Biotechnology Commons^™

Patterning Defects In Silkworm Embryos Analysed Through Cuticle Preparations, Amit Singh, Madhuri Kango-Singh, K. P. Gopinathan

Biology Faculty Publications

The mulberry silkworm, Bombyx mori, a holometabolous lepidopteran insect, has a metameric body plan. Due to its functional adaptation, B. mori presents some unique deviations in its pal/ern from the evolutionarily advanced dipteran insect, Drosophila melanogaster. Previous studies on mutant phenotype analysis in B. mori have been carried out in late stages of larval development. Here we employ, the cuticle preparation approach during embryonic development to study morphological landmarks associated with B. mori, Eri, another race a/silkworm, and pattern defects associated with Ekp mutant of B.mori. The homeotic mutant Ekp, generates ectopic abdominallegs, a feature …

A Discourse On The Public Nature Of Research In Contemporary Life Science: A Law-Policy Proposal To Promote The Public Nature Of Science In An Era Of Academia-Industry Integration, Michael J. Malinowski

Journal Articles

This article addresses the impact of integration of academia, industry, and government on the public nature of research. The article concludes that, while the integration has benefited science immensely, regulatory measures should be taken to restore the public nature of research in an age of integration.

A Cell Biology Laboratory Exercise To Study Sub-Cellular Organelles In Drosophila, Meghana Tare, Amit Singh

Biology Faculty Publications

The fast-changing scenario of undergraduate education puts emphasis on introducing students to hands-on techniques as part of their laboratory courses. In order to cater to large numbers of students and the time constraints involved with undergraduate level laboratory courses, there is a need for development of experiments that are cost effective and can be completed in a defined time frame. We have devised a laboratory exercise for teaching cell biology using the Drosophila melanogaster model. Drosophila can be reared in a short period of time in a cost effective manner. We used Drosophila tissue to study the sub-cellular organization of …

Molecular Characterisation Of A Bovine-Like Rotavirus Detected From A Giraffe, Emily Mulherin, Jill Bryan, Marijke Beltman, Luke O'Grady, Eugene Pidgeon, Lucie Garon, Andrew Lloyd, John Bainbridge, Helen O'Shea, Paul Whyte, Séamus Fanning

Department of Biological Sciences Publications

Background

Rotavirus (RV), is a member of the Reoviridae family and an important etiological agent of acute viral gastroenteritis in the young. Rotaviruses have a wide host range infecting a broad range of animal species, however little is known about rotavirus infection in exotic animals. In this paper we report the first characterisation of a RV strain from a giraffe calf.

Results

This report describes the identification and detailed molecular characterisation of a rotavirus strain detected from a 14-day-old Giraffe (Giraffa camelopardalis), presenting with acute diarrhea. The RV strain detected from the giraffe was characterized molecularly as G10P[11]. …

Spatial And Temporal Expression Profiling Of Cell-Wall Invertase Genes During Early Development In Hybrid Poplar, Thomas Canam, Sarah W.Y. Mak, Shawn D. Mansfield

Faculty Research & Creative Activity

Cell-wall invertase genes are spatially and temporally regulated in several plant species, including Daucus carota L., Lycopersicon esculentum L. and Solanum tuberosum L. However, few studies of cell-wall invertase genes of trees have been conducted, despite the importance of trees as a source of lignocellulosic biopolymers.We identified three putative cell-wall invertase genes in hybrid poplar (Populus alba L. × grandidentata Michx.) that showed higher homology to each other than to cell-wall invertases of other dicotyledonous species, with two of the genes (Pa×gINV2 and Pa×gINV3) appearing as a genomic tandem repeat. These genes are more similar to each other than to …

Rapid Detection Of Avian Eimeria Species Using Denaturing Gradient Gel Electrophoresis, Alexandra Martynova-Van Kley, A. Syvyk, I. Teplova, M. E. Hume, A. Nalian

Faculty Publications

A denaturing gradient gel electrophoresis (DGGE) assay was developed to rapidly discriminate species of avian Eimeria. Amplification by PCR of the small subunit ribosomal RNA gene (approximately 1,600 nucleotides) with Eimeria genus-specific primers followed by cloning and sequencing allowed us to carry out phylogenetic analyses and identify clone sequences to species level in most cases. Clones were subsequently used to amplify a smaller fragment (approximately 120 nucleotides) suitable for DGGE. The fragments were separated on denaturing gradient gel and bands with unique migration distances were mixed to obtain an identification ladder. The identification ladder and PCR products obtained from …

