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Effects of a Methylcholanthrene-Induced Lymphosarcoma on the Blood of DBA/1J Mice
This investigation was concerned with characterizing a tumor line induced and maintained in this laboratory. Various chemical assays, cell counts, and electron microscopy were the methods employed to characterize the blood of mice bearing the tumor at days 3, 6, 9, and 12 after injection of the 1.2 x 10^8 tumor cells.
Effects of a Methylcholanthrene-Induced Lymphosarcoma on Various Tissues of DBA/1J and Swiss White Mice
This investigation was concerned with characterizing effects of this tumor line on lipid metabolism in DBA/lJ mice and serum protein levels and cellular changes in DBA/lJ and Swiss white mice. Total lipids, lipid phosphorus, neutral lipids, and changes in fatty acids were determined in liver, spleen, skin, and tumor of DBA/lJ mice bearing the lymphosarcoma at various days after injection of tumor cells.
Biochemical Systematics of the Genus Sophora
Three unusual amino acids, y-amino-n-butyric acid, pipecolic acid, and 4-hydroxypipecolic acid, and an uncommon dipeptide, y-glutamyltyrosine, have been isolated and characterized from the seeds of members of the genus Sophora. Structural proof of these compounds was carried out by paper chromatography, thin-layer chromatography, column chromatography on amino acid analyzer, infrared, nuclear magnetic resonance, mass spectrometry, and C, H, N analysis. The presence and absence of these compounds was used as a criterion for the classification of 23 species of the genus Sophora. A phylogenetic classification which seems to follow the morphological taxonomy of this genus was carried out on the basis of seeds that contained pipecolic acid, those which did not contain pipecolic acid, and plants which contained both pipecolic acid and 4-hydroxypipecolic acids. Another chemical classification was also introduced based on the presence and absence of y-amino-n-butyric acid and y-glutamyltyrosine.
A Computer Assisted Micro-Dye Uptake Interferon Assay System
A new rapid computer assisted micro-titer plate interferon assay system was developed and characterized for use in high capacity clinical and research applications. The biological aspect of the assay was a modification of the assay methods of Finter, Armstrong and McManus. It was an application of spectrophotometric quantification of the reduction of viral cytopathic effect (CPE) as reflected by neutral red dye uptake by viable cells. A computer program was developed for the extrapolation of raw data to reference interferon units.
Postsynthetic Modifications of Glycolytic Enzymes of the Geriatric Immune System and in Fibroblasts from Premature Aging Diseases
During mitogen-induced transformation of human lymphocytes, phosphoglycerate kinase (PGK) exhibits new electrophoretic forms (pl=8.5-8.9). Electrophoresis and electrofocusing showed that the new forms are not due to expression of the autosomally linked isozyme found in semen (PGK-B; pl=9.7). The multiple electrophoretic forms are the result of protease modification of sex-linked PGK-A isozyme.When peripheral lymphocytes from young persons are stimulated in vitro with phytohemagglutinin, a selective increase in the levels of the glycolytic enzymes occurs concomitantly with blastogenesis. Human lymphocytes from a geriatric population were also subjected to mitogen stimulation. The initial levels of the enzymes were essentially identical in lymphocytes from young and old subjects as were mitogenfree cultured controls. However, during mitogen stimulation the cells from the old subjects failed to increase the glycolytic enzymes. This inability to activate glycolysis may be related to the decline in cell-mediated immunity which occurs with advancing age. Triosephosphate isomerase (TPI) has an increased thermolabile component in skin fibroblasts from patients with progeria (41.4 per cent)and Werner's syndrome (20.1 per cent) when compared with normal fibroblasts (0-3 per cent). The incorporation of various protease inhibitors failed to affect the percentages of heat-labile triosephosphate isomerases. The labile component appears to be identical to the deamidated form of the enzyme which accumulates in other aging cells. Isoelectric focusing demonstrated increased quantities of the deamidated TPI-A form in progeria and Werner's syndrome fibroblasts as compared to normal. The deamidated TPI-A was considerably more labile than the native TPI-B indicating the increased lability of triosephosphate isomerase in premature aging syndrome fibroblasts is due to an accumulation of the deamidated form of the enzyme. The levels of several proteases were found to be diminished in progeria fibroblast extracts as compared to normal. A deamidation mechanism of enzyme degradation plays a key role in the normal cellular catabolism of this ...
Posttranslational Modification of Proteins by ADP-ribosylation
This work presents the development of a highly sensitive and selective chemical assay for mono(ADP-ribose) residues covalently bound to proteins in vivo. An extensive review of the literature is presented in the introduction of this work. The physiological.functions of mono(ADP-ribosyl)transferase activities associated with certain bacterial toxins (e.g., diphtheria, cholera and pertussis toxins) are well established. However, the roles of endogenous vertebrate transferases are unknown. The elucidation of the roles of these cellular transferases will likely require identification of the physiologically relevant target proteins. Toward this end, it will also be important to identify the types of (ADP-ribose)-protein linkages present in vivo. ADP-ribosylation reactions catalyzed by the different bacterial and vertebrate transferases are specific for different amino acid acceptors in vitro. However, the vertebrate transferases that have been characterized thus far are NAD:arginine mono(ADP-ribosyl)transferases. The work presented here describes the development of a chemical assay for the detection of in vivo modified, ADP-ribosylated proteins containing N-glycosylic linkages to arginine. The assay was applied to the analysis of ADP-ribose residues in adult rat liver. The strategy employed for detection of protein-bound ADP-ribose residues eliminated potential artifacts arising from trapped nucleotides (or their degradation products), since the acid-insoluble material was completely dissolved in a strongly denaturing solution and freed of non-covalently bound nucleotides prior to chemical release from proteins. Thus, the studies presented here demonstrate the unambiguous detection and quantification of protein-bound ADP-ribose residues in adult rat liver. "Arginine-linked" mono(ADP-ribose) residues (31.8 pmol/mg protein) were present in vivo at a level almost 400-fold higher than poly(ADP-ribose). A minor fraction (23%) of the ADP—ribose residues detected were bound via a second more labile linkage with chemical properties very similar to those described previously for carboxlylate esterlinked ADP-ribose. After fractionation of rat liver proteins by gel filtration HPLC, the major peak of "arginine-linked" ADP-ribose residues ...
A New LC Column for the Separation and the Quantitation of Nucleotides
A new column, Dionex AS4A, (polystyrenedivinylbenzene matrix) used for the separation of ribonucleotides and deoxyribonucleotides for the first time, and previously used for ion analysis was found superior to conventional silica columns because it separates ribonucleotides and deoxyribonucleotides. Resolution of dGTP was not possible with the Dionex column and CTP and GDP often co-eluted. Using conventional silica columns, monophosphates separated from diphosphates and diphosphates from triphosphates. Using the new Dionex column resolves all three simultaneously. The Dionex column resolved nucleotides with sharper peaks than silica columns, and the longer its retention time the better was the resolution. This Dionex column is stable, with 80 runs possible without cleaning while resolving ribonucleotides and deoxyribonucleotides to the picomole level.
Regulation of Colony-Stimulating Factor-1 Biosynthesis
Recent studies suggest that synthesis of the Colony-stimulating factor (CSF) is a well regulated process. However, the molecular mechanisms of the signal transduction of the various inducers of CSF such as monokines and lymphokines are not well understood. Using Interleukin 1 (IL-1) stimulation of CSF-1 in the MIA PaCa-2 cell line as a model system, the involvement of G-protein has been studied. The IL-1 induction of CSF-1 synthesis can be inhibited by both Pertussis toxin and Cholera toxin, which are known to modify the Gᵢ and Gₛ proteins respectively, thus activating adenylate cyclase to release more cAMP. The toxin inactivation can be prevented by inhibitors of the ADP-ribosylation such as, benzamide and MBAMG. Addition of dibutyryl-cAMP inhibits the IL-1 induced CSF production. Both Theophylline and Forskolin which increase cAMP by inhibiting phosphodiesterase and stimulating adenylate cyclase respectively, also inhibit CSF-1 production. Results from these studies have shown that cAMP level inversely regulates the biosynthesis of CSF-1. Preincubation of MIA PaCa-2 cells with IL-1 and 5'- guanylylimidodiphosphate (GppNHp) prevents the inhibitory effect of pertussis toxin on CSF-1 production. These data are consistent with the hypothesis that IL-1 binds to its receptor and couples to Gᵢ∝ resulting in the inhibition of adenylate cyclase and reducing cAMP level. Lowering of the' cAMP level leads to the activation of CSF-1 gene expression. The activity of another inducer of CSF-1 production in this system, 12-0-tetradecanoylphorbol-13-acetate (TPA), can be abolished by 1- (5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), which is a specific inhibitor of protein kinase C. However, H-7 failed to inhibit IL-1 stimulated CSF-1 production. Other known activators of protein kinase C namely, Ca²⁺ and L-α-l-oleoyl-2-acetoyl-sn- 3-glycerol (OAG), also increase CSF production. On the other hand, Indomethacin which is known to inhibit prostaglandin E (PGE), stimulates CSF-1 production in MIA PaCa-2 cells. These data suggest that different mechanisms ...
