"Science, Faculty of"@en . "Microbiology and Immunology, Department of"@en . "DSpace"@en . "UBCV"@en . "Kropinski, Andrew Maitland Boleslaw"@en . "2011-03-04T17:55:52Z"@en . "1973"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "The nucleic acid of Pseudomonas acidovorans bacteriophage 0W-14 was isolated and characterized as double-stranded DNA. Unlike normal DNA molecules, a great disparity was found between the moles % GC calculated from CsCl buoyant density gradient data and Tm\r\ndetermination. The buoyant density in neutral CsCl was 1.666 g/cm\u00C2\u00B3 (4.5 moles % GC) and the Tm in SSC was 99.3 C (72.9 moles % GC). The buoyant density in Cs\u00E2\u0082\u0082SO\u00E2\u0082\u0084 was also unusually low (1.415 g/cm\u00C2\u00B3).\r\nAcid hydrolysates contained five bases: adenine, guanine, cytosine, thymine and an unknown. The concentration of thymine was approximately half that of adenine. Assuming that the unknown base made up for this deficiency in thymine, the moles % GC in 0W-14 DNA was calculated to be 56.2.\r\nThe unknown base, a pyrimidine, was isolated and purified by column chromatography. It was characterized by chemical analysis, NMR, IR and mass spectroscopy as N-thyminyl-putrescine: [5-(4-aminobutylamino methy1)uracil]. Chemical synthesis confirmed the structure of the compound."@en . "https://circle.library.ubc.ca/rest/handle/2429/32081?expand=metadata"@en . "f b ' 7 ' - : THE PHYSICO-CHEMICAL PROPERTIES OF BACTERIOPHAGE 0W-H DEOXYRIBONUCLEIC ACID by ANDREW MAITLAND BOLESLAW KROPINSKI B.Sc. (Bacter io logy and Biochemistry) Un ivers i ty of B r i t i s h Columbia, 1965 M.Sc. (Microbiology) U.B.C., 1969 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT OF MICROBIOLOGY WE ACCEPT THIS THESIS AS CONFORMING TO THE REQUIRED STANDARD THE UNIVERSITY OF BRITISH COLUMBIA MAY, 1973 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by h i s representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of /ffi/cAog/oto&y The University of B r i t i s h Columbia Vancouver 8, Canada Date / 2 V \u00C2\u00A5 , f/t I ABSTRACT The nuc le ic ac id of Pseudomonas acidovorans bacteriophage 0W-14 was i so la ted and character ized as double-stranded DNA. Unl ike normal DNA molecules, a great d i s p a r i t y was found between the moles % GC ca l cu la ted from CsCl buoyant density gradient data and Tm \u00E2\u0080\u00A23 determinat ion. The buoyant density in neutral CsCl was 1.666 g/cm (4.5 moles % GC) and the Tm in SSC was 99 -3 C (72.9 moles % GC). The buoyant density in Cs2S0^ was a l so unusually low (1.415 g/cm ). Ac id hydrolysates contained f i v e bases: adenine, guanine, cy tos ine, thymine and an unknown. The concentrat ion of thymine was approximately ha l f that of adenine. Assuming that the unknown base made up for th i s de f i c iency in thymine, the moles % GC in 0W-14 DNA was ca l cu la ted to be 56.2. The unknown base, a pyr imid ine, was i so la ted and p u r i f i e d by column chromatography. It was character ized by chemical ana l y s i s , NMR, IR and mass spectroscopy as N-thyminyl -putrescine: [5 -(4-aminobutylamino methy1)uraci1]. Chemical synthesis confirmed the s t ructure of the compound. TABLE OF CONTENTS Page INTRODUCTION 1 MATERIALS AND METHODS 6 Sect ion 1. General propert ies of phage DNA.... 6 Phage and bac te r i a l s t ra in s 6 Preparat ion of phage lysates and i s o l a t i o n of DNA 6 U l t r a v i o l e t specta 6 Ca lcu la t i on of DNA concentrat ions 6 Molar ex t i n c t i on c o e f f i c i e n t 6 Spectrophotometry t i t r a t i o n 7 Spectral propert ies of the DNA at pH 3. 7 Melting temperature experiments 9 Renaturation experiments 9 Buoyant dens i ty experiments 10 Determination of base compositions 11 Thin layer chromatography and solvent systems 12 MATERIALS AND METHODS 11* Sect ion 2. I so lat ion and propert ies of the unusual pyr imidine in 0W-14 DNA 1*t Growth of phage 1^ P u r i f i c a t i o n of 0W-14 1*t Table of Contents (Continued) Page P u r i f i c a t i o n of 0W-14 DNA 15 Enzymatic hydro lys i s of 0W-Ht DNA 15 DNase 1 d i ges t ion of 0W-Ht DNA 15 Snake venom phosphodiesterase d igest ion 16 DEAE-cel lu lose chromatography 16 P u r i f i c a t i o n of compound X 16 DNA hydro lys i s 16 CM Sephadex chromatography 17 Sephadex G-10 chromatography 17 Recry s ta l1 i z a t i on of compound X, 18 Chemical analyses 18 U l t r a v i o l e t spectra 18 Spectrophotometry t i t r a t i o n 18 Din i t ropheny lat ion of compound X 19 Ext inc t i on c o e f f i c i e n t 20 Infrared spectra 20 Nuclear magnetic resonance (NMR) spectra 20 Mass spectra 20 L i s t of chemicals 21 Table of Contents (Continued) iv Page Melting temperature analyses 24 Renaturation experiments 26 Buoyant density experiments 26 Neutral CsCl 26 Neutral C s ^ C y . . . 29 A l k a l i n e CsCl 29 Base composition of 0W-14 DNA 31 Chromatographic propert ies of bases 32 RESULTS 36 Sect ion 2 . I so lat ion and propert ies of the unusual pyr imidine in 0W-14 DNA 36 P u r i f i c a t i o n of compound X 36 Chemical ana lys i s of compound X 41 General propert ies of compound X 41 U l t r a v i o l e t spectra of compound X 43 Spectrophotometric t i t r a t i o n of compound X 46 Infrared spectra of compound X 46 NMR spectra of compound X 50 60 MHz spectra l data 52 Spin-sp in decoupling 53 Structure of compound X 55 V Table of Contents (Continued) Page Mass spectra l data on N-thyminylputrescine 55 RESULTS 60 Sect ion 3. Chemical synthesis of N-thyminylputrescine. 60 Synthesis of N-acetyl putresc ine 60 Spectral propert ies of N-acety lputresc ine 60 Synthesis of 5 _bromomethyluraci1 64 Spectral propert ies of 5-bromomethyluraci1 64 Chemical synthesis of N-thyminylputrescine 67 A l t e r n a t i v e synthet ic route 68 Proof of synthesis 69 Chemical analyses 69 Chromatographic propert ies 69 UV spectra l propert ies 69 IR spectrum 73 NMR spectra l data 73 GENERAL DISCUSSION 77 Incidence of unusual bases in DNA 77 Physico-chemical e f f e c t s of subs t i tu t i on 78 Polyamines in phage p a r t i c l e s 80 Models of 0W-14 DNA 81 Table of Contents (Continued) Page Theoret i ca l d i scuss ion of increased Tm and buoyant dens i ty 81 SUMMARY 91 BIBLIOGRAPHY 93 APPENDIX 1. A computer program to f a c i l i t a t e DNA Tm ca l cu l a t i on s 100 Introduction 100 Computer F a c i l i t i e s 101 Procedure 101 Program 105 Discussion 107 Summary 109 References 109 APPENDIX 2. Problems inherent in the production of fermen-t e r - s c a l e lysates of 0W-14 111 Preparat ion of high t i t r e lysates 111 1. E f f e c t of medium 111 2. E f f ec t of ant i foam agent 111 3. Aerat ion 112 k. M.0.1. and time of phage add i t ion 113 Table of Contents (Continued) v i i Page APPENDIX 3. Deta i led mass spectra l ana ly s i s of N-thyminyl-putresc ine 114 Mass spectra l ana ly s i s of N-thyminylputrescine 114 Fragmentation schemes 115 References 118 v i i i LIST OF TABLES Page Table l a . Nucleic ac ids of Pseudomonas phages 2 Table 1. Base composition of 0W-14 DNA 33 Table II. TLC of nuc le i c ac id bases 34 Table III. TLC of nuc le i c ac id bases 35 Table IV. P u r i f i c a t i o n scheme of compound X 40 Table V. Elemental ana ly s i s of compound X 42 Table VI. Spectral propert ies of compound X 45 Table VII. Chromatographic propert ies of synthet ic N-thyminylputrescine 71 Table VIII. UV spectra l propert ies of natural and syn-t h e t i c N-thyminylputrescine 72 Table IX. Physico-chemical propert ies of various modif ied DNA's 79 Table X. App l i ca t i on of the equation of Kohn and Spears (1967) 88 Table XI. Resume of propert ies of 0W-14 DNA 92 Table XII. Tm Computer Program 102 Table XIII. Computer card layout 104 Table XIV. Tm Computer Program data presentat ion 106 Ix LIST OF FIGURES Page Figure 1. Spectral propert ies of DNA at pH 3- 8 Figure 2 . Spectrophotometric t i t r a t i o n of 0W-14 DNA 23 Figure 3- Influence of SSC concentrat ion on Tm 25 Figure 4 . Renaturation k i ne t i c s of thermally denatured phage DNA 27 Figure 5 . Buoyant dens i ty of 0W-14 DNA in CsCl 28 Figure 6 a . C s^O^ density gradient of 0W-14 DNA 30 6b . A l k a l i n e CsCl i sopycnic gradient of phage DNA.. 30 Figure 7- E lu t i on p r o f i l e of compound X on CM-Sephadex... 38 Figure 8 . Chromatographic p u r i f i c a t i o n of compound X on G-10 Sephadex 39 Figure 9- UV spectra of compound X 44 Figure 10a. Spectrophotometric t i t r a t i o n of compound X 47 10b. Spectrophotometric t i t r a t i o n of compound X 48 Figure 11. IR spectrum of compound X 49 Figure 12. 60 MHzNMR spectrum of compound X 51 Figure 13. Decoupling of the 100 MHzNMR spectrum of compound X 54 Figure 14. S t ructure of compound X 56 Figure 15- Mass spectrum of compound X 57 LIST OF FIGURES (continued) Page Figure 16. Proposed fragmentation pattern of compound X. . . 59 Figure 17- IR spectra of N-acety lputresc ine 61 Figure 18. IR spectrum of putresc ine d ihydroch lor ide 62 Figure 19. 100 Mh^ NMR spectrum of N-acety lputresc ine 63 Figure 2 0 . IR spectrum of 5 -bromomethyluraci1 65 Figure 2 1 . IR spectrum of 5 _ hydroxymethyluraci 1 66 Figure 2 2 . Proposed a l t e r n a t i v e synthesis of N-thyminyl-putresc ine 70 Figure 2 3 . IR spectrum of synthet ic N-thyminylputresc ine.. 74 Figure 24. 100 MHzNMR spectrum of synthet ic N-thyminyl-putresc ine 75 Figure 2 5 . 100 MHzNMR spectrum of putresc ine d ihydro-ch lo r i de 76 Figure 2 6a . Atomic models of DNA 82 26b . \" 83 26c . \" 84 26d . 1 1 85 Figure 27- Computer p lot of Tm data 108 1 INTRODUCTION The nuc le i c acids of many Pseudomonas phages have been character ized with respect to t h e i r physico-chemical propert ies (see Table 1a). Unl ike the DNA's of ce r t a i n col iphages and Bac i l l u s phages, the DNA's of the Pseudomonas phages examined prev ious ly contain only adenine, guanine, cytos ine and thymine. Pre l iminary cha rac te r i za t i on of the DNA of Pseudomonas acidovorans phage 0W-14 (Kropinsk i , M.Sc. t he s i s , 1969) indicated the presence of an add i t iona l base. This thes i s describes in greater de ta i l the propert ies of 0W-14 DNA and i t s unusual base. 2 Table la . Propert ies of the nuc le i c acids of various Pseudomonas phages. Phage Host Bacterium Nucleic Acid Method Reference 2 SD1 PX2 PX3 PX7 CB3 B3 D3 E79 F116 HD2 HD3 HD7 HD11 HD16 HD24 P_. aeruginosa P_. aerug i nosa P_. aerug i nosa P_. aeruginosa f_. aerug i nosa f_. aerug inosa P_. aerug inosa P_. aerug inosa f_. aerug inosa f_. aeruginosa P_. aerug inosa P_. aeruginosa P_. aerug i nosa P_. aerug ? nosa P_. aerug inosa P_. aerug inosa ds DNA ds DNA ds DNA ds DNA ds DNA ds DNA ds DNA ds DNA ds DNA ds DNA ds DNA ds DNA ds DNA ds DNA ds DNA ds DNA 54.7 53.8 68.2 45.0 54.6 60.4 55.0 55.3 53.4 46.1 63.0 54.7 b,c a ,c a a a a b,c b,c b,c b,c b,c b,c 1 2 3 3 3 3 4 4 4 4 Table la - continued Phage Host Bacterium Nucleic Acid %GC Method Reference HD44 P. aerug inosa ds DNA 57.3 b,c 11 HD68 P_. aerug inosa ds DNA 54.8 b,c 11 HD95 f_. aerug inosa ds DNA 61.0 b,c 11 HD113 IP. aerug inosa ds DNA 61.8 b,c 11 352 P. aerug inosa ds DNA 55 .7 b,c 11 1214 f_. aerug i nosa ds DNA 60.1 b ,c 11 PB-1 f_. aerug i nosa ds DNA 12 0-MC P. aerug inosa ds DNA 45.5 a,c 13 02 P. aerug inosa ds DNA 51.0 13 1X1 f_. aerug inosa ds DNA 63.O 14 Pf f_. aeruginosa ss DNA 5 PP7 f_. aerug inosa ss RNA 6 7s P. aerug inosa ss RNA -t. J U .r. 50.4 c 7 PX4 f_. f1uorescens ds DNA 44 .4 a 3 PX10 f_. f1uorescens ds DNA 53.0 a 3 PX12 f_. f1uorescens ds DNA 55.8 a 3 4 T a b l e 1a - c o n t i n u e d Phage Host B a c t e r i u m N u c l e i c A c i d %GC Method R e f e r e n c e Pf P. pu t i d a ds DNA 62 .0 a ,b 8 PX1 P_. put i da ds DNA 52.5 a 3 gh-1 f_. put i d a ds DNA 57 .0 a , b ,c 9 P f 16 P. put i da ds DNA 47 . 5 a , c , d 16 PM2 P.. s t u t z e r i ds DNA 43 .0 a ,b 10 PX14 f_. g e n i c u l a t a ds DNA 53.8 a 3 06 P_. p h a s e d i c o l a ds RNA 17 ds ( d o u b l e - s t r a n d e d ) ; s s ( s i n g l e - s t r a n d e d ) ; RNA ( r i b o n u c l e i c a c i d ) ; DNA ( d e o x y r i b o n u c l e i c a c i d ) a ( d e t e r m i n e d f r o m Tm); b ( d e t e r m i n e d f r om buoyant d e n s i t y ) ; c ( d e t e r -mined by q u a n t i t a t i v e c h r o m a t o g r a p h y a f t e r a c i d h y d r o l y s i s ) ; d ( d e t e r m i n e d by q u a n t i t a t i v e c h r oma tog r aphy a f t e r e n z y m a t i c h y d r o l y s i s ) \" \" A d e n i n e ( 23 .8 mo le s % ) ; Guan ine (24 .6 mo le s % ) ; C y t o s i n e (25.8 mo les % ) ; U r a c i 1 (25 .8 mo le s % ) . 1. Grogan and Johnson (1964) 2. S h a r g o o l and Townsend (1966) 3. O l s e n , e ^ a l _ . (1968) 4. D a v i d s o n , et^ a_l_. (1964) 5. Takeya and Amako (1966) 6. B r a d l e y (1966) 7. F e a r y , et_ a l _ . (1963) 8. N i b l a c k and Gun sa l u s (1965) 9 . Lee and B o e z i (1966) 10. E s p e j o and C a n e l o (1968) 5 Table 1a - continued 11 . O'Callagah, et a|_. (1969) 12. Bradley and Robinson (1968) 13. Chow and Yamamoto (1969) 14. Marmur, et al_. (1964) 15. Semancik, et a l . (1972) 16. Nib l a c k (19^8 \"P 6 MATERIALS AND METHODS Section 1: General propert ies of phage 0W-14 DNA. Phage and bac te r i a l s t r a i n s . Phage 0W-14 was grown on P_. acidovorans s t r a i n 29 (Kropinski and Warren, 1 970 ) . Preparation of phage lysates and i s o l a t i o n of DNA. The condit ions of phage growth, l y s i s ( F r e i f e l d e r , 1966) and DNA i s o l a t i o n (Marmur, 1961) have been described prev ious ly (Kropinsk i , M.Sc. Thes i s , 1969)\u00E2\u0080\u00A2 U l t r a v i o l e t Spectra. A l l UV-spectra were obtained using a Unicam SP800 spectrophotometer (Pye Unicam L t d . , Cambridge, England.). Ca l cu la t ion of DNA concentrat ions . DNA concentrat ions were estimated 2 using an ex t i nc t i on c o e f f i c i e n t of 20 cm /mg based upon the op t i c a l dens i ty at 260 nm (Lee and Boezi , 1966). Molar ex t inc t i on c o e f f i c i e n t f E 2 6 0 n m ^ ^ A l l necessary glassware was rendered phosphate-free by b o i l i n g in g l a s s - d i s t i l l e d water. To each of three hydro lys i s v i a l s was added 0.10 ml of h ighly p u r i f i e d 0W-14 DNA so l u t i on , prev ious ly d ia lyzed against 0.0015 M NaCl, and 0.15 ml of 12 M HC1. The v i a l s were sealed and placed in a 100-110 C oven for 3 hr. They were then removed, c h i l l e d on i c e , opened and 0 .025 ml a l i quot s were used fo r the determination of inorganic phosphate (Chen, Tor ibara and Warner, 1956 ) . 