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Production, Bioassay, and partial purification of erogens from Tremelia mesenterica Fr Reid, Ian Duncan 1973

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PRODUCTION, BIOASSAY,  AND  PARTIAL  EROGENS FROM TREMELLA  PURIFICATION  MESENTERICA  FR.  "by IAN  DUNCAN REID  B. S c . , U n i v e r s i t y  of G u e l p h , I 9 6 9  A THESIS SUBMITTED IN PARTIAL FULFILMENT THE REQUIREMENTS FOR  OF  THE DEGREE OF  DOCTOR OF PHILOSOPHY in  t h e Department of Botany  We  accept t h i s  required  thesis  as c o n f o r m i n g t o t h e  standard  THE UNIVERSITY  OF BRITISH COLUMBIA  June,  1973  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r  an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e  and  study.  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may by h i s r e p r e s e n t a t i v e s .  be granted by  permission.  Department of Botany  The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada  Date  Jiine 28,  1973  Department or  I t i s understood t h a t copying or p u b l i c a t i o n  of t h i s t h e s i s f o r f i n a n c i a l g a i n written  the Head of my  s h a l l not be  allowed without  my  i ABSTRACT A defined thiamine,  medium c o n t a i n i n g  salts  and  glucose,  microelements  m e s e n t e r i c a F r . 2259-7-  A high  selectively  i n media  asparagine  slows causes  Conjugation mating tubes  type from  hormones aqueous They  slow  hormones  cells  of the  solution  partly  by  Porapak.  ionic  forces.  weight  materials  a r e s e p a r a t e d by  can be  of water  s e p a r a t e d by t h i n  based An  and  low  cell  for  conjugation  the  medium on p r o d u c t i o n  studied, been  density  erogens  The  have been  side  may  be  chains.  and  of c e l l s  and  solvents.  on t h e  adsorbed  on  indicates  forces  that  the  Three  active columns  silica  gel  two  active  components  activity  which  The  can be  foaming.  has  produce  effect  i n 2259 7 -  medium g i v i n g  amino a c i d s  polar  on  nitrogen  of e r o g e n s  hampered by  from,  chromatography.  production.  by  conjugation  t h a n 750-  o f 12 t o 18 h o u r s ,  erogens  each  p a r t l y by n o n - p o l a r  is less  w i t h a complex  from, c u l t u r e s  erogens  polar  tube  of  extracted  are a l s o  Ultrafiltration  i n ethanol,  time  a defined  selected.  purified  The  incubation  and  resins,  b i o a s s a y f o r erogen  tubes.  L~  conjugation  charcoal,  erogens  layer  of  not w i t h l e s s  chromatography  on t h e f r a c t i o n  cells  The  can be  activated  of t h e e r o g e n s  A quantitative oped,  type.  2259 7  exchange  of T r e m e l l a  amino a c i d s .  by h a p l o i d  _  The  growth  of m i c r o e l e m e n t s  i n d u c e t h e growth  on  sulphate,  growth.  other mating  adsorbed  cation  a gradient  containing  secreted  molecular  with  concentration  w i t h n - b u t a n o l , but  resin  a n i o n and  s u p p o r t s good  abnormal  ( e r o g e n s ) from s t r a i n  are s t r o n g l y  both  and  of T_. m e s e n t e r i c a  polystyrene  and  growth  ammonium  been  conjugation  a t 20°C,  pH  of compositon cultures  c o n c e n t r a t e d and  low r e c o v e r i e s or s h o r t  5-5;  source i s optimal  has  h i g h hormone y i e l d s  Attempts  devel-  of been  has  partially  to purify  the  of hormone  peptides with  activity.  non-  i i TABLE OF CONTENTS PAGE ABSTRACT TABLE LIST  i  OF CONTENTS  i i  OF TABLES  vi  LIST OF FIGURES  viii  ACKNOWLEDGMENT  x  INTRODUCTION  1 l6  GENERAL MATERIALS AND METHODS I. II.  Cultures  16  Media  16  CHAPTER ONE. THE NUTRITION AND GROWTH OF TREMELLA MESENTERICA Materials  2259-7  17  and Methods  I. N i t r o g e n II.  Vitamins  III.  A growth  IV. E f f e c t  17  source  17 18  curve  f o r 2259-7  of sodium a c e t a t e  19 on pH d r i f t  Results  19 21  I.  Nitrogen  source  21  II.  Vitamins  27  III.  Growth  curve  27  IV.  Effect  of sodium a c e t a t e  on pH d r i f t  Discussion  29  CHAPTER TWO. PRELIMINARY STUDIES ON THE EROGENS. Materials  and Methods  I. A q u a l i t a t i v e II. III. IV.  Production  Adsorption  VI. Adsorption  VIII. IX.  Adsorption  33 33  bioassay  33  o f hormone  E x t r a c t i o n with  V. A d s o r p t i o n  VII.  27  organic  3^ solvents  3^  on c a t i o n exchange r e s i n  35  on a n i o n  36  exchange r e s i n  on a c t i v a t e d c h a r c o a l  37  on P o r a p a k  39  Chromatography Ultrafiltration  on Sephadex G-10  39 kO  iii PAGE X. XI.  Chromatography Paper  on  columns  of s i l i c a  4l  gel  chromatography  42  o f c y c l i c - 3 *, 5 ' - a d e n o s i n e  XII. E f f e c t  monophosphate  44  Results I. E x t r a c t i o n with  organic  II.  Adsorption  on  III.  Adsorption  on a n i o n  IV. A d s o r p t i o n V. VI. VII. VIII.  Adsorption  44  solvents  44  c a t i o n exchange r e s i n  45  exchange r e s i n  on a c t i v a t e d c h a r c o a l  45  on P o r a p a k  45  Chromatography  on Sephadex G - 1 0  46 46  Ultrafiltration Chromatography  IX. Paper  on  silica  46  g e l columns  chromatography  X. E f f e c t  47  of c y c l i c - 3 5 - a d e n o s i n e 1  monophosphate  CHAPTER THREE. QUANTITATIVE Materials  BIOASSAY OF THE  55  EROGENS  55  and Methods  I. R e l a t i o n  of c o n j u g a t i o n  tube  length  to  erogen 55  concentration  III.  A bioassay Relation  based  on  of f r a c t i o n  conjugation IV. A b i o a s s a y conjugation  55  concentration....  56  of c e l l s  tubes t o erogen based  length..  conjugation  on f r a c t i o n  tube  bearing  of c e l l s  with 57  tubes  60  Results I. R e l a t i o n erogen  of c o n j u g a t i o n  tube  length  to 60  concentration  60  a. D a t a b. S t a t i s t i c a l II. III.  47 50  Discussion  II.  42  A bioassay Relation  IV. B i o a s s a y  based  on  of f r a c t i o n  conjugation  tubes  based  conjugation  60  analysis  65  concentration....  65  of c e l l s  t o erogen  on f r a c t i o n  tubes  length..  conjugation  tube  bearing  of c e l l s  with 65  i v PAGE Discussion  69  CHAPTER FOUR. THE RESPONSE TO THE EROGENS Materials  and Methods  I. Time and II.  course  75  of c o n j u g a t i o n  tube  initiation  growth  75  Distribution per  III.  75  o f number o f c o n j u g a t i o n  tubes  cell  Effect  IV. E f f e c t V. E f f e c t VI. E f f e c t  75  of temperature  16  o f pH  76  of c e l l  concentration  of n i t r o g e n  76  source  77  Results  78  I. Time and II.  of c o n j u g a t i o n  tube  initiation  growth  78  Distribution per  III.  course  o f number o f c o n j u g a t i o n  tubes  cell  Effect  IV. E f f e c t V. E f f e c t VI. E f f e c t  78  of temperature  78  o f pH  78  of c e l l  concentration  of n i t r o g e n  78  source  88  Discussion  90  CHAPTER F I V E . PRODUCTION Materials  OF THE EROGENS  95  and Methods  I. Time  course  95  of erogen p r o d u c t i o n  II.  Effect  of n i t r o g e n  III.  Effect  of medium c o n c e n t r a t i o n  IV. E f f e c t  VI.  on e r o g e n p r o d u c t i o n  96 96  of v a r y i n g t h e c o n c e n t r a t i o n of  individual V. L a r g e  source  95  medium, components  scale productions  of erogens  Foam  97 99 100  Results  103  I. Time  course  of erogen p r o d u c t i o n  II.  Effect  of n i t r o g e n  source  on e r o g e n  III.  Effect  o f medium c o n c e n t r a t i o n  103 productionl03 103  V  PAGE IV. E f f e c t  of v a r y i n g the c o n c e n t r a t i o n  individual V. VI.  Large  of 106  medium components  scale production  112  of t h e e r o g e n s  112  Foam  Discussion CHAPTER SIX.  PARTIAL PURIFICATION OF THE  Materials I. II. III.  114 119  EROGENS  119  and Methods  119  Instability First  attempt  119  at p u r i f i c a t i o n  I n t e r a c t i o n of the  erogenswwith-ioneexbhange 122  resins IV. F u r t h e r V. T h i n  attempts  124  at p u r i f i c a t i o n  126  l a y e r chromatography  129  Results I. II. III.  129  Instability First  attempt  129  at p u r i f i c a t i o n  I n t e r a c t i o n of t h e e r o g e n s  with  ion  exchange  131  res ins IV. F u r t h e r V. T h i n  attempts  132  at p u r i f i c a t i o n  132  l a y e r chromatography  137  Discussion SUMMARY AND  l4l  GENERAL DISCUSSION  144  BIBLIOGRAPHY  15 4  APPENDICES A. F r e q u e n t l y  used  B. Measurements  of c o n j u g a t i o n  concentrations C. A  computer  154  media tube  length  at  156  of e r o g e n  program  to process  various  bioassay  data  158  vi LIST  OF  TABLES  TABLE I.  PAGE Doubling in  II.  times  nitrogen  sources  Doubling  times  various The  Rf  IV.  and  nitrogen  various  VI.  VII.  the  of  concentration number  Analysis  of  paper  mesen-  chromatography  dilutions  cells  49 heterogeneity  factor  hormone b i o a s s a y experiment  with  on  68  the  effect  of  conjugation a t two  doses  tubes/the  total  of  with  erogen  sources  of v a r i a n c e  grown  mesenterica  i n GS. pIu;sXv.ar±ousci.H./  of t h e  counted  nitrogen  of T.  88  cells  tube  turbidities  cultures  27  48  from, t h e  conjugation  Final  macroelements) plus  caffeine  cell  on IX.  of  conjugation  various  on  distribution  of  of  erogen from T r e m e l l a  grown f o r s i x days  counts  The  for cultures  a t 20°C  one-tenth  Cell  number  VIII.  sources  f o r the  concentrations  for  pH's  solvents  Turbidities  Frequency  various 21  final  2259-7 o b t a i n e d  cultures  V.  2259-7  a t 20°C  i n b a s a l medium (low  values  terica in  mesenterica  b a s a l medium ( h i g h m i c r o e l e m e n t s ) p l u s  2259-7 grown  III.  for Tremella  88  for effect  of n i t r o g e n  source  production  and  erogen y i e l d s  in various  89 f o r 2259-7  concentrations  of  Gluc-AmS  medium X.  107  Analysis of  of  variance  erogen y i e l d s  Gluc-AmS and XI.  Final  x/2  turbidities  doubled  from 2259 7 -  and  components  cultures  base  two  grown i n  doubled  108 erogen y i e l d s  concentrations  media w i t h  t o the  media  X I I . A n a l y s i s of v a r i a n c e in  of l o g a r i t h m n s  of  individual  of l o g a r i t h m n s concentrations  i n media  with  components of of  erogen  108  yields  individual 109  vii TABLE  PAGE  XIII. F i n a l  turbidities  factorial  experiment  concentration, concentration XIV.  and e r o g e n y i e l d s on e f f e c t  microelement of r e s t  of v a r i a n c e  sulphate  concentration,  concentration  turbidity  of ammonium  sulphate  c o n c e n t r a t i o n , and  o f medium  Analysis and  from the  for effects  o f ammonium  microelement  of r e s t  and l o g a r i t h m n  XV. P r o g r e s s o f t h e e r o g e n  110 concentration  o f medium on of erogen  purification  final  yield  I l l 131  V I 1 1  LIST OF  FIGURES  FIGURE  PAGE  1.  Structure  2.  The  of some s e x u a l hormones f r o m f u n g i  relation  of t u r b i d i t y 2259-7  of T_. m e s e n t e r i c a 3-  Regression  of t u r b i d i t y  at  560  nm  to  k  concentration 22  cells on t h e  logarithmn  of  cell 23  concentration k.  Growth c u r v e s high at  5.  f o r T_. mes e n t e r i c a 2 2 5 9 - 7  microelements plus  various  nitrogen  with  sources  20°C  2k  R e l a t i o n of t u r b i d i t y  at 6k0  of T_. m e s e n t e r i c a 2 2 5 9 - 7 6.  i n BM  Growth c u r v e s low  nm  to  concentration 25  cells  f o r T_. m e s e n t e r i c a 2259~7  microelements  plus  various  i n BM  nitrogen  with  sources  at  20°G 7.  26  A growth c u r v e at  f o r T_. m e s e n t e r i c a 2 2 5 9 7 _  in  GM3  20°C  28  8.  R e l a t i o n of mean c o n j u g a t i o n  9.  Dependence  of v a r i a n c e  t u b e t o dose of e r o g e n  on mean f o r c o n j u g a t i o n  tube 62  l e n g t h measurements 10.  R e l a t i o n of a v e r a g e t r a n s f o r m e d l e n g t h t o dose  11. 12.  length  on t h e  Variation standard Time  with  i n the  response  on  the 66  slope  and  intercept  of t h e  bioassay 67  curve  course  various  into  conjugation  of e r o g e n dose  of  conjugation  times  after  tube  initiation  and 79  erogen  Frequency d i s t r i b u t i o n s at  cells  6k  erogen  growth i n r e s p o n s e t o 15.  of  conjugation  of e r o g e n dose  L i n e a r r e g r e s s i o n of t r a n s f o r m e d logarithmn  Ik.  63  logarithmn  a. R e l a t i o n of f r a c t i o n b.  tube  of e r o g e n  t u b e s t o dose of  13.  conjugation  L i n e a r r e g r e s s i o n of a v e r a g e t r a n s f o r m e d tube  6l  of  conjugation  inoculation  media c o n t a i n i n g 0 . 2  tube  of 2 2 5 9 - 6  lengths cells  u n i t s of e r o g e n s per  ml..  80  ix FIGURE 16.  PAGE Frequency d i s t r i b u t i o n s at  various  times  after  of c o n j u g a t i o n  17.  Frequency d i s t r i b u t i o n tubes  18.  Regression per  19. 20.  per c e l l ,  cell  on t h e e r o g e n of c e l l s  concentrations  of c o n j u g a t i o n  83  t o t h e erogen at  The a v e r a g e l e n g t h  of c o n j u g a t i o n  r e s p o n s e t o two c o n c e n t r a t i o n s of c e l l s  two c o n c e n t r a t i o n s  Time c o u r s e erogen  produced  o f e r o g e n s as a t o the erogens 86  as a f u n c t i o n o f pH of c e l l s  and a v e r a g e l e n g t h  concentration  with  conjugation  of c o n j u g a t i o n  t u b e s on 87  of c e l l s  of t u r b i d i t y  accumulation  i n c r e a s e , pH d r o p , and  i n 2259-7  c u l t u r e s i n Gluc-AmS  medium 24.  104  Time c o u r s e  of t u r b i d i t y  erogen accumulation  i n c r e a s e , pH d r o p , and  i n 2259-7  c u l t u r e s i n Gluc-AmS 105  medium 25-  Chromatography silica  26.  g e l with  Chromatography silica  84  85  responding  Dependence o f f r a c t i o n tubes,  tubes  of temperature  The f r a c t i o n  82  tubes  concentration responding  8l  conjugation  as a f u n c t i o n o f t e m p e r a t u r e . . . .  at  23.  erogen  of  two c o n c e n t r a t i o n s  function  22.  at three  cells  of erogens per ml...  o f t h e number  o f a v e r a g e number  The f r a c t i o n  in 21.  unit  lengths  of 2259-6  inoculation  i n t o media c o n t a i n i n g 1 . 0  tube  g e l with  of f i r s t  e r o g e n p r e p a r a t i o n on  a gradient of second  o f water  i n ethanol  e r o g e n p r e p a r a t i o n on  a gradient  o f water  i n ethanol  27-  Thin  layer  c h r o m a t o g r a p h y o f hormone I p r e p a r a t i o n .  28.  Thin  layer  chromatography  preparation  130  of second  133 134  erogen 135  X  ACKNOWLEDGMENT I wish  t o t h a n k D r . R. J . B a n d o n i f o r p r o v i d i n g l a b o r a t o r y  facilities, in  this  f o r suggesting  thesis,  and f o r r e v i e w i n g  Dr.  C. 0. P e r s o n ,  for  generously  making  equipment  for  their  acknowledged. helpful  am i n d e b t e d Research  staff  t o me.  after  willing  encouragement,  like  reading the manuscript, financial  I  The N a t i o n a l support.  t o t h a n k my w i f e , S h i r l e y ,  and f o r t e c h n i c a l  The a s s i s t a n c e  c o - o p e r a t i o n , and  t o t h e members o f my c o m m i t t e e .  also  thank  i n preparing the figures i s  C o u n c i l o f Canada p r o v i d e d  I would  I also  and D r . G. H. N. Towers  available  For their  suggestions  the research reported  the manuscript.  D r . E . B. T r e g u n n a ,  of t h e B i o l o g y D a t a C e n t e r gratefully  and e n c o u r a g i n g  f o r her constant  and s e c r e t a r i a l a s s i s t a n c e .  INTRODUCTION  Hormonal c o n t r o l frequently  of s e x u a l r e p r o d u c t i o n  i n fungi  has been  (1952, I967), K'dhler<  r e v i e w e d ; f o r example, by Raper  (I967), M a c h l i s and R a w i t s c h e r - K u n k e l (1967), B a r k s d a l e and  M a c h l i s (1966, I972).  (I972) has d i s c u s s e d t h e t e r m i n o l o g y used t o  Machlis describe  s e x u a l hormones, and has p r o p o s e d  indicate  their  stance  causes  an " e r o g e n "  sexual  chemotropic  an " e r o t a c t i n " of m o t i l e  growth  i n d u c e s or c o n t r o l s  genera  within  lished  activity:  t h r e e new words t o i s a sub-  gametes, an  o f some s e x u a l s t r u c t u r e ,  t h e d i f f e r e n t i a t i o n of  structures.  Three and,  biological  causing chemotactic a t t r a c t i o n  "erotropin" and  (I969),  o f f u n g i have b e e n  the l a s t  five  years, structures  f o r hormones f r o m each  from Allomyces,  antheridiol,  Achlya,  and t h e t r i s p o r i c  fungi.  Also,  iculosus  an e r o t a c t i n  (MUller  have been The  an e r o g e n  acids,  f r o m Mucor  from and r e l a t e d  f r o m t h e brown a l g a E c t o c a r p u s and a  sil-  conjugation-initiating  Blepharisma  have been  (Kubota  studied  inducing the d i f f e r e n t i a t i o n  phores)  and e r o t r o p i n s  e t a n g i a towards  e t a l 1973)  directing  one "another.  i n the Mucorales are  of progametangia  t h e growth  (zygo-  of compatible  progametangia t h e membrane.  of a c o l l o d i o n  were i n d u c e d and a t t r a c t e d He p o s t u l a t e d  that  B u r g e f f ' s experiments  and  (1937)-  Similar  t o one a n o t h e r a c r o s s  into  t h e medium b y e a c h  were r e p e a t e d b y V e r k a i k (1930)  studies  with s i m i l a r  results  on Phycomyces b l a k e s l e e a h u s by R o n s d o r f crystallinus  membrane,  p r o g a m e t a n g i a - i n d u e i n g and  s u b s t a n c e s were s e c r e t e d  strain.  on P i l o b o l u s  (1971)-  (1924) f o u n d t h a t i f c o m p a t i b l e s t r a i n s o f Mucor  mucedo were grown on o p p o s i t e s i d e s  -attracting  gam-  The p h y s i o l o g y o f sex i n t h e  has been r e v i e w e d by van den Ende and Stegwee  Burgeff  performed  and e r o t r o p i n  erogens  erogens  Kehl  the e r o t a c t i n  identified. hormones which  Mucorales  i n detail,-  have been e s t a b -  of t h e s e - - s i r e n i n ,  e t a l 1971),  s u b s t a n c e from, t h e c i l i a t e  investigated  by K r a f c z y k (1931,  were  (1931), and  1935)•  (1954) s u p p o r t e d B u r g e f f ' s h y p o t h e s i s by  Banbury that  t h e medium f r o m a mated  progametangia  i n an unmated  minus s t r a i n . under trates  from  active, types  on b o t h p l u s  cultures  type  progamone  type.  activity of  filtrates  that  were  He f o u n d t h a t  fil-  were n o t m o r p h o g e n e t i c a l l y m y c e l i a o f t h e two could  of the f i r s t - g r o w n  induce  "progamone". mating  Plempel attempted  On t h i s  was  only  The  strain  colony.  just  After  produces  i n the opposite  t h e gamones  ahead  four  the t e s t  hours,  The number  of progametangia  per u n i t  up t o a p p r o x i m a t e l y one m i c r o g r a m solution.  extensively  A l t h o u g h he p u r i f i e d  and o b t a i n e d i t i n c r y s t a l l i n e  a structure.  of B l a k e s l e a t r i s p o r a strongly, factors  Plempel a l s o  R h i z opus n i g r i c a n s  active  reported of t h a t  linearly  related  of gamone p e r  the a c t i v e form,  material  he was  that  found t h a t  mated  and Phycomy.ces ,b'lakesleeanus, and Abs i d i a  spinosa  i n t h e Mucor mucedo b i o a s s a y , and  f r o m M. mucedo gamone.  glauca  of t h e w e l l .  unable  t o s e p a r a t e t h e plus-gamone f r o m t h e minus-gamone  assign  able  a r e a was  a methanol  front  c o u n t s were made of  developed i n the v i c i n i t y  ml of t e s t  sol-  of t h e m y c e l i a l  of progametangia  t o t h e dose  from  progametangium-inducing  b i o a s s a y e d by p l a c i n g  cut i n the agar  or minus  t y p e , each  number  to  basis,  In t h e p r e s e n c e o f a  to isolate  of Mucor mucedo.  in a well  either  mating  progametangia  strain.  induces progametangia  of a s o l u t i o n  a plus  the  cultures  from the opposite  cultures  ution  cultures  and minus s t r a i n s .  sequentially  specific  "gamone", w h i c h  mated  t h e mated  (1963) s u g g e s t e d t h a t each s t r a i n c o n t i n u o u s l y p r o d u c e s  a mating  mating  mucedo would i n d u c e  although not i n the  culture  media i n w h i c h  had been grown  unmated  strain,  produced  strain  but c u l t u r e  Plempel  a  single  plus  of M.  (1957)^ by g r o w i n g  vigorous aeration,  effective  in  Plempel  culture  showing  or  cultures  and,  less  produced indistinguish-  R e c e n t l y , Ueyama (1972), has  extract  o f a mated  induces gametangial i n i t i a l s  culture  in single  strain  o f Abs i d i a cultures  fungus.  Van den Ende lea trispora  (I967) i s o l a t e d f r o m mated c u l t u r e s of B l a k e s -  a m a t e r i a l which  induces progametangia  i n Mucor  3 mucedo, and Trisporic from  suggested  acids  A,  B,  culture fluids  lioti  et  chemical  progametangium acids  and  B and  C  ( F i g . l ) had  Cainelli,  comparison  inducing  C  i t might "be t r i s p o r i c  of B_. t r i s p o r a and  (1967) and  al  Detailed  that  (van  den  Grasselli,  confirmed  the  I968).  acids  B and  C have b e e n  affdltb.er.•.sy_>b'_.et:i'ei "mater.iiasbsev.ere i n both mating  types  Plempel's has  been  of M.  concept  questioned  Stegwee 1971)  and  C are  of  capable  by  by  however, r e p o r t s B and and  strain.  only  Ende et  minus  on  cultures  to  the  of B.  free t r i s p o r i c  I f Plempel's  two  i t , and  progametangium  specific  al  I971)  active  gamones  den  Ende  1970).  minus  B  types  (I969),  Reschke  of t r i s p o r i c  strain.  t r i s p o r a can  induction.  and and  i n both mating  acids  Bu'Lock,  Drake and  hydrolyse  the  a c i d much more t h a n t h e e x i s t , i t seems  to t r i s p o r i c  the  (presumably t r i s p o r i c  gamones were b e i n g t h e y were The  trisporic This  a c i d by  acid  is  hypothesis  plus  probable  the  strain  responsible  explains  why  acid)  converted  f r o m mated  to t r i s p o r i c  cultures—the  acids  as  fast  as  produced. i n t e r a c t i o n s between t h e  synthesis  i a l l y _B.  of t r i s p o r i c  t r i s p o r a have been  production  ligible  et  (I963) i s o l a t e d o n l y m a t e r i a l a c t i v e i n b o t h m a t i n g  Plempel  acid  al  gamones do  to respond  to  Gooday  biologically  T r i s p o r i c acids  methyl esters  able  t o the  t o be  (Edwards  (I968, van  Ende  (1968).  e a c h gamone i s c o n v e r t e d  types  and  C f r o m Mucor mucedo.  mating-type  den  that for  Bu'Lock,  synthesized  progametangia  the  trisporic  (1972) have c o n f i r m e d t h i s o b s e r v a t i o n ,  Winstanley  methyl esters  den  that  C were a c t i v e  shown t h a t  van  Gooday  of Mucor mucedo (van  the  mucedo.  of two  inducing  Cag-  (I967).  Selva  t r i s p o r a with  Austin,  found  by  i d e n t i t y of  (I969) a l s o i s o l a t e d t r i s p o r i c a c i d s B and Trisporic  and  C.  teen i s o l a t e d  characterized  f a c t o r s f r o m B. Ende  earlier  acid  two  acids  studied  mating types  i n M.  in single  cultures  of  mu cedo and  intensively.  p r o c e e d s r e a d i l y i n mated  leading espec-  Trisporic  c u l t u r e s , but  e i t h e r mating type  (van  i s neg-  den  Ende  C O O H  Trisporic Trisporic Trisporic  ac i d A acid B acid C  X X X  H, 0 H,  Antheridiol  Fig.  1.  Structure  of  some s e x u a l hormones f r o m  fungi.  H OH  5 et  a l I970) .  thesis ated  In JB. t r i s p o r a ,  i s f o u n d when m y c e l i u m  i n culture f l u i d  Sutter, verted  precursor  to trisporic  Van  that  each s t r a i n  material  the  mating type  opposite  synthesis  strain  contribute  cultures. increases rate  secretes  small  An i n c r e a s e the rate  found t h a t both plus  i n t h e amount by t h e p l u s  acid  of p l u s  syn-  T h i s low Using  mycelia  and minus  acid synthesis  i n mated  mycelium  o f t r i s p o r at e : - - 3 y n t h e . s i s e by  of s y n t h e s i s  amounts  progametangia i n  o f _B. t r i s p o r a •  carbon t o t r i s p o r i c  con-  (1972) have  o f M. mucedo and t r i s p o r i c  they  studies  mycelium..  of i n d u c i n g  mating type  I97O,  i s being  i s not s e n s i t i v e t o cycloheximide.  grown on C - ^ - g l u c o s e ,  the  tracer  o f B. t r i s p o r a  capable  i n the opposite  strains  Radioactive  from the f i r s t  present  minus - m y c e l i u m ;  1  m y c e l i u m depends  on t h e  intensity  of s y n t h e t i c a c t i v i t y  o f t h e minus  high  of s y n t h e s i s  c u l t u r e s was i n h i b i t e d by  rate  cycloheximide  i n mated  strain.  Ende, Werkman and v a n den B r i e l  and  van den Ende  1972).  Recently  precursor  was p a r t i a l l y  layer  purified  strain  by s o l v e n t  mating type.  to radioactive  One o f t h e p r e c u r s o r s  may be 3 - m e t h y l - l - ( 2 ', 6 ', 6 ' - t r i m e t h y l -  3 - o x o - c y c l o h e x e n - 1 - y l ) - o c t a - 1 , 3 ~ d i e n - 7 - o n e which 1  (1972) have f o u n d as a d e g r a d a t i o n  D r a k e and W i n s t a n l e y  of m e t h y l t r i s p o r a t e C.  rate  ulated acid  and minus p r e c u r s o r s  of p r e c u r s o r  by t r i s p o r i c  synthesis  acid.  product  I n a d d i t i o n Werkman and van den Ende  (1973) d e v e l o p e d s p e c t r o p h o t o m e t r i c the  Bu'Lock,  1  amounts o f p l u s  mating  e x t r a c t i o n and t h i n  and shown t o be c o n v e r t e d  a c i d by t h e other  from the plus  acid biosynthesis.  m a t e r i a l from, s i n g l e c u l t u r e s o f each  chromatography,  trisporic  Werkman  (1973) have c l a r i f i e d t h e r o l e o f p r e c u r s o r  exchange between t h e two s t r a i n s i n t r i s p o r i c type  The  or 5 - f l u o r o u r a c i l ( v a n den Ende e t a l I97O, van  den  Labelled  syn-  i s incub-  (Sutter  a c i d by t h e s e c o n d  of p r e c u r s o r  rate  1971)-  acid  mating type  den Ende, Werkman, and van den B r i e l  confirmed  thesis  of t r i s p o r i c  of e i t h e r mating type  from the other  Capage, and H a r r i s o n  have shown t h a t  a low r a t e  methods f o r m e a s u r i n g t h e present,  and showed  by each m a t i n g t y p e  The s t i m u l a t o r y  i s negated by 5 - f l u o r o u r a c i l .  effect  was  that stim-  of t r i s p o r i c  6 It  appears,  then,  that  the  low  rate  of t r i s p o r i c  synthesfs  in culture f i l t r a t e s  i s caused  the  amounts  constitutively  small  mating  type.  positive The  The  precursor  high  f e e d b a c k by  sensitivity  formation that  of  to  rate  trisporic  of t r i s p o r i c  inhibitors  of  enzyme d e r e p r e s s i o n Besides  carotenoid  i n mated  inducing  synthesis  acid  acid  transformation produced  cultures on  and  precursor  MA  by  i s caused  stimulation  protein  is  by  acid of each  by  production.  of  precursor  synthesis  suggests  involved.  progametangia, i n minus  trisporic  cultures  of  B.  acids  stimulate  trispora  (Thomas  and  I967., Thomas e t a l I967, van den Ende I968, S u t t e r and R a f e l s o n I968). T h i s e f f e c t of t r i s p o r i c a c i d s i s a l s o i n h i b i t e d Goodwin  by  cycloheximide,  act  as  derepressors  carotenoid  retinyl  Whether  clear.  the  acetate,  synthesis  i t has  of  an  synthesis.  demonstrated  C.  and  or  Austin,  trisporic i t s effect Ende  and  of  not  B-carotene  biosynthesis  decrease  (1970) has  trisporic  synthesis ficient  i n mated  and  exogenous  of t r i s p o r i c  Feofilova  acid  acid  is stimulating  acids  synthesis  (J-carotene to t r i s p o r i c  acid  natural  and  found  that  synthesis of _B.  aeration  its  substrate  and  increase  t r i s p o r a.  depress  and  i s not  yet  the  acids for  does  the  precursors.  B-ionone  and  B  precursor  (1971) p o i n t o u t ,  Stegwee  for  from (J-carotene,  into t r i s p o r i c  carotenoid  i s the  cultures  excessive  on  acids  (1970) have  Drake  acid  den  that  Bu'Lock and  mevalonic  trisporic  is rate-limiting  and  transformation prove  enzyme w h i c h  that  of r a d i o a c t i v i t y  not  van  suggested  incorporation  through  As  been  the  acetic acid  carotenoid  Also yield  both of  insuf-  trisporic  acid .  ism)  The  mutual  was  also  repulsion  of  a t t r a c t i o n of  studied like  by  compatible  Banbury  progametangia  zygophores  (1954, 1955)and  a t t r a c t i o n of  p r o g a m e t a n g i a between m y c e l i a  w h i c h were not  and  concluded  stimulus  and  his  Plempel  that  associates  the  tropic  (Plempel  He  was  (zygotropdemonstrated  compatible  in liquid  airborne.  contact, Plempel  i960, P l e m p e l and Dawid 1961,*  I962) were u n a b l e t o d e m o n s t r a t e d i f f u s i o n of  zygotropic  7 substances  through  of  the  mating types  by  a m i c a membrane w i t h  the to  two  zygophores  agar  bearing and  through  air-borne  separated  in i t s aerial section,  i n c r e a s e d the  Once a g a i n ,  When c u l t u r e s  z y g o p h o r e s were  small holes  grew t o w a r d  zygotropic factors  phores .  d e s p i t e many a t t e m p t s .  the  holes.  Exposure  growth r a t e of t h e  zygotropic factors  zygo-  were  impli-  cated . In A l l o m y c e s , but  the  female  the  male gametes a r e  gametes a r e  vigorously motile,  s l u g g i s h and  remain  close to  the  female  garnet a n g i a - a f t e r - t h e i - r ^ d i s c h a r g e ... jv:M'&ehlis [l-95.8'a>  c) has  shown t h a t t h e  gametangia because of  an  and  erotactin,  A.  produce  for  of t h e  sirenin.  obtained  a male  strain  Sirenin  placing  the  i n a cup  bottom,  immersed  test  gametes w h i c h  measured lO "^  as  the  molar  -  solution  i n a suspension settled  gradient,  onment. sample  The cups,  depended the  and  the  response  level  By al  described this  with area  to  a given i n the and  by  number  as  the  was  low  sperm.  as The  concentra-  from t h e i r  sirenin  sample b i o a s s a y  gametes  membrane  contained  Machlis  used  membrane  of t h e  other  was  The  concentrations  apparatus  (Carlile  a single  of t h e  swimming up  and  bioassayed  a dialysis  remove s i r e n i n  of hormone  gametes were e x p o s e d  eliminate  by  irreversibly  male  of male gametes.  Sirenin  sirenin  arbuscula  strain  was  female  gametangia  t o p r o d u c e male  activity  a unit  o r i g i n a l bioassay  on t h e  (I969) has  et  and  to  female  A female  noticeable attraction  male gametes r e s p o n d tion  on  response.  caused  the  to the  predominantly  strains.  i n the b i o a s s a y .  of  use  and  attracted  From c r o s s e s between A.  female h y b r i d  sirenin  are  s e c r e t i o n by  macrogynus , M a c h l i s  predominantly to  male gametes  b,  several  concentration  cups t o  which  1965).  Machlis  apparatus  to  interaction.  processing  200  litres  of  culture f l u i d  a week,  Machlis  (I966) s u c c e e d e d i n i s o l a t i n g 2.5 grams of s i r e n i n .  structure (1968).  ( F i g . l ) was Racemic  Katzenellenbogen  envir-  deduced by  sirenin  was  N u t t i n g , Rapoport  s y n t h e s i z e d by  (I969), P l a t t n e r , B h a l e r a o ,  and  Machlis  Corey, Achiwa and  The  Rapoport  and  (I969,  8 Bhalerao,  Plattner,  and  Rapoport  I970), G r i e c o (I969), M o r i  and M a t s u i (I969), and C o r e y and A c h i w a material of  was  found  magnitude  achieved  as n a t u r a l  the  (l-sirenin  sirenin.  s y n t h e s i s of t h e  i s the n a t u r a l  configuration sirenin  t o have e r o t a c t i c  i s not  activity  Plattner  pure  and  active,  synthetic  and  same  d- and  determined  and  does not  order  (1971)  Rapoport  1-sirenin  the  absolute  (1972) r e p o r t s t h a t  Machlis  biologically  The  on t h e  enantiomers  product)  of 1 - s i r e n i n .  (1970).  d-  interfere  with 1 - s i r e n i n . J.  R.  control ized  Raper has  i n the  demonstrated He  s e x u a l hormones f r o n reaction for A,  (Raper  mycelia ulates The  1939)  induces with  the  that  d  then  antheridial  antheridial  two  by and  from  antheridia  the the  tubes  could  present  (Raper  then  hormone B,  any  by  a third  After  induces  Although  stim-  mycelia. and  considered  Fertilization  the  and  growth  evidence  the tubes  grow t o  of t h e  controlled, on t h i s  C,  develop,  b a s a l s e p t a t i o n of oogonia,  cause  hormone,  antheridia  also hormonally  experimental  hormone  Male  hyphae, Raper  sexual  evidence  which  on f e m a l e  antheridia.  p e n e t r a t e the  i s probably  of t h e  i n male m y c e l i a .  of o o s p h e r e s .  oospheres.  (summar-  diffusible  course  antheridial  hormonal  fertRaper  point  1940).  In  a more d e t a i l e d  developed, response  Smit  the  into  which  formation  ilization not  attract  hormone D,  fertilize  of  m y c e l i a produce  hyphae p r o d u c e  oogonial i n i t i a l s .  oogonia, and  hyphae  of  experimental  female  a c t i o n s were p e r f o r m e d  the  produce the  provided  The  to d i f f e r e n t i a t e  these  they  of  a n  f o r m a t i o n of o o g o n i a l i n i t i a l s  tips  produced  action  h i s o b s e r v a t i o n s of t h e  oogonial i n i t i a l s  their  in  p o s t u l a t e d the  f o u r s e p a r a t e hormones. which  e l a b o r a t e system  oogamous s e x u a l r e p r o d u c t i o n of A c h l y a  I952).  by R a p e r  an  based  on t h e number  t o t h e hormone  (1942) a t t e m p t e d  culture  although  study  filtrate,  still  to they  impure,  of hormone A,  of a n t h e r i d i a l  (Raper  isolate  1942a). mg  hyphae  Raper  hormone A.  o b t a i n e d 0.2  induced  a bioassay and  was  formed Haagen-  From lkk-0  litres  of m a t e r i a l w h i c h ,  a n t h e r i d i a l hyphae  at a  dilution  9 of  10"13.  The male p l a n t s  d e s i g n a t e d A', w h i c h 1942b).  He a l s o  found t h a t  female  plants  i n acetone, which  2  branching,  or i n c r e a s e s  soluble).  Male p l a n t s  system  secrete  Homothallic species similar  a factor,  secreted induces  (Raper  a second  antheridial  t h e r e s p o n s e t o hormone A ( a c e t o n e hormone A , a c e t o n e  d e c r e a s e s t h e r e s p o n s e t o A or A  1950a).  t o produce  augmented t h e r e s p o n s e t o hormone A  hormone, A , i n s o l u b l e  which  were f o u n d  in principle  of A c h l y a d i s p l a y e d to that  soluble,  or A p l u s A' (Raper  2  a  hormonal  of h e t e r o t h a l l i c  species  (Raper 1950b). Barksdale responded  (1963a) f o u n d t h a t  strains  of A c h l y a  t o hormone A by p r o d u c i n g a n t h e r i d i a l hyphae  hormone f r o m t h e medium, w h i l e u n r e s p o n s i v e s t r a i n s The  hormone c o u l d  with or  acetone.  n o t be r e c o v e r e d by e x t r a c t i o n  B a r k s d a l e (1963b) a l s o  polystyrene plastic  hormone A had been and  cause  particles  adsorbed,  antheridia  initials.  would  t o form.,  Thus t h e a c t i o n s  stimulatory  effect  McMorris of  et  the nutrient  Later  pure  and d e t e r m i n e d  e t a l I972).  a synthetic pure  ( B a r k s d a l e 1970)-  a structure  McMorris  (McMorris  the absolute  moderate y i e l d s  from  and 22,23-  2 3 - d e o x y a n t h e r i d i o l from a s t r a i n not appear  Green  have  developed  of s t e r e o c h e m i c a l l y  and S e s h a d r i 1971>  isolated  does  hormone A,  configuration  and c o l l a b o r a t o r s  and S e s h a d r i I972).  molecule  plants  ( F i g . l ) . Edwards  of a n t h e r i d i o l  achalam, This  from female  they accomplished the synthesis of  method g i v i n g  antheridiol  The  o f A can be  of c r y s t a l l i n e  a mixture  isoantheridiol. antheridiol  t o hormone  A r s e n a u l t e t a l (I968)  evidence, suggested  a l (I969) s y n t h e s i z e d  (Edwards  level  10 m i l l i g r a m s  t h e y named a n t h e r i d i o l .  spectroscopic  to oogonial  s o u r c e s o f hormone A.  and B a r k s d a l e (1967) i s o l a t e d  Achlya bisexualis  which  by R a p e r  o f hormone A' i n t h e p r e s e n c e  by a d j u s t i n g  purified  a n t h e r i d i a l ' .hyphae .  i n a manner s i m i l a r  attributed  d i d not.  polyvinyl  partially  attract  removed  of t h e mycelium  showed t h a t  on which  C c a n be a c c o m p l i s h e d by l o c a l i z e d mimicked  which  McMorris,  e t a l (1971) have  Arun-  also  o f A. b i s e x u a l i s .  t o have any e r o g e n i c  activity.  10 (I969) has  In p r e l i m i n a r y s t u d i e s , B a r k s d a l e hormone B i s s e c r e t e d by phase upon extracted  s t i m u l a t i o n by  or  A?>,  chromatographic  have y e t  appeared  significant  Achlya  growth  - T h i s , hormone _ can. be  methylene  chemistry  is correlated M u l l i n s I967,  uptake r a t e  (Warren and  cellulase phases  i s high  of t h e with  chloride,  and  of hormones  medium f r o m l a t e  an  M u l l i n s I969).  low  from the  in early  wall  induced  a rise  I969) and  in mycelia  of g r o w t h , but  cell  Antheridiol  (Thomas and  Wo  A^,  in early  Branching  i n response  supply  of  carbon,  The late  favour  vegetative branching  over  activity  inducibility l o g and  of  plateau  Incubation  inducibility Sells  r e q u i r e s an  energy.  of  i n oxygen  log mycelia.  antheridiol  n i t r o g e n and  branching  increase  l o g c u l t u r e s (Warren and to  polysaccharides  in endocellulase  log cultures increases  cellulase  of  197l)exogenous  High n i t r o g e n  levels  a n t h e r i d i a l branching  (Barks-  1970).  dale  Besides above,  the  relatively  w e l l s t u d i e d systems  s e x u a l hormones have b e e n p o s t u l a t e d or  but  not  has  been f o u n d  in  of t h e  properties to a n t h e r i d i o l .  on t h e  fraction  is cellulose.  male m y c e l i a  in  with  end  that  D.  A in  antheridiol.  from c u l t u r e f l u i d s  shows s i m i l a r reports  male p l a n t s a t t h e  found  fully  Achlya  c h a r a c t e r i z e d i n s e v e r a l other f o r a h o r m o n a l system, s i m i l a r  i n two  other  1940)  and  Dictyuchus  these  s y s t e m s have not  been  (Sherwood  studied  demonstrated,  fungi.  Evidence  to that  operating  water m o l d s — S a p r o m y c e s  monosporus  re ins c h i i  I966),  i n the  described  (Bishop  although  same d e t a i l  as  Achlya. Considerable of  a t t e n t i o n has  hormonal c o n t r o l  Saccharomyces which  of  in  cells  an  active  diffusion  of t h e  L e v i (1956)  occurred  minus m a t i n g  culture f i l t r a t e , of t h e  possibility  c o n j u g a t i o n between h a p l o i d s t r a i n s  cerevis iae.  c o n j u g a t i o n had  been p a i d t o the  inducer  could  type. or t o  across  claimed  induce He  was  copulatory unable  consistently  a membrane.  