Cloning and Mutagenesis: Tinkering with the Order of Things
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Chapter 14: Functional Genomics Learning Objectives
Chapter 14: Functional Genomics Learning objectives Upon reading this chapter, you should be able to: ■ define functional genomics; ■ describe the key features of eight model organisms; ■ explain techniques of forward and reverse genetics; ■ discuss the relation between the central dogma and functional genomics; and ■ describe proteomics-based approaches to functional genomics. Outline : Functional genomics Introduction Relation between genotype and phenotype Eight model organisms E. coli; yeast; Arabidopsis; C. elegans; Drosophila; zebrafish; mouse; human Functional genomics using reverse and forward genetics Reverse genetics: mouse knockouts; yeast; gene trapping; insertional mutatgenesis; gene silencing Forward genetics: chemical mutagenesis Functional genomics and the central dogma Approaches to function; Functional genomics and DNA; …and RNA; …and protein Proteomic approaches to functional genomics CASP; protein-protein interactions; protein networks Perspective Albert Blakeslee (1874–1954) studied the effect of altered chromosome numbers on the phenotype of the jimson-weed Datura stramonium, a flowering plant. Introduction: Functional genomics Functional genomics is the genome-wide study of the function of DNA (including both genes and non-genic regions), as well as RNA and proteins encoded by DNA. The term “functional genomics” may apply to • the genome, transcriptome, or proteome • the use of high-throughput screens • the perturbation of gene function • the complex relationship of genotype and phenotype Functional genomics approaches to high throughput analyses Relationship between genotype and phenotype The genotype of an individual consists of the DNA that comprises the organism. The phenotype is the outward manifestation in terms of properties such as size, shape, movement, and physiology. We can consider the phenotype of a cell (e.g., a precursor cell may develop into a brain cell or liver cell) or the phenotype of an organism (e.g., a person may have a disease phenotype such as sickle‐cell anemia). -
Mutational Signatures: Emerging Concepts, Caveats and Clinical Applications
REVIEWS Mutational signatures: emerging concepts, caveats and clinical applications Gene Koh 1,2, Andrea Degasperi 1,2, Xueqing Zou1,2, Sophie Momen1,2 and Serena Nik-Zainal 1,2 ✉ Abstract | Whole-genome sequencing has brought the cancer genomics community into new territory. Thanks to the sheer power provided by the thousands of mutations present in each patient’s cancer, we have been able to discern generic patterns of mutations, termed ‘mutational signatures’, that arise during tumorigenesis. These mutational signatures provide new insights into the causes of individual cancers, revealing both endogenous and exogenous factors that have influenced cancer development. This Review brings readers up to date in a field that is expanding in computational, experimental and clinical directions. We focus on recent conceptual advances, underscoring some of the caveats associated with using the mutational signature frameworks and highlighting the latest experimental insights. We conclude by bringing attention to areas that are likely to see advancements in clinical applications. The concept of mutational signatures was introduced or drug therapies, in an attempt to decipher causes7–9. in 2012 following the demonstration that analy However, the origins of many signatures remain cryp sis of all substitution mutations in a set of 21 whole tic. Furthermore, while earlier analyses reported single genomesequenced (WGS) breast cancers could reveal signatures, for example of UV radiation (that is, SBS7)2, consistent patterns of mutagenesis across tumours1 more recent studies reported multiple versions of these (FIG. 1a). These patterns were the physiological imprints signatures (that is, SBS7a, SBS7b, SBS7c and SBS7d)3, of DNA damage and repair processes that had occurred leading the community to question whether some find during tumorigenesis and could distinguish BRCA1null ings are reflective of biology or are simply mathemat and BRCA2null tumours from sporadic breast cancers. -
Gene Therapy Glossary of Terms
