Specific Binding of Transposase to Terminal Inverted Repeats Of

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The EMBO Journal Vol. I No. 12 pp. 1539-1544, 1982 Long terminal repeat-like elements flank a human immunoglobulin epsilon pseudogene that lacks introns Shintaro Ueda', Sumiko Nakai, Yasuyoshi Nishida, lack the entire IVS have been found in the gene families of the Hiroshi Hisajima, and Tasuku Honjo* mouse a-globin (Nishioka et al., 1980; Vanin et al., 1980), the lambda chain (Hollis et al., 1982), Department of Genetics, Osaka University Medical School, Osaka 530, human immunoglobulin Japan and the human ,B-tubulin (Wilde et al., 1982a, 1982b). The mouse a-globin processed gene is flanked by long terminal Communicated by K.Rajewsky Received on 30 September 1982 repeats (LTRs) of retrovirus-like intracisternal A particles on both sides, although their orientation is opposite to each There are at least three immunoglobulin epsilon genes (C,1, other (Lueders et al., 1982). The human processed genes CE2, and CE) in the human genome. The nucleotide sequences described above have poly(A)-like tails -20 bases 3' to the of the expressed epsilon gene (CE,) and one (CE) of the two putative poly(A) addition signal and are flanked by direct epsilon pseudogenes were compared. The results show that repeats of several bases on both sides (Hollis et al., 1982; the CE3 gene lacks the three intervening sequences entirely and Wilde et al., 1982a, 1982b). Such direct repeats, which were has a 31-base A-rich sequence 16 bases 3' to the putative also found in human small nuclear RNA pseudogenes poly(A) addition signal, indicating that the CE3 gene is a pro- (Arsdell et al., 1981), might have been formed by repair of cessed gene.
A Sleeping Beauty Mutagenesis Screen Reveals a Tumor Suppressor Role for Ncoa2/Src-2 in Liver Cancer

A Sleeping Beauty mutagenesis screen reveals a tumor suppressor role for Ncoa2/Src-2 in liver cancer Kathryn A. O’Donnella,b,1,2, Vincent W. Kengc,d,e, Brian Yorkf, Erin L. Reinekef, Daekwan Seog, Danhua Fanc,h, Kevin A. T. Silversteinc,h, Christina T. Schruma,b, Wei Rose Xiea,b,3, Loris Mularonii,j, Sarah J. Wheelani,j, Michael S. Torbensonk, Bert W. O’Malleyf, David A. Largaespadac,d,e, and Jef D. Boekea,b,i,2 Departments of aMolecular Biology and Genetics, iOncology, jDivision of Biostatistics and Bioinformatics, and kPathology, and bThe High Throughput Biology Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; cMasonic Cancer Center, dDepartment of Genetics, Cell Biology, and Development, eCenter for Genome Engineering, and hBiostatistics and Bioinformatics Core, University of Minnesota, Minneapolis, MN 55455; fDepartment of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030; and gLaboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 AUTHOR SUMMARY Emerging data from cancer ge- functions are altered. This ap- fi nome-sequencing studies have Sleeping Beauty (SB) transposase proach identi ed at least 16 demonstrated that human genes/loci that contribute to liver tumors exhibit tremendous Transposon array with gene trap (GT) tumor development. complexity and heterogeneity in We next validated that the the number and nature of iden- genes identified in the SB screen tified mutations (1). Based on contribute to tumor initiation these findings, there is an in- SB mobilization and/or progression using in vitro creasing need for in vivo vali- and in vivo cancer model sys- dation of genes whose altered tems.
RNA As a Source of Transposase for Sleeping Beauty-Mediated Gene Insertion and Expression in Somatic Cells and Tissues

