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Isolation of a cDNA for Drosophila melanogaster manganese superoxide dismutase Carmichael, Stuart 1995

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Isolation of a c D N A for Drosophila melanogaster Manganese Superoxide Dismutase. by Stuart Carmichael B. Sc. (Major) University of Victoria, 1989 A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in The Faculty of Graduate Studies Department of Biochemistry and Molecular Biology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1995 © Stuart Carmichael, 1995 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of gjoCffiS^l < f rj{ j g, flA OLE cu L-AA g(QL 0 6 Y r The University of British Columbia Vancouver, Canada Date TUNE Yi/ / 9 ? JT DE-6 (2/88) 11 ABSTRACT: Numerous phenomena in aging animals are thought to result from chemical damage by activated oxygen species. One of the prime candidates for a causative agent in aging is superoxide, a free radical species resulting from single electron transfer to molecular oxygen. Superoxide is produced in normal metabolism by occasional adventitious oxidation of electron carriers higher up in the electron-transport chain (ETC) by oxygen, and by the activities of several oxidative enzymes, such as xanthine oxidase. A cDNA for Drosophila manganese-containing matrix localized superoxide dismutase was cloned and sequenced. To study the effects on Drosophila life span of reducing mitochondrial matrix superoxide levels, protein over expression studies by introduction of extra copies of the gene in a genetically defined background population were planed. A partial gene for a putative manganese superoxide dismutase from an unknown organism was also isolated. Manganese and iron superoxide dismutase protein homology groups and sequence conservation are also explored. Ill TABLE OF CONTENTS: Abstract: ii Table of Contents: iii List of Tables: viii List of Figures: ix Abbreviations: x Acknowledgements: xiii Dedication: xiii INTRODUCTION: 1 The Aging Problem 1 A Universal Phenomenon 1 Theories of Aging: 1 Group-Specific Aging Theories Limitations of Group Specific Theories 2 Universal Aging Theories 3 Theories Specific to Higher Organisms 6 Aging: Studies and Experimentation 6 Rate of Dying Curves 6 Interspecies Correlates of Life Spans 8 Cell Biology of Aging 8 Procedures Which Extend Life Span 9 Caloric Restriction 9 Antioxidant Drugs 10 Other Drugs 11 Manipulations of the Neuroendocrine System 12 Melatonin 12 Growth Hormone 12 Genetic Alterations 13 iv Transformation with Elongation Factor 1 Alpha 13 Transformation with Cu-Zn SOD 14 Cu-Zn SOD and Downs Syndrome 15 Evaluation and Summary of the Various Theories and Research on Aging 16 The Free Radical Theory of Aging Revisited 18 Types of Metabolic Free Radicals (#1) 18 Sources of Metabolic Free Radicals (#2) 19 Free Radical Defenses (#3) 20 Small Molecules 20 Chelation of Metal Ions 21 Superoxide Dismutases 22 Peroxidases & Catalase 23 Damage Repair Mechanisms: 23 Targets of Oxidative Damage (#4) 23 Rate Limiting Steps (#5) 25 Mitochondrial Degeneration: a Hypothesis 26 Experimental Approaches 27 Membrane Character 27 Molecular Approaches 27 Catalase and Peroxidase 28 Superoxide Dismutases 29 Goals of This Research 29 MATERIALS: 31 Drosophila melanogaster Strains 31 Bacterial Strains 31 Cloning Vectors 31 Chemicals and Reagents 31 Enzymes 32 V Buffers 32 Buffers used in electrophoresis: 32 Loading Dyes for Acrylamide and Agarose Gels 32 Buffers for Blotting gels in Northerns and Southerns 33 Buffers for Extraction of DNA and RNA 33 Buffers for Enzymatic Reactions: 33 Restriction Endonucleases: 33 Incubation mixes for Polymerase reactions: 34 Buffers for Other DNA Modification Enzymes: 35 Bacterial Media: 36 Other Bacteriology Reagents: 36 DNA Probes 38 METHODS: 40 Methods I: Molecular Techniques 40 Isolation of Drosophila melanogaster DNA 40 Isolation of Drosophila melanogaster RNA 42 Plasmid DNA Isolations 43 ssDNA isolation (Ml3 phage): 44 Lambda Phage DNA Isolation From Broth Cultures: 45 Isolation of DNA from Agarose Gels 47 Oligonucleotide Purification 47 Polyacrylamide for DNA Precipitation 48 Nucleic Acid Quantitation by UV Absorption 48 Preparation of DNA Size Standards for Agarose Gels 49 Agarose Gel Electrophoresis: 51 Denaturing Agarose Gel Electrophoresis: 52 Preparation of Acrylamide Gels for DNA sequencing 52 Spun Column Preparation 52 vi PCR conditions for DNA and RNA samples 53 Cloning into M13 and BlueScript; 53 Plaque and Colony Lifts 53 DNA and RNA Blots 54 DNA Sequencing 54 METHODS: 55 Methods II: Bacterial Procedures 55 Competent Cell Preparation 55 Transformation of Competent Cells 55 Lambda Proceedures 56 Materials and Tips for Screening a lambda phage Library: 56 Titreing a lambda Phage Library: 57 Plating a Lambda Phage Library 57 Rescreening of Positive Phage Plaques 58 METHODS: 60 Methods III: Drosophila techniques 60 Paraquat Induction of Adult Flies for SOD mRNA 60 Paraquat Induction of Fly Larvae for SOD mRNA 60 Isolation of Fly Larvae from Fly Food 60 RESULTS: 61 Study premise 61 Jan90 Southern Blots 61 Studies with PCR 61 Isolation of an Archaebacterial SOD-like sequence 65 Plasmid Contaminations 69 Demonstration that BSC23 is not a Drosophila gene 71 Isolation of the Drosophila Mn SOD Gene 74 Library Screening 76 V l l Sequencing of the Drosophila Mn SOD cDNAs 80 Demonstration of a single copy gene in Drosophila 88 Analysis of the Mn SOD Sequences 90 Cryptic 181 amino acid open reading frame 90 DISCUSSION: 94 Opening Remarks 94 Comments on Methods: PCR 94 Isolation of the Copia-like Element 95 Isolation of BSC23; A Putative Archaebacterial Mn SOD 96 The Drosophila melanogaster Mn SOD gene 98 Polymorphisms in the DNA Sequence 98 Assignment of mRNA 5' and 3' Ends 100 Sequence Alignment of Known Mn SOD Genes 102 Previous Work With Cu-Zn SOD 110 CONCLUSIONS: 112 REFERENCES: 116 APPENDIX: 133 DNAABS Program 133 Copia 3' LTR 135 Vl l l LIST OF TABLES: Table #1: Reactions of superoxide 22 Table #2: Table of DNA Oligonucleotides 38 Table #3: U.V. Absorption Values for Nucleic Acids 49 Table #4: Molecular Size Standards for Agarose Gels 51 Table #5: Subclone Lineage's 82 Table #6: Mn SOD with Polymorphisms from Clones 2D/2F (Allele #1) 84 Table #7: Polymorphism effects in reverse complement open reading frame 92 Table #8: Sequence Polymorphisms Between Drosophila Alleles/Strains 100 Table #9: 5' End Assignment and Polyadenylation Site Usage of 5 Clones 101 Table #10: Polyadenylation Sites 101 ix LIST OF FIGURES: Figure #1: Rate of Dying Curves 7 Figure #2: Positions of Oligonucleotides Used in this Study 63 Figure #2: Positions of Oligonucleotides Used in this Study 64 Figure #3: PCR with S2, SCI and HEW 66 Figure #4: DNA Sequence and Amino Acid Translation of BSC23 67 Figure #5: Genomic Southern Blot with Klenow Fragment Labeled BSC23 70 Figure #6 Comparison of Hybridisations, Doolittle VS. BSC23 72 Figure #7: PCR of Drosophila Mn SOD partial DNA 75 Figure #8: Screening of Lambda GT10 Phage Library: Primary Screen 77 Figure #9: Gel Separation of Isolated Lambda GT10 Phage Insert DNAs 78 Figure #10: Gel Separation of pBlueScript Clones from Lambda Phage Positives 79 Figure #11: Gel of Hpa II and Taq I Digested Insert DNAs 81 Figure #12: Subcloning and Sequencing Strategy 82 Figure #13: DNA Sequence of Allele #1 with Sequence Polymorphisms 84 Figure #14: DNA Sequence and Amino Acid Translation of Allele #2 86 Figure #15: Genomic Southern Blot 89 Figure #16: 181 Amino Acid Open Reading Frame on Non Coding Strand 91 Figure #17: Amino Acid Sequence Alignment of Known Mn Superoxide Dismutases .... 104 X ABBREVIATIONS: ADP Adenosine diphosphate A G E Advanced glycation end products AMP Adenosine monophosphate ATP Adenosine triphosphate BlueScript A Col E l based plasmid containing genes for Ampicillin resistance, Beta-galactosidase, a MCS, and an M13 phage origin of replication. BSA Bovine serum albumin CR Caloric restriction Cu-Zn SOD Copper-Zinc containing superoxide dismutase DEPC Diethylpyrocarbonate DNA Deoxyribonucleic acid dsDNA double stranded DNA DTT Dithiothreitol EDRF Endothelium derived relaxing factor EDTA Ethylenediaminetetraacetic acid ETC Electron-Transport Chain F A D + or FADH2 Oxidized and reduced forms of Flavin adenine dinucleotide, respectively Fe SOD Iron containing superoxide dismutase GSH Reduced glutathione GPX Glutathione peroxidase HEPES N-2-Hydoxyethylpiperazine-N'-2-Ethanesulfonic acid Hi TE8 10 mM Tris HC1, 10 mM EDTA, pH 8.0 IPTG isopropylthiogalactoside Kb Kilobasepairs LB Luria-Bertani media LSP Life span potential xi M9 bacterial culture media M13-mpl8/19 Male specific + ssDNA virus of Escherichia coli, containing a multiple cloning site in the amino terminal region of the Beta-galactosidase gene alpha fragment with a galactose inducible promoter. MCS Multiple cloneing site Mn SOD Manganese containing superoxide dismutase MOPS 3-[N-Morpholino] propanesulfonic acid NAD+ Oxidized nicotinamide adenine dinucleotide NADH Reduced nicotinamide adenine dinucleotide NADP+ Oxidized nicotinamide adenine dinucleotide-phosphate NADPH Reduced nicotinamide adenine dinucleotide phosphate NTA Nitrilotriacetic Acid N Z C Y M bacterial culture medium NZY bacterial culture medium N Z Y M bacterial culture medium O/N overnight OAc Acetate •OH hydroxyl radical PAGE Polyacrylamide gel electrophoresis pBlueScript See BlueScript PBN N-tert-butyl-alpha-phenylnitrone pBR322 A Col E l based plasmid of Escherichia coli: contains genes for Ampicillin and Tetracycline resistance. (Saunders & Saunders, 1987.) PCR Polymerase Chain Reaction PEG Polyethylene glycol PFU Plaque forming units PNK Polynucleotide kinase Pq+2 Paraquat X l l RNA Ribonucleic acid ROS Reactive oxygen species RT Room Temperature sdH20 Sterile distiled water SDS Sodium Dodecyl Sulfate (Sodium Laryl Sulfate) SLG Superoxide dismutase Like Gene SM Tris, magnesium sulfate, gelatin, sodium chloride; Lambda phage dilution buffer SOB bacterial culture medium SOD Superoxide dismutase SSCP Single Stranded Conformational Polymorphism SSC Standard Saline Citrate (buffer) ssDNA single stranded DNA STETL Sodium chloride, Tris, EDTA, Triton X-100, Lysozyme TBE Tris Borate-EDTA TCA Tricarboxylic Acid TdT Terminal deoxynucleotidyl transferase TE8 10 mM Tris HC1, 1 mM EDTA, pH 8.0 TELT Triton X-100, EDTA, Lithium chloride, Tris TENT Tris, EDTA, NaCl, Triton X-100 T M G Tris, Magnesium sulfate, Gelatin; Lambda phage dilution buffer. Tris Tris (hydroxymethyl) amino methane UV Ultraviolet light wcDNA Whole cellular DNA wcRNA Whole cellular RNA X-gal 5-Bromo-4-chloro-3-indolyl-beta-D-galactoside Xll l ACKNOWLEDGMENTS: I would like to take this opportunity to express my gratitude to all the wonderful people who have guided and assisted me in this work: Gordon Tener, Peter Candido, Ivan Sadowski, Ian Gillam, Hiron Poon, and Don Jones. My special thanks to Ross MacGillivray, Dave Banfield, Helen Cote, Jeff Hewitt, Sandy Strumpfer, and all the other members of Ross' lab for their selfless provision of support, training, time, bench space, reagents, love, and insightful advice, without which none of this would have been possible. Thanks also to Rosy Redfield, Ye Te and my father, John William Carmichael, for their friendship, support, and much needed pokes, prods, and boots, judiciously applied, to get me moving in the right direction: something I owe the members of Ross' group a great deal of thanks for also. My special thanks also go to Don Sinclair and Hugh Brock for providing me with the Lambda GT10 cDNA Library used for isolation of the Drosophila cDNAs, for the opportunity to work in Hugh's lab under Don's direction plating the library, and to Don specifically for suggesting I contact Dr. Duttaroy. I would like to extend my special gratitude also to John Phillips, Atanu Duttaroy, and Arthur Hilliker for including my name on their paper. I feel it has been a privilege to work with and learn from all of you, and I wish you all the best for the future. I would be remiss in the extreme if I did not also mention my mother Dorthy Carmichael, as without her selfless and loving moral and financial support I would never have completed this manuscript or my degree. DEDICATION: I should like to dedicate this paper to those who have come before me, for the immeasurable gift of knowledge that makes all of what I have done possible, and fills the air of the future with ever greater possibilities. May we always show ourselves worthy of their sacrifices and achievements. Yours, Stuart Carmichael I INTRODUCTION: T H E A G I N G P R O B L E M C. O. Whitman (1894) once wrote, "Theory without fact is fantasy, but facts without theory is chaos". Until recently this quote was an apt description of the literature on aging. There were, and still are, many unrelated and currently unrelatable facts, as well as numerous theories with only the barest hints of fact to support them. Chaos has indeed reigned. In spite of this, aging research has made great strides in recent years. Certain aspects of cellular physiology and biochemistry have now presented more convincing credentials as candidates for causative agents of aging. Their separation from the incidental effects and subsequent results of aging has resulted in a more unified and comprehensive understanding. We still have a long way to go, but at least the direction is now clear. A UNIVERSAL PHENOMENON Cellular aging and senescence are a universal phenomenon in eukaryotic and possibly even prokaryotic organisms. Although rates differ between and within species, the intracellular effects of aging include phenomena common to all organisms. These common themes have led to the hypothesis that the causes of aging, in organisms as diverse as paramecia and Blue whales, are also common. Thus any useful theory of aging must encompass the universality of its effects, while still allowing for the large differences in rates which are observed. THEORIES OF AGING: GROUP-SPECIFIC AGING THEORIES Some theories have been put forward to explain various aspects of the aging process in different groups of plants and animals. These theories successfully explain the cause of death for certain organisms. Three such theories are: the programmed senescence theory, the wear and tear theory, and the accidental death theory. Introduction 2 The programmed senescence theory is a good explanation of aging for species such as cicadas, squid, salmon, bamboo, and century plant, in which senescence and death are closely coupled to reproduction, and ensue rapidly after it is completed. The cellular/genetic trigger involved in this phenomenon has not been investigated to my knowledge. The wear and tear theory holds that physical wearing out of body parts results in decline and death. Obvious examples of this theory are seen in elephants and some beetles, where the decline in tooth or mandible function results in malnutrition and eventual starvation. Another example is decline in wing structures of insects (butterflies, honey bees, etc.) where loss of flight ability results in death. Trees such as redwood and bristle cone pine seem to have indefinite life spans, where the cause of death is invariably drought, fire, wind storm, or predation. These trees follow the accidental death theory where death is invariably caused by external forces. Al l three of these theories work for specific species under their native conditions (for reviews see Finch 1990 and Schneider & Rowe, 1990). LIMITATIONS OF GROUP SPECIFIC THEORIES For most species however, old age and death are the result of a gradual decline in health and vitality for which there is no obvious explanation. These organisms include insects, mammals, birds, lizards, reptiles, amphibians, fish, plants, fungi, and others, and cover a wide variety of life histories and life spans. In addition, the previously mentioned theories for the aging and death of salmon, elephants, bamboo, and other organisms, only tell half the story: in all the animal cases mentioned, inhibition of the primary cause of death reveals it is simply preempting the more gradual decline found in other species, while in all the plant cases, seeming immortality is achieved by the continual generation of new tissue: none of the actively metabolizing, non-dividing cells in plants live more than 20-30 years. Theories put forward to explain this universal form of gradual decline do not have the same obvious examples or clear correlations found for the species specific Introduction 3 theories previously mentioned. As presently formulated, they rely on principles postulated as universal to all living things. These theories attempt to explain why all actively metabolizing cells that cease to divide, be they plant, animal, fungal, or bacterial, eventually grow old and die. UNIVERSAL AGING THEORIES Universal theories of aging can be divided into several categories according to the process or compound named as the cause of damage, and the target which is thought to be important in it. Those theories with universal applicability include the free radical theory, the glycation theory, the racemization theory, and the deamidation theory. Various branches of the free radical theory postulate that aging results from oxidative damage caused by transition metal catalysis or metabolically derived reactive oxygen species (ROS) acting on one or more of a variety of targets. These targets include nuclear DNA, mitochondrial DNA, mitochondrial membrane lipids, mitochondrial membrane proteins, plasma membrane proteins, or any of a host of other specific or general targets. The commonly cited damaging agents (i.e., ROS) include superoxide, hydrogen peroxide (H2O2), alkyl hydroperoxides, alkyl peroxyl radicals, hydroxyl radical (-OH), and also singlet oxygen and peroxynitrite. An example of this kind of theory is the protein oxidation theory postulated by Stadtman (1993). This theory postulates that aging results from oxidation of proteins by oxygen free radicals generated one of two ways: 1) metal catalyzed oxidation (MCO) by metals directly chelated to specific sites in proteins, as demonstrated in vitro for glutamine synthase, and 2) protein oxidation caused by electron transfers to oxygen by enzymes and electron carrier proteins which contain flavins, such as Xanthine oxidase, NADH/NADPH dehydrogenases, cytochrome p450 reductases, and quinone reductases. A specific example where protein oxidation could be of critical importance is the progressive inactivation of ribosomes in non dividing cells, which could result from a single oxidative reaction with a specific amino acid residue in one Introduction 4 of the numerous ribosomal proteins. The various free radical theories of aging are the focus of this work and will be discussed in greater detail later. For reviews see Bittles, (1992), Cutler, (1991), Dizdaroglu, (1992), Finch, (1990), Fleming et al. (1992), Fridovich (1989), Harman, (1992a), Harman, (1992b), Masoro et al. (1991), Pacifici & Davies, (1991), Pryor, (1986), Richter, (1992), Sohal & Brunk, (1992), Stadtman, (1993). The Glycation theory holds that aging is caused by glucose and other sugars reacting spontaneously with free amino groups present in lysine and arginine residues of proteins, and guanine, adenine and cytosine residues of DNA and RNA. These initially unstable reaction products,(Schiff base and Amadori rearrangement intermediates) are thought to progress to form more stable adducts known as A G E products (for Advanced Glycation End products). Glycation is known to progress as a function of age and serum sugar content; it affects erythrocyte membranes, extracellular matrix, and other extracellular and possibly intracellular biomolecules in man, rats, and other species. Its rate is affected by redox state, temperature, sugar type and conformation, and other poorly understood factors. While glycation levels have been shown to correlate with aging, they have not been demonstrated to play a causative role in senescence, and thus its significance is uncertain. For reviews see Masoro (1992), Schneider & Rowe (1990)(pl 16-127), and Finch (1990)(p400-406). A third theory with broad applicability is the racemization theory, which proposes that aging results from the gradual loss of stereo specific conformation in protein amino acid subunits. The rate at which amino acids undergo racemization is dependent on protein sequence and folding, pH, and the R group of the amino acid involved. The most rapid rates are associated with aspartate residues adjacent to glycine, and higher body temperatures. This theory is supported by circumstantial evidence, in that dextro aspartate and other dextro amino acids can be isolated from long lived matrix proteins in amounts increasing in proportion to the age of the organism. However there is no proven cause and effect relation between these Introduction 5 increases and organismal life span, and no explanation of why some animals and plants live so much longer than others. The rates of racemization should be slower in some of the shortest lived species like insects (low body temperature), and faster in longer lived organisms like man (high body temperature), but the corresponding relationship in life span potential is obviously missing. Similar arguments can be made for the deamidation theory. Reduced use of asparagine and glutamine in proteins of longer lived organisms tends to suggest that protein amide content is important in longer lived organisms (Finch 1990, pg. 408). A specific methylase has been described that "repairs" deamidated proteins, underlining the importance of the resulting Gin to Glu and Asn to Asp changes. This is not unexpected considering deamidation constitutes "random" addition of negative charges in proteins. For brief reviews of the racemization and deamidation theories, see Finch (1990)(406-409), Stadtman (1992), Stadtman etal. (1992). Most of these numerous theories for the causes of aging are not mutually exclusive. It is entirely possible that aging results from the interactions of two or even several of the effects covered by the various theories. A specific example of possible synergism is the glycation of erythrocyte Cu-Zn SOD (Finch, 1990 pg. 404), which may inactivate it, thus tying together the glycation and free radical theories of aging. Genetic experiments indicate that aging rates are subject to complex genetic control, (Hutchinson & Rose, 1991, and Rose, 1989-90) and many tradeoffs may be involved in controlling the various mechanisms suggested as causes for aging. The theory of aging by genetic mutations has its roots in studies on cancer. According to this theory, cells accumulate damage to their DNA during the life span of the organism, and when a critical component of the DNA or a specific level of damage is reached, the cell dies or loses its ability to divide or function correctly. The sources of DNA damage most often cited are free radicals, biochemicals, and natural background radiation. While this is an attractive theory, the evidence for it is somewhat contradictory. Cells and animals which are exposed to excess radiation at Introduction 6 various levels, with measurable amounts of DNA damage do not appear to age significantly faster than unexposed controls. (Finch, 1990, 430, and Rattan, 1989, 52). Perhaps the best arguments for this theory come from nuclear transplant experiments on ciliates, where cellular age was determined by the age of the DNA rather than the age of the cytoplasm (Finch, 1990, 125-26, Schneider & Rowe, 1990, 24-38). THEORIES SPECIFIC TO HIGHER ORGANISMS Still other theories, specific to complex organisms, hold that aging is the result of gradually evolving imbalances in the neuroendocrine system, loss of specific differentiated states in the cells of adult organisms, other specific degenerative events within cells, or result from a lack of further instructions needed to maintain organismal balance after the completion of the developmental program. All of these theories have some merit, and all have their proponents and detractors. An example of these is the autoimmune theory of aging. This theory postulates that aging results from loss of immunological self tolerance. Several autoimmune diseases probably result from immunological attack on long lived cell surface or extracellular matrix macro-molecules which have been altered by oxidation, glycation, racemization, or deamidation, and are no longer recognized as "self". Autoimmune ailments form a special case as they are not seen in all specimens or in all species: they are thus unlikely to constitute a universal cause of aging. AGING: STUDIES AND EXPERIMENTATION RATE OF DYING CURVES Studies of life span in different organism have resulted in a variety of different "rate of dying" curves. These come in three main types, parabolic, linear, and sigmoidal (see figure #1). Parabolic curves result when the rate of death for an organism is initially very high, but decreases continually as the organism approaches maturity. Linear die off results when the chances of dying remain constant throughout the life span of the organism. Sigmoidal curves are found for most species under ideal conditions: the risk of death starts off small, increases to reach Introduction 7 some maximum rate centered around some limiting age, usually very close to the mean life span of the species, and then declines again for those few individuals fortunate enough to live to a ripe old age. Thus the most significant value for life span potential (LSP) for most species is the mean life span under ideal conditions, as this is generally the point of maximum rate of dying due to old age (Finch, 1990, 25). Figure #1: Rate of Dying Curves The vertical axis is number of organisms still alive: the horizontal axis is organism's age. Parabolic Rate of Dying Numbers Alive Organism's Age Linear Rate of Dying Numbers Alive Organism's Age Sigmoidal Rate of Dying Numbers Alive Organism's Age Introduction 8 INTERSPECIES CORRELATES OF LIFE SPANS Numerous characteristics at the organismal, cellular, and biochemical levels, correlate with life spans between species. At the level of physical structure in animals, body size, brain to body ratio, low heart rate, low body temperature and low metabolic rate all have positive correlations with life span (Turturro & Hart, 1991). At the cellular and biochemical level, DNA content (Rattan 1989), levels and amounts of antioxidants such as urea, vitamin A and E, and ceruloplasmin, (Cutler 1991) and levels of enzymes such as Cu-Zn SOD and Mn SOD (Tolmasoff et al. 1980), as well as repair rates by various enzyme systems such as DNA repair (Turturro & Hart 1991, Cutler, 1991), all increase in longer lived organisms. Longer lived organisms also tend to have lower blood sugar levels (Finch 1990) and generate reduced amounts of