Life Sciences Commons^™

Tal Effector-Nucleotide Targeter (Tale-Nt) 2.0: Tools For Tal Effector Design And Target Prediction, Erin L. Doyle, Nicholas J. Booher, Daniel S. Standage, Daniel F. Voytas, Volker P. Brendel, John K. Vandyk, Adam J. Bogdanove

Nicholas J. Booher

Transcription activator-like (TAL) effectors are repeat-containing proteins used by plant pathogenic bacteria to manipulate host gene expression. Repeats are polymorphic and individually specify single nucleotides in the DNA target, with some degeneracy. A TAL effector-nucleotide binding code that links repeat type to specified nucleotide enables prediction of genomic binding sites for TAL effectors and customization of TAL effectors for use in DNA targeting, in particular as custom transcription factors for engineered gene regulation and as site-specific nucleases for genome editing. We have developed a suite of web-based tools called TAL Effector-Nucleotide Targeter 2.0 (TALE-NT 2.0;https://boglab.plp.iastate.edu/) that enables design …

Sumoylation, Oliver Kerscher

Oliver Kerscher

Eukaryotic cells utilise the dynamic addition and removal of SUMO, a small ubiquitin‐like modifier (UBL), to modulate protein functions, interactions and localisation. Protein SUMOylation involves a cascade of dedicated enzymes that facilitate the covalent modification of specific lysine residues on target proteins with monomers or polymers of SUMO. The cellular homeostasis of SUMOylated proteins is also regulated by SUMO proteases and SUMO‐targeted ubiquitin ligase (STUbLs). SUMO proteases cleave SUMO from modified proteins. In contrast, STUbLs ubiquitinate proteins modified with SUMO chains. Recent data suggests that ubiquitination via STUbLs effects the turnover of SUMOylated proteins as well as the spatio‐temporal composition …

Sumo-Targeted Ubiquitin Ligase (Stubl) Slx5 Regulates Proteolysis Of Centromeric Histone H3 Variant Cse4 And Prevents Its Mislocalization To Euchromatin, Kentaro Ohkuni, Yoshimitsu Takahashi, Alyona Fulp, Oliver Kerscher

Oliver Kerscher

Centromeric histone H3, CENP-ACse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-ACse4 restricts its localization to centromeres. Mislocalization of CENP-ACse4 is associated with aneuploidy in yeast and flies and tumorigenesis in human cells; thus defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast shows that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and …

Sumo-Targeted Ubiquitin Ligases (Stubls) Reduce The Toxicity And Abnormal Transcriptional Activity Associated With A Mutant, Aggregation-Prone Fragment Of Huntingtin, Kentaro Ohkuni, Nagesh Pasupala, Jennifer Peek, Oliver Kerscher

Oliver Kerscher

Cell viability and gene expression profiles are altered in cellular models of neurodegenerative disorders such as Huntington’s Disease (HD). Using the yeast model system, we show that the SUMO-targeted ubiquitin ligase (STUbL) Slx5 reduces the toxicity and abnormal transcriptional activity associated with a mutant, aggregation-prone fragment of huntingtin (Htt), the causative agent of HD. We demonstrate that expression of an aggregation-prone Htt construct with 103 glutamine residues (103Q), but not the non-expanded form (25Q), results in severe growth defects in slx5Δ and slx8Δ cells. Since Slx5 is a nuclear protein and because Htt expression affects gene transcription, we …

Sumo Targeting Of A Stress-Tolerant Ulp1 Sumo Protease, Jennifer Peek, Catherine Harvey, Dreux Gray, Oliver Kerscher

Oliver Kerscher

SUMO proteases of the SENP/Ulp family are master regulators of both sumoylation and desumoylation and regulate SUMO homeostasis in eukaryotic cells. SUMO conjugates rapidly increase in response to cellular stress, including nutrient starvation, hypoxia, osmotic stress, DNA damage, heat shock, and other proteotoxic stressors. Nevertheless, little is known about the regulation and targeting of SUMO proteases during stress. To this end we have undertaken a detailed comparison of the SUMO-binding activity of the budding yeast protein Ulp1 (ScUlp1) and its ortholog in the thermotolerant yeast Kluyveromyces marxianus, KmUlp1. We find that the catalytic UD domains of both ScUlp1 and …

