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Biosynthesis of ribosomes in Pseudomonas aeruginosa. Medveczky, Nicholas E. 1968

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- THE BIOSYNTHESIS OF RIBOSOMES IN PSEUDOMONAS AERUGINOSA . NICHOLAS E. MEDVECZKY B. Sc. (Honours Biochemistry) U n i v e r s i t y of B r i t i s h Columbia, 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE • I n the Department of . M i c r o b i o l o g y We accept t h i s t h e s i s as conforming t o the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA J u l y , 1968 In presenting th is thesis in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i sh Columbia, I agree that the Library sha l l make i t f ree ly ava i lab le for reference and Study. I further agree that permission for extensive copying of th is thesis for scholar ly purposes may be granted by the Head of my Department or by h.lis representat ives. It is understood that copying or publ icat ion of th is thesis for f inanc ia l gain shal l not be allowed without my wri t ten permission. Department of Microbiology The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada Date 6th September, 1968  ABSTRACT The s t r u c t u r e ribosomes of Pseudomonas aeruginosa and E s c h e r i c h i a c o l i were compared. The thermal.denatur-a t i o n p r o f i l e s of the P. aeruginosa ribosomes were c o n s i s t e n t l y at higher temperatures than the corresponding p r o f i l e s f o r those of E. c o l i . The d i f f e r e n c e s i n the thermal denaturation p r o f i l e s . o f the ribosomal RNAs were not as pronounced. Although the numbers of the p r o t e i n s r e s o l v e d from the ribosomal subunits of E. c o l i and P. aeruginosa were approximately the same, the d i s t r i b u t i o n of these p r o t e i n s i n the g e l was markedly d i f f e r e n t . The optimum c o n d i t i o n s f o r the r e s y n t h e s i s of r i b o -somes i n P. aeruginosa have been e s t a b l i s h e d . I t was demonstrated t h a t the r e s y n t h e s i s of ribosomes proceeds through a number of ribosomal pr e c u r s o r s . By isotope l a b e l l i n g experiments the r a t e of l a b e l l i n g of the RNA moiety of the ribosome was found t o be d i f f e r e n t from the r a t e of l a b e l l i n g of the p r o t e i n moiety. Furthermore both l a b e l s were found t o accumulate i n the ribosomes at the expense of the ribosomal precursors. The f o l l o w i n g precursors were r e s o l v e d by sucrose d e n s i t y gradient c e n t r i f u g a t i o n : 16s » 20S ^23S -? 25s ->28s - 30s •» 32s ^ 3^S 4os •»» 43S The l6s t o 28S precursors were not c h a r a c t e r i z e d w e l l enough t o be p l a c e d e i t h e r i n the 50S or 30S sequence. There was evidence t h a t the ribosomal RNA i t s e l f had precu r s o r s , 9S and 12S. i v TABLE OF CONTENTS Page INTRODUCTION LITERATURE REVIEW 2 I. Structure of Ribosomes 2 1. General structure 2 2. Ribosomal RMA k 3. Ribosomal proteins h k. Dissociation and re-association of ribosomes . 7 I I . Biosynthesis of Ribosomes .- 8 1. Radioactive isotope l a b e l l i n g of ribosomes of exponentially growing c e l l s 9 2. Ribonucleoprotein p a r t i c l e s 17 a. Chloramphenicol (CM) p a r t i c l e s 17 b. Relaxed p a r t i c l e s 22 c. Magnesium p a r t i c l e s 23 d. Potassium depletion (KD) p a r t i c l e s . . . 2h e. Puromycin (PM) p a r t i c l e s 24 f. 5-Fluorouracil (FU) p a r t i c l e s 25 3. Shi f t up cultures 26 MATERIAL AM) METHODS I. Organisms and Media 29 V Table of Contents (Continued) Page I I . P r e p a r a t i o n of Washed C e l l Suspensions . . . . 30 I I I . S t a r v a t i o n Medium and S t a r v a t i o n Conditions . . 30 IV. P r e p a r a t i o n of C e l l - f r e e E x t r a c t s 31 1. Sonic o s c i l l a t i o n 31 2. French press 31 V. P h y s i c a l F r a c t i o n a t i o n of C e l l Free E x t r a c t s . . 32 Ik V I . I n c o r p o r a t i o n of C - u r a c i l i n t o C e l l s . . . . 32 V I I . F r a c t i o n a t i o n of Ribosomes on Sucrose Density Gradients 3^ 1. P r e p a r a t i o n of the sucrose gradients . . . . 3^+ 2. The a n a l y s i s of the ribosomal m a t e r i a l . . . 37 a. The measurement of absorbance at 260 mu . 38 b. The measurement of r a d i o a c t i v i t y . . . . 38 V I I I . P r e p a r a t i o n of Samples f o r D i s c G e l E l e c t r o p h o r e s i s 39 1. P r e p a r a t i o n of ribosomes 39 2. P r e p a r a t i o n of ribosomal p r o t e i n s . . . . kO a. U r e a - L i C l kO b. A c e t i c a c i d e x t r a c t i o n kl IX. Polyacrylamide G e l E l e c t r o p h o r e s i s k2 v i Table of Contents (Continued) Page 1. Separation of ribosomes, ribosomal p r e c u r s o r s , ribosomal RNA, and ribosomal p r o t e i n s . . . . k-2 a. Separation of ribosomes and ribosomal RNA . h3 b. Separation of ribosomal p r o t e i n s . . . . kk X. L o c a l i z i n g the Substances Separated by Acrylamide G e l E l e c t r o p h o r e s i s *+5 1. S t a i n i n g w i t h s p e c i f i c reagents 1+5 2. The a n a l y s i s of the d i s t r i b u t i o n of the r a d i o -a c t i v i t y i n acrylamide g e l s 1+7 XI. Determination of the M e l t i n g Curves f o r Ribosomes and RNA hi 1. M e l t i n g curves f o r RNA 1+7 2. The m e l t i n g curves of the ribosomes . . . . 1+8 X I I . Chemical Analyses 1+9 1. The determination of RNA . 1+9 2. The determination of p r o t e i n 1+9 3. The determination of p r o t e i n and RNA i n the same ribosome sample 50 X I I I . The Determination of R a d i o a c t i v i t y . 51 1. The determination of r a d i o a c t i v i t y i n samples l a b e l l e d by a s i n g l e isotope 51 Table of Contents (Continued) Page 2. Determination of r a d i o a c t i v i t y i n samples doubly l a b e l l e d w i t h and \ 53 RESULTS AND DISCUSSION I. Separation of Ribosomes 55 1. Separation of ribosomes on agarose columns . . 55 2. The se p a r a t i o n of ribosomes on polyacrylamide g e l s 6k I I . Separation of the Ribosomal P r o t e i n s by Polyacrylamide g e l e l e c t r o p h o r e s i s 66 1. Determination of the number of p r o t e i n bands i n the v a r i o u s ribosomal subunits 66 2. Comparison of the ribosomal p r o t e i n s i n the v a r i o u s subunits t e s t e d i n E.. c o l i and P. aeruginosa . . 71 I I I . The Determination of the M e l t i n g Curves of Ribosomes and Ribosomal RNA's . 73 1. The m e l t i n g curve of ribosomal RNA 73 2. The m e l t i n g curve of ribosomes . . . . . . 8 l IV. The Determination of the R a t i o of RHA t o P r o t e i n i n Ribosomes 90 V. Determination of the S t a r v a t i o n and the Reincubation Conditions Optimal f o r the I n v e s t i g a t i o n of the Resynthesis of Ribosomes 92 Table of Contents (Continued) VI. Analyses of P. aeruginosa C e l l Free E x t r a c t s f o r Ribosomal Precursors 11+ 1. C e l l s l a b e l l e d w i t h C - u r a c i l l U 2. Analyses of c e l l s doubly l a b e l l e d by C - u r a c i l and - p r o t e i n hydrolyzate f o r ribosomal precursors a. The analyses of doubly l a b e l l e d c e l l s . . . . b. I s o l a t i o n of the regions around the 30S and 50S ribosomal peaks and the analyses of these f r a c t i o n s by sucrose d e n s i t y gradient c e n t r i -f u g a t i o n c. The analyses of the ribosomal RNA of the regions i s o l a t e d around the 30S and 50S ribosomal peaks d. Analyses of the ribosomal RNAs on d i s c g e l e l e c t r o p h o r e s i s e. A n a l y s i s of the ribosomal precursors on d i s c g e l e l e c t r o p h o r e s i s GENERALIZED DISCUSSION LITERATURE CITED LIST OF TABLES Table I . The Tm s of the Ribosomal RNA s of E. c o l i and P. aeruginosa Table I I . The Tm s of the Various Ribosomal F r a c t i o n s of E. c o l i and P. aeruginosa Table I I I . R e l a t i v e Q u a n t i t i e s of RHA and P r o t e i n i n Ribosomes X LIST OF FIGURES Page F i g . 1. The b i o s y n t h e s i s of ribosomes i n E. c o l i ik F i g . 2. The e f f e c t of c o n c e n t r a t i o n by the Amicon f i l t e r on the sedimentation r a t e s of 50S ribosomes. 57 F i g . 3. Comparison of the e l u t i o n p r o f i l e s of r i b o -somes passed through Sepharose 2B and kB columns. 59 F i g . k. C h a r a c t e r i z a t i o n of the e l u t i o n p r o f i l e of ribosomes passed through a Sepharose hB . column 6 l F i g . : 5. C h a r a c t e r i z a t i o n of the e l u t i o n p r o f i l e of ribosomes passed through a Sepharose 2B column 62 F i g . 6. Separation of 30S and 50S ribosomes on an a n a l y t i c a l acrylamide g e l e l e c t r o p h o r e s i s column 65 F i g . 7. A n a l y s i s of the p r o t e i n s of P. aeruginosa ribosomes by d i s c g e l e l e c t r o p h o r e s i s . 68 F i g . 8. A n a l y s i s of the p r o t e i n s of E. c o l i ribosomes by d i s c g e l e l e c t r o p h o r e s i s 69 F i g . 9* Comparison of the ribosomal p r o t e i n s of P. aeruginosa and those of E. c o l i 72 F i g . 10. Comparison of the ribosomal p r o t e i n s of the va r i o u s ribosomal f r a c t i o n s of P. aeruginosa t o the corresponding p r o t e i n s of E. c o l i 7^ F i g . 11. The thermal denaturation p r o f i l e of'P. aeruginosa l6s KNA 77 F i g . 12. The thermal denaturati#n p r o f i l e of P. aeruginosa 23S RWA 78 L i s t of Figu r e s (Continued) Page F i g . 13. The thermal denaturation p r o f i l e of E. c o l i l6s RNA 79 F i g . ik. The thermal denaturation p r o f i l e of E. c o l i 23S RNA 80 F i g . 15. The thermal denaturation p r o f i l e of P. aeruginosa 30S ribosomes 83 F i g . 16. The thermal denaturation p r o f i l e o f P. aeruginosa 50S ribosomes Qk F i g . 17. The thermal denaturation p r o f i l e of P. aeruginosa 70S ribosomes 85 F i g . 18. The thermal denaturation p r o f i l e o f E. c o l i 30S ribosomes 86 F i g . 19. The thermal denaturation p r o f i l e of E. c o l i 50S ribosomes 87 F i g . 20. The thermal denaturation p r o f i l e of E. c o l i 70S ribosomes 88 F i g . 21. Ribosome p r o f i l e of P. aeruginosa grown.' i n yeast-tryptone medium f o r 48 hr then starved i n 0.1 M t r i s HCI pH 7.h. 93 F i g . 22. Ribosome p a t t e r n of P. aeruginosa starved f o r kQ hr then reincubated w i t h l^C-glucose 95 F i g . 23. Ribosome p a t t e r n of P. aeruginosa starved f o r 2k hr then reincubated w i t h l ^ C - u r a c i l f o r kOO min. 97 F i g . 2k. The i n c o r p o r a t i o n of - ^ C - u r a c i l i n t o s t arved c e l l s of P. aeruginosa. 98 F i g . 25. Comparison of the s t a r v a t i o n b u f f e r s on the r a t e of uptake of -*-^C - u r a c i l 101 x i i L i s t of Figures (Continued) w i t h 1 >me p a t t e r n 01 r . aeruginosa remcuoai - 4 c-uracil and 3 H-protein hydrolyzate Page F i g . 26. The e f f e c t of the c o n c e n t r a t i o n of tryptone on the r a t e of i n c o r p o r a t i o n of l 4 c - u r a c i l i n t o the EUA of P. aeruginosa 102 F i g . 27. Ribosome p a t t e r n of P. aeruginosa reincubated w i t h l 4 c - u r a c i l f o r 60 min. 105 F i g . 28. Ribosome p a t t e r n of P. aeruginosa reincubated w i t h l ^ C - u r a c i l f o r 80 min. 106 F i g . 29. Ribosome p a t t e r n of P. aeruginosa reincubated w i t h l l + c - - u r a c i l f o r 100 .min. 107 F i g . 30. Ribosome p a t t e r n of P. aeruginosa reincubated w i t h l ^ c - u r a c i l f o r 120 min. 108 F i g . 31. Ribosome p a t t e r n of P. aeruginosa reincubated w i t h 14c - u r a c i l and 3 k - p r o t e i n h y d r o l y z a t e f o r 100 min. 112 F i g . 32. Ribosome p a t t e r n of P. aeruginosa reincubated f o r 120 min. 114 F i g . 33. Ribosome p a t t e r n of P. aeruginosa reincubated w i t h 1 4 c-uracil and 3H-protein h y d r o l y z a t e f o r 150 min. 115 F i g . 34. Ribosome p a t t e r n s of P. aeruginosa t o separate ribosomal regions. 117 F i g . 35. Ribosome p a t t e r n of 3D-100-30S to r e s o l v e ribosomal precursors 118 F i g . 36. Ribosome p a t t e r n of 3D-120-30S to r e s o l v e ribosomal precursors 120 ^ i g . 37. Ribosome p a t t e r n of 3D-150-30S t o r e s o l v e ribosomal precursors 121 x i i i L i s t of Figures (Continued) Page F i g . 38. Ribosome p a t t e r n of 3D-100-50S t o r e s o l v e ribosomal precursors 124 F i g . 39. Ribosome p a t t e r n of 3D-120-50S to r e s o l v e ribosomal precursors 125 F i g . 40. Ribosome p a t t e r n of 3D-150-50S to r e s o l v e ribosomal precursors 126 F i g . hi. Sedimentation p r o f i l e of the rRRA from 3D-100-30S 130 F i g . h2. Sedimentation p r o f i l e of the rRRA from 3D-120-30S 131 F i g . h3. Sedimentation p r o f i l e of the rRNA from 3D-150-30S 132 F i g . hh. Sedimentation p r o f i l e of the rRNA from 3D-100-50s 134 F i g . 45. Sedimentation p r o f i l e of the rRNA from 3D-120-50S 135 F i g . h6. Sedimentation p r o f i l e of the rRNA from 3D-150-50S 136 F i g . 47. D i s c e l e c t r o p h o r e t i c p a t t e r n of i h c - u r a c i l l a b e l l e d ribosomes 138 F i g . 48. Disc e l e c t r o p h o r e t i c p a t t e r n of l ^ C - l a b e l l e d u r a c i l l a b e l l e d rRNA 138 F i g . 49. D i s c e l e c t r o p h o r e t i c p a t t e r n of -^ C - p r o t e i n h y d r o l y z a t e l a b e l l e d ribosomes 139 F i g . 50. D i s c e l e c t r o p h o r e t i c p a t t e r n of the rRNAs from the 3D-100 s e r i e s 140 x i v L i s t of Figures (Continued) Page F i g . 51. D i s c e l e c t r o p h o r e t i c p a t t e r n of the rRNAs from the 3D-120 s e r i e s 141 F i g . 52. D i s c e l e c t r o p h o r e t i c p a t t e r n of the rRNAs from the 3D-150 s e r i e s l 4 l F i g . 53. D i s c e l e c t r o p h o r e t i c p a t t e r n of P. aeruginosa c e l l f r e e e x t r a c t s l a b e l l e d w i t h ~ L ^ C - u r a c i l f o r 100 min 139 F i g . 54. D i s c e l e c t r o p h o r e t i c p a t t e r n of P. aeruginosa c e l l f r e e e x t r a c t s l a b e l l e d with~l-^C - u r a c i l f o r 120 min 144 F i g . 55. D i s c e l e c t r o p h o r e t i c p a t t e r n of P. aeruginosa c e l l f r e e e x t r a c t s l a b e l l e d w i t h - u r a c i l f o r 150 min 144 ACKNOWLEDGEMENT I would l i k e t o express my si n c e r e g r a t i t u d e t o Dr. A. F. Gronlund f o r her i n t e r e s t , encouragement and c o n s t r u c t i v e c r i t i c i s m during the course of the e x p e r i -mental work and the p r e p a r a t i o n of t h i s t h e s i s . Thanks are a l s o extended t o Dr. J . J . E. Campbell f o r h i s encouragement and c a r e f u l e d i t i n g of t h i s t h e s i s . I would a l s o l i k e t o express my g r a t i t u d e to my w i f e J i l l f o r her encouragement during the course of these studies and f o r her help i n the p r e p a r a t i o n of t h i s t h e s i s . F i n a l l y I would l i k e t o extend my a p p r e c i a t i o n t o my f e l l o w students f o r the help they gave during the course of t h i s work, and t o Mrs. Rosemary Morgan f o r her ty p i n g of t h i s t h e s i s . INTRODUCTION Although the current research on ribosome b i o s y n t h e s i s has been extensive, o n l y a l i m i t e d number of t e c h n i c a l approaches have been employed. The methods f o r o b t a i n i n g ribosomal precursors have r e l i e d e i t h e r on the pulse l a b e l l i n g of e x p o n e n t i a l l y growing c u l t u r e s , or on the inbalance of RNA synthesis r e l a t i v e t o t h a t of p r o t e i n , i n the presence of an i n h i b i t o r of p r o t e i n s y n t h e s i s . The reason f o r o b t a i n i n g ribosomal precursors i s twofold: one i s to analyse the k i n e t i c s of ribosome b i o s y n t h e s i s , the other, i s to i s o l a t e and c h a r a c t e r i z e the ribosomal precursors themselves. Pulse l a b e l l i n g of e x p o n e n t i a l l y growing c u l t u r e s i s u s e f u l f o r k i n e t i c analyses, but does not y i e l d precursors i n s u f f i c i e n t q u a n t i t y f o r physico-chemical or biochemical c h a r a c t e r i z a t i o n . Immature r i b o n u c l e o p r o t e i n p a r t i c l e s can be obtained i n adequate q u a n t i t i e s by the use of agents which i n h i b i t p r o t e i n s y n t h e s i s , however, the k i n e t i c s of the eventual completion of the ribosomes from these precursor p a r t i c l e s are d i f f i c u l t to i n t e r p r e t . Studies of the endogenous metabolism of Pseudomonas aeruginosa revealed t h a t under s t a r v a t i o n c o n d i t i o n s , the ribosomes were r a p i d l y degraded. The r e s u l t i n g ribosome d e f i c i e n t c e l l s were v i a b l e and m e t a b o l i c a l l y a c t i v e (Gronlund and Campbell, 19&5; McKelvie, Campbell, and Gronlund, 1968). I t was thought t h a t a study of ribosome r e s y n t h e s i s i n ribosome depleted c e l l s of P. aeruginosa might y i e l d u s e f u l i n f o r m a t i o n regarding ribosome r e s y n t h e s i s . The synt h e s i s of RNA was monitored by the i n c o r p o r a t i o n of r a d i o a c t i v e u r a c i l t h a t of p r o t e i n , by the i n c o r p o r a t i o n of r a d i o a c t i v e p r o t e i n h y d r o l y s a t e , and the i s o l a t i o n and c h a r a c t e r i z a t i o n of the ribosomal precursors was attempted. Most of the work reported has been c a r r i e d out w i t h E s c h e r i c h i a c o l i , t h e r e f o r e i t was thought d e s i r a b l e to compare the ribosomes of E. c o l i and P. aeruginosa because of p o s s i b l e d i f f e r e n c e s i n ribosome b i o s y n t h e s i s that might e x i s t between the two microorganisms. 2 LITERATURE REVIEW I . S t r u c t u r e of Ribosomes 1. General s t r u c t u r e There are two fundamental subunits of the ribosome, the 50S and the 30S p a r t i c l e s ( T i s s i e r e s et a l . 1959). I n v i t r o -k at 10 M magnesium i o n c o n c e n t r a t i o n most of the ribosomes ++ e x i s t as the 50S and 30S subunits. I f the Mg con c e n t r a t i o n _2 i s r a i s e d to 10 M, the 50S and 30S p a r t i c l e s a s s o c i a t e t o form a 70S p a r t i c l e ; and the l a t t e r u n i t w i l l dimerize t o form 100S p a r t i c l e s i n the presence of 0 .1 M Mg + +. Gesteland (1966) ++ found t h a t the complete removal of Mg by EDTA causes the u n f o l d i n g of the ribosome without a corresponding l o s s of p r o t e i n . As the sedimentation r a t e i n d i c a t e s a hydrodynamic parameter i t i s , t h e r e f o r e , extremely dependent on secondary and t e r t i a r y s t r u c t u r e s r a t h e r than on the a c t u a l molecular s i z e and t h i s must always be kept i n mind. 6 The 50S subunit weighs 1.8 x 10 daltons and the 30S sub-u n i t , 0 .8 x 10^ daltons. Both subunits are r i b o n u c l e o p r o t e i n s , c o n t a i n i n g 63% RNA and 37$ p r o t e i n ( T i s s i e r e s et a l . 1959). The 50S subunit contains one molecule of 23S KNA and one of 5S KNA ( G a l i b e r t et_ a l . 1967; Comb and Ze h a v i - W i l l n e r , I967); the 30S subunit contains one l 6 s KNA molecule. The t e r t i a r y and quaternary s t r u c t u r e s of the ribosomes are not f u l l y e l u c i d a t e d as yet. Recently M c P h i e l and G-ratzer (1966) and Sarkar, Yang and Doty (1967) demonstrated by studying the o p t i c a l r o t a t o r y d i s p e r s i o n s p e c t r a of ribosomes and t h e i r c o n s t i t u e n t s , t h a t the t e r t i a r y s t r u c t u r e of the ribosomal KNA and ribosomal p r o t e i n i n s i d e the ribosomes was very s i m i l a r to t h a t i n the f r e e s t a t e . They a l s o showed t h a t no aromatic C.otton e f f e c t s were d i s c e r n i b l e and th a t the ribosomal p r o t e i n s were of the g l o b u l i n type w i t h approximately 25$ h e l i c a l content. The f i n e s t r u c t u r e of the ribosome s t i l l remains a mystery, however there i s a c t i v e research i n progress on the e l u c i d a t i o n of the f i n e s t r u c t u r e and sequence of the 5S KNA by Brownlee and Sanger (1967). Kaltschmidt et a l . (1967); C h e r s i et_ a l . (1968), as w e l l as Traut et_ a l . (1967) are sequencing some of the ribosomal p r o t e i n s u s i n g t r y p t i c peptide maps and determining the amino a c i d compositions of these peptides. 