DOCSLIB.ORG

Repair and Mutagenesis of Plasmid DNA Modified by Ultraviolet

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Repair and Mutagenesis of Plasmid DNA Modified by Ultraviolet Proc. Nati Acad. Sci. USA Vol. 79, pp. 4133-4137, July 1982 Genetics Repair and mutagenesis of plasmid DNA modified by ultraviolet irradiation or N-acetoxy-N-2-acetylaminofluorene (excision repair/postreplication repair/plasmid pKO482) S. E. SCHMID, M. P. DAUNE, AND R. P. P. FUCHS* Institut de Biologie Moleculaire et Cellulaire du Centre National de la Recherche Scientifique, Laboratoire de Biophysique, 67084 Strasbourg Cedex, France Communicated by James A. Miller, March 29, 1982 ABSTRACT Plasmid DNA was modified in vitro to various The plasmid used in this system, pKO482, contains a gene extents with N-acetoxy-N-2-acetylaminofluorene or UV irradia- coding for 3lactamase, which provides for ampicillin resis- tion. The modified plasmid DNAs were then used to transform tance, and a second gene, galK, which codes for the enzyme Escherichia coli strains having different repair capabilities. Both galactokinase. All of the recipient bacterial strains used were survival and mutagenesis frequencies of the plasmid were mea- ampicillin sensitive and unable to metabolize galactose due to sured as a function ofthe number oflesions per plasmid molecule. a galK mutation in their own genomic DNA. Transformed cells The majority ofN-2-acetylaminofluorene (AAF) adducts, like thy- were selected on MacConkey's agar containing ampicillin and mine dimers, were repaired by the excision (uvrA+-dependent) galactose. Complementation of the galK mutation of the host pathway. In rec' strains, dose-dependent mutagenesis occurred cell the of the allows in either AAF- or UV-modified plasmid DNA. This is in contrast by galactokinase gene plasmid galactose with results obtained in recA- strains, in which only AAF adducts fermentation and the formation ofa red colony on MacConkey's gave rise to a lower, but dose-dependent, mutagenesis frequency. agar. Mutation in the plasmid galK gene results in a visually In these recA- strains there was no UV mutagenesis. Unlike what distinguishable white colony. is observed with phages, induction of the "SOS" functions by UV We studied both plasmid survival and mutation frequency irradiation ofthe bacteria prior to transformation did not increase of the galK gene in pKO482 modified by either UV irradiation the survival or the mutagenesis of the plasmid. or N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF). Results obtained by using host cells with wild-type DNA repair capac- Two major types of DNA repair that have been found in Esch- ities are compared to those obtained by using uvrA, recA, and erichia coli are excision repair and postreplication repair (refer uvrA recA mutant strains. In addition, the effect of UV induc- to refs. 1-3 for excellent reviews). Excision repair is dependent tion of SOS functions was also studied. upon the product of the uvrA' gene along with several other gene products and is generally considered accurate and not in- MATERIALS AND METHODS volved in the formation of mutations. Postreplication repair is Bacteria and Plasmid DNA. The E. coli strains used were dependent on the recA+ gene product. This gene product ap- AB 1157 (wild-type strain), AB 1886 (uvrA-), AB 2463 (recA-), pears to be involved in the control ofexpression ofother genes and AB 2480 (uvrA- recA-) (6). All four strains carry a mutation coding for two interrelated processes that can operate to repair in the galK gene. Plasmid pKO482, 2.8 X 106 daltons, was con- gaps left in daughter strand DNA molecules after the replication structed (7) and generously supplied by K. McKenney, H. Shi- of DNA that contains damage left unrepaired by excision repair. matake, and M. Rosenberg (National Cancer Institute, Be- These two interrelated postreplication processes are recom- thesda, MD). binational and "SOS" repair. Recombinational processes are Preparation of Plasmid DNA. Plasmid DNA was purified thought to be error free. The dependence of UV-induced mu- either on a small scale (0.5 ml of culture) by the procedure of tations on the presence of a functional recA+ gene, however, Birnboim and Doly (8) or on a larger scale (1 liter ofculture) by has led to the hypothesis that there is a related SOS repair func- an adaptation ofthe procedures developed by Helinski and co- tion that is error prone. The recA+-dependent repair processes workers (9, 10). have been found to be induced by the presence of UV-damaged Modification of Plasmid DNA with UV Irradiation. Plasmid DNA-as well as by many other types of DNA damage. pKO482 DNA at a concentration of 20 jig/ml in 10 mM Tris- Although repair and mutagenesis have been studied for ge- HCVl10 mM CaCl2/10 mM MgCl2 (pH 7.0) was irradiated in nomic and phage DNA in E. coli, little is known about the ef- 1.0-ml portions in 54-mm-diameter Petri dishes with a germi- fects of the different repair systems of the host E. coli on plas- cidal lamp (15 W, Phillips). Radiant