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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, COLDSENSITIVE MUTANTS OF DROSOPHILA MELANOGASTER:  GENETIC AND  BIOCHEMICAL STUDIES. by HELEN MARGARET MAYOH B.A., U n i v e r s i t y o f B r i t i s h Columbia, 1953 M.A., U n i v e r s i t y o f Toronto, 1960 Candidate i n P h i l o s o p h y , U n i v e r s i t y o f C a l i f o r n i a , B e r k e l e y , 1969 B.L.S., U n i v e r s i t y o f A l b e r t a , 1973  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  i n the G e n e t i c s D i v i s i o n  We accept t h i s t h e s i s as conforming required  t o the  standard  THE UNIVERSITY OF BRITISH COLUMBIA September - 1973  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r  an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree the  L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e  and  that  study.  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may by h i s r e p r e s e n t a t i v e s .  be  granted by  the Head of my  Department or  I t i s understood t h a t copying or  publication  of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without written  permission.  Department The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada  Date  M<Hh/¥~  )  /773  my  ii  ABSTRACT Cold-sensitive  mutants o f E. c o l i have been  valuable  i n the study o f the s t r u c t u r e anat assembly o f b a c t e r i a l ribosomes.  Some c o l d ^ s e n s i t i v e mutations of E. c o l i  have  been mapped t o l o c i coding f o r ribosomal p r o t e i n s .  Other  c o l d - s e n s i t i v e mutations have been shown t o a l t e r the regulatory  properties  In c o n t r a s t ,  o f b a c t e r i a l enzymes.  l i t t l e was known about the  genetics  and  b i o c h e m i s t r y o f c o l d - s e n s i t i v e mutants o f D r o s o p h i l a  and  o t h e r eukaryotes.  somal p r o t e i n s  A l s o , the g e n e t i c  l o c i of r i b o -  o f D. me1anogaster were unknown although  the ribosomal RNA genes have been l o c a t e d on the X and 2nd chromosomes. asked:  T h e r e f o r e the f o l l o w i n g q u e s t i o n s were  Can c o l d - s e n s i t i v e l e t h a l s o f the X chromosomes  of D r o s o p h i l a melanogaster be i s o l a t e d ? t h e i r genetic proteins  properties?  I f so, what are  Do some have a l t e r e d r i b o s o m a l  as i n c o l d - s e n s i t i v e mutants o f b a c t e r i a ?  The  study was p a r t o f a g e n e r a l search f o r and 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 the chromosomes o f D. melanogaster. Among 3„919 EMS-treated X chromosomes,  25 were  as c o l d - s e n s i t i v e l e t h a l s o r s e m i - l e t h a l s .  That i s , more  than 20% o f the f l i e s c a r r y i n g a c o l d ^ - s e n s i t i v e survived  a t 22  o  C and none a t 17  semi-lethals,, > 30% s u r v i v e d  o  retained  lethal  C, and f o r c o l d - s e n s i t i v e  a t 22° c and <13%  a t 17° c .  The c o l d - s e n s i t i v e mutations were not randomly d i s t r i b u t e d ,  i i i  7 being right the  located  of  car.  phenotypes  been the  the  Over  permissive  As  at  X tip  half  temperature  presence  demonstrated ribosomal  of  in  3 being  exhibited and  characteristic  the  and  of  altered  ribosomal  from  2-dimensional  gel  this  method,  it  was  that  the  differences proteins  ribosomes  were  from  estimated  proteins  of  observed  of  -  72  Drosophila.  in  the  5 cold-sensitive,  and  gel  proteins No  controls By are  qualitative  patterns  sex-linked  has  bacteria,  electrophoresis. 69  at  mutations.  D r o s o p h i l a mutants  by  the  visible  c o l d - s e n s i t i v e mutants  proteins  to  sterility  bobbed and Minute  studied  in  female  7 exhibited  were  present  alleles  of  ribosomal  mutants  and  the  controls. A from  summary  other  given. that  the  biochemical  cold-sensitive  No b i o c h e m i c a l  cold-sensitive  ribosomes and  of  was  evidence  mutants  obtained.  recommendations  mutants  for  of  The future  testing of to  of  the  Drosophila support  Drosophila limitations research  the  have of  are  the  ribosomes is  also  hypothesis altered study  discussed.  iv  TABLE OF CONTENTS Page I. II.  III.  Introduction:  C o l d - s e n s i t i v e mutants . . . .  1-22  Genetic studies of sex-linked, r e c e s s i v e , c o l d s e n s i t i v e mutants o f D r o s o p h i l a melanogaster. Introduction . . . . 23M a t e r i a l s and methods 25Results 29Discussion 43C o l d - s e n s i t i v e , s e x - l i n k e d mutants o f D r o s o p h i l a melanogas t e r ; Two-dimensional g e l e l e c t r o p h o r e s i s o f ribosomal p r o t e i n s . Introduction Methods and m a t e r i a l s Results Discussion Bibliography  24 28 42 47  48-49 50- 64 65- 75 7 6 - 89 90-102  Appendix  103  Tables  104-113  Figures  114-133  V  LIST OF TABLES Table I  II  Title  Page  The e f f e c t o f 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 t o the Oregon-R c o n t r o l , f o r the cross," j?(T) /Yd* x C (T)RM or C(T)DXQ.  104-5  The e f f e c t of temperature on the f r e q u e n c i e s 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 o f mutant males from the c r o s s , i ( l ) / Y ^ X C(1)RM/Y o r C(I)DX/Yg  106  Male progeny a t 22° C and 17° C o f females heterozygous f s o r y cv v f_ c a r and mutations which have v i s i b l e pKehotypes  107  C S  III  IV  S u r v i v a l o f progeny i n t e s t s o f Jj(l)5 , 6 , 1 5 , and 16^ w i t h 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 p r o x i m a l heterochromatin o f the X chromosome. 108 cs  c s  V  VI  V i a b i l i t i e s and phenotypes o f t r a n s heterozygous and homozygous females i n complementation t e s t s of c o l d - s e n s i t i v e mutations 109 The e f f e c t o f temperature on the v i a b i l i t i e s of mutant males and homozygous females from the c r o s s , J?(1) /FM6 $$ x j ( l ) / Y t f V , as compared to the Oregon-R c o n t r o l . . . CS  VII  CS  s  c s  F e r t i l i t y a t 22° C o f females homozygous f o r cold-sensitive lethals  110 I l l  VIII  Male progeny o f females heterozygous f o r y cv v f c a r and mutant chromosomes a t 17° C . . 112  IX  C h a r a c t e r i s t i c s o f c o l d - s e n s i t i v e mutants o f the X chromosome 113  vi  L I S T OF FIGURES  Figure  1.  2.  Title  Page  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 and recovery o f sex-linked recessive c o l d s e n s i t i v e mutations  114  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 3.  4.  5.  6a.  6b.  116  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 t h e Ma-1 r e q i o n '-'.{Schalet a n d F i n n e r t y , 1968b) ~ .  118  Genetic p o s i t i o n s o f c o l d - s e n s i t i v e , heats e n s i t i v e and n o n - c o n d i t i o n a l lethal m u t a t i o n s on t h e X chromosome  120  Composite diagram o f two-dimensional e l e c t r o phorograms 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  122  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 Mg-HAc preparation o f Drosophila ribosomal proteins. Long r u n  124  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 Mg-HAc preparation o f Drosophila ribosomal proteins. Intermediate run  124  6c.  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 Mg-HAc preparation o f Drosophila ribosomal proteins. Short run . . . ; . 126  7a.  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 HC1acetone preparation o f Drosophila ribosomal proteins. Long r u n . .  126  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 HC1acetone preparation o f Drosophila ribosomal proteins. Intermediate run  128  7b.  8.  9  Two-dimensional g e l e l e c t r o p h o r e s i s ribosomal proteins  o f E. c o l i  Two-dimensional g e l e l e c t r o p h o r e s i s somal p r o t e i n s o f mutant 1 6  of ribo-  Two-dimensional g e l e l e c t r o p h o r e s i s somal p r o t e i n s o f m u t a n t 5 °  of ribo-  .  13.1  C s  10  5  129  131  vii  LIST  Figure  11  OF F I G U R E S  cont.  Title  Page  Two-dimensional gel electrophoresis somal p r o t e i n s of mutant 2 7  of  Two-dimensional gel electrophoresis somal p r o t e i n s of Oregon-R males  of  ribo133  c S  12  ribo133  1  1.  Aim  INTRODUCTION:  of research:  C o l d - S e n s i t i v e Mutants  In the p r e s e n t  study,  sex-linked cold-  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 were i s o l a t e d ^ c h a r a c t e r i z e d genetically ations.  and  The  thought  examined b i o c h e m i c a l l y f o r r i b o s o m a l  r e s e a r c h was  t h a t the  undertaken  b e c a u s e i t was  s e l e c t i o n o f c o l d - s e n s i t i v e mutants  D r o s o p h i l a might enhance t h e r e c o v e r y o f m u t a t i o n s ribosomal  p r o t e i n s as  i s the case  T h e s e m u t a n t s w o u l d be as:  near  the genes f o r r i b o s o m a l  scattered  Are  i n E. c o l i  t h e genes f o r r i b o s o m a l  throughout  RNA  on  larval  and  ribosomal  adult periods?  questions  proteins clustered  melanogaster?  ribosomes c h e m i c a l l y constant throughout  during  1970).  t h e X chromosome o r  t h e genome o f D.  Drosophila or are d i f f e r e n t  of  affecting  (Nomura,  i n v a l u a b l e i n answering  such  alter-  the  Are  l i f e cycle  of  genes t r a n s c r i b e d  Is the  temperature-  sensitive period of Drosophila temperature-sensitive mutants r e l a t e d gene p r o d u c t the product  to 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  or to a c r i t i c a l i s required  ( T a r a s o f f and  the assemblies  of the  interdependent  i n Drosophila?  bacterial ities  s t u d i e s and  s m a l l and  a l s o new  of a higher organism  ribosomal  mutants.  developmental  event  Suzuki,  i n which  1970)?  large ribosomal  Are  subunits  Thus p r o b l e m s examined i n ones r e l a t e d  c o u l d be  to the  investigated  complex-  with  2  Cold-sensitive  mutants o f m i c r o o r g a n i s m s ;  mutants a r e d e f i n e d  by t h e i r v i a b i l i t y  t e m p e r a t u r e s and i n v i a b i l i t y  for cold-sensitive  allosteric  control  ribosomes. were  a t "normal"  a t low t e m p e r a t u r e s .  types of molecular a l t e r a t i o n s far  Cold-sensitive  have been e s t a b l i s h e d  mutants o f b a c t e r i a :  o f enzymes  and d e f e c t i v e  Several cold-sensitive  assembly o f  auxotrophs o f E.  (O'Donovan  and Ingraham,  mutants o f b a c t e r i a  fail  than losses 1965).  coli  of  i n enzyme  Other  so  loss of  f o u n d t o have c h a n g e s i n f e e d b a c k c o n t r o l  m e t a b o l i c pathways r a t h e r  Two  activities  cold-sensitive  t o a s s e m b l e 30S and 50S  ribosomal  s u b u n i t s a t low t e m p e r a t u r e ( D a v i e s and Nomura, 1 9 7 2 ) . T h e s e a r e c a l l e d Sad o r s u b u n i t a s s e m b l y m u t a n t s and some have b e e n s t u d i e d elucidate  the i n vivo  of other cold-sensitive  have n o t b e e n so t h o r o u g h l y mutations of v i r u s e s ,  but (Cox  to  mutants Cold-sensitive  shown t o map  number o f g e n e s and t o a f f e c t v i r a l  and S t r a c k ,  1971;  Scotti,  bacteria1  mutants;  1968;  o f enzymes  A cold-sensitive  Dowell,  of  properties  The m u t a n t grew w e l l n o t a t 2 0 ° C.  (O'Donovan  I t required  1967).  cold-sensitive  altered  and Ingraham,  i n glucose-salts  done  mutant o f E. c o 1 i  shown t o h a v e a h i s t i d i n e enzyme w i t h  regulatory  in a  development  few b i o c h e m i c a l t e s t s o f t h e m u t a n t s h a v e b e e n  feedback c o n t r o l  but  investigated.  f o r example, were  Defective  was  intensively  assembly o f b a c t e r i a l ribosomes.  The m o l e c u l a r d e f e c t s  limited  defective  1965).  medium a t 3 7 ° C  h i s t i d i n e o n l y a t t h e low  3  temperature.  When e x t r a c t s o f t h e mutant and  s t r a i n were t e s t e d activity, values low  i t was  similar  temperature  f o r p h o s p h o r i b o s y l - ATP  had  no d e t e c t a b l e enzyme a c t i v i t y . i n the h i s t i d i n e  o f t h e p y r o p h o s p h o r y l a s e by  pathway and  i s p r e v e n t e d by  the end-product  Enzyme f r o m t h e m u t a n t , however, was 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  w i l d - t y p e enzyme, and B o t h t h e m u t a n t and  a t low  r e q u i r e m e n t a t low  inhibition  was  substitute 1963; alters  ATP  m u t a n t had  stock.  site.  t h e low  the  a t 2 0 ° C t h a n a t 37° o f the mutant  to result  in a  C.  enzyme  histidine  1964).  As  feedback  temperature  synthesis  i f the  regulatory  and  of  cannot (Martin,  analogue  o f t h e enzyme and n o t  predicted  an a l t e r e d  of  inhibition  Resistance to the site  derived  T h i s analogue  i n protein  thiazolealanine abolished at  -  irreversibly.  pyrophosphorylase but  for histidine  the r e g u l a t o r y  catalytic  than  t o t h i a z o l e a l a n i n e were  i s known t o c a u s e  Sheppard,  1000  temperature.  resistant  phosphoribosyl—  o f t h e pathway.  by h i s t i d i n e  sensitivity  sufficient  from the c o l d - s e n s i t i v e histidine  inhibition  t h e w i l d - t y p e enzyme were more  the i n c r e a s e d  temperature  This  normally  f o u n d t o be  bound t h e i n h i b i t o r  t o feedback  Mutants  pyrophosphorylase  t o t h o s e f o r w i l d - t y p e b u t m u t a n t grown a t  excess production of h i s t i d i n e  Apparently  parental  f o u n d t h a t m u t a n t grown a t 3 7 ° C g a v e  enzyme o c c u r s e a r l y  sensitive  the  site,  cold-sensitive resistance  the requirement the extreme  the  to  for histidine  sensitivity  4  to  histidine  It  was  stock the  inhibition  also  shown t h a t  selected  extreme  for  of  6  phosphoribosyl  enzyme  to  -  inhibition  cold-sensitive,  It  in  the  is  thought  first  proteins  with  repression  that  sensitive  appear  requiring  and  of  in  of  the  sites, will  it  be  vitro  Davies  the  50S  were  arginine  is  15  pathway.  inhibition  1965).  in changes Since  allosteric  predicted in  that  some  cold-  bacteria.  assembly of  biochemistry  and  mutants  altered  the  30S  Furthermore,  multimeric,  to  particles  parental  a l . ,  useful  70S  the  et  be  which  more  (O'Donovan,  Sad mutants  complex  was  conformational  the  outline  than  Out  produced  to  to  is  discussing  1972;  bacteria.  all  feedback  Before  (Kurland,  occur  due  induction  mutants  structure  of  1965).  altered  regulatory  and  seems t o  histidine  -  lost  c o l d - s e n s i t i v e mutants  enzyme  enzymes  also  cold-sensitive  inhibiton.  auxotrophs  Ingraham,  c o l d - s e n s i t i v e mutants  repressors  The  and  by  1965).  temperature  temperature,  arginine  in  allosteric  the  low  inhibition  low  Ingraham,  ATP p y r o p h o s p h o r y l a s e t h a t  (O'Donovan  blocked  at  at  of  feedback  isolated  histidine  sensitive  to  cold-sensitive  independently  required  the  protdrophy  feedback  in  and  derivatives  sensitivity  Increased frequently  (O'Donovan  and Nomura, which  subunits.  in of  ribosomes;  it  bacterial  1972).  small  coli  detail,  consist of The  E.  two  They  may  ribosomes are  subunits,  subunit  of  be  E.  the coli  5  c o n t a i n s one  16S  RNA  m o l e c u l e and  20-21  large subunit  contains  one  molecule,  27-34 u n i q u e p r o t e i n s .  RNA  subunits  o f E. c o l i  ribosomal  RNA  are d e r i v e d  occurs  proteins  f r o m 3OS  specific  ionic  40°  C  subunits  required before  and  system.  The 2 3S  RNA,  remaining  at high  5S  when 16S  The  extra  assembly.  ribosomal and  ribosomal  a r e mixed  RNA  10° C o r  under  accumulate.  ribosomal  a t low  intermediate  subunits occurs at  Thus, more  low. 5OS  subunits  from  proteins also involves  ribonucleoprotein particles  to completion  Heat i s  proteins attach.  ribosomal  50S  protein  intermediate  rearrangement of the  r e c o n s t i t u t i o n of  and  20  reassembled  in vitro  less,  subunits  ribosomal  30S  Such  a c t i v e i n an  temperatures  and  does  not  (Nomura  and  Erdmann, 1970;  Nomura, 1 9 7 2 ) .  Reconstitution studies  of  other multimeric  p r o t e i n s from w i l d  enzymes and  organisms have demonstrated t h a t the cold-sensitive  (Van  Holde,  1966;  2  subunits  The  RNA  o f E. c o l i  temperatures than  in vitro  intermediate proceed  At  of w i l d type  r e c o n s t i t u t i o n of readily  30S  Nomura, 1 9 6 9 ) .  for a structural  the  The  c o n d i t i o n s a t t e m p e r a t u r e s between  are b i o l o g i c a l l y  particles  forms.  r e c o n s t i t u t i o n of  subunits  molecule,  ribosomal  d u r i n g ribosome  spontaneously  (Traub  synthesizing 21S  i n the  are cleaved  in vitro  RNA  p r o t e i n s i n common.  from l a r g e r p r e c u r s o r  subunits  and  have no  s p e c i e s found  oligonucleotides The  and  5S  proteins  whereas t h e 23S  one  unique  type  assembly p r o c e s s  is  V a l e n t i n e e t a l , 1966).  6  Cold-sensitive defects  mutations  which  exaggerate  Cold-sensitive that  they  protein  studies  have  cooperative Only RNA.  attached  improved in  correct  if  vitro  proteins  studies  temperatures,  and  30S  directly  other  and  1970).  with  proteins binding  16S  are is  simultaneously.  the  information  the  structure  no  with  ribosomal defects;  the  Sad mutants  various  templates  or  1969a;  temperatures  they  produced  ribosomes  like  wild-type  ribosomes  from  E.  coli  cold-sensitive  in  an  (Guthrie  et  in  a l . ,  Ribosomal mutants  of  bacteria  ribonucleoprotein  a l . ,  the  in  of  i.e.  et  system  that  proteins  and Nomura,  present  contained  mutants  accumulated  (Guthrie,  is  imply  sense  sequential  Sometimes  are  cold.  aggregates.  combine if  the  as  for  of  the  auxiliary  needed.  Cold-sensitive  and  (Mizushima  bind  toward  changes of  process is  so o n .  themselves;  are  the  regarded  in  reconstitution  only  and  assembly  components enzymes  two  specific  on the  proteins  Some p r o t e i n s  already  Such  the  reaction  specific  character  of  be  within  shown t h a t  in  certain  may b e  conformational  interactions  subunits  therefore,  a normal  mutations  affect  Further  may,  Tai,  et  30S  a l . ,  and  strains.  vitro  grew  At  subunits  were  protein  poorly  1969).  50S  low  particles  Assembled  Sad mutants  At  not  high  and  70S  70S  functionally  synthesizing  1969b). may  c o l d - s e n s i t i v e mutants  constitute of  E.  coli.  a  large Out  portion of  100  of  7  c o l d - s e n s i t i v e mutants o f E . c o l i , gave i r r e g u l a r  sedimentation  one t h i r d  p a t t e r n s when c r u d e e x t r a c t s  were e x a m i n e d b y a n a l y t i c a l c e n t r i f u g a t i o n 1969a). linked  Nomura e s t i m a t e d  mutations closely  (Guthrie e t a l . ,  t h a t 20% o f t h e c s m u t a n t s were  t o the region o f the E. c o l i  spectinomycin  t o one h a l f  chromosome where  r e s i s t a n c e and s t r e p t o m y c i n r e s i s t a n c e  map.  linked  The 2 a n t i b i o t i c  resistance l o c i are  and code f o r r i b o s o m a l p r o t e i n s .  g e n e s i n t h e s p c - s t r r e g i o n may c o n s t i t u t e r i b o s o m a l p r o t e i n s and s u p e r n a t a n t with ribosomes  an o p e r o n o f  p r o t e i n s which  (Nomura and E n g b a c k , 1 9 7 2 ) .  The  combine  But ribosomal  p r o t e i n s have b e e n mapped t o o t h e r a r e a s o f t h e E . c o l i chromosome putative  (Bollen,  e t a l . , 1973).  Sad m u t a n t s o f E . c o l i  Only  a few o f t h e  mapping i n t h e 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 m u t a n t s o f S a l m o n e l l a t y p h i m u r i u m were s c r e e n e d to  by t r a n s d u c t i o n f o r t h e i r  linkage  t h e s t r e p t o m y c i n r e s i s t a n c e l o c u s , 7 showed s u c h  linkage  (Tai, for  e t a l . , 1969).  Only  ribosomal defects.  s u b u n i t s accumulated temperature  2 o f t h e l a t t e r were t e s t e d  Evidence  t h a t incomplete  ribosomal  i n t h e 2 S a l m o n e l l a mutants a t low  was d e r i v e d f r o m  double  label  experiments. 14  Mixtures and by  of wild  type  mutant p a r t i c l e s  ribosomes l a b e l e d with C 3 labeled with H  sucrose gradient sedimentation.  temperatures  with  u r a c i l were  sedimentation  analyzed  When grown a t h i g h  radioactive precursors, wild  mutants had s i m i l a r  uracil  profiles  type and  o f 70S, 50S  8  and  30S p e a k s .  When l a b e l e d w i t h r a d i o a c t i v e  uracil at  2 0 ° C, however, t h e m u t a n t s , b u t n o t t h e p a r e n t had  altered  sedimentation  profiles.  strain,  One S a l m o n e l l a  mutant h a d a 23S peak a n d t h e o t h e r mutant h a d a 30S peak w h i c h was i n c r e a s e d r e l a t i v e  t o t h e other peaks.  This  i n c r e a s e may have b e e n due t o an a c c u m u l a t i o n o f subparticles  a t t h e 26S and t h e 32S r e g i o n s  (Nomua, 1 9 7 0 ) .  Three p u t a t i v e r i b o s o m a l mutants o f E. c o l i , representatives of the 3 patterns of irregular  sedimentation,  were a n a l y z e d b y t e m p e r a t u r e  (Guthrie,  et a l . ,  ribosomal by  sucrose  experiments  C u l t u r e s grown a t 4 2 ° c ,  1969a and 1 9 6 9 b ) .  transferred  shift  t o 20° C and l a b e l e d w i t h H  supernatant  from  half  uracil.  The  o f t h e c u l t u r e was a n a l y z e d  gradient sedimentation.  subparticles  3  were  Thus r i b o n u c l e o p r o t e i n  s y n t h e s i z e d i n t h e c o l d were l a b e l e d a n d  identified  by t h e i r  other half  o f t h e c u l t u r e was washed a n d i n c u b a t e d a t 4 2 ° C  with cold  uracil.  unusual  Subparticles converted  u n i t s a t h i g h temperature 50S  peaks.  