2. Ribosomal RNA The ribosomal RNA (rRNA) contains a c h a r a c t e r i s t i c -a l l y asymmetrical base composition of h i g h guanosine and low cytosine content (Spahr and T i s s i e r e s , 1959)• As suggested by i t s asymmetrical base composition, the rRNA c o n s i s t s of a s i n g l e p o l y n u c l e o t i d e chain which apparently possesses a r a t h e r complicated secondary s t r u c t u r e . About 75$ of the rRNA i s i n hydrogen-bonded double h e l i c a l regions (Fresco, A l b e r t s and Doty, i 9 6 0 ) . A c r i t i c a l a n a l y s i s of the nu c l e o t i d e s obtained from the h y d r o l y s i s of r i g o r -o u s l y p u r i f i e d rRNA revealed the existence of methylated bases and r i b o s e , i n the rRNA (Gordon and Boman, I96I+). Nucleotide d i s t r i b u t i o n maps of rRNA were shown to be species s p e c i f i c and, t h e r e f o r e , the rRNA i t s e l f must a l s o be s p e c i f i c (Aronson, 1963) . 3. Ribosomal p r o t e i n s The p r o t e i n moiety of the ribosome was shown to be extremely heterogeneous by Traut et a l . (1967). Those workers and Gesteland and S t a e h e l i n (1967) were able to 5 i s o l a t e 20 p r o t e i n s from the 50S ribosome and 12 from the 30S ribosome. Furthermore, Traub et a l . (1967) showed d e f i n i t i v e p h y s i o l o g i c a l d i f f e r e n c e s between 30S ribosomal p r o t e i n s of E. c o l i , w i t h reference to the p r o t e i n s y n t h e s i z i n g a b i l i t i e s of p a r t i a l l y r e c o n s t i t u t e d 30S ribosomes i n an i n v i t r o system. Leboy et a l . (1964) found t h a t the 30S ribosomal subunits of E. c o l i K-12 s t r a i n s have one p r o t e i n which d i f f e r s e l e c t r o p h o r e t i c a l l y from the corresponding component of B s t r a i n s . Otaka, I t o h and Osawa (1968) u s i n g double l a b e l l i n g and co-chromato-graphy of ribosomal p r o t e i n s , found two such p r o t e i n s . Furthermore, they demonstrated t h a t there was on l y one p r o t e i n component of the 30S ribosome t h a t was e l e c t r o -p h o r e t i c a l l y i d e n t i c a l to a 50S ribosomal p r o t e i n from E. c o l i . They a l s o showed th a t the ribosomal p r o t e i n s , as w e l l as the rRNA's were species s p e c i f i c . Friedman et a l . (1968) subjected E. c o l i ribosomes to C s C l i s o p y c n i c c e n t r i f u g a t i o n and obtained p a r t i a l l y d e p r o t e i n i z e d 'core" p a r t i c l e s and ' s p l i t ' ribosomal p r o t e i n s . A n t i b o d i e s were prepared against the p r o t e i n s of 50S ribosomes and of E. c o l i . Friedman et a l . (1968) showed tha t a n t i b o d i e s against the p r o t e i n s of the 50S ribosome of E. c o l i d i d not cross react w i t h 50S core p a r t i c l e s , or w i t h 30S p a r t i c l e s . They, of course, do react w i t h the 50S and 70S ribosomes of E. c o l i , and als o w i t h 50S ribosomes i s o l a t e d from taxonomically r e l a t e d species. Immunoelectrophoretic a n a l y s i s demonstrated one pre-c i p i t i n band, suggesting t h a t the antiserum was s p e c i f i c f o r a 50S ' s p l i t ' p r o t e i n determinant. Estrup and Santer (1966) prepared antibodies against 50S E. c o l i ribosomes and.found t h a t they contained at l e a s t seven immunologically d i s t i n g u i s h a b l e p r o t e i n s . At l e a s t two of the 50S pr o t e i n s were homologous with two p r o t e i n s i n the 30S ribosome. I n a d d i t i o n , f o u r or f i v e of the 30S ribosomal p r o t e i n s cross reacted with antiserum prepared against r i g o r o u s l y p r u i f i e d 50S ribosomal p r o t e i n s , i n d i c a t i n g at l e a s t s i m i l a r i t i e s i n amino a c i d sequences, i f not complete homology. Santer et a l . (1968) have shown that antiserum against i n t a c t 50S ribosomes r e a c t s w i t h some sol u b l e cytoplasmic p r o t e i n s and, i n f a c t , one of these p r o t e i n s was found to be immunologically i d e n t i c a l to one of the 50S p r o t e i n s . The b i o s y n t h e t i c s i g n i f i c a n c e of t h i s f i n d i n g w i l l be discussed l a t e r . 7 k. D i s s o c i a t i o n and r e - a s s o c i a t i o n of ribosomes Lerman et a l . (1966) demonstrated t h a t E. c o l i ribosomes when subjected to i s o p y c n i c c e n t r i f u g a t i o n i n Cs C l l o s e p r o t e i n i n d i s c r e t e and r e v e r s i b l e steps. The f i r s t c e n t r i f u g a t i o n y i e l d s the 43S 'core' p a r t i c l e s (Lerman et a l . (1966) named 'core' p a r t i c l e s "A" p a r t i c l e s ) and ' s p l i t ' ribosomal p r o t e i n . On r e c e n t r i f u g a t i o n i n Cs C l the 'core' p a r t i c l e s l o s e more p r o t e i n and give r i s e to Chloramphenicol p a r t i c l e - l i k e p a r t i c l e s of 28s and 22S. (Chloramphenicol p a r t i c l e s (CM p a r t i c l e s ) w i l l be discussed i n a l a t e r s e c t i o n ) . 50S ribosome \ k3S 'A' p a r t i c l e •> 28S CM p a r t i c l e - l i k e p a r t i c l e 30S " * 28S " * 22S •<« (37$ p r o t e i n ) (30$ p r o t e i n ) (20$ p r o t e i n ) The f i r s t step of t h i s d i s s o c i a t i o n was described p r e v i o u s l y by Meselson et a l . (1964). The i n v i t r o r e c o n s t r u c t i o n of these p a r t i c l e s to form ribosomes argues s t r o n g l y f o r the s e l f assembly of ribosomes (Lerman et a l . 1966). This argument w i l l be developed f u r t h e r i n a l a t e r s e c t i o n . S t a e h e l i n and Meselson (1966) showed th a t ribosomes which were t r e a t e d w i t h 5 M C s C l ^ and then allowed to r e -assemble, were a c t i v e i n an i n v i t r o p r o t e i n s y n t h e s i z i n g system. Traub and Nomura (1968) d i s s o c i a t e d E. c o l i 30S ribosomes by i s o p y c n i c c e n t r i f u g a t i o n i n C s C l t o a 23S 'core' and ' s p l i t ' p r o t e i n s , then d i s s o c i a t e d the core t o l6s KNA by phenol e x t r a c t i o n and 'core' p r o t e i n s by urea L i C l e x t r a c t i o n . They could reassemble 30S ribosomes from the l6s KNA and the ' s p l i t 1 and 'core' p r o t e i n s . The p r o t e i n s of the r e c o n s t i t u t e d 30S p a r t i c l e s were e l e c t r o p h o r e t i c a l l y • i d e n t i c a l to those obtained from n a t i v e 30S p a r t i c l e s . The s e l e c t i v e removal of these p r o t e i n s p r i o r to the r e c o n s t r u c t i o n of the ribosomes and the subsequent measure of the a b i l i t y of these p a r t i a l l y reassembled 30S ribosomes to d i r e c t ribosome-dependent p r o t e i n s y n t h e s i s , allowed Traufc and Nomura (1968) to a s s i g n p h y s i o l o g i c a l f u n c t i o n s to some of the ' s p l i t ' p r o t e i n s . I I . B i o s y n t h e s i s of Ribosomes The e l u c i d a t i o n of the b i o s y n t h e s i s of such a complex u n i t as a ribosome i s o b v i o u s l y a d i f f i c u l t problem. . An Examination of ribosomal s t r u c t u r e i n d i c a t e s the n e c e s s i t y of the f o l l o w i n g sequence of events: a) the formation of the p o l y n u c l e o t i d e chain b) the m e t h y l a t i o n of the KNA c) the formation of the p r o t e i n d) the assembly of the ribosome This sequence of events has r e c e n t l y been the subject of extensive research and has r e s u l t e d i n a v a s t accumu-l a t i o n of i n f o r m a t i o n concerning ribosomes. However, these events have not been s a t i s f a c t o r i l y deciphered nor has the c o r r e l a t i o n of the sequence of events been demonstrated during ribosome b i o s y n t h e s i s . 1. R a d i o a c t i v e isotope l a b e l l i n g of ribosomes of e x p o n e n t i a l l y growing c e l l s . The analyses of the r a t e s of l a b e l l i n g of ribosomal precursors i n e x p o n e n t i a l l y growing c u l t u r e s can be mathe-m a t i c a l l y i n t e r p r e t e d t o y i e l d the k i n e t i c s and sequence of the b i o s y n t h e t i c r e a c t i o n s . B r i t t e n and McCarthy (1962) developed a mathematical model f o r the time course l a b e l l i n g of rKKA of e x p o n e n t i a l l y growing E. c o l i . The d e r i v a t i o n of the mathematical treatment n e c e s s i t a t e d t h a t the f o l l o w i n g r e s t r i c t i v e c o n d i t i o n s be a p p l i e d to the system under i n v e s t i g a t i o n : a) r e l a t i v e q u a n t i t i e s of intermediates should be constant w i t h time, t h a t i s , steady-state e x p o n e n t i a l growth must be maintained. b) from a mathematical p o i n t of view, c o n d i t i o n (a) should apply to each c e l l . This, of course, i s impossible i n a c u l t u r e whose members propagate by b i n a r y f i s s i o n , t h e r e f o r e i t i s assumed t h a t the p o p u l a t i o n of c e l l s i s random and t h a t a l l the e f f e c t s of v a r i a t i o n s of processes w i t h time, during the d i v i s i o n c y c l e , w i l l be o f f s e t by the analyses of random samples of the c e l l p o p u l a t i o n . c) the s p e c i f i c r a d i o a c t i v i t y and r a t e of u t i l i z a t i o n of the t r a c e r must be constant. d) t r a n s f e r of t r a c e r from one intermediate to the next must be u n i d i r e c t i o n a l . e.) any r e p r e s e n t a t i v e molecule of a p a r t i c u l a r intermediate has a constant chance of being t r a n s f e r r e d t o the next stage i n the sequence regardless of the le n g t h of time t h a t i t has been present. Under these r e s t r i c t i o n s , the i n c o r p o r a t i o n of l a b e l i n t o precursors and subsequently i n t o ribosomes, obeys exp o n e n t i a l growth k i n e t i c s . McCarthy, B r i t t e n and Roberts (1962) took advantage of t h i s mathematical treatment and l a b e l l e d l o g a r i t h m i c i l l -phase E. c o l i c e l l s w i t h C - u r a c i l i n order to determxne the sequence and the p o s s i b l e number of precursors en  route t o the ribosomes. They analysed the order of the ra t e of the i n c o r p o r a t i o n . I f the p o p u l a t i o n of c e l l s are l a b e l l e d under the above mentioned r e s t r i c t i o n s , then the order of the r a t e of the i n c o r p o r a t i o n of the r a d i o -a c t i v e isotope i n t o a ribosomal precursor i s a f u n c t i o n of the p o s i t i o n of the l a b e l l e d precursor i n the sequence of ribosome b i o s y n t h e s i s . They i n t e r p r e t e d t h e i r r e s u l t s i n the f o l l o w i n g manner: there are two cl a s s e s of precursors; eosomes neosomes ribosomes. They v i s u a l i z e d the eosomes as being a heterogeneous 'pool' of low molecular precursors of 8-20S. The f a c t t h a t t h i s m a t e r i a l was contaminated w i t h messenger RNA (mRWA) has now made the 'pool' nature of the eosome questionable. The mRNA, w i t h i t s c h a r a c t e r -i s t i c a l l y h i g h turnover r a t e , would demonstrate k i n e t i c s i d e n t i c a l to th a t of ribosome pr e c u r s o r s . Nevertheless, McCarthy and Aronson (1961) managed to i s o l a t e from E. c o l i c e l l f r e e e x t r a c t s , s m a l l 4-8S eosome-like RNA which was l a b e l l e d i n a manner which would be p r e d i c t e d f o r a r i b o -somal precursor. From k i n e t i c analyses, the 30S r e g i o n appeared to be heterogeneous, c o n t a i n i n g mature 3°S ribosomes, and a l s o the 30S neosome precursors of 5°S ribosomes. The 43S neosome was c l e a r l y demonstrated and o n l y one h a l f of the c a l c u l a t e d f l o w of the l a b e l to the 50S ribosome passed through the 43S neosome. This was f u r t h e r evidence f o r the existence of a 30S neosome. The k i n e t i c s of the i n c o r p o r a t i o n of l a b e l i n t o the 50S ribosome suggested t h a t no eosome was d i r e c t l y converted t o the 50S ribosome. The f o l l o w i n g sequence was p o s t u l a t e d by McCarthy, B r i t t e n and Roberts (1962): 8-20S eosome ^ 30S ribosome 30S neosome ^ 1+3S neosome ^ 50S ribosome B r i t t e n , McCarthy and Roberts (1962) a l s o s t u d i e d the synthesis of the p r o t e i n moiety of the E. c o l i ribosome by the i n c o r p o r a t i o n of r a d i o a c t i v e amino a c i d s . I n c o n t r a s t t o the p a t t e r n obtained from analyses of RHA-labelled c e l l s , the amino acids were d i r e c t l y i n c o r p o r a t e d i n t o the 30S and the 50S ribosomes. However, there was evidence of the l a b e l moving from the 1+3S neosome to the 50S ribosome. Ribosomal p r o t e i n was not l a b e l l e d when chloramphenicol (CM) was added to the c e l l s , and eosome and neosome l i k e p a r t i c l e s accumu-l a t e d . These combined p i c t u r e s l e d B r i t t e n , McCarthy and Roberts (1962) to propose the f o l l o w i n g model f o r ribosome b i o s y n t h e s i s ( F i g . l ) . One important f e a t u r e of t h i s model i s the f a c t t h a t the-a d d i t i o n of p r o t e i n and RNA occur i n time separated stages. This was f u r t h e r s u b s t a n t i a t e d by the i s o l a t i o n of RNA r i c h intermediates. The f a c t t h a t no l a b e l was observed i n the 11+ CM p a r t i c l e s when the c e l l s were incubated w i t h C-leucine l e d B r i t t e n , McCarthy and Roberts (1962) t o propose the i d e a of a ribosomal p r o t e i n p o o l . The development of modern techniques has allowed M a n g i a r o t t i et a l . (1968) to extend the research on the l a b e l l i n g of e x p o n e n t i a l l y growing E. c o l i . They used f r a g i l e c u l t u r e s ( M a n g i a r o t t i and Schlessinger, 1966) which could be l y s e d r a p i d l y and, i n the l y s a t e , the mRNA r e s i d e d e x c l u s i v e l y i n the polysome r e g i o n l e a v i n g the low molecular EOSOME NEOSOME RIBOSOME F i g . 1. The b i o s y n t h e s i s of ribosomes i n E s c h e r i c h i a c o l i . Copied from B r i t t e n , McCarthy and Roberts (1962). weight ribosomal precursors ujicontaminated except f o r t r a n s f e r KNA ( M a n g i a r o t t i and Schlessinger, 1967). The technique of RNA-DNA h y b r i d i z a t i o n allowed these workers to determine whether the KNA of a ribosomal precursor was of the l6s or 23S type. Despite the f a c t t h a t M a n g i a r o t t i et a l . (1968) found the l6s and 23S rKNA of t h e i r s t r a i n of E. c o l i c r o s s - h y b r i d i z e d a p p r e c i a b l y when i s o l a t e d by sucrose d e n s i t y gradient c e n t r i f u g a t i o n , they d e r i v e d mathematical equations f o r the determination of the t r u e h y b r i d i z a t i o n of the 23S and l6s KNA. This extensive study l e d to the f o l l o w i n g important conclusions: a) p r o t e i n i s added t o the KNA chain before the KNA chain i s completed. There are two l i n e s of evidence f o r t h i s c l a i m . One i s t h a t ribosomal precursors w i t h a sedimentation r a t e of l e s s than 26s y i e l d e d KNA on phenol e x t r a c t i o n , which moved slower than l6s. The second, a f a r more d i r e c t proof, i s the observation t h a t KNase which i s known to destroy the incomplete KNA chains of u n f i n i s h e d ribosomes, r e l e a s e s ribosomal p r o t e i n s a l s o . The f o l l o w i n g sequence of events was proposed: b) l6s RNA ->26S p a r t i c l e 30S ribosome 23S RNA ^  32S p a r t i c l e } 43S p a r t i c l e 50S ribosome This sequence of events i s i n agreement w i t h t h a t proposed by McCarthy, B r i t t e n and Roberts (1962). c) k i n e t i c a n a l y s i s of the i n c o r p o r a t i o n of r a d i o a c t i v e u r a c i l i n t o the RNA of the ribosomes revealed t h a t the time r e q u i r e d f o r the synthesis of the l6s and the 23S RNA was approximately the same. The p o s s i b i l i t y t h a t the 23S RNA molecule i s a dimer of two l6s molecules cannot be r u l e d out. This p o s s i b i l i t y was proposed by McCarthy, B r i t t e n and Roberts (1962) and f u r t h e r extended by Midgley (1965). d) ribosomal precursors w i t h sedimentation r a t e s of 26s, 32S and 43S were i s o l a t e d by sucrose d e n s i t y gradient c e n t r i f u g a t i o n . Since they were a l l sharp d i s t i n c t peaks, and furthermore were a l l e x p o n e n t i a l l y s a t u r a t e d w i t h l a b e l , they were presumed to be homogeneous. Such precursors could a r i s e , i n the authors' view, i f a r a r e component, such as 5S RNA or an infrequent ribosomal p r o t e i n became l i m i t i n g . . A f u r t h e r c o m p l i c a t i o n i n the scheme of ribosome b i o -synthesis i s the presence of n a t i v e 30S and 50S p a r t i c l e s . These p a r t i c l e s , f i r s t described by T i s s i e r e s et a l . (1959), do not a s s o c i a t e t o form 70S ribosomes i n the presence of 10~ M Mg . Nakada and K a j i (1967) found t h a t these " n a t i v e " ribosomes r e f e r r e d to as 30'S and 50'S became l a b e l l e d before the normal 30S and 50S ribosomes. For t h i s reason i t was proposed t h a t the 30'S and 50'S ribosomes were precursors t o 30S and 50S ribosomes. There i s no d i s c e r n i b l e d i f f e r e n c e between the ribosomal p r o t e i n s of 30'S and 30S and 50'S and 50S ribosomes. 2. R i b o n u c l e o p r o t e i n p a r t i c l e s a. Chloramphenicol (CM) p a r t i c l e s When CM s e n s i t i v e b a c t e r i a , such as E. c o l i or B a c i l l u s s u b t i l i s are incubated w i t h CM during growth, p r o t e i n synthesis i s s t r o n g l y i n h i b i t e d , but the r a t e of KNA synthesis i s not a f f e c t e d . E. c o l i , t r e a t e d w i t h CM gave r i s e to two new p a r t i c l e s 18S and 25S d i s t i n c t from the 30S and 50S ribosomes (Nomura and Watson, 1959; Pardee and Pestidge, 1957)* The CM p a r t i c l e s c o n s i s t of 76$ KNA and 2k<f0 p r o t e i n i n c o n t r a s t to 63$ KNA and 37$ p r o t e i n f o r normal ribosomes (Nomura and Watson, 1959)' Upon the removal of CM, the CM p a r t i c l e s were r a p i d l y degraded. When a s u f f i c i e n t l y h i g h r a t e of p r o t e i n s y n t h e s i s was evident, they were i n t e g r a t e d i n t o the ribosomes, even i n the presence of actinomycin D which i n h i b i t e d f u r t h e r RNA synthesis ( L e v i n t h a l et a l . 