reactive oxygen species (Sohal & Brunk 1992) compared to shorter lived species. These trends were the basis for many of the original theories in aging, and they gave many of the clues which direct aging research today. (Finch, 1990, 248-295, and Turturro & Hart, 1991) CE L L B IOLOGY O F AGING Many biochemical characteristics of cells and tissues change as an organism ages. Some of these changes include reductions in the number, volume, and integrity of the mitochondria (see Mutation Research, vol. 275, for reviews), a reduced respiration rate (Schneider & Rowe, 1992, 391-2), reduced ATP/ADP ratios (Pall, 1990), reduced levels and transport of adenine nucleotides (Kim, 1988), reduced mitochondrial transport of phosphate (Bittles, 1992), reduced protein degradation (Finch, 1990, 398), reductions in the rate of protein synthesis (cytosolic and mitochondrial) (Bailey & Webster, 1984), reduced or absent cell division (in some tissues) (Cutler, 1992, and Martin, 1992), accumulation of lipofuscin (Finch 1990, 409-10), increased H2O2 and O2- output by the mitochondria (Sohal & Brunk, Introduction 9 1992, Sohal 1991), and reduced mitochondrial efficiency with some carbon sources, notably glutamate (Vitorica et al., 1985). PROCEDURES WHICH EXTEND LIFE SPAN. Numerous attempts have been made, both in pre- and post-scientific times, to extend the life spans of man and animals. These attempts, both scientific and mystical, have been largely unsuccessful with only a few modest, and mostly recent, exceptions. Much of the evidence for the various theories of aging is derived from experimental attempts to extend life span, so they are briefly covered here to provide background information. CALORIC RESTRICTION Possibly the first treatment ever found which significantly increased the life span of any organism was partial starvation or "caloric restriction" (CR). This treatment can extend the life span of rats by up to about 30% for both mean and maximum (Masoro et al. 1991 pg. 345). Caloric restriction has been extensively studied in rats and mice, and numerous data are available on the physiological and biochemical effects of CR. (for examples see Laganiere & Yu, 1989a,b for reviews see Masoro et al. 1991, Pieri, 1991, and Turturro & Hart, 1991; in flies see Zwaan et al., 1991). The overall effects in rats are as follows. Reduced body size and mass, but no apparent reduction in the number of the cells in the animal. A slight increase in basal metabolic rate, (needed to partially compensate for increased heat loss due to altered surface area to volume ratio) a net decrease in oxygen consumption, lower resting body temperature, and lower serum glucose levels. (Schneider & Reed, 1985, Masoro, 1992) Caloric restriction may be a hidden effect of numerous other treatments that have been found to extend life span, such as larval crowding in Drosophila cultures, various endocrine surgical alterations and hormone treatments, and other experimental conditions. It is important to avoid confusing procedures that increase LSP in of themselves, and those that operate by causing coincidental CR (Schneider & Reed, 1985). It is noteworthy in this context to mention that large cell Introduction 10 size correlates with increased likelihood of senescent phenotype in studies of the Hayflick phenomenon: the inability of primary fibroblast cells to divide indefinitely in long term culture (Finch, 1990, Rattan, 1989). Every species has a characteristic fibroblast cell division limit, and this number is strongly correlated to species life span (Cutler, 1991, Goldstein et al. 1989). As cells which are larger just after cell division are more likely to be blocked for subsequent cell divisions than small cells (Schneider & Rowe, 1990, 135), and caloric restriction causes an overall reduction in cell size (Masoro, 1991), increased longevity in CR may be due to increased cell division potential in the smaller cells, resulting in increased cell replacement. Other possible effects of CR include i) reduced saturation of ETC electron carriers resulting in a lifetime reduction in oxygen radical generation, or ii) reduced cytoplasm to DNA ratio, resulting in slower accumulation of DNA damage (less metabolic activity/g of DNA). A N T I O X I D A N T D R U G S Numerous natural and artificial antioxidant compounds have been tested for anti-aging effects in rats and mice. These include vitamins A, , C, and E, carotenoids, butylated hydroxy toluene (Goldstein et al. 1989), 2-mercaptoethylamine (Goldstein et al. 1989, Finch, 1990, 523) Diethyl-hydroxylamine, ethoxyquin, 2-ethyl-6- methyl-3- hydroxypyridine, (Finch, 1990, 523), propyl gallate (Fleming et al., 1992) and others (for example, Xiao et al. 1993). Several of these compounds extend the life spans of rats and/or mice, and some also result in reductions in tumor incidence or atherogenesis. Carney et al. (1991) tried intra-peritoneal injection of N-tert-butyl-alpha-phenylnitrone (PBN) in gerbils over a shorter period of time (2 weeks). This resulted in increased glutamine synthase activity and reduced levels of oxidatively damaged proteins in the brains of older animals, as well as improving their performance in maze tests. These results indicate PBN may well act to extend LSP in rodents, and this could be tested by longer term injection studies. < > compared numerous studies on life span extension with a variety of different antioxidant drugs, and found that certain classes of antioxidants were Introduction 11 generally effective, while other classes were always ineffective: specifically, efficient free radical trapping agents and sulfhydryl reagents were generally effective, while reducing compounds, non radical trapping antioxidants, and quinones were ineffective. OTHER DRUGS Deprenyl: In addition to antioxidants, a number of other drugs have been tested for life span extending properties in experimental animals. One of the premier examples of this is the highly specific xanthine oxidase inhibitor, deprenyl (Knoll, 1988, Knoll et al., 1989, Milgram et al., 1990). Deprenyl increased mean life span in male rats by 30% when treated three times per week starting from the end of their second year of life (already senescent). This drug could be listed under antioxidant drugs, as xanthine oxidase may make substantial amounts of superoxide in some tissues. A true antiaging drug, deprenyl increased health and vitality, restored sexual potency, and renewed learning capacity in the treated rats, in addition to increasing their mean life span well beyond its previous maximum. Prostaglandin B oligomers: A second class of anti aging drugs which have recently been discovered are the phospholipase A2 (PLA2) inhibitors. The reason(s) for these drugs acting as anti aging agents is not well understood. Regelson and Franson (1991) report that in studies done by Sohal's group, oligomerised prostaglandin B (PGBx), a potent PLA2 inhibitor, extended the life span of house flies by almost 30%. Previous authors cited by them report that PGBx stabilizes mitochondrial membranes, and theoretical considerations suggest it should reduce the biosynthesis of free radicals by reducing the availability of free arachidonic acid for thromboxin and leukotriene metabolism. Prostaglandin B oligomers are also antioxidants, and this may explain a significant part of their life span enhancement properties. Numerous other beneficial effects have been reported for this compound, and it will be interesting to see what the actual biochemical basis of these effects is. Paraquat: Paraquat is a viologen used extensively in aging studies. This compound is used to increase oxidative stress in order to test the effectiveness of various other Introduction 12 protective treatments. It catalyses single electron transfers from the ETC to molecular oxygen, thus greatly increasing O2- production. For a more in depth look at the chemistry of viologens, see Pasi, 1978. M A N I P U L A T I O N S O F T H E N E U R O E N D O C R I N E S Y S T E M Several authors over the years have suggested that a major cause of degeneration in old age is a gradual loss of proper balance in the complex network of hormones and neurotransmitters which control or modulate most body, organ, and cellular functions from conception to adulthood. Aging and stress have very similar effects on the neuroendocrine system (Frolkis 1992) and anecdotal evidence suggests that prolonged stress accelerates the aging process. Melatonin One candidate for causing these imbalances is a reduction in innervation and functional capacity of the pineal gland, with subsequent reductions in the levels of melatonin and in the ratio of melatonin to serotonin. Experimental night feeding of mice with melatonin resulted in a 20 % increase in life span when started at older ages (18-20 months), while grafting pineal glands from young syngenic mice into the thymus gland of old recipients resulted in life span increases of over 30 % (Pierpaoli et al., 1991). For Reviews see Reiter (1992) and Grad & Rozencwaig (1993). Growth Hormone A second candidate is growth hormone (GH); normally secreted in surges or pulses, both the frequency and amplitude of pulsatile secretion decline in the later years of life in rats and man. Exogenous supplementation of GH results in increased protein synthesis in the skeletal muscles, and increases in the levels of insulin like growth factor I (ILG-I). This is similar to the effects of exercise in the elderly, which increase GH levels 3 to 7 fold, and results in an array of beneficial effects, such as increases in bone mass, vitamin D3, and somatomedin (Corpas et al., 1993). Old mice implanted with GH3 cells, a tumor cell line which secretes GH, showed rejuvenation of the thymus and increases in prolactin and insulin like growth factor. Introduction 13 Substantial improvements in immunological functions occurred. These effects were not observed for GH alone and may be a result of some other aspect of the GH3 cell line or the host reaction to it. For a review, see Corpas et al. (1993) and Finch (1990) (L-dopa and GH, 541, and GH3 cell engraftment, 551, and skeletal muscle, 383). I did not see any studies on the effects of exogenous GH on life span, possibly because the use of molecular biology techniques to produce it in inexpensive purified form is a recent innovation. GENETIC ALTERATIONS In the recent past genetic selection of Drosophila melanogaster has produced fly stocks with substantial increases in their life spans (Arking & Wells, 1990, Hutchinson et al., 1991, Hutchinson & Rose, 1991, Rose et al., 1992, Rose, 1989-90). Study of these stocks has resulted in identification of a few genes which are candidates for longevity determinants. Some authors have taken advantage of recent advances in molecular genetic techniques to create specific fly lines with increased expression of these genes. These studies determine whether these genes are involved in aging, or if their increased expression in long lived flies is just incidental. Genes so far identified as possible longevity determinants in long lived flies include Superoxide dismutases (Bartosz et al. 1979) and catalase (Fleming et al., 1992), and may include genes involved in lipid metabolism: longer lived fly strains have much higher levels of stored lipid (Rose, 1989-90). Transformation with Elongation Factor 1 Alpha One of the salient observations in older cells is a reduction in the rate of protein biosynthesis compared to younger cells (Schneider & Rowe, 1990, 97-107, Bailey & Webster, 1984). In an attempt to reverse this decline and demonstrate its importance in aging, Shepherd et al. (1989) used P element mediated transformation to add copies of the elongation factor 1 alpha (EFla) gene, under control of a heatshock promoter, (hsp70) to Drosophila embryos. Elongation factor 1 alpha mediates a crucial step in protein synthesis, and if increased EF la facilitated protein biosynthesis, life span might Introduction 14 be increased. The resulting flies had mean life spans 18% longer than untransformed controls at 25 °C, and this discrepancy increased to 41% at 29.5 °C where expression from the heatshock promoter is presumably increased. These data indicate that reductions in EF la synthesis are important in determining LSP, and that increased EF la transcription does increase LSP, at least in flies. As reduced protein biosynthesis is a common feature of aging cells in all organisms, it is possible this alteration will extend the life spans of most species in which it is undertaken. Transformation with Cu-Zn SOD Three groups have reported the transformation of Drosophila with Cu-Zn SOD: one with the Drosophila gene (Seto et al. 1990), the second with the bovine gene (Reveillaud, 1991), and the third with the Drosophila genes for both Cu-Zn SOD and catalase (Orr & Sohal, 1994). In all cases longevity increased in some of the transformed stocks, although in the case of Seto et al. (1990), no statistical analysis was conducted, and the increase was designated as insignificant. This result is not completely unexpected, as genetic selection in flies for enhanced longevity indicates that it is controlled by a complex system of genes (Rose 1989-90), and thus the alteration of one gene should not make a large difference in the overall life span. By far the greatest increase in longevity (up to 1/3 increase over controls) was reported for the Cu-Zn SOD plus catalase double transformants, even though transformation with catalase by itself had no effect (Orr & Sohal, 1992). One study on mouse tissue culture cells indicates that resistance to Paraquat (Pq+2), (and presumably to oxidative stress) is proportional to glutathione peroxidase but not to Cu-Zn SOD. Thus glutathione peroxidase may be a rate limiting enzyme in antioxidant defenses in mammalian cells (Kelner & Bagnell, 1990). As flies lack glutathione peroxidase entirely (Orr & Sohal, 1992), and catalase is absent from the mitochondrial matrix of higher eukaryotes (Rikans et al., 1992), mitochondrial matrix H2O2 and hydroxyl radical damage is probably the major cause of their short life spans. Introduction 15 CU-ZN SOD AND DOWNS SYNDROME Several lines of evidence suggest that overexpression of any SOD, (Cu-Zn SOD, Fe SOD or Mn SOD) may be toxic to mammalian and prokaryotic cells. Increases in Fe SOD (Scott et al., 1987), and Mn SOD (Bloch & Ausubel, 1986) in Escherichia coli resulted in increased sensitivity to Pq+2, and in tracing the lesion, Scott et al. (1987) found that reduced glutathione was depleted more rapidly and to a greater extent in the transformed bacteria. Mammalian tissue culture cells transformed to overexpress Mn SOD or Cu-Zn SOD have increased resistance to Pq+2, but also show increased levels of lipofuscin (Krall et al., 1988, Elroy-Stein et al., 1986, St. Clair et al., 1991). P element mediated transformation in Drosophila melanogaster with Cu-Zn SOD never resulted in greater than about 1.6x wild type levels: higher levels caused pupae to accumulate lipofuscin and die during eclosion (Fleming et al., 1992). Mice transgenic for a modified human Cu-Zn SOD gene driven by the actin promoter had increased Cu-Zn SOD activity, (1.9x normal) but also experienced increased lipid peroxidation and Mn SOD expression, indicating overexpression of Cu-Zn SOD resulted in increased oxidative stress. The mice developed lesions of the neuromuscular junctions similar to those seen in Downs syndrome and in normal aging (Ceballos-Picot et al., 1992a). All of these papers have suggested that the damage and decrease in reduced glutathione levels is the result of increased levels of H2O2. Liochev & Fridovich (1991) suggested increased oxygen sensitivity in bacteria results from reduced expression of other protective genes normally induced by superoxide, although this is not seen in mammalian cells (Ceballos-Picot et al 1992b). Two other possible effects of increased Cu-Zn SOD were not discussed. One concerns an interesting paper by Yim et al. (1993), who found that Cu-Zn SOD is able to catalyze the formation of OH. from H2O2 by interaction with other physiological substrates: if this has biological relevance, then increased levels of Cu-Zn SOD would have the extremely deleterious effect of increasing the rate of formation of this extremely reactive free radical. So far, Mn SOD and Fe SOD have not been tested for Introduction 16 this activity. Fe SOD is inactivated by H2O2, probably by hydroxyl radical formation at the active site (Beyer & Fridovich, 1987). External hydroxyl radical formation by Cu-Zn SOD could explain all of the above mentioned deleterious changes associated with increased levels of Cu-Zn SOD, and may be a factor in the pathologies seen in Downs syndrome and in normal aging. Distinct isoforms of Cu-Zn SOD seen by Mavelli et al. (1983) may be the result of this activity. The second possibility concerns the activity of O2- as a modulator of gene expression: in addition to its activity in turning on stress response genes, superoxide may act as an intra- and extra-cellular second messenger and mitogenic signal in normal metabolism in several diverse cell lines. It has already been demonstrated that NO, or something closely related to it (Murphy & Sies 1991), is the endothelium derived relaxing factor (EDRF) sought after for so many years by doctors as a treatment in arterial diseases. It is now postulated that CO, H2O2, O2-, and even hydroxyl radical (Nagy et al., 1993) are involved in sending signals between and within cells (Allen & Balin 1989). Two recent papers (Prasad et al., 1990, Church et al., 1993) have clearly demonstrated that O2- is a mitogen for melanoma cells, (but not smooth muscle, Boscoboinik et al., 1992), and deprivation of it results in terminal differentiation for these cells. This makes perfect sense if one thinks about the role of normal melanocytes in protecting the skin from ultraviolet light. One of the first biochemical indications of a need for melanocyte function is likely to be the production of light induced free radicals. Other cell lines may also rely on O2- as a mitogen. Melanocytes are a neural crest derived cell lineage, so low levels of O2- may also be involved in Downs syndrome mental retardation: inhibition of a O2- mediated neural developmental signal by high levels of Cu-Zn SOD could result in altered or attenuated neural development. EVALUATION AND SUMMARY OF THE VARIOUS THEORIES AND RESEARCH ON AGING The above experiments and observations on life span extension help us to identify some significant features of the aging process: Introduction 17 Racemization: The accumulation of dextro amino acids in long lived proteins, while it may play some roll in the aging process, is probably not of great significance. The diversity of life spans among organisms and the absence of clear correlations between senescence rates and the rate of dextro amino acid accumulation indicates they probably play a minor part in the generation of aging phenomena. Deamidation: Deamidation does occur, and longer lived proteins tend to have a reduced amide content (Finch, 1990, pg. 407). Scant mention of this theory appears in the literature. Short lived species should deamidate at rates similar to longer lived species, and no correlation has been reported so far between protein amide content and LSP. Specific methylases which "repair" deamidation products by O-methylation and the theoretical implications of addition of a negative charge to the protein structure indicate that deamidation is a significant event for the cell, at least in some cases. Once again however, there is no clear correlation between species LSP, protein amide content and body temperature, so this theory is also of dubious significance. This theory cannot be dismissed as yet, but so far it lacks any substantial evidence (Finch 1990, 408-409). In both of the above cases the increase in species life span which results from CR is the reverse of what would be predicted if these reactions were significant causes of aging. Caloric restriction should reduce the rate of protein turnover, thus accentuating the accumulation of dextro and deamidated amino acids. Glycation: The reduced sugar levels seen in longer lived organisms and in CR treatments indicate that AGE products may be of some importance in the aging process. However, the modest reductions in LSP in treated diabetics (who still suffer from substantial increases in lifetime exposures to sugar), and the differences in the effects of diabetes versus aging lead one to the conclusion that this also is a minor player in the overall scheme of things. Introduction 18 This leaves only the free radical theories among the mainstream "universal cause" aging theories, and evidence for the involvement of reactive oxygen species is now quite substantial. THE FREE RADICAL THEORY OF AGING REVISITED The basis of the free radical theory of aging is the idea that metabolically derived oxygen free radicals such as O2- and .OH, and reactive oxygen species such as H2O2 and singlet oxygen, react spontaneously with biomolecules to cause the degenerative damage which results in aging. Evidence for the free radical theory of aging includes numerous circumstantial and experimental data: correlations between increased antioxidant defenses and increasing LSP (Cutler 1991, 18), LSP and DNA content (the more "junk" DNA, the less chance the cell will loose a critical gene to a given oxidative event. (Rattan 1989)), reduced basal generation of ROS in longer lived organisms (Sohal 1991, Sohal & Brunk 1992, Sohal & Orr 1992, Sohal et al. 1993), the effects of caloric restriction, (lower metabolic rate per gram of DNA, reduced body temperature) (Masoro et al. 1991), the efficacy of dietary antioxidants (Vina et al. 1992, Deucher 1992, Fleming et al. 1992, Finch 1990, 523, 284-288), the activity of deprenyl (Knoll 1988), and experiments with increased oxygen in tissue culture (Sohal et al. 1989), all lead to the conclusion that ROS are major players in the aging process. Some of the various questions which then evolve are: #1 "What free radical species are involved?", #2 "Which parts of the cell generate these reactive oxygen species?", #3 "How does the cell protect itself from free radical damage?", #4 "What are the important targets of free radical/ROS damage?", and #5 "What is/are the rate limiting step(s) in this protection for various experimental species and for man?". TYPES OF METABOLIC FREE RADICALS (#1) The best suspect for question #1 is metabolically derived reactive oxygen species, particularly superoxide, hydrogen peroxide, and hydroxyl radical. Superoxide Introduction 19 is produced in normal basal metabolism, and accounts for about 1-2 % of total oxygen consumption in living tissues (Richter 1992). While in most instances this radical is relatively unreactive in aqueous solutions, in its protonated form (pKa about 4.7, Fridovich 1989) it can enter the bilayer to react with lipids, and in solution it is able to react with transition metal ions, hydrogen peroxide (Fridovich 1989), or nitric oxide (Beckman 1990) to produce the hydroxyl radical (OH.). Transition metals, nitric oxide, and hydrogen peroxide are all found in normal animal tissues. The hydroxyl radical is an extremely reactive species: it will attack any biomolecule it comes in contact with, including sugars, proteins (Stadtman 1992, Stadtman et al. 1992), lipids and nucleic acids (Simic 1992, Pacifici & Davies 1991). It has been demonstrated to react in vitro in several ways with each of the nucleic acid bases and the ribose sugar ring, resulting in a plethora of products, many of which are seen in vivo (Halliwell & Aruoma, 1991). Thus accumulating damage resulting from metabolically produced ROS has long been seen as a possible causative agent in aging (Harman 1956). SOURCES OF METABOLIC FREE RADICALS (#2) The major metabolic source of superoxide and H2O2 is the mitochondria (Sohal & Brunk 1992), with additional contributions from peroxisomes and cytosolic enzyme systems (Fridovich 1983). Membranes have a demonstrated ability to contain ROS and protect proteins located on the opposite side from oxidative damage (Hunt & Dean 1989). Thus it is no surprise the two largest producers of superoxide are membrane bound organelles with co-localized superoxide dismutases: Mn SOD in the mitochondria, and Cu-Zn SOD in the peroxisomes (Dhaunsi et al., 1992). Superoxide radicals produced within peroxisomes are rapidly reduced by Cu-Zn SOD to hydrogen peroxide, which is subsequently reduced to water and oxygen by catalase or peroxidase. In the mitochondria, Mn SOD reduces superoxide to H2O2, and this is reduced to water by glutathione peroxidase: this system requires a constant supply of reduced glutathione, and depletion of this compound is a possible problem under increased oxygen stress, or when levels of NADPH are low. Mitochondrial Introduction 20 superoxide radicals are generated primarily by single electron transfers to oxygen by the electron transport chain (Miquel et al. 1992). Experiments have determined that the primary "leakage" in the electron transport chain (ETC) occurs by direct oxidation of ubisemiquinone (or electron carriers above it) by molecular oxygen to produce superoxide and regenerate ubiquinone (Sohal & Brunk 1992). This side reaction of the ETC seems to be accelerated by aging, as mitochondrial output of superoxide and hydrogen peroxide rise sharply with age in every species tested to date (Sohal & Brunk 1992). In vivo evidence for the proximal location of electron leakage from the electron transport chain has been obtained in studies of yeast deficient in Mn SOD in conditions of high oxygen. Mn SOD deficient yeast cannot grow in 95% oxygen. However, when the electron transport chain is blocked above the level of ubiquinone, (Rho 0 state) they grow "normally"; even in 95% oxygen (Guidot et al. 1993). FREE RADICAL DEFENSES (#3) A further indication of the biological importance of free radicals is the presence in biological systems of numerous enzymes and small molecules which act to inhibit the formation of, reduce the concentrations of, or repair the damage caused by, various types of free radicals and other ROS. S M A L L M O L E C U L E S Biological systems contain numerous small molecules which act as free radical trapping agents or chain terminators, and reducing agents. These include uric acid in the lungs (Peden et al., 1990) saliva, and blood (reviewed by Becker, 1993), bilirubin in the blood and bile, ubiquinone, alpha tocopherol, and retinal in membranes (ubiquinone may be especially important in bacterial and mitochondria membranes where tocopherol may be absent), ascorbate and GSH in the blood and extracellular fluids, carnosine in brain and muscle, and ascorbate and glutathione in the cytoplasm (Ames & Gold 1991). Blood glutathione levels may vary considerably from species to species: levels in man are 1-2 uM, while in rat they reach 25 uM. Of this, greater than 95% is in the reduced form (Dolphin et al., 1989 pg. 355-6). This is backwards Introduction 21 from what one would expect from LSP, and this indicates differences in aging protective mechanisms in man and rat. CH E L A T I O N O F METAL IONS Transition metal ions such as vanadium, nickel, copper, iron, and manganese can catalyse hydroxyl radical formation (reaction 1) and NADPH depletion (reaction 2) in biological systems (Fridovich 1989, Dizdaroglu 1992). This chemistry is especially relevant to cellular free radical damage because these metals are often bound to polyanionic surfaces such as DNA and lipid membranes, so that the reactive species are produced in intimate contact with vulnerable cellular components. Reaction 2 gains special significance when we consider the requirement for NADPH in the regeneration of reduced GSH in the mitochondrial matrix: the same GSH required for reduction of H2O2 by GPX. Reactions 1 and 2 are inhibited when the metals are rendered inaccessible by chelation. In most biological systems, proteins made specifically to trap metal ions are present. These include metal specific proteins such as transferrin and lactoferrin, and nonspecific metal trapping proteins such as metalothionines. Some chemical chelating agents actually enhance the deleterious effects of iron (e.g. Fe-NTA, Fe-EDTA), although with other metals they are generally beneficial (Dizdaroglu 1992), and some chelating agents can reduce hydroxyl radical generation by iron (some examples include desferrioxamine, desferrithiocin, and phenanthroline, Halliwell & Aruoma 1991, also Desferal, a,a'-bipyridyl, and diethylenetriamine pentaacetic acid (pentenic acid) Beyer et al. 1991). Even traces of iron or copper can cause DNA fragmentation when incubated with H2O2 and superoxide in vitro (Burkitt et al, 1994). This highlights the threat these metals pose to the cell. Evidence for this kind of chemistry can also be demonstrated in vivo (Beyer et al. 1991). Introduction 22 Table #1: Reactions of superoxide. Reaction 1: Metal Catalyzed Formation of Hydroxyl Radical Fe(III) + 0 2 " < —> Fe(II) + 0 2 Fe(II) + H O O H < - - > Fe(I)-OOH + H+ Fe(I)-OOH < — > Fe(II)=O .