1:30 Introduction

1:45 cDNA ENCODING A 160-KDA SPECIAL LOBE-SPECIFIC SILK PROTEIN FROM SALIVARY GLANDS OF CHIRONOMUS PALLIDIVITTATUS

Steven T. Case* and Carol Cox, University of Mississippi Medical Center, Jackson, MS 39216

The aim of this project was to compare the primary structure of ssp160 (a 160-kDa special lobe-specific silk protein) from Chironomus pallidivittatus salivary glands to the homologous protein in Chironomus thummi. Existing C. thummi ssp160 antibody and cDNA probes react, respectively, with a corresponding C. pallidivittatus 160-kDa silk protein and 2.4-kb mRNA, both of which are limited to four cells that comprise the salivary gland's "special lobe." Corresponding near full-length cDNA was cloned and sequenced to ascertain the primary structure of the protein. C. pallidivittatus ssp160 is similar to C. thummi ssp160 including its mass, pI, and amino acid sequence (over 75% identity). The hydrophilic region used to elicit antibodies is virtually identical. Most noteworthy are domains encoding repetitive motifs (Asn-X-Ser/Thr) for N-linked glycosylation. Comparison of the amino acid sequence of proteins encoded by two allelic variants from both species indicate these motifs are more dynamic than any other region of the protein. We propose that short repetitive sequences encoding these motifs are prone to strand slippage or unequal crossing over that increases or decreases their number. Supported by National Science Foundation grant #MCB-9204837 and U.S. Army Research Office grant #DAAH04-95-1-0129.

2:00 GENES ENCODING CHIRONOMUS PALLIDIVITTATUS SSP160: ONE GENE OR TWO?

Carol Cox*, Aimee Anido, Sudha Govindachar, and Steven T. Case, University of Mississippi Medical Center, Jackson, MS 39216

The focus of this project was to determine the structure of the Chironomus pallidivittatus ssp160 gene. ssp160 is a 160-kDa "special silk protein" produced by larval salivary glands of various Chironomid species. The salivary glands are composed of some 40 cells; however, ssp160 synthesis is limited to the four cells which form the "special lobe" of the gland. ssp160 cDNA from Chironomus thummi was hybridized to northern blots made of special and main lobe RNAs of C. pallidivittatus. A single 2.4-kb special lobe-specific transcript was detected indicating that hybridization conditions were specific enough to screen a C. pallidivittatus cDNA library. A 2.4-kb cDNA was isolated and sequenced and, in turn, used to probe a C. pallidivittatus genomic library. Southern blots of total genomic DNA (isolated from many larvae) and individual genomic clones suggested two variants were present, as defined by an EcoRI restriction fragment length polymorphism. Transcripts from both variants were found in the cDNA library, therefore both genes are expressed. Genomic southern blots demonstrated individual larvae were either homo- or heterozygous for one or both variants, indicating that they are alleles of one gene. The overall sequence of the alleles and flanking regions was quite similar; however, deletions were found in intron 1 and downstream of the gene that may be due to strand-slippage or unequal crossing over amoung five base pair repeats found therein. Supported by U.S. Army Research Office grants DAAH04-95-1-0129 and DAAH04-95-1-0624.

2:15 A MOLECULAR EXPLANATION FOR THE LACK OF BR4 IN CHIRONOMUS TENTANS

Aimee Anido*, Carol Cox, and Steven T. Case, University of Mississippi Medical Center, Jackson, MS 39216

The object of this study was to provide a molecular explanation for the lack of Balbiani Ring (BR) 4 in Chironomus tentans salivary gland polytene chromosome IV. BRs are giant puffs on secretory cell polytene chromosomes which result from intensive transcription of one or more silk protein genes. Chironomus pallidivittus and C. tentans are sister species with nearly identical polytene chromosomes including BR1, BR2, and BR3 on chromosome IV; however, C. tentans lacks BR4 which forms in special lobe cells of C. pallidivittus. BR4 in C. pallidivittus contains the gene for a 160-kDa special lobe-specific silk protein (ssp160). We cloned and sequenced full-length 2.4-kb ssp160 cDNA and two allelic genes. We then used in situ hybridization to probe C. tentans polytene chromosomes with PCR products spanning 4 kb upstream and downstream of the C. pallidivittus ssp160 gene. Whereas all ssp160 upstream, gene, cDNA, and downstream probes map to Beermann's "SZ+" band in region 1-A on chromosome IV in C. pallidivittus (the band that forms BR4 in special lobe cells), only upstream and downstream probes hybridized to the homologous locus in C. tentans, suggesting a deletion occurred. An ssp160 upstream probe was used to obtain a corresponding C. tentans genomic clone. The DNA sequence of this clone confirmed in situ hybridization results: the lack of BR4 in C. tentans is due to the deletion of the ssp160 gene. Supported by U. S. Army Research Office grant #DAAH04-95-1-0642.

2:30 cDNA FOR A 195-KDA SILK PROTEIN FROM CHIRONOMUS TENTANS

Amy M. Donhardt¹*, Dennis Wong², Carol Cox³, and Steven T. Case³, ¹Mississippi College, Clinton, MS 39058, ²Millsaps College, Jackson, MS 39210, and ³University of Mississippi Medical Center, Jackson, MS 39216

The aim of this project was to clone and sequence cDNA encoding a 195-kDa silk protein (sp195) from larval salivary glands of Chironomus tentans. Dreesen et al. (J. Cell Biol. 106:21-27, 1988) reported a 120-bp cDNA sequence that encodes a peptide derived from sp195. Since the cDNA represented only a fraction (120/6500 bases) of the length of the mRNA as measured on Northern blots, we attempted to clone and sequence full-length sp195 cDNA. A LambdaZAP cDNA library that has yielded full-length cDNAs for other silk proteins was screened by plaque hybridization with a 21-base oligonucleotide derived from the existing 120-bp cDNA. Eight clones were selected and the size of their insert determined by restriction enzyme digestion: the largest cDNA was only 2.5 kb. Since restriction sites used to clone the cDNA were present at both flanks and partial sequencing suggested all had 3'-end poly(A) tails, it appears that the cDNA was never fully reverse transcribed. To preciesly locate these putative sites of reverse transcriptase termination, partial clones of various lengths will be sequenced. In addition, the 2.5-kb cDNA is being analysed by transposon-mediated DNA sequencing. Data thus far indicates the 3'-end of the cDNA contains 75-bp tandem repeats of protein coding sequences; however, the 5'-end of the clone contains non-repetitive DNA. This research was supported by the Howard Hughes Medical Institute (Undergraduate Sciences Education Grant #HHMI171195-538901) and U.S. Army Research Office (Grant #DAAH04-95-1-0642).

