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Graduate Summer Colloquium 2005: Abstracts

2005 Abstracts

Christopher Hittinger

“Pleiotropic functions of a conserved insect-specific HOX peptide motif”

The genetic regulatory networks governing development and morphology have been rewired throughout the course of animal evolution, but their protein-coding components have remained remarkably well conserved. A few cases have been described where the protein-coding regions of some of the most conserved developmental regulators have functionally evolved. Here, we have analyzed the function of the insect-specific but highly conserved "QA peptide motif in the HOX protein ULTRABITHORAX (UBX) by removing it genetically through precise allelic replacement in Drosophila melanogaster. The QA motif was originally characterized as involved in the repression of limb formation, but QA deletion homozygotes were viable and had a normal complement of limbs. However, at reduced dosages of Ubx and the abdominal-A (abd-A) Hox gene, ectopic limb primordia and sub-epidermal adult abdominal limbs formed when the QA motif was absent. These results indicate that functional redundancy and expression level play important roles in moderating the phenotypic consequences of HOX protein evolution. Deleting the QA motif had stronger effects in other tissues, indicating a large reduction in Ubx activity. The differences between tissues in their requirement for QA function suggest that this peptide motif is preferentially required for the regulation of a subset of UBX target genes. The overall subtlety of the homozygous phenotype demonstrates that even modest pleiotropic effects elicit intense purifying selection over vast periods of time.

Dominic Ebacher

“cut mutant Drosophila auditory organs differentiate abnormally and degenerate”

The Drosophila antenna is a sophisticated structure that functions in both olfaction and audition. Previous studies have identified Homothorax, Extradenticle, and Distal-less, three homeodomain transcription factors, as required for specification of antennal identity. Antennal expression of cut is activated by Homothorax and Extradenticle, and repressed by Distal-less. cut encodes the Drosophila homolog of human CAAT-displacement protein, a cell cycle-regulated homeodomain transcription factor. Cut is required for normal development of external mechanosensory structures and Malphigian tubules (kidney analogs). The role of cut in the Drosophila auditory organ, Johnston's organ, has not been characterized. We have employed the FLP/FRT system to generate cut null clones in developing Johnston's organ. In cut mutants, the scolopidial subunits that constitute Johnston's organ differentiate abnormally and subsequently degenerate. Electrophysiological experiments confirm that adult Drosophila with cut null antennae are deaf. We find that cut acts in parallel to atonal, spalt-major, and spalt-related, which encode other transcription factors required for Johnston's organ differentiation. We speculate that Cut functions in conjunction with these factors to regulate transcription of as yet unidentified targets.

Sang-Kyu Park

“Identification of Biomarkers of Aging And The Effect of Nutritional Supplementation on The Aging Process”

Understanding of the molecular basis of aging in mammals has progressed slowly, in part because we lack a large number of biomarkers that can be used to measure the aging process. Extensive research suggests that aging is associated with a state of increased oxidative stress caused by reactive oxygen species (ROS). ROS produced as a result of aerobic metabolism can be reduced enzymatically by antioxidant enzymes or non-enzymatically by antioxidant molecules. To establish a panel of transcriptional markers of aging, differential expression of genes from several strains of young and old mice (B6, 129, CBA, DBA, BalbC, C3H, and B6/C3H) were screened using high-density oligonucleotide arrays. Based on the microarray results, we identified a panel of molecular markers of aging that are common in multiple strains of mice. These molecular markers of aging were used to measure the effect of antioxidant supplementation on the aging process. Among antioxidants tested, lycopene, resveratrol, acetyl-L-carnitine and tempol were as effective as caloric restriction, the only intervention known to extend lifespan in many organisms, in preventing age-related transcriptional alteration of molecular markers of aging.

Emily Putiri

“mission impossible, a zebrafish maternal effect mutation that affects gastrulation”

In early animal development, the basic body plan is established during gastrulation: the primary axes are determined and the three embryonic germ layers, the ectoderm, mesoderm, and endoderm, are formed. These events require a complex coordination of correct cell fate specification and morphogenetic cell movements. Analyses of mutants that affect these events provide a framework for understanding the molecular controls of gastrulation. mission impossible (mis) is a zebrafish maternal effect mutation that reduces the induction of the mesendodermal germ layer during gastrulation. The mutation also disrupts morphogenetic cell movements, resulting in embryo lysis during late gastrulation. I will present progress on the phenotypic characterization and molecular identification of mis.

