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In one of the early steps in the initiation of translation, the cap-binding complex eIF4F binds to the 7-methyl-guanosine of the mRNA in preparation for translation. The eIF4F complex is composed of eIF4G, the scaffolding protein, and eIF4E, the cap-binding protein, and is conserved in mammals, fungi, and plants. Unique to plants is an alternative complex, eIFiso4F, made up of eIFiso4G and eIFiso4E. Having a comparable molecular weight and an amino acid sequence similarity of ~41%, eIFiso4E is very similar to eIF4E. The functional domains found in eIF4G are the same in eIFiso4G; however, eIFiso4G is lacking a significant portion of the N-terminus. Our hypothesis is that these differences indicate differing roles that the 2 complexes may have. To investigate these roles, we have carried out RNA-immunoprecipitation on wild type Arabidopsis thaliana using antibodies specific to either of the 2 complexes, Graduate
favored by one isoform over the other will be confirmed by in vitro translation assays.
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PREDICTING TETRACYCLINE RESISTANCE THROUGH MATHEMATICAL MODELINGAnisha Perez, Colin Bacorn, Erica Stoddard, Kimberly DeBruler, Yousif Shamoo.
Rice University, Houston, TX.
The US Department of Health & Human Services reports that healthcare-associated infections (HAIs) are one of the top 10 leading causes of death in the United States. Due to the widespread use of antibiotics, bacterial resistance has emerged as a growing threat to public health. We have demonstrated that under simple conditions, antibiotic resistance and its evolution can be accurately modeled in vitro. We use experimental evolution and biochemical
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assays to link cellular fitness (e.g., resistance) to the physicochemical properties of proteins, specifically to the enzyme TetX2. TetX2 is an enzyme that inactivates the tetracycline class of antibiotics including minocycline (MCN) and the first-line antibiotic tigecycline. Our mathematical model correlates the effect of increasing internal MCN concentrations on bacterial growth rates taking into account MCN inactivation by TetX2, and diffusion of the antibiotic across the membrane and periplasm. Most importantly, this relationship is reversible allowing us to predict kinetic protein properties from growth rates. Our goal now is to extend the model to include drug effluxer pumps specific to tetracycline resistance, TetA, and those indicated in general antibiotic resistance such as the tripartite pumps AcrAB, TolC, and AdeABC. The model can be used to deduce the physicochemical parameters of these membrane proteins without the difficult and arduous tasks of purification and kinetic assays.
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THE DEVELOPMENT OF IN VITRO MICELLES FOR DETERMINING THE KM OF LECITHIN CHOLESTEROL
ACYLTRANSFERASE FOR CHOLESTEROL IN THE PRESENCE OR ABSENCE OF JOJOBA OILJeremiah Heredia, Raymond Garcia.
California State University, Los Angeles, Los Angeles, CA.
Atherosclerosis is the leading cause of death in the United States. It is a progressive disease that causes the hardening of arteries due to excess cholesterol and fat deposits in the inner arterial wall. Elevated high density lipoprotein (HDL) concentrations are anti-atherogenic. Reverse cholesterol transport (RCT) is an important process for transporting cholesterol in HDL from peripheral tissues and macrophages to the liver for degradation. This transport system is regulated through various proteins including lecithin cholesterol acyltransferase (LCAT), which is an enzyme that esterfies free cholesterol to cholesteryl esters in HDL. When New Zealand White (NZW) rabbits were fed a cholesterol plus jojoba oil-rich diet, elevated HDL concentrations and enhanced LCAT activity were obtained.
We hypothesize that jojoba oil is regulating the biochemical mechanism action of LCAT by decreasing the Km of this enzyme for its cholesterol substrate. This hypothesis will be validated by developing in vitro micelles with different concentrations of free cholesterol. Two types of micelles, those with free cholesterol and those with free cholesterol plus jojoba oil, will be exposed to LCAT in the serum of NZW rabbits. The rate of esterification of free cholesterol to cholesteryl ester will be determined with an enzymatic cholesterol assay. If our hypothesis is correct, then the Km of LCAT for cholesterol will be lower with the cholesterol plus jojoba oil micelles than with the cholesterol micelles.
(Supported by NIH Grant GM61331.)
