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An investigation of RNA induction in amphibian tissues Biggin, William Philip 1964

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AN INVESTIGATION OF RNA INDUCTION IN AMPHIBIAN TISSUES by  WILLIAM PHILIP BIGGIN B.Sc.  (Honours), The U n i v e r s i t y o f B r i t i s h Columbia, 1962  A t h e s i s Submitted i n P a r t i a l F u l f i l m e n t o f the Requirements f o r the Degree o f MASTER OF SCIENCE i n the Department o f Zoology  We accept t h i s t h e s i s as conforming t o the required. staAdand  THE UNIVERSITY OF BRITISH COLUMBIA September, 1964  In presenting  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 of  the requirements for an advanced degree at the U n i v e r s i t y B r i t i s h Columbia, I agree that  of  the 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 reference.and study.  I f u r t h e r agree that  per-  m i s s i o n f o r extensive copying of 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 granted by the Head of my Department or  his representatives.  I t i s understood that,copying or p u b l i -  c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not without my  written  Department  of  permission*  P^O~^P^  The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada Date  by  Sg^z^W^/-  Iff^ (J&y  be  allowed  ii ABSTRACT Ribonucleic acid  (RNA)  i s o l a t e d and p u r i f i e d by The  from c a l f s p l e e n t i s s u e  a m o d i f i e d K i r b y phenol  was  procedure.  a b s o r p t i o n maximum o f the i s o l a t e o c c u r r e d at 260. m|t  i n d i c a t i n g the presence  o f n u c l e i c a c i d s and the a b s o r p t i o n  minima recorded at 230 m\l and 280 m\i i n d i c a t e d the absence o f p e p t i d e s and p r o t e i n s . the presence DNA  o f RNA  and carbohydrate  Colorimetric analyses i n d i c a t e d  and the absence o f p e p t i d e , p r o t e i n , contamination.  Chromatographic  i n d i c a t e d the absence o f carbohydrate  contamination  a f t e r the p u r i f i c a t i o n w i t h 2-methoxyethanol. RNA  prepared by the phenol method was  The  analysis only  spleen  undegraded and demon-  s t r a t e d three d i s t i n c t m o l e c u l a r s p e c i e s when analysed w i t h the u l t r a c e n t r i f u g e ; 8S fraction.  a 2 7 S fraction,  an I.8.S. f r a c t i o n and  Competent e a r l y g a s t r u l a ectoderm and  o f Xenopus l a e v i s exposed to undegraded s p l e e n RNA s t r a t e d no t i s s u e - s p e c i f i c i n d u c t i o n . in vitro  an  embryos demon-  However, i n both  the  and i n v i v o experimental s e r i e s an enhancement of  development was phenomena was  observed.  discussed.  A p o s s i b l e e x p l a n a t i o n of t h i s  vi  ACKNOWLEDGMENT  The author wishes t o acknowledge the guidance and encouragement o f Dr. C.V. Finnegan throughout the c u r r e n t investigation.  I am i n d e b t e d t o Dr. W.S. Hoar and Dr. P.  Ford f o r t h e i r comments. M.E.  A p p r e c i a t i o n i s expressed to Dr.  Reichmann o f the A g r i c u l t u r a l Research S t a t i o n o f the  Government o f Canada f o r t e c h n i c a l a s s i s t a n c e and the provision of research  facilities.  TABLE OF CONTENTS  Abstract T a b l e o f Contents L i s t o f Tables L i s t o f Figures Acknowledgment Introduction M a t e r i a l s and Methods Results Part I Part I I Discussion Summary References Appendix  iv  LIST OF TABLES page Table 1.  The i s o l a t i o n and p u r i f i c a t i o n o f c a l f spleen r i b o n u c l e i c a c i d (RNA).  Table 2.a.Spectrophotometry analysis s-RNA.  o f commercial l i v e r  k lk  2 . b . S p e c t r o p h o t o m e t r y a n a l y s i s o f c a l f s p l e e n RNA a f t e r one phenol e x t r a c t i o n .  lk  2 . c . S p e c t r o p h o t o m e t r y a n a l y s i s o f c a l f spleen RNA a f t e r three phenol e x t r a c t i o n s .  lk  Table 3.  Q u a l i t a t i v e experiments to demonstrate the presence or absence o f v a r i o u s m a t e r i a l s i n the i s o l a t e d sample.  16  T a b l e k.a.The e f f e c t o f i s o l a t e d c a l f spleen RNA on amphibian competent ectoderm i n v i t r o .  26  k.b.The e f f e c t o f i s o l a t e d c a l f spleen RNA on whole embryos o f Xenopus l a e v i s i n v i v o .  26  V  LIST OF FIGURES F a c i n g page F i g . 1.  F i g . 2.  F i g . 3* Fig. 4.  Sedimentation o f the v a r i o u s l a y e r s f o l l o w i n g homogenization and c e n t r i f u g a t i o n o f c a l f s p l e e n t i s s u e i n napthalene1 , 5 - d i s u l f o n i c a c i d and p h e n o l .  6  A stage 9+ Xenopus l a e v i s . The d o t t e d a r e a r e p r e s e n t s the competent ectodermal r e g i o n which was removed f o r the i n v i t r o study.  6  The u l t r a v i o l e t l i v e r s-RNA.  a b s o r p t i o n spectrum o f  commercial 12  The u l t r a v i o l e t a b s o r p t i o n s p e c t r a o f i s o l a t e d c a l f s p l e e n RNA f o l l o w i n g one phenol arid three phenol e x t r a c t i o n s .  13  F i g , 5»  Chromatographic a n a l y s i s o f p u r i f i e d RNA s o l u t i o n b e f o r e 2-methoxyethanol e x t r a c t i o n . A f t e r the e x t r a c t i o n no spot was observed. 19  Fig. 6.  Sedimentation diagram  Fig. 7.  S c h l i e r e n photograph o f a 0.1 M. NaCl s o l u t i o n of s p l e e n RNA a f t e r r e a c h i n g the speed o f 4 4 , 7 7 0 r.p.m. i n the 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 . From the meniscus, M, the peaks have sedimentation c o e f f i c i e n t s o f 8S, 18S, and 2 7 S .  21  F i g . 8.  Sedimentation diagram  22  Fig. 9 .  The combined graphs from the u l t r a c e n t r i f u g e a n a l y s i s o f c a l f s p l e e n RNA ( f i g . 7 ) . The s l o p e o f the curves i s a measure o f the sedimentation c o e f f i c i e n t s , which i s r e a d o f f on a s p e c i a l t r a n s p a r e n t p r o t r a c t o r designed by Markham ( i 9 6 0 )  o f commercial  l i v e r s-RNA  o f c a l f s p l e e n RNA  21  23  F i g . 1 0 . I s o l a t e d Xenopus ectoderm which had been c u l t u r e d i n a s o l u t i o n o f c a l f s p l e e n RNA f o r 5 days. No t i s s u e - s p e c i f i c i n d u c t i o n can be seen, but, i n c o n t r a s t to the c o n t r o l s e r i e s the c e l l s are b e t t e r developed and more numerous. 25 Fig. 11.  S e c t i o n through c o n t r o l Xenopus embryo c u l t u r e d i n an "unconditioned* medium.  28  S e c t i o n through experimental Xenopus embryo c u l t u r e d i n an RNA-enriched medium.  28  1  Fig. 12.  1 INTRODUCTION Niu and T w i t t y organizer material was explanted  (1953) demonstrated that when amphibian ( d o r s a l l i p o f b l a s t o p o r e - a x i a l mesoderm)  i n t o a non-nutrient  d i f f u s e d from the explant  medium, substances which  were capable o f i n i t i a t i n g  f e r e n t i a t i o n i n exposed competent ectoderm.  N i u (1956)  c h a r a c t e r i s e d the m a t e r i a l as r i b o n u c l e o p r o t e i n  and suggested  that the RNA f r a c t i o n was the a c t i v e component, T a k a t a (1955) obtained kidney and r e p o r t e d  dif-  Yamada and  d i f f e r e n t i a t i o n u s i n g guinea p i g  t h a t the i n d u c t i v e f r a c t i o n was r i c h i n  ribonucleoprotein. Niu  (1958a,b) u s i n g  c a l f thymus RNA, r e p o r t e d  successful  t i s s u e - s p e c i f i c i n d u c t i o n i n amphibian t i s s u e and concluded t h a t thymus RNA r e s u l t e d i n thymus-like  histogenesis.  Saxen and Toivonen (1962) i n d i s c u s s i n g Niu's r e p o r t s i d e r e d the d i f f e r e n t i a t i o n to be poor.  Yamada  con-  (1961)  repeated Niu's thymus RNA experiments u s i n g i d e n t i c a l  iso-  l a t i o n procedures and was unable to show any t i s s u e - s p e c i f i c e f f e c t on amphibian t i s s u e s w i t h c a l f thymus RNA. More r e c e n t l y , H i l l m a n  and N i u (l962a,b)  using  i s o l a t e d from embryonic c h i c k b r a i n and. notochord s p e c i f i c e f f e c t s on n e u r a l  and n o t o c h o r d a l  RNA  reported  histogenesis  both i n v i v o and i n v i t r o but no e f f e c t was observed with c h i c k l i v e r RNA.  In the i n v i v o experiments an enlargement  o r d u p l i c a t i o n o f b r a i n and n o t o c h o r d a l  s t r u c t u r e s was  observed. Butros (1963) found that RNA i s o l a t e d from embryonic c h i c k b r a i n and a d u l t r a t heart  resulted i n hyperplasia  2 of the  epidermis w h i l e adult  r a t l i v e r had  no  effect.  