INVESTIGATOR: Hovey, R. C.
ENDOCRINE REGULATION OF PORCINE MAMMARY GLAND DEVELOPMENT AND MORPHOGENESIS
NON-TECHNICAL SUMMARY: Sow lactation is a limiting factor for piglet growth and survival. This project seeks to define the hormonal factors that regulate mammary gland development in female pigs.
OBJECTIVES: The long term objective of the proposed research is to determine the factors that regulate mammary gland growth in gilts as a means to enhance this development and the subsequent milk production potential of sows. The objective of the proposed research is to determine the effects of ovarian and pituitary hormones on mammary gland growth, morphogenesis and hormone receptor expression in nulliparous female pigs.
APPROACH: This study will be conducted using nulliparous gilts. During sexual maturity they will be ovariectomized prior to treatment with various combinations of ovarian and pituitary hormones. Following treatment, mammary tissue will be obtained at the time of euthanasia for analysis of cell proliferation in the mammary gland. The levels of hormone receptors in the mammary gland will also be determined.
ACCESSION NO: 0199105
PROJ NO: VT-0047CG
START: 15 JAN 2004 TERM: 14 JAN 2007
INVESTIGATOR: Kerr, D. E.
TRANSGENIC APPROACH TO PREVENT BOVINE MASTITIS
NON-TECHNICAL SUMMARY: Mastitis is the most costly disease of dairy cattle and strategies for its prevention and cure remain frustratingly ineffective. The purpose of this project is to evaluate the use of transgenesis as a means to improving the mastitis resistance of animals. This technology has great potential for improving the efficiency of milk production and for reducing the need for antibiotics to control this disease.
OBJECTIVES: The long-term goal of our research is to produce transgenic cattle that are resistant to microbial infection of the mammary gland. Mastitis is the most costly disease of dairy cattle and strategies for its prevention and cure remain frustratingly ineffective. The transgenic approach that we are following relies on the production of new antibacterial enzymes by mammary epithelial cells. This strategy led us to the generation of transgenic mice that produce a bioactive variant of lysostaphin, a potent antistaphylococcal enzyme, in their milk. These mice quite resistant to infection from an intramammary challenge of Staphylococcus aureus, the major contagious mastitis-causing pathogen. However, the lactation-specific milk protein promoter in the transgene is constitutively expressed during the lactating period, and offers no protection during mastitis susceptible non-lactating periods.
APPROACH: Objective one is to develop a transgene promoter that will activate in response to mammary infection. Microarray analysis will be used to identify genes that are rapidly and substantially induced in primary cultures of bovine mammary epithelial cells in response to infection. Appropriate gene promoters will then be isolated, linked to our lysostaphin gene, and tested for infection responsiveness in cultured cells. Objective two will validate in transgenic mice the effectiveness of the promoter to enhance mastitis resistance by directing expression of lysostaphin in response to an intramammary challenge of S. aureus. Objective three is to continue our evaluation new antibacterial enzymes as candidates to complement lysostaphin in the killing of mastitis causing pathogens. The application of transgenesis to improving the mastitis resistance of dairy cattle has great potential for improving the efficiency of milk production and for reducing the need for antibiotics to control this disease. The strategy is dependent both on continuing efforts to improve the ability to generate transgenic dairy cows, and as importantly, on the development of appropriate gene constructs.
ACCESSION NO: 0195991
PROJ NO: VT-AE-037CG
START: 01 AUG 2003 TERM: 31 JUL 2005
INVESTIGATOR: Parsons, R. L.; Wood, L.; Jokela, W.; Meals, D.; Cassell, E. A.; Hughell, D.
INTERACTIVE SPATIALLY DYNAMIC FRAMEWORK FOR SUSTAINABLE WATERSHED PHOSPHORUS MANAGEMENT
NON-TECHNICAL SUMMARY: Watersheds find it almost impossible to estimate current phosphorus losses occuring in watershed and the impacts of a change in managment in part of the watershed. This project will combine a WEND modeling with GIS knowledge base to set up a framework to aid estimates of P losses in a watershed that can be useful for regional planning purposes.
OBJECTIVES: Objective 1: Adapt an existing WEND model for a model forested/dairy/cropped/urban watershed in the Lake Champlain Basin Objective 2: Construct a GIS process for classifying and quantifying contribution source areas and P-Index factors within a watershed. Objective 3: Extend Wend models to N parallel submodels based on P Index/CSA classification scheme. Objective 4: Link Wend Model and GIS Objective 5.Develop GIS processes for visualization and display of WEND outputs.