Ben Harrison

“Rooting around in the dirt for my thesis”

This seminar will focus on work that has further refined the role that the Arabidopsis proteins ARG1 and ARL2 play in early gravity signal transduction. Interestingly, ARG1 and ARL2 appear to mediate several events that follow shortly after a change in root orientation within the gravity vector. Results will be presented that show that ARG1 and ARL2 are required for the gravity-responsive relocalization of PIN3 within the gravity-sensing columella cells of the root tip. PIN3 is a component of the auxin efflux complex that may, in part, mediate the movement of auxin through the root tip and may mediate gravity-responsive redistribution of auxin toward the lower flank of gravity-stimulated roots. Such lateral redistribution of auxin has been postulated as a major and causal event in the downward curvature of gravity stimulated roots. These results will shock you. At least one plant physiologist will be on hand to soothe the awestricken and immobile.

Jamie Verheyden

“Conditional inactivation of Fgfr1 in mouse defines its role in limb bud establishment, outgrowth, and digit patterning.”

Previous work has demonstrated that Fibroblast Growth Factors expressed in the Apical Ectodermal Ridge (AER-FGFs) signal to the mesenchyme and are required for limb outgrowth. However, the precise nature of their role in the mesenchyme has not been well defined. Among the four FGF receptors (FGFR), Fgfr1 is the most likely candidate mediating AER-FGF function. Here we inactivate Fgfr1, using two different Cre recombinase expressing lines, and characterize the impact on limb formation. The Tcre line led to inactivation of Fgfr1 in the lateral plate mesoderm (LPM) including limb bud mesenchyme prior to limb bud initiation. The mutant phenotype demonstrates that Fgfr1 restricts mesenchymal cell number in the early limb bud, and is required for cell survival later during limb outgrowth. The Sonic Hedgehogcre (Shhcre) allele led to Fgfr1 inactivation in the posterior mesenchymal cells after limb bud initiation. This mutant offers a unique setting to address Fgfr1 function in gene expression regulation and patterning without the complication of changes in cell number. Our data show that Fgfr1 influences digit number and identity, which is likely through regulation of Shh expression. This study has led to a more complete understanding of the multiple roles of Fgfr1 in limb bud establishment, growth, and patterning

Allison Weber

“Association Mapping in Teosinte: How Natural Allelic Variation Controls Phenotypic Variation”

Understanding the inheritance of complex traits is a fundamental goal of quantitative genetics. Complex traits are controlled by a mixture of genetic and environmental factors. The identification of genes that control complex traits could lead to advances in plant breeding, human health, and the understanding of evolution. Our laboratory is interested in the evolution of maize from the wild Mexican grass teosinte (Zea mays ssp. parviglumis). In this project, we are using association mapping to study natural variation in teosinte. Our goal is to assess how natural allelic variation at candidate genes controls phenotypic variation in the population. One concern in association mapping is that population structure can cause false genotype-phenotype associations. Recent statistical advancements in association mapping control for underlying population structure, making association mapping a powerful tool. We are conducting association mapping in two different teosinte populations. In the first population, several candidate genes were found to have significant associations with measured traits. We are currently evaluating a second population. This research may also help to identify allelic variants useful in maize breeding.

Qiong Zhao

“Molecular Population Genetics of MADS-box Genes During Maize Domestication”

MADS-box genes encode transcription factors which are key regulators of plant vegetative, inflorescence, flower and fruit development. Evolutionary genetics studies showed that the duplication and diversification in sequence and function of the MADS-box genes were correlated with the origin of land plants, the establishment of certain reproductive structures in higher plants, and the increasingly diverse and complex flower structures in land plants. Given their important roles in regulating plant development and involvement in the evolution of plant morphology, we systematically examined nucleotide diversity in 32 MADS-box genes in the context of the domestication of maize from its wild ancestor, teosinte. Based on surveyed 32 MADS-box genes, maize domestication is associated with a greater loss of genetic diversity in maize at MADS-box genes than at randomly selected genes. Among the 32 MADS-box genes, seven genes were identified as putative targets of selection during domestication, and one additional gene was probably under selection during the modern improvement of maize. The candidate selected MADS-box genes were located in a wide range of maize chromosomes and distributed in different subfamilies of MADS-box transcription factors. In addition, severity of the domestication bottleneck was estimated conditioning on the sequence variations of presumably ?neutral? MADS-box genes. Analysis of coalescent simulation showed that the bottleneck associated with domestication was at a moderate level of severity.

