Cellular, Molecular and Developmental Biology.

Chair: Ross Whitwam, Mississippi University for Women 

Vicechair: Yuan Luo, University of Southern Mississippi

THURSDAY MORNING

Exhibition Hal B

Session I--And What Is It You Do Again? Investigating Molecular Functions and Effects

8:00 Introduction

8:15 TYROSINE PHOSPHORYLATION OF THE JAK2 ACTIVATION LOOP IS ESSENTIAL FOR A HIGH ACTIVITY CATALYTIC STATE, BUT DISPENSIBLE FOR A BASAL CATALYTIC STATE

Roy Duhe* and Kiranam Chatti, University of Mississippi Medical Center, Jackson, MS 39216

The phosphorylation of an "activation loop" within protein kinases is commonly associated with establishing catalytic competence, and phosphorylation of the Tyr1007 residue in the activation loop of JAK2 is essential for intracellular propagation of cytokine-initiated signalling. The transition of JAK2 from basal to high activity states can be partially described via two kinetically distinct ATP binding sites. Phosphorylation of the JAK2 activation loop was essential for conversion to the high activity state, which was characterized by high efficiency ATP utilization during autophosphorylation. Mutagenesis of activation loop tyrosine residues Tyr1007/1008 to phenylalanine residues severely impaired, but did not abolish, the enzyme's ability to autophosphorylate; the mutant enzyme remained in a basal activity state characterized by low efficiency ATP utilization during autophosphorylation. Mutagenesis of a critical Lys882 residue to a glutamate residue abolished all evidence of kinase activity, confirming that the observed phosphorylation of Tyr-to-Phe mutants was not a transphosphorylation catalyzed by another kinase. Our data are consistent with the proposal that JAK2 is an inefficient, but active, enzyme in the absence of activation loop phosphorylation, and is capable of autoconversion to a high activity state under physiological ATP concentrations, which would preclude the need for an extraneous trans-activating kinase.

8:30 THE DETERGENT-LIKE ACTION OF CYTlA, A DELTA-ENDOTOXIN FROM BACILLUS THURINGIENSIS VAR. ISRAELENSIS

Slobodanka Manceva (1 *), Marianne Pusztai-Carey (2), and Peter Butko (1), (1)University of Southern Mississippi, Hattiesburg, MS 39406, and (2) Case Western Reserve University, Cleveland, OH

Cyt1A is a mosquitocidal delta-endotoxin from Bacillus thuringiensis var. israelensis, whose mechanism of cytolysis is not fully resolved. Some data support the notion that CytlA forms pores in lipid bilayers, but other are consistent with a detergent-like dissolution of the membrane. We used epifluorescence microscopy to study changes in the size and morphology of fluorescently labeled giant unilamellar vesicles (GUV). In an osmotically balanced medium, Cyt1A induced a rapid disintegration of 5-micrometers GUVs into objects 100 times smaller, which the poreformation model cannot explain. Chemical cross-linking and SDS PAGE electrophoresis revealed that in the presence of lipid Cyt1A formed high-molecular-weight protein aggregates, which did not enter the gel. The pore-formation model postulates the existence of stoichiometric oligorners in the membrane. Thus, our results give support to the detergent-like model of the Cyt1A's action.

8:45 ANALYSIS OF THE SNXA1 MUTATION OF ASPERGILLUS NIDULANS

Sarah McGuire (*) and David Norris, Millsaps College, Jackson, MS 39210

In eukaryotic cells, the cyclin-dependent kinase Cdc2 is the master mitotic regulator: when active, cells can divide. The Aspergillus nidulans homolog of the Cdc2 gene is termed nimX. Mutations in nimX cause phenotypes in which the cells may or may not undergo division, depending upon the environmental conditions. In particular the nimX2 mutation prevents mitosis at temperature of 42[degrees]C or higher. Previous work has identified several extragenic suppressor mutations of nimX2. The first of these is termed snxA1 (for suppressor of nimX). It allows normal cell division to occur above the restrictive temperature but also confers a cold sensitivity when cultured below 20[degrees]C. Evidence from plate and plug growth assays performed as part of this experiment have confirmed the suppressor and cold-sensitive phenotypes of snxA1. Further, nuclear morphology assays conducted have shown that snxA is involved in one of the cell cycle checkpoints, likely between G2 and M. Transformation and cloning have thus far been unsuccessful, but future work will likely lead to the sequencing of the gene and subsequent identification of the protein for which it codes.

9:00 THE PROTECTION OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE BY THE SELECTIVELY PERMEABLE PROTEIN SHELL OF THE CARBOXYSOME

Eric Williams (*), Gordon C. Cannon, and Sabine Heinhorst, University of Southern Mississippi, Hattiesburg, MS 39406

Halothiobacillus neapolitanus carboxysomes are polyhedral proteinaceous micro-compartments that have been shown to contain much of the cell's ribulose-1,5bisphosphate carboxylase/oxygenase (RuBisCO). To date, RuBisCO is the only protein demonstrated to have enzymatic activity within carboxysomes. Cell growth under [CO.sub.2] limiting conditions results in an increase of carboxysomal RuBisCO levels. Further more, mutants whose RuBisCO is not packaged into carboxysomes require elevated [CO.SUB.2] levels for growth, indicating that sequestering of RuBisCO into carboxysomes is essential for efficient [CO.SUB.2] fixation. The molecular mechanism by which carboxysomes enhance the catalysis of [CO.SUB.2] fixation by RuBisCO is not clear. However, RuBisCO is a bi-functional enzyme that catalyzes [CO.SUB.2] fixation, in addition to the competitive and wasteful fixation of [O.sub.2]. Therefore, the carboxysomal shell may act as a selectively permeable membrane that excludes [O.sub.2] from RuBisCO inside the carboxysome . To test this hypothesis, carboxylation reaction was measured with intact, H. neapolitanus carboxysomes and with free RuBisCO in saturating levels of [O.sub.2]. As expected, [O.sub.2] acted as a competitive inhibitor of the carboxylation reaction in both free and carboxysome bound enzyme. However, the degree of inhibition was significantly less in intact carboxysomes. To further examine the ratio of carboxylase to oxygenase activity, carboxysomes were subjected to treatments that compromise the integrity of the shell. Preliminary experiments with treatments that disrupt carboxysomal shell suggest a reaction ratio shift towards oxygenation.

