Browsing by Subject "Department of Genetics, Cell Biology and Development"
Now showing 1 - 20 of 34
- Results Per Page
- Sort Options
Item Absence of Twisted Gastrulation (Twsg1) Limits the Population of Cranial Neural Crest Cells(2009-04-08) Mittelstaedt, GinaCraniofacial defects are among the most common birth defects with cranial neural crest cells (NCCs) playing a fundamental role in craniofacial development. The cranial NCCs migrate from dorsal neural folds to populate the branchial arches. Then they differentiate into the cells that form facial structures. For example, the mandible forms from the first branchial arch (BA1). Twisted gastrulation (Twsg1) is a gene that has been found to influence craniofacial development. A mutation in Twsg1 in mice produces a spectrum of craniofacial defects ranging from normal appearance to underdevelopment of the mandible, midline facial defects, and forebrain defects resulting in holoprosencephaly. Previous research has shown that Twsg1 acts as a bone morphogenetic protein (BMP) antagonist and thereby limits apoptosis in BA1, a key destination of cranial NCCs. Apoptosis is increased in BA1 in the absence of Twsg1, leading to a loss of BA1 derivatives. The hypothesis of this work is that the absence of Twsg1 also increases apoptosis in the cranial NCCs and thereby limits the population of NCCs prior to their migration to BA1. This study focused on determining whether the NCC population was depleted in Twsg1 knockout mice and whether the midbrain region itself was affected. We found that the midbrain markers were normal, but the markers specific for NCCs were significantly reduced. This reduction is consistent with a depletion of the NCC population. Future research will directly study proliferation, apoptosis, and BMP signaling in NCC populations in the absence of Twsg1.Item Candida albicans Mutagenesis: Response to Stress(2009-04-08) Bruck, David JoachimCandida albicans is a model eukaryotic yeast and an opportunist human pathogen. It generates novel drug resistance through mutation and mitotic recombination. The rate loss of heterozygosity, or rate LoH, is a measure of this mutation and recombination. Previous research has shown that some stresses like the anti-fungal drug fluconazole cause the yeast cells to increase their rate LoH while other stresses do not. On the assumption that fluconazole does not directly affect DNA replication, it is possible that the increased rate LoH is due to a stress response pathway. My UROP project aimed to create a list of computationally curated gene targets using microarray expression data analysis which would then be tested for mutant phenotype rate LoH. The majority of the time allocated to the project was spent doing ortholog searches of gene lists curated from another yeast, Saccharomyces cerevisiae, as well as studying any previous papers where any of the orthologs in question were directly examined.Item Cold Adaptation & Mitochondrial Function in S. cerevisiae(2010-04-21) Leeaw, PhoebeThe two current theories concerning the origin of life deliberate whether life originated from a hot stew or freezing ice. Today, life can be found throughout the entire globe, even at the poles. For survival to be possible, organisms must be capable of energy production under extreme temperatures. The main focus of this study is to determine whether the ability of a cell to grow at low temperatures Yeasts are capable of fermentation in cold environments but the effect of extreme temperatures on respiration is still under investigation. The objective of the experiment was to determine if S. cerevisiae mitochondrial function is important for growth at low temperatures. To test this hypothesis, we screened cold sensitive mutants for their ability to grow on media containing glycerol and ethanol, which required respiration. If the gene codes for a mitochondrial process, then its deletion should hinder oxidative phosphorylation. Yeast is grown in conditions at permissive and cold temperatures to observe differences in growth patterns. Results have demonstrated the yeast’s capability to produce ATP in cold and overall restrictive environments.Item Combating HIV: Exploring the Structure of APOBEC3G (A3G)(2009-04-08) Valesano, JohnThe human APOBEC3G (A3G) protein is a DNA deaminase that can inhibit HIV replication. In most forms of HIV though, the deaminase activity is limited by the viral protein VIF. While recent work has shed light on the three dimensional structure of A3G, little is known about its complete structure and the domains that mediate self-interaction. For instance, inside some types of cells human, A3G forms much larger aggregates with itself, RNA and other proteins, but it is not