flux was measured with a mid DNA (1). We report here the results of studies using a UVX Digital Radiometer (Ultra-Violet Products, San Gabriel, system recently developed by Kakefuda and co-workers (4, 5) CA). The number ofthymine dimers formed per plasmid mol- in which plasmid DNA is modified in vitro with DNA-damaging ecule was calculated by using apreviously published conversion agents and then used to transform strains ofE. coli with variable factor (11) of0.041% thymine bases converted from 10 J/m2 of DNA-repair capacities. In vitro modification ofthe DNA allows UV. for an accurate quantitative assessment ofthe number ofdamage Modification of Plasmid DNA with N-AcO-AAF. N-AcO- sites per DNA molecule prior to exposure to in vivo repair sys- [3H]AAF (173 Ci/mol; 1 Ci = 3.7 x 1010 becquerels) was syn- tems. In addition, this system has the advantage of excluding thesized as described (12) from 2-nitro[ring-3H]fluorene (Com- any toxic effects of the DNA damaging agent on other cell pro- missariat a l'Energie Atomique, Saclay, France). The N-AcO- cesses or structures. [3H]AAF (42.5 ,uM) was then allowed to react for various The publication costs ofthis article were defrayed in part by page charge Abbreviations: AAF, acetylaminofluorene; N-AcO-AAF, N-acetoxy-N- payment. This article must therefore be hereby marked "advertise- 2-acetylaminofluorene. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. * To whom reprint requests should be addressed. 4133 Downloaded by guest on September 25, 2021 4134 Genetics: Schmid et aL Proc. Natl. Acad. Sci. USA 79 (1982) lengths of time with plasmid DNA (50 Ag/ml) in 10 mM However, after transformation with pKO482, which contains Tris.HCV1 mM EDTA (pH' 8.0) containing 5% (vol/vol) a galK gene, these strains will normally form red colonies on ethanol. Removal ofunbound fluorene derivativeswas achieved MacConkey's agar due to restoration of the galactose metabo- by four successive ethanol precipitations. The number ofN-2- lism pathway and, consequently, production of an acidic envi- acetylaminofluorene molecules (AAF adducts) covalently bound ronment. A visually distinctwhite colony is formed ifa mutation per plasmid DNA molecule was determined by scintillation has occurred in the plasmid galK gene ofthe plasmid pKO482. counting of tritium radioactivity and measuring the nucleotide Verification ofthe presence of a plasmid galK gene mutation concentration from the absorbance at 260 nm. in thewhite colonieswas performedbyextraction ofthe plasmid Preparation and UV Irradiation of the Bacteria Prior to DNA as described above followed by (i) agarose gel electro- Transformation. Where indicated, suspensions ofE. coli in LB phoresis and (ii) retransformation into the AB 1157 strain to medium (2-4 X 108 cells per ml) were irradiated on ice with obtain only white colonies. Five percent ofthe more than 1,100 agermicidal lamp (15 W, Phillips). The dose ofUVwas adjusted mutants obtained in these experiments were randomly selected in each strain to give approximately 50% survival-i.e., 166 J/ for this procedure, and positive results were obtained in all m2 for AB 1157, 7.9 J/m2 for AB 1886, 2.3 J/m2 for AB 2463, cases. and 0.7 J/m2 for AB 2480. All subsequent procedures were car- Mutation frequency is expressed as the number ofwhite col- ried out under semidark conditions to minimize in vivo pho- onies pertotalnumberoftransformants. Thenumberofcolonies toreactivation of the thymine dimers. Cultures were next in- scored to obtain the mutation frequency for each experimental cubated for an additional 30 min at 370C to allow expression of condition and strain ranged between at least 20,000 for un- the SOS functions prior to the transformation step. These con- modified plasmid and at least 1,000 at the highest plasmid mod- ditions have been shown to be optimal for induction ofthe SOS ification levels reported. response in E. coli as measured by Weigle reactivation and mutagenesis in UV-irradiated phage A (13-15). RESULTS Transformation with Plasmid DNA and Selection for Trans- formed Cells. The E. coli were transformed with pKO482 by Relative Importance of uvrA+- and recA+-Dependent Re- the procedure of Mandel and Higa (16) as modified by Cohen pair Processes to Survival of Plasmid DNA Damaged by UV et aL (17). Selection for transformed cells was made by plating Irradiation or N-AcO-AAF Binding. Fig. 1 Left shows the de- on MacConkey's agar (18) containing 2% galactose and ampi- crease in survival ofplasmid pKO482 DNA containing increas- cillin at 50 Ag/ml and incubating overnight at 370C: the (3-lac- ing numbers ofthymine dimers when used to transform strains tamase gene of pKO482 provides ampicillin resistance to the of E. coli with different DNA repair capabilities. Thymine di- transformed cells. The transformation efficiency was found to mers are thought to be the major DNA lesion created by UV be a linear function of both plasmid and cell concentrations irradiation, although other less frequent types of UV lesions when the above conditions were used.
