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Biological Sciences. (Abstracts).

THE EFFECTS OF CHRONIC INORGANIC AND ORGANIC PHOSPHATE EXPOSURE ON ASPECTS OF MUSCLE PHYSIOLOGY OF THE NEARSHORE SEA URCHIN Lytechinus variegatus (ECHINODERMATA: ECHINOIDEA).

S. Anne Bottger *, David W. Kraus and James B. McClintock, Department of Biology, The University of Alabama at Birmingham, 1300 University Boulevard, Birmingham, AL, 35294-1170.

The common nearshore sea urchin Lytechinus variegatus is exposed to phosphate pollutants in its natural environment. Sea urchins were collected in April, 200 from Saint Joseph Bay in the northern Gulf of Mexico. Individuals were divided into a control (artificial seawater), seawater containing three concentrations of inorganic (sodium phosphate), or three concentrations of organic (triethyl phosphate) phosphate. Individuals were thus maintained for an 8-week period, dissected and the Aristotle's lantern protractor muscles extracted. Each muscle was attached to a strain gage, stimulated electrically, and muscle contractions recorded. Maximum force of muscle contraction (N force/ mm cross-sectional area), and rates of muscle contraction and relaxation (N force/ mm cross-sectional area/ sec) decreased with chronic exposure to increasing concentrations of organic phosphate. Chronic exposure to inorganic phosphates elicited no response except at the highest concentration, where the maximum force of muscular contra ction increased compared to controls. Acetyl cholinesterase activity, measured in both a static enzyme assay and by vesicular staining, displayed concentration-dependent declines of activity in individuals maintained in organic phosphate for four weeks. The activity of AChE was not adversely affected by exposure to inorganic phosphate or seawater controls over the experimental time period. These findings indicate that shallow-water populations of Lytechinus variegatus subjected to organic phosphate pollutants may display impaired muscular activity that is potentially related to the inhibition of the muscle relaxant enzyme AChE, and subsequently muscular overstimulation and fatigue.

APPLICATION OF SUBTRACTION HYBRIDIZATION APPROACH TO IDENTIFICATION OF GENETIC ELEMENTS IN PANDEMIC STRAINS OF VIBRIO PARAHAEMOLYTICUSO3:K6 SEROTYPE. Lisa Ann Blankinship and Asim Bej, Dept. of Biology, Univ. of Ala. at Birmingham, Birmingham, AL 35294.

Vibrio parahaemolyticus serotype O3:K6 a Gram negative microorganism that causes gastroenteritis in humans, emerged in 1996 as an epidemic strain in Calcutta, India and spread throughout Southeast Asia and North America. This study was conducted to identify genetic differences between V. paraphemolyticus nonpandemic pre-1996 strains and pandemic post-1996 strains and to identify novel genetic elements found in pandemic O3:K6 by subtractive hybridization methods. In this procedure, "Tester" DNA (pandemic O3:K6) was restricted with Rsai and split into two samples with different adaptors ligated onto each sample. "Driver" DNA (nonpandemic O3:K6) was similarly digested with Rsal before adding in excess to each Tester sample. Samples were allowed to hybridize overnight followed by mixing of the two samples and an additional round of hybridization was conducted. PCR was preformed on secondary hybridization products to selectively amplify only those fragments that contained two different adaptors, which represented unique Tester sequences. PCR products were cloned onto a Topo[TM] TA plasmid vector, sequenced, and made into probes to test Driver, Tester, and non O3:K6 V parahaemolyticus genomic DNA in a Dot Blot hybridization. Two fragments were found to be unique to V parahaemalyticus pandemic strain, and are currently being investigated to determine the possible origin of this pandemic strain and their use as targets for a PCR-based and/or gene probe-based specific rapid detection of this pathogen in marine water and shellfish.

ANTIMICROBIAL ACTIVITIES OF BACTERIAL ISOLATES FROM SALINE ENVIRONMENTS. Maria Martin and Donald Salter, University of West Alabama.

Our laboratory has cultured a significant number of bacterial isolates from three different aquatic saline environments: an inland salt spring near Jackson, AL, an estuarine environment near Pensacola Beach. FL, and from the Gulf off Pensacola Beach, FL. We have previously reported the inorganic requirements amid biological activities of some of these isolates. We have now screened a significant number of bacterial isolates from these diverse saline environments for antimicrobial activity and growth competition against known Grain-positive and Grain-negative bacterial species. Approximately twenty percent of time isolates appeared to produce measurable antimicrobial activity as assayed using a modified Kirby-Bauer technique. Further, many other isolates appear to directly compete with both Gram-positive and Gram-negative bacterial species for growth on the agar plates. These bacterial isolates have been re-tested against these and additional Gram-positive and Gram-negative bacteria to confirm and extend the initial results. Those isolates producing significant anti-microbial activity and growth competition will be used in future studies that will include optimizing antimicrobial production, determining antimicrobial stability, and developing a quantitative method for measuring growth competition.

ARCHAEBACTERIA AND EUBACTERIA CONCENTRATIONS IN AQUATIC AREAS OF THREE DIVERSE SALINE ENVIRONMENTS. Mandy Cook *. Donald Salter ** and Michael Lemke *, * University of Illinois at Springfield and ** University of West Alabama.

This study examines microorganism abundance in an extreme saline aquatic ecosystem and compares the Findings to abundances in other less saline aquatic systems. The study objectives were to determine the total number of bacteria and number of microorganisms in the Domains Archaea and Bacteria in these saline samples. Water samples (n=3) were collected from seven sites in Alabama and Florida between June - September 2000: A Ssaline seep (48ppt). bubbling springs (45 ppt). and lake-saline river inlet (10 ppt) from the Salt Springs area near Jackson, AL, two sites of a freshwater stream (Up-stream 3 ppt: Down-stream 0 ppt) near the Salt Springs area, and an estuarine environment (26 ppt) and the Gulf waters (37 ppt) near Pensacola Beach, FL. Total bacteria numbers, determined by the DAPI method, showed highest abundance in the estuary sample (3.01 x [10.sup.6]/ml) as compared to the Salt Springs samples (1.99x [10.sup.5]/ml). In-situ whole-cell hybridization with domain-specific fluorescent probes was used to de termine abundance of prokaryotes in cacti Domain. Both Domains had highest abundance at the estuary site (eubacteria at 6.51 x [10.sup.5]/ml; archaeabacteria at 3.72 x [10.sup.4]/ml) as compared to the Salt Springs (eubacteria at 1.19 x [10.sup.4]/ml; archaeabacteria at 1.73 x [10.sup.3]/ml). Active prokaryotes. (calculated as the number of hybridized cells/number of DAPI-stained cells) showed that three sites (estuary, sea shore. and the saline seep) had the highest cellular activity. These results show that, even though the most abundant site of study was the estuary, the most cellular activity is occurring in the three separate systems.