2:45 Break

3:00 ANALYSIS OF THE AMBIENT pH SIGNAL TRANSDUCTION GENE, palI, OF ASPERGILLUS NIDULANS

Wendy Mayer*, Alpa Goel* and Steven H. Denison, Mississippi College, Clinton, MS 39058

The filamentous ascomycete fungus, Aspergillus nidulans, possess a regulatory system that enables it to produce alkaline protease and phosphatase when growing in an alkaline environment and acid protease and phosphatase when growing in an acid environment. Production of pH-appropriate enzymes is under the transcriptional control of the pacC gene-encoded transcriptional regulator. Under conditions of alkaline ambient pH 1, the products of the pal genes, palA, B, C, F, H, and I, transduce a signal which results in the activation of the pacC transcription factor as an activator of alkaline-expressed genes and a repressor of acid-expressed genes. Genetic evidence suggests that the palI gene product may function as the sensor of ambient pH. We cloned the palI gene by the ability of an A. nidulans genomic fragment to restore wild type pH regulation to a palI30 mutant strain. Northern analysis demonstrates that the palI message is approximately 3 kb (kilobases). We have sequenced three palI cDNA clones, the largest of 2.4 kb and a palI-containing genomic fragment of 3.7 kb. An open reading frame of 556 amino acids has been identified. The predicted palI amino acid sequence shows strong similarity to a putative membrane-spanning protein of the yeast, Saccharomyces cerevisiae. This similarity strengthens the hypothesis that the palI protein functions in the cytoplasmic membrane as the ambient pH sensor in the pH signal transduction pathway.

3:15 GENERATION OF EXTRAGENIC SUPPRESSORS OF A NIMX^CDC2 MUTATION IN ASPERGILLUS NIDULANS

Dana L. Roe, Gene A. Lang, Sean P. Grace, and Sarah Lea McGuire*, Millsaps College, Jackson, MS 39210

In an attempt to identify proteins which interact with the nimX^CDC2 protein of the filamentous fungus Aspergillus nidulans, we have generated a series of 7,000 suppressors of the nimX^CDC2F223L mutation by chemical mutagenesis. This conditional mutation leads to an inability to traverse both the G2-M and G1-S transitions of the cell cycle at 42C and suppressor strains overcome this mutant phenotype, thereby allowing cells to grow and divide at 42C. Genes containing extragenic suppressor mutations frequently encode proteins that interact with the original mutant protein; extragenic suppressors of nimX^CDC2 mutations may thus encode proteins which bind to and regulate nimX^CDC2 or which are substrates of nimX^CDC2. We have screened the suppressors both for the presence of an easily scorable selectable phenotype (cold-sensitivity or sensitivity to DMSO, hydroxyurea, or MMS) which will facilitate future cloning of the suppressor genes, as well as for the presence of extragenic vs. intragenic suppressor mutations. 34 cold-sensitive, 4 hydroxyurea-sensitive, and 1 DMSO-sensitive extragenic suppressor strains have been identified and are being further analyzed genetically and phenotypically to identify candidates for cloning.

3:30 DNA BINDING PROTEINS OF CHLOROPLAST NUCLEOIDS

Rebecca S. Walters*, Gordon C. Cannon, and Sabine Heinhorst, University of Southern Mississippi, Hattiesburg, MS 39406-5043

Chloroplast DNA (ctDNA) is arranged into suborganellar structures called nucleoids that have dynamic conformations thought to be related to the developmental stage of the plastid and the rate at which the ctDNA is transcribed and replicated. Nucleoid proteins are believed to take on the role of binding to the ctDNA, compacting and arranging it into the appropriate conformation, but to date these have not been clearly classified, nor have their functions been elucidated. The goal of this research project is to determine which nucleoid proteins bind to DNA and change its topology. Purified nucleoids from soybean chloroplasts were subjected to SDS-PAGE, the proteins blotted onto nitrocellulose membranes and incubated with radiolabeled ctDNA fragments. Proteins with ctDNA binding affinity were identified by autoradiography. Preliminary results indicate three primary nucleoid proteins which exhibit ctDNA binding affinity. Further studies are underway to purify individual DNA binding proteins from chloroplast nucleoids to study their ability to compact and/or coil DNA.

3:45 DETECTION OF A PUTATIVE DNA-RENATURATION ACTIVITY IN P. SATIVUM CHLOROPLASTS

Brian S. Dunston*, Sabine Heinhorst, and Gordon C. Cannon, University of Southern Mississippi, Hattiesburg, MS 39406

Using a crude high salt/detergent extract prepared from isolated chloroplasts of Pisum sativum cv. Alaska plants, a heat-labile activity which promotes the renaturation of homologous single-stranded DNA molecules to the linear double-stranded DNA monomeric form was detected. The reaction is reminiscent of that catalyzed by E. coli RecA protein in that when Mg²⁺ and ATP were not present in the reaction system, only the double-stranded DNA monomer was formed. Another feature of the chloroplastic renaturation activity similar to that of RecA was the formation of high molecular weight DNA "networks" that are detergent-resistant in an ATP and Mg²⁺-dependent fashion. Efforts are underway to further characterize and to ultimately purify the chloroplastic DNA renaturation activity.

4:00 STIMULATION OF DNA SYNTHESIS ACTIVITY BY CHLOROPLAST NUCLEOID PROTEINS

Chad I. Case*, Sabine Heinhorst, and Gordon C. Cannon, University of Southern Mississippi, Hattiesburg, MS 39406-5043

Using the photomixotrophic soybean cell line SB-M, chloroplast DNA binding proteins were isolated that demonstrated an ability to coil chloroplast DNA. To determine the impact of this coiling on DNA synthesis, assays were performed using a chloroplast replication extract isolated from the same soybean cell line to measure the amount of [3H]TTP incorporation into chloroplast DNA. A direct correlation between binding protein concentration and the rate of nucleotide incorporation was discovered. These experiments suggest that the topology of chloroplast DNA affects the efficiency with which the replication extract can use this template in vitro. Further studies are aimed at purifying individual DNA binding proteins to determine the nature of their interaction with the chloroplast DNA synthesis machinery.