erable  label  considerable  sedimentation  resulted  3OS a n d / o r  68 h a d c o n s i d -  i n t h e 30S p e a k , none i n t h e 50S peak a n d i n a 43S peak  label  peak was g r e a t l y From s u c h Sad  The  t o normal sub-  i n labeled  F o r e x a m p l e , a t 2 0 ° C, mutant  ( G u t h r i e e t a l . , 1969a).  t r a n s f e r r e d t o 42° C with c o l d considerable  properties.  uracil,  When  t h e mutant had  i n t h e 30S a n d 50S p e a k s b u t t h e 4 3S  reduced.  s t u d i e s , Nomura  (1970) c o n c l u d e d  m u t a n t s o f E . c o l i were o f 3 t y p e s :  that the  9  (1)  mutants and  that  failed  accumulated  to  32S  synthesize  particles  50S  subparticles  (precursors  of  50S  subunits), (2)  mutants  that  failed  accumulated (3)  mutants  that  subunits at  20°  The  30S  of  shown t h a t  in  30S  mutant  subunit  and  50S  and  and  21S  and  30S  particles  No  mutants  been  Analyses  to  1970).  of  21S  a  to  et  the  be  in  (Davies  subparticles, ribosomal  and  43S  particles  contained  16S  The of  of  of  Sad  E.  .  Here of  30S  explains  coli 5OS 50S  subunit all  the  1972).  supports  occurs  an  vitro  normal  however,  subparticles  in  type  and Nomura,  assembly  independently  but  independent  on  had  1970).  mutant  1971)  which  50S  dependent  the  subunits  theory  of  presence  fourth  al. ,  vivo  studies  and Erdmann,  30S  in  assembled  apparent  (Rosset,  published  32S  vitro  A heat-sensitive  appeared  the  in  indicate  of  the  a s s e m b l y was  Recently  satisfactory  The the  Nomura  seemed  incomplete  mutants.  the  that  independently  5OS s u b u n i t s  1968;  formed  assembly  has  subunit  intermediates  assembly.  whereas  32S  proceeded  50S  isolated.  v/ere  that  subunits  both  suggested  mechanism not  (Nomura,  subunits  both  contrast,  30S  control  was  mutants  but In  both  accumulated  idea  of  3 Sad mutants  studies  synthesize  accumulated  and Nomura,  vivo  to  subunits  assembly.  type  50S  C.  assembly  (Traub  synthesize  particles,  failed  and  3 kinds  assembly subunit  43S  to  the  in  the  Sad  contained  23S  RNA  RNA  (Guthrie,  et  a l . ,  10  1969a).  These are the  s p e c i e s o f RNA  32S  and  4 3S  and  21S  s u b p a r t i c l e s a r e p r e c u r s o r s o f 30S  The by gel  subparticles  expected  a r e p r e c u r s o r s o f 5OS  p r o t e i n s i n the  carboxymethyl  21S  cellulose  a spectinomycin  sensitive  and  subunits.  tion.  The  The in  2IS  subunits support  also  intermediates of ribosomal  f o r the  2 m u t a n t s were  and  type latter cold-  50S  p r o t e i n s were the  prepara-  similar.  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  steps of the  (Mizushima the  3OS  30S  i n t e r m e d i a t e s d e p e n d i n g on  p r o t e i n s o f the  Two  r e s i s t a n c e m u t a n t w h i c h was  analyses  the e a r l y  1971).  The  N i n e t o 11 u n i q u e  i n the  2-dimensional  Spc-49-1.  accumulated  present  identified  chromatography or  e l e c t r o p h o r e s i s ( N a s h i m o t o , 'et a l . ,  subunits,  subunits.  p a r t i c l e s were  3 m u t a n t s were e x a m i n e d , Sad-410 and was  i f the  and  in vitro  r e c o n s t i t u t i o n of  Nomura, 1 9 7 0 ) .  idea of incomplete  Again  the  r i b o s o m a l assembly  30S  results in  Sad  mutants. M a p p i n g o f t h e Sad by  m u t a n t s was  accomplished  phage P-^ t r a n s d u c t i o n o f c o l d - s e n s i t i v i t y  mutant  strain,  recipient,  aroE  aroE"  spc  +  spc  r  str ,  s  str  r  s  to a  were s e l e c t e d sensitivity  and  natants  the from  examined.  the  1969a  Transductants  and  for  t e s t e d f o r g r o w t h a t 2 0 ° C as w e l l  to streptomycin  whether c o l d - s e n s i t i v i t y related,  from  cold-insensitive  (Guthrie, et a l . ,  1969b; N a s h i m o t o , e t aJL. , 1 9 7 1 ) .  mainly  and  and  sedimentation  spectinomycin.  t h e Sad  as test  p h e n o t y p e were  patterns of ribosomal  several cold-sensitive  To  aroE  super-  t r a n s d u c t a n t s were  +  11  F o u r Sad m u t a n t s of  the E.  (Sad-410,  coli  were mapped t o t h e s p c - s t r  area  chromosome:  Sad-38)  aroE  Sad-19  spcA  strA  k-protein  Spc-49-1 The  r e g i o n between a r o E and  and  50S  ribosomal proteins  resistant  (Dekio, 1971).  1969;  S5 and  S12,  B o l l e n and H e r z o g ,  (The r i b o s o m a l p r o t e i n s o f E . c o l i  numbering  S, and  2-dimensional gels.) located  in sites  the l a r g e ,  ( D a v i e s and Nomura, 1 9 7 2 ) . accumulated  32S  between a r o E and et  al.,  to  the l e f t  located  1969b).  Type  1971).  phenotypes resistance,  closely  subunits, linked  and  30S  RNA  region  Sad-19, a t y p e 1 m u t a n t  which  mapped  t o spcA  (Guthrie,  Sad-410 and Sad-38, mapped  (Nashimoto, e t a l . ,  o f t h e Spc-49-1 mutant,  m e d i a t e s o f 50S  proteins  f o r ribosomal  from the s p c - s t r  3 mutants,  cold-sensitivity  The  of the  o f a r o E . b u t a n o t h e r t y p e 3 mutant  i n t h e s p c gene  by  L, s u b u n i t s i n  i n t e r m e d i a t e s o f 50S spcA and was  1969).  generally accepted  'et ' a l . ,  The known g e n e s distant  ( D e k i o and  Ozaki, e t a l . ,  s y s t e m i s b a s e d on t h e m o b i l i t i e s  from the s m a l l ,  are  (Wittmann,  altered  are i d e n t i f i e d  Wittmann's n o m e n c l a t u r e w h i c h has been by r i b o s o m e r e s e a r c h e r s  have  respectively  1970;  30S  Spectinomycin  and s t r e p t o m y c i n r e s i s t a n t mutants  30S r i b o s o m a l p r o t e i n s , Takato,  spc A c o d e s f o r s e v e r a l  spc-49-1,  1971).  was  A l l the  i . e .spectinomycin  and a c c u m u l a t i o n o f  inter-  s u b u n i t s mapped t o t h e s p c  locus.  12  A l t h o u g h t h e p r o t e i n components mutants  Sad-410 and Spc-49-1 were s i m i l a r ,  m u t a t i o n s mapped i n d i f f e r e n t some  regions  (Nashimoto, e t a l . , 1971).  especially are  o f 2IS s u b p a r t i c l e s f r o m  f o r Spc-49-1,  t h e two  o f t h e chromo-  The mapping  results,  suggest that ribosomal  a l t e r e d i n t h e Sad m u t a n t s .  The r e s u l t s  proteins  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 causes non-assembly Generally,  of both  subunits.  cold-sensitivity  were a s s o c i a t e d  3 mutant  and t h e Sad  i n the transductants.  phenotype  Sad 68, a t y p e  mutant w h i c h a l s o mapped t o t h e s p c - s t r r e g i o n , was exception; late  the c o l d - s e n s i t i v e transductants  4 3S p a r t i c l e s  but synthesized  (Guthrie e t a l . , 1969a). this the  observation. recipient  One  strain  No may  speculate  sufficiently  an  d i d n o t accum-  n o r m a l 50S  explanation  2  was  subunits offered for  that modifiers i n  complemented  the mole-  c u l a r d e f e c t t o s u p p r e s s t h e Sad p h e n o t y p e i n t h e sedimentation test  test.  The  l a t t e r may  than t h e 20° C growth One  other  the  dominance  al.,  1969b).  using  genetic  test.  f a c t w h i c h may  be i m p o r t a n t  o f the c o l d - s e n s i t i v e phenotype A partial  diploid  a recombination-negative  t h e mutant  be a l e s s s e n s i t i v e  region.  The mutant  ( G u t h r i e , <et  o f Sad-19 was  formed  F' e p i s o m e w h i c h p h e n o t y p e was  was  covers  dominant  to t h e standcird phenotype. The mechanism Spc-49-1  affects  b y w h i c h a l t e r e d p r o t e i n S5 o f m u t a n t  ribosomal  assembly i s not c l e a r  13  (Nashimoto, e t a_l., 21S  l e a d i n g t o the  the presence  of altered  s t i t u t i o n mixture assembly o f RNA  i n the  formation  30S  had  S5  no  one  observations  altered  both  30S  Nomura,  50S  adverse  minor  particles.  Finally  effect  on  the  Whether t h e u s e  i s uncertain.  reconin vitro  o f mature  precursor  16S  16S  RNA,  between i n v i v o and The  protein results  subunits  Omission  a relatively  s t u d i e s , r a t h e r than  ribosomal  and  has  o f 28S  r e s p o n s i b l e f o r the discrepancy  vitro  i n the  f r o m Spc-49-1 i n t h e  subunits.  in vitro  not present  the mutant.  from r e c o n s t i t u t i o n m i x t u r e s  effect  and  S5 was  i n t e r m e d i a t e s a c c u m u l a t e d by  o f S5  is  1971).  mechanism by  which  i n non-assembly  i s a l s o not understood  in  of  (Davies  1972).  O t h e r a n t i b i o t i c m u t a n t s have a l s o been e x a m i n e d f o r cold-sensitivity  and  the  Sad  phenotype  Brownstein,  1969;  significant  f r a c t i o n of phenotypic  streptomycin  low  Other  and  temperatures  streptomycin  and  affect  pic  accumulated  26S  the  and  B i r g e and  ribosomal Kurland,  43S s u b p a r t i c l e s  map  p r o t e i n , S12  1969;  r e v e r t a n t s from s t r e p t o m y c i n  1970). dependence i n the  Hashimoto,  either  p r o t e i n s , S4  30S  ribosomal  1960).  dependence t o but  o f two  strA locus  (Ozaki, et a l . ,  r e m a i n u n c h a n g e d i n p r o t e i n S12 one  A  i n d e p e n d e n c e were  (Nashimoto and Nomura,  r e s i s t a n c e mutations  30S  and  r e v e r t a n t s from  s t u d i e s have shown t h a t s t r e p t o m y c i n  and  1969;,  Nomura, 1 9 7 0 ) .  dependence t o s t r e p t o m y c i n  cold-sensitive at  N a s h i m o t o and  (Lewandowski  independence  show a l t e r a t i o n s or  Phenoty-  S5  in  14  ( B i r g e and and  Kurland,  Brownstein,  streptomycin have e i t h e r latter  1970;  1972;  D e u s s e r , e t a l . , 1970;  Stoffler,  e t a l . , 1971).  independent mutants which are a l t e r e d p r o t e i n S5  i s r e s p o n s i b l e f o r the  Kreider  or a l t e r e d Sad  Presumably  cold-sensitive, S4  and  p h e n o t y p e and  one  of  the  cold-  sensitivity. Though much work s t i l l Sad  mutants, the  r e m a i n s t o be  following conclusions  done on  the  seem w a r r a n t e d :  (1) 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 a c c u m u l a t e intermediates ribosomal  of ribosomal  subunits.  p r o t e i n s are the only mutational  implicated.  Sad  mutations occur  some o f w h i c h a r e known r i b o s o m a l v i v o assembly o f r i b o s o m a l the  in vitro  p r o t e i n genes.  subunits  be  Genetic  of ribosomal RNA  have been i d e n t i f i e d  RNA  30S  by  50S  In  than  Some subunit  c o n t a i n reduced  amounts o f DNA  ribosomes  o f D r o s o p h i l a melanogas ter.;  bobbed  (bb)  p h e n o t y p e were shown t o c o m p l e m e n t a r y t o RNA  ( R i t o s s a , e t a l . , 1966).  s t u d i e s i n d i c a t e d t h a t the c l o s e to the  the heterochromatic  melanogaster  RNA-DNA h y b r i d i z a t i o n .  the  X chromosome was  and  genes o f D r o s o p h i l a  Drosophila with  cytological  (3)  involved.  ribosomal  Drosophila  loci  i s more complex  reconstitution studies indicated.  a s s e m b l y may loci  alterations  i n several genetic  c o n t r o l mechanism w h i c h c o o r d i n a t e s  The  (2) Thus f a r  region  nucleous  (Cooper,  bb  Previous  locus of  organizer  1959).  from  the  (NO)  Ritossa  in and  15  colleagues  regard  o f t h e NO  t h e bb  to r e s u l t  DNA.  polytene  from p a r t i a l  A s e c o n d bb  o f 5S  ribosomal  chromosome  total  l o c u s and  RNA  (Wimber and  RNA  t e s t s would p r o b a b l y  ( T a r t o f and Various  (Kiefer, allows  (Cooper,  RNA  used  The  was  reduced  (Mohan and  The  18S  28S  RNA,  c o d e s f o r 2 8S  and  18S  1970). XX,  results  R i t o s s a , 1970).  tergites,  XY  and  XXY,  indicate  only a s l i g h t recessive  contained  t h a t dosage r e g u l a t i o n  i . e . reduced  effect  lethals.  RNA less  on  RNA  from 1 t o  3  s y n t h e s i s , however, than  i n reduced  shorter b r i s t l e s  Alleles  RNA  1 complete  Combinations o f extreme  also resulted  1968).  the  and  i n bobbed m u t a n t s w i t h  s t o c k s have f i n e r ,  Grell,  As  the  i n t h e bobbed h y b r i d -  be m a i n l y  rate of ribosomal  i n females  indicated  a r e c l u s t e r e d on  equivalent production of ribosomal  regions.  1959).  gland  e q u i v a l e n t amounts o f r i b o s o m a l  1968).  NO  alleles  Perry,  g e n o t y p e s , XO,  approximately  etched  bb  are located  S t e f f e n s e n , 1970).  the n u c l e o l u s o r g a n i z e r probably  and  NO  to s a l i v a r y  ribosomal  extract of ribosomal  ization  bb  The  deletions in  chromosomes 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 a t t h e genes f o r t h i s  RNA  Atwood, 1 9 6 9 ) .  t h e Y chromosome o f D r o s o p h i l a m e l a n o g a s t e r  Hybridization  2nd  counterpart  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 on  the mutant  ( R i t o s s a , e t a l . , 1966;  mutants a r e thought t h e NO  l o c u s as  egg  bb  production.  than w i l d type  abdominal banding  NO  and  (Lindsley  v a r y c o n s i d e r a b l y ; some e x e r t  the phenotype whereas o t h e r s  Variation  i n the  intensity  of  are  the  16  mutant p h e n o t y p e o f bb  alleles  s i z e s of d e l e t i o n s but  some m u t a n t s do  interpretation assumed t h a t  (Atwood, 1969;  concomitant p r o t e i n synthesis  Previous  of  bb  attempts to  of  Drosophila.  no  other  Several  the  brings  the  Drosophila  and  numerous  identify  the  ribosomal protein  bb  loci  approaches used to  and  5S  RNA  have been  genes site,  identified.  locate ribosomal  protein  to Drosophila.  however, have been b r i e f  and  The preliminary  character. 1.  S t r a i n - and  Mapping o f  K-12  species-differences  ribosomal proteins  d i f f e r e n c e s was  first  (Leboy, e t a l . , 1 9 6 4 ) .  proteins  al.,  protein"  1964;  E.  The  two  or  B and  E.  s t r a i n s showed of  proteins.  species-  coli  banding patterns  recombinants of  the  two  their  coli one  ribosomal  strains indicated  l o c u s mapped n e a r t h e  Mayuga, e t a l . , 1 9 6 8 ) .  successfully using  strain-  i n ribosomal  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 .  of various "K  by  p e r f o r m e d w i t h E.  major d i f f e r e n c e i n the  the  It is  mutants.  Except f o r the  reports,  f i t this  about the  r i b o s o m a l genes o f D r o s o p h i l a of  various  ribosome production  genes i n b a c t e r i a h a v e b e e n a p p l i e d  in  not  K i e f e r , 1968).  a slower r a t e of  abnormalities  i s a t t r i b u t e d to  applied  to the  strA locus  This  mapping o f  dysenteriae  hybrids  E.  (Osawa, e t a l . , 1971;  D e k i o , e t a l . , 1970).  The  latter  that  (Leboy,  a p p r o a c h has  species  c o l i - S a Intone 1 l a t y p h i m u r i u m and  Gels  studies  et  been  differences  c o li - Shi gel 1 a  Dekio,  1971;  indicated  that  17  t h e genes f o r e i g h t 30S  r i b o s o m a l p r o t e i n s and  ribosomal p r o t e i n s are c l u s t e r e d between a r o E  and  i n the  nine  spc-str  50S  region  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 g e n o t y p e s o f D r o s o p h i l a were compared by electrophoresis  ( K i e f e r and  d i f f e r e n c e s were o b s e r v e d Samarkand g e n o t y p e s , but  XY,  Gross,  1969).  differences,  species,  pseudoobscura  D.  between two  XXY,  XO  and  2.  D.  and  melanogaster  Streptomycin  D.  me1anogaster,  bobbed  not  associated with  ties.  Ribosomes f r o m  protein  i n the presence  the  streptomycin  are i n h i b i t e d streptomycin altered  a s S12  first  bacterial  ribosomal  o f s t r e p t o m y c i n whereas  sensitive  protein proper-  ribosomes  (Str-S) p a r e n t a l  strains  Str-R ribosomes b i n d ( K a j i and  Tanaka,  r i b o s o m a l p r o t e i n o f s t r A m u t a n t s was  by  reconstitution  T r a u b and  Nomura, 1968>.  strA  Amherst.  S t r - R mutants c o n t i n u e t o s y n t h e s i z e  Str-S ribosomes  somal, s t r e p t o m y c i n  also  resistant  a change i n r i b o s o m a l  (Davies, 1964). than  two  reported.  Streptomycin  were t h e  and  Samarkand and  m u t a n t s d e m o n s t r a t e d t o have an a l t e r e d definitely  strains  Clear-cut  melanogaster  strains,  mutants.  ( S t r - R ) m u t a n t s o f E. c o l i  the  two  gel  qualitative  however, e x i s t e d b e t w e e n t h e  M a p p i n g o f t h e s e d i f f e r e n c e s was  The  No  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 f o u n d .  qualitative  from  1-dimensional  among t h e v a r i o u s D. XX,  and  studies  less 1968).  identified  ( O z a k i , e t a l . , 1969;  i t s h o u l d be  noted  r e s i s t a n t mutants, not  l o c u s , have a l s o b e e n o b t a i n e d  that  nonribo-  associated with  (Roberts  and  Reeve,  18  1970;  Yamanda and Drosophila  concentrations eye  color  sensitive  are of  generally  streptomycin  streptomycin  development i n the  a f f e c t adults  of  mapped t o t h e  3rd  ribosomes of  food.  In a r e c e n t  stocks  abstract,  that  bound 10  t i m e s more l a b e l e d  streptomycin  color  of  t h a t d r u g r e s i s t a n c e may  be  streptomycin (Breckenridge  The  trait  studies  of  reported.  larvae  Rasmuson o f D.  protein  Resistance  (1971)  melanogaster  than those  results indicated  was  from  that  synthesis  X chromosome.  in  the  ribosomal  ribosomal found t o A  full  protein be account  i n t e r e s t because i t i n d i c a t e s be  r i b o s o m a l p r o t e i n mutants o f Suppressors.  mutations  biochemical  l a r v a e have a t l e a s t one  o f t h i s work w o u l d be  stages  stock.  sensitivity  In two-dimensional g e l s of  mapped t o t h e  be  19 6 8 ) .  early larval  streptomycin  inhibited i n vivo  more t h a n S t r - R l a r v a e .  m u t a t i o n s can  No  were  Preliminary  Str-S  d o m i n a n t and  Glassman,  Drosophila  ribosomes from S t r - S  larvae.  found t o  L a m b e r t s s o n and  reported  Str-R larvae.  A scarlet  Glassman, 1963).  chromosome. the  high  resistance  dominant t o  G o r i n i , .1969; Duke and  3.  eye  u s u a l l y r e c e s s i v e ) , the  r e s i s t a n c e m u t a t i o n was  proteins,  the  b a c t e r i a l streptomycin  (which a r e  Str-S  in their  (Duke and  1%  d i d not  the  a f f e c t e d by  to  but  and  not  however, was  drug a r r e s t e d  the  1971).  (st) mutant s t o c k ,  The  Unlike  Davies,  a s u c c e s s f u l means o f  obtaining  Drosophila.  It i s well  suppressed a t the  established  that  translation level  in  19  b a c t e r i a by a l t e r e d the  early  transfer  i t has been s u s p e c t e d  may a l s o s u p p r e s s  mutations  (Gorini, e t al.,1966).  streptomycin  Streptomycin  presence effect  resulted  Strigini  ribosomes level  misreadings  (Davies,  1966). that  a number o f m u t a t i o n s Mutations  i n vivo.  can a l s o a f f e c t  i n vivo  to streptomycin i n vitro  i n the  of the streptomycin  Streptomycin  the efficiency  resistance  of suppression  ( A p i r i o n and S c h l e s s i n g e r , 1967)J  and G o r i n i ,  1970).  idea that ribosomal  These o b s e r v a t i o n s  led to  structure influences the f i d e l i t y  translation. R e c e n t l y mutants o f E. c o l i  suppressed  other mutations  ribosomes w i t h h i g h e r (Rosset called  and G o r i n i ,  which  i n v i v o and c o n t a i n e d  translation  1969; G o r i n i ,  ram o r r i b o s o m a l  were i s o l a t e d  ambiguity  m u t a n t s were i s o l a t e d  1971).  T h e s e were  m u t a n t s and mapped t o a  by l o o k i n g f o r m u t a t i o n s  counteract the r e s t r i c t i o n streptomycin  The ram that  o f s u p p r e s s i o n by  r e s i s t a n c e mutations  ram l o c u s o f E . c o l i  altered  errors i n vitro  l o c u s i n the; s p c - s t r r e g i o n o f t h e chromosome.  The  Since on  to the finding  and r e s t r i c t i o n  on s u p p r e s s i o n  t r a n s f e r RNAs  caused  i n less misreading  o f the drug  mutations  of  suppressed  e t a l . , 1966).  resistance  the  that altered  s y n t h e t i c m e s s e n g e r RNAs i n v i t r o  (Gorini,  1972).  a t the t r a n s l a t i o n  T h i s o b s e r v a t i o n seemed r e l a t e d  by  (Smith,  studies of the e f f e c t o f streptomycin  bacteria,  of  RNAs  on t r a n s l a t i o n  may code f o r t h e 30S  suppression.  ribosomal  20  p r o t e i n , S4 (Zimmermann, fet a l . , 1971).  I t has now been  concluded t h a t s u p p r e s s i o n i n b a c t e r i a may r e s u l t from a change i n e i t h e r t r a n s f e r RNAs o r ribosomes. Some mutations  o f D r o s o p h i l a have l i t t l e e f f e c t on  the phenotype by themselves appear c l o s e r t o w i l d type  but cause o t h e r mutants t o ( L i n d s l e y and G r e l l ,  1967).  They have been d e s i g n a t e d as suppressors but i t i s unknown whether t h e i r mechanism o f a c t i o n i s s i m i l a r t o that o f b a c t e r i a l suppressors.  A D r o s o p h i l a suppressor  may c o r r e c t some a l l e l e s but not o t h e r s and may c o u n t e r a c t mutations  at several l o c i .  At least  superficially,  D r o s o p h i l a suppressors resemble t h e b a c t e r i a l mutants. I t has been suggested  that Drosophila suppressors,  such as i s u ( f ) , may code f o r a l t e r e d t r a n s f e r RNAs o r a l t e r e d ribosomes but few suppressors have been  investi-  gated b i o c h e m i c a l l y ( S c h a l e t , 1973; Dudick and Wright, The mutation,  1973).  su(s) , which suppresses v e r m i l l i o n mutants  of D r o s o p h i l a has been c o r r e l a t e d w i t h an a l t e r a t i o n i n the chromatographic (Twardzik e t  p r o p e r t i e s o f a t y r o s i n e t r a n s f e r RNA  1971).  The e f f e c t o f the suppressor,  however, was not a t the t r a n s l a t i o n l e v e l and su(s)  is  not c o n s i d e r e d t o be the l o c u s o f t h e a l t e r e d t r a n s f e r RNA (Jacobson, 1971; White, e t a l ; 1973a).  