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The lens is a transparent tissue of the eye that serves to focus light on the retina for optimal visual acuity. Loss of lens transparency results in a disease termed “cataract” which affects ~77 million individuals and accounts for ~50% cases of blindness worldwide. Specialized lens cells termed “fiber cells” function to render the lens transparent by undergoing a terminal differentiation program that involves dramatic regulation of gene expression accompanied by cellular elongation and organelle degradation. We have identified an RNA binding protein and RNA granule component TDRD7, mutations in which are associated with posterior polar cataract in pediatric patients. We hypothesize that TDRD7 mediates post-transcriptional control of gene expression in fiber cells. Tdrd7 null mouse mutants closely phenocopy the human cataract and thus present an excellent resource for investigating TDRD7 function in the lens. We intend to perform RNA sequencing to identify differentially regulated transcripts and potential splice variants in Tdrd7 null mouse mutant lens. Furthermore, we intend to test if Tdrd7 nullizygosity affects the expression of small RNAs (e.g., miRNAs, piRNAs, and snoRNAs) in the lens. Additionally, to test if different RNAs are preferentially localized in different regions of the elongated fiber cells, we are using laser capture microdissection to isolate apical and basal regions of mouse fiber cells for gene expression profiling by microarrays.
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SCREEN FOR NUCLEAR-ENCODED SPLICING FACTORS FOR YEAST MITOCHONDRIAL GROUP II INTRONSRachel Wolf, Alan Lambowitz.
University of Texas at Austin, Austin, TX.
Hypothesized to be ancestors of eukaryotic spliceosomal introns, extant group II introns have been found in bacteria Archaea and in the mitochondria and chloroplast genomes of some eukaryotes. Sometimes referred to as molecular fossils, some of these catalytic RNAs may encode an intron-encoded protein (IEP) that has reverse-transcriptase, RNA splicing, DNA binding, and DNA endonuclease activities. Not all group II introns, however, encode an IEP, eliciting the question as to how these introns are spliced out. Two such model introns are the mitochondrial (mt) introns aI5γ and bI1 in Saccharomyces cerevisiae. Previous in vitro studies on the self-splicing activity of these introns have revealed requirements of nonphysiologically high salt concentration and temperature for catalytic activity, suggesting dependence upon proteins in vivo. With most mitochondrial proteins known to be encoded by the nucleus, Graduate
Previous searches for such mutants relied on a combination of 2 screens: looking for a glycerol (Gly) phenotype in the presence of a given intron and Gly+ phenotype in the intron’s absence. In contrast, this study employs a rapid method that uses northern hybridization to identify splicing defects in null mutants, allowing for recognition of proteins that would not have been detectable by other methods. Preliminary results suggest involvement of multiple proteins and a possible splicing complex situated at the surface of the mt inner membrane in close association with mt ribosomes.
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RNA-FLOURESCENCE IN SITU HYBRIDIZATION PROBE DESIGNLuis Flores, Enrique Sosa, John R. McCarrey.
University of Texas at San Antonio, San Antonio, TX.
RNA fluorescence in situ hybridization (RNA-FISH) is a powerful technique used to detect sites of de novo transcription in cells. RNA-FISH can be used in combination with immunofluorescence staining to detect gene expression in specific cell types. We have optimized the RNA-FISH protocol for use in spermatogenic cells. We have designed RNA-FISH probes to be used to determine the transcriptional timing of X-linked microRNA (miRNA) genes that escape the process of meiotic sex chromosome inactivation (MSCI) during prophase I of spermatogenesis. Thus far, we have successfully produced RNA-FISH probes for the control genes Ataxia telangiectasia and Rad3 related (Atr) and α-crystalline (Cryaa). Atr is a constitutively expressed autosomal gene and therefore serves as a positive control, while Cryaa is a gene that is terminally repressed in spermatogenic cells and serves as a negative control. We have also produced probes specific to X-linked miRNAs that escape MSCI. This approach will allow us to assess the extent to which X-linked miRNA genes escape MSCI during spermatogenesis and allow us to determine the precise timing of X-linked miRNA transcriptional activity in relation to normal X-linked mRNA gene inactivation due to MSCI.