7 The molar ex t inc t i on c o e f f i c i e n t was ca l cu la ted as fo l lows : E 2 6 0 n m ^ = ^ 2 6 0 n m U n ' t s ^ m ' x 10^ n moles Pi/mmole Pi n moles Pi/ml Spectrophotometric t i t r a t i o n . 0W-14 DNA d i s so lved in 0 . 5 M NaCI was very c a r e f u l l y t i t r a t e d with f re sh l y prepared 0.1 M NaOH - 0 . 5 M NaCI. At various pH values the ^gQrm w a s r e c o r c * e d and adjusted for the volume changes due to the add i t ion of the t i t r a n t . Spectral propert ies of the DNA at pH 3 . 0W-14 DNA, d ia lyzed extens ive ly against 0 .0015 M NaCI, was d i l u ted 1/20 into 0 .05 M a c e t i c a c i d . The resu l t ing pH was 3\u00C2\u00AB0 - 3 . 1 - U l t r a -v i o l e t absorpt ion spectra were recorded at room temperature and the absorbancy r a t i o ^260nm^^280nm w a S c a ^ c u ' a t e c ' ' The m \u00C2\u00B0 l e s 0/\u00C2\u00B0 AT were ca l cu la ted from a rep lot of Freder icq et_ aj_., (1961) r e s u l t s , using the fo l lowing equation: E 2 6 0 n m / E 2 8 0 n m = EE. AT (22,400) + mp_ GC (17,300) mp_ AT (9 ,300) + mp_ GC (20,500) where mp_ is the molar propert ion of e i ther adenine + thymine (AT) or guanine + cytos ine (GC). The resu l t s were p lot ted on log- log paper (F ig . 1 ) . \u00E2\u0080\u00A2I I I S I 1 I I L A rep lo t of the data of Freder icq et a l . (1961) on the spectra l propert ies of DNA at pH 3- T O theore t i ca l p l o t ; (2) p lot of experimental data. 9 Melting Temperature Experiments. The Tm experiments were c a r r i e d out e s s e n t i a l l y as described by Mandel and Marmur ( 1 9 6 8 ) . A G i l f o r d automatic recording spectrophotometer model 2400 equipped with model 2417 thermosensor (G i l f o rd Instrument Laborator ies Inc., Ober l i n , Ohio) was the bas ic unit used for running melting p r o f i l e s of the DNA preparat ions. A thermostat i ca l l y con t ro l l ed waterbath and pump (Haake constant temperature c i r c u l a t o r Model F) were used to heat and c i r c u l a t e the ethylene g lyco l to the inner thermo-spacers f l ank ing the cuvette chamber. The rate of heating was automat ica l ly con t ro l l ed by a va r i ab le speed motor and c o n t r o l l e r (Gerald K. He l le r Co., Las Vegas, Nevada). In order to study the inf luence of standard s a l i ne c i t r a t e (SSC) concentrat ion on the Tm the DNA was d ia lyzed against 0 .0015 M NaCI pr io r to d i l u t i o n into the appropr iate strength of SSC. Escher ich ia c o l i DNA (Worthington Biochemical Corp., Freehold, N.J.) was used as the reference DNA. The resu l t s of the Tm experiments were analyzed using a computer program (see Appendix 1). Renaturation Experiments. The DNA was sheared and prepared for the experiments as described by Se id le r and Mandel (1971). Sealed cuvettes were placed in the G i l f o r d spectrophotometer, and 10 allowed to e q u i l i b r a t e to Tm - 25 C. The recorder sca le was c a r e f u l l y zeroed. The cuvettes were then removed and the DNA thermally denatured in a bath of ethylene g lyco l heated to Tm + 5 C. A f t e r 5 minutes, the cuvettes were wiped dry and replaced in the cuvette chamber. Readings were i n i t i a t e d immediately. The renaturat ion curve was p lot ted sccording to the method of Wetmur ( 1 9 6 7 ) . Buoyant Density Experiments. The buoyant dens i t i e s of 0W-14 DNA and f_. acidovorans #14 DNA in neutral CsCl dens i ty gradients were determined according to the method of Mandel et^ aj_. ( 1 9 6 8 ) ; gradients were analyzed with an An-D rotor using a Beckman Model E a n a l y t i c a l u l t r a cen t r i f u ge (Beckman Instruments Inc., Palo A l t o , C a l i f . ) . The rotor was spun at 44 ,000 rpm for 22 hr at 20 C. IE. col i DNA (Worthington) was used as a \u00E2\u0080\u00A2i density marker at 1.710 g/cc (Schi ldkraut et^ aj_., 1 962 ) . Kodak Commercial F i lm (Eastman Kodak Co., Rochester, N.Y.) was used fo r the UV photography, and the negatives were developed with D-11 developer. The f i lms were scanned with a Joyce-Loebl double-beam recording microdensitometer. The buoyant density of the phage DNA was determined by the fo l lowing equation (Mandel e_t aj_., 1 968 ) : p = 1.710 - 0 .0089 ( r 2 - r Q 2 ) g/cm 3 where r and r are the peak distances from the centre of ro ta t ion of 11 the phage DNA and the E_. col i marker, r e spec t i ve l y . In the case of a l k a l i n e CsCl g rad ients , the mater ia l s were made up in 0.1 M t r i b a s i c sodium phosphate (Espejo, e_t al_., 1 9 69 ) . The gradients were analyzed as above and the buoyant dens i t i e s were c a l -culated from the fo l lowing equat ion: p = 1.772 - 0 .0089 ( r 2 - r Q 2 ) g/cm 3 The method of Szybalski (1968) was used to f i nd the buoyant density of the phage DNA in a 05250^ dens i ty grad ient . Determination of Base Compositions. Samples ( 1 .0 mg) of phage DNA were put into th i ck wal led Pyrex hydro lys i s tubes and 0.71 ml of 90% formic ac id was added to each. A f te r f reez ing the contents in d ry - i ce -acetone, the v i a l s were sealed under reduced pressure. Hydrolys i s was ca r r i ed out at approximately 175 C fo r 45 min, a f t e r which the v i a l s were refrozen and opened. The hydrolysates were placed in a warm water bath (approximately 40 C) and evaporated to dryness under a stream of H^. The residues were d i s so lved in 0 .06 ml of 0.1 N HC1, of which 0 .05 ml was appl ied to a sheet of Whatman #40 chromatography paper. A 0 .005 ml sample was d i l u ted with 0.1 N HC1 and the tota l op t i c a l dens i ty uni ts (at 260 nm) were determined spectrophotometr ica l ly . The bases were separated by descending chromatography using isopropanol-HCI-H\u00E2\u0080\u009E0 ( 6 5 : 1 7 : 1 8 , v/v) at 30 C. A f te r a time, s u f f i c i e n t to g ive good separat ion of the bases (18 - 2k h r ) , the papers were removed and d r i e d . The UV-absorbing areas on the chromatogram were cut out along with contro l areas, and were placed in c lean test - tubes with 5 ml of 0. 1 M HC1. A f te r thorough mixing to e lu te the bases, the c e l l u l o s e f i b r e s were allowed to s e t t l e out and the UV-spectral propert ies of the various so lut ions were determined. The bases were quant i tated using t h e i r molar ex t inc t i on c o e f f i c i e n t s . Thin Layer Chromatography and Solvent Systems. Stock so lut ions of the bases were prepared at a concentrat ion of 5 mg/ml using 0.1 M HC1 as the solvent in a l l cases except f o r guanine (1 MHC1), N-thyminylputrescine (d ist i1 led water), 5 _ carboxyuraci1 and 5-carboxymethyluraci1 (d i l u te NH^OH). Samples (0.002 ml) of each stock so lut ion were app l ied to sheets of 6064 c e l l u l o s e (Eastman Kodak Co., Rochester, N.Y.). The bases were separated by ascending chromatography at 30 C using f r e sh l y prepared solvent systems. When the solvent f ront had r i sen to with in one inch of the top, the sheet was removed and a i r d r i ed . The bases were located using a UV l i gh t source (Chromato-Vue, U l t r a - V i o l e t Products, Inc., San Gab r i e l , C a l i f o r n i a ) . The solvent systems employed in the TLC separation of the bases were: 1. n (w/v)NH.0H in water 2. Methanol-HC1-Water (70:20:10, v/v) 3. Butanol-Methanol-Water-NH^OH (60:20:20:10, v/v) (Randerath, 1965) 4. 2-Propanol-HCl-Water (65:17=18, v/v) (Bendich, 1957) 5. t -Butanol-Formic acid-Water (80:5:20, v/v) (Roberts, 1961) 6. 2-Propanol-NH^OH-Water (70:10:20, v/v) (Schwarz #24) 7. Butano l -G lac ia l Ace t i c Acid-Water (50 :25:25, v/v) (Schwarz #6) 8. t-Butanol-Methyl Ethyl Ketone-HCl-Water (40:30:10:20, v/v) (CIine, Fink and Fink, 1958) 9. 1 M Ammonium Acetate-Absolute Ethanol (35:70, v/v) 10. t-Butanol-Methyl Ethyl Ketone-Formic Acid-Water (40:30:15:15, v/v) (CI ine, Fink and F ink, 1958) For the paper chromatographic separat ion of bases, Whatman #40 chromatography paper was used with solvent systems 4_ (Bendich, 1957) and Schwarz #24. MATERIALS AND METHODS Sect ion 2: I so lat ion and propert ies of the unusual pyr imidine from 0W-14 DNA. Growth of phage. CAA-M medium, rather than MB (Kropinski and Warren, 1970) was used for the preparat ion of phage 0W-14 in fermenters. CAA-M conta ined, in g/1: casamino a c i d s , 12 .5; yeast ex t rac t , 2.0; mannitol , 5 .0 ; tryptophan, 0.05. Dow Corning C was used to suppress foaming. The c e l l s were grown at 30 C with high aerat ion (2 volumes air/volume medium/min). Phage was added when the cu l tu re reached an 0D 650 nm of 1.5 (see Appendix 2 fo r d e t a i l s on fermenter c u l t u r e of 0W-14). P u r i f i c a t i o n of 0W-14. The phage lysates were passed through a Sharpies high speed c e n t r i -fuge at a rate of 1 L/min to remove whole c e l l s and c e l l u l a r debr i s . The opalescent supernatant was c h i l l e d to 4 C, s o l i d NaCl was added to 0.5 M and the phage p rec ip i t a ted with polyethylene g lyco l (Yamamoto et a l . , 1970). The polyethylene g lyco l (PEG) 6000 was added to the s t i r r e d lysates to a f i n a l concentrat ion of 1% (w/v) and the lysates were l e f t at k C for several days u n t i l the f i ne p r e c i p i t a t e had se t t l ed out. This proved fa r more convenient than centrifugi ing large volumes. The c lea r supernatant f l u i d was siphoned o f f and d iscarded, and the p r e c i p i t a t e was purther p u r i f i e d by d i f f e r e n t i a l cen t r i f u ga t i on . The f i n a l phage p e l l e t was d i s so lved in phage buf fer (see Appendix 1). P u r i f i c a t i o n of 0W-14 DNA. For the i s o l a t i on of compound X from the DNA of very large numbers of p a r t i c l e s (approximately 1 0 ^ pfu) a pre l iminary d igest ion with nucleases or fur ther p u r i f i c a t i o n of phage was assumed to be unnecessary. Sodium dodecyl sulphate was added to the phage suspension to a f i n a l concentrat ion of 2% (w/v). A f te r l y s i s had v i s i b l y occurred, the so lu t ion was warmed to kS C and maintained for 15 min to ensure complete l y s i s . Phenol or chloroform-isoamyl alcohol depro te in i za t i on proved inapp l i cab le due to the large volume involved. The DNA was p rec ip i t a ted with ethanol and the product c o l l e c t e d on a g lass rod. The DNA was p a r t i a l l y p u r i f i e d by pronase d igest ion (50 yg/ml, 3 hr, 37 C) and a fu r ther ethanol p r e c i p i t a t i o n . This materia l was d i s so lved in 0.1 x SSC and used without fur ther p u r i f i c a t i o n . This was j u s t i -f i e d on the basis that compound X was prev ious ly found in DNA which had been highly p u r i f i e d . Enzymatic Hydrolys is of 0W-14 DNA. 1. DNAse 1 d i ges t ion of 0W-14 DNA. A stock so lu t ion of h ighly p u r i f i e d DNA in SSC was d ia lyzed over-n ight against ten volumes of 0.033 M magnesium acetate, pH 6.8. Then 100 ml of the s o l u t i o n , contain ing approximately 50 mg of DNA, was placed in a f l a s k at 37 C. Pancreat ic DNAse, d isso lved in a 16 minimal volume of the magnesium acetate s o l u t i on , was added to a f i n a l concentrat ion of 20 yg/ml. Digest ion was ca r r i ed out f o r 1.5 h. 2. Snake venom phosphodiesterase d i ge s t i on . The so lu t i on of DNA a f t e r d igest ion with DNAse 1 was adjusted to pH 8.5 with 2 M ammonium carbonate, snake venom phosphodiesterase was added to a f i n a l concentrat ion of approximately k ]ig/m], and d iges t ion was continued fo r a fur ther 2 hr at 37 C. The d igest was passed through a small Dowex-50 (NH^ +-form) column 2+ to remove Mg . The column was washed thoroughly with d i s t i l l e d water to recover a l l 0D 2 \u00C2\u00A3q ma te r i a l . 3 . DEAE-cel1ulose chromatography. 2+ The Mg - f r e e digest was l yoph i l i zed to dryness and the residue taken up in s u f f i c i e n t d i s t i l l e d water to render the conduct-i v i t y approximately 1 m i l l i MHO. This so lu t ion was appl ied to a DEAE-cel lu lose column, pH 8.0 (HCO^ -form; 1.2 x kk cm). The mononucleotides were e luted with a l inear gradient of NH^HCO^ (zero - 0.1 M). O l i go -nucleot ides were recovered by e l u t i on with 1.0 M NH^HCO^. P u r i f i c a t i o n of compound X. 1. DNA hydro l y s i s . Phage 0W-14 DNA was p rec ip i t a ted from 0.1 x SSC with two volumes of 35% ethanol . The f ibrous material was dehydrated with acetone and a i r d r i e d . One gram quant i t ie s of the dr ied material were introduced into hydro lys i s v i a l s with 16 ml of i ce - co ld 6 N HC1. Nitrogen was gent ly bubbled through the so lut ions for 10 min. The v i a l s were evacuated, sealed and placedin an oven at 100 - 110 C fo r 90 min. The v i a l s were then cooled and opened. The deep brown hydrolysate was f i l t e r e d through Whatman #1 paper to remove p rec ip i t a ted material which in ter fered with subsequent p u r i f i c a t i o n steps. The f i l t e r s were washed with several volumes of 1 H HC1 and the combined f i l t r a t e s were evaporated to dryness. Twice water was added to the residue and the so lu t ion taken to dryness. The residue was f i n a l l y d i s so lved in a small volume of water. This so lu t ion was adjusted to pH 6.8 with 0.01 M NH^OH. 2. Carboxymethyl (CM) Sephadex Chromatography. The neut ra l i zed hydrolysates were app l ied to a column of CM-50 Sephadex (2.5 x 100 cm; NH^ + form). The colour was removed by washing the column with several volumes of 0.005 M NH^OH. The compound X - r i c h f r a c t i o n was e luted with 0.1 M HC1. It was evaporated to dryness in a rotary evaporator. 3. Sephadex G-10 Chromatography. The dry material from the previous step was red isso lved in the minimum volume of 0.01 M HC1 and app l ied to a Sephadex G-10 column (2.5 x 100 cm) pre-equi1 ibrated with th i s a c i d . The column was developed with 0.01 M HC1 at 0.5 ml/min and f r ac t i on s of approx i -mately 3 ml were c o l l e c t e d . The f r ac t i ons contain ing compound X were pooled and evaporated to dryness. 4. R e c r y s t a l l i z a t i o n of compound X. The f i n a l product was s l i g h t l y yel low. The colour was re -moved by t r i t u r a t i o n with i ce - co ld absolute methanol: compound X was r e l a t i v e l y in so lub le , and a f t e r three extract ions the co lour le s s c r y s t a l s were c o l l e c t e d , dr ied and weighed. Approximately 160 mg of compound X were recovered from about 4 g of crude phage DNA. It was stored over anhydrous ^ 2^5 \" ^ small amount was rec ry s ta l1 i zed from methanol-1 M HC1 for chemical ana l y s i s . Chemical Analyses. A l l chemical analyses were c a r r i e d out by the A l f r e d Bernhardt Mikroanalyt isches Laboratorium (West Germany). U l t r a v i o l e t Spectra. A l l spectra were obtained using a Unicam SP800 spectrophotometer (Pye Unicam L t d . , Cambridge, England.). 