known how these aggregates are put together. In order to fully understand how A3G interacts with polynucleotides (single-stranded DNA and RNA), how it is inhibited by VIF and how it is regulated within the cell, it is necessary to know how two A3G proteins dimerize. I hypothesized that a distinct site within A3G nucleates or mediates dimerization. To test this hypothesis, a large library of full-length A3G mutants was created. These mutants were then tested in a well-known protein-protein interaction assay called the yeast two-hybrid system. Mutant A3G proteins were pitted against the C-terminal domain of A3G to screen for those that had lost the capacity to dimerize. Candidate dimerization mutants were then sequenced to determine the changes in the nucleotide bases that were responsible. I currently have over ten non-interacting candidates. In the coming months, theses candidates will be tested for their ability to deaminate DNA, dimerize in an independent protein-protein interaction assay and restrict HIV replication in a model cell-based system. My data will help reveal whether the functional unit of A3G is a monomer, a dimer or some more complex polymer.Item Comparative Analysis of Hs6st and Sulf1-mcherry Expression Patterns in Drosophila(2011-04-13) Uk, Samantha; Dang, AnHeparan sulfate proteoglycans (HSPGs) are molecules that are comprised of a core protein modified with heparan sulfate (HS), a negatively charged linear polysaccharide, consisting of uronic acid and N-acetyl-glucosamine (GlcNAc) disaccharide repeats. Generally, these molecules are located on the cell surface and the extracellular matrix. HSPGs have been known to be associated with various biological processes such as growth factor signaling, neuronal development, and cell adhesion. HS chains possess heterogeneous structures, and their diverse patterns of sulfation can determine the binding specificity for certain proteins. 6-O-sulfation of GlcNAc (or GlcNSO3) is the key modification of HS, since it can be dynamically regulated; heparan sulfate 6-sulfotransferase (Hs6st) catalyzes the transfer of sulfate groups of GlcNAc (or GlcNSO3), while heparan sulfate 6-O endosulfatase (Sulf1) removes it. However, how 6-O-sulfation is regulated during animal development remains largely unknown. In this poster, we will present expression analysis of these enzymes in the fruit fly Drosophila melanogaster. We generated a transgenic reporter fly for Sulf1 gene, and its expression pattern was compared with that of Hs6st gene using Hs6st-lacZ enhancer trapline. Our study will provide regulatory mechanisms of HS during the development of Drosophila as well as other multi-cellular organisms.Item Contact-dependent Growth Factor Signaling Mediated by Heparan Sulfate Proteoglycans in Drosophila in vivo(2011-04-13) Arashiro, TakeshiHeparan sulfate proteoglycans (HSPGs) are special type of glycoproteins which play important roles in development, such as regulation of growth factor signaling and maintenance of the stem cells. Nakato Lab recently found that Dally, a Drosophila HSPG is expressed in the niche cells and regulates BMP signaling in directly contacting stem cells in the germline stem cell niche of the Drosophila ovary. Thus, Dally can mediate signaling in trans in directly contacting cells. In order to search for tissues where Dally-expressing cells have direct contact with cells expressing growth factors such as bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs), we established a novel system to detect cell-cell contact using split-GFP (spGFP) method in vivo. The system effectively detected the interface of Dally-expressing and BMP-expressing cells.Item Determining the Location of Tektins within a Vital Molecular Machine(2011-04-13) Ries, MichaelFlagella and cilia play crucial roles in keeping life moving, one of the most easily recognizable flagella being a spermazoon’s tail. These remarkable molecular machines contain a ring of 9 outer doublet microtubules (hollow tubes, 30 nm in diameter) each composed of protofilaments (PFs). Within each microtubule is a hyper-stable, 3-PF “ribbon” containing the integral, filamentous protein tektin. Two hypotheses exist regarding the location of tektin: (i) the dynein-spoke-tektin filament and (ii) the partition model: (i) suggests that tektin forms one of the three PFs. (ii) suggests that a tektin filament lies on top of the ribbon and is not one of the PFs. In this project ribbons were first isolated from Strongylocentrotus purpuratus (sea urchins). Secondly, they were partially digested by several different enzymes and sized (showing degree of digestion) by gel electrophoresis. Electron microscopy verified that the ribbons were still partially