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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 PURIFICATION EROGENS FROM TREMELLA MESENTERICA FR. "by IAN DUNCAN REID B. Sc., U n i v e r s i t y of Guelph, I969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Botany We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1973 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by h i s representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Botany The University of B r i t i s h Columbia Vancouver 8, Canada Date Jiine 28, 1973 i ABSTRACT A d e f i n e d medium c o n t a i n i n g glucose, ammonium sulphate, thiamine, s a l t s and microelements supports good growth of T r e m e l l a  mesenterica F r . 2259-7- A h i g h c o n c e n t r a t i o n of microelements s e l e c t i v e l y slows growth i n media c o n t a i n i n g amino a c i d s . L~ asparagine causes slow and abnormal growth. Conjugation hormones s e c r e t e d by h a p l o i d c e l l s of each mating type of T_. mesenter i c a induce the growth of c o n j u g a t i o n tubes from c e l l s of the other mating type. The c o n j u g a t i o n hormones (erogens) from s t r a i n 2259 _7 can be e x t r a c t e d from, aqueous s o l u t i o n with n-butanol, but not with l e s s p o l a r s o l v e n t s . They are s t r o n g l y adsorbed on a c t i v a t e d c h a r c o a l , and on the p o l y s t y r e n e r e s i n Porapak. The erogens are a l s o adsorbed on both anion and c a t i o n exchange r e s i n s , p a r t l y by non-polar f o r c e s and p a r t l y by i o n i c f o r c e s . U l t r a f i l t r a t i o n i n d i c a t e s t h a t the molecular weight of the erogens i s l e s s than 750- Three a c t i v e m a t e r i a l s are separated by chromatography on s i l i c a g e l columns with a g r a d i e n t of water i n ethanol, and two a c t i v e components can be separated by t h i n l a y e r chromatography. A q u a n t i t a t i v e b i o a s s a y f o r erogen a c t i v i t y has been d e v e l -oped, based on the f r a c t i o n of c e l l s which produce conjugation tubes. An i n c u b a t i o n time of 12 to 18 hours, at 20°C, pH 5-5; and low c e l l d e n s i t y with a complex n i t r o g e n source i s optimal f o r c o n j u g a t i o n tube p r o d u c t i o n . The e f f e c t of compositon of the medium on p r o d u c t i o n of erogens i n 2259 -7 c u l t u r e s has been s t u d i e d , and a de f i n e d medium g i v i n g high hormone y i e l d s has been s e l e c t e d . The erogens can be concentrated and p a r t i a l l y p u r i f i e d from, c u l t u r e s by foaming. Attempts to p u r i f y the erogens have been hampered by low r e c o v e r i e s of hormone a c t i v i t y . The erogens may be amino ac i d s or short peptides with non-p o l a r s i d e chains. i i TABLE OF CONTENTS PAGE ABSTRACT i TABLE OF CONTENTS i i LIST OF TABLES v i LIST OF FIGURES v i i i ACKNOWLEDGMENT x INTRODUCTION 1 GENERAL MATERIALS AND METHODS l6 I. C u l t u r e s 16 I I . Media 16 CHAPTER ONE. THE NUTRITION AND GROWTH OF TREMELLA MESENTERICA 2259-7 17 M a t e r i a l s and Methods 17 I. N i t r o g e n source 17 I I . Vitamins 18 I I I . A growth curve f o r 2259-7 19 IV. E f f e c t of sodium acetate on pH d r i f t 19 R e s u l t s 21 I. N i t r o g e n source 21 I I . Vitamins 27 I I I . Growth curve 27 IV. E f f e c t of sodium acetate on pH d r i f t 27 D i s c u s s i o n 29 CHAPTER TWO. PRELIMINARY STUDIES ON THE EROGENS. 33 M a t e r i a l s and Methods 33 I. A q u a l i t a t i v e b i o a s s a y 33 I I . P r o d u c t i o n of hormone 3^ I I I . E x t r a c t i o n with organic s o l v e n t s 3^ IV. A d s o r p t i o n on c a t i o n exchange r e s i n 35 V. A d s o r p t i o n on anion exchange r e s i n 36 VI. A d s o r p t i o n on a c t i v a t e d c h a r c o a l 37 VII . A d s o r p t i o n on Porapak 39 V I I I . Chromatography on Sephadex G-10 39 IX. U l t r a f i l t r a t i o n kO i i i PAGE X. Chromatography on columns of s i l i c a g e l 4 l XI. Paper chromatography 42 XII. E f f e c t of c y c l i c - 3 *, 5 '-adenosine monophosphate 42 R e s u l t s 44 I. E x t r a c t i o n with organic s o l v e n t s 44 I I . A d s o r p t i o n on c a t i o n exchange r e s i n 44 I I I . A d s o r p t i o n on anion exchange r e s i n 45 IV. A d s o r p t i o n on a c t i v a t e d c h a r c o a l 45 V. A d s o r p t i o n on Porapak 45 VI. Chromatography on Sephadex G-10 46 V I I . U l t r a f i l t r a t i o n 46 V I I I . Chromatography on s i l i c a g e l columns 46 IX. Paper chromatography 47 X. E f f e c t of c y c l i c - 3 5 1 - a d e n o s i n e monophosphate 47 D i s c u s s i o n 50 CHAPTER THREE. QUANTITATIVE BIOASSAY OF THE EROGENS 55 M a t e r i a l s and Methods 55 I. R e l a t i o n of co n j u g a t i o n tube l e n g t h to erogen c o n c e n t r a t i o n 55 I I . A b i o a s s a y based on conjugation tube l e n g t h . . 55 I I I . R e l a t i o n of f r a c t i o n of c e l l s b e a r i n g c o n j u g a t i o n tubes to erogen c o n c e n t r a t i o n . . . . 56 IV. A b i o a s s a y based on f r a c t i o n of c e l l s with c o n j u g a t i o n tubes 57 R e s u l t s 60 I. R e l a t i o n of co n j u g a t i o n tube l e n g t h t o erogen c o n c e n t r a t i o n 60 a. Data 60 b. S t a t i s t i c a l a n a l y s i s 60 I I . A b i o a s s a y based on conjugation tube l e n g t h . . 65 I I I . R e l a t i o n of f r a c t i o n of c e l l s b e a r i n g c o n j u g a t i o n tubes to erogen c o n c e n t r a t i o n . . . . 65 IV. Bioassay based on f r a c t i o n of c e l l s with c o n j u g a t i o n tubes 65 i v PAGE D i s c u s s i o n 69 CHAPTER FOUR. THE RESPONSE TO THE EROGENS 75 M a t e r i a l s and Methods 75 I. Time course of con j u g a t i o n tube i n i t i a t i o n and growth 75 I I . D i s t r i b u t i o n of number of conjugation tubes per c e l l 75 I I I . E f f e c t of temperature 16 IV. E f f e c t of pH 76 V. E f f e c t of c e l l c o n c e n t r a t i o n 76 VI. E f f e c t of n i t r o g e n source 77 R e s u l t s 78 I. Time course of con j u g a t i o n tube i n i t i a t i o n and growth 78 I I . D i s t r i b u t i o n of number of conjugation tubes per c e l l 78 I I I . E f f e c t of temperature 78 IV. E f f e c t of pH 78 V. E f f e c t of c e l l c o n c e n t r a t i o n 78 VI. E f f e c t of n i t r o g e n source 88 D i s c u s s i o n 90 CHAPTER FIVE. PRODUCTION OF THE EROGENS 95 M a t e r i a l s and Methods 95 I. Time course of erogen p r o d u c t i o n 95 I I . E f f e c t of n i t r o g e n source on erogen p r o d u c t i o n 96 I I I . E f f e c t of medium c o n c e n t r a t i o n 96 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 of i n d i v i d u a l medium, components 97 V. Large s c a l e productions of erogens 99 VI. Foam 100 R e s u l t s 103 I. Time course of erogen p r o d u c t i o n 103 I I . E f f e c t of n i t r o g e n source on erogen productionl03 I I I . E f f e c t of medium c o n c e n t r a t i o n 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 of i n d i v i d u a l medium components 106 V. Large s c a l e p r o d u c t i o n of the erogens 112 VI. Foam 112 D i s c u s s i o n 114 CHAPTER SIX. PARTIAL PURIFICATION OF THE EROGENS 119 M a t e r i a l s and Methods 119 I. I n s t a b i l i t y 119 I I . F i r s t attempt at p u r i f i c a t i o n 119 I I I . I n t e r a c t i o n of the erogenswwith-ioneexbhange r e s i n s 122 IV. F u r t h e r attempts at p u r i f i c a t i o n 124 V. T h i n l a y e r chromatography 126 R e s u l t s 129 I. I n s t a b i l i t y 129 I I . F i r s t attempt at p u r i f i c a t i o n 129 I I I . I n t e r a c t i o n of the erogens with i o n exchange res ins 131 IV. F u r t h e r attempts at p u r i f i c a t i o n 132 V. Th i n l a y e r chromatography 132 D i s c u s s i o n 137 SUMMARY AND GENERAL DISCUSSION l 4 l BIBLIOGRAPHY 144 APPENDICES 15 4 A. F r e q u e n t l y used media 154 B. Measurements of co n j u g a t i o n tube l e n g t h at various c o n c e n t r a t i o n s of erogen 156 C. A computer program to process b i o a s s a y data 158 v i LIST OF TABLES TABLE PAGE I. Doubling times f o r T r e m e l l a mesenterica 2259-7 i n b a s a l medium (high microelements) plus v a r i o u s n i t r o g e n sources at 20°C 21 I I . Doubling times and f i n a l pH's f o r c u l t u r e s of 2259-7 grown i n b a s a l medium (low macroelements) plus v a r i o u s n i t r o g e n sources at 20°C 27 I I I . The Rf values f o r the erogen from T r e m e l l a mesen-t e r i c a 2259-7 obtained on paper chromatography i n v a r i o u s s o l v e n t s 48 IV. T u r b i d i t i e s of one-tenth d i l u t i o n s of T. mesenterica c u l t u r e s grown f o r s i x days i n GS. pIu;sXv.ar±ousci.H./ c o n c e n t r a t i o n s of c a f f e i n e 49 V. Frequency d i s t r i b u t i o n of the h e t e r o g e n e i t y f a c t o r f o r the c o n j u g a t i o n hormone b i o a s s a y 68 VI. C e l l counts from, the experiment on the e f f e c t of c e l l c o n c e n t r a t i o n 88 V I I . The number of c e l l s with conjugation tubes/the t o t a l number of c e l l s counted at two doses of erogen with v a r i o u s n i t r o g e n sources 88 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 of n i t r o g e n source on c o n j u g a t i o n tube p r o d u c t i o n 89 IX. F i n a l t u r b i d i t i e s and erogen y i e l d s f o r 2259-7 c u l t u r e s grown i n v a r i o u s c o n c e n t r a t i o n s of Gluc-AmS medium 107 X. A n a l y s i s of v a r i a n c e of logarithmns to the base two of erogen y i e l d s from 2259 -7 c u l t u r e s grown i n Gluc-AmS and x/2 media 108 XI. F i n a l t u r b i d i t i e s and erogen y i e l d s i n media with doubled 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 108 XII. A n a l y s i s of v a r i a n c e of logarithmns of erogen y i e l d s i n media with doubled 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 109 v i i TABLE PAGE X I I I . F i n a l t u r b i d i t i e s and erogen y i e l d s from the f a c t o r i a l experiment on e f f e c t of ammonium sulphate c o n c e n t r a t i o n , microelement 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 of r e s t of medium 110 XIV. A n a l y s i s of va r i a n c e f o r e f f e c t s of ammonium sulphate c o n c e n t r a t i o n , microelement 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 of r e s t of medium on f i n a l t u r b i d i t y and logarithmn of erogen y i e l d I l l XV. Progress of the erogen p u r i f i c a t i o n 131 V I 1 1 LIST OF FIGURES FIGURE PAGE 1. S t r u c t u r e of some sex u a l hormones from f u n g i k 2 . The r e l a t i o n of t u r b i d i t y at 560 nm to c o n c e n t r a t i o n of T_. mesenterica 2259-7 c e l l s 22 3- Regression of t u r b i d i t y on the logarithmn of c e l l c o n c e n t r a t i o n 23 k. Growth curves f o r T_. mes enter i c a 2259-7 i n BM with high microelements plus v a r i o u s n i t r o g e n sources at 20°C 2k 5 . R e l a t i o n of t u r b i d i t y at 6k0 nm to c o n c e n t r a t i o n of T_. mesenter i c a 2259-7 c e l l s 25 6. Growth curves f o r T_. mesenter i c a 2259~7 i n BM with low microelements plus v a r i o u s n i t r o g e n sources at 20°G 26 7. A growth curve f o r T_. mesenter i c a 2 2 5 9 _ 7 i n GM3 at 20°C 28 8 . R e l a t i o n of mean conj u g a t i o n tube to dose of erogen 6 l 9 . Dependence of v a r i a n c e on mean f o r c o n j u g a t i o n tube l e n g t h measurements 62 10 . R e l a t i o n of average transformed c o n j u g a t i o n tube l e n g t h to dose of erogen 63 11 . L i n e a r r e g r e s s i o n of average transformed conjugation tube l e n g t h on the logarithmn of erogen dose 6k 12 . a. R e l a t i o n of f r a c t i o n of c e l l s with conjugation tubes to dose of erogen b. L i n e a r r e g r e s s i o n of transformed response on the logarithmn of erogen dose 66 13 . V a r i a t i o n i n the slope and i n t e r c e p t of the b i o a s s a y standard curve 67 Ik. Time course of c o n j u g a t i o n tube i n i t i a t i o n and growth i n response to erogen 79 15 . Frequency d i s t r i b u t i o n s of c o n j u g a t i o n tube lengths at v a r i o u s times a f t e r i n o c u l a t i o n of 2259-6 c e l l s i n t o media c o n t a i n i n g 0 . 2 u n i t s of erogens per ml.. 80 i x FIGURE PAGE 16. Frequency d i s t r i b u t i o n s of con j u g a t i o n tube lengths at v a r i o u s times a f t e r i n o c u l a t i o n of 2259-6 c e l l s i n t o media c o n t a i n i n g 1 .0 u n i t of erogens per ml... 8 l 17. Frequency d i s t r i b u t i o n of the number of con j u g a t i o n tubes per c e l l , at three erogen c o n c e n t r a t i o n s 82 18. R e g r e s s i o n of average number of conjugation tubes per c e l l on the erogen c o n c e n t r a t i o n 83 19. The f r a c t i o n of c e l l s responding t o the erogen at two c o n c e n t r a t i o n s as a f u n c t i o n of temperature.... 84 2 0 . The average l e n g t h of con j u g a t i o n tubes produced i n response to two c o n c e n t r a t i o n s of erogens as a f u n c t i o n of temperature 85 2 1 . The f r a c t i o n of c e l l s responding t o the erogens at two c o n c e n t r a t i o n s as a f u n c t i o n of pH 86 2 2 . Dependence of f r a c t i o n of c e l l s with conjugation tubes, and average l e n g t h of conjugation tubes on c o n c e n t r a t i o n of c e l l s 87 2 3 . Time course of t u r b i d i t y i n c r e a s e , pH drop, and erogen accumulation i n 2259-7 c u l t u r e s i n Gluc-AmS medium 104 2 4 . Time course of t u r b i d i t y i n c r e a s e , pH drop, and erogen accumulation i n 2259-7 c u l t u r e s i n Gluc-AmS medium 105 25- Chromatography of f i r s t erogen p r e p a r a t i o n on s i l i c a g e l with a g r a d i e n t of water i n ethanol 130 2 6 . Chromatography of second erogen p r e p a r a t i o n on s i l i c a g e l with a gra d i e n t of water i n eth a n o l 133 27- T h i n l a y e r chromatography of hormone I p r e p a r a t i o n . 134 2 8 . T h i n l a y e r chromatography of second erogen p r e p a r a t i o n 135 X ACKNOWLEDGMENT I wish to thank Dr. R. J. Bandoni f o r p r o v i d i n g l a b o r a t o r y f a c i l i t i e s , f o r suggesting and encouraging the r e s e a r c h r e p o r t e d i n t h i s t h e s i s , and f o r reviewing the manuscript. I a l s o thank Dr. C. 0. Person, Dr. E. B. Tregunna, and Dr. G. H. N. Towers f o r generously making equipment a v a i l a b l e to me. The a s s i s t a n c e of the B i o l o g y Data Center s t a f f i n pr e p a r i n g the f i g u r e s i s g r a t e f u l l y acknowledged. For t h e i r w i l l i n g c o -operation, and f o r t h e i r h e l p f u l suggestions a f t e r r e a d i n g the manuscript, I am indebted to the members of my committee. The N a t i o n a l Research C o u n c i l of Canada provided f i n a n c i a l support. I would a l s o l i k e to thank my wife, S h i r l e y , f o r her constant encouragement, and f o r t e c h n i c a l 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 of sexual r e p r o d u c t i o n i n f u n g i has been f r e q u e n t l y reviewed; f o r example, by Raper (1952, I967), K'dhler< (I967), Machlis and Rawitscher-Kunkel (1967), Barksdale (I969), and Machlis (1966, I972). Machlis (I972) has d i s c u s s e d the terminology used to d e s c r i b e sexual hormones, and has proposed three new words to i n d i c a t e t h e i r b i o l o g i c a l a c t i v i t y : an " e r o t a c t i n " i s a sub-stance causing chemotactic a t t r a c t i o n of motil e gametes, an " e r o t r o p i n " causes chemotropic growth of some sexual s t r u c t u r e , and an "erogen" induces or c o n t r o l s the d i f f e r e n t i a t i o n of sexual s t r u c t u r e s . Three genera of f u n g i have been i n v e s t i g a t e d i n detail,-and, w i t h i n the l a s t f i v e years, s t r u c t u r e s have been estab-l i s h e d f o r hormones from each of t h e s e - - s i r e n i n , the e r o t a c t i n from Allomyces, a n t h e r i d i o l , an erogen and e r o t r o p i n from Achlya, and the t r i s p o r i c a c i d s , erogens from Mucor and r e l a t e d f u n g i . A l s o , an e r o t a c t i n from the brown a l g a Ectocarpus s i l -i c u l o s u s (MUller et a l 1971), and a c o n j u g a t i o n - i n i t i a t i n g substance from, the c i l i a t e Blepharisma (Kubota et a l 1973) have been i d e n t i f i e d . The hormones which have been s t u d i e d i n the Mucorales are erogens i n d u c i n g the d i f f e r e n t i a t i o n of progametangia (zygo-phores) and e r o t r o p i n s d i r e c t i n g the growth of compatible gam-eta n g i a towards one "another. The ph y s i o l o g y of sex i n the Mucorales has been reviewed by van den Ende and Stegwee (1971)-Burgeff (1924) found that i f compatible s t r a i n s of Mucor  mucedo were grown on opposite s i d e s of a c o l l o d i o n membrane, progametangia were induced and a t t r a c t e d to one another across the membrane. He p o s t u l a t e d that progametangia-indueing and - a t t r a c t i n g substances were sec r e t e d i n t o the medium by each s t r a i n . B u r g e f f ' s experiments were repeated by Verkaik (1930) and Kehl (1937)- S i m i l a r s t u d i e s with s i m i l a r r e s u l t s were performed on Phycomyces blakesleeahus by Ronsdorf (1931), and on P i l o b o l u s c r y s t a l l i n u s by Krafczyk (1931, 1935)• Banbury (1954) supported B u r g e f f ' s hypothesis by showing t h a t the medium from a mated c u l t u r e of M. mucedo would induce progametangia i n an unmated plus s t r a i n , although not i n the minus s t r a i n . Plempel (1957)^ by growing the mated c u l t u r e s under vigorous a e r a t i o n , produced c u l t u r e f i l t r a t e s t h a t were e f f e c t i v e on both plus and minus s t r a i n s . He found t h a t f i l -t r a t e s from s i n g l e s t r a i n c u l t u r e s were not morphogenetically a c t i v e , but c u l t u r e media i n which mycelia of the two mating types had been grown s e q u e n t i a l l y could induce progametangia i n unmated c u l t u r e s of the f i r s t - g r o w n s t r a i n . On t h i s b a s i s , Plempel (1963) suggested t h a t each s t r a i n c o n t i n u o u s l y produces a mating type s p e c i f i c "progamone". In the presence of a progamone from the opposite mating type, each s t r a i n produces a "gamone", which induces progametangia only i n the opposite mating type. Plempel attempted to i s o l a t e the gamones from mated c u l t u r e s of Mucor mucedo. The progametangium-inducing a c t i v i t y of a s o l u t i o n was bioassayed by p l a c i n g the t e s t s o l -u t i o n i n a w e l l cut i n the agar j u s t ahead of the m y c e l i a l f r o n t of a plus or minus colony. A f t e r four hours, counts were made of the number of progametangia developed i n the v i c i n i t y of the w e l l . The number of progametangia per u n i t area was l i n e a r l y r e l a t e d to the dose up to approximately one microgram of gamone per ml of t e s t s o l u t i o n . Although he p u r i f i e d the a c t i v e m a t e r i a l e x t e n s i v e l y and obtained i t i n c r y s t a l l i n e form, he was unable e i t h e r to separate the plus-gamone from the minus-gamone or to a s s i g n a s t r u c t u r e . Plempel a l s o found t h a t 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 and Phycomy.ces ,b'lakesleeanus, and, l e s s s t r o n g l y , Rhiz opus n i g r i c a n s and Abs i d i a s pinosa produced f a c t o r s a c t i v e i n the Mucor mucedo bioassay, and i n d i s t i n g u i s h -able from M. mucedo gamone. Rec e n t l y , Ueyama (1972), has r e p o r t e d that a methanol e x t r a c t of a mated c u l t u r e of Abs i d i a  g l auca induces gametangial i n i t i a l s i n s i n g l e s t r a i n c u l t u r e s of t h a t fungus. Van den Ende (I967) i s o l a t e d from mated c u l t u r e s of Blakes-l e a t r i s p o r a a m a t e r i a l which induces progametangia i n Mucor 3 mucedo, and suggested that i t might "be t r i s p o r i c a c i d C. T r i s p o r i c a c i d s A, B, and C ( F i g . l ) had e a r l i e r teen i s o l a t e d from c u l t u r e f l u i d s of B_. t r i s p o r a and c h a r a c t e r i z e d by Cag-l i o t i et a l (1967) and C a i n e l l i , G r a s s e l l i , and S e l v a (I967). D e t a i l e d chemical comparison confirmed the i d e n t i t y of the progametangium in d u c i n g f a c t o r s from B. t r i s p o r a with t r i s p o r i c a c i d s B and C (van den Ende I968). A u s t i n , Bu'Lock, and Gooday (I969) a l s o i s o l a t e d t r i s p o r i c a cids B and C from Mucor mucedo. T r i s p o r i c a cids B and C have been s y n t h e s i z e d (Edwards et a l I971) affdltb.er.•.sy_>b'_.et:i'ei "mater.iiasbsev.ere found to be b i o l o g i c a l l y a c t i v e i n both mating types of M. mucedo. Plempel's concept of two mating-type s p e c i f i c gamones has been questioned by van den Ende (I968, van den Ende and Stegwee 1971) and by Gooday (1968). T r i s p o r i c a c i d s B and C are capable of i n d u c i n g progametangia i n both mating types of Mucor mucedo (van den Ende et a l 1970). Reschke (I969), however, r e p o r t s that the methyl e s t e r s of t r i s p o r i c a cids B and C were a c t i v e only on the minus s t r a i n . Bu'Lock, Drake and Winstanley (1972) have confirmed t h i s o b s e r v a t i o n , and shown t h a t minus c u l t u r e s of B. t r i s p o r a can h y d r o l y s e the methyl e s t e r s to f r e e t r i s p o r i c a c i d much more than the plus s t r a i n . I f Plempel's two gamones do e x i s t , i t seems probable t h a t each gamone i s converted to t r i s p o r i c a c i d by the s t r a i n able to respond to i t , and the t r i s p o r i c a c i d i s r e s p o n s i b l e f o r progametangium i n d u c t i o n . T h i s hypothesis e x p l a i n s why Plempel (I963) i s o l a t e d only m a t e r i a l a c t i v e i n both mating types (presumably t r i s p o r i c a c i d ) from mated c u l t u r e s — t h e gamones were being converted to t r i s p o r i c a c i d s as f a s t as they were produced. The i n t e r a c t i o n s between the two mating types l e a d i n g to the s y n t h e s i s of t r i s p o r i c a c i d s i n M. mu cedo and espec-i a l l y _B. t r i s p o r a have been s t u d i e d i n t e n s i v e l y . T r i s p o r i c a c i d p r o d u c t i o n proceeds r e a d i l y i n mated c u l t u r e s , but i s neg-l i g i b l e i n s i n g l e c u l t u r e s of e i t h e r mating type (van den Ende C O O H T r i s p o r i c T r i s p o r i c T r i s p o r i c ac i d a c i d a c i d A B C X X X H, H 0 H, OH A n t h e r i d i o l F i g . 1. S t r u c t u r e of some sexual hormones from f u n g i . 5 et a l I970) . In JB. t r i s p o r a , a low r a t e of t r i s p o r i c a c i d syn-t h e s i s i s found when mycelium of e i t h e r mating type i s incub-ated i n c u l t u r e f l u i d from the other mating type ( S u t t e r I97O, S u t t e r , Capage, and H a r r i s o n 1971)- R a d i o a c t i v e t r a c e r s t u d i e s have shown t h a t p r e c u r s o r from the f i r s t s t r a i n i s being con-verted t o t r i s p o r i c a c i d by the second mycelium.. Van den Ende, Werkman, and van den B r i e l (1972) have confirmed t h a t each s t r a i n of B. t r i s p o r a s e c r e t e s s m a l l amounts of p r e c u r s o r m a t e r i a l capable of in d u c i n g progametangia i n the opposite mating type of M. mucedo and t r i s p o r i c a c i d syn-t h e s i s i n the opposite mating type of _B. t r i s p o r a • T h i s low r a t e s y n t h e s i s i s not s e n s i t i v e to cycloheximide. Using mycelia grown on C-^-glucose, they found that both plus and minus s t r a i n s c o n t r i b u t e carbon t o 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 . An i n c r e a s e i n the amount of plus mycelium present i n c r e a s e s the r a t e of t r i s p o r at e : - - 3 y n t h e . s i s e by 1 minus - mycelium; the r a t e of s y n t h e s i s by the plus mycelium depends on the i n t e n s i t y of s y n t h e t i c a c t i v i t y of the minus s t r a i n . The hi g h r a t e of s y n t h e s i s i n mated c u l t u r e s was i n h i b i t e d by cycloheximide or 5 - f l u o r o u r a c i l (van den Ende et a l I97O, van den Ende, Werkman and van den B r i e l 1972). R e c e n t l y Werkman and van den Ende (1973) have c l a r i f i e d the r o l e of precursor exchange between the two s t r a i n s i n t r i s p o r i c a c i d b i o s y n t h e s i s . L a b e l l e d p r e c u r s o r m a t e r i a l from, s i n g l e c u l t u r e s of each mating type was p a r t i a l l y p u r i f i e d by s o l v e n t e x t r a c t i o n and t h i n l a y e r chromatography, and shown to be converted to r a d i o a c t i v e t r i s p o r i c a c i d by the other mating type. One of the pre c u r s o r s from the plus s t r a i n may be 3-methyl-l- ( 2 ', 6 ', 6 ' - t r i m e t h y l -3 1-oxo-cyclohexen-1 1-yl)-octa-1,3~dien-7-one which Bu'Lock, Drake and Winstanley (1972) have found as a degradation product of methyl t r i s p o r a t e C. In a d d i t i o n Werkman and van den Ende (1973) developed spectrophotometric methods f o r measuring the amounts of plus and minus p r e c u r s o r s present, and showed that the r a t e of pr e c u r s o r s y n t h e s i s by each mating type was sti m -u l a t e d by t r i s p o r i c a c i d . The s t i m u l a t o r y e f f e c t of t r i s p o r i c a c i d i s negated by 5 - f l u o r o u r a c i l . 6 I t appears, then, that the low r a t e of t r i s p o r i c a c i d s y n t h e s f s i n c u l t u r e f i l t r a t e s i s caused by t r a n s f o r m a t i o n of the s m a l l amounts of p r e c u r s o r c o n s t i t u t i v e l y produced by each mating type. The high r a t e i n mated c u l t u r e s i s caused by p o s i t i v e feedback by t r i s p o r i c a c i d on p r e c u r s o r p r o d u c t i o n . The s e n s i t i v i t y of t r i s p o r i c a c i d s t i m u l a t i o n of p r e c u r s o r formation to i n h i b i t o r s of p r o t e i n and M A s y n t h e s i s suggests t h a t enzyme d e r e p r e s s i o n i s i n v o l v e d . Besides i n d u c i n g progametangia, t r i s p o r i c a c ids s t i m u l a t e c a r o t e n o i d s y n t h e s i s i n minus c u l t u r e s of B. t r i s p o r a (Thomas and Goodwin I967., Thomas et 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 ids i s a l s o i n h i b i t e d by cycloheximide, and i t has been suggested that t r i s p o r i c a c ids act as d e r e p r e s s o r s of an enzyme which i s r a t e - l i m i t i n g f o r c a r o t e n o i d s y n t h e s i s . A u s t i n , Bu'Lock and Drake (1970) have demonstrated the i n c o r p o r a t i o n of r a d i o a c t i v i t y from (J-carotene, r e t i n y l a c e t a t e , and mevalonic a c i d i n t o t r i s p o r i c a c ids B and C. Whether or not t r i s p o r i c a c i d i s s t i m u l a t i n g p r e c u r s o r s y n t h e s i s through i t s e f f e c t on c a r o t e n o i d s y n t h e s i s i s not yet c l e a r . As van den Ende and Stegwee (1971) p o i n t out, the t r a n s f o r m a t i o n of exogenous (J-carotene to t r i s p o r i c a c i d s does not prove that B-carotene i s the n a t u r a l s u b s t r a t e f o r the b i o s y n t h e s i s of t r i s p o r i c a c i d and i t s p r e c u r s o r s . F e o f i l o v a (1970) has found t h a t B-ionone and a c e t i c a c i d decrease t r i s p o r i c a c i d s y n t h e s i s and i n c r e a s e c a r o t e n o i d s y n t h e s i s i n mated c u l t u r e s of _B. t r i s p o r a. A l s o both i n s u f -f i c i e n t and e x c e s s i v e a e r a t i o n depress the y i e l d of t r i s p o r i c a c i d . The mutual a t t r a c t i o n of compatible zygophores (zygotrop-ism) was a l s o s t u d i e d by Banbury (1954, 1955)- He demonstrated r e p u l s i o n of l i k e progametangia and a t t r a c t i o n of compatible progametangia between mycelia which were not i n l i q u i d contact, and concluded t h a t the t r o p i c stimulus was a i r b o r n e . Plempel and h i s a s s o c i a t e s (Plempel i960, Plempel and Dawid 1961,* Plempel I962) were unable to demonstrate d i f f u s i o n of z y g o t r o p i c 7 substances through agar d e s p i t e many attempts. When c u l t u r e s of the two mating types b e a r i n g zygophores were separated by a mica membrane with s m a l l holes i n i t s a e r i a l s e c t i o n , the zygophores grew toward and through the h o l e s . Exposure to z y g o t r o p i c f a c t o r s i n c r e a s e d the growth r a t e of the zygo-phores . Once again, a i r - b o r n e z y g o t r o p i c f a c t o r s were i m p l i -cated . In Allomyces, the male gametes are v i g o r o u s l y m o t i l e , but the female gametes are s l u g g i s h and remain c l o s e to the female garnet angia-af t e r - t h e i - r ^ d ischarge ... jv:M'&ehlis [l-95.8'a> b, c) has shown t h a t the male gametes are a t t r a c t e d to the female gametangia because of the s e c r e t i o n by the female gametangia of an e r o t a c t i n , s i r e n i n . From crosses between A. a r b u s c u l a and A. macrogynus, Machlis obtained predominantly male and predominantly female h y b r i d s t r a i n s . A female s t r a i n was used to produce s i r e n i n and a male s t r a i n t o produce male gametes f o r use i n the b i o a s s a y . S i r e n i n a c t i v i t y was bioassayed by p l a c i n g the t e s t s o l u t i o n i n a cup with a d i a l y s i s membrane bottom, immersed i n a suspension of male gametes. The number of gametes which s e t t l e d on a u n i t area of the membrane was measured as the response. S i r e n i n c o n c e n t r a t i o n s as low as l O - " ^ molar caused n o t i c e a b l e a t t r a c t i o n of the sperm. The male gametes respond to s i r e n i n by swimming up the concentra-t i o n g r a d i e n t , and i r r e v e r s i b l y remove s i r e n i n from t h e i r e n v i r -onment. The o r i g i n a l b i o a s s a y apparatus contained s e v e r a l sample cups, and the response to a given s i r e n i n c o n c e n t r a t i o n depended on the l e v e l of hormone i n the other cups to which the gametes were exposed ( C a r l i l e and Machlis 1965). Machlis (I969) has d e s c r i b e d a s i n g l e sample b i o a s s a y apparatus to e l i m i n a t e t h i s i n t e r a c t i o n . By p r o c e s s i n g 200 l i t r e s of c u l t u r e f l u i d a week, Machlis et a l (I966) succeeded