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Sex-linked, recessive, cold-sensitive mutants of Drosophila melanogaster : genetic and biochemical studies Mayoh, Helen Margaret 1973

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SEX-LINKED, RECESSIVE, COLD-SENSITIVE MUTANTS OF DROSOPHILA  MELANOGASTER: GENETIC AND BIOCHEMICAL STUDIES. by HELEN MARGARET MAYOH B.A., University of B r i t i s h Columbia, 1953 M.A., University of Toronto, 1960 Candidate i n Philosophy, University of C a l i f o r n i a , Berkeley, 1969 B.L.S., University of Alberta, 1973 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Genetics D i v i s i o n We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September - 1973 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department The University of B r i t i s h Columbia Vancouver 8, Canada Date M<Hh/¥~) / 7 7 3 i i ABSTRACT Cold-sensitive mutants of E. c o l i have been valuable i n the study of the structure anat assembly of b a c t e r i a l ribosomes. Some cold^sensitive mutations of E. c o l i have been mapped to l o c i coding for ribosomal proteins. Other cold-sensitive mutations have been shown to a l t e r the regulatory properties of b a c t e r i a l enzymes. In contrast, l i t t l e was known about the genetics and biochemistry of cold-sensitive mutants of Drosophila and other eukaryotes. Also, the genetic l o c i of r i b o -somal proteins of D. me1anogaster were unknown although the ribosomal RNA genes have been located on the X and 2nd chromosomes. Therefore the following questions were asked: Can cold-sensitive l e t h a l s of the X chromosomes of Drosophila melanogaster be isolated? I f so, what are t h e i r genetic properties? Do some have altered ribosomal proteins as i n cold-sensitive mutants of bacteria? The study was part of a general search for and characterization of cold-sensitive l e t h a l s of a l l the chromosomes of D. melanogaster. Among 3„919 EMS-treated X chromosomes, 25 were retained as cold-sensitive l e t h a l s or semi-lethals. That i s , more than 20% of the f l i e s carrying a cold^-sensitive l e t h a l o o survived at 22 C and none at 17 C, and for cold-sensitive semi-lethals,, > 30% survived at 22° c and <13% at 17° c . The cold-sensitive mutations were not randomly d i s t r i b u t e d , i i i 7 b e i n g l o c a t e d a t t h e X t i p and 3 b e i n g a l l e l e s t o t h e r i g h t o f c a r . O v e r h a l f e x h i b i t e d f e m a l e s t e r i l i t y a t t h e p e r m i s s i v e t e m p e r a t u r e a n d 7 e x h i b i t e d v i s i b l e p h e n o t y p e s c h a r a c t e r i s t i c o f b o b b e d a n d M i n u t e m u t a t i o n s . A s t h e p r e s e n c e o f a l t e r e d r i b o s o m a l p r o t e i n s h a s b e e n d e m o n s t r a t e d i n c o l d - s e n s i t i v e m u t a n t s o f b a c t e r i a , t h e r i b o s o m a l p r o t e i n s f r o m D r o s o p h i l a m u t a n t s a n d c o n t r o l s w e r e s t u d i e d b y 2 - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s . B y t h i s m e t h o d , i t was e s t i m a t e d t h a t 69 - 72 p r o t e i n s a r e p r e s e n t i n t h e r i b o s o m e s o f D r o s o p h i l a . No q u a l i t a t i v e d i f f e r e n c e s w e r e o b s e r v e d i n t h e g e l p a t t e r n s o f r i b o s o m a l p r o t e i n s f r o m 5 c o l d - s e n s i t i v e , s e x - l i n k e d m u t a n t s a n d t h e c o n t r o l s . A summary o f t h e b i o c h e m i c a l t e s t i n g o f t h e r i b o s o m e s f r o m o t h e r c o l d - s e n s i t i v e m u t a n t s o f D r o s o p h i l a i s a l s o g i v e n . No b i o c h e m i c a l e v i d e n c e t o s u p p o r t t h e h y p o t h e s i s t h a t c o l d - s e n s i t i v e m u t a n t s o f D r o s o p h i l a h a v e a l t e r e d r i b o s o m e s was o b t a i n e d . T h e l i m i t a t i o n s o f t h e s t u d y a n d r e c o m m e n d a t i o n s f o r f u t u r e r e s e a r c h a r e d i s c u s s e d . i v TABLE OF CONTENTS Page I. Introduction: Cold-sensitive mutants . . . . 1-22 I I . Genetic studies of sex-linked, recessive, cold-sensitive mutants of Drosophila melanogaster. Introduction . . . . 23- 24 Materials and methods 25- 28 Results 29- 42 Discussion 43- 47 I I I . Cold-sensitive, sex-linked mutants of Drosophila melanogas ter; Two-dimensional gel electrophoresis of ribosomal proteins. Introduction 48-49 Methods and materials 50- 64 Results 65- 75 Discussion 76- 89 Bibliography 90-102 Appendix 103 Tables 104-113 Figures 114-133 V LIST OF TABLES Table T i t l e Page I The e f f e c t of temperature on the v i a b i l i t i e s of mutant males, r e l a t i v e to the Oregon-R control, for the cross," j?(T) /Yd* x C (T)RM or C(T)DXQ. 104-5 II The e f f e c t of temperature on the frequencies of the abnormal abdomen phenotype and r e l a t i v e v i a b i l i t i e s of mutant males from the cross, i ( l ) C S / Y ^ X C(1)RM/Y or C(I)DX/Yg 106 III Male progeny at 22° C and 17° C of females heterozygous fsor y cv v f_ car and mutations which have v i s i b l e pKehotypes 107 IV Survival of progeny i n tests of Jj(l)5cs, 6 C S, 15 c s, and 16^ s with duplications and d e f i c i e n c i e s of proximal heterochromatin of the X chromosome. 108 V V i a b i l i t i e s and phenotypes of trans heterozygous and homozygous females i n complementation tests of cold-sensitive mutations 109 VI The e f f e c t of temperature on the v i a b i l i t i e s of mutant males and homozygous females from the cross, J?(1)CS/FM6 $$ x j ( l ) c s / Y t f V , as compared to the Oregon-R control... 110 VII F e r t i l i t y at 22° C of females homozygous for cold-sensitive l e t h a l s I l l VIII Male progeny of females heterozygous for y cv v f car and mutant chromosomes at 17° C . . 112 IX Charact e r i s t i c s of cold-sensitive mutants of the X chromosome 113 v i LIST OF FIGURES F i g u r e T i t l e Page 1. Screening procedure f o r the d e t e c t i o n and recovery o f s e x - l i n k e d r e c e s s i v e c o l d -s e n s i t i v e mutations 114 2. C y t o l o g i c a l r e p r e s e n t a t i o n o f X chromosome scute i n v e r s i o n s . (adapted from Cooper. TT5TT 116 3. R e l a t i v e b r e a k p o i n t s o f d e f i c i e n c i e s and d u p l i c a t i o n s o f the Ma-1 r e q i o n '-'.{Schalet and F i n n e r t y , 1968b) ~ . 118 4. Gen e t i c p o s i t i o n s o f c o l d - s e n s i t i v e , heat-s e n s i t i v e and n o n - c o n d i t i o n a l l e t h a l mutations on the X chromosome 120 5. Composite diagram o f two-dimensional e l e c t r o -phorograms of the ribosomal p r o t e i n s o f D. melanogaster 122 6a. Two-dimensional g e l e l e c t r o p h o r e s i s o f Mg-HAc p r e p a r a t i o n o f D r o s o p h i l a ribosomal p r o t e i n s . Long run 124 6b. Two-dimensional g e l e l e c t r o p h o r e s i s of Mg-HAc p r e p a r a t i o n o f D r o s o p h i l a ribosomal p r o t e i n s . Intermediate run 124 6c. Two-dimensional g e l e l e c t r o p h o r e s i s o f Mg-HAc p r e p a r a t i o n o f D r o s o p h i l a r ibosomal p r o t e i n s . Short run . . . ; . 126 7a. Two-dimensional g e l e l e c t r o p h o r e s i s o f HC1-acetone p r e p a r a t i o n o f D r o s o p h i l a ribosomal p r o t e i n s . Long run. . 126 7b. Two-dimensional g e l e l e c t r o p h o r e s i s o f HC1-acetone p r e p a r a t i o n of D r o s o p h i l a r i b o s o m a l p r o t e i n s . Intermediate run 128 8. Two-dimensional g e l e l e c t r o p h o r e s i s o f E. c o l i r i b osomal p r o t e i n s . 129 9 Two-dimensional g e l e l e c t r o p h o r e s i s o f r i b o -somal p r o t e i n s o f mutant 1 6 C s 13.1 10 Two-dimensional g e l e l e c t r o p h o r e s i s o f r i b o -somal p r o t e i n s o f mutant 5 ° 5 131 v i i L I S T OF F IGURES c o n t . F i g u r e T i t l e P a g e 11 T w o - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s o f r i b o -s o m a l p r o t e i n s o f m u t a n t 2 7 c S 133 12 T w o - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s o f r i b o -s o m a l p r o t e i n s o f O r e g o n - R m a l e s 133 1 1. INTRODUCTION: C o l d - S e n s i t i v e Mutants Aim o f r e s e a r c h : In the p r e s e n t study, s e x - l i n k e d c o l d -s e n s i t i v e mutants of D r o s o p h i l a were i s o l a t e d ^ c h a r a c t e r i z e d g e n e t i c a l l y and examined b i o c h e m i c a l l y f o r ribosomal a l t e r -a t i o n s . The r e s e a r c h was undertaken because i t was thought t h a t the s e l e c t i o n of c o l d - s e n s i t i v e mutants of D r o s o p h i l a might enhance the re c o v e r y of mutations a f f e c t i n g ribosomal p r o t e i n s as i s the case i n E. c o l i (Nomura, 1970). These mutants would be i n v a l u a b l e i n answering q u e s t i o n s such as: Are the genes f o r ribosomal p r o t e i n s c l u s t e r e d near the genes f o r ribosomal RNA on the X chromosome or s c a t t e r e d throughout the genome of D. melanogaster? Are ribosomes c h e m i c a l l y constant throughout the l i f e c y c l e of D r o s o p h i l a or are d i f f e r e n t ribosomal genes t r a n s c r i b e d d u r i n g l a r v a l and a d u l t p e r i o d s ? Is the temperature-s e n s i t i v e p e r i o d o f D r o s o p h i l a t e m p e r a t u r e - s e n s i t i v e mutants r e l a t e d t o the i n i t i a l s y n t h e s i s of the d e f e c t i v e gene product or to a c r i t i c a l developmental event i n which the product i s r e q u i r e d ( T a r a s o f f and Suzuki, 1970)? Are the assemblies of the s m a l l and l a r g e ribosomal subunits interdependent i n D r o s o p h i l a ? Thus problems examined i n b a c t e r i a l s t u d i e s and a l s o new ones r e l a t e d t o the complex-i t i e s of a h i g h e r organism c o u l d be i n v e s t i g a t e d w i t h ribosomal mutants. 2 C o l d - s e n s i t i v e mutants of microorganisms; C o l d - s e n s i t i v e mutants are d e f i n e d by t h e i r v i a b i l i t y a t "normal" temperatures and i n v i a b i l i t y a t low temperatures. Two types of mo l e c u l a r a l t e r a t i o n s have been e s t a b l i s h e d so f a r f o r c o l d - s e n s i t i v e mutants of b a c t e r i a : l o s s o f a l l o s t e r i c c o n t r o l o f enzymes and d e f e c t i v e assembly o f ribosomes. S e v e r a l c o l d - s e n s i t i v e auxotrophs of E. c o l i were found t o have changes i n feedback c o n t r o l o f m e t a b o l i c pathways r a t h e r than l o s s e s i n enzyme a c t i v i t i e s (O'Donovan and Ingraham, 1965). Other c o l d - s e n s i t i v e mutants o f b a c t e r i a f a i l t o assemble 30S and 50S ribosomal subunits a t low temperature (Davies and Nomura, 1972). These are c a l l e d Sad o r subunit assembly d e f e c t i v e mutants and some have been s t u d i e d i n t e n s i v e l y t o e l u c i d a t e the i n v i v o assembly of b a c t e r i a l ribosomes. The molecular d e f e c t s o f o t h e r c o l d - s e n s i t i v e mutants have not been so thoroughly i n v e s t i g a t e d . C o l d - s e n s i t i v e mutations of v i r u s e s , f o r example, were shown to map i n a l i m i t e d number of genes and to a f f e c t v i r a l development but few b i o c h e m i c a l t e s t s o f the mutants have been done (Cox and S t r a c k , 1971; Scotti, 1968; Dowell, 1967). D e f e c t i v e feedback c o n t r o l o f enzymes o f c o l d - s e n s i t i v e  b a c t e r i a 1 mutants; A c o l d - s e n s i t i v e mutant o f E. c o 1 i was shown t o have a h i s t i d i n e enzyme w i t h a l t e r e d r e g u l a t o r y p r o p e r t i e s (O'Donovan and Ingraham, 1965). The mutant grew w e l l i n g l u c o s e - s a l t s medium a t 37° C but not a t 20° C. I t r e q u i r e d h i s t i d i n e o n l y a t the low 3 temperature. When e x t r a c t s o f the mutant and the p a r e n t a l s t r a i n were t e s t e d f o r p h o s p h o r i b o s y l - ATP pyrophosphorylase a c t i v i t y , i t was found t h a t mutant grown a t 37° C gave valu e s s i m i l a r t o those f o r w i l d - t y p e but mutant grown a t low temperature had no d e t e c t a b l e enzyme a c t i v i t y . T h i s enzyme occurs e a r l y i n the h i s t i d i n e pathway and normally excess p r o d u c t i o n of h i s t i d i n e i s prevented by i n h i b i t i o n o f the pyrophosphorylase by the end-product o f the pathway. Enzyme from the mutant, however, was found to be 1000 -f o l d more s e n s i t i v e to i n h i b i t i o n by h i s t i d i n e than the w i l d - t y p e enzyme, and bound the i n h i b i t o r i r r e v e r s i b l y . Both the mutant and the w i l d - t y p e enzyme were more s e n s i t i v e t o feedback i n h i b i t i o n a t 20° C than a t 37° C. Apparently the i n c r e a s e d s e n s i t i v i t y o f the mutant enzyme a t low temperature was s u f f i c i e n t t o r e s u l t i n a h i s t i d i n e requirement a t low temperature. Mutants r e s i s t a n t t o t h i a z o l e a l a n i n e were d e r i v e d from the c o l d - s e n s i t i v e s t o c k . T h i s analogue of h i s t i d i n e i s known to cause feedback i n h i b i t i o n o f p h o s p h o r i b o s y l — ATP pyrophosphorylase but cannot s u b s t i t u t e f o r h i s t i d i n e i n p r o t e i n s y n t h e s i s (Martin, 1963; Sheppard, 1964). R e s i s t a n c e to the analogue a l t e r s the r e g u l a t o r y s i t e o f the enzyme and not the c a t a l y t i c s i t e . As p r e d i c t e d i f the c o l d - s e n s i t i v e mutant had an a l t e r e d r e g u l a t o r y s i t e , r e s i s t a n c e to t h i a z o l e a l a n i n e a b o l i s h e d the requirement f o r h i s t i d i n e a t the low temperature and the extreme s e n s i t i v i t y 4 t o h i s t i d i n e i n h i b i t i o n ( O ' D o n o v a n a n d I n g r a h a m , 1 9 6 5 ) . I t was a l s o shown t h a t d e r i v a t i v e s o f t h e c o l d - s e n s i t i v e s t o c k s e l e c t e d f o r p r o t d r o p h y a t l o w t e m p e r a t u r e l o s t t h e e x t r e m e s e n s i t i v i t y t o f e e d b a c k i n h i b i t o n . I n c r e a s e d f e e d b a c k i n h i b i t i o n seems t o o c c u r f r e q u e n t l y i n c o l d - s e n s i t i v e a u x o t r o p h s o f b a c t e r i a . O u t o f 6 i n d e p e n d e n t l y i s o l a t e d c o l d - s e n s i t i v e m u t a n t s w h i c h r e q u i r e d h i s t i d i n e a t l o w t e m p e r a t u r e , a l l p r o d u c e d p h o s p h o r i b o s y l - ATP p y r o p h o s p h o r y l a s e t h a t was more s e n s i t i v e t o i n h i b i t i o n b y h i s t i d i n e t h a n t h e p a r e n t a l enzyme ( O ' D o n o v a n a n d I n g r a h a m , 1 9 6 5 ) . F u r t h e r m o r e , 15 c o l d - s e n s i t i v e , a r g i n i n e - r e q u i r i n g m u t a n t s w e r e b l o c k e d i n t h e f i r s t enzyme o f t h e a r g i n i n e p a t h w a y . I t i s t h o u g h t t h a t a l t e r e d f e e d b a c k i n h i b i t i o n i n t h e c o l d - s e n s i t i v e m u t a n t s i s due t o c o n f o r m a t i o n a l c h a n g e s i n a l l o s t e r i c e n z y m e s ( O ' D o n o v a n , e t a l . , 1 9 6 5 ) . S i n c e r e p r e s s o r s a l s o a p p e a r t o b e m u l t i m e r i c , a l l o s t e r i c p r o t e i n s w i t h r e g u l a t o r y s i t e s , i t i s p r e d i c t e d t h a t r e p r e s s i o n a n d i n d u c t i o n w i l l be a l t e r e d i n some c o l d -s e n s i t i v e m u t a n t s o f b a c t e r i a . T h e s t r u c t u r e a n d i n v i t r o a s s e m b l y o f E . c o l i r i b o s o m e s ; B e f o r e d i s c u s s i n g t h e S a d m u t a n t s i n d e t a i l , i t may b e u s e f u l t o o u t l i n e t h e b i o c h e m i s t r y o f b a c t e r i a l r i b o s o m e s ( K u r l a n d , 1 9 7 2 ; D a v i e s a n d N o m u r a , 1 9 7 2 ) . T h e y a r e c o m p l e x 70S p a r t i c l e s w h i c h c o n s i s t o f two s u b u n i t s , t h e 30S a n d t h e 50S s u b u n i t s . T h e s m a l l s u b u n i t o f E . c o l i 5 c o n t a i n s one 16S RNA molecule and 20-21 unique p r o t e i n s whereas the l a r g e subunit c o n t a i n s one 5S RNA molecule, one 23S RNA molecule, and 27-34 unique p r o t e i n s . The 2 subunits of E. c o l i have no p r o t e i n s i n common. The ribosomal RNA s p e c i e s found i n the ribosomal s u b u n i t s are d e r i v e d from l a r g e r p r e c u r s o r forms. The e x t r a o l i g o n u c l e o t i d e s are c l e a v e d d u r i n g ribosome assembly. The i n v i t r o r e c o n s t i t u t i o n of 30S ribosomal subunits occurs spontaneously when 16S RNA and ribosomal p r o t e i n s from 3OS subunits o f E. c o l i a r e mixed under s p e c i f i c i o n i c c o n d i t i o n s a t temperatures between 20 and 40° C (Traub and Nomura, 1969). Such reassembled subunits are b i o l o g i c a l l y 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. At 10° C or l e s s , i n t e r m e d i a t e 21S p a r t i c l e s of w i l d type s u b u n i t s accumulate. Heat i s r e q u i r e d f o r a s t r u c t u r a l rearrangement o f the i n t e r m e d i a t e b e f o r e the remaining ribosomal p r o t e i n s a t t a c h . Thus, r e c o n s t i t u t i o n of 30S r i b o s o m a l s u b u n i t s o c c u r s more r e a d i l y a t h i g h temperatures than a t low. The i n v i t r o r e c o n s t i t u t i o n of 5OS s u b u n i t s from 2 3S RNA, 5S RNA and 50S ribosomal p r o t e i n s a l s o i n v o l v e s i n t e r m e d i a t e 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 and does not proceed to completion a t low temperatures (Nomura and Erdmann, 1970; Nomura, 1972). R e c o n s t i t u t i o n s t u d i e s of enzymes and o t h e r m u l t i m e r i c p r o t e i n s from w i l d type organisms have demonstrated t h a t the assembly process i s c o l d - s e n s i t i v e (Van Holde, 1966; V a l e n t i n e e t a l , 1966). 6 C o l d - s e n s i t i v e m u t a t i o n s m a y , t h e r e f o r e , b e r e g a r d e d a s d e f e c t s w h i c h e x a g g e r a t e a n o r m a l r e a c t i o n t o w a r d c o l d . C o l d - s e n s i t i v e m u t a t i o n s may b e s p e c i f i c i n t h e s e n s e t h a t t h e y a f f e c t c o n f o r m a t i o n a l c h a n g e s o f p r o t e i n s a n d p r o t e i n i n t e r a c t i o n s w i t h i n s p e c i f i c a g g r e g a t e s . F u r t h e r s t u d i e s o n t h e r e c o n s t i t u t i o n o f 30S s u b u n i t s h a v e shown t h a t t h e p r o c e s s i s s e q u e n t i a l a n d c o o p e r a t i v e i n c h a r a c t e r ( M i z u s h i m a a n d N o m u r a , 1 9 7 0 ) . O n l y c e r t a i n o f t h e p r o t e i n s c o m b i n e d i r e c t l y w i t h 16S RNA. Some p r o t e i n s o n l y b i n d i f o t h e r p r o t e i n s a r e a l r e a d y a t t a c h e d a n d s o o n . S o m e t i m e s b i n d i n g i s i m p r o v e d i f two p r o t e i n s a r e p r e s e n t s i m u l t a n e o u s l y . S u c h i n v i t r o s t u d i e s i m p l y t h a t t h e i n f o r m a t i o n f o r c o r r e c t a s s e m b l y i s c o n t a i n e d i n t h e s t r u c t u r e o f t h e c o m p o n e n t s t h e m s e l v e s ; i . e . no t e m p l a t e s o r a u x i l i a r y e n z y m e s a r e n e e d e d . C o l d - s e n s i t i v e m u t a n t s w i t h r i b o s o m a l d e f e c t s ; A t l o w t e m p e r a t u r e s , t h e S a d m u t a n t s o f b a c t e r i a g rew p o o r l y a n d a c c u m u l a t e d v a r i o u s 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 ( G u t h r i e , e t a l . , 1 9 6 9 a ; T a i , e t a l . , 1 9 6 9 ) . A t h i g h t e m p e r a t u r e s t h e y p r o d u c e d 30S a n d 50S s u b u n i t s a n d 70S r i b o s o m e s l i k e w i l d - t y p e s t r a i n s . A s s e m b l e d 70S r i b o s o m e s f r o m E . c o l i S a d m u t a n t s w e r e n o t f u n c t i o n a l l y c o l d - s e n s i t i v e i n a n 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 s y s t e m ( G u t h r i e e t a l . , 1 9 6 9 b ) . R i b o s o m a l m u t a n t s may c o n s t i t u t e a l a r g e p o r t i o n o f t h e c o l d - s e n s i t i v e m u t a n t s o f E . c o l i . O u t o f 100 7 c o l d - s e n s i t i v e mutants of E. c o l i , one t h i r d t o one h a l f gave i r r e g u l a r sedimentation p a t t e r n s when crude e x t r a c t s were examined by a n a l y t i c a l c e n t r i f u g a t i o n (Guthrie e t a l . , 1969a). Nomura estimated t h a t 20% o f the cs mutants were l i n k e d t o the r e g i o n o f the E. c o l i chromosome where spectinomycin r e s i s t a n c e and streptomycin r e s i s t a n c e mutations map. The 2 a n t i b i o t i c r e s i s t a n c e l o c i are c l o s e l y l i n k e d and code f o r ribosomal p r o t e i n s . The genes i n the s p c - s t r r e g i o n may c o n s t i t u t e an operon o f ribo s o m a l p r o t e i n s and supernatant p r o t e i n s which combine w i t h ribosomes (Nomura and Engback, 1972). But rib o s o m a l p r o t e i n s have been mapped t o oth e r areas o f the E. c o l i chromosome ( B o l l e n , e t a l . , 1973). Only a few of the p u t a t i v e Sad mutants of E. c o l i mapping i n the s p c - s t r r e g i o n have been examined i n d e t a i l . When 45 c o l d - s e n s i t i v e mutants of Salmonella typhimurium were screened by t r a n s d u c t i o n f o r t h e i r l i n k a g e to the streptomycin r e s i s t a n c e l o c u s , 7 showed such l i n k a g e ( T a i , e t a l . , 1969). Only 2 of the l a t t e r were t e s t e d f o r r i b o s o m a l d e f e c t s . Evidence t h a t incomplete ribosomal subunits accumulated i n the 2 Salmonella mutants a t low temperature was d e r i v e d from double l a b e l experiments. 14 Mixtures o f w i l d type ribosomes l a b e l e d w i t h C u r a c i l 3 and mutant p a r t i c l e s l a b e l e d w i t h H u r a c i l were analyzed by sucrose g r a d i e n t s e d i m e n t a t i o n . When grown a t hig h temperatures with r a d i o a c t i v e p r e c u r s o r s , w i l d type and mutants had s i m i l a r sedimentation p r o f i l e s o f 70S, 50S 8 and 30S peaks. When l a b e l e d w i t h r a d i o a c t i v e u r a c i l a t 20° C, however, the mutants, but not the parent s t r a i n , had a l t e r e d sedimentation p r o f i l e s . One Salmonella mutant had a 23S peak and the oth e r mutant had a 30S peak which was i n c r e a s e d r e l a t i v e to the oth e r peaks. T h i s i n c r e a s e may have been due to an accumulation o f s u b p a r t i c l e s a t the 26S and the 32S r e g i o n s (Nomua, 1970). Three p u t a t i v e ribosomal mutants of E. c o l i , r e p r e s e n t a t i v e s o f the 3 p a t t e r n s o f i r r e g u l a r sedimentation, were analyzed by temperature s h i f t experiments ( G u t h r i e , e t a l . , 1969a and 1969b). C u l t u r e s grown a t 42° c, were t r a n s f e r r e d t o 20° C and l a b e l e d w i t h H 3 u r a c i l . The ribosomal supernatant from h a l f o f the c u l t u r e was analyzed by sucrose g r a d i e n t sedimentation. Thus r i b o n u c l e o p r o t e i n s u b p a r t i c l e s s y n t h e s i z e d i n the c o l d were l a b e l e d and i d e n t i f i e d by t h e i r unusual sedimentation p r o p e r t i e s . The othe r h a l f o f the c u l t u r e was washed and incubated a t 42° C with c o l d u r a c i l . S u b p a r t i c l e s converted to normal sub-u n i t s a t h i g h temperature r e s u l t e d i n l a b e l e d 3OS and/or 50S peaks. For example, a t 20° C, mutant 68 had c o n s i d -e r a b l e l a b e l i n the 30S peak, none i n the 50S peak and c o n s i d e r a b l e i n a 43S peak (Guthrie e t a l . , 1969a). When t r a n s f e r r e d t o 42° C w i t h c o l d u r a c i l , the mutant had c o n s i d e r a b l e l a b e l i n the 30S and 50S peaks but the 4 3S peak was g r e a t l y reduced. From such s t u d i e s , Nomura (1970) concluded t h a t the Sad mutants of E. c o l i were of 3 ty p e s : 9 (1) m u t a n t s t h a t f a i l e d t o s y n t h e s i z e 50S s u b p a r t i c l e s and a c c u m u l a t e d 32S p a r t i c l e s ( p r e c u r s o r s o f 50S s u b u n i t s ) , (2) m u t a n t s t h a t f a i l e d t o s y n t h e s i z e 50S s u b u n i t s a n d a c c u m u l a t e d 43S p a r t i c l e s , (3) m u t a n t s t h a t f a i l e d t o s y n t h e s i z e b o t h 50S a n d 30S s u b u n i t s a n d a c c u m u l a t e d b o t h 32S a n d 21S p a r t i c l e s a t 2 0 ° C . T h e 3 k i n d s o f m u t a n t s s u g g e s t e d t h a t t h e i n v i v o a s s e m b l y o f 30S s u b u n i t s p r o c e e d e d i n d e p e n d e n t l y o f 50S s u b u n i t a s s e m b l y b u t 50S s u b u n i t a s s e m b l y was d e p e n d e n t o n 30S a s s e m b l y . I n c o n t r a s t , t h e i n v i t r o s t u d i e s h a d shown t h a t b o t h 30S a n d 5OS s u b u n i t s a s s e m b l e d i n d e p e n d e n t l y ( T r a u b a n d N o m u r a , 1 9 6 8 ; Nomura a n d E r d m a n n , 1 9 7 0 ) . T h e t y p e 3 S a d m u t a n t s seemed t o i n d i c a t e t h e p r e s e n c e o f a n i n v i v o c o n t r o l m e c h a n i s m n o t a p p a r e n t i n t h e i n v i t r o s t u d i e s (Nomura , 1 9 7 0 ) . R e c e n t l y a f o u r t h t y p e o f S a d m u t a n t was i s o l a t e d . A h e a t - s e n s i t i v e m u t a n t o f E . c o l i a c c u m u l a t e d i n t e r m e d i a t e s o f 30S s u b u n i t s b u t n o r m a l 5OS s u b u n i t s v /ere f o r m e d ( R o s s e t , e t a l . , 1971) . H e r e 50S s u b u n i t a s s e m b l y a p p e a r e d t o b e i n d e p e n d e n t o f 30S s u b u n i t a s s e m b l y . No s a t i s f a c t o r y t h e o r y w h i c h e x p l a i n s a l l t h e m u t a n t s h a s b e e n p u b l i s h e d ( D a v i e s a n d N o m u r a , 1 9 7 2 ) . A n a l y s e s o f t h e s u b p a r t i c l e s , h o w e v e r , s u p p o r t s t h e i d e a t h a t i n c o m p l e t e r i b o s o m a l a s s e m b l y o c c u r s i n t h e S a d m u t a n t s . T h e 32S a n d 43S s u b p a r t i c l e s c o n t a i n e d 23S RNA w h e r e a s t h e 21S p a r t i c l e s c o n t a i n e d 16S RNA ( G u t h r i e , e t a l . , 10 1969a). These are the s p e c i e s o f RNA expected i f the 32S and 4 3S s u b p a r t i c l e s are p r e c u r s o r s o f 5OS s u b u n i t s , and 21S s u b p a r t i c l e s are p r e c u r s o r s o f 30S s u b u n i t s . The p r o t e i n s i n the 21S p a r t i c l e s were i d e n t i f i e d by carboxymethyl c e l l u l o s e chromatography or 2-dimensional g e l e l e c t r o p h o r e s i s (Nashimoto, 'et a l . , 1971). Two type 3 mutants were examined, Sad-410 and Spc-49-1. The l a t t e r was a spectinomycin r e s i s t a n c e mutant which was a l s o c o l d -s e n s i t i v e and accumulated i n t e r m e d i a t e s o f 30S and 50S s u b u n i t s . Nine to 11 unique 3OS ribosomal p r o t e i n s were presen t i n the 2IS i n t e r m e d i a t e s depending on the p r e p a r a -t i o n . The analyses f o r the 2 mutants were s i m i l a r . The p r o t e i n s o f the s u b p a r t i c l e s were a l l p r o t e i n s i n v o l v e d i n the e a r l y steps of the i n v i t r o r e c o n s t i t u t i o n of 30S subunits (Mizushima and Nomura, 1970). Again the r e s u l t s support the i d e a o f incomplete r i b o s o m a l assembly i n Sad mutants. Mapping of the Sad mutants was accomplished mainly by phage P-^  t r a n s d u c t i o n of c o l d - s e n s i t i v i t y from the mutant s t r a i n , a r o E + s p c s s t r r , t o a c o l d - i n s e n s i t i v e r e c i p i e n t , aroE" s p c r s t r s (Guthrie, e t a l . , 1969a and 1969b; Nashimoto, e t aJL. , 1971). Transductants f o r a r o E + were s e l e c t e d and t e s t e d f o r growth at 20° C as w e l l as s e n s i t i v i t y t o streptomycin and spectinomycin. To t e s t whether c o l d - s e n s i t i v i t y and the Sad phenotype were r e l a t e d , the sedimentation p a t t e r n s of ribosomal super-natants from s e v e r a l c o l d - s e n s i t i v e t r a n s d u c t a n t s were examined. 