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RNA INHIBITION OF LATE MITOTIC TARGETS THAT REDUCE MITOTIC SLIPPAGE IN DROSOPHILA S2 CELLSLuis Soto, Blake Riggs.
San Francisco State University, San Francisco, CA.
Chemotherapy is one of the most widely used cancer treatments; however, it is not effective against all types of cancer. It has been shown that some cancer cells escape the mitotic arrest caused by chemotherapeutic drugs and exit mitosis without completing chromosome segregation. This phenomenon is known as mitotic slippage and it can confer resistance to chemotherapy by allowing cancer cells to exit mitosis before the onset of apoptosis caused by a prolonged mitotic arrest. Recently, a study showed that blocking the regulatory machinery that drives mitotic exit eliminates mitotic slippage. However, this study relied on depleting a regulatory protein to levels below 5%, which may not be a practical therapeutic strategy. Therefore, our research aims to identify better targets for blocking mitotic slippage. I hypothesize that knockdown of late mitotic targets via RNA interference (RNAi) will block mitotic slippage and induce apoptosis in Drosophila S2 cells. To determine the efficiency of each knockdown, we will use time-lapse microscopy to visualize the number of cells that undergo apoptosis in the presence of a chemotherapeutic drug and compare these results to the number of cells that undergo mitotic slippage under the same conditions. Mitotic slippage will be determined by measuring DNA content of surviving cells via FACS. Cells that undergo mitotic slippage should be tetraploid because they exit mitosis prior to chromosome segregation. To conclude, the identification of better targets for blocking mitotic slippage could provide a way to improve current chemotherapeutic treatments and advance the field of cancer research.
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NINE POSTTRANSLATIONAL MODIFICATIONS DURING THE BIOSYNTHESIS OF CINNAMYCIN AND THE
TAILORING ENZYMESAyse Okesli1, Wilfred van der Donk2.
University of Illinois at Urbana-Champaign, Urbana, IL, 2Howard Hughes Medical Institute, University of Illinois at 1 Urbana-Champaign, Urbana, IL.
Lantibiotics are ribosomally synthesized and post-translationally modified antimicrobial peptides that are characterized by the thioether cross-linked amino acids lanthionine (Lan) and methyllanthionine (MeLan). Cinnamycin is a 19-amino-acid lantibiotic that contains one Lan and two MeLan. Cinnamycin also contains an unusual lysinoalanine (Lal) bridge formed from the ε-amino group of lysine 19 and a serine residue at position 6, and an erythro-3-hydroxyL-aspartic acid resulting from the hydroxylation of L-aspartate at position 15. These modifications are critical in mediating the interactions of cinnamycin with its target, phosphatidylethanolamine. Recently, the cinnamycin biosynthetic gene cluster (cin) from Streptomyces cinnamoneus cinnamoneus DSM 40005 was reported. Herein, we investigated the biosynthetic machinery using both in vitro studies and heterologous expression in Escherichia coli.
CinX is an R-ketoglutarate/iron(II)-dependent hydroxylase that carries out the hydroxylation of aspartate 15 of the precursor peptide CinA. In addition, CinM catalyzes dehydration of 4 Ser and Thr residues and subsequent cyclization of Cys residues to form the three MeLan bridges. The order of the post-translational modifications catalyzed by CinM and CinX is interchangeable in vitro. CinX did not require the leader sequence at the N-terminus of CinA for activity,
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but the leader peptide was necessary for CinM function. Although CinM dehydrated serine 6, it did not catalyze the formation of Lal. A small protein encoded by cinorf7 is critical for the formation of the cross-link between Lys19 and dehydroalanine 6 as shown by coexpression studies of CinA, CinM, CinX, and Cinorf7 in E. coli.
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DETERMINATION OF THE MEMBRANE TOPOLOGY OF PORCUPINERaymund Bueno1, Lisa Galli1, David Hernandez1, Vishwanath Lingappa2, Laura Burrus1.
San Francisco State University, San Francisco, CA, 2Prosetta Antiviral Inc., San Francisco, CA.
