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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 University of B r i t i s h Columbia, 1962 A thesis Submitted i n P a r t i a l Fulfilment of the Requirements f o r the Degree of MASTER OF SCIENCE i n the Department of Zoology We accept t h i s thesis as conforming to the required. staAdand THE UNIVERSITY OF BRITISH COLUMBIA September, 1964 In presenting this thesis i n p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make i t freely available for reference.and study. I further agree that per-mission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. I t i s understood that,copying or publi-cation of this thesis for f i n a n c i a l gain sh a l l not be allowed without my written permission* Department of P^O~^P^ The University of B r i t i s h Columbia, Vancouver 8, Canada Date Sg^z^W^/- Iff^ (J&y i i ABSTRACT Ribonucleic acid (RNA) from c a l f spleen tissue was i s o l a t e d and p u r i f i e d by a modified Kirby phenol procedure. The absorption maximum of the i s o l a t e occurred at 260. m|t i n d i c a t i n g the presence of nucleic acids and the absorption minima recorded at 230 m\l and 280 m\i indicated the absence of peptides and proteins. Colorimetric analyses indicated the presence of RNA and the absence of peptide, protein, DNA and carbohydrate contamination. Chromatographic analysis indicated the absence of carbohydrate contamination only a f t e r the p u r i f i c a t i o n with 2-methoxyethanol. The spleen RNA prepared by the phenol method was undegraded and demon-strated three d i s t i n c t molecular species when analysed with the ult r a c e n t r i f u g e ; a 2 7 S f r a c t i o n , an I.8.S. f r a c t i o n and an 8 S f r a c t i o n . Competent early gastrula ectoderm and embryos of Xenopus l a e v i s exposed to undegraded spleen RNA demon-strated no t i s s u e - s p e c i f i c induction. However, i n both the i n v i t r o and i n vivo experimental series an enhancement of development was observed. A possible explanation of t h i s phenomena was discussed. v i ACKNOWLEDGMENT The author wishes to acknowledge the guidance and encouragement of Dr. C.V. Finnegan throughout the current in v e s t i g a t i o n . I am indebted to Dr. W.S. Hoar and Dr. P. Ford f o r t h e i r comments. Appreciation i s expressed to Dr. M.E. Reichmann of the A g r i c u l t u r a l Research Station of the Government of Canada f o r technical assistance and the provision of research f a c i l i t i e s . TABLE OF CONTENTS Abstract Table of Contents L i s t of Tables L i s t of Figures Acknowledgment Introduction Materials and Methods Results Part I Part I I Discussion Summary References Appendix i v 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 of c a l f spleen k ribonucleic acid (RNA). Table 2.a.Spectrophotometry analysis of commercial l i v e r lk s-RNA. 2.b.Spectrophotometry analysis of c a l f spleen RNA lk a f t e r one phenol extraction. 2.c.Spectrophotometry analysis of c a l f spleen RNA lk a f t e r three phenol extractions. Table 3. Qualitative experiments to demonstrate the 16 presence or absence of various materials i n the i s o l a t e d sample. Table k.a.The ef f e c t of i s o l a t e d c a l f spleen RNA on 26 amphibian competent ectoderm i n v i t r o . k.b.The ef f e c t of i s o l a t e d c a l f spleen RNA on 26 whole embryos of Xenopus l a e v i s i n vivo. V LIST OF FIGURES Facing page Fig. 1. Sedimentation of the various layers following homogenization and centrifuga-t i o n of c a l f spleen tissue i n napthalene-1,5-disulfonic acid and phenol. 6 Fig. 2. A stage 9+ Xenopus l a e v i s . The dotted area represents the competent ectodermal region which was removed f o r the i n v i t r o study. 6 Fig. 3* The u l t r a v i o l e t absorption spectrum of commercial l i v e r s-RNA. 12 Fig. 4 . The u l t r a v i o l e t absorption spectra of i s o l a t e d c a l f spleen RNA following one phenol arid three phenol extractions. 13 Fig, 5» Chromatographic analysis of p u r i f i e d RNA solution before 2-methoxyethanol extraction. After the extraction no spot was observed. 19 Fig. 6 . Sedimentation diagram of commercial l i v e r s-RNA 21 Fig. 7 . Schlieren photograph of a 0.1 M. NaCl solution of spleen RNA a f t e r reaching the speed of 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 of 8S, 18S, and 2 7 S . 21 F i g . 8. Sedimentation diagram of c a l f spleen RNA 22 Fig. 9 . The combined graphs from the ultracentrifuge analysis of c a l f spleen RNA ( f i g . 7 ) . The slope of the curves i s a measure of the sedimentation c o e f f i c i e n t s , which i s read o f f on a special transparent protractor designed by Markham ( i 9 6 0 ) 23 F i g . 1 0 . Isolated Xenopus ectoderm which had been cultured i n a solution of c a l f spleen RNA f o r 5 days. No t i s s u e - s p e c i f i c induction can be seen, but, in contrast to the control series the c e l l s are better developed and more numerous. 25 Fig. 1 1 . Section through control Xenopus embryo cultured i n an "unconditioned* 1 medium. 28 Fig . 1 2 . Section through experimental Xenopus embryo cultured i n an RNA-enriched medium. 28 1 INTRODUCTION Niu and Twitty (1953) demonstrated that when amphibian organizer material (dorsal l i p of blastopore-axial mesoderm) was explanted into a non-nutrient medium, substances which diff u s e d from the explant were capable of i n i t i a t i n g d i f -f e r e n t i a t i o n i n exposed competent ectoderm. Niu (1956) characterised the material as ribonucleoprotein and suggested that the RNA f r a c t i o n was the active component, Yamada and Takata (1955) obtained d i f f e r e n t i a t i o n using guinea pig kidney and reported that the inductive f r a c t i o n was r i c h i n ribonucleoprotein. Niu (1958a,b) using c a l f thymus RNA, reported successful t i s s u e - s p e c i f i c induction i n amphibian tissue and concluded that thymus RNA resulted i n thymus-like histogenesis. Saxen and Toivonen (1962) i n discussing Niu's report con-sidered the d i f f e r e n t i a t i o n to be poor. Yamada (1961) repeated Niu's thymus RNA experiments using i d e n t i c a l i s o -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 effect on amphibian tissues with c a l f thymus RNA. More recently, Hillman and Niu (l962a,b) using RNA is o l a t e d from embryonic chick brain and. notochord reported s p e c i f i c e f f e c t s on neural and notochordal histogenesis both i n vivo and i n v i t r o but no effect was observed with chick l i v e r RNA. In the i n vivo experiments an