1999 NSF Participants-Department of Biological Sciences - Carnegie Mellon University

1999 National Science Foundation (NSF)-supported Participants

Charles AndersonCharles Anderson, University of Maryland, Baltimore County
(Advisor: Dr. Charles Ettensohn)

The Effects of Lithium Chloride on Primary Mesenchyme Cells During Embryonic Development Displayed by Sea Urchin Embryos
Previous research has shown that b-catenin provides the micromeres in sea urchin embryos with the ability to differentiate as primary mesenchyme cells, PMCs. As well, LiCl has been shown to inhibit the activity of a negative regulator of b-catenin, glycogen synthase kinase 3, GSK-3. Thus, it stands to reason that increasing levels of b-catenin by using the classical vegetalizing agent, LiCl, will cause an increase in the number of PMCs. Our research tackles this particular reasoning and hints at the relationship between b-catenin and LiCl using PMCs. As early as the 1800's LiCl has been found to be a vegetalizing agent, causing an excess of endodermal and mesodermal cell types at the expense of ectodermal cell types. How LiCl affects the particular mesodermal cell type, primary mesenchyme, is still unclear. We treated sea urchin embryos with differing amounts of a 0.5M LiCl solution and allowed the embryos to grow to a developmental stage characteristic for the primary mesenchyme cell ring. We used a mesenchyme specific protein marker, 6a9b10, specific for PMCs, to stain the cells for counting. Endo 1, a marker which labels hindgut and midgut, was used to confirm that endodermal tissue increases with increased levels of the vegetalizing agent. Indirect immunofluorescence allowed us to count PMCs and determine that treatment with LiCl causes an enhancement in the number of PMCs. Increased numbers of PMCs may mean that LiCl caused more b-catenin to accumulate in micromere nuclei, cells known to give rise to PMCs.

Rashad ArcementRashad Arcement, Xavier University of Louisiana, New Orleans, LA
(Advisor: Dr. John Pollock)

Western Blot Analysis of Runt Expression in Lozenge Mutants of Drosophila melanogaster
Drosophila melanogaster is known to contain three members of the runt homology domain family of transcription factor proteins; RUNT, RUNT-RELATED and LOZENGE. The runt homology domain contains a DNA binding site, a protein-protein interaction site, and a putative ATP binding site. Furtermore, these proteins end with the same VWRPY amino acid sequence. Runt works by forming heterodimers with other proteins. The proteins BROTHER and BIG BROTHER have been predicted to be b partners to form the dimer that makes RUNT active. The protein called GROUCHO on the other hand, is known to target the pentapeptide sequence (VWRPY) and represss transcription. The protein-protein interaction sites in RUNT and LOZENGE are so similar that they could be competing for the same subset of partner proteins. The Pollock lab has shown that both LOZENGE and RUNT are involved in eye development . I am interested in knowing how perturbations in LOZENGE expression may affect RUNT expression. Using the western blot technique I analyzed the pattern of RUNT expression in normal developing eyes as compared to different lozenge mutants including lzr1 and lzmr1. These studies complement additional in situ expression studies carried out on intact developing eyes.

Erin BaldwinErin Baldwin, Berry College, Mt. Berry, GA
(Advisor: Dr. Jonathan Minden)

Identifying Proteome Differences in Drug- and Alcohol-Intoxicated Drosophila melanogaster using DIGE
The biological processes involving drug and alcohol abuse have yet to be understood. Using Drosophila melanogaster as a model, it has been found that cocaine response pathways are evolutionarily conserved and that ethanol intoxicated flies have similar responses to those of intoxicated higher organisms (McClung 1998; Moore, et. al 1998). The goal of this project was to identify proteome differences in the brains of drug- and alcohol-intoxicated flies relative to untreated flies. Protein isolated from fly heads was analyzed using a two-dimensional gel electrophoresis technique called Difference Gel Electrophoresis (DIGE). This method compares two samples in the same gel, using different color fluorescent dyes to tag the proteins of the samples to be compared, such as the heads of flies on cocaine versus untreated fly heads. Images of the gels were taken at different fluorescent wavelengths to visualize the different samples. Proteins that are different between samples appear as spots composed of more than one dye than the other. Protein changes were found between drug- and alcohol-intoxicated flies and their respective untreated controls. Most of the differences were common across the samples; thus, it appears that protein degradation may have occurred. Mass spectrometry was used to generate a peptide fingerprint to identify the protein differences.