1 Wnt signaling is critical for proper embryonic development and adult tissue homeostasis. The palmitoylation of Wnt proteins by Porcn, a multitransmembrane spanning protein, is required for Wnt signaling. Porcn is localized to the endoplasmic reticulum (ER), Golgi, and cell surface. Mutations in Porcn cause dramatic defects in mice and humans. Though the developmental roles of Porcn are known, its topology is poorly understood. Although numerous bioinformatic algorithms that predict membrane topology are available, no clear prediction for Porcn has emerged.
Thus, we initiated experiments to experimentally determine the topology of Porcn. Our preliminary data orient the N and C terminus towards the lumenal and cytosolic sides of the ER, respectively. Moreover, introducing N-linked glycosylation sites into Porcn showed that an A174N point mutation results in glycosylation, indicating ER lumen localization. Cumulatively, our bioinformatic and experimental data led us to hypothesize that Porcn has 11 TM domains and that Cys 17 is the only cysteine residue oriented toward the ER lumen/cell surface. Immunostaining with a polyclonal antibody targeted against Porcn residues 281-301 localizes this region to the cytosol and is consistent with our hypothesis. Labeling of Porcn-expressing cells with biotin-maleimide further shows that at least one cysteine residue is present on the cell surface. We are now testing whether substitution of Cys 17 will remove all maleimide reactive sites in the Porcn protein that are localized to the cell surface. These data will provide strong evidence for the proposed model. Additional experiments will be carried out to further validate this model.
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CHEMICALLY DIVERSE MICROTUBULE STABILIZERS CAUSE DISTINCT DYSREGULATION OF KEY MITOTICPROTEINS Cristina Rohena, Susan Mooberry.
University of Texas Health Science Center at San Antonio, San Antonio, TX.
Microtubule stabilizers are some of the most useful and successful drugs used to treat adult solid tumors. However, the molecular events responsible for their antimitotic actions are not yet fully understood. In this study, we evaluated the mitotic defects and signaling events initiated by three structurally different microtubule stabilizers: taccalonolide AJ, laulimalide/fijianolide B, and paclitaxel. These agents cause the formation of aberrant, but morphologically distinct, mitotic spindles leading to the hypothesis that they initiate distinct mitotic signaling events. Each microtubule stabilizer caused different patterns of expression of key mitotic signaling proteins. Taccalonolide AJ caused centrosome separation failure to a much greater extent than paclitaxel or laulimalide. Taccalonolide AJ is also unique in that it was the only stabilizer to cause centrosome disjunction failure. These observations were consistent with the different defects in expression and activation of Plk1 and Eg5 caused by each stabilizer. Localization studies revealed that TPX2 and Aurora A are associated with each spindle aster formed by each stabilizer, which suggests a common mechanism of aster formation. However, taccalonolide AJ was the only one that caused pericentrin accumulation on every spindle aster. Pericentrin’s localization to every spindle aster could facilitate the stability of the highly focused asters formed by taccalonolide AJ. Laulimalide and paclitaxel caused completely different patterns of expression and activation of these proteins, as well as phenotypically different spindle phenotypes. Determining how chemically Graduate
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ASSOCIATIONS BETWEEN DEMERSAL FISHES AND STRUCTURE-FORMING INVERTEBRATES IN
TEMPERATE WATERS ON THE CONTINENTAL SHELF OF THE PACIFIC NORTHWESTElizabeth Lopez1, Sarah Henkel2, James Lindholm3.
University of San Diego, San Diego, CA, 2Oregon State University, Newport, OR, 3California State University, 1 Monterey Bay, Seaside, CA.
Plans are underway to deploy wave-energy capture devices off the coast of the Pacific Northwest as a solution to the region’s ever-growing demand for energy. Implementation of wave energy fields could have far-reaching effects on structure-forming invertebrates, which are slow growing, fragile, and sensitive to siltation. Impacts on these invertebrates could in turn have adverse effects on associated fish species by reducing available or preferred habitat. We aim to evaluate the extent to which fishes associate with the structure-forming invertebrate community at sites that may be impacted by wave energy generation. Video footage was collected for this study by the remotely operated vehicle (ROV) Hammerhead from Gray’s Bank, Washington, and Siltcoos Reef, Oregon, in late summer