Successful Enrichment And Recovery Of Whole Mitochondrial Genomes From Ancient Human Dental Calculus, Andrew T. Ozga, Maria A. Nieves-Colon, Tanvi P. Honap, Krithivasan Sankaranarayanan, Courtney A. Hofman, George R. Milner, Cecil M. Lewis Jr., Anne C. Stone, Christina Warinner

Andrew Ozga

Objectives

Archaeological dental calculus is a rich source of host‐associated biomolecules. Importantly, however, dental calculus is more accurately described as a calcified microbial biofilm than a host tissue. As such, concerns regarding destructive analysis of human remains may not apply as strongly to dental calculus, opening the possibility of obtaining human health and ancestry information from dental calculus in cases where destructive analysis of conventional skeletal remains is not permitted. Here we investigate the preservation of human mitochondrial DNA (mtDNA) in archaeological dental calculus and its potential for full mitochondrial genome (mitogenome) reconstruction in maternal lineage ancestry analysis.

Materials and …

Origins Of An Unmarked Georgia Cemetery Using Ancient Dna Analysis, Andrew T. Ozga, Raul Y. Tito, Brian M. Kemp, Hugh Matternes, Alexandra J. Obregon-Tito, Leslie Neal, Cecil M. Lewis Jr.

Andrew Ozga

Determining the origins of those buried within undocumented cemeteries is of incredible importance to historical archaeologists and, in many cases, the nearby communities. In the case of Avondale Burial Place, a cemetery in Bibb County, Georgia, in use from 1820 to 1950, all written documentation of those interred within it has been lost. Osteological and archaeological evidence alone could not describe, with confidence, the ancestral origins of the 101 individuals buried there. In the present study, we used ancient DNA extraction methods in well-preserved skeletal fragments from 20 individuals buried in Avondale Burial Place to investigate the origins of the …

Differential Preservation Of Endogenous Human And Microbial Dna In Dental Calculus And Dentin, Allison E. Mann, Susanna Sabin, Kirsten Ziesemer, Ashild J. Vagene, Hannes Schroeder, Andrew T. Ozga, Krithivasan Sankaranarayanan, Courtney A. Hofman, James A. Fellows Yates, Domingo C. Salazar-Garcia, Bruno Frohlich, Mark Aldenderfer, Menno Hoogland, Christopher Read, George R. Milner, Anne C. Stone, Cecil M. Lewis Jr., Johannes Krause, Corinne Hofman, Kirsten I. Bos, Christina Warinner

Andrew Ozga

Dental calculus (calcified dental plaque) is prevalent in archaeological skeletal collections and is a rich source of oral microbiome and host-derived ancient biomolecules. Recently, it has been proposed that dental calculus may provide a more robust environment for DNA preservation than other skeletal remains, but this has not been systematically tested. In this study, shotgun-sequenced data from paired dental calculus and dentin samples from 48 globally distributed individuals are compared using a metagenomic approach. Overall, we find DNA from dental calculus is consistently more abundant and less contaminated than DNA from dentin. The majority of DNA in dental calculus is …

Spotted Cotton Oligonucleotide Microarrays For Gene Expression Analysis, Joshua A. Udall, Lex E. Flagel, Foo Cheung, Andrew W. Woodard, Ran Hovav, Ryan Adam Rapp, Jordan M. Swanson, Jinsuk J. Lee, Alan R. Gingle, Dan Nettleton, Christopher D. Town, Z. Jeffrey Chen, Jonathan F. Wendel

Dan Nettleton

Microarrays offer a powerful tool for diverse applications plant biology and crop improvement. Recently, two comprehensive assemblies of cotton ESTs were constructed based on three Gossypium species. Using these assemblies as templates, we describe the design and creation and of a publicly available oligonucleotide array for cotton, useful for all four of the cultivated species. Synthetic oligonucleotide probes were generated from exemplar sequences of a global assembly of 211,397 cotton ESTs derived from >50 different cDNA libraries representing many different tissue types and tissue treatments. A total of 22,787 oligonucleotide probes are included on the arrays, optimized to target the …

Mu Transposon Insertion Sites And Meiotic Recombination Events Co-Localize With Epigenetic Marks For Open Chromatin Across The Maize Genome, Sanzhen Liu, Cheng-Ting Yeh, Tieming Ji, Kai Ying, Haiyan Wu, Ho Man Tang, Yan Fu, Daniel S. Nettleton, Patrick S. Schnable

