Genetics and Genomics Commons^™

Rnase Κ Promotes Robust Pirna Production By Generating 2',3'-Cyclic Phosphate-Containing Precursors, Megumi Shigematsu, Takuya Kawamura, Keisuke Morichika, Natsuko Izumi, Takashi Kiuchi, Shozo Honda, Venetia Pliatsika, Ryuma Matsubara, Isidore Rigoutsos, Susumu Katsuma, Yukihide Tomari, Yohei Kirino

Computational Medicine Center Faculty Papers

In animal germlines, PIWI proteins and the associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons. Here we report the extensive sequence and quantitative correlations between 2′,3′-cyclic phosphate-containing RNAs (cP-RNAs), identified using cP-RNA-seq, and piRNAs in the Bombyx germ cell line and mouse testes. The cP-RNAs containing 5′-phosphate (P-cP-RNAs) identified by P-cP-RNA-seq harbor highly consistent 5′-end positions as the piRNAs and are loaded onto PIWI protein, suggesting their direct utilization as piRNA precursors. We identified Bombyx RNase Kappa (BmRNase κ) as a mitochondria-associated endoribonuclease which produces cP-RNAs during piRNA biogenesis. BmRNase κ-depletion elevated transposon levels and disrupted a piRNA-mediated …

N-Terminal Domain Of Human Uracil Dna Glycosylase (Hung2) Promotes Targeting To Uracil Sites Adjacent To Ssdna-Dsdna Junctions, Brian P Weiser, Gaddiel Rodriguez, Philip A Cole, James T Stivers

Rowan-Virtua School of Osteopathic Medicine Faculty Scholarship

The N-terminal domain (NTD) of nuclear human uracil DNA glycosylase (hUNG2) assists in targeting hUNG2 to replication forks through specific interactions with replication protein A (RPA). Here, we explored hUNG2 activity in the presence and absence of RPA using substrates with ssDNA-dsDNA junctions that mimic structural features of the replication fork and transcriptional R-loops. We find that when RPA is tightly bound to the ssDNA overhang of junction DNA substrates, base excision by hUNG2 is strongly biased toward uracils located 21 bp or less from the ssDNA-dsDNA junction. In the absence of RPA, hUNG2 still showed an 8-fold excision bias …

Organelle_Pba, A Pipeline For Assembling Chloroplast And Mitochondrial Genomes From Pacbio Dna Sequencing Data, Aboozar Soorni, David Haak, David Zaitlin, Aureliano Bombarely

Kentucky Tobacco Research and Development Center Faculty Publications

Background: The development of long-read sequencing technologies, such as single-molecule real-time (SMRT) sequencing by PacBio, has produced a revolution in the sequencing of small genomes. Sequencing organelle genomes using PacBio long-read data is a cost effective, straightforward approach. Nevertheless, the availability of simple-to-use software to perform the assembly from raw reads is limited at present.

Results: We present Organelle-PBA, a Perl program designed specifically for the assembly of chloroplast and mitochondrial genomes. For chloroplast genomes, the program selects the chloroplast reads from a whole genome sequencing pool, maps the reads to a reference sequence from a closely related species, and …

Duplication And Remolding Of Trna Genes In The Mitochondrial Genome Of Reduvius Tenebrosus (Hemiptera: Reduviidae), Pei Jiang, Hu Li, Fan Song, Yao Cai, Jianyun Wang, Jinpeng Liu, Wanzhi Cai

Markey Cancer Center Faculty Publications

Most assassin bugs are predators that act as important natural enemies of insect pests. Mitochondrial (mt) genomes of these insects are double-strand circular DNAs that encode 37 genes. In the present study, we explore the duplication and rearrangement of tRNA genes in the mt genome of Reduvius tenebrosus, the first mt genome from the subfamily Reduviinae. The gene order rearranges from CR (control region)-trnI-trnQ-trnM-ND2 to CR-trnQ-trnI2-trnI1-trnM-ND2. We identified 23 tRNA genes, including 22 tRNAs commonly found in insects and an additional trnI (trnI2), which has high sequence similarity to trnM. We found several …