i n i s o l a t i n g 2.5 grams of s i r e n i n . The s t r u c t u r e ( F i g . l ) was deduced by N u t t i n g , Rapoport and Machlis (1968). Racemic s i r e n i n was s y n t h e s i z e d by Corey, Achiwa and Katzenellenbogen (I969), P l a t t n e r , Bhalerao, and Rapoport (I969, 8 Bhalerao, P l a t t n e r , and Rapoport I970), G r i e c o (I969), Mori and Matsui (I969), and Corey and Achiwa (1970). The s y n t h e t i c m a t e r i a l was found to have e r o t a c t i c a c t i v i t y on the same order of magnitude as n a t u r a l s i r e n i n . P l a t t n e r and Rapoport (1971) achieved the s y n t h e s i s of the pure enantiomers d- and 1 - s i r e n i n ( l - s i r e n i n i s the n a t u r a l product) and determined the absolute c o n f i g u r a t i o n of 1 - s i r e n i n . Machlis (1972) r e p o r t s t h a t d-s i r e n i n i s not b i o l o g i c a l l y a c t i v e , and does not i n t e r f e r e with 1 - s i r e n i n . J. R. Raper has demonstrated an e l a b o r a t e system of hormonal c o n t r o l i n the oogamous sexual r e p r o d u c t i o n of A c h l y a (summar-i z e d by Raper I952). He p o s t u l a t e d the a c t i o n of d i f f u s i b l e s e xual hormones f r o n h i s o b s e r v a t i o n s of the course of the sexual r e a c t i o n (Raper 1939) a n d then provided experimental evidence f o r f our separate hormones. The female mycelia produce hormone A, which induces a n t h e r i d i a l hyphae i n male mycelia. Male myce l i a with a n t h e r i d i a l hyphae produce hormone B, which s t i m -u l a t e s the formation of o o g o n i a l i n i t i a l s on female mycelia. The o o g o n i a l i n i t i a l s a t t r a c t the a n t h e r i d i a l hyphae, and cause t h e i r t i p s t o d i f f e r e n t i a t e i n t o a n t h e r i d i a . Raper considered t h a t these two a c t i o n s were performed by a t h i r d hormone, C, produced by the o o g o n i a l i n i t i a l s . A f t e r a n t h e r i d i a develop, they produce hormone D, which induces b a s a l s e p t a t i o n of the oogonia, and the formation of oospheres. F e r t i l i z a t i o n tubes from the a n t h e r i d i a then penetrate the oogonia, and grow to and f e r t i l i z e the oospheres. Although the growth of the f e r t -i l i z a t i o n tubes i s probably a l s o hormonally c o n t r o l l e d , Raper could not present any experimental evidence on t h i s p o i n t (Raper 1940). In a more d e t a i l e d study of hormone A, a b i o a s s a y was developed, based on the number of a n t h e r i d i a l hyphae formed i n response to the hormone (Raper 1942a). Raper and Haagen-Smit (1942) attempted to i s o l a t e hormone A. From lkk-0 l i t r e s of c u l t u r e f i l t r a t e , they obtained 0.2 mg of m a t e r i a l which, although s t i l l impure, induced a n t h e r i d i a l hyphae at a d i l u t i o n 9 of 10"13. The male p l a n t s were found to produce a f a c t o r , designated A', which augmented the response t o hormone A (Raper 1942b). He a l s o found t h a t female p l a n t s s e c r e t e d a second hormone, A 2, i n s o l u b l e i n acetone, which induces a n t h e r i d i a l branching, or in c r e a s e s the response to hormone A (acetone s o l u b l e ) . Male p l a n t s s e c r e t e hormone A , acetone s o l u b l e , which decreases the response to A or A 2 or A plus A' (Raper 1950a). Homothallic species of Ac h l y a d i s p l a y e d a hormonal system s i m i l a r i n p r i n c i p l e to that of h e t e r o t h a l l i c species (Raper 1950b). Barksdale (1963a) found t h a t s t r a i n s of Ac h l y a which responded to hormone A by producing a n t h e r i d i a l hyphae removed hormone from the medium, while unresponsive s t r a i n s d i d not. The hormone could not be recovered by e x t r a c t i o n of the mycelium with acetone. Barksdale (1963b) a l s o showed that p o l y v i n y l or p o l y s t y r e n e p l a s t i c p a r t i c l e s on which p a r t i a l l y p u r i f i e d hormone A had been adsorbed, would a t t r a c t anther i d i a l ' .hyphae . and cause a n t h e r i d i a to form., i n a manner s i m i l a r t o o o g o n i a l i n i t i a l s . Thus the a c t i o n s a t t r i b u t e d by Raper to hormone C can be accomplished by l o c a l i z e d sources of hormone A. The s t i m u l a t o r y e f f e c t of hormone A' i n the presence of A can be mimicked by a d j u s t i n g the n u t r i e n t l e v e l (Barksdale 1970)-McMorris and Barksdale (1967) i s o l a t e d from female p l a n t s of A c h l y a b i s e x u a l i s 10 m i l l i g r a m s of c r y s t a l l i n e hormone A, which they named a n t h e r i d i o l . A r s e n a u l t et a l (I968) from s p e c t r o s c o p i c evidence, suggested a s t r u c t u r e ( F i g . l ) . Edwards et a l (I969) s y n t h e s i z e d a mixture of a n t h e r i d i o l and 22,23-i s o a n t h e r i d i o l . Later they accomplished the s y n t h e s i s of pure a n t h e r i d i o l and determined the absolute c o n f i g u r a t i o n (Edwards et a l I972). McMorris and c o l l a b o r a t o r s have developed a s y n t h e t i c method g i v i n g moderate y i e l d s of s t e r e o c h e m i c a l l y pure a n t h e r i d i o l (McMorris and S e s h a d r i 1971> McMorris, Arun-achalam, and Se s h a d r i I972). Green et a l (1971) have a l s o i s o l a t e d 23-deoxyantheridiol from a s t r a i n of A. b i s e x u a l i s . T h i s molecule does not appear t o have any erogenic a c t i v i t y . 10 In p r e l i m i n a r y s t u d i e s , Barksdale (I969) has found t h a t hormone B i s s e c r e t e d by male p l a n t s at the end of the growth phase upon s t i m u l a t i o n by a n t h e r i d i o l . - This, hormone _ can. be e x t r a c t e d from c u l t u r e f l u i d s with methylene c h l o r i d e , and shows s i m i l a r chromatographic p r o p e r t i e s to a n t h e r i d i o l . Wo r e p o r t s have yet appeared on the chemistry of hormones A^, A?>, or D. A s i g n i f i c a n t f r a c t i o n of the c e l l w a l l p o l y s a c c h a r i d e s i n A c hlya i s c e l l u l o s e . A n t h e r i d i o l induced branching of male mycelia i s c o r r e l a t e d with a r i s e i n e n d o c e l l u l a s e a c t i v i t y (Thomas and M u l l i n s I967, I969) and an i n c r e a s e i n oxygen uptake r a t e (Warren and M u l l i n s I969). The i n d u c i b i l i t y of c e l l u l a s e i s high i n mycelia from the l a t e l o g and p l a t e a u phases of growth, but low i n e a r l y l o g mycelia. Incubation i n medium from l a t e l o g c u l t u r e s i n c r e a s e s i n d u c i b i l i t y of c e l l u l a s e i n e a r l y l o g c u l t u r e s (Warren and S e l l s 1 9 7 l ) -Branching i n response to a n t h e r i d i o l r e q u i r e s an exogenous supply of carbon, n i t r o g e n and energy. High n i t r o g e n l e v e l s favour v e g e t a t i v e branching over a n t h e r i d i a l branching (Barks-dale 1 9 7 0 ) . Besides the r e l a t i v e l y w e l l s t u d i e d systems d e s c r i b e d above, sexual hormones have been p o s t u l a t e d or demonstrated, but not f u l l y c h a r a c t e r i z e d i n s e v e r a l other f u n g i . Evidence has been found f o r a hormonal system, s i m i l a r to that o p e r a t i n g i n A c h l y a i n two other water molds—Sapromyces re ins c h i i (Bishop 1940) and Dictyuchus monosporus (Sherwood I 9 6 6 ) , although these systems have not been s t u d i e d i n the same d e t a i l as A c h l y a . C o n s i d e r a b l e a t t e n t i o n has been paid to the p o s s i b i l i t y of hormonal c o n t r o l of 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 of Saccharomyces c e r e v i s i a e . L e v i (1956) claimed t h a t agar on which co n j u g a t i o n had occurred could induce c o p u l a t o r y processes i n c e l l s of the minus mating type. He was unable to o b t a i n an a c t i v e c u l t u r e f i l t r a t e , or to c o n s i s t e n t l y demonstrate d i f f u s i o n of the inducer across a membrane. 11 Other i n v e s t i g a t o r s have used c e l l expansion as an i n d i -cator of sexual response. Duntze, MacKay and Manney (1970) r e p o r t e d the p a r t i a l p u r i f i c a t i o n of a f a c t o r , p o s s i b l y an o l i g o p e p t i d e , from the <s*--mating type which caused e l o n g a t i o n and i n h i b i t i o n of budding i n c e l l s of the a-mating type. Yanagishima (1969) i s o l a t e d from each mating type a s t e r o i d a l substance which induced c e l l expansion i n the other mating type. Actinomycin D, chloramphenicol, cycloheximide, and t r a n s - c i n -namic a c i d i n h i b i t e d the response. The a c t i o n of the hormone from the <*_-str a i n was simulated by t e s t o s t e r o n e , and e s t r a d i o l showed a-hormone a c t i v i t y (Yanagishima et a l 1 9 6 9 ). S t a t i o n a r y phase c u l t u r e s of S_. c e r e v i s i a e can be separated 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 i n t o hormone-sensitive ( p e l l e t ) and h o r m o n e - i n s e n s i t i v e ( s u r f a c e ) c e l l s (Yanagishima and Shimoda 1970) . Treatment of o<. c e l l s with _a hormone, or t e s t o s t e r o n e , s t i m u l a t e d s e c r e t i o n of a substance, i n s o l u b l e i n methylene c h l o r i d e , which s p e c i f i c a l l y expanded a, c e l l s (Yanagishima 1971) - A f t e r mating a_ and <=>*- s t r a i n s , the r e l e a s e of sugar and p r o t e i n from the c e l l s i n t o the medium r a p i d l y i n c r e a s e d . The i n c r e a s e i n a u t o l y t i c a c t i v i t y preceded the appearance of con j u g a t i n g c e l l s (Shimoda and Yanagishima I972). I t has been suggested t h a t the c e l l - e x p a n d i n g e f f e c t of the sexual hormones i s caused by a c t i v a t i o n of c e l l w a l l degrading enzymes (Yanagi-shima and Shimoda 1973)- The s e x u a l a g g l u t i n a b i l i t y of c e r t a i n _a-type s t r a i n s i s i n c r e a s e d by i n c u b a t i o n with a heat s t a b l e p r i n c i p l e from at c u l t u r e s (Sakai and Yanagishima 1 9 7 2 ) . B i s t i s ( I 9 5 6 , 1957> B i s t i s and Raper I963) has uncovered a s e r i e s of hormones c o n t r o l l i n g plasmogamy i n the discomycete Ascobolus s t e r c o r a r i u s . In t h i s fungus, mycelia of each mating type produce both the female organs, ascogonia, and the male elements, o i d i a , but are s e l f - s t e r i l e . Each s t r a i n produces a hormone which s e x u a l l y a c t i v a t e s o i d i a from the other mating type. A c t i v a t e d o i d i a s t i m u l a t e the development of ascogonia on the compatible mycelium, and c h e m o t r o p i c a l l y a t t r a c t the t r i c h o g y n e (the apex of the ascogonium). In a d d i t i o n t o the 12 o i d i a , p i eces of the v e g e t a t i v e mycelium can he s e x u a l l y a c t -i v a t e d t o act as males ( a n t h e r i d i a ) . The t r i c h o g y n e s are a t t r a c t -ed to and w i l l fuse with a c t i v a t e d o i d i a or a n t h e r i d i a of e i t h e r mating type. F i n a l l y , a f t e r plasmogamy, the ascogonium seems to s t i m u l a t e hyphal branching i n i t s v i c i n i t y , and chemotrop-i c a l l y a t t r a c t the branches to form a sheath around i t s e l f . Raper (l967) : bas p o s t u l a t e d a minimum, set of seven hormones to account f o r these o b s e r v a t i o n s . In another ascomycete, G l o m e r e l l a , some s t r a i n s are s e l f -f e r t i l e ( h o m o t h a l l i c ) and others are s e l f - s t e r i l e ( h e t e r o t h a l l i c ) . P e r i t h e c i a are produced along the i n t e r f a c e between two mated mycel i a i n crosses i n which at l e a s t one of the p a r t n e r s i s s e l f - f e r t i l e . Some p a i r s of s t r a i n s i n t e r a c t more weakly than o t h e r s . When a p a i r of w e a k l y - i n t e r a c t i n g s t r a i n s i s mated i n the presence of d i a l y s a t e from a s t r a i n which crosses v i g o r -o u s l y with one of the p a r t n e r s , a strong r e a c t i o n i s obtained (Markert l^ky)'. McGahen and Wheeler (1951) observed t h a t i n crosses between h o m o t h a l l i c and h e t e r o t h a l l i c s t r a i n s , the p e r i t h e c i a l i n i t i a l s always a r i s e on the h o m o t h a l l i c hyphae at or near the p o i n t where the hypha i s crossed by a h e t e r o -t h a l l i c hypha. L a t e r , D r i v e r and Wheeler (1955) showed t h a t c u l t u r e f i l t r a t e s from h o m o t h a l l i c s t r a i n s s t i m u l a t e d the development of p e r i t h e c i a i n the s e l f - s t e r i l e s t r a i n s . A l l of these r e s u l t s suggest t h a t p e r i t h e c i a l i n i t i a t i o n i s c o n t r o l l e d by d i f f u s i b l e hormones, but the exact nature of the i n t e r a c t i o n between the s t r a i n s i s not c l e a r . M achlis (1966) has reviewed evidence f o r hormonal a c t i o n i n the c o p u l a t i o n of the gametes of Synchytrium endobioticum ( C h y t r i d i a l e s ) and f o r chemotropic a t t r a c t i o n of t r i c h o g y n e s to spermatia i n Bombardia l u n a t a . In many of the m y c e l i a l ascomycetes and basidiomycetes, plasmogamy occurs by f u s i o n s between v e g e t a t i v e hyphae. B u l l e r (1933)^ a f t e r s t u d y i n g hyphal f u s i o n s i n many f u n g i , concluded t h a t hyphae s e c r e t e d substances which could induce l a t e r a l branching i n adjacent hyphae and c h e m o t r o p i c a l l y guide the 13 branches to meet t i p - t o - t i p . Raper (1952) suggested t h a t a l l p a r t i c i p a t i n g hyphae s e c r e t e and r e a c t to a s i n g l e substance. I f each hypha s e c r e t e d one f a c t o r and responded to another, an i n f i n i t e number of substances would need to be p o s t u l a t e d to account f o r the apparent c a p a c i t y of hyphae to fuse with any other hypha, sometimes of a d i f f e r e n t s p e c i e s . In cases where the hyphal f u s i o n b r i n g s together s e x u a l l y compatible n u c l e i , the branch i n d u c i n g and a t t r a c t i n g f a c t o r s could be c a l l e d s exual hormones. In Schizophyllum commune, Ahmad and M i l e s (I970) have shown t h a t chemotropic a t t r a c t i o n of hyphal t i p s occurs only i n compatible matings, and a g r e a t e r p r o p o r t i o n of hyphal encounters r e s u l t i n f u s i o n when the two hyphae have d i f f e r -ent a l l e l e s at the A i n c o m p a t i b i l i t y l o c u s than when they have the same A. When compatible s t r a i n s are grown one above the other, separated by cellophane, they are a c t i v a t e d i n some manner so that subsequent matings, even between s t r a i n s of the same mating type, have a high f u s i o n frequency. The substance which d i f f u s e s between the s t r a i n s , and enhances the a b i l i t y of the hyphae to fuse can be considered a sexual hormone. It seems d e s i r a b l e to extend M a c h l i s ' s (1972) d e f i n i t i o n of the term "erogen" to i n c l u d e substances which cause p h y s i o -l o g i c a l s e xual a c t i v a t i o n of t h e i r t a r g e t c e l l s (as i n As cobolus and Schizophyllum) as w e l l as those which c o n t r o l s exual morpho-genesis . In heterobasidiomycetes with y e a s t - l i k e h a p l o i d phases, c o p u l a t i o n of s e x u a l l y compatible c e l l s occurs by the i n i t i -a t i o n , growth, and f u s i o n of filamentous conjugation tubes. Bauch (I925) noted that c o n j u g a t i o n tubes were only formed i n U s t i l a g o bromivora when compatible s t r a i n s were grown t o g e t h e r . He suggested that each s t r a i n produces a hormone which induces c o n j u g a t i o n tubes i n the opposite mating type. Despite s e v e r a l attempts he was unable to o b t a i n conjugation tube i n d u c t i o n with c e l l - f r e e c u l t u r e f l u i d s . 14 Bandoni (I963) analyzed the mating system of T r e m e l l a  mesenterica, and found a modified t e t r a p o l a r p a t t e r n . The formation of con j u g a t i o n tubes i s c o n t r o l l e d by a g e n e t i c locus with two a l l e l e s , A and _a. In mixtures of A and _a c e l l s , c o n j u g a t i o n tubes are produced, but i n mixtures of A. with A, or _a with _a s t r a i n s , only v e g e t a t i v e growth ensues. E s t a b -lishment of a s t a b l e dikaryon with clamp connections depend on h e t e r o z y g o s i t y at a second l o c u s , B, with m u l t i p l e a l l e l e s . For convenience i n t h i s d i s c u s s i o n the B locus w i l l be ignored, and mating type w i l l r e f e r to A or a. Conjugation tubes are induced when s t r a i n s of d i f f e r e n t mating types are grown on opposite sides of a cellophane mem-brane, or when c e l l s of one mating type are i n o c u l a t e d on a d i a l y s i s p r i n t from the other mating type (Bandoni I965). T h i s i s evidence f o r p r o d u c t i o n by each mating type of d i f f u s i b l e erogens which s p e c i f i c a l l y induce conjugation tubes i n the other mating type. Bandoni showed t h a t the hormones were pro-duced i n the absence of the other mating type, and were not destroyed by a u t o c l a v i n g , or by storage at room temperature f o r p eriods up t o f i v e days. D i f f u s i b l e c o n j u g a t i o n hormones have a l s o been demonstrated i n T r e m e l l a subanomala and T_. encephala by F l e g e l (I968) and i n T_. b ambus i n a by Brough (I970). S i m i l a r s t u d i e s i n Rhodosporidium t o r u l o i d e s showed that c o n j u g a t i o n tubes are produced when the two compatible s t r a i n s are grown separated by cellophane. However, no hormone a c t i v i t y could be found i n d i a l y s i s p r i n t s or c e l l - f r e e c u l t u r e f l u i d of e i t h e r s i n g l e or mated c u l t u r e s ( I . Reid, unpublished d a t a ) . Apparently the con j u g a t i o n hormones i n t h i s s p e c i es are e i t h e r very u n s t a b l e , or produced only when both mating types are present, and then e f f i c i e n t l y scavenged from the medium by the r e a c t i n g s t r a i n . The m o r p h o l o g i c a l s i m i l a r i t y of con j u g a t i o n i n other species of Rhodospor idium, and i n the genera Sporob-olomyces, Leucosporidium, T i l l e t i a , and U s t i l a g o to con j u g a t i o n i n T r e m e l l a suggests t h a t c o n j u g a t i o n hormones are a c t i n g i n these f u n g i as w e l l (R. J . Bandoni, pers . comm..). 15 F l e g e l (I968) showed i n T_. mesenterica t h a t hormone must be c o n t i n u o u s l y present to maintain conjugation tube growth; i f hormone i s removed, the c e l l s r e v e r t to budding. He sug-gested t h a t the conjugation hormones were a c t i n g as inducers or r e p r e s s o r s of enzymes i n v o l v e d with c e l l w a l l metabolism. Bandoni (I965) observed that compatible conjugation tubes of T. mesenterica u s u a l l y met t i p - t o - t i p — c h a n g i n g growth d i r e c -t i o n i f necessary to accomplish t h i s . He suggested that the t i p s are c h e m o t r o p i c a l l y a t t r a c t e d to each other over a range of about 15 microns. S i m i l a r chemotropism of con j u g a t i o n tubes i n T_. bambusina i s r e p o r t e d by Brough (I970). Raper (1967) has questioned whether the chemotropic a t t r a c t i o n was due to a separate e r o t r o p i c hormone, or to a high c o n c e n t r a t i o n of the erogenic conjugation hormone, i n analogy with hormone A i n A c h l y a . . J . A. Berry and T. W. F l e g e l began s t u d i e s on the i s o l -a t i o n and c h a r a c t e r i z a t i o n of the erogen from T r e m e l l a mesen-t e r i c a s t r a i n 2259-6. Although they d i d not pursue t h i s study very f a r , they f e l t t h a t the hormone might be a peptide (Berry and F l e g e l , unpublished d a t a ) . R. J . Bandoni undertook p r e l i m -i n a r y s t u d i e s on the hormone from s t r a i n 2259~7 of the other mating type, with i n c o n c l u s i v e r e s u l t s (Bandoni, pers . comm..). The o b j e c t i v e of the present study was to i s o l a t e and c h e m i c a l l y c h a r a c t e r i z e the erogen from s t r a i n 2259-7 of T r e m e l l a mesenterica F r . T h i s g o a l has not been reached, but some inform-a t i o n about the chemical nature of the hormone has been accum-u l a t e d , and methods f o r producing, assaying, and p a r t i a l l y p u r i f y i n g the erogenic a c t i v i t y have been developed. GENERAL MATERIALS AND METHODS 16 I. C u l t u r e s The organisms used i n t h i s study were two s e x u a l l y com-p a t i b l e , h a p l o i d s t r a i n s of T r e m e l l a mesenterica F r . i s o l a t e d and i d e n t i f i e d by Dr. R. J . Bandoni. C u l t u r e RJB # 2259~7 (UBC # 559-7), mating type A B I ; [ I (Bandoni I963) was used to produce c o n j u g a t i o n hormones, and RJB jj= 2259-6 (UBC jj= 559-6) , mating type a B j j , t o assay hormone a c t i v i t y . 2259-6 was i n c l u d e d by Bandoni (I963) i n h i s o r i g i n a l study on c o n j u g a t i o n i n T_. mesenterica, and a l s o i n h i s demon-s t r a t i o n of conjugation hormones (Bandoni I965). Both 2259 _ 6 and 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 of c o n j u g a t i o n i n T r e m e l l a • Stock c u l t u r e s were maintained on Malt Yeast Peptone (MYP) s l a n t s at 4°C, and were t r a n s f e r r e d at l e a s t once every s i x months. A l l c u l t u r e s were grown at 20°C, unless otherwise spec-i f i e d . I I . Media Chemicals used i n media were obtained from M a l l i n c k r o d t Chemical Works, F i s h e r S c i e n t i f i c Company, The N i c h o l s Chemi-c a l Co., and J . T. Baker Chemical Co., and were of reagent grade. V i t a m i n - f r e e s a l t - f r e e c a s e i n h y d r o l y s a t e , and thiamine h y d r o c h l o r i d e were purchased from N u t r i t i o n a l Biochemicals Corp. Bacto Soytone, Bacto Malt E x t r a c t , and Bacto Yeast E x t r a c t came from D i f co L a b o r a t o r i e s . Agar was obtained from. D i f c o , or K & S L a b o r a t o r i e s , Vancouver, or N u t r i t i o n a l B i o -chemicals C o r p o r a t i o n . Water was g l a s s d i s t i l l e d once and stor e d i n a p o l y e t h y -lene carboy. The composition of c e r t a i n f r e q u e n t l y used media are l i s t e d i n Appendix A. Recipes f o r other media are given with the account 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 M a t e r i a l s and Methods 17 I. Nitrogen 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) was prepared a c c o r d i n g to the r e c i p e : Glucose 6.0 gm KHgPOl,. 1.0 gm MgS0^'7H20 0.5 gm CaCl 2-2H 20 0.1 gm NaCl 0.1 gm Microelement stock s o l u t i o n 1.3 ml Thiamine 100 \ig D i s t i l l e d water 1 l i t r e Separate 100 ml l o t s of b a s a l medium, were supplemented with 0.2 gm of one of the f o l l o w i n g n i t r o g e n sources: pot-assium n i t r a t e , ammonium n i t r a t e , L-asparagine, v i t a m i n - f r e e , s a l t - f r e e c a s e i n h y d r o l y s a t e , Bacto Soytone. One l o t was 1 supplemented with 0.2 gm of asparagine plus 0.2 gm of soytone. The media were dispensed i n 50 ml p o r t i o n s i n t o 250 ml E r l e n -meyer f l a s k s . The f l a s k s were capped with a double t h i c k n e s s of aluminum f o i l , and autoclaved f o r 15 minutes at 15 p s i p r e s s u r e . Each f l a s k was i n o c u l a t e d with 2.0 ml of a suspension of 2259-7 c e l l s which had been c e n t r i f u g e d from a two day o l d c u l t u r e i n Growth Medium # 3 (GM3) and washed with two 100 ml p o r t i o n s of s t e r i l e d i s t i l l e d water. To determine the r e l a t i o n between t u r b i d i t y and amount of c e l l s present, 1/5, 2/5, 3/5, 4/5 and then l/25, 2/25, 3/25, and 4/25 d i l u t i o n s of the inoculum suspension were prepared and t h e i r t u r b i d i t y measured. The c e l l s i n a sample of the l/25 d i l u t i o n were counted i n a haemacytometer. The i n o c u l a t e d f l a s k s were placed on a r e c i p r o c a t i n g shaker o p e r a t i n g at 100 strokes per minute with a t r a v e l of 3.5 cm, i n a 20°C incubator room. A f t e r 24 hours, a 2 ml 18 sample was taken from each f l a s k with a s t e r i l e p i p e t t e , and the f l a s k s were returned t o the shaker. The samples were d i l u t e d with 2 ml of d i s t i l l e d water, and t h e i r absorbance measured at a wave l e n g t h of 560 nanometers i n a Bausch and Lomb Spec-t r o n i c 20 Spectrophotometer using d i s t i l l e d water as a blank. Samples were taken at 2 4 hour i n t e r v a l s u n t i l 2l6 hours a f t e r i n o c u l a t i o n , and t h e i r t u r b i d i t y measured as above. T h i s experiment was repeated with the f o l l o w i n g m o d i f i -c a t i o n s . Only 0.13 ml of the microelement stock s o l u t i o n was added per l i t r e of b a s a l medium. Ammonium sulphate at 2 gm/l was i n c l u d e d as an e i g h t h treatment. Each l o t of medium was adjusted t o pH 4.5 with 0.1 N h y d r o c h l o r i c a c i d a f t e r adding the n i t r o g e n source. The inoculum c o n s i s t e d of washed c e l l s from a s t a t i o n a r y phase c u l t u r e of 2259-7 i n Gluc-AmS medium. Samples were taken d a i l y from each f l a s k , and d i l u t e d 1 to 10 with d i s t i l l e d water before measuring t h e i r t u r b i d i t y at 64-0 nm.. A f t e r 7 days, the pH of each c u l t u r e was measured with a Radio-meter pH meter model 28. I I . Vitamins The growth of 2259-7 i n s y n t h e t i c media c o n t a i n i n g thiamine as the only added v i t a m i n demonstrated that no other vitamins are r e q u i r e d by t h i s s t r a i n under the c o n d i t i o n s employed. To t e s t the n e c e s s i t y of thiamine, a medium s i m i l a r to the b a s a l medium, plus c a s e i n h y d r o l y s a t e d e s c r i b e d above, but o m i t t i n g thiamine, was prepared. In t h i s and subsequent experiments only 0.13 ml of microelement 3'olution was added per l i t r e of b a s a l medium. Two f l a s k s c o n t a i n i n g 50 m l of t h i s medium were supple-mented with 5 micrograms each of thiamine h y d r o c h l o r i d e , and along with two v i t a m i n - f r e e f l a s k s , i n o c u l a t e d with 1 ml each from a 3-day c u l t u r e of 2259-7 i n GM3 • A f t e r four days on the shaker, growth i n the f l a s k s was assessed v i s u a l l y , and a sample from each f l a s k checked m i c r o s c o p i c a l l y f o r contamination. 19 I I I . A growth curve f o r 2259-7 A more p r e c i s e growth curve f o r 2259-7 was determined using a C o u l t e r Counter. Three 200 ml p o r t i o n s of GM3 i n one l i t r e Erlenmeyer f l a s k s capped with B e l l c o s t a i n l e s s s t e e l f l a s k c l o s u r e s were i n o c -u l a t e d with 2.0 ml each from a 3-day c u l t u r e of 2259-7 i n GM3, and incubated as d e s c r i b e d above. Two ml samples were taken from each f l a s k a f t e r s i x hours and d a i l y t h e r e a f t e r f o r ei g h t days . The samples were d i l u t e d with 0. 9$> sodium c h l o r i d e to b r i n g the c e l l c o n c e n t r a t i o n between 40,000 and 100,000 c e l l s per m i l l i l i t r e . The s a l i n e d i l u e n t provided the e l e c t r o l y t e needed by the C o u l t e r Counter. Blank counts were c a r r i e d out on the d i l u t i o n f l u i d each day and s u b t r a c t e d from the sample counts. D i l u t i o n s used ranged from 1 to 20 to 1 to 2000. C e l l counts were made on a C o u l t e r Counter Model B f i t t e d with a 100 micron a p e r t u r e . The a m p l i f i c a t i o n was set at l/2, aperture current at l/2, the lower t h r e s h o l d at 7-5 and the upper t h r e s h o l d at i n f i n i t y . From each sample, d u p l i c a t e . d i l -u t i o n s were prepared, and f o u r 0-5 ml p o r t i o n s of each d u p l i c a t e were counted. The r e p o r t e d values have been c o r r e c t e d f o r background and c o i n c i d e n c e , and averaged. IV. E f f e c t of sodium, acetate on pH d r i f t Growth of 2259-7 on the medium c o n t a i n i n g ammonium was accompanied by a drop i n pH. The p o s s i b i l i t y of s t a b i l i z i n g the c u l t u r e pH by i n c l u d i n g sodium acetate i n the medium was t h e r e f o r e t e s t e d . B a s a l medium c o n t a i n i n g 2 grams of ammonium sulphate and 20 m i l l i g r a m s of bromocresol green per l i t r e was prepared. F i v e a l i q u o t s of t h i s medium were supplemented with 0, 1, 2, 3 or 4 grams of sodium acetate t r i h y d r a t e per l i t r e . The pH of each medium was adjusted with 5 N h y d r o c h l o r i c a c i d u n t i l the colour of the i n d i c a t o r matched the green of the medium without sodium acetate (pH 4.5)- The media were then dispensed i n two 20 ml 20 r e p l i c a t e s i n t o 125 ml Erlenmeyer f l a s k s , capped with f o i l , and a u t o c l a v e d . A f t e r c o o l i n g , each f l a s k was i n o c u l a t e d with 1.0 ml of a 5 day o l d shake c u l t u r e of 2259~7 grown i n medium c o n t a i n i n g 1 gm/l of sodium acetate t r i h y d r a t e . A f t e r f i v e days i n c u b a t i o n on the shaker at 20°C, the colour of the i n d i -cator i n each f l a s k was noted. The experiment was repeated, s u b s t i t u t i n g bromocresol purple f o r bromocresol green i n the media with 3 or k gm/l of sodium a c e t a t e . In a t h i r d experiment, a s e r i e s with 0, 2.2, 2.4, 2.6 or 2.8 grams of sodium acetate per l i t r e of BM plus ammonium sulphate and bromocresol purple was prepared. These media were adjusted to an i n i t i a l pH of 5-5> autoclaved and incubated as above. 21 R e s u l t s I. N i t r o g e n source A volume of .04 m i c r o l i t r e s i n the haemacytometer contained 603 c e l l s . T h i s corresponds to 1.5 X 10^ c e l l s per m i l l i l i t r e . The r e l a t i o n of the absorbance at 56O nm to the c o n c e n t r a t i o n of c e l l s i s presented i n F i g . 2. A p l o t of the A^^^ a g a i n s t the logarithmn of the c e l l c o n c e n t r a t i o n i s show i n F i g . 3- Growth curves, i n terms of c u l t u r e t u r b i d i t y , f o r 2259-7 i n BM plus v a r i o u s n i t r o g e n sources, are i l l u s t r a t e d i n F i g . 