t h a t agar to  of  on processes  obtain  demonstrate  11 Other cator  investigators  of s e x u a l r e s p o n s e .  reported  the p a r t i a l  oligopeptide, and  have used  substance which A c t i n o m y c i n D, namic a c i d  purification  of budding  (1969)  Yanagishima  Duntze,  f r o m t h e <s*--mating  inhibition  isolated  inhibited  cultures  .  1971)  -  which  and  protein  The  increase  suggested is  that  shima  principle  activity  of c e l l  Ascobolus  The  elements,  a hormone w h i c h  but  preceded effect  oidia  in  methylene  on t h e  c o m p a t i b l e mycelium,  of  I972).  increased. I t has  of been  s e x u a l hormones  fungus,  stimulate  I963) has  stable  uncovered discomycete  m y c e l i a of each Each  strain  produces mating  of a s c o g o n i a  chemotropically attract  ( t h e apex of t h e a s c o g o n i u m ) .  mating  t h e male  from the other  the development  certain  1972).  a s c o g o n i a , and  oidia  of  with a heat  plasmogamy i n t h e  organs,  and  sugar  the appearance  of t h e  and Raper  activates  Activated  Shimoda  (Yanagishima  and Y a n a g i s h i m a  are s e l f - s t e r i l e .  sexually  and  testosterone,  the r e l e a s e  incubation  In t h i s  type.  trichogyne  by  (Sakai  both the female  oidia,  and  sexual a g g l u t i n a b i l i t y  controlling  stercorarius.  type produce  density  w a l l d e g r a d i n g enzymes ( Y a n a g i -  1957> B i s t i s  o f hormones  estradiol Stationary  s e p a r a t e d by  and Y a n a g i s h i m a  i s increased  (I956,  and  i n t o t h e medium r a p i d l y  f r o m at c u l t u r e s  Bistis  trans-cin-  et a l 1969).  a, c e l l s  type.  o f t h e hormone  insoluble  expanded  cell-expanding  activation  _a-type s t r a i n s  a series  cells  Shimoda 1 9 7 3 ) -  and  action  (Yanagishima  a_ and < = > * - strains,  (Shimoda  the  cells  of a s u b s t a n c e ,  in autolytic cells  c a u s e d by  other mating  w i t h _a hormone, or  specifically  mating  can be  steroidal  into hormone-sensitive ( p e l l e t )  of o<. c e l l s  from the  conjugating  type.  type a  i n the  The  (Yanagishima  (surface)  secretion  After  each m a t i n g  an  elongation  of t h e a - m a t i n g  expansion  o f S_. c e r e v i s i a e  Treatment  chloride,  from  caused  indi-  (1970)  possibly  s i m u l a t e d by t e s t o s t e r o n e ,  centrifugation  stimulated  type which  the response.  was  hormone-insensitive 1970)  of a f a c t o r ,  c h l o r a m p h e n i c o l , c y c l o h e x i m i d e , and  showed a-hormone a c t i v i t y gradient  e x p a n s i o n as an  MacKay and Manney  in cells  induced c e l l  f r o m t h e <*_-str a i n phase  cell  the  In a d d i t i o n t o t h e  12 oidia,  pieces  of t h e  ivated  to  as  ed  act  t o and  will  mating type. to  Raper to  fuse  attract (l967)  account  the  bas  :  fertile  are  the  (Markert  one  diffusible  between t h e the  always  suggest  hyphae  branching  Wheeler  itself. hormones  and  of t h e  on  hypha and  the  i s not  the  secreted  i n adjacent  crosses  by  vigor-  obtained that  homothallic  in  the  hyphae  a  hetero-  (1955) showed t h a t  strains  stimulated  initiation nature  the  strains. is  of t h e  A l l of  controlled interaction  clear.  gametes  f o r hormonal a c t i o n  of S y n c h y t r i u m attraction  of  endobioticum trichogynes  lunata. and  basidiomycetes,  f u s i o n s between v e g e t a t i v e  studying  than  i s mated  strains,  i s crossed  m y c e l i a l ascomycetes  by  is  more w e a k l y  which  self-sterile  f o r chemotropic  i n Bombardia  mated  partners  reaction is  Wheeler  exact  self-  (heterothallic).  strains  heterothallic  arise  are  (1951) o b s e r v e d  that p e r i t h e c i a l  c o p u l a t i o n of t h e  after  seems  between two  interact  a strong  i n the  hormones, b u t  plasmogamy o c c u r s that  partners,  from h o m o t h a l l i c  In many of t h e (1933)^  one  (1966) has r e v i e w e d e v i d e n c e  spermatia  either  chemotrop-  around  self-sterile  interface  strains  Later, Driver  ( C h y t r i d i a l e s ) and to  are  the  p o i n t where t h e  strains  Machlis in  along  of p e r i t h e c i a  results  and  some s t r a i n s  of w e a k l y - i n t e r a c t i n g  initials  filtrates  by  others  McGahen and  culture  development  Glomerella,  i n which at l e a s t  of t h e  hypha.  ascogonium  of s e v e n  of d i a l y s a t e f r o m a s t r a i n  thallic  these  and  between h o m o t h a l l i c  or n e a r t h e  attract-  a n t h e r i d i a of  in i t s vicinity,  a minimum, s e t  ascomycete,  l^ky)'.  perithecial  or  are  observations.  When a p a i r  ously with crosses  postulated  sexually act-  trichogynes  plasmogamy, t h e  Some p a i r s of  presence  The  he  branches t o form a sheath  in crosses  others.  after  produced  self-fertile.  can  activated oidia  branching  (homothallic)  mycelia  at  with  f o r these  Perithecia  mycelium  (antheridia).  Finally,  In a n o t h e r  in  males  stimulate hyphal  ically  vegetative  hyphal  fusions  substances hyphae and  which  hyphae.  i n many f u n g i , could  induce  chemotropically  Buller  concluded  lateral  guide  the  13 branches  t o meet t i p - t o - t i p .  participating If  each  an  infinite  to  account  any  hyphae  s e c r e t e and  hypha s e c r e t e d one number  factor  apparent  sometimes  (1952) s u g g e s t e d  react  of s u b s t a n c e s  f o r the  o t h e r hypha,  Raper  and  to a single responded  to  called  the branch  capacity  species.  attracting  shown t h a t  chemotropic  compatible  encounters ent  alleles  the  same A.  other,  result  and  In  cases  compatible  factors  at the A  c o u l d be  incompatibility  have  are a c t i v a t e d  i n some  matings,  between t h e  even between  strains,  can be  to include  frequency. and  The  enhances t h e  of t h e  substance ability  (1972) d e f i n i t i o n  Machlis's  substances  of t h e i r  as w e l l  strains  the  c o n s i d e r e d a s e x u a l hormone.  t o extend  sexual activation  Schizophyllum)  when t h e y  cellophane, they  seems d e s i r a b l e  logical  differ-  above  subsequent  "erogen"  hyphal  hyphae have  l o c u s than  only  a r e grown one  of t h e hyphae t o f u s e  of t h e t e r m  occurs  strains  t y p e , have a h i g h f u s i o n  which d i f f u s e s  (I970) have  of h y p h a l t i p s  when t h e two  When c o m p a t i b l e  same m a t i n g  Miles  a g r e a t e r p r o p o r t i o n of  in fusion  s e p a r a t e d by  It  attraction  matings,  manner so t h a t  and  with  s e x u a l hormones.  In S c h i z o p h y l l u m commune, Ahmad and in  another,  o f hyphae t o f u s e  of a d i f f e r e n t  i n d u c i n g and  substance.  would need t o be p o s t u l a t e d  where t h e h y p h a l f u s i o n b r i n g s t o g e t h e r s e x u a l l y nuclei,  that a l l  target  which cells  as t h o s e w h i c h  cause  physio-  (as i n As c o b o l u s  control  s e x u a l morpho-  genesis . In h e t e r o b a s i d i o m y c e t e s copulation ation, Bauch  growth,  and  bromivora  suggested  that  conjugation  tubes  attempts  was  with  compatible  fusion  cells  haploid  o c c u r s by  phases,  the  initi-  of f i l a m e n t o u s c o n j u g a t i o n t u b e s .  (I925) n o t e d t h a t c o n j u g a t i o n t u b e s were o n l y formed i n  Ustilago He  of s e x u a l l y  with y e a s t - l i k e  he  cell-free  when c o m p a t i b l e each  strain  i n the  unable culture  strains  produces  were grown t o g e t h e r .  a hormone w h i c h  o p p o s i t e mating  type.  Despite  t o o b t a i n c o n j u g a t i o n tube fluids.  induces several  induction  14  (I963) a n a l y z e d t h e m a t i n g s y s t e m o f T r e m e l l a  Bandoni mesenterica, formation with  and f o u n d  of c o n j u g a t i o n tubes  two a l l e l e s ,  A and _a.  c o n j u g a t i o n tubes  on For  convenience  in this  and  mating type  will  brane,  dialysis is  evidence  duced  i n the absence  for  encephala  by F l e g e l  Similar conjugation  tubes  of the other days.  type  i n Tremella  tubes  or mated  or p r o d u c e d  prints  temperature hormones  subanomala and T_.  cultures ( I . hormones  in  species  of Rhodospor idium, that  in this  o n l y when b o t h  The m o r p h o l o g i c a l  Leucosporidium,  Reid,  scavenged  Tilletia,  activity fluid  unpublished  data).  species are e i t h e r  mating types are  f r o m t h e medium by t h e  similarity  of c o n j u g a t i o n  and i n t h e g e n e r a  Sporob-  and U s t i l a g o t o c o n j u g a t i o n  c o n j u g a t i o n hormones  (R. J . B a n d o n i ,  culture  that  strains  no hormone  or c e l l - f r e e  strain.  f u n g i as w e l l  pro-  and were n o t  conjugation  However,  reacting  suggests  This  i n the  when t h e two c o m p a t i b l e  efficiently  these  I965).  of d i f f u s i b l e  a t room  Diffusible  and t h e n  in Tremella  (Bandoni  mating type,  present,  olomyces,  mem-  a r e i n o c u l a t e d on a  conjugation  by c e l l o p h a n e .  the conjugation  very unstable,  other  of d i f f e r e n t  (I968) and i n T_. b ambus i n a b y B r o u g h (I970).  in dialysis  single  Apparently  ignored,  showed t h a t t h e hormones were  are produced  grown s e p a r a t e d either  be  s t u d i e s i n R h o d o s p o r i d i u m t o r u l o i d e s showed  c o u l d be f o u n d of  alleles.  s i d e s of a c e l l o p h a n e  mating type  induce  Bandoni  up t o f i v e  depend  multiple  when s t r a i n s  b y a u t o c l a v i n g , or b y s t o r a g e  periods  Estab-  connections  l o c u s , B, w i t h  o f one m a t i n g  have a l s o b e e n d e m o n s t r a t e d  are  clamp  are induced  specifically  mating type.  o f A. w i t h A,  t o A or a.  from the other  other  locus  o f A and _a c e l l s ,  f o r p r o d u c t i o n by e a c h m a t i n g t y p e  erogens which  destroyed  with  a r e grown on o p p o s i t e  print  by a g e n e t i c  d i s c u s s i o n the B locus w i l l  refer  tubes  o r when c e l l s  The  o n l y v e g e t a t i v e growth ensues.  h e t e r o z y g o s i t y at a second  mating types  pattern.  i s controlled  In m i x t u r e s  of a s t a b l e d i k a r y o n  Conjugation  tetrapolar  are produced, but i n mixtures  or _a w i t h _a s t r a i n s , lishment  a modified  are a c t i n g i n  p e r s . comm..).  15 (I968) showed i n T_. m e s e n t e r i c a t h a t hormone must  Flegel be  continuously present  if  hormone  gested  i s removed, t h e c e l l s  that the conjugation  or r e p r e s s o r s  conjugation  T. m e s e n t e r i c a i f necessary  tips  are chemotropically  that  t o accomplish  15 m i c r o n s .  this.  attracted  T_. b a m b u s i n a  i s r e p o r t e d b y Brough  has  questioned  whether t h e c h e m o t r o p i c  sug-  metabolism.  conjugation  tubes  He s u g g e s t e d  direc-  that the  over  a range  S i m i l a r chemotropism of c o n j u g a t i o n  (I970).  attraction  e r o t r o p i c hormone, or t o a h i g h conjugation  hormone,  Raper  tubes  (1967)  was due t o  c o n c e n t r a t i o n of  i n analogy  with  hormone A  Achlya. . J.  ation very  A. B e r r y strain  2259-6.  f a r , they Flegel,  inary  felt  Although  s t u d i e s on t h e i s o l -  they  data).  R. J . B a n d o n i u n d e r t o o k  The  inconclusive results  o b j e c t i v e of the present  chemically  Fr.  This  nature  the erogenic  activity  prelim-  p e r s . comm..).  2259 7 o f T r e m e l l a -  b u t some  o f t h e hormone has been  and methods f o r p r o d u c i n g ,  (Berry  was t o i s o l a t e and  g o a l has n o t been r e a c h e d ,  about t h e c h e m i c a l  purifying  (Bandoni,  c h a r a c t e r i z e the erogen from s t r a i n  mesenterica ulated,  study  study  2259~7 o f t h e o t h e r  s t u d i e s on t h e hormone f r o m s t r a i n with  mesen-  d i d not pursue t h i s  t h a t t h e hormone might be a p e p t i d e  unpublished  mating type,  ation  and T. W. F l e g e l began  and c h a r a c t e r i z a t i o n o f t h e e r o g e n f r o m T r e m e l l a  terica and  wall  t o each other  in  in  cell  compatible  about  erogenic  He  hormones were a c t i n g as i n d u c e r s  of  the  growth;  u s u a l l y met t i p - t o - t i p — c h a n g i n g g r o w t h  tion  a separate  tube  r e v e r t t o budding.  o f enzymes i n v o l v e d w i t h  (I965) o b s e r v e d  Bandoni of  to maintain  assaying,  have been  accum-  and p a r t i a l l y  developed.  inform-  GENERAL MATERIALS AND  METHODS  16 I.  Cultures The  organisms  patible, and  haploid  identified  produce mating  by Dr. R.  s t u d y were two  type a B j j , on  J . Bandoni.  type A B  C u l t u r e RJB  t o a s s a y hormone  i n c l u d e d by  jj=  isolated  2259-6 (UBC  used jj=  to  559-6),  activity.  Bandoni  (I963) i n h i s o r i g i n a l also  i n h i s demon-  ( B a n d o n i I965).  o f c o n j u g a t i o n hormones  com-  # 2259~7  I963) was  (Bandoni  I ; [ I  c o n j u g a t i o n i n T_. m e s e n t e r i c a , and  stration  sexually  of T r e m e l l a m e s e n t e r i c a F r .  c o n j u g a t i o n hormones, and RJB  2259-6 was study  in this  strains  # 559-7), m a t i n g  (UBC  Both  2259 6 _  2259-7 were s t u d i e d by F l e g e l (I968) i n h i s i n v e s t i g a t i o n  and of  used  conjugation in Tremella• Stock cultures  (MYP)  slants  were m a i n t a i n e d on M a l t Y e a s t  a t 4°C,  and  were t r a n s f e r r e d  at l e a s t  Peptone once  every  s i x months. All  cultures  were grown a t 20°C, u n l e s s o t h e r w i s e  spec-  ified . II.  Media Chemicals  used  i n media were o b t a i n e d f r o m  C h e m i c a l Works, F i s h e r cal  Co.,  grade.  and  J . T.  Baker  were p u r c h a s e d  Bacto Soytone,  Extract Difco,  Company, The  C h e m i c a l Co.,  Vitamin-free salt-free  hydrochloride Corp.  Scientific  and  Mallinckrodt Nichols  Chemi-  were of r e a g e n t  casein hydrolysate,  and  thiamine  from N u t r i t i o n a l Biochemicals  Bacto Malt E x t r a c t ,  came f r o m D i f co L a b o r a t o r i e s . or K & S L a b o r a t o r i e s ,  and  Agar  Vancouver,  Bacto Yeast  was  o b t a i n e d from.  or N u t r i t i o n a l  Bio-  chemicals C o r p o r a t i o n . Water was lene  distilled  once  and  stored  in a polyethy-  carboy. The  listed the  glass  c o m p o s i t i o n of c e r t a i n  i n Appendix  account  A.  f r e q u e n t l y used  media a r e  R e c i p e s f o r o t h e r media a r e g i v e n w i t h  of the a s s o c i a t e d  experiment.  i '  CHAPTER  ONE  THE NUTRITION AND  GROWTH OF  TREMELLA MESENTERICA  2259-7  17 Materials  and  I. N i t r o g e n  Methods  source  A "basal g l u c o s e - s a l t s - t h i a m i n e medium (BM) according  t o the  6.0  gm  KHgPOl,.  1.0  gm  MgS0^'7H 0  0.5  gm  CaCl -2H 0  0.1  gm  NaCl  0.1  gm  1.3  ml  2  2  2  Microelement  stock  solution  Thiamine  100 \ig  Distilled with  0.2  assium  gm  100  salt-free  1  lots  of t h e  gm  L-asparagine,  meyer f l a s k s .  The  of aluminum f o i l ,  i n 50  flasks and  Soytone.  of a s p a r a g i n e  m e d i a were d i s p e n s e d  supplemented  following nitrogen sources:  c a s e i n h y d r o l y s a t e , Bacto 0.2  litre  of b a s a l medium, were  ammonium n i t r a t e ,  supplemented with The  water  ml  of one  nitrate,  prepared  recipe:  Glucose  Separate  was  ml  portions into  were capped w i t h  autoclaved  f o r 15  vitamin-free,  One  p l u s 0.2  pot1  l o t was  gm  of  250  soytone.  ml  a double  Erlen-  thickness  minutes  at 15  psi  ml  suspension  pressure. Each f l a s k of 2259-7 c e l l s culture To  determine  present,  4/25  and  their The  3.5  cm,  been  1/5,  turbidity  relation  2/5,  3/5,  of t h e  were c o u n t e d  inoculated flasks  o p e r a t i n g at 100  and  of a  a two  day  washed w i t h two  old  100  ml  water. between t u r b i d i t y 4/5  and  inoculum  measured.  2.0  c e n t r i f u g e d from  3 (GM3)  distilled  the  dilutions  dilution  shaker  w h i c h had  of s t e r i l e  and l/25  inoculated with  i n Growth Medium #  portions cells  was  The  then  amount  l / 2 5 , 2/25,  suspension cells  and  were  3/25,  prepared  i n a sample  of  the  i n a haemacytometer. were p l a c e d  s t r o k e s per  i n a 20°C i n c u b a t o r room.  on  a  reciprocating  minute w i t h After  24  a travel  hours,  a 2  ml  of  of  18 sample the  t a k e n from each f l a s k  flasks  with at  was  2 ml  were r e t u r n e d of d i s t i l l e d  a wave l e n g t h  tronic  20  t o the  water,  of 560  with a s t e r i l e shaker.  and  their  nanometers  Spectrophotometer  inoculation, This  experiment  cations.  O n l y 0.13  added  litre  was  per  included  adjusted the  using  The  phase  After  7 days,  meter  pH  meter  model  2l6  as  hours  above. modifi-  solution  acid  inoculum consisted  after  was gm/l  l o t of medium  N hydrochloric  was  adding  o f washed  cells  2259-7 i n Gluc-AmS medium.  of  from each f l a s k ,  of each  Spec-  as a b l a n k .  until  stock  Each  b e f o r e measuring  t h e pH  Lomb  Ammonium s u l p h a t e a t 2  treatment.  culture  were t a k e n d a i l y water  and  measured  of the microelement  eighth  source.  distilled  turbidity  diluted  measured  water  intervals  and  were  r e p e a t e d with the f o l l o w i n g  4.5 w i t h 0.1  t o pH  with  culture  and  their was  diluted  turbidity  measured  1 to  10  a t 64-0  nm..  with a Radio-  28.  Vitamins The  as t h e are To  was  absorbance  distilled  o f b a s a l medium.  from a s t a t i o n a r y  II.  their  ml  as an  nitrogen  Samples  and  samples  i n a Bausch  Samples were t a k e n a t 2 4 h o u r after  The  pipette,  growth  only  required test  t h i a m i n e , was ml  along  added  vitamin  by t h i s casein  Two  hydrolysate  flasks  from  growth  each  flask  of  each  no  other vitamins  conditions  50  m  above,  subsequent added  l of t h i s  employed. to the  but  flasks, was  experiments  per l i t r e  of  only  basal  medium were s u p p l e -  inoculated After  and  w i t h 1 ml four  assessed v i s u a l l y ,  microscopically  basal  omitting  of t h i a m i n e h y d r o c h l o r i d e ,  2259-7 i n GM3 •  i n the f l a s k s checked  and  3 ' o l u t i o n was  vitamin-free  f r o m a 3-day c u l t u r e  the  described  In t h i s  containing  w i t h 5 micrograms  shaker,  under  that  of t h i a m i n e , a medium s i m i l a r  prepared.  w i t h two  demonstrated  strain  of microelement  medium. mented  -  the n e c e s s i t y  medium, p l u s 0.13  2259 7 i n s y n t h e t i c media c o n t a i n i n g t h i a m i n e  of  days and  each on t h e a  for contamination.  sample  19 III.  A growth  c u r v e f o r 2259-7  A more p r e c i s e using  a Coulter Three  200  c u r v e f o r 2259-7 was  growth  Counter. ml p o r t i o n s  o f GM3  i n one  with Bellco  stainless  ulated  w i t h 2.0  each f r o m a 3-day c u l t u r e  ml  i n c u b a t e d as d e s c r i b e d  f r o m each f l a s k  after  steel  litre  capped and  determined  flask  above.  s i x hours  Two  and  Erlenmeyer  closures  ml  daily  were  flasks  inoc-  o f 2259-7 i n  samples  GM3,  were t a k e n  thereafter  f o r eight  days . The bring  samples  the  cell  were d i l u t e d The  on t h e d i l u t i o n  fluid  Dilutions  Cell aperture  micron  current  threshold  utions  diluent  Counter.  Blank  each day  used  and  aperture.  The  subtracted  0-5  were c o u n t e d .  The  background  and  coincidence,  IV. E f f e c t  o f sodium, a c e t a t e on pH by  the  pH by  therefore  a drop  i n pH.  including  milligrams  aliquots  threshold  sample,  v a l u e s have been and  of t h i s  medium was acetate  was  sample fitted  s e t at l/2,  a t 7-5  ml p o r t i o n s  and  the  duplicate.dilof each  duplicate  corrected for  averaged. drift  The sodium  possibility  ammonium of  was  stabilizing  a c e t a t e i n t h e medium  was  litre  medium were s u p p l e m e n t e d acetate trihydrate  4.5)-  matched t h e g r e e n The  was  prepared.  w i t h 0,  per l i t r e .  adjusted with 5 N hydrochloric  indicator (pH  2 grams of ammonium s u l p h a t e and  of b r o m o c r e s o l green per  grams of sodium of t h e  out  tested.  B a s a l medium c o n t a i n i n g 20  electrolyte  from the  Growth o f 2259-7 on t h e medium c o n t a i n i n g accompanied  cells  Counter Model B  From each  four  reported  100,000  c o u n t s were c a r r i e d  amplification  at l/2, the lower at i n f i n i t y .  to  f r o m 1 t o 20 t o 1 t o 2000.  ranged  were p r e p a r e d , and  culture  chloride  provided the  c o u n t s were made on a C o u l t e r  w i t h a 100 upper  saline  by t h e C o u l t e r  counts.  sodium  c o n c e n t r a t i o n between 40,000 and  per m i l l i l i t r e . needed  w i t h 0. 9$>  acid  The  1, pH  until  2, of  Five  3 or 4 each  the  colour  o f t h e medium w i t h o u t  sodium  media were t h e n d i s p e n s e d i n two  20  ml  20 replicates and  into  125  autoclaved.  ml E r l e n m e y e r  After  cooling,  flasks,  each  capped  flask  was  1.0 ml of a 5 day o l d shake  culture  containing  acetate trihydrate.  days  incubation  cator  i n each The  purple of  of sodium  on t h e  sodium  experiment  was  repeated, s u b s t i t u t i n g  with  After  five  of t h e  indi-  bromocresol  i n t h e media w i t h 3 or k  gm/l  acetate. experiment,  grams of sodium  above.  green  inoculated  colour  noted.  a series  a c e t a t e per initial  pH  w i t h 0,  litre  and b r o m o c r e s o l p u r p l e was  were a d j u s t e d t o an as  a t 20°C, t h e  was  a third  sulphate  shaker  foil,  of 2259~7 grown i n medium  flask  f o r bromocresol  In 2.8  1 gm/l  with  of BM  2.2,  2.6  or  p l u s ammonium  prepared.  of 5-5>  2.4,  These  a u t o c l a v e d and  media incubated  21 Results I.  Nitrogen  source  A volume of 603  cells.  The  relation  of  cells  .04  This  microlitres  corresponds  of t h e  logarithmn  of t h e  i n terms  various  nitrogen sources,  of t h e  any  contamination  experiment,  are  microscopic  of t h e  cells  vacuolate.  Doubling  (Table  Table  against  In t h e  At  d i d not plus  the  reveal  soytone,  large, spherical,  were c a l c u l a t e d from, t h e  where g r o w t h was  plus  media c o n t a i n i n g  or a s p a r a g i n e  were e x t r a - o r d i n a r i l y times  Growth  f o r 2259 7 i n BM i n F i g . 4.  the  approximately  and  s e c t i o n s of  exponential  i) .  I. D o u b l i n g t i m e s f o r T r e m e l l a m e s e n t e r i c a 2259-7 i n b a s a l medium ( h i g h microelements") p l u s v a r i o u s n i t r o g e n s o u r c e s at 20°C.  Nitrogen  Source  Doubling  >2l6  Potassium n i t r a t e Ammonium, n i t r a t e Asparagine Casein hydrolysate Soytone Soytone plus asparagine A standard curves  obtained  Doubling the  listed  curve  times  i n the  The  cells  asparagine exper iment.  second  of t h e  time  (hours)  l/2  at 640  i s presented  nm  against  i n F i g . 5-  experiment  c a l c u l a t e d from the  f i n a l pH i n Table  12 59 47 27 49  of a b s o r b a n c e  of 2259-7 c e l l s  tration  and  concentration  -  examination  cultures.  some of t h e  curves  millilitre.  of t h e A^^^  illustrated  casein hydrolysate,  growth  per  to the  A plot  culture turbidity,  asparagine,  the  nm  cells  contained  c o n c e n t r a t i o n i s show i n F i g . 3-  curves, end  haemacytometer  X 10^  a t 56O  i n F i g . 2.  cell of  t o 1.5  absorbance  i s presented  i n the  are  The  growth  shown i n F i g .  e x p o n e n t i a l phase  cultures with  concen-  of  6.  growth,  each n i t r o g e n source  are  II. grown w i t h  showed t h e  casein hydrolysate  and  especially  " s w o l l e n " morphology n o t i c e d i n the  first  Fig.  3-  R e g r e s s i o n of t u r b i d i t y  concentration.  on t h e  logarithmn  of  cell  Fig.  h. Growth  curves  high  microelements  f o r T.  m e s e n t e r i c a 2259-7  plus various n i t r o g e n sources  i n BM  with  a t 20°C .  25  CONCENTRATION OF CELLS Fig. T.  5. R e l a t i o n o f t u r b i d i t y  mesenterica  2259-7 c e l l s .  a t 640 nm t o c o n c e n t r a t i o n o f  H O  <  OQ  •H  H- ' ON  O  -  o  Q  H  o  aCD  P"  0)  CD  c+tn  >d  H  O  <! fl>  en  cn  <  o  ii HO  «  en  B  £»  P dO CW CD  P  cn  o  o CD  01 03  Soytone  2  o*a.  CD  Ammonium, sulpha  CD cn CD  P dCD  HO 93  ro ro  vn MD  I  -<l  H-  c^-  P  ro o o o  W  •  Cas e i n hydrolysate  ro. ON  LLI U  1 CD ° • <  Potassium, n i t r a t e  s  si  H-  c+  0*Q 30*  +  40'  + GO*  +  80*  +  100'  + ISO*  +  140*  TIME FROM INOCULATION (HOURS)  160'  ISO.  27 Table  I I . D o u b l i n g t i m e s and f i n a l pH's f o r c u l t u r e s o f 2259-7 grown i n b a s a l medium ( l o w m i c r o e l e m e n t s ) p l u s v a r i o u s n i t r o g e n s o u r c e s a t 20°C.  Nitrogen  Source  Doubling  None Potassium n i t r a t e Ammonium n i t r a t e Ammonium s u l p h a t e Asparagine Casein hydrolysate Soytone Soytone plus asparagine  time  (hours)  F i n a l pH 4.1 4.1 2-3 2.4 4.7 3-9 4.0 4.4  13-3 12.8 38 15 14.7 30  II. Vitamins After turbid; the of  f o u r days, t h e f l a s k s  the f l a s k s  microscope,  without  with  thiamine  Growth The  t h e c u l t u r e s showed  clear.  only the yeast  very  Under  cells  typical  curve  number o f c e l l s  o f 2259~7 V  a l o g a r i t h m i c s c a l e a g a i n s t time phase  of growth, t h e d o u b l i n g  IV. E f f e c t After  of sodium a c e t a t e 5 days  b e l o w 4.0).  acetate  t i m e was 9 l / 4 h o u r s . on pH  containing bromocresol acetate  the indicator  i n the c u l t u r e s  t r i h y d r a t e had t u r n e d 3 °  r  was b l u e  the bromocresol  purple  i n the f l a s k s  t r i h y d r a t e was p u r p l e , yellow  experiment,  as b e f o r e ;  with  greenish yellow  and g r e e n i s h p u r p l e  2.6  gm/l were  gm/l or 2.8  purple.  In  i n the cultures turned  yellow.  The  3 o r 4 gm/l o f sodium a pH above 7-  c u l t u r e s without with  yellow  (pH above 5-5)-  green  indicating  flasks  with  4 gm/l o f sodium  0, 1, or 2 gm/l o f s o d i u m a c e t a t e  In t h e t h i r d turned  drift  In t h e c u l t u r e s w i t h  experiment,  i s p l o t t e d on  In t h e e x p o n e n t i a l  incubation, the indicator  trihydrate,  second  millilitre  eT  i n F i g . 7-  0, 1, or 2 gm/l o f sodium a c e t a t e  the  were a l m o s t  were  Tremella.  III.  (pH  added t h i a m i n e  2.2  sodium  gm/l and 2.4  acetate gm/l were  respectively; flasks  with  29 Discussion The number  and s i z e  closely in  turbidity  o f m i c r o b i a l c u l t u r e s depends on b o t h t h e  of t h e suspended  related to the concentration  relation  between t u r b i d i t y  i n 2259-7  cultures  markedly n o n - l i n e a r . logarithmn straight 0.4.  of c e l l  line,  In t h i s A plot  therefore  particularly  the  against  graph).  result  i n a linear  slopes  doubling  used  does  experiment,  is  of c e l l  d r y weight  con-  should  Examination  include  a linear  of F i g .  portion.  t h e c u l t u r e samples were d i -  measuring t h e t u r b i d i t y ,  experiments,  for this  increase  readings  so t h a t t h e  concentration  were used  ( F i g . 5)-  to calculate  t h e b a s a l medium w i t h o u t i n culture turbidity.  growth may have been  introduced  of the glassware. slightly  potassium n i t r a t e  indicate that  a l l , by T r e m e l l a  nitrate  In t h e f i r s t  used o r  experiment, i n the  i n t h e b a s a l medium.  c a n be u s e d 2259-7-  only  nitrogen  the inoculum,  more growth was f o u n d  than  mesenterica  added  The n i t r o g e n  with  come f r o m i m p u r i t i e s i n t h e c h e m i c a l s  not i n t h e second,  medium w i t h at  ( F i g . 4)  i n t h e upper t w o - t h i r d s of  inturbidity.  of t h e t u r b i d i t y  a small  contamination  data  time  times.  or may have but  against  would be p r o p o r t i o n a l t o c e l l  In b o t h supported  corre-  times.  Logarithmns doubling  concentration  that  s t r a i g h t s e c t i o n s were u s e d t o c a l c u l a t e  t e n times before  turbidity  i s greater  of t h e l o g a r i t h m s  e a c h growth c u r v e  of these  ( F i g . 2) was  o f O.326.  growth o f c e l l  increase  In t h e s e c o n d luted  of c e l l  (at least  Exponential  4 shows t h a t The  time  of t h e  ( F i g . 3) i s more n e a r l y a  i n absorbance to a plot  The  against the  when t h e a b s o r b a n c e  a doubling  i s more  d r y weight  experiment  of the t u r b i d i t y  of c u l t u r e t u r b i d i t y  equivalent  centration  i n the f i r s t  concentration  sponds t o an i n c r e a s e  of c e l l u l a r  ( A ^ Q ) and c o n c e n t r a t i o n  A plot  range,  and t h e r e f o r e ,  numbers ( M a l l e t t e I969).  the c u l t u r e than t o c e l l  cells  cells,  very  These  slowly, i f  The pH o f t h e s e  media  30 before  autoclaving  measured  pH was  inoculum,  was  4.1.  which  4-5This  was  At t h e end pH  d r o p may  of t h e e x p e r i m e n t , have been  grown i n Gluc-AmS and  had  the  c a u s e d by  a pH  of 2-3  the before  washing. In  the second  experiment,  ammonium s u l p h a t e showed than was  on  soytone  reached  and  similar  casein  drastically.  The  the f i r s t  and  in  the f i r s t  experiment,  on  ammonium n i t r a t e , The  of pH  In t h e  f o r growth  and  pH's  times  5-7  s o y t o n e was  growth  on  casein  i n growth either  fell  medium However,  much s l o w e r  h y d r o l y s a t e was  r a t e s between t h e s e  effects  of pH,  the  initial  experiment  pH  initial  two micro-  media  c a s e i n h y d r o l y s a t e had the  still  or o f t h e  experiment,  than  pH's  of a l l media  4.5F l e g e l (I968) has shown t h a t t h e optimum of 2259-7 i n a medium c o n t a i n i n g g l u c o s e , s o y t o n e  a  nd  i s about  7-2.  error,  as h i g h as  both  or  soytone  the  hydrolysate  experiment,  of m a c r o e l e m e n t s  importance  (pH 6.5,  no  appears  Also  rates  i s h i g h i n media leading  by a s p a r a g i n e .  of an was  ten  microelements the d o u b l i n g  microelements).  i n media w i t h of c e l l s  containing  with the normal t o s l o w growth  of m i c r o e l e m e n t s  growth  i n media w i t h a s p a r a g i n e  the f r a c t i o n  to i n t e r f e r e  A high concentration  of  added  a s p a r a g i n e are slower than  or s o y t o n e .  slower  because  i s suggested'by  t h e growth  T r e m e l l a i n some way growth  The rate  i n G-M3  " s w o l l e n " morphology Asparagine  with p r o g r e s s i v e l y  concentration  intended.  experiments plus  4.7^  In t h e f i r s t  of 9 l/4 h o u r s  In  of  on  d e t e r m i n i n g t h e growth  time  of  pH  were c o m p a r a b l e .  In t h e f i r s t  second  yeast extract  arithmetic in  and  culture  growth  have been  soytone  nitrate  adjusted to  and at  may  faster  turbidity  i n ammonium  i n ammonium n i t r a t e  experiments  concentration.  6.5-  was  second  slightly  A h i g h e r peak  ammonium, t h e  rates  differences  experiments containing  rates,  and  (NH^)^SO^ c o n t a i n s more ammonium t h a n  growth  in  element  initial  hydrolysate.  In m e d i a c o n t a i n i n g  slower.  on ammonium n i t r a t e  i n ammonium s u l p h a t e medium t h a n  medium, p o s s i b l y b e c a u s e NH^NO^-  growth  enhances  casein  with  asparagine. metabolism  and  large  cells.  the  inhibition  31 It  is i n t e r e s t i n g that  elements acids,  increases  but  not  amino a c i d s rate  of  entry  by  u s e d were c a p a b l e 2259-7 has  thiamine without in  the  as  the  source  not  on  that  than  He  Brough the  required  the  methods  requirements.  least  vitamin  to  20  containing  transfers,  vitamin  carry  the  on  sole  requirements.  levels.  of  pH  to r i s e  as  the  explanation  acids  the  on  pH  pH  by  nutrition  pH  d r o p can  of t h e be  This  i s the  prevented  to  d r o p and  organic  case  about 2-3  c l o s e t o 5-5-  of  medium,  a r e a d i l y metabolized  i n T_.  grams  Smaller  per  amounts  l a r g e r amounts  proceeds.  of t h e  c u l t u r e pH  required  ammonium s a l t s  t r i h y d r a t e at  growth  explicit  vitamin  i n the  1965)-  c u l t u r e pH  allow  and  but  species.  This  litre  sodium a c e t a t e  2259-7-  statement  source,  t h i a m i n e was  nitrogen  acetate  the  no  supported  of h i s  nitrogen  media c o n t a i n i n g  medium ( N i c h o l a s  maintains  part  over  (1970) f o u n d t h a t T_. bambusina  r e s u l t s i n a decrease  inhibitory  i n the  organic  vitamin  s y n t h e t i c media  any  There-  c l e a r response  f o r at  a l s o found t h a t  including a neutral salt  No  only  Therefore,  s i m i l a r i n t h e s e two  acids  cause the  their  were t a k e n t o r i d  casein hydrolysate  mesenterica—sodium of  on  here r e f u t e the  Therefore  Growth of f u n g i  acid  the  increase  i n T_. m e s e n t e r i c a 2259-6 and  ammonium as  fungus.  often to  i s the  vitamin  obscuring  simpler  nitrate.  strong  thus  major v i t a m i n  i n growth r a t e .  growth.  on  seems t o be  by  added  conjugation  with  grew w e l l  Possibly  medium s u g g e s t s t h a t  of d e t e c t i n g  r e s u l t s presented  dealing  amino  (I968) r e p o r t s , w i t h o u t f u r t h e r comment, t h a t  Flegel  The  and  However, t h e  from the  i n o c u l u m i s not  g r o w t h and  ammonium..  thiamine  2259~7-  only  micro-  amounts of v i t a m i n s .  been s u b c u l t u r e d  decrease  nitrogen  of t r a c e  claim that  of t h i a m i n e  of  i n media c o n t a i n i n g  s p e c i a l precautions  glassware  T_. m e s e n t e r i c a  Also,  concentration  cells.  s t u d y , no  I cannot  omission  times  i n media c o n t a i n i n g i n t o the  medium or  fore,  doubling  high  complex h e a v y m e t a l i o n s ,  In t h i s the  the  effect  of ammonium  have a p p e a r e d  i n the  and  liter-  32 ature.  I t i s known, however, t h a t  membrane  is freely  (ammonia), to  i n many f u n g i t h e c e l l  permeable t o t h e uncharged  and t o u n d i s s o c i a t e d  organic  NH^"" o r t o i o n i z e d c a r b o x y l i c a c i d s i n t h e medium b e c a u s e NH]^ Uptake  right,  +  = WH  o f NH^ b y t h e c e l l s  resulting  drives this  i n an a c c u m u l a t i o n  RCOOH = RCOO- + H Uptake  Organic  i o n i n the  acids  exist i n  and i o n i z e d  forms:  +  a c i d by t h e c e l l s  drives  and t h u s removes h y d r o g e n  this  i o n from,  e x t e r n a l medium, and r a i s e s t h e pH. The  organic  pH change i n a c u l t u r e c o n t a i n i n g b o t h acid w i l l  depend  by t h e c e l l s .  the  rate  Since  species,  culture  pH d e c r e a s e s  concentration will  on t h e r e l a t i v e the c e l l  of uptake w i l l  charged  depend  which  t h e pH r i s e s ,  on t h e c o n c e n t r a t i o n  the concentration Therefore,  be t a k e n up b y t h e c e l l s ,  the concentration  will  up, and t h e pH w i l l  pH w i l l  depend (NH^  +  be s e l f - c o r r e c t i n g .  on t h e r a t i o  organic  This  acid salt  tend  to rise.  o f NH^ i n c r e a s e s  and t h e  More NH^ and l e s s RCOOH tend  to f a l l .  allow  predicts that  t h e pH t o d r o p ,  Active transport  membrane, f o r example  i n exchange f o r K  complicate  the p r e d i c t i o n s of t h i s  of H +  model.  changes will  o f ammonium  of o r g a n i c  cause t h e pH t o r i s e .  Thus  pH m a i n t a i n e d  between t h e c o n c e n t r a t i o n  will  of t h e un-  o f NH^ and i n c r e a s e s t h e  The e x a c t  model c o r r e c t l y  by d i f f u s i o n ,  more RCOOH and l e s s RH^  p l u s RH^) and t h e c o n c e n t r a t i o n  p l u s RCOO").  taken  A decrease i n  and t h e pH w i l l  o f RCOOH d e c r e a s e s .  be t a k e n  o f each  i s crossed  i s a f u n c t i o n o f pH.  o f RCOOH.  ammonium and  quantity  membrane  concentration in  NH^ i s  equilibrium to the  of hydrogen  i n pH.  of u n d i s s o c i a t e d  equilibrium t o the l e f t , the  1968) .  of the e q u i l i b r i u m :  e q u i l i b r i u m between t h e u n d i s s o c i a t e d  As  (Burnett  + H+  3  e x t e r n a l medium, and a d r o p  up  a c i d s , b u t impermeable  1  present  s p e c i e s , NH^,  acid  (RCOOH  too l i t t l e  and t o o much +  across  (Rothstein  will  the c e l l  I965) w i l l  CHAPTER  TWO  PRELIMINARY STUDIES ON THE EROGEN  33 Materials I.  A qualitative For  activity  First, 1.5$  agar  cut  was  plates  originated  by R.  J . Bandoni  (CjM) s o l i d i f i e d  and d e p o s i t e d c e l l  s i d e up i n p l a s t i c  To each  assay dish,  4.0  ml of  one t o t e n d r o p s ) was  added.  o v e r n i g h t a t 20°C, and t h e n t h e c e l l s  The a s s a y s  an a s s a y r e q u i r i n g  eliminating propriate  dilution into  or p l a s t i c  of the t e s t a 16 X 125  culture  a two t o f o u r  medium.  After  One ml of an ap-  i n CjM or 0-5$  tube,  capped  soytone  with a  stainless  and a u t o c l a v e d f o r 15  cooling,  culture  s o l u t i o n and  the assay  tubes  Pasteur  pipette  o f 2259-6 i n QS  ,  The t u b e s were mixed by g e n t l e s h a k i n g , and i n c u b a t e d  from  assay tube  each  presence  was  a t 20°C.  The n e x t m o r n i n g ,  a drop  examined u n d e r t h e m i c r o s c o p e f o r  of c o n j u g a t i o n tubes.  the i n t e n s i t y  cells  test  closure,  day o l d shake  (12 t o 18 h o u r s )  of  developed.  w i t h one d r o p from, a s t e r i l e  overnight the  of the t e s t  solution  mm  tube  a t 15 p s i p r e s s u r e .  were i n o c u l a t e d of  less  t h e agar b l o c k s was  pipetted  minutes  p f con-  tubes.  Later,  steel  were  on t h e agar  b l o c k s wer,e.iexamined:'uhaermt.hecmi'cposcopetfor;:the^preserice  was  with  CjM, a l o n g w i t h an a p p r o p r i a t e volume o f t e s t  (usually  jugation  was  w i t h 2259-6, and i n c u b a t e d a t  5 cm i n d i a m e t e r .  liquid  incubated  absence  24 h o u r s , b l o c k s a p p r o x i m a t e l y 1 cm by 1 cm were  plates,  solution  or  of t h e  required.  