Kelly BoenemanKelly Boeneman, Alma College, Alma, MI
(Advisor: Dr. Javier Lopez)

Identification of trans-acting factors on alternative splicing of Ultrabithorax
Alternative splicing is an important strategy for gene regulation, but its mechanisms are poorly understood. The homeotic gene Ultrabithorax (Ubx) of Drosophila melanogaster is a useful model system that allows genetic approaches to this problem. Alternative splicing of Ubx transcripts produces six mRNA and protein isoforms that differ in their inclusion of three elements-mI, mII and B. The isoforms are produced in distinct ratios in different tissues as a consequence of competition between 5' splice sites that either retain or remove these elements. Previous investigations in the lab have identified several trans-acting factors required for inclusion of exon mI. Three of these, encoded by the genes sans-fille (snf), virilizer (vir) and fl(2)d are also required for the female-specific alternative splicing of Sex-lethal (Sxl) RNA. The product of sans-fille is a homolog of human splicesome components U1A and U2B''. Previous work in the lab had revealed a dominant female-specific lethal interaction between loss-of-function mutations in snf and vir or fl(2)d, suggesting that their products function at a common step, perhaps as a complex. To identify additional factors that may function at this step, I tested other mutations known to reduce female-specific splicing of Sxl (i.e., l(2)49DbTW6, hel25E1) or inclusion of mI in Ubx mRNA's (i.e., Df(3R)l26c, Df(3R)Rdl-2, Df(3L)29A6, Df(2L)TW158, DF(2L)TW203 and several EMS-induced mutations) for synthetic lethality with snfe8h. l(2)49DbTW6, hel25E, and EMS-28 exhibited a dominant female-specific lethal interaction with snfe8h. In addition, RT-PCR analysis revealed that hel25E, like snf, vir, and fl(2)d, is required for inclusion of mI in Ubx mRNA's, thus identifying a fourth component that is shared in the regulation of Ubx and Sxl splicing. The product of hel25E is a putative RNA helicase whose function has not been defined up to this point.

Shantay DinksShantay Dinks, University of Maryland, Eastern Shore
(Advisor: Dr. William Brown)

Improvement of kinetic parameters associated with abzymes used for polyurethane degradation
The production of polyurethanes results in large amounts of waste products that have no distinct environmentally conscious way of being bioremediated. One solution to this problem is the creation of a catalytic antibody termed a single chain variable fragment, or ScFv. Previously, seven ScFv's were isolated, and of these, ScFv #9 had the highest enzymatic activity to hydrolyze the carbamate bond in MDI-based polyurethanes. If the three-dimensional structure of one of the other low catalytic activity ScFv's was determined, it could be used as a comparison to help improve the catalytic activity of ScFv #9. Therefore, the goals of this study are to develop a method of purifying large quantities of ScFv #1, #8, and #12 in order to attempt crystallization and obtain the three-dimensional structures. In this study, each of these proteins was found to be located in inclusion bodies both before and after IPTG induction which led to the conclusion that denaturing agents have to be used to extract these proteins from the inclusion bodies. However, in previous studies, proteins extracted with the use of denaturing agents have been shown to have difficulties in refolding when these agents were removed. Consequently, a purification method using affinity chromatography was designed and applied to ScFv #1 and the protein seemed to have no difficulty refolding upon renaturation. This finding will allow for future large-scale purification and crystallization of ScFv #1 for comparison with ScFv #9.