Identification Of A Novel Gene On 10q22.1 Causing Autosomal Dominant Retinitis Pigmentosa (Adrp)., Stephen P Daiger, Lori S Sullivan, Sara J Bowne, Daniel C Koboldt, Susan H Blanton, Dianna K Wheaton, Cheryl E Avery, Elizabeth D Cadena, Robert K Koenekoop, Robert S Fulton, Richard K Wilson, George M Weinstock, Richard A Lewis, David G Birch

Faculty Publications

Whole-genome linkage mapping identified a region on chromosome 10q21.3-q22.1 with a maximum LOD score of 3.0 at 0 % recombination in a six-generation family with autosomal dominant retinitis pigmentosa (adRP). All known adRP genes and X-linked RP genes were excluded in the family by a combination of methods. Whole-exome next-generation sequencing revealed a missense mutation in hexokinase 1, HK1 c.2539G > A, p.Glu847Lys, tracking with disease in all affected family members. One severely-affected male is homozygous for this region by linkage analysis and has two copies of the mutation. No other potential mutations were detected in the linkage region nor were …

Quantitative Field Testing Heterodera Glycines From Metagenomic Dna Samples Isolated Directly From Soil Under Agronomic Production., Yan Li, Gary W. Lawrence, Shien Lu, Clarissa Balbalian, Vincent P. Klink

College of Agriculture & Life Sciences Publications and Scholarship

A quantitative PCR procedure targeting the Heterodera glycines ortholog of the Caenorhabditis elegans uncoordinated-78 gene was developed. The procedure estimated the quantity of H. glycines from metagenomic DNA samples isolated directly from field soil under agronomic production. The estimation of H. glycines quantity was determined in soil samples having other soil dwelling plant parasitic nematodes including Hoplolaimus, predatory nematodes including Mononchus, free-living nematodes and biomass. The methodology provides a framework for molecular diagnostics of nematodes from metagenomic DNA isolated directly from field soil.

Characteristics And Prediction Of Rna Structure., Hengwu Li, Daming Zhu, Caiming Zhang, Huijian Han, Keith A Crandall

Computational Biology Institute

RNA secondary structures with pseudoknots are often predicted by minimizing free energy, which is NP-hard. Most RNAs fold during transcription from DNA into RNA through a hierarchical pathway wherein secondary structures form prior to tertiary structures. Real RNA secondary structures often have local instead of global optimization because of kinetic reasons. The performance of RNA structure prediction may be improved by considering dynamic and hierarchical folding mechanisms. This study is a novel report on RNA folding that accords with the golden mean characteristic based on the statistical analysis of the real RNA secondary structures of all 480 sequences from RNA …

Phage Cluster Relationships Identified Through Single Gene Analysis., Kyle C Smith, Eduardo Castro-Nallar, Joshua Nb Fisher, Donald P Breakwell, Julianne H Grose, Sandra H Burnett

Computational Biology Institute

BACKGROUND: Phylogenetic comparison of bacteriophages requires whole genome approaches such as dotplot analysis, genome pairwise maps, and gene content analysis. Currently mycobacteriophages, a highly studied phage group, are categorized into related clusters based on the comparative analysis of whole genome sequences. With the recent explosion of phage isolation, a simple method for phage cluster prediction would facilitate analysis of crude or complex samples without whole genome isolation and sequencing. The hypothesis of this study was that mycobacteriophage-cluster prediction is possible using comparison of a single, ubiquitous, semi-conserved gene. Tape Measure Protein (TMP) was selected to test the hypothesis because it …

Archaeal Nucleosome Positioning In Vivo And In Vitro Is Directed By Primary Sequence Motifs, Narasimharao Nalabothula, Liqun Xi, Sucharita Bhattacharyya, Jonathan Widom, Ji-Ping Wang, John N. Reeve, Thomas J. Santangelo, Yvonne N. Fondufe-Mittendorf

Molecular and Cellular Biochemistry Faculty Publications

Background: Histone wrapping of DNA into nucleosomes almost certainly evolved in the Archaea, and predates Eukaryotes. In Eukaryotes, nucleosome positioning plays a central role in regulating gene expression and is directed by primary sequence motifs that together form a nucleosome positioning code. The experiments reported were undertaken to determine if archaeal histone assembly conforms to the nucleosome positioning code.

