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Identifying genetic interactions of the spindle checkpoint in Caenorhabditis elegans.

Description: Faithful segregation of chromosomes is ensured by the spindle checkpoint. If a kinetochore does not correctly attach to a microtubule the spindle checkpoint stops cell cycle progression until all chromosomes are attached to microtubules or tension is experienced while pulling the chromosomes. The C. elegans gene, san-1, is required for spindle checkpoint function and anoxia survival. To further understand the role of san-1 in the spindle checkpoint, an RNAi screen was conducted to identify genetic interactions with san-1. The kinetochore gene hcp-1 identified in this screen, was known to have a genetic interaction with hcp-2. Interestingly, san-1(ok1580);hcp-2(ok1757) had embryonic and larval lethal phenotypes, but the phenotypes observed are less severe compared to the phenotypes of san-1(ok1580);hcp-1(RNAi) animals. Both san-1(ok1580);hcp-1(RNAi) and san-1(ok1580);hcp-2(RNAi) produce eggs that may hatch; but san-1(ok1580):hcp-1(RNAi) larvae do not survive to adulthood due to defects caused by aberrant chromosome segregations during development. Y54G9A.6 encodes the C. elegans homolog of bub-3, and has spindle checkpoint function. In C.elegans, bub-3 has genetic interactions with san-1 and mdf-2. An RNAi screen for genetic interactions with bub-3 identified that F31F6.3 may potentially have a genetic interaction with bub-3. This work provided genetic evidence that hcp-1, hcp-2 and F31F6.2 interact with spindle checkpoint genes.
Date: May 2009
Creator: Stewart, Neil
Partner: UNT Libraries

Synthetic Peptides Model Instability of Cardiac Myosin Subfragment-2

Description: Hypertrophic cardiomyopathy (HCM), a heart-related abnormality, is the most prevalent cause of sudden death in young athletes at sporting events. A cluster of cardiomyopathy mutations are localized in β-cardiac myosin at the N-terminal region of subfragment-2. Using resonance energy transfer probes, a synthetic peptide model system was developed to study stability of the coiled coil (S2 fragment) structure by determining monomer-dimer equilibrium of the peptide. Fluorescence resonance energy transfer and MacroModel software suite were used to obtain distance measurements along with measurement of coiled coil formation. The model peptide was used to characterize the effects of disease-causing-mutations and examine potential candidate drugs (polyamines) to counteract effects of mutations causing HCM. Distance measurements between donor and acceptor probes obtained by computational simulation and fluorescence resonance energy transfer (FRET) were consistent. Measurements also agreed with simulations of unlabeled wildtype, indicating coiled coil structural stability of the peptide. Interaction of the site-specific antibody with the peptide strongly inhibited dimerization and destabilized coiled coil structure of the peptide. Presence of negatively charged glutamate residues in the region of subfragment-2 strongly suggested a potential interaction site for positively charged polyamines. Binding of certain polyamines, such as poly-L-Lysine 11 residues and poly-D-Lysine 17 residues, demonstrated the ability to enhance dimerization and improve stability of the coiled coil structure, while some other polyamines were shown to have insignificant impact on the structure. In an attempt to characterize the effect of HCM-causing-mutations, peptides containing E924K mutation and lethal mutation E930 deletion were synthesized. Fluorescence resonance probes were conjugated to the mutant peptides to determine coiled coil formation. Results obtained from both dynamic simulations and resonance energy transfer experiments indicated that these mutations strongly inhibit dimerization, and thus, destabilize coiled coil structure of the peptide. Further experiments were conducted using heterodimers containing a chain of wildtype and a chain ...
Date: August 2013
Creator: Taei, Nasrin
Partner: UNT Libraries

Characterization of Aspartate Transcarbamoylase and Dihydroorotase in Moraxella Catarrhalis