11 Four Sad mutants were mapped t o the s p c - s t r area of the E. c o l i chromosome: (Sad-410, Sad-38) aroE Sad-19 spcA s t r A k - p r o t e i n Spc-49-1 The r e g i o n between aroE and spc A codes f o r s e v e r a l 30S and 50S ribosomal p r o t e i n s (Dekio, 1971). Spectinomycin r e s i s t a n t and st r e p t o m y c i n r e s i s t a n t mutants have a l t e r e d 30S ribosomal p r o t e i n s , S5 and S12, r e s p e c t i v e l y (Dekio and Takato, 1969; B o l l e n and Herzog, 1970; Ozaki, e t a l . , 1969). (The ribosomal p r o t e i n s of E. c o l i are i d e n t i f i e d by Wittmann's nomenclature which has been g e n e r a l l y accepted by ribosome r e s e a r c h e r s (Wittmann, 'et ' a l . , 1971). The numbering system i s based on the m o b i l i t i e s of the p r o t e i n s from the s m a l l , S, and the l a r g e , L, s u b u n i t s i n 2-dimensional g e l s . ) The known genes f o r ribosomal RNA are l o c a t e d i n s i t e s d i s t a n t from the s p c - s t r r e g i o n (Davies and Nomura, 1972). Sad-19, a type 1 mutant which accumulated 32S i n t e r m e d i a t e s of 50S s u b u n i t s , mapped between aroE and spcA and was c l o s e l y l i n k e d t o spcA (Guthrie, e t a l . , 1969b). Type 3 mutants, Sad-410 and Sad-38, mapped to the l e f t of aroE.but another type 3 mutant spc-49-1, was l o c a t e d i n the spc gene (Nashimoto, e t a l . , 1971). A l l the phenotypes of the Spc-49-1 mutant, i . e . spectinomycin r e s i s t a n c e , c o l d - s e n s i t i v i t y and accumulation o f i n t e r -mediates of 50S and 30S sub u n i t s mapped to the spc l o c u s . 12 Although the p r o t e i n components o f 2IS s u b p a r t i c l e s from mutants Sad-410 and Spc-49-1 were s i m i l a r , the two mutations mapped i n d i f f e r e n t r e g i o n s of the chromo-some (Nashimoto, e t a l . , 1971). The mapping r e s u l t s , e s p e c i a l l y f o r Spc-49-1, suggest t h a t ribosomal p r o t e i n s are a l t e r e d i n the Sad mutants. The r e s u l t s a l s o show t h a t a s i n g l e c o l d - s e n s i t i v e mutation i n a type 3 mutant causes non-assembly of both s u b u n i t s . G e n e r a l l y , c o l d - s e n s i t i v i t y and the Sad phenotype were a s s o c i a t e d i n the t r a n s d u c t a n t s . Sad 68, a type 2 mutant which a l s o mapped to the s p c - s t r r e g i o n , was an ex c e p t i o n ; the c o l d - s e n s i t i v e t r a n s d u c t a n t s d i d not accum-l a t e 4 3S p a r t i c l e s but s y n t h e s i z e d normal 50S s u b u n i t s (Guthrie e t a l . , 1969a). No e x p l a n a t i o n was o f f e r e d f o r t h i s o b s e r v a t i o n . One may s p e c u l a t e t h a t m o d i f i e r s i n the r e c i p i e n t s t r a i n s u f f i c i e n t l y complemented the mole-c u l a r d e f e c t t o suppress the Sad phenotype i n the sedimentation t e s t . The l a t t e r may be a l e s s s e n s i t i v e t e s t than the 20° C growth t e s t . One other g e n e t i c f a c t which may be important was the dominance o f the c o l d - s e n s i t i v e phenotype (G u t h r i e , <et a l . , 1969b). A p a r t i a l d i p l o i d o f Sad-19 was formed u s i n g a recombination-negative F' episome which covers the mutant r e g i o n . The mutant phenotype was dominant to the standcird phenotype. The mechanism by which a l t e r e d p r o t e i n S5 of mutant Spc-49-1 a f f e c t s ribosomal assembly i s not c l e a r 13 (Nashimoto, e t a_l., 1971). S5 was not p r e s e n t i n the 21S i n t e r m e d i a t e s accumulated by the mutant. Omission of S5 from r e c o n s t i t u t i o n mixtures has a r e l a t i v e l y minor e f f e c t l e a d i n g t o the formation o f 28S p a r t i c l e s . F i n a l l y the presence of a l t e r e d S5 from Spc-49-1 i n the recon-s t i t u t i o n mixture had no adverse e f f e c t on the i n v i t r o assembly of 30S s u b u n i t s . Whether the use of mature 16S RNA i n the i n v i t r o s t u d i e s , r a t h e r than p r e c u r s o r 16S RNA, i s r e s p o n s i b l e f o r the d i s c r e p a n c y between i n v i v o and i n v i t r o o b s e r v a t i o n s i s u n c e r t a i n . The mechanism by which one a l t e r e d ribosomal p r o t e i n r e s u l t s i n non-assembly of both 30S and 50S s u b u n i t s i s a l s o not understood (Davies and Nomura, 1972). Other a n t i b i o t i c mutants have a l s o been examined f o r c o l d - s e n s i t i v i t y and the Sad phenotype (Lewandowski and Brownstein, 1969; Nashimoto and Nomura, 1970). A s i g n i f i c a n t f r a c t i o n of phenotypic r e v e r t a n t s from streptomycin dependence t o streptomycin independence were c o l d - s e n s i t i v e and accumulated 26S and 43S s u b p a r t i c l e s a t low temperatures (Nashimoto and Nomura, 1970). Other s t u d i e s have shown t h a t streptomycin dependence and streptomycin r e s i s t a n c e mutations map i n the s t r A l o c u s and a f f e c t the 30S ribosomal p r o t e i n , S12 (Ozaki, e t a l . , 1969;, B i r g e and Kurland, 1969; Hashimoto, 1960). Phenoty-p i c r e v e r t a n t s from streptomycin dependence to independence remain unchanged i n p r o t e i n S12 but show a l t e r a t i o n s i n e i t h e r one of two 30S ribosomal p r o t e i n s , S4 or S5 14 (Birge and Kurland, 1970; Deusser, e t a l . , 1970; K r e i d e r and Brownstein, 1972; S t o f f l e r , e t a l . , 1971). Presumably streptomycin independent mutants which are c o l d - s e n s i t i v e , have e i t h e r a l t e r e d p r o t e i n S5 or a l t e r e d S4 and one of the l a t t e r i s r e s p o n s i b l e f o r the Sad phenotype and c o l d -s e n s i t i v i t y . Though much work s t i l l remains t o be done on the Sad mutants, the f o l l o w i n g c o n c l u s i o n s seem warranted: (1) Some c o l d - s e n s i t i v e mutants of b a c t e r i a accumulate i n t e r m e d i a t e s of ribosomal s u b u n i t s . (2) Thus f a r ribosomal p r o t e i n s are the o n l y m u t a t i o n a l a l t e r a t i o n s i m p l i c a t e d . Sad mutations occur i n s e v e r a l g e n e t i c l o c i some of which are known ribosomal p r o t e i n genes. (3) In v i v o assembly of ribosomal s u b u n i t s i s more complex than the i n v i t r o r e c o n s t i t u t i o n s t u d i e s i n d i c a t e d . Some c o n t r o l mechanism which c o o r d i n a t e s 30S and 50S s u b u n i t assembly may be i n v o l v e d . Genetic l o c i o f ribosomal RNA of D r o s o p h i l a melanogas ter.; The ribosomal RNA genes of D r o s o p h i l a melanogaster have been i d e n t i f i e d by RNA-DNA h y b r i d i z a t i o n . D r o s o p h i l a w i t h the bobbed (bb) phenotype were shown to c o n t a i n reduced amounts of DNA complementary to RNA from D r o s o p h i l a ribosomes ( R i t o s s a , e t a l . , 1966). Prev i o u s c y t o l o g i c a l s t u d i e s i n d i c a t e d t h a t the bb l o c u s o f the X chromosome was c l o s e to the nucleous o r g a n i z e r (NO) i n the heterochromatic r e g i o n (Cooper, 1959). R i t o s s a and 15 c o l l e a g u e s regard the bb l o c u s as the mutant c o u n t e r p a r t of the NO ( R i t o s s a , e t a l . , 1966; Atwood, 1969). The bb mutants are thought to r e s u l t from p a r t i a l d e l e t i o n s i n the NO r e g i o n which c o n t a i n s a l a r g e s e r i e s of d u p l i c a t i o n s of ribosomal DNA. A second bb l o c u s and NO are l o c a t e d on the Y chromosome of D r o s o p h i l a melanogaster (Cooper, 1959). H y b r i d i z a t i o n of 5S ribosomal RNA to s a l i v a r y gland polytene chromosomes of D r o s o p h i l a melanogaster i n d i c a t e d t h a t the genes f o r t h i s ribosomal RNA are c l u s t e r e d on the 2nd chromosome (Wimber and S t e f f e n s e n , 1970). As the t o t a l e x t r a c t of ribosomal RNA used i n the bobbed h y b r i d -i z a t i o n t e s t s would probably be mainly 18S and 28S RNA, the n u c l e o l u s o r g a n i z e r probably codes f o r 2 8S and 18S RNA ( T a r t o f and P e r r y , 1970). V a r i o u s genotypes, XO, XX, XY and XXY, c o n t a i n e d approximately e q u i v a l e n t amounts of ribosomal RNA ( K i e f e r , 1968). The r e s u l t s i n d i c a t e t h a t dosage r e g u l a t i o n allows e q u i v a l e n t p r o d u c t i o n of ribosomal RNA from 1 t o 3 NO r e g i o n s . The r a t e o f ribosomal RNA s y n t h e s i s , however, was reduced i n bobbed mutants wi t h l e s s than 1 complete NO (Mohan and R i t o s s a , 1970). Combinations of extreme bb a l l e l e s i n females a l s o r e s u l t e d i n reduced egg p r o d u c t i o n . bb stocks have f i n e r , s h o r t e r b r i s t l e s than w i l d type and etched t e r g i t e s , i . e . reduced abdominal banding ( L i n d s l e y and G r e l l , 1968). A l l e l e s vary c o n s i d e r a b l y ; some e x e r t o n l y a s l i g h t e f f e c t on the phenotype whereas o t h e r s are r e c e s s i v e l e t h a l s . V a r i a t i o n i n the i n t e n s i t y o f the 16 mutant phenotype of bb a l l e l e s i s a t t r i b u t e d t o v a r i o u s s i z e s of d e l e t i o n s but some mutants do not f i t t h i s i n t e r p r e t a t i o n (Atwood, 1969; K i e f e r , 1968). I t i s assumed t h a t a slower r a t e of ribosome p r o d u c t i o n and concomitant p r o t e i n s y n t h e s i s b r i n g s about the numerous a b n o r m a l i t i e s of the bb mutants. Previous attempts to i d e n t i f y the ribosomal p r o t e i n genes  of D r o s o p h i l a . Except f o r the bb l o c i and 5S RNA s i t e , no other ribosomal genes of D r o s o p h i l a have been i d e n t i f i e d . S e v e r a l of the approaches used to l o c a t e ribosomal p r o t e i n genes i n b a c t e r i a have been a p p l i e d to D r o s o p h i l a . The D r o s o p h i l a r e p o r t s , however, have been b r i e f and p r e l i m i n a r y i n c h a r a c t e r . 1. S t r a i n - and s p e c i e s - d i f f e r e n c e s i n ribosomal p r o t e i n s . Mapping of ribosomal p r o t e i n s by s t r a i n - or s p e c i e s -d i f f e r e n c e s was f i r s t performed w i t h E. c o l i B and E. c o l i K-12 (Leboy, e t a l . , 1964). The two s t r a i n s showed one major d i f f e r e n c e i n the banding p a t t e r n s of t h e i r r i bosomal p r o t e i n s a f t e r one-dimensional g e l e l e c t r o p h o r e s i s . Gels o f v a r i o u s recombinants of the two s t r a i n s i n d i c a t e d t h a t the "K p r o t e i n " l o c u s mapped near the s t r A l o c u s (Leboy, e t a l . , 1964; Mayuga, e t a l . , 1968). T h i s approach has been s u c c e s s f u l l y a p p l i e d to the mapping of s p e c i e s d i f f e r e n c e s u s i n g E. c o l i - S a Intone 1 l a typhimurium and E. c o l i - S h i g e l 1 a  d y s e n t e r i a e h y b r i d s (Osawa, e t a l . , 1971; Dekio, 1971; Dekio, e t a l . , 1970). The l a t t e r s t u d i e s i n d i c a t e d t h a t 17 the genes f o r e i g h t 30S ribosomal p r o t e i n s and nine 50S ribosomal p r o t e i n s are c l u s t e r e d i n the s p c - s t r r e g i o n between aroE and spcA. Ribosomal p r o t e i n s from v a r i o u s s p e c i e s , s t r a i n s and genotypes of D r o s o p h i l a were compared by 1-dimensional g e l e l e c t r o p h o r e s i s ( K i e f e r and Gross, 1969). No q u a l i t a t i v e d i f f e r e n c e s were observed among the v a r i o u s D. me1anogaster, Samarkand genotypes, XY, XX, XXY, XO and two bobbed s t r a i n s but some q u a n t i t a t i v e d i f f e r e n c e s were found. C l e a r - c u t q u a l i t a t i v e d i f f e r e n c e s , however, e x i s t e d between the two s p e c i e s , D. pseudoobscura and D. melanogaster and a l s o between two D. melanogaster s t r a i n s , Samarkand and Amherst. Mapping o f these d i f f e r e n c e s was not r e p o r t e d . 2. Streptomycin mutants. Streptomycin r e s i s t a n t (Str-R) mutants of E. c o l i were the f i r s t b a c t e r i a l mutants demonstrated to have an a l t e r e d ribosomal p r o t e i n d e f i n i t e l y a s s o c i a t e d with a change i n ribosomal proper-t i e s . Ribosomes from Str-R mutants continue t o s y n t h e s i z e p r o t e i n i n the presence o f s t r e p t o m y c i n whereas ribosomes from the streptomycin s e n s i t i v e (Str-S) p a r e n t a l s t r a i n s are i n h i b i t e d (Davies, 1964). Str-R ribosomes b i n d l e s s streptomycin than Str-S ribosomes ( K a j i and Tanaka, 1968). The a l t e r e d ribosomal p r o t e i n of s t r A mutants was i d e n t i f i e d as S12 by r e c o n s t i t u t i o n s t u d i e s (Ozaki, e t a l . , 1969; Traub and Nomura, 1968>. i t should be noted t h a t n o n r i b o -somal, streptomycin r e s i s t a n t mutants, not a s s o c i a t e d w i t h the s t r A l o c u s , have a l s o been obtained (Roberts and Reeve, 18 1970; Yamanda and Davies, 1971). D r o s o p h i l a are g e n e r a l l y not a f f e c t e d by h i g h c o n c e n t r a t i o n s of streptomycin i n t h e i r food. A s c a r l e t eye c o l o r (st) mutant stock, however, was found t o be s e n s i t i v e t o 1% streptomycin (Duke and Glassman, 19 68). The drug a r r e s t e d development i n the e a r l y l a r v a l stages but d i d not a f f e c t a d u l t s of the eye c o l o r stock. U n l i k e the b a c t e r i a l streptomycin r e s i s t a n c e mutations (which are u s u a l l y r e c e s s i v e ) , the D r o s o p h i l a streptomycin r e s i s t a n c e mutation was dominant to s e n s i t i v i t y (Breckenridge and G o r i n i , .1969; Duke and Glassman, 1963). The t r a i t mapped t o the 3rd chromosome. No b i o c h e m i c a l s t u d i e s of the ribosomes of the s t o c k s were r e p o r t e d . In a r e c e n t a b s t r a c t , Lambertsson and Rasmuson (1971) r e p o r t e d t h a t ribosomes from S t r - S l a r v a e of D. melanogaster bound 10 times more l a b e l e d streptomycin than those from Str-R l a r v a e . P r e l i m i n a r y r e s u l t s i n d i c a t e d t h a t streptomycin i n h i b i t e d i n v i v o p r o t e i n s y n t h e s i s i n the S t r - S l a r v a e . In two-dimensional g e l s of r ibosomal p r o t e i n s , S t r - S l a r v a e have a t l e a s t one ribosomal p r o t e i n more than Str-R l a r v a e . R e s i s t a n c e was found t o be dominant and mapped to the X chromosome. A f u l l account of t h i s work would be of i n t e r e s t because i t i n d i c a t e s t h a t drug r e s i s t a n c e may be a s u c c e s s f u l means of o b t a i n i n g r i bosomal p r o t e i n mutants of D r o s o p h i l a . 3. Suppressors. I t i s w e l l e s t a b l i s h e d t h a t mutations can be suppressed a t the t r a n s l a t i o n l e v e l i n 19 b a c t e r i a by a l t e r e d t r a n s f e r RNAs (Smith, 1972). S i n c e the e a r l y s t u d i e s o f the e f f e c t o f streptomycin on b a c t e r i a , i t has been suspected t h a t a l t e r e d ribosomes may a l s o suppress mutations a t the t r a n s l a t i o n l e v e l ( G o r i n i , e t al.,1966). Streptomycin caused misreadings of s y n t h e t i c messenger RNAs i n v i t r o (Davies, 1966). T h i s o b s e r v a t i o n seemed r e l a t e d t o the f i n d i n g t h a t streptomycin suppressed a number of mutations i n v i v o ( G o r i n i , e t a l . , 1966). Mutations t o streptomycin r e s i s t a n c e r e s u l t e d i n l e s s misreading i n v i t r o i n the presence o f the drug and r e s t r i c t i o n of the streptomycin e f f e c t on supp r e s s i o n i n v i v o . Streptomycin r e s i s t a n c e mutations can a l s o a f f e c t the e f f i c i e n c y o f su p p r e s s i o n by t r a n s f e r RNAs ( A p i r i o n and S c h l e s s i n g e r , 1967)J S t r i g i n i and G o r i n i , 1970). These o b s e r v a t i o n s l e d to the i d e a t h a t ribosomal s t r u c t u r e i n f l u e n c e s the f i d e l i t y of t r a n s l a t i o n . Recently mutants of E. c o l i were i s o l a t e d which suppressed o t h e r mutations i n v i v o and co n t a i n e d a l t e r e d ribosomes w i t h h i g h e r t r a n s l a t i o n e r r o r s i n v i t r o (Rosset and G o r i n i , 1969; G o r i n i , 1971). These were c a l l e d ram or ribosomal ambiguity mutants and mapped t o a lo c u s i n the; s p c - s t r r e g i o n o f the chromosome. The ram mutants were i s o l a t e d by l o o k i n g f o r mutations t h a t c o u n t e r a c t the r e s t r i c t i o n o f su p p r e s s i o n by streptomycin r e s i s t a n c e mutations on t r a n s l a t i o n s u p p r e s s i o n . The ram locu s o f E. c o l i may code f o r the 30S ribosomal 20 protein, S4 (Zimmermann, fet a l . , 1971). I t has now been concluded that suppression i n bacteria may r e s u l t from a change i n either transfer RNAs or ribosomes. Some mutations of Drosophila have l i t t l e e f f e c t on the phenotype by themselves but cause other mutants to appear closer to wild type (Lindsley and G r e l l , 1967). They have been designated as suppressors but i t i s unknown whether t h e i r mechanism of action i s similar to that of b a c t e r i a l suppressors. A Drosophila suppressor may correct some a l l e l e s but not others and may counteract mutations at several l o c i . At lea s t s u p e r f i c i a l l y , Drosophila suppressors resemble the b a c t e r i a l mutants. It has been suggested that Drosophila suppressors, such as i s u ( f ) , may code for altered transfer RNAs or altered ribosomes but few suppressors have been i n v e s t i -gated biochemically (Schalet, 1973; Dudick and Wright, 1973). The mutation, su(s) , which suppresses v e r m i l l i o n mutants of Drosophila has been correlated with an a l t e r a t i o n i n the chromatographic properties of a tyrosine transfer RNA (Twardzik et 1971). The e f f e c t of the suppressor, however, was not at the tr a n s l a t i o n l e v e l and su(s) i s not considered to be the locus of the altered transfer RNA (Jacobson, 1971; White, e t a l ; 1973a). As yet no bio-chemical studies of the ribosomes of Drosophila suppressors have been reported although they are being considered (Wright, 1973). 21 I t i s i n t e r e s t i n g t o n o t e t h a t r i b o s o m a l m u t a n t s o f y e a s t h a v e bejen o b t a i n e d by s e l e c t i n g f o r s t r e p t o m y c i n s u p p r e s s i o n cind t h a t o n e o f t h e s e m u t a n t s was c o l d -s e n s i t i v e ( B c i y l i s s a n d V i n o p a l , 1971). L i k e o t h e r e u k a r -y o t e s , t h e y e a s t , S a c c h a r o m y c e s c e r e v i s i a e , i s i n s e n s i t i v e t o s t r e p t o m y c i n . I t was a s s u m e d t h a t t h e i n s e n s i t i v i t y was e q u i v a l e n t t o s t r e p t o m y c i n r e s i s t a n c e i n b a c t e r i a , i . e . d u e t o ai r i b o s o m a l p r o t e i n . M u t a n t s o f a h i s t i d i n e - r e q u i r i n g s t r a i n w e r e s e l e c t e d w h i c h w o u l d g r o w i n l o w c o n c e n t r a t i o n s o f s t r e p t o m y c i n (50 jiq/ml o r l e s s ) w i t h o u t h i s t i d i n e , i . e . s t r e p t o m y c i n s e n s i t i v e m u t a t i o n s w e r e s e l e c t e d w h i c h p e r m i t t e d t h e s u p p r e s s i o n o f t h e h i s t i d i n e r e q u i r e m e n t b y s t r e p t o m y c i n . U n l i k e t h e p a r e n t a l s t r a i n , t h e m u t a n t s w e r e s e n s i t i v e t o m o d e r a t e c o n c e n t r a t i o n s o f s t r e p t o m y c i n ( a b o u t 5 0 0 ^ i g / m l ) . One o f t h e s t r e p t o m y c i n s e n s i t i v e m u t a n t s was a l s o c o l d - s e n s i t i v e a n d h a d a n a l t e r e d r i b o s o m a l p r o f i l e i n s u c r o s e g r a d i e n t s when g r o w n a t l o w t e m p e r a t u r e . A s c o l d - s e n s i t i v i t y a n d s t r e p t o m y c i n s e n s i t i v i t y w e r e n o t s e p a r a t e d i n m a p p i n g p r o c e d u r e s , t h e two t r a i t s a r e a p p a r e n t l y d u e t o o n e m u t a t i o n . U n l i k e w i l d t y p e r i b o s o m e s , t h e m u t a n t r i b o s o m e s w e r e s e n s i t i v e t o s t r e p t o m y c i n i n a n 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 s y s t e m . I t a p p e a r s t h a t t h i s c o l d -s e n s i t i v e m u t a n t o f y e a s t h a s a n a l t e r e d r i b o s o m a l p r o t e i n . ^ * C o l d - s e n s i t i v e m u t a n t s . A s d i s c u s s e d p r e v i o u s l y , s e l e c t i o n f o r c o l d - s e n s i t i v i t y i s a n o t h e r means o f o b t a i n i n g 22 r i b o s o m a l m u t a n t s o f b a c t e r i a . B e c a u s e t h e b a c t e r i a l m u t a n t s mapped i n s e v e r a l r i b o s o m a l p r o t e i n . g e n e s i t was t h o u g h t t h a t a number o f d i f f e r e n t r i b o s o m a l m u t a n t s o f D r o s o p h i l a m i g h t be o b t a i n e d b y t h i s a p p r o a c h . A l s o , when t h e p r e s e n t s t u d y was ih - i - t ia fced , l i t t l e was known a b o u t c o l d - s e n s i t i v e m u t a n t s o f e u k a r y o t e s . T h e r e f o r e , t h e f o l l o w i n g q u e s t i o n s w e r e a s k e d : C a n c o l d - s e n s i t i v e l e t h a l s o f t h e X chromosome o f D r o s o p h i l a m e 1 a n o g a s t e r be i s o l a t e d ? I f s o , w h a t a r e t h e i r g e n e t i c p r o p e r t i e s ? Do some h a v e a l t e r e d r i b o s o m a l p r o t e i n s ? T h e s t u d y was p a r t o f a g e n e r a l s e a r c h f o r a n d c h a r a c t e r i z a t i o n o f c o l d -s e n s i t i v e l e t h a l s o f a l l t h e c h r o m o s o m e s o f D. m e l a n o g a s t e r ( R o s e n b l u t h , e t a J . , 1 9 7 2 ; Mayoh a n d S u z u k i , 1 9 7 3 ; T a s a k a a n d S u z u k i , 1 9 7 3 ) . 23 1 1 . G e n e t i c S t u d i e s o f S e x - r l i n k e d R e c e s s i v e C o l d -s e n s i t i v e M u t a n t s o f D r o s o p h i l a m e l a n o g a s t e r . T h e u t i l i t y o f c o n d i t i o n a l l y l e t h a l m u t a t i o n s i n a n a l y z i n g a v a r i e t y o f m o l e c u l a r , g e n e t i c a n d d e v e l o p -m e n t a l phenomena h a s b e e n e x t e n s i v e l y d o c u m e n t e d ( E p s t e i n e t c i l . , 1 9 6 3 ; H a r t w e l l , 1 9 6 7 ; S u z u k i , 1 9 7 0 ) . T e m p e r a t u r e -s e n s i t i v e ( t s ) l e t h a l s a r e p r o b a b l y t h e s i m p l e s t c l a s s o f c o n d i t i o n a l l y e x p r e s s e d m u t a t i o n s t o r e c o v e r a n d s t u d y i n d i p l o i d e u k a r y o t e s . R e m a r k a b l y , w h i l e h e a t - s e n s i t i v e l e t h a l s map e x t e n s i v e l y a r o u n d t h e p h a g e c h r o m o s o m e ( E d g a r a n d L i e l a u s i s , 1 9 6 4 ) , c o l d - s e n s i t i v e l e t h a l s a r e h i g h l y c l u s t e r e d w i t h i n a l i m i t e d number o f c i s t r o n s ( S c o t t i , 1 9 6 9 ) . T h e f u n c t i o n a l s i g n i f i c a n c e o f t h e d i f f e r e n c e b e t w e e n t h e two c l a s s e s o f m u t a t i o n s i s s u g g e s t e d by t h e f i n d i n g t h a t a l a r g e p r o p o r t i o n o f c o l d - s e n s i t i v e l e t h a l m u t a t i o n s i n E . c o l i a f f e c t t h e s e l f - a s s e m b l y o f r i b o s o m a l s u b u n i t s ( G u t h r i e , e t a l . , 1969 a , b ; T a i , e t c i l . , 1 9 6 9 ) . M o r e -o v e r , c o l d - s e n s i t i v e m u t a t i o n s c a n a l t e r t h e r e g u l a t o r y p r o p e r t i e s o f p r o t e i n s ( O ' D o n o v a n a n d I n g r a h a m , 1 9 6 5 b ) . C l e a r l y , c o l d - s e n s i t i v i t y a s t h e s e l e c t i v e p r o p e r t y o f m u t a t i o n s r e s t r i c t s t h e s i t e s a n d f u n c t i o n s o f l o c i d e t e c t a b l e i n m i c r o - o r g a n i s m s . T h e p o s s i b i l i t y o f e n r i c h -ment f o r r e g u l a t o r y a n d r i b o s o m a l d e f e c t s by s i m p l y s e l e c t -i n g f o r c o l d - s e n s i t i v i t y h a s g r e a t p o t e n t i a l f o r t h e s t u d y o f d e v e l o p m e n t a l m e c h a n i s m s i n e u k a r y o t e s . C o n s e q u e n t l y , we d e c i d e d t o d e t e r m i n e w h e t h e r c o l d - s e n s i t i v e l e t h a l 24 m u t a t i o n s a r e r e a d i l y r e c o v e r a b l e i n D r o s o p h i l a a n d w h e t h e r t h e i r g e n e t i c p r o p e r t i e s d i s t i n g u i s h them f r o m h e a t - s e n s i t i v e l e t h a l s . T h e a n s w e r s t o t h e s e q u e s t i o n s w i l l d e t e r m i n e w h e t h e r i t i s p o s s i b l e t o e n r i c h f o r r i b o s o m a l m u t a n t s a n d o t h e r d e f e c t s s u c h a s a l t e r e d f e e d -b a c k c o n t r o l o f e n z y m e s . 