Biology Commons^™

Correlated Confocal And Intermediate Voltage Electron Microscopy Imaging Of The Same Cells Using Sequential Fluorescence Labeling, Fixation, And Critical Point Dehydration, Lee D. Peachey, Harunori Ishikawa, Tohru Murakami

Scanning Microscopy

Confocal laser scanning microscopy (CLSM) and intermediate voltage transmission electron microscopy (IVEM) each has its own particular advantages. CLSM can examine living cells, but is particularly useful when applied to cells that have been lightly fixed, permeabilized, and stained with fluorescent-labeled antibodies for localization of specific molecular species at the resolution of the light microscope while still in the hydrated state. IVEM provides much higher resolution images, but requires more drastic preparation procedures, including dehydration. This paper presents methods for combining these complementary approaches to examine exactly the same cells sequentially by CLSM and IVEM. Cells are grown in culture …

Immunocytochemistry By Electron Spectroscopic Imaging Using Well Defined Boronated Monovalent Antibody Fragments, M. M. Kessels, B. Qualmann, W. D. Sierralta

Scanning Microscopy

Contributing to the rapidly developing field of immunoelectron microscopy a new kind of markers has been created. The element boron, incorporated as very stable carborane clusters into different kinds of peptides, served as a marker detectable by electron spectroscopic imaging (ESI) - an electron microscopic technique with high-resolution potential.

Covalently linked immunoreagents conspicuous by the small size of both antigen recognizing part and marker moiety are accessible by using peptide concepts for label construction and their conjugation with Fab' fragments. Due to a specific labeling of the free thiol groups of the Fab' fragments, the antigen binding capacity was not …

Long-Term Monitoring And Analyses Of Physical Factors Regulating Variability In Coastal Antarctic Phytoplankton Biomass, In Situ Productivity And Taxonomic Composition Over Subseasonal, Seasonal And Interannual Time Scales, Mark A. Moline, Barbara B. Prezelin

Biological Sciences

A 3 yr high-resolution temporal data base related to phytoplankton dynamics was collected during the austral spring/summer periods of 1991 to 1994 in shelf waters adjacent to Palmer Station, Antarctica. Here, the data base is used (1) to quantify the variability in phytoplankton biomass, in situ productivity and taxonomic composition over subseasonal, seasonal and interannual time scales; (2) to elucidate environmental mechanisms controlling these temporal patterns; and (3) to ascertain which phytoplankton markers are most suitable for detecting longer-term (i.e. decadal) trends in phytoplankton dynamics in coastal waters of the Southern Ocean. The Long-Term Ecological Research (LTER) coastal study sites …

In Situ Hybridization, In Situ Transcription, And In Situ Polymerase Chain Reaction, L. E. De Bault, J. Gu

Scanning Microscopy

In situ hybridization, in situ transcription, and in situ polymerase chain reaction (PCR) are techniques used to detect DNA and RNA sequences within a cell or tissue structure. These three in situ methodologies employ the principles of recombinant DNA to form double-stranded hybrids of DNA-DNA, DNA-RNA, or RNA-RNA. The essence of in situ hybridization (ISH) is the hybridization of a labeled probe to a complementary target sequence, whereas in situ transcription (IST) is the synthesis of complementary DNA incorporating a label directly on the target DNA or RNA within a cell or tissue. In the case of in situ PCR …

Preparation Of Samples For Polymerase Chain Reaction In Situ, Gerard J. Nuovo

Scanning Microscopy

The purpose of this paper is to describe the key variables in sample and reagent preparation needed for successful polymerase chain reaction (PCR) in situ. Tissue or cell preparations should be fixed in a cross linking fixative, such as 10% buffered formalin, preferably from 15 to 48 hours. Tissues should be embedded in paraffin; cell preparations can be fixed when near confluence, then physically removed and processed. When possible three samples (4 μM tissue sections or 1-5000 cells) should be placed on silane coated glass slides. Digestion in pepsin (2 mg/ml) for 30 min is adequate for DNA detection …

Microscopic Analysis Of Dna And Dna-Protein Assembly By Transmission Electron Microscopy, Scanning Tunneling Microscopy And Scanning Force Microscopy, T. Müller-Reichhert, H. Gross