were s t r e a k e d l i g h t l y  After  sterile  would r e v e a l t h e p r e s e n c e  o f C o n j u g a t i o n Medium  from the p l a t e s ,  petri  on t h e c h e m i c a l b e h a v i o u r  an a s s a y p r o c e d u r e  Petri  20°C.  studies  a bioassay that  hormone  used.  bioassay  preliminary  erogen, of  and Methods  of t h e r e a c t i o n ,  A subjective based  impression  on t h e p r o p o r t i o n of  b e a r i n g c o n j u g a t i o n t u b e s , t h e l e n g t h o f t h e t u b e s , and  the  number  of t u b e s  per yeast c e l l ,  was used  the  relative  amounts  of hormone  Similar  results  were o b t a i n e d f r o m t h e two a s s a y methods.  i n the t e s t  to roughly estimate solutions.  34 II.  Production  o f hormone  Conjugation 2259-7  i n shake  included shake of  or by scraping  o n MYP.  phase  volume  into  cells  three  portions.  The second  directly  or treated with  polysaccharide  butanol,  T h e pH o f t h e f i r s t 6 by measurement w i t h  p o r t i o n was a d j u s t e d  a c i d , and t h e t h i r d a t each  pH w e r e  ethyl acetate,  diethyl  ether,  emulsions  formed  water bath  ether  was h a s t e n e d  temperature  i n vacuo  40°C).  To  culture estimate  between water  indicator  t o pH 2 w i t h  concentrated con-  of the culture  t e n m l o f one o f nchloroform,  benzene,  (65-HO).  Persistent cases  b y c e n t r i f u g a t i o n . The  (Buchler  Each residue  10 m l o f C j M , a n d b i o a s s a y e d ,  original  pHydrion  e x t r a c t s and t h e aqueous r a f f i n a t e s t o dryness  cells.  was d i v i d e d  i n some o f t h e e x t r a c t i o n s , a n d i n t h e s e  of t h e phases  evaporated  equal  and r e s i d u a l  p o r t i o n t o pH 10 w i t h  shaken w i t h  c h l o r i d e , or petroleum  solvent  an  p o r t i o n was f o u n d  Ten ml samples  methylene separation  station-  solvents  ammonium h y d r o x i d e .  supernatant  from plate  into  f r o m a c u l t u r e o f 2259-7 i n GM3  centrated  along  with  were  separated,  rotary  evaporator,  was r e d i s s o l v e d a sample  of the  supernatant. the partition  and n - b u t a n o l ,  coefficient  a counter  of t h e erogens  current  distribution  carried out. A sample  extracting in  t o grow  The  hydrochloric  was  of P e t r i  organic  paper.  in  either  Extraction with supernatant  times  or e a r l y s t a t i o n a r y phases  from t h e surface  to precipitate  t o be a p p r o x i m a t e l y  and  at various  were removed b y c e n t r i f u g a t i o n . The  was e i t h e r u s e d  of acetone  by growing  I n o c u l u m was o b t a i n e d  The c u l t u r e s were a l l o w e d  and t h e c e l l s  supernatant  was p r o d u c e d Media used  cultures i nthe exponential  cultures  III.  c u l t u r e a t 20°C.  MYP, C j M , a n d GM3.  growth,  ary  hormone f o r s t u d y  GM3 w i t h  water).  of erogens  f o r the distribution  150 m l o f t h e s u p e r n a t a n t four  50 m l p o r t i o n s  The b u t a n o l  phases  were  was o b t a i n e d  by  f r o m , a c u l t u r e o f 2259-7  of n-butanol  (saturated  centrifuged briefly  with  t o remove  35 droplets vacuo.  of t h e aqueous phase, The  extract  n-butanol.  After  transferred  as  into was  a second  the  t o the  paper  each  plate.  adjacent  tube,  and  After  with butanol)  was  (Schleicher  and  was  disc  ten  trans-  w i t h 1-5$  i n a stream agar  overnight incubation  was  of warm  over  the  in a Petri  plate.  lawn of c e l l s ,  spaced  around  the  a t 20°C, t h e  were examined under  the  to  type  Inc.)  evenly spread  in  tubes  of t h e  Schuell,  dried  was  equally  of water  tube  repeated u n t i l paper  discs  ml  of b u t a n o l were- added  a filter  four  tube.  separated, the  added t o 2.0  ml  (saturated  i n the f i r s t  t h e phases  was  pipette  of n - b u t a n o l  were p l a c e d on t o p of t h i s  c e n t e r and  one  edge of cells  microscope  conjugation tubes.  hundred  2259-7 i n GM3 strong  acid  effluent  was  saved  water  and  cation  cation  at a f l o w r a t e  then t r e a t e d was  of d i s t i l l e d  The  w i t h 100  water,  and  i n 10  ml  i n GM3  for bioassay.  through  200  of d i s t i l l e d  200  ml  sodium  was ml  form  (Mallinckrodt A sample  bicarbonate  washed w i t h 50 of 10$  water,  of d i s t i l l e d  resin 100  water.  was ml  of t h e  solution  ml  of  distilled  aqueous ammonia.  of d i s t i l l e d The  of  of A m b e r l i t e IR-120  ml/hour.  re-evaporated.  1 t o 10  another  hydrogen  of 200  w i t h 10$ resin  of 20 ml  a culture  e v a p o r a t e d t o d r y n e s s _in v a c u o ,  dissolved ml  a bed  exchange r e s i n ,  neutralized  ammonia e l u a t e  resin  of s u p e r n a t a n t f r o m  were r u n t h r o u g h  f o r assay.  finally  exchange  millilitres  Chemical Works),  and  2.0  procedure  assays  t o the paper  Two  was  ml  t u b e was  each t u b e ,  IV. A d s o r p t i o n on  5 ml  (saturated  2.0  After  medium s o l i d i f i e d  discs  i n the  and  and  w i t h t h e b u t a n o l phase and  disc  for  tube  second  This  For  of GS  with a Pasteur  of water  -  surface The  ml  shaken.  second  tube.  of w a t e r - s a t u r a t e d  s e p a r a t e d , t h e b u t a n o l phase  A s u s p e n s i o n of 2259 6 c e l l s  air.  ml  b u t a n o l phase f r o m t h e f i r s t  for antibiotic  saturated  i n 2.0  added t o t h e aqueous phase  The  were i n u s e . used  second  i n the  tube.  first  Two  were t h e n  b u t a n o l phase ferred  t h e phases  tube.  w i t h w a t e r ) was  a third  dissolved  then evaporated to dryness i n  c o m p l e t e l y as p o s s i b l e  added t o t h e  Both tubes  was  and  The  dissolved  eluted  water,  The  and  in  material diluted  r e g e n e r a t e d by  of 2 N s u l p h u r i c  running acid,  36 In the  another  column,  experiment,  and t h e r e s i n  culture  supernatant  washed as above.  A  was r u n t h r o u g h concentrated  ammonium a c e t a t e b u f f e r ,  pH 1, was p r e p a r e d  glacial  100 ml o f c o n c e n t r a t e d  acetic  hydroxide. prepared  acid  with  by mixing  100 ml o f  (30$ NHg)  ammonium  Q u a r t e r - s t r e n g t h and h a l f - s t r e n g t h b u f f e r s were  by d i l u t i o n  from  the concentrated  solution.  One  h u n d r e d ml o f q u a r t e r - s t r e n g t h ammonium a c e t a t e was r u n t h o u g h the  column, f o l l o w e d b y 100 ml o f t h e h a l f - s t r e n g t h b u f f e r .  Finally  100 ml o f t h e f u l l  was p a s s e d dried in  through  s t r e n g t h ammonium  t h e column.  The t h r e e  t o remove ammonium a c e t a t e .  10 ml o f d i s t i l l e d  water,  acetate  solution  e l u a t e s were f r e e z e -  The r e s i d u e s were d i s s o l v e d  and d i l u t e d  1 t o h with  GM3 f o r  bioassay. In  a third  culture  experiment,  supernatant  after  pyridine  65 ml d i s t i l l e d  (25 ml g l a c i a l  a c e t a t e e l u a t e was f r e e z e - d r i e d .  V.  GM3  Adsorption  on a n i o n  supernatant  anion  and t h e n Samples  with  run through  acid,  10 ml  N, pH 5)-  The  The e l u t e d m a t e r i a l and d i l u t e d  1 t o 10  resin  were t a k e n  The a c e t i c  yellow.  acid  o f sodium  (Diamond Shamrock  culture  supernatant  and t h e n  the yellow front  a fresh receiver  was e v a p o r a t e d  water,  and r e - e v a p o r a t e d . water,  form  f o r bioassay.  water,  eluate  distilled  solution  bicar-  The r e s i n with  Chemical  and t h e was washed  100 ml o f 1 N  changed t h e c o l o u r o f t h e r e s i n  Just before  column,  a 10$  a 20 ml bed o f D u o l i t e A-k weak b a s e  chloride  50 ml o f d i s t i l l e d  acid.  water  with  200 ml o f a 3-day c u l t u r e o f 2259~7  of the o r i g i n a l  column e f f l u e n t  the  from  exchange r e s i n ,  Co.).  to  exchange  GM3 was a d j u s t e d t o pH 7 w i t h  bonate,  with  f o r bioassay.  The in  acetic  w a t e r ; a p p r o x . 1-75  was d i s s o l v e d i n 10 ml o f d i s t i l l e d with  the r e s i n  and w a s h i n g , t h e column was e l u t e d  a pyridine acetate solution pyridine,  treating  reached  was p u t i n p l a c e .  from  acetic grey  t h e bottom of The a c e t i c  acid  i n v a c u o , d i s s o l v e d i n 10 ml o f d i s t i l l e d The r e s i d u e was d i s s o l v e d i n 10 ml o f  and d i l u t e d  1 t o 10 w i t h GM3 f o r b i o a s s a y .  37 The tilled  column was  water,  distilled of  100  water,  distilled  ml  ml  on  ensure of  activated  A decolourizing  been was  of 2259-7 i n GM3• a reciprocating  15  coated with  one  washed w i t h two i n 50  minutes,  decanted After  ml  and  and  the  shaking  After 25  the  ml  rated of  The  distilled  a t 20°C f o r h a l f  on  reached.  added t o t h e  rinses  the  The the  ml  suspension  c h a r c o a l suspended  acid,  made t o 50  methanol,- e t h a n o l , and i n vacuo,  water.  then  The  was  i n 5 ml  was  i n 50  c h a r c o a l a d s o r p t i o n s t e p were b i o a s s a y e d concentrations.  The  solutions  e t h a n o l , acetone  20 w i t h GM3  and  ml  was  of  acetone.  acetone, i t of  pyridine,  distilled ml  freeze-  water.  the f i l t r a t e at t h e i r  evapo-  in 5  Samples from  the  original  of t h e m a t e r i a l s e l u t e d pyridine  cake then  e l u a t e s were  of d i s t i l l e d and  had  c e n t r i f u g e d , the  a c e t a t e e l u a t e was  supernatant,  gram,  of a b s o l u t e e t h a n o l .  ml w i t h  acetone  to  shaken f o r  the r e s i d u e s d i s s o l v e d  pyridine  dissolved culture  and  agi-  and  methanol supernatant i n 50  from  filter  (10 ml  acetic  was  water,  This suspension  c h a r c o a l suspended and  The  of d i s t i l l e d  of m e t h a n o l .  minutes,  Drug  a Buchner f u n n e l w h i c h  acetate solution  original  treated  ml  suspension,  with a pyridine  of  On  200  Then one  the  and  to  of  an hour  c e n t r i f u g e d from  dried,  methanol,  dis-  of s u p e r n a t a n t  been  to dryness  the  10 ml  f o r 15  of g l a c i a l  water).  out  ml  gram, of H y f l o S u p e r C e l .  c h a r c o a l had  treated  and  charcoal suspension  a i d was  centrifuged.  e t h a n o l decanted, was  The  shaker  charcoal f i l t e r e d  suspended  ml  charcoal (British  mixed w i t h 400  t h a t a d s o r p t i o n e q u i l i b r i u m was  the  of  charcoal  H y f l o Super C e l f i l t e r i n g  and  200  of 2 N h y d r o c h l o r i c a c i d ,  Houses, Canada L t d . ) was tated  hydroxide,  ml  water.  gram of N o r i t  a culture  "by w a s h i n g w i t h 200  of 2 N p o t a s s i u m  100  V I . A d s o r p t i o n on One  regenerated  with  a c e t a t e were d i l u t e d  1  for bioassay.  another w i t h 0.5  o c c a s i o n , 20 ml gm  of N o r i t  the  c h a r c o a l was  sedimented  was  decanted  the  and  A. by  of c u l t u r e After  half  supernatant an hour  centrifugation.  c h a r c o a l washed w i t h 20 ml  The of  of  was shaking,  supernatant distilled  38 water. water  The c h a r c o a l was t h e n  ( l t o l ) , 20 ml o f a c e t o n e - w a t e r  acetone.  The e l u a t e s were f i l t e r e d  evaporated for  t o dryness  bioassay.  natant  from  tested  supernatant after  were  with  t o adsorb  treated  with  i n 2 ml o f GM3  supernatant, were a l s o  and t h e s u p e r -  bioassayed.  Scientific  t h e hormone.  Fifty  coconut  after  another  0.1  Co.) was ml o f c u l t u r e  gm o f c o c o n u t  gm o f c o c o n u t  0.1  gm o f c h a r c o a l .  charcoal.  Samples o f t h e o r i g i n a l  medium, and t h e s u p e r n a t a n t charcoal treatments  from  were  the f i r s t ,  10 ml o f a c e t o n e ,  the acetone  was  decanted.  c h a r c o a l was washed t w i c e w i t h 40 ml o f d i s t i l l e d ml o f c u l t u r e  three  0.1  were t r e a t e d  gm p o r t i o n s o f t h e a c e t o n e  supernatants method  c h a r c o a l was p r e p a r e d  of A s a t o o r  10 gm o f N o r i t  and D a l g l i e s h  A in a solution  diluted  was c o l l e c t e d air  with  by f i l t r a t i o n ,  for half  an h o u r , water.  washed w i t h  acid,  U.S.P.,  and t h e n  The c h a r c o a l  distilled  w a t e r , and  gm. o f N o r i t  2259-7 c u l t u r e s u p e r n a t a n t were shaken  A, 0.1  gm o f d e a c t i v a t e d c h a r c o a l , or  gm o f d e a c t i v a t e d c h a r c o a l .  sions each  A suspension of  dried.  w i t h 0.1  for  c h a r c o a l , and t h e  gm o f s t e a r i c  4-50 ml o f d i s t i l l e d  F o r t y ml p o r t i o n s o f 0.5  as above w i t h  by a m o d i f i c a t i o n o f  (1956).  o f 0.4  50 ml o f e t h a n o l was s t i r r e d  slowly  washed  water.  bioassayed.  Deactivated the  supernatant  second,  bioassayed.  c h a r c o a l was wet w i t h  t h e c h a r c o a l had s e t t l e d ,  Fifty  in  culture  carbon,  c e n t r i f u g e d down, and t h e s u p e r n a t a n t  One gram o f c o c o n u t The  suspended  shaken f o r 15 m i n u t e s w i t h 0.1  a third  culture  third  and,  t o remove  charcoal (Fisher  c h a r c o a l was a l s o  spent  (3 t o l ) , and 20 ml o f  The c h a r c o a l was c e n t r i f u g e d o u t , and t h e s u p e r n a t a n t  sampling,  treated and  coconut  20 ml o f a c e t o n e -  and d i s s o l v e d  the c h a r c o a l treatment  for ability  charcoal.  i n vacuo,  The o r i g i n a l  Granular  This  washed w i t h  were  After  c e n t r i f u g e d and samples  bioassay.  of t h e s u p e r n a t a n t s  The c h a r c o a l s e d i m e n t s  of d i s t i l l e d  water  and t h e n  30 m i n u t e s t h e were washed w i t h  suspended  suspentaken 40 ml  i n 20 ml each o f  39 10$ aqueous  phenol.  centrifuged  and t h e s u p e r n a t a n t s were d e c a n t e d and e v a p o r a t e d  to  After  d r y n e s s _in v a c u o .  5 ml o f d i s t i l l e d for  These  water,  Adsorption One  and d i l u t e d  i n Canada  chromatography glass  the  i n a c e t o n e , and p o u r e d made f r o m a P a s t e u r  was washed w i t h  t h r o u g h t h e column  minute.  effluent  Samples  of  plus  and t h e n ' e l u t e d  5 ml a c e t o n e ) , bioassay,  0-5$  three  stopcock  .05 ml o f 10$  10 ml o f d i s t i l l e d  10 ml o f 50$ a c e t o n e (5 ml water  on Sephadex  grams o f Sephadex  hours  in distilled column  at the bottom  flowing  calibrate  solution  was added  t o 1 ml  t h e column,  (Difco  out u s i n g  water  ( P h a r m a c i a ) were and t h e n p o u r e d  (2 cm i n t e r n a l  o f t h e column while  a solution  the g e l surface,  Q.  soaked into a  diameter).  was opened,  containing  0.1  level  water  To  gm o f s o l u b l e c h l o r i d e per  i n t h e Sephadex  1 ml o f t h e s t a r c h - s a l t  The  so t h a t  the g e l s e t t l e d .  L a b o r a t o r i e s ) and 0.1 gm o f sodium When t h e water  Porapak  G-10  G-10  t h r o u g h t h e column  ml was p r e p a r e d . reached  with  .1 W p o t a s s i u m h y d r o x i d e ,  was washed w i t h  e x p e r i m e n t was c a r r i e d  chromatography  starch  medium and t h e  soytone.  Thirty  was  N i n e t y ml  10 ml o f 75$ a c e t o n e , and 10 ml o f a c e t o n e .  Chromatography  glass  with  o f f , and  water.  culture  with  3 drops of each e l u a t e  A similar VIII.  with  a miniature  o f a p p r o x i m a t e l y 2 ml  were s u p p l e m e n t e d  (w/v)' s o y t o n e p e r ml, n e u t r a l i z e d water,  Belleville,  2259-7 i n Gluc-AmS medium were  at a flow rate  The column  into  to drain  50 ml o f d i s t i l l e d  of the o r i g i n a l  f r o m t h e column  and b i o a s s a y e d .  Associates,  pipette, f i t t e d  The a c e t o n e was a l l o w e d  supernatant from a c u l t u r e  per  for  soytone  by Chromatographic S p e c i a l t i e s ,  column  wool p l u g .  resin  passed  of  1 t o k w i t h 0-5$  on P o r a p a k  O n t a r i o ) was s u s p e n d e d  For  were each d i s s o l v e d i n  gram o f P o r a p a k QS, 120-150 mesh ( W a t e r s  distributed  of  eluates  "bioassay.  VII.  a  15 m i n u t e s t h e s e s u s p e n s i o n s were  column  solution  was  ko applied  t o the t o p  of t h e  volume o f d i s t i l l e d the in  column a t O.k treated  potassium starch, of  ml  per  minute  iodide  . 5 gm,  chloral  culture  0.5  a separate nitrate  of  with  evaporated  to dryness  Two  ml  solution  of  O.k  five was  ml  water,  hundred  of c u l t u r e  The  distilled  effluent A 0 . 5 ml  prepared  "by  drop  extract  i n water.  of t h e  Sephadex  at a f l o w  collected  and  a  extraction  butanol  water  sample  to detect  with  dissolved  was  gm,  ion.  to the top  of 1 0 $ s o y t o n e  ml  filter  was  filtered  from 1.5  in  forty-  each  ml  rate  fraction  of  distilled  materials  with  Application and  w i t h two  1 ml  was  to  1 5 ml  of  retained  The  precipitate was  The  evaporated  of d i s t i l l e d type  i n diameter, filter  weight  solution  partially  into  solution  with d i s t i l l e d  suction  from  retains  solutes  with  1 5 ml  water.  a stream  of r e t a i n e d  water.  The  sample  m a t e r i a l s were d i l u t e d  filter  on  of water  and  the  1 t o 1 0 w i t h GM3  took  the  The  m a t e r i a l was  filtrate  retains  1250 (Millipore  the m a t e r i a l s r e t a i n e d  a beaker  This  washed t h r o u g h  p o r t i o n s of d i s t i l l e d and  dryness  completely  of t h e  was  was  PSWP membrane  greater than  Filtration  to  water.  under  7 5 0 and  greater than  dismantled,  were washed  w h i c h formed  Pellicon  AR-2l).  hours.  the  a Pellicon  2 5 mm  a molecular  Report  a wash b o t t l e .  assay.  weight  a half  apparatus filter  The  stirring  filtrate  through  to 200  were added w i t h  i n 1 5 ml  Ltd.),  aspirator.  and  The  redissolved  a molecular  filter  of a c e t o n e  supernatant,  (Millipore  a water with  ml  filtration.  vacuo and  solution  one  with  fraction  1.5  1 0 0 ml)  treated  The  and  of each  bioassayed.  removed by in  minute.  n-butanol.  collected  (iodine  water  was  was  through  Ultrafiltration Two  ml  sample  were a p p l i e d  eluted  with 0 . 1  and  1 0 0 gm,  i n vacuo,  5 minute f r a c t i o n s . mixed  IX.  and  per  sample  reagent  a small  passed  effluent  to detect chloride  was  column,  the  c o n j u g a t i o n hormone was  supernatant  of t h i s  ml  i n with  water was  A 0 . 5 ml  of M e l z e r ' s  0.1 N silver  G-10  and  a drop  and  washed  Distilled  with  A sample of  water.  10 minute f r a c t i o n s .  twenty  was  column and  the from  made  up  solution for bio-  41 X.  C h r o m a t o g r a p h y on Fifty  Ltd.),  columns of s i l i c a g e l  grams of s i l i c a g e l , 60  were s l u r r i e d  in absolute  chromatography tube, allowed  to  settle,  the  was  just  bed  and  dry.  i n diameter.  2 ml  of a b s o l u t e  column,  column, f o l l o w e d by  of 1-95  ml  per  and ml  was  The  distilled  finally  100  water), ml  ml ml  of 50$  ethanol.  100 One  ml  top  of  pipetted to  sink  mortar.  The  at a  flow  collected  of e t h a n o l had at t h e  was  i n a mortar  was  ethanol was  a  n-butanol  solvent allowed  in  passed  into  same f l o w r a t e  with  absolute  f o l l o w e d by  the  ground  suspension  effluent  continued  100 ml of 87.5$ e t h a n o l (87.5 ml  was  of a b s o l u t e  A f t e r 100  elution  the  until  washings from the  minute.  2 minute f r a c t i o n s . column,  100  This  into  s i l i c a gel  grams of f r e e z e - d r i e d  i n t o the  the  The  Chemicals  poured  0.2  ethanol.  eluted with  and  solvent drained  top  column was  of t h e  mesh (BDH  the  onto t h e  rate  ethanol  f r o m 2259~7 c u l t u r e s u p e r n a t a n t  extract with  2 cm  t o 120  12.5  e t h a n o l and  of 75$  hundred  ethanol  fractions  and were  collected. From each even-numbered d r o p s were a d s o r b e d The  d i s c s were d r i e d  with  at 20°C, t h e  on t h e  cells  and  placed  e t h a n o l was  column  on  ground w i t h  the  top  of t h e  absolute  on  a magnetic  0.3  The  in ethanol.  stirrer. inlet  The and  to a reservoir  extended connected  outlet  and  generate was  polyethylene  the  fitted  c o n t a i n i n g 50$  tubes absolute  preparation a  convex  gradient,  ml E r l e n m e y e r  tubes.  incu-  t r a n s f e r r e d to  eluted with  The  inlet  mixing  t u b i n g t o the  400  ml  flask  w i t h ,a r u b b e r  ethanol.  n e a r l y t o t h e b o t t o m of t h e with  spread  noted.  gram of hormone  i n a 500  flask  agar  of s i l i c a g e l i n  ethanol  To  disc.  of c o n j u g a t i o n  was  column was  e t h a n o l were p l a c e d  bearing  connected  above.  of a b s o l u t e  column.  of water  of  stopper  2 ml  as  six  After overnight  or a b s e n c e gm  8,  paper  a p l a t e of GS  c o n t a i n i n g 50  prepared  was  gradient  presence  with  i n c h diameter  c l o s e t o each p a p e r d i s c  Another  tube  a one-half  starting  2259-6 c e l l s , s i x d i s c s p e r p l a t e .  bation  was  on  fraction  top  tube The  was  outlet  flask, of t h e  and chroma-  42 tography ute, The  tube.  S o l v e n t was f e d t o t h e column a t 1 ml p e r min-  and t h e column e f f l u e n t even-numbered  t h e paper  disc  X I . Paper On  fractions  was c o l l e c t e d  method as above.  chromatography  various occasions during this chromatography  t o g r a p h y was c a r r i e d  out on s t r i p s  2 cm by 14 cm.  detection along  Sufficient  o f t h e hormone a f t e r  a line  2 cm f r o m  of t h e s t r i p s a paper  clip  tube  so t h a t  t h e p o i n t e d bottom  of t h e s o l v e n t s had  risen  stuck into  used  The s e c t i o n s  were e i t h e r  1 ml o f GS medium.  and i n o c u l a t e d  The t e s t  XII. E f f e c t The  line,  the solvent the s t r i p s  and t h e s o l v e n t into t e n equal  into test  o f GS tubes  overnight  sections  incubation,  induced  were  autoconju-  calculated.  o f c y c l i c - 3 5 ' - a d e n o s i n e monophosphate  t o t h e erogens  that  c y c l i c - 3 % 5'-AMP m e d i a t e s  of cyclic-3'>5 '-adenosine  B i o c h e m i c a l s C o r p . ) was s t e r i l i z e d  a sterile  a sterile  the response  was examined.  A 1 mM s o l u t i o n through  After  line  and R f v a l u e s f o r t h e e r o g e n s  possibility  (Nutritional  Composit i o n s  t u b e s were t h e n  After  a s s a y s were examined t o see w h i c h  gation tubes,  was immersed  p l a c e d on p l a t e s  or dropped  w i t h 2259-6.  test  i n a stream, o f c o o l a i r .  between t h e s t a r t  s p r e a d w i t h 2259-6 c e l l s ,  claved  into  III.  ends  suspended,  a 25 X 150 mm  of the tube.  and d r i e d  to allow  was  end o f t h e s t r i p i n Table  chromatography  The b o t t o m  strip  inside  Chroma-  was s t r e a k e d  were c u t p e r p e n d i c u l a r t o t h e l o n g a x i s  containing the  Each  a cork,  are l i s t e d  of the s t r i p s  sections. agar  chromatography  a p p r o x i m a t e l y 10 cm. above t h e s t a r t  parts  front  o f Whatman # 1  hormone s o l u t i o n  a t t h e bottom  were removed from, t h e t u b e s The  has been examined.  were c u t t o a p o i n t .  a p o o l of s o l v e n t  study, the behaviour of  one end o f t h e s t r i p s .  from in  fractions.  f r o m 6 t o 134 were b i o a s s a y e d b y  t h e hormone i n paper paper  i n 5 minute  Gelman membrane f i l t e r  flask.  The s t e r i l e  monophosphate by f i l t r a t i o n  w i t h 0.22 u p o r e s  solution  was added  aseptically  ^3 to  5.0  ml p o r t i o n s  to  give  final  1 X 10-5,  of GS  cyclic  5 x 10~ ,  and  under  Caffeine AMP  M.  The  flasks  were  10~5,  inoculated  3. i n c u b a t e d on t h e s h a k e r a t daily  f o r s i x days,  the microscope.  i s known t o i n h i b i t  the  cells.  GS  medium i n 125  2,  k, 8,  w i t h 1 ml After  -  an<  -6  5 X  cyclic  AMP  phosphodiesterases  I966) and t h e r e f o r e i t might cause a c c u m u l a t i o n o f c y c l i c  inside  20°C.  1 X 10  flasks  o f 1 X 10~\  Samples were t a k e n f r o m e a c h f l a s k  examined  (Wair  ml E r l e n m e y e r  concentrations  2259 6,  w i t h T_. m e s e n t e r i c a 20°C.  AMP and  6  medium i n 50  and  16  of a  Daily  ml E r l e n m e y e r mM.  After  2259 6 -  samples  s i x days,  with d i s t i l l e d  Caffeine  flasks and  t o 20  ml p o r t i o n s  at c o n c e n t r a t i o n s each f l a s k  was  of  of 0,  1,  inoculated  i n c u b a t e d on t h e s h a k e r at  f r o m each f l a s k and  added  autoclaving,  culture  a sample water  was  were examined  from each f l a s k i t s absorbance  was  microscopically.  diluted  measured  1 to  a t 5^0  10  nm.  Results I. E x t r a c t i o n w i t h The ether, ether  organic  m a t e r i a l e x t r a c t e d f r o m w a t e r "by e t h y l  chloroform,  benzene, methylene  a t any o f t h e pH's t r i e d  raffinates  from these  able t o the o r i g i n a l at  solvents  original  i n t h e amount acid,  activity.  o f hormone  corresponding  t o tubes  among t h e t u b e s 2-3  The  k t o 10.  1 and 8.  This  corresponds  exchange culture  tubes  Each off a  i n the assay  of t h e e l u t i n g  t h e column.  convenient The  erogenic  activity.  passage through  t h e i o n exchange  10$ ammonia  a low l e v e l  activity  was e l u t e d w i t h  strength  ammonium a c e t a t e  point  o f hormone  the half  solutions.  was t h e a c t i v i t y  supernatant.  induced  conju-  pigment  of colour i n the e f f l u e n t  m a t e r i a l e l u t e d f r o m t h e column w i t h  fractions  Neither the  cells.  The a p p e a r a n c e  showed  o f about  a p p l i e d t o the c a t i o n  b u f f e r s t e s t e d washed brown  erogenic  culture  coefficient  activity  resin  marker f o r t h e b r e a k t h r o u g h  ammonium a c e t a t e  eluate  o f hormone  was  and n - b u t a n o l .  column n o r t h e m a t e r i a l e l u t e d w i t h gation  current  intense response  to a partition  column showed h i g h after  detected  around t h e d i s c s  distribution  of c u l t u r e supernatant  supernatant  weaker t h a n t h e  from the counter  The most  on c a t i o n exchange  sample  extract  e x t r a c t e d by n - b u t a n o l a t  were f o u n d  f o r t h e e r o g e n s between water  II. Adsorption  compar-  from the b u t a n o l e x t r a c t i o n s  of t h e f r a c t i o n s  c o n j u g a t i o n tubes  i n samples  activity  The  pH.  distribution, found  petroleum  The b u t a n o l  slightly  diethyl  activity.  No d i f f e r e n c e was  activity  n e u t r a l or a l k a l i n e In t h e b i o a s s a y  activity,  The r a f f i n a t e  low hormone  or  showed no hormone  culture supernatant.  supernatant.  displayed  chloride,  e x t r a c t i o n s had e r o g e n i c  a l l pH's showed hormone  acetate,  was  of each b u f f e r . quarter  strength  activity.  More  s t r e n g t h and f u l l  I n none o f t h e t h r e e  as h i g h  as i n t h e o r i g i n a l  45 The  material eluted  from  the cation  pyridine  a c e t a t e showed hormone a c t i v i t y  original  culture  III. but  culture  none a f t e r  acetic  acid  exchange  supernatant  the anion  the f i r s t  experiment,  methanol,  e t h a n o l , and a c e t o n e  had  the second  acetone  experiment,  culture  first,  Hormone  activity, and t h i r d  the o r i g i n a l with  used  charcoal with  but the p y r i d i n e  culture from  50$,  supernatant  the c h a r c o a l  75$  w i t h the coconut  and  100$  i n the o r i g i n a l . acetone  treatments  The c o c o n u t  also  charcoal  and so d i d t h e s u p e r n a t a n t s  was n o t n o t i c e a b l y l e s s  with  coconut  from  charcoal.  i n the t r e a t e d  samples  c h a r c o a l w h i c h had been  d i d not reduce  t h e hormone  activity  the supernatants. Nor i t A a t 0.1  0.1  inactive,  The s u p e r n a t a n t  supernatant  second,  activity  washed w i t h of  after  The m a t e r i a l e l u t e d  and t h e m a t e r i a l s e l u t e d  showed" h i g h hormone  than  was  supernatant  were a l l i n a c t i v e .  The the  The  erogenic.  h i g h hormone a c t i v i t y .  adsorption,  culture  and t h e f i l t r a t e  had low hormone a c t i v i t y .  In  column.  activity.  the o r i g i n a l  erogenic a c t i v i t y  e l u a t e was h i g h l y  before,  charcoal  treatment acetate  activity  exchange  e l u a t e d i s p l a y e d h i g h hormone  showed m o d e r a t e  to the  resin  showed h i g h e r o g e n  passing through  IV. A d s o r p t i o n on a c t i v a t e d In  comparable  with  supernatant.  A d s o r p t i o n on a n i o n The  exchange r e s i n  and 0.5  gm p e r 40 ml c o m p l e t e l y  from, t h e c u l t u r e phenol  from  gm p e r 40 ml and d e a c t i v a t e d c h a r c o a l a t  supernatant.  removed e r o g e n i c  The m a t e r i a l s e l u t e d  any o f t h e c h a r c o a l samples  activity by  10$  d i d n o t show hormone  act i v i t y .  V. A d s o r p t i o n on P o r a p a k The hormone  original activity  culture was f o u n d  medium  was h i g h l y  i n the e f f l u e n t  e r o g e n i c , b u t no from  the Porapak  QS  kG column. a band  After  t h e medium had p a s s e d  of yellow  When 50$ a c e t o n e colour air  was p a s s e d  appeared  eluted  with  some a c t i v i t y as  was v i s i b l e through  moved down t h e column w i t h  bubbles  erial  pigment  50$ a c e t o n e  was p r e s e n t  t h e Porapak  column,  at t h e t o p of t h e r e s i n . t h e column, t h e y e l l o w  the acetone  i n t h e column b e h i n d had h i g h  front,  and numerous  the f r o n t .  erogenic  i n t h e 75$ and 100$  The mat-  a c t i v i t y , and  acetone  eluates  well. P o r a p a k Q gave r e s u l t s  P o r a p a k QS, e x c e p t eluted  VI.  with  was  very  little  on Sephadex  obtained  additional  and none w i t h  pure  minutes  and c h l o r i d e  after  t h e sample  hormone was  acetone.  the  fractions  was  applied.  eluted  from  was a p p l i e d .  from  column,  t h e column a f t e r  i n the f r a c t i o n from  a t t h e same f l o w r a t e ,  with  G-10  i n the f r a c t i o n eluted  100 m i n u t e s ,  VII.  s i m i l a r t o those  t h e c a l i b r a t i o n r u n o f t h e Sephadex G-10  found  sample  that  75$ a c e t o n e  Chromatography In  erogenic  80 t o  120 t o 170  In t h e r u n w i t h activity  200 t o 225 m i n u t e s  starch  a hormone  was f o u n d i n  after  t h e sample  Ultrafiltration • The u l t r a f i l t r a t e  showed h i g h hormone a c t i v i t y ,  retained  m a t e r i a l s washed  active.  Apparently  from  t h e erogens  PSWP membrane f i l t e r  (molecular  VIII.  on s i l i c a  Chromatography A band  apparently pigment was  through  of dark  was c o l l e c t e d  column, eluted  with  were  completely i n -  are not r e t a i n e d weight  cutoff  by a P e l l i c o n  approximately  1000).  g e l columns  brown pigment  unretarded,  changed t o  the f i l t e r  and t h e  t r a v e l l e d through  the absolute  in fractions  t h e column,  ethanol eluant.  8 t o 10.  This  When t h e e l u a n t  87.5$ e t h a n o l , more pigment moved down t h e  and was c o l l e c t e d more pigment  50$ e t h a n o l e l u t e d  which pigment  i n fractions emerged  35 t o 37-  in fractions  in fractions  The 75$  ethanol  63 t o 70, and t h e  87 t o 92.  47 Conjugation with  samples f r o m  est  response  and  88  fractions In from  IX.  t o 100 second  80  and 10,  34 36  paper d i s c s t o 100.  and  33  t o 53  The  38,  material in fractions  treated  60  high-  to  76,  8 t o 12  was  hormone I I , and  in  hormone I I I . run,  6 t o 18,  t o 134  Paper  8 and  the unretarded  i n f r a c t i o n 6.  in fractions  each  active  around t h e  8 t o 12,  to f r a c t i o n s  42  in fractions  The in  5h  fractions  tions  fractions  The  column  material  were f o u n d  hormone I, i n f r a c t i o n s  the  the  that  was  t o 100.  designated  in  tubes  42  Erogenic  t o 62,  6 t o 18 t o 62  brown pigment  and  was  80  emerged  a c t i v i t y was  t o 134.  The  found active  i d e n t i f i e d w i t h hormone  w i t h hormone I I , and  that  I,  in frac-  w i t h hormone I I I .  chromatography s o l v e n t s used  s o l v e n t are  and  listed  the Rf  of t h e  i n Table  erogenic  III.  The  Rf  activity values  obtained  were r e p e a t a b l e . X. E f f e c t The to  10 ^  cells  treated  M d i d not  -  cells  of c y c l i c - 3 % 5 ' - a d e n o s i n e  treated the  with  flask  with  caffeine  from  above 2 mM  turbidities caffeine  tubes  2 mM  c y c l i c AMP  at c o n c e n t r a t i o n s  morphological  cyclic  appear  Some of t h e  caffeine,  on  inhibited  from  the  the  of t h e but  s i x days  remained  Concentrations  growth of t h e of t h e  cells  particularly in  were e l o n g a t e d ,  dilutions  concentration after  any  cells,  conjugation tubes.  of o n e - t e n t h  differences  up  AMP.  d i d not  caffeine.  distinguishable  IV.  show any  grown w i t h o u t Conjugation  with  monophosphate  cells.  cultures  are p r e s e n t e d  at  of The  each  i n Table  48 Table  I I I . The R f v a l u e s f o r t h e e r o g e n f r o m T r e m e l l a mesent e r i c a 2259-7 o b t a i n e d on p a p e r c h r o m a t o g r a p h y i n v a r i o u s solvents.  Solvent  Composition (v/v/v)  n-butanol,  acetic  (8/1/1) (12/3/5) (4/1/5, upper  n-butanol,  acid,  (8/1/1) (1/1/1)  n-butanol ( s a t u r a t e d with water) c h l o r o f o r m , m e t h a n o l , water  (4/4/1) (4/2/5, l o w e r phase); acetic  acid,  (1/1/1) (2/1/25 (4/1/5, l o w e r p h a s e ) (5/4/1)  water  acetic  acid,  water  benzene,  acetic  acid,  a c e t o n e , water  ethyl  (10/6/10/3)  a c e t a t e , e t h a n o l , water ( 4 / l / 5 , upper p h a s e ) (h/2/5,  u p p e r phase).  (*A/2) phenol,  water w/w)  (4/l, methanol ethanol ethanol,  water  acetone,  water  (13/1) (1/1>  d i s t i l l e d water 2$ aqueous a c e t i c a c i d 0.1 N h y d r o c h l o r i c a c i d 0.1 N ammonia  Activity  •3-.5 .4-. 6 • 35--4 •1--3 .5-.8 0-.2 0  benzene,  CV3/1)  of E r o g e n  water  phase) p y r i d i n e , water  chloroform,  Rf  •1.0  .7-.9 0-. 15 0 •35--5 0 0-.2, 0 0-.5 0..1, .9-1.0 0 0  3- 6 (two z o n e s )  5 (two z o n e s )  .8-1.0 •95-1-0 0-.1, .2- 6 (two z o n e s ) .5--8 0-.1, .7- 9 (two z o n e s ) 0-.1, .6- 9 (two z o n e s )  49 Table  IV. T u r b i d i t i e s o f o n e - t e n t h d i l u t i o n s o f T. c u l t u r e s grown f o r s i x days i n GS medium p l u s c o n c e n t r a t i o n s of c a f f e i n e .  Caffeine  concentration  0 1 2 4 8  16  (mM)  Absorbance  mesenterica various  a t 540 nm  0.26 0 . 24 0.19 0.046 0.011 0.007  50 Discussion The be  substances  e x t r a c t e d from  with is  erogenic  less  aqueous  from, o t h e r culture  fungi.  and  ether  fact  t h a t the less  (van  p o l a r than  extracted acidic  resins  not  such pH  pH  with  On  little  as  erogens  evidence charges  ammonia,  on  cation  t h a t the  as w e l l as b e i n g  interpretation.  The  were o n l y p a r t i a l l y  attracted  The  importance  to the  has  are  of t h e  acid  may  ionic  the  on t h e  its  behaviour  been used  acetic  of  ionic  acid. and  on t h e  to non-polar  The by  carry  hormones  raising  sites.  with  suggest the  The this  non-ionic  the  et a l  anion  caused  exchange  by  experiments  non-ionic binding to erogens  of  supports  (Schroeder  Further  d e s c r i b e d i n Chapter  cation  exchange  i n a scheme f o r t h e  hormones f r o m  neutral polystyrene resin  i s due  simply  recovered  erogens  have been p a r t l y  s t r o n g a d s o r p t i o n of t h e  and  charcoal  elution  of t h e  exchange r e s i n s  of hormone  These r e s u l t s  ready  relative  common  anion  of p y r i d i n e t o d i s r u p t  i o n exchange r e s i n s  effect  they  p o l y s t y r e n e backbone  in eluting  ability  1 N acetic  and  and  I962). solvent  a r e not  exchange r e s i n  s e p a r a t i o n of p e p t i d e m i x t u r e s  with  the  amount  cation  preparative resin  and  the  of p y r i d i n e a c e t a t e  The  shows t h a t  a t a p p r o p r i a t e pH's.  t h e hormones were b i n d i n g t o t h e  to  the  hormone m o l e c u l e s  that  binding  extracted  compounds.  c o n c e n t r a t i o n s of ammonium a c e t a t e .  success  isolated  o t h e r hand,  on t h e  erogens  high  resin,  the  sugars  effect  hormone  I966,  a c i d s can be  into, n-butanol  s o l v e n t s , the  e l u t e d from 10$  not  were e x t r a c t e d from,  ( M a c h l i s et a l  1970).  metabolites  has  organic  negative  be  chloride  partition  i s presumptive and  but  shows t h a t t h e T r e m e l l a  I967),' t r i s p o r i c  a d s o r p t i o n of t h e  positive the  with  or b a s i c  The  could  Since  n-butanol,  antheridiol  Ende e t a l  erogens  amino a c i d s .  and  methylene  den  with  2259-7 can  s e x u a l hormones w h i c h have b e e n  Barksdale  with  This  Sirenin  media w i t h  McMorris  are  the  T_. m e s e n t e r i c a  solution  polar solvents.  more p o l a r t h a n  from  on  the the  ion  6. on  Porapak  exchange r e s i n .  activated  charcoal  i s c o n s i s t e n t with Adsorption  b i n d i n g f o r c e s and  on  is usually  51 strongest The is  f o r aromatic  t e n a c i t y with  substances  which the  remarkable—aromatic on  tures  ethanol  and  erogens b i n d  amino a c i d s  have been a d s o r b e d of water  (Asatoor  charcoal or  can  and  to  activated  small  charcoal  peptides  u s u a l l y be  acetone.  D a l g l i e s h I956).  and  eluted  Asatoor  and  which  with  Dalgliesh  (I956) have recommended d e a c t i v a t i o n of c h a r c o a l w i t h acid  e l u t i o n with  10$  aromatic  substances.  My  charcoal  r e t a i n s a high  10$  and  phenol  lack  of  adsorption  of  may  crosslinked QS,  with  differ  He  for  the  degree  been  atoms  i n the  deactivated  erogens,  f o r the  charcoal  and  that  hormones.  suggests that  some s p e c i a l s u r f a c e  ratio  of  styre.ne  non-polar  of t h e  chain.  and  two  the  property  of  as  a method  f r o m aqueous s o l u t i o n . side  the  chains,  number  amino a c i d s  of  were bound  less  s t r a i g h t chain  com-  saturated,  pounds.  Peptides  were a d s o r b e d  more s t r o n g l y t h a n t h e i r  stituent  amino a c i d s . charcoal  supernatants, Porapak with It  was  indicate  50$  the  molecular  water  hormones  as  weight  solvent,  than  sodium c h l o r i d e .  cates  that  the  valid  conclusions on  can  easily  i s as  hormones  conjugation  chromatography  later  tion  Porapak Q be  con-  eluted  effective  from, c u l t u r e from  the  acetone.  