Molecular Genetics Commons^™

Ecdysterone Regulatory Elements Function As Both Transcriptional Activators And Repressors., Leonard Dobens, Karen Rudolph, Edward M. Berger

Dartmouth Scholarship

A synthetic, 23-bp ecdysterone regulatory element (EcRE), derived from the upstream region of the Drosophila melanogaster hsp27 gene, was inserted adjacent to the herpes simplex virus thymidine kinase promoter fused to a bacterial gene for chloramphenicol acetyltransferase (CAT). Hybrid constructs were transfected into Drosophila S3 cells and assayed for ecdysterone-inducible CAT expression. In the absence of ecdysterone a tandem pair of EcREs repressed the high constitutive level of CAT activity found after transfection with the parent reporter plasmid alone. After hormone addition very high levels of CAT activity were observed. Insertion of the EcRE pair 3' of the CAT gene …

Physiology Of Murine B Lymphocytes Ii. Life-Spans Of Mitogen And Thymus-Independent Antigen (Type 2) Reactive B Lymphocytes From Aged Mice, Venkatachalam Udhayakumar, S. Niranjan Goud, Richard J. Cross, Subbarao Bondada

Microbiology, Immunology, and Molecular Genetics Faculty Publications

We showed earlier that life spans of murine B lymphocytes could be estimated by measuring the functional reactivities of normal B cells upon transfer into x-linked immunodeficient (xid) mice, which do not respond to anti-mouse IgM (anti-μ) antibodies and thymic-independent type -2 (TI-2) antigens. Here the same approach was adopted, to evaluate the life spans of B-lymphocytes from aged mice. Spleen cells from normal young and aged mice were transferred into young or aged xid recipients and the decay kinetics were followed by measuring the proliferative response to anti-μ and PFC response to TNP-Ficoll, a prototype TI-2 antigen. The results …

Characterization Of Hsd::Mudx128 Operon Fusion Mutant Of Escherichia Coli K-12, Marjorie Ann T. Reyno

Loma Linda University Electronic Theses, Dissertations & Projects

The study of the regulation of gene expression in complex genes can be facilitated by the use of operon fusions which place a well characterized lacZ gene under the control of a promoter of interest. An hsd::MudX128 operon fusion mutant of E. coli K-12 isolated by Prakash (1986) was observed to have a high β-galactosidase activity (1000 units), ten to twenty times higher than other hsd::MudX mutants. This β-galactosidase activity was suppressed to a very low level (25 units) by the introduction of an F’ plasmid, F'101, which carries the 98 min to 2 min region of the …

Macrophages But Not B Cells From Aged Mice Are Defective In Stimulating Autoreactive T Cells In Vitro, Aruna Seth, Mitzi Nagarkatti, Prakash S. Nagarkatti, Subbarao Bondada, Venkatachalam Udhayakumar

Microbiology, Immunology, and Molecular Genetics Faculty Publications

In the present study the effect of aging on the capacity of Ia⁺ cells to stimulate autoreactive T cells in the syngeneic mixed lymphocyte reaction (SMLR) was investigated. Using young CD4⁺ T cells as responders, it was observed that unseparated whole spleen cells from aged mice had normal stimulatory activity comparable to that of young spleen cells. Interestingly, however, when purified splenic adherant cells (SAC) enriched for macrophages or splenic B cells were used as stimulators, aged SAC but not aged B cells were found to be defective in stimulating autoreactive T cells. This defect in aged SAC …

Phenotypic And Functional Properties Of B Lymphocytes From Aged Mice, Subbarao Bondada, Joan Morris, Richard J. Kryscio

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Phenotypic and functional properties of B lymphocytes from individual young and old mice of different inbred strains were studied. B lymphocyte subpopulation defined by the ratios of the densities of cell surface IgM and IgD were found to be altered with age. However, such alterations in B cell subsets were found only in 30-40% of the old mice. B cell mitogenic responses to anti-μ and anti-Lyb2 antibodies were decreased in a majority of DBA/2 mice. Proliferative responses to LPS and antiμ were reduced only in a minority of CBA/Ca mice but there was a very good correlation in the responsiveness …