BACTERIAL SOURCE TRACKING IN SURFACE WATERS. Kim Autrey, Brianna Stansbury, and Brian S. Burnes, Dept. of Biology, Judson College, Marion, AL 36756.

Antibiotic resistance analysis (ARA) was used to determine sources of fecal coliform pollution in Dry Creek in south Perry County, AL. The Dry Creek watershed encompasses 5.329 acres and is predominantly agricultural with little urban or developed area A library of antibiotic resistance profiles based on 9 drugs was developed for 1,152 fecal coliform isolates from human, cattle, and deer sources. Discriminant functions were developed to classify each isolate as human, cattle or deer in origin. Correct classification rates were greater than 80% for each isolate. Antibiotic resistance profiles were then determined for 768 isolates from Dry Creek, collected during high or low water levels during January 2002, and compared to the known sources using discriminant analysis Human, cattle, and deer profiles were found in each sample, however, the isolates classified as human predominated in both samples. The results indicate fecal coliforms from both human and cattle sources contribute to fecal pollution in the Dry C reek watershed.

CELL SIGNALING PATHWAYS FOR ECDYSTEROIDOGENESIS IN BLUE CRAB (CALLINECTES SAPIDUS) Y-ORGANS. Deug Woo Han and R. Douglas Watson. Department of Biology, University of Alabama at Birmingham, AL 35294.

Synthesis of ecdysteroid molting hormones by crustacean Y-organs is regulated (negatively) by a neuropeptide, molt-inhibiting hormone (MIH). The underlying mechanisms that link MIH receptor occupancy to the subsequent suppression of ecdysteroidogenesis in Y-organs remain unresolved. Several lines of evidence indicate the effect of MIH is mediated by a rise in the intracellular level of one or more cyclic nucleotide second messenger (cAMP, cGMP, or both). We report here the results of experiments conducted in vitro to assess the possible roles of these key second messengers in cell signaling pathways for ecdysteroidogenesis in Y-organs of the blue crab, Callinectes sapidus. A Gs-protein activator (cholera toxin), cAMP analogs (dibutyryl cAMP or 8-bromo-cAMP), and forskolin (an adenylyl cyclase activator) each had no effect on ecdysteroid synthesis. By contrast, a cGMP analog (8-bromo-cGMP) dose-dependently suppressed ecdysteroid synthesis by Y-organs in vitro. The maximal level of suppression (~50%) was simila r to that observed when blue crab Y-organs were incubated with eyestalk extract containing native MIH. The results suggest that cGMP is likely' to be a physiologically relevant second messenger in regulation of ecdysteroid synthesis by Y-organs of C. sapidus, and that activation of the MIH receptor may be linked to regulation of guanylyl cyclase activity. Supported by MS/AL Sea Grant and the UAB Graduate School.

CHARACTERIZING A WATERSHED SEDIMENT EROSION POTENTIAL USING GIS TECHNOLOGY. Janna Owens. Ken Marion and Robert Angus. UAB, Dept. of Biology, Birmingham. AL 35294. Melinda Lalor, Civil and Environmental Engineering, UAB. Eric Meyer and Steve McKinney, SWMA, Inc.

To be effective, monitoring programs for nonpoint source contaminants must include the ability to identify the causes of water quality impairment within the watershed. Our study objective was to devise a water quality assessment protocol to evaluate sedimentation effects that incorporated habitat assessment and biological sampling with a sediment-erosion potential model based on geographical information system (GIS) data. The Cahaba River and its tributaries were investigated for water quality conditions and excessive sedimentation by utilizing benthic macroinvertebrate community structures as possible indicators. Various aspects of the biological status of the benthic community were then analyzed with a series of metrics. To quantify the upstream watershed sedimentation characteristics above the sample sites, a soil erosion potential model was constructed using Geographic Information Systems (GIS) and remote sensing technologies. The cartographic model consisted of selected data layers for the study area, in cluding soil types, multispectral satellite imagery, parcel level land use, and a digital elevation model. A Sedimentation Potential Index (SPI) was calculated for each watershed and correlated with site habitat conditions, sediment depths and upstream watershed land usage Strong associations were evident between the SPI scores and specific metrics that have previously been shown to be sensitive to sedimentation impacts, such as the EPT (Ephemeroptera, Plecoptera, Trichoptera), Sorenson Similarity and Hilsenhoff Biotic Indices.

DIATOM ASSEMBLAGES FROM A SALINE SPRING SYSTEM. CLARKE COUNTY. ALABAMA. Lisa A. Muellner and Roland R. Dute. Department of Biological Sciences, Auburn University. Auburn, AL 36849.

Diatoms (Bacillariophyceae) have recently gaincd the attention of ecologists and paleolimnologists as valuable indicators of water quality and environmental change. The Fred T. Stimpson Wildlife Sanctuary provides a unique environment in which to study the influence of environmental factors on diatom communities. The Sanctuary, located in Clarke County, Alabama, is comprised of a system of natural salt springs that flow into freshwater streams. giving rise to a salinity gradient. Preliminary data suggest that salinity measured at various stations along the gradient is stable over time and ranges from 0 to 48 ppt. Spring flooding. however, might result temporarily in a more homogenous, less saline environment throughout much of the system. Sediment samples taken from stations along the gradient consist of unique benthic diatom communities with varying levels of taxonomnic diversity. Dominant taxa include Achnanthes lanceolata de Brebisson ex Kutttzing (freshwater). Fragilaria pinnata var. intercedens (Grunow) Hustedt (14-17 ppt. salinity), and Amphora coffeaeformis (Agardh) Kuttzing (40-48 ppt. salinity). At present, 94 diatom species have been identified. Ongoing research is expected to clarify relationships between diatom communities within the system and environmental factors such as light, temperature, nutrients, and pll, as well as salinity ranges and salinity tiuctuatiomis. Funding is provided through the Auburn University Dean's Research Initiative. Auburn University Graduate Research Award, amid the Lands Division of the Alabama Department of Conservation and Natural Resources.

THE EFFECT OF TEMPERATURE ON CONSUMPTION AND GROWTH IN TUE SEA URCHIN LYTECHNIUS VARIEGATUS. Scott C. Hofer, Stephen A. Watts, Dept. of Biology, Univ. of Ala. at Birmingham, AL 35294-1170. John M. Lawrence, Univ. S. Florida, Tampa, FL 33620.

