Molecular Biology Commons^™

Effect Of Charged Residue Substitutions On The Membrane-Interactive Properties Of Signal Sequences Of The Escherichia Coli Lamb Protein., Jeffrey D. Jones, Lila Gierasch

Lila Gierasch

Although the central role of the signal sequence in protein export is well established, the molecular details underlying signal sequence in vivo function remain unclear. As part of our continuing effort to relate signal sequence phenotypes to specific biophysical properties, we have carried out an extensive characterization of the secondary structure and lipid interactions for a family of peptides corresponding to the wild-type E. coli LamB signal sequence, and mutants that harbor charged residue point mutations in the hydrophobic core region. We used membrane-resident fluorescence quenching according to the parallax method to determine the relative depth of insertion of tryptophan-labeled …

Effect Of Charged Residue Substitutions On The Thermodynamics Of Signal Peptide-Lipid Interactions For The Escherichia Coli Lamb Signal Sequence., Jeffrey D. Jones, Lila Gierasch

Lila Gierasch

We have used tryptophan fluorescence spectroscopy to characterize the binding affinities of an Escherichia coli LamB signal peptide family for lipid vesicles. These peptides harbor charged residue substitutions in the hydrophobic core region. Titrations of peptides with vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-3-phosphoglycerol (65:35 mol%), in conjunction with evaluation of peptide dissociation rates from these vesicles, were used to determine binding parameters quantitatively. We find that under low ionic strength conditions, point mutations introducing negatively charged aspartate residues substantially reduce peptide affinity relative to the wild-type peptide. However, the difference between wild-type and mutant peptide affinities was much lower under …

Lag-2 May Encode A Signaling Ligand For The Glp-1 And Lin-12 Receptors Of C-Elegans, Samuel T. Henderson, Dali Gao, Eric J. Lambie, Judith Kimble

Dartmouth Scholarship

The C. elegans lag-2 gene is required for several cell-cell interactions that rely on the receptors GLP-1 and LIN-12. In this paper, we report that lag-2 encodes a putative membrane protein with sequence similarity to Drosophila Delta, a proposed ligand for the Notch receptor. Furthermore, we show that the lag-2 promoter drives expression of a reporter protein in the signaling distal tip cell (DTC) of the DTC/germline interaction. By in situ hybridization, we have found that endogenous lag-2 mRNA is present in the DTC but not the germ line. One fusion protein, called LAG-2::beta-gal(intra), rescues a lag-2 null mutant and …

The Pha-4 Gene Is Required To Generate The Pharyngeal Primordium Of Caenorhabditis-Elegans, Susan E. Mango, Eric J. Lambie, Judith Kimble

Dartmouth Scholarship

In the 4-cell Caenorhabditis elegans embryo, two blastomeres are destined to generate pharyngeal cells, each by a distinct developmental strategy: one pathway is inductive, while the other is autonomous. Here, we identify the pha-4 locus. In animals lacking pha-4 activity, an early step in pharyngeal organogenesis is blocked: no pharyngeal primordium is formed and differentiated pharyngeal cells are absent. Most other tissues are generated normally in pha-4 mutants, including cells related to pharyngeal cells by cell lineage and position. Thus, pha-4 activity is required to form the pharyngeal primordium. We propose that pha-4 marks a convergence of the inductive and …

Heterologous Expression Of The Bchm Gene Product From Rhodobacter Capsulatus And Demonstration That It Encodes S-Adenosyl-L-Methionine: Mg-Protoporhyrin Ix Methyltransferase, David Bollivar

Scholarship

No abstract provided.

Structure-Substrate Binding Relationships Of Hiv-1 Reverse Transcriptase, Steve Chien-Wen Huang

Theses and Dissertations in Biomedical Sciences

Human Immunodeficiency Virus, type 1 (HIV-1), is the causative agent of the Acquired Immunodeficiency Syndrome (AIDS). HIV-1 reverse transcriptase (RT), a heterodimer p66/p51, has been the major target for treatment of AIDS. The significance of the p51 subunit and the RNase H domain of p66 in terms of their influence on the RNA-dependent DNA synthesis was investigated. Clones of the wildtype HIV-1 RT subunits, p66 and p51, and a recombinant C-terminal deletion mutant, p64, [Barr, P. J. (1987) Bio/Technoloav 5, 486-489] were employed to study the structure-substrate binding relationships of HIV-1 RT. The activity assays of RNA-dependent DNA synthesis on …