the other hand, an u n i d e n t i f i e d adult p a n c r e a t i c the development of the Yamada  RNA  On  enhanced  endodermal e p i t h e l i u m .  (1961, I962) and  have r e p o r t e d d e f i n i t e and  Yamada and  Takata  s p e c i f i c induction  mesodermal t i s s u e when competent ectoderm was  (1961)  of n e u r a l  and  exposed to  p r o t e i n f r a c t i o n s i s o l a t e d from guinea p i g bone marrow. However, Hillman and  Niu  specific histogenesis  (1963b) were unable to detect  i n t i s s u e s which had  i s o l a t e d embryonic c h i c k b r a i n  been exposed to  protein.  In the present i n v e s t i g a t i o n an attempt was i s o l a t e c a l f spleen r i b o n u c l e i c a c i d (RNA) K i r b y procedure (1962), and contaminants from the  i s o l a t e u l t r a v i o l e t (UV) and  qualitative tests for protein,  i s o l a t e d and  other  t e s t the p u r i t y of  carbohydrates and  the  desoxy-  Sedimentation analyses- of  samples were made to determine the degree of  d e g r a d a t i o n of the RNA. the  and  a b s o r p t i o n s p e c t r a were measured,  r i b o n u c l e i c a c i d were a p p l i e d . the  To  made to  by a m o d i f i e d  to remove p r o t e i n  isolate.  any-  possible  F i n a l l y , the b i o l o g i c a l a c t i v i t y  p u r i f i e d s p l e e n RNA  l a e v i s tissues i n both i n vivo  and  was  of  t e s t e d w i t h Xenopus  i n v i t r o systems.  MATERIALS AND METHODS I.  P r e p a r a t i o n o f c a l f spleen r i b o n u c l e i c a c i d  (RNA).  C a l f spleen was chosen as the source o f r i b o n u c l e i c acid  s i n c e t h i s organ,  a h i g h RNA content,  a c t i v e l y s y n t h e s i z i n g p r o t e i n , has  and i s r e l a t i v e l y f r e e o f connective  t i s s u e , thereby r e d u c i n g the p o l y s a c c h a r i d e  contamination  of t h e i s o l a t e d m a t e r i a l . Fresh s p l e e n t i s s u e was f r o z e n by b e i n g p l a c e d on s o l i d carbon d i o x i d e immediately slaughtered calves.  f o l l o w i n g removal from  Upon r e t u r n t o the l a b o r a t o r y , samples  of the f r o z e n t i s s u e were separated from the connective t i s s u e capsule and s l i c e d i n t o s m a l l p i e c e s lcm. x 2 c m . ) .  (approximately  R i b o n u c l e i c a c i d was i s o l a t e d from  t i s s u e , s l i c e s by a m o d i f i e d K i r b y procedure i n T a b l e I (see, a l s o , Appendix i ) .  these  (1962)  as shown  The t i s s u e was homo-  g e n i z e d i n 2 . 5 m l . o f 0 . 0 1 5 M. n a p t h a l e n e - 1 , 5 - d i s u l f o n a t e and 2 . 5 ml. o f 8 8 - 9 0 $ phenol  (Mallinckrodt l i q u i f i e d  phenol)/  gram wet weight o f t i s s u e s i m u l t a n e o u s l y f o r 2 minutes i n a Waring blender at room temperature.  Three ($) e x t r a c t i o n s  w i t h phenol were employed f o r the. i s o l a t i o n o f spleen RNA used i n the c u l t u r e experiments.  The. cloudy  supernatant  l a y e r s from the o r i g i n a l c e n t r i f u g a t e . and washings were carefully  s u c t i o n e d o f f to i n s u r e a g a i n s t contamination  i n t e r m e d i a t e l a y e r (see f i g u r e l ) .  from the  When p r e p a r a t i o n s were  p u r i f i e d by e x t r a c t i o n w i t h r e d i s t i l l e d 2-methoxy e t h a n o l the time o f d i a l y s i s was 6 - 1 2 hours i n the c o l d at k°-6° w i t h 2 changes o f c o l d s a l i n e  (pH a t 7 ) .  C.  Table I.  The i s o l a t i o n and p u r i f i c a t i o n o f c a l f spleen r i b o n u c l e i c a c i d (RNA). s p l e e n homogenized i n equal volumes o f 0 . 0 1 5 M. napthalene d i s u l f o n a t e and water-saturated phenol at room temperature. S t i r mixture 30 minutes, c e n t r i f u g e 1 hour at 0°C., 2 , 0 0 0 r.p.m.  cloudy aqueous supernatant wash with 1 v o l . phenol shake 10 minutes repeat twice c e n t r i f u g e 5»000 r.p.m.  supernatant  cloudy aqueous precipitate supernatant discard combine w i t h f i r s t supernatent  precipitate discard  make up to r e s p e c t to add 2 v o l . centrifuge r supernatant discard  supernatant discard  p r e c i p i t a t e (optional) wash w i t h 50 ml., napthalene disulfonate, centrifuge 5 , 0 0 0 r.p.m. f o r 15 min. Repeat at l e a s t twice.  2$ w i t h K+CH-jCOO" ethanol 2 , 0 0 0 r.p.m. f o r 20 min.  precipitate wash w i t h ethanol-water ( 3 s i ) centrifuge  precipitate d i s s o l v e i n 0 . 1 M. NaCl e x t r a c t phenol with e t h e r 3 times expel e t h e r w i t h N i t r o g e n gas (continued on the next page)  5 Table I.  (continued)  c l e a r aqueous s o l u t i o n  precipitate  discard  add equal v o l . 2 . 5 M. KgHPO^, 0 . 0 5 v o l . 3 3 . 3 $ H3PO4, 1 v o l . 2-methoxyethanol. separate lower l a y e r , wash w i t h 20 ml. o f top l a y e r from ( 1 : 1 : 1 : 0 . 0 5 ) . d i a l y s e against 2 : 1 . o f water c e n t r i f u g e c o n t e n t s o f bag.  precipitate  supernatant  discard  make up to 2$ K CH^C00p r e c i p i t a t e w i t h 2 v o l . 9 5 $ ethanol. centrifuge. +  r supernatant discard  1 precipitate wash RNA  6  F i g u r e 1.  Sedimentation of the v a r i o u s l a y e r s of c a l f spleen t i s s u e f o l l o w i n g homogenization  i n napthalene  d i s u l f o n a t e and p h e n o l .  Figure  2.  A stage 2+ Xenopus l a e v l s .  The  dotted area represents  the competent ectodermal r e g i o n which was f o r the i n v i t r o  study.  removed  CLOUDY AQUEOUS SUPERNATE INSOLUBLE PROTEIN PriENOL LAYER DNA-PROTEIN PRECIPITATE  r  RNA  L  -POLYSACCHARIDES  —  PHENOLIC NUCLEI  To assure the removal o f a l l p r e v i o u s contaminants and e s p e c i a l l y r i b o n u c l e a s e . (RNase) both glassware and equipment  were washed w i t h IN. NaOH.  Versene^, a c h e l a t i n g  agent, was used i n 2% c o n c e n t r a t i o n s to remove heavy metal i o n s which would p r e c i p i t a t e the b i o l o g i c a l rnacromolecules during i s o l a t i o n procedures. In the above i s o l a t i o n s the samples were c e n t r i f u g e d i n an I n t e r n a t i o n a l h i g h speed c e n t r i f u g e model HR-1 w i t h an #856 head.  A l l r e a d i n g s were i n r e v o l u t i o n s p e r minute  (r.p.m.). In some o f the c o n t r o l biochemical- and p h y s i c a l  tests  commercial l i v e r RNA, c o n s i s t i n g o f the a-RNA^ f r a c t i o n , was u t i l i z e d but t h i s m a t e r i a l (s-RNA) was n o t used i n the t i s s u e c u l t u r e or i n v i v o experiments concerned w i t h the possible b i o l o g i c a l function (induction) of t i s s u e - s p e c i f i c RNA. II.  UV a b s o r p t i o n  spectra  Spectrophotometric r e a d i n g s o f both the commercial ( l i v e r ) RNA and the i s o l a t e d  ( s p l e e n ) RNA s o l u t i o n s were  made u s i n g the Unicam Sp. 200 spectrophotometer and the Beckman spectrophotometer.  Prom t h i s d a t a t h e r a t i o o f  1.  versene = E.D.T.A. o r e t h y l e n e diamine t e t r a c e t i c  acid.  2.  s-RNA = s o l u b l e or t r a n s f e r RNA.  3.  S = Svedbers u n i t o r S v a l u e = 10-13 m./sec./dynes.  4.  C e t a v l o n = c e t y l t r i m e t h y l ammonium  '  bromide.  8  the  a b s o r p t i o n v a l u e s at 230 m|i, to 260 mji. and 280 m\i t o  260  m|i was c a l c u l a t e d t o i n d i c a t e the presence or absence  of n u c l e i c a c i d s (26O m(-l) , p r o t e i n s (230 III.  (280 m|i), and p e p t i d e s  m|i) (Morton, I 9 6 2 ) . C o l o r i r a e t r i c and chromatographic tests.. Solutions containing i s o l a t e d  s p l e e n RNA were t e s t e d  to determine t h e presence o f contaminating substances. The B i u r e t t e s t the  (Schneider, 1957) was a p p l i e d t o determine  presence o f p r o t e i n .  Dische's diphenylamine t e s t  Schneider, 1 9 5 7 ; C h a r g a f f and Davidson, 1955) was to  demonstrate  the presence or absence o f DNA.  o r c i n o l method ( C h a r g a f f and Davidson, 1955)  utilized  The B i a l s 1  was used to.  c o n f i r m the presence o f RNA i n s o l u t i o n (see Appendix I i ) . Dreywood's anthrone t e s t  ( M o r r i s , 1948) was used to demon-  s t r a t e the presence o f carbohydrates.. A chromatogram was. prepared to demonstrate t h e presence or absence o f carboh y d r a t e s ( i e . p o l y s a c c h a r i d e s ) b e f o r e and a f t e r w i t h 2-methoxyethanol IV.  extraction  (see Appendix I I I ) .  