Lytechinus variegatus are common in near-shore marine communities throughout much of the Caribbean and Gulf of Mexico regions. Adult individuals (ca.39 to 45mm diameter) collected at St. Joseph Bay, FL (22 C) were held at 16, 22 or 28 (' for 8 weeks and fed a prepared diet ad libitum. At the time of the initial collection and at the end of the study urchins were dissected for component analysis. Overall consumption was highest in those exposed to 22 C. Urchins exposed to 16 C showed partial acclimation (feeding rate) within one month. Dry matter absorption efficiency was 66, 67. and 76% at 16, 22 and 28 C. Individuals exposed to 22 C exhibited die highest total weigh gain (production) and increase in diameter. Total production efficiencies (g dry mass produced/g dry mass absorbed) were inversely proportional to the exposure temperature, ranging from 27 to 42 %. indicating that the energy requirement for maintenance and/or production increases with temperature. Gonad production was significantly higher in urch ins held at 22 C. Gonad production efficiencies were also inversely proportional to temperature and ranged from 9 to 15%. Interesting, gut mass was 2-fold larger in those exposed to 16 C than at 28 C. We hypothesize that the storage capacity of the gut is increased at low temperature. Alternatively, a larger gut may increase digestive capacity to compensate for the effects of reduced temperature on the rate of digestion. The temperature of maximal growth (22 C) is also the median temperature to which the urchins are exposed in the field annually.

The EFFECTS OF EPICUTICULAR WAX ON THE RNTE OF WATER LOSS OF SORGHUM BICOLOR. Mijitaba Harnissou, Department of Biology, Jacksonville State University, Al 36265 and Dale Weibel, Oklahoma State University, Ok 74078

Sorghum leaves are normally covered with an epicuticular wax. The amount of wax ranges from a heavy covering or 'bloom' to light covering or "sparse bloom". The condition where there is no visible wax covering is termed "bloomless." Wax prevents desiccation, minimizes mechanical damage, and protects against excessive ultra-violet radiation. A reduction of solar energy load on the plant through increased reflectance, an avoidance of reduced water potential, and maintenance of a more complete stomatal control over transpiration manifest the contribution of wax to the prevention of water loss. Physiological and anatomical features of the plant govern plant water loss. The objectives of this study are (I) to determine the difference in stomatal conductance among the bloom, the sparse-bloom, and the bloomless, (2) and to compare the rate of water loss between the flag leaf and the third leaf down.

Field and greenhouse experiments were conducted to determine the rate of water loss of three near-isogenic lines of sorghum variety of ROKY62 and ROKY78. Boot stage, stomatal conductance, photosynthetic and transpiration rate were measured on both flag leaf and the third leaf down. Leaf water potential was measured using thermocouple psychrometers.

Our data indicated that epicuticular wax plays an important role in protecting sorghum plants against stomatal water loss. We also found that bloomless sorghum plants had more negative water potential and lower photosynthetic rate than the other lines.

EFFECTS OF PHLOXINE B ON POPULATION GROWTH AND RESPIRATION RATES IN TETRAHYMENA SPP. Christa Collins, Charles Olander, Mark Meade, H. Blake Otwell, Dept. of Physical and Earth Sciences, and Dept. of Biology, Jacksonville State Univ., Jacksonville State Univ., Jacksonville, AL 36265.

Phloxine B is a halogenated Photoactive xanthene dye that is FDA approved for use in human cosmetics and drugs. Research shows Phloxine 13 to be a safe and effective pesticide for the treatment of various insects. Light activates the ingested dye inside the insects, where it forms a potent oxidizing agent that attacks their tissues. It is not contact toxic. Comparison of the toxicity and metabolic effects of the light independent and light dependent Phloxine 13 on Tetrahymena pyriformis are currently being conducted. Our data indicate that Phloxine 13 at 10ppm, lppm, 0.1ppm is detrimental to the growth and that Phloxine B at 10ppm has a significant effect on the oxygen consumption of Tetrahymena.

ERYSIPELOTHRIX IN A BOTTLENOSE DOLPHIN. Gerald T. Regan 4000 Dauphin Street, Mobile, AL 36608.

This grain-positive bacillus has been Found in bottlenose dolphins elsewhere in their geographic range, but its presence in Alabama coastal waters is now documented. The beach-cast specimen appeared on the Gulf of Mexico side of Dauphin Island on March 23, 2001. The freshly dead female had a total length of 218 cm and no external parasites, lesions or other signs of traumatic death. The usual protocol for the necropsy of marine mammals in Alabama was followed, excepting that a newly acquired form of liquefied protectant was used on the right-banded prosector's hands instead of latex gloves. The prosector had no contact with any other kind of specimen for several days prior to this necropsy. On the morning after the completion of the necropsy, two small, circular, inflamed areas appeared on the left hand of the prosector, one on the nail side of the terminal knuckle of the second finger and one at the base of the second ringer and part way to the third finger. The thumb is the first finger here. Throughout tha t same day, the inflamed areas widened and swelled and pain set in. On the second clay after the completion of the necropsy the prosector was hospitalized, laboratory tests were performed on fluid from one of the inflamed areas, and intravenous antibiotics were administered until the next morning, when the inflamed areas were no longer growing. The hospital laboratory found that the infection was caused by Erysipelothrix rhusiopathiae. In this form, evidence appeared for infection of the dolphin itself by the same bacteria. The help of C. Adrien Bodet. III, Patrick E. Nolan, and Stephen G. Alsip is gratefully acknowledged.

AN EVALUATION OF AGONISTIC BEHAVIORS OF SEVEN SPECIES OF CANTHIGASTER FISH AS A MECHANISM TO EVALUATE PHYLOGENETIC INTERRELATIONSHIPS. Yusheng Iluang. Dept. of Biology. Univ. of Ala. At Birmingham, Birmingham, Al 35294. Ilin-kiun Mok, Inst. Of Marine Biology, National Sun Yat-Sen Univ., Kaohsiung 804, Taiwan (R. O. C.)

Many studies, including those with fishes, reptiles. rodents and birds, have evaluated phylogenetic relationships by using behavioral traits. Agonistic behaviors of seven species of tropical ( 'anthigaster fish (( '. Amboinensis, C. hehhetti, C. compresus C. coronata, C. janthinoptera. C. solandri, and C. valentini) were analyzed by using cladistic methods to construct a consensus phylogenetic tree. Eleven agonistic behavioral patterns were recognized in the behavioral sequences of these seven fish species. While the degree of aggressiveness varied within a species (for example, female individuals of C. valentini were more aggressive than malee individuals), intraspecific variation in patterns of agonistic behaviors was very low. This consistency of agonistic behaviors within a species facilitated the construction of a phylogenetic tree. On the basis of behavioral characters, the tree was comprised of two monophyletic groups. One group consisted of C. hennetti. C. solandri. C. Coronata, C. valentini and C. co mpresus and the other group consisted of C. valentine and C. compresus. On the basis of the proposed phylogeny, it appears that both behavioral repertoire and the complexity of the stereotypic behavior increase over evolutionary time.