Scanning Microscopy

To investigate DNA and DNA-protein assembly, nucleic acids were adsorbed to freshly cleaved mica in the presence of magnesium ions. The efficiency of DNA adhesion and the distribution of the molecules on the mica surface were checked by transmission electron microscopy. In addition, various kinds of DNA-protein interactions including DNA wrapping and DNA super-coiling were analyzed using electron microscopy. In parallel, this Mg²⁺/mica method can be applied (1) to analyze embedded DNA by scanning tunneling microscopy, (2) to visualize freeze-dried, metal coated DNA-protein complexes by tunneling microscopy, and (3) to image DNA or DNA-protein interaction in air or …

Aspects Of Cryofixation And Cryosectioning For The Observation Of Bulk Biological Samples In The Hydrated State By Cryoelectron Microscopy, K. Richter

Scanning Microscopy

Cryoelectron microscopy allows the observation of hydrated samples at high spatial resolution, and it would be of great interest in biology to apply this method to cells and tissues. However, because of technical problems, the cryo-observation of frozen hydrated ultrathin sections of bulk material has not become an established method. The major limitations are due to the difficulty of achieving the vitrification of such material, and the structural deformation caused by ultrathin sectioning: 1. The vitrification of cells in a physiological environment requires high-pressure freezing. However, new results suggest that the pressure may alter the ultrastructure of the sample. 2. …

The Rna-Binding Site Of Bacteriophage Qβ Coat Protein, Francis Lim, Marc Spingola, David Peabody

Biology Department Faculty Works

The coat proteins of the RNA bacteriophages Qβ and MS2 are specific RNA binding proteins. Although they possess common tertiary structures, they bind different RNA stem loops and thus provide useful models of specific protein-RNA recognition. Although the RNA-binding site of MS2 coat protein has been extensively characterized previously, little is known about Qβ. Here we describe the isolation of mutants that define the RNA-binding site of Qβ coat protein, showing that, as with MS2, it resides on the surface of a large β-sheet. Mutations are also described that convert Qβ coat protein to the RNA binding specificity of MS2. …

The Rna-Binding Site Of Bacteriophage Qβ Coat Protein, Francis Lim, Marc Spingola, David S. Peabody

Marc Spingola

Evaluation Of The Success Of Constructed Wetlands In The Cave Run Lake Watershed, April Diane Haight

Morehead State Theses and Dissertations

A thesis presented to the faculty of the College of Science and Technology at Morehead State University in partial fulfillment of the requirements for the Degree of Master of Science by April Diane Haight on December 10, 1996.

Electro-Optical Imaging Of F-Actin And Endoplasmic Reticulum In Living And Fixed Plant Cells, Nina Stromgren Allen, Marty N. Bennett

Scanning Microscopy

Confocal and video micrographs of living and fixed alfalfa roots, onion epithelial and pear pollen cells illustrate the architecture of the cytoskeleton and endoplasmic reticulum in plant cells. Fixation of plant tissues to preserve cytoplasmic structure poses special problems. When possible, emphasis should be placed on the imaging of structures in stained living cells over time. The early events that occur when Nod factors or bacteria elicit nodule formation in alfalfa roots will illustrate several approaches to plant cell fixation, staining and imaging. The first observable events after Nod factor stimulation occur in root hairs and are changes in rates …

Parasexuality And Heterokaryosis In Fusarium Oxysporum Forma Specialis Cubense, Blanca R. Cortes

FIU Electronic Theses and Dissertations

Intra and Inter Vegetative Compatibility Group (VCG) heterokaryon formation was observed in Fusariurn oxysporum f.sp. cubense (Foc). Using the double-pick method to force heterokaryon formation via hyphal anastomosis, 10⁴ pairwise combinations were done between 100 auxotrophic mutants of Foe representing races 1, 2 and 4 in five VCGs. Inter formae speciales heterokaryon formation was also observed on pairings between benomyl resistant Wild Type Testers (WTT) and wild type isolates in 15 VCGs and four different formae speciales. Microconidia analysis of heterokaryons identified both parental phenotypes as well as diploid phenotypes. Colonies with altered and hybrid genotypes were …

Chemical Communication In House Mice (Mus Musculus): Can They Recognize Gender From The Anogenital, Harderian Gland Or Mouth/Nose Odor?, Dawn Michele Andrews

Dissertations and Theses

Identifying the sensory systems animals employ to communicate chemically and the function of the chemical signals facilitates further understanding of chemical communication. Increased knowledge of how animals use the olfactory and vomeronasal systems in order to interpret the meaning of body odors will aid in developing a more detailed organization of chemosensory pathways. The message that each body odor contains can change from species to species.

