Summer Undergraduate Research Symposium
Thursday, 8 August 2002
McKinly Laboratory
University of Delaware
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Abstracts for Oral Presentations in 061 McKinly Laboratory 1:00 – 3:00 PM

1:00 PM  Erin Hill  2:00 PM  Stephanie Miller
1:15 PM  Bevan Kirley 2:15 PM  Christopher Eller
1:30 PM  Arthur Suckow 2:30 PM  Brandy Heckman
1:45 PM  Alice Wong 2:45 PM  Dana Ungerbuehler

1:00   Nucleotide Sequence of the Apolipoprotein A-II Gene in Trachemys scripta elegans
Erin Hill, Lauren Baker, Liz Manning, Stacey Karr, and Robin Davis
(Sponsors - David Usher and Robert Hodson, Biological Sciences)

Apolipoproteins of the red-ear slider turtle Trachemys scripta elegans are being studied to compare the cDNA and gene sequences to other vertebrate species. By comparing sequences from such evolutionarily distant animals, conserved regions should indicate functionally important domains. The gene studied in this project is apolipoprotein A-II (apoA-II). Previously some of this gene’s sequence was determined, including in the 3’ direction, most of Intron 2, Exon 3, Intron 3 and Exon 4. The complete sequence has now been found for Exon 1, Intron 1, Exon 2 and Intron 2 using gene “walking” techniques, yielding a genomic sequence nearly 3 kb long. Comparisons of the genomic DNA of turtles and other animals have not shown much homology along the gene; however, alignments of the cDNA sequences have shown conserved regions, particularly throughout the coding region

1:15 P.M.    Genetic Characterization of HR Despite NOS Inhibitor (hdn) Mutants
Bevan Kirley Barb A. Farnworth, JoAnne Z. Lynch, and Hajime Sakai
(Sponsor - Allan Shapiro, Plant Science)

Activation tagging is a mutagenesis procedure by which a strong enhancer is inserted randomly throughout the genome.  The result is usually the overexpression of the gene closest to the insertion site.  Activation tagged mutants were selected that showed a hypersensitive response (HR) despite the presence of a nitric oxide synthase inhibitor (hdn mutants) that has been previously shown to block the HR.  This indicates that these  mutants are bypassing the hypothetical pathway leading to the HR.  The HR is a programmed cell death (PCD) response resulting in the controlled death of the affected area.  In one mutant, hdn-101, a likely candidate for the overexpressed gene has been identified and experiments are underway to confirm this.  Phenotypically, the HR is associated with irreverisble damage to the plant plasma membrane resulting in ion leakage from the cells.  This leakage can be measured macroscopically by measuring the conductivity of leaf discs in distilled water.  Wild-type plants display a characteristic sigmoid increase in conductivity with time after an initial ~3.5 h lag.  By contrast, mutant hdn-101 displays a much shorter lag phase (~2 h) and a much more rapid initial increase in ion leakage.  These results indicate that the rate-limiting step for committment to PCD has been overcome in this mutant.  However, the final level of PCD is similar to wild type.  This result highlights the function of negative feedback control in limiting the limiting the extent of programmed cell death.

1:30 P.M.   Ocular Expression of the JAM Family Members
Arthur Suckow (Sponsor - Melinda Duncan, Biological Sciences)

The three members of the junctional adhesion molecule (JAM) family of cell adhesion molecules (CAMs), JAM-1, JAM-2 and JAM-3, are all members of the immunoglobin superfamily.  Recently, our group became interested in this family of CAMs when a cDNA microarray analysis on transgenic mice overexpressing PAX-6 in the lens revealed that JAM-1 mRNA expression was 2.5 fold elevated over normal.  More recently, another group established that heterozygous small eye mice containing mutations for the PAX-6 gene lack functional cell adhesive properties in their corneas.  These data suggested that JAM-1 is present in both the lens and the cornea and that its gene expression is regulated by PAX-6, a transcription factor that is essential for normal development of eye tissue.  In order to confirm the presence of JAM-1, RNA was isolated from both the corneas and lenses of adult mice, as well as from 15-day embryos where the JAM-1 transcript is known to be expressed.  Primers were then designed and the presence of JAM-1, JAM-2 and JAM-3 RNA was confirmed in both the lens and the cornea by RT-PCR analysis.  This analysis also found that JAM-2 RNA was preferentially expressed in the lens and that JAM-3 RNA was preferentially expressed in the cornea.  At the protein level, the presence of the JAM-1 protein in the lens and the cornea was confirmed by both immunohistochemistry and western analysis.  Currently, a developmental expression study of the JAM-1 protein in eye tissue is underway.