U l t r a c e n t r i f u g a l analyses. Sedimentation a n a l y s e s were c a r r i e d out i n a Spinco  Model 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 . for  UV o p t i c s were  employed  an examination o f the commercial s-RNA p r e p a r a t i o n  from l i v e r and S c h l e i r e n o p t i c s were employed t i o n o f the i s o l a t e d c a l f s p l e e n RNA.  f o r examina-  Photographs were  taken a f t e r the speed o f 4 4 , 7 7 0 r.p.m. was. a t t a i n e d i n the case o f s p l e e n RNA and 55>770 r.p.m. i n the case o f l i v e r s-RNA, at 4 minute i n t e r v a l s .  Sedimentation c o e f f i c i e n t s  were determined by the method o f Markham  (i960, 1962)  (see Appendix I V ) . V,  Preparation of b i o l o g i c a l materials. Eggs o f Xenopus l a e v i S - (Daudin, 1802) were obtained  w i t h the method o f Brown and L i t t n a (1964) m o d i f i e d by u s i n g a "breeding medium" c o n t a i n i n g NaCl, KC1, MgSO^^HgO, Ca(N02)2*^ 2°» H  3 1 1 ( 1  s t r e p t o m y c i n s u l f a t e but not p e n i c i l l i n .  The pH o f the medium was maintained at. 7 and the l i g h t and dark p e r i o d s were a l t e r n a t e d every 12 hours.  Eggs were  removed from the b r e e d i n g medium when at stages 9 and 9+ (very e a r l y g a s t r u l a ) a c c o r d i n g to Nieuwkoop and Faber  (1956). A m o d i f i e d N i u - T w i t t y medium  (Douglas and Finnegan,  unpublished) was used t o c u l t u r e whole animals i n vivo. and ectodermal t i s s u e c u l t u r e s i n v i t r o T h i s medium was  (see Appendix  V) .  " c o n d i t i o n e d " by the a d d i t i o n o f 50 (Ag/ml.  o f p u r i f i e d s p l e e n RNA,  added immediately b e f o r e use i n  order t o minimize any p o s s i b l e RNA d e g r a d a t i o n e f f e c t s . Competent ectoderm  (see f i g u r e 2) was i s o l a t e d  from  stage 9+ animals w i t h c a r e taken to remove any c l i n g i n g m a t e r i a l from the ectodermal i s o l a t e .  S t e r i l e procedures  were used throughout the experiments.  E x p l a n t s developed  i n e i t h e r c o n t r o l o r c o n d i t i o n e d media f o r 5 days, b e f o r e they were f i x e d i n Carnoys, embedded i n p a r a f f i n wax and s e c t i o n e d at 5 V- f o r h i s t o l o g i c a l examination.  Similar  c u l t u r e and h i s t o l o g i c a l procedures were f o l l o w e d f o r the whole embryos though the l a t t e r were s e c t i o n e d at 8 (X.  10 The  ivhole embryos were s t a i n e d viith methyl-green Pyronin,  T o l u i d i n e b l u e and Haematoxylin and E o s i n .  The t i s s u e  c u l t u r e b a l l s were s t a i n e d w i t h Chromotrope.  RESULTS Part I .  I s o l a t i o n , p u r i f i c a t i o n and c h a r a c t e r i s a t i o n o f c a l f s p l e e n RNA.  Samples o f r i b o n u c l e i c a c i d (RNA), i s o l a t e d and p u r i f i e d by the m o d i f i e d K i r b y procedure,  were d i s s o l v e d  i n o . l M. NaCl s o l u t i o n and were examined w i t h the s p e c t r o photometer i n the u l t r a v i o l e t r e g i o n s to determine the presence 1962). liver  o f n u c l e i c a c i d s , p r o t e i n s and p e p t i d e s  (Morton,  A b s o r p t i o n s p e c t r a f o r a commercial p r e p a r a t i o n o f s-RNA as w e l l as the i s o l a t e d c a l f s p l e e n RNA are  r e p r e s e n t e d i n f i g u r e s 3 and 4 .  I t can be seen from  figure  3 t h a t the l i v e r s-RNA demonstrated an a b s o r p t i o n peak a t 265 m|i and as seen i n T a b l e s 2 a , 2 b , and 2c t h e r a t i o o f the a b s o r p t i o n v a l u e s 260 m|i/230 m\l was 2 . 2 0 and the r a t i o o f the v a l u e s 260 m|i/280 m\X was 1.82.  The former r a t i o o f  2 . 2 0 demonstrates the r e l a t i v e freedom o f the s-RNA from p e p t i d e contamination  and the l a t t e r r a t i o demonstrates  freedom from p r o t e i n contamination. When c a l f s p l e e n RNA was i s o l a t e d by the K i r b y p r o c e dure w i t h a s i n g l e phenol  e x t r a c t i o n an a b s o r p t i o n curve  as r e p r e s e n t e d i n f i g u r e 4 was produced i n which the r a t i o o f the a b s o r p t i o n v a l u e s 260 m|0,/23O mjl was 1.80 and the r a t i o o f 260 mfJ./280 mfX was 1.82 {see Table 2 b ) .  As i s  c h a r a c t e r i s t i c o f n u c l e i c a c i d s , the maximum a b s o r p t i o n occured  at 260 m|i and the minimum a b s o r p t i o n v a l u e s were  r e c o r d e d at 230 m|i and 280 mfl, t y p i c a l o f p e p t i d e s and proteins respectively  (Morton,  1962).  When c a l f spleen  12  F i g u r e 3»  The u l t r a v i o l e t l i v e r s-RNA.  a b s o r p t i o n spectrum o f commercial  13  F i g u r e 4.  The u l t r a v i o l e t a b s o r p t i o n s p e c t r a o f i s o l a t e d c a l f s p l e e n RNA phenol  f o l l o w i n g one phenol and  extraction(s).  three  14 Table 2 a .  Spectrophotometric  a n a l y s i s o f commercial  liver  s-RNA. wavelength (mfi)  r e a d i n g i n O.D.  230 260 280  G.266 0.588 0.421  Table 2b.  r a t i o 260/230  2.20  1.82  Spectrophotometric a n a l y s i s o f c a l f s p l e e n a f t e r one phenol e x t r a c t i o n .  wavelength (m|i)  r e a d i n g i n O.D.  r a t i o 260/230  230 260 280  0.690 1.240 0.680  1.80  Table 2 c .  260/28O  Spectrophotometric a f t e r t h r e e phenol  260/280  1.82  analysis of c a l f spleen extractions.  wavelength (m(i)  r e a d i n g i n O.D.  230 260 280  0.550 1.230 O.56O  RNA  RNA  r a t i o 260/230  260/280  2.23  2.20  15  RNA  was  e x t r a c t e d w i t h t h r e e phenol, washings an a b s o r p t i o n  spectrum  was  as seen i n f i g u r e 4 i n which the r a t i o  produced  of the a b s o r p t i o n v a l u e 260 m(l/230 m\x was  2.23  of the a b s o r p t i o n v a l u e 260 m|i/280 m(i was  2.20.  2c).  and the r a t i o  These r a t i o s i n d i c a t e that p r o t e i n and p e p t i d e conta-  m i n a t i o n was more e f f e c t i v e l y removed with the phenol  (See T a b l e  additional  extraction. The uv a b s o r p t i o n at 260 m\l i n d i c a t e s the  of a l l n u c l e i c a c i d s (DNA necessary to determine  as w e l l a s RNA) .  presence  Thus i t i s  more s p e c i f i c a l l y the nature o f the  c o n s t i t u e n t s of the i s o l a t e p a r t i c u l a r l y s i n c e contamination by substances o t h e r than r i b o n u c l e i c a c i d i n i s o l a t i o n s u s i n g the K i r b y procedure workers ( K i r b y , 1956,  has been r e p o r t e d by v a r i o u s  I960, 1962;  Huppert and Pelmont, 1962;  Laskov  et a l . . 1959;  and, Ralph and Bellamy,  1964).  To t h i s end v a r i o u s q u a l i t a t i v e t e s t s were employed to i n d i c a t e the presence constituents.  The  or absence o f s p e c i f i c  molecular  r e s u l t s of these t e s t s are r e p r e s e n t e d  i n T a b l e 3. The B i u r e t t e s t , to i n d i c a t e the presence when a p p l i e d to samples both o f one tions yielded negative r e s u l t s . complex was  observed  of protein,  and three phenol e x t r a c -  When the biuret-RNA  on the spectrophotometer  solution  at 550 m(i no  blue c o l o u r developed which would be i n d i c a t i v e of p e p t i d e s or p r o t e i n s .  However, the B i u r e t method cannot be c o n s i d e r e d  as s e n s i t i v e as an a n a l y s i s of the c o n s t i t u e n t s o f the s o l u t i o n with the spectrophotometer  (see T a b l e s 2b and  RNA 2c).  16 T a b l e 3»  Q u a l i t a t i v e experiments demonstrating presence  or absence o f v a r i o u s m a t e r i a l s i n  the i s o l a t e d Test 1.  sample.  