Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

A comparison of methods for the isolation of deoxyribonucleic acid from small amounts of tissue Mezei, Catherine 1960

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1960_A6_7 M3 C6.pdf [ 5.09MB ]
Metadata
JSON: 831-1.0106330.json
JSON-LD: 831-1.0106330-ld.json
RDF/XML (Pretty): 831-1.0106330-rdf.xml
RDF/JSON: 831-1.0106330-rdf.json
Turtle: 831-1.0106330-turtle.txt
N-Triples: 831-1.0106330-rdf-ntriples.txt
Original Record: 831-1.0106330-source.json
Full Text
831-1.0106330-fulltext.txt
Citation
831-1.0106330.ris

Full Text

A COMPARISON OF METHODS FOR THE ISOLATION OF DEOXYRIBONUCLEIC ACID FROM SMALL AMOUNTS OF TISSUE by CATHERINE MEZEI A Thesis Submitted i n P a r t i a l Fulfilment of The Requirements for the Degree of M A S T E R OF S C I E N C E i n the Department of Biochemistry We accept t h i s thesis as conforming to the' required standard THE UNIVERSITY OF BRITISH COLUMBIA September, i 9 6 0 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not, be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Columbia, Vancouver 8, Canada. i i ABSTRACT The chemical and physico - chemical properties of deoxyribonucleic acid preparations iso l a t e d from small amounts of l i v e r and i n t e s t i n a l mucosa of r a t (1-10 g.) 'by f i v e d i f f e r e n t procedures, have been compared. The f i r s t method (29), used for preparation of deoxyribonucleic acid was based on the separation of nuclei from tissue homogenates, followed by extraction and deproteinization of deoxyribonucleic acid with strong s a l t solutions. The second method (20, 31) consisted of the extraction anddeproteinization of nucleic acids by detergent solutions, and separation of ribonucleic acid and deoxyribonucleic acid by f r a c t i o n a l p r e c i p i t a t i o n with iso-propyi alcohol. In the t h i r d procedure crude deoxyribonucleic acid was isol a t e d from nu c l e i according to the f i r s t method and the crude product was further p u r i f i e d according to the second procedure. The fourth method (32) was based on the d i s i n t e g r a t i o n of tissues by high frequency sonic o s c i l l a t i o n s , extraction of nucleopro-t e i n from the nuclear fragments with strong s a l t solutions and deproteinization of deoxyribonucleic acid with chloroform -amyl alcohol mixtures. In the f i f t h method (36, 37) nucleic acids were extracted from tissues by hot, strong s a l t solutions, ri b o n u c l e i c acid and deoxyribonucleic acid were separated by a l k a l i treatment and deoxyribonucleic acid was precipitated with concentrated acid solutions. The advantages and shortcomings i i i of the d i f f e r e n t procedures with respect to y i e l d , purity and macromolecular state of the isol a t e d material have been discussed. An improved technique has been described for the e l u t i o n of purine and pyrimidine bases from paper chromatograms. iv ACKNOWLEDGMENTS. The author wishes to express her sincere thanks and appreciation to Dr. S.H. Zbarsky for his continual advice and encouragement. The personal assistance of the National Research Council i n the form of a Bursary and Studentship i s g r a t e f u l l y acknowledged. Dr. M. E. Reichmann kindly performed the molecular weight determinations by l i g h t scattering measurements on some DNA preparations. V TABLE OF CONTENTS Page T i t l e i A b s t r a c t i i Acknowledgement i v Table o f Contents v L i s t o f F i g u r e s v i i i L i s t o f Tables i x H i s t o r i c a l I n t r o d u c t i o n 1 C h a r a c t e r i z a t i o n o f D e o x y r i b o n u c l e i c A c i d (DNA) P r e p a r a t i o n s . 9 I . The Chemical C h a r a c t e r i z a t i o n o f DNA P r e p a r a t i o n s . 10 I I . The P h y s i c o - Chemical C h a r a c t e r i z a t i o n of DNA. 13 (a) The hyperchromic E f f e c t o f DNA S o l u t i o n s . 14 (b) The Anomalous V i s c o s i t y of DNA S o l u t i o n s . 15 E x p e r i m e n t a l 19 The P r e p a r a t i o n o f DNA from S m a l l I n t e s t i n a l Mucosa and L i v e r o f Rat by D i f f e r e n t P r o c e d u r e s . 19 I . I s o l a t i o n o f DNA by the Method of Emanuel and C h a i k o f f . 19 I I . I s o l a t i o n of DNA by the Method of Kay et a l . M o d i f i e d by Stevens and Duggan. 22 I I I . I s o l a t i o n of DNA by the Combined Methods of Emanuel and C h a i k o f f and Kay et a l . 24 I V . I s o l a t i o n of DNA by the Method of Zubay. 24 V . I s o l a t i o n o f DNA by the Methods o f Bendich et a l . and Tyner et a l . 26 V I Page The Chemical C h a r a c t e r i z a t i o n o f DNA . 28 The D e t e r m i n a t i o n o f N i t r o g e n Content by the M i c r o K j e l d a h l Procedure . 28 The D e t e r m i n a t i o n o f Phosphorous Content by the Method o f B a r t l e t t . 28 The D e t e r m i n a t i o n o f Base Composi t ion 34 I . H y d r o l y s i s o f DNA by Concentrated Formic A c i d S o l u t i o n . 34 I I . S e p a r a t i o n o f Pur ine and P y r i m i d i n e Bases by Paper Chromatography. 35 I I I . The Q u a n t i t a t i v e E s t i m a t i o n of P u r i n e and P y r i m i d i n e Bases Separated by Paper Chromatography. 36 Procedure 1. ( E x t r a c t i o n Method) . 38 Procedure 2 . (The Column E x t r a c t i o n Method) 39 Blank A b s o r p t i o n . 40 P u r i f i c a t i o n o f the P a p e r . 44 P u r i f i c a t i o n o f Glass Wool . 44 The D e t e c t i o n o f Amino A c i d Contaminat ion of DNA H y d r o l y s a t e s by Paper Chromatography. 5 2 The P h y s i c a l C h a r a c t e r i z a t i o n o f DNA. 5 3 I . The D e t e r m i n a t i o n o f £(p) Va lues o f DNA P r e p a r a t i o n s . 53 I I . The D e t e r m i n a t i o n of I n t r i n s i c V i s c o s i t y of DNA P r e p a r a t i o n s . 54 R e s u l t s and D i s c u s s i o n 5 7 N i t r o g e n and Phosphorous Content o f DNA P r e p a r a t i o n s . °3 The D e t e c t i o n o f P r o t e i n I m p u r i t i e s i n DNA P r e p a r a t i o n s by Paper Chromatography. 68 v i i Page The Ni t rogenous Base Composi t ion of DNA P r e p a r a t i o n s . 6 9 The C h a r a c t e r i z a t i o n o f DNA by U l t r a v i o l e t A b s o r p t i o n . 7 5 The V i s c o s i t y of DNA P r e p a r a t i o n s . 8 1 Summary 9 1 B i b l i o g r a p h y 9 5 v i i i LIST OF FIGURES Page 1. C a l i b r a t i o n Curve f o r Phosphorous D e t e r m i n a t i o n . 33 2. The Comparison o f R e c o v e r i e s of Thymine from Paper Chromatograms Us ing Procedure 1 and 2 f o r E l a t i o n . 46 3. The Comparison o f Recover ies of C y t o s i n e from Paper Chromatograms U s i n g Procedure I and 2 f o r E l u t i o n . 46 4. The Comparison o f Recover ies o f Adenine from Paper Chromatograms Us ing Procedure 1 and 2 f o r E l u t i o n . 47 5. The Comparison o f Recover ies o f U r a c i l from Paper Chromatograms Us ing Procedure 1 and 2 f o r E l u t i o n . 47 6. The Comparison of Recover ies of Guanine from Paper Chromatograms Us ing Procedure 2 f o r E l u t i o n . 48 7. C a l i b r a t i o n Curve o f Adenine U s i n g Procedure 2. 49 8. C a l i b r a t i o n Curve o f Guanine Us ing Procedure 2. 49 9. C a l i b r a t i o n Curve of C y t o s i n e U s i n g Procedure 2. 50 10.. C a l i b r a t i o n Curve o f Thymine Us ing Procedure 2. 50 11. C a l i b r a t i o n Curve of U r a c i l Us ing Procedure 2. 51 12. P l o t o f the Reduced V i s c o s i t y ( ~~Z ) Versus C o n c e n t r a t i o n ( g . / l O O m l . ) o f DNA Samples A-16 and A-17. 82 13. P l o t of the Reduced V i s c o s i t y ( — ~ ) Versus C o n c e n t r a t i o n ( g . / l O O m l . ) o f DNA Samples A-12 and A -21 . 83 14. P l o t o f the Reduced V i s c o s i t y ( ') Versus C o n c e n t r a t i o n ( g . / l O O m l . ) o f DNA Samples A-15 and A-18 84 15. P l o t o f the Reduced V i s c o s i t y ( - L _ - ) Versus C o n c e n t r a t i o n ( g . / l O O m l . ) of DNA Samples C - l , C-2 and C - 3 . 8 5 i x LIST OF TABLES Page I . Comparison o f the T h e o r e t i c a l and Exper imenta l ly -Determined N i t r o g e n Contents of D i f f e r e n t S t a n d a r d s . 29 I I . The D e t e r m i n a t i o n o f Phosphorous Content o f P h e n y l Disodium Phosphate Us ing D i f f e r e n t H y d r o l y s i s P r o c e d u r e s . 31 I I I . The Comparison o f T h e o r e t i c a l and E x p e r i m e n t a l l y Found Phosphorous Content of D i f f e r e n t Organic S t a n d a r d s . 31 I V . The Comparison of R f Va lues o f P u r i n e and P y r i m i d i n e Bases i n i s o - p r o p a n o l H y d r o c h l o r i c A c i d S o l v e n t . 37 V . The O p t i c a l D e n s i t y Va lues o f the B lanks o f N u c l e i c A c i d Bases Obtained from Readings at t h e i r A b s o r p t i o n Maxima. 41 V I . Comparison o f % Recover ies o f P u r i n e and P y r i m i d i n e Bases U s i n g Procedure 1 and Procedure 2 f o r E l u t i o n . 42 V I I . Y i e l d s o f DNA from L i v e r o f Rat I s o l a t e d by D i f f e r e n t Methods. 58 V I I I . Y i e l d s o f DNA from S m a l l I n t e s t i n a l Mucosa o f Rat I s o l a t e d by D i f f e r e n t Methods. 59 I X . The N i t r o g e n and Phosphorous Contents of DNA P r e p a r a t i o n s I s o l a t e d by D i f f e r e n t P r o c e d u r e s . 64 X . The Comparison o f the N i t r o g e n Contents o f DNA P r e p a r a t i o n s Determined by the Dumas and K j e l d a h l P r o c e d u r e s . 65 X I . The Comparison o f Amino A c i d Contents of DNA H y d r o l y s a t e s . 70 X I I . D i s t r i b u t i o n of P u r i n e s and P y r i m i d i n e s i n DNA. 74 X Page X I I I . The D i f f e r e n c e between the % of Recovery of Ni t rogenous Bases from DNA H y d r o l y s a t e s Us ing the E l u t i o n Technique of Chargaf f ( 3 8 ) and the Column Chromatographic E l u t i o n Method. 7 6 X I V . The U l t r a v i o l e t E x t i n c t i o n C o e f f i c i e n t of DNA P r e p a r a t i o n s Expressed as £f p ) V a l u e s . 7 9 X V . I n t r i n s i c V i s c o s i t i e s and Est imated M o l e c u l a r Weights o f D i f f e r e n t DNA P r e p a r a t i o n s . 8 7 1. Historical Introduction: Among the components of animal and plant cel l the nucleic acids occupy a position of unusual interest because of.their special position within the nucleus with their consequent relation to specific nuclear processes. In this respect deoxyribonucleic acid (DNA) the chief constituent of chromosomes plays a very important role in modern biochemistry. It is now generally realized that DNA is in a l l probability, chiefly responsible for determining the inheritable character-istics of bacterial viruses, bacteria and higher organisms (1) and that, therefore, the question of its structure and function as the genetic carrier must rank as one of the central problems of biology. In order to have a fair idea about the physico-chemical structure and physiological function of a naturally occuring compound, it is necessary to devise pro-cedures by which that compound can be isolated from its natural sources in a state similar to that in which it occures in the living c e l l . In the following section the history of isolation of DNA is reviewed briefly. In 1871 Friedrich Miescher announced the preparation of a material obtained from digest of pus cells . (2) He called the substance "nuclein". The isolation of this material was performed by treating pus cells with dilute hydrochloric acid for a period of weeks and the product was then shaken in a separatory funnel with ether. Part of the 2. s o l i d m a t e r i a l gathered i n the i n t e r f a c e between the ether and water and the second s o l i d l a y e r formed on the bottom of the s e p a r a t o r y f u n n e l . The l a t t e r c o n s i s t e d o f p r a c t i c a l l y pure n u c l e a r m a t e r i a l . A v e r y s i m i l a r substance r e s u l t e d by d i g e s t i n g pus c e l l s w i t h a r t i f i c a l g a s t r i c j u i c e . T h i s substance had p r a c t i c a l l y i d e n t i c a l p r o p e r t i e s w i t h those o f the sediment obta ined by the mechanical method. M i e s c h e r ' s s t u d i e s on t h i s substance convinced him that i t was a complex phosphorous-c o n t a i n i n g a c i d of h i g h molecular w e i g h t . The d i s c o v e r y of M i e s c h e r ' s " n u c l e i n " opened a new chapter i n the h i s t o r y of b i o c h e m i s t r y . A tremendous i n t e r e s t arose and s e v e r a l o ther s c i e n t i s t s cont inued the i n v e s t i g a t i o n s on n u c l e a r m a t e r i a l s o f d i f f e r e n t o r i g i n s . The term " n u c l e i c a c i d " was not used nor was a convenient and g e n e r a l method d e s c r i -bed f o r i t s p r e p a r a t i o n u n t i l the p b u l i c a t i o n o f Altmann i n 1889. (3). The newer methods of p r e p a r a t i o n o f n u c l e i c a c i d s are p r a c t i c a l l y a l l based on that developed by h i m . I n 1899 Neumann (4) p u b l i s h e d a new m o d i f i c a t i o n o f the o r i g i n a l Altmann method. I n t h i s p u b l i c a t i o n the o l d t r a -d i t i o n a l f e a r of u s i n g d r a s t i c methods f o r the s e p a r a t i o n of the n u c l e i c a c i d from the p r o t e i n was abandoned. Neumann's method e s s e n t i a l l y c o n s i s t e d o f hea t ing the minced organs i n a 3% s o l u t i o n o f sodium hydrox ide and p r e c i p i t a t i o n of the n u c l e i c a c i d s not as a sodium s a l t , which was water s o l u b l e , but as a water i n s o -l u b l e barium s a l t . Feulgen (5) and Levene (6,7) in t roduced 3 . f u r t h e r d r a s t i c methods f o r the i s o l a t i o n o f n u c l e i c a c i d s . The chemica l i n v e s t i g a t i o n of n u c l e i c a c i d p r e p a r a -t i o n s from many b i o l o g i c a l sources has demonstrated that they resembled e i t h e r yeast n u c l e i c a c i d and contained a pentose , i d e n t i c a l w i t h D - r i b o s e , or thymus n u c l e i c a c i d and contained a deoxypentose, i d e n t i c a l w i t h 2 - d e o x y - D - r i b o s e . A l t h o u g h i t was once thought (8 ,9) t h a t the pentose n u c l e i c a c i d s (ENA) were c h a r a c t e r i s t i c of p l a n t t i s s u e s , whereas the d e o x y r i b o -n u c l e i c a c i d (DNA) were conf ined to a n i m a l c e l l s , by 1924 an i d e a began to develop (10,11) t h a t both an imal and p l a n t t i s s u e s c o n t a i n DNA and RNA, the former being conf ined to c e l l n u c l e i , w h i l e RNA i s present mainly i n cytoplasm and n u c l e o l i . The t r a d i t i o n a l b e l i e f i n the unusual l a b i l i t y of n u c l e i c a c i d s was r e v i v e d i n 1924. From then on s c i e n t i s t s r e a l i z e d t h a t the n u c l e i c a c i d s i s o l a t e d by the d r a s t i c a l k a l i e x t r a c t i o n s and a c i d p r e c i p i t a t i o n s were p a r t i c u l a r l y degraded and d e n a t u r e d , and d i d not represent at a l l the n a t u r a l s t a t e as they are present i n c e l l s . Methods were devised to avoid exposing the t i s s u e s to a l k a l i or a c i d , and h e a t . A l l p r e p a r a -t i o n s were c a r r i e d out i n the c o l d . These p r e p a r a t i v e procedures f o r i s o l a t i n g DNA from mammalian t i s s u e s can be d i v i d e d i n t o f i v e g r o u p s . 1. E x t r a c t i o n o f t i s s u e s w i t h s t rong s a l t s o l u t i o n and d e p r o t e i n i z a t i o n o f n u c l e o p r o t e i n by s a t u r a t i o n w i t h sodium h chloride. This method was developed by Signer and Schwander ( 1 2 ) using the necleoprotein preparation of Mirsky and P o l l i s t e r ( 1 3 - 1 5 ) . This procedure consists of: a. mechanical mincing of the gland i n molar NaCT with addition of an enzyme i n h i b i t o r (usually sodium c i t r a t e ) . b. extraction of nucleoprotein into molar NaCl. c. p r e c i p i t a t i o n of the nucleoprotein by d i l u t i o n to 0 . 1 5 M sodium chloride. d. s p l i t t i n g of the nucleoprotein into protein and DNA by saturation with sodium chloride. !§. removal of the protein portion by f i l t r a t i o n , f . p r e c i p i t a t i o n of DNA by ethanol. 2 . Extraction of tissues with strong s a l t solution, deproteinization with chloroform - amyl alcohol mixture. This method was e s s e n t i a l l y developed by Gulland, Jordan and T h r e l f a l l ( l 6 ) . The nucleoprotein i s prepared by the method of Mirsky and P o l l i s t e r ( 1 3 - 1 5 ) . The r e s u l t i n g nucleoprotein i s repeatedly treated with a mixture of amyl alcohol and chloroform, and the denatured protein i s sedimented by centrifugation (17). 3 . Extraction of tissues with water and deproteinization either by saturation with sodium chloride or by using detergents. This i s o l a t i o n procedure i s based on the work of Crampton et a l . ( 1 8 ) . k. Extraction of tissues with anionic detergents. This 5 method used to prepare DNA from c a l f thymus, was developed by Kay et a l . ( 1 9 - 2 1 ) . In t h i s method the t i s s u e i s homogenized s e v e r a l t imes w i t h i c e - c o l d p h y s i o l o g i c a l s a l i n e , c o n t a i n i n g 0 . 