hoped t h a t  the  distilled  but  i n removing  that  that  carbon  corresponding  I have f o u n d  on  (1971) and  peptides  I971) with  Aromatic  Porapak Q  Niederwieser  hydrocarbon  increased  used,  Adsorption  Giliberti with  types  than the  Norit A  the  ethylvinylbenzene  strongly  as  The  components.  amino a c i d s and  and  adsorption side  The  i n v e s t i g a t e d by  cX-amino a c i d s of  adsorb  eluant  divinylbenzene.  (Niederwieser  found  an  coconut on  to  of  used.  i n the  recovering  indoles  as  i s a co-polymer  P o r a p a k Q has for  on  depend  charcoal  Porapak and  capacity  stearic  improve r e c o v e r i e s  r e s u l t s indicate that  is ineffective  adsorption brand  phenol to  mix-  hormones a r e  of t h e  the  Sephadex G-10  erogens.  However,  hormone a c t i v i t y  This being  about m o l e c u l a r  Sephadex g e l has  on  high  is  weight  on  can  the be  f r e q u e n t l y been n o t e d ,  with  eluted  r e t e n t i o n volume  adsorbed  would  indi-  g e l , and  drawn.  no  Adsorp-  especially for  52 aromatic of  substances  erogen  gests  activity  that  the  (Wolf "by t h e  at  least  pendently assay can  three  capable  strain.  be  by  ethanol  which w i l l  results  obtained  observed uities  the  whether t h e tion  or  of t h e  using  a gradient that  of t h e  the  solvent.  silica  As  of hormone  and  in The  result  activity  of d i s c o n t i n I t i s not  clear  i s a c t u a l l y adsorp-  g e l , or d i f f e r e n t i a l mixtures.  of t h e  the  concentration  solvent.  adsorption  that  of a b a s i c  the  three  structure.  f o r erogenic  activity  molecule,  and  of t h e  destroying of t h e  the  present the  rest  biological  hormone  f o r m by  Wot  i n the  solubility  a l l of  the  absolute the  ethanol  bulk  column g r a d u a l l y or p a r t i t i o n  f o r m , but  responding  production a c i d s A,  differ  by B,  i n the  can  hormone  of dimin-  solubility on  chemical  occupy  part  molecule  the  not  be  The  T_. m e s e n t e r i c a  Blakeslea and level  C. of  only can  be  are structures  of t h e  varied  hormone  without  A l t e r n a t i v e l y , some or a l l  readily  fungus.  components  Perhaps the  activity.  components may  j u g a t i o n hormones by  but  of  g e l column.  main phenomenon i s d i f f e r e n t i a l  necessary  sporic  inde-  in cells  e l u t i o n proceeds,  top  enhanced by  i s probable  variants  the  is  there  gel.  It  their  peaks  is soluble  m a t e r i a l at t h e  separation  silica  separation  silica  starting  with  tubes  were t h e  i n ethanol-water  P o s s i b l y the  of w h i c h  of water  eluting  mechanism of t h i s on  the  experiment  used  ishes.  conjugation  750-  of water  hormone p r e p a r a t i o n  undissolved  hormones--each  than  shown t h a t  minimum c o n c e n t r a t i o n  initial as  g e l columns has  less  the  strength  hormones  weight  sug-  polarities.,  first  partition  of r e t e n t i o n  PSWP membrane f i l t e r  e l u t e them f r o m t h e  by  lack  components have d i f f e r e n t  possibility  i n the  i n the  silica  inducing  These  complete  a molecular  distinct of  recognized  eliminate  on  The  Pellicon  hormone has  Chromatography are  I969).  trispora  erogenically active in be  converted  production  of t h r e e  2259 7 can be -  and  A l l of t h e s e  i n t o an  con-  compared  Mucor mucedo of have z y g o g e n i c  o x i d a t i o n at t h e  active  penultimate  to  tri-  activity, carbon  53 of t h e s i d e duction  chain  of s e v e r a l  activity  distinct  behaviour  consistent tion  of t h e erogens  with t h e i r  must  i n paper  solvent p a r t i t i o n  c o n t a i n a minimum amount  of the erogens.  into  are s i m i l a r  chromatography i s  characteristics.  o f water t o cause  t o those  f o r the aromatic  two z o n e s ,  in  migra-  f o r m u l t i p l e forms  C y c l i c - 3 S 5 '-adensine  f o r the (Smith  80$ p h e n o l i s a n o m a l o u s l y  supporting the evidence  an i n t r a c e l l u l a r  The  amino a c i d s  I n some s o l v e n t s y s t e m s t h e e r o g e n i c a c t i v i t y  tography  as  the p e n i c i l l i n s  s o l v e n t systems t h e Rf v a l u e s  I958), b u t t h e R f f o r t h e e r o g e n s high.  biological  I n t h e n - b u t a n o l - a c e t i c a c i d - w a t e r and  n-butanol-pyridine-water erogens  example,  Pro-  1967)-  (Abraham and Newton  solvent  compounds w i t h t h e same  i s common among f u n g i - - f o r  The  I972).  ( B u ' L o c k , D r a k e and W i n s t a n l e y  from  silica  i s resolved g e l chroma-  of t h e erogen. monophosphate has been  mediator  i n the responses  implicated  t o hormones o f  1971), and i n h i g h e r p l a n t s ( P o l l a r d I97O, Salomon and M a s c a r e n h a s I97I, Wood, L i n and B r a u n I972). I t has a l s o b e e n f o u n d t o s e r v e higher  animals  (Robison,  as t h e c h e m o t a c t i c  Butcher,  agent  acrasin  D i c t y o s t elium. d i s c o i d e u m The for  experiments  the involvement possibility.  i n the c e l l u l a r  (Barkley  of c y c l i c  AMP  but they  Exogenous  slime  mold,  1969)-  r e p o r t e d here  in Tremella mesenterica, the  and S u t h e r l a n d  do n o t p r o v i d e any e v i d e n c e i n c o n j u g a t i o n tube do n o t r i g o r o u s l y  cyclic  production  exclude  AMP may have been  inactive  because  i t could not p e n e t r a t e t h e c e l l s ,  or i t may have been  rapidly  d e s t r o y e d by h y d r o l y t i c  The t o x i c i t y  feine  show t h a t  ity  may have  way  not r e l a t e d  enzymes.  i t was a b l e t o e n t e r t h e c e l l s ,  interfered  with  to cyclic  phosphodiesterases  c o n j u g a t i o n tube  AMP m e t a b o l i s m .  may n o t be s e n s i t i v e  anion  exchange r e s i n s  both  weakly a c i d i c  tion  on a c t i v a t e d  suggests  and w e a k l y b a s i c  that  As w e l l ,  Tremella  to caffeine. on b o t h  the molecules  groups.  c h a r c o a l and P o r a p a k  but the t o x i c -  p r o d u c t i o n i n some  In summary, t h e a d s o r p t i o n o f t h e e r o g e n s and  of c a f -  Strong  indicate  that  cation contain  adsorpt h e erogen  54 molecules  include  bonding.  Because the  very may  strongly include  indicates expected  held  from  acids  erogens  are  activated rings.  summation be  adsorbed  charcoal,  The  solvent  with non-polar  or f r o m side  from  an  a small  chains.  of  hydrophobic  on Sephadex,  and  the non-polar partition  portions  behaviour  have medium p o l a r i t y ,  a molecular  expected  chain,  capable  of t h e n o n - p o l a r  suggests  could side  portions  t h e whole m o l e c u l e s  Ultrafiltration non-polar  on  aromatic  that  properties  non-polar  and  weight  ionic  portions.  b e l o w 750.  amino a c i d peptide  as  with a  These large  containing  amino  CHAPTER THREE QUANTITATIVE  BIOASSAY OF THE EROGENS  55 Materials  and  Methods  I . R e l a t i o n of A sample  conjugation  of e r o g e n s was  butanol  e x t r a c t was  solved  i n a mixture acetate.  m i x t u r e was  decanted  e t h y l acetate  was  freeze-dried. 22.2  ml  of GS  to  give  mg  evaporated of 10  ml  and  by  of d i s t i l l e d shaking,  discarded,  stock  tubes.  2220 m i c r o g r a m s was  dispensed  The  inoculated GS.  water  10  and  the  the  ml  diluted  222,  Kkk,  per  t u b e s were i n c u b a t e d  were d r i e d i n an  f o r 10  minutes,  at 20°C.  1$  GS  medium  888, ml.  Each  and  test  then  A f t e r 22  hours,  with  glass  cover  slips.  eyepiece  micrometer,  1  slide.  t o room  In each sample,  the  (l/l/l), lengths  t u b e s were measured,  at X1000 m a g n i f i c a t i o n  The  temperature,  Phloxine-3$ potassium h y d r o x i d e - g l y c e r o l  randomly s e l e c t e d c o n j u g a t i o n  a Filar  10.0  amounts i n t o t r i p l i c a t e  oven at 105°C, c o o l e d  of 20  phase  666,  slides  covered  phases.  with  p i p e t t e d onto a m i c r o s c o p e  and  acetate  2 d r o p s per t u b e f r o m a 23-hour c u l t u r e of 2259-6  with  with  of  dissolved in  d r o p f r o m each t u b e was stained  ml  aqueous  of hormone p r e p a r a t i o n  i n one  dis-  water-ethyl  separate  111,  t u b e s were a u t o c l a v e d  The  and  the  s o l u t i o n was  The  i n vacuo,  and  super-  n-butanol.  f r e e z e - d r i e d e x t r a c t was  This  concentration  e x t r a c t i n g the  to dryness  s o l u t i o n s c o n t a i n i n g 55-5,  and  erogen  with  centrifuged to  phase was  of t h e  medium.  dilution  in  prepared  A f t e r vigorous  The  1110,  length to  f r o m a c u l t u r e of 2259-7 i n GM3  natant  ethyl  tube  using  ( o i l immersion  ob j e c t i v e ) . II.  A bioassay The  tion was  linear  tube used The  I was  based  length as t h e  conjugation  dependence o f t h e on  the  logarithmn  tube  length  mean l o g a r i t h m n of e r o g e n  as  of  a standard.  hormone p r e p a r a t i o n  For  an  assay,  i s d i s s o l v e d i n 6.0  s o l u t i o n i s d i v i d e d i n t o two  procedure.  described 6.0  ml 3 ml  conjuga-.  concentration  basis for a quantitative bioassay  f r e e z e - d r i e d hormone p r e p a r a t i o n  used  standard  on  mg of GS  in section  of t h e  standard  medium.  portions  i n 16  The mm  56 test  tubes.  O n e - t h i r d and  by w i t h d r a w i n g it  t o 2.0  mixing, ml  ml  1.0  o f GS  of f r e s h ml  prepared  up  The  minutes  One  medium and  about  cycle, w i t h two  After  a microscope  slide,  dried,  as a b o v e . the  sum  against  cells  bearing  of the l e n g t h s  The Finney  For  data thus  (1964). that  of each t u b e  are f i t t e d the l i n e s  of t h e t e s t  calculated. is  used  The  confidence III.  Erogens The  culture  activated sional  variation  and  a t 20°C  of  on  25  i s measured  conjugation  tube,  i s recorded.  In t u b e  relative  of  length  w i t h the  From t h e  standard regression  of t h e t u b e  f o r potency  to  i s placed  sample  parallel.  and  set  p s i pressure  length  f o r average  must be  are l i n e a r  of f r a c t i o n  erogen  series  cooling  f o r each p r e p a r a t i o n  preparation  lines  limits  Relation to  i n each  horizontal  lines,  the  to the standard i s  length within  to estimate experimental error,  the r e g r e s s i o n  appropriate  o b t a i n e d a r e a n a l y z e d by t h e method  d i s t a n c e between t h e sample potency  The  more t h a n one  Regression lines  l o g dose  constraint  stained. tubes  solution.  incubation  a d r o p from, e a c h a s s a y t u b e  conjugation  i s with-  drops per tube from a  hours,  selected  2.0  series is  a t 15  after  thorough with  tube  a t an  18  randomly  mixed  dilutions  and,  adding  After  o f s t a n d a r d hormone  duplicate  and  and  standard d i l u t i o n  2 day shake c u l t u r e o f 2259-6 i n GS. for  and  are a u t o c l a v e d  on a d r y goods inoculated  tube,  are d i s s o l v e d  assay tubes  room, t e m p e r a t u r e ,  tube.  ml f r o m t h e t h i r d  portion  preparations  i n GS  10  from t h i s  A dulicate  second  concentration as above.  i n a second t e s t  tube.  discarded.  or more t e s t  for  GS  i s withdrawn  from the  are prepared  ml f r o m t h e s t a n d a r d s o l u t i o n ,  in a third  drawn and One  1.0  one-ninth d i l u t i o n s  test  parallel,  each  dose  the assumption and  that  calculate  ratios.  of c e l l s  bearing  conjugation  tubes  concentration  were p r e p a r e d by  growing  s u p e r n a t a n t s were mixed  c h a r c o a l per l i t r e ,  stirring.  The  and  c h a r c o a l was  2259 7 i n Gluc-AmS medium. -  w i t h 0-5  left  gm  of N o r i t  f o r an hour  r e c o v e r e d by  A  with occa-  centrifugation,  57 and  washed w i t h  distilled  eluted  from, t h e  (50  of g l a c i a l  ml  water.  charcoal with acetic  a pyridine acetate  acid,  20  ml  solution  of p y r i d i n e , and  distilled  in  vacuo, r e d i s s o l v e d i n a s m a l l volume of w a t e r , then  m a t e r i a l was  f r e e z e r s e c t i o n of a A  sample  0.5$  solution  Nine  serial  had  of t h e of  were a u t o c l a v e d  10  2 drops  After ined The  15  each  hours  5>  soytone, 2/3  on  of a 2 day  a t 20°C,  number  of  counted  i n 80  A bioassay  re-evaporated,  capped  The  vial  in  dissolved in a  as  i n which  great  a dry shake  as  goods  each  the  i n 16  of 1.0  mm  tubes  and  longer  number  than  of  inoculated  2259 6 i n was  of t h e cells  one  cell  cells  with  one,  tubes  -  X400 power  the  dilution  test  c y c l e and  c u l t u r e of  mg/ml.  preceding  a sample f r o m each d i l u t i o n  cells,  small  dryness  at a c o n c e n t r a t i o n  of each d i l u t i o n  minutes  or more c o n j u g a t i o n  IV.  pH  i n 0-5$  ml  ml  freeze-dried.  in a tightly  i n a h a e m a c y t o m e t e r under t h e total  and  to  50  refrigerator.  concentration Two  water  stored  soytone,  were p r e p a r e d .  evaporated  e r o g e n p r e p a r a t i o n was  dilutions  a hormone  with  e l u a t e was  dissolved in d i s t i l l e d  freeze-dried the  The  hormones were  of and  water).  Conjugation  GS. exam-  microscope.  with  diameter  one were  squares.  based  on  fraction  of  conjugation  tubes The that is  described  defined  the 10  standard as  standard mg  are  of  i n the  in section III. the  amount  .04  b o t t o m of a 16  ml mm  0.5$  soytone,  pH  unit  per  From t h i s  and ml  l/8  i n 0.5$  from the  contains  one  l/8 ml.  per  of  ml  test  dilution  i s the  of  same  conjugation  hormone i n 0.2  stock  aqueous  hormone mg  of  acetone  uses.  tube,  stock  and  solution  mixed w i t h  i s placed  2.0  ml  of  of  1  an  erogen  concentration  solution,  serial  dilutions  are  as  solutions containing  of 50$  standard  5 • 5. > to give soytone  unit  Standard  f r e e z e r between  assay,  ml.  One  conjugation  preparation  i n the  an  of  preparation.  standard  stored For  hormone p r e p a r a t i o n used  prepared  i s discarded  i n l6 mm so t h a t  test  of l / 2 ,  l/k  tubes.  One  each t e s t  tube  58 If units  the t e s t  of erogen  solution  i s expected  p e r ml, a sample  volume  o f 10$  (w/v) s o l u t i o n  sample  i s a d j u s t e d t o 5-5  to contain  i s supplemented  of soytone.  w i t h 0.1  as an i n d i c a t o r .  soytone  t o g i v e an e x p e c t e d  p e r ml. a test the  simultaneously. minutes  one d r o p  each  GS.  The t u b e s  cycle  100 t o 150  one c e l l cells  in  one day's  as  possible. The  against  diameter  statistical  ln(p/(l-p))  )•  series  Weighted  can be a s s a y e d inoculated  o f 2259-6' i -  n  i n a haemacytometer, with  lines  dose,  as s h o r t  increase  in deviation  test  from, t h e h o r i z o n t a l  (logit  with  of p = of response f o r the  and t h e n  a set  f o r a l l prepar-  of the data from the  p r e p a r a t i o n are t e s t e d t o  expected  test  of c e l l s  separately  preparation,  f r o m random, s a m p l i n g .  when p a r a l l e l  o f each  a period  for logit  t o be p a r a l l e l  the hypothesis that  erogenic potency  lines  are f i t t e d  f o r each  The  A l l o f t h e samples  the f r a c t i o n  The d e v i a t i o n s  i f they are g r e a t e r than  a t o t a l of  of t h e b i o a s s a y d a t a i s p a t t e r n e d  constrained lines  c o n j u g a t i o n tubes  Usually  sample.  within  regression  and f o r each  regression  to test  as f o r  at 4°C.  i s transformed t o i t s l o g i t  see  The  f o r each  F o r each  i s calculated.  separate  into  autoclaved f o r ten  culture  are counted.  analysis  (1971)-  of r e g r e s s i o n  prepared  cooling,  and c e l l s  t h e l o g a r i t h m n o f dose  standard  0.5$  are p i p e t t e d  solutions  i s mounted  cells,  i s counted  conjugation tubes  used  shake  b i o a s s a y are counted  Finney  ations  test  and, a f t e r  a sample  t h e number o f t o t a l than  with  a r e i n c u b a t e d a t 20°C o v e r n i g h t (13 t o 20  From e a c h t u b e ,  after  solution  and t h e n p l a c e d i n a r e f r i g e r a t o r  longer  a c i d or  i s diluted  a r e capped,  from, a 24 hour  1/20^  of a p p r o x i m a t e l y 1 u n i t  test  The a s s a y t u b e s  on a d r y goods  with  and  sample  From one t o f i f t e e n  10  or b r o m o c r e s o l  and l / 2 , l / 4 , and l / 8 d i l u t i o n s  standard.  with  N hydrochloric  potency  Two ml o f t h e d i l u t e d  tube,  hours)  The t e s t  than  The pH o f t h e t e s t  potassium hydroxide, u s i n g bromocresol green purple  less  lines  the l i n e s  are f i t t e d i s  really  are p a r a l l e l .  preparation i s estimated  d i s t a n c e between t h e r e g r e s s i o n  line  59 for  the t e s t  dence  preparation  intervals  and t h e s t a n d a r d  f o r the potency r a t i o  C.  and  95$  are c a l c u l a t e d .  program which performs t h e s e c a l c u l a t i o n s Appendix  line,  confiA  i s described in  comput  6o Results  I. R e l a t i o n of c o n j u g a t i o n a.  l e n g t h t o erogen  concentration  Data The  ious  length  measurements  concentrations  A p p e n d i x B. against  b.  tube  of t h e data  of the tube  a t low d o s e s .  sample  on l o g a r i t h m i c  has  a slope  the  variance  valid  o f 2.13•  the  line, ting  i n F i g . 9.  is significant  o f t h e mean.  The  concentration  e r o g e n dose  regression  concentration  (Fig.  of t h e data  tube  should  be  f o r data to i t s  The means o f lengths  i n F i g . 10. i s obtained  against  For  f o r each A  straight  by p l o t -  the logarithms  of  l l ) .  of average  is significant  two means t h a t  o f t h e mean.  i960).  of c o n j u g a t i o n  length  l e v e l , and  each measurement  and T o r r i e  of In tube  i n each  a t t h e .01$  more s u i t a b l e f o r a s s a y p u r p o s e s , the average  doses  i n t h e same  can be a c c o m p l i s h e d  by t r a n s f o r m i n g  are p l o t t e d against  length  of approximately  This  (Steel  erogen  The r e g r e s s i o n o f  analysis the variance  logarithmn  hormone  i s plotted  of t h e tube l e n g t h  the average tube  A slope  n a t u r a l logarithmns  sample  at h i g h  i s p r o p o r t i o n a l t o the square  s u c h as t h e p r e s e n t natural  f o r each sample  i s higher  scales  on l o g mean  statistical  independent  are l i s t e d i n  i n F i g . 8.  The v a r i a n c e  i s plotted against  variance  at var-  i n A p p e n d i x B shows t h a t t h e v a r -  length  sample log  hormones  tubes  analysis  Inspection than  length  concentration  Statistical  iability  of the conjugation  The mean t u b e  erogen  of t h e c o n j u g a t i o n  In tube  length  a t t h e .01$  on l o g hormone  level.  61  CSNCMDim Fig.  8. R e l a t i o n o f mean c o n j u g a t i o n  erogen. at  H13N31 3Bni NV3N  each  Bars r e p r e s e n t dose.  95$  tube  confidence  l e n g t h t o dose o f  intervals  f o r t h e mean  Fig.  9. Dependence  length  of v a r i a n c e  measurements.  on mean f o r c o n j u g a t i o n  tube  Fig.  10. R e l a t i o n  l e n g t h t o dose  of  of a v e r a g e erogen.  transformed conjugation  tube  Fig.  11.  Linear  tube  length  on  regression the  of  logarithmn  average transformed of  erogen  dose.  conjugation  65 II.  A bioassay The  based  bioassay  employed  a few  too  dence  intervals  on  tedious  assay  the  III.  The  total  number  150.  The  Bioassay  tion  bearing  of  a second  conjugation  cells  bearing  found  Confipotency.  procedure  based  tubes.  conjugation  tubes  2259-6 c e l l s i n 80 s m a l l s q u a r e s  of  of  cells  bearing  hormone c o n c e n t r a t i o n  the  bioassay  logarithmn  data  f o r the  conjugation  i n F i g . 12a.  of hormone  the  cells  to produce  (Fig.  13)-  last  fraction  tubes  The is  concentration  chapters  of t h e  slope  which are  regression  in  dispersion  calculated  to the  of t h e  can  data  conjuga-  provided  most  of  thesis.  of t h e  regression  causes  (ED50) have been  50$  greater  between t h e  than  slope  of  the  charted  ED50 show  standard  day-to-  their  and  the  d e v i a t i o n s from  the  explained  be  points  about t h e  linear.  a heterogeneity  by  recognized.  d e v i a t i o n expected  heterogeneity  has  tubes  errors  zero.  be  remains  producing  which  significantly  d e v i a t i o n can  of t h e  as  the  correlation  than  basic relation  deviation  slope  standard  and  conjugation  cells  of t h i s  f r e q u e n t l y show g r e a t e r  lines  types.of  the  tubes  ED50 i s e s s e n t i a l l y data  of  with  e x t e n s i v e l y , and  conjugation  The  of c e l l s  performed,  Both the  estimation.  bution  on  two  dilution  variations  The  fraction  based  each a s s a y  and  standard  on  b e e n used  lines  the  f o r r o u t i n e use.  been r e p l a c e d by  fraction  based  t u b e s has For  Two  was  I964) of t h e f r a c t i o n of c e l l s r e s p o n d i n g  against  The  of  lengths  was  12b.  Fig.  day  of t u b e  length  concentration  against  (Finney  plotted  tube  ±25$ t o ±70$ of t h e r e l a t i v e  from  cells  erogen  logit  the  of  length  conjugation  time-consuming  method has  to  plotted  IV.  and  on  tube  measurement  R e l a t i o n of f r a c t i o n  averaged is  but  ranged  fraction  conjugation  method b a s e d  times,  t o be This  on  The  sampling.  In some c a s e s ,  line ratio The  the  i s increased, of t h e  frequency  i s summarized  but  actual  f r o m random s a m p l i n g  factor.  factor  random  is  distri-  in Table  V.  4.  O  '3  b.  tfl  cj  ul  d  CU  iii o  <J* "iii o • o  3  ST• •  •  ^  -H  S3aru NDiivgnrNDD HUM NDioftu d d  aj  user ! 1  a O  °saaru roiSvgrmD  <H Fig. to  12a.  dose  of b.  logarithms  Relation  of f r a c t i o n  H?IM STTO 3D NDU3*S  of c e l l s with  c o n j u g a t i o n tubes  erogen. Linear regression of e r o g e n  dose.  of t r a n s f o r m e d  response  on  the  Fig.  13.  standard  Variation curve.  i n the  slope  and  intercept  of t h e  bioassay  68 In o t h e r  cases,  the  from e x t r a p o l a t i o n can he Table Value  seen  V. for  in Fig.  response of t h e  response  doses  at lower  i s lower  than  expected  doses..  This  effect  12.  F r e q u e n c y d i s t r i b u t i o n of t h e h e t e r o g e n e i t y t h e c o n j u g a t i o n hormone b i o a s s a y .  of h e t e r o g e n e i t y  1.00 1.50  at h i g h  2- 50 3- 50 4.50 5 .50 6.50 7.50 8.50 9-50  1A9 2. kg 3-kg k.kg  5 6.  7  Frequency  29 9 7 k  .kg kg -kg  Q.kg 9-kg  10.  factor  kg  3 k  l  2 0 .1  factor  6  9  D i s cus s i o n The ical  theory  assay  that the  variability,  may n o t a p p l y  both  reason  curve  vary  greatly  in  each a s s a y .  at  several dilutions  Measuring  f o r curvature  curve  standard  procedures  a specified  polation  and a l l o w s line.  i n terms  and M a c h l i s developed  1965  Units  weight of the  i n previous  one dose  )•  by R a p e r  meet  s e v e r a l doses  o f t h e unknown i s d e t e r m i n e d  hormone A i n A c h l y a  these  of s t a n d -  o f t h e unknown.  by g r a p h i c a l i n t e r -  In the b i o a s s a y f o r  (1942a), s e v e r a l dose  o f t h e unknown a r e t e s t e d , b u t o n l y one c o n c e n t r a t i o n  standard  hormone  i s used  to "calibrate"  the mycelia.  One u n i t o f  i s d e f i n e d as t h e amount p e r c . c . r e q u i r e d t o p r o d u c e  average  the  test  She  tests  o f 10 a n t h e r i d i a l b r a n c h e s  hyphae.  solution  Barksdale  several dilutions  dilution  produce  which  are assayed  the s e n s i t i v i t y  This  assay  method  i n t h e t e r m i n a l 3 mm o f  (1963a) h a s m o d i f i e d  this  o f t h e unknown, b u t n o t e s  causes  a n t h e r i d i a l branches.  that the  which t h e  response.  In t h e s i r e n i n b i o a s s a y ,  (Carlile  series  preparation  o f f u n g a l s e x u a l hormones do n o t c o m p l e t e l y  potency  lowest  with  w h i c h have b e e n used  The  an  to the test  p r e p a r a t i o n , r a t h e r than  p r e p a r a t i o n are t e s t e d along with  of  dilution  c a n be c a l c u l a t e d ,  ard  levels  For t h i s  s h o u l d be d e f i n e d as a c e r t a i n  amount needed t o p r o d u c e  requirements.  confirms  a particular  and n o n - p a r a l l e l i s m o f t h e t e s t  activity  on one  r e g r e s s i o n l i n e s and  increases the precision  tests  Bioassay  F i g . 13  t o another.  the response  i n biolog-  Because of  determined  t o include a standard  of the dose-response  a stable  (1964}.  r e q u i r e d t o produce  from, one a s s a y  slope  of b i o l o g i c a l  methods  a t some o t h e r t i m e .  the slope of the dose-response  i t i s necessary  studies  Finney  a standard  c o n c e n t r a t i o n of standard  response  of  of s t a t i s t i c a l  have been d e s c r i b e d ' b y  biological occasion  and p r a c t i c e  at l e a s t Three  simultaneously,  only the  25$ o f t h e hyphae t o dilutions  of a standard  a p p a r e n t l y as a check  o f t h e hyphae has n o t changed  i s only capable  assay.  drastically.  of measuring t h e potency of  unknown p r e p a r a t i o n t o w i t h i n a f a c t o r  o f two.  70 The  description  o f t h e b i o a s s a y f o r "gamones" i n Mucor  mucedo i s s k e t c h y , b u t i t a p p e a r s unknown p r e p a r a t i o n was t e s t e d , to  a standard curve determined  mine t h e z y g o g e n i c assayed,  acids.  activity  of s e v e r a l  and  allow the c a l c u l a t i o n  Despite  into  relative  erogen  bation  (See Chapter length  mone o v e r t h e r a n g e  at  short  mately  tubes  average  a t each tube  proportional  this  logical which  required f o r  e s t i m a t e of t h e such  as c h r o m a t o -  of the b i o a s s a y  I I , IV, and V.) after  22 h o u r s  of c o n j u g a t i o n hor-  variable  be e x p e c t e d ,  ( c o e f f i c i e n t of the v a r i a b i l i t y  than at l o n g average  l e n g t h s at each  dose  o f t h e mean t u b e  length ( F i g .  o f v a r i a n c e on mean v i o l a t e s t e c h n i q u e s such  tube  i s approxithe basic  as t h e a n a l y s i s  l e n g t h measurements were t r a n s f o r m e d t o  l o g a r i t h m n s t o make t h e v a r i a n c e a t e a c h constant.  incu-  However, t h e l e n g t h s o f  are h i g h l y  o f common s t a t i s t i c a l  natural  response  dose  t o the square  Such a dependence  approximately that  ( F i g . 8).  the safeguards  version  on t h e dose  lengths i s less  of v a r i a n c e , and t h e t u b e their  Six, sections  The v a r i a n c e o f t u b e  assumptions  t h e v a r i a b i l i t y of  i s generally  of c o n j u g a t i o n tubes  tested  seem t o  I n T r e m e l l a , my e x p e r i e n c e  a reduced  O.k t o 0.5), and as might  lengths. 9).  i s desired,  shows a s t r o n g dependence  variation  assays  and have e n a b l e d t h e  Possibly  procedure  was t a k e n as i s a good d e s i g n ,  above, t h e s e  assay designs.  were  limits.  c o n c e n t r a t i o n i n many samples,  average  individual  This  However, when an a p p r o x i m a t e  fractions,  The  noted  to the  compound  lines  of confidence  satisfactorily  t h e complete  results.  adequate.  activity.  materials.  more c a r e f u l  reliable  is  Bu'Lock, D r a k e and  was low enough t o make u n n e c e s s a r y  shown t h a t  graphic  purpose  of the a c t i v e  the responses has  biological  the d e f i c i e n c i e s  served t h e i r  isolation  compared  compounds r e l a t e d  and t h e s l o p e o f t h e d o s e - r e s p o n s e  would  o f an  was  S e v e r a l c o n c e n t r a t i o n s of each  of r e l a t i v e  built  and t h e r e s p o n s e previously.  a measure  have  o n l y one dose  (1972) u s e d a more s o p h i s t i c a t e d p r o c e d u r e t o d e t e r -  Winstanley trisporic  that  Comparison  dose  o f F i g . 10 t o F i g . 8 shows  t r a n s f o r m a t i o n a c c e n t u a t e s t h e c u r v a t u r e of t h e dosec u r v e , b u t does n o t a l t e r  assays  shape.  i t i s c o n v e n i e n t t o have a f u n c t i o n  the response  logarithms  i t s basic  is linearly  o f t h e dose  i s such  related.  For b i o o f dose t o  F i g . 11 shows t h a t t h e  a function.  71 This jugation assay.  preliminary information tube  should  be  dilution was Four  the  of dose  series  serial  linear  dilutions  a dose r a n g e would be  of 27  on t h e  be  sustained  at  erogen  these  this.  at h i g h  ratio  1 to  curve h-0  3,  even  doses  serial tim.es.  covering  to ensure t h a t the curve  the  the  A  at l e a s t  of  an  on  dose-response  erogen  initiation,  f o r a longer point  to design  response  of  con-  responses  i f small  errors  doses.  earlier  This  The  p o r t i o n of t h e of  of  computations,  a dose r a n g e  time than  concentrations,  factors.  used  logarithmic scale.  were used  tubes  of  i n the  at a d i l u t i o n  choice  because  growth low  on t h e  times,  conjugation  longer  used  over  linear  were made i n t h e The  i s t o be  accomplishes  t o be  relation  r e g r e s s i o n of t r a n s f o r m e d  e q u a l l y spaced  found  the  l e n g t h t o hormone dose was  Since  logarithmn  about  faster the  or b e c a u s e  is discussed  concentration  could  growth,  or  conjugation  tubes  of a c o m b i n a t i o n  of  f u r t h e r i n Chapter  Four . The  tube  measurements than  simply  tube. the  length bioassay of t u b e  determining  In p r a c t i c e ,  labour  the  i s of t h e into  without assay  the  variability  one  fraction  type  called  of two  or a b s e n c e of t h e  conjugation  tube.  are  dead  and  alive.  has  been developed  of  cells  Quantal  but  probit transformation  lengths  conjugation is  conjugation  tube  or  i s important  where t h e  has  two  available.  logit  of t h e  fraction  of t h e  erogen  concentration  categories  data. tube  b e e n r e p l a c e d by  explicit  i n the  probit analysis  conjugation  because  logarithmn  of t u b e  and  cell  transformation, The  conjugation  lengths  (1971)' f o r q u a n t a l  h i s methods t o t h e T r e m e l l a an  cell  each  known as  applied  the  tube  because  data  insecticides,  Finney  per  of a  producing  "quantal",  A technique by  because  advantage.  categories--with  of f u n g i c i d e s and  the  first  more i n f o r m a t i o n  presence  theoretical  D a t a about t h e placed  give  developed  r e q u i r e d t o measure l a r g e numbers  have overcome t h i s tubes  length  was  formula  f o r the  responding  I have counts,  the  logistic  latter  is  plotted against  closely  follows  a  72 straight erogen  line  except  doses,  the  a conjugation  the  fraction  of  cells  highest  line  doses.  a t low  by  other  Dose-response the  Tremella  (Finney  1971)  I964).  Two  shape  An  the  sites  to the  assumed t o be this  of t h e  Simonis,  and  of t h e  cells  cells  dose,  react.  fraction i s the  the  sites  Langmuir  cells  explain  cell  has  are  adsorption  responding  on t h e  whose  will  shown t h a t be  a  linear  197l)The  to produce also  isotherm. number  a  frequency  theory.  occupied  the  cells  active material increases, which  Rossum  postulated to  i n order  p r o p o r t i o n a l to the  admin-  attach a  response.  the  increases  I f the  of r e c e p t o r  response sites  r e g r e s s i o n of  logarithmn  the  of dose  (Ariens,  Rossum I964). the  dose  threshold  i n each  to that cell  to a given  to  van  i t can be  (Finney are  of  here  I f the  receptor  the  in toxicology  and  responding  dose  At  because  only those  i s l o g normal,  than the  presented  model e a c h  model a l s o p r e d i c t s a l i n e a r  van  per  first  of t h e  of t h e  dose  model assumes t h a t t h e receptors)  doses  preparation.  ( A r i e n s , Simonis  a c t i v e molecules  fraction  effective  hormone  dose w i l l  of t h e  inside  Essentially, the  the  approach  of r e c e p t o r  occupied,  low  probably  one  cell  error in estimating  d e v i a t i o n from  t o the  a given  logarithmn  concentration  according  logit  at  threshold  biologically  fraction is  of t h e  (or l o g i t )  to receptor  similar  i t is  a particular  the  decreased  low  and  a cause f o r c o n c e r n .  of t h e  In t h e  and  alternative  istered  the  At  short,  of models have been p r o p o s e d  curves.  of t h e  i s not  curves  are  i s l a r g e r at  response  i s lower than  probit  function  As  dose  doses.  or not  reason,  Therefore  pharmacology  types  distribution  tubes  components  threshold,  threshold  formed  whether this  low  e r o g e n s have f r e q u e n t l y been f o u n d  and  of t h e s e  response  the  with  dose t e s t e d t h e  inhibition  for  For  and  tubes  to decide  tube.  at medium or h i g h straight  extreme h i g h  conjugation  sometimes d i f f i c u l t has  at  cell  distribution i s the  same, and  the  i s v a r i a b l e , whereas t h e  effective  dose  i s v a r i a b l e , and  effective  model assumes t h a t  dose  (number the  of  receptor  occupied  sensitivity  i s constant.  sensitivity  of  T h e s e two  the concepts  73 are  not mutually e x c l u s i v e - - v a r i a t i o n  receptors in a  per c e l l  t h e number  are  c o u l d he teamed w i t h an i n d e p e n d e n t  variation  of o c c u p i e d r e c e p t o r s per c e l l , .necessary t o produce  assays  greater than  i n which  deviations  attention.  linear,  the heterogeneity f a c t o r  the  increased  indicates In  When t h e b a s i c  cause  i n shaken  inadequate  mixing  now employed,  bottom, o f t h e t u b e s . ably  less  cells  in static  remains  into  account  tubes. solutions  When a s s a y s were  most  i n shaken  large  of the c e l l s  C o n j u g a t i o n tube than  into  per-  clumps,  Under t h e s t a t i c  incu-  settle  tothe  p r o d u c t i o n i s not n o t i c e -  media, b u t t h e c o n c e n t r a t i o n s  a t t h e bottom, o f t h e a s s a y t u b e  environment  t o take  of the assay  c o n j u g a t i o n tubes  a c c u r a t e c o u n t i n g was i m p o s s i b l e . conditions  relation  t o provide continuous mixing, the c e l l s  became e n t a n g l e d by t h e i r bation  require  among t h e a s s a y  of h e t e r o g e n e i t y .  flasks  lines  Significant heterogeneity  non-uniformity of c o n d i t i o n s  may be a major  and  dose-response  c a n be used  estimation error.  the T r e m e l l a assay,  formed  from the r e g r e s s i o n  can be e x p l a i n e d by random s a m p l i n g  special  to  of occupied  response. Those  of  i n t h e number  could  i n a n o n - r e p r o d u c i b l e way and t h u s  alter  the l o c a l  effect  the response  the erogens. The  second  case,  i n which  the responses  anomalously  low, l e a d i n g  regression,  o c c u r s most f r e q u e n t l y  relatively are  impure  assayed,  These  or when t h e doses  i n t h e crude  limited  by some f a c t o r  omit doses  adopted  i n two s i t u a t i o n s - - w h e n as c r u d e  the r e s u l t s  f r o m t h e one or two h i g h e s t  higher than the response  bias  the response i s  h e r e has been t o examine t h e d a t a , and t o  of data a f t e r  introducing  substances  o t h e r t h a n hormone c o n c e n t r a t i o n . The  of a p r e p a r a t i o n i f the responses  Rejection  supernatant  by i n h i b i t o r y  p r e p a r a t i o n s , or because  from the a n a l y s i s  substantially  culture  o f t h e unknown a r e t o o h i g h .  a r e p r o b a b l y caused  present  procedure  t o c u r v a t u r e of the dose-response  p r e p a r a t i o n s such  low r e s p o n s e s  a t h i g h doses a r e  into  examination  the r e s u l t s ,  t o t h e s e doses at t h e next  carries  are not  lower  dose.  t h e danger o f  but the a l t e r n a t i v e s are  74 even  less  would  appealing.  produce  conform  To  a l l t h e doses  misleading results,  t o t h e assumed  a s s a y would  include  waste t h e  linear  "because  analysis  t h e d a t a would  regression.  information  i n the  To r e j e c t  available  not  the  from the  entire  lower  doses. The  d e s i g n of assays based  discussed doses  by F i n n e y  (1964).  of e a c h p r e p a r a t i o n ,  parallelism  He  recommends  to allow tests  of the dose-response  erogen b i o a s s a y , f o u r providing  on q u a n t a l r e s p o n s e s has  an  extra  doses  independent  For  the r e s u l t s  an a s s a y w i t h f o u r  doses  mends c h o o s i n g t h e doses ing  a r e about  cision.  0.15,  Because,  from  as m e n t i o n e d  of c e l l s  rates,  somewhat h i g h e r doses o f l/2 between  Crude contain tions  at l e a s t  or  convex,  similar types  and  the e r r o r  doses  The  response  gives  encountered. the proper s p a c i n g .  active  2259 7 -  Dose-response materials  dose-response  rela-  have b e e n  curves  observed  2259 7 have always been l i n e a r -  a r e showing  "simple  a l l t h r e e components  through the  same mechanism.  of i n t e r a c t i o n s  between t h e  components  curves which  pre-  i n the T r e m e l l a b i o a s s a y ,  components  In o t h e r words,  respond-  i n c o u n t i n g the  cells  dose-response  doses.  F i n n e y recom-  i s h i g h a t low  components.  the  severity  to give highest  are not n o r m a l l y  of b i o l o g i c a l l y  suggesting that the  sensitivity  of t h e h i g h e r  O.85  above,  successive  (1971)-  by  the expected f r a c t i o n s  p r e p a r a t i o n s from  action".  stimulating  or two  were used  three active  by F i n n e y  the erogen  the  dose,  p r e p a r a t i o n s f r o m T_. m e s e n t e r i c a  f o r mixtures  described for  erogen  fourth  a l s o reduces the  with c o n j u g a t i o n tubes  r e s p o n s e r a t e s b e l o w 0.25  A ratio  The  and  For the T r e m e l l a  of each p r e p a r a t i o n ,  O.65,  number and  one  so t h a t  O.35,  of l i n e a r i t y  datum,, r e d u c e s  of t h e a s s a y t o h e t e r o g e n e i t y , and of r e j e c t i n g  a minimum of t h r e e  regressions.  were u s e d .  been  are concave  are  More  should lead  (Finney  1971)•  complex to  CHAPTER FOUR THE RESPONSE TO THE EROGENS  75 Materials I.  