Construction of a Cloning Vector Based upon a Rhizobium Plasmid Origin of Replication and its Application to Genetic Engineering of Rhizobium Strains
Rhizobia are Gram-negative, rod-shaped, soil bacteria with the ability to fix atmospheric nitrogen into ammonia as symbiont bacteroids within nodules of leguminous plant roots. Here, resident Rhizobium plasmids were studied as possible sources of components for the construction of a cloning vector for Rhizobium species.
Subcloning and Nucleotide Sequence of Two Positive Acting Regulatory Genes, xy1R and xy1S, from the Pseudomonas putida HS1 TOL Plasmid PDK1
TOL plasmids of Pseudomonas putida encode enzymes for the degradation of toluene and related aromatics. These genes are organized into two operons regulated by the Xy1R and Xy1S transcriptional activators. Previous analysis of the TOL pDK1 catechol-2,3-dioxygenase gene (xy1E) and a comparison of this gene to xy1E from the related TOL plasmid pWW0, revealed the existance of a substantial level of sequence homology (82%).
Nucleotide Sequence Determination, Subcloning, Expression and Characterization of the xy1LT Region of the Pseudomonas putida TOL Plasmid pDK1
The complete nucleotide sequence of the region encoding the DHCDH function of the pDK1 lower operon was determined. DNA analysis has shown the presence of two open reading frames, one gene consisting of 777 nucleotides encoding a polypeptide of 27.85 kDa and another gene of 303 nucleotides encoding a polypeptide of 11.13 kDa. The results of enzymatic expression studies suggest that DHCDH activity is associated only with xy1L. However although the addition of xy1T cell-free extracts to xy1L cell-free extracts does not produce an increase in DHCDH activity, subclones carrying both xy1L and xy1T exhibit 300- 400% more DHCDH activity than subclones carrying only xy1L.
Isolation and Characterization of the Operon Containing Aspartate Transcarbamoylase and Dihydroorotase from Pseudomonas aeruginosa
The Pseudomonas aeruginosa ATCase was cloned and sequenced to determine the correct size, subunit composition and architecture of this pivotal enzyme in pyrimidine biosynthesis. During the course of this work, it was determined that the ATCase of Pseudomonas was not 360,000 Da but rather present in a complex of 484,000 Da consisting of two different polypeptides (36,000 Da and 44,000 Da) with an architecture similar to that of E. coli ATCase, 2(C3):3(r2). However, there was no regulatory polypeptide found in the Pseudomonas ATCase.
Regulation, Evolution, and Properties of the ato Qperon and its Gene Products in Escherichia coli
The regulation of short chain fatty acid metabolism has been examined. Metabolism of acetoacetate, and short chain fatty acids such as butyrate and valerate, is predicated upon the expression of genes of the ato operon. Acetoacetate induces expression of a CoA transferase (encoded by the atoDA genes) and expression of a thiolase (encoded by the atoB gene). Metabolism of saturated short chain fatty acids requires the activities of the transferase and thiolase and enzymes of 6-oxidation as well. Spontaneous mutant strains were isolated that were either constitutive or that were inducible by valerate or butyrate instead of acetoacetate.
Cell-Free Recovery and Isotopic Identification of Cyanide Degrading Enzymes from Pseudomonas Fluorescens
Cell-free extracts from Pseudomonas fluorescens NCIMB 11764 catalyzed the degradation of cyanide into products that included C02, formic acid, formamide and ammonia. Cyanide-degrading activity was localized to cytosolic cell fractions and was observed at substrate concentrations as high as 100 mM. Two cyanide degrading activities were identified by: (i) the determination of reaction products stoichiometries, (ii) requirements for NADH and oxygen, and (iii) kinetic analysis. The first activity produced CO2 and NH3 as reaction products, was dependent on oxygen and NADH for activity, and displayed an apparent Km for cyanide of 1.2 mM. The second activity generated formic acid (and NH3) pfus formamide as reaction products, was oxygen independent, and had an apparent Km of 12 mM for cyanide. The first enzymatic activity was identified as cyanide oxygenase whereas the second activity consists of two enzymes, a cyanide nitrilase (dihydratase) and putative cyanide hydratase. In addition to these enzymes, cyanide-grown cells were also induced for formate dehydrogenase (FDH), providing a means of recycling NADH utilized by cyanide oxygenase.
DNA Typing of HLA-B by PCR with Primer Mixes Utilizing Sequence-Specific Primers
The aim of this study was to design a resolution typing system for the HLA-B gene. This technique involves a one-step PCR reaction utilizing genomic DNA and sequence-specific primers to determine the specificity of each allele and to produce a larger primer data base ideal for serological analysis. The application of this technique to serological analysis can improve serology detection which is currently hindered by antibody cross-reactivity and the unavailability of useful typing reagents.
Subcloning and Nucleotide Sequence of the xylO/PUWCMA Region from the Pseudomonas putida TOL Plasmid pDK1
The TOL plasmids of Pseudomonas putida encode enzymes required for the oxidation of toluene and other related aromatic compounds. These genes are organized into two operons, the xylUWCMABN operon (upper), and the xylXYZLTEGFJQKIH operon (lower). Here we report the nucleotide sequence of a 7107 bp segment of the TOL pDK1 plasmid encoding the region just upstream of the "upper" operon through the genes encoding xylUWCMA. Sequence analysis, comparison of base-usage patterns, codon-usage patterns, and intergenic distances between genes help support the idea that the "upper" and "lower" operons have evolved independently in different genetic backgrounds and have only more recently been brought together in TOL and related catabolic plasmids.
Cloning of Carbonic Anhydrase from Cotton (Gossypium hirsutum L.)
Carbonic anhydrase is a ubiquitous zinc-metalloenzyme that catalyzes the interconversion of carbon dioxide and carbonate and has been found to play a wide range of roles in animals, plants and bacteria. Cotton genomic and cDNA libraries were screened for the plastidial isoform of carbonic anhydrase. The nucleotide sequences of two 1.2 Kb partial cDNA clones were determined. These clones exhibit high homology to carbonic anhydrases from other dicot plants and possess all the expected peptide motifs. For example, serine and threonine rich chloroplastic targeting peptide and conserved zinc binding residues are both present. These clones were utilized to isolate two carbonic anhydrase genes that were shown to encode different isoforms by PCR and RFLP analysis.
Mutagenized HLA DNA Constructs: Tools for Validating Molecular HLA Typing Methodologies
This study describes the development and validation of mutagenized cloned DNA constructs, which correspond to the polymorphic regions of the class II region of the HLA complex. The constructs were used to verify the allelic specificity of primers and probes in polymerase chain reaction (PCR)-based HLA typing assays such as Sequence Specific Primers (SSP) and Sequence Specific Oligonucleotide Probes (SSOP). The constructs consisted of the entire polymorphic region of exon 2 of class II HLA allele sequences that included primer annealing sites or probe hybridization sites. An HLA allele sequence was inserted into a plasmid, cloned, then mutagenized to match a specific HLA allele, and finally, the correct clone was verified by bidirectional sequencing of the insert. Thus, the construct created a cloned reference DNA sample for any specific allele, and can be used to validate the accuracy of various molecular methodologies.
Comparative Mitochondrial DNA Sequence Diversity in Isolated and Open Populations of Southern Flying Squirrels
Three populations of Southern flying squirrels were studied in the Ouachita Mountains of Arkansas to assess the impact of population subdivision-due to island formation--on the population genetics of Glaucomys volans. One island, one mainland, and one open population were investigated. A 367 nucleotide hypervariable region of mitochondrial DNA was sequenced in individuals from each population. Individuals and populations were compared to assess relatedness. Higher sequence diversity was detected in the open and island populations. One island individual shared characters with both the island and mainland populations. Results support the hypothesis that the mainland population may have reduced gene flow. Also, the island population may have been originally founded by at least two maternal lineages.