The purpose of this thesis was to study three previously untested body odors in house mice (M. musculus) for their role in gender recognition of conspecifics. These odors are the anogenital (feces, …

Expression Of Α4Β1 Integrin On Tissue Eosinophils In The August Rat, John C. Phipps

Masters Theses

The integrin α4β1 is expressed on a wide variety of peripheral leukocytes, and functions in recruitment of these cells to sites of inflammaiton. We infected august (AUG) rats with the helminth parasite Nippostrongylus brasiliensis and later used leukocytes from the peritoneum of the sensitized animals and antibodies against α4β1 integrin to localize that adhesion molecule on the cell surfaces after antigen challenge. We found that after antigen challenge, eosinophils, monocytes, and small lymphocytes, but not mast cells, expressed α4β1 integrin if the animals had been previously infected, but cells from uninfected animals did not show expression of this molecule. Our …

The Bradyrhizobium Japonicum Fega Gene Encodes An Iron-Regulated Outer Membrane Protein With Similarity To Hydroxamate-Type Siderophore Receptors., Kristin Levier, Mary Lou Guerinot

Dartmouth Scholarship

Iron is important in the symbiosis between soybean and its nitrogen-fixing endosymbiont Bradyrhizobium japonicum, yet little is known about rhizobial iron acquisition strategies. Analysis of outer membrane proteins (OMPs) from B. japonicum 61A152 identified three iron-regulated OMPs in the size range of several known receptors for Fe(III)-scavenging siderophores. One of the iron-regulated proteins, FegA, was purified and microsequenced, and a reverse genetics approach was used to clone a fegA-containing DNA fragment. Sequencing of this fragment revealed a single open reading frame of 750 amino acids. A putative N-terminal signal sequence of 14 amino acids which would result in a mature …

Biology: What One Needs To Know, Ursula Goodenough

Biology Faculty Publications & Presentations

Biology on this planet represents an astonishing experiment in carbon-based chemistry which, over billions of years, has generated billions of species adapted to countless major and minor fluctuations in ecological circumstances. In one sense there is no way to generalize about biology. While biological activities can all be ultimately explained by physical laws (like everything else in the universe), it is the emergent intensely particular properties of organisms that most interest us. This essay represents an attempt to describe some of the more prominent patterns that emerge from the sea of biological particularities, patterns that present many opportunities for religious …

Evagination Of The Thyroid Primordium Involves Novel Cell Behaviors, Gwendolyn M. Kinebrew, S. R. Hilfer

Biology

No abstract provided.

Expression And Regulation Properties Of Pseudomonas L-Arabinose Utilization Plasmids, Mary E. Barecki

Masters Theses

The L-arabinose utilization pathway in Pseudomonas fluorescens is a unique metabolic pathway. In Pseudomonas, L-arabinose is converted to the end product α-ketogluterate and does not involve phosphorylated intermediates. This differs from the well studied L-arabinose pathway found in Escherichia coli which contains phosphorylated intermediates.

The enzymes of the L-arabinose pathway have been shown to be induced in the presence of L-arabinose in P. saccharophila. The current study determined regulatory properties of previously cloned chromosomal fragments of P. fluorescens which confer an L-arabinose positive phenotype to the surrogate host, Pseudomonas putida. Cells of P. putida containing plasmids pPZ221, …

The Effects Of Benzo(A)Pyrene And Dimethylbenzanthracene On Cultured Porcine Aortic Endothelial Function, Michelle Lynn Kagey

Masters Theses

Polycyclic aromatic hydrocarbons are environmental contaminants which have been shown to initiate and promote atherosclerosis and carcinogenesis in various animal species. These findings have been based primarily on the increase in incidence and size of smooth muscle cell tumors in these animals were exposed to benzo(a)pyrene (BaP) or dimethylbenzanthracene (DMBA). The endothelium has also been identified as an important mediator in vascular disease and atherogenesis, although the endothelial response to these PAHs has not been investigated. Thus, the focus of this study was on characterization of the endothelial response to BaP and DMBA. Concentration/response relationships for these chemicals were examined, …