As y e t no b i o -  chemical s t u d i e s o f the ribosomes o f D r o s o p h i l a suppressors have been r e p o r t e d although they a r e b e i n g c o n s i d e r e d (Wright, 1973).  21  It yeast  is  interesting  have  bejen  suppression sensitive yotes, to  obtained  cind t h a t  the  yeast,  equivalent  i.e.  due  low  to  were  It  to  strain,  and had when  an  of  at  streptomycin  low  permitted  mutation.  Unlike  two  ^* selection  mutants  wild  of  in  bacteria,  of  a  jiq/ml  or  less)  the  to  the  moderate  500^ig/ml).  was  profile  grow  mutations  Unlike  also  in  One  not  sucrose  are  separated  apparently  ribosomes,  to  streptomycin  system. has  It an  cold-sensitivity  is  appears altered  in  due  the in  gradients and  mapping to  one  mutant an  that  in this  ribosomal  As d i s c u s s e d another  of  cold-sensitive  As c o l d - s e n s i t i v i t y  type  yeast  eukar-  insensitive  sensitive  (about  C o l d - s e n s i t i v e mutants. for  (50  of  insensitivity  sensitive  mutants  traits  is  suppression of  were  were  sensitive  mutant  other  the  streptomycin.  ribosomal  synthesizing  sensitive  by  the  temperature.  the  protein  Like  s e l e c t e d which would  streptomycin  sensitivity  were  cold-  Mutants  streptomycin  sensitive  procedures,  ribosomes  was  resistance  were  streptomycin  altered  grown  1971).  mutants  streptomycin  mutants  protein.  of  i.e.  the  streptomycin  for  assumed t h a t  strain  requirement  concentrations the  was  ai r i b o s o m a l  s e l e c t e d which  parental  these  streptomycin  histidine,  histidine  ribosomal  selecting  one o f  concentrations  without  by  that  Saccharomyces c e r e v i s i a e ,  histidine-requiring in  note  ( B c i y l i s s and V i n o p a l ,  streptomycin.  was  to  means  vitro coldprotein.  previously, of  obtaining  22  ribosomal mutants was of  that  the  a number  present  be  following of  of  isolated? some h a v e  s t u d y was  altered  sensitive  Suzuki,  et  of  aJ.,  1973).  all  bacterial  of  ribosomal  this ,  asked:  little  Also,  was  known  Therefore,  Can c o l d - s e n s i t i v e  genetic  properties?  The  study  and c h a r a c t e r i z a t i o n  1972;  mutants  D r o s o p h i l a me1anogaster  their  the  it  approach.  eukaryotes.  ribosomal proteins?  search for  lethals  (Rosenbluth,  are  by  ih-i-tiafced  X chromosome o f  I f s o , what  a general  different  obtained  q u e s t i o n s were  the  Because the  ribosomal p r o t e i n . genes  c o l d - s e n s i t i v e mutants  lethals  and  bacteria.  several  Drosophila might  about  of  of  mapped i n  thought  when  the  mutants  chromosomes o f  Mayoh and  Suzuki,  of D.  was  be Do  part  coldmelanogaster  1973;  Tasaka  23  11.  Genetic Studies of Sex-rlinked Recessive Colds e n s i t i v e Mutants of D r o s o p h i l a melanogaster.  The  utility  analyzing mental et  a variety  phenomena  cil. ,  of  1963;  sensitive  (ts)  of  genetic  has  molecular,  been  extensively  1967;  lethals  are  Suzuki,  probably  expressed mutations  diploid  eukaryotes.  lethals  map  1964),  within  a  around  number  functional  significance  of  two  classes  mutations  suggested  a  large  E.  coli  proportion  of  affect  self-assembly  (Guthrie, over,  et  of  Clearly, mutations  detectable ment ing  the  a l . ,  for for  in  a,b;  restricts  can  the  as  sites  micro-organisms. and  cold-sensitivity  of  developmental  we  decided  mechanisms  determine  in  whether  in  the  cil.,  (Edgar  highly 1969).  between  the  finding  that  mutations  in  subunits  1969). the  More-  regulatory  and  Ingraham,  1965b).  the  selective  property  functions  The  possibility  great  study  (Scotti,  and  ribosomal has  are  ribosomal  alter  of  chromosome  lethal  et  (O'Donovan  cold-sensitivity  regulatory  to  Tai,  mutations  proteins  of  by  class  heat-sensitive  difference  cold-sensitive  1969  cold-sensitive  properties  of  is  and  cistrons  The  of  the  Temperature-  lethals  of  (Epstein  simplest  phage  in  develop-  1970).  while  the  and  recover  cold-sensitive  limited  mutations  documented  the  to  Remarkably,  extensively  Lielausis,  clustered  lethal  Hartwell,  conditionally  and  conditionally  defects  by  potential  eukaryotes.  of  loci of  enrich-  simply  select-  for  the  study  Consequently,  cold-sensitive  lethal  24  mutations whether  are  readily  recoverable  their  genetic  properties  heat-sensitive  lethals.  will  whether  determine  ribosomal back  mutants  control  of  The it  is  and o t h e r  enzymes.  in  Drosophila  distinguish  them  answers  to  these  possible  to  enrich  defects  such  as  and from  questions for  altered  feed-  25  M A T E R I A L S AND METHODS  Mutant  S c r e e n and V i a b i l i t y  Tests.  cedure  for  recessive cold-sensitive  lethal  mutations  Males with ved 20  the  from an  detection  inbred  0.005M o r in  on the  (Lewis  (C (I) R M / Y )  defined The  can be  this  in  the  screens  in  step  which  a heterozygous Single y  C(1)RM/Y days,  at  and a f t e r  1  were  1).  vial,  the  greater  it  mutagenized  males  were mated  in  vials  transferred  dissol-  Approximately  which  the  at  22° C  are  not 1968).  recovery  F^ male  was more  of  offspring.  except  conditionals  efficient  chromosome i s  at  (Step to  22° C to  than  recovered  2,  Figure  fresh  3 or 1).  replicate  O f f s p r i n g emerged a f t e r 17° C .  one month a t without  When f e w replicate  was  (EMS)  bottle  lethals  screen,  hours  attached-X-bearing  permitted  all  1.  in  recipient.  proportion  strain  24  L i n d s l e y and G r e l l ,  females  the  the  17° C .  scanned b r i e f l y Figure  in  X chromosomes i n  of  females  they  cubated  F  20  and markers  procedure eliminated first  to  Figure  for  19 6 8 ) .  each q u a r t e r - p i n t  attached-X  mutagenized paternal As  crossed  found  fed  methanesulfonate  and B a c h e r ,  rearrangements  here  use of  in  screening pro-  shown i n  Oregon-R s t o c k were  E M S - t r e a t e d males were  (chromosomal  In  X chromosome i s  0.0125M e t h y l  1% s u c r o s e  females  the  of  The  or  vial of  some e x p e r i m e n t s ,  etherizing no m a l e s at  males  retained the  The  as  flies  were  2 2 ° C was  a putative in  vials  2 weeks  the  4  inat  22° C  were  (Step  observed in inspected.  occurred at  flies  After  17° C y i a l s the  4  22° C than  3, a  17° C  If  a  at  17°  cold-sensitive 17° C c u l t u r e s  C,  lethal. were  26  etherized  and examined  phenotypes,  i.e.,  an  for  conditionally  abnormal  phenotype  expressed at  1 7 ° C and  normal o  appearance  or  The m u t a t i o n s ^ ( l ) l by  c  an  s  ,  reduced penetrance  of  are  the  ^(1)2  C S  ,  designated,  in  etc.  They  have  number  of  abbreviation,  superscript Males  (i(l)2 of  retested  by  12  were  males  the  the  repeating  assessed  by  times  males  in  bottles.  bility  mated  test,  17° C but  the  within  to  of  each  referred and  at  as  to  the  trial.  cs  were  years.  C.  checked con-  X chromosome  females  lowest  2 2 ° C were  17°  The  at  observed in  the  to  The  some w e r e  on t h e  was  Six  2 2 ° C and  and  and C ( 1 ) D X / Y  at  C.  were  1).  chromosomes  three  yielding  values  22  detection  mutation  twice  When h e t e r o g e n e i t y  high  in  maintained  vials  been  (Figure  ratios  least  C(l)RM/y  those  3  mutant  a period were  also  of  at  ).  tested  sex  at  C S  order  the  2 and  the  the  tested  cs mutations  of  at  determing  s t o c k s were  firmed  steps  of  2  abnormality  cold-sensitive strains  individually  viabilities  several  be  putative  relative  All  will  c s  the  a  22° C via-  viabilities  used to  perpetuate  stock.  Testing  Female  Fertility.  Females homozygous f o r  the o  mutations  were  by  them w i t h  mating  generated  some e x p e r i m e n t s , were In  present  other  in  tests,  and  FM6/Y  one  to  for  and mutant three  each v i a l females  tested  were  males  females  along with mated  fertility at  1 to  22°  6 days  3 males  separately  of  at C.  C In  old  each  with  22  type.  the  27  two  types  for  each mutant.  progeny When  of  few  unhatched  dead  Mapping.  following  markers  positions: vermi 1i o n car  the  the  were  were  controls. 22°  y,  with  and  forked  -  f_,  the  males  f_ c a r  females,  20  the  tip  to  the  in  1 7 ° C were  the  -  proximal  region at  around  22°  C  females  at  Males  exhibiting  13.7;  euchromatin  crossed  bottles  mated  the  genetic  cv,  and the  days.  s t o c k were  at  of  were  of  and c a r n a t i o n  heterochromatic  CS  Those mutants  mapped u s i n g  56.7;  distal  i.e.,  adults.  symbols and  span most  the  or  crossveinless -  quarter-pint  lesthality  briefly  tunnelling  pupae  were  their  0.0;  scored for  Oregon-R w i l d - t y p e  at  cs mutants  cv v  were  and  or  The  testcrossed in  offspring  vials  and  e c l o s i o n began.  pupae,  JL(1) /Y  homozygous y  the  after  larvae  include  centromere.  9 days  tested  1 week  development,  These markers  do n o t  was  discarded after  occurrence of  -  females  during  X chromosome f r o m  but  25  lethality  listed  yellow  of  the  -' v , 3 3 . 0 ;  62.5.  area  to  signs of  eggs,  least  occurred,  medium w i t h o u t  Genetic  of  at  offspring for  An a v e r a g e A d u l t s were  scored for  scanned  the  males.  17° from  C. the  above  abnormal  mapped a t  Male  scheme  as  phenotypes both  temperatures. Mutations tested various  further  w h i c h mapped t o by  deletions  heterochromatin.  the  right  of  carnation  c o m b i n i n g them w i t h  a  duplication  that  encompass most  of  The  rearrangements  used were  the  were  or  proximal the  X-Y  28  insertion, Df(T)mal  1 2  Extensive  y_ ,  Y mal  Df(l)mal  Grell  All  were  crosses  demonstrating  phenotype  at  phenotype) phenotype tested  6  descriptions  Lindsleyand  to  ,  17°  with with  fell  duplication.  in  and  and  the  deleted  In(1)sc  of  the  4 L  sc  and  Schalet  carried  out  at  pseudodominant  the  (either  17°  and  or  duplication.  the  regions  None  c o v e r e d by  a  or of the  the  a  view  mutant  visible a  wild-type the  in  (1968a,b).  C with  expression of  deletion  the  22°  3).  found  and F i n n e r t y  lethality  proximal  ( F i g u r e s 2 and  8 R  chromosomes c a n be  (1968)  C  X chromosomes,  mutations  deletions  or  29  RESULTS  S c r e e n and V i a b i l i t y individually-tested as p o s s i b l e had  Tests. F^ m a l e s ,  than  Only  25  0.16  ratio  tests.  Of  g r e a t e r than  two  retained  a t 22°  viability  ratio  a t 2 2 ° C and flies  a t t h e two  i n at least  mutants w i t h a  1.32  (1,095 f l i e s i n vial's  1.37  The  (1,252 f l i e s  scored)  cold  ( T a b l e 1) .  which the r a t i o s  ( r^/r ) Q  i n t h e mutant c r o s s and  100 r  male t o scored)  These r a t i o s  where r ^ =  were  against  o f t h e m u t a n t s were compared.  the equation  little  a t 1 7 ° C when t h e  considered t o represent the standard v i a b i l i t y  ratio  two  verified.  temperatures.  o f t h e c o n t r o l was  were r a i s e d  done by  and  O r e g o n - RO*x C ('!') RM/Y<^ c o n t r o l c r o s s e s showed  change i n sex r a t i o female  32  test.  s e n s i t i v e p h e n o t y p i c e f f e c t were r e c o v e r e d and The  3,919  these, only 0.29  these requirements  In a d d i t i o n ,  of  s t o c k s were  a t 17° C i n the f i r s t  stocks f i t t e d  viability  151  c o l d - s e n s i t i v e mutants.  a male t o female  less  From t h e p r o g e n y  This  was  male/female  = male/female  ratio  in  o controls.  The  arbitrarily  s e t a t g r e a t e r than  2 2 ° and  17  o  less  limits  than  13%  f o r r e t e n t i o n o f t h e m u t a n t s were  a t 17°  21%  relative viability  at  C.  T w e l v e o f t h e 25 m u t a n t s r e t a i n e d were l e t h a l a t ° C and had v i a b i l i t i e s g r e a t e r t h a n 20% a t 22 C in  the f i r s t  viability  test  ( T a b l e s 1 and  13 were s e m i - l e t h a l w i t h g r e a t e r t h a n  11).  The  0% b u t  less  remaining than  13%  30  viability The at  at  17°  semilethals the  C and  showed a t  permissive  temperature.  greater  In  lethals  on  l e a s t 4.6  The  at  17°  lethals,  or before  subsequent t e s t s .  the  The  C(1)RM/Y and although the  C(1)RM/Y  females  only  be  surmised.  third  trial.  putative the  viability  The  mutants t o  first  viability  34  technical difficulty  selection for cold-  Viabilities  of  the  the  two  types  same m u t a n t ,  of  the  drastic decline  due  accurately  assessing  Consequently,  the  90  first  heat-sensitive  were c o n f i r m e d on  1967).  retest.  This  characteristic attributed  initial  the  are  For  l e t h a l s of first  no  retest  longer  to mosaicism  i n d u c e d by  (Jenkins,  1967;  In i t  of  EMS  and  Epler,  has  186  X chromosome, (Suzuki,  sub-  heat-sensitive  only  et a l . ,  u n r e p r o d u c i b i l i t y seems t o  of mutations  after  month-old  EMS,  example, out the  151  t h a t were  f o r h e a t - s e n s i t i v e m u t a n t s i n d u c e d by  a l s o b e e n n o t e d t h a t many s t o c k s  with  the  a t b o t h t e m p e r a t u r e s were p r o b a b l y r e t a i n e d .  putative  stocks  to  vital  i n the  in  can  stocks  in large part  C  of  from  kept at  lethals  22°  generally  stocks  cold-sensitive lethal  C.  at  or both  vials  screens  22°  exhibited  t e s t s were c a r r i e d o u t  initial  of  viability  restrictive  decreasing  instability  t e s t was  C.  f o r example, became s e m i -  viabilities  reasons f o r the  a t 22°  times g r e a t e r  C despite  C(1)DX/Y, o f  differed  The  viability  subsequent t e s t s , the mutants  a l s o changed, e i t h e r i n c r e a s i n g o r  stocks,  30%  temperature than at the  increasing v i a b i l i t i e s sensitivity.  than  be  has  been  1966).  31  The  instability  Habobracon  o f t s s t r a i n s has  ( S m i t h , 1968)  and  Other causes of v a r i a b i l i t y may  be  changes i n the  instability Wright that  the  Paramecium of  genetic  the  b a c k g r o u n d and  o f cs  of accumulation of genetic  genome.  L e a k i n e s s and  microorganism® has  Phenotypes.  modifiers  reversion of  a l s o been r e p o r t e d  Some o f t h e  the  and  a b n o r m a l abdomen, a .  m a l e s and  for  5 ,  6  C S  and  etched, missing the  15 or  ventral surface  exhibited pinching plate. and  The  c s  The  had  missing  of  the the  t h a n among t h e II). cs M u t a n t s 26 similar  to  frequent  5f:jV 6  27 C S  17°  in  of  Strack,  1971).  abnorbb,  f e m a l e s homozygous  reduced s t e r n i t e s , black  The  specks  mutants  permissive C a l s o had  on  sometimes  genital  more  restrictive  also exhibited and  severe at  In a d d i t i o n , f l i e s a rotated  at  elsewhere  the  frequent  temperatures  temperature e x t r a wing  veins,  cs and  and  at the  found  most o f t e n  segment n e a r t h e  o f f s p r i n g a t the Survivors  has  C exhibited  o c c u r r e n c e o f t h e s e t r a i t s was  e x t r e m e among s u r v i v o r s  (Table  or  abdomen. last  HRF.  phenotype o f bobbed,  a n g l e d t e r g i t e s , and of  and  and  mutants a t 22°  phenotypes which resembled  fact,  cs mutants  (Cox  mal  stocks  inherent  In  l e t h a l s are  for  1966).  cold-sensitive  made s i m i l a r o b s e r v a t i o n s  changes i n v i a b i l i t y  C S  (Igarashi,  of c o l d - s e n s i t i v e mutations.  (1973) has  result  a l s o been r e p o r t e d  15° 17°  s  and  the  C than a t  abdominal  t r a i t s were more 22°  c a r r y i n g mutant 2 6  genit-ial p l a t e .  abnormalties  c s  C  (Table  sometimes  A l t h o u g h mutants 2 6  c s  II). exhibited and  27  c s  32  were not  slightly lethal  less  and  phenotypic  therefore  mutants.  abdomen t r a i t (Table  viable  II)  in  17°  than  at  22° C,  referred  to  as  frequencies  of  the  are  The  these  a s was  at  5 mutants  noted  was  previously  were  cold-sensitive abnormal  extremely  for  they  their  variable viabilities. c s  The  visible  and  27  were  the  abnormal  c s  phenotypic  mapped a t  17° C,  abdomen t r a i t  Mapping  demonstrated  that  located  in  areas  the  other  and  15  c s  mutants  ).  mapped t o 27  c s  in  different  The the  the  abnormal  of  of  mutations  temperature  at  penetrant  (Table  these  two  mutations  were  from  one  another  abnormal  abdomen t r a i t y  X near  and c v , y.  as w e l l  abdomens  Note  of  (5  that  in  III).  as ,  C S  mutant  and t h a t  26  which  most  between  the  the  was  exhibiting  region  tip  abnormality  6  26  of  from C S  C S  mutant  the  cross  cs involving  26_  number when  ,  all  classes  compared w i t h  carrying  their  crossover  classes.  The b a s i s  uncertain  but  indicate  autosomal  mutation  combination  it  exhibited  21% o f  the  male  the  females,  the  various  was a  also  small  which  with cv.  spring  attributed  may  siblings.  crossover to  noted  a  for the  percentage  in of  observation  presence of  was  The fairly  classes  the  8% o f  appearance evenly  a  the  of  the  viability  attached-X  effect  in off-  compared the  could not  mutant  offspring  in  among  X chromosome.  of  to  trait  distributed  tests  female  is  segregating  female  as  and t h e r e f o r e , of  in  reciprocal  abdomen t r a i t  specific region  that  this  addition,  abnormal  however,  respective  reduced  produced a detrimental  In  the  c v were  be It c s  26  showed  33  the  a b n o r m a l abdomen t r a i t .  autosomal f a c t o r  i n mutant  A g a i n , t h e p r e s e n c e o f an 26  c s  i s indicated.  Clearly  cs mutant buted  26  i s c o m p l e d and i t s p r o p e r t i e s  to a single  c a n n o t be  attri-  mutation. c s  The s i m i l a r i t y  i n t h e phenotypes o f mutants  cs  5_,  &  cs and  15'  and t h e i n c r e a s e d p e n e t r a n c e o f t h e s e t r a i t s  17°  C, s u g g e s t e d t h a t t h e t h r e e m u t a n t s m i g h t be  w i t h the abnormal phenotype r e l a t e d lethality. ideas.  Indeed, t h e mapping o  Lethality  a t 17  each case  (Table 111).  men  occurring  flies  alleles  to the c o l d - s e n s i t i v e  data supported  these  C mapped t o t h e r i g h t  of car i n  M o r e o v e r , t h e few a b n o r m a l abdo-  a t 1 7 ° C were  consistent with this  at  location.  i n crossover  classes  Most o f the 22° C  mapping  data also  s u p p o r t e d t h e c o n c l u s i o n t h a t t h e a b n o r m a l abdo-  men  was  trait  located  number o f e x c e p t i o n s (2/1,199) were and of  may  f o r mutants 5  distributed  of car. C S  among a l l  factors  small 6  the crossover  t h e c s m u t a t i o n , o r t h e e x c e p t i o n s may of a d d i t i o n a l  The  (6/856) and  have b e e n due t o a s e c o n d c r o s s o v e r  selection the  t o the r i g h t  C S  classes  to the r i g h t  have b e e n due t o  d u r i n g the maintenance of  stocks.  cs cs cs The l o c a t i o n s o f m u t a t i o n s 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 m a p p i n g  •{Table IV) .  None o f them  fell  by t h e r e a r r a n g e m e n t s d i s c u s s e d  w i t h i n the region earlier.  spanned  O n l y two  small  s e c t i o n s o f t h e X chromosome were n o t c o v e r e d by t h e d u p l i c a t i o n - d e f i c i e n c y mapping;  the heterochromatic  region  34  immediately the  area  tions  5  adjacent  between ,  C S  6  15  mapping  procedures,  located  to  the  5  complementation mutations FM6/j.(l)  C S  it  ,  6  was  (Figure  of  car  in  C S  and  15  one  one C  of  V).  strain  at  trans-heterozygous bearing the V  females  was  the  low  viability  mal  abdomen t r a i t the  two  considered  0.0364  (the a  ratio  at  at  to  be of  at-17  r>  C and  45%  of  0.141  and  52%  gotes the  of  mutants  5  and  52%  respectively.  and  crossing  males  of  a  to  of  the  in  brackets in  high  then  the  FM6-  viability  the  in  c  5  abnormal from  of  mutants  the  Table  gave  comparable  two  of  calculation  frequency  example,  of  a  abnorto  both  were  trans-  had a v i a b i l i t y  ratio o a b d o m e n t r a i t . a t 22 C  the  reciprocal  abdomen).  The  cross,  had  homozy-  C S  and  trans-heterozygote;  0.0460  by  ratio  lethals  an e x t e n t  abnormal  CS  /Y  in  cold-sensitive  trans-heterozygote  and 1 5  S  same t r a n s - h e t e r o z y g o t e  ratio  a  For c  C S  regions.  alleles  The  involved  to  allelic. 5  two  C and a  22° C,  mutants  C.  figures  If  17°  17  the  flies  ratio.  the  above two like  muta-  were  Different  a measure  homozygous s t o c k s ,  heterozygote of  for  The  of  they  with ^ ( 1 )  and  used as  number  each v i a b i l i t y  of  females  and  o  22  trans-heterozygote.  indicate  of  both  all  trans-heterozygotes strain  2)  As the  in  the  behaved  S  ° different  3).  similarly  (Table  of  (Figure  concluded that  combined i n  females  centromere  behaved  s  tests  were  C S  c  right  Mutations  the  a n d .4.34  car  and  v S  to  62%  On t h e  15  had p r o p e r t i e s  viability  ratios  expression of other  hand,  of  abnormal  the  similar 0.0441  to and  abdomen,  trans-heterozygotes  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 cs cs c s 16 , 27 a n d 21 had h i g h v i a b i l i t y ratios, i.e., "*>  0.75  a n d <Cl%  tested, one  only  abnormal  mutants  ,  5  abdomen. 6  Of  and  C S  15  the did  seven not  ,  mutants  complement  another. cs All  flies  in  two  ted  fine  short  bristles  22  C.  