enlargement or duplication of brain and notochordal structures was observed. Butros (1963) found that RNA i s o l a t e d from embryonic chick brain and adult rat heart resulted i n hyperplasia 2 of the epidermis while adult rat l i v e r had no e f f e c t . On the other hand, an u n i d e n t i f i e d adult pancreatic RNA enhanced the development of the endodermal epithelium. Yamada (1961, I962) and Yamada and Takata (1961) have reported d e f i n i t e and s p e c i f i c induction of neural and mesodermal tissue when competent ectoderm was exposed to protein 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 (1963b) were unable to detect any-s p e c i f i c histogenesis i n tissues which had been exposed to i s o l a t e d embryonic chick brain protein. In the present investigation an attempt was made to i s o l a t e c a l f spleen ribonucleic acid (RNA) by a modified Kirby procedure (1962), and to remove protein and other contaminants from the i s o l a t e . To test the p u r i t y of the i s o l a t e u l t r a v i o l e t (UV) absorption spectra were measured, and q u a l i t a t i v e tests f o r protein, carbohydrates and desoxy-ribonucleic acid were applied. Sedimentation analyses- of the samples were made to determine the degree of possible degradation of the RNA. 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 of the i s o l a t e d and p u r i f i e d spleen RNA was tested with Xenopus  la e v i s tissues i n both i n vivo and i n v i t r o systems. MATERIALS AND METHODS I. Preparation of c a l f spleen ribonucleic acid (RNA). Calf spleen was chosen as the source of ribonucleic acid since t h i s organ, a c t i v e l y synthesizing protein, has a high RNA content, and i s r e l a t i v e l y free of connective tissue, thereby reducing the polysaccharide contamination of the iso l a t e d material. Fresh spleen tissue was frozen by being placed on s o l i d carbon dioxide immediately following removal from slaughtered calves. Upon return to the laboratory, samples of the frozen tissue were separated from the connective tissue capsule and s l i c e d into small pieces (approximately lcm. x 2 c m . ) . Ribonucleic acid was is o l a t e d from these tissue, s l i c e s by a modified Kirby procedure (1962) as shown i n Table I (see, also, Appendix i ) . The tissue was homo-genized i n 2 . 5 m l . of 0 .015 M. napthalene - 1 , 5 -disulfonate and 2.5 ml. of 8 8 - 9 0 $ phenol (Mallinckrodt l i q u i f i e d phenol)/ gram wet weight of tissue simultaneously f o r 2 minutes i n a Waring blender at room temperature. Three ($) extractions with phenol were employed for the. i s o l a t i o n of spleen RNA used i n the culture experiments. The. cloudy supernatant layers from the o r i g i n a l centrifugate. and washings were care-f u l l y suctioned o f f to insure against contamination from the intermediate layer (see figure l ) . When preparations were p u r i f i e d by extraction with r e d i s t i l l e d 2-methoxy ethanol the time of d i a l y s i s was 6 - 1 2 hours i n the cold at k°-6° C. with 2 changes of cold saline (pH at 7 ) . Table I. The i s o l a t i o n and p u r i f i c a t i o n of c a l f spleen ribonucleic acid (RNA). spleen homogenized i n equal volumes of 0.015 M. napthalene disulfonate and water-saturated phenol at room temperature. S t i r mixture 30 minutes, centrifuge 1 hour at 0°C., 2 , 0 0 0 r.p.m. cloudy aqueous supernatant wash with 1 v o l . phenol shake 10 minutes repeat twice centrifuge 5»000 r.p.m. pr e c i p i t a t e (optional) wash with 50 ml., napthalene disulfonate, centrifuge 5,000 r.p.m. for 15 min. Repeat at least twice. supernatant p r e c i p i t a t e discard make up to 2$ with respect to K+CH-jCOO" add 2 v o l . ethanol centrifuge 2 ,000 r.p.m. f o r 20 min. cloudy aqueous p r e c i p i t a t e supernatant discard combine with f i r s t supernatent r supernatant discard p r e c i p i t a t e wash with ethanol-water ( 3si) centrifuge supernatant discard p r e c i p i t a t e dissolve i n 0 . 1 M. NaCl extract phenol with ether 3 times expel ether with Nitrogen gas (continued on the next page) 5 Table I. (continued) cle a r aqueous solution p r e c i p i t a t e 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 layer, wash with 20 ml. of top layer from ( 1 : 1 : 1 : 0 . 0 5 ) . dialyse against 2 :1 . of water centrifuge contents of bag. supernatant p r e c i p i t a t e discard make up to 2$ K+CH^C00-pre c i p i t a t e with 2 v o l . 95$ ethanol. centrifuge. r supernatant discard 1 pr e c i p i t a t e wash RNA 6 Figure 1. Sedimentation of the various layers of c a l f spleen tissue following homogenization i n napthalene disulfonate and phenol. Figure 2. A stage 2+ Xenopus l a e v l s . The dotted area represents the competent ectodermal region which was removed f o r the i n v i t r o study. CLOUDY AQUEOUS SUPERNATE INSOLUBLE PROTEIN PriENOL LAYER r RNA L -POLYSACCHARIDES — PHENOLIC NUCLEI DNA-PROTEIN PRECIPITATE To assure the removal of a l l previous contaminants and e s p e c i a l l y ribonuclease. (RNase) both glassware and equipment were washed with IN. NaOH. Versene^, a chelating agent, was used i n 2% concentrations to remove heavy metal ions 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 centrifuged i n an International high speed centrifuge model HR-1 with an #856 head. A l l readings were i n revolutions per minute (r.p.m.). In some of the control biochemical- and physical tests commercial l i v e r RNA, consisting of 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 material (s-RNA) was not used i n the tissue culture or i n vivo experiments concerned with 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. I I . UV absorption spectra Spectrophotometric readings of both the commercial ( l i v e r ) RNA and the i s o l a t e d (spleen) RNA solutions were made using the Unicam Sp. 200 spectrophotometer and the Beckman spectrophotometer. Prom t h i s data the r a t i o of 1. versene = E.D.T.A. or ethylene diamine t e t r a c e t i c acid. 2. s-RNA = soluble or transfer RNA. ' 3. S = Svedbers unit or S value = 10-13 m./sec./dynes. 4. Cetavlon = cetyltrimethyl ammonium bromide. 