25 MATERIALS AND METHODS M u t a n t S c r e e n a n d V i a b i l i t y T e s t s . T h e s c r e e n i n g p r o -c e d u r e f o r t h e d e t e c t i o n o f r e c e s s i v e c o l d - s e n s i t i v e l e t h a l m u t a t i o n s o n t h e X c h r o m o s o m e i s shown i n F i g u r e 1 . M a l e s f r o m a n i n b r e d O r e g o n - R s t o c k w e r e f e d f o r 24 h o u r s w i t h 0 .005M o r 0 .0125M e t h y l m e t h a n e s u l f o n a t e (EMS) d i s s o l -v e d i n 1% s u c r o s e ( L e w i s a n d B a c h e r , 19 6 8 ) . A p p r o x i m a t e l y 20 E M S - t r e a t e d m a l e s w e r e c r o s s e d t o 20 a t t a c h e d - X - b e a r i n g f e m a l e s (C (I) RM/Y) i n e a c h q u a r t e r - p i n t b o t t l e a t 2 2 ° C ( c h r o m o s o m a l r e a r r a n g e m e n t s a n d m a r k e r s w h i c h a r e n o t d e f i n e d h e r e c a n b e f o u n d i n L i n d s l e y a n d G r e l l , 1 9 6 8 ) . T h e u s e o f a t t a c h e d - X f e m a l e s p e r m i t t e d t h e r e c o v e r y o f m u t a g e n i z e d p a t e r n a l X c h r o m o s o m e s i n F ^ m a l e o f f s p r i n g . A s t h i s p r o c e d u r e e l i m i n a t e d a l l l e t h a l s e x c e p t c o n d i t i o n a l s i n t h e f i r s t s t e p o f t h e s c r e e n , i t was m o r e e f f i c i e n t t h a n s c r e e n s i n w h i c h t h e m u t a g e n i z e d c h r o m o s o m e i s r e c o v e r e d i n a h e t e r o z y g o u s r e c i p i e n t . S i n g l e F m a l e s w e r e m a t e d a t 2 2 ° C t o 3 o r 4 y 1 C ( 1 ) R M / Y f e m a l e s i n v i a l s ( S t e p 2 , F i g u r e 1 ) . A f t e r 4 d a y s , t h e y w e r e t r a n s f e r r e d t o f r e s h r e p l i c a t e v i a l s i n -c u b a t e d a t 17° C . O f f s p r i n g e m e r g e d a f t e r 2 w e e k s a t 2 2 ° C a n d a f t e r one m o n t h a t 17° C . T h e 17° C y i a l s w e r e s c a n n e d b r i e f l y w i t h o u t e t h e r i z i n g t h e f l i e s ( S t e p 3 , F i g u r e 1 ) . When few o r no m a l e s w e r e o b s e r v e d i n a 17° C v i a l , t h e r e p l i c a t e v i a l a t 2 2 ° C was i n s p e c t e d . I f a g r e a t e r p r o p o r t i o n o f m a l e s o c c u r r e d a t 2 2 ° C t h a n a t 17° C , t h e s t r a i n was r e t a i n e d a s a p u t a t i v e c o l d - s e n s i t i v e l e t h a l . I n some e x p e r i m e n t s , t h e f l i e s i n t h e 17° C c u l t u r e s w e r e 26 e t h e r i z e d a n d e x a m i n e d f o r c o n d i t i o n a l l y e x p r e s s e d p h e n o t y p e s , i . e . , a n a b n o r m a l p h e n o t y p e a t 1 7 ° C a n d n o r m a l o a p p e a r a n c e o r r e d u c e d p e n e t r a n c e o f t h e a b n o r m a l i t y a t 22 C . T h e m u t a t i o n s a r e d e s i g n a t e d , i n t h e o r d e r o f d e t e c t i o n a s ^ ( l ) l c s , ^ ( 1 ) 2 C S , e t c . T h e y h a v e a l s o b e e n r e f e r r e d t o b y a n a b b r e v i a t i o n , t h e number o f t h e m u t a t i o n a n d t h e c s s u p e r s c r i p t ( i ( l ) 2 c s w i l l b e 2 C S ) . M a l e s o f t h e p u t a t i v e c o l d - s e n s i t i v e s t r a i n s w e r e r e t e s t e d b y r e p e a t i n g s t e p s 2 a n d 3 ( F i g u r e 1 ) . S i x t o 12 m a l e s w e r e i n d i v i d u a l l y t e s t e d i n e a c h t r i a l . T h e r e l a t i v e v i a b i l i t i e s o f t h e m u t a n t c h r o m o s o m e s w e r e a s s e s s e d b y d e t e r m i n g t h e s e x r a t i o s a t 2 2 ° C a n d 1 7 ° C . A l l s t o c k s w e r e t e s t e d a t l e a s t t w i c e and some w e r e c h e c k e d s e v e r a l t i m e s w i t h i n a p e r i o d o f t h r e e y e a r s . T h e c o n -f i r m e d c s m u t a t i o n s w e r e m a i n t a i n e d o n t h e X c h r o m o s o m e o f m a l e s m a t e d t o C ( l ) R M / y a n d C ( 1 ) D X / Y f e m a l e s a t 2 2 ° C i n b o t t l e s . When h e t e r o g e n e i t y was o b s e r v e d i n a v i a -b i l i t y t e s t , t h o s e v i a l s y i e l d i n g t h e l o w e s t v i a b i l i t i e s a t 1 7 ° C b u t h i g h v a l u e s a t 2 2 ° C w e r e u s e d t o p e r p e t u a t e t h e s t o c k . T e s t i n g F e m a l e F e r t i l i t y . F e m a l e s h o m o z y g o u s f o r t h e o m u t a t i o n s w e r e g e n e r a t e d a n d t e s t e d f o r f e r t i l i t y a t 22 C by m a t i n g them w i t h F M 6 / Y a n d m u t a n t m a l e s a t 2 2 ° C . I n some e x p e r i m e n t s , one t o t h r e e f e m a l e s 1 t o 6 d a y s o l d w e r e p r e s e n t i n e a c h v i a l a l o n g w i t h 3 m a l e s o f e a c h t y p e . In o t h e r t e s t s , f e m a l e s w e r e m a t e d s e p a r a t e l y w i t h t h e 27 two t y p e s o f m a l e s . A n a v e r a g e o f 25 f e m a l e s was t e s t e d f o r e a c h m u t a n t . A d u l t s w e r e d i s c a r d e d a f t e r 1 week a n d p r o g e n y s c o r e d f o r a t l e a s t 9 d a y s a f t e r e c l o s i o n b e g a n . When few o f f s p r i n g o c c u r r e d , t h e v i a l s w e r e b r i e f l y s c a n n e d f o r s i g n s o f l e t h a l i t y d u r i n g d e v e l o p m e n t , i . e . , u n h a t c h e d e g g s , d e a d l a r v a e o r p u p a e , a n d t u n n e l l i n g o f t h e medium w i t h o u t t h e o c c u r r e n c e o f p u p a e o r a d u l t s . G e n e t i c M a p p i n g . T h e c s m u t a n t s w e r e mapped u s i n g t h e f o l l o w i n g m a r k e r s l i s t e d w i t h t h e i r s y m b o l s a n d g e n e t i c p o s i t i o n s : y e l l o w - y , 0 . 0 ; c r o s s v e i n l e s s - c v , 1 3 . 7 ; v e r m i 1 i o n - ' v , 3 3 . 0 ; f o r k e d - f_, 5 6 . 7 ; a n d c a r n a t i o n -c a r 6 2 . 5 . T h e s e m a r k e r s s p a n m o s t o f t h e e u c h r o m a t i n o f t h e X chromosome f r o m t h e d i s t a l t i p t o t h e p r o x i m a l a r e a b u t do n o t i n c l u d e t h e h e t e r o c h r o m a t i c r e g i o n a r o u n d t h e c e n t r o m e r e . JL(1)CS/Y m a l e s w e r e c r o s s e d a t 2 2 ° C t o h o m o z y g o u s y c v v f_ c a r f e m a l e s , a n d t h e f e m a l e s w e r e t e s t c r o s s e d i n q u a r t e r - p i n t b o t t l e s a t 1 7 ° C . M a l e o f f s p r i n g w e r e s c o r e d f o r 20 d a y s . M a l e s f r o m t h e O r e g o n - R w i l d - t y p e s t o c k w e r e m a t e d i n t h e a b o v e scheme a s c o n t r o l s . T h o s e m u t a n t s e x h i b i t i n g a b n o r m a l p h e n o t y p e s a t 2 2 ° a n d l e s t h a l i t y a t 1 7 ° C w e r e mapped a t b o t h t e m p e r a t u r e s . M u t a t i o n s w h i c h mapped t o t h e r i g h t o f c a r n a t i o n w e r e t e s t e d f u r t h e r b y c o m b i n i n g them w i t h a d u p l i c a t i o n o r v a r i o u s d e l e t i o n s t h a t e n c o m p a s s m o s t o f t h e p r o x i m a l h e t e r o c h r o m a t i n . T h e r e a r r a n g e m e n t s u s e d w e r e t h e X - Y 28 i n s e r t i o n , y_ Y m a l , a n d t h e d e l e t e d X c h r o m o s o m e s , D f ( T ) m a l 1 2 , D f ( l ) m a l 6 a n d I n ( 1 ) s c 4 L s c 8 R ( F i g u r e s 2 a n d 3 ) . E x t e n s i v e d e s c r i p t i o n s o f t h e c h r o m o s o m e s c a n be f o u n d i n L i n d s l e y a n d G r e l l (1968) a n d S c h a l e t a n d F i n n e r t y ( 1 9 6 8 a , b ) . A l l c r o s s e s w e r e c a r r i e d o u t a t 1 7 ° a n d 2 2 ° C w i t h a v i e w t o d e m o n s t r a t i n g p s e u d o d o m i n a n t e x p r e s s i o n o f t h e m u t a n t p h e n o t y p e a t 1 7 ° C ( e i t h e r l e t h a l i t y o r a v i s i b l e p h e n o t y p e ) w i t h t h e p r o x i m a l d e l e t i o n o r a w i l d - t y p e p h e n o t y p e w i t h t h e d u p l i c a t i o n . None o f t h e m u t a t i o n s t e s t e d f e l l i n t h e r e g i o n s c o v e r e d b y t h e d e l e t i o n s o r d u p l i c a t i o n . 29 RESULTS Screen and V i a b i l i t y T e s t s . From the progeny of 3,919 i n d i v i d u a l l y - t e s t e d F^ males, 151 stocks were r e t a i n e d as p o s s i b l e c o l d - s e n s i t i v e mutants. Of these, o n l y 32 had a male to female r a t i o g r e a t e r than 0.29 a t 22° and l e s s than 0.16 a t 17° C i n the f i r s t v i a b i l i t y t e s t . Only 25 stocks f i t t e d these requirements i n a t l e a s t two v i a b i l i t y t e s t s . In a d d i t i o n , two mutants w i t h a c o l d s e n s i t i v e phenotypic e f f e c t were recovered and v e r i f i e d . The Oregon- RO*x C ('!') RM/Y<^  c o n t r o l c r o s s e s showed l i t t l e change i n sex r a t i o a t the two temperatures. The male t o female r a t i o of the c o n t r o l was 1.37 (1,252 f l i e s scored) a t 22° C and 1.32 (1,095 f l i e s scored) a t 17° C when the f l i e s were r a i s e d i n vial's (Table 1) . These r a t i o s were c o n s i d e r e d t o r e p r e s e n t the standard v i a b i l i t y a g a i n s t which the r a t i o s of the mutants were compared. T h i s was done by the equation ( r ^ / r Q ) 100 where r ^ = male/female r a t i o i n the mutant c r o s s and r = male/female r a t i o i n o c o n t r o l s . The l i m i t s f o r r e t e n t i o n of the mutants were a r b i t r a r i l y s e t a t g r e a t e r than 21% r e l a t i v e v i a b i l i t y a t 22° and l e s s than 13% a t 17° C. Twelve of the 25 mutants r e t a i n e d were l e t h a l a t o ° 17 C and had v i a b i l i t i e s g r e a t e r than 20% a t 22 C i n the f i r s t v i a b i l i t y t e s t (Tables 1 and 11). The remaining 13 were s e m i - l e t h a l w i t h g r e a t e r than 0% but l e s s than 13% 30 v i a b i l i t y a t 17° C and g r e a t e r than 30% v i a b i l i t y a t 22° C. The s e m i l e t h a l s showed at l e a s t 4.6 times g r e a t e r v i a b i l i t y a t the p e r m i s s i v e temperature than at the r e s t r i c t i v e temperature. In subsequent t e s t s , the mutants e x h i b i t e d i n c r e a s i n g v i a b i l i t i e s a t 17° C d e s p i t e s e l e c t i o n f o r c o l d -s e n s i t i v i t y . The l e t h a l s , f o r example, became semi-l e t h a l s on or b e f o r e the t h i r d t r i a l . V i a b i l i t i e s a t 22° C a l s o changed, e i t h e r i n c r e a s i n g or d e c r e a s i n g o r both i n subsequent t e s t s . The v i a b i l i t i e s of the two types of s t o c k s , C(1)RM/Y and C(1)DX/Y, o f the same mutant, g e n e r a l l y d i f f e r e d although the v i a b i l i t y t e s t s were c a r r i e d out w i t h C(1)RM/Y females The reasons f o r the i n s t a b i l i t y o f the s t o c k s can o n l y be surmised. The i n i t i a l d r a s t i c d e c l i n e from 151 p u t a t i v e mutants to 34 c o l d - s e n s i t i v e l e t h a l s t o c k s a f t e r the f i r s t v i a b i l i t y t e s t was due i n l a r g e p a r t t o the t e c h n i c a l d i f f i c u l t y of a c c u r a t e l y a s s e s s i n g the month-old v i a l s kept a t 22° C. Consequently, stocks t h a t were sub-v i t a l a t both temperatures were probably r e t a i n e d . In screens f o r h e a t - s e n s i t i v e mutants induced by EMS, i t has a l s o been noted t h a t many stock s are no longer h e a t - s e n s i t i v e l e t h a l s i n the f i r s t r e t e s t . For example, out o f 186 p u t a t i v e h e a t - s e n s i t i v e l e t h a l s of the X chromosome, o n l y 90 were confirmed on the f i r s t r e t e s t (Suzuki, e t a l . , 1967). T h i s i n i t i a l u n r e p r o d u c i b i l i t y seems t o be c h a r a c t e r i s t i c of mutations induced by EMS and has been a t t r i b u t e d to mosaicism (Jenkins, 1967; E p l e r , 1966). 31 The i n s t a b i l i t y o f t s s t r a i n s has a l s o been r e p o r t e d f o r Habobracon (Smith, 1968) and Paramecium ( I g a r a s h i , 1966). Other causes o f v a r i a b i l i t y o f the c o l d - s e n s i t i v e stocks may be changes i n the g e n e t i c background and i n h e r e n t i n s t a b i l i t y of c o l d - s e n s i t i v e mutations. In f a c t , HRF. Wright (1973) has made s i m i l a r o b s e r v a t i o n s and has found t h a t changes i n v i a b i l i t y of cs l e t h a l s are most o f t e n the r e s u l t of accumulation of g e n e t i c m o d i f i e r s elsewhere i n the genome. Leakiness and r e v e r s i o n o f cs mutants of microorganism® has a l s o been r e p o r t e d (Cox and S t r a c k , 1971). Phenotypes. Some of the mutants a t 22° C e x h i b i t e d abnor-mal phenotypes which resembled the phenotype of bobbed, bb, and abnormal abdomen, a. The males and females homozygous f o r 5 C S , 6 C S and 1 5 c s had m i s s i n g or reduced s t e r n i t e s , etched, m i s s i n g or angled t e r g i t e s , and b l a c k specks on the v e n t r a l s u r f a c e o f the abdomen. The mutants sometimes e x h i b i t e d p i n c h i n g of the l a s t segment near the g e n i t a l p l a t e . The occurrence of these t r a i t s was more fre q u e n t and extreme among s u r v i v o r s a t the r e s t r i c t i v e temperatures than among the o f f s p r i n g a t the p e r m i s s i v e temperature (Table I I ) . S u r v i v o r s a t 17° C a l s o had e x t r a wing v e i n s , c s cs Mutants 26 and 27 a l s o e x h i b i t e d abdominal abnormalties s i m i l a r to 5f : j V 6 C S and 1 5 ° s and the t r a i t s were more frequent and severe a t 17° C than a t 22° C (Table I I ) . In a d d i t i o n , f l i e s c a r r y i n g mutant 2 6 c s sometimes e x h i b i t e d a r o t a t e d g e n i t - i a l p l a t e . Although mutants 2 6 c s and 2 7 c s 32 w e r e s l i g h t l y l e s s v i a b l e a t 1 7 ° t h a n a t 2 2 ° C , t h e y w e r e n o t l e t h a l a n d t h e r e f o r e a r e r e f e r r e d t o a s c o l d - s e n s i t i v e p h e n o t y p i c m u t a n t s . T h e f r e q u e n c i e s o f t h e a b n o r m a l abdomen t r a i t i n t h e s e 5 m u t a n t s was e x t r e m e l y v a r i a b l e ( T a b l e I I ) a s was n o t e d p r e v i o u s l y f o r t h e i r v i a b i l i t i e s . c s T h e v i s i b l e p h e n o t y p i c a b n o r m a l i t y o f m u t a t i o n s 26 a n d 2 7 c s w e r e mapped a t 1 7 ° C , t h e t e m p e r a t u r e a t w h i c h t h e a b n o r m a l abdomen t r a i t was m o s t p e n e t r a n t ( T a b l e I I I ) . M a p p i n g d e m o n s t r a t e d t h a t t h e s e two m u t a t i o n s w e r e l o c a t e d i n d i f f e r e n t a r e a s f r o m o n e a n o t h e r a s w e l l a s f r o m t h e o t h e r m u t a n t s e x h i b i t i n g a b n o r m a l abdomens ( 5 C S , 6 C S a n d 1 5 c s ) . T h e a b n o r m a l abdomen t r a i t o f m u t a n t 2 6 C S mapped t o t h e r e g i o n b e t w e e n y a n d c v , a n d t h a t o f m u t a n t 2 7 c s i n t h e t i p o f t h e X n e a r y . N o t e t h a t i n t h e c r o s s c s i n v o l v i n g 26_ , a l l c l a s s e s c a r r y i n g c v w e r e r e d u c e d i n number when c o m p a r e d w i t h t h e i r r e s p e c t i v e r e c i p r o c a l c r o s s o v e r c l a s s e s . T h e b a s i s f o r t h i s o b s e r v a t i o n i s u n c e r t a i n b u t i t may i n d i c a t e t h e p r e s e n c e o f a s e g r e g a t i n g a u t o s o m a l m u t a t i o n w h i c h p r o d u c e d a d e t r i m e n t a l e f f e c t i n c o m b i n a t i o n w i t h c v . In a d d i t i o n , 8% o f t h e f e m a l e o f f -s p r i n g e x h i b i t e d t h e a b n o r m a l abdomen t r a i t a s c o m p a r e d t o 21% o f t h e m a l e s i b l i n g s . T h e a p p e a r a n c e o f t h e t r a i t i n t h e f e m a l e s , h o w e v e r , was f a i r l y e v e n l y d i s t r i b u t e d among t h e v a r i o u s c r o s s o v e r c l a s s e s a n d t h e r e f o r e , c o u l d n o t be a t t r i b u t e d t o a s p e c i f i c r e g i o n o f t h e X c h r o m o s o m e . I t c s was a l s o n o t e d t h a t i n t h e v i a b i l i t y t e s t s o f m u t a n t 26 a s m a l l p e r c e n t a g e o f a t t a c h e d - X f e m a l e o f f s p r i n g showed 33 the abnormal abdomen t r a i t . Again, the presence of an autosomal f a c t o r i n mutant 2 6 c s i s i n d i c a t e d . C l e a r l y cs mutant 26 i s compled and i t s p r o p e r t i e s cannot be a t t r i -buted to a s i n g l e mutation. c s c s The s i m i l a r i t y i n the phenotypes of mutants 5 _ , & c s and 15' and the i n c r e a s e d penetrance o f these t r a i t s a t 17° C, suggested t h a t the three mutants might be a l l e l e s w i t h the abnormal phenotype r e l a t e d t o the c o l d - s e n s i t i v e l e t h a l i t y . Indeed, the mapping data supported these o i d e a s . L e t h a l i t y at 17 C mapped t o the r i g h t o f c a r i n each case (Table 111). Moreover, the few abnormal abdo-men f l i e s o c c u r r i n g a t 17° C were i n c r o s s o v e r c l a s s e s c o n s i s t e n t w i t h t h i s l o c a t i o n . Most of the 22° C mapping data a l s o supported the c o n c l u s i o n t h a t the abnormal abdo-men t r a i t was l o c a t e d t o the r i g h t o f c a r . The s m a l l number of ex c e p t i o n s f o r mutants 5 C S (6/856) and 6 C S (2/1,199) were d i s t r i b u t e d among a l l the c r o s s o v e r c l a s s e s and may have been due t o a second c r o s s o v e r to the r i g h t of the cs mutation, or the e x c e p t i o n s may have been due t o s e l e c t i o n of a d d i t i o n a l f a c t o r s d u r i n g the maintenance of the s t o c k s . c s c s c s The l o c a t i o n s of mutations S , 6 , and 15 were more s p e c i f i c a l l y d e f i n e d by d u p l i c a t i o n - d e f i c i e n c y mapping •{Table IV) . None of them f e l l w i t h i n the r e g i o n spanned by the rearrangements d i s c u s s e d e a r l i e r . Only two s m a l l s e c t i o n s of the X chromosome were not covered by the d u p l i c a t i o n - d e f i c i e n c y mapping; the heterochromatic r e g i o n 34 i m m e d i a t e l y a d j a c e n t t o t h e c e n t r o m e r e ( F i g u r e 2) a n d t h e a r e a b e t w e e n c a r a n d .4.34 ( F i g u r e 3 ) . A s t h e m u t a -t i o n s 5 C S , 6 v S a n d 15 c s b e h a v e d s i m i l a r l y i n a l l t h e m a p p i n g p r o c e d u r e s , i t was c o n c l u d e d t h a t t h e y w e r e l o c a t e d t o t h e r i g h t o f c a r i n o n e o f t h e a b o v e two r e g i o n s . M u t a t i o n s 5 C S , 6 C S a n d 15 C S b e h a v e d l i k e a l l e l e s i n c o m p l e m e n t a t i o n t e s t s ( T a b l e V ) . D i f f e r e n t c o l d - s e n s i t i v e m u t a t i o n s w e r e c o m b i n e d i n t r a n s - h e t e r o z y g o t e s b y c r o s s i n g F M 6 / j . ( l ) C S f e m a l e s o f one s t r a i n w i t h ^ ( 1 ) C S / Y m a l e s o f a ° o d i f f e r e n t s t r a i n a t b o t h 22 a n d 17 C . T h e r a t i o o f t r a n s - h e t e r o z y g o u s f e m a l e s f o r t h e two l e t h a l s t o F M 6 -b e a r i n g f e m a l e s was u s e d a s a m e a s u r e o f t h e v i a b i l i t y o f t h e t r a n s - h e t e r o z y g o t e . T h e f i g u r e s i n b r a c k e t s i n T a b l e V i n d i c a t e t h e number o f f l i e s i n v o l v e d i n t h e c a l c u l a t i o n o f e a c h v i a b i l i t y r a t i o . I f a t r a n s - h e t e r o z y g o t e g a v e a l o w v i a b i l i t y r a t i o a t 1 7 ° C a n d a h i g h f r e q u e n c y o f a b n o r -m a l abdomen t r a i t a t 2 2 ° C , t o a n e x t e n t c o m p a r a b l e t o b o t h o f t h e two h o m o z y g o u s s t o c k s , t h e n t h e two m u t a n t s w e r e c o n s i d e r e d t o b e a l l e l i c . F o r e x a m p l e , t h e t r a n s -h e t e r o z y g o t e o f m u t a n t s 5 c S a n d 1 5 c 5 h a d a v i a b i l i t y r a t i o r> o o f 0 . 0 3 6 4 a t - 1 7 C a n d 45% a b n o r m a l abdomen t r a i t . a t 22 C ( t h e same t r a n s - h e t e r o z y g o t e f r o m t h e r e c i p r o c a l c r o s s , h a d a r a t i o o f 0 . 1 4 1 a n d 52% a b n o r m a l a b d o m e n ) . T h e h o m o z y -C S C S g o t e s o f m u t a n t s 5 a n d 15 h a d p r o p e r t i e s s i m i l a r t o t h e t r a n s - h e t e r o z y g o t e ; v i a b i l i t y r a t i o s o f 0 . 0 4 4 1 a n d 0 . 0 4 6 0 a n d 52% a n d 62% e x p r e s s i o n o f a b n o r m a l a b d o m e n , r e s p e c t i v e l y . On t h e o t h e r h a n d , t h e t r a n s - h e t e r o z y g o t e s 35 c s f o r m e d f r o m e i t h e r m u t a n t w i t h O r e g o n - R o r m u t a n t s 26 , c s c s c s 16 , 27 a n d 21 h a d h i g h v i a b i l i t y r a t i o s , i . e . , "*> 0 . 7 5 a n d <Cl% a b n o r m a l a b d o m e n . O f t h e s e v e n m u t a n t s t e s t e d , o n l y m u t a n t s 5 , 6 C S a n d 15 d i d n o t c o m p l e m e n t o n e a n o t h e r . c s c s A l l f l i e s i n two o t h e r s t o c k s , 16 a n d 20 , e x h i b i -t e d f i n e s h o r t b r i s t l e s a n d some e t c h i n g o f t e r g i t e s a t 22 C . I n a d d i t i o n , f l i e s c a r r y i n g 16 o c c a s i o n a l l y h a d c s i n d e n t e d a n d r e d u c e d e y e s a n d i n d e n t e d w i n g s . B o t h 16 c s o a n d 20 were; l e t h a l a t 17 C . T h e v i s i b l e p h e n o t y p e s o f t h e f i v e m u t a n t s s o f a r c o n s i d e r e d a r e s i m i l a r t o t h e e f f e c t s o f v a r i o u s a l l e l e s o f bb ( s e e L i n d s l e y a n d G r e l l , 19 6 8 ) . T h e known r o l e o f b b i n t h e s p e c i f i c a t i o n o f r i b o s o m a l RNA ( R i t o s s a , e t a l . , 1965) s u g g e s t s t h a t a b o b b e d p h e n o t y p e m i g h t r e f l e c t d e f e c t i v e r i b o s o m e p r o d u c -t i o n . c s In a d d i t i o n , 10 f l i e s when e x p o s e d t o c o l d s h o c k s d u r i n g d e v e l o p m e n t , e x h i b i t e d a b d o m i n a l t u m o r s , a s m a l l i n c i d e n c e o f p l e x u s a n d b l i s t e r e d w i n g s a n d o c c a s i o n a l l o s s o f e x t e r n a l a d u l t g e n i t a l i a (W. W i l l i s , p e r s o n a l c s c o m m u n i c a t i o n ) . 8 m a l e s a n d f e m a l e s h a d u p h e l d w i n g s a t b o t h 2 2 ° a n d 1 7 ° C ( e s c a p e r s ) . T h e a l u l a e o f t h e w i n g s a p p e a r n o r m a l a n d a r e n o t i n v o l v e d i n t h e m u t a n t p h e n o t y p e . A n y p h e n o t y p i c a b n o r m a l i t y o b s e r v e d w i t h i n a c s m u t a n t s t o c k c o u l d b e a p l e i o t r o p i c e f f e c t o f t h e m u t a t i o n b e i n g s t u d i e d o r t h e e x p r e s s i o n o f a s e c o n d m u t a t i o n 36 induced elsewhere on the chromosome. Consequently, i t i s necessary to map both the cs l e t h a l d e f e c t a t 17° C and the v i s i b l e phenotype a t 22° C (Table I I I ) . I t can be seen t h a t i n every case, the p o s i t i o n of the cs l e t h a l corresponds very c l o s e l y t o the r e g i o n i n which the mutation e x h i b i t i n g the v i s i b l e d e f e c t a l s o maps (Table I I I ) . V i a b i l i t y o f Mutant Females. T h e o r e t i c a l l y , the s c r e e n -i n g p r o t o c o l (Figure 1) should a l l o w the r e c o v e r y o f dominant as w e l l as r e c e s s i v e c o l d - s e n s i t i v e l e t h a l mutations. In f a c t , a l l o f the mutants i s o l a t e d proved u cs to be r e c e s s i v e as shown by the v i a b i l i t y o f a l l JC (1) /FM6 and JL(1)°S/+ females a t 17° C. T h i s i s not s u r p r i s i n g s i n c e a l l mutations but one s e l e c t e d by t h e i r dominant heat- o r c o l d - s e n s i t i v e l e t h a l i t y have been found t o be r e c e s s i v e l e t h a l s a t the p e r m i s s i v e temperature (Suzuki and P r o c u n i e r , 1 9 6 9 ; Rosenbluth, e t a l . , 1972; Holden and Suzuki, 1973). The absence of dominant cs mutants i n the X chromosome screen may, t h e r e f o r e , have been due t o l e t h a l i t y o f dominant cs mutations i n the hemizygous s t a t e a t the p e r m i s s i v e temperature. Moreover, a f t e r s e l e c t i o n of 400 r e c e s s i v e t s l e t h a l s i n y e a s t , H a r t w e l l (1967) found t h a t o n l y 4 behaved as dominant t s l e t h a l s i n d i p l o i d s . A comparison of s u r v i v a l o f J L(D heterozygotes and homozygotes was made by c r o s s i n g j£(T) C 5/FM6 females t o j f f ( l ) C S / Y males f o r 4 days a t 22° C then a t 17° C f o r a week. FM6/£( 1) o f f s p r i n g o c c u r r e d i n l a r g e numbers at both 37 t e m p e r a t u r e s w h e r e a s m a l e s a n d h o m o z y g o u s f e m a l e s g e n e r a l l y w e r e g r e a t l y r e d u c e d i n n u m b e r s a t 17° C ( T a b l e V I ) . T h e v i a b i l i t i e s o f t h e o f f s p r i n g f r o m t h e a b o v e c r o s s w e r e a s s e s s e d b y t h e r a t i o o f jH.(T) / Y m a l e s o r Jj( 1) f e m a l e s t o jff ( T ) ° S / F M 6 f e m a l e s . F M 6 / Y m a l e s w e r e n o t u s e d a s a s t a n d a r d b e c a u s e t h e y w e r e s u b v i t a l a t b o t h t e m p e r a -t u r e s . T h e v i a b i l i t y r a t i o s o f t h e O r e g o n - R c o n t r o l d i f -f e r e d l i t t l e a t t h e two t e m p e r a t u r e s ; f o r t h e m a l e s , t h e r a t i o s w e r e 0 . 9 2 a t 2 2 ° C a n d 0 . 