Biology Department

1999 Senior Thesis Abstracts

(CLASS OF 1999: If your thesis abstract is not currently included on this page and you would like it to be, please follow this link.)

Evaluation of the Possible Symbioses between Temperate Grass Crop Species and Nitrogen-fixing Bacteria Isolated from European Beachgrass (Ammophila arenaria)

Nicolas Gabriel Azios

(Advisor: Dalton)

Abstract

European beachgrass (Ammophila arenaria ) is a dune grass common to the Pacific Northwest coastline. The recent isolation of N₂-fixing bacteria from Ammophila suggests that the prolific success of this grass on nutrient-poor sand may be due in part to the ability to fix nitrogen through the action of endophytic bacteria. This project examines the possibility that similar symbioses might be established between bacterial isolates from Ammophila and various cereal and forage grasses taxonomically related to Ammophila.

Six species of bacteria (Agrobacterium radiobacter, A. rubi, Xanthomonas campestris celebensis, X. fragariae, Sphingomonas multivorum, and Roseomonas genomospecies 5) were isolated on N-free media. Acetylene reduction assays revealed low levels of nitrogenase activity for all isolates in pure culture. Three bacterial isolates (Xanthomonas campestris celebensis, Agrobacterium rubi and Agrobacterium radiobacter) were used to inoculate the cereal/forage grasses.

The genera of grasses tested include Triticum, Hordeum, Secale, Phleum, Avena, and Koeleria. Plants were grown from surface-sterilized seeds in heat-sterilized sand with (+) or (-) N nutrient solution and treated with (+) or (-) bacteria inoculations. Nitrogen-fixing associations were assessed by acetylene reduction and plant growth. All plant genera showed acetylene reduction in small amounts for all treatments. Possible bacteria-induced growth effects were observed for Koeleria, Hordeum, and Triticum. The presence of endophytic bacteria in roots of select plant samples were assessed by reisolation of bacteria on N-free media and verification was attempted by LM immunogold silver staining screens with an anti-nitrogenase antibody.

The potential for establishing endophytic N₂-fixing associations in grasses of agricultural importance could have profound agronomic/economic implications. This study was funded in part by a HHMI Undergraduate Research Program grant.

Apoptosis and the Cell Cycle: The Effects of Protein Kinase C Activation in Xenopus laevis Splenocytes and Thymocytes

Ashlee Beth Bergin

(Advisor: Ruben)

Abstract

Apoptosis, or programmed cell death (PCD), is an integral part of life. The apoptotic process removes unnecessary and/or unwanted cells. However, some cells may lose the ability to undergo apoptosis, and continue to proliferate. When unchecked, cell transformation to neoplasia may result. The incidence of spontaneous and chemically-induced cancer in non-isogenic amphibians is rare. Even injections of oncogens which bind directly to DNA result in little or no cancer formation. This would seem to suggest that amphibian cells may somehow be able to become apoptotic more easily than those of mammals.

Entrance into the cell cycle appears to be imperative in order for mammalian lymphocytes to undergo apoptosis. Thus, it has been suggested that apoptosis and the cell cycle are so closely linked that entry into the cell cycle is required for mammalian lymphocytes to become apoptotic. It is of interest then, to ask whether amphibian cells differ from mammalian lymphocytes in this regard, since cells that are more likely to undergo apoptosis should be less likely to become transformed into cancer.

In vitro, exposure of Xenopus splenocytes or thymocytes to PMA, a tumor promoting agent in mammals, or its weakly mitogenic isomer, MPMA, revealed that while both were capable of inducing apoptosis in splenocytes and thymocytes in Xenopus laevis, PMA was a more effective apoptogen than was MPMA with both cell types. However, when apoptosis and proliferation were assayed concurrently using the same cell populations, consistent levels of apoptosis in both PMA- and MPMA-treated cells were seen throughout the three day period, but proliferation, as measured by BrdU uptake, was high only when PMA was used. These results suggest that there is not a direct relationship between apoptosis and the cell cycle, since decreases in the percentage of apoptotic cells in splenocyte populations exposed to PMA were not evident when BrdU uptake was maximal. Apoptosis can be seen as early as three hours, but BrdU uptake is not apparent until many hours later. Thus, an increased susceptibility for apoptosis in Xenopus cells may account for the resistance to neoplasia seen in amphibians. This study was funded in part by a HHMI Undergraduate Research Program grant.

CO₂ Exchange, N₂ Fixation, and Growth of Lobaria oregana Transplants in an Old-growth Forest Canopy

Patrick James Brown

(Advisor: Dalton)

Abstract

Lobaria oregana is a nitrogen-fixing cyanolichen endemic to old-growth forests in the Pacific Northwest. Often the most abundant old-growth epiphyte, it fixes atmospheric N₂ at rates as high as 3.5 kg ha^-1 y^-1. However, it is usually absent from younger forest stands. Both microclimate and dispersal limitations have been proposed to explain the distribution of L. oregana.