4. At the end of the experiment, m i c r o s c o p i c examination d i d not r e v e a l any contamination of the c u l t u r e s . In the media c o n t a i n i n g asparagine, c a s e i n h y d r o l y s a t e , or asparagine plus soytone, some of the c e l l s were e x t r a - o r d i n a r i l y l a r g e , s p h e r i c a l , and v a c u o l a t e . Doubling times were c a l c u l a t e d from, the s e c t i o n s of the growth curves where growth was approximately e x p o n e n t i a l (Table i ) . Table I. Doubling times f o r T r e m e l l a mesenterica 2259-7 i n b a s a l medium (high microelements") plus v a r i o u s n i t r o g e n sources at 20°C. Nitr o g e n Source Doubling time (hours) Potassium n i t r a t e >2l6 Ammonium, n i t r a t e 12 l/2 Asparagine 59 Casein h y d r o l y s a t e 47 Soytone 27 Soytone plus asparagine 49 A standard curve of absorbance at 640 nm against concen-t r a t i o n of 2259-7 c e l l s i s presented i n F i g . 5- The growth curves obtained i n the second experiment are shown i n F i g . 6. Doubling times c a l c u l a t e d from the e x p o n e n t i a l phase of growth, and the f i n a l pH of the c u l t u r e s with each n i t r o g e n source are l i s t e d i n Table I I . The c e l l s grown with c a s e i n h y d r o l y s a t e and e s p e c i a l l y asparagine showed the "swollen" morphology n o t i c e d i n the f i r s t exper iment. F i g . 3- Regression c o n c e n t r a t i o n . of t u r b i d i t y on the logarithmn of c e l l F i g . h i g h h. Growth curves f o r T. mesenterica microelements plus v a r i o u s n i t r o g e n 2259-7 i n BM with sources at 20°C . 25 CONCENTRATION OF CELLS F i g . 5. R e l a t i o n of t u r b i d i t y at 640 nm to c o n c e n t r a t i o n of T. mesenterica 2259-7 c e l l s . H O < OQ •H H- ' ON O -o Q 0) H o CD a CD P" c+- O tn >d <! H fl> en cn < o £» i i H- « O en B CD P cn CD d- P d-O CD CW CD H-P O 93 cn o ro ro vn o MD CD I 01 -<l 03 H-c^ - P ro W o s o o si • H-c+ 2 o*a. CD LLI U 1 CD ° • < 0*Q Cas e i n h y d r o l y s a t e Soytone Ammonium, sulpha ro. ON Potassium, n i t r a t e + + + + + + 30* 40' GO* 80* 100' ISO* 140* TIME FROM INOCULATION (HOURS) 160' I S O . 27 Table I I . Doubling times and f i n a l pH's f o r c u l t u r e s of 2259-7 grown i n b a s a l medium (low microelements) plus v a r i o u s n i t r o g e n sources at 20°C. Nitr o g e n Source Doubling time (hours) F i n a l pH None 4.1 Potassium n i t r a t e 4.1 Ammonium n i t r a t e 13-3 2-3 Ammonium sulphate 12.8 2.4 Asparagine 38 4 .7 Casein h y d r o l y s a t e 15 3-9 Soytone 14.7 4.0 Soytone plus asparagine 30 4 .4 I I . Vitamins A f t e r four days, the f l a s k s with added thiamine were very t u r b i d ; the f l a s k s without thiamine were almost c l e a r . Under the microscope, the c u l t u r e s showed only the yeast c e l l s t y p i c a l of T r e m e l l a . I I I . Growth curve The number of c e l l s of 2259~7 VeT m i l l i l i t r e i s p l o t t e d on a l o g a r i t h m i c s c a l e against time i n F i g . 7- In the e x p o n e n t i a l phase of growth, the doubling time was 9 l / 4 hours. IV. E f f e c t of sodium acetate on pH d r i f t A f t e r 5 days i n c u b a t i o n , the i n d i c a t o r i n the c u l t u r e s with 0, 1, or 2 gm/l of sodium acetate t r i h y d r a t e had turned yellow (pH below 4.0). In the c u l t u r e s with 3 ° r 4 gm/l of sodium acetate t r i h y d r a t e , the i n d i c a t o r was blue (pH above 5-5)- In the second experiment, the bromocresol green i n the c u l t u r e s c o n t a i n i n g 0, 1, or 2 gm/l of sodium acetate turned yellow. The bromocresol purple i n the f l a s k s with 3 or 4 gm/l of sodium acetate t r i h y d r a t e was purple, i n d i c a t i n g a pH above 7-In the t h i r d experiment, c u l t u r e s without sodium acetate turned yellow as b e f o r e ; f l a s k s with 2.2 gm/l and 2.4 gm/l were gr e e n i s h yellow and gr e e n i s h purple r e s p e c t i v e l y ; f l a s k s with 2.6 gm/l or 2.8 gm/l were p u r p l e . 29 D i s c u s s i o n The t u r b i d i t y of m i c r o b i a l c u l t u r e s depends on both the number and s i z e of the suspended c e l l s , and t h e r e f o r e , i s more c l o s e l y r e l a t e d to the c o n c e n t r a t i o n of c e l l u l a r dry weight i n the c u l t u r e than to c e l l numbers ( M a l l e t t e I969). The r e l a t i o n between t u r b i d i t y ( A ^ Q ) and c o n c e n t r a t i o n of the c e l l s i n 2259-7 c u l t u r e s i n the f i r s t experiment ( F i g . 2) was markedly n o n - l i n e a r . A p l o t of the t u r b i d i t y a gainst the logarithmn of c e l l c o n c e n t r a t i o n ( F i g . 3) i s more n e a r l y a s t r a i g h t l i n e , p a r t i c u l a r l y when the absorbance i s gr e a t e r that 0.4. In t h i s range, a doubling of c e l l c o n c e n t r a t i o n c o r r e -sponds t o an i n c r e a s e i n absorbance of O.326. A p l o t of c u l t u r e t u r b i d i t y a gainst time ( F i g . 4) i s t h e r e f o r e e q u i v a l e n t to a p l o t of the log a r i t h m s of c e l l con-c e n t r a t i o n a g a i n s t time (at l e a s t i n the upper two-thirds of the graph). E x p o n e n t i a l growth of c e l l dry weight should r e s u l t i n a l i n e a r i n c r e a s e i n t u r b i d i t y . Examination of F i g . 4 shows t h a t each growth curve does i n c l u d e a l i n e a r p o r t i o n . The slopes of these s t r a i g h t s e c t i o n s were used to c a l c u l a t e doubling times. In the second experiment, the c u l t u r e samples were d i -l u t e d ten times before measuring the t u r b i d i t y , so th a t the t u r b i d i t y would be p r o p o r t i o n a l to c e l l c o n c e n t r a t i o n ( F i g . 5)-Logarithmns of the t u r b i d i t y readings were used to c a l c u l a t e doubling times. In both experiments, the b a s a l medium without added n i t r o g e n supported a small i n c r e a s e i n c u l t u r e t u r b i d i t y . The n i t r o g e n used f o r t h i s growth may have been introduced with the inoculum, or may have come from i m p u r i t i e s i n the chemicals used or contamination of the glassware. In the f i r s t experiment, but not i n the second, s l i g h t l y more growth was found i n the medium with potassium n i t r a t e than i n the b a s a l medium. These data i n d i c a t e that n i t r a t e can be used only very slowly, i f at a l l , by T r e m e l l a mesenterica 2259-7- The pH of these media 30 before a u t o c l a v i n g was 4-5- At the end of the experiment, the measured pH was 4.1. T h i s pH drop may have been caused by the inoculum, which was grown i n Gluc-AmS and had a pH of 2-3 before washing. In the second experiment, growth on ammonium n i t r a t e and ammonium sulphate showed s i m i l a r i n i t i a l r a t e s , s l i g h t l y f a s t e r than on soytone and c a s e i n h y d r o l y s a t e . A higher peak t u r b i d i t y was reached i n ammonium sulphate medium than i n ammonium n i t r a t e medium, p o s s i b l y because (NH^)^SO^ contains more ammonium than NH^NO^- In media c o n t a i n i n g ammonium, the c u l t u r e pH f e l l d r a s t i c a l l y . The growth r a t e s i n ammonium n i t r a t e medium i n the f i r s t and second experiments were comparable. However, i n the f i r s t experiment, growth on soytone was much slower than on ammonium n i t r a t e , and growth on c a s e i n h y d r o l y s a t e was s t i l l slower. The d i f f e r e n c e s i n growth r a t e s between these two experiments may have been e i t h e r e f f e c t s of pH, or of the micro-element c o n c e n t r a t i o n . In the f i r s t experiment, the media c o n t a i n i n g soytone and c a s e i n h y d r o l y s a t e had i n i t i a l pH's of 6.5- In the second experiment the i n i t i a l pH of a l l media was adjusted to 4.5- F l e g e l (I968) has shown t h a t the optimum pH f o r growth of 2259-7 i n a medium c o n t a i n i n g glucose, soytone and yeast e x t r a c t i s about 4.7^  with p r o g r e s s i v e l y slower growth at pH's 5-7 a n d 7-2. In the f i r s t experiment, because of an a r i t h m e t i c e r r o r , the c o n c e n t r a t i o n of macroelements was ten times as high as intended. The importance of microelements i n determining the growth r a t e i s suggested'by the doubling time of 9 l/4 hours i n G-M3 (pH 6.5, no added microelements). In both experiments the growth r a t e s i n media with asparagine or soytone plus asparagine are slower than i n media with c a s e i n h y d r o l y s a t e or soytone. A l s o the f r a c t i o n of c e l l s with "swollen" morphology i s high i n media c o n t a i n i n g asparagine. Asparagine appears to i n t e r f e r e with the normal metabolism of T r e m e l l a i n some way l e a d i n g to slow growth and l a r g e c e l l s . A h i g h c o n c e n t r a t i o n of microelements enhances the i n h i b i t i o n of growth by asparagine. 31 I t i s i n t e r e s t i n g that the high c o n c e n t r a t i o n of micro-elements i n c r e a s e s doubling times i n media c o n t a i n i n g amino a c i d s , but not i n media c o n t a i n i n g ammonium.. P o s s i b l y the amino a c i d s complex heavy metal ions, and thus i n c r e a s e t h e i r r a t e of entry i n t o the c e l l s . In t h i s study, no s p e c i a l precautions were taken to r i d the medium or glassware of t r a c e amounts of v i t a m i n s . There-f o r e , I cannot c l a i m that thiamine i s the only v i t a m i n r e q u i r e d by T_. mesenterica 2259~7- However, the c l e a r response to omission of thiamine from the medium suggests t h a t the methods used were capable of d e t e c t i n g major v i t a m i n requirements. A l s o , 2259-7 has been su b c u l t u r e d on s y n t h e t i c media c o n t a i n i n g thiamine as the only added vi t a m i n f o r at l e a s t 20 t r a n s f e r s , without decrease i n growth r a t e . T h e r e f o r e , v i t a m i n c a r r y over i n the inoculum i s not obscuring any v i t a m i n requirements. F l e g e l (I968) r e p o r t s , without f u r t h e r comment, that no n i t r o g e n source simpler than c a s e i n h y d r o l y s a t e supported growth and c o n j u g a t i o n i n T_. mesenterica 2259-6 and 2259-7-The r e s u l t s presented here r e f u t e the part of h i s statement d e a l i n g with growth. Brough (1970) found that T_. bambusina grew w e l l on ammonium as the s o l e n i t r o g e n source, but not on n i t r a t e . He a l s o found that thiamine was r e q u i r e d by t h a t fungus. Ther e f o r e the n i t r o g e n and v i t a m i n n u t r i t i o n seems to be s i m i l a r i n these two s p e c i e s . Growth of f u n g i on media c o n t a i n i n g ammonium s a l t s of strong a c i d s r e s u l t s i n a decrease i n the pH of the medium, of t e n to i n h i b i t o r y l e v e l s . T h i s pH drop can be prevented by i n c l u d i n g a n e u t r a l s a l t of a r e a d i l y metabolized organic a c i d i n the medium (N i c h o l a s 1965)- T h i s i s the case i n T_. m e s e n t e r i c a — s o d i u m acetate t r i h y d r a t e at about 2-3 grams per l i t r e maintains the c u l t u r e pH c l o s e to 5-5- Smaller amounts of sodium acetate allow the pH to drop and l a r g e r amounts cause the pH to r i s e as growth proceeds. No e x p l i c i t e x p l a n a t i o n of the e f f e c t of ammonium and organic a c i d s on the c u l t u r e pH have appeared i n the l i t e r -32 a t u r e . I t i s known, however, that i n many f u n g i the c e l l membrane i s f r e e l y permeable to the uncharged s p e c i e s , NH^, (ammonia), and to u n d i s s o c i a t e d organic a c i d s , but impermeable to NH^ "1" or to i o n i z e d c a r b o x y l i c acids (Burnett 1968) . NH^ i s present i n the medium because of the e q u i l i b r i u m : NH]^+ = WH3 + H+ Uptake of NH^ by the c e l l s d r i v e s t h i s e q u i l i b r i u m to the r i g h t , r e s u l t i n g i n an accumulation of hydrogen i on i n the e x t e r n a l medium, and a drop i n pH. Organic acids e x i s t i n e q u i l i b r i u m between the u n d i s s o c i a t e d and i o n i z e d forms: RCOOH = RCOO- + H + Uptake of u n d i s s o c i a t e d a c i d by the c e l l s d r i v e s t h i s e q u i l i b r i u m t o the l e f t , and thus removes hydrogen ion from, the e x t e r n a l medium, and r a i s e s the pH. The pH change i n a c u l t u r e c o n t a i n i n g both ammonium and organic a c i d w i l l depend on the r e l a t i v e q u a n t i t y of each taken up by the c e l l s . Since the c e l l membrane i s crossed by d i f f u s i o n , the r a t e of uptake w i l l depend on the c o n c e n t r a t i o n of the un-charged s p e c i e s , which i s a f u n c t i o n of pH. A decrease i n c u l t u r e pH decreases the c o n c e n t r a t i o n of NH^ and i n c r e a s e s the co n c e n t r a t i o n of RCOOH. Th e r e f o r e , more RCOOH and l e s s RH^ w i l l be taken up by the c e l l s , and the pH w i l l tend t o r i s e . As the pH r i s e s , the c o n c e n t r a t i o n of NH^ in c r e a s e s and the co n c e n t r a t i o n of RCOOH decreases. More NH^ and l e s s RCOOH w i l l be taken up, and the pH w i l l tend to f a l l . Thus changes i n pH w i l l be s e l f - c o r r e c t i n g . The exact pH maintained w i l l depend on the r a t i o between the c o n c e n t r a t i o n of ammonium (NH^ + plus RH^) and the c o n c e n t r a t i o n of organic a c i d (RCOOH plus RCOO"). T h i s model c o r r e c t l y p r e d i c t s t h a t too l i t t l e o r ganic a c i d s a l t w i l l allow the pH to drop, and too much w i l l cause the pH to r i s e . A c t i v e t r a n s p o r t of H + across the c e l l membrane, f o r example i n exchange f o r K + ( R o t h s t e i n I965) w i l l complicate the p r e d i c t i o n s of t h i s model. CHAPTER TWO PRELIMINARY STUDIES ON THE EROGEN M a t e r i a l s and Methods 33 I. A q u a l i t a t i v e b i o a s s a y For p r e l i m i n a r y s t u d i e s on the chemical behaviour of the erogen, a b i o a s s a y that would r e v e a l the presence or absence of hormone a c t i v i t y was r e q u i r e d . F i r s t , an assay procedure o r i g i n a t e d by R. J . Bandoni was used. P e t r i p l a t e s of Conjugation Medium (CjM) s o l i d i f i e d with 1.5$ agar were streaked l i g h t l y with 2259-6, and incubated at 20°C. A f t e r 24 hours, blocks approximately 1 cm by 1 cm were cut from the p l a t e s , and deposited c e l l s i d e up i n p l a s t i c p e t r i p l a t e s , 5 cm i n diameter. To each assay d i s h , 4.0 ml of s t e r i l e l i q u i d CjM, along with an a p p r o p r i a t e volume of t e s t s o l u t i o n ( u s u a l l y one to ten drops) was added. The assays were incubated overnight at 20°C, and then the c e l l s on the agar b l o c k s wer,e.iexamined:'uhaermt.hecmi'cposcopetfor;:the^preserice pf con-j u g a t i o n tubes. L a t e r , an assay r e q u i r i n g l e s s of the t e s t s o l u t i o n and e l i m i n a t i n g the agar blocks was developed. One ml of an ap-p r o p r i a t e d i l u t i o n of the t e s t s o l u t i o n i n CjM or 0-5$ soytone was p i p e t t e d i n t o a 16 X 125 mm t e s t tube, capped with a s t a i n l e s s s t e e l or p l a s t i c c u l t u r e tube c l o s u r e , and autoclaved f o r 15 minutes at 15 p s i pre s s u r e . A f t e r c o o l i n g , the assay tubes were i n o c u l a t e d with one drop from, a s t e r i l e Pasteur p i p e t t e of a two to fo u r day o l d shake c u l t u r e of 2259-6 i n QS , medium. The tubes were mixed by g e n t l e shaking, and incubated overnight (12 to 18 hours) at 20°C. The next morning, a drop from each assay tube was examined under the microscope f o r the presence of conjugation tubes. A s u b j e c t i v e impression of the i n t e n s i t y of the r e a c t i o n , based on the p r o p o r t i o n of c e l l s b e a r i n g c o n j u g a t i o n tubes, the l e n g t h of the tubes, and the number of tubes per yeast c e l l , was used to roughly estimate the r e l a t i v e amounts of hormone i n the t e s t s o l u t i o n s . S i m i l a r r e s u l t s were obtained from the two assay methods. 34 I I . P r o d u c t i o n o f hormone C o n j u g a t i o n hormone f o r s t u d y was p r o d u c e d b y g r o w i n g 2259-7 i n s h a k e c u l t u r e a t 20°C. M e d i a u s e d a t v a r i o u s t i m e s i n c l u d e d MYP, C j M , and GM3. I n o c u l u m was o b t a i n e d e i t h e r f r o m s h a k e c u l t u r e s i n t h e e x p o n e n t i a l o r e a r l y s t a t i o n a r y p h a s e s o f g r o w t h , o r b y s c r a p i n g c e l l s f r o m t h e s u r f a c e o f P e t r i p l a t e c u l t u r e s on MYP. The c u l t u r e s were a l l o w e d t o grow i n t o s t a t i o n -a r y p h a s e and t h e c e l l s were removed b y c e n t r i f u g a t i o n . The s u p e r n a t a n t was e i t h e r u s e d d i r e c t l y o r t r e a t e d w i t h an e q u a l v o lume o f a c e t o n e t o p r e c i p i t a t e p o l y s a c c h a r i d e and r e s i d u a l c e l l s . I I I . E x t r a c t i o n w i t h o r g a n i c s o l v e n t s The s u p e r n a t a n t f r o m a c u l t u r e o f 2259-7 i n GM3 was d i v i d e d i n t o t h r e e p o r t i o n s . The pH o f t h e f i r s t p o r t i o n was f o u n d t o be a p p r o x i m a t e l y 6 by measurement w i t h p H y d r i o n i n d i c a t o r p a p e r . The s e c o n d p o r t i o n was a d j u s t e d t o pH 2 w i t h c o n c e n t r a t e d h y d r o c h l o r i c a c i d , and t h e t h i r d p o r t i o n t o pH 10 w i t h c o n -c e n t r a t e d ammonium h y d r o x i d e . Ten m l s a m p l e s o f t h e c u l t u r e s u p e r n a t a n t a t e a c h pH were s h a k e n w i t h t e n ml o f one o f n-b u t a n o l , e t h y l a c e t a t e , d i e t h y l e t h e r , c h l o r o f o r m , b e n z e n e , m e t h y l e n e c h l o r i d e , o r p e t r o l e u m e t h e r (65-HO). P e r s i s t e n t e m u l s i o n s f o r m e d i n some o f t h e e x t r a c t i o n s , and i n t h e s e c a s e s s e p a r a t i o n o f t h e p h a s e s was h a s t e n e d b y c e n t r i f u g a t i o n . The s o l v e n t e x t r a c t s and t h e aqueous r a f f i n a t e s were s e p a r a t e d , and e v a p o r a t e d t o d r y n e s s i n v a c u o ( B u c h l e r r o t a r y e v a p o r a t o r , w a t e r b a t h t e m p e r a t u r e 40°C). E a c h r e s i d u e was r e d i s s o l v e d i n 10 ml o f C j M , and b i o a s s a y e d , a l o n g w i t h a s a m p l e o f t h e o r i g i n a l c u l t u r e s u p e r n a t a n t . To e s t i m a t e t h e p a r t i t i o n c o e f f i c i e n t o f t h e e r o g e n s b e t w e e n w a t e r and n - b u t a n o l , a c o u n t e r c u r r e n t d i s t r i b u t i o n was c a r r i e d o u t . A s a m p l e o f e r o g e n s f o r t h e d i s t r i b u t i o n was o b t a i n e d b y e x t r a c t i n g 150 ml o f t h e s u p e r n a t a n t from, a c u l t u r e o f 2259-7 i n GM3 w i t h f o u r 50 ml p o r t i o n s o f n - b u t a n o l ( s a t u r a t e d w i t h w a t e r ) . The b u t a n o l p h a s e s were c e n t r i f u g e d b r i e f l y t o remove 35 d r o p l e t s of the aqueous phase, and then evaporated to dryness i n vacuo. The e x t r a c t was d i s s o l v e d i n 2.0 ml of water-saturated n-butanol. A f t e r the phases separated, the b u t a n o l phase was t r a n s f e r r e d as completely as p o s s i b l e with a Pasteur p i p e t t e i n t o a second tube. Two ml of water ( s a t u r a t e d with butanol) was added to the second tube and 2.0 ml of n-butanol ( s a t u r a t e d with water) was added to the aqueous phase i n the f i r s t tube. Both tubes were then shaken. A f t e r the phases separated, the b u t a n o l phase i n the second tube was added to 2.0 ml of water i n a t h i r d tube. The b u t a n o l phase from the f i r s t tube was t r a n s -f e r r e d t o the second tube, and 2.0 ml of b u t a n o l were- added to the f i r s t tube. T h i s procedure was repeated u n t i l ten tubes were i n use. For each tube, a f i l t e r paper d i s c of the type used f o r a n t i b i o t i c assays ( S c h l e i c h e r and S c h u e l l , Inc.) was s a t u r a t e d with the b u t a n o l phase and d r i e d i n a stream of warm a i r . A suspension of 2259-6 c e l l s was evenly spread over the s u r f a c e of GS medium s o l i d i f i e d with 1-5$ agar i n a P e t r i p l a t e . The paper d i s c s were placed on top of t h i s lawn of c e l l s , one d i s c i n the center and four e q u a l l y spaced around the edge of each p l a t e . A f t e r overnight i n c u b a t i o n at 20°C, the c e l l s adjacent to the paper d i s c s were examined under the microscope f o r c o n j u g a t i o n tubes. IV. A d s o r p t i o n on c a t i o n exchange r e s i n Two hundred m i l l i l i t r e s of supernatant from a c u l t u r e of 2259-7 i n GM3 were run through a bed of 20 ml of Amberlite IR-120 strong a c i d c a t i o n exchange r e s i n , hydrogen form ( M a l l i n c k r o d t Chemical Works), at a flow r a t e of 200 ml/hour. A sample of the e f f l u e n t was n e u t r a l i z e d with 10$ sodium b i c a r b o n a t e s o l u t i o n and saved f o r assay. The r e s i n was washed with 50 ml of d i s t i l l e d water and then t r e a t e d with 100 ml of 10$ aqueous ammonia. The ammonia e l u a t e was evaporated to dryness _in vacuo, d i s s o l v e d i n 5 ml of d i s t i l l e d water, and re-evaporated. The e l u t e d m a t e r i a l was f i n a l l y d i s s o l v e d i n 10 ml of d i s t i l l e d water, and d i l u t e d 1 t o 10 i n GM3 f o r b i o a s s a y . The r e s i n was regenerated by running through 200 ml of d i s t i l l e d water, 100 ml of 2 N s u l p h u r i c a c i d , and another 200 ml of d i s t i l l e d water. 36 In another experiment, c u l t u r e supernatant was run through the column, and the r e s i n washed as above. A concentrated ammonium acetate b u f f e r , pH 1, was prepared by mixing 100 ml of g l a c i a l a c e t i c a c i d with 100 ml of concentrated (30$ NHg) ammonium hydroxide. 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 prepared by d i l u t i o n from the concentrated s o l u t i o n . One hundred ml of q u a r t e r - s t r e n g t h ammonium acetate was run though the column, f o l l o w e d by 100 ml of the h a l f - s t r e n g t h b u f f e r . F i n a l l y 100 ml of the f u l l s t r e n g t h ammonium acetate s o l u t i o n was passed through the column. The three e l u a t e s were f r e e z e -d r i e d to remove ammonium a c e t a t e . The r e s i d u e s were d i s s o l v e d i n 10 ml of d i s t i l l e d water, and d i l u t e d 1 to h with GM3 f o r bi o a s s a y . In a t h i r d experiment, a f t e r t r e a t i n g the r e s i n with c u l t u r e supernatant and washing, the column was e l u t e d with a p y r i d i n e acetate s o l u t i o n (25 ml g l a c i a l a c e t i c a c i d , 10 ml p y r i d i n e , 65 ml d i s t i l l e d water; approx. 1-75 N, pH 5)- The p y r i d i n e a cetate e l u a t e was f r e e z e - d r i e d . The e l u t e d m a t e r i a l was d i s s o l v e d i n 10 ml of d i s t i l l e d water and d i l u t e d 1 to 10 with GM3 f o r b i o a s s a y . V. A d s o r p t i o n on anion exchange r e s i n The supernatant from 200 ml of a 3-day c u l t u r e of 2259~7 i n GM3 was adjusted to pH 7 with a 10$ s o l u t i o n of sodium b i c a r -bonate, and then run through a 20 ml bed of D u o l i t e A-k weak base anion exchange r e s i n , c h l o r i d e form (Diamond Shamrock Chemical Co.). Samples of the o r i g i n a l c u l t u r e supernatant and the column e f f l u e n t were taken f o r b i o a s s a y . The r e s i n was washed with 50 ml of d i s t i l l e d water, and then with 100 ml of 1 N a c e t i c a c i d . The a c e t i c a c i d changed the colour of the r e s i n from grey t o yellow. Just before the yellow f r o n t reached the bottom of the column, a f r e s h r e c e i v e r was put i n p l a c e . The a c e t i c a c i d e l u a t e was evaporated i n vacuo, d i s s o l v e d i n 10 ml of d i s t i l l e d water, and re-evaporated. The r e s i d u e was d i s s o l v e d i n 10 ml of d i s t i l l e d water, and d i l u t e d 1 t o 10 with GM3 f o r b i o a s s a y . 37 The column was regenerated "by washing with 200 ml of d i s -t i l l e d water, 100 ml of 2 N potassium hydroxide, 200 ml of d i s t i l l e d water, 100 ml of 2 N h y d r o c h l o r i c a c i d , and 200 ml of d i s t i l l e d water. VI. A d s o r p t i o n on a c t i v a t e d c h a r c o a l One gram of N o r i t A d e c o l o u r i z i n g c h a r c o a l ( B r i t i s h Drug Houses, Canada Ltd.) was mixed with 400 ml of supernatant from a c u l t u r e of 2259-7 i n GM3• The c h a r c o a l suspension was a g i -t a t e d on a r e c i p r o c a t i n g shaker at 20°C f o r h a l f an hour to ensure 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 reached. Then one gram, of H y f l o Super C e l f i l t e r i n g a i d was added to the suspension, and the c h a r c o a l f i l t e r e d out on a Buchner f u n n e l which had been coated with one gram, of H y f l o Super C e l . The f i l t e r cake was washed with two 10 ml r i n s e s of d i s t i l l e d water, and then suspended i n 50 ml of methanol. T h i s suspension was shaken f o r 15 minutes, and c e n t r i f u g e d . The methanol supernatant was decanted and the c h a r c o a l suspended i n 50 ml of absolute e t h a n o l . A f t e r shaking f o r 15 minutes, the suspension was c e n t r i f u g e d , the e t h a n o l decanted, and the c h a r c o a l suspended i n 50 ml of acetone. A f t e r the c h a r c o a l had been c e n t r i f u g e d from the acetone, i t was t r e a t e d with a p y r i d i n e acetate s o l u t i o n (10 ml of p y r i d i n e , 25 ml of g l a c i a l a c e t i c a c i d , made to 50 ml with d i s t i l l e d water). The methanol,- ethanol, and acetone e l u a t e s were evapo-r a t e d to dryness i n vacuo, and the r e s i d u e s d i s s o l v e d i n 5 ml of d i s t i l l e d water. The p y r i d i n e acetate e l u a t e was f r e e z e -d r i e d , and then d i s s o l v e d i n 5 ml of d i s t i l l e d water. Samples of the o r i g i n a l c u l t u r e supernatant, and the f i l t r a t e from the c h a r c o a l a d s o r p t i o n step were bioassayed at t h e i r o r i g i n a l c o n c e n t r a t i o n s . The s o l u t i o n s of the m a t e r i a l s e l u t e d with methanol, ethanol, acetone and p y r i d i n e acetate were d i l u t e d 1 t o 20 with GM3 f o r b i o a s s a y . On another occasion, 20 ml of c u l t u r e supernatant was t r e a t e d with 0.5 gm of N o r i t A. A f t e r h a l f an hour of shaking, the c h a r c o a l was sedimented by c e n t r i f u g a t i o n . The supernatant was decanted and the c h a r c o a l washed with 20 ml of d i s t i l l e d 38 water. The c h a r c o a l was then washed with 20 ml of acetone-water ( l to l ) , 20 ml of acetone-water (3 to l ) , and 20 ml of acetone. The el u a t e s were f i l t e r e d to remove suspended carbon, evaporated t o dryness i n vacuo, and d i s s o l v e d i n 2 ml of GM3 f o r b i o a s s a y . The o r i g i n a l c u l t u r e supernatant, and the super-natant from the c h a r c o a l treatment were a l s o bioassayed. Granular coconut c h a r c o a l ( F i s h e r S c i e n t i f i c Co.) was t e s t e d f o r a b i l i t y to adsorb the hormone. F i f t y ml of c u l t u r e supernatant were shaken f o r 15 minutes with 0.1 gm of coconut c h a r c o a l . The c h a r c o a l was c e n t r i f u g e d out, and the supernatant a f t e r sampling, t r e a t e d with another 0.1 gm of coconut c h a r c o a l . T h i s c h a r c o a l was a l s o c e n t r i f u g e d down, and the supernatant t r e a t e d with a t h i r d 0.1 gm of c h a r c o a l . Samples of the o r i g i n a l spent c u l t u r e medium, and the supernatant from the f i r s t , second, and t h i r d coconut c h a r c o a l treatments were bioas s a y e d . One gram of coconut c h a r c o a l was wet with 10 ml of acetone, and, a f t e r the c h a r c o a l had s e t t l e d , the acetone was decanted. The c h a r c o a l was washed twice with 40 ml of d i s t i l l e d water. F i f t y ml of c u l t u r e supernatant were t r e a t e d as above with three 0.1 gm p o r t i o n s of the acetone washed c h a r c o a l , and the supernatants b i o a s s a y e d . D e a c t i v a t e d c h a r c o a l was prepared by a m o d i f i c a t i o n of the method of Asatoor and D a l g l i e s h (1956). A suspension of 10 gm of N o r i t A i n a s o l u t i o n of 0.4 gm of s t e a r i c a c i d , U.S.P., i n 50 ml of eth a n o l was s t i r r e d f o r h a l f an hour, and then slowly d i l u t e d with 4-50 ml of d i s t i l l e d water. The c h a r c o a l was c o l l e c t e d by f i l t r a t i o n , washed with d i s t i l l e d water, and a i r d r i e d . F o r t y ml p o r t i o n s of 2259-7 c u l t u r e supernatant were shaken with 0.1 gm. of N o r i t A, 0.1 gm of d e a c t i v a t e d c h a r c o a l , or 0.5 gm of d e a c t i v a t e d c h a r c o a l . A f t e r 30 minutes the suspen-sions were c e n t r i f u g e d and samples of the supernatants taken f o r b i o a s s a y . The c h a r c o a l sediments were washed with 40 ml each of d i s t i l l e d water and then suspended i n 20 ml each of 39 10$ aqueous phenol. A f t e r 15 minutes these suspensions were c e n t r i f u g e d and the supernatants were decanted and evaporated to dryness _in vacuo. These e l u a t e s were each d i s s o l v e d i n 5 ml of d i s t i l l e d water, and d i l u t e d 1 to k with 0-5$ soytone f o r "bioassay. VI I . A d s o r p t i o n on Porapak One gram of Porapak QS, 120-150 mesh (Waters A s s o c i a t e s , d i s t r i b u t e d i n Canada by Chromatographic S p e c i a l t i e s , B e l l e v i l l e , O n t a rio) was suspended i n acetone, and poured i n t o a miniature chromatography column made from a Pasteur p i p e t t e , f i t t e d with a g l a s s wool plug. The acetone was allowed t o d r a i n o f f , and the r e s i n was washed with 50 ml of d i s t i l l e d water. Ninety ml of supernatant from a c u l t u r e of 2259-7 i n Gluc-AmS medium were passed through the column at a flow r a t e of approximately 2 ml per minute. Samples of the o r i g i n a l c u l t u r e medium and the e f f l u e n t from the column were supplemented with .05 ml of 10$ (w/v)' soytone per ml, n e u t r a l i z e d with .1 W potassium hydroxide, and bioassayed. The column was washed with 10 ml of d i s t i l l e d water, and th e n ' e l u t e d with 10 ml of 50$ acetone (5 ml water plus 5 ml acetone), 10 ml of 75$ acetone, and 10 ml of acetone. For b i o assay, 3 drops of each el u a t e s o l u t i o n was added to 1 ml of 0-5$ soytone. A s i m i l a r experiment was c a r r i e d out using Porapak Q. V I I I . Chromatography on Sephadex G-10 T h i r t y grams of Sephadex G-10 (Pharmacia) were soaked f o r three hours i n d i s t i l l e d water and then poured i n t o a g l a s s chromatography column (2 cm i n t e r n a l diameter). The stopcock at the bottom of the column was opened, so that water was f l o w i n g through the column while the g e l s e t t l e d . To c a l i b r a t e the column, a s o l u t i o n c o n t a i n i n g 0.1 gm of s o l u b l e s t a r c h ( D i f c o L a b o r a t o r i e s ) and 0.1 gm of sodium c h l o r i d e per ml was prepared. When the water l e v e l i n the Sephadex column reached the g e l s u r f a c e , 1 ml of the s t a r c h - s a l t s o l u t i o n was ko a p p l i e d to the top of the column and washed i n with a s m a l l volume of d i s t i l l e d water. D i s t i l l e d water was passed through the column at O.k ml per minute and the e f f l u e n t was c o l l e c t e d i n twenty 10 minute f r a c t i o n s . A 0 . 