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THE ROLE OF A PUTATIVE ARABIDOPSIS THALIANA FLAVONOL SYNTHASE IN THE BIOREMEDIATION OF
PHENANTHRENEJuan Hernandez-Vega, Adan Colon-Carmona.
University of Massachusetts Boston, Boston, MA.
Polycyclic aromatic hydrocarbon (PAH) pollution is an issue with severe consequences to the environment. PAHs are a group of contaminants with 2 or more benzene rings fused together; they are very stable and resistant to degradation in soil and water environments. Current bioremediation strategies include the use of soil bacteria such as Pseudomonas to degrade these contaminants. These bacteria contain dioxygenases (DOXs) that modify PAHs by oxidizing their structure and increasing their reactivity. The objective of this research is to determine if plant DOXs have similar ability. In our laboratory, we use phenanthrene as a model PAH. Previous data confirmed the overexpression of 6 DOX genes in response to PAH exposure. The cDNA of one of these enzymes, flavonol synthase (AT5G05600), was cloned and expressed in Escherichia coli cells. These proteins were successfully purified from the cells’ extracts. Current experiments include in vitro enzymatic assays to determine possible chemical modifications of PAHs. Preliminary fluorescent emission results suggest that modifications of phenanthrene took place. Phenotypic analysis of knock-out mutant lines, compared with wild-type plants, showed that mutants were hyposensitive to phenanthrene, suggesting a possible role of this gene in PAH degradation in vivo. Moreover, preliminary results of transgenic lines over-expressing flavonol synthase showed a hypersensitive effect of these lines under phenanthrene treatment. Taken together, these results support the idea of the involvement of flavonol synthase in the catabolism of PAHs and the accumulation of toxic intermediates during its biotransformation in plants.
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INVESTIGATION OF NATURAL POPULATIONS OF CARICA PAPAYA’S MORPHOLOGICAL AND GENETIC
STRUCTURE THROUGHOUT MESOAMERICASandra Mardonovich1, Richard Moore1, Oscar Rocha2.
Miami University, Oxford, OH, 2Kent State University, Kent, OH.
1 Gene flow is an important process when looking at the population structure of a species and its evolutionary history.
This project aims to characterize the morphological and genetic diversity of natural Carica papaya populations throughout Mesoamerica to determine whether these populations are wild (populations derived from uncultivated stock) or feral (populations derived from individuals escaped from cultivation). A previous study on natural Costa Rican papaya populations illustrated a wide range of morphological diversity in reproductive structures and showed a high level of allelic diversity among these populations. This previous study, however, was limited in geographic scope because only populations from Costa Rica were sampled, while the range of natural papaya populations extends from Costa Rica north to Southern Mexico. An expansion of this project to include populations from Panama, Nicaragua, Honduras, Guatemala, and Southern Mexico combined with the data from Costa Rica will give a thorough analyses of gene flow among and within papaya populations. With 20 nuclear microsatellites, the structure and levels of genetic diversity will be measured to distinguish genetic relationships among populations and to untangle any shared genetic ancestry with cultivars. If results are similar to Costa Rican populations, we can infer there is some level of introgression from cultivated papaya into natural papaya populations. This could also provide more insight to the domestication history of this important tropical fruit crop.
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SMALL-SCALE GENOTYPIC DIVERSITY AND BREEDING DYNAMICS IN THE DESERT MOSS SYNTRICHIACANINERVIS Jennifer Rodriguez, Kristen Fisher.
California State University, Los Angeles, Los Angeles, CA.
Syntrichia caninervis is a dominant moss in the Mojave Desert, where it plays an important role in the composition of biotic soil crusts. While individual patches of this moss species are typically assumed to represent multiple branches of the same clone (individual genotype), preliminary genotyping efforts on S. caninervis have indicated that small patches may, in fact, comprise multiple genetically unique individuals. Furthermore, sexual reproduction is relatively uncommon in S. caninervis, since male and female gametangia are produced by separate individuals, and fertilization distances are presumably very short. We have located a population of S. caninervisin in the eastern San Gabriel Mountains that supports an unusually high frequency of sexual reproduction, as indicated by the presence of multiple sporophytes. Here, we characterize the fine-scale demographics of S. caninervis by densely sampled and genotype branches from the same patch as, or patches proximate to, sporophytes in order to answer these questions: How many individuals (unique genotypes) are present in a small patch of S.caninervis? Are individual branches within a small patch closely related (e.g. siblings)? What is the fertilization distance from male to female gametophyte? Are multiple sporophytes on a female gametophyte fathered by the same male?