Cassette Systems for Creating Intergeneric Hybrid ATCases
Cassette systems for creating intergeneric hybrid ATCases were constructed. An MluI restriction enzyme site was introduced at the carbamoylphosphate binding site within the pyrB genes of both Pseudomonas putida and Escherichia coli. Two hybrids, E. coli pyrB polar domain fused with P. putida pyrB equatorial domain and P. putida pyrB polar domain fused with E. coli pyrB equatorial domain, are possible. The intergeneric E. coli-P. putida hybrid pyrB gene was constructed and found to encode an active ATCase which complemented an E. coli Pyr- strain. These hybrids are useful for kinetic and expression studies of ATCase in E. coli.
Structural Analysis of the TOL pDK1 xylGFJQK Region and Partial Characterization of the xylF and xylG Gene Products
TOL plasmids encode enzymes responsible for utilization of toluene and related aromatic compounds by Pseudomonas putida, ultimately converting them to central metabolic intermediates. The nucleotide sequence for the 5.6 kb xylGFJQK region of the pDK1 TOL meta operon was determined. DNA sequence analysis revealed the presence of five open reading frames corresponding to xylG (1458 bp), xylF (846 bp), xylJ (783 bp), xylQ (936 bp) and xylK (1047 bp), encoding predicted protein products of 51.6, 31.3, 27.8, 32.8, and 36.6 kDa in size, respectively. The average G+C content of the xylLTEGFJQK region was 65.7%, somewhat higher than the 58.9% seen in the immediately upstream xylXYZ region and substantially more than the 50% G+C content reported for the upper TOL operon of this plasmid. Homology comparisons were made with genes and proteins of related catabolic plasmids. The dmpCDEFG and pWWO xylGFJQK regions exhibit consistently high levels of nucleotide and amino acid homology to pDK1 xylGFJQK throughout the entire region. In contrast, although the nucleotide sequence homology of the Acinetobacter atdCDE region to xylGFJ is high, the homology of atdFG to xylQK is markedly less. Such radical changes in homology between corresponding regions of different operons, combined with variable base and codon usage patterns within and between operons, provides additional support for the idea that the upper and lower operons encoding enzymes of aromatic pathways have evolved independently of one another and that these operons have continued to exchange genetic material with homologous expression units through a series of recombination events. Recombinant plasmids were constructed for individual expression of each of the xylGFJQK genes. HMSD (XylG) and HMSH (XylF) were partially purified and characterized with respect to substrate specificity and kinetic mechanism. Evidence was obtained suggesting that the HMSD reaction occurs via a steady state ordered mechanism or a random mechanism where ...
Mutation Rate Analysis of the Human Mitochondrial D-loop and its Implications for Forensic Identity Testing
To further facilitate mitochondrial DNA (mtDNA) sequence analysis for human identity testing, a better understanding of its mutation rate is needed. Prior to the middle 1990's the mutation rate applied to a forensic or evolutionary analysis was determined by phylogenetic means, This method involved calculating genetic distances as determined by amino acid or DNA sequence variability within or between species. The mutation rate as determined by this method ranged from 0.025-0.26 nucleotide substitutions/ site/ myr (million years). With the recent advent of mtDNA analysis as a tool in human identity testing an increased number of observations have recently come to light calling into question the mutation rate derived from the phylogenetic method. The mutation rate as observed from forensic analysis appears to be much higher than that calculated phylogenetically. This is an area that needs to be resolved in human identity testing. Mutations that occur within a maternal lineage can lead to a possible false exclusion of an individual as belonging to that lineage. A greater understanding of the actual rate of mutation within a given maternal lineage can assist in determining criteria for including or excluding individuals as belonging to that lineage. The method used to assess the mutation rate in this study was to compare mtDNA sequences derived from the HVI and HVII regions of the D-loop from several different maternal lineages. The sequence information was derived from five unrelated families consisting of thirty-five individuals. One intergenerational mutational event was found. This derives to approximately 1.9 nucleotide substitutions/ site/ myr. This mutation rate was very consistent with several other similar studies. This increased mutation rate needs to be considered by forensic testing laboratories performing mtDNA sequence analysis prior to formulating any conclusive results.
Isolation and analysis of cotton genomic clones encompassing a fatty acid desaturase (FAD2) gene
Polyunsaturated fatty acids are major structural components of plant chloroplast and endoplasmic reticulum membranes. Two fatty acid desaturases (designated FAD2 and FAD3) desaturate 75% of the fatty acids in the endoplasmic reticulum. The w -6 fatty acid desaturase (FAD2) may be responsible for cold acclimation response, since polyunsaturated phospholipids are important in helping maintain plant viability at lowered temperatures. To study regulation of FAD2 gene expression in cotton, a FAD2 gene was isolated from two genomic libraries using an Arabidopsis FAD2 hybridization probe and a cotton FAD2 5¢ -flanking region gene-specific probe, respectively. A cotton FAD2 gene was found to be in two overlapping genomic clones by physical mapping and DNA sequencing. The cloned DNA fragments are identical in size to cotton FAD2 genomic DNA fragments shown by genomic blot hybridization. The cotton FAD2 coding region has 1,155 bp with no introns and would encode a putative polypeptide of 384 amino acids. The cotton FAD2 enzyme has a high identity of 75% with other plant FAD2 enzymes. The enzyme has three histidine-rich motifs that are conserved in all plant membrane desaturases. These histidine boxes may be the iron-binding domains for reduction of oxygen during desaturation. To confirm that this FAD2 enzyme is functional, a plasmid construct containing the cotton FAD2 coding region was transformed into Saccharomyces cerevisiae. The transformed yeast cells were able to catalyze the conversion of oleic acid (C18:1) into linoleic acid (C18:2). The FAD2 gene contains an intron of 2,967 bp in its 5¢ -flanking region, 11 bp upstream from the initiation codon. The intron could be essential for transcriptional regulation of FAD2 gene expression. Several putative promoter elements occur in the 5¢ -flanking region of this gene. A potential TATA basal promoter element occurs at 41 bp upstream from the cap site. Two presumptive helix-loop-helix (bHLH) ...
A Novel Mechanism for Site-Directed Mutagenesis of Large Catabolic Plasmids Using Natural Transformation
Natural transformation is the process by which cells take up DNA from the surrounding medium under physiological conditions, altering the genotype in a heritable fashion. This occurs without chemical or physical treatment of the cells. Certain Acinetobacter strains exhibit a strong tendency to incorporate homologous DNA into their chromosomes by natural transformation. Transformation in Acinetobacter exhibits several unique properties that indicate this system's superiority as a model for transformation studies or studies which benefit from the use of transformation as an experimental method of gene manipulation. Pseudomonas putida is the natural host of TOL plasmids, ranging between 50 kbp and 300 kbp in size and encoding genes for the catabolism of toluene, meta-toluate, and xylene. These very large, single-copy plasmids are difficult to isolate, manipulate, or modify in vitro. In this study, the TOL plasmid pDKR1 was introduced into Acinetobacter calcoaceticus strains and genetically engineered utilizing natural transformation as part of the process. Following engineering by transformation, the recombinant DNA molecule was returned to the native genetic background of the original host P. putida strain. Specific parameters for the successful manipulation of large plasmids by natural transformation in Acinetobacter were identified and are outlined. The effects of growth phase, total transforming DNA concentration, transforming DNA conformation, and gene dosage on transformation efficiency are presented. Addition of Acinetobacter plasmid DNA sequences to the manipulated constructs did not have an effect on transformation rates. Results suggest that a broadly applicable and efficient method to carry out site-directed genetic manipulations of large plasmids has been identified. The ability to easily reintroduce the recombinant DNA molecules back into the original host organism was maintained.
A Possible Role of Ascorbate in Boron Deficient Radish (Raphanus sativa L. cv. Cherry Belle)
The most apparent symptom of boron deficiency in higher plants is a cessation of growth. Deficiency causes a reduction in ascorbate concentration and the absorption of nutrient ions. Addition of ascorbate temporarily relieves deficiency symptoms. In boron sufficient plants the addition of ascorbate to media causes an increased uptake of nutrients. In an attempt to discover if ascorbate addition to deficient plants causes increased ion uptake, radish plants were grown hydroponically in four different strengths of boron solution. A colorimetric assay for phosphorus was performed both before and after supplementation. Results, however, were inconclusive.