1. Spectrophotometric T i t r a t i o n . P u r i f i e d compound X was d i l u ted with 0.1 M HC1 to g ive an of approximately 1.4. A 20 ml sample of th i s so lu t ion was c a r e f u l l y t i t r a t e d with NaOH; samples were removed at frequent in terva l s and UV spectra recorded. The resu l t s were p lo t ted as (a) X max vs pH; (b) E 2 8 Q / E 2 6 0 vs pH; (c) E ^ / E ^ vs pH. D in i t ropheny lat ion of compound X. An attempt was made to estimate the ex t inc t i on c o e f f i c i e n t of compound X by d in i t ropheny la t ion using the method of Ghuysen et a 1. ( 1 9 6 6 ) . An aqueous so lu t ion of compound X (0.1 ml, 0.4 mg/ml) was added to 0.02 ml of e thano l i c d in i t ro f luorobenzene (0.13 ml plus 10 ml absolute ethanol) in 0.1 ml of 2% aqueous I^B^O^. A f te r incubation for 30 min at 60 C, 0.8 ml of 2 M HC1 was added. The d in i t ropheny lated der i va t i ves were extracted into ether (3 times with 1.5 ml) and the E420nm \u00C2\u00B0^ t ' i e c o m ' 3 ' n e c ' extracts was determined. The molar ex t i nc t i on c o e f f i c i e n t of the DNP de r i va t i ve was assumed to be the same as that fo r DNP amino a c i d s , i e . E M ( i , 2 0nm) = ^ 2 0 \u00C2\u00B0 ( G n u v s e n > \u00C2\u00A3 l i l - 1966). The ex t i nc t i on c o e f f i c i e n t (E^) was determined using the fo l lowing equation EM(260nm) c o m P o u n c l ^ (p h 1) = ^260nm c o m P o u n d x ' n m ^ sample ymoles -NH 2 group in 0.1 ml sample Chromatography of the DNP de r i va t i ve was c a r r i e d out on sheets of s i l i c a gel (Gelman Instrument Co., Ann Arbor, Michigan) of the I.T.L.C. type, using chloroform-methanol-g lac ia l a ce t i c ac id ( 8 5 : 1 4 : 1 , v/v) (Ghuysen et_ a K , 1 966 ) . 2. Ex t inc t ion C o e f f i c i e n t . A number of samples of rec ry s ta l1 i zed N-thyminylputrescine d ihydroch lor ide (approximate weight 0.6 mg) were weighed out. The samples were d i s so lved in 1.0 ml of d i s t i l l e d water and 0.05 ml a 1 i -quots were d i l u ted to 1.0 ml with f r e sh l y prepared 0.1 M HC1 for UV spectroscopy. Infrared Spectra. Infrared spectra were obtained using a Beckman IR-10 spectrophoto-meter (Beckman Instruments Corp., F u l l e r t o n , C a l i f o r n i a ) . The KBr d i sc technique was used in every case. Nuclear Magnetic Resonance Spectra. Nuclear magnetic resonance spectra were obtained of D^ O so lut ions of the various chemicals using an HA-60 or HA-100 instrument. The l a t t e r instrument was equipped with an Audio o s c i l l a t o r fo r decoupling work (Model 4204A Yokogawa-Hewlett-Packard, Tokyo, Japan). The 60 and 100 megacycle NMR instruments were products of Varian Assoc iates (Palo A l t o , C a l i f o r n i a ) . A l l chemical s h i f t s were recorded r e l a t i v e to an external tetramethy l s i lane (TMS) s i g n a l . Mass Spectra. A l l mass spectra were obtained using a Nucl ide 12-90-G mass spectrometer (Nuclide Corp., State Co l lege, Pennsylvania). The ion iza t ion voltage used in every case was 70 ev, 100 yA trap current . Samples were introduced into the source by means of a d i r ec t in ser t ion probe and the c r u c i b l e temperature was ra ised to 200 C to g ive adequate to ta l ion cur rent . L i s t of Chemicals. 5-Hydroxymethyluraci1 was purchased from Mann Research Laborator ies 5-hydroxymethylcytosine from Calbiochem; 5 _ carboxyuraci1 from A l d r i c h Chemical Co.; 5-aminouraci1 from Sigma Chemical Co. 5 - Carboxymethyl-u r a c i l was generously donated by Dr. B.G. Lane. E_. col i DNA, pan-c r e a t i c DNase arid snake venom phosphodiesterase were purchased from Worthington Biochemical Corp. Putresc ine f r e e base was purchased from J . T . Baker Chemical Co. RESULTS (SECTION 1) General Propert ies of 0W-14 DNA. The u l t r a v i o l e t spectrum in 0.1 x SSC showed a maximum adsorpt ion (Amax) at 258 nm with a minimum (Xmin) at 230 nm. Upon the add i t ion of NaOH to 0 .25 M, the X max sh i f ted to 263 nm with a 30% increase in the chromic i ty at 260 nm. The spectra l ra t io s ^ 2 3 0 ^ / ^ 2 6 0 \u00E2\u0084\u00A2 ! ! a n c ' E 2 8 0 / ' E 2 6 0 n m \u00C2\u00B0^ t h e n a t ' v e D N A were 0 .425 and 0 . 5 3 , r e spec t i ve l y . In no preparat ion of the phage DNA did the spectra l r a t i o s approach the values usua l ly given by DNA: 0 .450 (230/260) and 0 .515 (280/260) (Marmur, 1963 ) -The molar ex t inc t i on c o e f f i c i e n t ( E 2 6 0 n m ^ P ^ \u00C2\u00B0 ^ \" w _ 1 i * D N A w a s ca lcu la ted to be 6800 , a value well wi th in the range of other double-stranded DNAs, eg. Bac i l l u s sub t i l is phage SP50, 6650 (Biswal et a l . , 1967 ) , and well below that obtained with s ing le-s t randed DNAs, eg. E. c o l i phage 0X-174 87OO (Sinsheimer, 1959 ) . The spectrophotometric t i t r a t i o n of 0W-14 DNA is shown in Figure 2 . A sharp r i s e in the chromicity at 260 nm, i nd i ca t i ve of a he l i x to c o i l t r a n s i t i o n , was observed at pH 10.7 to 11.0 with a t r a n s i t i o n midpoint (pH M) at 1 0 . 85 . This value is cons iderably lower than the pH normally associated with s t rand-separat ion, i e . 11.7 (Dore and F r a n t a l i , 1 968 ) . 1-40-Figure 2. Spectrophotometric t i t r a t i o n of 0W-14 DNA. The DNA was d i s so lved in 0.5 M NaCI and the pH adjusted with 0.1 M NaOH- 0.5 M NaCI. Freder icq et^ aj_. (1961) estimated the moles % AT in var ious DNA preparations from the 260/280 r a t i o at pH 3 . 0 . The average 260/280 r a t i o from a number of experiments with 0W-14 DNA was 1.210, which corresponds to 40 .5 moles X AT o r , conversely, 59 -5 moles % GC. This value agreed well with the value of 56 .2 moles % GC derived from hydro lys i s experiments. Melting Temperature Analyses. The melting curve of phage 0W-14 DNA (see Appendix 1) showed no dev ia t ion from the normal steep sigmoidal shape c h a r a c t e r i s t i c of phage DNAs. Ana lys i s of the curve showed approximately kh% thermal induced hyperchromicity and a melt ing temperature (Tm) of 99 -75 C in SSC. Under s im i l a r cond i t i ons , E_. c o l i DNA exhib i ted a Tm of 92 .3 C, which agreed well with the l i t e r a t u r e value of 91 .85 C (Mendel et_ al_., 1 970 ) . The Tm of 0W-14 DNA was adjusted down 0 .45 C. This corresponds to 66 .3 moles % GC using the equation of Mandel et_ al_., ( 1 970 ) . 1. Moles % GC = 100 ( [Tm - 16-3 l og 1 ( ) Rel .SSC Conc . ]/50.2) - 0 .990 A more de ta i l ed examination of the melting propert ies of the phage DNA (Figure 3) showed that the Tm was less inf luenced by the sa l t concentrat ion than those of DNAs prev ious ly analyzed. In a recent de ta i l ed reassessment of the melting propert ies of various nuc le ic ac ids (Mandel e_t a_l_., 1 970 ) , i t was shown that for a t en - f o l d change in the SSC concentrat ion, there was a corresponding 16.3 C change in the Tm. Phage 0W-14 DNA showed a 13\u00E2\u0080\u00A20 C change; there fo re , equation I. VJ1 26 equation 1 was modif ied to g ive : 2. moles % GC = 100 ((Tm - 13-0 log 1 ( ) Rel.SSC Conc.)/50.2) - 0.990 Using th i s equat ion, a value of 72.9 moles % GC was ca l cu la ted for 0W-14 DNA. Chemical ana ly s i s showed that the DNA was a c t u a l l y 56.2 moles X GC, so that i t should have a Tm of 94.1 C. In a d d i t i o n , the DNA contained f i v e bases. Therefore, the presence of the unusual base had the e f f e c t of increasing the Tm 5-2 C. Renaturation Experiments. Pre l iminary studies on the renaturat ion of sheared, thermally denatured DNA indicated normal second order k i n e t i c s . A second order rate constant of 27 l i t res /mole-second was found using SSC as the so lvent , and 8 l i t res/mole-second with 0.5 x SSC (F ig . 4) , values which compared favourably with those obtained fo r the renaturat ion of T phage DNA (Wetmur, 1967). even r 3 \u00C2\u00BB Buoyant Density Experiments. A microdensitometer scan of a CsCl density gradient is shown in F i g . 5- \u00C2\u00A3 . col i DNA (1.7100 g/cm 3) (Schi 1 dkraut et a K , 1962) was used as the dens i ty standard in the determination of the buoyant density of the phage DNA. On the basis of four separate determinations, the buoyant density 3 3 of 0W-14 DNA was found to be 1.6659 g/crrT \u00C2\u00B1 0.0015 g/cm . This value was unaffected by the method of i s o l a t i on of the DNA or by Figure k. Renaturation kinetics (see Wetmur, 19&7) of 0W-14 DNA in SSC (Line A) and 0 . 5 x SSC (Line B) after fragmentation and thermal denaturation. 28 u> o z < CO K O co CO < 1 -667 M e n i s c u s 1-725 B U O Y A N T D E N S I T Y Figure 5. Microdensitometer scan of a neutral CsCl density gradient showing 0W-14 DNA (p 1.667 g/cm^), P_. acidovorans #14 DNA (p 1.725 g/cm3), and the E. c o l i DNA marker (p 1.710 g/cm3) the treatment of the DNA with t r y p s i n , pepsin or co ld ac id fol lowed by neu t r a l i z a t i on (Kropinsk i , M.Sc. thes i s , 1 969 ) . Employing the equation presented by de Ley ( 1 970 ) ; 3. % GC = 1020.6 ( p - 1.6606) a value of 4 .5 moles % GC was ca l cu la ted fo r th i s DNA. The except iona l ly low dens i ty compares with those obtained for rat l i v e r nuc leoprote in ; 1.66 g/cm 3 (Lehnert and Okada, 1965) and poly dAT:dAT; 1.672 - 1.679 g/cm 3 ( Szyba lsk i , 1 968 ) . As the DNA a c t u a l l y contained 56 .2 moles % GC, which should g ive 3 a buoyant dens i ty of 1.7157 g/cm , the presence of compound X caused 3 a decrease in the buoyant density of 49 .8 mg/cm . Denaturation of the DNA with sodium hydroxide fol lowed by n e u t r a l -i za t ion (Biswal et al_., 1967) or by heat fol lowed by rapid c h i l l i n g 3 resu l ted in an increase in the buoyant density of 14.2 mg/cm to g ive a value of 1.6801 g/cm 3 (Krop insk i , M.Sc. t he s i s , 1969)\u00E2\u0080\u00A2 A dens i ty increase of th i s magnitude agrees well with the value of 15 mg/ml in the denaturation obtained for E_. col i DNA (Lee and Boezi , 1 966 ) . A microdensitometer scan of a Cs2S0^ density gradient is pre-3 sented in F i g . 6 a . Using a value of 1.426 g/cm for the buoyant density of the \u00C2\u00A3 . c o l i DNA marker (Szyba lsk i , 1968) under these cond i t i ons , the dens i ty of the phage DNA was ca l cu la ted to be 1.415 g/cm . This value resembled c l o se l y with those obtained for poly dA:dT, 1.416 g/cm 3 ( Szyba l sk i , 1968) and poly d(A-T-C) :d(G-A-T) , 30 1-416 Figure 6a. Microdensitometer t rac ing of a f ^ S O ^ isopycnic gradient of 0W-14 DNA ( p 1.416 g/cm3) and E. c o l i DNA marker (p 1.426 g/cm 3 ) . Figure 6b. Microdensitometer t rac ing of an a l k a l i n e CsCl density grad ient . E. c o l i DNA (p 1.722 g/cm 3) and 0W-14 DNA (p 1.750 g/cm 3 ) . 1.418 g/cm 5 (Wells and Larson, 1970). In F i g . 6b the scan of an a l k a l i n e CsCl density gradient is 3 shown. Using a Value of 1.772 g/cm for the buoyant density of \u00C2\u00A3 . c o l i DNA (Vinograd e_t aj_., 1963) under these cond i t ions , a value of 3 1.750 g/cm was ca l cu la ted fo r 0W-14 DNA. This represents an increase 3 in dens i ty over that of the nat ive DNA, of 84 mg/cm , a value g reat ly 3 in excess of the 62 -63 mg/cm increase found for other DNAs (Vinograd et al_., 1 963 ) . Base Composition of 0W-14 DNA. Hydrolys is of the phage DNA with formic ac id fol lowed by paper chromatographic separat ion of the bases showed the presence of f i v e UV-absorbing components. Four of the components were i d e n t i f i e d by descending paper chromatography, using solvent systems (as adenine, guanine, cytos ine and thymine), 4 and 11 , and the i r spectra l proper-t i e s in 0.1 M HC1 and 0.1 M NaOH. The f i f t h base, compound X, mig-rated more slowly than guanine in solvent system #4. Determination of the to ta l ex t inc t i on of thymine and compound X in var ious hydroly-sates showed that the r a t i o of X/T var ied from 0.4 for formic ac id hydrolysates to 0 .8 f o r hydrochlor ic ac id hydrolysates ( 6 M_, 100 C, 4 h r ) . No compound X was recovered when the DNA was hydrolyzed with perch lo r i c ac id (70%, 100 C, 60 min). Quant i tat ion of the bases showed that A + G 1. The concentrat ion of thymine in s ix ind iv idua l experiments with formic ac id was approximately one-hal f that of adenine. Assuming that the f i f t h base was a pyrimidine which made up for the de f i c iency in thymine, the actual moles % GC in the DNA were ca l cu la ted from a mod i f i ca t ion of Py = 1. i e . A + G = 1. Pu T + C + X Using th i s modif ied equation, a value of 56 .2 moles % GC was ca l cu la ted for 0W-14 DNA (Table 1). That thymine resu l ted from the breakdown of compound X was un l i ke l y because a f t e r hydro lys i s of the DNA with e i ther formic ac id or HC1, the (T)/(A) r a t i o was always 0 .5 whi le the (X)/(T) ra t i o s d i f f e r e d by a f ac to r of two. The converse, that compound X is in some way produced from thymine during hydro ly s i s , seems a remote p o s s i b i l i t y . Chromatographic Propert ies of Bases. A small amount of compound X was p u r i f i e d by repeated descending paper chromatography on Whatman #k0 paper using solvent systems k and 11. The chromatographic propert ies of th i s material and several other nuc le i c ac id bases were checked by th in layer chromatography on c e l l u -lose using a v a r i e t y of solvent systems (see Tables II and III). Compound X showed very l i t t l e Rfs in the a c i d i c solvent systems and higher Rfs in the bas ic systems. This behaviour was comparable to that of 5-aminouraci1 and, to a lesser extent, cy tos ine. 33 Base Adenine Thymine Compound X Cytosine Guanine Moles % 22.1 \u00C2\u00B1 0.4 10.3 \u00C2\u00B1 0 .2 11.9 \u00C2\u00B1 0.5 28 .4 \u00C2\u00B1 0.8 27 .8 \u00C2\u00B1 0 .2 % GC = 56 .2 [T] / [A] = 46 .3 0 Based upon: [Pu] / [Py] = 1 \u00C2\u00B0\u00C2\u00B0\u00C2\u00B0 [K] = [A + G] - [T + C] Table I. The base composition of phage 0W-14 DNA as determined by formic ac id hydro ly s i s . T a b l e II. TLC o f N u c l e i c A c i d Bases Base Rf V a l u e s S o l v e n t S y s tem: 1 2 3 4 5 A d e n i n e 0, .53 0, .35 0, .52 0 .33 0 .41 G u a n i n e 0. .61 s 0. .17 .0, ,27s 0 .21 0 .26s C y t o s i n e 0, .77 0. .50 0. .48 0 .47 0 .40 5 - H y d r o x y m e t h y l c y t o s i n e 0, .82 0, .49 0, .38 0 .46 0 .35 Thymine 0, .87 0. .73 0, .64 0 .80 0 .74 U r a c i l 0, .90 0, .66 0. \u00E2\u0080\u00A2 50 0 .69 0 .63 5 _ H y d r o x y m e t h y l u r a c i 1 0, .93 0. .64 0. .44 0 .62 0 .53 5'-Am i n ou r ac i 1 0, .90 0, .29 0, .37 0 .22 0 .29 5 - C a r b o x y u r a c i 1 0 .96 0, .56 0, .14 0 .63 0 .59 5 - C a r b o x y m e t h y l u r a c i 1 0 .97 0, .73 0, .21 0 \u00E2\u0080\u00A2 70 0 .59 Compound X 0 .90 0, .17 0, .23 0 .15 0 .09 (P) - i s o l a t e d f r om phage DNA s - s t r e a k e d S o l v e n t S y s tem: 1. 