CELLULAR, MOLECULAR AND DEVELOPMENTAL BIOLOGY.

Chair: David Carson, Mississippi University for Women 

Vicechair: Peter Butko, University of Southern Mississippi

THURSDAY MORNING

Petit Bois

8:45 Introduction

David Carson, Mississippi University for Women, Columbus, MS 39701

9:00 SUPPRESSORS OF THE ASPERGILLUSNIDU-LANS NIMX2 MUTATION AFFECT SEPTATION, BRANCH1NG, DEVELOPMENT, AND NUCLEAR DIVISION

Brett Carter [*], Robert Carter, Peyton Hays, Angela Payne, Melanie Schrader, Chad Young, and Sarah Lea McGuire, Millsaps College, Jackson, MS 39210

The [p34.sup.cdc2] kinase is believed to trigger mitotic entry in all eukaryotes. In the filamentous fungus Aspergilus nidulans, the functional [p34.sub.cdc2] homolog, [nimX.sup.cdc2], is required for both the GI/S and G2/M transitions. To identify proteins that interact with [nimX.cdc2], we generated extragenic suppressors of the heat-sensitive nimX2 mutation. Four strains containing clonable extragenic suppressor mutations have been isolated. The suppressor genes were designated snxA-snxD (for suppressor of nimX). The snxA1 mutation halts the cell cycle in G 1 at 20[degrees]C, producing abnormal nuclei. snxA maps to chromosome II and is not allelic with any other Aspergullus cell cycle genes located on chromosome II. SnxB1 leads to a hyperbranching, hyperseptate phenotype, and preliminary evidence suggests that this maps to chromosome I or VI. SnxC1 maps to chromosome I and affects septation and conidiophore structure, leading to sparse conidiation. The conidiophores contain septae (absent in wild type) an d many have abnormal developmental structures such as hyphae growing from metulae. SnxD1 is similar to snxB1 in that it also is hyperseptate and hyper-branching; preliminary evidence suggests that it maps to chromosome VII or VIII. Further analysis and cloning of these [nimX.sup.cdc2] suppressors will lead to a better understanding of cell cycle regulation in Aspergilus nidulans and of how the cell cycle relates to development, branching and septation.

9:15 POLYSACCHARIDE CAPSULE OF GROUP B STREPTOCOCCUS IS NOT THE ANTIGENIC TARGET FOR THE PROTECTIVE NATURAL IgM IN MICE

Angela H. Adains [*] and Peter Butko, University of Southern Mississippi, Hattiesburg, MS 39406

It was previously found that normal mouse serum contains natural IgM antibody which is protective against infection by group B Streptococcus (Streptococcus agalactiae, GBS). The long-term objective of this work is to identify the molecular target for this IgM. The bacterial surface exhibits three types of molecules: capsular polysaccharide, surface proteins, and the cell wall peptidoglycan. The capsule, being the most conspicuous among the surface structures, was chosen first to be tested for 1gM binding. Three strains with varying degrees of encapsulation were used in this study: a highly encapsulated strain M781; strain COHl with medium encapsulation; and an acapsular mutant strain COHl-13. An enzyme-linked immunosorbent assay (ELISA) performed on the suspension of GBS with normal mouse serum was used to detect bound IgM. The amount of bound IgM was found to vary inversely with the amount of capsule expressed by the bacteria. It is concluded that the polysaccharide capsule is not the target for murine natu ral IgM, but rather it attenuates binding of the antibody to its target molecule, which may be a protein or peptidoglycan.

9:30 SURFACE ASSEMBLY OFANAMPHIPATHIC FUNGAL HYDROPHOBIN AND AN ASSOCIATED POLYSACCHARIDE

Paul Stroud [*], Gregory G. Martin, Gordon C. Cannon, and Charles L. McCormick, University of Southern Mississippi, Hattiesburg, MS 39406-5043

The hydrophobin protein, Sc3p, is excreted into the growth medium by the wood-rotting fungus Schizophyllum commune when grown as a monokaryon in liquid culture. Co-excreted into the growth medium with Sc3p is a high molecular weight polysaccharide, schizophyllan. Sc3p has many properties including the ability to self assemble at both hydrophilic surfaces such as mica and hydrophobic surfaces such as Teflon, rendering them hydrophobic and hydrophilic respectively after surface coverage. Interactions of Sc3p with schizophyllan have been shown to be necessary for stable hydrophilic surface coverage but is still unclear in the specific role schizophyllan plays in self assembly at the surfaces as well as the stability of the Sc3p in solution. An elucidation of the structure function relationship of the Sc3p for binding hydrophobic and hydrophilic surfaces has been undertaken, as well as the elucidation of the role the polysaccharide, schizophyllan, plays in binding these surfaces.

