A. Effect of acid pH on cyanobacteria. Cyanobacteria typically grow in alkaline waters, but lakes are becoming more acidic due to air pollution. We are looking at the effect of acid stress on protein synthesis, cell growth and specific enzymes. This project involves 2D gel electrophoresis and proteomics as well as growth and biochemical analyses. Other types of experiments to determine how cyanobacteria raise the pH of their environment to cope with this stress include NMR spectroscopy (in collaboration with Prof. Nancy Kolodny) and study of amino acid decarboxylases.
B. Biofilm capability of cyanobacteria. We have observed biofilm formation in stationary-grown acid-stressed cells. Experiments are underway to determine how, why and when biofilms may form, to observe pili by electron microscopy, and eventually to observe protein differences by proteomics.
C. Characterization of chromate and lead resistant bacteria from the Paint Shop pond area. High levels of lead and chromium were present in soil sampled from this contaminated area before the cleanup. Former students have isolated bacteria from the wetland area and we are characterizing them in terms of heavy metal and antibiotic resistance, as well as analyzing their plasmid content and amplifying the bacterial 16S rDNA for phylogenetic analysis (in collaboration with Prof. Andrea Sequeira).
Blue Light Signaling cyclins
in Arabidopsis thaliana
Gary Harris
The experiments our lab group will be conducting this summer focus on the identification and characterization of molecular components of a blue light signaling pathway that controls chloroplast positioning, stomatal opening and phototropism in Arabidopsis thaliana. The blue light receptors are called Phototropin 1 and Phototropin 2. We will be using two different experimental approaches in an effort to identify unknown components of this signal transduction pathway. One experimental approach involves the analysis of global gene expression using microarrays. By analyzing patterns of gene expression it is possible to predict which proteins are likely downstream components of the phototropin signaling cascade. During this project the students would learn methods of RNA extraction, RNA characterization, and labeling cDNAs with fluorescent dyes. In addition there are various protocols associated hybridizing the cDNAs with microarrays and analyzing the resultant data. We will also be taking a more direct biochemical approach in an effort to identify proteins that physically interact with the phototropins. To accomplish this we will use immunoprecipitation, an immunological procedure that exploits the specificity of antigen-antibody interactions. Briefly the protocol involves combining a specific antibody with a protein extract that contains the antigen of interest (i.e. Phototropin1). The resultant antigen-antibody complexes can be readily removed from the solution by a further incubation with beads of agarose linked to protein A. The collected antigen-antibody complexes and other associated proteins can then be analyzed by one and two-dimensional polyacrylamide gel electrophoresis. It is these associated proteins, those that are clearly physically interacting with target antigen, which may be components of the blue light signaling pathway. The individual proteins collected from the electrophoresis gels will be identified by a proteomic method called peptide mass fingerprinting. During this procedure proteins are digested by a protease (i.e. trypsin) that cuts at specific positions in the amino acid sequence generating a series of peptides from a given parent protein. The masses of these peptides can be very accurately determined by matrix assisted laser desorption time of flight (MALDI-TOF) mass spectrometry and utilized as a fingerprint to help identify the protein. Computer algorithms compare the experimentally determined peptides masses to theoretical cut patterns (masses) of known gene products and predict the most likely matches (MASCOT; www.matrixscience.com/). It is also possible to obtain information on protein post-translational modifications using these methods. For example, a key post- translational modification that is important is most signaling pathways is the addition of phosphate group to specific amino acids. Therefore using these methods it should be possible to both identify new proteins components of the blue light signaling pathway and to monitor critical post-translational modifications.
