DOCSLIB.ORG

Dissertation Expanding and Evaluating Sense Codon

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Dissertation Expanding and Evaluating Sense Codon DISSERTATION EXPANDING AND EVALUATING SENSE CODON REASSIGNMENT FOR GENETIC CODE EXPANSION Submitted by C. William Biddle Department of Chemistry In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Summer 2017 Doctoral Committee: Advisor: John D. (Nick) Fisk Chris Ackerson Charles Henry Tim Stasevich Copyright by C. William Biddle 2017 All Rights Reserved ABSTRACT EXPANDING AND EVALUATING SENSE CODON REASSIGNMENT FOR GENETIC CODE EXPANSION Genetic code expansion is a field of synthetic biology that aims to incorporate non-canonical amino acids (ncAAs) into proteins as though they were one of the 20 “natural” amino acids. The amino acids which naturally make up proteins are chemical limited, and ncAAs can carry new chemical functionality into proteins. Proteins are of interest because they are simple to produce with good consistency and have immense potential due to the diversity of structure and function. Incorporating ncAA into proteins expands the scope of function of proteins even further. Two methods have been widely used for genetic code expansion, global amino acid replacement and amber stop codon suppression. Global amino acid replacement exchanges one of the natural amino acids for a ncAA, producing an altered 20 amino acid genetic code. Amber stop codon suppression incorporates ncAA in response to the UAG stop codon making a 21 amino acid genetic code, but is limited in incorporation efficiency and producing proteins with multiple instances of a ncAA is challenging. We wanted to use a third genetic code expansion system called sense codon reassignment which has not been widely employed at all but should enable multisite incorporation of ncAAs. When the work presented in this dissertation was started, a single report of sense codon reassignment existed in the literature. We set out to improve and expand sense codon reassignment for the incorporation of multiple copies of ncAAs into proteins. We quickly discovered disparities in what was known regarding the variables that could be used to manipulate genetic code expansion, and the focus of our work shifted to systems for improving sense codon reassignment using quantitative measurements. The first chapter of this dissertation is an introduction to genetic code expansion and the processes of translation and gene expression that are likely involved or could be involved in genetic code expansion. ii The three following chapters will build upon the fundamentals described in Chapter 1. The second chapter is a complete story about how a screen to quantify sense codon reassignment was developed. The fluorescence based screen was used in a high throughput fashion to screen a directed evolution library of variants for increased sense codon reassignment efficiency at the Lys AAG sense codon. While evaluating various sense codons for potential reassignment efficiency, the AUG anticodon was found to be incapable of discriminating between the CAU and CAC codons. This was anomalous relative to the other anticodons we tested. Chapter 3 describes how unintended modifications to an engineered tRNA were identified and then how the fluorescence based screen was used to engineer the tRNA further for increased sense codon reassignment efficiency and to avoid the unintentional modification. Most applications of genetic code expansion rely on modifications to tRNAs but few reports actually consider them, The final chapter of this dissertation is a manuscript in preparation describing the reassignment of a rare sense codon to incorporate ncAAs. The chapter focuses on how improvements made in a system specific for an amino acid can be transferred to systems specific for other ncAAs. Over 150 different ncAAs have been incorporated into proteins using genetic code expansion technologies, but the extent to which the various systems are combinable has barely been evaluated. This dissertation is a story about developing sense codon reassignment to functional levels and quantifying the effects of different variables along the way. iii ACKNOWLEDGMENTS I am tremendously grateful to my advisor, Dr. Nick Fisk for his contagious enthusiasm for science. When I first sat in his office (and countless times since) his excitement about the possibilities in the world of science and engineering convinced me that he would be an excellent advisor for my first formal scientific pursuits. I was not wrong. I am grateful for Nick’s patience, support, inspiration, and guidance throughout my PhD. Thank you to Dr. Meg Schmitt who acted as an unofficial co-advisor, staff scientist and friend. Meg was always available for help with technical protocols or with dealing with the mundane challenges