9:45 Invited Speaker

CLONING AND CHARACTERIZING THE CALICHEAMICIN SELF-RESISTANCE GENE IN MICROMONOSPORA ECHINOSPORA

Ross E. Whitwam [1][*], Joachim Ahlert [2] , Theodore. R. Holman [3], Mark Ruppen [3], and Jon S. Thorson [2], (1.) Mississippi University for Women, Columbus, MS 39701; (2.) Memorial Sloan-Kettering Cancer Center, New York, NY 10021; and (3.) Wyeth-Ayerst Research Division of American Home Products, Pearl River, NY 10965

Calicheamicin (CLM) is a DNA-binding and cleaving enediyne antibiotic produced by the soil Actinomycete Micromonospora echinospora ssp. calichensis. While the oxidative mechanism by which CLM cleaves DNA is well understood, nothing is known of how M. echinospora synthesizes this architecturally complex compound or of how M. echinospora controls its reactive nature and toxic effects. Screening a cosmid library from M. echinospora revealed a clone resistant to high levels of CLM. Subcloning and screening localized CLM resistance to a single gene we have labeled calC. Expression, purification, and preliminary characterization of the corresponding protein as a maltose-binding protein fusion product reveals that CalC is a none-heme iron metalloprotein that can function in vitro to inhibit CLM-induced DNA cleavage.

10:15 Break

10:30 Divisional Poster Session

THE ECOLOGY AND MICROBIAL FLORA OF THE CARABID BEETLES

Monroe Parker [*] and Alfred Mikell, Alcorn State University, Alcorn State, MS 39096 and University of Mississippi, University, MS 38677

Ecology is the study of the relationship of organisms and their environments. In this research we examined three genera from the Carabid Family: the Brachinus, the Tiger Beetle, and the Chalaenius. These insects live near streams and rivers. Beetles eat mites and insect larvae. The Genus Brachinus is known as the Bombardier Beetle. When these small insects feel threatened, they release hydroquinone and hydrogen peroxide from a pair of glands in the tip of its abdomen. An inhibitor is also added to this mixture to keep the chemicals from exploding inside the beetle. When released all these chemicals are in the form of a gas. This mixture is very caustic from the number of reactive oxygen species generating during the course of this reaction. The focus of the study is to compare the bacteria found inside the intestinal flora of these beetles with the bacteria found on their surfaces. This comparison is necessary to determine if isolates from the Bombardier's spray are different and adapted to this extreme envi ronment. Also, the anaerobic process and the effects of oxidative stress on these amazing creatures were examined. Oxidative stress can cause damage to a certain molecule or the entire organism. It causes damage by reactive oxygen species. Once biochemical adaptations are established, they may well give us insights into coping with oxidative stress.

THE EFFECTS OF HEAVY METALS ON HYPERTHERMOPHILIC ARCHAEA 

Patrick D. Williams [1][*], Rafael Montalvo [2], and Paul Blum [2], (1.) Alcorn State University, Alcorn State, MS 39096-7500 and (2.) University of Nebraska-Lincoln, Lincoln, Nebraska 68588

Little is known about the effects of heavy metals on hyperthermophiles, specifically hyperthermophilic Archaea. This project is to compare two strains of S. solfataricus (98/2 and a natural isolate named CoSo 3) and a mutant. The mutant is a derivative of 98/2 with a 15 kb deletion. Inside the deletion is a putative heavy metal resistance gene. Studying the effects of cadmium and mercury toxicity will determine if the gene conveys resistance towards these two heavy metals. This experiment will also determine if these putative mercury and cadmium genes can be used as selectable markers on future genetic systems in S. solfataricus.

THE MEDICAL CONTRIBUTIONS OF THE BOMBARDIER BEETLE

Fameeka S. Jenkins [*] and Alfred Mikell, Alcorn State University, Alcorn State, MS 39096 and University of Mississippi, Oxford, MS 38677

Bombardier beetles are named for the explosive, caustic chemicals ejected from their abdomen. These chemicals represent a biochemically complex defense mechanism. The research explored the physiological aspects of the beetle's chemical defense mechanisms. The goal was to analyze the presence of bacteria in the abdomen of the Bombardier beetle. The hypothesis was that there may be an undiscovered species of bacteria that would lead to an advancement in medical knowledge. The discovery was hoped to provide further understanding of DNA agents and cancer mutations. Procedures in starting this process included the dissection of the beetle's abdominal-final glands and applications of sensitive techniques designed to detect the presence of bacteria. The determination of oxidative stress along with the tolerance level of heat were to be observed. Several trials were performed to compare the results and obtain conclusive data. Through the data obtained, the medical implications of the beetle's defense mechanism were to be determined.

EXPRESSION OF MUTANT P67 IN E. COLI

Quatrisa Douglas [1][*], Rekha Datta [2], and Bansidhar Datta [2], (1.) Alcorn State University, Alcorn State, MS 39096 and (2) Universtiy of Nebraska, Lincoln, NE 68588

The initiation of protein synthesis in mammals is largely regulated at the level of phosphorylation of eukaryotic initiation factor-2 (eIF2). When phosphorylated by eIF2 kinases, it is inactive in protein synthesis initiation. P67, a cellular glycoprotein, protects eIF2 from phosphorylation by its kinases. Understanding the interaction between p67 and eIF2 will help to elucidate the detail mqchanism of protection ofelF2 from phosphorylation. We introduced mutations at the putative active site of p67 and subcloned the mutant gene into an E. coli expression vector. Later, we wanted to isolate the mutant p67 protein and to study the interaction between eIF2 and p67 in vitro.