4:15 DNA POLYMERASE D FROM SOYBEAN: BIOCHEMICAL CHARACTERIZATION AND CDNA CLONING

Jeannie T.B. Collins, Sharon E. Jones*, Gordon C. Cannon, and Sabine Heinhorst, University of Southern Mississippi, Hattiesburg, MS 39402-5043

Like all eukaryotes, plants require multiple DNA polymerases and accessory proteins to replicate their nuclear DNA. While in other systems components of the DNA replication machinery have been well characterized with respect to their structure and function, almost nothing is known about these enzymes in plants, and what little information is available has been unable to resolve the question whether plants and other organisms replicate their genome by the same or different mechanisms. A double gradient elution approach allowed the successful separation of DNA polymerases and from soybean cells. Both enzymes were found to cross-react with antibodies raised against their mammalian counterparts, suggesting structural similarity. Using pools of degenerate primers that were designed based on highly conserved amino acid motifs found in replicative nuclear DNA polymerases of the B family, the central portion of the cDNA for DNA polymerase d was amplified by Touchdown PCR from a rapidly dividing soybean cell line. The 5' and 3' portions of the cDNA were obtained by the Rapid Amplification of cDNA Ends (RACE) technique. The amino acid sequence deduced from the full length cDNA sequence revealed strong homology to -type DNA polymerases from other sources.

8:30 NUCLEOLAR PROTEIN B23 HAS MOLECULAR CHAPERONE ACTIVITY

Attila Szebeni* and Mark O.J. Olson, University of Mississippi Medical Center, Jackson MS 39211

Molecular chaperones are a diverse group of proteins that share the common activity of modulating protein conformation. Their functions are to facilitate the post translational folding and transport of nascent polypeptides and to maintain existing proteins in a native state. Nucleolar protein B23 is an abundant, multifunctional and highly conserved phosphoprotein proposed to be a ribosome assembly factor. Protein B23 shows chaperone-like activity toward other proteins by preventing their aggregation or protecting them against thermal denaturation. Protein B23 acts as a chaperone towards the HIV-1 Rev protein by preventing formation of insoluble fibers and inhibiting Rev aggregation at low ionic strength. Temperature-dependent aggregation of Rev was measured using a zero angle light scattering (turbidity) assay. The inhibition of aggregation was proportional to the concentration of B23 added and this activity was saturable. Nearly complete inhibition of aggregation was seen when the molar ratio of B23:Rev was sightly above one. In competition studies, nuclear localization signal-containing peptides inhibited protein B23 chaperone activity suggesting that the interaction of the Rev NLS with protein B23 is necessary for the activity. Protein B23 phosphorylated with casein kinase II showed enhanced chaperone activity by inhibiting aggregation at a much lower molar ratio of B23:Rev (as low as 1/100) compared to non-phosphorylated protein. Protein B23 exhibits chaperone activity towards other proteins including carboxypeptidase A and liver alcohol dehydrogenase by protecting them from thermal denaturation and preserving enzyme activity. These studies suggest that protein B23 acts as a specialized molecular chaperone for proteins involved in ribosome assembly.

8:45 CREATING NUCLEI FOR AGGREGATION OF ALZHEIMER'S BETA-AMYLOID PROTEIN

Suzanne E. Wahrle* and Terrone L. Rosenberry, Millsaps College, Jackson, MS 39210, and Mayo Clinic Neurodegenerative Disease Research Center, Jacksonville, FL 32224

Aggregation of beta-amyloid protein (A) may cause Alzheimer's Disease. A aggregation has not been studied at physiologically relevant concentrations (picomolar) because aggregation occurs much too slowly. Seeding A with nuclei may increase the rate of aggregation to a level that can be studied. To examine this possibility, nuclei were made using two methods: 1) Two proteins known to promote A aggregation, Serum Amyloid P component (SAP) and complement protein Clq, were radiomethylated, then coupled to activated sepharose. SAP and Clq coupling efficiencies were both low. With increased sepharose activation the coupling efficiency of Clq increased and SAP remained the same. It appears that SAP did not couple to sepharose and that low percentages of Clq did couple. Increased sepharose activation may make Clq coupled sepharose an effective nucleus. 2) Pierce cross-linkers were used to cross-link A_1-40 and A_l-42 to SAP and Clq. The cross-linked products were analyzed using SDS-PAGE followed by immunoblotting. Products of A_l-42 cross-linking were either monomeric or had very high molecular weights. This indicates that A_1-42 aggregates were cross-linking. With A_1-40, complexes ranged from monomeric to a molecular weight proportional to the cross-linker concentration, which suggests that A_1-40 monomers were cross-linking. A to SAP or Clq cross-linked complexes, in particular A_1-42 cross-linked Clq, may be sufficiently stable, massive, and attractive to act as effective nuclei for A aggregation.

9:00 PDGF STIMULATES CHANGES IN THE PHOSPHORYLATION OF MUSCLE CELL PROTEINS

Alexia L. Smith* and James B. Hutchins, Millsaps College, Jackson, MS 39210, and University of Mississippi Medical Center, Jackson, MS 39216-4505

Platelet-derived growth factor (PDGF) is a protein which controls the differentiation and division of many cell types. In fibroblasts, PDGF controls the "C" point in the cell cycle, at the G₁/S transition, and allows cells to proceed through mitosis. It has recently been shown, in a variety of mitotic and non-mitotic cell types, that PDGF controls or influences not only mitosis but cytoskeletal transformations and ion channels as well. We are exploring these issues in the C₂C₁₂ mouse muscle cell line. In medium containing low glucose and 20% fetal bovine serum, these cells are myoblasts; when cultured in high glucose, 5% horse serum-containing medium, these cells undergo a morphological transformation to become multinucleate syncytia (myotubes). This parallels the in vivo development of muscle cells. It has been shown previously that PDGF receptors are expressed on rat L6 myoblasts as they undergo a similar transformation. These studies have two goals: first, to confirm that PDGF receptors are transiently expressed by C₂C₁₂ cells during differentiation into myotubes, as is seen in rat L6 myoblasts; second, to determine which proteins are differentially phosphorylated during C₂C₁₂ myoblast differentiation in either untreated controls or PDGF-stimulated experimental cultures. Semi-quantitative Western blotting using antibodies directed against PDGF receptors or downstream signal transduction elements and immunoprecipitation have been used to characterize the biochemical changes within C₂C₁₂ myoblasts during differentiation.

