Browsing by Subject "genes"
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Item Directed Evolution Of Xylose Isomerase For Improved Xylose Catabolism And Fermentation In The Yeast Saccharomyces Cerevisiae(2012-08) Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S.; Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S.The heterologous expression of a highly functional xylose isomerase pathway in Saccharomyces cerevisiae would have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways in S. cerevisiae suffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of the Piromyces sp. xylose isomerase (encoded by xylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing a gre3 knockout and tall and XKS1 overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.Item Forelimb-Hindlimb Developmental Timing Changes across Tetrapod Phylogeny(2007-10) Bininda-Emonds, Olaf R. P.; Jeffery, Jonathan E.; Sanchez-Villagra, Marcelo R.; Hanken, James; Colbert, Matthew; Pieau, Claude; Selwood, Lynne; ten Cate, Carel; Raynaud, Albert; Osabutey, Casmile K.; Richardson, Michael K.; Colbert, MatthewTetrapods exhibit great diversity in limb structures among species and also between forelimbs and hindlimbs within species, diversity which frequently correlates with locomotor modes and life history. We aim to examine the potential relation of changes in developmental timing (heterochrony) to the origin of limb morphological diversity in an explicit comparative and quantitative framework. In particular, we studied the relative time sequence of development of the forelimbs versus the hindlimbs in 138 embryos of 14 tetrapod species spanning a diverse taxonomic, ecomorphological and life-history breadth. Whole- mounts and histological sections were used to code the appearance of 10 developmental events comprising landmarks of development from the early bud stage to late chondrogenesis in the forelimb and the corresponding serial homologues in the hindlimb. Results: An overall pattern of change across tetrapods can be discerned and appears to be relatively clade- specific. In the primitive condition, as seen in Chondrichthyes and Osteichthyes, the forelimb/ pectoral fin develops earlier than the hindlimb/ pelvic fin. This pattern is either retained or re- evolved in eulipotyphlan insectivores (= shrews, moles, hedgehogs, and solenodons) and taken to its extreme in marsupials. Although exceptions are known, the two anurans we examined reversed the pattern and displayed a significant advance in hindlimb development. All other species examined, including a bat with its greatly enlarged forelimbs modified as wings in the adult, showed near synchrony in the development of the fore and hindlimbs. Conclusion: Major heterochronic changes in early limb development and chondrogenesis were absent within major clades except Lissamphibia, and their presence across vertebrate phylogeny are not easily correlated with adaptive phenomena related to morphological differences in the adult fore- and hindlimbs. The apparently conservative nature of this trait means that changes in chondrogenetic patterns may serve as useful phylogenetic characters at higher taxonomic levels in tetrapods. Our results highlight the more important role generally played by allometric heterochrony in this instance to shape adult morphology.Item Functional Assessment of Human 21st Chromosome Orthologs in Caenorhabditis elegans(2020-05) Smith, Sofia; Pierce, JonathanDown syndrome (DS) is a neurodevelopmental disorder caused by having an extra copy of the 21st chromosome. Every person with DS exhibits muscle weakness and ID, though the precise genotype-phenotype is not fully understood. Little is known about the individual genes that make up the human 21st chromosome. In attempting to fill this gap, I have worked with the laboratory of Dr. Jon Pierce to systematically characterize the genes of the 21st chromosome for a potential role in the nervous system or muscle function. To this end, the Pierce lab uses the nematode Caenorhabditis elegans to study orthologs, or equivalent genes, from the human 21st chromosome. The general approach is to identify genes conserved between human and C. elegans and conduct broad, unbiased screens to identify potentially important genes. C. elegans can be a useful model for taking this approach to DS, as it has been used to discover the function of hundreds of genes in the nervous system and muscle in humans and shares orthologs with many of the protein-coding genes on our 21st chromosome. We first conducted a loss-of-function screen, taking the C. elegans genes and systematically knocking them down to observe phenotypic defects. The C. elegans loss-of-function mutants underwent a series of behavioral and pharmacological assays to evaluate phenotypic defects. The loss-of-function screen resulted in the identification of 10 genes thought to be critical for synaptic or motor function. Several of these genes were not previously identified for a contribution to DS. We have since begun a gain-of-function screen using similar behavioral tests to empirically determine which genes are problematic when overexpressed. Preliminary results implicate a few genes in disrupting neuron-to-muscle circuitry. I have also verified transcriptional overexpression in mutants relative to control strains. I aim to uncover the roles of the individual genes involved in DS-associated phenotypes. Establishing which genes contribute to DS could clarify