9 1 a t 1 7 ° C , a n d f o r t h e h o m o z y g o u s f e m a l e s , 1 .02 a t 2 2 ° C a n d 1 .03 a t 1 7 ° C . T h e f o u r v a l u e s w e r e d e r i v e d f r o m 8 6 7 , 8 8 9 , 842 a n d 947 f l i e s , r e s p e c t i v e l y , . T h e r a t i o s f o r t h e O r e g o n - R c o n t r o l w e r e c o n s i d e r e d t o r e p r e s e n t 100% v i a b i l i t y . R e l a t i v e v i a -b i l i t i e s o f t h e m u t a n t s h a v e b e e n c a l c u l a t e d a s f o l l o w s : f o r m a l e s , (1(1) ° 5 / Y < f l / (FM6/Ji( 1) C S g ) x 100 (FM6/X$) / Oregon-R<$ a n d f o r f e m a l e s , (1(1)cs/Jj( 1 ) c 5 $ ) / ( F M 6 / j ( 1 ) c 5 g ) x 100 ( F M 6 / X f t ) / ( O r e g o n - R f f l . T h e f i g u r e s i n b r a c k e t s b e l o w t h e r e l a t i v e v i a b i l i t i e s o f T a b l e V I i n d i c a t e t h e number o f f l i e s i n v o l v e d i n t h e c a l c u l a t i o n o f e a c h v i a -b i l i t y r a t i o . , G e n e r a l l y , m a l e a n d f e m a l e v i a b i l i t i e s i n e a c h j j ( l ) c s s t o c k w e r e s i m i l a r b o t h a t 2 2 ° a n d 1 7 ° C ( T a b l e V I ) . T h e m o s t s t r i k i n g e x c e p t i o n t o t h i s g e n e r a l i z a t i o n was c s c s m u t a n t 2 . T h e r e l a t i v e v i a b i l i t y o f t h e h o m o z y g o u s 2 f e m a l e s was t w i c e a s g r e a t a s t h a t o f m a l e s a t 2 2 ° C a n d a l t h o u g h f e m a l e v i a b i l i t y d e c l i n e d m o d e r a t e l y a t 17° C , t h e v a l u e was s t i l l 13 t i m e s g r e a t e r t h a n t h a t f o r t h e 38 males a t 17 C. T h i s mutant was a l s o unusual i n t h a t the homozygous females were the most fr e q u e n t c l a s s of o f f -o o s p r i n g a t both 22 C and 17 C and exceeded the heterozy-gous females i n v i a b i l i t y . Sexual dimorphism has p r e v i o u s -l y been r e p o r t e d f o r h e a t - s e n s i t i v e mutants ( T a r a s o f f and Suzuki, 1970) and n o n - c o n d i t i o n a l l e t h a l s (Thompson 1921; R e d f i e l d , 1926) of D r o s o p h i l a . Whether such a mechanism c s i s i n v o l v e d w i t h mutant 2 remains to be determined by f u r t h e r t e s t s . Spoffor (J (1961) has shown t h a t g e n e t i c e x p r e s s i o n can be a f f e c t e d by the p a r e n t a l source of a chromosome. Indeed, f u n c t i o n i n g of ribosomal genes of maternal chromo-somes d u r i n g oogenesis has been e x t e n s i v e l y documented (Brown and Dawid, 1968). I f cs l e t h a l s do, i n f a c t , e n r i c h f o r s e l e c t i o n f o r ribosomal d e f e c t s , one might expect an e f f e c t of p a r e n t a l source of the mutation on i t s v i a b i l i t y . I t can be seen t h a t each J^(l) i s p a t e r n a l l y (Tables 1 and 11) and m a t e r n a l l y (Table VI) i n h e r i t e d so t h a t a comparison can be made. While v i a b i l i t y v a r i e s from t e s t t o t e s t (compare Tables 1 and V I ) , t h e r e i s no A cs s t r i k i n g p a r e n t a l e f f e c t on s u r v i v a l of X(D / Y males. Female F e r t i 1 1 t y . During oogenesis of some i n s e c t s and amphibians, extrachromosomal ribosomal RNA i s e x t e n s i v e l y a m p l i f i e d i n the oocyte (Brown and Dawid, 1968; G a l l , 1969). These molecules are then t r a n s c r i b e d t o p r o v i d e a ribosome-r i c h cytoplasm f o r the egg. In other organisms, where 39 t h e s y n t h e s i s o f RNA f o r t h e o o c y t e i s t a k e n o v e r by n u r s e c e l l s , s e l e c t i v e r e p l i c a t i o n o f o o c y t e r i b o s o m a l DNA i s l e s s common. I n D. m e l a n o g a s t e r , f o r e x a m p l e , 15 h i g h l y p o l y p l o i d n u r s e c e l l s a d j a c e n t t o t h e o o c y t e , s e r v e t o m u l t i p l y t h e rDNA f o r t r a n s c r i p t i o n b y t h o u s a n d s o f t i m e s ( K i n g , 1970) . A u t o r a d i o g r a p h i c s t u d i e s show t h a t RNA i s t r a n s p o r t e d o v e r c y t o p l a s m i c b r i d g e s b e t w e e n t h e n u r s e c e l l s a n d t h e o o c y t e . H e n c e g e n e t i c d e f e c t s i n r i b o s o m e p r o d u c t -i o n . may b e d e t e c t a b l e b y d e c r e a s e d p r o d u c t i o n o r s u r v i v -a l o f e g g s f r o m m u t a n t f e m a l e s . I t h a s b e e n s h o w n , f o r e x a m p l e , t h a t D r o s o p h i l a f e m a l e s w i t h e x t e n s i v e d e l e t i o n s i n N . O . r e g i o n s p r o d u c e f e w e r e g g s t h a n w i l d - t y p e f e m a l e s (Mohan a n d R i t o s s a , 1970) . S t e r i l i t y o r l o w f e r t i l i t y , t h e r e f o r e , cam be a n o t h e r c r u d e c r i t e r i o n f o r s e l e c t i o n o f a r i b o s o m a l d e f e c t . T h e r e s u l t s o f c r o s s e s o f i n d i v i d u a l f e m a l e s w i t h FM6/Y o r Ml)CS/Y m a l e s s e p a r a t e l y o r b o t h t o g e t h e r a r e s u m m a r i z e d i n T a b l e V I I . T h e a v e r a g e number o f p r o g e n y p e r f e m a l e i n a s e v e n d a y e g g l a y i n g p e r i o d i s shown w i t h t h e number o f f e m a l e s t e s t e d l i s t e d i n b r a c k e t s . O f t h e 27 s t o c k s , 8 p r o d u c e d an a v e r a g e o f 51 p r o g e n y o r m o r e p e r f e m a l e w h e r e a s 7 w e r e c o m p l e t e l y s t e r i l e . T h e r e s t c s v a r i e d f r o m a l m o s t c o m p l e t e s t e r i l i t y ( s e e m u t a n t 11 ) t o a b o u t a q u a r t e r t h e f e r t i l i t y o f t h e w i l d - t y p e c o n t r o l . T h u s , 70% (19/27) o f t h e m u t a t i o n s s e l e c t e d o n t h e b a s i s o f c o l d - s e n s i t i v i t y h a d r e d u c e d o r no f e r t i l i t y . T h i s 40 i s i n c o n t r a s t t o the o b s e r v a t i o n t h a t 57% (50/88) of a l l EMS-induced h e a t - s e n s i t i v e l e t h a l s produce v i a b l e and f e r t i l e homozygous females (Suzuki and P i t e r n i c k , 1972) . No eggs or o t h e r i n d i c a t i o n s of o f f s p r i n g were ob-\ cs served i n the v i a l s c o n t a i n i n g females mutant f o r 12 cs and 17 A l s o , a t l e a s t h a l f of these females were dead when the f e r t i l i t y t e s t v i a l s were c l e a r e d (9 days a f t e r the females had emerged). The i n f e r t i l i t y o f these females i s apparently; j u s t one aspect of t h e i r abnormal p h y s i o l o g y at the p e r m i s s i v e temperature. S t e r i l e mutants 5 C S , 1 3 c s c s and 15 produced eggs which f a i l e d t o hatch. Even a t the p e r m i s s i v e temperature, the eggs of these females f a i l e d t o support normal embryogenesis r e g a r d l e s s of the genotype of the f e r t i l i z i n g sperm. A l s o , mutant females 6 C S , T 6 C S , 2 ( ) c s and 2 4 c s which were p o o r l y f e r t i l e f r e -q u e n t l y l a i d eggs which f a i l e d t o hatch. S t e r i l e mutants 3 C S , 1 0 c s , l l c s and 2 6 c s e x h i b i t e d evidence t h a t develop-ment sometimes progressed as f a r as the l a r v a l and pupal stages. As more than one mutation may be p r e s e n t i n each stock, the observed i n f e r t i l i t y may not be due t o the mutation c a u s i n g c o l d - s e n s i t i v e l e t h a l i t y . Of those homozygous females which were f e r t i l e , a l l c s except mutant 23 y i e l d e d a l l expected types of o f f s p r i n g . Mutant 2 3 c s females, when cr o s s e d t o mutant males and FM6 males, o n l y y i e l d e d FM6/j(T)2 3 C S o f f s p r i n g . Jj(l) 2 3 c s 41 males and probably females f a i l e d t o develop normally. T h i s o b s e r v a t i o n suggests t h a t the a d d i t i o n of the w i l d -type a l l e l e t o the zygote a t f e r t i l i z a t i o n was necessary, even a t the p e r m i s s i v e temperature, to overcome some d e f i c i e n c y i:n the eggs l a i d by homozygous JL{1) 2 3 C S females. A s i m i l a r p a t t e r n of l i m i t e d f e r t i l i t y has been r e p o r t e d f o r the s e x - l i n k e d , r e c e s s i v e v i s i b l e mutants dor, f u and r (Counce, 1956a,b,c) and a t s l e t h a l ( T a r a s o f f and Suzuki, 1970). A l l the v i s i b l e phenotypes were r e c e s s i v e except f o r c s the abnormal abdomen t r a i t o f mutant 26 The l a t t e r can be c o n s i d e r e d semi-dominant because a t 22° C, 5% of 9 1 i d ) 2 6 C S / F M 6 females e x h i b i t e d the t r a i t , and a t 17° C, 48% of 62 females were a f f e c t e d . In the same c r o s s , 39% o f 106 homozygous females and 9% o f 91 hemizygous males e x h i b i t e d the t r a i t a t 22° C, whereas a t 17° C, 69% of the homozygous females (48) and 44% of the males (41) were a f f e c t e d . Mapping. Genetic l o c a l i z a t i o n o f the 27 mutants was c a r r i e d out a t 17° C r e l a t i v e t o the markers: y, cv, v, f and c a r (Tables I I I and V I I I ) . The r e g i o n between y and cv was designated as 1, between cv and v as 2, and so on. Twenty-four of the c o l d - s e n s i t i v e mutations were r e a d i l y l o c a l i z e d t o a r e g i o n of the X chromosome. Two mutants were too leaky ( i . e . , y i e l d e d s u r v i v i n g mutants a t 17° C) to map p r e c i s e l y , and two ot h e r mutants no 42 l o n g e r b e h a v e d a s l e t h a l s u p o n o u t c r o s s i n g t o t h e m a r k e r s t o c k . T h e l a t t e r o b s e r v a t i o n h a s b e e n r e p o r t e d f o r t s m u t a t i o n s ( S u z u k i / e t a l . , 1967) a n d i s p r e s u m e d t o r e s u l t f r o m t h e i n t r o d u c t i o n o f g e n e t i c m o d i f i e r s f r o m c s t h e m a r k e r s t o c k . A l t h o u g h m u t a n t 19 b e l o n g e d t o t h e l a t t e r g r o u p , i t s t i l l c o u l d be mapped b e c a u s e s u r v i v i n g m a l e s c a r r y i n g t h e m u t a t i o n e m e r g e d 9 d a y s l a t e r t h a n t h o s e w i t h t h e w i l d - t y p e a l l e l e ( T a b l e V I I I ) . T h e c r o s s o v e r d i s t a n c e s a t 1 7 ° C i n e a c h r e g i o n o f t h e O r e g o n - R c o n t r o l w e r e s i m i l a r t o t h e s t a n d a r d v a l u e s ( L i n d s l e y a n d G r e l l , 1 9 6 8 ) . T h e m u t a n t s , h o w e v e r , f r e q e u n t l y showed d i s c r e p a n c i e s when c o m p a r e d w i t h t h e c o n t r o l v a l u e s . In s u c h c a s e s , c r o s s o v e r v a l u e s w e r e c h e c k e d i n f e m a l e s a n d a l l showed v a l u e s s i m i l a r t o s t a n d -a r d v a l u e s . None o f t h e c s - b e a r i n g c h r o m o s o m e s g a v e a n y e v i d e n c e o f chromosome r e a r r a n g e m e n t . 43 D ISCUSSION T h e f r e q u e n c y o f s e x - l i n k e d l e t h a l i n d u c t i o n b y EMS c a n be e s t i m a t e d b y c o m p a r i n g t h e s e x - r a t i o s o f p r o g e n y o f t r e a t e d a n d u n t r e a t e d m a l e s c r o s s e d t o a t t a c h e d - X -b e a r i n g f e m a l e s . On t h i s b a s i s , i t was f o u n d t h a t a b o u t 36% o f a l l X c h r o m o s o m e s e x p o s e d t o 0 . 0 0 5 M EMS c a r r y a n i n d u c e d l e t h a l . A t t h i s l e v e l o f m u t a t i o n - i n d u c t i o n , a p r o p o r t i o n o f t h e l e t h a l c h r o m o s o m e s w i l l c a r r y m o r e t h a n 1 n e w l y - i n d u c e d l e t h a l m u t a t i o n . M a p p i n g s t u d i e s show t h a t t h e c s l e t h a l s a r e r e c o v e r e d a s s i n g l e m u t a t -i o n e v e n t s . T h u s , a n e s t i m a t e o f t h e r a t e o f c s p r o d u c t -i o n s h o u l d b e b a s e d o n t h e i r p r o p o r t i o n among t h o s e c h r o m o s o m e s c a r r y i n g o n l y o n e i n d u c e d m u t a t i o n ( B a i l l i e e t a l , 1 9 6 8 ) . In p r e v i o u s t e s t s o f E M S - i n d u c e d l e t h a l s , i t was e s t i m a t e d t h a t o f t h e c h r o m o s o m e s b e a r i n g a s i n g l e m u t a t i o n , 10-12% w e r e h e a t - s e n s i t i v e ( S u z u k i e t a l . , 1 9 6 7 ; B a i l l i e e t a _ l » , 1 9 6 8 ) . I n t h e p r e s e n t e x p e r i m e n t s i t was c a l c u l a t e d t h a t 1 . 5 - 3 . 0 % o f a l l s i n g l e l e t h a l c h r o m o s o m e s a r e c o l d - s e n s i t i v e . (See a p p e n d i x f o r c a l c u l a t i o n . I I t i s o b v i o u s t h a t s e x - l i n k e d c s l e t h a l s w e r e much l e s s f r e q u e n t t h a n h e a t - s e n s i t i v e l e t h a l s ( 1 . 5 - 3% a s c o m p a r e d t o 10 - 12%). P a r t o f t h e g r e a t e r p r o p o r t i o n o f h e a t - s e n s i t i v e l e t h a l s , h o w e v e r , may r e f l e c t t h e l a r g e r t e m p e r a t u r e r a n g e u s e d t o s c r e e n ( 2 9 ° t o 1 7 ° a n d 2 2 ° C f o r h e a t - s e n s i t i v e s , 2 2 ° t o 1 7 ° C f o r c o l d -s e n s i t i v e s ) . 44 T h e m o s t m e a n i n g f u l c o m p a r i s o n o f f r e q u e n c i e s o f t s m u t a n t s come f r o m r e c e n t e x p e r i m e n t s w h e r e t h e same s e t o f m u t a g e n ! z e d 3 r d c h r o m o s o m e s was s c r e e n e d f o r b o t h h e a t - a n d c o l d - s e n s i t i v i t y ( T a s a k a a n d S u z u k i , 1 9 7 3 ) . F l i e s c a r r y i n g e a c h m u t a g e n i z e d c h r o m o s o m e w e r e t e s t e d f o r v i a b i l i t y a t 1 7 ° , 2 2 ° , a n d 2 9 ° C . T w e n t y r e c e s s i v e m u t a t i o n s w e r e l e t h a l a t 2 9 ° C b u t v i a b l e a t 1 7 ° C , i . e . w e r e h e a t s e n s i t i v e , a n d a n e q u a l number o f m u t a t i o n s w e r e l e t h a l a t 1 7 ° b u t v i a b l e a t 2 9 ° C , i . e . w e r e c o l d -o s e n s i t i v e . A l l t h e s e m u t a n t s w e r e v i a b l e a t 22 C . T h r e e o t h e r m u t a t i o n s w e r e v i a b l e a t o n e o f t h e e x t r e m e t e m p e r a t u r e s b u t w e r e l e t h a l a t t h e o t h e r two t e m p e r a t u r e s . In t o t a l , 21 h e a t - s e n s i t i v e m u t a n t s , 22 c o l d - s e n s i t i v e s , a n d 10 m u t a n t s s e n s i t i v e t o b o t h 1 7 ° a n d 2 9 ° C b u t v i a b l e o a t 22 C w e r e f o u n d . H e n c e t h e f r e q u e n c i e s o f r e c e s s i v e c o l d - s e n s i t i v e a n d h e a t - s e n s i t i v e m u t a t i o n s w e r e v e r y s i m i l a r o n t h e t h i r d chromosome o f D r o s o p h i l a . C o m p a r i s o n s o f t h e g e n e t i c d i s t r i b u t i o n s o f d o m i n a n t a u t o s o m a l h e a t - (DTS) a n d c o l d - s e n s i t i v e (DCS) l e t h a l s c a n b e m a d e . On c h r o m o s o m e 3 , DTS l e t h a l s w h i c h map a t a number o f s i t e s h a v e b e e n r e c o v e r e d ( H o l d e n a n d S u z u k i , 1973) w h e r e a s no DCS l e t h a l s h a v e b e e n f o u n d ( R o s e n b l u t h e t a l . , 1972; T a s a k a a n d S u z u k i , 1 9 7 3 ) . On c h r o m o s o m e 2 , a l a r g e c l u s t e r o f DTS l e t h a l s was f o u n d n e a r d p i n 2L b u t o t h e r s i t e s w e r e a l s o f o u n d ( S u z u k i a n d P r o c u n i e r , 1 9 6 9 ) . 45 DCS l e t h a l s were found on chromosome 2 only i n the c l u s t e r (Rosenbluth et aJL., 1972). C l e a r l y , DCS l e t h a l s are more rare and d i s t r i b u t e d quite non-randomly i n contrast to DTS mutations. On the X chromosome, about 4% of EMS-induced non-conditional and heat-sensitive l e t h a l s map to the r i g h t of car (Suzuki and P i t e r n i c k , 1972) whereas 12% (3/24) of the cs mutants mapped f e l l to the r i g h t of car. Of the heat-sensitive l e t h a l s , 7.4% (7/95) mapped at the t i p of the X chromosome (Suzuki and Piternick, 1972) whereas 29% (7/24) of the cs l e t h a l s map i n that region (Figure 4). Clearly, selection for c o l d - s e n s i t i v i t y of l e t h a l mutations yielded a d i f f e r e n t d i s t r i b u t i o n of s i t e s from l e t h a l s selected by h e a t - s e n s i t i v i t y . cs cs cs The 3 cs mutations (5 , 6 and 15 ) mapping to the r i g h t of car were a l l e l i c (Table VIII). Mutation 15° s was recovered i n a completely d i f f e r e n t experiment from 5 C S and 6 C S and the l a t t e r are phenotypically d i s t i n c t (Table IX). I t was concluded, therefore, that each a l l e l e represented a d i f f e r e n t induction event. Moreover, a s i m i l a r cs mutant mapping to the r i g h t of car has been i s o l a t e d i n another lab (Wright, 1973). Among the 7cs mutants i s o l a t e d by Wright, a f i n e b r i s t l e mutant sim i l a r to 16° S and 20 c s, was also found. Comple-mentation tests of the 2 sets of mutants are now i n progress (T. Wright, personal communication). I t appears 4 6 t h a t s e l e c t i o n f o r c o l d - s e n s i t i v i t y o n t h e X c h r o m o s o m e e n r i c h e s f o r m u t a t i o n s i n some s i t e s . W h e t h e r c o l d - s e n s i t i v i t y i n f a c t e n r i c h e s f o r r i b o -s o m a l o r r e g u l a t o r y d e f e c t s i n D r o s o p h i l a m u s t be d e t e r m i n e d b y b i o c h e m i c a l t e s t s . B i o c h e m i c a l a n d g e n e t i c e v i d e n c e t h a t t h e b o b b e d l o c u s o n t h e X c h r o m o s o m e c o d e s f o r r i b o -s o m a l RNA i s v e r y g o o d ( R i t o s s a e t E L I . , 1 9 6 5 ; Mohan a n d R i t o s s a , 1 9 7 0 ) . M u t a n t s f o r e x t r e m e a l l e l e s o f b o b b e d a r e i n v i a b l e a n d l e s s e x t r e m e a l l e l e s may p r o d u c e a p h e n o t y p e o f s l e n d e r b r i s t l e s , e t c h i n g o f a b d o m i n a l t e r g i t e s a n d p r o l o n g e d d e v e l o p m e n t . T h i s h a s l e d t o t h e s u g g e s t -i o n t h a t M i n u t e m u t a t i o n s w h i c h e x h i b i t some o f t h e s e p h e n o t y p e s may h a v e a l t e r e d tRNAs a n d t h e r e b y a f f e c t t h e t r a n s l a t i o n a l a p p a r a t u s . T h u s , i t i s t e m p t i n g t o s u g g e s t t h a t t h e p h e n o t y p e s o f bb a n d M i n u t e f l i e s m i g h t b e c h a r a c t e r i s t i c o f t r a n s l a t i o n d e f e c t s . S e v e n o f t h e 2 7 , . . , , c s , c s 1 c c s , , c s ..cs o r c s „ c s c s m u t a t i o n s (5 , 6 , 1 5 , 1 6 , 2 0 , 2 6 , 2 7 ) e x h i b i t e d t e r g i t e e t c h i n g a n d / o r s l e n d e r b r i s t l e s . M a t e r n a l l y - p r o d u c e d r i b o s o m e s c o n s t i t u t e t h e b u l k o f t h e r i b o s o m e s p r e s e n t d u r i n g e a r l y e m b r y o g e n e s i s (Brown a n d D a w i d , 1 9 6 8 ) . D e f e c t i v e r i b o s o m e s y n t h e s i s i n a f e m a l e m i g h t b e r e f l e c t e d i n f e m a l e s t e r i l i t y d u e t o t h e p r o d u c t i o n o f e g g s w h i c h c a n n o t d e v e l o p . I n f a c t , 7 o f t h e 2 7 c s m u t a t i o n s r e t a i n e d y i e l d e d s t e r i l e f e m a l e s a n d 1 0 more p r o d u c e d f e m a l e s w i t h l o w f e r t i l i t y ( T a b l e v ) . c s M o r e o v e r , a l l b u t o n e ( 2 7 ) o f t h e m u t a t i o n s w h i c h h a d a b r i s t l e o r a b d o m i n a l p h e n o t y p e w e r e a l s o s t e r i l e o r 47 p o o r l y f e r t i l e . (See T a b l e IX f o r a summary o f t h e p r o p e r t i e s o f t h e m u t a n t s . ) T h e p o s s i b i l i t y t h a t t h e c o m b i n e d p r o p e r t i e s o f s p e c i f i c v i s i b l e p h e n o t y p e s , f e m a l e s t e r i l i t y a n d c s l e t h a l i t y a r e s u f f i c i e n t t o d e f i n e many d e f e c t s i n r i b o s o m a l p r o t e i n s i s a n i n -t r i g u i n g o n e w h i c h demands f u r t h e r b i o c h e m i c a l s t u d i e s f o r v e r i f i c a t i o n . U n l i k e h e a t - s e n s i t i v e l e t h a l s , t h e c s m u t a t i o n s r e p o r t e d a r e q u i t e v a r i a b l e i n p h e n o t y p e a n d a p p a r e n t l y q u i t e s e n s i t i v e t o g e n e t i c a n d e n v i r o n m e n t a l d i f f e r e n c e s . V i a b i l i t y r a t i o s r e c o v e r e d i n d i f f e r e n t e x p e r i m e n t s p e r -f o r m e d w i t h d i f f e r e n t s t o c k s o r a t d i f f e r e n t t i m e s c a n v a r y w i d e l y . O u t c r o s s i n g c s l e t h a l s t o c k s c a n a l s o g r e a t l y a f f e c t v i a b i l i t y o f t h e m u t a n t c h r o m o s o m e ( W r i g h t , 1 9 7 3 ) . We w o u l d s u g g e s t t h a t c s l e t h a l s m u s t o be r i g i d l y s e l e c t e d f o r t h e i r c o m p l e t e l e t h a l i t y a t 17 C a n d s t a b i l i t y f r o m t e s t t o t e s t . O t h e r w i s e , t h e v a r i a -b i l i t y a n d t h e v e r y l o n g g e n e r a t i o n t i m e a t 1 7 ° C r e n d e r t h e c s c l a s s o f m u t a t i o n s v e r y d i f f i c u l t t o w o r k w i t h . 48 I I I , C o . l d ^ s e n s i t i v e S e x - r l i n k e d M u t a n t s o f D r o s o p h i l a m e l a n o g a s t e r : T w o - d i m e n s i o n a l G e l E l e c t r o p h o r e s i s o f R i b o s o m a l P r o t e i n s . INTRODUCTION C o l d - s e n s i t i v e m u t a n t s o f D r o s o p h i l a may p r o d u c e a l t e r e d r i b o s o m a l p r o t e i n s , e n z y m e s , o r o t h e r d e f e c t i v e c o m p o n e n t s . T h o u g h m u t a n t e n z y m e s w o u l d b e o f i n t e r e s t , t h e i r b i o c h e m i c a l d e t e c t i o n w o u l d p r o b a b l y b e d i f f i c u l t . W h i c h o f t h e many known e n z y m e s s h o u l d one a s s a y ? U n -l e s s some g e n e r a l p r e t e s t s c o u l d b e d e v i s e d t o d e t e c t f a u l t y b i o c h e m i c a l p a t h w a y s , c h o o s i n g a r e l e v a n t enzyme a s s a y w o u l d b e a m a t t e r o f c h a n c e . On t h e o t h e r h a n d , N o m u r a ' s s t u d i e s i n d i c a t e d t h a t r i b o s o m a l m u t a n t s c o n -s t i t u t e a l a r g e p o r t i o n o f c o l d - s e n s i t i v e l e t h a l s o f b a c t e r i a ( G u t h r i e , e t a l . , 1 9 6 9 b ) . A n o t h e r a d v a n t a g e o f i n v e s t i g a t i n g r i b o s o m e s i s t h a t r i b o s o m a l a s s a y s s u c h a s s e d i m e n t a t i o n i n s u c r o s e g r a d i e n t s o r g e l e l e c t r o p h o r e s i s o f r i b o s o m a l p r o t e i n s , t e s t many c o m p o n e n t s i n o n e p r o c e d u r e . R i b o s o m a l p r o t e i n s f r o m f i v e o f t h e c o l d - s e n s i t i v e D r o s o p h i l a m u t a n t s a n d O r e g o n - R c o n t r o l s h a v e b e e n e x a m i n e d b y t w o - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s . T h e s e l e c t e d m u t a n t s e x h i b i t e d f e m a l e s t e r i l i t y a n d / o r p h e n o t y p e s s i m i l a r t o b o b b e d m u t a n t s . A s t h e l a t t e r m u t a n t s a r e d e f i c i e n t i n DNA c o d i n g f o r r i b o s o m a l RNA, i t was t h o u g h t t h a t c o l d - s e n s i t i v e m u t a n t s w i t h s i m i l a r p h e n o t y p e s b u t m a p p i n g i n d i f f e r e n t a r e a s o f t h e X c h r o m o s o m e , m i g h t h a v e 49 a l t e r e d r i b o s o m a l p r o t e i n s . T w o - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s was c h o s e n b e c a u s e i t h a s b e e n a u s e f u l t o o l i n d e m o n s t r a t i n g a l t e r e d r i b o s o m a l p r o t e i n s i n m u t a n t s a n d v a r i o u s s t r a i n s o f b a c t e r i a ( K a l t s c h m i d t , e t a l . , 1 9 7 0 c ; D e u s s e r , e t a l . , 1 9 7 0 ; B o l l e n , e t a l . , 1 9 7 3 ) . O n e - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s was p r e v i o u s l y u s e d t o i d e n t i f y a n d g e n e t i c a l l y map t h e "K r i b o s o m a l p r o t e i n " o f E . c o l i K 12 ( L e b o y e t a l . , 1 9 6 4 ) . T w o - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s i s a r e c e n t a d a p t a t i o n o f o n e - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s a n d h a s a b o u t d o u b l e t h e r e s o l v i n g c a p a c i t y o f t h e o l d e r m e t h o d ( K a l t s c h m i d t a n d W i t t m a n n , 1 9 7 0 b ) . E . c o l i r i b o s o m a l p r o t e i n s c a n be s e p a r a t e d i n t o a b o u t 25 b a n d s i n o n e -d i m e n s i o n a l g e l s . I n t w o - d i m e n s i o n a l g e l s , h o w e v e r , m o r e t h a n 50 p r o t e i n c o m p o n e n t s a r e e v i d e n t . 50 METHODS AND MATERIALS I n t r o d u c t i o n . . A p r e v i o u s l y r e p o r t e d p r o c e d u r e f o r t h e i s o l a t i o n o f r i b o s o m e s f r o m D r o s o p h i l a l a r v a e was u s e d ( B o s h e s , 1 9 7 0 ) . T h e m e t h o d s e p a r a t e d " s o l u b l e " o r " f r e e r i b o s o m e s " f r o m r i b o s o m e s a t t a c h e d t o t h e e n d o p l a s m i c r e t i c u l u m . B o t h t h e " f r e e r i b o s o m a l " a n d " a t t a c h e d r i b o s o m a l " p r e p a r a t i o n s w e r e shown t o c o n t a i n p o l y s o m e s a n d r i b o s o m e s a s w e l l a s 18S a n d 28S RNA. T h e " f r e e r i b o s o m a l " p r e p a r a t i o n , h o w e v e r , was f o u n d t o a c c u m u l a t e r a d i o a c t i v e u r i d i n e i n t o 18S a n d 28S RNA more r a p i d l y t h a n t h e " a t t a c h e d r i b o s o m a l " p r e p a r a t i o n . T h e l a t t e r f r a c t i o n e x h i b i t e d a h i g h e r r a t e o f a m i n o a c i d i n c o r p o r a t i o n . T h e r e s u l t s s u g g e s t t h a t " s o l u b l e r i b o s o m a l " p r e p a r a t i o n s may r e p r e s e n t n e w l y f o r m e d r i b o s o m e s a n d p o l y s o m e s n o t y e t e n g a g e d i n p r o t e i n s y n t h e s i s . G r e a t e r p r o t e i n s y n t h e s i s b y t h e " a t t a c h e d p o l y s o m e s " t h a n by t h e s o l u b l e p o l y s o m e s h a s b e e n o b s e r v e d i n n u m e r o u s o r g a n i s m s a n d i t i s t h o u g h t t h a t t h e " a t t a c h e d " f r a c t i o n s y n t h e s i z e s p r o t e i n s f o r t r a n s p o r t t h r o u g h t h e e n d o p l a s m i c r e t i c u l u m ( B i r n i e , 1 9 7 2 ) . I n t h e p r e s e n t s t u d y o f r i b o s o m a l p r o t e i n s , " s o l u b l e r i b o s o m a l " p r e p a r a t i o n s w e r e u s e d b e c a u s e t h e y may be r e l a t i v e l y f r e e o f n a s c e n t p r o t e i n s . T h e m e t h o d a l s o s e p a r a t e s m i t o c h a n d r i a f r o m " s o l u b l e r i b o s o m e s " . T h o u g h B o s h e s p r o c e d u r e was -adopted, f o r o u r u s e , n a s c e n t p r o t e i n s a r e n o t c o n s i d e r e d t o b e a s e r i o u s s o u r c e o f c o n t a m i n a t i o n o f r i b o s o m e s . T h e t i s s u e s e x a m i n e d 51 w e r e o f d i v e r s e t y p e s a n d t h e i r r i b o s o m e s w e r e , no d o u b t , e n g a g e d i n t h e s y n t h e s i s o f many p r o t e i n s a t n u m e r o u s s t a g e s o f p a r t i a l c o m p l e t i o n . I t i s u n l i k e l y t h a t t h e c o n c e n t r a t i o n o f a n y o n e o f t h e s e p r o t e i n s o r p a r t i a l p r o t e i n s w o u l d b e l a r g e e n o u g h t o show up i n t h e g e l s o f t h e r i b o s o m a l p r o t e i n s . T h e m e t h o d o u t l i n e d b e l o w , w h i c h i s an a d a p a t i o n o f B o s h e s m e t h o d , was p r e v i o u s l y u s e d i n t h i s l a b o r a t o r y t o i s o l a t e r i b o s o m e s f r o m a d u l t D r o s o p h i l a . Some o f B o s h e s ' 1 o b s e r v a t i o n s w e r e r e p e a t e d (M. G o u l d - S o m e r o , p e r s o n a l c o m m u n i c a t i o n ) . A d u l t s w e r e f e d r a d i o a c t i v e u r a c i l o r l a b e l e d a m i n o a c i d o v e r n i g h t . The s e d i m e n -t a t i o n o f p a r t i c l e s i n t h e p o s t - m i t o c h o n d r i a l s u p e r n a t a n t w e r e e x a m i n e d i n s u c r o s e g r a d i e n t s . P a r t i c l e s w i t h s e d i m e n t a t i o n p r o p e r t i e s l i k e r i b o s o m e s a n d p o l y s o m e s h a d i n c o r p o r a t e d t h e p r e c u r s o r s o f RNA a n d p r o t e i n . I t seems r e a s o n a b l e t o a s s u m e t h a t t h e m e t h o d o u t l i n e d i s o l a t e s r i b o s o m e s f r o m a d u l t D r o s o p h i l a . T h e d e g r e e o f p u r i t y o f t h e r i b o s o m a l p r e p a r a t i o n , h o w e v e r , i s u n c e r t a i n . I s o l a t i o n o f r i b o s o m e s . I n t h e p r e l i m i n a r y e x p e r i m e n t s , r i b o s o m e s w e r e i s o l a t e d f r o m m i x e d s a m p l e s o f O r e g o n - R , a d u l t m a l e s a n d f e m a l e s . A s f e m a l e s h o m o z y g o u s f o r some o f t h e c o l d - s e n s i t i v e m u t a t i o n s p r o d u c e d few o r no o f f -s p r i n g e v e n a t t h e p e r m i s s i v e t e m p e r a t u r e , m u t a n t m a l e s f r o m s t o c k s m a i n t a i n e d a t 2 2 ° C w i t h C ( 1 ) R M / Y f e m a l e s , w e r e u s e d . A l s o , O r e g o n - R m a l e s a n d t h e i r C C D R M / Y s i b s 52 w e r e s e p a r a t e l y e x a m i n e d . E t h e r i z e d o r GC^ a n e s t h e t i z e d , a d u l t D r o s o p h i l a m e l a n o g a s t e r w e r e s o r t e d a c c o r d i n g t o s e x a n d c o l l e c t e d i n v i a l s c o o l e d i n D r y I c e and e t h a n o l . T h e f l i e s w e r e s t o r e d a t - 3 0 ° C f o r a week t o 7 m o n t h s b e f o r e b e i n g u s e d f o r r i b o s o m e i s o l a t i o n s . No c h a n g e , i n t h e p a t t e r n o f r i b o s o m a l p r o t e i n s i n t h e t w o - d i m e n s i o n a l g e l s was o b s e r v e d w i t h i n c r e a s i n g s t o r a g e t i m e s . R i b o -somes w e r e p r e p a r e d i n a c o l d r o o m a t 5 ° C a n d a l l e q u i p -ment a n d s o l u t i o n s w e r e p r e - c h i l l e d . F i v e t o 20 gm o f f l i e s w e r e h o m o g e n i z e d i n f r e s h l y p r e p a r e d T K M - S ( 0 . 