UBC Theses and Dissertations

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UBC Theses and Dissertations

An immunofluorescent demonstration of nuclear proteins David, Lyle Anacletus 1968

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AN  I M M U N O F L U ORES C E N T OF N U C L E A R  DEMONSTRATION  PROTEINS  by  L Y L E  ANACLETUS  B.Sc,  U n i v e r s i t y of A l b e r t a ,  1962  M.Sc,  U n i v e r s i t y of A l b e r t a ,  1965  A THESIS S U B M I T T E D THE  i n the  FOR  THE  OF PHILOSOPHY Division  IN  of M e d i c a l  a c c e p t t h i s t h e s i s as  required  THE  IN P A R T I A L , F U L F I L M E N T  REQUIREMENTS  DOCTOR  We  DAVID  DEGREE  OF  GENETICS Genetics  c o n f o r m i n g to  the  standard  UNIVERSITY  OF BRITISH  N O V E M B E R  1968  COLUMBIA  OF  In p r e s e n t i n g an the  thesis  advanced degree at Library  I further for  this  shall  the  his  of  this  agree that  written  University  of  permission  representatives. thesis  f u l f i l m e n t of  make i t f r e e l y  s c h o l a r l y p u r p o s e s may  by  in p a r t i a l  be  available  g r a n t e d by  gain  of  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  the  It i s understood  for financial  for  for extensive  permission.  Department  British  Columbia  shall  requirements  Columbia,  Head o f my  be  I agree  r e f e r e n c e and copying of  that  not  the  that  Study.  this  thesis  Department  copying or  for  or  publication  allowed without  my  i ABSTRACT  This thesis fluorescent technique  describes  the d e v e l o p m e n t of a n i m m u n o -  and its a p p l i c a t i o n to a c y t o c h e m i c a l study  of the i n t r a c e l l u l a r d i s t r i b u t i o n of a b a s i c p r o t e i n which satisfies  s e v e r a l c r i t e r i a u s e d to identify  histone.  The technique was developed in three  1.  component  steps:  P r o d u c t i o n of A n t i b o d y  T h e m e t h o d of n u c l e a r p r o t e i n e x t r a c t i o n , , the a r a t i o n of the i n o c u l u m , the i m m u n i s a t i o n p r o c e d u r e species  prep-  and  the  of a n t i g e n d o n o r a n d r e c i p i e n t w e r e a l l c o n s i d e r e d  c o n t r i b u t o r y of the  2.  s u c c e s s f u l p r o d u c t i o n of a n t i b o d y .  A n a l y s e s of A n t i g e n a n d A n t i b o d y  Electrophoretic and chromatographic techniques e m p l o y e d to c h a r a c t e r i s e  the n u c l e a r p r o t e i n a n t i g e n s .  a n t i s e r u m was purified by i m m u n o l o g i c a l absorption and graphy.  The antigen-antibody responses were  were  The chromato-  studied using  electrophoretic and immunodiffusion techniques,  both singly and  in combination.  3.  A p p l i c a t i o n to C e l l  Preparations  Fluorescent antibody provided a protein tracer  with which  ii t o d e m o n s t r a t e the p r e c i s e c y t o l o g i c a l l o c a t i o n of a n a l y t i c a l l y d e f i n e d nuclear  proteins. T h e a n t i b o d y w a s p r o d u c e d i n the c h i c k e n a g a i n s t  protein f r o m calf thymus nuclei. c h r o m o s o m e s but was neither  It w a s s p e c i f i c f o r n u c l e i  organ nor species  specific.  basic and  iii ACKNOWLEDGEMENTS  The author  g r a t e f u l l y a c k n o w l e d g e s the  direction provided by his committee of t h e  chairmen,  F a c u l t y of M e d i c i n e , , a n d D r . C . P e r s o n ,  support  and  Dr. J. R. of the  Miller,  Botany  Department. Special thanks are continued enthusiasm  g i v e n to D r . M . C o r e y for  and interest  i n d i r e c t i n g the  project and assisting i n preparation The m e m b e r s  Dr.  J.  research  thesis.  of the a d v i s o r y c o m m i t t e e  t h e i r t i m e to p r o v i d e a s s i s t a n c e . to D r .  of the  Appreciation is  G e r w i n g f o r p r o v i d i n g the a m i n o a c i d  gave f r e e l y of expressed  analyses.  C . Finnegan was also especially helpful with his  of h i s t o c h e m i c a l  her  knowledge  techniques.  Technical assistance was received f r o m several i n the  Zoology Department,  a n d the D e p a r t m e n t  people  T h e U n i v e r s i t y of B r i t i s h C o l u m b i a ,  of P a e d i a t r i c s ,  Vancouver General Hospital.  H e l p w i t h s e v e r a l p h a s e s of the p r o j e c t w a s g i v e n b y M i s s A n n e W r i g h t to w h o m I a m e s p e c i a l l y  grateful.  T h a n k s a l s o go to C a n a d a P a c k e r s the b o v i n e t i s s u e s ,  to the P a t h o l o g y D e p a r t m e n t  G e n e r a l H o s p i t a l for autopsy Poultry Science,  L t d . for providing  specimens  of the  Vancouver  a n d the D e p a r t m e n t  T h e U n i v e r s i t y of B r i t i s h C o l u m b i a , f o r  of  the  iv chickens. of l u p u s  A l s o to D r . R . B . L o w r y f o r a r r a n g i n g the c o l l e c t i o n serum. Dr.  Alberta,  R.  F . Ruth, Department  of Z o o l o g y ,  U n i v e r s i t y of  E d m o n t o n , p r o v i d e d valuable advice on s e v e r a l T h i s w o r k w a s done u n d e r  M e d i c a l R e s e a r c h C o u n c i l of C a n a d a .  occasions.  a g r a n t p r o v i d e d b y the  V.  TABLE  OF  CONTENTS  Page ABSTRACT  i  ACKNOWLEDGEMENTS  iii  INTRODUCTION  1  LITERATURE  3  REVIEW  I  C h a r a c t e r i z a t i o n of H i s t o r i e s  3  II  E a r l y Studies of N u c l e a r P r o t e i n s  4  III  Preparative  6  IV  H i s t o n e s as S t r u c t u r a l P r o t e i n s  10  V  Acidic Nuclear Proteins  13  VI  H i s t o c h e m i c a l C h a r a c t e r i z a t i o n of H i s t o n e s  15  VII  A n t i g e n i c i t y of N u c l e i c A c i d s  METHODS I  II  and Analytical P r o c e d u r e s  and  Nuclear Proteins  17  AND MATERIALS  21  Immunisation Procedure  21  1.  I s o l a t i o n of C a l f  21  2.  Nuclear Protein  21  3.  P r e p a r a t i o n of I n o c u l u m  22  4.  Immunisation  22  Protein Analysis  23  1.  23  Thymus Nuclei  Chromatography A. B.  D e s c r i p t i o n of C h r o m a t o g r a p h i c Columns Column Packing  23 24  vi  2.  C.  Protein Fractionation  26  D.  Protein Concentration  26  Disc Electrophoresis  28  A.  Acidic Proteins  .28  B.  Basic Proteins  28  3.  Amino Acid Analysis  30  4.  Immunodiffusion  30  A.  Serum Proteins  30  B.  Nuclear Proteins  31  C.  I m m u n o l o g i c a l A n a l y s i s of . Electrophoretic Fractions  5.  Precipitin Tests  32 33  III  Absorption  34  IV  Non-Immune  V  Lupus Erythematosis  VI  Fluorescent  VII  Histological Techniques  35  1.  Mammalian Cells  35  A.  Peripheral Blood Cultures  35  B.  Buccal Smears  36  C.  Blood Smears  36  D.  Organ Imprints  36  Serum  34 Serum  34  D y e - P r o t e i n Conjugation  35  vii 2. I n s e c t M a t e r i a l A.  Polytene  B.  Protozoan  37  Chromosomes  37  Cells  37  3.  Plant Materials  38  4.  Antibody Application  39  5.  A c r i d i n e Orange Staining  40  6.  Biebrich Scarlet  40  Staining  VIII  Microscopy  40  IX  Photography  41  RESULTS  43  I  E x t r a c t i o n of N u c l e a r P r o t e i n s  45  II  P r o d u c t i o n of A n t i s e r u m  48  1.  Schedule  of I m m u n i s a t i o n s  and  Precipitin Tests 2.  3. III  Immunodiffusion  48 Tests  52  A.  Micro-immunodiffusion  52  B.  Macro-immunodiffusion  56  A b s o r p t i o n of A n t i s e r u m  62  A n a l y s i s of N u c l e a r P r o t e i n E x t r a c t  65  1.  Disc Electrophoresis  65  2.  Immuno Disc Electrophoresis  68  3.  B i e b r i c h Scarlet Staining  71  4.  A b s o r p t i o n of A n t i g e n  71  viii IV  V  Chromatographic  S e p a r a t i o n of N u c l e a r  Protein  76  A n a l y s i s of C h r o m a t o g r a p h i c F r a c t i o n s  84  1.  Disc Electrophoresis  84  2.  Amino Acid Composition  87  3. I m m u n o d i f f u s i o n T e s t  89  VI  P r e p a r a t i o n of F l u o r e s c e n t S e r u m F r a c t i o n s  92  VII  A n a l y s i s of F l u o r e s c e n t S e r u m F r a c t i o n s  97  1.  Disc Electrophoresis  97  2.  Immunodiffusion Test  101  3.  Precipitin Tests  104  VIII  I m m u n o f l u o r e s c e n t T e s t s of the C e l l u l a r L o c a l i s a t i o n and D i s t r i b u t i o n of N u c l e a r Protein Antigen  104  1.  Bovine Cells  105  A.  Calf  105  B.  Liver  C.  Spleen Cells  110  D.  Testis Cells  113  E.  C h r o m o s o m e s of C u l t u r e d L e u k o c y t e s  116  2.  Thymus Imprints Cells  110  Human Cells  119  A.  Buccal Cells  1.19  B.  Thymus Cells  119  C.  Testis Cells  120  D.  C h r o m o some s of  Cultured Leukocytes  12 3  ix  I X  3.  Insect  Chromosomes  4.  Plant  5.  Protozoan  6.  Chicken Blood  126  Cells  Human  Lupus  Against  Calf  129 Cells  132  Cells  Erythematosis Thymus  Nuclear  Antigens  132 Antibody  Activity  Protein 138  SUMMARY  141  DISCUSSION  145  BIBLIOGRAPHY  15 6  APPENDIX  162  I. II. III.  Chromosomal Buccal  Preparation  (Smears  Acridine Orange  162 165  Staining  167  X  LIST OF  Table I  TABLES  IMMUNOCHEMICAL PROCEDURE  FOR THE  DEMONSTRATION  PROTEIN  OF NUCLEAR  ANTIGEN AT THE C E L L U L A R  T a b l e II  AMINO ACID COMPOSTION PROTEIN  T a b l e III  PREPARATION  FRACTIONS NUCLEAR  OF  1  Figure 2  OF  3)'  47  THREE  CHROMATOGRAPHED 88  PROTEIN  LIST OF Figure  OF NUCLEAR .  (BATCH  AMINO ACID COMPOSITION  44  LEVEL.  FIGURES  P r e c i p i t i n T e s t o f A n t i s e r a 333 a n d 312  51  M i c r o - i m m u n o d i f f u s i o n T e s t s of AntibodyA c t i v i t y of C h i c k e n S e r u m A g a i n s t  Nuclear  Protein Antigen Figure  3  55  Macro-immunodiffusion  T e s t of A n t i b o d y  A c t i v i t y of C h i c k e n S e r u m A g a i n s t  Nuclear  Protein Antigen Figure 4  Immunodiffusion  58 T e s t of A n t i s e r a  Against Cytoplasm, Protein  Histone and  333,  312  Nuclear 61  XI  Figure  5  Immunodiffusion  T e s t of A n t i s e r u m  Absorbed with Cytoplasm,  64 67  6  Comparative  Figure  7  Immunodiffusion  Disc Electrophoresis T e s t of Two C h i c k e n  Antisera Against Calf Proteins, 8  Nuclear  P r o t e i n and Cytoplasm Figure  Figure  Against  312,  Thymus  Basic  Nuclear  F r a c t i o n e d by D i s c E l e c t r o p h o r e s i s  Disc Electrophoresis  of N u c l e a r  70  Protein  Stained for P r o t e i n and Histone  74  Figure 9  Nuclear Protein Chromatography  80  Figure  Chromatography  10  of a M o l e c u l a r  Weight  Mixture  80 83  Figure  11  Nuclear Protein Chromatography  Figure  12  Disc Electrophoretic Chromatographed  Figure  13  Immunodiffusion Chromatographic  A n a l y s i s of  Nuclear Proteins T e s t of U n f r a c d o n a t e d Fractions  of  86 and  Nuclear  Protein Figure  14  91  Sephadex Chromatography Absorbed, Serum  Immune,  of a S a m p l e  Fluorescent  of  Chicken 96  Xll  Figure  15  Disc Electrophoresis Fluorescent,  of  Absorbed,  Immune Serum  312.  Before  and After Chromatography Figure  16  M i c r o - i m m u n o d i f f u s i o n T e s t of Fluorescent  Figure  17  100  Calf DNA,  Absorbed,  Immune Chicken Serum  Thymus  312  I m p r i n t s S h o w i n g P r e s e n c e of  Histone and B a s i c N u c l e a r  Protein  Antigen in Nuclei Figure  18  109  Bovine L i v e r and Spleen I m p r i n t s  Showing  P r e s e n c e of B a s i c N u c l e a r P r o t e i n  Antigen  and Histone i n N u c l e i Figure  19  112  B o v i n e T e s t i s I m p r i n t s S h o w i n g the  Presence  of D N A , H i s t o n e a n d B a s i c N u c l e a r  Protein  Antigen in Meiotic N u c l e i and Sperm Heads F i g u r e 20  Bovine Acid Alcohol F i x e d  Nuclear  P r o t e i n a n d N u c l e i c A c i d i n the S a m e S i t e s H u m a n C e l l s ( U n f i x e d ) S h o w i n g the  Human Chromosomes  S h o w i n g the  Chromosomes  of C h i r o n o m u s  122  Presence  of B a s i c N u c l e a r P r o t e i n A n t i g e n F i g u r e 23  118  Presence  of B a s i c N u c l e a r P r o t e i n A n t i g e n i n N u c l e i F i g u r e 22  115  Chromosomes  S h o w i n g the P r e s e n c e of B a s i c  F i g u r e 21  103  tentans  12 5 Showing  the P r e s e n c e of B a s i c N u c l e a r P r o t e i n A n t i g e n  128  X1X1  F i g u r e 24  P l a n t C e l l s S h o w i n g the P r e s e n c e of B a s i c Nuclear Protein Antigen in Some Nuclei  Figure  2-5  A n I n s e c t P a r a s i t e ( H o l o m a s t i g o t o i d e s) Labelled With Fluorescent  F i g u r e 26  Antibody  134  Chicken Blood'Cells Labelled With Fluorescent  F i g u r e 27  131  Antibody  Immunodiffusion of L . E . Protein  137  T e s t of A n t i b o d y  Plasma Against Basic  Activity  Nuclear 140  1 INTRODUCTION  P r o t e i n s m a k e u p a p p r o x i m a t e l y 70% of the n e t w o r k of i n t e r p h a s e  nuclei (Busch,  i n t e g r a l c o m p o n e n t of c h r o m o s o m e s Histones,  chromatin  1 9 6 5 , p . 5) a n d a r e ( M i r s k y and R i s ,  1947).  the D N A - a s s o c i a t e d b a s i c n u c l e a r p r o t e i n s ,  about 3 5 % of the t o t a l p r o t e i n i n n u c l e i .  an  comprise  The r e m a i n d e r  is made  up  of h i g h l y i n s o l u b l e a c i d i c p r o t e i n s w h i c h a r e a l s o p r e s e n t i n cytoplasm. proteins  B o t h the  s t r u c t u r a l a n d f u n c t i o n a l r o l e s of n u c l e a r  are u n r e s o l v e d but it has been suggested that  histones  a r e i n v o l v e d i n the c o n t r o l of D N A a c t i v i t y ( S t e d m a n a n d 1950). histones  Both acidic nuclear proteins (Zubay and Doty,  i n the m a i n t e n a n c e  ( M i r s k y a n d R i s , 1947) a n d  1959) h a v e b e e n p r o p o s e d to be  of c h r o m o s o m e  now i n use are  s t u d y of h i s t o n e s ,  inadequate  but the c y t o c h e m i c a l t e c h n i q u e s  highly specific and sensitive.  different  chromosomes.  The immunofluorescent technique  studies  are  f o r d i s t i n g u i s h i n g b e t w e e n the  histone fractions i n cells and  involved  structure.  S e v e r a l extraction and separation techniques a v a i l a b l e f o r the  Stedman,  (Coons,  1951) i s  It h a s n o t b e e n w i d e l y a p p l i e d to  of n u c l e a r p r o t e i n s b e c a u s e of the d i f f i c u l t i e s i n v o l v e d i n  the p r o d u c t i o n of f l u o r e s c e n t a n t i b o d y a g a i n s t n u c l e a r have long been c o n s i d e r e d non-antigenic  proteins,which  ( W i l s o n et a l . ,  1966).  T h i s p r o j e c t w a s d e s i g n e d t o a t t e m p t the a p p l i c a t i o n of  2 a n i m m u n o f l u o r e s c e n t t e c h n i q u e to a s t u d y of n u c l e a r p r o t e i n s  in  cellular and c h r o m o s o m a l preparations.  the  Since techniques  p r e p a r a t i o n a n d c h a r a c t e r i z a t i o n of a c i d i c n u c l e a r p r o t e i n s received scant attention, the a n t i g e n i c  material.  basic nuclear proteins were  for  have  chosen  as  3 LITERATURE  I.  CHARACTERIZATION OF  REVIEW  HISTONES  H i s t o n e s a r e defined as b a s i c p r o t e i n s that at t i m e are a s s o c i a t e d w i t h D N A ( M u r r a y , 1964). includes protamines which are proteins.  This  some  definition  c o n s i d e r e d to be analogous  »  The nomenclature u s e d for v a r i o u s histone fractions  d i f f e r e n t l a b o r a t o r i e s d e p e n d s o n the m e t h o d of a n a l y s i s preparation.  T h i s h a s l e d to s e v e r a l s y s t e m s  electrophoretic mobility,  and sedimentation rate (Busch,  1965, p. 40).  or  of c l a s s i f i c a t i o n ,  based on a m i n o a c i d c o m p o s i t i o n , N H £ - t e r m i n a l a m i n o precipitation behaviour,  by  acid,  chromatography  M u r r a y (1964) l i s t s  t w e n t y - s e v e n f r a c t i o n s that have b e e n d e s c r i b e d i n the  literature  u s i n g a v a r i e t y of s y m b o l s t h a t s h o w a l m o s t n o c o r r e l a t i o n w i t h each other.  T h e m o l a r r a t i o s of l y s i n e to a r g i n i n e f o r  these  f r a c t i o n s f o r m a c o n t i n u o u s r a n g e o f v a l u e s f r o m 0 . 0 9 t o 1. 3 5 . He  s u g g e s t s t h a t t h i s r a t i o be a d a p t e d as a n i n i t i a l d e s c r i p t i o n of a  histone f r a c t i o n followed by as complete a d e s c r i p t i o n as T h i s s y s t e m c o u l d be u s e d i n c o n j u n c t i o n w i t h the m o r e and widely used classifications, v e r y l y s i n e - r i c h , slightly l y s i n e - r i c h and a r g i n i n e - r i c h .  general  lysine-rich,  S i n c e no single  component has been i s o l a t e d i n a pure f o r m ,  possible.  histone  c h a r a c t e r i z a t i o n of  h i s t o n e f r a c t i o n s a r e d e s c r i p t i o n s of m i x t u r e s of p r o t e i n s .  This  4 h a s l e d to m u c h c o n f u s i o n i n the  II.  literature.  E A R L Y STUDIES OF N U C L E A R  PROTEINS  Studies i n v o l v i n g p r o t e i n s as e s s e n t i a l components the c e l l n u c l e u s b e g a n one h u n d r e d y e a r s  ago i n the  laboratories  of D r . E r n s t H o p p e - S e y l e r a t t h e U n i v e r s i t y of S t r a s b o u r g and T S ' O , 1964). of h i s s t u d e n t s , preparation, (Meischer, phorous  of  (Bonner  T h e e a r l i e s t w o r k w a s p e r f o r m e d i n 1869 b y Johan F r i e d r i c h M e i s c h e r , who isolated a  crude  w h i c h he c a l l e d " n u c l e i n " , f r o m pus c e l l n u c l e i 1897).  T h i s m a t e r i a l w a s f o u n d to be r i c h i n p h o s -  a n d w a s p r e s e n t i n a l l the n u c l e i he e x a m i n e d .  M e i s c h e r again isolated a D N A like substance, salmon spermatozoa,  of H o p p e - S e y l e r ' s a s s i s t a n t s ,  I n 1872  this time  along with a nitrogen rich basic  w h i c h he c a l l e d " p r o t a m i n e " .  from  protein  This w o r k was continued by  another  Albrecht Kossel.  D u r i n g the p e r i o d f r o m 1872 to 1910 K o s s e l p u b l i s h e d a  series  one  of p a p e r s on the  c h e m i s t r y of the c e l l n u c l e u s  which  c u l m i n a t e d i n h i s r e c e i v i n g the N o b e l P r i z e i n m e d i c i n e . s h o w e d t h a t the p r o t a m i n e s not p r e s e n t i n u n r i p e  w e r e unique to g e r m c e l l s and  salmon testes,  but another  basic  He were  protein  s u b s t a n c e w h i c h he c a l l e d " h i s t o n e " w a s p r e s e n t i n n u c l e i f r o m somatic cells. nucleoproteins  He was able to d i s t i n g u i s h between o n the b a s i s  of a r g i n i n e content,  the  these protamines  containing high amounts  of a r g i n i n e ,  the h i s t o n e s m u c h l e s s .  In  1884 he i s o l a t e d h i s t o n e s f r o m g o o s e  erythrocyte nuclei by u s i n g  water  a n d c e n t r i f u g a t i o n to  to d i s r u p t t h e c e l l m e m b r a n e s  o u t the n u c l e a r  separate  mass.  B y 1910 K o s s e l h a d s h o w n t h a t h i s c l a s s i c w e r e a c t u a l l y a c o m p l e x of p r o t e i n s w i t h different amino acid compositions. c o m m o n features,  preparations  solubilities and  A l l proteins i n this c l a s s had  however.  They were  some  s i m i l a r to p r o t a m i n e s  h a v i n g a h i g h i s o e l e c t r i c p o i n t a n d t h e a b i l i t y to c o m b i n e w i t h  in acids  in fact they w e r e a l w a y s found i n a s s o c i a t i o n w i t h n u c l e i c a c i d s . They were  t h o u g h t to c o n t a i n 1 7 - 3 0 % a r g i n i n e i n c o m p a r i s o n to  80% a r g i n i n e f o r p r o t a m i n e .  K o s s e l h y p o t h e s i z e d that  were larger,  m o l e c u l e s than p r o t a m i n e s ,  more  complex,  they w e r e d e g r a d e d d u r i n g s p e r m a t o g e n e s i s b y the l o s s of n o n - b a s i c a m i n o  to f o r m  and  that  protamines  acids.  Another important advance i n early histone was  histones  chemistry  the i s o l a t i o n o f m a m m a l i a n h i s t o n e s f r o m c a l f t h y m u s  cells.  T h i s w a s done b y L i l i e n f i e l d w h o w a s a s t u d e n t of K o s s e l i n 1894. This procedure made  p o s s i b l e the p r e p a r a t i o n of l a r g e a m o u n t s  nuclear protein f r o m an easily obtainable  source.  This material  w a s c h o s e n b e c a u s e the t h y m u s i s c o m p o s e d a l m o s t e n t i r e l y of loosely packed cells, a predominant nucleus  of  s i m i l a r to s m a l l l y m p h o c y t e s ,  which  have  s u r r o u n d e d by a thin l a y e r of c y t o p l a s m .  6 (Busch,  1965,  p.  12).  T h e study of n u c l e a r p r o t e i n s many years  after  for  the i n i t i a l w o r k b y E u r o p e a n b i o l o g i s t s a n d  not taken up a g a i n u n t i l m o r e interest i n histones  recent  times.  of the o x , f o w l ,  They studied histones cod,  did find some m i n o r interspecies suggest that h i s t o n e s m i g h t act as  Nature  from  salmon and human  u s e d a r g i n i n e content as their m e a s u r e of s p e c i f i c i t y . they c o u l d f i n d no d i f f e r e n c e b e t w e e n  was  The p r e s e n t wave of  s t e m s f r o m a s h o r t c o m m u n i c a t i o n of  b y S t e d m a n l a n d S t e d m a n i n 1950. different tissues  remained dormant  and  Although  their m a i n fractions  differences.  they  This led them  gene s u p p r e s s o r s ,  since  to  they  k n e w that although c e l l s w i t h i n an o r g a n i s m c o n t a i n e d i d e n t i c a l sets of g e n e s , IIL  they w e r e  c a p a b l e of p r e f o r m i n g different  PREPARATIVE A N DANALYTICAL  functions.  PROCEDURES  I n 19 5 4 D a l y a n d M i r s k y d e v i s e d a p r o c e d u r e , strong acids were avoided, calf thymus nuclei. from  for removing histones f r o m  The cytoplasmic constituents  t h y m o c y t e s b y h o m o g e n i z i n g the t h y m u s  were  in which  isolated removed  tissue in citric  acid.  T h e h i s t o n e s w e r e e x t r a c t e d b y a d d i n g s o d i u m c h l o r i d e to t h e  nuclear  s u s p e n s i o n at v a r i o u s m o l a r i t i e s .  a  A t low salt concentrations  h i s t o n e f r a c t i o n w a s e x t r a c t e d that h a d a l y s i n e - a r g i n i n e r a t i o of a p p r o x i m a t e l y 9 : 1 a n d c o n t a i n e d 3 5 . 9% l y s i n e . decreased  to 1 : 1 a s t h e  This  ratio  salt concentration was increased.  They  7 obtained l y s i n e - r i c h histones f r o m as w e l l ,  calf l i v e r and turtle  red-cells  a n d c o n c l u d e d t h a t the l y s i n e - r i c h h i s t o n e s w e r e  c o n s t a n t n u c l e a r c o m o n e n t that w a s m o r e  a  readily dissociable  n u c l e i c a c i d t h a n w a s the a r g i n i n e - r i c h h i s t o n e s .  This new  from  histone,  b e i n g s o l u b l e at p H 10. 6 (the i s o e l e c t r i c r e g i o n at w h i c h a r g i n i n e rich histones precipitate), supernatent.  This  h a d p r e v i o u s l y b e e n d i s c a r d e d i n the  study e m p h a s i s e d that h i s t o n e s w e r e a c o m p l e x  of b a s i c p r o t e i n s that w o u l d r e q u i r e  special methods for  separation  and analysis. D u r i n g the e a r l y 1 9 5 0 ' s two n e w t e c h n i q u e s w e r e to t h e  study of histones:  resis.  column chromatography and  applied  electropho-  C r u f t a n d h i s c o l l e a g u e s ( C r u f t et aL. 1957) u s e d m o v i n g  boundary electrophoresis  to s e p a r a t e h i s t o n e s f r o m  o n the b a s i s of t h e i r e l e c t r o p h o r e t i c m o b i l i t y , three f r a c t i o n s they obtained as alpha,  several  sources  and designated  beta and gamma.  the  Amino  a c i d a n a l y s i s s h o w e d that these f r a c t i o n s c o u l d a l s o be d e s i g n a t e d lysine-rich,  argine-rich,  a n d s l i g h t l y l y s i n e - r i c h ( C r u f t et a L  T h e y a l s o o b s e r v e d that h i s t o n e s at p H ' s a b o v e 4. methods of  1958).  t e n d to f o r m m o l e c u l a r a g g r e g a t e s  T h i s p l a c e d a l i m i t a t i o n on those  that r e q u i r e d r a s t i c changes  chromatographic  i n pH for elution and  separation  histones. However,  as  i n 1955 C r a m p t o n et a l , a c h i e v e d f r a c t i o n -  a t i o n of the l e s s b a s i c c a l f t h y m u s h i s t o n e s o n c o l u m n s of  the  8  carboxylic acid continuously protein of  r e s i n 1 RC - 50 u s i n g b a r i u m a c e t a t e b u f f e r  increasing ionic  fractions  amino a c i d  strength.  They o b t a i n e d  w h i c h w e r e c h a r a c t e r i z e d as h i s t o n e s  content.  Improved r e s u l t s  guanadinium c h l o r i d e b u f f e r  (Busch,  were o b t a i n e d  1965, p.  of  three on the  basis  with  78).  