doi:10.1016/j.ymthe.2005.10.014 SHORT COMMUNICATION RNA as a Source of Transposase for Sleeping Beauty-Mediated Gene Insertion and Expression in Somatic Cells and Tissues Andrew Wilber,1 Joel L. Frandsen,1 Jennifer L. Geurts,1 David A. Largaespada,1 Perry B. Hackett,1,2 and R. Scott McIvor1,* 1The Arnold and Mabel Beckman Center for Transposon Research, Gene Therapy Program, Institute of Human Genetics, and Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA 2Discovery Genomics, Inc., Minneapolis, MN 55455, USA *To whom correspondence and reprint requests should be addressed at The Department of Genetics, Cell Biology, and Development, 6-160 Jackson Hall, 321 Church Street SE, University of Minnesota, Minneapolis, MN 55455, USA. Fax: +1 612 625 9810. E-mail: [email protected]. Available online 20 December 2005 Sleeping Beauty (SB) is a DNA transposon capable of mediating gene insertion and long-term expression in vertebrate cells when co-delivered with a source of transposase. In all previous reports of SB-mediated gene insertion in somatic cells, the transposase component has been provided by expression of a co-delivered DNA molecule that has the potential for integration into the host cell genome. Integration and continued expression of a gene encoding SB transposase could be problematic if it led to transposon re-mobilization and re-integration. We addressed this potential problem by supplying the transposase-encoding molecule in the form of mRNA. We show that transposase-encoding mRNA can effectively mediate transposition in vitro in HT1080 cells and in vivo in mouse liver following co-delivery with a recoverable transposon or with a luciferase transposon.
Licensing RNA-Guided Genome Defense

TIBS-1023; No. of Pages 10 Review Recognizing the enemy within: licensing RNA-guided genome defense Phillip A. Dumesic and Hiten D. Madhani Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158, USA How do cells distinguish normal genes from transpo- RNAi-related RNA silencing pathways are deeply con- sons? Although much has been learned about RNAi- served genome defense mechanisms that act in organisms related RNA silencing pathways responsible for genome from protist to human to recognize and suppress transpo- defense, this fundamental question remains. The litera- sons [8]. In all of these pathways, 20–30 nucleotide small ture points to several classes of mechanisms. In some RNAs act in complex with Argonaute family proteins to cases, double-stranded RNA (dsRNA) structures pro- silence complementary transcripts. Depending on the duced by transposon inverted repeats or antisense inte- pathway and context, silencing proceeds by a variety of gration trigger endogenous small interfering RNA mechanisms, which include RNA degradation, translation- (siRNA) biogenesis. In other instances, DNA features al repression, and the establishment of repressive histone associated with transposons – such as their unusual modiﬁcations [9,10]. The pathways also differ in the means copy number, chromosomal arrangement, and/or chro- by which they generate small RNAs. In the canonical RNAi matin environment – license RNA silencing. Finally, re- pathway, RNase-III-type Dicer enzymes convert long cent studies have identiﬁed improper transcript dsRNA into small interfering RNA (siRNA). In contrast, processing events, such as stalled pre-mRNA splicing, PIWI-interacting RNA (piRNA) pathways do not require as signals for siRNA production.
Exploring the Interplay of Telomerase Reverse Transcriptase and Β-Catenin in Hepatocellular Carcinoma

cancers Review Exploring the Interplay of Telomerase Reverse Transcriptase and β-Catenin in Hepatocellular Carcinoma Srishti Kotiyal and Kimberley Jane Evason * Department of Oncological Sciences, Department of Pathology, and Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-801-587-4606 Simple Summary: Liver cancer is one of the deadliest human cancers. Two of the most common molecular aberrations in liver cancer are: (1) activating mutations in the gene encoding β-catenin (CTNNB1); and (2) promoter mutations in telomerase reverse transcriptase (TERT). Here, we review recent findings regarding the interplay between TERT and β-catenin in order to better understand their role in liver cancer. Abstract: Hepatocellular carcinoma (HCC) is one of the deadliest human cancers. Activating muta- tions in the telomerase reverse transcriptase (TERT) promoter (TERTp) and CTNNB1 gene encoding β-catenin are widespread in HCC (~50% and ~30%, respectively). TERTp mutations are predicted to increase TERT transcription and telomerase activity. This review focuses on exploring the role of TERT and β-catenin in HCC and the current findings regarding their interplay. TERT can have contradictory effects on tumorigenesis via both its canonical and non-canonical functions. As a critical regulator of proliferation and differentiation in progenitor and stem cells, activated β-catenin drives HCC; however, inhibiting endogenous β-catenin can also have pro-tumor effects. Clinical studies revealed a significant concordance between TERTp and CTNNB1 mutations in HCC. In Citation: Kotiyal, S.; Evason, K.J. stem cells, TERT acts as a co-factor in β-catenin transcriptional complexes driving the expression Exploring the Interplay of Telomerase of WNT/β-catenin target genes, and β-catenin can bind to the TERTp to drive its transcription.
A Survey of Transposable Element Classification Systems Â