1963). The f a c t t h a t RNA contains methylated bases and r i b o s e poses the question of whether met h y l a t i o n occurs p r i o r to or proceeding the completion of the p o l y n u c l e o t i d e chain, t h a t i s , at the mononucleotide or at the p o l y n u c l e o t i d e l e v e l . Rurland, Nomura and Watson (1962) observed t h a t the CNV. p a r t i c l e s had rRNA; th a t i s , l6s and 23S RNA. Pardee et a l . (1957) found t h a t the base composition of the RNA was s i m i l a r to t h a t of t o t a l c e l l u l a r RNA. A more c r i t i c a l a n a l y s i s by Dubin and Gunalp (1966) revealed a s l i g h t l y f a s t e r sediment-a t i o n r a t e and l e s s a f f i n i t y on MAK columns f o r CM RNA than f o r normal rRNA. Gordon, Boman and Isaksson (196b-) demon-s t r a t e d t h a t the d i f f e r e n c e between CM RNA and rRNA was due t o submethylation of the CM RNA. Dubin and Gunalp (1966) found t h a t i n a d d i t i o n t o the d r a s t i c i n h i b i t i o n of methylation, CM als o prevented the formation of pseudouridine r e s i d u e s . The m e t h y l a t i o n of the ribosomal RNA was i n v e s t i g a t e d by Hayashi, Osawa and Miura (1966). These workers double-l a b e l l e d the ribosomal RNA of E. c o l i w i t h ^C-Me-methionine and H-adenosine. The r e s u l t i n g C/ H r a t i o was assumed to i n d i c a t e the extent of m e t h y l a t i o n of KNA. I t was found th a t l6s KNA was 60$ more methylated than the 23S KNA and that m e t h y l a t i o n was s i g n i f i c a n t l y i n h i b i t e d by CM. There-f o r e , i t appeared t h a t CM p o s s i b l y has an e f f e c t on KNA synthes i s as w e l l as on p r o t e i n s y n t h e s i s . An a d d i t i o n a l unsolved c o m p l i c a t i o n i n t h i s area has been introduced by Hecht and Woese (1968), who analysed normal KNA, r a p i d l y l a b e l l e d KNA and CM-KNA on polyacrylamide g e l s . To minimize the e f f e c t s of the s u p e r c o i l i n g of the KNA, the s o l u t i o n s were denatured p r i o r to t h e i r a p p l i c a t i o n t o the g e l , Thevjfound t h a t both the r a p i d l y l a b e l l e d KNA and the CM KNA had s i g n i f i c a n t l y slower m o b i l i t i e s on the g e l than those of the corresponding mature KNAs. The authors i n t e r -p r e t e d t h i s d i f f e r e n c e i n m o b i l i t i e s as being an index of s i z e d i f f e r e n c e s , and c a l c u l a t e d i t to be 130,000 daltons f o r the l6s KNA s e r i e s . The d i f f e r e n c e f o r the 23S s e r i e s was not as pronounced. The authors p o s t u l a t e d t h a t upon maturation the precursor KNA lo s e s a segment of the p o l y n u c l e o t i d e chain to become mature rKNA. This e x p l a n a t i o n was suggested to account f o r the o r i g i n of the 5S rRNA (Hecht and Woese, 1968). Of course, the p o s s i b i l i t y t h a t the d i f f e r e n c e s i n the m o b i l i t i e s could be due to some f a c t o r s other than s i z e d i f f e r e n c e s , cannot be r u l e d out at t h i s time. Whether or not the pulse l a b e l l e d rRNA i s indeed i d e n t i c a l to the CM RNA remains to be c l a r i f i e d . One i n t e r e s t i n g question that arose from the study of CM p a r t i c l e s was the o r i g i n of the CM p a r t i c l e p r o t e i n . Kurland and Maaloe (1962) proposed t h a t t h i s p r o t e i n o r i g i n a t e d from a p r e - e x i s t i n g p r o t e i n p o o l . Osawa (1965) demonstrated t h a t l i m i t e d p r o t e i n synthesis does occur during CM treatment, and t h a t the CM p a r t i c l e s r e a d i l y accepted the newly synthesized p r o t e i n . The 'protein p o o l ' hypothesis r e c e i v e d f u r t h e r support from the work of Santer et a l . (1968), who prepared antibodies against p u r i f i e d 50S E. c o l i ribosomes and found t h a t one p r o t e i n of the 50S ribosome was immunologically i d e n t i c a l , by Ouchterlony t e s t s , to one p r o t e i n present i n the 100,000 g supernatant f l u i d s of c e l l f r e e e x t r a c t s . Santer et a l (1968) p o s t u l a t e d the existence of a p o o l of ribosomal p r o t e i n s , 14 12 whose h a l f l i f e was c a l c u l a t e d to be 0.1 generations by C - C isotope s h i f t experiments. They succeeded i n demonstrating t h a t t h i s p o o l was not a degradation product, and t h a t the p o o l was r a p i d l y depleted when r e l a x e d c o n t r o l s t r a i n s were subjected to amino a c i d s t a r v a t i o n . Most, i f not a l l of the p r o t e i n s of the r e l a x e d c o n t r o l (RC) p a r t i c l e s were shown to be derived from t h i s p o o l . Dalgarno and Gros (1968) demonstrated t h a t e a r l y RC and CM p a r t i c l e s could mature to 30S and 50S ribosomes during i n h i b i t i o n of p r o t e i n s y n t h e s i s . Their i n t e r p r e t a t i o n of r e s u l t s assumed the existence of a sm a l l p o o l of ' l a t e ' ribosomal p r o t e i n s i n E. c o l i . Otaka, I t o h and Osaka (1967) demonstrated t h a t a kOS CM p a r t i c l e (obtained by the use of low concentrations of CM) i s m i s s i n g f o u r s p e c i f i c 50S ribosomal p r o t e i n s . These same fou r p r o t e i n s appear t o be d i s s o c i a t e d by 1.25 M L i C l . S e l l s and Davis (1968) t r e a t e d J H - l e u c i n e Ill-l a b e l l e d E. c o l i w i t h CM, and added C-leucine to the r e -covering c e l l s . They found marked v a r i a t i o n s i n the lkc/hl r a t i o s i n the i s o l a t e d ribosomal p r o t e i n s . S e l l s and Davis (1968) i n t e r p r e t these r e s u l t s t o i n d i c a t e t h a t c e r t a i n ribosomal p r o t e i n s are newly synthesized and are added to the ribosomes i n a nonrandom manner. Another important c o n t r i b u t i o n to t h i s aspect of ribosome synthesis was the discovery, by Wagata and Mau.ro (1967), of an i n v i t r o ribosome s y n t h e s i z i n g system c a r r i e d out by spheroplast membranes of E. c o l i . They found t h a t t h i s system was o p e r a t i o n a l even i n the presence of chloramphenicol at s u f f i c i e n t l y h i g h concentrations to i n h i b i t p r o t e i n s y n t h e s i s . The e x p l a n a t i o n of t h i s system a l s o i n v o l v e s the existence of a ribosomal p r o t e i n p o o l . M e t h y l a t i o n or any other step i n maturation of the CM M A was not explained by e i t h e r Dalgarno and Gros (1968) or by Nagata and Mauro (1967). b. Relaxed p a r t i c l e s Most E. c o l i amino a c i d auxotrophs stop the synthesis of RNA immediately a f t e r the medium has become depleted of the r e q u i r e d amino a c i d . However, some E. c o l i s t r a i n s , continue to synthesize RNA under these c o n d i t i o n s (Borek, Ryan and Rochenback, I96I; Stent and Brenner, 1961). These mutant s t r a i n s have been designated r e l a x e d c o n t r o l (RC1'6"'"), as opposed to the normal s t r i n g e n t c o n t r o l (RC S"^ r) mutants, r e l RC s t r a i n s accumulate subribosomal RNA p a r t i c l e s during amino a c i d s t a r v a t i o n . Dagley et a l . (1962) and Nakada, Anderson and Magasanik (1964) showed an accumulation of l8S and 28S p a r t i c l e s when t h e i r s t r a i n of an E, c o l i (RC met") was subjected to methionine s t a r v a t i o n . F u r t h e r -more, Nakada (1965) showed t h a t the a d d i t i o n of methionine t o these starved c e l l s i n a step-down medium r e s u l t e d i n p r e f e r e n t i a l ribosomal p r o t e i n s y n t h e s i s , however the syn-t h e s i s of KNA was almost completely a r r e s t e d . RC p a r t i c l e s accepted t h i s newly synthesized ribosomal p r o t e i n t o form 30S and 50S ribosomes, at l e a s t the RNA moiety of the RC p a r t i c l e s was i n c o r p o r a t e d d i r e c t l y i n t o ribosomes. Wakada (1967) has found t h a t the p r o t e i n s o f the RC p a r t i c l e s are d e r i v e d mostly from p r e - e x i s t i n g p r o t e i n s i n the c e l l . These p r o t e i n s are e l e c t r o p h o r e t i c a l l y i d e n t i c a l to some of the normal ribosomal p r o t e i n s . When the amino a c i d i s r e s t o r e d t o the s t a r v i n g c u l t u r e , the r e l a x e d p a r t i c l e s combine w i t h p r e f e r e n t i a l l y synthesized new ribosomal p r o t e i n to form mature ribosomes. Kamiryo, Tagaki and Maruo (1966) found by another l i n e of evidence \ tha t the RC p a r t i c l e s combine w i t h newly synthesized ribosomal p r o t e i n s . c. Magnesium p a r t i c l e s E. c o l i , grown i n a Mg d e f i c i e n t medium, were shown to l o s e most of t h e i r ribosomes without a concommitant l o s s of v i a b i l i t y (McCarthy, 1962; Suzuki and Hayashi, 1^6h). The a d d i t i o n of Mg to such c u l t u r e s r e s u l t e d i n a s h i f t up e f f e c t , t h a t i s , an immediate resumption of KNA synthesis occurred, but a l a g of about 3-6" hours i n the resumption of p r o t e i n synthesis and i n the resumption of growth was evident. During the s h i f t up, l6s and 23S RNAs accumulated i n r i b o n u c l e o p r o t e i n p a r t i c l e s of l8S and 25S r e s p e c t i v e l y . These p a r t i c l e s appeared to be very s i m i l a r t o , i f not i d e n t i c a l t o , CM p a r t i c l e s (Suzuki and Hayashi, I96I+). d. Potassium d e p l e t i o n (KD) p a r t i c l e s Ennis and Lubin (1965) described r i b o n u c l e o p r o t e i n s t h a t were v e r y s i m i l a r t o CM, RC and Mg p a r t i c l e i n K depleted E. c o l i . S i m i l a r to the other r i b o n u c l e o p r o t e i n p a r t i c l e s described, the K p a r t i c l e s matured i n t o ribosomes on the a d d i t i o n of K without evidence of p r i o r degradation. e. Puromycin (PM) p a r t i c l e s S e l l s (196J+) observed t h a t PM at a con c e n t r a t i o n s u f f i c i e n t l y high t o i n h i b i t p r o t e i n s y n t h e s i s , allowed the s y n t h e s i s of rRNA which accumulated i n the FM p a r t i c l e s . These p a r t i c l e s matured i n t o ribosomes on the removal of PM, even when- f u r t h e r RNA synthesis was i n h i b i t e d by pro-f l a v i n e . ( S e l l s , I966). f . 5 - F l u o r o u r a c i l (FU) p a r t i c l e s Horowitz and Chargaff (1959) found t h a t E. c o l i i n c o r p o r a t e d FU i n t o i t s RNA. Osawa (1965) found t h a t when E. c o l i was incubated w i t h FU, r i b o n u c l e o p r o t e i n p a r t i c l e s of 28S and 32S and c o n t a i n i n g l 6 s and 23S RNA r e s p e c t i v e l y , accumulated. The reason why there was more p r o t e i n i n the FU p a r t i c l e s was probably due to the f a c t t h a t p r o t e i n synthesis occurred at an appreciable r a t e w i t h the concentrations of FU used (Osawa, 1965). I t would be u s e f u l to c r i t i c a l l y compare the RNAs of the p r e v i o u s l y described p a r t i c l e s t o determine the p r e c i s e c o r r e l a t i o n between the s i z e , the meth y l a t i o n of the rRNA and the p r o t e i n content. Such an approach should y i e l d v a l u a b l e i n f o r m a t i o n on the p o s s i b l e b i o -s y n t h e t i c s i g n i f i c a n c e of the p r e v i o u s l y described r e c o n s t i t u t i o n experiments of Traut et a l . (1968) and Traut and Nomura (1968). 3. S h i f t up c u l t u r e s When b a c t e r i a l c e l l s were t r a n s f e r r e d from a n u t r i t i o n a l l y poor medium t o a n u t r i t i o n a l l y r i c h medium, except f o r an i n i t i a l b u r s t of extremely r a p i d r a t e , the synthe s i s of the RNA i s inc r e a s e d t o the r a t e observed i n the new medium. However, the r a t e of synthesis of p r o t e i n and DNA was u n a l t e r e d f o r q u i t e some time (Kjel g a a r d , Maaloe and Schaester, 1958; Nomura and Watson, Neidhart and Magasanik, i960). I n other words, s h i f t up c o n d i t i o n s mimicked the a c t i o n of i n h i b i t o r s of p r o t e i n synthesis and r e s u l t e d i n the r a p i d appearance of t r a n s i e n t RNA-rich intermediates. Kono and Osawa (196I+) s t u d i e d ribosome b i o s y n t h e s i s i n E. c o l i by pulse Ik l a b e l l i n g s h i f t up c u l t u r e s w i t h C - u r a c i l . They f r a c t i o n a t e d the intermediates by z o n a l c e n t r i f u g a t i o n i n a sucrose d e n s i t y gradient and designated them as l8S, 2 2 S , 25S, 28S and 32S r i b o n u c l e o p r o t e i n precursor p a r t i c l e s Because the l8S and 25S ribosome precursor p a r t i c l e s , obtained from the s h i f t up experiments, were s i m i l a r t o CM p a r t i c l e s , and because the 28s and 32S precursors were s i m i l a r to FU p a r t i c l e s , Kono and Osawa (1964) p l a c e d CM and. FU p a r t i c l e s into the normal sequence of ribosome biosynthesis. CM FU l6s RNA -» 18S —^ 28S —> 3 0 S ribosome 23S RNA -> 25S _^ 32S _> 28S 43S _^ 50S ribosome This sequence of biosynthetic events i s , of course, an oversimplification of the true picture. Sedimentation rates do not indicate the actual primary structure of RNA because they are dependent on the supercoiling of the RNA, further, they do not necessarily indicate the exact time sequence of the addition of protein. The discrete S values determined f o r the intermediates may indicate a change i n conformation of the ribosome pre-cursor rather than p a r t i c u l a r additions to i t . A good example of t h i s existing ambiguity i s the description of a 43S ribonucleoprotein p a r t i c l e i n addition to the 30S and 50S ribosomes (Lewandowski and Brownstein, 1966). The 1+3S p a r t i c l e was found to contain 23S RNA exclusively, but no characterization of the protein moiety of the p a r t i c l e has been reported . Because of the f a c t that t h i s 4-3S p a r t i c l e existed i n a steady s t a t e c u l t u r e , and no other p r e c u r s o r - l i k e p a r t i c l e s were evident, the H-3S p a r t i c l e could very w e l l be a 50S p a r t i c l e w i t h an a l t e r e d conformation. This completes a b r i e f survey of the research t h a t has been c a r r i e d out on m i c r o b i a l ribosome b i o s y n t h e s i s . The current research i n t h i s f i e l d i s extensive i n volume and impressive i n content. With the development of more s o p h i s t i c a t e d a n a l y t i c a l and p r e p a r a t i v e techniques the f i n a l e l u c i d a t i o n of the complete sequence of ribosome synthesis i s i n e v i t a b l e . MATERIALS AND METHODS I . Organisms and Media The organisms used throughout the course of t h i s study were P. aeruginosa ATCC 9027 and E. c o l i B / r / l . Both microorganisms were r o u t i n e l y c u l t u r e d i n Roux f l a s k s from a 1$ l 6 hr inoculum. The c u l t u r e medium c o n s i s t e d of 1$ KH PO^, 0 .5$ tryptone, and 0 .5$ yeast e x t r a c t . The pH of the medium was adjusted to 7 .2 w i t h 5 N KOH and s e p a r a t e l y s t e r i l i z e d glucose and MgSO^* TH^O were added t o give concen-t r a t i o n s of 0 .5$ and 0 .05$, r e s p e c t i v e l y . P. aeruginosa was grown f o r 12 hours at 30 C, E. c o l i f o r l 6 hours at 37 C. The P. aeruginosa c u l t u r e s were p e r i o d i c a l l y p l a t e d on t o King's agar t o check f o r p u r i t y and f o r the a b i l i t y of the c e l l s t o produce pyocyanine. E. c o l i was streaked on to p l a t e count agar (PCA) t o check p u r i t y . Stock c u l t u r e s were maintained f o r both organisms i n s m a l l screw cap v i a l s . P. aeruginosa was grown on minimal agar (Kay, 1968) f o r 2k hours then s t o r e d at 6 C. E. c o l i was grown f o r 2k hr on PCA and then s t o r e d at 6 C. I I . P r e p a r a t i o n of Washed C e l l Suspensions C e l l s were harvested by c e n t r i f u g a t i o n at 5,000 x g f o r 7 min at room temperature to avoi d cold-shock (McKelvie, Gronlund, and Campbell, I968) and washed three times w i t h 0 .85$ s a l i n e (pH 7 . 2 ) . The washed c e l l p e l l e t was resuspended t o the d e s i r e d c o n c e n t r a t i o n e i t h e r i n 0 .05 M t r i s - ( h y d r o x y m e t h y l ) -aminomethane hydr o c h l o r i d e ( t r i s HC1 b u f f e r ) (pH 7.2) c o n t a i n i n g -k -2 e i t h e r 10 or 10 M MgCl^, or i n s t a r v a t i o n medium, described i n the f o l l o w i n g s e c t i o n . I I I . S t a r v a t i o n Medium and S t a r v a t i o n Conditions When P. aeruginosa was t o be analysed f o r ribosomal p r e -cursors, c e l l s were s t a r v e d i n ammonium-phosphate b u f f e r of the f o l l o w i n g composition: 0 .3$ NHjH^PO^ and 0.2$ K^HPO^ adjusted to pH 7.2 w i t h 5 N K0H ( M P b u f f e r ) . The s t a r v a t i o n suspension was incubated at 30 C e i t h e r i n l a r g e Warburg cups (20 ml cup, 70 mg dry weight of c e l l s / m l ) on a Warburg respirometer, or i n 250 ml Erlenmeyer f l a s k s (50 ml f l a s k , kO mg dry weight of c e l l s / m l ) i n a shaking water bath. IV. P r e p a r a t i o n of C e l l - f r e e E x t r a c t s 1. Sonic o s c i l l a t i o n A B r o n w i l l B i o s o n i k BP I I sonic probe was used t o prepare c e l l - f r e e e x t r a c t s (CFX) of both P. aeruginosa and E. c o l i when the c e l l c o n c e n t r a t i o n was too great t o use the French press ( i n excess of 20 mg dry weight of c e l l s / m l ) , when ribosomes were d e s i r e d f o r chemical a n a l y s i s or f o r the i s o l a t i o n of ribosomal p r o t e i n s . The c e l l suspensions were pl a c e d i n c e l l u l o s e . n i t r a t e c e n t r i f u g e tubes of the appro-p r i a t e s i z e and these were pla c e d i n t o an i c e bath f o r the du r a t i o n of the s o n i c a t i o n , which was s i x 30 sec b u r s t s f o r P. aeruginosa and fo u r 30 sec b u r s t s f o r E. c o l i at i n t e n s i t y s e t t i n g of 87 watts. 2. French press A drop of 2.mg/ml, e l e c t r o p h o r e t i c a l l y pure (ribonuelease (KNase) f r e e ) deoxyribonuclease (DNase) was added to the c e l l suspension before i t was plac e d i n the.French press. Each suspension was passed twice through the c e l l dropwise at 18,000 l b / i n 2 . The e x t r a c t s , obtained by e i t h e r method, were f r e e d of i n t a c t c e l l s by c e n t r i f u g a t i o n at 3,000 x g f o r 7 min at 6 C. The c e l l f r e e e x t r a c t s were e i t h e r used immediately a f t e r p r e p a r a t i o n or immediately f r o z e n at -20 C. V. P h y s i c a l F r a c t i o n a t i o n of C e l l Free E x t r a c t s The procedure used to p h y s i c a l l y f r a c t i o n a t e c e l l - f r e e e x t r a c t s was t h a t of Campbell, Hogg and Strasdine (1962). The CPX was c e n t r i f u g e d at 20,000 x g,to remove the membranes and the 20,000 x g supernatant f l u i d was c e n t r i f u g e d a t 10,000 x g f o r 2 hours t o o b t a i n the ribosome p e l l e t . The ribosomes were resuspended w i t h a t i s s u e homogenizer, i n -k -? 0.05 M t r i s HC1 b u f f e r (pH 7.2) w i t h e i t h e r 10 or 10 M MgCl 2. Ik V I . I n c o r p o r a t i o n of C - u r a c i l i n t o C e l l s Ik Although the i n c o r p o r a t i o n of C - u r a c i l i n t o the c o l d TCA p r e c i p i t a b l e f r a c t i o n of the c e l l s does not n e c e s s a r i l y index the r a t e of ribosomal RNA syn t h e s i s , i t i s a r a p i d and convenient measure of the t o t a l KNA s y n t h e s i z i n g a b i l i t y of the c e l l s . This method was t h e r e f o r e e x p l o i t e d t o determine the optimum s t a r v a t i o n c o n d i t i o n s of c e l l suspensions p r i o r t o ribosome r e s y n t h e s i s . The s t a r v i n g c e l l s (80 mg dry weight of c e l l / m l ) were harvested at the d e s i r e d time i n t e r v a l s and resuspended (40 mg dry weight of c e l l / m l ) i n NPK b u f f e r c o n t a i n i n g 0.2$ * 14 glucose, 0.05% MgS0^*7H20 and 0.5 ucuries of C u r a c i l . The c e l l suspensions were incubated at 30 C f o r the d e s i r e d time 14 and the i n c o r p o r a t i o n of C - u r a c i l was p e r i o d i c a l l y analysed i n the f o l l o w i n g manner: one ml of the c u l t u r e was c e n t r i -fuged and the supernatant was c a r e f u l l y removed w i t h a Pasteur p i p e t t e . The c e l l s were resuspended i n one ml of d i s t i l l e d water, and one ml of 10$ TCA was added t o the suspension. A f t e r standing on i c e f o r twenty minutes the p r e c i p i t a t e was removed by c e n t r i f u g a t i o n a t 15,000 x g f o r 10 min at 6 C, and the supernatant f l u i d was removed w i t h a Pasteur p i p e t t e . The p r e c i p i t a t e was resuspended i n 0.1 H K0H and 0.1 ml of each f r a c t i o n was added t o 5 ml toluene-ethanol s c i n t i l l a t i o n f l u i d (TESF, described i n a l a t e r s e c t i o n ) . 