Molecular Biology Commons^™

Saccharomyces Cerevisiae Sec59 Cells Are Deficient In Dolichol Kinase Activity, Loree Heller, Peter Orlean, W. Lee Adair Jr.

Bioelectrics Publications

The temperature-sensitive Saccharomyces cerevisiae mutant sec59 accumulates inactive and incompletely glycosylated protein precursors in its endoplasmic reticulum at the restrictive temperature. O-mannosylation and glycosyl phosphatidylinositol membrane anchoring of protein are also abolished, consistent with a deficiency in dolichyl phosphate mannose. Membranes prepared from sec59 cells that had been shifted to the restrictive temperature, however, made normal amounts of dolichyl phosphate mannose when exogenous dolichyl phosphate was supplied, but dolichyl phosphate mannose synthesis was severely depressed in the absence of exogenous dolichyl phosphate. Quantitative measurements of dolichyl phosphate in sec59 cells showed that the levels were decreased to 48% of wild …

The Structure Of Residues 7-16 Of The Aɑ-Chain Of Human Fibrinogen Bound To Bovine Thrombin At 2.3 Å Resolution, Philip D. Martin, William Robertson, Dusan Turk, Robert Huber, Wolfram Bode, Brian Fp Edwards

Biochemistry and Molecular Biology Faculty Publications

The tetradecapeptide Ac-D-F-L-A-E-G-G-G-V-R-G-P-R-V-OMe, which mimics residues 7f-20f of the A alpha-chain of human fibrinogen, has been co-crystallized with bovine thrombin from ammonium sulfate solutions in space group P2(1) with unit cell dimensions of a = 83.0 A, b = 89.4 A, c = 99.3 A, and beta = 106.6 degrees. Three crystallographically independent complexes were located in the asymmetric unit by molecular replacement using the native bovine thrombin structure as a model. The standard crystallographic R-factor is 0.167 at 2.3-A resolution. Excellent electron density could be traced for the decapeptide, beginning with Asp-7f and ending with Arg-16f in the active …

Structural Changes That Accompany The Reduced Catalytic Efficiency Of Two Semisynthetic Ribonuclease Analogs, V. Srini J. De Mel, Philip D. Martin, Marilynn S. Doscher, Brian Fp Edwards

Biochemistry and Molecular Biology Faculty Publications

The structures of two catalytically defective semi-synthetic RNases obtained by replacing aspartic acid 121 with asparagine or alanine have been determined and refined at a resolution of 2.0 A (R = 0.186 and 0.172, respectively). When these structures are compared with the refined 1.8-A structure (R = 0.204) of the fully active aspartic acid-containing enzyme (Martin, P.D., Doscher, M.S., and Edwards, B. F. P. (1987) J. Biol. Chem. 262, 15930-15938), numerous and widespread changes, much greater in number and magnitude than the small structural variations noted previously between the semisynthetic complex and RNase A, are found to have occurred. These …

Glossary For Chemists Of Terms Used In Biotechnology, B. Nagel, H. Dellweg, Lila Gierasch

Lila Gierasch

The glossary contains definitions and explanatory notes, if needed, of over 230 terms frequently used in publications in the multidisciplinary field of biotechnology. The glossary was developed as a step to help facilitate communication between chemists, chemical engineers, biologists and bioengineers and to make biotechnology and its methodologies more accessible to the chemical profession. The interrelation between chemistry, chemical engineering and the burgeoning areas of molecular biology will be especially important as chemical industrial processes begin to incorporate recombinant DNA techniques, for example. The range of terms includes microbiology, genetic engineering, biochemistry, molecular biology, biochemical engineering, bioprocessing and general concepts …

Mouse Mast Cell Proteases: Induction, Molecular Cloning, And Characterization, Wei Chu

Electronic Theses and Dissertations

Tryptase, a mast cell-specific serine protease with trypsin-like specificity, has been identified in a mouse mast cell line (ABFTL-6) based on it's enzymatic activity, inhibition properties, and cross-reactivity to a human mast cell tryptase antibody. The effects of fibroblast-conditioned medium and sodium butyrate on ABFTL-6 mast cell differentiation and tryptase expression have been examined. ABFTL-6 mouse mast cells undergo phenotypic changes upon culturing in media supplemented with fibroblast-conditioned media at 50% or 1 mM sodium butyrate. The induced cells increased in size, had larger and more metachromatic cytoplasmic granules, and increased their total cellular protein about four-fold. Tryptase activity increased …