In  addition,  i n d e n t e d and r e d u c e d cs a n d 20 were; l e t h a l the  five  effects  mutants of  19 6 8 ) .  so  various  The  ribosomal  other and  at far  17  carrying  o  and  The  considered  are  (see  role  of  bb  in  the  (Ritossa,  et  al. ,  reflect  ,  exhibi-  tergites  wings.  visible  bb  might  20  Both  similar  to  Lindsley  Grell, of  that  ribosome  of  the  and  suggests  defective  had cs 16  phenotypes  specification  1965)  at  occasionally  indented  C.  of  16  of  bobbed phenotype  and  some e t c h i n g  flies eyes  16  alleles  known  RNA  stocks,  c s  a  produc-  tion. cs In during  addition,  development,  incidence loss  of  of  plexus  external  communication). at  both  wings  10  22°  and  appear  flies exhibited  8  normal  wings  genitalia  males C  exposed to  abdominal  and b l i s t e r e d  adult cs  17°  when  and  (W.  females  (escapers).  and  are  not  The  involved  cold  tumors, and  personal  upheld  alulae in  small  occasional  Willis,  had  a  shocks  of  the  wings the  mutant  phenotype. Any mutant being  phenotypic  stock studied  abnormality  c o u l d be or  the  observed within  a pleiotropic expression  of  effect a  second  of  a  the  cs mutation  mutation  36  induced elsewhere is and be  on t h e chromosome.  n e c e s s a r y t o map the v i s i b l e seen  that  both the cs l e t h a l  phenotype  Viability  a t 22° C  the v i s i b l e  i n which  d e f e c t a l s o maps  ( F i g u r e 1)  d o m i n a n t as w e l l mutations.  a t 17° C  (Table I I I ) .  to the r e g i o n  o f Mutant Females.  ing protocol  defect  I t can  i n every case, the p o s i t i o n of the cs  corresponds very c l o s e l y exhibiting  Consequently, i t  the  mutation  (Table I I I ) .  Theoretically,  the screen-  should allow the recovery of  as r e c e s s i v e c o l d - s e n s i t i v e  In f a c t ,  lethal  lethal  a l l o f the mutants i s o l a t e d  proved  u t o be  r e c e s s i v e as shown by  and JL(1)° /+ S  females  since a l l mutations  a t 1 7 ° C. b u t one  heat- or c o l d - s e n s i t i v e recessive  lethals  Procunier, 1969;  and  S u z u k i , 1973).  The  absence  et a l . ,  ts lethals  homozygotes was C S  F M 6 / £ ( 1)  (Suzuki Holden  i n the hemizygous  Moreover,  after  to state  selection  i n y e a s t , H a r t w e l l (1967)  of s u r v i v a l of JL(D  made by c r o s s i n g  j f f ( l ) / Y males f o r 4 days  1972;  be  t h e r e f o r e , h a v e b e e n due  t h a t o n l y 4 b e h a v e d as d o m i n a n t t s l e t h a l s A comparison  dominant  o f dominant c s mutants i n  o f dominant c s mutations  recessive  their  /FM6  surprising  have b e e n f o u n d t o  Rosenbluth,  at the permissive temperature. 400  i s not  s e l e c t e d by  lethality  t h e X chromosome s c r e e n may,  of  This  o f a l l JC (1)  a t the p e r m i s s i v e temperature  and  lethality  the v i a b i l i t y  cs  in diploids.  heterozygotes  j£(T) / F M 6 C 5  found  females  and  to  a t 2 2 ° C t h e n a t 1 7 ° C f o r a week.  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 a t b o t h  37  temperatures  whereas  were  r e d u c e d i n numbers  greatly The  were  viabilities  assessed  females as  males  of  and homozygous f e m a l e s  o f jH.(T)  females.  S  a standard because they  tures. fered  The v i a b i l i t y little  at  were  0.92  ratios  homozygous four  v a l u e s were  considered bilities for  of  males,  x  100  below  (1(1)  bility  ratio.,  Generally,  most  females  striking c s  value  female  as  100  1)  c 5  of Table  as g r e a t  to  via-  follows:  VI  in  g)  brackets  indicate  in  c 5  the via-  each VI).  g e n e r a l i z a t i o n was  viability  o f t h e homozygous 2  of males  at  declined moderately  13 t i m e s  were  2 2 ° and 1 7 ° C (Table  this  as t h a t  flies,  $)/(FM6/j( 1)  viabilities at  The  (FM6/X$) /  The f i g u r e s  both  exception  was s t i l l  Relative  i n the c a l c u l a t i o n o f each  similar  the  17° C.  842 a n d 9 4 7  x  cs  and female  viability  g)  (1(1) /Jj(  viabilities  The r e l a t i v e  was t w i c e  although the  .  889,  at  have been c a l c u l a t e d C S  dif-  1 7 ° C , and f o r the  f o r the Oregon-R c o n t r o l  5  s t o c k were  2  867,  ° / Y < f l / (FM6/Ji( 1)  male  at  f o r the males,  100% v i a b i l i t y .  involved  tempera-  the Oregon-R c o n t r o l  (FM6/Xft)/(Oregon-Rffl.  flies  cross  o r Jj( 1)  at both  2 2 ° C and 1.03  and f o r f e m a l e s ,  of  mutant  subvital  of  from  The r a t i o s  the mutants  number  The  at  derived  the r e l a t i v e  c s  1.02  t h e above  / Y males  2 2 ° C and 0.91  to represent  Oregon-R<$  jj(l)  at  VI).  FM6/Y males were n o t used  were  ratios  (Table  from  t h e two t e m p e r a t u r e s ;  females,  respectively,.  17° C  the o f f s p r i n g  by the r a t i o  t o jff ( T ) ° / F M 6  at  generally  greater  than  that  2 2 ° C and at  17° C ,  for the  c s  38  m a l e s a t 17  C.  T h i s m u t a n t was  a l s o unusual  i n that  the  homozygous f e m a l e s were t h e most f r e q u e n t c l a s s o f o f f o spring a t both gous f e m a l e s  22  o C and  17  C and  in viability.  exceeded  the heterozy-  Sexual dimorphism  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 Suzuki,  1970)  Redfield, is  and n o n - c o n d i t i o n a l l e t h a l s  1926)  of Drosophila. cs  i n v o l v e d w i t h mutant 2  further  (Tarasoff  (Thompson  1921;  Whether s u c h a mechanism  remains  (1961) has  t o be d e t e r m i n e d  by  shown t h a t g e n e t i c e x p r e s s i o n c a n  be a f f e c t e d by t h e p a r e n t a l  s o u r c e o f a chromosome.  f u n c t i o n i n g o f r i b o s o m a l genes o f m a t e r n a l  somes d u r i n g o o g e n e s i s has b e e n e x t e n s i v e l y (Brown and enrich  and  tests.  Spoffor(J  Indeed,  has p r e v i o u s -  Dawid, 1 9 6 8 ) .  for selection  I f cs l e t h a l s  e x p e c t an e f f e c t o f p a r e n t a l viability.  I t c a n be  ( T a b l e s 1 and  11)  documented  do,  f o r ribosomal defects,  chromo-  i n fact, one  might  s o u r c e o f t h e m u t a t i o n on i t s  s e e n t h a t e a c h J^(l)  and m a t e r n a l l y ( T a b l e VI)  i s paternally inherited  t h a t a comparison  c a n be made.  from t e s t  (compare T a b l e s 1 and V I ) , t h e r e i s no  to test  While v i a b i l i t y  so  A  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 o f X(D Female F e r t i 1 1 t y . amphibians, amplified These rich  varies  cs /  males.  Y  D u r i n g o o g e n e s i s o f some i n s e c t s  extrachromosomal  i n the oocyte  r i b o s o m a l RNA  (Brown and  c y t o p l a s m f o r t h e egg.  i s extensively  Dawid, 1968;  molecules are then t r a n s c r i b e d  and  Gall,  to provide a  In other organisms,  1969).  ribosomewhere  39  the  synthesis  cells, less  common.  (King,  the  transported and the  example, in  N.O.  (Mohan  over  that  cytoplasmic  The  produce  be  oocyte,  by  serve  defects  in  It  fewer  has  with  eggs  the  Sterility crude  times RNA  ribosome  been  low  criterion  for  cells  product-  or  surviv-  shown,  for  deletions  wild-type or  is  nurse  extensive  than  to  of  show t h a t  between  is  highly  15  thousands  studies  nurse  DNA  decreased production  1970).  of  crosses  males  CS  summarized  in  Table  per  female  in  a  the  number  of  females  female  ribosomal  by  females  fertility, selection  of  defect.  results  stocks,  over  example,  the  bridges  females  another  FM6/Y o r Ml) /Y  27  to  females.  Drosophila  cam  ribosomal  by  taken  oocyte  genetic  detectable  Ritossa,  is  transcription  Hence  regions  therefore, a  adjacent  from mutant  and  of  Autoradiographic  be  eggs  oocyte  melanogaster,for  rDNA f o r  oocyte.  . may of  D.  cells  1970).  the  replication  In  nurse  multiply  al  RNA f o r  selective  polyploid  ion  of  VII.  The  seven day  egg  tested  7 were  individual  separately  8 p r o d u c e d an  whereas  of  or  both  average laying  completely  of  of  is  are  progeny  shown  in  brackets.  51  progeny  sterile.  with  together  number  period  listed  average  females  Of  or  The  with the  more  per  rest cs  varied to  about  Thus, of  from  70%  almost  a quarter  complete the  (19/27) o f  cold-sensitivity  fertility  the had  sterility of  mutations reduced  or  (see  the  wild-type  selected no  mutant  on the  fertility.  11  )  control. basis This  40  is  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%  all  EMS-induced h e a t - s e n s i t i v e  lethals  and  fertile  ( S u z u k i and  homozygous f e m a l e s  (50/88) o f  produce  viable  Piternick,  1972) . No  eggs o r o t h e r i n d i c a t i o n s o f o f f s p r i n g were \ i n the v i a l s  served  c o n t a i n i n g females  mutant f o r  ob-  12  cs  cs and  17  Also, at least  h a l f of these  females  when t h e 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 the  females  had  emerged).  The  is  a p p a r e n t l y ; j u s t one  at  the p e r m i s s i v e temperature.  were d e a d  (9 d a y s  after  i n f e r t i l i t y of these  aspect of t h e i r  females  abnormal p h y s i o l o g y  S t e r i l e mutants 5  C S  ,  13  c s  cs and  15  produced  eggs w h i c h f a i l e d  the permissive temperature, failed  C S  T6  C S  quently 3  C S  ,  10  t h e eggs o f t h e s e  ,  2()  laid c s  ,  c s  and  24  c s  sperm.  eggs w h i c h f a i l e d  l l  c  s  and  26  c s  to hatch.  exhibited  that  stages.  be  As more t h a n one  stock, the observed  mutation  mutation  may  i n f e r t i l i t y may  causing cold-sensitive t h o s e homozygous f e m a l e s  fre-  Sterile  evidence  the  females  w h i c h were p o o r l y f e r t i l e  larval  Of  females  A l s o , mutant  ment sometimes p r o g r e s s e d a s f a r a s t h e  each  Even a t  t o support normal embryogenesis r e g a r d l e s s o f  genotype of the f e r t i l i z i n g 6 ,  to hatch.  mutants  develop-  and  pupal  present i n  n o t be  due  to  the  lethality. w h i c h were f e r t i l e , a l l  cs e x c e p t mutant 23 Mutant 2 3  c s  yielded  a l l expected  types of  offspring.  f e m a l e s , when c r o s s e d t o m u t a n t m a l e s and  males, o n l y y i e l d e d  FM6/j(T)2 3  C S  offspring.  Jj(l)  23  FM6 c s  41  m a l e s and p r o b a b l y  females f a i l e d  to develop  normally.  This observation suggests that the a d d i t i o n of the w i l d type a l l e l e  t o t h e z y g o t e a t f e r t i l i z a t i o n was  necessary,  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 , t o overcome deficiency A similar for  i:n t h e eggs l a i d  by homozygous JL{1) 2 3  pattern of l i m i t e d  the s e x - l i n k e d ,  (Counce, 1956a,b,c)  some  fertility  recessive visible and a t s l e t h a l  h a s been  females.  C S  reported  m u t a n t s d o r , f u and r  (Tarasoff  and  Suzuki,  1970). All  the v i s i b l e  p h e n o t y p e s were r e c e s s i v e e x c e p t f o r cs  t h e a b n o r m a l abdomen t r a i t c a n be c o n s i d e r e d 9  1  id)26  C S  /FM6  o f mutant  The  latter  s e m i - d o m i n a n t b e c a u s e a t 2 2 ° C,  females e x h i b i t e d  the t r a i t ,  48% o f 62 f e m a l e s were a f f e c t e d . of  26  5% o f  and a t 1 7 ° C,  I n t h e same c r o s s ,  106 homozygous f e m a l e s and 9% o f 91 h e m i z y g o u s  exhibited  the t r a i t  a t 2 2 ° C, whereas  a t 1 7 ° C,  39%  males  69% o f  t h e homozygous f e m a l e s (48) and 44% o f t h e m a l e s  (41) were  affected. Mapping. carried and c a r  Genetic l o c a l i z a t i o n o u t a t 17° C r e l a t i v e (Tables  and c v was so o n . readily  t o the markers:  I I I and V I I I ) .  was  y, cv, v, f  The r e g i o n between  d e s i g n a t e d a s 1, between  c v and v as 2,  y  and  T w e n t y - f o u r 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 were localized  to a region  m u t a n t s were t o o l e a k y at  o f t h e 27 m u t a n t s  1 7 ° C) t o map  (i.e.,  precisely,  o f t h e X chromosome. yielded  surviving  Two  mutants  and two o t h e r m u t a n t s  no  42  longer  behaved  stock.  The  mutations result  as  lethals  latter  (Suzuki/  from  the  upon o u t c r o s s i n g t o  observation et  a l . ,  has  1967)  introduction  of  been  and  is  the  reported presumed  genetic  marker for  ts  to  modifiers  from  cs the  marker  latter  stock.  group,  it  Although still  c o u l d be  males  carrying  the  those  with  wild-type  The the  the  (Lindsley  control  and  freqeuntly  Grell,  values.  checked  in  values.  evidence  of  were  at  9 days  (Table 17°  similar  1968).  females None  In  The  such cases,  and of  chromosome  all the  belonged  mapped b e c a u s e  emerged  allele  19  to  than  the  region  standard  values  compared w i t h  showed v a l u e s  of  however,  crossover values  rearrangement.  the  surviving  later  each  mutants,  cs-bearing  to  VIII).  C in  showed d i s c r e p a n c i e s when  control  ard  mutation  crossover distances  Oregon-R  mutant  similar  the were  to  stand-  chromosomes gave  any  43  DISCUSSION  The can of  be  frequency  estimated  treated  bearing 36%  of  of  by  comparing  and u n t r e a t e d  females. all  induced  sex-linked  lethal. of  At  the  lethal  than  1 newly-induced  show  that  the  cs  this  basis,  lethal  lethals  are  Thus,  ion  s h o u l d be  b a s e d on t h e i r  chromosomes  an  carrying  only  al,  1968).  it  was  estimated  that  single  mutation,  10-12% w e r e  et  are  of  that  of  tests  the  attached-Xfound  that  carry  Mapping as  the  single  rate  of  of  product-  those (Baillie  EMS-induced  the  lethals,  of  all  cold-sensitive.  et  lethal  appendix  for  calculation.I It less  is  frequent  compared of  to  10  than -  temperature  C for  that  sex-linked  heat-sensitive  12%).  heat-sensitive  larger 22°  obvious  Part  lethals, range  22°  to  lethals  lethals the  however,  used to  heat-sensitives,  sensitives) .  of  cs  were  (1.5  greater  -  3%  reflect  the  screen  (29° to  17°  C for  much as  proportion  may  17°  a l . ,  experiments  single  (See  a  (Suzuki  present  a  mutat-  cs  among  heat-sensitive  1.5-3.0%  an  studies  chromosomes b e a r i n g  In  about  more  induced mutation  a_l»,1968).  calculated  chromosomes  previous  progeny  mutation-induction,  proportion  one  of  b y EMS  0 . 0 0 5 M EMS c a r r y  recovered  estimate  et  Baillie  In  of  was  mutation.  events.  was  it  to  chromosomes w i l l  ion  it  crossed  level  induction  sex-ratios  X chromosomes exposed t o  proportion  1967;  the  males  On t h i s  lethal  cold-  and  44  The most m e a n i n g f u l mutants of  come f r o m  mutagen!zed  comparison of  recent  3 rd  experiments  c h r o m o s o m e s was  heat-  and c o l d - s e n s i t i v i t y  Flies  carrying  for  viability  each mutagenized at  17°,  mutations  were  were  sensitive,  heat  lethal  at  (Tasaka  22°,  lethal  1 7 ° but  at  and  29°  and an  viable  where  the  screened for and  29°  Suzuki,  C.  C but  at  number  of  i.e.  were  29° C,  ts  set  both  1973).  Twenty  viable  of  same  chromosome were  equal  at  frequencies  tested recessive  17°  C,  i.e.  mutations  were  coldo  sensitive. Three  All  other  these  mutations  mutants were  viable  temperatures  but  In  heat-sensitive  total,  and  10  21  mutants  were  lethal  sensitive  were  to  at  viable  at  the  one  both  of  other  mutants,  at  22  the  two  C. extreme  temperatures.  22 c o l d - s e n s i t i v e s ,  1 7 ° and  29° C but  viable  o at  22  C were  found.  Hence  the  cold-sensitive  and h e a t - s e n s i t i v e  similar  third  on the  Comparisons autosomal  heat-  can be made. a number 1973)  a l . ,  a  large  1972;  genetic  have  been  T a s a k a and  sites  of  were  distributions  also  Suzuki,  found  (DCS)  DTS l e t h a l s  have  very  1973).  found  dominant  lethals  and  at  Suzuki,  (Rosenbluth  On c h r o m o s o m e  found near  (Suzuki  of  w h i c h map  (Holden  been  was  recessive  Drosophila.  recovered  DTS l e t h a l s  were  mutations  and c o l d - s e n s i t i v e  n o DCS l e t h a l s  cluster  other  the  (DTS)  sites  of  chromosome o f  On c h r o m o s o m e 3 ,  whereas  et  but  of  of  frequencies  dp  in  2,  2L  and P r o c u n i e r ,  1969).  45  DCS  l e t h a l s were found on chromosome 2 only i n the  (Rosenbluth e t aJL., r a r e and  1972).  C l e a r l y , DCS  cluster  l e t h a l s are more  d i s t r i b u t e d q u i t e non-randomly i n c o n t r a s t to  DTS  mutations. On  the  conditional  X chromosome, about 4% of EMS-induced and  heat-sensitive  of car  (Suzuki and  of the  cs mutants mapped f e l l to the  the h e a t - s e n s i t i v e of the 29%  yielded  by  The  r i g h t of c a r .  i n that  1972)  region  tip  whereas (Figure  4).  (5  cs  , 6  cs  and  15  cs  ) mapping to  (Table V I I I ) .  Mutation  recovered i n a completely d i f f e r e n t experiment and  C S  distinct  6  C S  and  the  (Table IX).  l a t t e r are  I t was  phenotypically  concluded, t h e r e f o r e ,  each a l l e l e represented a d i f f e r e n t i n d u c t i o n Moreover, a s i m i l a r cs mutant mapping to the been i s o l a t e d i n another lab  Among the  7cs  S  mentation t e s t s of the  and  2 0 , was c s  that  event. right  of  (Wright, 1973).  mutants i s o l a t e d by Wright, a f i n e  mutant s i m i l a r to 1 6 °  progress  Of  heat-sensitivity.  3 cs mutations  was  car has  (3/24)  f o r c o l d - s e n s i t i v i t y of l e t h a l mutations  r i g h t of car were a l l e l i c  from 5  right  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  s  whereas 12%  Piternick,  cs l e t h a l s map  selection  to the  (7/95) mapped a t the  X chromosome (Suzuki and  Clearly,  15°  1972)  l e t h a l s , 7.4%  (7/24) of the  the  Piternick,  l e t h a l s map  non-  also  bristle  found.  2 s e t s of mutants are now  (T. Wright, p e r s o n a l communication).  Complein I t appears  46  that  selection  enriches  for  by  or  the  somal  regulatory  are  RNA i s  very  inviable of  and p r o l o n g e d ion  that  may  translational the  , ..  mutations  exhibited  tergite  ribosomes  and  Dawid,  female  1  production the 10  27 more  present  of  be  cs mutations  to  of  a  these  affect  tempting  flies  to  might  be  Seven of  the  bristles.  female  „c s 27  the  synthesis  sterility  develop.  yielded low  ,  sterile  fertility  )  of  (Brown in  due  In  27  bulk  embryogenesis  ribosome  with  cs  26  constitute  early  cannot  ,  slender  ribosomes  in  20  the  suggest  and/or  ,  tergites  suggest-  o r  16  and  bobbed  the  thereby  is  ribo-  Mohan  some o f  defects.  ,  retained  females  it  for  abdominal  led  and  Minute  Defective  eggs which  evidence  produce  of  exhibit  determined  c s , , c s ..cs  during  reflected  produced  c  has  tRNAs  and  15  etching  1968).  might  This which  bb  ,  etching  Thus,  Maternally-produced the  1965;  may  bristles,  6  ELI. ,  alleles  translation ,  et  ribo-  be  genetic  extreme  of  for  D r o s o p h i l a must  alleles  ,,cs ,cs  (5  enriches  extreme  apparatus.  of  fact  for  altered  phenotypes  in  (Ritossa  mutations have  characteristic cs  less  X chromosome  X chromosome c o d e s  development.  Minute  phenotypes  that  good  slender  the  sites.  in  the  Mutants  and  on  B i o c h e m i c a l and  l o c u s on  1970).  phenotype  some  defects  tests.  bobbed  Ritossa,  in  cold-sensitivity  biochemical  that  cold-sensitivity  mutations  Whether somal  for  a  to  fact,  of  7  females (Table  the  and  v).  cs Moreover, bristle  or  all  but  one  abdominal  (27  )  of  phenotype  the were  mutations also  which  sterile  or  had  a  47  poorly  fertile.  properties  of  (See the  sterility  define  many  triguing for  IX  mutants.)  combined p r o p e r t i e s female  Table  of  one w h i c h  The  a  summary o f  possibility  specific visible  and c s  defects  for  in  lethality  are  further  that  the  phenotypes,  sufficient  ribosomal proteins  demands  the  is  to  an  biochemical  instudies  verification. Unlike  reported quite  heat-sensitive  are  sensitive  Viability  in  recovered  cs  phenotype  in  of  We w o u l d  mutations  and  different  apparently  lethal  the  differences.  experiments  stocks or at different  viability  1973).  the  and e n v i r o n m e n t a l  Outcrossing cs  affect  (Wright,  genetic  different  widely.  greatly  variable  to  ratios  formed w i t h vary  quite  lethals,  times  stocks can  mutant  per-  can  also  chromosome  suggest that  cs  lethals  must o  be and  rigidly  selected  stability  from  bility  and the  the  class  cs  of  very  for test  their to  long  mutations  complete  test.  generation very  lethality  Otherwise,  the  time  at  17° C  difficult  to  work  at  17  variarender with.  C  48  III,  Co.ld^sensitive Sex-rlinked Mutants of Drosophila melanogaster: Two-dimensional Gel 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 .  INTRODUCTION  Cold-sensitive altered  ribosomal  components.  mutants  proteins,  biochemical  detection  Which  of  known  many  some g e n e r a l  faulty assay  the  would  Nomura's stitute  be  a  bacteria  large  portion  (Guthrie,  et  sedimentation  in  Drosophila by  is  chance. that  that  cold-sensitive  mapping  in  different  mutants areas  other  hand,  lethals  of  1969b). that  Another  ribosomal  many  or  five  of  the  with of  the  as  have The  one  procedure.  been  examined  selected  phenotypes  latter RNA,  similar  in  cold-sensitive  and/or  ribosomal  such  of  gelelectrophoresis  controls  As the  advantage  assays  components  sterility  for  enzyme  cold-sensitive  bobbed mutants. DNA c o d i n g  detect  con-  electrophoresis.  in  to  Un-  mutants  gel  deficient  difficult.  