9:15 EFFECT OF PLATELET-DERIVED GROWTH FACTOR (PDGF) ON MITOCHONDRIAL FUNCTION IN NEURONS AND FIBROBLASTS

Robert L. Moore* and James B. Hutchins, University of Mississippi Medical Center, Jackson, MS 39216-4505

A well-studied effect of platelet-derived growth factor (PDGF) is in controlling the transition from the G₁ phase to the S phase of the cell cycle in fibroblasts. Recently, several new functions for PDGF have been identified. Our laboratory has been studying the effects of stimulating the tyrosine kinase activity of the PDGF receptor on downstream proteins in E14 embryonic mouse cortical neurons maintained in primary culture. Zhang, Pan and Hutchins reported the results of a screen for PDGF-stimulated protein phosphorylation using 2D PAGE/autoradiography to identify candidate proteins which were then microsequenced from a blotted membrane. They identified one such protein whose phosphorylation is up-regulated by PDGF and demonstrated that it is apparently identical to the mitochondrial ATP synthase (F₁F₀ ATPase) subunit. Cheng and Mattson showed that PDGF promotes partial neuronal survival in the absence of glucose, a treatment which normally results in the death of all neurons. This led us to hypothesize that PDGF, by stimulating F₁F₀ ATPase subunit phosphorylation, increases the amount of ATP available to the cell. We are currently studying whether F₁F₀ ATPase subunit phosphorylation can be demonstrated in a mouse fibroblast cell line. If it is, we will determine if cellular ATP levels are altered by PDGF treatment, and if ATP production is increased in isolated mitochondria from PDGF-treated fibroblasts compared to untreated controls.

9:30 CHARACTERIZATION AND IDENTIFICATION OF FACTORS THAT INTERACT WITH YPT1

Everett Allen Roark¹*, Sara Jones², and Nava Segev², ¹Jackson State University, Jackson, MS

39217, and ²University of Chicago, Chicago, IL 60617

Yeast is used as a model system in our studies because there is a high degree of functional conservation in the secretory pathway of all eukaryotic organisms. In yeast, there is a family of small GTPases known as the Rab family. One essential member of the Rab family, Ypt1, is involved in the regulation of vesicles traveling from the endoplasmic reticulum (ER) to the Golgi. Mutation of Yptl prevents vesicles from fusing with the Golgi thus causing an accumulation of vesicles in the cytoplasm, which leads to death. A suppression screen was performed to detect factors which, when over expressed, allow the yeast cells to survive lost of Yptl expression. The suppressors that were identified are SLY1, SLY2, SLY12, SLY20, and SLY41. A second screen was done which produced another suppressor, Hs252. The plasmid I Is252 contained multiple genes. The gene suspected of being a true suppressor is SLY41-like (SFL1). The SFL1 gene was isolated and tested for suppression. Knock-out versions of SLY41 and SFL1 were constructed and the transformed into yeast to determine if they were essential. The results indicated that SFL1 is a suppressor of lost of Yptl expression and SFL1 is not essential nor is the combination of SFL1 and SLY-41. In order to further understand the function of Yptl a two hybrid screen was performed to identify factors that interact with Yptl. Twelve possible candidates were identified.

9:45 Break

10:00 THERMAL INACTIVATION STUDIES OF NATIVE AND MUTANT RECOMBINANT HUMAN LIVER PHOSPHOFRUCTOKINASE-1 EXPRESSED FROM GENETICALLY ENGINEERED ESCHERIA COLI

Lakesia M. Sutton¹*, John Trujillo², and Randy Willink², ¹Jackson State University, Jackson, MS 39217, and ²University of New Mexico, Albuquerque, NM 87131

The Liver L type isozyme phosphofructokinase-1 (PFK-1) is a key regulatory enzyme of glycolysis and gluconeogenesis in carbohydrate metabolism. PFK-1 is allosterically regulated by ATP and mainly resides in the liver cell in an inhibited state under the influence of ATP. The enzyme is activated by fructose 2,6-bisphosphate and AMP and inhibited by citrate. These experiments involve three enzyme preparations of human liver PFK-1 and two mutant enzymes; one where arginine (470) had been changed to glycine and the other where lysine (474) had been changed to glycine. The mutant enzymes are found to be insensitive to ATP inhibition and exhibit hyperbolic kinetics as compared to the native enzyme which exhibits sigmoid kinetics (determined by kinetic analysis). The heat stability of the native versus the mutant enzymes through the use of thermal inactivation kinetics have been determined over time. The data show that the native enzyme was found to be less stable to thermal inactivation than the mutant enzymes 470 and 474. In the presence of ATP, the native enzyme is less stable than 470 and 474. This may be due to the fact that the ATP binding site had been altered on 470 and 474. Further studies will be done to determine if the loss of stability is in fact due to the fact that the ATP binding site had been altered on 470 and 474.

10:15 A SCREEN FOR MUTANTS AFFECTING KINESIN RELATED PROTEINS IN DROSOPHILA MELAGNOSTER

Ciju John¹*, Maura McGrail², Aaron Bowman², and Lawrence Goldstein², ¹Mississippi University for Woman, Columbus, MS 39701, and ²University of California, San Diego, CA 92093

First identified and purified in 1985, kinesin and kinesin-like protein have been found to be the main components responsible for large-scale intracellular transport. There are over 40 known kinesin like proteins, some of which are required for organelle transport in neurons, others that are thought to be involved in various aspects of mitosis and cell division. In order to identify mutations in kinesin-related proteins that act in cell division, we screened through a collection of Ethyl Methy Sulfonate (EMS) induced lethal mutations for mutants that disrupt mitosis. Imaginal discs presence or absence was noted and brain squashes of third instar homozygous lethal mutants were examined for possible mitotic phenotypes such as monopolar metaphase, polyploidy, hypercondensation and tripolar metaphase. In our screens we found out of a total of 10 samples, 4 with non or reduced imaginal discs. The brain squashes showed quite a variation in their mitotic counts but at least 2 mutant samples were found with no mitotic structures at all. The mutants found with these phenotypes were then analyzed for further Mendelian complementation tests with all the known kinesin like protein gene sequences.