Dana Somers

“A Plasma Membrane Histidine Kinase Is Essential For Osmotolerance and Seed Desiccation”

Because they are immobile, plants have evolved unique coping mechanisms to tolerate physical and chemical extremes.

Lan Yi

“Understanding transcription factor function in mouse lung branching and differentiation”

The mammalian lung is highly efficient gas exchange machinery that is vital for survival. It originates from a small group of cells in embryonic foregut and develops into a mature organ through elaborate morphogenesis and differentiation. Many questions remain regarding how this sophisticated design of the lung is achieved. I am particularly interested in the role of key molecules such as transcription factors (TFs) in this developmental process, as they perform essential functions in multiple other processes through out development. The overall hypothesis of this work is that TFs perform important function in lung formation. Taking the advantage of the completed mouse genome sequence, I propose to establish a 3-D TF expression pattern database of genes with expression in distinct cell types in the developing lung. The database will serve as a rich source for functional study Meis1 using advanced mouse genetic technology.

Erin Patterson

“Analysis of FKH1-mediated silencing in yeast”

Silencing in S. cerevisiae represses transcription in multiple areas of the genome, including the cryptic mating type loci, HML and HMR. Silencing at the HM-loci requires four specialized factors, silent information regulator (Sir) 1-4 proteins. Sir1p plays a role in the establishment of the silenced state at the HMRa locus. The gene forkhead homologue one(FKH1) was identified as a high-copy suppressor of the silencing defect observed in a strain lacking SIR1. FKH1 encodes a transcription factor that plays a role in regulation of genes necessary for normal cell cycle progression. The mechanism by which FKH1 is able to bypass the SIR1 requirement for silencing at HMR is not clear. I am using a genome-wide approach, exploiting FKH1-mediated silencing, to gain insight into the relationships between the cell cycle, chromatin structure and gene expression.

Jennifer Dingwall

“Searching For A Eukaryotic Sigma”

Transcription in bacteria requires that core RNA polymerase forms a complex with a sigma factor. This sigma factor allows the RNA polymerase holoenzyme to specifically recognize promoter DNA, melt the DNA at the promoter, and efficiently initiate transcription. Bacteria cannot carry out transcription and, therefore, cannot survive without the sigma factors. Eukaryotic transcription also requires an RNA polymerase. Eukaryotic homologues for each of the bacterial core RNA polymerase subunits (alpha, beta, beta-prime, and omega) have been identified. However, no homologue of sigma has yet been recognized. Because the sigma factor is so essential in bacteria, it is crucial to our understanding of transcription that we investigate which, if any, eukaryotic factor is undertaking the role of sigma. The Burgess laboratory has characterized many of the interactions between the bacterial RNA polymerase and the sigma factors. Using some of the same biochemical techniques used to characterize the bacterial system and a parallel bioinformatics approach, I have been investigating whether there is indeed a eukaryotic sigma equivalent.

Sarah Duellman

“Manipulating transcription in cancer cells: Protein purification and mAb characterization”

The complex mechanisms involved in regulating gene expression incorporate intricate processes at many levels, including transcription, translation, and post-translational modification. Abnormal regulation of eukaryotic gene expression can often lead to disease, including cancer. As a result, the ability to control gene expression would be a powerful tool both in basic research and therapeutic development. The goal of my project is to manipulate transcription in cancerous cells by inhibiting the function of oncogenic transcription factors. The general strategy involves killing transformed cells in culture by introducing a monoclonal antibody (mAb) into the cell through the use of a protein transduction domain (PTD) peptide. By using inhibitory mAbs to interfere with the function of Epstein Barr virus nuclear antigen 1 (EBNA-1), we hope to inhibit the survival and proliferation of Burkitt?s lymphoma cells. I will discuss work on purifying EBNA-1 and generating and characterizing a library of mAbs against this antigen.

