Thursday, 8 August 2002 McKinly Laboratory University of Delaware |
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.