Spatial And Temporal Expression Profiling Of Cell-Wall Invertase Genes During Early Development In Hybrid Poplar, Thomas Canam, Sarah Mak, Shawn Mansfield

Faculty Research & Creative Activity

Cell-wall invertase genes are spatially and temporally regulated in several plant species, including Daucus carota L., Lycopersicon esculentum L. and Solanum tuberosum L. However, few studies of cell-wall invertase genes of trees have been conducted, despite the importance of trees as a source of lignocellulosic biopolymers.We identified three putative cell-wall invertase genes in hybrid poplar (Populus alba L. × grandidentata Michx.) that showed higher homology to each other than to cell-wall invertases of other dicotyledonous species, with two of the genes (Pa×gINV2 and Pa×gINV3) appearing as a genomic tandem repeat. These genes are more similar to each other than to …

Comparative Functional Analysis Of Aquaporins/Glyceroporins In Mammals And Anurans, Carissa M. Krane, David L. Goldstein

Biology Faculty Publications

Maintenance of fluid homeostasis is critical to establishing and maintaining normal physiology. The landmark discovery of membrane water channels (aquaporins; AQPs) ushered in a new area in osmoregulatory biology that has drawn from and contributed to diverse branches of biology, from molecular biology and genomics to systems biology and evolution, and from microbial and plant biology to animal and translational physiology. As a result, the study of AQPs provides a unique and integrated backdrop for exploring the relationships between genes and genome systems, the regulation of gene expression, and the physiologic consequences of genetic variation. The wide species distribution of …

Selective Cholesterol Dynamics Between Lipoproteins And Caveolae/Lipid Rafts, Stephen M. Storey, Adalberto M. Gallegos, Barbara P. Atshaves, Avery L. Mcintosh, Gregory G. Martin, Rebecca D. Parr, Kerstin K. Landrock, Ann B. Kier, Judith M. Ball, Friedhelm Schroeder

Faculty Publications

Although low-density lipoprotein (LDL) receptor-mediated cholesterol uptake through clathrin-coated pits is now well understood, the molecular details and organizing principles for selective cholesterol uptake/efflux (reverse cholesterol transport, RCT) from peripheral cells remain to be resolved. It is not yet completely clear whether RCT between serum lipoproteins and the plasma membrane occurs primarily through lipid rafts/caveolae or from non-raft domains. To begin to address these issues, lipid raft/caveolae-, caveolae-, and non-raft-enriched fractions were resolved from purified plasma membranes isolated from L-cell fibroblasts and MDCK cells by detergent-free affinity chromatography and compared with detergent-resistant membranes isolated from the same cells. Fluorescent sterol …

Over-Expression Of Udp-Glucose Pyrophosphorylase In Hybrid Poplar Affects Carbon Allocation, Heather Coleman, Thomas Canam, Kyu-Young Kang, David Ellis, Shawn Mansfield

Faculty Research & Creative Activity

The effects of the over-expression of the Acetobacter xylinum UDP-glucose pyrophosphorylase (UGPase) under the control of the tandem repeat Cauliflower Mosaic Virus promoter (2335S) on plant metabolism and growth were investigated in hybrid poplar (Populus alba3grandidentata). Transcript levels, enzyme activity, growth parameters, leaf morphology, structural and soluble carbohydrates, and soluble metabolite levels were quantified in both transgenic and wild-type trees. Transgenic 2335S::UGPase poplar showed impaired growth rates, displaying reduced height growth and stem diameter. Morphologically, 2335S::UGPase trees had elongated axial shoots, and leaves that were substantially smaller in size when compared with wild-type trees at equivalent developmental stages. Biochemical analysis …

Full-Length, Glycosylated Nsp4 Is Localized To Plasma Membrane Caveolae By A Novel Raft Isolation Technique, Stephen M. Storey, Thomas F. Gibbons, Cecelia V. Williams, Rebecca D. Parr, Friedhelm Schroeder, Judith M. Ball