+ OH" Fe(II)=0 + H + < —> Fe(III)-OH Fe(III)-OH — > Fe(III) + O H ' Reaction 2: Metal Catalyzed Oxidation of NADPH and NADH Mn(II) + 0 2 - < — > Mn(I)-00 Mn(I)-00 + N A D P H <---> Mn(I)-OOH + N A D P -Mn(I)-OOH + H + < — > Mn(II) + H O O H NADP- + 0 2 < - - > NADP+ + 0 2 " S U P E R O X I D E D I S M U T A S E S Another method of reducing the damage caused by ROS is by enzymatic dismutation of the reactive species. Superoxide dismutase is the first line of defense in these systems, as superoxide is the first reactive oxygen species commonly produced. Superoxide dismutases are metaloenzymes of three types from two different evolutionary systems: the Cu-Zn SODs which contain one atom each of copper and zinc per subunit, the holoenzyme being either a homo dimer or a homo tetramer, and the Fe SODs and M n SODs, which are structurally related homo tetrameric enzymes with one metal ion of iron or manganese per subunit. Some of these enzymes have the novel ability to function at comparable activities with either iron or manganese, depending on metal availability. The most versatile SOD discovered so far is the manganese-iron-copper SOD isolated from Propionibacterium shermanii, which has now been show to function with any one of these three different metals at the active site in vivo (Meier et al. 1994). It is structurally closely related to the other Fe and M n SODs. The majority of these enzymes have tight specificity for a particular metal ligand: thus control of specificity and the lack of it in SODs like the one from P. shermanii are an area of active interest in research. Introduction 23 P E R O X I D A S E S & C A T A L A S E Peroxidases, NADPH peroxidases, and glutathione peroxidases reduce organic hydroperoxides and H2O2 to less reactive forms (Flohe, 1988). Catalase reacts specifically with H2O2 (Beyer & Fridovich, 1988), and one report indicates that Cu-Zn superoxide dismutase may act on peroxynitrite to reduce its toxicity as well (Murphy & Sies 1991). The importance of such protection is confused by studies like those of Lopez-Torres et al. (1993) who have demonstrated increased LSP in frogs by suppression of catalase activity. However, most of the evidence directly supports the idea that catalase/peroxidase activity is essential to normal LSP and antioxidant defense. Lopez-Torres attributes increased LSP in catalase depleted frogs to the up-regulation by hydrogen peroxide of other antioxidant defense systems. DAMAGE RE P A I R ME C H A N I S M S : Numerous enzymes and systems in cells are designed to overcome the effects of oxidative damage to host macromolecules. These include protein degradation systems (Pacifici & Davies 1991, Schneider & Rowe 1990, Stadtman 1992, Stadtman et al. 1992), DNA repair mechanisms (Pacifici & Davies 1991, Turturro & Hart 1991, Rattan 1989), lipid turnover by phospholipases (Pacifici & Davies 1991), and the repair activities of glutathione peroxidase and other peroxidases (Flohe, 1988, 663). The activities of these enzymes are designed to ensure that the products of oxidative damage do not build up to toxic levels within the cell. Rapid rate of mitochondrial turnover is probably a direct response to high levels of oxidative damage occurring in them. The shorter life spans of some organisms may result from a slower rate of mitochondrial turnover and/or more rapid damage accumulation in them. TARGETS OF OXIDATIVE DAMAGE (#4) Probably the point of greatest speculation and contention in aging research is the identity of the targets of degenerative changes which results in cellular senescence. Numerous age related changes have been documented over the years, but the consequential importance of any given alteration has rarely been established. Prime Introduction 24 candidates include genomic DNA, Ribosomes, mitochondrial DNA, and the lipids and proteins of the mitochondrial inner membrane. Because the mitochondria are the predominant source of oxygen radicals within the cell, they are an attractive candidate for the role of "primary target" in aging. Many of the changes associated with aging can be traced to defects in the mitochondria. These include reduced energy metabolism (Schneider & Rowe 1990, 391-392), declines in ATP/ADP ratios (Pall 1990) , and morphological changes in the mitochondria, such as reduced number of mitochondria, reductions in the number and integrity of the cristae, alterations in morphology (Bittles 1992), increased free radical output (Sohal & Brunk 1992, Sohal 1991) , production of lipofuscin (glycation and, Finch 1990 400-405, ascorbate and, Yin & Brunk 1991, Cu-Zn SOD activity and, Ceballos-Picot et al. 1992a, free radicals and, Harman 1992), reduced protein synthesis (Bailey & Webster 1984) and a large number of damaged DNA bases: steady state levels of base damage are about 16x higher in mitochondrial DNA as compared to genomic DNA (Richter 1992). Damage to the mitochondrial inner membrane results in numerous changes, these include alterations in fluidity and composition: an increased content of longer chain saturated fatty acids, increases in cholesterol (almost absent in young mitochondria) (Kim et al. 1988), a pronounced increase in thermal transition temperature, (Pieri 1991, 356), declines in cardiolipin content (Bittles 1992), and reduced membrane containment of monovalent cations such as potassium (Von Zglinicki 1987). Old mitochondria maintain their pH gradient, but their electrochemical gradient declines, probably due to leakage of potassium (Von Zglinicki 1987). This is a further indication of lost membrane integrity. It may be that the loss of the electrochemical gradient is the final disaster for the cell, because this drastically reduces the ability of the mitochondria to import cytosolic proteins (Hard et al. 1989, Maduke & Roise 1993, Glick et al. 1992), and thus the generation of new, viable mitochondria is curtailed. As mitochondrial turnover rates in healthy cells run at about 10% per day (Holmes et al. 1992), the loss of protein import capacity should result in rapid depletion of the cells mitochondrial Introduction 25 population, as is seen (Bittles 1992). The alteration of membrane composition and character in old mitochondria may well be responsible for the increase in superoxide and hydrogen peroxide production (Sohal 1991, Sohal & Brunk 1992), as it would alter the ability of the membrane to dissolve both oxygen and ubisemiquinone, and might thus be responsible for the increased reactivity between them in old mitochondria. For numerous reviews of mitochondrial involvement in aging, see Bittles, 1992, Richter, 1992, Linnane et al., 1992 or other articles in volume 275 of Mutation Research. A great many of the changes seen with age directly involve or can be traced to the mitochondria. Things like reduced respiration, declines in cell division, decreased thermal regulation and loss of vitality can all be traced to reduced mitochondrial size, numbers, substrate utilization, and integrity. Thus it is possible that many or even all age related declines result from imperfect protection of the mitochondria by antioxidant defenses. In yeast (Guidot et al. 1993) and animal cell cultures, (Schneider & Rowe, 1990 ?) when conditions are arranged so that only anaerobic metabolism takes place, extensive protection from oxidative stress is realized, even in conditions of high oxygen. Mitochondrial involvement is also indicated in studies by Guidot et al. (1993) in Mn SOD deficient yeast: normal growth in 95 % oxygen was only possible when the cells were completely blocked in electron transport (Rho 0 state). Blocking the ETC at cytochrome c had no effect. RATE LIMITING STEPS (#5) With all of the targets for oxidative damage which can be demonstrated, it is difficult to distinguish which target or combination of targets is rate limiting in the aging process. Genomic and mitochondrial DNA, membrane lipids, long lived cellular RNA's, ribosomes, electron transport proteins, proteins involved in protection or repair of cellular components, or specific combinations of them, could all play roles in determining the aging process. In order to determine which process is rate limiting, it is essential to know what the key target(s) are. One likely target group is the lipids and proteins of the mitochondrial inner membrane. Introduction 26 MITOCHONDRIAL DEGENERATION: A HYPOTHESIS From the forgoing discussion, we can now begin to assemble a rough scenario for the progression of aging in the mitochondria. Initially, the inner membrane is almost devoid of cholesterol, longer chain fatty acids, and oxidatively damaged proteins and lipids: it is also high in cardiolipin. Once the cell ceases to divide, a prolonged period of gradual degeneration sets in as protein synthesis and mitochondrial turnover rates decline: cholesterol, (possibly due to inactivation of ribosomes and/or changes in nuclear histones) longer chain saturated fatty acids, and oxidative damage start to accumulate, and cardiolipin levels drop. As these changes occur, the character of the mitochondrial inner membrane changes, and the membrane solubility of oxygen, ubiquinone and ETC proteins changes, allowing greater leakage of electrons from the ETC to oxygen. Respiration rates are reduced by the slowing of adenosine nucleotide exchange and phosphate transport due to the increased rigidity of the mitochondrial membrane. Reduced electron flow through the ETC results in increases in the electron saturation levels of the ETC electron carriers resulting in further increases in adventitious oxidation by molecular oxygen. The increases in ROS result in accelerated rates of membrane damage and continued declines in membrane integrity. Reduced rates of ATP synthesis result in declines in other protective mechanisms, and further reductions in protein synthesis. The drops in protein synthesis result in further reductions in mitochondrial turnover, and deterioration of the mitochondria continues. When the rate of damage reaches critical levels, the osmotic integrity of the inner membrane is lost, and the mitochondria shrink and lose the ability to use some carbon sources: deterioration reaches the point where delta psi is lost or reduced, and protein import is no longer possible: mitochondrial decline is now irreversible, as generation of new mitochondria requires new cytosolic proteins, and these are no longer accessible. Production of H2O2 overwhelms the capacity of the mitochondria to regenerate reduced glutathione; ever increasing levels of H2O2 and O2- start to leak out: the internal enzymatic defenses are inactivated by sugars or ROS and not replaced. Glutamate, Introduction 27 acetate, ethanol, and other compounds from glycolysis and other sources begin to accumulate in the cell as the mitochondria lose the ability to take up or metabolize these carbon sources. Eventually the elevated levels of free radicals and other compounds becomes toxic, and the affected cells and tissues start to degenerate. Declines in cell and tissue vitality reduce the capacity of the organism to withstand stress, and when decline reaches critical levels, some stress or shock results in loss of homeostasis, and the organism dies. EXPERIMENTAL APPROACHES. The above hypothesis is in general agreement with the known information on cell death and aging. While some parts of this scenario are likely to be wrong or irrelevant, the central aspects of this theory are open to experimental investigation by numerous techniques. M E M B R A N E CHARACTER The importance of cholesterol, cardiolipin, and long chain saturated fatty acids could be tested by measuring mitochondrial superoxide and H2O2 production before and after introduction of additional amounts of these compounds into the mitochondrial membranes in vitro. Experimentation with mitochondrial lipid cross linking has already been done (Sohal 1992), and the results are in agreement with this model: reduced fluidity results in increased H2O2 and O2- production. M O L E C U L A R A P P R O A C H E S The importance of accumulations in oxidative damage could be addressed by incorporation of additional antioxidant enzymes into mitochondria by molecular genetic techniques. Because the rate limiting step in antioxidant defenses is not known, all of the different stages in the elimination of free radical damage would have to be tested: one of the evidences in favor of this theory is the effectiveness of increased protein synthesis by incorporation of additional genes for elongation factor T alpha into Drosophila, which indicates that increased levels of protein turnover are beneficial. Candidate genes for study by overexpression include Mn SOD, catalase, glutathione Introduction 28 peroxidase, and glutathione reductase, as well as enzymes involved in maintenance of mitochondrial membrane lipids and mitochondrial matrix protein synthesis and DNA repair. C A T A L A S E A N D P E R O X I D A S E The importance of H2O2 in mitochondrial degeneration could be tested by several strategies. Increasing the level of glutathione peroxidase, glutathione reductase or both should result in an increase in the level of antioxidant defense in mammals. As flies do not contain GPX in their mitochondria (Orr & Sohal 1992), and according to Rikans et al. (1992) there is no catalase in the mitochondria of any species, introduction of mitochondrial GPX and GRX genes should be at least as successful in flies as treatments with antioxidant drugs. Redesigning the enzyme catalase for transport into, or coding within, the mitochondrial matrix could help conserve matrix pools of reduced glutathione, resulting in increased protection. A problem with this idea may lie in the extreme sensitivity of the animal heme-containing catalases to superoxide (Fridovich 1987): normal levels of matrix superoxide could result in their rapid inactivation, thus making them a useless addition. That fly mitochondria do not contain any form of matrix localized catalase or peroxidase is thus more easily understood. It would be extremely interesting to see what effect introduction of a bacterial Mn containing catalase would have on the vitality of tissue culture cells and transgenic organisms: the Mn containing catalases are never more than 50% inactivated by superoxide (Fridovich 1987), and could thus continue to function no matter what level of superoxide was present. It is possible there is a reason other than evolutionary oversight for the omission of catalase from animal mitochondria, so the results of including it could be very instructive, both in flies (no GPX) and in mice or some other mammal (with mitochondrial GPX). If experiments with cholesterol addition to membranes indicate cholesterol is an important factor, then transformation with additional genes for cholesterol acyl Introduction 29 transferase or other enzymes involved in cholesterol metabolism could also prove to be beneficial. S U P E R O X I D E D I S M U T A S E S Over expression of mitochondrial manganese superoxide dismutase could also be beneficial. Reduced levels of superoxide could produce the desired protective effect by reducing the level of membrane damage caused directly by superoxide. Although overexpression of Cu-Zn SOD was not successful in producing extensive increases in LSP (Fleming et al. 1992, Reveillaud et al. 1991, Seto et al. 1990), the localization of M n SOD in the mitochondria and possible differences in direct production of hydroxyl radical make it worth exploring, as the effects of overexpression of this gene may have quite different effects compared to overexpression of the Cu-Zn SOD gene. Experimental overexpression in bacteria and tissue culture indicates that increased levels of Mn SOD increases the levels of oxidative stress in living cells. Thus it is possible that M n SOD is also a producer of hydroxyl radical, or that it increases stress by raising H2O2 levels, and experimental overexpression may result in similar effects to those seen for Cu-Zn SOD overexpression. In either case it is worthwhile to explore the effect of altered expression of Mn SOD in order to determine the role(s) it may play in several different aspects of cellular metabolism: 1) in reducing or regulating lipid and protein damage in the mitochondrial membrane, 2) as a possible agent in the production of hydroxyl radical and the accumulation of lipofuscins, and 3) as a regulator of developmental events by regulation of superoxide levels. GOALS OF THIS RESEARCH This project was designed to test the effect of overexpression of native M n SOD in Drosophila melanogaster. The first step, isolation of a complete Drosophila M n -SOD cDNA, was achieved by screening a Drosophila melanogaster cDNA library with a partial genomic clone obtained by PCR. The second half, reintroduction of the Introduction 30 cDNA under the control of a new promoter by P element mediated transformation, was not attempted by this author. 31 M A T E R I A L S : DROSOPHILA MELANOGASTER STRAINS DNA and RNA extractions were carried out primarily on wild type isogenic Oregon R flies provided by Dr. G. M. Tener. Some of the DNA extractions included Cu-Zn SOD mutants, curly wing, balancer stock, and others in the extractions as available. No attempt was made initially to keep these separate. Later work included only wild type flies in DNA isolations. BACTERIAL STRAINS Bacteria used for all transformations were JM101 or JM103 Echerichia coli, maintained on M9 agar plates with 1 \il of 2.5 ug/ml thiamine added per ml of agar. They were obtained as gifts from the labs of Dr. Sadowski and Dr. McGillivray. The plasmid BlueScript 11+ was a gift from Don Jones in Dr. Candido's lab, in a frozen culture of Escherichia coli strain XLl-Blue (Stratagene). C600 Escherichia coli used in Lambda phage plating were a gift from Dr. Dennis's lab. CLONING VECTORS Cloning Vectors used were: pBR322, M13 mpl8, M13 mpl9, Lambda GT10, and BlueScript 11+. The Lambda GT10 cDNA library from Drosophila imaginal disc tissue culture was a gift from Dr. Don Sinclair in Dr. Brock's lab (Department of Zoology, U.B.C.). CHEMICALS AND REAGENTS Chemicals and reagents were purchased from Pharmacia, BDH, Boehringer-Mannheim, Aldrich, Fischer, Eastman Kodak, ICN Pharmaceuticals Inc., Matheson Coleman & Bell (MCB), Baker & Adamson/Allied Chemicals, Sigma, and others. SeaPlaque Agarose was purchased from FM C BioProducts, 5 Maple St., Rockland, M E 04841-2994, USA. Radioisotopes and nylon filters were purchased from Materials 32 Amersham. Plastic Tubes, Ependorf tubes, and Pipet Tips were from Falcon, Elkay Products Inc. and others. E N Z Y M E S Enzymes were purchased from Pharmacia, Boehringer-Mannheim, BRL (Bethsada Research Laboratories), Bio-Rad, New England Biolabs, Promega Biotec, and others. BUFFERS B U F F E R S U S E D I N E L E C T R O P H O R E S I S : TBE Tris Borate. EDTA lx (Maniatis et al. 1982) 89 mM Tris, 89 mM boric acid, 0.2 mM EDTA. Made up as a 5x, lOx, or 20x stock solution, used at lx in DNA sequencing gels. At lOx or 20x it is desirable to filter it to avoid precipitate formation after several days standing. MOPS Buffer lOx (Davis et al. 1983) 200 mM 3-[N-morpholine]propanesulfonic acid, 50 mM sodium acetate, 10 mM EDTA. Made as a lOx stock and autoclaved. It may yellow with age, but is still useful. Used for RNA separation on denaturing formaldehyde-agarose gels. Tris Acetate EDTA 50x: (TAB) (Maniatis et al. 1982) 2.0 M Tris base, 0.1 M EDTA, 1.0 M acetic acid pH to 8.2 with glacial acetic acid, then make up to volume. Made as a 50x stock, used at lx on agarose gels (for use with Sea Plaque agarose gels see Struhl, 1985, and Dhananjaya et al. 1991). L O A D I N G D Y E S F O R A C R Y L A M I D E A N D A G A R O S E G E L S Northern Loading Dye (Davis et al. 1983) 80 pi 4 g/100 ml bromophenol blue, 260 pi deionized formaldehyde, 720 pi deionized formamide, 180 ul DEPC treated distilled H 2 0 , 160 pi lOx MOPS buffer (see electrophoresis buffers), 100 pi 80 g/100 ml glycerol. Materials 33 Loading Dye for DNA Sequencing (Maniatis et al. 1982) 8 ml deionized formamide, 0.5 ml 1.0 M Tris HC1, pH 8.3, 50 ul 500 mM EDTA, 10 saturated bromophenol blue, 10 ul saturated xylene cyanol. B U F F E R S F O R B L O T T I N G G E L S I N N O R T H E R N S A N D S O U T H E R N S . Standard Saline Citrate (SSQ: (Maniatis et al. 1982) 150 mM sodium chloride, 15 mM sodium citrate: pH to 7.0 with HCl/NaOH. Made as a 20x stock solution, filtered, and autoclaved. Used for blotting DNA and RNA from agarose gels to filters, hybridization of filters with probe, washing filters after hybridization. Gel Denaturing Solution: (Maniatis et al. 1982) 1.5MNaCl, 0.5 M NaOH Gel Neutralizing Solution: (Maniatis et al. 1982) 1.5 M NaCl, 1.0 M Tris HC1 pH 8.0 B U F F E R S F O R E X T R A C T I O N O F D N A A N D R N A TENT Buffer (Nuclei Isolation) (Henry et al., 1990) 10 mM Tris HC1 (pH 7.4), 25 mM EDTA, 10 mM NaCl, 0.5 % Triton X-100 STET Buffer 8% Sucrose, 5 % Triton x-100, 50 mM Tris HC1 pH 8.0, 50 mM EDTA pH 8.0 Solution D (RNA extraction) (Chomczynski & Sacchi, 1987) 4.0 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5 % Sarcosyl, 0.1 M 2-mecaptoethanol. B U F F E R S F O R E N Z Y M A T I C R E A C T I O N S : R e s t r i c t i o n Endonucleases: Maniatis Buffers (Maniatis et al. pg. 104) lOx Low: 100 mM Tris HC1 (pH 7.5), 100 mM MgCl 2 , 10 mM DTT lOx Medium: 500 mM NaCl, 100 mM Tris HC1 (pH 7.5), 100 mM MgCl 2 , 10 mM DTT lOx High: 1.0 M NaCl, 500 mM Tris HC1 (pH 7.5), 100 mM MgCl 2 , 10 mM DTT Materials 34 Pharmacia Buffers L, M, and H Pharmacia buffers are identical to Maniatis Low, Medium, and High, respectively; in addition, they recommend K M and K H for some enzymes: lOx K M Buffer: 500 mM KCI, 100 mM Tris HC1 (pH 7.5), 100 mM MgCl 2 , 10 mM DTT lOx K H Buffer: 1.0 M KCI, 100 mM Tris HC1 (pH 8.0), 100 mM MgCl 2 , 10 mM DTT Pharmacia now makes "One-Phor-All Buffer PLUS" for use with all of their enzymes: the contents of this buffer (lOx) is 100 mM Tris-acetate, pH 7.5, 100 mM Mg(OAc) 2, 500 mM KOAc. Incubation mixes for Polymerase reactions: lOx T4 DNA Polymerase Buffer Incubation Mixture (Berger & Kimmel. 1987. pg. 107) 330 mM Tris acetate (pH 7.9), 660 mM potassium acetate, 100 mM magnesium acetate. Used with nucleotides at 3:1 (v/v, nucleotides to DNA + Polymerase) as follows: 50 mM dATP, dTTP, dCTP, dGTP, 33 mM Tris acetate (pH 7.9), 66 mM potassium acetate, 10 mM magnesium acetate, 14 % (v/v) glycerol T7 DNA polymerase Incubation Mixture: 20 mM Tris HC1 (pH 7.5), 10 mM MgCl 2 , 25 mM NaCl, 2.5 mM DTT, 150 pM dATP, dTTP, dGTP, and dCTP lOx Tag DNA Polymerase Incubation Mixtures: ("PCR Buffers) Two different sets of buffer conditions, A and C series, were used: the A buffers work well with most of the Taq polymerases I have used, the C buffers works extremely well with the Promega Taq, and gave results similar to the A buffers with BRL Taq. However, the C series requires ultra pure KCI be used in formulation, or the reaction will be inhibited (Don Jones, personal communication). Type A (Tse & Forget, 1990) Materials 35 lOx buffer is 500 mM Tris HC1 (pH 8.4), 500 mM KC1, 15 to 50 mM MgCl 2 * Type C (Promega, 1993) lOx buffer is 100 mM Tris HC1 (pH 8.8), 500 mM KC1, 1% Triton X-100, 15-50 mM MgCl 2 * * individual magnesium concentrations were 15, 20, 25, 30 and 50 mM MgCl 2 in lOx stock solutions. Buffers for Other DNA Modification Enzymes: lOx T4 Polynucleotide Kinase (PNK) Incubation Mixture 670 mM Tris HC1, pH 8.3, 100 mM MgCl 2 , 100 mM DTT (Berger and Kimmel, 1987, pg. 438: see also alternate buffer conditions pg. 100-101, and in Maniatis et al. 1982, pg. 122,124, 127) T4 DNA Ligase Incubation Mixtures See Berger and Kimmel (1987, pg. 109) for reaction conditions for T4 DNA Ligase: different conditions are used depending on the nature of the DNA to be ligated. 