Result Biuret  (a) one  Comment phenol  no c o l o r change (b) three  (a) one  absence o f p r o t e i n s arid p e p t i d e s  phenol  very p o s i t i v e green c o l o r ( b ) three  3. Diphenylamine (a) one phenol (Dische's t e s t ) s l i g h t blue c o l o r developed (b) t h r e e  i n d i c a t e s the presence o f RNA large quantity  in  phenols  very p o s i t i v e  RNA  present  presence o f small q u a n t i t y of DNA  phenols  no c o l o r change k. Anthrone (Dreywood* s)  absence of p r o t e i n s and p e p t i d e s  phenols  no c o l o r change 2. O r c i n o l (Bial's test)  the  no DNA  detectable  (a) b e f o r e methoxy s l i g h t change o f carbohydrate c o l o u r from y e l l o w to b l u e green  present  (b) a f t e r methoxy ho  c o l o u r change  no d e t e c t a b l e carbohydrate  17  The presence o f r i b o n u c l e i c a c i d (RNA)  c o u l d be  confirmed by the method o f B i a l , which employs o r c i n o l reagent and i s s p e c i f i c f o r p u r i n e r i b o n u c l e o t i d e s ( C h a r g a f f and Davidson, spleen RNA  1955)*  A f t e r the s o l u t i o n c o n t a i n i n g i s o l a t e d  and o r c i n o l reagent was  heated f o r 20 minutes at  100°C a d e f i n i t e green c o l o u r developed, s o l u t i o n s c o n t a i n i n g RNA.  c h a r a c t e r i s t i c of  The i s o l a t e f o l l o w i n g three phenol of 0 . 3 0 .  e x t r a c t i o n s gave a r e a d i n g on the spectrophotometer  Since an a b s o r p t i o n maximum at 260 m\X c o u l d r e p r e s e n t u n p r e c i p i t a t e d DNA  as w e l l as the demonstrated  Dische diphenylamine  RNA,  the  t e s t , which i s s p e c i f i c f o r DNA,  (2-  desoxyribpse and 2-deoxyxylose) was .employed.  When the .  cloudy aqueous l a y e r ^ r e s u l t i n g from one phenol  extraction  were t e s t e d w i t h diphenylamine  reagent a p o s i t i v e blue  c o l o u r developed i n d i c a t i n g that DNA  was  present.  The  s p e c t r o p h o t o m e t r i c r e a d i n g o f t h i s mixture at 595 m|J, was 0.026.  Since l i t t l e  care was  taken to remove the upper l a y e r  without contamination from the i n t e r f a c i a l l a y e r , the s m a l l q u a n t i t y o f DNA  which r e a c t e d w i t h the reagent  c o u l d be  a t t r i b u t e d to the p h e n o l i c n u c l e i which have been  demonstrated  i n t h i s i n t e r m e d i a t e l a y e r by Georgietf et a l . , ( i 9 6 0 ) . the o t h e r hand a f t e r t h r e e phenol e x t r a c t i o n s and removal  complete  o f the i n t e r f a c i a l m a t e r i a l the same t e s t was  i n d i c a t i n g the absence o f DNA  On  negative  contamination i n any d e t e c t -  able q u a n t i t y (see T a b l e 3 ) . Since any p o s s i b l e r o l e o f carbohydrates such as mucop o l y s a c c h a r i d e s i n i n d u c t i o n i s unknown i t i s c o n s i d e r e d i m p e r a t i v e to remove a l l t r a c e s o f these substances. p o l y s a c c h a r i d e contamination e x i s t s ,  That  even a f t e r e x t e n s i v e  18 phenol e x t r a c t i o n s has been demonstrated by K i r b y 1962)  and Ralph and Bellamy  (1964).  Two  (195-6,  methods were  employed to i n d i c a t e the presence o f contaminating carbohydrates.  One method was  the Anthrone  t e s t o f Dreywood  ( M o r r i s , 1 9 4 8 ) , which i s s p e c i f i c f o r c a r b o h y d r a t e s . RNA  The  s o l u t i o n , a f t e r t h r e e phenol e x t r a c t i o n s , developed a  b l u e - g r e e n c o l o u r w i t h anthrone reagent at room  temperature.  The r e a d i n g on the spectrophotometer at 620 m|i was i n d i c a t i n g the d e f i n i t e presence o f c a r b o h y d r a t e s . methoxyethanol  e x t r a c t i o n the RNA  solution  O.O65 After  demonstrated  no c o l o u r change w i t h the anthrone r e a g e n t .  Thus the  carbohydrate contamination appears t o have been d r a s t i c a l l y reduced by the p u r i f i c a t i o n procedure. A second method o f demonstrating carbohydrate i n the i s o l a t e was  accomplished w i t h paper chromatography.  chromatographic  study o f the i s o l a t e d RNA  w i t h 2-methoxyethanol and one  demonstrated  before p u r i f i c a t i o n  traces of polysaccharide  spot corresponded to the c o n t r o l spot c o n t a i n i n g  g l u c o s e (see f i g u r e 5 ) . may  A  be r i b o s e .  The  second spot i n the i s o l a t e  F o l l o w i n g e x t r a c t i o n w i t h methoxyethanol  no  t r a c e o f carbohydrate c o u l d be d e t e c t e d . These r e s u l t s i n d i c a t e that the i s o l a t e i n the absence o f DNA,  c o n t a i n s RNA  p e p t i d e , p r o t e i n and carbohydrate  contamination o n l y a f t e r t h r e e phenol e x t r a c t i o n s p u r i f i c a t i o n w i t h methoxyethanol  extraction.  and  However, these  d a t a have g i v e n no c l u e as to the s i z e or c o n d i t i o n (degraded) o f the molecules o f i s o l a t e d  RNA.  To o b t a i n i n f o r m a t i o n on the s i z e and c o n d i t i o n o f both commercial  l i v e r s-RNA and K i r b y i s o l a t e d c a l f s p l e e n  BEFORE liETHOXYETHANOL EXTRACTION GLUCOSE CONTROL  AFTER METHOXYETHANOL EXTRACTION  19  F i g u r e 5-  Chromatographic a n a l y s i s o f p u r i f i e d RNA. s o l u t i o n b e f o r e 2-methoxyethanol  e x t r a c t i o n . A f t e r the  e x t r a c t i o n no spot was observed.  20 RNA,  analyses o f these molecules were c a r r i e d out using, the  analytical ultracentrifuge.  The sedimentation p a t t e r n s  o b t a i n e d by t h i s procedure are shown i n f i g u r e s 6, 7» and The q u a n t i t y o f commercial uv o p t i c s were employed.  s m a l l and thus  The diagram o b t a i n e d i n f i g u r e 6  r e p r e s e n t s the l i v e r RNA f o r 20 minutes.  s-RNA was  8.  i n 0.1 M. NaCl at 55,770 r.p.m.  As can be seen from f i g u r e 6, there was  apparent movement o f the molecules. that the molecule o f s-RNA was  This indicates  no  either  a l a r g e molecule which  had  become degraded, p o s s i b l y by the i s o l a t i o n procedures employed or by methods o f storage, or that the s-RNA molecule was  quite small.  The l a t t e r e x p l a n a t i o n would appear to be  c o n s i s t e n t w i t h the p r e s e n t knowledge c o n c e r n i n g the s i z e o f s o l u b l e RNA  (Spirin,  1963).  The a n a l y s i s o f i s o l a t e d c a l f spleen RNA o p t i c s demonstrated  using Schlieren  a d e f i n i t e m o l e c u l a r s p e c i e s ( f i g u r e 7).  The sedimentation c o e f f i c i e n t s expressed i n Svedberg or S u n i t s f o r these molecules were c a l c u l a t e d from the graph ( f i g u r e 8) by the method o f Markham ( i 9 6 0 ) . f o r the 3 s p e c i e s o f RNA (SgQ ) w  were 27S,  f o r water at 20°C.  18S,  The v a l u e s  and 8S  respectively,  The f a s t e s t moving component.,  the 27S f r a c t i o n , xvas a l s o present i n the h i g h e s t c o n c e n t r a t i o n . T h i s sedimentation p a t t e r n f o r s p l e e n RNA  s e r v e d to demon-.  s t r a t e that the i s o l a t e d molecules were r e l a t i v e l y and not degradod  by the i s o l a t i o n procedure.  m a t e r i a l which was  intact  It i s this  used i n the i n d u c t i o n experiments.  7-9 S  1  ?7S  21  F i g u r e 6. Sedimentation diagram o f commercial liver-RNA.  F i g u r e 7. S c h l i e r e n photograph o f a 0.1M spleen-RNA  NaCl s o l u t i o n o f  a f t e r r e a c h i n g the speed o f 44,770 r.p.m.  i n the. 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 . From the meniscus, M,  the peaks have sedimentation co-  e f f i c i e n t s o f 8S, 18S, and 27S.  22  F i g u r e 8.  Sedimentation diagram o f c a l f s p l e e n  RNA.  5-9 I  1  1  1  2  I 3 EXPOSURE  I 4  ANGLE  i • • i 5  6  23  F i g u r e 9«  The  combined graphs from the u l t r a c e n t r i f u g e  a n a l y s i s o f c a l f spleen RNA  (fig.  7 ) . The  slope of the curves i s a measure o f the sedimentation  c o e f f i c i e n t s , which i s read o f f  on a s p e c i a l t r a n s p a r e n t p r o t r a c t o r designed by Markham ( i 9 6 0 ) .  24  Part I I .  The i n v i t r o and i n v i v o e f f e c t s o f c a l f  spleen  RNA on competent ectoderm and embryos o f Xenopus laevis. The  i s o l a t e d , undegraded and uncontaminated c a l f  RNA was t e s t e d f o r s p e c i f i c b i o l o g i c a l a c t i v i t y duction)  spleen  (i.e. in-  by exposing the t i s s u e s o f Xenopus l a e v i s embryos  under both i n v i t r o and i n v i v o c o n d i t i o n s t o the RNA at. a concentration vitro  o f 50 Jig/ml.  (Niu, 1 9 5 8 ; Yamada,  196l).  studies.  When e x c i s e d competent ectoderm from stage 9+ embryos was  p l a c e d e i t h e r i n RNA-conditioned o r c o n t r o l media there  was  no adhesion o f the g a s t r u l a ectoderm to the g l a s s  s l i p s i n the c u l t u r e d i s h e s the o p e r a t i o n  and w i t h i n one-half  the ectodermal explant  These ectodermal explants  cover-  an hour o f  had r o l l e d i n t o a b a l l .  