Time  and Methods  course  Two  5-0  Erlenmeyer units/ml, After five  of c o n j u g a t i o n tube  ml portions  flasks  were  drops  sample  each  drop  from  number  sample  of cells  Another  drop  with  slide.  tubes  were measured  II. of  preparation.  were  inoculated  sample i n each  with  flask,  1 4 , a n d 24 h o u r s .  and s t a i n e d  each One  i n a haemacytometer,  was d r i e d  A  and t h e f l a s k s  Samples were t a k e n f r o m  was m o u n t e d  and t h e  was c o u n t e d . on a m i c r o -  selected conjugation  of the stained  slides  f r o m t h e 8,  samples.  o f number  supernatant from  of Porapak  washed  eluted  with  with  concentrated then  of conjugation tubes one l i t r e  Q at a flow rate  per c e l l  of a stationary  100 m l o f 50$ i n vacuo  diluted  i n 1.0  until  phase  culture  dilutions  for a bioassay.  cells  a 0.5$  solution 1/2,  secondary  slides  Comparison  pH 5-5>  l / 4 , l / 8 , l / l 6 , l/32,  seventeen  were p r e p a r e d  p o w d e r was  T h i s s o l u t i o n was  soytone  autoclaved, inoculated After  dilutions.  T h e e l u a t e was  of soytone,  i n 0-5$  were  had e v a p o r a t e d , and  water.  producing conjugation tubes  stained  and t h e erogens  acetone.  t h e acetone  dilutions  were  water,  The Porapak  One m.g o f t h e f r e e z e - d r i e d  primary diltuion  l/l28 s e c o n d a r y  These  aqueous  ml of d i s t i l l e d  1 t o 50 w i t h  from t h i s  o f 300 m l / h o u r .  100 m l o f d i s t i l l e d  was f r e e z e - d r i e d .  dissolved  and  each  a n d 0.2  2259-7 i n G l u c - A m S m e d i u m was p a s s e d t h r o u g h a b e d o f 10  was  as  unit/ml  o f 2259-6 i n GS.  T h e l e n g t h s o f 35 r a n d o m l y  Distribution  grams  and  a t 20°C.  l 4 , a n d 24 h o u r  The  from  i n 25 m l  and w i t h o u t c o n j u g a t i o n tubes  of each  scope  10, 12,  the flasks  2, 4, 5, 6, 1, 8, 10, 12,  each  pH 5-0, 1.0  of a two day o l d c u l t u r e  was t a k e n a s e p t i c a l l y after  with  and growth  of t h e standard erogen  and c o o l i n g ,  were p l a c e d on a s h a k e r flask  soytone,  supplemented  respectively,  autoclaving  o f 0-5$  initiation  were  and  l/64,  prepared.  and i n c u b a t e d  hours, t h e f r a c t i o n of  a t each  dilution  from t h e l/2, with  was m e a s u r e d ,  l / 8 , a n d l/32  a standard  dilution  76 series of  assayed  20.5  simultaneously indicated  u n i t s / m l i n the primary d i l u t i o n .  a p p r o x i m a t e l y 200 numbers  w i t h 0,  cells  1, 2,  III.  of temperature  Five w i t h 1.0  test unit  w i t h 0.25  tubes  u n i t s / m l were at  containing  i n c u b a t e d a t each  were a l s o  6.0,  with  and 8.0  p e r ml and f i v e After  5-0,  tubes  each  tube  0.25  20°C, 25°C and 28°C.  with c o n j u g a t i o n tubes determined.  N hydrochloric  w i t h 0.1  Stained  randomly measured.  a t 20°C.  w i t h 1.0  or 0.25  and a u t o c l a v e d . After  concentration  ml o f 0.5$  the  s t a n d a r d erogen  six  test  tubes.  soytone,  pH 5-5,  the t o t a l  -  i  GS, and  n  of c e l l s  Sterile ml.  GS was  with  determined.  1.0  one t u b e was  w i t h 0.02,  volume t o 1.1  inoculated  unit  p e r ml were d i s t r i b u t e d  autoclaving,  inoculated  ml o f t h e c u l t u r e .  of 2259 6>  containing  w i t h 0.01 ml o f a t h r e e day o l d c u l t u r e o t h e r t u b e s were  or p o t a s s i u m  of t h e s t a n d a r d  The t u b e s were  t u b e was  preparation  After  units  17 h o u r s , t h e f r a c t i o n  V. E f f e c t  of c e l l  acid  4.0,  of t h e medium, a t each pH i n t e s t  i n each t e s t  bring  15°C,  of the c e l l s  c o n j u g a t i o n tubes  0.10  autoclaving  o f 10°C,  1 d r o p f r o m a one day o l d c u l t u r e  Six  pH  similar  f r o m each t r e a t m e n t were  One ml p o r t i o n s  preparation,  incubated  soytone,  s o y t o n e were a d j u s t e d t o pH 3-0,  t u b e s were s u p p l e m e n t e d erogen  ml of 0.5$  p r e p a r e d , and t h e l e n g t h s of 25  o f 0.5$  7-0,  hydroxide.  points  o f pH  Aliquots 5.0,  growing  counted.  c o m b i n a t i o n was  c o n j u g a t i o n tubes  IV. E f f e c t  and t h e  One t u b e w i t h 1 u n i t / m l and one t u b e w i t h  each d o s e - t e m p e r a t u r e  selected  slides,  w i t h one d r o p f r o m a one day o l d c u l t u r e o f  18 h o u r s , t h e f r a c t i o n  slides  1.0  u n i t s / m l were p r e p a r e d .  2259-6 i n GS. After  were  of s t a n d a r d erogen  inoculated  On t h e s t a i n e d  3> • • • > 8 c o n j u g a t i o n t u b e  f r o m one y e a s t c e l l  Effect  concentration  were examined a t each dose,  originating  was  an e r o g e n  of 2259-6 0.04,  0.06,  of into  inoculated i n GS. 0.08  The and  added t o each t u b e t o  The t u b e s were  incubated at  77 20°C f o r 18 of  cells  test  hours.  The  were  counted  were p r e p a r e d , and  VI.  were  Effect  solution sulphate soytone with  gm  litre  was  directly  per  and  0.13  litre.  The  pH  ml  e a c h medium a t c o n c e n t r a t i o n s o f 1.0 2 ml p o r t i o n s were p i p e t t e d  p r e p a r e d by  centrifuging,  f r o m a two  cells  w i t h a volume o f s t e r i l e  volume  of s t e r i l e  s u s p e n s i o n was incubated  old culture  volume,  and  tubes  hours.  tube, Samples  were mounted  i n the haemacytometer,  g a t i o n tubes  and  were  counted.  without  and  drop and  c o n j u g a t i o n tubes  units/ml.  concenInoculum,  tube,  washing  the  the  equal to the cells  i n another  o f t h e washed the tubes  from the  aliquots  0.25  centrifuge  resuspending the One  to  erogen  5-5  sample  autoclaved.  d i s t i l l e d water  t o each t e s t  a t 20°C f o r 18  and  A  added  of 2259-6 i n GS,  d i s t i l l e d water.  added  was  u n i t / m l and  in a sterile  cells  culture  day  from Porapak  test  medium  adjusted to  o f each medium a t b o t h into  con-  stock  basal  or p o t a s s i u m h y d r o x i d e .  eluted  but  h y d r o l y s a t e or  of e a c h medium, was  of  original  i n each  gram of ammonium,  grams of c a s e i n  acid  One,  of t h i s  w i t h one  c o n j u g a t i o n hormones  was  slides  of microelement  Aliquots  or s u p p l e m e n t e d or f i v e  of Chapter  of  trations  Stained  each  c o n j u g a t i o n tubes  t o t h e BM  prepared.  N hydrochloric  Duplicate  number  source  of D - g l u c o s e ,  per l i t r e ,  0.1  the  measured.  per  were used  and  i n a known volume f r o m  t h e l e n g t h s o f 25  of n i t r o g e n  10  of c e l l s ,  i n a haemacytometer.  A b a s a l medium s i m i l a r taining  number  with c o n j u g a t i o n tubes  tube  sample  total  were  each t e s t  cells i n 400  cell  tube  with conjusmall  squares  78 Results  I . Time c o u r s e The is  of c o n j u g a t i o n  fraction  plotted  tube  of c e l l s w i t h  against  time  t h e 8,  10,  of  the frequency  presented Fig  12,  II.  distributions  Distribution  erogen  units,  samples  of tube  o f number  frequency  per c e l l  a r e shown i n F i g . 17-  tubes  per c e l l  i s plotted  III.  was  Effect The  average  significant  conjugation  tube  temperature  IV. E f f e c t  o f pH  in  fraction  of erogen  lengths  a similar  in  each t e s t  plotted  against  of  cell  conjugation  The a v e r a g e number o f i n F i g . 18.  The  level.  tubes,  and t h e against  respectively.  tubes  t h e pH i n F i g . 21. a t each pH  of c e l l s  at each The  varied  responding.  concentration t h e counts along  Confidence  with  of t o t a l c e l l s , t h e volumes  limits  and t h e a v e r a g e  with  i n which t h e c e l l s  by a s s u m i n g  counts.  against the t o t a l c e l l  and c e l l s  f o r the concentration  t u b e were c a l c u l a t e d  tubes,  tubes,  conjugation  tubes produced  f o r the t o t a l c e l l  conjugation  per  a t each dose a r e p l o t t e d  manner t o t h e f r a c t i o n  counted.  tribution  tubes  e r o g e n dose  of c e l l s p r o d u c i n g  VI l i s t s  conjugation were  u.  i n F i g . 19 and F i g . 20  i s plotted  of c e l l  Table  and i n  Tube l e n g t h s a r e  conjugation  length  of t h e c o n j u g a t i o n  V. E f f e c t  tubes  Histograms  concentration,  1.^6  a t t h e 10$  of c e l l s with  incubation  dose  concentrations.  of temperature  fraction  The  against  tubes  a t each t i m e a r e  o f t h e number  tubes  regression  erogen  length  of c o n j u g a t i o n  distributions  conjugation  a r e shown.  concentration.  which e q u a l  and g r o w t h  of t h e c o n j u g a t i o n  i n F i g . 15 f o r t h e low e r o g e n  i n eyepiece  The  length  14 and 24 hour  16 f o r t h e h i g h  given  recognizable  i n F i g . 14 f o r b o t h  On t h e same g r a p h , t h e a v e r a g e in  initiation  a Poisson  The f r a c t i o n  conjugation  concentration  of c e l l s dis-  of c e l l s  tube  in Fig.  with  length are 22.  79  •  (nd3)  H13N31 • 3ETLL • NDI1VE3TFNCD • •  SBETll NDIIVDTTMT) Fig. in 0.2  HUM STT3D  JO NDIlDVyj  Ik . Time c o u r s e o f c o n j u g a t i o n tu"be i n i t i a t i o n  response unit/ml.  t o erogen.  Upper  curves 1 . 0 unit/ml,  Bars r e p r e s e n t 9 5 $ c o n f i d e n c e  NVBN •  and  growth  lower  curves  intervals.  80  0-4  8  hours  10  hours  o . a .  0-3.  0*B 0-1 -I  to  >-  !• 0»5i.  3«  5*  12  |  4=1-  |_JQ»Q  7-  9* 0«  !•  3*  5*  hours  7«  +  9«  14 h o u r s  0-3.  0-0.  H  bo.  1—i-Q-Q  0-4.. o - a .  24 h o u r s  o-i  i i i i n  O'Q  1. Fig.  3-  times  containing  0.2  7-  9-  i  i  i  11. 13. 15. 17- 19.  CONJUGATION TUBE LENGTH (EPU)  15. F r e q u e n c y  various  5-  i  distributions  after units  inoculation of erogens  of c o n j u g a t i o n of  2259-6  p e r ml.  cells  tube  lengths at  into  media  81  0-! 0-4.  8 hours  10  hours  0.3L. o.«  o.ii 0-0. CSi.  _Q.Q  i«  3.  0*4.  5*  7*  9*  o-r  1.  3«  5*  0.4.  12 h o u r s  7*  9*  14 h o u r s  0-3. M  0-1  0-1  0*0.  -Q.Q  o»:  24 h o u r s  IFig.  n. 3*  times a f t e r  containing  1.0  4=f 5-  7«  9*  Ch  11« 13- 15. 17- 19.  CONJUGATION TUBE LENGTH (EPU)  16. F r e q u e n c y  various  n  CH  unit  distributions inoculation of erogens  of c o n j u g a t i o n  tube  lengths at  o f 225-9-6 c e l l s  into  media  p e r ml.  82  2.6  units/ml  5.1  units/ml  LU >  NUMBER OF CONJUGATION TUBES^PER CELL Fig. tubes  17.  Frequency d i s t r i b u t i o n  per c e l l ,  at three  of  t h e number o f c o n j u g a t i o n  erogen c o n c e n t r a t i o n s .  83  0*  S>  4.  6»  B«  10.  12.  CONCENTRATION OF EROGENS (UNITS/ML)  Fig. per  18. R e g r e s s i o n cell  o f a v e r a g e number  on t h e e r o g e n  concentration.  of c o n j u g a t i o n  tubes  Qk  l»Qi.  o . a .  0*4.  0-0.  + 15'  +  10.  +  +  3D.  30-  i  TEMPERATURE (DEG- C)  Fig.  19- The f r a c t i o n  concentrations 1.0  as a f u n c t i o n  u n i t / m l ; lower  confidence  of c e l l s  curve  intervals.  0.25  responding  t o t h e erogens  of temperature. units/ml.  Bars  Upper  a t two  curve—  represent  85  B»..  6-..  H  A : .  +  +  +  10.  15*  +  SO.  TEMPERATURE (DEG- C)  Fig.  20. The a v e r a g e  response  of c o n j u g a t i o n  to the concentrations  temperature. unit/ml.  length  Bars  Upper  curve—1.0  represent  95$  of erogens unit/ml;  confidence  tubes  produced i n  as a f u n c t i o n o f  lower  curve—0.25  intervals.  30.  86  1-0+  Fig. two  21.  The f r a c t i o n  concentrations  unit/ml;  lower  confidence  of c e l l s  responding  as a f u n c t i o n of pH.  curve--0.25  intervals.  units/ml.  t o the erogens at Upper  Bars  curve—1.0  represent  95$  Fig. and  22.  Dependence  average  length  of f r a c t i o n of  of  conjugation  cells tubes  with on  conjugation  concentration  tubes, of  cells.  88 T a b l e V I . C e l l counts from c e l l concentration. Volume o f inoculum i n ml  Number o f c e l l s with conjugation tubes  0 .01 0 .02  T o t a l number of c e l l s  0.06 0.08 0 .10 VI. E f f e c t  of n i t r o g e n  numbers o f c e l l s  numbers  of c e l l s  nitrogen  source  on t h e e f f e c t o f Volume i n w h i c h c e l l s were c o u n t e d nanolitres  186 324 331 622 458 373  162 275 201 299 208 176  0.0k  The  the experiment  100 100 40 40 40 20  source with  counted  conjugation tubes,  i n each  combinations  replicate  are given  and t h e t o t a l  of t h e erogen  dose-  i n Table V I I .  T a b l e V I I . The number o f c e l l s w i t h c o n j u g a t i o n t u b e s / t h e t o t a l number o f c e l l s c o u n t e d a t two doses o f e r o g e n w i t h v a r i o u s nitrogen sources. Nitrogen source  0.25 u n i t s o f e r o g e n /ml  None Ammonium s u l p h a t e Casein hydrolysate Soytone  22/851, 34/910 136/445, 133/670 350/851, 252/610  The  fractions  square  roots  O/28O,  as a 4 X 2 f a c t o r i a l analysis The  0/500  were t r a n s f o r m e d  ( S t e e l and T o r r i e experiment  1.0 u n i t o f e r o g e n /ml  i960) and t h e d a t a was a n a l y z e d with 2 r e p l i c a t i o n s .  of v a r i a n c e i s p r e s e n t e d  sources  tubes  with  soytone  was s i g n i f i c a n t  0/400  t o the a r c s i n e s of t h e i r The  i n Table VIII.  c o n t r a s t between t h e f r a c t i o n  gation  0/485,  43/300, 68/680 260/274, 172/330 359/443, 312/400  of c e l l s  producing  conju-  and c a s e i n h y d r o l y s a t e as n i t r o g e n  a t t h e 2.5$  level.  8 Table  9  V I I I . A n a l y s i s of v a r i a n c e f o r e f f e c t on c o n j u g a t i o n t u b e p r o d u c t i o n .  Source of variation  Sum o f squares  Main e f f e c t of n i t r o g e n source  1.942  Main e f f e c t of e r o g e n concentration  O.278I  Interact ion Error  Degrees of freedom.  Mean s guar e  of n i t r o g e n F  source  Probability a larger F  0.6474  84.7  0.000003  1  0.2781  36.4  0.0003  0.1013  3  0.03378  0 .06112  8  0 .00764  4.42  0.041  of  90 Discussion The  effects  of some e n v i r o n m e n t a l  v a r i a b l e s on  of  c o n j u g a t i o n tubes  i n response  to  determine  c o n d i t i o n s f o r the  First,  optimum the  kinetics  of  to  the  erogens.  a l a g of tubes  observed  erogens  increased  more t h a n  initiated.  per  ml,  the  rapidly  erogens  tubes  per  rose  the  than  the  a t Ik  formation  original  ml,  the  yeast  After of  or by  are  tube  switched  growing  cells sion times in  of t h e  buds a t t h e  i n c u b a t i o n times  erogen  conjugation apices longer  concentrations. use  up  of t h e  the  male m y c e l i a  d a l e 1963a).  (1968) has growing  tubes.  Bandoni  18  this  tubes  view.  from t h e i r  of A c h l y a  from  the  tube conju-  to maintain  conju-  occurs  tubes  at  the  I have f r e q u e n t l y tubes, and  especially  a t low  initial  antheridiol  the  Rever-  incubation  phenomenon has and  that  respond.  at l o n g  medium ( C a r l i l e up  by  shown t h a t  sexual hormones—sperm  take  caused  (1965) has s u g g e s t e d  A similar  conis  conjugation  hours,  to budding  units  tubes  conjugation  conjugation  than  fungi reacting to  remove s i r e n i n  of  erogen  budding  c o n j u g a t i o n hormones as t h e y  conjugation  supports  other  by  continuously present with  then  conjugation  i s probably  r e v e r s i o n of  i f cells  of  tubes  and  conjugation  t o h o r m o n e - f r e e medium, b u d d i n g  point  observed at  growth;  with  with  at b o t h  either  Flegel  conjugation  cells  decrease  cells,  unit  medium c o n t a i n i n g 0.2  hours,  cells  This  yeast  2k  suggests  conjugation  inoculation,  p r o p o r t i o n of  g a t i o n hormones must be  any  medium c o n t a i n i n g 1.0  In t h e  tubes  i n F i g . 13  after  hours.  cells,  curves  and  2259-6 c e l l s  of t h e  7 hours  growing p o i n t s t o budding. gation  exposure  with  fraction  of new  conjugation  cells  hours.  examined  initiation  of  more s l o w l y .  centrations, lower  fraction  Ik  tube  3 hours b e f o r e  In t h e  until  more s l o w l y u n t i l of  after  were  bioassay.  Recognizable  E x t r a p o l a t i o n of t h e  slightly  were  hours  k  erogens  conjugation  g r o w t h a t 20°C were s t u d i e d . were f i r s t  to the  production  been  of  Machlis  found  Allomyces  I965)- and  irreversibly  (Barks-  91 In F l e g e l ' s hormone p u l s i n g e x p e r i m e n t t u b e s were f o u n d hormone.  2 hours  of was  study  He a l s o c a r r i e d  of c o n j u g a t i o n  2259-6 and 2259-7initiated  a constriction  cell. but as  a bud, and l a t e r  of  erogens  At  0.2  1.5  units/ml  least  t o longer  initiated  lengths,  tubes  by 14 h o u r s , average  length  at h i g h  than  average  growth r a t e  concentration. to  24 h o u r s ,  gation  established no  longer  average at  being  length  very  with  is faster length  0.2  hormone  tubes  still  time.  These  data  i s at  few r e c e n t units/ml,  concentration.  suggesting at h i g h  that the  hormone with  of tube  or  conju-  growth of  new t u b e s  do n o t t e l l  a r e some t u b e s  faster  f r o m 14 h o u r s  of c e l l s  i n c r e a s i n g a t 24 h o u r s ,  are very broad--there  the  progresses,  increases  while  The d i s t r i b u t i o n s  few o f t h e  is similar  increases  continued  than  concentrations  lengths  concentration,  tubes  initiated. was  are s h o r t e r  does n o t n e c e s s a r i l y mean t h a t  conjugation  some e a r l i e r  24 h o u r s  of tube  at the higher  of the tubes  p e r ml.  By 12 h o u r s ,  i n t h e medium  The a v e r a g e t u b e This  unit  at 1 u n i t  As t i m e  hormone  i n contrast to the f r a c t i o n  tubes.  length  e r o g e n dose  initiation.  initiated  tube.  Therefore,  at high  of t h e c o n j u g a t i o n  a t low hormone  tube  length range.  the d i s t r i b u t i o n  distribution  s t r u c t u r e was  as a t 0.2  and b r o a d e n .  are found  o f buds,  a conjugation  f o r both  e a r l y tube  t o the yeast  of the tubes  of t h e tubes  distributions  t o t h e 8 hour The  length  cultures recorded  This  but at 1 u n i t / m l  c a u s e d by e a r l i e r  frequency  shift and  partly  into  a large fraction  average  3 hours.  is characteristic  as g r e a t  are i n t h i s  tube  photo-  o f t h e two s t r a i n s , b u t  conjugation  epu ( r e c e n t l y i n i t i a t e d ) ,  greater  ly  the average  tubes  conjugation  Perhaps t h i s  transformed  p e r ml i s t w i c e  conjugation  the  tubes.  lapse  i n mixed  of a t t a c h m e n t  constriction  to the  or t h e c o n c e n t r a t i o n  production  mixing  conjugation  of t h e c e l l s  out a t i m e  at a tube b e f o r e  not of c o n j u g a t i o n  8 hours,  after  at i t s p o i n t  Such a b a s a l  By  tube  The e a r l i e s t  $0 m i n u t e s  was n o t r e c o g n i z a b l e has  exposure  He does n o t s p e c i f y t h e s t r a i n ,  of t h e hormone u s e d . graphic  after  (1968),  were  whether t h e stopped  lengths at  as s h o r t  as 2 epu,  92 and  as  long  as  1 unit/ml. time,  the  of t h e  of  The  at  units/ml, probably  long tubes  some i n t e r m e d i a t e time,  average  l e n g t h of  suggest  that  The the  nized,  conjugation  but  cells.  a l l cells not  so  long  Incubation  times  data  epu  at an  growing u n t i l  at  early  the  end  stopped  presented  here  s e p a r a t i o n of t h e  growth s t o p p i n g  are  effects  time  on  the  a t v a r i o u s hormone d o s e s ,  but  important.  going long  allow  i s long  enough t o be accumulation  between 12  enough t o  t o r e a c t have done so,  and  18  easily of new  hours  and  recogyeast  should  response  time  for Tremella  i s longer  are  read  after  kO  where b i o a s s a y s  are  read  after  2 hours  (Barksdale  after  hours  Mucor where b i o a s s a y s As  the  read  on t h e  tubes  are  produced,  These  effects  s u r f a c e of t h e and  c a u s e an  the  meet  r e g r e s s i o n of a v e r a g e number  points  per  tested  ( F i g . 18)  cell  This  on  possibility  has  Among t h e  temperatures  produced  conjugation  cells good.  However, t h e those  20°C was Longer  formed  selected  as t h e  i n c u b a t i o n might  this  would not  ( F i g . 17)-  suggests  not  been  be  at  standard  of p r a c t i c a l  with  formed  conju-  tube  the  growing  range  bioassay  fraction  temperatures.  a higher  interest  of  20°C a l m o s t  a t 20°C were  temperature  have a l l o w e d  of  linearity  a third  largest  a t 15°C, other  conjugation  investigated.  t e s t e d , the  and  over  number  increase.  The  of c o n j u g a t i o n  c o n j u g a t i o n tubes  a t 15°C  tubes,  and  1963)-  a v e r a g e number  and  tubes  1963a);  at which  c o n c e n t r a t i o n of e r o g e n s  is striking,  Allomyces,  1958a), A c h l y a ,  (Plempel  of t h e  i n c r e a s e i n the cell  (Machlis  cells  branching  g r o w i n g p o i n t s per  for  i s i n c r e a s e d , the  yeast  of the  method.  k  c o n c e n t r a t i o n of e r o g e n s  of p o i n t s  g a t i o n tube  are  minutes  than  where b i o a s s a y s  but  18  criteria. The  than  as  growing  for a bioassay  are  as t o  and  are  which are tubes  The  tubes  i n c u b a t i o n time  long  medium l e n g t h t u b e s  a clear  factors  as  stopped  time.  growth r a t e ,  a l l three  best  ensure t h a t  the  conjugation  and  continued  enough t o p e r m i t  initiation  these  at 0.2  short tubes  extremely  extensive  that  epu  i n c u b a t i o n p e r i o d , and  growing not  13  longer'  Therefore  f o r the response  f o r the  as  bioassay. at  10°C,  bioassay.  93 At  an e r o g e n dose  responding but  decreased  dose at  was a l m o s t  of 1 u n i t / m l , constant  a t pH's 3 and 8.  the response  pH' s t e s t e d .  of c e l l s  t h e pH r a n g e f r o m 5 t o 7,  a t pH k, and was z e r o  o f 0.25 u n i t s / m l ,  other  over  the f r a c t i o n  At a  a t pH 6 was h i g h e r  than  A pH o f 5-5 was s e l e c t e d f o r t h e b i o -  (1968) f o u n d maximum c o n j u g a t i o n t u b e f o r m a t i o n i n mixed c u l t u r e s o f 2259-6 and 2259-7 a t pH 4.7, no t u b e s  assay. at  Flegel  pH 7-2, b u t some s h o r t b r a n c h e d  different of  b u f f e r systems  pH a r e c o n f o u n d e d  buffer tides  salts.  the  at d i f f e r e n t  with  In the present  study,  so t h a t t h e e f f e c t s effects  of the  t h e amino a c i d s  and pepThe d i s -  1  between F l e g e l ' s r e s u l t s  c u l t u r e s and pure  p H s,  He u s e d  as b u f f e r s a t a l l p H s.  d i f f e r e n c e i n b u f f e r systems,  mixed  a t pH 8. 1  possible specific  o f s o y t o n e were used  crepancies  tubes  and mine may be c a u s e d by  o r by t h e d i f f e r e n c e between  c u l t u r e s with  added p a r t i a l l y  purified  erogens. At  high  of  the c e l l s  er  than  possible produce  concentrations produce  a t lower  the  cell  receive less of  share  per hundred  source  casein hydrolysate  s o y t o n e medium.  effect.  The  the c e l l s  cell  are short-  2259-6 c e l l s of t h i s  could  could  inhibitor  and t h u s  decrease  compete f o r t h e  concentrations,  tubes  fraction  a r e a t l e a s t two  concentrations,  each  cell  and t h e r e f o r e  i s designed  would respond  t h e amount o f t o give  a density  picolitres. i s included  a r e formed.  utilization  Conjugation  i n t h e assay  medium,  Ammonium s u l p h a t e  for conjugation  c o n t r a s t t o i t s ready by 2259-7-  There  n e a r maximal r e s p o n s e  source  tubes  and t h e t u b e s  o f t h e e r o g e n dose,  To a l l o w  conjugation  production in  At high  no n i t r o g e n  poor n i t r o g e n in  cell  added t o t h e a s s a y  250 c e l l s If  no  a smaller  a smaller  The c o n c e n t r a t i o n  Alternatively,  strongly.  inoculum  for this  at high  erogen molecules.  tubes,  concentrations.  a self-inhibitor.  response.  2259-6 c e l l s ,  conjugation  explanations  would be h i g h e r  of  is a  tube p r o d u c t i o n f o r growth  and e r o g e n  tube p r o d u c t i o n  medium, and s i g n i f i c a n t l y  I t appears t h e r e f o r e t h a t  b y 2259-6,  i s better  greater i n  amino a c i d s  stimulate  9  the  response  casein  t o the erogens.  hydrolysate  may a r i s e  k  The s u p e r i o r i t y  of soytone  f r o m more f a v o u r a b l e  to  proportions  of amino a c i d s , or from, some o t h e r  stimulatory substances.  importance  that protein synthesis i s  involved that  i n the response  cycloheximide,  fungi,  which  d i d not i n h i b i t  slightly that  o f amino a c i d s s u g g e s t s  decreased  inhibits  conjugation  i n some  tube p r o d u c t i o n .  I t only  erogens,  have p r e v e n t e d  w h i c h might  (1968). f o u n d  protein synthesis  i t was n o t p e n e t r a t i n g t h e c e l l s  factors  Flegel  t h e growth r a t e of t h e c e l l s ,  Time l i m i t a t i o n s other  t o the erogens.  The  very  effectively.  investigation  i n f l u e n c e the response  and a l s o o f t h e i n t e r a c t i o n s  suggesting  o f many  t o the  between v a r i o u s  factors.  CHAPTER  FIVE  PRODUCTION OF THE EROGENS  95 Materials I.  Time  and  Methods  course of erogen  For  a pilot  production  s t u d y of the k i n e t i c s of 2259-7 c u l t u r e s ,  d u r i n g t h e growth  Gluc-AmS medium i n 1 l i t r e on a s h a k e r a t 20°C.  imately and  a t 640  after  previous fuged  o f 12  n a t i o n a l Equipment ml p o r t i o n s  at f u l l Co.,  Model  o f e a c h sample  o f 10$  s o y t o n e , and  potassium hydroxide.  continued  until  pH  5-5>  until The  120  after  Sampling  Three  pH  and  samples 24,  36  immediately a f t e r after  turbidity  tration.  48 The  hours  1 litre  (Inter-  w i t h 0.1  l / 2 , l / 4 , and of t h e  W  l/8 bio-  cultures  was  flasks  containing  ml e a c h of a  The  were  flasks  ml samples and  were t a k e n  a t 12  hour and  168  were measured,  and  the  The  samples original  h o u r s were d i l u t e d  from  intervals  t h e n a t 144  and  were b i o a s s a y e d a t t h e i r t a k e n a t 58  plan  w i t h 3-0  inoculation,  inoculation,  used t o  Erlenmeyer  Five  of t h e samples  samples  centri-  centrifuge  t o 5-5  s t u d y was  p r e p a r e d f o r b i o a s s a y as above. and  were  inoculation.  from the p i l o t  on a s h a k e r at 20°C. hours  from the  were p r e p a r e d , and t h e  7 day o l d c u l t u r e of 2259-7 i n Gluc-AmS. each f l a s k  taken  stored  morning  adjusted  ml e a c h o f Gluc-AmS were i n o c u l a t e d  incubated  Samples were  to  was  samples  t o sediment  their  out as u s u a l .  200  water  with  carried  experiment.  t h e thawed  Bausch  s u p e r n a t a n t were s u p p l e m e n t e d  a s s a y was  a second  in a  sample  model 28.  From t h e s e samples,  hours  The  Two  s o y t o n e , pH  information  of each  approx-  the c e l l s .  i n 0-5$  The  pH  ml  incu-  of  with d i s t i l l e d  in a clinical  HN)  dilutions  168  determined  from that  speed  were  samples  inoculation  day,  e v e n i n g , and t h e samples  f o r 5 minutes  ml  The  Each  flasks  hours,  was  after  w i t h 2.0  from each f l a s k .  pH meter  hours  in a freezer.  The  dilution  0-5-  a Radiometer  odd m u l t i p l e s  overnight  0.1  20  t h e a b s o r b a n c e below  measured u s i n g at  E v e r y 12  nm. of t h e samples  Lomb S p e c t r o n i c  keep  were i n o c u l a t e d  5 ml were t a k e n a s e p t i c a l l y  turbidity  production  200 ml p o r t i o n s of  two  of 2259-7 i n GS.  f r o m a 4-day c u l t u r e bated  flasks  of e r o g e n  hours.  taken at concen1 to  2;  96 the  t a k e n a t 72  samples  96 hour 168  samples  hour  before  hours 1  were d i l u t e d  samples  were d i l u t e d  were d i l u t e d  t o 8; 1  t o 4;  the 1 0 8 ,  and  t o 16  1  with 0 . 5 $  120,  determine  the r a t i o 2259 7  dry weight,  between t u r b i d i t y  Gluc-AmS were washed t h r e e t i m e s w i t h d i s t i l l e d 1  diluted  _  in distilled F o r t y ml  a d r y , weighed  an oven  iccator  Petri  at 105°C,  Effect  and  of n i t r o g e n  1.33 g  cooled  with 1  bated  m  minutes  1.0  ml  III.  o f a 4 day f o r 10  i n a des-  section  minutes  at f u l l  of  acetate trihydrate  casein hydrolysate,  Each  flask  of 2 2 5 9 - 7  old culture After  gm  VI  of  or  each  were p r e p a r e d and a u t o c l a v e d  cycle.  at 20°C.  o f medium  4 times t h e i r  seven  days  speed  Co.,  i n GS,  inoculated and  incu-  the c u l t u r e s  in a c l i n i c a l  M o d e l HN,  diluted  was  centri-  angle head).  1 to 4 with 0-5$  were The  soytone,  concentration  containing  normal  regular  part  of t h i s  Gluc-AmS.  the  components  c o n c e n t r a t i o n was  w i t h an e q u a l volume  medium, and give  pipetted  and b i o a s s a y e d .  Effect  diluted  suspension  dried overnight  50 ml r e p l i c a t e s  flasks  were d e c a n t e d ,  A medium ( 4 x ) at  d i s h was  and  i t s turbidity  t o room t e m p e r a t u r e  Three  ( i n t e r n a t i o n a l Equipment  5-5,  The  salt-free  on a d r y goods  on a s h a k e r  supernatants pH  of t h i s  s u s p e n s i o n were  2 grams o f sodium  of v i t a m i n - f r e e ,  centrifuged fuge  and  culture  water,  of ammonium s u l p h a t e , or 1  gm  medium i n 2 5 0 ml E r l e n m e y e r with  water,  of the  dish.  of s o y t o n e p e r l i t r e . 15  A sample  phase  s o u r c e on e r o g e n p r o d u c t i o n  ammonium s u l p h a t e p l u s  for  water.  o f t h e b a s a l medium, o f C h a p t e r F o u r ,  were s u p p l e m e n t e d  2 gm  5-5,  before weighing.  Portions  or  from a s t a t i o n a r y  t o 50 w i t h d i s t i l l e d  a t 6 4 0 nm.  measured  II.  and  concentration  in  then resuspended  cells  and  cell  in  144  s o y t o n e , pH  of  into  and  bioassaying.  To  was  t h e 84  was  of d i s t i l l e d diluted  Duplicate  o f Gluc-AmS medium  prepared. water  to give  with d i s t i l l e d  5 0 ml  aliquots  Part 2X  water  of 4 x  was  and  to 2X,  97 and t r i p l i c a t e ml E r l e n m e y e r thickness  50 ml a l i q u o t s flasks.  culture  ity  and at  flask  of t h e c u l t u r e s  on samples  was  speed  supernatants  after  of samples  f r o m each f l a s k  were a d j u s t e d  clinical  s o y t o n e , pH 5-5^ four  125  on a s h a k e r a t 20°C.  one  of e a c h medium, and t h e t u r b i d i t i e s  turbidity  a t 640  soytone, being in  nm  adjusted  pH 5-5>  e a c h medium  diluted  flasks  of h a l f - s t r e n g t h w i t h 0.5  Samples were t a k e n d a i l y days.  measured.  On t h e f i f t h of samples  and b i o a s s a y e d . were i n a d v e r t e n t l y  1 t o 16 w i t h  While  the assay c e l l s  Peak flask  were 0-5$  t h e a s s a y s f o r one  left  and  from  f r o m each  diluted  containing  ml  day, t h e  of t h e 6 u n d i s t u r b e d c u l t u r e s  t o pH 5-5>  counted, the tubes  The  of 2259-7 i n Gluc-AmS,  of l / l O d i l u t i o n s  measured, and samples  centrifuged,  N KOH,  flasks  incubated  four  640  f o r 5 minutes  ml E r l e n m e y e r  and f o u r  old culture  was r e a c h e d a f t e r  at  was measured,  were p r e p a r e d , a u t o c l a v e d , i n o c u l a t e d  turbidity  1 days  and b i o a s s a y e d .  from, a 31 hour  was  and a f t e r  centrifuge.  w i t h 0.1  measure-  turbid-  The t u r b i d i t y  per f l a s k flask  ml f r o m a  The  were c e n t r i f u g e d  25 ml e a c h of Gluc-AmS  (x/2)  on a  turbidity  flasks.  inoculation,  t o pH 5-5  In a s e p a r a t e e x p e r i m e n t Gluc-AmS  w i t h 1.0  f r o m each f l a s k  i n an IEC model HN  1 t o 16 w i t h 0.5$  with a double  and i n c u b a t e d on a s h a k e r a t 20°C.  were removed f r o m t h e s h a k e r .  5 ml samples  containing  inoculated  f r o m one o f t h e Gluc-AmS  of l / l O d i l u t i o n s full  capped  was m o n i t o r e d by d a i l y  r e a c h e d a maximum 5 days  the f l a s k s nm  Each  were  were d i s p e n s e d i n 250  and a u t o c l a v e d f o r 15 m i n u t e s  o f 2259~7 i n Gluc-AmS,  Progress ments  The f l a s k s  o f aluminum f o i l ,  d r y goods c y c l e .  o f Gluc-AmS  were culture  a t room t e m p e r a t u r e f o r  two h o u r s . IV. E f f e c t  of v a r y i n g  the concentration  of i n d i v i d u a l  medium  components Aliquots of D - g l u c o s e  1.0  of Gluc-AmS (+G), or 1.0  gm o f KH P0^, p l u s 0-5  (+S),  2  or w i t h a l l t h r e e  medium  were s u p p l e m e n t e d  w i t h 10  gm  gm o f ammonium s u l p h a t e (+N), or  gm o f MgS0^-7Hg0 p l u s 0.1  (+GNS) p e r l i t r e .  Duplicate  gm  CaCl -2H 0  50 ml  2  2  9  portions  of r e g u l a r  Gluc-AmS,  8 +G medium, +N medium, +S medium,  and +GNS medium, were d i s p e n s e d i n t o After  autoclaving  each f l a s k  flask  incubated in  o f Gluc-AmS  on a s h a k e r  the t h i r d f l a s k  ity  was  as d e s c r i b e d In of  was  inoculated  days  were sampled  inoculation,  for turbidity  solution  (+M) p e r l i t r e  o f Gluc-AmS,  meyer f l a s k s flask  Gluc-AmS  at  20°C, and t h e t u r b i d i t y and on t h e s i x t h  To t e s t yield, used  w i t h 100  Duplicate i n 250  50 ml ml  the e f f e c t  along with a t h i r d  f r o m a 3 day o l d  culture  were i n c u b a t e d on a s h a k e r flask  was  was r e a c h e d f o u r  after  inoc-  day samples  of l o w e r  and m i c r o e l e m e n t s  days  were t a k e n f r o m each  flask  c o n c e n t r a t i o n s of ammonium  and t h e i r  a 2 X 2 X 2 factorial  had  0.25  had  O.O65 ml o f m i c r o e l e m e n t  interaction  experiment  o f Gluc-AmS.  gm  was  Media  of ammonium s u l p h a t e p e r l i t r e ;  erogen The media  d e s i g n a t e d N/2 c o n t a i n e d  t h o s e d e s i g n a t e d N/4  stock solution  0.033 ml of m i c r o e l e m e n t s p e r l i t r e .  on  s e t up.  gm o f ammonium s u l p h a t e p e r l i t r e .  Similarly  of t h e e i g h t  IR),  (N/2, M/4,  concen-  2 R media c o n t a i n e d t h e s e components  twice the concentration  each  media  IR media c o n t a i n e d D - g l u c o s e ,  2  as i n Gluc-AmS;  M/2  and M./h media had  KHgPO^, MgSO^•7H 0, CaClg^HgO and t h i a m i n e a t t h e same  meyer f l a s k s .  Erlen-  of t h e t h i r d Gluc-AmS  0.5  at  ug  measurements and b i o a s s a y .  were v a r i a t i o n s  tration  fifth  ml of t h e m i c r o e l e m e n t  prepared.  The f l a s k s  Peak t u r b i d i t y  turbidity  sulphate  was  supplemented  w i t h 1.0. ml p e r f l a s k  2259-7 i n Gluc-AmS.  for  turbid-  r e a d i n g s and b i o a s s a y s  +T medium, and +M medium  of  ulation,  Peak  and on t h e  were a u t o c l a v e d and i n o c u l a t e d ,  o f Gluc-AmS,  monitored.  culture  i n section I I I .  a separate experiment,  portions  were  of t h e  was measured d a i l y .  after  ml f r o m  The f l a s k s  a t 20°C and t h e t u r b i d i t y  t h i a m i n e h y d r o c h l o r i d e (+T) or 0.13  stock  w i t h 1.0  flasks .  At t h e same t i m e ,  inoculated.  o f Gluc-AmS  reached four  day t h e f l a s k s  ml E r l e n m e y e r  o f 2259-7 i n Gluc-AmS.  a k day o l d c u l t u r e a third  was  250  i n Gluc-AmS.  combinations  Two  26 ml p o r t i o n s o f  i . e . , (N/2, M/2,  I R ) , and so on, were p r e p a r e d The e x p e r i m e n t  was  divided  into  I R ) , (N/4,  i n 125 two  ml  M/2,  Erlen-  replicates  99 which was  were r u n a t d i f f e r e n t  included  i n each r e p l i c a t e .  w i t h 0.5 ml each and  f r o m a 3-day  i n c u b a t e d on a s h a k e r  were sampled cribed  To  flask  The f l a s k s  were  medium  inoculated  of 2259-7 i n Gluc-AmS,  culture  a t 20°C.  o f each  After  five  days,  the c u l t u r e s  f o r t u r b i d i t y measurements and b i o a s s a y s as  scale  p r o d u c t i o n of  produce  cation,  four  amounts  5 gallon  Co.) were employed. stopper,  through  des-  3 mm  tubes  were l o o s e l y  Each  bottle  passed  reached tube  packed  inside  t o another  outlet  of the erogens was  the b o t t l e  was b e n t  fitted  tube,  Scientific  w i t h a #13  tubes--an 10 mm  by a s h o r t  rubber  a i r inlet  i.d.  Both  The a i r i n l e t  tube  l e n g t h of rubber  t u b i n g of t h e same  t o the bottom over  for purifi-  (Fisher  w i t h c o t t o n wool.  p i e c e of g l a s s  almost  sufficient  two g l a s s  i . d . , and an a i r o u t l e t  connected  tubing  erogens  polypropylene bottles  which  tube,  which  One  above.  V. L a r g e  was  times.  of t h e b o t t l e .  o u t s i d e the b o t t l e  diameter The a i r  through  an a n g l e  of 120°. The and  were l o a d e d w i t h 12 l i t r e s  then the i n g r e d i e n t s  sugar to  bottles  and s a l t s  prevent  position,  f o r 12  caramelization. and t h e b o t t l e s  leave the b o t t l e s Inoculum  in  1 litre  cultures  The r u b b e r  each  the r e s t  the  bottles tube  bubbled  cooling  flasks  close  used  before  cycle.  I t was  ml b a t c h e s  on a s h a k e r was  to inoculate  used  a t 20°C.  connected  t o a compressed  through the cultures  medium,  The fresh  After  a i r tap.  at 1 t o 2 l i t r e s  at  necessary  l o g phase.  to start  1 bottle.  into  inoculation.  of Gluc-AmS  t o t h e end of t h e i r  The  autoclaving  usually  were p l a c e d i n a 20°C i n c u b a t o r room, was  medium.  s t o p p e r s were w i r e d  overnight before  inoculum f l a s k  was  water,  were a u t o c l a v e d f o r 30 m i n u t e s  grown i n 200  Erlenmeyer  and  inlet  was  were u s e d  10 ml f r o m  of Gluc-AmS  must be a d e q u a t e l y d i s p e r s e d  15 p s i p r e s s u r e on a d r y goods to  litres  of d i s t i l l e d  per  inoculum, Generally inoculum,  inoculation,  and t h e a i r A i r was minute.  100 After tube  five  days  the  continuous f l o w attachment  Culture speed  flow rate  was  was  about  15,000 rpm,  and  of t h e s u p e r n a t a n t was was  culture  w i t h 6.0  treated  was  i n a S o r v a l l RC2-B  300  ml/min, t h e  the temperature  gm  of N o r i t vigorously  charcoal,  f o r two  hours  centrifuged The a 50 the  ml  tubes  glass  charcoal  pyridine, for  15  30  portions vacuo,  The  and  and  dissolved  determine  original  out t h e  charcoal  culture. from the  stored  by (20  suspending ml  water),  leaving  charcoal.  was  The  resuspended  A total  into  of  in  five  f o r each  l o t of  evaporated to dryness  of d i s t i l l e d  was  culture  the  specific  water,  water,  re-evaporated,  and  freeze-dried.  