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Wolfe Hall Room 3246 Saturday April 15, 2000 9:15AM- 3:00PM

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Chart Abstracts 5th URS 5th URS Photos  UT Biological Sciences


(click on the presenter name for abstract)

Time

Speaker

Presentation Title

Advisor

9:15

Dr. Margaret Wheelock

Welcome

 

9:20

Plenary Session- Dr. Dorothea Sawiki, Medical College of Ohio “The Needs of A Simple RNA Virus"

10:00

 Coffee Break

Session I

10:30

Alisha K. Anderson

Identification of virus protein-protein interactions

Dr. Scoff Leisner

10:50

 Thomas Baltzell

Investigation of LLS1 function by multiple protein alignment and protein interactor hunt

Dr. John Gray

11:10

Omar S. Hajibrahim

Specific monoclonal antibodies against Strabismus

Drs. Margaret Wheelock and Keith Johnson

11:30

Sarah Jackson

P300 1-117 and its interaction with cDNA library of p19 cells.

Dr. Lirim Shemshedini

11:50

Nathan Pennings

Characterization of the perivitelline fluid proteins in Ascaris suum and their possible roles in hatching

Drs. Patricia Komuniecki and Emilo Duran

12:00

Pizza lunch

Session II

12:45

Kimberly Stookey

The effects of gene Il frameshiff mutation on symptom severity caused by CaMV strains CM1841 and Bari-I

Dr. Scoff Leisner

1:05

Fahim Malik

Differential expression of heat shock proteins as nematode community bioindicators

Dr. Deborah Neher

1:25

Andrea L. Woolley

Effect of hormones on cadherin expression in the vulvar carcinoma cell line A43 1

Dr. Jani Lewis

1:45

Wuraola Omotosho

Expression of a GFP-intermediate filament fusion protein in tissue fragility mutants of the nematode Caenorhabditis elegans

Dr. John Plenefisch

2:05

Christopher Buehrer

Screening for LLS1 partner proteins by the yeast two-hybrid assay

Dr. John Gray

2:25

 Break

2:45

Awards ceremony


Abstract 5th URS


Alisha K. Anderson
Identification of virus protein-protein interactions
Advisor: Dr. Scott Leisner

Many plant and animal viruses are similar. An understanding of resistance to plant viruses may lead to new strategies for controlling animal pathogens and vice-versa. Cauliflower Mosaic Virus (CaMV) is a plant virus that is similar to the human viruses hepatitis B (Hep B) and human immune virus (HIV). The CaMV genome contains six genes. Gene III encodes a protein involved in viral particle formation. In the absence of gene VI, the protein encoded by gene III (pIII) is unstable. The prediction was that pIII is stabilized by the gene VI product (pVI) through a direct interaction. If these two proteins interact, the disruption may lead to virus inhibition. We have employed the yeast two-hybrid system to examine possible pVI-plll interactions. Gene III was amplified by polymerase chain reaction (PCR) and inserted into pJG4-5. The plasmid attached pill to the activation domain of a transcription factor used in the yeast two-hybrid system. Gene VI had previously been inserted into pEG2O2, thereby attached to the DNA binding domain of a transcription factor. The yeast two-hybrid system indicates that pIll and pVI do not appear to directly interact. Hence, stabilization of pill by pVl must be more complex than simple protein-protein interaction.