the mechanism for the neuromuscular symptoms associated with DS. Doing so can provide a foundation for other DS researchers and inform efforts toward a more precise mechanism of action. The hope is that these genes might later serve as therapeutic targets for alleviating neuromuscular symptoms in people with DS.Item miR-503 Represses Human Cell Proliferation and Directly Targets the Oncogene DDHD2 by Non-Canonical Target Pairing(2015-02) Polioudakis, Damon; Abell, Nathan S.; Iyer, Vishwanath R.; Polioudakis, Damon; Abell, Nathan S.; Iyer, Vishwanath R.The pathways regulating the transition of mammalian cells from quiescence to proliferation are mediated by multiple miRNAs. Despite significant improvements in our understanding of miRNA targeting, the majority of miRNA regulatory networks are still largely unknown and require experimental validation. Results: Here we identified miR-503, miR-103, and miR-494 as negative regulators of proliferation in primary human cells. We experimentally determined their genome wide target profiles using RNA-induced silencing complex (RISC) immunoprecipitations and gene expression profiling. Analysis of the genome wide target profiles revealed evidence of extensive regulation of gene expression through non-canonical target pairing by miR-503. We identified the proto-oncogene DDHD2 as a target of miR-503 that requires pairing outside of the canonical 5' seed region of miR-503, representing a novel mode of miRNA-target pairing. Further bioinformatics analysis implicated miR-503 and DDHD2 in breast cancer tumorigenesis. Conclusions: Our results provide an extensive genome wide set of targets for miR-503, miR-103, and miR-494, and suggest that miR-503 may act as a tumor suppressor in breast cancer by its direct non-canonical targeting of DDHD2.Item Prediction of gene–phenotype associations in humans, mice, and plants using phenologs(BMC Bioinformatics, 2013-06-21) Woods, John O.; Singh-Blom, Ulf Martin; Laurent, Jon M.; McGary, Kriston L.; Marcotte, Edward M.Background: Phenotypes and diseases may be related to seemingly dissimilar phenotypes in other species by means of the orthology of underlying genes. Such “orthologous phenotypes,” or “phenologs,” are examples of deep homology, and may be used to predict additional candidate disease genes. Results: In this work, we develop an unsupervised algorithm for ranking phenolog-based candidate disease genes through the integration of predictions from the k nearest neighbor phenologs, comparing classifiers and weighting functions by cross-validation. We also improve upon the original method by extending the theory to paralogous phenotypes. Our algorithm makes use of additional phenotype data — from chicken, zebrafish, and E. coli, as well as new datasets for C. elegans — establishing that several types of annotations may be treated as phenotypes. We demonstrate the use of our algorithm to predict novel candidate genes for human atrial fibrillation (such as HRH2, ATP4A, ATP4B, and HOPX) and epilepsy (e.g., PAX6 and NKX2-1). We suggest gene candidates for pharmacologically-induced seizures in mouse, solely based on orthologous phenotypes from E. coli. We also explore the prediction of plant gene–phenotype associations, as for the Arabidopsis response to vernalization phenotype. Conclusions: We are able to rank gene predictions for a significant portion of the diseases in the Online Mendelian Inheritance in Man database. Additionally, our method suggests candidate genes for mammalian seizures based only on bacterial phenotypes and gene orthology. We demonstrate that phenotype information may come from diverse sources, including drug sensitivities, gene ontology biological processes, and in situ hybridization annotations. Finally, we offer testable candidates for a variety of human diseases, plant traits, and other classes of phenotypes across a wide array of species.Item Rare Key Functional Domain Missense Substitutions in MRE11A, RAD50, and NBN Contribute to Breast Cancer Susceptibility: Results From a Breast Cancer Family Registry Case-Control Mutation-Screening Study(2014) Damiola, Francesca; Pertesi, Maroulio; Oliver, Javier; Le Calvez-Kelm, Florence; Voegele, Catherine; Young, Erin L.; Robinot, Nivonirina; Forey, Nathalie; Durand, Geoffroy; Vallee, Maxime P.; Tao, Kayoko; Roane, Terrell C.; Williams, Gareth J.; Hopper, John L.; Southey, Melissa C.; Andrulis, Irene L.; John, Esther M.; Goldgar, David E.; Lesueur, Fabienne; Tavtigian, Sean V.; Roane, Terrell C.The MRE11A-RAD50-Nibrin (MRN) complex plays several critical roles related to repair of DNA double-strand breaks. Inherited mutations in the three components predispose to genetic instability disorders and the MRN genes have been implicated in breast cancer susceptibility, but the underlying data are not entirely convincing. Here, we address two related questions: (1) are some rare MRN variants intermediate-risk breast cancer susceptibility alleles, and if so (2) do the MRN genes follow a BRCA1/BRCA2 pattern wherein most susceptibility alleles are protein-truncating variants, or do they follow an ATM/CHEK2 pattern wherein half or more of the susceptibility alleles are missense substitutions? Methods: Using high-resolution melt curve analysis followed by Sanger sequencing, we mutation screened the coding exons and proximal splice junction regions of the MRN genes in 1,313 early-onset breast cancer cases and 1,123 population controls. Rare variants in the three