APPROACH: This project will develop a modeling/GIS analytical framework to facilitate understanding and evaluation of long-term spatial and temporal dynamics of alternative management scenarios for watershed P watershed management. This framework will account for all P sources, sinks, and fluxes, quantity linkages among sources and human activities and support evaluation of management strategies to achieve water quality objectives. We will combine Watershed Ecosystem Nutrient Dynamics (WEND) modeling with the concept of critical P source areas to analyze P dynamics and response to management over time and space. GIS analysis will be used to identify intersections of hydrologically active areas and high soil P areas. WEND modeling will be expanded to reflect the spatial variation and P dynamics in a watershed. A GIS application will provide for visualization of spatial and temporal dynamics of alternative P management scenarios. The framework will be applied to a representative model watershed in Vermont's Champlain Valley. This project will develop and provide local and regional stakeholders a framework for assessment of management policies and alternative scenarios for P management in the model watershed.
ACCESSION NO: 0186427
PROJ NO: VT-AS-034CG
START: 01 DEC 2000 TERM: 30 NOV 2004
INVESTIGATOR: Kerr, D. E.; Plaut, K.; Bramley, A. J.
TRANSGENIC APPROACH TO PREVENT MASTITIS
NON-TECHNICAL SUMMARY: Approximately 30 percent of dairy cows will experience a course of mastitis during their annual lactation cycle. The estimated cost of this disease to the US dairy industry is two billion dollars per year. The long-term goal of our research is to develop dairy cattle with enhanced resistance to microbial infection of the mammary gland. Our approach is to use transgenic animal technology to enable mammary cells to produce additional anti-bacterial enzymes. Transgenic cattle are very expensive to generate and thus proposed new genes are tested in transgenic mice. We have generated transgenic mice that contain the bacterial gene for lysostaphin, a potent antibacterial enzyme. This gene is under the control of a milk protein regulatory region such that lysostaphin is only produced by lactating mammary cells. These mice secrete lysostaphin into their milk and have markedly enhanced resistance to staphylococcal mastitis. The first objective of the current proposal is to document the effects, if any, of lysostaphin on the production, composition, and nutritional quality of milk produced by these mice. Our second objective is to examine a new gene regulatory unit that appears to be relatively silent during normal lactation but will potentially direct lysostaphin production in response to infection and during the mastitis-prone time periods shortly before and immediately following a lactation period. The regulatory unit will be isolated from the bovine genome and will be used for the generation of new transgenic mice that will then be evaluated for mastitis resistance.
OBJECTIVES: Perform further characterization of mammary gene expression and the milk produced by transgenic mice that contain a modified lysostaphin gene under control of the beta-lactoglobulin 5'-flanking region. Determine the effects of lysostaphin production on milk yield and on the ability of the milk to support growth of mouse pups. Generate, and characterize transgenic mice that contain the modified lysostaphin gene under control of a bovine regulatory region that directs expression of lysostaphin in the non-lactating period and in response to infection.
APPROACH: Transgenic mice that secrete lysostaphin into their milk have been produced. Additional characterization will be performed. This characterization includes an evaluation of more milk samples for lysostaphin content and activity, total protein, and protein profiles (PAGE). Northern blot analysis of lactating mammary tissue for lysostaphin and other milk proteins (whey acidic protein, b-casein) will be performed as will tissue distribution of lysostaphin mRNA. To assess milk yield a weigh-suckle-weigh experiment comparing the performance of non-transgenic controls and lysostaphin expressing transgenic mice will be conducted. A second weigh-suckle-weigh experiment will compare the growth of non-transgenic pups suckling transgenic or non-transgenic mice. For objective two, a bovine regulatory region that is induced by infection and involution will be cloned from a bovine genomic library. The regulatory region will be ligated to the modified lysostaphin gene and the resulting construct used to generate transgenic mice. Milk will be obtained from these mice before and 24 hours after pup removal to assess the ability of the regulatory region to direct expression of lysostaphin following involution. In a similar fashion, milk will be obtained before and 24 hours after a bacterial challenge to assess the infection responsiveness of the regulatory region.
ACCESSION NO: 0193788
PROJ NO: VT-AS-036CG
START: 01 NOV 2002 TERM: 31 OCT 2004
INVESTIGATOR: Hovey, R.
CORE FACILITY MICROPLATE READER FOR MULTIPLE USERS
NON-TECHNICAL SUMMARY: A core group of lactation and mammary gland biologists at the University of Vermont require access to high throughput, accurate facilities for luminometry, fluorimetry, and UV/visible absorbance. The project will provide this group of researchers with a core facility microplate reader that will address their experimental needs.
OBJECTIVES: The objective of this proposal is to provide a group of lactation and mammary gland biologists at the University of Vermont with access to a state-of-the-art microplate reader. This reader will provide high throughput capacity (to 384-wells) to measure luminescence, fluorescence, and visible/UV absorbance.