Thomas Baltzell
Investigation of LLS1 function by multiple protein alignment and protein interactor hunt
Advisor: Dr. John Gray

LLS1 is a protein produced in maize whose purpose(s) has not been determined. Mutations of lls1 produce lesions on plant leaves, mimicking lesions produces by many plant diseases. Gray, et al. (1997) hypothesized that LLS1 lies in the cell death pathway of maize since mutation of lls1 ultimately leads to death of the entire leaf containing the mutation. Protein motif alignment studies indicate related function to methyl monoxygenases (i.e. chlorophyll a oxygenase), phenolic dioxygenases, and Tic55. Therefore, LLS1 is suspected to interact with other proteins in a fashion similar to these enzymes. Study of interactions with other novel proteins is one way to characterize a protein. The two-hybrid system is one system employed to detect interaction between LLS1 and other novel proteins encoded in the maize genome. The experiment involves the creation of a fusion protein between LLS 1 and Gal4-AD, as well as a fusion protein between the maize library encoded-proteins and Gal4-AD. The fusion proteins are expressed using yeast as a "test-tube." Binding of LLS1 to a novel maize protein brings the binding (BD) and activating (AD) domains into close proximity to form a complete Gal4 transcriptional activating unit capable of activating lacZ. The current experiment remains in progress with the bait construct (pTB-1) successfully created. The activating domain requires the creation of a maize cDNA library, which has not been completed. Therefore, the experiment remains a work in progress, and interaction between LLS1 and other proteins has not been evaluated.

Omar S. Hajibrahim
Speciflc monoclonal antibodies against Strabismus and the search for B-catenin interacting proteins
Advisors: Drs. Margaret Wheelock and Keith Johnson

The Wnt signaling pathway plays a critical role in cell fate determination during embryogenesis in animals, and tissue polarity in Drosophila. Mutations in this pathway can lead to its uncontrolled activation and give rise to formation of various human cancers. Mutations in frizzled, a receptor for the Wnt ligand, as well as Strabismus, alter tissue polarity and genetically link Strabismus to the Wnt signaling pathway. The first part of our experiment attempted to make a specific monoclonal antibody against Strabismus. A fusion protein was made with a piece of human Strabismus and then injected into mice. Antibody-secreting mouse B-lymphocytes were fused with myeloma cancer cells to produce hybridomas. Unfortunately, the hybridomas did not secrete a specific antibody against Strabismus. The second focus of our experiment was identifying novel protein interactions with B-catenin, specifically using the carboxy terminus. B-catenin is another protein that is involved in the Wnt pathway. Cell-cell adhesion is dependent on B-catenin, which forms a protein complex and ties the cytosolic domain of cadherins to the cytoskeleton. B-catenin also plays a significant role in cellular communication. Intracellular levels of B-catenin are controlled by Wnt signaling. Activation of the Wnt pathway results in increased levels of B-catenin. Phage display was used to identify potential B-catenin binding partners, and we found a novel interaction with a PDZ domain protein, TIP-1. The possible interaction was then verified by using in vitro binding assays.

Sarah Jackson
P300 1-117 and its interaction with cDNA library of p19 cells
Advisor: Dr. Lirim Shemshedini

p300ICBP is a co-integrator of several signaling processes leading to activation of various transcriptional activators. One portion of p300, namely amino acids 1-117, has been shown to interact with nuclear receptors during transactivation. In an attempt to better understand this activity of p300, we used a yeast functional screen to look for proteins that modulate the activity. Since p300 1-117 harbor a strong activation domain, we looked for proteins that interfered with this domain. The screen utilized a reporter plasmid encoding the B-Galactosidase gene and the results were several white colonies among a sea of blue colonies. We then made attempts to isolate the library plasmid from five white colonies in an effort to eventually sequence the portion of cDNA interacting with p300 1-117. Analysis of our five putative positive clones is still underway and the identification of the library cDNA is unknown at this time.