AN EXAMINATION OF THE EFFECTS OF QUANTITATIVE AND QUALITATIVE DIFFERENCES IN FOOD AVAILABILITY ON REGENERATION IN PLANKTOTROPHIC LARVAE OF THE SEA STAR PISASTER OCHRACEUS Minako S. Vickery and James B. McClintock, Department of Biology, The University of Alabama at Birmingham, Birmingham, Alabama 35294-1170.

The role of food quantity and quality on regeneration in planktotrophic larvae of the sea star Pisaster ochracens was examined. Surgically bisected late bipinnaria/early brachiolaria larvae were either starved, fed three concentrations of monospecific diets of the phytoplankton Chactocerous calcitrans. Dnnalte ha tertiolecta, or Isocrysis galbana, or fed three concentrations of mixed diets comprised of equal numbers of cells of die three phytoplankton species over a 20-day experimental period. Control non-bisected larvae were fed the highest concentration of mixed phytoplankton. Regeneration occurred regardless of a complete lack of food or differences in food concentrations or food mixtures. Anterior portions of surgically bisected larvae starved or maintained on low or medium concentrations of single or mixed diets generally showed no growth or decreased in length over the experimental period. In contrast, while posterior portions of surgically bisected larvae starved or fed the lowest concentration of phyt oplankton did not grow, those fed medium or high levels of single or mixed diets attained sizes equivalent to non-bisected fed control larvae, and also grew larger than posterior portions starved or fed low concentrations of food. These patterns of shrinkage or growth are likely related to the retention of a digestive system in the posterior portion of larvae post-bisection, whereas anterior portions required at least one week for regeneration of a functional gut. These results suggest regeneration occurs in damaged larvae in the natural environment even under conditions of food limitation.

FEMALE-BIASED SEX RATIO IN JUVENILE KEMP'S RIDLEY SEA TURTLES CAPTURED NEAR CEDAR KEYS, FL. Alyssa Geis, Dept. of Biology, Univ. of AL-Birmingham, Birmingham, AL 35294. Jaime Barichivich, U.S.G.S. Gainesville, FL 32653. and Thane Wibbels, Dept. of Biology, Univ. of AL-Birmingham, Birmingham, AL 35294.

The Kemp's ridiley sea turtle, Lepidochelys kempi is the most endangered sea turtle in the world. It possesses temperature-dependent sex determination (TSD), where the incubation temperature of the egg determines the sex of the hatchling. Past studies have suggested that TSD has the potential of producing highly biased sex ratios, which may not be advantageous to the recovery of endangered sea turtle populations. In that respect, it is important to monitor sex ratios within sea turtle populations. However, determining the sex of juvenile sea turtles is difficult, due to the fact that they lack the secondary sex characteristics commonly used to identify sex in adult turtles. In this study, a testosterone radioimmunoassay (RIA) was validated and used to predict the sex of juvenile Kemp's ridileys that were captured near Cedar Keys, Florida The results show a significant female bias (3.7:1, female: male) currently exists in this group of juvenile Kemp's ridleys. The biased sex ratio predicted in the current stud y exemplifies the need to monitor sex ratios in sea turtle populations. Such information is a prerequisite to the development of an effective management strategy for endangered populations.

The Female Mosquitofish Anal Fin as a Biomarker for Androgen Exposure: a Dose-Response Study Comparing Characteristics of Fin Morphology and Measures of Reproductive Fitness. Jason P. Stanko, R. Douglas Watson, and Robert A. Angus, Dept. of Biology, Univ. of Ala. at Binningham, Birmingham, AL 35294.

Abstract

The anal fin rays of female mosquitofish can be induced to form a normally male-limited trait (gonopodiuin) if exposed to androgens. This external morphological trait can be used as a sensitive biomarker of environmental androgen exposure. This study characterized the dose-response relationship between androgen exposure and female axial fin masculinization and, and correlated changes in the biomarker with effects on gonad development and vitellogenesis. Female Gambusia affinis were masculinized through dietary administration of four concentrations of 17[alpha]-methyltestosterone over a six-week period. Significant changes in fin ray morphology, an indicator of masculinization, occurred at the two higher dose levels. The mean gonadosomatic index value, however, did not change significantly until the highest treatment dose. A similar pattern was observed in vitellogenin expression. Histological analysis of ovarian sections from females treated with the highest dose revealed a decrease in total ovarian area due to a reduction in the number and frequency of late-stage vitellogenic follicles. This study demonstrates that the anal fin of female mosquitotish is a sensitive biomarker for environmental androgens. The mosquitofish anal fin may be used as a gauge of environmental androgen exposure in field studies and to identify exposed populations before effects on reproductive fitness become important. This work supported by EPA Grant R82630-01-0.

FLOWER ANATOMY AND MORPHOLOGY OF CYRILLA RACEMIFLORA. Roland R. Dute, Debbie R. Folkerts, Auburn Univ. 36849, James E. Watkins, Univ. of Florida, Milam E. Saxon and Heather A. Pritchard, Auburn University.

Cyrilla racemiflora is a typical component of the wetlands ecosystem in the Southeast. During our study of its pollination biology, the need for a thorough study of Cyrilla's flower anatomy became evident. This was accomplished using light and scanning electron microscopy. At flowering. the pollen receptive organ (the stigma) is of the "wet" type with lipids on the surface. Lipid in material is also manufactured in the stigmatoid tissue with in the style--the identical path way followed by growing pollen tubes. After flowering, petals and stamens abscise, hut sepals remain. The sepals have stomates, and it is thought that the openings provide gas exchange fur photosynthesis to support the enlarging fruit. At anthesis (flowering) time ovary is surrounded by a bright green belt thought to be a nectary. Sugar is probably manufactured at the site or brought in by vascular tissue. Nectar is then secreted through open stomates. The adaxial surfaces of the flower petals are thickened. While these sites might represe nt additional nectaries, there is no convincing evidence to support this hypothesis. Pollen grains, formed within the anthers, are two--celled at the time of their release. Calcium crystals are associated with the sites of pollen release from the anthers. This feature might represent a method of sequestering calcium from degrading anther walls prior to pollen release. Present studies of this species are focused on pollination biology and effectiveness of cross versus self-pollination.

