Browsing by Author "Department of Biology, School of Science"

Now showing 1 - 10 of 51
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    Acetylation regulates DNA repair mechanisms in human cells
    (Informa UK (Taylor & Francis), 2016-06-02) Piekna-Przybylska, Dorota; Bambara, Robert A.; Balakrishnan, Lata; Department of Biology, School of Science
    The p300-mediated acetylation of enzymes involved in DNA repair and replication has been previously shown to stimulate or inhibit their activities in reconstituted systems. To explore the role of acetylation on DNA repair in cells we constructed plasmid substrates carrying inactivating damages in the EGFP reporter gene, which should be repaired in cells through DNA mismatch repair (MMR) or base excision repair (BER) mechanisms. We analyzed efficiency of repair within these plasmid substrates in cells exposed to deacetylase and acetyltransferase inhibitors, and also in cells deficient in p300 acetyltransferase. Our results indicate that protein acetylation improves DNA mismatch repair in MMR-proficient HeLa cells and also in MMR-deficient HCT116 cells. Moreover, results suggest that stimulated repair of mismatches in MMR-deficient HCT116 cells is done though a strand-displacement synthesis mechanism described previously for Okazaki fragments maturation and also for the EXOI-independent pathway of MMR. Loss of p300 reduced repair of mismatches in MMR-deficient cells, but did not have evident effects on BER mechanisms, including the long patch BER pathway. Hypoacetylation of the cells in the presence of acetyltransferase inhibitor, garcinol generally reduced efficiency of BER of 8-oxoG damage, indicating that some steps in the pathway are stimulated by acetylation.
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    Alpha-ring independent assembly of the 20S proteasome
    (Nature Publishing Group, 2015-08-19) Panfair, Dilrajkaur; Ramamurthy, Aishwarya; Kusmierczyk, Andrew R.; Department of Biology, School of Science
    Archaeal proteasomes share many features with their eukaryotic counterparts and serve as important models for assembly. Proteasomes are also found in certain bacterial lineages yet their assembly mechanism is thought to be fundamentally different. Here we investigate α-ring formation using recombinant proteasomes from the archaeon Methanococcus maripaludis. Through an engineered disulfide cross-linking strategy, we demonstrate that double α-rings are structurally analogous to half-proteasomes and can form independently of single α-rings. More importantly, via targeted mutagenesis, we show that single α-rings are not required for the efficient assembly of 20S proteasomes. Our data support updating the currently held "α-ring first" view of assembly, initially proposed in studies of archaeal proteasomes, and present a way to reconcile the seemingly separate bacterial assembly mechanism with the rest of the proteasome realm. We suggest that a common assembly network underpins the absolutely conserved architecture of proteasomes across all domains of life.
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    Assembly of proteasome subunits into non-canonical complexes in vivo
    (Elsevier, 2017-01) Hammack, Lindsay J.; Kusmierczyk, Andrew R.; Department of Biology, School of Science
    Proteasomes exist in all domains of life. In general, they are comprised of a compartmentalized protease whose activity is modulated by one or more regulatory complexes with which it interacts. The quaternary structure of this compartmentalized protease, called the 20S proteasome, is absolutely conserved and consists of four heptameric rings stacked coaxially. The rings are made of structurally related α and β subunits. In eukaryotes, assembly factors chaperone the α and β subunits during 20S biogenesis. Here we demonstrate that proteasome subunits can assemble into structures other than the canonical 20S proteasome in vivo. Specifically, the yeast α4 subunit forms high molecular weight complexes whose abundance increases when proteasome function is compromised. Results from a disulfide crosslinking approach are consistent with these complexes being ring-shaped. Though several eukaryotic α subunits can form rings when expressed recombinantly in bacteria, this is the first evidence that such non-canonical complexes exist in vivo.
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    The Axolotl Fibula as a Model for the Induction of Regeneration across Large Segment Defects in Long Bones of the Extremities
    (Public Library of Science, 2015) Chen, Xiaoping; Song, Fengyu; Jhamb, Deepali; Li, Jiliang; Bottino, Marco C.; Palakal, Mathew J.; Stocum, David L.; Department of Biology, School of Science
    We tested the ability of the axolotl (Ambystoma mexicanum) fibula to regenerate across segment defects of different size in the absence of intervention or after implant of a unique 8-braid pig small intestine submucosa (SIS) scaffold, with or without incorporated growth factor combinations or tissue protein extract. Fractures and defects of 10% and 20% of the total limb length regenerated well without any intervention, but 40% and 50% defects failed to regenerate after either simple removal of bone or implanting SIS scaffold alone. By contrast, scaffold soaked in the growth factor combination BMP-4/HGF or in protein extract of intact limb tissue promoted partial or extensive induction of cartilage and bone across 50% segment defects in 30%-33% of cases. These results show that BMP-4/HGF and intact tissue protein extract can promote the events required to induce cartilage and bone formation across a segment defect larger than critical size and that the long bones of axolotl limbs are an inexpensive model to screen soluble factors and natural and synthetic scaffolds for their efficacy in stimulating this process.
