA gene sequences have been obtained.  Through the analysis of the secondary structures of the RNAs encoded by theses sequences, along with those of others obtained over time, the RNase P database may be enhanced for further comparison of the variation in the structure and evolution of this RNA. Finally, these sequences may also be placed on the phylogenetic tree in an attempt to identify the organisms from which they were obtained.
 

Student Author(s):	Shaikh, Faten F.
Department(s):	Biochemistry
Research Mentor(s):	Frederick J. Fuller/Microbiology, Pathology, and Parasitology
Title of Presentation:	Does an Equine Infectious Anemia Virus Induce Apoptosis in a Cell?

Apoptosis, or programmed cell death, is an active process that plays a major role during cellular differentiation, development of multi-cellular organisms, tissue homeostasis, metamorphosis and cancer.  During development, the process of Apoptosis helps sculpture the body, shape the organs, and carve out fingers and toes.  Apoptosis is necessary to tightly control cell numbers and tissue size, and to protect the organism from rogue cells that threaten homeostasis.  Apoptotic cell death is recognized by specific visible changes caused by Caspases, or Cysteine proteases. Caspases selectively cleave a restricted set of target proteins, thus activating a DNA nuclease.  This nuclease cuts the genomic DNA between nucleosomes to generate DNA fragments with lengths corresponding to multiple integers of approximately 180 base pairs. Some of the morphological changes observed during Apoptosis include chromatin condensation, reduction in nuclear size, shrinkage of the cell, membrane blebbing, and breakdown of the cell into membrane-bound apoptotic bodies. Such a critical process has to be strictly regulated as too little or too much cell death may lead to birth defects, auto-immune diseases, or cancer.  In this project, the ELISA technique was used to determine whether a viral infection activates the process of Apoptosis within the infected cell. After performing the ELISA assay, we are able to conclude that with a 95% confidence, the cells infected with a P17 strain of the Equine Infectious Anemia virus are undergoing Apoptosis. Thus the virus in the infected cells induces the process of Apoptosis within the cells.  To confirm the results of the ELISA assay, we will use the Annexin V-FITC assay.  This assay is similar to ELISA in that it is detecting Apoptosis; however, instead of detecting nuclear nucleosomes as evidence of cells undergoing Apoptosis, the Annexin V assay detects the presence of this specific protein on cell membranes of cells undergoing Apoptosis.

Student Author(s):	Shiver, Erin C.
Department(s):	Biological Sciences
Research Mentor(s):	Clay Clark/Biochemistry  Cristina Pop/Biochemistry
Title of Presentation:	Creation and Expression of Monomeric Human Procaspase-3 (V 266 E)

Human procaspase-3 is a member of the caspase family (caspase stands for "cysteinyl aspartate-specific protease").  Procaspase-3 is a zymogen of caspase-3, part of a family of endoproteases involved in a cascade leading to apoptosis (programmed cell death). By this morphological cell suicide, damaged or infected cells are removed without involving inflammation or necrosis.  Deciphering the mechanism of caspase folding and assembly is important for developing specific medication against cancer and neurodegenerative disorders.  Caspase-3 is a homodimer of heterodimeric units, composed of two large and two small subunits, where the dimer interface is positioned mainly between the small subunits.  The recombinant procaspase-3 (C163S), mutated at the active site to prevent autoproteolysis, is a dimer in vitro and is believed to have a similar dimer interface with the one from mature caspase-3, whose crystal structure is known. Val266/Val266' from the small subunits establishes one of the most important hydrophobic contacts within the caspase-3 dimer. To find out whether procaspase-3 dimerizes using the same interface, an additional mutation in procaspase-3 (C163S) at Val266 has been introduced, changing it to a glutamate. The C-terminus-His-tagged protein was purified onto a Ni-column, followed by DEAE-Sepharose ion exchange chromatography. The protein was checked by MALDI-TOF spectrometry. Gel filtration studies and analytical ultracentrifugation experiments have shown that procaspase-3 (C163S/V266E) is a monomer. Therefore, the dimeric proprieties of procaspase-3 (C163S) were disrupted after making a point mutation within the dimer interface.  The result suggests that procaspase-3 and mature caspase-3 fold in a similar manner, using the same interface during dimerization.

