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Aprtl, 1979 a Study of the Metabolisi4 of a Novel Dinuclioside Polyphosphatë (Hs3) Found in Mammalian Cills : Possiblt Regulat]On of Nucltic Ac]D Biosynthisis

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Aprtl, 1979 a Study of the Metabolisi4 of a Novel Dinuclioside Polyphosphatë (Hs3) Found in Mammalian Cills : Possiblt Regulat]On of Nucltic Ac]D Biosynthisis THE UI'IIVERS TTY OF MANITOBA A STUDY OF THE ù1ETABOLISM OF A NOVEI, DINUCLEOSIÐE POLYPHOSPHATE (HS3) FOUND IN ITAPIT4AL IAN CELLS: POSS]BLE REGULATTON OF TTUC],EÏC ACTD BIOSYNTHESÏS BY HS3 DURING STËP-DOWN GRO¡]TH CONDITIONS BY SI{EE HAN GOH A THES]S SUBI'IITTED TO THE FACULTY OF GR.ADUATE STUDIES IN PART]AL FULLFTLIqENT OF THE REOUTREUENTS FOR THE DEGREE OF DOCTOR OF PHTLOSOPHY . DEPARTMENT OF I{ICROBÏOI,OGY WTNNIPEG , 1UANTTOBA APRTL, 1979 A STUDY OF THE METABOLISI4 OF A NOVEL DINUCLIOSIDE POLYPHOSPHATË (HS3) FOUND IN MAMMALIAN CILLS : POSSIBLT REGULAT]ON OF NUCLTIC AC]D BIOSYNTHISIS BY HS3 DURING STEP.DOI,,IN GROWTH CONDITIONS. BY SI.IEE HAN GOH A dissertation slrbmitted to the Ftculty of Gradtrate Studies ol the University of Marlitoba in partial fulfillment of tlre requirerrrents ol the degree of DOCTOR OI. PI{ILOSOPHY o! 1979 Pemrission has bcen granted to the LIBRARY OF TIIE UNIVER- SITY OF MANITOBA to lend or sell copies of thìs dissertätion, to the NATIONAL LIIIRARY OF CANADA to microfilni this dissertation and to lend or sell copies of the film' and UNIVERSITY MICROFILMS to publish an abstrâct ol'this dissertation' 'l'lìc author reserves otlrer publicatiorr rights, and neither the dissertation nor extensive extracts frorìl it rnay be printed or other- wise reproduced witlìout the autl'ìor's writtell pernrission To Siew See and my parents Acknowledgements I would like to lhank Ðr. Herb B- LeJohn for his advice, interest and patience during the course of this research progranme. The friendship and. constructive criticisms of Linda, Renate, Dave, Glen, Rob and. other members of the Dept. of }licrobiology are gratefully acknowledged - I would also wish to express my deepest gratitude to Dr. Jím Vtright for his generosity and inval"uable advice \¡rith man¡nalian t.issue culture teihniques and the gift of numerous cell lines used for this study. The counsel of Drs. I. Suzuki and PeÈer Ï,oevren is much appreciated. ABS TRACT l-v A novef dinucleoside polyphosphat.e (HS3) prevously found ín various fungi has now been detected in numerous cultured mammalian ce11 lines. Physical and chemj.caf analyses of HS3 (McNaughton et. aI' 1978) show that the molecule consists of a glutamyl,-ADP- sugar moiety which is covalently linked to a UDP-mannitol-- tetraphosphate . Physiological studi.es demonstrated that as long as de novo purine biosynthesis was bfocked (by nutrient deprivation, drugs or mutation) and no exogenous purines (anil some pyrimidines) \^rere supplied, I1S3 accumulation took place. For exampfe, the addition of de novo purine inhibitors (50 or biosynthesis Lf-azaserine ¡:g,/ml) MTx (f .Ju¡a) I resulted in HS3 synthesis as did the \,¡ith- drawal- of glutamine (2 mM). The addition of hypoxanthine, inosine, adenine, adenosine, guancsine, uridine and cytidine, but not thymidine, (al-l- at 0.3 mM) strongÌy suppressed HS3 accumulation in g lutamine- starved cHo cells. CHO purine salvage nutants (HGPRT-) failed to do likewise when supplied v/ith high concentrations of hypoxanthine, guanosine and inosine (0.3-2. 