|David Carlson, Dickinson College, Carlisle, PA
(Advisor: Dr. Gordon Rule)
An Investigation into the Structure and Expression of the Human Class Pi Glutathione Transferase
Human glutathione s-transferase pi is a member of a family of proteins involved in cellular detoxification. These proteins catalyze a nucleophilic attack on many electrotrophilic compounds, including various carcinogens, by the reduced glutathione (GSH). In our investigation it was first necessary to increase the level of expression of human glutathione transferase PI-1 in E. coli. This was done by transforming E. coli. JM109 Ca competent cells with a plasmid known for its high-level bacterial expression of GST-PI-1 (Rdiger, K. H., et al. Protein Exp. and Purif. 6, 265-271, 1995). Next, the structure will be investigated by using high-resolution nuclear magnetic resonance spectroscopy. Of special interest will be mapping the substrate-binding site of PI.
|Kara Coval, Carnegie Mellon University
(Advisor: Dr. Jonathan Minden)
The Effect of Cell Death in Embryonic Head Development of Drosophila melanogaster
During Drosophila embryogenesis, an embryo develops through seventeen stages lasting approximately 24 hours. During this time, an embryo transforms from a single cell layered sac to a multilayered larvae. In particular, the larval head is formed by a process called head involution in which the anterior half of the embryo is completely internalized by the anterior sliding of a structure called the dorsal ridge. Embryonic cell death has been suggested to play a significant role in head involution. Although found throughout the embryo, embryonic cell death is especially abundant in the embryonic head region. In addition, embryos homozygous for the H99 deficiency, which uncovers three genes (reaper, head involution defective, and grim) that induce programmed cell death, lack embryonic cell death and do not undergo head involution.
In order to address the role of apoptosis in head involution, I first developed a method to quantitate the head involution process. Sex Combs Reduced protein (Scr ) is expressed in the labial and T1 segments which form the frontal boundary of head involution and move anteriorly as the head develops. Therefore, in order to observe the location of the T1 segment, and thus the level of head involution, indirect immunodetection (antibody staining) of Scr was used, and the position of the anterior boundary of Scr expression was measured in Wild type and H99 deficient embryos. In the H99 deficient embryos, the boundary failed to move as far anteriorly as the wild-type. Furthermore, to observe if cell death within a certain area is important for head involution, I inhibited cell death in small regions of the head and measured the effect on head development. Cell death was inhibited locally by expressing P35, a viral cell death inhibitor, in embryos carrying a UAS-P35 transgene. In these embryos, GAL4, a transcriptional activator, can induce the expression of P35. I used a technique call the photoactivated gene expression which allows UV light dependent activation of modified GAL4 protein. Modified, thus inactive, GAL4 protein was injected into embryos, and its activity was selectively activated in a small area within the head by UV light, allowing the expression of P35. When P35 was expressed at the anterior tip of the embryo, there was no profound effects evident. However, when P35 was expressed at the cephalic furrow, there was a slight difference in the position of the Scr expression compared to the control.
|Erica Eichers, Willamette University, Salem, OR
(Advisor: Dr. John Pollock)
Alternative transcripts of lozenge in Drosophila
The lozenge gene in Drosophila melanogaster encodes a transcription factor necessary for development. Both lozenge and its human homolog AML1 (acute myeloid leukemia) impact the differentiation of sensory organs and hematopoetic cells. AML1 is known to have nine different splice isoforms that exert both positive and negative regulation of key genes. In lozenge, we know of only two alternative transcripts, yet have reason to believe others exist. Previous experimentation has suggested that particular exons of the lozenge gene are duplicated within the genomic DNA. Specifically, we are investigating the possibility of a duplicate exon VI. It is thought that an extra exon VI may exist farther downstream in the gene. Southern Blot analysis was done with various digests of cosmid clones representing over 30 kb of lozenge genomic DNA probed with exon VI specific sequences. No definitive evidence was seen to prove the existence of an additional exon VI, but further analysis must be done before a conclusion can be made. Identifying the alternative transcripts of lozenge and studying the locations and extent to which each is expressed offers avenues for learning more about cell fate determination and neural development. The results can also be used to complement studies with AML1.
|Laura Goodwin, Washington & Lee Univ., Lexington, VA
(Advisor: Dr. Adam Linstedt)
The Role of MEK-1 in Golgi Fragmentation During Cell Division
Our aim is to address the mechanisms by which the organization of the Golgi membranes is regulated during the cell cycle. Mitotic phosphorylation is likely to play a key role in this organization. In vitro studies with a selective inhibitor (PD98059) have shown that Golgi fragmentation depends on the activity of cytosolic mitogen-activated protein kinase kinase 1 (MEK-1). However, our experiments utilizing the same inhibitor demonstrate that MEK-1 activity is not required for Golgi breakdown in vivo. HeLa cells were cell-cycle synchronized with a thymidine double block and then released in the presence or absence of PD98059. An examination of Golgi breakdown in control and experimental cells by simultaneous staining of the Golgi, microtubules and DNA revealed that the Golgi stacks of the PD98059-treated mitotic cells undergo fragmentation indistinguishable from the fragmentation of control cells. Inhibitor activity was verified by demonstrating that PD98059 prevented the spindle assembly checkpoint-mediated arrest of cells in nocodazol. It has been shown previously in Xenopus extracts that this checkpoint requires the MEK-1 pathway. Taken together our results demonstrate that MEK-1 activity is required for checkpoint mediated mitotic arrest but not for mitotic Golgi breakdown in vivo.