1% (v/v) aqueous NH^OH; 30 min deve l opmen t t i m e (D.T.) 2. m e t h a n o l ~ H C 1 - w a t e r ( 7 0 : 2 0 : 1 0 , v / v ) ; 75 min D.T. 3. b u t a n o l - m e t h a n o l - w a t e r - c o n c . NH^OH ( 6 0 : 2 0 : 2 0 : 1 , v / v ) ; 120 min D.T. 4. 2 - p r o p a n o l - H C l - w a t e r ( 6 5 : 1 7 : 1 8 , v / v ) ; 270 min D.T. 5. t - b u t a n o l - f o r m i c a c i d - w a t e r ( 8 0 : 5 : 2 0 , v / v ) ; 270 min D.T. 35 T a b l e III. TLC o f N u c l e i c A c i d Bases Base Rf V a l u e s S o l v e n t S y s tem: 6 7 8 c I 10 Aden i n e 0.63 0.51 0 . 32 0 . 68 0.40 Guan i ne 0 . 3 8 s \" 0.40 0. 22 0. 51 0 .27s C y t o s i ne 0 .60 0.45 0. 43 0. 67 0 .39 5 - H y d r o x y m e t h y l c y t o s i ne 0.55 0.43 0 . 43 0. 63 0 .35 Thymine 0.77 0.76 0 . 86 0 . 79 0.71 U r a c i l 0.63 0.66 0. 73 0. 76 0.57 5 - H y d r o x y m e t h y l u r a c i1 0.61 0.59 0 . 68 0. 70 0.45 5 _Am i nou rac i1 0 .49 0.42 0. 23 0. 61 0.24 5 - C a r b o x y u r a c f l 0 .24 0.56 0 . 75 0. 29 0 .50 5 - C a r b o x y m e t h y 1 u r a c i 1 0 .40 0 .60 0. 76 0. 42 0.53 Compound X 0.53 0.34 0. 10 0. 62 0 .16s (P)_ - i s o l a t e d f r om phage DNA s - s t r e a k e d S o l v e n t Sy s tem: 6. 2 - p r o p a n o l - c o n c . NH^OH-water ( 7 0 : 1 0 : 2 0 , v / v ] ; 165 mm D.T. 7.. b u t a n o l - g l a c i a l a c e t i c a c i d - w a t e r ( 5 0 : 2 5 : 2 5 , v / v ) ; 135 min D.T. 8. t ^ b u t a n o l - m e t h y l e t h y l k e t o n e - H C 1 - w a t e r ( 4 0 : 3 0 : 1 0 : 2 0 , v / v ) ; 180 min D.T. 9 . 1M NH^ a c e t a t e - a b s o l u t e e t h a n o l ( 3 5 : 7 0 , v / v ) ; 135 min D.T. 10. t - b u t a n o l - m e t h y l e t h y l k e t o n e - f o r m i c a c i d - w a t e r ( 4 0 : 3 0 : 1 5 : 1 5 , v / v ) ; 105 min D.T. 36 RESULTS (SECTION 2) P u r i f i c a t i o n of Compound X. I n i t i a l attempts to pur i fy the unusual base from 0W-14 DNA i n -volved the enzymatic d iges t ion of the phage DNA with pancreat ic deoxyribonuclease (DNase 1) and snake venom phosphodiesterase fol lowed by the chromatographic separation of the mononucleotides on columns of DEAE-cel1ulose (HCO^ form, pH 8 . 0 ) , using a l i near gradient of ammonium bicarbonate. A f r a c t i o n accounting fo r approximately 30% of the 0&260 u n ' t s app l ied t o the column was e luted from the column at a molar i ty of >0.15. This tended to ru le out the p o s s i b i l i t y of a d inuc leo t ide f r a c t i o n (Lehman, 1960 ) . Redigestion of th i s o l i g o -nuc leot ide f r a c t i o n with snake venom phosphodiesterase f a i l e d to re lease fur ther mononucleotides. It was assumed that the o l i gonuc leot ides were terminated ( 5 1 terminus) by compound X un i t s , and that the X bases conferred p a r t i a l res i s tance to nuclease d iges t ion in a manner s im i l a r to g lucosy la t ion of T e v e n phage DNA (Sinsheimer, 195^ ; Vo l k i n , 195 2*; J e s a i t i s , 1 957 ) . Further work using other nucleases, such as Lehman's phosphodiesterase (Lehman, 1 960 ) , or l i m i t d igest ion using the two prev ious ly mentioned nucleases might re su l t in the re lease of the X nuc leot ide. The next method for pur i f y ing compound X was that of HC1 hydro lys i s of the phage DNA fol lowed by the paper chromatographic p u r i f i c a t i o n of the base using a number of d i f f e r e n t solvent systems. This method proved of l i t t l e value in the large sca le p u r i f i c a t i o n because (a) HC1 hydro lys i s resu l ted in cons iderable degradation of the deoxyribose r i n g , and the dark brown degradation products migrated up the paper with the solvent and obscured the slower moving UV-absorbing products; and (b) guanine, which is r e l a t i v e l y inso luble and has an Rf value c lose to that of compound X, streaked badly with the isopropanol-HC1 solvent system and contaminated compound X. Repeated runs were necessary to overcome these problems. When the chromatograms were checked fo r amino ac id contamination using ninhydrin spray, i t was found that compound X gave a po s i t i ve reac t i on . This led to the development of the p u r i f i c a t i o n procedure described in the Methods sec t i on . Figures 7 and 8 show, re spec t i ve l y , the p u r i f i c a t i o n of compound X on CM- and G-10 Sephadex, and Table IV gives the resu l t s of a t yp i ca l p u r i f i c a t i o n . Most of the UV-absorbing materia l passed through the CM-Sephadex column and the crude compound X was recovered by e lu t i on with d i l u t e HC1. This material was resolved into two peaks by passage through a Sephadex G-10 column, the f i r s t of which was compound X. The materia l from the second peak had Rfs s im i l a r to adenine in solvent systems k and 11 and developed yel low colour with ninhydrin-cadmium aceta te . It was poss ib le that th i s unknown materia l 0 10 20 3 0 40 5 0 6 0 70 8 0 9 0 1 0 0 F R A C T I O N N U M B E R Figure 7. E lu t i on p r o f i l e of p a r t i a l l y p u r i f i e d kropinos ine on CM-Sephadex. Absorbance at 260 nm ( so l id l i n e ) ; pH (dashed-l i n e ) . Peak 2 contains kropinos ine whi le an u n i d e n t i f i e d base is present in peak 1. 1 5 1 1 0 10 20 3 0 4 0 5 0 6 0 7 0 80 9 0 1 0 0 F R A C T I O N N U M B E R Figure 8. E l u t i on p r o f i l e of CM-Sephadex peak 2 mater ia l on a column of G-10 Sephadex. The vaoid volume (V Q) of the column is i nd i ca ted . Peak 1 contains krop inos ine; the u n i d e n t i f i e d base is present in peak 2. T a b l e . IV. T y p i c a l p u r i f i c a t i o n scheme o f compound X f rom a c i d h y d r o l y s a t e s o f 0W-14 DNA. P u r i f i c a t i o n S tep Volume T o t a l E260 Xmax. J-Am i n. 250/260 \" 280/260 \" 290/260 \" N e u t r a l i z e d h y d r o l y s a t e 2000 ml 28 ,000 267 234 0.81 0.74 0 .34 CM-Sephadex e l u a t e 2034 21 ,560 267 234 0 .82 0.76 0 .35 0.005M NH^OH wash 870. 5,310 267 234 0 .79 0.77 0.33 0.1M HC1 e l u a t e 58 1,880 262 230 0.75 0.43 0 .15 G-10 peak 1. 261 229 0 .75 0 .29 0.013 G-10 peak 2. 282.5 241 0 .65 2.16 1.94 2 9 1 \" \" 2 5 4 \" \" 0.91 \" 3 . 0 6 \" J- J, 3 . 6 7 \" \" t h e s p e c t r a l p r o p e r t i e s were o b t a i n e d u s i n g 0.1M HC1 as t h e s o l v e n t , w i t h t h e e x c e p t i o n o f t h o s e marked * * i n w h i c h c a s e 0.1M NaOH was u s e d . was a degradation product of adenine, which is qu i te l a b i l e to HC1 hydrolys i s. Compound X from the G-10 column appeared to be pure. It was homogeneous in th in layer chromatography with several solvent systems and the ninhydrin-cadmium acetate spray reagent reacted only with the UV-absorbing area of each chromatogram to g ive a pink co lour . Of other nuc le ic ac id bases l i s t e d in Table II, only 5 - aminouraci1 was found to react with th i s reagent to g ive an orange co lour . Chemical Ana lys i s of Compound X. A sample of r e c r y s t a l1 i z ed compound was subjected to elemental ana lys i s (Table V) . A molecular formula of CgH^0 2N^*2HCI was c a l c u -lated g iv ing a molecular weight of 285. It should be noted that the molecular weight of thymine is only 126. The chemical ana lys i s a l so indicated that the materia l possessed a high degree of pu r i t y . General Propert ies of Compound X. P u r i f i e d compound X was a white c r y s t a l l i n e substance which was extremely water so lub le . This was in marked contrast to the s o l u b i l i t y of other nuc le i c ac id bases; in p a r t i c u l a r , guanine, which are in general poorly water so lub le . Upon heating i t decomposed at 255 C. D in i t ropheny lat ion was used in an attempt to determine the molar ex t inc t i on c o e f f i c i e n t of compound X and from th i s value to estimate the molecular weight. Unl ike DNP-derivatives of amino ac id s , the DNP-de r i va t i ve of compound X was not water so lub le , and so lu t ion could E L E M E N T P e r c e n t a g e by W e i g h t F o u n d C a l c u l a t e d M O L A R R A T I O C a r b o n H y d r o g e n O x y g e n -N i t r o g e n o C h l o r i n e 38 08 6-43 11-41 19-51 24-57 3 7 - 8 2 6 - 3 2 11 -23 1 9 - 6 5 24 -91 9 18 2 4 2 M o l e c u l a r f o r m u l a C 9 H 1 6 \u00C2\u00B0 2 N 4 2 H \u00C2\u00B0 ' M o l e c u l a r w e i g h t 2 8 5 C h l o r i n e b y d i f f e r e n c e T a b l e V. E l e m e n t a l a n a l y s i s o f r e c r y s t a l 1 i z e d compound X i s o l a t e d f rom 0W-14 DNA. The c a l c u l a t e d p e r c e n t a g e s were d e r i v e d f rom t h e m o l e c u l a r f o r m u l a . 43 only be obtained by ext ract ion into ether . Using th i s method an EM(420nm) \u00C2\u00B0^ ->>900 w a s c a l cu l a ted . Because th i s value is s i g n i f i -cant ly lower than that of other pyr imid ines, the extract was checked by TLC on s i l i c a gel p l a tes . This revealed the presence of seven yel low bands of d i f f e r e n t i n t e n s i t i e s . The most in teres t ing bands displayed Rfs of 0.11 and O.76 ( p r i nc i p l e band). By decreasing the r a t i o of DNFB: compound X in the react ion mixture, the main band was found at 0.10 with a minor band at 0 . 72 . It was concluded, that the low Rf product represented a monosubstituted d e r i v a t i v e (N-DNP-compound X) whi le the high Rf der i va t i ves was the d i subs t i tu ted product (N,N-diDNP compound X). U l t r a v i o l e t Spectra of Compound X. The UV spectra of compound X in 0.1 M HC1 and 0.1 M NaOH are pre -sented in Figure 9 - The absorpt ion maxima, minima and spectra l r a t i o s are recorded in Table VI. The spectra indicated that the pyr imidine r ing of compound X was of the 2 ,4 -d icarbony l (u rac i l ) type rather than the 4-amino-2-carbonyl type (cytos ine) , s ince the X increased with pH. m 3 x In the case of cytos ine and re lated compounds, the Xmax is at a lower wavelength at pH 7 than at pH 1 or 14 (Dunn and H a l l , 1970). The spectra l propert ies of compound X were almost i dent i ca l to those of 5-hydroxymethyluraci1 (Dunn and H a l l , 1970) at pH 7 , and only s l i g h t l y d i f f e r e n t at pH 13. k5 X S p e c t r a l P rope r t y pH 1 pH7 p H l 3 Max nm 261 262 288 -5 Min nm 229 2 3 0 - 5 246 o I s o b e s t i c p o i n t s 274 238 5 R a t i o s E X l W a x / E X M i n 6 0 0 4-50 3 \u00E2\u0080\u00A228 E 26 0 P H 1 3 / E 260 p H 7 / E 26 0 P H 1 0-40 : 0 9 5 : 1 E \ l W a x p H l 3 / E X l V l a x pH7 / E X M a x p H 1 0-79 : I 0-96 : 1 2 3 0 / 2 6 0 0-17 0-27 1 \u00E2\u0080\u00A289 2 4 0 / 2 6 0 0-39 0-39 0 \u00E2\u0080\u00A274 2 5 0 / 2 6 0 0-76 0-74 0 \u00E2\u0080\u00A264 2 7 0 / 2 6 0 0-80 0-84 1 39 2 8 0 / 2 6 0 0-29 0-36 1 \u00E2\u0080\u00A269 2 9 0 / 2 6 0 0-02 0-07 1 \u00E2\u0080\u00A293 x pH 1 = 0-1N HCI pH 7 = 0 - 0 5 M K H P O - N a O H 2 4 p H 1 3 = 0-1N N a O H o I s o b e s t i c p o i n t s - pH 1 and 13 s p e c t r a Table VI. UV spectra l propert ies of compound X. at pH 1 , 7 and.13. 46 The ex t i nc t i on c o e f f i c i e n t at the X of a \% (w/v) so lu t ion max 1 12 (Figure 1 0 a ) . These resu l t s would ind icate a pK considerably lower than that observed with the other pyr imid ines. Thymine has a pK at 9 - 9 , u r a c i l at pH 9 . 5 , and 5-hydroxymethyluraci1 shows a pK at 9 . 4 . Infrared Spectra of Compound X. The in f rared spectrum (Figure 11) of the d ihydroch lor ide sa l t of 290-4 285-28 OH < S 275 A 270H 265H 260 / 1 3 \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 \u00E2\u0080\u0094 e \u00E2\u0080\u0094 \u00E2\u0080\u00A2 \u00E2\u0080\u0094 o* ,K 9-0 \u00E2\u0080\u0094I\u00E2\u0080\u0094 10 - i H\u00E2\u0080\u0094r 11 13 P H Figure 10a. SpectrophotometrIc t i t r a t i o n of compound X d ihydroch lor ide Isolated from 0W-14 DNA. Figure 10b. Spectrophotometric t i t r a t i o n of compound X, dI hydrochlor ide Isolated from 0W-1V DNA. p H 50 compound X showed carbonyl absorption bands at 1740 cm and 1685 cm corresponding to the C=0 s t retch ing frequencies of the carbonyl groups at pos i t ions 4 and 2 of the pyrimidine r ing (Parker, 1971). The subst i tuent group did not have a hydroxy l - , methy l - , pheny l - , carboxyl or carboxylate moiety, as indicated by the lack of d i s t i n c t absorption bands at 1385-1375 cm 1 (C-H symmetrical banding of a -CH^ group); 1200-1000 (C-0H s t r e t c h ) ; 710-690 cm 1 (C-H out -o f -p l ane deformation of an unsubst ituted phenyl group); 3500 cm 1 (C-0H s t re tch of a -C00H moiety); 1610-1550 cm\" 1 and 1400-1300 cm\" 1 (-C00~ r a d i c a l ) . This confirmed the f ind ings of the chemical ana l y s i s . The medium to sharp band at 3260 cm 1 was assigned to an NH^ + s t r e t c h , and the bands in the region of 1650-1500 cm 1 to NH^ + and N H 2 + deformations. The sharp to medium band at 1480 cm 1 was assigned to the C-H sc i s so r ing of a -CH 2 ~ group. The exact locat ion of the primary and secondary amino adsorption bands could be determined more exact ly using the f ree base of deuterium exchange. Nuclear Magnetic Resonance (NMR) Spectra of Compound X. The NMR spectra l data together with the chemical ana lys i s prev ious ly discussed o f fe red the best c lues to the s t ructure of compound X. A 60MHZ spectrum of a D20 so lu t ion of compound X is presented in Figure 12. If one ignored the large peak at approximately 4.8 ppm, which was due to the solvent (DH0) s i g n a l , there were four p r i n c i p l e s igna ls Subsequent to w r i t i n g , we have noticed an inconsistency between the 60 and 100 MHZ chemical s h i f t . This we a t t r i b u t e to a technica l er ror during the recording of the 60MHZ spectrum, s ince a 100 MHZ spectrum of the natural product showed the same 6 values as the synthet ic product. 4-78 3-99 \u00E2\u0080\u0094 J 1 1 1 1 1 I I I 8-0 7-0 6-0 5-0 4-0 3-0 2-0 1-0 0-0 P P M Figure 12. 