THE EFFECT OF MET-ENKEPHALIN AND RELATED PEPTIDES ON 1NTERLEUKIN-2 (IL-2) PRODUCTION

Corey Montgomery [*], Natalia Gordonov, and Robert C. Sizemore, Alcorn State University, Alcom State, MS 39096

This project will determine the effect of metenkephalin, Tyr-Gly-Gly (YGG) and Tyr-Gly (YG) on the in vitro production of the cytokine, IL-2. Murine splenocytes placed in serum-free medium will be stimulated with concanavalin A with or without phorbol 12-myristate 13-acetate (PMA). Various concentrations of met-enkephalin, or the first two (YG) or three (YGG) amino acids of the enkephalins, will then be added to determine if the immune parameter (IL-2 production) is enhanced or suppressed. The amount of IL-2 in the tissue cultures will be measured by Enzyme-Linked Immunosorbent Assay (ELISA). These studies will help elucidate the relationship between the central nervous system and the immune system by examining the importance of endogenous peptides on the regulation of immune responses. Supported by NIH grant # 3 SO6 GM55356-02S1.

CLONING AND EXPRESSION OF TWO COTTON FIBER cDNAS ENCODING MYB-TYPE PROTEINS

Chuan-Yu Hsu [1][*], Roy G. Creech [1], Johnie N. Jenkins [2], and Din-Pow Ma [1], (1.) Mississippi State University and (2.) USDA/ARS Crop Science Research Laboratory, Mississippi State, MS 39762

Plant MYB-type proteins contain DNA-binding domains which regulate both general and specific gene transcription. Two cotton cDNA clones (GhMYB1 and GhMYB2), which encoded MYB-like proteins, have been isolated from 15 DPA (days post anthesis) fiber mRNAs by using 5' and 3' RACEs (rapid amplification of cDNA ends). The derived amino acid sequences from these two full-length fiber myb cDNAs show that the basic N-terminal DNA-binding domains of cotton fiber MYBs are highly homologous to other plant MYB-type proteins. Based on the comparison of cDNA and genomic sequences, each of the two myb genes contains two introns located in the coding region of the gene. The expression patterns of these two myb genes were studied by Northern blotting analysis using total RNA from different cotton tissues. The GhMYB 1 and GhMYB2 cDNAs were cloned into an expression vector, pET-32b(+), and the expressed His-MYB fusion proteins were purified and used for DNA-binding studies.

CHARACTERIZATION OF AJUBA, A NOVEL LIM PROTEIN, BY PROTEIN-PROTEIN INTERACATION SCREENING

Dayle Houston [1][*], Henry Outlaw [1], and Gregory D. Longmore [2], (1.) Delta State University, Cleveland, MS 38733 and (2.) Washington University, ST. Louis, MO 63130

Ajuba is novel group 3 LIM protein along with zyxin, paxillin, trip6 and others. It is 55 kDa in size and is characterized by a proline-rich pre-LIM region containing two potential SH3 recognition sites, and three tandemly arranged LIM domains located at the c-terminus. So far, it has been determined that ajuba functions to stimulate MAP kinase activity, interact with Grb2 in a serum-dependent manner, and promote meiotic maturation in Xenopus oocytes. Its stimulation of MAP kinase activity is of interest because it occurs in serum-starved cells, indication that molecules besides Grb2 may aid in this function. Enhancement of MAP kinase activity has also been shown to be the mechanism by which ajuba promotes meiotic maturation. Ajuba has been found in tissues of adult mouse skin, nervous system, and genitourinary tract and all mouse embryonic germ layers. My work has been directed toward using the yeast two-hybrid selection system to screen a 9day mouse embryo cDNA library for molecules that interact with ajub a. Though the screening is still in progress, two positive interactions have been identified: myosin Va and profilin.

BACTERIAL MOTILITY AND THE GENERAL SECRETORY PATHWAY

Yang Ruifeng [*], Steven E. Passmore, and Donna L. Marykwas, University of Southern Mississippi, Hattiesburg, MS 39406

The bacterial motor protein FliM interacts with the General Secretory Pathway protein GspE in a two-hybrid screen for protein-protein interactions. FliM is required for flagellum assembly, but the role of the General Secretory Pathway in Escherichia coli is not known. To test the role of GspE in bacterial motility, the gene coding for GspE was deleted from E. coli. A mutant in which the gspE gene bore a large insertion was nonmotile, but when the insertion was resolved by homologous recombination to yield a deletion within gspE, this deletion mutant turned out to be motile. This suggests one possibility, that the insertion mutant could be polar, turning off the expression of genes downstream of gspE in the same operon, and that one of these downstream genes is required for motility. However, there are two other chromosomal gene products in E. coli that are highly similar to GspE. This suggests a second possibility, that one of these GspE homologues (not GspE itself) is actually the one involved in motility, or a third possibility, that GspE and its homologues share redundant functions. To test these possibilities, we are constructing mutations (transposon insertions) within each of these three genes and in the neighboring genes of their respective operons. If the single gene knockouts all swim, we will construct each double mutant and the triple mutant and test their motility.

