CELLULAR, MOLECULAR AND DEVELOPMENTAL BIOLOGY.

Chair: Peter Butko, University of Southern Mississippi

Vicechair: Roy J. Duhe, University of Mississippi Medical Center 

THURSDAY MORNING

Room 3

8:45 Introduction--Peter Butko

9:00 IDENTIFICATION OF CELLULAR PHOSPHATIDYLCHOLINE SPECIES AND PRODUCTION AND IDENTIFICATION OF CELLULAR PHOSPHATIDYL ALCOHOLS USING ELECTROSPRAY MASS SPECTROMETRY

Patrick B. [Kyle.sup.*] and Rodney Baker, University of Mississippi Medical Center, Jackson, MS 39216

Phosphatidylcholine is an important membrane phospholipid that plays an integral role as a second messenger in intercellular signaling cascades of most cell types. Phosphatidylcholine is hydrolyzed by phospholipase D to produce phosphatidic a ubiquitous intercellular messenger. Ethanol and other alcohols are utilized by phospholipase D as co-substrates in a transphosphatidylation reaction to produce phosphatidyl(alcohol). The inclusion of phosphatidyl alcohols into the cell alters the normal phospholipid composition and consequently alters phospholipid dependent cell signaling processes. Phospholipase D was used to produce several phosphatidyl alcohols, which were identified using a tandem mass spectrometer (PE SCIEX API 365, triple quadrupole). We used the same instrument to identify the normal phosphatidylcholine species in a macrophage (RAW 264.7) cell line. This cell line was treated with ethanol, ethylene glycol, and two ethylene glycol ethers to produce phosphatidyl alcohols that compared to the normal phosphatidyl choline species.

9:15 IN VITRO EVOLUTION OF NEW RIBOZYMES WITH THIOESTER SYNTHASE ACTIVITY

Lijun [Zhang.sup.*] and Faqing Huang, University of Southern Mississippi, Hattiesburg, MS 39406

The "RNA world" hypothesis, which assumes that the chemical processes leading to the appearance of life were carried out by RNA molecules, has stimulated interests in catalytic reactions involving oligonucleotides such as catalytic RNA (ribozyme). Thioesters are important intermediates in the metabolism of modern organisms, and might have played important roles in the origin and early evolution of life. If there had been an RNA world before our RNP (ribonucleoprotein) world, thioester biosynthesis would have been carried out by RNA catalysts in the RNA world. Demonstration of RNA-catalyzed thioester synthesis might provide direct experimental evidence that thioesters could have been synthesized and played important functional roles in the RNA world and possibly in a pre-RNA world. Here an iterative in vitro selection procedure is being performed to isolate a new class of catalytic RNAs with thioester synthase activity from a large pool of 60N random-sequence coenzyme A-linked RNA molecules. Biotinyl adenylat e was synthesized chemically and used as the reactant for the thioesterification. The new ribozymes will catalyze the covalent C-S bond formation between a sufhydryl group and a carboxyl group.

9:30 ISOLATION OF RNA WITH NOVEL CATALYTIC ACTIVITY BY IN VITRO SELECTION

Tricia M. [Coleman.sup.*] and Faqing Huang, University of Southern Mississippi, Hattiesburg, MS 39406

The 'RNA world' hypothesis claims that ancient life forms employed a variety of different catalytic RNA in their biosynthetic machinery. In the quest for novel catalytic activity, we are investigating the use of polyphosphate as a potential energy source for these life forms. Employing an in vitro selection method, RNA capable of catalyzing its own phosphorylation from polyphosphate is explored using a library with a random region of 60N in length. Following nine rounds of selection, UV shadowing revealed an unexpected band in the gel corresponding to an apparently much larger RNA. This band is found to make up 38% of the total RNA upon selection procedure modification and additional selection cycles. The structure and function of this RNA is being examined.

9:45 EXPRESSION OF FLUORESCENT PROTEINFUSION OF RAT JANUS KINASE 2 AND HUMAN THIOREDOXIN IN COS-7 CELLS

Sheeyong [Lee.sup.*], John K. Smith, J.S. Vig Parminder, and Roy J. Duhe, University of Mississippi Medical Center, Jackson, MS 39216

The Janus protein-tyrosine kinases (JAKs) are important in cytokine signaling. However, because they are widely expressed and have low apparent specific activities, it is difficult to confirm expression of recombinant JAKs by conventional assays. The enhanced green fluorescent protein (GFP)-fusion proteins is a well-developed tool for visualizing the production of recombinant proteins. We constructed a chimeric pEGFP:rat JAK2 (rJAK2) vector in two step steps. First, we generated a cDNA fragment that encodes the amino terminal domain of rJAK2 and subcloned it into pEGFP-C1 vector. Second, we subcloned the cDNA fragment encoding the carboxy terminal domain into the above vector. pEGFP:hJAK3 vector was generated in same strategy. We built red fluorescent protein (RFP):human thioredoxin (hTRX)expression vector for co-transfection to investigate whether the reductive enhancement of JAKs can account for the mitogenic "helper effect" of thioredoxin. We are generating site-directed mutant vectors for GFP-fusion JAKs and REP-fusion TRXs. These inactive proteins can be used as negative control. Transfection was performed with effectine transfection reagents and cells were sorted via fluorescent activated cell sorter. This approach should provide major technical advantages including the easy isolation of transfected cells from non-transfected cells. Furthermore, the expression of mutagenized JAKs can help evaluate physiological function of modified JAKs in signal transduction.