60 MHZNMR spectrum of a D20 so lut ion of compound X d ihydroch lor ide . The so l i d l i n e represents the integrator scan. U1 in the spectrum at S = 7.77 ppm, 3-99 ppm, 3-09 ppm and 1.77 ppm. The r e l a t i v e i n t e n s i t i e s of these peaks as ca l cu la ted from the integrator scan indicated the presence of one, two, four and four protons, re spec t i ve l y . The s ing le t at 6 = 7.77 ppm (1H) was ass igned, by comparison with the NMR spectra of other pyrimidines (Bhacca et_ aj_., 1963), to the proton on pos i t i on s ix (Hg) of the pyrimidine r i ng . If the base was subst i tuted at pos i t i on 6^ with a proton at pos i t ion 5_ (as in o r o t i c acid) or a methyl group at pos i t i on 5_ (as in a 6-subst i tuted thymine) there would have been a s ignal at approximately 5 ppm in the former case and no s ignal at a l l in the l a t t e r . The lack of s p l i t t i n g of th i s peak, due to sp in - sp in in te rac t ions , a l so indicated that there were no adjacent hydrogens. Th i s , coupled with the UV spectra l evidence, confirmed that the base was subst i tuted at pos i t i on 5. rather than pos i t ion 6_. The s ing le t at 6=3-99 ppm (2H) was t en ta t i ve l y ass igned, by com-parison with the NMR spectrum of 5 _carboxymethyluraci1 (Gray and Lane, 1969), to a methylene group at of the pyrimidine r i ng , which was i nd i ca t i ve of the base being subst i tuted thymine. The lack of s p l i t -t ing of th i s group showed that the adjacent group contained e i ther no hydrogens or only those exchangeable (-0H, -Nh^, etc) with the so lvent, D 20. The absorption of these protons at qu i te low f i e l d indicated that an adjacent group(s) had some deshie ld ing p o t e n t i a l . The integrator showed eight protons in the s ide cha in. The s igna ls at 1.77 ppm and 3.09 ppm were at too high f i e l d to be due to hydrogens bonded to C=C, s ince the l a t t e r absorb at 6=4-5 ppm; the i r chemical s h i f t s resembled those of methylene groups. There was no -CH^ group in the molecule; i t s protons would absorb at approximately 1 ppm, with the s ignal appearing as a s i n g l e t , doublet (1 :1 ) or t r i p l e t ( 1 : 2 : 1 ) , depending upon the number of protons on adjacent groups. A port ion of 100 MHz NMR spectrum is reproduced in Figure 13\u00C2\u00AB Though the spectrum resembled that at 60 MNz, the reso lut ions of the peaks, p a r t i c u l a r l y the mu l t ip le t at 3.09 ppm, were increased. The s ignal at 6=3.09 ppm was probably due to overlapping t r i p l e t s , r e s u l t -ing from the sp in - sp in coupl ing of the 3~protons to the a-protons in a 3 a s t ructure such as X- CH 2 ~ CH 2 - (X = two d i f f e r e n t nonprotonated groups). Sp in-sp in decoupling was used so that the chemical s h i f t s of the ind iv idua l groups could be observed f ree from sp in - sp in i n t e r -ac t i ons . Saturat ion of the protons at 6 -3 .09 ppm resu l ted in the co l l apse of the mu l t i p l e t at 1.77 ppm into a s i n g l e t , ind icat ing that the four hydrogens were arranged: -CH 2 - CH 2 - . Saturat ion of these protons by i r r a d i a t i o n at 3222 Hz resu l ted in the co l l apse of the other mu l t i p l e t into a pair of s i ng le t at 6=3.54 and 6=3.62, with a peak separat ion of 8 cps. It was concluded that the s ide chain was: R -X - CH -CV-(!H - ft-L-X' (R = thyminyl group). The chemical ana lys i s 55 and the NMR data showed that groups X and X' were primary and secondary amines, r e spec t i ve l y . Thus the s ide chain was re lated to putresc ine (1,4-diaminobutane). The mu l t ip le t at 3.09 ppm was assigned to the a , a 1 protons. The Structure of Compound X. The empir ica l formula of the material i so lated from the DNA of 0W-14 was ^g^|8^2^^^12\" ^ e u l travIol et spectrum was typ i ca l of a u r a c i l d e r i v a t i v e , so that a l lowing C 2 j H 2\u00C2\u00B02 N 2 ^ o r t ' i e P v r ' m ' c ' ' n e r m 9 > the subst i tuent groups could be represented as C^H^N^I,,. The compound was strongly bas i c , contained an amino group(s), and was probably a d ihydroch lor ide s a l t . This l e f t C 5 H 1 ^ N 2 f o r t h e s u b s t ' t u e n t group(s). The NMR spectrum showed a proton at Cg and was cons is tent with the group -CH 2NH(CH' 2)/ tNH2 at C,.. Therefore, compound X appeared to be an adduct of thymine and putresc ine (1,4-diaminobutane)(Figure 14). The t r i v i a l name N-thyminylputrescine was assigned (for precedent see C l i n e , Fink and Fink, 1959)\u00E2\u0080\u00A2 Mass Spectral Data on N-thyminylputrescine. The fragmentation pattern of N-thyminylputrescine in the mass spectrometer at 70 ev is shown in Figure 15. A parent ion (M+) was not found, which, though unfortunate, is a r e l a t i v e l y common occurrence with amines s ince the molecular ion (M+) is unstable. Several unsuccess-fu l attempts were made to obtain an M + peak. These included lowering the temperature of the ion chamber. 56 1 f . V x C H - N - C H - C H - CH - CH - NH n HN C 2 2 2 2 2 2 I II 0 * C \ / C v H N H 5 - ( 4 - a m i n o b u t y l l a m t n o m e t h y f y u r a c i l 5- ( 4 - a m i n o b u t y l ) a m i n o m e t h y l ) - ( 2 , 4 - p y r i m i d i n e d i o l ) N - t h y m i n y l p u t r e s c i n e Compound X Figure 14. Proposed structure and nomenclature of compound X. 57 Figure 15. Mass spectrum of compound X ( f ree base) at 70 ev. A 1 I 5 N 3 1 N I 3 A I 1 V 1 3 U Analys i s of the spectrum confirmed the presence of a primary amine. A very intense peak at m/e 30 resu l t s from the 3~cleavage of primary a Iky I amines (Pasto and Johnson, 1969)- The peaks at m/e 54 and 82 may be ind i ca t i ve of an existence of a thyminyl moiety in the molecule, s ince these are a l so observed in mass spectra of thymine and 5-hydroxymethyluraci1 (Rice, Dudek and Barber, 1965). The proposed fragmentation pattern f o r th i s compound is presented in Figure 16. It i s , in genera l , based upon hindsight s ince the s t ructure had been proposed already on the basis of NMR data and the chemical a n a l y s i s , but i t serves as another conf irmat ion of s t ructure and is included as such. The s i g n i f i c a n c e of the m/e 112 peak is not yet understood; i t s s i z e indicates that i t is of some importance in the fragmentation process (see Appendix 3 fo r more de ta i l ed mass spectra l in terpretat ion) The mass spectrum of an analogue of N-thyminylputrescine, 5 -methyl aminomethyluracil (Carbon, David and S tud ier , 1968), shows prominent peaks at m/e 140 (M- CH^) and at m/e 70. Thus, i t is poss ib le that the m/e 70 peak does not re su l t from the break-up of the putresc ine moiety but from fragmentation of the pyrimidine r i ng . Figure 16. Proposed fragmentation pattern of compound X ( f ree base) as derived from mass spectra l (70 ev) evidence. H N . C H 2 - N - C H 2 - C H 2 - C H 2 - C H 2 - N H 2 M + ( 2 1 2 m / e ) H N H N C O 43 H N= + - C O 28 - 8 7 - 1 2 5 - 168 - 1 8 2 0 II H - H m / 4 l 2 5 , + H N - - ( C H 2 ) - N H 2 m / e \" [ \u00C2\u00BB 7 ) M \" H 2 C = +\u00E2\u0080\u00A2 : C - N H H 2 * C H 2 (5 6-3) - N H 3 17 m / e 82 +\u00E2\u0080\u00A2 H H N - C H - C H 2 - C = C H 3 ^ 7 0 H 2 C = N H 2 7 ^ 3 0 H C = C - C = N H 2 H m / e 5 4 O T H E R I O N S O H C H - N H 2 H \" % 1 4 0 H N C H - N = C H , 2 H m / \u00C2\u00A3 1 5 4 RESULTS (SECTION 3) Chemical Synthesis of N-thyminylputrescine. Synthesis of N-acety lputresc ine Monohydrochloride. N-acety lputresc ine monohydrochloride was synthesized by the method of Tabor, Tabor and Bachrach (1964). It was converted to the f ree base by adsorpt ion on a column of Dowex-50 (H +-form) and e l u t i on with 2M NH^OH. The e luate was l yoph i l i zed to dryness. Spectral Propert ies of N-Acety lputresc ine. The IR spectrum of N-acetylputrescine-HCl (Figure 17) showed the presence of a sharp band at 1640 cm 1 due to the C=0 s t re tch (Amide 1 band). The Amide II band (NH stretch) was observed as a sharp peak at 1550 cm 1 , whi le NH deformations (Amide V band) appeared as a broad but weak band at 690 cm 1 . The band of medium in tens i t y at 1380 cm 1 in the spectra of both N-acetylputrescine-HCl and putresc ine 2HC1 (Figure 18) was assumed to be due to symmetrical deformations of the -NH^ + group. The bands in the 3000 - 3400 cm 1 region of the spectrum are due to the amide and NH^ + s t re tch ing modes. The NMR spectrum of N-acetylputrescine-HCl is shown in Figure 19-The sharp s ing le t (3H) at 2.46 ppm was assigned to the CH^- protons on the acety l group. The broad t r i p l e t (4H) at 6= 2.12 ppm was assigned Figure 17. 1. Infrared spectrum of N-acetylputrescine-HCl (KBr d i s c ) . 2. Repeat scan at a higher concentrat ion. 0 0 II I \u00E2\u0080\u00A2 H C - C - N - C H - C H - C H - C H - N D CI 3 2 2 2 2 3 \u00E2\u0080\u00A2 b c o d N - A e \u00C2\u00BB t y l p u t r \u00C2\u00AB \u00C2\u00BB e l n \u00C2\u00AB - H C I 5-13 3-50 2-46 2-12 I 7 0 I 6-0 r~ 5-0 4-0 I 3-0 I 2-0 1-0 P P M Figure 19. 100 MHZ NMR spectrum of a D20 so lut ion of N-acety lputresc ine-HCl . to the protons on the internal ethylene moiety ( -Ch^-C^ - ) of the putresc ine cha in . The c l o se l y associated t r i p l e t s at 3-5 ppm and 3.66 ppm contained a to ta l of four protons and were assigned to the -Ch^-ND^Cl and -C^-ND-CO protons, r e spec t i ve l y . The sp in - sp in s p l i t t i n g of these peaks was equivalent to 6 -7 cps, which was probably i nd i ca t i ve of 1 ,3 -coupl ing. These resu l t s confirmed the synthesis of the monoacetyl de r i v a t i ve of putresc ine. Synthesis of 5-Bromomethyluraci1. 5-Bromomethyluraci1 (5-BrmUra) was synthesized from 5\"hydroxy-methy lurac i l (5-HmUra) by a modi f i cat ion of the method of Carbon (1960) To 1 g of 5-HmUra (7 mmoles) was added 25 ml of 32% HBr in g l a c i a l a c e t i c a c i d . The mixture was ref luxed gently using a condenser and drying tube for 6 hr in a fume hood. A f ter cool ing the react ion vessel to room temperature, s i x volumes of anhydrous ethyl ether were added. The p r e c i p i t a t e was allowed to separate out in the c o l d ; the supernatant was d i scarded, and the p r e c i p i t a t e washed a fur ther four times with 100 ml of ether . The f i n e l y granular product ( 1 .4 g; 37% y i e l d ) was dr ied in vacuum over KOH p e l l e t s . Spectral Propert ies of 5~Bromomethyluraci1. The examination of the IR spectra of 5-BrmUra (Figure 20) and 5-HmUra(Figure 21) confirmed that the subs t i tu t i on had occurred. The spectrum of 5~BrmUra showed none of the bands associated with Figure 20. Infrared spectrum of 5-bromomethyluraci1 (KBr d i s c ) . z to S o a S <0 3 > < % o J->-LA E ZJ \u00E2\u0080\u00A2U u Q. tn \u00E2\u0080\u00A2o 3DNV J.1 I WSNVU1 lN30H3d 3 67 -OH groups, eg. the sharp bands at 3500 cm and 3370 cm charact -e r i s t i c of the -OH s t retch ing modes of f ree and intermoiecular ly bonded groups, and the band at 1060 cm 1 due to the C-0 s t r e t c h . Though the spectrum lacked a band at approximately 650 cm \ which is c h a r a c t e r i s t i c of the C-Br s t re tch (Bellamy, 1 958 ) , the f a r - 1 8 region of the spectrum (not i l l u s t r a t e d ) showed a sharp band at 500 cm 1 which was not found in the spectrum of 5\"Hmllra. Bromination of 5-Hmllra resu l ted in the accentuation of the carbonyl region of the spectrum with the emergence of two bands at 1670 and 1740 c m \" 1 . Chemical Synthesis of N-thyminylputrescine. I n i t i a l attempts to synthesize N-thyminylputrescine involved a nuc l eoph i l i c subs t i tu t i on react ion between 5\"BrmUra and N-acety l -putresc ine. The l a t t e r compound was chosen s ince a preparat ive method ex i s t s in the chemical l i t e r a t u r e (Tabor, Tabor and Bachrach, 1964) and equimolar amounts of the reactants could be used without too much d i s u b s t i t u t i o n occur r ing . The react ion did not work as well as ex-pected, s ince the s o l u b i l i t y of N-acety lputresc ine is greatest in polar solvents such as water and methanol, which w i l l a l so react with the 5-BrmUra. Second, the removal of the N-acetyl protect ing group requires vigorous hydro lys i s condit ions which would a l so tend to destroy the base. Therefore, th i s appraoch was abandoned a f t e r several unsuccessful 68 attempts. The IR and NMR spectra of N-acetylputrescine-HCl are recorded in Figures 17 and 19, re spect i ve ly and are included in th i s thes is because a search of the chemical l i t e r a t u r e f a i l e d to locate ei ther. 5-Bromomethyluraci1 (1 .4 g, 7 mmoles) was added d i r e c t l y , with s t i r r i n g , to 3 ml (30 mmoles) of putresc ine. In ce r t a in cases, i t was d i s so lved in a minimal volume of dr ied dimethylformamide p r i o r to a d d i t i o n . The mixture was kept at room temperature fo r 30 min then a c i d i f i e d with 5 ml (60 mmoles) of concentrated HC1. The p r e c i p i t a t e was d i s so lved by the add i t ion of a small volume of d i s t i l l e d water, and any inso lub le ma te r i a l , assumed to be 5\"HmUra or d i subs t i tu ted putresc ine, was removed by c e n t r i f u g a t i o n . The supernatant was appl ied to a Sephadex G-10 column ( 2 .5 x 80 cm) and eluted with 0.01 M HC1. Fract ions contain ing the monothyminylated product were combined, con-centrated and app l ied to a Bio-Gel P-2 column. Though the react ion appeared to g ive a good y i e l d of product, as jusged by the low level of 5-HmUra present, the product from the column was heav i ly contaminated with putresc ine and many runs were required to d i l u t e th i s material out. Because of t h i s , the f i n a l y i e l d was very low. The pooled products of three runs y ie lded approximately 0 .5 g which represents an approximate 10% o ve ra l l y i e l d . Further attempts at separation included s e l e c t i v e ext rac t ion at high pH with butanol (Raina, 1963) and chromatography on columns of Dowex-50 (H +-form) and Amberl i te IRC-50 K + - form (Tabor, Rosenthal and Tabor, 1 958 ) . Although th i s method provided an unequivocal synthesis of N-thyminylputresince, i t is not a p r a c t i c a l method fo r synthesis of the base unless a more convenient procedure for product separat ion is devised. Figure 22 i l l u s t r a t e s a proposed a l te rna te approach to the synthesis of th i s base which has the added benef i t that the s t a r t i ng ma te r i a l , 5 _ cyanouraci1 , can be produced in high y i e l d and inexpensively by the method of Shaw (1955)\u00E2\u0080\u00A2 Proof of Synthesis of N-thyminylputrescine. 1. Chemical Ana ly s i s . The chemical ana lys i s of the synthet ic product showed (per-cent) C, 3 7 - 7 0 ; H, 6.42; 0 , 11 . 