3h V I I . F r a c t i o n a t i o n of Ribosomes on Sucrose Density Gradients 1. P r e p a r a t i o n of the sucrose gradients L i n e a r 5-20$ sucrose d e n s i t y gradients were obtained by the use of a continuous gradient making apparatus analogous to t h a t of B r i t t e n and Roberts ( i960). Gradients were prepared i n chamber mixing v e s s e l s of two s i z e s , k.9 ml and 29 ml . The s m a l l v e s s e l was s u i t a b l e f o r preparing sucrose gradients i n a L u s t e r o i d c e n t r i f u g e tube which f i t t e d the SW 39 r o t o r of the Spinco Model L p r e p a r a t i v e u l t r a c e n t r i f u g e . Twenty per cent sucrose (2.5 nil)? c o n t a i n i n g the d e s i r e d b u f f e r and magnesium conc e n t r a t i o n was added t o the compart-ment w i t h the o u t l e t , and f i v e per cent sucrose w i t h the same b u f f e r and magnesium c o n c e n t r a t i o n (2.U ml) was added t o the other chamber. The two compartments were connected by a sm a l l U-tube f i l l e d w i t h 5$ sucrose and the s o l u t i o n s were mixed on a magnetic s t i r r e r . K i n d l y s u p p l i e d by Dr. R. 5feee-Smith, Department of A g r i -c u l t u r e Research S t a t i o n . Samples were g e n t l y l a y e r e d on the top of the gra d i e n t by i n t r o d u c i n g them, s l i g h t l y below the surface of the gradient, w i t h a syringe f i t t e d w i t h a bent #18 needle. As much as 0.25 ml of sample co u l d be w e l l r e s o l v e d by t h i s technique. The tubes were then c e n t r i f u g e d i n the Spinco Model L f o r the times i n d i c a t e d i n a f o l l o w i n g l i s t f o r the appropriate times at 37,500 rpm. At the t e r m i n a t i o n of the c e n t r i f u g a t i o n , the c e n t r i f u g e tubes were p l a c e d i n a s p e c i a l l y constructed p l e x i g l a s s holder and the bottom of the tube was punctured w i t h a #22 needle, which protruded approximately 1 mm i n t o the tube. The contents were dispensed dropwise i n t o approximately 22 f r a c t i o n s . Twenty-nine ml gradients were prepared s i m i l a r l y . The chamber w i t h the o u t l e t contained 15 ml of 20$ sucrose, the other chamber, ik ml of 5$ sucrose, w i t h the appropriate b u f f e r and magnesium concentration. The tubes were p l a c e d i n a Spinco SW 25 r o t o r and c e n t r i f u g e d f o r the appropriate times and speeds. The contents of these tubes, a f t e r the t e r m i n a t i o n of the c e n t r i f u g a t i o n , were f r a c t i o n a t e d i n the f o l l o w i n g manner: the tube was c l o s e d by a two-holed s o f t rubber stopper. Through one hole of the stopper, a s o l u t i o n of kCffo sucrose was introduced through a g l a s s tube extending to approximately 2 mm from the bottom of the tube. The kCffo sucrose s o l u t i o n was s t o r e d i n a r e s e r v o i r above the tube t h a t was being f r a c t i o n a t e d and the r a t e of f l o w of the sucrose was a p p r o p r i a t e l y c o n t r o l l e d . The gradient was f o r c e d out of the tube through the other hole i n the stopper which was connected, by g l a s s and Tygon tubing, t o the p h o t o c e l l of a f r a c t i o n c o l l e c t o r . The contents of the tube were f r a c t i o n a t e d dropwise i n t o approximately 36 samples. The kofo sucrose s o l u t i o n was c o l o r e d w i t h pyronine r e d dye, and thus the end of the g r a d i e n t c o u l d e a s i l y be seen. The boundary between the c o l o r l e s s gradient and the r e d sucrose s o l u t i o n was always sharp, i n d i c a t i n g t h a t the gradient d i d not mix w i t h the 4-0$ sucrose s o l u t i o n . I t should be noted at t h i s time t h a t when p l o t t e d , the analyses of the s m a l l gradient tubes y i e l d e d a graph i n which the d e n s i t y of the g r a d i e n t decreased w i t h r e s -pect t o the number of f r a c t i o n s c o l l e c t e d , whereas the d e n s i t y of the g r a d i e n t i n c r e a s e d w i t h the number of f r a c t i o n s c o l l e c t e d from the l a r g e g r a d i e n t tubes. These gradients served t o f r a c t i o n a t e not o n l y ribosomes, but ribosomal RNA as w e l l . I n t h i s case 0.5$ sodium dodecyl s u l f a t e (Duponol C) was added to the sample p r i o r t o a p p l i c a t i o n t o the gradient which contained 0.5$ Duponol C as w e l l ( B r i t t e n and Roberts, i960) . The f o l l o w i n g l i s t s the v a r i o u s b u f f e r s and c o n d i t i o n s f o r the f r a c t i o n a t i o n of the i n d i c a t e d p a r t i c l e s : D e s i r e d B u f f e r f o r sample and C e n t r i f u g a t i o n time (hr) p a r t i c l e s sucrose s o l u t i o n s 37,500 rpm 23 ,000 rpm 70S ribosomes 0.05 M t r i s HCI b u f f e r 1 . 5 a 4.5 (pH 7.2) w i t h 0 . 0 1 M Mg C l g . 30S + 50S; 50S 0.05 t r i s HCI (pH 7.2) 2 . 5 a 1 0 . 0 ribosomes w i t h 10-k M MgClg. 30S ribosomes 0.05 M t r i s HCI (pH 7-2) 4.0 w i t h 1 0 - ^ M M g C l 2 < 23S + l6s rRNA; 0.05 M t r i s HCI (pH 7.2) 6 . 0 a 23S'rRNA w i t h 0.05$ Duponol C 16S rRNA . 0.05 M t r i s HCI (pH 7.2) 1 0 . 0 h w i t h 0.05$ Duponol C a) d e r i v e d from B r i t t e n and Roberts (i960) 2. The a n a l y s i s of the ribosomal m a t e r i a l The d i s t r i b u t i o n of the ribosomal m a t e r i a l i n the c o l l e c t ' f r a c t i o n s from sucrose d e n s i t y gradients was monitored i n two ways: a. The measurement of absorbance at 260 mu The absorbance at 260 mu of the v a r i o u s f r a c t i o n s was determined on appropriate d i l u t i o n s of each s e r i e s of samples e i t h e r w i t h a Beckman model DU, or a Beckman model DBG spectrophotometer. The samples were d i l u t e d w i t h d i s t i l l e d water. b. The measurement of r a d i o a c t i v i t y . When the c e l l s were l a b e l l e d w i t h a r a d i o a c t i v e isotope p r i o r t o t h e i r f r a c t i o n a t i o n on a sucrose d e n s i t y g r a d i e n t , i n a d d i t i o n t o l o c a l i z i n g the u l t r a v i o l e t (uv) absorbing m a t e r i a l , the d i s t r i b u t i o n of r a d i o a c t i v i t y throughout the gradient was a l s o determined. A p r e c i s e a l i q u o t of each sample was added t o 5 ml of toluene T-riton s c i n t i l l a t i o n f l u i d ( described i n a l a t e r s e c t i o n ) and the r a d i o a c t i v i t i e s of the samples were measured i n a Nuclear Chicago s c i n t i l l a t i o n spectrophotometer model 72S. VIII.. P r e p a r a t i o n of Samples f o r D i s c G e l E l e c t r o p h o r e s i s 1. P r e p a r a t i o n of ribosomes The ribosome samples f o r e l e c t r o p h o r e s i s were prepared according t o the method of Hjerten, J e r s t e d t and T i s e l i u s (1965). I n order t o remove l a r g e p a r t i c l e s which may have clogged the pores of the polyacrylamide g e l , the resuspended ribosome p e l l e t , or the CFX, destined t o be analyzed f o r ribosomes by t h i s method, was c e n t r i f u g e d at 20,000 x g f o r 30 min at 0 C. G l y c e r o l was added t o the 20,000 x g supernatant f l u i d t o give a f i n a l c o n c e n t r a t i o n of kO'fo. The purpose of t h i s a d d i t i o n was twofold: f i r s t l y , t h i s s o l u t i o n could be a p p l i e d t o the g e l d i r e c t l y , because the presence o f . g l y c e r o l rendered the samples s u f f i c i e n t l y dense such t h a t they would l a y e r smoothly on the top of the g e l . Secondly, t h i s s o l u t i o n could be st o r e d at -20 C without f r e e z i n g the samples. This allowed repeated use of the samples without the n e c e s s i t y of f r e e z i n g and thawing which would p o s s i b l y have introduced some degradation of the ribosomal m a t e r i a l . Ribosomal RNA was prepared from the ribosomes by the a d d i t i o n of Duponol t o 0.5$ and sucrose to 10$ to the samples p r i o r t o t h e i r a p p l i c a t i o n to the g e l s ( B r i t t e n and Roberts, i960; Bishop, Claybrook and Spiegelman, 1967). 2. P r e p a r a t i o n of ribosomal p r o t e i n s The ribosomal p r o t e i n s of the two organisms were prepared by two procedures (Gesteland and S t a e h e l i n , 1967). a. U r e a - L i C l Ribosomes were f r a c t i o n a t e d by sucrose d e n s i t y c e n t r i -f u g a t i o n , the appropriate f r a c t i o n s were pooled and c e n t r i f u g e d at 10,000 x g a f t e r the magnesium co n c e n t r a t i o n was in c r e a s e d to 0.1 M. The ribosomal p e l l e t s were resuspended i n d i s t i l l e d water and an equal volume of 8 M urea c o n t a i n i n g k M L i C l was added. The s o l u t i o n was st o r e d f o r 10-13 hours a t 6 C and the p r e c i p i t a t e d RNA. was removed by c e n t r i f u g a t i o n at 15,000 x g f o r 30 min at 6 C. The r e s u l t a n t supernatant f l u i d was s u i t a b l e f o r a p p l i c a t i o n t o the g e l . The k M urea 2 M L i C l s o l u t i o n was, of course, s u f f i c i e n t l y dense to a l l o w l a y e r i n g of the sample on top of the g e l , and the i o n i c s t r e n g t h was s u f f i c i e n t l y h i g h t o a l l o w storage at -20 C without f r e e z i n g . b. A c e t i c a c i d e x t r a c t i o n This method, a m o d i f i c a t i o n of t h a t of Waller and H a r r i s (1964), was much simpler i n execution. The appropriate f r a c t i o n s , from sucrose d e n s i t y g r adients c o n t a i n i n g the d e s i r e d ribosomes, were pooled and s u f f i c i e n t g l a c i a l a c e t i c a c i d was added to produce a 67$ a c e t i c a c i d s o l u t i o n . This s o l u t i o n was st o r e d overnight at 6 C and the p r e c i p i t a t e d RNA was removed by c e n t r i f u g a t i o n at 15,000 x g f o r 10 min at 6 C. Two and a h a l f volumes of i c e c o l d acetone were added t o the c a r e f u l l y removed super-natant ' f l u i d . The acetone caused the c o p r e c i p i t a t i o n of the sucrose and the ribosomal p r o t e i n s . A f t e r 2h hours at 6 C the amorphous p r e c i p i t a t e s e t t l e d t o the bottom of the beaker, and the supernatant f l u i d was removed by decanting. The d r i e d p r e c i p i t a t e was then r e d i s s o l v e d i n the minimum volume of d i s t i l l e d water, and enough 100$ TCA was added t o give a f i n a l c o n c e n t r a t i o n of 8$ TCA. A f t e r standing on i c e f o r 20 min, the suspension was c e n t r i f u g e d at 10,000.x g f o r 10 min at 6 C and the p r e c i p i t a t e was d i s s o l v e d i n the minimum volume of 8M urea. This p r o t e i n p r e p a r a t i o n was s u i t a b l e f o r a p p l i c a t i o n t o the g e l . DC. Polyacrylamide G e l E l e c t r o p h o r e s i s 1. Separation of ribosomes, ribosomal precursors, ribosomal KNA, and ribosomal p r o t e i n s , These experiments were performed i n g l a s s tubes, 0.6 cm ( i . d . ) x 10 cm, f i x e d t o the upper v e s s e l of a s p e c i a l l y constructed p l e x i g l a s s e l e c t r o p h o r e s i s apparatus ( s i m i l a r t o t h a t described by Davis, (j.^6k). The cathode of the Heathkit powerpack was connected t o the upper b u f f e r r e s e r v o i r ; the anode, t o the lower r e s e r v o i r . The current a p p l i e d w i l l be i n d i c a t e d f o r the i n d i v i d u a l g e l s described. A monolayer g e l system was used throughout these s t u d i e s because i t i s a much more r a p i d and convenient method and i t does not s a c r i f i c e any of the r e s o l u t i o n of the t r a d i t i o n a l three g e l system (Dr. G. W. Dixon, p e r s o n a l communication). a. Separation of ribosomes and ribosomal RNA. The g e l was prepared by the a d d i t i o n of the f o l l o w i n g i n g r e d i e n t s i n the i n d i c a t e d amounts. I n order t o avoi d premature p o l y m e r i z a t i o n of the g e l , the components were always added i n the sequence given and the po l y m e r i z i n g s o l u t i o n was mixed i n a red. Erlenmeyer f l a s k . The g e l was a m o d i f i c a t i o n of the one described by Bishop, Claybrook and Spiegelman (1967): Component Quantity D i s t i l l e d water 9.0 ml 1 M t r i s HCI (pH 7.4) w i t h 0.03 M M g C l 2 1.0 ml • 40$ acrylamide 1.0 ml 2$ N,N' -methylenebisacrylamide ( b i s ) 1.0 ml N,N, N',N',-tetramethylethylenediamine (TEMED) 10 u l Ammonium per s u l f a t e (NHj^SgOg) 10 mg When the g e l was used to r e s o l v e the ribosomal RNAs of Duponol t r e a t e d samples, 0.5$ Duponol C was a l s o added t o the g e l . The g l a s s tubes, which were c l o s e d at the bottom end w i t h serum stoppers, were r i n s e d twice w i t h one ml of the p o l y -m e r i z i n g l i q u i d , then f i l l e d to a height of 7 cm w i t h the same m a t e r i a l . A s m a l l volume of water was g e n t l y l a y e r e d on the top of the acrylamide s o l u t i o n t o ensure t h a t the g e l would have a f l a t surface. A f t e r the p o l y m e r i z a t i o n was completed, (judged by a sharp i n t e r f a c e between the top of the g e l and the water l a y e r ) , the tubes were p l a c e d i n t o the e l e c t r o p h o r e s i s apparatus so tha t the g e l was i n an u p r i g h t p o s i t i o n . Both the anode and cathode compartments were f i l l e d w i t h 500 ml of 0.60. M t r i s HCI (pH f.k) w i t h 3 x 10~^ M MgClg. The samples t o be analyzed were g e n t l y l a y e r e d on the top of the g e l w i t h the a i d of a m i c r o p i p e t t e . The current was a p p l i e d and the amperage was s l o w l y i n c r e a s e d t o the d e s i r e d value. The g e l s were removed a f t e r the t e r m i n a t i o n of the e l e c t r o -phoresis by adding water between the g e l and the g l a s s tube w i t h a syringe f i t t e d w i t h a #22 needle. b. Separation of ribosomal p r o t e i n s The e l e c t r o p h o r e s i s of the ribosomal p r o t e i n s was c a r r i e d out i n a s i m i l a r manner. The g e l used was a modi-f i c a t i o n of tha t used by Leboy, Cox and F l a k s (1964); E e i s f e l d , Lewis and W i l l i a m s (1962); Gesteland and S t a e h e l i n (1967): Component Quantity 8M urea 5.5 ml Potassium f e r r o c y a n i d e (K^Fe(CN)) 5 mg (NH^Og 15 mg 1+0$ acrylamide 1.0 ml 2$ b i s 1.0 ml TEMED 25 u l -alanine adjusted t o pH k.5 w i t h g l a c i a l 1.0 ml a c e t i c a c i d c o n t a i n i n g hU urea The connections of the two compartments of the e l e c t r o -p h o resis apparatus were reversed, the anode was connected to the top compartment; the cathode, t o the bottom. D i l u t e r ibosomal p r o t e i n s o l u t i o n s were added t o tubes c o n t a i n i n g pyronine r e d i n d i c a t o r to serve as the t r a c k i n g dye. The b u f f e r was O.067 M (!> -alanine adjusted t o pH 4.5 w i t h g l a c i a l a c e t i c a c i d (Gesteland and S t a e h e l i n , 1967). X. L o c a l i z i n g the Substances Separated by Acrylamide Cfel  E l e c t r o p h o r e s i s 1. S t a i n i n g w i t h s p e c i f i c reagents Because the ribosome i s composed of RKA and a la r g e v a r i e t y of b a s i c p r o t e i n s , s e v e r a l procedures are a v a i l a b l e t o s t a i n the ribosome and i t s components. The RNA moiety or the f r e e rRNA can be s t a i n e d by 1$ a c r i d i n e orange i n 15$ a c e t i c a c i d , a m o d i f i c a t i o n of the method of Gould.(19^7) and Hindley (1967), or by 1$ t o l u i d i n e b lue i n 1$ a c e t i c a c i d (Clarke, 196I+). The gels were exposed t o the s t a i n s f o r ih hrs at room temperature and the excess dye was leached out w i t h water. The p r o t e i n moiety of the ribosome or the f r e e r i b o -somal p r o t e i n were s t a i n e d by 0.5$ amido b l a c k i n 7$ a c e t i c a c i d . The ge l s were immersed i n t h i s s t a i n f o r 20 min and the excess dye was removed by soaking the s t a i n e d g e l i n batches of 7$ a c e t i c a c i d . On occasions the gels were exposed to the s t a i n f o r lh hrs and then the g e l was e l e c t r o p h o r e t i c a l l y destained i n 7$ a c e t i c a c i d . When e l e c t r o p h o r e t i c d e s t a i n i n g was employed, the a c e t i c a c i d used i n the procedure was s l i g h t l y c o l o r e d w i t h the s t a i n t o avoi d the complete d e c o l o r i z a t i o n o f t t h e p r o t e i n bands (Canalco t e c h n i c a l l i t e r a t u r e ) . The b a s i c p r o t e i n s , or more s p e c i f i c a l l y , the f r e e amino groups of l y s i n e and a r g i n i n e , were s t a i n e d by a 1$ aqueous s o l u t i o n of f a s t green adjusted to pH 8.0 (Repsis, 1967). The g e l s were s t a i n e d i n t h i s s o l u t i o n f o r 1 hour and then the excess dye was removed by soaking the g e l s i n d i l u t e 0.01 M t r i s HCI pH 8 .0 . 2. The a n a l y s i s of the d i s t r i b u t i o n of the r a d i o a c t i v i t y i n acrylamide g e l s , When the d i s t r i b u t i o n of the r a d i o a c t i v i t y i n the g e l s was to be determined, the g e l s were f r o z e n at -20 C immediately a f t e r removal from the e l e c t r o p h o r e s i s tube. This treatment of the g e l does not d i s t u r b the bands formed during the e l e c t r o -p h o resis (Bishop, Claybrook, and Spiegelman, 1967). ^ h e f r o z e n g e l was then s l i c e d i n t o 1.5 mm s l i c e s w i t h a s c a l p e l on a f r o z e n p l a s t i c block. The s l i c e s were p l a c e d i n s c i n t i l -l a t i o n v i a l s and d r i e d w i t h an i n f r a - r e d heat lamp. F i v e ml of toluene s c i n t i l l a t i o n f l u i d (TSF) was added t o each v i a l and the r a d i o a c t i v i t y was measured i n the s c i n t i l l a t i o n spectrometer. XT.. Determination of the M e l t i n g Curves f o r Ribosomes and RNA 1. M e l t i n g curves f o r RNA E. c o l i and P. aeruginosa CFX's were t r e a t e d w i t h 0.5$ Duponol C and the 23S and l6s rRNAs were i s o l a t e d by z o n a l c e n t r i -f u g a t i o n i n sucrose d e n s i t y gradients as described p r e v i o u s l y . The appropriate f r a c t i o n s from the gradients were pooled, and the contents were d i a l y z e d (1.5 ml/sample) three times against 1000 volume batches of 0.01 M KH^PO^ -K^HPO^ b u f f e r (pH 7.0) c o n t a i n i n g 10~^ M M g C l 2 (Stenesh and Holazo, 1966). A f t e r d i a l y s i s , the absorbance of the KNA s o l u t i o n was measured at 35 C and the change i n absorbance a t 260 mu w i t h the change i n temperature was measured i n a G i l f o r d 2000 spectrophotometer equipped w i t h an automatic temperature advance, set at 0.5 c/min. 2. The m e l t i n g curves of the ribosomes The m e l t i n g curves f o r ribosomes were determined by a s i m i l a r technique, however the d i a l y s i s b u f f e r was 0.01 M t r i s HC1 (pH 7-2) w i t h 0.01 M Mg CI (Suzuki and K i l g o r e , 1967). The spectrophotometer was made a v a i l a b l e through the courtesy of Dr. S. H. Zbarsky. Dr. I . Hynea's a s s i s t -ance i n operating the spectrophotometer i s g r a t e f u l l y acknowledged. X I I . Chemical Analyses 1. The determination of RNA The c o n c e n t r a t i o n of KNA was determined by the method of Schneider (1957) which measures purine bound pentoses. The procedure was mo d i f i e d as f o l l o w s : the reagents used were 0.2$ FeCl^'S^O i n 100 ml concentrated HC1 and 1.0 g of o r c i n o l i n 10 ml of 95$ ethanol. These s o l u t i o n s were combined immediately p r i o r t o c a r r y i n g out the assay and a 1.0 ml sample was added to 1.0 ml of the combined reagent. The assay mixtures were heated i n a b o i l i n g water bath f o r 4-5 min a f t e r which the tubes were cooled i n running water and the absorbance a t 665 mu was read w i t h a Beckman model B or model DBG spectrophotometer. This m o d i f i c a t i o n of the assay had the working range of 15-80 ug of KNA f o r the assay mixture. 