GENE THERAPY GLOSSARY OF TERMS A • Phase 3: A phase of research to describe clinical trials • Allele: one of two or more alternative forms of a gene that that gather more information about a drug’s safety and arise by mutation and are found at the same place on a effectiveness by studying different populations and chromosome. different dosages and by using the drug in combination • Adeno-Associated Virus: A single stranded DNA virus that has with other drugs. These studies typically involve more not been found to cause disease in humans. This type of virus participants.7 is the most frequently used in gene therapy.1 • Phase 4: A phase of research to describe clinical trials • Adenovirus: A member of a family of viruses that can cause occurring after FDA has approved a drug for marketing. infections in the respiratory tract, eye, and gastrointestinal They include post market requirement and commitment tract. studies that are required of or agreed to by the study • Adeno-Associated Virus Vector: Adeno viruses used as sponsor. These trials gather additional information about a vehicles for genes, whose core genetic material has been drug’s safety, efficacy, or optimal use.8 removed and replaced by the FVIII- or FIX-gene • Codon: a sequence of three nucleotides in DNA or RNA • Amino Acids: building block of a protein that gives instructions to add a specific amino acid to an • Antibody: a protein produced by immune cells called B-cells elongating protein in response to a foreign molecule; acts by binding to the • CRISPR: a family of DNA sequences that can be cleaved by molecule and often making it inactive or targeting it for specific enzymes, and therefore serve as a guide to cut out destruction and insert genes. -
The Limitations of DNA Interstrand Cross-Link Repair in Escherichia Coli
Portland State University PDXScholar Dissertations and Theses Dissertations and Theses 7-12-2018 The Limitations of DNA Interstrand Cross-link Repair in Escherichia coli Jessica Michelle Cole Portland State University Follow this and additional works at: https://pdxscholar.library.pdx.edu/open_access_etds Part of the Biology Commons Let us know how access to this document benefits ou.y Recommended Citation Cole, Jessica Michelle, "The Limitations of DNA Interstrand Cross-link Repair in Escherichia coli" (2018). Dissertations and Theses. Paper 4489. https://doi.org/10.15760/etd.6373 This Thesis is brought to you for free and open access. It has been accepted for inclusion in Dissertations and Theses by an authorized administrator of PDXScholar. Please contact us if we can make this document more accessible: [email protected]. The Limitations of DNA Interstrand Cross-link Repair in Escherichia coli by Jessica Michelle Cole A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biology Thesis Committee: Justin Courcelle, Chair Jeffrey Singer Rahul Raghavan Portland State University 2018 i Abstract DNA interstrand cross-links are a form of genomic damage that cause a block to replication and transcription of DNA in cells and cause lethality if unrepaired. Chemical agents that induce cross-links are particularly effective at inactivating rapidly dividing cells and, because of this, have been used to treat hyperproliferative skin disorders such as psoriasis as well as a variety of cancers. However, evidence for the removal of cross- links from DNA as well as resistance to cross-link-based chemotherapy suggests the existence of a cellular repair mechanism. -
Transposon Insertion Mutagenesis in Mice for Modeling Human Cancers: Critical Insights Gained and New Opportunities
International Journal of Molecular Sciences Review Transposon Insertion Mutagenesis in Mice for Modeling Human Cancers: Critical Insights Gained and New Opportunities Pauline J. Beckmann 1 and David A. Largaespada 1,2,3,4,* 1 Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; [email protected] 2 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA 3 Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA 4 Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA * Correspondence: [email protected]; Tel.: +1-612-626-4979; Fax: +1-612-624-3869 Received: 3 January 2020; Accepted: 3 February 2020; Published: 10 February 2020 Abstract: Transposon mutagenesis has been used to model many types of human cancer in mice, leading to the discovery of novel cancer genes and insights