ribosomal  two-dimensional  to  interest,  assay?  On t h e  Oregon-R  similar  of  choosing a relevant  from  female  defective  be  devised  and  exhibited  be  s h o u l d one  mutants  mutants  produce  other  would  c o u l d be  test  proteins  or  may  probably  sucrose gradients  proteins,  Ribosomal  of  a l . ,  ribosomes  ribosomal  of  indicated  investigating  of  would  pathways,  a matter  studies  enzymes  enzymes  pretests  biochemical  Drosophila  enzymes,  Though mutant  their  less  of  mutants it  was  are thought  phenotypes  but  X chromosome, might  have  49  altered  ribosomal  proteins.  Two-dimensional because  it  has  ribosomal bacteria Bollen, was "K  proteins  et  a l . ,  previously ribosomal  double  the  used  protein" gel  proteins  can  dimensional than  50  et  in  and  a l . ,  various  1970c;  identify of  E.  and  coli  separated  gels. protein  In  of  the  1970b). into  about  are  gel  (Leboy  is  a  and  altered of  et  a l . ,  1970;  electrophoresis map  et  has  the  a l . ,  recent  older  1964).  adaptation about  method  E.  coli  25  bands  two-dimensional  components  strains  genetically  electrophoresis  capacity  chosen  Deusser,  K 12  electrophoresis gel  was  demonstrating  One-dimensional  and Wittmann, be  tool  mutants  to  resolving  (Kaltschmidt  more  in  1973).  one-dimensional  electrophoresis  a useful  (Kaltschmidt,  Two-dimensional of  been  gel  gels,  evident.  ribosomal in  one-  however,  50  METHODS AND M A T E R I A L S  Introduction.. isolation (Boshes,  A previously  of  ribosomes  1970).  ribosomes"  Both  ribosomal" and  ribosomes the  r i b o s o m e s as w e l l  ribosomal"  preparation,  radioactive than  uridine  and  however, 18S  and  the  "attached  ribosomal"  fraction  exhibited  a higher  The  results  may  represent  engaged by  the  has  in  the  through  present  ribosomal"  separates  study  of  of  were  contamination  are  from  of  not  found  28S  RNA m o r e  The to  "free  accumulate rapidly The  acid  ribosomal"  by  the  latter  incorporation.  preparations  synthesis  soluble  polysomes  and  it  is  for  proteins,  "soluble  used because  they  1972).  be  also  ribosomes".  considered to The  may  The method  was - a d o p t e d , f o r  ribosomes.  thought  (Birnie,  "soluble  yet  protein  reticulum  proteins.  Boshes procedure  proteins  "free  polysomes  synthesizes proteins  ribosomal  nascent  was  organisms  endoplasmic  mitochandria  Though  of  numerous  the  RNA.  Greater  polysomes" than in  or  "attached  contain  amino  used  r i b o s o m e s and polysomes not  synthesis.  preparations  relatively free  nascent  "soluble  formed  and  28S  of  was  the  endoplasmic  preparation.  rate  "attached"fraction  transport In  protein  observed  the  that  newly  "attached  been  that  suggest  the  shown t o  18S  into  to  for  "soluble"  ribosomal"  were  as  larvae  separated  attached  "free  preparations  procedure  Drosophila  The method  from  reticulum.  from  reported  be  a  tissues  our  use,  serious  source  examined  51  were  of  diverse  engaged stages  in of  the  partial  proteins  would  Boshes  to  isolate  Boshes'  1  any  be  was  to  below,  amino  in  sedimentation  used  in  this  the  the  in  acid the  of  to  of  ribosomes  were  from  from  females.  the  As  permissive  from  stocks maintained  were  used.  Also,  at  radioactive The  the  method  sedimen-  22°  supernatant with  the  The  however,  preliminary  mixed  temperature,  Oregon-R males  and  degree  is  Oregon-R,  or  no  mutant  C(1)RM/Y their  of  uncertain.  homozygous f o r  p r o d u c e d few  C with  It  experiments,  samples o f  females  had  outlined  Drosophila.  c o l d - s e n s i t i v e mutations at  fed  RNA a n d p r o t e i n .  that  In  of  Gould-Somero,  Particles  preparation,  isolated  Some  r i b o s o m e s and p o l y s o m e s  of  adult  ribosomes.  and  (M.  overnight.  like  assume  laboratory  post-mitochondrial  precursors  ribosomal  Isolation  even  gels  adapation  Drosophila.  sucrose gradients.  ribosomes  males  the  partial the  an  A d u l t s were  properties  reasonable  of  in  is  previously  communication).  examined  isolates  doubt,  that  or  which  repeated  incorporated  no  numerous  unlikely  show up  were  particles  spring  enough  is  at  proteins  observations  of  the  these  adult  tation  of  It  from  labeled  adult  proteins  ribosomes  or  purity  many  of  outlined  uracil  seems  one  large  method,  personal  were  ribosomes were,  proteins.  The method of  their  completion.  of  ribosomal  and  synthesis of  concentration  the  types  some  offmales  females,  CCDRM/Y  sibs  52  were  separately  adult  examined.  Drosophila melanogaster  sex  and c o l l e c t e d  The  flies  before the  were  being  pattern  gels  was  and  prepared  for  Twenty  20  TKM-S 0.007  ml  of  tissue.  This  strokes  high  with  homogenizer remove  flies  were  ratio less  of  a  a week  in  the  and  to  7  ethanol. months  No c h a n g e ,  in  two-dimensional  storage 5°  times. C and  and  Ribo-  all  filtrate  B sized,  to  ensure  that  hard  tissue  by  0.007  added  solution  equip-  freshly  M at  top  speed.  e a c h gram tissue  homogenate  was  most  then  pestle  cells  were  7.8,  mercaptoethanol)  for to  pH  of  was  of  seemed  ribosomal pellet  remove  teflon  in  M Tris-HCl,  operated  The  mesh t o  homogenized 0.03  colored  homogenates.  The  Ice  to  pre-chilled.  Omni-mixer  coarse nylon  anesthetized,  according  Dry  room a t  M sucrose,  an  GC^  isolations.  increasing  M Mg a c e t a t e ,  a clearer,  exoskelleton.  to  gm o f  in  proteins  a cold  or  sorted  C for  TKM-S s o l u t i o n was  concentrated through  cooled  ribosome  in  (0.26  were  -30°  ribosomal  seconds i n  produce  for  prepared  to  KC1, 20  at  s o l u t i o n s were  Five  0, 1M  vials  stored  used of  in  observed with  somes were ment  Etherized  than  a  the  insect  Dounce  made  centrifuged  at  the  five  glass  ruptured.  filtering  did  filtered  homogenized by in  to  Failure  latter  step  difficult. The minutes material  homogenate in  a  fixed  on t o p  of  was  angle, the  Sorvall  supernatant  rotor was  18,000  rpm  (39,100 removed  for  g). and  30 Fatty  the  53  precipitate  was  discarded.  supernatant  was  treated with  detergent  per  10  s o m a l membrane  ml  The 1 ml  supernatant  present.  post-mitochondrial of  to  10%  Triton-X-100  solubilize  The m i x t u r e  was  any  left  micro-  for  1/2  to  o 1 hour  at  fat-like The 42,000  C and a g a i n  topping clear  or  90  g).  minutes  The  thoroughly  drained.  dried  Kimwipes  possible was  from  usually  the  frozen  in  the  The to  was  Any  tubes  were of  at  angle  translucent  with  rotor  pellet  inverted the  and  tubes  a s much s u p e r n a t a n t  ribosomal p e l l e t . overnight  rpm.  centrifuged  inside walls  remove  18,000  Bechman 5 0 . 1  orange,  centrifuge  at  was d i s c a r d e d .  supernatant  A slightly  obtained.  with  centrifuged  precipitate  orange  rpm f o r  (167,000 was  5  were  as  The r i b o s o m a l  a  small  amount  pellet  of  TMA _2  s o l u t i o n ( 0 . 0 1 M T r i s - H C l , pH 7 . 6 , 0.001 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 Solubilization (1)  in  however, C(l) 10  ribosomal  HCl-acetone.  somal p r o t e i n s tested  of  for  initial was  RM f e m a l e s  gm t i s s u e  S e v e r a l methods  experiments. the  suspended i n  Cold  1 M HCl  (0.47  give  a  concentration  was  final  stirred  flocculent  at  5°  et  ml)  C for  precipitate  was  of  preparing  electrophoresis  The  HCl-acetone  M  a l . ,  1970).  total  0.25  the  The  were  and  Ribosomes  1.4  ml  chilled  M HCl.  2 hour.  RNA w h i c h  of  The  from  cold TMAH suspension mixture  white,  f o r m e d was  ribo-  method,  Oregon-R males,  added t o  1 to of  a  of  gel  mutants,  (Lambertsson,  were  5 X 10  proteins.  2-dimensional  used f o r  M MgCl^  removed  . to  54  by  centrifuging  The  at  supernatant  extracted smaller  To  20  saved and  the  precipitate  0.6  ml  of  (11.5  ml).  of  reduced  ml)  was  supernatant  The  protein  pellet  dried  at  room  (2)  was  95%  gm t i s s u e w a s  pH  7.8,  acetic stirred of  for  RNA w a s  supernatant In  To  0.45  (Hardy, an  hour  the  Sorvall.  reWhen  volumes  of  5 volumes  the  speed  cold was  and  for  cold  washings  an h o u r  separated  or  from  centrifugation.  washed w i t h twice  of  with  cold  acetone,  ether,  and  air-  ribosomal pellet  overnight acid, ml  in  0.01  2  a l . ,  in  an low  M MgCl^,  then  1969).  ice  (0.01  M  tris,  and  15 mM  ribosomes suspended i n  1 M MgCl ,  et  TSM  from  9.0  ml  cold  The m i x t u r e  bath.  TSM,  The w h i t e  speed c e n t r i f u g a t i o n  glacial  was precipitate  and  the  saved.  initial  trials,  sample g e l  Wittmann  (1970b).  as  pressure  the  supernatant  s u g g e s t e d by  To keep  ribosomal protein  greater  was  supernatant  in  The  4.5  ml  removed by  against  and  low  acid.  M. s u c c i n i c  added  acid  the  TMA I I .  precipitate  then  frozen  mercaptoethanol). first  in  temperature.  20  was  by  the  left  ethanol,  Mg-acetic  0.003  was  protein  colored  cold  used,  added to  the  with  M HCl in  ribosomal proteins,  The w h i t e  twice  minutes  proportionally.  The m i x t u r e  longer.  0.25  t i s s u e were  precipitate  acetone (2.3  with  were  for  was  amounts  reagents  15,00 0 rpm  small,  inside  the  the  was  dialyzed  Kaltschmidt  final  volume  d i a l y s i s was dialysis  and  of  sample  performed  sac than  gel  with  outside.  a  55  The  procedure  a n d was the  replaced  supernatant  acetic 5%  acid  (v/v)  for  24  16  was to  acid  added  8 0% o f  gelatinous  ml  This  for  from  (4)  without  1-2  (4  was  ribosomal  of  mercaptoethanol  The  final  from  RNA w a s  gm  0.0015  c o l d ribosome  The  was  dialyzate  M MgCl , 2  suspension allowed  orange  low  dialyzed  was  give  by  tissue  was  large  removed  supernatant  reported  ribosomes. 10  M KCl,  cold.  to  liver  The m i x t u r e  reticulocyte  re agent.  gm t i s s u e  M LiCl, was  left  mixing.  moved by  ml  a  speed  against  freeze-dried.  complete  ribosomes  recovery (Mathias  of and  1964).  from  reagent  (0.05  then  for  rat  2.3  the  reported  rabbit  hours,  1 mM  pellet  trials,  (v/v)  from  4 M LiCl.  The  Cleland's  reagent  pellet  was  66%  24  1971).  hands  later  p r o c e d u r e was  TKM To  The  28 hours.  Williamson,  pellet  ml  in  for  my  This  ribosomal  precipitate  procedure  proteins  a l . ,  The  40 hours  centrifugation. water  et  protein  cold  In  against  against  the  2.3  in  1 mM m e r c a p t o e t h a n o l  finally  pH 7 . 8 ) .  2.3 for  dialysis.  freeze-dried.  M Tris,  difficult  dialyzed  and  and  LiCI.  stand  The  first  resuspended in  0.001  standard  (Welfle,  was  (3)  technically  1 mM m e r c a p t o e t h a n o l  hours,  release  was  and  hours  dialyzate  by was  acetic  further  to  proved  8 M urea  prepared in  was  proteins,  added and  with  ribosomal  centrifugation was  a  and  the  saved.  small 50  0.005  immediately  contact The  To  the  ribosomal  lambda M  of  dithiothreital).  before reagent  using.  supernatant,  The  overnight  RNA p r e c i p i t a t e  The  Cleland's  was  re-  containing  supernatant  was  mixed  56  w i t h 0.2 was  ml of sample g e l and  0.1  to 0.15  ml of the mixture  a p p l i e d to the 1-dimension g e l column.  Two-dimensional Gel E l e c t r o p h o r e s i s .  Two-dimensional g e l  e l e c t r o p h o r e s i s of the e x t r a c t e d ribosomal was  performed as d e s c r i b e d by Kaltschmidt  (1970a) except f o r some minor changes. i n the procedure were as f o l l o w s . were p l a c e d  i n the middle o f a g e l  proteins and  Wittmann  The main  The  extracted  cm).  Unlike  v e n t i o n a l methods, t h i s arrangement allowed the  The  g e l column was  still  8.6)  con-  proteins  remain on the g e l .  removed from the tube and e q u i l i b r a t e d  i n b u f f e r f o r the second e l e c t r o p h o r e s i s . column was  proteins  (8% a c r y l a m i d e , pH  polymerized i n a g l a s s tube (14 x 0.5  to migrate to e i t h e r pole and  steps  attached  along  (14 cm x 10 cm x 0.5  cm)  The  gel  the top edge of a g e l s l a b composed of 18%  a c r y l a m i d e , pH  A f t e r e l e c t r o p h o r e s i s i n the second dimension,  the  p r o t e i n s were s t a i n e d w i t h Amido Black or Coumassie  c Brilliant S  Blue.  The  con e n t r a t i o n s of g e l s and  the  pH  values o f the b u f f e r s were s e l e c t e d to emphasize separat i o n by change i n the f i r s t dimension, and  by  s i z e i n the  second dimension. F i r s t dimension e l e c t r o p h o r e s i s . reagents were used 1970a) .  The  (Addendum, Kaltschmidt  following  and  Wittmann,  4.6.  57  Separation 'gel, 54  gm  pH  8.6  urea  6.0  gm a c r y l a m i d e  0.2  gm N ,  (recrystallized  in  N'-methylenebisacrylamide  1 . 2 gm d i s o d i u m s a l t acid (EDTA-Na )  of  chloroform) (bisacrylamide).  ethylenediaminetetraacetic  2  4.8  gm b o r i c  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  0.45 The  ml  N,  acid.  N,  N*,  the  up  to  above m i x t u r e ,  ammonium p e r s u l f a t e Sample  N'-tetramethylethylenediamine  s o l u t i o n was made  polymerize  (Tris).  was  1.5  14 8 . 5 ml  (TEMED).  ml w i t h w a t e r .  freshly  made  To  7%  required.  gel  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 0 . 3 2 gm b o r i c a c i d 0 . 0 6 m l TEMED 2  The  s o l u t i o n was made u p  polymerize 0.5  the  above  mg r i b o f l a v i n  water  was  to  solution,  and  a  99  ml w i t h w a t e r .  freshly  To  made m i x t u r e  5 mg ammonium p e r s u l f a t e  in  1  of  ml  added.  Electrode  buffer,  360.0  gm  pH  8.6  urea  2 . 4 gm E D T A - N a 9 . 6 gm b o r i c a c i d 1 4 . 5 5 gm T r i s 2  The reagents  s o l u t i o n w a s made u p t o  1 liter  were  two  weeks.  0.5  cm i n n e r  Glass  kept  tubes,  at 18  4°  C up t o  cm l o n g  and  with water.  diameter,  The  58  were  thoroughly  washed w i t h  dilute  Photoflow  solution  dry.  The  were  caps  and  gel. of  filled  The  water  After  the  Two  but  a  to  losses  of  was  light. gel  The gel  and  were the  kept  protein  to  added up tubes  of  to  were  the upper  Four  top  The  a  and  in  drained  rubber  separation a  few  curved  removed  gel  inferior  water  and  drops surface.  with  of  the  using or  dissolved in separation was  from  not  small  results.  the  gel  quantitative  the  The  tube,  0.1  equipment.  decreasing  and p o l y m e r i z e d  water  sample  with  U.V.  separation  cm.  tubes gel  the  were  shorter  8.5  was  samples were  of  Canalco  into  E.  1-dimensional  so t h a t the  section,  s a m p l e was  section  in  inserted  projected  When t h e  section,  was  transfer  electrophoresed  The  lower  the  sample  of  14  separation  chamber.  to  proteins  a minimum by  with  Electrophoresis C.  was  dipped  with  with  forming  water  tube.  removal  chamber  5°  from  the  produced  overlayered  cathode  the  bottom  overlayered  gel  applied  volume  of  and  the  ml water)  first-dimension  ribosomal  capillary  section  protein,  the  3 mg o f  equipment.  anode  at  9 cm w i t h  s o l u t i o n was  After  was  closed  polymerized  gel  Increasing  gel  (.1 m l / 2 0 0  rinsed,  paper.  long  amount  to  prevent  gel  sample  with  gel  to  absorbant  ml  tubes  detergent,  into coli  separation  gel  the  longer  bottom the  top  ribosomal was  5.5  cm  cm.  carried run  in  out  in  a  c o l d room  each experiment.  at Three  59  mamp c u r r e n t  was  The  current  was  initially  pH o f  the  applied  was  kept  per  constant  110-150 v o l t  electrode  tube  buffer  at  for 12  10  to  23  mamp.  hours.  The  but  increased with  was  unchanged at  voltage  time.  the  The  end o f  a  run. The them  columns were  under water  gauge, The  gel  4  gel  filled  removed  and moving  inches)  gently  a  then  be  removed  rubber  bulb  on  the  was  routinely  and  treated  Second Kaltschmidt  if  glass  intact  the  the  tubes  by  placing  hypodermic needle  the  end of  successful  as  long  between  could  from  by  the  and  applying  tube.  glass  the  tubes  had  gel. a  The  (22  water-  procedure been  washed  described.  dimension  electrophoresis.  and Wittmann  (1970a)  The  reagents  were m o d i f i e d  as  of  indicated  below. Equilibration  buffer  300 gm u r e a 0 . 6 7 gm KOH Approximately to  make  the  The quantity ing  of  volume gave  gel of  urea  was  acid  Separation gm  was  urea  pH  up  decreased  to  gel  varied  4.6  acetic  acid,  i.e.  sufficient  4.6.  made  and V a r i a b l e gel,  glacial  pH  was  columns  a. h i g h  360  solution  solution of  3 ml  to  1 liter  from  s l a b s was because pH.  480  with water. gm b e c a u s e  improved.  the  original  The  anneal-  The amount  60  180 gm a c r y l a m i d e ( e l e c t r o p h o r e s i s lized) 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 sufficient The  60 m l g l a c i a l  grade,  not r e c r y s t a l -  acetic acid, i . e . ,  t o make t h e s o l u t i o n pH 4.6.  s o l u t i o n was made up t o 967 ml w i t h  Thirty-three polymerize  water.  ml o f 10% ammonium p e r s u l f a t e was added t o  t h e above m i x t u r e .  acrylamide r e c r y s t a l l i z e d  from  the q u a l i t y o f the reagent  In i n i t i a l  experiments,  c h l o r o f o r m was u s e d .  d i d n o t have an o b v i o u s  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 e x p e n s i v e consuming, e l e c t r o p h o r e s i s  grade  As effect  and t i m e -  a c r y l a m i d e was u s e d a s  purchased. Electrode  buffer,  140 gm  glycine  Approximately sufficient The The  15 m l g l a c i a l  stored  acetic acid, i . e .  t o make t h e s o l u t i o n pH 4.6.  s o l u t i o n was made up t o 10 l i t e r s  equilibration buffer  prepared  v  pH 4.6  and s e p a r a t i o n  j u s t before use.  with  g e l were  The e l e c t r o d e  water. generally  b u f f e r was  f o r s e v e r a l weeks a t 5 ° C.  Amido B l a c k  Stain  5.5 gm Amido B l a c k 50 m l g l a c i a l a c e t i c The  acid  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 ,  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  repeatedly  to stain several  Coumassie B r i l l i a n t  gels.  Blue  0.3 gm C o u m a s s i e B r i l l i a n t 15 m l m e t h a n o l 980  ml 10% t r i c h l o r o a c e t i c a c i d  The  d y e was d i s s o l v e d  added t o t h e TCA s o l u t i o n . The  s t a i n was u n s t a b l e  each  Blue (TCA).  i n m e t h a n o l w h i c h was t h e n The m i x t u r e was  filtered.  and h a d t o be made up f r e s h f o r  experiment. In p r e p a r a t i o n  f o r t h e second e l e c t r o p h o r e s i s , the  1 - d i m e n s i o n a l g e l c o l u m n s were e q u i l i b r a t e d i n pH 4.6 buffer in  f o r 1 t o 3 h o u r s a t room t e m p e r a t u r e .  t h e t i m e b e t w e e n t h e end o f t h e f i r s t  and  start The  two-dimensional g e l e l e c t r o p h o r e s i s  except that  x  to that o f Kaltschmidt  4 g e l chambers, e a c h  10 cm h i g h  were p r e s e n t  0.5 cm x 20 cm.  of  five  plastic  four rectangular The the  electrophoresis  o f t h e s e c o n d h a d no a p p a r e n t e f f e c t o n r e s u l t s .  was i d e n t i c a l  and  Variations  apparatus  and Wittmann  14 cm l o n g  x 0.5 cm w i d e  i n s t e a d o f 5 chambers,  E s s e n t i a l l y the apparatus  forms w h i c h when b o l t e d  20 cm  consisted  together  created  chambers open a t t o p and b o t t o m .  b o t t o m s o f t h e g e l chambers were s e a l e d  assembled  (1970a)  apparatus  depth o f separation  i n a tray containing  g e l and p e r s u l f a t e .  w i t h water as p r e s c r i b e d  by K a l t s c h m i d t  by p l a c i n g  a half  inch  (Overlayering and Wittmann was  o m i t t e d a s t h e p r o c e d u r e was d i f f i c u l t and u n n e c e s s a r y . ) After  t h e bottom l a y e r o f g e l had p o l y m e r i z e d , g e l  62  mixture gel  was  poured  into  the  heat  from  the  polymerization  subsided,  the  1-dimensional  along  top  edges  the  During  these  of  solution  gel  the  flack  while  in  they  to  protein  to  form  to  attach  the  into  were  gel  gel  from  the  chcimber  buffer.  The; a n o d e  cathode  at  in  C cold  the  applied  left  gel.  in  the  gel  the  by  keeping  by  flask  gel  slabs  Kaltschmidt  The  And  mixture.  of  the  prescribed  seal  gel  columns  extensive  columns  containing was  room. 12  bottom  apparatus.  at  bottom.  for  the  to  It  chilled  the  top  or  of  A constant 22  hours.  The  electrode  buffer  changed d u r i n g  long  fresh  buffer.  the  other  the  first  electrophoresis,  the  10  gel  carried 200  As t h e  slabs  it  of  in  chambers  voltage,  hours  chambers the  electrode  of  rung,  gel  placed  gel  current  time.  within  then  E l e c t r o p h o r e s i s was  increased with  replaced  the  pH 4 . 6  volts,  times  of  was  100  After  to  unsuccessful.  attached  gel  delayed  columns  as  fluid  used to  electrode  to  the  was  had  occurred.  trimmed  5°  the  were  more  polymerization  polymerizing  (1970a)  reaction  columns were  slabs with  persulfate  water.  sink  Excess  a  the  ice  residues  artifacts  was  and  were  and Wittmann tended  of  gel  manipulations,  Attempts  60  chambers  slabs. After  was  sealed  a  were  and  out  mamps initially  pH o f was run  the  somewith  removed  from  63  the  apparatus.  were  bolts  l o o s e n e d and w a t e r  slabs  and The  the  an  plastic  s l a b s were  They were for  The  placed  hour  in  was  walls  the  running  room t e m p e r a t u r e .  