in a tightly  46.6  dissolved  of 0-5$  i n 2.0  activity  s u p e r n a t a n t , 10 ml  were f r e e z e - d r i e d .  capped  vial  ml  mg  of the erogens  of c u l t u r e  of t h e f r e e z e - d r i e d soytone,  pH  5-5,  i n the  supernatant material  was  and b i o a s s a y e d .  Foam To  determine  i f different  aeration  could  a series  of experiments  (New  ml d i s t i l l e d  of d i s t i l l e d  material  then  freezer. To  VI.  30  the  and  water  acetate solution  were combined,  i n 10 ml  The  stirring,  were e l u t e d  acetate solution.  i n 10 ml  sample  remainder  charcoal.  a c e t a t e s o l u t i o n were u s e d  eluates  dissolved  and  A  scraped from the f i l t e r  centrifuging  decanted,  freeze-dried a  then  the  as t h e o r i g i n a l  Erogens  of p y r i d i n e  of p y r i d i n e  The  and  tube.  rotor  2 t o 3°C.  washed w i t h d i s t i l l e d  acetic acid,  was ml  ml  of p y r i d i n e  charcoal. again  ml  with occasional  a filter,  centrifuge  minutes,  another  was  centrifuge.  to evenly d i s t r i b u t e the  same c o n d i t i o n s  onto  i n 30  50  supernatant  in  the  recovered charcoal  centrifuge  in  under  was  A activated  stirred  eight  centrifuge  t a k e n f o r b i o a s s a y , and  s u s p e n s i o n was left  p a s s e d t h r o u g h an  Brunswick  a culture stirrer  increase  the  erogen  using  Scientific  Co.)  volume of 5 l i t r e s ,  speed,  conditions  a i r flow rate,  yield  of a g i t a t i o n in large  scale  a Microferm laboratory was  begun.  This  and  facilities  and  temperature.  and cultures,  fermenter  apparatus  for  has  controlling  To  p r e v e n t foam  101 from  o v e r f l o w i n g onto  "by p l a s t i c plug  neck of a 1 l i t r e  pipette,  with the  stirrer fifth,  n  speed and  contents  a t 0,  sixth  In a n o t h e r rpm, for  a i r flow  t r a p was 300  rpm  and  had  of Gluc-AmS  temperature  and  a i r flow  the  the  k,  5,  a fresh  i n the sterile  bioassayed.  Serial  the b i o a s s a y  series.  of t h e  test  the  stirrer  and  flasks  Microferm of  litres/min,  of t h e  fermentor  bioassayed. s e t at  300  at 20°C.  Samples  inoculation.  collected  at 0.5  was  s e t up  litres/min.  with The  i n the  foam t r a p . 1 litre  500 The  flask,  dilutions  mis  trap flask and  foam  the  t h a t the  cells  carried  of e r o g e n s ,  of 2259-7 i n Gluc-AmS,  and  incubated  each f l a s k  of t h e  i n c u b a t o r room, and  After  a total  five foam  replaced foam  two  over  was  i n the  2800 ml  e a c h of Gluc-AmS were from  flasks the  of s i x days of  were b i o a s s a y e d ,  was  200  poorly aerated condition  ml  i n the  was  After  collected  5-0  One  at  down t o 1/2048 were i n c l u d e d i n  s t i m u l a t i n g the  days.  inoculum  of c o n d e n s e d  i n o c u l a t e d with  was  stirring  of 2259-7 i n Gluc-AmS.  ml  20°C f o r t h r ee shaker.  along  the f o u r t h ,  was  6 days a f t e r  c o n t a i n i n g 500  Each f l a s k  bench  speed  temperature  foam t o s y n t h e s i z e l a r g e q u a n t i t i e s  culture  On  samples  w h i c h had  culture  possibility  foam t r a p was  Fernbach  set at 2  at 20°C.  and  liquid  old culture  collected  To  flask,  culture  c e n t r i f u g e d , and  of i n c u b a t i o n , a p p r o x i m a t e l y  with  The  i n the  o l d shake  inoculation,  aseptically,  Microferm  o f a 2 day  days  a c o t t o n wool  flask.  a i r f l o w r a t e was  experiment,  day,  connected  assayed.  A third ml  The  at 2 l i t r e s / m i n ,  seventh  was  t u b i n g were a u t o c l a v e d  of a h day  50 ml  b i o a s s a y were t a k e n the  Erlenmeyer  5 litres  days a f t e r  were t a k e n  a i r outlet  i t s contents.  experiment,  Gluc-AmS.  the  p i p e t t e h e l d by  connecting  and  i n o c u l a t e d with  2259-7 i  On  and  fermentor  In one was  floor,  tubing to a Pasteur  i n the  Pasteur  the  starting  a 3 day  was  on  o l d shake  a shaker  then  o t h e r was  prepared. at  moved t o a  left  on  the  i n c u b a t i o n , samples with  a l/l6  dilution.  from  102 To by  the  litres in  determine  c u l t u r e s c o u l d be of  (N/2,  rpm,  and  foam, and  of t h e t o t a l  recovered  the  from  and  main c u l t u r e  centrifuged.  bioassayed diluted  at  The  The  its original  1 t o 100  with  erogens  collected  section  i n o c u l a t e d with  a i r f l o w a t 200 the  i n the  I R ) medium ( s e e  2259-7 i n Gluc-AmS.  of  were t a k e n  was  M/2,  the Microferm,  culture 300  what f r a c t i o n  200  ml/min.  speed  After the  soytone,  pH  was  old  s e t at  s i x days,  from  the  the  5-5>  samples  collected  main volume  c o n c e n t r a t i o n , and 0.5$  prepared  of a 3 day  volume, and  sample f r o m  foam, 5  I V ) was  ml  stirrer  produced  foam  was sample  before b i o -  as s a y i n g . The bottles bottle  a i r outlet was  was  sterilized, of  connected loaded  by  with  12  on  one  plastic litres  i n o c u l a t e d , and  section V.  collected.  tube  of t h e  5 gallon  polypropylene  t u b i n g t o a foam t r a p . of  (N/2,  incubated  After  7 days,  125  ml  T h i s was  diluted  l/400  M/2,  I R ) medium,  a c c o r d i n g to the  of condensed and  The  foam  bioassayed.  methods had  103 Results I . Time  course  The the  of e r o g e n  c u l t u r e pH, c u l t u r e age  for  second  the  than  0.1  unit  The  dry weight  of 1.0  corresponds  II. Effect The without  cells  cells  supernatant  tubes  samples  the  or  dilution turbidity  per  ml.  production pellet  during  media formed  centri-  a  loosely  were examined, t h e  samples  and  soytone  fraction  impossible. formed  supernatants than  The  the  d i f f e r e n c e was plus  presence cells  lengths of  conjugation  from the those  The  of 2259-6  l/l6 dilution  visually.  large  p r e s u m a b l y 2259~7  Therefore,  i n the  plus  contained  centrifugation.  of t h e  f r o m ammonium, s u l p h a t e  Effect The  No  l/50  a culture  conjugation tubes,  were l o n g e r  samples.  hydrolysate,  III.  to  tubes  c u l t u r e s grown i n  formed  observed  i n response between  sodium a c e t a t e ,  the  casein  soytone.  turbidities  and  the  2259-7 c u l t u r e s grown i n 4 x ,  erogen  2X,  and  of  each c u l t u r e  of medium, c o n c e n t r a t i o n  final  24  more  suspension  c o n t a i n i n g ammonium, s u l p h a t e  were compared  i n response  other  bioassays  made c o u n t i n g  ammonium s u l p h a t e the  other  been removed by  tubes  vary  Fig.  cell  dry weight  a compact  grown i n t h e  without  conjugation conjugation  formed  media  not  and  medium c o n t a i n i n g ammonium, s u l p h a t e  casein hydrolysate,  w h i c h had  formed  Therefore,  When t h e  sodium a c e t a t e ,  the  0.248.  erogen  of t h e  d i d not  of t h e  Q  on  pellet.  of t h e s e  Ag^  of n i t r o g e n s o u r c e  cells  plotted  study,  washed  cell  from assays  cells  of t h e The  from the  are  and  replicates.  of  the  of  values  mg/ml.  t o 0-75  sodium a c e t a t e  numbers  ml  pilot  mg  cells  fugation; packed  was  pH  X dilution),  supernatant  f o r the  of t h e  i n 40.0  or 9.3  suspension  i n the The  between any  0.3725 gm,  (measured Ag^Q  i n F i g . 23  experiment.  of t h e  with  turbidity  c o n c e n t r a t i o n of e r o g e n s  against  was  production  concentrations Gluc-AmS media  of are  to  EROGEN CONCENTRATION (UNITS/ML) -tjOT  •mnxpsiH  ru  8  SUiy-orLLf)  U  T  ssjn^xrio  L-6<nZZ T U  notq.'BTrmiriooe  CULTURE PH 8 S TURBIDITY AT B40 NM  EROGEN CONCENTRATION (UNITS/ML)  ut  8  lo6 shown the  i n T a b l e IX.  concentration  erogen yield  of t h e medium  to decrease.  turbidities  increases,  i n t h e second  block  experiment  have b e e n s p l i t  c o n t a i n s t h e assays which  two h o u r s  analysis  erogen y i e l d s  grown  than  of v a r y i n g  experiment  of  experi-  i n Gluc-AmS  and  The  two b l o c k s - - t h e s e c o n d a t room  i n the f i r s t  temperature block.  f o r the l o g a r i t h m s t o the base  in this  as  erogen  i n T a b l e IX.  were l e f t  longer than the assays  of v a r i a n c e  IV. E f f e c t  into  i n average  greater  are i n c l u d e d  increase  but the y i e l d  The d i f f e r e n c e  e r r o r . The d a t a -.from t h e c u l t u r e s  bioassay results for  culture  between media i s n o t s i g n i f i c a n t l y  mental x/2  appears  The f i n a l  two of t h e  i s presented i n Table  the concentration  An  of i n d i v i d u a l  X.  medium  component s The ments  final  turbidities  on t h e e f f e c t  of i n c r e a s i n g  ammonium s u l p h a t e , s a l t s , in Table XI. The  The a n a l y s i s  difference  first  second  for  comparing  the c o n c e n t r a t i o n  of v a r i a n c e  of g l u c o s e ,  highly  Application  a r e shown  i s presented i n Table XII. significant  and on t h e b o r d e r l i n e  experiment.  from the e x p e r i -  t h i a m i n e and m i c r o e l e m e n t s  between m e d i a was  experiment,  the  and b i o a s s a y r e s u l t s  i n the  of s i g n i f i c a n c e i n  of Dunnett's  t r e a t m e n t means w i t h a c o n t r o l  procedure  (Steel  and T o r r i e  i960) showed t h a t t h e e r o g e n y i e l d s i n t h e +N medium and t h e +GNS medium yield  were s i g n i f i c a n t l y  i n Gluc-AmS.  significantly  different  from t h e second medium  than  The XIII.  and  from the y i e l d  experiment  indicate  i n t h e +T medium  The a n a l y s i s  and  of v a r i a n c e  on t h e f i n a l  yield  i s shown  replicates  t h e main e f f e c t  and +S m e d i a were n o t  i n Gluc-AmS.  a lower y i e l d  experiment  The d a t a  i n t h e +M  is listed  f o r the e f f e c t s  turbidity culture  confidence) than the  Gluc-AmS.  of t h e  i n Table medium  and on t h e l o g a r i t h m n s of t h e  i n T a b l e XIV.  on f i n a l  (95$  i n t h e +G  data from the f a c t o r i a l  components erogen  The y i e l d s  less  The main e f f e c t s  turbidity  of N on e r o g e n y i e l d  of N, R  are s i g n i f i c a n t .  is significant.  Only  Table  IX. F i n a l t u r b i d i t i e s and e r o g e n y i e l d s f o r 2259-7 c o n c e n t r a t i o n s o f Gluc-AmS medium. Medium  First e x p e r iment  Second e x p e r iment  Final turbidity at 640 nm  cultures  Erogen y i e l d (units/ml)  grown i n v a r i o u s  95$ c o n f i d e n c e limits  7  9 3 k 5  7 6, 11 2 3 6, 5 6  2X  5 0 5 2  5 9 7 4  6. 1,  Gluc-AmS  3 4 3 3  11 8 10 6  9 8  Gluc-AmS  3 8 3 8 3 8  11 3 f i r s t b l o c k 10 5 9 0 second b l o c k  9 5, 13 3 8 9, 12 3 7 6, 10 5  x/2  3 0 3 3 3 1  9 0 f i r s t block 8 8 6 1 second b l o c k  7 6, 10 5 7 5, 10 3 7 1 5 l  4X  6 0  5  4 1,  9, Q,  ;  7 2 8 8 14 1  12 5  io a Table  X. A n a l y s i s o f v a r i a n c e o f l o g a r i t h m n s t o t h e base two of e r o g e n y i e l d s from. 2 2 5 9 - 7 c u l t u r e s grown i n Gluc-AmS and x/2 media.  Source of var i a t i on  Degrees of freedom.  Blocks Media Error  Table  1 1 3  Sum o f squares  Mean s guare  0.2296 0.2166 0.0295  0.2296 0.2166 0.00983  Final turbidity  Erogen concentration u n i t s /ml  0  O O 0) CQ  <D Pt X 0)  with  95$ c o n f i d e n c e lira.it s  13 1 14 6  1 1 1, 13 0,  14 7 16 3  +G  4 0 3 8  13 6 14 6  12 1 , 12 9,  15 16  +N  4 l 4 0  7 0 6 8  6 1, 5 9,  +S  3 4 3 5  13 13  +GNS  4 7 h 7  12 0 10 . 8  10 1, 9 5,  13 5 12 2  Gluc-AmS  3 5 3 6  18 0 23 1  14 6, 18 8 ,  22 5 29 0  +T  3 7 3 5  21 7 19 5  17 16  6, 2,  27 3 24 0  +M  3 7 3 6  9 8 5 3  8 0, 4 3,  12 0 6 5  8 9  12 12  of  0.017 0.018  3 4 3 4  -p C! 0)  23.4 22.0  Gluc-AmS  •p  •H CO (D U Pi •H X  Probability a larger F  X I . F i n a l t u r b i d i t i e s and e r o g e n y i e l d s i n media d o u b l e d c o n c e n t r a t i o n s o f i n d i v i d u a l components. Medium  &  F  2, 3,  3 4  7 9 7 7 15 15  4 7  Table  X I I . A n a l y s i s of v a r i a n c e o f l o g a r i t h m n s o f e r o g e n d o u b l e d c o n c e n t r a t i o n s of i n d i v i d u a l components.  Source First  of v a r i a t i o n  experiment Media  Error  Second e x p e r i m e n t Media Error  Degrees freedom  of  Sum o f squares  Mean square  h  ±.<?6K  5  O.O2895  0-3909 O.OO579  2 3  3-020 0A758  I.510 O.I586  yields  P  67.5  9-52  i n media w i t h  P r o b a b i l i t y of a larger F  0.0001  0.050  110  Table  X I I I . F i n a l t u r b i d i t i e s and e r o g e n y i e l d s f r o m f a c t o r i a l e x p e r i m e n t on e f f e c t o f ammonium s u l p h a t e c o n c e n t r a t i o n , m i c r o e l e m e n t c o n c e n t r a t i o n , and c o n c e n t r a t i o n o f r e s t o f med ium. Medium  N/2,  M/2,  IR  N/4,  M/2,  IR  M/4, N/4, M/4,  IR  N/2,  M/2,  2R  N/4,  M/2,  2R  N/2, -p  «5 u  -p - H CQ H Pi •H <L>  U  0 -p OS  >-l ~ O H 0 ft d) 0) tQ  U  Final turbidity  2R  N/2, N/4,  M/2,  IR  M/2,  IR  M/4, N/4, M/4,  IR  W/2,  M/2,  2R  N/4,  M/2,  2R  M/4, N/4, M/4,  2R  N/2,  N/2,  A7 •05  IR  M/4, N/4, M/4, N/2,  .09  2R  IR  2R  ,46  2- 75 3- 27 2-55  3 32 59 3 37 2 75 3 4l 2 88 3 81 2 88 2  Erogen y i e l d units/ml  11 8 9 11 12 7 13 8  9570 c o n f i d e n c e limits  8-9, 7-2, 7-6, 9.1, 10.1, 6.3, 10.7, 6.3,  13 6 14  0  17 7 15 6 12 8  8 3 9 0  8 3  14.1  10.7 11-7 13-9 15 .6 9-5 16.4  9.9  18 19 13 2, 24 5 4, 9 11 5, 21 4 1, " 8 9 3, 17 6 0, 10 9  10  9, 3,  3 0  1 5 8 0  3 9  Table  XIV. A n a l y s i s of v a r i a n c e f o r e f f e c t s o f ammonium s u l p h a t e c o n c e n t r a t i o n , m i c r o e l e m e n t c o n c e n t r a t i o n , and c o n c e n t r a t i o n o f r e s t o f medium on f i n a l t u r b i d i t y and l o g a r i t h m n of e r o g e n y i e l d .  Source a.  of v a r i a t i o n  Turbidity Main e f f e c t s N M R Replicates I n t e r a c t ions NM NR MR NMR Error  b. Erogen y i e l d s Main e f f e c t s N M R R eplicates I n t e r a c t ions NM NR MR NMR E r r or  Sum o f squares  Mean square  1 1  O.OI879 0.00001157 0.002352 0.002305  0.01879 0.00001157 0 .002352 0.002305  119 0.07 14 . 9  0.00001 0.79 0 .0062 0.0065  1 1 1 1  0.000053 O.OOOO69 O.OOOOO82 O.OOOI76  0.34 0.44 0.05  7  0.001104  0.000053 0.000069 0.0000082 0.000176 0.0001578  0.58 0.53 0.83 0.33  1 1 1 1  1.458 0.00226 0.1105 0.0827  1.458 0.00226 0.1105 0.0827  11.1 0.017  0.013 0.90 0.39 0.45  1 1 1 1  O.OOOOO625  0.00000625 0.2835 0.0315 0.135 0.1315  0.00005 2.16 0 . 24 1.03  Degrees of freedom  1 1  7  O.2835 0.0315 0.135 0.9209  P r o b a b i l i t y of a larger F  14.6  1.12  0.84  0.63  0-99 0.19 0.64  0.3^  112 V.  Large  scale production  After  filter  Reducing the The varied  days  i n c u b a t i o n i n the  and e s c a p e a i r flow  erogen  around  the  r a t e d i d not  erogen  2259-7 c u l t u r e s u p e r n a t a n t  the  methods d e s c r i b e d .  was  125^000 u n i t s , The  potency  1.005)-  units/mg.  a recovery  yield  corresponds  charcoal  treat-  supernatant  and s i x t e e n  was . litres  and p r o c e s s e d b y  of concentrated  erogen  a t 23-3 mg/ml had an  (95$ c o n f i d e n c e  to a specific  The hormone c o n c e n t r a t e  prepared  activity  the  and e l u t i o n  charcoal adsorption  problem.  o f a p p r o x i m a t e l y 80$.  o f O.85 u n i t s / m l  This  stopper.  supernatants  After  were p r o d u c e d  c h a r c o a l had a s p e c i f i c  VI.  culture  F i v e hundred  The t o t a l  rubber  eliminate this  f r e e z e - d r i e d c u l t u r e supernatant  erogenic  cation  of the  c o n c e n t r a t i o n i n the  g e n e r a l l y b e l o w 0.05 u n i t s / m l .  and  edges  c o n c e n t r a t i o n i n the  residual  large bottles, the  The foam would wet t h e a i r  between 0.25 and 0-5 u n i t s / m l .  ment, t h e of  erogens  o f 2259-7 began t o foam.  cultures outlet  three  o f the  limits  steps  o f 0.037  activity  by e l u t i o n  o f 5-0 u n i t s / m g .  O.7O8  of the  Therefore  accomplished  a purifi-  o f 135 f o l d .  Foam In t h e  culture  first  fluid  confidence  experiment, the  after  limits  was  O.92 u n i t s / m l  the  sixth  4 days  2-5 and 3-7)-  Foam s t a r t e d  t o pass  day  day s a m p l i n g s .  In t h e units/ml  second  sample 0.55 u n i t s / m l s i x day sample  and  0.37)-  day  showed h i g h  tration  into  the  O.56 and 1-3), limits  f o u r d a y sample  activity  four 0.23  f i v e day  0.31 and O.82)  (95$ c o n f i d e n c e  condensed  and on  assayed  O.O76 and O.38), t h e limits  potency  0.14 and O.57).  the  0.33 u n i t s / m l  (l/l6).  day,  foam t r a p between t h e  (95$ c o n f i d e n c e  erogenic  of the  the  limits  The c o l l e c t e d ,  tested  limits  fifth  (95$ c o n f i d e n c e  experiment,  (95$ c o n f i d e n c e  the  On t h e  (95$ c o n f i d e n c e over  potency  i n c u b a t i o n was 3-1 u n i t s / m l (95$  day 0.37 u n i t s / m l  and f i v e  erogenic  foam a s s a y e d  down t o t h e  limits on t h e  lowest  and  0.29 seventh  concen-  113 The  collected  concentration  foam i n t h e t h i r d  experiment  (95$ c o n f i d e n c e  o f I96 u n i t s / m l  had an e r o g e n  limits,  174 and  220);. , I n t h e c o m p a r i s o n between t h e shaken the  shaken  culture  (95$  confidence  left  on t h e b e n c h  an e r o g e n  1.4  had an e r o g e n  and s t a t i c  concentration  cultures,  o f 13 u n i t s / m l  7 - 4 and 19), and t h e c u l t u r e w h i c h was  limits  for the last  concentration  three  days  o f 3-5 u n i t s / m l  o f i n c u b a t i o n had  (95$ c o n f i d e n c e  limits  and 5.5).. In t h e e x p e r i m e n t  carried densed  over with  (95$  t h e foam, t h e volume  foam, a f t e r  erogenic  potency  confidence  o f t h e main  amount  (95$ c o n f i d e n c e  of erogens  left  and t h e p o t e n c y limits  units/ml of t h e  148 and I91). The  i n t h e main c u l t u r e was t h e r e f o r e  5,000 ml X O.57 u n i t s / m l » 2850 u n i t s . erogen  con-  was 250 m l . The  c u l t u r e volume was 0.57  0.49 and 0 . 6 4 )  limits  of t h e erogens  of the c o l l e c t e d ,  c e n t r i f u g i n g out t h e c e l l s ,  foam was 170 u n i t s / m l total  t o determine the f r a c t i o n  The t o t a l  i n t h e foam was 250 ml X 170 u n i t s / m l  amount o f  - 42,000  u n i t s , or  92$ o f t h e t o t a l . The culture  volume  foam c o l l e c t e d  o f 2259-7 i n t h e 5 g a l l o n b o t t l e  concentration confidence  of erogen  limits  erogen y i e l d culture  o f condensed  f r o m t h e 12 l i t r e  was 125 ml- The  i n t h e foam was 1,360 u n i t s / m l (95$  994 and 4300).  This  corresponds t o a t o t a l  o f 170,000 u n i t s or 14.2 u n i t s / m l  volume.  of the o r i g i n a l  Ilk  Discussion The  rise  i n erogen  in  Gluc-AmS f o l l o w s  in  turbidity,  but  imum e r o g e n i c turbidity. the  a curve  potency  data  about  i s reached  T h e r e may  be  one  not  a small decrease  i s decreasing,  experiment,  c o n s i s t e n t l y l o w e r hormone a c t i v i t y storage  production  that  stopped  e r o g e n was  produced  may  be  monitored reached  after  the  one  or two  specific is  not  pH  was  the  pH  Millis  to  fell  the  this.  ikk  hours  adjacent during  of hormone of  primary  mainly  I965).  after Most  b e l o w 2.5.  of  the  This  erogen  peak t u r b i d i t y  ensure t h a t  peak  destroyed  experiments,  best  of t h e  yields of  a l l cultures  nitrogen  A d d i t i o n of  decreased  c u l t u r e with  of a c e t a t e ,  the had  was  the  sources  sodium a c e t a t e  hormone y i e l d .  sodium a c e t a t e ,  inhibiting  or  erogen  to  the  Whether  some more  production  clear.  Logarithmns  of t h e  values  were u s e d  erogen  concentrations  logarithmns, distributed the  appear  max-  the  and  the  products  should  increase  yield.  the  of t h e  effect  are  days a f t e r  reached,  maximum  stabilize  higher  kinetics  c u l t u r e pH  f o r erogen p r o d u c t i o n .  medium t o  The  erogens  the  than  2259-7  during  but  108  e r o g e n was  In s u b s e q u e n t  Ammonium s u l p h a t e tested  from. Qk,  ( A i b a , Humphrey and  c u l t u r e was  their  the  metabolites  a coincidence.  were d e t e r m i n e d  samples  freezer.  i n d i c a t e that  metabolism—secondary  the  some of t h e  i n the  growth has  the  p r e c i s e enough t o f i r m l y . e s t a b l i s h  showed  overnight  as  i n potency  second  indicating  to the  However, t h e  same t i m e  In t h e  samples,  c u l t u r e s of  parallel  day.  at t h e  culture turbidity  are  i n shake  approximately  l a g g i n g by  t i m e when t h e  bioassay  concentration  f o r the  and on  erogen y i e l d s  a n a l y s i s of v a r i a n c e  are  hence  estimated  e r r o r s of  can  be  expected  i n the  to  Also  the  because  bioassay  estimation w i l l  a logarithmic scale.  erogen y i e l d s  r a t h e r than  the  as  be  absolute the their  normally  variability  i n c r e a s e with  the  of  average  yield. Increasing the  final  the  concentration  concentration  of t h e  of Gluc-AmS d i d not  erogens,  and  appears t o  increase have  115 actually icantly  decreased  i t , although  greater than  concentration experiment, supports  the  experimental  significantly  the  division  the  to  f o r erogen  concentration, other rather  of t h e  components  components The  reduced  d i d not  might be  found  erogen y i e l d .  results  of t h e the  or  i f the  highly significant  stock  effect  significant, icant  slightly  smaller,  i s close to  not  produce  phase  The  effect,  second  ammonium s u l p h a t e  only f a c t o r  and  on  the  of t h e  yield  rest  without  effect.  is  optimal  near  of t h e An  not  realize  from the  medium, and  bioassay at the  bottle  how  individual  factorial  a l s o had  suggesting  that  concentration.  the  was  a  one The  i n the  sampled  first  of signif-  two at  a  replicate.  a  This  erogen  Concentration  t h a t had  their  con-  of  significant of  interactions,  microelements were  c o n c e n t r a t i o n of 0-5 and  the  (w/2,  gm/l  M/2,  c h o i c e f o r hormone  production.  of t h e  begun b e f o r e  procedure  time  Further  were v a r i e d .  R factor  f o r s y n t h e s i s of e r o g e n s , a good  of  concentrations  ammonium s u l p h a t e  scale production  quantitative  The  was  erogens  c u l t u r e s were.  was  developed.  abnormally  in  main l i m i t i n g n u t r i e n t  replicates.  of e r o g e n .  medium a p p e a r s t o be Large  the  micro-  individually  d i f f e r e n c e i n average  two  in  Increases  of e r o g e n s .  replicate  of g r o w t h t h a n  a significant was  increased  average t u r b i d i t i e s  c o n c e n t r a t i o n between t h e effect  is close  c u l t u r e s i n the  a limiting  means t h a t t h e  earlier  of t h e gm/l.  d i f f e r e n c e between t h e  replicates did  although  components  blocks  of ammonium s u l p h a t e  i n d i c a t e s t h a t n i t r o g e n i s the a t 0.25  and  solution  experiment  its  turbidity  at l e a s t  this  blocks.  initially  concentrations  on  growth,  two  were t e s t e d  centration for  In  medium were d o u b l e d  yield  salts  microelement  final  into  erogen y i e l d s .  change t h e  i f the  i n a group, of t h e  medium  d i f f e r e n c e between  assay  signif-  production.  significantly  than  the  i n c r e a s e d ammonium s u l p h a t e  components  effects  reduced  H a l v i n g the  medium c o n c e n t r a t i o n c h o s e n  When i n d i v i d u a l elements  d i f f e r e n c e s were not  error.  highly significant  Apparently optimal  the  low  the  Therefore erogen  IR) the  I did  yields  116 Three erogens  methods f o r r e c o v e r y and  from  large  volumes  extraction  with n-butanol,  adsorption  on  activated  rejected  because  oration  of l a r g e  tion  on  the  culture  ing  sites  better  charcoal.  A d s o r p t i o n on  charcoal,  purification. and  The  gave 80$  purification.  would  Both  been h i g h e r  i f the  supernatant  had  washed w i t h t h e  thought  that  The  erogen  tions the  yields  of s t i r r i n g  the  erogens,  the  The  erogens  erogens  i f the  erogen  from  in aeration  mixing  sharp  foam, was The  might  have  culture the  been  from the  shake  and  the  of t h e  of s t i r r i n g  pre-  5  gallon  cultures,  agitation  12  litre  cultures. and  might  first be might oxygen,  Experiments  c o u l d be  initially  I  cultures  a i r flow rate  parameters  cultures  were begun  easily  merely  to  for  conneatness.  at v a r i o u s c o n d i -  a i r f l o w were c o m p a r a b l e t o t h e y i e l d s In t h e f i r s t  experiment  d r o p between t h e f i r s t  s u g g e s t i o n o f S c o t t Redhead,  activity.  135-fold  from  c h a r c o a l had  w i t h t h e b e g i n n i n g o f foam c a r r y  collected  for bind-  i n the  eluted  yields  from, t h e M i c r o f e r m  bottles. The  with  purification  c o n c e n t r a t i o n i n t h e medium d e c r e a s e d  with time. cided  the  where t h e s e  and  of  was  Foam t r a p s were i n c l u d e d  5 gallon  erogen  At  effect  the Microferm,  trolled.  erogen  or t o o h i g h a c o n c e n t r a t i o n of d i s s o l v e d  provide inadequate the  components  c h a r c o a l a d s o r p t i o n procedure  Bubbling a i r through  t o o low  fewer  over  and  than those  differences  responsible.  favoured  extrac-  acetate eluant.  that  were much l o w e r  in  c h a r c o a l was  c o n c e n t r a t i o n of t h e  bottles  investigate  made c o n t i n u o u s  r e c o v e r y of the  pyridine  was  to higher recoveries  been g r e a t e r .  and  of n - b u t a n o l made e v a p -  and  t h e r e c o v e r y and  When I r e a l i z e d  or  point  compete w i t h t h e  c h a r c o a l would have been p u r e r  provide  Solvent extraction  leading  of t h e  i o n exchange r e s i n ,  i o n exchange r e s i n s "because  supernatants  purification  media were c o n s i d e r e d -  a d s o r p t i o n on  volum.es d i f f i c u l t ,  on t h e  practical,  of c u l t u r e  the high b o i l i n g  impractical.  adsorption  primary  assayed,  comparison  and  over  i n the  found  between t h e  in  d e s c r i b e d , the  rather two into  second  than i n c r e a s e d  samples  coin-  t h e foam. t r a p . experiment  t o have h i g h e r o g e n i c shaken and  static  flask  the  117 cultures  confirmed  the  culture  main  synthesized culture, the  by  92$  the  foam i n d i c a t e s able  to that  earlier  cells The  erogens as  produced  well.  The  and  was  bottle  cultures  Collection  of t h e  foam f r o m t h e  cultures  method f o r p r e l i m i n a r y  concentration  and  lates  Such a h i g h  i n the  residual  foam t r a p  hormone i n t h e  Selective f o a m i n g has cation  to  water.  in  received  at  and  non-polar  responsible culture as  supernatant  foamy as  fractionation operating that  can  the  the  be  or  has  of  been  the  "wet"  completely. a higher  of  are  i n the  the  Tremella  and  high  cultures.  solution from  by  appliwaste  material  evidence  which i s  for  both  discussed  low  The  only  to  a  predict of  "dry"  adsorbed  purification  optimum condition from  rates most  foams, w h i c h  give  substances  in  the  i n a c t i v e mater-  g i v i n g a compromise of t h e  is  foam  materials  from s u r f a c e  rate  of  erogens  High a i r flow adsorbed  bed  content,  operating  concentration  flow  the  through  erogen  are  principally  When  passed  i n order  separation  intermediate  recovery  accumu-  t h e o r e t i c a l basis  produce  surface  better  the  the  p r o b a b l y not  very  The  I968).  rates  any  The  remove s u r f a c e  of  of  i t s possible  foam a r e  investigated  a i r flow  foam, and  C l e a r l y an  tween h i g h for  Low  found  excellent  discard  2259-7 c u l t u r e s .  solution.  and  yields  erogen molecules  erogens  (Lemlich  concentration  condensed ials .  foams,  the  compar-  activity  detergents  2259-7 c u l t u r e s i s t h e a i r f l o w r a t e . produce  in  erogen molecules themselves  condensed  readily varied  i s an  erogen  agents,  i n the  that  original  low  purification  of  removed.  sections  foaming  erogens  discarded.  f r o m aqueous  e f f l u e n t , w h i c h has  condition be  active  However, t h e  f o r the  of P o r a p a k Q,  can  suggests  active.  solutes removal  surface  a surface  C h a p t e r Two  surface  as  of as  the  in  culture.  a t t e n t i o n because  problems  well  of t h e  typical  erogens  cultures  i t i s p r a c t i c a l to  main  transport  such  As  adsorbed polar  that  of  when foam was  percentage  a  concentrated  bottle  explains  from  being  In  removes t h e  f o r the  erogens.  than  trap.  amount  from the  cultures,  transported  foam., r a t h e r  foam e f f e c t i v e l y  erogen y i e l d  i n shake  the  a f t e r t h e y r e a c h the  cultures an  e r o g e n s were b e i n g  t r a p by  total  foam.  bottle  the  i n t o the  of t h e  condensed  from the  that  erogens  is  bebest  118 In t h e s e been measured.  experiments,  only the t o t a l  The p r o p o r t i o n s  ents  may  vary  with  they  may  have d i f f e r e n t  erogen  of the i n d i v i d u a l  c o n c e n t r a t i o n has active  compon-  t h e age o f t h e c u l t u r e and t h e medium, and affinities  f o r t h e foam.  CHAPTER SIX  PARTIAL PURIFICATION  OF THE EROGENS  119 Materials  and  Methods  I. I n s t a b i l i t y 4-7-1 in  1.0  ml  mg  of  the  of  10$  ammonia.  1 N hydrochloric potassium vials on  chloride goods  to  was  adjusted  to  II.  First 5l6  mg  mg  solution. for  5-5  with  attempt litres  distilled  The  cooling,  the  solution  2  X 20  cm  had  cm  of  cold  acetic resin  and  was  changed reached The  distilled  distilled  pH  6.5-  adsorbed  pH  the  the  at  of  volumetric 0.1  ml  soytone,  and  grams  A-4 M  of  1 N  screw  cap  pressure, each  vial  flask  of  each  the  pH  and diluted  was  effluent.  regenerated  ml  of  .IN  water,  and  250  ml  the  eluted  of  concentrate, ml  adjusted  to  The  eluate by  350  ml  down t h e The  acetic  the no  more  s o l u t i o n was  washing w i t h  0.2  anion  M  of  ml  cold  column, acid  1  N  the  eluate  acid  pigment freeze-  400  ml 400  concentrate  exchange r e s i n ,  pH  300  ammonium a c e t a t e  gm. of  a  which  yellow  potassium hydroxide, of  6.5  neutral-  washed w i t h  time t h a t  of  ml/hour t h r o u g h  with  yellow.  e r o g e n s f r o m 20 from the  i n 20  exchange r e s i n  column u n t i l  The  200  50  progressed  the  the  of  eluted  from grey to  column was  solution.  column was  acid  b o t t o m of  methods  erogen  s o l u t i o n was  anion  then  acetic  of  the  ammonium a c e t a t e b u f f e r ,  The  and  by  were d i s s o l v e d  a flow rate  just before  i n the  and  ml  of  ml  steam  contents  ml  units/mg.  of  4°C.  water,  colour  In t o t a l on  5-0  w i t h 0.2  the  water,  of  of  25  concentrate  the  room a t As  0-5$  psi  medium p r o c e s s e d  column of D u o l i t e  acid.  dried.  of  V yielded  passed  distilled  visible  ml  i n 1.0  in small  15  water.  sodium b i c a r b o n a t e  c o l l e c t e d from  front  distilled  culture  10$  in a cold  a 10  i n 1.0  bioassay.  been e q u i l i b r a t e d  6.5,  to  dissolved  at p u r i f i c a t i o n  water,  ized  solutions  After  2.0  section  of  dissolved at  grams of t h i s  addition  was  was  dissolved  mintures  a specific activity Five  by  for  was  10  mark w i t h  added t o  Chapter F i v e ,  was  38-8  40.0  cycle.  the  solution  was  erogen p r e p a r a t i o n  quantitatively transferred  diluted  of  acid.  were a u t o c l a v e d  a dry  was  standard  in  of ml buffer,  were four  120 "batches.  The f r e e z e - d r i e d e l u a t e m a t e r i a l was combined and  weighed.  5.k mg was s e t a s i d e f o r b i o a s s a y .  The  remainder  ml  of d i s t i l l e d  to  approximately  of t h e e l u a t e  water.  ( l . O gm) was s u s p e n d e d  The pH o f t h e s u s p e n s i o n  seven with  was  10$ s o d i u m b i c a r b o n a t e  i n 100  adjusted  solution,  whereupon some o f t h e d i s s o l v e d m a t e r i a l p r e c i p i t a t e d . precipitate  was removed by c e n t r i f u g a t i o n ,  supernatant  was d e c a n t e d .  portions This  of d i s t i l l e d  supernatant  saturated butanol  with  water,  solution  water,  briefly  vacuo  just  was e x t r a c t e d w i t h  t o remove water  water  swirling,  some o f t h e d r i e d  into  not d i s s o l v e .  a c e n t r i f u g e tube,  flask. acetone. separate The  dissolve..in  acetate,  formed  and t h e n  by  centrifugation, The  ed  a t 0.0024 mg/ml.  solution butanol  acetate  of the e l u a t e  acid.  was  decanted  exchange  mg/ml.  The e t h y l  d i d not  w a t e r , n o r i n 50$  was c e n t r i f u g e d t o phase was  decanted.  i n vacuo t o remove w h i c h had  solution  dissolved  mostly  m a t e r i a l was removed  was f r e e z e - d r i e d .  e l u a t e was b i o a s s a y e d a t  The r e d i s s o l v e d p r e c i p i t a t e The n - b u t a n o l acetate  e x t r a c t was  1 t o 50 f o r b i o a s s a y .  e x t r a c t i o n was a l s o d i l u t e d  bioassay-  e x t r a c t was e v a p o r a t e d  and r e d i s s o l v e d i n 10 ml o f 50$  was d i l u t e d  vigorous  The p r e c i p i t a t e  Undissolved  and t h e s u p e r n a t a n t  a t 0.27  i n vacuo,  mixture  concentrated  c o n c e n t r a t i o n o f O.O36 mg/ml.  dryness  saturated with  acetate  f r e e z e - d r i e d anion  was b i o a s s a y e d  10 ml o f  of t h e f l a s k  mixture  i n the f l a s k  freeze-dried.  on n e u t r a l i z a t i o n  20 ml o f 1 N a c e t i c  a  n-butanol  and t h e e t h y l  in  in  f o l l o w e d by water w a s h i n g s f r o m t h e  The w a t e r - e t h y l the phases,  Despite  acetate  centri-  evaporated  m a t e r i a l on t h e w a l l s  aqueous phase was b r i e f l y  ethyl  acetate.  The u n d i s s o l v e d m a t e r i a l l e f t  completely  n-butanol  e x t r a c t s were  d r o p l e t s , and t h e n  The w a t e r - e t h y l  supernatant.  50 ml p o r t i o n s o f n-  The b u t a n o l  and 20 ml o f e t h y l  brown  two 20 ml  100 ml o f  The r e s i d u e was t r e a t e d w i t h  distilled would  w h i c h was added t o t h e  water.  to dryness.  and t h e c l e a r  was washed w i t h  f o l l o w e d by f i v e  saturated with  fuged  The p e l l e t  The  acetone.  The r a f f i n a t e  1 t o 50 f o r b i o a s s a y .  to  This from the  121 The in  100  r e d i s s o l v e d m a t e r i a l from  ml  extracted extracts  of d i s t i l l e d  water  with  ml  five  a c e t a t e , and  dried  butanol  was  1/25O  for bioassay. The  d i s s o l v e d i n l60 butanol  freeze-dried a mortar. absolute  with  of water section  X.  ml  The  poured The  two  freez.e-  thoroughly  mixed.  acetone.  This solution  remainder  of t h e b u t a n o l  aqueous a c e t o n e ,  i n 1 ml  on  of a b s o l u t e  g e l (BDH)  into  was  top  of t h e  by  t h e method  collected  for bioassay.  a d r y goods  ml  The  cycle,  with and  tubes  a gradient the  then  Two, effluent  From  each  soytone,  were  cm  eluted  of C h a p t e r  of 0-5$  and  in  erogen  i n 5 minute f r a c t i o n s .  were added t o 1.0  diluted  column, 2  of t h e  1 ml/min,  ex-  the  slurried  then  was  and  column, and  e t h a n o l , and  6.1  ethanol in  a chromatography  e l u a n t f l o w r a t e was  tube  minutes  The  ethanol suspension  of a b s o l u t e  f o u r drops  in a test 10  ground  a p p l i e d t o the  column was  fraction, for  50  butanol  r e d i s s o l v e d i n water-  and  of 25$  grams of s i l i c a  diameter. was  ml  i n ethanol, generated  the  5-5,  powder was  e t h a n o l , and  preparation  The  and  e x t r a c t s were a g a i n f r e e z e - d r i e d , and  Fifty  internal  from  of 20$  suspended  The  i  l/5000 f o r b i o a s s a y .  was  first  ml  was  n-butanol,  p o r t i o n s of n - b u t a n o l .  e x t r a c t s were combined  tract  in  saturated with  f r e e z e - d r i e d as b e f o r e .  d i s s o l v e d i n 6.0  diluted  precipitate  were c e n t r i f u g e d , e v a p o r a t e d ,  ethyl mg  50  the  pH  autoclaved  i n o c u l a t e d with  1 d r o p e a c h f r o m a 30 hour o l d c u l t u r e of 2259-6 i n (4,4) medium. After  12  hours  i n c u b a t i o n a t 20°C, t h e  c o n j u g a t i o n tubes Fractions dryness stored II,  i n each a s s a y  5 t o 18  tube  were p o o l e d  to dryness  e t h a n o l and  s t o r e d i n the  was  through  silica  with  run  fractions  evaporated ethanol.  the 77  t o 84  to dryness I t would not  and  freezer.  g e l column,  as hormone I I I .  i n v a c u o , and completely  as much as p o s s i b l e of t h e a vial  of a b s o l u t e  s t o r e d i n the  ethanol  were p o o l e d  i n v a c u o , d i s s o l v e d i n 3 ml  aqueous  and  100 the  ml  as of  effluent  to  and hormone 50$  of 50$  This solution  ethanol combined  was  the r e s i d u e t r e a t e d with  dissolve,  insoluble freezer.  with  determined.  F r a c t i o n s 41 t o j6  freezer.  evaporated  was  of c e l l s  as hormone I, e v a p o r a t e d  i n vacuo, d i s s o l v e d i n 3 ml i n the  fraction  and  the  solution  50$ and  m a t e r i a l were t r a n s f e r r e d  to  122 III.  Interaction To  in  clarify  o f t h e erogens-.with-'ion . exchange!" r e s i n s the importance  t h e a d s o r p t i o n of t h e erogens  equilibrium and  distribution  the s o l u t i o n Amberlite  converted  to the K  washed  of t h e erogens  IR-120 c a t i o n  water,  distilled  water  capped  Three into  until  1.0  screw  The r e s i n acid  and t h e m o i s t  resin  +  i tto settle, equilibrated  0.1 M KHgPO^.  samples the r e s i n  were i n v e r t e d  draining,  5-0  been e q u i l i b r a t e d was s e t up. Porapak  over  samples  After  the r e s i n  The s e c o n d of r e s i n  sample  which  direction  tube,  shaker, w i t h t h e i r  of t h e erogens  buffer,  resin  w i t h which  capped'  l o n g axes  a t 20°C f o r 1-5  acetone,  hours  between t h e s o l u t i o n  i n the After the  the r e s i n to drain.  t h e r e s i n had  A control  of erogens  were  was  o f a c e t o n e and  t o w e l f o r 5 minutes  20 u n i t s  The t u b e s  of K  with  The p r o c e d u r e +  containing  ml o f t h e .05 M KHgPO^^O^  of motion,  equilibrated  had been l e f t  was added t o each t u b e .  