Purification of Aspartate Transcarbamoylase from Moraxella (Branhamella) catarrhalis
The enzyme, aspartate transcarbamoylase (ATCase) from Moraxella (Branhamella) catarrhalis, has been purified. The holoenzyme has a molecular mass of approximately 510kDa, harbors predominantly positive charges and is hydrophobic in nature. The holoenzyme possesses two subunits, a smaller one of 40 kDa and a larger one of 45 kDa. A third polypeptide has been found to contribute to the overall enzymatic activity, having an approximate mass of 55 kDa. The ATCase purification included the generation of cell-free extract, streptomycin sulfate cut, 60 °C heat step, ammonium sulfate cut, dialysis and ion, gel-filtration and hydrophobic interaction chromatography. The enzyme's performance throughout purification steps was analyzed on activity and SDS-PAGE gradient gels. Its enzymatic, specific activities, yield and fold purification, were also determined.
The regulatory roles of PyrR and Crc in pyrimidine metabolism in Pseudomonas aeruginosa
The regulatory gene for pyrimidine biosynthesis has been identified and designated pyrR. The pyrR gene product was purified to homogeneity and found to have a monomeric molecular mass of 19 kDa. The pyrR gene is located directly upstream of the pyrBC' genes in the pyrRBC' operon. Insertional mutagenesis of pyrR led to a 50- 70% decrease in the expression of pyrBC', pyrD, pyrE and pyrF while pyrC was unchanged. This suggests that PyrR is a positive activator. The upstream regions of the pyrD, pyrE and pyrF genes contain a common conserved 9 bp sequence to which the purified PyrR protein is proposed to bind. This consensus sequence is absent in pyrC but is present, as an imperfect inverted repeat separated by 11 bp, within the promoter region of pyrR. Gel retardation assays using upstream DNA fragments proved PyrR binds to the DNA of pyrD, pyrE, pyrF as well as pyrR. This suggests that expression of pyrR is autoregulated; moreover, a stable stem-loop structure was determined in the pyrR promoter region such that the SD sequence and the translation start codon for pyrR is sequestered. β-galactosidase activity from transcriptional pyrR::lacZ fusion assays, showed a two-fold in increase when expressed in a pyrR- strain compared to the isogenic pyrR+ strain. Thus, pyrR is negatively regulated while the other pyr genes (except pyrC) are positively activated by PyrR. That no regulation was seen for pyrC is in keeping with the recent discovery of a second functional pyrC that is not regulated in P. aeruginosa. Gel filtration chromatography shows the PyrR protein exists in a dynamic equilibrium, and it is proposed that PyrR functions as a monomer in activating pyrD, pyrE and pyrF and as a dimeric repressor for pyrR by binding to the inverted repeat. A related study discovered that the catabolite repression ...
Characterization of Moraxella bovis Aspartate Transcarbamoylase
Aspartate transcarbamoylase (ATCase) catalyzes the first committed step in the pyrimidine biosynthetic pathway. Bacterial ATCases have been divided into three classes, class A, B, and C, based on their molecular weight, holoenzyme architecture, and enzyme kinetics. Moraxella bovis is a fastidious organism, the etiologic agent of infectious bovine keratoconjunctivitis (IBK). The M. bovis ATCase was purified and characterized for the first time. It is a class A enzyme with a molecular mass of 480 to 520 kDa. It has a pH optimum of 9.5 and is stable at high temperatures. The ATCase holoenzyme is inhibited by CTP > ATP > UTP. The Km for aspartate is 1.8 mM and the Vmax 1.04 µmol per min, where the Km for carbamoylphosphate is 1.05 mM and the Vmax 1.74 µmol per min.
Characterization of cDNA and Genomic Clones for a Palmitoyl-acyl Carrier Protein Thioesterase and an Osmotin-Like PR5 Protein in Gossypium Hirsutum.
Putative cotton cDNA clones and cognate genomic clones for a palmitoyl-acyl carrier protein (ACP) thioesterase (PATE) and an osmotin-like pathogenesis-related 5 (PR5) protein have been isolated and characterized. PATE is a class B fatty acid thioesterase with specificity for saturated long-chain fatty acids such as palmitate, and is implicated as a key enzyme to be targeted for regulation of fatty acid synthesis in order to alter cotton seed oil profiles. A nearly full-length 1.7-kb cDNA clone was isolated using a hybridization probe derived from an Arabidopsis PATE cDNA clone designated TE 3-2. A 17-kb genomic segment encompassing the PATE gene was also isolated, which has six exons and five introns with high sequence identity with other FatB cDNA/gene sequences. The deduced PATE preprotein amino acid sequence of 413 residues has putative signal sequences for targeting to the chloroplast stroma. PR5 proteins called osmotins are made in response to fungal pathogen stress or osmotic stress (water deprivation or salt exposure). Osmotins may actually form pores in fungal membranes, leading to osmotic rupture and destruction of the fungal cells. A cotton osmotin-like PR5 cDNA insert of 1,052 base-pairs was isolated and shown to encode a preprotein of 242 amino acids and is predicted to be secreted to the extracellular matrix as a neutral isoform. The deduced amino acid sequence has 16 cysteine residues that are highly conserved in osmotin-like proteins and are important in stabilizing the three-dimensional structure seen in thaumatin, zeamatin, and PR5-d. The intronless cognate cotton genomic clone has two putative ethylene response elements (GCC boxes) found in other PR5 gene promoter regions, as well as several tentative promoter/enhancer elements possibly involved in spatial/temporal gene expression.
Dna Profiling of Captive Roseate Spoonbill (Ajaia Ajaja) Populations As a Mechanism of Determining Lineage in Colonial Nesting Birds.
Roseate spoonbills are colonial nesting birds with breeding grounds extending from the United States Gulf coast to the pampas of Argentina. The U.S. population suffered a severe bottleneck from 1890 to 1920. The population's recovery was slow and partially credited to migrations from Mexican rookeries, but a gene pool reduction would be expected. Five polymorphic Spoonbill autosomal short tandem repeat (STR) loci [three (GAT)n, one (AAAG)n and one (GT)n] and one Z/W-linked microsatellite exhibiting sex-specific dimorphism were isolated and characterized. The Z/W-linked STR locus accurately confirmed the sex of each bird. Allelic profiles for 51 spoonbills obtained from Dallas (Texas), Fort Worth (Texas) and Sedgwick County (Kansas) zoos revealed a non-continuous distribution of allele frequencies, consistent with the effects of a population bottleneck. Allelic frequencies also differed significantly between the isolated zoo populations. Although extra-pair copulations were suspected and difficult to document, zoos commonly used observational studies of mating pairs to determine familial relationships among adults and offspring. STR parentage analysis of recorded family relationships excluded one or both parents in 10/25 cases studied and it was further possible to identify alternative likely parents in each case. Mistaken familial relationships quickly lead to the loss of genetic variability in captive populations. Here, a decreased heterozygosity (HO) in 2nd generation captive-bred birds was observed at 3 out of 4 loci evaluated. Although these results could not be statistically validated because of the small number of individuals available for study (15 wild birds with no offspring vs. eight 2nd generation captive birds), they are considered biologically important, as decreased HO is an indicator of inbreeding and this apparent decrease occurred within two generations of removal from the wild. Collectively, the evidence obtained from this study suggests that captive spoonbill populations are experiencing rapid loss of diversity from an already depleted wild gene ...
Molecular cloning and analysis of the genes for cotton palmitoyl-acyl carrier protein thioesterase (PATE) and Δ-12 fatty acid desaturase (FAD2-3) and construction of sense and anti-sense PATE plasmid vectors for altering oilseed composition of transgenic cotton plants.