9:15 Break

9:30 MODULATION OF CELL STRESS RESPONSE AND REACTIVE OXYGEN SPECIES PRODUCTION N AMYLOID-BETA SECRETING NEUROBLASTOMA CELLS BY THE GINKGO BILOBA EXTRACT EGb761

Julie Vining Smith (*), Adam J. Burdick, and Yuan Luo, University of Southern Mississippi, Hattiesburg, MS 39406

Standardized Ginkgo biloba extract (EGb761), used extensively in clinical trials, is popularly used in the USA as a natural dietary supplement for memory enhancement, and is being prescribed in European countries for the treatment of cerebral insufficiency, age-related deterioration of mental functions such as that commonly found in Alzheimer's Disease (AD). Recent findings suggest links between AD and deposition of Amyloid Beta-peptide (ABeta), oxidative stress, and apoptosis. Existing pharmacological data and clinical trials show EGb761 to be a potent antioxidant with neuroprotective effects, however specific mechanisms of protection are still being investigated. We examined potential mechanisms of neuroprotection using N2a neuroblastoma cells (wild type), or the ABeta-secreting N2a cell line stably expressing double Swedish mutant human APP695 and exon-9 deleted mutant PS1 (swe/D9). In a recent publication, we showed that in these cells EGb761 caused a marked decrease in caspase-3 activity, a key enzyme in the apoptosis cell-signaling cascade, as well as inhibition of ABeta aggregation. We now additionally suggest that EGb761 provides neuroprotection by modulating multiple cell signaling pathways, including prevention of intracellular oxygen free radical accumulation, and regulating the activity of the Caspase-12 protein, an endoplasmic reticulum-specific caspase capable of initiating a separate, stress-induced apoptosis cascade in response to toxic insults to the cell.

9:45 APOPTOTIC MACHINERY IN DIFFERENTIATED PC12 CELLS IS MODULATED BY GINKGO BILOBA EXTRACT EGb761

Adam J. Burdick (1) (*), Julie Vining Smith (1), Paul Golik (2), Douglas Wallace (2), and Yuan Luo (1), University of Southern Mississippi (1), Hattiesburg, MS 39406, and Emory University (2), Atlanta, GA 30322

Recent literature suggests links between oxidative stress and nerve cell death occurring in a variety of neurological disorders, including Alzheimer's Disease. Therefore, the determination of cellular mechanisms of known neuroprotectants such as EGb761, the standardized Ginkgo biloba extract, is important in understanding the molecular basis of neurodegeneration. However, the mechanisms of protection remain unclear. Differentiated PC 12 cells are a well-established cellular model for studying apoptosis and potential neuroprotective mechanisms. We have determined the effects of EGb761 on cellular apoptotic machinery following cellular insult with staurosporine or juglone on PC 12 cells, using assays of intracellular levels of free radicals, cytochrome c release, DNA microarray, and Bcl-2 western blotting. Our results show that pre-treatment with EGb761 attenuates the levels of cytochrome c in the cytosol. Data from DNA microarray indicates that transcription of multiple apoptosis-related genes such as Bcl-2 li ke protein and caspase-12 was up- and down-regulated respectively, in cells treated with EGb761. The results of microarray were confirmed with Bcl-2 western blotting indicating that Bcl-2 immunoreactivity is increased in cells pre-treated with EGb761. Free radical analysis indicates a decrease in intracellular hydrogen peroxide in cells pretreated with EGb761. These results suggest that modulation of multiple targets of the cellular apoptotic machinery may, at least in part, mediate the neuroprotective effects of EGb761.

10:00 EGb76L INCREASES OXIDATIVE STRESS RESISTANCE, EXTENDS LIFE SPAN, AND ATEENUATES THE HEAT SHOCK PROTEIN HSP-16 EXPRESSION IN CAENORFIABDITIS ELEGANS

Amy Strayer (*), Zhixin Wu, Astrid Gutierrez-Zepeda, and Yuan Luo, University of Southern Mississippi, Hattiesburg, MS 39402

EGb761, a standard extract of Gingko biloba, has been known to enhance cognition, stress resistance, and longevity in mammals; however, its cellular and molecular mechanisms are still unclear. Using the model nematode organism Caenorhabditis elegans, we were able to demonstrate the positive pharmacological effects of EGb761 on oxidative and thermal (heat) stress. EGb761 extended the wild type (N2) worms' mean and maximum life span when exposed to a mild oxidative stress. EGb761 also increased the N2s resistance to a more severe oxidative stress by 33% and thermal stress by 25%. The effects of EGb761 on the expression of the small heat shock protein hsp-16 was directly observed by the co-expressing gene, hsp: GFP (this strain of worm is known as CL2070). We observed a decrease in the expression of this gene in the nematode when treated with EGb761, indicating that EGb761 aided in protection where heat shock proteins are normally used. Under oxidative stress, CL2070 showed a 21% increase in mean survival when p retreated with EGb761. These results suggest that EGb761, through antioxidative and anti-stress properties, holds protective effects for C. elegans under oxidative and thermal stress.

10:15 ANALYSIS OF THE ACTIVATOR CPER DURING CHROMATIC ADAPTATION

Lee Peeples (*) and David Kehoe, Alcorn State University, Alcorn State, MS 39069, and Indiana University, Bloomington, IN

The cyanobacterium Fremyella diplosiphon undergoes complementary chromatic adaptation (CCA) in response to changes in ambient light color. This photo-reversible process works optimally in red and green light conditions, is controlled at the transcriptional level, and is regulated by a complex signal transduction pathway. CCA begins with the perception of light by the photoreceptor, RcaE, which initiates a chain of events leading to the expression of either cpcB2A2 in red-light (RL) or cpeBA in green-light (GL). The products of these genes, phycocyanin and phycoerythrin, respectively, accumulate in the light-harvesting complexes of F. diplosiphon cells, increasing their ability to absorb photons. A class of mutants was generated which showed no significant accumulation of CpeBA protein in GL while exhibiting normal regulation of cpcB2A2 in RL. Complementation of one of these Turquoise mutants revealed a gene designated cpeR. This gene may act as a global regulator. Mutation of this gene results in no transcrip t accumulation of either CpeBA or the GLactivated pebAB. To further study CpeR and its role in CCA, it was overexpressed in E. coli. CpeR was purified and used to generate antibodies which will be used to further analyze CpeR function. Western hybridization will be used to examine in what light condition(s) the protein is present. The current hypothesis is that CpeR acts as an activator of cpeBA and pebAB expression and thus should be present in GL and absent in RL. Study of CpeR will lead to a better understanding of its role in CCA and elucidating the controlling signal transduction pathway.