The Role Of Queuine Deficient Trna In Translational Control, Kathryn Lenore Hart

Theses and Dissertations in Biomedical Sciences

The purpose of this research was to study the effect of the level of tRNA modification with respect to queuine on translational efficiency. Transfer RNA from neoplastic cells is generally deficient with regard to certain hypermodified nucleosides found in normal cells. The hypothesis that was examined is whether or not the altered efficiency of translation can be correlated to the presence of queuine hypomodified tRNA. The loss of queuine in the anticodon may change the interaction of the tRNA's anticodon with its codon. Therefore, the translational complex may stall looking for a rare tRNA, or the stability of the complex …

A Temperature-Sensitive Mutant Of Escherichia Coli Affected In The Alpha Subunit Of Rna Polymerase, Majid Mehrpouyan

Electronic Theses and Dissertations

A temperature-sensitive mutant of Escherichia coli affected in the alpha subunit of RNA polymerase has been investigated. Gene mapping and complementation experiments placed the mutation to temperature-sensitivity within the alpha operon at 72 min on the bacterial chromosome. The rate of RNA synthesis in vivo and the accumulation of ribosomal RNA were significantly reduced in the mutant at 44$\sp\circ$C. The thermostability at 44$\sp\circ$C of the purified holoenzyme from mutant cells was about 20% of that of the normal enzyme. Assays with T7 DNA as a template showed that the fraction of active enzyme competent for transcription was reduced as a …

Separation Of Transfer Rna Isoacceptors By Reversed Phase W-Porex C-4 High Pressure Liquid Chromatography, Christine D. Witte

Chemistry & Biochemistry Theses & Dissertations

Transfer RNA (tRNA), is a complex class of small molecules which have been proven to play a major role in protein synthesis. Transfer RNA is composed of 20 families of isoacceptors which may recognize only one particular amino acid. This family of isoacceptors may differ from each other by primary sequence or modified nucleosides. Queuine is a modified nucleoside which is found in the anticodon regions of tRNA isoacceptors for asparagine, aspartic acid, tyrosine and histidine. Queuine has gained the interest of many researchers since queuine deficient tRNA was found in many neoplastic and undifferentiated cell-lines. It is often difficult …

The Role Of The Voltage Gradient In The Agarose Gel Electrophoresis Of Dna, David Wheeler

Theses and Dissertations in Biomedical Sciences

In Part I of this dissertation, empirical equations for predicting DNA mobility during agarose gel electrophoresis (AGE) from voltage gradient are developed from the data of McDonnel (36) for electrophoresis in a 1.6% agarose gel. These equations represented the data well for DNA between 2 and 10 kilobase pairs (KBp) in length. A computer program, called GELSIM, which incorporates these equations is described in Part II. GELSIM was designed to allow researchers to analyze electrophoresis data by predicting the effect on DNA migration of altering the voltage of electrophoresis. In this way, electrophoretic banding patterns produced using different voltages could …

A Retinoic Acid Responsive Gene, Mk, Produces A Secreted Protein A With Heparin Binding Activity, Masaru Nakamoto

Biology Faculty Publications

MK is a gene whose expression increases transiently during retinoic acid-induced differentiation of embryonal carcinoma cells. MK polypeptide was secreted by differentiating HM-1 embryonal carcinoma cells and by L-cells transfected with an MK cDNA under the control of the S-actin promoter and Rous sarcoma virus enhancer. MK polypeptide was found to have heparin binding activity. Conditioned medium of the transfected L-cells promoted growth of PC-12 pheochromocytoma cells. These findings support the view that MK polypeptide is a secreted factor involved in regulation of growth and differentiation.

Isoprenylation Is Required For The Processing Of The Lamin A Precursor, Michael Sinensky, L. A. Beck, T. J. Hosick

Michael Sinensky

The nuclear lamina proteins, prelamin A, lamin B, and a 70-kD lamina-associated protein, are posttranslationally modified by a metabolite derived from mevalonate. This modification can be inhibited by treatment with (3-R,S)-3-fluoromevalonate, demonstrating that it is isoprenoid in nature. We have examined the association between isoprenoid metabolism and processing of the lamin A precursor in human and hamster cells. Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase by mevinolin (lovastatin) specifically depletes endogenous isoprenoid pools and inhibits the conversion of prelamin A to lamin A. Prelamin A processing is also blocked by mevalonate starvation of Mev-1, a CHO cell line auxotrophic for mevalonate. …

Isoprenylation Is Required For The Processing Of The Lamin A Precursor, Michael Sinensky, L. A. Beck, T. J. Hosick