Molecular Phylogenetics and Evolution 86 (2015) 90–109 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Review A survey of transposable element classification systems – A call for a fundamental update to meet the challenge of their diversity and complexity ⇑ ⇑ Benoît Piégu a, Solenne Bire a,b, Peter Arensburger a,c, , Yves Bigot a, a UMR INRA-CNRS 7247, PRC, Centre INRA de Nouzilly, 37380 Nouzilly, France b Institute of Biotechnology, University of Lausanne, Center for Biotechnology UNIL-EPFL, 1015 Lausanne, Switzerland c Biological Sciences Department, California State Polytechnic University, Pomona, CA 91768, United States article info abstract Article history: The increase of publicly available sequencing data has allowed for rapid progress in our understanding of Received 24 December 2014 genome composition. As new information becomes available we should constantly be updating and Revised 11 March 2015 reanalyzing existing and newly acquired data. In this report we focus on transposable elements (TEs) Accepted 12 March 2015 which make up a significant portion of nearly all sequenced genomes. Our ability to accurately identify Available online 20 March 2015 and classify these sequences is critical to understanding their impact on host genomes. At the same time, as we demonstrate in this report, problems with existing classification schemes have led to significant Keywords: misunderstandings of the evolution of both TE sequences and their host genomes. In a pioneering pub- Transposon lication Finnegan (1989) proposed classifying all TE sequences into two classes based on transposition Mobility Host range mechanisms and structural features: the retrotransposons (class I) and the DNA transposons (class II).
Characterization of the First Cultured Representative of Verrucomicrobia Subdivision 5 Indicates the Proposal of a Novel Phylum

The ISME Journal (2016) 10, 2801–2816 OPEN © 2016 International Society for Microbial Ecology All rights reserved 1751-7362/16 www.nature.com/ismej ORIGINAL ARTICLE Characterization of the first cultured representative of Verrucomicrobia subdivision 5 indicates the proposal of a novel phylum Stefan Spring1, Boyke Bunk2, Cathrin Spröer3, Peter Schumann3, Manfred Rohde4, Brian J Tindall1 and Hans-Peter Klenk1,5 1Department Microorganisms, Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany; 2Department Microbial Ecology and Diversity Research, Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany; 3Department Central Services, Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany and 4Central Facility for Microscopy, Helmholtz-Centre of Infection Research, Braunschweig, Germany The recently isolated strain L21-Fru-ABT represents moderately halophilic, obligately anaerobic and saccharolytic bacteria that thrive in the suboxic transition zones of hypersaline microbial mats. Phylogenetic analyses based on 16S rRNA genes, RpoB proteins and gene content indicated that strain L21-Fru-ABT represents a novel species and genus affiliated with a distinct phylum-level lineage originally designated Verrucomicrobia subdivision 5. A survey of environmental 16S rRNA gene sequences revealed that members of this newly recognized phylum are wide-spread and ecologically important in various anoxic environments ranging from hypersaline sediments to wastewater and the intestine of animals. Characteristic phenotypic traits of the novel strain included the formation of extracellular polymeric substances, a Gram-negative cell wall containing peptidoglycan and the absence of odd-numbered cellular fatty acids. Unusual metabolic features deduced from analysis of the genome sequence were the production of sucrose as osmoprotectant, an atypical glycolytic pathway lacking pyruvate kinase and the synthesis of isoprenoids via mevalonate.
Molecular Architecture of a Eukaryotic DNA Transposase