10:30 Break

Session II--Is Anybody Else Feeling a Bit Whoozy? Molecular Aspects of Toxicology

11:00 ASSESSMENT OF THE DEVELOPMENTAL TOXICITY OF PEROXISOME PROLIFERATING AGENTS IN THE JAPANESE MEDAKA (ORYZIAS LATIPES)

Mary L. Haasch, University of Mississippi, University, MS 38677

The majority of studies assessing the developmental toxicity of peroxisome proliferating agents (PPAs) have been done in mammals. Little is known regarding possible adverse effects to aquatic species. Environmental chemical contamination has been suggested to be a possible causative agent in the decline of wild fish populations. PPA environmental chemical contaminants include plasticizers, solvents, herbicides, lubricants, and pharmaceuticals. The Japanese medaka (Oryzias latipes) has previously been used as a developmental model because development within the egg is external, the egg has a clear chorion allowing visualization and the time to hatch is relatively short (10-12 d), yet long enough to easily discern abnormalities. The medaka embryolarval assay (MELA) was used to assess the developmental toxicity of 1 and 10 ppm di-n-butyl phthalate (DBP; plasticizer; 3.59 and 35.9 nM), perchloroethylene (PCE; tetrachloroethylene; solvent; 6.03 and 60.3 nM), 2,4-dichlorophenoxyacetic acid (2,4-D; herbicide; 4.52 a nd 45.2 nM) or WY14643 (3.09 and 30.9 nM), a potent model PPA and representative of pharmaceuticals. Developmental abnormalities increased over time and with increasing concentration. DBP produced changes in eye pigmentation, spinal deformities, enlarged gall bladders and cardiovascular abnormalities including tube heart and blood clot formation and delayed or prevented hatching. DBP has been reported to have a mean bioconcentration factor of 167 mg/g. Concentrations of DBP in secondary sewage effluent of 450 ppm have been reported. Taken together these findings suggest that PPAs, when present at concentrations found in secondary sewage effluent, may be an important determinant in declining wild fish populations. Supported by ES07929.

11:15 INVESTIGATION OF COPPER TOXICITY ON ZEBRAFISH

Carloas Wilson (1)(*), Mudlagiri B. Goli (1), Joseph M. Wahome (1), Mike Mattie (2), and Jonathan Freedman (2), (1) Mississippi Valley State University, Itta Bena, MS 38941, and (2) Duke University, Durham, NC 27708

A study was carried out to observe the effect of copper sulfate ([CuSO.sub.4]) on the fast growing embryos of Zebrafish. Embryos were collected approximately three hours after fertilization and were examined for 15 days. The embryos of non-exposed, control embryos started hatching on the third day (13.6% hatched) and eventually 100% hatched by the fifth day. A delay in hatching was observed in embryos that were incubated in copper sulfate. Concentrations of 250 mg [CuSO.sub.4]/L or higher were toxic for the embryos. None of the embryos hatched when the concentration of the metal reached 250 mg/L and higher. A detailed study of the toxic effect of the metal on hatching characteristics will be presented in the meeting.

11:30 BACTERIA ISOLATED FROM SOIL CONTAMINATED WITH POLYCYCLIC AROMATIC HYDROCARBONS ARE RESISTANT TO THE HIGHER MOLECULAR WEIGHT REPRESENTATIVES

Stephen I.N. Ekunwe, Rochelle D. Hunter (*), Huey-Min Hwang, and Lynette Ekunwe, Jackson State University, Jackson, MS 39217

Polycyclic Aromatic Hydrocarbons (PAHs), environmental contaminants made up of fused benzene rings, are byproducts of incomplete combustion of organic matter. Other sources of PAHs include motor vehicle emissions, cigarette smoke and petroleum production and processing. Because many PAHs are toxic and mutagenic, their cleanup is necessary. Low molecular weight PAHs, e.g., naphthalene, phenanthrene, are readily degraded by many soil bacteria, but higher molecular weight PAHs, e.g., pyrenes, are not. Standard bacteria isolation protocol was followed. Partial characterization of the 1 liter pure cultures obtained was done utilizing their Gram staining properties, morphology, and fluorescence. Growth of isolates in the presence or absence of 1-aminopyrene (I-AP) and 1-hydroxypyrene (1-HP) has been evaluated. Results indicate 7 Gram negative and 4 Gram positive rod shaped isolates. Isolates tested so far are resistant to I-AP and 1-HP at the concentration of 10 [micro]g/ml. This may suggest potential degradation a bilities of the isolates. According to literature, PAH degradation involves an upper and a lower pathway. In the upper pathway, hydroxylation of an aromatic ring to a cis-dihydrodiol is catalyzed by dioxygenase, a multicomponent enzyme consisting of reductase, ferrodoxin, and large and small iron-sulfur proteins (ISP). ISP sequence presence may be used to predict PAH degradation capability. Five isolates were found to contain plasmids (12.2 kb). PCR is being used to probe DNA isolated from these bacteria for presence of ISP sequence. Acknowledgements: This work was supported by a grant from the Army Research Office (Grant No. DAAD 19-01-1-0733), awarded to Jackson State University.

THURSDAY AFTERNOON

Exhibition Hall B

Session III--Come Out, Come Out Wherever You Are. Searching for Genes and Proteins

1:30 Introduction

1:45 RECOMBINANT EXPRESSION OF SULFITE REDUCTASE FROM ARABIDOPSIS THALIANA

Steve Adamson (*), Cecilia Chi-Ham, Sabine Heinhorst, and Gordon C. Cannon, University of Southern Mississippi, Hattiesburg, MS 39406

Plastids are organelles that serve a variety of functions depending on the cell type in which they reside. Replication and expression of their DNA are believed to be involved in coordinating the maturation of immature proplastids into the green chloroplasts of photosynthetic tissue. During this development, dynamic structural changes of the organellar nucleoid have been observed that likely accompany differences in function of the protein-DNA complexes. A major constituent of plastid nucleoids is sulfite reductase (SiR), a siroheme enzyme that also participates in the sulfur assimilation pathway. The protein compacts DNA and inhibits DNA synthesis in vitro. To further explore cellular strategies that coordinate which one(s) of the two roles SiR plays in response to internal and external cues, antibodies are needed to locate and quantitate the protein. To obtain large amounts of protein, recombinant SiR from Arabidopsis thaliana was expressed in E. coli as a fusion protein with an intein tag that contains a ch itin-binding domain. After purification of the fusion protein on a chitin column, the intein is removed under reducing conditions and recombinant SiR eluted. The chicken antisera against recombinant A. thaliana SiR protein are currently being characterized with respect to specificity and cross-reactivity towards nucleoid proteins from Glycine max.