10:30 STRUCTURE AND EXPRESSION OF THE COTTON LIPID TRANSFER PROTEIN GENE LTP3

Hsi-Chou Liu* and Din-Pow Ma, Mississippi State University, Mississippi State, MS 39762

A cotton (Gossypium hirsutum L. cv DES119) cDNA clone (GH3 ) encoding a lipid transfer protein was previously isolated and characterized in our laboratory. Northern analysis showed that GH3 was differentially expressed in fiber cells in a temporal manner and in a spatial pattern. The GH3 corresponding gene (Ltp3) along with its 5'- and 3'-flanking regions were cloned using a PCR-based genomic DNA walking method. The Ltp3 gene has an identical nucleotide sequence with that of GH3 except its open reading frame is interrupted by an intron of 80 bp located in the region corresponding to the C-terminal ofthe protein. Basal promoter elements TATA and CAAT boxes were found in the 5'-flanking region of the Ltp3 gene. To assay the promoter activity of Ltp3, the 5'-flanking region (614 bp) of Ltp3 was fused to the GUS (-glucuronidase) reporter gene in a binary plasmid vector pBI101, and the chimeric plasmid then introduced into tobacco via an Agrobacterium-mediated transformation method. Both the histochemical staining method and the fluorometric assay were used to determine the expression of the GUS gene. The GH3 cDNA was also amplified by PCR and cloned into an expression vector pMAL downstream from the malE gene, which encodes maltose binding protein (MBP). The expressed MBP-LTP fusion protein was injected into rabbits to produce polyclonal antibody, which was then used in Western analysis to determine the LTP protein level during fiber development.

10:45 ANALYSIS OF PROMOTER ACTIVITY OF THE COTTON LIPID TRANSFER PROTEIN GENE LTP6 IN TRANSGENIC TOBACCO PLANTS

Chuan-Yu Hsu* and Din-Pow Ma, Mississippi State University, Mississippi State, MS 39762

A genomic clone (1.7 kb DNA insert) harboring the lipid transfer protein gene Ltp6 was isolated from a cotton genomic library and characterized. The Ltp6 gene contains an open reading frame of 360 bp, which is interrupted by a single intron of 136 bp. The Ltp6 is specifically expressed in cotton fibers (seed hairs) and is developmentally regulated. By using PCR amplification, DNA fragments with a series of 5' deletion of the Ltp6 promoter were generated and then cloned into the pBI101 plasmid upstream of the GUS (-glucuronidase) reporter gene. These constructs were introduced into Agrobacterium tumefaciens LBA4404 using a freeze-thaw method. Leaf discs of tobacco (Nicotiana tabacum) were transformed with Agrobacterium tumefaciens LBA4404 carrying the various promoter/GUS pBI101 plasmids. The expression of GUS in transgenic tobacco plants was determined by a histochemical localization method and the fluorometric assay. Histochemical analyses of the transgenic tobacco seedlings indicated that the Ltp6 promoter directed the GUS expression only in trichomes (hair cells). Fluorometric GUS assays showed that the promoter activity of the undeleted Ltp6 promoter (nt -447 to -1) was 500 times weaker than that of the 35S promoter of cauliflower mosaic virus (CaMV). Sequential deletions of the promoter gradually decreased the level of expression of the GUS gene. No GUS activity was observed when the 5' deletion of the Ltp6 promoter reached to nt -86, which removed the CAAT and TATA putative promoter elements.
 
 

11:00 RAPID DETECTION OF ESCHERICHIA COLI O157:H7 USING STANDARD POLYMERASE CHAIN REACTION (PCR) AND NESTED PCR

Kirby Harvey*, Jeffrey Evans, and Newton Fawcett, University of Southern Mississippi, Hattiesburg, MS 39406

Escherichia coli O157:H7 presents a potential health problem to consumers since it is mainly found in ground beef. Standard methods for detection and identification may take several days. We have tested one set of PCR primers using standard PCR protocols and have been able to detect as few as 10 CFU/mL in seven-and-one-half hours. We have designed another set of PCR primers. These primers amplify a region within the fragment already amplified by the first set of primers. This is called "nesting." Using this nested PCR method we have seen greater sensitivity. Initial results show detection, via gel electrophoresis and ethidium bromide staining, as few 65 cells/mL in a dilution series. With our nested PCR protocol we hope to achieve detection in less than four hours.
 
 

11:15 Divisional Business Meeting

11:30 Divisional Poster Session

SEARCHING FOR HYDROPEROXIDASE GENES IN HALOBACTERIUM HALOBIUM

Shinon Long* and Marvin L. Salin, Mississippi State University, Mississippi State, MS 39762

Hydroperoxidase is a class of heme-containing enzymes which has been shown to contain both catalase as well as peroxidase activity. These proteins remove H₂O₂ either by peroxidation of H₂O₂ (catalase) or by peroxidation of an organic peroxide (peroxidase). Previous work in our laboratory has resulted in the purification and characterization of a catalase-peroxidase from Halobacterium halobium as well as a mesohalic catalase that is induced in the halophile when it is grown in a low salt medium. Both enzymes have blocked N-termini thereby not allowing us to determine the N-terminal amino acid sequence. However partial internal amino acid sequences have been obtained through CN-Br fragment generation. Degenerate oligonucleotides were constructed to correspond to these amino acid sequences. In addition oligonucleotides were constructed to correspond to presumed conserved regions in catalases. Through the use of polymerase chain reactions (PCR), using combinations of the oligonucleotide probes, DNA fragments of various lengths were amplified from genomic as well as cDNA obtained from H. halobium. The DNA products were cloned into the pGEM-T Easy sequencing vector and the sequences of the clones determined by an ABI Prism 310 Genetic Analyzer. Results of the sequencing and data base search analysis will be presented.