2 6 M s u c r o s e , 0 . 0 3 M T r i s - H C l , p H 7 . 8 , 0, 1M K C 1 , 0 . 0 0 7 M Mg a c e t a t e , a n d 0 . 0 0 7 M m e r c a p t o e t h a n o l ) f o r 20 s e c o n d s i n a n O m n i - m i x e r o p e r a t e d a t t o p s p e e d . T w e n t y m l o f T K M - S s o l u t i o n was a d d e d f o r e a c h g r a m o f t i s s u e . T h i s h i g h r a t i o o f s o l u t i o n t o t i s s u e seemed t o p r o d u c e a c l e a r e r , l e s s c o l o r e d r i b o s o m a l p e l l e t t h a n d i d c o n c e n t r a t e d h o m o g e n a t e s . T h e h o m o g e n a t e was f i l t e r e d t h r o u g h c o a r s e n y l o n mesh t o r e m o v e m o s t o f t h e i n s e c t e x o s k e l l e t o n . T h e f i l t r a t e was t h e n h o m o g e n i z e d b y f i v e s t r o k e s w i t h a B s i z e d , t e f l o n p e s t l e i n a D o u n c e g l a s s h o m o g e n i z e r t o e n s u r e t h a t c e l l s w e r e r u p t u r e d . F a i l u r e t o r e m o v e h a r d t i s s u e by f i l t e r i n g made t h e l a t t e r s t e p d i f f i c u l t . T h e h o m o g e n a t e was c e n t r i f u g e d a t 1 8 , 0 0 0 rpm f o r 30 m i n u t e s i n a f i x e d a n g l e , S o r v a l l r o t o r ( 3 9 , 1 0 0 g ) . F a t t y m a t e r i a l o n t o p o f t h e s u p e r n a t a n t was r e m o v e d a n d t h e 53 p r e c i p i t a t e was d i s c a r d e d . T h e p o s t - m i t o c h o n d r i a l s u p e r n a t a n t was t r e a t e d w i t h 1 m l o f 10% T r i t o n - X - 1 0 0 d e t e r g e n t p e r 10 m l s u p e r n a t a n t t o s o l u b i l i z e a n y m i c r o -s o m a l membrane p r e s e n t . T h e m i x t u r e was l e f t f o r 1 /2 t o o 1 h o u r a t 5 C a n d a g a i n c e n t r i f u g e d a t 1 8 , 0 0 0 r p m . A n y f a t - l i k e t o p p i n g o r p r e c i p i t a t e was d i s c a r d e d . T h e c l e a r o r a n g e s u p e r n a t a n t was c e n t r i f u g e d a t 4 2 , 0 0 0 rpm f o r 90 m i n u t e s i n t h e Bechman 5 0 . 1 a n g l e r o t o r ( 1 6 7 , 0 0 0 g ) . A s l i g h t l y o r a n g e , t r a n s l u c e n t p e l l e t was o b t a i n e d . T h e c e n t r i f u g e t u b e s w e r e i n v e r t e d a n d t h o r o u g h l y d r a i n e d . T h e i n s i d e w a l l s o f t h e t u b e s w e r e d r i e d w i t h K i m w i p e s t o r e m o v e a s much s u p e r n a t a n t a s p o s s i b l e f r o m t h e r i b o s o m a l p e l l e t . T h e r i b o s o m a l p e l l e t was u s u a l l y f r o z e n o v e r n i g h t w i t h a s m a l l amount o f TMA _2 s o l u t i o n ( 0 . 0 1 M T r i s - H C l , pH 7 . 6 , 0 . 0 0 1 M M g C l ^ 5 X 10 M NH C I , a n d 0 . 0 0 7 M m e r c a p t o e t h a n o l ) . 4 S o l u b i l i z a t i o n o f r i b o s o m a l p r o t e i n s . (1) H C l - a c e t o n e . S e v e r a l m e t h o d s o f p r e p a r i n g r i b o -s o m a l p r o t e i n s f o r 2 - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s w e r e t e s t e d i n i n i t i a l e x p e r i m e n t s . T h e H C l - a c e t o n e m e t h o d , h o w e v e r , was u s e d f o r t h e m u t a n t s , O r e g o n - R m a l e s , a n d C ( l ) RM f e m a l e s ( L a m b e r t s s o n , e t a l . , 1 9 7 0 ) . R i b o s o m e s f r o m 10 gm t i s s u e w e r e s u s p e n d e d i n a t o t a l o f 1 .4 m l c o l d T M A H . C o l d 1 M H C l ( 0 . 4 7 ml ) was a d d e d t o t h e c h i l l e d s u s p e n s i o n t o g i v e a f i n a l c o n c e n t r a t i o n o f 0 . 2 5 M H C l . T h e m i x t u r e was s t i r r e d a t 5 ° C f o r 1 t o 2 h o u r . T h e w h i t e , f l o c c u l e n t p r e c i p i t a t e o f RNA w h i c h f o r m e d was r e m o v e d 54 b y c e n t r i f u g i n g a t 1 5 , 0 0 0 rpm f o r 20 m i n u t e s i n t h e S o r v a l l . T h e s u p e r n a t a n t was s a v e d a n d t h e p r e c i p i t a t e was r e -e x t r a c t e d w i t h 0 . 6 m l o f 0 . 2 5 M H C l i n TMA I I . When s m a l l e r a m o u n t s o f t i s s u e w e r e u s e d , t h e v o l u m e s o f r e a g e n t s w e r e r e d u c e d p r o p o r t i o n a l l y . T o p r e c i p i t a t e r i b o s o m a l p r o t e i n s , 5 v o l u m e s o f c o l d a c e t o n e ( 1 1 . 5 ml ) was a d d e d t o t h e s u p e r n a t a n t a n d w a s h i n g s ( 2 . 3 m l ) . T h e m i x t u r e was l e f t i n t h e c o l d f o r a n h o u r o r l o n g e r . T h e w h i t e p r o t e i n p r e c i p i t a t e was s e p a r a t e d f r o m t h e c o l o r e d s u p e r n a t a n t b y l o w s p e e d c e n t r i f u g a t i o n . T h e p r o t e i n p e l l e t was t h e n w a s h e d w i t h c o l d a c e t o n e , t w i c e w i t h c o l d 95% e t h a n o l , t w i c e w i t h e t h e r , a n d a i r -d r i e d a t r o o m t e m p e r a t u r e . (2) M g - a c e t i c a c i d . T h e r i b o s o m a l p e l l e t f r o m 20 gm t i s s u e was f r o z e n o v e r n i g h t i n TSM ( 0 . 0 1 M t r i s , pH 7.8, 0 . 0 0 3 M. s u c c i n i c a c i d , 0 . 0 1 M M g C l ^ , a n d 15 mM m e r c a p t o e t h a n o l ) . T o 4 . 5 m l r i b o s o m e s s u s p e n d e d i n T S M , was f i r s t a d d e d 0 . 4 5 m l 1 M M g C l 2 , t h e n 9 . 0 m l c o l d g l a c i a l a c e t i c a c i d ( H a r d y , e t a l . , 1 9 6 9 ) . T h e m i x t u r e was s t i r r e d f o r an h o u r i n an i c e b a t h . T h e w h i t e p r e c i p i t a t e o f RNA was r e m o v e d b y l o w s p e e d c e n t r i f u g a t i o n a n d t h e s u p e r n a t a n t s a v e d . I n i n i t i a l t r i a l s , t h e s u p e r n a t a n t was d i a l y z e d a g a i n s t s a m p l e g e l a s s u g g e s t e d b y K a l t s c h m i d t a n d W i t t m a n n ( 1 9 7 0 b ) . T o k e e p t h e f i n a l v o l u m e o f s a m p l e g e l a n d r i b o s o m a l p r o t e i n s m a l l , d i a l y s i s was p e r f o r m e d w i t h a g r e a t e r p r e s s u r e i n s i d e t h e d i a l y s i s s a c t h a n o u t s i d e . 55 T h e p r o c e d u r e p r o v e d t e c h n i c a l l y d i f f i c u l t i n my h a n d s a n d was r e p l a c e d b y s t a n d a r d d i a l y s i s . I n l a t e r t r i a l s , t h e s u p e r n a t a n t was f i r s t d i a l y z e d a g a i n s t 66% ( v / v ) a c e t i c a c i d a n d 1 mM m e r c a p t o e t h a n o l f o r 24 h o u r s , t h e n 5% ( v / v ) a c e t i c a c i d a n d 1 mM m e r c a p t o e t h a n o l f o r a f u r t h e r 24 h o u r s , a n d f i n a l l y a g a i n s t 1 mM m e r c a p t o e t h a n o l f o r 16 h o u r s ( W e l f l e , e t a l . , 1 9 7 1 ) . T h e f i n a l d i a l y z a t e was f r e e z e - d r i e d . T h i s p r o c e d u r e was r e p o r t e d t o r e l e a s e 8 0% o f t h e p r o t e i n f r o m r a t l i v e r r i b o s o m e s . (3) L i C I . T h e r i b o s o m a l p e l l e t f r o m 10 gm t i s s u e was r e s u s p e n d e d i n 2 . 3 m l TKM ( 0 . 0 5 M K C l , 0 . 0 0 1 5 M M g C l 2 , 0 . 0 0 1 M T r i s , p H 7 . 8 ) . T o 2 . 3 m l c o l d r i b o s o m e s u s p e n s i o n was a d d e d 2 . 3 m l c o l d 4 M L i C l . T h e m i x t u r e was a l l o w e d t o s t a n d f o r 4 0 h o u r s i n t h e c o l d . T h e l a r g e o r a n g e g e l a t i n o u s p r e c i p i t a t e o f RNA was r e m o v e d b y l o w s p e e d c e n t r i f u g a t i o n . T h e s u p e r n a t a n t was d i a l y z e d a g a i n s t w a t e r f o r 2 8 h o u r s . T h e d i a l y z a t e was f r e e z e - d r i e d . T h i s p r o c e d u r e was r e p o r t e d t o g i v e c o m p l e t e r e c o v e r y o f p r o t e i n s f r o m r a b b i t r e t i c u l o c y t e r i b o s o m e s ( M a t h i a s a n d W i l l i a m s o n , 1 9 6 4 ) . (4) C l e l a n d ' s r e a g e n t . T o a s m a l l r i b o s o m a l p e l l e t f r o m 1-2 gm t i s s u e was a d d e d 50 l a m b d a o f C l e l a n d ' s r e a g e n t (4 M L i C l , 8 M u r e a a n d 0 . 0 0 5 M d i t h i o t h r e i t a l ) . T h e r e a g e n t was p r e p a r e d i m m e d i a t e l y b e f o r e u s i n g . T h e p e l l e t was l e f t i n c o n t a c t w i t h t h e r e a g e n t o v e r n i g h t w i t h o u t m i x i n g . T h e r i b o s o m a l RNA p r e c i p i t a t e was r e -moved b y c e n t r i f u g a t i o n a n d t h e s u p e r n a t a n t , c o n t a i n i n g r i b o s o m a l p r o t e i n s , was s a v e d . T h e s u p e r n a t a n t was m i x e d 56 with 0.2 ml of sample gel and 0.1 to 0.15 ml of the mixture was applied to the 1-dimension gel column. Two-dimensional Gel Electrophoresis. Two-dimensional gel electrophoresis of the extracted ribosomal proteins was performed as described by Kaltschmidt and Wittmann (1970a) except for some minor changes. The main steps in the procedure were as follows. The extracted proteins were placed i n the middle of a gel (8% acrylamide, pH 8.6) polymerized i n a glass tube (14 x 0.5 cm). Unlike con-ventional methods, t h i s arrangement allowed the proteins to migrate to either pole and s t i l l remain on the g e l . The gel column was removed from the tube and equilibrated i n buffer for the second electrophoresis. The gel column was attached along the top edge of a gel slab (14 cm x 10 cm x 0.5 cm) composed of 18% acrylamide, pH 4.6. After electrophoresis i n the second dimension, the proteins were stained with Amido Black or Coumassie c B r i l l i a n t Blue. The con entrations of gels and the pH S values of the buffers were selected to emphasize separa-ti o n by change i n the f i r s t dimension, and by size i n the second dimension. F i r s t dimension electrophoresis. The following reagents were used (Addendum, Kaltschmidt and Wittmann, 1970a) . 57 S e p a r a t i o n ' g e l , pH 8 . 6 54 gm u r e a 6 . 0 gm a c r y l a m i d e ( r e c r y s t a l l i z e d i n c h l o r o f o r m ) 0 . 2 gm N , N ' - m e t h y l e n e b i s a c r y l a m i d e ( b i s a c r y l a m i d e ) . 1 .2 gm d i s o d i u m s a l t o f e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d ( E D T A - N a 2 ) 4 . 8 gm b o r i c a c i d . 7 . 3 gm t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e ( T r i s ) . 0 . 4 5 m l N , N , N * , N ' - t e t r a m e t h y l e t h y l e n e d i a m i n e ( T E M E D ) . T h e s o l u t i o n was made up t o 14 8 . 5 m l w i t h w a t e r . T o p o l y m e r i z e t h e a b o v e m i x t u r e , 1 . 5 m l f r e s h l y made 7% ammonium p e r s u l f a t e was r e q u i r e d . S a m p l e g e l 48 gm u r e a V0 gm a c r y l a m i d e 0 .2 gm b i s a c r y l a m i d e 0 . 0 8 5 gm E D T A - N a 2 0 . 3 2 gm b o r i c a c i d 0 . 0 6 m l TEMED T h e s o l u t i o n was made u p t o 99 m l w i t h w a t e r . T o p o l y m e r i z e t h e a b o v e s o l u t i o n , a f r e s h l y made m i x t u r e o f 0 . 5 mg r i b o f l a v i n a n d 5 mg ammonium p e r s u l f a t e i n 1 m l w a t e r was a d d e d . E l e c t r o d e b u f f e r , pH 8 . 6 3 6 0 . 0 gm u r e a 2 . 4 gm E D T A - N a 2 9 . 6 gm b o r i c a c i d 1 4 . 5 5 gm T r i s T h e s o l u t i o n was made u p t o 1 l i t e r w i t h w a t e r . T h e r e a g e n t s w e r e k e p t a t 4 ° C up t o two w e e k s . G l a s s t u b e s , 18 cm l o n g a n d 0 . 5 cm i n n e r d i a m e t e r , 58 w e r e t h o r o u g h l y w a s h e d w i t h d e t e r g e n t , r i n s e d , d i p p e d i n d i l u t e P h o t o f l o w s o l u t i o n (.1 m l / 2 0 0 m l w a t e r ) a n d d r a i n e d d r y . T h e t u b e s w e r e c l o s e d a t t h e b o t t o m w i t h r u b b e r c a p s a n d f i l l e d t o 9 cm w i t h f i r s t - d i m e n s i o n s e p a r a t i o n g e l . T h e g e l s o l u t i o n was o v e r l a y e r e d w i t h a few d r o p s o f w a t e r t o p r e v e n t t h e g e l f r o m f o r m i n g a c u r v e d s u r f a c e . A f t e r t h e g e l p o l y m e r i z e d t h e w a t e r was r e m o v e d w i t h a b s o r b a n t p a p e r . Two t o 3 mg o f r i b o s o m a l p r o t e i n s was d i s s o l v e d i n 0 .1 m l s a m p l e g e l a n d a p p l i e d t o t h e t o p o f t h e s e p a r a t i o n g e l w i t h a l o n g c a p i l l a r y t u b e . T h e t r a n s f e r was n o t q u a n t i t a t i v e b u t l o s s e s w e r e k e p t t o a min imum b y u s i n g s m a l l e q u i p m e n t . I n c r e a s i n g t h e v o l u m e o f s a m p l e g e l o r d e c r e a s i n g t h e amount o f p r o t e i n p r o d u c e d i n f e r i o r r e s u l t s . T h e s a m p l e g e l was o v e r l a y e r e d w i t h w a t e r a n d p o l y m e r i z e d w i t h U . V . l i g h t . A f t e r r e m o v a l o f w a t e r f r o m t h e t u b e , s e p a r a t i o n g e l was a d d e d up t o 14 c m . T h e t u b e s w e r e e l e c t r o p h o r e s e d i n C a n a l c o 1 - d i m e n s i o n a l g e l e q u i p m e n t . T h e t u b e s w e r e i n s e r t e d s o t h a t t h e l o n g e r s e c t i o n o f s e p a r a t i o n g e l p r o j e c t e d i n t o t h e b o t t o m c a t h o d e c h a m b e r a n d t h e s h o r t e r s e c t i o n , i n t o t h e t o p a n o d e c h a m b e r . When t h e s a m p l e was E . c o l i r i b o s o m a l p r o t e i n , t h e l o w e r s e c t i o n o f s e p a r a t i o n g e l was 5 . 5 cm a n d t h e u p p e r s e c t i o n , 8 . 5 c m . E l e c t r o p h o r e s i s was c a r r i e d o u t i n a c o l d r o o m a t 5 ° C . F o u r s a m p l e s w e r e r u n i n e a c h e x p e r i m e n t . T h r e e 59 mamp c u r r e n t was a p p l i e d p e r t u b e f o r 10 t o 23 h o u r s . T h e c u r r e n t was k e p t c o n s t a n t a t 12 mamp. T h e v o l t a g e was i n i t i a l l y 1 1 0 - 1 5 0 v o l t b u t i n c r e a s e d w i t h t i m e . T h e pH o f t h e e l e c t r o d e b u f f e r was u n c h a n g e d a t t h e e n d o f a r u n . T h e g e l c o l u m n s w e r e r e m o v e d f r o m t h e t u b e s b y p l a c i n g them u n d e r w a t e r a n d m o v i n g a l o n g h y p o d e r m i c n e e d l e (22 g a u g e , 4 i n c h e s ) g e n t l y b e t w e e n t h e g l a s s a n d t h e g e l . T h e g e l c o u l d t h e n be r e m o v e d i n t a c t b y a p p l y i n g a w a t e r -f i l l e d r u b b e r b u l b o n t h e e n d o f t h e t u b e . T h e p r o c e d u r e was r o u t i n e l y s u c c e s s f u l i f t h e g l a s s t u b e s h a d b e e n w a s h e d a n d t r e a t e d a s d e s c r i b e d . S e c o n d d i m e n s i o n e l e c t r o p h o r e s i s . T h e r e a g e n t s o f K a l t s c h m i d t a n d W i t t m a n n (1970a) w e r e m o d i f i e d a s i n d i c a t e d b e l o w . E q u i l i b r a t i o n b u f f e r 300 gm u r e a 0 . 6 7 gm KOH A p p r o x i m a t e l y 3 m l g l a c i a l a c e t i c a c i d , i . e . s u f f i c i e n t t o make t h e s o l u t i o n pH 4 . 6 . T h e s o l u t i o n was made up t o 1 l i t e r w i t h w a t e r . T h e q u a n t i t y o f u r e a was d e c r e a s e d f r o m 480 gm b e c a u s e a n n e a l -i n g o f g e l c o l u m n s t o g e l s l a b s was i m p r o v e d . T h e v o l u m e o f a c i d was v a r i e d b e c a u s e t h e o r i g i n a l amount g a v e a. h i g h a n d V a r i a b l e p H . S e p a r a t i o n g e l , pH 4 . 6 360 gm u r e a 60 180 gm acrylamide ( e l e c t r o p h o r e s i s grade, not r e c r y s t a l -l i z e d ) 5.0 gm b i s a c r y l a m i d e 2.7 gm KOH 5.8 gm TEMED Approximately 60 ml g l a c i a l a c e t i c a c i d , i . e . , s u f f i c i e n t to make the s o l u t i o n pH 4.6. The s o l u t i o n was made up to 967 ml w i t h water. T h i r t y - t h r e e ml of 10% ammonium p e r s u l f a t e was added t o polymerize the above mixture. In i n i t i a l experiments, acrylamide r e c r y s t a l l i z e d from c h l o r o f o r m was used. As the q u a l i t y of the reagent d i d not have an obvious e f f e c t on r e s u l t s and r e c r y s t a l l i z a t i o n was expensive and time-consuming, e l e c t r o p h o r e s i s grade acrylamide was used as purchased. E l e c t r o d e b u f f e r , pH 4.6 v 140 gm g l y c i n e Approximately 15 ml g l a c i a l a c e t i c a c i d , i . e . s u f f i c i e n t t o make the s o l u t i o n pH 4.6. The s o l u t i o n was made up to 10 l i t e r s w i t h water. The e q u i l i b r a t i o n b u f f e r and s e p a r a t i o n g e l were g e n e r a l l y prepared j u s t b e f o r e use. The e l e c t r o d e b u f f e r was s t o r e d f o r s e v e r a l weeks a t 5° C. Amido Black S t a i n 5.5 gm Amido Black 50 ml g l a c i a l a c e t i c a c i d The s o l u t i o n was made up t o 1 l i t e r w i t h water, mixed w e l l and f i l t e r e d . The same s o l u t i o n was used 61 r e p e a t e d l y t o s t a i n s e v e r a l g e l s . Coumassie B r i l l i a n t Blue 0.3 gm Coumassie B r i l l i a n t Blue 15 ml methanol 980 ml 10% t r i c h l o r o a c e t i c a c i d (TCA). The dye was d i s s o l v e d i n methanol which was then added t o the TCA s o l u t i o n . The mixture was f i l t e r e d . The s t a i n was u n s t a b l e and had to be made up f r e s h f o r each experiment. In p r e p a r a t i o n f o r the second e l e c t r o p h o r e s i s , the 1-dimensional g e l columns were e q u i l i b r a t e d i n pH 4.6 b u f f e r f o r 1 to 3 hours a t room temperature. V a r i a t i o n s i n the time between the end of the f i r s t e l e c t r o p h o r e s i s and s t a r t of the second had no apparent e f f e c t on r e s u l t s . The two-dimensional g e l e l e c t r o p h o r e s i s apparatus was i d e n t i c a l t o t h a t o f K a l t s c h m i d t and Wittmann (1970a) except t h a t 4 g e l chambers, each 14 cm long x 0.5 cm wide and 10 cm hig h were pr e s e n t i n s t e a d o f 5 chambers, 20 cm x 0.5 cm x 20 cm. E s s e n t i a l l y the apparatus c o n s i s t e d o f f i v e p l a s t i c forms which when b o l t e d together c r e a t e d f o u r r e c t a n g u l a r chambers open a t top and bottom. The bottoms of the g e l chambers were s e a l e d by p l a c i n g the assembled apparatus i n a t r a y c o n t a i n i n g a h a l f i n c h depth o f s e p a r a t i o n g e l and p e r s u l f a t e . ( O v e r l a y e r i n g w i t h water as p r e s c r i b e d by Ka l t s c h m i d t and Wittmann was omitted as the procedure was d i f f i c u l t and unnecessary.) A f t e r the bottom l a y e r o f g e l had polymerized, g e l 62 m i x t u r e was p o u r e d i n t o t h e s e a l e d c h a m b e r s t o f o r m t h e g e l s l a b s . A f t e r h e a t f r o m t h e p o l y m e r i z a t i o n r e a c t i o n h a d s u b s i d e d , t h e 1 - d i m e n s i o n a l g e l c o l u m n s w e r e a t t a c h e d a l o n g t h e t o p e d g e s o f t h e s l a b s w i t h m o r e g e l m i x t u r e . D u r i n g t h e s e m a n i p u l a t i o n s , p o l y m e r i z a t i o n o f t h e f l a s k o f g e l s o l u t i o n a n d p e r s u l f a t e was d e l a y e d b y k e e p i n g t h e f l a c k i n i c e w a t e r . A t t e m p t s t o a t t a c h t h e g e l c o l u m n s t o t h e g e l s l a b s w h i l e t h e y w e r e p o l y m e r i z i n g a s p r e s c r i b e d b y K a l t s c h m i d t a n d W i t t m a n n (1970a) w e r e u n s u c c e s s f u l . T h e g e l c o l u m n s t e n d e d t o s i n k i n t o t h e f l u i d g e l . A n d e x t e n s i v e p r o t e i n r e s i d u e s w e r e l e f t i n t h e g e l c o l u m n s o r o t h e r a r t i f a c t s o c c u r r e d . E x c e s s g e l u s e d t o s e a l t h e b o t t o m o f t h e g e l c h a m b e r s was t r i m m e d f r o m t h e a p p a r a t u s . I t was t h e n p l a c e d i n t h e e l e c t r o d e chcimber c o n t a i n i n g c h i l l e d pH 4 . 6 e l e c t r o d e b u f f e r . The; a n o d e was a t t h e t o p o f t h e g e l c h a m b e r s a n d c a t h o d e a t t h e b o t t o m . E l e c t r o p h o r e s i s was c a r r i e d o u t i n a 5 ° C c o l d r o o m . A c o n s t a n t c u r r e n t o f 200 mamps was a p p l i e d f o r 12 t o 22 h o u r s . T h e v o l t a g e , i n i t i a l l y 60 t o 100 v o l t s , i n c r e a s e d w i t h t i m e . A s t h e pH o f t h e e l e c t r o d e b u f f e r c h a n g e d d u r i n g l o n g r u n g , i t was s o m e -t i m e s r e p l a c e d w i t h i n t h e f i r s t 10 h o u r s o f a r u n w i t h f r e s h b u f f e r . A f t e r e l e c t r o p h o r e s i s , t h e g e l s l a b s w e r e r e m o v e d f r o m 63 t h e a p p a r a t u s . T h e b o l t s h o l d i n g t h e p a r t s t o g e t h e r w e r e l o o s e n e d a n d w a t e r was i n j e c t e d b e t w e e n t h e g e l s l a b s a n d t h e p l a s t i c w a l l s w i t h a h y p o d e r m i c s y r i n g e . T h e s l a b s w e r e u s u a l l y s t a i n e d w i t h A m i d o B l a c k . T h e y w e r e p l a c e d i n t h e s t a i n f o r 15 m i n u t e s a n d r i n s e d f o r an h o u r i n r u n n i n g w a t e r . T h e b a c k g r o u n d was d e -s t a i n e d b y p l a c i n g t h e g e l s i n w a r m , 1% a c e t i c a c i d f o r s e v e r a l h o u r s , a n d t h e n i n f r e s h 1% a c e t i c a c i d f o r s e v e r a l d a y s a t r o o m t e m p e r a t u r e . P l a s t i c s t a c k i n g t r a y s ( 8 . 5 i n c h e s x 5 . 5 x 2) o b t a i n e d f r o m a h a r d w a r e s h o p w e r e u s e -f u l f o r s t a i n i n g a n d s t o r i n g t h e g e l s . G e l s s t a i n e d w i t h A m i d o B l a c k f a d e d o n l y a f t e r s e v e r a l m o n t h s a n d c o u l d b e r e a d i l y r e s t c i i n e d . A l s o t h e g e l s , i f p r o p e r l y d e s t a i n e d , p h o t o g r a p h e d w e l l w i t h b l a c k a n d w h i t e f i l m . S t a i n i n g w i t h C o u m a s s i e B r i l l i a n t B l u e (CBB) was f a s t e r b u t t h e r e s u l t s w e r e n o t a s s a t i s f a c t o r y a s w i t h A m i d o B l a c k . T h e g e l s l a b s w e r e p l a c e d i n CBB s t a i n o v e r -n i g h t . T h e d y e s o l u t i o n was p o u r e d o f f a n d r e p l a c e d b y warm 10% TCA.. T h e l a t t e r s h a r p e n e d t h e s t a i n e d s p o t s a n d d e s t a i n e d t h e b a c k g r o u n d . A s e c o n d c h a n g e o f T C A was n e c e s s a r y f o r maximum d e s t a i n i n g . T h e p r o t e i n s a p p e a r e d a s b r i g h t b l u e s p o t s o n t h e g e l s w h i c h h a d a g r e y c o a t o n t h e s u r f a c e . T h e s p o t s w e r e c l e a r l y d i s t i n g u i s h a b l e v i s u a l l y b u t showed up i n d i s t i n c t l y i n b l a c k a n d w h i t e p h o t o g r a p h s . A l s o t h e g e l s f a d e d a n d s h r a n k w i t h i n a m o n t h . 64 T h e g e l s w e r e b e s t v i e w e d o n a f r o s t e d l i g h t t a b l e . H o w e v e r , a t t e m p t s t o p h o t o g r a p h them w i t h t r a n s m i t t e d l i g h t w e r e u n s u c c e s s f u l b e c a u s e o f t h e u n e v e n e s s o f t h e l i g h t . T h e g e l s w e r e b e s t p h o t o g r a p h e d w i t h s p e c i a l o v e r h e a d l i g h t i n g a p p a r a t u s ( L i n h o f , s e t t i n g 1 /2 ) t h a t p r o d u c e d a n e v e n l i g h t i n t e n s i t y . T h e c a m e r a was p l a c e d c l o s e t o t h e g e l s , a b o u t 8 . 5 i n c h e s b e t w e e n l e n s a n d g e l , i n o r d e r t o r e d u c e shadow a r t i f a c t s i n t h e p h o t o g r a p h s . F i n e g r a i n , b l a c k a n d w h i t e f i l m (16 D I N , Kodak) was u s e d . U n d e r t h e s e c o n d i t i o n s , c a m e r a s e t t i n g s o f 3 f a n d 1 /250 g a v e t h e b e s t n e g a t i v e s . H i g h c o n t r a s t , #4 p a p e r g a v e t h e b e s t p r i n t s . 65 RESULTS P r e l i m i n a r y e x p e r i m e n t s . T h e f o l l o w i n g m e t h o d s o f p r e -p a r i n g D r o s o p h i l a r i b o s o m a l p r o t e i n s f o r g e l e l e c t r o -p h o r e s i s w e r e t r i e d : (1) L i C l , (.2) H C l - a c e t o n e , (3) M g - a c e t i c a c i d a n d (4) C l e l a n d ' s r e a g e n t . T h e p r o t e i n r e s i d u e f r o m t h e L i C l e x t r a c t i o n d i s s o l v e d w e l l i n s a m p l e g e l s o l u t i o n i n i t i a l l y b u t l a t e r p r e c i p i t a t e d o u t . In a d d i t i o n , t h e s a m p l e g e l s o l u t i o n f a i l e d t o p o l y m e r i z e . M e t h o d s 2 a n d 3 p r o d u c e d s u i t a b l e r e s i d u e s w h i c h d i s s o l v e d w e l l i n s a m p l e g e l s o l u t i o n a n d p e r m i t t e d p o l y m e r i z a t i o n . M e t h o d 4 was c o n s i d e r e d u n s u i t a b l e b e c a u s e d i f f u s e b a n d s o f p r o t e i n w e r e p r o d u c e d i n t h e f i r s t d i m e n s i o n . T o d e t e r m i n e t h e s u i t a b i l i t y o f t h e K a l t s c h m i d t a n d W i t t m a n n g e l s y s t e m f o r s t u d y i n g D r o s o p h i l a r i b o s o m a l p r o t e i n s , s a m p l e s w e r e p l a c e d i n t h e c e n t r e s o f f i r s t d i m e n s i o n g e l c o l u m n s a n d e l e c t r o p h o r e s e d f o r 16 h o u r s a t pH 8 . 6 . When t h e g e l c o l u m n s w e r e s t a i n e d , i t was f o u n d t h a t m o s t o f t h e p r o t e i n s h a d m i g r a t e d t o t h e c a t h o d e . On t h e a n i o n i c h a l f o f t h e c o l u m n s , a few f a i n t b a n d s c l o s e t o t h e o r i g i n w e r e o b s e r v e d . T h e e x p e r i m e n t was p e r f o r m e d w i t h H C l - a c e t o n e a n d C l e l a n d ' s r e a g e n t p r e p a r a t i o n s . T h e r e a f t e r , t h e o r i g i n f o r D r o s o p h i l a s a m p l e s was s h i f t e d t o t h e a n i o n i c s i d e o f t h e g e l s o t h a t a n i n c r e a s e d a r e a o f t h e g e l s l a b was d e v o t e d t o p r o t e i n s m i g r a t i n g t o t h e c a t h o d e i n t h e f i r s t d i m e n s i o n ( F i g . 5 ) . T i m e s t u d i e s w e r e c o n d u c t e d t o f i n d o p t i m a l e l e c t r o -p h o r e t i c c o n d i t i o n s f o r D r o s o p h i l a p r o t e i n s o n g e l s l a b s 66 o f 10 cm x 14 c m . A t l e a s t two r u n s w e r e n e c e s s a r y t o c h a r a c t e r i z e t h e D r o s o p h i l a r i b o s o m a l p r o t e i n s , a l o n g r u n t o f u l l y d e v e l o p s l o w - m o v i n g s p o t s a n d a s h o r t r u n t o k e e p f a s t - m o v i n g p r o t e i n s o n t h e g e l s ( F i g . 6a t o 7 C ) . C o m p a r i s o n o f D r o s o p h i l a a n d E . c o l i r i b o s o m a l p r o t e i n s . T h a t D r o s o p h i l a a n d E , c o l i r i b o s o m a l p r o t e i n s a r e d i f f e r e n t i s e v i d e n t i n 2 - d i m e n s i o n a l g e l s ( F i g . 8 , 6 b , a n d 6 C ) . T h e m i g r a t i o n p a t t e r n shown h e r e f o r E . c o l i ( F i g . 