Student Author(s):	Southerland, Andrew M.
Department(s):	Zoology
Research Mentor(s):	Robert M. Grossfeld/Zoology
Title of Presentation:	Stress Protein Synthesis and its Correlation to the Protective Response of Oyster Gill Tissue Following Hyperthermia

Bivalves perform a major role in the marine ecosystem, food chain, and economy of our coastal waters. Since these shellfish are sessile ectotherms, they must be able to adapt to dynamic environmental conditions. In the last thirty years, however, oyster populations have experienced depletions by infection with the protozoa Perkinus marinus and Haplosporidium nelsoni, both of which thrive in areas of high water temperature and salinity. Oysters, like all organisms, produce constitutive levels of stress proteins, believed to play a protective role in maintaining cellular metabolism and integrity. Marine bivalves, like other organisms, also produce an inducible stress protein during times of stress. The purpose of our research was to determine if there is a correlation between inducible levels of stress protein and the protective response of the American oyster, C. virginica, as revealed through measurements of protein synthesis following hyperthermia in vitro. Past experiments on many organisms have shown increased tissue protection after a non-lethal, conditioning heat stress. Our results revealed that oyster gill tissue showed partial protection of protein synthesis if pre-conditioned at 41.5 deg. C and then allowed to recover before a second more severe heat-shock at 45 deg. C. SDS-PAG electrophoresis and corresponding autoradiographs of protected tissue samples revealed induction of proteins of 70kDa, corresponding in size to the major family of heat-shock proteins (HSP70) which is known to contribute to cellular stress tolerance.

Student Author(s):	Sullivan, Marshall W.
Department(s):	Center for Applied Aquatic Ecology Botany
Research Mentor(s):	Brant W. Touchette/Center for Applied Aquatic Ecology, Botany Howard B. Glasgow Jr./Center for Applied Aquatic Ecology, Botany JoAnn M. Burkholder/Center for Applied Aquatic Ecology, Botany
Title of Presentation:	Fish Habitat Value of American Water Willow in a Piedmont Reservoir

The emergent wetland plant, American water willow (Justicia americana L.) is the dominant species of aquatic vegetation in the Narrows Reservoir (located in the western Peidmont of North Carolina).  In this study, we sampled forty-five different locations seasonally within the reservoir in order to assess the habitat value of water willow for fishes.  The locations were differentiated based on habitat structure and included:  (i) sheltered water willow, (ii) exposed water willow, (iii) forested wetlands, (iv) mixed-species wetland, (v) tree edge, (vi) piers and boat docks, and (vii) retaining walls (rip-rap and concrete slabs).  Seasonal fish collections (Spring, Summer, Autumn, and Winter) were conducted using seine nets and traps in order to determine fish diversity and abundance.  The data demonstrate the importance of water willow as a habitat for fish. Furthermore, the relative value of different habitats is seasonally dependent; wherein fish preferred sheltered areas (e.g., water willow, mixed-species wetlands, and forested wetlands) during the spring and more open areas during the summer.  It is likely that these habitat preferences are associated with the life cycles of the resident fishes (eg., open areas during breeding season in sunfish).  Overall, our data illustrate the value of water willow and other littoral habitats in providing habitat for a variety of fish species.
 