8 the absorption values at 230 m|i, to 260 mji. and 280 m\i to 260 m|i was calculated to indicate the presence or absence of nucleic acids (26O m(-l) , proteins (280 m|i), and peptides (230 m|i) (Morton, I 9 6 2 ) . I I I . Coloriraetric and chromatographic tests.. Solutions containing i s o l a t e d spleen RNA were tested to determine the presence o f contaminating substances. The Biuret test (Schneider, 1957) was applied to determine the presence of protein. Dische's diphenylamine test Schneider, 1957; Chargaff and Davidson, 1955) was u t i l i z e d to demonstrate the presence or absence of DNA. The B i a l 1 s o r c i n o l method (Chargaff and Davidson, 1955) was used to. confirm the presence of RNA i n solution (see Appendix I i ) . Dreywood's anthrone test (Morris, 1948) was used to demon-strate the presence of carbohydrates.. A chromatogram was. prepared to demonstrate the presence or absence of carbo-hydrates ( i e . polysaccharides) before and af t e r extraction with 2-methoxyethanol (see Appendix I I I ) . IV. U l t r a c e n t r i f u g a l analyses. Sedimentation analyses were carr 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 . UV optics were employed for an examination of the commercial s-RNA preparation from l i v e r and Schleiren optics were employed f o r examina-t i o n of the i s o l a t e d c a l f spleen RNA. Photographs were taken after the speed of 44,770 r.p.m. was. attained i n the case of spleen RNA and 55>770 r.p.m. i n the case of 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 of Markham (i960, 1962) (see Appendix IV). V, Preparation of b i o l o g i c a l materials. Eggs of Xenopus l a e v i S - (Daudin, 1802) were obtained with the method of Brown and L i t t n a (1964) modified by using a "breeding medium" containing NaCl, KC1, MgSO^^HgO, Ca(N02)2*^ H2°» 3 1 1 ( 1 streptomycin sulfate but not p e n i c i l l i n . The pH of the medium was maintained at. 7 and the l i g h t and dark periods were alternated every 12 hours. Eggs were removed from the breeding medium when at stages 9 and 9+ (very early gastrula) according to Nieuwkoop and Faber (1956). A modified Niu-Twitty medium (Douglas and Finnegan, unpublished) was used to culture whole animals i n vivo. and ectodermal tissue cultures i n v i t r o (see Appendix V) . This medium was "conditioned" by the addition of 50 (Ag/ml. of p u r i f i e d spleen RNA, added immediately before use i n order to minimize any possible RNA degradation e f f e c t s . Competent ectoderm (see figur e 2) was i s o l a t e d from stage 9+ animals with care taken to remove any cl i n g i n g material from the ectodermal i s o l a t e . S t e r i l e procedures were used throughout the experiments. Explants developed i n either control or conditioned media f o r 5 days, before they were f i x e d i n Carnoys, embedded i n p a r a f f i n wax and sectioned at 5 V- f o r h i s t o l o g i c a l examination. Similar culture and h i s t o l o g i c a l procedures were followed for the whole embryos though the l a t t e r were sectioned at 8 (X. 10 The ivhole embryos were stained viith methyl-green Pyronin, Toluidine blue and Haematoxylin and Eosin. The tissue culture b a l l s were stained with Chromotrope. RESULTS Part I. Isolation, p u r i f i c a t i o n and characterisation of c a l f spleen RNA. Samples of ribonucleic acid (RNA), i s o l a t e d and p u r i f i e d by the modified Kirby procedure, were dissolved i n o . l M. NaCl solution and were examined with the spectro-photometer i n the u l t r a v i o l e t regions to determine the presence of nuclei c acids, proteins and peptides (Morton, 1 9 6 2 ) . Absorption spectra f o r a commercial preparation of l i v e r s-RNA as well as the i s o l a t e d c a l f spleen RNA are represented i n figures 3 and 4 . It can be seen from figure 3 that the l i v e r s-RNA demonstrated an absorption peak at 265 m|i and as seen i n Tables 2 a , 2b, and 2c the r a t i o of the absorption values 260 m|i/230 m\l was 2 .20 and the r a t i o of the values 260 m|i/280 m\X was 1.82. The former r a t i o of 2.20 demonstrates the r e l a t i v e freedom of the s-RNA from peptide contamination and the l a t t e r r a t i o demonstrates freedom from protein contamination. When c a l f spleen RNA was i s o l a t e d by the Kirby proce-dure with a single phenol extraction an absorption curve as represented i n figure 4 was produced i n which the r a t i o of the absorption values 260 m|0,/23O mjl was 1.80 and the ra t i o of 260 mfJ./280 mfX was 1.82 {see Table 2 b ) . As i s ch a r a c t e r i s t i c of nucleic acids, the maximum absorption occured at 260 m|i and the minimum absorption values were recorded at 230 m|i and 280 mfl, t y p i c a l of peptides and proteins respectively (Morton, 1 9 6 2 ) . When c a l f spleen 12 Figure 3» The u l t r a v i o l e t absorption spectrum of commercial l i v e r s-RNA. 13 Figure 4. The u l t r a v i o l e t absorption spectra of i s o l a t e d c a l f spleen RNA following one phenol and three phenol extraction(s). 14 Table 2 a . Spectrophotometric analysis of commercial l i v e r s-RNA. wavelength (mfi) reading i n O.D. r a t i o 260/230 260/28O 230 G.266 260 0.588 280 0 . 4 2 1 2.20 1.82 Table 2b. Spectrophotometric analysis of c a l f spleen RNA afte r one phenol extraction. wavelength (m|i) reading i n O.D. r a t i o 260/230 260/280 230 0 . 6 9 0 260 1 .240 280 0.680 1.80 1 .82 Table 2 c . Spectrophotometric analysis of c a l f spleen RNA af t e r three phenol extractions. wavelength (m(i) reading i n O.D. r a t i o 260/230 260/280 230 0 . 5 5 0 260 1.230 280 O.56O 2 . 2 3 2.20 15 RNA was extracted with three phenol, washings an absorption spectrum was produced as seen i n figure 4 i n which the r a t i o of the absorption value 260 m(l/230 m\x was 2 .23 and the r a t i o of the absorption value 260 m|i/280 m(i was 2 . 2 0 . (See Table 2 c ) . These r a t i o s indicate that protein and peptide conta-mination was more e f f e c t i v e l y removed with the additional phenol extraction. The uv absorption at 260 m\l indicates the presence of a l l nucleic acids (DNA as well as RNA) . Thus i t i s necessary to determine more s p e c i f i c a l l y the nature of the constituents of the i s o l a t e p a r t i c u l a r l y since contamination by substances other than ribonucleic acid i n i s o l a t i o n s using the Kirby procedure has been reported by various workers (Kirby, 1956, I960, 1962; Laskov et a l . . 1959; Huppert and Pelmont, 1962; and, Ralph and Bellamy, 1964). To t h i s end various q u a l i t a t i v e tests were employed to indicate the presence or absence of s p e c i f i c molecular constituents. The r e s u l t s of these tests are represented i n Table 3. The Biuret test, to indicate the presence of protein, when applied to samples both of one and three phenol extrac-tions yielded negative r e s u l t s . When the biuret-RNA solution complex was observed on the spectrophotometer at 550 m(i no blue colour developed which would be i n d i c a t i v e of peptides or proteins. However, the Biuret method cannot be considered as sensitive as an analysis of the constituents of the RNA solution with the spectrophotometer (see Tables 2b and 2 c ) . 