Faculty Publications, Biological Sciences

The nuclear lamina proteins, prelamin A, lamin B, and a 70-kD lamina-associated protein, are posttranslationally modified by a metabolite derived from mevalonate. This modification can be inhibited by treatment with (3-R,S)-3-fluoromevalonate, demonstrating that it is isoprenoid in nature. We have examined the association between isoprenoid metabolism and processing of the lamin A precursor in human and hamster cells. Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase by mevinolin (lovastatin) specifically depletes endogenous isoprenoid pools and inhibits the conversion of prelamin A to lamin A. Prelamin A processing is also blocked by mevalonate starvation of Mev-1, a CHO cell line auxotrophic for mevalonate. …

Regulation Of Queuine Insertion Into Transfer Rna: Effects Of Tumor Promoters, Bonnie Jean Brooks

Chemistry & Biochemistry Theses & Dissertations

The purpose of this study was to treat normal human fibroblasts with known tumor promoters and observe queuine modification of tRNA. The queuine insertion enzyme, tRNAguanine ribosyltransferase, was studied in vivo and in vitro. Tumor promoter-treated human fibroblast cultures exhibited variable queuine-insertion rates with a transient inhibition that correlated with variable levels of queuine modified tRNA over time in culture from passages 3 — 8. In contrast the in vitro studies showed that phorbol cetera and saccharin actually increased insertion with strong evidence indicating phosphorylation as a positive modulating force of the enzyme activity. It is proposed that chronic stimulation …

Microwell Based Competitive Enzyme Immunoassay For Quantifying Serum Triiodothyronine And Thyroxine Levels, Yiwen Yang

Chemistry & Biochemistry Theses & Dissertations

A microwell based competitive enzyme immunoassay for determination of total thyroxine and triiodothyronine in human serum was developed with use of horseradish peroxidase conjugate. The assay, which can be performed in only 1.5 hours at room temperature, involves simultaneous incubation of T₃- or T₄- HRPO conjugate and serum sample in anti-T₃ or anti-T₄ gamma globulin fraction coated polystyrene microwells.

Isolation of anti-T₃ and anti-T₄ gamma globulin fractions were accomplished from whole antiserum by using DEAE-cellulose chromatograph. A carbodiimide coupling method was described to prepare the T₄- and T₃-HRPO …

Hypoxanthine-Induced Differentiation Of Cultured Human Leukemia Cells, Gayle Jennette Singleton

Chemistry & Biochemistry Theses & Dissertations

Human cultured leukemia cells appear to have a decreased amount of inosine in their tRNA. When cells with inosine deficient tRNA are placed in a hypoxanthine fortified media, they incorporate hypoxanthine into their tRNA by the action of the enzyme tRNA-hypoxanthine ribosyl transferase. This generates the nucleoside inosine in the tRNA. The cultured human leukemia cell lines, CCRF-CEM, HL-60, and HGPRT(-) HL- 60, incorporate hypoxanthine into their tRNA, as determined by tRNA isolation, hydrolysis, and HPLC analysis. Hypoxanthine treatment dramatically inhibited cell growth in conjunction with partial induction of differentiation in the CCRF-CEM, HL-60, and HGPRT ( - ) HL-60 …

The Three-Dimensional Structure Of Bovine Platelet Factor 4 At 3.0-Å Resolution, Robert St. Charles, Daniel A. Walz, Brian Fp Edwards

Biochemistry and Molecular Biology Faculty Publications

Platelet factor 4 (PF4), which is released by platelets during coagulation, binds very tightly to negatively charged oligosaccharides such as heparin. To date, six other proteins are known that are homologous in sequence with PF4 but have quite different functions. The structure of a tetramer of bovine PF4 complexed with one Ni(CN)42− molecule has been determined at 3.0 Å resolution and refined to an R factor of 0.28. The current model contains residues 24–85, no solvent, and one overall temperature factor. Residues 1–13, which carried an oligosaccharide chain, were removed with elastase to induce crystallization; residues 14–23 and presumably 86–88 …

A Flow Cytometric Approach To Assessing The Environmental And Physiological Status Of Phytoplankton, Serge Demers, Kimberly Davis, Terry L. Cucci

CCPO Publications

No abstract provided.