c u l t u r e d i n the RNA-conditioned  medium f o r 5 days demonstrated no s p e c i f i c i n d u c t i o n . N e i t h e r were any i n d i c a t i o n s o f h i s t o g e n e s i s evident i n the c o n t r o l s e r i e s c u l t u r e d i n the balanced s a l t s o l u t i o n which i n c i d e n t a l l y i s d e f i c i e n t i n both p r o t e i n and RNA. However, the enhanced development o f the e x p l a n t s c o n d i t i o n e d medium was most evident Table  4a).  i n the  (see f i g u r e 10 and  When the s e c t i o n e d m a t e r i a l s were compared,  the s i n g l e - l a y e r e d c i l i a t e d ectodermal c e l l s o f the e x p l a n t s i n unconditioned  medium c o n t r a s t e d w i t h the s u p e r f i c i a l  c e l l s o f the RNA-enhanced explants. which were two to: f o u r l a y e r s i n t h i c k n e s s and w i t h c e r t a i n , o f the c e l l s columnar r a t h e r than c u b o i d a l . explants  -appearing  I n a d d i t i o n ectodermal  i n RNA-medium were observed t o s u r v i v e b e t t e r than  those grown i n the u n c o n d i t i o n e d  medium.  \ 1  25  F i g u r e 10.  I s o l a t e d Xenopus ectoderm which had been i n a s o l u t i o n o f c a l f s p l e e n RNA No t i s s u e - s p e c i f i c i n d u c t i o n i n contrast  cultured  f o r 5 days.  can be seen, but  to the c o n t r o l s e r i e s the c e l l s are  b e t t e r developed and more numerous.  26 T a b l e 4a.  Effect  o f i s o l a t e d calf, spleen RNA on  competent ectoderm  in vitro.  number o f cases controls experimentals N.B.  T a b l e 4b.  amphibian  inductive effect  no e f f e c t  6  0  6  18  0  18  Although 6 c o n t r o l animals and 18 experimental animals were observed throughout the experiment, only 2 o f each s e r i e s , chosen at random, were sectioned.  Effect  o f i s o l a t e d c a l f spleen RNA on whole  embryos o f Xenopus l a e v i s i n v i v o . number o f cases  inductive effect  no e f f e c t  controle  8  0  8  experimentals  8  0  8  N.B.  A l l animals i n t h i s s e r i e s were s e c t i o n e d .  27 2.  In v i v o  studies.  Stage 9+ embryos were p l a c e d i n RNA-medium and c o n t r o l , medium  and allowed to develop f o r 5 days.  Embryos developed  w e l l i n both c o n t r o l and c o n d i t i o n e d media.  However.,, the  experimental animals were at a l l times s l i g h t l y , but cons i s t e n t l y , a c c e l e r a t e d i n organogenesis as compared to the. c o n t r o l animals (see f i g u r e s 11 and 12 and T a b l e 4 b ) .  Also,  w h i l e t h e r e was no i n c r e a s e d h i s t o g e n e s i s o f s p l e e n - l i k e or mesodermal t i s s u e i n the experimental animals at the t e r m i n a t i o n o f the c u l t u r e p e r i o d , the o t i c v e s i c l e s demons t r a t e d a more pronounced d i f f e r e n t i a t i o n o f the v e n t r o medial e p i t h e l i u m and were becoming d i v i d e d i n t o the p a r s i n f e r i o r and the p a r s s u p e r i o r , the t o r s i o n o f the i n t e s t i n e was more obvious and the h i s t o l o g i c a l development o f the g a s t r i c glands more d i s t i n c t  than i n the c o n t r o l s .  28a  F i g u r e 11.  S e c t i o n through c o n t r o l Xenopus embryo c u l t u r e d i n an "unconditioned"  medium.  V  28b  Figure 1 2 .  S e c t i o n through, experimental Xenopus embryo c u l t u r e d i n an RNA-enriched medium.  29  DISCUSSION The  i s o l a t i o n of c a l f spleen RNA  phase p a r t i t i o n system (1956) i n s u r e l e s s contaminated  by the K i r b y  r e q u i r e d m o d i f i c a t i o n to.  and undegraded RNA  the subsequent i n d u c t i o n experiments  molecules f o r  s i n c e RNA  t h i s method has been shown to be contaminated components ( K i r b y , 1956, 1964).  i960,  1962;  isolated  by  w i t h other  Ralph and  Bellamy,  F o l l o w i n g the f i r s t phenol washing the aqueous phase  can be demonstrated to c o n t a i n RNA, 1956;  two-  and T a b l e 4 ) , p r o t e i n  (Ralph and Bellamy,  amino a c i d s (Niu e t a l , 1 9 6 l ) , which may Hayashi  polysaccharides (Kirby,  and DNA  1964),  (Table 4 ) .  Any  lipids  be p r e s e n t i n the i s o l a t e have been shown by  (1959)  to be unimportant  i n i n d u c t i o n and are not  c o n s i d e r e d i n t h i s work. In the p r e s e n t i n v e s t i g a t i o n an u l t r a v i o l e t  absorption  a n a l y s i s of i s o l a t e s o b t a i n e d with one phenol washing and w i t h t h r e e phenol washings has demonstrated that, p r o t e i n and p e p t i d e c ontamination  was  no s i g n i f i c a n t r e d u c t i o n i n RNA  c o n s i d e r a b l y reduced content  However, Huppert and Pelmont ( 1 9 6 2 ) ,  with  (Table 2b and  2c).  u s i n g the more s e n s i t i v e  method o f Lowry f o r p r o t e i n d e t e r m i n a t i o n , found a p r o t e i n content between 20 and 50 |0.g/ml. a f t e r three phenol e x t r a c tions.  Thus i t i s d i f f i c u l t  ( G i e r e r , 1957; Niu,  1958a,  b;  to c o n s i d e r , as do many workers  G i e r e r and Schramm, 1956a, et a l , 196l;  Niu  and, Butros, 1963)» i s completely absent phenol washings.  b; and K i r b y , 1962;  H i l l m a n and  that p r o t e i n or p e p t i d e f o r the i s o l a t e d RNA  Niu,  1963a,  b;  contamination  even a f t e r t h r e e  Very s m a l l q u a n t i t i e s of p r o t e i n would not  be d e t e c t e d w i t h the B i u r e t t e s t and the r a t i o s o f the v a l u e s  30 at 260 m|i/230 m|A and 260 mfi/280 mjx o n l y serve to i n d i c a t e the q u a l i t a t i v e absence o f p e p t i d e s and p r o t e i n s from the i s o l a t e d sample.  C e r t a i n l y the low l e v e l o f p e p t i d e  p r o t e i n contamination  p o s s i b l y remaining  a f t e r three phenol  e x t r a c t i o n s i n the c u r r e n t experiments was to act i n d u c t i v e l y . The  DNA,  and  insufficient  present- i n the RNA  solution  a f t e r one phenol e x t r a c t i o n and a t t r i b u t e d - t o "phenolic . n u c l e i " (Georgiev,  i 9 6 0 ) i n the i n t e r f a c i a l m a t e r i a l , i s  removed w i t h the subsequent phenol e x t r a c t i o n s . d r a t e s or p o l y s a c c h a r i d e contamination  Carbohy-  (see F i g u r e 5) i s  not removed with phenol but with 2-methoxyethanol e x t r a c t i o n . Laskov et a l (1959) found that RNA  i s o l a t e d by  the  phenol water method ( K i r b y , 1956; G i e r e r and Schramm, 1956a, b) r e s u l t e d i n d e g r a d a t i o n m o l e c u l a r weight RNA nate was  was  of the molecule.  obtained i f n a p t h a l e n e - 1 , 5 - d i s u l f o -  employed i n p l a c e o f water.  i s o l a t e d RNA  A higher  The  i n s t a b i l i t y o f the  molecule has been demonstrated by Huppert  and  Pelmont (1962), K u b i n s k i and Koch (1963) and Amos and Moore (1963) who  have shown that when the i s o l a t e was  room temperature d e g r a d a t i o n was  began w i t h i n 130 minutes  d r a s t i c w i t h i n 24 hours.  A f t e r s e v e r a l days no  mentable f r a c t i o n s were observable I962).  l e f t at  E q u a l l y p e r t i n e n t , was  and  sedi-  (Huppert and Pelmont,  the o b s e r v a t i o n o f Amos and  Moore (1963) and Amos ejt a l (1964) who  r e p o r t e d t h a t , while  they were able to o b t a i n an a c t i v e p r o p a r a t i o n of RNA  only  i r r e g u l a r l y , the a c t i v e p r e p a r a t i o n r e t a i n e d i t s b i o l o g i c a l a c t i v i t y when s t o r e d at temperatures o f -20°C. over a p e r i o d o f s e v e r a l weeks. spleen RNA  As a r e s u l t of these  u t i l i s e d i n the present  s t u d i e s the  experiments was  employed  31 e i t h e r immediately  f o l l o w i n g i s o l a t i o n o r w i t h i n the. next  7 days a f t e r storage at -20°C. on the assumption that the b i o l o g i c a l a c t i v i t y o f the i s o l a t e , i f e x i s t i n g , would have been  maintained. A p o s s i b l e source o f RNA degradation i s by r i b o n u c l e a s e  a c t i v i t y d u r i n g o r a f t e r i s o l a t i o n procedures.  Kirby  (1956,  i 9 6 0 ) demonstrated that r i b o n u c l e a s e was i n h i b i t e d by phenol reagent, molecule.  