Results: Eukaryotic nucleosome positioning is favored and directed by phased helical repeats of AA/TT/AT/TA and CC/GG/CG/GC dinucleotides, and disfavored by longer AT-rich oligonucleotides. Deep sequencing of genomic DNA protected from micrococcal nuclease digestion by assembly into archaeal nucleosomes has established that …

Borrelia Burgdorferi Cp32 Bpab Modulates Expression Of The Prophage Nucp Nuclease And Ssbp Single-Stranded Dna-Binding Protein, Alicia M. Chenail, Brandon L. Jutras, Claire A. Adams, Logan H. Burns, Amy Bowman, Ashutosh Verma, Brian Stevenson

Microbiology, Immunology, and Molecular Genetics Faculty Publications

The Borrelia burgdorferi BpaB proteins of the spirochete's ubiquitous cp32 prophages are DNA-binding proteins, required both for maintenance of the bacteriophage episomes and for transcriptional regulation of the cp32 erp operons. Through use of DNase I footprinting, we demonstrate that BpaB binds the erp operator initially at the sequence 5′-TTATA-3′. Electrophoretic mobility shift assays indicated that BpaB also binds with high affinity to sites located in the 5′ noncoding regions of two additional cp32 genes. Characterization of the proteins encoded by those genes indicated that they are a single-stranded DNA-binding protein and a nuclease, which we named SsbP and NucP, …

Adventures In The Enormous: A 1.8 Million Clone Bac Library For The 21.7 Gb Genome Of Loblolly Pine., Zenaida V. Magbanua, Seval Ozkan, Benjamin D. Bartlett, Philippe Chouvarine, Christopher A. Saski, Aaron Liston, Richard C. Cronn, C. Dana Nelson, Daniel G. Peterson

College of Agriculture & Life Sciences Publications and Scholarship

Loblolly pine (LP; Pinus taeda L.) is the most economically important tree in the U.S. and a cornerstone species in southeastern forests. However, genomics research on LP and other conifers has lagged behind studies on flowering plants due, in part, to the large size of conifer genomes. As a means to accelerate conifer genome research, we constructed a BAC library for the LP genotype 7-56. The LP BAC library consists of 1,824,768 individually-archived clones making it the largest single BAC library constructed to date, has a mean insert size of 96 kb, and affords 7.6X coverage of the 21.7 Gb …

Help3 Directly Modulates The Expression Of Hsp70 Gene In Hela Cells Via Hat Activity, Fen Li, Jixian Ma, Yu Ma, Yanyan Hu, Shuhuan Tian, Richard E. White, Guichun Han

PCOM Scholarly Papers

Human Elongator complex, which plays a key role in transcript elongation in vitro assay, is incredibly similar in either components or function to its yeast counterpart. However, there are only a few studies focusing on its target gene characterization in vivo. We studied the effect of down-regulation of the human elongation protein 3 (hELP3) on the expression of HSP70 through antisense strategy. Transfecting antisense plasmid p1107 into HeLa cells highly suppressed hELP3 expression, and substantially reduced expression of HSP70 mRNA and protein. Furthermore, chromatin immunoprecipitation assay (ChIP Assay) revealed that hElp3 participates in the transcription elongation of HSPA1A in HeLa …

Sialic Acid Transport And Catabolism Are Cooperatively Regulated By Siar And Crp In Nontypeable Haemophilus Influenzae, Jason W. Johnston, Haider Shamsulddin, Anne-Frances Miller, Michael A. Apicella

Microbiology, Immunology, and Molecular Genetics Faculty Publications

BACKGROUND: The transport and catabolism of sialic acid, a critical virulence factor for nontypeable Haemophilus influenzae, is regulated by two transcription factors, SiaR and CRP.