Student Author(s):	Taylor, Duncan R.
Department(s):	Molecular & Structural Biochemistry
Research Mentor(s):	Paula Brown/National Institute of Enviromental Health Sciences: Gamete Biology, Laboratory of Reproductive and Developmental Toxicology E.M. Eddy/National Institute of Enviromental Health Sciences: Gamete Biology, Laboratory of Reproductive and Developmental Toxicology John Vandenbergh/Zoology
Title of Presentation:	Use of ESTs to Characterize an mRNA Present in Mouse Pachytene Spermatocytes

We screened a testis cDNA expression library with a monoclonal antibody and isolated several cDNAs encoding proline-rich proteins.  Theses cDNAs were used to probe Northern blots of RNA from 13 different mouse tissues and one of these (designated ATC 4.1) identified a 2.6 kb mRNA transcript present only in the testis.  The transcript was first detected in juvenile mouse testis on postnatal day 14, coincident with the initial appearance of pachytene spermatocytes.  The transcript was also present in round spermatids but was not detected in condensing spermatids or Sertoli cells.  Further molecular analysis was performed to isolate and sequence the full length ATC 4.1 cDNA, but was not successful due to termination of reverse transcription by a GC-stretch near the 5’-end.  Current studies have taken advantage of the EST project to determine the sequence of the ATC 4.1 transcript.  A search of the GenBank Mouse EST database using the known 2.1 kb sequence of ATC 4.1 identified mouse and human clones with significant homology to different regions of the ATC 4.1 sequence.  Based upon homology and position of alignment of the EST sequence, several mouse clones were selected for further analysis.  Sequencing of the EST clones and aligning them with the known ATC 4.1 sequence allowed us to compile a consensus sequence that verified the known 3’-end of ATC 4.1 and provided additional sequence on the 5’-end.  The deduced protein structure does not contain any known functional motifs and the role of this protein in pachytene spermatocytes remains to be determined.

Student Author(s):	Thomas, Walter E.
Department(s):	Crop Science
Research Mentor(s):	John Wilcut/Crop Science Janet F. Spears/Crop Science
Title of Presentation:	Biology and Herbicidal Control of Slender Amaranth (Amaranthus viridis L.)

Slender amaranth (Amaranthus viridis L.) is becoming more prevalent in cotton fields in North Carolina, Tennessee, and Mississippi.  If slender amaranth becomes more common in cotton, it is likely to be problematic in rotational crops such as corn, peanut, and soybean.  Slender amaranth is not listed in Cooperative Extension weed control recommendations or herbicide labels that control other Amaranthus spp.  Since little information is available about slender amaranth, a series of experiments were designed to evaluate the germination, emergence, and herbicidal control of this newly emerging weed species.  Germination was evaluated using treatments of temperature, pH, and osmotic potential.  Germination was studied at six constant temperatures and four diurnal temperature regimes representative of May, June, July, and August in North Carolina. Osmotic potential and pH were evaluated in the four alternating temperature regimes.  Depth of seedling emergence was also evaluated.  The optimum constant temperature was 34 C.  In depth of emergence trials, emergence declined linearly with increasing planting depths.  The second objective was to evaluate control of slender amaranth using postemergence herbicides registered in corn, cotton, peanut, and soybean.  Good control was generally found with herbicides that are registered to control other Amaranthus spp.

Student Author(s):	Warren, Erin N.
Department(s):	Molecular and Structural Biochemistry
Research Mentor(s):	Carla Mattos/Molecular and Structural Biochemistry
Title of Presentation:	Expression and Purification of the Small GTPase, R-Ras

Signal transduction pathways involve complex communication networks that are responsible for eliciting intracellular responses upon stimulation by extracellular chemical signals.  The Ras family of GTPases is an essential aspect to this communication pathway and provides an intermediate link between external signals and genetic regulation of cellular growth, proliferation and survival.  Certain mutant Ras proteins propagate a persistent signal for the cell to divide independently of appropriate extracellular stimulation, a condition otherwise known as cancer.  R-Ras belongs to the Ras family of proteins, but has exhibited lower transforming capabilities than its closely related family members, Ras and TC21. Given the close homology in their amino acid sequence and three-dimensional structure, it is of interest to uncover the mechanism that accounts for the different transforming capabilities of R-Ras and its highly oncogenic family members.  My project involves optimizing the expression and purification of R-Ras using traditional purification techniques to obtain an abundance of pure, biologically active protein suitable for growing crystals.  These crystals will ultimately be used to determine the three-dimensional structure and ligand binding sites for R-Ras, and the results will be compared to other related Ras GTPases.  My preliminary results were achieved by expressing R-Ras in Escherichia coli cells using a pAT vector and inducing the cells with 0.5 mM IPTG at 37°C for four hours.  Soluble protein was then harvested from the cells via sonication and centrifugation.  Next, soluble R-Ras was purified in a 20-40% cut of ammonium sulfate and then eluted during anion exchange at a NaCl concentration of 200-275 mM on a Pharmacia Hi Load 26/10 Q Sepharose FF column.  After the ion exchange, Western blot analysis and polyacrylamide gel electrophoresis confirmed the presence of R-Ras accompanied by some high molecular weight contaminants.  I am currently optimizing a gel filtration step for further purification, as well as determining the best strain of E.coli for high-level expression.