16 Table 3» Qualitative experiments demonstrating the presence or absence of various materials i n the i s o l a t e d sample. Test 1. Biuret 2. Orcinol ( B i a l ' s test) Result (a) one phenol no color change (b) three phenols no color change (a) one phenol very p o s i t i v e green color ( b ) three phenols very p o s i t i v e 3. Diphenylamine (a) one phenol (Dische's test) s l i g h t blue color developed (b) three phenols no color change (a) before methoxy k. Anthrone (Dreywood* s) Comment absence of proteins and peptides absence of proteins arid peptides indicates the presence of RNA i n large quantity RNA present presence of small quantity of DNA no DNA detectable s l i g h t change of colour from yellow to blue-green (b) a f t e r methoxy ho colour change carbohydrate present no detectable carbohydrate 17 The presence of ribonucleic acid (RNA) could be confirmed by the method of B i a l , which employs or c i n o l reagent and i s s p e c i f i c f o r purine ribonucleotides (Chargaff and Davidson, 1955)* After the solution containing i s o l a t e d spleen RNA 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 colour developed, c h a r a c t e r i s t i c of solutions containing RNA. The i s o l a t e following three phenol extractions gave a reading on the spectrophotometer of 0 . 3 0 . Since an absorption maximum at 260 m\X could represent unprecipitated DNA as well as the demonstrated RNA, the Dische diphenylamine test, 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 tested with diphenylamine reagent a p o s i t i v e blue colour developed i n d i c a t i n g that DNA was present. The spectrophotometric reading of t h i s mixture at 595 m|J, was 0 . 0 2 6 . Since l i t t l e care was taken to remove the upper layer without contamination from the i n t e r f a c i a l layer, the small quantity of DNA which reacted with the reagent could be attributed to the phenolic nuclei which have been demonstrated i n t h i s intermediate layer by Georgietf et a l . , ( i 9 6 0 ) . On the other hand a f t e r three phenol extractions and complete removal of the i n t e r f a c i a l material the same test was negative i n d i c a t i n g the absence of DNA contamination i n any detect-able quantity (see Table 3 ) . Since any possible r o l e of carbohydrates such as muco-polysaccharides i n induction i s unknown i t i s considered imperative to remove a l l traces of these substances. That polysaccharide contamination exists, even a f t e r extensive 18 phenol extractions has been demonstrated by Kirby (195-6, 1962) and Ralph and Bellamy ( 1 9 6 4 ) . Two methods were employed to indicate the presence of contaminating carbo-hydrates. One method was the Anthrone test of Dreywood (Morris, 1948), which i s s p e c i f i c f o r carbohydrates. The RNA solution, a f t e r three phenol extractions, developed a blue-green colour with anthrone reagent at room temperature. The reading on the spectrophotometer at 620 m|i was O.O65 i n d i c a t i n g the d e f i n i t e presence of carbohydrates. After methoxyethanol extraction the RNA solution demonstrated no colour change with the anthrone reagent. Thus the carbohydrate contamination appears to 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 of demonstrating carbohydrate i n the i s o l a t e was accomplished with paper chromatography. A chromatographic study of the i s o l a t e d RNA before p u r i f i c a t i o n with 2-methoxyethanol demonstrated traces of polysaccharide and one spot corresponded to the control spot containing glucose (see figure 5 ) . The second spot i n the i s o l a t e may be ribose. Following extraction with methoxyethanol no trace of carbohydrate could be detected. These r e s u l t s indicate that the i s o l a t e contains RNA i n the absence of DNA, peptide, protein and carbohydrate contamination only a f t e r three phenol extractions and p u r i f i c a t i o n with methoxyethanol extraction. However, these data have given no clue as to the size or condition (de-graded) of the molecules of i s o l a t e d RNA. To obtain information on the size and condition of both commercial l i v e r s-RNA and Kirby i s o l a t e d c a l f spleen BEFORE liETHOXYETHANOL EXTRACTION GLUCOSE CONTROL AFTER METHOXYETHANOL EXTRACTION 19 Figure 5- Chromatographic analysis of p u r i f i e d RNA. solution before 2-methoxyethanol extraction. After the extraction no spot was observed. 20 RNA, analyses of these molecules were carried out using, 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 . The sedimentation patterns obtained by t h i s procedure are shown i n figures 6, 7» and 8. The quantity of commercial s-RNA was small and thus uv optics were employed. The diagram obtained i n figure 6 represents the l i v e r RNA i n 0.1 M. NaCl at 55,770 r.p.m. for 20 minutes. As can be seen from figure 6, there was no apparent movement of the molecules. This indicates either that the molecule of s-RNA was a large molecule which had become degraded, possibly by the i s o l a t i o n procedures employed or by methods of storage, or that the s-RNA molecule was quite small. The l a t t e r explanation would appear to be consistent with the present knowledge concerning the size of soluble RNA (S p i r i n , 1963). The analysis of i s o l a t e d c a l f spleen RNA using Schlieren optics demonstrated a d e f i n i t e molecular species (figure 7). The sedimentation c o e f f i c i e n t s expressed i n Svedberg or S units for these molecules were calculated from the graph (figure 8) by the method of Markham (i960). The values for the 3 species of RNA were 27S, 18S, and 8S respectively, (SgQ w ) for water at 20°C. The fas t e s t moving component., the 27S f r a c t i o n , xvas also present i n the highest concentration. This sedimentation pattern f o r spleen RNA served to demon-. strate that the i s o l a t e d molecules were r e l a t i v e l y i n t a c t and not degradod by the i s o l a t i o n procedure. It i s t h i s material which was used i n the induction experiments. 7-9 S 1 ? 7 S 21 Figure 6. Sedimentation diagram of commercial liver-RNA. Figure 7. Schlieren photograph of a 0.1M NaCl solution of spleen-RNA a f t e r reaching the speed of 44,770 r.p.m. in 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 of 8S, 18S, and 27S. 22 Figure 8. Sedimentation diagram of c a l f spleen RNA. 5-9 I 1 1 I I i • • i 1 2 3 4 5 6 EXPOSURE ANGLE 23 Figure 9« The combined graphs from the ultracentrifuge analysis of c a l f spleen RNA ( f i g . 