Specialization of cyclins
in cell cycle regulation
Jennifer Hood-DeGrenier
My lab studies the regulation of cell division in the model eukaryote Saccharomyces cerevisiae, or budding yeast, using genetic, microscopic, and biochemical approaches. I am particularly interested in the role of subcellular protein localization in regulating the cell cycle. Current projects in the lab address the ways in which various members of a key class of regulatory proteins, the cyclins, direct their catalytic partner, a cyclin-dependent kinase, to phosphorylate specific substrate proteins, resulting in the events that drive cell cycle transitions. One ongoing project employs a genetic screen to investigate the function of the cytoplasmic population of a particular mitotic cyclin, the protein Clb2p. Another project is investigating the mechanistic basis for the hypersensitivity of a yeast strain that does not express one of two S phase cyclins, the Clb5p protein, to the growth suppressive drug rapamycin. Rapamycin inhibits a central cell growth regulator, a kinase called TOR; we are interested in discovering how the TOR signaling pathway may intersect with cell cycle events regulated by Clb5p. We are studying this question by characterizing the effect of rapamycin on cell cycle progression in wildtype and ?CLB5 cells using microscopy and flow cytometery and by observing the transcriptional response of these two strains to rapamycin treatment using DNA microarray experiments.
Cancer Researchin Drosophila melanogaster
Brett Pellock
I perform basic cancer research using the fruit fly, Drosophila melanogaster, as a model organism to identify and characterize genes that restrict cell growth, cell division, and cell survival. The overall goal of this research is to understand how deregulating conserved signaling pathways that control cell growth and survival can contribute to human cancer. This approach is viable because many of the signaling networks that control cell growth and survival are well conserved. Additionally, a wide array of powerful genetic tools is available in Drosophila for in vivo analysis of gene function. Using some of these tools, we have performed a series of genetic screens for mutations that allow patches of mutant tissue to outgrow the neighboring wild type tissue. These screens have successfully identified an abundance of both known and novel tumor suppressors, many of which are mutated in human cancers. There are several ongoing investigations in my lab that are available for student involvement. For example:
1) A novel tumor suppressor that functions in the ubiquitin pathway. Mutations in a gene named bendless result in overproliferation in our growth and proliferation screens. The Bendless protein is a highly conserved E2 ubiquitin conjugating enzyme previously implicated in neural development and immune response. However, its role in growth control is a novel discovery. I am using genetic, immunohistochemical, and biochemical approaches to determine which growth pathway(s) Bendless regulates, what other proteins Bendless may interact with, and whether Bendless-mediated ubiquitination results in degradation of the target proteins or modulates their activity in a degradation-independent manner.
2) Differentiate or die. During organismal development cells that do not differentiate properly or in a timely fashion are eliminated via apoptotic cell death. However, little is known about how these cells are detected and eliminated. One of the overgrowth mutations identified in my screens also allows cells in the developing eye to survive death that results from delayed differentiation. I am using genetic and immunohistochemical approaches to determine how non-differentiating cells are signaled to die and how the death of these cells is executed. I am particularly interested in discovering what distinguishes these surviving mutant cells from normal cells, since one hallmark of cancer is resistance to cell death signals.
Both projects may involve travel (to museums or
the field) to examine and/or collect specimens.
Plant cytokinesis
T. Kaye Peterman
Cell division is a central event in the development of all multicellular organisms but it is especially important for plant development. Because plant cells are surrounded by a rigid cellulose cell wall that renders them incapable of movement, the spatial and temporal patterns of cell division play a central role in defining the ultimate architecture of the organism. While the events of plant cytokinesis are known in detail at an ultrastructural level, our understanding at the molecular level is quite limited. Furthermore despite the striking differences between cytokinesis in plants and animals, recent studies have revealed a common requirement for membrane trafficking. Consequently insights into the cellular mechanisms of plant cytokinesis are likely to shed light on vesicle trafficking events that occur during cytokinesis in both plant and animal cells.
The focus of our work this summer will be on patellin1 (PATL1), a protein first described in our laboratory, which functions during the late stages of cytokinesis in plants. PATL1 is found associated with punctate cytoplasmic structures and localizes during cytokinesis to the expanding and maturing cell plate. In vesicle binding assays, PATL1 binds to specific phosphoinositides (PtdIns(5)P, PtdIns(4,5)P2 and PtdIns(3)P), important regulators of membrane trafficking and cytoskeletal dynamics. Based on its localization, biochemical properties, and sequence similarity to Sec14p (a Saccharomyces cerevisiae protein that is essential for secretion) and GOLD domain membrane trafficking proteins, we hypothesize that patellin1 plays a critical role in membrane trafficking events during plant cytokinesis. We are using molecular genetic, biochemical and cell biological approaches to test this hypothesis.