Effect Of Nesting Structure Density On Clutch Success Of Canada Geese, Branta Canadensis, Nesting In Constructed Wetlands In The Cave Run Lake, Kentucky, Area, Teresa L. Caudill

Morehead State Theses and Dissertations

A thesis presented to the faculty of the College of Science and Technology at Morehead State University in partial fulfillment of the requirements for the Degree of Master of Science in Biology by Teresa L. Caudill on November 26, 1996.

A Scanning Electron Microscopy Study Of Taxus Leaves As Related To Taxonomy, G.A. Strobel, W. M. Hess

Scanning Microscopy

Scanning electron microscopy, when applied to the surfaces of the needles of Taxus spp. (yew) revealed features that appear useful in the taxonomy of this tree species which yields the important anticancer drug, taxol. For instance, all of the four North American species have 3-5 rows of stomata on one-half of the abaxial leaf surface, whereas all of the others, including those from Europe and Asia, have 7-10 rows of stomata. The appearance of individual or fused papilliform epidermal cells and their arrangement on the leaf surface also is a feature that varies between species. Patterns of wax formation appeared …

Structure Of The Gdp-Pi Complex Of Gly203→Ala G(Iα1): A Mimic Of The Ternary Product Complex Of Galpha-Catalyzed Gtp Hydrolysis, Albert M. Berghuis, Ethan Lee, André S. Raw, Alfred G. Gilman, Stephen R. Sprang

Biological Sciences Faculty Publications

Background: G proteins play a vital role in transmembrane signalling events. In their inactive form G proteins exist as heterotrimers consisting of an α subunit, complexed with GDP and a dimer of βγ subunits. Upon stimulation by receptors, G protein α subunits exchange GDP for GTP and dissociate from βγ. Thus activated, α subunits stimulate or inhibit downstream effectors. The duration of the activated state corresponds to the single turnover rate of GTP hydrolysis, which is typically in the range of seconds. In G(iα1), the Gly203→Ala mutation reduces the affinity of the substrate for Mg²⁺, inhibits a key …

Structures Of The Extracellular Domain Of The Type I Tumor Necrosis Factor Receptor, James H. Naismith, Tracey Q. Devine, Tadahiko Kohno, Stephen R. Sprang

Biological Sciences Faculty Publications

Background: Tumor necrosis factor (TNF) is a powerful cytokine that is involved in immune and pro-inflammatory responses. Two TNF receptors that belong to the cysteine-rich low affinity nerve growth factor receptor family (TNF-R1 and TNF-R2) are the sole mediators of TNF signalling. Signalling is thought to occur when a trimer of TNF binds to the extracellular domains of two or three receptor molecules, which permits aggregation and activation of the cytoplasmic domains. The complex is then internalized within an endocytic vesicle, whereupon it dissociates at low pH. Structure of the soluble extracellular domain of the receptor (sTNF-R1) both in the …

The Chromatin Structure Of Well-Spread Demembranated Human Sperm Nuclei Revealed By Atomic Force Microscopy, M. J. Allen, E. M. Bradbury, R. Balhorn

Scanning Microscopy

The fundamental structure formed when genomic DNA is packaged by protamine in the human sperm nucleus still remains essentially unresolved. It is known that the binding of protamine, a small arginine-rich protein, to DNA generates a large dense, hydrophobic complex making the sperm chromatin structure difficult to study microscopically. To visualize the internal nuclear structures, isolated human sperm nuclei were swollen extensively in saline buffer using only a reducing agent. The nuclei were swollen during deposition onto coverglass and then imaged in the atomic force microscope (AFM). The two main results obtained from imaging individual well-spread nuclei indicate that native …

Selection Of Populations Of Puccinia Recondita F. Sp. Tritici For Shortened Latent Period On A Partially Resistant Wheat Cultivar, Jeffrey Lehman