intact. Finally, flanking tubulin fragments still loosely associated with the tektin filament were to be identified by mass spectrometry. By comparing these short fragments of tubulin to their known PF structure, we would be able to deduce whether the tektin filament binds to the sides of the flanking tubulin PFs (as in model i) or binds along the inside surface of the ribbon (as in model ii). The optimal digestion has yet to be achieved; future studies aim to optimize these conditions, through use of different digestive enzymes and digestion times, and subsequently distinguish which model is indeed a more accurate representation of tektin location.Item Determining the Meiotic Stability of HRAS1 Minisatellite Tract(2011-01-05) Raja, MasoomMinisatellites are repetitive DNA sequences with repeat units that range from 16 to 100 base pairs in length. These sequences are stable during mitosis but are highly unstable during meiosis with alteration in both length and sequence compositions. One class of minisatellite, rare alleles, have been correlated with cancers, including primary tumors of the brain, lung, ovaries, colon, bladder, and breast. Due to the difficulty of studying meiosis in humans, a novel minisatellite system in the yeast Saccharomyces cerevisiae using the common A1 allele, of the human HRAS1 minisatellite integrated into its genome adjacent to the HIS4 gene was used in this research. After the insertion of the HRAS1 minisatellite into the yeast genome, it was found to exhibit the same phenotypes as in mammalian cells. Our strains have a rare HRAS1 minisatellite allele isolated from breast cancer cells inserted into the promoter region of HIS 4. Two strains, namely .slx1 and .slx4 mutants of this his4-H10 HRAS1 rare minisatellite allele, were used to collect data. Data from other researchers showed that SLX4 andSLX1 play a vital role in resolving Holliday junctions recombination. Hence, to determine the factors involved in minisatellite stability, we determined the frequency at which the HRAS1 minisatellite repetitive tract undergoes alterations during meiosis, and the nature of the alterations, as well as calculated recombination and crossover frequency of this minisatellite in the our mutants.Item The Effect of Saturated Fatty Acids on Lipogenic Gene Expression in Rat Primary Hepatocytes(2009-04-08) Best, AmandaConsumption of saturated fat has been associated with the development of obesity, diabetes, hepatic steatosis and related diseases. Additionally, animal studies have shown that consumption of a diet high in saturated fat results in a rapid upregulation of hepatic lipogenic genes. Thus, to determine if saturated fatty acids have a direct effect on hepatic gene expression, rat primary hepatocytes were cultured and treated with various fatty acids. RNA was harvested from the hepatocytes and cDNA was subsequently prepared to analyze for lipogenic gene expression. Lipogenic enzymes included fatty acid synthase (FAS), acetyl-coA carboxylase (ACC) isoforms alpha and beta, stearoyl-coenzyme A desaturase 1 (SCD1) and fatty acid elongase 6 (Elovl6). Although lipogenic gene expression was altered in response to the presence of insulin and glucose, saturated fatty acids did not appear to significantly upregulate gene expression of lipogenic genes. These data suggest that the effects of saturated fatty acids on lipogenic gene expression in the liver are not direct. Further experiments will explore whether signals from the adipose tissue or inflammatory signals from microphages are necessary for saturated fatty acids to cause changes in de novo lipogenesis.Item The Effects of Hypoxia and Cytotoxic Stress on β-cells During Transplantation(2009-04-08) Mian, IstiaqType 1 diabetes is a devastating disorder resulting from the autoimmune destruction of insulin-producing β-cells within the islets of Langerhans. Complications from hyperglycemia lead to a lower quality of life characterized by seizures, heart attack, kidney failure or retinopathy. Recent advances in islet cell transplantation have presented a possible cure for Type 1 diabetes, however the viability of a graft is drastically lowered by hypoxia and cytotoxic stress during isolation and transplantation. In this study, we mimicked the process of islet transplantation by exposing rat INS-1 cells to hypoxia (1% oxygen) and three different concentrations of three cytokines (IL-1β, TNF-α and IFN-gamma) for 24 hours and analyzed their effects via oxygen consumption rate (OCR), ATP and caspase assay. Results from this study would be able to characterize the extent of the effects of hypoxia on graft viability and could implicate the most potent cytokine at a specific concentration. Results could also suggest