5 ml sample of each f r a c t i o n was t r e a t e d with a drop of Melzer's reagent ( i o d i n e 1 .5 gm, potassium i o d i d e .5 gm, c h l o r a l 1 0 0 gm, water 1 0 0 ml) to d e t e c t s t a r c h , and a separate 0 . 5 ml sample was t r e a t e d with a drop of 0 . 1 N s i l v e r n i t r a t e to detect c h l o r i d e i o n . A sample of conjugation hormone was prepared "by e x t r a c t i o n of c u l t u r e supernatant with n-butanol. The b u t a n o l e x t r a c t was evaporated to dryness i n vacuo, and d i s s o l v e d i n water. Two ml of t h i s s o l u t i o n were a p p l i e d to the top of the Sephadex G-10 column, and e l u t e d with d i s t i l l e d water at a flow r a t e of O.k ml per minute. The e f f l u e n t was c o l l e c t e d i n f o r t y -f i v e 5 minute f r a c t i o n s . A 0 . 5 ml sample from each f r a c t i o n was mixed with 0 . 1 ml of 1 0 $ soytone and 1 .5 ml of d i s t i l l e d water, and bioassayed. IX. U l t r a f i l t r a t i o n Two hundred ml of acetone were added with s t i r r i n g to 2 0 0 ml of c u l t u r e supernatant, and the p r e c i p i t a t e which formed was removed by f i l t r a t i o n . The f i l t r a t e was evaporated to dryness i n vacuo and r e d i s s o l v e d i n 15 ml of d i s t i l l e d water. T h i s s o l u t i o n was f i l t e r e d through a P e l l i c o n type PSWP membrane f i l t e r ( M i l l i p o r e L t d . ) , 25 mm i n diameter, under s u c t i o n from a water a s p i r a t o r . The P e l l i c o n f i l t e r p a r t i a l l y r e t a i n s s o l u t e s with a molecular weight g r e a t e r than 7 5 0 and completely r e t a i n s m a t e r i a l s with a molecular weight g r e a t e r than 1 2 5 0 ( M i l l i p o r e A p p l i c a t i o n Report A R - 2 l ) . F i l t r a t i o n of the 15 ml sample took one and a h a l f hours. The s o l u t i o n was washed through the f i l t e r with two 1 ml p o r t i o n s of d i s t i l l e d water. The f i l t e r apparatus was dismantled, and the m a t e r i a l s r e t a i n e d on the f i l t e r were washed i n t o a beaker with a stream of water from a wash b o t t l e . The s o l u t i o n of r e t a i n e d m a t e r i a l was made up to 15 ml with d i s t i l l e d water. The f i l t r a t e and the s o l u t i o n of r e t a i n e d m a t e r i a l s were d i l u t e d 1 to 10 with GM3 f o r b i o -assay. 41 X. Chromatography on columns of s i l i c a g e l F i f t y grams of s i l i c a g e l , 60 to 120 mesh (BDH Chemicals L t d . ) , were s l u r r i e d i n absolute ethanol and poured i n t o a chromatography tube, 2 cm i n diameter. The s i l i c a g e l was allowed to s e t t l e , and the s o l v e n t drained u n t i l the top of the bed was j u s t dry. 0.2 grams of f r e e z e - d r i e d n-butanol e x t r a c t from 2259~7 c u l t u r e supernatant was ground i n a mortar with 2 ml of absolute e t h a n o l . T h i s suspension was p i p e t t e d onto the top of the column, and the s o l v e n t allowed to sink i n t o the column, f o l l o w e d by washings from the mortar. The column was e l u t e d with 100 ml of absolute ethanol at a flow r a t e of 1-95 ml per minute. The e f f l u e n t was c o l l e c t e d i n 2 minute f r a c t i o n s . A f t e r 100 ml of ethanol had passed i n t o the column, e l u t i o n was continued at the same flow r a t e with 100 ml of 87.5$ ethanol (87.5 ml absolute e t h a n o l and 12.5 ml d i s t i l l e d water), f o l l o w e d by 100 ml of 75$ ethanol and f i n a l l y 100 ml of 50$ e t h a n o l . One hundred f r a c t i o n s were c o l l e c t e d . From each even-numbered f r a c t i o n s t a r t i n g with 8, s i x drops were adsorbed on a one-half inch diameter paper d i s c . The d i s c s were d r i e d and p l a c e d on a p l a t e of GS agar spread with 2259-6 c e l l s , s i x d i s c s per p l a t e . A f t e r overnight i n c u -b a t i o n at 20°C, the presence or absence of conjugation tubes on the c e l l s c l o s e to each paper d i s c was noted. Another column c o n t a i n i n g 50 gm of s i l i c a g e l i n absolute e t h a n o l was prepared as above. 0.3 gram of hormone p r e p a r a t i o n was ground with 2 ml of absolute ethanol and t r a n s f e r r e d to the top of the column. The column was e l u t e d with a convex g r a d i e n t of water i n e t h a n o l . To generate the g r a d i e n t , 400 ml of absolute ethanol were placed i n a 500 ml Erlenmeyer f l a s k on a magnetic s t i r r e r . The f l a s k was f i t t e d with ,a rubber stopper b e a r i n g i n l e t and o u t l e t tubes. The i n l e t tube was connected to a r e s e r v o i r c o n t a i n i n g 50$ e t h a n o l . The o u t l e t tube extended n e a r l y to the bottom of the mixing f l a s k , and was connected with p o l y e t h y l e n e t u b i n g to the top of the chroma-42 tography tube. Solvent was fed to the column at 1 ml per min-ute, and the column e f f l u e n t was c o l l e c t e d i n 5 minute f r a c t i o n s . The even-numbered f r a c t i o n s from 6 t o 134 were bioassayed by the paper d i s c method as above. XI. Paper chromatography On v a r i o u s occasions d u r i n g t h i s study, the behaviour of the hormone i n paper chromatography has been examined. Chroma-tography was c a r r i e d out on s t r i p s of Whatman # 1 chromatography paper 2 cm by 14 cm. S u f f i c i e n t hormone s o l u t i o n to allow d e t e c t i o n of the hormone a f t e r chromatography was streaked along a l i n e 2 cm from one end of the s t r i p s . The bottom ends of the s t r i p s were cut to a p o i n t . Each s t r i p was suspended, from a paper c l i p stuck i n t o a cork, i n s i d e a 25 X 150 mm t e s t tube so th a t the pointed bottom end of the s t r i p was immersed i n a po o l of so l v e n t at the bottom of the tube. Composit ions of the s o l v e n t s used are l i s t e d i n Table I I I . A f t e r the so l v e n t had r i s e n approximately 10 cm. above the s t a r t l i n e , the s t r i p s were removed from, the tubes and d r i e d i n a stream, of c o o l a i r . The p a r t s of the s t r i p s between the s t a r t l i n e and the sol v e n t f r o n t were cut p e r p e n d i c u l a r t o the long a x i s i n t o ten equal s e c t i o n s . The s e c t i o n s were e i t h e r placed on p l a t e s of GS agar spread with 2259-6 c e l l s , or dropped i n t o t e s t tubes c o n t a i n i n g 1 ml of GS medium. The t e s t tubes were then auto-claved and i n o c u l a t e d with 2259-6. A f t e r overnight i n c u b a t i o n , the assays were examined t o see which s e c t i o n s induced conju-g a t i o n tubes, and Rf values f o r the erogens were c a l c u l a t e d . XII. E f f e c t of c y c l i c - 3 5 ' - a d e n o s i n e monophosphate The p o s s i b i l i t y t h a t c y c l i c - 3 % 5'-AMP mediates the response to the erogens was examined. A 1 mM s o l u t i o n of cyclic-3'>5 '-adenosine monophosphate ( N u t r i t i o n a l Biochemicals Corp.) was s t e r i l i z e d by f i l t r a t i o n through a s t e r i l e Gelman membrane f i l t e r with 0.22 u pores i n t o a s t e r i l e f l a s k . The s t e r i l e s o l u t i o n was added a s e p t i c a l l y ^3 to 5.0 ml p o r t i o n s of GS medium i n 50 ml Erlenmeyer f l a s k s to give f i n a l c y c l i c AMP c o n c e n t r a t i o n s of 1 X 10~\ 5 X 10~5, 1 X 10-5, 5 x 10~6, and 1 X 10 - 6 M. The f l a s k s were i n o c u l a t e d with T_. mesenterica 2259 -6, an<3. incubated on the shaker at 20°C. Samples were taken from each f l a s k d a i l y f o r s i x days, and examined under the microscope. C a f f e i n e i s known to i n h i b i t c y c l i c AMP phosphodiesterases (Wair I966) and t h e r e f o r e i t might cause accumulation of c y c l i c AMP i n s i d e the c e l l s . C a f f e i n e was added to 20 ml p o r t i o n s of GS medium i n 125 ml Erlenmeyer f l a s k s at c o n c e n t r a t i o n s of 0, 1, 2, k, 8, and 16 mM. A f t e r a u t o c l a v i n g , each f l a s k was i n o c u l a t e d with 1 ml of a 2259-6 c u l t u r e and incubated on the shaker at 20°C. D a i l y samples from each f l a s k were examined m i c r o s c o p i c a l l y . A f t e r s i x days, a sample from each f l a s k was d i l u t e d 1 to 10 with d i s t i l l e d water and i t s absorbance measured at 5^ 0 nm. R e s u l t s I. E x t r a c t i o n with organic s o l v e n t s The m a t e r i a l e x t r a c t e d from water "by e t h y l acetate, d i e t h y l ether, chloroform, benzene, methylene c h l o r i d e , or petroleum ether at any of the pH's t r i e d showed no hormone a c t i v i t y . The r a f f i n a t e s from these e x t r a c t i o n s had erogenic a c t i v i t y compar-able t o the o r i g i n a l c u l t u r e supernatant. The bu t a n o l e x t r a c t at a l l pH's showed hormone a c t i v i t y , s l i g h t l y weaker than the o r i g i n a l supernatant. The r a f f i n a t e from the bu t a n o l e x t r a c t i o n s d i s p l a y e d low hormone a c t i v i t y . No d i f f e r e n c e was detected i n the amount of hormone a c t i v i t y e x t r a c t e d by n-butanol at a c i d , n e u t r a l or a l k a l i n e pH. In the b i o a s s a y of the f r a c t i o n s from the counter current d i s t r i b u t i o n , c o n j u g a t i o n tubes were found around the d i s c s corresponding to tubes k to 10. The most intense response was found i n samples 1 and 8. T h i s d i s t r i b u t i o n of hormone a c t i v i t y among the tubes corresponds to a p a r t i t i o n c o e f f i c i e n t of about 2-3 f o r the erogens between water and n-butanol. I I . A d s o r p t i o n on c a t i o n exchange r e s i n The sample of c u l t u r e supernatant a p p l i e d to the c a t i o n exchange column showed high erogenic a c t i v i t y . N e i t h e r the c u l t u r e supernatant a f t e r passage through the ion exchange column nor the m a t e r i a l e l u t e d with 10$ ammonia induced conju-g a t i o n tubes i n the assay c e l l s . Each of the e l u t i n g b u f f e r s t e s t e d washed brown pigment o f f the column. The appearance of colour i n the e f f l u e n t was a convenient marker f o r the breakthrough p o i n t of each b u f f e r . The m a t e r i a l e l u t e d from the column with quarter s t r e n g t h ammonium acetate showed a low l e v e l of hormone a c t i v i t y . More erogenic a c t i v i t y was e l u t e d with the h a l f s t r e n g t h and f u l l s t r e n g t h ammonium acetate s o l u t i o n s . In none of the three e l u a t e f r a c t i o n s was the a c t i v i t y as high as i n the o r i g i n a l c u l t u r e supernatant. 45 The m a t e r i a l e l u t e d from the c a t i o n exchange r e s i n with p y r i d i n e acetate showed hormone a c t i v i t y comparable to the o r i g i n a l c u l t u r e supernatant. I I I . A d s o r p t i o n on anion exchange r e s i n The c u l t u r e supernatant showed high erogen a c t i v i t y b e f o r e , but none a f t e r p a s s i n g through the anion exchange column. The a c e t i c a c i d e l u a t e d i s p l a y e d high hormone a c t i v i t y . IV. A d s o r p t i o n on a c t i v a t e d c h a r c o a l In the f i r s t experiment, the o r i g i n a l c u l t u r e supernatant showed moderate erogenic a c t i v i t y and the f i l t r a t e a f t e r c h a r c o a l treatment had low hormone a c t i v i t y . The m a t e r i a l e l u t e d with methanol, ethanol, and acetone was i n a c t i v e , but the p y r i d i n e a c etate e l u a t e was h i g h l y e r o g e n i c . In the second experiment, the o r i g i n a l c u l t u r e supernatant had high hormone a c t i v i t y . The supernatant from the c h a r c o a l a d s o r p t i o n , and the m a t e r i a l s e l u t e d with 50$, 75$ and 100$ acetone were a l l i n a c t i v e . The c u l t u r e supernatant used with the coconut c h a r c o a l showed" high hormone a c t i v i t y , and so d i d the supernatants from the f i r s t , second, and t h i r d treatments with coconut c h a r c o a l . Hormone a c t i v i t y was not n o t i c e a b l y l e s s i n the t r e a t e d samples than i n the o r i g i n a l . The coconut c h a r c o a l which had been washed with acetone a l s o d i d not reduce the hormone a c t i v i t y of the supernatants. Nor i t A at 0.1 gm per 40 ml and d e a c t i v a t e d c h a r c o a l at 0.1 and 0.5 gm per 40 ml completely removed erogenic a c t i v i t y from, the c u l t u r e supernatant. The m a t e r i a l s e l u t e d by 10$ phenol from any of the c h a r c o a l samples d i d not show hormone act i v i t y . V. A d s o r p t i o n on Porapak The o r i g i n a l c u l t u r e medium was h i g h l y erogenic, but no hormone a c t i v i t y was found i n the e f f l u e n t from the Porapak QS kG column. A f t e r the medium had passed through the Porapak column, a band of yellow pigment was v i s i b l e at the top of the r e s i n . When 50$ acetone was passed through the column, the yellow colour moved down the column with the acetone f r o n t , and numerous a i r bubbles appeared i n the column behind the f r o n t . The mat-e r i a l e l u t e d with 50$ acetone had high erogenic a c t i v i t y , and some a c t i v i t y was present i n the 75$ and 100$ acetone eluates as w e l l . Porapak Q gave r e s u l t s s i m i l a r to those obtained with Porapak QS, except t h a t very l i t t l e a d d i t i o n a l hormone was el u t e d with 75$ acetone and none with pure acetone. VI. Chromatography on Sephadex G-10 In the c a l i b r a t i o n run of the Sephadex G-10 column, s t a r c h was found i n the f r a c t i o n e l u t e d from the column a f t e r 80 t o 100 minutes, and c h l o r i d e i n the f r a c t i o n from 120 to 170 minutes a f t e r the sample was a p p l i e d . In the run with a hormone sample at the same flow r a t e , erogenic a c t i v i t y was found i n the f r a c t i o n s e l u t e d from 200 to 225 minutes a f t e r the sample was a p p l i e d . V I I . U l t r a f i l t r a t i o n • The u l t r a f i l t r a t e showed high hormone a c t i v i t y , and the r e t a i n e d m a t e r i a l s washed from the f i l t e r were completely i n -a c t i v e . A pparently the erogens are not r e t a i n e d by a P e l l i c o n PSWP membrane f i l t e r (molecular weight c u t o f f approximately 1000). V I I I . Chromatography on s i l i c a g e l columns A band of dark brown pigment t r a v e l l e d through the column, ap p a r e n t l y unretarded, with the absolute ethanol e l u a n t . T h i s pigment was c o l l e c t e d i n f r a c t i o n s 8 t o 10. When the eluant was changed to 87.5$ ethanol, more pigment moved down the column, and was c o l l e c t e d i n f r a c t i o n s 35 t o 37- The 75$ ethanol e l u t e d more pigment which emerged i n f r a c t i o n s 63 to 70, and the 50$ ethanol e l u t e d pigment i n f r a c t i o n s 87 to 92. 47 Conjugation tubes were found around the paper d i s c s t r e a t e d with samples from f r a c t i o n s 8 to 12, and 34 to 100. The h i g h -est response was to f r a c t i o n s 8 and 10, 36 and 38, 60 to 76, and 88 to 100. The a c t i v e m a t e r i a l i n f r a c t i o n s 8 to 12 was designated hormone I, i n f r a c t i o n s 33 to 53 hormone I I , and i n f r a c t i o n s 5h to 100 hormone I I I . In the second run, the unretarded brown pigment emerged from the column i n f r a c t i o n 6. Erogenic a c t i v i t y was found i n f r a c t i o n s 6 t o 18, 42 to 62, and 80 to 134. The a c t i v e m a t e r i a l i n f r a c t i o n s 6 t o 18 was i d e n t i f i e d with hormone I, t h a t i n f r a c t i o n s 42 to 62 with hormone I I , and t h a t i n f r a c -t i o n s 80 to 134 with hormone I I I . IX. Paper chromatography The s o l v e n t s used and the Rf of the erogenic a c t i v i t y i n each s o l v e n t are l i s t e d i n Table I I I . The Rf values obtained were r e p e a t a b l e . X. E f f e c t of c y c l i c - 3 % 5'-adenosine monophosphate The c e l l s t r e a t e d with c y c l i c AMP at c o n c e n t r a t i o n s up to 10-^ M d i d not show any m o r p h o l o g i c a l d i f f e r e n c e s from the c e l l s grown without c y c l i c AMP. Conjugation tubes d i d not appear on any of the c e l l s t r e a t e d with c a f f e i n e . Some of the c e l l s , p a r t i c u l a r l y i n the f l a s k with 2 mM c a f f e i n e , were elongated, but remained d i s t i n g u i s h a b l e from c o n j u g a t i o n tubes. Concentrations of c a f f e i n e above 2 mM i n h i b i t e d the growth of the c e l l s . The t u r b i d i t i e s of one-tenth d i l u t i o n s of the c u l t u r e s at each c a f f e i n e c o n c e n t r a t i o n a f t e r s i x days are presented i n Table IV. 48 Table I I I . The Rf values f o r the erogen from T r e m e l l a mesen-t e r i c a 2259-7 obtained on paper chromatography i n v a r i o u s s o l v e n t s . Solvent Composition (v/v/v) n-butanol, a c e t i c a c i d , water (8/1/1) (12/3/5) (4/1/5, upper phase) n-butanol, p y r i d i n e , water (8/1/1) (1/1/1) n-butanol ( s a t u r a t e d with water) chloroform, methanol, water (4/4/1) (4/2/5, lower phase); chloroform, a c e t i c a c i d , water (1/1/1) (2/1/25 (4/1/5, lower phase) (5/4/1) benzene, a c e t i c a c i d , water CV3/1) benzene, a c e t i c a c i d , acetone, water (10/6/10/3) e t h y l a c e t a t e , ethanol, water (4/l/5, upper phase) (h/2/5, upper phase). (*A/2) phenol, water (4/l, w/w) methanol et h a n o l ethanol, water (13/1) acetone, water (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 Rf of Erogen A c t i v i t y •3-.5 .4-. 6 • 35--4 •1--3 .5-.8 0-.2 •1.0 0 3-.7-.9 0-. 15 0 •35--5 0 0-.2, 0 0-.5 0..1, .9-1.0 0 0 .8-1.0 •95-1-0 0-.1, .2-.5--8 0-.1, .7-0-.1, .6-6 (two zones) 5 (two zones) 6 (two zones) 9 (two zones) 9 (two zones) 49 Table IV. T u r b i d i t i e s of one-tenth d i l u t i o n s of T. mesenterica c u l t u r e s grown f o r s i x days i n GS medium plus various c o n c e n t r a t i o n s of c a f f e i n e . C a f f e i n e c o n c e n t r a t i o n (mM) Absorbance at 540 nm 0 0.26 1 0 . 24 2 0.19 4 0.046 8 0.011 16 0.007 50 D i s c u s s i o n The erogenic substances from T_. mesenterica 2259-7 can be e x t r a c t e d from aqueous s o l u t i o n with n-butanol, but not with l e s s p o l a r s o l v e n t s . T h i s shows that the T r e m e l l a hormone i s more p o l a r than the sexual hormones which have been i s o l a t e d from, other f u n g i . S i r e n i n and a n t h e r i d i o l were e x t r a c t e d from, c u l t u r e media with methylene c h l o r i d e (Machlis et a l I966, McMorris and Barksdale I967),' t r i s p o r i c a c ids can be e x t r a c t e d with ether (van den Ende et a l 1970). On the other hand, the f a c t t h a t the erogens p a r t i t i o n into, n-butanol shows that they are l e s s p o l a r than such m e t a b o l i t e s as sugars and the common amino a c i d s . Since pH has l i t t l e e f f e c t on the amount of hormone e x t r a c t e d with organic s o l v e n t s , the erogens are not simply a c i d i c or b a s i c compounds. The a d s o r p t i o n of the erogens on c a t i o n and anion exchange r e s i n s i s presumptive evidence that the hormone molecules c a r r y p o s i t i v e and negative charges at a p p r o p r i a t e pH's. The hormones could not be e l u t e d from the c a t i o n exchange r e s i n by r a i s i n g the pH with 10$ ammonia, and were only p a r t i a l l y recovered with high c o n c e n t r a t i o n s of ammonium a c e t a t e . These r e s u l t s suggest t h a t the hormones were b i n d i n g to the p o l y s t y r e n e backbone of the r e s i n , as w e l l as being a t t r a c t e d to the i o n i c s i t e s . The success of p y r i d i n e acetate i n e l u t i n g the erogens supports t h i s i n t e r p r e t a t i o n . The a b i l i t y of p y r i d i n e to d i s r u p t n o n - i o n i c b i n d i n g to ion exchange r e s i n s has been used i n a scheme f o r the p r e p a r a t i v e s e p a r a t i o n of peptide mixtures (Schroeder et a l I962). The ready e l u t i o n of the hormones from the anion exchange r e s i n with 1 N a c e t i c a c i d may have been p a r t l y caused by the s o l v e n t e f f e c t of the a c e t i c a c i d . F u r t h e r experiments on the r e l a t i v e importance of i o n i c and n o n - i o n i c b i n d i n g to ion exchange r e s i n s are d e s c r i b e d i n Chapter 6. The strong a d s o r p t i o n of the erogens on a c t i v a t e d c h a r c o a l and on the n e u t r a l p o l y s t y r e n e r e s i n Porapak i s c o n s i s t e n t with i t s behaviour on the c a t i o n exchange r e s i n . A d s o r p t i o n on c h a r c o a l i s due to non-polar b i n d i n g f o r c e s and i s u s u a l l y 51 s t r o n g e s t f o r aromatic substances (Asatoor and D a l g l i e s h I956). The t e n a c i t y with which the erogens bind to a c t i v a t e d c h a r c o a l i s r e m a r k a b l e — a r o m a t i c amino acids and s m a l l peptides which have been adsorbed on c h a r c o a l can u s u a l l y be e l u t e d with mix-ture s of water and ethanol or acetone. Asatoor and D a l g l i e s h (I956) have recommended d e a c t i v a t i o n of c h a r c o a l with s t e a r i c a c i d and e l u t i o n with 10$ phenol to improve r e c o v e r i e s of aromatic substances. My r e s u l t s i n d i c a t e t h a t d e a c t i v a t e d c h a r c o a l r e t a i n s a high c a p a c i t y to adsorb erogens, and that 10$ phenol i s i n e f f e c t i v e as an eluant f o r the hormones. The l a c k of a d s o r p t i o n on coconut c h a r c o a l suggests t h a t the a d s o r p t i o n may depend on some s p e c i a l s u r f a c e p r o p e r t y of the brand of c h a r c o a l used. Porapak i s a co-polymer of styre.ne and e t h y l v i n y l b e n z e n e c r o s s l i n k e d with d i v i n y l b e n z e n e . The two types used, Porapak Q and QS, d i f f e r i n the r a t i o of the components. A d s o r p t i o n on Porapak Q has been i n v e s t i g a t e d by Niederwieser as a method f o r r e c o v e r i n g non-polar amino acids and peptides (1971) and i n d o l e s (Niederwieser and G i l i b e r t i I971) from aqueous s o l u t i o n . He found f o r cX-amino acids with hydrocarbon si d e chains, that the degree of a d s o r p t i o n i n c r e a s e d with the number of carbon atoms i n the s i d e chain. Aromatic amino acids were bound l e s s s t r o n g l y than the corresponding s a t u r a t e d , s t r a i g h t chain com-pounds. Peptides were adsorbed more s t r o n g l y than t h e i r con-s t i t u e n t amino a c i d s . I have found t h a t Porapak Q i s as e f f e c t i v e as N o r i t A c h a r c o a l i n removing co n j u g a t i o n hormones from, c u l t u r e supernatants, but the hormones can e a s i l y be e l u t e d from the Porapak with 50$ acetone. It was hoped that chromatography on Sephadex G-10 would i n d i c a t e the molecular weight of the erogens. However, with d i s t i l l e d water as s o l v e n t , the hormone a c t i v i t y i s e l u t e d l a t e r than sodium c h l o r i d e . T h i s high r e t e n t i o n volume i n d i -cates t h a t the hormones are being adsorbed on the g e l , and no v a l i d c o n c l u s i o n s about molecular weight can be drawn. Adsorp-t i o n on Sephadex g e l has f r e q u e n t l y been noted, e s p e c i a l l y f o r 52 aromatic substances (Wolf I969). The complete l a c k of r e t e n t i o n of erogen a c t i v i t y "by the P e l l i c o n PSWP membrane f i l t e r sug-gests t h a t the hormone has a molecular weight l e s s than 750-Chromatography on s i l i c a g e l columns has shown that there are at l e a s t three d i s t i n c t hormones--each of which i s inde-pendently capable of i n d u c i n g conjugation tubes i n c e l l s of the assay s t r a i n . These components have d i f f e r e n t p o l a r i t i e s . , and can be r e c o g n i z e d by the minimum c o n c e n t r a t i o n of water i n ethanol which w i l l e l u t e them from the s i l i c a g e l column. The r e s u l t s obtained by using a g r a d i e n t of water c o n c e n t r a t i o n e l i m i n a t e the p o s s i b i l i t y t h a t the peaks of hormone a c t i v i t y observed i n the f i r s t experiment were the r e s u l t of d i s c o n t i n -u i t i e s i n the s t r e n g t h of the e l u t i n g s o l v e n t . I t i s not c l e a r whether the mechanism of t h i s s e p a r a t i o n i s a c t u a l l y adsorp-t i o n or p a r t i t i o n on the s i l i c a g e l , or d i f f e r e n t i a l s o l u b i l i t y of the hormones i n ethanol-water mixtures. Wot a l l of the i n i t i a l hormone p r e p a r a t i o n i s s o l u b l e i n the absolute ethanol used as s t a r t i n g s o l v e n t . As e l u t i o n proceeds, the bulk of u n d i s s o l v e d m a t e r i a l at the top of the column g r a d u a l l y dimin-i s h e s . P o s s i b l y the main phenomenon i s d i f f e r e n t i a l s o l u b i l i t y with s e p a r a t i o n enhanced by a d s o r p t i o n or p a r t i t i o n on the s i l i c a g e l . It i s probable t h a t the three hormone components are v a r i a n t s of a b a s i c s t r u c t u r e . Perhaps the chemical s t r u c t u r e s necessary f o r erogenic a c t i v i t y occupy only part of the hormone molecule, and the r e s t of the molecule can be v a r i e d without d e s t r o y i n g b i o l o g i c a l a c t i v i t y . A l t e r n a t i v e l y , some or a l l of the hormone components may not be e r o g e n i c a l l y a c t i v e i n t h e i r present form, but can r e a d i l y be converted i n t o an a c t i v e form by the responding fungus. The p r o d u c t i o n of three con-j u g a t i o n hormones by T_. mesenterica 2259-7 can be compared to the p r o d u c t i o n by B l a k e s l e a t r i s p o r a and Mucor mucedo of t r i -s p o r i c a c i d s A, B, and C. A l l of these have zygogenic a c t i v i t y , but d i f f e r i n the l e v e l of o x i d a t i o n at the penultimate carbon 53 of the side chain (Bu'Lock, Drake and Winstanley I972). Pro-d u c t i o n of s e v e r a l d i s t i n c t compounds with the same b i o l o g i c a l a c t i v i t y i s common among f u n g i - - f o r example, the p e n i c i l l i n s (Abraham and Newton 1967)-The behaviour of the erogens i n paper chromatography i s c o n s i s t e n t with t h e i r s o l v e n t p a r t i t i o n c h a r a c t e r i s t i c s . The sol v e n t must c o n t a i n a minimum amount of water t o cause migra-t i o n of the erogens. In the n - b u t a n o l - a c e t i c acid-water and n-butanol-pyridine-water s o l v e n t systems the Rf values f o r the erogens are s i m i l a r to those f o r the aromatic amino acids (Smith I958), but the Rf f o r the erogens i n 80$ phenol i s anomalously high. In some s o l v e n t systems the erogenic a c t i v i t y i s r e s o l v e d i n t o two zones, s u p p o r t i n g the evidence from s i l i c a g e l chroma-tography f o r m u l t i p l e forms of the erogen. C y c l i c - 3 S 5 '-adensine monophosphate has been i m p l i c a t e d as an i n t r a c e l l u l a r mediator i n the responses t o hormones of higher animals (Robison, Butcher, and Sutherland 1971), and i n higher p l a n t s ( P o l l a r d I97O, Salomon and Mascarenhas I97I, Wood, L i n and Braun I972). I t has a l s o been found to serve as the chemotactic agent a c r a s i n i n the c e l l u l a r slime mold, D i c t y o s t elium. d i s coideum ( B a r k l e y 1969)-The experiments r e p o r t e d here do not provide any evidence f o r the involvement of c y c l i c AMP i n conjugation tube p r o d u c t i o n i n T r e m e l l a mesenterica, but they do not r i g o r o u s l y exclude the p o s s i b i l i t y . Exogenous c y c l i c AMP may have been i n a c t i v e because i t could not penetrate the c e l l s , or i t may have been r a p i d l y destroyed by h y d r o l y t i c enzymes. The t o x i c i t y of c a f -f e i n e show t h a t i t was able to enter the c e l l s , but the t o x i c -i t y may have i n t e r f e r e d with conjugation tube p r o d u c t i o n i n some way not r e l a t e d t o c y c l i c AMP metabolism. As w e l l , T r e m e l l a phosphodiesterases may not be s e n s i t i v e to c a f f e i n e . In summary, the a d s o r p t i o n of the erogens on both c a t i o n and anion exchange r e s i n s suggests that the molecules c o n t a i n both weakly a c i d i c and weakly b a s i c groups. Strong adsorp-t i o n on a c t i v a t e d c h a r c o a l and Porapak i n d i c a t e t hat the erogen 54 molecules i n c l u d e non-polar p o r t i o n s capable of hydrophobic bonding. Because the erogens are adsorbed on Sephadex, and very s t r o n g l y h e l d on a c t i v a t e d c h a r c o a l , the non-polar p o r t i o n s may i n c l u d e aromatic r i n g s . The s o l v e n t p a r t i t i o n behaviour i n d i c a t e s t h a t the whole molecules have medium p o l a r i t y , as expected from summation of the non-polar and i o n i c p o r t i o n s . U l t r a f i l t r a t i o n suggests a molecular weight below 750. These p r o p e r t i e s could be expected from an amino a c i d with a l a r g e non-polar si d e chain, or from a sma l l peptide c o n t a i n i n g amino acids with non-polar si d e chains. CHAPTER THREE QUANTITATIVE BIOASSAY OF THE EROGENS M a t e r i a l s and Methods 55 I. R e l a t i o n of c o n j u g a t i o n tube l e n g t h to erogen c o n c e n t r a t i o n A sample of erogens was prepared by e x t r a c t i n g the super-natant from a c u l t u r e of 2259-7 i n GM3 with n-butanol. The b u t a n o l e x t r a c t was evaporated to dryness i n vacuo, and d i s -s olved i n a mixture of 10 ml of d i s t i l l e d water and 10 ml of e t h y l a c e t a t e . A f t e r vigorous shaking, the w a t e r - e t h y l acetate mixture was decanted and c e n t r i f u g e d to separate the phases. The e t h y l acetate phase was d i s c a r d e d , and the aqueous phase was f r e e z e - d r i e d . 