8) i s s i m i l a r t o p h o t o g r a p h s p u b l i s h e d b y K a l t s c h m i d t a n d W i t t m a n n ( 1 9 7 0 b ) . A l t h o u g h m o s t o f t h e E . c o l i a n d D r o s o p h 1 1 a p r o t e i n s h a v e i s o e l e c t r i c p o i n t s h i g h e r t h a n pH 8 . 6 a n d m i g r a t e t o t h e c a t h o d e i n t h e f i r s t d i m e n s i o n , t h e b a s i c p r o t e i n s o f t h e two s p e c i e s p r o d u c e q u i t e d i f f e r e n t m i g r a t i o n p a t t e r n s . S e v e r a l m a j o r c o m p o n e n t s o f t h e E . c o l i s a m p l e s , h o w e v e r , o c c u r o n t h e a n i o n i c s i d e o f t h e g e l ( F i g . 8 ) . I n c o n t r a s t , t e n f a i n t l y s t a i n i n g s p o t s a r e f o u n d o n t h i s s i d e o f t h e D r o s o p h i l a g e l s a n d f o r m a d i f f e r e n t p a t t e r n t h a n t h a t f o r t h e E . c o l i p r o t e i n s ( F i g . 6c a n d 7 c ) . T h e e l e c t r o p h o r e t i c p a t t e r n p r e s e n t l y f o u n d f o r D r o s o p h i l a r i b o s o m a l p r o t e i n s i s s i m i l a r , b u t n o t i d e n t i c a l , t o r e c e n t l y p u b l i s h e d p h o t o g r a p h s o f D r o s o p h i l a 2 - d i m e n s i o n a l g e l s ( L a m b e r t s s o n , 1 9 7 2 ) . A s i n t h e p r e s e n t s t u d y , L a m b e r t s s o n u s e d H C l - a c e t o n e p r e p a r a t i o n s o f r i b o s o m a l p r o t e i n s f r o m a d u l t D. m e l a n o g a s t e r . I n h i s g e l p r o c e d u r e , h o w e v e r , he e x c l u d e d p r o t e i n s w i t h i s o e l e c t r i c p o i n t s 67 lower than pH 8.6. because they o c c u r r e d i n low concen-t r a t i o n . Some d i f f e r e n c e s i n Lambertsson's g e l s and the pre s e n t ones are a l s o apparent on the c a t i o n i c s i d e of the g e l near the o r i g i n . Lambertsson found 52 b a s i c p r o t e i n s whereas the present study suggests 58. The d i f f e r e n c e i n number seems t o be due mainly t o minor components. Whether these are contaminants or ribosomal p r o t e i n s p r e s e n t i n low c o n c e n t r a t i o n i s u n c e r t a i n . As i n the p r e s e n t study, the degree o f p u r i t y of the ribosomes used by Lambertsson i s unknown. Comparison o f HCl-acetone and Mg-acetic a c i d p r e p a r a t i o n s  of ribosomal p r o t e i n s . HCl-acetone and Mg-acetic a c i d p r e p a r a t i o n s o f D r o s o p h i l a ribosomal p r o t e i n s produced s i m i l a r , but not i d e n t i c a l p a t t e r n s of m i g r a t i o n i n 2-dimensional g e l s ( F i g . 6a t o 7 c ) . Three major d i f f e r e n c e s were noted: spots 24 and 26, 5 and 10, and 6 and 17. The d i f f e r e n c e s i n the g e l p a t t e r n s may be a t t r i b u t e d t o d i f f e r e n c e s i n the e x t r a c t i b i l i t y o f p r o t e i n s by the two procedures or to a l t e r a t i o n s of p r o t e i n s by the two t r e a t -ments. In p r o t e i n s t u d i e s , a r t i f a c t s can be c r e a t e d through d i s u l f i d e bond formation; aggregation of monomers i n t o dimers, t r i m e r s , e t c . ; a l t e r a t i o n of amino a c i d r e s i d u e s by i s o c y a s a t e which i s a d e g r a d a t i o n product of urea; and so on (Nomura, 1970). A d e f i n i t e c o n c l u s i o n about the nature o f the d i f f e r e n c e s cannot be reached but an i n t e r p r e t c i t i o n can be attempted. 68 T h e a p p e a r a n c e a n d l o c a t i o n s o f members o f t h e two p a i r s , s p o t s 24 a n d 26 a n d s p o t s 5 a n d 1 0 , s u g g e s t s t h a t e a c h p a i r may r e p r e s e n t o n e u n i q u e p r o t e i n , i . e . o n e p o l y p e p t i d e c h a i n , a n d t h a t 2 s p o t s may b e p r e s e n t b e c a u s e o f a c o n v e r s i o n . S p o t s 24 a n d 26 a r e a d j a c e n t t o o n e a n o t h e r ( P i g , , 7a) a n d s p o t s 5 a n d 10 a r e a l s o v e r y c l o s e t o o n e a n o t h e r ( F i g . 6 a ) . I n 2 - d i m e n s i o n a l g e l s o f E . c o l i r i b o s o m a l p r o t e i n s , d i s p l a c e m e n t s o f a b o u t t h i s e x t e n t a r e o b t a i n e d when m u t a n t p r o t e i n s a r e c o m p a r e d w i t h w i l d t y p e p r o t e i n s ( D e u s s e r , e t a l . , 1 9 7 0 ; B o l l e n , e t a l . , 1 9 7 3 ) . In s u c h c a s e s , a s i n g l e a m i n o a c i d d i f f e r e n c e ( K a h a n , e t a l . , 1973) o r a s h o r t e n i n g o f t h e p o l y p e p t i d e c h a i n ( F u n a t s u , e t a l . , 1972) h a s l a t e r b e e n d e m o n s t r a t e d by c o l u m n c h r o m a t o -g r a p h y a n d c h e m i c a l a n a l y s i s . I n t h e p r e s e n t s t u d y , t h e c l o s e n e s s o f t h e members o f e a c h p a i r s u g g e s t e d t o me t h a t t h e members o f e a c h p a i r m i g h t b e c h e m i c a l l y r e l a t e d . T h e i n v e r s e r e l a t i o n s h i p b e t w e e n members o f a p a i r a l s o s u g g e s t e d t h a t t h e two members w e r e r e l a t e d . I n t h e M g - a c e t i c a c i d g e l , s p o t 24 was l a r g e a n d d a r k a n d s p o t 26 was b a r e l y v i s i b l e ( F i g . 6 a ) . I n t h e H C l -a c e t o n e g e l , s p o t 24 was c l e a r l y d i m i n i s h e d a n d s p o t 26 was c l e a r l y i n c r e a s e d ( F i g . 7 a ) . T h i s r e l a t i o n s h i p s u g g e s t e d t o me t h a t s p o t s 24 a n d 26 may r e p r e s e n t a l t e r n a t e f o r m s o f o n e u n i q u e p r o t e i n a n d t h a t a c o n v e r s i o n may o c c u r b e t w e e n t h e two f o r m s . S p o t s 5 a n d 10 a l s o e x h i b i t e d a n i n v e r s e r e l a t i o n s h i p b u t i t i s l e s s c l e a r l y d e m o n s t r a t e d i n t h e p h o t o g r a p h s t h a n t h e p r e v i o u s e x a m p l e . In t h e 69 H C l - a c e t o n e g e l , s p o t 5 was r e l a t i v e l y l a r g e a n d d a r k a n d s p o t 10 was b a r e l y v i s i b l e ( F i g . 7 a ) . I n t h e M g - a c e t i c a c i d g e l , s p o t s 5 a n d 10 w e r e s m a l l a n d a b o u t e q u a l i n s i z e ( F i g . 6 a ) . S p o t s 5 a n d 10 may a l s o r e p r e s e n t a c o n v e r s i o n b e t w e e n two f o r m s o f one u n i q u e p r o t e i n . B u t one c a n a l s o a s s u m e t h a t t h e two members o f a p a i r r e p r e s e n t two u n i q u e p r o t e i n s a n d t h a t t h e i r c l o s e -n e s s a n d i n v e r s e r e l a t i o n s h i p a r e f o r t u i t o u s . I n t h i s c a s e t h e d i f f e r e n c e s w o u l d b e a t t r i b u t e d t o d i f f e r e n c e s i n t h e s o l u b i l i t i e s o f t h e members i n t h e two e x t r a c t i o n s y s t e m s . F o r e x a m p l e , one c o u l d a s s u m e t h a t p r o t e i n 24 i s w e l l e x t r a c t e d by t h e M g - a c e t i c a c i d p r o c e d u r e a n d p r o t e i n 26 i s b a r e l y s o l u b l e . I n t h e H C l - a c e t o n e p r o c e d u r e , t h e s o l u b i l i t y o f p r o t e i n 24 w o u l d be d e c r e a s e d a n d t h a t o f p r o t e i n 26 w o u l d be c o r r e s p o n d i n g l y i n c r e a s e d . S i m i l a r e x p l a n a t i o n s c o u l d be o f f e r e d f o r s p o t s 5 a n d 1 0 . T h e s o l u b i l i t y h y p o t h e s i s seems more c o n t r i v e d t o me t h a n t h e a r t i f a c t h y p o t h e s i s , b u t o b v i o u s l y n e i t h e r t h e o r y i s p r o v e d b y t h e o b s e r v a t i o n s . A s d i s c u s s e d l a t e r , a r t i f a c t s c a n be d e t e c t e d more r e a d i l y b y c o l u m n c h r o m a t -o g r a p h y a n d c h e m i c a l a n a l y s e s t h a n by g e l e l e c t r o p h o r e s i s . S o l u b i l i t y d i f f e r e n c e s , h o w e v e r , may be a more r e a s o n a b l e e x p l a n t i o n f o r s p o t s 6 a n d 17 t h a n a r t i f a c t f o r m a t i o n . I n t h e H C l - a c e t o n e g e l , s p o t 6 was a b s e n t a n d s p o t 17 s t a i n e d s t r o n g l y ( F i g . 7a) b u t when t h e M g - a c e t i c a c i d m e t h o d o f p r o t e i n e x t r a c t i o n was u s e d , s p o t 6 was p r o m i n e n t a n d s p o t 17 was f a i n t . A g a i n an i n v e r s e 70 r e l a t i o n s h i p i s e v i d e n t a n d c o n v e r s i o n o f o n e u n i q u e p r o t e i n t o two f o r m s c o u l d b e i n v o k e d . B u t t h e two s p o t s a r e l o c a t e d i n r e l a t i v e l y d i s t a n t a r e a s o f t h e g e l s . D i s p l a c e m e n t s o f t h i s e x t e n t w e r e n o t o b s e r v e d w i t h m u t a n t r i b o s o m a l p r o t e i n s o f b a c t e r i a . U n l e s s o n e a s s u m e s t h a t a d r a s t i c c h e m i c a l c h a n g e o c c u r r e d , c o n v e r s i o n seems a n u n -l i k e l y e x p l a n a t i o n f o r s p o t s 6 a n d 1 7 . T h e p r e s e n c e o f two d i f f e r e n t p o l y p e p t i d e c h a i n s w i t h q u i t e d i f f e r e n t m i g r a t i o n p r o p e r t i e s a n d s o l u b i l i t i e s seems t o be a more p l a u s i b l e t h e o r y . A g a i n t h e h y p o t h e s i s c a n n o t be p r o v e d by g e l e l e c t r o p h o r e s i s a l o n e . T h r e e m a j o r d i f f e r e n c e s w e r e a p p a r e n t i n g e l s s o l u b i l i z e d b y t h e two m e t h o d s b u t t h e o v e r a l l p a t t e r n s f o r t h e m a j o r i t y o f s p o t s w e r e r e m a r k a b l y s i m i l a r ( F i g . 6a t o 7 c ) . T h e M g - a c e t i c a c i d m e t h o d r e m o v e s a b o u t 80% o f t h e p r o t e i n f r o m r a t l i v e r r i b o s o m e s ( W e l f l e , e t a l . , 1 9 7 1 ) . No e v a l u a t i o n o f t h e H C l - a c e t o n e m e t h o d was f o u n d i n t h e l i t e r a t u r e . T h e s i m i l a r i t y s u g g e s t s t h a t t h e H C l - a c e t o n e a n d M g - a c e t i c a c i d m e t h o d s e x t r a c t a b o u t t h e same number a n d k i n d s o f p r o t e i n s f r o m D r o s o p h i l a r i b o s o m e s . T h e t h r e e d i f f e r e n c e s i n t h e H C l - a c e t o n e and M g - a c e t i c a c i d g e l s , h o w e v e r , i l l u s t r a t e how 2 - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s c a n r e v e a l d i f f e r e n c e s i n p r o t e i n s a m p l e s . Number o f D r o s o p h i l a r i b o s o m a l p r o t e i n s . P r o t e i n s t h a t r e m a i n e d a t t h e o r i g i n i n t h e f i r s t d i m e n s i o n a n d m i g r a t e d d i r e c t l y t o w a r d t h e c a t h o d e i n t h e s e c o n d d i m e n s i o n , h a v e 71 g e n e r a l l y not been r e p r e s e n t e d i n the schematic diagram, except f o r spots 4, 9 and 65 ( F i g . 5 ) . Such spots c o u l d be due to p r o t e i n s w i t h i s o e l e c t r i c p o i n t s equal t o 8.6 and hence f a i l t o migrate i n the f i r s t dimension. Or a p r o t e i n may e x h i b i t low s o l u b i l i t y a t pH 8.6 i n the f i r s t dimension and not move from the o r i g i n . I t may then e x h i b i t i n c r e a s e d s o l u b i l i t y a t pH 4.3 i n the second dimension and move away from the o r i g i n . The l a t t e r i n t e r p r e t a t i o n seems more a p p r o p i a t e than the former, e s p e c i a l l y f o r spots 4 and 9. They are connected t o spots 5 and 10 by s t r e a k s which are i n d i c a t i v e o f i n s o l u b i l i t y . T h i s type o f a r t i f a c t has been observed p r e v i o u s l y i n 2-dimensional g e l e l e c t r o p h o r e s i s (Sherton and Wool, 1972). Spot 65 a t the t i p of the c e n t r a l a x i s , however, may be a unique p r o t e i n w i t h an i s o e l e c t r i c p o i n t c l o s e t o pH 8.6 as i t i s not rep r e s e n t e d by a corres p o n d i n g spot t o one s i d e o f the c e n t r a l a x i s i n the second dimension ( F i g . 9 and 10). In e s t i m a t i n g the number o f unique p r o t e i n s p r e s e n t i n the D r o s o p h i l a ribosomal p r e p a r a t i o n s , the p r e v i o u s l y d i s c u s s e d p a i r s o f spots (24 and 26, 5 and 10, 6 and 17) can be regarded as 6 unique p r o t e i n s or 3 unique p r o t e i n s and 3 a r t i f a c t s . The t h r e e spots 52, 55 and 59 have been counted as one p r o t e i n because they may be a r t i f a c t s ( F i g . 5 and 7b). A s e r i e s arrangement l i k e t h i s i n the g e l s of r a t l i v e r r i bosomal p r o t e i n s was r e p l a c e d by one spot a f t e r a. change i n pretreatment o f the ribosomes 72 ( S h e r t o n a n d W o o l , 1 9 7 2 ) . I n t h e H C l - a c e t o n e p r e p a r a t i o n s o f D r o s o p h i l a r i b o s o m a l p r o t e i n s , t h e r e w e r e 42 - 45 m a j o r a n d 16 m i n o r p r o t e i n s w i t h i s o e l e c t r i c p o i n t s g r e a t e r t h a n 8 . 6 , 1 p r o t e i n w i t h a n i s o e l e c t r i c p o i n t c l o s e t o 8 .6 , , a n d p o s s i b l e 10 m i n o r p r o t e i n s w i t h i s o e l e c t r i c p o i n t s l e s s t h a n 8 . 6 ( F i g . 5 ) . A t o t a l o f 69 - 72 p r o t e i n c o m p o n e n t s v /ere p r e s e n t . C o m p a r i s o n o f 2 - d i m e n s i o n a l g e l s o f r i b o s o m a l p r o t e i n s  f r o m m a l e s a n d f e m a l e s . T h e g e l p a t t e r n s f o r O r e g o n - R m a l e s a n d C ( I ) R M / Y f e m a l e s w e r e q u i t e s i m i l a r t o o n e a n o t h e r a n d a l s o t o t h o s e f o r m i x e d s a m p l e s f r o m O r e g o n - R m a l e s a n d f e m a l e s . D i f f e r e n c e s , h o w e v e r , i n t h e d e g r e e o f s t a i n i n g o f some s p o t s w e r e o b s e r v e d . P r e v i o u s s t u d i e s o f D r o s o p h i l a r i b o s o m a l p r o t e i n s by 1 - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s a l s o i n d i c a t e d t h a t m a l e s a n d f e m a l e s p r o d u c e d s i m i l a r b a n d i n g p a t t e r n s a l t h o u g h q u a n t i t a t i v e d i f f e r e n c e s w e r e o b s e r v e d ( K i e f e r a n d G r o s s , 1 9 6 9 ; M. G o u l d S o m e r o , p e r s o n a l c o m m u n i c a t i o n ) . S u c h q u a n t i t a t i v e d i f f e r e n c e s a r e p r o b a b l y d u e t o v a r i a t i o n w i t h i n t h e p r o c e d u r e a n d do n o t r e p r e s e n t r e a l d i f f e r e n c e s i n t h e s a m p l e s . 2 - d i m e n s i o n a l g e l e l e c t r o -p h o r e s i s i s n o t i n t e n d e d t o be a q u a n t i t a t i v e m e t h o d . S t a i n i n g c o n d i t i o n s a r e n o t n e c e s s a r i l y o p t i m a l a n d t h e amount o f r e s i d u e a t t h e o r i g i n o r a l o n g t h e a x e s v a r i e s . T h e f a c t t h a t t h e p a t t e r n s f o r m a l e s a n d f e m a l e s w e r e a l i k e s u g g e s t s t h a t t h e i s o l a t i o n m e t h o d p r o d u c e s r i b o s o m e s 73 t h a t a r e r e a s o n a b l y f r e e o f e x t r a n e o u s p r o t e i n s . S e x d i f f e r e n c e s c a n c r e a t e p r o b l e m s i n i s o l a t i n g r i b o s o m e s . E g g p r o t e i n s i n f e m a l e c r i c k e t s , f o r e x a m p l e , w e r e f o u n d t o a d s o r b t o t h e r i b o s o m e s a n d p r e v e n t t h e i r i s o l a t i o n b y c u s t o m a r y m e t h o d s ( K a v l e n a s , 1 9 7 0 ) . When f e m a l e s w e r e f r e e d o f e g g s , r i b o s o m e s c o u l d b e i s o l a t e d b y t h e same m e t h o d u s e d f o r m a l e s . 2 - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s o f r i b o s o m a l p r o t e i n s f r o m  c o l d - s e n s i t i v e m u t a n t s . F o r e a c h s a m p l e , one p r e p a r a t i o n o f r i b o s o m e s was i s o l a t e d a n d a t l e a s t two g e l s w e r e d o n e f o r e a c h p r e p a r a t i o n . In t h e 2 - d i m e n s i o n a l g e l s o f t h e c s c s c s c s c s D r o s o p h i l a m u t a n t s , 5 , 1 5 , 1 6 , 2 6 a n d 27 , no new s p o t s o r d i s p l a c e d o n e s w e r e d e t e c t e d . I n t h e g e l s o f c s c s m u t a n t s 5 a n d 2 7 some o f t h e f a i n t l y s t a i n i n g s p o t s ( 5 8 , 5 2 , 5 5 , a n d 59) w e r e a b s e n t ( F i g . 10 a n d 11) b u t w e r e p r e s e n t i n t h e g e l s o f t h e m i x e d s a m p l e o f O r e g o n - R m a l e s a n d f e m a l e s , O r e g o n - R m a l e s a l o n e , C ( 1 ) R M f e m a l e s , a n d t h e t h r e e o t h e r m u t a n t s ( F i g . 9 a n d 1 2 ) . T h e a b s e n c e o f t h e f a i n t l y s t a i n i n g s p o t s i s p r o b a b l y due t o v a r i a b i l i t y i n t h e m e t h o d s . T h e y may be c o n t a m i n a n t s w h i c h a r e f r e q u e n t l y b u t n o t a l w a y s p r e s e n t i n t h e i s o l a t e d r i b o s o m e s . O r t h e y may be r i b o s o m a l p r o t e i n s o c c u r r i n g i n l o w c o n c e n t r a t i o n s a n d may s o m e t i m e s be l o s t d u r i n g p r o t e i n e x t r a c t i o n o r g e l e l e c t r o p h o r e s i s . T h o u g h a c o n s t a n t amount o f s a m p l e was a p p l i e d t o t h e g e l s , t h e e f f e c t i v e c o n c e n t r a t i o n o f t h e v a r i o u s p r o t e i n s w h i c h 74 m i g r a t e o n t o a g e l may v a r y d u e t o p r e c i p i t a t i o n a t t h e o r i g i n o r a l o n g t h e a x e s . s. A n a l t e r e d r i b o s o m a l p r o t e i n f r o m a n E . c o l i m u t a n t i s d e t e c t e d b y i t s d i s p l a c e m e n t f r o m t h e n o r m a l p o s i t i o n f o u n d i n g e l s o f t h e w i l d type. A d e c r e a s e d amount o f s i n g l e p r o t e i n i s g e n e r a l l y n o t o b s e r v e d . However , o n e E . c o l i " r e v e r t a n t " f r o m s t r e p t o m y c i n d e p e n d e n c e h a d a d i s p l a c e d S4 s p o t w h i c h s t a i n e d v e r y f a i n t l y i n 2 -d i m e n s i o n a l g e l s ( D e u s s e r , et a l . , 1 9 7 0 ; F u n a t s u , e t a l . , 1 9 7 2 ) . T h i s p r o t e i n n o r m a l l y p r o d u c e s a d a r k l y s t a i n i n g s p o t . O t h e r " r e v e r t a n t s " h a d d i s p l a c e d S4 s p o t s b u t t h e y w e r e n o t r e d u c e d i n a m o u n t . T h e d e c r e a s e d s t a i n i n g o f S4 f r o m t h i s p a r t i c u l a r m u t a n t was a t t r i b u t e d t o a c h a n g e i n s o l u b i l i t y o f t h e p r o t e i n a n d l o s s d u r i n g g e l e l e c t r o -p h o r e s i s . P r o t e i n S4 o f t h e m u t a n t was l a t e r i s o l a t e d by c o l u m n c h r o m a t o g r a p h y a n d f o u n d t o b e a l t e r e d i n e n d g r o u p , t r y p t i c d i g e s t f i n g e r p r i n t s , a n d m o l e c u l a r w e i g h t ( F u n a t s u , , e t S L L . , 1 9 7 2 ) . I t was c o n c l u d e d t h a t t h e m u t a n t p r o t e i n was s h o r t e r b y 9% c o m p a r e d t o t h e w i l d -t y p e p r o t e i n , , I t i s u n l i k e l y t h a t t h e q u a n t i t a t i v e d i f f e r e n c e s c s c s o b s e r v e d i n t h e g e l s o f 5 a n d 27 a r e s i m i l a r t o t h e a b o v e e x a m p l e f o r t h e f o l l o w i n g r e a s o n s : (1) S e v e r a l d e c r e a s e s i n s p o t s w e r e n o t e d . A s i n g l e c h a n g e i n a p r o t e i n w o u l d be e x p e c t e d t o r e s u l t f r o m a m u t a t i o n i n t h e s t r u c t u r a l g e n e o f a r i b o s o m a l p r o t e i n . (2) T h o u g h t h e 75 p a t t e r n o f p r o t e i n m i g r a t i o n i n t h e g e l s was f o u n d t o be c o n s t a n t , t h e d e g r e e o f s t a i n i n g o f s p o t s was v a r i a b l e . (3) T h e d e g r e e o f p u r i t y o f t h e r i b o s o m a l p r e p a r a t i o n s c s c s i s u n c e r t a i n . . (4) T h o u g h m u t a n t s 5 a n d 27 h a v e a b n o r m a l abdomen p h e n o t y p e s , t h e y a r e n o t r e l a t e d g e n e t i -c a l l y . T h e m u t a t i o n s map t o d i f f e r e n t a r e a s o f t h e X c s c s chromosome a n d t h e t r a n s h e t e r o z y g o t e s o f 5 a n d 27 l o o k l i k e w i l d t y p e . 76 D ISCUSSION C h e m i c a l a n a l y s i s o f r i b o s o m e s . Much e f f o r t h a s g o n e i n t o t h e c h e m i c a l a n a l y s i s o f E.~ c o l i r i b o s o m e s ( K u r l a n d , 1 9 7 2 ) . A n a c c u r a t e a c c o u n t i n g o f r i b o s o m a l c o n s t i t u e n t s i s a n e c e s s a r y p r e l i m i n a r y t o u n d e r s t a n d i n g how e a c h c o m p o n e n t o f t h e r i b o s o m e s f u n c t i o n s i n p r o t e i n s y n t h e s i s . On t h e b a s i s o f 2 - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s , c o l u m n c h r o m a t o g r a p h y , a n d r e c o n s t i t u t i o n s t u d i e s , i t h a s b e e n c o n c l u d e d t h a t t h e s m a l l s u b u n i t c o n t a i n s 2 0 - 2 1 p r o t e i n s ( W i t t m a n n , e t a l . , 1 9 7 1 ) . T h e number o f r i b o s o m a l p r o t e i n s i n t h e l a r g e s u b u n i t i s s t i l l u n c e r t a i n a n d may b e a s l o w a s 28 o r a s h i g h a s 34 ( M o r a , e t a l . , 1 9 7 1 ; D z i o n o r a , e t a l . , 1 9 7 0 ; K a l t s c h m i d t a n d W i t t m a n n , 1 9 7 0 b ) . T h e m a i n d i f f i c u l t y i n s u c h s t u d i e s h a s b e e n t o d i s t i n g u i s h r i b o s o m a l p r o t e i n s f r o m c o n t a m i n a n t s a n d a r t i f a c t s . By ammonium s u l f a t e p r e c i p i t a t i o n o f r i b o s o m e s , d i e t h y l a m i n o e t h y l c h r o m a t o g r a p h y a n d ammonium c h l o r i d e w a s h i n g o f r i b o s o m e s , i t was f o u n d t h a t much e x t r a n e o u s p r o t e i n c o u l d b e r e m o v e d f r o m r i b o s o m e s w i t h o u t i m p a i r i n g t h e i r b i o l o g i c a l a c t i v i t y ( K u r l a n d , e t a l . , 1 9 6 6 : N o m u r a , 1 9 7 0 ) . T h e p r o b l e m o f d i s t i n g u i s h i n g u n i q u e p r o t e i n s f r o m a r t i f a c t s was s o l v e d b y i s o l a t i n g a n d a n a l y z i n g e a c h c o m p o n e n t o f p u r i f i e d , b i o l o g i c a l l y a c t i v e r i b o s o m a l p r e p a r a t i o n s . T w o - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s o f p u r i f i e d E . c o l i r i b o s o m e s , f o r e x a m p l e , i n d i c a t e d t h a t 21 p r o t e i n c o m p o n e n t s w e r e p r e s e n t i n t h e s m a l l s u b u n i t 77 and 34 i n the large subunit (Kaltschmidt and Wittmann, 1970). As some of the protein components were f a i n t l y staining they could be ribosomal proteins present i n fewer copies than the major components or a r t i f a c t s created by secondary modification of proteins. Minor protein fractions were also obtained when ribosomal proteins were separated by column chromatography. The advantage of th i s method, however, i s that the fractions can be analyzed. End group analysis, t r y p t i c digest f i n g e r p r i n t s , molecular weight determinations and immunological tests have shown that many protein components of the ribosome have s p e c i f i c c h a r a c t e r i s t i c s and therefore are unique proteins (Mora, et a l . , 1971; S t o f f l e r and Wittmann, 1971). On the other hand, some protein components are probably a r t i f a c t s because t h e i r t r y p t i c f ingerprints etc. are similar to those of other fractions (Mora, et aJL., 1971). As already indicated, ribosomal mutants and the i n v i t r o reconstitution of subunits are also important methods of establishing the i d e n t i t y of ribosomal proteins. The a n a l y t i c a l studies have indicated that i s o l a t e d ribosomes of E. c o l i are heterogeneous (Kurland, 1972). Twelve of the proteins of the 30S subunits, for example, are present i n approximately one copy per ribosome while eight " f r a c t i o n a l " proteins are present i n 0.5 or fewer copies per ribosome (Voynow and Kurland, 1971). Obviously is o l a t e d ribosomes cannot be uniform i n composition. 78 One p r o t e i n o f t h e 50S s u b u n i t i s u n u s u a l i n t h a t i t a p p e a r s t o be p r e s e n t i n 2 o r 3 c o p i e s p e r r i b o s o m e ( M o l l e r , e t a l . , 1 9 7 2 ; T e r h o r s t / e t a l . , 1 9 7 2 ) . A number o f i n t e r p r e t a t i o n s o f t h e h e t e r o g e n e i t y h a v e b e e n a d v a n c e d . One i s t h a t h e t e r o g e n e i t y i s an a r t i f a c t a n d d o e s n o t o c c i i r i n v i v o . A more i n t e r e s t i n g t h e o r y i s t h a t h e t e r o g e n e i t y r e p r e s e n t s d i f f e r e n t f u n c t i o n a l s t a t e s t h a t t h e r i b o s o m e u n d e r g o e s d u r i n g p r o t e i n s y n t h e s i s ( K u r l a n d , 1 9 7 2 ) . T h e " f r a c t i o n a l " r i b o s o m a l p r o t e i n s , l i k e t h e s u p e r n a t a n t f a c t o r s , may s h u t t l e t o a n d f r o among t h e r i b o s o m e s d u r i n g i n i t i a t i o n , p r o p a g a t i o n , a n d t e r m i n a -t i o n o f p r o t e i n s y n t h e s i s . P r o t e i n s o f D r o s o p h i l a r i b o s o m e s . A b o u t 10 p r o t e i n s w i t h i s o e l e c t r i c p o i n t s l o w e r t h a n pH 8 . 6 w e r e o b s e r v e d i n t h e p r e s e n t s t u d y o f D r o s o p h i l a r i b o s o m e s . T h o u g h t h e s e p r o t e i n s o c c u r i n l o w c o n c e n t r a t i o n , t h e y s h o u l d n o t b e i g n o r e d a s c o n t a m i n a n t s u n t i l t h i s i s p r o v e d . L i k e t h e l i g h t l y s t a i n i n g s p o t s o n t h e c a t i o n i c s i d e o f t h e g e l , t h e y may b e f r a c t i o n a l r i b o s o m a l p r o t e i n s . T o h e l p d i s t i n g u i s h b e t w e e n t h e two p o s s i b i l i t i e s , D r o s o p h i l a r i b o s o m e s s h o u l d b e p u r i f i e d b y t h e m e t h o d s p r e v i o u s l y d i s c u s s e d . T h e e f f e c t i v e n e s s o f t h e p r o c e d u r e s i n r e m o v i n g e x t r a n e o u s p r o t e i n s w h i l e m a i n t a i n i n g t h e b i o l o g i c a l a c t i v i t y o f t h e r i b o s o m e s s h o u l d be e s t a b l i s h e d . U n f o r t u n a t e l y t h e s u i t a b i l i t y o f many E . c o l i p r o c e d u r e s h a s n o t b e e n e s t a b l i s h e d f o r D r o s o p h i l a . 