REPRODUCTIVE EFFECTS OF ESTROGENIC AND ANTIESTROGENIC CHEMICALS ON SHEEPSHEAD MINNOWS

Arthur Karels [*] and Marius Brouwer, University of Southern Mississippi, Institute of Marine Sciences Ocean Springs, MS 39566-7000

Accumulating scientific data suggests that many man-made chemicals, such as alkylphenmols/PCBs, have potential to adversely affect the endocrine system of humans/wildlife. Some of these compounds have estrogenic/antiestrogenic effects, collectively they are called endocrine disrupting chemicals (EDCs). These compounds can activate genes of the reproductive system, such as endogenous vitellogenin (a precursor to eggyolk protein), by activating the estrogen receptor (ER). Since little is known about effects of environmental estrogens/antiestrogens on reproductive fitness of estuarine/marine fish, we exposed adult lab-reared male sheepshead minnows (Cyprinodon variegatus) to estrogenic 4-tert-octylphenol (OP) and females to antiestrogenic cadmium (Cd) in aquaria to examine 1) fecundity of [F.sub.0] generation, 2) embryonic development/egg hatching/survival rate of [F.sub.1] generation fly, 3) vitellogenin levels in male/female blood sera to determine correlation to adverse changes in reproductive function and 4 ) gonadal development. A similar 3 week field exposure was set up using cages near an outfall of a sewage treatment plant. Initial results appear to show that higher levels of exposure adversely affect fecundity. Vitellogenin/histological analysis are underway. In addition to reproductive studies, we have isolated an ER cDNA fragment from total RNA of gravid female fish livers, which will be used for cloning of complete ER cDNA for future bioassay development. Sequence of partial ER cDNA shows homology to related fish.

FUNCTION OF PROTEIN KINASE (PKB) GENE IN DICTYOSTELIUM DISCOIDEUM

Kesi L. Gaskin [*] and Alice M. Powell, Alcorn State University, Alcorn State, MS 39096

Dictyostelium discoideum (Dd) is a eukaryotic organism with a relatively simple life cycle that comprises a vegetative growth phase and a differentiation phase. Chemotaxis provides a means for microorganisms to respond to environmental gradients of potential nutrients and toxins. Usually, this phenomenon results in motility towards or away from substances. In this experiment, a small population assay was used to determine the effects of cAMP and folic acid on the chemotactic movement of Dd. Three different cell lines of Diclyostelium discoideum was used in this assay: AX-3 (wild-type), 2D2 (knockout), and Carl/Car3-. Car1-/Car3- cells was used as the control. The cells were exposed to folic acid and cAMP for zero, ten, and twenty minute time intervals. With a concentration of one micrometer for cAMP and 100 micrometers for folic acid, we determined that the AX-3 cells and the 2D2 cells did not show very much movement towards the stimulus. However, due to the way the cells were set after each time limit, it w as hard to tell which cells moved the greatest distance or if they moved at all.

THE EFFECT OF HEAT ON THE HEAT SHOCK RESPONSE OF INDUCED WOUNDS IN RATS

April Watson [*], Leroy Johnson, and Alice M. Powell, Alcorn State University, Alcorn State, MS 39096

This study was to determine the effect of heat stress on the healing rate of induced wounds in rats. All wounds were superficial with uniformed lengths and depths. The outcomes of this study show an increased wound healing rate and a higher quality of wound heal. Animal studies (Riberro et al., 1995) have shown that the response is attributable to the elicitation of the "housekeeping" heat shock proteins. This research was an effort to examine the possible change in the total protein profiles using SDS-PAGE in heat treated animals. The increased or sudden appearance of new bands indicated possible heat shock protein response to the particular stress. Rat tissues were minced with sterile surgical blades and incubated in lysis buffer for thirty minutes on ice. The proteins were stored at -20[degrees]C and used as needed. Rats heat treated show an increase in the expressed profile of a total protein extract run on polyacrylamide gel electrophoresis. These proteins may not be chaperonins, but there is a correlat ion to the expression of these proteins and induction by heat stress.

THURSDAY AFTERNOON

Petit Bois

1:45 SIGNALING PATHWAYS INVOLVED IN SCATTER FACTOR-MEDIATED C-MET INDUCTION IN HUMAN GLIOBASTOMA CELLS

Claretha Nichols [1][*], Srikanth Ranganathan [2], Roger Abouader [2], and John Laterra [2], (1.) University of Southern Mississippi, Hattiesburg, MS 39406-5043 and (2.) Johns Hopkins University School of Medicine, Baltimore, MD 21205

Scatter Factor (SF) is a multifunctional growth factor that exerts its action through its only receptor, the tyrosine kinase c-met. SF and c-met play a role in tumorigenesis and malignant progression of a wide variety of tumors, including human glioblastomas. SF and c-met expression in human gliomas correlate with tumor grade. Over-expression of SF in glioblastomas increases their malignancy, whereas; knock-down of SF/c-met expression inhibits glioblastoma growth in vivo and in vitro. C-met in glioblastomas is induced by SF. Parts of the signaling pathway involved in c-met induction by SF have previously been characterized by our lab. This study shows that Ras and AP-1 are also involved in the signaling pathway of c-met induction by SF.