Biology and Earth Science Faculty Scholarship

Wild-type fungal population 851-WT was selected for shortened latent period on cv. CI 13227 for five uredinial generations to study the adaptation of Puccinia recondita f. sp. tritici to partially resistant wheat cultivars. Differences among wild-type and selected populations for traits contributing to parasitic fitness (i.e., latent period, infection frequency, and uredinium area and growth rate) were assessed in monocyclic infection experiments on susceptible cv. Monon and partially resistant cvs. Suwon 85, Sw 72469-6, L-574-1, and CI 13227. Differences were greatest among fungal populations on cv. CI 13227. The mean latent period of selected population 851-C5 was 2 days shorter …

Genetic And Social Control Of Male Maturation In Phallichthys Quadripunctatus (Pisces: Poeciliidae), Gita R. Kolluru, David N. Reznick

Biological Sciences

Age and size at maturity can have significant fitness consequences. Selection often favors early-maturing individuals because of their higher survival to maturity and greater relative contribution to population growth rate, but it may also favor delayed maturation if fitness increases with age or size at maturity. Males of several poeciliid fishes exhibit variation in age and size at maturity primarily controlled by a sex-linked gene called the P-locus. Wild-caught Phallichthys quadripunctatus males show a bimodal size distribution, which is often associated with a P-locus polymorphism in other poeciliids. We conducted two experiments to evaluate the inheritance of male age and …

High-Resolution Time-Series Data For 1991/1992 Primary Production And Related Parameters At A Palmer Lter Coastal Site: Implications For Modeling Carbon Fixation In The Southern Ocean, Mark A. Moline, Barbara B. Prezelin

Biological Sciences

Our goal was to provide a high-resolution temporal data base for modeling primary production in shelf waters adjacent to Palmer Station, Antarctica. Here, the resulting 1991/1992 data base is used to: (1) determine in situ productivity over a range of seasonal to subseasonal time scales; (2) identify time scales of significant variability in marine productivity during the peak growing season; (3) identify environmental, experimental and analytical factors that can significantly impact the accuracy of daily, weekly and seasonal productivity estimates; and (4) integrate our findings with previous studies of Antarctic coastal primary pro- duction. Data were gathered every 2–3 days …

Trade-Offs, Food Web Structure, And The Coexistence Of Habitat Specialists And Generalists, Mark A. Mcpeek

Dartmouth Scholarship

Species differ greatly in the breadth of their environmental distributions. Within the same collection of habitats, some species occur in many habitats, while others are only able to exist in one of a few. Trade-offs in the abilities of species to perform in various ecological interactions are important both to facilitating species coexistence within a habitat and to limiting the distributions of species among habitats. In this article I use a food web model to explore how in the same collection of habitats some species may be limited by trade-offs to occupying only one habitat, while other species may face …

A Re-Evaluation Of Hotspot Settlement In Lekking Sage Grouse, Robert M. Gibson

School of Biological Sciences: Faculty Publications

Recent analyses of avian leks have come to conflicting conclusions concerning the role of male settlement on female traffic hotspots. This issue was re-examined in the sage grouse, Centrocercus urophasianus, using data on prenesting movements of radio-tagged females and the dispersion of lekking males collected during a 10-year field study. As expected with hotspot settlement, leks were preferentially located in areas through which females traveled between wintering and nesting ranges before mating. In addition, the distribution of males among leks was related proximately to variation in numbers of females visiting each lek during the mating period and ultimately to …

A Highly Sensitive Plant Hybrid Protein Assay System Based On The Spm Promoter And Tnpa Protein For Detection And Analysis Of Transcription Activation Domains, Michael Schläppi, Ramesh Raina, Nina V. Fedoroff

Biological Sciences Faculty Research and Publications

TnpA is a multifunctional DNA binding protein encoded by the maize Suppressor-mutator (Spm) transposable element. TnpA is required for transposition and is a repressor of the unmethylated Spm promoter. While analyzing protein domains using a yeast GAL4-based hybrid system in transiently transformed tobacco cells, we found that TnpA represses the >10-fold transcriptional activation observed when the GAL4 DNA-binding domain is used alone. By contrast, compared to the backgroundless TnpA DNA-binding domain alone, 33- to 45-fold activation of the Spm promoter was observed when the VP16 activation domain was fused to it. TnpA-binding sites, but no TATA box, …