10:00 IN VITRO TOOLS TO STUDY REGULATION OF JANUS KINASE

Kiranam [Chatti.sup.*] and Roy J. Duhe, University of Mississippi Medical Center, Jackson MS 39216

Janus Kinases (JAKs) are intracellular receptor associated tyrosine kinases, essential for cytokine signaling. We constructed three short forms of rat JAK2 with N-terminal GST tags, and successfully used the baculovirus expression system to overproduce them in insect cells. These short forms GST:(NdeltaJH2)rJAK2, GST:(Ndelta66l) rJAK2 and GST:(NdeltaJH1)rJAK2, along with full length (GST:rJAK2) and kinase inactive (GST:(Cdelta795) rJAK2) forms are being used to elucidate the molecular mechanisms regulating JAK2 catalytic activity. One study involves the "relief of autoinhibition" hypothesis, based on earlier observations that short forms lacking N-terminal domains appear to be "hyperactive." By sequential immunodepletion, we separated the tyrosine-phosphorylated (PY) pool of purified full-length GST:rJAK2 from the non-PY pool, and compared their kinase activities. Our results indicate that non-PY GST:rJAK2 has negligible kinase activity. We are attempting to determine whether removal of the "autoinhibitory d omain" via partial proteolysis is sufficient to activate the non-PY GST:rJAK2. Complete purification of the three new GST-tagged forms is under way, and will allow rigorous comparison of their tyrosine phosphorylation activities to that of the full-length form, and further study of the apparent JAK hyperactivation phenomenon. Another study concerns sensitivity of full-length JAK2 to redox agents. We found that activity of two short forms is sensitive to ortho-iodosobenzoate(o-IBZ), which is reversible with dithiothreitol (DTT). Assuming that cysteine residues are responsible for such sensitivity, they can be identified using the short forms by site-directed mutagenesis.

10:30 Divisional Poster Session

PATERNITY TESTING AND THE ASSESSMENT OF STICKLEBACK FISH MATING SYSTEM

Fameeka [Jenkins.sup.*] and Guillermo Orti, Alcorn State University, Alcorn State, MS 39096 and University of Nebraska, Lincoln, NE

This study will characterize the mating system of Gasterosteus aculeatus, a territorial fish with parental care. During the mating season, territorial male stickleback compete for females to lay eggs in their nest. But some males adopt an alternative strategy ("sneaker" males) and steal fertilizations from another male's nest and allow that male to raise the offspring. In order to assess the success of these alternative strategies, a collection of 50 nests from a freshwater lake, containing all the fertilized eggs and their guarding males will be tested for paternity. Success will be measured as the proportion of the offspring in a nest inferred to be actually fertilized by the guarding male. Paternity tests for 20 eggs per nest based on microsatellite genotyping will determine the proportion of illegitimate offspring per nest. Five or more of the 50 nests were assayed over an eight week period. The preliminary results show that the percentage of illegitimate offspring range from 0% to 56%. This value will b e correlated to variation of body form and external features of the guarding males.

INHIBITION OF ESTROGEN INDUCED PROLIFERATION OF BREAST CANCER CELLS BY INDIRECT- ACTING ANTIESTROGENS

Beverly N. [Cruthirds.sup.*] [1], Emely Casto-Rivera [2], and Stephen Safe [2], (1.) Jackson State University, Jackson, MS 39217 and (2.) Texas A&M University, College Station, TX 77801

Breast cancer cells in culture have been used as a model to understand the effect of 17B-estradiol (E2 or estrogen) on cell growth. In addition, we have also investigated the inhibitory interaction of several compounds. They developed as indirect acting antiestrogens. These compounds which include 9-cis-retinoic acid (9CRA), AlI-transretinoic acid (ATRA), Indole-3-carbinol (13C) and diindolylmethane (DIM) analogues do not bind to the estrogen receptor (ER), but block E2 action through cross talk between signaling pathways. Results of initial studies showed that 10 nM E2 induced a 4 to 90 fold increase in proliferation in MCF7 and ZR75 human breast cancer cells. In contrast, the 9CRA, ATRA, 13C and DIM analogues alone exhibited minimal estrogen-like activity in the cell lines. In cell lines co-treated with 10 nM E2 plus different concentration (10 nM to 10 [micro]M), inhibition ([sim]90% to 30%) of cell proliferation was observed for all compounds. Current studies on the mechanism associated with this inhibit ory cross talk are currently being investigated in these cell lines.

INHIBITION OF PROLIFERATION BY VINCA ALKALOIDS IN P19 AND MCF-7 CELL LINES

Erin A. [Propst.sup.*], LaSharon D. Mosley, Barbara P. Rogers, and Sharon Lobert, University of Mississippi Medical Center, Jackson, MS 39216

Previous in vitro drug receptor experiments demonstrate differential activity for five vinca alkaloids. The purpose of this research was to determine IC50 values in two cell lines for five vinca alkaloids. The experiments were conducted by plating the cell lines in 96-well and 24-well plates, and exposing the cells to a range of drug concentrations from 30 nM to 1 nM. The P19 cells were exposed to each drug for 24 hours. The MCF-7 cells were exposed to each drug for 48 hours. The proliferation assay used was a standard NADH assay. The drug titration data were fit by exponential decay to obtain the IC50 values. Activity against both cell lines was found to be similar and will be presented.