Load more

Recommended publications
  • 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).
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • 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).
    [Show full text]
  • Molecular Biology and Applied Genetics
    MOLECULAR BIOLOGY AND APPLIED GENETICS FOR Medical Laboratory Technology Students Upgraded Lecture Note Series Mohammed Awole Adem Jimma University MOLECULAR BIOLOGY AND APPLIED GENETICS For Medical Laboratory Technician Students Lecture Note Series Mohammed Awole Adem Upgraded - 2006 In collaboration with The Carter Center (EPHTI) and The Federal Democratic Republic of Ethiopia Ministry of Education and Ministry of Health Jimma University PREFACE The problem faced today in the learning and teaching of Applied Genetics and Molecular Biology for laboratory technologists in universities, colleges andhealth institutions primarily from the unavailability of textbooks that focus on the needs of Ethiopian students. This lecture note has been prepared with the primary aim of alleviating the problems encountered in the teaching of Medical Applied Genetics and Molecular Biology course and in minimizing discrepancies prevailing among the different teaching and training health institutions. It can also be used in teaching any introductory course on medical Applied Genetics and Molecular Biology and as a reference material. This lecture note is specifically designed for medical laboratory technologists, and includes only those areas of molecular cell biology and Applied Genetics relevant to degree-level understanding of modern laboratory technology. Since genetics is prerequisite course to molecular biology, the lecture note starts with Genetics i followed by Molecular Biology. It provides students with molecular background to enable them to understand and critically analyze recent advances in laboratory sciences. Finally, it contains a glossary, which summarizes important terminologies used in the text. Each chapter begins by specific learning objectives and at the end of each chapter review questions are also included.
    [Show full text]
  • Statement by the Group of Chief Scientific Advisors
    Statement by the Group of Chief Scientific Advisors A Scientific Perspective on the Regulatory Status of Products Derived from Gene Editing and the Implications for the GMO Directive On 25 July 2018, the Court of Justice of the European Biotechnology’ (SAM, 2017a), we have examined the Union ('the Court') decided that organisms obtained GMO Directive taking into account current by the new techniques of directed mutagenesis are knowledge and scientific evidence. genetically modified organisms (GMOs), within the meaning of the Directive 2001/18/EC on the release 1. The Ruling of the Court of Justice of genetically modified organisms into the On request by the French Conseil d'État, the Court environment ('GMO Directive')1,2, and that they are was asked to determine whether organisms subject to the obligations laid down by the GMO obtained by mutagenesis4 should be considered Directive. GMOs and which of those organisms are exempt New techniques of directed mutagenesis include according to the provisions of the GMO Directive. In gene editing such as CRISPR/Cas9 methodologies. particular, the Court was asked to determine The legal status of the products of such techniques whether organisms obtained by new directed was uncertain, because it was unclear whether they mutagenesis techniques are exempt from the fell within the scope of the GMO Directive. obligations imposed by the GMO Directive, as are those obtained by conventional, random These techniques enable the development of a wide mutagenesis techniques that existed before the range of agricultural applications and the ethical, adoption of the Directive, or are regulated like those legal, social and economic issues of their use are obtained by established techniques of genetic discussed intensively.
    [Show full text]
  • Harnessing the Power of Mutagenesis and Adaptive Laboratory Evolution for High Lipid Production by Oleaginous Microalgae and Yeasts
    sustainability Review Harnessing the Power of Mutagenesis and Adaptive Laboratory Evolution for High Lipid Production by Oleaginous Microalgae and Yeasts Neha Arora 1, Hong-Wei Yen 2 and George P. Philippidis 1,* 1 Patel College of Global Sustainability, University of South Florida, 4202 E. Fowler Avenue, Tampa, FL 33620, USA; [email protected] 2 Department of Chemical and Materials Engineering, Tunghai University, Taichung City 407302, Taiwan; [email protected] * Correspondence: [email protected] Received: 8 April 2020; Accepted: 19 June 2020; Published: 23 June 2020 Abstract: Oleaginous microalgae and yeasts represent promising candidates for large-scale production of lipids, which can be utilized for production of drop-in biofuels, nutraceuticals, pigments, and cosmetics. However, low lipid productivity and costly downstream processing continue to hamper the commercial deployment of oleaginous microorganisms. Strain improvement can play an essential role in the development of such industrial microorganisms by increasing lipid production and hence reducing production costs. The main means of strain improvement are random mutagenesis, adaptive laboratory evolution (ALE), and rational genetic engineering. Among these, random mutagenesis and ALE are straight forward, low-cost, and do not require thorough knowledge of the microorganism’s genetic composition. This paper reviews available mutagenesis and ALE techniques and screening methods to effectively select for oleaginous microalgae and yeasts with enhanced lipid yield and understand the alterations caused to metabolic pathways, which could subsequently serve as the basis for further targeted genetic engineering. Keywords: oleaginous; microalgae; yeast; lipid; mutagenesis; adaptive laboratory evolution 1. Introduction Microorganisms with the inherent ability to synthesize and accumulate lipids to over 20% of their dry cell weight (DCW) are termed oleaginous.