ARTICLES Molecular architecture of a eukaryotic DNA transposase Alison B Hickman1, Zhanita N Perez1, Liqin Zhou2, Primrose Musingarimi1,4, Rodolfo Ghirlando1, Jenny E Hinshaw3, Nancy L Craig2 & Fred Dyda1 Mobile elements and their inactive remnants account for large proportions of most eukaryotic genomes, where they have had central roles in genome evolution. Over 50 years ago, McClintock reported a form of stress-induced genome instability in maize in which discrete DNA segments move between chromosomal locations. Our current mechanistic understanding of enzymes catalyzing transposition is largely limited to prokaryotic transposases. The Hermes transposon from the houseﬂy is part of the eukaryotic hAT superfamily that includes hobo from Drosophila, McClintock’s maize Activator and Tam3 from snapdragon. We http://www.nature.com/nsmb report here the three-dimensional structure of a functionally active form of the transposase from Hermes at 2.1-A˚ resolution. The Hermes protein has some structural features of prokaryotic transposases, including a domain with a retroviral integrase fold. However, this domain is disrupted by the insertion of an additional domain. Finally, transposition is observed only when Hermes assembles into a hexamer. Genome sequencing efforts have yielded many unanticipated results, particularly interested in the biochemistry and mechanism of the among them the realization that large proportions of many eukaryotic Hermes hAT transposase from the houseﬂy Musca domestica7. Hermes genomes originated as mobile elements. These mobile elements are is a 2,749-bp element that contains 17-bp terminal inverted repeats discrete pieces of DNA that can either move from one place to another and encodes a 70-kDa transposase7.
The Molecular Coupling Between Substrate Recognition and ATP Turnover in A

bioRxiv preprint doi: https://doi.org/10.1101/2020.10.21.345918; this version posted October 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. The molecular coupling between substrate recognition and ATP turnover in a AAA+ hexameric helicase loader Neha Puri1,2, Amy J. Fernandez1, Valerie L. O’Shea Murray1,3, Sarah McMillan4, James L. Keck4, James M. Berger1,* 1Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, MD 21205 2Bristol Myers Squibb, 38 Jackson Road, Devens, MA 01434 3Saul Ewing Arnstein & Lehr, LLP, Centre Square West, 1500 Market Street, 38th Floor, Philadelphia, PA 19102 4Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, WI, 53706 *Corresponding author Email: [email protected] Keywords: DNA replication, AAA+ ATPase, Helicase, Meier-Gorlin Syndrome 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.21.345918; this version posted October 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. ABSTRACT In many bacteria and in eukaryotes, replication fork establishment requires the controlled loading of hexameric, ring-shaped helicases around DNA by AAA+ ATPases. How loading factors use ATP to control helicase deposition is poorly understood.
Structure of Moloney Murine Leukemia Viral DNA: Nucleotide Sequence Of

Proc. Nati. Acad. Sci. USA Vol. 77, No. 6, pp. 3307-3311, June 1980 Biochemistry Structure of Moloney murine leukemia viral DNA: Nucleotide sequence of the 5' long terminal repeat and adjacent cellular sequences (strong-stop DNA/recombinant DNA/DNA sequence determination/inverted repeat/transposons) CHARLES VAN BEVEREN*, JANICE G. GODDARD*, ANTON BERNSt, AND INDER M. VERMA* *Tumor Virology Laboratory, The Salk Institute, P.O. Box 85800, San Diego, California 92138; and tLaboratory of Biochemistry, University of Nijmegen, Nijmegen, The Netherlands Communicated by E. Peter Geiduschek, March 31, 1980 ABSTRACT Some unintegrated and all integrated forms tRNAPrO viral DNA contain terminal of murine leukemia long repeats 5' gag pol end @ ib3 (LT s). The entire nucleotide sequence of the LTR and adjacent L-,, i Genomic terminal cellular sequences at the 5' end of a cloned integrated proviral ViralRredundancy DNA obtained from BALB/Mo mouse has been determined. Viral RNA (45-66cbp) It was compared to the nucleotide sequence of the LTR at the Reverse transcription 3' end. The results indicate: (i) a direct 517-nucleotide repeat at yT the 5' and 3' termini; (ii) 145 nucleotides out of 517 nucleotides represent sequences between the 5'-CAP nucleotide and 3' end ent 3' 5 {Cellular DNA of the primer tRNA (strong-stop DNA); (ifi) an li-nucleotide ,-Strong-stop DNA , -Strong-stop DNA inverted repeat is present at the ends of the 5'-LTR and a total (145 bp) of 17 out of21 nucleotides at the termini are inverted repeats; 5'-LTR (145 bp) 3'-LTR-LTR (iv) sequences CAATAAAAG (at positions -24 to -31) and Integrated viral DNA CAATAAAC (at positions +46 to +53) resembling the hypo- thetical DNA-dependent RNA polymerase II promoter site can FIG.
Measuring Error Rates of High-Fidelity DNA Polymerases by NGS Determining Fidelity of Phusion Plus and Phusion Hot-Start II DNA Polymerases