7). The slope of the curves i s a measure of the sedimentation c o e f f i c i e n t s , which i s read o f f on a special transparent protractor designed by Markham (i960). 24 Part I I . The i n v i t r o and i n vivo effects of c a l f spleen RNA on competent ectoderm and embryos of Xenopus  l a e v i s . The i s o l a t e d , undegraded and uncontaminated c a l f spleen RNA was tested 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 ( i . e . i n -duction) by exposing the tissues of Xenopus l a e v i s embryos under both i n v i t r o and i n vivo conditions to the RNA at. a concentration of 50 Jig/ml. (Niu, 1958; Yamada, 1 9 6 l ) . v i t r o studies. When excised competent ectoderm from stage 9+ embryos was placed either i n RNA-conditioned or control media there was no adhesion of the gastrula ectoderm to the glass cover-s l i p s i n the culture dishes and within one-half an hour of the operation the ectodermal explant had r o l l e d into a b a l l . These ectodermal explants cultured i n the RNA-conditioned medium for 5 days demonstrated no s p e c i f i c induction. Neither were any indications of histogenesis evident i n the control series cultured i n the balanced s a l t solution 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 protein and RNA. However, the enhanced development of the explants i n the conditioned medium was most evident (see figure 10 and Table 4 a ). When the sectioned materials were compared, the single-layered c i l i a t e d ectodermal c e l l s of the explants i n unconditioned medium contrasted with the s u p e r f i c i a l c e l l s of the RNA-enhanced explants. which were two to: four layers i n thickness and with certain, of the c e l l s -appearing columnar rather than cuboidal. In addition ectodermal explants i n RNA-medium were observed to survive better than those grown i n the unconditioned medium. \ 1 25 Figure 10. Isolated Xenopus ectoderm which had been cultured i n a solution of c a l f spleen RNA f o r 5 days. No t i s s u e - s p e c i f i c induction can be seen, but i n contrast to the control series the c e l l s are better developed and more numerous. 26 Table 4a. E f f e c t of i s o l a t e d calf, spleen RNA on amphibian competent ectoderm i n v i t r o . number of cases inductive effect no effect controls 6 0 6 experimentals 18 0 18 N.B. Although 6 control animals and 18 experimental animals were observed throughout the experiment, only 2 of each series, chosen at random, were sectioned. Table 4b. E f f e c t of i s o l a t e d c a l f spleen RNA on whole embryos of Xenopus la e v i s i n vivo. number of cases inductive effect no effect controle 8 0 8 experimentals 8 0 8 N.B. A l l animals i n t h i s series were sectioned. 27 2. In vivo studies. Stage 9+ embryos were placed i n RNA-medium and control, medium and allowed to develop f o r 5 days. Embryos developed well i n both control and conditioned media. However.,, the experimental animals were at a l l times s l i g h t l y , but con-s i s t e n t l y , accelerated i n organogenesis as compared to the. control animals (see figures 11 and 12 and Table 4b). Also, while there was no increased histogenesis of spleen-like or mesodermal tissue i n the experimental animals at the termination of the culture period, the o t i c v e s i c l e s demon-strated a more pronounced d i f f e r e n t i a t i o n of the ventro-medial epithelium and were becoming divided into the pars i n f e r i o r and the pars superior, the torsion of 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 of the ga s t r i c glands more d i s t i n c t than i n the controls. 28a Figure 11. Section through control Xenopus embryo cultured i n an "unconditioned" medium. V 28b Figure 1 2 . Section through, experimental Xenopus embryo cultured 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 by the Kirby two-phase p a r t i t i o n system (1956) required modification to. insure less contaminated and undegraded RNA molecules f o r the subsequent induction experiments since RNA i s o l a t e d by t h i s method has been shown to be contaminated with other components (Kirby, 1956, i 9 6 0 , 1962; Ralph and Bellamy, 1964). Following the f i r s t phenol washing the aqueous phase can be demonstrated to contain RNA, polysaccharides (Kirby, 1956; and Table 4 ) , protein (Ralph and Bellamy, 1964), amino acids (Niu et a l , 1 9 6 l ) , and DNA (Table 4 ) . Any l i p i d s which may be present i n the i s o l a t e have been shown by Hayashi (1959) to be unimportant i n induction and are not considered i n t h i s work. In the present investigation an u l t r a v i o l e t absorption analysis of i s o l a t e s obtained with one phenol washing and with three phenol washings has demonstrated that, protein and peptide c ontamination was considerably reduced with no s i g n i f i c a n t reduction i n RNA content (Table 2b and 2 c ) . However, Huppert and Pelmont (1962), using the more sensitive method of Lowry f o r protein determination, found a protein content between 20 and 50 |0.g/ml. a f t e r three phenol extrac-tions. Thus i t i s d i f f i c u l t to consider, as do many workers (Gierer, 1957; Gierer and Schramm, 1956a, b; and Kirby, 1962; Niu, 1958a, b; Niu et a l , 1 9 6 l ; Hillman and Niu, 1963a, b; and, Butros, 1963)» that protein or peptide contamination i s completely absent f o r the i s o l a t e d RNA even after three phenol washings. Very small quantities of protein would not be detected with the Biuret test and the r a t i o s of the values 30 at 260 m|i/230 m|A and 260 mfi/280 mjx only serve to indicate the q u a l i t a t i v e absence of peptides and proteins from the i s o l a t e d sample. Certainly the low l e v e l of peptide and protein contamination possibly remaining after three phenol extractions i n the current experiments was i n s u f f i c i e n t to act inductively. The DNA, present- i n the RNA solution after one phenol extraction and attributed-to "phenolic . n u c l e i " (Georgiev, i960) i n the i n t e r f a c i a l material, i s removed with the subsequent phenol extractions. Carbohy-drates or polysaccharide contamination (see Figure 5) i s not removed with phenol but with 2-methoxyethanol extraction. Laskov et a l (1959) found that RNA i s o l a t e d by the phenol water method (Kirby, 1956; Gierer and Schramm, 1956a, b) resulted i n degradation of the molecule. A higher molecular weight RNA was obtained i f napthalene-1,5-disulfo-nate was employed i n place