THE USE OF TRANSGENIC PLANTS AS A MODEL FOR THE STUDY OF STRESS TOLERANCE: INTRODUCTION OF AN FE-SOD GENE

Christina Chan-Weiher* and Marvin L. Salin, Mississippi State University, Mississippi State, MS 39762

The gene encoding the iron-containing superoxide dismutase (Fe-SOD) from the alga, Chlamydomonas reinhardtii, cloned and sequenced previously in our laboratory, consists of an open-reading frame (ORF) of approximately 700 nucleotides. We have identified consensus sequences corresponding to several upstream and downstream control elements. By the use of the polymerase chain reaction (PCR) we have amplified two regions of the gene. The first PCR product represents the ORF and certain downstream control elements to include the polyadenylation site. It is 1.5 kilobases in length. The second PCR product encompasses the entire sequence of the cloned gene and is 3.0 kb in length. This product would presumably contain the upstream promoter and control elements, the structural gene and the termination signals. Both PCR products were amplified using internal primers designed to introduce unique restriction sites thereby facilitating the unidirectional cloning into the vector's multicloning site. The PCR fragments were initially cloned into pUC18 and sequentially subcloned into the binary vector, pBI121. Tobacco leaf disks were transformed with these constructs using Agrobacterium tumefaciens. PCR amplification has detected the presence of the inserted Fe-SOD gene. The activity of the Fe-SOD has been enhanced two fold compared to wildtype as detected by tube gel analysis and selective staining. We aim to conduct studies on the stress tolerance of transformants to high salt, high light intensity and heat, all of which are encountered in southeastern agricultural conditions.

TOXIC EFFECTS OF MICROBIAL BYPRODUCTS ON THE FIRE ANT POPULATION

Marcus T. Harris* and Lewis R. Brown, Mississippi State University, Mississippi State, MS 39762

Fire ants are responsible for causing problems which range from death in cattle to sickness in people. Because of the large number of fire ants, it is extremely difficult to wipe out the entire population. Over the years, several chemical agents have been synthesized in attempt to eradicate the fire ant population. Although there are several pesticides registered for ant control, there is no product available that is effective. To over come this problem, there is a need for a product that has delayed toxicity on the fire ant population. With a product that has a delayed toxicity, this will allow the entire mound to be exposed to the toxin by virtue of transporting food to the queen. To accomplish this, microorganisms will be inoculated on to bacteriological agar that contains either .5% maltose, dextrose, glycerol, or sucrose. Once efficient growth has occurred, the microorganisms will be inoculated into broth solutions of the same carbon source. After the microorganisms have acclimated to the broth and growth is apparent, the broth cultures will be centrifuged. The supernate will be introduced to the fire ants through their food. The fire ants will be monitored over a period of 96 hours and the number of live ants will be recorded every 24 hours. An effective toxin that has a delayed toxicity on the fire ant population will be sought with the use of microorganisms.

REPRODUCTIVE AND BIOCHEMICAL EFFECTS OF 4-TERT-OCTYLPHENOL ON SMALL FISH

Suzanne Gronen* and Marius Brouwer, University of Southern Mississippi, Institute of Marine Science, Ocean Springs, MS 39566-7000

An increasing body of scientific evidence suggests that a wide variety of chemicals introduced into the environment could be causing adverse health effects in humans and wildlife species by disrupting endocrine system function. A metabolite of alkylphenolic ethoxylate surfactants, 4-tert-octylphenol, is a suspected endocrine disrupting chemical (EDC) which has estrogenic properties. It can be found in many household items such as detergents, paints, pesticides, cosmetics, and shampoos. Experiments conducted on both Japanese Medaka (Oryzias latipes) and Sheepshead Minnow (Cyprinodon variegatus) following a three week exposure to 4-tert-octylphenol determined that vitellogenin, a yolk protein normally found only in females was present in serum of male fish. Vitellogenin was detected by Western blot in Cyprinodon species at environmentally realistic concentrations of 20 ppb and at 50 ppb in the Medaka. Presence of vitellogenin in male fish could be a useful tool as a biomarker for environmental estrogens. A signifigant correlation (p=0.011) between 4-tert-octylphenol concentration in exposure aquaria and fertilization success of exposed male Medaka was observed.

MECHANISM OF BIDRIN-INDUCED TOXICITY TO RENAL TUBULAR EPITHELIAL CELLS: ROLE OF REACTIVE OXYGEN METABOLITES AND PROTECTIVE EFFECTS OF ANTIOXIDANTS

Vandana S. Poovala¹*, Vijaya Kanji², and Abdulla K. Salahudeen², ¹Jackson State University, Jackson, MS 39217, and ²University of Mississippi Medical Center, Jackson, MS 39216

Bidrin is an organophosphate (OP) insecticide. Due to their reputation for low environmental persistence, OP pesticides are often used indiscriminately. This has brought about detrimental exposure to humans and other non-target species from various environmental matrices and occupational sources. The primary mechanism of OP toxicity is through inhibition of acetylcholinesterase (AChE), which impedes conduction of nerve impulses. However, OP-induced pathophysiological manifestations have been observed in renal proximal tubules. These alterations have been shown to be unrelated to the degree of AChE suppression, which points to the existence of additional pathways of toxicity. This study examines the role of reactive oxygen species (ROS) in Bidrin-induced renal tubular epithelial cell (LLC-PK₁) toxicity. LLC-PK₁ cell death, determined by lactate dehydrogenase (LDH) release, was observed to increase concentration-dependently following exposure of the cells to 750, 1000, 1250, and 1500 ppm of Bidrin. A time-course study (24, 48, 72, and 96 hrs) exhibited a different trend. The first two concentrations displayed a significant decline in LDH (% of total) following incubation of cells for 96 hrs. At 1250 ppm of Bidrin, LDH leakage remained almost constant over time. The highest concentration demonstrated a slight elevation after 96 hrs. The cell damage following incubation with 1250 ppm of Bidrin for 24 hrs was found to be decreased in a concentration-related manner by antioxidants 2-Methyl aminochroman (2-MAC: 0.3-2.5 µM) and Desferrioxamine (DFO: 0.25-2 mM). The greatest reductions in LDH (n=3, ANOVA, p<0.05, mean±SE) were engendered by DFO 2 mM and 2-MAC 2.5 µM, both significantly lower then Bidrin alone (DFO: 29.67±3.29 vs. Bidrin: 47.67±1.46; 2-MAC: 26.67±2.61 vs. Bidrin: 47.67±1.46, p<0.05). These results implicate ROS and accompanying lipid peroxidation, at least in part, in the toxic effects of Bidrin on kidney cells. Therefore, we suggest that oxidative stress may contribute to acute proximal tubular necrosis and renal dysfunction noted in many OP-intoxication incidents involving humans.