Faculty Publications

Rotavirus NSP4, initially characterized as an endoplasmic reticulum intracellular receptor, is a multifunctional viral enterotoxin that induces diarrhea in murine pups. There have been recent reports of the secretion of a cleaved NSP4 fragment (residues 112 to 175) and of the association of NSP4 with LC3-positive autophagosomes, raft membranes, and microtubules. To determine if NSP4 traffics to a specific subset of rafts at the plasma membrane, we isolated caveolae from plasma membrane-enriched material that yielded caveola membranes free of endoplasmic reticulum and nonraft plasma membrane markers. Analyses of the newly isolated caveolae from rotavirus-infected MDCK cells revealed full-length, high-mannose glycosylated …

Over-Expression Of Udp-Glucose Pyrophosphorylase In Hybrid Poplar Affects Carbon Allocation, Heather D. Coleman, Thomas Canam, Kyu-Young Kang, David D. Ellis, Shawn D. Mansfield

Faculty Research & Creative Activity

The effects of the over-expression of the Acetobacter xylinum UDP-glucose pyrophosphorylase (UGPase) under the control of the tandem repeat Cauliflower Mosaic Virus promoter (2335S) on plant metabolism and growth were investigated in hybrid poplar (Populus alba3grandidentata). Transcript levels, enzyme activity, growth parameters, leaf morphology, structural and soluble carbohydrates, and soluble metabolite levels were quantified in both transgenic and wild-type trees. Transgenic 2335S::UGPase poplar showed impaired growth rates, displaying reduced height growth and stem diameter. Morphologically, 2335S::UGPase trees had elongated axial shoots, and leaves that were substantially smaller in size when compared with wild-type trees at equivalent developmental stages. Biochemical analysis …

The Fat Cadherin Acts Through The Hippo Tumor-Suppressor Pathway To Regulate Tissue Size, Maria Willecke, Fisun Hamaratoglu, Madhuri Kango-Singh, Ryan Udan, Chiao-Lin Chen, Chunyao Tao, Xinwei Zhang, Georg Halder

Biology Faculty Publications

Background: The Hippo tumor-suppressor pathway has emerged as a key signaling pathway that controls tissue size in Drosophila. Merlin, the Drosophila homolog of the human Neurofibromatosis type-2 (NF2) tumor-suppressor gene, and the related protein Expanded are the most upstream components of the Hippo pathway identified so far. However, components acting upstream of Expanded and Merlin, such as transmembrane receptors, have not yet been identified.

Results: Here, we report that the protocadherin Fat acts as an upstream component in the Hippo pathway. Fat is a known tumor-suppressor gene in Drosophila, and fat mutants have severely overgrown imaginal discs. We …

Lobe And Serrate Are Required For Cell Survival During Early Eye Development In Drosophila, Amit Singh, Xiao Shi, Kwang-Wook Choi

Biology Faculty Publications

Organogenesis involves an initial surge of cell proliferation, leading to differentiation. This is followed by cell death in order to remove extra cells. During early development, there is little or no cell death. However, there is a lack of information concerning the genes required for survival during the early cell-proliferation phase. Here, we show that Lobe (L) and the Notch (N) ligand Serrate (Ser), which are both involved in ventral eye growth, are required for cell survival in the early eye disc. We observed that the loss-of-ventral-eye phenotype in L or Ser mutants is due to …

Expression Of The Primary Carbohydrate Component Of The Bordetella Bronchiseptica Biofilm Matrix Is Dependent On Growth Phase But Independent Of Bvg Regulation, Yasuhiko Irie, Andrew Preston, Ming H. Yuk

Biology Faculty Publications

We previously showed that the Bvg virulence control system regulates biofilm formation in Bordetella bronchiseptica (Y. Irie, S. Mattoo, and M. H. Yuk, J. Bacteriol. 186:5692-5698, 2004). Analyses of the extracellular components of B. bronchiseptica biofilm matrix revealed that the major sugar component in the matrix was xylose, and linkage analysis indicated a majority of it to be in a 4-linked polymeric form. The production of xylose was independent of Bvg regulation but instead was dependent on bacterial growth phase. In addition, N-acetyl-glucosamine in the matrix was found to be important for the initial development of the biofilm. These …