2:00

CLONING OF TWO GENES OF UNKNOWN FUNCTION FROM THE CARBOXYSOME OPERON OF HALOTHIOBACILLUS NEAPOLITANUS AND DEVELOPMENT OF AN EXPRESSION CONSTRUCT

Devon Ingram (1) *, Kristi Budzinski (2), Gordon C. Cannon (2), and Sabine Heinhorst (2), (1)Cedar Crest College, Allentown, PA 18104, and (2) University of Southern Mississippi, Hattiesburg, MS 39406

Carboxysomes are the protein bodies of many chemotropic and phototrophic bacteria that enhance the catalytic properties of the resident enzyme, riboluse-1,5 bisphosphate carboxylase/oxygenase, by an as yet ill-defined mechanism. While most of the carboxysomal proteins can easily be purified and have been assigned a structural function, the protein products of two open reading frames (orfA and orfB) in the carboxysome gene cluster of Halothiobacillus neapolitanus have not been identified. To be able to determine the role these rather small proteins of apparently low abundance play in carboxysome structure, function and assembly, they have to be overexpressed in a suitable host. To that end, the coding sequences of orfA and orfB were amplified by the polymerase chain reaction (PCR) using primers that were designed to contain appropriate restriction sites for the pTYB1 vector. After restriction digestion, the amplified fragments were inserted to yield a C-terminal in-frame translational fusion with an intein tag that features a chitin-binding domain. The ligation products were introduced into E. coli DH5[alpha] cells and the presence of the insert was verified by restriction digestion and PCR amplification.

2:15

ANALYSIS OF KNOCKOUT LINES FOR INSERTION MUTANTS IN THE SULFITE REDUCTASE GENE OF APABIDOPSIS THALIANA

Sudha Sankaran (*), Sabine Heinhorst, and Gordon C. Cannon, University of Southern Mississippi, Hattiesburg, MS 39406

Chloroplasts are semi-autonomous organelles that have their own genome. The organellar DNA is complexed with DNA binding proteins into structures termed nucleoids. Composition and morphology of these nucleoids are vital to the different roles these complexes play in the replication and transcription of the chloroplast genome. One nucleoid associated protein is DCP68, which was identified from Glycine max suspension cells was identified as ferredoxin:sulfite reductase. To further delineate the roles this bifunctional protein plays in the chloroplast, T-DNA insertion mutants of Arabidopsis thaliana were screened for an insertion in the sulfite reductase gene. Two candidate lines were identified and are currently being characterized. In parallel, post-transcriptional silencing of the sulfite reductase gene is being attempted using an RNA interference (RNAi) construct that is designed to trigger specific degradation of the homologous RNA in the transformants and to allow one to make transgenic plants with a funct ional knockout of the gene in question. It is hoped that the RNAi approach will reveal important information about the biological effects of a sulfite reductase knockout and of the mechanisms used by the plant to coordinate the two functions of sulfite reductase protein in the chloroplast.

2:30

Break

2:45

DEVELOPING A HUMAN LIBRARY SCREEN FOR HUNTINGTIN (HTTQ 103) TOXICITY ANTAGONIZERS IN YEAST

Heather Vanpelt (1) *, Kavita Bapat (2), and Yuri O. Chernoff (2), (1) Mississippi University for Women, Columbus, MS 39701, and (2) Georgia Institute of Technology, Atlanta, GA 30332

HttQ 103 is a mutant form of the human huntingtin protein which contains an abnormally-long stretch of 103 glutamines. When HttQ 103 is transformed into yeast, the glutamine repeats cause a misfolding and aggregation of the protein, resulting in toxic prion formation in the yeast similar to that found in human Huntington's disease. Due to the evolutionary conservation of mechanisms, we believe that there may be compounds similar to those which have been found in yeast (e.g., Hsp 104) relieving huntingtin toxicity in human cells. We hope to find human proteins, which when transformed into HttQ 103-expressing yeast, can antagonize the toxicity and allow the yeast cells to resume normal growth. If a cDNA can be found in the Human Brain Library which negates the toxicity, it could potentially be used as a gene therapy for patients with Huntington's disease or other prion-associated diseases. The HttQ103 gene along with cDNAs from the Human Brain Library were transformed into yeast and some of these colonies grew to normal size, appearing to not show the effects of huntingtin toxicity. Colonies with normal growth were checked for the spontaneous loss of prion-forming capability. Those that retained the capability had the cDNA-containing plasmid isolated, transformed into E. coli, re-isolated from E. coli, and transformed back into yeast with the Q103 plasmid to check for reproducibility of the huntingtin toxicity-negating effects.

3:00 EXPRESSION OF OPEN READING FRAMES A AND B (ORFA AND ORFB) FROM THE CARBOXYZOME OPERON OF HALOTHIOBACILLUS NEAPOLITANUS

Kristi Budzinski (1) *, Devon Ingram (2), Gordon C. Cannon, (1) and Sabine Heinhorst, (1) (1) University of Southern Mississippi, Hattiesburg, MS 39406, and (2) Cedar Crest College, Allentown, PA 18104

Carboxysomes are polyhedral protein nanocompartments found in many chemoautotrophs and in cyanobacteria. Approximately 60% of the carboxysomal protein consists of the [CO.sub.2]-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, indicating that the carboxysome serves a function in carbon metabolism. Genetic studies of the carboxysome operon in Halothiobacillus neapolitanus have led to the discovery of two open reading frames (orfA and orfB) that could encode small polypeptides. However, these gene products have not yet been identified in H neapolitanus and do not seem to be structural components of the carboxysome. To develop antibodies against the orfA and orfB) polypeptides and use the antisera to detect the proteins in vivo and in vitro, the orfA and orfB genes were cloned into the pTYB1 vector and heterogously-expressed in E. coli as an in-frame translational fusion with a self-cleavable intein with a chitin-binding domain. Transformants were screened for the presence of the orfA and orfB gene s by restriction digestion. Positive clones were grown in liquid culture under different conditions and expression of orfA and orfB was induced by exposure of the cells to IPTG. The fusion protein was purified by chromatography on a chitin affinity column.

3:15 METHYL PARATHION INTOXICATION ALTERS THE NEUROCHEMISTRY OF THE SEPTOHIPPOCAMPAL PATHWAY

Ben Quilter *, N.T. Averett, and J.B. Hutchins, University of Mississippi Medical Center, Jackson, MS 39216

Enzymes called cholinesterases remove acetylcholine from the cholinergic synapse. Methyl parathion (MP), which is converted by the liver into the active metabolite methyl paraoxon, can permanently inhibit these enzymes. MP is also used as a pesticide, and has been used illegally in private residences. Exposure of children to MP has been reported to cause problems with learning and memory. We think that the expression of acetylcgolinesterase messenger RNA will be increased in the septal nuclei of mice treated chronically with dermal MP compared to vehicle-treated controls. The expression of cholinergic receptor message, particularly for the m2 muscarinic receptor, should be decreased in the hippocampus of mice treated chronically with dermal MP as compared to vehicle-treated controls. Adult mice (C3H and B6C3F1 strains) were treated daily for seven days using a 10 mg/ml mixture of MP dissolved in ethyl alcohol. The controls are dosed with alcohol. The MP dose is calculated to give 10 mg/kg body weight. The mic e were then perfused with fixative for immunohistochemical analysis, or they were killed and their septum or hippocampus were removed and frozen for further analysis. We carried out gene array analysis to determine which genes were up- or down- regulated by MP in the septum and hippocampus. We found a number of transmitter-related and apoptosis-related genes that changed their expression levels after MP treatment. Support provided by the Howard Hughes Medical Institute 51000122 and Centers for Disease Control and Prevention R06/CCR419466.