5x Terminal deoxynucleotidyl Transferase (TdT) Incubation Mixture. (from Berger & Kimmel, 1987, pg. 339) 500 mM potassium cacodylate, 10 mM CoCl 2 , 1 mM DTT. Note: C0PO4 and C0P2O7 are insoluble, and will precipitate: avoid enzyme preparations and salts containing phosphate or pyrophosphate. The commercially supplied buffer which comes with the enzyme worked better than that which I made myself. Materials 36 BACTERIAL MEDIA: Note: all recipes (except that for fly food) are for 1 liter and may be found in Maniatis et al. (1982) M9 minimal medium 6 g Na 2 HP0 4 , 3 g KH 2 P0 4 , 0.5 g NaCl, 1 g NH4C1 pH to 7.4 with HC1 or NaOH, autoclave, then add (sterile), 2 ml 1 M MgCl 2 , 10 ml 20% glucose, 100 pi 1 M CaCl 2 and any other requirements of the bacterial strain in question: for example, for E. coli JM101 , add 600 pi of 0.25 mg/ml thiamine HC1, for E. coli C600, add 600 pi of 0.25 mg/ml thiamine HC1 and appropriate amounts of the amino acids Leu and He. NZY: 10 g NZ amine, 5 g NaCl, 5 g Bacto-yeast extract adjust pH to 7.5 with NaOH and autoclave for 30 min. NZYM: identical to NZY but with 2 g MgS0 4 :7H 2 0 NZCYM: identical to NZY but with 2 g MgS0 4 :7H 2 0 and 1 g casamino acids. Luria-Bertani (LB) medium 10 g Bacto-tryptone, 5 g Bacto-yeast extract, 10 g NaCl adjust pH to 7.5 with NaCl and autoclave for 30 min. Other Bacteriology Reagents: Agar Plates To make agar plates, use 20 g/1 Bacto-agar in any of the above liquid media, autoclave as before, and pour when the temperature reaches about 50-55°C. Top Agar/Agarose If plates are to be lifted, then agarose is desirable as it shows less tendency to stick to the filters, otherwise agar may be used. Use 7 g of agar or agarose per liter. TMG (lambda phage dilution buffer) 10 mM Tris HC1 (pH 8.0), 10 mM MgS0 4 , 0.01 % gelatin SM: As TMG, but add 100 mM NaCl Materials 37 Fly Food. (Gordon Tener, personal communication) 1) mix in pot, 100 g Low fat soy flour 20 g Difco yeast extract 17 g Agar 1 g citric acid 9 g Trisodium citrate 1000 ml tap water 2) microwave (650 watts) on "hi" for 12 minutes, pause to stir at 8 minutes; microwave until it boils. 3) reduce power to "4" and continue to boil for 15 minutes. 4) stir in 40 g sucrose 40 g glucose 15 ml methyl parahydroxy benzoate (10% solution in 95% EtOH) 50 mg Ampicillin 5) microwave for 2 minutes more. 6) Cool to 60 °C and pour into fly bottles. Materials 38 DNA PROBES Table #2: Table of DNA Oligonucleotides. DNA oligonucleotides were synthesized by Jeff Hewitt in Dr. Ross McGillivray's lab, and by the oligonucleotide synthesis facility of the U.B.C. Biochemistry Department. The probes used are listed below in alphabetical order. The information on each probe is set out in the format specified at the top of the table. The oligonucleotide T17XSP is a generic primer used for reverse transcription of poly A+ RNA. It contains a 3' 17 base stretch of thymidine and a 5* region with 3 restriction sites. Materials Designation Numbering i n AA Sequence Lineup Strand to match Amino A c i d Sequence NH3+/C00- d i r e c t e d DNA Sequence Si z e Used For Designer. 39 6-90 206 205 204 203 202 201 Template Strand W N I V N W NH3+ 5' 5' CCA GTT GAT GAC GTT CCA 3' PCR, Screening Southerns 18 bases Stuart Carmichael GSGW 129 130 131 132 133 134 135 136 Coding Strand G V Q G S G W GAV COO- 3' 5' GGC GTG CAG GGT TCN GGN TGG G 3' 22 bases PCR Stuart Carmichael GT1 15 16 17 18 19 20 21 22 23 24 Coding Strand E P H I N A Q I M Q COO- 3' 5'GAR CCN CAY ATN ARY GCN GAR ATN ATG CA 3' C 29 bases PCR, Screening Southerns. Gordon Tener HEW 187 186 185 184 183 182 181 Template Strand L Y Y A H E W NH3+ 5' 5 1 AG GTA GTA TGC RTG YTC CCA 3' 20 bases PCR Stuart Carmichael Jan 90 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 Template H L Q M I Q/E A N/S I H P E L A G Y D Y P NH3+ 5' 5'TG CAG CTG CAT GAT CTG GGC GTT GAT GTG IGG CTC CAG GGC ICC AAG CTG CTA IGG 3' C T 56 bases Screening Southerns Stuart Carmichael S2 8 9 10 11 12 13 Coding Strand P Y D Y G A Toward COO- Term 5 1 CCN TAY GAY TAY GGN GC 3' PCR, Screening genomic Southerns. 17 bases Shizu Hayashi SCI 29 30 31 32 33 34 35 36 Coding Strand K H H A A/T Y V N Toward COO- Term 5'AAG CAT CAT GCG GCG TAT GTG AA 3' 23 bases PCR, Screening Southerns Gordon Tener SOD 5' 21 22 23 24 25 26 27 Coding Strand E I M E L H H 5' NNN AAG CTT G GAG ATC ATG GAG CTG CAT CAC C 3' 32 bases PCR J e f f Hewitt SOD 3' 133 132 131 130 129 128 127 126 Template S t r . K G S K K N F G 5' NNN CTG CAG TT GCC CGA CTT CTT GTT GAA GC 3 1 32 bases PCR J e f f Hewitt 40 M E T H O D S : METHODS I: MOLECULAR TECHNIQUES Isolation of Drosophila melanogaster DNA Procedure I: Extraction from Nuclei DNA was isolated by nuclei extraction according to the methods of Henry et al. (1990). This involves freezing flies in liquid nitrogen (placing them in a -70 °C freezer for 5 min. seems to work just as well), grinding them to a powder with a mortar and pestle, suspension of the fly powder in TENT buffer (see Buffers for Extraction of DNA and RNA), filtration through glass wool, cheese cloth, or Miracloth, centrifugation and resuspension of the nuclear pellet (50 mM Tris HC1 pH 8.0, 25 mM EDTA), and addition of proteinase K and SDS (0.3 %) for overnight digestion at 37 °C. DNA was then extracted with T E saturated phenol/2-butanol (5:1), T E saturated phenol, twice with T E saturated phenol/chloroform (1:1), and twice with chloroform before precipitation with isopropanol followed by three washes with 70% ethanol. 2-Butanol was included in the first phenol extraction as it seems to enhance removal of Drosophila eye pigments, which can inhibit some enzymes used in DNA manipulations. This method can be scaled up or down as desired. It is fast, (the steps up to the start of the overnight incubation at 37 °C can be completed in less than 20 minutes with previously frozen flies) and the DNA is amenable to enzymatic activity by various restriction enzymes. It is possible to eliminate most of the RNA by stirring in loose balls of glass wool prior to filtering: most of the preparations I did with this technique were nearly RNA free. Upon discovery that reluctant DNA pellets can be resuspended by adding ethanol and/or acetone to the mixture, I came up with the following procedure, which gives very clean, soluble DNA using less phenol and chloroform. I have not had time Methods I: Molecular Techniques 41 to thoroughly test this method, but it seems to give better results with less fuss than procedure I. Procedure II: Parti 1) collect 5 ml (about 1.5 g) of adult flies in a 15 ml Falcon tube and cap: place the flies, a mortar and pestle, and a scoopula in the -70°C freezer for > 5 min. 2) place on ice, 15 ml of TENT buffer in a 50 ml Falcon tube, and 2 empty 50 ml Falcon tubes. 3) remove the flies, mortar, and pestle from the freezer: rap the Falcon tube on the bench to loosen the flies, then pour them into the mortar and grind to a fine powder. 4) remove the scoopula from the freezer, and carefully scoop the fly powder into the TENT buffer: add 10 ml of room temperature TENT and gently swirl or stir the contents until all of the powder is in suspension. Stir in a loose wad of glass wool (speeds filtering and helps remove RNA and debris). 5) place a funnel with a glass wool plug in the top of one of the empty 50 ml Falcon tubes, and pour the TENT/fly mix through the glass wool. This process can be speeded up by substituting Miracloth (Calbiochem-Berhing corp.) for the glass wool, but the glass wool has the advantage of somehow removing most of the RNA from the preparation, especially if it is refiltered 2 or 3 times. 6) divide the filtrate equally between the two 50 ml Falcon tubes, and, spin in a clinical centrifuge at "6" for 3 min. 7) pour off the supernatant and resuspend the nuclei in 3.5 ml per tube of 50 mM Tris pH 7.5, /25 mM EDTA. 8) transfer to a single 15 ml Falcon tube, stir in about 0.5 to 1 cubic mm of proteinase K powder, add 210 ul of 10% SDS, swirl briefly, and place at 37 °C for at least 12 hours. Methods I: Molecular Techniques 42 Part II 9) add 5 ml of T E saturated phenol and 5 ml of chloroform, shake or invert repeatedly until well mixed, then place On ice for 15 minutes. 10) spin down at "10" in the clinical centrifuge for 10 min. at room temp or 4 °C. Separate aqueous fraction into a new tube. 11) add 700 pi 2.0 M NaOAc, pH 5.0, mix, then add 8 ml of isopropanol and mix slowly by inversion. Cut the end off of a 1 ml plastic pipette tip so as to obtain a very large bore size, and suck up the ball of D N A with a 1 ml Pipetman: transfer the ball to a 1.5 ml Eppendorf tube, and allow it to settle into the bottom of the tube. Remove as much of the supernatant as possible. 12) wash the D N A ball with 70 % ethanol, then add 400 pi of 10 m M Tris, 10 m M E D T A , and redissolve the D N A by adding 100 to 150 pi ethanol and 100 pi acetone and placing at 37 °C. The D N A may take anywhere from 5 minutes to overnight to dissolve. 13) extract the redissolved D N A on ice for 5 to 20 minutes each with 500 pi of T E saturated phenol, twice with 500 pi of T E saturated phenol: CHC13 (1:1) and twice with CHC13. 14) precipitate the D N A with 300 pi of 8.0 M N H 4 O A c , and 700 pi of isopropanol, mixing by inversion. 15) wash the resulting ball of D N A twice with 70 % ethanol, twice with 95 % ethanol, and air dry on the bench under a bell jar. Do not centrifuge the D N A pellet: after each alcohol wash, carefully remove the excess with a pipette. This avoids compacting the D N A ball, and makes resuspension easier. Leave the D N A ball on the side of the tube as spread out as possible, as this aids in resuspension. Resuspend in 100-1000 pi of 10 mM Tris (pH 8.0), 1.0 mM E D T A . Isolation of Drosophila melanogaster RNA Total cellular Drosophila RNA was isolated by the acid guanidinium thiocyanate-phenol-chloroform extraction procedure employed by Chomczynski and Methods I: Molecular Techniques 43 Sacchi (1987) first described by < >. The procedure was modified by the use of 0.3 volumes of D lysis buffer (4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5 % Sarkosyl, 0.1 M 2-mercapto ethanol) instead of 0.1 volumes for the first redisolution of the extracted RNA. Flies were flash frozen in liquid nitrogen, ground to partial homogeneity in a precooled mortar & pestal, and added at 100 mg/ml directly to D lysis buffer (as above) in a Dounce tissue grinder, and mixed with a few strokes. To this is added sequentially, 0.1 volumes 2 M NaOAc (pH 4.0), 1 volume of T E saturated phenol, and 0.2 volumes of CHC13:isoamyl alcohol, with mixing by inversion after each addition. The final suspension is shaken vigorously for 10 seconds, placed on ice for 15 minutes, and centrifuged at 10,000 g for 20 minutes. The top phase was then removed to a new tube and precipitated with 1 volume of isopropanol for > 1 hour at -20 °C. The RNA pellet was then redissolved in 0.3 volumes of solution D and reprecipitated with isopropanol for > 1 hour at -20 °C. The RNA was then redissolved in T E and used for PCR and Northern analysis. Plasmid DNA I s o l a t i o n s Several Plasmid DNA isolation procedures were tried. The fastest, simplest, and most successful was the STET rapid boiling mini prep, which worked well as a preparative method for dsDNA sequencing in addition to being restriction enzyme amenable. It does not require the use of phenol. STET Rapid Boiling Mini prep. Note: Always make a freezer culture of any new clones before using them up: 300 ul cell culture 200 ul 50% glycerol freeze at -70 °C 1) spin down 5 ml of an LB Amp overnight cell culture and discard supernatant. 2) resuspend cells in 600 ul of STET buffer and transfer to a 1.5 ml Eppendorf tube. This should be done on ice: it is important to complete this step in a minimum of time, as the cells may start to lyse if left too long in this solution. Methods I: Molecular Techniques 44 3) equilibrate on ice for 2 minutes. 4) place in boiling water bath for EXACTLY 2 minutes. NOTE: Even 10 seconds off will result in poor yield of plasmid DNA (not long enough) or contamination with bacterial genomic DNA (too long). In this context it is important to understand that anything which affects the heating curve of the tube contents, e.g. liquid volume, temperature at start of heating, poor immersion, etc. will adversely affect the results of this procedure. 5) place in ice water bath for at least one minute with mild agitation. 6) spin down in microfuge for 15 minutes 7) remove protein/lipid glob from tube with tooth pick. This is best done by stabbing the pellet and rotating the tooth pick to loosen the pellet before trying to remove it. 8) add 600 pi of isopropanol, vortex, and set on bench for 5-10 minutes, until a floculent precipitate is observed. 9) spin down DNA for 15 minutes, discard supernatant. 10) wash pellet 2x with 70% ethanol: drain off as much as possible. 11) dry pellet in Rotovap (or other centrifugal vacuum evaporator) for 10 minutes. This step is essential for plasmid to be used for DNA sequencing. 12) dissolve in 50 pi of TE or sdH 20: use 2 pi for digests, 18 pi for dsDNA sequencing reactions. ssDNA i s o l a t i o n (Ml3 phage) : Numerous simple isolations for M13 phage particles can be found. Most use precipitation of the phage particles with PEG and NaCl or NH 4 OAc. The following procedure is a shortened version from Glover, (1985) "DNA Cloning, a Practical Approach, volume 1". 1) grow phage overnight on an appropriate host at 37 °C (2 to 200 ml culture) 2) spin down the bacteria: save bacteria for preparation of RF DNA if desired. Decant phage containing supernatant into a new tube. Methods I: Molecular Techniques 45 3) add 4 g PEG 6000 and 3 g NaCl per 100 ml culture, dissolve PEG and salt and store at 4 °C for 4 hours to over night. 4) spin down precipitated phage for 15 minutes at 4 °C. 5) pour off the supernatant and drain tube by inversion for 5-10 minutes: remove as much of the residual supernatant as possible. 6) resuspend phage in 0.75 ml per 25 ml original volume of 10 mM Tris pH 8.0, 50 mM NaCl, 1.0 mM EDTA. 7) add an equal volume of 4% PEG 6000, 6% NaCl, mix well, and place at 4 °C for one hour or more. 8) pellet phage in a microfuge at 4 °C. Carefully remove the supernatant then spin briefly again and remove residual supernatant. 10) resuspend phage in 0.5 ml of 10 mM Tris pH 8.0, 50 mM NaCl, 1.0 mM EDTA. 11) extract three times with one volume each of T E saturated phenol, T E saturated phenol:CHC13, and CHC13, vortexing each time for 10 seconds. 12) precipitate DNA with 200 ul of 7.5 M NH 4 OAc and 700 ul of isopropanol. 13) spin down DNA, wash pellet twice with 70% ethanol, once with 95% ethanol, and air dry under a beaker on the bench. 14) dissolve DNA in 10 ul per ml of original culture, use 1 to 5 ul per attempt for sequencing. Lambda Phage DNA Isolation From Broth Cultures: This method was adapted from a protocol provided by Don Jones. It is the simplest and fastest I've run across. Preparation of phage 1) grow a confluent overnight plate culture of the desired phage. 2) inoculate 40 ml of N Z Y M or N Z C Y M with 1.0 ml of previously prepared 4x plating stock (see bacterial techniques, page 46), and grow at 37 °C with shaking for 1.5 to 2 hours. While the bacteria are growing, pour 10 ml of T M G onto the confluent over night plate culture and shake gently for 1.5 to 2 hours. Methods I: Molecular Techniques 46 3) inoculate the bacterial culture with 1.0 ml of the plate culture supernatant, swirl vigorously, and set on the bench for 15 minutes. 4) grow at 37 °C with vigorous shaking for 1.5 to 5 hours, by which time complete or nearly complete lysis should occur. 5) add 0.1 to 0.2 ml of CHC13 and shake for another 3-5 minutes. 6) transfer to a 30 ml Corex tube and centrifuge in the ss34 rotor for 10 minutes at 10,000 RPM. 7) carefully pour the supernatant into a new 30 ml Corex tube, and add 6 ml 50% PEG 6000, and 3 ml of 5 M NaCl. Mix well. 8) set for 4 hours to over night at 4 °C. 9) centrifuge as in 6), saving the phage pellet. Isolation of Lambda phage DNA 10) resuspend the phage pellet in 0.5 ml of DNase buffer (50 mM HEPES, pH 7.5; 5 mM MgCk; 0.5 mM CaCk) by pipetting up and down with a 1 ml pipette. 11) transfer the suspension to a 1.5 ml Eppendorf tube and extract with CHC13 12) add 5 pg of DNase I and 50 ug of RNase A (5 pi 1 mg/ml DNase, 5 pi 10 mg/ml RNase) 13) incubate at 37 °C for 20 to 30 minutes, or until viscosity disappears. 14) add 50 pi of 10X SET (5 % SDS, 0.2 M EDTA, 0.1 M Tris, pH 7.5), 20 pi 5 mg/ml proteinase K, and incubate 45-60 minutes at 68 °C. 15) spin down and discard debris (transfer to a new tube). 16) add 250 pi T E saturated phenol, vortex, add 250 pi CHC13, and vortex again. 17) centrifuge and withdraw the organic phase with a disposable plastic transfer pipette: this process can be made easier by fire drawing the tip of the pipette, giving a finer point. 18) extract the aqueous phase again three times with CHC13 (about 400 pi each time). Methods I: Molecular Techniques 47 19) transfer the aqueous phase to a fresh tube and add 200 ul of 7.5 M N H 4 O A C and 700 ul of isopropanol. Place at -20 °C for 2 or more hours, and centrifuge in the microfuge at 4 °C for 15 minutes. 20) wash DNA pellet twice with 70% ethanol and twice with 95% ethanol, dry under a beaker, and resuspend in 10 mM Tris HC1, pH 8.0, 10 mM EDTA, pH 8.0. I s o l a t i o n of DNA from Agarose Gels The following procedures start with a common theme; separate the DNA of interest from other extraneous DNA by agarose gel electrophoresis arid cut out the gel block containing the band of interest. method I: Isolation by Electroelution. The gel fragment is placed in a piece of dialysis tubing filled with lx T A E buffer, and all the air is removed prior to sealing. The bag is placed in the electrophoresis tank and the current is switched on for 10-15 minutes. The current is reversed for 30 seconds, and then the T A E in the bag is carefully removed to an Eppendorf tube. A quick spin in the microcentrifuge pellets any remaining gel fragments, and the supernatant is removed to a fresh Eppendorf tube. The DNA is then precipitated for further use. method II: Isolation using Glass Milk. This procedure was done using Qiagen's QIAEX DNA isolation Kit, or the Gene Clean Nal DNA isolation kit, following the instructions provided. Oligonucleotide P u r i f i c a t i o n . Oligonucleotides were used at 3 different levels of purification. These were: 1) untreated as they arrived from the oligonucleotide synthesis lab, 2) purified by C18 Sep-pak chromatography, and 3) separation from the attenuated reaction products by acrylamide gel electrophoresis followed by C18 Sep-pak purification. For the procedures for 2) and 3) see Tom Atkinson's unpublished protocol, or see Atkinson & Smith (1984). Methods I: Molecular Techniques 48 Polyacrylamide for DNA P r e c i p i t a t i o n Polyacrylamide prepared without N,N' methylene-bisacrylamide can be sheared by drawing through a fine needle and used as an inert carrier to aid in the precipitation of low concentrations of DNA. This was done as follows: 400 pi of 1M Tris HC1 (pH 8.0), 200 pi of NaOAc (pH 7.9), 40 pi of 500 mM EDTA, 0.5 g acrylamide, 100 pi 10% ammonium sulfate, and 10 pi N,N,N',N'-tetramethylene bisacrylamide (Temed), were mixed, and the volume was made up to 10 ml (the solution was not degassed before running the reaction). The resulting solution was left at room temperature for 30 minutes until it became viscous, and then mixed with 3.33 ml of 0.3 M NaOAc. 500 pi of the resulting solution was sheared with a fine needle and syringe, then precipitated with 2 vol. of 95% ethanol, pelleted in the microfuge, washed twice with 70% ethanol, dried and weighed. The dry pellet was then resuspended in sdfbO at 0.25% wt/vol. Five microliters of the resulting solution was then used as required for precipitation of low concentration solutions of DNA or RNA or as a visible marker for the location of small DNA or RNA pellets. This procedure was provided by Don Jones in Dr. Peter Candido's lab. Nucleic Acid Quantitation by UV Absorption. DNA, RNA, oligonucleotides, and nucleotide triphosphates were quantitated by their absorption spectra of 280 and 260 nm light. Samples were diluted to 1 ml and their absorptions were compared with the absorption of 1 ml of the appropriate buffer. Concentrations were calculated from absorption using the molar absorption values in Table 3. Methods I: Molecular Techniques Table #3: U.V. Absorption Values for Nucleic Acids. 49 N u c l e i c A b s o r p t i o n A c i d V a l u e (260 N u c l e i c A c i d nm) C o n c e n t r a t i o n dsDNA 1.0 wcRNA 1. 0 dATP 15,500 dGTP 14,300 dCTP 13,600 dTTP 9,400 50 ug/ml 4 0 ug/ml 1 M o l a r 1 M o l a r 1 M o l a r 1 M o l a r For s i n g l e s t r a n d e d o l i g o n u c l e o t i d e s : pAp 15,200 pGp 12,010 pCp 7,050 pTp 8,4 00 1 M o l a r *1 1 M o l a r *1 1 M o l a r *1 1 M o l a r *1 *1 These are the absorption values for one base of this type in the chain of the oligonucleotide (Berger & Kimmel, 1987): thus a 1 Molar concentration of the oligonucleotide GGCTGAA would have an absorption of 3(12,010) + 1(7,050) + 1(8,400) + 2(15,200) = 51,480 ABS or 0.05148 ABS/uM. One absorption unit equals 19.4 uM. The DNAABS program in the Appendix may be used to calculate this value. Preparation of DNA Size Standards for Agarose Gels. Molecular size standards were prepared using restriction digests of Lambda, pBR322, and BlueScript as follows: Lambda Ladder: 4.0 ug Lambda DNA in 20 ul was treated with 2.5 ul of medium buffer (50 mM NaCl, 10 mM Tris HC1 (pH 7.5), 10 mM MgCl 2 , 1 mM DTT; (Maniatis et al. 1982, pg. 104) and 2.0 ul of Hind III (15 U/ul)) restriction endonuclease for 2 hours and 45 min. at 37 °C. 10 ul of loading dye was then added, and the resulting ladder was tested at 4 ul in one well. This proved far more concentrated than necessary, so the remaining 30 ul was diluted with 80 ul of sdH 20 and 20 ul of loading dye. The resulting mixture was loading on agarose gels containing genomic digests at 4 ul per well. pBR322: 2.0 ug pBr322 DNA in 20 ul was treated with 2.5 ul of medium buffer (Maniatis pg. 104, as above) and 2.0 ul of Hinf I (15 U/ul) restriction endonuclease for Methods I: Molecular Techniques 50 90 min. at 37 °C. The resulting ladder was test loaded at 4 ul after dilution with 15 ul of loading dye and 10 ul of sdEkO, and used at this concentration subsequently. pBlueScript (BSII+): Approximately 15 pg of BSII+ DNA in 20 pi was diluted with 15 pi of sdfkO and 4 pi of 10X one-phor-all buffer (Pharmacia) and then digested with 1 pi of Hinf I (15 U/ul). Four microliters and 1 pi of the resulting ladder were mixed with 3 pi of sdH20 and 3 pi of loading dye and tested on a gel. The remaining 35 pi was diluted with 50 pi of loading dye and 100 pi of sdHaO, and used at 3 pi per loading. The resulting molecular size standards give bands as described in Table #4: Methods I: Molecular Techniques 51 Table #4: Molecular Size Standards for Agarose Gels. L a m b d a H i n d I I I b a n d l e n g t h l o g 1 23 . 130 k b 4 . 3 6 4 2 2 9 . 4 1 6 k b 3 . 9 7 3 9 3 6 . 5 5 7 k b 3 . 8167 4 4 . 4 6 1 k b 3 . 6 2 9 6 5 2 . 3 2 2 k b 3 . 3 6 5 9 6 2 . 0 2 7 k b 3 . 3 0 6 9 7 0 . 5 6 4 k b 2 . 7 5 1 3 1 k b L a d d e r b a n d l e n g t h l c g 1 12 . 2 1 6 k b 4 . 0 8 6 9 2 11 . 198 k b 4 . 0491 3 10 . 1 8 0 k b 4 . 0 0 7 7 4 9 . 162 k b 3 . 9 6 2 0 5 8 . 144 k b 3 . 9018 6 7 . 1 2 6 k b 3 . 8528 7 6 . 108 k b 3 . 7 8 5 9 8 5 . 0 9 0 k b 3 . 7 0 6 7 p B R 3 2 2 H i n f I b a n d l e n g t h l o g 1 1 . 6 3 0 k b 3 . 2 1 2 2 2 . 5 1 7 k b 2 . 7 1 3 5 3 . 3 9 6 k b 2 . 5 9 7 7 4 . 3 4 4 k b 2 . 5 3 6 6 5 . 2 9 8 kb 2 . 4742 6 . 2 2 1 kb 2 . 3444 6 . 2 2 0 kb 2 . 3424 7 . 154 kb 2 . 1 8 7 5 * 8 . 075 kb 1 . 8 7 5 1 * b a n d l e n g t h l o g 9 4 . 0 7 2 kb 3 . 6098 10 3 . 0 5 4 kb 3 . 4 8 4 9 11 2 . 0 3 6 kb 3 . 3088 12 1 . 635 kb 3 . 2 1 3 5 13 1 . 018 kb 3 . 0 0 7 7 14 5 1 6 - 5 0 6 b p 2 . 7084 15 . 394 kb 2 . 5 9 5 5 16 . 344 kb 2 . 5 3 6 6 p B l u e S c r i p t H i n f I b a n d l e n g t h l o g 1 1 . 0 7 7 k b 3 . 0 3 2 2 2 . 5 1 7 k b 2 . 7 1 3 5 3 . 4 5 6 k b 2 . 6 5 9 0 4 . 3 9 6 k b 2 . 5 9 7 7 5 . 3 5 6 k b 2 . 5 5 1 4 6 . 075 k b 1 . 8 7 5 1 * 7 . 065 k b 1 . 8 1 2 9 * 8 . 022 k b 1 . 3 4 2 4 * b a n d l e n g t h l o g 17 . 2 9 8 k b 2 . 4 7 4 2 18 . 2 2 0 k b 2 . 3 4 2 4 19 . 2 0 0 k b 2 . 3 0 1 0 20 . 154 k b 2 . 1 8 7 5 21 . 142 k b 2 . 1 5 2 3 22 . 075 k b 1 . 8 7 5 1 * 1 kb ladder was purchased from Pharmacia (?) * not visible on most gels. Agarose Gel Electrophoresis: Most of the agarose gels were run on the BIO-RAD mini gel apparatus, with equipment modifications on some gels. Gels were poured using 50 ml of 0.5x TBE or l.Ox T A E with the appropriate amount of agarose added (0.2 g for a 0.4% gel, 0.5 g for a 1.0% gel, etc.). The increase in volume due to the addition of agarose was assumed to be negligible. The agarose was melted by heating to a boil on a stirring hot plate. After melting, the volume was checked again and distilled water (dH20) was added if significant volume loss was detected. The agarose was then cooled with stiring for about 5-7 minutes.. Just prior to pouring the gel, 5 pi of 1 % ethidium bromide was added to stain the DNA in the gel. Ethidium bromide was also added to the running buffer at 5 pl/100 ml. Note: the 1% ethidium stock solution contained 20% ethanol to prevent bacterial growth. Methods I: Molecular Techniques 52 Electrophoresis was generally carried out at between 50 and 90 mV for between 1 and 3 hours. Longer running times were required for lower voltages and higher percentage gels. Equipment modifications included additionsl well formers (combs), and a 14 cm gel bed: the 14 cm gel bed required about 5 hours to run (a gel of this type was used for the genomic Southern, figure #25). Denaturing Agarose Gel Electrophoresis: Agarose gels for the separation of RNA 1 s were prepared as above in lx MOPS buffer, with the addition of 2.7 ml of 37 % deionized formaldehyde per 50 ml of gel after the addition of the ethidium. Formaldehyde addition and pouring and cooling of the gel are performed in a fume hood. The procedures used are otherwise as stated in Davis etal. (1986). Preparation of Acrylamide Gels for DNA sequencing Sequencing gels were prepared as per Maniatis et al. 1982. In some cases using the modifications as per Gelinas (1987). Spun Column Preparation Spun columns were prepared using a 1 ml plastic pipette tip, an Eppendorf tube cut in half horizontally, and a 5 ml Lucite test tube, by the following procedure: 1) Sephedex G-25 was hydrated in T E 8 and autoclaved for 30 min. on liquid cycle. 2) The 1 ml plastic pipette tip was plugged at the small end with a 1-2 mm tuft of glass wool, which was tamped in place with a pasture pipette. 