Freshwater Fishes Of Shipstern Nature Reserve, Caspar F. A. Bijleveld

Physiology Faculty Research Publications

A survey of fishes in and around Shipstern Nature Reserve in Northern Belize, Central America, was carried out over a period of eight months, from February to September 1990. Its purpose was to establish a preliminary list of the fishes of Shipstern Nature Reserve, including descriptions, distribution and collecting data and additionally to describe some of the habitats in which the fishes were found. This study focuses on fishes considered to be freshwater species. These occurred for the greater part in brackish habitats. Nevertheless, a few fish considered as marine species were caught in the waters of the reserve and …

Caribbean Island Zoogeography: A New Approach Using Mitochondrial Dna To Study Neotropical Bats, Carleton J. Phillips, Dorothy E. Pumo, Hugh H. Genoways, Phillip E. Ray

University of Nebraska State Museum: Mammalogy Papers

Genetic analysis of animal mitochondrial DNA is a new and valuable addition to the battery of techniques available to zoogeographers. This paper describes characteristics of mitochondrial DNA (mtDNA) that make it applicable for the study of island zoogeography.

Some traditional zoogeographic questions are examined using mtDNA from the Neotropical fruit bat, Artibeus jamaicensis. The specific questions are: 1) To what extent are island populations isolated (that is, does interbreeding occur between the insular subspecies)? 2) Can a single founding female account for the mitochondrial genomes on specific islands in the Antilles? 3) Is there a correlation between the genomic …

Isolation And Immunological Characterization Of A Group Of New B Lymphomas From Cba Mice, Venkatachalam Udhayakumar, Peter H. Brodeur, M. S. Rajagopalan, Stephen Zimmer, Karen E. Pollok, Subbarao Bondada

Microbiology, Immunology, and Molecular Genetics Faculty Publications

We have isolated and characterized a new series of B lymphomas which occurred spontaneously in a group of CBA N mice that were transferred with spleen or lymph node cells from 24-month-old CBA Ca mice. Tumor cell lines from six CBA N mice that received spleen cells were rescued and designated as BKS-2, BKS-3, BKS-4, BKS-5, BKS-6, and BKS-7. Also, tumor cells from a recipient of lymph node cells were rescued and the resulting cell line was designated BKL. These tumor cells expressed membrane immunoglobulin (μ,κ), major histocompatibility complex Class I and Class II molecules, B220, Lyb8, Fc receptors, J11d, …

A Specific Defect In The Proliferative Capacity Of B Cells From Old Mice Stimulated With Autoreactive T Cells, Prakash S. Nagarkatti, Aruna Seth, Mitzi Nagarkatti, Natarajan Muthusamy, Barbara Rychlik, Subbarao Bondada

Microbiology, Immunology, and Molecular Genetics Faculty Publications

B lymphocytes from aged mice were found to be defective in their ability to proliferate in response to stimulation with an autoreactive T cell clone D1.4. The differentiative response leading to antibody secretion was also impaired in the auto D1.4 T cell-stimulated B cells from old mice in comparison to similarly stimulated B cells from young mice. The B cells from old mice were competent in activating the autoreactive T cells such that the T cells were induced to proliferate. The B cell defect appears to be restricted to a certain phase of B cell activation, since old mouse B …

Genetic Transformation In Salmonella Typhimurium, Siao Ping Tasi

Loma Linda University Electronic Theses, Dissertations & Projects

Plasmid transformation is an essential step in many gene cloning experiments. In Salmonella tynhimurium, mutants of rough strain have shown improved transformation frequency previously. In this work a stable restriction-minus (r^-) and modification-plus (m⁺) strain and its derivatives were constructed. Effect of restriction barrier on transformation of plasmid pBR322 was tested. Several factors which may affect transformation frequency have been systematically examined to determine the optimum conditions for transformation in S. tvphimurium. Efforts were made to simplify the procedure.