APPROACH: The equipment proposed is a Biotek Synergy HT multi-detection microplate reader. This equipment is capable of measuring luminescence, fluorescence, and continual visible/UV absorbance in multiplate format (6 to 384 well format). The reader will be applied to numerous applications within the Department of Animal Science that encompasses six laboratories. Multiple assays will be performed in high throughput and require this equipment, including DNA and RNA quantification, luciferase promoter analyses, ELISA assays and PCR-product quantification.
ACCESSION NO: 0185918
PROJ NO: VT-BO-033CG
START: 01 SEP 2000 TERM: 31 AUG 2004
INVESTIGATOR: Stratton, D. A.
QTL ANALYSIS OF TRAITS THAT LIMIT ECOLOGICAL RANGE EXPANSION IN CATTAILS.
NON-TECHNICAL SUMMARY: Genetic changes in tolerance limits may allow plant species to invade previously unoccupied environments. Two models for the genetic control of tolerance curves make different predictions about the long-term limits to range expansion in invading species. One model assumes tradeoffs in performance in different habitats. Another assumes conditional expression of deleterious mutations at different loci in different environments. Our recent ability to identify regions of the genome (QTL) that cause differences in performance across environments now allows us to differentiate those models by testing patterns of expression of QTL along environmental gradients. Two species of common cattail, Typha latifolia and Typha angustifolia, are aggressive dominants in wetland communities. Cattails represent an unusually well-studied system where the environmental controls on distribution are understood and can be easily manipulated. By understanding the genetic forces that shape the tolerance limits of these species, we will better understand the evolutionary processes that limit the distribution and abundance of plants in general. For example, if most loci that control distribution limits are conditionally expressed, then it may be possible for species to expand into new habitats without reducing their fitness in the original environment.
OBJECTIVES: Genotype recombinant F2 offspring from a cross between two cattail species, Typha latifolia and Typha angustifolia. Construct a genetic linkage map for molecular markers in those cattail species. Identify QTL that affect drought tolerance, flooding tolerance, and salinity tolerance of cattails.
APPROACH: We will use amplified fragment length polymorphisms (AFLP), allozymes, and microsatellites to genotype the F2 progeny. Rhizomes of those individuals will be spit and replicates of each genotype will be grown at different levels of water and salinity. Growth rate and biomass will be used to assess the relative performance of different genotypes. Statistical associations between the molecular markers and performance in the different environments will be used to identify gene regions that control the ecological distribution of these cattail species.
ACCESSION NO: 0196322
PROJ NO: VT-BO-038CG
START: 01 SEP 2003 TERM: 31 AUG 2006
INVESTIGATOR: Molofsky, J.
COMPARISON OF PUTATIVE INVASIVE TRAITS IN EUROPEAN AND NORTH AMERICAN POPULATIONS OF PHALARIS ARUNDINACEA (REED CANARY GRASS)
NON-TECHNICAL SUMMARY: Invasive species are recognized as a major component of global change. To effectively prevent the further spread of invasive species, we need a better understanding of the characteristics that make a species invasive. The purpose of this project is to determine the characteristics that enable a species' ability to invade new habitats.
OBJECTIVES: Invasive species are recognized as a major component of global change. To effectively prevent the further spread of invasive species, we need a better understanding of the characteristics that make a species invasive. Here, we propose to use a single species known to vary in its 'invasion potential' to experimentally test several commonly stated hypotheses about what enables a species to become invasive. Phalaris arundinacea (reed canary grass) will be used as our model system because it is known to vary in its aggressiveness and individual genotypes can be easily determined through electrophoresis. Individuals of Phalaris arundinacea will be sampled from within its native range in Europe and in its introduced range in the United States. Phalaris' current distribution suggests that it is unable to grow in more southern climates. We will experimentally test whether individual genotypes taken from both European and North American populations can survive and grow when planted into a range of habitats along a North-South transect in the Eastern United States. Based on the results from this experiment, we will develop a hierarchical invasiveness index for all genotypes. Subsequent experiments will determine specific characteristics of these individuals. Phenotypically plastic will be tested in a 2-way factorial experiment with temperature and moisture as the variables. The relative investment in sexual and asexual reproduction will be assessed. In the field, we will determine if invasive individuals are more susceptible to herbivores than their less invasive counterparts. Finally, we will determine if invasive individuals are better competitors than their less invasive counterparts. The greenhouse and field studies will be integrated into one final analysis that will provide a complete picture of the traits that are associated with invasiveness. In addition we will investigate the following specific hypotheses. Hypothesis 1: More invasive individuals have higher phenotypic plasticity than less invasive individuals. Hypothesis 2: More invasive individuals allocate a greater proportion of biomass to reproduction (seed set) than less invasive individuals. Hypothesis2A: More invasive individuals invest more heavily in vegetative reproduction (rhizomes and tillers) than less invasive individuals. Hypothesis 3A: More invasive individuals invest less in defense than less invasive individuals (assessed by level of herbivory in the field). Hypothesis 3B: The effects of herbivory on survivorship and growth are less severe in more invasive than less invasive individuals. Hypothesis 4A: More invasive individuals are better competitors than less invasive individuals (assessed by growth rate). Hypothesis 4B: The effects of competition on growth are less severe in more invasive than less invasive individuals. Taken together, these experiments will advance our knowledge regarding what enables some individuals to become invasive. Moreover, this research will explicitly examine how genetic, biotic and abiotic factors contribute to the range expansion of an invasive plant species.