of water. The i n s t a b i l i t y of the i s o l a t e d RNA molecule has been demonstrated by Huppert and Pelmont (1962), Kubinski and Koch (1963) and Amos and Moore (1963) who have shown that when the i s o l a t e was l e f t at room temperature degradation began within 130 minutes and was d r a s t i c within 24 hours. After several days no sedi-mentable f r a c t i o n s were observable (Huppert and Pelmont, I962). Equally pertinent, was the observation of Amos and Moore (1963) and Amos ejt a l (1964) who reported that, while they were able to obtain an active proparation of RNA only i r r e g u l a r l y , the active preparation retained i t s b i o l o g i c a l a c t i v i t y when stored at temperatures of -20°C. over a period of several weeks. As a r e s u l t of these studies the spleen RNA u t i l i s e d i n the present experiments was employed 31 either immediately following i s o l a t i o n or within the. next 7 days after 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 of the i s o l a t e , i f existing, would have been maintained. A possible source of RNA degradation i s by ribonuclease a c t i v i t y during or after i s o l a t i o n procedures. Kirby (1956, i960) demonstrated that ribonuclease was i n h i b i t e d by phenol reagent, which apparently acts to denature the ribonuclease molecule. However, when the endogenous ribonuclease i n the. tissues has been destroyed by phenol a c t i v i t y there i s s t i l l the danger of contamination from glassware used i n the previous i s o l a t i o n s . Extreme care was employed throughout the present i s o l a t i o n s to reduce as much as possible any such sort of degradation. Since these experiments were terminated Ralph and Bellamy (1964) introduced a modification of the Kirby procedure i n which RNA i s recovered as the cetyltrimethylammoniura s a l t . The modification i s based on the f i n d i n g of Dutta et a l (1953) that a difference i n s o l u b i l i t y of the Cetavlon^ s a l t s of the nucleic acids permits the separation of RNA from DNA, This method of Ralph and Bellamy i s rapid and removes the d i a l y s i s step which i t s e l f produces an opportunity f o r ribonuclease a c t i v i t y . The sedimentation evidence, presented i n figures 7, 8, and 9» demonstrates that the i s o l a t e d spleen RNA was composed of three molecular species; a 27S f r a c t i o n , an 18.S f r a c t i o n and an 8S f r a c t i o n . The 27S and. 18S fractions would appear to represent undegraded RNA molecules, the ribosomal-f r a c t i o n ( S p i r i n , I963). 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 ascites tumor c e l l s 32 and s i m i l a r r e s u l t s were reported by Ralph and Bellamy (1964) with RNA i s o l a t e d from rat l i v e r , Chinese cabbage, and tobacco c e l l s ; by H a l l and Doty (1959) with c a l f l i v e r microsomes; and by Cheng ( l959» 19^0) with mouse brain. Jiang ( 1 9 6 4 ) , i s o l a t i n g ribonucleic acid from mammalian p i t u i t a r i e s demonstrated three fr a c t i o n s , 28S, 18S, and 4S. These values correspond to those obtained i n the present analysis so that the 8S f r a c t i o n found here may not be a. degradation product. The sedimentation c o e f f i c i e n t s of the ribosomal RNA (27S and 18S) appear to d i f f e r s l i g h t l y depending upon the b i o l o g i c a l source of the preparation ( S p i r i n , 1963). Demonstration of the two high molecular weight species of RNA from c a l f spleen isolate- tends to support the idea that the RNA molecules obtained from the modified procedure were i n t a c t and undegraded when i n t r o -duced into culture medium f o r the induction experiments.. The procedure f o r the i s o l a t i o n of ribonucleic acid as employed by Niu (1958 a,b), Niu et, a l (I96I) , Hillman and Niu (1963 a,b), Yamada (1961), and Butros (1963) were e s s e n t i a l l y the method proposed by Kirby i n 195^, which has been demonstrated (Laskov ejb a l . , 1959) to y i e l d a rather degraded product. The shorter procedure also used by these workers, i n which the methoxyethanol p u r i f i c a t i o n procedure was deleted, would tend to reduce the amount of degradation of RNA but since only one phenol extraction was performed the protein and peptide as well as the poly-saccharide contamination of t h e i r product might be s i g n i f i c a n t . Niu (1958b) dialysed his sample against running water over-night, a procedure that may allow considerable ribonuclease 33 contamination, and apparently no attempt was made to remove polysaccharide material even though l i v e r and muscle tissues were the source material used. In the present experiments c a l f spleen tissue 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 with a large quantity of nucleic acid and because Yamada ( l 9 6 l ) and Yamada and Takata ( l 9 6 l ) were able to obtain such d e f i n i t e inductions using the RNP and protein f r a c t i o n s of bone marrow ti s s u e . How-ever, both the i n v i t r o and i n vivo r e s u l t s have indicated the absence of any t i s s u e - s p e c i f i c ( i . e . mesodermal) i n -ductive response when i s o l a t e d and p u r i f i e d c a l f spleen RNA was presented to competent embryonic material. Niu et a l . (1961) and Amos and Moore (1964) have demonstrated that RNA molecules are taken up by embryonic c e l l s . Since no inductive e f f e c t was observed i t i s necessary to consider as a possible f a c t o r the exposure time of the test material. In the f i r s t p lace )5 days of culture appeared s u f f i c i e n t to r e s u l t i n any observable histogenesis since the tissues and embryos of Xenopus l a e v i s are exceedingly rapid i n t h e i r development. With regard to the i n i t i a l exposure time, Yamada (1962) has demonstrated that a 55$ inductive response was achieved i n amphibian, ectoderm afte r 30 minutes exposure to a protein medium and that this, could be increased to with a 180 minute treatment. There-fore, induction under these test conditions, should occur within the f i r s t hour(s) of exposure of the. competent tissue to the inducer. I t seems l i k e l y that i t i s p r e c i s e l y during t h i s time when i n t a c t RNA would be present i n the test 34 medium since Huppert and Pelmont (1962) have shown that degradation of i s o l a t e d RNA i s only i n i t i a t e d within the f i r s t 130 minutes at room temperature (20°C) and these cultures were maintained at a temperature of 12°C. This would tend to decrease the rate of degradation so that t o t a