0 1 M sodium c i t r a t e . The sediment a f t e r c e n t r i f u g a t i o n i s taken up i n 1 . 5 M s o d i u m - c h l o r i d e - 0 . 0 1 M c i t r a t e s o l u t i o n and the p r o t e i n s are p r e c i p i t a t e d by the a d d i t i o n of sodium x y l e n e s u l p h o n a t e . DNA i s p r e c i p i t a t e d w i t h 98% i s o - p r o p y l a l c o h o l . 5 . L i b e r a t i o n of DNA from t i s s u e s by the a c t i o n of c e r t a i n s a l t s o l u t i o n s and p h e n o l . The i s o l a t i o n o f DNA from mammalian t i s s u e s by the phenol method was developed by K i r b y ( 2 1 - 2 3 ) . DNA i s f r e e d from p r o t e i n by the a c t i o n o f phenol and the s a l t s o l u t i o n s . Contaimnat ing RNA i s removed by r i b o n u c l e a s e t r e a t -ment. T h i s method has been f u r t h e r improved by S . K i t (24) and G . P . Georgiev ( 2 5 ) . The above mentioned g e n t l e methods have s e v e r a l d i s -advantages , which can be summarized as f o l l o w s : 1 . Most o f them use r a t h e r l a r g e q u a n t i t i e s o f f r e s h t i s s u e as a s t a r t i n g m a t e r i a l f o r DNA. 2 . They are time consuming and cumbersome. 3 . S p e c i a l p r e c a u t i o n s and m o d i f i c a t i o n s become necessary on a p p l i c a t i o n to a wide v a r i e t y , o f t i s s u e s . 4 . DNA i s o l a t e d from the same t i s s u e by d i f f e r e n t methods does not have e x a c t l y the same p h y s i c a l and chemica l p r o p e r t i e s . 6 . The aim of the present study was to overcome some of these d i f f i c u l t i e s , and f i n d a method s u i t a b l e f o r s m a l l q u a n t i t i e s o f t i s s u e ( 1 - 1 0 g . ) which g i v e s r e l a t i v e l y h i g h y i e l d s o f DNA i n h i g h l y polymerized " n a t i v e " f o r m . I n order to ach ieve t h i s aim s e v e r a l p r e p a r a t i v e procedures were t r i e d on two k i n d s of t i s s u e , namely, l i v e r and mucosa o f s m a l l i n t e s t i n e o f r a t . These t i s s u e s were chosen because the former shows a h i g h degree of metabol i c a c t i v i t y w i t h respec t to n u c l e i c a c i d s and was p a r t i c u l a r l y s t u d i e d i n t h i s l a b o r a t o r y ( 2 6 - 2 8 ) whereas the l a t t e r has a low n u c l e a r / c y t o p l a s m i c r a t i o , i . e . i t i s r i c h i n RNA and other non-nuc lear c o n s t i t u e n t s ( p o l y s a c c h a r i d e s ) . A f t e r the p r e p a r a t i o n of DNA from these t i s s u e s by d i f f e r e n t methods, the i s o l a t e d m a t e r i a l was c h a r a c t e r i z e d by chemica l and p h y s i c a l means i n order to compare the p r o p e r t i e s o f d i f f e r e n t p r e p a r a t i o n s and eva luate the best p o s s i b l e p r o -cedure f o r f u r t h e r i n v e s t i g a t i o n s . Four procedures seemed to be e s p e c i a l l y promis ing and a p p l i c a b l e to r e l a t i v e l y s m a l l q u a n t i t i e s o f t i s s u e s . I . A r a p i d method f o r p r e p a r i n g polymerized DNA developed by Emanuel and C h a i k o f f ( 2 9 ) . These i n v e s t i g a t o r s c la imed that the procedure i s s p e c i a l l y f a v o r a b l e f o r a p p l i -c a t i o n to t i s s u e s hav ing low n u c l e a r / c y t o p l a s m i c r a t i o s . I t i s based on the removal o f n u c l e i from the t i s s u e by means o f c o n t r o l l e d homogenizat ion and subsequent s e p a r a t i o n of n u c l e i from extraneous c e l l u l a r elements i n the homogenate by a b s o r p -t i o n on C e l i t e (diatomaceous e a r t h ) . A r a p i d method f o r p r e p a r a t i o n o f a homogenate w i t h a h i g h y i e l d of n u c l e i i s d e s c r i b e d by these workers ( 3 0 ) . They used a h y d r a u l i c homo-g e n i z e r f o r the c o n t r o l l e d r e l e a s e of c e l l u l a r components from v a r i o u s t i s s u e s . I n the present s tudy no such h y d r a u l i c homo-g e n i z e r was a p p l i e d , but a combined homogenizat ion w i t h a T e f l o n homogenizer and short d i s i n t e g r a t i o n w i t h a S e r v a l l omni-mixer was a t tempted . The absorbed n u c l e i were then d i s p e r s e d w i t h a s t rong s a l t s o l u t i o n (NaBr) and t h e i r DNA was separated from i t s b a s i c p r o t e i n which adheres to the absorbing C e l i t e . The sodium n u c l e a t e were f i l t e r e d o f f or c e n t r i f u g e d from the p r o t e i n - C e l i t e m i x t u r e . S e v e r a l s a l t s a t u r a t i o n and p r e c i p i t a t i o n steps were used f o r f u r t h e r p u r i f i c a t i o n . 2 . The second procedure was based on the o r i g i n a l work of Kay et a l . (20) u s i n g the m o d i f i c a t i o n s a p p l i e d by Stevens and Duggan ( 3 1 ) . In t h i s method the n u c l e i c a c i d s were e x t r a c t e d w i t h sodium d o d e c y l su lphate and sodium x y l e n e sulphonate s o l u t i o n c o n t a i n i n g some e t h y l e n e - d i a m i n e - t e t r a -ace ta te (Versene) i n order to i n h i b i t any deoxyr ibonuc lease a c t i o n . T h e i r p r o t e i n was removed by the detergent treatment combined w i t h adjustment of the pH o f the s o l u t i o n to somewhat lower v a l u e s (pH 4 . 3 ) . A f t e r r e a d j u s t i n g the pH o f the p r o t e i n -f r e e sodium n u c l e a t e s o l u t i o n RNA and DNA were separated by 8 . f r a c t i o n a l p r e c i p i t a t i o n w i t h i s o - p r o p y l a l c o h o l . 3 . The methods o f Emanuel and Chai l teof f and Kay et a l . ( 2 9 , 2 0 ) were combined i n t h i s procedure . The n u c l e i were i s o l a t e d a c c o r d i n g to Emanuel and C h a i k o f f , crude DNA was obta ined by d i s r u p t i n g n u c l e i w i t h s a l t s a t u r a t i o n and t h i s crude product was f u r t h e r p u r i f i e d by the prev ious d e t e r -gent method. 4. The f o u r t h procedure was based on the work of Zubay ( 3 2 ) . In t h i s method the t i s s u e s were d i s i n t e g r a t e d by h i g h f requency sound, the n u c l e a r fragments were separated by c e n t r i f u g a t i o n and the n u c l e o p r o t e i n was e x t r a c t e d w i t h s t rong s a l t s o l u t i o n ( 1 3 - 1 5 ) . DNA was d e p r o t e i n i z e d a c c o r d i n g to the procedure o f Sevag et a l . ( 1 7 ) . Zubay a p p l i e d h i s method to a number o f mouse t i s s u e s ( s p l e e n , l i v e r , lymphoma) u s i n g v e r y s m a l l q u a n t i t i e s of s t a r t i n g m a t e r i a l s ( 1 - 3 g . ) . Al though s e v e r a l workers found ( 3 3 - 3 5 ) t h a t ultrasoundvwav.es are capable of damaging DNA i n s o l u t i o n s by caus ing the d i s r u p t i o n of hydrogen bonds i n the DNA molecule and thus degrading i t Into s m a l l e r fragments having lower molecular weight than the o r i g i n a l n a t i v e DNA, Zubay claimed that when DNA i s i n the form of n u c l e o p r o t e i n and i n s o l u t i o n , i t i s w e l l protec ted from t h i s damaging e f f e c t . He f u r n i s h e d some experiment a l l e v i d e n c e , showing that DNA p r e p a r a t i o n s by h i s method had v e r y h i g h i n t r i n s i c v i s c o s i t i e s a l though he admitted that DNA p r e p a r a t i o n s of v i r a l o r i g i n l o s t t h e i r t r a n s -forming a c t i v i t i e s . 9. I n order to o b t a i n a DNA p r e p a r a t i o n which presumably y i e l d s v e r y degraded and denatured DNA and thereby enable the comparison o f the p r o p e r t i e s o f a degraded m a t e r i a l w i t h those of n a t i v e ones the procedure of Bendich et a l . ( 3 6 ) and Tyner et a l . ( 3 7 ) was t r i e d . This procedure i s based on the e x t r a c t -i o n o f t i s s u e s w i t h hot ( 8 5 ° C ) 10% sodium c h l o r i d e f o r s e v e r a l h o u r s , p r e c i p i t a t i o n o f n u c l e i c a c i d s w i t h a l c o h o l , s e p a r a t i o n of RNA from DNA by i n c u b a t i n g the p r e c i p i t a t e w i t h 0.1 N NaOH, and f i n a l l y r e p r e c i p i t a t i o n of DNA from the b a s i c mixture w i t h concentrated H C I . Thus the procedure seemed to be d r a s t i c enough f o r y i e l d i n g denatured DNA. C h a r a c t e r i z a t i o n o f DNA P r e p a r a t i o n s . At present i t i s not known whether even a most c a r e f u l l y I s o l a t e d DNA can i n a l l r e s p e c t s be i d e n t i c a l w i t h the DNA as i t e x i s t e d i n the l i v i n g c e l l . S t r i c t l y speaking no compound, once i t i s i s o l a t e d from the c e l l , can be c o n s i d e r -ed as n a t i v e . However the s e r i e s o f d e g r a d a t i v e changes to which i t may be exposed, i n the course of i t s i s o l a t i o n w i l l u s u a l l y be g r a d u a l , and w h i l e i t may not yet be p o s s i b l e to d e f i n e the p e r f e c t compound, the b a d l y degraded one can be e a s i l y r e c o g n i z e d . ( 3 8 ) There are a c t u a l l y three main methods by which DNA can be c h a r a c t e r i z e d ; I . Chemical methods based upon the d e t e r m i n a t i o n of DNA c o n t e n t , base c o m p o s i t i o n , and n i t rogen-phosphorous 10. contents of the preparation. 2. Physical methods by which the i n t e g r i t y of macromolecular state of DNA can be defined. 3. The determination of i t s b i o l o g i c a l a c t i v i t y (39). One b i o l o g i c a l a c t i v i t y which can be demonstrated i n ce r t a i n DNA preparations i s th e i r transforming a c t i v i t y . Unfortunately at present Only a few b a c t e r i a l DNAs lend themselves tothe assay for transforming a c t i v i t y . During the course of t h i s study chemical and physical methods were used to compare the properties of DNAs prepared by d i f f e r e n t procedures from the same source to decide whether the compound i s i n native, undegraded state. 1. The Chemical Characterization of the DNA Preparations. The chemical composition of DNA i s less l i k e l y to change on mild denaturation. A very good i n d i c a t i o n can be obtained, however, by chemical means about the degree of purity of such preparations. The investigator may encounter three very important impurities i n DNA samples; the presence of RNA, proteins and polysaccharides. For example the presence of protein can be evaluated from the N/P r a t i o of the DNA pre-paration. Owing to the several s t r u c t u r a l r e g u l a r i t i e s i n a l l DNAs, the N and P contents are c l o s e l y similar for d i f f e r e n t preparations from d i f f e r e n t sources. The N/P value calculated on the basis of Wat son-Crick (40) model of DNA i s 1 .65. Any 11 . h igher v a l u e than t h i s would s u r e l y i n d i c a t e p r o t e i n contami-n a t i o n . Therefore n i t r o g e n and phosphorous d e t e r m i n a t i o n s were c a r r i e d out on the I s o l a t e d DNA samples, u s i n g the micro K j e l d a h l method (41) f o r N and the c o l o r i m e t r i c procedure of B a r t l e t t (42) f o r P e s t i m a t i o n s . S i m i l a r l y p r o t e i n contaminat ion can be demonstrated by the complete h y d r o l y s i s of samples f o l l o w e d by paper c h r o -matography of the h y d r o l y s a t e f o r f r e e amino a c i d s . Only q u a l i t a t i v e i d e n t i f i c a t i o n of amino a c i d s was attempted i n t h i s s tudy ( 4 3 , 4 4 ) . One o f the most important chemica l c h a r a c t e r i z a t i o n o f DNA i s the d e t e r m i n a t i o n o f I t s base c o m p o s i t i o n . By the year 1930 i t was d e f i n i t e l y known t h a t DNA on h y d r o l y s i s y i e l d e d phosphoric a c i d , a sugar (deoxyr ibose) and f o u r n i t r o g e n o u s bases namely; adenine , guanine , c y t o s i n e and thymine ( 3 8 ) . The e a r l y work of Levene and Jones suggested t h a t DNA prepared by e x t r a c t i o n w i t h a l k a l i was composed o f e q u i v a l e n t p r o p o r t i o n s o f the four n u c l e o t i d e s d e r i v e d from adenine , guanine , c y t o s i n e and thymine . When i t was recognized t h a t the n u c l e i c a c i d s could be obta ined i n the form of p a r t i c l e s of extremely h i g h mole-c u l a r w e i g h t , the t e t r a n u c l e o t i d e h y p o t h e s i s had to be m o d i f i e d . The development o f the methods o f chromatography encouraged the a p p l i c a t i o n of s i m i l a r techniques f o r the quan-t i t a t i v e d e t e r m i n a t i o n o f the product formed on c leavage o f the 12... nucleic acids. It was found that from hydrolysates of DNAs the purine and pyrimidine bases could be easily separated by paper chromatographic methods using different solvent systems ( 3 8 ) . [ Several procedures for hydrolysing DNA can be used. The most suitable for quantitative measurements appears to be the formic acid procedure originally developed by Vischer and Chargaff (45). A convenient solvent for separating the purine and pyrimidine bases on paper chromatograms was recommended by Myatt (46). The description of an arrangement permitting the easy demonstration of the purine and pyrimidine spots and the application of a commercially available ultraviolet lamp (47) facilitated the performance of analyses. For quantitative estimation spots were ;.cut out from the chromatograms add eluted by soaking them in a given volume of 0 . 1 N H C 1 . According to Chargaff (38) i f this elution is allowed to proceed overnight at room temperature, with shaking at the beginning and at the end it is quantitative. It was found in this laboratory however, (48) that this elution technique Is not necessarily quantitative, even with several (three-times) extractions of spots with aliquot portions of 0 . 1 N H C 1 . Therefore a new elution procedure was tried (49) and compared with the old technique. This method wil l be described and discussed later. The application of the above mentioned paper 1 3 chromatographic method toDNA p r e p a r a t i o n s of d i f f e r e n t c e l l u l a r o r i g i n soon demonstrated s i g n i f i c a n t chemica l d i f f e r e n c e s between these compounds ( 5 0 - 5 3 ) . Thus the d e t e r m i n a t i o n of base c o m p o s i t i o n proved to be a u s e f u l mean to c h a r a c t e r i z e DNA p r e p a r a t i o n s s p e c i a l l y w i t h respect to the p u r i t y o f such samples . One of the most troublesome i n p u r i t i e s i n DNA i s the presence of RNA as a l r e a d y mentioned above. The paper c h r o -matographic a n a l y s i s o f DNA h y d r o l y s a t e s would s u r e l y i n d i c a t e the presence of any such i m p u r i t y . Non-nuclear contaminants such as p o l y s a c c h a r i d e s or s a l t s could be a l s o e a s i l y demonstrated by the d e t e r m i n a t i o n of the DNA content o f the samples. Most o f these e s t i m a t i o n s are based however, on the d e t e r m i n a t i o n of the deoxyr ibose content o f the m a t e r i a l o r on spectophotometr ic e s t i m a t i o n s , compared w i t h t h a t o f a " r e l i a b l e " standard ( 3 8 ) . No such r e l i a b l e r e f e r e n c e standard n u c l e i c a c i d p r e p a r a t i o n i s a v a i l -ab le at p r e s e n t , t h e r e f o r e d i r e c t DNA e s t i m a t i o n s were not p e r -formed d u r i n g t h i s s t u d y . I I . Physdco-Chemical C h a r a c t e r i z a t i o n o f DNA. The c r i t e r i a of i n t e g r i t y o f a macromolecular substance o f n a t u r a l o r i g i n are not easy to d e f i n e , but as regards to DNA c e r t a i n f e a t u r e s can be d e s c r i b e d . 1 . I t has a v e r y h i g h and, w i t h i n the s p e c i e s u n i f o r m 1> molecular weight as shown by d i f f u s i o n experiments (5V - 56), sedimentation i n the ultracentrifuge (55? 56), and determination of v i s c o s i t y and streaming birefringance. (57). 2. The character of monodispersity i s l o s t even when the specimen i s prepared under very mild conditions i f p a r t i a l enzymatic attack i n the course of i s o l a t i o n i s not avoided. 3. Both the value and the uniformity of the mole-cular weight are affected i f the i s o l a t i o n i s carried out under degradative conditions (58) or i f the preparation i s exposed subsequently !to degradation by chemical or physical means. h. It has an anomalous amphoteric behavior on t i t r a t i o n . 5. The molecules posses a high and stable asymmetry. Solutions of undegraded DNA exhibit double r e f r a c t i o n of flow (59) and very considerable v i s c o s i t y . 