A canopy crane was used for in situ measurement of L. oregana's physiological activity. Two different sites were used: an old-growth forest and a 90 year-old stand of Douglas-fir. Litterfall was hung in "pendants", left to acclimatize for 4 months, and assayed at 3-week intervals for CO₂ exchange and N₂ fixation. Transplants in the top (62 m) and middle (37 m) levels of old-growth grew 15% in six months, the highest rate of growth yet reported for L. oregana. Transplants in the old-growth understory (2 m) became discolored and showed little CO₂ exchange or N₂ fixation. Transplants in the 90 year-old Douglas-fir understory (2 m) remained healthy but showed signs of disturbance from birds or small mammals. Physiological activity of transplants was directly related to hydration (%), which is expressed as a percentage of a lichen's dry weight. Hydration (%) was higher in winter than in spring and higher in the understory than the upper canopy. Net photosynthesis was highest between 120-200% hydration and was greatly reduced at high hydration levels. N₂ fixation increased linearly with hydration, and was much higher in fresh material than in litterfall or lichens that were dried and rewetted. N₂ input into forest ecosystems may be substantially affected by the population dynamics of symbiotic and free-living cyanobacteria in forest canopies. This study was funded in part by a HHMI Undergraduate Research Program grant.

Biotic Interactions and Invasion Resistance: A Study of the Introduced Green Crab (Carcinus maenas) on the West Coast of North America

Eric Russell Buhle

(Advisor: Childress)

Abstract

Long-range transportation technology has greatly accelerated the rate of invasion of communities by exotic species, often with disastrous ecological and economic consequences. Recognition of the impacts of introduced species has fueled interest in making the field of invasion ecology more predictive. Among the oldest and most controversial hypotheses of invasion ecology is the idea that high species diversity or strong biotic interactions enable a community to resist the establishment of self-sustaining populations by an invader. I investigated this hypothesis, using as a case study the ongoing invasion of marine communities on the West Coast of North America by the European green crab Carcinus maenas. Diet and habitat use patterns of native and introduced C. maenas populations suggest that strong competition for bivalve prey from the native sea star Pisaster ochraceus may prevent the establishment of green crabs in wave-exposed rocky intertidal habitats on the West Coast. I performed laboratory experiments to test two predictions of this hypothesis. To test the prediction that competition will cause Carcinus and Pisaster to partition prey resources by size, I compared the frequencies of three size classes of the mussel Mytilus californianus eaten by each predator in isolation and by both predators when kept together. Contrary to prediction, green crabs' prey size distributions in the presence and absence of Pisaster did not differ significantly, with the smallest mussels consistently preferred. Pisaster's prey size distribution was significantly affected by competition from Carcinus, with a reduced proportion of small mussels eaten in the presence of crabs. In a second experiment, I tested the prediction that there will be a difference in the relative strengths of intraspecific competition between green crabs and interspecific competition by Pisaster against green crabs, with implications for potential invasion success. I measured feeding rates of Carcinus on mussels in isolation and in the presence of another crab or a sea star. There were no significant differences in green crab feeding rates among competition treatments, but in the presence of a crab, Pisaster was found to feed at a significantly higher rate than Carcinus. These experiments produced no strong evidence to confirm or refute the hypothesis that competitive interactions prevent the invasion of the rocky intertidal by Carcinus, although observations of the foraging strategies of green crabs and sea stars provide some insight into possible mechanisms of competition. This study was funded in part by a HHMI Undergraduate Research Program grant.

Elicitation and Enhancement of Antibody Production Using a Pavlovian Conditioning Paradigm

Bridget A. Dyer

(Advisors: Ruben, Dickinson)

Abstract

There is little research on the conditioned activation of the immune response to antigenic challenge. Some authors suggested that a conditional stimulus can elicit antibody production in the absence of additional antigen. Others reported that a conditional stimulus cannot elicit antibody production without additional antigen, but that it can enhance antibody production in the presence of a minimally immunogenic antigen dose. Both of the above hypotheses were tested in Wistar rats, using a single conditioning trial. Subjects were exposed to the taste of saccharin, the conditional stimulus, followed by injection of TNP-OVA, the unconditional stimulus. Fourteen weeks later, subjects were re-exposed to the conditional stimulus, either with or without additional antigen. Conditioned elicitation of antibody production was not observed. Results suggested that conditioned enhancement of antibody production occurred, although they only approached significance. Subjects developed an unexpected conditioned taste aversion to saccharin.