3:15 Session IV--Poster Carnivale!

MATURATION OF DENDRITIC CELLS FOR CANCER IMMUNOTHERAPY

Antoinette L. Walker (1) *, James Kobie, (2) and Emmanuel Akporiaye (2), (1) Tougaloo College, Tougaloo, MS 39174, and (2) University of Arizona, Tucson, AZ 85721

Cancer is a group of diseases characterized by uncontrolled growth and spread of abnormal cells. Dendritic cells (DCs) are antigen-presenting cells that stimulate immune responses. This experiment was designed to develop a more efficient method to generate mature dendritic cells for cancer immunotherapy. Mature DCs express high levels of major histocompatability complex (MHC) class I and II molecules and co-stimulatory molecules (CD80, CD86, and CD40). Mature DCs are effective at presenting tumor antigens to T cells. The stimulated T cells develop into cytotoxic T cells, which encounter the tumor antigens and kill the tumor cells. Tumor Necrosis Factor Alpha (TNF-[alpha]) is a cytokine that contributes to the maturation of dendritic cells. Prostaglandin E2 (PGE2) is an immunomodulator that upregulates the expression of MHC molecules and co-stimulatory molecules on DCs. The murine DCs were treated with maturation factors TNF-[alpha], PGE2, or TNF-[alpha] + PGE2 and analyzed by flow cytometry. Dendritic cells t reated with the maturation factors had a higher expression of co-stimulatory molecules and MHC class II molecules than the DCs that remained untreated.

CORRELATION BETWEEN GROWTH PROCESSES AND GENETIC ENDPOINTS

Babu P. Patlolla (1) *, Anita K. Patlolla (2), and B.S. Sekhon (2), (1) Alcorn State University, Alcorn State, MS 39096, and (2) Jackson State University, Jackson, MS 39217

Halogenated aliphatic hydrocarbons have long been regarded as pharmacological and toxicological entities. Human exposure to complex mixtures of chlorinated compounds has been extensive and effects following long-term exposure to them can be investigated through toxicological laboratory studies. In the present study the effects of three chlorinated hydrocarbons (1,1-dichloroethane; 1,1,1-trichloroethane and 1,1,2,2-tetrachioroethane) on growth in plants are compared with the effects of the same chlorinated compounds on genetic endpoints in mice. Four different concentrations of each chemical were used in this study. Correlation coefficients (r-values) were calculated to establish the correlation and further confirmed by the Z-values. There was a strong negative correlation between the shoot length of Zea mays and chromosomal aberrations in mice bone marrow for 1,1-dichloroethane (r-value = -0.9400), for 1,1,1-trichloroethane (r-value = -0.9249) and for 1,1,2,2-tetrachloroethane (r-value = -0.8925). The results of this investigation indicated that observing their effects on shoot length, thus reducing the unwanted usage of animals, could monitor the harmful effects of chlorinated hydrocarbons.

A SIMPLE METHOD FOR HIGH RESOLUTION RANDOM AMPLIFIED POLYMORPHIC DNAs ASSAYS IN PLANT POPULATIONS

Janet Marie Gibson McDaniel* and Robert G. Hamilton, Mississippi College, Clinton, MS 39058

Using 43 cm x 0.45 mm 5% polyacrylamide gels and silver staining has been a high quality method for the detection and analysis of random amplified polymorphic DNAs (RAPDs). This method is, however, time consuming and tedious. We developed 8 cm x 1.0 mm 5% polyacrylamide gels as an equally high quality method for the analysis of RAPDs fragments. The 8 cm x 1 mm gels can be purchased pre-made, and do not have to be electrophoresed at high power levels. Electrophoresis at 4 mA for 11 hours produced very high quality data on the 8 cm x 1.0 mm gels for DNA fragments ranging in size from about 400 bp to about 1000 bp in length. The range in size of fragments that can be visualized on a single gel is the major limitation of the 8 cm gel over the 45 cm gel. Silver staining the 8 cm x 1.0 mm gels is much easier than silver staining the 43 cm x 0.45 cm gels, as there is no requirement to separate the plates such that the gel remains adhered to one plate. Measurement of fragment sizes can be completed manually using sem i log graph paper with just one logarithmic series, as all DNA fragment sizes are in the hundreds of base pairs. The use of an 8 cm x 1.0 mm polyacrylamide gel allows high quality visualization and analysis of RAPDs to be completed under a much greater diversity of laboratory conditions, as it requires far preparation and less technical expertise.

THE EXPRESSION OF THE TROL GENE'S THIRD DOMAIN, TROL III

Caronda Moore (1) *, Sumana Datta (2), and Derek Crunk (2), (1) Jackson State University, Jackson, MS 39217, and (2) Texas A&M University, College Station, TX 77843

The research project was a part of an ongoing project to express the five domains of the trol gene, a mutant gene in the fruit fly Drosphilia melano gaster. The project focused on the third domain in order to determine what role does the third domain of the trol gene play in the stimulation of the growth hormone FGF. Studies have shown that the FGF growth hormone is needed for stem cell division in early development. The procedure for expressing the third domain were to put together a DNA construct and inject it into the neuroblast of the fly. ANA cDNA, which is about 2.5 kb, was purified out of a pBS vector as the insert. pUAST, which is about 9kb, was purified as the cloning vector with flanking restriction sites. The ANA insert was ligated into the pUAST vector, and the ANA coding sequence was cut out leaving behind a signal peptide which will release the trol III (received from yellow white genomic DNA) from the cell therefore expressing it. The ANA and pUAST were ligated together, but it was noticed that the ANA insert was turned the wrong way (viewed through gel electrophoresis). This problem was approached with several different techniques, including trying to recut the transformed ANA/pUAST with restriction enzymes. The trol III sequence was successfully PCRed but not yet ligated and transformed with the ANA/pUAST. The construct has not been injected into the neuroblast.