Dan Hesselson

“GON-1 and Fibulin have Antagonistic Roles in Control of Organ Shape”

The GON-1 ADAMTS metalloprotease is required for migration of the distal tip cell (DTC), which controls both size and shape of the developing gonad in the nematode C. elegans. To investigate the role of the extracellular matrix (ECM) in DTC migration, we genetically removed individual ECM components from gon-1 mutants and discovered one gon-1 suppressor, called fibulin (fbl-1). Whereas the DTC fails to migrate in gon-1 null mutants, it migrates extensively in fbl-1 gon-1 double mutants. Therefore, wild-type fibulin must block DTC migration when GON-1 is absent. In fbl-1 single mutants, DTC migration is essentially normal, but the gonad is thicker than usual. Therefore, wild-type fibulin constrains gonadal width. Removal of GON-1 from fbl-1 mutants restores the gonad to its normal width, suggesting that GON-1 and fibulin have antagonistic, but balanced effects on gonadal width. I am now interested in learning about the function of fbl-1 at the molecular level. I am using structure/function studies to identify the domains within fbl-1 that regulate DTC migration and forward genetic screens to identify additional molecules that interact with fbl-1 to regulate organ shape.

Anongpat Suttangkakul

“Dissection of the Autophagic Process in Arabidopsis thaliana”

Autophagy is a mechanism for bulk protein degradation, which involves the engulfment of cytoplasm and cellular constituents into double membrane-bound vesicles and the deposition of these vesicles into the lytic vacuole of the cell. This degradative process allows for nutrient remobilization during periods of starvation and facilitates developmental remodeling via programmed cell death. In yeast, the autophagic process utilizes two protein tags, ATG8 and ATG12, in a conjugation pathway that is similar to ubiquitination. This conjugation pathway regulates proper formation of the autophagic vesicles by machinery that also functions in vesicle trafficking under non-starvation conditions. The TOR-activated ATG1-kinase regulatory complex activates the switch to the starvation-induced ATG8/12 pathway. Although important during various stages of growth and development, the molecular mechanisms underpinning autophagy in plants are just beginning to be uncovered. We have identified the Arabidopsis homologs for most members of the ATG8/12 conjugation and ATG1-kinase regulatory pathways based on the protein sequence similarity to their yeast counterparts. Several T-DNA insertion libraries have been screened for mutations in the Arabidopsis autophagy genes. We have characterized two mutants disrupting ATG7 and ATG5, genes encoding the enzyme required for ATG8/12 activation and the target of ATG12 conjugation. These mutants are hypersensitive to nitrogen and carbon starvation and display premature senescence. In addition, GFP-ATG8 lines have been developed as molecular makers for monitoring the autophagic process in planta. These makers allow, for the first time, the visualization of autophagic bodies inside the vacuole. We are continuing to use a combination of reverse genetics, biochemistry, and cytological techniques to further characterize the conjugation and regulatory pathways of autophagy in Arabidopsis.

Sheng Gao

“Characterization of Dpp responsive silencing complexes in Drosophila melanogaster”

Decapentaplegic (Dpp), a transforming growth factor-beta superfamily member, controls critical processes throughout the development of Drosophila. The signal is transduced by nuclear accumulation of transcription factor Mothers-against-Dpp (Mad) and Medea (Med), Drosophila homologs of the Smad1 and Smad4 proteins. Target genes can be activated or repressed depending on the specific responsive elements. Small silencer elements have been uncovered in the brinker (brk) and bag-of-marbles (bam) genes, repressing transcription in response to Dpp signaling. We found that these silencer elements contain both Mad and Medea binding sites, and require strict configuration of the sites, including their orientations and the spacing in between. These silencers are bound by a heterotrimer containing two Mad subunits and one Medea subunit. The DNA binding domains (MH1 domains) of all three subunits contribute to sequence-specific DNA contact. The Medea MH1 domain binds to canonical Smad box (GTCT), while the Mad MH1 domains bind to a GC-rich site. The Dpp responsive silencing through these silencers also requires Schnurri (Shn), a big zinc-finger protein, as co-repressor, though a C-terminal segment of Shn (ShnCT) is sufficient to confer repression in vivo. These results have led to a more complete understanding of how Dpp pathway recognize and control its targets.