2. The determination of p r o t e i n The method of Dowry et a l . (1951) was used t o deter-mine p r o t e i n concentrations. This procedure depends on the r e d u c t i o n of the phosphomolybdic-phosphotungstic reagent by the copper t r e a t e d a l k a l i n e p r o t e i n s o l u t i o n . The volume of the t o t a l r e a c t i o n mixture was reduced t o 1.1 ml throughout the course of t h i s study. This modi-f i c a t i o n of the assay was adequate f o r p r o t e i n con-c e n t r a t i o n s of 20-125 M-g/reaction mixture. 3. The determination of p r o t e i n and RNA. i n the same ribosome sample I t was found t h a t although RNA i n t e r f e r e s w i t h the Lowry p r o t e i n determination (100 ug of RNA i s equivalent t o 4.5 ug p r o t e i n , i . e . 4.5$ i n t e r f e r e n c e ) p r o t e i n does not r e a c t w i t h the o r c i n o l reagent. Therefore both the p r o t e i n and the RNA conc e n t r a t i o n could be determined on the same ribosomal s o l u t i o n without the need of f r a c t i o n -a t i n g the samples. For the determination of the RNA and p r o t e i n i n 30S, 50S and 70S ribosomes, the ribosomes were i s o l a t e d as described p r e v i o u s l y and c o l l e c t e d by the c e n t r i f u g a t i o n of the sucrose s o l u t i o n s a f t e r the Mg co n c e n t r a t i o n was brought t o 0.1 M. The ribosomal p e l l e t s were resuspended i n d i s t i l l e d water and the p r o t e i n and KNA concentrations were determined as described, and the necessary c o r r e c t i o n s f o r the KNA i n t e r f e r e n c e i n the p r o t e i n assays were c a l c u l a t e d . X I I I . The Determination of R a d i o a c t i v i t y 1. The determination of r a d i o a c t i v i t y i n samples l a b e l l e d by a s i n g l e isotope. The procedures used t o o b t a i n the samples to be assayed f o r r a d i o a c t i v i t y have a l r e a d y been mentioned. A l l the r a d i o a c t i v i t y measurements were c a r r i e d out w i t h a Nuclear Chicago l i q u i d s c i n t i l l a t i o n s p e c t r o -meter, model 725. The s c i n t i l l a t i o n f l u i d s t h a t were used were TSF, TESF and TTSF. TSF was prepared by the' a d d i t i o n of k g 2 , 5-diphenyloxazole (PPO) which was used as the primary s c i n t i l l a t o r , and 50 mg of 1 , 4-di ( 2 - ( 5-phenyloxazoyl))-benzene, the secondary s c i n t i l l a t o r , t o 1 l i t e r of toluene. TESF had the f o l l o w i n g composition per l i t e r of s c i n t i l l a t i o n f l u i d : 600 ml toluene, 400 ml absolute ethanol, 7 g PPO and 50 mg P0P0P. The TESF (Ok i t a , S p r a t t , and LeRoy, 1956) would inc o r p o r a t e 4$ of i t s volume as an aqueous sample, a l b e i t w i t h considerable quenching. The amount of quenching, however, was constant w i t h respect to the amount of water added, and w i t h respect t o the nature of the quenching agent used. I t was found, i n these s t u d i e s , t h a t w i t h the volumes of samples used, the amount of quenching f o r water, 5$ TCA and 0.1 N KOH were the same. Unfor t u n a t e l y , sucrose i s not s o l u b l e i n t h i s s c i n t i l l a t i o n f l u i d and, t h e r e f o r e , the f r a c t i o n s obtained from sucrose d e n s i t y gradients could not be d i r e c t l y analyzed i n t h i s s c i n t i l l a t i o n f l u i d . This shortcoming of TESF was f o r t u n a t e l y overcome by the use of TTSF. TTSF was prepared by a more l a b o r i o u s t e c h -nique. The commerical T r i t o n X 100 detergent was p u r i f i e d by the procedure of P a t t e r s o n and Green, (1965) and Benson ( l 9 6 6),with the exception t h a t the step i n v o l v i n g the passing of the decarbonized s o l u t i o n through an a c t i v a t e d s i l i c a column was omitted. B r i e f l y , the procedure was as f o l l o w s : 250 ml of toluene was added t o 250 mlT r i t o n X 100 and 2 g of N o r i t e . This s o l u t i o n was incubated at 50 C f o r 30 min. I t was found t h a t the removal of c h a r c o a l was f a c i l i t a t e d t o a great extent by a l l o w i n g the suspension t o stand at 6 C f o r Ik h r s . The c h a r c o a l was removed by f i l t r a t i o n , and the f i l t r a t e was adjusted t o c o n t a i n a f i n a l c o n c e n t r a t i o n of 667 ml toluene, 333 ml T r i t o n and 7 g PPO and 75 mg POPOP per l i t e r . 2. Determination of r a d i o a c t i v i t y i n samples doubly The simultaneous determination of the r a d i o a c t i v i t y of two isotopes i s p o s s i b l e i n a three channelled s c i n t i l -l a t i o n counter. The s c i n t i l l a t i o n counter was adjusted according t o the advice of the Nuclear Chicago consultant. Channel A was adjusted t o measure t r i t i u m t o an e f f i c i e n c y of o n l y 0.05$. The r a t i o of the counts per minute f o r Ik C was then determined i n channels A and C. Sample c a l c u l a t i o n s : l a b e l l e d w i t h C and \ SAMPLE T r i t i u m T r i t i u m + Channel A 10 20,000 1,000 Channel C 20,000 12,000 1,000 .The r a d i o a c t i v i t y , i n channel A caused by was Ill-assumed t o be i n s i g n i f i c a n t . Therefore, C cpm=?1000 (channel A) which i n t e r f e r e s to an extent of 600 cpm i n channel C. The H cpm i s t h e r e f o r e 1000-600 cpm= 400 cpm. RESULTS AMD DISCUSSION I . Separation of Ribosomes 1. Separation of ribosomes on agarose columns The purpose of these experiments was to separate r i b o -somal precursors i n q u a n t i t i e s adequate f o r physico-chemical and b i o l o g i c a l c h a r a c t e r i z a t i o n . G el f i l t r a t i o n through an agarose column seemed to be the i d e a l method. P r e l i m i n a r y s t u d i e s were necessary to a s c e r t a i n t h a t the agarose g e l i n f a c t i s able to r e s o l v e the 30S and 50S ribosomal subunits. Sepharose (Pharmacia's trade name f o r agarose) i s a c r o s s - l i n k e d dextran polymer. The degree of cross l i n k i n g , which depends on the co n c e n t r a t i o n of the agarose i n the g e l , determines the pore s i z e . Because the cross l i n k i n g i s e n t i r e l y due to hydrogen bonding, the pore s i z e s a v a i l a b l e are c o n s i d e r a b l y greater i n the Sepharose s e r i e s than i n the Sephadex s e r i e s . The fact, t h a t g e l f i l t r a t i o n i n v a r i a b l y caused d i l u t i o n of the recovered sample, n e c e s s i t a t e d the i n t r o d u c t i o n of a convenient method f o r the c o n c e n t r a t i o n of ribosomes. F i l t r a t i o n through an Amicon membrane f i l t e r (10,000 raw. pore s i z e ) appeared t o be the method of choice. P r i o r to r o u t i n e l y employing t h i s f i l t e r , i t was necessary to determine t h a t the f i l t r a t i o n procedure d i d not r e s u l t i n ribosomal degradation. A d i l u t e s o l u t i o n of 50S ribosomes was concentrated by the Amicon f i l t e r and both the d i l u t e and the concen-t r a t e d 50S ribosomes were analyzed by sucrose d e n s i t y gradient c e n t r i f u g a t i o n . The examination of these gradients r e v e a l l e d t h a t c o n c e n t r a t i o n of ribosomes by the Amicon f i l t e r d i d not r e s u l t i n t h e i r degradation P r e l i m i n a r y experiments compared the e l u t i o n p r o f i l e s of the two g e l s used, Sepharose 2B and Sepharose kB. The ( F i g . 2). Pharmacia l i t e r a t u r e describes these two g e l s i n the f o l l o w i n g manner: Type of g e l Approximate agarose co n c e n t r a t i o n P a r t i c l e s i z e i n swollen s t a t e Approximate f r a c t i o n a t i o n range M-w Sepharose KB 3 x 10 - 3 x 10 Sepharose 2B 2 x 10 - 25 x 10 J I I I 8 12 16 2 0 T U B E N O The e f f e c t of con c e n t r a t i o n by the Amicon f i l t e r ' on the sedimentation r a t e s of 50S ribosomes. The con centr a t e d ribosomes are represented by (0 - 0) and the unconcentrated ribosomes by (• - • ) . Two columns, each 0.6 cm ( i . d . ) were f i l l e d w i t h Sepharose 4B and Sepharose 2B r e s p e c t i v e l y , to a height of 50 cm. The b u f f e r used t o pack, wash and e l u t e the column was 0.1 M t r i s - H C l (pH 7 .4) , 0.1 M KC1, 1 0 M M g C l 2 and 10 M spermine (TKMS b u f f e r ) . The e l u t i o n p r o f i l e s of the two columns are p l o t t e d i n f i g u r e 3. Tlae s m a l l peak e l u t e d immediately p r i o r to the major u.v. absorbing peak was assumed t o c o n s i s t of membranes and other p a r t i c u l a t e m a t e r i a l , because there was considerable o p a c i t y i n t h a t f r a c t i o n . The major u.v. absorbing peak i s assumed t o be ribosomes. The recovery of the u.v. absorbing m a t e r i a l from both columns was o n l y 80$ of t h a t a p p l i e d . The g e l s were washed w i t h a l i t r e of 2 M t r i s -HCl b u f f e r at pH 8.0 but no more u.v. absorbing m a t e r i a l could be recovered. Extreme pH's and h i g h i o n i c strengths had to be avoided, because the hydrogen bonded cross l i n k s of the g e l are l a b i l e under these c o n d i t i o n s . N e i t h e r the Sepharose 2B, nor the Sepharose 4B column was able t o r e s o l v e the ribosomes t o the expected 30S and 50S peaks. I n order to achieve some r e s o l u t i o n s , the same experiment was repeated except t h a t 100 cm gels i n s t e a d 59 T U B E NO F i g . 3. The comparison of the e l u t i o n p r o f i l e s of ribosomes through Sepharose 2B and kB columns. Column s i z e , 50 cm x 0.6 cm. A. Sepharose kB column, 250 A 2 5o u n i t s a p p l i e d ; 200 •  recovered. B. Sepharose 2B column, 200 A 2go u n i t s a p p l i e d ; 170 recovered. of the previous 50 cm gels were used. The e l u t i o n p r o f i l e of the 100 cm Sepharose 4B column i s shown i n f i g u r e 4 a . The major u.v. absorbing peak has a shoulder t r a i l i n g which could v e r y w e l l be 30S ribosomes since 30S ribosomes would be expected t o be r e t a r d e d to a greater extent by the g e l than would the 50S ribosomes. An i n s p e c t i o n of f i g u r e 4 b , which shows the analyses of the concentrated peaks i n a sucrose d e n s i t y g r a d i e n t , r e v e a l e d t h a t t h i s was not the case. Peak 1 contained both 30S and 50S ribosomes, and peak 2 contained low molecular weight i m p u r i t i e s . This column t h e r e f o r e , d i d separate the ribosome p e l l e t s o l u t i o n i n t o the membrane f r a c t i o n , ribosome f r a c t i o n and low molecular weight f r a c t i o n , even though i t d i d not r e s o l v e the 30 and the 50 ribosomes. The same experiment was repeated u s i n g a 100 cm Sepharose 2B column. Due t o the higher f r a c t i o n a t i o n range of Sepharose 2B b e t t e r r e s o l u t i o n was expected. The e l u t i o n p r o f i l e depicted i n f i g u r e 5a shows t h a t the shoulder t r a i l i n g the main peak i s i n f a c t b e t t e r r e s o l v e d (compare t o f i g u r e 4a) but as i s evident from f i g u r e 5b, these peaks are i d e n t i c a l to the corresponding ones i n f i g u r e 4 b . 61 e o ID OJ I-< UJ o < o CO CO < A F i g . 4. C h a r a c t e r i z a t i o n of the e l u t i o n p r o f i l e of ribosomes passed, through a Sepharose kB column. ••' . AV- E l u t i o n p r o f i l e of ribosomes passed through a 100 cm x 0.6 cm column. B. Ribosome p a t t e r n s of concentrated peaks: peak 1 -(0 - 0); peak 2 - (• - • ) . •i 62 10 15 2 0 25 . 3 0 35 4 0 cr. 0 5 10 15 2 0 25 T U B E N O . F i g . 5. C h a r a c t e r i z a t i o n of the e l u t i o n p r o f i l e of ribosomes passed through a Sepharose 2B column. A. . E l u t i o n p r o f i l e of ribosomes passed through a 100 cm x 0.6 'cm column. B. Ribosome p a t t e r n s of concentrated peaks: p e a k . l (0 - 0) ; peak I I ( t - • ) . The se p a r a t i o n of the 30S from the 50S ribosomes, which was the u l t i m a t e aim of the preceding s e r i e s of experiments, was not r e a l i z e d . I t was however p o s s i b l e to p u r i f y the ribosome p e l l e t through these columns, e s p e c i a l l y through the 100 cm Sepharose 2B column. Due t o a more uniform g e l , and a slower f l o w r a t e (2.0 ml per h r ) , the sep a r a t i o n of the ribosomes from the membrane f r a c t i o n i n t h i s work was b e t t e r and the peaks were much sharper than those obtained by H j e r t e n (1962). Due to the f a c t t h a t the s e p a r a t i o n ranges of both the Sepharose g e l s were too low t o r e s o l v e the 30S and 50S ribosomes, t h i s method of f r a c t i o n a t i n g the c e l l f r e e e x t r a c t s f o r the analyses of ribosomal precursors had t o be abandoned. The p o s s i b i l i t y t h a t Sepharose g e l s w i t h a higher f r a c t i o n a t i o n range w i l l be developed s t i l l remains, and then of course g e l f i l t r a t i o n c o u l d become a v e r y con-venient and powerful technique t o r e s o l v e ribosomal sub-u n i t s w i t h both a n a l y t i c a l and p r e p a r a t i v e a p p l i c a t i o n s . 2. The sepa r a t i o n of ribosomes on polyacrylamide g e l s The o n l y reference t o date which describes the separ a t i o n of ribosomes i s the system of Hjerten, J e r s t e d t , and T i s e l i u s (1965). They used a 4$ acrylamide g e l w i t h 1$ c r o s s l i n k i n g , which s u c c e s s f u l l y r e s o l v e d the 30S and 50S ribosomes. I n a d d i t i o n t o the ribosomes,they obtained 8 other f a i n t l y c o l o r e d u n i d e n t i f i e d bands when they s t a i n e d t h e i r g e l w i t h amido bl a c k . The e l e c t r o p h o r e t i c m o b i l i t i e s obtained by Hje r t e n , J e r s t e d t and T i s e l i u s were 1.0 cm f o r the 50S ribosome and 1.08 cm f o r the 30S ribosome. The e l e c t r o p h o r e t i c m o b i l i t i e s obtained i n these s t u d i e s were 0.5 cm f o r the 50S ribosomes and 0.7 cm f o r the 30S ribosomes. F i g u r e 6 shows a 3.6$ acrylamide g e l which was-vused to r e s o l v e the ribosomes at 2.5 mA/tube f o r k hours. The g e l was s t a i n e d w i t h a c r i d i n e orange and the excess dye removed as described. The 50S and 30S ribosomes are w e l l separated, even though t h e i r m o b i l i t i e s are not as great as described by Hje r t e n , J e r s t e d t and T i s e l i u s (1965). There i s a t h i r d band, which i s w e l l separated from the ribosomes at a m o b i l i t y of 1.70 cm. This band i s a l s o 65 F i g . 6. Separation of 30S and 50S ribosomes on an a n a l y t i c a l acrylamide g e l e l e c t r o p h o r e s i s column. Column dimensions 0.6 x 7 cm Gel composition: 3.6$ acrylamide, 5$ c r o s s -l i n k i n g agent (with respect t o acrylamide). E l e c t r o p h o r e s i s b u f f e r : 10"2 M t r i s HC1 pH 7.k w i t h 3 x 10~^ M M g C l ^ . Current 2.5 mA/tube f o r 2 hours. apparent when the g e l i s s t a i n e d by t o l u i d i n e blue, but not when the g e l i s s t a i n e d by e i t h e r amido bla c k , or by f a s t green. The l a t t e r two s t a i n s , s p e c i f i c f o r p r o t e i n s , s t a i n o n ly the 30S and the 5°S ribosomes. The nature of the t h i r d band (band 'X') i s not understood. I t i s probably an a c i d i c macro-molecule ( s t a i n i n g p r o p e r t i e s ) which could o r i g i n a t e from the membrane f r a c t i o n contam-i n a t i n g the ribosomal p e l l e t . The reason why Hj e r t e n , J e r s t e d t and T i s e l i u s (1965), obtained 8 bands other than the ribosomes i s probably due to the f a c t t h a t they were u s i n g 6 mA./tube which could have r e s u l t e d i n some ribosomal degradation. R a d i o a c t i v e ribosomes as w e l l as the ribosomal RNAs were separated on t h i s g e l . The r e s u l t s of these experiments w i l l be discussed i n a l a t e r s e c t i o n . I I . Separation of the Ribosomal P r o t e i n s by Polyacrylamide  g e l e l e c t r o p h o r e s i s 1. Determination of the number of p r o t e i n bands i n the v a r i o u s ribosomal subunits The d i s t r i b u t i o n of the ribosomal p r o t e i n s i n the 30S, 50S and 79S ribosomes of P. aeruginosa i s shown i n Figu r e 7. The 30S ribosome has l 6 p r o t e i n bands; the 50S, 21; and the 70S, 31 p r o t e i n bands. These numbers must be taken w i t h r e s e r v a t i o n s because these p r o t e i n s are defined by on l y one parameter, t h e i r m o b i l i t y i n the polyacrylamide g e l . The comparative p a t t e r n s f o r the ribosomal p r o t e i n s of E. c o l i are shown i n f i g u r e 8. The 30S ribosome of E. c o l i has 15 bands; the 50S ribosome, 21 bands; and the 70S ribosome has 31 hands. These numbers agree w e l l w i t h the r e s o l u t i o n r e p o r t e d by Cox and F l a k s (1964). They res o l v e d , by g e l e l e c t r o p h o r e s i s , 13 p r o t e i n s from E. c o l i 30S ribosomes and 21 p r o t e i n s from the 50S r i b o -somes. Gesteland and S t a e h e l i n (1967) succeeded i n i s o l a t i n g 15 p r o t e i n s from the 30S ribosomes of E. c o l i ; 20 p r o t e i n s from the 50S ribosomes; and 31 p r o t e i n s from the 70S ribosomes. Otaka, I t o h and Osawa (1968), described the se p a r a t i o n of at l e a s t 15,and 11 p r o t e i n components i n the 50S and 30S ribosomes r e s p e c t i v e l y of E. c o l i , along w i t h minor proteinaceous components. These p r o t e i n s were f i r s t f r a c t i o n a t e d by carboxymethyl-c e l l u l o s e chromatography, and the i s o l a t e d peaks were 30S • < - ; 50 S 70S F i g . 7. A n a l y s i s of the p r o t e i n s of the ribosomes of P. aeruginosa by d i s c g e l e l e c t r o p h o r e s i s . Conditions of the e l e c t r o p h o r e s i s 13.3$ acrylamide g e l , M/15 P-alanine-acetate b u f f e r (pH 4 .5) , d u r a t i o n of e l e c t r o p h o r e s i s , 5 hr at 2.5 mA/tube. 6 9 -6 9 70S F i g . 8. Ana lys is of the proteins of the ribosomes of Escher ich ia  c o l i by d isc ge l e lect rophoresis . See legend fo r F i g . 7. subjected t o e l e c t r o p h o r e s i s . By the use of H - l a b e l l e d lit 50S ribosomal p r o t e i n s , and C - l a b e l l e d 30S ribosomal p r o t e i n s Otaka, I t o h and Osawa (1968) c o n c l u s i v e l y showed t h a t there were no p r o t e i n components common t o both the 50S and the 30S ribosomes. S e v e r a l groups r i g o r o u s l y proved t h a t the bands appearing on the g e l s from the ribosomes of E. c o l i at l e a s t , have d i f f e r e n t primary s t r u c t u r e s . Traut et a l . (1967) employed ribosomal p r o t e i n s and t h e i r t r y p t i c d i g e s t s 35 35 which had been l a b e l l e d by S-methionine and S-cysteine t o show t h a t they gave r i s e t o d i f f e r e n t i a l l y l a b e l l e d components. Moore et a l . (1968) c a r e f u l l y p u r i f i e d 13 p r o t e i n s from the 30S ribosome of E. c o l i and c h a r a c t e r i z e d them by amino a c i d composition, sedimentation e q u i l i b r i u m molecular weight and t r y p t i c f i n g e r p r i n t s . No s i m i l a r i t i e s were found between the v a r i o u s 30S p r o t e i n s t h a t were i s o l a t e d , hence each was assumed t o possess a unique amino a c i d sequence. These r e s u l t s along w i t h the a l r e a d y described p h y s i o -l o g i c a l c h a r a c t e r i z a t i o n of some of the 30S ribosomal p r o t e i n s (Traub et a l . 1967; Traub and Nomura, 1968) d e f i n i t i v e l y designate each of the acrylamide g e l separated p r o t e i n s t o be unique. I n a l l l i k e l i h o o d , t h e r e f o r e , the ribosomal p r o t e i n s of P. aeruginosa r e s o l v e d by acrylamide g e l e l e c t r o p h o r e s i s are a l s o d i f f e r e n t from each other. 