2:00 DETERMINATION OF cDNA SEQUENCES OF TWO CADMIUM-INDUCIBLE AND ONE COPPER-SPECIFIC METALLOTHIONEIN IN THE BLUE CRAB, CALLINECTES SAPIDUS, AND THEIR POTENTIAL AS BIOINDICATORS OF TOXIC TRACE METALS

Rachel A. Syring [*], Thea Hoexum-Brouwer, and Marius Brouwer, University of Southern Mississippi, Gulf Coast Research Laboratory, Ocean Springs, MS 39564

Copper is an essential metal which has the potential to be toxic, like cadmium and mercury, when occurring in excess. The blue crab, Callinectes sapidus, has two cadmium-inducible (CdMT-I and CdMT-II) and two copper-inducible (CuMT-I and CuMT-II) metallothionein (MT) isoforms comprising a mechanism of defense against metal-mediated oxidative damage. CdMT-I and CuMT-I are the same protein containing different metals, while CuMT-II is a unique, copperspecific isoform. The objective of this project was to determine the complementary DNA (cDNA) sequences of CdMT-I/CuMT-I, CdMT-II, and CuMT-II. The cDNA was isolated from hepatopancreas tissue of cadmium and copper exposed blue crabs using degenerate gene specific primers synthesized from the N-terminal amino acid sequence. The cDNA was PCR amplified using 3' Rapid Amplification of cDNA Ends (RACE), cloned, and sequenced. Upon 3' determination, reverse and complementary gene specific primers were synthesized and the 5' end was PCR amplified using 5' RACE, cloned, and sequenced. The 3' and 5' sequences were combined to obtain the complete cDNA sequences. Alignment of the cDNA species indicates that each isoform is encoded by a distinct gene. CuMT-II appears to have evolved prior to the CdMTs in view of its greater similarity to molluscan MTs rather than other crustacean MTs.

2:15 EXPRESSION OF GLUTAMINYL CYCLASE IN DROSOPHILA MELANOGASTER SCHNEIDER 2 CELLS

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

Glutaminyl cyclase converts an N-terminal glutamine to pyroglutamic acid to biologically activate many proteins and peptides. It has previously been expressed in several Eschrichia coli systems; however, production of the protein is not sufficient enough for x-ray crystallization studies. A Drosophila Expression System (DES) was used for the expression of glutaminyl cyclase (QC). PCR primers were designed to amplify the QC coding region including the signal sequence. Following analysis by gel electrophoresis, the PCR product was cloned into the pMT/V5-His-TOPO vector. The insert and flanking vector regions were sequenced for confirmation. This vector was then transformed into Drosophila Schneider 2 (S2) cells. After induction of the metallothionein promoter with copper sulfate, the protein will be purified from the cells using anion-exchange chromatography. Finally, a stable cell line will be generated from cotransfection of this QC expression vector and a selection vector, pCoHYGRO. The cell line will produ ce the milligram amounts of protein needed for x-ray crystallization studies.

2:30 FUNDAMENTAL MECHANISMS OF JANUS KINASE ACTIVATION AND INACTIVATION

Roy J. Duhe, University of Mississippi Medical Center, Jackson, MS 39216

Janus protein-tyrosine kinases (JAKs) serve as essential early mediators of cytokine-initiated signal transduction. Precise control of the amplitude and duration of JAK catalytic activity is crucial for regulating normal physiological responses; hyper- or hypo-activation of JAKs can result in leukemias or in immunosuppressive disorders, respectively. I will describe the research I conducted at the National Cancer Institute--Frederick Cancer Research and Development Center to elucidate two independent mechanisms of JAK regulation. My lab will continue to investigate the molecular foundations of both the autoinhibitory and the redox-regulated mechanisms of controlling JAK activity. Ongoing efforts to improve the speed and quantitative accuracy of assays for JAK activity may not only help to better define the role of JAK dysregulation in disease etiology, but also help to rapidly screen candidate drugs for disease therapy.

2:45 Break

3:00 ISOLATION AND CHARACTERIZATION OF A COTTON FIBER cDNA ENCODING A AUX/IAA PROTEIN

Shaohua Yu [1][*], Johnie N. Jenkins [2], Roy G. Creech [1], and DinPow Ma [1], (1.)Mississippi State University, and (2.)USDA/ARS, Crop Science Research Laboratory, Mississippi State, MS 39762

Auxin is an important phytohormone which controls cotton fiber development and differentiation. The auxin-inducible Aux/IAA gene family, a group of early auxin-response genes, have been shown to be essential in auxin signaling. Based on the conserved peptide sequences of AUX/IAA proteins, QVVGWP and WMLVGDVPW, two sets of oligonucleotides were synthesized and used as PCR primers to amplify cDNAs synthesized from 15 DPA (days postanthesis) fiber mRNAs. The amplified PCR prodjucts ([sim]350 bp) were cloned into the pGEM-T easy vector and sequenced. Sequencing data verified that these PCR products are cDNAs encoding parts of AUX/IAA proteins. Additional primers based on the partial IAA cDNA sequences were then synthesized and used in 5' and 3' RACEs (rapid amplification of cDNA ends) to clone the full length cDNAs. The derived amino acid sequence from a full-length IAA cDNA revealed the encoded AUX/IAA protein consists of four highly conserved domains (domains I, II, III, and IV). Domain III contains an amphipat hic [beta][alpha][alpha]-fold, found in [beta]-ribbon DNA-binding domains of prokaryotic repressor proteins.