Optimal Prey Selection by Invasive European Green Crabs, Carcinus maenas, and the Potential Impact on the Pacific Northwest's Commercial Oyster Industry

Robert Stater Foster

(Advisor: Childress)

Abstract

The arrival of the European green crab, Carcinus maenas, is likely to be one of the most ecologically detrimental invasions the west coast of North America has seen. Of particular concern with the invasion is the impact on commercial shellfish crops of the Pacific Northwest, a 44 million dollar a year industry. Employing Optimal Foraging Theory as a model, this study focused on the interaction between the European green crab and the commercially important giant Pacific oyster, Crassostrea gigas. I estimated the handling time and energetic values for a range of oyster sizes in order to predict the optimal prey size for three size classes of crabs. When given a choice of three distinct sizes of oysters, crab selectivity was not significantly different from either expected ratios of optimal prey selection or random prey selection. Although the results do not distinguish if crabs forage optimally, a comparison to previous work on alternative prey items suggests that green crabs may not have a significant impact on oyster cultures of the Pacific Northwest. This study was funded in part by a HHMI Undergraduate Research Program grant.

Inversion of the Cytoplasmic Contents of Xenopus laevis Embryos During the First Cell Cycle Results in an Inverted Phenotype Displaying a 180° Shift in the Expression Pattern of VegT

Lev Valentine Hofmann

(Advisor: Black)

Abstract

Inversion of the cytoplasmic contents of the eggs of the African clawed frog Xenopus laevis early in the first cell cycle results in an inverted phenotype in which the anterio-posterior axis is reversed from that specified by the normal distribution of maternal determinants. In Xenopus the body axes are generated via a complex system of interactions whose correct spatial and temporal expression is necessary for realization of the body plan. One of the key steps in this process is the induction of mesoderm in the marginal zone, which lies in the vegetal hemisphere near the equator. It is in this region that the dorsal organizer, which is responsible for the generation of both major body axes and marked at gastrulation by the blastopore, will form. The inversion procedure causes the blastopore to form in the animal hemisphere and gastrulation to proceed in the vegetal direction, the reverse of that observed in untreated embryos. This phenotypic result suggests that mesoderm is induced to form in the equatorial zone on the animal side. This thesis sought to determine the location of zygotic transcripts of two T-box transcription factors (VegT and Xbra) necessary for mesoderm induction, in gastrulae manipulated to display the inverted phenotype. The zygotic expression pattern of VegT in functionally inverted embryos is a mirror image of that observed in their normal counterparts, suggesting that the inverted phenotype arises from relocation of cytoplasmic contents, which than cause a 180° shift in the pattern of gene expression. This study was funded in part by a HHMI Undergraduate Research Program grant.

Characterization of Proliferative Senescence and Telomerase Activity in Chicken Embryonic Fibroblasts

Mojgan Hosseini-Varnamkhasti

(Advisor: Shampay)

Abstract

Telomeres and telomerase have been implicated in both replicative senescence and immortalization. In the absence of telomerase, telomere length decreases by progressive cell division as a result of the end replication problem. The erosion of telomeres results in the process of cellular senescence, which is associated with inhibition of cell proliferation, morphological changes and altered patterns of gene expression in cells. Telomerase, the enzyme responsible for maintenance of telomere length, has been demonstrated to play a role in increasing the proliferative capacity of cells.

Chicken embryonic fibroblasts (CEF) were passaged 1:10 till they became senescent, which happened at PDL 14 and PDL 19 in two different cultures. Cells from two other cultures which were passaged 1:3, were not senescent at PDL 18. This suggests that life-span of CEF's in culture is partly dependent on the density at which the cells are seeded when passaging. Senescent cells were shown to express senescence-associated ß-Galactosidase activity at pH 6.

Low passage number CEF's were infected with viruses expressing either human c-myc gene or a doubly truncated chicken myb gene and were assayed for presence of telomerase activity. In myc-expressing cells, telomerase activity was detectable one day post infection, suggesting that telomerase is under direct activation by c-Myc. Telomerase activity increased within the next few passages as the number of infected cells increased. Telomerase activity was very low or undetectable in myb-expressing cells or control (uninfected or virus without oncongene) cells. Myc activation of telomerase is only one of the pathways through which telomerase activity is regulated. Future studies are required to better characterize the nature of telomerase regulation.