3) The pipette tip was filled with the Sephedex suspension, and the T E 8 was sucked out the bottom with a vacuum hose until the meniscus was just above the surface of the packed beads. The filled pipet tip was then hung inside the 5 ml plastic Lucite tube using the top half of an Eppendorf tube (with the lid removed) as an adapter sleeve. The column was then centrifuged for 5 min. at approximately 5-6 on a clinical centrifuge. The resulting liquid fraction was then discarded. The sample was loaded on top of the column in a volume of about 50 ul and the column was spun again using the same conditions as the first spin. Methods I: Molecular Techniques 53 PCR conditions for DNA and RNA samples. Conditions for PCR were similar to those described by Linz et al (1990). Buffers were as described by Linz or according to those outlined by Promega. Typical reactions contained 200 uM dNTPs, 50 to 200 pM of each oligonucleotide primer, about 500 ng of template (DNA or whole cellular RNA), 1/10 volume of lOx buffer and 1 unit of Taq polymerase in a volume of 50 ul. Reactions were set up by combining all of the non enzyme parts, covering with 20 to 30 ul of Stanley's light mineral oil, and then heating to > 90 °C before addition of the Taq DNA polymerase. The temperature was then cycled through three phases, denaturation, annealing, and extension. A typical series might be; 3 cycles of 92 °C, 48 °C, and 65 °C for 60 sec, 300 sec, and 90 sec, respectively, followed by 30 cycles of 92 °C for 30 sec, 55 °C for 90 sec, and 70 °C for 60 sec. This kind of protocol was successful with both A series and C series buffers (see Buffers, Taq Polymerase) and various primer pairs (see results). The band 2-2-2 was produced using a lower annealing temperature, (42 °C in the first 3 cycles, 48 °C in subsequent cycles), wcRNA as a template, and A series buffers with 125 mM Tris instead of 50 mM Tris. Cloning into M13 and BlueScript Cloning was done using standard techniques: see Maniatis et al. (1982). Plaque and Colony L i f t s Plaque and colony lifts used an identical procedure as follows: Hybond N nylon filters (Amersham) were carefully placed without prior preparation onto the plates using sterile forceps. After a 45-60 second incubation to adsorb the phage and/or bacteria, the filters were removed and placed phage/bacteria side up on a filter paper soaked in gel denaturing solution on a plastic tray, and left for 7 minutes. The filter was likewise placed on filter paper soaked in gel neutralizing solution twice for 2 minutes, then washed briefly in 2x SSC before partial drying and cross linking for 3 minutes with the U.V. transilluminator. Methods I: Molecular Techniques 54 DNA and RNA Blots DNA blots were prepared using standard methods on Hybond N nylon filters. Briefly, this involved separation of restriction endonuclease digested DNA's on a lx TAE , 0.4 to 1.8 % agarose gel followed by capillary blotting with lOx SSC and crosslinking for 3 minutes with the UV transilluminator. Filters were then prehybridized with either heparin (for [32P] dATP labeled probes) or skim milk powder plus 25 mM EDTA (for Digoxigenin labeled probes: see Boehringer-Mannheim Digoxigenin DNA detection kit), and hybridized with the probe overnight at an appropriate temperature. DNA Sequencing. Single and double stranded sequencing of Ml3 and pBlueScript DNA was done according to the procedures laid out in the Pharmacia T7 DNA polymerase DNA sequencing kit. Sequencing of double stranded pBlueScript DNA clones was done according to the procedures current in Ross MacGillivray's lab, briefly as follows: 1) Combine in a tube, 18 pi of miniprep pBlueScript DNA (STET procedure), 2 pi of sequencing primer, 5 pi of sdH 20, and 2 pi of freshly made 2 M NaOH 5 mM EDTA. 2) tap to mix, then place in boiling water bath for 2 minutes. 3) Quickly add 2 pi of 3 M NaOAc (pH 5.2) and 65 pi of 95 % EtOH: place tubes on ice. Spin down DNA 5 minutes, rinse 2x with 70 % EtOH, decant supernatant, spin briefly, and pipette out last of 70 % EtOH. 4) Resuspend the DNA in 10 pi of lx sequencing buffer and anneal at 37 °C for 10 minutes: place tubes on ice (or freeze for later use). The remainder of the procedure follows that outlined by the Pharmacia T7 instructions. M E T H O D S : METHODS II: BACTERIA PROCEDURES Competent Cell Preparation Competent Cells were prepared according to a protocol provided by Ivan Sadowski's lab. Briefly; cells from several discrete colonies were picked from an overnight LB streak plate grown at 37 °C, and inoculated into 250 ml of SOB in a 2.0 liter flask (the large flask was used to increase aeration of cells). Cells were grown at 37 °C with vigorous shaking and the absorption of the culture was monitored at 650 nm. When the absorption reaches 0.6 to 0.8 the cells were chilled on ice for 15 min., then centrifuged at 4 °C to pellet the cells. The pellet was drained and as much of the liquid as possible was removed. The pellet was then broken up by brief vortexing, resuspended in 83 ml of pre-chilled RF1 (100 mM RbCl, 50 mM MnCb, 30 mM KOAc, 10 mM CaCL:, and 15% glycerol, pH 5.8, filter sterilized), and placed on ice for 30 min. Cells were then re-pelleted by centrifugation at 4 °C and resuspended in 20 ml of RF2 (10 mM RbCl, 10 mM MOPS, 75 mM CaCl 2, and 15% glycerol, pH 6.8, filter sterilize). Cells were left on ice 15 min., divided into 1 ml aliquots in pre-chilled Eppendorf tubes, and flash frozen in liquid nitrogen, an ethanol-dry ice bath, or the -70 °C freezer. Competence was tested by transformation of 200 ul of cells with 0.1 ng of supercoiled pUC 18 and plating 50 ul of cells. This resulted in between 50 and 500 ampicillin resistant colonies, or 10,000,000 to 100,000,000 transformants per ug plasmid DNA. Transformation of Competent C e l l s . This procedure was provided by Ivan Sadowski; briefly, 1 ml of competent cells were removed from the -70 °C freezer and thawed on ice for about 15 to 20 min. The cells were then divided into 200 ul aliquots in pre-cooled Eppendorf tubes, vector DNA was added in a volume of less than 20 ul, and the tube was swirled and placed on Methods II: Bacterial Procedures 56 ice for 40 min. The cells were then heat shocked by placing at 37 °C for 2 min., and then replaced on ice for 2 min. SOC, LB or other complete medium (800 pi) was then added, and the cells were set to incubate at 37 °C for 30 min. before plating on LB agar with 100 pg/ml ampicillin. LA M B D A PR O C E E D U R E S Materials and Tips for Screening a lambda phage Library: Required Items: *1 agar plates: N Z C Y M plates were used, although any of NZY, NZYM, or N Z C Y M should work. 10 cm plates for secondary screening, 14 cm plates for primary screens. Thicker plates result in larger phage plaques containing greater numbers of phage (Ginn & Rapp, 199_). Richer medium may also increase plaque size and phage numbers. *2 top agar: N Z Y M was used, although again, any of NZY, NZYM, or N Z C Y M should work. Top agar contained 6 g /100 ml electrophoresis grade agarose. *4 4x concentration O/N bacterial culture in 10 mM MgS0 4 1) add 10 ml 1.0 M MgS0 4 and 5 ml 10% maltose to 500 ml of LB medium (keep sterile) 2) inoculate 25 ml of culture with c600 or other suitable bacteria. 3) grow bacteria over night at 37 °C with shaking. 4) pellet cells at 2000 G for 10 min. and pour off medium. 5) resuspend cells in 1/4 volume (6 ml) of 10 mM MgS0 4 : place cells at 4 °C. Prepared cells can be used for up to one month. *6 The agar plates should be warm just prior to pouring the top agar. An easy way to keep them warm is to fill a styrofoam shipping box 2/3 full of hot tap water, and cover it with a glass plate, then place the agar plates on top of the glass plate. This keeps the plates at a warm, even temperature for 30-60 minutes. Methods II: Bacterial Procedures 57 T i t e r i n g a lambda Phage Library: 1) In Eppendorf tubes, dilute 1 ul of phage stock into 999 ul of TMG, followed by four 100:1 serial dilutions (10 ul into 1.0 ml). A good phage library contains about 10 u PFU (plaque forming units; a measure of the number of active phage.) per ul, so 10-100 ul from one of the last 3 dilutions should give a countable plate. 2) 10 ul of phage was removed from each of the last 3 dilutions and placed in an Eppendorf tube. 3) plates were place in the 37 °C oven to warm, and the top agar was melted. 4) 200 ul aliquotes of bacterial plating stock were added to each of the Eppendorf tubes prepared in 2), and incubate for 5-15 minutes at room temperature. 5) Three 3.5 ml aliquots of top agar were measured into 3 sterile 15 ml tubes, and place in a 50-55 °C water bath. 6) One at a time the phage-bacteria mixes from 4) were combined with 3.5 ml of molten top agar at 50-55 °C, mixed briefly, and poured onto the prewarmed plates (*6, above). The top agar was spread by tilting the plate in a circular motion, and the plate was then set to cool on a level surface. 8) Once the top agar had set, the plates were placed at 37 °C for 10 to 12 hours, and then counted or transfer to 4 °C until time was available to count them. P l a t i n g a Lambda Phage Library 1) The phage library was titered (see above) and 14 cm plates, top agar, bacterial culture (*4), and other reagents were prepared before hand. 2) Plates were prewarmed (*6) and the top agar was melted and place in a water bath at 50-55 °C. 3) Phage stock was diluted in TMG to a final concentration of 50,000 PFU/ul or less, and mixed with C600 E. coli 4x bacterial culture as follows: - 10 ul (500,000 PFU) phage - 6 ml bacterial culture (plating stock, *4) and incubate for 5-15 minutes at room temperature. Methods II: Bacterial Procedures 58 4) Ten 10 ml aliquots of top agar were measured out into ten sterile 15 ml tubes, these were then placed in a water bath at 50-55 °C. 5) One at a time, 600 pi aliquotes of phage/bacterial culture were pipeted into the previously prepared tubes of top agar, mixed briefly, and poured out on plates taken from the 37 °C incubator. The top agar was spread by tilting the plate around in a circular motion, and the plate was then placed on a level spot on the bench to cool. 6) Once the top agar was set, the plates were placed at 37 °C for 10 to 12 hours, by which time the near complete lysis of the bacterial lawn had occurred. The plates were then transfer to 4 °C prior to being used to prepare lifts for screening. See methods I, "Plaque and Colony Lifts". Rescreening of Positive Phage Plaques 1) Positive signals on the plaque lift filters were identified by screening with the Boerhringer-Mannheim digoxygenin nonradioactive detection kit. Positive plaques were located on the phage plates by superimposing the plate on the filter (NBT was used in screening. This resulted in clearly readable brown spots on the filters at each positive signal: all primary filters were done in duplicate). 2) Using the sterilized wide end of a Pasteur pipette a plug of agar was removed from each of the positive sites and transferred to an Eppendorf tube containing 1 ml of T M G and 10 pi of CHC1 3. The tube was left for at least 2 hours at room temperature or over night at 4 °C to allow the phage time to diffuse out of the gel. 3) The first tube with the agar plug was assumed to be a 1:10 dilution (agar into TMG): 1 pi of agar plug supernatant was then diluted into 99 pi of TMG, followed by three 10:1 serial dilutions of 10 pi into 100 pi. Thus the 2nd tube was 1:103, followed by L10 4 , 1:10s, and 1:106. Agar plugs typically contained about 106 to 107 phage per pi, so plating of 10 pi of each of the last three dilutions (L10 4, 1:10s, 1:106,) generally resulted in one plate with a countable number of plaques. Methods II: Bacterial Procedures 59 4) 10 ul aliquotes of each of dilutions hlO 4 , 1:105, and 1:106 from each of the phage plugs to be rescreened were placed in Eppendorf tubes. Plating then followed the same procedure as that outlined in steps 3) to 8) of "Titering a Phage Library" above. 60 METHODS: METHODS III: DROSOPHILA TECHNIQUES Paraquat Induction of Adult Flies for SOD mRNA. Adult flies were treated with Paraquat (Pq+2) and MnCl 2 to induce synthesis of the Mn SOD gene for later RNA isolation. This was done by placing the flies in an empty fly bottle containing a 3 cm x 10 cm piece of blotting paper soaked with 500 ul of a solution containing one or more of 5.0 mM Pq+2, 1.0 mM M n C l 2 , and 10 % sucrose. Flies were left in the bottles overnight then dumped directly into D lysis buffer (4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% Sarkosyl, 0.1 M 2-mercapto ethanol) and immediately ground up with a Dounce homogenizer. Four sets of conditions were tried for Mn SOD RNA induction; Mn, Mn/sucrose, Pq+2/sucrose, and Mn/Pq+2/sucrose. RNA was isolated from flies in each treatment group separately. RNA from all four treatments was used in separate reactions for RNA directed PCR. Paraquat Induction of Fly Larvae for SOD mRNA. Isolated Drosophila larvae were treated with Paraquat (Pq+2) by suspending the larvae in 4.5 ml of dH 2 0 and adding 200 ul of 100 mM Pq+2 and 100 ul of 100 mM MnCl 2 . This was left at room temperature for about 1 hour, and then placed at -70 °C overnight. The next day the frozen larvae were ground to a powder in a precooled mortar and pestle, and stirred into 8 ml of D lysis buffer (see above) in a 15 ml plastic tube. RNA extraction then proceeded as per the RNA isolation procedure mentioned earlier. Isolation of Fly Larvae from Fly Food. Fly larvae were isolated by heating the bottom of the fly bottle around the fly food in hot water. The larvae move to the coolest place they can find: on top of the fly food away from the walls of the bottle. They are then scooped up with a scoopula and suspended in distilled water, where the remaining fly food can be washed away, and the larvae recovered by panning and decanting off the water. 61 RESULTS: STUDY PREMISE In three previous studies (Seto et al. 1990 , Reveillaud, 1 9 9 1 , and Orr & Sohal, 1994) attempts were made to extend the LSP of Drosophila by overexpression of the cytosolic and peroxisomal Cu-Zn SOD. These studies did not result in spectacular increases in LSP for any of the transformed lineages: indeed, except in the case of Orr and Sohal ( 1994 ) , who co-overexpressed catalase in the same flies, the increases in LSP were undetectable except by careful statistical analysis. However, the strong hydration sphere of superoxide in solution, coupled with its extreme reactivity in the absence of solvent makes it impossible for it to cross intact biological membranes. Moreover, most of the superoxide produced in the cell is made in the mitochondrial matrix. For these reasons, mitochondrial matrix suppression of superoxide levels may have a far greater effect on LSP. This project was designed to isolate the mitochondrial matrix Mn SOD of Drosophila melanogaster for subsequent over-expression studies. JAN90 SOUTHERN BLOTS In an initial attempt to isolate the Drosophila Mn SOD gene by direct hybridization to a Lambda phage library, the synthetic oligonucleotide JAN 9 0 was created using protein similarities between known Mn SOD and Fe SOD proteins. Restriction digests of Drosophila genomic DNA were hybridized with alpha [32 P] dATP labeled JAN 9 0 , but only nonspecific binding was observed. At this time the direct hybridization approach was dropped in favor of the new technique, PCR. STUDIES WITH PCR In an attempt to isolate a partial sequence of the Drosophila Mn SOD gene for use as a probe for library screening, several PCRs were tried. They used existing and newly designed synthetic oligonucleotides based on protein sequence similarities between known Mn SOD and Fe SOD proteins. Primer combinations included Results 62 GT1.6-90, S2.6-90, SCl:6-90, SC1:T17XSP, and GSGW:HEW on DNA and wcRNA templates (see figure 2 for primer locations and orientations). These studies resulted in the isolation of a copia-like element 3* LTR (S2.6-90) and other undetermined sequences, but did not result in the isolation of any Mn SOD gene sequences, (data not shown). Results 63 Figure # 2: Positions of Oligonucleotides Used in this Study. This figure was constructed to allow analysis of the various PCR experiments done in this study. It shows the positions of the various oligonucleotides used relative to the sequences they were meant to bind to. Mis-matches are identified by lower case letters in the oligonucleotide sequence. The added restriction sites, Hind III on SOD 5' and Pst I on SOD 3' are in lower case and underlined. In the probe Jan 90, two sites are replaced by asterix (*). The one at the 3' end of the probe, gTc g*A, is either A or T, while the second at egg *TC is either C or G. Results 64 Figure # 2 Positions of Oligonucleotides Used in this Study. -17 -15 ,-11 +C GAC TTA GCC TTA TTA GCA GTC GAA TAA AAC GCA GAT ATG TTC GTG GCC CGT AAA ATT TCG 120 Met Phe Val A l a Arg Lys l i e Ser -5 -G . -1 +1 5 10 > S 2 5'CCN TAY GAY TAY CAA ACT GCA AGC CTG GCG GTG CGT GGC AAG CAC ACC CTA CCG AAG CTG CCC TAC GAC TAT 180 Gin Thr A l a Ser Leu A l a Val Arg Gly Lys His Thr Leu Pro Lys Leu Pro Tyr Asp Tyr GGI ATc gTc g*A < JAN-90 15 20 25 30 > S 0 D 5 / > 5' nnn aag c t t G GAG ATC ATG GAG CTG CAT CAC C 3' —S2-> > GT1 > > SC1-GgN GC 3" 5' GAR CCN caY ATN aRY gcN GAR ATN ATG cA 3' 5'AAG CAt CAt GCC GCC CTG GAG CCT ATC ATC TGC CGG GAG ATC ATG GAG CTG CAT CAC CAG AAG CAC CAC 24 0 A l a A l a Leu Glu Pro l i e H e Cys Arg Glu H e Met Glu Leu His His Gin Lys His His -JAN 90-35 SCI > gcG gCg TAt GTg AA 3' 55 75 95 115 -GSGW' > 5' Ggc GTg CAG ACG CTG ACC GTG GCG GTC CAG Thr Leu Thr Va l A l a V a l Gin 135 GGC AAA CTG CAA CTG GCC Gly Lys Leu Gin Leu A l a CCG TT gac gtc nnn 5' --< 155 ATC CCG CTC TTC GGC ATC H e Pro Leu Phe Gly H e 175 CGT CCC TCC TAC GTG GAG Arg Pro Ser Tyr Val Glu egg *TC TAG TAC gTC GAC GT --< 40 45 50 GCC GCC GAG GAG CAG CTG GAG GAG GCC AAG TCG AAG 300 A l a Ala Glu Glu Gin Leu Glu Glu A l a Lys Ser Lys 60 65 70 CTG GCT CCT GCC CTG CGT TTC AAT GGC GGT GGC CAC 360 Leu A l a Pro Ala Leu Arg Phe Asn Gly Gly Gly His 80 85 90 AAC CTC TCG CCC AAC AAG ACC CAG CCC AGC GAT GAT 420 Asn Leu Ser Pro Asn Lys Thr Gin Pro Ser Asp Asp 100 105 110 TGG AAG AGC CTC GAG GAG TTC AAA AAG GAG CTG ACC 480 Trp Lys Ser Leu Glu Glu Phe Lys Lys Glu Leu Thr 120 125 130 1 -> TCN GGN TGG G 3 TCC GGC TGG GGC TGG CTG GGC TTC AAC AAG AAG TCG 540 Ser Gly Trp Gly Trp Leu Gly Phe Asn Lys Lys Ser 3 CG AAG TTG TTC TTC AGC <— SOD 3' — 140 145 150 CCC AAC CAG GAT CCC CTG GAG GCC TCC ACC GGC CTG 600 Pro Asn Gin Asp Pro Leu Glu Ala Ser Thr Gly Leu 160 165 170 TGG GAG CAC GCC TAC TAT CTG CAG TAC AAG AAC GTG 660 Trp Glu His Ala Tyr Tyr Leu Gin Tyr Lys Asn Val ACC CTY GTR CGt ATG ATg GA 5' < -HEW- --< 180 185 190 TGG GAC ATC GCC AAC TGG GAT GAC ATC TCG TGC CGC 720 Trp Asp H e Ala Asn Trp Asp Asp H e Ser Cys Arg ACC tTG cAG taG TTG ACC 5' < 6--90— < 195 200 TTC CAG GAG GCC AAG AAG CTC GGT TGC TAA GCA CAG CCG GCG ATT GCG TAT GTT TAA TCT 780 Phe Gin Glu A l a Lys Lys Leu Gly Cys OCH Results 65 ISOLATION OF AN ARCHAEBACTERIAL S O D LIKE SEQUENCE In another attempt to obtain a partial sequence for use as a probe for l ibrary screening, another P C R was tried using oligonucleotides S2, S C I and H E W (see figure 2). Protein similarities indicate that, in the absence o f introns, amplif ication o f a M n S O D gene with S C I and H E W should result in a fragment o f about 426 bases, or with S2 and H E W about 485 bases. T h e initial P C R resulted in the production o f two prominent products, one about 700 bases and one around 430 bases (see figure 3). A second P C R , undertaken to determine pr imer use in the formation o f these products, demonstrated that both D N A fragments were the products o f S C I and H E W , and that no other primers alone or in pairs could give this result (data not shown). In order to determine i f the 430 base P C R product was derived from a M n S O D gene, both strong bands were subcloned into pBlueScript and a clone o f the 430 base band was subjected to sequence analysis. T h e resulting D N A sequence was read as protein in the same reading frame as that o f the primers and found to code for a M n S O D like protein (see figure 4). Sequence alignment with other known M n S O D proteins revealed it was most similar to the M n S O D s from halophil ic Archaebacteria, casting doubt that this clone even originated from Drosophila. Results 66 Figure #3: PCR with S2. SCI and HEW In an attempt to isolate a fragment of the Drosophila Mn SOD gene, PCR was tried using oligonucleotides S2, SCI and HEW on adult Drosophila DNA from DNA isolation 14.1. A strong band (arrow B, lanes e, g, and h) of the correct size (about 420 bases) for a putative Mn SOD cDNA fragment from SCI and HEW was produced in 3 of the reactions. Three clones of this band were obtained, and designated as BSC 17, BSC20, and BSC23. One clone of the band at arrow A in lanes e, g, and h was also obtained; this clone was designated BSC4. Lanes a-c are from another similar experiment. Lanes d, e, g, h, and i are PCR reactions with Drosophila DNA 14.1 and 1.5, 2.0, 2.5, 2.5, and 5.0 mM MgCl 2 , respectively. Lane j is PCR with sheared salmon sperm DNA. Lane f contains pBlueScript cut with Hinf I as a size standard (size in Kb recorded on the right of the photograph. Results 67 Figure #4: DNA Sequence and Amino Acid Translation of BSC23 In order to determine if clone BSC23 was a Mn SOD gene fragment, a tentative sequence of the inserted DNA was obtained. The resulting DNA sequence was translated in the reading frame corresponding to the original primer oligonucleotides (the primer sequences have been removed in this figure) and compared to other Mn SOD genes to determine sequence similarity. The resulting amino acid sequence spans the entire fragment and is very similar to other Mn SODs and Fe SODs, particularly those from Archaebacteria. This figure shows the DNA sequence and amino acid translation in 3 letter code, and the putative protein sequence in single letter code at the bottom. Results 68 1 9 18 27 36 45 GGA TTA AAT AAA GCT GAA GAA GAG ATT TAT TAC CGT AAT CAT GAA Gly Leu Asn Lys Ala Glu Glu Glu l i e Tyr Tyr Arg Asn His Glu + - + (+) -54 63 72 81 90 CCG GAT ATG TTA AGA CAT TGG TTA CGG GAG CAG GCT TTC AAC GGA Pro Asp Met Leu Arg His Trp Leu Arg Glu Gin Ala Phe Asn Gly + (+) + -108 135 TCA GGG CAT CTG CTT CAC TCT GTT TTT TGG AAA AAT ATG ACC CCA Ser Gly His Leu Leu His Ser Val Phe Trp Lys Asn Met Thr Pro (+) (+) + 153 180 TAC TCA TCA AAA GTA CCA AGC AAA CAG ATA GAG CAA TGG ATT AAT Tyr Ser Ser Lys Val Pro Ser Lys Gin l i e Glu Gin Trp l i e Asn + + -198 225 AGG GAC TTT TGG CTT CTT TGC CAT TTT AAG GAA GGT TTT ACA AAT Arg Asp Phe Trp Leu Leu Cys His Phe Lys Glu Gly Phe Thr Asn + - (+) + -243 270 GTC GCC 7A7AA AGT GTC CAA GGT CCG GGC TGG GCT GGA TTA TTG TAC val Ala Lys Ser Val Gin Gly Pro Gly Trp Ala Gly Leu Leu Tyr + 288 315 GAT CCA GTT AAC CAC CGG CTT GTA ATT GAA TCA ATT GAG AAA CAT Asp Pro Val Asn His Arg Leu Val l i e Glu, Ser l i e Glu Lys His ( + ) + - - + ( + ) 333 360 CAG CAG AAT CAC CTC GTT TCG ATG ATT CCG CTC CTT GTC CTT GAT Gin Gin Asn His Leu Val Ser Met l i e Pro Leu Leu Val Leu Asp ( + ) 363 ATG met 10 20 30 40 50 GLNKA EEEIY YRNHE PDMLR HWLRE QAFNG SGHLL HSVFW KNMTP YSSKV 60 70 80 90 100 PSKQI EQWIN RDFWL LCHFK EGFTN VAKSV QGPGW AGLLY DPVNH RLVIE 110 120 SIEKH QQNHL VSMIP LLVLD M Results 69 PLASMID CONTAMINATIONS In order to determine if this clone represented a Drosophila gene fragment, several Southern blots were conducted. For the first blots, labeled probe was prepared by linearising the plasmid clone (BSC23) with Eco RI and labelled with digoxygenin, according to the instructions given (Boehringer-Mannheim, non-radioactive detection kit). These blots gave a strong band (arrow A figure 5) of about 3 Kb for every 6 base recognition sequence restriction endonuclease tested, including Pst I, Eco RI, and Hind III (Bam HI gave a band at the same location as Pst I in a separate gel, data not shown). This band was tentatively assigned as plasmid contamination of Drosophila DNA isolation 16. The Hind III digest in figure 5 was incomplete, and shows additional bands at about 6 Kb and a weak pair of bands at 9 Kb, which suggests that the plasmid sequence is in the form of a tandem repeat, probably integrated into the Drosophila genome. Results 70 Figure #5: Genomic Southern Blot with Klenow Fragment Labeled BSC23 In order to determine if BSC23 represented a single copy gene from Drosophila, a genomic Southern blot was conducted using Drosophila DNA cut with Pst I, Hind III, Eco R I, Hin F l , Sau 3A I, and Hha I (lanes a to d, f, and g, respectively). The blot was probed with plasmid BSC23, linearised with Eco RI and labeled with Klenow DNA polymerase and digoxygenin labeled nucleotides. Lane e contains a Lambda Hind III digest as size markers. Results 71 DEMONSTRATION THAT BSC23 is NOT A DROSOPHILA GENE. In order to avoid interference from the plasmid sequences contained in the first probe and in the Drosophila genomic DNA, a second probe was created using PCR of the Eco RI linearised BSC23 with the original primers, SCI and HEW. This probe was used for several additional genomic Southern blots. The results were varied. After numerous highly ambiguous Southern blots, it was finally realized that all of the positive signals from the BSC23 probe were from restriction digests of DNA from isolations 15 or 16. Subsequent blots with the known Drosophila melanogaster Mn S O D gene (see below) gave positive results only with later DNA isolations. From this it was concluded that (1) DNA isolations 15 and 16 consisted primarily of contaminating DNA not from Drosophila, and (2) clone BSC23 was the product of that contamination and not a Drosophila gene. As a final demonstration of the mutual exclusivity of the two DNAs, two Southern blots of the same restriction digests, run side by side on the same gel, were conducted (see figure 6). Lanes a, b, f, and g show clearly that the binding of the probes is exclusive, with positives from one probe complemented by the absence of binding by the other for a given DNA isolation, as was observed in previous blots with both probes (data not shown). Lanes c and h in figure 6 are short of DNA and the digests appear incomplete, while the DNA in lane i is extensively degraded. As a result these data were not deemed useful. Results 72 Figure #6: Comparison of Hybridizations, Doolittle VS. BSC23 BSC23's ambiguous experimental results and extensive similarity to Archaebacterial Mn SODs gave rise to the suspicion that it might be derived from a contamination of the DNA isolations (DNA isolations 14, 15, and 16) used in its generation. To determine if this was true, two Southern blots of the same restriction digests, run side by side on the same gel were carried out. The filter on the left was hybridized with the Drosophila melanogaster digoxygenin labelled probe created by PCR with SOD 5' and SOD 3'. The filter on the right was hybridized with the BSC23 digoxygenin labelled probe created by PCR of Eco RI cut BSC23 with SCI and HEW. The results from lanes a, b, f, and g show that BSC23 does hybridize to DNA isolation 16, but not to the later DNA isolations, while the reverse is true for the Drosophila Mn SOD. Lanes c, d, h, and i failed to give a clear cut result. The digests and expected results were as follows: E x p e c t e d B i n d i n g : O b s e r v e d B i n d i n g : L A N E S : E n z y m e DNA F l y SOD B S C 2 3 F l y SOD B S C 2 3 L a n e a a n d f : Bam H I 2 8 . 