2. Comparison of the ribosomal p r o t e i n s i n the v a r i o u s subunits t e s t e d i n E. c o l i and P. aeruginosa Comparison of the p r o t e i n s of the v a r i o u s ribosomal subunits of P. aeruginosa i s shown i n f i g u r e 9* The d i s t r i b u t i o n of the p r o t e i n s of the 50S and the 30S ribosomes was d i f f e r e n t . I t i s , of course, impossible t o d e f i n i t i v e l y s t a t e at t h i s p o i n t whether or not there are any common p r o t e i n s of the two ribosomes. For t h i s reason the com-parisons were made of the p a t t e r n of d i s t r i b u t i o n and the r e l a t i v e i n t e n s i t i e s of the bands i n the two ribosomes. The p r o t e i n s of the 70S ribosome had the appearance of a combination of the 50S and 30S ribosomal p r o t e i n s ( F i g . 9)' Comparison of the ribosomal p r o t e i n s of the 30S and the 50S ribosomes of E. c o l i a l s o r e v e a l e d d i f f e r e n c e s ( F i g . 9)> and the 70S ribosomal p r o t e i n d i s t r i b u t i o n p a t t e r n looked l i k e a combination of the two ( F i g . 9)« 30S 50S 70S 30S 50S 70S qpHP B F i g . 9 . Comparison of the ribosomal p r o t e i n s of P . aeruginosa and those of E . c o l i . P . aeruginosa ( A ) ; E . c o l i [ B J See legend f o r F i g . 7. The comparison of the ribosomal p r o t e i n d i s t r i b u t i o n of the 30S ribosome of E. c o l i and P. aeruginosa r e v e a l e d d i f f e r e n t p a t t e r n s ( F i g . 10) as d i d the pa t t e r n s of the 50S ribosomes ( F i g . 10) and the 70S ribosomes ( F i g . 10) of the two species. The most s i g n i f i c a n t a p p l i c a t i o n of polyacrylamide g e l e l e c t r o p h o r e s i s t o the study of the ribosomal p r o t e i n s has r e c e n t l y been s t a r t e d by S t o f f l e r , R u d l o f f and Wittmann (1968). They are i n the process of developing p r e p a r a t i v e acrylamide g e l e l e c t r o p h o r e s i s , aiming t o r e s o l v e and p u r i f y ribosomal p r o t e i n s i n q u a n t i t i e s from which p o i n t t h e i r f u l l c h a r a c t e r -i z a t i o n , both physico-chemical and biochemical, w i l l be imminent. I I I . The Determination of the M e l t i n g Curves of Eibosomes and  Ribosomal RNA's 1. The m e l t i n g curve of ribosomal RNA The m e l t i n g curves of the ribosomal RNA's were determined as described. To f a c i l i t a t e comparison between the thermal denaturation p r o f i l e s of the RNAs of the two species, F i g . 10. Comparison of the ribosomal p r o t e i n s of the v a r i o u s ribosomal f r a c t i o n s of P. aeruginosa to the corresponding p r o t e i n s of E. c o l i . P. aeruginosa (A); E. c o l i (B). See legend f o r F i g . 7. P. aeruginosa and. E. c o l i , the r a t i o of the absorbance at 260 mu was c a l c u l a t e d at each temperature r e l a t i n g t o the absorbance at 35 C. This r a t i o was then p l o t t e d against the i n c r e a s i n g temperature and the m e l t i n g p o i n t (Tm) was c a l c u l a t e d by p l o t t i n g the f i r s t d e r i v a t i v e of the r a t i o of absorbances. I n other words, the change i n the r a t i o of the absorbance against the temperature. (This procedure i s e x a c t l y the same as the one used t o determine the pK values of amino acid s by t i t r a t i o n ) . The b u f f e r t h a t was used i n the determination of the m e l t i n g p o i n t s of the ribosomal KNA s was t h a t of Stenesh and Holazo (1967). These workers compared the Tm s of t h e r m o p h i l i c and m e s o p h i l i c B a c i l l u s species. They defined the Tm i n t h e i r p u b l i c a t i o n as the temperature at which 50$ of the hyperchromic s h i f t was observed. For three mesophiles, the - Tm s c a l c u l a t e d by t h i s method were 64-65 C; and f o r three thermophiles, the Tm s were between 69-71 C. C a l c u l a t e d by t h i s method the m e l t i n g p o i n t s of the E. c o l i and P. aeruginosa are: (from Table I ) 68 C f o r the l 6 s KNA and 65 C f o r the 23S RNA of P. a e r u g i -nosa and 6 l C f o r the l6s RNA and 6h C f o r the 23S KNA of E. c o l i . There i s a s l i g h t d i f f e r e n c e between the average Tm value f o r P. aeruginosa KNA, 67.5 C and E. c o l i KNA, 62.5 C. The hyperchromic s h i f t s were around 1.35; Stenesh and Holazo (1966) r e p o r t e d the hyperchromic s h i f t s of t h e i r KNAs as around 1.32. The f o l l o w i n g t a b l e l i s t s the d i f f e r e n t species of rRNA used and the Tm s c a l c u l a t e d by drawing the f i r s t d e r i v a t i v e s of the graphs of the m e l t i n g p r o f i l e s from f i g u r e s 11, 12, 13 and Ik. Table I: The Tm s of the Ribosomal KNA s of E. c o l i and P. aeruginosa rRNA Tm ( C) Tm ( C) 70 f i r s t h y p e rchromicity d e r i v a t i v e Reference P. aeruginosa l6s 68.0 23S 65.O 69.O 68.0 F i g . 11 F i g . 12 E. c o l i 16S 23S 6l.O 64.0 66.0 66.0 F i g . 13 F i g . Ik The m e l t i n g p o i n t s of the two P. aeruginosa KNA' s f o r a l l purposes are the same, as are the m e l t i n g p o i n t s of the two E. c o l i KNA s. I t i s d i f f i c u l t t o argue s t r o n g l y about 4 r-1-3 o m < 1-2 -\ o —^ CM ^. I • I 0 o-cro-o Tm = -69° C 3 0 4 0 5 0 6 0 7 0 TEMP. 8 0 9 0 F i g . 11. The thermal d e n a t u r a t i o n p r o f i l e of P. aeruginosa l6s rRNA. The r e l a t i v e absorbance .(•the r a t i o of the absorbance of the heated sample and the absorbance at 35 C i s p l o t t e d :against the temperature of the:sample. ; See M a t e r i a l s and Methods). i -3 - a 1-4 TEMP. F i g . 12. The thermal.denaturation p r o f i l e of P. aeruginosa 23S rRNA.. See. legend f o r F i g . 6. F i g . 13. The-thermal denaturation p r o f i l e of E. c o l l l6s rRNA. See legend f o r F i g . 6. 1-4 TEMP. F i g . Ik. The thermal denaturation p r o f i l e of E. c o l i 23S rRNA. See legend f o r F i g . 11. the d i f f e r e n c e s between the m e l t i n g p o i n t s of the E. c o l i K NAs (average 66 C) and the P. aeruginosa KNA s (average 68.5 C), because the m e l t i n g p r o f i l e s t h a t one obtains f o r K M i s not n e a r l y as sharp as one obtains from DNA. The f o l l o w i n g conclusions c o u l d be drawn, however, i f the a c t u a l m e l t i n g p r o f i l e s , r a t h e r than the Tm s were compared: I f one superimposes the m e l t i n g p r o f i l e s of the corresponding l6s KNAs f o r E. c o l i ( F i g . 8) and f o r P. aeruginosa ( F i g . 6) , then there i s a d e f i n i t e d i f f e r e n c e between the m e l t i n g p r o f i l e s of the two KNA s. A s i m i l a r s u p erimposition of the m e l t i n g p r o f i l e of the P. aeruginosa 23S KNA ( F i g . 7) against the E. c o l i 23S KNA ( F i g . 8) r e v e a l e d the same degree of d i f f e r e n c e . I t can t h e r e f o r e j u s t i f i a b l y be concluded t h a t the E. c o l i rKNA s have correspondingly lower m e l t i n g p o i n t s than those of the corresponding P. aeruginosa KNA s. 2. The m e l t i n g curve of ribosomes The Tm values f o r the v a r i o u s ribosomal f r a c t i o n s of the two organisms tested,are l i s t e d i n Table 2. The Tm values of the ribosomes were d e r i v e d by the i d e n t i c a l method used f o r the c a l c u l a t i o n of the Tm values of the KNAs. Table II: The Tm s of the Various Ribosomal F r a c t i o n s of E. c o l i and P. aeruginosa Organism Ribosomes Tm ( C) References P. aeruginosa 30S 6k C F i g . 15 50S 72 C F i g . 16 70S 82 C F i g . 17 E. c o l i 30S 5k C F i g . 18 50S 54 C F i g . 19 70S ik C F i g . 20 There are s i g n i f i c a n t d i f f e r e n c e s between the Tm s of the 30S, 50S and 70S ribosomes of P. aeruginosa ( F i g . 15, 16, 17 r e s p e c t i v e l y ) . The Tm increases as a-function of the s i z e of the molecules i n v o l v e d . Because there i s no d i f f e r e n c e i n the Tm values of l 6 s and 23S RNA e i t h e r i n P. aeruginosa or i n E. c o l i (Table I ) , the d i f f e r e n c e s of the Tm values of the ribosomes must be due t o a p r o p e r t y of the ribosomes themselves r a t h e r than to the d i f f e r e n c e between the ribosomal RNAs. I n the E. c o l i s e r i e s of 30S, 50S and 70S ribosomes, there i s no d i f f e r e n c e between the Tm values of the 30S and 50S F i g . 17. The thermal denaturation p r o f i l e of P. aeruginosa 70S ribosomes. See legend f o r F i g . 11. CO F i g . 19. The thermal d e n a t u r a t i o n p r o f i l e of E. c o l i 50S ribosomes. See legend fl>r f i g u r e 11. ribosomes (Fig. 18, 19) respectively), however there i s a large increase i n the Tm value of the 70S ribosome (Fig. 20). The Tm values of the P. aeruginosa ribosomes are consistently higher than the corresponding Tm's for those of E. c o l i (Table I I ) . This could be due to the fa c t that the forces responsible for the t e r t i a r y structure of the P. aeruginosa ribosomes are stronger than those holding the t e r t i a r y structure of the E. c o l i ribosomes. The 70S ribosome would be expected to have a higher Tm for the same reason because i t would, of course, be more compact than the 30S or 50S ribosomes, therefore, the t e r t i a r y structure and the double h e l i c a l regions of the ribosomal RNA should be stable to a higher temperature i n the 70S ribosome than i n either the 30S or 50S ribosome. The difference i n Tm values between the 30S and 50S ribosomes i s harder to explain. I t i s possible of course that i n the P. aeruginosa ribosomes the 30S ribosome i s held together by weaker forces than the 50S; therefore the 30S ribosome would melt at a lower temperature. This difference i n the Tm's of the 30S and 50S ribosomes i s not observed i n E. c o l i , therefore i t would be assumed that the 30S ribosomes and 50S ribosomes i n E. c o l i are held together by the same magnitude of force. The b u f f e r t h a t was used i n the preceding s e r i e s of experiments was tha t of Suzuki and K i l g o r e (1967). They reported t h a t the Tm of 70S ribosomes of E. c o l i was 70 C but they took readings at 5 C i n t e r v a l s , and the change i n the r a t i o of absorbance on t h e i r graph i s greater between 70-75 C than between 65-70 C. The Tm value obtained by Suzuki and K i l g o r e (1967) t h e r e f o r e , i n a l l p r o b a b i l i t y , i s the same as the one c a l c u l a t e d i n our experiments. The hyperchromic s h i f t s obtained i n these experiments (approximately 1.65) i s a l s o c l o s e t o t h a t r e p o r t e d by Suzuki and Kilgore', (1967) f o r the E. c o l i B 70S ribosomes (1.60). IV. The Determination of the R a t i o of RNA to P r o t e i n i n Ribosomes P. aeruginosa was grown as described i n yeast e x t r a c t -tryptone medium f o r 12 hours at 30 C. E. c o l i was grown i n the same medium at 37 C f o r 16 hours. The c e l l s were resuspended t o 80 mg dry weight of c e l l s / m l i n 0.05 M t r i s HC1 (pH 7.2) and the CPX was prepared by sonic o s c i l l a t i o n . The CPXs were f r a c t i o n a t e d on the appropriate 29 ml sucrose d e n s i t y gradients t o y i e l d the d e s i r e d ribosomal subunits. The appropriate tubes were pooled and the con c e n t r a t i o n of ++ Mg adjusted to 0.1 M. The ribosomes were c o l l e c t e d and resuspended as described. The co n c e n t r a t i o n of p r o t e i n and RNA were determined f o r the a p p r o p r i a t e l y d i l u t e d samples. i The r e s u l t s of these experiments are t a b u l a t e d i n Table I I I . Table I I I : R e l a t i v e Q u a n t i t i e s of RNA and P r o t e i n i n Ribosomes 30S ribosomes 50S ribosomes 70S ribosomes P. aeruginosa io RNA % p r o t e i n 60.5 39-5 60.0 hQ.Q 60.0 1+0.0 E. c o l i $ RNA i p r o t e i n 6l.5 38.5 62.5 37.5 6l.O 39.0 The $ of RNA i n E. c o l i i s given as 63$ ( T i s s i e r e s e t , a l . 1959)« The $ values i n Table I I I are w i t h i n experimental e r r o r of the 63$ RNA t o 37$ p r o t e i n value e s t a b l i s h e d by T i s s i e r e s et a l . (1959)-There i s no s i g n i f i c a n t d i f f e r e n c e i n the RNA/protein r a t i o of E. c o l i and P. aeruginosa. V. Determination of the Starvation and the Reincubation  Conditions Optimal for the Investigation of the Re- synthesis of Ribosomes I t was reasonable to suppose that a population of c e l l s whose ribosomes were depleted but which were metabolically active, would recommence the resynthesis of t h e i r ribosomes i f placed i n a suitable medium. R. McKelvie (Ph.D. Thesis) observed that the ribosomes of. P. aeruginosa were depleted when c e l l suspensions were starved f o r 1+8 hrs. The c e l l s used i n McKelvie's experiment were grown for 48 hr at 30 C i n the tryptone-yeast extract medium described. Preliminary experiments confirmed McKelvie's observations. In the present work, P. aeruginosa was grown for 48 hr, then harvested and resuspended i n 0.1 M t r i s HC1 (pH 7.4) as described at 70 mg dry weight of cells/ml. Samples of 6 ml were taken after 0, 24 and 48 hr starvation at 30 C i n large Warburg cups, and the respective c e l l free extracts (1.5 ml) were analyzed f o r ribosomes by sucrose density gradient centrifugation. The ribosome patterns obtained are plotted i n figure 21. The ribosomes i n the 48 hr starved c e l l s were completely degraded and there was also considerable degradation of the ribosomes i n the 24 hr c e l l s . 1-0 T U B E NO F i g . 21. Ribosome p r o f i l e of P. aeruginosa grown i n ye a s t -tryptone medium f o r 4~8 hours, and then starved i n 0.05'M t r i s HC1 pH 7.4 (0 - 0) 0 hr s t a r v a t i o n (& - A) 24 hr s t a r v a t i o n , and (• - •) 48 hr s t a r v a t i o n . I t was t h e r e f o r e reasoned t h a t the 48 hr st a r v e d c e l l s should provide a s a t i s f a c t o r y system f o r the study of the r e s y n t h e s i s of the ribosomes. A p r e l i m i n a r y ex-periment to a s c e r t a i n whether these c e l l s were i n f a c t able t o r e s y n t h e s i z e t h e i r f u l l ribosomal complement was c a r r i e d out by-the f o l l o w i n g procedure: The starved c e l l s were harvested and resuspended at 70 mg dry weight of c e l l s / m l i n 100 ml of MPK b u f f e r c o n t a i n i n g 0.5$ , v lb-glucose, 0.1% MgCl^ and 1% tryptone w i t h 10 u-c C-glucose, and incubated i n a 1000 ml Erlenmeyer f l a s k i n a 30 C incubator. A f t e r 6 hours of i n c u b a t i o n the c e l l s were l y s e d , and were mixed w i t h a CPX of P. aeruginosa grown f o r 48 hr t o serve as a source of c a r r i e r ribosomes. The combined CPX was f r a c t i o n a t e d on a sucrose d e n s i t y gradient, and the ribosomes were l o c a l i z e d as described. The ribosome p a t t e r n p l o t t e d i n f i g u r e 22 shows th a t these 14 c e l l s were unable to inco r p o r a t e the C-glucose i n t o t h e i r ribosomes, even a f t e r 6 hrs i n c u b a t i o n . These c e l l s are t h e r e f o r e not as a c t i v e as one would want f o r the study of ribosome r e s y n t h e s i s , and shorter s t a r v a t i o n c o n d i t i o n s w i l l have t o be i n v e s t i g a t e d . 95 i « I 1 1 0 5 10 15 20 25 TUBE NO F i g . 2 2 . Ribosome p a t t e r n of P. aeruginosa starved • • f o r 48 hours, then reincubated w i t h 100 p-curies of C l ^ - g i u c o s e . The c e l l s were, reincubated • f o r 6 hours, both the absorbance (0 - 0) and the r a d i o a c t i v i t y (• - •) were measured. C e l l s grown f o r k8 hours were harvested and st a r v e d f o r 2k hours as described i n the previous s e c t i o n . They were reincubated i n the same medium f o r kOO min and the ribosomes were f r a c t i o n a t e d . No c a r r i e r ribosomes were added t o t h i s CPX. The r e s u l t s are p l o t t e d i n f i g u r e 23. Ik These c e l l s i n c o r p o r a t e d C-glucose but the i n c o r p o r a t i o n was s t i l l v e r y s m a l l c o n s i d e r i n g the le n g t h of time allowed f o r ribosome r e s y n t h e s i s . I t appeared t h a t the s t a r v a t i o n time s t i l l had to be decreased. I n order t o survey a range of s t a r v a t i o n lb-times j the i n c o r p o r a t i o n of .. C - u r a c i l i n t o the c e l l s upon r e i n c u b a t i o n was measured. The c e l l s were grown as des-c r i b e d , except t h a t the i n c u b a t i o n p e r i o d was decreased t o 2k hours. The s t a r v i n g c e l l s at 50 mg dry weight of c e l l s / m l were sampled at 3, 6, 9 a n ( i 12 h r s , harvested, and reincubated Ik as described i n the presence of 10 |_ic of C - u r a c i l , The i n c u b a t i o n was c a r r i e d out f o r 180 .min a f t e r which time the c e l l s were harvested and f r a c t i o n a t e d as described. The r a d i o a c t i v i t i e s of the r e s p e c t i v e f r a c t i o n s were measured and the r e s u l t s p l o t t e d i n f i g u r e 2k. This f i g u r e d e p i c t s the changes of the r a d i o a c t i v i t i e s of the s p e c i f i c f r a c t i o n s , and the r e s u l t s are expressed as the percentage of the t o t a l r a d i o a c t i v i t y at t h a t time. 97 I L —1 ! I I 0 5 10 15 20 25 T U B E NO F i g . -23,- Ribosome p a t t e r n of P. aeruginosa starved f o r 2k hr then reincubated with, l 4 c - u r a c i l f o r 400 min. 98 100 r < 80 --.50 2 CL o 2 CL o T I M E i 14 'Fig.. 24. The i n c o r p o r a t i o n of C - u r a c i l i n t o s t a r v e d c e l l s of P. aeruginosa. C e l l s were starved f o r the i n d i c a t e d times and reincubated w i t h ' v C - u r a c i l as described f o r 180 min. The r a d i o a c t i v i t y was measured i n the supernatant f l u i d (A - A) the c o l d TCA s o l u b l e f r a c t i o n (0 - 0) and the c o l d TCA p r e c i p i t a t e (• - #). The i n c o r p o r a t i o n of the - u r a c i l i s p l o t t e d as the percent of the t o t a l r a d i o -a c t i v i t y at the time o f the sampling. The corresponding r a d i o a c t i v i t i e s of the 3 and the 6 hour f r a c t i o n s were the same. The c o l d TCA s o l u b l e f r a c t i o n stayed a low l e v e l of r a d i o a c t i v i t y throughout the experimental p e r i o d . The r a d i o a c t i v i t y i n the super-nate increased as the time of s t a r v a t i o n was extended beyond 6 hrs and the percentage r a d i o a c t i v i t y i n the TCA p r e c i t i b l e f r a c t i o n , i . e . the KNA, correspondingly decreased. I t i s obvious from f i g u r e 2h t h a t as the s t a r v a t i o n i s prolonged beyond 6 hours the a b i l i t y of the c e l l s t o i n c o r -lk porate C - u r a c i l i s decreased, t h e r e f o r e the a b i l i t y of the c e l l s t o synthesize KNA must be decreased. About t h i s time s e v e r a l disadvantages of t r i s b u f f e r f o r b i o l o g i c a l work w i t h t h i s organism became apparent, and the n e c e s s i t y t o t e s t a l t e r n a t e s t a r v a t i o n b u f f e r s arose; Since phosphate b u f f e r s are by f a r the most adequate i n the pH range used (around pH 7.2) the most l i k e l y choice was NPK b u f f e r c o n s i s t i n g of 0.3$ NH^PO^ and 0.2$ K^HPO^, adjusted to pH 7.2 w i t h 5 N KOH. C e l l s were grown f o r 20 h r s , harvested and resuspended i n t o two suspensions, one i n 0.1 M t r i s HC1, the other i n NKP b u f f e r , and st a r v e d f o r h hours, At the end of the s t a r v a t i o n p e r i o d , the c e l l s were resuspended at 60 mg dry weight of c e l l s / m l as p r e v i o u s l y described and incubated 11+ at 30 C. Ten ucuries of C - u r a c i l was added t o both the t r i s s t a r v e d and KPK starved c e l l s . The i n c o r p o r a t i o n of l 4 the C - u r a c i l i n t o the c e l l s was then determined at 15 min i n t e r v a l s f o r 90 min and p l o t t e d along w i t h the r a d i o a c t i v i t y i n the supernatant f l u i d against time ( F i g . 