Yi Cai

“Screen the corepressors of Drosophila zinc-finger protein Schnurri”

In Drosophila Dpp signaling pathway, the signal transducer proteins Mad and Medea will assembly on the Dpp-responsive silencing element in the brinker gene and further recruit the zinc-finger protein Schnurri (Shn) to repress the transcription upon Dpp signaling. Recently, a 110-aa repression domain in Shn (ShnRD) has been identified. We are interested in the mechanism of transcriptional repression mediated by the ShnRD. To identify corepressors required by the ShnRD, we used coimmunoprecipitation (CoIP) assay to screen candidate corepressors for interaction with the ShnRD. Unexpectedly, many general transcriptional corepressors like HDACs, dCtBP and Groucho (Gro) can bind ShnRD.To examine which protein-protein interactions are functional, we started a genetic screen by conducting RNAi in Drosophila S2 cells. We used RNAi to knockdown the expression of transcriptional corepressors individually and look for corerpessors required for the repression of a GAL4DBD-ShnRD fusion protein. After two rounds of screening, we identified Groucho as a candidate corepressor required for the repression mediated by ShnRD. Currently we are generating point mutations in ShnRD to disrupt its ability to repress transcription. We will test whether these mutants lose their ability to recruit Groucho.

Amy Hubert

“Progress toward cloning laf-1, a C. elegans sex-determination gene”

C. elegans has two sexes, male and hermaphrodite. Hermaprodites are essentially female animals, but they produce sperm in addition to oocytes and are consequently self-fertile. Sex determination in C. elegans is a complex process requiring precise temporal and spatial expression of many genes. tra-2 encodes a large transmembrane protein and acts in the sex determination pathway to promote female development. tra-2 is translationally repressed at specific stages in the hermaphrodite to allow spermatogenesis, and this repression is carried out by binding of factors (such as GLD-1) to elements in the tra-2 3?UTR. Proper tra-2 regulation requires laf-1, another gene involved in sex determination. laf-1 mutations are homozygous lethal, and a proportion of laf-1/+ animals are feminized (that is, they fail to produce sperm). In this talk, I will describe recent developments in the effort to clone and characterize laf-1

John Stanga

“Identifying enhancers of root gravitropism defects in Arabidopsis altered response to gravity 1 (arg1) mutants”

Plant organs are able to alter their growth patterns in response to a variety of stimuli. Roots must be able to perceive light, gravity, barriers, minerals, and water, then coordinate a directional growth response. The response to gravity is characterized by four distinct phases: gravity perception, signal transduction, signal transmission, and differential growth. An Arabidopsis thaliana mutant, altered response to gravity 1 (arg1), exhibits a significant delay in its reorientation when roots are rotated 90

Jeffrey Berger

“Molecular characterization of a Drosophila temperature sensitive paralytic mutant that affects synaptic morphology at the larval neuromuscular junction”

Neurons communicate with each other via specialized structures called synapses. However, synapses are not static structures. Developmental events and experiences lead to changes in synaptic morphology and strength. These changes are an essential part of learning and memory. I would like to better understand the molecular events that are required for these changes to occur. To answer this question, the Ganetzky lab has used a forward genetic strategy of isolating temperature sensitive paralytic mutants as a way of identifying genes that are essential for neuronal function. Examining the neuromuscular junction in this group of temperature sensitive paralytic mutants revealed that they are enriched for mutants with unusual synaptic morphology. The Drosophila neuromuscular junction has been an excellent model system for studying synaptic morphology and plasticity because of its large size and easy accessibility to histological and electrophysiological studies. One of these mutants, 9-76, displays a significant increase in synaptic bouton number and in synaptic branching. Using deficiency mapping, I was able to map the 9-76 mutation to a small region of the second chromosome. This region contained two candidate genes, a glutamate receptor encoded by the gene clumsy and a WD40 domain containing protein encoded by the gene CG8678. Clumsy is a particularly attractive candidate because glutamate is the neurotransmitter at the Drosophila neuromuscular junction. Multiple alleles of clumsy fail to complement the temperature sensitive paralysis phenotype of 9-76. Therefore, I believe that 9-76 is an allele of the glutamate receptor, clumsy. Clumsy belongs to the kainate family of ionotropic glutamate receptors. In mammals, kainate receptors are known to affect synaptic lasticity via a protein kinase C dependant pathway. I hope to use the powerful genetics of Drosophila to better understand the role that kainate receptors play in determining synaptic morphology and plasticity.