Identification Of Genes That Interact With Glp-1, A Gene Required For Inductive Cell Interactions In Caenorhabditis Elegans, Eleanor M. Maine, Judith Kimble

Biology - All Scholarship

The glp-1 gene functions in two inductive cellular interactions and in development of the embryonic hypodermis of C. elegans. We have isolated six mutations as recessive suppressors of temperature-sensitive (ts) mutations of glp-1. By mapping and complementation tests, we found that these suppressors are mutations of known dumpy (dpy) genes; dpy genes are required for development of normal body shape. Based on this result, we asked whether mutations previously isolated in screens for mutants defective in body shape could also suppress glp-1(ts). From these tests, we learned that unselected mutations of eight genes required for normal C. elegans morphogenesis, including …

A Method For The Large Scale Purification Of Hcg (Human Chorionic Gonadotropin) From The Urine Of Pregnant Women, Chun-Shyan Kuo

Chemistry & Biochemistry Theses & Dissertations

A large scale preparation of human chorionic gonadotropin (HCG) from the urine of pregnant women was accomplished by using kaolin adsorption, NH₄OH extraction and DEAE-cellulose ion exchange. A recovery about 81% of HCG and a specific activity in the 2,000-3,000 IU/mg range was achieved using this purification method.

Two HCG activity fractions, HCG I & HCG II, were isolated from DEAE-cellulose chromatography by a stepwise sodium chloride gradient elution. HCG I was eluted at 0.01-0.02 M NaCl while HCG II was eluted at 0.12-0.02 M NaCl.

The differences between HCG I and HCG II have been studied. The …

Molecular Study Of The B19 (Human) Pathogenic Parvovirus, Jamshed Ayub

Theses and Dissertations in Biomedical Sciences

The B19 (human) parvovirus is a small single stranded DNA virus of 5.4 kilobases. B19 is specific for erythroid progenitor cells and has been propagated in vitro only with human erythroid bone marrow. Replication of viral DNA and the viral protein products of B19 appear similar to those of other animal parvoviruses. However, B19 differs from other parvoviruses in some important aspects, which include the initiation of all transcripts at a strong left side promoter (p6) and the absence of a functional internal promoter. B19 has an unusual transcription map which is described in this study.

The transcription map of …

Modulation Of Queuine Uptake In Cultured Human Fibroblasts By Phorbol Esters And Interferons, Debra L. Crane

Chemistry & Biochemistry Theses & Dissertations

Queuosine, a nucleoside found exclusively in the first position of the anticodon of transfer RNA (tRNA), is generated post-transcriptionally in an exchange of guanine for queuine by the modifying enzyme tRNA-guanine ribosyltransferase. Phorbol ester tumor promoters were shown to induce queuine hypomodified tRNA, and that phorbol ester action was due, in part, to inhibition of queuine transport across the cell membrane. An unidentified protein designated conditioned medium factor (CMF) that potentiated a phorbol-induced inhibition of queuine uptake was also documented. We suggest here that phorbol ester-induced inhibition of queuine uptake is not a significant factor in inducing queuine-deficient tRNA although …

Incorporation Of A Product Of Mevalonic Acid Metabolism Into Proteins Of Chinese Hamster Ovary Nuclei, Michael Sinensky, L. A. Beck, T. Hosick

Michael Sinensky

We have examined the nuclear localization of isoprenylated proteins in CHO-K1 cells labeled with [14C]mevalonate. Nuclear proteins of 68, 70, and 74 kD, posttranslationally modified by an isoprenoid, are also components of a nuclear matrix-intermediate filament preparation from CHO cells. Furthermore, the 68-, 70-, and 74-kD isoprenylated polypeptides are immunoprecipitated from cell extracts with two different anti-lamin antisera. Based on exact two-dimensional comigration with lamin B, both from rat liver lamin and CHO nuclear matrix-intermediate filament preparations, and its immunoprecipitation with anti-lamin antisera, we conclude that the 68-kD isoprenylated protein found in nuclei from [14C]mevalonate-labeled CHO cells is lamin B. …

Incorporation Of A Product Of Mevalonic Acid Metabolism Into Proteins Of Chinese Hamster Ovary Nuclei, Michael Sinensky, L. A. Beck, T. Hosick

Faculty Publications, Biological Sciences

We have examined the nuclear localization of isoprenylated proteins in CHO-K1 cells labeled with [14C]mevalonate. Nuclear proteins of 68, 70, and 74 kD, posttranslationally modified by an isoprenoid, are also components of a nuclear matrix-intermediate filament preparation from CHO cells. Furthermore, the 68-, 70-, and 74-kD isoprenylated polypeptides are immunoprecipitated from cell extracts with two different anti-lamin antisera. Based on exact two-dimensional comigration with lamin B, both from rat liver lamin and CHO nuclear matrix-intermediate filament preparations, and its immunoprecipitation with anti-lamin antisera, we conclude that the 68-kD isoprenylated protein found in nuclei from [14C]mevalonate-labeled CHO cells is lamin B. …