22.2 mg of the f r e e z e - d r i e d e x t r a c t was d i s s o l v e d i n 10.0 ml of GS medium. T h i s stock s o l u t i o n was d i l u t e d with GS medium to give s o l u t i o n s c o n t a i n i n g 55-5, 111, 222, Kkk, 666, 888, 1110, and 2220 micrograms of hormone p r e p a r a t i o n per ml. Each d i l u t i o n was dispensed i n one ml amounts i n t o t r i p l i c a t e t e s t tubes. The tubes were autoclaved f o r 10 minutes, and then i n o c u l a t e d with 2 drops per tube from a 23-hour c u l t u r e of 2259-6 i n GS. The tubes were incubated at 20°C. A f t e r 22 hours, 1 drop from each tube was p i p e t t e d onto a microscope s l i d e . The s l i d e s were d r i e d i n an oven at 105°C, cooled to room temperature, s t a i n e d with 1$ Phloxine-3$ potassium h y d r o x i d e - g l y c e r o l ( l / l / l ) , and covered with g l a s s cover s l i p s . In each sample, the lengths of 20 randomly s e l e c t e d c o n j u g a t i o n tubes were measured, u s i n g a F i l a r eyepiece micrometer, at X1000 m a g n i f i c a t i o n ( o i l immersion ob j e ct ive ) . I I . A b i o a s s a y based on c o n j u g a t i o n tube l e n g t h The l i n e a r dependence of the mean logarithmn of conjuga-. t i o n tube l e n g t h on the logarithmn of erogen c o n c e n t r a t i o n was used as the b a s i s f o r a q u a n t i t a t i v e b i o a s s a y procedure. The f r e e z e - d r i e d hormone p r e p a r a t i o n d e s c r i b e d i n s e c t i o n I was used as a standard. For an assay, 6.0 mg of the standard hormone p r e p a r a t i o n i s d i s s o l v e d i n 6.0 ml of GS medium. The standard s o l u t i o n i s d i v i d e d i n t o two 3 ml p o r t i o n s i n 16 mm 56 t e s t tubes. One-third and one-ninth d i l u t i o n s are prepared by withdrawing 1.0 ml from the standard s o l u t i o n , and adding i t to 2.0 ml of f r e s h GS i n a second t e s t tube. A f t e r thorough mixing, 1.0 ml i s withdrawn from t h i s tube, and mixed with 2.0 ml of GS i n a t h i r d tube. One ml from the t h i r d tube i s with-drawn and d i s c a r d e d . A d u l i c a t e standard d i l u t i o n s e r i e s i s prepared from the second p o r t i o n of standard hormone s o l u t i o n . One or more t e s t p r e p a r a t i o n s are d i s s o l v e d at an a p p r o p r i a t e c o n c e n t r a t i o n i n GS medium and d u p l i c a t e d i l u t i o n s s e r i e s set up as above. The assay tubes are autoclaved at 15 p s i pressure f o r 10 minutes on a dry goods c y c l e , and, a f t e r c o o l i n g to room, temperature, i n o c u l a t e d with two drops per tube from a 2 day shake c u l t u r e of 2259-6 i n GS. A f t e r i n c u b a t i o n at 20°C f o r about 18 hours, a drop from, each assay tube i s placed on a microscope s l i d e , d r i e d , and s t a i n e d . The l e n g t h of 25 randomly s e l e c t e d c o n j u g a t i o n tubes i n each sample i s measured as above. For c e l l s b e a r i n g more than one conjugation tube, the sum of the lengths of each tube i s recorded. The data thus obtained are analyzed by the method of Finney (1964). Reg r e s s i o n l i n e s f o r average In tube l e n g t h a g a i n s t l o g dose are f i t t e d f o r each p r e p a r a t i o n with the c o n s t r a i n t that the l i n e s must be p a r a l l e l . From the h o r i z o n t a l d i s t a n c e between the sample and standard r e g r e s s i o n l i n e s , the potency of the t e s t p r e p a r a t i o n r e l a t i v e to the standard i s c a l c u l a t e d . The v a r i a t i o n of the tube l e n g t h w i t h i n each dose i s used to estimate experimental e r r o r , t e s t the assumption that the r e g r e s s i o n l i n e s are l i n e a r and p a r a l l e l , and c a l c u l a t e confidence l i m i t s f o r potency r a t i o s . I I I . R e l a t i o n of f r a c t i o n of c e l l s b e a r i n g c o n j u g a t i o n tubes to erogen c o n c e n t r a t i o n Erogens were prepared by growing 2259-7 i n Gluc-AmS medium. The c u l t u r e supernatants were mixed with 0-5 gm of N o r i t A a c t i v a t e d c h a r c o a l per l i t r e , and l e f t f o r an hour with occa-s i o n a l s t i r r i n g . The c h a r c o a l was recovered by c e n t r i f u g a t i o n , 57 and washed with d i s t i l l e d water. Conjugation hormones were e l u t e d from, the c h a r c o a l with a p y r i d i n e acetate s o l u t i o n (50 ml of g l a c i a l a c e t i c a c i d , 20 ml of p y r i d i n e , and 50 ml of d i s t i l l e d water). The e l u a t e was evaporated to dryness i n vacuo, r e d i s s o l v e d i n a s m a l l volume of water, re-evaporated, and then d i s s o l v e d i n d i s t i l l e d water and f r e e z e - d r i e d . The f r e e z e - d r i e d m a t e r i a l was s t o r e d i n a t i g h t l y capped v i a l i n the f r e e z e r s e c t i o n of a r e f r i g e r a t o r . A sample of the erogen p r e p a r a t i o n was d i s s o l v e d i n a 0.5$ s o l u t i o n of soytone, pH 5> at a c o n c e n t r a t i o n of 1.0 mg/ml. Nine s e r i a l d i l u t i o n s i n 0-5$ soytone, i n which each d i l u t i o n had a hormone c o n c e n t r a t i o n 2/3 as great as the preceding one, were prepared. Two ml of each d i l u t i o n i n 16 mm t e s t tubes were autoclaved 10 minutes on a dry goods c y c l e and i n o c u l a t e d with 2 drops each of a 2 day shake c u l t u r e of 2259-6 i n GS. A f t e r 15 hours at 20°C, a sample from each d i l u t i o n was exam-ined i n a haemacytometer under the X400 power of the microscope. The t o t a l number of c e l l s , and the number of c e l l s with one or more con j u g a t i o n tubes longer than one c e l l diameter were counted i n 80 s m a l l squares. IV. A b i o a s s a y based on f r a c t i o n of c e l l s with conjugation tubes The standard hormone p r e p a r a t i o n used i s the same as that d e s c r i b e d i n s e c t i o n I I I . One u n i t of conjugation hormone i s d e f i n e d as the amount of c o n j u g a t i o n hormone i n 0.2 mg of the standard p r e p a r a t i o n . Standard stock s o l u t i o n s c o n t a i n i n g 10 mg of standard p r e p a r a t i o n per ml of 50$ aqueous acetone are s t o r e d i n the f r e e z e r between uses. For an assay, .04 ml of standard stock s o l u t i o n i s placed i n the bottom of a 16 mm t e s t tube, and mixed with 2.0 ml of 0.5$ soytone, pH 5 • 5 .> to give an erogen c o n c e n t r a t i o n of 1 u n i t per ml. From t h i s s o l u t i o n , s e r i a l d i l u t i o n s of l / 2 , l/k and l/8 i n 0.5$ soytone are prepared i n l6 mm t e s t tubes. One ml from the l/8 d i l u t i o n i s d i s c a r d e d so t h a t each t e s t tube contains one ml. 58 I f the t e s t s o l u t i o n i s expected to co n t a i n l e s s than 10 u n i t s of erogen per ml, a sample i s supplemented with 1/20^ volume of 10$ (w/v) s o l u t i o n of soytone. The pH of the t e s t sample i s adjusted to 5-5 with 0.1 N h y d r o c h l o r i c a c i d or potassium hydroxide, using bromocresol green or bromocresol purple as an i n d i c a t o r . The t e s t sample i s d i l u t e d with 0.5$ soytone t o give an expected potency of approximately 1 u n i t per ml. Two ml of the d i l u t e d t e s t s o l u t i o n are p i p e t t e d i n t o a t e s t tube, and l/2, l / 4 , and l/8 d i l u t i o n s prepared as f o r the standard. From one to f i f t e e n t e s t s o l u t i o n s can be assayed s i m u l t a n e o u s l y . The assay tubes are capped, autoclaved f o r ten minutes on a dry goods c y c l e and, a f t e r c o o l i n g , i n o c u l a t e d with one drop each from, a 24 hour shake c u l t u r e of 2259-6' i - n GS. The tubes are incubated at 20°C overnight (13 to 20 hours) and then placed i n a r e f r i g e r a t o r at 4°C. From each tube, a sample i s mounted i n a haemacytometer, and the number of t o t a l c e l l s , and c e l l s with conjugation tubes longer than one c e l l diameter are counted. U s u a l l y a t o t a l of 100 to 150 c e l l s i s counted f o r each sample. A l l of the samples i n one day's b i o a s s a y are counted w i t h i n as short a p e r i o d as p o s s i b l e . The s t a t i s t i c a l a n a l y s i s of the bi o a s s a y data i s patterned a f t e r Finney (1971)- For each dose, the f r a c t i o n of c e l l s with co n j u g a t i o n tubes i s transformed t o i t s l o g i t ( l o g i t of p = l n ( p / ( l - p ) ) )• Weighted r e g r e s s i o n l i n e s f o r l o g i t of response ag a i n s t the logarithmn of dose are f i t t e d s e p a r a t e l y f o r the standard s e r i e s and f o r each t e s t p r e p a r a t i o n , and then a set of r e g r e s s i o n l i n e s c o n s t r a i n e d t o be p a r a l l e l f o r a l l prepar-a t i o n s i s c a l c u l a t e d . The d e v i a t i o n s of the data from the separate r e g r e s s i o n l i n e s f o r each p r e p a r a t i o n are t e s t e d to see i f they are g r e a t e r than expected from random, sampling. The i n c r e a s e i n d e v i a t i o n when p a r a l l e l l i n e s are f i t t e d i s used to t e s t the hypothesis t h a t the l i n e s r e a l l y are p a r a l l e l . The erogenic potency of each t e s t p r e p a r a t i o n i s estimated from, the h o r i z o n t a l d i s t a n c e between the r e g r e s s i o n l i n e 59 f o r the t e s t p r e p a r a t i o n and the standard l i n e , and 95$ c o n f i -dence i n t e r v a l s f o r the potency r a t i o are c a l c u l a t e d . A comput program which performs these c a l c u l a t i o n s i s d e s c r i b e d i n Appendix C. 6o R e s u l t s I. R e l a t i o n of con j u g a t i o n tube l e n g t h to erogen c o n c e n t r a t i o n a. Data The l e n g t h measurements of the conjugation tubes at var-ious c o n c e n t r a t i o n s of the conjugation hormones are l i s t e d i n Appendix B. The mean tube l e n g t h f o r each sample i s p l o t t e d a g a i n s t erogen c o n c e n t r a t i o n i n F i g . 8. b. S t a t i s t i c a l a n a l y s i s I n s p e c t i o n of the data i n Appendix B shows t h a t the var-i a b i l i t y of the tube l e n g t h i s higher at high erogen doses than at low doses. The v a r i a n c e of the tube l e n g t h i n each sample i s p l o t t e d against the average tube l e n g t h i n the same sample on l o g a r i t h m i c s c a l e s i n F i g . 9. The r e g r e s s i o n of l o g v a r i a n c e on l o g mean i s s i g n i f i c a n t at the .01$ l e v e l , and has a slope of 2.13• A slope of approximately two means that the v a r i a n c e i s p r o p o r t i o n a l t o the square of the mean. For v a l i d s t a t i s t i c a l a n a l y s i s the va r i a n c e of the data should be independent of the mean. T h i s can be accomplished f o r data such as the present by t r a n s f o r m i n g each measurement to i t s n a t u r a l logarithmn ( S t e e l and T o r r i e i960). The means of the n a t u r a l logarithmns of conjugation tube lengths f o r each sample are p l o t t e d a g a i n s t erogen dose i n F i g . 10. A s t r a i g h t l i n e , more s u i t a b l e f o r assay purposes, i s obtained by p l o t -t i n g the average of In tube l e n g t h against the logarit h m s of hormone c o n c e n t r a t i o n ( F i g . l l ) . The r e g r e s s i o n of average In tube l e n g t h on l o g hormone c o n c e n t r a t i o n i s s i g n i f i c a n t at the .01$ l e v e l . 61 CSNCMDim H13N31 3Bni NV3N F i g . 8. R e l a t i o n of mean conjugation tube l e n g t h to dose of erogen. Bars represent 95$ confidence i n t e r v a l s f o r the mean at each dose. F i g . 9. Dependence of vari a n c e on mean f o r conjugation tube l e n g t h measurements. F i g . 10. R e l a t i o n of average transformed conjugation tube l e n g t h t o dose of erogen. F i g . 11. Linear r e g r e s s i o n of average transformed conjugation tube l e n g t h on the logarithmn of erogen dose. 65 I I . A b i o a s s a y based on conjugation tube l e n g t h The b i o a s s a y method based on conjugation tube l e n g t h was employed a few times, but measurement of tube lengths was found to be too t e d i o u s and time-consuming f o r r o u t i n e use. C o n f i -dence i n t e r v a l s ranged from ±25$ to ±70$ of the r e l a t i v e potency. T h i s assay method has been r e p l a c e d by a second procedure based on the f r a c t i o n of c e l l s b e a r i n g conjugation tubes. I I I . R e l a t i o n of f r a c t i o n of c e l l s b e a r i n g conjugation tubes to erogen c o n c e n t r a t i o n The t o t a l number of 2259-6 c e l l s i n 80 s m a l l squares averaged 150. The f r a c t i o n of c e l l s b e a r i n g conjugation tubes i s p l o t t e d a gainst hormone c o n c e n t r a t i o n i n F i g . 12a. The l o g i t (Finney I964) of the f r a c t i o n of c e l l s responding i s p l o t t e d a gainst the logarithmn of hormone c o n c e n t r a t i o n i n F i g . 12b. IV. Bioassay based on f r a c t i o n of c e l l s with conjugation tubes The b i o a s s a y based on f r a c t i o n of c e l l s producing conjuga-t i o n tubes has been used e x t e n s i v e l y , and has provided most of the data f o r the l a s t two chapters of t h i s t h e s i s . For each assay performed, the slope of the r e g r e s s i o n l i n e s and the d i l u t i o n of the standard which causes 50$ of the c e l l s to produce co n j u g a t i o n tubes (ED50) have been charted ( F i g . 13)- Both the slope and the standard ED50 show day-to-day v a r i a t i o n s which are s i g n i f i c a n t l y g r e a t e r than t h e i r e r r o r s of e s t i m a t i o n . The c o r r e l a t i o n between the slope and the standard ED50 i s e s s e n t i a l l y zero. The data f r e q u e n t l y show gr e a t e r d e v i a t i o n s from the r e g r e s s i o n l i n e s than can be explained by random sampling. Two t y p e s . o f d e v i a t i o n can be r e c o g n i z e d . In some cases, the d i s p e r s i o n of the data p o i n t s about the l i n e i s i n c r e a s e d , but the b a s i c r e l a t i o n remains l i n e a r . The r a t i o of the a c t u a l d e v i a t i o n to the d e v i a t i o n expected from random sampling i s c a l c u l a t e d as a h e t e r o g e n e i t y f a c t o r . The frequency d i s t r i -b u t i o n of the h e t e r o g e n e i t y f a c t o r i s summarized i n Table V. 4. O b . tfl cj ul d iii <J* a O <H CU "iii 3 S T -• • • • o -H ^ aj S3aru NDiivgnrNDD HUM NDioftu dd u s e r 1 ! '3 o o °saaru roiSvgrmD H?IM STTO 3D NDU3*S F i g . 12a. R e l a t i o n of f r a c t i o n of c e l l s with conjugation tubes to dose of erogen. b. L i n e a r r e g r e s s i o n of transformed response on the logar i t h m s of erogen dose. F i g . 13. V a r i a t i o n i n the slope and i n t e r c e p t of the b i o a s s a y standard curve. 68 In other cases, the response at high doses i s lower than expected from e x t r a p o l a t i o n of the response at lower doses.. T h i s e f f e c t can he seen i n F i g . 12. Table V. Frequency d i s t r i b u t i o n of the h e t e r o g e n e i t y f a c t o r f o r the conjugation hormone b i o a s s a y . Value of h e t e r o g e n e i t y f a c t o r Frequency 1.00 1.50 2- 50 3- 50 4.50 5 .50 6.50 7.50 8.50 9-50 1A9 2. kg 3-kg k.kg 5 .kg 6. kg 7 -kg Q.kg 9-kg 10. kg 29 9 7 k 3 k l 2 0 .1 6 9 D i s cus s ion The theory and p r a c t i c e of s t a t i s t i c a l methods i n b i o l o g -i c a l assay have been described'by Finney (1964}. Because of b i o l o g i c a l v a r i a b i l i t y , a standard curve determined on one occa s i o n may not apply at some other time. F i g . 13 confirms t h a t both the slope of the dose-response r e g r e s s i o n l i n e s and the c o n c e n t r a t i o n of standard r e q u i r e d to produce a p a r t i c u l a r response vary g r e a t l y from, one assay to another. For t h i s reason i t i s necessary t o i n c l u d e a standard d i l u t i o n s e r i e s i n each assay. Measuring the response to the t e s t p r e p a r a t i o n at s e v e r a l d i l u t i o n s i n c r e a s e s the p r e c i s i o n with which the slope of the dose-response curve can be c a l c u l a t e d , and allows t e s t s f o r curvature and n o n - p a r a l l e l i s m of the t e s t l i n e . U n i t s of b i o l o g i c a l a c t i v i t y should be de f i n e d as a c e r t a i n weight of a s t a b l e standard p r e p a r a t i o n , r a t h e r than i n terms of the amount needed to produce a s p e c i f i e d response. Bioassay procedures which have been used i n previous s t u d i e s of f u n g a l sexual hormones do not completely meet these requirements. In the s i r e n i n b i oassay, s e v e r a l doses of stand-ard p r e p a r a t i o n are t e s t e d along with one dose of the unknown. The potency of the unknown i s determined by g r a p h i c a l i n t e r -p o l a t i o n ( C a r l i l e and Machlis 1965 )• In the bi o a s s a y f o r hormone A i n Achlya developed by Raper (1942a), s e v e r a l dose l e v e l s of the unknown are t e s t e d , but only one c o n c e n t r a t i o n of standard i s used to " c a l i b r a t e " the mycelia. One u n i t of hormone i s de f i n e d as the amount per c.c. r e q u i r e d t o produce an average of 10 a n t h e r i d i a l branches i n the t e r m i n a l 3 mm of the t e s t hyphae. Barksdale (1963a) has modified t h i s assay. She t e s t s s e v e r a l d i l u t i o n s of the unknown, but notes only the lowest d i l u t i o n which causes at l e a s t 25$ of the hyphae to produce a n t h e r i d i a l branches. Three d i l u t i o n s of a standard s o l u t i o n are assayed simultaneously, a p p a r e n t l y as a check t h a t the s e n s i t i v i t y of the hyphae has not changed d r a s t i c a l l y . T h i s assay method i s only capable of measuring the potency of the unknown p r e p a r a t i o n to w i t h i n a f a c t o r of two. 70 The d e s c r i p t i o n of the b i o a s s a y f o r "gamones" i n Mucor  mucedo i s sketchy, but i t appears that only one dose of an unknown p r e p a r a t i o n was t e s t e d , and the response was compared to a standard curve determined p r e v i o u s l y . Bu'Lock, Drake and Winstanley (1972) used a more s o p h i s t i c a t e d procedure t o d e t e r -mine the zygogenic a c t i v i t y of s e v e r a l compounds r e l a t e d to the t r i s p o r i c a c i d s . S e v e r a l c o n c e n t r a t i o n s of each compound were assayed, and the slope of the dose-response l i n e s was taken as a measure of r e l a t i v e b i o l o g i c a l a c t i v i t y . T h i s i s a good design, and would allow the c a l c u l a t i o n of confidence l i m i t s . D e spite the d e f i c i e n c i e s noted above, these assays seem to have served t h e i r purpose s a t i s f a c t o r i l y and have enabled the i s o l a t i o n of the a c t i v e m a t e r i a l s . P o s s i b l y the v a r i a b i l i t y of the responses was low enough to make unnecessary the safeguards b u i l t i n t o more c a r e f u l assay designs. In Tr e m e l l a , my experience has shown t h a t the complete procedure i s g e n e r a l l y r e q u i r e d f o r r e l i a b l e r e s u l t s . However, when an approximate estimate of the r e l a t i v e erogen c o n c e n t r a t i o n i n many samples, such as chromato-graphic f r a c t i o n s , i s d e s i r e d , a reduced v e r s i o n of the b i o a s s a y i s adequate. (See Chapter S i x , s e c t i o n s I I , IV, and V.) The average l e n g t h of con j u g a t i o n tubes a f t e r 22 hours i n c u -b a t i o n shows a strong dependence on the dose of con j u g a t i o n hor-mone over the range t e s t e d ( F i g . 8). However, the lengths of i n d i v i d u a l tubes at each dose are h i g h l y v a r i a b l e ( c o e f f i c i e n t of v a r i a t i o n O.k to 0.5), and as might be expected, the v a r i a b i l i t y at short average tube lengths i s l e s s than at long average tube l e n g t h s . The va r i a n c e of tube lengths at each dose i s a p p r o x i -mately p r o p o r t i o n a l to the square of the mean tube l e n g t h ( F i g . 9). Such a dependence of varia n c e on mean v i o l a t e s the b a s i c assumptions of common s t a t i s t i c a l techniques such as the a n a l y s i s of v a r i a n c e , and the tube l e n g t h measurements were transformed to t h e i r n a t u r a l logarithmns to make the varia n c e at each dose approximately constant. Comparison of F i g . 10 to F i g . 8 shows th a t t h i s t r a n s f o r m a t i o n accentuates the curvature of the dose-response curve, but does not a l t e r i t s b a s i c shape. For b i o -l o g i c a l assays i t i s convenient to have a f u n c t i o n of dose to which the response i s l i n e a r l y r e l a t e d . F i g . 11 shows that the loga r i t h m s of the dose i s such a f u n c t i o n . 71 T h i s p r e l i m i n a r y i n f o r m a t i o n about the r e l a t i o n of con-j u g a t i o n tube l e n g t h to hormone dose was used to design an assay. Since the r e g r e s s i o n of transformed response on the logarithmn of dose i s to be used i n the computations, the doses should be e q u a l l y spaced on the l o g a r i t h m i c s c a l e . A s e r i a l d i l u t i o n s e r i e s accomplishes t h i s . The dose-response curve was found to be l i n e a r over a dose range of at l e a s t h-0 tim.es. Four s e r i a l d i l u t i o n s at a d i l u t i o n r a t i o of 1 to 3, covering a dose range of 27 times, were used to ensure t h a t the responses would be on the l i n e a r p o r t i o n of the curve even i f s m a l l e r r o r s were made i n the choice of doses. The c o n j u g a t i o n tubes at high erogen c o n c e n t r a t i o n could be longer because of e a r l i e r i n i t i a t i o n , f a s t e r growth, or growth s u s t a i n e d f o r a longer time than the c o n j u g a t i o n tubes at low erogen c o n c e n t r a t i o n s , or because of a combination of these f a c t o r s . T h i s p o i n t i s d i s c u s s e d f u r t h e r i n Chapter Four . The tube l e n g t h b i o a s s a y was developed f i r s t because measurements of tube l e n g t h give more i n f o r m a t i o n per c e l l than simply determining the presence or absence of a conjugation tube. In p r a c t i c e , the v a r i a b i l i t y of the tube lengths and the labour r e q u i r e d to measure l a r g e numbers of tube lengths have overcome t h i s t h e o r e t i c a l advantage. Data about the f r a c t i o n of c e l l s producing conjugation tubes i s of the type c a l l e d "quantal", because each c e l l i s placed i n t o one of two c a t e g o r i e s - - w i t h conjugation tube or without c o n j u g a t i o n tube. Quantal data i s important i n the assay of f u n g i c i d e s and i n s e c t i c i d e s , where the two c a t e g o r i e s are dead and a l i v e . A technique known as p r o b i t a n a l y s i s has been developed by Finney (1971)' f o r quantal data. I have a p p l i e d h i s methods to the T r e m e l l a conjugation tube counts, but the p r o b i t t r a n s f o r m a t i o n has been r e p l a c e d by the l o g i s t i c t r a n s f o r m a t i o n , because an e x p l i c i t formula f o r the l a t t e r i s a v a i l a b l e . The l o g i t of the f r a c t i o n responding p l o t t e d against the logarithmn of the erogen c o n c e n t r a t i o n c l o s e l y f o l l o w s a 72 s t r a i g h t l i n e except at extreme high and low doses. At low erogen doses, the conjugation tubes formed are short, and i t i s sometimes d i f f i c u l t t o decide whether or not a p a r t i c u l a r c e l l has a c o n j u g a t i o n tube. For t h i s reason, the e r r o r i n e s t i m a t i n g the f r a c t i o n of c e l l s with tubes i s l a r g e r at low doses than at medium or high doses. The r e f o r e the d e v i a t i o n from the s t r a i g h t l i n e at low dose i s not a cause f o r concern. At the highest dose t e s t e d the response decreased probably because of i n h i b i t i o n by other components of the hormone p r e p a r a t i o n . Dose-response curves s i m i l a r to the one presented here f o r the T r e m e l l a erogens have f r e q u e n t l y been found i n t o x i c o l o g y (Finney 1971) and pharmacology ( A r i e n s , Simonis and van Rossum I964). Two types of models have been proposed to e x p l a i n the shape of these curves. In the f i r s t model each c e l l has a response t h r e s h o l d , and at a given dose, only those c e l l s whose t h r e s h o l d i s lower than the dose w i l l r e a c t . I f the frequency d i s t r i b u t i o n of the t h r e s h o l d i s l o g normal, i t can be shown that the p r o b i t (or l o g i t ) of the f r a c t i o n responding w i l l be a l i n e a r f u n c t i o n of the logarithmn of the dose (Finney 197l)-An a l t e r n a t i v e approach i s the r e c e p t o r theory. The admin-i s t e r e d b i o l o g i c a l l y a c t i v e molecules are p o s t u l a t e d to a t t a c h to r e c e p t o r s i t e s i n s i d e the c e l l s i n order to produce a response. As the c o n c e n t r a t i o n of the a c t i v e m a t e r i a l i n c r e a s e s , the f r a c t i o n of r e c e p t o r s i t e s which are occupied a l s o i n c r e a s e s a c c o r d i n g to the Langmuir a d s o r p t i o n isotherm. I f the response i s assumed to be p r o p o r t i o n a l to the number of r e c e p t o r s i t e s occupied, t h i s model a l s o p r e d i c t s a l i n e a r r e g r e s s i o n of the l o g i t of the f r a c t i o n responding on the logarithmn of dose ( A r i e n s , Simonis, and van Rossum I964). E s s e n t i a l l y , the t h r e s h o l d d i s t r i b u t i o n model assumes t h a t the e f f e c t i v e dose i n each c e l l i s the same, and the s e n s i t i v i t y of the c e l l s to t hat dose i s v a r i a b l e , whereas the r e c e p t o r model assumes t h a t the e f f e c t i v e dose (number of occupied r e c e p t o r s ) per c e l l i s v a r i a b l e , and the s e n s i t i v i t y of the c e l l s to a given e f f e c t i v e dose i s constant. These two concepts 73 are not mutually e x c l u s i v e - - v a r i a t i o n i n the number of occupied r e c e p t o r s per c e l l could he teamed with an independent v a r i a t i o n i n the number of occupied r e c e p t o r s per c e l l , .necessary to produce a response. Those assays i n which d e v i a t i o n s from the r e g r e s s i o n l i n e s are g r e a t e r than can be exp l a i n e d by random sampling r e q u i r e s p e c i a l a t t e n t i o n . When the b a s i c dose-response r e l a t i o n remains l i n e a r , the h e t e r o g e n e i t y f a c t o r can be used to take i n t o account the i n c r e a s e d e s t i m a t i o n e r r o r . S i g n i f i c a n t h e t e r o g e n e i t y i n d i c a t e s non-uniformity of c o n d i t i o n s among the assay tubes. In the T r e m e l l a assay, inadequate mixing of the assay s o l u t i o n s may be a major cause of h e t e r o g e n e i t y . When assays were per-formed i n shaken f l a s k s to provide continuous mixing, the c e l l s became entangled by t h e i r c o n j u g a t i o n tubes i n t o l a r g e clumps, and accurate counting was im p o s s i b l e . Under the s t a t i c i n c u -b a t i o n c o n d i t i o n s now employed, most of the c e l l s s e t t l e to the bottom, of the tubes. Conjugation tube p r o d u c t i o n i s not n o t i c e -a b l y l e s s i n s t a t i c than i n shaken media, but the co n c e n t r a t i o n s of c e l l s at the bottom, of the assay tube could a l t e r the l o c a l environment i n a non-reproducible way and thus e f f e c t the response to the erogens. The second case, i n which the responses at hig h doses are anomalously low, l e a d i n g t o curvature of the dose-response r e g r e s s i o n , occurs most f r e q u e n t l y i n two situations--when r e l a t i v e l y impure p r e p a r a t i o n s such as crude c u l t u r e supernatant are assayed, or when the doses of the unknown are too high. These low responses are probably caused by i n h i b i t o r y substances present i n the crude p r e p a r a t i o n s , or because the response i s l i m i t e d by some f a c t o r other than hormone c o n c e n t r a t i o n . The procedure adopted here has been to examine the data, and to omit from the a n a l y s i s the r e s u l t s from the one or two highest doses of a p r e p a r a t i o n i f the responses to these doses are not s u b s t a n t i a l l y higher than the response at the next lower dose. R e j e c t i o n of data a f t e r examination c a r r i e s the danger of i n t r o d u c i n g b i a s i n t o the r e s u l t s , but the a l t e r n a t i v e s are 74 even l e s s a p p e a l i n g . To i n c l u d e a l l the doses i n the a n a l y s i s would produce m i s l e a d i n g r e s u l t s , "because the data would not conform to the assumed l i n e a r r e g r e s s i o n . To r e j e c t the e n t i r e assay would waste the i n f o r m a t i o n a v a i l a b l e from the lower doses. The design of assays based on quantal responses has been d i s c u s s e d by Finney (1964). He recommends a minimum of three doses of each p r e p a r a t i o n , t o allow t e s t s of l i n e a r i t y and p a r a l l e l i s m of the dose-response r e g r e s s i o n s . For the T r e m e l l a erogen b i o a s s a y , four doses were used. The f o u r t h dose, by p r o v i d i n g an e x t r a independent datum,, reduces the s e n s i t i v i t y of the assay to h e t e r o g e n e i t y , and a l s o reduces the s e v e r i t y of r e j e c t i n g the r e s u l t s from one or two of the higher doses. For an assay with four doses of each p r e p a r a t i o n , Finney recom-mends choosing the doses so t h a t the expected f r a c t i o n s respond-ing are about 0.15, O.35, O.65, and O.85 to give h i g h e s t pre-c i s i o n . Because, as mentioned above, the e r r o r i n counting the