25). C l e a r l y the c e l l s t h a t have been incubated i n the HPK b u f f e r i n c o r -1k porated the C - u r a c i l to a greater extent and at a much f a s t e r r a t e . Therefore, NPK b u f f e r appears to be more s a t i s f a c t o r y f o r the s t a r v a t i o n of the c e l l s than does t r i s b u f f e r . The medium th a t was used f o r the i n c u b a t i o n of the starved c e l l s contained one percent tryptone. I t would be d e s i r a b l e t o see whether the tryptone could be removed w i t h -l 4 out a f f e c t i n g the a b i l i t y of the c e l l s t o inco r p o r a t e C-u r a c i l . A tryptone f r e e mediumecertainly would be more d e s i r a b l e when one i s attempting to l a b e l the ribosomal p r o t e i n s . The c e l l s were st a r v e d at a conc e n t r a t i o n of 40 mg dry weight of c e l l s / m l i n M T b u f f e r w i t h 0.5$ glucose, o . l $ MgCl^ and v a r i o u s concentrations of tryptone ( F i g . 26), 14 w i t h 1.5 M-curies of C - u r a c i l were added to each f l a s k . 101-28 24 20 to i 2 16 X o |2 8 70 60 50 r o X - 30 20 a. a 20 40 60 80 100 M I N F i g . 25. Comparison of the s t a r v a t i o n b u f f e r s on the r a t e of uptake of ^ C - u r a c i l . P. aeruginosa were sta r v e d i n the i n d i c a t e d b u f f e r s f o r k hr then reincubated w i t h Ike - u r a c i l . The r a d i o a c t i v i t i e s of the whole c e l l s s t arved i n KPK b u f f e r (• - §) and t r i s b u f f e r ( A - A) and of the supernatant f l u i d s s t arved i n NPK b u f f e r (0 - 0) and t r i s b u f f e r ( * - A.) were determined. l2--.5r 0 5 10 15 2 0 MIN Figure 26. The. e f f e c t of the co n c e n t r a t i o n of tryptone on the r a t e of i n c o r p o r a t i o n of. - ^ C - u r a c i l i n t o the RNA of P. aeruginosa. C e l l s were starved f o r k hr i n NPK b u f f e r and reincubated w i t h l ^ C - u r a c i l as described. The f o l l o w i n g concentrations of tryptone were used: 0% (° - 0 ) ; °-2$ (^-&) a n d ' 1% ( # - • ) . Only the c o l d TCA p r e c i t i b l e radio-, a c t i v i t i e s were measured. \ The r a t e s of C - u r a c i l i n c o r p o r a t i o n f o r each of the tryptone concentrations are p l o t t e d i n f i g u r e 26. The removal of tryptone o b v i o u s l y enhances the i n c o r p o r a t i o n of 14 the C - u r a c i l as i s evident from the i n s p e c t i o n of the r a t e s from f i g u r e 26. I n c o n c l u s i o n then, i t appears t h a t f o r the o p t i m a l l 4 r a t e of the i n c o r p o r a t i o n of the C - u r a c i l extensive degradation of the ribosomes i s not needed. VI. Analyses o f P. aeruginosa C e l l Free E x t r a c t s f o r  Ribosomal Precursors 14 1. C e l l s l a b e l l e d w i t h C - u r a c i l The l a b e l l i n g of precursors by the i n c o r p o r a t i o n of r a d i o a c t i v e substrates i s a d i f f i c u l t process, both t h e o r e t i c a l l y and p r a c t i c a l l y . The precursors i n a dynamic system would e x i s t only i n s m a l l q u a n t i t i e s , t h e r e f o r e i t i s necessary t o i n c o r p o r a t e the maximum amount of l a b e l i n the- s h o r t e s t time p o s s i b l e . P r e l i m i n a r y experiments showed t h a t the r e s y n t h e s i s of ribosomes i s a r e l a t i v e l y long process. The s t a r v a t i o n and r e i n c u b a t i o n media developed i n the previous s e c t i o n were used. A twelve hour c u l t u r e of P. aeruginosa was sta r v e d at 4 x growth c o n c e n t r a t i o n f o r 4 hours i n NPK 14 b u f f e r , then reincubated w i t h 15 ucuries of C - u r a c i l at 2 x the o r i g i n a l c e l l d e n s i t y . This corresponds t o a dry weight of c e l l s of 40 mg/ml and a t o t a l volume of 100 ml c u l t u r e . The c e l l s were reincubated at 25 C and samples were taken at 6o, 80, 100 and 120 min. The ribosome patte r n s are recorded i n f i g u r e s 27, 28, 29 and 30, r e s p e c t i v e l y f o r the 60, 80, 100 and 120 min c e l l s . The 60 min ribosome p a t t e r n ( F i g . 27) showed t h a t no ribosomes were l a b e l l e d at t h a t time, although there was a s m a l l r a d i o a c t i v e peak immediately i n f r o n t of the 30S ribosome peak (peak A) which was l o c a t e d by measuring the abs o r p t i o n of the f r a c t i o n a t e d gradient at 26o mu. An i n s p e c t i o n of the 80 min ribosome p a t t e r n ( F i g . 28) showed t h a t there was indeed a r a d i o a c t i v e peak i n f r o n t of the 3 0 S ribosome, and one j u s t a f t e r the 30S ribosome (peaks A and B i n F i g . 28) There i s a r a d i o a c t i v e peak (peak C) i n f r o n t of the 50S ribosome peak a l s o . I n a d d i t i o n t o these precursor peaks, a s l i g h t amount of r a d i o a c t i v i t y i n the ribosomal regions was a l s o detected. The l a b e l l i n g of the ribosomes i s much 0-6 r to-5 o ID (VI 0-4 < o 0-3 or o < 0 * I 0 8 12 16 20 TUBE NO 24 24 20 16 8 4 28 32 F i g . ' 2 7 . Ribosome p a t t e r n of'P. aeruginosa reincubated w i t h - u r a c i l f o r 60 min. The J ribosomes are l o c a t e d by absorption at 260 mu; 'the ribosomal precursors, by' r a d i o a c t i v i t y . T h e ' f r a c t i o n s were d i l u t e d l / l O f o r absorbance (0 - 0) and 3/4 f o r r a d i o a c t i v i t y measurement ( § - • ) . ' . ro i O 12 ~* Q_ O O I I I I I I I I I 0 5 10 15 20 25 30 35 40 F i g . 28. Ribosome p a t t e r n of P. aeruginosa r e i n c u b a t e d w i t h . C - u r a c i l f o r .80 min. See legend f o r Fig. ' 2 7 . I 1 1 — 1 I I I I J 0 4 8 12 16 20 24 28 32 TUBE NO F i g - . 29.. Ribosome p a t t e r n of E. aeruginosa reincubated w i t h - C - u r a c i l f o r 100 min. See legend f o r Fig. '27,. -. • . T U B E NO F i g . 30. Ribosome p a t t e r n of P. aeruginosa r e i n c u b a t e d w i t h C - u r a c i l ; for- 120 min. f gee legend f o r - F i g . 27. more pronounced i n the 100 min ribosome p a t t e r n . Peaks A, B and C ( i n Fig.. 29) are s t i l l apparent, but the 30S and 50S l a b e l l e d ribosome peaks were a l s o r e s o l v e d . An a d d i t i o n a l peak co u l d be seen between the 30S and the 50S ribosomal regions (peak D). The ribosomal peaks were qu i t e pronouncedly l a b e l l e d at 120 min as apparent from f i g u r e 30. There could p o s s i b l y be two ribosomal p r e -cursors i n f r o n t of the 30S ribosome, peaks A and E. The r a d i o a c t i v i t y around the 30S regions (peak A) was one tube ahead of the ribosomes. This s e r i e s of experiments demonstrated the gradual i n c o r p o r a t i o n of l a b e l i n t o the ribosomes through the time course stud i e d . S e v e r a l ribosomal precursors could a l s o be demonstrated. Because the lower molecular weight ribosomal precursors are masked by mKNA and tRNA, o n l y the l a r g e r precursors w i l l be considered. I n a l l cases the r a d i o a c t i v i t y i s l o c a t e d j u s t ahead of the 30S ribosomes. This r e g i o n appeared t o be heterogeneous i n a l l the r i b o -somes p a t t e r n s , suggesting t h a t these peaks contained p o s s i b l e 30S and 50S precursors, as w e l l as mature 30S ribosomes. I n the 80 and the 100 min ribosome p a t t e r n s there were two common peaks between the 30S and the 50S ribosomal regions. One was j u s t a f t e r the 30S ribosomes, (peak B), and the other, j u s t before the 50S ribosome (peak C). The 100 min p a t t e r n contained an a d d i t i o n a l r a d i o a c t i v e peak (peak D) about halfway between the 30S and the 50S ribosomes. This peak (peak D) was a l s o apparent i n the 120 min ribosome p a t t e r n ( F i g . 28, 29 and 30). ' The sedimentation values t h a t are quoted i n t h i s s e c t i o n are merely d e s c r i p t i v e , i . e . they are not a r e s u l t of u l t r a -c e n t r i f u g a t i o n a l analyses, but are designated by t h e i r p o s i t i o n i n the sucrose d e n s i t y gradient. The f o l l o w i n g precursors appeared i n the CPXs analyzed: 25S, 28s, 30S, 32S, 43S and k&S ( i n a d d i t i o n t o 30S and 50S ribosomes). The f l o w of l a b e l would probably be: 25S (peak E) + 28S (peak A) -> 30S 32S (peak B) k3S (peak D) •» kQS (peak C) 4 50S Ik 2. Analyses of c e l l s doubly l a b e l l e d by C - u r a c i l and - p r o t e i n h y d r o l y z a t e f o r ribosomal precursors a. The analyses of doubly l a b e l l e d c e l l s The r e s u l t s of the previous s e c t i o n suggested t h a t the sampling of c e l l suspensions from 100 min on would c o n t a i n both l a b e l l e d ribosomal precursors as w e l l as mature ribosomes. The use of double l a b e l l i n g should y i e l d v a l u a b l e i n f o r m a t i o n about the time of the a d d i t i o n of the p r o t e i n t o the ribosomes. These experiments were c a r r i e d out i n a manner s i m i l a r to t h a t of the previous s e c t i o n . The f i n a l i n c u b a t i o n mixture (100 ml) contained hO mg dry weight c e l l s / m l w i t h 20 lk 3 u c u r i e s of C - u r a c i l and 50 ucuries of H-protein h y d r o l y z a t e . The c e l l s were analyzed f o r ribosomal precursors and ribosomes as described (absorbance f o r ribosomes, r a d i o a c t i v i t y f o r ribosomal p r e c u r s o r s ) . The c e l l suspension was analyzed at 100, 120 and 150 min. An examination of the 100 min ribosome p a t t e r n r e v e a l e d t h a t lk 3 there were s m a l l amounts of both the C and the H isotopes i n c o r p o r a t e d around the 30S and the 50S ribosome peaks. The r a d i o a c t i v i t y appeared t o be heterologous around the ribosomal regions and t o be composed of both ribosomal precursors and lk ribosomes. I n f r o n t of the 30S ribosomes, a C peak analogous to the A peaks i n the previous s e r i e s ( F i g . 31) "was detected and designated A \ 3 A s l i g h t i n f l e c t i o n appeared i n the H p r o f i l e immediately ahead of peak A \ The r e g i o n around the 50S ribosomes, ! 0 5 10 15 2 0 2 5 30 35 I . TUBE NO • ' . Fig.-31. Ribosome p a t t e r n of P. aeruginosa reincubated w i t h C - u r a c i l and H-protein . - j h y d r o l y z a t e f o r 100: min. See legend f o r F i g . 27. Peaks are not l a b e l l e d to -; " • • { ' . a v o i d f u r t h e r confusion i n an already crowded diagram. although'not r e s o l v e d too w e l l , d i d i n d i c a t e the existence of a peak (analogous to peak C F i g . 28) and of 5°S ribosomes. There was a l s o a t r i t i u m peak corresponding t o peak c \ The 120 min ribosome p a t t e r n ( F i g . 32) showed Ik the same p r o f i l e except f o r b e t t e r r e s o l u t i o n i n the C-3 p r o f i l e . The H - p r o f i l e was not as w e l l r e s o l v e d . There 3 1 was an i n f l e c t i o n i n the H p r o f i l e i n f r o n of peak A . The 50S r e g i o n appeared to c o n t a i n 2 peaks, one analogous t o peak C i n F i g . 28, c a l l e d C"*" and a 50S ribosome peak. The 50S r a d i o a c t i v e peak was one tube heavier than the corresponding ribosome peak l o c a t e d by u.v. absorbance. The 150 min ribosome p a t t e r n contained a w e l l r e s o l v e d peak A"*" as w e l l as 30S ribosomes ( F i g . 33). Peak A"1" and the 30S ribosomes contained both l a b e l s . There a l s o appeared a w e l l r e s o l v e d peak C"'", and the ribosomal peak, c o n t a i n i n g both l a b e l s , was again s l i g h t l y h e a v i e r than the c o r r e s -ponding non-radioactive ribosome. b. I s o l a t i o n of the regions around the 30S and 50S ribosomal peaks and the analyses of these f r a c t i o n s by sucrose d e n s i t y gradient c e n t r i -f u g a t i o n . 0 10 15 2 0 2 5 T U B E NO 3 0 3 5 F i g . 32.• Ribosome p a t t e r n o f P. aeruginosa r e i n c u b a t e d with. ^ C - u r a c i l and 3 H - p r o t e i n h y d r o l y z a t e f o r 120 min. See legend f o r F i g . 27. 0 10 2 0 3 0 T U B E NO F i g . 33. Ribosome p a t t e r n of P. aeruginosa reincubated w i t h C-uracil and 3pl-protein h y d r o l y z a t e f o r 150 min. See legend f o r F i g . 27.. \jt. The o b j e c t i v e of the f o l l o w i n g s e r i e s of experiments was to o b t a i n b e t t e r r e s o l u t i o n around the ribosomal r e g i o n s . The previous experiment was repeated but i n order t o achieve b e t t e r l a b e l l i n g of the ribosomes, 3 100 ucuries of H-protein hydrolyzate and 25 pcuries of l ) +C - u r a c i l was added t o the 100 ml, 40 mg dry weight c e l l s / m l suspension. (33 ml samples were taken at 100, 120 and 150 min concentrated 10 x t o y i e l d 3.3 ml CPX). The ribosomes were l o c a t e d by monitoring the f r a c t i o n s on a spectrophotometer and the regions depicted i n f i g u r e 34 were pooled, adjusted t o 0.1 M MgCl^ and c e n t r i f u g e d at 100,000 g f o r 4 h r . The p e l l e t s were resuspended i n 0.05 M t r i s HC1 (pH 7 -2) and f r a c t i o n a t e d by c e n t r i f u g a t i o n i n a sucrose d e n s i t y g r a d i e n t . The a n a l y s i s of the 30S r e g i o n of the 100 min sample (3D-100-30S described i n F i g . 34, 35) i n d i c a t e d t h a t there 14 was a considerable amount of C - l a b e l l e d 30S ribosomes present and t h a t there were two i n f l e c t i o n s suggesting 14 C - l a b e l l e d precursors l i g h t e r than 30S. There was one 3. 14 ""H-labelled precursor corresponding t o the heavier C-l a b e l l e d precursor. There appeared to be a ^ H - l a b e l l e d peak immediately a f t e r the 30S peak, and undoubtedly t h i s 34. Ribosome patte r n s of P. aeruginosa to separate ribosomal regions.^ The c e l l s were reincubated w i t h 14^ 1 - u r a c i l and 3H-protein hydrolyzate f o r 100 min (0 - 0) , 120 min (A - A ) and 150 min ( t - • ) . The contents of the f o l l o w i n g tubes were pooled and the ribosomal m a t e r i a l i s o l a t e d from: Code name 100 .min tubes 7-18 19-26 f o r 30S ribosomes 50S ribosomes 3D-100-30S 3D-100-50s 120 min tubes 8-20 21-30 f o r 30S ribosomes 50S ribosomes 3D-120-30S 3D-120-50S 150 min tubes 7-18 19-30 f o r 30S ribosomes 50S ribosomes 3D-150-3OS 3D-150-50S These f r a c t i o n s were c e n t r i f u g e d i n 0.1 M MgCl^ at 110,000 g f o r 4 hr and resuspended i n 0.05 M t r i s HC1 (pH 7.2) w i t h 10-4 M MgCl 2. The code numbers r e f e r t o these resuspended f r a c t i o n s . 0 5 10 15 20 25 T U B E NO F i g . 35. Ribosome-pattern., of 3D-100-30S to resolve ribosomal precursors. The 30S ribosome (or the 50S ribosome f o r the 50S series) i s -located by absorption at 260 mu. and the precursors by the l ^ r j _ and the % l a b e l l e d p r o f i l e . Code described i n Fig. 3k. H CO "^H-labelled m a t e r i a l contained some ^ H - l a b e l l e d 30S ribosomes as w e l l as some precursors en route to 5°S ribosomes. Further down the gradient a double l a b e l l e d precursor of the 50S ribosomes was evident. I n the 3D-120-30S ribosome p a t t e r n a somewhat d i f f e r e n t d i s t r i b u t i o n of the l a b e l occurred ( F i g . 36). There Ik was o n l y one C - l a b e l l e d precursor, but there were 3 three H - l a b e l l e d precursors. Again although there 3 was not a d i s c r e t e H peak d i r e c t l y at the l o c a t i o n of 3 the ribosomes, there were two H peaks on e i t h e r s i d e , 3 which probably masked the 30S H - l a b e l l e d ribosomes. 3 The H peak j u s t a f t e r the 30S ribosome peak migrated towards the 5°S ribosome i n r e l a t i o n t o the corresponding precursor i n the 3D-100-30S sample. The double l a b e l l e d p recursor towards the bottom of the gradient was again evident although i n a smaller q u a n t i t y . The 30-150-30S 1 ribosome p a t t e r n ( F i g . 37) showed t h a t the bu l k of the r a d i o a c t i v i t y was present as the 3°S ribosomes. There was o n l y a s m a l l peak i n f r o n t of the 30S ribosomes and t h i s was doubly l a b e l l e d . A comparison of the 3°S ribosome p a t t e r n s shows then that:-... i ) there was a gradual b u i l d u p of both " ^ C - l a b e l l e d 3 and H - l a b e l l e d ribosomes i n the c e l l s during the p e r i o d of the experiment. i i ) there appeared to be a d i f f e r e n c e i n the r a t e of 14 3 accumulation of 30S C - l a b e l l e d ribosomes and H - l a b e l l e d ribosomes. Ribosomes comprised a l a r g e p o r t i o n of the l4 C - l a b e l even as e a r l y as 100 min, whereas precursor peaks 3 masked the H - l a b e l l e d ribosome peak u n t i l 150 min. i i i ) there appeared t o be a d i f f e r e n c e i n time between Ik the accumulation and m i g r a t i o n of the C - l a b e l l e d r i b o -3 somal precursors and the H - l a b e l l e d ribosomal p r e c u r s o r s . An example of t h i s was the m i g r a t i o n of the ^H-peak from j u s t behind the 30S ribosome i n the 100 min sample towards the 50S ribosomes i n the 120 min sample and then completely i n t o the 50S ribosomes at 150 min. i v ) t h i s method of f r a c t i o n a t i o n r e v e a l e d a number of precursors: N.B. These S. values are o n l y designated from the p o s i t i o n of the peaks i n the g r a d i e n t . 100 min l6s 23S 30S kOS . 50S % 23s 25s 30s 32s 4os 50s 120 min "^C 20S 23S 30S 3^S 40S 50S 3H 20S 23S 28S 3kS kOS 50S 14 150 min C 23S 30S 23S 30S Although these S values are t e n t a t i v e they do i n d i c a t e the m i g r a t i o n of the l a b e l through the r i b o -somal precursors and i n t o the ribosomes. The ribosome p a t t e r n s of the 50S s e r i e s are depicte d i n f i g u r e s 38, 39 and kO f o r the 3D-100-50S, 3D-120-50S and the 3D-150-50S ribosome p a t t e r n s r e s p e c t i v e l y . The 3D-100-50S ribosome p a t t e r n ( F i g . 38) showed two precursors en route t o the 50S ribosomes lk 3 f o r both the C - l a b e l l e d and the H - l a b e l l e d p r o f i l e s . ll+ I n a d d i t i o n there was a w e l l r e s o l v e d C - l a b e l l e d and a 3 H - l a b e l l e d peak at the l o c a t i o n of the 50S ribosomes. I n the 3 D-120-50S ribosome p a t t e r n ( F i g . 39)? o n l y one precursor was apparent, and i t migrated c l o s e r to the 50S ribosome. Only a t r a c e amount of t h i s precursor was evident i n the 3D-150-50S ribosome p a t t e r n ( F i g . kO). However, the H peak i n f i g u r e 3 ^ i s one tube l i g h t e r lk than the C and ribosome peaks; t h e r e f o r e these could be an a d d i t i o n a l precursor. A comparison of these ribosome p a t t e r n s i n d i c a t e d : i ) there was an increase i n the l a b e l l i n g of the ribosome throughout the time course of the experiment. i i ) there i s a d i f f e r e n c e i n the r a t e of the 0 5 10 15 2 0 2 5 TUBE NO Fig-. 38. . Ribosome p a t t e r n of 3 D - 1 0 0 - § 0 S to r e s o l v e ribosomal .pre-cursors. See.legend f o r ' F i g . 35. 125 T U B E NO F i g . 39.. • Ribosome p a t t e r n of 3D-120-50S t o r e s o l v e ribosomal precursors. See legend f o r F i g . 35. 