In Vitro Expression And Purification Of Class I Mhc Molecules, Loi Cheng

Honors Scholar Theses

The major histocompatibility complex (MHC) is a gene family responsible for many critical functions of the immune system in most vertebrates. The MHC consists of three classes differentiated by their structure and function, and MHC class I encodes antigen binding proteins as well as chaperone and accessory proteins such as tapasin. The purpose of this project is to reconstitute several human MHC class I molecules in their peptide-filled and peptide-deficient forms, and to purify these proteins for biochemical study. The expressed proteins include wild type and mutant variants of the fusion protein human leukocyte antigen HLA-B*0801-fos, and human beta-2-microglobulin (β2m). …

Selective Male Mortality In The Red Imported Fire Ant, Solenopsis Invicta, Gary Fritz, Robert Vander Meer, Catherine Preston

Faculty Research & Creative Activity

Males in polygyne populations of Solenopsis invicta are primarily sterile diploids and thought to not express the Gp-9 gene coding for a pheromone-binding protein affecting complex social behavior.We examined an aspect of the breeding system hitherto not considered—male Gp-9 genotypes in relation to sperm stored in queens. Four sites with varying frequencies of sympatric monogyne and polygyne colonies were sampled, including sexuals, workers, and broods from four colonies. Most queens were heterozygotes storing B sperm. Although predicted to be common, only 14 of 504 males were B or BB genotypes, suggesting strong selection. Increased frequency of polygyne colonies at each …

Selective Male Mortality In The Red Imported Fire Ant, Solenopsis Invicta, Gary N. Fritz, Robert K. Vander Meer, Catherine A. Preston

Faculty Research & Creative Activity

Males in polygyne populations of Solenopsis invicta are primarily sterile diploids and thought to not express the Gp-9 gene coding for a pheromone-binding protein affecting complex social behavior.We examined an aspect of the breeding system hitherto not considered—male Gp-9 genotypes in relation to sperm stored in queens. Four sites with varying frequencies of sympatric monogyne and polygyne colonies were sampled, including sexuals, workers, and broods from four colonies. Most queens were heterozygotes storing B sperm. Although predicted to be common, only 14 of 504 males were B or BB genotypes, suggesting strong selection. Increased frequency of polygyne colonies at each …

The Rotavirus Enterotoxin Nsp4 Directly Interacts With The Caveolar Structural Protein Caveolin-1, Rebecca D. Parr, Stephen M. Storey, Deanne M. Mitchell, Avery Mcintosh, Minglong Zhou, Kiran D. Mir, Judith M. Ball

Faculty Publications

Rotavirus nonstructural protein 4 (NSP4) is known to function as an intracellular receptor at the endoplasmic reticulum (ER) critical to viral morphogenesis and is the first characterized viral enterotoxin. Exogenously added NSP4 induces diarrhea in rodent pups and stimulates secretory chloride currents across intestinal segments as measured in Ussing chambers. Circular dichroism studies further reveal that intact NSP4 and the enterotoxic peptide (NSP4_114-135) that is located within the extended, C-terminal amphipathic helix preferentially interact with caveola-like model membranes. We now show colocalization of NSP4 and caveolin-1 in NSP4-transfected and rotavirus-infected mammalian cells in reticular structures surrounding the nucleus …

Eye Development At The Houston "Fly Meeting", Amit Singh

Biology Faculty Publications

Meeting report: The 47th Annual Drosophila Research Conference or "Fly Meeting" took place at Houston, Texas, USA from March 29th- April 2nd, 2006, under the aegis of the Genetics Society of America. The Fly Meeting provides an excellent opportunity for fly researchers to present their work and to get a snapshot of recent developments and upcoming trends in their research field. The fruit fly, Drosophila melanogaster, is a very versatile model to study growth, patterning, neural development, evolution, systemetics and various other facets of biomedical science. The topics presented in the meeting covered a very broad spectrum of fly research. …

Empirical Analysis Of The Str Profiles Resulting From Conceptual Mixtures, David R. Paoletti, Travis E. Doom, Carissa M. Krane, Michael L. Raymer, Dan E. Krane