6. Undegraded DNA shows a t y p i c a l "hyperchromic" e f f e c t i n solution. The following two c h a r a c t e r i s t i c physico-chemical properties were studied during t h i s experiment: (a) The Hyperchromic E f f e c t of DNA Solutions. The depolymerization or denaturation of nucleic acids i s associated with i n t e n s i f i c a t i o n of their absorption of u l t r a v i o l e t l i g h t . In other words the e x t i n c t i o n of in t a c t 1 5 . p r e p a r a t i o n s i s lower than would correspond to the sum of t h e i r c o n s t i t u e n t mononucleot ides . T h i s e f f e c t a p p l i e s to RNA, DNA and s y n t h e t i c p o l y n u c l e o t i d e s . The i n t e n s i f i c a t i o n brought about by deoxyr ibonuc lease on DNA was f i r s t d e s c r i b e d by K u n i t z ( 6 0 ) . S i m i l a r e f f e c t s produced by a c i d , h e a t , a l k a l i or the a d d i t i o n of s a l t s have been s tudied i n g r e a t e s t d e t a i l by Thomas ( 6 1 ) and more r e c e n t l y by Schack ( 6 2 ) . A c c o r d i n g to Chargaf f et a l . ( 3 8 ) the e x t i n c t i o n o f the maximum i s almost constant f o r d i f f e r e n t DNA p r e p a r a t i o n s . When the e x t i n c t i o n at the maximum and at pH 7 i s expressed as the atomic e x t i n -c t i o n c o e f f i c i e n t w i t h respect to phosphorous and designated as ( 6 3 ) , p r e p a r a t i o n s i s o l a t e d c a u t i o u s l y from a l a r g e v a r i e t y o f sources w i l l show s u r p r i s i n g l y l i t t l e d ivergence from the v a l u e of _ 6 6 0 0 . A c c o r d i n g to Chargaf f ( 3 8 ) an«- €(p^ v a l u e h igher than about 7 2 0 0 i s a s i g n o f d e n a t u r a t i o n of DNA sample. (b) The Anomalous V i s c o s i t y o f DNA S o l u t i o n . P h y s i c o - c h e m i c a l s t u d i e s on DNA are l a r g e l y concerned w i t h a number o f p r o p e r t i e s a s s o c i a t e d w i t h the n a t i v e hydrogen bonded macromolecular s t a t e of the n u c l e a t e (40 ) . The h i g h l y anomalous v i s c o s i t y of DNA has been sub jec t o f c o n s i d e r a b l e s t u d y . The sthiking e f f e c t of e l e c t r o l y t e s ( 5 9 , 6 4 ) and of a c i d and a l k a l i ( 5 9 , 5 4 ) i n r e d u c i n g the v i s c o s i t y of DNA s o l u t i o n s p r o v i d e important i n f o r m a t i o n w i t h respec t to i t s p h y s i c o -chemica l s t a t e . The d e s t r u c t i o n of the secondary s t r u c t u r e 16 of DNA by hea t , a c i d or a l k a l i e s at constant molecular weight produces a p a r t i c l e p r o c e s s i n g a g r e a t l y decreased (Newtonian) v i s c o s i t y . In order to perform a measurement which i s meaningful ^ o - v i s c o s i t y of s o l v e n t must be measured i n s o l u t i o n s which are d i l u t e enough to a l l o w independent molecular motions and then e x t r a p o l a t e d to zero i n the low c o n c e n t r a t i o n range , and the e x t r a p o l a t e d v a l u e i s c a l l e d the i n t r i n s i c v i s c o s i t y . where c • number o f grams o f polymer i n 100 m l . o f s o l u t i o n . The i n t r i n s i c v i s c o s i t y may be used as a q u a l i t a t i v e measure of the molecular s i z e and presumably o f the molecular w e i g h t . S i n c e the DNA molecule i n s o l u t i o n i s probab ly not a s imple l i n e a r polymer but a s t rand composed of two p o l y n u c l e o t i d e c h a i n s , one would expect no s imple r e l a t i o n s h i p between the i n t r i n s i c v i s c o s i t y and the extent o f d e g r a d a t i o n . However, f o r a q u a l i t a t i v e comparison of d i f f e r e n t p r e p a r a t i o n s the i n t r i n s i c v i s c o s i t y p r o v i d e s a s e n s i t i v e measure o f d i f f e r e n c e s . Moreover s i n c e the v i s c o s i t y i s a m o n o t o n i c a l l y d e c r e a s i n g f u n c t i o n of the g r a d i e n t , the L ^ ] ] measured at some higher more c ( 6 5 ) c o n c e n t r a t i o n . P l o t s o f 1 7 . e x p e r i m e n t a l l y a c c e s s i b l e average g r a d i e n t ( c a . 1 0 0 0 sec."'*') can a l s o be u t i l i z e d to q u a l i t a t i v e l y detec t changes i n molecular c o n f i g u r . a t i o n ( 3 9 ) . One of the e s s e n t i a l parameters which c h a r a c t e r i z e s a h i g h molecular weight polymer compound such as DNA i s i t s molecular w e i g h t . The e s t i m a t i o n of the l a t t e r i s a d i f f i c u l t problem s o l v i n g o f which i n v o l v e s complicated apparatus such as f o r i n s t a n c e a n a l y t i c a l u l t r a c e n t r i f u g e , setups f o r d e t e r -m i n a t i o n o f the c o e f f i c i e n t . o f d i f f u s i o n and the measurement o f l i g h t s c a t t e r i n g . In a d d i t i o n to t h e s e , f o r a s e r i e s of h i g h molecular weight compounds the molecular weight has been determined by means of u t i l i z a t i o n of the simple v i s c o s i m e t r i c t e c h n i q u e . The f o l l o w i n g e q u a t i o n expresses the r e l a t i o n between i n t r i n s i c v i s c o s i t y and the molecular weight of the h i g h polymer ( 6 5 ) : l/y\] . KM ^ equ. ( 3 ) where M = molecular weight of h i g h polymer . The c o n s t a n t s ^ - and K depend upon the type o f polymer, the s o l v e n t , and the temperature of the v i s c o s i t y d e t e r m i n a t i o n . The v a l u e s o f K and ^ are known f o r s e v e r a l s y n t h e t i c a l polymers . U t i l i z i n g equat ion ( 3 ) . S p l t k o v s k i i suggested a s i m i l a r formula f o r the d e t e r m i n a t i o n of the molecular weight o f DNA ( 6 7 ) : 0]1 - 3 3 . 2 2 x 1 0 " 4 M ° - 6 1 6 equ.(4) The v a l u e s f o r oC = 0 . 6 1 6 and K = 3 3 . 2 2 x I f f 4 were d e r i v e d by him from t h e o r e t i c a l c o n s i d e r a t i o n s . U s i n g a s e r i e s of da ta 18. of molecular weight and the corresponding c h a r a c t e r i s t i c v i s c o s i t i e s found i n the l i t e r a t u r e , S p l t k o v s k i i was ab le to prove the v a l i d i t y o f h i s e q u a t i o n . A v e r y s a t i s f a c t o r y agreement was found between the da ta i n l i t e r a t u r e and mole-c u l a r w e i g h t s , c a l c u l a t e d a c c o r d i n g to equat ion ( 4 ) . Thus h i s v i s c o s i m e t r i c method seemed to be q u i t e promis ing f o r o b t a i n i n g e s t i m a t i o n s of the molecular weight of DNA p r e p a r -a t i o n s i s o l a t e d d u r i n g t h i s s t u d y . 1 9 EXPERIMENTAL The P r e p a r a t i o n of DNA from S m a l l I n t e s t i n a l Mucosa and L i v e r  of Rat by D i f f e r e n t Procedures . I . I s o l a t i o n of DNA by the Method o f Emanuel and  C h a i k o f f ( 2 9 ) . Reagents ; C e l i t e ( J o h n s - M a n v i l l e C o . ) Saturated Sodium Bromide S o l u t i o n Potass ium Arsenate S o l u t i o n A 0 . 2 M s o l u t i o n of potassium dihydrogen arsenate was prepared by d i s s o l v i n g 3 .6 g . o f potassium dihydrogen arsenate ( t e c h n i c a l grade) i n 100 m l . d i s t i l l e d water and the pH was ad justed to 7 by t i t r a t i o n w i t h concentrated potassium hydrox ide s o l u t i o n and t h i s n e u t r a l s o l u t i o n was then d i l u t e d w i t h water u n t i l i t s m o l a r i t y was 0 .014 . Ho mo g en i z at i o n Medium. 1 1 . 5 g . o f s o l i d potassium c h l o r i d e was d i s s o l v e d i n 1000 m l . o f 0.014 M potassium dihydrogen arsenate s o l u t i o n and the mixture was d i l u t e d to 2000 m l . T h i s y i e l d e d a medium which was 0 . 0 0 7 M i n arsenate i o n and 0.038 M i n c h l o r i d e i o n . The purpose o f adding arsenate i o n i n the homogenizat ion medium was to i n h i b i t deoxyr ibonuc lease a c t i o n d u r i n g i s o l a t i o n procedure . P r o c e d u r e ; The p r e p a r a t i o n o f t i s s u e s ; 20 V/istar r a t s weighing 200 g each, from the co lony o f B r i t i s h Columbia were used i n the experiments. . The animals were k i l l e d by blow on the head and d e c a p i t a t e d . The l i v e r was removed and f r o z e n by dropping i t i n t o l i q u i d n i t r o g e n . The s m a l l i n t e s t i n e was removed and cut i n t o 10 cm. segments which were f l u s h e d f r e e of contents w i t h the c o l d homogenizat ion medium. The segments were s p l i t open, a p p l i e d to a c h i l l e d g l a s s p l a t e . The mucosal e p i t h e l i u m was then scraped from the m u s c u l a r i s w i t h the edge o f microscope s l i d e and placed i n l i q u i d n i t r o g e n . Both t i s s u e s were s tored at -15°C. P r o c e d u r e . The t i s s u e s were homogenized w i t h 40 m l . arsenate b u f f e r f o r 10 minutes w i t h a g l a s s t i s s u e g r i n d e r . This homogenizer c o n s i s t e d o f a p i s t o n - t y p e T e f l o n p e s t l e and a g r i n d i n g v e s s e l o f Pyrex brand g l a s s , and w i l l be r e f e r r e d h e r e a f t e r as T e f l o n homogenizer. The homogenizat ion was cont inued f o r another two minutes i n a S e r v a l l Omni M i x e r . The mixture was then f i l t e r e d through a s i n g l e l a y e r of m u s l i n c l o t h . The r e t a i n e d connec t ive t i s s u e was washed w i t h 20 m l . of homogenizat ion medium. The f i l t r a t e s were c o l l e c t e d i n a beaker immersed i n i c e , 4 .2 g . o f d r y C e l i t e was added and the mixture was s t i r r e d at h i g h speed f o r about 10 seconds w i t h a magnetic s t i r r e r . The mixture was poured i n t o a 10 cm. cooled Buchner f u n n e l c o n t a i n i n g a f i l t e r paper o v e r l a i d X w i t h 2 .9 g . o f C e l i t e which had been suspended i n water and 21 sucked v e r y d r y . The pad was washed w i t h c h i l l e d homogenizat ion medium u n t i l the f i l t r a t e was c o l o r l e s s , then .-another 100 m l . > o f t h i s s o l u t i o n was sucked through the pad . As a r u l e a t o t a l o f 300 m l . o f s o l u t i o n s u f f i c e s to remove non-nuc lear m a t e r i a l s . The C e l i t e pad w i t h i t s adsorbed n u c l e i was l i f t e d out o f the f u n n e l and s u f f i c i e n t sa tura ted sodium bromide s o l u t i o n (15-20 m l . ) was added to i t w i t h s t i r r i n g . Dur ing the s t i r r i n g a s m a l l amount o f s o l i d sodium bromide was added to i n s u r e s a t u r a t i o n of the mixture w i t h the s a l t . The suspension was next f i l t e r e d on a 5 .5 cm. Buchner f u n n e l and the f i l t r a t e was c o l l e c t e d i n a s u c t i o n f l a s k . Two sheets o f Whatman No. 1 f i l t e r paper o v e r l a i d w i t h 2mm. o f C e l i t e were used f o r f i l t -r a t i o n . To remove r e s i d u a l n u c l e i c a c i d s , the pad was washed 3 t imes w i t h 4 m l . p o r t i o n o f c h i l l e d sa tura ted sodium bromide s o l u t i o n . The v e r y v i s c o u s c l e a r f i l t r a t e was d i l u t e d then w i t h one part water to f i v e part o f f i l t r a t e . Whi le the s o l u t i o n was being s w i r l e d by hand, 2 or 3 volumes of 95% e t h a n o l were added to i t and the p r e c i p i t a t e d f i b r o u s DNA was removed w i t h a g l a s s r o d . The crude DNA was d i s s o l v e d i n 20 m l . o f homogenizat ion medium. The mixture was s t i r r e d u n t i l a l l o f the n u c l e i c a c i d s d i s s o l v e d . 0 . 5 g . C e l i t e was added to the s o l u t i o n , f o l l o w e d by s o l i d sodium bromide s a t u r a t i o n . The mixture was c e n t r i f u g e d , the c l e a r supernatant was d i l u t e d w i t h one par t o f water to each f i v e par t o f s o l u t i o n and DNA was p r e c i p i t a t e d w i t h 2 or 3 volumes of e t h a n o l (95%). The 22 f i b r o u s DNA was washed s e v e r a l t imes w i t h 7 5 $ e t h a n o l then d r i e d i n vacuo at room temperature over phosphorous p e n t o x i d e . D r i e d DNA was then s tored i n a vacum d e s s i c a t o r at - 15°C. I I . I s o l a t i o n of DNA by the method of Kay et a l . (20) m o d i f i e d by Stevens and Duggan ( 3 D . Reagents? S o l u t i o n A . Arsenate b u f f e r , (descr ibed on p a g e ' ^ ) S o l u t i o n B . Sodium l a u r y l su lphate - Versene s o l u t i o n . 2 g . sodium d o d e c y l su lphate - 8 m l . 5 M Versene s o l u t i o n . S o l u t i o n C . Ace ta te b u f f e r ( pH 7 . ) Anhydrous sodium ace ta te 1 2 . 3 g . A c e t i c a c i d 0 . 4 m l . D i s t i l l e d water up to 5 0 0 m l . A d j u s t pH to 7 . 0 . S o l u t i o n D . Ace ta te b u f f e r - Versenate s o l u t i o n 0 . 0 1 M. 0 . 3 7 2 g . Versene i n 100 m l . ace ta te b u f f e r . S o l u t i o n E . Versene , potassium c h l o r i d e , Sodium Xylene S u l p h o n -ate s o l u t i o n . (Nease Chem. Comp.) 10 m M Versene 0 . 3 7 2 g . 0 . 2 M potassium c h l o r i d e 1.492 g . 12 % sodium x y l e n e sulphonate 1 2 . 0 0 g . d i s t i l l e d water up to 100 m l . T h i s s o l u t i o n should be kept i n r e f r i g e r a t o r . S o l u t i o n E. 0 . 0 5 M Versene s o l u t i o n 18.6 g . Versenate/1000 m l . d i s t i l l e d w a t e r . 23 S o l u t i o n G. 0 . 0 1 M Versene s o l u t i o n . 3 . 7 2 g . V e r s e n e / 1000 m l . d i s t i l l e d w a t e r . S o l u t i o n H . 80% w/v sodium x y l e n e sulphonate s o l u t i o n . P r e p a r a t i o n of t i s s u e s : L i v e r s and i n t e s t i n a l mucosa were obta ined as d e s c r i b e d p r e v i o u s l y from r a t s fed ad l i b i t u m . P r o c e d u r e : The t i s s u e was blended f o r two minutes i n t e n m l . o f s o l u t i o n B . The r e s u l t i n g g e l r e c e i v e d a l i t t l e to luene f o r p r e s e r v a t i v e . The pH was ad justed to approx imate ly 7 . 5 w i t h ammonium hydrox ide u s i n g pH paper . The g e l was s tored o v e r n i g h t at room temperature . Four volumes o f c h i l l e d s o l u t i o n E was added, and a f t e r 3 0 second b l e n d i n g i n i c e ba th the mixture was t r a n s f e r r e d to a beaker i n an i c e b a t h . The pH was ad justed to 4.3 w i t h g l a c i a l a c e t i c a c i d . The p r o t e i n detergent r e s i d u e was d i s c a r d e d , a f t e r c e n t r i f u g i n g the mixture at 4 5 0 0 RPM f o r 4 5 minutes at 0 °C. The pH of the supernatant was ad justed to 7 . 0 w i t h ammonium hydrox ide s o l u t i o n , and was s t i r r e d d u r i n g the a d d i t i o n o f 90% i s o - p r o p y l < a l c o h o l . The p r e c i p i t a t e d n u c l e i c a c i d s were recovered by c e n t r i f u g a t i o n . Crude n u c l e i c a c i d s were d i s s o l v e d i n 5 - 1 0 m l . o f s o l u t i o n F and an equal volume of s o l u t i o n H was added. The mixture was s t i r r e d f o r an h o u r , and 4 volumes of s o l u t i o n G. were added w i t h cont inous s t i r r i n g . Whi le the s o l u t i o n was c h i l l e d i n an i c e bath the pH was ad justed to 4.3 w i t h g l a c i a l a c e t i c a c i d . 24 The p r o t e i n r e s i d u e was c e n t r i f u g e d f o r 6 0 minutes at 4°C at 1 6 5 0 0 RPM and the pH of the c l e a r supernatant was then ad justed to pH 7 . 0 w i t h ammonium h y d r o x i d e . An equal volume o f 90% i s o - p r o p y l a l c o h o l was added. The n u c l e i c a c i d r e s i d u e was recovered by c e n t r i f u g a t i o n at 4 °C . The r e s i d u e was d i s s o l v e d i n 3 to 5 m l . of s o l u t i o n D and s l o w l y i s o - p r o p y l a l c o h o l was added to a f i n a l c o n c e n t r a t i o n o f 30% ( v / v ) . The p r e c i p i t a t e d RNA was d i s c a r d e d , a f t e r c e n t r i f u g a t i o n . To the c l e a r supernatant i s o - p r o p y l a l c o h o l was added to a f i n a l c o n c e n t r a t i o n of 34 to 40% v / v . The p r e c i p i t a t e d DNA was recovered by c e n t r i f u g a t i o n and the DNA p r e c i p i t a t e was washed w i t h 95% e t h a n o l , then d r i e d and s t o r e d as d e s c r i b e d on page 2.2. I I I . I s o l a t i o n o f DNA by the Combined Methods o f Emanuel and  C h a i k o f f (29) and Kay et a l . ( 2 0 . 3 1 ) . Procedure ; I t was f e l t , that a s u i t a b l e procedure could be obta ined by combining c e r t a i n d e s i r a b l e f e a t u r e s of the methods o f Emanuel and C h a i k o f f and Kay e t . a l . ( 2 9 , 3 1 ) . The procedure o f Emanuel and C h a i k o f f was f o l l o w e d u n t i l the f i r s t p r e c i -p i t a t i o n o f DNA was o b t a i n e d , then crude DNA was d e p r o t e i n i z e d a c c o r d i n g to the method of Kay et a l . and Stevens et a l . ( 2 0 , 3 1 ) I V . I s o l a t i o n o f DNA by the Method o f Zubay (32 ) . 25 Reagents : S o l u t i o n A . Arsenate b u f f e r (descr ibed on page W ) S o l u t i o n B . N e u t r a l s a l i n e - Versene s o l u t i o n 0 . 1 5 M sodium c h l o r i d e 8 . 7 7 7 g. 0 . 0 1 M e thylenediamine t e t r a a c e t i c a c i d N a . 3 . 7 2 g . D i s t i l l e d water up to 1 0 0 0 m l . S o l u t i o n C . 0 . 1 5 M sodium c h l o r i d e s o l u t i o n 8 . 