Description: Bacterial aspartate transcarbamoylases (ATCase's) are divided into three classes that correspond to taxonomic relationships within the bacteria. The opportunistic pathogen Moraxeila catarrhalis has undergone several reclassifications based on traditional microbiological criteria. The previously uncharacterized ATCase from M. catarrhalis was purified to homogeneity and its chemical properties characterized. The ATCase from M. catarrhalis is a class C ATCase with an apparent molecular mass of 480-520 kDa. The M. catarrhalis ATCase is a dodecomer composed of six 35 kDa polypeptides and six 45 kDa polypeptides. The enzyme has an unusually high pH optimum of greater than pH 10. The enzyme exhibited hyperbolic kinetic with a Km for aspartate of 2 mM. A single, separate 78 kDa dihydroorotase from M. catarrhalis was identified and it was not associated with ATCase. These data support the reclassification of M. catarrhalis out of the Neisseriaceae family.
Date: May 1998
Creator: Fowler, Michael A. (Michael Allen), 1961-
Partner: UNT Libraries

Identification and Characterization of the Pyrimidine Biosynthetic Operon in Streptomyces griseus

Description: To further understand the ATCase/DHOase bifunctional complex formed in Streptomyces, the genes encoding these and other pyrimidine enzymes were identified and characterized. Polymerase chain reaction (PCR) was utilized in this effort. Primers were constructed by selecting conserved regions of pyrimidine genes from known gene and protein sequences of a wide variety of organisms. These sequences were then optimized to Streptomyces codon usage. PCR products were obtained from internal sites within pyrimidine genes and also from primer combinations of different genes. The size, orientation, and partial sequence of the resulting products shows that Streptomyces has a gene organization of pyrR followed by pyrB, pyrC, carA, carB, and pyrF in an operon similar to that found in other Gram-positive bacteria.
Date: May 1998
Creator: Hooten, Jody J. (Jody Jeran)
Partner: UNT Libraries

Contemporary Biology Curriculum for Non-majors

Description: The proposed biology curriculum for non-majors has one main objective, namely to improve scientific literacy among college students. The National Science Education Standards defines scientific literacy as "the knowledge and understanding of scientific concepts and processes required for personal decision making, participation in civic and cultural affairs, and economic productivity". The suggested strategies to accomplish this goal are to limit the number of topics covered, introduce relevant scientific terminology, emphasize general biological concepts and themes, and hone critical thinking and problem-solving skills. Activities such as group projects, written and oral assignments, and class discussions are effective tools to assess student ability to communicate scientifically. It is also important for students to make connections between the course subject matter and how it affects real life events.
Date: August 1998
Creator: Smallwood, Susan
Partner: UNT Libraries

Use of tRNA Gene Probes to Identify Polymorphic Loci in the Bovine Genome

Description: A 30-mer oligonucleotide probe encoding the "A box" and anticodon loop regions of a human glycine tRNA gene was used to isolate a 581bp DNA fragment from a bovine genomic DNA library. Although the cross-hybridizing segment of DNA was found not to encode any tRNA gene or pseudogene, a region with homology to the "C-element" of the "BOV-tA" type Alulike artiodactyl retroposons was identified. This cross-hybridization was determined to be the result of conserved RNA polymerase III promoter elements in the probe portion of the tRNA gene and these repetitive elements. A microsatellite repeat (TC) was also found associated with this element. Future screening for bovine tRNA genes will require the use of a) longer probes and higher stringency hybridization conditions or b) the simultaneous screening with probes from the 5' and 3' ends of the gene which avoid the conserved Pol III promoter boxes.
Date: August 1998
Creator: Shariat, Parvaneh
Partner: UNT Libraries