GEOGRAPHIC DISTINCTNESS OF ESCHERICHIA COLIIN SEAGULLS BETWEEN GULFPORT, MS, AND DAUPHIN ISLAND, AL, AS DETERMINED BY BOXPCR DNA FINGERPRINTING

David Oliver *, Wail M. Hassan, Dawn Rebarchik, R.D. Ellender, and Shiao Y. Wang, University of Southern Mississippi, Hattiesburg, MS 39406

Fecal coliform bacteria is an environmental pollutant and high counts cause the closure of recreational waters and oyster harvest areas. Bacterial source tracking methods compare bacteria from environmental samples to isolates of known animal source to ascertain the likely animal origin of the environmental sample. Because the tracking method compares environmental isolates to those in a database, an important consideration is geographic distinctness of isolates. If fecal coliform bacteria from an animal species appear similar over a wide geographic area, information in the database can then be used to infer the animal origin of isolates from the same broad area. The present study compares the BOX-PCR DNA fingerprints of Escherichia coli isolates from seagulls in Gulfbort, Mississippi and Dauphin Island, Alabama to determine whether seagulls harbored distinct E. coli populations based on geographic location. A total of 32 seagull samples and 168 E. coli isolates were included in the study. The two most basal branches of the dendrogram using BOX-PCR DNA fingerprints showed distinctive clustering of isolates based on geographic location. In one, 31 of 37 isolates (84%) were from Gulfport seagulls. In the other, 47 of 55 isolates (85%) were from Dauphin Island seagulls. Our results suggest that geographic location should be considered when BOX-PCR is used in bacterial source tracking.

TARGETED DELIVERY OF THERMALLY SENSITIVE CYTOTOXIC POLYPEPTIDES

Gene L. Bidwell III, Melissa A. Skertich, and Drazen Raucher (*), University of Mississippi Medical Center, Jackson, MS 39216

To improve specificity and efficacy, while at the same time reduce toxicity caused by the current anticancer treatments, we developed a thermally targeted polypeptide-mediated therapeutic delivery vehicle. The design of this therapeutic delivery vehicle is based on elastin-like polypeptide (ELP), which is soluble in aqueous solution below physiological temperature (37[degrees]C), but aggregates when the temperature is raised above 41[degrees]C. Therefore, intravenously delivered ELP polymers are rapidly cleared under physiological conditions, but they accumulate at targeted diseased sites where local heat (41-43[degrees]C) is applied. We modified the coding sequence for ELP by addition of penetratin and a cytotoxic peptide, and expressed and purified from E. coli. Penetratin represents a membrane translocating sequence from helix 3 of Antennapedia, which mediates intracellular uptake of large ELP aggregates and increases its uptake by more than 100-fold in HeLa cells. The cytotoxic peptide is derived from he lix 1 of c-Myc. It has been shown that this peptide prevents c-Myc DNA binding and inhibits cell proliferation. The cytotoxicity, uptake, and localization of this ELP construct are examined by MTS assay, flow cytometry, and confocal fluorescence microscopy, respectively.

REGULATION OF THE MN/CA9 PROMOTER IN RENAL CELL CARCINOMA 

Shenekia A. Wells (*) and Thomas A. Gardner, Alcorn State University, Alcorn State, MS 39069, and Indiana University School of Medicine, Indianapolis, IN 46202

MN/CA9 is a tumor-associated antigen that was originally detected in human cervical carcinoma HeLa cells. It is a member of the carbonic anhydrase (CA) family and is speculated to be a potential marker and key target for gene therapy of the most common kidney cancer, Renal Cell Carcinoma (RCC). The MN/CA9 antigen is expressed in more than 90% of RCC and other cancer cells with malignant phenotypes in the cervix and ovaries of females. Despite all of the remarkable ways of using this antigen, the mechanism controlling the regulation in RCC has yet to be determined. This research explores the usage of the MN/CA9 promoter in gene therapy for RCC and mechanisms controlling its expression in RCC.

EFFECTS OF MONOTERPENES ON THE SERUM-INDUCED MORPHOLOGICAL CHANGE OF CANDIDA ALBICANS

Roger D. Holloway (*) and Paul McGeady, Alcom State University, Alcom State, MS 39069, and Clark Atlanta University, Atlanta, GA 30314

Perillyl alcohol and carveol were shown to inhibit the transformation of Candida albicans to a filamentous form, but not at a higher concentration than was previously shown for the corresponding aldehyde (perillyl aldehyde) and ketone (carvone). This morphological change is associated with C. albicans pathogenthcity; hence these naturally occurring monoterpenes are not suitable lead compounds in the development of therapeutic agents against C. albicans infections.

USING RAPID IDENTIFICATION TEST TO CATALOG THE DIFFERENT STRAINS OF E. COLI

Valarie Acoff (*), Al Mikell, and Trent Fivecoat, University of Mississippi, University, MS 38677

Recently, pathogenic strains of Escherichia coli have emerged. They are common in feces of warm-blooded terrestrial animals. E. coli is known as an indicator bacteria and strain for fecal pollution. It is an enterotoxigenic organism that is spread by fecal contamination of animal or human origin to food and water. This research was based on a series of procedures where seven samples were collected from seven domesticated and wildlife animals. Growth and isolation of the different bacteria were conducted on selective media to the third generation where suspected E. coli development was detected. By using commercial kit system identification, different strains of E. coli and other bacteria were identified and characterized. In addition to identification, a B-antigen specific test for O157:H7 enterohaemorrhagic E. coli strain was conducted on all E. coli samples. This test is called the Pro-Lab E. coli O157 Latex Test Reagent Kit. It is an agglutination test kit specifically for identifying the serogroup O157 a ntigen on selective media.

Once this experiment was conducted, the results identified specific virulence factors, bacteria, and strains. Overall, this research identified and cataloged no pathogenic strains of E. coli.

HUNTINGTONS AGGREGATES VISUALIZED IN LIVING COLORS

Akita Evans * and Lois Greene, Tougaloo College, Tougaloo, MS 39174, and National Institute of Health, National Heart, Lung, and Blood Institute, Bethesda, MD 20892

Huntington's Disease (HD) is a fatal neurodegenerative disease with clinical manifestations including progressive dementia, psychiatric symptoms, and movement disorder. It is characterized by an unusually long CAG expansion on the huntingtin gene giving rise to a very long polyglutamine repeat at its amino end. The affect of HD aggregates in cellular pathogenesis is not known. Through the use of spectro-proteins with different polymerization properties--Green Fluorescent Protein (GFP), a monomer, and DsRed, a tetramer--the effect of huntingtin aggregation with the two spectral variants was determined. DsRed huntingtin formed many aggregates throughout the cytosol of cells, whereas GFP huntingtin formed aggregates in the nucleus and usually one large one at the MTOC. In addition, DsRed promoted the aggregation of GFP huntingtin. With these findings, future studies can be done relating the lethality of the huntingtin to aggregation.