RAT OFFSPRINGLeon Clah, Ignacio Camarillo.
Purdue University, West Lafayette, IN.
Breast cancer is the most frequently diagnosed cancer among women. Lifestyle factors such as physical activity and diet play a role in attenuating the incidence of breast cancer. Current research shows that exercise during pregnancy can convey long-term health benefits to offspring. The goal of this project was to determine if maternal exercise during pregnancy could lead to reduced mammary tumor development in female offspring. Female rats were divided into 2 groups, sedentary and exercise, with the exercise group given access to a running wheel only during pregnancy.
Female pups were weaned at 21 days of age, were fed a high fat (HF) diet, and did not have access to an exercise wheel. At 6 weeks of age, pups were given a single injection of N-Methyl-N-nitrosourea (MNU) intraperitoneally
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at 50 mg/kg. The study was conducted for 15 weeks, and the developing tumors were palpated and measured with calipers. Endpoint analyses revealed that pups from exercised dams (Exercise) had a lower tumor incidence (42.86%), as compared to pups from sedentary dams (Sedentary), having 100% tumor incidence. Exercise pups had a higher frequency of tumors at 1.67 tumors/rat than Sedentary at 1.17 tumors/rat. In addition to tumor monitoring, the intraperitoneal (IP) fat was collected and weighed against the weight of the animal at time of euthanasia. Exercise pups had an increase in IP fat (2.67%) over the Sedentary pups (1.60%). Collectively, these are the first data to demonstrate that short-term exercise during pregnancy can lead to reduced tumor development in offspring.
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VISUAL CONFIRMATION OF DE NOVO GENE TRANSCRIPTION OF X-LINKED MIRNA GENES THAT ESCAPE
MEIOTIC SEX CHROMOSOME INACTIVATIONEnrique Sosa1, James Turner2, Wei Yan3, John R. McCarrey1.
University of Texas at San Antonio, San Antonio, TX, 2Division of Stem Cell Biology and Developmental Genetics, 1 Medical Research Council-National Institute for Medical Research, London, UK, 3University of Nevada, Reno, Reno, NV.
MicroRNAs (miRNAs) are small, non-coding RNAs known to regulate gene expression at the post-transcriptional level, and potentially at the transcriptional level as well. Studies with gene knockouts have demonstrated that miRNAs are critically required for normal spermatogenesis and male fertility in mammals. We have previously shown that ~20% of testicular miRNAs map to the X-chromosome and that ~40% of these display testis-specific or testispreferential expression. Surprisingly, real-time qPCR further revealed an increase in the expression of a majority of the X-linked miRNA transcripts in spermatocytes at a time when all 364 X-linked messenger RNA-encoding genes studied to date decline in expression due to meiotic sex chromosome inactivation (MSCI). Many of these genes also remain repressed by post meiotic sex chromatin (PMSC) in spermatids. We hypothesized that elevated levels of X-linked miRNA transcripts in primary spermatocytes are due to ongoing, active transcription, indicating escape of these genes from the repressive effects of MSCI. To test this hypothesis, we used immunofluorescence staining, DNA-fluorescence in situ hybridization (FISH), and RNA-FISH on spermatogenic cells from testes of adult mice. Our data confirm that type I, II, and III X-linked miRNAs suppressed by MSCI, escaping MSCI, or escaping MSCI and PMSC, respectively. Three-dimensional analyses obtained by reconstructing confocal images of spermatocyte nuclei indicate type II and III X-linked miRNA genes that escape MSCI undergo de novo transcription only at the periphery of the heterochromatic XY body. Further studies are under way to characterize the molecular mechanisms involved in escape of X-linked miRNA genes from MSCI or PMSC.
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CYTOKINE STIMULATION OF MUC16 EXPRESSION IN HUMAN ENDOMETRIAL AND OVARIAN EPITHELIALCELL LINES Micaela Morgado1, Margie N Sutton2, Mary Simmons3, Lu Zhen2, Robert Bast2, Daniel D Carson1.
Rice University, Houston, TX, 2University of Texas MD Anderson Cancer Center, Houston, TX, 3University of Texas 1 Southwestern Medical School, Dallas, TX.