genes were pooled using bioinformatics methods similar to those previously applied to ATM, BRCA1, BRCA2, and CHEK2, and then assessed by logistic regression. Results: Re-analysis of our ATM, BRCA1, and BRCA2 mutation screening data revealed that these genes do not harbor pathogenic alleles (other than modest-risk SNPs) with minor allele frequencies > 0.1% in Caucasian Americans, African Americans, or East Asians. Limiting our MRN analyses to variants with allele frequencies of < 0.1% and combining protein-truncating variants, likely spliceogenic variants, and key functional domain rare missense substitutions, we found significant evidence that the MRN genes are indeed intermediate-risk breast cancer susceptibility genes (odds ratio (OR) = 2.88, P = 0.0090). Key domain missense substitutions were more frequent than the truncating variants (24 versus 12 observations) and conferred a slightly higher OR (3.07 versus 2.61) with a lower P value (0.029 versus 0.14). Conclusions: These data establish that MRE11A, RAD50, and NBN are intermediate-risk breast cancer susceptibility genes. Like ATM and CHEK2, their spectrum of pathogenic variants includes a relatively high proportion of missense substitutions. However, the data neither establish whether variants in each of the three genes are best evaluated under the same analysis model nor achieve clinically actionable classification of individual variants observed in this study.Item Transcription Restores DNA Repair to Heterochromatin, Determining Regional Mutation Rates in Cancer Genomes(2014-11) Zheng, Christina L.; Wang, Nicholas J.; Chung, Jongsuk; Moslehi, Homayoun; Sanborn, J. Zachary; Hur, Joseph S.; Collisson, Eric A.; Vemula, Swapna S.; Naujokas, Agne; Chiotti, Kami E.; Cheng, Jeffrey B.; Fassihi, Hiva; Blumberg, Andrew J.; Bailey, Celeste V.; Fudem, Gary M.; Mihm, Frederick G.; Cunningham, Bari B.; Neuhaus, Isaac M.; Liao, Wilson; Oh, Dennis H.; Cleaver, James E.; LeBoit, Philip E.; Costello, Joseph F.; Lehmann, Alan R.; Gray, Joe W.; Spellman, Paul T.; Arron, Sarah T.; Huh, Nam; Purdom, Elizabeth; Cho, Raymond J.; Blumberg, Andrew J.Somatic mutations in cancer are more frequent in heterochromatic and late-replicating regions of the genome. We report that regional disparities in mutation density are virtually abolished within transcriptionally silent genomic regions of cutaneous squamous cell carcinomas (cSCCs) arising in an XPC-/- background. XPC-/- cells lack global genome nucleotide excision repair (GG-NER), thus establishing differential access of DNA repair machinery within chromatin-rich regions of the genome as the primary cause for the regional disparity. Strikingly, we find that increasing levels of transcription reduce mutation prevalence on both strands of gene bodies embedded within H3K9me3-dense regions, and only to those levels observed in H3K9me3-sparse regions, also in an XPC-dependent manner. Therefore, transcription appears to reduce mutation prevalence specifically by relieving the constraints imposed by chromatin structure on DNA repair. We model this relationship among transcription, chromatin state, and DNA repair, revealing a new, personalized determinant of cancer risk.Item Unique Patterns of Transcript and miRNA Expression in the South American Strong Voltage Electric Eel (Electrophorus Electricus)(2015-03) Traeger, Lindsay L.; Volkening, Jeremy D.; Moffett, Howell; Gallant, Jason R.; Chen, Po-Hao; Novina, Carl D.; Phillips, George N., Jr.; Anand, Rene; Wells, Gregg B.; Pinch, Matthew; Gueth, Robert; Unguez, Graciela A.; Albert, James S.; Zakon, Harold; Sussman, Michael R.; Samanta, Manoj P.; Zakon, HaroldWith its unique ability to produce high-voltage electric discharges in excess of 600 volts, the South American strong voltage electric eel (Electrophorus electricus) has played an important role in the history of science. Remarkably little is understood about the molecular nature of its electric organs. Results: We present an in-depth analysis of the genome of E. electricus, including the transcriptomes of eight mature tissues: brain, spinal cord, kidney, heart, skeletal muscle, Sachs' electric organ, main electric organ, and Hunter's electric organ. A gene set enrichment analysis based on gene ontology reveals enriched functions in all three electric organs related to transmembrane transport, androgen binding, and signaling. This study also represents the first analysis of miRNA in electric fish. It identified a number of miRNAs displaying electric organ-specific expression patterns, including one novel miRNA highly over-expressed in all three electric organs of E. electricus. All three electric organ tissues also express three conserved miRNAs that have been reported to inhibit muscle development in mammals, suggesting that miRNA-dependent regulation of gene expression might play an important role in specifying an electric organ identity from its muscle precursor. These miRNA data were supported using another complete miRNA profile from muscle and electric organ tissues of a second gymnotiform species. Conclusions: Our work on the E. electricus genome and eight tissue-specific gene expression profiles will greatly facilitate future research on determining the coding and regulatory sequences that specify the function, development, and evolution of electric organs. Moreover, these data and future studies will be informed by the first comprehensive analysis of miRNA expression in an electric fish presented here.