APPROACH: Invasion success will be assessed experimentally by determining survivorship, growth and reproduction in common garden field transplant experiments with genotypes taken from the four locations (Czech Republic, France, Vermont and North Carolina) at four locations along a North-South transect in the Eastern United States (from Vermont to Florida). We will establish a rank order of invasiveness for all genotypes based on each genotype's relative performance at the field sites. The proposed experiment will also allow us to answer several interesting questions about invasiveness. Are there genotypes that do well across all environments? Or is invasive success context-dependent , i.e. it depends upon genotype by environment interactions? Are the traits associated with invasion success the same across the European and North American plants or are there multiple ways to be invasive? Are the traits the confer invasive ability the same at the center of a species range as compared to the margin of a species range? In addition, we will test the breadth of phenotypic response of our genotypes when subject to a range of temperature and moisture conditions. The experimental design will consist of two temperature and two moisture treatments (four treatments). Four genotypes from each of the three populations in the four locales (Czech Republic, France, Vermont and North Carolina, 48 genotypes total) will be used in the experiment. Each treatment combination will be replicated five times for a total of 960 plants. This experiment will also be used to assess differences in reproductive allocation among genotypes. Field experiments using paired genotypes with and without herbivory and with and without neighbors will be used to determine the genotypic response to herbivory and competition, respectively.
ACCESSION NO: 0196877
PROJ NO: VT-BO-045CG
START: 01 SEP 2003 TERM: 31 AUG 2004
INVESTIGATOR: Tierney, M. L.
STRUCTURAL ANALYSIS OF ATPRP3
NON-TECHNICAL SUMMARY: The AtPRP3 protein localizes to the wall of the growing root hair tip and becomes insolubilized within mature root hair cell walls. In order to begin to understand the molecular interactions through which ATPRP3 functions, it will be necessary to determine the structure of this protein after secretion into the plant cell wall. The objectives of this project include overexpression and purification of ATPRP3 and its PRP domain, biochemical analysis of the purified recombinant proteins and identification of conditions that favor crystal formation. If crystals are generated, they will be used in crystallization studies to solve the structure of this cell wall protein.
OBJECTIVES: Epitope-tag and overexpress ATPRP3 and its PRP domain, using the Pichia pastoris expression system. Purify recombinant proteins and analyze them using circular dichroism and molecular light scattering to access the polydispersity of these protein constructs. In parallel, engineer a selenomethionyl variant of the full length AtPRP3 protein and its PRP domain for crystallization trials. If crystals are obtained from either the wild type or selenomethionyl protein constructs, solve the structure using multiwavelength anomalous diffraction (MAD).
APPROACH: We will evaluate Pichia pastoris as an overexpression system for ATPRP3 and its PRP domain. All biochemical studies on the AtPRP3 proteins will be accompanied by circular dichroism and dynamic light scattering. This will be done to assess the aggregation state and polydispersity of these protein constructs. A strong correlation has been established between a protein being monodisperse and its ability to crystallize. For crystallization, we will first use the commercially available crystallization screens as a way to evaluate the solubility of the protein with regards to different buffers, salts, and pHs. We will then design incomplete factorial screens (Carter and Carter, 1979) based on the information gleaned from the commercial screens. The full length AtPRP sequence contains four cysteines, all of which are localized within the non-PRP domain. Cysteines are choice targets for binding heavy metals such as Mercury or Gold. Diffraction data will be collected on the resulting derivatized crystals and will be used for multiple isomorphous replacement phasing. In parallel, we propose to engineer the selenomethionyl variant of the full length AtPRP3 protein and its PRP domain. These sequence modifications should provide enough phasing power to solve the structure of iether protein construct using multiwavelength anomalous diffraction (MAD) of the selenomethionyl proteins.