E. COLI MOTILITY AND PATHOGENESIS

Donna L. Marykwas, University of Southern Mississippi, Hattiesburg, MS 39406

A clockwise versus counterclockwise switch controls the direction of rotation of each E. coli flagellum, and this in turn controls the cell's swimming behavior. I have demonstrated direct interactions between the known components of the switch (FliG, FliM, FliN) using the two-hybrid system developed in yeast for the identification of interacting proteins. Using this method, I have identified FliG/FliM, FliM/FliM, and FliM/FliN switch protein interactions, and I have shown that the switch is attached to the MS ring, the presumed rotor, via interaction between FliG and FliF (the MS ring protein). Regions of proteins important for several of these interactions were identified by mutational analysis. This included the generation of fliG mutants that produce proteins defective in their interaction with FliM. These mutants have revealed amino acid residues of FliG that are important for this interaction and that are genetically suppressible by compensating changes in FliM. Thus, I have begun to build a molecular model for motor architecture. Unexpected protein-protein interactions were identified in two-hybrid screens. These included interactions between proteins of the switch, a protein involved in transcription, and a protein involved in extracellular protein secretion. These findings suggest common control points through which flagellar motility (often a virulence factor) and pilus-mediated host cell adhesion (an early step of infection) might be coordinately controlled, at the levels of gene expression, protein secretion and cell surface protein assembly.

CLONING AND CHARACTERIZATION OF A NOVEL FORM OF CYTOSOLIC SUPEROXIDE DISMUTASE IN THE BLUE CRAB, CALLINECTES SAPIDUS

Walter D. Grater* and Marius Brouwer, University of Southern Mississippi, Institute for Marine Sciences, Ocean Springs, MS 39564

Superoxide dismutase (SOD), which catalyzes the dismutation of the superoxide radical, is present in the cytosol and mitochondria of all oxygen-respiring eukaryotes. The cytosolic form of all eukaryotes studied to date contains copper and zinc (CuZnSOD), whereas the mitochondrial form contains manganese (MnSOD). The later protein is synthesized in the cytosol as a MnSOD precursor, containing a N-terminal mitochondrial-targeting sequence. Purification and amino acid sequence analyses of cytosolic and mitochondrial SOD from the blue crab have revealed the mitochondrial form is similar to MnSOD from other eukaryotes, while the cytosolic form is a novel form of MnSOD with a unique mitochondrial-targeting sequence. This unique targeting-sequence prevents the protein from being imported into mitochondria. We have cloned the complementary DNA (cDNA) of the cytosolic MnSOD gene to investigate the functional and regulatory properties of this novel antioxidant system and its evolutionary origin. Cytosolic MnSOD cDNA was isolated using 3' random amplification of cDNA ends (RACE) and 5' RACE, and the nucleotide sequence has been determined. The derived amino acid sequence shows the protein, starting with residue 23, is similar to eukaryotic mitochondrial MnSODs. The amino acids that constitute the Mn-containing active site of blue crab cytosolic MnSOD are identical to the conserved residues that make up the active site of the mitochondrial SODs. Cloning of mitochondrial MnSOD is currently underway.

CELL PROLIFERATION IN ELASMOBRANCH AND BONY FISH TISSUES

Nancy J. Brown-Peterson* and William E. Hawkins, University of Southern Mississippi, Institute of Marine Sciences, Ocean Springs, MS 39564

Cell proliferation is important in cellular growth, damage and repair. The proliferating cell nuclear antigen (PCNA) immunohistochemical assay identifies proliferating cells and is important for determining baseline and abnormal proliferative rates in tissues. The PCNA assay has been primarily used with mammalian tissues; little information exists regarding its use in lower vertebrates. We used the PCNA assay to compare baseline cell proliferation in liver, kidney and gill tissues from nine phylogenetically distinct fish species: blacktip shark, Carcharhinus limbatus; Atlantic stingray, Dasyatis sabina; spotted gar, Lepisosteus oculatus; hardhead catfish, Arius felis; guppy, Poecilia reticulata; Japanese medaka, Oryzias latipes; spot, Leiostomus xanthurus; tilapia, Tilapia mossambica x nilotica and southern flounder, Paralichthys lethostigma. Liver proliferative labelling indices (LLI) were significantly different among species (p=0.002), with guppy showing LLI significantly higher than shark, ray, medaka and tilapia. Kidney tubule proliferative labelling indices (KLI) were significantly different among species (p=0.0001), with guppy KLI significantly higher than all other species. The greatest taxonomic proliferative differences were evident in gill tissue. Elasmobranch secondary lamellae were highly proliferative at the tips, whereas bony fishes showed no proliferation at the tips and moderate proliferation at the bases. These data show that the PCNA assay is effective in a wide range of fish species and suggest some taxonomic differences in gill tissue proliferation.

1:30 IMMUNOCYTOLOCALIZATION OF GLUTAMINYL CYCLASE IN ALFALFA BY LIGHT AND ELECTRON MICROSCOPY

Sandy West*, Robert C. Bateman, Jr., and Kenneth J. Curry, University of Southern Mississippi, Hattiesburg, MS 39406

Glutaminyl cyclase (QC) is a post-translational enzyme responsible for the cyclization of N-terminal glutamine into pyroglutamic acid with the additional release of ammonia. Alfalfa seedling tissues were fixed and embedded in ERL4206 (Spurr's resin). Tissues were sectioned for light and electron microscopy and sections were probed with a rabbit antibody raised against QC purified from papaya latex. A commercial goat-anti-rabbit IgG gold conjugate was used as a second antibody to visualize the primary antibody. Initially, alfalfa seedlings between 24 hrs and 14 days were probed at the light level. At this level, QC was seen to be abundant in early cotyledon tissue and 7 day hypocotyl. We determined that the germinating cotyledon tissues were the most promising for a QC study. Examination of early cotyledon tissue indicates that QC is associated with protein bodies and intercellular material between cell walls. This helps support the hypothesis that QC is involved in the export of proteins from cotyledon protein storage to other parts of the growing plant.