QUANTITATION OF PROSTATE APOPTOSIS RESPONSE-4 (Par-4) IN RAT COLONIC TISSUE

Lauretta A. [Ansah.sup.*] [1], Laurie Davidson [2], and Robert S. Chapkin [2], (1.) Jackson State University, Jackson, MS 39217 and (2.) Texas A&M University, College Station, TX 77843

Colon cancer is the third leading cancer diagnosed in both men and women in the United States. Scientists are currently investigating why this cancer is so prevalent in our society. Inhibition of apoptosis is now thought to be an integral component of the genesis of colorectal adenomas and carcinomas. Apoptosis is a programmed cell death that is needed to destroy cells that are a threat to the integrity of the organism. It has also been suggested that reduced apoptotic ability may predispose individuals to an increased risk for cancer, implying that apoptosis rates may be useful in identifying "at risk" subjects. Par-4 is a pro-apoptotic protein that plays a pivotal role in the down-regulation of cancer cell formation with respect to the regulation of apoptosis. However, Par-4 localization and expression within the colon has not been determined, therefore, we quantified patterns of Par-4 expression during colonic tnmorigenesis. Sprague-Dawley rats were provided with one of two diets (fish oil or corn oi l) and animals were injected with the carcinogen, azoxymethane, and the distal colon was removed at 0 and 9 hr later. Immunohistochemical analysis for Par-4 was performed using paraffin-embedded colonic sections. Fifteen crypts (divided into tertiles) from each animal were taken and quantified using fluorescence microscopy. The results showed an increase of Par-4 protein in the colonic sections from carcinogen-treated animals (P [less than]0.05). In addition, carcinogen-treated colon sections had the highest levels of apoptosis in the bottom one-third of the crypt compared with the non-carcinogen treated colon sections, which had higher levels of expression in the top one third of the crypt (P [less than] 0.05). This is significant since stem cells develop in the bottom one-third of the crypt, and apoptosis could therefore delete damaged/mutated cells before they divide and populate the crypt. These results are consistent with previous findings that show apoptosis plays a major role in the development of colo n cancer.

CELLULAR RESPONSE, CYTOTOXICITY, AND P53 EXPRESSION OF ARSENIC AND ATRAZINE USING HEPG2 CELL LINES

Reshuna Y. [Durden.sup.*], Barbara Wilson, Paul Tchounwou, and Ali Ishaque, Jackson State University, Jackson, MS 39217

Atrazine and arsenic are among the leading chemicals that are used in America for agricultural purposes. Atrazine is a manmade herbicide used for the control of broadleaf and grassy weeds. It is persistent in the environment and contaminates ground waters and surface waters that are sources for human consumption and recreation. Lifetime exposure to atrazine, at levels above 0.003 mg/L, has the potential of causing cancer. Arsenic is a carcinogen not only for skin, but also for internal organs such as the lung and bladder. Health effects associated with arsenic exposure include diabetes, cardiovascular disease, hearing loss, and neurological and neurobehavioral effects. The maximum contaminant level for arsenic exposure is 50 [micro]g/L. The goals of this project were: 1) to determine the toxicity of arsenic trioxide and atrazine using the Lactate dehydrogenase (LDH) assay and 2) to determine the cellular response mechanism of arsenic and atrazine in human hepatic carcinomal cell (HepG2) lines. To conduct thi s experiment, HepG2 cells were seeded at [10.sup.6] cells/ml and exposed to the chemicals for 48 hours. LDH analysis was used to determine the lethal concentration at which fifty-percent of the cell population (LC50) would die. Total protein concentration was determined using the Bradford Assay, and Western Blot Analysis evaluated p53 cellular protein expression. The results indicated that atrazine alone was non-toxic to HepG2 cells in the concentration ranges tested. The LC50 value for arsenic trioxide was shown to be l2ppm. The tumor suppressor gene product p53, which is a critical mediator of the cellular response to DNA damage, was expressed after treatment of HepG2 cells with 12 ppm of arsenic. This research was supported by NIH- RCMI Grant G122RR13459 and NIMH-COR grant MH-16926.