    [Show full text]
  • Lecture 3 Mutagens and Mutagenesis A. Physical and Chemical
    Lecture 3 Mutagens and Mutagenesis 1. Mutagens A. Physical and Chemical mutagens B. Transposons and retrotransposons C. T-DNA 2. Mutagenesis A. Screen B. Selection C. Lethal mutations Read: 508-514 Figs: 14.23-28 1 Transposon (transposable element) as a mutagen Transposon (or TE): DNA segment that can move from one position to another (1) Retrotransposons Copia Drosophila (LTR-type) Ty1 Yeast (LTR-type) LINEs Human (non-LTR-type) SINEs (Alu) Human (non-LTR-type) (2) Transposons Ac/Ds Maize P-element Drosophila 2 Retrovirus Figure 5-73. The life cycle of a retrovirus. The retrovirus genome consists of an RNA molecule of about 8500 nucleotides; two such molecules are packaged into each viral particle. The enzyme reverse transcriptase first makes a DNA copy of the viral RNA molecule and then a second DNA strand, generating a double-stranded DNA copy of the RNA genome. The integration of this DNA double helix into the host chromosome is then catalyzed by a virus-encoded integrase enzyme. This integration is required for the synthesis of new viral RNA molecules by the host cell RNA polymerase, the enzyme that transcribes DNA into RNA. (From Molecular Biology of the Cell by Alberts et al.) 3 Fig. 14.26 4 5 Figure 5-76. Transpositional site- specific recombination by a nonretroviral retrotransposon. Transposition by the L1 element (red) begins when an endonuclease attached to the L1 reverse transcriptase and the L1 RNA (blue) makes a nick in the target DNA at the point at which insertion will occur. This cleavage releases a 3′-OH DNA end in the target DNA, which is then used as a primer for the reverse transcription step shown.
    [Show full text]
  • ADRIAMYCIN® Solution for Injection
    PRODUCT INFORMATION ADRIAMYCIN® Solution for Injection WARNINGS 1. For intravenous or intravesical use only. Severe local tissue necrosis will occur if there is extravasation during administration. ADRIAMYCIN must not be given by the intramuscular or subcutaneous route. 2. Serious irreversible myocardial toxicity with delayed congestive failure often unresponsive to any cardiac supportive therapy may be encountered as total dosage approaches 550 mg/m2. This toxicity may occur at lower cumulative doses in patients with prior mediastinal irradiation or on concurrent cyclophosphamide therapy. 3. Dosage should be reduced in patients with impaired hepatic function. 4. Severe myelosuppression may occur. 5. ADRIAMYCIN should be administered only under the supervision of a physician who is experienced in the use of cancer chemotherapeutic agents. NAME OF MEDICINE Non-proprietary name: doxorubicin hydrochloride The structural formula of doxorubicin hydrochloride is shown below: CAS Registry Number: 25316-40-9 Chemical name: (8S,10S)-10-[(3-Amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]- 6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-7,8,9,10- tetrahydrotetracene-5,12-dione hydrochloride Molecular formula: C27H29NO11,HCl Molecular weight: 580.0 Version: pfpadrii10612 Supersedes: pfpadrii10909 1 of 18 DESCRIPTION Doxorubicin is a cytotoxic anthracycline antibiotic isolated from cultures of Streptomyces peucetius var. caesius. Doxorubicin hydrochloride is an orange-red, crystalline, hygroscopic powder that is soluble in water and slightly soluble in methanol. ADRIAMYCIN Injection is a red coloured, clear solution and should be stored at 2°C - 8°C. ADRIAMYCIN Injection is available in vials containing the active ingredient doxorubicin hydrochloride 10 mg/5 mL, 20 mg/10 mL, 50 mg/25 mL and 200 mg/100 mL.
    [Show full text]