TECHNICAL NOTE Phusion Plus DNA Polymerase Measuring error rates of high-fidelity DNA polymerases by NGS Determining fidelity of Phusion Plus and Phusion Hot-Start II DNA Polymerases Introduction High-fidelity DNA polymerases are widely used in the same assay, to account for differences in assay applications such as cloning and site-directed mutagenesis methodology, template DNA, and reporting units. to help ensure replication of template sequences without introducing errors. Preserving accuracy of DNA sequences Here, we report a molecular-barcoding method when amplifying the template by PCR is important for such for determining polymerase fidelity. This approach applications because one misincorporated nucleotide introduces unique molecular identifiers (UMIs) during may change a codon, resulting in an incorrect amino acid MuA fragmentation in NGS library preparation. and possible dysfunction of the encoded protein due to Using this method, we determined the error rates of misfolding. Researchers can help minimize troubleshooting Thermo Scientific™ Phusion™ Plus DNA Polymerase, in downstream applications by using a higher-fidelity DNA Thermo Scientific™ Phusion™ Hot-Start II DNA Polymerase, polymerase, and can benefit from comparing reported and Taq DNA polymerase. values of polymerase fidelity when selecting enzymes. Several methods are available to measure polymerase Phusion high-fidelity DNA polymerases fidelity, such as blue-white colony screening assays; The family of Phusion high-fidelity DNA polymerases denaturing gradient gel electrophoresis
Repeated Horizontal Transfers of Four DNA Transposons in Invertebrates and Bats Zhou Tang1†, Hua-Hao Zhang2†, Ke Huang3, Xiao-Gu Zhang2, Min-Jin Han1 and Ze Zhang1*

Tang et al. Mobile DNA (2015) 6:3 DOI 10.1186/s13100-014-0033-1 RESEARCH Open Access Repeated horizontal transfers of four DNA transposons in invertebrates and bats Zhou Tang1†, Hua-Hao Zhang2†, Ke Huang3, Xiao-Gu Zhang2, Min-Jin Han1 and Ze Zhang1* Abstract Background: Horizontal transfer (HT) of transposable elements (TEs) into a new genome is considered as an important force to drive genome variation and biological innovation. However, most of the HT of DNA transposons previously described occurred between closely related species or insects. Results: In this study, we carried out a detailed analysis of four DNA transposons, which were found in the first sequenced twisted-wing parasite, Mengenilla moldrzyki. Through the homology-based strategy, these transposons were also identified in other insects, freshwater planarian, hydrozoans, and bats. The phylogenetic distribution of these transposons was discontinuous, and they showed extremely high sequence identities (>87%) over their entire length in spite of their hosts diverging more than 300 million years ago (Mya). Additionally, phylogenies and comparisons of transposons versus orthologous gene identities demonstrated that these transposons have transferred into their hosts by independent HTs. Conclusions: Here, we provided the first documented example of HT of CACTA transposons, which have been so far extensively studied in plants. Our results demonstrated that bats had continuously acquired new DNA elements via HT. This implies that predation on a large quantity of insects might increase bat exposure to HT. In addition, parasite-host interaction might facilitate exchanging of their genetic materials. Keywords: Horizontal transfer, CACTA transposons, Mammals, Recent activity Background or isolated species.