N-Acylethanolamines and Plant Phospholipase D

Description: Recently, three distinct isoforms of phospholipase D (PLD) were identified in Arabidopsis thaliana. PLD α represents the well-known form found in plants, while PLD β and γ have been only recently discovered (Pappan et al., 1997b; Qin et al., 1997). These isoforms differ in substrate selectivity and cofactors required for activity. Here, I report that PLD β and γ isoforms were active toward N-acylphosphatidylethanolamine (NAPE), but PLD α was not. The ability of PLD β and γ to hydrolyze NAPE marks a key difference from PLD α. N-acylethanolamines (NAE), the hydrolytic products of NAPE by PLD β and γ, inhibited PLD α from castor bean and cabbage. Inhibition of PLD α by NAE was dose-dependent and inversely proportional to acyl chain length and degree of unsaturation. Enzyme kinetic analysis suggested non-competitive inhibition of PLD α by NAE 14:0. In addition, a 1.2-kb tobacco (Nicotiana tabacum L.) cDNA fragment was isolated that possessed a 74% amino acid identity to Arabidopsis PLD β indicating that this isoform is expressed in tobacco cells. Collectively, these results provide evidence for NAE producing PLD activities and suggest a possible regulatory role for NAE with respect to PLD α.
Date: December 1998
Creator: Brown, Shea Austin
Partner: UNT Libraries

Cloning of Carbonic Anhydrase from Cotton (Gossypium hirsutum L.)

Description: 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.
Date: December 1998
Creator: Local, Andrea
Partner: UNT Libraries

Forensic DNA Extraction Strategies for PCR Analysis

Description: There is a transition nationwide on the analysis of forensic evidentiary stains containing biological material from traditional serology to Polymerase Chain Reaction (PCR) methodologies. The increased sensitivity of PCR, the limited number of alleles at each locus, and the necessity of producing unambiguous data for entry into the FBI's Combined DNA Index System make this study of extraction procedures of utmost importance. A "single tube" extraction procedure for blood stains collected onto FTA™ paper and a modified differential nonorganic extraction method from spermatozoa containing mixed stains were analyzed and compared. The extraction success was evaluated by amplification and typing of the amplified fragment length polymorphism, D1S80. These modifications of the nonorganic method utilized gave an improved separation of the spermatozoa-containing mixed stains.
Date: May 1998
Creator: Van Winkle, Carolyn
Partner: UNT Libraries

Oral Microbiology

Description: Recent studies regarding research on oral microorganisms and the oral diseases are presented. The normal flora of the mouth and the oral environment are first described. Dental plaque and dental caries are primary causes of oral disease. Streptococcus mutans is the major contributor in the initiation and progression of dental caries. Lactobacillus, Actinomyces, and Veillonella are other genera of bacteria linked to dental caries. Periodontitis and gingivitis are periodontal diseases that are caused by oral microorganisms. New research has indicated that various antimicrobial agents and techniques to eliminate or lessen the severity of periodontal diseases. Premature delivery of low birth weight babies in pregnant women has been strongly linked to periodontal disease. Present and future microbiological tests are available to easily determine the causative organisms for most oral diseases that help in diagnosis and treatment of a particular disease.
Date: August 1998
Creator: Osman, Shaiesta
Partner: UNT Libraries

Cassette Systems for Creating Intergeneric Hybrid ATCases

Description: 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.
Date: December 1999
Creator: Simpson, Luci N.
Partner: UNT Libraries

Analysis and expression of the cotton gene for the D-12 fatty acid desaturases 2-4 (FAD2-4)

Description: A genomic clone containing a 16.9-kb segment of cotton DNA was found to encompass a D-12 fatty acid desaturases (FAD2-4) gene. The FAD2-4 gene has a single, large intron of 2,780 bp in its 5'-untranslated region, just 12 bp upstream from the ATG initiation codon of the FAD2-4 opening reading frame. A number of prospective promoter elements, including several light-responsive sequences, occur in the 5'-flanking region. The coding region of the gene is 1155 bp with no introns, and would encode a FAD2-4 polypeptide of 384 amino acids. The putative protein had four membrane-spanning helices, hallmarks of an integral membrane protein, and would probably be located in the endoplasmic reticulum. The FAD2-4 gene is indeed a functional gene, since yeast cells transformed with a plasmid containing the coding region of the gene synthesize an appreciable amount of linoleic acid (18:2), not normally made in wild-type yeast cells. The FAD2-4 gene has many structural similarities to the cotton FAD2-3 gene that was also analyzed in this laboratory.
Date: August 2003
Creator: Park, Stacy J.
Partner: UNT Libraries

Adherence and Haemagglutination of Moraxella Catarrhalis.