1 + + L a n e b a n d g : E c o R I 1 6 . 2 + + L a n e c a n d h : H i n d I I I 2 9 . s i + -L a n e d a n d i : P s t 1 1 6 . 2 + +/--the column marked "DNA" shows the number of the specific DNA isolation used in these lanes. Results Figure #6: Comparison of Hybridizations, Doolittle VS. BSC23 a b c d e f g h i Results 74 ISOLATION OF THE DROSOPHILA MN SOD GENE In January of 1992 Smith and Doolittle published several partial sequences of Mn SODs obtained by PCR, including one for Drosophila. In order to obtain a probe for library screening, 2 PCR oligonucleotides matching the ends of the published sequence, SOD 5' and SOD 3', were created (see figure 2). Subsequent PCR of Drosophila genomic DNA resulted in the isolation of a band of the expected size (see figure 7), which was cloned into pBlueScript. A preliminary sequence was obtained from one end of the clone, which demonstrated that the PCR product matched Smith and Doolittle1 s (1992) published sequence. Results 75 Figure #7: PCR of Drosophila Mn SOD partial D N A In 1992 Smith and Doolittle (1992) published a PCR derived partial sequence of the Drosophila M n SOD gene. In order to obtain this sequence for use as a probe, two oligonucleotides (SOD 5' and SOD 3') based on their sequence were synthesized and used for PCR of Drosophila genomic D N A . This gel shows the results of the first successful PCR with these primers. The resulting D N A was cloned into pBlueScript and a partial sequence was obtained in order to demonstrate that the Drosophila Mn SOD gene fragment had been obtained. The band at arrow B is the full lenght PCR product. The band at arrow A is undetermined. The lanes contained as follows: c and h: 1 Kb ladder D N A size standards (sizes at right), lanes a, b, g, i , j , Drosophila D N A subjected to PCR with SOD 5':SOD 3' (a and j), SOD 3' and H E W (g), and SOD 5' and H E W (i), lanes b,d, e, f, used 1 pi of a previous PCR with Drosophila D N A and SOD 5':SOD 3' with 10% DMSO. They contained 1.5, 2.0, 2.5 and 5.0 mM M g C l 2 , respectivly. a b c d e f g h i j 12.2--1.64 1.02 Results 76 LIBRARY SCREENING An agarose gel block of the original PCR product was eluted by diffusion and used as a PCR template to make a digoxygenin labeled probe using SOD 5* and SOD 3'. The resulting digoxygenin labeled DNA was used to probe a Lambda cDNA library derived from a Drosophila imaginal disc tissue culture (see figure 8). Twenty-one positive plaques were identified in an initial screen of part of the library. Eight clones were purified to homogeneity, and 7 were grown up in broth culture to isolate DNA. The insert DNA was subsequently cut out with Eco RI and ligated into the Eco RI site of pBlueScript (see figures 9 and 10). The pBlueScript clones were named with the first number and letter copied from the phage isolate from which they are obtained, and the last number differentiating each independent clone of that phage insert. Size variability among the inserts indicated that at least 5 independent clones were represented by the seven isolates. The descending order of sizes was coincidental (note that 10A is larger than 9A, and could be equivalent to 8F). The band sizes show that 6A and 8A could also be equivalent. The Eco RLBam HI digest in figure 9 shows that cDNA 9A differs form the rest in a fragment more or less common to all the others (this turns out to be a 66 base deletion form the 5' end of the cDNA). Results 77 Figure #8: Screening of Lambda GT10 Phage Library: Primary Screen In order to obtain a complete clone of the Drosophila Mn SOD gene, a Lambda GT10 cDNA library was screened using the digoxygenin labeled probe produced by P C R with SOD 5' and SOD 3'. This photograph shows filter 2A, one of seven phage lifts screened. Thirteen positives are visible on this filter, 5 of which were picked and eluted for secondary screening. Results 78 Figure #9: Gel Separation of Isolated Lambda GT10 Phage Insert DNAs In order to obtain phage insert D N A for subcloning and gain more evidence that the correct clones had been obtained, isolated phage D N A was cut with Eco R I to release the cDNA insert. Because the sequence determined by Smith and Doolittle (1992) contains a Bam HI site near the 3' end of the published sequence and about 2/3 of the way through the expected protein coding region, phage D N A was also cut with Eco RI and Bam HI for comparison,. Lanes a-e, g and h, in the left hand gel contain Eco RI digests of clones 2D, 2F, 6A, 8A, 8F, 9A, and 10A, as indicated at the top of the gel photograph. The right hand gel contains Bam HI/Eco RI digests of clones 2D, 2F, 6A, 8A, 8F, and 9A as indicated at the top of the gel photograph. The length in kilo bases of the 1Kb D N A size markers are noted to the right of each photograph. Results 79 Figure #10: Gel Separation of pBlueScript Clones from Lambda Phage Positives The seven cDNA isolates from the Lambda library were subcloned into pBlueScript in order to facilitate D N A sequencing. This gel shows Eco RI digests of various phage derived pBlueScript clones done to demonstrate the presence of phage derived insert D N A . Lanes a to m were, 1 Kb ladder, 2F3, 6A1, 6A2, 8A1, 8A2, 8F1, 8F3, 9A1, 9A2, 10A1, pBlueScript Hinf I ladder, and 1 Kb ladder, respectively. The lower right side of the gel photograph came out very dark on reprinting, and for this reason the smaller bands of the two ladders on the right hand side are virtually invisible. The bands sizes in kilobases of the 1 Kb ladder are displayed on the left hand side of the gel photograph. rH CO r H C N J r H O J r H C O r H C s l r f ' CM V O V £ > G O G O O O a O O * \ C r v , - H a b c d e f g h i j k l m 1 2 . 2 -Results 80 SEQUENCING OF THE DROSOPHILA MN SOD cDNAs Preliminary sequencing revealed that the clones were divided into two alleles, both carrying two poly A addition signals. Both alleles were subcloned as smaller fragments by digestion of Eco RI isolated clone DNA with Hpa II or Taq I (figure 11) followed by ligation into the Cla I site of pBlueScript. Sequences obtained from these subclones were then compiled into two final sequences, one for each allele (see figure 12 for sequencing strategy). Clones 2D and 2F were designated as allele 1 (figure 13) and clones 6A, 9A, and 10A were designated as allele 2 (figure 14). The final sequences of both DNA and Protein were the compared with those obtained by Dutteroy et al., (1994) and Smith and Doolittle, (1992) (figures 13 and 14, and tables 6 and 8). Careful examination of the sequence data indicates that allelic differences between alleles 1 and 2 in regions between bases 300-340, 572-579, and 800-840 may have been missed as none of the sequences from clones 2D and 2F cover these regions (see figure 12) As the clone isolated by Duttaroy et al. (1994) is incomplete on the ends, information on polymorphisms in these regions is lacking (see figure 13). Results 81 Figure #11: Gel of Hpa II and Tag I Digested Insert DNAs In order to prepare the cloned cDNAs for more exhaustive sequence analysis, several subclones were created. This gel shows separation of the isolated plasmid insert cDNAs 2D1 (lanes a & d), 2F3 subclone 3.1 (lanes b & e), and 6A1 (lanes c & f), after digestion with Taq I (lanes a-c) and Hpa II (lanes d-f). The bands on the far right are pBlueScript/Cla I and pBlueScript/Cla LEco R I (lanes g and h, respectively) prepared as vector to accept the new inserts for subcloning. g 1 2 . 2 - 3 . 0 5 Results 82 Figure #12: Subcloning and Sequencing Strategy To obtain an accurate sequence of the cloned cDNAs, various subclones were generated, from these a series of sequencing gels, covering both strands of the cDNA, were generated. The diagram below shows the locations of the various subclones. Important restriction sites are shown across the top of the diagram. Arrows showing sequences obtained are displayed below. Clones taken from allele 1 are shown as bold lines, while those from allele 2 are shown with finer lines. The individual subclones were obtained from the original phage clones as described in Table #5: o r i g i n a l c l o n e : d e r i v e d c l o n e s : Table #5: Subclone Lineage's. 2D1 2F3 € TBC2 KF# C TGI 3.# I* 6A1 A MS 2 KB# HW# TYZ4 TV HXY3 TW1 10A ME# DA HB1 KD# BA l.# HC3 Results Figure #12: Subcloning and Sequencing Strategy as © S £ ? 5 o 2-5 ~ « 5v K fl I fl £ lid £ £ — I 1 1 I i I L L J I TGI TW1/TBC2 HXY3 HB1 ME5 HW1 KB1 MSI BA/CA/DA 3.1/3.2 KD/KF Clone 9A Clones 2D and 2F Clone 6A Clone 10A compiled sequence from 5' end compiled sequence from 3' end : > < Results 84 Figure #13: DNA Sequence of Allele #1 with Sequence Polymorphisms This figure shows the DNA sequence for cDNA allele number 1, (clones 2D and 2F) in parallel with the sequence for the cDNA obtained by Duttaroy et al. (1994). This alignment allows rapid location of individual allelic differences between allele #1, allele #2, and the sequences obtained by Duttaroy et al. (1994) and Smith and Doolittle (1992). These differences are noted in table 6 below and in the sequence by bold uppercase letters in Dr. Duttaroy's sequence. Frame shifted sequences are underlined. Table #6: Mn SOD with Polymorphisms from Clones 2D/2F (Allele #n Mn SOD with Polymorphisms from clones 2D/2F (Allele #11 l o c a t i o n # 121 138 168 402 468 475 567 767 820 850 860 Clone: A l l e l e #1 T G G C T T A G T (2D/2F) A l l e l e #2 G G A C C C A G G (6A/9A/10A) A l l e l e # 3 +C -G G T A C C C G -G *1 A l l e l e #4 G *2 *1 Duttaroy et a l . , 1994. *2 Smith & D o o l i t t l e , 1992. The bold lower case "g" in the top sequence at base 850 was inferred from close scrutiny of the gels coupled with experiments demonstrating that Sph I does not cut in this location. Results 85 10 20 30 40 50 • • • • • • • • • • G AAC AAG C GA AAT AAC GAGA ACGTAAGCCT TGTTTTATTT GATATAAAAT 50 CTGATATCGG GACTTAGCCT TATTAGCAGT CGAATAAAAC GCAGATATGT 100 cg g g c c t t a t t a g c a g t cgaataaaac g c a g a t a t g t TCGTGGCCCG TAAAATTTCG CAAACTGCAA GCCTGGCGGT GCGTGGCAAG 150 t c g t g g c c c g taaaatttcgCCAAACTGCAA GCCTGGC gt gcgtggcaag CACACCCTAC CGAAGCTTCC CTACGACTAT GCCGCCCTGG AGCCTATCAT 2 00 c a c a c c c t a c cgaagctGcc c t a c g a c t a t g c c g c c c t g g a g c c t a t c a t CTGCCGGGAG ATCATGGAGC TGCATCACCA GAAGCACCAC CAGACCTACG 2 50 ct g c c g g g a g a t c a t g g a g c t g c a t c a c c a gaagcaccac c a g a c c t a c g TCAACAATCT AAATGCCGCC GAGGAGCAGC TGGAGGAGGC CAAGTCGAAG 300 t c a a c a a t c t a a a t g c c g c c gaggagcagc tggaggaggc c a a g t c g a a g AGCGACACCA CCAAGCTGAT TCAGCTGGCT CCTGCCCTGC GTTTCAATGG 350 agcgacacca c c a a g c t g a t t c a g c t g g c t c c t g c c c t g c g t t t c a a t g g CGGTGGCCAC ATCAACCACA CCATCTTCTG GCAGAACCTC TCGCCCAACA 4 00 cg g t g g c c a c a t c a a c c a c a c c a t c t t c t g g c a g a a c c t c t c g c c c a a c a AGACCCAGCC CAGCGATGAT CTGAAGAAGG CCATCGAGTC GCAGTGGAAG 4 50 aTacccagcc c a g c g a t g a t ctgaagaagg ccat-cgagtc gcagtggaag AGCCTCGAGG AGTTCAAGAA GGAGCTGACC ACGCTGACCG TGGCGGTCCA 500 ag c c t c g a g g a g t t c a a A a a ggagCtgacc a c g c t g a c c g t g g c g g t c c a GGGCTCCGGC TGGGGCTGGC TGGGCTTCAA CAAGAAG TCG GGCAAACTGC 550 ggg c t c c g g c t g g g g c t g g c t g g g c t t c a a c a a g a a g t c g g g c a a a c t g c AACTGGCCGC CCTGCCTAAC CAGGATCCCC TGGAGGCCTC CACCGGCCTG 600 a a c t g g c c g c c c t g c c C a a c c a g g a t c c c c t g g a g g c c t c c a c c g g c c t g ATCCCGCTCT TCGGCATCGA TGTCTGGGAG CACGCCTACT ATCTGCAGTA 650 a t c c c g c t c t t c g g c a t c g a t g t c t g g g a g c a c g c c t a c t a t c t g c a g t a CAAGAACGTG CGTCCCTCCT ACGTGGAGGC CATCTGGGAC ATCGCCAACT 7 00 caa g a a c g t g c g t c c c t c c t acgtggaggc c a t c t g g g a c a t c g c c a a c t GGGATGACAT CTCGTGCCGC TTCCAGGAGG CCAAGAAGCT CGGTTGCTAA 750 gggat g a c a t c t c g t g c c g c t t c c a g g a g g ccaagaagct c g g t t g c t a a GCACAGCCGG CGATTGTGTA TGTTTAATCT ATAAGCATCT GCGGATCGGA 800 gcacagccgg c g a t t g C g t a t g t t t a a t c t a t a a g c a t c t gcggatcgga GATCCTAAAT TGTAGTCTAA GTTGCGGCTA ATAAATTGGT ACCAGCTAGg 850 g a t c c t a a a t t g t a g t c t a G g t t g c g g c t a a t a a a t t g g t a c c a g c t a g _ CAACTAATTT TTATAATTTT TATGAATGGG AATAAAACAA TTTAAAACATA 901 c a a c t G Results 86 Figure #14: DNA Sequence and Amino Acid Translation of Allele #2 This figure shows the relative placement of the amino acid coding region, 5' untranslated region, and poly A addition signals on the DNA sequence of allele number 2 (clones 6A, 9A, & 10A). Sites of single base polymorphism are underlined in bold. Three frame shifts in Duttaroy et al. (1994) relative to this clone were noted, one single base insertion, and two single base deletions; these positions are underlined with the inserted and deleted bases noted above. The two poly A addition signals are marked with "—#—>•' over top of the A A T A A A consensus sequence. The first site is used by clone 9A, while the second site is used by clones 2D, 2F, and 10A. Clone 6A terminates without a poly A tail at a point past the first addition site. Amino Acid Translation. Single letter amino acid code representation of the Drosophila Mn SOD gene allele #1 & #2, with amino acid sequence polymorphisms from Duttaroy et al. (1994) and Smith and Doolittle (1992) signal peptide: KHTLPKLPYD YAALEPIICR KSDTTKLIQL APALRFNGGG KSLEEFKKEL TTLTVAVQGS V *2 LIPLFGIDVW EHAYYLQYKN *1 Duttaroy et al. 1994. *2 Smith and Doolittle, 1992. MF VARKI SQTAS LAVRG 0 PNCK PG *1 EIMELHHQKH HQTYVNNLNA AEEQLEEAKS 50 HINHTIFWQN LSPNKTQPSD DLKKAIESQW 100 N *1 GWGWLGFNKK SGKLQLAALP NQDPLEASTG 150 VRPSYVEAIW NIANWDDISC RFQEAKKLGC 2 00 Results Figure #14: MnSOD cDNA Sequence and Amino Acid Translation of Allele #2 GAA CAA GCG AAA TAA CGA GAA CGT AAG CCT TGT TTT ATT TGA TAT AAA ATC TGA TAT CGG 60 -17 -15 -11 +C GAC TTA GCC TTA TTA GCA GTC GAA TAA AAC GCA GAT ATG TTC GTG GCC CGT AAA ATT TCG 120 Met Phe Val A l a Arg Lys H e Ser -5 -G - 1 + 1 5 ' 10 CAA ACT GCA AGC CTG GCG GTG CGT GGC AAG CAC ACC CTA CCG AAG CTG CCC TAC GAC TAT 180 Gin Thr A l a Ser Leu A l a V a l Arg Gly Lys His Thr Leu Pro Lys Leu Pro Tyr Asp Tyr 15 20 25 30 GCC GCC CTG GAG CCT ATC ATC TGC CGG GAG ATC ATG GAG CTG CAT CAC CAG AAG CAC CAC 24 0 A l a A l a Leu Glu Pro H e H e Cys Arg Glu H e Met Glu Leu His His Gin Lys His His 35 40 45 50 CAG ACC TAC GTC AAC AAT CTA AAT GCC GCC GAG GAG CAG CTG GAG GAG GCC AAG TCG AAG 300 Gin Thr Tyr V a l Asn Asn Leu Asn A l a A l a Glu Glu Gin Leu Glu Glu A l a Lys Ser Lys 55 60 65 70 AGC GAC ACC ACC AAG CTG ATT CAG CTG GCT CCT GCC CTG CGT TTC AAT GGC GGT GGC CAC 360 Ser Asp Thr Thr Lys Leu H e Gin Leu A l a Pro A l a Leu Arg Phe Asn Gly Gly Gly His 75 80 85 90 ATC AAC CAC ACC ATC TTC TGG CAG AAC CTC TCG CCC AAC AAG ACC CAG CCC AGC GAT GAT 4 20 H e Asn His Thr H e Phe Trp Gin Asn Leu Ser Pro Asn Lys Thr Gin Pro Ser Asp Asp 95 100 105 110 CTG AAG AAG GCC ATC GAG TCG CAG TGG AAG AGC CTC GAG GAG TTC AAA AAG GAG CTG ACC 4 80 Leu Lys Lys A l a H e Glu Ser Gin Trp Lys Ser Leu Glu Glu Phe Lys Lys Glu Leu Thr 115 120 , 125 130 ACG CTG ACC GTG GCG GTC CAG GGC TCC GGC TGG GGC TGG CTG GGC TTC AAC AAG AAG TCG 54 0 Thr Leu Thr V a l A l a V a l Gin Gly Ser Gly Trp Gly Trp Leu Gly Phe Asn Lys Lys Ser 135 140 145 150 GGC AAA CTG CAA CTG GCC GCC CTG CCC AAC CAG GAT CCC CTG GAG GCC TCC ACC GGC CTG 600 Gly Lys Leu Gin Leu A l a A l a Leu Pro Asn Gin Asp Pro Leu Glu A l a Ser Thr Gly Leu 155 160 165 170 ATC CCG CTC TTC GGC ATC GAT GTC TGG GAG CAC GCC TAC TAT CTG CAG TAC AAG AAC GTG 660 H e Pro Leu Phe Gly H e Asp Val Trp Glu His A l a Tyr Tyr Leu Gin Tyr Lys Asn Val 175 180 185 190 CGT CCC TCC TAC GTG GAG GCC ATC TGG GAC ATC GCC AAC TGG GAT GAC ATC TCG TGC CGC 7 20 Arg Pro Ser Tyr V a l Glu A l a H e Trp Asp H e A l a Asn Trp Asp Asp H e Ser Cys Arg 195 200 TTC CAG GAG GCC AAG AAG CTC GGT TGC TAA GCA CAG CCG GCG ATT GCG TAT GTT TAA TCT 780 Phe Gin Glu A l a Lys Lys Leu Gly Cys OCH — ft — > ATA AGC ATC TGC GGA TCG GAG ATC CTA AAT TGT AGT CTA AGT TGC GGC TAA TAA ATT GGT 84 0 - G — « — > ACC AGC TAG GCA ACT AAT TTG TAT AAT TTT TAT GAA TGG GAA TAA AAC AAT TTA AAA CAT A 90 Results 88 DEMONSTRATION OF A SINGLE COPY GENE IN DROSOPHILA In order to demonstrate that there is one and only one copy of the Mn SOD gene in Drosophila a Southern blot was done using several restriction enzymes and enzyme pairs and the original digoxygenin labelled probe (figure 15). This resulted in a single band present for each restriction enzyme and enzyme pair, thus demonstrating that only one copy of this gene exists in Drosophila. An attempt was also made on this blot to reveal the presence of any introns which might be present within the coding region for this gene. This experiment failed because the putative Sph I site anticipated at the 3' end of the gene was not cut by treatment with Sph I. Careful examination of the sequencing autoradiographs shows an ambiguity in the sequence: it was thought to be CCAGCTAGC, however, I now believe the correct sequence is CCAGCTAGgC, which would interrupt the Sph I restriction site. As a result of this no determination of the presence or absence of introns was possible using Sph I. Eco RV treatment of plasmid 2F3 releases a 61 base restriction fragment from the 5' end of the cDNA (which is not detected by the probe) but does not release the bulk of the cDNA. The heavy band at arrow g in figure 15 is thus 3862 bases; the sum of the vector and cDNA insert, minus the 61 bases cut out by Eco RV. The two positively hybridizing bands (The band at 1.64 Kb in the 1Kb ladder and the 2F3 plasmid band of 3.86 Kb at arrow d, lane d) of known size can be used to help determine the sizes of the other positives signals from the genomic digests (all greater than 4.0 Kb). The double band in lane f is the result of uneven mixing of the DNA in the loading well, coupled with overloading of this lane. The fainter upper band is likely a more accurate measure of the band size, as it does not appear to be affected by overloading. Results 89 Figure #15: Genomic Southern Blot As a final demonstration that the recovered cDNA represents a single copy gene from Drosophila melanogaster, and in an attempt to reveal possible introns in the genomic D N A , a genomic Southern blot was prepared. This figure shows the agarose gel (right side) and autolumiograph (left side) from this Southern blot. The blot was probed with the PCR derived digoxygenin labelled probe used for library screening. The gel contains genomic D N A cut with Xba I, Xba I + Sst I, Sst I, Eco R V , Eco R V + Sph I, Sph I, Hind III + Xba I, and Hind III (lanes a-c, e, f, and h-j, respectively): lane d contains 1 Kb ladder; the 1.64 Kb band of the ladder gives an adventitious positive signal (arrow d) Lane g contains plasmid 2F3 D N A treated with Eco R V and Sph I, restriction sites expected near the 5' and 3 ' ends of the cDNA sequence respectively. Results 90 ANALYSIS OF THE MN SOD SEQUENCES Comparison of the various sequences shows a total of 11 allelic differences between the four known Drosophila Mn SOD sequences. The sequence isolated by Dutteroy et al., (1994) is identical to allele 2 of this study in 5 of 11 polymorphic sites, with one site for which no information was obtained (base 860, figure 13). The most dramatic change is the single base insertion and single base deletion at bases 121 and 138 respectively. These result in an 18 base frame shift in the carboxy terminal end of the signal peptide which alters the amino acid sequence from Ser-GLN-THR-ALA-SER-LEU-ALA-Val in alleles 1 and 2 to Ser-PRO-ASN-CYS-LYS-PRO-GLY-Val in Dutteroy et al. (1994) (see figure 14. The guanine base missing from Dutteroy et al. (1994) in the 3' non-coding region (base 850) may be a sequencing artifact. The extreme difficulty in demonstrating the existence of this base (see above) may indicate it was simply missed by Duttaroy et al., (1994), and thus it may not represent and allelic difference. CRYPTIC 181 AMINO ACID OPEN READING FRAME Reverse complementation and translation in all 3 reading frames of the cDNA sequence revealed the existence of a cryptic open reading frame of 181 amino acids coded for by the strand complementary to the Mn SOD coding region (see figure 16). Sequence similarity searches using this amino acid sequence, undertaken to determine if this sequence represented a real transcribed gene, were inconclusive. Thus the significance of this sequence is open to debate. Results 91 Figure #16: 181 Amino Acid Open Reading Frame on Non Coding Strand Curiosity prompted me to try reverse complementation and amino acid translation of the Mn SOD cDNA. This revealed a 181 amino acid (543 base) open reading frame on the complementary strand, present in all known alleles of the gene, covering the central portion of the Mn SOD coding sequence. Allele #1 is shown here: alterations in amino acid sequence for other alleles are shown in table 7 below. Locations of polymorphisms are shown by "***" above the site, and changes in case for the DNA base and the amino acid code affected. The protein sequence is repeated in one letter code at the bottom with changes in the putative amino acid sequence due to Polymorphisms shown underneath. The frame shift found in Dr. Duttaroy's sequence is depicted by locating the shifted sequence (-ag get tgc agtttg+) over top of the sequence for allele #1, with "-" indicating the missing C residue, and + indicating the inserted G (bases 766 to 780, lower right). A poly A addition signal is noted in the 3' region by "—#—>" located over top of the DNA sequence. Results 92 Table #7: Polymorphism effects in reverse complement open reading frame. This table details sequence polymorphisms between the four known alleles of Drosophila melanogaster Mn SOD genes as viewed from the opposite strand. The column "surrounding sequences," details the DNA sequence around each polymorphic site. The "#" symbols indicate bases which are variable between alleles. The "/" symbol indicates a site at which there is an insertion in the DNA sequence of some alleles relative to alleles 1 and 2. A lower case letter in the sequence indicates a base which is deleted in some alleles relative to alleles 1 and 2. Specific changes for each allele are listed under "Allele Number": here the symbol "/" indicates no insertion, "+G" indicates the insertion of a G residue into the DNA sequence, and " -C" indicates a deletion. A horizontal bar ("-") indicates there is no sequence data available for this site from this clone. The "Effect" column tells the effect of each DNA sequence change relative to alleles 1 and 2 on the protein coding sequence of the cryptic open reading frame. b a s e # S u r r o u n d i n g b a s e # A l l e l e # s i t e ( f o r ) S e q u e n c e ( r e v ) 1 2 3 4 E f f e c t 1 860 T T C A T A # A A A T T A 32 A C 2 850 T A G T T G C C T A G C T 42 C C -c n o n c o d i n g 3 820 C G C A A C # T A G A C T 81 T T C n o n c o d i n g 4 767 A C A T A C # C A A T C G 134 A G G H i s -> A r g 5 - 567 C T G G T T # G G C A G G 334 A G G G A r g - > G l y 6 4 7 5 T G G T C A # C T C C T T 426 G G G C G i n == G i n 7 468 C T C C T T # T T G A A C 433 C T T T L e u - > P h e 8 402 C T G G G T # T T G T T G 500 C C A C L e u - > H e 9 168 G T A G G G # A G C T T C 733 A c C n o n c o d i n g 10 138 C A C C G C c A G G C T T 763 C c - C n o n c o d i n g 11 120 A G T T T G / C G A A A T 781 / / +G n o n c o d i n g Allele #1, this study. Allele #2, this study. Allele #3, Duttaroy et al. (1994). Allele #4, Smith & Doolittle (1992). Amino Acid Sequence of reverse complement ORF DLRSADAYRL NIHNRRLCLA TELLGLLEAA RDVIPVGDVP DGLHVGGTHV 50 R LVLQIVGVLP DIDAEERDQA GGGLQGILVR QGGQLQFARL LVEAQPAPAG 100 G ALDRHGQRGQ LLLELLEALP LRLDGLLQII AGLGLVGRQV LPEDGWDVA 150 Q F I TAIETQGRSQ LNQLGGVALR LGLLQLLLGG I 181 Results 93 NOVEL 181 AMINO ACID OPEN READING FRAME ON NON-CODING STRAND OF MnSOD cDNA. ^ * * * ATG TTT TAA ATT GTT TTA TTC CCA TTC ATA aAA ATT ATA AAA ATT AGT TGC CTA GCT GGT 61 *** 91 . / l /5 ACC AAT TTA TTA GCC GCA ACt TAG ACT ACA ATT TAG GAT CTC CGA TcC GCA GAT GCT TAT AMB AMB Asp Leu Arg Ser Ala Asp Ala Tyr 121./10 *** /15 151./20 /25 AGA TTA AAC ATA CaC AAT CGC CGG CTG TGC TTA GCA ACC GAG CTT CTT GGC CTC CTG GAA Arg Leu Asn H e HIS Asn Arg Arg Leu Cys Leu Ala Thr Glu Leu Leu Gly Leu Leu Glu 181./30 211./40 GCG GCA CGA GAT GTC ATC CCA GTT GGC GAT GTC CCA GAT GGC CTC CAC GTA GGA GGG ACG A l a A l a Arg Asp Val H e Pro Val Gly Asp Val Pro Asp Gly Leu His Val Gly Gly Thr 241./50 271./60 CAC GTT CTT GTA CTG CAG ATA GTA GGC GTG CTC CCA GAC ATC GAT GCC GAA GAG CGG GAT His V a l Leu Val Leu Gin H e Val Gly Val Leu Pro Asp H e Asp Ala Glu Glu Arg Asp 301./70 331.*** CAG GCC GGT GGA GGC CTC CAG GGG ATC CTG GTT aGG CAG GGC GGC CAG TTG CAG TTT GCC Gin A l a Gly Gly Gly Leu Gin Gly H e Leu Val ARG Gin Gly Gly Gin Leu Gin Phe Ala 361./90 391/100 CGA CTT CTT GTT GAA GCC CAG CCA GCC CCA GCC GGA GCC CTG GAC CGC CAC GGT CAG CGT Arg Leu Leu Val Glu A l a Gin Pro A l a Pro Ala Gly A l a Leu Asp Arg His Gly Gin Arg 421 . *** *** 451/120 GGT CAg CTC CTT cTT GAA CTC CTC GAG GCT CTT CCA CTG CGA CTC GAT GGC CTT CTT CAG Gly GLN Leu Leu LEU Glu Leu Leu Glu Ala Leu Pro Leu Arg Leu Asp Gly Leu Leu Gin 481/130 *** 511/140 ATC ATC GCT GGG CTG GGT cTT GTT GGG CGA GAG GTT CTG CCA GAA GAT GGT GTG GTT GAT H e H e A l a Gly Leu Gly LEU Val Gly .Arg Glu Val Leu Pro Glu Asp Gly Val Val Asp 541/150 571/160 GTG GCC ACC GCC ATT GAA ACG CAG GGC AGG AGC CAG CTG AAT CAG CTT GGT GGT GTC GCT Val A l a Thr A l a H e Glu Thr Gin Gly Arg Ser Gin Leu Asn Gin Leu Gly Gly Val A l a 601/170 631/180 stop CTT CGA CTT GGC CTC CTC CAG CTG CTC CTC GGC GGC ATT TAG ATT GTT GAC GTA GGT CTG Leu Arg Leu Gly Leu Leu Gin Leu Leu Leu Gly Gly H e AMB 661 691 GTG GTG CTT CTG GTG ATG CAG CTC CAT GAT CTC CCG GCA GAT GAT AGG CTC CAG GGC GGC 721 *** 751 -ag get tgc agtttg+ ATA GTC GTA GGG aAG CTT CGG TAG GGT GTG CTT GCC ACG CAC CGC CAG GCT TGC AGT TTG 781 811 CGA AAT TTT ACG GGC CAC GAA CAT ATC TGC GTT TTA TTC GAC TGC TAA TAA GGC TAA GTC 841 —#--> 871 CCG ATA TCA GAT TTT ATA TCA AAT AAA ACA AGG CTT ACG TTC TCG TTA TTT CGC TTG TTC 9H D I S C U S S I O N : Opening Remarks. This work has completed the first step necessary to study the over expression of Mn SOD in Drosophila. Subsequent steps, including mapping the location of the gene in the Drosophila genome, conducting genetic studies to determine the effect of homozygous deletion of the gene, reintroduction of the gene for overexpression, and life span studies of over- and under-expressing strains, will have to wait for the efforts of others. The gene has now been mapped by Duttaroy et al to region 53E on polytene chromosomes (Atanu Duttaroy, personal communication, August, 1993). Comments on Methods: PCR Polymerase chain reaction was used extensively in the pursuit of this gene, and it seems germane to discuss here some of the parameters which affect the outcome of this method: 1) Oligonucleotide length: PCR can be carried out with oligonucleotides as short as 17 bases on either a DNA (Erlich 1989) or an RNA template (Tse & Forget 1990). However, for efficient priming under standard salt conditions the probe should be at least 20-21 bases (Dave Banfield, personal communication). The unintentional cloning of the Copia-like element LTR demonstrates that priming on RNA templates with 17 and 18 base oligonucleotides will work provided higher salt buffer conditions are used (125 mM vs. 50 mM Tris HC1, pH 8.4 at 20 C). High salt buffers were not tried with 17 base primers and DNA templates. 