number of c e l l s with c o n j u g a t i o n tubes i s high at low response r a t e s , somewhat higher doses were used i n the T r e m e l l a bioassay, and response r a t e s below 0.25 are not normally encountered. A r a t i o of l/2 between s u c c e s s i v e doses gives the proper spacing. Crude erogen p r e p a r a t i o n s from T_. mesenterica 2259-7 c o n t a i n at l e a s t three a c t i v e components. Dose-response r e l a -t i o n s f o r mixtures of b i o l o g i c a l l y a c t i v e m a t e r i a l s have been d e s c r i b e d by Finney (1971)- The dose-response curves observed f o r the erogen p r e p a r a t i o n s from 2259-7 have always been l i n e a r or convex, suggesting that the components are showing "simple s i m i l a r a c t i o n " . In other words, a l l three components are s t i m u l a t i n g the c e l l s through the same mechanism. More complex types of i n t e r a c t i o n s between the components should l e a d t o dose-response curves which are concave (Finney 1971)• CHAPTER FOUR THE RESPONSE TO THE EROGENS M a t e r i a l s and Me t h o d s 75 I . Time c o u r s e o f c o n j u g a t i o n t u b e i n i t i a t i o n and g r o w t h Two 5-0 m l p o r t i o n s o f 0-5$ s o y t o n e , pH 5-0, i n 25 ml E r l e n m e y e r f l a s k s were s u p p l e m e n t e d w i t h 1.0 u n i t / m l and 0.2 u n i t s / m l , r e s p e c t i v e l y , o f t h e s t a n d a r d e r o g e n p r e p a r a t i o n . A f t e r a u t o c l a v i n g and c o o l i n g , t h e f l a s k s were i n o c u l a t e d w i t h f i v e d r o p s e a c h o f a two day o l d c u l t u r e o f 2259-6 i n GS. A sa m p l e was t a k e n a s e p t i c a l l y f r o m e a c h f l a s k , and t h e f l a s k s were p l a c e d on a s h a k e r a t 20°C. S a m p l e s were t a k e n f r o m e a c h f l a s k a f t e r 2, 4, 5, 6, 1, 8, 10, 12, 14, and 24 h o u r s . One d r o p f r o m e a c h s a m p l e was mounted i n a h a e m a c y t o m e t e r , and t h e number o f c e l l s w i t h and w i t h o u t c o n j u g a t i o n t u b e s was c o u n t e d . A n o t h e r d r o p o f e a c h s a m p l e was d r i e d and s t a i n e d on a m i c r o -s c o p e s l i d e . The l e n g t h s o f 35 r a n d o m l y s e l e c t e d c o n j u g a t i o n t u b e s were m e a s u r e d i n e a c h o f t h e s t a i n e d s l i d e s f r o m t h e 8, 10, 12, l4, and 24 h o u r s a m p l e s . I I . D i s t r i b u t i o n o f number o f c o n j u g a t i o n t u b e s p e r c e l l The s u p e r n a t a n t f r o m one l i t r e o f a s t a t i o n a r y p h a s e c u l t u r e o f 2259-7 i n Gluc-AmS medium was p a s s e d t h r o u g h a b e d o f 10 grams o f P o r a p a k Q a t a f l o w r a t e o f 300 m l / h o u r . The P o r a p a k was washed w i t h 100 ml o f d i s t i l l e d w a t e r , and t h e e r o g e n s were e l u t e d w i t h 100 ml o f 50$ aqueous a c e t o n e . The e l u a t e was c o n c e n t r a t e d i n v a c u o u n t i l t h e a c e t o n e had e v a p o r a t e d , and t h e n was f r e e z e - d r i e d . One m.g o f t h e f r e e z e - d r i e d powder was d i s s o l v e d i n 1.0 ml o f d i s t i l l e d w a t e r . T h i s s o l u t i o n was d i l u t e d 1 t o 50 w i t h a 0.5$ s o l u t i o n o f s o y t o n e , pH 5-5> and f r o m t h i s p r i m a r y d i l t u i o n 1/2, l / 4 , l / 8 , l / l 6 , l/32, l / 6 4 , and l/l28 s e c o n d a r y d i l u t i o n s i n 0-5$ s o y t o n e were p r e p a r e d . T h e s e d i l u t i o n s were a u t o c l a v e d , i n o c u l a t e d and i n c u b a t e d as f o r a b i o a s s a y . A f t e r s e v e n t e e n h o u r s , t h e f r a c t i o n o f c e l l s p r o d u c i n g c o n j u g a t i o n t u b e s a t e a c h d i l u t i o n was m e a s u r e d , and s t a i n e d s l i d e s were p r e p a r e d f r o m t h e l / 2 , l / 8 , and l/32 s e c o n d a r y d i l u t i o n s . C o m p a r i s o n w i t h a s t a n d a r d d i l u t i o n 76 s e r i e s assayed s i m u l t a n e o u s l y i n d i c a t e d an erogen c o n c e n t r a t i o n of 20.5 u n i t s / m l i n the primary d i l u t i o n . On the s t a i n e d s l i d e s , approximately 200 c e l l s were examined at each dose, and the numbers with 0, 1, 2, 3> • • • > 8 conjugation tube growing p o i n t s o r i g i n a t i n g from one yeast c e l l were counted. I I I . E f f e c t of temperature F i v e t e s t tubes c o n t a i n i n g 1.0 ml of 0.5$ soytone, pH 5-0, with 1.0 u n i t of standard erogen per ml and f i v e s i m i l a r tubes with 0.25 u n i t s / m l were prepared. A f t e r a u t o c l a v i n g each tube was i n o c u l a t e d with one drop from a one day o l d c u l t u r e of 2259-6 i n GS. One tube with 1 u n i t / m l and one tube with 0.25 u n i t s / m l were incubated at each of 10°C, 15°C, 20°C, 25°C and 28°C. A f t e r 18 hours, the f r a c t i o n of the c e l l s with conjugation tubes at each dose-temperature combination was determined. Sta i n e d s l i d e s were a l s o prepared, and the lengths of 25 randomly s e l e c t e d c o n j u g a t i o n tubes from each treatment were measured. IV. E f f e c t of pH A l i q u o t s of 0.5$ soytone were adjusted to pH 3-0, 4.0, 5.0, 6.0, 7-0, and 8.0 with 0.1 N h y d r o c h l o r i c a c i d or potassium hydroxide. One ml p o r t i o n s of the medium, at each pH i n t e s t tubes were supplemented with 1.0 or 0.25 u n i t s of the standard erogen p r e p a r a t i o n , and au t o c l a v e d . The tubes were i n o c u l a t e d with 1 drop from a one day o l d c u l t u r e of 2259-6> i n GS, and incubated at 20°C. A f t e r 17 hours, the f r a c t i o n of c e l l s with co n j u g a t i o n tubes i n each t e s t tube was determined. V. E f f e c t of c e l l c o n c e n t r a t i o n Si x ml of 0.5$ soytone, pH 5-5, c o n t a i n i n g 1.0 u n i t of the standard erogen p r e p a r a t i o n per ml were d i s t r i b u t e d i n t o s i x t e s t tubes. A f t e r a u t o c l a v i n g , one tube was i n o c u l a t e d with 0.01 ml of a three day o l d c u l t u r e of 2259-6 i n GS. The other tubes were i n o c u l a t e d with 0.02, 0.04, 0.06, 0.08 and 0.10 ml of the c u l t u r e . S t e r i l e GS was added t o each tube to b r i n g the t o t a l volume to 1.1 ml. The tubes were incubated at 77 20°C f o r 18 hours. The t o t a l number of c e l l s , and the number of c e l l s with conjugation tubes i n a known volume from each t e s t tube were counted i n a haemacytometer. S t a i n e d s l i d e s were prepared, and the lengths of 25 conjugation tubes i n each sample were measured. VI. E f f e c t of n i t r o g e n source A b a s a l medium s i m i l a r to the BM of Chapter One, but con-t a i n i n g 10 gm of D-glucose, and 0.13 ml of microelement stock s o l u t i o n per l i t r e was prepared. A l i q u o t s of t h i s b a s a l medium were used d i r e c t l y or supplemented with one gram of ammonium, sulphate per l i t r e , or f i v e grams of c a s e i n h y d r o l y s a t e or soytone per l i t r e . The pH of each medium, was adjusted to 5-5 with 0.1 N h y d r o c h l o r i c a c i d or potassium hydroxide. A sample of c o n j u g a t i o n hormones e l u t e d from Porapak was added to a l i q u o t s of each medium at c o n c e n t r a t i o n s of 1.0 u n i t / m l and 0.25 u n i t s / m l . D u p l i c a t e 2 ml p o r t i o n s of each medium at both erogen concen-t r a t i o n s were p i p e t t e d i n t o t e s t tubes and autoclaved. Inoculum, was prepared by c e n t r i f u g i n g , i n a s t e r i l e c e n t r i f u g e tube, the c e l l s from a two day o l d c u l t u r e of 2259-6 i n GS, washing the c e l l s with a volume of s t e r i l e d i s t i l l e d water equal to the o r i g i n a l c u l t u r e volume, and resuspending the c e l l s i n another volume of s t e r i l e d i s t i l l e d water. One drop of the washed c e l l suspension was added to each t e s t tube, and the tubes were incubated at 20°C f o r 18 hours. Samples from each t e s t tube were mounted i n the haemacytometer, and the c e l l s with conju-g a t i o n tubes and without conjugation tubes i n 400 sma l l squares were counted. R e s u l t s 78 I. Time course of con j u g a t i o n tube i n i t i a t i o n and growth The f r a c t i o n of c e l l s with r e c o g n i z a b l e conjugation tubes i s p l o t t e d a g a i n s t time i n F i g . 14 f o r both erogen c o n c e n t r a t i o n s . On the same graph, the average l e n g t h of the con j u g a t i o n tubes i n the 8, 10, 12, 14 and 24 hour samples are shown. Histograms of the frequency d i s t r i b u t i o n s of tube l e n g t h at each time are presented i n F i g . 15 f o r the low erogen c o n c e n t r a t i o n , and i n F i g 16 f o r the high erogen c o n c e n t r a t i o n . Tube lengths are given i n eyepiece u n i t s , which equal 1.^6 u. I I . D i s t r i b u t i o n of number of con j u g a t i o n tubes per c e l l The frequency d i s t r i b u t i o n s of the number of conjugation tubes per c e l l are shown i n F i g . 17- The average number of tubes per c e l l i s p l o t t e d a g a i n s t erogen dose i n F i g . 18. The r e g r e s s i o n was s i g n i f i c a n t at the 10$ l e v e l . I I I . E f f e c t of temperature The f r a c t i o n of c e l l s with conjugation tubes, and the average co n j u g a t i o n tube l e n g t h at each dose are p l o t t e d a gainst i n c u b a t i o n temperature i n F i g . 19 and F i g . 20 r e s p e c t i v e l y . IV. E f f e c t of pH The f r a c t i o n of c e l l s producing conjugation tubes at each dose of erogen i s p l o t t e d a gainst the pH i n F i g . 21. The lengths of the con j u g a t i o n tubes produced at each pH v a r i e d i n a s i m i l a r manner to the f r a c t i o n of c e l l s responding. V. E f f e c t of c e l l c o n c e n t r a t i o n Table VI l i s t s the counts of t o t a l c e l l s , and c e l l s with c o n j u g a t i o n tubes, along with the volumes i n which the c e l l s were counted. Confidence l i m i t s f o r the c o n c e n t r a t i o n of c e l l s i n each t e s t tube were c a l c u l a t e d by assuming a Poisson d i s -t r i b u t i o n f o r the t o t a l c e l l counts. The f r a c t i o n of c e l l s with c o n j u g a t i o n tubes, and the average conjugation tube l e n g t h are p l o t t e d a g a i n s t the t o t a l c e l l c o n c e n t r a t i o n i n F i g . 22. 7 9 (nd3) H13N31 3ETLL NDI1VE3TFNCD NVBN • • • • • • SBETll NDIIVDTTMT) HUM STT3D JO NDIlDVyj F i g . Ik . Time course of c o n j u g a t i o n tu"be i n i t i a t i o n and growth i n response to erogen. Upper curves 1 . 0 u n i t / m l , lower curves 0 . 2 u n i t / m l . Bars r e p r e s e n t 9 5 $ confidence i n t e r v a l s . 0-4 0-3. 0*B 0-1 0-3. 0-0. 0-4.. o - a . o - i O ' Q 80 8 hours o . a . to -I | |_JQ»Q 10 hours 4=1- + !• 3« 5* 7- 9* !• 3* 5* 7« 9« >- 0»5i. 0« 12 hours H 1 — i - Q - Q 14 hours bo. 24 hours i i i i n i i i i 1. 3- 5- 7- 9- 11. 13. 15. 17- 19. CONJUGATION TUBE LENGTH (EPU) F i g . 15. Frequency d i s t r i b u t i o n s of conjugation tube lengths at various times a f t e r i n o c u l a t i o n of 2259-6 c e l l s i n t o media c o n t a i n i n g 0.2 u n i t s of erogens per ml. 81 M 0-! o.« o.ii 0-0. 0*4. 0-1 0*0. 8 hours 0-4. 0.3L. _ Q . Q 10 hours i« 3. 5* 7* 9* 1. 3« 5* 7* 9* C S i . o-r 12 hours 0.4. 0-3. 0-1 - Q . Q 14 hours CH n o»: n. 24 hours 4=f Ch I- 3* 5- 7« 9* 11« 13- 15. 17- 19. CONJUGATION TUBE LENGTH (EPU) F i g . 16. Frequency d i s t r i b u t i o n s of conjugation tube lengths at vari o u s times a f t e r i n o c u l a t i o n of 225-9-6 c e l l s i n t o media c o n t a i n i n g 1.0 u n i t of erogens per ml. 82 LU > 2.6 u n i t s / m l 5.1 u n i t s / m l NUMBER OF CONJUGATION TUBES^PER CELL F i g . 17. Frequency tubes per c e l l , at d i s t r i b u t i o n three erogen of the number of c o n c e n t r a t i o n s . conjugation 83 0* S> 4. 6» B« 10. 12. CONCENTRATION OF EROGENS (UNITS/ML) F i g . 18. Reg r e s s i o n of average number of conjugation tubes per c e l l on the erogen c o n c e n t r a t i o n . l » Q i . Qk o . a . 0*4. 0-0. + + + + 10. 15' 3D. i TEMPERATURE (DEG- C) 30-F i g . 19- The f r a c t i o n of c e l l s responding to the erogens at two con c e n t r a t i o n s as a f u n c t i o n of temperature. Upper c u r v e — 1.0 u n i t / m l ; lower curve 0.25 u n i t s / m l . Bars represent confidence i n t e r v a l s . 85 B».. 6-.. H A : . + + + + 10. 15* SO. 30. TEMPERATURE (DEG- C) F i g . 20. The average l e n g t h of conjugation tubes produced i n response to the co n c e n t r a t i o n s of erogens as a f u n c t i o n of temperature. Upper c u r v e — 1 . 0 u n i t / m l ; lower curve—0.25 u n i t / m l . Bars represent 95$ confidence i n t e r v a l s . 86 1-0+ F i g . 21. The f r a c t i o n of c e l l s responding to the erogens at two c o n c e n t r a t i o n s as a f u n c t i o n of pH. Upper curve—1.0 u n i t / m l ; lower curve--0.25 u n i t s / m l . Bars represent 95$ confidence i n t e r v a l s . F i g . 22. Dependence of f r a c t i o n of c e l l s with conjugation tubes, and average l e n g t h of conjugation tubes on c o n c e n t r a t i o n of c e l l s . 88 Table VI. C e l l counts from the experiment on the e f f e c t of c e l l c o n c e n t r a t i o n . Volume of inoculum i n ml  0 .01 0 .02 0.0k 0.06 0.08 0 .10 Number of c e l l s with c o n j u g a t i o n tubes  162 275 201 299 208 176 T o t a l number of c e l l s 186 324 331 622 458 373 Volume i n which c e l l s were counted n a n o l i t r e s  100 100 40 40 40 20 VI. E f f e c t of n i t r o g e n source The numbers of c e l l s with c o n j u g a t i o n tubes, and the t o t a l numbers of c e l l s counted i n each r e p l i c a t e of the erogen dose-n i t r o g e n source combinations are given i n Table V I I . Table V I I . The number of c e l l s with conjugation tubes/the t o t a l number of c e l l s counted at two doses of erogen with v a r i o u s n i t r o g e n sources. N i t r o g e n source None Ammonium sulphate Casein h y d r o l y s a t e Soytone 0.25 u n i t s of erogen /ml O/28O, 0/500 22/851, 34/910 136/445, 133/670 350/851, 252/610 1.0 u n i t of erogen /ml 0/485, 0/400 43/300, 68/680 260/274, 172/330 359/443, 312/400 The f r a c t i o n s were transformed t o the a r c s i n e s of t h e i r square r o o t s ( S t e e l and T o r r i e i960) and the data was analyzed as a 4 X 2 f a c t o r i a l experiment with 2 r e p l i c a t i o n s . The a n a l y s i s of varia n c e i s presented i n Table V I I I . The c o n t r a s t between the f r a c t i o n of c e l l s producing conju-g a t i o n tubes with soytone 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 sources was s i g n i f i c a n t at the 2.5$ l e v e l . 8 9 Table V I I I . A n a l y s i s of va r i a n c e f o r e f f e c t of n i t r o g e n source on co n j u g a t i o n tube p r o d u c t i o n . Source of v a r i a t i o n Main e f f e c t of n i t r o g e n source I n t e r a c t ion E r r o r Sum of Degrees of Mean squares freedom. s guar e 1 . 9 4 2 Main e f f e c t of erogen c o n c e n t r a t i o n O . 2 7 8 I 0 . 1 0 1 3 0 .06112 1 3 8 0 . 2 7 8 1 0 . 0 3 3 7 8 0 .00764 F P r o b a b i l i t y of a l a r g e r F 0 .6474 84.7 0.000003 3 6 . 4 0 . 0 0 0 3 4 . 4 2 0 . 0 4 1 90 D i s c u s s i o n The e f f e c t s of some environmental v a r i a b l e s on p r o d u c t i o n of c o n j u g a t i o n tubes i n response to the erogens were examined to determine optimum c o n d i t i o n s f o r the b i o a s s a y . F i r s t , the k i n e t i c s of conjugation tube i n i t i a t i o n and growth at 20°C were s t u d i e d . Recognizable conjugation tubes were f i r s t observed k hours a f t e r exposure of the 2259-6 c e l l s to the erogens. E x t r a p o l a t i o n of the curves i n F i g . 13 suggests a l a g of s l i g h t l y more than 3 hours before any conjugation tubes were i n i t i a t e d . In the medium c o n t a i n i n g 1.0 u n i t of erogens per ml, the f r a c t i o n of c e l l s with conjugation tubes i n c r e a s e d r a p i d l y u n t i l 7 hours a f t e r i n o c u l a t i o n , and then more slowly u n t i l Ik hours. In the medium c o n t a i n i n g 0.2 u n i t s of erogens per ml, the p r o p o r t i o n of c e l l s with c o n j u g a t i o n tubes rose more sl o w l y . A f t e r 2k hours, at both erogen con-c e n t r a t i o n s , the f r a c t i o n of c e l l s with conjugation tubes i s lower than at Ik hours. T h i s decrease i s probably caused by the formation of new yeast c e l l s , e i t h e r by budding from the o r i g i n a l yeast c e l l s , or by r e v e r s i o n of conjugation tube growing p o i n t s to budding. F l e g e l (1968) has shown that conju-g a t i o n hormones must be c o n t i n u o u s l y present to maintain conju-g a t i o n tube growth; i f c e l l s with growing conjugation tubes are switched t o hormone-free medium, budding occurs at the growing poin t of the conjugation tubes. I have f r e q u e n t l y observed buds at the apices of conjugation tubes, e s p e c i a l l y at i n c u b a t i o n times longer than 18 hours, and at low i n i t i a l erogen c o n c e n t r a t i o n s . Bandoni (1965) has suggested that the c e l l s use up the conjugation hormones as they respond. Rever-s i o n of the conjugation tubes to budding at long i n c u b a t i o n times supports t h i s view. A s i m i l a r phenomenon has been found i n other f u n g i r e a c t i n g to sexual hormones—sperm of Allomyces remove s i r e n i n from t h e i r medium ( C a r l i l e and Machlis I965)- and male mycelia of Achlya take up a n t h e r i d i o l i r r e v e r s i b l y (Barks-dale 1963a). 91 In F l e g e l ' s hormone p u l s i n g experiment (1968), conjugation tubes were found 2 hours a f t e r exposure of the c e l l s to the hormone. He does not s p e c i f y the s t r a i n , or the c o n c e n t r a t i o n of the hormone used. He a l s o c a r r i e d out a time lapse photo-graphic study of conjugation tube p r o d u c t i o n i n mixed c u l t u r e s of 2259-6 and 2259-7- The e a r l i e s t c o njugation tube recorded was i n i t i a t e d $0 minutes a f t e r mixing of the two s t r a i n s , but was not r e c o g n i z a b l e at a tube before 3 hours. T h i s e a r l y tube has a c o n s t r i c t i o n at i t s point of attachment t o the yeast c e l l . Such a b a s a l c o n s t r i c t i o n i s c h a r a c t e r i s t i c of buds, but not of conjugation tubes. Perhaps t h i s s t r u c t u r e was i n i t i a t e d as a bud, and l a t e r transformed i n t o a conjugation tube. By 8 hours, the average conjugation tube l e n g t h at 1 u n i t of erogens per ml i s twice as great as at 0.2 u n i t per ml. At 0.2 u n i t s / m l a l a r g e f r a c t i o n of the tubes are s h o r t e r than 1.5 epu ( r e c e n t l y i n i t i a t e d ) , but at 1 u n i t / m l very few of the conj u g a t i o n tubes are i n t h i s l e n g t h range. T h e r e f o r e , the g r e a t e r average l e n g t h of the tubes at high erogen dose i s at l e a s t p a r t l y caused by e a r l i e r i n i t i a t i o n . As time progresses, the frequency d i s t r i b u t i o n s f o r both hormone c o n c e n t r a t i o n s s h i f t t o longer l e n g t h s , and broaden. By 12 hours, few r e c e n t -l y i n i t i a t e d tubes are found i n the medium with 0.2 u n i t s / m l , and by 14 hours, the d i s t r i b u t i o n of tube lengths i s s i m i l a r t o the 8 hour d i s t r i b u t i o n at the higher hormone c o n c e n t r a t i o n . The average l e n g t h of the conjugation tubes i n c r e a s e s f a s t e r at high than at low hormone c o n c e n t r a t i o n , suggesting that the average growth r a t e of the tubes i s f a s t e r at high hormone c o n c e n t r a t i o n . The average tube l e n g t h i n c r e a s e s from 14 hours to 24 hours, i n c o n t r a s t to the f r a c t i o n of c e l l s with conju-g a t i o n tubes. T h i s does not n e c e s s a r i l y mean t h a t growth of e s t a b l i s h e d conjugation tubes continued while new tubes were no longer being i n i t i a t e d . These data do not t e l l whether the average l e n g t h was s t i l l i n c r e a s i n g at 24 hours, or stopped at some e a r l i e r time. The d i s t r i b u t i o n s of tube lengths at 24 hours are very broad--there are some tubes as short as 2 epu, 92 and as long as 13 epu at 0.2 u n i t s / m l , and as long as 18 epu at 1 u n i t / m l . The short tubes probably stopped growing at an e a r l y time, the extremely long tubes continued growing u n t i l the end of the i n c u b a t i o n p e r i o d , and the medium l e n g t h tubes stopped growing at some intermediate time. The data presented here are not extensive enough to permit a c l e a r s e p a r a t i o n of the e f f e c t s of i n i t i a t i o n time, growth r a t e , and growth stopping time on the average l e n g t h of c o n j u g a t i o n tubes at v a r i o u s hormone doses, but suggest that a l l three f a c t o r s are important. The best i n c u b a t i o n time f o r a b i o a s s a y i s long enough to ensure t h a t a l l c e l l s which are going to r e a c t have done so, and that the c o n j u g a t i o n tubes are long enough to be e a s i l y recog-n i z e d , but not so long as to allow accumulation of new yeast c e l l s . Incubation times between 12 and 18 hours should meet these c r i t e r i a . The response time f o r T r e m e l l a i s longer than f o r Allomyces, where bioa s s a y s are read a f t e r kO minutes (Machlis 1958a), Achlya, where bioa s s a y s are read a f t e r 2 hours (Barksdale 1963a); and Mucor where bioa s s a y s are read a f t e r k hours (Plempel 1963)-As the c o n c e n t r a t i o n of erogens i s i n c r e a s e d , the number of p o i n t s on the s u r f a c e of the yeast c e l l s at which conjugation tubes are produced, and the branching of the tubes, i n c r e a s e . These e f f e c t s cause an i n c r e a s e i n the average number of conju-g a t i o n tube growing p o i n t s per c e l l ( F i g . 17)- The l i n e a r i t y of the r e g r e s s i o n of average number of c o n j u g a t i o n tube growing p o i n t s per c e l l on c o n c e n t r a t i o n of erogens over the range t e s t e d ( F i g . 18) i s s t r i k i n g , and suggests a t h i r d b i o a s s a y method. T h i s p o s s i b i l i t y has not been i n v e s t i g a t e d . Among the temperatures t e s t e d , the l a r g e s t f r a c t i o n of c e l l s produced co n j u g a t i o n tubes at 15°C, with 20°C almost as good. However, the c o n j u g a t i o n tubes formed at 20°C were longer' than those formed at 15°C and at other temperatures. T h e r e f o r e 20°C was s e l e c t e d as the standard temperature f o r the b i o a s s a y . Longer i n c u b a t i o n might have allowed a higher response at 10°C, but t h i s would not be of p r a c t i c a l i n t e r e s t f o r the b i o a s s a y . 93 At an erogen dose of 1 u n i t / m l , the f r a c t i o n of c e l l s responding was almost constant over the pH range from 5 to 7, but decreased at pH k, and was zero at pH's 3 and 8. At a dose of 0.25 u n i t s / m l , the response at pH 6 was higher than at other pH' s t e s t e d . A pH of 5-5 was s e l e c t e d f o r the b i o -assay. F l e g e l (1968) found maximum conjugation tube formation i n mixed c u l t u r e s of 2259-6 and 2259-7 at pH 4.7, no tubes at pH 7-2, but some short branched tubes at pH 8. He used d i f f e r e n t b u f f e r systems at d i f f e r e n t pH 1 s, so th a t the e f f e c t s of pH are confounded with p o s s i b l e s p e c i f i c e f f e c t s of the b u f f e r s a l t s . In the present study, the amino a c i d s and pep-t i d e s of soytone were used as b u f f e r s at a l l pH 1 s. The d i s -c repancies between F l e g e l ' s r e s u l t s and mine may be caused by the d i f f e r e n c e i n b u f f e r systems, or by the d i f f e r e n c e between mixed c u l t u r e s and pure c u l t u r e s with added p a r t i a l l y p u r i f i e d erogens. At high c o n c e n t r a t i o n s of 2259-6 c e l l s , a smaller f r a c t i o n of the c e l l s produce conjugation tubes, and the tubes are s h o r t -er than at lower c e l l c o n c e n t r a t i o n s . There are at l e a s t two p o s s i b l e e x p l a n a t i o n s f o r t h i s e f f e c t . The 2259-6 c e l l s could produce a s e l f - i n h i b i t o r . The c o n c e n t r a t i o n of t h i s i n h i b i t o r would be higher at high c e l l c o n c e n t r a t i o n s , and thus decrease the response. A l t e r n a t i v e l y , the c e l l s could compete f o r the erogen molecules. At high c e l l c o n c e n t r a t i o n s , each c e l l would r e c e i v e a smal l e r share of the erogen dose, and t h e r e f o r e respond l e s s s t r o n g l y . To allow near maximal response the amount of inoculum added t o the assay tubes i s designed to give a d e n s i t y of 250 c e l l s per hundred p i c o l i t r e s . I f no n i t r o g e n source i s i n c l u d e d i n the assay medium, no c o n j u g a t i o n tubes are formed. Ammonium sulphate i s a poor n i t r o g e n source f o r c o n j u g a t i o n tube p r o d u c t i o n by 2259-6, i n c o n t r a s t to i t s ready u t i l i z a t i o n f o r growth and erogen p r o d u c t i o n by 2259-7- Conjugation tube p r o d u c t i o n i s b e t t e r i n c a s e i n h y d r o l y s a t e medium, and s i g n i f i c a n t l y g r e a t e r i n soytone medium. I t appears t h e r e f o r e that amino a c i d s s t i m u l a t e 9k the response t o the erogens. The s u p e r i o r i t y of soytone to cas e i n h y d r o l y s a t e may a r i s e from more fa v o u r a b l e p r o p o r t i o n s of amino a c i d s , or from, some other s t i m u l a t o r y substances. The importance of amino a c i d s suggests that p r o t e i n s y n t h e s i s i s i n v o l v e d i n the response t o the erogens. F l e g e l (1968). found t h a t cycloheximide, which i n h i b i t s p r o t e i n s y n t h e s i s i n some f u n g i , d i d not i n h i b i t c o n j u g a t i o n tube p r o d u c t i o n . I t only s l i g h t l y decreased the growth r a t e of the c e l l s , suggesting t h a t i t was not p e n e t r a t i n g the c e l l s very e f f e c t i v e l y . Time l i m i t a t i o n s have prevented i n v e s t i g a t i o n of many other f a c t o r s which might i n f l u e n c e the response t o the erogens, and a l s o of the i n t e r a c t i o n s between v a r i o u s f a c t o r s . CHAPTER FIVE PRODUCTION OF THE EROGENS M a t e r i a l s and Methods 95 I. Time course of erogen p r o d u c t i o n For a p i l o t study of the k i n e t i c s of erogen p r o d u c t i o n during the growth of 2259-7 c u l t u r e s , two 200 ml p o r t i o n s of Gluc-AmS medium i n 1 l i t r e f l a s k s were i n o c u l a t e d with 2.0 ml from a 4-day c u l t u r e of 2259-7 i n GS. The f l a s k s were i n c u -bated on a shaker at 20°C. Every 12 hours, samples of approx-i m a t e l y 5 ml were taken a s e p t i c a l l y from each f l a s k . The t u r b i d i t y at 640 nm. of the samples was determined i n a Bausch and Lomb S p e c t r o n i c 20 a f t e r d i l u t i o n with d i s t i l l e d water to keep the absorbance below 0-5- The pH of each sample was measured u s i n g a Radiometer pH meter model 28. Samples taken at odd m u l t i p l e s of 12 hours a f t e r i n o c u l a t i o n were s t o r e d overnight i n a f r e e z e r . Each day, the thawed samples from the previous evening, and the samples from t h a t morning were c e n t r i -fuged f o r 5 minutes at f u l l speed i n a c l i n i c a l c e n t r i f u g e ( I n t e r -n a t i o n a l Equipment Co., Model HN) to sediment the c e l l s . Two ml p o r t i o n s of each sample supernatant were supplemented with 0.1 ml of 10$ soytone, and t h e i r pH adjusted to 5-5 with 0.1 W potassium hydroxide. From these samples, l/2, l/4, and l/8 d i l u t i o n s i n 0-5$ soytone, pH 5-5> were prepared, and the b i o -assay was c a r r i e d out as u s u a l . Sampling of the c u l t u r e s was continued u n t i l 168 hours a f t e r i n o c u l a t i o n . The i n f o r m a t i o n from the p i l o t study was used to plan a second experiment. Three 1 l i t r e Erlenmeyer f l a s k s c o n t a i n i n g 200 ml each of Gluc-AmS were i n o c u l a t e d with 3-0 ml each of a 7 day o l d c u l t u r e of 2259-7 i n Gluc-AmS. The f l a s k s were incubated on a shaker at 20°C. F i v e ml samples were taken from each f l a s k immediately a f t e r i n o c u l a t i o n , and at 12 hour i n t e r v a l s u n t i l 120 hours a f t e r i n o c u l a t i o n , and then at 144 and 168 hours. The pH and t u r b i d i t y of the samples were measured, and the samples prepared f o r b i o a s s a y as above. The samples taken at 24, 36 and 48 hours were bioassayed at t h e i r o r i g i n a l concen-t r a t i o n . The samples taken at 58 hours were d i l u t e d 1 to 2; 96 the samples taken at 72 hours were d i l u t e d 1 to 4; the 84 and 96 hour samples were d i l u t e d 1 to 8; and the 1 0 8 , 1 2 0 , 144 and 168 hour samples were d i l u t e d 1 to 16 with 0 . 5 $ soytone, pH 5 -5 , before b i o a s s a y i n g . To determine the r a t i o between t u r b i d i t y and c o n c e n t r a t i o n of c e l l dry weight, 2 2 5 9 _ 7 c e l l s from a s t a t i o n a r y phase c u l t u r e i n Gluc-AmS were washed three times with d i s t i l l e d water, and then resuspended i n d i s t i l l e d water. A sample of t h i s suspension was d i l u t e d 1 to 50 with d i s t i l l e d water, and i t s t u r b i d i t y measured at 6 4 0 nm. F o r t y ml of the suspension were p i p e t t e d i n t o a dry, weighed P e t r i d i s h . The d i s h was d r i e d overnight i n an oven at 105°C, and cooled t o room temperature i n a des-i c c a t o r before weighing. I I . E f f e c t of n i t r o g e n source on erogen p r o d u c t i o n P o r t i o n s of the b a s a l medium, of Chapter Four, s e c t i o n VI were supplemented with 1 gm of ammonium sulphate, or 1 gm of ammonium sulphate plus 2 grams of sodium acetate t r i h y d r a t e or 1.33 g m of v i t a m i n - f r e e , s a l t - f r e e c a s e i n h y d r o l y s a t e , or 2 gm of soytone per l i t r e . Three 50 ml r e p l i c a t e s of each medium i n 2 5 0 ml Erlenmeyer f l a s k s were prepared and autoclaved f o r 15 minutes on a dry goods c y c l e . Each f l a s k was i n o c u l a t e d with 1 . 