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    Break-Induced Replication is a Source of Mutation Clusters Underlying Kataegis
    (Elsevier B.V., 2014-06) Sakofsky, Cynthia J.; Roberts, Steven A.; Malc, Ewa; Mieczkowski, Piotr A.; Resnick, Michael A.; Gordenin, Dmitry A.; Malkova, Anna; Department of Biology, School of Science
    Clusters of simultaneous multiple mutations can be a source of rapid change during carcinogenesis and evolution. Such mutation clusters have been recently shown to originate from DNA damage within long single-strand (ss) DNA formed at resected double-strand breaks and dysfunctional replication forks. We identify here double-strand break (DSB)-induced replication (BIR) as another powerful source of mutation clusters that formed in nearly half of wild-type yeast cells undergoing BIR in the presence of alkylating damage. Clustered mutations were primarily formed along the track of DNA synthesis and were frequently associated with additional breakage and rearrangements. Moreover, the base specificity, strand coordination and strand bias of the mutation spectrum was consistent with mutations arising from damage in persistent ssDNA stretches within unconventional replication intermediates. Together, these features closely resemble kataegic events in cancers, suggesting that replication intermediates during BIR may be the most prominent source of mutation clusters across species.
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    Cell-specific actions of a human LHX3 gene enhancer during pituitary and spinal cord development
    (The Endocrine Society, 2013-12) Park, Soyoung; Mullen, Rachel D.; Rhodes, Simon J.; Department of Biology, School of Science
    The LIM class of homeodomain protein 3 (LHX3) transcription factor is essential for pituitary gland and nervous system development in mammals. In humans, mutations in the LHX3 gene underlie complex pediatric syndromes featuring deficits in anterior pituitary hormones and defects in the nervous system. The mechanisms that control temporal and spatial expression of the LHX3 gene are poorly understood. The proximal promoters of the human LHX3 gene are insufficient to guide expression in vivo and downstream elements including a conserved enhancer region appear to play a role in tissue-specific expression in the pituitary and nervous system. Here we characterized the activity of this downstream enhancer region in regulating gene expression at the cellular level during development. Human LHX3 enhancer-driven Cre reporter transgenic mice were generated to facilitate studies of enhancer actions. The downstream LHX3 enhancer primarily guides gene transcription in α-glycoprotein subunit -expressing cells secreting the TSHβ, LHβ, or FSHβ hormones and expressing the GATA2 and steroidogenic factor 1 transcription factors. In the developing nervous system, the enhancer serves as a targeting module active in V2a interneurons. These results demonstrate that the downstream LHX3 enhancer is important in specific endocrine and neural cell types but also indicate that additional regulatory elements are likely involved in LHX3 gene expression. Furthermore, these studies revealed significant gonadotrope cell heterogeneity during pituitary development, providing insights into the cellular physiology of this key reproductive regulatory cell. The human LHX3 enhancer-driven Cre reporter transgenic mice also provide a valuable tool for further developmental studies of cell determination and differentiation in the pituitary and nervous system.
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    Chemical Assay for the Detection of Vertebrate Fecal Metabolites in Adult Blow Flies (Diptera: Calliphoridae)
    (Oxford, 2018-06-06) Owings, Charity G.; Skaggs, Christine; Sheriff, Winyu; Manicke, Nicholas; Picard, Christine J.; Department of Biology, School of Science
    Filth flies are commonly implicated in pathogen transmission routes due to their affinity for vertebrate waste and their synanthropic associations. However, solidifying the link between flies and infected feces in the wild can be difficult, as interpretations made solely from microbial culturing or sequencing methods may represent an incomplete picture of pathogen acquisition. We present an analytical assay using high performance liquid chromatography tandem mass spectrometry (HPLC MS/MS) to detect vertebrate fecal metabolites (urobilinoids) in adult blow fly guts. Proof of concept experiments consisted of controlled feeding in which flies were grouped into three treatments (unfed, exposure to beef liver tissue, and exposure to canine feces; N = 20/treatment) using the black blow fly Phormia regina Meigen (Diptera: Calliphoridae). It was revealed that only feces-related samples exhibited peaks with an m/z of 591 and MS/MS spectra consistent with urobilinoids. These peaks were not seen for beef liver tissue, flies exposed to beef liver tissue, or unfed flies. Samples taken directly from beef liver tissue and from feces of several animals were also tested. To test this assay in wild flies, 216 flies were additionally analyzed to determine whether they had ingested vertebrate feces. About 13% of the wild flies exhibited these same peaks, providing a baseline measure of blow flies collected in urban and residential areas consuming feces from the environment. Overall, this assay can be used for P. regina collected in an applied setting and its integration with microbial culturing and sequencing methods will help to improve its use.