in graduate school. A special thank you to my PhD committee, Dr. Chris Ackerson, Dr. Chuck Henry, and Dr. Tim Stasevich. I owe Dr. Ackerson for several occasions where he was available to discuss my research, or sometimes more importantly, the academic world outside my lab and how my research fit into a larger scientific frame. Dr. henry was always available to help, whether I needed supplies for an analytical procedure or logistics help getting to and from conferences. Working between two departments was sometimes a challenge and Chuck was there to help. And thank you to Dr. Stasevich, who agreed last minute to fill in on and increase the biological perspective on my committee. I owe a debt of gratitude to all of the professors in the Chemistry Department for encouraging, inspiring and educating me, but I especially want to thank the late Dr. Michael Elliott. Mike inspired me to not only be a better chemist and think more carefully, but also to try and inspire others around me without worrying about what people will think. Dr. Elliott was his own person and left a lasting impact on my impression of what a scientist and a good human being should be. Thank you, Mike, and I miss you. Special thanks go to my brother Hovis, with whom I had several late night conversations where he encouraged me to keep pursuing my research. His passion for science is tangible and I was able to hold on iv to that passion when the tedium of research sometimes seemed too much. Thank you to the rest of my family as well who supported me emotionally and physically throughout not only my PhD, but also through my undergraduate experience. Thank you to my parents who nourished a natural curiosity in me from an early age. And finally, thank you to all my friends who have become my family abroad. You have helped me continue exploring the world around us. My colleagues from grad school have become my most trusted of friends. v TABLE OF CONTENTS ABSTRACT .................................................................................................................................................. ii ACKNOWLEDGEMENTS ......................................................................................................................... iv LIST OF TABLES ..................................................................................................................................... viii LIST OF FIGURES ..................................................................................................................................... ix CHAPTER 1 – INTRODUCTION: EXPANDING THE GENETIC CODE ............................................... 1 CHEMICAL DIVERSITY OF NATURAL PROTEINS .................................................................... 1 FUNDAMENTALS OF TRANSLATION ......................................................................................... 3 Transcription ............................................................................................................................. 5 Translation ................................................................................................................................. 6 COMMONLY USED METHODS FOR GENETIC CODE EXPANSION ..................................... 18 Global Amino Acid Replacement ........................................................................................... 19 Nonsense Codon Suppression ................................................................................................. 21 Sense Codon Reassignment: Breaking the Degeneracy of the Genetic Code ......................... 24 OTHER METHODS FOR INCREASING CHEMICAL DIVERSITY OF PROTEINS ................. 27 Solid Phase Synthesis .............................................................................................................. 27 Cell Free Translation Systems ................................................................................................. 28 QUANTIFYING GENETIC CODE EXPANSION .......................................................................... 29 DISSERTATION OUTLINE ............................................................................................................ 31 REFERENCES ........................................................................................................................................... 34 CHAPTER 2 – EVALUATING SENSE CODON REASSIGNMENT WITH A ASIMPLE FLUORESCENCE SCREEN ..................................................................................................................... 43 OVERVIEW ..................................................................................................................................... 43 INTRODUCTION............................................................................................................................