Using pBR322 plasmid as an experimental model, the r^- strain demonstrated 10⁵ to …

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

School of Medical Diagnostics & Translational Sciences Publications

We have isolated ribosomal RNA gene (rDNA) chromatin from Physarum polycephalum using a nucleolar isolation procedure that minimizes protein loss from chromatin and, subsequently, either agarose gel electrophoresis or metrizamide gradient centrifugation to purify this chromatin fraction (Amero, S. A., Ogle, R. C., Keating, J. L., Montoya, V. L., Murdoch, W. L., and Grainger, R. M. (1988) J. Biol. Chem. 263, 10725-10733). Metrizamide-purified rDNA chromatin obtained from nucleoli isolated according to the new procedure has a core histone/DNA ratio of 0.77:1. The major core histone classes comigrate electrophoretically with their nuclear counterparts on Triton-acid-urea/sodium dodecyl sulfate two-dimensional gels, although they …

Mitochondrial Dna Polymorphism In Three Antillean Island Populations Of The Fruit Bat, Artibeus Jamaicensis, Dorothy E. Pumo, Everett Z. Goldin, Beth Elliot, Carleton J. Phillips, Hugh H. Genoways

University of Nebraska State Museum: Mammalogy Papers

The Neotropical fruit bat, Artibeus jamaicensis, occurs throughout Latin America and on many islands in the Caribbean. Populations from Jamaica (in the Greater Antilles) to Barbados (in the Lesser Antilles) have been classified as a subspecies (A. j. jamaicensisi separate from that on the Lesser Antillean island of St. Vincent (A. j. schwartzi). Mitochondrial DNA (mtDNA) was isolated from 54 individuals collected on these islands, analyzed by digestion with restriction endonucleases, and the restriction sites were mapped. Three different mtDNA genotypes (16,000 ± 200 bp) were identified: J-1 (16 animals from Jamaica, one from St. Vincent, …

Properties Of Anti-Lyb-2-Mediated B-Cell Activation And The Relationship Between Lyb-2 Molecules And Receptors For B-Cell Stimulatory Factor-1 On Murine B Lymphocytes, Subbarao Bondada, Joan Morris, A. R. Baluyut

Microbiology, Immunology, and Molecular Genetics Faculty Publications

The murine B-cell differentiation antigen Lyb-2 has been shown to be involved in B-lymphocyte activation and has been postulated by some to be related to a receptor for B-cell stimulatory factor I (BSF-1) (H. Yakura et al., J. Immunol. 137, 1475, 1986). Here we have demonstrated that monoclonal antibody (mAB) to Lyb-2 resembles BSF-1 in its ability to activate small resting B cells and enhancement of surface Ia. Anti-Lyb-2 antibodies bound B cells with very high avidity and were able to induce mobilization of cytosolic-free calcium. Anti-Lyb-2 mAB differs from BSF-1 in that BSF-1 but not anti-Lyb-2 is able to …

Impaired Autoreactive T Cell-Induced T Cell-T Cell Interaction In Aged Mice, Venkatachalam Udhayakumar, Subbarao Bondada, Aruna Seth, Mitzi Nagarkatti, Prakash S. Nagarkatti

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Self-Ia-reactive (autoreactive) L3T4⁺ T cell clones have been shown earlier to stimulate the proliferation of syngeneic naive L3T4⁺ T cells and initiate a T cell-T cell (T-T) interaction leading to the generation of immunoregulatory circuits. Since aging has been shown to be associated with a decline of the immune responsiveness, age-related alterations in the T-T interaction was investigated in the present study. Using several I-E^d-specific autoreactive T cell clones isolated from 2- to 3-month-old (young) DBA/2 mice as stimulators, it was observed that L3T4⁺ T cells from 22- to 24-month-old (aged) DBA/2 mice, failed to …

Partial Characterization Of Lpss From Rhizobium Fredii Usda 205 Before And After Nod Gene Induction, Bradley Lawrence Reuhs

