136 Matching Results

Search Results

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

Role of GPR17 in Thrombocyte Aggregation in Adult Zebrafish

Description: GPR17, a uracil nucleotide cysteinyl leukotriene receptor, belongs to the GPCR (G protein coupled receptor) family. It has been shown recently that inhibiting this protein in the nervous system in mice can lead to blockage of oligodendrocyte maturation, which supports myelin repair. Interestingly, our laboratory found GPR17 in thrombocytes. However, we do not know whether it has any function in thrombocyte aggregation or the nature of the ligand. In this paper, we studied the role of GPR17 in hemostasis, which is a fundamental defense mechanism in the event of injury. Using zebrafish as a model system, our laboratory has studied specifically thrombocytes, which play a significant role in hemostasis. The major reasons to use zebrafish as a model system are that their thrombocytes are functionally equivalent to human platelets, the adult fish are amenable to knockdown experiments, and they are readily available in the market. This study was performed by using a piggy back knockdown method where we used a chemical hybrid of control morpholino and an antisense oligonucleotide sequence leads to the degradation the mRNA for GPR17. After knockdown GPR17 in thrombocytes, the percent difference of the thrombocytes aggregation between the control and knockdown blood samples was measured by flow cytometry. We used various thrombocyte agonists to study differences in aggregation between the control and knockdown blood samples. The study showed that knockdown of GPR17 resulted in no significant differences in percent thrombocyte aggregation between control and agonist treated samples except for a slight increase in collagen-treated samples. Thus, it appears that GPR17 has no significant role in hemostasis.
Access: This item is restricted to UNT Community Members. Login required if off-campus.
Date: December 2015
Creator: Bohassan, Maruah Hejey
Partner: UNT Libraries

The Role of Cysteinyl Leukotriene Receptor 2 in Thrombocyte Aggregation

Description: Cysteinyl leukotriene receptor 2, a G-protein coupled receptor known to be expressed and functional on human platelets. However, it seems that upon ligand activation the cysteinyl leukotriene receptor 2 activates a variety of signaling pathways in multiple cell types among different species. Previously, a former laboratory member Vrinda Kulkarni found cysteinyl leukotriene receptor 2 to be expressed on the surface of adult zebrafish thrombocytes. In this work I studied the characteristics of aggregation in adult zebrafish thrombocytes with the knockdown of cysteinyl leukotriene receptor 2. I used a newly developed knockdown method to study the function of cysteinyl leukotriene receptor 2. Knockdown of the cysteinyl leukotriene was confirmed using RT-PCR results showed p=.001, reduced sell surface level of expression of the cysteinyl leukotriene receptor 2 results showed that p=.002. I found that the knockdown of cysteinyl leukotriene receptor 2 results in prothrombotic thrombocytes by using flow cytometry p=.0001.
Date: December 2015
Creator: Reyna, Julianna
Partner: UNT Libraries

Development of von Willebrand Factor Zebrafish Mutant Using CRISPR/Cas9 Mediated Genome Editing

Description: von Willebrand factor (VWF) protein acts in the intrinsic coagulation pathway by stabilizing FVIII from proteolytic clearance and at the site of injury, by promoting the adhesion and aggregation of platelets to the exposed subendothelial wall. von Willebrand disease (VWD) results from quantitative and qualitative deficiencies in VWF protein. The variability expressivity in phenotype presentations is in partly caused by the action of modifier genes. Zebrafish has been used as hemostasis animal model. However, it has not been used to evaluate VWD. Here, we report the development of a heterozygote VWF mutant zebrafish using the genome editing CRISPR/Cas9 system to screen for modifier genes involved in VWD. We designed CRISPR oligonucleotides and inserted them into pT7-gRNa plasmid. We then prepared VWF gRNA along with the endonuclease Cas9 RNA from Cas9 plasmid. We injected these two RNAs into 1-4 cell-stage zebrafish embryos and induced a mutation in VWF exon 29 of the zebrafish with a mutagenesis rate of 16.6% (3/18 adult fish). Also, we observed a germline transmission with an efficiency rate of 5.5% (1/18 adult fish). We obtained a deletion in exon 29 which should result in truncated VWF protein.
Date: May 2017
Creator: Toffessi Tcheuyap, Vanina
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