Ecology of migratory songbirds in a north temperate forest:
demographic, experimental, and modeling studies
Nicholas L. Rodenhouse
Our research will have three main emphases: the effects of climate change on migratory songbirds, competition between birds and invertebrate predators for a common prey type--caterpillars, and the bioaccumulation and biomagnification of mercury in the food webs of which songbirds are a part. To document the effects of climate change we are intensively monitoring a population of Black-throated Blue Warblers (Dendroica caerulescens) that breeds across a 600 m elevation gradient within the Hubbard Brook Experimental Forest of north central New Hampshire (see http://www.hubbardbrook.org/research/current/discipline/animals/bird/holmes-intro03.htm). At high, middle and low elevations, representing a 2 oC difference in mean annual temperature, we are quantify the breeding activities of pairs, which includes netting and banding birds, finding nests, monitoring activity at the nest, and weighing and banding nestlings. Habitat quality is also being measured, including food abundance, weather, predator abundance, and vegetation composition and structure. Competition between birds and invertebrate predators, such as wasps, spiders and stinkbugs for caterpillars will be examined experimentally by using exclosures, enclosures and predator-prey experiments. Bioaccumulation and biomagnification of mercury in food webs will involve sampling all levels of the food web in the field, e.g., insects that feed on live or dead plant material (e.g., caterpillars), and their predators and parasitoids (e.g., wasps). Field-collected samples will be analyzed for mercury content in the lab. Summer research students will develop individual projects associated with one of the various aspects of the overall research program. Results of this research are contributing to basic ecological theory and will have direct management applications for migratory songbirds, some of which are of conservation concern.
Colleagues and students at Dartmouth College, the Smithsonian Institution, the University of Vermont, University of Montana, Keene State College and Washington State University are collaborating on various aspects of this study. Students working on the project will be living and working in New Hampshire with the research team, totaling between 15 and 20 research assistants, graduate students and post-doctoral fellows. Our studies are a part of the broader Hubbard Brook Ecosystem Studies (see http://www.hubbardbrook.org/research/current/new_current.htm); hence, students will have many opportunities to learn about the ecological research of others at this Long Term Ecological Research (LTER) site.
Colleagues and students at
Dartmouth
College
and
the
Smithsonian
Migratory
Bird
Center
are
collaborating with this study, and students working on the project will
be living and working in
New Hampshire
with
this joint research team.
Molecular tools to answer evolutionary and conservation questions in the Galápagos archipelago
Andrea Sequeira
The focus of this laboratory is to understand how and why new species form. Our objects of study are endemic Galápagos flightless weevils. Oceanic islands serve as crucial sites for evolutionary studies providing model substrates for studying the process of divergence and speciation. One of the most famous archipelagoes that have played a fundamental role in the history of evolutionary biology is certainly Galápagos. Unfortunately, there has been an increase of 59% in the number of unintentional insect introductions to Galápagos since 1998. A close relative of the wingless endemics object of our study has recently been formally listed as an invasive species in the archipelago.
Student projects will involve research on endemic and invasive Galápagos weevils. High polymorphism and the relative ease of scoring represent the two major features that make microsatellites of large interest for many genetic studies. All of the material critical for this project has already been assembled and is available in the laboratory. The laboratory is fully equipped for performing DNA extraction, cloning, amplification by PCR and sequencing. We will devote the summer to genotyping (using the microsatellite markers developed in our laboratory) to track the demographic history of introduced populations and the evolutionary history of endemic populations of the endemics to the youngest and most topologically and geologically complex island (Isabela). These studies will address specific hypothesis and questions pertaining promoters of speciation in these weevils. From a conservation genetics stand point the introduced species will provide a relevant comparison of patterns of genetic variation and migration.
Summer projects will involve:
1) Genotyping and reconstructions of patterns of differentiation and demographic history of introduced populations of Galapaganus howdenae in the agricultural area of the island of Santa Cruz, Galápagos.
2) Optimization of additional microsatellite markers for the endemic species G. williamsi and genotyping of multiple populations from distinctive volcanoes in the island of Isabela.