CHARACTERIZATION OF METAL BINDING TO A SINGLE AMINO ACID RESIDUE MUTANT, C11G CADC; A METAL-RESPONSIVE TRANSCRIPTIONAL REPRESSOR FROM STAPHYLOCOCCUS AUREUS P1258

Patricia [Gordon.sup.*] [1], Laura Busenlehner [2], and David Giedroc [2], (1.) Jackson State University, Jackson, MS 39217 and (2.) Texas A&M University, College Station, TX 77843

Staphylococcus aureus p1258 CadC is a metalresponsive transcriptional repressor of the cad operon. In vivo, Cd(II), Pb(II), Zn(II), and Bi(III) all stimulate the expression of an efflux pump to efficiently remove toxic metal ions. C11G CadC is a protein that is produced by changing the 11th amino acid in CadC from cysteine to glycine, a mutation from a metal binding residue to one which can not bind metal. CadC is derived from a spherical bacterium called Staphylococcus aureus. This bacterium contains strains that are capable of producing a highly heatstable protein toxin that causes illness in humans. The CadC protein from the CadA cadmium resistance operon of Staphylococcus aureus plasmid p1258 regulates transcription of this metal detoxification system in vivo. The CllG CadC protein was over-produced in Escherichia coli cells and purified. Titrations with known aliquots of Cd(II), Pb(II), and Bi(III) reveal that, compared to wild type CadC, Cl1G CadC binds less equivalents of metals, as well as having les s metal-to-ligand charge transfer absorbance which indicates that cysteine 11 is a metal bindng residue. Further experiments in which all cysteines are mutated should reveal which residues are involved in metal binding, and whether or not cysteines play a role in metal specificity.

MOVEMENT OF tmRNA GENE FRAGMENTS IN THE SALMONELLA GENOME

Shenekia A. [Wells.sup.*] and Kelly Williams, Alcorn State University, Alcorn State, MS 39096 and Indiana University, Bloomington, IN 47405

Salmonella, a well-known pathogen with widespread effects, has affected many lives throughout recent years. This study takes a closer look at tmRNA gene fragments that have been identified in genome sequencing projects for multiple Salmonella species and strains. tmRNA is a recently discovered bacterial RNA with characteristics of both tRNA and mRNA, which helps cells solve problems from stalled ribosomes. This gene is often used by bacteriophages as an attachment site. While this is true in Salmonella, what is unusual is that many of 45 nucleotides at the 3' end fragment of the gene have also been found at different sites in the genome, but are always flanked on at least one side by bacteriaphage related sequence. This project's aim is to analyze these fragments and their locations through a series of tests. These tests are run to see if there is a specific pattern of sequences for why these tmRNA fragments are in those locations. To further explore the distribution of this fragment throughout the Salmonell a genome, 37 DNA from different strains of Salmonella are being tested along with an Escherichia coli strain. In a series of polymerase chain reactions (PCR), Salmonella sequences will be amplified, by cycles of denaturing, primer annealing, and extension by Taq DNA polymerase. For the tmRNA gene and each of the gene fragment sequences available from the genomic projects, PCR primers were designed, and the expected PCR product sizes were tabulated. Genomic DNA was prepared from 37 Salmonella strains that span the diversity of the genus. All 10 PCRs will be performed on each DNA sample. This work will reveal the pattern of distribution of tmRNA gene fragments among Salmonella and may allow reconstructing of the sequence of bacteriophage mobilization.

mRNA EXPRESSION OF AGGRECAN IN NEURONAL AND ASTROCYTIC CULTURES IN CHICK BRAIN

Quartrisa [Douglas.sup.*] [1], Miriam Domowicz [2], and Nancy Schwartz [2], (1.) Alcorn State University, Alcorn State, MS 39096 and (2.) University of Chicago, Chicago, IL 60637

Expression patterns of aggrecan, other major chondroitin sulfate proteoglycans (CSPGs) and tenascin-C, which has been reported to be glia product, were investigated in brain tissue sections via in situ hybridization. Expression of aggrecan and brevican was low in early cultures, doubled days later, and declined by day seven. Some cultures were treated with chondroitinase ABC, an enzyme that digest chondroitin sulfate chains. The expression of each CSPG increased with chondroitinase treatment. Astrocytic cultures were also in situ hybridized. The expression patterns differed with the various CSPGs in that expression was more or less based on morphology. Taken together, these experiments offer a possible explanation to the discrepancy observed in the spatial-temporal pattern of expression of aggrecan in vivo and the presence of aggrecan in neuronal and astrocytic cultures.

MONOCLONAL ANTIBODY PURIFICATION: REDUCTION OF CONTAMINANT CONCENTRATIONS TO LESS THAN ONE PART PER MILLION OF ANTIBODY

Steve Blaisdell and Kristi [Isaac.sup.*], Schering-Plough Research Institute, Union, NJ 07108

Monoclonal antibodies are proteins of a single known specificity and homogeneous structure that are produced in tissue culture. These antibodies are produced from hybridomas of an antibody-secreting cell and a myeloma cell. Monoclonal antibodies are very important in medicine. Antibodies are used in a number of different treatments fighting diseases such as autoimmune diseases, cancer, and asthma. Animal and plant proteins required for the production and purification of the antibody need to be reduced to non-detectable levels. This can be done by using a series of chromatography and filtration steps.