79 S e p a r a t i o n o f r i b o s o m a l s u b u n i t s may a l s o h e l p t o r e m o v e e x t r a n e o u s p r o t e i n s . I n 2 - d i m e n s i o n a l g e l s o f p u r i f i e d r a t l i v e r " 8 0 S " r i b o s o m e s , f o r e x a m p l e , a maximum o f 6 p r o t e i n s was o b s e r v e d o n t h e a n i o n i c s i d e o f t h e g e l . When t h e r i b o s o m a l s u b u n i t s w e r e e x a m i n e d , 3 o f t h e a n i o n i c s p o t s , b u t no o t h e r s p o t s w e r e a b s e n t . A s t h e p r o c e d u r e p r o d u c e d s u b u n i t s t h a t s t i l l f u n c t i o n e d n o r m a l l y i n v i t r o , o n l y t h e 3 a n i o n i c p r o t e i n s r e m a i n i n g a r e by d e f i n i t i o n , r i b o s o m a l p r o t e i n s . S i n c e 11 r i b o s o m a l p r o t e i n s f r o m E . c o l i s u b u n i t s o c c u r o n t h e a n i o n i c s i d e o f 2 - d i m e n s i o n a l g e l s , i t i s t h o u g h t t h a t t h e s e p r o t e i n s may o c c u r more f r e q u e n t l y i n p r o k a r y o t i c r i b o s o m e s t h a n i n e u k a r y o t i c o n e s . T h e p r e s e n t s t u d y i n d i c a t e d t h a t t h e number o f p r o t e i n s i n D r o s o p h i l a r i b o s o m e s i s 69 t o 7 2 . I n o n e -d i m e n s i o n a l g e l s d e v e l o p e d a t a c i d i c pH v a l u e s , 2 2 - 3 0 p r o t e i n b a n d s a r e e v i d e n t ( K i e f e r a n d G r o s s , 9 6 9 ; L a m b e r t s s o n , e t a l . , 1 9 7 0 : M. G o u l d S o m e r o , p e r s o n a l c o m m u n i c a t i o n ) . T h o u g h t w o - d i m e n s i o n a l g e l e l e c t r o -p h o r e s i s r e s o l v e d t h e D r o s o p h i l a p r o t e i n s t o a g r e a t e r e x t e n t t h a n o n e - d i m e n s i o n a l g e l s , i t i s o b v i o u s f r o m t h e p r e v i o u s d i s c u s s i o n t h a t t h e number 6 9 - 7 2 i s s t i l l o n l y a n a p p r o x i m a t i o n . T h e i s o l a t i o n p r o c e d u r e p r o d u c e s 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 o f unknown p u r i t y a n d t h e s e w e r e n o t t e s t e d f o r b i o l o g i c a l a c t i v i t y . M o r e o v e r , t h o u g h o n e c a n make r e a s o n a b l e a s s u m p t i o n s a b o u t a r t i f a c t s i n 2 - d i m e n s i o n a l g e l s , t h e s e c a n n o t be p r o v e d e x c e p t by 80 c h e m i c a l a n a 3 . y s i s . D e s p i t e some v a r i a t i o n i n e s t i m a t e s , i t i s g e n e r a l l y f o u n d t h a t " 8 0 S " e u k a r y o t i c r i b o s o m e s c o n t a i n m o r e p r o t e i n s t h a n 70S E . c o l i r i b o s o m e s . P u r i f i e d p r e p a r a -t i o n s o f r a t l i v e r r i b o s o m e s , f o r e x a m p l e , c o n t a i n e d 6 8 - 7 2 p r o t e i n c o m p o n e n t s i n 2 - d i m e n s i o n a l g e l s ( S h e r t o n a n d W o o l , 1 9 7 2 ) . E s t i m a t e s f o r o t h e r e u k a r y o t e s b y t h i s m e t h o d v a r y f r o m 70 t o 76 r i b o s o m a l p r o t e i n s . H e n c e t h e p r e s e n t e s t i m a t e o f 69 -72 p r o t e i n s f o r 82S D r o s o p h i l a r i b o s o m e s i s r e a s o n a b l e ( L a m b e r t s s o n , e t a l . , 1 9 7 0 ) . In f a c t , t h e number o f D r o s o p h i l a r i b o s o m a l p r o t e i n s may h a v e b e e n s l i g h t l y u n d e r e s t i m a t e d d u e t o i n c o m p l e t e r e s o l u t i o n o f t h e p r o t e i n s i n a s y s t e m d e s i g n e d f o r E . c o l i . T h r e e p a i r s o f p r o t e i n s f r o m t h e " 8 0 S " r i b o s o m e s o f r a t l i v e r , f o r e x a m p l e , w e r e n o t s e p a r a t e d u n t i l t h e g e l c o n c e n t r a t i o n a n d pH i n t h e f i r s t d i m e n s i o n w e r e c h a n g e d f r o m t h e s t a n d a r d c o n d i t i o n s u s e d f o r E . c o l i ( S h e r t o n a n d W o o l , 1 9 7 2 ) . One p a i r o f p r o t e i n s was s e p a r a t e d o n l y when t h e s u b u n i t s w e r e e x a m i n e d . E x a m i n a t i o n o f r i b o s o m e s f r o m c o l d - s e n s i t i v e m u t a n t s o f  D r o s o p h i l a . T h e a d v a n t a g e o f 2 - d i m e n s i o n a l g e l e l e c t r o -p h o r e s i s i s t h a t i t r e q u i r e s a s m a l l s a m p l e a n d r e s o l v e s m o s t r i b o s o m a l p r o t e i n s i n o n e , r e l a t i v e l y q u i c k , p r o c e d u r e . T h e m e t h o d s h o u l d d e t e c t c h a n g e s i n t h e c h a r g e o r s i z e o f r i b o s o m a l p r o t e i n s i f t h e s e o c c u r i n t h e c o l d - s e n s i t i v e m u t a n t s . H o w e v e r , f i v e s e x - l i n k e d , c o l d - s e n s i t i v e m u t a n t s 81 examined by the method, e x h i b i t e d no d i s p l a c e d p r o t e i n s . Q u a n t i t a t i v e d i f f e r e n c e s observed i n g e l s of mutants 5 C S and 2 7 c s were a t t r i b u t e d to v a r i a b i l i t y of the method. I t was concluded t h a t t h e r e was no d e t e c t a b l e change i n c s c s c s a d u l t ribosomal p r o t e i n s caused by mutations 5 , 15 , 16 , cs cs 26 c and 27 . c s As a p r e c a u t i o n , mutants 5 and 2 7 c s should be r e t e s t e d , p r e f e r a b l y by an independent method such as sedimentation of ribosomal p a r t i c l e s produced at low temperature. I f the q u a n t i t a t i v e changes are due t o contaminants or v a r i a b i l i t y of the e l e c t r o p h o r e s i s method, the sedimentation p r o f i l e s f o r the mutants should be l i k e t h a t of w i l d - t y p e r ibosomal p a r t i c l e s . Ribosomal p r o t e i n s from numerous c o l d - s e n s i t i v e mutants were examined by 1-dimensional g e l e l e c t r o p h o r e s i s (M. Gould Somero, p e r s o n a l communication). These i n c l u d e d 8 o t h e r r e c e s s i v e , c o l d - s e n s i t i v e l e t h a l s of the X chromosomes; 25 dominant, c o l d - s e n s i t i v e l e t h a l s of the 2nd chromosomes; 11 dominant, c o l d - s e n s i t i v e developmentally r e t a r d e d mutants of the 2nd chromosomes; and 3 r e c e s s i v e , c o l d - s e n s i t i v e l e t h a l s of the 3rd chromosomes. The dominant r e t a r d e d mutations are not l e t h a l s but p r o l o n g development from 5 t o 10 days at 17° C and 1-3 days a t 22° C (Rosenbluth, e t a l . , 1972). Heterozygous females of the t e s t e d dominant, r e t a r d e d mutants were s t e r i l e or p o o r l y v i a b l e a t 22° C. Although i r r e g u l a r i t i e s were sometimes noted i n the g e l s of some of the mutants, none were 82 r e p r o d u c i b l e (M. G o u l d S o m e r o , p e r s o n a l c o m m u n i c a t i o n ) . I t was c o n c l u d e d t h a t t h e r e was no e v i d e n c e o f c h a n g e i n t h e r i b o s o m a l p r o t e i n s f r o m t h e m u t a n t s . T h e s e d i m e n t a t i o n p r e p e r t i e s o f r i b o s o m a l p a r t i c l e s f r o m a few o f t h e c o l d - s e n s i t i v e m u t a n t s w e r e a l s o s t u d i e d (M. G o u l d S o m e r o , p e r s o n a l c o m m u n i c a t i o n ) . T h e s e i n c l u d e d 5 r e c e s s i v e , c o l d - s e n s i t i v e l e t h a l s o f t h e X c h r o m o s o m e s w h i c h h a d b e e n e x a m i n e d b y 1 - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s . o 3 A d u l t m a l e s w e r e f e d o v e r n i g h t a t 17 C H u r i d i n e w i t h D r o s o p h i l a f o o d . R i b o s o m a l p r e p a r a t i o n s w e r e s e d i m e n t e d i n s u c r o s e g r a d i e n t s . A l l t h e f l i e s i n c o r p o r a t e d l a b e l i n t o r i b o s o m e s a t t h e low t e m p e r a t u r e . T h e r e was no a p p a r e n t d i f f e r e n c e b e t w e e n t h e s e d i m e n t a t i o n p r o f i l e s o f r i b o s o m e s f r o m t h e m u t a n t s a n d w i l d - t y p e . L i m i t a t i o n s o f t h e s t u d y a n d some r e c o m m e n d a t i o n s . Do some c o l d - s e n s i t i v e m u t a n t s o f D r o s o p h i l a h a v e a l t e r e d r i b o s o m a l p r o t e i n s ? No d i r e c t e v i d e n c e t o s u p p o r t t h e h y p o t h e s i s was o b t a i n e d . On t h e o t h e r h a n d , t h e n e g a t i v e r e s u l t s a r e n o t c o n c l u s i v e f o r t h e f o l l o w i n g r e a s o n s . (1) A c o l d - s e n s i t i v e m u t a n t may h a v e a n a l t e r e d a m i n o a c i d s e q u e n c e i n a r i b o s o m a l p r o t e i n b u t t h e e l e c t r o p h o r e t i c p r o p e r t i e s o f t h e p r o t e i n may n o t b e c h a n g e d . S l i g h t a l t e r a t i o n s i n c h a r g e o r s i z e w o u l d be p a r t i c u l a r l y d i f f i c u l t t o d e t e c t i n 1 - d i m e n s i o n a l g e l s . A s t u d y o f s t r e p t o m y c i n i n d e p e n d e n c e m u t a n t s o f E . c o l i i l l u s t r a t e s t h e p r o b l e m ( D e u s s e r , e t a l . , 1 9 7 0 ) . O u t o f 13 m u t a n t s , 83 4 e x h i b i t e d c h a n g e s i n p r o t e i n S4 i n 2 - d i m e n s i o n a l g e l s . T h r e e o t h e r m u t a n t s h a d S4 p r o t e i n s w h i c h w e r e i n d i s t i n g u i s h -a b l e f r o m w i l d - t y p e i n 2 - d i m e n s i o n a l g e l s b u t w e r e c l e a r l y d i f f e r e n t i n i m m u n o l o g i c a l p r o p e r t i e s . I f c o l d -s e n s i t i v e r i b o s o m a l m u t a n t s o f D r o s o p h i l a o c c u r i n l o w f r e q u e n c y , t h e l i m i t a t i o n s o f t h e m e t h o d w o u l d be c r i t i c a l . (2) A s e r i o u s p r o b l e m i n t h e b i o c h e m i c a l t e s t i n g o f t h e d o m i n a n t , c o l d - s e n s i t i v e m u t a n t s was t h e n e c e s s i t y o f e x a m i n i n g them i n t h e h e t e r o z y g o u s c o n d i t i o n . H o m o z y -g o t e s o f t h e d o m i n a n t , c o l d - s e n s i t i v e l e t h a l s a r e i n v i a b l e e v e n a t t h e p e r m i s s i v e t e m p e r a t u r e . A n d h o m o z y g o t e s o f t h e d o m i n a n t , r e t a r d e d m u t a n t s c o u l d n o t be o b t a i n e d b e c a u s e o f t h e s t e r i l i t y o r l o w f e r t i l i t y o f t h e h e t e r o z y -g o u s f e m a l e s . I f a d o m i n a n t , c o l d - s e n s i t i v e m u t a n t h a d an a l t e r e d r i b o s o m a l p r o t e i n , t h e c h a n g e may n o t h a v e b e e n d e t e c t e d i n 1 - d i m e n s i o n a l g e l s b e c a u s e o f t h e p r o d u c t i o n o f t h e n o r m a l r i b o s o m a l p r o t e i n f r o m t h e w i l d - t y p e a l l e l e . A l t e r e d r i b o s o m a l p r o t e i n s i n h e t e r o z y g o t e s may be d i f f i c u l t t o d e t e c t i n o t h e r t e s t s . I -am u n c e r t a i n w h e t h e r a b n o r m a l s u b p a r t i c l e s w o u l d be d e t e c t e d i n s e d i -m e n t a t i o n p r o f i l e s i f n o r m a l r i b o s o m e s a n d s u b u n i t s w e r e p r o d u c e d a t t h e same t i m e . A c h a n g e i n t h e e l e c t r o -p h o r e t i c p r o p e r t i e s o f a m a j o r r i b o s o m a l p r o t e i n , h o w e v e r , w o u l d p r o b a b l y be d e t e c t a b l e i n h e t e r o z y g o t e s by 2 -d i m e n s i o n a l g e l e l e c t r o p h o r e s i s . B o t h t h e n o r m a l a n d a l t e r e d a l l e l i c r i b o s o m a l p r o t e i n s o f an E . c o l i m u t a n t , 84 f o r e x a m p l e , w e r e d i s t i n g u i s h a b l e i n 2 - d i m e n s i o n a l g e l s o f r i b o s o m a l p r o t e i n s f r o m t h e h e t e r o z y g o u s m e r o d i p l o i d ( B o l l e n , e t a l . , 1 9 7 3 ) . (3) A n o t h e r l i m i t a t i o n i n t h e t e s t i n g o f t h e c o l d -s e n s i t i v e m u t a n t s i s t h a t o n l y a d u l t s w e r e e x a m i n e d . I f r i b o s o m a l p r o t e i n s i n t h e v a r i o u s d e v e l o p m e n t a l s t a g e s d i f f e r , t h e n m u t a n t s w i t h d e f e c t s i n l a r v a l o r p u p a l r i b o -somes w o u l d h a v e b e e n o v e r l o o k e d . T h e r e i s g o o d e v i d e n c e t h a t some c o m p o n e n t s o f t h e p r o t e i n s y n t h e s i z i n g s y s t e m c h a n g e d u r i n g i n s e c t d e v e l o p -m e n t . Numerous q u a n t i t a t i v e c h a n g e s i n t h e i s o a c c e p t i n g f o r m s o f t - R N A s w e r e o b s e r v e d i n 1 s t a n d 3 r d i n s t a r l a r v a e a n d a d u l t s o f D r o s o p h i l a ( W h i t e , et a l . , 1 9 7 3 ) . I t h a s a l s o b e e n shown t h a t p u p a e o f T e n e b r i o m o l e t o r p r o d u c e a new s p e c i e s o f t - R N A a n d a new s y n t h e t a s e ( I l a n , 1 9 6 9 ; I l a n , e t a l . , 1 9 7 0 ) . In a d d i t i o n , s t a g e - s p e c i f i c i n i t i a t i o n f a c t o r s f o r p r o t e i n s y n t h e s i s w e r e f o u n d i n t h i s i n s e c t ( I l a n a n d I l a n , 1 9 7 1 ) . Two t y p e s o f e x p e r i m e n t s s u g g e s t t h a t t h e p r o t e i n c o n s t i t u e n t s o f D r o s o p h i l a r i b o s o m e s c h a n g e d u r i n g d e v e l o p -m e n t . T h e r i b o s o m a l p r o t e i n s f r o m 2 l a r v a l a n d 2 p u p a l s t a g e s , a s w e l l a s f r o m a d u l t s , w e r e c o m p a r e d b y 2 -d i m e n s i o n a l g e l e l e c t r o p h o r e s i s ( L a m b e r t s s o n , 1 9 7 2 ) . Numerous d i f f e r e n c e s i n t h e p r o t e i n p a t t e r n s w e r e a p p a r e n t . Some s p o t s i n c r e a s e d i n s t a i n i n g i n t e n s i t y f r o m o n e s t a g e t o a n o t h e r w h i l e o t h e r s f a d e d . T h e o b s e r v a t i o n s s u g g e s t t h a t some r i b o s o m a l p r o t e i n s may b e s t a g e s p e c i f i c . T h e 85 evidence i s not c o n c l u s i v e , however, because the observed d i f f e r e n c e s may be due to (1) contaminants u n r e l a t e d to p r o t e i n s y n t h e s i s , (2) supernatant f a c t o r s such as i n i t a t o r s of p r o t e i n s y n t h e s i s which become attached t o ribosomes momentarily and (3) secondary changes i n ribosomal p r o t e i n s produced by p r o t e o l y t i c enzymes i n the d i f f e r e n t developmental stages. The o t h e r experiments suggesting t h a t ribosomes of l a r v a e and a d u l t s d i f f e r , were d i s c u s s e d i n the i n t r o d u c t i o n . Larvae, but not a d u l t s , of the s c a r l e t eye stock were s u s c e p t i b l e to streptomycin (Duke and Glassman, 1968). And ribosomes from streptomycin s e n s i t i v e l a r v a e bound more streptomycin than those from streptomycin r e s i s t a n t l a r v a e (Lambertsson and Rasmuson, 1971). (4) An obvious omission are the c o l d - s e n s i t i v e mutants s t i l l remaining to be t e s t e d . These i n c l u d e 14 r e c e s s i v e , c o l d - s e n s i t i v e l e t h a l s on the X chromosomes and 29 r e c e s s i v e , c o l d - s e n s i t i v e l e t h a l s of the 3rd chromosomes (Tasaka and Suzuki, 1973). (5) More worrisome are the c o l d - s e n s i t i v e mutants t h a t may have been missed i n the X chromosome s c r e e n . The p r e s e n t screen was designed on the b a s i s of the g e n e t i c c h a r a c t e r i s t i c s of the b a c t e r i a l r ibosomal mutants, i . e . the Sad mutations are c o l d ^ - s e n s i t i v e l e t h a l s and dominant to the normal a l l e l e . By o b s e r v i n g the e f f e c t s o f low temperature i n D r o s o p h i l a w i t h the genotype X/Y, where the X chromosome has been exposed t o mutagen, the f o l l o w i n g 86 types of c o l d - s e n s i t i v e mutants were expected: (a) Dominant c o n d i t i o n a l l e t h a l s i n genes t h a t are p r e s e n t on both the X and Y chromosomes. (b) Dominant c o n d i t i o n a l l e t h a l s i n genes pres e n t on the X chromosome o n l y , (c) Recessive c o n d i t i o n a l l e t h a l s i n genes pres e n t o n l y on the X chromosome and hence r e v e a l e d i n the hemizygous c o n d i t i o n . As w e l l as p r o v i d i n g f o r t h r e e types o f p o s s i b l e mutants, the screen i n v o l v e d l e s s work than schemes which i n t r o d u c e the mutagenized chromosome i n t o heterozygous females. In f a c t , the screen gave o n l y (c) type c o l d - s e n s i t i v e mutants, i . e . r e c e s s i v e l e t h a l s d e t e c t a b l e i n the hemizy-gous c o n d i t i o n . I t i s now known t h a t almost a l l dominant heat- and c o l d - s e n s i t i v e mutations of D r o s o p h i l a are l e t h a l when homozygous a t the p e r m i s s i v e temperature (Suzuki and P r o c u n i e r , 1969; Rosenbluth, e t a l . , 1972; Holden and Suzuki, 1973). Thus s c r e e n i n g v i a hemizygosity may s e l e c t a g a i n s t dominant mutations. A l s o , dominant mutations of types (a) and (b) may not have been i s o l a t e d because they occur i n f r e q u e n t l y and i n s u f f i c i e n t numbers of chromosomes were t e s t e d . A f o u r t h type of p o s s i b l e mutant, i . e . r e c e s s i v e l e t h a l s i n genes pres e n t on both the X and Y, c o u l d not be d e t e c t e d by our sc r e e n . Such mutants may be important i f the c h a r a c t e r i s t i c s o f the ribosomal p r o t e i n genes are l i k e those of the ribosomal RNA genes. The bobbed 87 mutations, which are thought t o be d e l e t i o n s i n the redundant r e g i o n s coding f o r ribosomal RNA, are r e c e s s i v e and map on the X and Y chromosomes ( R i t o s s a , e t a l . , 1966; L i n d s l e y and G r e l l , 1967). Although the genes f o r r i b o -somal p r o t e i n s occur i n s i n g l e c o p i e s i n E. c o l i , t h i s s i t u a t i o n i s not n e c e s s a r i l y t r u e f o r D r o s o p h i l a (Davies and Nomura, 1972). Eukaryotes, f o r example, have more r e p e t i t i v e DNA than b a c t e r i a ( B r i t t e n and Kohn, 1968). Furthermore a r e c e n t study of ribosomes from D. melanogaster «•> D. simulaps h y b r i d s by 1-dimensional g e l s suggests t h a t genes f o r ribosomal p r o t e i n s are l o c a t e d on the X chromosome near bb ( S t e f f e n s e n , 1973). I t may, t h e r e f o r e , be worthwhile t o r e s c r e e n f o r c o l d -s e n s i t i v e mutations on the X chromosomes by another s e l e c -t i o n scheme. A l l f o u r types of mutants should be o b t a i n -a b l e i n a t r a d i t i o n a l s c r e e n where the mutagenized X chromosome i s i n t r o d u c e d i n t o a heterozygous female and then t e s t e d i n both the homozygous and heterozygous con-d i t i o n (Suzuki, e t a l . , 1967; Tasaka and Suzuki, 1973). (6) B i o c h e m i c a l examination of the mutants was almost e x c l u s i v e l y by g e l e l e c t r o p h o r e s i s of ribosomal p r o t e i n s . I t i s p o s s i b l e t h a t the sedimentation of ribosomal p a r t i c l e s from c o l d - t r e a t e d D r o s o p h i l a c o u l d have d e t e c t e d d i f f e r e n c e s where e l e c t r o p h o r e s i s cannot. Although s u b p a r t i c l e s which do not g r e a t l y d i f f e r i n s i z e from normal s u b u n i t s are d i f f i c u l t t o d e t e c t by t h i s method, i t was s u c c e s s f u l l y used t o i d e n t i f y s e v e r a l Sad mutants of 88 b a c t e r i a ( T a i , e t a l . , 1 9 6 9 ; N o m u r a , 1 9 7 0 ) . None o f t h e D r o s o p h i l a m u t a n t s seemed t o b e s u s c e p t i b l e t o t h e l e t h a l e f f e c t o f c o l d a s a d u l t s ( R o s e n b l u t h , e t a l . , 1 9 7 2 , T a s a k a , , p e r s o n a l c o m m u n i c a t i o n ) . H e n c e t h e e f f e c t o f c o l d o n s u b u n i t a s s e m b l y c a n be r e a d i l y s t u d i e d i n a d u l t s . T o a p p l y t h i s a p p r o a c h t o p u p a e a n d l a r v a e , h o w e v e r , w o u l d r e q u i r e p r e l i m i n a r y s t u d y o f t h e d e v e l o p -ment o f t h e m u t a n t s . The l e t h a l p h a s e a n d t e m p e r a t u r e -s e n s i t i v e p e r i o d o f e a c h m u t a n t s h o u l d b e d e t e r m i n e d . T h e l e t h a l p h a s e i s t h e d e v e l o p m e n t a l s t a g e a t w h i c h t h e o r g a n i s m d i e s a f t e r c o n t i n u o u s e x p o s u r e t o t h e r e s t r i c t i v e t e m p e r a t u r e ( S u z u k i , 1 9 7 0 ) . T h e t e m p e r a t u r e - s e n s i t i v e p e r i o d p r e c e d e s t h e l e t h a l p h a s e a n d i s c o n s i d e r e d t o be t h e i n t e r v a l when t h e g e n e p r o d u c t i s p r o d u c e d , a c t i v a t e d o r f i r s t n e e d e d f o r a c r i t i c a l d e v e l o p m e n t a l p r o c e s s . H e n c e i f a m u t a n t i s k e p t a t t h e n o n - p e r m i s s i v e t e m p e r a t u r e d u r i n g t h e t e m p e r a t u r e - s e n s i t i v e p e r i o d , i t w i l l l a t e r d i e e v e n i f s h i f t e d t o t h e p e r m i s s i v e t e m p e r a t u r e . T h e t e m p e r a t u r e - s e n s i t i v e p e r i o d o f t h e c o l d - s e n s i t i v e m u t a n t s may b e t h e d e v e l o p m e n t a l s t a g e when a d e f e c t i n s u b u n i t a s s e m b l y i s m o s t e v i d e n t . E s t a b l i s h m e n t o f t h e t e m p e r a t u r e - s e n s i t i v e p e r i o d a n d l e t h a l p h a s e w o u l d a l s o be a u s e f u l means o f c o m p a r i n g c o l d - s e n s i t i v e m u t a n t s a n d t h e p r e v i o u s l y s t u d i e d h e a t - s e n s i t i v e m u t a n t s . C o n c l u s i o n . T h e h y p o t h e s i s t h a t r i b o s o m e a s s e m b l y d e f e c t s s h o u l d o c c u r i n c o l d - s e n s i t i v e m u t a n t s i s b a s e d o n c h e m i c a l 89 p r i n c i p l e s . I t s h o u l d , t h e r e f o r e , a p p l y t o t h e r i b o s o m e s o f o t h e r o r g a n i s m s a s w e l l a s t h o s e o f s E . c o l i : A l t h o u g h c o n s i d e r a b l e w o r k was d o n e i n t e s t i n g t h e t h e o r y o n D r o s o p h i l a , no e v i d e n c e f o r o r a g a i n s t t h e h y p o t h e s i s was f o u n d . B e c a u s e o f t h e g r e a t e r c o m p l e x i t y o f D r o s o p h i l a , t h e t h e o r y h a s s t i l l b e e n i n c o m p l e t e l y t e s t e d . 90 BIBLIOGRAPHY A p i r i o n , D. a n d S c h l e s s i n g e r , D. 1 9 6 7 . T h e l o s s o f p h e n o t y p i c s u p p r e s s i o n i n s t r e p t o m y c i n - r e s i s t a n t m u t a n t s . , P r o c . N a t . A c a d . S c . U . S . 5 8 : 2 0 6 - 1 2 . A t w o o d , K . C . 1 9 6 9 . Some a s p e c t s o f t h e b o b b e d p r o b l e m i n D r o s o p h i l a G e n e t i c s , S u p p l e m e n t 6 1 : 319-327 , , 4 B a i l l i e , D . , S u z u k i , D . T . , a n d T a r a s o f f , M. 1 9 6 8 . T e m p e r a t u r e - s e n s i t i v e m u t a t i o n s i n D r o s o p h i l a m e l a n o g a s t e r . 1 1 . F r e q u e n c y among s e c o n d c h r o m o s o m e r e c e s s i v e l e t h a l s i n d u c e d b y e t h y l m e t h a n e s u l f o n a t e . C a n . J . G e n e t . C y t o l . 1 0 : 4 1 2 - 4 2 C B a y l i s s , F . T . , a n d V i n o p a l , R . T . 1 9 7 1 . S e l e c t i o n o f r i b o s o m a l m u t a n t s by a n t i b i o t i c s u p p r e s s i o n i n y e a s t . S c i e n c e 1 7 4 : 1 3 3 9 - 1 3 4 1 . B i r g e , E . A . , C r a v e n , G . R . , H a r d y , S . J . S . , K u r l a n d , C . G . , V o y n o w , P . 1 9 6 9 . S t r u c t u r e d e t e r m i n a n t o f a r i b o s o m a l p r o t e i n : K. l o c u s . S c i e n c e 1 6 4 : 1285-86 , , B i r g e , K . A . a n d K u r l a n d , C . G . 1 9 6 9 . A l t e r e d r i b o s o m a l p r o t e i n i n s t r e p t o m y c i n - d e p e n d e n t E s c h e r i c h i a c o l i . S c i e n c e 1 6 6 : 1282 . -1284 . B i r g e , E . A . a n d K u r l a n d , C . G . 1 9 7 0 . R e v e r s i o n o f a s t r e p t o m y c i n d e p e n d e n t s t r a i n o f E s c h e r i c h i a c o l i . M o i . G e n . G e n e t . 1 0 9 : 3 5 6 - 6 9 . B i r n i e , G . D . ( e d . ) 1 9 7 2 . " S u b c e l l u l a r c o m p o n e n t s , p r e p a r a t i o n a n d f r a c t i o n a t i o n . " L o n d o n , B u t t e r w o r t h s . p 2 0 6 - 2 1 i . B o l l e n , A . , D a v i e s , J . , O z a k i , M . , a n d M i z u s h i m a , S . 1 9 6 9 . R i b o s o m a l p r o t e i n c o n f e r r i n g s e n s i t i v i t y t o t h e a n t i b i o t i c s p e c t i n o m y c i n i n E s c h e r i c h i a c o l i . N a t u r e 1 6 5 : 8 5 - 8 6 . B o l l e n , A . a n d H e r z o g , A . 1 9 7 0 . T h e r i b o s o m a l p r o t e i n a l t e r e d i n s p e c t i n o m y c i n r e s i s t a n t E s c h e r i c h i a c o l i . F E B S L e t t e r s 6 : 6 9 - 7 2 . B o l l e n , A . , e t a l^ . 1 9 7 3 . T h e s t r u c t u r a l g e n e f o r t h e ribosomaT p r o t e i n S18 i n E s c h e r i c h i a c o l i . 1. G e n e t i c s t u d i e s o n a m u t a n t h a v i n g a n a l t e r a t i o n i n t h e p r o t e i n S 1 8 . J . M o i . B i o l . 7 6 : 4 6 3 - 4 7 2 . 0 91 B o s h e s , R . A . 1 9 7 0 . D r o s o p h i l a r i b o s o m e s . T h e c h a r a c t e r i z a t i o n o f two p o p u l a t i o n s by c e l l f r a c t i o n -a t i o n a n d i s o t o p i c l a b e l i n g w i t h n u c l e i c a c i d a n d p r o t e i n p r e c u r s o r s . J . C e l l B i o l o g y ' £ 6 : 4 7 7 - 4 9 0 . B r e c k e n r i d g e , L . a n d G o r i n i , L . 1 9 6 9 . T h e d o m i n a n c e o f s t r e p t o m y c i n s e n s i t i v i t y r e - e x a m i n e d . P r o c . N a t . A c a d . S c . U . S . A . 62_: 9 7 9 - 8 5 . B r i t t e n , R . J . a n d K o h n e , D . E . 1 9 6 8 . R e p e a t e d s e q u e n c e s i n DNA. S c i e n c e 1 6 1 : 5 2 9 - 5 4 0 . B r o w n , D . D . , a n d D a w i d , I . B . 1 9 6 8 . S p e c i f i c g e n e a m p l i f i c a t i o n i n o o c y t e s . S c i e n c e 1 6 0 : 2 7 2 - 2 8 0 . C o o p e r , K .W. 1 9 5 9 . C y t o g e n e t i c a n a l y s i s o f m a j o r h e t e r o c h r o m a t i c e l e m e n t s ( e s p e c i a l l y Xh a n d Y) i n D r o s o p h i l a m e l a n o g a s t e r a n d t h e t h e o r y o f H e t e r o -c h r o m a t i n . Chromosoma 1 0 : 5 3 5 - 5 8 8 . C o u n c e , S . J . 1 9 5 6 a . S t u d i e s on f e m a l e - s t e r i l i t y g e n e s i n D r o s o p h i l a m e l a n o g a s t e r . 1. T h e e f f e c t s o f t h e g e n e d e e p o r a n g e o n e m b r y o n i c d e v e l o p m e n t . Z*#" i n d u k t . A b s t a m m . V e r e r b u n g s . 87_: 44 3 - 4 6 1 . C o u n c e , S . J . 1 9 5 6 b . S t u d i e s o n f e m a l e - s t e r i l i t y g e n e s i n D r o s o p h i l a m e l a n o g a s t e r . 1 1 . T h e e f f e c t s o f t h e g e n e f u s e d o n e m b r y o n i c d e v e l o p m e n t . Z . i n d u k t . A b s t a m m . V e r e r b u n g s . 87_: 4 6 2 - 4 8 1 . C o u n c e , S . J . 1 9 5 6 c . S t u d i e s o n f e m a l e - s t e r i l i t y g e n e s i n D r o s o p h i l a m e l a n o g a s t e r . 1 1 1 . T h e e f f e c t s o f t h e g e n e r u d i m e n t a r y o n e m b r y o n i c d e v e l o p m e n t . Z . i n d u k t . A b s t a m m . V e r e r b u n g s . 87_: 4 8 2 - 4 9 2 . C o x , J . H . a n d S t r a c k , H . B . 1 9 7 1 . C o l d - s e n s i t i v e m u t a n t s o f b a c t e r i o p h a g e X« G e n e t i c s 67_: 5 - 1 7 . C r a v e n , G . R . , V o y n o w , P . , H a r d y , S . J . S . , a n d K u r l a n d , C . G . 1 9 6 9 . T h e r i b o s o m a l p r o t e i n s o f E s c h e r i c h i a c o l i . 1 1 . C h e m i c a l a n d p h y s i c a l c h a r a c t e r i z a t i o n o f t h e 30S r i b o s o m a l p r o t e i n s . B i o c h e m . 8_: 2 9 0 6 - 2 9 1 5 . D a v i e s , J . 