which a p p a r e n t l y a c t s to denature  the r i b o n u c l e a s e  However, when the endogenous r i b o n u c l e a s e i n the.  t i s s u e s has been destroyed by phenol the danger o f contamination previous i s o l a t i o n s .  a c t i v i t y there i s s t i l l  from glassware  used i n the  Extreme care was employed  throughout  the present i s o l a t i o n s to reduce as much as p o s s i b l e any such s o r t o f d e g r a d a t i o n . terminated Ralph  Since these experiments were  and Bellamy (1964) i n t r o d u c e d a m o d i f i c a t i o n  o f the K i r b y procedure  i n which RNA i s r e c o v e r e d as the  cetyltrimethylammoniura  salt.  The m o d i f i c a t i o n i s based on  the f i n d i n g o f D u t t a et a l (1953) t h a t a d i f f e r e n c e i n s o l u b i l i t y o f the C e t a v l o n ^ s a l t s o f the n u c l e i c a c i d s permits the s e p a r a t i o n o f RNA from DNA,  T h i s method o f Ralph and  Bellamy i s r a p i d and removes the d i a l y s i s step which i t s e l f produces an o p p o r t u n i t y f o r r i b o n u c l e a s e a c t i v i t y . The  sedimentation  evidence, p r e s e n t e d i n f i g u r e s 7,  8, and 9» demonstrates t h a t the i s o l a t e d spleen RNA was composed o f three m o l e c u l a r f r a c t i o n and an 8S f r a c t i o n .  s p e c i e s ; a 27S f r a c t i o n , an 18.S The 27S and. 18S f r a c t i o n s would  appear to r e p r e s e n t undegraded RNA molecules, fraction  (Spirin,  I963).  the ribosomal-  Huppert and Pelmont (1962)  obtained  RNA i n two f r a c t i o n s when i s o l a t e d from a s c i t e s tumor c e l l s  32  and s i m i l a r r e s u l t s were r e p o r t e d by Ralph and (1964) w i t h RNA  i s o l a t e d from r a t l i v e r ,  and tobacco c e l l s ; by H a l l and Doty  Bellamy  Chinese cabbage,  (1959) w i t h c a l f  liver  microsomes; and by Cheng ( l 9 5 9 » 19^0) w i t h mouse b r a i n . J i a n g ( 1 9 6 4 ) , i s o l a t i n g r i b o n u c l e i c a c i d from mammalian p i t u i t a r i e s demonstrated  t h r e e f r a c t i o n s , 28S,  18S,  and  4S.  These v a l u e s correspond to those o b t a i n e d i n the present a n a l y s i s so that the 8S f r a c t i o n found here may degradation product. the r i b o s o m a l RNA  The  not be a.  sedimentation c o e f f i c i e n t s o f  (27S and 18S)  appear to d i f f e r  slightly  depending upon the b i o l o g i c a l source o f the p r e p a r a t i o n (Spirin, weight  1963).  Demonstration o f the two h i g h m o l e c u l a r  s p e c i e s o f RNA  from c a l f s p l e e n i s o l a t e - tends to  support the i d e a that the RNA  molecules o b t a i n e d from the  m o d i f i e d procedure were i n t a c t and undegraded  when i n t r o -  duced i n t o c u l t u r e medium f o r the i n d u c t i o n experiments.. The procedure f o r the i s o l a t i o n o f r i b o n u c l e i c  acid  as employed by N i u (1958 a,b), N i u et, a l ( I 9 6 I ) , H i l l m a n and N i u (1963 a,b), Yamada ( 1 9 6 1 ) ,  and Butros (1963) were  e s s e n t i a l l y the method proposed by K i r b y i n 195^, has been demonstrated  (Laskov ejb a l . , 1959)  r a t h e r degraded p r o d u c t . by these workers, procedure was  to y i e l d  a  s h o r t e r procedure a l s o used  i n which the methoxyethanol  purification  d e l e t e d , would tend to reduce the amount o f  d e g r a d a t i o n o f RNA was performed  The  which  but s i n c e o n l y one phenol  extraction  the p r o t e i n and p e p t i d e as w e l l as the p o l y -  s a c c h a r i d e contamination o f t h e i r product might  be  significant.  Niu (1958b) d i a l y s e d h i s sample a g a i n s t r u n n i n g water overnight,  a procedure that may  allow c o n s i d e r a b l e r i b o n u c l e a s e  33 contamination, and a p p a r e n t l y no attempt was made to remove p o l y s a c c h a r i d e m a t e r i a l even though l i v e r and muscle  tissues  were the source m a t e r i a l used. In  the p r e s e n t experiments c a l f spleen t i s s u e  was  u t i l i z e d as the source tissue, because, i t i s an organ of. completely mesodermal o r i g i n w i t h a l a r g e q u a n t i t y o f n u c l e i c a c i d and because Yamada ( l 9 6 l ) and Yamada and T a k a t a ( l 9 6 l ) were able t o o b t a i n such d e f i n i t e i n d u c t i o n s u s i n g the  RNP  and p r o t e i n f r a c t i o n s o f bone marrow t i s s u e .  How-  ever, both the i n v i t r o and i n v i v o r e s u l t s have i n d i c a t e d the  absence o f any t i s s u e - s p e c i f i c  (i.e.  mesodermal) i n -  d u c t i v e response when i s o l a t e d and p u r i f i e d c a l f spleen was p r e s e n t e d to competent  embryonic m a t e r i a l .  RNA  Niu e t a l .  (1961) and Amos and Moore (1964) have demonstrated that molecules are taken up by embryonic  RNA  cells.  Since no i n d u c t i v e e f f e c t was observed i t i s n e c e s s a r y to  c o n s i d e r as a p o s s i b l e f a c t o r the exposure time o f the  test material.  I n the f i r s t p l a c e 5 days o f c u l t u r e appeared )  s u f f i c i e n t to r e s u l t i n any o b s e r v a b l e h i s t o g e n e s i s s i n c e the  t i s s u e s and embryos o f Xenopus l a e v i s are e x c e e d i n g l y  r a p i d i n t h e i r development.  With r e g a r d to the i n i t i a l  exposure time, Yamada (1962) has demonstrated t h a t a 55$ i n d u c t i v e response was  a c h i e v e d i n amphibian, ectoderm  a f t e r 30 minutes exposure to a p r o t e i n medium and that c o u l d be i n c r e a s e d to  this,  w i t h a 180 minute treatment.  There-  f o r e , i n d u c t i o n under these t e s t conditions, s h o u l d occur w i t h i n the f i r s t  h o u r ( s ) o f exposure o f the. competent  to  I t seems l i k e l y that i t i s p r e c i s e l y d u r i n g  the i n d u c e r .  t h i s time when i n t a c t RNA would be p r e s e n t i n the t e s t  tissue  3  4  medium s i n c e Huppert and Pelmont (1962) have shown that degradation of i s o l a t e d RNA first  i s only i n i t i a t e d w i t h i n the  130 minutes at room temperature  c u l t u r e s were maintained would tend to decrease  (20°C) and  at a temperature  these  of 12°C.  This  the r a t e of d e g r a d a t i o n so that  t o t a l degradation o f the RNA  molecules  i n the t e s t medium  would not be complete f o r more than 2k hours. some time a f t e r i n t r o d u c i n g the i n t a c t RNA  Thus, at  molecules  and  the competent ectoderm i n t o the medium, and a f t e r the. time when the RNA  i n d u c t i o n c o u l d have o c c u r r e d , t h e r e probably  would be present a s o l u t i o n o f n u c l e o t i d e s and n u c l e o s i d e s i n s t e a d o f the i n t a c t RNA  molecules.  While the  presence  o f a s s o c i a t e d p r o t e i n i n the t e s t media might f u n c t i o n as suggested  by Niu (1958b) to s t a b i l i z e the RNA  molecule f o r  a s u f f i c i e n t time i n an i n d u c t i v e system, i t i s a l s o p o s s i b l e that the i n v i t r o i n d u c t i o n observed by Niu and T w i t t y and N i u (1958b) may response  (1953)  have been the r e s u l t o f a s p e c i f i c  by competent t i s s u e to a h i g h c o n c e n t r a t i o n of  o l i g o n u c l e o t i d e s (Lash jet a l , 1962) o r RNP  (Yamada, 1958>  1961, I962) p r e s e n t i n the c o n d i t i o n e d medium. While no i n d u c t i o n was v e s t i g a t i o n , t h e r e was  evidenced  observed  an enhancement o f t i s s u e  development both i n the ectodermal embryos.  i n the p r e s e n t i n -  e x p l a n t s and i n the. whole  T h i s e f f e c t w i t h s p l e e n RNA  i n v i t r o and i n v i v o  appears to be s i m i l a r to the r e s u l t s d e s c r i b e d by (1955, 1958, 1962.  Ambellan  1963) i n which s o l u t i o n s o f v a r i o u s  n u c l e o t i d e s were found to a c c e l e r a t e n e u r a l - t u b e i n amphibian embryos.  A s i m i l a r mechanism may  formation  be o p e r a t i n g  i n the enhancement o f d i f f e r e n t i a t i o n achieved w i t h panc r e a t i c , h e a r t and b r a i n RNA  by Butros  (1963).  