|Fiona Mack, Cornell University, Ithaca, NY
(Advisor: Dr. David Hackney)
The Effects of Kinesin Tail Domain from Position 841-952 on Dimerization
Kinesin is a microtubule-associated ATPase containing two 120 kD heavy chains and two 64 kD light chains. The heavy chain of this molecular motor is a 975 amino acid protein consisting of a globular head domain connected to a small nonhelical tail region by an alpha helical coiled-coil stalk. Deletion of the 81 C-terminal amino acids results in the loss of folding ability, indicating that these regions are likely to bind to the head domain. A His-tagged construct for a fusion protein between thioredoxin and amino acids 841-952 of kinesin was created by PCR amplification with appropriate primers and transfected into competent cells. The lysate from induced cells was run through a nickel column and the protein eluted with a high imidazole solution. Peak fractions from the nickel column were then gel filtered through an S-300 column, the results of which showed that construct Trx 841-952 is not only dimeric in solution, but also highly asymmetric. This indicates that the amino acids residues 841-952 are sufficient for forming a coiled-coil segment.
|Tiffany Miles, Washington & Lee Univ., Lexington, VA
(Advisor: Dr. Tracy Ripmaster)
Regulation of Gene Expression - A Different Approach: Inhibition of Protein Synthesis by Steric Blockage of the Ribosome by Theophyline
The ability to control the expression of a gene product experimentally is an important tool used to study the function of genes. Currently, transcriptional control systems are used for temporal expression of gene products. The drawbacks to these approaches include replacement of the naturally occurring promoter of the gene to be expressed with a transcriptionally regulated promoter and often the expression of a protein that allows control of the engineered promoter to be repressed or activated. The use of a translational repression/activation system that could be controlled by the presence or absence of a small molecule would simplify gene expression systems by eliminating the requirement to replace the cognate promoter and to express an inhibitor/activator protein. Recent RNA selection experiments have identified a small RNA molecule capable of binding theophyline with very high affinity and specificity. To test whether this short RNA sequence could block expression of a gene in the presence of theophyline in vivo, this RNA sequence was inserted in between the galactose promoter and the lacZ gene in a vector designed to be expressed in Saccharomyces cerevisiae. After transformation into yeast cells, enzymatic assays were performed to determine if this RNA sequence inhibited production of beta-galatosidase when yeast were grown in the presence of theophyline. Future experiments will be designed to determine if inhibition of enzyme production is due strictly to translational control.
|Therese Mitros, University of Notre Dame, South Bend, IN
(Advisor: Dr. Javier Lopez)
Studies on cis- and trans-acting Components that Regulate Alternative Splicing of Ultrabithorax RNA
Ultrabithorax (Ubx) is a homeotic gene which controls the development of the posterior thoracic and abdominal regions of Drosophila melanogaster. Ubx RNA is alternatively spliced into 6 isoforms which differ in the inclusion of three internal elements: the B element, which results from an alternative splice site at the end of the 5' exon, and two internal microexons (mI and mII). This alternative splicing is regulated spatially and temporally during development. We are interested in the cis- and trans-acting components involved in regulation of mI. Sequences within mI itself are required for its inclusion in Ubx mRNAs. To determine whether downstream intron sequences also play a role I created deletions immediately downstream of the microexon I 5' splice site. Coupled reverse transcription-PCR analyses showed that proximal intron 2 sequences are required for the retention of mI in Ubx mRNAs. Previous work has also shown that microexon II is required for retention of mI in Ubx mRNAs. To determine whether this involves the function of special sequences in mII, I analyzed the splicing of mI in RNAs where mII was replaced with an artificial exon of similar size but unrelated sequence. RT-PCR showed that the artificial exon could substitute functionally for mII to retain mI. Previous genetic analysis has shown that deletions in the region which codes for the splicing factor Rbp-1, a member of the SR family of RNA-binding proteins, cause exclusion of exon mI from Ubx mRNAs. Rbp-1 is a phosphoprotein. Deletions of the Rbp-1 region were tested for synthetic lethality or visible phenotypes in combination with mutations in the gene for an SR protein kinase, Darkener of apricot (Doa); however, no interaction was found. A possible effect of Doa on Ubx splicing is being tested by RT-PCR.