Load more

Recommended publications
  • Addressing Evolutionary Questions with Synthetic Biology
    Addressing evolutionary questions with synthetic biology Florian Baier and Yolanda Schaerli Department of Fundamental Microbiology, University of Lausanne, Biophore Building, 1015 Lausanne, Switzerland Correspondence: [email protected]; [email protected] Abstract Synthetic biology emerged as an engineering discipline to design and construct artificial biological systems. Synthetic biological designs aim to achieve specific biological behavior, which can be exploited for biotechnological, medical and industrial purposes. In addition, mimicking natural systems using well-characterized biological parts also provides powerful experimental systems to study evolution at the molecular and systems level. A strength of synthetic biology is to go beyond nature’s toolkit, to test alternative versions and to study a particular biological system and its phenotype in isolation and in a quantitative manner. Here, we review recent work that implemented synthetic systems, ranging from simple regulatory circuits, rewired cellular networks to artificial genomes and viruses, to study fundamental evolutionary concepts. In particular, engineering, perturbing or subjecting these synthetic systems to experimental laboratory evolution provides a mechanistic understanding on important evolutionary questions, such as: Why did particular regulatory networks topologies evolve and not others? What happens if we rewire regulatory networks? Could an expanded genetic code provide an evolutionary advantage? How important is the structure of genome and number of chromosomes? Although the field of evolutionary synthetic biology is still in its teens, further advances in synthetic biology provide exciting technologies and novel systems that promise to yield fundamental insights into evolutionary principles in the near future. 1 1. Introduction Evolutionary biology traditionally studies past or present organisms to reconstruct past evolutionary events with the aim to explain and predict their evolution.
    [Show full text]
  • And Chemical-Activated Nucleosides and Unnatural Amino Acids. (Under the Direction of Dr
    ABSTRACT LIU, QINGYANG. Synthesis of Photo- and Chemical-Activated Nucleosides and Unnatural Amino Acids. (Under the direction of Dr. Alexander Deiters). Synthetic oligonucleotides coupled with photolabile caging groups have been developed to regulate a variety of biological processes in a spatial and temporal fashion. A UV-cleavable caging group was installed on deoxyadenosine and two morpholino oligonucleotide (MO) monomers of which the morpholino core synthesis was also investigated. The synthesis of a two-photon caging group was optimized and two chromophores with > 400 nm absorption maximum were applied to cage thymidine. These caged monomers can serve as light-triggers of oligonucleotide function upon incorporation. Two phosphine-labile azido thymidine derivatives were synthesized as orthogonal small molecule-triggers to the above light-triggers. Additionally, two coumarin linkers were synthesized, which can cyclize a linear MO so as to inactivate MO activity until > 400 nm light irradiation. These two linkers have been applied to the wavelength-selective regulation of zebrafish embryo development. An azide linker was also synthesized to control MOs using phosphines, as well as a UV-cleavable phosphoramidite to regulate DNA oligonucleotide activities. On the regulation of proteins, a two-photon caged lysine, four azido lysines and an azido tyrosine were synthesized to control protein function with either light or small molecules. The phosphine-induced cleavage of the azido groups were investigated on a coumarin reporter. A fluorescent lysine and an isotope labeled lysine were also synthesized as additional biophysical probes to label protein. These unnatural amino acids have been or will be incorporated into proteins through exogenous tRNA-aaRSs pairs.
    [Show full text]
  • Peritoneal Cells
    Immunology 1976 30 741 Two distinct lymphocyte-stimulating soluble factors (LAF) released from murine peritoneal cells I. THE CELLULAR SOURCE AND THE EFFECT OF cGMP ON THEIR RELEASE T. DIA MANTSTEIN & A. UL MER Immunological Research Unit, Klinikum Steglitz Freie Universitdt, Berlin, Germany Received 26 September 1975; acceptedfor publication 30 October 1975 Summary. Culture fluids of murine peritoneal cells similar lymphocyte (predominantly thymocyte)- contain two distinct, non-dialysable principles activating factor(LAF) and lymphocyteproliferation- (LAF) with thymocyte proliferation-stimulating inhibiting factor (LIF) can be obtained by incubat- properties. One of them is elaborated from phago- ing normal peritoneal mouse cells (rich in non- cytic cells (presumably macrophages), the other is stimulated macrophages) in the presence of cyclic- released by non-phagocytic cells (lymphocytes). 3',5'-guanosine monophosphate (cGMP). This paper cGMP added exogenously stimulates the production reports in detail on this phenomenon with respect and/or release of LAF from phagocytic cells, but not to the specificity of cGMP as an inducer for LAF from non-phagocytic cells. The phagocytic cells (but release and the identity of the cells releasing LAF in not the lymphocytes) require intact RNA and pro- presence and absence ofcGMP. tein synthesis for LAF release. The action of LAF differs from those of cGMP itself by being non- dialysable, and unable to prevent the inhibitory MATERIALS AND METHODS action of cAMP on mitogen-stimulated lymphocyte proliferation.