FUNGAL GROWTH AND HOST RESPONSE ASSOCIATED WITH AMBROSIA BEETLE ATTACK ON CERCIS CANADENSIS. Roland R. Dute, Michael E. Miller, Auburn Univ. and the Alabama Agricultural Experiment Station, Micheal A. Davis, University of Southern Mississippi, Floyd M. Woods and Kathy S. McLean, Auburn Univ. and the Alabama Agricultural Experiment Station

Micro-organisms introduced by Asian ambrosia beetles (Xylosandrus crassiusculus) into redbud trees (Cercis canadensis) cause wilting and death of the host and economic losses to nursery owners. Female beetles bore into tree trunks in the spring. Investigation of the tunnels shows the presence of both bacteria and fungi. 'the fungi infect the host wood, and their hyphae spread from the site of infection. Fungi grow from one host cell to another through perforations of the xylem vessels, through pit membranes connecting cells. and even directly through secondary cell walls. Microscopy indicates the hyphae to be surrounded by a sheath and to contain considerable amounts of stored food. Fungi, isolated and cultured from infected trees, consist of various genera from the Deuteromycetes/Ascomycetes. In particular, the genus Ambrosiella is always associated with infection. In response to invasion by beetles and fungi, the host parenchyma cells degrade their inner walls. The resulting gel moves into and blocks the wa ter-conducting vessels. This mechanism might inhibit spread of the fungi. Studies are underway to identify the species of Ambrosiella and to reinfect healthy trees using fungal cultures. 'this project is funded by the Alabama Agricultural Experiment Station.

HOOP-TRAP SURVEY OF FRESI IWA'FER FISH COMMUNITIES ALONG COASTAL ALABAMA. David H. Nelson, William M. Turner and T. Joshua Meyer, Dept. of Biological Sciences, Univ. of South Alabama, Mobile, AL 36688.

Hoop traps were deployed for four summers in freshwater habitats in the Mobile-Tensaw delta and along the major coastal Alabama rivers. In 1994. traps set out in the lower delta (north of Interstate 10) yielded 236 specimens of 13 species (684 trap days). In 1995, the middle delta yielded 718 specimens of24 species (1806 trap days). In 1997, traps in the upper delta (north of Interstate 65) yielded 231 specimens of 21 species (779 trap days). In 1996, the major coastal rivers (from Mississippi to Florida) and the Mobile Bay causeway were trapped to reveal 249 specimens of 24 species (1151 trap days). A total of 1434 captures were made of 38 species of fishes (4420 total trap days). Six species of Fishes accounted for 80.5% of the captures: largemouth bass (Micropterus salmoides) 21.6% black crappie (Pomoxis nigromaculatus) 18.7%. spotted gar (Lepisosteus oculatus) 16.6%, smallmouth buffalo (lctobius bubalus) 14.2%, southern flounder (Paralichthys lethostigma) 4.8%, and bluegill (Lepomis macrochirus) 4.6%. The bowfin (Amia calva) and the alligator gar (Lepisosteus spatula) occurred (2.3% and 2.0%, respectively) within both the delta and coastal rivers. The fresh water drum (Aplodinotus grunniens) occurred (1.7%) in the delta but not in coastal rivers. The introduced common carp (Cyprinus carpio) and grass carp (Ctenopharyngodon idella) accounted for 0.5% and 0.1% of total catches, respectively.

INFLUENCE WEATHER ON THE SEX RATIO OF HATCHLING HAWKSBILL SEA TURTLES INHABITING BUCK ISLAND REEF NATIONAL MONUMENT, U. S. VIRGIN ISLANDS. Jennifer M. Estes. Thane Wibbels, Dept of Biology, Univ. of AL-Birmingham, Birmingham, AL 35294. Zandy Hillis-Starr, National Park Service. Christiansted, St. Croix 00820-4611, U. S. Virgin Islands and Brendalee Phillips. U. S. G. S. Christiansted. St. Croix 00820-4611, U.S. Viigiii Islands

The hawksbill sea turtle. Erethmochelys imbricala. possesses temperature-dependent sex determination (TSD) in which the incubation temperature of the egg (during the approximate middle third of incubation) determines die sex of lie hatchling. This form of sex determination has the potential of producing a wide variety of sex ratios. Hatchling sex ratios resulting from TSD are of conservational interest, since they can affect die recovery of endangered populations. Buck Island represenis a major natural nesting beach in the Caribbean for endangered hawksbill sea turtles. The purpose of the current study was to estimate hatchling sex ratios of hawksbill sea turtles on Buck Island Reef dining the 1997, 1998, and 1999 nesting seasons and further determine possible influence of weather on these sex ratios. Incubation temperatures were monitored in a total of 48 nests over the 1997-1999 nesting seasons. The average incubation temperature during the middle third of incubation was used as a predictor of die overall c lutch sex ratio. The results suggest an overall female-bias during each of the nesting seasons. However. the results also suggest that some male-biased clutches were produced due to major tropical weather systems passing through the U.S. Virgin Islands.

MOLECULAR ANALYSIS OF ALUMINUM TOXICITY TO ARABIDOPSIS THALIANA. Fleather Morefield, Tomiko Goodman, and Mijitaba Hamissou, Department of Biology, Jacksonville State University, AL 36265

Aluminum, (Al), is the most abundant metal on the earth's crust. Aluminum interferes with the uptake, transport, and use of other ions required for metabolic activities. Research has indicated that Al disrupts cellular calcium homeostasis, rapidly represses root elongation, and interacts with the cell wall, the plasma membrane, and the root symplasm. Al was also shown to inhibit DNA synthesis. It also affects the nutrient status of the root zones due to its competitive interaction with Mg. One solution to Al toxicity to plants is the development of transgenic plants. Arabidopsis thaliana is a small flowering plant widely used as a model organism in plant molecular biology. Member of the mustard (Brassicaceae) family, it is characterized by a small genome and a rapid life cycle making it ideal for biological studies. The objective of this study is to investigate the molecular responses of arabidopsis plants to Al toxicity, and to assess on its encapsulation in the plant body.

Arabidopsis seeds were planted in pots containing mixture of vermiculite and sand. Germinated plants were kept in a growth chamber, watered and fertilized for four weeks. Plants were deprived of water for four days then watered with different concentrations of Al solution. Samples of leaves were taken at different time intervals for protein extractions and analysis.

The data indicated that arabidopsis plants exposed to higher Al concentration repress several classes of cytoplasmic proteins and express new classes of protein in the range of 80-85 Kd.

MONOCLONAL ANTIBODIES AGAINST ECDYSONE-BINDING YOLK POLYPEPTIDE IN THE CRICKET, ACHETA DOMESTICUS. Jeremy W. Bishop, Barbara H. Estridge, and James T. Bradley, Department of Biological Sciences, Auburn University, AL 36849.