39 ; N, 19.42; CI, 24.77- The values ca l cu la ted for a molecular formula C g H ^ O ^ ^ ^ H C l a re : C, 3 7 - 8 2 ; H, 6 . 3 2 ; 0 , 1 1 . 2 3 ; N, 1 9 . 6 5 ; CI, 24 .91- Therefore, the synthet ic product was ident i ca l in composition to the natural product. 2 . Chromatographic P roper t ie s . The synthet ic and natural products could not be d i s t ingu i shed by th in layer chromatography (Table VI I). 3- UV Spectral P roper t ies . The u l t r a v i o l e t spectra of the synthet ic and natural products were e s s e n t i a l l y i dent i ca l (Table VI I l ) . It is of in teres t that the spectra l r a t i o s of compound X more c l o s e l y resemble those of 5-HmUra Figure 22. Proposed a l t e r n a t i v e from 5 -cyanouraci1. synthet ic route fo r compound X o H ^ H y d r a z i n e Compound X 71 Table VII. Thin layer chromatographic propert ies of natural and synthet ic N-thyminylputrescine. N-thyminylputrescine Rf Values-Solvent System; 2 6. 7_ 8_ 9_ Natural product 0 .22 0.61 0.3 1 * 0 .12 0 .67 Synthet ic product 0.22 0.60 0.36 0.13 0.67 Solvent Systems: 2. methanol-HC1-water (70 :20:10, v/v) 6. 2-propanol-conc. NH^OH-water (70 :10:20, v/v) 7. butanol - a c e t i c ac id - water (50:25:25, v/v) 8. t -butanol-methyl ethyl ketone-HCl-water (40:30:10:30, v/v) 9. 1M NH, acetate-ethanol (35 :70, v/v) Table V I M . UV spectra l propert ies of Compound X derived from 0W-14 DNA and chemical ly synthes ized. Spectral Property pH 1 Natura1 Synthet ic pH 7 Natural Synthet ic pH 13 Natural Syntheti c X Max nm X Min nm Isosbest ic points Spectral r a t i o s : 230/260 240/260 250/260 270/260 280/260 290/260 261 229 0 .17 0.39 0.76 0 .80 0.29 0 .02 261 262 230 230.5 274.5 (274) 0.18 0 .39 0.75 O.83 O.32 0 .02 NaOH 0.27 0.39 0.74 0.84 O.36 0.07 262 288.5 288 .5 230.5 246 245.5 238.5 (238.5) 0.24 1.89 1.94 0.37 0.74 0.76 0.73 0.64 0 .63 0.86 1.39 1-42 O.38 1.69 1.78 0.06 1.93 1-97 pH 1 = 0.1M HC1 pH 7 = 0.05M Kh^PO^-pH 13 = 0.01M NaOH Isosbest ic points were ca l cu la ted from the pH 1 and pH 13 spectra, Values in brackets are f o r natural product. 73 than those of a s t ruc tura l analogue, 5-methylaminomethyluraci1 (Dunn and Ha 11 , 1970 ) . k. IR Spectrum The IR spectra of the synthet ic and natural products were ident i ca l (Figure 2 3 ) . An in teres t ing point concerning the spectrum is the lack of a sharp absorption band in the region 720 -750 cm 1 corresponding to the ske leta l v ib ra t i ons of a - ( C ^ J ^ - c h a i n . The only bands found in th i s region are a l so found in model pyr imidines. 5. NMR Spectral Data on Synthetic N-thyminylputrescine. A 100 MHz NMR spectrum of deuterium exchanged synthet ic N-thyminlyputrescine is shown in Figure 2k. Although i t is i dent i ca l to the spectrum of the natural product, i t is included because i t is a better spectrum and accurate measurement of the r e l a t i v e chemical s h i f t s were made from i t . A l l measurements were made r e l a t i v e to an external TMS s i g n a l . The H^ proton was observed at 825 Hz (6 = 8 .25 ppm) downfield from TMS. The protons at C r of the pyr imidine r ing absorbed 5 at kk7 Hz (<5 = k.k7). The internal ethylene protons of the putresc ine moiety exhib i ted a s ignal at 225 Hz (6 = 2 . 2 5 ) , and the external methy-lene protons absorbed 256 Hz downfield from TMS. It is of in terest that the NMR spectrum of putrescine*2HCl is more than s u p e r f i c i a l l y s im i l a r to that of N-thyminylputrescine (Figure 2 5 ) . The protons have e s s e n t i a l l y the same chemical s h i f t s as are found in N- thyminy lputresc ine\u00C2\u00AB2HC1 (235.9 Hz and 266 .5 Hz) and the peak separa-t ion distances are i dent i ca l (131 Hz). / o z < t -1 0 0 - 1 9 0 - | 8 0 1 6 0 H \u00C2\u00AB SO 4 0 H 30H u a. 2 0 - j 1 0 0 S Y N T H E T I C N ~ 1 M Y M I N V L P U T R E S C I N E \u00E2\u0080\u0094I 1 2 0 0 I\u00E2\u0080\u0094 8 0 0 n 1 r soo 1 0 0 ( - 9 0 \u00E2\u0080\u00A2 8 0 - 7 0 - 6 0 - S O - 4 0 - 3 0 - 2 0 - 1 0 - 0 4 0 0 0 1 3 0 0 0 1 2 0 0 0 1 6 0 0 W A V E N U M B E R C M Figure 23. Infrared spectrum of chemical ly synthesized compound X^d lhydroch lo r i de (N - thyminy lpu t re sc ine\u00C2\u00AB2HC l ) (KB r d i s c ) . GENERAL DISCUSSION Phage 0W-14 DNA is unusual in a number of respects , a l l of which appear to be the consequence of the pa r t i a l replacement of thymine with N-thyminylputrescine. Unusual bases are qu i te common in phage DNA, but N-thyminylputresince e f f ec t s the propert ies of the DNA (Tm and buoyant density) much more markedly than any base change reported so f a r . It is a l so s i g n i f i c a n t to note that i t represents the f i r s t unusual base to be found in the DNA of a pseudomonas phage. The f i r s t unusual base to be found in phage DNA was 5 _ hydroxy-methylcytosine (HmCyt) in the T e v e n phages of E_. col i (Wyatt and Cohen, 1952). Later i t was shown that some of the HmCyt residues were g luco-sy lated (Sinsheimer, 1954). HmCyt has a l so been demonstrated in other col iphages (Kay and F i l d e s , 1962), and in the DNA of a K l e b s i e l l a phage (Anisymova et_ aj_., 1969) . In a l l these cases there is complete rep lace -ment of the cytos ine residues by HmCyt. Studies on Bac i l l u s phages have shown that in many of them the thymine residues are completely replaced by e i ther u r a c i l (phages PBS1, PBS2 and SP90) or 5 _hydroxymethyluraci1 (HmUra) residues (phages SPO-1 and SP8) (see Shapiro, 1970). Minor const i tuents found in phage DNA include 6-methyladenine, which is present at 2 moles % in Streptomyces gr iseus phage C20 (Dunn and Smith, 1958), and 5 _ methylcytos ine (5-MeCyt) which is present at 0.13 moles? in the DNA of E. col i phage DD7 (Nikolskaya et al_., 1968).. In Xanthomonas oryzae phage XP12, 5-MeCyt completely replaces the cytos ine residues and amounts to 33-4% of the DNA bases (Kuo, Huang and Teng, 1968). It is of in teres t that 5 _MeCyt a l so occurs in the DNA of many Tracheophyta and ce r t a i n Thai lophyta at leve l s as high as 7 moles? (Shapiro, 1970). The e f f e c t s of the base subs t i tu t ions on the melting temperatures and buoyant dens i t i e s of the phage DNAs contain ing modif ied bases are l i s t e d in Table IX. As can be seen, demethylation (Thy -\u00C2\u00BB\u00E2\u0080\u00A2 Ura) hydroxy-l a t i on (Thy -*\u00E2\u0080\u00A2 HmUra) , hydroxymethylation (Cyt -*\u00E2\u0080\u00A2 5_HmCyt) and g lucosy-l a t i on (5-HmCyt \u00E2\u0080\u00A2+\u00E2\u0080\u00A2 Glc-5 -HmCyt) cause s p e c i f i c changes in the buoyant propert ies of the DNA as indicated by the normalized r a t i o s . N-thyminyl-putresc ine produces e f f ec t s which are both q u a l i t a t i v e l y and quant i -t a t i v e l y d i f f e r e n t from those seen with other subs t i tu t i on s . While in a l l other cases there is an increase in the buoyant density and a decrease in the Tm of the DNA, the modi f i cat ion in 0W-14 DNA resu l t s in the oppos i te e f f e c t s . The normalized dens i ty change r a t i o (Kohn and Spears, 1967) of 0W-14 DNA most c l o se l y resembles that of a DNA which has undergone a base-pair s u b s t i t u t i o n . The only na tu ra l l y occurr ing DNA to possess a buoyant density s im i l a r to that of 0W-14 DNA is that of Baci1lus cereus phage PR (Levin and Compans, 1968) but the cause of the low density (1.659 g/cm ) was not fur ther invest i gated. Table IX. Physico-chemical propert ies of various modified DNAs. 1. Buoyant density changes - g/cm DNA Base subs t i tu t ion % GC CsCl Ap C s 2 SO^ Ap Ratio Actual Theoret. Actual Theoret \u00E2\u0080\u00A2 SPO-1 HmUra for Thy 43 1.742 1.703 +0.039 1.455 1.424 +0.031 1.30 PBSI Ura for Thy 28 1 .722 1.687 +0.035 1.433 1.417 +0.016 0 .76 Ts(o)S HmCyt for Cyt 34 1.706 1.695 +0.011 1.431 1.422 +0.009 1.34 T2 GlcHmCyt fo r Cyt 34 1.701 1.695 +0.006 1.443 1.422 +0.011 2.90 0W-14 Thy+KX- = Ade 56 1.666 1.716 -0.050 1.415 1.428 - 0 . 0 13 0.41 J L *\ norma 1i zed dens i ty change r a t i o = ( A / C s ^ O ^ / ^ C s ^ - p Water) (A^CsCD/^CsCl - p Water) 2. Melting temperature (Tm) changes - C DNA Base subs t i tu t ion % GC Tm ATm Actual Theoret ica l SP8 HmUra for Thy 43 76.5 87.6 - 11.1 AR9 Ura for Thy 27.7 74.5 80.5 - 6.0 (3) T2 GlcHmCyt fo r Cyt 34 83 83 0.0 T even HmCyt for Cyt degree of gl ucosy lat ion has no i nf1uence 0W-14 Thy+ X = Ade 56 99.3 94.1 + 5.2 (1) most of these data were derived from Shapiro, 1971 (2) Kohn and Spears, 1967 (3) Vanyushin, et a l . , 1970. ^ 80 Compound X is an example of a hypermodified pyrimidine (Ha l l , 1971). The presence of mu l t ip le bases in phage DNAs is an uncommon occurrence, having only been demonstrated in T col iphage DNAs ' 3 1 even v a where one f inds 5\"HmCyt, g lucosy lated 5_HmCyt, and d ig lucosy la ted 5-HmCyt, and in Ser ra t i a marcescens phage n (Pons, 1967) where some of the guanine residues are replaced by an as yet un iden t i f i ed pur ine. Part of the problem in v e r i f y i n g new resu l t s based upon chemical hydrolyses is that the resu l t s may be due to a modifying act ion of the hydro l y t i c agent on known or novel bases. An example of th i s type of problem is that of Bac i l l u s phage Vx which i n i t i a l l y showed a T:A r a t i o of 0.22 (ikeda et_ aj_., 1965). It was l a te r shown (Shimizu et al . , 1970) that the thymine was formed from HmUra during ac id hydro ly s i s . It is unfortunate that enzymatic d igest ion f a i l e d to re lease a nucleot ide de r i va t i ve of N-thyminylputrescine, but th i s is not unusual with modif ied DNAs (see phage PR; Levin and Compans, 1968). It is poss ib le that by using d i f f e r e n t nucleases, a nucleot ide could be r e -1 13 leased. H or C magnetic resonance spectroscopy may prove of use in confirming the chemical nature of compound X in intact DNA. The f ind ing of polyamines assoc iated with phage DNA, though not cova lent ly bonded as in the case of 0W-14 DNA, is not without pre-cedent. The presence of polyamines, inc luding putresc ine, in p u r i f i e d The p a r t i a l replacement of thymine by an un ident i f i ed pyrimidine in B. subti 1 is phage SP-15 DNA was reported recent ly (Konvicka and Mandel, 1972) but the physico-chemical e f f e c t s of the replacement resu l t in an increase in the buoyant dens i ty and a decrease in the Tm of the DNA. 81 phage p a r t i c l e s has been commented on by a number of workers (Ames and Dubin, 1958; Ames ert a K , 1960; Kay and F i l d e s , 1962). Coliphages T2 and T4 conta in , re spec t i ve l y , 132 and 164 mmoles of polyamine per mole of DNA phosphorus (Ames et_ aj_., 1960). In T4 spermidine accounts for 20% while putresc ine makes up the bulk of the polyamine component. The presence of spermine and monoacetylspermine was indicated in Salmonella typhimurium phage PLT-22 (Ames et_ al_., I960). Ten co l i f o rm bacteriophages i so la ted from sewage contained putresc ine and spermidine (Kay and F i l d e s , 1962). Because 0W-14 DNA contains cova lent ly bonded putresc ine, i t s physico-chemical propert ies should be compared with those of model systems involv ing polyamines and DNA. An increase in the Tm of DNA has been observed when DNA is incubated with basic o l igo-L-amino ac ids (Kawashima, et^ aj_., 1969); diamines (Frazer and Mahler, 1958; Mahler e_t a_l_., 1961; Frazer and Mahler, 1961; Mahler and Mehrota, 1962); spermine (Mandel, 1962); spermidine (Tabor, 1962); and ce r t a in a n t i b i o t i c s , inc luding a c r i d i n e orange and p ro f l a v i n (Kersten, Kersten and Szyba l sk i , 1966). The increased Tm of 0W-14 DNA can be read i l y explained in terms of polyamine in teract ions with nuc le ic a c id s . CPK models have been constructed to show the spa t i a l re l a t ionsh ips of the putresc ine s ide chains to the rest of the double he l i x in 0W-14 DNA (see F i g . 26a,b, c and d) . Contrary to a l l proposed model systems involv ing polyamines and DNA (Cohen, 1971), i t is observed that the s ide chains bridge the major 82 Figure 26 a) Atomic model o f DNA. The methyl groups on thymldyl lc ac id uni ts are I l l u s t r a ted with arrows (\u00E2\u0080\u00A2*-). Figure 26 b) Atomic model of DMA. The two methyl groups ind icated in F i g . 26 a) are subst i tuted with the putresc ine chains (\u00C2\u00AB-). 