A cotton PATE cDNA clone has a 1.7-kb insert with an coding region for 410 amino acids, lacking codons for the three N-terminal amino acids. The predicted amino acid sequence of the PATE preprotein has a characteristic stromal-targeting domain and a 63% identity to the Arabidopsis FatB1 thioesterase sequence. A cotton genomic clone containing a 17.4-kb DNA segment was found to encompass a palmitoyl-ACP thioesterase (FatB1) gene. The gene spans 3.6 kb with six exons and five introns. The six exons are identical in nucleotide sequence to the open reading frame of the corresponding cDNA, and would encode a preprotein of 413 amino acids. The preprotein is identified as a FatB thioesterase from its deduced amino acid sequence similarity to those of other FatB thioesterase preproteins. A 5'-flanking region of 914 bp was sequenced, with the potential promoter/enhancer elements including basic helix-loop-helix elements (E box). Alkaline blot hybridization of cotton genomic DNA suggests the presence at least two FatB1 thioesterase genes in cotton. Four plasmid constructs for both constitutive and seed-specific anti-sense RNA suppression and gene-transgene co- suppression of PATE gene expression were successfully generated. Two overlapping cotton genomic clones were found to encompass a Δ-12 fatty acid desaturase (FAD2-3) gene. The continuous FAD2-3 coding region is 1,155 bp and would encode a protein of 384 amino acids. The FAD2-3 gene has one large intron of 2,967 bp entirely within its 5'-untranslated region. Several potential promoter/enhancer elements, including several light responsive motifs occur in the 5'-flanking region. Yeast cells transformed with a plasmid construct containing the cotton FAD2-3 coding region accumulate an appreciable amount of linoleic acid (18:2), not normally present in wild-type yeast cells, indicating that the gene encodes a functional FAD2 enzyme.
Construction of a Pseudomonas aeruginosa Dihydroorotase Mutant and the Discovery of a Novel Link between Pyrimidine Biosynthetic Intermediates and the Ability to Produce Virulence Factors
The ability to synthesize pyrimidine nucleotides is essential for most organisms. Pyrimidines are required for RNA and DNA synthesis, as well as cell wall synthesis and the metabolism of certain carbohydrates. Recent findings, however, indicate that the pyrimidine biosynthetic pathway and its intermediates maybe more important for bacterial metabolism than originally thought. Maksimova et al., 1994, reported that a P. putida M, pyrimidine auxotroph in the third step of the pathway, dihydroorotase (DHOase), failed to produce the siderophore pyoverdin. We created a PAO1 DHOase pyrimidine auxotroph to determine if this was also true for P. aeruginosa. Creation of this mutant was a two-step process, as P. aeruginosa has two pyrC genes (pyrC and pyrC2), both of which encode active DHOase enzymes. The pyrC gene was inactivated by gene replacement with a truncated form of the gene. Next, the pyrC2 gene was insertionally inactivated with the aacC1 gentamicin resistance gene, isolated from pCGMW. The resulting pyrimidine auxotroph produced significantly less pyoverdin than did the wild type. In addition, the mutant produced 40% less of the phenazine antibiotic, pyocyanin, than did the wild type. As both of these compounds have been reported to be vital to the virulence response of P. aeruginosa, we decided to test the ability of the DHOase mutant strain to produce other virulence factors as well. Here we report that a block in the conversion of carbamoyl aspartate (CAA) to dihydroorotate significantly impairs the ability of P. aeruginosa to affect virulence. We believe that the accumulation of CAA in the cell is the root cause of this observed defect. This research demonstrates a potential role for pyrimidine intermediates in the virulence response of P. aeruginosa and may lead to novel targets for chemotherapy against P. aeruginosa infections.
Analysis of a Cotton Gene Cluster for the Antifungal Protein Osmotin
Three overlapping genomic clones covering 29.0 kilobases of cotton DNA were found to encompass a cluster of two presumptive osmotin genes (OSMI and OSMII) and two osmotin pseudogenes (OSMIII and OSMIV). A segment of 16,007 basepairs of genomic DNA was sequenced from the overlapping genomic clones (GenBank Accessions AY303690 and AF304007). The two cotton osmotin genes were found to have open reading frames of 729 basepairs without any introns, and would encode presumptive osmotin preproteins of 242 amino acids. The open reading frames of the genes are identical in sequence to two corresponding cDNA clones (GenBank Accessions AF192271 and AY301283). The two cDNA inserts are almost full-length, since one lacks codons for the four N-terminal amino acids, and the other cDNA insert lacks the coding region for the 34 N-terminal amino acids. The cotton osmotin preproteins can be identified as PR5 proteins from their similarities to the deduced amino acid sequences of other plant osmotin PR5 preproteins. The preproteins would have N-terminal signal sequences of 24 amino acids, and the mature 24 kilodalton isoforms would likely be targeted for extracellular secretion. Prospective promoter elements, including two ethylene response elements, implicated as being positive regulatory elements in the expression of a number of PR-proteins, occur in the 5'-flanking regions. The mature osmotin proteins accumulate in cotton plants treated with the inducers ethephon and hydrogen peroxide. Thus, the two cotton osmotin genes encode osmotin proteins. The coding regions of the two genes have been expressed and isolated as fusion polypeptides in a bacterial expression system. Binary constructs containing the open reading frames of the two osmotin genes under the control of the 35S CaMV promoter have been generated for eventual production of transgenic Arabidopsis and cotton plants for potential constitutive expression of the osmotin proteins for increased resistance against fungal pathogens.
Influence of Cholesterol Import on Aspergillus fumigatus Growth and Antifungal Suscepibility
Invasive pulmonary aspergillosis is a life-threatening fungal infection commonly observed in immunocompromised patients and has a mortality rate approaching 100% once the disease is disseminated. Aspergillus fumigatus is the most common pathogen. Early diagnosis improves the prognosis but is very difficult since most signs and symptoms are nonspecific. Antifungal therapy, usually based on sterol biosynthesis inhibitors, is also of limited efficacy. In my attempts to discover a diagnostic sterol marker for aspergillosis, I observed that A. fumigatus incorporates large amounts of cholesterol from serum-containing medium. This observation suggested the hypothesis that exogenous cholesterol from the host can be imported by A. fumigatus and used as a substitute for ergosterol in the cell membrane. This proposed mechanism would reduce the efficacy of antifungal drugs that act as sterol biosynthesis inhibitors. Experiments to test this hypothesis were designed to determine the effects of serum-free and serum-containing medium on growth of A. fumigatus in the presence and absence of azole antifungal agents. The results showed a marked increase in growth in the presence of human serum. Cultures in media containing cholesterol but no serum also showed enhanced growth, a result indicating that a non-cholesterol component of serum is not primarily responsible for the increased growth. However, sterol analysis of A. fumigatus cultured in the absence of inhibitors showed little or no change in ergosterol levels. This result suggested that the imported cholesterol was not being used as membrane sterol. However, in parallel experiments using Itraconazole™, an antifungal agent that attenuates sterol biosynthesis by inhibiting the sterol 14a-demethylase (ERG11), ergosterol levels decreased with increasing doses of inhibitor. Moreover, serum-containing medium partially rescued A. fumigatus from the effects of Itraconazole™, and a similar rescue effect was observed with serum-free media containing cholesterol. From the preceding results, it can be concluded that human serum enhances A. ...
Pyrimidine Genes in Pseudomonas Species
This thesis is a comparative study of gene arrangements in Pseudomonas species, and is organized into three major sections. The first section compares gene arrangements for different pathways in Pseudomonas aeruginosa PAO1 to determine if the gene arrangements are similar to previous studies. It also serves as a reference for pyrimidine gene arrangements in P. aeruginosa. The second part compares the physical, and genetic maps of P. aeruginosa PAO1 with the genome sequence. The final section compares pyrimidine gene arrangements in three species of Pseudomonas. Pyrimidine biosynthesis and salvage genes will be aligned for P. aeruginosa PAO1, P. putida KT2440, and P. syringae DC3000. The whole study will gives insight into gene patterns in Pseudomonas, with a focus on pyrimidine genes.