Inquiry-based science for high school students: a forensic unit

Description: This project constitutes an instructional unit for honors biology that involves the use of science in the field of criminal investigation and forensics. Before beginning the unit, the learners should have mastered basic laboratory skills, including use of the microscope. They should also have an understanding of the basic structure and function of DNA and its role in heredity and protein synthesis. The standard time frame is 24 days with 70-minute periods, but can be easily adjusted to meet classroom needs. Several instructional strategies enhance student learning and make science fun. The unit is inquiry-driven and activity-based. Students are surprised by the crime, gather and analyze evidence, and work towards proposing an explanation. This real world problem involves the use of cooperative learning and a variety of assessment techniques.
Access: This item is restricted to UNT Community Members. Login required if off-campus.
Date: August 2000
Creator: Apple, Kendra Kea
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.
Access: This item is restricted to UNT Community Members. Login required if off-campus.
Date: August 2000
Creator: Kosterman, Edward, III
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

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

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

The Relationship of Force on Myosin Subfragment 2 Region to the Coiled-Coiled Region of the Myosin Dimer

Description: The stability of myosin subfragment 2 was analyzed using gravitational force spectroscopy. The region was found to destabilize under physiological force loads, indicating the possibility that subfragment 2 may uncoil to facilitate actin binding during muscle contraction. As a control, synthetic cofilaments were produced to discover if the observations in the single molecule assay were due to the lack of the stability provided by the thick filament. Statistically, there was no difference between the single molecule assay data and the synthetic cofilament assay data. Thus, the instability of the region is due to intrinsic properties within subfragment 2.
Date: December 2011
Creator: Hall, Nakiuda M.
Partner: UNT Libraries

Linkage of a nitrilase-containing Nit1C gene cluster to cyanide utilization in Pseudomonas fluorescens NCIMB 11764.

Description: Pseudomonas fluorescens NCIMB 11764 (Pf11764) is uniquely able to grow on the poison cyanide as its sole nitrogen source. It does so by converting cyanide oxidatively to carbon dioxide and ammonia, the latter being assimilated into cellular molecules. This requires a complex enzymatic machinery that includes nitrilase and oxygenase enzymes the nature of which are not well understood. In the course of a proteomics analysis aimed at achieving a better understanding of the proteins that may be required for cyanide degradation by Pf11764, an unknown protein of 17.8 kDa was detected in cells exposed to cyanide. Analysis of this protein by ESI-coupled mass spectrometry and bioinformatics searches gave evidence of strong homology with a protein (Hyp1) of unknown function (hypothetical) present in the bacterium Photorhabdus luminescens subsp. laumondii TTO1 (locus plu_1232). A search of available microbial genomes revealed a number of Hyp1 orthologs the genes of which are found in a conserved gene cluster known as Nit1C. Independent studies revealed that in addition to Hyp1, Pf11764 possesses a gene (nit) specifying a nitrilase enzyme whose closest homologue is a nitrilase found in Nit1C gene clusters (77% amino acid identity). DNA sequence analysis has further revealed that indeed, hyp1Pf11764 and nitPf11764 are contained in a cluster that includes also a gene specifying an oxygenase. Given the possible connection of Nit1C-endoded nitrilase and oxygenase enzymes to enzymatic cyanide degradation, there is strong reason for thinking that the genes specifying these enzymes contribute to bacterial growth on cyanide in those bacteria containing the Nit1C cluster. Because the biological function of the Hyp1 protein is currently unknown, it was cloned and the protein expressed in E. coli so that its properties could further be explored. Unfortunately, the expression of the protein in an insoluble form complicated these analyses. However, at least two lines of ...
Date: May 2009
Creator: Ghosh, Pallab
Partner: UNT Libraries

Comparison of Aspartate Transcarbamoylase Activity Between Pseudomonas Aeruginosa Which Has One Chromosome and Burkholderia Cepacia Which Has Three Chromosomes

Description: The pyrimidine biosynthetic pathway is essential and similar in all bacteria. The pathway from Pseudomonas is regulated by nucleotides which bind to the upstream region of the pyrBC’ gene complex. Work in our lab mapped the genes and showed that the pyrB and pyrC’ were part of an overlap complex. The Pseudomonas aeruginosa has one circular chromosome. A former Pseudomonas now called Burkholderia cepacia is similar to P. aeruginosa except that it contains three circular chromosomes (CI, CII, CIII) and one large plasmid. The primary chromosome named CI contains the pyrBC’. To our knowledge there has been no report of the activity of ATCase in Pseudomonas and contrasted with that of Burkholderia. Here, we compare the activity of ATCase in P. aeruginosa and B .cepacia. Cells of both organisms were grown in Pseudomonas minimal medium and in Enriched medium. The ATCase was extracted and partially purified from each sample. It is hypothesized that the B. cepacia has greater activity for ATCase than do the Pseudomonas.
Date: August 2012
Creator: Nusair, Arwa Y.
Partner: UNT Libraries