Biology Faculty Publications

Samples containing DNA from two or more individuals can be difficult to interpret. Even ascertaining the number of contributors can be challenging and associated uncertainties can have dramatic effects on the interpretation of testing results. Using an FBI genotypes dataset, containing complete genotype information from the 13 Combined DNA Index System (CODIS) loci for 959 individuals, all possible mixtures of three individuals were exhaustively and empirically computed. Allele sharing between pairs of individuals in the original dataset, a randomized dataset and datasets of generated cousins and siblings was evaluated as were the number of loci that were necessary to reliably …

Atypical Pkciota Contributes To Poor Prognosis Through Loss Of Apical-Basal Polarity And Cyclin E Overexpression In Ovarian Cancer, Astrid M. Eder, Xiaomei Sui, Daniel G. Rosen, Laura K. Nolden, Kwai Wa Cheng, John P. Lahad, Madhuri Kango-Singh, Karen H. Lu, Carla L. Warneke, Edward N. Atkinson, Isabelle Bedrosian, Khandan Keyomarsi, Wen-Lin Kuo, Joe W. Gray, Jerry C. P. Yin, Jinsong Liu, Georg Halder, Gordon B. Mills

Biology Faculty Publications

We show that atypical PKCι, which plays a critical role in the establishment and maintenance of epithelial cell polarity, is genomically amplified and overexpressed in serous epithelial ovarian cancers. Furthermore, PKCι protein is markedly increased or mislocalized in all serous ovarian cancers. An increased PKCι DNA copy number is associated with decreased progression-free survival in serous epithelial ovarian cancers. In a Drosophila in vivo epithelial tissue model, overexpression of persistently active atypical PKC results in defects in apical-basal polarity, increased Cyclin E protein expression, and increased proliferation. Similar to the Drosophila model, increased PKCι proteins levels are associated with …

Group 13 Hox Proteins Interact With The Mh2 Domain Of R-Smads And Modulate Smad Transcriptional Activation Functions Independent Of Hox Dna Binding Capability, Thomas M. Williams, Melissa E. Williams, Joanne H. Heaton, Thomas D. Gelehrter, Jeffrey W. Innis

Biology Faculty Publications

Interactions with co-factors provide a means by which HOX proteins exert specificity. To identify candidate protein interactors of HOXA13, we created and screened an E11.5–E12.5, distal limb bud yeast two-hybrid prey library. Among the interactors, we isolated the BMP-signaling effector Smad5, which interacted with the paralogous HOXD13 but not with HOXA11 or HOXA9, revealing unique interaction capabilities of the AbdB-like HOX proteins. Using deletion mutants, we determined that the MH2 domain of Smad5 is necessary for HOXA13 interaction.

This is the first report demonstrating an interaction between HOX proteins and the MH2 domain of Smad proteins. HOXA13 and HOXD13 …

The Bvg Virulence Control System Regulates Biofilm Formation In Bordetella Bronchiseptica, Yasuhiko Irie, Seema Mattoo, Ming H. Yuk

Biology Faculty Publications

Bordetella species utilize the BvgAS (Bordetella virulence gene) two-component signal transduction system to sense the environment and regulate gene expression among at least three phases: a virulent Bvg⁺ phase, a nonvirulent Bvg⁻ phase, and an intermediate Bvgⁱ phase. Genes expressed in the Bvg⁺ phase encode known virulence factors, including adhesins such as filamentous hemagglutinin (FHA) and fimbriae, as well as toxins such as the bifunctional adenylate cyclase/hemolysin (ACY). Previous studies showed that in the Bvgⁱ phase, FHA and fimbriae continue to be expressed, but ACY expression is significantly downregulated. In this report, we determine …

Drosophila As An Emerging Model To Study Metastasis, Madhuri Kango-Singh, Georg Halder

Biology Faculty Publications

Metastasis is the primary cause of human cancer-related deaths. Two recent studies describe a system for testing how multiple genetic events synergize to promote neoplastic growth and metastasis in Drosophila, paving the way for systematic approaches to understanding metastasis using the powerful tools of Drosophila genetics.