Role of Two Species of Endophytic Bacteria in the Nitrogen Nutrition of European Beachgrass (Ammophila arenaria)

Forest Kaser

(Advisor: Dalton)

Abstract

Ammophila arenaria is a beach-dwelling perennial also known as European beachgrass and marram grass. Its ability to thrive in the nitrogen poor sands of coastal areas around the world suggests that it may have evolved the ability to associate with nitrogen-fixing bacteria as a way of meeting its nitrogen requirements. Previous studies suggest that the intercellular spaces of A. arenaria are extensively colonized by bacteria and that the nitrogenase enzyme is present in substantial amounts as well (Old and Nicolson, 1975; Fusaro, 1997). If A. arenaria forms a nutritionally significant relationship with nitrogen-fixing endophytes, it would be the first known temperate monocot to do so. The bacteria involved would be of tremendous agricultural interest, since the genetic and environmental similarities between beachgrass and many crop grasses are much higher than they are between crop species and existing models of associative nitrogen fixation. The bacteria in beachgrass could potentially be used as agricultural inoculants in a more environmentally sensible fertilization strategy. It may also be possible to modify the bacteria to be pathogenic, thereby providing a new approach to controlling the spread of beachgrass on the west coast of North America, which is a major ecological concern. To further investigate the possibility that A. arenaria forms an endophytic association with diazotrophic bacteria and to evaluate the nutritional significance that such a relationship may have, A. arenaria seedlings grown from surface sterilized seeds were inoculated with two bacterial species isolated from the surface-sterilized field beachgrass. Following a growth period under aseptic, controlled environmental conditions, the nutritional significance of the bacteria was assessed by comparing root and shoot weights and nitrogen contents of inoculated and uninoculated seedlings. To assess the potential of the inoculant bacteria to fix nitrogen under field-like conditions, the nitrogenase activities of intact, cultivated beachgrass/soil systems were determined using the acetylene reduction assay. No significant differences were found in the growth of inoculated and uninoculated plants, but there were some indications that at least one of the two species of bacteria used as inoculants can fix nitrogen in association with European beachgrass. This study was funded in part by a HHMI Undergraduate Research Program grant.

Associative Nitrogen Fixation in Temperate Coastal Grasses

Sasha Bryn Kramer

(Advisor: Dalton)

Abstract

Recent discoveries at Reed College indicate that Ammophila arenaria (European beachgrass) associates with nitrogen-fixing bacteria which colonize the interior of the shoots and rhizomes. This association represents the first evidence for endophytic nitrogen fixation in a temperate grass. The objective of this thesis was to determine whether this type of bacterial association is present in other temperate grasses which are subjected to the same ecological constraints as A. arenaria. Results from acetylene reduction assays and immunolocalization demonstrate that the three grasses screened for nitrogenase activity, Ammophila brevigulata , Elymus mollis , and Uniola paniculata, all appear to engage in associations with endogenous diazotrophs. Resin-embedded stem and rhizome sections from these grasses were screened for the presence of the nitrogense enzyme using silver-enhanced immunogold labeling and fluorescent Cy3 immonolocalization with a anti-nitrogenase antibody. Immunolocalization results indicate that the bacteria preferentially colonize the apoplast (intercellular spaces and middle lamella), cell wall, and the outer layer of the plasma membrane in Elymus mollis and Uniola paniculata. Plant tissue from Ammophila brevigulata and Elymus mollis was tested for nitrogense activity using the acetylene reduction assay. For both plants it appears that endogenous and rhizospheric diazotrophs contribute to overall acetylene reduction rates, as evidenced by differences between surface-sterilized and unsterilized tissue. Acetylene reduction rates were highest for unsterilized rhizome and root tissue, although low rates were also observed for surface-sterilized rhizome and stem tissue (sterilized and unsterilized). In nitrogen-deficient sand dune ecosystems, associative nitrogen fixation by coastal grasses could play an important role in the nitrogen economy of, not only the grasses themselves, but of the ecosystem as a whole. This study was funded in part by a HHMI Undergraduate Research Program grant.

Extracellular Calcium is Necessary for Exocytosis in the Bag Cell Organs of Aplysia californica

Jinah Lee

(Advisor: Arch)

Abstract

An increase in intracellular calcium is necessary for exocytosis to occur. It is generally believed that this increase in internal calcium level is the result of an influx of extracellular calcium. However, recent studies have challenged this idea by proposing that internal stores of calcium can release sufficient amounts of calcium to trigger exocytosis. The neuropeptidergic bag cell organs (BCOs) of the marine mollusk Aplysia californica were used to examine this issue. Bag cells were radiolabeled with tritiated leucine to measure secretion and high-potassium (100 mM) medium was used as a depolarizing stimulus. After exposure to calcium-free media, the bag cells failed to respond to the high-potassium challenge. When the BCOs were placed in media containing normal levels of calcium, the high-potassium challenge increased levels of secretion. Chelating internal free calcium with the membrane permeant BAPTA-AM, in calcium-free medium, had no effect. When a high-potassium challenge was applied in the calcium chelation experiment, again, the BCOs failed to respond. The BCOs can secrete when they are placed in media containing normal levels of calcium, but they are not competent to secrete after being in a calcium-free environment. This strongly suggests that extracellular calcium is necessary for exocytosis to occur and calls into question the recent reports of exocytosis driven solely by internal calcium reserves.