2) Probe specificity: The most important feature in PCR probe design was to get an exact match for the last 9 bases at the 3' end of the primer. If any mismatches occur in this region, priming will be curtailed or eliminated. Bases 5' of this region need not all match exactly to obtain efficient priming. The greater the degeneracy of the primer, the more difficult it was to obtain efficient polymerization of the desired sequence. For these reasons it was desirable to make probes which cover all of the Discussion: 95 possible codon combinations for the last nine bases, and use a single non degenerate DNA sequence for the 5' region covering only the most likely possible amino acid codons. Probe concentration should be increased for highly degenerate probes. 3) Restriction endonuclease sites: It is extremely helpful to include suitable restriction endonuclease sites in the 5' ends of PCR primer sequences to facilitate cloning of PCR products. The added expense is more than compensated for by the savings in time and effort that result from the inclusion of these sites. 4) In some cases it may be desirable to do four cutter restriction digests or SSCP on PCR products to determine if more than one sequence of the same length was being amplified: i.e. to determine if overlapping bands are present. 5) A useful control which can be run both on the original template and on the resulting PCR products is PCR with primer combinations not expected to result in a product. This helps to identify specious products and false positives obtained, for example, when one primer binds to both ends of a piece of DNA, or when two primers, both meant to bind to one end of the DNA of interest, amplify a piece of random DNA independently. I S O L A T I O N O F T H E C O P I A - L I K E E L E M E N T . The first PCR experiments resulted in isolation of a Copia-like element LTR from wcRNA. This suggests that these elements are actively transcribed during oxidative stress in Drosophila. It might be interesting to repeat these experiments to determine which types of stress will induce Copia transcription and also the background levels under unstressed conditions. PCR may provide a simple method for addressing these questions. Comparison of the sequence for a Copia-like element from the EMBL database with the sequence obtained here indicates that one of the two PCR priming sites, using primer 6-90, was contained in the 3* end of the last protein coding sequence of the Copia element, while the other priming site was contained in the Drosophila host DNA, and could be using either (or both) of the primers S2 and 6-90. The EMBL data base sequence of the last protein coding sequence in the Copia-like Discussion: 96 element has an exact match for the last 9 bases of 6-90. This is probably the site of amplification, as no better site is found in the entire Copia-like element, and amplification from this site results in a fragement of about the correct size. This once again demonstrates the importance of the last 9 bases for successful PCR (see "Copia-like element" in the Appendix). I S O L A T I O N O F BSC23; A P U T A T I V E A R C H A E B A C T E R I A L MN SOD. Subsequent PCR experiments with primers SCI and HEW on a purified DNA template resulted in isolation of clones BSC4, 17, 20, and 23. Amino acid translation of the sequences from clones BSC 17, 20, and 23 in frame with the amino acids coded for by the PCR primers resulted in a continuous open reading frame of 356 bases or 118 2/3 amino acids, excluding the primers. The resulting sequence has greatest sequence similarity with Mn SODs from the halophilic Archaebacteria, but it was the most divergent of all the known Archaebacterial and mycobacterial sequences. The occurrence of proline in place of serine in the highly conserved GSGW (amino acids 82-84, figure 4 and amino acids 132-135 in figure 18) sequence of the gene was unique among the more than 60 known Mn and Fe SODs. Attempts to isolate this gene from a Drosophila cDNA library resulted in months of confusion for the following reasons: 1) the Lambda cDNA library probed contained phage with plasmid vector as inserts. The probe used was labelled by treating the entire plasmid with the Klenow fragment, resulting in labelling of both the plasmid and the insert. This resulted in the isolation of two clones from the library, both of which contained only plasmid and lambda vector DNA. Attempts to subclone the plasmid vector sequences into pBlueScript for sequencing were uniformly unsuccessful because of the extensive sequence identity involved, and as a result for a long time the nature of the insert DNA remained a mystery. At about the time that I finally tentatively determined what the problem was, Don Sinclair informed me that others before me had also isolated plasmid vectors from this Lambda library, thus confirming my diagnosis. Discussion: 97 2) Drosophila stocks used in preparation of DNA for genomic Southern blots were contaminated with a plasmid vector (see figure 5), likely derived from multicopy tandem repeats in the fly genomic DNA from Nina Seto's Cu-Zn SOD transformation experiments. This plasmid must closely resemble the vector sequences labelled in preparation of the probe, resulting in the same strong band in genomic Southern blots at about 3 Kb. The ladder of hybridizing bands in the incomplete Hind III digest in figure 5, which run at about 3 Kb, 6 Kb, and 9 Kb give additional evidence that this band results from tandemly repeated DNA. Further genomic Southern blots using a PCR generated probe containing only putative BSC23 Mn SOD sequences gave very confusing mixed results. A careful review of several Southern blots using the BSC23/PCR and Doolittle/PCR probes eventually led to the realization that DNA isolations 14, 15, and 16 were responsible for all the BSC23 hybridizing digests, while DNA isolations 22, 23, 27, 29, 30, 31, 32 were uniformly negative with BSC23. Reviewing the dates flies were collected for DNA isolations 14, 15, and 16 led to the suspicion that the flies used in the preparation of the positive digests might have been heavily contaminated with some kind of bacteria, fungi, or yeast. These flies were collected during an extremely hot period of the summer, when temperatures in the lab were routinely over 25 °C. The fly bottles were often overgrown with some form of pale beige-yellow microorganism. At that time, when harvesting flies, no distinction was made between live and recently dead (within 24 hours) flies: thus many dead flies were included in the preparation of these DNA stocks. It is believed that the obtained Mn SOD clone BSC23 came from overgrowth by an Archaebacterium of fly cadavers included in the isolations. Seeding NZY or LB plates (with or without 100 ug/ml ampicillin) with fly cadavers from flies grown at 25 °C, with subsequent incubation at 25 °C for 24-48 hours, resulted in extensive microbial growth of colonies similar to those seen in the fly bottles (fly food also included 100 ug/ml ampicillin). Microscopic examination of the resulting colonies revealed a gram negative rod similar to Escherichia coli. Incubation Discussion: 98 at 37 °C resulted in negligible bacterial growth after 48 hours. No further determination was made as to whether or not this organism was responsible for clone BSC23. Isolation of the bacterium from which BSC23 was derived may be possible by repeating the conditions used. These would include the BSC23 probe or recloning of it with the original oligonucleotides (SCI and HEW), the same DNA isolation procedure, and growing flies on the same fly food at the same 25 °C incubation temperature. T H E DROSOPHILA MELANOGASTER M N S O D G E N E With the publication of the PCR derived partial sequence for Drosophila Mn SOD by Smith and Doolittle (1992), the cloning of the complete cDNA became relatively straight forward. cDNA clones were obtained from a Drosophila melanogaster imaginal disc tissue culture derived Lambda phage library after making PCR primers matching the ends of the Smith and Doolittle sequence, and using them to create a digoxygenin labelled probe by PCR. Several complete cDNAs of the two alleles present in the library were isolated, and these were compared to the two partial alleles derived by Smith and Doolittle (1992), and Duttaroy et. al. (1994). See figure #20, page 78-79. P O L Y M O R P H I S M S I N T H E D N A S E Q U E N C E Ten polymorphic sites were found in the cDNA sequences of the four different alleles of Drosophila Mn SOD looked at to date (see table 8). The five sites polymorphic between alleles 1 and 2 do not change the amino acid sequence. Allele 4 (Smith & Doolittle, 1992) has a C to G change at base 475 resulting in a coding change from Leu to Val. This difference indicates either allele 4 was a separate allele with a conservative amino acid substitution or it was a copy of allele 2 with a PCR error at base 475: as Smith and Doolittle do not mention sequencing several independent clones the possibility of a PCR error cannot be excluded by this author. The amino acids found at this location in other animal species are Leu (7x), Met (2x), and Phe (lx). Discussion: 99 Leucine predominates in this location in animal Mn SOD genes, but conservation was not complete at this site or in the adjacent ones, so valine is likely to be a viable substitution (see amino acid 123, figure 26). It is interesting, though probably not significant, that all but one of the five base substitutions from clone 1 to clone 2 serve to increase the G+C content of the DNA. Duttaroy et al. (1994) produced the sequence for allele #3 from a. Drosophila melanogaster Canton S cDNA obtained in Dr. Phillips lab (personal communication). This sequence contains a six amino acid compensated frame shift in the signal peptide, in addition to the amino acid substitution of asparagine for lysine at amino acid position 85 (base 402) and a silent substitution at position 820: it is otherwise identical to allele 2 of this study. The absence of any significant alteration in phenotype resulting from the altered signal peptide once again demonstrates the lack of specific sequence requirements for this region of the protein. Alignment of the putative amino acid sequence for Drosophila Mn SOD with the other Mn containing SODs in the SwissProt protein database resulted in closest similarity with Caenorhabditis eleagans and sea cucumber, (Synapta sp.). Discussion: 100 Table #8: Sequence Polymorphisms Between Drosophila Alleles/Strains. This table details sequence polymorphisms between the four known alleles of Drosophila melanogaster M n SOD genes. The column "surrounding sequences," details the D N A sequence around each polymorphic site. The "#" symbols indicate bases which are variable between alleles. The "/" symbol indicates a site at which there is an insertion in the D N A sequence of some alleles relative to alleles 1 and 2. A lower case letter in the sequence indicates a base which is deleted in some alleles relative to alleles 1 and 2. Specific changes for each allele are listed under "Allele Number": here the symbol "/" indicates no insertion, "+C" indicates the insertion of a cytosine into the D N A sequence, and " -G" indicates a deletion. A horizontal bar ("-") indicates there is no sequence data available for this site from this clone. The "Effect" column tells the effect of each change in the D N A sequence relative to alleles 1 and 2 on the protein coding sequence. Surrounding Base A l l e l e Number Sit e Sequence Number 1 2 3 4 E f f e c t 1 ATTTCG/CAAACT 120 / / +c - frameshift 2 CCTGGCgGTGCGT 138 G G -G - frameshift 3 GAAGCT#CCCTAC 168 T G G - s i l e n t 4 CAACAA# AC C CAG 402 G G T G Lys -> Asn 5 GTTCAA#AAGGAG 468 G A, A A s i l e n t 6 AAGGAG#TGACCA 475 c c c G Leu -> Val 7 CCTGCC#AACCAG 567 T c c c s i l e n t 8 CGATTG#GTATGT 767 T c c - non coding 9 AGTCTAftGTTGCG 820 A A G - non coding 10 AGCTAGgCAACTA 850 G G -G - non coding 11 TAAT T T # TATAAT 860 T G - - non coding - no sequence information Allele #1 this study. Allele #2 this study. Allele #3 Duttaroy et al. (1994). Allele #4 Smith & Doolittle (1992). A S S I G N M E N T O F M R N A 51 A N D 3 1 E N D S Assignment of the 5' end of the mRNA was made based on the 5' sequences of 4 clones; 2D, 2F, 9A, and 10A. Three of these clones ended in the same 5' sequence, with 2F and 10A containing a differing additional base at the 5' end. The additional base was probably an artefact; an addition to the 5' end by reverse transcriptase or one of the other D N A modifying enzymes used in preparation of this library. Thus, this sequence was tentatively assigned as the mature 5' end of the mRNA (see Table 9), although it is possible that reverse transcription ended at this site coincidentally or due to secondary structure, and the two differing bases represent differences between allele 1 (2F) and allele 2 (10A). Discussion: 101 Sequencing of the 3' ends of cDNA clones 2D, 2F, 6A, 9A, and 10A indicates that at least 2 different polyadenylation signals are used. These result in 3' sequences as shown in table 8. Table #9: 5' End Assignment and Polyadenylation Site Usage of 5 Clones. This table shows the 5' cDNA sequences obtained in this study at the point where they connected to the Eco RI linker. It also lists the poly A addition site used by each clone (#1 is the 5' most poly A site: see figure 14) and the length of the poly A tail contained in that clone. The sequence G A A C A A was tentatively assigned as the 5' end. Clone A l l e l e 5' seq: Assignment of 5 1 base Poly A Sit e Used Poly A Length 2D 1 GAACAA #1 #2 • 61 2F 1 AGAACAA #1 #2 54 6A 2 — — past #1 missing 9A 2- TTTCGC #66 #1 17 10A 2 TGAACAA #1 #2 6 Table #10: Polyadenylation Sites. This table shows the relationship between the two poly A addition signals found in both alleles and there respective poly A addition sites. The sequence 3' of the first R N A poly addenlyation site (site #1) is shown, and happens to correspond to the start of the sequence showing site #2. S i t e #1: (clone 9A only) Base # 821 825 830 835 840 845 850 855 866 DNA : : : : : : : : : sequence GTTGCGGCTAATAAATTGGTACCAGCTAGgCAACTAAAAA(n) : A(n) s i t e > A : RNA s p l i c e junction: ATTTGTATAA Sit e #2: (clones 2D, 2F, and 10A) Base # 866 870 875 880 885 890 895 901 DNA : : : : : : : : s equence AT TTT TAT GAAT G G GAATAAAACAAT T TAAAACATAAAAA(n) A(n) s i t e > A Removed pre mRNA sequence not known: NNNNNNNNNN The last experiment attempting to determine the presence of introns in the D N A sequence by Southern blot was a failure, as the expected Sph I site at the 3' end of the gene is interrupted by a G residue which was not initially noticed on the sequencing gels. Thus the expected size of the individual gene fragments in the absence of introns is not known for any of the restriction digests. Discussion: 102 SE Q U E N C E A L I G N M E N T O F KNOWN MN SOD GENES . , AH of the M n SOD and Fe SOD protein sequences contained in the E M B L gene library and SwissProt protein database were assembled, together with the new sequences for BSC23 and Drosophila Mn SOD from this study, and aligned in groups using the P C G E N E program C L U S T A L . In cases where two or more sequences were identical or nearly identical, only one sequence was retained (for example, in the case of Synechococcus sp. and Anacystis nidulans, only 3 differences exist in the amino acid sequence, so only A. nidulans was retained). The resulting alignments were compared, and new alignments tried. After several cycles of this treatment, a series of specific similarity groups were derived which roughly follow species and class lines, with one or two surprises. The six major groups derived were labelled as (1) the Animal M n SODs, (2) the Plant Mn SODs, (which includes the budding yeast, Saccharomyces cerevisiae) (3) the Archaebacterial Mn SODs (which includes the two known Mycobacterial M n SODs because of their close similarity to the Archaebacterial consensus sequence), (4) the Eubacterial Mn SODs, (5) the Plant Fe SODs (including the Cyanobacterial Fe SOD from Anacystis nidulans) and (6) the Bacterial Fe SODs, including the two protista Fe SOD sequences from Entamoba and Tetrahymena. The Cyanobacterial and Protista Fe SODs, and the fungal and yeast Mn SODs, may make up separate divisions, but not enough sequences are available to define separate categories for them. The two Bacteroides species, Methylomonas J , and Streptococcus mutans, which are able to use both Fe and Mn as metal cofactors, were placed according to the group with which they had greatest homology: the two Bacteroides species with the bacterial Fe SODs, and the other two with the bacterial Mn SODs. The consensus sequences for these derived groups contain specific elements which allow the assignment of new sequences without knowing the species they are derived from (as in the case of BSC23). Some of these sequence characteristics are detailed in the alignment of consensus sequences included in the Appendix. Some examples include the sequence F N N A A Q , which is specific for Fe containing SODs, or Discussion: 103 the replacement of lysine with threonine at position 32, which is specific to the Archaebacteria. One of the most striking of these is the conservation of the hydrogen bond found by Stoddard et al. (1990) in the Fe SOD of Pseudomonas ovalis between threonine 171 and cysteine 90 (see sequence line-up, figure 18). This unusual hydrogen bond is replaced by a more common threonine 171 to serine 90 hydrogen bond in some of the other Fe SODs, but all of them with the possible exception of Tetrahymena appear to have a hydrogen bond structure at this location. The only other exceptions are the four SODs which can use either metal, Mn or Fe. Thus it is possible that this bond is involved in metal selectivity for the two types of SOD. In this context Tetrahymena deserves special mention: the original alignment of the Tetrahymena Fe SOD placed a serine at position 93: adjusting the sequence gaps to place it at position 90 makes little difference to the overall fit, so this was done. Then it was noticed that neither the original, nor the serine adjusted alignments placed the conserved histidine ligand in the correct location: it was also noticed that the sequence " F N L G G H V N H " (numbers 76-87 in the alignment) which contains the putative ligand (# 87) is a much closer match for the Mn SODs from Mycobacterium leprae and Mycobacterium tuberculosis than it is for the bacterial Fe containing SODs. A third point relating it to the Mn containing SODs is the conserved sequence "NQD" (154-156 in the alignment) which is found exclusively in the Eubacterial and eukaryotic M n SODs, except for Tetrahymena, where it appears from 157 to 159 in the alignment. The very substantial divergence in primary sequence seen here indicates that Tetrahymena is likely to have a rather novel crystal structure, and this may be worth pursuing. It also underlines the likelihood that the Protista Mn/Fe SODs are a class unto themselves. Discussion: 104 Figure #17: Amino Acid Sequence Alignment of Known Mn Superoxide Dismutases The overall sequence alignment shown, here was derived by examining the results of numerous mixed alignments of most of the iron and manganese superoxide dismutase sequences in the Swissprot protein database together with the new sequences for Drosophila and BSC23 using the C L U S T A L algorithm of the program P C G E N E (many thanks to Ross MacGillivray and Jeff Hewitt for their help, instructions, and use of their computer). Initial alignments were followed by resorting of the sequences into groups according to their greatest similarities, realignment of each group, and overall alignment of the group alignments by eye on the word processor Microsoft Word. Signal peptides were removed prior to alignment, and are not included in the figure. At six points in the alignment sequences which were particular to a single organism were looped out and replaced with a " |"; these sequences are included with a dash (-) at each end either over top of the grouping after the group title, or before the organism's name at the end of the line. The second proline residue in the plant sequences at position 101/102 was left out of the numbering system at the top of the page, as were the sequences which were "looped out" as mentioned. The four mixed metal Fe/Mn SODs, which are able to use either Fe or Mn as a metal cofactor (Martin et. al, 1984) were included under the class to which they bore the greatest similarity. The two Bacteriodes SODs listed (numbers 42 and 43) are listed under the bacterial Fe SODs because they share the greatest similarity with the Fe SOD sequences; the sequences for Methylamonas J and Streptococcus mutatis were included under the bacterial M n SODs because they were most similar to them. The sequences for a few of the known M n and Fe SODs were left out of the final sequence alignment. They were removed because their sequences so closely match other sequences which were included it was felt no new information would be gained by including them. These included the sequences for Synechococcus sp. (only 3 amino acids different from Anacystis nidulans) and two sequences from the bacterial Mn SODs. It should be noted that the sequence for Bacillus stearothermophilus contains at least two differences Discussion: 105 in the database sequence v.s. that reported for the X-ray structure: position 107 changes from E to D and 191 from I to L (Parker & Blake, 1988b). The alpha helix and beta sheet domains for four SODs, Bacillus stearothermophilus (Mn), Thermus thermophilus (Mn), Escherichia coli (Fe), and Pseudomonas ovalis (Fe), are known from X-ray crystal structures. These are marked on the sequence alignment with dashed lines over top of the appropriate sequence, "a" for alpha helix, and "b" for beta sheet. The metal ligands are marked by "*" in these lines. Two single amino acid substitutions between alleles 1 and 2 of Drosophila melanogaster Oregon R (this study), the sequence for D. melanogaster Canton S, reported by Duttaroy, et al. (1994), and the partial sequence for D. melanogaster reported by Smith and Doolittle (1992), occur. In this sequence alignment they correspond to position 97 (lys -> asp) (Duttaroy et al. 1994) and 123 (leu -> val) (Smith and Doolittle, 1992). The sequence for Mycobacterium tuberculosis is included under the archaebacterial M n SODs, although it is reported to bind iron preferentially for its activity (Zhang et al. 1991). Jo6 Animal Mn SODs I 5 10 15 20 25 30 35 40 45 50 55 60 I I I I I I h i I I I I I 1 KHSLPDLPYDYGALEPHINAQIMQLHHSKHHAAYVNNLNVTEEKYQEALAKGDVTAQ 2 KHSLPDLPYDYGALEPHINAQIMQLHHSKHHATYVNNLNVTEEKYHEALAKGDVTTQ 3 KHSLPDLPYDYGALEPHINAQIMQLHHSKHHAAYVNNLNATEEKYHEALAKGDVTTQ 4 HINAQIMQLHHSEHHAAYVNNLNWEEKYQEALKKGDVTAQ 5 HISAEIMQLHHSKHHAAYVNNVNWEEKLAEALGKGDVNTQ 6 HISANIMQLHHSKHHATYVNNLNVAEQKLAEAVAKGDVTAE 7 ; VISAEIMQVHHQKH HAT Y VN N LN AAE E QLAEAIH KQ DVT KM 8 KHSLPDLPYDYADLEPVISHEIMQLHHQKHHATYVNNLNQIEEKLHEAVSKGNVKEA 9 KHTLPKLPYDYAALEPIICREIMELHHQKHHQTYVNNLNAAEEQLEEAKSKSDTTKL 10 VIIGDIMELHHKKHHATYTNNLNAAEEKLAAAHAEGDIGGM 11 HISEMIMQIHHTKHHQAYINNLKACTEKLKQAEQANDVAAM Plant Mn SODs 12 -LQTFSLPDLPYDYGALEPAISGDIMQLHHQNHHQTYVTNYNKALEQLHDAISKGDAPTV 13 -LHVFTLPDLAYDYGALEPVISGEIMQIHHQKHHQTYITNYNKALEQLHDAVAKADTSTT 14 -VTTVTLPDLSYDFGALEPAISGEIMRLHHQKHHATYVANYNKALEQLETAVSKGDASAV 15 -KVTLPDLKWDFGALEPYISGQINELHYTKHHQTYVNGFNTAVDQFQELSDLLAKEPS man rat mouse p i g h a g f i s h lamprae l a n c e l e t C. elegans Drosophila melanogaster sea cucumber l o b s t e r tobacco pea corn yeast -HSNP- Haloarcula A r c h a e b a c t e r i a l Mn SODs -LEKKF- Methanobacterium thermoautotrophicum 16 MNDIYELPELPYPYDALEPHISREQLTIHHQKHHQAYVDGANALLRKLDEARESDTDVDI 17 MAE-YTLPDLDWDYGALE PHI SGQINELHHSKHHAT YVKGANDAVAKLEEARAKEDHSAI 18 VAE-YTLPDLDWDYAALEPHISGEINEIHHTKHHAAYVKGVNDALAKLDEARAKDDHSAI 19 MSE|-ELPPLPYDYDALEPHISEQVLTWHHDTHHQGYVNGLESAEETLAENRDAGDFGSS 20 MSY—ELDPLPYEYDALEPHISEQVLTWHHDTHHQGYVNGWNADDETLAENREAGEFGSS 21 MSEY-ELPPLPYDYDALEPHISEQVLTWHHDTHHQGYVNGWNDAEETLAENRETGDHAST 22 MSQH-ELPSLPYDYDALEPHISEQAVTWHHDTHHQSYVDGLNSAEENLAGNRETGDHAST 23 MSQH-ELPSLPYDYDALEPHISEQWTWHHDTHHQSYVDGLNSAEETLAENRETGDHAST 24 GLNKAEEEIYYRNHEPD--ML E u b a c t e r i a l Mn SODs 2 Methanobacterium t. Mycobacterium tuberculosis Mycobacterium leprae Haloarcula marismortui Haloferax volcanii Halobacterium cutirubrum Halobacterium sp. sig Halobacterium halobium BSC23 * — a 1 --a 25 MPF—ELPALPYPYDALEPHIDKETMNIHHTKHHNTYVTNLNAALEGHPDLQNKSLEELL 26 MTY—ELPKLPYTYDALEPNFDKETMEIHYTKHHNTYVTKLNEAVAGHPELASKSAEELV 27 MSY—TLPSLPYAYDALEPHFDKQTMEIHHTKHHQTYVNNANAALESLPEFANLPVEELI 28 -PYPFKLPDLGYPYEALEPHIDAKTMEIHHQKHHGAYVTNLNAALEKYPYLHGVEVEVLL 2 9 -AYT—LPPLDYAYTALE PHI DAQTMEIHHTKHHQTYINNVNAALEGTS FANE P-VEALL 30 MAI—LLPDLPYAYDALEPYIDAETMTLHHDKHHATYVANANAALEKHPEIGE-NLEVLL Eukaryotic Fe SODs 31 MSY—ELPALPFDYTALAPYITKETKE FHHDKHHAAYVNNYNNAVKDTDLDGQPIEAVIK 32 KFELQPPPYPMDALEPHMSSRTFEFHWGKHHRAYVDNLNKQI-DGTELDGKTLEDII 33 -TANYVLKPPPFALDALEPHMSKQTLEFHWGKHHRAYVDNLKKQV-LGTELEGKPLEHII 34 -NAKFELKPPPYPLNGLEPVMSQQTLEFHWGKHHKTYVENLKKQV-VGTELDGKSLEEII B a c t e r i a l Fe SODs 35 MTF—TLPQLPYALDALAPHVSKETLEYHYGKHHNTYVTNLNKLIPGT EFESMTL 36 MAH—TLPPLPYALDALAPRISKETLEFHYGKHHQTYVTNLNNLVPGT EFENLSL *--a 1 37 MSF—ELPALPYAKDALAPHISAETIEYHYGKHHQTYVTNLNNLIKGT AFEGKSL 38 -AF—ELPALPFAMNALEPHISQETLEYHYGKHHNTYWKLNGLVEGT ELAEKSL 39 MAF—ELPDLPYKLNALEPHISQETLEYHHGKHHRAYVNKLNKLIEGT PFEKEPL *--a 1 4 0 -AF—ELPPLPYAHDALQPHISKETLEYHHDKHHNTYWNLNNLVPGT—PEF--EGKTL 41 MSF—QLPQLPYAYNALEPHISKETLEFHHDKHHATYVNKLNGLV-KGTEQEHKTLEELI 42 MTH—ELISLPYAVYALAPVISKETVEFHHGKHLKTYVDNLNKLIIGT—EFE—NADLN 43 MTY—EMPKLPYANNALEPVISQQTIDYHYGKHLQTYVNNLNSLVPGT--EYE—GKTVE 44 LNYEYSDLEPVLSAHLLSFHHGKHHQAYVNNLN AT--YEQIAAATKE Bacil. stearothermophilus L i s t e r i a monocytogenes. Echerichia coli Thermus thermophilus Methylomonas Streptococcus mutans Anacystis nidulans Tobacco Arabidopsis Soybean Legionella pnemophila? Bordetella pertusis Echerichia coli Photobacterium leiognathi Coxiella b Pseudomonus oval is Entamoeba Bacteroides g i n g i v i l u s Bacteroides f r a g i l i s Tetrahymena pyriformis Discussion: 107 Animal Mn SODs 65 70 75 80 85 90 95 100 105 110 115 120 I I I I I I I I I I I I 1 IALQPA LKFNGGGHINHSIFWTNLSP—NGGGEP KGELLEAIKRDFGSFDKFK man 2 VALQPA LKFNGGGHINHSIFWTNLSP--KGGGEP KGELLEAIKRDFGSFEKFK r a t 3 VALQPA LKFNGGGHINHTIFWTNLSP--KGGGEP KGELLEAIKRDFGSFEKFK mouse 4 VALQPA LKFNGGGHINHSIFWTNLSP--NGGGEP KGELLEAIKRDFGSFEKFK p i g 5 VSLQPA FRFNGGGHINHSIFWRNLSP—SGGGQP CGDLLKAIENDFGSVDKLR h a g f i s h 6 IALQPA IKFNGGGHINHSIFWTNLSP—NGGGAP TGDLQKAIETDFGSFTKLQ lamprae 7 IALQSA IKFNGGGHINHSIFWNNLCP—SGGGEP TGPLAEAITRDFGSFEAFK l a n c e l e t 8 IALQPA —LKFNGGGHINHSIFWTNLAK—DGG-EP SAELLTAIKSDFGSLDNLQ C. elegans 9 IQLAPA LRFNGGGHINHTIFWQNLSP.