During vitellogenesis, the fat body of Acheta domesticus produces and secretes two multisubunit vitellogenins (VGs; 352 kDa and 357 kDa) into the hemolymph. These are taken up by vitellogenic oocytes via receptor-mediated endocytosis and become egg vitellins (VTs). The five yolk polypeptide (YP) subunits (50-186 kDa) comprising VG/VT undergo developmental stage-specific processing during embryogenesis. One of these (YPIII; 53 kDa) reacts with anti-ecdysone and is believed to carry the steroid hormone into the egg during vitellogenesis. Preparative SDS-PAGE and affinity chromatography (Seize-X, Pierce Biochem. Co.) were used to purify egg YPIII which was used as an antigen to inoculate mice for MAB production. Six MABs were obtained from single-cloned fused cell lines. Three MABs reacted only with YPIII in Western blots and two reacted with YPIII and YPIIb. IgG MABs and anti-ecdysone showed similar patterns of reactivity with YPs in developing eggs from oviposition through day 7 of embryogenesis. At this point , the reactivity between anti-ecdysone and YPIII decreases, suggesting that ecdysone, during this period is cleaved from YPIII. This is further supported by the reactivity between the IgG MABs against YPIII, which does not decrease until day 13 of embryogenesis. In addition to this, Western blotting shows slight decreases in molecular weight of YPIII when it is incorporated into the oocyte from the hemolymph. These and the four IgM MABs obtained will be valuable for examining the entry of ecdysone into developing oocytes and its biological significance during embryogenesis.

MORPHOMETRIC VARIATION IN BLUEGILL SUNFISH FROM LOTIC ND LENTIC ENVIRONMENTS IN SUMTER COUNTY, ALABAMA. Sarah Branson and John McCall. Dept. of Biological and Environmental Sciences, University of West Alabama, Livingston, AL 35470.

Current velocity can be expected to be a significant factor affecting the fitness of fish. For a fish species that is found in both stream and pond environments, morphometric variation might well be expected between populations in these respective habitats. Bluegill sunfish (Lepomis macrochirus) collected in lotic and lentic environments in Sumter County, Alabama, were examined for variation in body and fin dimensions. Similarly-sized bluegill were sampled from non-flowing habitats within the Alamuchee Creek drainage and from a flowing environment in Ponkabia Creek. Body and fin dimensions were measured arid ratios developed to allow comparison of body and fin shape. Statistical analysis revealed that fin shape differed significantly between the two populations. Both pectoral and pelvic fins were relatively longer in bluegill collected in lotic environments. Differences existed, as well, in the shape of the dorsal, anal, and caudal fins.

NESTING ACTIVITY OF THE ALABAMA REDBELLY TURTLE (PSEUDEMYS ALABAMENSIS). David H. Nelson, William M. Turner and T. Joshua Meyer, Dept. of Biological Sciences, Univ. ofSouth Alabama, Mobile, AL 36688.

A study of nesting activity in the Alabama redbelly turtle was conducted in the vicinity of the Mobile Bay Causeway during the summer of 2001. Vegetative analysis of 47 depredated nests that turtles may prefer sites having some vegetation (when compared to unvegetated or heavily-vegetated areas). Nests were laid from 29 May to 7 August; hatchlings emerged from 3 September to 3 November, Clutch sizes for 16 nests ranged from 7 to 15 eggs (x=10.8). Developmental periods ranged from 69 to 117 days (x=92.3) The numbers of hatchlings from each of 16 nests ranged from 0 to 15 (x=8.3) The numbers of undeveloped eggs ranged from 0 to 14 eggs per nest (x=8.0). Virtually all hatchlings manifested mandibular cusps, eyebars and reddish plastrons (with vermiculations). Measurements of 91 hatchlings (from 12 clutches) disclosed a x carapace length of 38.7mm, a carapace width of 38.7mm, a plastron width of 36.1mm, a x tail length of 13.3mm and a x wet body weight of 11.4 grams. Nest predation by raccoons, fish crows, and bo at-tailed grackles was very high. Predator-excluder covers were used to protect eggs until they successfully completed development.

PATTERN OF STEROIDOGENIC FACTOR-1 EXPRESSION IN THE RED-EARED POND SLIDER TURTLE. Chris Murdock and Thane Wibbels, Dept. of Biology, U of Ala-Birmingham Birmingham, AL 35294.

A variety of reptiles possess temperature-dependent sex determination (TSD). The incubation temperature during the approximate middle-third of incubation, known as the thermosensitive period (TSP), determines the sex of the developing embryo. Studies have suggested that estrogen production during the TSP may control female sex determination. Therefore, the production of transcription factors or enzymes related to estrogen synthesis could represent temperature-sensitive elements. Steroidogenic factor-1 (SF1) represents a transcription factor of interest since it regulates of the expression of various steroidogenic enzymes (e.g., P450 hydroxylase enzymes). The reported study utilized a quantitative competitive RT-PCR for measuring SF1 mRNA levels in the adrenal-kidney-gonad complexes of developing red-eared pond slider turtle embryos, at both male-and female-producing incubation temperatures. Results from these quantitative competitive RT-PCRs indicated that the pattern of SF1 expression was almost identical, t hroughout development, between embryos incubated at male-and female-producing temperatures. Only at stage 19 (a stage corresponding to the very end of the TSP) were significantly higher (t = -5.3, P = 0.027) SF-1 mRNA levels observed from embryos incubated at female-producing temperatures. These results suggest that the expression of SF1, in the developing adrenal-kidney-gonad complexes, is not a sex-specific regulatory step in the sex determination cascade of these reptiles.

POPULATION DYNAMICS FOR TWO POPULATIONS OF AMBYSTOMA MACULATUM. Eric A. Blackwell, Robert A. Angus, and Ken R. Marion. The University of Alabama at Birmingham, Birmingham, AI 35294-1170.

Primarily pond breeders, ambystomatid salamanders migrate from their terrestrial habitats to nearby ponds during breeding season. In northeast Alabama the migration of die spotted salamander (Ambystoma maculatum) is a conspicuous event that occurs during late winter. For three breeding seasons (2000 - 2002) an intensive mark-recapture study was conducted on two breeding populations of A. maculatum in northeastern Alabama. Drift fences completely encircling an ephemeral and a permanent pond were used to capture all adult salamanders migrating into and out of die ponds. All captured salamanders were marked for identification with passive integrate transponders (PIT tags). The Triple Catch method of population estimation was used to estimate population size, gains and survival rates for each population. Both populations had pronounce female biased size dimorphism. Each population had high survivorship and modest gain indicating stable populations. More salamanders used the ephemeral pond than the permanent pond.

REAL-TIME PCR DETECTION OF Vibrio parahaemolyticus 03:K6 Michael L. Myers and Asim K. Bej, Dept. of Biology, Univ. of Alabama at Binningham, AL 35294-1170.