This  35 enhancement o f development, may have been the r e s u l t o f t i s s u e exposure to both i n t a c t RNA and RNA fragments  ( i . e . nucleo-  t i d e s ) s i n c e both would be present i n the e a r l y p e r i o d o f these experiments. F i n a l l y i t must be noted that the. absence o f s p e c i f i c i n d u c t i v e e f f e c t s i n any o f these t e s t media.may have been due t o the absence o f a b i o l o g i c a l l y  "active" preparation  as suggested by Amos and Moore (1963) and Amos et a l . ( 1 9 6 4 ) .  36 SUMMARY 1.  R i b o n u c l e i c a c i d (RNA)  from c a l f spleen t i s s u e  was  i s o l a t e d and p u r i f i e d by a m o d i f i e d K i r b y procedure. 2.  The u l t r a v i o l e t was measured.  a b s o r p t i o n spectrum The  of the RNA  solution  a b s o r p t i o n maximum occured at 260 m(i  i n d i c a t i n g the presence o f n u c l e i c a c i d s while the a b s o r p t i o n minima observed at 230 mU  and 280 mH  indicated  the absence o f p e p t i d e s and p r o t e i n s . 3.  C o l o r i m e t r i c a n a l y s e s i n d i c a t e d the absence o f p e p t i d e s , p r o t e i n s , DNA, of  4.  and carbohydrates as w e l l as the presence  RNA.  Chromatographic i s o l a t e d RNA  a n a l y s i s i n d i c a t e d that the samples o f  c o n t a i n e d t r a c e s o f carbohydrate b e f o r e  e x t r a c t i o n w i t h methyoxyethanol but were f r e e  from  carbohydrate contamination a f t e r the e x t r a c t i o n . 5.  A sedimentation a n a l y s i s was  performed  that the i s o l a t e c o n t a i n e d t h r e e RNA fraction,  an 18S  demonstrated  fractions:  that the RNA  Competent ectoderm  27S  I t was  i s o l a t e d i n the present  undegraded.  e x c i s e d from stage 9+ Xenopus l a e v i s  and c u l t u r e d i n RNA-conditioned  medium demonstrated  t i s s u e - s p e c i f i c i n d u c t i o n but d i d demonstrate development when compared to the c o n t r o l 7.  a  component and an 8S component.  experimental s e r i e s was 6.  which i n d i c a t e d  no  enhanced  series.  S i m i l a r l y , when stage 9 + Xenopus were grown i n  RNA-  e n r i c h e d medium the experimental animals were s l i g h t l y advanced i n development over the c o n t r o l animals. no t i s s u e - s p e c i f i c i n d u c t i o n was  observed.  Again,  37  REFERENCES Ambellan, E. 1955. E f f e c t o f adenine mononucleotides on n e u r a l tube f o r m a t i o n o f f r o g embryo. Proc. 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Hall,  B.D.  and Doty, P. 1959. The p r e p a r a t i o n and p h y s i c a l chemical p r o p e r t i e s o f r i b o n u c l e i c a c i d from microsomal p a r t i c l e s . J . Mol. B i o l . Ii 111-126.  Hayashi, Y. 1959• S i g n i f i c a n c e o f l i p i d s i n i n d u c t i o n by t i s s u e and by a pentose n u c l e o p r o t e i n sample i s o l a t e d from t i s s u e . Develop. B i o l . JL: 3 4 3 - 3 6 3 . H i l l m a n , N. and Niu, M.C. 1 9 6 3 a . Chick c e p h a l o g e n e s i s . I . The e f f e c t o f RNA on e a r l y c e p h a l i c development. P r o c . N a t l . Acad. S c i . (USA).. jjO: 4 8 6 - 4 9 3 . 1963b. The e f f e c t o f e x t r a c t e d n o t o c h o r d a l RNA and b r a i n RNA on c u l t u r e d c h i c k embryos. Anat. Rec. 145: 2 4 0 . Huppert,  J . and Pelmont, J . I 9 6 2 . U l t r a c e n t r i f u g e s t u d i e s o f r i b o n u c l e i c a c i d d e g r a d a t i o n . Arch. Biochem. Biophys. 98:  214-223.  J i a n g , N-S. 1964. R i b o n u c l e i c a c i d from bovine, o v i n e , and porcine p i t u i t a r i e s . Biochim. Biophys. A c t a . 87? 347-348.  K i r b y , K.S. 1956. A new method f o r the i s o l a t i o n o f r i b o n u c l e i c a c i d s from mammalian t i s s u e s . Biochem. J . 46: 405-408. i960. F r a c t i o n a t i o n and c o u n t e r c u r r e n t d i s t r i b u t i o n of r i b o n u c l e i c a c i d . Biochim. Biophys. A c t a . 80:  ——  193-205.  1962. R i b o n u c l e i c a c i d s I I Improved p r e p a r a t i o n of r a t l i v e r r i b o n u c l e i c a c i d . Biochim. Biophys. A c t a . ji5_: 545-446.  K u b i n s k i , H. and Koch, G. I96J. I n s t a b i l i t y o f an R N A - f r a c t i o n from P o l i o v i r u s i n f e c t e d c e l l s . J . Mol. B i o l . 6: 102-104.  39 Lash, J.W., Hommes, F.A., and Z i l l i k e n , F. 1962. I n d u c t i o n of c e l l d i f f e r e n t i a t i o n . The i n v i t r o i n d u c t i o n o f v e r t e b r a l c a r t i l a g e w i t h a low-molecular-weight t i s s u e component. Biochim. Biophys. A c t a . 56:  313-319.  Laskov,  R., M a r g o l i a s h , E., L i t t a u e r , U.Z., and E i s e n b e r g , U. 1959• High-molecular-weight r i b o n u c l e i c a c i d from rat l i v e r . Biochim. Biophys. Acta. 33_: 2 4 7 - 2 4 8 . <  -  Markham, R. i 9 6 0 . A g r a p h i c a l method f o r the r a p i d d e t e r mination o f sedimentation c o e f f i c i e n t s . Biochem.  j.  22' 516-519.  1962. The 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 as a t o o l f o r the i n v e s t i g a t i o n o f p l a n t v i r u s e s . In, Advances i n V i r u s Research. V o l . 9. Ed. by K.M. :Smith and M.A. L a u f f e r . Academic P r e s s , pp. 241-270. M o r r i s , D.L. 1948. Q u a n t i t i v e d e t e r m i n a t i o n o f carbohydrates w i t h Dreywood's anthrone reagent. S c i e n c e . 107j  254-255.  Morton, R.A. 1962. Spectrophotometry i n the u l t r a v i o l e t and v i s i b l e r e g i o n s . I n , Comprehensive B i o c h e m i s t r y V o l . 3 . ed. by M. F l o r k i n and E.H. S t a t z . Elsevier P u b l i s h i n g Company, New York. pp. 66-132. Nieuwkoop, P.D. and Faber, J . 1956. Normal Table o f Xenopus L a e v i s (Daudin). North-Holland P u b l i s h i n g Company, Amsterdam. Niu, M.C. 1956. New approaches to the problem o f embryonic induction. In, C e l l u l a r Mechanisms i n D i f f e r e n t i a t i o n and Growth, ed. by D. R u d n i l k . U n i v e r s i t y Press, P r i n c e t o n , pp. 155-171. 1958a. The r o l e o f r i b o n u c l e i c a c i d i n embryonic differentiation. Anat. Rec. 131: 585. 1958b. Thymus r i b o n u c l e i c a c i d and embryonic differentiation. Proc. N a t l . Acad. S c i . (USA).  44: 1264-1274.  Niu, M.C, and T w i t t y , V.C. 1953. The d i f f e r e n t i a t i o n o f g a s t r u l a ectoderm i n medium c o n d i t i o n e d by a x i a l mesoderm. Proc. N a t l . Acad. S c i . (USA) . 21'' 985-989. Niu, M.C., Cordova, C.C., and Niu, L.C. I 9 6 I . R i b o n u c l e i c a c i d induced changes i n mammalian c e l l s . Proc. N a t l . Acad. S c i . (USA). 42: 1689-1700. Ralph, R.K., and Bellamy, A.R. 1964. I s o l a t i o n and p u r i f i c a t i o n of undegraded r i b o n u c l e i c a c i d s . Biochim. Biophys. Acta. 82: 9-16. Saxen, L. and Toivonen, S. 1962. Primary Embryonic I n d u c t i o n . P r e n t i c e - H a l l . I n c . , New J e r s e y .  4o  Schneider, W.C. 1957» Determination of n u c l e i c acids o f t i s s u e s by pentose analysis.. I n , Methods i n Enzymology. V o l . 3« ed by S.P. Colowick and N.O. Kaplan. Academic Press, New York. pp. 680-684. S p i r i n , A.S. I 9 6 3 . Some problems concerning the macromolecul a r strecture o f r i b o n u c l e i c acids. In Progress i n . N u c l e i c A c i d Research. V o l . 1. ed. by J.N. Davidson, and W.E. Cohn. Academic Press, New York. pp. 301-345* Yamada, T. 1 9 5 8 . I n d u c t i o n o f s p e c i f i c d i f f e r e n t i a t i o n by samples o f p r o t e i n s and n u c l e o p r o t e i n s i n the i s o l a t e d ectoderm o f T r i t u r u s - g a s t r u l a e . E x p e r e n t i a 14: 8 1 - 8 7 . 1 9 6 I . A chemical approach to the problem o f the o r g a n i s e r . I n , Advances i n Morphogenesis. V o l . 1. Ed. by M. Abererombie and J . B r a c h e t . Academic Press, New York. pp. 1 - 5 3 . 1962. The i n d u c t i v e phenomenon as a t o o l f o r understanding the b a s i c mechanism o f d i f f e r e n t i a t i o n . J . C e l l . Comp. P h y s i o l . 60 (Suppl. l ) : 49-64. Yamada, T. and Takata, 1955• An a n a l y s i s o f spino-caudal i n d u c t i o n by t h e guinea p i g k i d n e y i n the i s o l a t e d ectoderm o f the T r i t u r u s - g a s t r u l a . J . E x p t l . Z o o l .  128: 2 9 1 - 3 3 2 .  Yamada, T. and Takata, K. I 9 6 I . A technique f o r t e s t i n g macromolecular samples i n s o l u t i o n f o r morphogenetic e f f e c t s on the i s o l a t e d ectoderm o f the amphibian gastrula. Develop. B i o l . Ji: 4 1 1 - 4 2 3 .  