into the mechanism of tumorigenesis. For this review, we identified over twenty types of human cancer that have been modeled in the mouse using Sleeping Beauty and piggyBac transposon insertion mutagenesis. We examine several specific biological insights that have been gained and describe opportunities for continued research. Specifically, we review studies with a focus on understanding metastasis, therapy resistance, and tumor cell of origin. Additionally, we propose further uses of transposon-based models to identify rarely mutated driver genes across many cancers, understand additional mechanisms of drug resistance and metastasis, and define personalized therapies for cancer patients with obesity as a comorbidity. Keywords: animal modeling; cancer; transposon screen 1. Transposon Basics Until the mid of 1900’s, DNA was widely considered to be a highly stable, orderly macromolecule neatly organized into chromosomes. -
The Repertoire of Mutational Signatures in Human Cancer
Article The repertoire of mutational signatures in human cancer https://doi.org/10.1038/s41586-020-1943-3 Ludmil B. Alexandrov1,25, Jaegil Kim2,25, Nicholas J. Haradhvala2,3,25, Mi Ni Huang4,5,25, Alvin Wei Tian Ng4,5, Yang Wu4,5, Arnoud Boot4,5, Kyle R. Covington6,7, Dmitry A. Gordenin8, Received: 18 May 2018 Erik N. Bergstrom1, S. M. Ashiqul Islam1, Nuria Lopez-Bigas9,10,11, Leszek J. Klimczak12, Accepted: 18 November 2019 John R. McPherson4,5, Sandro Morganella13, Radhakrishnan Sabarinathan10,14,15, David A. Wheeler6,16, Ville Mustonen17,18,19, PCAWG Mutational Signatures Working Group20, Published online: 5 February 2020 Gad Getz2,3,21,22,26, Steven G. Rozen4,5,23,26*, Michael R. Stratton13,26* & PCAWG Consortium24 The number of DBSs is proportional to the number of SBSs, with few exceptions Open access Somatic mutations in cancer genomes are caused by multiple mutational processes, each of which generates a characteristic mutational signature1. Here, as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium2 of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), we characterized mutational signatures using 84,729,690 somatic mutations from 4,645 whole-genome and 19,184 exome sequences that encompass most types of cancer. We identifed 49 single-base-substitution, 11 doublet-base-substitution, 4 clustered-base-substitution and 17 small insertion-and-deletion signatures. The substantial size of our dataset, compared with previous analyses3–15, enabled the discovery of new signatures, the separation of overlapping signatures and the decomposition of signatures into components that may represent associated—but distinct—DNA damage, repair and/or replication mechanisms. -
A Highly Efficient Recombineering-Based Method for Generating Conditional Knockout Mutations Pentao Liu, Nancy A
Methods A Highly Efficient Recombineering-Based Method for Generating Conditional Knockout Mutations Pentao Liu, Nancy A. Jenkins, and Neal G. Copeland1 Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA Phage-based Escherichia coli homologous recombination systems have recently been developed that now make it possible to subclone or modify DNA cloned into plasmids, BACs, or PACs without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombineering, has many different uses for functional genomic studies. Here we describe a new recombineering-based method for generating conditional mouse knockout (cko) mutations. This method uses homologous recombination mediated by the phage Red proteins, to subclone DNA from BACs into high-copy plasmids by gaprepair,and together with Cre or Flpe recombinases, to introduce loxPorFRT sites into the subcloned DNA. Unlike other methods that use short 45–55-bpregions of homology for recombineering, our metho d uses much longer regions of homology. We also make use of several new E. coli strains, in which the proteins required for recombination are expressed from a defective temperature-sensitive prophage, and the Cre or Flpe recombinases from an arabinose-inducible promoter. We also describe two new Neo selection cassettes that work well in both E. coli and mouse ES cells. Our method is fast, efficient, and reliable and makes it possible to generate cko-targeting vectors in less than 2 wk. This method should also facilitate the generation of knock-in mutations and transgene constructs, as well as expedite the analysis of regulatory elements and functional domains in or near genes. -