Nathan Pennings
Characterization of the perivitelline fluid proteins in Ascaris suum and their possible roles in hatching
Advisors: Drs. Patricia Komuniecki and Emilo Duran

Ascaris lumbricoides is a parasitic nematode that lives in the human small intestine and is currently estimated to infect over 1.5 billion people worldwide. It is very closely related to A. suum, a much easier worm to obtain and study, which inhabits the small intestines of pigs. Methods of controlling Ascaris chemo­therapeutically are presently being investigated. One such target involves manipulating the hatching process of the infective third-stage larva (L3). The L3s, and earlier embryonic and larval stages, reside within a very resistant eggshell and are bathed with perivitelline fluid (PF). The escape from the eggshell involves a complicated hatching process linking PF proteins and environmental triggers within the host. Two-dimensional gel electrophoresis analysis of the ascarid PF has revealed that it contains at least eight major proteins. One of these, As-p18, has been characterized as a novel, developmentally regulated, secreted fatty acid binding protein. In addition, the N-terminal amino acid sequence of another protein, As-p16, has been determined and it appears to be a transthyretin. As-p16 oligonucleotide primers and A. suum larval cDNA, which are both necessary to perform the polymerase chain reaction, have been prepared. Current efforts are moving towards finding the specific nucleic acid sequence of the gene which codes for this protein so that its structure and possible role in development and/or hatching of the A. suum larva may be better understood.

Kimberly Stookey
The effects of gene II frameshift mutation on symptom severity caused by Ca MV strains CM1841 and Bari-1
Advisor:  Dr. Scott Leisner

Viruses infect nearly all organisms, with disease characterized by the formation of symptoms within a host. Cauliflower mosaic virus [CaMV] is a plant DNA pararetrovirus containing seven genes that encode for seven different proteins. Gene II encodes PII, an 18 kDa protein whose primary function is to facilitate viral transmission by aphids, but this protein may also relate to symptom severity. To enhance our knowledge of symptom formation and severity, two strains of CaMV with mild symptomatology, CM1 841 and Bari-1, were used. Gene II of the viral clones was disrupted by restriction enzyme digestion and fill-in with DNA polymerase. The mutant viral genome was then excised from its vector and inoculated onto turnip plants. These experiments will permit us to determine the role of gene II in symptom formation.

Fahim Malik
Differential expression of heat shock proteins as nematode community bioindicators
Advisor Dr. Deborah Neher

Sensitivity of nematodes to chemical and metal pollution make them good ecological indicators of soil quality. Heat shock proteins (HSPs) may be used as biomarkers because they are induced by exposure to heat, metal ions or organic toxins. We hypothesize that environmental stress alters nematode growth and reproduction. Caenorhabditis elegans serves as reference to other bacterial feeding nematode species that represent different successional maturity values. C. elegans is transformed with a green fluorescent protein (GFP) gene inserted after the HSP promoter. In principle, the nematode will fluoresce green under ultraviolet light when stress is applied. However, stable transgenic lines have not been achieved due to complications in injection-quality DNA. Once stable transformants are produced, growth rate and fecundity of the transformed C. elegans will be quantified with and without heat and chemical stress. Growth and development will be compared between transformed and wild-type C. elegans and with other nematode species that have a similar habitat as C. elegans.

 

Andrea L. Woolley
Effect of hormones on cadherin expression in the vulvar carcinoma cell line A431
Advisor: Dr. Jani Lewis

A major characteristic in the development of cancer at the cellular level is loss of cell-cell interactions. Our research examined the adhesion molecules known as cadherins, specifically E- and P­cadherin. Mostly expressed in epithelial tissue, these cadherins are prime mediators of cell adhesion. Previous studies demonstrate the synthetic steroid, dexamethasone, downregulates E- and P-cadherin in the vulvar carcinoma-derived cell line A431. Since the hormones estrogen and progesterone are commonly used for HRT and birth control, the aim of this study was to determine if these hormones were capable of downregulation of E- and P-cadherin in A431 cells. Cell extracts were examined for the presence of estrogen and progesterone receptor expression by western blot using commercially available antibodies. These results indicated that A431 cells do express the B-form of the estrogen receptor but do not express the a-form or the progesterone receptor. Based on these findings, we hypothesized that treatment of carcinoma-derived cells with estrogen may downregulate cadherin expression in these cells; however, progesterone would not downregulate E- and P-cadherin. To test these hypotheses, cells were grown in presence and absence of different concentrations of estrogen, progesterone or combinations of both. At various timepoints after hormone exposure, cells were analyzed by immunofluorescence and Western blot to determine presence of E- and P-cadherin. Our preliminary data indicate that none of the concentrations of hormones are capable of downregulating E- or P- ­cadherin.