1:45 EVIDENCE FOR A NOVEL FORM OF GLUTAMINYL CYCLASE IN BOVINE SPLEEN

Paul A. Sykes*, Stephanie J. Watson, and Robert C. Bateman, Jr., University of Southern Mississippi, Hattiesburg, MS 39406-5043

Glutaminyl cyclase (QC) catalyzes the posttranslational conversion of N-terminal glutaminyl residues to pyroglutamyl residues in numerous mammalian bioactive peptides. The bovine pituitary QC was originally discovered in 1987 and its cDNA sequence and mRNA expression in various bovine tissues have been reported. However, no QC mRNA expression was observed in spleen, a major source for IgG which contains a pyroglutamyl residue terminating the heavy chain sequence. We have detected QC catalytic activity in spleen and have partially purified the spleen enzyme by ammonium sulfate fractionation and anion exchange chromatography. Spleen QC appears to be more soluble in ammonium sulfate and to have a significantly higher pH optimum than the previously reported pituitary enzyme. Preliminary experiments also suggest that the spleen QC may have an altered specificity for glutaminyl-peptide substrates. These apparent variations in properties suggest the possibility of multiple forms of QC, though it is not yet certain whether these differences are the product of separate genetic coding regions or posttranslational processing.

2:00 A CHEMILUMINESCENT ASSAY FOR DIAMINES IN SPOILED SEAFOOD

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

A quick, sensitive chemiluminescent assay was developed to measure the levels of cadaverine and putrescine. Elevated levels of these diamines are produced during the decomposition of both finfish and shellfish. The coupled reaction begins with the production of hydrogen peroxide due to the interaction of the diamine and diamine oxidase. This hydrogen peroxide in turn is reacted with luminol in the presence of horseradish peroxidase to output relative amount of light. The amount of diamine can thus be indirectly determined by measuring the amount of light generated from the luminol reaction. This reaction is significantly affected by variations in buffer type, pH levels, incubation time, and concentrations of HRP and luminol. These conditions were optimized thus generating a linear relationship between the light intensity and the concentration of diamine in the 10-100*M range.

2:15 THE EFFECTS OF CORTICOSTERONE ON BEHAVIOR

DeMeka R. Bean* and Rebecca Holberton, University of Mississippi, University, MS 38677

Corticosterone, the main hormone of stress in birds, acts to obtain energy in the form of glucose from skeletal muscle when fat stores are depleted. Long term use of this process destroys the muscle and lessens the mobility of an animal. This study sought to find if a rise in corticosterone level during periods of stress also increased the locomotor activity and pecking behavior in birds. An increase in these variables would increase the animal's chances of finding food, thus eliminating the body's need for gluconeogenesis (break down of skeletal muscle to glucose) and salvaging needed skeletal muscle. This experiment was broken down into two separate experiments, (1) organizational effects of corticosterone of feeding behavior and locomotor activity: exposure during early development in chick embryos, and (2) activational effects of corticosterone on feeding behavior and locomotor activity: exposure after hatching. In experiment one, chick eggs were injected with high, low, and no levels of corticosterone early in development. This particular portion is still in progress, so no results have been achieved. In experiment two, 7-day old chicks were injected with high, and no corticosterone and their steps and pecking counted respectively. There was no significant difference between the experimental chicks and control chicks' locomotor activity, though the experimental groups displayed slightly more activity. The pecking behavior of the experimental chicks was lower than the controls, though this difference was not significant enough to reject the null hypotheses (Null Hypotheses: There is no difference between chicks treated with corticosterone and ones that were not.)

2:30 Break

2:45 PATHOGENESIS AND MORPHOGENESIS OF WHITE SPOT VIRUS (WSV) IN PENAEID SHRIMPS FROM SOUTH CAROLINA

R.M. Krol¹* , William E. Hawkins¹, Robin M. Overstreet¹, K.K. Vijayan², and J.M Lotz¹, ¹Institute of Marine Sciences, Gulf Coast Research Laboratory, Ocean Springs, MS 39566, and ²Central Institute of Brackishwater Aquaculture, Madras, India

White spot virus (WSV) , a significant disease of cultured shrimp in Asia, has rarely been reported in the U.S. In 1996, a presumptive WSV was detected in cultured shrimps from South Carolina. Gill, cuticle, and lymphoid organ from five Penaeus vannamei and five P. setiferus from affected raceways were examined by transmission electron microscopy (TEM). No virions were found by TEM, but three P. setiferus were positive for the virus by bioassay. One of those three shrimp was the WSV source of virus for experimental studies on P. vannamei. In several experimental exposures, gill, epidermis, lymphoid organ, foregut, heart, muscle, and antennal gland were positive for WSV when examined by TEM. WSV was a rod-shaped, enveloped, non-occluded virus that replicated in hypertrophied nuclei containing a central virogenic stroma and marginated chromatin. The virogenic stroma contained membranous profiles, stacks of tubules, small empty capsids surrounded by open-ended envelopes, and large, dense virions, with a nipple-like projection, sometimes lined up in parallel arrays. Lysed cells released virions. Clumps of virions occured in the cytoplasm of lymphoid organ cells. This study showed that the pathogenesis and ultrastructure of the South Carolina virus resembled Chinese and Indian WSV-infected P. vannamei from experimental infections. Supported by USDA, CSREES Grant 96-38808-2580.

3:00 SDS--PAGE ANALYSIS OF FLAGELLA FROM TRICHOMONAS VAGINALIS AND TRITRICHOMONAS FOETUS

LaNassa P. Barber* and Phillip F. Jemilohun, Jackson State University, Jackson, MS 39217

Trichomonas vaginalis and T. foetus are parasitic protozoans causing a sexually transmitted disease known as trichomoniasis in humans and animals respectively. About 200 million people are infected annually world wide. And trichomoniasis in animals has been a source of great economic lost to the farmers. In this study the flagella of both parasites will be isolated and purified by sucrose density centrifugation. The flagellar proteins will be analyzed on an SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) for the comparision of protein bands of the two parasites.

3:15 INCORPORATING INTERNET COMPUTATIONAL TOOLS INTO THE BIOCHEMISTRY TEACHING LABORATORY

Jeffrey Temple*, Jeffrey Evans, and Robert C. Bateman, Jr., University of Southern Mississippi, Hattiesburg, MS 39406-5043

The internet has allowed biochemists around the world free access to many powerful computational tools for analysis of biological macromolecules. We have begun to incorporate these tools into one of our biochemistry teaching laboratories through a homepage which provides hands-on experimental protocols as well as exercises in protein and nucleic acid analysis using internet tools. The student can use these tools on any computer with access to the world wide web and thereby will experience a sample of the same analysis a researcher will perform on a newly discovered biopolymer sequence. The homepage is open to the public and is available at http://ocean.st.usm.edu/~jstemple/biochemlab/BIOCHEM.html.