Incision by Uvrabc Excinuclease Is a Step in the Path to Mutagenesis By
Proc. Nati. Acad. Sci. USA Vol. 86, pp. 3982-3986, June 1989 Biochemistry Incision by UvrABC excinuclease is a step in the path to mutagenesis by psoralen crosslinks in Escherichia coli (plasmid mutagenesis/pSV2-gpt/homologous recombination/angelicin/deletions) FRANCES M. SLADEK*t, AGUSTIN MELIANt, AND PAUL HOWARD-FLANDERS§ Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511 Communicated by Fred Sherman, February 21, 1989 (receivedfor review June 10, 1988) ABSTRACT 4,5',8-Trimethylpsoralen (psoralen) plus yield of SOS-dependent mutations (15, 16), which tend to be near UV light produces interstrand crosslinks and monoad- base-pair substitutions (17-19). ducts in DNA, both ofwhich are mutagenic. InEscherichia coil, The repair of crosslinks appears to occur by a sequential crosslinks are incised by UvrABC excinuclease, an event that excision-recombination mechanism (20, 21). In vitro studies can lead to homologous recombination and repair. To deter- show that crosslinked DNA is incised by the UvrABC exci- mine whether UvrABC incision of crosslinks is a step in the nuclease (22, 23) so that an 11-base oligonucleotide containing path to mutagenesis as well as repair, the effect of DNA the crosslink is left attached to an intact DNA strand (24). They homologous to a target gene on a plasmid was determined. also show that RecA-mediated strand exchange can proceed pSV2-gpt DNA was treated with psoralen and transformed into past the crosslinked oligonucleotide (25) and that a second a pair of hosts: one was gpt', the other was A(gpt-ac)5. The round of incision by UvrABC can release the psoralen moeity DNA was extracted and transformed into a tester strain from the DNA (25, 26). -
A Homologous Recombination-Based Method of Genetic Engineering
PROTOCOL Recombineering: a homologous recombination-based method of genetic engineering Shyam K Sharan1, Lynn C Thomason2, Sergey G Kuznetsov1 & Donald L Court3 1Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland 21702, USA. 2SAIC-Frederick Inc., Gene Regulation and Chromosome Biology Laboratory, Basic Research Program, Frederick, Maryland, 21702, USA. 3Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland 21702, USA. Correspondence should be addressed to S.K.S. ([email protected]) or D.L.C. ([email protected]). Published online 29 January 2009; doi:10.1038/nprot.2008.227 Recombineering is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in Escherichia coli. This method circumvents the need for most standard in vitro cloning techniques. Recombineering allows construction of DNA molecules with precise junctions without constraints being imposed by restriction enzyme site location. Bacteriophage homologous recombination proteins catalyze these recombineering reactions using double- and single-stranded linear DNA substrates, natureprotocols so-called targeting constructs, introduced by electroporation. Gene knockouts, deletions and point mutations are readily made, gene / m tags can be inserted and regions of bacterial artificial chromosomes or the E. coli genome can be subcloned by gene retrieval using o c . recombineering. Most of these constructs can be made within about 1 week’s time. e r u t a n . INTRODUCTION w w Recombineering is an in vivo method of genetic engineering used This protocol will emphasize modification of bacterial artificial w / / 1–5 : primarily in Escherichia coli that uses short 50 base homologies . -
Bacterial Artificial Chromosome Mutagenesis Using Recombineering
Hindawi Publishing Corporation Journal of Biomedicine and Biotechnology Volume 2011, Article ID 971296, 10 pages doi:10.1155/2011/971296 Review Article Bacterial Artificial Chromosome Mutagenesis Using Recombineering Kumaran Narayanan1, 2 and Qingwen Chen2 1 Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY 10029, USA 2 School of Science, Monash University, Sunway Campus, Room 2-5-29, Bandar Sunway, 46150, Malaysia Correspondence should be addressed to Kumaran Narayanan, [email protected] Received 2 August 2010; Accepted 21 October 2010 Academic Editor: Masamitsu Yamaguchi Copyright © 2011 K. Narayanan and Q. Chen. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Gene expression from bacterial artificial chromosome (BAC) clones has been demonstrated to facilitate physiologically relevant levels compared to viral and nonviral cDNA vectors. BACs are large enough to transfer intact genes in their native chromosomal setting together with flanking regulatory elements to provide all the signals for correct spatiotemporal gene expression. Until recently, the use of BACs for functional studies has been limited because their large size has inherently presented a major obstacle for introducing modifications using conventional genetic engineering strategies. The development of in vivo homologous recombination strategies based on recombineering in E. coli has helped resolve this problem by enabling facile engineering of high molecular weight BAC DNA without dependence on suitably placed restriction enzymes or cloning steps. These techniques have considerably expanded the possibilities for studying functional genetics using BACs in vitro and in vivo. -
Genetic Engineering Using Homologous Recombination1
17 Oct 2002 13:49 AR AR174-GE36-14.tex AR174-GE36-14.SGM LaTeX2e(2002/01/18) P1: IBC 10.1146/annurev.genet.36.061102.093104 Annu. Rev. Genet. 2002. 36:361–88 doi: 10.1146/annurev.genet.36.061102.093104 GENETIC ENGINEERING USING HOMOLOGOUS RECOMBINATION1 Donald L. Court, James A. Sawitzke, and Lynn C. Thomason Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland 21702; e-mail: [email protected], [email protected], [email protected] Key Words DNA replication forks, strand annealing, in vivo cloning, oligo recombination, recombineering ■ Abstract In the past few years, in vivo technologies have emerged that, due to their efficiency and simplicity, may one day replace standard genetic engineering tech- niques. Constructs can be made on plasmids or directly on the Escherichia coli chromo- some from PCR products or synthetic oligonucleotides by homologous recombination. This is possible because bacteriophage-encoded recombination functions efficiently re- combine sequences with homologies as short as 35 to 50 base pairs. This technology, termed recombineering, is providing new ways to modify genes and segments of the chromosome. This review describes not only recombineering and its applications, but also summarizes homologous recombination in E. coli and early uses of homologous recombination to modify the bacterial chromosome. Finally, based on the premise that phage-mediated recombination functions act at replication forks, specific molecular models are -
Improved Bacterial Recombineering by Parallelized Protein Discovery
Improved bacterial recombineering by parallelized protein discovery Timothy M. Wanniera,1, Akos Nyergesa,b, Helene M. Kuchwaraa, Márton Czikkelyb, Dávid Baloghb, Gabriel T. Filsingera, Nathaniel C. Bordersa, Christopher J. Gregga, Marc J. Lajoiea, Xavier Riosa, Csaba Pálb, and George M. Churcha,1 aDepartment of Genetics, Harvard Medical School, Boston, MA 02115; and bSynthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre, Szeged HU-6726, Hungary Edited by Susan Gottesman, National Institutes of Health, Bethesda, MD, and approved April 17, 2020 (received for review January 30, 2020) Exploiting bacteriophage-derived homologous recombination pro- recombineering, is the molecular mechanism that drives MAGE cesses has enabled precise, multiplex editing of microbial genomes and DIvERGE (4, 32, 33). This method was first described in and the construction of billions of customized genetic variants in a E. coli, and is most commonly promoted by the expression of bet single day. The techniques that enable this, multiplex automated ge- (here referred to as Redβ) from the Red operon of Escherichia nome engineering (MAGE) and directed evolution with random ge- phage λ (5, 6, 34). Redβ is an ssDNA-annealing protein (SSAP) nomic mutations (DIvERGE), are however, currently limited to a whose role in recombineering is to anneal ssDNA to compli- handful of microorganisms for which single-stranded DNA-annealing mentary genomic DNA at the replication fork. Although im- proteins (SSAPs) that promote efficient recombineering have been provements to recombineering efficiency have been made (7–9), identified. Thus, to enable genome-scale engineering in new hosts, the core protein machinery has remained constant, with Redβ efficient SSAPs must first be found.