1 9 6 4 . S t u d i e s o n t h e r i b o s o m e s o f s t r e p t o m y c i n s - s e n s i t i v e a n d r e s i s t a n t s t r a i n s o f E s c h e r i c h i a c o l i . P r o c . N a t . A c a d . S c i . U . S . A . i r n — s v t t c — D a v i e s , J . 1 9 6 6 . S t r e p t o m y c i n a n d t h e g e n e t i c c o d e . C o l d S p r i n g H a r b o r Symp. Q u a n t . B i o l . 3 1 : 6 6 5 - 6 6 9 . 92 D a v i e s , J . a n d N o m u r a , M. 1 9 7 2 . T h e g e n e t i c s o f b a c t e r i a l r i b o s o m e s . A n n u a l R e v i e w o f G e n e t i c s £ : 2 0 3 - 2 3 4 . D e k i o , S . a n d T a k a t o , R. 1 9 6 9 . G e n e t i c s t u d i e s o f t h e r i b o s o m a l p r o t e i n s o f E s c h e r i c h i a c o l i . I I . A l t e r e d 3OS r i b o s o m a l p r o t e i n c o m p o n e n t s p e c i f i c t o s p e c t i n -o m y c i n r e s i s t a n t m u t a n t s . M o i . G e n . G e n e t i c s . T O 5 ; 2 1 9 - 2 4 . 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T h e s u b u n i t s t r u c t u r e a s e x a m i n e d b y e l e c t r o n m i c r o s c o p y . B i o c h e m i s t r y . 5_: 3 1 1 1 - 3 1 1 6 . V a n H o l d e , K . E . |<ffob. T h e m o l e c u l a r a r c h i t e c t u r e o f m u l t i c h a i n p r o t e i n s i n " M o l e c u l a r A r c h i t e c t u r e a n d C e l l P h y s i o l o g y " H a y a s h i , T . a n d S z e n t G y o r g y i , A . G . e d . E n g l e w o o d C l i f f s , N . J . : P r e n t i c e - H a l l : 1 9 6 6 . V o y n o w , P . a n d K u r l a n d , C . G . 1 9 7 1 . S t o i c h i o m e t r y o f t h e 30S r i b o s o m a l p r o t e i n s o f E s c h e r i c h i a c o l i . B i o c h e m . 1 0 : 5 1 7 - 2 4 . W e l f l e , H . , S t a h l , J . a n d B i e l k a , H . S t u d i e s o n p r o t e i n s o f a n i m a l r i b o s o m e s . V I I I . T w o - d i m e n s i o n a l p o l y a c r y l a m i d e g e l e l e x t r o p h o r e s i s o f r i b o s o m a l p r o t e i n s o f r a t l i v e r . B i o c h i m i c a e t B i o p h y s i c a A c t a 2_4_3: 4 1 6 - 4 1 9 ( 1 9 7 1 ) . W h i t e , B . N . , T e n e r , G . M . , H o l d e n , J . , a n d D . T . S u z u k i . 1 9 7 3 a . A c t i v i t y o f a tRNA m o d i f y i n g enzyme d u r i n g t h e d e v e l o p m e n t o f D r o s o p h i l a a n d i t s r e l a t i o n s h i p t o t h e s u ( s ) l o c u s . J . M o i . B i o l . 7_4: 6 3 5 - 6 5 1 . W h i t e , B . N . , H o l d e n , J . , S u z u k i , D . T . a n d T e n e r , G . M . 1 9 7 3 b . A n a l y s i s o f tRNA d u r i n g t h e d e v e l o p m e n t o f D r o s o p h i l a . D e v e l . B i o l . 33/: 1 8 5 - 1 9 5 . W i m b e r , D . E . a n d S t e f f e n s e n , D . M . 1 9 7 0 . L o c a l i z a t i o n o f 5S RNA g e n e s o n D r o s o p h i l a c h r o m o s o m e s b y RNA-DNA hybridi2 : a t i o n . S c i e n c e 1 7 0 : 6 3 9 - 4 1 . W i t t m a n n , H . G . , ' e t a l . 1 9 7 1 . C o r r e l a t i o n o f 30S r i b o s o m a l p r o t e i n s o f E s c h e r i c h i a c o l i i s o l a t e d i n d i f f e r e n t l a b o r a t o r i e s " ! M o i . G e n . G e n e t . I l l : 3 2 7 - 3 3 . W r i g h t , T . 1 9 7 3 . T h e r e c o v e r y , p e n e t r a n c e , a n d p l e i o t r o p y > o f X - l i n k e d , c o l d - s e n s i t i v e m u t a n t s i n D r o s o p h i i a . M o l e c . G e n . G e n e t . 1 2 2 : 1 0 1 - 1 1 8 . Y a m a d a , T . a n d D a v i e s , J . 1 9 7 1 . A g e n e t i c a n d b i o -c h e m i c a l s t u d y o f s t r e p t o m y c i n - a n d s p e c t i n o m y c i n -r e s i s t a n c e i n S a l m o n e l l a t y p h i m u r i u m . M o i . G e n . G e n e t . 1 1 0 : 1 9 7 - 2 1 0 . Z immermann , R . A . , G a r n i n , R . T . , Gorini, L . 1 9 7 1 . A l t e r a t i o n o f a 30S r i b o s o m a l p r o t e i n a c c o m p a n y i n g t h e ram m u t a t i o n i n E s c h e r i c h i a c o l i . P r o c . N a t . A c a d . S c . U . S . 6 8 : 2 2 6 3 - 6 7 . 103 Appendix C a l c u l a t i o n of the frequency of cs l e t h a l s as a percentage of s i n g l e h i t s . XXY x X/Y i n b o t t l e s no. treatment:, u / 0 =•" r r v ~ 1.01 •+• 463 O o t r e a t e d w i t h 0.005 H EMS, °/g - •—• = 0 . 6 4 To c a l c u l a t e the frequency of s i n g l e h i t s P = X n J l T X n i n. where n - number of h i t s and X - frequency of h i t . When n = 0, i . e . no h i t , P = .64 - X°'lTX - ™ o i . J l X = . 64 X = .46 , Frequency of s i n g l e h i t s pi = -^6 L (.64) = . 2904 -.1. ! • . 18 Frequency of cs l e t h a l s - Y~^97~ x ~ .004436 where no, of t e s t e d d'e^ = 2,597, no. of cs l e t h a l s ='18, and frequency of no h i t </<^  = .64 % s i n g l e h i t s e x - l i n k e d l e t h a l s t h a t are c o l d - s e n s i t i v e = * ~ x 100 -1.5% on the average. I n a s i n g l e experiment, 9 cs l e t h a l s were found out of 672 tested chromosomes. The maximum % s i n g l e h i t s e x - l i n k e d l e t h a l s that were c o l d -s e n s i t i v e = - ~ — x .64 x = 3%. 6 / Z . / Q — 104 TABLE I The effect of temperature on the viabilities of mutant males, relative to the Oregon-R control, from the cross ,X (1) CS/Y x C(1)RM or C(1)DX£. Stock* 22°C 17°C $ 9 Test Viability (%) Total Flies Viability (%) Total Flies OR 1 100 1252 100 1095 ^cs RM 1 81.8 795 0 347 2 72 192 0 63 3 71.8 375 1 232 RA 1 69.6 340 0.5 288 2cs RM 1 42.0 249 0.5 163 RA 1 34 187 0.9 86 3 C S RM 1 35 118 0 42 2 37.7 341 1 148 3 41 190 0.6 263 4 102 252 0 110 RA 1 43 208 0.4 180 . cs 4 RM 1 49.9 421 3.3 340 2 16 268 2 259 3 14 283 3 140 4 42 166 2 296 RA 1 49.4 265 3 428 7 C S RM 1 78.1 275 6.1 267 2 87.6 303 6.2 253 3 89.1 277 11 352 8cs RM 1 47 167 2 98 2 46 87 3 104 RA 1 56.6 284 0.3 368 9cs RM 1 61 171 0 225 2 60 106 11 121 i o c s RM 1 31 147 1 129 2 22 177 0 156 3 56.0 212 3 146 11 C S RM 1 61.2 305 2 322 2 78.1 261 8.6 117 3 70.9 286 3 263 4 58 196 23 177 RA 1 60.6 366 3.3 388 1 2cs RM 1 50.1 253 0 319 2 28 134 0.3 266 3 48 183 0 51 4 36 116 0 76 105 Table I (cont'd) 13 C S RM 1 66.5 449 0.2 362 RA 1 34.1 336 1 300 2 5 68 0 62 14 C S RM 1 33.3 259 7.3 226 2 36.7 299 1 225 3 45.0 244 9.3 146 4 45.4 292 12 245 5 31.1 489 2 292 16 C S RM 1 92.0 307 0 226 2 47 135 0 38 3 10 135 1 366 RA 1 68.6 225 0.5 160 1 ?cs RM 1 45 74 0 83 2 57.3 266 2.4 322 3 62.4 308 0.8 99 RA 1 87 140 9.4 163 18 C S RM 1 76.6 951 12.5 746 RA 1 79.6 333 12 330 2 76.6 958 15.1 794 19 C S RM 1 52.6 222 0 103 2 52.8 355 4 143 3 80 94 15 122 . RA 1 85.4 351 18 340 20 C S RM 1 47 131 0 40 2 71.4 637 0 326 RA 1 67.5 1007 0 486 21 C S RM 1 69 124 7 24 2 98.5 1167 1 545 RA 1 76.6 1362 0.3 601 22 C S RM 1 100 155 2 103 2 82.5 370 13 373 RA 1 65.7 228 4.8 266 23 C S RM 1 29 249 0 59 2 100 340 1 230 RA 1 63.4 381 0.3 266 24" RM 1 48.9 379 2 88 2 52.1 372 4.7 323 3 60.3 347 2.4 414 RA 1 85.4 284 3 229 25 C S RM 1 21 261 0 63 2 75.2 393 12 496 RA 1 50.5 653 17.1 704 Viability at 22°C = (mutant ) //(C(1)RM/Y ??) x 100/1.37 Viability at 17°C = (mutant )//(C(1)RM/Y ?$) x 100/1.32 RM = C(1)RM/Y f email =s RA = C(1)DX, y f/Y females OR = Oregon-R *Mutants 1 to 18 were derived from 2,597 X chromosomes treated with 0.005M EMS; mutants 19 to 27, from 1,322 chromosomes treated with 0.0125M EMS. 106 TABLE I I The effect of temperature on the frequencies of the abnormal abdomen Jhenotype and relative v i a b i l i t i e s of mutant males from the cross, ( I ) c s / Y # x c(l)RM/Y or C(l)DX/YO. . Stock 22°C 17°C crV 99 Test %V* Total Flies % aa %v** Total Flies % aa OR 1 100 1252 0 100 1095 0 , - C S RM 1 66.8 249 - 0 306 -2 67.4 273 - 2 165 -3 55.5 310 49 3.4 304 100 RA 1 64 133 40 11 71 78 2 67.4 458 33 9.8 359 54 , cs o RM 1 54.7 357 - 5.1 316 -2 70 159 - 6.5 227 -3 69.3 341 56 6.8 327 70 RA 1 72.5 291 - 21 268 46 2 83 147 65 6 55 100 3 78.8 540 28 18 400 61 1 5 c s RM 1 70.9 339 - 1 166 -2 52 210 - 0.9 83 -3 51 121 - 7.4 . 135 -4 75.9 270 77 12 235 81 RA 1 65.0 225 - 21 183 70 2 58.8 251 37 6 88 50 3 60.9 457 37 18 476 55 26 C S RM 1 73 170 - 63 68 -2 95.6 629 - 52.9 489 49 RA 1 78.8 521 29 55.8 224 86 27 C S RM 1 83.9 308 - 69 126 67 2 75.2 209 28 41.4 385 48 3 81.0 430 12 71.6 667 14 4 72.2 350 44 52 137 61 RA 1 81.8 529 6.8 84.8 560 18 2 84 159 42 57 121 60 %V* = relative v i a b i l i t y at 22°C = (mutant //(C(1)RM/Y $ $) x 100/1.37 %v**= relative v i a b i l i t y at 17°C = (mutant^)//(C(1)RM/Y gg) x 100/1.32 RM = C(1)RM/Y females RA = C(1)DX, y f/Y females OR = Oregon-R %aa = percentage of showing abnormal abdomen traits TABLE III Male progeny at 22°C and 17°C of females heterozygous for y cv v f car and mutations which have visible phenotypes. M u t a n t 5 15" 16 C l a s s e s ( C O . ) R e g i o n O r e g o n - R M P * + MP + MP + MP + MP + MP + P a r e n t : a l y c v v f_ c a r 0 1 7 2 0 72 1 1 8 9 0 82 0 244 0 1 2 9 0 1 5 1 + + + + + 1 6 5 1 1 85 1 0 8 1 3 0 9 5 1 8 9 55 0 1 5 2 0 £ i 7: z l e •f c v v f c a r 1 4 1 0 10 1 4 2 0 1 7 1 i 5 1 2 28 0 21 i + + + + 3 3 1 0 15 21 2 0 8 4 3 2 0 1 2 0 + + v f c a r 2 69 0 27 1 72 0 35 0 1 1 4 4 29 16 21 y _ c v -f + + 5 1 0 0 32 3 1 0 0 28 70 1 24 5 24 + + + _ f. c a r 3 75 0 4 7 1 9 0 0 6 0 1 116 9 0 56 0 X £Y- X + 6 3 0 0 4 2 15 0 0 46 4 8 0 54 1 72 + + * c a r 4 32 0 1 0 2 25 0 13 0 26 1 0 9 0 y_ c v v _£ -r 16 0 0 9 4 0 0 9 8 0 7 0 15 M u l t i p l e + c v 1** 11 0 1 1 0 1 22 X + 1 7 0 1 1 0 19 2 1 + V 2 2 4 0 4 0 3 1 15 4 3 C_v T 16 0 4 0 6 1 2 0 3 1 0 + f 3 2 6 0 6 0 18 0 5 V + 24 0 2 0 0 0 27 + car 4 9 0 7 0 1 1 0 4 0 13 2 9 f + 7 0 0 4 2 1 0 7 4 1 2 T O T A L eV e23 1 8 6 856 248 1 1 9 9 3 8 8 6 0 9 * >!P EUtant p h e n o t y p e * * M u l t i p l e CO c l a s s , r e g i o n 1 , i n c l u d e s a l l d* o f f s p r i n g w i t h a c . o . i n r e g i o n 1 a n d a 2 n d c . o . i n a n y o t h e r r e g i o n . 1 7 ° C 2 2 ° C 1 7 ° C : • • ' ' 2 2 ° C 1 7 ° C + 2 ° C 1 7 ° C MP + MP + + (= • MP + MP + MP >5> 0 128 0 1 9 0 . 137 0 28S 299 1 107 7 4 2 7 0 1 3 6 3 3 58 1 5 7 0 3 0 1 0 1 97 0 349 3 6 6 36 13}. 0 21 0 3 5 4 0 0 59 21 0 18 5 4 1 17 3C> 2 0 1 39 0 6 0 0 1 26 0 13 75 0 4 5 0 4 1 0 78 62 0 1 0 9 1 4 9 0 50 67 93 21 53 1 0 9 29 0 74 0 0 1 2 0 1 ' 1 1 6 0 65 0 64 0 1 0 1 35 5 4 1 242 1 39 86 1 7 5 1 0 7 •-0 0 16 44 29 3 1 67 0 26 0 o 144 0 97 0 8 0 16 0 99 0 0 6 0 36 45 5 27 0 0 3 6 10 0 3 0 34 0 3 0 4;-0 0 12 2 6 3.5 6 10 0 17 10 26 3 65 0 33 1 0 16 9 27 32 i i 2 0 13 3 8 3 35 0 19 0 19 1 2 5 1 0 3 3 0 22 S 67 0 27 1 2 18 9 25 0 8 0 26 2S 6 .16 0 1 2 0 1 1 0 1 5 1 2 23 0 31 0 0 3 7 9 13 0 8 8 13 4 7 3 0 3 8 5 7 353 1 2 5 3 7 9 1 4 2 5 22 7', 9 0 t-* o ! TABLE I V Survival of progeny in tests of j ( l ) 5 C S, 6 C S, 15 c s, and 1 6 c s with d u p l i c a t i o n s and d e f i c i e n c i e s of proximal heterochromatin of the X chromosome. Mutant °C (a) inal^  deficiency . ,106 and max duplication (fe) . ,12 , mai det] Lciencv 4 fc) q sc deficiency FM6 id) cs FM6 Xa)cs Mifs o In49mal't mal12Df iu) c s ' FMS FK6 Mi ) c s mal6Df mal6Df Yra l106Dp Yn.al106Dp mal12Df In49mal2 Y Y sc 4 Lsc 8 RDf s c4L s cSR D f Y V MP* 88 4- M P * .i. l c s 22 134 160 12 108 24 41 23 0 22 20 13 8 13 17 190 208 0 0 90 21 36 0 0 30 21 17 19 0 6CS 22 180 150 82 4 ' 130 28 38 28 0 135 108 90 49 84 17 263 238 0 0 141 45 50 16 0 109 82 72 13 4 15cs 22 205 219 118 2 145 53 59 40 0 76 77 60 52 31 17 270 294 0 0 105 56 56 14 0 119 69 86 7 1 16CS 22 179 155 76 52 136 63 61 48 0 113 116 82 19 36+ 17 266 270 0 0 138 45 55 18 0 206 211 65 0 0 Oregon-R 22 166 194 0 149 130 66 79 64 0 46 46 48 0 42 17 213 227 0 176 109 . 74 75 4 0 49 35 16 0 44 cross: (a) g° &1)CS/FM6 X &IrA9Btu- v mal6Df/y+Y mal i uW (b) £° Df(l)cial12sc8B/In49 v snx2mal2 X^j(l) c s/Y MP* = mutant phenotype (c) Ogl(l)CS/FM6 XJ'j'lnCDsc^sc811, £ cv v f/Y + = wild type phenotype M o 00 TABLE V V i a b i l i t i e s and phenotypes of trans heterozygous and homozygous females in complementation tes t s of c o l d - s e n s i t i v e mutations j j ( l ) c /Fm oo i>(i) o s y/v - c # T5 To Ore-R 26 15 16 27 21 27 21 VR 2 2 ° C VR 170C % aa VR 2 2 ° C VR 17QC % aa VR 2 2 ° C VR 1 7 ° C % aa VR 2 2 ° C VR 1 7 ° C % aa VR 2 2 ° C VR 1 7 ° C % aa VR 2 2 ° C VR 1 7 ° C % aa VR 2 2 ° C VR 1 7 ° C % aa VR 2 2 ° C VR 1 7 ° C % aa 1.02(842) 0.905(232) 1.03(9-17) 0.960(343) 0 (426) 0.S (134) 0.947(296) 1.16(197) 0.792(95) 0.774(110) 1 (144) 39 (106) 0.862(229) 1.11(268) 0.792(172) 0.752(198) 0 (105) 0.7 (141) 0.754(328) 1.00(176) 1.01(310) 0.725(226) 0.7 (141) 0 (88) 1.24(273) 1.04(411) 0 (151) 1.00(134) 0.824(166) 1.02(107) 0.690(147) 0 (67) 0 (75) 0.993(287) 0.899(338) 0 (143) 1.09 (192) 0.678(97) 2 (100) 1.05(302) 0.0441(71) 52 (155) 0.722(303) 0.141(194) 52 (127) 0.933(290) 0.106(238) 60 (140) 0.973 (14S) 0.547(147) 0 (73) 1.18(305) 1.01(423) 0 (155) 1.04(188) 0.667(95) 3 (96) 0.617(228) 0.0364(114) 45 (87) 0.915(316) 0.0460(182) 62 (151) 0.783(321) 0.161(332) 25 (141) 1.27(204) 0.969(250) 0 (114) 0.892(210) 0.820(344) 0 (99) 1.16 (158) 0.792(85) 4 (85) 1.12(269) 0.96(179) 0.7 (147) 1.18(308) 1.04(421) 0.5 (167) 1.18(203) 0 (152) 72 (110) 0.829(267) 1.00 (364) 0 (121) 1.01 (229) 0.931(253) 2 (115) 0.783(246) 0.0513(62) 43 (108) 0.849(233) 0.236(131) 56 (107) 0.933(290) 0.919(378) 0.7 (140) 0.950(156) 0.164(170) 28 (76) 1.24(224) 0.913 (241) 0 (124) 0.938(312) 1.18 (533) 0 (151) 0.778(176) 1.36(125) 4 (77) 1.05(170) 1.09(138) 0 (87) 1.17(419) 1.39(421) 0 (226) 0.339(205) 0.958(417) 0 (94) 1.22(307) 0.975 (480) 0 (169) 1.13(207) 0.814(312) 0 (110) 1.02(333) 1.00(455) 0.6 (156) 0.917(232) 0.814(155) 3 (111) 0.973(219) 1.00(126) 0 (103) 0.865(209) 1.21(199) 0 (97) 1.02(266) 1.10(369) 0 (134) 1.07(290) 1.02(503) 0 (150) 1.06(107) 0.949(308) 2 (55) 1.33(119) 0 (77) 0 (63) VR ( v i a b i l i t y r a t i o ) = I 11 )""/In ) c s y p 0 / / J n ^ c s y / F M K op % aa = percent of Jitl)csx/P l^csVnr, showing the abnormal abdomen t r a i t a t 22°C 110 TABLE VI The effect of temperature on the v i a b i l i t i e s of mutant males and homozygous females from the cross, j(1) C S/FM6ggx l(l) c s/Yd<f, as compared to the Oregon-R control. Chr* Relative V i a b i l i t i e s Chr* Relative V i a b i l i t i e s 22 °C 17°C 22°C 170C 9 ? OR** 100% 100% 100% 100% 1 4 c s 49.0 36.5 12 11 (867) (842) (889) (947) (522) (494) (379) (381) •j^CS 111 101 0 0 1 5 c s 45 44 0.8 1 (565) (569) (159) (159) (244) (250) (281) (283) 5cs 85 186 9 117 16 c s 44 30 0 0.4 (174) (279) ( 97) (201) (265) (248) (279) (280) 3cs 36 22 0 0 17 c s 49 57.0 0 0 (101) ( 93) ( 88) ( 88) (294) (321) (123) (123) 4 c s 75.5 70.1 5 5 1 8 c s 62.4 75.3 12 44.9 (672) (681) (408) (413) (494) (555) (205) (269) 5 C S 86.3 102 4 2 1 9 c s 87.6 109 37 43 (456) (493) (270) (266) (285) (333) (134) (144) 6cs 99.3 74.4 6 4 2 0 c s 73.6 40.0 0 0 (262) (241) (183) (180) (439) (369) (114) (114) gCS 89 78 3 2 2 1 c s 98 111 0 0 (213) (210) (158) (156) (196) (219) ( 77) ( 77) gCS 111 88.9 0 0 22 C S 87.3 106 11 75 (305) (288) ( 98) ( 98) (353) (407) (226) (363) 1 0 c s 68.3 54.9 6 4 23 c s 113 76.9 0 0 (340) (326) (204) (201) (274) (239) (161) (161) l l c s 94.0 103 7 9.1 24CS 91.3 98.0 64 35 (339) (374) (219) (226) (261) (284) (128) (110) 1 2 c s 100 34.4 0 0 2 5 c s 89 94 28 6 (593) (417) (264) (264) (122) (131) (111) ( 94) 13 c s 23 51 12 16 26 c s 109 118 73 75 (213) (267) (106) (111) (182) (197) (103) (110) 21CS 99.1 108 76.2 79.0 (185) (207) (291) (312) Relative Vi a b i l i t y of: oVat 22°C = C£(DCS/Y) //(FM6/j(l) c s) x 100/0.918 oVat 17°C = U(1) C S/Y) //(Fm/JiTf 7^") x 100/0.908 Q Q a t 220c = 5^ at 170C = Chr* = Chromosome OR** = Oregon-R T7cs7i( 1) CS77/(FM4/Jj( 1) cs) x 100/1.02 r c r r ) x 100/1.03 I l l TABLE VII F e r t i l i t y at 22°C of females homozygous for cold-sensitive l e t h a l s . Progenyy/$ when mated to: 5 FM6/Y FM6/Y + Fert-"1F S/YPPSA- i l i t y * Progeny/? when mated to: ? FM6/Y FM6/Y + Fert-ft)cs/Y i l i t y OR ^cs 2 C S  3 C S 4 C S 5 C S  6 C S 8 C S gCS 1 0 c s 11 cs 12 cs 86 ( 6) 95 ( 6) 14 (12) 39 ( 4) 11 (18) 35 (18) 0 ( 8) 0.9 (12) 0 ( 7) 13 cs 75 (30) 0 (27) 63 (21) 0 (24) 64 ( 6) 76 (10) 1.7 (12) 66 ( 4) 7 (18) 15 (18) 0 ( 8) 3.5 (12) 0 ( 8) F F F Ss F St Ss Ss F St Ss St St 1 4 c s 1 5 c s 0 (18) 1 6 c s 15 (18) 1 7 c s 0 (12) 18 cs 1 9 C S 52 ( 3) 2 0 c s 4 (18) 21 cs 22 cs 23 cs 24 cs 25 cs 26 cs 27 cs 69 (30) 18 (18) 14 (18) 7** (21) 13 (26) 11 (24) 0 (36) 70 (15) 51 ( 6) 0 (18) 2 (18) 0 (12) 28 ( 3) 3 (21) St Ss St F Ss Ss Ss Ss Ss Ss St * F = f e r t i l e ; Ss = semi-sterile; St viable adult o f f s p r i n g . ** only FM6$^Ff_ offspring recovered OR = Oregon-R - s t e r i l e , i . e . produces no \ TABLE VIII Male progeny of females heterozygous for y_ cv v f_ car and mutant chromosomes at 17°c. Crossover (CO.) CO. ; j(D C S Mutation Number Classes Region Oregon-R 1 2 3 4 7 9 11 12 13 14 18 19** 21 "22 23,-' 25 Parental y_ cy_ v f_ car + + + + + 172 165 69 0 78 1 66 0 97 3 122 2 179 5 166 4 90 0 90 0 83 3 65 15 . 204/63 0/174 72 4 152 10 145 0 59 8 Single + cy_ v f_ car y_ + + + + 1 41 33 1 3 3 19 0 15 1 14 0 2 44 0 55 0 44 0 24 4 27 17 0 19 8 24/7 0/45 15 4 28 1 0 0 16 1 + + v f_ car _y_ cy+ + + 2 69 51 0 26 29 0 35 0 37 1 0 46 1 59 3 47 0 24 0 45 32 0 28 0 52/11 0/35 35 0 34 47 49 0 27 0 + + + f_ car y cv v + + 3 75 63 0 3 3 41 1 1 29 48 4 0 58 0 82 0 59. 0 29 0 35 10 3 25 . 0 46/22 13/33 1 34-1 69 46 23 17 10 + + + + car y_ cv y_ f_ + 4 32 16 0 6 1 8 0 '•7 0 4 0 17 0 15 0 23 0 7 0 15 0 14 7 ' 4 0/19 17/3 0 13 1 13 0 8 0 8 " Multiple + cv Y_ + 1* 11 17 1 10 2 14 8 3 0 6 0 24 0 55 0 9 0 18 1 18 2 3 2 8 4/9 10/7 10 3 14 0 0 0 3 1 + v cv + 2 24 16 2 10 5 5 14 1 8 3 0 6 5 21 2 8 2 6 4 a 3 1 2 9 4/7 7/3 11 0 6 12 10 5 0 3 + £ V + 3 26 24 14 1 15 1 1 18 9 1 22 2 29 •3 12 2 15 . 0 12 2 1 11 2 17/6 2/14 0 16 5 ' 14 6 5 2 + car f + 4 9 7 4 0 1 5 2 3 0 7 10 0 29 0 2 0 7 1 10 0 1 3 5 1 0/3 6/1 0 3 4 3 0 6 2 TOTAL <SV 828 189 202 178 224 '323 458 365 198 244 170 194 819 200 385 292 149 • * multiple c.o. class, region 1 includes a l l offspring with a c.o. in region 1 and a 2nd c.o.(s) * n a n y other region(s) . ** late emerger, not lethal - 1st 9 days/last 11 days -/ a l l offspring y_ because mothers were y_ cv v f_ car/y + + J?(l) c s- + + to 113 TABLE IX Characteristics of cold-sensitive mutants of the X chromosome. RV — F e r t i l i t y of Visible phenotvpe Genetic Mutant 22°C 17°C j ( l ) c s / j ( l ) c s $ of mutation" Position 1 82 0 F None 0,6 2 42 0,5 F None 57 3 35 0 Low F None 34 4 50 3 P None 65 5 67 0 S Reduced sternites at centro' and etched or dis- mere or turbed tergites, extra wing veins be twee: car & 6 55 5 Low F Like 5 but pene-trance lower than 5 al l e l e 5 & 15 7 8 78 47 6 2 F None Upheld wings 0,4 22-25 9 61 0 F None 0 10 31 1 S None -11 61 2 Low F None 0 12 50 0 S None 0 13 67 0.2 S None 1.4 14 33 7 F None 51 15 71 1 S Like 5 allele 5 & 6 16 92 0 Low F Short, fine bristles and etched tergites 46-49 17 45 0 S None -18 77 13 F None 61 19 53 0 F None 52** 20 47 0 Low F Like 16 57 21 69 7 Low F None 34 22 23 100 29 2~ 0 Low Low F p* None None 22 48 24 49 2 Low F None ~ 25 21 0 Low F None 45 26 73 63 S Abnormal abdomen traits like 5 5/ 27 84 69- F Abnormal abdomen traits like 5 low penetrance 0 * only ^(1) C S/FM6 progeny recovered ** late emerger; not lethal when mapped / another mutation present in this stock <?'RVs relative v i a b i l i t y of males, see Table I and II ?- f e r t i l i t y : see Table VII Genetic locations: see Tables III and IV. 114 FIGUPE 1 Step 1 jnass mating d/Y ?? X/Y oV (EMS-treated) 22°C p a i r mating A" X*/Y d" score f o r s u r v i v a l or w i l d - t y p e phenotype of males XX/Y ?? k days at 22°C, then . t r a n s f e r to 17°C 17°C score f o r mutant phenotype or absence of males * denotes EMS-treated chromosome. F i g u r e 1. Screening procedure f o r the d e t e c t i o n and r e c o v e r y of sex-l i n k e d r e c e s s i v e c o l d - s e n s i t i v e mutations. 115 F i g u r e 2 . C y t o l o g i c a l r e p r e s e n t a t i o n o f X c h r o m o s o m e s c u t e i n v e r s i o n s , ( a d a p t e d f r o m C o o p e r , 1 9 5 9 ) . EH = e u c h r o m a t i c - h e t e r o c h r o m a t i c j u n c t i o n w i t h h e t e r o c h r o m a t i n s h a d e d a n d e u c h r o m a t i n o p e n . NO = n u c l e o l u s o r g a n i z e r K = c e n t r o m e r e XR = r i g h t a rm o f X h A , h B , h C f hD = m a i n h e t e r o c h r o m a t i c s e g m e n t s o f l e f t a rm o f X . I n ( l ) s c 4 L s c ° R o r i g i n a t e s f r o m a c r o s s o v e r b e t w e e n I n ( l ) s c 4 a n d I n ( l ) s c ^ 1 1 6 F i g u r e 2 sc 8 EH NO a n m m m m ® hD hC hB hA XR N o r m a l X sc NO HE MmWMMB ® 4R hD hC hB hA XR In (Dsc NO hA hB hC hD sc8K © 8R hA In (Dsc 4 sc hD 8 sc K hA T A , N 4 1 8 R In (Dsc sc 117 F i g u r e 3 . R e l a t i v e b r e a k p o i n t s o f d e f i c i e n c i e s a n d d u p l i c a t i o n s o f t h e M a - J r e g i o n ( S c h a l e t a n d F i n n e r t y , 1 9 6 8 b ) . NO = n u c l e o l a r o r g a n i z e r EH = e u c h r o m a t i c - h e t e r o c h r o m a t i c j u n c t i o n 118 F i g u r e 3 Df(1)ma-l 12 Dp,y Yma- l 106 Df(1)ma-l I 20 Sch alet 1 97, 1137 Novitski -1114 Novitsk i -1DCB1-35C Kaplan ce ntro m e re -bb (NO) -su-f -I 22 Schalet IA7 Kaplan — E H -135 Finnerty lgluful-2 Novitski 1DCA 3-19 Kaplan Kaplan -It 2-14a Kaplan "1152 Novitski Hi moe -ma -I m e l s w •ot -1 34 Schalet •M (1)n •car 119 Figure 4. Genetic positions of cold-sensitive, heat-sensitive and non-conditional lethal mutations on the X chromosome. 3 Cold-sensitive <orj Heat-sensitive lethals lethals 5*2. O < < -->r r j?7?77777777zZcZa o 7ZZZ& m CD • Q. ' c n o. O IT o • m cn i n C L c o <D Q. in m cn • Q. C o <D Q. o cn cn" Non-conditional lethals 120 H O a 121 F i g u r e 5. C o m p o s i t e d i a g r a m o f t w o - d i m e n s i o n a l e l e c t r o p h -o r o g r a m s o f t h e r i b o s o m a l p r o t e i n s o f D. m e l a n o g a s t e r A d u l t m a l e s a n d f e m a l e s . T h e s o l i d s p o t s a r e a l w a y s s e e n . T h e c r o s s - h a t c h e d s p o t s a r e f a i n t l y s t a i n i n g a n d may s o m e -t i m e s b e a b s e n t . T h e o p e n s p o t s i n d i c a t e d i f f e r e n c e s b e -t w e e n H C l - a c e t o n e a n d M g - a c e t i c a c i d p r e p a r a t i o n s o f r i b o -s o m a l p r o t e i n s . A . r e p r e s e n t s a l o n g r u n t o r e s o l v e t h e s l o w m o v i n g s p o t s . T h e f a s t m o v i n g s p o t s h a v e m i g r a t e d o f f t h e g e l i n t o t h e e l e c t r o d e b u f f e r . S e e F i g u r e s 6a a n d 7a f o r e x a m p l e . B . r e p r e s e n t s a s h o r t r u n . T h e s l o w m o v i n g s p o t s a r e n o t f u l l y r e s o l v e d b u t t h e f a s t m o v i n g s p o t s a r e r e t a i n e d o n t h e g e l s l a b . O n l y t h e f a s t m o v i n g s p o t s a r e shown i n t h e d i a g r a m a l o n g w i t h n e i g h b o u r i n g s p o t s s o t h a t A a n d B may be r e l a t e d . S e e F i g u r e s 6 c , 7 c , a n d 9 f o r e x a m p l e s . 122 F i g u r e 5 123 Figure 6a. Two-dimensional g e l e l e c t r o p h o r e s i s of Mg-HAc preparation of Drosophila ribosomal p r o t e i n s . Long Run 3 mg p r o t e i n , Oregon-R males and females. F i r s t dimension: 23 hr. at 12 mamp/4 gels and 110 - 195 v o l t . Second dimension: 20 hr. at 200 mamp/4 gels and 65 - 140 v o l t . Figure 6b. Two-dimensional g e l e l e c t r o p h o r e s i s of Mg-HAc prepara t i o n of Drosophila ribosomal p r o t e i n s . Intermediate Run 3 mg p r o t e i n , Oregon-R males and females. F i r s t dimension: 14.5 hr. at 12 mamp/4 gels and 150-310 v o l t . Second dimension: 16.5 hr. at 2 00 mamp/4 gels and 60-100 v o l t . Figure 6a 124 125 Figure 6c. Two-dimensional gel electrophoresis of Mg—HAc preparation of Drosophila ribosomal proteins. Short Run 3 mg protein,Oregon-R males and females. F i r s t Dimension: 10 hr. at 12 mamp/4 gels and 152-185 v o l t . Second dimension: 15.5 hr. at 200 mamp/4 gels and 65-170 v o l t . Figure 7a. Two-dimensional gel electrophoresis of HCl-acetone preparation of Drosophila ribosomal proteins. Long Run 3 mg protein, Oregon-R males and females. F i r s t dimension: 2 3 hr. at 12 mamp/4 gels and 110 - 19 5 v o l t . Second Dimension: 20 hr. at 200 mamp/4 gels and 65 - 140 v o l t . 127 Figure 7b. Two-dimensional g e l e l e c t r o p h o r e s i s of HCl-acetone p r e p a r a t i o n of Drosophila ribosomal p r o t e i n s . Intermediate Run 3 mg p r o t e i n , Oregon-R males and females. F i r s t dimensions 15.5 hr. at_12 mamp/4 gels and 150-185 v o l t . Second dimension: 15.5 hr. at 200 mamp/4 ge l s and 65-170 v o l t . Figure 7c. Two-dimensional g e l e l e c t r o p h o r e s i s of HCl-acetone p r e p a r a t i o n of Drosophila ribosomal p r o t e i n s . Short Run 3 mg p r o t e i n , Oregon-R males and females. F i r s t dimension; 10 hr. at 12 mamp/4 gels and 152-185 v o l t . Second dimension: 3.5.5 hr. at 200 mamp/4 ge l s and 65-170 v o l t . 128 Figure 7b Figure 7c o 129 F i g u r e 8 F i g u r e 8, Two-dimensional g e l e l e c t o r p h o r e s i s of E . c o l i r i bosomal p r o t e i n s . 3 mg p r o t e i n . Mg-HAc p r e p a r a t i o n . F i r s t d imension: 15 h r s . , 12 mamps/4 ge l s and 130-230 v o l t s . Second d imens ion : 12 h r s . , 200 mamp5/4gels and 63-14 5 v o l t s . Some o f the p r o t e i n s on the r i g h t s ide of the g e l have s t a r t e d to migra te i n t o the cathode buf fe r of the f i r s t d imens ion . 6 = o r i g i n , A n i o n i c s i d e of g e l on l e f t , c a t i o n i c . s i de on r i g h t . 130 F igu re 9. Two-dimensional g e l e l e c t r o p h o r e s i : p r o t e i n s of mutant 1 6 c s of r ibosomal 3 mg p r o t e i n , HCl-acetone p r e p a r a t i o n . Mutant males . F i r s t " d i m e n s i o n : 10.5 h r . a t 12 mamp/4 g e l s and 125-182 v o l t . Second d imens ion: 12 h r . a t 170-175 mamp/4 g e l s and 90-180 v o l t . F igu re 10. Two-dimensional g e l e l e c t r o p h o r e s i s of r i bosoma l p r o t e i n s of mutant 5 3 mg p r o t e i n , mutant males . HCl-acetone p r e p a r a t i o n . F i r s t d imens ion: 10.5 h r . a t .12 mamp/4 ge l s and 125-182 v o l t . Second d imens ion: 12 h r . at 170-175 mamp/4 g e l and 90-180 v o l t . 131 132 F i g u r e 1 1 . T w o - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s o f r i b o s o m a l p r o t e i n s o f m u t a n t 2 7 c s 3 mg p r o t e i n , H C l - a c e t o n e p r e p a r a t i o n . M u t a n t m a l e s . F i r s t d i m e n s i o n : 23 h r . a t 7 -12 mamp/4 g e l s a n d 1 3 0 - 2 0 0 v o l t . S e c o n d d i m e n s i o n : 2 1 . 5 h r . a t 200 mamp/4 g e l s a n d 1 0 0 - 2 0 0 v o l t . F i g u r e 1 2 . T w o - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s o f r i b o s o m a l p r o t e i n s o f O r e g o n - R m a l e s . 3 mg p r o t e i n , O r e g o n - R m a l e s . H C l - a c e t o n e p r e p a r a t i o n . F i r s t d i m e n s i o n : 14 h r . a t 12 mamp/4 g e l a n d 1 2 5 - 2 1 0 v o l t . S e c o n d d i m e n s i o n : 13 h r . a t 200 mamp/4 g e l s a n d 7 5 - 1 5 0 v o l t . 133 F i g u r e 11 F i g u r e 12 

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