41 Appendix I E x t r a c t i o n o f c a l f spleen 1.  I s o l a t i o n o f spleen The  RNA ( K i r b y , 1956, 1962)  RNA.  t i s s u e was homogenized i n 0 . 0 1 5 M. naptha-  spleen  lene-1,5-disulfonate  ( a t a pH o f 6 . 8 to 7 « 0 ) and water  s a t u r a t e d phenol ( 2 . 5 ml. o f 0 . 0 1 5 M, napthalene d i s u l f o n a t e 2 . 5 ml. o f 8 8 - 9 0 $ phenol / gram wet weight o f t i s s u e ) f o r  and  2 minutes i n a Waring b l e n d e r at room temperature.  The  homogenate was f i l t e r e d through a s i n g l e l a y e r o f gauze i n a T h i s mixture was s t i r r e d f o r 30 minutes  Buckner f u n n e l . and  centrifuged  f o r 1 hour at 0°C at 2 , 0 0 0 r.p.m.  p r e c i p i t a t e obtained  The  was washed w i t h 100 ml. o f 0 . 0 1 5 M.  napthalene d i s u l f o n a t e and c e n t r i f u g e d f o r 15 minutes at 5 , 0 0 0 r.p.m. twice.  T h i s washing procedure was r e p e a t e d at l e a s t  The cloudy supernatant l a y e r s from the o r i g i n a l  c e n t r i f u g a t e and the washings, were c a r e f u l l y s u c t i o n e d o f f to i n s u r e a g a i n s t  contamination from the intermediate  layer  (see f i g u r e 1 . ) .  The supernatant l a y e r s were pooled t o g e t h e r  and were washed w i t h 0 . 5 t o 1 volumes o f phenol and shaken gently  f o r 10 minutes b e f o r e c e n t r i f u g i n g at 5 , 0 0 0 r.p.m.  f o r 15 minutes. up  The aqueous l a y e r s were removed and made  to 2$ w i t h r e s p e c t  The  t o potassium acetate  (K*CH^C00~).  RNA was p r e c i p i t a t e d from the aqueous l a y e r w i t h 2  volumes o f c o l d 95$ e t h a n o l f o r 20 minutes.  and c e n t r i f u g e d  at 2,000. r.p.m.  The aqueous l a y e r was d i s c a r d e d  and the  p r e c i p i t a t e washed i n an e t h a n o l - d i s t i l l e d water s o l u t i o n (3sl)« salt three  The p r e c i p i t a t e was f i n a l l y d i s s o l v e d i n 0 . 1 M. NaCl  s o l u t i o n (pH at 7»0 t o 7»2),« consecutive  Phenol was removed by  e x t r a c t i o n s w i t h equal volumes o f e t h e r .  hz E t h e r was e x p e l l e d by p a s s i n g n i t r o g e n gas through the aqueous l a y e r . 2.  P u r i f i c a t i o n o f spleen RNA. Equal volumes o f 2.5 M. I^HPO^, r e d i s t i l l e d 2-methoxy-  e t h a n o l * and 0.05 v o l . o f  33.3$  H-jPO^ (upper phase, lower  phase 5 s i ) were added t o the aqueous p r e p a r a t i o n o f s p l e e n RNA.  The lower l a y e r was separated  and washed with 10-20 ml.  o f the top l a y e r from a mixture o f 2 - m e t h o x y e t h a n o l - d i s t i l l e d water-2.5 M.  K HP0^-33.3^ 2  H3PO4 (1:1:1:0.05) by volume.  combined top l a y e r s c o n t a i n e d a l l the RNA. supernatant  The  The, c l e a r  l a y e r s were added t o g e t h e r and these, were  d i a l y s e d a g a i n s t 2 l i t e r s o f d i s t i l l e d i;'ater (changed twice) f o r 6 t o 12 hours i n the c o l d at 4 - 6 ° C .  The d i a l y s a t e  was c e n t r i f u g e d , made up to 2% w i t h r e s p e c t t o potassium a c e t a t e and p r e c i p i t a t e d by 2 volumes o f 95$ e t h a n o l . The p r e c i p i t a t e was washed i n e t h a n o l and the p r e c i p i t a t e d spleen RNA s t o r e d i n 95$ e t h a n o l at -20°C.  •methoxyethanol = b.p. 122-124°C.  43  Appendix I I , 1,  The B i u r e t method f o r p r o t e i n , 1.5 g. o f c u p r i c s u l f a t e  sodium potassium t a r t r a t e  (Schneider,  1957).  (CuS0^»5H 0) and 6.0 g. o f 2  (Na KC^H^Og•4^0) were d i s s o l v e d  i n 500 ml. o f d i s t i l l e d water.  300 ml. o f 10$ NaOH was  added t o t h i s w i t h constant s w i r l i n g and the mixture was then d i l u t e d to 1 l i t e r .  T h i s served as b i u r e t  reagent.  4.0 ml. o f b i u r e t reagent was added to 1 ml. o f s o l u t i o n c o n t a i n i n g RNA and the mixture was allowed t o stand at room temperature  f o r 30 minutes.  A control  c o n s i s t e d o f 4.0 ml. o f b i u r e t reagent, and 1.0. ml. o f The a b s o r p t i o n was r e a d between 540 mji and 560 mil.  water. 2.  O r c i n o l t e s t f o r RNA.  (Schneider, 1957; C h a r g a f f and  1955).  Davidson,  1.0 g. o f p u r i f i e d o r c i n o l was d i s s o l v e d  immediately  b e f o r e use i n 100 ml. o f c o n c e n t r a t e d HC1. c o n t a i n i n g 5 g. 0 . 2 ml. o f RNA s o l u t i o n was d i l u t e d to 1.5 ml.  of FeCl^.  and heated w i t h 1.5 ml* o f o r c i n o l reagent f o r 20 minutes i n b o i l i n g water. wavelength 3.  The i n t e n s i t y o f the green c o l o u r at a  o f 660 mU was r e a d on the spectrophotometer.  Dische's diphenylamine  1957;  t e s t f o r DNA.  C h a r g a f f and Davidson,  (Schneider,  1955).  1.0 g« o f p u r i f i e d diphenylamine was d i s s o l v e d i n 100 ml. o f reagent g l a c i a l a c e t i c a c i d and 2 . 7 5 ml*- o f reagent sulfuric acid.  10 ml, o f the s o l u t i o n c o n t a i n i n g RNA was  mixed w i t h 2 ml, o f diphenylamine 10 minutes  i n b o i l i n g water.  c o l o u r at 595 4,  reagent and heated f o r  The i n t e n s i t y o f the. blue  was read on the spectrophotometer.  Dreywood's anthrone t e s t f o r c a r b o h y d r a t e s . 1,0  (Morris,  g, o f anthrone was d i s s o l v e d i n 500 ml, o f 95$  1948).  sulfuric  a c i d (prepared by a d d i t i o n  t r a t e d reagent s u l f u r i c  o f 500 ml. o f concen-  a c i d to 25 ml, o f d i s t i l l e d  and a l l o w i n g the mixture to c o o l ) .  1 ml. o f the  water,  RNA  s o l u t i o n was mixed w i t h 2 ml. o f the Anthrone. reagent and the mixture was 10 minutes.  The  p e r m i t t e d to stand at room temperature f o r i n t e n s i t y o f the b l u e c o l o u r at 650 rati was  read on the spectrophotometer.  45  Appendix I I I Chromatographic a n a l y s e s . The  s o l v e n t used "was. n-butanol  which a l s o c o n t a i n e d 2.5  s a t u r a t e d w i t h water  &• °£ p h t h a l i c a c i d i n 100  1 volume o f p h t h a l i c a c i d s o l u t i o n was o f b u t a n o l and the b u t a n o l l a y e r was  ml.  shaken w i t h 4 volumes  separated to be  used  i n the chromatography j a r . A f t e r the run, the Whatman #1 paper was  sprayed w i t h  a n a l i n e (2$ i n e t h y l e t h e r ) , allowed to dry at room temperature the s p o t s .  and then heated to 100°C i n an oven t o develop  46  Appendix IV U l t r a c e n t r i f u g a l analyses. Samples o f RNA NaCl s o l u t i o n .  The  to be analyses were d i s s o l v e d temperature  throughout  i n 0.1  the analyses  H. was  22.4°C. The  sedimentation c o e f f i c i e n t s * were read o f f the  ultracentrifuge plates  (see f i g u r e s 7» 8, and 9) and  plotted  on l o g a r i t h m i c  graph paper a c c o r d i n g to Markham ( i 9 6 0 , I 9 6 2 ) .  The p r o t r a c t o r  (designed by Markham) was p l a c e d on the graph  paper with i t s a b s c i s s a  s c a l e along a h o r i z o n t a l graph  l i n e , w i t h the l i n e corresponding to the r o t o r speed at the i n t e r s e c t i o n o f the experimental curve w i t h the l a t t e r .  The  experimental curve then i n t e r s e c t s the o r d i n a t e s c a l e o f the protractor  at the c o r r e c t  S value.  *The sedimentation c o e f f i c i e n t o f S o f r i b o n u c l e i c a c i d i s the r a t e at which i t would sediment through the suspending medium i n a f i e l d o f 1 dyne (or 1/981 per second, (S2o )» tW  o f 1.0  gram) i n centimeters  and i s g e n e r a l l y r e f e r r e d to water at 20°C.  A more u s e f u l term o r u n i t i s the Svedberg or' S  v a l u e which i s 10~ ^cm./sec./dyne. 1  Svedberg ~ S.  (Markham, 1962).  47  Appendix V Preparation Finnegan,  of modified  Niu-Twitty  (Douglas and  unpublished).  C o n t r o l medium;  a d e f i n e d medium was prepared as f o l l o w s :  NaCl KC1 CaN0„«4H 0 MgSOWB^O 1 N. HC1 g l a s s d i s t i l l e d water T r i s * buffer 2  The  medium  3.400g. 0.050g. 0.080g. O.lOOg. 4 ml. 996 ml. 0.560g.  1,000 ml. s o l u t i o n was b o i l e d f o r not l e s s than  2 minutes and then cooled o f streptomycin  to room temperature.  2 . 5 nig.  s u l f a t e was added t o the medium which was  then s t o r e d f o r as short a p e r i o d as p o s s i b l e at 7 ° C  * T r i s = hydroxy methyl amino methane o r 2-amino-2-hydroxymethyl-1,3-propanediol).  

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