Diverging from Your Ancestors: Chloroplast DNA Differences in Delphinium leucophaeum and Delphinium nuttallii Populations in Oregon and Washington

Michael Benjamin Lerner

(Advisor: Karoly)

Abstract

We are faced with the greatest loss of biodiversity since the age of the dinosaurs. In an effort toward conservation, morphological and molecular techniques are commonly used to identify and protect threatened and endangered species. This study focuses on whether chloroplast DNA (cpDNA) can be used to distinguish the Oregon endangered species Delphinium leucophaeum from the more common Delphinium nuttallii. Polymorphisms in the chloroplast genome of Delphinium leucophaeum and Delphinium nuttallii were detected by restriction site studies of PCR-amplified fragments. A total of five cpDNA fragments were amplified and digested with eight different restriction enzymes: Hinf I, Dde I, Hae III, Mse I, Dpn II, Taq I, Rsa I, and Hha I. 21.2 kb of cpDNA were amplified, and 816 bp observed by restriction analysis. Polymorphisms were identified in four of the fragments. This chloroplast DNA study did not provide a source of unique variation to support distinguishing D. leucophaeum and D. nuttallii. However, differences in the amount of divergence between D. leucophaeum and D. nuttallii may indicate recent speciation of D. nuttallii. Sharing of genetic markers within D. nuttallii was also found over wide geographic distances. This suggests that gene flow may be occurring via environmental vectors such as river flow.

It's All in the Display: Dominance Hierarchies and Recognition in the Red Swamp Crayfish, Procambarus clarkii

Elizabeth Anne Morgan

(Advisor: Childress)

Abstract

Aggressive behaviors frequently occur in animal populations due to competition over limited resources. However, since aggressive behaviors may be costly, in terms of energetic costs and risk of injury or death, mechanisms should evolve that decrease the need for escalated contests. Dominance hierarchies are one means that may have evolved to decrease the need for high levels of aggression in populations. Dominance hierarchies may form through individual recognition, in which familiarity results in behavioral changes, or dominance recognition, in which individuals respond to the dominance status of an opponent. The crayfish Procambarus clarkii form linear dominance hierarchies, but how they recognize individuals remains largely unknown. In this study, P. clarkii were paired together in order to establish dominance hierarchies consisting of four individuals. P. clarkii appeared to behave according to the relative rank of their opponent, with dominant individuals exhibiting greater numbers of aggressive behaviors than subordinate individuals. There is no evidence that P. clarkii recognized individuals because their behaviors did not change across trials, with familiar or unfamiliar opponents. Assessment appears to influence the outcome of contests, since prior aggressive level and length of chelae are correlated with winning contests. This supports the idea that aggressive level and chela size are indicators of the resource holding potential, or fighting ability, of P. clarkii and that they play an important role in dominance recognition. Overall, these results support the hypothesis that dominance recognition, rather than individual recognition, is the mechanism under which linear dominance hierarchies form in P. clarkii. This study was funded in part by a HHMI Undergraduate Research Program grant.

Phosphatidylserine Exposure on Apoptotic Lymphocytes of Xenopus laevis, the South African Clawed Toad

Marie Sherleen Estrella Nera

(Advisor: Ruben)

Abstract

Apoptosis is a fundamental feature of many biological processes which culminate in cell death. It depends on early recognition and removal of the dying cells by phagocytes. Several methods are available to detect apoptotic cells, e.g. failure to exclude large molecular dyes like propidium iodide and DNA fragmentation assayed by agarose gel electrophoresis, are two used at the Ruben laboratory to monitor apoptosis. These methods, however, measure only the late stages of the process.

Here, a rapid and reliable assay for detecting early stages of apoptosis in lymphocytes of Xenopus laevis, the South African clawed toad, has been employed. The assay takes advantage of the translocation of phosphatidylserine (PS) from the inner face of the plasma membrane to the cell surface of mammalian cells. Fluorescein isothiocyanate (FITC)-labeled Annexin V, a protein with a strong natural affinity for binding PS is used to identify external PS expression by flow cytometry. Apoptosis of Xenopus lymphocytes is of special interest, as differences may exist from mammalian cells that could provide a basis for understanding amphibian resistance to spontaneous and chemically induced cancer development in wildtype adult populations (Ruben et al., 1995). Cells that are more easily induced to undergo apoptosis, may be less likely to be transformed into cancer.