THURSDAY AFTERNOON

Room 3

1:45 bis-ANS BINDING AND SURFACE HYDROPHOBICITY OF BACILLUS THURINGIENSIS d-ENDOTOXIN CYT1A

Slobodanka D. [Manceva.sup.*] and Peter Butko, University of Southern Mississippi, Hattiesburg, MS 39406

In order to elucidate mode of action of the toxin Cyt1A from Bacillus thuringiensis var. israelensis, we studied changes in its surface hydrophobicity as a function of pH and ionic strength, using a polarity-sensitive fluorescence probe bis-ANS (4, 4'-dianilino-1, 1'-binaphthy1-5, 5'-disulfonic acid). An increase in the bis-ANS fluorescence was observed upon lowering the pH, with an apparent pK of 4.5, which is indicative of increased surface hydrophobicity. Scatchard plots were biphasic at both pH 7.5 and 4.2. In accord with previous studies on bis-ANS binding to many other proteins, the plots were interpreted in terms of two classes of binding sites. At pH 7.5, the two sites bound 0.8 [+ or -] 0.1 and 5.7 [+ or -] 3.0 bis-ANS molecules per molecule of Cyt1A (dissociation constants 3.4 [+ or -] 0.4 and 70 [+ or -] 30 mM, respectively). At pH 4.2, these values were 1.6 [+ or -] 0.1 and 9.1 [+ or -] 9.8 (0.3 and 18 [+ or -] 15 mM, respectively). These results support the hypothesis that decreased pH induces c onformation changes in the toxin that make the latter more hydrophobic, with increased affinity toward lipid membranes. In contrast with the smooth, two-State transitions observed in the pH titration, fluorescence of Cyt1A-bound bis-ANS varied with ionic strength in a complex multiphasic manner. This suggests that the probe fluorescence is simultaneously modulated by several processes, such as electrostatic effects on the probe/protein binding and salt-induced unfolding of the protein.

2:00 HYDROPHOBIN Sc3 SELF-ASSEMBLES AT INTERFACES VIA [beta]-SHEET STACKING

Justin P. [Buford.sup.*], J. Shawn Goodwin, Paul Stroud, Charles L. McCormick, Gordon C. Cannon, and Peter Butko, University of Southern Mississippi, Hattiesburg, MS 39406

Amphipathic fungal proteins called hydrophobins are able to self-assemble into insoluble supramolecular structures at hydrophobic/hydrophilic interfaces, but the molecular mechanism and underlying protein conformation changes are not known. Many amyloidogenic proteins self-assemble into insoluble amyloid fibrils while undergoing a change to an all-[beta] conformation. Stacked [beta]-sheets can be very specifically detected by binding of two dyes, thioflavin T (ThT) and Congo red (CR). Secondary-structure prediction and circular dichroism data indicated that hydrophobin Sc3 is an all-[beta] protein. In this study, spectral changes in fluorescence of ThT and in absorption of CR were measured in the presence of Sc3, amyloid [beta] peptide (as a positive control) or an all-[alpha] protein apolipophorin-III (as a negative control). It was found that ThT interacts with Sc3 assemblies in the same manner as with the amyloid [beta]-sheet fibrils. CR did not show a discernible signal at neutral pH. However, when the e lectrostatic repulsion between Sc3 and CR was decreased by low pH, CR exhibited the same spectral changes in the presence of assembled Sc3 as those observed in the presence of assembled amyloid [beta] peptide. It is concluded that Sc3, and probably other hydrophobins too, self-assemble at interfaces in the same manner as amyloidogenic proteins, i.e., through [beta]-sheet stacking.

2:15 PURIFICATION AND CHARACTERIZATION OF RECOMBINANT GLUTAMINYL CYCLASE PRODUCED IN DROSOPHILA SCHNEIDER 2 CELLS

Rachell E. [Booth.sup.*] and Robert C. Bateman, Jr., University of Southern Mississippi, Hattiesburg, MS 39406-5043

Peptides including neurotransmitters and hormones are processed along the secretory pathway by processing enzymes. For example, glutaminyl cyclase (QC) cyclizes N-terminal glutamine to pyro-glutamine to produce a biologically active peptide. In an effort to determine the mechanism of action and important structural characteristics of QC, we have expressed human QC in Drosophila Schneider 2 cells (DES QC). In this system, the protein is secreted into the media at levels approaching 50 mg/L. This recombinant form of the protein has been purified by anion-exchange chromatography and characterized to validate its representation of the native form. The pH activity optimum and kinetic constants for DES QC were found to be similar to that of the native bovine pituitary enzyme. Thus, the recombinant QC is a good representation to be used as a model for further structural studies. Preliminary gel shift assay studies indicate that the DES QC is glycosylated and phosphorylated. We are currently examining the specific s ites where these post-translational modifications occur and determining if they are essential for activity or correct structure formation.

2:30 INHIBITION STUDIES OF GLUTAMINYL CYCLASE

Stephanie A. [Misquitta.sup.*] and Robert C. Bateman, Jr., University of Southern Mississippi, Hattiesburg, MS 39406-5043

Glutaminyl cyclase (QC) catalyzes the conversion of N-terminal glutamine to pyroglutamic acid in certain peptide hormones and neurotransmitters. We sought to obtain inhibitors for glutaminyl cyclase with a view to understanding the mechanism of enzyme action and as a tool for investigating the role of QC in cell culture. We studied the inhibition of the recombinant human QC using a series of imidazole derivatives. We determined the type of inhibition and the inhibitor constant for those that were found to inhibit the enzyme, with the best inhibitors exhibiting Ki values in the low micromolar range. Finally we are immobilizing these inhibitors in order to develop an affinity column for the rapid purification of QC.