Dan Nettleton

The Mu transposon system of maize is highly active, with each of the ∼50–100 copies transposing on average once each generation. The approximately one dozen distinct Mutransposons contain highly similar ∼215 bp terminal inverted repeats (TIRs) and generate 9-bp target site duplications (TSDs) upon insertion. Using a novel genome walking strategy that uses these conserved TIRs as primer binding sites, Mu insertion sites were amplified from Mu stocks and sequenced via 454 technology. 94% of ∼965,000 reads carried Mu TIRs, demonstrating the specificity of this strategy. Among these TIRs, 21 novel Mu TIRs were discovered, revealing additional complexity of …

Identification Of Differentially Expressed Gene Categories In Microarray Studies Using Nonparametric Multivariate Analysis, Dan Nettleton, Justin Recknor, James M. Reecy

Dan Nettleton

The field of microarray data analysis is shifting emphasis from methods for identifying differentially expressed genes to methods for identifying differentially expressed gene categories. The latter approaches utilize a priori information about genes to group genes into categories and enhance the interpretation of experiments aimed at identifying expression differences across treatments. While almost all of the existing approaches for identifying differentially expressed gene categories are practically useful, they suffer from a variety of drawbacks. Perhaps most notably, many popular tools are based exclusively on gene-specific statistics that cannot detect many types of multivariate expression change. We have developed a nonparametric …

Differentially Expressed Genes In Blood From Young Pigs Between Two Swine Lines Divergently Selected For Feed Efficiency: Potential Biomarkers For Improving Feed Efficiency, Haibo Liu, Yet T. Nguyen, Daniel S. Nettleton, Jack C. M. Dekkers, Christopher K. Tuggle

Dan Nettleton

The goal of this study was to find potential gene expression biomarkers in blood of piglets that can be used to predict pigs’ future feed efficiency. Using RNA-seq technology, we found 453 genes were differentially expressed (false discovery rate (FDR) ≤ 0.05) in the blood of two Yorkshire lines of pigs divergently selected for feed efficiency (FE) based on residual feed intake (RFI). Genes involved in several biosynthetic processes were overrepresented among genes more highly expressed in the low RFI line compared to the high RFI line. Weighted gene co-expression network analysis (WGCNA) also revealed genes involved in some of …

Promoter Library Designed For Fine-Tuned Gene Expression In Pichia Pastoris, Franz S. Hartner, Claudia Ruth, David Langenegger, Sabrina N. Johnson, Petr Hyka, Geoff P. Lin-Cereghino, Joan Lin-Cereghino, Karin Kovar, James Cregg, Anton Glieder

Joan Lin-Cereghino

Although frequently used as protein production host, there is only a limited set of promoters available to drive the expression of recombinant proteins in Pichia pastoris. Fine-tuning of gene expression is often needed to maximize product yield and quality. However, for efficient knowledge-based engineering, a better understanding of promoter function is indispensable. Consequently, we created a promoter library by deletion and duplication of putative transcription factor-binding sites within the AOX1 promoter (P_AOX1) sequence. This first library initially spanned an activity range between ∼6% and >160% of the wild-type promoter activity. After characterization of the promoter library employing …

Blood Mercury Levels Of Zebra Finches Are Heritable: Implications For The Evolution Of Mercury Resistance, Kenton A. Buck, Claire W. Varian-Ramos, Daniel A. Cristol, John P. Swaddle

Daniel Cristol

Mercury is a ubiquitous metal contaminant that negatively impacts reproduction of wildlife and has many other sub-lethal effects. Songbirds are sensitive bioindicators of mercury toxicity and may suffer population declines as a result of mercury pollution. Current predictions of mercury accumulation and biomagnification often overlook possible genetic variation in mercury uptake and elimination within species and the potential for evolution in affected populations. We conducted a study of dietary mercury exposure in a model songbird species, maintaining a breeding population of zebra finches (Taeniopygia guttata) on standardized diets ranging from 0.0–2.4 μg/g methylmercury. We applied a quantitative genetics …

Saccharomyces Genome Database & Uniprot Bioinformatics Analysis, Ray A. Enke

Ray Enke Ph.D.