0 ml of a 4 day old c u l t u r e of 2 2 5 9 - 7 i n GS, and i n c u -bated on a shaker at 2 0°C. A f t e r seven days the c u l t u r e s were c e n t r i f u g e d f o r 10 minutes at f u l l speed i n a c l i n i c a l c e n t r i -fuge ( i n t e r n a t i o n a l Equipment Co., Model HN, angle head). The supernatants were decanted, d i l u t e d 1 to 4 with 0 - 5 $ soytone, pH 5 - 5 , and bioassayed. I I I . E f f e c t of medium c o n c e n t r a t i o n A medium ( 4 x ) c o n t a i n i n g the components of Gluc-AmS medium at 4 times t h e i r normal c o n c e n t r a t i o n was prepared. Part was d i l u t e d with an equal volume of d i s t i l l e d water to give 2 X medium, and part of t h i s was d i l u t e d with d i s t i l l e d water to give r e g u l a r Gluc-AmS. D u p l i c a t e 50 ml a l i q u o t s of 4 x and 2 X , 97 and t r i p l i c a t e 50 ml a l i q u o t s of Gluc-AmS were dispensed i n 250 ml Erlenmeyer f l a s k s . The f l a s k s were capped with a double t h i c k n e s s of aluminum f o i l , and autoclaved f o r 15 minutes on a dry goods c y c l e . Each f l a s k was i n o c u l a t e d with 1.0 ml from a c u l t u r e of 2259~7 i n Gluc-AmS, and incubated on a shaker at 20°C. Progress of the c u l t u r e s was monitored by d a i l y t u r b i d i t y measure-ments on samples from one of the Gluc-AmS f l a s k s . The t u r b i d -i t y reached a maximum 5 days a f t e r i n o c u l a t i o n , and a f t e r 1 days the f l a s k s were removed from the shaker. The t u r b i d i t y at 640 nm of l / l O d i l u t i o n s of samples from each f l a s k was measured, and 5 ml samples from each f l a s k were c e n t r i f u g e d f o r 5 minutes at f u l l speed i n an IEC model HN c l i n i c a l c e n t r i f u g e . The supernatants were adjusted to pH 5-5 with 0.1 N KOH, d i l u t e d 1 to 16 with 0.5$ soytone, pH 5-5^  and bioa s s a y e d . In a separate experiment four 125 ml Erlenmeyer f l a s k s c o n t a i n i n g 25 ml each of Gluc-AmS and four f l a s k s of h a l f - s t r e n g t h Gluc-AmS (x/2) were prepared, autoclaved, i n o c u l a t e d with 0.5 ml per f l a s k from, a 31 hour o l d c u l t u r e of 2259-7 i n Gluc-AmS, and incubated on a shaker at 20°C. Samples were taken d a i l y from one f l a s k of each medium, and the t u r b i d i t i e s measured. Peak t u r b i d i t y was reached a f t e r four days. On the f i f t h day, the t u r b i d i t y at 640 nm of l / l O d i l u t i o n s of samples from each f l a s k was measured, and samples of the 6 undisturbed c u l t u r e s were c e n t r i f u g e d , adjusted t o pH 5-5> d i l u t e d 1 t o 16 with 0-5$ soytone, pH 5-5> and bioassayed. While the assay c e l l s were being counted, the tubes c o n t a i n i n g the assays f o r one c u l t u r e i n each medium were i n a d v e r t e n t l y l e f t at room temperature f o r two hours. 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 of i n d i v i d u a l medium components A l i q u o t s of Gluc-AmS medium were supplemented with 10 gm of D-glucose (+G), or 1.0 gm of ammonium sulphate (+N), or 1.0 gm of KH2P0^, plus 0-5 gm of MgS0^-7Hg0 plus 0.1 gm CaCl 2-2H 20 (+S), or with a l l three (+GNS) per l i t r e . D u p l i c a t e 50 ml 9 8 p o r t i o n s of r e g u l a r Gluc-AmS, +G medium, +N medium, +S medium, and +GNS medium, were dispensed i n t o 250 ml Erlenmeyer f l a s k s . A f t e r a u t o c l a v i n g each f l a s k was i n o c u l a t e d with 1.0 ml from a k day o l d c u l t u r e of 2259-7 i n Gluc-AmS. At the same time, a t h i r d f l a s k of Gluc-AmS was i n o c u l a t e d . The f l a s k s were incubated on a shaker at 20°C and the t u r b i d i t y of the c u l t u r e i n the t h i r d f l a s k of Gluc-AmS was measured d a i l y . Peak t u r b i d -i t y was reached f o u r days a f t e r i n o c u l a t i o n , and on the f i f t h day the f l a s k s were sampled f o r t u r b i d i t y readings and bioassays as d e s c r i b e d i n s e c t i o n I I I . In a separate experiment, Gluc-AmS supplemented with 100 ug of thiamine h y d r o c h l o r i d e (+T) or 0.13 ml of the microelement stock s o l u t i o n (+M) per l i t r e was prepared. D u p l i c a t e 50 ml p o r t i o n s of Gluc-AmS, +T medium, and +M medium i n 250 ml E r l e n -meyer f l a s k s were autoclaved and i n o c u l a t e d , along with a t h i r d f l a s k of Gluc-AmS, with 1.0. ml per f l a s k from a 3 day o l d c u l t u r e of 2259-7 i n Gluc-AmS. The f l a s k s were incubated on a shaker at 20°C, and the t u r b i d i t y of the t h i r d Gluc-AmS f l a s k was monitored. Peak t u r b i d i t y was reached four days a f t e r i n o c -u l a t i o n , and on the s i x t h day samples were taken from each f l a s k f o r t u r b i d i t y measurements and b i o a s s a y . To t e s t the e f f e c t of lower c o n c e n t r a t i o n s of ammonium sulphate and microelements and t h e i r i n t e r a c t i o n on erogen y i e l d , a 2 X 2 X 2 f a c t o r i a l experiment was set up. The media used were v a r i a t i o n s of Gluc-AmS. Media designated N/2 contained 0.5 gm of ammonium sulphate per l i t r e ; those designated N/4 had 0.25 gm of ammonium sulphate per l i t r e . S i m i l a r l y M/2 media had O.O65 ml of microelement stock s o l u t i o n and M./h media had 0.033 ml of microelements per l i t r e . IR media contained D-glucose, KHgPO^, MgSO^•7H20, CaClg^HgO and thiamine at the same concen-t r a t i o n as i n Gluc-AmS; 2R media contained these components at twice the c o n c e n t r a t i o n i n Gluc-AmS. Two 26 ml p o r t i o n s of each of the eight combinations i . e . , (N/2, M/2, IR), (N/4, M/2, IR), (N/2, M/4, IR), and so on, were prepared i n 125 ml E r l e n -meyer f l a s k s . The experiment was d i v i d e d i n t o two r e p l i c a t e s 99 which were run at d i f f e r e n t times. One f l a s k of each medium was i n c l u d e d i n each r e p l i c a t e . The f l a s k s were i n o c u l a t e d with 0.5 ml each from a 3-day c u l t u r e of 2259-7 i n Gluc-AmS, and incubated on a shaker at 20°C. A f t e r f i v e days, the c u l t u r e s were sampled f o r t u r b i d i t y measurements and bio a s s a y s as des-c r i b e d above. V. Large s c a l e p r o d u c t i o n of erogens To produce amounts of the erogens s u f f i c i e n t f o r p u r i f i -c a t i o n , four 5 g a l l o n polypropylene b o t t l e s ( F i s h e r S c i e n t i f i c Co.) were employed. Each b o t t l e was f i t t e d with a #13 rubber stopper, through which passed two gl a s s tubes--an a i r i n l e t tube, 3 mm i . d . , and an a i r o u t l e t tube, 10 mm i . d . Both tubes were l o o s e l y packed with cotton wool. The a i r i n l e t tube was connected i n s i d e the b o t t l e by a short l e n g t h of rubber t u b i n g to another piece of g l a s s t u b i n g of the same diameter which reached almost to the bottom of the b o t t l e . The a i r o u t l e t tube was bent over o u t s i d e the b o t t l e through an angle of 120°. The b o t t l e s were loaded with 12 l i t r e s of d i s t i l l e d water, and then the i n g r e d i e n t s f o r 12 l i t r e s of Gluc-AmS medium. The sugar and s a l t s must be adequately d i s p e r s e d before a u t o c l a v i n g to prevent c a r a m e l i z a t i o n . The rubber stoppers were wired i n t o p o s i t i o n , and the b o t t l e s were autoclaved f o r 30 minutes at 15 p s i pressure on a dry goods c y c l e . I t was u s u a l l y necessary to leave the b o t t l e s c o o l i n g overnight before i n o c u l a t i o n . Inoculum was grown i n 200 ml batches of Gluc-AmS medium, i n 1 l i t r e Erlenmeyer f l a s k s on a shaker at 20°C. The inoculum, c u l t u r e s were used c l o s e to the end of t h e i r l o g phase. G e n e r a l l y 10 ml from each inoculum f l a s k was used to s t a r t f r e s h inoculum, and the r e s t was used to i n o c u l a t e 1 b o t t l e . A f t e r i n o c u l a t i o n , the b o t t l e s were placed i n a 20°C incubator room, and the a i r i n l e t tube was connected to a compressed a i r tap. A i r was bubbled through the c u l t u r e s at 1 to 2 l i t r e s per minute. 100 A f t e r f i v e days the c u l t u r e was passed through an ei g h t tube continuous flow attachment i n a S o r v a l l RC2-B c e n t r i f u g e . C u l t u r e flow r a t e was about 300 ml/min, the c e n t r i f u g e r o t o r speed was 15,000 rpm, and the temperature was 2 to 3°C. A sample of the supernatant was taken f o r bioassay, and the remainder was t r e a t e d with 6.0 gm of N o r i t A a c t i v a t e d c h a r c o a l . The suspension was s t i r r e d v i g o r o u s l y to evenly d i s t r i b u t e the c h a r c o a l , l e f t f o r two hours with o c c a s i o n a l s t i r r i n g , and then c e n t r i f u g e d under the same c o n d i t i o n s as the o r i g i n a l c u l t u r e . The recovered c h a r c o a l was washed with d i s t i l l e d water from the c e n t r i f u g e tubes onto a f i l t e r , and scraped from the f i l t e r i n t o a 50 ml g l a s s c e n t r i f u g e tube. Erogens were e l u t e d by suspending the c h a r c o a l i n 30 ml of p y r i d i n e a c etate s o l u t i o n (20 ml p y r i d i n e , 50 ml a c e t i c a c i d , and 30 ml d i s t i l l e d water), l e a v i n g f o r 15 minutes, and then c e n t r i f u g i n g out the c h a r c o a l . The supernatant was decanted, and the c h a r c o a l was resuspended i n another 30 ml of p y r i d i n e acetate s o l u t i o n . A t o t a l of f i v e p o r t i o n s of p y r i d i n e acetate s o l u t i o n were used f o r each l o t of c h a r c o a l . The e l u a t e s were combined, evaporated to dryness i n vacuo, d i s s o l v e d i n 10 ml of d i s t i l l e d water, re-evaporated, again d i s s o l v e d i n 10 ml of d i s t i l l e d water, and f r e e z e - d r i e d . The f r e e z e - d r i e d m a t e r i a l was s t o r e d i n a t i g h t l y capped v i a l i n a f r e e z e r . To determine the s p e c i f i c a c t i v i t y of the erogens i n the o r i g i n a l c u l t u r e supernatant, 10 ml of c u l t u r e supernatant were f r e e z e - d r i e d . 46.6 mg of the f r e e z e - d r i e d m a t e r i a l was d i s s o l v e d i n 2.0 ml of 0-5$ soytone, pH 5-5, and bioassayed. VI. Foam To determine i f d i f f e r e n t c o n d i t i o n s of a g i t a t i o n and a e r a t i o n could i n c r e a s e the erogen y i e l d i n l a r g e s c a l e c u l t u r e s , a s e r i e s of experiments u s i n g a Microferm l a b o r a t o r y fermenter (New Brunswick S c i e n t i f i c Co.) was begun. T h i s apparatus has a c u l t u r e volume of 5 l i t r e s , and f a c i l i t i e s f o r c o n t r o l l i n g s t i r r e r speed, a i r flow r a t e , and temperature. To prevent foam 101 from o v e r f l o w i n g onto the f l o o r , the a i r o u t l e t was connected "by p l a s t i c t u b i n g to a Pasteur p i p e t t e held by a cotton wool plug i n the neck of a 1 l i t r e Erlenmeyer f l a s k . The f l a s k , Pasteur p i p e t t e , and connecting t u b i n g were autoclaved along with the fermentor and i t s contents. In one experiment, 5 l i t r e s of Gluc-AmS i n the Microferm was i n o c u l a t e d with 50 ml of a h day o l d shake c u l t u r e of 2259-7 i n Gluc-AmS. The a i r flow r a t e was set at 2 l i t r e s / m i n , s t i r r e r speed at 0, and temperature at 20°C. On the f o u r t h , f i f t h , and s i x t h days a f t e r i n o c u l a t i o n , samples of the fermentor contents were taken a s e p t i c a l l y , c e n t r i f u g e d , and bioassayed. In another experiment, the s t i r r e r speed was set at 300 rpm, a i r flow at 2 l i t r e s / m i n , and temperature at 20°C. Samples f o r b i o a s s a y were taken k, 5, and 6 days a f t e r i n o c u l a t i o n . On the seventh day, the l i q u i d which had c o l l e c t e d i n the foam t r a p was assayed. A t h i r d Microferm c u l t u r e was set up with s t i r r i n g at 300 rpm and a i r flow at 0.5 l i t r e s / m i n . The inoculum was 200 ml of a 2 day o l d c u l t u r e of 2259-7 i n Gluc-AmS. A f t e r f i v e days of i n c u b a t i o n , approximately 500 mis of condensed foam had c o l l e c t e d i n the foam t r a p . The t r a p f l a s k was r e p l a c e d with a f r e s h s t e r i l e 1 l i t r e f l a s k , and the c o l l e c t e d foam was bioassayed. S e r i a l d i l u t i o n s down to 1/2048 were i n c l u d e d i n the b i o a s s a y s e r i e s . To t e s t the p o s s i b i l i t y t h a t the p o o r l y aerated c o n d i t i o n of the foam t r a p was s t i m u l a t i n g the c e l l s c a r r i e d over i n the foam to 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 of erogens, two 2800 ml Fernbach f l a s k s c o n t a i n i n g 500 ml each of Gluc-AmS were prepared. Each f l a s k was i n o c u l a t e d with 5-0 ml from a 3 day o l d shake c u l t u r e of 2259-7 i n Gluc-AmS, and incubated on a shaker at 20°C f o r t h r ee days. One of the f l a s k s was then moved to a bench i n the incubator room, and the other was l e f t on the shaker. A f t e r a t o t a l of s i x days of i n c u b a t i o n , samples from each f l a s k were bioassayed, s t a r t i n g with a l / l 6 d i l u t i o n . 102 To determine what f r a c t i o n of the t o t a l erogens produced by the c u l t u r e s could be recovered i n the c o l l e c t e d foam, 5 l i t r e s of (N/2, M/2, I R ) medium (see s e c t i o n I V ) was prepared i n the Microferm, and i n o c u l a t e d with 200 ml of a 3 day o l d c u l t u r e of 2259-7 i n Gluc-AmS. The s t i r r e r speed was set at 300 rpm, and the a i r flow at 200 ml/min. A f t e r s i x days, samples were taken from the main c u l t u r e volume, and from the c o l l e c t e d foam, and c e n t r i f u g e d . The sample from the main volume was bioassayed at i t s o r i g i n a l c o n c e n t r a t i o n , and the foam sample was d i l u t e d 1 t o 100 with 0.5$ soytone, pH 5-5> before b i o -as s aying. The a i r o u t l e t tube on one of the 5 g a l l o n polypropylene b o t t l e s was connected by p l a s t i c t u b i n g to a foam t r a p . The b o t t l e was loaded with 12 l i t r e s of (N/2, M/2, I R ) medium, s t e r i l i z e d , i n o c u l a t e d , and incubated according to the methods of s e c t i o n V . A f t e r 7 days, 125 ml of condensed foam had c o l l e c t e d . T h i s was d i l u t e d l/400 and bioassayed. 103 R e s u l t s I. Time course of erogen p r o d u c t i o n The c u l t u r e pH, t u r b i d i t y (measured Ag^Q X d i l u t i o n ) , and the c o n c e n t r a t i o n of erogens i n the supernatant are p l o t t e d a g a i n s t c u l t u r e age i n F i g . 23 f o r the p i l o t study, and F i g . 24 f o r the second experiment. The pH values d i d not vary more than 0.1 u n i t between any of the r e p l i c a t e s . The dry weight i n 40.0 ml of the washed c e l l suspension was 0.3725 gm, or 9.3 mg/ml. The Ag^ Q of the l/50 d i l u t i o n of the suspension was 0.248. T h e r e f o r e , a c u l t u r e t u r b i d i t y of 1.0 corresponds to 0-75 mg of c e l l dry weight per ml. I I . E f f e c t of n i t r o g e n source on erogen p r o d u c t i o n The c e l l s from the medium c o n t a i n i n g ammonium, sulphate without sodium acetate formed a compact p e l l e t during c e n t r i -f u g a t i o n ; the c e l l s grown i n the other media formed a l o o s e l y packed p e l l e t . When the bioassays were examined, the samples from assays of the media c o n t a i n i n g ammonium, sulphate plus sodium a c e t a t e , c a s e i n h y d r o l y s a t e , and soytone contained l a r g e numbers of c e l l s without c o n j u g a t i o n tubes, presumably 2259~7 c e l l s which had not been removed by c e n t r i f u g a t i o n . The presence of these c e l l s made counting of the f r a c t i o n of 2259-6 c e l l s with c o n j u g a t i o n tubes i m p o s s i b l e . T h e r e f o r e , the lengths of the c o n j u g a t i o n tubes formed i n the l / l 6 d i l u t i o n of each c u l t u r e supernatant were compared v i s u a l l y . The conjugation tubes formed i n response to supernatants from the c u l t u r e s grown i n ammonium sulphate were longer than those formed i n response to the other samples. No d i f f e r e n c e was observed between the samples from ammonium, sulphate plus sodium ac e t a t e , c a s e i n h y d r o l y s a t e , or soytone. I I I . E f f e c t of medium, c o n c e n t r a t i o n The f i n a l t u r b i d i t i e s and the erogen c o n c e n t r a t i o n s of the 2259-7 c u l t u r e s grown i n 4 x , 2X, and Gluc-AmS media are EROGEN CONCENTRATION (UNITS/ML) -tjOT • m n x p s i H S U i y - o rLL f ) U T s s j n ^ x r i o L-6<nZZ UT notq.'BTrmiriooe ru 8 CULTURE PH 8 S TURBIDITY AT B40 NM ut 8 EROGEN CONCENTRATION (UNITS/ML) lo6 shown i n Table IX. The f i n a l c u l t u r e t u r b i d i t i e s i n c r e a s e as the c o n c e n t r a t i o n of the medium i n c r e a s e s , but the y i e l d of erogen appears t o decrease. The d i f f e r e n c e i n average erogen y i e l d between media i s not s i g n i f i c a n t l y g r e a t e r than e x p e r i -mental e r r o r . The data -.from the c u l t u r e s grown i n Gluc-AmS and x/2 i n the second experiment are i n c l u d e d i n Table IX. The b i o a s s a y r e s u l t s have been s p l i t i n t o two b l o c k s - - t h e second b l o c k contains the assays which were l e f t at room temperature f o r two hours longer than the assays i n the f i r s t b l o c k . An a n a l y s i s of v a r i a n c e f o r the logarithms to the base two of the erogen y i e l d s i n t h i s experiment i s presented i n Table X. 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 of i n d i v i d u a l medium component s The f i n a l t u r b i d i t i e s and b i o a s s a y r e s u l t s from the e x p e r i -ments on the e f f e c t of i n c r e a s i n g the c o n c e n t r a t i o n of glucose, ammonium sulphate, s a l t s , thiamine and microelements are shown i n Table XI. The a n a l y s i s of v a r i a n c e i s presented i n Table XII. The d i f f e r e n c e between media was h i g h l y s i g n i f i c a n t i n the f i r s t experiment, and on the border l i n e of s i g n i f i c a n c e i n the second experiment. A p p l i c a t i o n of Dunnett's procedure f o r comparing treatment means with a c o n t r o l ( S t e e l and T o r r i e i960) showed t h a t the erogen y i e l d s i n the +N medium and the +GNS medium were s i g n i f i c a n t l y l e s s (95$ confidence) than the y i e l d i n Gluc-AmS. The y i e l d s i n the +G and +S media were not s i g n i f i c a n t l y d i f f e r e n t from the y i e l d i n Gluc-AmS. The data from the second experiment i n d i c a t e a lower y i e l d i n the +M medium than i n the +T medium and Gluc-AmS. The data from the f a c t o r i a l experiment i s l i s t e d i n Table X I I I . The a n a l y s i s of v a r i a n c e f o r the e f f e c t s of the medium components on the f i n a l t u r b i d i t y and on the logarithmns of the erogen y i e l d i s shown i n Table XIV. The main e f f e c t s of N, R and r e p l i c a t e s on f i n a l c u l t u r e t u r b i d i t y are s i g n i f i c a n t . Only the main e f f e c t of N on erogen y i e l d i s s i g n i f i c a n t . Table IX. F i n a l t u r b i d i t i e s and erogen concentrations of Gluc-AmS Medium F i n a l t u r b i d i t y at 640 nm F i r s t 4X 6 0 exper iment 5 7 2X 5 0 5 2 Gluc-AmS 3 4 3 3 Second Gluc-AmS 3 8 exper iment 3 8 3 8 x/2 3 0 3 3 3 1 y i e l d s f o r 2259-7 c u l t u r e s grown i n var i o u s medium. Erogen y i e l d 95$ confidence (units/ml) l i m i t s  9 3 7 6, 11 2 k 5 3 6, 5 6 5 9 4 1, 7 2 7 4 6. 1, 8 8 11 8 9 9, 14 1 10 6 8 Q, 12 5 11 3 f i r s t b l o c k 9 5, 13 3 10 5 8 9, 12 3 9 0 second b l o c k 7 6, 10 5 9 0 f i r s t b l o c k 7 6, 10 5 8 8 7 5, 10 3 6 1 second block 5 l ; 7 1 io a Table X. A n a l y s i s of v a r i a n c e of logarithmns t o the base two of erogen 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 Blocks Media E r r o r Degrees of freedom. 1 1 3 Sum of squares 0 . 2 2 9 6 0 . 2 1 6 6 0 . 0 2 9 5 Mean s guare 0 . 2 2 9 6 0 . 2 1 6 6 0 . 0 0 9 8 3 F P r o b a b i l i t y of a l a r g e r F  2 3 . 4 0 . 0 1 7 2 2 . 0 0 . 0 1 8 Table XI. F i n a l t u r b i d i t i e s and erogen y i e l d s i n media with doubled 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. Medium F i n a l Erogen t u r b i d i t y c o n c e n t r a t i o n u n i t s /ml 95$ confidence lira.it s •p & •H CO (D U Pi •H X Gluc-AmS 3 4 13 1 11 1, 14 7 3 4 14 6 13 0, 16 3 +G 4 0 13 6 12 1, 15 3 3 8 14 6 12 9, 16 4 +N 4 l 7 0 6 1 , 7 9 4 0 6 8 5 9, 7 7 +S 3 4 13 8 12 2 , 15 4 3 5 13 9 12 3 , 15 7 +GNS 4 7 12 0 10 1, 13 5 h 7 10 . 8 9 5, 12 2 Gluc-AmS 3 5 18 0 14 6, 22 5 -p 3 6 23 1 18 8 , 29 0 C! 0) 0 +T 3 7 21 7 17 6 , 27 3 3 5 19 5 16 2, 24 0 O <D O Pt 0) X +M 3 7 9 8 8 4 0 , 12 0 CQ 0) 3 6 5 3 3, 6 5 Table XII. A n a l y s i s of variance of logarithmns of erogen y i e l d s i n media with doubled concentrations of i n d i v i d u a l components. Source of v a r i a t i o n Degrees of Sum of Mean P P r o b a b i l i t y of freedom squares square a l a r g e r F F i r s t experiment Media h ±.<?6K 0-3909 67.5 0.0001 E r r o r 5 O.O2895 O.OO579 Second experiment Media 2 3-020 I.510 9-52 0.050 E r r o r 3 0A758 O.I586 1 1 0 Table X I I I . F i n a l t u r b i d i t i e s and erogen y i e l d s from f a c t o r i a l experiment on e f f e c t of ammonium sulphate c o n c e n t r a t i o n , microelement 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 of r e s t of med ium. -p «5 u -p -H CQ H U P i •H <L> Medium N / 2 , M/2, I R N/4, M/2, I R N / 2 , M/4, I R N/4, M/4, I R N / 2 , M/2, 2R N/4, M/2, 2R N / 2 , M/4, 2R N/4, M/4, 2R F i n a l Erogen y i e l d 9570 confidence t u r b i d i t y u n i t s / m l l i m i t s  .09 A 7 •05 ,46 2- 75 3- 27 2-55 11 8 9 11 12 7 13 8 8-9, 7-2, 7-6, 9.1, 10.1, 6.3, 10.7, 6.3, 14.1 10.7 11-7 13-9 15 .6 9-5 16.4 9.9 N/2, M / 2 , N/4, M/2, N / 2 , M/4, N/4, M/4, I R 3 32 13 6 9 9, 18 3 I R 2 59 1 4 0 1 0 3, 19 0 0 I R 3 37 17 8 13 2, 24 1 -p OS I R 2 75 7 3 5 4, 9 5 W/2, M/2, N/4, M/2, N / 2 , M/4, N/4, M/4, 2R 3 4l 15 9 11 5, 21 8 >-l ~ O H 2R 2 88 6 0 4 1, "8 0 0 ft d) 0) 2R 3 81 12 8 9 3, 17 3 tQ U 2R 2 88 8 3 6 0, 10 9 Table XIV. A n a l y s i s of variance f o r e f f e c t s of ammonium sulphate c o n c e n t r a t i o n , microelement c o n c e n t r a t i o n , and co n c e n t r a t i o n of r e s t of medium on f i n a l t u r b i d i t y and logarithmn of erogen y i e l d . Source of v a r i a t i o n a. T u r b i d i t y Main e f f e c t s N M R R e p l i c a t e s Degrees of freedom 1 1 1 1 Sum of squares O.OI879 0.00001157 0.002352 0.002305 Mean square 0.01879 0.00001157 0 .002352 0.002305 119 0.07 14 . 9 14.6 P r o b a b i l i t y of a l a r g e r F 0.00001 0.79 0 .0062 0.0065 I n t e r a c t ions NM NR MR NMR E r r o r 1 1 1 1 7 0.000053 O.OOOO69 O.OOOOO82 O.OOOI76 0.001104 0.000053 0.000069 0.0000082 0.000176 0.0001578 0.34 0.44 0.05 1 . 1 2 0.58 0.53 0.83 0.33 b. Erogen y i e l d s Main e f f e c t s N M R R e p l i c a t e s 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 . 8 4 0.63 0.013 0.90 0.39 0.45 I n t e r a c t ions NM NR MR NMR E r r or 1 1 1 1 7 O.OOOOO625 O.2835 0.0315 0.135 0.9209 0 . 0 0 0 0 0 6 2 5 0 . 2 8 3 5 0 . 0 3 1 5 0 . 1 3 5 0 . 1 3 1 5 0 . 0 0 0 0 5 2 . 1 6 0 . 24 1 . 0 3 0-99 0.19 0.64 0.3^ 112 V. Large s c a l e p r o d u c t i o n of the erogens A f t e r three days i n c u b a t i o n i n the l a r g e b o t t l e s , the c u l t u r e s of 2259-7 began t o foam. The foam would wet the a i r o u t l e t f i l t e r and escape around the edges of the rubber stopper. Reducing the a i r flow r a t e d i d not e l i m i n a t e t h i s problem. The erogen c o n c e n t r a t i o n i n the c u l t u r e supernatants v a r i e d between 0.25 and 0-5 u n i t s / m l . A f t e r c h a r c o a l t r e a t -ment, the r e s i d u a l erogen c o n c e n t r a t i o n i n the supernatant was . g e n e r a l l y below 0.05 u n i t s / m l . F i v e hundred and s i x t e e n l i t r e s of 2259-7 c u l t u r e supernatant were produced and processed by the methods d e s c r i b e d . The t o t a l y i e l d of concentrated erogen was 125^000 u n i t s , a recovery of approximately 80$. The f r e e z e - d r i e d c u l t u r e supernatant at 23-3 mg/ml had an erogenic potency of O.85 u n i t s / m l (95$ confidence l i m i t s O.7O8 and 1.005)- T h i s corresponds t o a s p e c i f i c a c t i v i t y of 0.037 units/mg. The hormone concentrate prepared by e l u t i o n of the c h a r c o a l had a s p e c i f i c a c t i v i t y of 5-0 units/mg. The r e f o r e the c h a r c o a l a d s o r p t i o n and e l u t i o n steps accomplished a p u r i f i -c a t i o n of 135 f o l d . VI. Foam In the f i r s t experiment, the erogenic potency of the c u l t u r e f l u i d a f t e r 4 days i n c u b a t i o n was 3-1 u n i t s / m l (95$ confidence l i m i t s 2-5 and 3-7)- On the f i f t h day, the potency was O.92 u n i t s / m l (95$ confidence l i m i t s O.56 and 1-3), and on the s i x t h day 0.37 u n i t s / m l (95$ confidence l i m i t s 0.14 and O.57). Foam s t a r t e d t o pass over i n t o the foam t r a p between the four day and f i v e day samplings. In the second experiment, the four day sample assayed 0.23 un i t s / m l (95$ confidence l i m i t s O.O76 and O.38), the f i v e day sample 0.55 u n i t s / m l (95$ confidence l i m i t s 0.31 and O.82) and the s i x day sample 0.33 u n i t s / m l (95$ confidence l i m i t s 0.29 and 0.37)- The c o l l e c t e d , condensed foam assayed on the seventh day showed high erogenic a c t i v i t y down to the lowest concen-t r a t i o n t e s t e d ( l / l 6 ) . 113 The c o l l e c t e d foam i n the t h i r d experiment had an erogen c o n c e n t r a t i o n of I96 u n i t s / m l (95$ confidence l i m i t s , 174 and 220);. , In the comparison between the shaken and s t a t i c c u l t u r e s , the shaken c u l t u r e had an erogen c o n c e n t r a t i o n of 13 u n i t s / m l (95$ confidence l i m i t s 7-4 and 19), and the c u l t u r e which was l e f t on the bench f o r the l a s t three days of i n c u b a t i o n had an erogen c o n c e n t r a t i o n of 3-5 u n i t s / m l (95$ confidence l i m i t s 1.4 and 5.5).. In the experiment to determine the f r a c t i o n of the erogens c a r r i e d over with the foam, the volume of the c o l l e c t e d , con-densed foam, a f t e r c e n t r i f u g i n g out the c e l l s , was 250 ml. The erogenic potency of the main c u l t u r e volume was 0.57 u n i t s / m l (95$ confidence l i m i t s 0.49 and 0.64) and the potency of the foam was 170 u n i t s / m l (95$ confidence l i m i t s 148 and I91). The t o t a l amount of erogens l e f t i n the 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 . The t o t a l amount of erogen i n the foam was 250 ml X 170 u n i t s / m l - 42,000 u n i t s , or 92$ of the t o t a l . The volume of condensed foam c o l l e c t e d from the 12 l i t r e c u l t u r e of 2259-7 i n the 5 g a l l o n b o t t l e was 125 ml- The co n c e n t r a t i o n of erogen i n the foam was 1,360 u n i t s / m l (95$ confidence l i m i t s 994 and 4300). Th i s corresponds to a t o t a l erogen y i e l d of 170,000 u n i t s or 14.2 u n i t s / m l of the o r i g i n a l c u l t u r e volume. Ilk D i s c u s s i o n The r i s e i n erogen c o n c e n t r a t i o n i n shake c u l t u r e s of 2259-7 i n Gluc-AmS f o l l o w s a curve approximately p a r a l l e l to the i n c r e a s e i n t u r b i d i t y , but l a g g i n g by about one day. However, the max-imum erogenic potency i s reached at the same time as the peak t u r b i d i t y . There may be a s m a l l decrease i n potency during the time when the c u l t u r e t u r b i d i t y i s decreasing, but the b i o a s s a y data are not p r e c i s e enough to f i r m l y . e s t a b l i s h t h i s . In the second experiment, the samples from. Qk, 108 and ikk hours showed c o n s i s t e n t l y lower hormone a c t i v i t y than the adjacent samples, i n d i c a t i n g t h a t some of the erogen was destroyed during overnight storage i n the f r e e z e r . The k i n e t i c s of hormone p r o d u c t i o n i n d i c a t e that the erogens are products of primary m etabolism—secondary m e t a b o l i t e s should appear mainly a f t e r growth has stopped (Aiba, Humphrey and M i l l i s I965). Most of the erogen was produced a f t e r the c u l t u r e pH f e l l below 2.5. T h i s may be a c o i n c i d e n c e . In subsequent experiments, erogen y i e l d s were determined one or two days a f t e r the peak t u r b i d i t y of the monitored c u l t u r e was reached, to ensure that a l l c u l t u r e s had reached t h e i r maximum y i e l d . Ammonium sulphate was the best of the n i t r o g e n sources t e s t e d f o r erogen p r o d u c t i o n . A d d i t i o n of sodium acetate to the medium to s t a b i l i z e the pH decreased the hormone y i e l d . Whether the higher pH of the c u l t u r e with sodium acetate, or some more s p e c i f i c e f f e c t of a c e t a t e , was i n h i b i t i n g erogen p r o d u c t i o n i s not c l e a r . Logarithmns of the erogen y i e l d s r a t h e r than the absolute values were used f o r the a n a l y s i s of v a r i a n c e because the erogen c o n c e n t r a t i o n s are estimated i n the b i o a s s a y as t h e i r logarithmns, and hence e r r o r s of e s t i m a t i o n w i l l be normally d i s t r i b u t e d on a l o g a r i t h m i c s c a l e . A l s o the v a r i a b i l i t y of the erogen y i e l d s can be expected to i n c r e a s e with the average y i e l d . I n c r e a s i n g the c o n c e n t r a t i o n of Gluc-AmS d i d not i n c r e a s e the f i n a l c o n c e n t r a t i o n of the erogens, and appears to have 115 a c t u a l l y decreased i t , although the d i f f e r e n c e s were not s i g n i f -i c a n t l y g r e a t e r than experimental e r r o r . H a l v i n g the medium c o n c e n t r a t i o n s i g n i f i c a n t l y reduced the erogen y i e l d . In t h i s experiment, the h i g h l y s i g n i f i c a n t d i f f e r e n c e between blocks supports the d i v i s i o n of the assay r e s u l t s i n t o two b l o c k s . Apparently the medium c o n c e n t r a t i o n chosen i n i t i a l l y i s c l o s e to o ptimal f o r erogen p r o d u c t i o n . When i n d i v i d u a l components of the medium were doubled i n c o n c e n t r a t i o n , i n c r e a s e d ammonium sulphate and i n c r e a s e d micro-elements s i g n i f i c a n t l y reduced the erogen y i e l d s . Increases i n other components