7 7 g . N a C l / 1 0 0 0 m l . d i s t i l l e d water S o l u t i o n D . 3 M sodium c h l o r i d e s o l u t i o n 1 7 5 . 3 5 g . sodium c h l o r i d e / 1 0 0 0 m l . d i s t i l l e d water S o l u t i o n E . Chloroform - amyl a l c o h o l mixture Chloroform 3 p a r t s Amyl a l c o h o l 1 p a r t P r e p a r a t i o n o f T i s s u e s : S i x male r a t s were s tarved and t h e i r l i v e r s and i n t e s t i n a l mucosa were obta ined and f r o z e n as d e s c r i b e d on page 2o . These t i s s u e s were used f o r the i s o l a t i o n o f DNA by procedures IV and V . P r o c e d u r e ; S t a r t i n g m a t e r i a l : 3 g . o f i n t e s t i n a l mucosa and 6 g . o f l i v e r The t i s s u e was homogenized s h o r t l y ( 1 0 - 2 0 s t r o k e s ) w i t h a T e f l o n homogenizer i n a f i n a l t o t a l volume of about 6 0 m l . The samples o f c e l l suspens ion were exposed to sonic i r r a d i a t i o n 2 6 i n a Raytheon s o n i c o s c i l l a t o r ( 9 k . c y c l e s / s e c ) f o r 1 . 5 minute . The n u c l e a r fragments obta ined a f t e r d i s i n t e g r a t i o n o f t i s s u e s were washed 6 - 8 t imes w i t h 6 0 ml. s o l u t i o n B l by a l t e r n a t e c e n t r i f u g a t i o n f o r 8 minutes at 3 5 0 0 RPM i n a S e r v a l l r e f r i g e r a t e d c e n t r i f u g e . The washed n u c l e a r fragments were resuspended i n 4 5 m l . o f c o l d s o l u t i o n G. i n a S e r v a l l t o p - d r i v e Omni Mixer and r u n at about 3 0 0 0 r e v / m i n u t e . 9 0 ml. of s o l u t i o n D was at once added and b l e n d i n g was cont inued f o r 2 0 minutes at 3°C and at a same low speed. The d i s s o c i a t e d s o l u t i o n was then e m u l s i f i e d up to 8 t imes s u c c e s s i v e l y , w i t h 3 0 - 4 5 ml. o f Chloroform - amyl a l c o h o l m i x t u r e , each e m u l s i -f i c a t i o n was c a r r i e d out i n a v i b r a t o r y shaker f o r 1 0 minutes and was then a l t e r n a t e d w i t h c e n t r i f u g a t i o n f o r 1 0 minutes at 4 5 0 0 RPM on S e r v a l l c e n t r i f u g e . When no f u r t h e r i n t e r f a c i a l f i l m of p r o t e i n was produced by e r a u l s i f i c a t i o n , the s o l u t i o n was d i l u t e d w i t h an equal volume o f water and DNA p r e c i p i t a t e d by a d d i t i o n of equal volume of e t h a n o l . T h e . p r e c i p i t a t e d DNA was d i s s o l v e d a g a i n i n minimum amount of s o l u t i o n G and c e n t r i f u g e d i f necessary to o b t a i n a c r y s t a l c l e a r s o l u t i o n . DNA was p r e c i p i t a t e d from the supernatant as p r e v i o u s l y . DNA was washed d r i e d and stored as d e s c r i b e d on page 22 . V . I s o l a t i o n of DNA by the Methods o f Bendich et a l . ( 3 6 )  and Tyner et a l . ( 3 7 ) . 2 7 Reagents : S o l u t i o n A . 10% sodium c h l o r i d e s o l u t i o n S o l u t i o n B . 5% sodium c h l o r i d e s o l u t i o n S o l u t i o n C . A p p r o x i m a t e l y 0 . 1 N sodium hydrox ide s o l u t i o n S o l u t i o n D . Ether (anhydrous) P r o c e d u r e ; 3 g . of f r e s h t i s s u e was homogenized s h o r t l y ( 1 0 - 2 0 s t r o k e s ) w i t h a T e f l o n homogenizer, w i t h 2 0 m l . o f 10% sodium c h l o r i d e s o l u t i o n . The t i s s u e s were e x t r a c t e d w i t h the 2 0 m l . 10% sodium c h l o r i d e s o l u t i o n f o r 6 hours at 8 5 ° C . The mixture was then c e n t r i f u g e d at 2 5 0 0 RPM. and the r e s i d u e was d i s c a r d e d . To the supernatant 3 volumes o f 95% e t h a n o l were added and the p r e c i p i t a t e d n u c l e i c a c i d s were l e f t o v e r n i g h t i n the f r i d g e . The n u c l e i c a c i d s were c e n t r i f u g e d and the obta ined p r e c i p i t a t e was washed w i t h 2 m l . o f 95% e t h a n o l then w i t h 2 m l . e t h e r , and d r i e d i n vacuo . The d r y p e l l e t s were d i s s o l v e d i n 1 0 volumes o f 5% sodium c h l o r i d e and s t i r r e d f o r 1 0 to 1 5 minutes at 85°C u n t i l a l l muc le i c a c i d s went i n t o s o l u t i o n . The s o l u t i o n was then c e n t r i f u g e d . To the supernatant 3 volumes o f 95% e t h a n o l were added and c h i l l e d i t f o r couple h o u r s . The n u c l e i c a c i d s were recovered by c e n t r i f u g a t i o n and washed w i t h 95% e t h a n o l , 5 0 : 5 0 e t h a n o l - ether m i x t u r e , then w i t h e t h e r . The r e s i d u e was d r i e d i n vacuo at room temperature . The n u c l e i c a c i d r e s i d u e was weighed and 1 m l . o f 0 . 1 N . sodium h y d r o x i d e 28 s o l u t i o n was added f o r each 10 mg of dry p r e c i p i t a t e . The s o l u t i o n was then incubated a t 3 7 . 5 ° f o r 20-22 hours . A f t e r i n c u b a t i o n , the s o l u t i o n was n e u t r a l i z e d w i t h concentra ted h y d r o c h l o r i c a c i d drop by drop u n t i l good p r e c i p i t a t e formed. The mixture was c h i l l e d and l e f t o v e r n i g h t i n f r i d g e . Next day the mixture was c e n t r i f u g e d and the DNA r e s i d u e washed 2 t imes w i t h 0 . 1 N h y d r o c h l o r i c a c i d , then w i t h e t h a n o l and 5 0 : 5 0 e t h a n o l - e ther m i x t u r e , and ether and f i n a l l y d r i e d i n vacuo at room temperature . The Chemical Characterisation of DNA. The d e t e r m i n a t i o n of n i t r o g e n content by the micro K j e l d a h l procedure was performed a c c o r d i n g to r e f . ( 4 1 , 6 8 ) . S t a n d a r d s : Urea r e c r y s t a l l l z e d ( a n a l y t i c a l grade) ( F i s c h e r . S c . C o . ) Guanine h y d r o c h l o r i d e ( N u t r i t i o n a l B i o c h e m i c a l s C o r p . ) p u r i f i e d by i o n exchange chromatography ( 6 9 ) . D e o x y r i b o n u c l e i c a c i d ( C a l i f o r n i a C o r p . f o r Biochem. R e s . ) For t e s t i n g the n i t r o g e n r e c o v e r i e s by t h i s procedure u r e a , guanine h y d r o c h l o r i d e and commercial DNA were used as n i t r o g e n s t a n d a r d s . The r e s u l t of the ana lyses are shown i n Table I . The Determination of Phosphorous, Content of DNA bv the Method of B a r t l e t t . ( 42 ) . 2 9 TABLE I Comparison of the T h e o r e t i c a l and E x p e r i m e n t a l l y Determined N i t r o g e n Contents of D i f f e r e n t Standards • Name o f Compound T h e o r e t i c a l NJ5 M i c r o K j e l d a h l N$ found e x p e r i m e n t a l l y Urea 46 . 6 7 46 . 5 8 it it 46 . 5 9 Guanine h y d r o c h l o r i d e 37.34 37.39 II ii 37.41 DNA commercial 1 5 . 2 (38) 14 . 9 9 ti II 1 5 . 0 9 3 0 Reagents: Solution A. Phosphorous Standard: 1 /ugP/ml. 2 . 1 9 3 5 g potassium dihydrogen phosphate i n 5 0 0 ml. d i s t i l l e d water. 1 ml. = 1 mg P 1 ml. of this s o l u t i o n was di l u t e d to 1 0 0 0 ml. and the resu l t i n g s o l u t i o n contained 1 /t-g P/ml. Solution B. 1 0 N sulphuric acid solution. Solution C. 5% ammonium molybdate solution Solution D. Fiske - Subba-Row reagent. 0 . 5 g. of l-amino-2-naphtol- 1+-sulphonic acid was dissolved with s t i r r i n g i n 2 0 0 ml. fr e s h l y prepared 15% sodium bisulphite solution, followed by the addition of 1 g. anhydrous sodium sulphite. The solution was f i l t e r e d and stored i n a dark bottle and f r e s h l y prepared weekly. Adenosine 5 ' Phosphate (AMP) (Pabst. Fine Chemicals) Thymidine 5 ' Phosphate (TMP) (Pabst Fine Chemicals) Procedure. 1 - 2 mg. previously dried DNA samples were digested i n a Kjeldahl f l a s k with 1 ml. concentrated sulphuric acid for L+-5 hours. The digest was transferred quantitatively into a 5 0 or 1 0 0 ml. volumetric f l a s k and 3 - 5 ml. aliquots corresponding to 4 - 6Mg phosphorous were taken f o r phosphorous analyses. The aliquots of unknown were pipetted into a test tube, calibrated to 1 0 ml., 1 ml. 1 0 N sulphuric acid, O.h ml. 5% ammonium 3 1 TABLE I I The D e t e r m i n a t i o n o f Phosphorous Content o f P h e n y l :Q i s o d i u m fhosphate itJsing ' D i f f e r e n t H y d r o l y s i s P r o c e d u r e s . Method of H y d r o l y s i s % P found e x p e r i m e n t a l l y % P t h e o r e t i c a l 1 ml 70% HCI0 4 1 0 . 5 1 14.2 II 1 0 . 3 5 1 1 ml CC.H2SO4 1 3 . 8 1 u 1 3 . 8 9 1 TABLE I I I Comparison o f T h e o r e t i c a l and E x p e r i m e n t a l l y Found Phosphorous Contents o f D i f f e r e n t Organic S t a n d a r d s . Name of Compound % P found e x p e r i m e n t a l l y %P t h e o r e t i c a l AMP 8.86 8 . 9 2 1 8 . 8 1 TMP 8 . 6 2 8 . 5 9 1 8 . 3 1 1 F r u c t o s e - l , 6 - d i phosphate 1 1 . 4 10.01 1 1 1 . 5 I  ti 1 1 . 3 I  3 2 molybdate 0 . 4 m l . F iske-Subba-Row reagent were added, i n the order d e s c r i b e d and the s o l u t i o n was then made up to 1 0 m l . w i t h d i s t i l l e d w a t e r . In order to o b t a i n u n i f o r m readings i t was found to be v e r y i m p o r t a n t , to s t i r the s o l u t i o n a f t e r a d d i t i o n of each reagent . The s o l u t i o n was heated f o r 7 minutes at 1 0 0 ° C and the c o l o u r red at 8 3 0 m ^ i n a Beckman D . U . s p e c t r o -photometer, aga ins t a reagent b l a n k . The c o l o u r produced was p r o p o r t i o n a l to the conj c e n t r a t i o n o f phosphorous up to 8yu-g /10 m l . of the r e a c t i o n m i x t u r e . A c a l i b r a t i o n curve was cons t ruc ted ( F i g . 1 ) which shows the c o n c e n t r a t i o n dependence of the c o l o u r produced f o r standard phosphorous s o l u t i o n s . The c o l o u r o f the s o l u t i o n s was s t a b l e f o r at l e a s t 24 h o u r s . To t e s t the v a l i d i t y o f the procedure , the phosphorous content of s e v e r a l o r g a n i c substances were determined by t h i s method. A comparison between the p e r c h l o r i c a c i d ( 7 0 ) and concentrated s u l p h u r i c a c i d h y d r o l y s i s of phosphorous c o n t a i n i n g , s u b s t a n c e s was a l s o performed. Table I I summarizes the r e s u l t s o f such exper iments . I t can be seen that s u l p h u r i c a c i d h y d r o l y s i s gave h igher phosphorous v a l u e s , and these v a l u e s were a c t u a l l y c l o s e r to the t h e o r e t i c a l ones . Table I I I summarizes the r e s u l t s obta ined u s i n g d i f f e r e n t o r g a n i c s t a n d a r d s . These substances were hydro lysed w i t h concentrated s u l p h u r i c a c i d . The experiments show, t h a t the phosphorous r e c o v e r i e s i n the case o f n u c l e o t i d e s AMP. and 33 Optical Density 1-0.9 Uo.6 ho? F i g . I. C a l i b r a t i o n c u r v e f o r Phosphorous, D e t e r m i n a t i o n . 34 TMP were v e r y s a t i s f a c t o r y . The a n a l y s i s of f r u c t o s e -1 . 6 d iphosphate gave somewhat h igher v a l u e s than the t h e o r e t i c a l one , however the p u r i t y o f t h i s sample was d o u b t f u l . The D e t e r m i n a t i o n of Base Composi t ion of DNA Reagents ; Guanine h y d r o c h l o r i d e ( N u t r i t i o n a l B i o c h e m i c a l C o r p . ) P u r i f i e d by Ion. ex change chromatography ( 6 9 ) . Adenine ( N . B . C . ) P u r i f i e d by s u b l i m a t i o n i n vacuo at 2 2 0 ° C . ( 7 1 ) C y t o s i n e h y d r o c h l o r i d e . ( N . B . C . ) P u r i f i e d by i o n exchange chromatography. ( 6 9 ) Thymine ( N . B . C . ) P u r i f i e d by r e c r y s t a l l i z a t i o n from hot w a t e r . U r a c i l ( N . B . C . ) P u r i f i e d by r e c r y s t a l l i z a t i o n from hot w a t e r . W y a t t ' s s o l v e n t . I s o - p r o p y l a l c o h o l 1 7 0 m l . Concentrated h y d r o c h l o r i c a c i d 41 m l . D i s t i l l e d water up to 2 5 0 m l . Procedure ; I . H y d r o l y s i s of DNA by concentrated f o r m i c a c i d  s o l u t i o n ( 7 2 ) . 35 1 - k mg. of DM previously dried i n vacuo over phosphorous pentoxide were accurately weighed on an a n a l y t i c a l micro balance and placed i n a pyrex glass tube. The tube was sealed arid placed into the oven and heated for two hours at 161 - l63°C. At the end of two hours hydrolysis the sealed tubes were cooled i n dry ice-ethanol mixture and opened cautiously, by melting the tips of sealed ends i n flame. The contents of tubes were poured i n 10 ml. beakers, cooled again i n dry ice - ethanol mixture, and evaporated to dryness i n vacuo over s o l i d potassium hydroxide. The purine and pyrimidine bases were extracted with three successive portions of 2 ml. O.lNhydrochloric a c i d , followed by centrifugation to remove charred p a r t i c l e s . The 0.1 N hydrochloric acid extracts were com-bined and evaporated i n vacuo. The dry residue was then taken up with a known quantity of 0.01 N hydrochloric a c i d . Usually the f i n a l volume was made up to exactly 1.0 ml. i n a volumetric f l a s k . 1 1 • Separation of Purine and Pyrimidine Bases hv Paner Chromatography (46). P r o q e d i a r f i : Strips of Whatman No. 1 paper were used. 5 0 - 2 0 0 X of hydrolysates containing 50-300/<g DNA were applied on chromatograms using calibrated micro-pipettes. The chromato-grams were placed into an a l l glass chromatographic cabinet, 36 and some solvent was placed at the bottom of cabinet to get saturation with respect to the vapours of solvent. A descending technique was applied and the chromatograms were run about 3 2 - 3 6 hours. At the end of t h i s period the solvent l i n e almost reached the bottom of the paper. The paper s t r i p s were taken out from the cabinet, dried i n a i r , and the purine and pyrimidine spots were outlined with the aid of untraviolet l i g h t ( 4 7 ) . The R(f) values of the unknown spots were compared with the values of standards run on the same paper and at the same time, as the unknown samples. The R(f) values of the purine and pyrimidine bases using the iso-propanol-hydrochloric acid solvent are summarized i n Table TV. The values are compared with those found i n r e f . ( 3 8 ) Table IV, shows that the R( f) values obtained during t h i s investigation are somewhat larger than i n r e f . ( 3 8 ) . This may be due to the large temperature f l u c t u a t i o n i n t h i s laboratory, or the s l i g h t composition difference i n solvent systems. I I I . The Quantitative Estimation of Purine and Pyrimidine Bases  Separated by Paper Chromatography. For quantitative e l u t i o n of the bases from the spots outlined under the u l t r a v i o l e t l i g h t , two techniques were t r i e d and the r e s u l t s compared. It was mentioned before on page II that the elution of spots with 0.1 N hydrochloric acid for 24 3 7 TABLE IV The Comparison of Rf Values of Purine and PPyrimidine Bases i n iso-propanol Hydrochloric acid Solvent. Name of Compound Rf Values found experimentally . Rf Values from r e f . ( 3 8 ) Adenine 0 . 3 2 Guanine 0 . 2 5 0 . 2 2 Cytosine 0 . 4 7 o.kk U r a c i l 0 . 6 9 0 . 6 6 Thymine 0 . 7 8 0 . 7 6 3 8 hours was not quantitative even i f several extractions were performed. Therefore a nev; technique was t r i e d , which was based on the personal communication of I. Csizmadia (49). The des c r i p t i o n of the two elution techniques i s as follows: Procedure I. (Extraction Method) The paper spots were cut into very small pieces and placed i n a test tube, 2 ml. of 0 . 1 N hydrochloric acid was added and the mixture allowed to stand overnight. Next day the test tube was shaken i n a shaker for 3 0 minutes and after standing most of the 0 . 1 N hydrochloric acid extract was decanted. Another 2 ml. portion of 0 . 1 N hydrochloric acid was pipetted into the test tube and the previous procedure was repeated twice. The combined extracts were centrifuged and transferred into a small beaker. The contents of beaker were evaporated to dryness, and the residue was taken up i n an exact volume of 0 . 1 N hydrochloric acid appropriate for the c e l l s i n which the extinctions were to be read. The u l t r a -v i o l e t absorption curves were recorded i n a Beckman Automatic D.K. 2 spectrophotometer. To allow for u l t r a v i o l e t absorbing substances i n the paper, blanks were cut equal i n area to the spots and at equal distances from the star t i n g l i n e , and were eluted and read at the same wavelenghts as the corresponding spots. The extinction c o e f f i c i e n t s used i n estimating nucleic acid derivatives by the absorption at their maxima were 3 9 determined previously i n t h i s laboratory. Procedure 2 . (The column extraction method.) U l t r a v i o l e t absorbing areas on paper chromatograms were cut into very fi n e pieces and these were placed intubes resembling Pasteur pipettes. The diameter of tubes were about 7 mm., and the ends were drawn out to form a veryfine and narrow outlet with an approximately 1 mm. diameter and 2 0 cm. lenght. The upper part of the tubes was widened i n order to f a c i l i t a t e the packing. The glass wool was packed very t i g h t l y i n the bottom of the wide part of the tube up to about 1 - 1 . 