—NKT-QP SDDLKKAIESQWKSLEEFK Drosophila melanogaster 10 IALQPA LKFNGGGFINHCIFWTNLSP--QGGGVP EGDLADAINRDFGSFDSFK sea cucumber 11 NALLPA IKFNGGGHINHTIFWTNMAP--SAGGEP AGPVADAITKEFGSFQAFK l o b s t e r Plant Mn SODs 12 AKLHSA IKFNGGGHINHSIFWKNLAPVREGGGEPPKGSLGWAIDTNFGSLEALV tobacco 13 VKLQNA IKFNGGGHINHSI FWKNLAPVSEGGGEPPKESLGWAIDTNFGSLEALI pea 14 VQLQAA IKFNGGGHVNHSIFWKNLKPISEGGGEPPHGKLGWAIDEDFGSFEALV corn 15 PANARKMIAIQQNIKFHGGGFTNHCLFWENLAPESQGGGEPPTGALAKAIDEQFGSLDELI yeast A r c h a e b a c t e r i a l Mn SODs 16 K AALKELSFHVGGYVLHLFFWGNMGPADECGGEP SGKLAEYIEKDFGSFERFR Methanobacterium t. 17 L LNEKNLAFNLAGHVNHTIWWKNLSPNG--GDKP TGELAAAIADAFGSFDKFR Mycobacterium tuberculosis 18 F LNEKNLAFHLGGHVNHSIWWKNLSPNG—GDKP TGGLATDIDETFGSFDKFR Mycobacterium leprae 19 A AAWVNVTHNGCGQDLHTLFWENMDPNG—GGEP EGELLDRIEEDFGSYEGWK Haloarcula marismortui 20 A GAVRNVTHNGSGHILHDLFWQNMSPEG—GDEP EGLLAERIAEDFGSYEAWK Haloferax volcanii 21 A GALGDVTHNGSGHILHTLFWQSMSPAG--GDEP SGALADRIAADFGSYENWR Halobacteriurn cutirubrum 22 A GALGDVTHNGCGHYLHTMFWEHMSPDG—GGEP SGALADRIAADFGSYENWR Halobacterium sp. sig 23 A GALGDVTHNGCGHYLHTMFWEHMSPDG—GGEP SGALADRIAADFGSYENWR Halobacterium halobium 24 R HWLREQAFNGSGHLLHSVFWKNMTPYS--SKVP SKQIEQWINRDFWLLCHFK BSC23 E u b a c t e r i a l Mn SODs 25 SNLEALPESIRTAVRNNGGGHANHSLFWTILSPNG—GGEP TGELAEAINKKFGSFTAFK Bacil. stearothermophilus 26 TNLDSVPEDIRGAVRNHGGGHANHTLFWSILSPNG—GGAP TGNLKAAIESEFGTFDEFK L i s t e r i a monocytogenes. 27 TKLDQLPADKKTVLRNNAGGHANHSLFWKGL—KK--GTTL QGDLKAAIERDFGSVDNFK Echerichia coli 28 RHLAALPQDIQTAVRNNGGGHLNHSLFWRLLTPGG--AKEP VGELKKAIDEQFGGFQALK Thermus thermophilus 29 QKLDSLPENLRGPVRNNGGGHANHSLFWKVLTPNG--GGEP KGALADAIKSDIGGLDTFK Methylomonas 30 ADVEQIPADIRQSLINNGGGHLNHALFWELLSPEK--TK-V TAEVAAAINEAFGSFDDFK Streptococcus mutans Eukaryotic Fe SODs 31 AIAGDASKAG LFNNAAQAWNHSFYWNSIKPNG--GGAP TGALADKIAADFGSFENFV Anacystis nidulans 32 LVTYNK—GAPLPAFNNAAQAWNHQFFWESMKPNG--GGEP SGELLELINRDFGSYDAFV Tobacco 33 HSTYNN—GDLLPAFNNAAQAWNHEFFWESMKPGG--GGKP SGELLALLERDFTSYEKFY Arabidopsis 34 VTSYNK—GDILPAFNNAAQVWNHDFFWECMKPGG—GGKP SGELLELIERDFGSFVKFL Soybean B a c t e r i a l Fe SODs 35 EEIIMKAKGG 1FNNAAQVWNHTFYWHSMSPNG—GGEP KGRLAEAINKSFGSFAAFK Legionella pnemophila? 36 EEIVKKSSGG VFNNAAQVWNHTFYWNSLSPNG—GGEP SGALADAIKAKWGSVDAFK Bordetella pertusis 37 EEIIRSSEGG VFNNAAQVWNHTFYWNCLAPNA—GGEP TGKVAEAIAASFGSFADFK Echerichia coli 38 EEIIKTSTGG VFNNAAQVWNHTFYWNCLAPNA—GGEP TGEVAAAIEKAFGSFAEFK Photobacterium leiognathi 39 EEIIRKSDGG 1FNNAAQHWNHTFYWHCMSPDG—GGDP SGELASAIDKTFGSLEKFK Coxiella b a 2 * a 3 a 40 EEIVKSSSGG 1FNNAAQVWNHTFYWNCLSPDG—GGQP TGALADAINAAFGSFDKFK Pseudomonus o v a l i s 41 KQ—KPTQAIYNNAAQAWNHAFYWKCM-CGC--GVKP SEQLIAKLTAAFGGLEEFK Entamoeba 42 TIVQKS-EGG 1FNNAGQTLNHNLYFTQFRPGK--GGAP KGKLGEAIDKQFGSFEKFK Bacteroides g i n g i v i l u s 43 AIVASAPDGA IFNNAGQVLNHTLYFLQFAPKPA-KNEP AGKLGEAIKRDFGSFENFK Bacteroides f r a g i l i s 44 NDAHKIATLQSALRFNLGGHV-NHWIYWDNLAPVKSGGGV| HSPLTKAIKEKWGSYENFI -LPDE- Tetrahymena p y r i f o r Discussion: 108 Animal Mn sods 121 125 130 135 140 145 150 155 160 165 170 175 180 I I I I I I I I I I I I I 1 E K L T A A S V G V Q G S G W G W L G F N K Q R G H L Q I A A C P N Q D P L Q G T T G — L I P L L G I D V W E H A Y Y man 2 E K L T A V S V G V Q G S G W G W L G F N K E Q G R L Q I A A C S N Q D P L Q G T T G — L I P L L G I D V W E H A Y Y r a t 3 E K L T A M S V G V Q G S G W G W L G F N K E Q G R L Q I A A C S N Q D P L Q G T T G — L I P L L G I D V W E H A Y Y mouse 4 E K L T A V S V G V Q G S G W G W L G F N K E Q G R L Q I A A C S N Q D P L Q G T T G — L V P L L G I D V p i g 5 E K L V A A A V G V Q G S G W A W L G F N K E S K R L Q I A T C A N Q D P L Q G T T G — L F P L L G I D V h a g f i s h 6 E K M S A V S V A V Q G S G W G W L G Y D K E T G R L R I A A C A N Q D P L Q A T T G — L I P L L G I D V lamprae 7 E K M T A A T V A V Q G S G W G W L G L D P T S K K L R I V A C P N Q D P L E G T T G — L K P L L G I D V l a n c e l e t 8 K Q L S A S T V A V Q G S G W G W L G Y C P K G K I L K V A T C A N Q D P L E A T T G — L V P L F G I D V W E H A Y Y C. elegans 9 K E L T T L T V A V Q G S G W G W L G F N K K S G K L Q L A A L P N Q D P L E A S T G — L I P L F G I D V W E H A Y Y Drosophila melanogaster 10 T T L T A A T V A I Q G S G W G W L G F D P K T H H L K I A T C V N Q D P L Q A T T G — M V P L F G I D V sea cucumber 11 . D K F S T A S V G V K G S G W G W L G Y C P K N D K L A V A T C Q N Q D P L Q L T H G - - L I P L L G L D V l o b s t e r Plant Mn SODs 12 Q K M N A E G A A L Q G S G W V W L G V D K E L K R L V I E T T A N Q D P L V - S K G A N L V P L L G I D V W E H A Y Y tobacco 13 Q K I N A E G A A L Q A S G W V W L G L D K D L K R L W E T T A N Q D P L V - T K G A S L V P L L W I D V W E H A Y Y pea 14 K K M N A E G A A L Q G S G W V W L A L D K E A K K V S V E T T A N Q D P L V - T K G A S L V P L L G I D V W E H A Y Y corn 15 K L T N T K L A G V Q G S G W A F I V K N L S N G G K L D W Q T Y N Q D T V - T - G P - L V P L V A I D A W E H A Y Y yeast A r c h a e b a c t e r i a l Mn SODs 16 K E F S Q A A I S A E G S G W A V L T Y C Q R T D R L F I M Q V E K H N V N V - 1 P H F R - - 1 L L V L D V W E H A Y Y Methanobacterium t. 17 A Q F H A A A T T V Q G S G W A A L G W D T L G N K L L I F Q V Y D H Q T N F - P L G I V - - P L L L L D M W E H A F Y Mycobacterium tuberculosis 18 A Q F S A A A N G L Q G S G W A V L G Y D T L G N K L L T F Q L Y D Q Q A N V - S L G I I - - P L L Q V D M W E H A F Y Mycobacterium leprae 19 G E F E A A A S A — A G G W A L L V Y D P C A K Q L R N V P V D K H D Q G A - L W G S H — P I L A L D V W E H S Y Y Haloarcula marismortui 20 G E F E A A A G A — A A G W A L L V Y D S F S N Q L R N V W D K H D Q G A - L W G S H — P I L A L D V W E H S Y Y Haloferax volcanii 21 A E F E A A A S A - — A S G W A L L V Y D S H S N T L R N V A V D N H D E G A - L W G S H — P I L A L D V W E H S Y Y Halobacterium cutirubrum 22 A E F E V A A G A — A S G W A L L V Y D P V A K Q L R N V A V D N H D E G A - L W G S H — P I L A L D V W E H S Y Y Halobacterium sp. sig 23 A E F E V A A G A — A S G W A L L V Y D P V A K Q L R N V A V D N H D E G A - L W G S H - - P I L A L D V W E H S Y Y ifalobacterium halobium 24 E G F T N V A K S V Q G P G W A G L L Y D P V N H R L V I E S I E K H Q Q N H - L V S M I - - P L L V L D M BSC23 E u b a c t e r i a l Mn SODs 5 — b l —b2b2- b3 * * > 25 D E F S K A A A G R F G S G W A W L W N N - G - - E L E I T S T P N Q D S P I M E G K T — P I L G L D V W E H A Y Y Bacil. stearothermophilus 26 E K F N A A A A A R F G S G W A W L W N D - G — K L E I V S T A N Q D S P L S D G K T — P V L G L D V W E H A Y Y L i s t e r i a monocytogenes. 27 A E F E K A A A S R F G S G W A W L V L K G - D — K L A W S T A N Q D S P L M G E A I S I P I M G L D V W E H A Y Y - G A S G F - E. coli a 5 — b l __ b 2-b2- ---b3—-*- * > 28 E K L T Q A A M G R F G S G W A W L V K D P F G — K L H V L S T P N Q D N P V M E G F T - - P I V G I D V W E H A Y Y Thermus thermophilus 2 9 E A F T K A A L T R F G S G W A W L S V T P E K — K L W E S T G N Q D S P L S T G N T — P I L G L D V W E H A Y Y Methylomonas 30 A A F T A A A T T R F G S G W A W L W D K E G — K L E V T S T A N Q D T P I S Q G L K — P I L A L D V W E H A Y Y Streptococcus mutans Eukaryotic Fe SODs -ARKFDGENVANPPSP- (soybean) 31 T E F K Q A A A T Q F G S G W A W L V L — I D N G T L K I - K T T G N A D T P I A H - G Q T P L L T I D V W E H A Y Y Anacystis nidulans 32 K E F K A A A A T Q F G S G W A W L A Y K P E E - K K L A L - V K T P N A E N P L V L G - Y T P L L T I D V W E H A Y Y Tobacco 33 E E F N A A A A T Q F G A G W A W L A Y S N - E — K L K V - V K T P N A V N P L V L G - S F P L L T I D V W E H A Y Y Arabidopsis 34 D E F K A A A A T Q F G S G W A W L A Y R I D E D N K L W - L K S P N A V N P L V W G G Y Y P L L T I D V W E H A Y Y Soybean B a c t e r i a l Fe SODs 35 E Q F S Q T A A T T F G S G W A W L V Q — D Q S G A L K I - I N T S N A G T P M T E T G L N A L L T C D V W E H A Y Y Legionella pnemophila? 36 E A F N K S A A G N F G S G W T W L V K — K A D G T L D I - V N T S N A A T P L T T - A D K A L L T C D V W E H A Y Y Bordetella pertusis 5 b l __ b 2>b2— --b3-*- * 37 A Q F T D A A I K N F G S G W T W L V K — N S D G K L A I - V S T S N A G T P L T T - D A T P L L T V D V W E H A Y Y Echerichia coli 38 A K F T D S A I N N F G S S W T W L V K — N A N G S L A I - V N T S N A G C P I T E E G V T P L L T V D L W E H A Y Y Photobacterium leiognathi 39 A L F T D S A N N H F G S G W A W L V K — D N N G K L E V - L S T V N A R N P M T - E G K K P L M T C D V W E H A Y Y Coxiella b 4 bl/b2 > b2/3 * * 40 E E F T K T S V G T F G S G W A W L V K A D G S L A L - C S T I G A G A P L T S - G D T P L L T C D V W E H A Y Y Pseudomonus o v a l i s 41 K K F T E K A V G H F G S G W C W L V E H D G K L E I - I D T H D A V N P M T - N G M K P L L T C D V W E H A Y Y Entamoeba 42 E E F D T A G T T L F G S G W V W L A S — D A N G K L S I - E K E P N A G N P V - R K G L N P L L G F D V W E H A Y Y Bacteroides g i n g i v i l u s 43 K E F N A A S V G L F G S G W A W L S V - - D K D G K L H I - T K E P N G S N P V - R A G L K P L L G F D V W E H A Y Y Bacteroides f r a g i l i s 44 T L F N T R T A A I Q G S G W G W L G Y - - D T V S K S L R - L F E L G N Q D M P E W S S I V P L L T I D V W E H A Y Y Tetrahymena pyriformis Discussion: Animal Mn SODs 181 185 190 195 200 205 210 220 225 230 2 I I I I I I I I I I 1 LQYKNVRPDYLKAIWN-VINWENVTERYMACKK--2 LQYKNVRPDYLKAIWN-VINWENVSQRYIVCKK--3 LQYKNVRPDYLKAIWN-VINWENVTERYTACKK— 4 5 6 7 8 LQYKNVRPDYVNAIWK-IANWKNVSERFAKAQQ— 9 LQYKNVRPSYVEAIWN-IANWDDISCRFQEAKKLGC 10 H Plant MnSODs 12 LQYKNVR PDYLKNIWK-VMNWKYAN EVY EKEC P 13 LQYKNVRPDYLKNIWK-VINWKHASEVYEKESS 14 LQYKNVRPDYLNNIWK-VMNWKYAGEVYENVLA 15 LQYQNKKADYFKAIWN-WNWKEASRRFDAGKI A r c h a e b a c t e r i a l Mn SODs 16 IDYRNVR PDYVEAFWN-1VNWKEVEKR 17 LQYKNVKVDFAKAFWN-WNWADVQSR 18 LQYKNVKADYVKAFWN-WNWADVQSR 19 YDYGPARGDFIDAFFE-WDWDKAAEE 20 HDYGPARGDFVSAFFE-WDWDEPAAR 21 YDYGPDRGSFVDAFFE-WDWDEPTER 22 YDYGPDRGSFVDAFFE-VIDWDPIAAN 23 YDYGPDRGTFVDAFFE-VIDWDPIAAN 24 E u b a c t e r i a l Mn SODs a 6— a 7 — a 7 25 LKYQNRRPEYIAAFWN-IVNWDEVAKRYSEAKAK 26 LKFQNRRPEYIETFWN-VINWDEANKRFDAAK— 27 LKFQNRRPDYIKEFWN-WNWDEAAARFAAKK— a 6 a 7 a 7 28 LKYQNRRADYLQAIWN-VLNWDVAEE FFKKA 29 LKYQNRRPEYIGAFFN-WNWDEVSRRYQEALA-30 LNYRNVRPNYIKAFFE-VINWNTVARLYAEALTK Eukaryotic Fe SODs 31 LDYQNRRPDYISTFVEKLANWDFASANYA 32 LDFQNRRPDYISIFMEKLVSWEAVSSRL KA—ATA 3 3 LDFQNRRP DY IKT FMTNLVSWEAVSARLEAAKA- -ASA 34 LDFQNRRPDYISVFMDKLVSWDAVSSRLEQAKALITSA E u b a c t e r i a l Fe SODs 35 IDYRNRRPDYIEAFWS-LVNWDFAS S 36 I DY RNAR PKY L E N FWA- LVNWE FAA K a 6 — a 7-7 37 IDYRNARPGYLEHFWA-LVNWEFVA K 38 IDYRNLRPSYMDGFWA-LVNWDFVS K 39 IDTRNDRPKYVNNFWQ-WNWDFVM K a 5/g > 4 0 IDYRNLRPKYVEAFWN-LVNWAFVAEEGK 41 IDTRNNRAAYLEHWWN-WNWKFVEEQL-42 LTYQNRRADHLKDLWS-IVDWDIVES 43 LDYQNRRADDVNKLWE-IIDWDWEK 4 4 LDYQNLRPKYLTEVWK-IVNWREVEKRYLQAIE 109 35 240 I I man ra t mouse p i g h a g f i s h lamprae l a n c e l e t C. elegans Drosophila melanogaster sea cucumber l o b s t e r tobacco pea corn yeast Methanobacteriurn thermoautotrophicum Mycobacterium tuberculosis Mycobacterium leprae Haloarcula marismortui Haloferax volcanii Halobacterium cutirubrum Halobacterium sp. sig Halobacterium halobium BSC23 B a c i l l u s stearothermophilus Listeria monocytogenes. Echerichia coli Thermus thermophilus Methylomonas Streptococcus mutans Anacystis nidulans Tobacco Arabidopsis Soybean Legionella pnemophila ? Bordetella pertusis Echerichia coli Photobacterium leiognathi Coxiella b Pseudomonus oval is Entamoeba Bacteroides g i n g i v i l u s Bacteroides fragilis Tetrahymena pyriformis 110 PR E V I O U S WORK WITH CU - ZN SOD. Four experimental attempts have been made using direct genetic manipulation to extend LSP in Drosophila (Shepherd et al. 1989, Seto et al. 1990, Reveillaud et al. 1991, and Orr & Sohal 1994). Of these, three have involved increasing the flies' natural defences to oxygen radicals by increasing the levels of Cu Zn SOD. The only experiment not based on increased free radical protection was that by Shepherd et al. (1989) who introduced additional copies of the gene for eFIa. Several researchers have found that overexpression of Cu Zn SOD is toxic above certain levels, (about 1.6x wild type in flies Fleming et al. 1992) which may explain the inability of various groups to gain significant life span extension with this enzyme alone. The recent experiment by Orr & Sohal (1994) demonstrates that a concomitant increase in protection from H2O2 makes a significant improvement over higher levels of Cu Zn SOD alone: flies co-transformed with Cu Zn SOD and catalase showed significant increases in LSP over flies transformed with Cu Zn SOD alone. Yim et al. (1993) have shown that Cu Zn SOD is able to react with H2O2 and other physiological substrates to form hydroxyl radical. This may account for inactivation of Cu Zn SOD by H 2 0 2 , and may also account for the toxic affects of Cu Zn SOD overexpression. Alternatively the increased levels of Cu Zn SOD could simply result in higher levels of H2O2 with a resulting increase in toxicity. The possible roles of NO. O2-, and H 2 02 as mitogens in cell division and morphogens in embryogenesis may make complete protection from these agents impractical for the cell, and juvenile or constitutive expression of excessive antioxidant enzymes may result in growth inhibition or fatal teratogenic effects. Thus specific adult induction of SOD and catalase to test for life span extension may be preferable to ectopic expression. A third possibility is that increased Cu Zn SOD does not increase LSP in flies because of its subcellular localization to the peroxisomes (Dhaunsi et al., 1992). The predominant cellular source of superoxide is the mitochondria: thus it may be that they are the most important target for antioxidant protection. In this regard the reported Discussion: 111 absence of catalase in the mitochondrial matrix (Rikans et al. 1992) and the complete absence of glutathione peroxidase in Drosophila (Orr & Sohal, 1992) are probably major contributors to shortened LSP in Drosophila. H I C O N C L U S I O N S : We can now conclude that cellular aging is, at least in part, an oxidative process. We can see that it is held in check by cell division, and is controlled by both the quantity and quality of free radical defences, but even more so by the rate at which reactive oxygen species are produced, especially in the mitochondria (Sohal et al. 1989). Drosophila may lack any intramitochondrial defences against H2O2 (Rikans et al. 1-992, Orr & Sohal 1992), and thus transformation with a gene for some form of mitochondrial matrix peroxidase or catalase should yield large increases in LSP, especially i f that enzyme is resistant to inactivation by superoxide. Increased levels of glutathione reductase in the mitochondria, or anything that slows or reverses the alterations in mitochondrial membrane integrity that accompany aging (Pieri 1991, 356, Sohal & Brunk 1992, 299), should slow aging in higher eukaryotes. The overall scenario for most higher eukaryotes probably runs something like this: 1) After the cessation of cell division, chronic oxidative alterations to membrane components and/or other (hormonal?) effects result in a decline in the fluidity of the mitochondrial inner membrane: this results in reduced transmembrane "flipping" by ubiquinone, and alters its surface exposure and availability to oxygen. Membrane alterations also reduce the transport rates for inorganic phosphate and A D P . Biosynthesis of new cytosolic ribosomes also declines with the cessation cell division, and oxidative and chemical inactivation begins to reduce the number of active ribosomes. 2) With the reduction in availability of ADP and P i , (which are normally rate limiting), oxidative metabolism slows, and deficits in ATP biosynthesis begin to accumulate; the ETC begins to back up, and electron saturation of ubiquinone, F A D H 2 and other electron carriers increases. This results in more univalent and divalent electron transfers to free oxygen, resulting in increases in superoxide and H2O2 in the mitochondrial matrix. 3) Declines in protein synthesis and other cellular activities requiring A T P begin to take a toll: the rate of mitochondrial turnover in the cell slows Conclusions: 113 113 as a result, and the problems with the mitochondrial membrane become progressively worse. Reduced turnover and increasing levels of damage begin to snowball as old age approaches. 4) As H2O2 and organic peroxide damage increases, the mitochondrial matrix stores of reduced GSH become depleted: repair of oxidative damage, and resistance to increases in H2O2 levels becomes increasingly compromised. 5) As mitochondrial damage becomes more severe, the mitochondria lose the ability to metabolize specific substrates, the result of inhibition of specific redox enzymes by membrane alterations or an inability to passage electrons through N A D H or one of the other initial electron carriers in the ETC. This results in the accumulation of toxic levels of these substrates and in substrate inhibition of the last steps in glycolysis and the T C A cycle. At some point the electrochemical gradient across the inner membrane is lost, and virtually all externally supplied protein replacement, and thus all mitochondrial turnover, ceases. Cell division potential has long since been lost: the cell can no longer generate the required threshold levels of ATP. 6) In the final days, the cell can no longer keep up its duties to the rest of the body: the complex web of bodily functions begins to unravel: gastric juices are less acidic, neurotransmitter levels drop, gastric enzymes decline, liver activity, kidney function and cell replacement are reduced or cease all together. Declines in gastric effectiveness result in starvation for energy and essential nutrients; any insult, no matter how inconsequential, is now enough to cause death, as no reserves of energy or repair potential are left. 7) If nothing comes along, eventually the heart muscle or the innervation to it becomes critically compromised; the organism can no longer supply enough blood to its tissues: death comes creeping in as it measures the time until its victim is too exhausted to resist, or too demoralised to care. Part 2) of this scheme explains why metabolism and energy consumption both decline with age in higher eukaryotes (Schneider & Rowe 1990, Finch 1990), even after the electrochemical gradient is lost. The tight coupling of oxidative phosphorylation with the electron transport chain (Zubay 1983, pg. 383) also fits well Conclusions: (|4 114 with this theory by explaining the life extending effects of semi-starvation; in well fed animals, most of the ubiquinone and other sites of electron leakage to oxygen are maintained in the reduced state, as A D P levels are limiting. This results in higher rates of spurious oxidation by molecular oxygen and thus higher levels of superoxide and H2O2. In semi-starvation, ETC electron carriers spend more time in the oxidized state, and spurious electron transfers to oxygen are reduced, resulting in decreased damage rates over the animal's lifetime. Alterations in mitochondrial membrane fluidity may also explain the reduced utilisation of glutamate as a carbon source. Glutamate uptake is dependent on specific membrane bound transporters for entry into the mitochondria, and is dehydrogenated via the N A D H pathway. Utilisation of succinate is not substantially affected by aging, but it uses a different transporter for entry, and succinate dehydrogenation uses the F A D H 2 pathway. Thus we can see several factors which should enhance longevity in any organism: 1) inducers of cell division and growth should increase longevity by diluting the effects of free radical damage and increasing the population of younger more vital cells. 2) Agents which protect or improve the character of the mitochondrial membrane will result in a reduction in the biosynthesis of superoxide, which should slow the rate of free radical damage and restore mitochondrial function, and 3) agents which increase the levels of reduced glutathione in the mitochondrial matrix or by other means increase the rate of removal of hydrogen peroxide in the matrix should result in reduced rates of membrane damage and an increased level of protection for the cell. I have tried in this thesis to make as clear and eclectic a presentation as possible of the field of aging research. The constraints of space, time, and the continued publication of new material, make true completeness a hopeless task, however. Conclusions: 115 115 As a final note of hope, I will now refer the reader to the words of Isaiah, where the prophet writes from God foretelling a time to come: "But be glad and rejoice forever in what I will create, for I will create Jerusalem to be a delight and its people a joy. I will rejoice over Jerusalem and take delight in my people; the sound of weeping and of crying will be heard in it no more. 'Never again will there be in it an infant who lives but a few days, or an old man who does not live out His years; he who dies at a hundred will be thought a mere youth; he who fails to reach a hundred will be considered accursed.'" Isaiah, chapter 65 verses 18-20 116 REFERENCES: Abe, Y . , and Okazaki, T. (1987). Purification and Properties of the Manganese Superoxide Dismutase from the Liver of Bullfrog, Rana catesbeiana. Archives of Biochemistry and Biophysics, vol. 253 (1), 241-248. Allen, G.R. , and Balin, A . K . (1989). 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Heredity, vol. 66, 29-39. 133 APPENDIX: D N A A B S PROGRAM 20 WIDTH "LPT1:",120:CLS 30 O P E N "LPT1:" FOR OUTPUT AS #1 50 INPUT"sequence to be searched (OLD calls up the list of old probe sequnces)";SEQ$ 51 IF SEQ$ = " O L D " OR SEQ$ = "old" T H E N GOTO 900 60 A G = 12010:AA = 15200:AT=8400:AC=7050:AI=12000:AY=7725:AR = 13605:AN=10665 70 W G = 150.13:WA = 134.13:WT=125.11:WC = 110.1:WI = 135.11:WR=142.13:WY = 117.61: W N = 129.8 80 INPUT "DO Y O U W A N T THIS PRINTED? IF SO, START PRINTER NOW" ;PRNTED 100 N M = N M + 1 120 BSE$=MID$(SEQ$,NM,1):IF BSE$ = "" T H E N GOTO 500 130 IF BSE$ = " Y " T H E N Y = Y + 1 140 IF BSE$ = "R" T H E N R=R+1 150 IF BSE$ = "T" T H E N T=T+1 160 IF BSE$ = "C" T H E N C = C + 1 170 IF BSE$ = " A " T H E N A = A + 1 180 IF BSE$ = " G " T H E N G=G+1 190 IF BSE$ = " N " T H E N N = N + 1 . 195 IF BSE$ = "I" T H E N 1=1+1 200 GOTO 100 500 • C A L OF M O L WT— 510 W T = T * W T + C * W C + G * W G + A * W A + Y * W Y + R * W R + P W I + N * W N : T O L = T + C + A + G + Y + R + N + I 600 ' C A L OF M O L A R EXTINCTION COEFFICIENT 605 PRINT SEQ$ 610 E P = A T * T + A C * C + A A * A + A G * G + Y * A Y + R * A R + I * A I + N * A N : P R I N T "E="EP " (MOLAR EXTINCTION COEFICIENT) M W T = " W T 615 CON=1/EP*1000000! 620 PRINT" ABS/"EP" = [] I N MOLES/LITER. W H E N ABS = 1, [PROBE] =" C O N "uM/L per ABS 650 P R I N T ' B A S E COMPOSITION IS: T"T" C"C" A " A " G"G" I T ' Y " Y " R"R" N " N " T O T A L ' T O L 670 TM=A*2+T*2+C*4+G*4+Y*2+R*2+N*2+I*2 680 PRINT "TM~=A*2+T*2+C*4+G*4+Y*2+R*2+N*2+I*2: in 1.0M Na+, the min Tm, Tmin="TM "C" 690 TEX=TOL-23:IF T E X > 0 T H E N T M = T M - ( T E X * 3 ) : T M = T M + T E X * . 4 700 PRINT" tm for sequence > 23 bases is " T M " C " 790 GOTO 1010 "IF SEQ$ = "OLD" T H E N GOTO 1010 Appendix 134 800 E N D 900 RESTORE 910:FOR J = l TO 9:READ OSQ$(J):NEXT J:J=0 910 D A T A jan90, un90 , 6-90 , s2, GT1, sci ,Hind III linker, open, space, here 920 PRINT" old sequences" 1000 J=J+1:PRINT " #"J OSQ$(J)" ";:IF J<9 T H E N :GOTO 1000 1010 INPUT "WHICH O L D SEQUENCE (start printer now if printout desired)";OLD 1020 O N O L D GOSUB 2000,2010,2020,2030,2040,2050,2060,2070,2080,2090:GOTO 60 2000 SEQ$ = "jan90: aa8-28 GGI A T G CTG A T G CCI C G G G A C CTC GGI GTG T A G TTG C G G GTC T A G T A C GTC G A C GT": R E T U R N 2010 SEQ$ = "un90 aa 206-198 C C A GTT G A T G A C GTT C C A G A T G G C C T T " : R E T U R N 2020 SEQ$ = "6-90 aa 206-201 C C A GTT G A T G A C GTT C C A " : R E T U R N 2030 SEQ$ = "s2 aa8-13 C C N T A Y G A Y T A Y G G N G C " : R E T U R N 2040 SEQ$ = "gtl aalO-19 G A R C C N C A Y A T N A R Y G C N Gc A R A T N A T G C A " : R E T U R N 2050 SEQ$ = "scl aa 29-36 5 A A G C A T C A T G C G G C G T A T GTG A A 3 " 2060 SEQ$ = "hind i i i linker G C C C A T G G G C " :RETURN Appendix 135 C OPIA 3 ' L T R Sequence of the Copia-like element 3' long terminal repeat aligned with the tentative sequence of band 2-2-2 D N A obtained by PCR of whole cellular R N A with oligos S2 and 6-90. top line: Copia-like element from Drosophila, E M B L # D M I S 1 7 6 lower line: band 2-2-2 preliminary sequence AAAACTA7AAA TTAAGAAAAA ATCAAAATCA AAACACAGCA CAACAAATAG 6850 5* CC AAATGGAAGA CGTTCCATTA CCCCTACTAT ATCCATCAAT CCCAGCCCAA 6900 I | || || I I I I I I I d i r e c t matches (|) CAGTTGATGA CGTTCCA 3' 6-90 Probe sequence GTATAGGCTT CTCTTTAAGG GAAGGGAAGT GAC AT AT T C A CATACA7AAAC 6950 CACATAACGT AGAGTAAACA TATTGAAAAG CCGCATACGT CAACAATAAG 7 000 T GAC CAC CAT GCTAATGTGG ATCAAATAAC AA7A7AATATCC ACTCTGCATT 7050 TTGACACCCC CATACTGTAT GCCATCTGCG CAGTATGCAT TCTAATAAAC 7100 I I I I I I I I I M I N I M I || I I I I I I I I Band 2-2-2 3'GGTAGACGC GTCATACGTA AGATTATTTG AAATTCTTTG ACAG CGG CAC TTAGCCATTC TTGTAAACAA ATCTTAAAGT 7150 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I TTTA GAA C TGTCGC GTG AATCGGTAAG 7AACATTTGTT TAGAATTTCA CTGCCTGCTC TCTCTGAGGC TTCTCCTCCA CTTAAGAATC CAAGAGCAAT 7200 I I I I I I I I I I M I I I I I M I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I GACGGACGAG AGAGACTCCG AAGAGGAGGT GAATTCTTAG GTTCTCGTTA GCTCTCCCAA AAACACTAAC ATATTCTTTA AGCAAGCACA GAGGCTTCTC 72 50 I I I I I I I I I I I I I I I I CGAGAGAGTT TTATGAATTC GTAGATTCTC GTTACGAGAG GGTTTTTGTG CTCATTTTCA CTTTCATTTG ATTTTCAGTC TTAAGCTGAA CGTTAATCAA 7300 I I I I I I I I I I I I I I ATTGTATAAG AAATTCGTGT CTCCGAAGAG GAGTAAAAGT GAAAGTA7AAC TAAACAACAC AATCGATACC GAAATTTTGA TTCGTTTTAT TTTGGCAAAA 7350 I I I I I I I I I I TAAAAATCAG AATTCGACTT GCAATTAGTT ATTTGTTGTG TTAGCTAG 5' CTCAATTTTC AGCGTTGGTC TTAGTTCATA TTCGGAACGG TCCATTTAAT 7 400 AGACTCAAAA CTATTTATTG CAACCATTTA TTTGCAATT 74 39 

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