Michelle Rickard-Taylor

“Liver Targeting and the Identification of Hepatocyte-Specific Membrane Proteins”

Liver is one of the most important target tissues for gene therapy and drug delivery given its central role in metabolism and in the secretion of circulating proteins. Liver targeting entails the delivery of a therapeutic agent to hepatocytes (parenchymal cells). Greater efficacy and decreased toxicity would be gained by delivering therapeutic agents more selectively to hepatocytes while avoiding non-parenchymal liver cells. The overall objective of the proposed research is to identify receptors present on hepatocytes and absent on non-parenchymal liver cells. These receptors can then be used to identify ligands that can selectively target therapeutic agents to hepatocytes

Wei Zheng

“The degradation behavior of nonsense ash1 mRNA”

In eukaryotes, messenger RNA's containing a premature stop codon (nonsense mRNA's) are rapidly degraded by the nonsense mediated mRNA decay (NMD) pathway. In the yeast Saccharomyces cerevisiae, nonsense transcripts bound to either the nuclear or cytoplsmic cap-binding complexes can be degraded by NMD, which can occur during the first (pioneer) round of translation or during subsequent rounds of translation. The degradation of nonsense transcripts during pioneer translation occurs during or immediately after nuclear export. For normal transcripts lacking a PTC, the pioneer ribosome displaces nuclear mRNP proteins and may remove factors that would otherwise trigger NMD.

Xiaoyan Ge

“Characterizing the role of the maternal-effect gene nebel in cytokinesis and gastrulation”

We present progress on the characterization of a mutation identified in an early pressure-based screen for maternal-effect mutations. nebel is a recessive, strictly maternal gene involved in cytokinesis. nebel mutant embryos exhibit defects in cell adhesion and membrane deposition, which result from defects of microtubule reorganization at the forming furrow. We have found that nebel mutant embryos exhibit a strong reduction in the slow calcium waves that accompany furrow formation. We are currently testing whether nebel mutants are defective in specific calcium channels in the early embryo. nebel-dependent function during cellularization has been shown to be temperature-sensitive. Even when nebel mutant embryos escape their early cellularization defects under permissive temperature, they die within a day after fertilization. This late defect may reflect a second essential function for this gene during gastrulation. We are currently carrying out expression analysis to understand the cause of this late phenotype. We are also using a positional cloning approach to determine the molecular identity of this gene. Progress on the positional cloning will be presented.

Labib Rouhana

“Vertrbrate GLD-2 poly(A) polymerases in the germline and brain”

Cytoplasmic polyadenylation is important in the control of mRNA stability and translation. It is critical during early development and for synaptic plasticity. Here we focus on vertebrate poly(A) polymerases that are members of the recently described GLD-2 family. We identify and characterize two closely related GLD-2 proteins in Xenopus oocytes, and analyze the regulation of GLD-2 during oocyte maturation. We show that both homologues possess PAP activity, and that they bind known polyadenylation factors and target mRNAs. We propose that two distinct polyadenylation complexes exist in oocytes: one contains GLD-2 and the polyadenylation factors, CPSF and CPEB; the other lacks GLD-2, but contains CPEB and components associated with translational repression, including maskin and Pumilio. We show that an isoform of GLD-2 mRNA is translationally activated during oocyte maturation, by virtue of regulatory sequences present uniquely in its 3?UTR. These data suggest a self-reinforcing positive feedback loop operating at the level of polyadenylation. Mammalian GLD-2 homologs ectopically expressed in frog oocytes physically interact with the endogenous cytoplasmic polyadenylation machinery, demonstrating that these interactions are conserved. The expression of GLD-2 is widespread. In the mouse and human brain, mRNAs encoding the proteins are expressed in anatomical regions necessary for long-term cognitive and emotional learning. In the hippocampus, mGLD-2 mRNA co-localizes with CPEB-1 and Pumilio mRNAs, both of which are likely involved in synaptic plasticity. In the cerebellum, mGLD-2 mRNA is present within the major neuronal cell populations, coincident with the distribution of other CPEB isoforms. We suggest that mammalian GLD-2 poly(A) polymerases are critical in local and sustained activation of synaptic translation in the brain, and that positive feedback acting on mammalian GLD-2 mRNA may be a key component of that role.