The u s e o f c a r b o x y m e t h y l c e l l u l o s e c o l u m n s was p i o n e e r e d by D a v i s o n  ( 1 9 5 7 ) who u s e d i n c r e a s i n g c o n c e n t r a t i o n s  NaCl to separate a l y s i n e - r i c h the h i s t o n e s .  He was a b l e  chromatographed by t h i s  t o a c h i e v e 90% r e c o v e r y o f t h e  t e c h n i c a l problems  need f o r a method o f a n a l y s i s o f h i s t o n e  d i s t i n g u i s h between m i x t u r e s o f p r o t e i n s a s i n g l e component. applied  erythrocyte histone N e e l i n and N e e l i n bands at  To t h i s  end N e e l i n  the method o f e l e c t r o p h o r e s i s  by S m i t h i e s ( 1 9 5 5 ) .  pH 4 . 9 ,  remainder o f histones  method.  One o f t h e m a j o r the  f r a c t i o n from the  of  They r e p o r t e d  the  this  t i m e was  fractions  and f r a c t i o n s and C o n n e l l  i n a starch  which  would  containing  (1959)  gel  introduced  fractionation of chicken  i n t o s i x t e e n bands at  pH 4 . 1 ,  achieved electrophoresis  again substantiating  at  In  of c a l f  that histones  1960  thymus i n t o were  complex. C o r r e l a t i o n of chromatographic f r a c t i o n s , phoretic patterns Johns  et  and a m i n o a c i d  a l . , i n 1961.  electro-  a n a l y s i s was b r o u g h t  They c h r o m a t o g r a p h e d  calf  about  thymus  by  histone  18  9  o n c a r b o x y m e t h y l c e l l u l o s e u s i n g s o d i u m a c e t a t e b u f f e r at p H 4 . 2 and N a C l to elute t h r e e p e a k s . F3,  were  characterised  arginine-rich.  These peaks,  as l y s i n e - r i c h ,  Further,  called F l , F2  and  slightly lysine-rich  and  each fraction produced a  electrophoretic banding pattern which, r e p r o d u c e d the o r i g i n a l p a t t e r n  when  of w h o l e  characteristic  recombined,  histone.  In o r d e r to s e p a r a t e h i s t o n e on the b a s i s of m o l e c u l a r s i z e a n d a v o i d a g g r e g a t i o n of h i s t o n e m o l e c u l e s ,  c o l u m n s of  S e p h a d e x G - 7 5 w e r e f i r s t u s e d b y C r u f t (1961) a n d a m o r e a n a l y s i s o f t h i s m e t h o d m a d e b y J o h n s o n et a l .  (1964).  detailed  Cruft  a c h i e v e d 1 0 0 % r e c o v e r y o f h i s t o n e u s i n g 0 . 02 N H C 1 a s t h e solution.  H e w a s able to r e c o v e r  3 peaks  containing  that c o r r e s p o n d e d to 3 b a n d i n g g r o u p s on s t a r c h g e l  eluting  fractions electrophoresis.  Johnson and his c o - w o r k e r s found that satisfactory  separations  c o u l d be a c h i e v e d on Sephadex u s i n g s o d i u m c i t r a t e buffer p H 7. 0.  A s l o n g as the  s o d i u m c o n c e n t r a t i o n w a s k e p t at  a b o v e 0. 0 2 M the p r e s e n c e  of s o d i u m i o n s p r e v e n t e d  histone on the Sephadex g r a n u l e s formation.  and d i d not a l l o w  at or  a d s o r p t i o n of aggregate  T h e y concluded that Sephadex r e s o l v e d histones  four or five incompletely resolved, Another  recent  heterogeneous  into  fractions.  advance in histone characterisation  the u s e of p o l y a c r y l a m i d e g e l s as an e l e c t r o p h o r e t i c  is  medium.  T h i s m e t h o d h a s t h e a d v a n t a g e of a l l o w i n g g e l p o l y m e r i z a t i o n at  10  p H v a l u e s as l o w as 2. 3 t h e r e b y d e c r e a s i n g the p o s s i b i l i t y of histone aggregation.  A l s o the u s e of g e l s h a v i n g v a r i a b l e  pore  sizes and v e r y thin starting zones results i n high r e s o l u t i o n during relatively short runs.  McAllister,  Wan and Irvin  a n d S h e p h e r d a n d G u r l e y (1965) m o d i f i e d the o r i g i n a l  (1963)  procedures  and adapted a single l o w p H gel with a p o r o s i t y appropriate e l e c t r o p h o r e t i c s e p a r a t i o n of h i s t o n e s .  Shepherd and  experimented w i t h acid extracted calf thymus histone produced highly complex banding patterns.  Gurley which  The number  and their m o b i l i t i e s v a r i e d considerably when different of s a m p l e s w e r e u s e d . a simple,  sensitive,  McAllister,  Nevertheless,  of  amounts  and highly reproduce able > technique. buffer  s y s t e m and c o m p a r e d the banding p a t t e r n s p r e s e n t w h e n  IV.  several  of calf t h y m u s h i s t o n e s w e r e t e s t e d .  also achieved high resolution and reproduceability, w h i c h felt was  bands  they concluded that it w a s  W a n and I r v i n used a slightly different  different preparations  for  s u p e r i o r to that of s t a r c h g e l  HISTONES AS S T R U C T U R A L  They they  electrophoresis;  PROTEINS  C h r o m o s o m e s of h i g h e r o r g a n i s m s c o n t a i n a p p r o x i m a t e l y equal amounts bonds.  of D N A and histone h e l d together  T h e y c a n be s e p a r a t e d  of s a l t s o r a c i d s .  by  b y e x p o s u r e to h i g h  electrostatic concentrations  O n r e m o v a l of the s a l t s o r a c i d s b y d i a l y s i s the  11  DNA and h i s t o n e as  r e - f o r m a n u c l e o h i s t o n e complex which  a gel i n concentrated  solutions, suggesting  behaves  that histone  acts  a molecular bridge l i n k i n g nucleoprotein molecules together well  as  f o r m i n g more i n t i m a t e a s s o c i a t i o n s w i t h DNA.  diffraction regularly  patterns  a l o n g the  suggest large  that histone  b r i d g e s b e t w e e n DNA m o l e c u l e s a r e to  the  sixty  large  g r o o v e o f DNA w i t h  degrees  to  the  involves histones during in  cell  i n the maintenance  structure  (Busch,  is  interphase  at  that  as  variance with acid  92).  involved nuclei.  This  concept  structure  the  finding  of  extraction of histones  a structural  1965, p .  arginine-rich histones acid  an a n g l e o f  organization of chromatin during Somers, d i d not  That p r o t e i n o f  affect  some  c o m p o n e n t h a s b e e n shown b y following  In a recent  showed c o n v i n c i n g e v i d e n c e t h a t fraction,  at  lie parallel  o f chromosome  c o m p l e t e d i s r u p t i o n o f chromosomes  trypsin  in  long axis  o f metaphase chromosomes.  nature i s necessary the  and i n t h e  This i s  C o l e and Hsu (1963) the  their  less  Histone  l o n g a x i s o f DNA ( Z u b a y , 1 9 6 4 ) .  division  terphase.  also proposed to  as  X-ray  i s b o u n d more o r  g r o o v e o f t h e DNA h e l i x .  one h i s t o n e  treatment  article Littau component,  i n holding chromatin together  with et  the  al.  from c a l f  thymus n u c l e i ,  i n clumps  using salt  The n u c l e i w e r e e x a m i n e d b y  (1965)  lysine-rich  T h e y s e l e c t i v e l y r e m o v e d s i n e - r i c h and  extraction procedures.  as  and  electron  m i c r o s c o p y f o r d e p l e t i o n o f c h r o m a t i n a g g r e g a t e s on r e m o v a l of  histones  a n d r e a g g r e g a t i o n w h e n h i s t o n e s w e r e added b a c k to  nuclei.  T h e y r e p o r t that the d e n s e c h r o m a t i n of t h y m o c y t e  is inactive i n R N A synthesis  and is held i n a condensed  c r o s s - l i n k a g e of l y s i n e - r i c h h i s t o n e .  nuclei  state by  A r g i n i n e - r i c h histone  c o m b i n e s w i t h c h r o m a t i n f i b r i l s but does not c r o s s l i n k Since histones  the  also  them.  a r e attached to D N A t h r o u g h salt l i n k a g e ,  i t i s of p a r t i c u l a r i n t e r e s t that s e v e r a l h i s t o n e f r a c t i o n s that  have  been studied contained sufficient b a s i c a m i n o a c i d s to saturate phosphate groups histones  of D N A ,  were extracted  phosphate groups  p r e s e n t i n the n u c l e i f r o m w h i c h  ( V e n d r e l y et a l . ,  of D N A a r e  I960).  Since  the  the  r e g u l a r l y spaced and histones  p r o p o s e d to l i e a l o n g the m a j o r  g r o o v e i n the D N A h e l i x ,  the  were  it was  e x p e c t e d that the b a s i c a m i n o a c i d s of h i s t o n e s m i g h t s h o w a complementary the n u m b e r  spacing.  P h i l l i p s a n d S i m p s o n (1962)  of n o n - b a s i c a m i n o a c i d s that w e r e p r e s e n t  lysine and arginine residues, histone.  investigated between  u s i n g a t r y p t i c d i g e s t of c a l f  S i n c e a p p r o x i m a t e l y one q u a r t e r  of the a m i n o  thymus  acids  p r e s e n t w e r e b a s i c they l o o k e d for e v e n s p a c i n g of l y s i n e and arginine separated  by three non-basic amino acids.  They found  that the b a s i c a m i n o a c i d s w e r e i r r e g u l a r l y s p a c e d a n d v a r i e d f r o m b e i n g i n j u x t a p o s i t i o n to b e i n g s e p a r a t e d amino acids.  by seven  non-basic  It a p p e a r s t h a t m o s t h i s t o n e - D N A i n t e r a c t i o n s  do  not fit a s i m p l e m o d e l of phosphate n e u t r a l i s a t i o n by r e g u l a r l y  13  spaced basic amino acids lying i n p a r a l l e l (Busch,  V.  ACIDIC  1965, p.  108).  NUCLEOPROTEINS  H i s t o n e s have b e e n e x t e n s i v e l y s t u d i e d i n the p a s t b e c a u s e of t h e i r c l o s e a s s o c i a t i o n w i t h n u c l e i c a c i d a n d the r e l a t i v e  ease  w i t h w h i c h t h e y c a n be e x t r a c t e d f r o m n u c l e i i n a s o l u b l e f o r m . However,  t h e y m a k e up o n l y about one t h i r d of the t o t a l p r o t e i n of  the n u c l e u s .  The remainder  of the n u c l e o p r o t e i n s  are insoluble in  a c i d s o l u t i o n s a n d m a k e up 45 t o 50 % of the d r y w e i g h t of the T h e s e a c i d i c n u c l e o p r o t e i n s have not been c h a r a c t e r i s e d , b i o c h e m i c a l l y o r f u n c t i o n a l l y , b e c a u s e of t h e i r p e c u l i a r characteristics.  nucleus.  either solubility  They are insoluble i n m o s t aqueous reagents  for conventional studies  on proteins,  used  being soluble only i n salt  s o l u t i o n s of h i g h m o l a r i t y o r d i l u t e a l k a l i (0. 0 5 N N a O H ) . T h i s g r o u p of p r o t e i n s i s thought to c o m p r i s e a v a r i e t y of m o l e c u l a r s p e c i e s  coming f r o m different nuclear  having different functions,  but a r e g r o u p e d t o g e t h e r b e c a u s e of  t h e i r h i g h c o n c e n t r a t i o n i n the n u c l e u s , characteristics,  the p r e s e n c e  glutamic amino acids,  common  of h i g h amounts  solubility  of a s p a r t i c  a n d the h i g h r a t e at w h i c h t h e y  labelled amino acids in vivo (Busch,  1965, p . p .  S t e e l e a n d B u s c h (1963) p r e p a r e d extracts  structures,  and  incorporate  197 - 3 2 6 ) .  salt and a l k a l i n e  of a c i d i c n u c l e o p r o t e i n s f r o m n u c l e i of n o r m a l l i v e r  a Walker tumour i n a rat.  They compared these proteins for  and amino  14  acid content,  N H ^ - t e r m i n a l amino acids and synthetic  ( i n c o r p o r a t i o n of l a b e l l e d l y s i n e ) i n v i v o . t e n t s of t h e i r f r a c t i o n s w e r e aspartic were  activity  The amino acid con-  s i m i l a r , b e i n g n o t a b l y h i g h (23%) i n  and glutamic acids.  E n d group a n a l y s i s indicated that t h e r e  at l e a s t f i v e d i f f e r e n t p r o t e i n s  i n e a c h of t h e i r  subfractions.  14 T h e d a t a o n i n c o r p o r a t i o n of l y s i n e - C p r o t e i n s t u r n o v e r at d i f f e r e n t tumour.  "In the t u m o r ,  showed that a c i d i c n u c l e o -  r a t e s i n n u c l e i of l i v e r a n d W a l k e r  approximately two-thirds  of the  label  e n t e r e d the histone f r a c t i o n and o n e - t h i r d w a s i n c o r p o r a t e d the a c i d i n s o l u b l e p r o t e i n s . (Steele and B u s c h ,  In the l i v e r ,  the r e v e r s e w a s  isolating chromosomes removing histones  and studying their  with high molar  " r e s i d u a l c h r o m o s o m e s " that  A n earlier  s t u d y on the n a t u r e of the  p r o t e i n had s h o w n that it w a s an a c i d i c p r o t e i n as it w a s i n I M N a C l a t p H 10 ( M i r s k y a n d P o l l i s t e r , a b o u t 1% t r y p t o p h a n e . chromosome  for  chemical composition.  from chromosomes  and l o w p H they d e s c r i b e d D N A and p r o t e i n .  true"  1963).  M i r s k y a n d R i s (1947) d e v e l o p e d a p r o c e d u r e  After  into  1946) a n d  salt  contained residual soluble  contained  T h i s r e s i d u a l p r o t e i n m a d e up 8 - 10% of the  ( M i r s k y and R i s ,  1947).  If h i s t o n e a n d D N A w e r e  r e m o v e d w i t h H C 1 and D N A - a s e the r e m a i n i n g r e s i d u a l  protein  f o r m e d c o i l e d f i l a m e n t s that c o u l d be s e e n i n the e l e c t r o n m i c r o scope. These  R e m o v a l of h i s t o n e d i d n o t affect observations  chromosome  w e r e the b a s i s f o r a m o d e l of  structure.  chromosome  15  structure i n w h i c h residual or acidic proteins about w h i c h D N A i s c o i l e d .  H i s t o n e s a r e thought to f o r m  linkage with D N A phosphate groups of the i n t e r a c t i o n b e t w e e n  on the outer  acidic proteins  the p r e s e n c e of s u b s t a n t i a l l e d to the  f o r m a central  surface.  but  a m o u n t s of s e r i n e i n a c i d i c p r o t e i n  has  1965, p . p .  VI.  CHARACTERISATION OF  91 -  staining has been evident.  the u s u a l m e t h o d for staining histones  method,  however,  interest  Until  recently  w a s the a c i d fast  green  s t r u c t u r e s s u c h as r i b o s o m e s  mucopolysaccharide inclusions (Cowden,  s e l e c t i v e s t a i n i n g of h i s t o n e  formalin ammoniacal staining technique.  This  basic  and  1966).  R e c e n t l y B l a c k and A n s l e y (1965, a m e t h o d for the  histones  form in cell nuclei.  l a c k s s p e c i f i c i t y as i t w i l l s t a i n other  protein-containing  is  HISTONES  s e q u e n c e w h i c h d e p e n d s on the e x t r e m e l y b a s i c n a t u r e of i n a concentrated  esters  119).  In the f i e l d of h i s t o c h e m i s t r y c o n s i d e r a b l e  and their occurrence  nature  and D N A is unknown,  one p o s s i b i l i t y ( B u s c h ,  in differential histone  salt The  s u g g e s t i o n that the f o r m a t i o n of p h o s p h o - s e r i n e  HISTOCHEMICAL  core  1966) h a v e c a l l e d the  This method  developed postresults  i n l y s i n e - r i c h histone being stained y e l l o w and a r g i n i n e - r i c h histone being stained black. stains  This differential colour  reaction  calf thymus n u c l e i yellow and l i v e r nuclei b r o w n i s h - b l a c k ,  indicating that different histones  are present i n different  cell  types.  16  They also r e p o r t e d that t h y m o c y t e s f r o m m i c e injected w i t h antigen s t a i n e d b l a c k i n c o m p a r i s o n to u n t r e a t e d  mouse  thymocytes  w h i c h stained y e l l o w and suggested that this r e p r e s e n t e d r e s p o n s e to antigen,  that is a'discharge  of h i s t o n e ,  a cellular  reflecting a  change i n c e l l function. Spicer  (1962) d e v e l o p e d a n o t h e r  specific for basic proteins, protamines  in cell nuclei.  to the f i x a t i o n p r o c e d u r e s c h a n g e s i n p H of the  staining technique,  which selectively stains histones This method requires  strict  and  adherence  d e s c r i b e d and i s h i g h l y s e n s i t i v e to  s t a i n i n g s o l u t i o n (0. 04% B i e b r i c h  scarlet).  T h i s m e t h o d h a s b e e n u s e d t o s t u d y t h e b a s i c p r o t e i n c o n t e n t of cell components by D o u g l a s , S p i c e r and B a r t e l s At the c h r o m o s o m e l e v e l , S w i f t  (1966).  (1964) u s e d a c i d  fast  g r e e n to stain b a s i c p r o t e i n bands i n D r o s o p h i l a s a l i v a r y gland chromosomes.  T h e a m o u n t of s t a i n i n g d u r i n g f o u r  s t a g e s of puff  f o r m a t i o n at the e n d of c h r o m o s o m e 2 w a s m e a s u r e d w i t h a microdensitometer.  T h e y could find no m e a s u r a b l e  difference  i n the a m o u n t of s t a i n a b l e b a s i c p r o t e i n p e r b a n d d u r i n g the puffing p r o c e s s . histones  H o w e v e r , it has been shown that D N A and  o c c u p y a c l o s e s p a t i a l r e l a t i o n s h i p i n the c h r o m a t i n of  Drosophila polytene chromosomes. chromosome preparations  H o r n a n d W a r d (1957)  stained  for D N A (Feulgen reaction) and for  b a s i c p r o t e i n (acid fast green).  T h e y found that each band or  c h r o m o m e r e that stained for D N A a l s o had a h i g h concentration of b a s i c p r o t e i n . lesser  VII.  T h e i n t e r c h r o m e r i c r e g i o n s s t a i n e d to a m u c h  extent.  ANTIGENICITY  OF NUCLEIC ACIDS A N D N U C L E A R  PROTEINS I m m u n o l o g i c a l t e c h n i q u e s f o r the c h a r a c t e r i s a t i o n of p r o t e i n s h a v e b e e n a p p l i e d to the scattered instances. autoimmune disease ( S . L . E . ).  s t u d y of n u c l e a r c o n s t i t u e n t s  This has m a i n l y been i n reference in humans;  In this disease  the  systemic lupus  Alteration  of n u c l e i of m e s e n c h y m a l c e l l s b y a u t o a n t i b o d y i n the  1950).  The subsequent use  globulin  D N A , histone  a n d a D N A - h i s t o n e c o m p l e x ( K u n k e l et a l . , I 9 6 0 ) .  s e r u m was first demonstrated  to a n  erythematosis  s e r u m contains a g a m m a  w h i c h has a n t i b o d y a c t i v i t y a g a i n s t the p a t i e n t ' s  in  patient's  c y t o c h e m i c a l l y ( K l e m p e r e r et a l . ,  of the f l u o r e s c e n t a n t i b o d y t e c h n i q u e  showed c l e a r l y that c e l l i n j u r y was caused by an i n t e r a c t i o n between S . L . E . suspensions  s e r u m and nuclear  w e r e e x p o s e d to S . L . E .  components.  If c e l l s  s e r u m and then  treated  w i t h f l u o r e s c e n t a n t i - h u m a n g l o b u l i n the n u c l e i s h o w e d m a r k e d fluorescence. presence  T h i s f l u o r e s c e n c e i s n o w k n o w n to be due to  of s e v e r a l a n t i - n u c l e a r f a c t o r s p r e s e n t i n  globulin which are globulin,  counter stained by fluorescent  p r o d u c e d i n the h o r s e  or rabbit.  the  S.L.E.  anti-human  The predominant  factor  18  a p p e a r s to be a n t i b o d y to w h o l e n u c l e o p r o t e i n ( K u n k e l et a l . , I960).  T h e a n t i - n u c l e a r a c t i v i t y i n S. L . E . s e r u m h a s  f o u n d at t i t e r s i n e x c e s s of 1 : 4, 0 0 0 . own cells,  It r e a c t s w i t h t h e  g l o b u l i n ( G o o d m a n et a l . ,  also directed against antigen present has been demonstrated  by Krooth,  serum is  in metaphase  Tobie,  chromosomes  Tjio and G o o d m a n .  of S. L . E . f a c t o r to a l l the c h r o m o s o m e s  Chinese hamster  as w e l l as c h r o m o s o m e s  antigens  show  of h u m a n from  fibroblasts.  A l t h o u g h the histones  cases,  gamma  T h e y u s e d the f l u o r e s c e n t a n t i b o d y t e c h n i q u e to  fibroblasts and leukocytes,  cells,  I960).  T h a t the a n t i - n u c l e a r a c t i v i t y i n S. L . E .  attachment  patient's  c e l l s f r o m other donors and other m a m m a l i a n  a n d a p p e a r s t o b e r e s t r i c t e d to t h e 6. 6S f r a c t i o n of  (1961).  been  are  and D N Anon-antigenic,  c o n s i d e r e d to be w e a k  it has been  possible, in  some  to p r o d u c e a n t i - h i s t o n e a n t i b o d i e s i n e x p e r i m e n t a l  animals.  W i l s o n et a l . (1966) r e p o r t e d t h a t D N A - h i s t o n e  calf thymus was neither antigenic nor haptenic i n rabbits prolonged immunisation.  However, a DNA-histone  from after  complex  f r o m a canine t u m o u r injected into rabbits gave positive complement fixation tests although other negative.  The authors  serological tests  were  i n t e r p r e t e d t h e i r r e s u l t s to m e a n that  some components  of D N A - p r o t e i n s a r e  elicit a response,  but a r e p r e s e n t  s u f f i c i e n t l y a n t i g e n i c to  in very small  I n a n e a r l i e r s t u d y M e i s c h e r et a l .  quantities.  (I960) u s e d  calf  t h y m u s n u c l e i and n u c l e o p r o t e i n to i m m u n i s e guinea pigs rabbits.  A b o u t h a l f the a n i m a l s r e a c t e d p o s i t i v e l y to  antibodies detectable  by complement fixation,  produce  passive  a n a p h y l a x i s a n d the f l u o r e s c e n t a n t i b o d y t e c h n i q u e . p r o t e i n s w e r e f o u n d to be w e a k l y a n t i g e n i c a n d the  cutaneous  Nucleosera  produced showed cross reactivity between rabbit and pig cells.  They also examined tissues  and  guinea  of i m m u n i s e d a n i m a l s  f o r e v i d e n c e of a u t o i m m u n i t y a g a i n s t the a n i m a l s o w n n u c l e i but could find  none. In a short  communication R u m k e and Sluyser  r e p o r t e d a m e t h o d of p r e p a r a t i o n i n w h i c h h i s t o n e s liver are  s t r o n g l y a n t i g e n i c to r a b b i t s .  (1966)  from  B r i e f heat treatment  (5 m i n u t e s a t 8 0 ° C ) o f t h e t i s s u e b e f o r e h i s t o n e e x t r a c t i o n c a r r i e d out to i n a c t i v a t e p r o t e o l y t i c e n z y m e s breakdown. acid,  and prevent  The histones were extracted with N a C l ,  H C 1 a n d e t h a n o l ( S l u y s e r et a l . ,  rat  1965).  A  was histone  perchloric  lysine-rich  f r a c t i o n w a s f o u n d to be m o s t a n t i g e n i c a n d p r o d u c e d a s i n g l e p r e c i p i t a t i o n l i n e i n the O u c h t e r l o n y t e s t w h i c h d i d not react with other histone fractions antibody studies contents  or with rat  serum.  crossFluorescent  r e v e a l e d that this a n t i s e r u m r e a c t e d w i t h  of n u c l e i of r a t l i v e r s e c t i o n s a n d c e l l  suspensions.  Another l y s i n e - p o o r histone fraction was less antigenic. a n t i s e r u m d i d not r e a c t w i t h the l y s i n e - r i c h h i s t o n e , weakly with rat serum.  the  but  It p r o d u c e d t w o d i s t i n c t l i n e s of  The reacted  20 p r e c i p i t a t e a g a i n s t the i n o c u l u m .  The authors  conclude that  l i v e r h i s t o n e s a r e a n t i g e n i c to the r a b b i t a n d c a n be into i m m u n o c h e m i c a l l y distinct fractions.  rat  separated  21  METHODS AND MATERIALS  I.  IMMUNISATION PROCEDURE  1.  Isolation of Calf  F o l l o w i n g the fresh  thymus  procedure  t i s s u e was o b t a i n e d ,  and p l a c e d i n the  cold.  o f D a l y and M i r s k y  (1955),  1 to  slaughter,  The t i s s u e  a n d as much c o n n e c t i v e t i s s u e mately  Thymus N u c l e i  3 hours  was c l e a r e d o f f a t ,  as p o s s i b l e r e m o v e d .  150 gm o f t i s s u e i*as c o m b i n e d w i t h  crushed  i c e and 400 m l 0 . 0 0 5 M c i t r i c  gauze and c e n t r i f u g e d centrifuge  at  acid,  f o r 20 m i n u t e s .  c o n t a i n i n g c y t o p l a s m i c and t i s s u e p l a c e d i n a f r o z e n s t o r a g e at washed i n 0 . 0 1 M c i t r i c  acid until  the  Approxi-  strained  through  refrigerated  The  proteins,  -20°C.  minced,  an e q u a l v o l u m e o f  i n 250 m l t u b e s i n a  low speed  after  supernatant, was d e c a n t e d  The n u c l e a r  and  sediment  s u p e r n a t a n t was f r e e  protein.  2.  Nuclear  Protein  Nuclear proteins nuclear preparation  xvere e x t r a c t e d  from  the  u s i n g a m o d i f i c a t i o n o f one o f  several  was of  22  procedures  ( p r e p a r a t i o n 8) d e s c r i b e d b y D a l y a n d M i r s k y  A n e q u a l a m o u n t of 0. 2 M c i t r i c a c i d ,  1. 2 5 M N a C l  (1955).  ( p H 2 . 0) w a s  added to the n u c l e a r p r e p a r a t i o n and s t i r r e d f o r one h o u r i n a n ice bath.  T h e p r e p a r a t i o n w a s c e n t r i f u g e d a t 1, 5 0 0 R P M t o  p e l l e t the n u c l e a r m a s s and s e p a r a t e l i p i d m a t e r i a l . w a s c a r e f u l l y s k i m m e d off a n d the  s t o r a g e at  separated  lipid  supernatant decanted.  c o n c e n t r a t i o n w a s d e t e r m i n e d w i t h the r e f r a c t o m e t e r solution was  The  Protein  and  the  into 5 m l aliquots and p l a c e d i n f r o z e n  -20°C.  3.  P r e p a r a t i o n of I n o c u l u m  The whole nuclear protein solution was prepared i n j e c t i o n b y d i a l y s i s of 5 m l s a m p l e s  enclosed in 27/32" dialysis  t u b i n g w i t h a w a l l t h i c k n e s s of 0. 001 i n c h e s Chicago,  111. ) a n d p l a c e d i n a l i t r e b e a k e r  the r e f r i g e r a t o r .  