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Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by fibroblast and collagen deposition and irreversible lung destruction. There is a genetic contribution to the development of IPF, including telomererelated genes. Moreover, the development of IPF has been correlated with a shorter telomere length compared to reference populations without IPF. We investigated the telomere lengths of individuals with IPF with the hypothesis that heterogeneity in telomere length will inform genetic discovery in IPF. We extracted DNA from 180 individuals with IPF. The DNA samples were plated with a total volume of 50 µL per sample, which consisted of a diluted 1 ng/mL DNA sample and 20 ng/mL of total yeast RNA with water. The samples were then analyzed using a monochrome multiplex quantitative PCR (MMQPCR) producing a high abundance of tandem repeats (T) and low abundance of single copy genes (S). The ratio of (T/S) represents an average telomere length per cell. The average telomere length of the 180 individuals with IPF is 0.826 ± 0.182. Due to our interest in small telomere length, we conducted preliminary analysis of the data in the lowest 25%, which consisted of the 40 smallest values with an average telomere length of 0.604 ±
0.078. There is a large telomere length spectrum (0.397 to 1.38), which indicates the samples are heterogeneous in telomere length. Shorter telomere lengths may suggest a pathology that is related to telomere biology. Investigation of this group may lead to discovery of genetic contributions to IPF and telomere length regulation.
IDENTIFYING THE ENHANCER REGION FOR SINGLES BAR EXPRESSION IN DROSOPHILA MELANOGASTERMYOBLASTS Brayon Fremin Sr., Tonya Brunetti, Richard Cripps.
University of New Mexico, Albuquerque, NM.
In Drosophila melanogaster, singles bar (sing) codes for a MARVEL domain trans-membrane protein found in all myoblasts that is essential for myoblast fusion. Myoblast fusion is critical for proper muscle formation and is conserved between flies and humans. Studying singles bar and other genes that control myoblast fusion in flies therefore has broad applications to humans. During myoblast fusion, myoblasts come together to form a prefusion complex. The myoblasts later fuse together to form a syncytial myofiber. Without sing expression, however, progression does not occur past the prefusion complex. Preliminary data have shown that sing is downregulated in myocyte enhancer factor 2 (MEF2) knockdown flies. MEF2 is a transcription factor with many target genes involved in muscle development. This study has demonstrated that MEF2 is a direct regulator of singles bar and provided a novel mechanism by which MEF2 regulates myoblast fusion. By creating a transgenic line of flies using an enhancer region of sing-lacZ fusion construct, we have illustrated mesoderm-specific lacZ expression. The β-galactocidase produced by lacZ was visualized via antibody and DAB staining of Drosophila embryos. Moreover, we have shown that MEF2 is capable of binding to the enhancer region of singles bar via gel mobility shift assays. These studies enhance our understanding of how sing expression and myoblast fusion are controlled in flies and will likely have applications for treating heart disease and muscular diseases in humans.
CHARACTERIZATION OF ROOT UV-B SENSETIVE2 PROMOTER IN ARABIDOPSIS THALIANAMichelle Wallace, Zheng-Hui He.
San Francisco State University, San Francisco, CA.
Ultraviolet B light (UV-B, 280 to 320 nm) can either harm or help organisms depending on its fluence level. Low fluence levels serve as signals for plant morphogenesis, while high fluence levels can increase mutation and damage macromolecules such as DNA, RNA, and protein. Thus far, little is known about how low-fluence UV-B regulates plant growth and development. Our lab has discovered an Arabidopsis mutant, called rus2 (root uv-b sensetive2), that is hypersensitive to very low-fluence (VLF) UV-B. Under VLF UV-B, rus2 is stunted and pale, suggesting RUS2 plays important roles in early seedling UV-B response. To understand how RUS2 functions, we studied RUS2 promoter activities at various developmental stages. The RUS2 promoter region was PCR amplified from Arabidopsis genomic DNA, cloned in a pGEM vector and confirmed by sequencing. This fragment was then fused to the reporter gene
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beta-glucoronidase (GUS) to create a RUS2::GUS construct, then ligated into the pBI101 transformation vector. We are placing the confirmed plasmid into Agrobacterium tumerfacien to transform wild-type Arabidopsis. RUS2 promoter activities will be analyzed by incubating transgenic plants with X-Gluc, a colorless substrate converted by GUS to a blue product. The amount of GUS activities found in Arabidopsis seedlings is a direct reflection of RUS2 promoter activity. RUS2::GUS transgenic seedlings of various ages will be subjected to various treatments (with or without stresses related to UV-B, osmotic, drought, and temperature), and RUS2::GUS activities will be assayed. We expect to obtain a detailed RUS2 expression profile, providing important insights to how RUS2 functions in early seedling UV-B responses.