Student Author(s):	Wood, Jamie L.
Department(s):	Genetics
Research Mentor(s):	Stephanie E. Curtis/Genetics
Title of Presentation:	Characterization of a Sequence Up-regulated During Heterocyst Development Encoding a Putative Signal Transduction System

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that produces a nitrogen fixing cell called the heterocyst in response to nitrogen starvation.  We are interested in identifying genes important for heterocyst development, and understanding how these genes are regulated during the differentiation process.  A screen for sequences up-regulated at the transcript level after nitrogen starvation was conducted as a way of identifying genes that may be involved in heterocyst development.  One 890 bp sequence selected for further study, denoted AD206.1, hybridized to transcripts that increased approximately two-fold after nitrogen starvation. To characterize this clone further, a 4.0 kb fragment of Anabaena genomic DNA containing AD206.1 was cloned, sequenced, and analyzed.  The region contains three open reading frames (ORFs) that appear to encode a sensor molecule and two regulator molecules of a signal transduction system.  Probes specific to each ORF were used to analyze the expression of each gene across a developmental time course.  Future experiments will involve the mutagenesis of the ORFs to determine whether they are required for heterocyst differentiation.

Student Author(s):	Younger, Joseph W.
Department(s):	Zoology
Research Mentor(s):	John R. Godwin/Zoology
Title of Presentation:	Filled to the Gills:  The Search for a Correlation Between White Flank Patch Size and Polymorphisms in the 11-beta-hydroxylase Gene in Thalassoma bifasciatum

The enzyme 11-beta-hydroxylase plays a key role in androgen production in many vertebrates, including humans and fishes.  Androgens are directly responsible for primary and secondary sexual characteristics in males.  Defects in the gene that code for this enzyme cause the most common inherited endocrine disorder in humans, or Adrenal Hyperplasia Type IV.  Point mutations, or changes in a single base pair in the DNA molecule, in the gene encoding this enzyme may cause variation in secondary sexual characteristics of the bluehead wrasse (Thalassoma bifasciatum).  This common coral reef inhabitant of the Caribbean has two alternative male mating morphs, a drab initial phase male and a colorful terminal phase (TP) male.  The TP male has a blue head separated from the rest of the body by a white bar bordered on each side by a black bar.  Males with more white in the patch have increased mating success, suggesting that this trait is under sexual selection.  The major androgen 11-ketotestosterone (11KT) is responsible for the development of color in the TP males and 11-beta-hydroxylase is critical for production of 11KT.  Because genetic variability must be present for sexual selection to occur, it follows that polymorphisms in the 11-beta-hydroxylase gene may correlate to variations in the expressed pattern of the flank patch in the TP males.  The first step in investigating this possible correlation is identifying the exons, or expressed coding regions of the gene, and the introns, or noncoding regions of the gene.  We used PCR amplification followed by cloning in a plasmid recombinant DNA vector before sequencing to identify the exons of the 11-beta-hydroxylase gene in T. bifasciatum.  The next step will be to compare individual 11-beta-hydroxylase genes from individual bluehead wrasses and test for possible associations between genetic polymorphisms and male patch size.

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