2:45 IDENTIFICATION OF THE CHLOROPLAST NUCLEOID PROTEIN, DCP68, AS A SULFITE REDUCTASE 

Cecilia L. Chi-[Ham.sup.*], Gordon C. Cannon, and Sabine Heinhorst, University of Southern Mississippi, Hattiesburg, MS 39406

Plastids are semiautonomous organelles that perform different functions depending on the cell type in which they reside: Their specific function is closely coordinated with the replication and expression of its DNA, which may be correlated to changes in nucleoid structure. In an effort to understand how the dynamic changes of the nucleoid may affect the organelle's function we have begun to characterize nucleoid proteins. We have previously shown that one of these proteins, DCP68, is able to compact DNA and inhibit DNA synthesis in vitro. N-terminal amino acid analysis and the absorption spectrum of the purified protein suggest that DCP68 is a sulfite reductase, a siroheme enzyme involved in the assimilation of sulfur for amino acid biosynthesis. The association of DCP68 with the nucleoid has been confirmed by in organello formaldehyde cross-linking of protein/DNA complexes and immuno-colocalization using antibodies against recombinant sulfite reductase from Arabidopsis thaliana. These findings suggest that DCP68 may be a bifunctional protein that plays a role in plastid DNA compaction and sulfur assimilation.

3:00 Break

3:15 A cDNA CLONE OF DCP68, A MAJOR DNA-BINDING PROTEIN OF SOYBEAN CHLOROPLAST NUCLEOIDS

Mignon A. [Keaton.sup.*], Gordon C. Cannon, and Sabine Heinhorst, University of Southern Mississippi, Hattiesburg, MS 39406

Chloroplasts, like mitochondria, are semi-autonomous organelles that contain their own DNA while also relying on nuclear encoded gene products. The chloroplast genome is associated with proteins into compact structures called nucleoids. During organellar development, nucleoid morphology and protein composition vary suggesting that nucleoid structure plays a role in the regulation of replication and transcription of the chloroplast genome. Therefore, identifying proteins that regulate nucleoid structure may lead to a better understanding of these processes. Previously in our lab, a 68 kDa DNA-binding protein (DCP68) was isolated from chloroplast nucleoids of Glycine max (soybean). Purified DCP68 is able to compact DNA and inhibit DNA synthesis in vitro. N-terminal sequencing and heme absorption peaks identified this protein as a sulfite reductase. The purpose of this project is to isolate a cDNA clone of DCP68 for further studies. Plant sulfite reductase cDNA sequences were used to design primers for the isol ation and amplification of the cDNA of the soybean sulfite reductase. To date, approximately half of the corresponding sequence has been identified, cloned, and sequenced. The deduced amino acid sequence, which shows over 90% homology with other plant sulfite reductases, contains a potential transit peptide and the N-terminal sequence previously obtained by microsequencing of purified DCP68.

3:30 AMPLIFICATION, CLONING, AND SEQUENCING OF EXONS ENCODING THE HUMAN ANDROGEN RECEPTOR

Christopher H. [Wyatt.sup.*], Randall S. Hines, and Steven T. Case, University of Mississippi Medical Center, Jackson, MS 39216

Mutations in the gene encoding the androgen receptor, an androgen activated, DNA-binding protein, frequently result in synthesis of a dysfunctional receptor and clinical manifestations termed androgen insensitivity syndrome (AIS.) The objective of this work was to develop and validate a method to screen human androgen receptor exons for mutations. About 2.5 [micro]g of genomic DNA was extracted from 240 [micro]L of whole blood. DNA was precipitated and resuspended at a concentration of 7.8 ng/[micro]L. 100 [micro]L PCR reactions were constructed using the genomic DNA as template. Pairs of opposing PCR primers corresponding to intron sequences flanking exons were designed. PCR parameters were optimized for each primer pair using a 15:1 mixture of KlentaqI and Pfu thermostable DNA polymerases. Successful PCR amplification resulted in a single band corresponding to the known exon length. PCR products were purified using a Wizard Plus Miniprep Kit (Promega), quantified, and cloned into the blunt-end plasmid vect or, pT7Blue-3. Confirmation of cloning was provided by PCR. Plasmid DNA was subsequently prepped from the remainder of liquid cultures exhibiting PCR products whose size corresponded to that expected for each corresponding exon. Cycle DNA sequencing was performed on each strand using Sequitherm Excel II DNA Polymerase (Epicentre) and opposing infrared-labeled M13 primers. Products were analyzed on a LICOR Model 4000L Automated DNA Sequencer. Resulting sequences were compiled and compared using DNASIS (Hitachi). Using the above method, all the exons from control DNA had 100% sequence identity with wild-type androgen receptor exons.