Reorganization Of Lipid Domain Structure In Membranes By A Transmembrane Peptide: An Esr Spin Label Study On The Effect Of The Escherichia Coli Outer Membrane Protein A Signal Peptide On The Fluid Lipid Domain Connectivity In Binary Mixtures Of Dimyristoyl Phosphatidylcholine And Distearoyl Phosphatidylcholine., M. B. Sankaram, D. Marsh, Lila Gierasch, T. E. Thompson

Lila Gierasch

The effect of a transmembrane peptide on the domain structure of a two-component, two-phase lipid bilayer composed of dimyristoyl phosphatidylcholine (DMPC) and distearoyl phosphatidylcholine (DSPC) was examined by spin label electron spin resonance (ESR) spectroscopy. The peptide, pOmpA, is the hydrophobic, 25-residue signal sequence of the outer membrane protein A from Escherichia coli. Nitroxide derivatives of the phospholipid DSPC, 16-DSPCSL, and of the pOmpA signal peptide, pOmpA-IASL, were used as probes. The first-derivative lineshapes of the ESR spectra were analyzed using a normalized intensity ratio, R, that gives information on the average sizes of the disconnected fluid domains and their …

The Phosphate Transporter From Pea Mitochondria (Isolation And Characterization In Proteolipid Vesicles), Cecilia A. Mcintosh, David J. Oliver

David J. Oliver

The phosphate transporter from mitochondria will exchange matrix phosphate for cytosolic phosphate and facilitate either phosphate/proton symport or phosphate/hydroxyl ion antiport. The phosphate transported into the matrix by this carrier is either used for ATP synthesis or exchanges back out to the cytosol on the dicarboxylate transporter, permitting entry of malate and succinate into the matrix. The phosphate transporter was solubilized from etiolated pea (Pisum sativum L. cv Alaska) mitochondrial membranes with Triton X-114, purified approximately 500-fold by hydroxylapatite chromatography, and reconstituted into azolectin vesicles that were preloaded with 0.1 or 10 mM phosphate. Phosphate transport was measured as the …

Production Of Interleukin 10 By Islet Cells Accelerates Immune-Mediated Destruction Of Beta Cells In Nonobese Diabetic Mice., Lise Wogensen, Myung-Shik Lee, Nora Sarvetnick

Journal Articles: Regenerative Medicine

The T helper type 2 (Th2) cell product interleukin 10 (IL-10) inhibits the proliferation and function of Th1 lymphocytes and macrophages (M phi). The nonobese diabetic mouse strain (NOD/Shi) develops a M phi and T cell-dependent autoimmune diabetes that closely resembles human insulin-dependent diabetes mellitus (IDDM). The objective of the present study was to explore the consequences of localized production of IL-10 on diabetes development in NOD/Shi mice. Surprisingly, local production of IL-10 accelerated the onset and increased the prevalence of diabetes, since diabetes developed at 5-10 wk of age in 92% of IL-10 positive I-A beta g7/g7, I-E- mice …

Translational Regulation Of The C-Jun Proto-Oncogene, Anil Sehgal

Theses and Dissertations in Biomedical Sciences

The v-jun oncogene was originally isolated from the ASV17 virus in 1987. Ever since its isolation, extensive work has been done to understand the role of the v-jun oncogene in cell transformation. The c-Jun protein is a transcription factor which binds to the DNA target TGACTCA. The c-Jun protein binds to DNA in the form of dimers. It can form homodimers with itself and heterodimers with Jun family (JunB and JunD), Fos family (FosB, Fra1 and Fra2), or with CREB family members through the leucine zipper motif. Because the c-jun proto-oncogene plays an important role in cell transformation, extensive work …

Altering The Rna Binding Specificity Of A Translational Repressor, F Lim, Marc Spingola, D Peabody

Biology Department Faculty Works

The coat proteins of RNA phages MS2 and GA are specific RNA-binding proteins which function to encapsidate viral RNA and to translationally repress synthesis of the viral replicase. The two proteins have highly homologous amino acid sequences, yet they show different RNA binding specificities, recognizing RNA stem-loop structures which differ primarily in the nucleotide sequences of their loops. We sought to convert MS2 coat protein to the RNA binding specificity of GA through the introduction of GA-like amino acid substitutions into the MS2 coat protein RNA-binding site. The effects of the mutations were determined by measuring the affinity of the …

Altering The Rna Binding Specificity Of A Translational Repressor, F Lim, Marc Spingola, D S. Peabody