84 rather than the minor groove without causing any not iceab le s t r a i n to the secondary s t ructure of the double he l i x , and in teract with the phosphage groups on the complementary chains. D irect measurements from the model ind icate that the -N+ . . . -0-P-0 d is tance is approx i -o mately 3.3A. This compares favourably with the 2.8A recorded by L iquor i et a l . ( l967)for the distances corresponding to the optimum interact ions of spermine and spermidine with DNA. Not only w i l l e l e c t r o s t a t i c in teract ions contr ibute to the supra - s tab i1 i ty of the phage DNA but there is a l so the p o s s i b i l i t y of hydrogen bonding i ( -NH\u00E2\u0080\u009E . . ,0=P -0 ) and the neu t r a l i z a t i on of repu l s ive forces between 1 i complementary negat ively-charged chains. From the content of N-thyminylputresc ine. In phage 0W-14 DNA, th i s base could neu t ra l i ze between 12 (only 1\u00C2\u00B0 amine involved) and 2k% (both amines involved) of the negat ively-charged phosphate groups in the DNA. It would be of in teres t to determine i f the phage p a r t i c l e s a l so contain a noncovalently bonded polyamine component. The unusual r e l a t i on sh ip of Tm to SSC concentrat ion probably r e -su l t s from the inf luence of the solvent concentrat ion on the polyamine-DNA i n te rac t i on s . It appears (Horacek and bWnohorsky, 1968) that polyamines are most e f f e c t i v e at s t a b i l i z i n g DNA, as measured by an increase in the Tm over that of a polyamine-free c o n t r o l , at low SSC concentrat ion (eg. 0.01 x SSC), with the induced increase being minimal in SSC. This resu l t s from competit ion between the pos i t i ve ly -charged 87 phosphate groups. S imi lar resu l t s were noted with 0W-14 DNA. A dens i ty decrease is observed when DNA is a l ky la ted with n i t r o -gen mustard (Kohn and Spears, 1967) and upon the i n te r ca l a t i on of c e r t a i n a n t i b i o t i c s , inc luding nogalamycin and mithramycin (Kersten, Kersten and Szyba l sk i , 1966 ) -The unusually low density of phage 0W-14 DNA could be due to a number of f a c to r s : (a) competit ion between the -NH^ + moiet ies of the putresc ine s ide chains and the C s + cat ions for the \"PO/j groups on the DNA backbone; (b) increased hydration of the phage DNA with a con-comitant decrease in dens i ty ; (c) the low dens i ty of the s ide chains on the thymine moiet ies . The p o s s i b i l i t y that the protonated amino group(s) can act as counter ion(s) f o r the negatively-charged phosphate groups and as such exclude potent ia l C s + counterions can probably be ignored s ince the high so lute (CsCl) molar i ty would preclude these in teract ions (see Horacek and Cernohorsky, 1968). The p o s s i b i l i t y of increased hydration was invest igated using the method of Kohn and Spears (1967). who showed that the a l k y l a t i o n of DNA with nitrogen mustard resu l ted in a hypodensity which apparently was mainly due to an increased hydration of the DNA. The app l i c a t i on of t h e i r c a l cu l a t i on s (equations 1, 2 and 3) to a v a r i e t y of modif ied DNA's is summarized in Table X. 1. h = M - pV pV - M W ! Table X. App l i ca t ion of the equation of Kohn and Spears (1967) to various modif ied DNAs. DNA Fract ion of bases mod i f i ed Density change (P \" P ' ) h_ AM AV Y Ah Nitrogen mustard 0.044 0.050 7.4 174 150.4 6.3 25.9 0W-14 0.12 0.050 7.4 f159 131.7 a 5.0 6.2 1 89 100.3 b 6.51 4.56 PBSI 0.36 -0.035 5.9 -14 - 17 .8 C -1.3 -1.2 SPO-1 0.285 -0.039 5.4 16 - 5 . l 4d 1.9 5.2 12 0.17 -0.006 6.5 192 133 e 2.7 3.6 Ts(o)S 0.17 -0.011 6.3 30 40 .5 f 3.1 4.8 a AV was based upon the molar volume (Vm) of putresc ine plus the Vm of two CJP ions - 15.7 cmVmole based upon the ana lys i s of X-ray c rys ta l lographic data of ch lo r ide sa l t s ( ). Kohn and Spears (1967) used the value 15.4 cm3/mole. AM and AV assuming no CI\" ions. The Vm of a -CH, moiety was ca lcu la ted from the AVms of a number of methylated compounds and re lated nonmethylated compounds - e.g. toluene and benzene. The Vm of an -OH group was ca lcu la ted in a s im i l a r manner except that alkanes and t h e i r corresponding a l ky l a lcohols were compared. The Vm of the -CH2 _ 0-g lucose residue was based upon the Vm of a-methylg lucos ide. The Vm of the -CH2-OH group was assumed to be s im i l a r to that of methanol. CRC Handbook of Chemistry and Physics, 49th e d i t i o n (p. B-308). 2. y = p'AV - AM ( P ' \" P w ) V w 3. Ah = (M - p V) [ p - p' - x {p'AV - AM } ] [ ( p - p w ) (p ' - p j M - p w V { p ' - p w > 3 xVw Where Ah is the increase in the number of molecules of water of hydration per DNA nuc leot ide upon mod i f i ca t i on ; p, the buoyant density of the unsubst ituted DNA; p ' , the buoyant dens i ty of the modif ied DNA; p , the density of water; x, w the proport ion of subst i tuted bases; M, the molecular weight of an average CsDNA nucleot ide (442 g/mole); V, the molar volume of an anhydrous CsDNA nucleot ide unit (212 cm /mole); AM and AV, the changes in molecular weight and molar volume due to sub s t i t u t i on ; V and M , the volume and mass of w w water; h, the number of water molecules (hydration) per nuc leot ide un i t ; Yi the buoyancy due to anhydrous mass and volume changes upon mod i f i ca t ion per nuc leot ide (in uni ts equivalent to the number of hydration water molecules) . Unl ike the case of nitrogen mustard-treated DNA, the buoyancy of 0W-14 DNA as indicated by the density r a t i o (pCs,S0,/pCsCl) is i dent i ca l 90 to that of t h e o r e t i c a l l y normal DNA ( r a t i o : 0.84). Therefore, i t was f e l t that the counterions (CI and S0^ ) did not inf luence the hydrat ion of the phage DNA abnormally, and the i r presence should not be taken into cons iderat ion when ca l cu l a t i n g the AM. Thus, the AM of the protonated s ide chain was taken to be 89 (g/mole). Only nitrogen mustard-treated DNA showed a major d i f f e rence between h and Ah, i nd i ca t i ve of an increase in hydrat ion. The other DNAs, with the exception of PBS1 DNA, showed a s l i g h t increase in hydrat ion. It appeared that the hypodensity change in the case of 0W-14 DNA was a consequence of the anhydrous mass and volume changes caused by the presence of the subst i tuent group (see y column). Although these ca l cu l a t i on s provide an theore t i ca l approach to the ana ly s i s of the e f f ec t s of nuc le i c ac id a l t e r a t i on s on the phys ico-chemical propert ies of DNA, an inves t i ga t ion of the actual degree of hydration of 0W-14 and other modif ied phage DNAs using the methods of Hearst and Vinograd (1961) and Tunis and Hearst (1968), would prove of cons iderable va lue. Exper imental ly, i t is known that the buoyant dens i ty of DNA \u00E2\u0080\u00A23 usua l ly increases by 61 - 63 mg/cm in a l k a l i n e CsCl (Vinograd et a l . , 3 1963). The density of 0W-14 DNA increased by 84 mg/cm . The expected 3 buoyant dens i ty in a l k a l i n e CsCl would have been e i ther 1.728 g/cm 3 (based upon the density in neutral CsCl) or 1.778 g/cm (based upon the dens i ty expected f o r 56 moles % GC DNA). The reason f o r the unusual dens i ty s h i f t in a l k a l i i s , at present, not f u l l y understood. SUMMARY The propert ies of f_. acidovorans phage 0W-14 DNA are summarized in Table XI. 92 Table XI. Summary of propert ies of 0W-14 DNA. Value expected Indicated Observed Value fo r 56 moles moles % GC % GC E260nm / E280nm a t p H 3 - \u00C2\u00B0 Melt ing temperature i n : SSC 6.1 x SSC \u00E2\u0080\u00A23 Buoyant density (g/cm ) in : ( i) neutral CsC l , na t i ve : denatured: ( i i ) a l k a l i n e CsCl ( i i i ) neutral Cs2S0^ Base composit ion, chemical ana lys i s 1.210 1.255 59.5 99-3 94.1 66.3 (72.9) 86.3 77.8 1.666 1.716. 4.5 1.680 1.731 1.750 1.778 1.415 1.428 56.2 JU Af te r co r rec t i on fo r the abnormal e f f e c t of the SSC concentrat ion on the melting temperature of 0W-14 DNA. BIBLIOGRAPHY Anisymova, N.I., I.M. Gabr i l ov i ch , N.V. Soshlna and S.N. Cherenkevich. 1969. 5-Hydroxymethylcytosine-containing K l e b s i e l l a bac te r i o -phage. Biochim. Biophys. Acta 190:225-227. Ames, B.N. and D.T. Dubin. 1960. 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Yamamoto, K.R., B.M. A l b e r t s , R. Benzinger, L. Lawhorne and G. T r e i b e r . 1970. Rapid bacteriophage sedimentation in the presence of po ly -ethylene g lyco l and i t s a p p l i c a t i o n to l a rge - sca le v i ru s p u r i f i c a -t i o n . V i ro logy 40 :734 -744 . APPENDIX 1 A COMPUTER PROGRAM TO FACILITATE DNA Tm CALCULATIONS* INTRODUCTION Two of the most commonly used methods fo r analyzing the base composition of DNA are: (a) equ i l i b r ium dens i ty gradient c e n t r i f u -gat ion (Sueoka et^ aj_., 1959) and (b) u l t r a v i o l e t absorbance-temperature p r o f i l e (Marmur and Dory, 1959) experiments. Both of these methods g ive estimates of the moles percent guanine + cytos ine (?GC) in the DNA. As part of a de ta i l ed study of the DNA of Pseudomonas acidovorans phage 0W-14 (Kropinski and Warren, 1970), i t became necessary to carry out numerous melting temperature (Tm) experiments. The l a t t e r are not only time consuming with respect to execution time but a l so to the time required to analyze the data. Recently, a computer program which analyzes the resu l t s o f CsCl i sopycnic gradient experiments was published by Quetler e_t aj_. (1969). This program permits the simultaneous c a l c u l a t i o n of the buoyant dens i ty , % GC, and the minimum molecular weight of the DNA samples used from data der ived from microdensitometer scans. It was to th i s end, namely to f a c i l i t a t e data ana l y s i s , that we designed the fo l lowing computer program. It is wr i t ten in Watfor, an e a s i l y understood computer language, and permits the graphical as well as tabular l i s t i n g of the resu l t s from Tm experiments along with ca l cu l a t i on s of the Tm and the % GC. 101 Computer F a c i l i t i e s . The f a c i l i t i e s of the student terminal at the Computer Centre (Un ivers i ty of B r i t i s h Columbia) included IBM 29 card punch un i t s , a card reader, IBM 2 9 4 4 1 1 data channel repeater, 2821 contro l un i t , and 1403 p r i n t e r . PROCEDURE The bas ic program is composed of two sets of computer cards: A. a set of 165 cards which comprises the master program, and which is l a i d out numerical ly in Table XII; B. a set of 80 \"TEMP-STEF\" cards bearing temperatures (TEMP) from 25C-105C and the corresponding thermal expansion fac tor s (STEF). The l a t t e r can be read i l y made using the table of values provided in Mandel and Marmur's a r t i c l e ( 1 9 6 8 ) . A t h i r d set of cards, the \"data ca rds \" , is constructed for each experiment as described in Table XIII: card numbers 1, 2 and 3 plus a ser ies of cards car ry ing the chart readings (CHRD) at 25C and the de-s i red sequence of temperatures to be analyzed. These l a t t e r cards are a l ternated with the \"TEMP-STEF\" cards as descr ibed in Table XIII, and the whole set is assembled behind the DATA card of the master program deck. The add i t i on of two \"nonsense\" data cards: descr ibed in master program cards 20 and 2 1 : e f f ec t s the correct termination sequence of the computation. Table XII. Tm computer program T I HE s 1 2 2 1 4 : 2 5 U N I v t * s : r y O F a . c . c o M p j r i N s C E N T R E * C O M P I L \u00C2\u00A3 K R O i M N S i a - M C D O N A l J 0AT f c : 0 2 - 0 3 - 7 1 I M P L I C I T l N I f c G t K I I ) , C H A R A C T E * \u00C2\u00BB 4 1 X , B ) 01 MtiMSUi 1 * T ( 90 ) ,A3 I 9 0 ) . 0 1 9 ) . T F I 4 1 ) , A B F 1 4 1 ) , B 1 4 2 ) . B F ( 2 1 ) , L ( 4 1 ) , L B L A 8 9 10 11 12 13 1'. 15 16 i ; _ 1 8 _ 19 2 0 2 I 22 23 2 5 26 27 28 29 3 0 _ \" 31 32 33 34 35 36 1 0 0 1 5 0 5 165 2 6 0 I N * ( 1 2 ) x b = ' ' X = ' * \u00E2\u0080\u00A2 X T = ' \u00C2\u00AB ' x r F = > \u00C2\u00BB \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 R E A D . M E X P N O , S O L K E A J < 6 | 1 0 0 ) D WiU I t [ o , ISO) . ' l EXP.M, SUL t i k i IE ( 1 5 b ) I 0< 1 ) , 1= 1 , 9 ) FORMAI I VA4) FORMAT ( \u00E2\u0080\u00A2 0 ' t ' E < P t R I ^ E N T N O . 1 _ F 1 2 . 4 ) . _ _ FORMA 11 \" 1 4 , 5 X , ' S O L V E N T ( S S C C O N C E N T R A T I O N ) * ' i 1 5 5 J R M A K ' U ' , ' U M A OSE J - \u00E2\u0080\u00A2 , 5 X , 9 A * ) R t A J . S I W P J O . u X P A N . S i N C r t R MK1 I t l o , If.O) 1 6 0 F j R H A l l ' U ' , ' I d r t P E R A I U K b ( O E G C) \u00C2\u00BB \u00E2\u0080\u00A2 , 5 X , ' REL A l l VE A B S O R B A N C E ' ) 1 = 0 200 u t A J , i t t v , s r t F R E A J t C r H J IF I 1 . C J . V J l i d TO 260 1 F 1 T E M P . t d . 0 ) 1 , 0 f J 2 6 0 1=1 + 1 2bRuV = Cr l i \0 -$ l.NCMR _S1 N C U j = Z tRO\ / * L X P AN \" A C i oo =~ I s I Vci/u \u00C2\u00BB s i N i ' u o i * s r t F I F U t M P . t O . 2 i ) A t r U U A = A C T Q O IF I 1 . u . i . A ! 4 J . T t M P . N t . 2 5 )GU 10 9 0 0 R b L A b = A L I J O / A L T 0 J A rfKI 11 ( o . i a S ) f fcMP.RELAB _F; j R .1 AT I ' ' , 3 X , 1 5 , 1 2 X , F 1 5 . 4 ) 1 I I ) = 1 I I * A o l 1 ) = R L L A B 00 TU 2uU HI 1 H = I R t L A J - 1 . 0 ) * 1 0 0 VI-IRtLAb/2.0)>0.50 00 2 50 M=1 i i 37 38 39 4 0 41 _ 4 2 _ 4 3 44 4 5 4 6 4 7 4 8 l\u00C2\u00AB = M I F I P r . L c . A u l M ) )G0 TO 2 7 0 2 5 0 CON Tl ,SOt 2 7 0 N A = N - l S j i r F = A u ( N I - A i J ( N A ) TO I f F - l i . N ) - T ( N A ) S T u I F F = F L O A l I T L . 1 F F ) P I A = 1 P T - A J ( N A ) ) / .>0 IFr P T M L L T = ( P T A * S T u I F F ) * F L U A T ( T I N A ) ) TMAX = I F l X l f T M t L r H - 2 0 T M l i * = l F l X ( P I M b L T > - 2 0 1 F ( I M A X . O r . l l J ) T M A X - l 1 0 a an Table XII. Tm computer program Table XII - continued ( s 4 9 50 51 52 5 3 54 I F ( r M I , l . L r . 2 > ) IMIN=2S NUM=0 l id 2 8 0 I A = L.I I M I I l A l . L l . I H I M I G J . TO 2 8 0 I F ( 11 I A I . G T . T M A X I G U 1 0 2 8 0 1 55 I F I A i M l A I . G f . V l G O I J 2dO \ 5 6 NUM-I-iOM* 1 5 7 TF(.NUi-t)=T( IAI i 5 8 AtJF ( KJM) =Ad< I A l 1 5 9 L( .JUM) =ijji-t . 6 0 2 80 CJ.NT I f lUt 61 S U L A = $ U L / 0 . 10 i 6 2 PCE i t I - 1 1 (P tMEL r-\u00C2\u00BB 1 6 . > * A L 0 ^ 1 0 ( SOLA ) ) ) / S O . 2) - 0 . 9 9 0 1 * 1 0 0 . 0 j 6 3 w K i i t i o , i u 7 ) p r i H , p r M f c L 7 , P C E ^ r 64 1 0 7 F U r l r t A r i ' J 1 , ' U / O INJUCC i ) H Y P E f i C H K a M l . C i r y = < , K J c G C 1 \u00E2\u0080\u00A2 , F d . 1 , 3 X , \u00C2\u00AB 0 / 0 G . C . * I , F 1 0 . 2 ) F 1 2 . 2 , 5 X t ' M E L T l N G T E M P . i 6 5 IP=1 j 6 6 U M H I . N - l 6 7 L lUr, = NUH j 68 L dL A N K ( 1 ) ' l 6 9 iMU:lb = N U U - l 1 70 H=0 j 71 15 l=rt 72 13 1FI I . t J . f i J M J l G U r u l b 73 1= 1*1 74 IF< A u F l U 1 I ) - L T . A b F l L < > IGO TO 13 j 75 M=I 76 14 K = L l l ) i 77 L l l l = L I | t l l i 73 1. 1 79 1 = 1-1 \u00E2\u0080\u00A2 8 0 I F ( I . c u . U l i i J TU 15 81 1F{ A i l F I L I 1 1 ) . U T . A U F ( L ( I + l ) ) l & J TO 14 j 82 Ciu TU IS | 83 16 0 0 I'll M = l , 4 2 84 d l r t ) = X o ! 85 2 9 2 ; 86 CiJ TU Hi 87 2 9 0 I F = H - l 88 IF 1 I F . c J . O l G O TO 2 * 6 89 UU 2'*4 * = 1 , 1 F 9 0 K = L L > L A . I I U , 1 ) 91 8 1 K ) - X d j 9 2 2 9 4 CU.NTl.NUc 93 291 L/U 2 V i ,1=1,21 94 UF ( M) =Xi) 9 5 2 9 3 CU.4T INJC 9 6 2 9 6 V - V - U . J 1 U ; 97 NS TAi-X \"3 It 11 St - z ~. -> z o II I H \u00E2\u0080\u0094 M _> a. o \"3 U -VI XI - J + .-1 o * II < * \u00E2\u0080\u0094 * -* M \u00E2\u0080\u0094 x. \u00E2\u0080\u0094 x < X < \u00E2\u0080\u00A2 > \u00E2\u0080\u00A2 \u00E2\u0080\u00A2> -\u00C2\u00BB f i < U4 * \u00E2\u0080\u00A2 a i \u00C2\u00BB u + ! \u00E2\u0080\u0094 ' J -\u00C2\u00BB _) i N N O j \u00E2\u0080\u0094 < \u00E2\u0080\u0094 < * + -o t rO -C -\u00C2\u00BB II O \u00E2\u0080\u0094 < \u00E2\u0080\u0094 \u00E2\u0080\u00A2* it ii o . n u. r x X \u00E2\u0080\u0094 a o O 3 : a -3 JJ M -M T O -\u00C2\u00AB \u00E2\u0080\u00A2 n - i \u00E2\u0080\u00A2> vo J \u00E2\u0080\u0094 /> ' : \u00E2\u0080\u0094 H . >! -> Z '\u00E2\u0080\u00A2 \u00E2\u0080\u00A2 II . i < -* I II + \u00C2\u00ABJI\"vl \"3 \"> i -\u00C2\u00BB \u00E2\u0080\u00A2 -J a \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 j ^ \u00E2\u0080\u00A2 > \u00E2\u0080\u0094 -T i : \u00E2\u0080\u0094 n . . u. *- . \u00E2\u0080\u0094 X U . -4 O -t -4 i N J \u00E2\u0080\u0094 \u00E2\u0080\u00A2 . - j < a. -\u00E2\u0080\u00A2 \u00E2\u0080\u0094 t II I \u00E2\u0080\u0094 . I XJ \u00E2\u0080\u00A2 \u00E2\u0080\u00A2\u00C2\u00A3 1- \u00E2\u0080\u00A2 II . It -\u00C2\u00BB . ~ T. 2 ~f C \u00E2\u0080\u0094 <- i i a. Ojjc j fi: o . \u00E2\u0080\u0094 (j \u00E2\u0080\u00A2 T * x J. \u00C2\u00AB 3 j 1/1 Ifl I O O - ^ I N i ^ ^ i i A ' O N c O ^ ' O - Z M ^ ^ . t ^ ^ ^ ffl^QH IN rn IT O f . a\"> C O f\ i Hi >\u00C2\u00BB\u00E2\u0080\u00A2 4 M O \u00C2\u00BB 0 \" \u00C2\u00AB N ^ 0 < ^ ^ - ^ ^ ^ ^ i ^ ^ ^ ^ ^ r s j u j r g a j f \ i : f \ j f g r N j r s ( f N r g ( ^ f T i m m i\u00C2\u00AB m m m i\u00C2\u00AB m ^ ^ ^ , j - \u00C2\u00BBr |^ *f wt i f tf-> *r> -3 X * 3C \u00E2\u0080\u00A2-u * 3 > . O - \u00C2\u00BB II J> . 