    [Show full text]
  • Secretariat of the CBD Technical Series No. 82 Convention on Biological Diversity
    Secretariat of the CBD Technical Series No. 82 Convention on Biological Diversity 82 SYNTHETIC BIOLOGY FOREWORD To be added by SCBD at a later stage. 1 BACKGROUND 2 In decision X/13, the Conference of the Parties invited Parties, other Governments and relevant 3 organizations to submit information on, inter alia, synthetic biology for consideration by the Subsidiary 4 Body on Scientific, Technical and Technological Advice (SBSTTA), in accordance with the procedures 5 outlined in decision IX/29, while applying the precautionary approach to the field release of synthetic 6 life, cell or genome into the environment. 7 Following the consideration of information on synthetic biology during the sixteenth meeting of the 8 SBSTTA, the Conference of the Parties, in decision XI/11, noting the need to consider the potential 9 positive and negative impacts of components, organisms and products resulting from synthetic biology 10 techniques on the conservation and sustainable use of biodiversity, requested the Executive Secretary 11 to invite the submission of additional relevant information on this matter in a compiled and synthesised 12 manner. The Secretariat was also requested to consider possible gaps and overlaps with the applicable 13 provisions of the Convention, its Protocols and other relevant agreements. A synthesis of this 14 information was thus prepared, peer-reviewed and subsequently considered by the eighteenth meeting 15 of the SBSTTA. The documents were then further revised on the basis of comments from the SBSTTA 16 and peer review process, and submitted for consideration by the twelfth meeting of the Conference of 17 the Parties to the Convention on Biological Diversity.
    [Show full text]
  • Chapter 23 Nucleic Acids
    7-9/99 Neuman Chapter 23 Chapter 23 Nucleic Acids from Organic Chemistry by Robert C. Neuman, Jr. Professor of Chemistry, emeritus University of California, Riverside [email protected] <http://web.chem.ucsb.edu/~neuman/orgchembyneuman/> Chapter Outline of the Book ************************************************************************************** I. Foundations 1. Organic Molecules and Chemical Bonding 2. Alkanes and Cycloalkanes 3. Haloalkanes, Alcohols, Ethers, and Amines 4. Stereochemistry 5. Organic Spectrometry II. Reactions, Mechanisms, Multiple Bonds 6. Organic Reactions *(Not yet Posted) 7. Reactions of Haloalkanes, Alcohols, and Amines. Nucleophilic Substitution 8. Alkenes and Alkynes 9. Formation of Alkenes and Alkynes. Elimination Reactions 10. Alkenes and Alkynes. Addition Reactions 11. Free Radical Addition and Substitution Reactions III. Conjugation, Electronic Effects, Carbonyl Groups 12. Conjugated and Aromatic Molecules 13. Carbonyl Compounds. Ketones, Aldehydes, and Carboxylic Acids 14. Substituent Effects 15. Carbonyl Compounds. Esters, Amides, and Related Molecules IV. Carbonyl and Pericyclic Reactions and Mechanisms 16. Carbonyl Compounds. Addition and Substitution Reactions 17. Oxidation and Reduction Reactions 18. Reactions of Enolate Ions and Enols 19. Cyclization and Pericyclic Reactions *(Not yet Posted) V. Bioorganic Compounds 20. Carbohydrates 21. Lipids 22. Peptides, Proteins, and α−Amino Acids 23. Nucleic Acids **************************************************************************************
    [Show full text]
  • UNIVERSITY of CALIFORNIA, IRVINE an Expanded Genetic Code
    UNIVERSITY OF CALIFORNIA, IRVINE An expanded genetic code for the characterization and directed evolution of tyrosine-sulfated proteins DISSERTATION submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILSOPHY in Biomedical Engineering by Xiang Li Dissertation Committee: Assistant Professor Chang C. Liu, Chair Associate Professor Wendy Liu Associate Professor Jennifer A. Prescher 2018 Portion of Chapter 2 © John Wiley and Sons Portion of Chapter 3 © Springer Portion of Chapter 4 © Royal Society of Chemistry All other materials © 2018 Xiang Li i Dedication To My parents Audrey Bai and Yong Li and My brother Joshua Li ii Table of Content LIST OF FIGURES ..................................................................................................................VI LIST OF TABLES ................................................................................................................. VIII CURRICULUM VITAE ...........................................................................................................IX ACKNOWLEDGEMENTS .................................................................................................... XII ABSTRACT .......................................................................................................................... XIII CHAPTER 1. INTRODUCTION ................................................................................................ 1 1.1. INTRODUCTION .................................................................................................................