RESULTS: Using a mutagenesis approach, glucosamine-6-phosphate (GlcN-6P) was identified as a co-activator for SiaR. Evidence for the cooperative regulation of both the sialic acid catabolic and transport operons suggested that cooperativity between SiaR and CRP is required for regulation. cAMP was unable to influence the expression of the catabolic operon in the absence of SiaR but was able to induce catabolic operon expression when both SiaR and GlcN-6P were present. Alteration of helical phasing supported …

Bpab, A Novel Protein Encoded By The Lyme Disease Spirochete's Cp32 Prophages, Binds To Erp Operator 2 Dna, Logan H. Burns, Claire A. Adams, Sean P. Riley, Brandon L. Jutras, Amy Bowman, Alicia M. Chenail, Anne E. Cooley, Laura A. Haselhorst, Alisha M. Moore, Kelly Babb, Michael G. Fried, Brian Stevenson

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Borrelia burgdorferi produces Erp outer surface proteins throughout mammalian infection, but represses their synthesis during colonization of vector ticks. A DNA region 5′ of the start of erp transcription, Operator 2, was previously shown to be essential for regulation of expression. We now report identification and characterization of a novel erp Operator 2-binding protein, which we named BpaB. erp operons are located on episomal cp32 prophages, and a single bacterium may contain as many as 10 different cp32s. Each cp32 family member encodes a unique BpaB protein, yet the three tested cp32-encoded BpaB alleles all bound to the same DNA …

Characterization Of Conserved Properties Of Hemagglutinin Of H5n1 And Human Influenza Viruses: Possible Consequences For Therapy And Infection Control, Veljko Veljkovic, Nevena Veljkovic, Claude P. Muller, Sybille Müller, Sanja Glisic, Vladimir Perovic, Heinz Köhler

Microbiology, Immunology, and Molecular Genetics Faculty Publications

BACKGROUND: Epidemics caused by highly pathogenic avian influenza virus (HPAIV) are a continuing threat to human health and to the world's economy. The development of approaches, which help to understand the significance of structural changes resulting from the alarming mutational propensity for human-to-human transmission of HPAIV, is of particularly interest. Here we compare informational and structural properties of the hemagglutinin (HA) of H5N1 virus and human influenza virus subtypes, which are important for the receptor/virus interaction.

RESULTS: Presented results revealed that HA proteins encode highly conserved information that differ between influenza virus subtypes H5N1, H1N1, H3N2, H7N7 and defined an …

Borrelia Burgdorferi Ebfc Defines A Newly-Identified, Widespread Family Of Bacterial Dna-Binding Proteins, Sean P. Riley, Tomasz Bykowski, Anne E. Cooley, Logan H. Burns, Kelly Babb, Catherine A. Brissette, Amy Bowman, Matthew L. Rotondi, M. Clarke Miller, Edward Demoll, Kap Lim, Michael G. Fried, Brian Stevenson

Microbiology, Immunology, and Molecular Genetics Faculty Publications

The Lyme disease spirochete, Borrelia burgdorferi, encodes a novel type of DNA-binding protein named EbfC. Orthologs of EbfC are encoded by a wide range of bacterial species, so characterization of the borrelial protein has implications that span the eubacterial kingdom. The present work defines the DNA sequence required for high-affinity binding by EbfC to be the 4 bp broken palindrome GTnAC, where ‘n’ can be any nucleotide. Two high-affinity EbfC-binding sites are located immediately 5′ of B. burgdorferi erp transcriptional promoters, and binding of EbfC was found to alter the conformation of erp promoter DNA. Consensus EbfC-binding …

Genic Regions Of A Large Salamander Genome Contain Long Introns And Novel Genes, Jeramiah J. Smith, Srikrishna Putta, Wei Zhu, Gerald M. Pao, Inder M. Verma, Tony Hunter, Susan V. Bryant, David M. Gardiner, Timothy T. Harkins, S. Randal Voss

Biology Faculty Publications

BACKGROUND: The basis of genome size variation remains an outstanding question because DNA sequence data are lacking for organisms with large genomes. Sixteen BAC clones from the Mexican axolotl (Ambystoma mexicanum: c-value = 32 x 10(9) bp) were isolated and sequenced to characterize the structure of genic regions.