A relatively rapid and quantitative method was developed for the detection of a microbial pathogen, Vibrio parahaemolyticus 03:K6, by using real-time polymerase chain reaction (PCR). Oligonucleotide primers were designed to amplify a 368-bp segment of the open reading frame B (ORF8) DNA sequence that is found specifically in V. parahaemolyticus 03:K6. The PCR reaction was optimized with the FastStart DNA Master SYBR Green I Kit (Roche) by adjusting magnesium chloride concentration, cycling times, and cycling temperatures. This kit provides a mix with all PCR reaction components except for template DNA and primers. Optimization was also performed separately by using Platinum Taq DNA polymerase (Invitrogen) and adjusting the concentrations of magnesium chloride, SYBR Green I fluorescent dye, and gene-specific primers, as well as cycling times and temperatures. Consistent and sensitive detection was obtained with Platinum Taq DNA polymerase and SYBR Green I dye. The sensitivity of detection using Platinum Taq DN A polymerase was determined to be 10 pg. of purified genomic DNA, as opposed to 0.1 ng. with the SYBR Green I Kit. The applicability of this method for detection of this pathogen in the environment was tested by seeding Gulf water with serially diluted V parahaemolyticus 03:K6 cells. The detection level observed in a 1% (w/v) agarose gel was [10.sup.3] cells/100 ml Gulf water. Comparison of boiled template DNA from seeded Gulf water and of purified DNA using commercially available kits exhibited promising results for the applicability of real-time PCR for the detection of this marine pathogen in the environment.

REPRODUCTIVE CYCLE OF THE SEA URCHIN LYTECHINUS VARIEGATUS FROM A SHELTERED BAY IN FLORIDA: A PRELIMINARY DESCRIPTION. Adele W. Cunningham and Stephen A. Watts. Dept. of Biology, Univ. of Ala. at Birmingham. Birmingham, AL 35294-1170.

The reproductive cycle of Lytechinus variegatur was monitored for thc last 11 months in a north Florida bay. For both males and females, average gonad mass increased substantially from November to April. An additional peak in average gonad mass occurred in females in Septcmber. Qualitative and quantitative analysis of gonad histology is being used to assess the storage capacity of thc nutritive phagocytes and the developmental state of the gametes in the gonad. In April and June. urchins were actively producing gametes and have the capacity to spawn. In July. urchins have recently spawned. contained relict gametes. and were renewing nutritive phagocytes. In females. germinal epithelium was the thickest in April. declined in June. and was greatly reduced in July. In females and males, the percent of gonad occupied by gametes was also highest in April. declined in June. and was lowest in July. Size-frequency analysis of oocyte diameters showed the highest frequency of large ooeytcs in April. the highest frequen cy of small oocytes in June. and the fewest oocytes at all sizes in July. The presence of immature and mature oocytes in all three months and the range of reproductive stages in each month suggests spawning is initiated at least by April and continues through July. The reproductive state is currently being evaluated for other months. The success of L. variegatus in nearshore environments may be a consequence of its ability to produce and release gametes over an extended period. thus, maximizing recruitment and increasing its overall fitness.

RESTRICTION ENZYME SURVEY OF HALOPHILC AND HALOTOLERANT BACTERIAL ISOLATES FROM DIVERSE SALINE ENVIRONMENTS. Pat Graham and Donald Salter, University of West Alabama.

Using a high NaCI concentration selective complex growth medium, we have cultured a number of unique and interesting bacterial isolates from three different saline ecosystems: an inland salt spring near Jackson, AL. an estuary near Pensacola Beach, FL, and seawater off Pensacola Beach, FL. Our laboratory has previously characterized their ability to produce different exoenzymes and to grow in a complex growth medium containing several different inorganic components at different concentrations, These lialophilic and haloterant isolates may also produce potentially useful biological molecules such as restriction enzymes with unique properties and unique nucleotide sequence sites. We investigated three extract preparation methods: sonication of broth grown cells, TritonX100/lysozyme lysis of agar grown cells, and Triton-X100/lysozyme lysis of agar grown cells with subsequent viscosity reduction by related passing through a 26-30 gauge needle. The extracts were assayed by incubating with lambda DNA in a commercia l restriction enzyme buffer at 37[degrees]C for 1.5 hours, and then analyzed for products of DNA degradation by agarose gel electrophoresis and ethidium bromide staining. Twenty-four isolates were screened by these three extract preparation methods and the standard assay. Extracts prepared by the third cell breaking method seem to be give more consistent results, was easier to perform, and required less time to complete. One isolate, PG9A, prepared by the third cell breaking technique produced a restriction enzyme that excises lambda DNA into 10 or more DNA fragments. Future research will emphasize re-testing the isolates with the third extract preparation method and determine whether any of the restriction enzymes are unique and potentially useful tools for the biotechnology industry.

SILTATION EITECTS ON FISH COMMUNITIES IN THE CAHABA WATERSHED. Jaideep Honavar, Robert Angus and Ken Marion. Dept. of Biology, Univ of Alabama in Birmingham, AL 35294.

Excessive siltation of streams has been identified as the leading cause of fish habitat alteration in the southeastern. Although some siltation is natural, anthropogenic sources greatly increase the volume of sediment entering the water body. Increases in turbidity and deposition reduce light penetration and cause local habitat alterations. Fish are sensitive indicators of aquatic ecosystem quality. The Cahaba river system supports a very diverse ichthyofaunal assemblage. Rapid urbanization with associated increases in siltation is occurring in the upper Cahaba basin. Six sites of varying sedimentation impact in the upper Cahaba drainage were assessed in order to measure the effect of siltation on fish community structure. Habitat quality was evaluated using EPA Rapid Bioassessment Protocol. Fish collections were carried out by seining and electroshocking during fall and spring of 2000 and 2001. Fish data were analyzed using an Index of Biotic integrity (IBI), which is a fish assemblage quantification scheme based on abundance and trophic and taxonomic composition of the fish community. Comparing the habitat assessment values with the fish metrics showed a distinct relation that habitat alteration due to siltation led to an alteration in the fish community structure within the assemblage. The IBI was negatively correlated with decreased habitat quality and increased siltation. Sediment -- impacted streams also showed decreases in darter abundance, crevice spawning species and sensitive minnows

SUCCESSION IN FISH COMMUNITIES OF AN EPHEMERAL STREAM IN SUMTER COUNTY, ALABAMA. Kathryn Alexander and John N. McCall, Dept. of Biological and Environmental Sciences, University of West Alabama, Livingston, AL. 35470.