Structure-Function Studies on Aspartate Transcarbamoylase and Regulation of Pyrimidine Biosynthesis by a Positive Activator Protein, PyrR in Pseudomonas putida
The regulation of pyrimidine biosynthesis was studied in Pseudomonas putida. The biosynthetic and salvage pathways provide pyrimidine nucleotides for RNA, DNA, cell membrane and cell wall biosynthesis. Pyrimidine metabolism is intensely studied because many of its enzymes are targets for chemotheraphy. Four aspects of pyrimidine regulation are described in this dissertation. Chapter I compares the salvage pathways of Escherichia coli and P. putida. Surprisingly, P. putida lacks several salvage enzymes including nucleoside kinases, uridine phosphorylase and cytidine deaminase. Without a functional nucleoside kinase, it was impossible to feed exogenous uridine to P. putida. To obviate this problem, uridine kinase was transferred to P. putida from E. coli and shown to function in this heterologous host. Chapter II details the enzymology of Pseudomonas aspartate transcarbamoylase (ATCase), its allosteric regulation and how it is assembled. The E. coli ATCase is a dodecamer of two different polypeptides, encoded by pyrBI. Six regulatory (PyrI) and six catalytic (PyrB) polypeptides assemble from two preformed trimers (B3) and three preformed regulatory dimers (I2) in the conserved 2B3:3I2 molecular structure. The Pseudomonas ATCase also assembles from two different polypeptides encoded by pyrBC'. However, a PyrB polypeptide combines with a PyrC. polypeptide to form a PyrB:PyrC. protomer; six of these assemble into a dodecamer of structure 2B3:3C'2. pyrC' encodes an inactive dihydroorotase with pyrB and pyrC' overlapping by 4 bp. Chapter III explores how catabolite repression affects pyrimidine metabolism. The global catabolite repression control protein, Crc, has been shown to affect pyrimidine metabolism in a number of ways. This includes orotate transport for use as pyrimidine, carbon and nitrogen sources. Orotate is important because it interacts with PyrR in repressing the pyr genes. Chapter IV describes PyrR, the positive activator of the pyrimidine pathway. As with other positive activator proteins, when pyrimidine nucleotides are depleted, PyrR binds to ...
Bacterial Cyanide Assimilation: Pterin Cofactor and Enzymatic Requirements for Substrate Oxidation
Utilization of cyanide as the sole nitrogen source by Pseudomonas fluorescens NCIMB 11764 (Pf11764) occurs via oxidative conversion to carbon dioxide and ammonia, the latter satisfying the nitrogen requirement. Substrate attack is initiated oxygenolytically by an enzyme referred to as cyanide oxygenase (CNO), which exhibits properties of a pterin-dependent hydroxylase. The pterin requirement for Pf11764 CNO was satisfied by supplying either the fully (tetrahydro) or partially (dihydro) reduced forms of various pterin compounds at catalytic concentrations (0.5 µM). These compounds included, for example, biopterin, monapterin and neopterin, all of which were also identified in cell extracts. A related CNO-mediated mechanism of cyanide utilization was identified in cyanide-degrading P. putida BCN3. This conclusion was based on (i) the recovery of CO2 and NH3 as enzymatic reaction products, (ii) the dependency of substrate conversion on both O2 and NADH, and (iiii) utilization of cyanide, O2 and NADH in a 1:1:1 reaction stoichiometry. In contrast to findings reported for Pf11764, it was not possible to demonstrate a need for exogenously added pterin as a cofactor for the PpBCN3 enzyme system. However, results which showed that cells of PpBCN3 contained approximately seven times the amount of pterin as Pf11764 (of which a significant portion was protein-bound) were interpreted as indicating that sufficient bound CNO-cofactor exists, thus eliminating any need for a supplemental source.
Cyanide Assimilation in Pseudomonas Fluorescens: Characterization of Cyanide Oxygenase as a Pterin-Dependent Multicomponent Enzyme Complex
Cyanide utilization in Pseudomonas fluorescens NCIMB 11764 occurs via oxidative conversion to carbon dioxide and ammonia, the latter satisfying the nitrogen requirement. Substrate attack is initiated by an enzyme referred to as cyanide oxygenase (CNO), previously shown to require components in both high (H) (>30 kDa) and low (L) (<10 kDa) molecular weight cell fractions. In this study, tetrahydrobiopterin (H4biopterin) was identified as a cofactor in fraction L, thus making CNO appear as a pterin- dependent hydroxylase. CNO was purified 150-fold (specific activity 0.9 U/mg) and quantitatively converted cyanide to formate and ammonia as reaction products. When coupled with formate dehydrogenase, the complete enzymatic system for cyanide oxidation to carbon dioxide and ammonia was reconstituted. CNO was found to be an aggregate of known enzymes that included NADH oxidase (Nox), NADH peroxidase (Npx), cyanide dihydratase (CynD) and carbonic anhydrase (CA). A complex multi-step reaction mechanism is proposed in which Nox generates hydrogen peroxide which in turn is utilized by Npx to catalyze the oxygenation of cyanide to formamide accompanied by the consumption of one and two molar equivalents of oxygen and NADH, respectively. The further hydrolysis of formamide to ammonia and formate is thought to be mediated by CynD. The role of H4biopterin and of the enzyme CA in the proposed process remains unclear, but the involvement of each in reactive oxygen and radical chemistry is consistent with the proposed formation of such species in the catalytic process. H4biopterin may additionally serve as a protein stabilizing agent along with a protein co-purifying with CynD identified as elongation factor Tu, a known chaperone. At least two of the CNO components (Nox and CynD) are complex oligomeric proteins whose apparent association with Npx and CA appears to be favored in bacterial cells induced with cyanide allowing their purification in toto as a ...
Callus Development and Organogenesis in Cultured Explants of Cowpea (Vigna unguiculata (L.) Walp
Cowpea, Vigna unguiculata (L.) Walp is an excellent source of protein, vitamins and minerals and a major food crop many parts of Africa. Optimal production levels are hampered by insect pests and diseases. Biotechnological techniques such as tissue culture and genetic engineering can aid in the development of varieties with resistance to insect pests and diseases. The objective of this study was to investigate conditions necessary for the development of a reproducible tissue culture system that can be applied to regenerate transformed cells from culture. The in vitro manipulation of cowpea using Murashige and Skoog (MS) medium, auxins and cytokinins resulted in the formation of callus and rhizogenesis. Calli that were formed were separated into six classes based on color and texture. Yellowish friable callus, yellowish compact, soft yellowish callus and green and white were composed of largely vacuolated cells and were non-regenerative. Friable green callus was the most prevalent callus type and could form of roots in some hormone combinations. Green spots were formed on hard compact green callus. The green spots became nodular, forming root primordia and ultimately giving rise to roots. None of the six calli types gave rise to the formation of shoots. Embryogenic callus was induced from cowpea explants cultured on MS medium supplemented with dicamba and picloram. Embryogenic suspension cultures were initiated from callus induced on MS supplemented with 3.0 mg/L dicamba or picloram and conditions for maintenance of embryogenic suspension cultures were evaluated. Somatic embryos were formed in suspension cultures. Attempts to convert and germinate the somatic embryos resulted in the formation of callus or formation of appendages on the somatic embryos or in the death of the embryos. The appendages formed roots on prolonged culture. Further research is needed to determine appropriate optimal conditions for embryo conversion and germination and ultimately plant ...
Isolation of a Pseudomonas aeruginosa Aspartate Transcarbamoylase Mutant and the Investigation of Its Growth Characteristics, Pyrimidine Biosynthetic Enzyme Activities, and Virulence Factor Production
The pyrimidine biosynthetic pathway is an essential pathway for most organisms. Previous research on the pyrimidine pathway in Pseudomonas aeruginosa (PAO1) has shown that a block in the third step of the pathway resulted in both a requirement for exogenous pyrimidines and decreased ability to produce virulence factors. In this work an organism with a mutation in the second step of the pathway, aspartate transcarbamoylase (ATCase), was created. Assays for pyrimidine intermediates, and virulence factors were performed. Results showed that the production of pigments, haemolysin, and rhamnolipids were significantly decreased from PAO1. Elastase and casein protease production were also moderately decreased. In the Caenorhabditis elegans infection model the nematodes fed the ATCase mutant had increased mortality, as compared to nematodes fed wild type bacteria. These findings lend support to the hypothesis that changes in the pyrimidine biosynthetic pathway contribute to the organism's ability to effect pathogenicity.