    [Show full text]
  • Alternative Biochemistries for Alien Life: Basic Concepts and Requirements for the Design of a Robust Biocontainment System in Genetic Isolation
    G C A T T A C G G C A T genes Review Alternative Biochemistries for Alien Life: Basic Concepts and Requirements for the Design of a Robust Biocontainment System in Genetic Isolation Christian Diwo 1 and Nediljko Budisa 1,2,* 1 Institut für Chemie, Technische Universität Berlin Müller-Breslau-Straße 10, 10623 Berlin, Germany; [email protected] 2 Department of Chemistry, University of Manitoba, 144 Dysart Rd, 360 Parker Building, Winnipeg, MB R3T 2N2, Canada * Correspondence: [email protected] or [email protected]; Tel.: +49-30-314-28821 or +1-204-474-9178 Received: 27 November 2018; Accepted: 21 December 2018; Published: 28 December 2018 Abstract: The universal genetic code, which is the foundation of cellular organization for almost all organisms, has fostered the exchange of genetic information from very different paths of evolution. The result of this communication network of potentially beneficial traits can be observed as modern biodiversity. Today, the genetic modification techniques of synthetic biology allow for the design of specialized organisms and their employment as tools, creating an artificial biodiversity based on the same universal genetic code. As there is no natural barrier towards the proliferation of genetic information which confers an advantage for a certain species, the naturally evolved genetic pool could be irreversibly altered if modified genetic information is exchanged. We argue that an alien genetic code which is incompatible with nature is likely to assure the inhibition of all mechanisms of genetic information transfer in an open environment. The two conceivable routes to synthetic life are either de novo cellular design or the successive alienation of a complex biological organism through laboratory evolution.
    [Show full text]
  • An Introduction to Recurrent Nucleotide Interactions in RNA Blake A
    Overview An introduction to recurrent nucleotide interactions in RNA Blake A. Sweeney,1 Poorna Roy2 and Neocles B. Leontis2∗ RNA secondary structure diagrams familiar to molecular biologists summarize at a glance the folding of RNA chains to form Watson–Crick paired double helices. However, they can be misleading: First of all, they imply that the nucleotides in loops and linker segments, which can amount to 35% to 50% of a structured RNA, do not significantly interact with other nucleotides. Secondly, they give the impression that RNA molecules are loosely organized in three-dimensional (3D) space. In fact, structured RNAs are compactly folded as a result of numerous long-range, sequence-specific interactions, many of which involve loop or linker nucleotides. Here, we provide an introduction for students and researchers of RNA on the types, prevalence, and sequence variations of inter-nucleotide interactions that structure and stabilize RNA 3D motifs and architectures, using Escherichia coli (E. coli) 16S ribosomal RNA as a concrete example. The picture that emerges is that almost all nucleotides in structured RNA molecules, including those in nominally single-stranded loop or linker regions, form specific interactions that stabilize functional structures or mediate interactions with other molecules. The small number of noninteracting, ‘looped-out’ nucleotides make it possible for the RNA chain to form sharp turns. Base-pairing is the most specific interaction in RNA as it involves edge-to-edge hydrogen bonding (H-bonding) of the bases. Non-Watson–Crick base pairs are a significant fraction (30% or more) of base pairs in structured RNAs. © 2014 John Wiley & Sons, Ltd.