Functional Neural Toxicity and Endocrine Responses in Mice Following Naphthalene Exposure

Description: Polycyclic aromatic hydrocarbons (PAHs) are a well studied and diverse class of environmental toxicants. PAHs act via the aryl hydrocarbon receptor (AhR), and studies have suggested that PAHs may elicit neurological and estrogenic effects. Doses of PAHs between 50 to 150 ppm may elicit neurotoxicity in rodent models. The present study investigated the effects of naphthalene on in vivo steroidogenesis in Swiss Webster male mice, and in vitro neural function of Balb-C/ICR mice frontal cortex neurons. These data suggest that naphthalene may not elicit steroidogenic effects at concentrations ranging from 0.2 to 25 mg/kg/day, following a 7 day subcutaneous dosing regime. In addition, naphthalene may cause functional toxicity of frontal cortex neurons at concentrations of 32 to 160 ppm naphthalene.
Date: August 2010
Creator: Colbert, Crystal
Partner: UNT Libraries

Structural Analysis of the Genes Encoding the Oxalocrotonate Branch of the Pseudomonas putida TOL Plasmid pDKI meta-cleavage Pathway and the Expression of the xy1G Gene Product in Escherichia coli

Description: Three overlapping DNA fragments from the lower operon of Pseudomonas putida TOL plasmid pDK1, covering the xy1IH genes and downstream flanking region, were cloned into pUC19. They include a 2.8 kbp XhoI fragment, a 2.7 kbp PstI fragment and a 2.0 kbp EcoRI-HindIII fragment. They were subjected to DNA sequence analysis. The xy1I (4-oxalocrotonate decarboxylase) and xy1H (4-oxalocrotonate tautomerase) genes were found to possess coding regions of 792 and 189 nucleotides, respectively. A possible transcriptional terminator resembling E. coli rho-independent terminators was identified downstream of the translational stop of xy1H. An additional stem and loop structure was found in the intergenic region between xy1I and xy1H. The individual ORF's of the oxalocrotonate branch (xy1G, xy1I and xy1H) have been cloned into pUC18/19. The expression of the xy1G gene in E. coli was successfully assayed spectrophotometrically.
Date: December 1992
Creator: Luo, Xuebin
Partner: UNT Libraries

Environmental Correlates to Genetic Variation in the Coral Reef Fish, Thalassoma bifasciatum

Description: Genetic variation was examined in bluehead wrasse, Thalassoma bifasciatum, populations along the Florida Keys. Interpretion of mean heterozygosity (H), percent polymorphic loci per population (P), genetic similarity, and F and G statistics demonstrated a clear division of wrasse populations into "northern" and "southern" groups. Correlation and cluster analyses indicated the six reefs can be grouped in a similar fashion based on genetic and environmental data. The conclusion from this analysis of wrasse populations in the Florida Keys is that substantial population subdivision occurs in response to differing selective pressures created by heterogeneous environments.
Date: December 1988
Creator: Pirkle, Michelle S. (Michelle Serpas)
Partner: UNT Libraries

Site Directed Mutagenesis Of Dienelactone Hydrolase

Description: The role of individual amino acid residues of the enzyme dienelactone hydrolase was investigated. Using the polymerase chain reaction (PCR), a 1.9 kbp clcD fragment was amplified and subcloned yielding a 821 bp BamHI to EcoRI clcD subclone in the plasmid pUC19. Site-specific mutants of dienelactone hydrolase were created using mismatched oligonucleotides to prime DNA synthesis. Specifically modified proteins from mutated clcD genes (Arg 81 to alanine, Tyr 85 to phenylalanine and Arg 206 to alanine), were encoded by the mutant clones. Enzyme assays showed that dienelactone hydrolase activity of the mutants Arg 81 and Arg 206 was totally abolished. The DLHase enzyme activity of mutant Tyr 85 is greatly decreased by approximately two thirds.
Date: December 1992
Creator: Chen, Wei, 1965-
Partner: UNT Libraries

Molecular and biochemical characterization of phospholipase D in cotton (Gossypium hirsutum L) seedlings.