Cell surface exposure of PS in splenocytes and thymocytes of Xenopus has been initially studied here in lymphocytes treated with the calcium ionophore, A23187, a widely used apoptogen. The calcium flux stimulated the expression of PS on Xenopus lymphocytes. Dosage dependency and the kinetics of PS expression were also examined. Finally, the expression of surface PS was explored following lymphocyte exposure to a variety of apoptogens effective with cells from both species. All induce the appearance of surface PS on Xenopus lymphocytes. The Annexin V assay will prove useful in future investigations of apoptosis with this species. This study was funded in part by a HHMI Undergraduate Research Program grant.

Stromal Regulation of Proliferative Effects of 17ß-estradiol and Epidermal Growth Factor on Cultured Epithelial Cells from the Feline Uterus

Sarah Louise Peters

(Advisor: McClellan)

Abstract

The classical model of estrogen action proposes that cellular receptors that act as transcription factors mediate the effects of estradiol-17ß (E₂) on uterine growth through changes in expression of genes such as epidermal growth factor (EGF). However, stromal/epithelial interactions are also known to play an integral role in the control of E₂- and EGF-stimulated cellular proliferation in female reproductive tract tissues. Specifically, E₂- and EGF-induced stromal factors have been proposed to indirectly regulate epithelial proliferation. Using culture medium conditioned by purified uterine stromal cells incubated with or without E₂- and EGF, I tested this paracrine model. My results are the first demonstration of a proliferative effect of paracrine stromal factors on uterine epithelial cells. Surprisingly, E₂- and EGF acted directly on epithelial cells to promote growth when combined with stromal CM, and both ligands overcame direct inhibitory effects of the antiestrogen, 4 hydroxy tamoxifen. Taken together, my results prompt a reconsideration of current models of estrogen action. My data can be explained by a complex interplay between stromal, paracrine factors and direct effects of E₂ and EGF on the proliferation of uterine epithelial cells. This study was funded in part by a HHMI Undergraduate Research Program grant.

Towards the Replacement of the S. cerevisiae M1 dsRNA virus ORF with the URA3 ORF and Luc ORF

Erroll Hiett Rueckert

(Advisor: Russell)

Abstract

In order to study the encapsidation, replication, and transcription of the Saccharomyces cerevisiae dsRNA L-A and satellite viruses, the long-term goal of the laboratory is to recombine an M1 cDNA into the k1 and k2 plasmids of the Kluyveromyces lactis killer system replicating in S. cerevisiae. In the end, we expect transcription of M1 RNA from the recombinant plasmids, and establishment of the M1 satellite life cycle in the presence of L-A and its protein products. Once this goal is accomplished, mutation of subsequent M1 cDNA constructs will lead to mutated M1 RNA viruses, and analysis of the subsequent killer phenotype, encapsidation and transcription efficiency, and survival rate will help us to understand which regions of the M1 RNA are responsible for each activity. This thesis work involved two divergent paths of research. On the one hand, four shuttle vector plasmids were tested for integration into the k1 plasmid and subsequent expression of luciferase activity. These plasmids were the putative pAR.UCS6.Luc, pAR.UCS6.Cul, pAR.UCS6.M1.Luc, and pAR.UCS6.M1.Cul. Unfortunately, sequencing data showed that these clones did not contain the UCS6 promoter, and must be constructed anew. On the other hand, a new project was initiated during the fall with the goal of replacing the M1 ORF with the URA3 ORF and the Luc ORF, which will provide a broader range of assays for future M1 mutagenesis assays. Work with one of these M1 ORF replacement constructs is progressing rapidly, and experiments using selection on -ura plates for M1-URA3 replacement ORF transformants can be expected soon. This study was funded in part by a HHMI Undergraduate Research Program grant.

Thermal Acclimation and Compensation: The Effects of Temperature Shifts and Initial Size on Metamorphic Traits in Bombina orientalis

Autumn Ashworth Salamack

(Advisor: Kaplan)

Abstract

The environment experienced by amphibians during the larval period may greatly impact both time to and size at metamorphosis. This study investigated how temperature switches influenced length of the larval period and size at metamorphosis in Bombina orientalis. Embryos were raised individually, and temperature was either increased or decreased at stages 18 or 20 (Gosner 1960). All changes in temperature, regardless of the switch stage, altered both the time to metamorphosis and the size at metamorphosis. In general, those larvae reared in the warm environment reached metamorphosis in less time and at a smaller size than those reared in the cold environment. However, this pattern was inverted with respect to the switch groups. Those larvae which spent more time in the warm environment took longer to metamorphose, and did so at a slightly smaller size, than those larvae which spent more time in the cold environment. This suggests a model whereby larvae are thermally compensating (in terms of physiological processes) differently at different stages in response to changing environmental temperatures. A wide range of initial egg sizes were represented in each temperature treatment as well. Although initial size was found to impact time to metamorphosis, no interaction effects were noted with temperature treatment. This indicates that variation in initial size did not affect how larvae responded to temperature switches. Variation in initial size and environmental temperature are often experienced in nature and can greatly impact larval survivorship and adult fitness indirectly through expression of time to metamorphosis and size at metamorphosis. As the larval environments experienced by amphibians are both uncertain and continually changing, variation in developmental rates may prove a crucial ability for an individual's survival.