IDENTIFYING GENES THAT MODULATE VIRAL REPLICATION IN C. ELEGANS
University of California, Riverside, Riverside, CA.
Defense against viruses is an important human health problem. We are using the nematode C. elegans as a model to study host-virus interactions. We have previously shown that the flock house virus (FHV), a (+)-strand RNA virus, can replicate in C. elegans, although this is not its natural host. Normal FHV carries two RNAs: RNA1 encodes and RNA-dependent RNA polymerase (RdRP) that copies the RNA and the B2 protein, which suppresses host RNA interference (RNAi). RNA2 encodes the viral capsid. We are working with a C. elegans strain that we have engineered to express FHV RNA1, in which the B2 coding region is replaced with that of GFP. When this transgene is induced by heat shock, GFP expression indicates viral replication. Using chemical mutagenesis and RNA interference, we have identified genes that, when mutated or silenced by RNAi, enhance or suppress replication of FHV in C. elegans. Many of the genes found by RNAi-mediated knockdown play roles in lipid storage and autophagy. While previous studies have shown a relationship between these pathways and longevity in C. elegans, we now have good evidence that autophagy and lipid storage also play roles in regulating a defense response against viral replication.
HR REPAIR PROTEIN SILENCING AND HETEROCHROMATIN FORMATIONChristopher Still II, Leo Brueggeman, Jake Kirkland, Rohinton Kamakaka.
University of California, Santa Cruz, Santa Cruz, CA.
Nearly all diseases have a genetic component to them and many diseases arise due to failure in an organism’s ability to repair and maintain its DNA. With this in mind, it is evident there is a need to better understand the proteins and functions of DNA repair and maintenance. Our research investigates the interactions between DNA repair proteins and heterochromatin formation in the budding yeast, Saccharomyces cerevisiae. To investigate this relationship we created a reporter strain with nonfunctional silencers flanking a reporter gene. Specific repair proteins can be recruited to the silencers via tethering to sequence specific DNA binding proteins and the reporter gene can be assayed for silencing. We created additional strains with gene knockouts in repair proteins, using site-directed homologous recombination and crossing. This allows us to better determine the pathway that repair proteins are using to silence genes and form heterochromatin. We have determined that some homologous recombination (HR) repair proteins are sufficient for silencing the reporter gene and that the HR proteins require Sir3, a heterochromatin protein for silencing.
We have also determined that HR proteins are sufficient in tethering a locus to the nuclear periphery where silencing occurs. With the results from this research it is believed that we can not only better understand how S. cerevisiae ensures genome integrity and thus create more elaborate experiments for further research, but also that these results may be used as groundwork in design of therapies and medications for diseases.
BRACHYPODIUM DISTACHYON: AN ANALYSIS OF QUANTITATIVE TRAITSJose Villarreal Jr., Kyle Hernandez.
University of Texas at Austin, Austin, TX.
Brachypodium distachyon is a cereal from the temperate regions and is widely used as a genomic model for economically important grasses such as maize, wheat, barley, etc. It is a relatively practical model due to its small genome and feasibility for experimental manipulation. Using a recombinant inbred-line (RIL) population grown under common greenhouse conditions (BD3-1 x BD21), we explored the quantitative genetics underlying a series of economically important traits including flowering time, physiology, and seed shattering. In addition, we used quantitative trait loci (QTL) mapping to localize regions of chromosomes associated with these traits. Preliminary
A MUTAGENESIS SCREEN TO IDENTIFY GENES REQUIRED FOR DEVELOPMENTAL ETHANOL RESPONSE INDROSOPHILA Jodie Wu, Rachael French, Peter Luu, Janet Lafler, Elizabeth Henn, Audrey Ford, Theresa Logan, David Do, Clare Wadsworth, Hilal Jarrar, Anthony Bortolazzo, Sabrina Lopez, Martin Koch.