5 cm._ height. The tubes were f i l l e d with the f i n e l y cut pieces of paper to form a f a i r l y uniform and t i g h t packing. Tnen c another 2 cm. layer of t i g h t l y packed glass wool was placed on the top of the paper columns. The tubes were placed on a stand which consisted of a piece of f a i r l y t h i n wood containing several 3-4 mm. diameter wholes which allowed through the lower c a p i l l a r y end of tubes but kept the upper part i n a straight and s o l i d p o s i t i o n . The end of the c a p i l l a r y outlets reached a 1 0 ml. small beaker, to c o l l e c t the eluates. About 2 0 - 3 0 tubes were eluted at the same time by f i l l i n g up the columns continuously with 0 , 1 N hydrochloric acid. The rate of eflux was about 0 . 1 6 ml./minute. For each column about 9 ml. of eluate were collected and theh the contents of the beakers were evaporated i n vacuo over 40 sodium hydroxide p e l l e t s and concentrated sulphuric acid. The dry residues were taken up i n an appropriate volume of 0.1 N hydrochloric acid, and the u l t r a v i o l e t spectra were recorded as described i n Procedure I on page ^ . Blank spots were eluted and read i n the same manner as described above. To show the differences between procedure 1 and 2, a standard solution was made up containing known amounts of each of the nitrogenous bases. To construct a c a l i b r a t i o n curve known and increasing amounts of t h i s stock solution were applied on paper s t r i p s i n duplicates and the separation of bases were performed as described on page ^ . The spots were eluted with both procedures 1 and 2. Blank absorption. It was found that very high blank readings were obtained using both procedures. These blanks might obscure the o p t i c a l density values of standards e s p e c i a l l y at the low concentration region. Table V. summarizes the blank readings for each of the bases obtained at th e i r absorption maxima. It can be seen that higher blank values were obtained by the column extraction method. In order to determine the cause of the high blank absorption i n case of procedure 2, a chromato-graphic column was packed with glass wool and eluted with 0.1 N hydrochloric acid. The u l t r a v i o l e t examination of the eluate gave a curve that was characterized by a continous increase of absorption at decreasing wavelenghts. It was concluded that glass wool contained some unknown, u l t r a v i o l e t absorbing material 41 TABLE V The Optical Density Values of the Blanks of Nucleic Acid Bases, Obtained from Readings at t h e i r Absorption Maxima. Name of Blank Optical Density / 1 ml. of solution by procedure 1 . Optical Density / 1 ml. of solution by procedure 2 . Adenine 0 . 2 3 6 0 . 6 5 4 Guanine 0 . 2 6 6 0 . 6 2 2 Cytosine 0 . 1 6 7 0 . 4 5 0 U r a c i l 0 . 2 5 0 0 . 6 5 6 Thymine 0 . 2 2 1 0.646 42 TABLE VI Comparison of % Recoveries of Purine and Pyrimidine Bases Using Procedure 1 and Procedure 2 f o r E l u t i o n . Name of Compound Applied on paper i n M. Found exp. by proc. 1 i n M. % Recovery Found exp. by proc. 2 i n M. /o Recovery Adenine 1 . 8 5 x 1 0 " 2 1 . 2 3 x 1 0 " 2 6 6 . 6 1 . 2 2 x 1 0 " 2 6 5 . 8 3 . 7 0 2 . 3 5 6 3 . 5 5 . 1 5 1 3 9 . 3 1 1 . 0 8 8.53 7 6 . 9 9 . 4 4 8 5 . 2 i 14 . 7 8 1 2 . 7 1 8 6 . 0 1 1 . 8 8 8 0 . 4 1 8 . 4 * 1 6 . 5 ? 8 Q . 4 18 . 2 ^ 0 8 . 7 U r a c i l 2 . 1 7 1 . 0 3 4 7 . 5 . 7 8 3 6 . 6 8 . 6 7 5 . 9 6 6 8 . 7 3 . 9 2 4 5 . 2 1 3 . 0 1 1 0 . 5 8 ' 8 1 . 4 1 8 . 6 1 1 4 3 . 0 17 . 3 4 17 . 2 5 9 9 . 5 16 . 1 9 9 3 . 4 21.68 22.1+Q 10^ . 7 2 ^ . 8 8 110.1 Cytosine 1 . 7 5 . 9 5 5 4 . 5 2 . 1 2 1 2 1 . 1 3 . 5 1 2 . 5 4 72 . 4 4 . 3 0 122.7 7 . 0 2 5 . 5 4 7 9 . 0 8 . 0 3 114 . 5 1 0 . 5 2 8.64 8 2 . 1 9 . 5 0 9 0 . 3 14 . 0 3 1 3 . 7 5 9 7 . 9 1 2 . 0 8 8 6 . 1 17 . 5 4 1 8 . 0 0 102 .6 19 . 7 4 112 . 5 Thymine 1 . 7 1 1 . 6 3 95.7 1 . 6 2 9 4 . 7 6 . 8 2 3 . 4 4 5 0 . 4 3 . 6 7 5 3 . 8 1 0 . 2 3 4 . 9 3 48 . 2 8 . 6 3 84 . 3 13.64 1 0 . 8 7 7 9 . 7 1 1 . 8 8 8 7 . 1 17.05 13.53 79.^ 19.25 116.4 Note: Unpurlfied glass wool was used f o r procedure 2 and unwashed paper for procedure 1 and 2 . 43 (perhaps v e r y f i n e l y powdered g l a s s ) and t h i s substance caused the h i g h b lank r e a d i n g s i n procedure 2 . Because procedure 1 g i v e s a l s o f a i r l y h i g h blank r e a d i n g s , t h i s par t o f the a b s o r -bance must be due e n t i r e l y to some u l t r a v i o l e t absorbing m a t e r i a l i n paper . Table V I summerizes the r e c o v e r i e s o f n i t rogenous bases u s i n g procedure I and 2 . The v a l u e s f o r guanine were not i n c l u d e d , because d u r i n g the p r e p a r a t i o n o f standard s o l u t i o n a decomposi t ion of t h i s base took p l a c e , which was shown by the d i s t o r t e d u l t r a v i o l e t a b s o r p t i o n curve obta ined f o r t h i s compound, a f t e r the s e p a r a t i o n o f the bases o f standard s o l u t i o n by paper chromatography. Comparing the r e s u l t s of t a b l e V I the f o l l o w i n g o b s e r v a t i o n s can be made: 1 . There was a l a r g e d e v i a t i o n i n r e c o v e r i e s o f p u r i n e and p y r i m i d i n e bases u s i n g both p r o c e d u r e s . 2 . The average percentage r e c o v e r y was 7 0 - 8 0 $ u s i n g procedure I , t h e r e f o r e t h i s method d i d not e f f e c t the q u a n t i -t a t i v e e x t r a c t i o n o f p u r i n e and p y r i m i d i n e bases from paper . 3 . In some cases even h igher then 1 0 0 $ r e c o v e r i e s were obta ined by u s i n g procedure 2 . T h i s was maybe due to the f a c t t h a t the amount o f g l a s s wool used f o r packing was not the same i n each cases , and depending on the q u a n t i t y of g l a s s wool u s e d , d i f f e r e n t amount o f u l t r a v i o l e t absorbing m a t e r i a l was e l u t e d . Because the column e x t r a c t i o n method seemed to be 44 more promising with respect to the quantitative extraction of the nitrogenous bases, a new standardization curve was constructed, using washed paper for separation of the bases and p u r i f i e d glass wool for the e l u t i o n technique. The p u r i f i -cation procedures and the preparation of a new standard solution i s described as follows: P u r i f i c a t i o n of the paper Whatman No. I paper s t r i p s were washed with 0.1 N hydrochloric acid for 12 hours i n a chromatographic cabinet. The papers were dried, and washings were repeated with d i s t i l l e d water and with Wyatt's solvent. P u r i f i c a t i o n of glass wool A large a l l glass chromatographic column was packed t i g h t l y with glass wool (pyrex. lab. glassware). The amount of the glass wool was about 200 g. The column was eluted with about 4 l i t e r of 0.1 N hydrochloric acid (eflux'time about 4 ml. /minute) and then with 2 l i t e r of d i s t i l l e d water. The e l u t i o n was continued u n t i l no more u l t r a v i o l e t absorbing material was present i n the eluate at 240 m/*.. Preparation of standard stock solution: A new standard stock solution was prepared from the pu r i f i e d purine and pyrimidine bases. Guanine was dissolved 4 5 f i r s t i n 2 N hydrochloric acid and then transferred to the stock solution containing the other four bases i n 0 . 0 1 N hydrochloric acid. The r e s u l t s of procedure 2 using purified paper and glass wool are represented graphically i n Figures ( 2 - 6 ) . In these graphs the applied amounts of purine and pyrimidine bases were plotted versus the recovered amounts. In the case of procedure 2 f a i r l y straight l i n e s were obtained proving that the deviation from the t h e o r e t i c a l values were the same i n a l l concentration regions. In contrast, at the low con-centration range the recoveries of procedure 1 were always lower, than those of procedure 2 , and approached the theore-t i c a l l i n e at higher concentrations. The reason for t h i s could be that the obscuring effect of paper blank absorption was r e l a t i v e l y smaller at higher concentrations. Typical standard curves of purine and pyrimidine bases are shown i n Figures 7 - 1 1 using procedure 2 with p u r i f i e d paper and glass wool. It was concluded from these experiments; 1 . The hydrolysis product of nucleic acids obtained after separation by paper chromatography can be eluted almost qu a n t i t a t i v e l y from the paper using the column extraction method (procedure 2 ) . 2 . The general recoveries of the bases approached 9 0 $ , 4 6 Rg.l -3.The Comparison ofRecov&nes of Thymine and Cytosine from Paper C h r o m a t o g r a m s U s i n g P r o c e d u r e I and 2 t o r E l u t i o n . E x p e r i m e n t a l ^ F o u n d ConceniVahon in /UmolesxIO Experimentally Found Concentration in ,umo\esx 10" 4 8 N o t e : ...Theoretical Recovery •=Procedure2. A p p l i e d Concentration is zo i n /A/rmole«>x 10 F i g 6 . T h e C o m p a r i s o n of R e c o v e r i e s of G u a n i n e f r o m Paper C K r o m a t o g r a m s Usincj P r o c e d u r e 2. f o r E l u a t i o n . 4 9 Optical Density F.g.7. NOTE: Fig. 7= Adenine F i g . f t - G u a n i n e < C o n c e n t r a t i o n i n ° 1 4 6 8 io I* i 6 , 8 z o f / A m o l e s / 4 m | ^ x i o - 2 Optical Density 0.4 F i g . 8 . ________________ Concentration in • i H 16 te zoC /U-mole&y4.m| ,j x I O - 2 Fig.7o.nd 8. Calibration Curve of A d e n i n e a n d G u a n i n e U s i n g P r o c e d u r e 2. . s o Optical Density 0 1 + Optica \ Dens i ty 0/H NOTE-. Fig. Cytos-me Fig. 10 -Thymine 12 It lb Concentration irv ~i& 20 (yii, naoles/4nal 10" P g . 10 Con c e n t r a t i o n in o I 4 I I Jb" \z ilf ie ig 2o f ^ . m o l e s / 4 m 0 x l 0 " F i g . 9 a n d 10. C a l i b r a t i o n Curve of C y t o s i n e a n d T h y m i n e Using P r o c e d u r e % . 5\ O.D. 0.5 1 C o n c e n t r a t i o n i n ^ naole&x 10 /4ml F i g - II C a l i b r a t i o n Curve of U r a c i l U s i n g P r o c e d u r e 2 . 5 2 except of thymine where recoveries averaged 8 6 $ . 3 . This method proved to be very s a t i s f a c t o r y provided that both paper and the glass wool used during the procedure were p u r i f i e d as described above. By these p u r i f i c a t i o n procedures blank readings were reduced considerably and did not obscure the evaluation of unknowns, even when working i n the very low concentration range (4 - 6 x 1 0 yaM/4 ml.) 4. The procedure was time saving and more e f f e c t i v e as compared with procedure 1 . The Detection of Amino Acid Contamination of DNA  Hydrolysates by Paper Chromatography. Reagents: A. Amino acid standards: Glycine, Alanine, Serine, Cysteine, Cystine, Threonine, Methionine, Valine, Leucine, iso-Leucine, Aspartic acid, Glutamic acid, Lysine, Arginine, Phenylalanine, Tyrosine, Tryptophan, Proline and H i s t i d i n e . ( N u t r i t i o n a l Biochemicals Corp.) B. Butanol-acetic acid solvent. To 5 0 0 ml. of a f r e s h l y shaken mixture of equal volumes of water and n-butanol i s added 6 0 ml. of g l a c i a l acetic acid. After the layers separated the upper layer i s used as the moving phase. An aliquot of the lower layer ( 2 5 - 1 0 0 ml.) i s placed i n the chromatogram chamber. 5 3 C. Ninhydrin reagent. 0.25% w/v Ninhydrin i n acetone. Procedure; The formic acid hydrolysates of DNA. samples were applied on large Mhatman No. 1 paper sheets ( 5 8 x 48 cm). The chromatograms were run for about 24 hours, then a i r dried and sprayed with ninhydrin reagent and heated at 65°C for 3 0 minutes, ^he spots were i d e n t i f i e d with the aid of standard amino acids, run at the same time and on the same paper. The bands were outlined i n pencil as fading of the colour took place after few days. The Physical Characterization of DNA 1 . The Determination of £'(p) values of DNA Preparations. Reagents: 0 . 0 2 M sodium chloride solution. 1 . 1 6 9 g. sodium c h l o r i d e / 1 0 0 0 ml. d i s t i l l e d water. Procedure: Accurately weighed DNA samples ( about 1 mg.) were transferred into a 2 5 ml. volumetric f l a s k . The samples were dissolved i n 0 . 0 2 M Sodium chloride solution, by allowing them to stand overnight at 0°C. When a l l the DNA was dissolved, 54 the volume of solution was made up to exactly 2 5 ml. The u l t r a v i o l e t absorption spectra of the solutions were then recorded with a Beckman D.K. 2 spectrophotometer. From the o p t i c a l density value at maximum absorption (usually around 2 5 7 . 5 n$ the £(p) value of the sample was calculated according to equation: ^ ( P ) = 0 D. at max.A, - 0 D x 30 . 9 8 C x d P cc i n g . / l x 1 where ^(p) = the atomic extinction c o e f f i c i e n t with respect to phosphorous. 0 D = O p t i c a l Density of the solution C r Phosphorous concentration of the solution i n moles/liter d = Internal c e l l lenght i n cm. 2 . The Determination of I n t r i n s i c V i s c o s i t y of DNA Preparations Reagent: 0 . 2 M sodium chloride solution 11 . 6 9 g. sodium chloride/1000 ml. d i s t i l l e d water. Apparatus: Ostwald type of c a p i l l a r y viscometer;;.'. Procedure: Accurately weighed DNA samples were transferred into a 2 5 ml. volumetric f l a s k , and dissolved i n 0 . 2 M sodium chloride 55 solution by allowing them to stand overnight at 0°C and by occasional inversion of the volumetric flasks at room temper-ature for 4 -5 hours. $hen the contents of the flas k s seemed to be clear, the solutions were centrifuged at 2 0 0 0 R'PM for 5 minutes. The clear supernatant was used as a stock solution. The stock solution and three d i f f e r e n t d i l u t i o n s of i t were used for viscosimetric measurements. The flow time of these solutions were determined as follows: The clear and dry viscometer was clamped v e r t i c a l l y i n the thermostat bath ( 2 5°C) i n such a pos i t i o n that i t could be viewed e a s i l y , and 4 ml. of the solution was added from § pipette. A dust free rubber tube loosely plugged with cotton was attached to the smaller tube and the l i q u i d was drawn up into the enlarged bulb and above the upper mark. The l i q u i d was then allowed to flow down through the c a p i l l a r y and the stop watch was started when the meniscus passed the upper mark and stopped when i t passed the lower mark. Two or three check determinations on the time of outflow,were made. The flow time for the solvent ( 0 . 2 M sodium chloride) was s i m i l a r l y determined. Since i t was found that the densities of the d i l u t e DNA solutions were not s i g n i f i c a n t l y d i f f e r e n t from that of the solvent, the density terms were not used for the ca l c u l a t i o n of r e l a t i v e v i s c o s i t i e s . The r e l a t i v e v i s c o s i t i e s were calculated from equation. 5 6 b e c o u s e d — d o 1^  j(me of OUT flow o\ aolMtion /Vj _^ and s.olv/enV vespedw«ly ^ ° d <J - d e ^ s ' A i c s of s.olu4i 'ori a n d -sotv/e.^ veJ-pechve.y The i n t r i n s i c v i s c o s i t i e s were determined graphically as described on page Ik , and calculated from the following equation ( £>): i c ' vwne-re. oC - — — z L'*)^!" viscoi\iy concenMi"©*, C Z\\ {- viscosity c»+ conceinfy'oJVon C 2 57 RESULTS AND DISCUSSION In defining the properties of the DNA of a given tissue i t i s important that the i s o l a t i o n be as quantitative as possible, that degradation and impurities be reduced to minimum. Losses i n preparation might y i e l d a product whose properties are not representative of the whole, while degrad-ation by such agents as acids, a l k a l i , enzymes and heat might destroy some of the unique properties that would di s t i n g u i s h that p a r t i c u l a r substance. The presence of impurities i n the isolated DNA would also change i t s physical-chemical structure and composition. Tables VII and VIII summarize the y i e l d s of DNA from l i v e r and small i n t e s t i n a l mucosa of rat obtained by the f i v e d i f f e r e n t procedures used during t h i s study. On inspection the data on Tables VII and VIII one can conclude: 1. The y i e l d of DNA was i n a l l cases higher from i n t e s t i n a l mucosa, than from l i v e r . This fact i s i n accordance with the findings that the nuclear/cytoplasmic r a t i o i s much higher i n i n t e s t i n a l mucosa than i n l i v e r . ( 3 8 ) 2 . In both tissues the highest y i e l d s were obtained by the detergent method ( 2 0 ) . It i s now almost generally accepted that the procedure of Kay et a l . gives the highest y i e l d among other methods ( 3 8 , 7 3 ) . 3 . In case of small i n t e s t i n a l mucosa somewhat similar y i e l d s were obtained by the following three procedures 5 8 TABLE VII Different, Methods, Method of preparation Sample No. Y i e l d of DNA i n mg /10 g of fresh tissue Average y i e l d of DNA i n mg/10 g of fresh t i ssue Procedure I. Emanuel and Chaikoff ( 2 9 ) A - l A - 2 M 5 . 7 11 A - 3 7 . 6 It A-lf 6 . - 5 . 7 11 A-5 6 . 5 It A - 6 5 . -11 A - 7 5 . 2 tl A - l l V . 5 Procedure I I . Detergent method of Kay et a l ( 2 0 ) A - 1 6 A - 1 7 2 3 . 