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    Climatic and geographic predictors of life history variation in Eastern Massasauga (Sistrurus catenatus): A range-wide synthesis
    (Plos, 2017-02-14) Hileman, Eric T.; King, Richard B.; Adamski, John M.; Anton, Thomas G.; Bailey, Robyn L.; Baker, Sarah J.; Bieser, Nickolas D.; Bell Jr., Thomas A.; Bissell, Kristin M.; Bradke, Danielle R.; Campa III, Henry; Casper, Gary S.; Cedar, Karen; Cross, Matthew D.; DeGregorio, Brett A.; Dreslik, Michael J.; Faust, Lisa J.; Harvey, Daniel S.; Hay, Robert W.; Jellen, Benjamin C.; Johnson, Brent D.; Johnson, Glenn; Kiel, Brooke D.; Kingsbury, Bruce A.; Kowalski, Matthew J.; Lee, Yu Man; Lentini, Andrew M.; Marshall, John C.; Mauger, David; Moore, Jennifer A.; Paloski, Rori A.; Phillips, Christopher A.; Pratt, Paul D.; Preney, Thomas; Prior, Kent A.; Promaine, Andrew; Redmer, Michael; Reinert, Howard K.; Rouse, Jeremy D.; Shoemaker, Kevin T.; Sutton, Scott; VanDeWalle, Terry J.; Weatherhead, Patrick J.; Wynn, Doug; Yagi, Anne; Department of Biology, School of Science
    Elucidating how life history traits vary geographically is important to understanding variation in population dynamics. Because many aspects of ectotherm life history are climate-dependent, geographic variation in climate is expected to have a large impact on population dynamics through effects on annual survival, body size, growth rate, age at first reproduction, size-fecundity relationship, and reproductive frequency. The Eastern Massasauga (Sistrurus catenatus) is a small, imperiled North American rattlesnake with a distribution centered on the Great Lakes region, where lake effects strongly influence local conditions. To address Eastern Massasauga life history data gaps, we compiled data from 47 study sites representing 38 counties across the range. We used multimodel inference and general linear models with geographic coordinates and annual climate normals as explanatory variables to clarify patterns of variation in life history traits. We found strong evidence for geographic variation in six of nine life history variables. Adult female snout-vent length and neonate mass increased with increasing mean annual precipitation. Litter size decreased with increasing mean temperature, and the size-fecundity relationship and growth prior to first hibernation both increased with increasing latitude. The proportion of gravid females also increased with increasing latitude, but this relationship may be the result of geographically varying detection bias. Our results provide insights into ectotherm life history variation and fill critical data gaps, which will inform Eastern Massasauga conservation efforts by improving biological realism for models of population viability and climate change.
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    Coiled-coil domain containing 42 (Ccdc42) is necessary for proper sperm development and male fertility in the mouse
    (Elsevier, 2016-04-15) Pasek, Raymond C.; Malarkey, Erik; Berbari, Nicolas F.; Sharma, Neeraj; Kesterson, Robert A.; Tres, Laura L.; Kierszenbaum, Abraham L.; Yoder, Bradley K.; Department of Biology, School of Science
    Spermiogenesis is the differentiation of spermatids into motile sperm consisting of a head and a tail. The head harbors a condensed elongated nucleus partially covered by the acrosome-acroplaxome complex. Defects in the acrosome-acroplaxome complex are associated with abnormalities in sperm head shaping. The head-tail coupling apparatus (HTCA), a complex structure consisting of two cylindrical microtubule-based centrioles and associated components, connects the tail or flagellum to the sperm head. Defects in the development of the HTCA cause sperm decapitation and disrupt sperm motility, two major contributors to male infertility. Here, we provide data indicating that mutations in the gene Coiled-coil domain containing 42 (Ccdc42) is associated with malformation of the mouse sperm flagella. In contrast to many other flagella and motile cilia genes, Ccdc42 expression is only observed in the brain and developing sperm. Male mice homozygous for a loss-of-function Ccdc42 allele (Ccdc42(KO)) display defects in the number and location of the HTCA, lack flagellated sperm, and are sterile. The testes enriched expression of Ccdc42 and lack of other phenotypes in mutant mice make it an ideal candidate for screening cases of azoospermia in humans.
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    Data on the identity of non-canonical complexes formed from proteasome subunits in vivo
    (Elsevier, 2016-11-22) Hammack, Lindsay J.; Kusmierczyk, Andrew R.; Department of Biology, School of Science
    The dataset presented here represents analysis supplied by the local proteomics core facility on samples submitted to it in support of the article "Assembly of proteasome subunits into non-canonical complexes in vivo" Hammack and Kusmierczyk (2016) [1]. This article provides the detailed protein contents of gel slices, cut from non-denaturing polyacrylamide gels, containing distinct protein complexes visualized following gel staining. The identification of the protein contents of these complexes was carried out by liquid chromatography tandem mass-spectrometry (LC-MS/MS).