3:45 TRANSLATIONAL REGULATION MEDIATED BY THE PROXIMITY OF AN RNA APTAMER-LIGAND COMPLEX TO THE INITIATION CODON

Arpna [Vajpayee.sup.*] and Charles Wilson, University of California, Santa Cruz, CA 95060

Previous research has shown that after the insertion of a malachite green binding motif into a pRS306-derived plasmid, progression through the cell cycle was dramatically slowed. The malachite green binder was cloned into the 5'-untranslated region of CLB2 gene, which codes for a cell cycle protein that directs transition from G2 to mitosis. The aptamer-ligand decreased transcript translatability of the CLB2, but not its stability. This confirmed that translational initiation is blocked by ligand-induced conformational changes in the 5'-UTR. The purpose of the experiment was to confirm that aptamer-mediated translational regulation depends upon the proximity of the malachite green aptamer to the start codon. The first step was to design, synthesize, and purify malachite green oligosaccharides. Next, the pGem-3Z-luciferase was digested with the restriction enzyme BamHI. Then, the malachite green aptamer was ligated into the expression vector. After isolating the plasmid construct, an analytical agarose gel co nfirmed that the thirty-one nucleotide malachite-green aptamer insert was present. Another restriction enzyme digestion was performed using the enzymes NarI and XhoI. After isolating the two colonies of the digestion, the in vitro transcription procedure was performed. Future experiments will involve optimizing in vitro translation tests with the construct.

4:00 MOLECULAR GENETIC ANALYSIS OF trqB FUNCTIONS

Lee R. Peeples [III.sup.*], and David Kehoe, Alcorn State University, Alcorn State, MS 39096 and Indiana University, Bloomington, IN 47405

Complementary chromatic adaptation is the ability of some cyanobacteria to alter the synthesis of certain proteins that make up the outer portion (rods) of their light harvesting complex (phycobilisome) with changing light quality. Several mutants have been characterized and complemented, revealing a putative sensor-RcaE. It is believed that this sensor receives photons of light and autophosphorylates, and passes this signal to different response regulators. This ultimately results in altered synthesis of phycocyanin and phycoerythrin, the pigmented phycobiliproteins that make up the rods of the phycobilisome. A novel class of mutants to the cyanobacterium Fremyella diplosiphon has been characterized. Unlike wild type cells, which produce the red pigment phycoerythrin when exposed to green light, these mutants are turquoise in color, revealing a change in the production of phycoerythrin. DNA sequencing has found there to be an insertion in the gene trqB for some of these mutant. It is believed that the lesio n in trqB is the cause of this mutant phenotype. Therefore, by complementing the mutants with trqB from wild type, the hope is to discover if polymorphism in trqB is the cause of these turquoise mutants.

FRIDAY MORNING

Room 3

8:45 Introduction--Peter Butko

9:00 PLASMID SEQUENCES OF EDWARDSIELLA ICTALURI

Vee Yang [Tan.sup.*] and John Boyle, Mississippi State University, Mississippi State, MS 39762

Edwardsiella ictaluri causes enteric septicemia in channel catfish. It is responsible for loss of about 10% of the catfish crop in Mississippi each year. The molecular cause of the virulence is unknown. We have sequenced the two plasmids of this bacterium in order to search for putative virulence factors. The sequences show that one plasmid is of the p 15a family while the other is related to colE2 in E. coli. There are multiple open reading frames and one has a leu rich repeat domain seen in a virulence factor found in Salmonella and Shigella species.

9:15 INTERFERON-GAMMA (IFN-g) PRODUCTION BY MURINE CD8 AND CD4 CELLS IN RESPONSE TO SPECIFIC ANTIGEN AND NON-SPECIFIC FACTORS FROM TRYPANOSOMA BRUCEI RHODENSIENSE

Caleph B. [Wilson.sup.*] [1], Karen Demick [2], and John M. Mansfield [2], (1.) Alcorn State University, Alcorn State, MS 39096 7500 and (2.) University of Wisconsin, Madison, WI 53706

Lymphocytes from uninfected and Trypanosoma brucei rhodesiense infected mouse strains were examined for the production of IFN-g following in vitro exposure to (1) the major surface antigen, variant surface glycoprotein (VSG), (2) a non-antigenic stimulating factor present in trypanosome whole cell extracts (Tex), and (3) a panspecific T cell antigen, Concanavalin A (Con A). Spleen cells from four murine strains were cultured onto 96 well nitrocellulose microtiter plates as well as in 24-well tissue culture plates. The nitrocellulose plates were processed for the ELISPOT assay, which detects the number of cells secreting IFN-g after various incubation periods at 37[degrees]C. The 24-well tissue culture plates were incubated at 37[degrees]C and supernatant fluids were harvested and examined for IFN-g via the enzyme-linked immunosorbent assay (ELISA). Data indicated that the most effective stimulant for IFN-g  in uninfected splenocytes (expect the RAG-l mice) was Con A. Tex was the secon d most effective stimulant in the uninfected cells. In the infected splenocytes, Con A and Tex presented comparable stimulation levels, while VSG induced the least. The overall results suggest that CD8 T lymphocytes may contribute to overall IFN-glevels after exposure to factors(s) present in trypanosome cells.