for  ( Visking Corp. ,  of d i s t i l l e d w a t e r  T h e d i a l y s a t e w a s c h a n g e d e v e r y 24 h o u r s  t h r e e d a y s a f t e r w h i c h t h e p H of t h e d i a l y s a t e w a s at  in for  neutrality  a n d the p r o t e i n s w i t h i n the d i a l y s i s bag had f o r m e d a w h i t e precipitate.  The precipitate  and supernatant w e r e diluted i n  p h o s p h a t e b u f f e r e d i s o t o n i c s a l i n e to a c o n c e n t r a t i o n of a p p r o x i mately 4 m g / m l .  4.  Immunisation  M a t u r e L e g h o r n hens w e r e obtained f r o m the  Depart-  23  merit of P o u l t r y S c i e n c e , They were housed wing tags.  singly i n w i r e cages and identified by  as f o l l o w s :  1 m l i n t r a m u s c u l a r l y i n the right l e g and 3 m l  This procedure the 8th w e e k  was repeated 3 times  10 m l o f b l o o d w e r e  and placed i n tubes i n a water  During  d r a w n f r o m the b r a c h i a l v e i n s  b a t h at 4 0 ° C f o r  12 h o u r s t o a l l o w  After  clot  retraction  blood was obtained by cardiac puncture, absent or s c a r r e d beyond  PROTEIN  In s o m e  to  frozen.  r o u t i n e l y t a k e n off f o o d f o r 24  p r i o r to b l e e d i n g to l o w e r s e r u m l i p i d content.  hours  cases  when wing veins  were  use.  ANALYSIS  Chromatography  A.  D e s c r i p t i o n of C h r o m a t o g r a p h i c  Chromatographic ing manner.  wing.  placed i n 2 m l storage bottled and stored  The chickens were  1.  of the  r e m o v e d f r o m the tubes w i t h a c a t h e t e r attached  a 10 m l s y r i n g e ,  II.  intra-  a t 14 d a y i n t e r v a l s .  c l o t f o r m a t i o n w i t h o u t h e m o l y s i s to o c c u r . sera were  nuclear  1 m l i n t r a m u s c u l a r l y i n the  v e n o u s l y t h r o u g h the b r a c h i a l v e i n on the u n d e r s i d e  the  metal  T h e c h i c k e n s w e r e i n o c u l a t e d w i t h 5 m l of the  protein preparation breast,  T h e U n i v e r s i t y of B r i t i s h C o l u m b i a .  columns were prepared  Columns  i n the  A #4 T e f l o n s t o p c o c k a n d a # 2 4 / 4 0 P y r e x  follow-  glass  24  j o i n t w e r e f i t t e d to e i t h e r e n d of a 2 . 5 c m g l a s s t u b e , length.  34 c m i n  A p o r o u s g l a s s d i s c w a s f i t t e d i n the b o t t o m of the  to support the g e l bed.  tube  Two such columns were fabricated by  Vancouver Scientific Glassblowing L t d . , Vancouver, B . C. could be u s e d singly or connected i n t a n d e m .  They  Each column had  3 a b e d v o l u m e of 1 9 5 . 0 c m  .  When l a r g e r columns were required, two 1 l i t r e graduated d i s p e n s i n g c y l i n d e r s h a v i n g an i n s i d e d i a m e t e r 2. 5 c m and 70. 0 c m i n length w e r e used.  These are  equipped  w i t h s t o p c o c k s a n d c a n b e s e a l e d at t h e t o p w i t h a d r i l l e d stopper.  of  rubber  A s m a l l a m o u n t of g l a s s w o o l o n t o p of a p o r o u s p l a s t i c  d i s c w a s p l a c e d i n the b o t t o m of t h e c o l u m n s to s u p p o r t the  gel  bed. B.  Column  Packing  Sephadex G-75 and G - 2 0 0 w e r e obtained f r o m Fine Chemicals,  Inc. , i n the bead f o r m .  The Sephadex was  p e n d e d i n a l a r g e e x c e s s of e l u t i n g buffer and a l l o w e d to completely and settle.  Pharmacia  The excess buffer was  hydrate  F"siphom@'<i o f f t o  r e m o v e fine p a r t i c l e s , the Sephadex w a s suspended i n buffer s t o r e d at r o o m  sus-  and  temperature.  Two methods  of p a c k i n g the c o l u m n s w e r e  employed.  In the f i r s t m e t h o d a 1 l i t r e s e p a r a t o r y funnel w a s attached to  the  25  c o l u m n to p r o v i d e a r e s e r v o i r homogeneous buffer  of S e p h a d e x  by an electric s t i r r e r .  suspension,  The column was filled with  and a f e w m l of S e p h a d e x a l l o w e d to s e t t l e u n d e r  p r o v i d e a b a s e for the gel bed.  kept  g r a v i t y to  T h e c o l u m n w a s t h e n a l l o w e d to  run freely and a steadily r i s i n g l e v e l bed was maintained, out s t o p p i n g u n t i l the d e s i r e d h e i g h t w a s r e a c h e d . filter-paper  disc was  it when applying  with-  Finally,  a l l o w e d to s e t t l e o n top of the b e d to  a protect  samples.  Since this method results  in a tightly packed  w h i c h s o o n c e a s e s to f l o w at a s u f f i c i e n t r a t e ,  column  a second  method  was e m p l o y e d w h i c h does not bind the h y d r a t e d gel beads tightly together.  A glass  cylinder slightly larger in  so  diameter  t h a n the c o l u m n w a s f i t t e d to the top of the c o l u m n u s i n g a  rubber  seal.  filled  The column,  and the r e s e r v o i r thus p r o v i d e d , w e r e  w i t h a s u s p e n s i o n of e q u a l v o l u m e s of h y d r a t e d S e p h a d e x buffer.  The Sephadex was  out b u f f e r  elution.  gravity with-  W h e n it had c o m p l e t e l y settled the buffer  a l l o w e d to f l o w s l o w l y , r e s e r v o i r and excess added,  a l l o w e d to settle under  and  c a u s i n g the b e d to p a c k e v e n l y .  gel were  removed,  was  The  a s m a l l amount  of  buffer  a n d the top of the b e d s t i r r e d to a d e p t h of about 2 i n c h e s  and a l l o w e d to  resettle.  The columns were tested for even packing by eluting a s a m p l e o f 0 . 1% d e x t r a n b l u e ,  a high molecular weight  indicator  26  w h i c h p a s s e s t h r o u g h the c o l u m n i n a single band without  hin-  drance.  C.  Protein Fractionation  To introduce  s e r u m or nuclear protein samples  for  f r a c t i o n a t i o n the buffer l e v e l w a s l o w e r e d i n the c o l u m n to top of the g e l b e d .  The s a m p l e was l a y e r e d on c a r e f u l l y and  a l l o w e d to s i n k into the g e l u n t i l the l i q u i d l e v e l w a s w i t h the top of the b e d .  again  even  The c o l u m n was then filled w i t h buffer  a n d a 2 0 l i t r e b u f f e r t a n k i n s t a l l e d 12 i n c h e s a b o v e t h e and connected w i t h p l a s t i c tubing. n e c t e d to a p e r i s t a l t i c p u m p  column  The c o l u m n outlet was  con-  (Sigmamotor Inc. , M i d d l e p o r t ,  N . Y . ) w h i c h provided a constant flow rate. were  the  Effluent  fractions  c o l l e c t e d i n 10 m l t u b e s i n a n I s c o m o d e l 2 7 0 f r a c t i o n  collector  (Instrumentation  5 minute intervals, flow rate selected. was read,  Specialties Co. , Lincoln,  the amount i n e a c h tube depending T h e o p t i c a l d e n s i t y of the d i l u t e  at t h e a p p r o p r i a t e  spectrophotometer  wave length,  on the fractions  on a U n i c a m  Sp-500  (Unicam Instruments Ltd. , Cambridge,  gland) and plotted against tube  D.  N e b r . ) at  . Protein  Protein fractions  En-  number.  Concentration  c o m p r i s i n g s e v e r a l tubes (50-200  ml)  27  were reconcentrated  by two methods.  Initially,  were reconcentrated  using a vacuum dialysis apparatus  f r o m Schleicher and Schuell C o . , Keene, N . H . c o n t a i n i n g the d i l u t e p r o t e i n ,  is suspended  an a i r t i g h t s e a l b e t w e e n the i n n e r and outer v a c u u m p u m p i s connected to the outer pressure forces water bag.  serum  purchased  A collodion bag,  in distilled water chambers.  chamber  with  When a  atmospheric  and salts t h r o u g h the w a l l s of the d i a l y s i s  A t a v a c u u m p r e s s u r e of 20 c m m e r c u r y the  rate decreased  proteins  concentration  f r o m 4 - 6 m l p e r h o u r t o 0. 25 - 0. 5 m l p e r h o u r  concentration proceeded.  Although this method was  the c o n c e n t r a t i o n of s e r u m p r o t e i n f r a c t i o n s , weight proteins,  suitable  as  for  small molecular  p r e s e n t i n the n u c l e a r p r o t e i n f r a c t i o n s ,  passed  t h r o u g h the w a l l s of the d i a l y s i s b a g . A more  efficient concentrator  The A m i c o n Corporation (Cambridge, This concentrator membranes  has  was purchased  from  M a s s . ), D i a f l o m o d e l 5 0 .  a 50 m l c a p a c i t y a n d m a k e s u s e - o f  gel  U M - 1 and U M - 2 w h i c h w i l l a l l o w p a s s a g e of m o l e c u l e s  having a " M W  l a r g e r t h a n 1, 0 0 0 a n d 1 0 , 0 0 0 r e s p e c t i v e l y .  a p r e s s u r e of 50 P S I p r o v i d e d b y a n i t r o g e n c y l i n d e r t h e  Under  concen-  t r a t i o n r a t e v a r i e d f r o m a p p r o x i m a t e l y 20 m l p e r h o u r to 5 m l p e r hour with increasing concentration.  T h i s concentration has  added advantage that the m e m b r a n e s  w i l l not p l u g w i t h  use.  the  repeated  28  2.  Disc  A.  Electrophoresis  . Acidic  Proteins  A model 6 disc electrophoresis  apparatus and  were purchased from Canalco Corporation, standard for  a c r y l a m i d e gels and buffer  Md.  (Ornstein,  Serum protein samples were  the s t a c k i n g gel i n the e l e c t r o p h o r e s i s that the standard  order  columns.  This  required  of p o l y m e r i z i n g the g e l s be r e v e r s e d .  of m o r e d i l u t e s o l u t i o n s .  proteins made  the  of l a r g e r  T h e u s e of f l u o r e s c e n t  it p o s s i b l e to o b s e r v e the p r o g r e s s  The  sucrose-  T h i s m e t h o d h a s t h e a d v a n t a g e of r e d u c i n g  t i m e for an a n a l y s i s and a l l o w i n g the e l e c t r o p h o r e s i s samples  the  and l a y e r e d o n top of  c o l u m n s w e r e t h e n f i l l e d b y l a y e r i n g buffer o n top of the protein solution.  mixed  For  in nuclear protein preparations  s a m p l e s w e r e d i l u t e d i n 0. 1 m l , I ' M s u c r o s e  used  1965) w i t h  w i t h a s a m p l e g e l as i n s t r u c t e d i n the C a n a l c o m a n u a l . a n a l y s i s of a c i d i c p r o t e i n s  The  ( 7 % , p H 8 . 9 - 9 . 5) w e r e  the a n a l y s i s of s e r u m p r o t e i n f r a c t i o n s  the f o l l o w i n g m o d i f i c a t i o n s .  Bethesda,  chemicals  serum  of e l e c t r o -  p h o r e t i c s e p a r a t i o n u s i n g the u l t r a v i o l e t l a m p and on c o m p l e t i o n of a r u n t h e t u b e s c o u l d be p h o t o g r a p h e d fixation,  directly without  further  s t a i n i n g and de s t a i n i n g .  B.  Basic  Proteins  The electrophoretic  separation  of b a s i c n u c l e a r  proteins  requires  the u s e of l o w p H g e l s of s m a l l e r p o r e  W a n and Irvin,  1963; S h e p h a r d a n d G u r l e y ,  p o s s i b l e to a c h i e v e  size  1965).  (McAllister,  It w a s  not  s e p a r a t i o n o f n u c l e a r p r o t e i n u s i n g the  method  d e s c r i b e d i n t h e C a n a l c o m a n u a l f o r l o w p H g e l s (15%, p H 5. 0 - 4 . 3) until glycine was as  s u b s t i t u t e d f o r b e t a a l a n i n e i n the r u n n i n g  d e s c r i b e d by M c A l l i s t e r ,  W a n a n d I r v i n (19 6 3 ) .  It w a s  buffer  also  found that b a s i c n u c l e a r p r o t e i n s w o u l d not m i g r a t e if a p p l i e d i n a sample  g e l , but that they m u s t be a p p l i e d i n a 1 M s u c r o s e  medium.  S i n c e the n u c l e a r p r o t e i n s w e r e n o t l a b e l l e d w i t h fluorescent  d y e t h e g e l s w e r e f i x e d i n 7% a c e t i c a c i d a n d  o v e r n i g h t i n 0. 1% A m i d o  Black  1 0 B , i n 7% a c e t i c a c i d s o l u t i o n .  A n o t h e r u s e f u l m o d i f i c a t i o n w a s f o u n d to b e r e v e r s a l o f electrodes  stained  d u r i n g the d e s t a i n i n g p r o c e d u r e  the  so t h a t u n b o u n d  w a s m o v e d u p w a r d t h o u g h the c o l u m n s a n d into the u p p e r chamber.  T h i s a v o i d s c o n t a m i n a t i o n o f the d e s t a i n i n g  dye  buffer  solution  w i t h dye d u r i n g the i n i t i a l p e r i o d of d e s t a i n i n g . S h e r i d a n a n d S t e r n (1967) a n a l y s e d t h e h i s t o n e o f p l a n t c e l l s u s i n g a f u r t h e r m o d i f i c a t i o n of the t e c h n i q u e of M c A l l i s t e r , W a n and Irvin, acrylamide,  w h i c h p r o v i d e s e v e n s m a l l e r g e l p o r e s s i z e s (18%  10% c a t a l y s t ) .  nuclear proteins and Irvin, greater  T h i s m e t h o d d i d not separate  as efficiently as  the m e t h o d o f M c A l l i s t e r ,  but p r o v i d e d w i d e r bands w h i c h s e p a r a t e d by a  distance.  basic Wan  30  Basic nuclear proteins,  electrophoresed  i f i c a t i o n of S h e r i d a n and S t e r n (1967),  were  u s i n g the  s t a i n e d f o r the  presence  of h i s t o n e u s i n g a m o d i f i c a t i o n of a h i s t o c h e m i c a l t e c h n i q u e oped by Douglas, Spicer and B a r t e l s  (1966).  mod-  devel-  The  acrylamide  cylinders w e r e fixed i n C a r n o y ' s fluid for 6 hours  and stained  n i g h t i n a 0. 0 4 % s o l u t i o n of B i e b r i c h s c a r l e t i n I N g l y c i n e adjusted  to p H 9. 5 w i t h I N N a O H .  Destaining was  over-  buffer,  accomplished,  u s i n g 7% a c e t i c a c i d , i n 9 0 m i n u t e s .  T h e stained d i s c s of p r o t e i n  were photographed  destaining,  i m m e d i a t e l y after  since  p a r t i a l l y d i f f u s e d out of t h e a c r y l a m i d e i n a b o u t 2 4  3.  Amino Acid  chicken i m m u n i s a t i o n s and fractions p r e p a r e d were  by  of M i c r o b i o l o g y ,  Vancouver,  B . C . , using a B e c k m a n , m o d e l 120C  amino acid  analyser.  Sephadex-75  T h e U n i v e r s i t y of B r i t i s h C o l u m b i a ,  Serum  automatic  Proteins  T h e c o n t e n t s of s e r u m f r a c t i o n s , were  for  Immunodiffusion  A.  tography,  used  a n a l y s e d for a m i n o a c i d content i n the  Department  4.  hours.  Analysis  The whole nuclear protein preparation,  chromatography,  they  prepared  by  chroma-  also analysed i m m u n o l o g i c a l l y on M i l l i p o r e  31  cellulose acetate m e m b r a n e s  using a M R P - 2 ,  Immunodiffusion K i t (National Instruments Rockville,  M d . ).  Briefly,  NIL-Saravis  L a b o r a t o r i e s , Inc. ,  t h e m e t h o d c o n s i s t s of r e a c t i n g l a m b d a  a m o u n t s of antibody ( a n t i - c h i c k e n s e r u m or a n t i - c h i c k e n globulin) in a central well against antigen (chicken s e r u m proteins, surrounding wells,  o v e r a d i s t a n c e of 8 m m .  place in a saline-saturated  in 4  The reactions  cellulose acetate m e m b r a n e ,  w i c h e d between a r e s i l i e n t base and an o v e r l y i n g p l a s t i c into w h i c h the w e l l s have been m a c h i n e d .  Under  sandtemplate  standard  c o n d i t i o n s the p r e c i p i t i n l i n e s r e q u i r e 48 h o u r s to d e v e l o p w h i c h t i m e they were  stained and fixed,  and  s u p p l i e d b y the  The m e m b r a n e was then mounted on a glass  c l e a r e d i n an a c i d - a l c o h o l bath.  It c o u l d t h e n b e  slide  photographed,  p r i n t e d d i r e c t l y on to p h o t o g r a p h i c p a p e r i n the e n l a r g e r or as a p e r m a n e n t  after  using a Ponceau-S dye,  trichlorecetic-sulfosalycylic acid preparation, manufacturer.  take  stored  record.  B.  Nuclear Proteins  A l t h o u g h p r e c i p i t i n l i n e s c o u l d be obtained w i t h the m i l l i p o r e m e t h o d u s i n g a n t i b o d y p r o d u c e d i n the c h i c k e n calf t h y m u s n u c l e a r proteins and a c o m m e r c i a l histone (Nutritional Biochemical Co. , Cleveland,  against preparation  O h i o ) the l i n e s of  p r e c i p i t a t e w e r e diffuse a n d c o u l d not a l w a y s be r e p r o d u c e d i n the  32  same  pattern. A s an alternative,  immunodiffusion tests were  out u s i n g the O u c h t e r l o n y t e c h n i q u e (Ionagar N o . 2,  powdered  at p H 5. 0 u s i n g W a l p o l e ' s a c e t i c  (Culling,  agar was  u n t i l the  1963,  p.  cylinders  of 1:10, 0 0 0 .  a r o u n d the  were  was  added to g i v e a f i n a l  and a l l o w e d to s o l i d i f y .  A further  cylinders.  by drying for  159) i n i s o t o n i c s a l i n e .  T h e a g a r s o l u t i o n (10 m l ) w a s  (1/4" diameter)  sample wells.  1 hour  acid-sodium  at r o o m  C.  boiling  con-  Small hollow porous  w e r e p o s i t i o n e d o n the a g a r to f o r m 10 m l o f a g a r s o l u t i o n w a s t h e n  Excess water was at 3 7 ° C .  a  pipetted  The s e r u m and histone  the  poured  r e m o v e d f r o m the  plates  samples  a p p l i e d to the w e l l s w i t h a s y r i n g e and the r e a c t i o n s  to p r o c e e d  The  It w a s r e m o v e d f r o m the h e a t a n d  s t o c k s o l u t i o n o f 1% m e r t h i o l a t e  into P e t r i dishes  Baltimore,  s t i r r e d into the b o i l i n g solvent and kept  agar had d i s s o l v e d .  centration  A s o l u t i o n o f 1% a g a r  Baltimore Biological Laboratories,  M d . ) was buffered acetate buffer  (1948).  carried  allowed  temperature.  I m m u n o l o g i c a l A n a l y s i s of  Electrophoretic  Fractions  Acrylamide cylinders, proteins  were  c o n t a i n i n g d i s c s of b a s i c  r e m o v e d f r o m the e l e c t r o p h o r e s i s  i m b e d d e d i n 1% I o n a g a r b u f f e r e d  at p H 5. 0.  columns  nuclear and  A n t i s e r u m troughs  were formed,  p a r a l l e l to the a c r y l a m i d e c y l i n d e r , b y i m b e d d i n g  3/16" diameter  g l a s s r o d s of the  after the g e l h a d s o l i d i f i e d .  s a m e length and r e m o v i n g  The plates were dried for  them  1 hour  at  o 37  C and c h i c k e n a n t i n u c l e a r p r o t e i n s e r u m added to the  troughs.  A control cylinder, prepared  at t h e s a m e t i m e , w a s f i x e d  stained b y the u s u a l m e t h o d .  The precipitin lines could then  c o r r e l a t e d w i t h the 5.  stained  Precipitin  The presence  Tests  of p r e c i p i t a t i n g antibody i n the  s e r u m of carried  Nuclear protein solution, buffered  p H 7. 0, w a s d r a w n i n t o t h e t u b e s f o r a d i s t a n c e of about capillary action.  1 i n c h by  The end of the tube w a s p l u g g e d w i t h  P l a s t i c e n e and the tube i n v e r t e d and stood upright i n a T h i s p r o v i d e d an a i r - l i q u i d m e n i s c u s  the tube on w h i c h p r e c i p i t a t e s of the  to  T h i s w a s f o l l o w e d b y an e q u a l a m o u n t of the  a n t i s e r u m to be t e s t e d .  block.  be  discs.  immunised chickens was determined by a m i c r o method out i n h e m a t o c r i t t u b e s .  and  n e a r the b o t t o m of It a l s o c a u s e d  mixing  s e r u m and n u c l e a r p r o t e i n as the m o r e dense s e r u m  settled  to the b o t t o m .  could settle.  Plasticene  A m e a s u r e of the  s t r e n g t h of the a n t i s e r a  selected  w a s p r o v i d e d b y t i t r a t i n g i t a g a i n s t d o u b l i n g d i l u t i o n s of the protein solution.  nuclear  34  III.  ABSORPTION  Unwanted reactions against cytoplasmic proteins r e m o v e d f r o m the a n t i s e r u m by m i x i n g 5 m l aliquots of  were  serum  w i t h 1 m l of c y t o p l a s m i c s o l u t i o n o b t a i n e d f r o m the c i t r i c  acid  w a s h of calf t h y m o c y t e s .  The cytoplasm was prepared for  sorption by dialysis  phosphate buffered isotonic saline (pH  7.0).  The c y t o p l a s m - s e r u m solution was agitated slowly on a  Fisher bath,  with  C l i n i c a l Rotator ( F i s h e r Scientific C o . ) for 1 hour i n an ice  t h e n c e n t r i f u g e d at a m o d e r a t e  The reaction was repeated precipitin  IV.  ab-  s p e e d to r e m o v e p r e c i p i t a t e s .  as m a n y as 3 t i m e s to r e m o v e a l l  activity.  NON-IMMUNE  SERUM  T w o c h i c k e n s w e r e m a i n t a i n e d t h r o u g h o u t the c o u r s e of the e x p e r i m e n t s to p r o v i d e a s u p p l y of s e r u m w h i c h d i d not h a v e activity against calf thymus cell constituents. labelled with fluorescent dye, i n the s a m e m a n n e r  This serum  chromatographed and  as i m m u n e s e r u m .  was  concentrated  T h i s p r o v i d e d s a m p l e s of  w h o l e s e r u m and f l u o r e s c e n t g l o b u l i n f r a c t i o n s to be u s e d as for i m m u n e  V.  controls  reactions.  LUPUS ERYTHEMATOSIS  Human L . E .  SERUM  s e r u m was obtained f r o m patients  at  the  35  Vancouver  General Hospital.  It w a s t e s t e d f o r a n t i - c a l f t h y m u s  h i s t o n e a c t i v i t y b y p r e c i p i t i n t e s t s a n d i m m u n o d i f f u s i o n i n 1% Ionagar.  VI.  FLUORESCENT DYE-PROTEIN  The conjugation procedure t h a t o f R i g g s et a l . , ( 1 9 5 8 ) , the r e a c t i o n m i x t u r e s thiocyanate Los  CONJUGATION  followed was  except that a c e t o n e w a s not added  (Cohen e t a l . ,  I960).  California,  Laboratories,  i n a stable p o w d e r e d f o r m w h i c h  b e s t o r e d f o r l o n g p e r i o d s at r o o m t e m p e r a t u r e .  A  s e r u m b u f f e r e d at p H 9. 0 w i t h 0. 1 M c a r b o n a t e  added to the ratio.  buffer,  the  0. 3 m l  F o r e a c h m g of p r o t e i n 0. 05 m g of F I T C  surface  of the  s e r u m to p r o v i d e a 1 : 20  was  dye-protein  The r e a c t i o n m i x t u r e w a s agitated s l o w l y i n an i c e bath on  the c l i n i c a l r o t a t o r ,  at s p e e d s n o t e x c e e d i n g 70 R P M i n o r d e r  avoid foaming, for 6 - 8  VII.  could  refractometer  reading w a s taken to d e t e r m i n e p r o t e i n concentration and  p e r m l of s e r u m .  to  Fluorescein iso-  (FITC) was purchased f r o m Hyland  Angeles,  essentially  HISTOLOGICAL  1.  hours.  TECHNIQUES  Mammalian Cells  A.  Peripheral Blood  Cultures  Bovine and h u m a n c h r o m o s o m e p r e p a r a t i o n s  were  to  36  obtained f r o m l e u k o c y t e c u l t u r e s u s i n g a m o d i f i c a t i o n of Hirschhorns microtechnique (Montagu,  1961, p p 46 - 4 7 ,  See  A p p e n d i x I).  B.  Buccal  Smears  H u m a n epithelial cells were prepared for  fluorescent  m i c r o s c o p y b y a m o d i f i c a t i o n of a s t a n d a r d t e c h n i q u e used for sex chromatin determination (Barr,  routinely  1959, See A p p e n d i x  ID.  C.  Blood  Smears  Uncultured leukocytes were prepared by making o f f r e s h w h o l e b l o o d a n d a l l o w i n g t h e m t o d r y at r o o m  smears  temperature.  E r y t h r o c y t e s w e r e r e m o v e d b y i m m e r s i n g t h e s l i d e s b r i e f l y i n 1% acetic acid and thence into i s o t o n i c buffered  D.  Organ  s a l i n e , p H 7. 0.  Imprints  Organs were prepared for imprinting by slicing  the  t i s s u e w i t h a r a z o r b l a d e to p r o v i d e a flat s u r f a c e .  Imprints  were  m a d e b y p r e s s i n g the  slide and  air  surface  gently against a glass  d r y i n g o v e r n i g h t at r o o m t e m p e r a t u r e t o ^ s t i c k t h e c e l l s t o  the  slide. Bovine organs w e r e obtained,  1-2  h o u r s after  slaughter,  37  from Canada Packers Ltd. , Vancouver, B . C.  Slides were  p r e p a r e d i m m e d i a t e l y f r o m the f r e s h t i s s u e and the  remainder  o s t o r e d f r o z e n at - 2 0  C.  Human organs were made Laboratory,  available by The Autopsy  Vancouver General Hospital,  w h e n they w e r e i m p r i n t e d and the organs 2.  Insect  A.  2 - 3  days after  death,  stored frozen.  Material  Polytene  Chromosomes  Salivary glands w e r e dissected f r o m live C h i r o n o m u s tentans l a r v a e by amputating the head w i t h a pointed s c a l p e l and s q u e e z i n g out the contents needle.  of the s e c o n d s e g m e n t w i t h a d i s s e c t i n g  T h e g l a n d s w e r e f i x e d b r i e f l y i n a d r o p of I N H C 1 a n d  s q u a s h e d b y t h e w e i g h t of t h e c o v e r g l a s s . on the m i c r o s c o p e under l o w p o w e r  The slide was placed  (3. 5 X ) a n d the c o v e r  r o t a t e d gently to b r e a k up the c e l l m e m b r a n e s chromosomes,  and expose  u s u a l l y l y i n g o n top of a m e m b r a n e  the  segment.  c e l l u l a r m a t e r i a l w a s t h e n q u i c k f r o z e n by p l a c i n g the b l o c k of d r y i c e .  glass  The  slide on a  T h i s a l s o c a u s e d the c o v e r g l a s s to d i s l o d g e .  T h e p r e p a r a t i o n w a s a l l o w e d to t h a w a n d a i r d r y at r o o m  temper-  a t u r e i n p r e p a r a t i o n f o r a p p l i c a t i o n of f l u o r e s c e n t a n t i b o d y . f i x a t i o n w i t h H C 1 the c h r o m o s o m e s  After  c o u l d be e x a m i n e d d i r e c t l y w i t h  t h e p h a s e m i c r o s c o p e o r s t a i n e d w i t h 0 . 