l degradation of the RNA molecules i n the test medium would not be complete f o r more than 2k hours. Thus, at some time aft e r introducing the in t a c t RNA molecules and the competent ectoderm into the medium, and a f t e r the. time when the RNA induction could have occurred, there probably would be present a solution of nucleotides and nucleosides instead of the i n t a c t RNA molecules. While the presence of associated protein i n the test media might function 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 inductive system, i t i s also possible that the i n v i t r o induction observed by Niu and Twitty (1953) and Niu (1958b) may have been the r e s u l t of a s p e c i f i c response by competent tissue to a high concentration of oligonucleotides (Lash jet a l , 1962) or RNP (Yamada, 1958> 1961, I962) present i n the conditioned medium. While no induction was evidenced i n the present i n -vestigation, there was observed an enhancement of tissue development both i n the ectodermal explants and i n the. whole embryos. This e f f e c t with spleen RNA i n v i t r o and i n vivo appears to be s i m i l a r to the r e s u l t s described by Ambellan (1955, 1958, 1962. 1963) i n which solutions of various nucleotides were found to accelerate neural-tube formation i n amphibian embryos. A s i m i l a r mechanism may be operating i n the enhancement of d i f f e r e n t i a t i o n achieved with pan-cr e a t i c , heart and brain RNA by Butros (1963). This 35 enhancement of development, may have been the resu l t of tissue exposure to both intact RNA and RNA fragments ( i . e . nucleo-tides) since both would be present i n the early period of these experiments. F i n a l l y i t must be noted that the. absence of s p e c i f i c inductive e f f e c t s i n any of these test media.may have been due to the absence of 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 al.(1964) . 36 SUMMARY 1. Ribonucleic acid (RNA) from c a l f spleen tissue was i s o l a t e d and p u r i f i e d by a modified Kirby procedure. 2. The u l t r a v i o l e t absorption spectrum of the RNA solution was measured. The absorption maximum occured at 260 m(i i n d i c a t i n g the presence of nucleic acids while the absorption minima observed at 230 mU and 280 mH indicated the absence of peptides and proteins. 3. Colorimetric analyses indicated the absence of peptides, proteins, DNA, and carbohydrates as well as the presence of RNA. 4. Chromatographic analysis indicated that the samples of i s o l a t e d RNA contained traces of carbohydrate before extraction with methyoxyethanol but were free from carbohydrate contamination a f t e r the extraction. 5. A sedimentation analysis was performed which indicated that the i s o l a t e contained three RNA f r a c t i o n s : a 27S f r a c t i o n , an 18S component and an 8S component. I t was demonstrated that the RNA i s o l a t e d i n the present experimental series was undegraded. 6. Competent ectoderm excised from stage 9+ Xenopus laevis and cultured i n RNA-conditioned medium demonstrated no t i s s u e - s p e c i f i c induction but did demonstrate enhanced development when compared to the control series. 7 . S i m i l a r l y , when stage 9 + Xenopus were grown i n RNA-enriched medium the experimental animals were s l i g h t l y advanced i n development over the control animals. Again, no t i s s u e - s p e c i f i c induction was observed. 37 REFERENCES Ambellan, E. 1955. E f f e c t of adenine mononucleotides on neural tube formation of frog embryo. Proc. Natl . Acad. S c i . (USA). 4 l : 428-432. 1958. Comparative ef f e c t s of mono-, d i - , and triphosphorylated nucleosides on amphibian morpho-genesis. J . Embryo1. Exptl. Morphol. 6: 86-93. Ambellan, E. and Webster, G. 1962. E f f e c t s of nucleotides on neurulation i n amphibian embryos. Develop. B i o l . 452-467. 1963. 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Niu, M.C. 1956. New approaches to the problem of 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. Rudnilk. University Press, Princeton, pp. 155-171. 1958a. The role of ribonucleic acid i n embryonic d i f f e r e n t i a t i o n . Anat. Rec. 131: 585. 1958b. Thymus ribonucleic acid and embryonic d i f f e r e n t i a t i o n . Proc. Natl. Acad. S c i . (USA). 44: 1264-1274. Niu, M.C, and Twitty, V.C. 1953. The d i f f e r e n t i a t i o n of gastrula ectoderm i n medium conditioned by a x i a l mesoderm. Proc. Natl. Acad. S c i . (USA) . 21'' 985-989. Niu, M.C., Cordova, C.C., and Niu, L.C. I96I. Ribonucleic acid 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 ribonucleic acids. Biochim. Biophys. Acta. 82: 9-16. Saxen, L. and Toivonen, S. 1962. Primary Embryonic Induction. Prentice-Hall. Inc., New Jersey. 4o Schneider, W.C. 1957» Determination of nucleic acids of tissues by pentose analysis.. In, Methods i n Enzymology. Vol. 3« ed by S.P. Colowick and N.O. Kaplan. Academic Press, New York. pp. 680-684. Spi r i n , A.S. I 963 . Some problems concerning the macromolecu-l a r strecture of ribonucleic acids. In Progress i n . Nucleic Acid Research. Vol. 1. ed. by J.N. Davidson, and W.E. Cohn. Academic Press, New York. pp. 301-345* Yamada, T. 1958. Induction of 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 of proteins and nucleoproteins i n the is o l a t e d ectoderm of Triturus-gastrulae. Experentia 14: 81 -87. 196I. A chemical approach to the problem of the organiser. In, Advances i n Morphogenesis. Vol. 1. Ed. by M. Abererombie and J . Brachet. Academic Press, New York. pp. 1 -53 . 1962. The inductive phenomenon as a tool f o r understanding the basic mechanism of d i f f e r e n t i a t i o n . J. C e l l . Comp. Physiol. 60 (Suppl. l ) : 49-64. Yamada, T. and Takata, 1955• An analysis of spino-caudal induction by the guinea p i g kidney i n the is o l a t e d ectoderm of the Triturus -gastrula. J . Exp t l . Zool. 128: 291-332. 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 solution f o r morphogenetic eff e c t s on the i s o l a t e d ectoderm of the amphibian gastrula. Develop. B i o l . Ji: 411-423. 41 Appendix I Extraction of c a l f spleen RNA (Kirby, 1956, 1962) 1. I s o l a t i o n of spleen RNA. The spleen tissue was homogenized i n 0.015 M. naptha-lene - 1 , 5-disulfonate (at a pH of 6 . 8 to 7«0) and water saturated phenol ( 2 . 5 ml. of 0.015 M, napthalene disulfonate and 2 . 