3 < . x x o >- a . t \u00C2\u00AB. i 3 \u00C2\u00BB O : x t_5 - \u00C2\u00AB x - 4 < ^ w i o o o CC c: Table XIII. Layout of data and \"TEMP-STEF\" cards. Data Card Number Data required Computer Terminology Explanation 0 $DATA i n i t i a t i o n card 1 MEXPNO, SOL experiment number; solvent concentrat ion r e l a t i v e to SSC 2 D type of DNA used 3 SINPOD, EXPAN, SINCHR DNA concentrat ion in OD260 un i t s /ml ; instrument chart expansion f a c t o r ; i n i t i a l chart reading k TEMP, STEF temperature (25 C) ; thermal expansion fac tor (1.000) 5 CHRD chart reading at 25 C ( iden-t i c a l to SINCHR value) 6,8 etc TEMP, STEF temperature of experimental in terest and t h e i r correspond-ing thermal expansion values 7,9 e tc CHRD charti readings corresponding to above TEMP values 105 The time required to punch out twenty data cards, a l t e rna te them with the cor rect \"TEMP-STEF\" cards and assemble the whole program deck takes approximately 5 minutes. The tota l computer time used in compil ing and executing the program is s l i g h t l y over three seconds. PROGRAM The program is wr i t ten in WATFOR (verson 1 level 1 January 1 1 970 ) , a read i l y understood computer language. The computer c a l cu l a t i on of the r e l a t i v e absorbance (RELAB) values is la id-out on cards 2 3 - 26 and 28 of the master program. Each chart reading (CHRD) is adjusted to a common basel ine of zero to wompensate fo r the instrumental o f f s e t used. The zeroed readings (ZEROV) are then mu l t i p l i ed by the instrumental expansion f ac to r (EXPAN) employed to give an unadjusted increment value (SINCHR), When the l a t t e r value is added to the number of O D 2 6 o n m u n ' t s / m l (SINPOD) and mu l t i p l i ed by the appro-p r i a te thermal expansion f ac to r (STEF) the actual number of 0 u 260nm u n ' t s / ' ml (ACTOD) are obtained. Each ACTOD value is d iv ided by the ACTOD at 25 C g iv ing the r e l a t i v e absorbance (RELAB) va lues. These values and the i r corresponding temperatures are displayed in Table XIV along with the percent thermal ly induced hyperchromicity (PTIH). The l a t t e r value is ca l cu la ted using the fo l lowing formula: PTIH = 100 (RELAB - 1.0) Table XIV. P r i n t - o u t of melt ing curve data. z> 3i ^ -sj \u00E2\u0080\u0094> 13 -3 f \"> 3 - * M ' r -\u00C2\u00BB :> 10 r\u00C2\u00BB t -j -4 \u00E2\u0080\u0094\u00C2\u00AB -3 -\u00C2\u00AB M *\ I -\u00C2\u00BB\u00E2\u0080\u0094 J \u00E2\u0080\u00A2 J * 3 I I ; \u00E2\u0080\u00A2 \u00E2\u0080\u0094\u00E2\u0080\u00A2 R l _ l \u00E2\u0080\u00A2\u00E2\u0080\u0094 O \u00C2\u00AB3 \u00E2\u0080\u0094 */- .J -t ti. U - +i u -(. t ' : ; i O ~ t Master program cards 61 and 62 are concerned with the c a l c u l a t i o n of the moles percent guanine plus cytos ine using the equation of Mandel et a l . ( 1 9 7 0 ) ; % GC = 100 { [ T m s s c - 16.3 l o g 1 Q (SSC X/SSC 0 ])]/S0.Z} - 0.990 X E s s e n t i a l l y the remainder of the program is concerned with the graphical representat ion of the data presented in Table XIV (see Figure 27). DISCUSSION The program, as presented, in i t s long form allows numerous modi-f i c a t i o n s to be made as des ired by the ind iv idua l researcher or by the a v a i l a b i l i t y of computer f a c i l i t i e s . Some of the poss ib le modi f icat ions w i l l be out l ined below. If the graphical representat ion of the re su l t s is not needed, the master program can be shortened to include only cards 1-64 and 160-165. This mod i f i ca t ion w i l l permit normal termination of the program. If the researcher possesses core storage in the computer, the master program can be e a s i l y modif ied for use with an outs ide terminal such that the only data to be punched out would be those contained in Table XII I - cards 1,2 and 3 _ and a ser ies of cards bearing the temp-108 F i g u r e 27. Computer p l o t o f m e l t i n g curve o f DNA. 3 I <\i \u00C2\u00BB\ * \ l I\J <\. 'N> . M I ' O ' <\u00E2\u0080\u0094 l_> V, U U eratures and t h e i r corresponding CHRDs. As over one-ha l f the program is concerned with the graphical representat ion of the r e s u l t s , the a v a i l a b i l i t y of computer p l o t t e r f a c i l i t i e s permits a fur ther mod i f i -c a t i o n . Though th i s program was wr i t ten fo r Tm experiments employing SSC, i t may be used for other solvent systems a l so by the de le t ion or modi-f i c a t i o n s of master program cards numbered 12, 61 and 6 2 . SUMMARY A s imple, rapid and e a s i l y modi f iab le computer program fo r the ana lys i s of DNA melting curve data has been developed. It al lows the graphical as well as tabular presentat ion of resu l t s which include the Tm and ?GC. REFERENCES Krop insk i , A.M.B. and Warren, R.A.J. J . Gen. V i r o l . 6, 85 ( 1 970 ) . Marmur, J . and Doty, P. Nature, 183, 1427 ( 1 959 ) . Quet ier , F., G u i l l e , E. and Lejus, L. Arch. Biochem. Biophys. 130, 685 ( 1 969 ) . Mandel, M. and Marmur, J . J_n \"Methods in Enzymology\", V o l . 12B, Grossman and Moldave, (ed.) , Academic Press, New York, ( 1 9 6 8 ) . Mandel, M., Igambi, L., Bergendal, J . , Dobson, M.L. and Scheltgen, E. . J . B a c t e r i o l . 101 , 333 ( 1 970 ) . 110 Krop insk i , A.M.B. and R.A.J. Warren. 1970. I so lat ion and propert ies o f a Pseudomonas acidovorans bacteriophage. J . gen. V i r o l . 6_; 8 5 - 9 3 Mandel, M. and J . Marmur. 1968. Use of u l t r a v i o l e t absorbance-temp-erature p r o f i l e for determining the guanine plus cytos ine content of DNA. Methods Enzymol. 12B, 195 -206 . Academic Press, New York. Mandel, M., L. Igambi, J . Bergendahl, M.L. Dobson and E. Scheltgen. 1970. Cor re la t ion of the melting temperature and cesium ch lo r i de buoyant dens i ty of bac te r i a l deoxyr ibonucle ic a c i d . J . B a c t e r i o l . 101 ,333 -338 . Marmur, J . and P. Doty. 1959- Heterogeneity in deoxyr ibonucle ic a c id s . I. Dependence on composition of the conf i gurat iona l s t a b i l i t y of deoxyr ibonucle ic a c i d . Nature 183, 1427 -1429 . Quet ier , F., E. G u i l l e and L. Lejus. 1969. Equ i l ibr ium dens i ty gradient u l t r a c e n t r i f u g a t i o n : a simple computer program for routine DNA ana l y s i s . Arch. Biochem. Biophys. 130, 6 85 - 687 . Sueoka, N., J . Marmur and P. Doty. 1959- Heterogeneity in deoxyribo-nuc le i c ac ids . II. Dependence of the dens i ty of deoxyr ibonucle ic ac id on guanine-cytos ine content. Nature 183, 1429 -1431 . APPENDIX 2 PROBLEMS INHERENT IN THE PRODUCTION OF FERMENTER-SCALE LYSATES OF 0W-14. 111 Preparat ion of high t i t r e lysates of 0W-14. Attempts to obta in high t i t r e lysates using fermenters or large bot t les of medium aerated through sparging rocks usua l ly y ie lded l i t t l e or not phage as compared with preparations grown in small volumes as descr ibed prev ious ly (Kropinsk i , M.Sc. t he s i s , 1969). This problem was invest igated to some extent and the gen-eral observations from a number of experiments were c o r r e l a t e d , but the problem was not overcome completely. 1. E f f e c t of Medium. The mannitol broth (MB), based upon the composition of Lur ia broth, was discarded s ince P_. acidovorans is nonproteo lyt ic and therefore cannot u t i l i z e f u l l y the polymeric const i tuents of t r yp -tone and yeast ex t rac t . A l so , c e l l s tend to clump in MB u n t i l past the mid-log phase of growth, and the high prote in content of the medium resu l t s in excess ive foaming during growth. A new medium (CAA-M) conta in ing casamino acids enriched with tryptophan, to a c e r t a i n extent overcame these problems while s t i l l supporting rapid growth of the organism. 2. E f fect of the Antifoam Agent. I n i t i a l l y fermenter experiments, using t r i b u t y l c i t r a t e (TBC) as the antifoam agent, gave very low phage y i e l d s , although the c e l l s had indeed l y sed . TBC had no adverse a f f e c t on the phage p a r t i c l e s per se, but i t may have induced premature l y s i s of the in fected c e l l s . Dow Corning antifoam agents A and C were e f f e c t i v e in that reasonable phage y i e l d s could be obtained in t h e i r p re -sence, but vast amounts were required to prevent the foaming assoc ia ted with c e l l l y s i s . Besides los ing 1/2 to 2/3 of the contents of a fermenter vessel w i th in a few minutes of the onset of foaming, the foam a l so caused f r o t h f l o a t a t i o n : the phage t i t r e in the foam.was s i g n i f i c a n t l y higher than that in the under ly ing f l u i d . A major f ac to r in the large sca le production of 0W-14 i s , in my op in ion , an e f f e c t i v e means of foam c o n t r o l . 3. Aerat ion. Although a f low rate of 0.6 volumes of a i r /volume of c u l t u r e / min was s u f f i c i e n t to g ive normal growth k i ne t i c s of P_. acidovorans w i th in the l i m i t s s tud ied , optimum phage production was obtained at rates of 2 volumes/volume/min or g rea ter . Th i s high rate of aera t ion posed two problems: (a) e f f e c t i v e s t e r i l i z a t i o n of the in f luent and e f f l u e n t gases, and (b) foam c o n t r o l . V io len t foaming occurs when a i r is pumped through 25L of l y s ing phage preparat ion at a rate of 50L/min and antifoam agents, in economical q u a n t i t i e s , a re i n s u f f i c i e n t to prevent t h i s . 4. M u l t i p l i c i t y of Infect ion and Time of Addi t ion of Phage. For a m u l t i p l i c i t y of i n fec t i on (MO 1) of one, the phage y i e l d was d i r e c t l y proport ional to the c e l l density up to a density of approximately 2 x 10 ce l l s /ml (3 O D ^ Q ) \u00E2\u0080\u00A2 Very high phage y i e l d s were obtained using a MOI of 0.01 and a c e l l density of approx i -9 mately 10 ce l l s /m l (1.5 0D 650 nm). However, although a very high phage y i e l d was obtained ( i e . 1 0 1 1 p fu/ml) , the lysate was so viscous that i t was impossible to remove the c e l l u l a r debris by c e n t r i f u g a t i o n . APPENDIX 3 DETAILED MASS SPECTRAL ANALYSIS OF COMPOUND X. 114 Mass spectra l ana lys i s of N-thyminylputrescine. Mass spectrum (70eV) m/e ( r e l a t i v e in tens i ty ) 154 (4); 140 (15) 125 ( 1 0 ) ; 112 (11); 97 (5); 82 ( 1 5 ) ; 81 (5); 72 (8); 71 ( 1 5 ) ; 70 (41); 69 (11); 68 (5); 59 (25); 56 ( 1 0 ) ; 55 (11); 54 ( 1 2 ) ; 53 ( 5 ) ; 45 ( 2 5 ) ; 44 ( 6 1 ) ; 43 ( 1 0 0 ) ; 42 ( 2 8 ) ; 41 (23); 40 ( 9 ) ; 39 (11); 32 ( 1 9 ) ; 31 ( 5 6 ) ; and 30 (\u00C2\u00B0\u00C2\u00B0). The ion at m/e 30 was of much greater in tens i ty than any other ; there fore , the ion of m/e 43 was used as the base peak ( r e l a t i v e in tens i ty 1 0 0 ) . A parent ion (M+) was not detected in the mass spectra of N-thyminylputrescine. This is r e l a t i v e l y common with amines, fo r which the parent ions are unstable. The major ions, m/e 30 (a) and m/e 43 (b) are formed by f r a g -mentation of the putrescyl s ide chain according to Scheme 1. Scheme 1. e p c \u00E2\u0080\u009E r N H - C H f C H 2 V ^ o Z + H2C = NH 2 ^ 3 0 (a} V3,H H N M / \ / \ Z H 2 C ~ C H 2 H2C C H 2 m ^ 4 3^b) m / e 4 4(b') a-Cleavage at the secondary amine gives r i s e to the fragment ion m/e 87 (c) which generates the proposed ion d (m/e 70) by the loss of NH^. A metastable peak is observed at m/e 56 .3 confirming the conversion c_-Scheme 2. C H 2 \" ^ \" C H 2 \" C H 2 \" C H 2 \" C H 2 \" N H 2 * H N - C H 2 - C H 2 - C H 2 - C H 2 - N H + m. 8 7 ( c ) M ' & - N H 3 | \u00C2\u00AB m * 56-31 + H m, 7 0 Further fragmentations of the putrescyl s ide chain by 3~ and the a l te rna te a-cleavage at the secondary amine g ive r i s e to the highest observed mass fragment (m/e 154) and the r e l a t i v e l y intense ion at m/e 140 ( f ) , r e spec t i ve l y . An ion at m/e 140 was a l so found in the mass spectra of 5-methylaminomethyluraci1 (Carbon, David and Studier 1968 ) . o o ii H N 7 V C V H = C H 2 H N / V C H 2 \" N H H H m. 154 m . 14 0 Ionizat ion l o c a l i z e d on the he te rocyc l i c r ing i n i t i a t e s a ser ies of fragmentations which are summarized in Scheme 3. The metastable ion at 53\u00E2\u0080\u00A2 7 confirms the conversion g Hi. Scheme 3. CH - N H - ( C H I - N H 2 2 4 2 M m y 1 2 5 ( g l H C = C - C H = N H \u00C2\u00AB -2 m y 5 4 (i) CO - H N C O X\ H N 1 C H \u00E2\u0080\u009E 2 m . 8 2 ( h ) The intense ion at m/e 112 was assigned to u r a c i l and probably a r i ses d i r e c t l y from M + by the highly favoured loss of the e n t i r e s ide chain as a neutral fragment according to the fo l lowing rearrange-ment: 4<2 H i N - ( C H 2 l 4 \" N H 2 my 1 1 2 By an analagous rearrangement the putrescyl s ide chain gives r i s e to the propylamine fragment ion at m/e 5 9 . This ion is a l so found in the spectrum of putresc ine d ihydroch lor ide (Tobari and Tchen, 1971) H ^ l 2 - \u00E2\u0080\u00A2 H 3 C - , C V 2 - N H 2 S C H - ( C H I - N H \u00E2\u0080\u009E 2 2 2 2 M i n , 59 The ion j_ (m/e 97) may poss ib ly a r i s e by the loss of CO from the ion g_ (m/e 125) in Scheme 2 by the fo l lowing proposed concerted rearrangement: 0-k j H - C O ' r j \ Jl H 9V ( j ) N H ( g ) H REFERENCES Carbon, J . , H. David, and M.H. Studier. 1968. Thiobases in Escherichia col? transfer RNA: 2-thiocytosine and 5 -methyl-amino-methyl-2-thiouraci1. Science 161: 1146-1147. Kropinski, A.M.B., R.J. Bose and R.A.J. Warren. 5\"(4-amino-butyl-aminomethyl)uraci1, an unusual pyrimidine from the deoxyribo-nucleic acid of bacteriophage 0W-14. Submitted to Biochemistry. Tobari, J . and T.T. Tchen. 1971- Identification of (+)-hydroxy-putrescine (1,4-diaminobutan-2-ol) from a Pseudomonas species. J . B iol . Chem. 246: 1262-1265. "@en . "Thesis/Dissertation"@en . "10.14288/1.0101060"@en . "eng"@en . "Microbiology and Immunology"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Physico-chemical properties of bacteriophage OW-14 deoxyribonucleic acid"@en . "Text"@en . "http://hdl.handle.net/2429/32081"@en .