5% a c e t o - o r c e i n .  38  Another alternative, scopy,  i n preparation for phase  w a s to p l a c e the g l a n d i n 1/15  fixative,  M phosphate buffer,  to s w e l l the c e l l s o s m o t i c a l l y .  the w e i g h t of the c o v e r g l a s s w a s  microwithout  Under these conditions  s u f f i c i e n t to s p r e a d the  s o m e s w h i l e k e e p i n g the n u c l e a r m e m b r a n e  intact.  chromo-  More  h y p o t o n i c s o l u t i o n s w e r e f o u n d t o c a u s e c o m p l e t e d i s i n t e g r a t i o n of the c e l l s ,  i n c l u d i n g the  B.  chromosomes.  Protozoan Cells  Preparations  of the i n t e s t i n a l p a r a s i t e  w e r e m a d e b y s q u e e z i n g the i n t e s t i n a l contents Heterotermes  on to a glass  membranes.  of the  termite  s l i d e and a l l o w i n g t h e m to a i r d r y .  i n s u r e that f l u o r e s c e n t antibody m a d e d r o p of 1/15  Holomastigotoides  To  contact w i t h the n u c l e u s  M p h o s p h a t e buffer w a s added to r u p t u r e the  a  outer  F l u o r e s c e n t a n t i b o d y w a s added d i r e c t l y to the  wet  preparation.  3.  Plant Materials  M i t o t i c plant c e l l s of L i l l i u m and A l l i u m w e r e  prepared  b y r e m o v i n g the r o o t t i p s a n d i n c u b a t i n g t h e m f o r 30 m i n u t e s 3 7 ° C i n a 2 . 0% s o l u t i o n o f p e c t i n a s e Company,  Cleveland,  and S h a r m a ,  1965, p.  (Nutritional  Biochemical  Ohio) to soften and s e p a r a t e the c e l l s 106).  The root tips were  at  macerated,  (Sharma  39  spread on glass slides and a i r d r i e d .  4.  Antibody  Application  M a t e r i a l s that had been p r e p a r e d w i t h o r g a n i c fixatives w e r e b r o u g h t to an aqueous m e d i u m b y p a s s a g e t h r o u g h i n c r e a s i n g d i l u t i o n s o f t h e f i x a t i v e u s e d a n d f i n a l l y i n t o 0 . 02 M buffered isotonic saline.  phosphate  M a t e r i a l s that w e r e fixed by d r y i n g  p l a c e d d i r e c t l y i n the buffer for s e v e r a l m i n u t e s . s l i d e s w e r e k e p t w e t t h r o u g h o u t the r e s t of the  In both cases  with absorbent paper  surrounding  and a d r o p of f l u o r e s c e n t  s p r e a d o v e r the s u r f a c e of the t e s t m a t e r i a l s .  The slides  antibody were  p l a c e d i n a P e t r i d i s h c o n t a i n i n g a w e t p i e c e of f i l t e r p a p e r provide a saturated  atmosphere.  the  procedure.  E x c e s s buffer w a s r e m o v e d f r o m the s l i d e s , the t i s s u e s ,  were  to  The slides w e r e rotated gently on  the c l i n i c a l r o t a t o r f o r 30 m i n u t e s ,  r e m o v e d and r i n s e d i n buffer,  t h e n w a s h e d f o r 30 m i n u t e s i n 3 c h a n g e s of b u f f e r ,  on the  rotator.  A c o v e r g l a s s w a s a p p l i e d u s i n g the buffer as m o u n t i n g m e d i u m . The s l i d e s could be v i e w e d d i r e c t l y or sealed w i t h m e l t e d p a r a f f i n and s t o r e d i n the wet P e t r i d i s h e s .  In either case they w e r e  photo-  g r a p h e d at t h e e a r l i e s t c o n v e n i e n c e a n d d i s p o s e d of. S e v e r a l s l i d e s of e a c h t y p e of m a t e r i a l w e r e r o u t i n e l y conjugated with fluorescent antibody.  A t l e a s t one s l i d e of e a c h  s e r i e s w a s t r e a t e d w i t h f l u o r e s c e n t n o n - i m m u n e g l o b u l i n to  serve  40  as a c o n t r o l a n d s h o w the k i n d s a n d a m o u n t of n o n - s p e c i f i c fluorescence  5.  present.  Acridine Orange Staining  Blood smears,  unfixed c h r o m o s o m a l preparations  organ i m p r i n t s w e r e stained with A c r i d i n e , orange to the m e t h o d of V o n B e r t a l a n f f y (1961).  and  (AO) according  This is a limited  publi-  c a t i o n , (See A p p e n d i x III).  6.  Biebrich Scarlet Staining  F r e s h tissue imprints were stained with  Biebrich  s c a r l e t a c c o r d i n g to the m e t h o d of D o u g l a s , S p i c e r and B a r t e l s (1966).  T h e i m p r i n t s w e r e f i x e d f o r 24 h o u r s i n C a r n o y ' s f l u i d  (1:1:1,  V / V , absolute ethanol - c h l o r o f o r m - glacial acetic acid).  A f t e r f i x a t i o n the s l i d e s w e r e h y d r a t e d t h r o u g h g r a d e d a l c o h o l s a n d s t a i n e d f o r 60 m i n u t e s i n a 0. 04% s o l u t i o n of B i e b r i c h i n I N g l y c i n e b u f f e r a d j u s t e d to p H 9. 5 w i t h I N N a O H .  .scarlet  The  i m p r i n t s w e r e then dehydrated with two r a p i d i m m e r s i o n s i n absolute a l c o h o l a n d c l e a r e d r a p i d l y i n 1:1 a l c o h o l - x y l o l f o l l o w e d b y x y l o l . F i n a l l y they were mounted i n P e r m o u n t .  VIII.  MICROSCOPY  A Z e i s s - P h o t o m i c r o s c o p e w a s u s e d throughout the  course  41  of t h e s e i n v e s t i g a t i o n s .  It w a s e q u i p p e d f o r p h a s e c o n t r a s t  fluorescence microscopy.  and  T h i s p e r m i t s the o b s e r v a t i o n of  f l u o r e s c e n t c e l l s w h i c h c a n t h e n be v i e w e d b y p h a s e c o n t r a s t w i t h o u t m o v i n g the slide or changing objectives.  F o r fluorescence  micro-  s c o p y B G / 3 a n d B G 38 w h i c h m a k e u p e x c i t e r f i l t e r II w e r e u s e d t o l i m i t e x c i t e r l i g h t t r a n s m i s s i o n to the 3 0 0 0 - 4 0 0 0 A ° r a n g e . filter  Barrier  5 3 / 4 4 w h i c h t r a n s m i t s above 5250 A ° was u s e d i n c o m b i n a t i o n .  It w a s f o u n d t h a t t h e b e s t r e s u l t s w e r e o b t a i n e d u s i n g t h e 4 5 X o b j e c t i v e w i t h the c o n d e n s e r  IX.  set at the  " J " position.  PHOTOGRAPHY  F l u o r e s c e n t c e l l s w e r e p h o t o g r a p h e d b y d i r e c t i n g a l l the light up the c a m e r a tube, Ektachrome  d i r e c t l y on to the f i l m .  Kodak High Speed  ( A S A 120, d a y l i g h t t y p e ) w a s u s e d t h r o u g h o u t a n d d e v e l -  oped c o m m e r c i a l l y to p r o v i d e c o l o u r s l i d e s .  Exposure times  d e t e r m i n e d b y t a k i n g test e x p o s u r e s to p r o v i d e the e x p o s u r e i n w h i c h the different m a t e r i a l s c o u l d be p h o t o g r a p h e d . photographs,  over this range,  were routinely taken.  w h i c h v a r i e d f r o m 5 to 240 s e c o n d s ,  were range  Three  Exposure times,  provided a measure  of the  s t r e n g t h of f l u o r e s c e n c e . The  colour photographs were machine printed by  methods in most cases, enlargements  d i r e c t l y f r o m the c o l o u r s l i d e s .  of p o r t i o n s of a s l i d e o r s p e c i a l m e t h o d s to  standard  Where combat  42  over-exposure were required, Mr.  J.  Deitrich,  of A l b e r t a ,  Head,  Edmonton.  the p r i n t s w e r e hand  Department  developed  of P h o t o S e r v i c e ,  Univers  43  RESULTS  The source  o± i m m u n o g e n i c m a t e r i a l u s e d i n t h i s  was nuclear protein extracted f r o m calf thymus 1955).  (Daly and  Mirsky,  This protein preparation was injected into chickens  their i m m u n o l o g i c a l response studied.  The resultant  study  and  antiserum  w a s u s e d to s t u d y the n u c l e a r p r o t e i n e x t r a c t i m m u n o l o g i c a l l y (Ouchterlony,  1948).  The extract was also analysed  g r a p h i c a l l y ( J o h n s o n et a l . , Wan and Irvin,  1964),  chromato-  electrophorectically  (McAllister,  1963), b y a m m o a c i d a n a l y s i s a n d b y a d i f f e r e n t i a l  staining technique  (Douglas, Spicer and B a r t e l l s ,  1966).  The a n t i s e r u m was a b s o r b e d to r e m o v e unwanted logical activity.  It w a s p r e p a r e d a s a p r o t e i n t r a c e r  immuno-  by labelling  w i t h f l u o r e s c e n t a n t i b o d y , w a s a p p l i e d t o a v a r i e t y of c e l l s t o d e m o n s t r a t e the d i s t r i b u t i o n of the n u c l e a r p r o t e i n a n t i g e n a n d results  photographed.  procedures  T a b l e 1 s h o w s the  and analytical tests  employed.  s e q u e n c e of  the  preparative  TABLE  I  IMMUNOCHEMICAL PROCEDURE FOR THE FLUORESCENT DEMONSTRATION OF NUCLEAR PROTEIN ANTIGEN AT THE CELLULAR L E V E L CALF THYMUS NaCl  . ANALYSES •< amino a c i d c o m p o s i t i o n disc electrophoresis chromatography differential staining  TISSUE Citric  \  /  nuclear protein  acid  cytoplasm  precipitate  •ANALYSES disc electrophoresis immunodiffusion t e s t s  \ chicken  precipitin tests-, _ immunodiffusion t e s t s  antiserum vs. nuclear p r o t e i n & ~* impurities  precipitin test immunodiffusion  antiserum vs. nuclear protein  test  J  absorption  1  l a b e l w i t h FITC and chromatograph  spectrophotometry immunodiffusion t e s t s disc electrophoresis precipitin tests  f l u o r e s c e n t ^antibody— immunofluorescent c a l f thymus c e l l s ' bovine l i v e r c e l l s bovine spleen c e l l s bovine t e s t i s c e l l s b o v i n e chromosomes  human human human human  tests  thymus c e l l s buccal c e l l s testis cells chromosomes  "insect c e l l s plant cells chicken c e l l s  45 I.  EXTRACTION  OF NUCLEAR  PROTEINS  Nuclear proteins were prepared u s i n g 150 g m of c a l f t h y m u s e a c h t i m e .  Batches  p r e p a r e d d u r i n g the e a r l y s t a g e s of t h e s e i  These preparations  sations and w e r e not  on three  occasions  1 and 2 were  experiments,  w e r e u s e d i n the i n i t i a l i m m u n i -  analysed.  The third extraction was considered most as m o r e  extensive washing was possible.  satisfactory  In this case  the  c y t o p l a s m i c f r a c t i o n w a s s t o r e d f o r l a t e r a n a l y s i s a n d the n u c l e i w a s h e d t h r e e t i m e s i n 6,  250 m l centrifuge t u b e s .  This  r e s u l t e d i n c o m p l e t e r e m o v a l of c y t o p l a s m i c p r o t e i n a s mined by a refractometer the w a s h s o l u t i o n .  test for protein,  standardised  procedure deter against  T h e f i n a l y i e l d o f B a t c h 3 w a s 16 0 m l of  n u c l e a r p r o t e i n s o l u t i o n a t a c o n c e n t r a t i o n o f 4 . 3 g m % a n d 120 n i l of c y t o p l a s m i c p r o t e i n ( 1 . 5  gm%).  M i c r o s c o p i c e x a m i n a t i o n of the n u c l e i a f t e r c y t o p l a s m w a s c o m p l i c a t e d b y the p r e s e n c e the h i g h d e n s i t y of the n u c l e a r p e l l e t .  of t i s s u e  r e m o v a l of debris  and  H o w e v e r , a n i m p r i n t of  calf t h y m o c y t e s was e x a m i n e d m i c r o s c o p i c a l l y after  being treated  w i t h 0. 005 M c i t r i c a c i d f o r 30 m i n u t e s a n d r i n s e d i n b u f f e r e d saline.  T h i s s h o w e d the c o m p l e t e r e m o v a l of c y t o p l a s m l e a v i n g  bare nuclei,  as r e p o r t e d b y L i t t a u et a l . ,  (1965).  46 A m i n o a c i d a n a l y s i s of B a t c h 3 w a s c a r r i e d out. results  are  parison.  s h o w n i n T a b l e II a l o n g w i t h s t a n d a r d  The slight differences  acids are attributable  values for  i n p e r c e n t a g e s of b a s i c  to d i f f e r e n c e s  in extraction  The com-  amino  methods.  C r a m p t o n e t a l . , u s e d 2 . 6 M N a C l to e x t r a c t a n a r g i n i n e - r i c h fraction.  A 1. 2 5 M N a C l  solution was u s e d i n this project  extract a lysine-rich fraction.  to  A l t h o u g h the t o t a l m o l e s of  basic amino acids in Batch 3 are  2 . 1% l e s s  v a l u e t h e r a t i o o f l y s i n e to a r g i n i n e i s  t h a n the  higher.  standard  47 T A B L E AMINO ACID  COMPOSITION OF NUCLEAR PREPARATION  The values are acids.  II  (BATCH  PROTEIN  3)  p e r c e n t a g e s of total m o l e s of  T h e s t a n d a r d v a l u e s a r e those of C r a m p t o n  ( l 9 5 5 ) . a c c e p t e d as to f r a c t i o n a t i o n  standard for calf thymus histones  (Busch,  Amino Acid  1965 p .  prior  43). Standard  Nuclear  Values  Protein  14. 7  Lysine  amino et a l . ,  15. 5  Histidine  2.  Arginine  8. 3  5.4  4. 8  6. 1  11. 6  11. 5  Aspartic  Acid  Threonine  and  Serine  Glutamic  Acid  0  1. 6  8. 2  9.7  Proline  4.9  4.6  Glycine  8. 3  10. 8  Alanine  13. 5  15. 3  Valine  6.2  5.4  Methionine  0.9  0. 8  Isoleucine  4. 3  2.9  Leucine  7. 7  6.4  Tyrosine  2. 5  1.7  Phenylalanin e  2. 2  2. 1  23.0 13. 0  20. 9 15. 8 2. 8  L y s i n e and  Arginine  A s p a r t i c and  Glutamic  Lysine/Arginine  1.8  48 II.  PRODUCTION OF ANTISERUM  B e f o r e the n u c l e a r p r o t e i n p r e p a r a t i o n c o u l d be i n g r e a t e r d e t a i l it was n e c e s s a r y to determine i m r n u n o g e n i c to the c h i c k e n .  analysed  whether it was  Immunisations were  c a r r i e d out a n d  the c h i c k e n s e r a t e s t e d f o r antibody a c t i v i t y b y p r e c i p i t i n t e s t s , immunodiffusion tests on millipore membranes,  and i m m u n o -  d i f f u s i o n t e s t s i n 1% a g a r .  1.  S c h e d u l e of I m m u n i s a t i o n s a n d P r e c i p i t i n  Calf, t h y m u s n u c l e a r p r o t e i n ( B a t c h e s p r e c i p i t a t e d a n d i n j e c t e d i n t o 22 L e g h o r n h e n s . chickens were  starved overnight,  Tests  1 a n d 2) w a s After  14 d a y s  5 m l s e r u m samples taken,  the c h i c k e n s r e i n j e c t e d w i t h the i n o c u l u m .  After a further  14 d a y s ,  and  The s e r a of 7 b i r d s  gave w e a k l y p o s i t i v e p r e c i p i t i n t e s t s a g a i n s t the n u c l e a r solution.  the  protein  20 m l s e r u m s a m p l e s  were  t a k e n f r o m the b i r d s w h i c h h a d p r o d u c e d p r e c i p i t i n s and 6 b i r d s again injected,  one h a v i n g d i e d i n the i n t e r v a l .  of 20 m l p e r b i r d w a s p e r f o r m e d after  A final bleeding  the s i x t h w e e k .  The  sera  f r o m 4 of the s e l e c t e d b i r d s s t i l l c o n t a i n e d w e a k p r e c i p i t i n a c t i v i t y w h i c h w a s not evident i n d i l u t i o n s g r e a t e r than  1:1.  C o n t r o l s e r a f r o m n o n i m m u n i s e d chickens gave a negative Two  result.  c h i c k e n s ( n u m b e r e d 3 1 2 a n d 3 3 3 ) : p r o d u c e d l i t r e s o f 1:32  1:16 a s s h o w n i n F i g u r e 1.  These birds were reinjected  with  and  49 n u c l e a r p r o t e i n ( B a t c h 3) a n d b l e d a f t e r successive  a 28 d a y i n t e r v a l on t w o  o c c a s i o n s a t 8 a n d 10 m o n t h s .  p r o v i d e d a s t o c k of 120 m l of  antiserum.  These  procedures  50  Figure  1  P r e c i p i t i n T e s t of A n t i s e r a 333 and 312  - Tube 1 contains a n t i s e r u m and nuclear a n t i g e n 1:1.  Successive tubes  protein  represent  d o u b l i n g d i l u t i o n s of a n t i s e r u m . . P r e c i p i t a t e s c a n be s e e n r e s t i n g on m e n i s c u s  at  air-liquid  interface.  A - A n t i s e r u m 333, tube 6 i s negative.  Titre  B - A n t i s e r u m 312, tube 6 w e a k l y p o s i t i v e .  1:16.  Titre  1:32  • 5  52 2.  Immunodiffusion A.  Tests  Micro-immunodiffusion  I n a n e f f o r t t o q u a n t i t a t e the i m m u n e r e s p o n s e b y the i m m u n i s a t i o n p r o c e d u r e were  two m e t h o d s  employed using unfractionated  elicited  of i m m u n o d i f f u s i o n  a n t i s e r u m a n d the  nuclear  protein solution. The first method chosen was m i c r o - i m m u n o d i f f u s i o n on m i l l i p o r e m e m b r a n e s .  This method is reported  advantages of r e q u i r i n g only l a m b d a amounts high resolving power, be  of reagents,  the having  providing sharp precipitin lines which  s t a i n e d to p r o v i d e a p e r m a n e n t  to p r e p a r e .  to h a v e  slide, and being relatively  T h i s w a s f o u n d to b e t h e c a s e w h e n a n a l y s i n g  can easy  serum  proteins. However,  t h e m e t h o d d o e s n o t a p p e a r to b e w e l l  to t h e i m m u n o l o g i c a l a n a l y s e s o f s m a l i ' m o l e c u l a r w e i g h t , proteins. are  Twelve plates were prepared.  illustrated in Figure 2 where  obtained r a t h e r than sharp  The strongest  z o n e s of p r e c i p i t a t e  reactions  were  in close proximity  to t h e a n t i s e r u m w e l l s p r o v i d e d t h e f i r s t e v i d e n c e t h a t t h e  assumed  basic  lines.  T h e p o s i t i o n of the p r e c i p i t i n z o n e s ,  components  suited  m i g r a t e at a f a s t e r  r a t e t h a n the a n t i b o d y .  to b e d u e to t h e a n t i g e n i c m o l e c u l e s h a v i n g  m o l e c u l a r d i m e n s i o n s t h a n the a n t i b o d y Figure 2 A serves  antigenic  This is  smaller  molecules.  to d e m o n s t r a t e ,  by another  the i m m u n o l o g i c a l a c t i v i t y of d i l u t e d a n t i s e r u m ,  method,  first observed in  the p r e c i p i t i n t e s t s .  F i g u r e 2 B indicates that this antibody a c t i v i t y  is d i r e c t e d against a n antigen that is p r e s e n t i n c o m m e r c i a l h i s t o n e a s w e l l as the 3 n u c l e a r p r o t e i n p r e p a r a t i o n s .  This  i n d i c a t e d b y the c o n t i n u i t y of the r i n g of p r e c i p i t a t e a r o u n d central well.  T h e i n c r e a s e d d e n s i t y of the p r e c i p i t a t e s  w e l l 4 indicates that m o r e t h a n i n the other  samples.  is the  opposite  of the a n t i g e n w a s p r e s e n t i n B a t c h 3  54  Figure 2  Micro-irnrnunodiffusion  T e s t s of A n t i b o d y A c t i v i t y of  Chicken Serum Against Nuclear Protein Antigen.  A  - The c i r c u l a r wells contained doubling dilutions of 2 a n t i s e r a w h i l e the a n t i g e n w a s p r e s e n t i n the c e n t r a l t r o u g h . increasing  Precipitates fainter  with  dilution.  B - The c e n t r a l w e l l contained the  antiserum.  Outer wells contained 4 nuclear protein preparations.  Precipitates f o r m a continuous  a r o u n d w e l l 5,  concentrated opposite w e l l 4.  Z o n e s of p r e c i p i t a t e o c c u r r e d after  72 h o u r s ,  n e a r e s t the a n t i s e r u m w e l l s i n a l l e a s e s . membranes were on glass.  stained,  The  c l e a r e d and mounted  T h e m e m b r a n e i t s e l f w a s u s e d as  negative i n the e n l a r g e r to p r o v i d e the graphs i n w h i c h the stained p r e c i p i t a t e s white.  ring  Enlarged 6X.  a  photoappear  55  A. W e l l No.  B. Reagent  W e l l No,  Reagent  1  A n t i s e r u m 3 1 2 , 1:1  1  2  A n t i s e r u m 3 1 2 , 1:2  2  Nuclear P r o t e i n ,  Batch  3  A n t i s e r u m 3 1 2 , 1:4  3  Nuclear P r o t e i n ,  Batch 2  4  A n t i s e r u m 3 3 3 , 1:1  4  Nuclear P r o t e i n ,  Batch  5  A n t i s e r u m 3 3 3 , 1:2  5  6  Antiserum 333,  7  Nuclear  1:4  Protein  Commercial Histone  Antiserum  312  1  3  56 B.  Macro-immunodiffusion  T h e s e c o n d m e t h o d of a n a l y s i n g the c h i c k e n for reactivity against nuclear protein antigen was i m m u n o d i f f u s i o n i n 1% a g a r ,  in Petri dishes.  antiserum  macro-  This  method  r e q u i r e s m o r e of e a c h r e a g e n t ( a p p r o x i m a t e l y 0. 5 m l ) b u t p r o v i d e d d e f i n i t e l i n e s of p r e c i p i t a t e .  T h e m e t h o d w a s u s e d to test the  sera  f r o m s e v e r a l c h i c k e n s , that had g i v e n p o s i t i v e r e s u l t s i n the precipitin test,-for antigens.  antibody activity against nuclear protein  T h e s e r a f r o m c h i c k e n s 312 and 333 gave the  strongly positive results. proteins,  These s e r a w e r e tested against  c o m m e r c i a l histone,  they reacted positively.  most nuclear  and c y t o p l a s m i c proteins to w h i c h  Antibodies against c y t o p l a s m i c antigens  w e r e a b s o r b e d out of t h e s e 2 a n t i s e r a a n d t h e y w e r e a g a i n t e s t e d for  antibody activity against nuclear protein antigens. F i g u r e 3 A shows the r e s u l t s of an i n i t i a l attempt i n  w h i c h a n t i s e r a f r o m c h i c k e n s 312 a n d 333 b o t h p r o v i d e d 2 l i n e s of p r e c i p i t a t e a g a i n s t the n u c l e a r p r o t e i n a n t i g e n s , for 5 days.  after d e v e l o p i n g  In o r d e r to a s c e r t a i n w h e t h e r both c h i c k e n s w e r e  p r o d u c i n g antibodies against the same antigens the outer  wells  w e r e m o v e d c l o s e r to each other and the c e n t r a l w e l l ( F i g u r e 3 B ) . T h e c o n t i n u o u s l i n e s of p r e c i p i t a t e s u r r o u n d i n g the c e n t r a l w e l l show that the antigens i n v o l v e d are i d e n t i c a l . were prepared, wells.  Three such plates  i n o r d e r to d e t e r m i n e the o p t i m u m d i s t a n c e  between  57  Figure 3  Macro-immunodiffusion  T e s t of A n t i b o d y A c t i v i t y  of C h i c k e n S e r u m A g a i n s t N u c l e a r P r o t e i n  Antigen.  A - Two antisera, present i n outer w e l l s ,  have  diffused  a s h o r t d i s t a n c e to c o m b i n e w i t h 2  antigens f r o m the  c e n t r a l w e l l to p r o d u c e 2  l i n e s of p r e c i p i t a t e o p p o s i t e e a c h w e l l .  B - A n t i s e r a 312 a n d 333 h a v e r e a c t e d w i t h nuclear  p r o t e i n antigen and p r o d u c e d  precipitin lines w h i c h are joined. serum  2  Anti-  357 d i d not p r o d u c e l i n e s of  precipitate.  It w a s w e a k l y p o s i t i v e i n the p r e c i p i t i n t e s t .  B y m o v i n g the  antiserum wells  closer  t o g e t h e r the p r e c i p i t i n l i n e s p r e s e n t i n A c a n be s e e n i n B to be c o n t i n u o u s , t h a t a n t i s e r a 333 and 312 c o n t a i n against 2 identical antigens. c l o s e s t to the  indicating antibodies  The lines  a n t i s e r u m wells i n both  are cases.  '  So  A.  1  U  B.  Well No.  Feagent  W e l l No.  p  eaqent  1  Antiserum 312  1  A n t i s e r u m 312  2  A n t i s e r u m 312  2  Antiserum 312  3  Antiserum 333  3  Antiserum 333  4  Antiserum 333  4  Antiserum 333  5  N u c l e a r n r o t e in Batch #3  5  Antiserum 357  6  Antiserum 357  7  "Tuclear D r o t e i n Batch #3  In o r d e r to further  define the i m m u n o l o g i ca l  reactions  of t h e s e a n t i s e r a t h e y w e r e t e s t e d a g a i n s t a c o m m e r c i a l h i s t o n e preparation,  the n u c l e a r p r o t e i n f r a c t i o n and the c y t o p l a s m i c f r a c t i o n  obtained f r o m the p r e p a r a t i v e p r o c e d u r e . similar positive reactions F i g u r e 4.  Both antisera  against a l l 3 antigens,,  as i l l u s t r a t e d i n  Two straight immunodiffusion lines are present  the a n t i s e r u m and the n u c l e a r p r o t e i n f r a c t i o n , narrow.  gave  between  one w i d e and  one  T h i s i s m o s t c l e a r l y e v i d e n t i n F i g u r e 4 A , w e l l 6.  s i m i l a r but w e a k e r l i n e s are p r e s e n t between the a n t i s e r u m the c o m m e r c i a l histone  ( F i g u r e 4 B , w e l l 5).  The  Two and  antiserum  r e a c t e d w i t h the c y t o p l a s m i c p r o t e i n to p r o d u c e a l i n e of i d e n t i t y w i t h the other two s y s t e m s  and a crescent  shaped line  super-  i m p o s e d on i t ( F i g u r e 4 A , w e l l 4). O n the b a s i s of the r e s u l t s  shown i n F i g u r e 4 and by  studying four other plates w h i c h produced s i m i l a r patterns it was concluded that a l l 3 antigenic preparations component i n v a r y i n g amounts  contained a  common  (the c o m m o n w i d e l i n e ) .  nuclear protein preparation contained a unique antigenic of s m a l l e r w e i g h t t h a n t h e c o m m o n c o m p o n e n t c l o s e s t to the antigen w e l l ) .  component  (the n a r r o w l i n e  Another different antigenic  w a s p r e s e n t i n t h e c y t o p l a s m (the c u r v e d l i n e ) .  The  species  60  Figure 4  Immunodiffusion  T e s t s of A n t i s e r a 3 3 3 ,  Against Cytoplasm,  Histone and N u c l e a r  A n t i s e r a w e r e a l l o w e d to diffuse adding antigens. a p p e a r e d after the  24 h o u r s  5 days.  before  After a further 4 days  s i n g l e l i n e c l o s e to w e l l s 3,  are heavier  Protein.  Central line linking all antigens  c u r v e d l i n e ( w e l l s 1 a n d 4) a p p e a r e d ,  by the  6.  333.  followed  Lines  i n B indicating m o r e antibody  i n a n t i s e r u m 312 t h a n  s  312  present  61  B.  A. W e l l No.  Reagent  rc.ea^ent  " e l l No.  