RESULTS: Annotation of genes within BACs showed that axolotl introns are on average 10x longer than orthologous vertebrate introns and they are predicted to contain more functional elements, including miRNAs and snoRNAs. Loci were discovered within BACs for two novel EST transcripts that are differentially expressed during spinal cord regeneration and skin …

Leptospira Interrogans Endostatin-Like Outer Membrane Proteins Bind Host Fibronectin, Laminin And Regulators Of Complement, Brian Stevenson, Henry A. Choy, Marija Pinne, Matthew L. Rotondi, M. Clarke Miller, Edward Demoll, Peter Kraiczy, Anne E. Cooley, Trevor P. Creamer, Marc A. Suchard, Catherine A. Brissette, Ashutosh Verma, David A. Haake

Microbiology, Immunology, and Molecular Genetics Faculty Publications

The pathogenic spirochete Leptospira interrogans disseminates throughout its hosts via the bloodstream, then invades and colonizes a variety of host tissues. Infectious leptospires are resistant to killing by their hosts' alternative pathway of complement-mediated killing, and interact with various host extracellular matrix (ECM) components. The LenA outer surface protein (formerly called LfhA and Lsa24) was previously shown to bind the host ECM component laminin and the complement regulators factor H and factor H-related protein-1. We now demonstrate that infectious L. interrogans contain five additional paralogs of lenA, which we designated lenB, lenC, lenD, lenE and lenF …

Similar Levels Of X-Linked And Autosomal Nucleotide Variation In African And Non-African Populations Of Drosophila Melanogaster, Nadia D. Singh, J. Michael Macpherson, Jeffrey D. Jensen, Dmitri A. Petrov

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Background: Levels of molecular diversity in Drosophila have repeatedly been shown to be higher in ancestral, African populations than in derived, non-African populations. This pattern holds for both coding and noncoding regions for a variety of molecular markers including single nucleotide polymorphisms and microsatellites. Comparisons of X-linked and autosomal diversity have yielded results largely dependent on population of origin.

Results: In an attempt to further elucidate patterns of sequence diversity in Drosophila melanogaster, we studied nucleotide variation at putatively nonfunctional X-linked and autosomal loci in sub-Saharan African and North American strains of D. melanogaster. We combine our experimental …

Drosophila Unpaired Encodes A Secreted Protein That Activates The Jak Signaling Pathway, Douglas A. Harrison, Patricia E. Mccoon, Richard Binari, Michael Gilman, Norbert Perrimon

Biology Faculty Publications

In vertebrates, many cytokines and growth factors have been identified as activators of the JAK/STAT signaling pathway. In Drosophila, JAK and STAT molecules have been isolated, but no ligands or receptors capable of activating the pathway have been described. We have characterized the unpaired (upd) gene, which displays the same distinctive embryonic mutant defects as mutations in the Drosophila JAK (hopscotch) and STAT (stat92E) genes. Upd is a secreted protein, associated with the extracellular matrix, that activates the JAK pathway. We propose that Upd is a ligand that relies on JAK signaling to stimulate transcription of pair-rule genes in a …

Characterization Of Experimentally Induced, Nonaflatoxigenic Variant Strains Of Aspergillus Parasiticus., Shubha Kale Ireland, J. W. Cary, D. Bhatnagar, J. W. Bennett

Faculty and Staff Publications

Six previously isolated, nonaflatoxigenic variants of Aspergillus parasiticus, designated sec mutants, were characterized morphologically by electron microscopy, biochemically by biotransformation studies with an aflatoxin precursor, and genetically by Northern (RNA) hybridization analysis of aflatoxin biosynthetic gene transcripts. Scanning electron micrographs clearly demonstrated that compared with the parental sec+ forms, the variant sec forms had an abundance of vegetative mycelia, orders of magnitude reduced number of conidiophores and conidia, and abnormal metulae. Conidiospores were detected in sec cultures only at higher magnifications (x500), in contrast to the sec+ (wild-type) strain, in which abundant conidiospores (masking the vegetative mycelia) were observed even …