Description: M. catarrhalis is a gram-negative diplococci frequently associated with infections of the upper respiratory tract. During the past decade, some preliminary studies have attempted to elucidate mechanisms of adherence and haemagglutination of M. catarrhalis. These studies have reported, in many cases, inconsistent results. There are two purposes of this research. First, identify mechanisms that may potentially be associated with the adherence and haemagglutination of M. catarrhalis. Second, suggest research directions that may be fruitful in clarifying these mechanisms.
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Date: August 2000
Creator: Kosterman, Edward, III
Partner: UNT Libraries

Inherent Problems Associated with the Identification of Genes Responsible for Allowing B. cepacia to Adhere to Human Lung Carcinoma A549 Cells

Description: In this project a bacteria's ability to bind to human lung tissue was investigated. To carry out this study Pseudomonas aeruginosa, Eschericia coli and Burkholderia cepacia were used. B. cepacia served as the bacterium of interest. Isolating the gene which confers upon this bacterium the ability to bind to lung tissue was the main objective of this study. P. aeruginosa has been identified as being the bacteria most responsible for causing serious lung infections that can result in cystic fibrosis. This bacterium therefore served as the positive control in this study. On the contrary, E. coli does not possess this binding ability and served as the negative control. This paper gives a detailed outline of the different procedures necessary for the successful completion of this project. Firstly, a broad guideline of the important steps involved are explored. This is followed by a discussion on potential problems and possible solutions. Throughout the document, illustrations of expected results are indicated so as to further guide the researcher.
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Date: May 2001
Creator: Nesty, Gilda S.
Partner: UNT Libraries

Pyrimidine Metabolism in Streptomyces griseus

Description: Salvage of pyrimidine nucleosides and bases by S. griseus and the regulation of aspartate transcarbamoylase (ATCase) were studied. The velocity-substrate curve for S. griseus ATCase was hyperbolic for both aspartate and carbamoylphosphate. The enzyme activity was diminished in the presence of ATP, CTP, or UTP. The synthesis of ATCase was repressed in cells grown in the presence of exogenous uracil. The specific activity of cells grown with uracil was 43 percent of that for cells grown in minimal medium only. Maximal ATCase and dihydroorotase activities were found in the same column fraction after size-exclusion chromatography, suggesting that both activities could reside in the same polypeptide. The pyrimidine salvage enzymes cytosine deaminase and uridine phosphorylase were identified in S. griseus using HPLC reversed-phase chromatography.
Date: August 1994
Creator: Hughes, Lee E. (Lee Everette)
Partner: UNT Libraries

Comparison of Aspartate Transcarbamoylase and Pyrimidine Salvage in Sporosarcina urea, Sprolactobacillus inulinus, Lactobacillus fermentum, and Micrococcus luteus

Description: The enzyme that catalyzes the committed step in pyrimidine biosynthesis, aspartate transcarbamoylase, has been compared in selected endospore-forming organisms and in morphologically similar control organisms. The ATCases and pyrimidine salvage from Sporosarcina ureae, Sporolactobacillus inulinus, Lactobacillus fermentum, and Micrococcus luteus were compared to those of Bacillus subtilis. While the ATCases from Sporosarcina ureae, Sporolactobacillus inulinus, and L. fermentum were found to exhibit characteristics to that of Bacillus with respect to molecular weight and kinetics, M. luteus ATCase was larger at approximately 480 kDa. Furthermore, pyrimidine salvage in Sporosarcina ureae and M. luteus was identical to those of B. subtilis, while pyrimidine salvage of Sporolactobacillus inulinus and L. fermentum resembled that of the pseudomonads.
Date: August 1994
Creator: Barron, Vincent N. (Vincent Neal)
Partner: UNT Libraries