Wuraola Omotosho
Expression of a GFP-intermediate filament fusion protein in tissue fragility mutants of the nematode Caenorhabditis elegans
Advisor: Dr. John Plenefisch

The body-wall of Caenorhabditis elegans is made up of four layers: the body wall muscle, the basal lamina or basement membrane, the hypodermis and the cuticle. These layers are connected to each other and to muscle by a cell-cell and cell-matrix linkage system. In wild-type C. elegans, locomotion is achieved by the transmission of contractile force from the four longitudinal bands of muscle, through the basal lamina and the hypodermis to the cuticle. Tissue fragility mutants of Caenorhabditis elegans exhibit a post-embryonic flaccid paralysis, which results from the deficiency or disruption of cell-cell or cell-matrix linkages. These animals have mutations in genes that are required for the integrity of muscle attachment during post-embryonic growth and are denoted as mua for "muscle attachment defective". In particular, intermediate filaments in the hypodermis are involved in the transmission of muscle force to the cuticle through hemidesmosome-­like cell-matrix linkage sites. It is therefore expected that intermediate filaments in mua animals might be either absent or displaced. To test this possibility, I developed strains of mua animals that express a GFP-Intermediate Filament extrachromosomal array. The GFP- Intermediate Filament fusion protein gives off a greenish glow when exposed to ultraviolet light and was used a reporter protein. Examination of the transgenic mua animals under ultraviolet light revealed variations in the normal striated pattern of intermediate filaments that were specific to each mutation.

Christopher Buehrer
Screening for LLS1 partner proteins by the yeast two-hybrid assay
Advisor: Dr. John Gray

LLS1 (lethal leaf spot) is a gene found in corn, which when mutated, causes a disease lesion mimic phenotype. Through the investigation of alignment studies, I examined LLS1's relationship to other proteins that contain Fe-binding motifs. LLS1 does not appear to be more closely aligned with any one of the studied protein groups. A yeast two-hybrid study was performed to determine if any protein interactions existed with LLS1. Linking the open reading frame of the LLS1 gene to the binding domain of the Gal4 protein from yeast by cloning it into the pEG2O2 vector created a new plasmid, pCB- 1. An arabidopsis leaf cDNA library was obtained in which each sequence is linked to the activation domain of the Gal4 protein in the pJG4-5 vector. The pCB-1 plasmid and the arabidopsis leaf cDNA library were electroporated into yeast cells containing a lac Z reporter plasmid. The library was screened for interacting proteins by selecting for growth in the absence of leucine. If a protein interaction occurred, some colonies should grow in the absence of leucine while activating the lac Z gene, causing the colonies to turn blue. The controls suggested the protein constructs had been successfully inserted, but no blue colonies were observed. The results suggested either no proteins interact with LLS1, or the proteins from the arabidopsis library interact too weakly with LLS1.

5th URS Photos


Student Speakers
5th Presenters
Left to right, front row: Alisha Anderson, Sarah Jackson, Kimberly Stookey, Andrea Woolley, Wuraola Omotosho.  Back row: Omar Hajibrahim, Nathan Pennings, Christopher Buehrer, Thomas Baltzell, Fahim Malik.


Symposium Winners
5th Winners
Left to right: Omar Hajibrahim (1st place), Andrea Woolley (2nd place), Plenary Lecturer, Dr. Dorothea Sawiki of Medical College of Ohio, Thomas Baltzell (2nd place), Wuraola Omotosho (!st place), Alisha Anderson (2nd place), Dr. Margaret Wheelock, Symposium Coordinator.

last updated 29 Dec 2005
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