1:45 P.M. Receptors for the Binding of Eristostatin to Melanoma Cells
Alice Wong and Carrie Paquette-Straub (Sponsor - Mary Ann McLane, Medical Technology)

Eristostatin, a disintegrin isolated from the viper Eristocophis macmahoni, can interact with many types of cells, including platelets, endothelial cells, and melanoma cells, via integrin receptors expressed on the cell surface. Eristostatin’s ability to inhibit melanoma metastasis may therefore involve an integrin. Human MV3, 1205 LU, WM164, and C8161 melanoma cells were incubated with fluorescent-labeled antibodies to the integrin ?vb3, the integrin subunits alpha-2, alpha-4, alpha-6, and beta-1, and observed by confocal microscopy. All cells express alpha-2 while none express detectable levels of alpha-6. The integrin subunit alpha-4 is expressed by 1205LU, MV3, and WM164, but not by C8161. MV3 is the only cell line not to express alpha-vb3. Studies were also done to assess the binding of eristostatin and a panel of mutations to anti-alpha-4. These results suggest that while the critical residues involved in the binding of eristostatin to the melanoma cells vary, eristostatin requires an intact RGD sequence in order to inhibit binding of anti-alpha-4 to the cells. Additionally, flow cytometry and immunoblotting techniques are being used to examine the binding of eristostatin to melanoma cells.

2:00  P.M.  Effects of HIP/RPL29 Knock-down on In Vitro Chondrocyte Differentiation
Stephanie Miller (Sponsors Mary C. Farach-Carson andCatherine Kirn-Safran, Biological Science)

Heparin/heparan sulfate interacting protein (HIP) is a small, highly basic protein identical to ribosomal protein L29 believed to participate in multiple cell processes, such as cell adhesion, protein synthesis and potentiation of growth factor activity. In situ hybridization and immunohistochemistry showed that HIP/RPL29 is tightly expressed during chondrocyte terminal differentiation. To investigate the role of HIP/RPL29 normal expression during cartilage formation, we designed a ribozyme approach to knock-down HIP/RPL29 expression in a cell culture model for chondrogenesis. The multipotent mouse embryonic skin fibroblast cell line C3H/10T1/2 was stably transfected with an expression vector driving high ubiquitous nuclear expression of a ribozyme flanked by either HIP/RPL29-targeted or control scrambled sequences.  Clones obtained after zeocin selection were further analyzed for integration and expression of the ribozyme constructs using PCR and RT-PCR, respectively.  Semi-quantitative analysis of HIP/RPL29 by Northern and Western Blotting identified at least two clones in which HIP/RPL29 expression levels are perturbed at both mRNA and protein levels.  Preliminary studies showed in one ribozyme-transfected clone that reduced levels of HIP/RPL29 inhibits cell growth and accelerates differentiation of C3H/10T1/2 into cartilage-like cells. These data suggest a role for HIP/RPL29 as a regulator supporting cartilage growth.  Additional clones will be analyzed to demonstrate that reduced expression of HIP/RPL29 is associated with fast progression towards a more differentiated chondrocytic state. Studies on the effect of HIP/RPL29 knock-down will shed light on cartilage formation and maturation of adult cartilage.