Genetic Characterization of Central and South American Populations of Scarlet Macaw (Ara macao)

Description: The wild populations of the Scarlet Macaw subspecies native to southern Mexico and Central America, A. m. cyanoptera, have been drastically reduced over the last half century and are now a major concern to local governments and conservation groups. Programs to rebuild these local populations using captive bred specimens must be careful to reintroduce the native A. m. cyanoptera, as opposed to the South American nominate subspecies (A. m. macao) or hybrids of the two subspecies. Molecular markers for comparative genomic analyses are needed for definitive differentiation. Here I describe the isolation and sequence analysis of multiple loci from 7 pedigreed A. m. macao and 14 pedigreed A. m. cyanoptera specimens. The loci analyzed include the 18S rDNA genes, the complete mitogenome as well as intronic regions of selected autosomally-encoded genes. Although the multicopy18S gene sequences exhibited 10% polymorphism within all A. macao genomes, no differences were observed between any of the 21 birds whose genomes were studied. In contrast, numerous polymorphic sites were observed throughout the 16,993 bp mitochondrial genomes of both subspecies. Although much of the polymorphism was observed in the genomes of both subspecies, subspecies-specific alleles were observed at a number of mitochondrial loci, including 12S, 16S, CO2 and ND3. Evidence of possible subspecies-specific alleles were also found in three of four screened nuclear loci. Collectively, these mitochondrial and nuclear loci can be used as the basis to distinguish A. m. cyanoptera from the nominate subspecies, A. m. macao, as well as identify many hybrids, and most importantly will contribute to further reintroduction efforts.
Date: May 2016
Creator: Kim, Tracy
Partner: UNT Libraries

Rapid Isolation and Purification of Plasmid DNA Using High Performance Liquid Chromatography

Description: High Performance Liquid Chromatography (HPLC) has been employed as an analytical tool for the purification and separation of nucleic acids. A Nucleogen DEAE 4000-10 weak anion exchange column, prepacked with modified silica gels, was used to purify and separate a number of Escherichia coli plasmids. Plasmid DNAs were extracted by the alkaline lysis method. The cleared lysate was injected directly onto the Nucleogen column, and the peaks were collected, desalted and analysed by gel electrophoresis. On the chromatogram, the pBR322 formed a distinctive peak at 27 minutes and partial separation was made for the E. coli V517 plasmids. Plasmid pBR322 showed a clear band without any detectable contamination on agarose gel. This purified plasmid DNA is biologically active for enzymatic reaction commonly used in genetic engineering techniques.
Date: May 1987
Creator: Nam, Kiebang
Partner: UNT Libraries

Anatomy and Physiology Syllabus for Community Colleges

Description: This syllabus includes both lecture notes and laboratory activities for a two-semester anatomy and physiology community college course. The syllabus is based on a 16-week semester that is comprised of a three-hour lecture section and a one-hour laboratory class each week. Both the lecture course and laboratory are necessary to fulfill the requirement for anatomy and physiology. Laboratory activities coincide with lectures to enhance understanding of each topic by providing visual and hands-on experiments for the concepts learned in the lecture. Laboratory quizzes will be given each week to help students maintain a working knowledge of the material learned in the laboratory. This course is appropriate for the typical anatomy and physiology student and should benefit both students who plan to major in biology and also those who are non-biology majors. Because subject matter in anatomy and physiology is quite difficult, the importance of attendance and good study skills is stressed.
Date: May 2000
Creator: Schulz, Leslie
Partner: UNT Libraries