Cy top l a s m i c Fraction  Cytoplasmic Fraction Cytoplasmic Fraction  4  Cytoplasmic Fraction  Commercial Histone  2  Commercial Kistone Commercial Histone  Commercial Histone 3  Nuclear protein Batch #3  3  6  Nuclear protein Batch #3  6  7  Antiserum 333  7  Nuclear nrotein Batch #3 v  u c l e a r protein Batch #3 A n t i s e r u r 312  62 3.  A b s o r p t i o n of A n t i s e r u m  In o r d e r to r e m o v e the a n t i b o d i e s that w e r e  reacting  w i t h c y t o p l a s m i c a n t i g e n s the a n t i s e r a w e r e m i x e d w i t h amounts  small  of c y t o p l a s m a n d a l l o w e d to r e a c t u n t i l p r e c i p i t a t e s  longer appeared.  F i g u r e 5 s h o w s t h e r e s u l t of t e s t i n g  no  antiserum  312 a g a i n s t the n u c l e a r a n d c y t o p l a s m i c p r o t e i n f r a c t i o n s  after  absorption with cytoplasm. T h e s t r a i g h t l i n e s of p r e c i p i t a t e b e t w e e n w e l l s 5 and 5 a n d 2 i n d i c a t e the p r e s e n c e  i n s e r u m 312 of a n t i b o d y a g a i n s t  1, a  s i n g l e a n t i g e n i c c o m p o n e n t i n the n u c l e a r p r o t e i n p r e p a r a t i o n . T h e a n t i g e n s w e r e a p p l i e d 2 4 h o u r s a f t e r the a n t i s e r u m a n d the l i n e s a r e e q u i d i s t a n t b e t w e e n the w e l l s .  The absence  of p r e c i p i t i n  l i n e s a n d free d i f f u s i o n of the a n t i s e r u m t o w a r d s w e l l s 3 a n d 4 i n d i c a t e the a b s e n c e  of a n t i b o d y a g a i n s t c y t o p l a s m i c p r o t e i n s i n  s e r u m 312, w h i c h has been absorbed with c y t o p l a s m .  63  Figure 5  Immunodiffusion  T e s t of A n t i s e r u m 312,  with Cytoplasm,  Against Nuclear Protein  Absorbed and  Cytoplasm.  L i n e s of p r e c i p i t a t e a r e  equidistant between  w e l l s 5 a n d 1,. 5 a n d 2 .  Antigens introduced  h o u r s after  antiserum.  24  64  Reagent v  u c l e a r protein Batch #3  Nuclear protein Batch #3 Cytoplasmic n r o t e i n Batch #3 Cytoplasmic p r o t e i n Batch #3 Absorbed Antiserum 312  65 III.  ANALYSIS OF NUCLEAR 1.  Disc  The  PROTEIN  Electrophoresis  nuclear protein preparation was  several fractions, by disc electrophoresis. e x t r a c t c o u l d be d i v i d e d into 8 m a j o r s h o w s the 3 m a j o r cathode  EXTRACT  separated  The nuclear protein,  components.  F i g u r e 6B  discs present which migrate towards  the  a t a r a t e s i m i l a r to t h a t o f a c i d i c s e r u m p r o t e i n s  the s a m e c o n d i t i o n s .  F i g u r e 6 C i l l u s t r a t e s the 5 m o r e  for  the  Figure  These conditions have  s e p a r a t i o n of s m a l l m o l e c u l a r weight,  under  diffuse  b a n d s p r e s e n t u n d e r c o n d i t i o n s of l o w p H , s m a l l e r p o r e and reversed polarity.  into  size  been.devised basic  proteins.  6 D s h o w s the e l e c t r o p h o r e t i c p a t t e r n o b t a i n e d w h e n a  s a m p l e of c o m m e r c i a l histone ( a c i d extracted) basic proteins.  T h i s p r e p a r a t i o n contained 3 d i s c s ( A , B and C)  not p r e s e n t i n o u r extract. to b o t h p r e p a r a t i o n s nuclear proteins  was tested for  D i s c s 2 a n d 3 a p p e a r to b e  w h i l e d i s c s 1,  extracted in our  common  4 a n d 5 a r e u n i q u e to  laboratory.  the  66  Figure 6  Comparative Disc  Electrophoresis  A - 3 u l (150 ug) s a m p l e , time,  5 M A per  5 M A per  C - 0. 1 m l s a m p l e , time,  5 M A per  D - 0. 1 m l s a m p l e , time,  5 M A per  running  tube  B - 7 u l (90 u g ) s a m p l e , time,  90 m i n u t e s  90 m i n u t e s  running  tube B a t c h 3,  2 hours  running  tube (90 u g ) ,  2 hours  running  tube  Tube A i s i n c l u d e d to show the r e l a t i v e p o s i t i o n s of d i s c s of s e r u m p r o t e i n a n d a c i d i c n u c l e a r D i s c s 2 and 3 i n tubes C and D are  a s s u m e d to  i d e n t i c a l b e c a u s e of t h e i r c o r r e s p o n d i n g and appearance. Black 10B.  protein.  A l ltubes stained with  be  positions Amido  A. Serum Protein  B. Acidic Nuclear Protein  S t a n d a r d G e l , pH 8.5  » Basic Nuclear Protein  D. Commercial Histone  c  15^  G e l , pH  5.0  68 2.  Immuno Disc  Electrophoresis  In an attempt to d e t e r m i n e w h i c h component components  of the b a s i c n u c l e a r p r o t e i n f r a c t i o n w e r e  to the c h i c k e n the techniques phoresis were combined. unstained,  or antigenic  of i m m u n o d i f f u s i o n and d i s c  I m m e d i a t e l y after  electro-  electrophoresis  an  unfixed, acrylamide cylinder was imbedded in agar.  A c o n t r o l c y l i n d e r , r u n at t h e  same time,  to d e t e r m i n e the p o s i t i o n s of the  was fixed and  stained  discs.  T h e s i n g l e p r e c i p i t i n l i n e p r o d u c e d ( F i g u r e 7) i s ciated w i t h the  slowest moving component  f r a c t i o n ( d i s c 1).  of the n u c l e a r  T h i s l i n e w a s f a i n t l y v i s i b l e after  c o n t i n u e d t o d e v e l o p f o r 10 d a y s ,  asso-  protein  72 h o u r s  at w h i c h p o i n t i t w a s  photographed.  T h i s l e n g t h y t i m e i n t e r v a l a n d the p o s i t i o n of the l i n e c l o s e t o a c r y l a m i d e . c y l i n d e r i s thought to r e f l e c t the  surrounding gel matrix.  s u r r o u n d i n g the a n t i s e r u m troughs s e r u m components, sera were period,  pore  The concentric lines  a r e due to s e p a r a t i o n of a n t i -  w h i c h h a v e d i f f e r e n t r a t e s of d i f f u s i o n .  added to the troughs  daily,  to a t o t a l of T . 5 m l e a c h .  s u c c e s s f u l l y on one other  the  s l o w e r r a t e of  diffusion f r o m the a c r y l a m i d e , due to its h a v i n g a s m a l l e r size than the  and  d u r i n g the  development  This result was  occasion.  repeated  Anti-  69  Figure 7  I r r r m u n o d i f f u s i o n T e s t of T w o C h i c k e n A n t i s e r a Against Calf Thymus, Fractionated by D i s c  The unstained  Basic Nuclear  Electrophoresis.  cylinder has been r e m o v e d  facilitate photography. c y l i n d e r i s on the right. precipitate  Proteins,  A stained  to  control  T h e s i n g l e l i n e of  (arrow) is associated with disc  1.  Both antisera have produced a s i m i l a r p r e c i pitin line, serum  which is more pronounced with  312.  71 3.  B iebrich Scarlet Staining  The nuclear proteins were  also electrophoresed using a  m o d i f i e d t e c h n i q u e d e v e l o p e d b y S h e r i d a n and S t e r n (1966) f o r s t u d y of p l a n t h i s t o n e s .  T h i s m e t h o d m a k e s u s e of p o l y a c r y l a m i d e  gels having a smaller pore technique  separated  diffuse pattern.  the  s i z e (18% g e l ,  10% c a t a l y s t ) .  This  the d i s c s m o r e w i d e l y but r e s u l t e d i n a  D i s c s 2 and 3 c o u l d not be s e p a r a t e d  more  using this  method. T u b e A , . F i g u r e 8, w a s i n the u s u a l fashion.  stained with A m i d o Black  10B  The a c r y l a m i d e c y l i n d e r i n tube B was  stained for the p r e s e n c e  of h i s t o n e w i t h B i e b r i c h s c a r l e t  using  a m o d i f i c a t i o n of the c y t o c h e m i c a l t e c h n i q u e d e s c r i b e d by D o u g l a s , S p i c e r and B a r t e l s (1966).  Disc 1 stained faintly.  D i s c s 2 and 3  stained m o r e d e e p l y w h i l e d i s c s 4 and 5 d i d not accept the  4.  A b s o r p t i o n of A n t i g e n  A further  t e s t of a n t i s e r u m s p e c i f i c i t y f o r  d i s c s of b a s i c n u c l e a r p r o t e i n w a s p e r f o r m e d . c o r o l l a r y of the one j u s t d e s c r i b e d ,  electrophoretic  This test,  antigenic proteins before  the  involved reacting a small  a m o u n t of a n t i s e r u m w i t h the n u c l e a r p r o t e i n p r e p a r a t i o n remove  stain.  to  electrophoresis.  A f t e r five d r o p s of a b s o r b e d  antiserum were  0. 5 m l of t h e n u c l e a r p r o t e i n f r a c t i o n n o m o r e  added  precipitates  to  72 occurred.  A 0. 2 m l s a m p l e of t h i s a b s o r b e d n u c l e a r p r o t e i n w a s  electrophoresed  to d e t e r m i n e w h i c h b a s i c p r o t e i n c o m p o n e n t s  b e e n r e m o v e d b y the  had  antibody.  The resultant  d i s c s w e r e v e r y faint due to d i l u t i o n of  the n u c l e a r p r o t e i n s d u r i n g the a b s o r p t i o n p r o c e s s .  However,  disc 1 was c o m p l e t e l y absent while d i s c s 2 and 3 w e r e present i n trace amounts.  D i s c s 4 and 5 w e r e  sharply  visible.  73  Figure 8  D i s c E l e c t r o p h o r e s i s of N u c l e a r P r o t e i n S t a i n e d for P r o t e i n and H i s t o n e .  B o t h t u b e s c o n t a i n 0 . 1 m l (90 u g ) s a m p l e s nuclear protein,  B a t c h 3,  t i m e 5 M A per tube.  r u n at the  Running time  of  same - 4  hours.  Tube A stained with A m i d o Black 10B. T u b e B s t a i n e d w i t h B i e b r i c h s c a r l e t at p H 9. 5 a f t e r  Disc  1 has  fixation in Carnoys  )(_  fluid.  stained faintly with 13iebrich scarlet,  d i s c s 2 and 3 stained m o r e of d i s c s 4 a n d 5.  deeply.  No  staining  \f  75 On the b a s i s of r e s u l t s precipitin tests, observations  o b t a i n e d from amino a c i d  immunodiffusion and d i s c e l e c t r o p h o r e s i s ,  several  c o u l d be made.  Nuclear p r o t e i n s ,  s a l t extracted  from c a l f  thymus, were  immunogenic to some, but not to a l l , chickens when i n j e c t e d precipitate.  The r e s u l t a n t  was d i r e c t e d a g a i n s t  cytoplasmic f r a c t i o n .  nuclear  p r e p a r a t i o n , and the  Another a n t i b o d y was d i r e c t e d a g a i n s t  component unique to the  types  One type  an a n t i g e n shared by the s a l t - e x t r a c t e d  a commercial a c i d e x t r a c t e d h i s t o n e  antigenic  as a  a n t i s e r u m c o n t a i n e d at l e a s t t h r e e  of p r e c i p i t a t i n g a n t i b o d y demonstrable by i m m u n o d i f f u s i o n .  protein,  analysis,  cytoplasm.  A third  an  antibody  r e a c t e d w i t h an a n t i g e n demonstrable o n l y i n the n u c l e a r p r o t e i n preparation.  The a n t i b o d i e s  against  the shared a n t i g e n and the  c y t o p l a s m i c a n t i g e n c o u l d be removed from the a n t i s e r u m by absorbing i t with cytoplasm.  The a n t i s e r u m continued to  w i t h the n u c l e a r p r o t e i n a n t i g e n a f t e r  absorption.  T h i s a n t i g e n had s e v e r a l unique 1. not  I t was p r e s e n t  i n the c y t o p l a s m i c 2.  agar,  characteristics:  in salt-extracted  n u c l e a r p r o t e i n , but  fraction.  I t migrated more s l o w l y  than the shared a n t i g e n i n 1%  i n d i c a t i n g t h a t i t had l a r g e r m o l e c u l a r 3.  I t was p r e s e n t  dimensions.  i n the slowest moving of 5 d i s c s  p r o t e i n produced i n p o l y a c r y l a m i d e d i s c e l e c t r o p h o r e s i s conditions  designed  react  f o r d e m o n s t r a z t i o n of the presence  of  under of  histones.  76 This i n d i c a t e d that it had a r e l a t i v e l y l a r g e m o l e c u l a r weight or i n the l e a s t p o s i t i v e l y c h a r g e d g r o u p of p r o t e i n s , The  presence  is  or both.  of the antigen i n d i s c 1 w a s  demonstrable  by p r o d u c t i o n of a p r e c i p i t i n l i n e a s s o c i a t e d w i t h t h e p o s i t i o n of t h i s d i s c a n d b y t h e a b i l i t y of the a b s o r b e d a n t i b o d y to r e m o v e d i s c p r i o r to 4.  electrophoresis. It s t a i n e d f a i n t l y w i t h B i e b r i c h  selectively stains histones under  IV.  CHROMATOGRAPHIC  scarlet,  a dye w h i c h  specific conditions.  SEPARATION OF NUCLEAR  Although several chromatographic methods for  this  are  the f r a c t i o n a t i o n of n u c l e a r p r o t e i n p r e p a r a t i o n s ,  PROTEIN  available  Sephadex  w a s u s e d b e c a u s e i t r e s u l t s i n the l e a s t d e n a t u r a t i o n of a n t i g e n i c proteins. S e p h a d e x c h r o m a t o g r a p h y w a s s t u d i e d (17 r u n s ) u s i n g f i r s t a s m a l l c o l u m n (9 r u n s ) a n d t h e n a l a r g e r o n e (8 r u n s ) u n d e r v a r y i n g chromatographic conditions. separation, for  F r a c t i o n s , f r o m runs giving best  w e r e pooled and re concentrated.  They were  analysed  p r o t e i n content and antigenicity b y d i s c e l e c t r o p h o r e s i s ,  amino  acid analysis and immunodiffusion. The  preparative  s e p a r a t i o n of n u c l e a r p r o t e i n s  Sephadex G-75 was investigated using techniques Cruft  (1961) a n d J o h n s o n ,  D r i e d g e r and M a r k o ,  in  described by (1964).  77 The i n i t i a l attempts w e r e m a d e w i t h 2, connected i n tandem. G-75 using a stirrer  They were packed with hydrated and r e s e r v o i r  90 m l a n d the u n m e t e r e d A 1 m l sample  g r a p h e d a n d the effluent  3  columns  Sephadex  and equilibrated with potassium  c i t r a t e b u f f e r p H 3 . 14 a n d ( K + ) = 0 . 05 M .  minute,  195 c m  The void volume  c o l u m n h a d a f l o w r a t e o f 1. 6 m l o f n u c l e a r p r o t e i n ( B a t c h 2) w a s collected i n 2. 5 m l aliquots.  was per  chromato-  Optical  2 d e n s i t y w a s r e a d at 276 m u u s i n g 1 c m were then pooled into three lots,  quartz  cuvettes.  c o r r e s p o n d i n g to p e a k s ,  The  tubes  and  an  a t t e m p t w a s m a d e to c o n c e n t r a t e t h e m u s i n g v a c u u m d i a l y s i s . This proved unsuccessful, proteins,  however,  as s m a l l  present i n a l l three fractions,  collodion bags and were lost.  molecular  passed through  A t this point a pressure  weight the  dialysis  u n i t w a s p u r c h a s e d h a v i n g g e l m e m b r a n e s of v a r y i n g p o r o s i t y . F i g u r e 9 s h o w s the r a t h e r p o o r s e p a r a t i o n o b t a i n e d i n a t y p i c a l r u n ( N o . 8) u s i n g t h i s m e t h o d .  S i n c e r e d u c t i o n of the  sample  size  d i d not i m p r o v e r e s o l u t i o n i t w a s c o n c l u d e d that the c o l u m n w a s too long i n r e l a t i o n to its d i a m e t e r  and had too fast a flow  rate.  B e f o r e abandoning this c o l u m n it w a s u s e d to obtain a r e l a t i v e e s t i m a t e of the m o l e c u l a r s i z e s of the n u c l e a r involved,  A m i x t u r e o f 1 m l o f 1% b l u e d e x t r a n ,  rescent chicken albumin,  proteins  1 m l of f l u o -  1 m l o f n u c l e a r p r o t e i n ( B a t c h 2) a n d  1 m g F I T C was chromatographed.  T h e p o s i t i o n s of d e x t r a n  a n d F I T C c o u l d be s e e n as b a n d s of c o l o u r i n the c o l u m n a n d  blue  78 their volumes measured  as they w e r e eluted.  of the p r o t e i n s o l u t i o n s w e r e in Figure  r e a d at 276 m u .  The optical density The result is  10. Blue dextran,  which is a polysaccharide polymer having  a m o l e c u l a r w e i g h t of a p p r o x i m a t e l y 2 m i l l i o n ,  was eluted first.  T h i s w a s f o l l o w e d i m m e d i a t e l y by f l u o r e s c e n t a l b u m i n i n a b a n d ( M W 68, 000).  A l m o s t a l l the a l b u m i n w a s e l u t e d  the f i r s t n u c l e a r p r o t e i n a p p e a r e d . dye ( M W 389).  These results  nuclear proteins  extracted were  small,  weight f r o m over  1, 0 0 0 to a b o u t 6 0 , 0 0 0 ,  overlapping subgroups.  Further,  proteins were intermediate peak.  sharp  before  T h i s was eluted i n three  followed by free  central  shown  i n d i c a t e d that  ranging i n molecular and f a l l i n g into  the m a j o r i t y o f the  in size,  three  nuclear  a s e v i d e n c e d b y the  large  peaks the  79 Figure 9  Nuclear Protein  A  Chromatography.  1 m l s a m p l e of n u c l e a r p r o t e i n .  C o l u m n v o l u m e 390 m l . per  B a t c h 2.  F l o w r a t e 1. 6 m l  minute.  The shallow troughs  b e t w e e n the p e a k s  a h i g h d e g r e e of o v e r l a p b e t w e e n the graphic fractions  indicated  chromato-  and incomplete separation.  Peak  1 c o n t a i n e d the l a r g e s t m o l e c u l a r s i z e s of n u c l e a r protein.  Peak 2 was intermediate  contained smaller  Figure  and peak  3  proteins.  10 C h r o m a t o g r a p h y o f a M o l e c u l a r W e i g h t M i x t u r e . .  P e a k s 2,  3 and 4 c o r r e s p o n d to the 3 p e a k s  p r e s e n t i n F i g u r e 3 and show the incomplete separation  s a m e k i n d of  of n u c l e a r p r o t e i n .  s h a r p n e s s of t h e f i r s t p e a k ,  The  containing albumin,  i n d i c a t e s the r e l a t i v e h o m o g e n e i t y of this, s u b stance as c o m p a r e d to n u c l e a r p r o t e i n w h i c h has  again been separated  fractions  a s i n F i g u r e 9.  into 3 overlapping  80  81 The next series  of 9 c h r o m a t o g r a p h i c  experiments  made using a 1 litre column, packed with Sephadex gravity method.  G - 7 5 by the  A p e r i s t a l t i c p u m p w a s u s e d to c o n t r o l f l o w  rate and the fractions c o n d i t i o n s w e r e the  c o l l e c t e d i n 10 m l t u b e s .  s a m e as w i t h t h e s m a l l e r  D u r i n g the varied,  were  course  Otherwise columns.  of t h e s e t r i a l s the f l o w r a t e  w i t h i n the l i m i t s of our t i m e d c o l l e c t o r s y s t e m ,  a v a r i a b l e speed p e r i s t a l t i c p u m p p l a c e d between the and the c o l l e c t o r .  The protein sample  size was  1 to 5 m l i n o r d e r to a s c e r t a i n the p r o p e r found that a 3 m l sample  was by using  column  also varied  column load.  from  It w a s  (130 m g ) of p r o t e i n w a s t h e l a r g e s t  l o a d the c o l u m n c o u l d a c c o m m o d a t e .  outlet  ,  A f l o w r a t e of 9 m l p e r  w a s found to be o p t i m a l f o r o u r c o l l e c t o r s y s t e m .  The  hour  column  h a d a v o i d v o l u m e of 350 m l . Figure  11 i l l u s t r a t e s t h e i m p r o v e d s e p a r a t i o n  with this column,  although there is still considerable  b e t w e e n the t h r e e p e a k s of p r o t e i n c o n c e n t r a t i o n . contained i n each peak w e r e p o o l e d as concentrated 2 ml  The  overlap fractions  s h o w n i n the f i g u r e  i n t h e p r e s s u r e d i a l y s e r t o v o l u m n s of 3,  respectively.  possible  and  5 and  82  Figure  11 N u c l e a r P r o t e i n C h r o m a t o g r a p h y .  A 3 m l s a m p l e of B a t c h 3. 1, 0 0 0 m l . deeper  Column volume  F l o w rate 9 m l per hour.  troughs indicate more  The  complete  s e p a r a t i o n of the t h r e e m a i n m o l e c u l a r s p e c i e s of n u c l e a r  protein.  84 V.  ANALYSIS OF CHROMATOGRAPHIC  1.  Disc  FRACTIONS  Electrophoresis  The three fractions  of c h r o m a t o g r a p h e d  concentrated  n u c l e a r p r o t e i n w e r e a n a l y s e d f o r b a s i c p r o t e i n u s i n g 15% g e l at p H 5. 0. while  Eight electrophoretic runs were made  v a r y i n g the  The most  sample load, buffer  systems  of t h e s e  fractions  and gel p o r o s i t y .  s a t i s f a c t o r y r e s u l t i s s h o w n i n F i g u r e 12.  The  second  c h r o m a t o g r a p h i c p e a k c o n t a i n e d a l l the d i s c s p r e s e n t i n the original sample.  P e a k 1 c o n t a i n e d a t r a c e of d i s c s 1 a n d 2 w h i c h  suggests that these are  c o m p o s e d of s o m e l a r g e r m o l e c u l e s i n  that they are eluted f i r s t f r o m the Sephadex c o l u m n and t r a v e l s l o w e s t i n the a c r y l a m i d e g e l .  That these discs are  geneous was evidenced by their presence  hetero-  i n p e a k s 2 and 3.  4 a n d 5 a p p e a r to be of s m a l l e r t h a n a v e r a g e  molecular  Discs  dimensions  as t h e y w e r e e l u t e d i n the s e c o n d and t h i r d c h r o m a t o g r a p h i c and t r a v e l e d fastest i n the a c r y l a m i d e g e l .  Disc 3 which is  p r e s e n t o n l y i n p e a k 2 a p p e a r s to be a l e s s h e t e r o g e n e o u s having m o l e c u l a r d i m e n s i o n s w h i c h l i e between the other groups.  peaks  fraction two  85  Figure  12  D i s c E l e c t r o p h o r e t i c A n a l y s i s of C h r o m a t o g r a p h e d Nuclear  Proteins.  0 . 1 m l (90 u g ) s a m p l e s . 5 M A per tube.  Running time 2 hours.  15% g e l s ( p H 5. 0).  Amido  Black 10B stain. . Peak 2 shows a pattern v e r y s i m i l a r to the s t a r t i n g  sample.  Unfractionated Nuclear P r o t e i n  Peak 1  Pe*k 2  Peak 3  87 2.  Amino Acid Composition  R e s u l t s of the a m i n o a c i d a n a l y s i s of the 3 f r a c t i o n s chromatographed observation,  nuclear protein are  shown i n Table 3.  The  f r o m d i s c e l e c t r o p h o r e t i c a n a l y s i s , t h a t the  proteins have not been fractionated to any great degree by graphy,  of  basic chromato-  i s c o n f i r m e d b y the a m i n o a c i d a n a l y s e s .  Fraction 2 is  s i m i l a r i n a m i n o a c i d c o m p o s i t i o n to the o r i g i n a l  unfractionated  sample some  (Table 2).  Chromatography does,  however,  result in  s e p a r a t i o n of a c i d i c p r o t e i n s f r o m b a s i c p r o t e i n s as  evidenced  b y the h i g h e r p e r c e n t a g e s of a s p a r t i c a n d g l u t a m i c a c i d s i n f r a c t i o n s 1 and 3.  88 TABLE A M M O ACID COMPOSITION OF  CHROMATOGRAPHED  Values are Fractions has  1 and  III OF THREE NUCLEAR  FRACTIONS PROTEIN.  p e r c e n t a g e s of t o t a l m o l e s of a m i n o  acids  3 contain high values for acidic amino  acids.  v a l u e s c l o s e to t h a t o f the u n f r a c t i o n a t e d n u c l e a r Amino Acid  present. Fraction  2  protein.  Fraction 1  Fraction 2  Lysine  5.6  15.2  8.7  Histidine  2.5  1.8  1.5  Arginine  4.4  5. 2  5. 8  Aspartic Acid  9-9  6.2  11.7  Threonine  9. 5  13. 3  10.2  12.6  6.0  12.6  Proline  6.7  5.3  2.4  Glycine  10.7  13.8  11.2  Alanine  10.8  15.1  12.6  Valine  8.6  5.0  5.8  Methionine  1.7  0.8  0.0  Isoleucine  4.5  2.8  5. 3  Leucine  7.7  6. 2  8.3  Tyrosine  2.3  1.5  1.0  Phenylalanine  2.0  1.7  2.4  Lysine + Argine  9.9  20.4  14.6  22.5  12.2  24.3  + Serine  Glutamic Acid  Aspartic + Glutamic Lysine / Arginine  1.3  2. 9  Fraction  1.5  3  89 3.  Immunodiffusion  Test  The three chromatographed, w e r e t e s t e d for a n t i g e n i c i t y as formed  reconcentrated  shown i n F i g u r e 13.  fractions  The  s i n g l e l i n e s of p r e c i p i t a t e w i t h the u n f r a c t i o n a t e d  and w i t h f r a c t i o n 2 but not w i t h f r a c t i o n s the a n t i g e n i c c o m p o n e n t i s i n f r a c t i o n 2.  antiserum protein  1 and 3, i n d i c a t i n g that  90  Figure  13  Immunodiffusion  T e s t of U n f r a c t i o n a t e d  Chromatographic  Fractions  of N u c l e a r  1% a g a r m a t r i x .  Reactions developed  5 days.  Single precipitation lines  4 a n d 5,  2 and  5.  and Protein  after  between  11 N o .  Reagent  1  Fraction  1  2  Fraction  2  3  Fraction  3  4  Unfr a c t i o n ated Nuclear Protein  5  Absorbed A n t i s e r u m 312  92 The  c h r o m a t o g r a p h i c a n a l y s i s of n u c l e a r p r o t e i n d i d not  s e p a r a t e the a n t i g e n i c c o m p o n e n t as d e s i r e d .  It d i d p r o v i d e a d d i -  t i o n a l i n f o r m a t i o n about the c o m p o s i t i o n of t h e n u c l e a r p r e p a r a t i o n and the e f f i c i e n c y of the  1.  techniques.  The nuclear proteins have m o l e c u l a r weights  i n t e r m e d i a t e between free dye and a l b u m i n and are weight  protein  small molecular  proteins. 2.  T h e n u c l e a r p r o t e i n p r e p a r a t i o n c a n be  i s e d as h a v i n g 3 b r o a d m o l e c u l a r s i z e  character-  subgroups.  F r a c t i o n 1 - larger molecular weight,  acidic proteins.  F r a c t i o n 2 - intermediate molecular weight,  basic  proteins. F r a c t i o n 3 - smaller molecular weight,  acidic  proteins. 3.  The antigenic component,  not d e s t r o y e d d u r i n g the p r o c e s s  p r e s e n t i n f r a c t i o n 2,  of c h r o m a t o g r a p h y and  is  reconcen-  tration. 4. for  VI.  S e p h a d e x G - 7 5 does not p r o v i d e sufficient r e s o l u t i o n  f i n e s e p a r a t i o n of n u c l e a r  PREPARATION  The  proteins.  OF FLUORESCENT  SERUM  FRACTIONS  a b s o r b e d a n t i s e r a f r o m c h i c k e n s 333 a n d 312 a n d  s a m p l e s of n o n - i m m u n e c h i c k e n s e r u m w e r e p r e p a r e d for u s e  as  two  93 f l u o r e s c e n t p r o t e i n t r a c e r s t o s t u d y t h e c e l l u l a r l o c a l i s a t i o n of basic nuclear protein. u s e d b y the author  The p r o c e d u r e was based on  i n a p r e v i o u s study (David,  involving fluorescent  techniques  R u t h and L a w ,  chicken isoantibody.  