a specific pathway that a certain cytokine uses. Implications from this study can lead to improved methods of transplanting islet cells, perhaps via additives such as tocopherols, antioxidants, or JNK inhibitors that may increase islet protection throughout the transplantation process. If graft viability can be increased, this can increase the availability of islet cells and quality of life for Type 1 diabetic patients.Item Effects of Kinetochore Depletion in Candida Albicans(2009-04-08) Applen, ShellyCentromere DNA and the numerous associated kinetochore proteins, are found in our cells attaching sister chromatids together and are essential for normal chromosomal segregation. The objective of this project is to characterize kinetochore mutants in Candida albicans and determine which kinetochore proteins are essential and which are necessary for chromosome stability. With GRACE (gene replacement and conditional expression) strain analysis using a tet-off promoter to regulate gene expression, we have concluded that the gene products of Dad2, Spc19 and Cse4 are absolutely essential for the growth of C. albicans, while depletion of Nuf2 and Skp1 gene products resulted in a growth defect. The growth phenotypes following depletion of other kinetochore proteins are yet to be determined. We quantified cell viability with FUN-1 vital stain and found that after prolonged depletion of Nuf2, Dad2, Skp1, and Cse4 gene products, most, but not all, cells have undergone cell death rather than remaining in a dormant state. As a second method of obtaining a knock-out strain to determine essentiality, we are transforming a Uridine-Arginine-Uridine (UAU) construct, selecting for the integration of this construct into both genome copies, and determining if all surviving cells still contain a wild-type allele resulting from triosomy or a gene duplication event. This phase is currently in progress. We also plan to determine cell size as a measure of polyploidy which could give insight as to how some cells are adapting and escaping cell death. This work will provide a better understanding of how cells react to stresses, such as kinetochore malfunction.Item Effects of NOM1 on Ribosome Biogenesis(2010-04-21) Zimmerman, RheanneRibosomes translate genetic information from messenger RNA into proteins, and are therefore necessary for cell growth. Disruption of ribosome biogenesis leads to arrest in cell growth and replication, and has been identified as a precursor to some cancers. Eukaryotic ribosomes are composed of 40S and 60S subunits. The 40S subunit includes one segment of ribosomal RNA and approximately 30 proteins. The 60S subunit is composed of three segments of rRNA and approximately 50 proteins. Both are necessary to translate proteins from mRNA. NOM1 was identified by the Conklin lab because of its location at a breakpoint on chromosome 7 associated with acute myeloid leukemia. Functional studies of NOM1 have demonstrated that it: is required for cell growth and cell replication; localizes to the nucleolus; interacts with and targets several proteins to the nucleolus including Protein Phosphatase I, the oncogene MSP58 and the RNA helicase eIF4AIII; is required for production of 40S ribosomes.Item Exploring the Mechanism of Ara-C Resistance in Acute Myeloid Leukemia(2010-04-21) Ellingson, Alexandra M.Acute myeloid leukemia (AML) is the most common and most deadly type of leukemia in adults, affecting approximately 3 people per 100,000. AML is typically treated with a cocktail of chemotherapeutic drugs, most often involving the pharmaceutical agent cytosinearabinoside (Ara-C). Treatment with Ara-C will almost always cause remission in AML patients. However, developed resistance to Ara-C becomes a problem for many patients suffering from the disease, and many relapse within a few years of remission. We are using an in vitro system to model the Ara-C resistance in AML cell lines.Item Expression and purification of GxcA, a c-type cytochrome involved with metal respiration by the bacterium, Geothrix fermentans(2011-04-13) Liu, JoanneThe study of bacteria capable of respiring oxidized metals offers insights into the geochemical cycling of metals, including toxic heavy metal contaminants. These dissimilatory metal-reducing organisms couple oxidation of organic compounds with the reduction of substrates to gain energy. Improving understanding of the various bacterial metal-reducing strategies will increase our ability to produce renewable energy using microbial fuel cell technologies. The bacterium Geothrix fermentanshas long been ignored in the study of metal respiration, but it has recently been shown to employ a unique strategy involving more than one biochemical pathway that