ALTERATION OF PROTEIN EXPRESSION IN MURINE IMMUNE CELLS DUE TO ESTRADIOL AND BISPHENOL A EXPOSURE

Derese Getnet, Ella Lazo, and Rebecca Roberts *, Ursinus College, Collegeville, PA 19426

The discovery of estrogen receptors (ER) on lymphocytes and antigen presenting cells (APCs) has sparked a growing interest in the role of estrogen (E2) in cell-mediated immunity. Antigen presentation in uterine and vaginal cells of rats has been demonstrated to be under the regulation of estrogen. The mechanisms of action by which estrogen and estrogenic endocrine-disrupting chemicals (EEDCs) affect cellular function is not fully understood. Bisphenol-A (BPA), an EEDC, is used as a monomer in polycarbonate plastic production. Here, we report the effect of E2 and BPA on the protein expression of splenocytes and dendritic cells. Splenocytes from three strains of mice and a murine dendritic cell line were exposed to BPA and E2 in culture. Protein expression in treated samples was altered compared to controls. However, expression of the endosomal cysteine protease cathepsin L did not appear to be affected.

FRIDAY MORNING

Exhibition Hall B

Session V--Special Joint Session with Mississippi Functional Genomics Network

8:30 BRIN Meeting and Presentations

See program on page 25.

FRIDAY AFTERNOON

Exhibition Hall B

Session VI--It's an RNA World, We Just Live in It. Ribozymes

1:00 Introduction

1:15 PROBING RNA CONFORMATION CHANGES BY FLUORESCENCE SPECTROSCOPY

Peter Butko * and Michelle Magee, University of Southern Mississippi, Hattiesburg, MS 39406, and Centenary College, Shreveport, LA 71134

Flavine adenine dinucleotide (FAD) exhibits fluorescence due to its isoalloxazine conjugated ring structure. In principle, this fluorescence can be used as a tool to detect conformation changes in FAD-coupled macromolecules. We tested this with the recently synthesized RNA ribozymes, able to catalyze the synthesis of flavine adenine dinucleotide (FAD) from flavine mononucleotide (FMN). It is known from kinetic data that these ribozymes require the presence of divalent cations [Mn.sup.++] and possibly [Ca.sup.++]. We used fluorescence spectroscopy of FADRNA to study the possible effect of the cations on the structure of the ribozyme. None of the tested cations influenced fluorescence of free FAD, but [Mn.sup.++] caused a significant increase in fluorescence of FAD-RNA, which is ascribed to a Mn-induced conformation change in RNA. We conclude that FAD fluorescence can be used to monitor conformation changes in FAD-ribozymes. The current results will be confirmed and extended by time-resolved fluorescence spectr oscopy.

1:30 RJBOZYME KINETICS

Jason Manning *, Tricia M. Coleman, and Faqing Huang, University of Southern Mississippi, Hattiesburg, MS 39406

The 'RNA world' hypothesis posits that early life forms employed RNA as both an informational and catalytic molecule. In this preprotein world, RNA served the role of both DNA (informational) and proteins (catalytic). A technique known as in vitro selection has been used in the isolation of numerous artificial catalytic RNA molecules in various research laboratories. Although catalytic RINA (ribozymes) cannot enhance catalytic rates as efficiently as protein enzymes, they can increase the rate of reactions significantly. Kinetic characterization of ribozymes can give valuable information about reaction mechanisms and a standard for the "fitness" of a ribozyme. A series of thioester synthase ribozymes have previously been isolated in our laboratory from size heterogeneous pools containing random regions of 30, 60, 100, and 140 nucleotides. Ribozymes isolated from the 100 N size group (N = number of nucleotides) were characterized kinetically by standard Michaelis-Menten kinetics. Each active thioester synthase ribozyme sequence was allowed to react with different concentrations of substrate Biotin-AMP for varying times to yield apparent first order rates. These rates were then plotted versus substrate concentrations to give kinetic parameters of [K.sub.cat] and [K.sub.cat]. The kinetic parameters for each sequence from this size group were compared to each other to determine the most efficient ribozymes. Reaction rates for individual ribozymes from the 100 N group could then be compared to ribozymes from the other three size groups in order to form conclusions about the size versus activity relationship for thioester synthesis by RNA.

1:45 IN VITRO SELECTION OF A MINIMUM RNA LIGASE 

Guocan Wang * and Faqing Huang, University of Southern Mississippi, Hattiesburg, MS 39406

The "RNA world" hypothesis proposes that RNA performed both genomic and catalytic functions in the very early history of life on earth. In such an RNA world, RNA should have performed similar biochemical reactions as protein enzymes do in modem biology. Relatively large RNA can form complex structures and can potentially perform sophisticated catalytic functions. However, synthesis of large sizes of RNA was thought to be very difficult by RNA alone, based on known efficiency and accuracy of RNA catalysis. Therefore, there exists a fundamental problem of how the first large RNA arose. Large RNA can be made by two different mechanisms: (1) stepwise addition of nucleotides to an existing RNA chain or (2) ligation of two or more short RNA pieces. The first mechanism is used by current biology to make RNA. However, we believe that the second mechanism may be advantageous to synthesize large RNA by primitive catalytic RNA, and ribozymes with RNA ligation activity might have been an essential catalytic activity in t he RNA world. We are using SELEX to isolate minimum RNA ligase sequences. Unlike conventional SELEX, an 18-nucleotide complete random RNA library without constant flanking primer sequences is being used in order to remove the effect of primer regions. The selection process involves a combination of RNA/DNA ligation, dephosphorylation, phosphorylation, and restriction enzyme digestion, in addition to reverse transcription, PCR, and in vitro transcription. Isolated ribozymes are expected to catalyze the formation of RNA dimer, trimer, etc. Our results will define the minimum sequence requirement for an RNA ligase, which can be used to evaluate the complexity of an early RNA world.

2:00 RNA-CATALYZED PEPTIDE FORMATION FROM ACYL CoA

Na Li * and Faqing Huang, University of Southern Mississippi, Hattiesburg, MS 39406

Many toxins and antibiotics, such as [alpha]-amanitin, cyclosporin, vancomycin, contain both D- and L-forms of amino acids connected by peptide bonds. These peptide bonds are made not by ribosomes, but rather by large protein complexes called nonribosomal polypeptide synthetases (NRPS). The NRPS, by modular design, define the sequence of antibiotics. In the process of antibiotic peptide synthesis, amino acids are first activated and attached to pantetheinyl groups as thioesters, followed by peptide bond formation. Having succeeded in isolating ribozymes making CoA and its thioesters, we are isolating new ribozymes by SELEX from random RNA libraries to catalyze the peptide bond formation from acyl CoA. The experiment was designed according to the mechanism of NRPS. The results will provide evidence to support our hypothesis of RNA-based metabolic pathways (metabolic ribozymes). Integration of our in vitro-generated various ribozyme activities may lead to novel antibiotic synthesis through rational modular ribo zyme design. It is conceivable that modular multi-component RNA systems can be engineered (based on Watson-Crick base pairing) to produce different products, once each ribozyme module's function is defined. Due to drug resistance of bacteria, current available antibiotics are becoming less effective. Production of new antibiotics has become a serious challenge. An RNA-based system for antibiotics synthesis may provide a new solution.