Purification and Characterization of Proteolytic Aspartate Transcarbamoylase (ATCase) from Burkholderia cepacia 25416 and Construction of a pyrB1 Knock-out Mutant
Burkholderia cepacia is a common soil bacterium of significance in agriculture and bioremediation. B. cepacia is also an opportunistic pathogen of humans causing highly communicable pulmonary infections in cystic fibrosis and immunocompromized patients. The pyrB gene encoding ATCase was cloned and ATCase was purified by the glutathione S-transferase gene fusion system. The ATCase in B. cepacia has been previously classified as a class A enzyme by Bethell and Jones. ATCase activity gels showed that B. cepacia contained a holoenzyme pyrBC complex of 550 kDa comprised of 47 kDa pyrB and 45 kDa pyrC subunits. In the course of purifying the enzyme, trimeric subunits of 140 kDa and 120 kDa were observed as well as a unique proteolysis of the enzyme. The 47 kDa ATCase subunits were cleaved to 40 kDa proteins, which still demonstrated high activity as trimers. The proteolysis site is between Ser74 and Val75 residues. To confirm this, we converted the Ser74 residue to an Ala and to an Arg by site-directed mutagenesis. After this primary sequence changed, the proteolysis of ATCase was not observed. To further investigate the characteristics of B. cepacia pyrB gene, a pyrB knock-out (pyrB-) was constructed by in vitro mutagenesis. In the assay, the 550 kDa holoenzyme and 140 kDa and 120 kDa trimers disappeared and were replaced with a previously unseen 480 kDa holoenzyme pyrB- strain. The results suggest that B. cepacia has two genes that encode ATCase. ATC1 is constitutive and ATC2 is expressed only in the absence of ATC1 activity. To check for the virulence of these two strains, a eukaryotic model virulence test was performed using Caenorhabditis elegans (C. elegans). The pyrB1+pyrB2+ (wild type) B cepacia killed the nematode but pyrB1-pyrB2+ B. cepacia had lost its virulence against C. elegans. This suggests that ATC1 (pyrB1) is involved in virulence ...
Characterization of Infection Arrest Mutants of Medicago Truncatula and Genetic Mapping of Their Respective Genes.
In response to compatible rhizobia, leguminous plants develop unique plant organs, root nodules, in which rhizobia fix nitrogen into ammonia. During nodule invasion, the rhizobia gain access to newly divided cells, the nodule primordia, in the root inner cortex through plant-derived cellulose tubes called infection threads. Infection threads begin in curled root hairs and bring rhizobia into the root crossing several cell layers in the process. Ultimately the rhizobia are deposited within nodule primordium cells through a process resembling endocytosis. Plant host mechanisms underlying the formation and regulation of the invasion process are not understood. To identify and clone plant genes required for nodule invasion, recent efforts have focused on Medicago truncatula. In a collaborative effort the nodulation defect in the lin (lumpy infections) mutant was characterized. From an EMS-mutagenized population of M. truncatula, two non-allelic mutants nip (numerous infections with polyphenolics) and sli (sluggish infections) were identified with defects in nodule invasion. Infection threads were found to proliferate abnormally in the nip mutant nodules with only very rare deposition of rhizobia within plant host cells. nip nodules were found to accumulate polyphenolic compounds, indicative of a host defense response. Interestingly, nip was also found to have defective lateral root elongation suggesting that NIP has a role in both nodule and lateral root development. NIP was found to map at the upper arm of chromosome 1. In sli, infection threads were observed to bring rhizobia from infection threads to newly divided nodule primordium cells in the roots inner cortex. Polyphenolic accumulation in sli nodule/bumps was found. Lateral roots in sli were found to be clustered at the top of the root, indicating that sli like nip may be defective in lateral root development.
Impaired virulence factor production in a dihydroorotate dehydrogenase mutant (pyrD) of Pseudomonas aeruginosa.
Previous research in our laboratory showed that when knockout mutations were created in the pyrB and pyrC genes of the pyrimidine pathway in Pseudomonas aeruginosa, not only were the resultant mutants auxotrophic for pyrimidines but they were also impaired in virulence factor production. Such a correlation had not been previously reported for P. aeruginosa, a ubiquitous opportunistic pathogen in humans. In an earlier study it was reported that mutants blocked in one of the first three enzymes of the pyrimidine pathway in the non-pathogenic strain P. putida M produced no pyoverdin pigment while mutants blocked in the later steps produced copious amounts of pigment, just like the wild type. This study probed for the same connection between pyrimidine auxotrophy and pigment production applied in P. aeruginosa. To that end a knockout mutation was created in pyrD, the fourth step in the pyrimidine pathway which encodes dihydroorotate dehydrogenase. The resulting mutant required pyrimidines for growth but produced wild type pigment levels. Since the pigment pyoverdin is a siderophore it may also be considered a virulence factor, other virulence factors were quantified in the mutant. These included casein protease, hemolysin, elastase, swimming, swarming and twitching motility, and iron binding capacity. In all cases these virulence factors were significantly decreased in the mutant. Even supplementing with uracil did not attain wild type levels. Starvation of the pyrimidine mutant for uracil caused increased specific activity of the pyrimidine enzymes, suggesting that regulation of the pyrimidine pathway occurred at the level of transcription. This effect has also been reported for P. oleovorans. The present research consolidates the idea that pyrimidine auxotrophs cause decreased pathogenicity in P. aeruginosa. Such a finding may open the search for chemotherapy targets in cystic fibrosis and burn victims where P. aeruginosa is an infecting agent.
Isolation and Characterization of Polymorphic Loci from the Caribbean Flamingo (Phoenicopterus ruber ruber): New Tools for Wildlife Management
Methods to determine genetic diversity and relatedness within populations are essential tools for proper wildlife management. Today the approach of choice is polymerase chain reaction-based microsatellite analysis. Seven new polymorphic loci were isolated from a microsatellite-enriched Caribbean flamingo genomic library and used to characterize survey populations of Caribbean and African greater flamingos. In addition, four of these loci were used to verify parentage relationships within a captive-breeding population of African greater flamingos. Parentage predictions based upon gamekeeper observations of breeding and nesting did not always agree with genetic-based parentage analyses of the nine suggested family groups. Four family groups were supported (groups I, II, III and VI) by there results. However, an analysis of the remaining five suggested groups, with a total of eight offspring/dam and eight offspring/sire suggested relationships, yielded seven exclusions of the suggested dam and six exclusions of the suggested sire. This put the overall suggested dam exclusion rate at 35% and exclusion rate for suggested sires at 29%. Although the keeper observation data for our family groups must be considered a variable of concern at this time, these findings are certainly suggestive that more carefully controlled studies may reveal that flamingos are not monogamous as long accepted, but rather socially monogamous or even promiscuous. Thus we have now been able to both characterize and demonstrate the utility of our polymorphic microsatellite loci. We hope these results will interest additional wildlife facilities in further parentage and behavioral studies that will collectively aid to improve monitoring and maintenance of genetic diversity, and as provide better insight into breeding habits of both wild and captive populations.
Microsatellite-based genetic profiling for the management of wild and captive flamingo populations.
Flamingo species generate tremendous interest whether they are small captive groups or wild populations numbering in the thousands. Genetic pedigrees are invaluable for maintaining maximum genetic diversity in captive, as well as wild, populations. However, presently there is a general lack of genetic data for flamingo populations. Microsatellites are loci composed of 2-6 base pair tandem repeats, scattered throughout higher eukaryotic genomes, often exhibiting high levels of polymorphism and heterozygosity. These loci are thus important genetic markers for identity, parentage and population studies. Here, six microsatellite loci were isolated from a microsatellite-enriched Caribbean flamingo partial genomic library. Two are compound complex repeats and four are perfect trinucleotide repeats. Each locus was amplified from Caribbean, African greater, Chilean and lesser flamingo genomic DNAs. Heterozygosity frequencies were calculated for Caribbean (range 0.12-0.90) and African greater flamingos (range 0.23-0.94) loci. All six microsatellite loci were found to be in Hardy-Weinberg equilibrium and linkage disequilibrium analyses did not suggest linkage for any pair of two greater flamingo subspecies (African and Caribbean) loci. At least five of the loci also exhibit polymorphism in Chilean and lesser flamingos, but due to small sample numbers, relevant allele/heterozygosity frequency calculations could not be estimated. Nucleotide sequence comparisons of the amplicons derived from the four flamingo groups reveal a high level of sequence conservation at all loci. Although small sample numbers again limit the data for lesser flamingos and to some degree for the Chilean birds, the sequences of the two greater flamingo subspecies were identical and the number of nonconserved nucleotides appears to be higher for lesser/greater comparisons than for Chilean/greater comparisons. This is consistent with Chilean flamingos being a different species within the same genus as the greater flamingos, while lesser flamingos belong to a separate genus. Parentage analyses on suggested African greater flamingo family groups from ...
Identification and characterization of an incomplete root hair elongation (IRE)-like gene in Medicago truncatula (L.) root nodules.