Cuauhtemoc Gomez BarrosoCuauhtemoc Gomez Barroso, Instituto Tecnologico Y de Estudios Superiores de Monterrey, Monterrey, Mexico (Advisor: Dr. Frederick Lanni)

Effect of fibronectin content in a collagen model ECM on the attachment and behavior of fibroblasts
Fibroblasts build an actin-based cytoskeleton for particular tasks and according to molecular signals in the extracellular fluid (ECF) and the extracellular matrix (ECM). In response, the action of the cells modifies or remodels the ECM. We have been studying a well-defined cytomechanical task: the contraction of collagen gels by 3T3 fibroblasts, in order to discover the elementary steps of this process. Biologically, collagen contraction occurs in wound-healing and in the development of embryonic tissue. It is also relevant to the growth of artificial (replacement) tissues and organs. The experiments in this project comprised two main lines of work:

The first part centered on the growth of 3T3 cells on collagen (I) gel surfaces with known amounts of superfibronectin, a functionally distinct form of fibronectin. Animal cells use receptors, called integrins, to bind to extracellular matrix proteins, such as collagen, fibronectin, and laminin. The type of collagen constituting the gels (type I) accounts for 90% of body collagen, and it can be found in bone, skin, internal organs, and other tissue. The concentration of superfibronectin in the collagen gels ranged from 0% to 0.01%. Twenty of the thirty-six specimens obtained were fixed at different stages of growth with para-formaldehyde, and then stained with rhodamine phalloidin, in order to study them by means of fluorescence microscopy. Also, all thirty-six specimens were studied using phase contrast microscopy. No clear and unequivocal relationship was found between the amount of superfibronectin in the gels and the cells’ behavior on them, at these concentrations of superfibronectin. Future directions in this area will most probably include increasing the concentration of superfibronectin in the collagen gels on which the fibroblasts are to be grown.

The second part of the project focused on launching the process of making GFP-b -actin-expressing 3T3 cells, so as to enable the study of their cytoskeleton in vivo. The GFP-b -actin vector was successfully introduced into E. coli, which effected its amplification. The plasmid DNA was then purified, and approximately 200 nanograms of it were obtained. This DNA will later be used to transfect 3T3 cells, which may then be used for studies in collagen gel systems in which microinjection is difficult.

Alexander LangeAlexander Lange, Muskingum College, New Concord, OH
(Advisor: Dr. Amy Csink)

Intraspecific Variation in Satellite DNA Among Natural Populations of the Melanogaster Species Subgroup
Highly repetitive satellite DNA is found in the heterochromatic region of the chromosomes of Drosophila. Although earlier studies have been limited, they do suggest that satellite DNA qualitatively varies greatly between closely related species, but does not vary within a species. The study compared satellite DNA sequences of five natural populations of D. melaongaster and five of D. simulans from throughout the world to determine if any intraspecific variation in these sequences may account for speciation. The populations used in the study are ancestral or are thought to be undergoing speciation. Ten different satellite sequences were examined by means of in-situ hybridization of mitotic chromosomes. The results of the study showed very little intraspecific variation. Further studies and analysis of this variation may determine if it is significant enough to account for speciation.

Carlos (De-Juan) RuffinCarlos (De-Juan) Ruffin, Lyon College, Batesville, AR
(Advisor: Dr. Robert F. Murphy)