2:15 P.M. Substrate Assisted Protein Folding in Pseudouridine Synthase Mutants
Christopher Eller (Sponsor - Eugene Mueller, Chemistry and Biochemistry)

Pseudouridine synthases catalyze the conversion of uridine to pseudouridine (?) in RNA.  Four families of ? synthases have been identified, represented by TruA, TruB, RluA, and RsuA in Escherichia coli.  While homologous, the four families have no statistically significant sequence similarity.  A single aspartic acid residue is the only absolutely conserved residue, and it is essential for catalysis [Ramamurthy V., Swann, S.L., Paulson, J.L., Spedaliere, C.J., and Mueller, E.G. (1999) J. Biol. Chem. 274, 22225-22230].  Three of the families share a short sequence motif that contains adjacent lysine and proline residues.  Mutagenesis of the conserved lysine and proline residues in TruB and RluA resulted in proteins with circular dichroism (CD) spectra of significantly reduced intensities, suggesting a loss of secondary structure associated with protein unfolding.    The lysine and proline mutants are less stable, as indicated by storage behavior and a decreased melting point.  Despite the apparent loss of structure, the catalytic activity of these altered enzymes was only mildly impaired [Spedaliere, C.J., Hamilton, C.S., and Mueller, E.G. (2000) Biochemistry 39, 9459-9465]. In light of these observations, CD spectra and melting curves were recorded for both wild-type TruB and the P20G mutant in the presence of a 17-mer RNA substrate, the T-arm stem loop of tRNAPhe, to determine if RNA binding to the unfolded protein stabilizes the native conformation, which will be detected as an increase in the CD spectra intensity and the melting temperature.

2:30  P.M. Genomic Applications of Stabilized Synaptic Complexes
Brandy Heckman, Michael Rice, and Michael Usher
(Sponsor - Eric Kmiec, Biological Sciences and the Delaware Biotechnology Institute)

The recombination protein Escherichia coli RecA has been shown to catalyze strand exchange between both single and double-stranded DNA.  In vitro experiments have shown RecA forms a stable filament when added to single stranded DNA with the use of ATPgS, or other non-hydrolyzable ATP analogs.  This filament, when incubated with a larger double-stranded homologous target, forms a triplex nucleic acid structure known as a displacement loop or D-loop.  The addition of a second modified oligonucleotide, complementary to the initial incoming oligonucleotide, results in the formation of a complement stabilized double displacement loop or double D-loop.  Upon the removal of RecA protein, evidence has shown D-loops remain stable only in supercoiled DNA targets.  Double D-loops, however, have been shown to be much more stable not only in supercoiled DNA, but also in linear and genomic DNA targets.  Because of its increased stability, double D-loops help to provide a number of opportunities for diagnostic applications.

2:45  P.M.   Investigating Mixed Chain Kinetics of P22 Tailspike
         Dana Ungerbuehler (Sponsor - Anne Robinson, Chemical Engineering)

Misfolded and aggregated proteins are implicated in several disorders such as Alzheimer’s, Cystic fibrosis, and prion diseases.  The lack of productive folding is thought to play a major role in the progression of these diseases, and some treatments are being sought in minimizing the tendency towards misfolding or maximizing the efficiency of these proteins.  The kinetics of protein folding is an area of great interest in biochemical engineering and pharmaceutical industries, and are extremely complex to model in vivo.  P22 tailspike is a protein-folding model due to its complex molecular interactions and oligomeric structure.  While the native trimeric structure is not disulfide bonded, evidence exists for a folding intermediate with oxidized sulfhydryl groups.  A C-terminal truncation revealed that the cysteines at 496, 613, and 635 were the most likely sites of sulfhydryl activity.  Cysteine to serine point mutations at the 496, 613, and 635 residues were purified in trimer form.  Initial analysis of the single serine mutants revealed that they folded 2-3 times slower than wild type and with only 65-80% of wild type yields.  In vitro refolding studies were performed by mixing different single mutants to investigate if wild type yield and assembly kinetics could be recovered.  Combining mutant chains has shown qualitative improvement in the yields, however the kinetics of folding appears to remain unchanged.  Thus, allowing mixed chain interactions do not produce more rapid folding, yet may produce more efficient folding.  More research will be conducted to characterize these interactions and investigate more favorable conditions for productive folding.

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Created 1 August 2002, Last revised 5 August 2002 by Hal White
Copyright 2002 Harold B. White, Department of Chemistry and Biochemistry, University of Delaware