The s e r a w e r e l a b e l l e d w i t h fluorescent dye and tographed  chroma-  on Sephadex G - 2 0 0 to r e m o v e n o n - g l o b u l i n p r o t e i n s  free dye.  This procedure  fractions:  h i g h m o l e c u l a r w e i g h t (19S) g l o b u l i n s , s m a l l e r  s e p a r a t e s the  globulins and a n o n - g l o b u l i n fraction, et, a l . ,  1964).  antigens.  (7S)  mostly albumin (Fahey  The fractions were reconcentrated  The globulin fractions  and  analysed  of a n t i s e r u m ,  which  were prepared  i n the  same way for use  as a c o n t r o l .  a n t i s e r u m 333 w a s  sera  Non-immune  fractionated  once and a n t i s e r u m 312, w h i c h had the h i g h e s t t i t e r s w a s  frac-  times.  The four peaks  obtained are  s h o w n i n F i g u r e 14.  f i r s t p e a k to be e l u t e d f r o m the c o l u m n c o n t a i n e d the molecules,  as  S i m i l a r g l o b u l i n f r a c t i o n s of n o n - i m m u n e  serum was fractionated twice,  tionated four  as  contain  w e r e tested for i m m u n o l o g i c a l activity and used  protein tracers.  and  s e r u m into 3 m a i n  e l e c t r o p h o r e t i c a l l y and i m m u n o l o g i c a l l y u s i n g the f r a c t i o n s  antibody,  1966)  mostly lipoproteins.  peak i s the a l b u m i n f r a c t i o n .  largest  The second peak i s the  g l o b u l i n f r a c t i o n and the t h i r d i s the 7S.  The  The fourth and  19S largest  94 The tubes containing each peak w e r e pooled and t r a t e d b y v a c u u m d i a l y s i s to a f i n a l v o l u m e of 1 m l .  The  concenconcen-  t r a t e d p r o t e i n w a s r e m o v e d f r o m the d i a l y s i s tubes w i t h a l e n g t h of c a t h e t e r t u b i n g a t t a c h e d to a s y r i n g e .  The tubes w e r e  then  w a s h e d b y p l a c i n g 0. 5 m l i s o t o n i c s a l i n e i n t h e m a n d a g i t a t i n g thirty minutes  on the c l i n i c a l rotator.  and added to the p r o t e i n  fractions.  This wash was  removed  for  95  Figure  14  S e p h a d e x C h r o m a t o g r a p h y of a S a m p l e of A b s o r b e d , Immune,  Fluorescent Chicken Serum.  A 5 m l s a m p l e of a n t i s e r u m 3 1 2 . 1 liter.  F l o w rate 6 m l per  Column volume  hour.  A n t i b o d y a c t i v i t y w a s found to be a s s o c i a t e d p e a k 3 w h i c h c o n t a i n t h e 75 g l o b u l i n  with  component.  TUBE RTTBKB Tool 1  Tool  2  '  fool  3  "  Tool U  97 VII.  ANALYSIS OF FLUORESCENT  SERUM  FRACTIONS  T h e f r a c t i o n s w e r e a n a l y s e d f o r p u r i t y and the t y p e of proteins present by disc electrophoresis on M i l l i p o r e  membranes.  and m i c r o - i m m u n o d i f f u s i o n  They were tested for  anti-nuclear  p r o t e i n a c t i v i t y b y the p r e c i p i t i n t e s t a n d b y t h e i r a b i l i t y to specifically with nuclear fluorescent technique  1.  Disc  Figure  components  of C o o n s  a c c o r d i n g to the  immuno-  (1951).  Electrophoresis  15 s h o w s t h e r e s u l t s  of d i s c e l e c t r o p h o r e s i s  f l u o r e s c e n t l a b e l l e d w h o l e s e r u m and f r a c t i o n s by Sephadex chromatography. is not i n c l u d e d .  combine  2,  3 and 4,  obtained  F r a c t i o n 1 containing lipoproteins  T h i s p h o t o g r a p h w a s m a d e by p l a c i n g the  electro-  p h o r e s i s tubes d i r e c t l y on the u l t r a - v i o l e t l a m p i m m e d i a t e l y electrophoresis. d e s t a i n the  gels. some lipoprotein,  s m a l l a m o u n t o f 7S g l o b u l i n , Tube 3 contains  19S g l o b u l i n s a n d a  slightly m o r e than i n whole  serum.  a t r a c e of 19S a n d a h i g h c o n c e n t r a t i o n of 7S  It i s f r e e  mostly albumin.  of n o n - g l o b u l i n p r o t e i n s .  Tube 4  contains  T h e other d i s c s i n T u b e 4 a r e a s s u m e d to  small molecular weight proteins Sephadex c o l u m n after  as t h e y w e r e  (lipoproteins,  eluted f r o m  be the  7S g l o b u l i n s , w i t h the a l b u m i n f r a c t i o n .  L a r g e m o l e c u l a r weight proteins that w e r e first  after  T h i s e l i m i n a t e s the n e e d to f i x , s t a i n and  Tube 2 contains  globulin.  of  eluted f r o m the  19S g l o b u l i n s ) , h a v e m i g r a t e d t h e  column  shortest  distance  98 i n the g e l . further.  T h e 7S g l o b u l i n s ( M W  A l b u m i n ( M . W.._ 6 8 , 0 0 0 ) w h i c h w a s e l u t e d f r o m  column last,  has the  smallest molecular weight,  n e g a t i v e l y c h a r g e d of the m a j o r furthest  100, 000) h a v e t r a v e l l e d  i n the g e l .  serum proteins.  the  and i s the It h a s  most  migrated  99  Figure  15  D i s c E l e c t r o p h o r e s i s of A b s o r b e d , Immune  S e r u m 312.  B e f o r e and  Fluorescent,  After  Chromatography.  5 ul samples, gels.  r u n n i n g t i m e 90 m i n u t e s ,  P h o t o g r a p h e d w i t h H . S.  minute exposure, contain samples Figure  14.  f. 5. 6.  Ektachrome,  Tubes 2,  f r o m fractions  2,  1  3 and 4 3 and 4,  M o s t of the 7S g l o b u l i n i s i n  F r a c t i o n 3.  Tubes 2 and 3 are  of a l b u m i n .  I d e n t i f i c a t i o n of the d i s c s  reference  standard  completely  free  by  to an a n a l y s i s m a d e b y O r n s t e i n  (1964).  100  lipoprotein 19S  globulin  7S  globulin  transferrin  albumin  Whole serum  2  3  101 2.  Immunodiffusion  Test  S a m p l e s of the c h i c k e n s e r u m w e r e a l s o a n a l y s e d b y micro-immunodiffusion  ( F i g u r e 16).  T h i s w a s done by u s i n g  f o u r f r a c t i o n s of c h i c k e n a n t i s e r u m a s a n t i g e n s with rabbit anti-chicken  the  and reacting  them  serum.  T h e d i f f e r e n t m o l e c u l a r s i z e s of 19S a n d 7S g l o b u l i n s and a l b u m i n cause different i n the m i l l i p o r e m e m b r a n e .  r a t e s of d i f f u s i o n of t h e s e  T h i s i s r e f l e c t e d i n the p o s i t i o n s o f  the p r e c i p i t i n l i n e s .  A l b u m i n has m i g r a t e d furthest  to t h e c e n t r a l w e l l .  T h e 7S g l o b u l i n i s i n t e r m e d i a t e  globulin,  b e i n g the l a r g e s t ,  molecules  and is a n d the  h a s m i g r a t e d the s h o r t e s t  2 a n d 3 shows that these c o m p o n e n t s  are  19S l i n e i s h e a v i e s t o p p o s i t e w e l l 2,  w h e r e i t i s i n the  concentrated. crosses  19S  distance.  T h e c o n t i n u i t y o f the 7 S a n d 19S l i n e s b e t w e e n  concentration.  closest  i n both fractions.  wells The  greatest  T h e 7S l i n e i s h e a v i e s t o p p o s i t e w e l l 3 w h e r e i t i s The albumin line is associated only with well 4 and  the 7S l i n e s h o w i n g that t h e s e a r e  T h e r e i s n o a l b u m i n i n f r a c t i o n s 2 a n d 3. o r a l b u m i n i n f r a c t i o n 1.  different  antigens.  T h e r e a r e no g l o b u l i n s  102  F i g u r e 16  M i c r o - i m m u n o d i f f u s i o n T e s t of 4 F r a c t i o n s of Absorbed, Immune  Fluorescent,  Chromatographed,  C h i c k e n S e r u m 312.  The lines are identified by reference Johnson,  W e s t w o o d and B e a u l i e u (1964).  . P h o t o g r a p h p r o d u c e d by p l a c i n g the m e m b r a n e d i r e c t l y i n the e n l a r g e r printing.  to  Enlarged 4X.  mounted and  103  W e l l No.  Reagent  1  Fraction 1  2  fraction 2  3  Fraction 3  4  Fraction 4 Rabbit a n t i chicken serum  104 3.  Precipitin  Tests  W h e n the f r a c t i o n s w e r e t e s t e d f o r a n t i b o d y a c t i v i t y b y the p r e c i p i t i n t e s t o n l y the 7S f r a c t i o n , chromatography,  gave p o s i t i v e r e a c t i o n s  obtained a g a i n s t the  protein preparation.  Some activity appeared  d u r i n g the p r o c e s s e s  of c y t o p l a s m i c absorption,  tography and reconcentration for, was  by.Sephadex nuclear  to h a v e b e e n labelling,  a l t h o u g h the p r o t e i n  s e v e r a l times higher i n these fractions  t h e n i n the  original  t h e t i t r e s h a d d r o p p e d to 1:16 a n d 1:8 f o r c h i c k e n s  a n d 333  respectively.  IMMUNOFLUORESCENT  TESTS OF THE  LOCALISATION A N D DISTRIBUTION OF PROTEIN  C E L L U L A R NUCLEAR  r e s u l t e d i n the  of 5 m l o f a b s o r b e d a n d p u r i f i e d f l u o r e s c e n t  production  g l o b u l i n k n o w n to  contain antibody against a basic nuclear protein antigen. f r o m c h i c k e n 312,  Four  w e r e u s e d e x c l u s i v e l y i n the  i m m u n o f l u o r e s c e n t t e s t s to f o l l o w .  T w o m l of f l u o r e s c e n t  i m m u n e g l o b u l i n w e r e a l s o p r e p a r e d b y the reagents were  312  ANTIGEN.  The foregoing procedures  m l of this,  chroma-  concentration  serum,  VIIL  lost  same method.  nonThese  stored frozen i n s m a l l aliquots.  F l u o r e s c e n t m i c r o s c o p y w a s e m p l o y e d to s t u d y c e l l u l a r localisation, o r g a n distribution and species  the s p e c i f i c i t y of  calf thymus basic nuclear protein antigen using fluorescent  anti-  body as a p r o t e i n t r a c e r  a  (Coons,  1951).  Biebrich scarlet,  105 histone stain (Douglas, Spicer and B a r t e l s ,  1966) w a s u s e d  to  d e t e r m i n e w h e t h e r h i s t o n e s a n d the n u c l e a r p r o t e i n a n t i g e n o c c u p i e d the s a m e s i t e s .  C o m p a r i s o n of the  fluorescence  o b s e r v e d w i t h the p r e s e n c e  of D N A a n d R N A w a s m a d e b y s t a i n i n g  similar cells with Acridine  orange  Phase-contrast  (Von Bertalanffy,  1961).  m i c r o s c o p y w a s u s e d as a n a l t e r n a t i v e m e t h o d of  viewing fluorescent cells.  T h e i m m u n o l o g i c a l s p e c i f i c i t y of  fluorescent reactions was tested,  i n a l l cases,  cent n o n - i m m u n e globulin to c o n t r o l s l i d e s .  by applying fluores-  Control globulin was  p r e p a r e d f r o m n o n - i m m u n e c h i c k e n s u s i n g the s a m e m e t h o d s  as  for i m m u n e globulins. Cellular localisation and organ specificity were by a p p l y i n g these methods to calf thymus and testis cells and bovine c h r o m o s o m e s . studied using bovine cells, and protozoan  1.  cells,  studied  bovine liver,  Species specificity  human cells,, plant cells,  spleen was  insect cells  cells.  Bovine A.  Cells Calf Thymus  Imprints  Comparative studies on calf thymocytes are presented Figure  in  17. Figure  1 7 A s h o w s the y e l l o w f l u o r e s c e n c e ,  w i t h D N A , . of t h y m o c y t e n u c l e i .  The r e d fluorescence  be s e e n i n the c y t o p l a s m of s o m e c e l l s . of R N A o v e r - l a y the n u c l e u s ,  associated of R N A c a n  Where sufficient  the n u c l e i a p p e a r m o r e  amounts  green.  106 Such Acridine  orange  stained materials  m e t h o d of v i e w i n g c e l l p r e p a r a t i o n s p r e l i m i n a r y to l a b e l l i n g the antibodies.  also provided a  under ultra-violet illumination  s a m e t y p e of m a t e r i a l s w i t h  General morphology,  o p t i m u m conditions for  cent m i c r o s c o p y and the r a n g e s of e x p o s u r e t i m e s f o r photography were  fluorescent  studied using A c r i d i n e  fluores-  colour  orange.  B i e b r i c h s c a r l e t w a s a l s o u s e d to s t a i n c e l l i m p r i n t s . T h i s p r o v i d e d a c y t o c h e m i c a l m e a n s of s h o w i n g t h e l o c a t i o n of basic protein in cell nuclei, made with fluorescent  as a c o m p a r i s o n to the  antibody.  The presence  observation  of b a s i c p r o t e i n i n  t h y m o c y t e n u c l e i i s i n d i c a t e d b y the b r o w n to g r e y c o l o u r in Figure 17B. present in  The dark brown cytoplasmic inclusions  shown  are  granulocytes. Figure  17C shows the r e s u l t s  of a p p l y i n g f l u o r e s c e n t  a n t i b o d y to an i m p r i n t of u n f i x e d c a l f t h y m o c y t e s . i s a s s o c i a t e d w i t h n u c l e i and does not h a v e the  The  same  fluorescence  density  throughout. Treatment  of c a l f t h y m o c y t e s w i t h 0. 005 M c i t r i c  ( i m m e r s i o n and gentle w a s h i n g for  15 m i n u t e s ) b e f o r e  w i t h F I T C i m m u n e g l o b u l i n r e s u l t e d i n the n u c l e i b e i n g and m u c h  brighter fluorescence,  a b i l i t y of the n u c l e a r m e m b r a n e s 17D).  A further  acid  labelling condensed*  p e r h a p s due to i n c r e a s e d  perme-  a n d r e m o v a l of c y t o p l a s m ( F i g u r e  t r e a t m e n t w i t h 0 . 01 M c i t r i c a c i d - 1 . 2 5  M  NaCl  107 for three m i n u t e s , a l s o c a r r i e d out.  w h i c h i s k n o w n to r e l e a s e  p r o t e i n s as h a l o s of f l u o r e s c e n c e  of the r e l e a s e d  nuclear  s u r r o u n d i n g the n u c l e i ( F i g u r e  1 7 F i s of a c o n t r o l s l i d e s h o w i n g the r e s u l t s  fluorescent spot is a c e l l fragment.  That cells are present  s l i d e c a n be s e e n i n the p h a s e - c o n t r a s t  17E,).  of l a b e l l i n g  unfixed thymocytes with non-immune fluorescent globulins.  (Figure 17G).  was  Subsequent air drying and labelling with f l u o r e s -  cent i m m u n e g l o b u l i n s h o w s the p r e s e n c e  Figure  nuclear proteins,  i m a g e of the s a m e  The o n the  material  108 Figure  17  Calf Thymus Imprints Showing Presence  of D N A ,  Histone and Basic Nuclear P r o t e i n Antigen in Nuclei.  A  - Acridine orange  stain.  D N A is  yellow;  R N A which is r e d and present i n c y t o p l a s m s h o w s the a m o u n t of c y t o p l a s m i n t h e s e cells. B  Exposure time  15  seconds.  - Biebrich scarlet stain.  Brown  i n d i c a t e s the p r e s e n c e  of h i s t o n e  nuclei.  colour  Dark brown inclusions  granulocytes.  Automatic  in are  exposure.  C - F l u o r e s c e n t l a b e l l i n g of u n f i x e d Nuclear fluorescence  d i s t r i b u t i o n of b a s i c n u c l e a r antigen.  cells.  showing uneven  E x p o s u r e t i m e 60  protein seconds.  D  - F l u o r e s c e n t l a b e l l i n g of c i t r i c a c i d treated cells. Nuclei are condensed and show m u c h brighter fluorescence. E x p o s u r e t i m e 45 s e c o n d s .  E  - F l u o r e s c e n t l a b e l l i n g of s a l t cells.  N u c l e a r p r o t e i n has  released from nuclei,  treated been  in some  cases  completely (dark nuclei), and is as a h a l o of d i f f u s e  fluorescence  surrounding cells.  Exposure  60 F  seen  time  seconds.  - F l u o r e s c e n t l a b e l l i n g of u n f i x e d c e l l s with non-immune be s e e n f a i n t l y .  globulin.  A cell fragment  seen fluorescing brightly. t i m e 60 G - Phase  Cells  can is  Exposure  seconds.  contrast photography  f i e l d as i n F ,  of s a m e  showing cells present.  109  5Q  WVM,  110 B.  Liver  Cells  The presence  of b a s i c p r o t e i n i n l i v e r c e l l s i s i n d i c a t e d  b y the p i n k c o l o u r a s s o c i a t e d w i t h n u c l e i i n F i g u r e 1 8 B , f r o m an imprint stained with B iebrich Figure  scarlet.  1 8 A , C s h o w s the r e s u l t s  of l a b e l l i n g a s i m i l a r ,  unfixed imprint, with fluorescent immune globulin. nuclei have distinctive nucleoli w h i c h were m o r e b r i g h t l y t h a n the  surrounding nuclear  This photograph serves  cell  fluoresce  components.  t o d e m o n s t r a t e the  increased  technique.  Spleen Cells  Figure p r e p a r e d b y the  Liver  o b s e r v e d to  r e s o l u t i o n p o s s i b l e w i t h the i m m u n o f l u o r e s c e n t  C.  taken  18E and Figure 18F are  same methods  nuclear fluorescence  of s p l e e n i m p r i n t s  as w e r e the l i v e r c e l l s .  p r e s e n t a g a i n d e m o n s t r a t e s the  of b a s i c n u c l e a r p r o t e i n s  in interphase  i n c l u s i o n of b a c k g r o u n d m a t e r i a l .  The presence  n u c l e i w i t h o u t the  Ill  Figure  18  Bovine L i v e r and Spleen Imprints Showing P r e s e n c e of B a s i c N u c l e a r P r o t e i n A n t i g e n a n d H i s t o n e i n Nuclei.  A,  C - F l u o r e s c e n t l a b e l l i n g of u n f i x e d l i v e r cells.  Bright fluorescence  E x p o s u r e t i m e 45 B  - Biebrich  of n u c l e o l i .  seconds.  scarlet stain.  P i n k c o l o u r of  n u c l e i i n d i c a t e s the p r e s e n c e D  - Phase  c o n t r a s t i m a g e of the  of h i s t o n e . same  cells  as i n C , showing n u c l e o l i . E  - F l u o r e s c e n t l a b e l l i n g of u n f i x e d s p l e e n cells.  Some nuclei fluoresce  brightly than others. 30 F  more  Exposure time  seconds.  - Biebrich  scarlet stain.  P i n k c o l o u r of  n u c l e i s h o w s the p r e s e n c e  of h i s t o n e i n  v a r y i n g d e g r e e s of i n t e n s i t y .  112  50  wyi  4  D.  Testis  Cells  Testis imprints provided excellent cytological material and were  studied more  s h o w e d the p r e s e n c e  extensively.  Staining with Beibrich  of b a s i c p r o t e i n i n i n t e r p h a s e  undergoing meiotic division and s p e r m heads, erythrocytes.  nuclei,  but absent  from  i s i n c l u d e d to s h o w the p r e s e n c e visible using other  were  (Figure  1 9 F , a p h a s e c o n t r a s t p h o t o g r a p h of a n u n s t a i n e d  19).  imprint,  of s p e r m f l a g e l l a w h i c h w e r e  not  techniques.  The presence  of D N A i n p o s i t i o n s c o r r e s p o n d i n g to t h a t  of b a s i c n u c l e a r p r o t e i n i n n u c l e i i s d e m o n s t r a t e d with Acridine  cells  T h e s a m e p a t t e r n s of s t a i n i n g a n d i n t e n s i t y  p r e s e n t w h e n u s i n g the f l u o r e s c e n t a n t i b o d y t e c h n i q u e Figure  Scarlet  orange.  D N A fluorescence. unlike liver nucleoli,  i n 19B,  stained  Two s p e r m heads are labelled S and  N u c l e o l i c a n be s e e n i n s e v e r a l c e l l s , d i d not show i n c r e a s e d f l u o r e s c e n c e  labelled with fluorescent  antibody.  show which  when  114 Figure  19  B o v i n e T e s t i s I m p r i n t s S h o w i n g the P r e s e n c e ,  of  D N A , Histone and Basic Nuclear P r o t e i n Antigen in M e i o t i c Nuclei and S p e r m Heads.  A  - Biebrich  scarlet  stain.  M  - nucleus i n meiotic  S  - sperm  E  -  division  head  erythrocyte  T h e p i n k s t a i n i n g of n u c l e i i n d i c a t e s present. B  S p e r m heads stain m o r e  - A c r i d i n e lorange stain.  histone  darkly.  Red fluorescence  i n d i c a t e s h i g h a m o u n t of R N A p r e s e n t . S - s p e r m head showing yellow fluoresc e n c e of D N A . C,  E - Fluorescent labelling.  S p e r m heads  fluoresce m o r e brightly than E x p o s u r e t i m e 60 D  - Phase as  C.  c o n t r a s t i m a g e of the s a m e Stained for nuclei with  o r c e i n after F  - Phase  nuclei.  seconds.  fluorescence  field  aceto-  photography.  c o n t r a s t i m a g e of u n s t a i n e d  cells.  S p e r m f l a g e l l a p r e s e n t w h i c h do not  stain  for b a s i c n u c l e a r p r o t e i n and do not fluoresce when labelled with antibody.  fluorescent  115  116 E.  C h r o m o s o m e s of C u l t u r e d L e u k o c y t e s  T h e a n t i g e n i c i t y of b a s i c p r o t e i n s of p r e p a r a t i o n of m e t a p h a s e c h r o m o s o m e s  s u r v i v e d the  techniques  of c u l t u r e d l y m p h o c y t e s .  F i g u r e 20 i l l u s t r a t e s the o b s e r v a t i o n t h a t the  chromo-  s o m e s d i d not f l u o r e s c e e v e n l y but s h o w e d a s t i p p l e d p a t t e r n . c h r o m o s o m e s f l u o r e s c e d w i t h e q u a l i n t e n s i t y a n d the p h a s e p h o t o g r a p h i s i n c l u d e d to s h o w that a l l c h r o m o s o m e s c e n c e i n c l u d i n g the s e x  were  contrast  show fluores-  chromosomes.  Control slides, labelled with non-immune globulin,  The  fluorescent  were routinely prepared with each fluorescent test and  negative. It w a s f o u n d t h a t B i e b r i c h ' s c a r l e t d i d n o t s t a i n c h r o m o -  somes prepared i n this manner extent o n the u s e chromosomal  as the r e a c t i o n d e p e n d s to a  of s p e c i f i c f i x a t i v e s w h i c h a r e n o t u s e d i n  preparation.  large  117  F i g u r e 20  Bovine,  Acid-Alcohol Fixed,  S h o w i n g the P r e s e n c e  of B a s i c N u c l e a r P r o t e i n  a n d N u c l e i c A c i d i n the S a m e  A  Chromosomes  - Fluorescent labelling.  Sites.  Note stippled  a p p e a r a n c e of c h r o m o s o m e s i n d i c a t i n g u n e v e n d i s t r i b u t i o n of b a s i c protein antigen.  A l l chromosomes  show fluorescence.  Arrow  submetacentric  chromosomes.  sex  E x p o s u r e t i m e 90  B  nuclear  indicates  seconds.  - The same field stained for nucleic acid w i t h a c e t o - o r c e i n after photography.  fluorescence  118  2.  Human  Cells  F l u o r e s c e n t a n t i b o d y w a s a l s o a p p l i e d to t h r e e t y p e s of human cells and tissues  A.  and human  Buccal  chromosomes.  Cells  E p i t h e l i a l c e l l s f r o m the l i n i n g of the m o u t h w e r e a n d s t a i n e d f o r the d e m o n s t r a t i o n of B a r r b o d i e s w i t h i n the Similar cells treated with fluorescent immune globulin  prepared nucleus.  showed  bright nuclear fluorescence and some cytoplasmic fluorescence (which was also present in control slides).  The fluorescent  of B a r r b o d i e s c o u l d n o t be a s c e r t a i n e d a s t h e w h o l e  labelling  nucleus  fluoresced brightly and evenly (Figure 21A).  B.  A death,  Thymus  Cells  s a m p l e of h u m a n t h y m u s w a s o b t a i n e d ,  f r o m the c a d a v e r  o f a 13 y e a r o l d g i r l .  three days  This tissue  after  had  d e t e r i o r a t e d b a d l y , p r o b a b l y due to the n o r m a l r e g r e s s i o n of t h i s organ and autolysis.  When imprints were made  and treated  fluorescent antibody some nuclear fluorescence was evident suitable photographs  c o u l d n o t be  with but  obtained.  The spleen f r o m this patient was i n better condition. d e m o n s t r a t i o n of f l u o r e s c e n t n u c l e i w a s a c h i e v e d u s i n g the techniques  (Figure 21C).  usual  The  120 C.  A  Testis  Cells  s a m p l e of h u m a n t e s t i s w a s o b t a i n e d f r o m a 45  old patient s h o r t l y after  death and provided m a t e r i a l for  d e m o n s t r a t i o n of f l u o r e s c e n t n u c l e a r p r o t e i n s few mature  s p e r m were present i n this tissue.  s p e r m heads,  the  (Figure 2IE). However,  Very  two  a s w e l l a s n u c l e i i n v a r i o u s s t a g e s of d i v i s i o n ,  be s e e n to f l u o r e s c e .  year  can  121  F i g u r e 21  Human Cells  (Unfixed) S h o w i n g the P r e s e n c e  of  Basic Nuclear'Protein Antigen in Nuclei.  A  - F l u o r e s c e n t l a b e l l i n g of b u c c a l Brilliant nuclear fluorescence, over-exposed. fluorescence  B  - Phase  Some  smear. slightly-  cytoplasmic  present.  c o n t r a s t i m a g e of the s a m e  field  as A . C - F l u o r e s c e n t l a b e l l i n g of s p l e e n i m p r i n t . E x p o s u r e t i m e 60 D  - Phase the  E  c o n t r a s t i m a g e of a n o t h e r  same slide as  field  on  C.  - F l u o r e s c e n t l a b e l l i n g of t e s t i s i m p r i n t . Fluorescence sperm heads.  F  seconds.  - Phase as E .  of m e i o t i c n u c l e i a n d t w o E x p o s u r e t i m e 60  contrast image  of the  same  Note presence  of f l a g e l l a .  seconds. field  122  5Q mu.  123 D.  C h r o m o s o m e s of C u l t u r e d L e u k o c y t e s  F i g u r e 2 2 A w a s t a k e n f r o m a s l i d e of h u m a n p r e p a r e d b y the s t a n d a r d t e c h n i q u e , fixation,  including methanol-acetic  and labelled with fluorescent antibody.  the f i l m p h o t o g r a p h s  c o u l d be o b t a i n e d ,  fluorescent anti-chicken globulins.  chromosomes  B y over-exposing  w i t h o u t the u s e  of a d d i t i o n a l  Special colour printing tech-  n i q u e s d e s c r i b e d i n the l e g e n d w e r e n e c e s s a r y to p r o d u c e 22C.  acid  Figure  T h a t s u c h c h r o m o s o m a l f l u o r e s c e n c e i s not a n a r t i f a c t of  f i x a t i o n w a s d e t e r m i n e d b y the n e g a t i v e r e s u l t s  obtained with  control non-immune globulin and by labelling unfixed w i t h f l u o r e s c e n t antibody as i n F i g u r e 2 2 D .  