0 m.l'!) . A cHo mutant (GAT -auxotrophic for adenosine, thymidine and glycine) with blocked de novo purine biosynthesis, accumulated HS3 only when adenosine (0.1 mI'I) , but not glutamine, was withdrardn f r'om its grohrth medium. Thus HS3 accumulation may be related to a lack of either precursors for purine nucleotide biosynthesis or of purinê nucleotides themselves. The rate of HS3 synthesis in CHO l,tT ceLls increased 5-6 fold sholtly after gluta¡nine withdrarval and hras maintained at that Level for at least 6 hr before declínÍng slowly back to the control- rate by 22 hr. The pool sizes of IIS3 increased and decreased in concert with rate changes. The replenishment of glutamine and adenosine to starved CHO WT and CHO GAT- celfs respect- ive]y, resulted in an immediate depletion of the accumulated IIS 3. An inverse relationship r^¡as observed betr\'een nucleic acid (DNA and RNA) synthesis and HS3 accumulation. When DNA and RNA synthesis decreased, HS3 synthesis increased and vice versa. No such correlation was apparent for protein synthesis. The inhibition of protein synthesis by either puromycin (100 ¡¡9,/n1) or cycloheximide (10 ¡rg,/m1) or that of Rl¡A synthesis by actinomycin D (1 ¡¡glml), stimulated HS3 depletion in glutanine- starved CHO M cel1s. Both fungal- and mammaLian HS3 v/ere found to be equally potent inhlbitors of in vitro and in vivo v.l manì¡nalian RNA synthesis. AIso, studies by Lewis et. al. (1977) showed that HS3 strongly inhibited partially purified ribonucleotide reductase from CHO celLs. Thus the decrease in DNA synthesis during periods of rapid HS3 accumualtion may be the indirect result of a deficiency in deoxyribonucfeotides. The data suggest t,hat HS3 may be involved in the regulation of nucLeic acid biosynthesis in mammalian cells. TÃ3LE OF CONTENTS PAGE Acknowledgements . iii Abstract. v Tabre of contents. viii List of Tables' xvii List of Figures. xix Abbreviations. xxii rntroduction''''' ' xxvi H i stori ca l- Tissue Culture. 2 Development of Tissue Culture Techniques. 2 Growth Requirements and Characteristics of cultured Marûnalian Cell-s. 5 Marnmalian Cells: RNA Biosynthesis and Amino Acid Withdrar¡a1. 9 Physiology, Biochemistry and Possible Roles of Polyphosphates and Unique Phosphory- lated Nucleosides in Procaryotic and Eucaryotic Cellutar Regulation. 11 vr- 11 PAGE Polyphosphates... 12 Adenosine 3' :5' Cyclic Monophosphate. 16 Biosynthesis, Catabolism and possi- ble Regul-atory Ro1e(s).... l-6 Cyclic AMP in Bacteria. 18 Cycti-c AIUP in Èhe Fungi. 20 Cyclic AMP in Manmalian Ce11s in culture. 22 Guanosine 3':5' Cyclic Monophosphate.. 24 Bacterial MS (ppGpp and pppGpp) Nucleotides 26 The stringent Responbe . 26 fn Vivo Synthesis of ppGpp and Pppcpp. 27 In Vitro Synthesis of ppGpp and pPpcpp. 32 Effects of ppcpp and pppcpp on CeIl- ular Enzymes, Translation and Transcripti.on In Vitro 34 Influence of Carbon and Nitrogen Sources on ppcpp, pppcpp and rRNÄ Metabolism. 37 HPN Compounds.... 39 Diguanosine Polyphosphates. .. 4l ix PAGE Diadenosine Polyphosphate s . 42 Triadenosine Pentaphosphate. 43 Dinucl-eoside Polyphosphates (HS) ..... 44 Other Pol-yphosphorylated Nucleos j-des. 48 Mammal-ian Purine.and Pyrimidine l4etabolism. 49 Regulation of De Novo Purine Biosynth- etic Enzynes in J"lammalian cells and Tissues. 