appears tuned to use of high potential metals, such as uranium and manganese. Membranes isolated from Fe(III)-respiring G. fermentans contain high levels of a decahemecytochrome, known as GxcA, which is suspected in electron transfer by G. fermentans. As a genetic system for G. fermentans is not yet available, GxcAwas targeted for expression and purification. The DNA sequence for GxcA, containing an in-frame hexahistidinesequence, was first cloned into E. coli, using the inducible expression vector pETlite. Then, the recombinant plasmid was co-transformed into E. coli with pEC86, a plasmid that contains genes for the ccmc-type cytochrome maturation system. Colonies were screened for c-type cytochromes by redox difference absorption analysis and hemestain analysis. Future work will involve GxcApurification for redox and localization experiments to determine GxcA’spotential role in G. fermentans.Item Identification of Genes Required for Minisatellite Instability(2009-04-08) Swanlund, SethRare alleles of minisatellites, repetitive DNA tracts whose repeat units range from 15 to 100 base pairs in length, have been correlated with human diseases including breast, colon, and urinary cancer. Little is known about the regulation of minisatellite stability. The Kirkpatrick lab conducted a color-based screen that identified factors involved in minisatellite stability during stationary phase using the yeast Saccharomyces cerevisiae. Factors were identified by screening for mutants that destabilized a minisatellite repeat construct in the coding region of the ADE2 gene. One of the factors identified in this screen was the zinc transporter gene ZRT1. I followed up these findings by identifying and sequencing mutations that suppressed the minisatellite instability phenotype in the ZRT1 null strains. To identify a suppressor gene, I transformed a yeast genomic plasmid library into a suppressor mutant stain, selected transformants that reverted to the parental phenotype, then rescued the candidate plasmids and sequenced them. I also identified specific mutations in the suppressor genes PET112 and IFM1 by amplification and sequencing. Both of these genes are involved in mitochondrial function. This suppressor screen has shown that many processes are involved in minisatellite stability during stationary phase. Further investigation is needed to identify other genes involved in the ZRT1 pathway, and to determine how these genes affect minisatellite stability. Understanding the regulation of minisatellite stability in yeast could lead to effective treatment and prevention for many human diseases.Item The Identification of Girk Channel Domains that Facilitate Rapid and Efficient Coupling to G Protein- Coupled Receptors(2011-04-13) Young, DanieleG protein-gated inwardly rectifying potassium (GIRK or Kir3) channels constitute one subfamily of potassium-selective ion channels that regulate neuronal activity and heart rate. GIRK channels have been implicated in many biological processes, including pain perception, learning and memory, food intake, and reward. Structurally, GIRK channels consist of 4 subunits, able to form homotetramers or heterotetramers within the cell membrane. Activation of these channels occurs through a signal transduction cascade originating from ligandstimulated G-protein coupled receptors (GPCRs). This results in hyperpolarization of the cell membrane. With the variety of biological processes GIRK channels are involved in, understanding the interaction of the GPCR signaling cascade with GIRK channels could provide a beneficial therapeutic target. Using a variety of molecular biology techniques, I synthesized various chimeric proteins to investigate the GIRK channel and GPCR relationship, specifically focusing on amino acid residues promoting the coupling of GIRK1 with the GABAB receptor. Functional and biochemical assays in native systems suggest the Cterminal region and pore residue of the GIRK1 subunit are necessary to mediate a large receptor-induced response from the GIRK channel. Investigating this phenomenon further can determine how the identified GIRK1 channel domains are mediating efficient GABAB receptor coupling.Item Identification of the fla4 Mutation in Chlamydomonas reinhardtii(2009-04-08) Tank, DamienThe unicellular green alga Chlamydomonas reinhardtii is commonly used as a model organism to study flagella and their associated proteins. The fla4 mutant exhibits normal flagellar function at a permissive temperature of 21C, but sheds its flagella at a restrictive temperature of 32C. Transformation with large insert BAC clones had previously identified a candidate FLA4 gene. I confirmed the identity of the FLA4 gene by transformation with a