Marc Spingola

Pancreatic Islet Production Of Murine Interleukin-10 Does Not Inhibit Immune-Mediated Tissue Destruction., Myung-Shik Lee, Lise Wogensen, Judith Shizuru, Michael B.A. Oldstone, Nora Sarvetnick

Journal Articles: Regenerative Medicine

IL-10 inhibits macrophage-dependent antigen presentation, cytokine production, and generation of allospecific cells in vitro. These findings have lead to the widespread expectation that IL-10 may be a useful immunosuppressive agent to inhibit allograft rejection or autoimmunity in vivo. We used two experimental paradigms to study effects of murine IL-10 on in vivo immune responses. First, fetal pancreata or adult pancreatic islets from transgenic mice expressing IL-10 in pancreatic beta cells (Ins-IL-10 mice) were grafted across the MHC barrier to examine if IL-10 could inhibit allograft rejection. Second, Ins-IL-10 mice were crossed with transgenic mice expressing lymphocytic choriomeningitis virus (LCMV) antigens …

The Role Of Alternative Splicing In The Regulation Of Cathepsin B Expression In Human Tumors, Qiuming Gong

Dissertations

No abstract provided.

The Effect Of Acute Ethanol Exposure On The Male Rodent Reproductive Axis, Margaret Mary Halloran

Dissertations

No abstract provided.

Ribosomal Protein S3: A Multifunctional Eukaryotic Dna Repair Enzyme Active On Ultraviolet And Oxidative Dna Damage, David M. Wilson

Dissertations

No abstract provided.

Properties Of A Novel Early Lymphoid Activation Gene (Elag) Isolated From A Hodgkin's-Disease Cell Line, John Scott Bennett

Dissertations

No abstract provided.

Molecular Genetic Analysis Of Terminal Steps In Bacteriochlorophyll A Biosynthesis: Characterization Of A Rhodobacter Capsulatus Strain That Synthesizes Geranylgeranoil-Esterified Bacteriochlorophyll A, David Bollivar

Scholarship

No abstract provided.

Expression Of Prelamin A Confers Sensitivity Of Dna Biosynthesis To Lovastatin On F9 Teratocarcinoma Cells, Michael Sinensky, T. Mclain, K. Fantle

Michael Sinensky

No abstract provided.

The Processing Pathway Of Prelamin A, Michael Sinensky, K. Fantle, M. Trujillo, T. Mclain, A. Kupfer, M. Dalton

Michael Sinensky

The conversion of mammalian prelamin A to mature lamin A proceeds through the removal of 18 amino acids from the carboxyl terminus. The initial step in this processing is the isoprenylation of a CAAX box cysteine. This proteolytic event is distinctive for prelamin A among the known prenylated mammalian proteins. Since the carboxyl terminus of prelamin A is removed during maturation, it is not obvious that this protein would undergo the two reactions subsequent to prenylation observed in other CAAX box proteins-the endoproteolytic removal of the carboxyl-terminal 3 amino acids and the subsequent methylation of the now carboxyl-terminal cysteine. To …

The Processing Pathway Of Prelamin A, Michael Sinensky, K. Fantle, M. Trujillo, T. Mclain, A. Kupfer, M. Dalton

Faculty Publications, Biological Sciences

The conversion of mammalian prelamin A to mature lamin A proceeds through the removal of 18 amino acids from the carboxyl terminus. The initial step in this processing is the isoprenylation of a CAAX box cysteine. This proteolytic event is distinctive for prelamin A among the known prenylated mammalian proteins. Since the carboxyl terminus of prelamin A is removed during maturation, it is not obvious that this protein would undergo the two reactions subsequent to prenylation observed in other CAAX box proteins-the endoproteolytic removal of the carboxyl-terminal 3 amino acids and the subsequent methylation of the now carboxyl-terminal cysteine. To …

Binding Of Matrix Attachment Regions To Lamin Polymers Involves Single-Stranded Regions And The Minor Groove., M. E. Eva Ludérus, Jan L. Den Blaauwen, Oncko J. De Smit, Duane A. Compton, Roel Van Driel

Dartmouth Scholarship

Chromatin in eukaryotic nuclei is thought to be partitioned into functional loop domains that are generated by the binding of defined DNA sequences, named MARs (matrix attachment regions), to the nuclear matrix. We have previously identified B-type lamins as MAR-binding matrix components (M. E. E. Ludérus, A. de Graaf, E. Mattia, J. L. den Blaauwen, M. A. Grande, L. de Jong, and R. van Driel, Cell 70:949-959, 1992). Here we show that A-type lamins and the structurally related proteins desmin and NuMA also specifically bind MARs in vitro. We studied the interaction between MARs and lamin polymers in molecular detail …

Expression Of Prelamin A Confers Sensitivity Of Dna Biosynthesis To Lovastatin On F9 Teratocarcinoma Cells, Michael Sinensky, T. Mclain, K. Fantle

Faculty Publications, Biological Sciences

No abstract provided.