The Purification Of Ribosomal Rna Gene Chromatin From Physarum Polycephalum, Sally A. Amero, Roy C. Ogle, John L. Keating, Vicky L. Montoya, Wendy L. Murdoch, Robert M. Grainger

Medical Diagnostics & Translational Sciences Faculty Publications

We have undertaken the purification of ribosomal RNA gene (rDNA) chromatin from the slime mold Physarum polycephalum, in order to study its chromatin structure. In this organism rDNA exists in nucleoli as highly repeated minichromosomes, and one can obtain crude chromatin fractions highly enriched in rDNA from isolated nucleoli. We first developed a nucleolar isolation method utilizing polyamines as stabilization agents that results in a chromatin fraction containing far more protein than is obtained by the more commonly used divalent cation isolation methods. The latter method appears to result in extensive histone loss during chromatin isolations. Two methods were then …

The Refined Crystal Structure Of A Fully Active Semisynthetic Ribonuclease At 1.8 Å Resolution, Philip D. Martin, Marilynn S. Doscher, Brian Fp Edwards

Biochemistry and Molecular Biology Faculty Publications

A fully active, semisynthetic analog of bovine ribonuclease A, comprised of residues 1-118 of the molecule in a noncovalent complex with the synthetic peptide analog of residues 111-124, has been crystallized in space group P3(2)21 from a solution of 1.3 M ammonium sulfate and 3.0 M cesium chloride at pH 5.2. The crystallographic structure was determined by rotation and translation searches utilizing the coordinates for ribonuclease A reported by Wlodawer and Sjolin (Wlodawer, A., and Sjolin, L. (1983) Biochemistry 22, 2720-2728) and has been refined at 1.8-A resolution to an agreement factor of 0.204. Most of the structure of the …

Comparison Of Surface Polysaccharides Of R. Leguminosarum Anu57 (Exo-1), R. Leg. Anu57(Pbr1an) (Nod+, Nif-), And R. Leg. Anu57(Pjb5j1) (Nod+, Nif-), Frank G. Hustmyer

Masters Theses

Extracellular polysaccharides (EPS) and lipopolysaccharides (LPS) are suspected to be involved in the legume/Rhizobium symbiotic process. Polysaccharide compositions from the EPS, LPS, capsular polysaccharide (CPS), and small polysaccharide (SmPS) were compared between Rhizobium leguminosarum ANU57 (parent, Exo^-1) and two mutants R. leg. ANU57(pBR1AN) (nod⁺, nif^-) and R. leg. ANU57(pJB5J1) (nod⁺, nif^-) both containing a Sym (symbiotic) plasmid insertion. R. leg. ANU57(pBR1AN) and R. leg. ANU57(pJB5J1) are nod⁺ on clover and peas, respectively. The nod⁺ mutants produce larger amounts of EPS and decreased amounts of LPS compared to …

Determination Of Erythrocyte Zinc Concentrations Using Zeeman Effect Atomic Absorption Spectroscopy, Tanya Maranda Lewis

Chemistry & Biochemistry Theses & Dissertations

The purpose of this research project was to develop assay procedures for determination of zinc in erythrocytes. Three methodologies were developed. The methods were evaluated on the basis of within-run and between-run precision. The most reproducible method, the 213.856 nm flame methodology, was used to determine red blood cell zinc concentrations in more than 200 samples obtained from the Neonatal Intensive care Unit at the Children's Hospital of the King's Daughters.

This study demonstrated that erythrocyte zinc levels in premature infants were similar to those of healthy adults. A linear relation between plasma and red blood cell zinc concentrations was …

Distribution Of Limonin During The Growth And Development Of Leaves And Branches Of Citrus Paradisi, Cecilia Mcintosh, Richard L. Mansell

Cecilia A. McIntosh

An Analysis Of The Limonin And Naringin Content Of Grapefruit Juice Samples Collected From Florida State Test Houses, Richard L. Mansell, Cecilia Mcintosh, Susan E. Vest

Cecilia A. McIntosh

Isomers Of 5.8s Ribosomal Rna, David Smith

Chemistry & Biochemistry Theses & Dissertations

When whole cell RNA was separated by high resolution PAG (i.e., polyacrylamide gel) electrophoresis, 5.8S rRNA migrated as three higher mobility intense bands, and three faint bands of lower mobility. The three intense bands represent conformational isomers which migrated as a single band under denaturing conditions on PAG. The three faint bands represent conformational isomers of a minor form of 5.8S rRNA which is elongated at the 5' terminus of the molecule. This minor form migrates as a single band of lower mobility than the major form when electrophoresed on a denaturing PAG. The minor form was determined to have …