Description: N-Acylethanolamines (NAEs) are enriched in seed-derived tissues and are believed to be formed from the membrane phospholipid, N-acylphosphatidylethanolamine (NAPE) via the action of phospholipase D (PLD). In an effort to identify a functional NAPE-PLD in cotton seeds and seedlings, we have screened a cotton seedling cDNA (cotyledon mRNA from 48 h dark grown seedlings) library with a 1.2 kb tobacco partial cDNA fragment encoding the middle third of a putative PLDβ/γ (genbank accession, AF195614) isoform. Six plaques were isolated from the Uni-ZAP lambda library, excised as pBluescript SK(-) phagemids and subjected to nucleotide sequence analysis. Alignment of derived sequences with Arabidopsis PLD family members indicated that the cDNAs represent six different PLD gene products -three putative PLD β isoforms and three putative PLD δ isoforms. The PLD β isoforms, designated Ghpldβ1a, GHpldβ1b and a truncated Ghpldβ1b isoform. Both the full-length PLD β proteins contained characteristic HKxxxxD catalytic domains, a PC-binding domain, a PIP2-binding domain and a C2 domain. In addition both cotton PLD β isoforms had a N-terminal "SPQY" rich domain which appeared to be unique to these PLDs. The three PLD δ isoforms, designated Ghpldδ1a, Ghpldδ1b and Ghpldδ1b-2 encode full-length PLDδ proteins, and like the above PLDs, contained the characteristic catalytic and regulatory domains. The expression of Ghpldδ1b showed hydrolytic and transphosphatidylation activity toward radiolabelled phosphatidylcholine (PC) but it appears Ghpldδ1b does not utilize NAPE as a substrate to produce NAEs nor does it seem to be suppressed by NAEs.
Access: This item is restricted to UNT Community Members. Login required if off-campus.
Date: May 2005
Creator: McHugh, John
Partner: UNT Libraries

Pyrimidine Nucleoside Metabolism in Pseudomonads and Enteric Bacteria

Description: Metabolic differences in the strategies used for pyrimidine base and nucleoside salvage were studied in the pseudomonads and enteric bacteria. Fluoro--analogs were used to select mutant strains of E. coli, S. typhimurium, P. putida, and P. aeruginosa blocked in one or more of the uracil and uridine salvage enzymes. HPLC analysis of cell-free extracts from wild-type and mutant strains examined the effectiveness of the selections. Evidence was found for cytidine kinase in Pseudomonas and for an activity that converted uracil compounds to cytosine compounds. Using media supplemented with 150 μg of orotic acid per ml, P. putida SOC 1, a Pyr, upp mutant which utilizes orotic acid as a pyrimidine source was isolated for the first time in any study.
Date: December 1991
Creator: Scott, Allelia Worrall
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

Map-based cloning of the NIP gene in model legume Medicago truncatula.

Description: 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 ...
Date: May 2007
Creator: Morris, Viktoriya
Partner: UNT Libraries

Physical Map between Marker 8O7 and 146O17 on the Medicago truncatula Linkage Group 1 that Contains the NIP Gene

Description: The Medicago truncatula NIP gene is located on M. truncatula Linkage Group 1. Informative recombinants showed crossovers that localize the NIP gene between markers 146O17 and 23C16D. Marker 164N9 co-segregates with the NIP gene, and the location of marker 164N9 is between markers 146O17 and 23C16D. Based upon data from the Medicago genome sequencing project, a subset of the model legume Medicago truncatula bacterial artificial chromosomes (BACs) were used to create a physical map on the DNA in this genetic internal. BACs near the potential NIP gene location near marker 164N9 were identified, and used in experiments to predict the physical map by a BAC-by-BAC strategy. Using marker 164N9 as a center point, and chromosome walking outward, the physical map toward markers 146O17 and 23C16D was built. The chromosome walk consisted of a virtual walk, made with existing sequence of BACs from the Medicago genome project, hybridizations to filters containing BAC DNA, and PCR reactions to confirm that predicted overlapping BACs contained DNA that yielded similar PCR products. In addition, the primers which are made for physical mapping via PCR could be good genetic markers helpful in discovering the location of the NIP gene. As a result of efforts repotted here, gap in physical map between marker 164N9 and 146O17 was closed.
Date: December 2007
Creator: Lee, Yi-Ching
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

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