126 i 4 3 accumulation of the C - l a b e l l e d and the H - l a b e l l e d l 4 50S ribosomes. C - l a b e l l e d ribosomes as i n the 30S 3 s e r i e s , accumulate before the H - l a b e l l e d ribosomes do. i i i ) there were not as many precursors apparent f o r the 50S ribosomes as f o r the 30S. Nevertheless two Ik 3 C - l a b e l l e d and three H - l a b e l l e d precursors were detected en route t o the 50S ribosome i n the 3D-100-5QS sample. These precursors were towards the top of the gradient and could be contamination from l i g h t e r weight pre c u r s o r s . I n the 120 min sample there was a 45S 14 3, precursor which was l a b e l l e d both by C and a and 3 p o s s i b l y a 48S H - l a b e l l e d precursor. A t r a c e of t h i s 45S precursor was d e t e c t i b l e i n the 150 min ribosome p a t t e r n . c. The analyses of the ribosomal RNA of the regions i s o l a t e d around the 30S and 50S ribosomal peaks Hecht and Woese (1968) demonstrated t h a t when a mixture of normal rRNA and e i t h e r r a p i d l y l a b e l l e d rRNA or CM RNA i s analyzed by acrylamide g e l e l e c t r o p h o r e s i s , the CM RNA and the r a p i d l y l a b e l l e d RNA had slower m o b i l i t i e s i n the g e l than d i d the normal rRNA. Hecht and Woese (1968) i n t e r p r e t e d these d i f f e r e n c e s i n m o b i l i t i e s to be a d i f f e r e n c e i n s i z e between the precursor and the normal rRNA. This d i f f e r e n c e i s greater i n the l 6 s rRNA s e r i e s , where the precursor RNA i s 130,000 daltons heavier than the corresponding mature rRNA. M a n g i a r o t t i et a l . (1968) on the other hand, described the process of ribosome synthesis as an a d d i t i o n of p r o t e i n t o p o l y n u c l e o t i d e chains whose primary s t r u c t u r e i s s t i l l incomplete at t h i s time. The ribosomal RNAs were f r a c t i o n a t e d t h e r e f o r e both by sucrose d e n s i t y gradient c e n t r i f u g a t i o n and by d i s c g e l e l e c t r o p h o r e s i s (described i n a l a t e r s e c t i o n ) . The samples used i n the previous s e r i e s of e x p e r i -ments 3D-100-30S and -50S; 3D-120-30S and -50S and 3D-150-30S and - 5 0 S were adjusted to co n t a i n 0.5$ Duponol C. Sucrose d e n s i t y gradients of k.9 ml co n t a i n i n g 0.5$ Duponol.C were prepared. The samples were f r a c t i o n a t e d and analyzed as described. The r e s u l t s of the 30S s e r i e s w i l l be examined f i r s t . The p r o f i l e of the 100 min 30S rRNA was depict e d i n f i g u r e 1+1. I n a d d i t i o n to the l 6 s rENA ( l o c a t e d by absorbance at 260 mu) there appeared t o be two other species of rENA, one of 12S the other 9S. The 120 min 30S sample a l s o contained three species of rENA. The r a t i o of the l6s rENA to the l i g h t e r molecular weight rRNAs was however greater i n the 120 min 30S sample than i n the 100 min 30S sample ( F i g . 1+2). The 150 min 30S sample contained o n l y one peak, the l6s rRNA and on l y t r a c e s of the lower molecular weight rENAs remained ( F i g . 1+3). The m i g r a t i o n of the l a b e l through the 9 s a n d the 12S rENAs i n t o the l 6 s rENA and the accumulation of the 16S rENA at the expense of the 9S and 12S rENAs was c l e a r l y demonstrated. Both the 3D-100-30S and the 3D-120-30S 14 ribosome p a t t e r n s ( F i g . 35, 36) contained two C - l a b e l l e d precursors to the 30S ribosome. P o s s i b l y these precursors contained the 9S and the 12S rENAs. There were c o r r e s -ponding ^ H - l a b e l l e d peaks i n the 120 and the 100 min ribosome p a t t e r n s , t h e r e f o r e i t appeared l i k e l y t h a t the a d d i t i o n of the ribosomal p r o t e i n to the ribosomes commenced p r i o r to the completion of the p o l y n u c l e o t i d e chain of the rRNA. ? 1-0 - i 1250 9 F i g . 41. Sedimentation p r o f i l e of the rRNA. from 3D-100-30S. The l 6 s rRNA (23S RNA f o r the 50S s e r i e s ) i s l o c a t e d by i t s absorbance at 260 mu. The rRNA from the ribosomal pre-cursors i s l o c a t e d by i t s r a d i o a c t i v i t y . Code i s described i n F i g ; 34. • 1 3 1 132 F i g . 43.-' Sedimentation p r o f i l e of the rRNA from 3B-150-30S." See legend f o r ' Fig..4 1 . The examination of the rKNAs from the 1 0 0 min, 1 2 0 min and 1 5 0 min 50S samples r e v e a l e d t h a t most of the lk C - l a b e l r e s i d e d i n the 23S rKNA i n a l l three samples. There were s m a l l peaks of r a d i o a c t i v i t y i n f r o n t of the 23S rKNAs f o r the 1 0 0 min ( F i g . kk) sample, which could be l8S prec u r s o r s . An a d d i t i o n a l 2 0 S precursor appeared i n the 1 2 0 min sample ( F i g . 1+5). However, these p o s s i b l e precursors had e s s e n t i a l l y disappeared by 150 min ( F i g . 1+6). The ribosome patt e r n s f o r t h i s s e r i e s ( F i g . 38, 39 and 1+0) demonstrated t h a t most of the l a b e l was present i n the 50S ribosomes. The observation t h a t most of the rRNA occurred as the 23S rKNA was i n good accord w i t h e a r l i e r observations t h a t most of the l a b e l was present as the 5 0 S ribosomes. The ribosomal precursors c o u l d p o s s i b l y c o n t a i n the incomplete ribosomal rKNA. d. Analyses of the ribosomal KNAs on d i s c g e l e l e c t r o p h o r e s i s The system used to separate the ribosomal KNAs has already been described. I n order to f i r s t a s c e r t a i n whether the ribosomes and the ribosomal KNAs could be 0 5 15 20 25 TUBE NO F i g . kk. Sedimentation p r o f i l e of the rRNA from 3D-100-50S. See legend f o r F i g . .kl. F i g . 45. Sedimentation p r o f i l e of the rKNA from 3D-120-50S. See legend f o r F i g . 4l. • 136; 9* 20 r - i 10'® A 8 ro o H 6 H 4 H 2 10 15 TUBE NO 25 2 CL O F i g . 1+6V Sedimentation p r o f i l e of the rKNA from 3D-150-50S. .See legend f o r F i g . kl. r e s o l v e d by the d i s c g e l e l e c t r o p h o r e s i s , the p r e l i m i n a r y l4 experiments compared the m o b i l i t i e s of C - u r a c i l l a b e l l e d l4 ribosomes, C-protein h y d r o l y z a t e l a b e l l e d ribosomes and 14 C - u r a c i l l a b e l l e d ribosomal RNAs. The ribosomes were indeed w e l l separated from the ribosomal RNAs. Fi g u r e 47 14 shows t h a t the C - u r a c i l l a b e l l e d ribosomes are w e l l r e s o l v e d , the 50S being at tube f i v e , the 30S at tube ten. 14 The C-protein hydr o l y z a t e l a b e l l e d ribosomes have the same m o b i l i t i e s ( F i g . 49). I n f a c t t h i s experiment confirms the r e s u l t s i n f i g u r e 6 which showed the photo-graph of the s t a i n e d ribosomes separated by the g e l . The ribosomal KNAs are r e s o l v e d as w e l l ( F i g . 48). The ^ C 23S RNA i s l o c a t e d at tube 18; the l6s RNA, at tube 21. The examination of the d i s t r i b u t i o n of the rRNAs i n the 100 min sample ( F i g . 50) showed th a t there was h a r d l y any mature rRNA present. The major peak was l o c a t e d at tube 30 f o r both the 30S and the 50S samples. There appeared t o be some heterologous RNA peaks dispersed throughout the g e l . The 120 min 30S sample had a s m a l l amount of 23S RNA ( F i g . 51), and some RNA between the 23S and the major l6s RNA peak. A peak disappeared at tube 3 2 , s l i g h t l y ahead of the l6s RNA peak. The 320 min 50S .. 1+7. D i s c e l e c t r o p h o r e t i c p a t t e r n of C u r a c i l l a b e l l e d ribosomes.-O O O O r 8 0 0 0 -6 0 0 0 -4 0 0 0 -2 0 0 0 2 3 S 10 2 0 T U B E NO F i g . 1+8. D i s c e l e c t r o p h o r e t i c pattern, of u r a c i l l a b e l l e d rRNA. • 5 0 0 r 4 0 0 h 3 0 0 h 2 0 0 h 1 0 0 TUBE NO F i g . 53." D i s c electroxihore.tic p a t t e r n of?, F. aeruginosa c e l l f r e e e x t r a c t s l a b e l l e d w i t b - 1 ^ - u r a c i l f o r 100. min.-' 140 TUBE NO •' F i g . 50. • Disc e l e c t r o p h o r e t i c p a t t e r n of the rRNAs from the. 3D-100 series.' • 30S (0 - 0) and 50S (•---•). 2 3 S 2 0 3 0 T U B E N O . •Fig." "51.., Disc electrophoretic pattern- -cf the- rRNAs from the 3D-120/ 'series. 30S, (0 - 0) and ,5QS , (• Fig . 52. Disc electrophoretic pattern of ' :;. 'the rRNAs\ from-the-'.'3D-150 series''.. • • ) • ; 30S (0 - 0) and 503. (• - • ) . sample contained mostly 23S RNA (Fig. 52), but also contained some heavier RNA and some l6s RNA along with two faster moving peaks. The 150 min 30S sample (Fig. 52) contained mostly l6s RNA, along with some 23S RNA and a s l i g h t l y slower and a s l i g h t l y faster peak. The 150 min 50S sample (Fig. 52) contained mostly 23S RNA with some slow moving radioactive peaks. The analyses of the rKNAs showed more heterogen e i t y when the KNAs were fractionated on acrylamide gel" electrophoresis. The nature of the peaks other than the l6s and the 23S KNAs was not understood. Although t h i s explanation i s speculative, i t i s possible that the rKNAs could f o l d into t h e i r hydrogen bonded t e r t i a r y conformation before a l l the primary sequence i s completed. This would explain why more of the t o t a l rKNA existed as l6s and 23S rRNa i n the sucrose density gradient fractionated rKNAs (even at 100 min, Fig . hi, a s i g n i f i c a n t f r a c t i o n of the rRNA is o l a t e d from the 30S f r a c t i o n occurred as l6s rRNA, and the majority of the rKNA from the 50S f r a c t i o n occurred as 23S rRNA, F i g . hh). e. A n a l y s i s of the ribosomal precursors on d i s c g e l e l e c t r o p h o r e s i s The f o l l o w i n g samples were a l s o analyzed by d i s c g e l e l e c t r o p h o r e s i s : The c e l l f r e e e x t r a c t s t h a t the 3D-100-30S and -50S , the 3D-120-30S and -50S and the 3D-150-30S and -50S f r a c t i o n s were i s o l a t e d and f r e e d of membranes by c e n t r i -f u g a t i o n at 22,000 g f o r 30 min at 0 C. A s o l u t i o n of hQPJo g l y c e r o l was added t o the c a r e f u l l y removed super-natant f l u i d and the samples were f r a c t i o n a t e d by d i s c g e l e l e c t r o p h o r e s i s . The d i s t r i b u t i o n of the r a d i o a c t i v i t y of the 100 min supernatant f l u i d ( F i g . 53) showed some 30S and 50S r i b o -somes, as w e l l as two peaks which migrated between the ribosomes and the ribosomal RNAs. There was evidence of dispersed r a d i o a c t i v i t y around the r e g i o n of the rRNAs. The 120 min sample contained more r a d i o a c t i v i t y i n the ribosomes, and a l s o contained the two rRNAs along • w i t h other r a d i o a c t i v e peaks i n the same area ( F i g . 54). The same p a t t e r n was evident i n the 150 min sample ( F i g . 54) The o n l y way these peaks can be c h a r a c t e r i z e d at t h i s time i s by t h e i r r e l a t i o n to the ribosome standards and rKNA standards t h a t have been separated by t h i s system.. Although t h i s i s only a d e s c r i p t i v e c h a r a c t e r -i z a t i o n nevertheless there are some i n t e r e s t i n g observ-a t i o n s . ' There are, of course, more low molecular weight substances i n these g e l s because a 2 0 , 0 0 0 g supernatant f l u i d was used as the m a t e r i a l t o be f r a c t i o n a t e d . There was evidence of l a b e l l e d precursors m i g r a t i n g between the ribosomal regions and the KNA regions. I t i s not p o s s i b l e to say u n e q u i v o c a l l y t h a t the peaks w i t h m o b i l i t i e s s i m i l a r to those of the rKNAs were i n f a c t . rKNAs. They could p o s s i b l y be r i b o n u c l e o p r o t e i n p a r t i c l e s w i t h the m o b i l i t y of rKNA. I t i s p o s s i b l e of course t o v i s u a l i z e the matur-a t i o n of the ribosomes as a process during which the immature ribosome could e x i s t i n the t e r t i a r y s t r u c t u r e of the normal ribosome p r i o r to the completion primary s t r u c t u r e . I n other words, there could be precursor ribosomes w i t h the hydrodynamic p r o p e r t i e s of mature ribosomes, but whose primary s t r u c t u r e ( l e n g t h of p o l y -n u c l e o t i d e chain and ribosomal p r o t e i n complement) is'', not yet completed. I n c o n c l u s i o n then i t was p o s s i b l e t o c h a r a c t e r i z e some ribosomal precursors, d e s c r i p t i v e l y , at l e a s t . The f l o w of the l a b e l was demonstrated through • these; precursors i n t o ribosomes. I t was p o s s i b l e t o demonstrate t h a t the ribosomal precursors as w e l l as the ribosomal KKAs could be f r a c t -ionated by d i s c g e l e l e c t r o p h o r e s i s as w e l l as by sucrose d e n s i t y gradient c e n t r i f u g a t i o n . GENERALIZED DISCUSSION The e l u c i d a t i o n of the sequence of events l e a d i n g t o the u l t i m a t e b i o s y n t h e s i s of mature ribosomes i s made d i f f i c u l t by s e v e r a l f a c t o r s . F i r s t l y one should study the l a b e l l i n g of the ribosomal precursors and t h e i r maturation i n t o ribosomes. By the v e r y f a c t t h a t i n t e r -mediate compounds i n an enzymatic sequence are present i n o n l y minor amounts our methods of d e t e c t i o n and i d e n t i f i c a t i o n are taxed to t h e i r l i m i t s . Secondly these intermediates should be i s o l a t e d i n p r e p a r a t i v e q u a n t i t i e s i n order to perform physico-chemical and b i o -l o g i c a l c h a r a c t e r i z a t i o n . Again because of the minute amounts of m a t e r i a l a v a i l a b l e these are d i f f i c u l t o b j e c t i v e s . The precursors t h a t have been described i n t h i s study were de r i v e d by sucrose d e n s i t y gradient c e n t r i -f u g a t i o n and by polyacrylamide g e l e l e c t r o p h o r e s i s . The p i c t u r e t h a t emerged from the analyses of sucrose d e n s i t y g r adients i n d i c a t e d t h a t there were a number of precursors i n the process of being converted i n t o the ribosomes. Even though the S values t o these i n t e r -mediates of ribosome synthesis were designated so l e l y by t h e i r r e l a t i v e positions on the sucrose density gradients, some interesting correlations can be made. Precursors were te n t a t i v e l y designated as l 6 s , 20S, 23S, 25S, 28S, 30S, 32S, 34S, 43S, 48S and 50S. Although no further characterization of these pre-cursors were carried out, t e n t a t i v e l y they can be grouped into two pathways: l6s 20S y 23S > 25S > 28S •> 30S ribosomes 23S * 25S -b 28S v32S * 34S 43S ^ 48S 50S ribosomes Iwabuchi et a l . (1965) proposed the following sequence: 16S rRNA * 18S » 28S * 30S ribosome 23S rRNA -> 25S } 32S 38S > 43S -> 50S ribosome Because the l8S and 28S precursor p a r t i c l e s were similar to the l6s rRNA containing CM and FU p a r t i c l e s , respectively, Iwabuchi et a l . (1965) placed them into the sequence of 30S ribosome synthesis. Simiarly the 25S and the 32S p a r t i c l e s were c l a s s i f i e d as precursors to the 50S ribosome. The sequence proposed i n t h i s study of course cannot r e l y on CM or FU p a r t i c l e s , therefore the p a r t i c l e s that could be precursors to either ribosome by S value considerations, were placed i n both sequences. I t i s d i f f i c u l t to v i s u a l i z e the process of r i b o -somal biosynthesis as the enlargement of the molecule by material that i s equivalent to several S values. The p o s s i b i l i t y i s of course that there are undetected precursors. The precursors that have been described so far i n the l i t e r a t u r e could have arisen by l i m i t i n g material required to progress to the next step. The d i f f i c u l t y with t h i s type of description of the ribosomal precursors i s that the S values may indicate changes i n the conformation of the maturing ribosome rather than actual additions to i t equivalent to the change i n S value. For example, the t r a n s i t i o n from 34s to 43S could be caused by the addition of a ribosomal protein of approximately 1 0 , 0 0 0 daltons which would cause a ti g h t e r folding of the ribosome precursor, r e s u l t i n g i n the change i n S values. There was good evidence that the RNA i t s e l f under-goes changes i n the course of the addition of protein to the ribosome. I t was shown that 1 2 S and 9S rRNAs occur i n appreciable quantities inthe 1 0 0 and 1 2 0 min samples. The f l o w of l a b e l i s a l s o demonstrated through these rRNAs t o the l 6 s and p o s s i b l y the 23S rRNAs by the accumulation of l a b e l i n the l 6 s and 23S rRNAs at the expense of the precursor rRNAs. Of course i t cannot be c o n c l u s i v e l y s a i d t h a t the 9S and 12S rRNAs were RNAs w i t h incomplete p o l y -n u c l e o t i d e sequences t o which p r o t e i n could be added, (analogous t o the system described by M a n g i a r o t t i et a l . 1968). They could p o s s i b l y be l 6 s or- 23S RNA i n an 'unwound' conformation, and i t c e r t a i n l y i s a good p o s s i b i l i t y t h a t the a d d i t i o n of the p r o t e i n c o u l d cause the rRNA to assume a t i g h t e r conformation. The i n t e r p r e t a t i o n of the acrylamide g e l e l e c t r o -phoresis r e s u l t s i s very s p e c u l a t i v e , due t o the f a c t t h a t the o n l y way the peaks can be described at t h i s time i s by t h e i r l o c a t i o n r e l a t i v e t o the 30S and 50S ribosomes, and the l 6 s and 23S rRNAs. The assumption t h a t the g e l e l e c t r o p h o r e s i s i n t h i s case i s an accurate d e s c r i p t i o n of the molecular s i z e s of the precursors i s somewhat s p e c u l a t i v e because there could very w e l l be charge d i f f e r e n c e s during the maturation of the ribosomes. The d i s t r i b u t i o n of the rKNAs i n t h i s case gave an extremely heterologous p i c t u r e , although peaks w i t h considerable r a d i o a c t i v i t y and w i t h m o b i l i t i e s f a s t e r than t h a t of the l6s rRNA c o n s i s t e n t l y appeared. In a d d i t i o n there were i n d i c a t i o n s of rKNAs w i t h m o b i l i t i e s slower than the 23S rRNA. Slow moving shoulders on the l6s" rRNAs also appeared. Therefore there could be a stage i n the maturation of the rRNAs at which the RNA i s a c t u a l l y l a r g e r than the l6s and the 2 3 S rRNAs (Hecht and Woese, 1968). This could a r i s e by the maturation of the 9S and the 12S rRNAs t o the l6s and the 23S rKNAs. Sucrose d e n s i t y gradient, c e n t r i f u g a t i o n i n d i c a t e d t h a t the CM RNAs have s e d i -mentation constants of l6s amd 23S, even though they appear t o be l a r g e r when analyzed by g e l e l e c t r o p h o r e s i s (Hecht and Woese, 1968). The f a c t t h a t these d i f f e r e n c e s could a r i s e by d i f f e r e n c e s i n charge can not be con-c l u s i v e l y r u l e d out at t h i s time. As i s evident i n the l i s t of references an enormous-volume of new m a t e r i l has been presented i n t h i s f i e l d . With the accumulation of t h i s wealth of in f o r m a t i o n , b e t t e r and more meaningful c o r r e l a t i o n s w i l l be p o s s i b l e , which, w i t h the co i n c i d e n t advances i n a n a l y t i c a l techniques w i l l soon r e s u l t i n the e l u c i d a t i o n of the ribosomal b i o s y n t h e s i s . This study i s merely a s t a r t i n the eventual deciphering of the events of the b i o s y n t h e s i s o f P. aeruginosa ribosomes, however, a number of i n t e r e s t i n g problems d i d a r i s e from t h i s work: a) The c o n t r o l of the b i o s y n t h e s i s of ribosomes. The process of ribosome b i o s y n t h e s i s was v e r y slow and furthermore there was a d i f f e r e n c e i n the k i n e t i c s of the l a b e l l i n g of the RNA and of the p r o t e i n m o i e t i e s of the ribosomes. b) Could t h i s discrepancy i n the l a b e l l i n g of the RNA and the p r o t e i n be caused by the p r e f e r e n t i a l degradation of the RNA during the s t a r v a t i o n p e r i o d , r e s u l t i n g i n a ribosomal p r o t e i n pool? c) I f a p r e p a r a t i v e g e l e l e c t r o p h o r e s i s system can be developed f o r the s e p a r a t i o n of ribosomal precursors (Hjerten, J e r s t e d t and T i s e l i u s , 1965 'described one f o r ribosomes) or agarose gels w i t h higher f r a c t i o n a t i o n ranges are developed, then the ribosomal precursors described i n t h i s study could be f u l l y c h a r a c t e r i z e d . 153 LITEEATUKE CITED 1. Aronson, A.I. 1 9 6 3 . 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