3:15 CLONING OF THE cDNA FOR THE 68 KDA DNA BINDING PROTEIN OF GLYCINE MAX CHLOROPLAST NUCLEOLDS

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

Chloroplast DNA (ctDNA) is compacted by association with various proteins into structures called nucleoids. Nucleoid structure varies during development from proplastids into mature chloroplasts, suggesting that it plays a role in the regulation of DNA replication and transcription of ctDNA. Identifying the function of the proteins that are associated with nucleoids may lead to a better understanding of these processes. Previously, a 68 kDa protein had been isolated from chloroplast nucleoids of Glycine max that shows DNA compaction in vitro. The N-terminal 15 amino acids showed homology with several ferredoxin sulfite reductases of higher plants. These enzymes reduce sulfite possibly for sulfur assimilation in cysteine biosynthesis. This sequence was used to design degenerate primers for reverse transcription of soybean RNA and PCR amplification of the cDNA. A complete cDNA clone will provide insights into structure, function, expression patterns and possible post-translational modifications of the protein, and will allow creation of antisense and over expressing transgenic Arabidopsis lines.

3:30 CLONING AND CHARACTERIZATION OF CANINE-LIVER UDP-GLUCURONOSYL TRANSFERASES

Bernita M. Finley [*] and Christopher King, Alcorn State University, Alcorn State, MS 39096 and Merck Pharmaceutics, Rahway, NJ 08817

Dog liver microsomes contain UDP-glucuronsyl transferases which are important in detoxifying many endogenous and exogenous compounds such as morphine, bilirubin, and estrodiol. Studies were designed to isolate and clone a UGT-specific gene from canine liver, using an antibody and/or an oligonucleotide probe. Many positive plaques were seen, and they are being isolated. Secondly, another study was done to reveal substrates involved in glucuronidation in dog liver microsomes. Using glucuronidation assays, a significant amount of morphine and estrodiol glucuronide was seen. UGT assays reveal that dog liver microsomes metabolizes morphine to morphine-3-O glucuronide as well as morphine-6-O glucuronide to most potent form of morphine. Lastly, western analysis show that UGT1A1, -2B7, and -1A6 are UGT like isoform may be present in dog liver microsomes. These studies suggest that dog have similar UGT-isoforms to rat and human.

3:45 ISOLATION OF AN INSULIN-LIKE GROWTH FACTOR-I (IGF-I) COMPLEMENTARY DNA FROM CHANNEL CATFISH (ICTALURUS PUNCTATUS)

L.A. Clay [1,2][*] G.C. Waldbieser [2], and S.Y. Wang [1], (1.) University of Southern Mississippi, Hattiesburg, MS 39406 and (2.) U.S. Department of Agriculture-ARS, Stoneville, MS 38776

Insulin-like growth factor-I (IGF-I), previously known as somatomedin C, has been shown to be essential for normal development and growth in several species. This gene encodes a single chain 70-residue basic peptide with 3 intra-chain disulfide bridges. IGF-I is produced in the liver, brain and other tissues, and is considered to be the principal mediator of growth hormone action on growth and development. In order to better understand the endocrine regulation of growth in catfish, we have cloned and sequenced a 516 bp IGF-I cDNA in channel catfish. We constructed degenerate primers based upon mammalian and fish IGF-I amino acid sequence and used this information to amplify channel catfish IGF-I via 3'-and 5'-Rapid Amplification of cDNA Ends (RACE). Several clones were obtained from total RNA from adult liver and brain. A partial open reading frame in the cDNA encoded 109 amino acids of channel catfish IGF-I which shared 91% identity with the common carp amino acid sequence. Results from this study will be ut ilized to determine tissue- and developmental stage-specific levels of expression in channel catfish in order to assess the role of IGF-I in catfish growth.

4:00 TRANSFECTION STUDIES WITH A CANDIDATE HUMAN IMIDAZOLINE RECEPTOR cDNA

Michael Chen [*], Mary Elise Lutrick, He Zhu, James Baldwin, Victor Stuckey, and John Piletz, University of Mississippi Medical Center, Jackson, MS 39216