    [Show full text]
  • Bio 102 Practice Problems Genetic Code and Mutation
    Bio 102 Practice Problems Genetic Code and Mutation Multiple choice: Unless otherwise directed, circle the one best answer: 1. Choose the one best answer: Beadle and Tatum mutagenized Neurospora to find strains that required arginine to live. Based on the classification of their mutants, they concluded that: A. one gene corresponds to one protein. B. DNA is the genetic material. C. "inborn errors of metabolism" were responsible for many diseases. D. DNA replication is semi-conservative. E. protein cannot be the genetic material. 2. Choose the one best answer. Which one of the following is NOT part of the definition of a gene? A. A physical unit of heredity B. Encodes a protein C. Segement of a chromosome D. Responsible for an inherited characteristic E. May be linked to other genes 3. A mutation converts an AGA codon to a TGA codon (in DNA). This mutation is a: A. Termination mutation B. Missense mutation C. Frameshift mutation D. Nonsense mutation E. Non-coding mutation 4. Beadle and Tatum performed a series of complex experiments that led to the idea that one gene encodes one enzyme. Which one of the following statements does not describe their experiments? A. They deduced the metabolic pathway for the synthesis of an amino acid. B. Many different auxotrophic mutants of Neurospora were isolated. C. Cells unable to make arginine cannot survive on minimal media. D. Some mutant cells could survive on minimal media if they were provided with citrulline or ornithine. E. Homogentisic acid accumulates and is excreted in the urine of diseased individuals. 5.
    [Show full text]
  • Investigation of Overhauser Effects Between Pseudouridine and Water Protons in RNA Helices
    Investigation of Overhauser effects between pseudouridine and water protons in RNA helices Meredith I. Newby* and Nancy L. Greenbaum*†‡ *Department of Chemistry and Biochemistry and †Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4390 Communicated by Michael Kasha, Florida State University, Tallahassee, FL, August 8, 2002 (received for review July 2, 2002) The inherent chemical properties of RNA molecules are expanded by posttranscriptional modification of specific nucleotides. Pseudouridine (␺), the most abundant of the modified bases, features an additional imino group, NH1, as compared with uri- dine. When ␺ forms a Watson–Crick base pair with adenine in an RNA helix, NH1 is positioned within the major groove. The pres- ence of ␺ often increases thermal stability of the helix or loop in which it is found [Hall, K. B. & McLaughlin, L. (1992) Nucleic Acids Res. 20, 1883–1889]. X-ray crystal structures of transfer RNAs [e.g., Arnez, J. & Steitz, T. (1994) Biochemistry 33, 7560–7567] have depicted water molecules bridging ␺NH1 groups and nearby phos- phate oxygen atoms, but direct evidence for this interaction in solution has not been acquired. Toward this end, we have used a rotating-frame Overhauser effect spectroscopy-type NMR pulse sequence with a CLEAN chemical-exchange spectroscopy spin-lock pulse train [Hwang, T.-L., Mori, S., Shaka, A. J. & van Zijl, P. C. M. Fig. 1. Schematic structures of U and ␺ bases. R, ribose. The ␺ base is a uracil ␺ (1997) J. Am. Chem. Soc. 119, 6203–6204] to test for NH1–water rotated about the 3–6 ring axis, so that is has a COC base–sugar linkage and cross-relaxation effects within two RNA helices: (i) a complemen- an additional protonated ring nitrogen.