San Jose State University, San Jose, CA.
Fetal alcohol syndrome (FAS) is a spectrum disorder brought on by maternal alcohol consumption during pregnancy.
While it has been clear for over 2 decades that genetic factors are involved in the susceptibility to and severity of FAS, no genes mediating these effects have been identified. In addition, very little is known about the targets of ethanol during fetal development. Drosophila larvae reared in ethanol mirror the detrimental effects of FAS; we are therefore using flies as a genetic model to identify developmental ethanol targets. In order to identify the genes involved in larval response to ethanol exposure in an unbiased fashion, we generated approximately 1,000 novel transposoninduced mutations and screened them for altered survival and development time when reared in ethanol. Analysis is ongoing, but based on our current data, we expect to recover approximately 30 new mutations. We will discuss the genes identified as well as their ethanol-induced phenotypes. Additionally, we will show the expression patterns of a subset of the identified genes and how expression is affected by developmental ethanol exposure. Finally, mutants will be tested for changes in sedation and tolerance behaviors to ethanol relative to the wild-type Drosophila. The genes we identify will help us understand the molecular and cellular changes that underlie the deleterious effects of developmental ethanol exposure and, ultimately, may provide targets for the treatment of FAS.
GENETIC VARIATION OF THE ASSIMINEA INFIMA, AN ENDANGERED MOLLUSK OF THE RIO GRANDEREGION Jordan Thompson Thompson, Hisu-Ping Liu.
Metropolitan State University of Denver, Denver, CO.
The goal of this project is to evaluate the species status of the North American Assiminea infima species in the Death Valley of the lower Colorado River. Twenty-three total snails were collected from 3 sites from Blue Point Spring, Saratogo Spring, and Kings Pool in the Death Valley. DNA was extracted from each individual snail using the CTAB method. Two mitochondrial genes, cytochrome c oxidase subunit I and 16S ribosomal RNA, were sequenced. The molecular data suggest that the Blue Point Spring population is divergent from all other assiminneid populations, suggesting it should be described as a new species.
FINDING THE ANCESTRAL LINK AMONG WOMEN WITH TRIPLE-NEGATIVE BREAST CANCERNuria Perez Varela, Leticia Marquez-Magaña.
San Francisco State University, San Francisco, CA.
Triple negative breast cancers (TNBC) are characterized by the absence of estrogen receptor, progesterone receptor, and HER-2 expression, making them especially deadly. This is because these receptors are the targets for highly successful treatments for breast cancer, and their absence makes it difficult to treat TNBC. TNBC is found in 15% of US women, with African-American women having the highest incidence. In Sub-Saharan Africa, the rate of TNBC appears to be much higher. A recent study of 75 breast cancer patients in Ghana found that 82% had TNBC. The high rates of TNBC in African-American and Ghanaian women suggest it may have a genetic ancestral component.
The purpose of this study is to determine the mtDNA type of 100 Latinas with and without TNBC to determine if an ancestral link exists among patients diagnosed with this form of cancer. Latinas are a human subpopulation having European, Native American, and African ancestry. The ancestral nature of their maternal lineage can be determined by mtDNA typing. Therefore, the mtDNA type of 100 Latinas, 31 with TNBC, 34 with triple positive breast
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cancer, and 35 unaffected controls will be determined. Preliminary results show that a TNBC cell line has an mtDNA type indicative of African ancestry. A triple-positive cell line has an mtDNA type indicative of European ancestry.
Consequently, we predict that TNBC samples will display greater African ancestry. The identification of an ancestral link among women with TNBC may allow for development of methods for its early detection and treatment.
DOES THE GENETICS OF PARAQUAT RESISTANCE IN MOUSE STEM CELLS PREDICT INCREASED LIFE
EXPECTANCY IN NEMATODESJerome Castillon, James Cypser, Patricia Tedesco, Thomas Johnson.
University of Colorado Boulder, Boulder, CO.