Sterol Carrier Protein-2 Directly Interacts With Caveolin-1 In Vitro And In Vivo, Minglong Zhou, Rebecca D. Parr, Anca D. Petrescu, H. Ross Payne, Barbara P. Atshaves, Ann B. Kier, Judith M. Ball, Friedhelm Schroeder

Faculty Publications

HDL-mediated reverse-cholesterol transport as well as phosphoinositide signaling are mediated through plasma membrane microdomains termed caveolae/lipid rafts. However, relatively little is known regarding mechanism(s) whereby these lipids traffic to or are targeted to caveolae/lipid rafts. Since sterol carrier protein-2 (SCP-2) binds both cholesterol and phosphatidylinositol, the possibility that SCP-2 might interact with caveolin-1 and caveolae was examined. Double immunolabeling and laser scanning fluorescence microscopy showed that a small but significant portion of SCP-2 colocalized with caveolin-1 primarily at the plasma membrane of L-cells and more so within intracellular punctuate structures in hepatoma cells. In SCP-2 overexpressing L-cells, SCP-2 was detected …

A Method Based On Conserved Multiple Amino Acid Properties To Predict Amino Acid Substitutions Which Maintain The Protein Structure, Avinash M. Baktula

Masters Theses & Specialist Projects

A METHOD BASED ON CONSERVED MULTIPLE AMINO ACID PROPERTIES TO PREDICT AMINO ACID SUBSTITUTIONS WHICH MAINTAIN THE PROTEIN STRUCTURE Avinash M. Baktula September 16, 2004 1-117 Directed by: Claire A. Rinehart, Doug McElroy and Sigrid Jacobshagen Department of Biology Western Kentucky University Proteins often contain several domains, each with a distinct structure. Such domains have evolved as units that, when combined in various arrangements, produce proteins of unique structure. This study was conducted to identify amino acid substitutions that don’t change structure. Amino acid properties which were conserved in proteins with identical structures were used to predict a set of …

Morphological Characteristics And Species Separation Of Hawaiian Postlarval Amphidromous Fishes, M. Eric Benbow, Albert J. Burky, Carl M. Way

Biology Faculty Publications

There are five Hawaiian amphidromous fishes (Gobiidae: Lentipes concolor, Awaous guamensis, Sicyopterus stimpsoni, Stenogobius hawaiiensis; Eleotridae: Eleotris sandwicensis). Amphidromous adults deposit eggs on the stream bottom. After hatching, larvae drift to the ocean for growth followed by postlarval migration back into the streams. Postlarvae were collected to construct a dichotomous identification key based on 12 morphological measures and ray counts from four fins. Overall, the presense of fused pelvic fins, standard length (SL), and fin ray numbers were the most useful in species separation. Gobies were separated from the eleotrid by the former having fused …

Initial State Of The Drosophila Eye Before Dorsoventral Specification Is Equivalent To Ventral, Amit Singh, Kwang-Wook Choi

Biology Faculty Publications

Dorsoventral (DV) patterning is crucial for eye development in invertebrates and higher animals. DV lineage restriction is the primary event in undifferentiated early eye primordia of Drosophila. InDrosophila eye disc, a dorsal-specific GATA family transcription factor pannier (pnr) controls Iroquois-Complex (Iro-C) genes to establish the dorsal eye fate whereas Lobe (L), which is involved in controlling a Notch ligand Serrate (Ser), is specifically required for ventral growth. However, fate of eye disc cells before the onset of dorsal expression of pnr and Iro-C is not known. We show that L/Ser …

Life Cycle Of A Torrenticolous Hawaiian Chironomid (Telmatogeton Torrenticola): Stream Flow And Microhabitat Effects, M. Eric Benbow, Albert J. Burky, Carl M. Way

Biology Faculty Publications

In this study we documented the instar densities and life cycle of Telmatogeton torrenticola Terry (Chironomidae : Telmatogetoninae) from Kinihapai Stream, Maui, Hawaii. Greatest larval densities of this midge are found on substrates of high velocity, shallow flows of cascades, and splash zones of waterfalls, with lower densities in riffles. In the summer of 1994 we compared the effects of two microhabitats (termed optimal and suboptimal) on inter-instar density and relative abundance. In a second year (1995), we evaluated the effect of long-term reduced stream flow on these variables only in optimal microhabitats. A significant reduction in stream flow from …