Imidazoline receptors (IR) are involved in blood pressure and stress responses from brain nuclei. We have previously cloned a candidate [IR.sub.1] cDNA by screening from a human hippocampal cDNA expression library with two IR-selective antisera (J. Autonomic Nervous System, 72: 98--110, 1998; DNA and Cell Biology, submitted). That cDNA, designated imidazoline receptor antisera-selected (iras-1) cDNA, contains 5,131 bp and is predicted to encode a 167 kDal protein. Transfection of iras-1 cDNA into CHO (Chinese hamster ovary) cells results in a three-fold increase in the [B.sub.max] of [[[blank].sup.251]I]-p-iodoclonidine for [I.sub.1] sites; with a high-affinity component revealed only in transfected CHO cells for the [I.sub.1] ligands, moxonidine and rilmenidine. In PC-12 (phaeochromocytoma) cells, over 80 stably-transfected subclones were screened both by Western blotting and PCR, and six of these showed at least a two-fold increase in [I.sub.1] [B.sub.max] values. Transfected CHO and PC-12 cells showed a 1 67 kDal band as well as smaller bands ([sim] 85 kDal) on Western blots. On the other hand, transient trasfections into COS-7 and Sf9 cells failed to result in an increase in [I.sub.1] binding sites, even though there was an abundance of the [sim] 167 kDal protein made. To determine if there is any possible interaction between [I.sub.1] sites and [alpha.sub.2A] adrenergic sites, CHO cells stably transfected with the human [alpha.sub.2A] adrenergic receptor cDNA were transiently transfected with iras-1. These cells produced both [alpha.sub.2A] and [I.sub.1] receptors (immunologically) with the appropriate binding sites observed, and the results provide support for studies aimed at characterizing the pharmacological and functional interactions between [alpha.sub.2]-adrenergic and [I.sub.1] receptors. Supported by NIMH grant NM49248-06 and grants from Eli Lilly and Solvay Phannaceutical Companies.

FRIDAY MORNING

Petit Bois

9:00 UVA LIGHT-INDUCED FORMATION OF DNA COVALENT ADDUCTS AND DNA SINGLE STRANDED CLEAVAGE BY 1-HYDROXY-PYRENE

Shiming Dong [*], Laketa Halloway, Huey-Min Hwang, Xiaochun Shi, and Hongtao Yu [*], Jackson State University, Jackson, MS 39217

This research examines the light-induced damages to DNA by 1-hydroxypyrene (HOP). HOP is often found in the urine of human or animals exposed to polycyclic aromatic hydrocarbons (PAHs) and it is an important biomarker for studying PAH exposure. It has shown that HOP is both acute toxic and genotoxic. In this research we found that upon UVA light irradiation, HOP causes DNA single strand cleavages and forms HOP-DNA covalent adducts. The UVA light-induced cleavage of the supercoiled plasmid [phi]X 174 DNA is dependent upon both HOP concentration and UVA dosage. Longer irradiation time or higher HOP concentration induces more DNA cleavage. The DNA cleavage results done in the presence of reactive oxygen species scavengers indicate that superoxide free radical and singlet oxygen are both likely involved in causing DNA cleavages. The photocleavage is inhibited by the presence of an excited singlet state quencher, KI, indicating that it is an excited state reaction. Along with light-induced DNA cleavage, HOP also forms DNA covalent adducts efficiently while being degraded. Light-induced degradation of 20 mM HOP follows a first order reaction kinetics in 10% methanolic buffer (10 mM phosphate) solution in the absence or presence of 100 mM ct-DNA, with degradation half-lives of 20 min or 27 min, respectively. The longer degradation half-life in the presence of DNA is due to the formation of HOP-DNA covalent adduct which is light stable. The formation of the HOP-DNA covalent adduct is evidenced by comparing the UV-Vis spectra of the irradiated HOP/DNA solution before and after dialysis. While HOP from a non-irradiated solution containing HOP and DNA diffuses freely out of the dialysis membrane, the HOP from the irradiated sample remained in the membrane, indicating that the HOP is covalently bound to DNA that cannot diffuse out of the membrane. Acknowledgements: This research is supported by the National Institutes of Health through generous grants: NIH-RCMI 1G12RR12459-01 and NIH-MBRS S06GM08047.

9:15 ISOLATION OF A CATALYTIC RNA CAPABLE OF COENZYME SYNTHESIS

Wally Bugg [*] and Faqing Huang, University of Southern Mississippi, Hattiesburg, MS 39406-5043

Coenzymes, such as coenzyme A (CoA), nicotinamide adenine dinucleotide (NAD), and flavin adenine dinucleotide (FAD), play important roles in the metabolism of modem organisms. These coenzymes have the same biosynthetic pathways and the same functions among drastically different organisms, from bacteria to humans. Therefore, they must have been present in the last common ancestor. The presence of a ribonucleotide adenosine in these chemically complex coenzymes suggests that they might have arisen in a pre-protein world the so-called RNA world. In order to demonstrate the ability of RNA to catalyze the synthesis of coenzymes from their precursors, catalytic RNA is being isolated by powerful in vitro selection/amplification techniques from a pool of random RNA sequences. After 12 rounds of selection/amplification, the resulting RNA has been shown to synthesize a variety of RNA-linked molecular structures, including coenzymes NAD and FAD. The demonstration of RNA catalysts to synthesize coenzymes strongly suppor ts that coenzymes are ancient molecules, which might have existed before the first protein was made.