Cloning and molecular characterization of new genes constitutes a useful approach in studying the symbiotic interactions between the model plant Medicago truncatula and Synorhizobium meliloti. Large numbers of expressed sequence tags (ESTs) available for Medicago truncatula, along with numerous cDNA, oligonucleotides, and Affimetrix DNA microarray chips, represent useful tools for gene discovery. In an attempt to identify a new gene that might be involved in the process of nodulation in Medicago truncatula, preliminary data reported by Fedorova et al. (2002), who identified 340 putative gene products or tentative consensus sequences (TCs) expressed only in nodules, was used. This research was focused on TC33166 (TC103185), which has 3 ESTs in the TC, and whose strongest BLASTX hit of TC103185 is the incomplete root hair elongation (IRE) protein kinase-like protein (NP_192429) from Arabidopsis thaliana. The Arabidopsis IRE gene is required for normal root hair growth, and a role in apical growth was suggested (Oyama et al., 2002). Infection thread growth can be looked at as an inward growth of the root hair. Thus, TC103185 was a good candidate for identifying a gene that may be involved in early events of nodulation. MtIRE (GenBank accession AC122727) is organized in 17 exons and 16 introns, similarly to the Arabidopsis IRE gene. MtIRE is a new member of the IRE family and it is a putative Ser/Thr protein kinase. MtIRE is a nodule- and flower-specific gene, suggesting that nodulation may have recruited it from other developmental processes. MtIRE is likely to be involved in the invasion process, or in the maturation of the symbiosome, or of the cells that contain rhizobia, rather than infection thread initiation and elongation or in nitrogen fixation. Nodule invasion precedes the onset of MtIRE expression and the expression pattern changes in time within the nodule. RNA interference results support MtIRE ...
Regulation of pyrimidine biosynthesis and virulence factor production in wild type, Pyr- and Crc- mutants in Pseudomonas aeruginosa.
Previous research in our laboratory established that pyrB, pyrC or pyrD knock-out mutants in Pseudomonas aeruginosa required pyrimidines for growth. Each mutant was also discovered to be defective in the production of virulence factors. Moreover, the addition of exogenous uracil did not restore the mutant to wild type virulence levels. In an earlier study using non-pathogenic P. putida, mutants blocked in one of the first three enzymes of the pyrimidine pathway produced no pyoverdine pigment while mutants blocked in the fourth, fifth or sixth steps produced copious quantities of pigment, just like wild type P. putida. The present study explored the correlation between pyrimidine auxotrophy and pigment production in P. aeruginosa. Since the pigment pyoverdine is a siderophore it may also be considered a virulence factor. Other virulence factors tested included casein protease, elastase, hemolysin, swimming, swarming and twitching motilities, and iron binding capacity. In all cases, these virulence factors were significantly decreased in the pyrB, pyrC or pyrD mutants and even in the presence of uracil did not attain wild type levels. In order to complete this comprehensive study, pyrimidine mutants blocked in the fifth (pyrE) and sixth (pyrF) steps of the biosynthetic pathway were examined in P. aeruginosa. A third mutant, crc, was also studied because of its location within 80 base pairs of the pyrE gene on the P. aeruginosa chromosome and because of its importance for carbon source utilization. Production of the virulence factors listed above showed a significant decrease in the three mutant strains used in this study when compared with the wild type. This finding may be exploited for novel chemotherapy strategies for ameliorating P. aeruginosa infections in cystic fibrosis patients.
Analysis of the Expression Profiles of Two Isoforms of the Antifungal Protein Osmotin from Gossypium hirsutum
The expression of two cotton osmotin genes was evaluated in terms of the mRNA and protein expression patterns in response to chemical inducers such as ethylene, hydrogen peroxide, and sodium chloride. Reverse transcriptase-polymerase chain reactions (RT-PCR) indicated that osmotin mRNAs are expressed constitutively in root tissues of cotton plants, and that they are rapidly induced in leaf and stem tissues upon ethylene treatment. Real time RT-PCR indicated that osmotin transcript levels were induced 2 to 4 h after treatment with ethephon. The osmotin mRNA levels appear to increase 12 h after treatment, decrease, and then increase again. The osmotin protein expression patterns were analyzed in Western blot analyses using an anti-osmotin antibody preparation. A 24-KDa protein band was detected from cotton plants treated with the inducers. The 24-KDa osmotin proteins were induced 4 h after treatment with ethephon, while down-regulated 96 h after treatment. Multiple osmotin isoforms were observed to be induced in cotton plants upon treatment with ethephon by two-dimensional gel electrophoresis. One goal of this dissertation research was to genetically engineer two cotton osmotin genes to routinely overproduce their antifungal proteins in transgenic Arabidopsis and cotton plants as a natural defense against fungal infections, using co-cultivation with Agrobacterium tumefaciens cells harboring pCAMBIA 2301 vector constructs containing the osmotin genes. Many transgenic Arabidopsis and cotton plants were generated. However, genomic blotting analyses indicated the absence of the osmotin transgenes, but the presence of GUS genes from the vector cassette. Alkaline blot analyses of the vector DNAs from transformed Agrobacterium cells confirmed that an anomalous DNA structural rearrangement or aberrant recombination event probably occurred in the Agrobacterium cells, interdicting the integration of osmotin transgenes into the Arabidopsis and cotton plants. This research provides crucial baseline information on expression of cotton osmotin mRNAs and proteins.
Map-based cloning of the NIP gene in model legume Medicago truncatula.
Large amounts of industrial fertilizers are used to maximize crop yields. Unfortunately, they are not completely consumed by plants; consequently, this leads to soil pollution and negative effects on aquatic systems. An alternative to industrial fertilizers can be found in legume plants that provide a nitrogen source that is not harmful for the environment. Legume plants, through their symbiosis with soil bacteria called rhizobia, are able to reduce atmospheric nitrogen into ammonia, a biological nitrogen source. Establishment of the symbiosis requires communication on the molecular level between the two symbionts, which leads to changes on the cellular level and ultimately results in nitrogen-fixing nodule development. Inside the nodules hypoxic environment, the bacterial enzyme nitrogenase reduces atmospheric nitrogen to ammonia. Medicago truncatula is the model legume plant that is used to study symbiosis with mycorrhiza and with the bacteria Sinorhizobium meliloti. The focus of this work is the M. truncatula nodulation mutant nip (numerous infections and polyphenolics). The NIP gene plays a role in the formation and differentiation of nodules, and development of lateral roots. Studying this mutant will contribute knowledge to understanding the plant response to infection and how the invasion by rhizobia is regulated. Previous genetic mapping placed NIP at the top of linkage group 1 of the M. truncatula genome. A NIP mapping population was established with the purpose of performing fine mapping in the region containing NIP. DNA from two M. truncatula ecotypes A17 and A20 can be distinguished through polymorphisms. Positional mapping of the NIP gene is based on the A17/A20 genetic map of M. truncatula. The NIP mapping population of 2277 plants was scored for their nodulation phenotype and genotyped with flanking molecular genetic markers 146o17 and 23c16d, which are located ~1.5 cM apart and on either side of NIP. This resulted in the identification ...
Evaluation of Zinc Toxicity Using Neuronal Networks on Microelectrode Arrays: Response Quantification and Entry Pathway Analysis
Murine neuronal networks, derived from embryonic frontal cortex (FC) tissue grown on microelectrode arrays, were used to investigate zinc toxicity at concentrations ranging from 20 to 2000 mM total zinc acetate added to the culture medium. Continual multi-channel recording of spontaneous action potential generation allowed a quantitative analysis of the temporal evolution of network spike activity generation at specific zinc acetate concentrations. Cultures responded with immediate concentration-dependent excitation lasting from 5 to 50 min, consisting of increased spiking and enhanced, coordinated bursting. This was followed by irreversible activity decay. The time to 50% and 90% activity loss was concentration dependent, highly reproducible, and formed linear functions in log-log plots. Network activity loss generally preceded morphological changes. 20% cell swelling was correlated with 50% activity loss. Cultures pretreated with the GABAA receptor antagonists bicuculline (40 mM) and picrotoxin (1 mM) lacked the initial excitation phase. This suggests that zinc-induced excitation may be mediated by interfering with GABA inhibition. Partial network protection was achieved by stopping spontaneous activity with either tetrodotoxin (200 nM) or lidocaine (250 mM). However, recovery was not complete and slow deterioration of network activity continued over 6 hrs. Removal of zinc by early medium changes showed irreversible, catastrophic network failure to develop in a concentration-dependent time window between 50% and 90% activity loss. Investigation of entry routes suggested the L-type but not N-type calcium channels to be the main entry pathway for zinc. Data are presented implicating the chloride channel to be an additional entry route.