The Role of Rab4a in the Suppression of the Receptor Recycling Defect in TfT1.11
The process of endocytosis is characterized by the recycling of receptors in the endosome back to the cellular membrane while their ligands continue to be processed throughout the cell. TfT1.11 is a temperature-sensitive mutant of CHO cell line WTB. At the non-permissive temperature, a decrease in TfR (receptor) recycling is observed. Previous work has shown that the introduction and overexpression of GTP-binding protein rab4b in mutant TfT1.11 cells results in a partial rescue of the receptor-recycling defect. Rab4b specific RT-PCR, as well as 3' RACE (Rapid Amplification of cDNA Ends) and 5' RACE were previously used to sequence the DNA of rab4b in TfT1.11 and WTB cell lines. There was no observable mutation in rab4b in the TfT1.11 line. The fact that overexpressing rab4b in TfT1.11 only partially rescued receptor recycling suggests the possibility that TfT1.11 may be defective in one or more rab proteins other than rab4b. Hence, the goal of this project was to sequence rab4a, another GTP-binding protein, in WTB and TfT1.11 cells. Probes for rab4a specific RT-PCR were designed to determine whether Rab4a is expressed in CHO cells and, if so, to obtain an internal sequence. The results indicated that rab4a is expressed in both WTB and TfT1.11 and the sequences obtained were identical between the two cell lines. They also closely matched the previously published sequence of rat rab4a. To finish sequencing, primers for 3' RACE and 5' RACE were designed using the internal sequence. A comparison of the sequences obtained will be used to determine whether TfT1.11 has a mutation in this gene. In addition, the results provide the first evidence that both rab4a and rab4b may be expressed in the same cell type, suggesting the possibility that they are redundant isoforms.

Christine TorborgChristine Torborg, Adolphus College, St. Peter, MN
(Advisor: Dr. Gordon Rule)

Effects of Methionine 108 Mutation on the Kinetic and Structural Parameters of GSTM2-2
Glutathione S-transferases (GSTs) are a family of enzymes that detoxify chemical carcinogens by catalyzing the nucleophilic addition of reduced glutathione to a number of hydrophobic compounds. In this study the role of methionine 108 in binding the hydrophobic substrate was investigated by comparing the kinetic and structural parameters of a methionine 108 to alanine mutant to that of the wild-type enzyme. Enzyme kinetics were monitored using UVspectroscopy to measure the production of glutathione-2, 4-dinitrobenzene from 1-chloro-2, 4-dinitrobenzene. Kinetic data suggest that both the mutant and wild-type enzymes proceed through an ordered steady-state mechanism with no significant difference in Kia and KmA between the two enzymes, suggesting no change in the binding of glutathione to either enzyme. However, the mutant enzyme did have significantly higher KmB and Vmax than the wild-type, which suggests that the hydrophobic substrate is bound less tightly to the mutant protein. Crystals of both enzymes are currently being grown for X-ray crystallography, which will be used to determine their crystal structures. A comparison of these structures will indicate whether observed kinetic changes are due to the disruption of direct protein-substrate interactions or changes in the conformation of the hydrophobic binding site.

Shewanda VinesShewanda Vines, Tuskegee University, Tuskegee, AL
(Advisor: Dr. David Hackney)

E. coli MukB protein NH2 Head Terminal Domain Potential as a Mechanical Transducer for Motor Protein
The E. coli Muk B protein is a potential motor protein functioning in movement during chromosomal partition. The Muk B protein has an anti-parallel alpha helical coiled coil motif forming to rigid rod-like structures extending from a non-coiled hinge segment. Paired at each end of this rod is an NH2 and COOH terminal domain. This arrangement allows the protein to form two identical functional units at each end. With the presence of a Walker A motif at the N-terminal domain, the secondary structure of the Muk B protein closely resembles motor proteins kinesin and myosin. Presence of the Walker A motif suggests Muk B potential to hydrolyze ATP through mechanochemical activity. The possibility of ATP hydrolysis was researched using two N-terminal constructs: A) Muk B monomer and B) Muk B dimer. Constructs were cloned into plasmids and induced to express the protein in fusion. Cloned constructs contained HIS-Tags for nickel column purification fused with Thioredoxin that aided to ensure further purification from contaminating proteins through thiobond column purification. The protein was abundantly expressed after IPTG induction but was fairly insoluble. Abundance of the protein remained after nickel column purification but did not bind well to thiobond column. Traces of contaminating protein that could possibly drive ATP reactions remained in Muk B fractions after thiobond purification. On the basis of obtained impure Muk B protein, ATPase activity could not be tested to define Muk B as a mechanochemical transducer for motor proteins.