Stephan Woditschka

“Rexinoids, Tamoxifen and Celecoxib in Breast Cancer Chemoprevention and Biomarker Identification”

The field of chemoprevention represents a promising aspect in the battle against breast cancer. In our laboratory, we are using the neu-induced retroviral rat mammary carcinogenesis model to study chemoprevention in the laboratory rat. The mammary carcinomas of this model exhibit a mixed hormonal response in the same proportion as has been observed in women. In intact rats, approximately 50% of mammary carcinomas are hormonally responsive and can be prevented by anti-estrogenic tamoxifen treatment. In ovariectomized animals the mammary carcinomas are hormonally non-responsive and cannot be prevented by tamoxifen. We evaluated the chemoprevention agents bexarotene (a retinoic-X receptor selective retinoid), tamoxifen (a selective estrogen response modulator) and celecoxib (a cyclooxygenase-2 inhibitor) for their efficacy in preventing mammary carcinomas in our neu-induced rat models. The RXR-specific agonist bexarotene is a potent chemoprevention compound which shows similar reductions in tumor multiplicity (84% and 86%) in intact and ovariectomized rat models, respectively. This suggests that the chemoprevention properties of bexarotene are independent of ovarian hormone interactions or estrogen receptor status. The chemopreventive effects of celecoxib and tamoxifen parallel one another in both hormonal models. In the intact model, both compounds cause an approximately 50% reduction in tumor multiplicity. In ovariectomized rats the tumor multiplicity of celecoxib fed and tamoxifen treated animals do not differ significantly from control animals. These observations suggest that the chemopreventive modes of action of the two agents share or utilize similar biochemical pathways. Additionally, we aim to identify secreted biomarkers that can be used as surrogate, intermediate endpoints for evaluating the efficacy of chemopreventive agents. Such markers could aid in testing a variety of emerging chemopreventive compounds in clinical phase II settings. Using Affymetrix microarray technology, a set of 1166 genes was identified to be differentially expressed in the rat mammary gland in response to treatment with another RXR-selective retinoid, LG100268. Within this set, secreted proteins were identified by informatic screens which examine the amino acid structures for the presence of a 5? signal peptides. Currently, we are investigating the expression of a final set of 49 secreted genes in control and LG100268-treated mammary glands and tumors by quantitative RT-PCR.

Joshua Gnerer

“Identification of a Drosophila mutation that causes TS paralysis, shortened lifespan and neurodegeneration”

For more than 25 years the Ganetzky lab has studied temperature sensitive (TS) paralytic mutants in Drosophila with the goal of identifying genes necessary for neuronal growth, function, and viability. These mutants rapidly lose motor control at elevated temperature (38C). Recently, this collection was found to be enriched for mutations that also cause neurodegeneration. My talk will focus on one of these mutations that I mapped to the gene that encodes the glycolytic enzyme, triosephosphate isomerase (TPI). In humans, mutations in tpi lead to the neurodegenerative disease, TPI deficiency. TPI deficiency has been studied for 50 years but the underlying mechanism of neurodegeneration is still unknown and there is no therapy available. Several hypotheses have been proposed to explain the degeneration, but until now they have not been adequately tested. To investigate the cellular and biochemical basis of TPI deficient neurodegeneration, I am using the Drosophila model of this disorder to test the various proposed hypotheses. The results thus far favor the idea that neurodegeneration results from the accumulation of toxic metabolites behind the enzyme block. The data gathered from these studies will not only enhance our understanding of TPI deficiency but should also increase our knowledge of basic pathways that could be applicable to other human neurodegenerative diseases.