Masters Theses

The lipopolysaccharides (LPS) from Rhizobium fredii USDA 205 and its nod- mutant, HC 205, which has been cured of the 192-MDa symbiotic plasmid, were isolated by the phenol-water extraction method. The LPS from the wild-type strain which was grown in association with host-plant root extract or a flavone analog to a constituent of the root extract, which is required for the induction of at least a portion of the nodulation genes, was also isolated. The conditions required for the induction of the nod genes were determined by work with a nod- mutant of R. fredii USDA 201 which …

Karyotypic Analysis Of Five Rodents And A Marsupial From Belize, Central America, David W. Burton, John W. Bickham, Hugh H. Genoways, Timothy J. Mccarthy

University of Nebraska State Museum: Mammalogy Papers

Karyotypes and chromosomal banding patterns of six species of small mammals from Belize are presented and discussed in light of relevant data in the literature. Individuals of Heteromys desmarestianus had FN values of 72, which differs from previous reports. G-bands were obtained and compared to fluorescent bands (DAPI) and with the location of NOR sites. The karyotypes of Peromyscus mexicanus, Sigmodon hispidus, Oryzomys couesi, and Ototylomys phyllotis are identical to the karyotypes for these species described in the literature. Active NORs were located on four pairs of chromosomes in O. phyllotis and fluorescent bands obtained with the …

Early Events In B-Cell Activation: Anti-Lyb2, But Not Bsf-1, Induces A Phosphatidylinositol Response In Murine B Cells, Stephan A. Grupp, Judith A. K. Harmony, Arthur R. Baluyut, Subbarao Bondada

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Previous studies have indicated that the murine surface antigen Lyb2 is involved in an activation pathway that apparently does not involve the surface immunoglobulin receptor. As sIg has been shown to transduce its activation signal through the breakdown of phosphatidylinositol (PI), and since activation via Lyb2 does not involve sIg, it was of interest to determine if binding to Lyb2 generates a PI response. We have demonstrated that an allele-specific monoclonal antibody to Lyb2 (anti-Lyb2 mab), which has previously been shown to drive B cells into S, also activated PI metabolism in these cells. This activation occurred in a dose-dependent …

The Effects Of Strain Differences On The Binding Of Concavalian A To Mouse Sperm, Lisa A. Mckay

Honors Theses

The current study, lectins were used as genetic probes to determine the phenotype of the sperm from different strains of mice. Lectins are proteins of non-immune origin, which bind to specific carbohydrates (McCoy, 1986). Being that virtually all cell membranes contain carbohydrate components, lectins have been used to identify all types of cells. In this study, a biotinylated lectin Concavalian A (Con A) derived from Canavalia ensiformis, was used and binding was detected using an avidin-biotin peroxidase (ABP) complex technique. The object of the current research was to 1) determine if mouse strain differences have an effect on lectin binding, …

A Comparison Of The Acidic Extracellular Polysaccharides From Rhizobium Trifolii Mutants Affected In Trifolium Repens Root Hair Curling, Brian Andrew Hanley

Masters Theses

The compositions and sugar linkages of the acidic extracellular polysaccharide (EPS) from several Rhizobium trifolii mutants were compared. These mutants were derived from a wild type Rhizobium trifolii, ANU843, and are unable to cause normal root hair curling, Hac-, or nodulate clover, Nod-. Three of the mutants contain a Tn5 insertion in a 5.3 kb BglII fragment of the symbiotic plasmid (pSym). A fourth mutant is missing the pSym, which includes much of the 5.3 kb BglII fragment, both of these deletions result in Hac-Nod- phenotypes. When the 5.3 kb BglII fragment is cloned (pRT150) and reinserted into the …

Seasonal Responses Of Filtration Rates To Temperature, Oxygen Availability, And Particle Concentration Of The Freshwater Clam, Musculium Partumeium (Say), Albert J. Burky, Richard B. Benjamin, Denis G. Conover, Jennifer Detrick