    [Show full text]
  • Expanding the Genetic Code Lei Wang and Peter G
    Reviews P. G. Schultz and L. Wang Protein Science Expanding the Genetic Code Lei Wang and Peter G. Schultz* Keywords: amino acids · genetic code · protein chemistry Angewandte Chemie 34 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/anie.200460627 Angew. Chem. Int. Ed. 2005, 44,34–66 Angewandte Protein Science Chemie Although chemists can synthesize virtually any small organic molecule, our From the Contents ability to rationally manipulate the structures of proteins is quite limited, despite their involvement in virtually every life process. For most proteins, 1. Introduction 35 modifications are largely restricted to substitutions among the common 20 2. Chemical Approaches 35 amino acids. Herein we describe recent advances that make it possible to add new building blocks to the genetic codes of both prokaryotic and 3. In Vitro Biosynthetic eukaryotic organisms. Over 30 novel amino acids have been genetically Approaches to Protein encoded in response to unique triplet and quadruplet codons including Mutagenesis 39 fluorescent, photoreactive, and redox-active amino acids, glycosylated 4. In Vivo Protein amino acids, and amino acids with keto, azido, acetylenic, and heavy-atom- Mutagenesis 43 containing side chains. By removing the limitations imposed by the existing 20 amino acid code, it should be possible to generate proteins and perhaps 5. An Expanded Code 46 entire organisms with new or enhanced properties. 6. Outlook 61 1. Introduction The genetic codes of all known organisms specify the same functional roles to amino acid residues in proteins. Selectivity 20 amino acid building blocks. These building blocks contain a depends on the number and reactivity (dependent on both limited number of functional groups including carboxylic steric and electronic factors) of a particular amino acid side acids and amides, a thiol and thiol ether, alcohols, basic chain.
    [Show full text]
  • Cytotoxic and Biochemical Effects of Thymidine and 3-Deazauridine on Human Tumor Cells1
    [CANCER RESEARCH 44, 2534-2539. June 1984] Cytotoxic and Biochemical Effects of Thymidine and 3-Deazauridine on Human Tumor Cells1 Arnold Lockshin, John T. Mendoza, Beppino C. Giovanella,2 and John S. Stehlin, Jr. St. Joseph Hospital Laboratory for Cancer Research, Houston, Texas 77002 ABSTRACT cells, thymidine-resistant human malignant B-cells have much lower ratios of these activities (6, 19). The cause of thymidine- Cytotoxicity and perturbations of the deoxyribonucleoside tri- induced inhibition of DNA synthesis in mammalian cells has been phosphate pools caused by thymidine were studied in thymidine- ascribed to depletion of the intracellular dCTP pool brought about sensitive and -resistant human tumor cells. Incubation with 1 mw by allosteric effects of dTTP on ribonucleotide reducíase(2, 33, thymidine reduced cell viability by more than 90% in the three 36). Other investigators have concluded that the decrease in the sensitive cell lines (two melanomas and one adrenal carcinoma) dCTP pool is too small to account for growth inhibition of L1210 and reduced the growth rate without decreasing the viability of murine leukemia cells (14) and that high levels of dTTP (and/or resistant LO melanoma cells. Thymidine (1 HIM)greatly increased other dNTPs3) interact with a regulatory protein for DNA polym- the ratio of the deoxythymidine 5'-triphosphate to deoxycytidine 5'-triphosphate pools in the sensitive cells compared to LO cells erase (37). Yet another interpretation is that imbalance of the pyrimidine dNTP pools brought about by supranormal levels of and also caused larger relative increases in the pool sizes of deoxyguanosine 5'-triphosphate and deoxyadenosine 5'-tri- exogenous thymidine causes misincorporation of bases into DNA, which leads to mutagenicity and cytotoxicity (4).
    [Show full text]