To each  of d i s t i l l e d  o f 50$ a c e t o n e .  the tubes  a paper  and each  i n 15 ml o f t h i s  ml o f t h e s o l u t i o n  Q,)' were added.  reciprocating  ina  15 ml o f 2 N p o t a s s i u m  with a 1 t o 1 mixture  The sample  5-0  water  were weighed  and d e c a n t i n g t h e b u f f e r .  wash had b e e n d e c a n t e d ,  containing  converted  was s t o r e d  resin  was t h e n  f o r m was washed w i t h t h r e e changes  samples  water,  with  Two o f t h e r e s i n  form by adding  +  was r e p e a t e d t h r e e t i m e s .  bution  resin  and s h a k i n g v i g o r o u s l y .  One o f t h e K  similarly  with  Excess  sample was washed w i t h t h r e e 15 ml p o r t i o n s  allowing  After  was t h e n  and washed  of t h e moist  cap t u b e s .  .05 M KHgPO^ by s u s p e n d i n g  third  more d i s t i l l e d  t o t h e b o t t o m , t h e s u p e r n a t a n t was d e c a n t e d  water.  +  water.  gram p o r t i o n s  h y d r o x i d e t o each,  H  was washed  t h e w a s h i n g s were n e u t r a l .  were c o n v e r t e d t o t h e K  resin  conditions.  jar.  16 X 125 mm  settled  various  phase  f o r m w i t h 2 N p o t a s s i u m h y d r o x i d e , and  +  was removed b y f i l t r a t i o n , tightly  under  acid,  form with 2 N h y d r o c h l o r i c  +  "binding  between t h e r e s i n  exchange r e s i n  2 W hydrochloric  again with d i s t i l l e d  the H  and n o n - p o l a r  i n i o n exchange r e s i n s , t h e  phase was d e t e r m i n e d  distilled  to  of i o n i c  tube  b u t no r e s i n ,  (eluted  from  and p l a c e d on a  parallel  t o the  t o allow the d i s t r i and t h e r e s i n t o  123 reach the  equilibrium.  supernatants  The r e s i n  each  supernatant  5-5,  and b i o a s s a y e d .  2 N potassium  water, acid, of  was d i l u t e d  A-k a n i o n  and d i s t i l l e d  dry r e s i n  hydroxide,  i n screw  i n 15  each  washing  changes  with three  was e q u i l i b r a t e d the  other  50$  acetone.  were added  t o each  a second  resin  One res-in  The t u b e s  with  containing  which they  and t h e n had been  5 - 0 ml o f 0 . 1 M 100 units  s e t up. were  sample  o f 0 . 1 M KgHPOlj., and  were d r a i n e d as above,  was a l s o  base  h y d r o x i d e , and.  2  Then t h e r e s i n  experiment,  were w e i g h e d  samples  of  the r e s i n samples  portions  capped  K HP0^2  of  and l e f t  was a l l o w e d  erogens on t h e  to settle,  a d j u s t e d t o pH 5 - 5 ,  each  were  into  screw  with three converted  samples  of d i s t i l l e d  0 - 5 gm samples cap t u b e s .  changes  and d i l u t e d  base  water, water.  form  with  The o t h e r  tubes  were a g i t a t e d  resin  was a l l o w e d  adjusted  ml p o r t i o n s  5 - 0 ml o f 50$ a c e t o n e , of e r o g e n s  were added  t o pH 5-5.?  The o t h e r  as above.  w i t h t h r e e 15  resin  sample  o f 50$ a c e t o n e , A control  o f each  tube  con-  and t h e capped  at 20°C  type  drained,  t o each  tube,  ml  i n 5-0  was  prepared.  f o r 3 hours.  and t h e s u p e r n a t a n t s  and d i l u t e d  One  b u t no r e s i n ,  on t h e s h a k e r  to settle,  o f t h e samples  d r a i n e d , and r e s u s p e n d e d  i n 5 - 0 ml o f 50$ a c e t o n e .  units  form  were washed  and  taining  Two  0 . 1 M KgHPO^ and one o f  t h r e e 15  resuspended  of d r y D u o l i t e  o f 0 . 1 M KgHPOi,..  t o t h e free' base  equilibrated  i n the free  of d i s t i l l e d  four  was washed w i t h  50  t o the free  of 0 . 1 M K H P 0 ^  containing  were d e c a n t e d ,  samples  f o r bioassay.  two  ml  tube.  hours.  were e q u i l i b r a t e d  the  tube  b u t no r e s i n ,  f o r 1.5  In A-k  A control  supernatants  l/20  samples  pH  acetic  Two 0 - 5 gm  water.  ml changes  sample  distilled  1 N  ml o f 2 N p o t a s s i u m  15  A  with  water,  converted  i n 5 . 0 ml o f t h e same b u f f e r  acetone,  the  were  ml changes  The r e s i n  equilibrated.  shaker  distilled  of d i s t i l l e d  three 15  with three  suspended 50$  with  was washed  and a i r - d r i e d .  cap t u b e s  form, b y s u s p e n d i n g  t o s e t t l e , and  0 . 5 $ soytone,  l/k w i t h  exchange r e s i n  water,  allowed  and a d j u s t e d t o pH 5 . 5 .  were d e c a n t e d  from  Duolite  was t h e n  l / l O f o r bioassay.  were  Then t h e decanted,  124 IV. F u r t h e r  attempts  at p u r i f i c a t i o n  E r o g e n s were p r o d u c e d Fernbach of and  flasks,  incubated  i n 500 ml l o t s on a s h a k e r  t h e c u l t u r e s a t 64-0 nm was measured t h e c u l t u r e s were h a r v e s t e d  turbidity. minutes angle  The c e l l s  at f u l l  head):.  a t 20°C.  on samples t a k e n  one day a f t e r  in a clinical  The s u p e r n a t a n t s  were p a s s e d  diameter  a t a f l o w r a t e o f 200 m l / h o u r .  carded.  The r e s i n  t h e e r o g e n s were e l u t e d w i t h  eluate over, in  was washed w i t h  was c o n c e n t r a t e d and t h e n  a tightly  washed w i t h  capped  vial  through  for 5  water  2 cm i n i n t e r n a l  The e f f l u e n t  dryness of  acetate  i n vacuo. water  mixture  i n a freezer.  ate from diluted  was  and 4 ml o f e t h y l  acetone,  The w a t e r - e t h y l  the phases,  and t h e  4 ml p o r t i o n o f e t h y l  l/50 f o r b i o a s s a y .  extraction,  The  raffin-  and t h e aqueous s o l u t i o n o f  m a t e r i a l s were made up t o 50 ml w i t h  w i t h b u t a n o l was t h e n  evaporated t o  a c e t a t e e x t r a c t s were made up t o  and d i l u t e d  l/50 f o r b i o a s s a y .  50 ml  The e x t r a c t s were  acetate.  a second  five  were d i s s o l v e d i n 8 ml  was c e n t r i f u g e d t o s e p a r a t e ethyl  i n 50 ml  and e x t r a c t e d w i t h  The e x t r a c t e d s o l i d s  the butanol  extracted  distilled  The P o r a p a k r e s i n  s a t u r a t e d with water.  The combined  50 ml w i t h  The  f o l l o w e d by 500 ml o f d i s t i l l e d  aqueous phase r e - e x t r a c t e d w i t h acetate.  water,  no more a c e t o n e  t o remove water d r o p l e t s and t h e n  distilled  dis-  reuse.  of n - b u t a n o l  centrifuged  was  100 ml o f 50$ a c e t o n e .  saturated with n-butanol,  portions  HW,  a bed o f 10  100 mg o f t h e f r e e z e - d r i e d e l u a t e was s u s p e n d e d of  peak  The f r e e z e - d r i e d e l u a t e was s t o r e d  100 ml o f a c e t o n e  water b e f o r e  they reached  200 ml o f d i s t i l l e d  i n vacuo u n t i l  freeze-dried.  daily,  c e n t r i f u g e ( I E C model  grams o f P o r a p a k Q i n a c h r o m a t o g r a p h y t u b e  and  The t u r b i d i t y  were removed by c e n t r i f u g a t i o n  speed  i n 2800 ml  o f Gluc-AmS  The s o l u t i o n  concentrated  50$ a c e t o n e , and  of m a t e r i a l s e x t r a c t e d  i n v a c u o t o remove  acetone,  and f r e e z e - d r i e d . At batches  another  time  of c u l t u r e  t h e 50$ a c e t o n e  supernatant  v a c u o t o remove a c e t o n e ,  P o r a p a k e l u a t e s from, s e v e r a l  were combined,  and t h e n  concentrated i n  e x t r a c t e d f o u r times  w i t h two  125 volumes and in  of n - b u t a n o l .  evaporated 10  ml  acetate up  mixture ml  centration The  was  with  acetone  1 ml  to dryness  of water p l u s  t o 50  The  The  was  10  from  and  solution  freeze-dried ethanol  solute  The  ethanol.  Two  and  minute f r a c t i o n s  from  t o 17k  40  assay  vacuo t o d r y n e s s . The Gluc-AmS  and  was  and  The  aqueous s o l u t i o n then  extracts  with  f o u r 50  distilled 50  ml  water,  ml) ml  by with  water p l u s  as ml  centrifugation, 50$  acetone  u.1  soytone,  pH  day  old  fractions  u l added t o  and  each  evaporated  i n 10  ml  of  of 2259-7 c u l t u r e  a bed  of 10  in 75$  and  erogens three  The  cells.  resin  extracted with  and  completely  100  of e t h y l and  the  diluted  ml  ml The  of  The n-butanol,  butanol in_ v a c u o .  p o s s i b l e i n 10  acetate.  100  c u l t u r e s were  to dryness as  The  was  were e l u t e d w i t h 5 litre  evaporated  in  grams of P o r a p a k Q  p o r t i o n s of n - b u t a n o l .  10  ml/min, 50  i n v a c u o t o remove a c e t o n e . was  dissolved  the  0.25  c e n t r i f u g e d t o remove  e l u a t e s from  were c e n t r i f u g e d and  r e s i d u e was  separated  (170  ab-  freezer.  through  concentrated  by  bioassay,  o n l y 10  of  ethanol,  even numbered  g l a s s f u n n e l 6 cm. i n d i a m e t e r .  distilled  of  of a one  dissolved  foam. from. 5 l i t r e s  passed  acetone.  combined,  1 drop  again with  i n t h e M i c r o f e r m . was  washed w i t h  ml  The  r e s i d u e was  s t o r e d i n the  a sintered 50$  The  collected  supernatant  to  of 0-5$  1.0  medium.  were a s s a y e d  ml  in  top  gel in  f l o w r a t e was  F r a c t i o n s 42-174 were combined  tube.  acetone,  (k,k)  100  with  i n o c u l a t e d with n  gm. of s i l i c a  For  and  -  a p p l i e d t o the  collected.  autoclaved,  con-  e x t r a c t s were g r o u n d and  The  diluted  of 2259 6 i  of  were  made  was f r e e z e - d r i e d .  i n ethanol generated  X.  was  l/200 f o r b i o a s s a y .  eluted with  was  culture  The  section  water-ethyl  e x t r a c t s o l u t i o n by  c o n t a i n i n g 50  each f r a c t i o n  5-5>  and  the  The  dissolved  aqueous l a y e r  diluted  i n a mortar,  a g r a d i e n t of water  of C h a p t e r  acetate.  butanol  column was  method  of  the  and  r e s i d u e was  the  removed  column  on  of e t h y l  then  a chromatography  of  ml  The  acetone,  of a b s o l u t e  20  i n vacuo.  c e n t r i f u g e d , and  i n vacuo,  by  e x t r a c t s were c e n t r i f u g e d ,  50$  combined  followed  n-butanol  The  ml  phases  aqueous phase was  made  l/200 f o r b i o a s s a y .  were up The  126 raffinate  from the b u t a n o l e x t r a c t s  for bioassay.  The a c e t o n e was  extract  solution  V. T h i n  layer  Thin  and t h e e x t r a c t  of c e l l u l o s e  ml of d i s t i l l e d  1 minute, drying,  acetone-0.1  t h e hormone  were p r e p a r e d by s p r e a d i n g a  was  cellulose  suspended  mixed  ( J . T.  Baker  i n an e l e c t r i c b l e n d e r  After  on 20 X 20 cm. g l a s s  i n section  p r e p a r e d by s i l i c a IV.  The p l a t e  drying,  f r o m each zone ml of 0 . 5 $  was  was  developed i n 0.1  was  divided  scraped into  into nine  a test and  of 2 2 5 9 - 6 .  plates  spotted  were d e v e l o p e d acid  (4o/4o/20)  zones.  17  i n each  w i t h samples  in ethyl and  inoculated  chloroform-methan-  The a r e a s between t h e o r i g i n  the  solvent  i n t o t e n zones,  off  and a s s a y e d as  were d i v i d e d  per in  plate)'.  described  The s t a r t  chloroform-methanol-0.1 by s c r a p i n g  were s c r a p e d  w i t h 0.5 ml each o f t h e  were s t r e a k e d line  i n section  was  N ammonia  o f f zones  assaying the scrapings.  1  IV ( i . e .  compressed  50$ a c e t o n e , and t h e n t h e p l a t e s  located  which  and  above.  F o u r TLC p l a t e s hormone p r e p a r a t i o n  of  acetate-  •:oiT-0etLcN ,'amm.onia ( 4 0 / 2 5 / 2 . 5 ) front  front  t u b e and  After  with conjugation tubes  Similarly,  ethanol-0.1 N hydrochloric  N  The t u b e s were  on a d r y goods c y c l e ,  t h e two e r o g e n p r e p a r a t i o n s  chapter  g e l chromatography  s o y t o n e , pH 5 - 5 -  of c e l l s  assay wasdetermined.  I I of t h i s  t h e a r e a between t h e s o l v e n t  1 d r o p e a c h f r o m a one day c u l t u r e  hours, the f r a c t i o n  in  2 cm f r o m t h e bottom, w i t h 25 u l of described  f o r 10 m i n u t e s  plates.  (l/l).  f o r each p r e p a r a t i o n  i n 1.0  autoclaved  acid  spotted  i n section  and t h e o r i g i n  water,  cellulose  were washed by a s c e n d i n g development  aqueous ammonia.  with  freeze-dried.  the layers  10 u l of t h e e r o g e n s  The  from, t h e  750 u t h i c k  I preparation  as d e s c r i b e d  i n vacuo  in a layer  N hydrochloric  One p l a t e and  was  3/l00  diluted  chromatography  layers  Co.) i n 175 After  distilled  of 35 grams of m i c r o c r y s t a l l i n e  slurry for  (140 ml) was  1150  by b r i e f  units  development  were d e v e l o p e d t w i c e i n  (40/25/2-5)-  cm h i g h and 5 mm  The c e l l u l o s e  The e r o g e n s wide,  and  from. R f 0 . 0 5 t o 0 . 5 0  were  127 was  scraped  ture  o f f , powdered  chromatography  erogens  i n a mortar,  and p o u r e d  3 / l 6 " glass  column made f r o m  cellulose  f r o m R f O.85 t o O.95 was s i m i l a r l y  powdered,  and e l u t e d  H-l in  solution,  and a n o t h e r  zones,  The  cellulose  f r o m R f 0 . 0 5 t o O.3O  off,  powdered,  left  half,  and t h e H-2  the plate  15/25)-  pyridine-water before  use).  Zones 1  for  u l of t h e erogens  generated  drive  potassium  iodide  in  layer,  and s p r a y e d  from from,  w i t h 50$  g e l chromatography  with a 0.5$ s o l u t i o n  a t 105°C f o r 10 m i n u t e s .  was h e a t e d  A  p r e p a r a t i o n was p l a c e d c o n t a i n i n g c h l o r i n e gas  permanganate.  acid  After  with  removal  a t 105°C f o r 15 m i n u t e s t o  with a s o l u t i o n starch  c o n t a i n i n g 0.2$  (Rydon- a n d / S m i t h  I952).  were s p o t t e d w i t h 50 u l o f e r o g e n p r e p a r a t i o n  one c o r n e r , 2 cm. from, each  first  just  1/800 f o r b i o a s s a y .  sprayed  of p o t a s s i u m  and 0 . 1 $ s o l u b l e  Two TLC p l a t e s  i n n-butanol-  and e l u t e d  p r e p a r e d by s i l i c a  25 p i o f e r o g e n  the plate  o f f chlorine,  The c e l l u l o s e  5 ml o f c o n c e n t r a t e d h y d r o c h l o r i c  5 ml o f a 10$ s o l u t i o n from, t h e t a n k ,  o f a TLC p l a t e ,  The c e l l u l o s e  o f f , powdered,  i n a chromatography tank  by m i x i n g  to the  was r e d i s t i l l e d  f o r assay.  n i n h y d r i n i n acetone, and h e a t e d 5 minutes  scraped  o f t h e p l a t e was s c r a p e d  The e l u a t e ( 2 ml) was d i l u t e d  p l a t e b e a r i n g another  and t h e  cm wide and 5 mm h i g h were s c r a p e d  were s p o t t e d on a c e l l u l o s e of  half,  as u s u a l .  (thepyridine  0 . 6 4 t o 0 . 7 3 was s c r a p e d  25  and b i o a s s a y e d .  were  w h i c h was d e v e l o p e d  a r e a f r o m R f 0.4-5 t o O.9O  acetone.  plate  The e l u a t e was s t r e a k e d a l o n g t h e  TLC p l a t e ,  (30/30/30)  (4o/4o/lO).  acid  plate  developed  i n n-butanol-acetic acid-water ( 6 0 /  t o 0.40 on t h e H - l s i d e  of another  were  on t h e H - l p l a t e  were l o c a t e d  powdered, and e l u t e d .  origin  Rf  zones  scraped o f f ,  was s t r e a k e d w i t h t h e  each  eluate to the right  The  The  The H - l e l u a t e was a p p l i e d  was d e v e l o p e d  The e r o g e n  f r o m R f O.3O  the  and e l u t e d .  tubing.  The p l a t e s  f r o m R f 0 . 3 5 t o 0 . 7 0 on t h e H-2  cellulose  off,  w i t h t h e H-2.  5 mm. wide, were s c r a p e d f r o m  Ten  and  One TLC p l a t e  acetate-ethanol-0.1 N hydrochloric  ethyl  a minia-  w i t h 50$ a c e t o n e .  ( d e s i g n a t e d H-l)- were e l u t e d (H-2).  into  edge.  i n chloroform-methanol-0.1  The p l a t e s  N ammonia  were  developed  (4o/25/2.5)>  and t h e n  128 at r i g h t angles (40/4o/l5).  One  heated  f o r 15  heated  and  i n ethyl acetate-ethanol-0.1 K hydrochloric p l a t e was  minutes.  sprayed  The  sprayed  w i t h 0.5$  other p l a t e  with s t a r c h - i o d i d e  was  ninhydrin exposed t o  solution.  acid  and chlorine,  129 Results I.  Instability The  erogen  hydroxide per  I f no  attempt  When t h e  brown c o l o u r was  acid,  the  and  column.  when t h e anion  extract after  weighing The  More pigment  resin  more brown c o l o u r  e l u t e d from the 105  was  exchange  was  the  potency  applied to  was  The  f r e e z e - d r y i n g was  with  weight  column was  the  washed  e l u t e d by  regenerated  came o f f .  precipitate  activity  raffinate units.  of t h e  mater-  yellow  The  material  of about from, t h e  The  ethyl  and 1  the  anion  exchange r e s i n ,  butanol  unit/ml  the  e x t r a c t a l l showed  at the  concentrations  assayed.  butanol  e x t r a c t i o n contained  a total  acetate  e x t r a c t had  hormone  very  low  eroof  act i v i t y . The  combined b u t a n o l  125  units/mg,  and  purification  in Table The of  and  activity  at e a c h s t e p  fraction  each f r a c t i o n  number  a total  e x t r a c t s had of  of t h e  a specific  activity  30,500 u n i t s . procedure  are  The  of  yield  summarized  XV.  to r e l a t i v e  N  potassium  I.O92 grams.  a fluffy  1  the  mg.  m a t e r i a l e l u t e d from the  redissolved  .06  limits  purification some of t h e  through  auto-  unit/ml.  exchange r e s i n ,  butanol  1250  1  about  sample  been d e s t r o y e d ,  solution  hydroxide,  The  at  had  hormone  0.023 units  confidence  concentrate  acetic  genic  activity  The  hormone  straight  ial  erogen  .058).  (95$  potassium  conjugation  ammonia had  and  units/ml  f o r e a c h sample was  First  anion  0.15  had  .002  limits  showed no  i n 10$  autoclaved  confidence  .25)-  expected  II.  sample  in 1 N  been a u t o c l a v e d  N hydrochloric acid  i n 1 N KC1  claved and  The  (95$  ml  1  and  activity.  samples w h i c h had  of  from the  erogen  in Fig.  cells  25-  with silica  conjugation  tubes  g e l column has  c o n c e n t r a t i o n , and  plotted  i n the  been  against  assay  converted fraction  130  a  a  a  CD  ("lN/SlINTI) NDIlVaiN3DfvDD N C J I - C  Fig.  25.  Chromatography  with  a gradient  confidence  of water  intervals.  of f i r s t  erogen p r e p a r a t i o n  in ethanol.  Bars  represent  on  silica  gel  131 Table  XV. P r o g r e s s o f t h e e r o g e n  Stage  purification  T o t a l erogen activity u n i t s / ml  Specific activity u n i t s /mg  Original culture supernatant  124,000  0.037  X 1  Charcoal  100,000  5-0  x 135  81  eluate  Purification  Yield  100  A n i o n exchange eluate  59,000  54  x 1470  48  Butanol  30,500  125  x 34oo  25  III.  extract  Interaction  the K  times 50$  acetone  I.9)  times  These  a c t i v i t y as t h e c o n t r o l .  as much e r o g e n  data i n d i c a t e  that  resin  +  adsorption  was a l m o s t  .05 M KH P02j. c o n t a i n i n g 2  experiment  The .05 M KHgPO^-  gave s i m i l a r  exchange r e s i n  of t h e erogens  complete,  50$ a c e t o n e .  b u t was  2  limits  from 0.05 M negligible  Repetition  of t h e  results.  c o n t a i n e d no d e t e c t a b l e  .1 M K H P 0 ^ - 5 0 $ a c e t o n e  The 50$  had no e r o g e n a c t i v i t y .  .1 M KgHPOij. s u p e r n a t a n t f r o m t h e D u o l i t e  confidence  resin  .01 and . 0 5 )  a c t i v i t y as t h e c o n t r o l .  supernatant from the H  The  exchange  s u p e r n a t a n t had I . 3 6 ( 9 5 $ c o n f i d e n c e l i m i t s 1.0 and  KHgPOi,. o n t o t h e r e s i n from  resins  form c o n t a i n e d .03 (95$ c o n f i d e n c e l i m i t s  +  as much e r o g e n  acetone  w i t h i o n exchange  . 0 5 M KHgPO^ s u p e r n a t a n t f r o m t h e c a t i o n  The in  of t h e erogens  erogen  A-4 anion  activity.  The  s u p e r n a t a n t , however, had . 8 6 ( 9 5 $  . 6 1 and l . l ) t i m e s  as much e r o g e n a c t i v i t y  as t h e c o n t r o l . In  t h e second  natants The  base  0.11 and 0 . 1 6 )  as much e r o g e n  A-4 r e s i n  .1 M KgHPOij. c o n t a i n e d 0. 14 times  The 50$ a c e t o n e  f o r m had 0.15  almost  with  as much e r o g e n  super-  a c t i v i t y as t h e c o n t r o l .  which had  (95$ c o n f i d e n c e  a c t i v i t y as t h e  supernatant from the r e s i n  (95$ c o n f i d e n c e l i m i t s  completely adsorbed  water  had v e r y low e r o g e n a c t i v i t y .  supernatant from the D u o l i t e  been e q u i l i b r a t e d control.  t h e two d i s t i l l e d  A-4 r e s i n  from the D u o l i t e  50$ a c e t o n e  limits  experiment,  i n the free  .11 and . 1 8 )  times  Thus t h e e r o g e n s a r e  on t h e a n i o n exchange r e s i n  from  132 distilled erogens 0.1  o r . 1 M KgHPO^.  water  a r e adsorbed  M-K2HP04-50$  F r o m 5 0 $ a c e t o n e , 85$ o f t h e a t pH 7 o r h i g h e r ,  on t h e r e s i n  acetone,  15$  only  or less  but from  of t h e erogens a r e  adsorbed. IV.  Further The  attempts  at p u r i f i c a t i o n  specific activity  Q adsorption  from  shake  (95$ c o n f i d e n c e l i m i t s n-butanol erogenic 1375  extraction, activity.  units  25,000  43 a n d 5 4 ) .  The erogens  per  mg  of  units,  combined  2259-7  culture  Porapak  specific and  I670).  only by  activity  Porapak  development  o f 1500 u n i t s / m g  from  had a  total  o f 250  units  of 54,000  litres  of a c t i v i t y .  units,  and a  (95$ c o n f i d e n c e l i m i t s  1355  condensed  t h e hormone  the  erogen  of t h e erogens  prepared  f o a m was 660 u n i t s / m g  (95$  596 a n d 7 2 4 ) .  chromatography  i n the three  for  acetic  i n Fig.  f r o m t h e f o a m f r o m 15  activity  The d i s t r i b u t i o n o f e r o g e n  solvent  42 t o 174  The s p e c i f i c a c t i v i t y  limits  V. T h i n l a y e r  I preparation  preparation  activity  solvent  systems  of section of erogen  o f H - l were r e c o v e r e d a f t e r  systems.  on t h e TLC p l a t e s i s shown  after  i n F i g . 27  I I , ' a n d i n F i g . 28 f o r  of section IV.  t h e o r i g i n a l 4600 u n i t s  units  g e l c o l u m n i s shown  c o n t a i n e d 162,000 u n i t s  had a t o t a l  adsorption  confidence  100  activity  The r a f f i n a t e from t h e b u t a n o l e x t r a c t i o n c o n t a i n e d  1260 u n i t s .  Of  contained  228 a n d 2 7 2 ) .  eluates  i n Gluc-AmS  The b u t a n o l e x t r a c t  showed l o w  solution contained  and a s p e c i f i c a c t i v i t y  (95$ c o n f i d e n c e l i m i t s  The  extract  butanol extract  pooled from f r a c t i o n s  of 23,000  activity  units/mg  The d i s t r i b u t i o n o f e r o g e n i c  among t h e f r a c t i o n f r o m t h e s i l i c a 26.  acetate  The n - b u t a n o l e x t r a c t The second  Porapak  The r a f f i n a t e f r o m t h e  and t h e e t h y l  of erogens.  prepared.by  s u p e r n a t a n t s was 48  culture  of erogens.  units  of t h e erogens  The a c t i v i t y  o f H-2  activity,  preparative  less  than  TLC i n t h e f o u r  a f t e r TLC i n n - b u t a n o l -  a c i d - w a t e r was v e r y l o w , a n d a p p e a r e d  at Rf 0.4 t o 0.6.  133  o  INTO f^IlVc^N33vtD N Fig.  26.  Chromatography  gel with a gradient confidence  of s e c o n d  of water  intervals.  erogen p r e p a r a t i o n  i n ethanol.  Bars  on  represent  silica  tn  1.Q4.  134 0.1 N ammonia  go., M r— 0-4i  ^ 0-i O.Q 0.0  w  » o-al  cn  0.2 ethyl  0.4  0*6  O'B  1.0  acetate-ethanol-0.1 N hydrochloric acid (40/4o/20)  - o-si E 0*4i O.E  H  0-0  o-s  0.4  0.6  O.B  1-0  LOU.  O'B  c h l o r o f or m-me t h a n ol-OraioN (40/25/2.5)  "  ammonia  O'E 0>4  J  0-S O.Q 0*0 27.  Thin layer  +  0*2  +  0*4  chromatography  RF  + 0*6  +  O'B  o f hormone I p r e p a r a t i o n .  1*0  jL  135 O.l N  ammonia  go.. M  3 o.oJ o*s ethyl  M  tn -  0-6  0*4  0*B  1*0  a c e t a t e - e t h a n o l - 0 .1 N. h y d r o c h l o r i c a c i d (40/40/20)  0-(  & o.a  S  O-Q 0*0 1'Qj.  0-2  0*4  O.B  0-6  1.0  c h l o r o f o r m - m e t h a n o l -1@m3lo W1 ammo n i a  0-B  (40/25/2.5)  ^  O.E 0-4 0.£ 0*Q 0*0 28.  Thin  layer  +  0*S  +  0-4  chromatography  +  RF  0*6  1  0*B  1*0  of s e c o n d e r o g e n p r e p a r a t i o n .  136 The and  erogen p r e p a r a t i o n  a "bluish p u r p l e  dimensional  colour  gave a p i n k  colour  i n the Rydon-Smith  TLC, n e i t h e r n i n h y d r i n  a colour  r e a c t i o n i n the area  expected  t o be.  with test.  ninhydrin, After  nor the Rydon-Smith  where t h e e r o g e n a c t i v i t y  test was  two gave  137 Dis cussion The either cates in  complete  1 N hydrochloric that  high 98$  1 N KG1  increased claving high dry  destroyed the  85$.  The  s t a t e at f r e e z e r temperatures  In t h e f i r s t was  increased  A-k  anion  The  five  the  capacity  lost  11 f o l d  by  of c o n c e n t r a t e  of t h e r e s i n ,  and  effluent.  out a t k°C,  of exposure  have  eluted  Further silica  and  The  purification  initial  only  retarded  achieved.  The  of t h e e r o g e n s rational o f 50$  design  erogens  kept  and  i n the  by  could  of a c t i v i t y could  have  and  was have  elution was  o f pH  and  steps removed the  the 1 N a c e t i c a c i d  may  from the  resin.  the erogen  and  little  buffers. was  separation  was  on  adsorption  to allow  schemes.  from Porapak  was  activity  solvents  purification  after  chromatography  r e s i n s were s t u d i e d  The  more ability  r e s i n s suggested  the non-polar b i n d i n g  backbone  by  be-  organic  eliminate  been  lost  pyridine acetate  tried  low.  exceeded  may  have been  erogens  and  by t h e r e s i n ,  t o the p o l y s t y r e n e  e l u t i o n from D u o l i t e  c a t i o n exchange  i o n exchange  might  activity  of t h e hormone I I p r e p a r a t i o n  of i o n exchange  solvent  used  Also,  acetone t o e l u t e the erogens  this  pH  d e s t r u c t i o n of t h e e r o g e n s  conditions  effects on  been  the a c e t i c a c i d eluant  ammonium a c e t a t e  Under a l l t h e slightly  has  adsorption  a l l of the adsorbed  using  a t extreme  some o f t h e e r o g e n s  to the a c i d .  g e l chromatography  attempted,  auto-  been  the s p e c i f i c  on and  However, some o f t h e a c t i v i t y  cause  that  attempt,  but the r e c o v e r y  by f r e e z e - d r y i n g , t o m i n i m i z e acid.  not  have  f o r s e v e r a l months w i t h o u t  adsorption  resin,  gram p o r t i o n s  i n t h e column  may  activity. purification  exchange  were c a r r i e d  not  preparation  pH  during  has  avoided  ammonia  at n e u t r a l  l o s s of a c t i v i t y are u n s t a b l e  indibe  concentration  of the b i o a s s a y  the standard  l o s s of  salt  should  i n 10$  autoclaving  high  A l t h o u g h the erogens  noticeable  o f pH  Autoclaving  and  The  conditions  temperature,  on a u t o c l a v i n g i n  extremes  of t h e e r o g e n s .  the d e s t r u c t i o n .  measured.  and  of the a c t i v i t y  under  activity  a c i d or 1 N p o t a s s i u m h y d r o x i d e  temperatures  the h a n d l i n g  destroyed in  l o s s of e r o g e n  of t h e i o n exchange  of t h e resins.  133 , Comparison and  of the a d s o r p t i o n  of t h e erogens  0.05 M KR" P0^-50$ a c e t o n e  0.1 M K HP0]^ and 0.1 M K HP0i .-50$ a c e t o n e support  2  this  interpreted adsorption preted  view.  from s o l u t i o n s  containing  attraction  f r o m 50$ a c e t o n e  that  t h e erogens  alone.  onto t h e H  can a c q u i r e  erogen  adsorption petition  adsorpindicates  charge at s u f f i c i e n t l y isoelectric  A p p a r e n t l y t h e erogens  of t h e erogens  The e q u a l i t y  i s w e l l below  2  f o r t h e i o n exchange  sites  of t h e erogen  of a d s o r p t i o n  7-  The d e c r e a s e d  c o u l d be c a u s e d by com-  by p h o s p h a t e .  activity  In a l l cases  i s adsorbed,  components may be bound t o a d i f f e r e n t distribution  a r e neg-  form suggests that the  i n 0.1 M K HP02j.-50$ a c e t o n e  ment o f t h e e r o g e n  inter-  50$ a c e t o n e r e d u c e s b u t does n o t  adsorption.  point  where o n l y p a r t active  can be  form of t h e r e s i n  +  a t pH 7 and i n t h e f r e e base  isoelectric  can be  The c o m p l e t e  c h a r g e d a t pH 7 and above.  on r e s i n  A-4  b i n d i n g , and  50$ a c e t o n e  a positive  On D u o l i t e A-4 r e s i n ,  atively  onto D u o l i t e  The e r o g e n m o l e c u l e s may have an a c i d i c  eliminate  and from.  A d s o r p t i o n f r o m aqueous s o l u t i o n  tion  point.  J  as t h e sum o f n o n - p o l a r and i o n i c  as i o n i c  low pH.  2  onto A m b e r l i t e IR-120,  2  2  f r o m 0.05 M KH P0i,.  each  degree.  between t h e r e s i n  of t h e  Measure-  phase  and 50$  a c e t o n e a t more pH's c o u l d be used t o d e t e r m i n e t h e i s o e l e c t r i c pH o f t h e e r o g e n s ,  and t o check t h a t  anion  and c a t i o n  could  p r o v i d e an i n d e p e n d e n t  of t h e c h a r g e  on t h e e r o g e n s  Extraction n-butanol The  increases  activity  i s held  solved An large cells  Electrophoresis and magnitude  a t v a r i o u s pH's. f r o m aqueous s o l u t i o n s activity,  i s not l e f t  but gives  i n the r a f f i n a t e .  i s destroyed during  with low r e c o v e r y .  Possibly  e v a p o r a t i o n of t h e b u t a n o l ,  of the r e s i d u e  which  cannot be r e - d i s -  e v a p o r a t i o n of the b u t a n o l .  abbreviated  f o r m o f t h e b i o a s s a y has been  number o f samples  O n l y one dose  obtained with  e s t i m a t e of the s i g n  the s p e c i f i c  i n the part  after  are consistent.  of t h e erogens  missing a c t i v i t y  this or  exchangers  the r e s u l t s  arising  o f e a c h sample  used  from chromatographic  f o r the  separation.  i s a s s a y e d , and t h e f r a c t i o n o f  producing conjugation tubes  i s c o n v e r t e d t o erogen  concen-  '139 tration  using  values  f o r the regression  ED50 f r o m a s t a n d a r d is  less  the  accurate  assay  of h i g h e r  separated  numbers  on s i l i c a  two peaks  g e l as d e s c r i b e d  of a c t i v i t y ,  peak was u n r e t a r d e d  and  by t h e column,  i n the assays  four  of a l l these  t h e e r r o r s of e s t i m a t i o n  on s i l i c a  erogen a c t i v i t y the  e l u t i o n volume e x p e c t e d preparation The  silica After  relation  f r a c t i o n s was n e a r maximum,  I peak a p p e a r e d . peak,  f o r hormone I I .  at  described  development  0 . 1 ET ammonia, b o t h  with  erogen  R f 0 - 7 5 t o 1.0.  On TLC i n e t h y l  N hydrochloric acid  (40/4o/20),  imate R f ' s 0 . 7 5 n d O . 9 5 were a  components  with  Rf 0 . 0 5 , i n both  other  Rf's  of t h e s e c o n d they  acetate-ethanol-0.1 acetic  acid-water  approx-  preparation.  (40/25/2.5) d e m o n s t r a t e d  The hormone I p r e p a r a t i o n  polar  component,  has more of t h e more p o l a r (H-l)  and l e s s  erogen p r e p a r a t i o n  showed d i s t i n c t  with  0 - 3 and O . 9 5 , and p o s s i b l y a t h i r d  more o f t h e l e s s  erogen p r e p a r a t i o n  and a s e c o n d  acetate-ethanol-  two components,  i n d i c a t e d i n each  preparations.  When t h e more p o l a r TLC,  R f about  i n c h l o r of orm-met h a n o l - 0 . 1 EF ammonia  seems t o c o n t a i n  during  preparations  0.1,  component  with  change  that  o f s e c t i o n IV was i n v e s t i g a t e d by T L C . with  active  approximately  i n section I I .  i n t o a component  TLC  A l l of t h e  It i s possible  were s e p a r a t e d 0.1  However, t h e  between hormone I and t h e e r o g e n peak f r o m t h e  g e l chromatography  with  on f r a c t i o n 3 3 -  c a u s e d by some c h e m i c a l  procedures  i n the  and emerged w i t h t h e  sub-peaks.  i n one b r o a d  hormone I i s an a r t i f a c t , the  The m a t e r i a l  of a  o f s e c t i o n IV was c h r o m a t o -  g e l , no hormone  was f o u n d  but allows  are l a r g e .  When t h e hormone p r e p a r a t i o n graphed  method  a suggestion  T h e r e may be a s m a l l peak c e n t e r e d  main peak a p p e a r s t o c o n t a i n  response  This  i n section II  with  first The  standard  of samples.  peak a t t h e end o f t h e chromatogram. volume.  and  procedure,  third void  slope  simultaneously.  than the f u l l bioassay  Chromatography clearly  s e r i e s assayed  line  polar  N hydrochloric (60/15/25)-  acid  component.  (H-2) components  were s e p a r a t e d  Rf's i n the solvent  and t h e  by p r e p a r a t i v e  systems  ethyl  ( 4 0 / 4 o / l O ) and  T h e r e was no s e p a r a t i o n  n-butanolof e i t h e r  140 H-l  or H-2  into  solvents. tography may  The  more components yield  i n four  solvent  incompletely The  test  colour  indicate  systems  eluted  chromatography  was  amino a c i d s  were s e p a r a t e d f r o m t h e r e a c t i n g The  lack  and/or  material.  primary p u r i f i c a t i o n  supernatants. gentle  as  and  from  procedure  Porapak  Q,  culture  (660  f o r the erogens  eluting  a c e t o n e , and  The  erogens  have  effective  from crude in high  been  method  culture  yield on  under columns  of acetone i n  purification. a c h i e v e d by activity  foaming  of e r o g e n s  s u p e r n a t a n t s (48  can  be  prepared  units/mg)  units/mg). the best  appears  i n t h e foam f r o m t h e  foam t o remove  could  concentration  of t h e s p e c i f i c  foam  gel  f o r m a t i o n i n the  of t h e e r o g e n s  of t h e e r o g e n s from  silica  peptides.  and  eluted  p r o v i d e good  present information,  erogens  condensed the  adsorption  condensed  With the  might  comparison  Porapak  can be  Chromatography  purification  seen from  of the erogens  an a p p r o p r i a t e  solvent,  The by  using  Q, i s a r a p i d  erogens  conditions.  of P o r a p a k , water  The  the Rydon-Smith  colour  c a u s e d by  for  have  m a t e r i a l s "by two d i m e n s i o n a l  of d e t e c t a b l e  on P o r a p a k  erogens  or t h e y may  after  were e x p e c t e d t o be  Adsorption  chroma-  cellulose.  and  a r e a where t h e e r o g e n s insufficient  The  chromatography,  with ninhydrin  i n these  successive  v e r y low.  the erogen p r e p a r a t i o n  contains  chromatography.  after  f r o m t h e powdered  reactions  that  chromatography  of erogen a c t i v i t y  have "been d e s t r o y e d d u r i n g  "been  during  cells,  the erogens  extracting  preliminary  to include culture,  passing w i t h 50$  purification  concentration  centrifuging  of  the  the supernatant through acetone, d i s t i l l i n g  t h e aqueous s o l u t i o n  off  with n-butanol.  SUMMARY AND GENERAL DISCUSSION  Ikl Tremella as  the sole nitrogen  microelements acids. is  selectively  causes  the only  vitamin  nitrogen  sulphate,  in  The  A defined salts  Porapak,  t h e growth solution  into  solution  other  Thiamine  requirement.  i n cultures using supports  can be d e t e c t e d  to  attraction.  t o be b e l o w k.5.  n-butanol,  ammonium ammonium,  good  growth  tubes from the c e l l s  The  and on t h e p o l y s t y r e n e 50$  f o r c e s , and p a r t  suggests three  G-10,  by g e l chroma-  a molecular  of s i l i c a  appears  on Sephadex  cannot be e s t i m a t e d  separated  exchange  pH o f t h e e r o g e n s  are a l s o adsorbed  on columns  acetone.  c a t i o n and a n i o n  i s due t o n o n - p o l a r  weight  i n ethanol  solvents.  can be e l u t e d w i t h on b o t h  The e r o g e n s  Chromatography  c o e f f i c i e n t of  on a c t i v a t e d c h a r c o a l , r e q u i r i n g  However, u l t r a f i l t r a t i o n o f water  organic  The i s o e l e c t r i c  molecular  by t h e i r  but are not e x t r a c t e d  f o r desorption,  of the b i n d i n g  so t h a t t h e i r  amino  amino  medium c o n t a i n i n g g l u c o s e ,  polar  from which they  Part  gradient  with  shows a  of c o n j u g a t i o n  are a l s o adsorbed  below 7 5 0 .  containing  The e r o g e n s have a p a r t i t i o n  resins.  tography.  c o n c e n t r a t i o n of  i n media  and m i c r o e l e m e n t s  amounts by l e s s  acetate  ionic  A high  fungus  t h e pH d r o p  are s t r o n g l y adsorbed  erogens  ammonium b u t n o t n i t r a t e  e  2259-7-  2259-6.  detectable  pyridine  s  or i n c o m b i n a t i o n  -  f r o m aqueous  resin  alone  source.  t o induce  erogens  u  growth  f o r which t h i s  thiamine,  strain  2-3  n  erogens produced by 2 2 5 9 7  The of  a  f o r growth.  slows  counteracts  of T. m e s e n t e r i c a  ability  c  s l o w and a b n o r m a l growth o f t h e c e l l s .  Sodium a c e t a t e as  source  L-asparagine,  acids,  2259-7  mesenterica  weight  g e l with a  active  erogenic  components.  S i m i l a r chromatography  of a l a t e r  p r e p a r a t i o n of  erogens  only  Thin  chromatography  gave  demonstrated  at l e a s t  preparation. with  non-polar The  fraction length  one a c t i v e peak.  layer  two a c t i v e components  i n each  The e r o g e n s may be amino a c i d s side  or s h o r t  peptides  chains.  concentration of 2259-6  erogen  o f e r o g e n s has been  cells  of the conjugation  which produce tubes,  shown t o a f f e c t t h e  conjugation  and t h e number  tubes, the  of c o n j u g a t i o n  142" tubes per  cell.  A statistically  bioassay  f o r the  of  which produce  cells  unit  of  erogen  a standard  has  activity  pH  established.  and  reached  cell  best  nitrogen  tration  same t i m e  source  of v a r i o u s  adjusted  as t h e  sources and  i n the as  components  for highest  erogen  for  preliminary concentration  large  scale  through the  to p u r i f y  hormone a c t i v i t y . cation  of 0.2  incubation  s u c h as  time,  are  best  2259-7 c u l t u r e s  of  peak t u r b i d i t y .  and  Ammonium  defined  The  is  The  erogens  Foaming  concen-  are  removed  high  research  m a t e r i a l can  will  be  purified  tube  number  the  of d i s t i n c t  three,  process.  peaks  some of t h e s e separated  activity  studies  of T.  TLC  on  and  mesenterica,  cells  of  into  clarify the  of  the  purifi-  Then  using  thesis,  on t h e  mechanism  from budding T_.  to  mesenterica  silica be  at l e a s t  of  activity two  the  relation  the from  active  components  Monitoring the  suggests  artifacts  erogen The  gel  from, separation  between  components r e s o l v e d by  conjugation and  on  may  values.  may  the  in this  p r o d u c e d by  Chromatography  show s i m i l a r R f  of e r o g e n  type  erogens  l a y e r chromatography.  g e l column by  Parallel mating  thin  peaks  a silica  in  begin.  I n d i v i d u a l peaks  g e l column a r e  components by different  although  can  yields  develop  studies  g r o w t h of t h e  production  2259-7 i s s t i l l u n c l e a r .  silica  erogens  f o r chemical c h a r a c t e r i z a t i o n .  redirect  which they  low  erogens.  