9:30 CHARACTERIZATION OF A COENZYMESYNTHESIZING RIBOZYME 

Shannon Williams [*] and Faqing Huang, University of Southern Mississippi, Hattiesburg, MS 39406-5043

Coenzymes are essential molecules for cell functioning. For example, the three most common coenzymes, coenzymes A (CoA), nicotinamide adenine dinucleotide (NAD), and flavin adenine dinucleotide (FAD), participate in numerous biochemical reactions. To understand the origin and evolution of these coenzymes, we have isolated, by in vitro selection and amplification techniques, RNA catalysts that synthesize the coenzymes from their corresponding precursors. The RNA has been cloned and sequenced. The RNA-synthesized coenzyme product has been identified by enzymatic digestion, chromatography, and mass spectroscopy. In order to understand the RNA activity, structure characterization of the isolated RNA is being carried out. Further studies of the kinetics are also being investigated. Reaction rates under different conditions, such as pH, temperature, metal ion concentrations, and substrate concentrations are being measured in order to under the nature of RNA-catalyzed synthesis of coenzymes.

9:45 ESSENTIAL RESIDUES IN GLUTAM1NYL CYCLASE

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

Glutaminyl cyclase catalyses the conversion of Nterminal glutamine to pyroglutamic acid in certain peptide hormones, and neurotransmitters. In order to understand the mechanism of enzyme action, it is necessary to determine the residues involved in catalysis. DEPC inactivation of the recombinant enzyme showed that histidine is an important residue for catalysis. Sequence alignment showed eleven histidine residues that are conserved. The histidines at positions 140, 307, 319 and 330 were mutated. Kinetic studies indicate that the Km and Vmax for His307 and His319 are similar. The Km of these two mutants is higher than that for the native human enzyme, indicating that these residues are important for substrate binding. The Km for His 330 was lower, indicating that His 330 does not play such an important role in substrate binding while the His 140 was totally inactive indicating that it is an important residue for catalysis.

10:00 Break

10:15 SOYBEAN CHLOROPLASTNUCLEOID PROTEINS AND THEIR INTERACTIONS WITH DNA

Cecilia Chi-Ham [*], Gordon C. Cannon, and Sabine Heinhorst, University of Southern Mississippi, Hattiesburg, MS 39406-5043

Chloroplast DNA is organized by DNA-binding proteins into complexes called nucleoids. Plastid nucleoid structure and protein composition vary during the development of proplastids to chloroplasts, suggesting that genome packaging may play a crucial role in the regulation of replication, transcription, and recombination. In an effort to understand the role plastid nucleoid proteins play in determining nucleoid structure and its effect on gene expression and development of the organelle, we have purified several nucleoid proteins. One plastid nucleoid protein (DCP68) can associate with DNA into a form that does not support DNA replication in vitro. The amino terminal sequence of the purified DCP68 shows homology to ferredoxin-dependent sulfite reductases from Arabidopsis thaliana, Zea mays, and Nicotiana tabacum. We are currently studying the interaction of DCP68 with DNA and with other nucleoid proteins in order to obtain a better understanding of the role this protein plays in determining nucleoid structure and function.

10:30 SELF-ASSEMBLY OF THE SC3 HYDROPHOBIN PROTEIN

J. Shawn Goodwin [*], Charles L. McCormick, and Gordon C. Cannon, University of Southern Mississippi, Hattiesburg, MS 39406-5043

The Sc3 hydrophobin protein from Schizophyllum commune self-assembles in an aqueous environment to form vesicles with hydrophobic interior and hydrophilic exterior surfaces. We are studying the protein's ability to entrap a hydrophobic material from an aqueous environment into stable vesicles and the mechanism by which this occurs. Utilizing aradiolabeled [[blank].sup.14] C-hexadecane sequestration assay, we are able to quantify the amount of hydrophobic material that can be removed from an aqueous environment by hydrophobin. Epifluorescence microscopy studies using Nile Red, calcein, and Marina Blue confirm that hydrophobic molecules are within Sc3 hydrophobin protein vesicles. Insight into the mechanism of hydrophobin protein self-assembly can be determined using circular dichroism and fluorescence spectroscopy.

10:45 STRUCTURAL CHARACTERIZATION OF A SELF-CAPPING RNA CATALYST

Ping Zhang [*] and Faqing Huang, University of Southern Mississippi, Hattiesburg, MS 39406-5043

A previously isolated RNA catalyst, named Iso6, has multiple catalytic functions, such as RNA capping, phosphoryl coupling, pyrophosphotase, decapping, and cap-exchange activities. Among the known ribozymes, Iso6 RNA possesses many unique properties, such as multifunction, specific GTP binding site, [Ca.sup.2+] as the metal cofactor, and binding multiple small-molecule substrates and building larger products from them, etc. These unusual properties of Iso6 RNA coupled with its biological and evolutionary relevance, well established reaction center at its 5 alpha phosphate, makes it an excellent model system to study RNA structure-function relationship and mechanism of RNA catalysis. We are currently investigating the structural elements of the RNA that are essential for its functions. By using substrate-linked probes, such as EDTA and photo-crosslinking reagents, we hope to define the KNA sequence that constitutes the substrate binding site as well as the metal ion binding site. The results will enhance our understanding of RNA-substrate-metal ion interactions and RNA catalysis in general.

11:00 Divisional Business Meeting and Presentation of Awards