smaller subclone to rescue the mutant phenotype. The flagella of two transformed strains functioned normally at the restrictive temperature. Using reverse transcriptase-polymerase chain reactions (RT-PCR), gel electrophoresis, and sequence comparisons between wild-type and fla4 DNA, I determined the complementary DNA (cDNA) sequence of the wild-type FLA4 gene and identified the specific mutation in fla4. The predicted amino acid sequence corresponds to a conserved TPR repeat protein. This protein shows significant similarity to proteins found in many higher organisms, such as human and mouse. In animals, this protein has been associated with defects in nervous system development, but its cellular function is unknown. Further study of its function in C. reinhardtii may provide insight into its role in higher organisms.Item Investigating the Effects of NOM1 Deficiency on Human Cell Growth(2009-04-08) Fares, NancyNOM1 (Nucleolarprotein with MIF4G domain 1) is a MIF4G/MA3 protein identified at the chromosome 7q36 breakpoint involved in 7;12 translocations associated with certain acute childhood leukemias. Hypothesis: Since NOM1 homologs are essential in both Saccharomyces cerevisiae and Caenorhabditis elegans, and since NOM1 interacts with a number of proteins involved in ribosome biogenesis, we predict that NOM1 is essential in human cells and that NOM1 deficiency will lead to decreased cell growth.Item Mechanisms of Androgen-Mediated Repression of the Maspin Tumor Suppressor Gene in Prostate Cancer(2009-04-08) Bader, DavidIt is estimated that one in six men in North America will be diagnosed with prostate cancer (PCa) during his lifetime. Localized PCa is often treated using surgery and radiation. Advanced and metastatic PCa can be treated by blocking the production or action of androgens, the male sex hormones. This androgen depletion therapy is only temporarily successful because PCa frequently returns in an androgen-refractory form that is resistant to hormonal manipulations and capable of growing in an androgen-depleted environment. Androgen receptor (AR) is a nuclear receptor transcription factor necessary for normal prostate cell growth and function as well as for growth of PCa. Androgens activate the AR, which translocates to the nucleus where it transcriptionally activates or represses target genes. One such gene is the Maspin tumor suppressor. Maspin is a proteinase inhibitor that serves to prevent proteinase degradation of the extracellular matrix, which is prerequisite to tumor invasion and metastasis. Androgens transcriptionally repress Maspin, but the mechanisms have not yet been fully characterized. To investigate the mechanisms of Maspin repression, a plasmid containing the luciferase reporter gene under the control of the Maspin promoter was constructed and transfected into VCaP and LNCaP PCa cell lines. Transfected cells were treated with dihydrotestosterone (a natural androgen) or mibolerone (a synthetic androgen) for 24 hours. Luciferase activity was subsequently measured by dual luciferase assay. These experiments have indicated that the AR may not directly repress Maspin transcription. Ongoing research will utilize real time PCR to determine whether AR inhibits Maspin transcription via a direct or indirect mechanism.Item Multicultural Genetic Counseling(2010-12-21) Zuck, TaylorConduct a preliminary investigation of multicultural issues and diversity in Genetic Counseling by searching for literature relevant to this topic. Currently, Bonnie LeRoy, my faculty mentor for this project and the director of the genetic counseling program here at the University of Minnesota is planning to write a text book on multicultural genetic counseling with Pat McCarthy Veach, professor of Educational Psychology and Associate Director for Research in the genetic counseling program. My project is to help in their endeavor in an exploratory research effort that will gather necessary resources related to the subject. Goals: To establish a better understanding of the prevalence of journal articles, research and other sources concerning diversity in genetic counseling. To identify specific sources and potential authors to be used for a multicultural genetic counseling textbook. Specifically, on the following 12 topics: Gender Race/Ethnicity Sexual Orientation/Lifestyle Poverty, International/Transnational Diversity in the Profession Multicultural Supervision The Culture of Living with a Genetic Condition/Disability Multicultural Curricular Pedagogy Religion/Philosophical Beliefs Values/Value Conflicts Defining Culture/Diversity/Models of Multicultural Practice. To determine where information and research is lacking in regards to multicultural genetic counseling.