chromosomes  124  F i g u r e 22  H u m a n C h r o m o s o m e s S h o w i n g the P r e s e n c e Basic Nuclear'Protein Antigen.  A  - P r e p a r e d using fixatives and labelled with fluorescent antibody.  colour over-  exposure,  make  which was necessary  the p h o t o g r a p h .  are  escing throughout. seconds.  o r c e i n after colour  s e e n t o be  fluor-  Exposure time  Incomplete  - Phase-contrast.  to  B y comparison with B  all chromosomes  B  The  of t w o l y m p h o c y t e n u c l e i i n d i c a t e s  120  metaphase.  Stained with  aceto-  fluorescent labelling  and  photography.  C - P r e p a r e d using fixative and labelled with fluorescent antibody. graph was made  This  colour slide, m a k i n g a negative  seconds. D  Exposure time  Complete  the  using  a y e l l o w f i l t e r a n d p r i n t i n g the fluorescence.  photo-  by over-exposing  green 120  metaphase.  - P r e p a r e d without fixative and labelled with fluorescent antibody. g r a p h p r e p a r e d i n the Figure 22C.  This  same manner  The c h r o m o s o m e s  i n d i s t i n c t u s i n g this m e t h o d but fluorescence those i n C .  of the  photo-  show  same intensity  Exposure time  120  as  are as  seconds.  of  125  •  1  1  1  &  0  * J|»*  * f  B  A  r  t  ** + T C  >  D  J"  •  3.  Insect  Chromosomes  I n F i g u r e 2 3 A the p o l y t e n e s a l i v a r y g l a n d  chromosomes  f r o m the l a r v a of the d i p t e r a n C h i r o n o m u s t e n t a n s a r e they appear  after  b e i n g s t a i n e d w i t h 0. 5% a c e t o - o r c e i n f o r D N A  and viewed under phase contrast m i c r o s c o p y . spread by moderate  A similar  nucleus,  osmotic swelling and flattened by a cover  glass is shown in Figure 23B. viewed by phase  s h o w n as  This is an unstained  preparation  contrast.  In F i g u r e 2 3 F c h r o m o s o m e s p o r t i o n of a c e l l m e m b r a n e .  c a n be s e e n a d h e r i n g to a  This material was prepared  by  m e c h a n i c a l l y r u p t u r i n g the c e l l after  brief fixation in 1 N H C 1 .  After  same m a t e r i a l was  p h a s e c o n t r a s t p h o t o g r a p h y the  with fluorescent n o n - i m m u n e globulin as a c o n t r o l . non-specific fluorescence the c e l l m e m b r a n e  The  treated resulting  (Figure 23E) is associated equally with  a n d the c h r o m o s o m a l m a t e r i a l .  h i g h a f f i n i t y of s e r u m g l o b u l i n s f o r t h e s e m e m b r a n e s  The  rather  was a  constant  finding. F i g u r e s 2 3 C a n d 2 3 D w e r e p r e p a r e d i n the s a m e  manner  e x c e p t t h a t f l u o r e s c e n t i m m u n e g l o b u l i n w a s u s e d to l a b e l the chromosomes  in C.  T h i s i s a s p e c i f i c r e a c t i o n thought to be  to the p r e s e n c e  of b a s i c p r o t e i n ,  chromosomes.  T h e r e i s s o m e n o n - s p e c i f i c b i n d i n g to the  o n the u p p e r p o r t i o n of the  associated with D N A ,  figure.  due  i n the membrane  127  F i g u r e 23  C h r o m o s o m e s of C h i r o n o m u s t e n t a n s S h o w i n g the Presence  A  of B a s i c N u c l e a r P r o t e i n A n t i g e n .  - Aceto-orcein staining. photography.  Phase  100X objective  Squash preparation.  Four  contrast lens.  chromosomes  p r e s e n t s h o w i n g p a t t e r n s of b a n d i n g chromosomal B  puffs.  - Unstained. Phase contrast photography. 100X objective lens. Nucleus has been swelled and flattened under cover glass. Sharp banding patterns are visible.  C - Labelled with fluorescent  antibody.  C h r o m o s o m e s have been exposed, to l a b e l l i n g , brane.  b y r u p t u r i n g the c e l l  F l u o r e s c e n c e i s 1/4 the  t i m e 240  prior mem-  intensity  of t h a t of b o v i n e c h r o m o s o m e s .  Exposure  seconds.  D - The s a m e field as i n C photographed phase E  - Labelled with non-immune  fluorescent  The fluorescence  non-specific fluorescence membrane. t i m e 240  of the  cell are  Exposure  seconds,  contrast image  as i n E .  present is  That chromosomes  p r e s e n t c a n be seen, i n F .  - Phase  by  contrast.  globulin.  F  and  of the  same  field  128  129  4.  Plant Cells  The immunofluorescent some s p e c i a l problems.  study o f p l a n t c e l l s p r o v i d e d  The l i g n i f i e d m a t e r i a l i n c e l l w a l l s  h i g h a f f i n i t y f o r serum g l o b u l i n s It  and f l u o r e s c e d  non-specifically.  a l s o p r o v i d e d a b a r r i e r to the e n t r y of f l u o r e s c e n t  the c e l l .  Pectinase  the c e l l s ,  but o l d e r c e l l s were s t i l l  globulin.  Younger c e l l s ,  s e r v e d to s o f t e n the c e l l w a l l s  were permeable and showed d e f i n i t e types of tips  cells  of L i l y  are p r e s e n t e d  fluorescent  present  is  taken  with associated  strong a f f i n i t y of a s p i r a l  evident.  o f dust p a r t i c l e s .  vessel  The r e d d i s h f l u o r e s c e n c e at lower c e n t r e  is  due  No n u c l e a r f l u o r e s c e n c e  at to  was  slides.  F i g u r e 24C, a l s o of Onion root t i p fluorescent  associated  T h i s was  Some f l u o r e s c e n c e  due to a u t o f l u o r e s c e n c e  i n such c o n t r o l  fluorescence  cell.  root t i p material treated  and the  element f o r serum g l o b u l i n i s  autofluorescence  immune g l o b u l i n .  shown i n F i g u r e 24D.  non-immune g l o b u l i n .  is  Both  prepared i n the same manner, gave  of n o n - s p e c i f i c  (LiHum)  w i t h c e l l u l a r components  lower c e n t r e  a thick w a l l ,  as shown i n F i g u r e 24A of a s i n g l e  w i t h such p l a n t m a t e r i a l i s from a s l i d e o f L i l y  fluorescent  i n F i g u r e 24B, p r e p a r e d from r o o t  Onion r o o t t i p c e l l s ,  The h i g h l e v e l  into  separate  nuclear fluorescence.  and t r e a t e d w i t h f l u o r e s c e n t  comparable r e s u l t s ,  antibody  and  impermeable to  which had not developed  had a  immune g l o b u l i n , i s  presented  t h a t n u c l e o l i i n p l a n t c e l l s d i d not  (Alium). treated with  to support the  fluoresce.  observation  130  F i g u r e 24  P l a n t C e l l s S h o w i n g the P r e s e n c e Protein Antigen i n Some  A  of B a s i c N u c l e a r  Nuclei.  - Onion root tip cell labelled with antibody.  fluorescent  Plant cells show some  fluorescence.  N u c l e o l i a r e not  cytoplasmic fluorescent.  T h i s i s a young c e l l w h i c h has not a thick wall.  Exposure time  developed  180  seconds.  B  - L i l l y root tip cells labelled with fluorescent antibody. The young cell i n centre which does not have a t h i c k w a l l shows n u c l e a r f l u o r e s c e n c e ^ s antibody could penetrate. The other two cells show only non-specific f l u o r e s c e n c e of c e l l w a l l m a t e r i a l . E x p o s u r e t i m e 180 s e c o n d s .  C  - Onion root tip cells labelled with antibody. more  The nucleoli i n these cells  e v i d e n t a n d doi  Exposure time D  fluorescent  180  not  seconds.  - L i l l y root tip cells labelled with non-immune  globulin.  specific fluorescence, wall material,  present.  fluorescent  There is some  non-  associated with  cell  The s p i r a l  ture i s a v e s s e l element w h i c h has affinity for s e r u m globulin. time  180  seconds.  are  fluoresce.  strucstrong  Exposure  131  132 5.  Protozoan  Cells  This unicellular parasite for the p r e s c e n c e  (Holomastigotoides) was  tested  of b a s i c p r o t e i n i n the n u c l e u s b y r u p t u r i n g  the  p e l l i c l e o s m o t i c a l l y and l a b e l l i n g w i t h f l u o r e s c e n t g l o b u l i n i n the usual manner.  The nucleus,  which is Feulgen positive for D N A ,  i s l a b e l l e d (N); i n F i g u r e 2 5 B t a k e n w i t h p h a s e c o n t r a s t fluorescent  before  labelling.  As  c a n be s e e n i n F i g u r e 2 5 A the p e l l i c l e a n d  c e l l i n c l u s i o n s , as w e l l as the n u c l e u s , cent l a b e l l i n g .  are v i s i b l e after  T h i s i s a n o n - s p e c i f i c effect as the  was obtained with fluorescent non-immune  globulin.  same  several fluoresresult  133  F i g u r e 25  A n Insect Parasite  (Holomastigotoides) Labelled with  Fluorescent Antibody.  A  - C e l l has been ruptured antibody to penetrate. some  o s m o t i c a l l y to a l l o w The outer  c e l l u l a r i n c l u s i o n s a n d the  a l l show non-specific affinity for globulins.  pellicle, nucleus serum  S i m i l a r result obtained with  non-immune  globulin.  Exposure time  seconds.  B  - Phase N  contrast  = nucleus.  i m a g e of the s a m e  cell.  240  134  135 6.  Chicken Blood  Cells  A s a f i n a l t e s t of the i m m u n o f l u o r e s c e n t s p e c i f i c i t y of the f l u o r e s c e n t ,  chromatographed,  absorbed,  immune chicken  globulin it was tested against chicken blood cells obtained the s a m e results  immunised chicken.  F i g u r e 2 6 A s h o w s the  obtained after fluorescent labelling.  from  negative  The companion  photograph (Figure 26B)shows that there were c e l l s present this  field.  in  136  F i g u r e 26  Chicken Blood Cells Labelled with  Fluorescent  Antibody.  A  - F l u o r e s c e n t spot is n o n - s p e c i f i c  attachment  to d i r t p a r t i c l e a s s o c i a t e d w i t h the cell (arrow in B).  B  - Phase  ruptured  E x p o s u r e t i m e 60  contrast i m a g e of s a m e f i e l d .  seconds.  Most  of the c e l l s a r e n u c l e a t e d e r y t h r o c y t e s . l e a s t one l y m p h o c y t e (L); i s  present.  At  138 IX.  H U M A N LUPUS ERYTHEMATOSA ANTIBODY AGAINST  ACTIVITY  C A L F THYMUS NUCLEAR PROTEIN  ANTIGENS.  Lupus Erythematosis is an autoimmune disease which is k n o w n to cause the p r o d u c t i o n of a n t i b o d i e s a g a i n s t the own  n u c l e a r p r o t e i n ( K r o o t h et a l . ,  patients  1961).  patient's  The plasma from  5  diagnosed as having L u p u s E r y t h e m a t o s i s was tested  for  its i m m u n o l o g i c a l a c t i v i t y against calf thymus nuclear p r o t e i n by a precipitin test and immunodiffusion.  W h i l e the r e s u l t s  of these  t e s t s c a n be c o m p a r e d to the a n t i b o d y a c t i v i t y , p r o d u c e d e x p e r i m e n t a l l y i n the c h i c k e n against the s a m e antigens, conclusions m u s t await a m o r e detailed study. these tests are,  therefore,  further  The results  presented as observations  P l a s m a N (well 3,  of  only.  F i g u r e 27);was the o n l y one to  p o s i t i v e l y i n the p r e c i p i t i n t e s t .  The l i n e s of precipitate  react  in  F i g u r e 27 i n d i c a t e that the p l a s m a s r e a c t e d a g a i n s t one o r b o t h of two  a n t i g e n s p r e s e n t i n the n u c l e a r p r o t e i n p r e p a r a t i o n .  B and L r e a c t e d w i t h a fast m o v i n g component to produce n e a r the p l a s m a w e l l s .  lines  P l a s m a B also reacted with a slower  m o v i n g component to produce another  l i n e n e a r the c e n t r a l  This line is continuous opposite wells 2 and 4 containing N and C .  Plasmas  well.  plasmas  T h e s e l i n e s do not a p p e a r t o c o i n c i d e w i t h the p r e c i p i t i n  line opposite w e l l 5 containing absorbed chicken anti-nuclear serum.  protein  S i n c e o n l y one plate w a s p r e p a r e d t h i s i s a p r e l i m i n a r y  observation  only.  139  F i g u r e 27  I m m u n o d i f f u s i o n T e s t of A n t i b o d y A c t i v i t y of L . E . Plasma Against Basic Nuclear Protein.  — Plasma,  chicken antiserum and antigen a l l  added at the s a m e t i m e . wells  Lines closest  1 and 3 indicate an immune  between plasmas  to  reaction  B and L against a s m a l l  molecular weight nuclear protein antigen. L i n e c l o s e t o w e l l 6 o p p o s i t e w e l l s 1, and 4 indicates plasmas  B , N and C  reacting with a different larger weight  antigen.  2 are  molecular  T T  e l l No.  Reagent  1  Plasma B  2  Plasma N  3  Plasma L  4  Plasma C  5  C h i c k e n serum  312  6  Basic Nuclear P r o t e i n , B a t c h #3  141 SUMMARY The proteins f r o m calf thymus  cells were  separated  into a c y t o p l a s m i c and n u c l e a r f r a c t i o n u s i n g a c i t r i c and weak salt extraction procedure.  acid  A n a l y s i s o f the  nuclear  p r o t e i n r e v e a l e d a n a m i n o a c i d c o m p o s i t i o n c h a r a c t e r i s t i c of a crude,  lysine-rich,  histone preparation.  of the e x t r a c t w a s d e m o n s t r a t e d  The  heterogeneity  by s e p a r a t i o n into 3 a c i d i c  and 5 basic fractions by disc electrophoresis  on polyacrylamide  gels.  on Sephadex  These fractions  columns,  c o u l d not be  separated  G-75  i n d i c a t i n g that a l l 8 f r a c t i o n s w e r e of a s i m i l a r ,  small molecular  size.  N u c l e a r protein was precipitated by salt  removal  through d i a l y s i s against d i s t i l l e d water and injected into chickens.  F i v e o f 22 c h i c k e n s p r o d u c e d a n t i b o d i e s  by p r e c i p i t a t i o n and immunodiffusion. a s the b e s t a n t i b o d y p r o d u c e r s , repeated  detectable  Two chickens,  selected  w e r e hyper i m m u n i s e d by  i n j e c t i o n s of the p r e c i p i t a t e d n u c l e a r p r o t e i n  extract.  The s e r u m obtained f r o m these chickens contained precipitating antibodies against  3 antigenic components  of the i n o c u l u m ,  2 o f w h i c h w e r e a l s o p r e s e n t i n the c y t o p l a s m . against2  cytoplasmic antigens  were  Antibodies  r e m o v e d f r o m the  sera  a b s o r p t i o n w i t h the c y t o p l a s m i c f r a c t i o n of c a l f t h y m u s c e l l s . absorbed antisera  reacted with a single electrophoretic  of the b a s i c n u c l e a r p r o t e i n  preparation.  by The  fraction  142 The antisera  were conjugated with fluorescein  i s o t h i o c y a n a t e a n d the a n t i b o d y c o n t a i n i n g g l o b u l i n f r a c t i o n was isolated by Sephadex G - 2 0 0 chromatography. electrophoresis,  Disc  immunodiffusion and p r e c i p i t i n tests indicated  t h a t the a n t i b o d y a c t i v i t y w a s p r e s e n t i n a 7 S g l o b u l i n f r a c t i o n , of n o n - g l o b u l i n p r o t e i n .  S e r a o b t a i n e d f r o m c h i c k e n s that had  not b e e n i n j e c t e d w i t h n u c l e a r p r o t e i n s w a s p r e p a r e d same manner  n u c l e i of i m p r i n t s of a n u m b e r  specifically  of b o v i n e t i s s u e s :  with  thymus,  liver,  spleen,  testis,  epithelium:  liver,  spleen,  testis:  plant s m e a r s of root tip c e l l s ;  all chromosomes  a  tests.  The fluorescent antibody reacted  human tissues;  thymus, lily,  in fixed and unfixed human lymphocyte  cultures; fixed bovine lymphocyte cultures and insect gland cells,  i n the  astthe i m m u n e globulin f r a c t i o n and u s e d as  control in immunofluo re scent  onion:  salivary  The antibody d i d not r e a c t w i t h c h i c k e n c e l l s .  Control preparations,  'labelled with non-immune  negative i n a l l cases.  Exposure times,  the f l u o r e s c e n c e  human cells,  plant cells,  fluorescence  is comparable  globulin  necessary  indicated a decreasing affinity  antibody and test m a t e r i a l s  and insect cells.  to  were  photograph  between  i n the f o l l o w i n g o r d e r :  bovine  cells,  If t h e a m o u n t o f  to i n t e r s p e c i f i c s e r o l o g i c a l c r o s s -  reactivity it indicates an antigenic  similarity which exists  a w i d e phylogenetic range but d e c r e a s e s w i t h phylogenetic sepa ration.  free  over  143  T h i s common a n t i g e n i c i d e n t i f i e d as a h i s t o n e a.  tentatively  a c c o r d i n g to the f o l l o w i n g  I t was i s o l a t e d acid values  component was  from a p r e p a r a t i o n h a v i n g amino  consistent  preparations.  criteria:  with standard histone  T h i s p r e p a r a t i o n was,  a mixture of p r o t e i n s ,  however,  any one of which c o u l d be  the a n t i g e n i n q u e s t i o n . b.  I t m i g r a t e d i n p o l y a c r y l a m i d e g e l s under c o n d i t i o n s designed  f o r the electrophoresis  i n d i c a t i n g that  it  is  of  histones,  a small molecular weight,  basic protein. c.  It  stained,  scarlet,  i n polyacrylamide gels, with Biebrich  a dye known to s e l e c t i v e l y  stain histones  under a p p r o p r i a t e c o n d i t i o n s . d.  It occurred s o l e l y by  e.  i n the n u c l e i o f c e l l s ,  as seen  immunofluorescence.  I t was found to be i n c l o s e a s s o c i a t i o n w i t h DNA i n chromosomes,  as seen by comparison o f  aceto-orcein  s t a i n e d chromosomes and immunofluorescence. f.  It  occupied s i m i l a r s i t e s  nuclei  to h i s t o n e  in  and to protamines i n sperm h e a d s ,  comparison of B i e b r i c h s c a r l e t immunofluorescence.  cell as seen by  s t a i n i n g and  144 Immunodiffusion t e s t s showed a p r e c i p i t i n l i n e continuous between the b a s i c n u c l e a r p r o t e i n extract  and a commercial h i s t o n e  preparation,  i n d i c a t i n g the same a n t i g e n was present samples.  The p o s s i b i l i t y  i n both  remains t h a t both  p r e p a r a t i o n s c o u l d have c o n t a i n e d a common antigenic  contaminant. The author wishes to p o i n t out  these are i n d i r e c t p i e c e s o f evidence  and do  not prove t h a t the a n t i g e n i s  a histone  they do i n d i c a t e  a strong  that  this  is  that  although possibility.  145 DISCUSSION T h e p r i m a r y o b j e c t i v e o f t h i s s t u d y w a s to  develop  a n i m m u n o f l u o r e s c e n t t e c h n i q u e w h i c h c o u l d b e a p p l i e d to s t u d i e s of the l o c a t i o n a n d d i s t r i b u t i o n o f s p e c i f i c n u c l e a r proteins.  However,  n u c l e u s they w e r e  s i n c e o n l y t h e h i s t o n e s a r e u n i q u e to  s e l e c t e d a s the m o s t a p p r o p r i a t e  m a t e r i a l f o r this study.  H i s t o n e s h a v e the f u r t h e r  that t h e y h a v e b e e n the o b j e c t o f i n t e n s e fifteen y e a r s procedures  (Butler,  antigenic advantage  s t u d y d u r i n g the  1965) a n d c o n s e q u e n t l y  the  past  extraction  and analytical techniques for calf thymus  preparation are well developed (Busch,  1965,  In contrast,  d i f f i c u l t to i s o l a t e  acidic nuclear proteins are  have r e c e i v e d little attention (Busch, The major immunological  58-90).  ( W i l s o n et a l . ,  generally  19 6 6 ) .  and  1 9 7 - 2 2 6).  disadvantage of u s i n g histones  studies i s that they a r e  to b e p o o r a n t i g e n s  p. p.  p. p.  histone  in  considered  However,  strong  i m m u n e r e a c t i o n s a g a i n s t h i s t o n e s a r e p r o d u c e d i n the autoimmune  disease  systemic lupus erythematosis  I 9 60) a n d i t h a s b e e n p o s s i b l e ,  in some cases,  histone antibodies i n experimental animals.  to  (Kunkel, produce  M e i s c h e r et a L ,  (1960) i m m u n i s e d g u i n e a p i g s a n d r a b b i t s w i t h c a l f  thymus  n u c l e o p r o t e i n s a n d f o u n d t h e m to b e w e a k l y a n t i g e n i c .  More  146 recently, from  W i l s o n et a l . , (1966) r e p o r t e d that h i s t o n e s  calf thymus w e r e not antigenic i n rabbits after  immunisation.  extracted  prolonged  O n e a p p a r e n t d i f f e r e n c e b e t w e e n the  successful  e x p e r i m e n t s r e p o r t e d b y M e i s c h e r ( I 9 60) a n d i n t h i s s t u d y a n d the u n s u c c e s s f u l a t t e m p t s o f W i l s o n (1966) i s i n the m e t h o d s of h i s t o n e e x t r a c t i o n a n d p r e p a r a t i o n o f the a n t i g e n s . Wilson used an acid extracted preparation,  salt  extracted  nuclear proteins were u s e d by M e i s c h e r and this Chargaff  Whereas  author.  (1955) s u g g e s t e d that a c i d e x t r a c t e d h i s t o n e s  are  s h o r t - c h a i n c l e a v a g e p r o d u c t s of the n a t i v e h i s t o n e m o l e c u l e . T h i s h a s b e e n s u p p o r t e d b y H u a n g a n d B o n n e r (19 66) w h o found that a c i d e x t r a c t e d h i s t o n e s f r o m pea b u d n u c l e i h a d m o l e c u l a r w e i g h t s o f a p p r o x i m a t e l y 2 0, 0 0 0 ; o n e - t e n t h of the n a t i v e h i s t o n e m o l e c u l e .  that  If a c i d e x t r a c t i o n c l e a v e s  histone m o l e c u l e s into fragments,  the g e n t l e r m e t h o d o f  w e a k s a l t e x t r a c t i o n u s e d i n the p r e s e n t e x p e r i m e n t ,  and by  Meischer,  isolation  s h o u l d l i m i t d e n a t u r a t i o n a n d r e s u l t i n the  of a l a r g e r m o l e c u l e .  A s the m i n i m u m  s i z e f o r a p r o t e i n to  b e i m m u n o g e n i c i s c o n s i d e r e d to b e 2 0 , 0 0 0 ( C a r p e n t e r p.  41) i t s e e m s r e a s o n a b l e  to e x p e c t t h a t t h e s a l t  histones w o u l d be m o r e i m m u n o g e n i c .  I n the  1965,  extracted  present  experiments immunogenieity m a y have been further  enhanced by  the u s e of a r e l a t i v e l y c r u d e n u c l e a r p r o t e i n p r e p a r a t i o n injected as a precipitate since antigens introduced i n a particulate f o r m are usually m o r e i m m u n o g e n i c than if  147 injected as  solutes (Fischer,  19 6 4 ) .  R u m k e a n d S l u y s e r (19 66) h a v e r e p o r t e d a m e t h o d of p r e p a r a t i o n i n w h i c h s a l t e x t r a c t e d h i s t o n e s f r o m were  rat  s t r o n g l y a n t i g e n i c to r a b b i t s a n d a c i d e x t r a c t e d  were weakly antigenic.  liver  histones  T h e y a t t r i b u t e d t h e i r s u c c e s s to a  b r i e f h e a t t r e a t m e n t ( 5 m i n u t e s at 80*C) of the t i s s u e h i s t o n e e x t r a c t i o n to i n a c t i v a t e p r o t e o l y t i c e n z y m e s prevent histone breakdown.  before and  T h i s p r o c e d u r e m a y be  more  e f f e c t i v e t h a n the u s u a l m e t h o d of i n a c t i v a t i n g e n z y m e s by e x t r a c t i n g h i s t o n e s at 4 C ( D a l y a n d M i rsky ^ 1955). Although i n previous experiments calf n u c l e o p r o t e i n s w e r e injected into other  fflammals  etal.>  1964f  1959; Black,  Ansley and Mandl;  thymus (Meischer  W i l s o n et a l . ,  1965) the c h i c k e n w a s u s e d a s the r e c i p i e n t i n t h i s  project.  T h e i m p o r t a n c e of the c h o i c e of a n t i b o d y p r o d u c e r s indicated,  a s the o b s e r v a t i o n s o f o t h e r s h a d s h o w n that  histones from  different species  (Stedman and Stedman,  1951;  show r e m a r k a b l e  N e e l i n and Butler,  If i n s p i t e o f t h i s i n t e r s p e c i f i c s i m i l a r i t y , differences  was  similarity 1961).  slight antigenic  do e x i s t , t h e c h a n c e o f f i n d i n g v a r i a t i o n s h o u l d  i n c r e a s e i n v e r s e l y w i t h the d e g r e e of b i o l o g i c a l r e l a t i o n s h i p b e t w e e n the a n t i g e n d o n o r a n d r e c i p i e n t ( C a r p e n t e r , p.  65).  T h e u s e of the b o v i n e a n d a v i a n s p e c i e s m a y  19 6 5 , have  148 c o n t i r b u t e d to s u c c e s s f u l a n t i b o d y p r o d u c t i o n b y p r o v i d i n g a wide  species  separation. To date,  experiments using nuclear  components  as antigenic m a t e r i a l have been p r i m a r i l y concerned with p o s s i b i l i t y of p r o d u c i n g a n i m m u n e r e s p o n s e  and little  e m p h a s i s h a s b e e n p l a c e d o n c h a r a c t e r i s a t i o n of the I n i t i a l a t t e m p t s to a n a l y s e t h e a n t i g e n - a n t i b o d y made use of I m m u n o e l e c t r o p h o r e s i s R u m k e and Sluyser,  1966).  antigens.  responses  ( W i l s o n et_al, ,  1966;  Wilson compared immune  to D N A , D N A - p r o t e i n a n d h i s t o n e ,  responses  c o n c l u d i n g that only D N A -  p r o t e i n c o n t a i n e d t r a c e a m o u n t s of a n t i g e n i c m a t e r i a l s . a n d S l u y s e r w e r e a b l e to d e m o n s t r a t e  the p r e s e n c e  i m m u n o c h e m i c a l ^ distinct histone extracts.  lysine-rich preparation.  i n the p r e s e n t  Rumke  of three  Their  reaction produced a single precipitin line against a extracted,  strongest salt-  The antibody obtained  experiments also produced a single p r e c i p i t i n  l i n e a g a i n s t the l y s i n e - r i c h p r e p a r a t i o n .  