2:15 Break

Session VII--And If All Else Fails, Give It a Really Hard Whack! Developing Molecular Tools

2:30 Introduction

2:45 AN INVESTIGATION INTO THE SIZE-ACTIVITY RELATIONSHIP OF RNA CATALYSIS

Tricia M. Coleman *, Jason Manning, and Faqing Huang, University of Southern Mississippi, Hattiesburg, MS 39406

A technique known as in vitro selection (SELEX) makes it possible to isolate individual RNA sequences with novel biochemical properties from large pools ([10.sup.13] - [10.sup.15] different sequences) of random RNA molecules. It has been argued that longer sequences are more desirable for the selection process because RNA can then form into more complex secondary and tertiary structures, which would in turn allow for more complex and rare activities to be isolated. The known replication inefficiency of longer sequences, however, puts longer sequences at a disadvantage in a competitive situation. This effect would have been especially significant in the harsh conditions of the RNA world. To determine whether shorter sequences are advantageous in selection experiments, we have used size heterogeneous RNA pools to isolate a series of thioester synthase ribozymes. RNA molecules with random regions of 30, 60, 100, and 140 nucleotides were allowed to compete under identical conditions during selection. The selectio n therefore mimics natural selection in that RNA molecules compete for representation, and only those with the correct balance between replication efficiency and catalytic efficiency survive. At the end of the selection the relative size distribution was 30N > 60N [much greater than] 100N, 140N. We have also conducted a comprehensive kinetic analysis on the sequences isolated from each of the four size groups. These results indicate that there is an optimal size for catalytic activity centered around 60 nucleotides.

3:00 A STUDY OF GLOMERULAR DEVELOPMENT IN THE OLFACTORY BULB OF ZEBRAFISH, DANIO RERIO

Antony Schwartz * and Christine A. Byrd, Jackson State University, Jackson, MS 39217, and Western Michigan University, Kalamazoo, MI 49008

Glomeruli are anatomical and possibly functional modules in the vertebrate olfactory bulb. These spherical neuropilar structures are a prominent and ubiquitous feature of both invertebrate and vertebrate olfactory bulbs and represent the first relay stations in the olfactory pathway. A typical adult zebrafish olfactory bulb contains a small number of about 80 glomeruli that have a stereotyped configuration. In this study our aim was to identify markers that specifically label glomeruli in zebrafish embryos and then quantify glomeruli at different stages of development. DiI labeling and whole-mount immunocytochemistry procedures were utilized to study glomerular labeling under a confocal laser microscope. Alpha-tubulin and anti-keyhole limpet hemocyanin (Alpha-KLH) antibodies were used in the immunocytochemistry procedure. We found that DiI, alpha-tubulin and alpha-KLH labeled the olfactory receptor neurons, but only alpha-KLH labeled the axons leading away from the olfactory receptor and going toward the olfa ctory bulb. This indicates that alpha-KLH has potential, with adequate testing, as a labeling tool to study glomerular development in zebrafish embryos. This study was made possible by REU grant from NSF.

3:15 THE DEVELOPMENT OF A REP-PCR DNA FINGERPRINT DATABASE FOR BACTERIAL SOURCE TRACKING IN MISSISSIPPI

Wail M. Hassan *, Mary Phares, David Oliver, Brian Robinson, Dawn Rebarchik, R.D. Ellender, and Shiao Y. Wang, University of Southern Mississippi, Hattiesburg, MS 39406

The goal of the present project is to develop a bacterial DNA fingerprint database against which unknown environmental samples can be compared for identifying sources of fecal pollution in Mississippi. A total of 387 fecal samples were collected from human, cow, chicken, deer, dog, horse, and seagull (57, 73, 38, 39, 9, and 59 samples, respectively). Over a thousand isolates each of Escherichia coli (EC) and Enterococcus spp. (EN) were isolated and analyzed by a PCR-based fingerprinting method that amplifies repetitive sequences in the bacterial genome. Two different sets of primers (REP and BOX) were tested and the usefulness of EC as an indicator of fecal pollution was compared to that of EN. To compare the reliability between the two primer sets and the two indicator bacteria, Jackknife analysis was used to calculate the rate of correct assignment (RCA). The RCA values for REP-PCR using EC and EN were 64% and 62%, respectively. The RCA values for BOX-PCR using EC and EN were 62% and 84%, respectively. We c onclude that BOX-PCR using EN is the method of highest fidelity. Currently, the reliability of the DNA fingerprint database for bacterial source tracking in south Mississippi is being evaluated using a blind test.

3:30 THE DEVELOPMENT OF A REAL-TIME RT-PCR METHOD FOR THE QUANTIFICATION OF TAURA SYNDROME VIRUS IN SHRIMP

Zhiming Cao *, Anne Marie Flowers, Verlee Breland, Jeffrey M. Lotz, and Shiao Y. Wang, University of Southern Mississippi, Hattiesburg, MS 39406

Taura syndrome virus (TSV) is an enveloped single-stranded RNA virus in the family Picornaviridae. It is one of the most detrimental shrimp viral pathogens in the western hemisphere, causing high mortalities at shrimp aquaculture farms. To better understand progression of the TSV disease process, a sensitive procedure to quantify TSV in shrimp is urgently needed. A reproducible real-time RT-PCR assay to quantify TSV in shrimp hemolymph was developed in the present study. The assay relies on the use of Molecular Beacon technology and a Cepheid Smartcycler real-time detection system. The assay is sensitive to 2 x [10.sup.4] RNA copies per reaction with a dynamic range between [10.sup.4] and [10.sup.9] RNA copies. The coefficient of variation of threshold cycle (Ct) values in intra- and inter-runs were less than 2.0 1% and 2.61%, respectively. In a study of changes in viral titer over time, we observed large variations among shrimp. The highest viral titers occurred 4 days after infection with titers ranging from 7.7 x [10.sup.5] to 2.2 x [10.sup.9] copies/[micro]1 hemolymph (n = 10). On day 61 of the study when the last surviving shrimps were assayed, four of the six shrimp were still TSV-positive and the highest titer was 1 .1 x [10.sup.7], indicating that persistent infections take place with TSV.

Session VIII--Awards and Elections