Nicholas Shera

“Structural Variation in Cancer Genomes Discerned by Single Molecule Analysis ”

Pharmacogenomics relates patient genotypes with clinical outcome. I have chosen to work with a brain tumor (oligodendroglioma) as a model system to connect detailed genomic analysis with response to drug therapies. More specifically, loss of heterozygosity at 1p/19q (tumor) directly correlates with responsiveness to multiple types of treatment and results in longer survival rates for those patients. Although cytogenetic evidence has provided this linkage, molecular details are lacking, despite FISH, CGH and expression analysis. As such, I propose to construct detailed physical maps (~18 kb resolution), using the Optical Mapping System to identify and functionally annotate aberrant loci (insertions, deletions, translocations, and +/- restriction sites) covering ?genomes? that comprise this tumor. Aberrant loci will then be correlated with other experimental datasets to identify likely candidates involved in drug responsiveness.

Andrew Jauregui

“Characterization of C. elegans NPHP-1 and NPHP-4 and their role in cilia and male sensory behavior”

Nephronophthisis (NPH) is an autosomal recessive disorder characterized by formation of renal tubule cysts, tubular basement membrane disintegration, and renal interstitial fibrosis, and is the most frequent genetic cause of end stage renal disease (ESRD) in children. NPH is caused mutation in one of at least five different genes, with each disease locus varying in the age of onset of ESRD. The nephrocystins, the protein products of the NPH genes, localize to cilia. NPH is one of several cystic kidney diseases that may be caused by ciliary defects. The cellular and molecular functions of the nephrocystins remain largely unknown. We are using the nematode Caenorhabditis elegans as a model system for nephronophthisis. NPHP-1 and NPHP-4 are the C. elegans homologs of human nephrocystin-1 and nephrocystin-4. We find that nphp-1 and nphp-4 are expressed in ciliated sensory neurons. Furthermore, we find that NPHP-1 and NPHP-4 colocalize with the polycystins LOV-1 and PKD-2 in cilia of male-specific CEM, RnB, and HOB sensory neurons. nphp-1 and nphp-4 do not appear to be required for cilium formation. We find that nphp-1 (ok500); nphp-4(tm925) double mutants, but not single mutants, are defective in the response sub-behavioral step of male mating. We propose that NPHP-1 and NPHP-4 proteins play a role in facilitating ciliary sensory signal transduction. We will test the hypothesis that the C. elegans nephrocystins and the C. elegans polycystins share a common defect in cilary signaling. Watnick T. Nature Genetics. 34(4):355-6, 2003 Aug. Hildebrandt F. Pediatric Nephrology. 16(2):168-76, 2001 Feb.

Zachary Larson

“The Strange Case of the Trichome Pock”

Plant morphogenesis requires the coordination of cell division and cell expansion. These two mechanisms are balanced, such that one can react to and compensate for vagaries or defects of the other. In the leaf of Arabidopsis, for example, epidermal cells divide and expand to cover the photosynthesizing tissue, and differentiate to produce stomata, trichomes and their socket cells. bilfu is a mutant that shows a novel defect in leaf morphogenesis: trichomes are produced within an indentation, or pock, on the otherwise flat leaf surface. So far the culprit cause of this phenotype eludes us, although leaf cell expansion and the division or recruitment of trichome socket cells are prime suspects. We have reason to believe that the hormone brassinolide is implicated, suggesting local intercell communication defects.

Stephanie Nelson

“Mystery of the Pharoah or the Sphinx: Looking for a causative SNP in a Haplotype block that has an association with breast cancer risk in women”

We have used a quantitative trait locus (QTL) identified in the rat, that reduces DMBA induced tumor numbers, as a guide to find single nucleotide polymorphisms (SNPs) that associate with breast cancer risk in the human region of DNA orthologous to our rat QTL. We used the linkage disequilibrium present in the human genome as a guide to determine the haplotype block structure in the region homologous to the rat QTL. We tested the haplotype block that covers the orthologous rat region that contains the mammary tumor resistance phenotype in the rat. A tag SNP with a minor allele frequency of 0.12 in one human haplotype block has associated with breast cancer risk in two populations. The first population tested includes 1,500 women diagnosed with breast cancer and 1,500 frequency age matched controls from Wisconsin. The second population includes ~4,500 cases and ~4,500 controls from Europe. The combined odds ratio for the two populations is 0.86 (95%CI 0.79-0.94) for heterozygotes and the odds ratio is 0.77 (95% CI 0.57-1.04) for homozygotes for the minor allele. We are currently attempting to identify the specific genetic polymorphism that is responsible for the association observed in the two populations.