developed  by  preparative  of t h e  need t o  r e c o v e r i e s of t h e  e r o g e n s become a v a i l a b l e ,  least  when  i s a u s e f u l method  e r o g e n s have g i v e n  When p u r i f i e d  The  the  medium have been  purification  methods f o r e r o g e n p r o d u c t i o n  conjugation  of  have been  soytone  supernatant  of t h e  mg  ammonium i s p o o r .  cultures.  the  Future  methods w i t h  sufficient  the  of  The  production.  Attempts  on  activity bioassay  production.  i s bubbled  the  response.  t e s t e d f o r erogen p r o d u c t i o n .  air  at  the  fraction  2259-7 c u l t u r e s by t h e foam w h i c h forms s p o n t a n e o u s l y  from  the  u s i n g the  as  d e n s i t y f o r the  tube p r o d u c t i o n ,  at t h e  practical quantitative  Optimum c o n d i t i o n s  Maximum, e r o g e n a c t i v i t y is  tubes  i s defined  Complex n i t r o g e n  conjugation  and  been d e v e l o p e d ,  conjugation  preparation.  temperature, for  erogens  valid  hormones f r o m t h e  from other  species  of  TLC. other  Tremella  143 would the  be  of  erogens  Interest of  2259-7  for  the  that  similarities  they  might  to  show.  and  differences  from  1  BIBLIOGRAPHY  144 Abraham, E . P., and G. G. E . Newton. I967. P e n i c i l l i n s and c e p h a l o s p o r i n s , i n A n t i b i o t i c s V o l . I I , D. G o t t l i e b and P. D. Shaw, E d . S p r i n g e r - V e r l a g . pp l - l 6 . Ahmad, S. S., and P. G. M i l e s . 1970. H y p h a l f u s i o n s i n t h e w o o d - r o t t i n g f u n g u s S c h i z o p h y l l u m commune. Genet. Res. Camb. 15_: I9-28. Aiba,  S., A. E . Humphrey, and N. F. M i l l i s . Engineering. A c a d e m i c P r e s s , New Y o r k .  1965. Biochemical 333 pp.  A r i e n s , E . J . , A. M. S i m o n i s , and J . M. van Rossum. I964 . The mode o f a c t i o n of b i o l o g i c a l l y a c t i v e compounds, i n M o l e c u l a r P h a r m a c o l o g y , E . J . A r i e n s , E d . A c a d e m i c P r e s s , New Y o r k . 503 PPArsenault,  G. P., K. Biemann, A. W. B a r k s d a l e , and T. C. M c M o r r i s . The s t r u c t u r e of a n t h e r i d i o l , a s e x hormone i n A c h l y a bisexualis. J . Am. Chem. S o c . _9_0: 5 6 3 5 - 5 6 3 6 .  I968.  A s a t o o r , A., and C. E . D a l g l i e s h . 1956. The u s e o f d e a c t i v a t e d charcoals f o r the i s o l a t i o n of aromatic substances. J. Chem. S o c . I 9 5 6 : 2291-2299A u s t i n , D. J . , J . D. Bu'Lock, and G. ¥ . Gooday. 1969. Trisporic a c i d s : S e x u a l hormones f r o m Mucor mu cedo and B l a k e s l e a trispora. N a t u r e 2 2 3 : II78-II79. A u s t i n , D. J . , J . D. Bu'Lock, and D. D r a k e . 1970. The b i o s y n t h e s i s o f t r i s p o r i c a c i d s f r o m / 3 - c a r o t e n e v i a r e t i n a l and trisporol. E x p e r i e n t i a 15.:. 348-349. B a n b u r y , G. H. 1954. P r o c e s s e s c o n t r o l l i n g zygophore f o r m a t i o n and z y g o t r o p i s m i n Mucor mu c e d o . N a t u r e 173'• ^99Banbury, G. H. 1955P h y s i o l o g i c a l studies i n the Mucorales. I I I . The z y g o t r o p i s m o f z y g o p h o r e s o f Mucor mucedo. J . E x p t l . B o t a n y 6_: 2 3 5 - 2 4 4 . Bandoni,  R. J .  1963-  Can. J . Bot .  4-1:  Conjugation  467-474.  i n Tremella  mesenterica•  B a n d o n i , R. J . 1 9 6 5 Secondary c o n t r o l of c o n j u g a t i o n i n Tremella mesenterica. Can. J . B o t . _43: 6 2 7 - 6 3 0 . B a r k l e y , D. S. as a c r a s i n  I65 :  1969. Adenosine-3'>5'-phosphate: identification i n a s p e c i e s o f c e l l u l a r s l i m e mold. Science  1133-1134.  B a r k s d a l e , A. W. 1963aThe u p t a k e o f exogenous hormone A by c e r t a i n s t r a i n s of A c h l y a . M y c o l o g i a 55_: 164-171-  145 B a r k s d a l e , A. W. conjugation  1963bThe r o l e of hormone A d u r i n g s e x u a l i n A c h l y a a m b i s e x u a l i s • M y c o l o g i a _5_5_: 627-632..  B a r k s d a l e , A. W. I969. fungi. S c i e n c e 66_:  S e x u a l hormones of A c h l y a 83I-837 .  and  other  B a r k s d a l e , A. W. 1970N u t r i t i o n and a n t h e r i d i o l - i n d u c e d b r a n c h i n g i n A c h l y a a m b i s e x u a l i s . M y c o l o g i a _6_2: 411-420. Bauch, R. 1925U n t e r s u c h u n g e n ttber d i e E n t w i c k l u n g s g e s c h i c h t e und S e x u a l p h y s i o l o g i e der U s t i l a g o b r o m i v o r a und U s t i l a g o g r a n d i s . Z. B o t a n . 17_: 129-177. B h a l e r a o , U. T., J . J . P l a t t n e r , and H. R a p o p o r t . 1970Synt h e s i s of d l - s i r e n i n and d l - i s o s i r e n i n . J . Am. Chem. Soc. 92 : 3429-3433B i s h o p , H. 1940. A s t u d y of s e x u a l i t y M y c o l o g i a _3_2: 505-529-  i n Sapromyces  reinschii.  B i s t i s , G. N. I956. S e x u a l i t y i n A s c o b o l u s s t e r c o r a r i u s I . M o r p h o l o g y of t h e a s c o g o n i u m ; plasmogamy; e v i d e n c e f o r a s e x u a l hormone mechanism. Am. J . B o t . _4_3_: 389-394. B i s t i s , G. N. 1957S e x u a l i t y i n Ascobolus s t e r c o r a r i u s I I . P r e l i m i n a r y e x p e r i m e n t s on v a r i o u s a s p e c t s of t h e s e x u a l process. Am. J . B o t . 4-4: 436-443B i s t i s , G. N., sexuality 88O-89I.  and J . R. R a p e r . 1963H e t e r o t h a l l i s m and i n As c o b o l u s s t e r c o r a r i u s . Am. J . B o t . _5_0:  Brough, S. G. 1970The B i o l o g y of T r e m e l l a bambusina Ph.D. T h e s i s , 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 .  Sacc.  B u l l e r , A. H. R. 1933A c t i o n at a d i s t a n c e i n v e g e t a t i v e h y p h a l f u s i o n s and i t s t h e o r e t i c a l e x p l a n a t i o n , i n R e s e a r c h e s on F u n g i V o l . 5: 68-72. Bu'Lock, J . D., D. D r a k e , and D. J . W i n s t a n l e y . 1972. Speci f i c i t y and t r a n s f o r m a t i o n s of t h e t r i s p o r i c a c i d s e r i e s of f u n g a l sex hormones. Phytochemistry 11: 2011-2018. B u r g e f f , H. 1924. U n t e r s u c h u n g e n Uber S e x u a l i t a t und s i t i s m u s b e i Mucorineen I . B o t a n . Abhandl.. _4: (cited in Machlis, I966). B u r n e t t , J . H. I968. F u n d a m e n t a l s of M y c o l o g y . A r n o l d ( P u b l i s h e r s ) L t d . , London. 5 4 6 pp.  Para5-135-  Edward  C a g l i o t i , L., G. C a i n e l l i , B. C a m e r i n o , R. M o l d e l l i , A. P r i e t o , A. Q u i l i c o , T. S a l v a t o r i , and A. S e l v a . 1967The s t r u c t u r e of t r i s p o r i c - C - a c i d . Tetrahedron Suppl. 7: 175-187-  11+6  C a i n e l l i , G., P. G r a s s e l l i , arid A. S e l v a . I967, S t r u t t u r a d e l l ' a c i d o t r i s p o r l c o B. Chim. I n d . ( M i l a n ) 49: 628-  629.  C a r l i l e , M. J . , and L. M a e h l i s . 1 9 6 5 . The r e s p o n s e o f male gametes o f A l l o m y c e s t o t h e s e x u a l hormone s i r e n i n . Am. J . B o t . _5_2~ 4 7 8 - 4 8 3 . C o r e y , E . J . , K. A c h i w a , and J . A. K a t z e n e l l e n b o g e n . 1969. T o t a l s y n t h e s i s of d l - s i r e n i n . J . Am. Chem. S o c . 91: 43I8-432O. C o r e y , E . J . , and K. A c h i w a . I97O. A s i m p l e s y n t h e t i c r o u t e to d l - s i r e n i n . Tetrahedron Lett. 2_6: 2245-2246. D r i v e r , C. H., and H. E . W h e e l e r . 1 9 5 5 - A s e x u a l hormone i n Glomerella. M y c o l o g i a hj_: 3 I I - 3 1 6 . D u n t z e , W., V. MacKay, T. R. Manney. 1 9 7 0 . Saccharomyces c e r e v i s i a e : A d i f f u s i b l e sex f a c t o r . S c i e n c e 168: 1 4 7 2 Edwards, J . A., J . S. M i l l s , J . Sundeen, and J . H. F r i e d . The . s y n t h e s i s o f t h e f u n g a l s e x hormone a n t h e r i d i o l . Chem. S o c . £ l : 1 2 4 8 - 1 2 4 9 .  I969.  J . Am.  Edwards, J . A.,-V. Schwarz, J . F a j k o s , M. L. Maddox, and J . H. Fried. 1 9 7 1 - F u n g a l s e x hormones. The s y n t h e s i s o f ( i ) 7 ( t ) , 9 ( t ) - t r i s p o r i c a c i d B m e t h y l e s t e r . The s t e r e o c h e m i s t r y a t C-9 o f t h e t r i s p o r i c a c i d s . Chem. Comm. 1 9 7 1 '• 292-293. Edwards, J . A., J . Sundeen, W. Salmond, T. Iwadare, and J . H. Fried. 1 9 7 2 . A new s y n t h e t i c r o u t e t o t h e f u n g a l s e x hormone a n t h e r i d i o l , and t h e d e t e r m i n a t i o n o f i t s a b s o l u t e stereochemistry. Tetrahedron Lett. 1972: 791-794. Ende, H. v a n den. 1967. 215 : 211-212.  Sexual  factor  of the Mucorales.  Ende, H. v a n den. I968. R e l a t i o n s h i p between carotene synthesis i n B l a k e s l e a t r i s p o r a .  1298-1303.  Nature  s e x u a l i t y and J . Bact. 96:  Ende, H. van den, A. H. C. A. Wiechmann, D. J . Reyngond, and T. Hendriks. 1970. Hormonal i n t e r a c t i o n s i n Mucor mucedo and B l a k e s l e a t r i s p o r a . J . Bact. 101: 423-428. Ende, H. v a n den, and D. Stegwee. 1971Mucorales. B o t . R e v . _3J_: 22-36. Ende,  Physiology  of sex i n  H. v a n den, B. A. Werkman, and M. L. v a n den B r i e l . 1 9 7 2 T r i s p o r i c a c i d s y n t h e s i s i n mated c u l t u r e s o f t h e f u n g u s Blakeslea trispora. A r c h . M i k r o b i o l . 86: 175-184.  ihi F e o f i l o v a , E . P. 1970. C o n d i t i o n s of f o r m a t i o n of t r i s p o r i c a c i d s i n combined c u l t u r e s of p l u s s t r a i n s and minus s t r a i n s of B l a k e s l e a t r i s p o r a . M i c r o b i o l o g y 39: 266-270. F i n n e y , D. J . 1964. Second e d i t i o n . 668 pp.  S t a t i s t i c a l Method i n B i o l o g i c a l A s s a y . C l a r k s G r i f f i n & Company L t d . , London.  F i n n e y , D. J . 1971Probit Analysis. U n i v e r s i t y Press. 333 PP-  Third edition.  Cambridge  F l e g e l , T. W. I968. Some a s p e c t s of c o n j u g a t i o n i n t h e genus T r e m e l l a D i l l ex F r . MSc. T h e s i s , U n i v e r s i t y of B r i t i s h Columbia. Gooday, G. W. I968. Hormonal c o n t r o l of s e x u a l r e p r o d u c t i o n i n Mucor mucedo • New P h y t o l . 6j_: 815-821. Green, D. M., J . A. Edwards, A. ¥. B a r k s d a l e , and T. C. M c M o r r i s . 1971. The i s o l a t i o n and s t r u c t u r e of 2 3 - d e o x y a n t h e r i d i o l and t h e s y n t h e s i s of i t s C-22 epimer. Tetrahedron 27: H99  -  1203-  G r i e c o , P. A. I969. Chem. Soc. 91: Kehl,  The t o t a l s y n t h e s i s of d l - s i r e n i n . 566O-566I.  J.  Am.  H. .1937- E i n B e i t r a g zur M o r p h o l o g i e und P h y s i o l o g i e der Z y g o p h o r e n von Mucor mucedo. Arch. M i k r o b i o l . 8: 379-406. — ( c i t e d i n M a c h l i s , 1966).  K'dhler, K. 1967D i e c h e m i s c h e n G r u n d l a g e n der B e f r u c h t u n g (Gamone), i n Handbuch der P f l a n z e n p h y s i o l o g i e V o l . 18, ¥. R u h l a n d , E d . Springer-Verlag, Berlin. pp 2 8 2 - 3 2 0 . K r a f c z y k , H. I93I. D i e Z y g o s p o r e n b i l d u n g b e i P i l o b o l u s c r y s t a l l i n u s • Ber . D e u t . B o t a n . Bes . 4_9: 141-146 . ( c i t e d i n M a c h l i s , I966). K r a f c z y k , H. 1935D i e B i l d u n g und Keimung der Z y g o s p o r e n von P i l o b o l u s c r y s t a l l i n u s und s e i n h e t e r o k a r y o t i s c h e s M y z e l . B e i t r . B i o l . P f l a n z . _23_: 349-396. ( c i t e d i n M a c h l i s , I966) K u b o t a , T., T. Tokoroyama, Y. T s u k u d a , H. Koyama, and A. M i y a k e . 1973I s o l a t i o n and s t r u c t u r e d e t e r m i n a t i o n of b l e p h a r i s m i n , a c o n j u g a t i o n i n i t i a t i n g gamone i n t h e c i l i a t e Blepharisma. S c i e n c e 179: 400-402. L e m l i c h , R. 1968. P r i n c i p l e s of foam f r a c t i o n a t i o n , , i n P r o g r e s s i n S e p a r a t i o n and P u r i f i c a t i o n V o l . 1, E. S. P e r r y , E d . I n t e r s c i e n c e P u b l i s h e r s ( d i v i s i o n of J o h n W i l e y & S o n s ) , New York. pp I-56.  148 Levi,  J . D.  1956.  754.  Mating r e a c t i o n i n yeast.  Nature  177'  753-  McGahen, J . W., and H. E . W h e e l e r . 1951G e n e t i c s of G l o m e r e l l a IX. P e r i t h e c i a l d e v e l o p m e n t and plasmogamy. Am. J . B o t .  38:  610-617.  M c M o r r i s , T. C., and A. W. B a r k s d a l e . I967. Isolation sex hormone f r o m t h e water mold A c h l y a b i s e x u a l i s .  215:  of a Nature  320-321.  M c M o r r i s , T. C., and R. S e s h a d r i . 1971. antheridiol. Chem. Comm. I971:  Synthetic 1646.  studies  on  M c M o r r i s , T. C , T. A r u n a c h a l a m , and R. S e s h a d r i . 1972. A p r a c t i c a l s y n t h e s i s of a n t h e r i d i o l . Tetrahedron Lett.  26 :  2673-2676.  M a c h l i s , L. 1958aE v i d e n c e f o r a s e x u a l hormone i n P h y s i o l . P l a n t arum 11.: I8I-I92 . M a c h l i s , L. Nature  1958b. A procedure f o r p u r i f i c a t i o n l8l: 1790-1791.  of  Allomyces. sirenin.  M a c h l i s , L. 1958c. A s t u d y of s i r e n i n , t h e c h e m o t a c t i c sexual hormone from, t h e w a t e r m o l d A l l o m y c e s • P h y s i o l . P l a n t a r u m 11: 845-854. M a c h l i s , L. I966. Sex hormones i n f u n g i , i n The F u n g i V o l . I I , G. C. A i n s w o r t h and A. S. Sussman, E d . Academic P r e s s , New York. pp 4 1 5 - 4 3 3 . M a c h l i s , L. 1969. Zoospore P h y s i o l . P l a n t a r u m 22: M a c h l i s , L. 1972. M y c o l o g i a 6_4: Machlis,  chemotaxis I26-I39.  The coming 235-247.  of age  i n the  of sex  watermold  hormones  Allomyces.  in plants.  L.,  W. H. N u t t i n g , M. W. W i l l i a m s , and H. R a p o p o r t . P r o d u c t i o n , i s o l a t i o n , and c h a r a c t e r i z a t i o n of sirenin. B i o c h e m i s t r y 5: 2147-2152.  I966.  M a c h l i s , L., and E . R a w i t s c h e r - K u n k e l . 1967. Mechanisms of g a m e t i c a p p r o a c h i n p l a n t s , i n F e r t i l i z a t i o n V o l . 1, C. Metz and A. Monroy, E d . A c a d e m i c P r e s s , New Y o r k . pp  161.  M a l l e t t e , M. F. I969. E v a l u a t i o n of growth by p h y s i c a l and c h e m i c a l means, i n Methods i n M i c r o b i o l o g y V o l . 1, J . R. N o r r i s and D. W. R i b b o n s , E d . A c a d e m i c P r e s s , New Y o r k ,  pp  521-566.  B. 117-  149 M a r k e r t , C. L. I949. S e x u a l i t y i n t h e fungus, N a t u r a l i s t _8_3: 227-231. Mori,  K., and M. M a t s u i . p l a n t s e x hormone.  I969. S y n t h e s i s Tetrahedron Lett.  Glomerella.  Am.  of racemic s i r e n i n , 51: 44-35-4438.  a  M U l l e r , D. G., L. J a e n i c k e , M. D o n i k e , and T. A k i n t o b i . 1971. Sex a t t r a c t a n t i n a brown a l g a : chemical s t r u c t u r e . S c i e n c e 17_1: 8 l 5 - 8 l 6 . Nair,  K. G. I966. P u r i f i c a t i o n and p r o p e r t i e s o f 3**5 ' - c y c l i c n u c l e o t i d e p h o s p h o d i e s t e r a s e f r o m dog h e a r t . Biochemistry  5_:  150-157-  N i c h o l a s , D. J . D. 1965U t i l i z a t i o n of i n o r g a n i c n i t r o g e n compounds and amino a c i d s by f u n g i , i n The F u n g i V o l . I , G. C. A i n s w o r t h and A. S. Sussman, E d . A c a d e m i c P r e s s , New Y o r k . pp 349-376. N i e d e r w i e s e r , A. 1971Use o f n e u t r a l p o l y s t y r e n e r e s i n f o r r a p i d d e s a l t i n g and f r a c t i o n a t i o n , o f n o n - p o l a r amino a c i d s and n o n - p o l a r o l i g o p e p t i d e s . J . Chromatog. _6l: 81-94. N i e d e r w i e s e r , A., and P. G i l i b e r t i . I97I. S i m p l e e x t r a c t i o n o f i n d o l e d e r i v a t i v e s f r o m aqueous s o l u t i o n b y a d s o r p t i o n on neutral polystyrene resin. J . Chromatog. 6l: 95-99N u t t i n g , W. H., H. R a p o p o r t , and L. M a c h l i s . I968. The s t r u c t u r e of s i r e n i n . J . Am. Chem. S o c . _9_0: 6434-6438. P l a t t n e r , J . J . , U. T. B h a l e r a o , and H. R a p o p o r t . I969. S y n t h e s i s of d l - s i r e n i n . J . Am. Chem. S o c . _9_1: 4933-  4934. P l a t t n e r , J . J . , and H. R a p o p o r t . 1971The s y n t h e s i s o f dand 1 - s i r e n i n and t h e i r a b s o l u t e c o n f i g u r a t i o n s . J . Am. Chem. S o c . _9_1 1758-1761. :  P l e m p e l , M. 1957D i e S e x u a l s t o f f e der Mucoraceae. Mikrobiol. 2_6: 151-174.  Arch.  P l e m p e l , M. i960. D i e z y g o t r o p i s che R e a k t i o n I . M i t t e i l u n g . P l a n t a 5_5_: 254-258.  b e i Mucor i n eenrz.  P l e m p e l , M. I962. D i e z y g o t r o p i s c h e R e a k t i o n III. P l a n t a 58.: 5O9-52O.  b e i Mucorineen  P l e m p e l , M. 1963D i e chemischen Grundlagen b e i Zygomyceten. P l a n t a _59_: 492-508.  der S e x u a l r e a k t i o n  P l e m p e l , M., and W. Dawid. 1961. D i e z y g o t r o p i s c h e R e a k t i o n b e i M u c o r i n e e n I I . M i t t e i l u n g . P l a n t a 56: 438-446.  150 P o l l a r d , C. J . 1 9 7 0 . I n f l u e n c e o f g i b b e r e l l i c a c i d on t h e i n c o r p o r a t i o n o f 8--^C a d e n o s i n e i n t o a d e n o s i n e 3 ' , 5 c y c l i c phosphate i n b a r l e y aleurone l a y e r s . Biochim. B i o p h y s . A c t a 201: 5 1 1 - 5 1 2 . , _  R a p e r , J . R. evidence  Bot.  1939S e x u a l hormones i n A c h l y a • I. I n d i c a t i v e f o r a h o r m o n a l c o - o r d i n a t i n g mechanism.. Am.. J .  2_6:  639-650.  R a p e r , J . R. I9U0. S e x u a l hormones i n A c h l y a . II. Distance r e a c t i o n s , c o n c l u s i v e evidence f o r a hormonal c o - o r d i n a t i n g  mechanism.  Am. J . B o t .  _2J_: 162-I73.  R a p e r , J . R. 1942a. S e x u a l hormones i n A c h l y a . I I I . Hormone A and t h e i n i t i a l male r e a c t i o n . Am. J . B o t . _29: I59-I66. R a p e r , J . R. 1942b. S e x u a l hormones i n A c h l y a . V. Hormone A', a male s e c r e t e d augmenter or a c t i v a t o r o f hormone A. Proc. Wat. A c a d . S c i . _2§_: 5 0 9 - 5 1 6 . R a p e r , J . R. 1950a. S e x u a l hormones i n A c h l y a . V I . The hormones o f t h e A-complex. P r o c . N a t . A c a d . S c i . _3_6: 5 2 4 - 5 3 3 R a p e r , J . R. 1950b. S e x u a l hormones i n A c h l y a • V I I . The hormona l mechanism i n h o m o t h a l l i c s p e c i e s . B o t . Gaz . 112: 1 - 2 4 . R a p e r , J . R. 1952. C h e m i c a l r e g u l a t i o n o f s e x u a l p r o c e s s e s i n t h e T h a l l o p h y t e s . B o t . Rev. _ l 8 : 4 4 7 - 5 4 5 R a p e r , J . R. 1967The r o l e o f s p e c i f i c s e c r e t i o n s i n t h e i n d u c t i o n and d e v e l o p m e n t o f s e x u a l o r g a n s and i n t h e d e t e r m i n a t i o n o f s e x u a l a f f i n i t y , i n Handbuch d e r P f l a n z e n p h y s i o l o g i e V o l . 1 8 , W. R u h l a n d , E d . S p r i n g e r - V e r l a g , Berlin. pp 2 1 4 - 2 3 4 . R a p e r , J . R., and A. J . H a a g e n - S m i t . 1942. S e x u a l hormones i n Achlya. I V . P r o p e r t i e s o f hormone A o f A. b i s e x u a l i s . J . B i o l . Chem. JL4_3: 311-320. R e s c h k e , T. 1 9 6 9 . D i e Gamone aus B l a k e s l e a t r i s p o r a . hedron L e t t . 39_: 3 4 3 5 - 3 4 3 9 R o b i s o n , G. A., R. W. B u t c h e r , and E . W. S u t h e r l a n d . C y c l i c AMP. A c a d e m i c P r e s s , New Y o r k . 532 pp.  Tetra1971.  R o n s d o r f , L. 1 9 3 1 liber d i e c h e m i s c h e n B e d i n g u n g e n v o n Wachstum und Z y g o t e n b i - l d u n g b e i Phycomyces b l a k e s l e e a n u s . P l a n t a 14: 482-514. (cited i n Machlis, I966). R o t h s t e i n , A. 1 9 6 5 U p t a k e and t r a n s l o c a t i o n . I . Uptake, i n The F u n g i V o l . I , G. C. A i n s w o r t h and A. S. Sussman, E d . A c a d e m i c P r e s s , New Y o r k . pp 4 2 9 - 4 5 6 . Rydon and S m i t h .  1952-  see p. 153-  151 S a k a i , K., and N. Y a n a g i s h i m a . 1972. Mating r e a c t i o n i n Saccharomyces c e r e v i s i a e . I I . Hormonal r e g u l a t i o n of a g g l u t i n a b i l i t y of a t y p e c e l l s . Arch. M i k r o b i o l . 8 4 : 191-  I98.  Salomon, D., and J . P. M a s c a r e n h a s . 1971Auxin-induced s y n t h e s i s of c y c l i c 3S5 ' - a d e n o s i n e monophosphate i n Avena coleoptiles. L i f e S c i e n c e s _10: 879-885. S c h r o e d e r , W. A., R. T. J o n e s , J . C o r m i c k , and K. M c C a l l a . I962. C h r o m a t o g r a p h i c s e p a r a t i o n of p e p t i d e s on i o n exchange resins. S e p a r a t i o n of p e p t i d e s f r o m e n z y m a t i c hydrolysates of t h e , p> , and X c h a i n s of human h e m o g l o b i n s . A n a l . Chem. _3_4: 1570-1575Sherwood, W. A. 1966. E v i d e n c e f o r a s e x u a l hormone i n t h e w a t e r mold D i c t y u c h u s . M y c o l o g i a . 5_8 : 215-220. Shimoda, C., and N. Y a n a g i s h i m a . 1972. Mating r e a c t i o n i n Saccharomyces c e r e v i s i a e . I I I . Changes i n a u t o l y t i c activity. A r c h . M i k r o b i o l . _8_5_: 3IO-318. S m i t h , I. 1958. Chromatographic Techniques. M e d i c a l Books, L t d . , London. 3 0 9 pp.  W i l l i a m Heinemann  S t e e l , R. G. D., and J . H. T o r r i e . i960. P r i n c i p l e s and Prodedures of S t a t i s t i c s . M c G r a w - H i l l Book Company, L t d . , New Y o r k . 48l pp. S u t t e r , R. P. trispora.  1970. T r i s p o r i c acid synthesis Science l 6 8 : I59O-I592.  in Blakeslea  S u t t e r , R. P., and M. E . R a f e l s o n , J r . I968. S e p a r a t i o n of b e t a f a c t o r s y n t h e s i s from s t i m u l a t e d beta-carotene s y n t h e s i s i n mated c u l t u r e s of B l a k e s l e a t r i s p o r a . J. Bacteriol. 95: 426-432. S u t t e r , R. P., D. Capage, and T. L. H a r r i s o n . 1971Aspects of t r i s p o r i c a c i d s y n t h e s i s i n B l a k e s l e a t r i s p o r a . Abstr. F i r s t I n t e r n . M y c o l o g i c a l C o n g r e s s , p. 92~ (cited in Werkman and van den Ende, 1 9 7 3 ) Thomas, D. des S., and J . T. M u l l i n s . 1967w a l l s o f t e n i n g i n plant morphogenesis: in Achlya. Science 156: 84-85-  R o l e of e n z y m a t i c Hormonal i n d u c t i o n  Thomas, D. des S., and J . T. M u l l i n s . I969. C e l l u l a s e i n d u c t i o n and w a l l e x t e n s i o n i n t h e w a t e r mold A c h l y a a m b i s e x u a l i s . Physiol.. Plant. 22: 347-353-  152 Thomas, D. M., and T. ¥. Goodwin. 1967S t u d i e s on c a r o t e n o genesis i n Blakeslea t r i s p o r a . I. General observations on s y n t h e s i s i n mated and unmated s t r a i n s . Phytochemistry  6.:  355-360.  Thomas, D. M., R. C. H a r r i s , J . T. 0 . K i r k , and T. W. Goodwin. 1967S t u d i e s on c a r o t e n o g e n e s i s i n B l a k e s l e a t r i s p o r a . I I . The mode o f a c t i o n of t r i s p o r i c a c i d . Phytochemistry  6_:  361-366.  Ueyama, A. 1972. Chemical b i o l o g y of s e x u a l f a c t o r s i n the fungi. I . I n d u c t i o n o f t h e gamete i n i t i a l s b y t h e c u l t u r e e x t r a c t s i n n e t e r o t h a l l i c A b s i d i a g l a u c a Hagem. Trans. M y c o l . S o c . J a p . 13_: 66-70. V e r k a i k , C. 1930Uber das E n t s t e h e n von Z y g o p h o r e n von Mucor mu cedo ( + ) u n t e r B e e i n f l u s s u n g e i n e s von Mucor mu cedo (-) abgeschiedenen S t o f f e s . K o n i n k l . Ned. Akad. Wetenschap., Proc. C 33.: 6 5 6 - 6 5 8 . ( c i t e d i n M a c h l i s , I966). Warren, C. 0 . , and J . T. M u l l i n s . I969. R e s p i r a t o r y metabolis.m in Achlya ambisexualis. Am. J . B o t . jj6_: 1135-1142. Warren, C. 0 . , and B. H. S e l l s . 1971Cellulase induction d u r i n g s t a n d a r d i z e d v e g e t a t i v e growth i n A c h l y a . J . Gen. Microbiol. 6j_: 3 6 7 - 3 6 9 . Werkman, T. A., and H. v a n den E n d e . 1973T r i s p o r i c acid synthesis i n Blakeslea trispora. I n t e r a c t i o n between p l u s and minus m a t i n g t y p e s . Arch. M i k r o b i o l . (in press). W o l f , F. J . I969. Biochemistry.  S e p a r a t i o n Methods i n O r g a n i c C h e m i s t r y A c a d e m i c P r e s s , New Y o r k . 237 PP•  and  Wood, H., M. L i n , and A. B r a u n . 1972. The i n h i b i t i o n o f p l a n t and a n i m a l a d e n o s i n e 3 ' > 5 ' - c y c l i c monophosphate p h o s p h o d i e s t e r a s e s by a c e l l - d i v i s i o n - p r o m o t i n g substance from t i s s u e s of a higher p l a n t s p e c i e s . Proc. Nat. Acad. S c i . U.S.A. _6_9_: 403-406. Y a n a g i s h i m a , N.  110-118.  I969.  Sexual  hormones i n y e a s t .  Planta 87:  Y a n a g i s h i m a , N. 1971I n d u c e d p r o d u c t i o n o f a s e x u a l hormone in yeast. Physiol. Plant. 2_4: 26O-263. Y a n a g i s h i m a , N., C. Shimoda, K. S a k a i , and N. T a k a o . I969. Mating r e a c t i o n i n yeast with s p e c i a l r e f e r e n c e t o sexual hormones. XI I n t . B o t . C o n g r . A b s t r a c t p. 2 4 5 Y a n a g i s h i m a , N., and C. Shimoda. 1970S e p a r a t i o n o f hormones e n s i t i v e y e a s t c e l l s by d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n . P l a n t and C e l l P h y s i o l . 1 1 : 971-974.  153 Y a n a g i s h i m a , N. , and C. Shimoda. Rev. _39: 1-14.  1973-  Auxin  and y e a s t .  Bot  Rydon, H. N., and P. ¥. G. S m i t h . 1952. A new method f o r t h e t e c t i o n o f p e p t i d e s and s i m i l a r compounds on p a p e r chroma tograms. N a t u r e 169 : 922-923.  i  APPENDICES  15 4 A p p e n d i x A. F r e q u e n t l y Malt  Y e a s t Peptone  used  media.  (MYP).  Bacto Malt E x t r a c t  7.0  Bacto Yeast E x t r a c t  0 . 5 gm,  Bacto Soytone  1.0  Distilled  1  water  gm  gm  litre  Glu'cps-eeS oyib:one Medium (GS) :  D-Glucose  4.0 gm.  Bacto Soytone  4.0  Distilled  1  water  gm  litre  3 (GM3);  Growth Medium # D-Glucose  4.0 gm  Bacto  4.0 gm  Soytone  KH P0^  1.0 gm  2  MgS0 7H 0  0-5 gm  CaCl -2H 0  0 .1 gm.  v  2  2  2  Microelement Citric  Stock  Solution  acid-H 0 2  5 - 0 gm.  ZnSOj^HgO  5-0 gm  FeS0^-7H 0  0.71  CuS0^-5H 0  0 . 2 5 gm  MnS0^-H 0  0 . 0 5 gm  H^BO^  0 . 0 5 gm  2  2  2  gm.  Ra Mo0^-2H 0  0 . 0 5 gm  Distilled  95 ml  2  stored  2  water  in refrigerator  C o n j u g a t i o n Medium ( C j M ) D-Glucose  2.0 gm  Bacto  2.0 gm.  Soytone  Distilled  water  1  litre  155 Glucose-Ammonium  S u l p h a t e Medium (Gluc-AmS).  D-Glucose  10.0 gm.  (NH4) S0^  1.0  gm  KH POi .  1.0  gm  MgS0j^-7H 0  0.5  gm  CaCl -2H 0  0.1  gm  Thiamine-HC1  100  ugm  2  2  4  2  2  2  Microelement  stock  solution Distilled  water  .13 1  ml  litre  156 A p p e n d i x B.. Measurements o f c o n j u g a t i o n c o n c e n t r a t i o n s of erogen. per  UJ.QAP  14a 10* - 20. 19*  5o  „  length  Doses a r e e x p r e s s e d i n m i c r o g r a m s o f hormone ml. L e n g t h s a r e measured i n m i c r o n s .  __2.22.Q_e.0_ llo 17a 19e 6« 12J> 22« 18e 15-« 12* 12e 6-e lid  tube  l-l-i! 14e 6a _L_ 15. He 9-o  _ 12e 9e 4a 12a . 9.o 13e 9e 7J» , 4o 12e JL4-6 lie 10 a • .1.4 .o 9e 5a Aa 6e 13e 4. a.  LQ_  8« 26a 13e 18 a  21 o 14a 9e 6a 19a  12e 5© l l o l i e 13. 5e 14a 4« l l o 6 0 — ~4e 14e l i e 4a 16a 9-e 3<s 18o 5e l l o 4 , 7.e . . .6.0- _ 5o 10* I4e 18*. 1JL_ l i u He 13a 21a 14 9o l i e  _ 1 J L J  15o 18e 1 5 * — 16e 16e 14-e 14e 9* X* 12* 13* As 19e 14a 1.8.6  7e 18e 4 © — 8e 7e 8..e 13e 17o  e  15e 7o _ll_e 14« 4e 8e  0  6 66-«Q 12e 7o 4e 8o 6s 15 e 7a 5a 3_s 8__ 2a 9o 4» 8e 5.e ,_9_*. He 7o 15* 15e 6-» 3JJ 7a 5e 9a 7e 13J> JL6.a 4a 2e He -6a lift .,4s 8e '5e 7a 4o 8_e__ 5_.e_  ;  various  preparation  MSJLQ  :  l l o 18e 18-* 4a 13.  XI* 2e 4e 16e 14a 14a 4e 1-L* l i e 13& _9.* 21* 4s 2.a 9a l i e 7A 5« 6e l i e  3e 4a -1-2 e l i e 6e -13.e 5o 12e 1.5.e 7o l i e 9_c 12e l l o 5B  4A_M>-0— 5o 5e 7e 2e 4a 5© 7a 8c 8_» 8j» 9e 4o 3e 5a . Ao . 4o ' 4a 4 e 4e 10a 8_a l.n 4a 8• 10. 11. : 5 el 1.1. * 5e 4e 5a 4e _8_e JL2_e 7 a . 8e 8 c i l 0 e ; 9a 5_a  at  '— 5e 3* 8« 6a 4 e_ . 3o 4e 8_» . 4a 5 Ao. 4e 4a l i e 10a 4e 5„a 7e 7a 4e e  13e 9© 4e 11« 6_s 3e 3e  18e 2o 4e 3a 7_o_ 11*  9o 8-e~ 6e 7o __6JL_ 9e 5e it.«_. 4e 10* :5_e_ 4e 5o 6»__  l i t 4e 5_e 2* l i e 2J» 4s 9a 10. l i e 6 .  1A  2-2-2-e-O 5© 6e> 5e 5a 4e 4o 5e §.*, 3a 4 Ae 4o 4a 5_e 4« 5o 8a 4s 5a _5 a e  4a 4e 3o 2e 4o 4a 2a . la 5e A* 4e 4a JU 4a 4a la la 3c  4_o  6e 2s 5« 2a 3_»... 4e lo 3J>_ 6a la A_o_ 4a 4a 6a 5a 1 _e_ 7e 4e 4a  157  IJJJLQ  3s 4* 4 « 4 e 3 o  3o 4o 8a 4© 3« 7_» 3e 2e 4_« 2s 4 e Aa 4 s 2a  _3_9  4 o 2e -3.0 4 o 4 e -5_e 3c 3 e Ae. 5o 4* _2_B_  4s '4«  -  3s 3o 3e 5e 4 e 4e 4e lo  ___Ao 2* 4o kA 3o 2o  2_o_  3e 3e 3_s_ 4 e 2o  2©  3e 3e  2o 2e 2«  lb  2o 2s 4e  lo  2c 4 o  4.0  lo  4e 3..*_^ 3s 1e 4_s_._ 2o  lo  2.o___ 2o 3e 3J 2o  le  IJS So 3a 2a 4o 2.a 26 2o  2d 2@ 4e 4e 2s  A«  4» 2o 4,s 4o  l,e  2e 2e  __£§n5  .  lo  lo  3.© 2o  4o 6o  Is  2_e 4e 2 o  lo L  3jt 2e "3 e  158 A p p e n d i x G. A computer This 1130  IBM  British  program t o p r o c e s s  bioassay  data.  p r o g r a m has been w r i t t e n i n F o r t r a n IV, and r u n on an computer  at the B i o l o g y Data Center,  U n i v e r s i t y of  Columbia.  Input: Data 1.  cards  A first  required are:  card, s p e c i f y i n g  t h e number  (1-99)  °f a s s a y s  t o be  processed. 2.  F o r each assay, ations  3.  an i n i t i a l  card g i v i n g  ( 1 - 2 0 ) , i n 12 f o r m a t ,  assayed  F o r each p r e p a r a t i o n a c a r d g i v i n g in  I I format,  ation  and t h e d a t e t h e number  (12),  a s e q u e n c e number  t h e number o f p r e p a r -  the d i l u t i o n  of  cells  with  of  cells  counted  (0, -1,  :  t o t h e b a s e two  - 2 , e t c . ) i n Fh.l f o r m a t , (F4.0),  c o n j u g a t i o n tubes  (l-5)  o f doses  t h e name o f t h e p r e p a r -  (Ah), and f o r e a c h dose, t h e l o g a r i t h m n  of  (Ah) .  t h e number  and t h e t o t a l  number  (F4.0).  Output: The  program, f i r s t  with the c a l c u l a t e d the  logit  the  regression lines  the  chi-squared  sion are  of these  line  lists  the data  f o r each p r e p a r a t i o n  fraction  of c e l l s  with  fractions (Y3).  c o n j u g a t i o n tubes ( P ) ,  ( Y ) , and t h e e x p e c t e d The s l o p e  b y random  Next  comes a l i s t with  merit  sampling. o f a c t u a l and e x p e c t e d  a l l lines  parallel.  f o r the p r e c i s i o n  deviations tests  The v a l u e  lines. is  of the data from  of t h e assay.  logits  from the  and c o n f i d e n c e w i t h G, a f i g u r e  An a n a l y s i s o f t h e  the r e g r e s s i o n l i n e s  f o r d e v i a t i o n s g r e a t e r than  sampling  the regres-  that the deviations  i n t e r v a l f o r t h e common s l o p e a r e p r i n t e d , a l o n g of  from  o f t h e r e g r e s s i o n l i n e and  are given along with the p r o b a b i l i t y  regression  logits  f o r t h e d e v i a t i o n s of the data from  caused  along  follows  with  c a n be e x p l a i n e d b y random  ( h e t e r o g e n e i t y ) , and n o n - p a r a l l e l i s m o f t h e r e g r e s s i o n I f heterogeneity  calculated,  and used  is significant,  a heterogeneity  t o broaden the confidence  factor  intervals for  159 the  parameter  ED50, two  with  estimates.  95$  of r e l a t i v e  standard,  with  Finally,  confidence potency 95$  limits,  (M),  confidence  and  f o r each p r e p a r a t i o n , the the  the  limits,  logarithmn potency are  to the  relative  listed.  to  base the  PAGE  1  I, REID I. REID  // JOB T LOG DRIVE OOOO ' _  VZ MIC  CART SPEC 0001 ACTUAL  8K  CART AVAIL 0001 CONFIG  _.*EQUATIPRNTZ.PRNTY)  1  PHY DRIVE 0000  8K _  UNIVERSITY OF BRITISH'COLUMBIA BIOLOGY DATA CENTRE ON FRIDAY MAY 111 1973  USER JOB 11  TOTAL JOBS  227  // FOR *LIST SOURCE PROGRAM .... *IOCSICARD.1132 PRINTER) *LOAD+GQ C-ERRS...STNG.C  F O R T R A N  S O U R C E  S T A T E M E N T S  IDENTFCN  DIMENSION ALABI20)iD(5.20).R(5.20)»T(5.20)tP(20.5)IY(20.5).N(201 .X 1 BAR 120) . YBAR 1 20 ) . Y4 (-20 > 5 ) . Y 3 ( 20 . 5 ) > CH 111 2 0 ) . NF1 ( 2 0 ) . B ( 20 ) 1CHITI20).DCHi120).ED50I20).CLL(2C).CLU(20).STT(7) DATA STT/12.7.4.3,3.2.2.8.2.6.2.5.2.4/ TRIP)=0.5*ALOG(P/(1.-P)) READ(2.2)NR 00002 F O R M A T ! 1 2 ) DO 100 L=1.NR READ!2.3)NP.DATE 00003 F O R M A T ( I 2 .A4 ) • . WRITEI3.601DATE 60 FORMAT( 1 . TREMELLA HORMONE BIOASSAY PERFORMED .AA.//>6X PREP DO 1SE R T P Y Y3'l DO 101 J=1.NP SY = 0. sx*o. SX2-0. SXY=0. ITER=1 READI2.1)ND.ID.ALABIID).(DlI.ID).R(I.ID).T1 I.ID)tI=1.ND) 1 FORMAT 1 I 1 . 12 .A4.5 (F4.1 .2F4.0 ) ) DO 4 1 = 1 .ND P 1 I D . I ) = R I I . I D ) / T ( I . ID ) YI ID.I)=TR(P(ID.I )) SY = S Y + Y ( ID.I 1 sx*b>:+D( 11 ID) SX2=SX2+D!I. I D ) * * 2 SXY = SXY + D( I.ID)*Y(ID.I) 4 CONTINUE MIO)=ND AND=ND XBAR(ID)=SX/AND YBAR(ID)=SY/AND SSX=£X2-SX**2/AND SSXY=SXY-SX»SY/AND B1=SSXY/SSX 27 SN-=0. SX=0. SX2"=0. 1  v.  1  1  1  1  *»COMPILER .SSNI  MESSAGES**  20 I .  i o  -PAGE  - ! - . - « E-IDF O R T R A N  C - E R R S . ..STNO.C.  IDENTFCN  S T A T E M E N T S  S O U R C E  **COMPILER MESSAGES**  SY = 0. SY? = 0.._ SXY<=0. C YBAR(IDI-B1*XBAR<ID) DO, 11 I=1.ND Y4(ID.I)=C+B1*D(I.ID) Z= Y4(ID.I) _ U Z = I ../XL..+.EXE1.2._*ZJJ YW' = Z T ( P { I D . I i +QZ-1. ) / 12. »(1.-QZ)*QZI t.' = 4 . * l l . - Q Z ) * Q Z WN-.-;*T ( I . I D ) B  SN = Si;+v;N  SX=SX+WM«D< I . I D ) _S.Y_=.SYJ-J«J*X1J  SX2"SX2 + tiN*D ( I » IU >*«2 SY2=SY2+WN#YW**2 ... SXY»SXY*WN»PI I >IO.)»Y_W. 11 COfiT I i'JUE YBAR(IDI=SY/SN XBAR.Li a U S K / . S H  SSX=SX2-SX»*2/SN SSY=SY2-SY*»2/SN SSXY=SXY-SX*5Y/SN U2=SSXY/SSX C=YBAR< I0)-B2*XBAR( ID) _D.O_2J.._I.E.ljj^P Y3I ID.I )=C+fc2*D<I >ID) DIr-ABS(Y3IID.II-Y4IID.I))  H  CA  H  21 COftTIKUe GOTU 30 2b ITER=ITER+1 Bi = B2 IF(ITER-20127.27.28 . .26 WRITE13.29)ID 2V FORMAT l.'O'.5X12. ' DOES HOT CONVERGE ) GOTO 101 3 0 CHI I ( ID) = S.SY-.SSXY*»2/SSX 1  !!F 1 ( ID) =h0-2  PR = PCHI (CHIK ID) .NFK ID) ) R(ID)=B2 WRITE(3.5)ID.ALAB(ID) FORMAT('0'. 1XI2.3XA4) DO 10? 1=1.ND WRITE(3.6)D(I.ID).R<l.ID),T(l.ID).PtID.I).Y(ID.l).Y3UD I ) 6 FORMAT( .10XF5.1.2I2XF4.0).2XF5.3.2(2XF6.3 I) 102 COriT IMUE V.'KI TE (3.7) ITER .B2 .CHI 1 ( i D ) . NF1 ( I D ) . PR 7 FORMAT ( 0 ' . 'NUMBER OF ITERATIONS* '.12/' SLOPE" '.F7.4/ HETEROGEN 1FITY CHISO" ' » F8•2 . WITH '.13.' DEGREES OF FREEDOM ANDPROB= l F 7 1.4) 101 CO.NTK4UE WRITE(3.64) 64 FORMAT( OPREP DOSE Y Y4'./.' ') ITER"! 1  1  1  1  1  1  f  ptr,F  I . RFID  •>,  R T R A N  C-ERRS.  S O U R C E  ••COMPILER  MESSAGES**  34 DO 61 1=1iNP ND = f, ( ; ] DO C l J=l.ND 61 Y 4 ( : . J ) = Y 3 ( I . J ) S=,XY = n. ssx = c . SSY=0.  r  DO 104 1=1, NP Sis =0. <;x =n. SY*O. SX2=C.  ... .  SXY=0. ND=NII) .DO 105 J = l , ND 2=Y4(I.J)  CZ=1./(1.+EXP(2.*Z)) Ytt=Z+(P( I .Jl+OZ-l. ! /(?.*( 1.-0 J)*QZ) WN=4.*(1.-QZ)*OZ*T(J,I) SN = SN + Wi; SX = SX+V.'i-J*D( J. I ) SX2 = SX2fV.'l'i*D( J » I ) **2 SY«SY*WK*YW SY2=SY2friN*YW**2 SXY=SXY+WN*D(J.I)*YW 105 CONTINUE SSN( I)=SN YoAP.I i ) = SY/SN XEAR1 I)=SX/SN GS.. = GSN + SN SSXT=SX2-SX«*2/SN SSYT=SY2-SY**2/SN SSXYT=SXY-SX*SY/SN CHIT(I 1=SSYT-B1*SSXYT SSX=SSX+SSXT &SY«£iY*iSYT SSXY=SSXY+SSXYT 104 CONTINUE B2=SSXY/SSX :  '  '  '  31 32 20 63  "  "  ~  DO 20 1=1.NP C = YBAR(I )-B2*XBAR( I ) ND=N ( I ) DO 20 J=1.ND Y31I.J)=C+B2*D(J.I) DIF=ABS(Y3(I.J)-Y4(I.J)) IFID1F-.001) 31.31.32 GOTO 20 K=2 CONTINUE GOTOI33.63)>K ITER=ITER+1 B1-B2  '  !  163  O <ri _J  U)  O  _  f- •  > J  JO_!r-  H*  oj  -i ™  :><___ ) u> _> ^  —j -3"  _a Q_  . -sj * V  +  : <j __  OJ  -  OJ  I—  a— ac  .-n  —i _J  <£ .  3_'  U  -1  - h-j - r- _ H o u_ ; i o 4 < . o j a u r_ : u_ a. o s> 3a, a iu• •o a: i— y— _ x u jX _O_ __ _ >o a5J_q—x•< i-r i • O U. ii. : u uO < _ > „_ >LiJ'a.:__t•_. j,3i— n u'tl_ i O I — I . q <o j t \J r4 U  ZZ — O J rs: _) 11  LU  -1  U  L  :q_'  *-4  II  CL  <-< ^  2  11  CL < II  - J  1  ____-»<  - J  ^  -  I  ______  •di  c  i f j cn -  ' ™ i # r—4 <—I t CO I I  K  II CL. *-*  1  L  \  H L  I t— _t .it -  i—• r—4  - OJ t— OJ  I— ul  LO O  < ui  r  I—  O  i  _r> ;  I. RI-'ID C-ERRS. ..STNO.C.  0 R T R A N  SOURCE  S T A T E M E N T S  IDENTFCN  ••COMPILER MESSAGES**  1DOM AND PROB= '.F7.4/' HETEROGENEITY FACTOR* '.F9.4) V;R I . t E i i . i S J X h U P_..i'JEP_i.ERE  4a FORMAT!' PARALLELISM CHlSQc '>F9.4»' WITH '•121 DEGREES OF FREEDO 1M AND PRCB = ' ,F7.4) ... .. IFIPRP-.C5149.50.50 49 WR1TE13.51) 51 FORMAT( 0 PREP ND CHISQ CONTRIBUTION') 0 0_i _i_I_=l J ii£ , 5 3 WhlTe(3.52)1.ALAB( I ) ,M(I I .DCHI( I) 52 FORMAT I ' ' . 12.2XA4.14.5XF9.4) 50 DO 54 1 = 1 .NP ... GSN = 5SN( I ) ED50II I= XBAR( I)-YBAR(I)/B2 T1=ED50( I 1+6*(EPbOt I )-XBAR(I ) ) / ( ! . T2=ST/B2/1 1 .-G)*SQRT ( AH* i ( l.-G) /GSN-r ( E D 5 0 t 1 1-X3AR ( I ) )**2/SSX) ) CLL(I)=T1-T2 CLU.I.I ! = TltT2 CONTINUE WRITE!3.55) _f!0.a;aAlJ_LOPfiEP ED_5_0 CONFIDENCE LIMITS RELATIVE POTENCY CONFI ICtNCE LIMITS' ) (.RITE I 3 . 56) ED? 0! 1 I .CLLI 1 ) .CLU! 1 ) FORMAT ! ' S TD ' , 2XF 5 . 2 . 3XF5 . 2 ._2XF 5 . 2 ) IF(NP-iI 100.100.5 7 DO 5 8 1=2.NP Ai-l=EOaO 1 1_ir.EJir_0 i l l =XoAR(1J-XBAR!I) GSN=SSN(ll+SSN!I) T3=ST/B2*SQ«T ( ( (i.-G) /GSN+J AM-AD Lt^2/SSX )_*AH_) CL=AD+(AM-AD-T3)/Il.-G) CL'=AD+ ( AM-AD+T3)/ ( l.-G) _Rf:i2..*iAM RL=2.**CL F.U = 2 .**CU 58 WRITE! 3 .59I_ALAB( I ) .ED50! I U CLL ( I )_. CLU ( i ) .AM. RMj RLj_RU 59' FORMAT! • ' . A4. 1XF6. 2 . 1XF6.2 i 1XF6".~2 . 2XF6.2 > 5XF 7 . 3 • 7XF 7 . 3 . 2XF~7 . 3 f 100 CGNTINUE CALL EXIT END 1  t  FEATURES SUPPORTED ONE WORD INTEGERS' STANDARD PRECISION _Lp AD GO ICCS1132 PRINTER CARD  _ "  • " •  CORE REQUIREMENTS FOR COMMON0. VARIABLES AND TEMPORARIES-  "  2016.  CONSTANTS AND PROGRAM-  COMPILATION // XEQ  «• COMMENCE PROGRAM EXECUTION ••  2496  H  CA -f=-  TREMELLA HOBt-'ONEttl-OASSAY-BEKFOft-iEOfe5 PREP  1  DOSE  Y3  R  STD  0.0 237. 274. _!.._ 12-6.. _IX0.. -2.0 97. 142. -3.0 98. 214.  NUMBER OF I T E R A T I O N S * SLOPE* 0.324'. -H_I_RO_U.E-IJ-_.XH 14U«_  0.0. -i.'J -2.0  0.864 0.74-1 0.683 0.457  0.923 0.92 0 0 .__,__ _D-.-5V.5_ 0.384 0.271 -0.084 -0.053  _2_-4.l_wXT.H_ _2_D£ ORE ES_OF_J.liEEDQl__A.__  J ____!_  123.... 263.......0.4S7_ -0 . C64...„-0 .05 3.. _ 108. 338. 0.319 -0.377 -0.398 41. 230. 0.178 -0.764 -0.742  NUMBER OF ITERATIONS SLGPE= 0.3445 n£T£RCGEi.£lT.Y.CHlSa„_ 11  .  ..0.21. WITH  l_DEGREES_OP l:RE.ED0»_A/S ___R.0.3« .0_.-64.8J_  FB __0 ..0_ 15.6.. _2-9.3_. 0...2._2 -1.0 125. 414. .0.301 -2.0 49. 261. ,0.187 DUMBER OF ITERATIONS* SLGPE= 0.4110 _t_-__KU5 E ;.£lXY...ai !_.(_• PREP  JOSE  il  WITH  OJJJ.6.4.  -0.419 -0.732  0...0.3.7  -0.373 -0.784  ,  H  1 OFGREES OF FREEDOM AND PR06 =  0.2387  Y  0.968 0*0 0.928 0.526 0.616 - i .0 0.364 0.263 -2..0 -0.064 -3.0 -0.038 "l 2 0.0 -0.064 -0.0-4 7 2 . - i • 0 ., -0.3 77. .. -0.399 2 -2.0 -0.764 -0.751 ' 0.0 0.064 -0.011 3 -a -1..Q -__._i__ _Q...3.6.3 3 -2.0 -C.732 -0.716 1  OF IT ERA T Of-.S - 3 SLOPE= 0.3523.-.'IT.i CONFIDENCE LIMITS 0.4014 AND C.3031 0= 0.0 194 -ri-J-PX-E-EJLIY .H.I.SU" 4j__0.__W._Itl 4 DEGREES OF FREEDOM AND PROB* 0.4053 HETEROGENEITY FACTOR* 1.0000 PARALLELISM CHlSO" 2.0972 WITH 2 DEGREES OF FREEDOM AND PROSJ- 0.3515 PREP ED50 CONFIDENCE LIMITS M RELATIVE POTENCY STD -2.74 -3.04 -2.49 7 0_J._ _0.._0_ (jj_t_ _2_28_ FB 0.03 -0.18 0.28 .78 0. 145  CONFIDENCE LIMITS _0__U__  0.114  0.175  

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