9 2 ^ . 5 2 ^ . 2 Procedure I I I . Emanuel and Chaikoff 4. detergent method. A - 1 3 A - 2 1 7 . -1 3 . - 1 0 . ^ Procedure IV. sonication method(32) C -3 1 5 . -Procedure V. hot 1 0 $ NaCl extraction method ( 3 6 , 3 7 ) B-3 5 . -5 9 TABLE VIII Y i e l d s of DNA from Small Intestinal-Mucosa of Rat Isolated by Different Methods. Method of preparation Sample No. Y i e l d of DNA i n mg/10 g of fresh tissue Average y i e l d of DNA i n mg/10 g of fresh t i ssue Procedure I. Emanuel and Chaikoff ( 2 9 ) A - 8 A - 9 1 3 . 6 1 0 . - 1 3 . 9 A - 1 0 1 8 . -Procedure I I . detergent method of Kay et a l ( 2 0 ) A - 1 5 A - 1 8 8 2 . 2 A 6 5 . 8 6 5 . 8 * * A - 2 0 6 5 . 9 Procedure I I I . Emanuel and Chaikoff + detergent method A - 1 2 A-li+ A - 1 9 1 3 . 3 1 0 . -1 3 . 5 1 2 . 2 Procedure IV. „ AAA C-l 7 7 . -sonication method ( 3 2 ) 2 6 . 3 Procedure IV. hot 10% N A C 1 extraction method (36, 3 7 ) B-l B - 2 1 3 . 3 1 2 . 6 1 2 . 9 % o t e : Preparations A - 1 8 and A - 2 0 were deproteinized three times with detergent solution, preparation A - 1 5 only two times. **The y i e l d of preparation A - 1 5 was not used for calculations of average y i e l d by procedure TI. *«*Preparation C - 1 was deproteinized by shaking with chloroform-amyl alcohol mixture f i v e times and preparation C - 2was deproteinized by shaking with chloroform - amyl alcohol mixture seven times. 66 Emanuel and Chaikoff (Procedure I ) , the Emanuel and Chaikoff method combined with the detergent method of Kay et a l . (Procedure III) and the hot 10% Sodium chloride extraction method (Procedure V). A l l of these procedures are characterized by a very considerable loss of DNA, taking the average DNA content of i n t e s t i n a l mucosa as 1 2 9 mg/10 g. of fresh tissue ( 3 8 ) . 4. With l i v e r tissue quite similar y i e l d s were obtained by Procedure I and V. Somewhat higher y i e l d s resulted by Procedure I I I . The best y i e l d s were obtained with Procedure II, e s p e c i a l l y i f one considers the values given for l i v e r tissue i n r e f . ( 3 8 ) . In t h i s reference the DNA content of rat l i v e r i s given as 24 mg./IO g. of fresh t i s s u e . If one considers that appreciable losses might occur s p e c i a l l y working with small amount of ti s s u e , the y i e l d of Procedure I I . seems to be quite u n r e a l i s t i c . It i s very u n l i k e l y that any bio-chemical i s o l a t i o n process should give 1 0 0 $ y i e l d . Only one preparation was performed according to Procedure IV therefore no general conclusion can be formed about the e f f i c i e n c y of recovery of DNA from l i v e r tissue by t h i s method. The y i e l d of 1 5 mg./IO g. fresh tissue seems to be quite promising comparing i t that lower values of procedures I, III , and V. Considering the p o s s i b i l i t i e s , where the losses of DNA May occur during d i f f e r e n t i s o l a t i o n procedures, i t i s not surprising that appreciable amount of DNAs, were lo s t with Procedures I, and I I I , using both i n t e s t i n a l and l i v e r t i s s u e s . It was expected, that Procedure I would give the lowest possible y i e l d , because no such "controlled homogenization" was performed as described i n the o r i g i n a l publication ( 30 ) . Emanuel and Chaikoff used a hydraulic homogenizer i n the i r study for the controlled release of muclei from other c e l l u l a r elements. These workers claimed that much higher y i e l d s of DNAs were obtained by using the hydraulic homogenizer ( 3 0 ) . The amount of DNA obtained from 10ng. of fresh rat l i v e r tissue was 22 mg. compared with that of 10 mg. using the Teflon homogenizer alone. The average y i e l d s i n Table VII are even lower 5-7 mg./IO g.. of t i s s u e . The appreciable loss of DNA nirnaybe due to the fact that considerable part of nu c l e i were damaged during the homogenization with the Teflon homo-genizer and. blending with the S e r v a l l Omni Mixer. Once n u c l e i were damaged and the i r nucleoprotein content released, they no longer were retained by the negatively charged diatomaceous earth. As a matter of fact Zamenhof (74) advises avoiding the use of any stainl e s s s t e e l dissintegrator because according to him, minute;' traces of rust (Fe ion) would cause rapid degradation, and high speed mixing can effect the breakage of macromolecules into shorter fragments. However i n t h i s experiment i t was necessary to use short blending periods (one minute) at low speed with the 6;a S e r v a l l Omni Mixer s p e c i a l l y for preparations of rat l i v e r homogenate. If the Teflon homogenizer were used alone incomplete homogenization were resulted and t h i s prolonged the time required for i s o l a t i o n of n u c l e i , because the d i a -tomaceous earth was obstructed by c e l l u l a r material and the f i l t r a t i o n rate was thereby slowed down. Another effect of the incomplete homogenization would be the contamination of DNA with cytoplasmic RNA and other extraneous non-nuclear materials. It was found quite surprising that Procedure V did not give higher y i e l d s . Usually quite large amounts of nucleic acids were precipitated f i r s t , but most of the DNA seemed to be lo s t during the several p u r i f i c a t i o n processes. The combination of Procedures I and II did not show any appreciable improvement of y i e l d s i n case of i n t e s t i n a l pre-parations; some increase was found with l i v e r t i s s u e s . In the preparation of biochemical substances i t is usually necessary to make a choice between a high y i e l d and a pure, high q u a l i t y product. Pure qu a l i t y may re s u l t because of change i n the natural structure of the material caused by some d r a s t i c treatment during the preparation. If on.= the other hand a mild method i s used, and a large amount of substance i s obtained, t h i s may contain considerable amount of impurity. In order to conclude which procedure gives highly 63. polymerized pure product the properties of the di f f e r e n t DNA samples were compared by physical-and chemical means. Nitrogen and Phosphorous Content of DNA Preparations. The nitrogen and phosphorous contents and the N/P ra t i o s are shown i n Table IX. Before making any conclusions about the r e s u l t s demonstrated i n Table IX i t has to be ment-ioned that the a n a l y t i c a l data obtained for N content of DNA samples are somewhat doubtful and have to be interpreted very cautiously. Four DNA preparations were sent to Dr. Manser who performed nitrogen analyses according to the combustion method of Dumas. Table X compares the percentage of nitrogen i n DNA samples determined by the combustion and the micro-K j e l d a l procedures. It can be seen that the micro-Kjeldal determination gives considerably lower valuess for N content. This finding i s i n accordance with Chargaff 1s opinion (38) that the r e s u l t of the K j e l d a l determination i s lower than that of Dumas'. However t h i s concept cannot be accepted e n t i r e l y , because very good agreement was found between the t h e o r e t i c a l and experimental N values of several standards used for testing the r e l i a b i l i t y of K j e l d a l procedure. These findings are demonstrated i n Table _ l Page 29 . (experimental p a r t ) . In comparing the average nitrogen and phosphorous contents of DNA samples shown i n Table IX the following general 64 TABLE IX The Nitrogen and Phosphorous Contents of DNA Preparations Isolated "by Different Procedures. Method of Preparation Source of tissue Sample No. P% Atomic N/P ». N/P Proc.1 ( 2 9 ) it tt ti tt II Liver ti tt II II ti A-3 A-4 A-5 A-6 A-7 A - 1 7 1 0 . 2 3 10.64 11.08 12.85 12.63 12.64 avg.11.67 4.99 5 . 2 5 5.73 5.61 5 . 6 7 6.04 avg. £.54 4 . 5 3 4.48 4.28 5.07 4.93 4.63 2 . 0 5 2 . 0 3 1.93 2.4 2.23 2 . 0 9 avg. 2.12 Proc.11(20) II Liver ti A-16 A - 1 7 10.32 11.7 avg. 11.01 6.45 6.73 avg. 6.^9 3.54 3.84 1.6 It 7 4 avg. 1.67 P r o c . I l l II it it A-13 A - 2 1 1 2 . 3 5 1 2 . 6 1 avg. 12.48 6.8 avg. 7 . 5 8 4.02 3.33 1 . 8 2 1.51 avg. 1.66 Proc.IV ( 3 2 ) it C-3 12 . 1 5 8.56 3.14 1.42 Proc.V(36,37) II B-3 13.77 8.36 3.64 1.65 Proc.I H u Intestinal mucosa tt ti A - 8 A-9 A-10 1 3 . 2 8 12 . 9 2 11 . 9 2 avg .12.7 7.47 6.28 avg. 6.^2 3.93 4.57 3.92 1 . 7 8 2 . 0 7 1 . 7 7 avg. 1.87 Proc.II II tt ti it II A-15 A-18 A-20 11.78 11 . 2 3 9.76 avg. 11.5 6.2 6.6 5.66 avg. 6.4 4.2 3 . 7 6 1.9 1.7 1.73 avg. 1.77 P r o c . I l l II II II ti ti A-12 A-14 A-19 12.24 12.69 11.61 avg. 1 2 .18 7.39 7 . 9 8 7.55 avg. 7.64 3.66 3 . 5 2 3.4 1.66 1.59 1.54 ave. 1.59 Proc.IV II tt II C-I C-2 8.6b 11.38 6.22 7.32 3 . 0 9 3.44 1.4 1.56 Proc.V II ti it B - l B - 2 15.7 I f , 15 avg.15.42 9.49 8.9 avg. 9.19 3.66 3 . 7 6 1.65 1.7 avg. 1.6'/ Note:* Preparation A - 2 0 was not used for the c a l c u l a t i o n of average values. 6 5 TABLE X The Comparison of the N i t r o g e n Contents o f DNA P r e p a r a t i o n s Determined by the Dumas and K j e l d a h l P r o c e d u r e s . Sample No. % N by Dumas' method $N by K.ieldahl 'si ' .method A - 1 3 13.24 1 2 . 3 5 A - 1 5 13.80 1 1 . 7 8 A - 1 7 1 3 . 6 4 1 1 . 7 0 A - 1 8 1 2 . 6 5 1 1 . 2 3 66; observations are noticeable: 1. Procedure I gives the highest N/P rations for both l i v e r and i n t e s t i n a l preparations. As i t was mentioned i n the introduction N/P values higher than 1 . 6 5 would indicate protein contamination i n DNA. Procedure I uses s a l t saturation for breaking the linkages between protein and DNA. The high N/P values, 2 . 3 6 for l i v e r and I . 8 7 for i n t e s t i n a l DNAs would indicate that t h i s deproteinization procedure i s not completely s a t i s f a c t o r y . F r i c k ( 7 3 ) i n h i s c r i t i c a l study discussed the p o s s i b i l i t y that separation of protein and nucleic acid by saturated sodium chloride may be dependent upon autolysis which has already been carried out by c e l l u l a r enzymes. At low temperatures ( 0°C) these enzymes have l i t t l e e f f e c t . If however, the nucleoprotein i s allowed to stand in contact with the extraction l i q u i d for a longer time and at a higher temper-ature, the protein and the nucleic acid can be more e a s i l y separated. In the case of Procedure I the nucleoprotein i s i n contact with the saturated sodium chloride solution only for a short time and at a low temperature, therefore, the chance for autolysis by enzymes i s greatly reduced. 2 . Procedures I I , III, and V give values which are very close to the theorethical N/P r a t i o ( 1 . 6 5 ) . However, t h i s 6 7 does not necessary mean that the samples are completely free from protein contamination, e s p e c i a l l y i f one considers the low N values obtained by the micro-Kjeldal determination. For instance i f the N values of samples A -13 , A - 1 5 , A-17, A - 1 8 given by the Dumas' procedure were used for the ca l c u l a t i o n of N/P r a t i o s , much higher values would be obtained. 3 . DNA preparations of l i v e r and i n t e s t i n a l mucosa isolated by Procedure IV have su r p r i s i n g l y low N/P r a t i o s . These values are lower than the theorethical one. Two explan-ations may be possible: a, The percentage of nitrogen found by the micro K j e l d a l procedure i s low. b, The presence of a phosphorous containing non-nucleic acid impurity In DNA samples. 4. The nitrogen and phosphorous contents isolated by Procedures I, I I , I I I , and IV are i n a l l cases lower than the generally accepted values ( 3 8 ) . This may be due to the presence of some inert impurity (polysaccharides, inorganic sa l t s ) i n these samples. 5 . The best a n a l y t i c a l values for phosphorous and nitrogen contents of l i v e r and i n t e s t i n a l DNAs were obtained by using Procedure V. This method i s a very d r a s t i c one, and surely cleaves o f f impurities from DNA preparations. In summary, the presence of protein and other impur-i t i e s i n DNA samples were indicated i n some cases by the high N/P r a t i o s and the generally low values of phosphorous and 6 8 nitrogen contents. The Detection of Protein Impurities i n  DNA Preparations by Paper Chromatography To get some better and more d e f i n i t e information about protein impurities i n DNA samples, some q u a l i t a t i v e amino acid demonstrations were performed i n DNA hydrolyzates, as described on page 52 (experimental part) Table XI shows the r e s u l t s of these investigations. Unfortunately Table XI does not give a complete picture about the composition of protein impurities i n a l l DNA preparations. However some generalizations can be made on the basis of experimental findings i n Table XI. 1. The presence of d e f i n i t e protein contamination was shown i n a number of DNA preparations obtained from l i v e r and i n t e s t i n a l mucosa using Procedures I, II, I I I . Thus neither salt saturation nor detergent treatment could effect the complete removal of protein from DNA preparations. According to K i t ( 8 6 ) the d i s s o t i a t i o n of DNA from l i p o p r o t e i n by anionic deter-gents i s slow without the hydrolytic assistance of either mitochondrial deoxyribonuclease or heat. In Procedures II and III the action of.both heat and deoxyribonuclease were excluded, because a l l steps were performed i n cold and i n the presence of the enzyme i n h i b i t o r , Versene. 2. A l l DNA preparations having protein contamination y i e l d aspartic acid and leucine on hydrolysis. The presence of cysteine 6 9 or cystine and glutamic acid were also demonstrated i n the majority of cases. The fact that a c i d i c amino acids predominate i n the hydrolysates of DNAs support the findings of Butler ( 7 5 ) that the contaminating protein i n DNA preparations i s not a basic histone. 3 . Glycine was found i n a l l DNA samples tested for amino acids. This amino acid may ar i s e from the destruction of some purine bases during hydrolysis. The res u l t s i n Table XI have to be interpreted very cautiously because only one dimension chromatographic technique was used for separation of amino acids from DNA hydrolysates. However i n generally one can conclude that majority of DNA samples tested were contaminated by protein. The Nitrogenous Base Composition of DNA Preparations. The r e s u l t s of a comparative study of the composition of'many preparations show that the compsoition of DNA i s ch a r a c t e r i s t i c of these species from which i t i s derived, but within the l i m i t s of present a n a l y t i c a l methods the DNA of di f f e r e n t tissues of the same species have the same o v e r a l l composition ( 3 8 ) . Chargaff points out however, that t h i s r e s u l t does not rule out the p o s s i b i l i t y that there may be differences i n the sequence of arrangement of the nucleotides among nucleates of the same o v e r a l l compositon. A remarkable c o r r e l a t i o n emerges fro'm the analyses TABLE XI Comparison of Amino Acid Contents of DNA Hydrolysates. Method of Prep. Source of tis s u e Sample No. Alanine Asparf acid Asparagine Cysteine or Cystine Clutamic acid Glycine Leucine Methi-onine Valine Proc. 11(20) Liver A - 1 6 _ 4- •• II ti A - 1 7 4- 4> 4- 4> 4- - - 4-Proc. 1 1 1(29, 20) II A - 1 3 + - *• . 4- 4- 4- - -Proc. K29) Intestinal mucosa A-9 — 4- — 4> _ *• Proc. 11(20) II A - 1 5 4> — f 4- 4. *• ti II A-18 *. 4- 4> 4. 4. - 4. Proc. I l l (20,29) it A-12 f f 4- 4. *. 4- 4- - -7 1 of a large number of d i f f e r e n t DNAs ( 5 3 , 7 6 - 7 8 ) : the adenine/ thymine and guanine/cytosine mole r a t i o s are, i n the great majority of cases, equal to one, within the error of analyses. Much of the a n a l y t i c a l data on v i r a l ( 7 9 ) and b a c t e r i a l ( 8 0 ) DNAs maintain t h i s c o r r e l a t i o n . When the composition of many specimens of DNA from d i f f e r e n t c e l l u l a r sources i s compared, a very s t r i k i n g feature emerges, that two p r i n c i p a l groups can be distinguised, namely the adenine-thymine type (A.,T.) i n which adenine and thymine predominate, and the guanine-cytosine type (G., C.) i n which guanine and cytosine are the major constituents ( 3 8 ) . A l l t o t a l DNA preparations from animal sources described up to thi s time, belong to the A.T. type. The G.C. type has been encountered i n several micro-organisms and viruses. The nitrogenous base composition of several DNA pre-parations are shown i n Table XII. Unfortunately not a l l DNA samples isolated were characterized by th i s method. A few observations can be made on inspections the data i n Table XII: 1. In case of l i v e r preparations Procedure I yielded DNA samples, whose base composition was i n agreement with those reported i n l i t e r a t u r e . ( 5 3 , 7 6 - 7 8 ) . The DNA of rat l i v e r i s A.T. type as indicated by the r a t i o s of adenine * thymine/ guanine r cytosine. The ra t i o s of adenine/thymine and guanine/ cytosine are close to unity. 