Biology Faculty Publications

Seasonal filtration rates of a pond population of the freshwater pisidiid clam, Musculium par­tumeium (Say), were assessed by measuring the clearance of 2.02 µ.m latex beads from suspension and expressed in terms of FR = a(AFDW}b where FR = filtration rate (ml H20 · hr- 1 ) , AFDW = mg ash-free dry weight of a whole clam and "a" and "b" are constants. The a-values ( = FR of a 1-mg AFDW clam} vary seasonally with the highest rates corresponding to periods of maximum growth and reproduction in the spring and fall. Changes in "b" reflect the influence of …

Activation Of B Lymphocytes By Monovalent Anti-Lyb-2 Antibodies, Subbarao Bondada, D. E. Mosier

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Small resting B lymphocytes are activated when their cell surface immuno-globulin (Ig) receptors interact with specific antigen or anti-Ig antibodies. Such a receptor-ligand interaction leads to enlargement and proliferation of B lymphocytes. These activated B cells can subsequently mature into Ig-secreting cells in the presence of appropriate accessory cell signals (1-3). However, monovalent anti-Ig antibodies cannot induce B cell activation even though they can bind to the surface Ig (4-7). It has thus been concluded that membrane Ig has to be cross-linked to induce early steps in B cell activation (membrane depolarization; 8) as well as later steps (proliferation). T …

Interpopulation Variation In Calcareous And Proteinaceous Shell Components In The Stream Limpet, Ferrissia Rivularis, Carl M. Way, Daniel J. Hornbach, Albert J. Burky

Biology Faculty Publications

Natural populations of the North American stream limpet, F. rivularis, were studied in upstate New York [USA], in a set of localities whose waters have a 15-fold range of dissolved Ca (4.6-67.6 mg/liter) and also range from oligotrophy to eutrophy. Shell component analyses (CaCO3, total organic C and total N) are reported as component mass-fractions (mg/g or .mu.g/g dry weight) and as values for a standard limpet shell of 35 mm aperture length (AL). More than 2-fold differences occur between populations in all 3 components, with relatively little variation occurring within each population. Expressed per standard limpet, CaCO3 values for …

Seasonal Metabolism Of The Sphaeriid Clam, Musculium Partumeium (Say), From A Permanent And A Temporary Pond, Carl M. Way, Daniel J. Hornbach, Albert J. Burky

Biology Faculty Publications

Seasonal metabolic rates were examined for the sphaeriid clam, Musculium partumeium (Say) from a permanent and a temporary pond. For the fall-born generation from the permanent pond and for the single generation from the temporary pond, metabolic rates peak during periods of greatest growth and reproduction. Metabolic rates were consistently higher for the permanent pond population. These rates for Musculium partumeium extend the available information on the family Sphaeriidae to the genus Musculium.

Growth Of Pisidium Casertanum (Poli) In West Central Ohio, Albert J. Burky, Daniel J. Hornbach, Carl M. Way

Biology Faculty Publications

Clarke (1973) states that Pisidium casertanum (Poli) is perhaps the most common species of Pisidium and is truly cosmopolitan, occurring throughout Eurasia, Africa, Australia, and South, Central and North America. Populations of these clams are reported in habitats ranging from ephemeral ponds to benthic zones of deep lakes. Our study describes Manuscript received 29 May 1979 and in revised form 25 July 1980 (#79-34). 2 Present address: Department of Biology, University of Virginia, Charlottesville, VA 22901. 3 Present address: Department of Zoology, Miami University, Oxford, OH 45056. 41 aspects of the life-cycle of P. casertanum in a spring-fed stream with …

Role Of A Nonimmunoglobulin Cell Surface Determinant In The Activation Of B Lymphocytes By Thymus-Independent Antigens*, Subbarao Bondada, Donald E. Mosier, Aftab Ahmed, James J. Mond, Irwin Scher, William E. Paul

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Lyb 5 is a B-cell alloantigen which is expressed on 50-60% of B cells. It was defined originally on the basis of cytotoxicity. We have described a new reactivity within the anti-Lyb 5 serum on the basis of selective inhibition of antibody responses in vitro by this antiserum in the absence of complement. This inhibitory activity of anti-Lyb 5.1 serum appears to be due to recognition of antigenic determinants different from the prototype antigens detected in the cytotoxicity assay. Anti-Lyb 5 serum incorporated into spleen cell cultures selectively inhibits antibody responses to a class of thymus-independent antigens (TI-2) previously characterized …