ful  x  for  Amido  5.5  x  staining  Black  readily  Amido  and  but  only  well  Black.  night.  The  w a r m 10%  TCA..  with  solution The  maximum  as  blue  but  photographs. month.  spots The  as  Blue  (CBB)  in  gels  faded  and  over-  replaced  of  by  spots  a grey  appeared coat  distinguishable black shrank  and  T C A was  proteins had  with  CBB s t a i n  A second change The  was  as  stained  in  be  film.  the  indistinctly  with  destained,  sharpened  clearly  use-  could  and  s p o t s were  the  and  properly  placed  which  (8.5  stained  off  gels  several  trays  poured  on the  for  shop were  months if  de-  for  satisfactory  destaining.  showed up Also  was  latter  for  not  rinsed  acid  acid  Gels  and w h i t e  s l a b s were  gel  necessary  visually  gels,  The  background.  surface.  hardware gels.  were  and  stacking  several  the  Black.  1% a c e t i c  Coumassie B r i l l i a n t  the  the  a  results  dye  minutes  gel  syringe.  Amido  1% a c e t i c  the  black  destained  bright  from  the  b a c k g r o u n d was  Plastic  after  Also  with  the  fresh  storing  restciined.  Staining  in  obtained  faded  photographed  faster  2)  15  warm,  days  inches  for  gels  hours,  together  between  with  The  several  then  stained  in  parts  a hypodermic  water.  by  and  with  stain  stained  at  the  the  injected  usually  in  placing  holding  and  white  within  a  on  64  The However, light  gels  were  attempts  were  The  overhead  lighting  produced  an  close  the  in  order  Fine  grain,  Under gave the  to  these the  best  to  viewed  on a  photograph  gels  even  were  light  reduce  about  (Linhof,  8.5  shadow  inches  film  camera High  (16  light  uneveness with  the  DIN,  settings  of  contrast,  of  the  special 1/2)  camera  between in  table.  transmitted  setting The  artifacts  and w h i t e  negatives.  prints.  the  intensity.  conditions,  best  of  photographed  apparatus  gels,  black  best  frosted  them w i t h  u n s u c c e s s f u l because  light.  to  best  that  was  lens  placed  and  gel,  photographs. Kodak) 3 f #4  was  and  paper  used. 1/250  gave  65  RESULTS  Preliminary paring  Drosophila  phoresis  were  Mg-acetic residue gel  experiments.  from  addition,  the  well  sample  of  4 was  protein To  but  solution  produced the  for  When  columns the  the  anionic  to  origin  were  the  anionic  of  the  gel  Time phoretic  slab the  of  side was first  studies  of  gel to  few  to  Drosophila  ribosomal of  for  16 it  the  first hours was  at  found  cathode.  faint  bands  was  close  performed  s a m p l e s was an  proteins  conducted  and  preparations.  so t h a t  (Fig.  bands  Kaltschmidt  to  reagent  the  dissolved  diffuse  experiment  Drosophila  dimension  for  a  for  devoted  were  conditions  The  In  polymerization.  stained,  columns,  sample  out.  centres  had migrated  the  in  which  Drosophila  columns were  Cleland's  origin  the  the  protein  dimension.  electrophoresed  observed.  and  in  of  (3)  polymerize.  because  first  studying  and  to  permitted  the  placed  gel  half  the  to  in  and  in  proteins  HCl-acetone  cathode  precipitated  suitability  pH  Thereafter,  dissolved well  residues  gel  pre-  electro-  The  suitable  gel  with  reagent.  later  of  HCl-acetone,  3 produced  dimension  the  (.2)  gel  failed  system  On t h e  for  solution  were  of  methods  gel  samples were  most  LiCl,  extraction  proteins,  that  proteins  considered unsuitable  gel  8.6.  following  Cleland's  LiCl  determine  Wittmann  (4)  sample  2 and  Method  (1)  initially  Methods in  and  the  solution  ribosomal  tried:  acid  The  shifted  increased  migrating  area  to  the  5). find  optimal  proteins  electro-  on g e l  slabs  66  of  10  cm x  14  cm.  At  characterize  the  run  develop  to  keep  fully  fast-moving  Comparison That  of  6C).  is  proteins  and  8)  is  E,  evident  similar  and Wittmann  runs  and E .  Although  have  pH 8 . 6  to  the  cathode  of  the  two  the  and m i g r a t e  basic  proteins  different  migration  of  coli  the  side  E.  of  the  gel  staining  spots  gels  and  form  coli  proteins The  recently  gels  found  (Fig.  6c  ribosomal  on  from  pattern  however,  he  excluded  D.  Kaltschmidt  E.  coli  higher  and  than  dimension, quite components  the  anionic  ten  faintly  the  Drosophila  that  and  by  major  of  than  is  presently similar,  photographs  1972).  adult  side  6b,  coli  first  on  8,  for  the  E.  7c).  As i n  used HCl-acetone  proteins  occur  pattern  and  the  contrast,  this  proteins  published  (Lambertsson,  Lambertsson  In  a different  electrophoretic  Drosophila to  are  in  7C).  are  E.  the  points  Several  however,  8).  of  to  proteins.  species produce  patterns.  (Fig.  for  run  to  (Fig.  published  isoelectric  samples,  gels  most  Drosoph11aproteins  6a  to  long  short  proteins  shown h e r e  photographs  a  a  ribosomal  2-dimensional  (1970b).  and (Fig.  ribosomal  pattern to  gels  coli  necessary  proteins,  spots  on the  coli  in  were  ribosomal  slow-moving  The m i g r a t i o n  (Fig.  two  Drosophila  Drosophila  Drosophila  different  least  of the  proteins  with  but  not  Drosophila present  preparations  melanogast er.  found  In  of  for identical, 2-dimensional  study, ribosomal  his  isoelectric  gel  procedure,  points  67  lower  t h a n pH  tration.  8.6.  because  t h e y o c c u r r e d i n low  Some d i f f e r e n c e s  i n Lambertsson's  p r e s e n t ones a r e a l s o a p p a r e n t g e l near  the o r i g i n .  on  whereas t h e p r e s e n t s t u d y s u g g e s t s number seems t o be  due  these are contaminants  mainly  is  found  52 b a s i c  58.  The  As  proteins  difference in Whether  i n the present  o f p u r i t y o f the ribosomes  used  by  study,  Lambertsson  unknown.  of ribosomal proteins.  and M g - a c e t i c HCl-acetone  acid preparations  and  Mg-acetic  preparations of Drosophila ribosomal proteins similar,  but not i d e n t i c a l  dimensional gels were n o t e d :  differences  procedures ments.  26,  5 and  i n the e x t r a c t i b i l i t y  10,  and be  i n t o dimers, r e s i d u e s by o f u r e a ; and  studies,  differences  6 and  17.  attributed  artifacts  t h e two  to two  treat-  c a n be c r e a t e d  bond f o r m a t i o n ; a g g r e g a t i o n o f monomers  trimers,  etc.; alteration  o f amino  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 so  2-  o f p r o t e i n s by t h e  o r t o a l t e r a t i o n s o f p r o t e i n s by  In p r o t e i n  produced  Three major  i n t h e g e l p a t t e r n s may  through d i s u l f i d e  about  and  acid  patterns of migration i n  ( F i g . 6a t o 7 c ) .  s p o t s 24  differences  an  the  or ribosomal proteins present i n  Comparison o f HCl-acetone  The  the  side of  t o m i n o r components.  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 . the degree  g e l s and  the c a t i o n i c  Lambertsson  concen-  on  (Nomura, 1 9 7 0 ) .  attempted.  product  A definite conclusion  t h e n a t u r e o f t h e d i f f e r e n c e s c a n n o t be  i n t e r p r e t c i t i o n c a n be  acid  reached  but  68  The pairs,  appearance  spots  each p a i r  one  coli  (Pig,,  type  or  a  one  has  24  and  closeness  chemical of  members The  also  the of  the  spot  acetone  26  was  gel,  clearly  suggested of  one  occur  between  an  inverse  in  the  24  of  me t h a t unique the  be  clearly  24  and  forms.  than  that Spots  but  it  is  the  previous  close of  E. extent  et  (Funatsu,  column  a l . , et  chromato-  study,  the  me  that  related. of  a  pair  related.  large  6a).  and In  dark  the  and  HCl-  spot  26  relationship  may  represent  alternate  c o n v e r s i o n may  5 and  less  1973).  (Kahan,  members  a  wild  a l . ,  suggested to  was  26  one  this  et  present  This  and  gels  chain  diminished  7a).  to  about  by  were  (Fig.  because  very  chemically  24  that  one  present  Bollen,  between  spot  protein  two  of  the  members  (Fig.  i.e.  difference  In  might  spots  suggests  also  polypeptide  visible  relationship  photographs  1970;  acid  two  compared w i t h  each p a i r  two  was  are  are  the  adjacent  demonstrated  gel,  barely  increased  forms  the  been  acid  spot  to  a l . ,  relationship  Mg-acetic  10  proteins  of  the  are  of  2-dimensional  analysis.  suggested that  10,  protein,  26  5 and  amino  each p a i r  inverse  and  In  et  members  5 and  displacements  mutant  later  members  2 s p o t s may b e  6a).  single  of  unique  that  (Deusser, a  spots  spots  shortening  1972)  graphy  was  (Fig.  such cases,  al.,  and  and  and  when  proteins  1973)  In  and  Spots  7a)  another  obtained  the  26  ribosomal proteins,  are  In  chain,  a conversion.  another to  and  locations  may r e p r e s e n t  polypeptide of  24  and  10  clearly  example.  also  exhibited  demonstrated In  the  69  HCl-acetone  gel,  spot  10  barely  acid  gel,  size  (Fig.  was  spots  two  also  pair  represent  ness  and  inverse  case  the  differences  the  is  well  protein  For  26  is  and  that  Similar The  of  artifacts ography  of  the  by  protein  by  c a n be  and  reasonable  26  In  spot  17  stained  acid  method  prominent  of  and  in  could  In  be  be  seems more but  the  protein spot  spots  17  (Fig.  by  gel, 7a)  faint.  was  this in  extraction protein  24  and  HCl-acetone would  by  be  decreased increased.  spots  5 and to  10.  me  neither  theory  later,  column  chromat-  gel  electrophoresis.  may  be  17 spot  but  a  differences  contrived  6 and  extraction was  In  that  for  however,  HCl-acetone  strongly  to  obviously  than  a  close-  procedure  24  readily  in  of  their  As d i s c u s s e d  more  differences,  equal  correspondingly  observations.  for  the  offered  analyses  Mg-acetic  members  two  acid  and  protein.  that  the  protein  hypothesis,  detected  two  and  dark  represent  unique  assume  Mg-acetic  would  the  fortuitous.  could  of  also  the  and  about  attributed  soluble.  explantion  formation.  one  the  chemical  Solubility  may  are  be  In  and  one  members  hypothesis  the  10  proteins  solubility  artifact  proved  small  of  large  7a).  assume t h a t  would  explanations  the  forms  unique  barely  solubility  than  were 5 and  example,  the  (Fig.  relationship  extracted  procedure,  is  two  solubilities  systems.  10  Spots  can  relatively  visible  between  one  5 was  5 and  6a).  conversion But  spot  than  more artifact  6 was  when used,  Again  a  an  the spot  absent  and  Mg-acetic 6  inverse  was  70  relationship protein are  to  is  two  located  forms  in  Displacements  evident  and  c o u l d be  relatively of  this  extent  proteins  of  a  chemical  change  likely two  explanation  different  polypeptide  migration  properties  plausible  theory.  by  gel  major  solubilized for  the  to  7c).  of  the  by  The  two  from  the  rat  liver of  the  same  and k i n d s  Mg-acetic gel  acid  gels,  electrophoresis  gels. mutant that  seems  an  presence different  seems  to  be  cannot  apparent  but  of  the  a  be  un-  of  more proved  removes  reveal  in  (Fig.  about  method  the  6a  80% et  a l . ,  was  suggests  from  illustrate  patterns  (Welfle,  methods  proteins  gels  similar  similarity  differences  in  overall  remarkably  acid  however, can  spots  quite  HCl-acetone  The  and M g - a c e t i c  three  two  assumes  The  ribosomes  the  HCl-acetone  The  one  17.  a c i d method  the  ribosomes.  the  conversion  with  were  were  literature.  number  the  observed with  hypothesis  methods  spots  No e v a l u a t i o n in  chains  unique  alone.  Mg-acetic  protein  6 and  the  of  Unless  occurred,  differences  of  not  solubilities  Again  the  majority  1971). found  and  electrophoresis  Three  were  spots  But  areas  bacteria.  for  one  invoked.  distant  ribosomal drastic  conversion of  extract  that about  Drosophila HCl-acetone  how  and  2-dimensional  differences  in  protein  proteins.  Proteins  samples.  Number  of  Drosophila  remained  at  the  directly  toward  ribosomal  origin the  in  the  cathode  in  first the  dimension  and  that migrated  second dimension,  have  71  g e n e r a l l y n o t been r e p r e s e n t e d i n t h e s c h e m a t i c e x c e p t f o r s p o t s 4, 9 a n d 65 ( F i g . 5 ) . be due t o p r o t e i n s w i t h i s o e l e c t r i c and  hence f a i l t o m i g r a t e  p r o t e i n may e x h i b i t dimension exhibit  Or a  a t pH 8.6 i n t h e f i r s t I t may  then  a t pH 4.3 i n t h e s e c o n d  a n d move away f r o m t h e o r i g i n .  interpretation especially  dimension.  a n d n o t move f r o m t h e o r i g i n .  increased s o l u b i l i t y  dimension  Such s p o t s c o u l d  p o i n t s e q u a l t o 8.6  i n the f i r s t  low s o l u b i l i t y  diagram,  The l a t t e r  seems more a p p r o p i a t e t h a n t h e f o r m e r ,  f o r s p o t s 4 and 9.  They a r e c o n n e c t e d  t o spots  5 a n d 10 by s t r e a k s w h i c h a r e i n d i c a t i v e o f i n s o l u b i l i t y . This  type o f a r t i f a c t  2-dimensional Spot  g e l electrophoresis  65 a t t h e t i p o f t h e c e n t r a l  unique as  has been o b s e r v e d p r e v i o u s l y i n ( S h e r t o n and Wool,  1972).  a x i s , however, may b e a  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  i t i s n o t r e p r e s e n t e d by a c o r r e s p o n d i n g s p o t t o one  side of the central axis  i n t h e second  dimension  (Fig. 9  proteins  present  and 1 0 ) . In in  e s t i m a t i n g t h e number o f u n i q u e  the Drosophila ribosomal preparations, the previously  discussed p a i r s o f spots  (24 and 26, 5 a n d 10, 6 and 17)  c a n be r e g a r d e d a s 6 u n i q u e and  3 artifacts.  been c o u n t e d (Fig.  gels of r a t l i v e r  proteins  T h e t h r e e s p o t s 52, 55 a n d 59 h a v e  a s one p r o t e i n b e c a u s e  5 and 7 b ) .  spot a f t e r  p r o t e i n s o r 3 unique  A series  t h e y may be  arrangement l i k e t h i s  artifacts i n the  r i b o s o m a l p r o t e i n s was r e p l a c e d b y one  a. change i n p r e t r e a t m e n t o f t h e r i b o s o m e s  72  (Sherton of  and W o o l ,  Drosophila  major  and  greater close  16  69  -  72  8.6,  8.6,,  isoelectric  and  protein  of  from males  and  and  another males of  and  studies gel  of  with  an  possible less  to  Somero,  also  within  the  differences  in  phoresis  is  not  Staining  conditions  amount  of  The alike  the  suggests  45  points  proteins  point  with  5).  A total  ribosomal  patterns  quite  mixed  of  similar  samples  patterns (Kiefer  from  by  Oregon-R  the  degree  1-dimensional  males  although and  one  Previous  proteins that  Oregon-R to  in  observed.  indicated  proteins  for  however,  differences  procedure  samples.  intended  residue fact  -  and  females  quantitative  Gross,  1969;  M.  Gould  communication).  Such q u a n t i t a t i v e variation  were  ribosomal  observed  personal  of  gel  s p o t s were  42  present.  gels  for  preparations  isoelectric  (Fig.  Differences,  banding  were  8.6  The  those  some  similar  minor  v/ere  females  Drosophila  differences  10  than  females.  electrophoresis  produced  were  1 protein  2-dimensional  of  there  isoelectric  females.  staining  proteins,  components  also  HCl-acetone  with  C(I)RM/Y  and  the  proteins  points  Comparison  males  In  ribosomal minor  than  to  1972).  are  at  that that  not  the  the the  to  are  and  probably  do n o t  origin  patterns  gel  a quantitative  necessarily or for  isolation  males  method  the and  real  electro-  method.  optimal  along  to  represent  2-dimensional be  due  and  axes  the  varies.  females  produces  were  ribosomes  73  that  are  reasonably  differences  free  of  extraneous  can  create  problems  Egg p r o t e i n s  in  female  crickets,  to  the  adsorb to  customary freed  of  method  used  2-dimensional  spots  or  mutants. isolated  mutants,  (58,  52,  were  and 55,  and  the  probably  the  be  lost  Though the  in  the  and  at  found  isolation  females by  the  In cs  the cs  , 1 5  ribosomal  sample,  least  by  were same  two  cs  , 1 6  cs  ,26  proteins  from  one  preparation  gels  were  2-dimensional  gels  and  27  In  the  the  staining  done  of  cs  detected.  due  to  gels  absent of  mutants the  which  are  (Fig. mixed  in  protein  (Fig.  in  low  Or  the  ,  the no  gels  new  of  sample  concentration  the  but  alone,  C(1)RM  females,  9 and  12). spots  methods. but  not may  or  gel  s a m p l e was of  11)  Oregon-R  concentrations  of  and  spots  of  they  extraction  amount  10  staining  frequently  ribosomes.  constant  the  faintly  variability  occurring  effective  were  faintly  Oregon-R males  absence of  during a  59)  other  isolated  proteins  each  some o f  females,  contaminants in  5  27  and  three  The  were  c s  present  males and  5  When  of  For  d i s p l a c e d ones were cs  mutants  their  isolated  electrophoresis  each p r e p a r a t i o n .  Drosophila  ribosomes.  example,  1970).  Sex  males.  r i b o s o m e s was  for  (Kavlenas,  gel  cold-sensitive of  for  r i b o s o m e s c o u l d be  for  isolating  r i b o s o m e s and p r e v e n t  methods eggs,  in  proteins.  is T h e y may  always be  be  present  ribosomal  a n d may  sometimes  electrophoresis.  applied  various  to  the  proteins  gels,  which  74  migrate origin  onto or  a gel  along  may v a r y  the  due  to  precipitation  at  the  axes.  s. An a l t e r e d mutant  is  ribosomal protein  detected  by  position  found  gels  amount  single  of  However, one had  E.  coli  1972).  This  spot. were  gels  not  from  this  in  solubility  phoresis.  is  the  Protein  group,  tryptic  (Funatsu,, mutant type  digest SLL. ,  protein  the and  shorter  observed.  streptomycin very  loss  found  by  dependence  faintly  in  Funatsu, darkly  et  was  during was  to  be  2a l . ,  staining  spots but  attributed  mutant  It  normal  decreased staining  fingerprints,  1972).  was  the  A decreased  not  1970;  was  and  coli  to  a  gel  later  of  change  electro-  isolated  altered  in  and m o l e c u l a r concluded that  9% c o m p a r e d t o  they  the  end  weight the wild-  protein,, It  is  observed above  et  The  protein  column chromatography  from  produces a  mutant  S4 o f  E.  h a d d i s p l a c e d S4  amount.  particular of  a l . ,  normally  "revertants" in  from  stained  et  an  type.  wild  generally  spot which  (Deusser,  reduced  S4  the  "revertant"  protein  Other  displacement  of  protein  a d i s p l a c e d S4  dimensional  by  in  its  from  unlikely  in  the  example  decreases protein  in  would  structural  that  gels  for  the  of  gene  5  and  noted.  expected of  quantitative cs  following  s p o t s were be  the cs  to  27  are  reasons: A single  result  from  a ribosomal protein.  differences  similar (1)  to  Several  change  in  a mutation (2)  the  a in  Though  the the  75  pattern  of  constant, (3)  The  protein the  migration  degree  degree  of  of  in  the  staining  purity  of  the  of  gels  was  found  s p o t s was  ribosomal  uncertain..  (4)  Though mutants  5  abnormal  abdomen p h e n o t y p e s ,  they  cally.  T h e m u t a t i o n s map t o  different  chromosome look  like  and wild  the  trans  type.  are  heterozygotes  be  variable.  preparations  cs is  to  c s and  not  27  have  related  areas of cs of  5  geneti-  the  and  27  X cs  76  DISCUSSION  Chemical the  analysis  chemical  An a c c u r a t e necessary of  the  basis  preliminary  that  (Wittmann,  as  28  et  large or  1970;  as  the  The main distinguish  as  protein  c o u l d be  (Mora,  in  The  of  E.  protein  removed  of  coli  from  been  ribosomal  a n d may  be  proteins as  low  Dzionora,  has  been  contaminants  and  ammonium  that  et  isolating  for  the  proteins  precipitation  biologically  were  has  et  a l . ,  much  gel  a l . ,  active  ribosomes,  chloride  impairing  1966:  Nomura,  proteins  analyzing  in  the  each  ribosomal  electrophoresis  example,  present  and  extraneous  unique and  to  of  ribosomes without  Two-dimensional  components  it  1971;  distinguishing  ribosomes,  On  20-21  of  studies  (Kurland,  s o l v e d by  purified,  a l . ,  found  from  activity  was  preparations.  such  was  component  1970b).  chromatography  problem  artifacts  et  a  column  studies,  contains  into  1972).  is  synthesis.  number  sulfate  it  how e a c h  uncertain  proteins  ribosomes,  biological  component  21  34  B y ammonium  of  purified  still  difficulty  washing  from  is  The  and Wittmann,  diethylaminoethyl  1970).  subunit  gone  (Kurland,  electrophoresis,  1971).  ribosomal  artifacts.  gel  protein  reconstitution  subunit high  in  has  constituents  understanding  small  a l . ,  Kaltschmidt  their  to  ribosomes  ribosomal  functions  and  Much e f f o r t  E.~ c o l i  of  2-dimensional  concluded  the  of  accounting  chromatography,  in  ribosomes.  analysis  ribosomes of  of  indicated small  of that  subunit  77  and  34 i n the l a r g e subunit  1970).  (Kaltschmidt and Wittmann,  As some o f the p r o t e i n components were f a i n t l y  s t a i n i n g they c o u l d be ribosomal p r o t e i n s present i n fewer c o p i e s than the major components o r a r t i f a c t s c r e a t e d by secondary m o d i f i c a t i o n o f p r o t e i n s .  Minor p r o t e i n  f r a c t i o n s were a l s o obtained when ribosomal p r o t e i n s were separated by  column chromatography.  