The reestablishment of the fish community in an ephemeral stream in Sumter County, Alabama was followed over a period of l2 months in 2000-2001. Ponkabia Creek is part of the Sucarnoochee River drainage in west-central Alabama. In the early fall of 2000, the stream bed east of Sumter County Highway 28 was completely dry. Rains in late fall of that year flooded the stream bed, and allowed reestablishment of the fish community to begin. Monthly sampling was conducted of the fish community at five sites within the stream. Environmental parameters were also measured. Although a relatively diverse community was established at most sites by the following summer, community composition and diversity continued to fluctuate widely at all sites throughout the course of the study. This suggests that a stable community structure had not been achieved, and that short-term environmental fluctuations may exert significant control on community composition.

STRUCTURAL ANALYSIS OF GREENBUG FEEDING ON SORGHUM PLANTS UNDER DROUGHT STRESS. Mijitaba Hamissou, Biology Department, Jacksonville State University, AL 36265 and Paul E. Richardson, Oklahoma State University, Stillwater, OK 74078

Sorghum bicolor (L.) Moench is grown in countries of Africa and Asia as a principal staple food source. In the United States, sorghum production is centered in the Great Plains States. In these areas, sorghum production is hampered by drought and insect infestation. One of the most important insect pests in sorghum is the aphid greenbug, Schizaphis graminum (Rondani). Greenbugs exhibit antixenosis for the bloomless sorghum. Sorghum leaves are normally covered with a powdery wax termed "bloom". The condition where there is no wax covering is termed "bloomless." Wax cover was shown to reduce plant's water loss. The objective of this study is to determine the effects drought and greenbug feeding activities on the ultrastructure of sorghum.

Isogenic lines of sorghum, (ROKY62 Bin, ROKY62 bin, ROKY78 Bin, and ROKY78 bm) were grown under controlled environment and filed conditions. Drought stress was imposed by withholding water and by measuring plants relative water contents. Greenbug feeding activities were monitored electronically.

Our data indicated that drought stress interferes with the ability of greenbugs to successfully commit to phloem feeding and that salivation frequencies were higher in bloom plants than in bloomless plants under similar conditions. We also observed starch and plastoglobuli built up in the chloroplasts and an increase in the number of mitochondria in plant cells as a response to the aphid feeding.

VERTEBRATE ROAD-KILL SURVEY OF THE MOBILE BAY CAUSEWAY. David H. Nelson, Dept. of Biological Sciences, Univ. of South Alabama, Mobile, AL 36688.

A systematic, road-kill survey was conducted (by bicycle or automobile) on the Mobile Bay Causeway each week during 2001 to assess the numbers of vertebrates killed by vehicular traffic. More than 500 organisms representing 85 species of vertebrates were encountered: 8 amphibians, 25 reptiles, 39 birds and 13 mammals. Southern Leopard frogs (Rana utricularia) were the most abundant amphibian (n=8). "Endangered" Alabama redbelly turtles (Pseudemys alabamensis) were the most frequently encountered reptile (n=71); most specimens were hatchlings. Laughing gulls (Larus atricill, n=42) and American coots (Fulica americana, n=32) were the most commonly-encountered birds. Raccoons (Procyon lotor, n=47), opossums (Didelphis marsupialis, n=41), and nutria (Myocastor coypus, n=30) were the most abundant mammals. Data were analyzed by taxon and season. The most significant finding in this ongoing study has been the emergence of 51 hatchlings of Pseudemys alabamensis during the month of April. These data confirm overwinte ring in the nest by the Alabama redbelly turtle.

TELOMERASE (hTERT) PROMOTER REGULATION DURING NEOPLASTIC TRANSFORMATION OF WI-38 HUMAN FETAL LUNG FIBROBLASTS. Mark A. Casillas, Scott L. Brotherton, Lucy G. Andrews, J. Michael Ruppert, and Trygve O. Tollefsbol, University of Alabama at Birmingham.

Elucidation of the mechanisms governing expression of the human telomerase reverse transcriptase (hTERT) is important for understanding cancer pathogenesis. Approximately 90% of tumors express hTERT, the major catalytic component of telomerase. Activation of telomerase is an early event, and high levels of this activity correlate with poor prognosis. Recent studies have shown that the transcription factors c-Myc and Madl activate and repress hTERT, respectively. It is not clear how these transcription factors compete for the same recognition sequence in the hTERT core promoter region. Studies have shown that the combined expression of 5V40 large T antigen (T-Ag), hTERT, and H-Ras is able to transform human cells. We recapitulated these studies in a distinct human cell type, WI-38 fetal lung fibroblasts, a cell culture system widely used for senescence studies. We transduced cells and emphotropic retroviral constructs containing SV40 T antigen, hTERT, and activated H-ras. Transduced cells exhibited anchorage-independence in soft agar and expressed increased levels of c-Myc and endogenous hTERT. These effects were observed after more than 25 population doublings (PDs) following the establishment of the neoplastic cell line. During the process of transformation, we observed a switch from Madl/Max to c-Myc/Max binding to oligonucleotide sequences containing the hTERT distal and proximal E-boxes. c-Myc can bind specifically to the hTERT promoter in vitro, indicating that c-Myc expression in tumors may account for the increased expression of hTERT observed in vivo. This experimental model provides a means for evaluating mechanisms of cancer initiation, promotion, and progression in WI-38 fibroblasts.

TELOMERASE MAINTENANCE AND CONTROL IN AGING HUMAN CELLS. Nathaniel J Hansen Nadejda Lopatina, Joseph C. Poole, Lucy C. Andrews, and Trygve 0. Tollefsbol, Dept. of Biology, University of Alabama at Birmingham, Birmingham, AL, 35294.

Telomerase, the enzyme that maintains chromosomal ends, has been the subject of significant attention for its role in neoplastic transformation and cellular senescence. The subunit responsible for the catalytic action of telomerase, hTERT, is expressed in some differentiating tissues and in 85-95% of human tumors. However, it is not present in the majority of somatic cell lines, and the mechanisms of transcriptional regulation in the hTERT gene have been sought as potential control points of telomerase function. A portion of the hTERT gene from WI-38 human fibroblasts was targeted for assessment with bisulfite methylation sequencing. Following sodium bisulfite-mediated conversion of cytosine residues to uracil, the presence of unconverted 5-methylcytosine is detected in bisulfite-treated oligonucleotides through genomic sequencing. Using this method, an hTERT target PCR product has been amplified for sequencing. Results in a human teratocarcinoma cancer cell line during a twelve-day course of retinoic acid-in duced cellular differentiation have shown a marked increase in hTERT promoter methylation as well as a concomitant decrease in hTERT mRNA expression. While these data suggest a strong association between promoter methylation and silencing of the hTERT gene, published reports indicate that methylation may only play a minor role in a more complex telomerase silencing mechanism in other cancer cell types. Elucidation of tissue-specific modes of hTERT regulation should prove vital for the understanding of tumorigenesis and aging in human cells.
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Publication:Journal of the Alabama Academy of Science
Date:Apr 1, 2002
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