Tritium (3h) Radiolabeling Of Protein A And Antibody To High Specific Activity: Application To Cell Surface Antigen Radioimmunoassays, Ronald L. Wilder, Catherine C. Yuen, Subbarao Bondada, Virgil L. Woods, Cornelius B. Alexander, Rose G. Mage

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Staphylococcal protein A and several different immunoglobulins have been radio-labeled to high specific activities (> 106 cpm/pg) by reductive methylation with tritiated (3H) sodium borohydride. The proteins retain excellent functional and antigenic properties. The utility of these reagents in a variety of assays for cell surface antigens is illustrated. The results indicate that this radiolabeling procedure may become the method of choice for many cell surface and solution immunoassays.

Buoyancy Changes As Related To Respiratory Behavior In An Amphibious Snail, Pomacea Urceus (Müller), From Venezuela, Kathleen A. Burky, Albert J. Burky

Biology Faculty Publications

The ampullariid Pomacea urceus uses its ctenidium and lung in respiration. The snails reach the surface with their siphon and ventilate their lung by withdrawing their head-foot in a pumping action. The lung gas also serves to change the buoyancy of the snails. After a ventilation a snail does not necessarily return to the same buoyancy level. They may achieve overall specific gravities of <1 to > 1 after a ventilation. They may remain submerged (s.g. > 1) or float (s.g.

Inactivation Of Brain Hexokinase By An Adenosine 5'-Triphosphate Analog, Subbarao Bondada, Umakant W. Kenkare

Microbiology, Immunology, and Molecular Genetics Faculty Publications

An analog of ATP, 6-mercapto-9+nribofuranosylpurine 5’-triphosphate (SH-TP), functions as a phosphoryl donor for the reaction catalyzed by hexokinase. Incubation of the enzyme with millimolar concentrations of this reagent led to its rapid inactivation. However, the corresponding monophosphate derivative of this reagent was much less effective. A plot of the initial rate of inactivation versus the concentration of SH-TP exhibited saturation, suggesting the formation of a reversible complex between the enzyme and SH-TP prior to the inactivation reaction. The dissociation constant of this enzyme-inhibitor complex was the same as the K, of this reagent with respect to hexokinase in the phosphoryl …

Reaction Of Brain Hexokinase With Tetranitromethane: Oxidation Of Essential Thiol Groups, Subbarao Bondada, Umakant W. Kenkare

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Treatment of bovine brain mitochondrial hexokinase with a fivefold molar excess of tetranitromethane (TNM) at pH 8.0 results in a complete loss of activity of the enzyme. Spectral measurements and amino acid analysis showed that only cysteinyl residues were modified by TNM under these conditions. The products of the reaction of TNM with the enzyme were cysteic acid and disulfides as found by amino acid analysis of the carboxymethylated hexokinase. Failure of 2-mercaptoethanol to reverse the effect of TNM suggested that the thiol group that is oxidized to cysteic acid is the one essential for enzyme activity. Substrates glucose and …

Molecular Weight Determinations Of Some Salmonella Potsdam Bacteriophage Dna's, Winston H. Richards

Loma Linda University Electronic Theses, Dissertations & Projects

Two group A phages, P9c/2 and P4/2, were studied. Their density, sedimentation velocity, and biochemical properties were compared. Their densities were determined by density gradient centrifugation in 38.3% cesium chloride. Sedimentation velocity was used to determine their molecular weights. To accomplish this, the DNA of P9c/2 and P4/2 was labeled with tritium and T₂ DNA, labeled with ³²P, was used as a standard. The DNA of P9c/2 and P4/2 was separately mixed with T₂ DNA and centrifuged in a 0-15% sucrose gradient. The molecular weights were calculated using the formula D₂/D₁= (M_{2 …}