72 DNA samples A - l 6 and A-17 isolated by the detergent method were very heavily contaminated by RNA as indicated by the presence of u r a c i l and by the preponderance of guanine and cytosine. Their adenine r thymine/guanine r cytosine r a t i o s are less than unity, which i s not c h a r a c t e r i s t i c for mammalian ti s s u e s . The presence of contaminating RNA i n DNA preparations obtained by procedure II can be explained by the f a c t , that no previous washings of the tissue homogenates with 0.15 M sodium chloride solutions were performed, thus both RNA and DNA were extracted and precipitated from the tis s u e s . The f r a c t i o n a l p r e c i p i t a t i o n of RNA with iso-propyl alcohol did not effect the complete separation of the two nucleic acids. The preparations obtained by Procedure III were characterized by an adenine *• thymine/ guanine+cytosine r a t i o greater than unity. However the numerical value of t h i s r a t i o (1.13) i s lower than those obtained for samples isolated by Procedure I (1.24-1.29). According to Chargaff (38) a good DNA preparation i s characterized by adenine/thymine and guanine/ cytosine r a t i o s of 1 • 0.05. This requirement was not f u l -f i l l e d with preparations A-13, and A-21 where the guanine/ cytosine r a t i o s were greater than 1.05. This finding indicated a preponderance of guanine i n the samples mentioned. Procedure IV yielded a DNA preparation which showed the c h a r a c t e r i s t i c features of mammalian DNAs. However the 73 mole percent of guanine was also s l i g h t l y higher than i n samples A - l and A-2. Considerable RNA contamination was found i n sample C-3 indicated by the presence of 3.96 mole percent u r a c i l . 2. The base compositions of DNAs of small i n t e s t i n a l mucosa isolated by Procedure II and III are generally character-ized by the presence of heavy u r a c i l contamination. This finding indicates a considerable amount of RNA impurity i n the preparations. According to Chargaff (38) the a n a l y t i c a l r e s u l t s on preparations, containing more than three percent of t h i s contaminant, "command l i t t l e confidence" and t h i s was the case with, regard to preparations A - 1 5 , A-18, A-14 and A-19. None of these preparations showed the r e g u l a r i t i e s de-scribed by Chargaff(38). Only sample A-20 yielded the c h a r a c t e r i s t i c features of pure DNA samples. No u r a c i l contamination was found i n t h i s case. On comparing the base composition of t h i s pre-paration with those of "good" DNA preparations of l i v e r (A-2, A - l and C-3), no s i g n i f i c a n t difference can be observed, which i s i n agfeement with the findings reported i n l i t e r a t u r e (38). 3. The general base recovery was quite low i n almost a l l of the cases, which would indicate the presence of some inert impurity i n DNA preparations. TABLE X I I D i s t r i b u t i o n of P u r i n e s and P y r i m i d i n e s i n DNA. P r o p o r t i o n i n Moles of Nitrogeneous Const i tuents/100 g atoms of Phosphorous. Method of p r e p . Source of t i s s u e Sample No. A G C. T •U A4-T G+C A T G C i Rec . P r o c . I. (zs) L i v e r 1 A - l A-2 28. k$ 28.27 21.56 21.23 23.1 22.53 26.88 27.98 - 1.2*t 1.29 1.05 1.01 0.93 0.9k 6-7.1 7^.9 P r o c . I I (20) »t t» A-16 A-17 19.87 28.35 25.73 25.57 26.61 20.66 20.61 17.70 7.18 7.72 0.96 0.99 0.97 1.6 0.97 1.2*+ 8 1 . -71.3 P r o c . I l l (ii,") tt 1 A-13 A-21 26.88 26.7k 2k. 98 26.55 21.87 20.38 26.27 26.31 - 1.13 1.13 1.02 1.01 l.lk 1.3 76.0 7*+.3 P r o c . Iv (3i) C-3 27.95 22.01 19.13 26.97 3.96 1.33 1.0k 1.15 75.5 P r o c . I I M ti i n t . mucosa tt tt A - i 5 A-18 A-20 2^.18 19.66 23.23 20.63 22.28 21.66 25.08 22.02 20.96 23.99 19.97 29.13 6.12 6.07 •1.05 0. 89 1. t+6 1.01 0.98 0.95 0.93 1.01 1.03 98.9 99.5 63.2 P r o c . I l l (>0$ tt tt A-lk A-19 23.17 26.5 2*f.2 22.92 21.81+ 13.8 30.78 20.2*+ 16.51 1.17 1.27 0.77 1.31 1.1 1.66 85.6 92.7 Note : The Data i n t a b l e X I I are average values o b t a i n e d from d u p l i c a t e a n a l y s e s . The p r o p o r t i o n s of n i t rogenous bases are c o r r e c t e d f o r hundred percent r e c o v e r y . The f o l l o w i n g a b b r e v i a t i o n s were used: Adenine (A) Guanine (G) C y t o s i n e ( C ) Thymine (T) U r a c i l ( U ) . 7 5 It should be mentioned that a l l DNA hydrolysates were chromatographed i n duplicate and eluted by the two d i f f e r e n t methods as described i n experimental part page "33 . However, in most of the cases, unwashed paper wase used for paper chromatography which greatly obscured the r e s u l t s , s p e c i a l l y when very small amount of DNA samples were applied on paper chromatograms. To overcome these d i f f i c u l t i e s a few DNA samples were re-hydrolysed and chromatographed on washed papers and eluted according to Procedure 2 (Experimental part p a g e ^ ). A s i g n i f i c a n t increase i n percentage recoveries were found with these samples as compared with the percentage recoveries using the elution method of Chargaff ( 3 8 ) . These findings are summarized i n Table XIII. The same res u l t s were obtained for the base composition of RNA contaminated samples by using Procedures 1 or 2 for e l u t i o n the nitrogenous constituents from paper chromatograms. The Characterization of DNA by U l t r a v i o l e t Absorption. As i t was mentioned i n the introduction, a great deal of information concerning the intact macromulecular state of DNA can be gained by measuring the extinction of i t s solution i n u l t r a v i o l e t l i g h t . Both nucleic acids and t h e i r purine and pyrimidine derivatives absorb strongly u l t r a v i o l e t l i g h t i n the neighbourhood of 2 6 0 m^. However, i t was found that nucleic acids show anomalously low extinctions ( 8 1 , 8 2 ) . This absorption anomaly probable denotes some considerable degree 76 TABLE XIII The Difference between the % of Recovery of Nitrogenous Bases from DNA Hydrolysates Using the E l u t i o n Technique of Chargaff (38) and the Column Chromatographic El u t i o n Method. Sample No. % Recovery bv proc. I s % Recovery by proc. 2 A % Increase by using proc. I I . A-14 7 6 . 1 8 5 . 6 9 . 5 A-16 69.8 8 1 . 0 1 1 . 2 A - I 7 5 9 . 8 7 1 . 3 U . 5 A - 1 8 8 7 . 5 9 9 . 5 1 2 . 0 A - 2 I 6 1 . 8 7 4 . 3 1 2 . 5 aver. % 1 1 . 3 increase 7 7 of intarmolecular organization. A dir e c t c o r r e l a t i o n has been shown to exist between the number of hydrogen-bonds joining the bases of a pair of DNA chains and the magnitude of the "hyperchromia" effect ( 8 3 ) . Rupture of the hydrogen bonds due to i o n i z a t i o n of the basic groups due to thermal denaturation, or to transfer to a medium of low io n i c strenght i s associated with considerable enhancement of absorption i n the u l t r a v i o l e t , whereas a ce r t a i n degree of depolymerization involving shortening of the chains, but without the rupture of interchain hydrogen bonds, appears.not to give t h i s e f f e c t . Kunitz ( 6 0 ) observed that depolymerization of DNA at pH 5 . by deoxyribonuclease was accompanied by ultimate increase i n absorption at 2 6 0 mjui of nearly 30%, and increases of similar order were reported by other investigators ( 6 2 , 8 2 ) . The findings that high molecular weight DNA can undergo a denatur-ation change by the actions of heat, acid, a l k a l i and deoxy-ribonuclease, and t h i s change i s manifested as about 33% increase of i t s u l t r a v i o l e t absorption became the basis for estimation the degree of denaturation of DNA samples. According to Chargaff et a l . ( 6 3 ) the atomic extincti o n c o e f f i c i e n t s with respect to phosphorous ^ -(p) of DNA preparation i s a good in d i c a t i o n of i t s state of degrad-ation. Table XIV summarizes these values for the d i f f e r e n t DNA preparations investigated during t h i s study. The data i n Table XIV show that: 7 8 1 . D e f i n i t e l y high ( £cp ) values (above 7 2 0 0 ) were obtained by using Procedure I, II and V. for preparing DNA from l i v e r t i s s u e s . In the case of i n t e s t i n a l mucosa only Procedure V. yi e l d s DNA with an C(p)higher than 7 2 0 0 . According to Chargaff ( 3 8 ) an £(p) value higher than 7 2 0 0 i s considered as a sign of denaturation of DNA. It i s not surprising that Procedure V. which involves hot 1 0 $ sodium choride ex-tr a c t i o n of tiss u e , and exposes nucle i c acids to the action of a l k a l i and acid, gives high 6 ^ ) values for both l i v e r and i n t e s t i n a l DNA preparations. 2 . The ^ (p) values of DNA samples from i n t e s t i n a l mucosa isolated by Procedures I., I I . , m . and IV. are lower, ranging from 6 3 0 0 to 6 9 0 0 . S i m i l a r l y lower values were obtained for l i v e r DNAs using Procedure I I I . 3 . For two DNA preparations, one obtained from l i v e r and the other from i n t e s t i n a l mucosa, very low values of £-(j=> ) ( 5 6 8 7 , 4413 respectively) were found by using Procedure IV. However, sample C -2 isolated by the same Procedure and from i n t e s t i n a l mucosa, shows an £<,pRvalue which i s i n very good agreement with the average value reported by Chargaff ( 3 8 ) . Perhaps the reason for the above mentioned very low values, i s , that those samples ( C - l and C - 3 ) were not properly p u r i f i e d , and may be they contained some inert phosphorous containing material, which does not absorb u l t r a v i o l e t l i g h t at the r.egion of 2 6 0 iyu 79 TABLE XIV The U l t r a v i o l e t E x t i n c t i o n C o e f f i c i e n t of DNA Preparations Expressed as £{ PA Values. Method of Preparation Source of Tissue Sample Number Procedure I. (29) ti ti it Liver tt tt tt A-k A-5 A - 6 7553 77W 75kQ a v e . 7-6H Procedure 11 . (20) tt tt tt A -16 A-17 75^0 6997 ave. 7268 Procedure 111(29,20) n tt tt A-13 A - 2 1 6128 a v e . 6?cf7 Procedure IV. (32) tt C-l 1687 Procedure V. (36.^7) tt B-3 7^70 Procedure I. i! II I n t e s t i n a l mucosa tt tt A - 8 A - 9 A -10 59*+8 6»+77 6560 aver. 6^?8 Procedure I I . ti u tt it tt A-15 A-18 A - 2 0 6789 723* 6737 ave. 6020 Procedure I I I . ti tt ti tt tt A-12 A-11+ A-19 6536 6371 76h6 avg. 68%1 Procedure IV. tt tt tt C - l C-2 66?l+ Procedure V. tt tt tt B - l B-2 7533 7895 a v g . 7711+ 8 0 With the same reasoning the apparently good values obtained for i n t e s t i n a l samples using Procedures I, I I , III , IV, and for l i v e r samples Procedure I I I , cannot be taken as evidence for the intactness of the secondary structure of these pre-parations, e s p e c i a l l y i f one considers the nitrogen and phosphorous contents of the samples. It has to be recalled (Table IX) that the percentages of nitrogen and phosphorous of DNA preparations obtained by a l l procedures except method V were much lower, than those reported i n the l i t e r a t u r e ( 3 8 ) therefore the samples must contain some impurities. Although i t was observed by Chargaff et a l . ( 8 4 ) that the e x t i n c t i o n of nucleoprotein i s not less than that of free nucleic acid, and the ^ ) values remained close to 6 5 0 0 the presence of some phosphorous containing impurity may obscure the £ [ j o)vaIues i n DNA preparations. Keeping i n mind the above discussed l i m i t a t i o n of Chargaff's £(p)value, i t i s now realized that a truer picture would have been gained about the state of denaturation of DNA samples by the application of the simple test described i n reference ( 3 8 ) v o l . 1 , page. 5 2 6 . A similar and valuable procedure for the estimation of DNA was described by Schack ( 6 2 ) . 8 1 The V i s c o s i t y of DNA Preparations. Important information can be obtained about the size and shape of macromolecules by the application o f simple viscosimetric measurements. Unfortunately only a limited number of samples could be characterized by t h i s method i n th i s study. Graphs 1 2 - 1 5 show the determination o f C ^ o f iy\ spec d i f f e r e n t DNA preparations by plotting- 1^-versus concentration. Table XV summarizes the i n t r i n s i c v i s c o s i t i e s of some DNA preparations. The molecular weights were estimated from equation ( 4 ) using the values determined experimentally. A few general statements can be made evaluating the data i n Table XV: 1 . A l l i n t r i n s i c v i s c o s i t i e s were very low. 2 . Highest i n t r i n s i c v i s c o s i t y values were obtained with DNA preparations C - l , C>2, C - 3 . isolated by the sonication method of Zubay ( 3 2 ) . 3 . Because of the low L / ^ l values the molecular weights of DNAs calculated from equation ( H ) were very low. 4 . No measurable v i s c o s i t i e s were found for preparations of DNAs isolated by the hot 1 0 $ sodium chloride extraction method. C F . Thomas reports some Devalues for DNAs isolated by the procedures of Schwander and Signer ( 1 2 ) and by the detergent method of Kay et a l . ( 2 0 ) . These values range from 4 8 to 5 7 d l . / g . Reichmann et a l . ( 8 5 ) performed also some 8 2 e c c 13 i 12-f4-ID-S' 8-7-.4 - r -NOTE-. No. 1= R-16 Mo. 2= R-17 No.2 —i— .8 -T— 1.0 -T 1.2. -T- -r L6 18 i 24 20 i-2 c o n c e n t r a t i o n in g .x lo'^ioo ml 26 Fig. 12. Plot of the. " R e d u c e d V i s c o s i t y (^ Sc e c) V e r s u s Concentration (9/100ml-) of D N A S a m p l e s R- |6 o n d R-|7. o 5 -2L2£ No.l Nofe- . No i = R - I Z No.*=R-ll ConcervtraHon in g x Kf^loOml. F i g . 13. Plot of the "Reduced V i s c o s r t y (^^£) V e r s u s C o n c e n t r a t i o n ( g / i o o m l ) of DNR S a m p l e s R-12 a n d B-2|. 8 4 H\ SP<?c, c u ° NOTE:No.l= A-15. No.2=A-l8-__Q O Na 2 -O o — Concentration in i 4 I s io 12. • h j$ jg 20 g*lO"/IOOmt. Fig. 14. Plot of the Reduced Viscosity ( ' T l ^ > e c - ) versus Concentration (g/lOO ml.) of D N A Samples A—15 and A-18. 8* • Mo. I N O T E - N o . U C - 2 No. 2= C-3 No3= C-l N o ! -Mo.3 10 16 16 10 Concerto-lion in "J2 9 Xlo ' ^ ioowi . F i g . 15 . Plot of the R e d u c e d V i s c o s i t y C " 1 ? * 0 ) V e r s u s , C o n c e n t r a t i o n (g/ioo m l ) of D N f l S a m l e t C - l , C - l , C-3 86: ° v i s c o s i t y measurements on supposedly high molecular weight samples, and the i r values were i n the neighbourhood of - 50. Just recently S. Kit (86) reported similar£/*p values for l i v e r DNAs isolated by the modification of Kirby (22,23) procedure. Comparing the data of Table XV with the above mentioned values, one would conclude that a l l preparations isolated during t h i s study are degraded. However t h i s i s not n e c e s s a r i l y the case. On inspection of the a r t i c l e s pub-lished by the above mentioned investigators, one can see that a l l t h e i r measurements were made on a series of low v e l o c i t y gradients, and the values were extrapolated to zero gradient. Up to recently no standard values were given i n the l i t e r a t u r e for the v i s c o s i t y of c a r e f u l l y prepared specimens. This was due to the fact that investigators did not r e a l i s e the gradient dependence of DNA solutions. Greenstein et a l . (64) studied i n d e t a i l the nature of v i s c o s i t y of c a l f thymus preparations. They concluded that the v i s c o s i t y of thymonucleate was a function of v e l o c i t y gradient, i n other words solutions of t h i s substance possessed anomalous or s t r u c t u r a l v i s c o s i t i e s ..; This s t r u c t u r a l v i s c o s i t y i s due to a high degree of molecular asymmetry of sodium nucleate. The pressure time product for solutions of DNA are not independent of the applied pressure ^ e x c e p t for either very d i l u t e solutions of DNA or for very high pressures. Since the present t h e o r e t i c a l status of shear independence allows interpretation of the i n t r i n s i c v i s c o s i t y only when Brownlan motion i s overhelming, v i s c o s i t y measurements 8 7 TABLE XV I n t r i n s i c V i s c o s i t i e s and Estimated Molecular Weights of Different DNA Preparations. Method of Preparation Source of Tissue Sample No. from graphs i n dl./g. i n dl./g. calculated from equ.(6) Molecular weights estimated according to equ.(£) Proc. I I . Liver it A - 1 6 A - 1 7 9 . 7 6 . 0 9 . 6 5 . 8 1 + 2 ^ , ^ 0 0 1 9 ^ , 5 0 0 Proc. I I I . II tt n A - 1 3 A-21 1 0 . 2 k.o 9 . 6 1 0 0 , 7 0 0 J+ 6 0 , 5 0 0 Proc. I V . ( 3 2 ] ti c - 3 16 .1 1 6 . ^ 9 6 6 , ^ 0 0 Proc. I I II ti Small i n t e s t . mucosa A - 1 5 A - 1 8 A - 2 0 *f . 3 3.1 3 . 2 8 . 6 1 0 8 , 1 0 0 6 6 , 0 0 0 3 ^ 9 , 0 0 0 Proc. I I I . • A - 1 2 1 1 . 0 1 1 . 0 5 2 0 , 3 0 0 Proc. IV. » " C - l C -2 1 3 . 2 2 1 . 3 1 3 . 2 2 1 . 