Aspartate Transcarbamoylase of Aeromonas Hydrophila

Description: This study focused on the enzyme, aspartate transcarbamoylase (ATCase) from A. hydrophila, a Gram-negative bacterium found in fresh water. The molecular mass of the ATCase holoenzyme from A. hydrophila is 310 kDa. The enzyme is likely composed of 6 catalytic polypeptides of 34 kDa each and 6 regulatory polypeptides of 17 kDa each. The velocity-substrate curve for A. hydrophila ATCase is sigmoidal for both aspartate and carbamoylphosphate. The Km for aspartate was the highest to date for an enteric bacterium at 97.18 mM. The Km for carbamoylphosphate was 1.18 mM. When heated to 60 ºC, the specific activity of the enzyme dropped by more than 50 %. When heated to 100 ºC, the enzyme showed no activity. The enzyme's activity was inhibited by ATP, CTP or UTP.
Date: December 2000
Creator: Higginbotham, Leah
Partner: UNT Libraries

Syllabus for Advanced Placement Biology

Description: The purpose of this syllabus is to provide a working copy to those teachers of the advanced placement biology course taught at the high school level. Reference materials used were the Texas Education Agency ( TEA ) approved Campbell text Biology and the College Board's, Advanced Placement Biology Laboratory Manual. The syllabus is divided into major topics with outlined notes and includes laboratory exercises as recommended by the College Board. The AP biology course is intended to be equivalent to college biology. College freshman biology courses can differ among colleges and among teachers within the same college. This syllabus is intended to serve as an aid to AP teachers, to cover the topics and experiments as set out by the College Board, and to the high school student, the necessary material to successfully complete the AP examination while providing freshman biology equivalence.
Date: August 1999
Creator: Turk, Cathy Christine
Partner: UNT Libraries

General Biology Lecture and Laboratory Curriculum Outline in a Two or Four-Year College

Description: In July of 1999, I wrote to 24 Texas junior and community colleges (and one four-year institution) describing my thesis agenda of a general biology lecture and laboratory syllabus for introductory biology students. I requested the titles and authors of the general biology textbooks and laboratory manuals they were currently using, the publishers of these texts, and the edition of said texts. I then contacted publishers of the various textbooks who, in turn, directed me to the Dallas-area representatives for further inquiries. I assimilated the various authors' general biology topics into a two-semester syllabus of lecture and one semester of laboratory. The document is not a text manuscript, but an all-inclusive listing of a general biology syllabus broken down by subject.
Access: This item is restricted to UNT Community Members. Login required if off-campus.
Date: August 2000
Creator: Moreland, Amy L.
Partner: UNT Libraries

Mutagenized HLA DNA Constructs: Tools for Validating Molecular HLA Typing Methodologies

Description: 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.
Date: May 1999
Creator: Schulte, Kathleen Q.
Partner: UNT Libraries

Regulation of an S6/H4 Kinase in Crude Lymphosarcoma P1798 Preparations

Description: Purified S6/H4 kinase (Mr 60,000) requires autophosphorylation for activation. A rabbit anti-S6/H4 kinase peptide (SVIDPVPAPVGDSHVDGAAK) antibody recognized both the S6/H4 kinase holoenzyme and catalytic domain. Immunoreactivity with p60 kinase protein, and S6/H4 kinase activity were precisely correlated in fractions obtained from ion exchange chromatography of P1798 lymphosarcoma extracts. An enzyme which catalyzed the MgATP-dependent phosphorylation and activation of S6/H4 kinase coeluted with immunoreactivity from Mono 5, but not Mono Q chromatography. Since S6/H4 kinase is homologous with rac-activated PAK65, the observation that phosphorylation is also required for activation suggests a complex mechanism for in vivo activation of the S6/H4 kinase.
Date: December 1998
Creator: Taylor, Allison Antoinette
Partner: UNT Libraries