Love Thy Brother: Evidence for Kin Recognition in the Trinidadian Guppy, Poecilia reticulata

Cameron Kyle Scott

(Advisor: Childress)

Abstract

Kin recognition is the ability to distinguish related from non-related individuals for purposes of directing nepotistic behavior. The Trinidadian guppy, Poecilia reticulata, has been studied by many branches of science, but its kin recognition abilities remain largely unknown. This study examined if lab raised guppies could distinguish between and prefer to school with groups comprised of kin over non-kin and if this preference was linked to amount of relatedness shared between fish. Both inbred and outcrossed lines of fish were bred in order to produce three different levels of relatedness in each line. These fish were then allowed visual and olfactory contact with a related and an unrelated school in a choice tank containing a confined predator. The amount of time spent schooling with each groups was recorded and compared. Fish from the outcrossed lines displayed a preference to school with kin that was not correlated with relatedness level. Males and females from the outcrossed lines differed in their ability to recognize and school with kin, possibly due to the dichotomy between the sexes in body coloration. Fish from the inbred line showed no preference for kin, regardless of relatedness level. These results indicate that guppies do possess kin recognition behavior and that this trait may be affected by inbreeding depression and differences in the visual cues available to males versus females. This study was funded in part by a HHMI Undergraduate Research Program grant.

Regulation of Neuroendocrine Exocytosis by Cyclic Nucleotides and Their Effectors

Tobia Charles Seeger

(Advisor: Arch)

Abstract

Cyclic adenosine monophosphate (cAMP) is a second messenger formed when an external ligand activates a G-protein coupled receptor. This study concerned the downstream consequences of cAMP action in the stimulus-secretion pathway of the bag cells of Aplysia. Application of dibutyryl cAMP and theophylline increases secretion from the neurons. Superfusion with theophylline (a phosphodiesterase inhibitor) alone is not sufficient to elicit secretion. One probable action of cAMP is the activation of PKA; thus the enzyme was inhibited and secretion monitored following addition of cAMP. No elevation in secretion was detected. It appears, therefore, that the role of cAMP in promoting secretion is through its activation of PKA. The obvious implication of this outcome is that peptide secretion from the bag cells is contingent on the phosphorylation of one or more substrates within the docking, priming, and fusion apparatus. This study was funded in part by a HHMI Undergraduate Research Program grant.

The Effects of an Artificial Oxygen Carrier on Nitrogen Fixation in Soybean (Glycine max)

Clare Elizabeth Stockert

(Advisor: Dalton)

Abstract

Nitrogen-fixation is an energy-intensive process, but nitrogenase, the enzyme responsible for fueling this reaction, is irreversibly inactivated by O₂. To combat the damaging effects of O₂, nitrogen-fixing systems have developed mechanisms to tightly regulate O₂ exposure to nitrogenase. In legume root nodules, the energy demands of the bacterial symbiont (e.g. Bradyrhizobium japonicum in soybeans) are met by leghemoglobin, an abundant hemeprotein that transports O₂ to the bacteroids in such a fashion that nitrogenase is not inactivated. Legumes that either lack leghemoglobin or contain a mutant, inactive form are incapable of fixing nitrogen. The goal of this project is to see if perfluorochemicals (PFCs) can fill a similar role to that of leghemoglobin by enhancing O₂ delivery in soybean (Glycine max cv Williams) nodules formed with effective (Bradyrhizobium japonicum) and ineffective (Rhizobium japonicum strain 61A24) rhizobia. PFCs are used in humans and other mammals as a synthetic blood substitute that floats freely through the plasma and passively transports O₂ from the lungs to the body. In this experiment, PFCs were delivered to legume root nodules through intravascular stem infusion and by direct injection of the nodules. Plants were measured for changes in growth over the treatment period and nodules were tested for activities of nitrogenase (acetylene reduction) and ascorbate peroxidase, a key enzyme required for antioxidant defenses. Other tests were performed to measure total protein content and total leghemoglobin content. Positive results from assays of acetylene reduction and the total protein content of ineffective nodule extracts receiving a 25% PFC infusion treatment indicate that PFCs are capable of recovering nitrogen fixation in leghemoglobin deficient systems.