7 700 , 0 0 0 r , 5 2 6 ; o o o Proc. V. • B - 2 not measurable 8 8 must be performed over a range of shears and then extrapolated to zero shear. Thus i t i s necessary to make v i s c o s i t y measure-ments i n very d i l u t e solutions and at very low rates of shear, and t h i s condition present experimental d i f f i c u l t i e s . In t h i s study none of these d i f f i c u l t i e s were over-come, using only a simple Ostwald type of viscosimeter. The recent application of the Coette viscometers ( 8 7 ) greatly f a c i l i t a t e s viscosimetric measurements. Knowing the fact that the ordinary Ostwald viscometers give lower values forT^ , the values summarized i n Table XV do not necessary mean that a l l preparations were badly denatured. According to Reichmann et a l . ( 8 5 ) the r a t i o o f C^lat zero gradient and at 1 0 0 0 sec.~*- i s 2 . 5 . Most of the simple Ostwald viscometers have an average v e l o c i t y gradient around 1 0 0 0 s e c - 1 therefore multiplying the data i n Table XV by t h i s factor would give much higherC"?^values. However for a q u a l i t a t i v e comparison of d i f f e r e n t preparations the i n t r i n s i c v i s c o s i t i e s obtained by the Ostwald apparatus provide some measure of differences between the DNA preparations. The values obtained for DNA samples isolated by Procedures II and III , are too low, even multiplying them with the factor of 2 . 5 . The change of physico-chemical properties of these DNA preparations may be due to four important f a c t o r s : 89 a. During the course of deproteinization DNA preparations of Procedure II and III were exposed to somewhat lower pH values (4.3). It was mentioned before (54, 59) that acide or a l k a l i e s may cause the rupture of hydrogen bondings i n DNA molecules. b. The presence of contaminating protein may influence the v i s c o s i t y of the i r solutions. It was demonstrated by several investigators (88,89) that as the protein content of DNA increased the c h a r a c t e r i s t i c v i s c o s i t y of i t s solution decreased, having a sharp drop of approximately half of the o r i g i n a l v i s c o s i t y value, when protein content reached about 10%. c. The effect of storage of DNA preparations i n vacuo over phosphorous pentoxide may cause some deformation i n the macromolecular state of the samples. Zamenhof (90,91) demon-strated that storing over phosphorous pentoxide i n vacuo of the transforming p r i n c i p l e s of Hemophilus influenzae resulted i n 80$ i n a c t i v a t i o n . P a r a l l e l experiments with c a l f thymus DNA. also resulted the decrease of v i s c o s i t y of i t s solutions. Nothing i s known about the nature of changes accompanying the dehydratation of DNA, but i t seems probably that breaking of a few l a b i l e bonds, such as hydrogen bonds, takes place during the storage of DNA preparations i n vacuo. d. The presence of heavy RNA contamination may influence the molecular weights and v i s c o s i t y properties of these pre-parations. Sample B-2 prepared according to Procedure V does not y i e l d any measurable v i s c o s i t y , showing that the secondary 90 structure of DNA.is completely destroyed by t h i s d r a s t i c method. An interesting observation should be mentioned here: the effect of starvation of animals on the DNA preparation. A l l DNA specimens isolated by Procedure II and III, were obtained from non-starved animals. In most of these cases the macroscopic appearance of DNA was non fibrous. Thus starvation seemed to be influence the macromolecular state of DNA. This phenomenon cannot be explained at present, but i t would be very interesting to speculate that i t i s connected with the high metabolic a c t i v i t y of i n t e s t i n a l mucosa, or with the l e v e l of glycogen i n l i v e r . The best values were obtained by the sonication procedure. This i s quite surprising because several investigators found that ultra-sound waves damage the hydrogen bondings i n DNA molecules. It has to be mentioned that the sonicator used i n t h i s experiment works only at the region of 9 k.cycles/sec. which i s far from the ultra-sound range (above 1 6 k.cycles/sec.) The interpretation of the i n t r i n s i c v i s c o s i t y i n terms of size and shape requires that the value at zero gradient be known, therefore the molecular weights estimated from the apparent L?V)U of DNA samples are not r e l i a b l e . Dr. Reichmann performed a molecular weight determination by l i g h t scattering measurement on DNA sample A - 2 0 . The molecular weight of t h i s preparation was found to be 6 0 0 , 0 0 0 which was somewhat higher than the value given i n Table XV. 9 1 SUMMARY 1 . Deoxyribonucleic acid (DNA) has been isolated from small amounts of l i v e r and i n t e s t i n a l mucosa of rat ( 1 - 1 0 g.) by the following procedures; a. The f i r s t method ( 2 9 ) consisted of the removal of nuc l e i from the tissue by means of controlled homogenization, and subsequent deparation of n u c l e i from extraneous c e l l u l a r elements by adsorbtion on diatomaceous earth. DNA was deproteinized by salt saturation. b. In the second procedure ( 2 0 , 3 1 ) the nucleic acids were extracted and deproteinized by detergent solutions. Ribonucleic acid (RNA) and DNA were separated by f r a c t i o n a l p r e c i p i t a t i o n with iso-propyl alcohol. c. In the third procedure crude DNA was obtained according to the f i r s t method and the crude product was further p u r i f i e d by the detergent treatment of the second procedure. d. The fourth method ( 3 2 ) was based on the d i s i n t e -gration of tissues by high frequency sonic o s c i l l a t i o n s , separation of nuclear fragments by centrifugation, extraction of nucleoprotein with strong salt solution, and deprotein-i z a t i o n with chloroform-amyl alcohol mixtures. e. In the f i f t h method ( 3 6 , 3 7 ) nucleic acids were extracted from tissues by hot 10% sodium chloride solutions, RNA and DNA were separated by incubating the mixture with 0 . 1 N sodium hydroxyde, and DNA was precipitated from the basic solution by ne u t r a l i z a t i o n with concentrated 92 hydrochloric ac i d . The chemical and physico - chemical properties of DNA preparations isolated during t h i s study, have been compared. On the basis of experimental findings the procedures used for preparing DNA have been evaluated and discussed. The method of Emanuel arid Chaikoff (29) appeared to be-very s a t i s f a c t o r y i n three respects: . a. DNA preparations could be accomplished i n nine hours, b. DNA was never exposed to heat, acid a l k a l i , or low ionic strength. c. The p o s s i b i l i t y of RNA contamination was greatly reduced by the previous i s o l a t i o n of n u c l e i from homogenates. • • However, some d i f f i c u l t i e s have not been overcome by t h i s procedure. F i r s t a l l DNA samples were heavily contaminated with protein. Second, some sign of denaturation have been demonstrated i n l i v e r preparations and t h i r d , very low y i e l d s of DNA were obtained both from l i v e r and i n t e s t i n a l t i s s u e s . Becausd the actual quantities of DNAs were very small, the p u r i f i c a t i o n and handling of these samples, were d i f f i c u l t . Best y i e l d s of DNA have been obtained both from l i v e r and i n t e s t i n a l mucosa by the detergent method of Kay et a l . '(30) and Stevens et a l . ( 3 D . The preparations contained considerable protein and RNA impurities, indicated by 9 3 q u a l i t a t i v e aminoi-acid tests and by the determination of the base composition. Some macromolecular damage was observed by using v i s c o s i t y measurements, and i n the case of DNA of l i v e r t i s s u e , the high values obtained, also indicated some degree of denaturat ion. 5 . The combination of the methods of Emanuel and Chaikoff ( 2 9 ) and Kay et a l . ( 2 0 ) have resulted i n some improvements over the Emanuel and Chaikoff procedure, indicated by the general decrease of protein contaminations and s l i g h t l y higher yi e l d s of DNAs from l i v e r preparations. Although the deproteinization of DNA by detergent appeared to be more e f f e c t i v e , than the salt saturation alone, the con-siderable losses of DNA during the preparation, the complete removing of impurities and avoiding degradation of samples have not been overcome by t h i s procedure. 6 . .DNA preparations isolated by the method of Bendich et a l . ( 3 6 ) and Tyner et a l . . ( 3 7 ) have' been characterized by a high 'degree of chemical purity, however the secondary macro-molecular structure of DNA was completely destroyed by the d r a s t i c heat and acid - a l k a l i treatments. 7 . The procedure of Zubay ( 3 2 ) have been appeared the most _ promising for obtaining DNA from small amount of tissue, provided that the procedure i s improved by further p u r i f i -cations. This method had the following advantages: 94 a. DNA preparations could be accomplished i n ten hours. b. The yie l d s of DNA were quite high. c. Contaminating RNA was greatly reduced by the previous washings of nuclear fragments with physiological saline solutions. d. The secondary structure of DNA was not effected appreciably by the high frequency sonic o s c i l l a t i o n s shown by.the r e l a t i v e l y high£VJ values and low u l t r a v i o l e t extinction c o e f f i c i e n t s . The presence o f some impurities were indicated by the low nitrogen and phosphorous contents of these preparations. An improved technique has been described for the elu t i o n of purine and pyrimidine bases from paper chromatograms. •Generally. 10$ -increase i n recoveries of nitrogenous bases have been found by t h i s procedure compared to the extraction methods used previously. 95 BIBLIOGRAPHY 1. S i n s h e i m e r , R . L . , Sc ience 125, 1123 (1957). 2 . M i e s c h e r , F . , H o p p e - S e y l e r 1 s Med. Chem. U n t e r s . 441 (1871) . 3. Al tmann, R . , A r c h . A n a t . P h y s i o l . , P h y s i o l . A b t . 524 (1889). 4 . Neumann, A . , A r c h . A n a t . P h y s i o l . 552 (1899). 5. F e u l g e n , R . , Z . P h y s i o l . Chem. 20, 261 (1914) 6. Levene, P . A . , Z . P h y s i o l . Chem. 4 £ , 370 (1905) 7. Levene, P . A . , J . B i o l . Chem. $1, 441 (1922) 8. Jones , W., " N u c l e i c A c i d s - T h e i r Chemical P r o p e r t i e s and P h y s i o l o g i c a l C o n d u c t . " 2nd E d . Longmans Green & C o . , London, (1920) . 9. Levene, P . A . , Bass , L . W . , " N u c l e i c A c i d s " Chemical C a t a l o g Comp., New Y o r k , (1931) . 10. Jones , V/ . , P e r k i n s , M . E . , J . B i o l . Chem. 62, 290 (1924-25). 11 . J o r p e s , E . , A c t a Med. Scand. 68, 253 (1928) . 12. S i g n e r , R . , Schwander, H . , H e l v . Chim. A c t a 1521 (1950) 13 . M i r s k y , A . E . , and P o l l i s t e r , A . M . , J . Gen. P h y s i o l . ^ 0 , I I7. (1946) . 14. P o l l i s t e r , A . W . , M i r s k y , A . E . , J . Gen. P h y s i o l . 30, 101 (1946) . 15. M i r s k y , A . E . , and P o l l i s t e r , A . M . , P r o c . N a t l . A c a d . S c i . U . S . 28 , 344 (1942) . 16 . G u l l a n d , J . M . , J o r d a n , D . O . , T h r e l f a l l , G . J . , J . Chem. Soc . 1129, (1947) . 17. Sevag, M . G . , Lackman, D . B . , Smolens, J . , J . B i o l . Chem. 124, 425 (1938) . 18. Grampton, C . F . , L i p s h i t z , R . , C h a r g a f f , E . , J . B i o l . Chem. 206. 499 (1954) . 19. Marko, A . M . , B u t l e r , G . C . , J . B i o l . Chem. 190. 165 (1951). 96 2 0 . Kay, E.R.M., Simmons, N.S., Dounce, A. A., J. Am. Chem. Soc. 2 4 , 1 7 2 4 ( 1 9 5 2 ) 2 1 . Simmons, N.S., Chavos, S., Orbach, H.K., Federations Proc. 1 1 , 396 ( 1 9 5 2 ) . 2 2 . Kirby, K.S., Biochem. J. 6 6 , 4 9 5 ( 1 9 5 7 ) . 23. Kirby, K.S., Biochim. et Biophys. Acta 36, 1 1 7 ( 1 9 5 6 ) 2 4 . K i t , S., J. B i o l . Chem. 2 1 5 , 1 7 5 6 ( I 9 6 0 ) 25. Georgiev, G.P., Biochemistry U.S.S.R. 2 4 , 4 4 3 (1959) 26. Paterson, R.P.P., and Zbarsky, S.H., Canadian J . of Biochem. and Physiol. 16 , 755 ( 1 9 5 8 ) 27. Nixon, J.C., Zbarsky, S.H., Canadian J . Biochem. and Physiol. 1 2 , 1 4 0 5 (1959). 28. Zbarsky, S.H., Hori, A., and Findlay. B.S., Canadian J. Biochem. and Physiol. 16 , 1 1 8 ? ( 1 9 5 8 ) . 29. Emanuel, C.F., Chaikoff, I.L., Biochim. et Biophys. Acta 2 4 , 2 6 1 ( 1 9 5 7 ) . 3 0 . Emanuel, C.F., Chaikoff, I.L., Biochim. et Biophys. Acta 24, 2 5 4 ( 1 9 5 7 ) . 3 1 . Stevens, V.L., and Duggan, E.L., J. Am. Chem. Soc. 7 9 . 5 7 0 3 ( 1 9 5 7 ) . 3 2 . Zubay, G., Biochim et Biophys. Acta H, 2 4 4 ( 1 9 5 9 ) . 3 3 . Goldstein, G., and Stern, K.G., J . Polymer. Science, V, 6 8 7 ( 1 9 5 0 ) . 3 4 . Guild, R.J., De f i l i p p e s , H., Biochim et Biophys. Acta 2 6 , 2 4 1 ( 1 9 5 7 ) . 3 5 . Oster, J . , Gen. Physiol., 1 1 , 89 ( 1 9 4 7 ) . 3 6 . Bendich, A., Russell, P.J. J r . , Brown, G.B., J . B i o l . Chem. 2 2 0 , 3 0 5 ( 1 9 5 3 ) 3 7 . Tyner, E.P., Heidelberger, C , and LePage, G.A., Cancer Research, 1 1 , 1 8 6 ( 1 9 5 3 ) . 3 8 . Chargaff, E., Davidson, J.N., "The Nucleic Acids" Acad. Press. Inc., New York, ( 1 9 5 5 ) . 97 39. Colowick, S.P., Kaplan, NuO. , "Methods i n Enzymology" v o l . I I I . 703, Acad. Press Inc., New York, (1957). 40. Watson, J.D.,.. Crick, F.H.C., Nature, 1£L, 737 (1953). 41. Hawk, P.B., Oser, B.L., Summerson, W.H., " P r a c t i c a l P h y siological Chemistry" 13th Ed. McGraw. H i l l Co. New York (1947) . 42. B a r t l e t , G.R., J . B i o l . Chem., 224, 466 (1959) . 43. Lederer, M., Lederer, E., "Chromatography" 2nd Ed. Elsevi e r Pub. Co. New York, (1957) . 44. P h i l l i p s , D.M.P., Biochim. et Biophys Acta, 3,, 341 (1949). 45. Wischer, E., Chargaff, E.J. Biol.-Chem., 1Z6, 715 (1948) . 4 6 . Wyatt, G.R., Biochem. J., 48, 584 ( 1 9 5 D . 4 7 . Chargaff, E., Magasanik, B., Doniger, R., Vischer, E., J. Am. Chem. Soc. 21*. 1513 (1949). 4 8 . Personal Communication from Dr. S.H. Zbarsky. 4 9 . Personal Communication from I. Csizmadia. 50. Vischer, E., Chargaff, E., J . B i o l . Chem., 168, 78I (1947) . 51. Vischer, E., Chargaff, E., J. B i o l . Chem., 1Z6, 703 (1948) . 52. Hochkiss, R.D., J . B i o l . Chem., 175, 315 (1948) . 53. Chargaff, E., Vischer, E., Doniger. R., Green, C., Misani, F., J. B i o l . Chem. 122, 405 (1949) . 54. Vilbrandt, C.F., Tennent, H.G., J . Am. Chem. S o c , 6 £ , 1806 (1943) . 55. C e c i l , R., Ogston, A.G., J. Chem. S o c , 1382 (1948) . 56. Kahler, H., J. Phys. and C o l l o i d . Chem. $2, 676 (1948) . 57. Signer, R., Caspersson, T., Hammarsten, E., Nature., 141, 122 (1938) . 58. Tennent, H.G., v'ilbrandt, C.F., J. Am. Chem. S o c , 6£, 424 (1943) . 59. Creeth, J.M., Gulland, J.M., Jordan, D.O., J . Chem. S o c , 1141 (1947) . 9 8 60. Kunitz, M., J. Gen. Physiol., 3J.f 363 (1950). 61 . Thomas, R., Biochim. et Biophys. Acta, 14, 231 (1954) . 62. Schack, J., J . B i o l . Chem., &77 (1958). 63. Chargaff, E., Zamenhof, S., J. B i o l Chem. 17_L 327 (1948) . 64. Greenstein, J.P.. Jenrett, W.V., J . Natl. Cancer. Inst. 1, 77 (1940) . 65. Daniels, F., Mathews, J.H., Williams, J.W., Bender, P., Alberty, R.A., "Experimental Physical Chemistry" McGraw-Hill. Book. Co. New York, (1956). 66. Kraemer, G., Ind. Eng. Chem., 30, 1200 (1938) . 67. S p i t k o v s k i i , D.M., Biophysics U.S.S.R. 1, 319 (1956). 68. Pregl, F., "Quantitative Organic Microanalysis" 5th ed. Ju l i u s Grant. London ( 1 9 5 D . 6 9 . Cohn, W.E., Science 109., 377 (1949) . 70. King, J., Biochem J. 26, 292 (1932). 7 1 . Paterson, A.R.P.,: The Metabolism of 2 - C 1 4-Adenine i n the Adult Male Rat. Master of Art Thesis, U.B.C. (1952) . 72. J e r v e l l , K.F., Di n i z , C.R., Mueller, G.C., Arch. Biochem. and Biophys. £8? 157 (1958). 73. F r i c k , G., Biochim et Biophys. Acta, 1 ,^ 374 (1954). 74. Zamenhof S., "Biochemical Preparations" v o l . VI. John Wiley & Sons Inc. New York, (1958). 75. Butler, J.A.V., P h i l l i p s , D.M., and Shooter, K.V., Arch. Biochem. and Biophys. 7_1, 423 (1957). 76. Woodhouse, D.L., Biochem. J. £6, 349 (1954). 77. Chargaff, E., L i p s h i t z , R., J. Am. Chem. S o c , 2li 3658 (1953) . 78. Chargaff, E., L i p s h i t z , R., Green, C , Hodes, M.E., J . B i o l . Chem. 122, 223 ( 1 9 5 D . 9 9 7 9 . Cohen, S.S., Advances i n Virus. Res. 3., 1 (1953). 80. Vischer, E., J . B i o l . Chem., 177. 429 (1949). .81. Oster, G., and Grimsson, H., Arch. Biochem. and Biophys., 24, 1 1 9 (1949). 82. Tsuboi, K.K., Briochim. et Biophys. Acta, 6 , 202, ( 1 9 5 0 ) . 8 3 . S p i r i n , S., Gavrilova, L.P., B e l o z e r s k i i , A.N., Biochemistry :;U;S.S:iR. 24, 5 5 6 (1959,). 84. Magasanik, B., and Chargaff, E., Biochim. et Biophys. Acta, Z> 3 9 6 (1951). 8 5 . Reichmann, M.E., Rice, S.A., Thomas C.A., Doty, P., J. Am. Chem. S o c , 26, 3047 (1954). 86. K i t . S., Arch. Biochem. and Biophys. 82, 3 1 8 (I960).' 87. Pouyet, J ., J . Chim. Phys. 48, 9 0 ( 1 9 5 D . 8 8 . Tongur, V.S., Diskina, B.S., Sp i t k o v s k i i , D.M., Bio-chemistry, U.S.S.R. 22, 879 (1957). 8 9 . Diskina, B.S., S p i t k o v s k i i , D.M., Tongur, V.S., Bio-chemistry, U.S.S.R. 2 1 , 3 6 0 ( 1 9 5 8 ) . 90. Zamenhof, S., Alexander, H.E., and Leidy, G.C., J . Experimental Med. %8, 373 (1953). 91. Zamenhof, S., Griboff, G., Marul'lo, N., Biochim. et Biophys. Acta, 11, 4 5 9 ( 1 9 5 4 ) . 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0106330/manifest

Comment

Related Items