An Analysis Of Mitochondrial Dna In Rett Syndrome And Other Neurodegenerative Disorders, Catherine Erickson Burgess

Theses and Dissertations in Biomedical Sciences

Mitochondrial dysfunction resulting from mutations on mitochondrial DNA (mtDNA) is being recognized in a growing spectrum of diseases. These diseases, resulting from single base mutations, large deletions, or insertions, have been largely neuromuscular in origin. However, as an understanding of the effects of mtDNA mutations progresses, attention is now focusing on neurodegenerative diseases. Rett Syndrome (RS), a progressive neurodegenerative disease with predominantly female cases, demonstrates morphologic mitochondrial changes, mitochondrial enzyme deficiencies and maternal inheritance (characteristic of mtDNA diseases). No investigation of mtDNA involvement has been previously conducted and, to date, no biological marker exists for this disorder.

Our preliminary studies …

Global Phylogeography Of Yellowfin Tuna, Thunnus Albacares, And Mackerels Of The Genus Scomber, Daniel R. Scoles

Dissertations, Theses, and Masters Projects

Intraspecific genetic relationships within yellowfin tuna, Thunnus albacares, and three mackerels of the genus Scomber were studied by restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA). The comparison of these scombrids, with different larval distributions, adult distributions, and vagilities, served to investigate the hypothesis that population structure in marine fishes results from geographic and physical oceanographic processes that limit dispersal of early life history stages. Samples of 20 yellowfin tuna were examined from each of five Pacific locations and one Atlantic location. MtDNA analysis with 12 informative restriction endonucleases demonstrated considerable genetic variation, as evidenced by overall nucleon …

Mitochondrial Dna Variation In Natural And Cultured Populations Of The Bay Scallop, Argopecten Irradians (Lamarck) And The Calico Scallop, Argopecten Gibbus (Dall), Sandra Grace Blake

Dissertations, Theses, and Masters Projects

No abstract provided.

Novel Suicide Elements For Gene Disruption In Escherichia Coli, Brian Christopher Nicholson

Dissertations, Theses, and Masters Projects

No abstract provided.

Multisite Microprobes For Electrochemical Recordings In Biological Dynamics, G. Sreenivas, S. S. Ang, R. M. Ranade, A. S. Salian, W. D. Brown

Journal of the Arkansas Academy of Science

For over 30 years, techniques have been developed that allow for the microscale (10-30 /mum) measurement of chemical signals with high temporal resolution (1-200 Hz). Such measurements, called in vivo electrochemical recordings, allow for the direct determination of neurotransmitter molecules and related compounds in biological systems. Multiple recordings, simultaneously performed at different, closely spaced, well defined locations throughout a three-dimensional tissue volume in the brain, are of interest in neuroscience. Developments in microelectronic techniques enable the fabrication of multi-electrode microprobes for recording extracellular action potentials generated by individual neurons simultaneously. A high-yield microfabrication process has been successfully developed for the …

Xenopus Laevis Ribosomal Protein S1: Evidence For Regulation At The Transcriptional Level, Cleve Ray-Dean Sinor

Dissertations, Theses, and Masters Projects

No abstract provided.

Subunit Interactions Of Recombinant Hiv-1 Reverse Transcriptase With Mutations At L289, Jacquelyn R. Smith

Theses and Dissertations in Biomedical Sciences

Reverse transcriptase (RT) is a dimeric enzyme required for replication of the human immunodeficiency virus (HIV). If the subunits of the RT dimer are dissociated, the enzyme is no longer active; therefore, identification of subunit binding sites could lead to potential targets for antiviral therapy. In order to identify where subunit binding of RT occurs, mutations were made at leucine (L) 289, a residue believed to be involved in dimerization through hydrophobic interactions with other leucines. L289 is the central leucine of a leucine repeat sequence which resembles a leucine zipper protein-DNA binding motif. Two mutations, leucine to arginine (L289R) …