A further  fraction-  a t i o n of this e x t r a c t w a s a c h i e v e d by d i s c e l e c t r o p h o r e s i s an antibody was subtractions.  s h o w n to b e d i r e c t e d a g a i n s t o n e o f t h e  R u m k e and S l u y s e r d i d not r e p o r t  to d e m o n s t r a t e  heterogeneity  proteins by u s i n g fractions,  of the b a s i c  obtained by different  and  five  analysis  of the a n t i g e n i c c o m p o n e n t s o f t h e i r h i s t o n e e x t r a c t s , attempted  the  but nuclear  extraction  149 procedures,  as antigens.  T h e i r evidence for  immuno-  chemically distinct fractions was based on precipitin lines against l y s i n e - r i c h and lysine-poor preparations.  The  p o s s i b i l i t y o f c h a r a c t e r i s i n g the b a s i c n u c l e a r p r o t e i n s cytoplasmic contaminants  p r e s e n t i n the  and  lysine-rich  p r e p a r a t i o n u s e d i n this study w a s e x p l o r e d u s i n g i m m u n o logical,  electrophoretic and chromatographic Sephadex chromatography  nuclear proteins  c a n be  h a s the a d v a n t a g e  separated under  salt c o n c e n t r a t i o n that l i m i t d e n a t u r a t i o n 1964).  However,  chromatographic ation neither  techniques.  conditions of p H and ( J o h n s o n et a l . ,  the r e s u l t s o f d i s c e l e c t r o p h o r e s i s f r a c t i o n s i n d i c a t e d that Sephadex  separated  the n u c l e a r p r o t e i n s into  of  the b a s i c n u c l e a r p r o t e i n r e t a i n e d i t s a n t i g e n i c i t y  preparative  electrophoresis  our results  discrete Since  after  indicate  on p o l y a c r y l a m i d e gels  that could  b e e m p l o y e d to s e p a r a t e n u c l e a r p r o t e i n s i n t o s e p a r a t e in amounts logical  sufficient for amino acid analyses  the  fraction-  f r a c t i o n s n o r r e m o v e d the c y t o p l a s m i c c o n t a m i n a n t s .  analytical disc electrophoresis,  that  fractions  and i m m u n o -  tests. The fluorescent antibody obtained was  for nuclei and c h r o m o s o m e s  specific  but w a s neither o r g a n n o r  species  150 specific.  The antibody reacted with nuclei f r o m calf  and other bovine organs  and with bovine s p e r m heads and  leukocyte chromosomes. several human tissues, chromosomes. fluorescence,  thymus  It a l s o r e a c t e d w i t h n u c l e i f r o m plant nuclei and insect  However,  polytene  a d e c r e a s e i n the i n t e n s i t y o f  r o u g h l y p r o p o r t i o n a l to t h e i n c r e a s e  diversity was observed.  These observations  in  species  suggest that an  a n t i g e n i c a l l y s i m i l a r p r o t e i n w a s p r e s e n t i n the n u c l e i f r o m a l l these  species. The presence  in different  of a n t i g e n i c a l l y s i m i l a r  s p e c i e s i s not u n c o m m o n .  have been demonstrated  proteins  Classically,  by s e r o l o g i c a l tests for  these  cross-  r e a c t i o n s i n w h i c h the d e g r e e of i n t e r s p e c i f i c  protein  s i m i l a r i t y i s m e a s u r e d by the  reaction.  s t r e n g t h o f the  I n 19 34 H o o k e r a n d B o y d p r o p o s e d t h a t s u c h were  d u e to t h e p r e s e n c e  determinants  o f at l e a s t two t y p e s of a n t i g e n i c  as a n i n t e g r a l p a r t of the c o m p o s i t i o n a n d  s t r u c t u r a l c o n f i g u r a t i o n of p r o t e i n m o l e c u l e s . determinant  O n e type of  i s s p e c i f i c f o r the p a r t i c u l a r p r o t e i n i n q u e s t i o n  a n d the o t h e r  t y p e i s c o m m o n to a g r o u p o f r e l a t e d  Antibodies produced against react most  cross-reactions  such a protein class  s t r o n g l y w i t h the o r i g i n a l a n t i g e n ,  proteins. could  forming  i m m u n e c o m p l e x e s w i t h b o t h t y p e s of d e t e r m i n a n t s .  The  151 s a m e antibody w o u l d c r o s s - r e a c t w i t h a v a r i e t y of r e l a t e d p r o t e i n s that s h a r e the  same or similar determinants,  to the p r o t e i n s a s a g r o u p . numerous  common  This theory is supported by  r e p o r t s of i n t e r s p e c i f i c s e r o l o g i c a l  cross-  r e a c t i v i t y b e t w e e n soluble p r o t e i n antigens (see L a n d s t e i n e r , 1945,  f o r r e v i e w ) a n d the e x i s t e n c e o f a n t i g e n i c  determinants  h a s b e e n d e m o n s t r a t e d b y W e i g l e ( 1 9 61) w h o s h o w e d t h a t c r o s s - r e a c t i o n s of e l e v e n heterologous m a m m a l i a n s e r u m a l b u m i n s w e r e d u e to the p r e s e n c e o f f r o m  t w o to f i v e a n t i -  genic determinants  species.  s h a r e d b y the d i f f e r e n t  The o b s e r v a t i o n that f l u o r e s c e n t antibody with bovine nuclei and nuclei f r o m other  species,  v a r y i n g d e g r e e s of i n t e n s i t y , i n d i c a t e s that there  reacted  with are  antigenic d e t e r m i n a n t s o n b o v i n e histone m o l e c u l e s that f o r e i g n to t h e c h i c k e n .  Antibodies produced against  foreign determinants will react with bovine histones c r o s s - r e a c t with histones from other same or similar determinants.  these and  s p e c i e s that have  the  C o m p a r i s o n s of the  i n t e n s i t y of f l u o r e s c e n c e c a n thus be c o n s i d e r e d as a of the n u m b e r of s h a r e d d e t e r m i n a n t s . presumes  are  measure  This explanation  that the a n t i b o d y i s d i r e c t e d a g a i n s t a s i n g l e  histone molecule which varies from  s p e c i e s to s p e c i e s .  e v i d e n c e f o r t h i s i s b a s e d o n the d e m o n s t r a t i o n that  The  the  a n t i b o d y r e a c t s w i t h a s i n g l e e l e c t r o p h o r e t i c f r a c t i o n o f the  152 basic nuclear protein preparation.  Histones from a  single  c e l l type a r e heterogeneous,  the n u m b e r of f r a c t i o n s  v a r y i n g w i t h the p r o c e d u r e .  T h e r e i s no m e a n s of d e t e r m i n i n g  w h e t h e r o r not the f r a c t i o n s a r e h o m o g e n e o u s .  obtained  If the  antigenic fraction does contain different m o l e c u l e s , r e a c t i v i t y m i g h t b e d u e to t h e n u m b e r o f c o m m o n  single  cross-  molecules  of h i s t o n e a n d n o t the n u m b e r of s h a r e d d e t e r m i n a n t s  on a  single molecule. E i t h e r of these s y s t e m s of i n t e r s p e c i f i c v a r i a t i o n c o u l d a c c o u n t f o r the f a c t that the a n t i b o d y d i d not r e a c t chicken cells.  If t h e c h i c k e n l a c k s a n a n t i g e n i c  o r m o l e c u l a r type,  c o m m o n to a l l t h e o t h e r  determinant  species,  w o u l d p r o d u c e a n a n t i b o d y to t h i s f o r e i g n e n t i t y . possible,  however,  be  protein  to s o m e o f i t s o w n  p r o t e i n s and that autoantibodies  were produced  combined with antigen and were  catabolised,  a n t i b o d i e s to f o r e i g n p r o t e i n s i n t h e  it  It m a y  that i m m u n i s a t i o n w i t h n u c l e a r  c a u s e d the c h i c k e n to l o s e i t s t o l e r a n c e  with  serum.  which  leaving only In  general,  auto-immunity is detected by its association with  disease  s t a t e s b u t the d e t e c t i o n of the a c t u a l a u t o a n t i b o d y  probably  d e p e n d s o n the r e l a t i v e a m o u n t s p r e s e n t i n the  serum (Weigle,  of a n t i g e n and  1965).  A s the  antibody nuclear  proteins probably m a k e up a considerable portion of  the  153 of the p r o t e i n s r e l e a s e d i n t o the b o d y f l u i d s d u r i n g the normal processes  of a u t o l y s i s ,  auto-antibodies produced  s m a l l q u a n t i t i e s w o u l d be a b s o r b e d by e x c e s s a n t i g e n s removed from  the s e r u m .  Such auto-antibodies  neither cause auto-immune any s e r o l o g i c a l tests.  in  and  would  s y m p t o m s n o r be detectable  by  A s the c h i c k e n s s h o w e d no i l l e f f e c t s  f r o m i m m u n i s a t i o n and a u t o - a n t i b o d y w a s not detectable f l u o r e s c e n t t e s t s i t i s i m p o s s i b l e to d e t e r m i n e w h e t h e r not auto-antibodies w e r e  or  produced.  L a r g e amounts of auto-antibodies against components are  by  detectable both s y m p t o m a t i c a l l y and  logically in lupus erythematosis.  K r o o t h et a L ,  o b t a i n e d l u p u s s e r a h a v i n g t i t r e s as h i g h as  nuclear sero-  (19 61)  1:4, 0 0 0 .  These  antisera were unfractionated and probably contained antibodies against several nuclear antigens including  histones.  T h e a n t i s e r a w e r e s i m i l a r to t h e c h i c k e n a n t i b o d y f r a c t i o n s u s e d i n this p r o j e c t i n that they s p e c i f i c a l l y l a b e l l e d and chromosomes with fluorescence, chromo somescomplement.  nuclei  r e a c t i n g w i t h the  T h i s i n d i c a t e s that the  p r e s e n t i n l u p u s h a v e a s i m i l a r d i s t r i b u t i o n to t h e  full  antigens single  a n t i g e n i c c o m p o n e n t of b a s i c n u c l e a r p r o t e i n u s e d i n this study.  154 N e i t h e r the l u p u s s e r a n o r the c h i c k e n was  species specific.  Krooth demonstrated  antisera  that the  lupus  sera reacted with nuclei and chromosomes from  three  m a m m a l i a n species (human,  hamster,  The  chicken  antibody reacted with nuclei,  not only f r o m  different  species,  but even different phyla.  mouse).  These experiments provide a  u n i q u e i m m u n o l o g i c a l v e r i f i c a t i o n o f the r e p o r t s of  others  using electrophoretic and chromatographic techniques, a c i d analyses and D N A - h i s t o n e annealing studies 1965,  p. p.  39-90) that histone f r o m a n i m a l ,  nuclei have a basic similarity. add a further  amino  (Busch,  plant and insect  S i m i l a r i t i e s of antigenicity  d i m e n s i o n to t h e r e p o r t s o f o t h e r s i n t h a t  i m p l y that the p r o t e i n s i n q u e s t i o n h a v e amino acid sequences  and tertiary  they  s i m i l a r i t i e s of  structure  (Fischer,  T h e e x t e n s i v e c r o s s - r e a c t i v i t y of b a s i c  1964).  nuclear  p r o t e i n a n t i g e n has not b e e n r e p o r t e d f o r any o t h e r c l a s s of proteins over such a wide phylogenetic range.  In this  respect  h i s t o n e s c o u l d b e c o m p a r e d to t h e n u c l e i c a c i d s w i t h w h i c h they a r e i n v a r i a b l y a s s o c i a t e d .  The presence  of s u c h a  c o m m o n basic structure over a wide phylogenetic  range  s u g g e s t s that d u r i n g the p r o c e s s o f e v o l u t i o n the i n t e g r i t y of h i s t o n e m o l e c u l e s h a s b e e n r i g i d l y m a i n t a i n e d .  155 The results and observations i n this thesis  are  b a s e d o n t h e p r o d u c t i o n o f a n t i b o d y to o n l y o n e o f s e v e r a l f r a c t i o n s of n u c l e a r p r o t e i n s techniques developed here  E x t e n s i o n of the a n a l y t i c a l  s h o u l d v m a k e i t p o s s i b l e to  c h a r a c t e r i s e o t h e r a n t i g e n i c f r a c t i o n s of the n u c l e u s cytoplasm.  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In "The Nucleohistones",  Bonner and P .  San Vendrely,  23:  Francisco,  Ts'o,  London,  R. , K n o b l o c h - M a z e n ,  p.p.  169-192,  E d s . , H o l d e n - D a y Inc. , Amsterdam.  A . , and Vendrely,  Donnees biochemiques  C. ,  (1960):  recentes sur la relation entre  a c i d d e s o x y r i b o n u c l e i q u e et p r o t e i n e s b a s i q u e s le noyeau. Von Bertalanffy,  Biochem.  E . , (1961):  Pharmacol.  Acridine orange  microscope procedure  fluorescence  f o r the d i a g n o s i s of m a l i g n a n t  cells by exfoliative cytology. Medical College,  dans  4_: 1 9 - 2 7 .  Dept.  of Anatomy,  U n i v e r s i t y of M a n i t o b a .  161  Weigle,  W. O. ,  (19 6 1 ) :  Immunochemical properties  cross-reactions albumins. Weigle,  J.  W . O. . , (1965): rabbits  between  Imm.  Wilson,  R. H, ,  a n t i - B S A and  heterologous  T h e i n d u c t i o n of auto i m m u n i t y i n thyroglobulin.  J.  or  Exp.  Med.  289-308. Jurevics,  Rencz,  V. , Delaney,  K. , and Schram,  antigenic  characteristics  protein f r o m a canine Nature 209: G . , (1964):  of  T. ,  Comparative  nucleoproteins:  of d e o x r i b o n u c l e i c  acid  haemangiopericytoma.  1102-1105.  Nucleohistone  "The Nucleohistones", Ts'o,  R. , Ritter,  A . , (1966):  immunochemical properties  Zubay,  the  599-607.  f o l l o w i n g i n j e c t i o n of h e t e r o l o g o u s  altered homologous 120:  87:  of  structure and function In p. p.  95-106. ,  Bonner  and  E d s . , H o l d e n - D a y Inc. , San F r a n c i s c o ,  California. Zubay,  G. , and Doty,  P.,  (1959):  Isolation and propertie s  of d e o x y r i b o n u c l e o p r o t e i n p a r t i c l e s single nucleic acid molecules. 1:  1-20.  J.  containing Mol.  Biol.  162  APPENDIX  I.  CHROMOSOMAL  PREPARATION  M i c r o c u l t u r e s of b o v i n e and h u m a n b l o o d , mosome  studies,  w e r e p r e p a r e d u s i n g the f o l l o w i n g  reproduced here from  for  chro-  procedure,  The Gibco P r i c e and Reference  Manual,  Grand Island Biological Company, Oakland, California  (1967) p .  77.  Chromosome Medium 1A P r o c e d u r e - M i c r o - c u l t u r e for C h r o m o s o m e Studies:  1.  F r o m free-flowing puncture  (70% e t h y l a l c o h o l ,  then acetone  of c l e a n s e d and d r y  or ether and let dry) aspirate  skin 0. 1 -  0 . 2 m l of b l o o d w i t h s t e r i l e d i s p o s a b l e t u b e r c u l i n s y r i n g e and n e e d l e , p l a c e 2 s m a l l d r o p s of b l o o d i n t o c u l t u r e tube of G I B C O  Chromosome  M e d i u m 1A, and m i x w e l l .  2.  K e e p at 3 7 ° C f o r t h r e e  3.  At 66-70 hours,  days.  a d d 0. 1 c c of V e l b a n ,  E l i Lilly  ( v i n c a l e u c o b l a s t i n e ) m a d e up as a s t o c k s o l u t i o n of 0. 5 u g / c c k e e p at 3 7 ° C f o r 2 h o u r s . No.  521,  If p r e f e r r e d ,  can be substituted.  4.  To harvest  Colcemid Solution,  and Cat.  Use 0.25 m l / 5 m l m e d i u m .  s p i n c u l t u r e s at 8 0 0 R P M f o r 5 m i n u t e s .  163  5.  Discard supernate,  and suspend s l o w l y drop b y d r o p  i n a p p r o x i m a t e l y 5 c c o f 1:5 F e t a l C a l f S e r u m i n d i s t i l l e d  water.  6.  P l a c e s u s p e n s i o n a t 3 7 ° f o r 15 m i n u t e s .  7.  A d d 1 d r o p of f r e s h l y p r e p a r e d f i x a t i v e (3:1  alcohol:glacial acetic acid),  absolute  a n d s p i n at 4 0 0 R P M f o r 2 m i n u t e s .  (Absolute alcohol = methanol or ethanol. )  8. cells.  D i s c a r d s u p e r n a t e and l e a v e one l a r g e d r o p o v e r  M i x t h e d r o p a n d c e l l s a n d t h e n s l o w l y a d d 1. 5 c c  drop by drop with constant m i l d  9.  the  fixative  agitation.  P l a c e t u b e s at 4 ° C i n r e f r i g e r a t o r f o r 20 m i n u t e s .  (Tubes should be stoppered w h e n i n r e f r i g e r a t o r . )  10.  S p i n at 4 0 0 R P M f o r 5 m i n u t e s .  11.  D i s c a r d supernate and save a large drop over  cells:  DO N O TM I X .  12.  Layer  pipette) over cells, Wait 2 minutes  0 . 5 c c o f f i x a t i v e ( 0 . 5 c c = 1/2  b e i n g c a r e f u l not to d i s r u p t c e l l s .  and repeat 1 - 2  times.  DO N O TM I X .  Remove excess fixative,  and then add d r o p b y drop to obtain p r o p e r cell button i s disrupted,  Pasteur  c o n c e n t r a t i o n of c e l l s .  a d d f i x a t i v e t o 1. 5 c c , r e c e n t r i f u g e ,  as above one t i m e or put d i r e c t l y on c o v e r s l i p . )  mix, (If  and w a s h  164  13.  Prepare c l e a n cover s l i p s ,  of s u s p e n s i o n on i t .  and p l a c e t i n y drops  Spread evenly by b l o w i n g .  14.  When d r y , s t a i n as below.  15.  S t a i n i n g Procedure f o r Phas M i c r o s c o p y :  When d r y , i n v e r t i n t o a drop o f 0.5C a c e t i c o r c e i n JJ.0.5 gm o r c e i n i n 45 ml g l a c i a l a c e t i c  acid,  r e f l u x 1/2 hour, add 55 ml  d i s t i l l e d water w h i l e warm, r e f l u x 1/2 h o u r , l e t stand 24 h o u r s , filter  (and f i l t e r  fresh before using)_/  on a c l e a n s l i d e ,  suck out  excess s t a i n xri.th f i l t e r paper and s e a l w i t h K r o e n i g ' s cement. permanent s l i d e s a r e d e s i r e d , t r e a t f o l l o w i n g sequence: acid (dip u n t i l seconds),  0.5% a c e t i c  dry cover s l i p p r e p a r a t i o n i n  o r c e i n (30 m i n u t e s ) ,  f r e e o f excess s t a i n ) ,  xylene  (1 m i n u t e ) ,  ( t h i n n e d w i t h xylene) on c l e a n  45% a c e t i c  tertiary butyl alcohol  1:1 t e r t i a r y b u t y l a l c o h o l , xylene  (1 m i n u t e ) ,  If  (1 m i n u t e ) ,  (15  xyline  and i n v e r t wet i n t o Permount slide.  ( I f b r i g h t f i e l d microscopy i s to be u s e d , use 1% a c e t i c o r c e i n , made as above, but w i t h 1.0 gm o r c e i n . stains,  2% a c t i c o r c e i n i s  F o r permanent  recommended.)  T h i s method was used i n the C y t o g e n e t i c s  Laboratory,  D i v i s i o n o f M e d i c a l G e n e t i c s , Department of P a e d i a t r i c s , Vancouver General H o s p i t a l , Vancouver, B . C . , inhere c o n s i s t e n t r e s u l t s were a c h i e v e d u s i n g Gibco Chromosome Medium 1A #167,  165  C o l c e m i d S o l u t i o n #521,  A c e t o O r c e i n S t a i n 2 . 0% # 5 3 9 .  Unfixed c h r o m o s o m a l preparations were also  prepared  f r o m h u m a n blood by t e r m i n a t i n g the culture p r o c e d u r e and m a k i n g smears  of the c u l t u r e d c e l l s .  After  at s t e p 6  air drying a brief  p a s s a g e t h r o u g h 1. 0% a c e t i c a c i d r e m o v e d r e d c e l l s f r o m  the  slides.  and  T h e y w e r e t h e n b u f f e r e d at p H 7 . 0 f o r 10 m i n u t e s  c o n j u g a t e d w i t h f l u o r e s c e n t a n t i b o d y as d e s c r i b e d i n s e c t i o n V I I , 4. When fixed preparations the  s l i d e s w e r e brought to w a t e r  w e r e tested for  fluorescence  and left i n the buffer  solution for  1 hour before antibody application.  II.  BUCCAL  SMEARS  H u m a n e p i t h e l i a l c e l l s w e r e p r e p a r e d by the  following  method, routinely used for sex chromatin determination: 1. stainless  steel  S c r a p e t h e i n s i d e of the c h e e k w i t h the e d g e of a spatula.  2.  S m e a r the m u c o s a l c e l l s on a c l e a n s l i d e .  3.  Immerse  i n 9 5 % E t h y l a l c o h o l f o r at l e a s t 30  minutes  (may be left for a day or two).  A.  4.  Temporary Slides  R e m o v e f r o m a l c o h o l a n d a l l o w e x c e s s a l c o h o l to d r y .  166  5. on the  slide and cover w i t h a c o v e r  6. two,  P u t a g e n e r o u s d r o p of s t a i n ( a c e t o - o r c e i n )  put the  After  directly  slip.  a l l o w i n g the s t a i n to p e n e t r a t e f o r a m i n u t e  slide between  or  s e v e r a l l a y e r s of p a p e r t o w e l s and p r e s s  hard.  7.  S e a l the c o v e r  B.  slip w i t h m e l t e d paraffin or  . Permanent  vaseline.  Slides  4.  T r a n s f e r to 70% a l c o h o l for 2  5.  T r a n s f e r to tap w a t e r  6.  S t a i n w i t h e a r b o l - f u c h s i n f o r 90  7.  R i n s e off e x c e s s  for 2  minutes.  minutes.  stain i n absolute  seconds.  a l c o h o l (5 o r 6  quick dips).  8.  Differentiate i n absolute  9.  C l e a r i n t w o c h a n g e s of x y l o l ,  10.  a l c o h o l f o r one  2 minutes  minute.  each.  M o u n t i n neutral b a l s a m or D P X mountant.  Stains  1. G.  T.  Gurr,  C a r b o l f u c h s i n (dye s o u r c e : London).  Basic fuchsin from  167  Stock  Solution  a.  3% b a s i c  fuchsin  b.  5% c a r b o l i c a c i d i n  Staining Solution  6 ml of g l a c i a l  of a.  (5% a.  A l l o w to stand f o r one day b e f o r e  -  Store  i n a dark  Aceto-orcein:  and 95% b . ) .  (37%).  -  2.  use.  container.  2% o r c e i n i n 60% a c e t i c a c i d (dye  N a t u r a l o r c e i n from G . T . G u r r , London). Heat 60 ml of g l a c i a l  2 gm o r c e i n .  precipitate  a c e t i c a c i d to b o i l i n g p o i n t .  Warm and shake g e n t l y f o r 2 minutes and allow  D i l u t e to 100 c c .  III.  and b .  months)  acetic acid.  -6 ml of formaldehyde  source:  water.  (keeps f o r 4 - 5  -45 ml of s o l u t i o n  -  i n 70% ETOH.  w i t h d i s t i l l e d water and f i l t e r .  and r e q u i r e frequent  to  The s t a i n  Add cool. will  refiltering.  ACRIDINE ORANGE STAINING  P r e p a r a t i o n of  1.  the A c r i d i n e Orange (AO)  solutions:  A stock s o l u t i o n o f 0.1% AO i s  prepared i n  distilled  168  water.  This keeps indefinitely,  e s p e c i a l l y i f stored i n the  refrig-  erator.  2. w i t h 1/15 AO.  For  s t a i n i n g a p o r t i o n of a b o v e s o l u t i o n i s  M p h o s p h a t e b u f f e r t o o b t a i n a 0. 0 1 % s t a i n i n g s o l u t i o n of  A l s o this  solution keeps for a long  time.  Phosphate buffer:  T h i s i s a c o m b i n a t i o n of 1/15  p h o s p h a t e a n d 1/15  M potassium dihydrophosphate  distilled water,  The solutions  distilled water. at p H 6,  KH2PO4.  m a d e up i n  to p H 6.  9.465 g m N a H P 0 2  4  are  d i s s o l v e d i n 1, 0 0 0 m l  water.  2.  buffer  and m i x e d i n proportions  M disodium acid  a r e p r e p a r e d as f o l l o w s :  1. distilled  diluted  9. 0 7 2 g m K H  2  P 0  T o o b t a i n 1/15 40 m l N a ^ P O ^  4  are  d i s s o l v e d i n 1, 0 0 0 m l  M potassium-sodium  s o l u t i o n are m i x e d w i t h 230 m l  S o l u t i o n s k e e p i n d e f i n i t e l y e v e n at r o o m  C a l c i u m chloride solution:  phosphate  T h i s i s n e c e s s a r y to  temperature.  produce  differentiation between R N A and D N A fluorescence.  It i s  an  a q u e o u s 0 . 10 M c a l c i u m c h l o r i d e s o l u t i o n p r e p a r e d b y d i s s o l v i n g 1 1 . 099  g m of C a C l  keeps  indefinitely.  2  i n 1, 0 0 0 m l d i s t i l l e d w a t e r .  The  solution  169  T h e s t e p s i n the p r o c e s s i n g of s m e a r s  for fluorescence  cyto-  diagnosis:  The smears  a r e r a p i d l y p a s s e d t h r o u g h one change e a c h of  1.  80% alcohol  2.  70% a l c o h o l  3.  50% a l c o h o l  4.  distilled  5.  1% a c e t i c a c i d (to p r e v e n t f a d i n g o f f l u o r e s c e n c e )  6.  distilled  water  water  T h e s e steps r e q u i r e i n a l l about a m i n u t e : u n n e c e s s a r y to count  7.  it is  "dips".  The smears  are  stained i n the A c r i d i n e  Orange  solution for 3 minutes.  8.  They are passed into phosphate  1 m i n u t e to r e m o v e e x c e s s d y e . for  In the buffer,  several hours if p r o c e s s e d i n batches  o r i f r e - e x a m i n a t i o n of s m e a r s  o n the  b u f f e r f o r at smears  and s c r e e n e d  same day is  may  least remain  successively,  desired.  ) 170  9.  The smears  a r e d i f f e r e n t i a t e d f o r 1 to 2 m i n u t e s  i n the c a l c i u m c h l o r i d e s o l u t i o n , u n t i l m o s t t y p e s of n u c l e i e s p e c i a l l y t h o s e of g r a n u l o c y t e s ) a p p e a r green colour.  in a clear,  (and  translucent  The t i m e for differentiation depends p r i m a r i l y on  the p r e p a r a t i o n of A O , and after  a f e w t r i a l s c a n be e a s i l y  standardised.  10. smears  W i t h the a i d of a p o l y e t h y l e n e d i s p e n s i n g bottle  are r i n s e d with phosphate buffer,  w i t h a c o v e r glass and are now, f r o m the f i x a t i v e ,  still wet are  6 to 7 m i n u t e s after  r e a d y for s c r e e n i n g w i t h the  the  mounted  being removed  fluorescence  microscope.  It w a s f o u n d t h a t t h e s t a i n i n g t i m e s i n 0 . 0 1 % A O s o l u t i o n had to be adjusted for e a c h t i s s u e e x a m i n e d .  A constant p r o b l e m  was o v e r s t a i n i n g w h i c h caused the r e d f l u o r e s c e n c e , w i t h R N A , to appear  associated  throughout.  T h e t e c h n i q u e p r o v i d e d a s o u r c e of b r i g h t l y f l u o r e s c i n g m a t e r i a l w i t h w h i c h to adjust the f l u o r e s c e n c e m i c r o s c o p e as  well  as p r o v i d i n g an i n d i r e c t m e t h o d of c o m p a r i n g i m m u n o l o g i c a l  fluo-  r e s c e n c e w i t h the l o c a l i s a t i o n of D N A a n d R N A i n c e l l s c h r o m o s o m e s.  and  

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