The advantage o f  t h i s method, however, i s t h a t the f r a c t i o n s can be analyzed. End group a n a l y s i s , 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 , molecular weight d e t e r m i n a t i o n s and immunological  t e s t s have  shown t h a t many p r o t e i n components o f 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 t h e r e f o r e are unique p r o t e i n s (Mora, e t a l . ,  1971; S t o f f l e r and Wittmann, 1971).  On  the o t h e r hand, some p r o t e i n components a r e probably a r t i f a c t s because t h e i r t r y p t i c f i n g e r p r i n t s e t c . a r e s i m i l a r t o those o f o t h e r f r a c t i o n s  (Mora, e t aJL., 1971).  As a l r e a d y i n d i c a t e d , ribosomal mutants and the i n v i t r o r e c o n s t i t u t i o n o f subunits are a l s o important methods o f e s t a b l i s h i n g the i d e n t i t y o f ribosomal p r o t e i n s . The a n a l y t i c a l s t u d i e s have i n d i c a t e d t h a t i s o l a t e d ribosomes o f E. c o l i a r e heterogeneous  (Kurland, 1972).  Twelve o f the p r o t e i n s o f the 30S s u b u n i t s , f o r example, are p r e s e n t i n approximately one copy per ribosome w h i l e e i g h t " f r a c t i o n a l " p r o t e i n s are present i n 0.5 o r fewer c o p i e s per ribosome  (Voynow and Kurland, 1971).  Obviously  i s o l a t e d ribosomes cannot be uniform i n composition.  78  One p r o t e i n appears  to  (Moller, number  be  et  of  of  the  present  a l . ,  One occiir  is  not  that  heterogeneity  that  the  of  of  isoelectric present proteins  of  as  lightly  staining  may  distinguish  low  fractional between  discussed.  The  extraneous  proteins  ribosomes  suitability  of  established  for  have  been  artifact  shuttle  and  theory  is states  synthesis  ribosomal to  proteins, and  propagation,  than  pH  the  until  this  on the  two  while  fro  and  among  termina-  is  coli  Drosophila.  Though  proteins.  the of  the  the  gel,  help  Drosophila previously  procedures  established. procedures  the  To  methods  the  be  Like  of  the  these  proved. side  with  in  should not  possibilities, by  proteins  observed  they  maintaining  s h o u l d be  10  were  cationic  ribosomal  purified  E.  8.6  ribosomes.  effectiveness  many  About  concentration,  spots  s h o u l d be  the  A  functional  protein  ribosomes.  lower  ribosomes  of  may  initiation,  contaminants  be  an  interesting  during  Drosophila  in  ignored  they  ribosome  1972).  is  different  it  synthesis.  points  occur  per  that  heterogeneity  A more  factors,  in  a l . ,  "fractional"  Drosophila  study  et  heterogeneity  undergoes The  unusual  3 copies  the  represents  during  protein  of  vivo.  supernatant  Proteins  2 or  is  Terhorst/  that  1972).  ribosomes  tion  in  ribosome  (Kurland,  the  in  1972;  does  the  subunit  interpretations  advanced.  like  50S  in  biological  removing activity  Unfortunately has  not  been  the  79  Separation remove  of  extraneous  purified  rat  ribosomal proteins.  liver  "80S"  maximum o f  6 proteins  the  When  gel.  the  of  the  anionic  As  the  procedure  normally are  by  E.  o c c u r more  eukaryotic  2-dimensional  gels  of  for  in  the  functioned  still  proteins  proteins.  o c c u r on the  is  thought  that  frequently  in  prokaryotic  study  indicated  developed  are et  evident  a l . ,  at  is  acidic  (Kiefer  1970:  that  M.  69  ribosomal  anionic  side  proteins  ribosomes  resolved  than  discussion that  ribonucleoprotein tested  c a n make  2-dimensional  The  the  than  in  and G r o s s ,  it  number  of  biological  reasonable gels,  gels,  is  unknown  cannot  be  a  is  greater  proved  from  still  the  only  produces  purity  assumptions about  these  electro-  obvious  activity.  22-30  personal  to  procedure  one-  969;  gel  69-72  of In  pH v a l u e s ,  Gould Somero,  isolation  particles  for  72.  Drosophila proteins  one-dimensional  approximation.  number  to  the  one  11  these  the  phoresis  not  remaining  Since  Though t w o - d i m e n s i o n a l  were  3  that  communication).  an  examined,  of  absent.  it  gels  bands  previous  side  s p o t s were  3 anionic  subunits  a  anionic  s u b u n i t s were  Drosophila ribosomes  Lambertsson,  extent  example,  ones.  dimensional protein  to  no o t h e r  ribosomal  gels,  The p r e s e n t proteins  but  only  may  o b s e r v e d on t h e  ribosomal  coli  2-dimensional  may  help  produced subunits  vitro,  from  In  also  ribosomes,  was  spots,  definition,  proteins of  in  subunits  and  these  Moreover, artifacts except  by  though in  80  chemical  ana3.ysis.  Despite found  that  some v a r i a t i o n  "80S"  proteins  than  tions  of  rat  68-72  protein  and W o o l , method present  may  69-72  reasonable the  been of  the  pairs for  from  (Sherton  the  separated  only  Examination  of  Drosophila. phoresis most  is  ribosomal  The method ribosomal mutants.  of  in  should detect proteins However,  if  other  five  from not  gels  82S et  (Sherton  from  due  by  1970). proteins  incomplete  "80S"  separated  were  until  for  a  E.  coli  proteins  was  examined.  2-dimensional small  one,  sample  relatively  changes occur  in in  sex-linked,  the  were  c o l d - s e n s i t i v e mutants  of  E.  ribosomes  dimension  of  the  Drosophila  to  used  this  Hence  a l . ,  the  first  subunits  these  contained  eukaryotes  for  One p a i r  in  prepara-  proteins.  conditions  requires  proteins  more  system designed f o r  the  advantage it  a  were  1972). the  generally  Drosophila ribosomal  proteins  ribosomes The  for  underestimated  standard  when  that  of  a n d pH i n  and W o o l ,  example,  proteins  example,  concentration  for  ribosomal  proteins  is  Purified  (Lambertsson,  number  slightly  liver,  changed  76  it  2-dimensional  Estimates  of  is  ribosomes.  in  estimate  estimates,  ribosomes contain  ribosomes,  components  Three  rat  coli  to  have  gel  liver  fact,  coli.  E.  70  resolution  of  70S  from  ribosomes In  eukaryotic  1972).  vary  in  the the  gel and  electroresolves  quick,  charge  of  or  procedure. size  of  cold-sensitive  cold-sensitive  mutants  81  e x a m i n e d by  the  method, e x h i b i t e d  no  displaced  Quantitative  d i f f e r e n c e s observed  5  were a t t r i b u t e d t o v a r i a b i l i t y  C S  and  I t was  27  c s  concluded that  there  was  i n gels of  no  proteins.  mutants of the  detectable  method.  change i n cs  adult ribosomal proteins cs cs 26 and 27 .  c a u s e d by  mutations  5  cs , 15  cs , 16  ,  c  cs As  a precaution,  retested, preferably sedimentation of temperature.  mutants 5 by  an  be  like  I f the  should  q u a n t i t a t i v e changes a r e of  the  be as  low  due  to  electrophoresis  sedimentation p r o f i l e s  that of wild-type  f o r the  ribosomal  mutants  should  particles.  f r o m numerous c o l d - s e n s i t i v e  m u t a n t s were e x a m i n e d by  1-dimensional g e l  G o u l d Somero, p e r s o n a l  8 other  c s  ribosomal p a r t i c l e s produced at  Ribosomal p r o t e i n s  (M.  27  i n d e p e n d e n t method s u c h  contaminants or v a r i a b i l i t y method, t h e  and  electrophoresis  communication).  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  These the  included  X  chromosomes; 25  dominant, c o l d - s e n s i t i v e l e t h a l s o f  chromosomes; 11  dominant, c o l d - s e n s i t i v e d e v e l o p m e n t a l l y  retarded  mutants o f  cold-sensitive retarded from 5 t o  the  l e t h a l s of  mutations are 10  2nd  days a t  the  not  17°  chromosomes; and  C and  dominant, r e t a r d e d  viable  at  22°  noted i n the  C.  1-3  prolong  days a t 22°  A l t h o u g h i r r e g u l a r i t i e s were  gels of  some o f  The  the  dominant  development C  Heterozygous females of  m u t a n t s were s t e r i l e  2nd  recessive,  chromosomes.  l e t h a l s but  (Rosenbluth, e t a l . , 1972). tested  3rd  3  the  or  the  poorly  sometimes  m u t a n t s , none w e r e  82  reproducible It  was  the  ribosomal  from (M.  a  Gould Somero,  concluded that  The  5  (M.  was  from  the  proteins  of  the  recessive,  evidence  personal  examined  of  males  Drosophila in  were  fed  lethals  of  food.  ribosomes at  apparent  the  difference  ribosomes  from  Limitations  of  cold-sensitive  mutants  the  study  mutants  obtained.  On t h e  acid  C  sequence  in  of  alterations to  streptomycin problem  the  in  H  the  There  hand,  charge  detect  in  or  to  et  the  not  the  negative  no  profiles  of  Do  some  ribosomal  hypothesis  results  was  are  reasons.  protein be  would  mutants a l . ,  label  was  altered  support  1-dimensional  independence (Deusser,  size  sedimented  wild-type.  following  may  with  some r e c o m m e n d a t i o n s .  evidence  protein  were  D r o s o p h i l a have  a ribosomal  uridine  sedimentation  and  and  electrophoresis.  incorporated  A c o l d - s e n s i t i v e m u t a n t may  properties  the  of  other  conclusive for  difficult  17  flies  the  included  3  temperature.  the  No d i r e c t  (1)  low  the  between  proteins?  not  All  studied  X chromosomes  gel  Ribosomal preparations  sucrose gradients.  into  at  also These  the  1-dimensional  overnight  in  particles  were  communication).  by  change  ribosomal  o Adult  of  mutants.  preperties  cold-sensitive  had been  no  communication).  c o l d - s e n s i t i v e mutants  Gould Somero,  which  there  sedimentation  few  personal  of  1970).  have but  an  the  altered  electrophoretic  changed. be  Slight  particularly  gels. E.  amino  A study  coli Out  of  of  illustrates 13  mutants,  83  4  exhibited  Three able  other  changes  in  mutants  had  from w i l d - t y p e  protein S4  in  sensitive  ribosomal mutants  (2) the  A  dominant,  examining gotes even the  of at  the  gous  the  Altered  whether  to  the  or  low  properties  probably gel  allelic  be  critical. of  necessity  of  Homozy-  the  c h a n g e may  from in  the  tests. would  be  in  ribosomal  in  electrophoresis.  Both  ribosomal  of  an  allele.  may  in  were  electro-  protein,  E.  be  sedi-  subunits  the  the  been  uncertain  heterozygotes  proteins  have  wild-type  and  had  production  detected  ribosomes  a major  not  the  I -am  of  heterozy-  heterozygotes  A change  detectable  inviable  obtained  of  because of  protein  normal  be  are  c o l d - s e n s i t i v e mutant  gels  same t i m e . of  low  testing  condition.  fertility  the  subparticles  phoretic  the  could not  other  at  altered  mutants  detect  if  be  biochemical was  in  And homozygotes  proteins  profiles  dimensional  method would  the  ribosomal in  cold-  temperature.  protein,  ribosomal  produced  would  in  clearly  lethals  1-dimensional  abnormal  mentation  the  a dominant,  ribosomal  If  were  cold-sensitive  sterility If  but  gels.  indistinguish-  Drosophila occur  heterozygous  retarded  normal  difficult  the  permissive  in  of  problem  dominant,  females.  detected of  in  the  altered  of  were  gels  c o l d - s e n s i t i v e mutants  dominant, of  which  properties.  limitations  them  the  because  an  immunological  serious  2-dimensional  2-dimensional  in  the  in  proteins  different  frequency,  S4  by  however, 2-  normal coli  and  mutant,  84  for of  example,  were  ribosomal  (Bollen,  et  (3)  distinguishable  proteins a l . ,  from  the  limitation  sensitive  mutants  is  ribosomal  proteins  then  somes w o u l d There  in  have  ment.  Numerous  and  adults  also new et  t-RNAs  been  of  a l . ,  factors (Ilan  and Two  The  stages,  as  dimensional  Some to  another  that  the  cold-  examined.  defects  in  larval  If  stages  or  pupal  ribo-  overlooked. that  some c o m p o n e n t s  changes in  (White,  pupae  of  and  new  a  during  1st et  of  insect  the  develop-  in  the  isoaccepting  and  3rd  instar  a l . ,  Tenebrio  1973).  moletor  synthetase  stage-specific  protein  synthesis  were  found  in  larvae  It  has  produce  (Ilan,  addition,  a  1969;  Ilan,  initiation this  insect  1971). of  of  experiments  Drosophila  ribosomal well gel  as  ribosomes from  adults,  electrophoresis  increased while  suggest  proteins  from  differences  spots  were  In  types  ment.  adults  of  with  observed  t-RNA  Ilan,  constituents  Numerous  were  testing  developmental  quantitative  1970).  the  system change  shown t h a t  for  gels  merodiploid  various  Drosophila  species of  in  only  good e v i d e n c e  synthesizing  of  heterozygous  the  been  protein  forms  that  mutants  is  2-dimensional  1973).  Another  differ,  in  2  compared  (Lambertsson,  protein  in  staining  intensity  faded.  The  may  be  protein  during  larval  the  proteins  the  change  in  others  some r i b o s o m a l  were  that  patterns  and  2  by  pupal  2-  1972). were  from  apparent.  one  observations stage  develop-  stage  suggest  specific.  The  85  evidence  i s n o t c o n c l u s i v e , however, b e c a u s e t h e  d i f f e r e n c e s may protein  be  due  synthesis,  initators  to  (1) c o n t a m i n a n t s u n r e l a t e d  (2) s u p e r n a t a n t  p r o t e i n s p r o d u c e d by developmental  (3)  stages.  The  introduction.  scarlet and  such  as  eye  changes i n  other experiments adults d i f f e r ,  Larvae,  but  not  And  different  suggesting  were d i s c u s s e d  adults, of  ribosomes from  l a r v a e bound more s t r e p t o m y c i n  streptomycin  resistant  larvae  to  ribosomal  the  s t o c k were s u s c e p t i b l e t o s t r e p t o m y c i n  Glassman, 1968).  sensitive  secondary  p r o t e o l y t i c enzymes i n t h e  t h a t r i b o s o m e s o f l a r v a e and the  factors  to  o f p r o t e i n s y n t h e s i s w h i c h become a t t a c h e d  r i b o s o m e s m o m e n t a r i l y and  in  observed  (Duke  streptomycin than  those  ( L a m b e r t s s o n and  from  Rasmuson,  1971). (4) still  An  obvious  remaining  cold-sensitive recessive, (Tasaka (5) t h a t may present  to  Sad  t o be  and  lethals  Suzuki,  are the c o l d - s e n s i t i v e  tested. on  cold-sensitive  t h e X chromosomes and lethals  designed  chromosomes  cold-sensitive  on  the b a s i s of the ribosomal  are cold^-sensitive  the normal a l l e l e .  temperature  3rd  By  observing  i n Drosophila with  t h e X chromosome has  mutants  i n t h e X chromosome s c r e e n .  of the b a c t e r i a l  mutations  of the  29  1973).  have b e e n m i s s e d s c r e e n was  mutants  T h e s e i n c l u d e 14 r e c e s s i v e ,  More w o r r i s o m e a r e t h e  characteristics the  omission  genetic  mutants, i . e .  lethals the  The  and  dominant  effects of  t h e g e n o t y p e X/Y,  low  where  b e e n e x p o s e d t o mutagen, t h e f o l l o w i n g  86  t y p e s o f c o l d - s e n s i t i v e m u t a n t s were e x p e c t e d : Dominant both  conditional  lethals  i n genes t h a t  t h e X a n d Y chromosomes.  lethals (c)  (a)  are present  (b) Dominant  on  conditional  i n g e n e s p r e s e n t on t h e X chromosome o n l y ,  Recessive conditional  lethals  i n g e n e s p r e s e n t o n l y on  t h e X chromosome and h e n c e r e v e a l e d i n t h e h e m i z y g o u s condition.  As w e l l  as p r o v i d i n g f o r t h r e e t y p e s o f p o s s i b l e  m u t a n t s , t h e s c r e e n i n v o l v e d l e s s work t h a n i n t r o d u c e t h e m u t a g e n i z e d chromosome i n t o  schemes  which  heterozygous  females. In f a c t ,  t h e s c r e e n gave o n l y  mutants, i . e . r e c e s s i v e l e t h a l s gous c o n d i t i o n . heat-  I t i s now  (c) t y p e  d e t e c t a b l e i n the hemizy-  known t h a t  and c o l d - s e n s i t i v e m u t a t i o n s  almost  Holden may  and S u z u k i , 1 9 7 3 ) .  select  mutations  Rosenbluth,  because they  occur  o f chromosomes were  (a) and  (b) may  infrequently  A l s o , dominant  and i n s u f f i c i e n t  numbers  i n g e n e s p r e s e n t on b o t h  i . e . recessive  t h e X and Y, c o u l d n o t  S u c h m u t a n t s may  be  the c h a r a c t e r i s t i c s of the ribosomal p r o t e i n  like  isolated  tested.  be d e t e c t e d by o u r s c r e e n . if  e t a l . , 1972;  n o t have b e e n  A f o u r t h t y p e o f p o s s i b l e mutant, lethals  temperature  Thus s c r e e n i n g v i a h e m i z y g o s i t y  a g a i n s t dominant m u t a t i o n s . of types  a l l dominant  of Drosophila are  l e t h a l when homozygous a t t h e p e r m i s s i v e ( S u z u k i and P r o c u n i e r , 1969;  cold-sensitive  t h o s e o f t h e r i b o s o m a l RNA  genes.  The  important genes a r e  bobbed  87  mutations, which redundant and map  t o be d e l e t i o n s  r e g i o n s c o d i n g f o r r i b o s o m a l RNA,  on t h e X and Y chromosomes  Lindsley somal  a r e thought  and G r e l l ,  1967).  i n the are recessive  (Ritossa,  et a l . ,  A l t h o u g h t h e genes f o r r i b o -  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 ,  situation  i s not necessarily  and Nomura, 1 9 7 2 ) . r e p e t i t i v e DNA Furthermore  true  E u k a r y o t e s , f o r e x a m p l e , h a v e more  than b a c t e r i a  a recent  ( B r i t t e n and Kohn,  study o f ribosomes  genes f o r r i b o s o m a l p r o t e i n s  It  (Steffensen, may,  this  f o r D r o s o p h i l a (Davies  1968).  f r o m D. m e l a n o g a s t e r «•>  D. s i m u l a p s h y b r i d s by 1 - d i m e n s i o n a l g e l s  n e a r bb  1966;  are located  suggests on t h e X  that chromosome  1973).  therefore,  be w o r t h w h i l e  to rescreen f o r cold-  s e n s i t i v e m u t a t i o n s o n t h e X chromosomes by a n o t h e r tion  scheme.  A l l f o u r t y p e s o f m u t a n t s s h o u l d be  able i n a t r a d i t i o n a l chromosome then t e s t e d dition  into  a heterozygous  f e m a l e and  1967; T a s a k a  and S u z u k i ,  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  I t i s possible  ribosomal p a r t i c l e s  that  of ribosomal  Drosophila could  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  from normal it  was  do n o t g r e a t l y  subunits are d i f f i c u l t  successfully  was  the sedimentation of  from c o l d - t r e a t e d  Although s u b p a r t i c l e s which  con-  1973).  B i o c h e m i c a l e x a m i n a t i o n o f t h e mutants  proteins.  X  i n b o t h t h e homozygous and h e t e r o z y g o u s  (Suzuki, e t a l . ,  (6)  obtain-  s c r e e n where t h e m u t a g e n i z e d  i s introduced  selec-  cannot.  differ  i n size  t o d e t e c t by t h i s  used t o i d e n t i f y  several  method,  Sad m u t a n t s o f  88  bacteria  (Tai,  None to  the  1972, of  of  on  of  sensitive The  the or  if  during even  after  the  when  needed  lethal  the  for  a mutant  the  and  is  to  most  phase  at  and  permissive period  means  occur  The in  of  of  stage  comparing  cold-sensitive  develop-  to  at  which  the  the  restrictive  is  considered  produced,  to  be  activated  process.  non-permissive  temperature  period,  later  it  will  temperature.  when  a defect  lethal  of  mutants  in  is  mutants  subunit  the  phase would  ribosome  die  The  cold-sensitive  cold-sensitive  that  in  determined.  heat-sensitive  hypothesis  effect  temperature-  stage  is  the  and  a l . ,  larvae,  the  and  Establishment  period  studied  and of  et  the  studied  developmental  the  susceptible  temperature-sensitive  product  evident.  previously  Conclusion. should  kept  developmental  is  phase  The  critical  the  study  exposure  temperature-sensitive  a useful the  gene  a  temperature-sensitive be  1970).  Hence  pupae  developmental  be  (Rosenbluth,  s h o u l d be  continuous  (Suzuki,  to  lethal  the  shifted  assembly  The  to  readily  preliminary  is  temperature-sensitive may b e  approach  phase  dies  seemed  adults  c a n be  each mutant  the  if  as  of  interval  Hence  this  require  precedes  first  cold  1970).  communication).  mutants.  temperature period  mutants  assembly  apply  Nomura,  period  lethal  organism  of  personal  would the  1969;  Drosophila  subunit To  however,  a l . ,  effect  Tasaka,,  adults.  ment  the  lethal  cold  et  also  mutants  mutants.  assembly based on  defects chemical  89  principles. of  other  It  organisms  considerable Drosophila, found. the  should,  work  as w e l l was  done  no e v i d e n c e  Because of  theory  has  therefore,  s t i l l  the  for  to  of  coli:  as  those  in  testing  or  greater  been  apply  against  s  E. the the  complexity  incompletely  the  theory  ribosomes Although on  hypothesis of  tested.  was  Drosophila,  90  BIBLIOGRAPHY  A p i r i o n , D. a n d phenotypic mutants.,  S c h l e s s i n g e r , D. 1967. 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XXY x X/Y i n b o t t l e s no. treatment:, Oo  u  /0 =•" r r v •+• 463  ~ 1.01 °/g  t r e a t e d w i t h 0.005 H EMS,  - •—•  =0.64  To c a l c u l a t e t h e f r e q u e n c y o f s i n g l e h i t s  P  X JlT  =  n  n  X  i  n.  where n - number o f h i t s and X - f r e q u e n c y o f h i t . When n = 0, i . e . no h i t , P  = .64 - X°'lT - ™ o i . X  Jl  X  X  =  . 64  =  .46 ,  Frequency o f s i n g l e h i t s p  i = - ^ 6 (.64) =  . 2904  L  -  .1. !  •  .  18 Frequency o f c s l e t h a l s -  Y~^97~  x  ~ .004436  where no, o f t e s t e d d'e^ = 2,597, no. o f c s l e t h a l s ='18, and f r e q u e n c y o f no h i t </<^ = .64 % single h i t sex-linked  l e t h a l s that are c o l d - s e n s i t i v e =  * ~ x 100 -  1.5% on t h e a v e r a g e . I n a s i n g l e e x p e r i m e n t , 9 c s l e t h a l s were found out o f 672 t e s t e d chromosomes.  The maximum % s i n g l e h i t s e x - l i n k e d  sensitive = - ~ — 6/Z  = 3%.  