5 ml. of 8 8 - 9 0 $ phenol / gram wet weight of tissue) for 2 minutes i n a Waring blender at room temperature. The homogenate was f i l t e r e d through a single layer of gauze i n a Buckner funnel. This mixture was s t i r r e d f o r 30 minutes and centrifuged f o r 1 hour at 0°C at 2 ,000 r.p.m. The pr e c i p i t a t e obtained was washed with 100 ml. of 0.015 M. napthalene disulfonate and centrifuged f o r 15 minutes at 5,000 r.p.m. This washing procedure was repeated at least twice. The cloudy supernatant layers from the o r i g i n a l centrifugate and the washings, were c a r e f u l l y suctioned o f f to insure against contamination from the intermediate layer (see figure 1 . ) . The supernatant layers were pooled together and were washed with 0 . 5 to 1 volumes of phenol and shaken gently f o r 10 minutes before centrifuging at 5 , 0 0 0 r.p.m. for 15 minutes. The aqueous layers were removed and made up to 2$ with respect to potassium acetate (K*CH^C00~). The RNA was p r e c i p i t a t e d from the aqueous layer with 2 volumes of cold 95$ ethanol and centrifuged at 2,000. r.p.m. for 20 minutes. The aqueous layer was discarded and the pr 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 solution ( 3 s l ) « The p r e c i p i t a t e was f i n a l l y dissolved i n 0.1 M. NaCl salt solution (pH at 7»0 to 7»2),« Phenol was removed by three consecutive extractions with equal volumes of ether. hz Ether was expelled by passing nitrogen gas through the aqueous layer. 2. P u r i f i c a t i o n of spleen RNA. Equal volumes of 2.5 M. I^HPO^, r e d i s t i l l e d 2-methoxy-ethanol* and 0.05 v o l . of 3 3 . 3 $ H-jPO^ (upper phase, lower phase 5si) were added to the aqueous preparation of spleen RNA. The lower layer was separated and washed with 10-20 ml. of the top layer from a mixture of 2-methoxyethanol-distilled water-2.5 M. K 2 H P 0 ^ - 3 3 . 3 ^ H3PO4 (1:1:1:0.05) by volume. The combined top layers contained a l l the RNA. The, clear supernatant layers were added together and these, were dialysed against 2 l i t e r s of d i s t i l l e d i;'ater (changed twice) f o r 6 to 12 hours i n the cold at 4 - 6 ° C . The dialysate was centrifuged, made up to 2% with respect to potassium acetate and p r e c i p i t a t e d by 2 volumes of 95$ ethanol. The p r e c i p i t a t e was washed i n ethanol and the p r e c i p i t a t e d spleen RNA stored i n 95$ ethanol at -20°C. •methoxyethanol = b.p. 122-124°C. 43 Appendix I I , 1, The Biuret method f o r protein, (Schneider, 1957). 1.5 g. of cupric sulfate (CuS0^»5H 20) and 6.0 g. of sodium potassium t a r t r a t e (Na KC^H^Og•4^0) were dissolved i n 500 ml. of d i s t i l l e d water. 300 ml. of 10$ NaOH was added to t h i s with constant swirling and the mixture was then d i l u t e d to 1 l i t e r . This served as biuret reagent. 4.0 ml. of biuret reagent was added to 1 ml. of solution containing RNA and the mixture was allowed to stand at room temperature f o r 30 minutes. A control consisted of 4.0 ml. of biuret reagent, and 1.0. ml. of water. The absorption was read between 540 mji and 560 mil. 2. Orcinol test f o r RNA. (Schneider, 1957; Chargaff and Davidson, 1955). 1.0 g. of p u r i f i e d o r c i n o l was dissolved immediately before use i n 100 ml. of concentrated HC1. containing 5 g. of FeCl^. 0 .2 ml. of RNA solution was d i l u t e d to 1.5 ml. and heated with 1.5 ml* of o r c i n o l reagent f o r 20 minutes i n b o i l i n g water. The i n t e n s i t y of the green colour at a wavelength of 660 mU was read on the spectrophotometer. 3. Dische's diphenylamine test f o r DNA. (Schneider, 1957; Chargaff and Davidson, 1955). 1.0 g« of p u r i f i e d diphenylamine was dissolved i n 100 ml. of reagent g l a c i a l acetic acid and 2.75 ml*- of reagent s u l f u r i c acid. 10 ml, of the so l u t i o n containing RNA was mixed with 2 ml, of diphenylamine reagent and heated f o r 10 minutes i n b o i l i n g water. The i n t e n s i t y of the. blue colour at 595 was read on the spectrophotometer. 4 , Dreywood's anthrone test f o r carbohydrates. (Morris, 1 9 4 8 ) . 1 ,0 g, of anthrone was dissolved i n 500 ml, of 95$ s u l f u r i c acid (prepared by addition of 500 ml. of concen-trated reagent s u l f u r i c a c i d to 25 ml, of d i s t i l l e d water, and allowing the mixture to cool). 1 ml. of the RNA solution was mixed with 2 ml. of the Anthrone. reagent and the mixture was permitted to stand at room temperature f o r 10 minutes. The i n t e n s i t y of the blue colour at 650 rati was read on the spectrophotometer. 45 Appendix I I I Chromatographic analyses. The solvent used "was. n-butanol saturated with water which also contained 2.5 &• °£ phthalic acid i n 100 ml. 1 volume of phthalic acid solution was shaken with 4 volumes of butanol and the butanol layer was separated to be used i n the chromatography j a r . After the run, the Whatman #1 paper was sprayed with analine (2$ i n ethyl ether), allowed to dry at room temperature and then heated to 100°C i n an oven to develop the spots. 46 Appendix IV U l t r a c e n t r i f u g a l analyses. Samples of RNA to be analyses were dissolved i n 0.1 H. NaCl solution. The temperature throughout the analyses 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 figures 7» 8, and 9) and pl o t t e d on logarithmic graph paper according to Markham (i960, I962). The protractor (designed by Markham) was placed on the graph paper with i t s abscissa scale along a horizontal graph l i n e , with the l i n e corresponding to the rotor speed at the i n t e r s e c t i o n of the experimental curve with the l a t t e r . The experimental curve then i n t e r s e c t s the ordinate scale of the protractor at the correct S value. *The sedimentation c o e f f i c i e n t of S of ribonucleic acid i s the rate at which i t would sediment through the suspending medium i n a f i e l d of 1 dyne (or 1/981 of 1.0 gram) i n centimeters per second, and i s generally r e f e r r e d to water at 20°C. (S2o t W)» A more useful term or unit i s the Svedberg or' S value which i s 10~ 1^cm./sec./dyne. (Markham, 1962). Svedberg ~ S. 47 Appendix V Preparation of modified Niu-Twitty medium (Douglas and Finnegan, unpublished). Control medium; a defined medium was prepared as follows: The 1,000 ml. solution was b o i l e d f o r not less than 2 minutes and then cooled to room temperature. 2.5 nig. of streptomycin sulfate was added to the medium which was then stored f o r as short a period as possible at 7 ° C * T r i s = hydroxy methyl amino methane or 2-amino-2-hydroxy-methyl-1,3-propanediol). NaCl KC1 CaN0„«4H 20 MgSOWB^O 1 N. HC1 glass d i s t i l l e d water T r i s * buffer 3.400g. 0.050g. 0.080g. O.lOOg. 4 ml. 996 ml. 0 . 5 6 0 g . 

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