|Miguel Nogueras, University of Puerto Rico, Mayaguez, PR
(Advisor: Dr. Susan Henry)
The Effects of the OPI1 Gene Products on the Transcriptional Activity Mediated by the Yeast Ino2p Transcription Factor
In the yeast Saccharomyces cerevisiae, the biosynthesis of phospholipids is regulated in response to the level of precursors such as inositol and choline. Inositol is produced from inositol-1-P, which is in turn produced from glucose-6-P in a reaction that is catalized by the INO1 gene product: inositol-1-P synthase. The INO1 gene is the most highly regulated gene among the coordinately regulated yeast genes encoding enzymes of phospholipid biosynthesis. It is regulated by two classes of regulators: positive regulators such as Ino2p and Ino4p; and negative regulators such as Opi1p. Ino2p and Ino4p interact to form a heterodimer that activates the expression of the INO1 gene through UASIno elements located in the promoter. In the presence of inositol, INO1 gene expression is repressed and when inositol is not present the Ino2p-Ino4p complex activates the expression of INO1. In opi1 mutants, INO1 is constitutively expressed regardless of the inositol level. The repression requires Opi1p, but the mechanism of Opi1p action is not known at present. There is evidence that Opi1p does not bind to either Ino2p or Ino4p directly. Furthermore overexpression of the OPI1 gene does not affect the interaction intensity between Ino2p-Ino4p as measured in the two hybrid assay. In this study, OPI1 was overexpressed in the two hybrid system in order to see how Opi1p affects the transcriptional activity mediated by the yeast Ino2p transcription factor. Here we used the yeast SFY526 ino2 ino4 strain to overexpress OPI1. The transcriptonal activity of the Ino2p was measured by the activity of the Lac Z reporter gene using a b-galactosidase liquid assay. We observed no changes between our control cells containing the vector and the cells overexpressing OPI1. We also assayed activation mediated by Ino2p in SFY526 ino2 ino4 opi1D to exclude the potential effects caused by endogenous OPI1. The results were very similar to those obtained in cells containing endogenous OPI1 gene. Thus we conclude that Opi1p does not act to repress INO1 gene expression by altering the activation potential of Ino2p.
|Zandra Ruiz, Oglethorpe University, Atlanta, GA
(Advisor: Dr. Charles Ettensohn)
Effects of Proteoglycan Biosynthesis Inhibitors on Primary Mesenchyme Cell-Extracellular Matrix Interactions in the Sea Urchin Embryo
Normal skeletogenesis in the sea urchin embryo is contingent upon the presence and successful migration of primary mesenchyme cells (PMCs) to form a subequatorial ring and ventrolateral clusters. Primary mesenchyme cell migration occurs through the dynamic interactions of the filopodial processes of the PMCs with the extracellular matrix (ECM) fibers deposited on the basal lamina of the blastocoel wall. Previous research has revealed that the inhibition of proteoglycan synthesis, by way of treatment with exogenous xylosides or sulfate deprivation, result in PMC migration-defective embryos. It has been hypothesized that the defect in migration may be attributable to the inability of the primary mesenchyme cells to produce or properly express certain proteoglycans. In the present study, indirect immunofluorescent techniques are utilized in an effort to observe the effects of exogenous xyloside treatment and sulfate deprivation on the interactions between PMCs and ECM3 fibers in L. variegatus embryos. The results obtained with the PMC-specific antibodies 6a9 and 6e10, as well as ECM3-specific antibodies indicate that both sulfate deprivation and xyloside treatment do not interfere with the ability of the PMCs to bind with the ECM3 fibers, and do not alter molecular components which are required for the ingression and migration of the PMCs. Despite the ability of the PMCs to migrate, these cells were unable to form the subequatorial ring characteristic of early development. In addition, the patterning and distribution of the ECM3 fibers of the extracellular matrix were disrupted in the presence of exogenous xyloside. This finding indicates that ECM3 is possibly a proteoglycan molecule, consistent with the sequence similarity between ECM3 and a mammalian chondroitin sulfate.
|Eric Slivka, University of Richmond, Richmond, VA
(Advisor: Dr. Fred Lanni)
Model Extracellular Matrix for Studying Cell Growth of 3T3 Fibroblasts
The development of model extracellular matrices (ECMs) has important applications in both the study of isolated cells and in the development of artificial tissues using cells taken from a donor. Research in this lab focuses on the forces exerted on the ECM by actin fibers within the cell. Specifically, my work examines improved techniques for creating model ECMs to allow for better and easier examination of cellular forces. The incorporation of collagen, a major component of the ECM in skin and connective tissue, into an inert polyacrylamide hydrogel provides cells with a more native environment on which they can thrive. A protein crosslinking agent known as sulfo-SANPAH was used to bind collagen to the surface of the polyacrylamide gels. Swiss 3T3 fibroblasts adhered to the surface of the modified gel were subjected to a serum-deprived environment, causing the cells to become quiescent. Upon stimulation with serum, the cells experienced cytoskeletal contractions for a period of approximately one hour before resuming normal activity. Time lapse images were taken in order to observe the contractions as they took place.