x .64 x ./Q  —  l e t h a l s t h a t were c o l d -  104  TABLE I The effect of temperature on the viabilities of mutant males, relative to the Oregon-R control, from the cross ,X (1) /Y x C(1)RM or C(1)DX£. CS  Stock*  22°C $ 9  OR ^cs  RM  Test  Viability (%)  Total Flies  1  100  1252  Viability (%) 100  Total Flies 1095  81.8 72 71.8 69.6  795 192 375 340  0 0 1 0.5  347 63 232 288  RA  1 2 3 1  cs  RM RA  1 1  42.0 34  249 187  0.5 0.9  163 86  3  RM  1 2 3 4 1  35 37.7 41 102 43  118 341 190 252 208  0 1 0.6 0 0.4  42 148 263 110 180  1 2 3 4 1  49.9 16 14 42 49.4  421 268 283 166 265  3.3 2 3 2 3  340 259 140 296 428  RM  1 2 3  78.1 87.6 89.1  275 303 277  6.1 6.2 11  267 253 352  RM  1 2 1  47 46 56.6  167 87 284  2 3 0.3  98 104 368  RM  1 2  61 60  171 106  0 11  225 121  RM  1 2 3  31 22 56.0  147 177 212  1 0 3  129 156 146  RM  1 2 3 4 1  61.2 78.1 70.9 58 60.6  305 261 286 196 366  2 8.6 3 23 3.3  322 117 263 177 388  1 2 3 4  50.1 28 48 36  253 134 183 116  0 0.3 0 0  319 266 51 76  2  CS  . cs  4  RA RM  RA 7  8  CS  cs  RA 9  cs  io  11  c s  CS  RA 1 2  17°C  cs  RM  105  Table I (cont'd)  13  CS  RM RA  1 1 2  66.5 34.1 5  449 336 68  0.2 1 0  362 300 62  14  CS  RM  1 2 3 4 5  33.3 36.7 45.0 45.4 31.1  259 299 244 292 489  7.3 1 9.3 12 2  226 225 146 245 292  16  CS  RM  1 2 3 1  92.0 47 10 68.6  307 135 135 225  0 0 1 0.5  226 38 366 160  1 2 3 1  45 57.3 62.4 87  74 266 308 140  0 2.4 0.8 9.4  83 322 99 163  RA 1 ?  cs  RM RA  18  CS  RM RA  1 1 2  76.6 79.6 76.6  951 333 958  12.5 12 15.1  746 330 794  19  CS  RM  1 2 3 1  52.6 52.8 80 85.4  222 355 94 351  0 4 15 18  103 143 122 . 340  1 2 1  47 71.4 67.5  131 637 1007  0 0 0  40 326 486  1 2 1  69 98.5 76.6  124 1167 1362  7 1 0.3  24 545 601  1 2 1  100 82.5 65.7  155 370 228  2 13 4.8  103 373 266  1 2 1  29 100 63.4  249 340 381  0 1 0.3  59 230 266  1 2 3 1  48.9 52.1 60.3 85.4  379 372 347 284  2 4.7 2.4 3  88 323 414 229  RA 20  CS  RM RA  21  CS  RM RA  22  CS  RM RA  23  CS  RM RA  24"  RM RA  1 21 261 0 63 2 75.2 12 393 496 1 RA 50.5 704 653 17.1 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 RA = C(1)DX, y f/Y females RM = C(1)RM/Y f email=s 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. 25  CS  RM  106  TABLE I I The effect of temperature on the frequencies of the abnormal abdomen henotype 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 ) c s / # c(l)RM/Y or C(l)DX/YO. .  J  Y  x  Stock  crV  22°C  99  1  OR RM  , - C S  RA , cs o  RM RA  1 5  cs  RM  RA  26  CS  RM RA  27  Test  C S  RM  RA  17°C  %V*  Total Flies  100  1252  0  -  % aa  1 2 3 1 2  66.8 67.4 55.5 64 67.4  249 273 310 133 458  1 2 3 1 2 3  54.7 70 69.3 72.5 83 78.8  357 159 341 291 147 540  1 2 3 4 1 2 3  70.9 52 51 75.9 65.0 58.8 60.9  339 210 121 270 225 251 457  1 2 1  73 95.6 78.8  170 629 521  29  1 2 3 4 1 2  83.9 75.2 81.0 72.2 81.8 84  308 209 430 350 529 159  28 12 44 6.8 42  49 40 33  56  -  65 28  77  -  37 37  -  %v**  Total Flies  100  1095  0  0 2 3.4 11 9.8  306 165 304 71 359  -  5.1 6.5 6.8 21 6 18  316 227 327 268 55 400  1 0.9 7.4 12 21 6 18  166 83 . 135 235 183 88 476  63 52.9 55.8  68 489 224  49 86  69 41.4 71.6 52 84.8 57  126 385 667 137 560 121  67 48 14 61 18 60  % aa  100 78 54  -  70 46 100 61  -  81 70 50 55  -  %V* = r e l a t i v e v i a b i l i t y at 22°C = (mutant //(C(1)RM/Y $ $) x 100/1.37 **= r e l a t i v e 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 t r a i t s %v  TABLE Male progeny at 22°C and  III  17°C of females heterozygous for  y cv v f car and mutations which have visible  17°C Classes  (CO.)  Region  Oregon-R  +  MP*  16  15"  Mutant 5  MP  +  MP  +  +  MP  MP  +  +  MP  MP  +  0 3  phenotypes.  22°C MP  +  128  0  190  3  58  157  0 2  21 0  0 1  0 1  41 0  0 0  17°C  :•• ' ' 2 2 ° C  2°C  17°C  +  17°C  • MP + ( =  +  137  0  28S  299  1  107  7  427  0  136  0  301  0  1  97  0  349  366  36  13}.  35 39  40 0  0 60  59 0  21 1  0 26  18 0  5 13  41 75  17 0  3C> 45  0 9  78 29  62 0  0 74  109 0  149 0  0 12  50  67 1 '  93 116  21 0  53 65  64 0  0 16  101 44  35 29  5 31  41 67  242 0  1 26  39 0  86  o  175 144  10 0  97  0 0  8 0  0 3  16 6  0 10  99 0  0 30  0 34  6 0  0  36 0  45 4;-  5  27  0 0  0 17  12 10  26  6 3  3.5 65  6 0  10 33  1 0  0 13  16 3  9 8  27 3  32 35  i 0  i 2 19  0 1  19 2  12 18  0 0  12 0  +  MP  >5>  MP  Parent: a l  y  v  cv  f_ c a r  0  + +++ + £i  72  1  189  0  82  0  244  car  i +++ + + v f car y _ c v -f + + +  _. car  +  f  152  2 2  28 0  0 12  114 70  4 1  29 24  16 5  21 24  1  116 48  9 0  0 54  56 1  0  46  1 0  0 7  9 0  0 19  1 2  22 1  0 0  3 6  1 1  15 20  0 0  18 0  0 0  5 27  0 1  4 0  2 1  9 2  1  85  108  13  0  95  41 33  0 1  10 0  1 15  42 21  0 2  17 0  1 8  2  69 51  0 0  27 0  1 32  72 31  0 0  35 0  0 28  3  75 63  0 0  47 0  1 42  90 15  0 0  60 0  X £Y- X  32 16  0 0  10 0  2 9  25 4  0 0  13 0  0 9  1**  11 17  0 0  1 11  1 0  2  24 16  0 0  4 4  3  26 24  0 0  6 2  4  9 7  0 0  7 0  +  4  * car y_ c v v _£ -r +  151  0  129 0  1  1  0 55  165  +  +  0  189  7: z l e  •f c v v f  +  172  i  51 43  26 8  0  21 0  72 0 15  .  Multiple +  cv  X  + + V  C_v  T  + f V  +  + car f+ * >!P EUtant TOTAL  e V  phenotype  e23  11 2  0 7  13 4  248  1199 856 class, region 1, i n c l u d e s a l l d* o f f s p r i n g a 2nd c. o . i n any o t h e r r e g i o n .  186  CO  ** M u l t i p l e r e g i o n 1 and  0 4  with  388 a c.o. in  4 3  3 10  609  303  0 3  11 7 857  3  2  5 9  10 25  0 9 353  0  1253  7 •-  33 0  0 8  22 0  S 26  67 2S  0 6  27 .16  1 13  5 0  1 8  2 8  23 13  0  31 7  791  425  22 7',  4  90  t-* o  !  TABLE I V Survival  Mutant  °C  c  6  s  CS  id)  malDf  malDf  22  134  160  17  190  208  0  0  22  180  150  82  4 0  17 15  cs  16  CS  Oregon-R  cross:  C S  . ,106 duplication (a) inal^ deficiency and max cs FM6 FM6 Y al Dp 4 MP* 12 88  6  6  l  i n t e s t s o f j ( l ) 5 , 6 , 1 5 , and 1 6 with duplications o f p r o x i m a l h e t e r o c h r o m a t i n o f t h e X chromosome.  o f progeny  106  ra  263  238  0  Yn.al Dp 106  C S  c s  (fe) Xa)  . ,12 , mai det]Lciencv  mal Df  In49mal  o In49mal'  Mif  cs  s  12  and d e f i c i e n c i e s  c s  t  2  mal Df 12  Y  Y  fc)c s  4 q sc deficiency  iu)  sc  4 L  sc  8 R  Df  s c  ' FMS 4L SR s c  FK6 D f  Y  Mi ) M P  *  cs  V  .i.  13  108  24  41  23  0  22  20  13  8  90  21  36  0  0  30  21  17  19  ' 130  28  38  28  0  135  108  90  49  84  141  45  50  16  0  109  82  72  13  4  0  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  22  179  155  76  52  136  63  61  48  0  113  116  82  19  17  266  270  0  0  138  45  55  18  0  206  211  65  0  0 42 44  22  166  194  0  149  130  66  79  64  0  46  46  48  0  17  213  227  0  176  109  . 74  75  4  0  49  35  16  0  (a) g° &1) /FM6 X &IrA9B v mal6Df/y+Y mal W (b) £° Df(l)cial sc B/In49 v sn mal X^j(l) /Y  MP*  = mutant phenotype  (c) Ogl(l) /FM6  +  = wild type phenotype  CS  CS  iu  tu  12  8  36+  x2  2  c s  XJ'j'lnCDsc^sc , £ cv v f/Y 811  M  o 00  TABLE V V i a b i l i t i e s and phenotypes o f t r a n s heterozygous and homozygous females i n complementation t e s t s o f c o l d - s e n s i t i v e mutations jj(l)  c  /Fm oo  i>(i)  o s y  /v-c#  T5 VR 2 2 ° C VR 170C % aa  1.02(842) 1.03(9-17) 0 (426)  0.905(232) 0.960(343) 0.S (134)  0.993(287) 0.899(338) 0 (143)  1.18(305) 1.01(423) 0 (155)  VR 2 2 ° C VR 17QC % aa  0.947(296) 1.16(197) 0.792(95) 0.774(110) 1 (144) 39 (106)  1.09 (192) 0.678(97) 2 (100)  VR 2 2 ° C VR 1 7 ° C % aa  0.862(229) 0.792(172) 0 (105)  1.11(268) 0.752(198) 0.7 (141)  15  VR 2 2 ° C VR 1 7 ° C % aa  0.754(328) 1.01(310) 0.7 (141)  1.00(176) 0.725(226) 0 (88)  16  VR 2 2 ° C VR 1 7 ° C % aa  1.24(273) 1.04(411) 0 (151)  Ore-R  26  VR 2 2 ° C VR 1 7 ° C % aa  VR  27  VR 2 2 ° C VR 1 7 ° C % aa  21  VR 2 2 ° C VR 1 7 ° C % aa  (viability  1.00(134) 1.02(107) 0 (67)  r a t i o ) = I 11 )""/In  % a a = p e r c e n t o f Jitl) /P csx  l^ Vnr, cs  0.824(166) 0.690(147) 0 (75) )csyp  0 / /  J  n  To  27  21  0.892(210) 0.820(344) 0 (99)  0.829(267) 1.00 (364) 0 (121)  0.938(312) 1.18 (533) 0 (151)  1.02(333) 1.00(455) 0.6 (156)  1.04(188) 0.667(95) 3 (96)  1.16 (158) 0.792(85) 4 (85)  1.01 (229) 0.931(253) 2 (115)  0.778(176) 1.36(125) 4 (77)  0.917(232) 0.814(155) 3 (111)  1.05(302) 0.0441(71) 52 (155)  0.617(228) 0.0364(114) 45 (87)  1.12(269) 0.96(179) 0.7 (147)  0.783(246) 0.0513(62) 43 (108)  1.05(170) 1.09(138) 0 (87)  0.973(219) 1.00(126) 0 (103)  0.722(303) 0.141(194) 52 (127)  0.915(316) 0.0460(182) 62 (151)  1.18(308) 1.04(421) 0.5 (167)  0.849(233) 0.236(131) 56 (107)  1.17(419) 1.39(421) 0 (226)  0.865(209) 1.21(199) 0 (97)  1.18(203) 0 (152) 72 (110)  0.933(290) 0.919(378) 0.7 (140)  0.339(205) 0.958(417) 0 (94)  1.02(266) 1.10(369) 0 (134)  1.22(307) 0.975 (480) 0 (169) 1.13(207) 0.814(312) 0 (110)  1.07(290) 1.02(503) 0 (150) 1.06(107) 0.949(308) 2 (55) 1.33(119) 0 (77) 0 (63)  0.933(290) 0.106(238) 60 (140)  0.783(321) 0.161(332) 25 (141)  0.950(156) 0.164(170) 28 (76)  0.973 (14S) 0.547(147) 0 (73)  1.27(204) 0.969(250) 0 (114)  1.24(224) 0.913 (241) 0 (124)  ^csy/  F M K  op  showing the abnormal abdomen t r a i t a t 2 2 ° C  110  TABLE 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) /FM6ggx l(l) /Yd<f, as compared to the Oregon-R c o n t r o l . CS  Chr*  Relative V i a b i l i t i e s 17°C 22°C  ?  9  OR**  100% 100% 100% 100% (867) (842) (889) (947)  •j^CS  111 (565) 85 (174)  0 0 101 (569) (159) (159) 117 186 9 (279) ( 97) (201)  5  cs  3  cs  0 0 36 22 (101) ( 93) ( 88) ( 88)  4  cs  5 5 75.5 70.1 (672) (681) (408) (413)  5CS  4 2 86.3 102 (456) (493) (270) (266)  6  cs  gCS  4 6 99.3 74.4 (262) (241) (183) (180) 2 3 78 89 (213) (210) (158) (156)  1 4  cs  11 49.0 36.5 12 (494) (379) (381) (522)  1 5  cs  1 44 0.8 45 (244) (250) (281) (283) 0.4 0 30 44 (265) (248) (279) (280)  16  c s  17  c s  cs  44.9 62.4 75.3 12 (494) (555) (205) (269)  1 9  cs  37 43 87.6 109 (285) (333) (134) (144)  2 0  cs  0 0 73.6 40.0 (439) (369) (114) (114)  2 1  cs  0 0 111 98 (196) (219) ( 77) ( 77)  22  CS  cs  4 6 68.3 54.9 (340) (326) (204) (201)  23  c s  9.1 7 94.0 103 (339) (374) (219) (226)  24CS  1 2  c  s  cs  13  c s  0 34.4 0 100 (264) (264) (417) (593) 16 51 12 23 (106) (267) (213) (111)  0 0 57.0 49 (294) (321) (123) (123)  1 8  0 0 88.9 111 (305) (288) ( 98) ( 98)  ll  Relative V i a b i l i t i e s 170C 22°C  Chr*  gCS 1 0  2 5  75 11 106 (407) (226) (363)  87.3 (353)  0 0 76.9 113 (274) (239) (161) (161) 35 98.0 64 91.3 (261) (284) (128) (110)  c s 89 6 94 28 (122) (131) (111) ( 94)  26  cs  75 73 118 109 (182) (197) (103) (110) 76.2 79.0 99.1 108 (185) (207) (291) (312)  21  CS  Relative V i a b i l i t y o f :  oVat oVat  22°C = 17°C =  Q Q a t 220c =  C£(D /Y) / / ( F M 6 / j ( l ) ) x 100/0.918 CS  cs  T7cs7i( 1) CS77/(FM4/Jj( 1) cs) 100/1.02 U ( 1 ) / Y ) //(Fm/JiTf ^") x ) 100/0.908 x 100/1.03  5^ at 170C = Chr* OR** = = Chromosome Oregon-R  CS  cs  x  7  r c r r  Ill  TABLE V I I  Fertility lethals.  a t 22°C o f females homozygous  Progenyy/$ when mated t o : FM6/Y +  FM6/Y " 1 F / Y P P S A S  5  64 ( 6)  86 ( 6)  OR ^cs  75 (30)  Progeny/? when mated t o : FM6/Y + ft) /Y FM6/Y  Fert-  ility*  ?  cs  cs  F  1 4  F  15  F  16  c s  2  C S  95 ( 6)  76 (10)  3  C S  14 (12)  1.7 (12)  Ss  17  39 ( 4)  66 ( 4)  F  18 cs  St  19  4CS  c s  c s  51 ( 6) 0  0 (18)  (18)  15 (18)  2 (18)  0 (12)  St Ss St  0 (12)  69 (30)  C S  6  C S  11 (18)  7 (18)  Ss  20  8  C S  35 (18)  15 (18)  Ss  21 cs  18 (18)  Ss  F  22 cs  14 (18)  Ss  0 ( 8)  St  23 cs  7** (21)  Ss  Ss  24 cs  13 (26)  Ss  (12)  3.5 (12)  0 ( 7)  0 ( 8)  St  25 cs  11 (24)  Ss  St  26 cs  0 (36)  St  27 cs  70 (15)  cs  11 cs 12 cs 13 cs  0 (27)  63 (21) 0 ( 8) 0.9  0 (24)  C S  c s  52 ( 3)  28 ( 3)  F  4 (18)  3 (21)  Ss  F = f e r t i l e ; Ss = s e m i - s t e r i l e ; St - s t e r i l e , viable adult offspring. ** o n l y FM6$^Ff_ o f f s p r i n g recovered OR = Oregon-R *  Fertility  5  gCS 1 0  for cold-sensitive  i . e . produces no  \  TABLE VIII Male progeny of females heterozygous for y_ cv v f_ car and mutant chromosomes at Crossover (CO.) Classes Parental y_ cy_ v f_ car + + + + + Single + cy_ v f_ car y_ + + + + + + v f_ car _y_ cy+ + + + + + f_ car y cv v + + + + + + car y_ cv y_ f_ + Multiple + cv Y_ + + v cv + + £ V  CO. Region  172 165 1 2 3  4 1* 2 3  +  + car f + TOTAL <SV  Oregon-R  4  41 33 69 51 75 63 32 16  1 69 0  1 3 3  0 26  2  3  78 1  66  19  15 1 35 0 1 29  0  29 0  6  41 1 1 8  11 17  1 10  2 14  24 16  2 10  5 5  26 24  14 1  15 1  9 7 828  0  3 3 0  4 0  189  ;  1 5 202  j(D  4  0  97 3  14 0 37 1 48 4  CS  Mutation Number 12 13 14  145 0  59 8  24/7 0/45 52/11 0/35 46/22 13/33 0/19 17/3  15 4  0 0  16 1  35 0 1 340 13  28 1 34 47 1 69 1 13  49 0 46 23 0 8  2 8 2 9  4/9 10/7  10 3  14 0  0 0  3 1  4/7 7/3  11 0  17/6 2/14  0 16  10 5 6 5  0 3  11 2 5 1  6 12 5' 14  0/3 6/1  0 3  4 3  0 6  2  292  149  90 0  90 0  83 3  65 15 .  0 55 1 59 0 82 0 15  0 44 3 47 0 59. 0 23  0 24 0 24 0 29 0 7  4 27 0 45 0 35 0 15  17 0 32 0 10 3 0 14  19 8 28 0 25 . 0 7 ' 4  1 18 4 a 12  2 3  0 55  0 9  0 18  14 1  8  5 21  2 8  2 6  1 18  9 1  0 6 22 2  29 •3  12 2  15 . 0 7 1 198  0 7 224  152 10  166 4  0 24  2 3 178  72 4  179 5  0 6 3  204/63 0/174  122 2  8 3  "22  11  4  10 0 '323  29 0 458  2 0 365  10 0 244  3 1 2 1 1 3 170  23,-'  21  9  '•7  0  19**  7  2 44 0 46 0 58 0 17  0  18  17°c.  194  * multiple c.o. class, region 1 includes a l l offspring with a c.o. in region 1 and a 2nd c.o.(s) * region(s) . ** late emerger, not l e t h a l - 1st 9 days/last 11 days -/ a l l offspring y_ because mothers were y_ cv v f_ car/y + + J?(l) - + +  n  a n  819 y  200  385  25  27 0 17 10 0 8 "  2  •  other  cs  to  113  TABLE IX Characteristics  Mutant  — 22°C  RV  of cold-sensitive  F e r t i l i t y of 17°C j ( l ) / j ( l ) $ c s  c s  mutants of the X chromosome.  V i s i b l e phenotvpe of mutation"  Genetic Position  None None None None Reduced sternites and etched or d i s turbed tergites, extra wing veins Like 5 but penetrance lower than 5  0,6 57 34 65 at centro' mere or be twee: car & allele 5 & 15  1 2 3 4 5  82 42 35 50 67  0 0,5 0 3 0  F F Low F P S  6  55  5  Low F  7 8 9 10 11 12 13 14 15  78 47 61 31 61 50 67 33 71  6 2 0 1 2 0 0.2 7 1  F F S Low F S S F S  16  92  0  Low F  Short, fine b r i s t l e s and etched tergites  17 18 19 20  45 77 53 47  0 13 0 0  S F F Low F  None None None Like 16  21 22 23 24 25 26  69 100 29 49 21 73  7  2 0 63  Low Low Low Low Low S  27  84  69-  F  None None None None None Abnormal abdomen traits like 5 Abnormal abdomen traits like 5 low penetrance  2~  0  F F p* F F  None Upheld wings None None None None None None Like 5  * only ^(1) /FM6 progeny recovered ** l a t e emerger; not l e t h a l when mapped / another mutation present i n t h i s stock <?'RVs r e l a t i v e 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 I I I and IV. CS  0,4 22-25 0  -0 0 1.4 51 allele 5 & 6 46-49  -  61 52** 57 34 22 48 ~  45 5/ 0  114  FIGUPE  Step 1  d/Y  jnass mating  ??  1  X/Y oV  (EMS-treated)  22°C A"  X*/Y d"  p a i r mating  XX/Y  ??  k days a t 22°C, then . t r a n s f e r t o 17°C  score f o r s u r v i v a l or w i l d - t y p e phenotype of males  17°C s c o r e f o r mutant phenotype o r absence of males  * denotes EMS-treated chromosome.  Figure  1.  S c r e e n i n g p r o c e d u r e f o r the d e t e c t i o n and r e c o v e r y o f sexlinked recessive cold-sensitive mutations.  115  Figure  2.  Cytological representation scute i n v e r s i o n s , (adapted  o f X chromosome from Cooper, 1959).  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 junction w i t h h e t e r o c h r o m a t i n shaded and e u c h r o m a t i n open. NO = n u c l e o l u s o r g a n i z e r K = centromere XR = r i g h t a r m o f X hA, hB, h C 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 arm o f X . In (l)sc sc° o r i g i n a t e s from a c r o s s o v e r between I n ( l ) s c and I n ( l ) s c ^ f  4  L  R  4  116  Figure  2  8  NO  EH  sc  m m m m  a n  hD  hC  hB  ®  hA  XR  Normal X  NO  sc  MmWMMB ® 4R  HE  hD hC  hB  hA  XR  In (Dsc  sc K  NO hA  hB  8  hC  © 8R  hA  hD  In (Dsc  sc  8 sc K  4  hA  hD  T  A , N  4  1  In (Dsc sc  8  R  117  Figure  3.  Relative break points of d u p l i c a t i o n s of the Ma-J and F i n n e r t y , 1968b). NO EH  = =  d e f i c i e n c i e s and region (Schalet  nucleolar organizer euchromatic - heterochromatic  junction  118  Figure  3 ce ntro m e re - b b (NO)  -su-f -I 22  Schalet  I 20  Sch alet  1 97,  1137  -1114  Novitski  Novitsk i  -1DCB1-35C IA7 135  12  Kaplan — E H Finnerty  lgluful-2  Df(1)ma-l  Kaplan  1DCA  Novitski  3-19  Kaplan  Kaplan  Df(1)ma-l -It  2-14a  "1152  Dp,y Y m a - l  Kaplan Novitski  106  Hi moe  -ma -I mel sw •ot -1 34  Schalet  •M (1)n •car  119  Figure 4.  Genetic positions of c o l d - s e n s i t i v e , heat-sensitive and non-conditional l e t h a l mutations on the X chromosome. 3  Cold-sensitive lethals  <orj Heat-sensitive lethals 5*2.  120  rj?7?77777777zZcZa  O  <  H  O a  <  -->r  m  CD •  ZZZ&  • m  m  cn i  cn •  Q. n CL 'c c n o o. <D Q. O  Q. C o <D Q.  IT  cn"  7  o  o  in  Non-conditional lethals  o cn  121  Figure  5.  Composite diagram of two-dimensional electropho 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. melanogas t e r  A d u l t males and f e m a l e s . The s o l i d s p o t s a r e always seen. 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 be a b s e n t . The open s p o t s i n d i c a t e d i f f e r e n c e s between H C l - a c e t o n e and 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 somal p r o t e i n s .  A.  represents a long run The f a s t moving s p o t s the e l e c t r o d e b u f f e r .  to r e s o l v e the slow moving s p o t s . have m i g r a t e d o f f the g e l into S e e F i g u r e s 6a a n d 7a f o r example.  B.  represents a short run. The slow moving s p o t s are not f u l l y r e s o l v e d but the f a s t moving spots are retained on the g e l s l a b . Only the f a s t moving spots are 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 b e r e l a t e d . See F i g u r e s 6 c , 7c, and 9 f o r examples.  122  Figure  5  123  F i g u r e 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 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 f e m a l e s . F i r s t dimension: 23 h r . a t 12 mamp/4 g e l s and 110 - 195 v o l t . Second d i m e n s i o n : 20 h r . a t 200 mamp/4 g e l s and 65 - 140 v o l t .  F i g u r e 6b.  Two-dimensional g e l e l e c t r o p h o r e s i s o f Mg-HAc preparation of Drosophila ribosomal p r o t e i n s . I n t e r m e d i a t e Run  3 mg p r o t e i n , Oregon-R males and f e m a l e s . F i r s t dimension: 14.5 h r . a t 12 mamp/4 g e l s and 150-310 v o l t . Second dimension: 16.5 h r . a t 2 00 mamp/4 g e l s and 60-100 v o l t .  Figure 6a  124  125  Figure  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 ribosomal p r o t e i n s . Short Run  3 mg protein,Oregon-R males and females. F i r s t Dimension: 10 h r . a t 12 mamp/4 g e l s and 152-185 v o l t . Second dimension: 15.5 h r . a t 200 mamp/4 g e l s and 65-170 v o l t .  F i g u r e 7a.  Two-dimensional g e l e l e c t r o p h o r e s i s o f HClacetone p r e p a r a t i o n o f D r o s o p h i l a r i b o s o m a l proteins. Long Run  3 mg p r o t e i n , Oregon-R males and females. F i r s t dimension: 2 3 hr. a t 12 mamp/4 g e l s and 110 - 19 5 v o l t . Second Dimension: 20 h r . a t 200 mamp/4 g e l s and 65 - 140 v o l t .  127  F i g u r e 7b.  Two-dimensional g e l e l e c t r o p h o r e s i s o f H C l acetone p r e p a r a t i o n o f D r o s o p h i l a r i b o s o m a l proteins. I n t e r m e d i a t e Run  3 mg p r o t e i n , Oregon-R males and females. F i r s t dimensions 15.5 h r . at_12 mamp/4 g e l s and 150-185 v o l t . Second d i m e n s i o n : 15.5 h r . a t 200 mamp/4 g e l s and 65-170 v o l t .  F i g u r e 7c.  Two-dimensional g e l e l e c t r o p h o r e s i s o f H C l acetone p r e p a r a t i o n o f D r o s o p h i l a r i b o s o m a l proteins. S h o r t Run  3 mg p r o t e i n , Oregon-R males and females. F i r s t dimension; 10 h r . a t 12 mamp/4 g e l s and 152-185 v o l t . Second dimension: 3.5.5 h r . a t 200 mamp/4 g e l s and 65-170 v o l t .  128  Figure 7b  Figure 7c o  129 Figure 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 ribosomal proteins.  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 dimension: 15 h r s . , 12 mamps/4 g e l s and 130-230 v o l t s . Second dimension: 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 i d e o f the g e l have s t a r t e d t o m i g r a t e i n t o the cathode b u f f e r o f the f i r s t dimension. 6 = o r i g i n , A n i o n i c s i d e o f g e l on l e f t , c a t i o n i c . s i d e on r i g h t .  130  F i g u r e 9.  Two-dimensional g e l e l e c t r o p h o r e s i : of ribosomal p r o t e i n s o f mutant 1 6 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 . Mutant m a l e s . First"dimension: 10.5 h r . a t 12 mamp/4 g e l s and 125-182 volt. Second d i m e n s i o n : 12 h r . a t 170-175 mamp/4 g e l s and 90-180 v o l t .  F i g u r e 10.  Two-dimensional 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 o f mutant 5  3 mg p r o t e i n , mutant m a l e s . HCl-acetone preparation. F i r s t dimension: 10.5 h r . a t .12 mamp/4 g e l s and 125-182 volt. Second d i m e n s i o n : 12 h r . a t 170-175 mamp/4 g e l and 90-180 v o l t .  131  132  Figure  11.  Two-dimensional g e l electrophoresis p r o t e i n s o f mutant 2 7  of  ribosomal  c s  3 mg p r o t e i n , H C l - a c e t o n e F i r s t dimension: 23 h r . volt. Second dimension: and 100-200 volt.  Figure  12.  preparation. Mutant males. a t 7 - 1 2 mamp/4 g e l s a n d 1 3 0 - 2 0 0 2 1 . 5 h r . a t 200 m a m p / 4 g e l s  Two-dimensional g e l electrophoresis p r o t e i n s of Oregon-R males.  of  ribosomal  3 mg p r o t e i n , O r e g o n - R m a l e s . HCl-acetone preparation. F i r s t dimension: 14 h r . a t 12 mamp/4 g e l a n d 1 2 5 - 2 1 0 volt. Second dimension: 13 h r . a t 200 m a m p / 4 g e l s a n d 75-150 volt.  133 Figure  Figure  11  12  

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