9:30 PEPTIDOGLYCAN FROM GROUP B STREPTOCOCCUS AS A TARGET FOR NATURAL 1gM ANTIBODIES IN MICE

Andrea A. [Krell.sup.*] and Peter Butko, University of Southern Mississippi, Hattiesburg, MS 39406

Group B Streptococcus (GBS) bacteria can colonize healthy adults without causing infection, but when present in newborn infants, the elderly, and immuno compromised adults the bacteria can cause sepsis and meningitis, resulting in disability and sometimes death. It has been discovered previously that normal mouse serum (NMS) contains natural IgM antibodies to GBS. This work is part of a project aimed at identification of the molecular target for these antibodies. Potential binding sites include fibrous C and R proteins on the cell surface, the peptidoglycan cell wall, and the cell's polysaccharide capsule. Previous research indicated that the most probable of these suspects is the peptidoglycan cell wall. Peptidoglycan (PG) from GBS was isolated and its potential for binding anti GBS IgM was determined. Cell walls were prepared by treatment with DNAse, RNAse, and sodium dodecyl sulfate, followed by homogenization. A modified Enzyme-Linked ImmunoSorbent Assay (ELISA) was used to determine the extent of antibo dy binding to the whole GBS cells and to PG. Results indicate that the isolated PG bound the mouse anti-GBS IgM as efficiently as whole bacterial cells. Furthermore, serum preabsorbed with PG exhibited approximately 20% lower activity than NMS in ELISA. The results thus support the notion that GBS cell wall is the antigenic target for natural anti-GBS IgM in mice.

9:45 Break

10:00 CLONING AND OVER-EXPRESSION OF A MOLD-SPECIFIC GENE IN THE PATHOGENIC FUNGUS HIS TOPLASMA CAPSULATUM

Xianbin [Tian.sup.*] and Glenmore Shearer, Jr., University of Southern Mississippi, Hattiesburg, MS 39406

Mold/yeast dimorphism is an important feature in pathogenesis of the fungus Histoplasma capsulatum. The multicellular mold form, which is found in soil, shifts to the pathogenic yeast form in people who inhale spores or mold fragments. Thus an understanding of the molecular genetics of dimorphism is valuable from a developmental biology aspect as well as for potential development of therapeutic and diagnostic tools. Here we report the cloning of MS8, a single copy gene strongly upregulated in the mold form but transcriptionally silent in the yeast. The MS8 cDNA was 1250 bp long with an open reading frame of 612 bp. A GenBank homology search revealed no genes in the database with significant similarity to MS8. Forced expression of MS8 in the yeast form was accomplished by transforming cells with a vector carrying MS8 fused to the strong constitutive Tef1 promoter. Yeast cells expressing MS8 maintained the yeast morphology but exhibited clumping in liquid media and altered colony texture on agar plates. MS8 kn ockouts are being constructed to further study the role of this gene in dimorphism.

10:15 ANALYSIS OF THE rDNA CLUSTER IN THE PATHOGENIC FUNGUS HISTOPLASMA CAPSULATUM

Sally L. [Faherty.sup.*] and Glenmore Shearer, Jr., University of Southern Mississippi, Hattiesburg, MS 39406

The dimorphic fungus Histoplasma capsulatum (Hc) is the etiologic agent of the common and serious disease histoplasmosis which infects an estimated 500,000 Americans each year. The organism grows in soil as a saprophytic multicellular mold. When mold fragments or spores are inhaled, the organism shifts to a unicellular budding yeast form in the lungs. This mold-to-yeast shift is required for the disease to progress. In our studies to understand the molecular genetics of this dimorphic process, we have isolated several dimorphism regulated genes. Unfortunately, genetic knockouts are quite difficult to construct in Histoplasma, which makes experiments to test the role of these genes very expensive and time consuming. We are exploring the use of antisense ribosomes, containing small sections of target gene antisense, as a post transcriptional gene silencing alternative to gene knockouts. Here we report the first step in these studies: the isolation and analysis of the Histoplasma rDNA cluster. In contrast to mo st organisms, Hc appears to have a single copy of the rDNA. By comparison of the Hc rDNA sequence to other organisms, we have identified several putative dispensable regions suitable for target gene antisense insertion.

10:30 ANALYSIS OF THE MOLD-SPECIFIC GENE M46 AND THE YEAST-SPECIFIC GENE Y56 FROM HISTOPLASMA CAPSULATUM

Vani [Naraharisetty.sup.*] and Glenmore Shearer, Jr., University of Southern Mississippi, Hattiesburg, MS 39406

The dimorphic fungus Histoplasma capsulatum is the etiologic agent of the respiratory disease histoplasmosis which infects an estimated 500,000 people each year in the United States. The organism grows in the soil (or in the lab at 25[degrees]C) as saprophytic multicellular mold and converts to the pathogenic unicellular yeast form in the infected host (or in the lab at 37[degrees]C). In the attempt to understand the molecular biology of this morphological conversion, genes specific to the yeast and mold phase are being isolated in our lab. Two such genes, M46 specific to the mold phase and Y56 specific to the yeast phase, were isolated from a subtracted cDNA library, prepared with a PCR based normalizing method, which enriches for differentially expressed low abundance sequences. Northern blots showed M46 was expressed in mold form but undetectable in yeast cells. Y56 was expressed in yeast cells but was undetectable in mold phase cells. Partial sequence of M46 showed no homology to any sequence in the GenB ank database. Y56 however is a gene previously shown to encode a strongly antigenic protein in Histoplasma. Genetic knockout experiments are underway to help determine the role of these genes in dimorphism.

10:45 Divisional Business Meeting