Sub-nuclear Targeting and Protein-Protein Interactions of the A-Kinase Anchoring Protein, AKAP95

Justin Worthington Taraska

(Advisors: Shampay and Coghlan, NSI-OHSU)

Abstract

A-kinase anchoring protein, AKAP95, is a nuclear matrix protein that binds the regulatory subunit (RII) of cAMP-dependent protein kinase (PKA). I investigated the sub-nuclear targeting and protein-protein interactions of AKAP95. Indirect immunofluorescence studies of mouse sarcoma fibroblasts demonstrated that AKAP95 was distributed throughout the nucleus during interphase, but was excluded from nucleoli. AKAP95 was additionally shown to be a component of the insoluble nuclear matrix. Differential cellular extraction revealed that the association of AKAP95 with the nuclear matrix was calcium dependent. In mitotic cells, AKAP95 was targeted to the spindle midzone. Expression of AKAP95-GFP truncation mutants in monkey kidney fibroblasts demonstrated that the pre-zinc finger region of AKAP95 was necessary and sufficient for targeting to the spindle midzone during anaphase. Furthermore, three components of rat brain nuclear matrices were identified that bind AKAP95. Collectively, my findings suggest that AKAP95 may play a role in regulating the nuclear functions of PKA during interphase and mitosis. This study was funded in part by a HHMI Undergraduate Research Program grant.

Predator Inspection and Female Mate Choice: Do Female Guppies Prefer the "Bold" or the Beautiful?

Jennifer Lyn Wonner

(Advisor: Childress)

Abstract

Female mate choice for male secondary sexual traits may operate through a number of mechanisms. One proposed mechanism is known as the handicap principle, whereby females prefer a trait that actually reduces the male's chance of survival, but is favored because it is a good indicator of the male's genetic quality. It has been proposed that the red-orange coloration in guppies (Poecilia reticulata) is a trait that females prefer. If colorful red-orange males spend more time swimming up to and inspecting potential predators than less colorful males, this "bold" behavior may be an indication of superior genetic quality. In this study, I examine the relationship between color, "boldness" and female preferences in an attempt to understand the function of "boldness" in guppy courtship behaviors. I found no significant female preference for colorful males, nor for males that spent more time inspecting a predator. Colorful males were no more likely to inspect predators than dull males, however, this may be due to the significant correlation among paired males during predator inspections. These results do not support the assertions that "boldness" is preferred by females as predicted by the handicap principle. This study was funded in part by a HHMI Undergraduate Research Program grant.

Analysis of Nucleic Acid Annealing and Strand Exchange Functions of Nucleocapsid and Capsid-Nucleocapsid Proteins From Moloney Murine Leukemia Virus (Mo-MLV)

Weiyi Zhao

(Advisor: Shampay)

Abstract

Current molecular evidence suggest the importance of the nucleocapsid (NC) protein during the retroviral life cycle. NC has been demonstrated previously to possess strong binding affinity for single-stranded nucleic acids and the ability to facilitate hybrid annealing between complementary nucleic acids. This annealing ability may contribute to the processes of viral genomic RNA packaging, annealing of tRNA primer to the RNA primer binding site, as well as minus strand strong stop DNA (-sssDNA) and plus strand strong stop DNA (+sssDNA) strand transfer during retroviral reverse transcription. The focus of this thesis was on the nucleic acid annealing function of Moloney murine leukemia virus (Mo-MLV) NC and its two domain Gag precursor capsid-nucleocapsid (CANC) proteins. The goals here were: 1) to determine whether or not CANC proteins share similar annealing abilities as NC; 2) to investigate possible nucleic acid sequence preferences for both NC and CANC proteins and 3) to characterize the mechanism by which the annealing and strand transfer events take place. To pursue these aims, I employed nucleic acid annealing assays using ³²P-gamma-ATP labeled 28 nucleotide (nt) sense strand DNAs with their complementary 28 nt antisense and excess 18 nt antisense oligonucleotides, in the presence or absence of viral proteins. Results from my experiments show that the Mo-MLV NC and CANC proteins both possess DNA annealing and strand exchange abilities. The NC protein also exhibited preferences for the alternating TG sequence and single-stranded RNA. Based on these findings, the proposed mechanism for NC facilitated annealing process is one where NC proteins stoichiometrically bind to single-stranded nucleic acids, or single-stranded portions of nucleic acid duplexes. This binding is proposed to shift hybrid hybridization thermodynamics to favor single-stranded forms, effectively lowering hybrid melting temperatures and permitting the formation of the most stable double stranded duplexes.