49 PRPP Synthetase and IntracelLufar PRPP Leve.ls 52 5 -P ho spho r i bo s y I - 1-Pyrophosphate Amidotrans fera se. 54 Regulation of A-tvlP and GI'IP Synthesis from IMP. 57 Induction, Repression and Derepression of De Novo Purine Biosynthetic Pathway Enzymes. 58 Regulatj-on of De Novo Pyrimidine Bio- synthesis in lrlammal-ian Cells and Tissues. 59 lluman Genetic Disease and Defective Purine and Pyrimidine Metabofism. -.. - 63 PAGE cout . 63 Lesch-Nyhan Syndrome. 64 Immunodeficiency Syndromes. 65 Oroticaciduria and Xanthinuria.... 69 Platerials and Methods Organisms. 72 Mainrnal ian Cel1 lines . 72 Fungal Cells. 72 Gror'Jth Media, Sera and Cul ture-rrare s. t5 Radioisotopes. ¡ 73 Miscellaneous Materials..... 73 MeÈhods. 75 crowth of Cell Lines. 75 -'n2a crowth, - Orthophosphate Labelling and Formic Acid Extraction of Cell-s for Analysis of HS PoLyphosphorylated NucLeotides During Various Growth Conditions. 76 chromatography of "p-LabeÌled Formic Acid Extract from Àtamrnalian ce]Ls... '7 '7 DNA, RNÀ and Protein Analysis. 78 DNA PuÌse Labelling Experiments. 79 XI PAGE Protein Pulse Labelling Experiments 79 continuous RNA Labelling Experiments 80 Det.ermination ot -1lC-adenine rncor- poration into Nucleosides and Nucleotides 81 Determination of ATP Levels Using the Firefly Luciferin/luc ifera se lqethod.. 81 Isolation of HS3 From Ach]ya and CHO I{T cells. 83 Isolation of DNA-Dependent RNA Pofymerases From CHo ÌlT Cells. 84 Cel1 Permeabi l-i zation and Assay for RN.A synthesis. 87 Assay of RNÀ synthesis By Permeabilized CHo t^tT cells In the Presence of Achlya HS3... BB Re sul ts Hs3 Synthesis by cHo wT cells. 90 Effect of Varying Concentrations of L-Glutamine on HS3 and GTP Synthesis by cHo v,lT cetts. 99 xii PÀGE Effect of L-clutanine or !-fsol-eucine Deprivation on HS3 MetaboLism by CHO WT Cel1s. tO2 Effect of L-Glutarnine, Adencsine and Thymidine Deprivation on HS3 Metabol"ism by CHO GAT- Cells, IO2 Effect of L-Glutamine Withdrawal and the Replenishment with purines on HS3 Metabolism by CHO lfT and CHO purine Salvage Mutant Cel1s. IO5 Influence of purine and pyrimidine Compounds, MethoÈrexate. and Azaserine on the AccumulaÈion and Depletion of HS3 in Three CHO CeIt Lines Incubated in the presence or Absence of L-clutamine l_09 Determination of ATp Level-s in CHO VtT Cells Àfter Exposure to Various Grovrth Conditions and Metabolic Inhibitors. II0 Effects of Gtutamine Deprivation or Sufficiency on'"c-Adeninea^ ïncorporation into Adenine Nucleoside xii i P1\GE and Nucleotides by CHO yHD 13 Cells.. tI3 Effects of L-Glutamine and Adenosine Deprívation and Replenishment on Rate of AccumuLation and pool Size of HS3.. 115 a) FaÈe of --P-Labelled HS3 Accumulated by CHO WT Cell-s During cLutamine Deprivation Follor^'ing 32ní *ith- drawal From the Incubation lrledium..... !25 Time Course Studies of the Effects of s-Fluorouracil on Elevated Hs3 Levels Accumulated During L-GLutamine Starvation or Methotre*ate Treatment.. I28 Effect of L-Glutamine and Adenosine Starvation on Protein Synthesis. 132 Effect of L-clutamine and Adenosine Ðeprivation on Rate of 3H-Thymidine Incorporation lnto DNA... 135 Effect of L-Glutamine or Adenosine t{ith- drawal and Replenishment on RNA Accumulation by CHO l{T and CIiO GAT CeÌIs. 139 Effects of L-Glutamine and Adenosine Deprivation and Replenishment on the Grovrth of CHO WT and CHO GÀT Cells.. 140 xiv PAGE Effects of AchLya and CHO vtT HS3 on partially purified CHO Ì{T Cel,1 DNA_ Dependent RNA polymerases.
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