d i d not change the y i e l d of erogens. F u r t h e r e f f e c t s might be found i f the s a l t s were t e s t e d i n d i v i d u a l l y r a t h e r than i n a group, or i f the 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 of the microelement stock s o l u t i o n were v a r i e d . The h i g h l y s i g n i f i c a n t e f f e c t of ammonium sulphate con-c e n t r a t i o n on f i n a l t u r b i d i t y of the c u l t u r e s i n the f a c t o r i a l experiment i n d i c a t e s that n i t r o g e n i s the main l i m i t i n g n u t r i e n t f o r growth, at l e a s t at 0.25 gm/l. The R f a c t o r a l s o had a s i g n i f i c a n t , although smaller, e f f e c t , suggesting that one of i t s components i s c l o s e to a l i m i t i n g c o n c e n t r a t i o n . The s i g n i f -i c a n t d i f f e r e n c e between the average t u r b i d i t i e s i n the two r e p l i c a t e s means t h a t the second r e p l i c a t e was sampled at a s l i g h t l y e a r l i e r phase of growth than the f i r s t r e p l i c a t e . T h i s did not produce a s i g n i f i c a n t d i f f e r e n c e i n average erogen c o n c e n t r a t i o n between the two r e p l i c a t e s . C o n c e n t r a t i o n of ammonium sulphate was the only f a c t o r t h a t had a s i g n i f i c a n t e f f e c t on the y i e l d of erogen. The c o n c e n t r a t i o n s of microelements and of the r e s t of the medium, and t h e i r i n t e r a c t i o n s , were without e f f e c t . An ammonium sulphate c o n c e n t r a t i o n of 0-5 gm/l i s near optimal f o r s y n t h e s i s of erogens, and the (w/2, M/2, IR) medium appears to be a good choice f o r hormone p r o d u c t i o n . Large s c a l e p r o d u c t i o n of the erogens was begun before the q u a n t i t a t i v e b i o a s s a y procedure was developed. T h e r e f o r e I d i d not r e a l i z e at the time how abnormally low the erogen y i e l d s from the b o t t l e c u l t u r e s were. 116 Three methods f o r re c o v e r y and primary p u r i f i c a t i o n of the erogens from l a r g e volumes of c u l t u r e media were c o n s i d e r e d -e x t r a c t i o n with n-butanol, a d s o r p t i o n on ion exchange r e s i n , and a d s o r p t i o n on a c t i v a t e d c h a r c o a l . Solvent e x t r a c t i o n was r e j e c t e d because the high b o i l i n g p o i n t of n-butanol made evap-o r a t i o n of l a r g e volum.es d i f f i c u l t , and made continuous e x t r a c -t i o n i m p r a c t i c a l . A d s o r p t i o n on c h a r c o a l was favoured over a d s o r p t i o n on ion exchange r e s i n s "because fewer components of the c u l t u r e supernatants would compete with the erogen f o r b i n d -ing s i t e s on the c h a r c o a l , l e a d i n g t o higher r e c o v e r i e s and b e t t e r p u r i f i c a t i o n . The c h a r c o a l a d s o r p t i o n procedure was p r a c t i c a l , and gave 80$ r e c o v e r y of the erogens, with 135-fold p u r i f i c a t i o n . Both the r e c o v e r y and the p u r i f i c a t i o n might have been higher i f the c o n c e n t r a t i o n of the erogens i n the c u l t u r e supernatant had been g r e a t e r . The erogens e l u t e d from the c h a r c o a l would have been purer i f the c h a r c o a l had been pre-washed with the p y r i d i n e acetate e l u a n t . When I r e a l i z e d t h a t the erogen y i e l d s from the 5 g a l l o n b o t t l e s were much lower than those from shake c u l t u r e s , I f i r s t thought that d i f f e r e n c e s i n a e r a t i o n and a g i t a t i o n might be r e s p o n s i b l e . Bubbling a i r through the 12 l i t r e c u l t u r e s might provide too low or too high a c o n c e n t r a t i o n of d i s s o l v e d oxygen, or provide inadequate mixing of the c u l t u r e s . Experiments to i n v e s t i g a t e the e f f e c t of s t i r r i n g and a i r flow r a t e were begun i n the Microferm, where these parameters could be e a s i l y con-t r o l l e d . Foam t r a p s were i n c l u d e d i n i t i a l l y merely f o r neatness. The erogen y i e l d s from, the Microferm c u l t u r e s at v a r i o u s condi-t i o n s of s t i r r i n g and a i r flow were comparable to the y i e l d s i n the 5 g a l l o n b o t t l e s . In the f i r s t experiment d e s c r i b e d , the erogen c o n c e n t r a t i o n i n the medium decreased r a t h e r than i n c r e a s e d with time. The sharp drop between the f i r s t two samples c o i n -cided with the beginning of foam c a r r y over i n t o the foam. t r a p . At the suggestion of S c o t t Redhead, i n the second experiment the c o l l e c t e d foam, was assayed, and found to have high erogenic a c t i v i t y . The comparison between the shaken and s t a t i c f l a s k 117 c u l t u r e s confirmed t h a t the erogens were being t r a n s p o r t e d from the main c u l t u r e i n t o the t r a p by the foam., r a t h e r than being s y n t h e s i z e d by the c e l l s a f t e r they reach the t r a p . In a t y p i c a l c u l t u r e , 92$ of the t o t a l erogens produced was concentrated i n the condensed foam. The foam e f f e c t i v e l y removes the erogens from the b o t t l e c u l t u r e s as w e l l . The amount of erogens i n the foam i n d i c a t e s an erogen y i e l d from the b o t t l e c u l t u r e s compar-able to that i n shake c u l t u r e s , and e x p l a i n s the low y i e l d s found e a r l i e r f o r the b o t t l e c u l t u r e s when foam was d i s c a r d e d . C o l l e c t i o n of the foam from the c u l t u r e s i s an e x c e l l e n t method f o r p r e l i m i n a r y c o n c e n t r a t i o n and p u r i f i c a t i o n of the erogens. Such a high percentage of the erogen a c t i v i t y accumu-l a t e s i n the foam t r a p t h a t i t i s p r a c t i c a l to d i s c a r d the r e s i d u a l hormone i n the main c u l t u r e . S e l e c t i v e t r a n s p o r t of s o l u t e s from aqueous s o l u t i o n by foaming has r e c e i v e d a t t e n t i o n because of i t s p o s s i b l e a p p l i -c a t i o n to such problems as removal of detergents from waste water. As w e l l as s u r f a c e a c t i v e agents, any m a t e r i a l which i s adsorbed at a s u r f a c e can be removed. The evidence f o r both p o l a r and non-polar s e c t i o n s i n the erogen molecules d i s c u s s e d i n Chapter Two suggests t h a t the erogen molecules themselves are s u r f a c e a c t i v e . However, the erogens are probably not p r i n c i p a l l y r e s p o n s i b l e f o r the foaming of 2259-7 c u l t u r e s . When the c u l t u r e supernatant or condensed foam are passed through a bed of Porapak Q, the e f f l u e n t , which has very low erogen content, i s as foamy as the o r i g i n a l s o l u t i o n . The t h e o r e t i c a l b a s i s of foam f r a c t i o n a t i o n has been i n v e s t i g a t e d i n order to p r e d i c t optimum op e r a t i n g c o n d i t i o n (Lemlich I968). The only o p e r a t i n g c o n d i t i o n that can be r e a d i l y v a r i e d i n the c o n c e n t r a t i o n of erogens from 2259-7 c u l t u r e s i s the a i r flow r a t e . High a i r flow r a t e s produce "wet" foams, and remove su r f a c e adsorbed m a t e r i a l s most completely. Low a i r flow r a t e s produce "dry" foams, which give a higher c o n c e n t r a t i o n of s u r f a c e adsorbed substances i n the condensed foam, and b e t t e r s e p a r a t i o n from s u r f a c e i n a c t i v e mater-i a l s . C l e a r l y an intermediate flow r a t e g i v i n g a compromise be-tween high r e c o v e r y and high p u r i f i c a t i o n of the erogens i s best f o r the T r e m e l l a c u l t u r e s . 118 In these experiments, only the t o t a l erogen c o n c e n t r a t i o n has been measured. The p r o p o r t i o n s of the i n d i v i d u a l a c t i v e compon-ents may vary with the age of the c u l t u r e and the medium, and they may have d i f f e r e n t a f f i n i t i e s f o r the foam. CHAPTER SIX PARTIAL PURIFICATION OF THE EROGENS 119 M a t e r i a l s and Methods I. I n s t a b i l i t y 4-7-1 mg of the standard erogen p r e p a r a t i o n was d i s s o l v e d i n 1.0 ml of 10$ ammonia. 38-8 mg was d i s s o l v e d i n 1.0 ml of 1 N h y d r o c h l o r i c a c i d . 40.0 mg was d i s s o l v e d i n 1.0 ml of 1 N potassium c h l o r i d e s o l u t i o n . The s o l u t i o n s i n s m a l l screw cap v i a l s were autoclaved f o r 10 mintures at 15 p s i steam pressure, on a dry goods c y c l e . A f t e r c o o l i n g , the contents of each v i a l was q u a n t i t a t i v e l y t r a n s f e r r e d to a 10 ml v o l u m e t r i c f l a s k and d i l u t e d to the mark with d i s t i l l e d water. 0.1 ml of each d i l u t e d s o l u t i o n was added to 2.0 ml of 0-5$ soytone, and the pH was adjusted to 5-5 f o r b i o a s s a y . I I . F i r s t attempt at p u r i f i c a t i o n 5l6 l i t r e s of c u l t u r e medium processed by the methods of Chapter F i v e , s e c t i o n V y i e l d e d 25 grams of erogen concentrate, with a s p e c i f i c a c t i v i t y of 5-0 units/mg. F i v e grams of t h i s concentrate were d i s s o l v e d i n 20 ml of d i s t i l l e d water, and the pH of the s o l u t i o n was adjusted to 6.5 by a d d i t i o n of 10$ sodium b i c a r b o n a t e s o l u t i o n . The n e u t r a l -i z e d s o l u t i o n was passed at a flow r a t e of 50 ml/hour through a 2 cm X 20 cm column of D u o l i t e A-4 anion exchange r e s i n which had been e q u i l i b r a t e d with 0.2 M ammonium acetate b u f f e r , pH 6.5, i n a c o l d room at 4°C. The column was washed with 300 ml of c o l d d i s t i l l e d water, and then e l u t e d with 350 ml of c o l d 1 N a c e t i c a c i d . As the a c e t i c a c i d progressed down the column, the r e s i n changed colour from grey to yellow. The a c e t i c a c i d e l u a t e was c o l l e c t e d from j u s t before the time t h a t the yellow a c i d f r o n t reached the bottom of the column u n t i l no more pigment was v i s i b l e i n the e f f l u e n t . The e l u a t e s o l u t i o n was f r e e z e -d r i e d . The column was regenerated by washing with 400 ml of d i s t i l l e d water, 200 ml of . I N potassium hydroxide, 400 ml of d i s t i l l e d water, and 250 ml of 0.2 M ammonium acetate b u f f e r , pH 6.5- In t o t a l the erogens from 20 gm. of concentrate were adsorbed on and e l u t e d from the anion exchange r e s i n , i n 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 set aside f o r bi o a s s a y . The remainder of the e l u a t e ( l . O gm) was suspended i n 100 ml of d i s t i l l e d water. The pH of the suspension was adjusted to approximately seven with 10$ sodium b i c a r b o n a t e s o l u t i o n , whereupon some of the 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 . The p r e c i p i t a t e was removed by c e n t r i f u g a t i o n , and the c l e a r brown supernatant was decanted. The p e l l e t was washed with two 20 ml p o r t i o n s of d i s t i l l e d water, which was added to the supernatant. T h i s supernatant s o l u t i o n was e x t r a c t e d with 100 ml of n-butanol s a t u r a t e d with water, f o l l o w e d by f i v e 50 ml p o r t i o n s of n-bu t a n o l s a t u r a t e d with water. The b u t a n o l e x t r a c t s were c e n t r i -fuged b r i e f l y to remove water d r o p l e t s , and then evaporated i n vacuo j u s t to dryness. The r e s i d u e was t r e a t e d with 10 ml of d i s t i l l e d water and 20 ml of e t h y l a c e t a t e . Despite vigorous s w i r l i n g , some of the d r i e d m a t e r i a l on the walls of the f l a s k would not d i s s o l v e . The w a t e r - e t h y l acetate mixture was decanted i n t o a c e n t r i f u g e tube, f o l l o w e d by water washings from the f l a s k . The u n d i s s o l v e d m a t e r i a l l e f t i n the f l a s k d i d not completely d i s s o l v e . . i n n-butanol s a t u r a t e d with water, nor i n 50$ acetone. The w a t e r - e t h y l acetate mixture was c e n t r i f u g e d to separate the phases, and the e t h y l acetate phase was decanted. The aqueous phase was b r i e f l y concentrated i n vacuo to remove e t h y l a c e t a t e , and then f r e e z e - d r i e d . The p r e c i p i t a t e which had formed on n e u t r a l i z a t i o n of the elu a t e s o l u t i o n mostly d i s s o l v e d i n 20 ml of 1 N a c e t i c a c i d . U n d i s s o l v e d m a t e r i a l was removed by c e n t r i f u g a t i o n , and the supernatant was f r e e z e - d r i e d . The f r e e z e - d r i e d anion exchange el u a t e was bioassayed at a c o n c e n t r a t i o n of O.O36 mg/ml. The r e d i s s o l v e d p r e c i p i t a t e was bioassayed at 0.27 mg/ml. The n-butanol e x t r a c t was b i o a s s a y -ed at 0.0024 mg/ml. The e t h y l acetate e x t r a c t was evaporated t o dryness i n vacuo, and r e d i s s o l v e d i n 10 ml of 50$ acetone. T h i s s o l u t i o n was d i l u t e d 1 to 50 f o r bi o a s s a y . The r a f f i n a t e from the bu t a n o l e x t r a c t i o n was a l s o d i l u t e d 1 to 50 f o r bi o a s s a y . 121 The r e d i s s o l v e d m a t e r i a l from the p r e c i p i t a t e was suspended i n 100 ml of d i s t i l l e d water s a t u r a t e d with n-butanol, and e x t r a c t e d with f i v e 50 ml p o r t i o n s of n-butanol. The b u t a n o l i e x t r a c t s were c e n t r i f u g e d , evaporated, r e d i s s o l v e d i n water-e t h y l a c e t a t e , and f r e e z e - d r i e d as b e f o r e . The two freez.e-d r i e d b u t a n o l e x t r a c t s were combined and thoroughly mixed. 6.1 mg was d i s s o l v e d i n 6.0 ml of 20$ acetone. T h i s s o l u t i o n was d i l u t e d l/5000 f o r b i o a s s a y . The remainder of the b u t a n o l ex-t r a c t was d i s s o l v e d i n l60 ml of 25$ aqueous acetone, and d i l u t e d 1/25O f o r b i o a s s a y . The b u t a n o l e x t r a c t s were again f r e e z e - d r i e d , and the f r e e z e - d r i e d powder was ground i n 1 ml of absolute ethanol i n a mortar. F i f t y grams of s i l i c a g e l (BDH) was s l u r r i e d i n absolute ethanol, and poured i n t o a chromatography column, 2 cm i n i n t e r n a l diameter. The e t h a n o l suspension of the erogen p r e p a r a t i o n was a p p l i e d to the top of the column, and e l u t e d f i r s t with 50 ml of absolute ethanol, and then with a g r a d i e n t of water i n ethanol, generated by the method of Chapter Two, s e c t i o n X. The eluant flow r a t e was 1 ml/min, and the e f f l u e n t from the column was c o l l e c t e d i n 5 minute f r a c t i o n s . From each f r a c t i o n , four drops were added to 1.0 ml of 0-5$ soytone, pH 5-5, i n a t e s t tube f o r b i o a s s a y . The tubes were autoclaved f o r 10 minutes on a dry goods c y c l e , and then i n o c u l a t e d with 1 drop each from a 30 hour old c u l t u r e of 2259-6 i n (4,4) medium. A f t e r 12 hours i n c u b a t i o n at 20°C, the f r a c t i o n of c e l l s with c o n j u g a t i o n tubes i n each assay tube was determined. F r a c t i o n s 5 to 18 were pooled as hormone I, evaporated to dryness i n vacuo, d i s s o l v e d i n 3 ml of absolute ethanol and s t o r e d i n the f r e e z e r . F r a c t i o n s 41 to j6 were pooled as hormone I I , evaporated to dryness i n vacuo, d i s s o l v e d i n 3 ml of 50$ aqueous et h a n o l and s t o r e d i n the f r e e z e r . 100 ml of 50$ ethanol was run through the s i l i c a g e l column, and the e f f l u e n t combined with f r a c t i o n s 77 to 84 as hormone I I I . T h i s s o l u t i o n was evaporated to dryness i n vacuo, and the r e s i d u e t r e a t e d with 50$ e t h a n o l . I t would not completely d i s s o l v e , and the s o l u t i o n and as much as p o s s i b l e of the i n s o l u b l e m a t e r i a l were t r a n s f e r r e d to a v i a l and s t o r e d i n the f r e e z e r . 122 I I I . I n t e r a c t i o n of the erogens-.with-'ion . exchange!" res ins To c l a r i f y the importance of i o n i c and non-polar "binding i n the a d s o r p t i o n of the erogens i n ion exchange r e s i n s , the e q u i l i b r i u m d i s t r i b u t i o n of the erogens between the r e s i n phase and the s o l u t i o n phase was determined under v a r i o u s c o n d i t i o n s . Amberlite IR-120 c a t i o n exchange r e s i n was washed with d i s t i l l e d water, 2 W h y d r o c h l o r i c a c i d , more d i s t i l l e d water, converted to the K + form with 2 N potassium hydroxide, and washed again with d i s t i l l e d water. The r e s i n was then converted to the H + form with 2 N h y d r o c h l o r i c a c i d and washed with d i s t i l l e d water u n t i l the washings were n e u t r a l . Excess water was removed by f i l t r a t i o n , and the moist r e s i n was stored i n a t i g h t l y capped j a r . Three 1.0 gram p o r t i o n s of the moist r e s i n were weighed i n t o 16 X 125 mm screw cap tubes. Two of the r e s i n samples were converted t o the K + form by adding 15 ml of 2 N potassium hydroxide to each, and shaking v i g o r o u s l y . A f t e r the r e s i n s e t t l e d t o the bottom, the supernatant was decanted and each r e s i n sample was washed with three 15 ml p o r t i o n s of d i s t i l l e d water. One of the K + r e s i n samples was then e q u i l i b r a t e d with .05 M KHgPO^ by suspending the r e s i n i n 15 ml of t h i s b u f f e r , a l l o w i n g i t t o s e t t l e , and decanting the b u f f e r . The procedure was repeated three times. The second sample of K + r e s i n was s i m i l a r l y e q u i l i b r a t e d with a 1 to 1 mixture of acetone and 0.1 M KHgPO^. The sample of r e s i n which had been l e f t i n the H + form was washed with three changes of 50$ acetone. A f t e r the t h i r d wash had been decanted, the tubes c o n t a i n i n g the r e s i n samples were i n v e r t e d over a paper towel f o r 5 minutes to d r a i n . A f t e r d r a i n i n g , 5-0 ml of the s o l u t i o n with which the r e s i n had been e q u i l i b r a t e d was added t o each tube. A c o n t r o l tube c o n t a i n i n g 5-0 ml of the .05 M KHgPO^^O^ acetone, but no r e s i n , was set up. To each tube, 20 u n i t s of erogens ( e l u t e d from Porapak Q,)' were added. The tubes were capped' and placed on a r e c i p r o c a t i n g shaker, with t h e i r long axes p a r a l l e l to the d i r e c t i o n of motion, at 20°C f o r 1-5 hours t o allow the d i s t r i -b u t i o n of the erogens between the s o l u t i o n and the r e s i n to 123 reach e q u i l i b r i u m . The r e s i n was then allowed to s e t t l e , and the supernatants were decanted and adjusted t o pH 5 . 5 . A sample from each supernatant was d i l u t e d l/k with 0 . 5 $ soytone, pH 5 - 5 , and bioassayed. D u o l i t e A-k anion exchange r e s i n was washed with d i s t i l l e d water, 2 N potassium hydroxide, d i s t i l l e d water, 1 N a c e t i c a c i d , and d i s t i l l e d water, and a i r - d r i e d . Two 0 - 5 gm samples of dry r e s i n i n screw cap tubes were converted t o the f r e e base form, by suspending each i n 15 ml of 2 N potassium hydroxide, and. washing with three changes of d i s t i l l e d water. One res-in sample was e q u i l i b r a t e d with three 15 ml changes of 0 . 1 M KgHPOlj., and the other with three 15 ml changes of 0 . 1 M K 2 H P 0 ^ c o n t a i n i n g 50$ acetone. The r e s i n samples were drained as above, and then suspended i n 5 . 0 ml of the same b u f f e r with which they had been e q u i l i b r a t e d . A c o n t r o l tube c o n t a i n i n g 5 - 0 ml of 0 . 1 M K 2 H P 0 ^ -50$ acetone, but no r e s i n , was a l s o set up. 1 0 0 u n i t s of erogens were added t o each tube. The tubes were capped and l e f t on the shaker f o r 1 . 5 hours. Then the r e s i n was allowed t o s e t t l e , the supernatants were decanted, adjusted to pH 5 - 5 , and d i l u t e d l / 2 0 f o r b i o a s s a y . In a second experiment, four 0 - 5 gm samples of dry D u o l i t e A-k r e s i n were weighed i n t o screw cap tubes. Two of the samples were e q u i l i b r a t e d with three changes of 0 . 1 M KgHPOi,.. The other two samples were converted to the free' base form as above. One of the r e s i n samples e q u i l i b r a t e d with 0 . 1 M KgHPO^ and one of the samples i n the f r e e base form were washed with three 15 ml p o r t i o n s of d i s t i l l e d water, drained, and resuspended i n 5 - 0 ml each of d i s t i l l e d water. The other r e s i n sample of each type was washed with three 15 ml p o r t i o n s of 50$ acetone, drained, and resuspended i n 5 - 0 ml of 50$ acetone. A c o n t r o l tube con-t a i n i n g 5 - 0 ml of 50$ acetone, but no r e s i n , was prepared. 50 u n i t s of erogens were added t o each tube, and the capped tubes were a g i t a t e d on the shaker at 2 0°C f o r 3 hours. Then the r e s i n was allowed t o s e t t l e , and the supernatants were decanted, adjusted to pH 5-5.? and d i l u t e d l / l O f o r b i o a s s a y . 124 IV. F u r t h e r attempts at p u r i f i c a t i o n Erogens were produced i n 500 ml l o t s of Gluc-AmS i n 2800 ml Fernbach f l a s k s , incubated on a shaker at 20°C. The t u r b i d i t y of the c u l t u r e s at 64-0 nm was measured on samples taken d a i l y , and the c u l t u r e s were harvested one day a f t e r they reached peak t u r b i d i t y . The c e l l s were removed by c e n t r i f u g a t i o n f o r 5 minutes at f u l l speed i n a c l i n i c a l c e n t r i f u g e (IEC model HW, angle head):. The supernatants were passed through a bed of 10 grams of Porapak Q i n a chromatography tube 2 cm i n i n t e r n a l diameter at a flow r a t e of 200 ml/hour. The e f f l u e n t was d i s -carded. The r e s i n was washed with 200 ml of d i s t i l l e d water, and the erogens were e l u t e d with 100 ml of 50$ acetone. The eluat e was concentrated i n vacuo u n t i l no more acetone d i s t i l l e d over, and then f r e e z e - d r i e d . The f r e e z e - d r i e d e l u a t e was st o r e d i n a t i g h t l y capped v i a l i n a f r e e z e r . The Porapak r e s i n was washed with 100 ml of acetone f o l l o w e d by 500 ml of d i s t i l l e d water before reuse. 100 mg of the f r e e z e - d r i e d e l u a t e was suspended i n 50 ml of water s a t u r a t e d with n-butanol, and e x t r a c t e d with f i v e 50 ml p o r t i o n s of n-butanol s a t u r a t e d with water. The e x t r a c t s were c e n t r i f u g e d to remove water d r o p l e t s and then evaporated to dryness i n vacuo. The e x t r a c t e d s o l i d s were d i s s o l v e d i n 8 ml of d i s t i l l e d water and 4 ml of e t h y l a c e t a t e . The w a t e r - e t h y l acetate mixture was c e n t r i f u g e d to separate the phases, and the aqueous phase r e - e x t r a c t e d with a second 4 ml p o r t i o n of e t h y l a c e t a t e . The combined e t h y l acetate e x t r a c t s were made up to 50 ml with acetone, and d i l u t e d l/50 f o r bi o a s s a y . The r a f f i n -ate from the bu t a n o l e x t r a c t i o n , and the aqueous s o l u t i o n of e x t r a c t e d m a t e r i a l s were made up to 50 ml with 50$ acetone, and d i l u t e d l/50 f o r bi o a s s a y . The s o l u t i o n of m a t e r i a l s e x t r a c t e d with b u t a n o l was then concentrated i n vacuo t o remove acetone, and f r e e z e - d r i e d . At another time the 50$ acetone Porapak e l u a t e s from, s e v e r a l batches of c u l t u r e supernatant were combined, concentrated i n vacuo t o remove acetone, and then e x t r a c t e d f o u r times with two 125 volumes of n-butanol. The n-butanol e x t r a c t s were c e n t r i f u g e d , and evaporated to dryness i n vacuo. The r e s i d u e was d i s s o l v e d i n 10 ml of water plus 10 ml of e t h y l a c e t a t e . The w a t e r - e t h y l acetate mixture was c e n t r i f u g e d , and the aqueous l a y e r was made up t o 50 ml with 50$ acetone, and d i l u t e d l/200 f o r b i o a s s a y . The acetone was then removed from the e x t r a c t s o l u t i o n by con-c e n t r a t i o n i n vacuo, and the s o l u t i o n was f r e e z e - d r i e d . The combined f r e e z e - d r i e d b u t a n o l e x t r a c t s were ground i n 1 ml of absolute e t h a n o l i n a mortar, and a p p l i e d to the top of a chromatography column c o n t a i n i n g 50 gm. of s i l i c a g e l i n ab-s o l u t e e t h a n o l . The column was e l u t e d with 100 ml of ethanol, f o l l o w e d by a g r a d i e n t of water i n ethanol generated by the method of Chapter Two s e c t i o n X. The flow r a t e was 0.25 ml/min, and 20 minute f r a c t i o n s were c o l l e c t e d . For bioassay, 50 u.1 of each f r a c t i o n was d i l u t e d with 1.0 ml of 0-5$ soytone, pH 5-5> autoclaved, and i n o c u l a t e d with 1 drop of a one day o l d c u l t u r e of 2259-6 i n (k,k) medium. The even numbered f r a c t i o n s from 40 t o 17k were assayed again with only 10 u l added to each assay tube. F r a c t i o n s 42-174 were combined and evaporated i n  vacuo to dryness. The r e s i d u e was d i s s o l v e d i n 10 ml of 75$ acetone, and s t o r e d i n the f r e e z e r . The c o l l e c t e d foam. from. 5 l i t r e s of 2259-7 c u l t u r e i n Gluc-AmS i n the Microferm. was c e n t r i f u g e d to remove c e l l s . The supernatant was passed through a bed of 10 grams of Porapak Q on a s i n t e r e d g l a s s f u n n e l 6 cm. i n diameter. The r e s i n was washed with d i s t i l l e d water, and erogens were e l u t e d with 100 ml of 50$ acetone. The e l u a t e s from three 5 l i t r e c u l t u r e s were combined, and concentrated i n vacuo to remove acetone. The aqueous s o l u t i o n (170 ml) was e x t r a c t e d with 100 ml of n-butanol, and then with four 50 ml p o r t i o n s of n-butanol. The b u t a n o l e x t r a c t s were c e n t r i f u g e d and evaporated to dryness in_ vacuo. The r e s i d u e was d i s s o l v e d as completely as p o s s i b l e i n 10 ml of d i s t i l l e d water plus 10 ml of e t h y l a c e t a t e . The phases were separated by c e n t r i f u g a t i o n , and the aqueous phase was made up to 50 ml with 50$ acetone and d i l u t e d l/200 f o r b i o a s s a y . The 126 r a f f i n a t e from the b u t a n o l e x t r a c t s (140 ml) was d i l u t e d 3 / l 0 0 f o r b i o a s s a y . The acetone was d i s t i l l e d i n vacuo from, the e x t r a c t s o l u t i o n and the e x t r a c t was f r e e z e - d r i e d . V. T h i n l a y e r chromatography T h i n l a y e r s of c e l l u l o s e were prepared by spreading a s l u r r y of 35 grams of m i c r o c r y s t a l l i n e c e l l u l o s e ( J . T. Baker Co.) i n 175 ml of d i s t i l l e d water, mixed i n an e l e c t r i c blender f o r 1 minute, i n a l a y e r 750 u t h i c k on 20 X 20 cm. gla s s p l a t e s . A f t e r d r y i n g , the l a y e r s were washed by ascending development i n a c e t o n e - 0 . 1 N h y d r o c h l o r i c a c i d ( l / l ) . One p l a t e was spotted 2 cm from the bottom, with 25 u l of the hormone I p r e p a r a t i o n d e s c r i b e d i n s e c t i o n I I of t h i s chapter and 10 u l of the erogens prepared by s i l i c a g e l chromatography as d e s c r i b e d i n s e c t i o n IV. The p l a t e was developed i n 0 . 1 N aqueous ammonia. A f t e r d r y i n g , the area between the so l v e n t f r o n t and the o r i g i n f o r each p r e p a r a t i o n was d i v i d e d i n t o nine zones. The c e l l u l o s e from each zone was scraped i n t o a t e s t tube and suspended i n 1 .0 ml of 0 . 5 $ soytone, pH 5 - 5 - The tubes were autoclaved f o r 10 minutes on a dry goods c y c l e , and i n o c u l a t e d with 1 drop each from a one day c u l t u r e of 2 2 5 9 - 6 . A f t e r 17 hours, the f r a c t i o n of c e l l s with conjugation tubes i n each assay wasdetermined. S i m i l a r l y , p l a t e s spotted with samples of the two erogen p r e p a r a t i o n s were developed i n e t h y l 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 ( 4 o / 4 o / 2 0 ) and chloroform-methan-•:oiT-0etLcN ,'amm.onia ( 4 0 / 2 5 / 2 . 5 ) - The areas between the o r i g i n and the s o l v e n t f r o n t were d i v i d e d i n t o ten zones, which were scraped o f f and assayed as above. Four TLC p l a t e s were streaked with 0.5 ml each of the hormone p r e p a r a t i o n d e s c r i b e d i n s e c t i o n IV ( i . e . 1150 u n i t s per plate)'. The s t a r t l i n e was compressed by b r i e f development i n 50$ acetone, and then the p l a t e s were developed twice i n chloroform-methanol - 0 . 1 N ammonia ( 4 0 / 2 5 / 2 - 5 ) - The erogens were l o c a t e d by s c r a p i n g o f f zones 1 cm high and 5 mm wide, and assa y i n g the s c r a p i n g s . The c e l l u l o s e from. Rf 0 . 0 5 to 0 . 5 0 127 was scraped o f f , powdered i n a mortar, and poured i n t o a minia-t u r e chromatography column made from 3 / l 6 " g l a s s t u b i n g . The erogens (designated H-l)- were e l u t e d with 50$ acetone. The c e l l u l o s e from Rf O .85 to O .95 was s i m i l a r l y scraped o f f , powdered, and e l u t e d (H-2). One TLC p l a t e was streaked with the H-l s o l u t i o n , and another with the H-2. The p l a t e s were developed i n e t h y l 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 ( 4 o / 4 o/lO). Ten zones, 5 mm. wide, were scraped from each p l a t e and bioassayed. The c e l l u l o s e from Rf 0 . 0 5 to O.3O on the H - l p l a t e and the c e l l u l o s e from Rf 0 . 3 5 to 0 . 7 0 on the H-2 p l a t e were scraped o f f , powdered, and e l u t e d . The H - l elua t e was a p p l i e d to the l e f t h a l f , and the H-2 e l u a t e to the r i g h t h a l f , of a TLC p l a t e , and the p l a t e was developed i n n - b u t a n o l - a c e t i c acid-water ( 6 0 / 1 5 / 2 5 ) - The erogen zones were l o c a t e d as u s u a l . The c e l l u l o s e from Rf O.3O t o 0.40 on the H - l s i d e of the p l a t e was scraped o f f , powdered, and e l u t e d . The elu a t e was streaked along the o r i g i n of another TLC p l a t e , which was developed i n n-butanol-p y r i d i n e - w a t e r ( 3 0 / 3 0 / 3 0 ) (the p y r i d i n e was r e d i s t i l l e d j u s t b e fore use). Zones 1 cm wide and 5 mm high were scraped from the area from Rf 0.4-5 to O.9O f o r assay. The c e l l u l o s e from, Rf 0 . 64 t o 0 . 7 3 was scraped o f f , powdered, and e l u t e d with 50$ acetone. The elu a t e ( 2 ml) was d i l u t e d 1/800 f o r bi o a s s a y . 25 u l of the erogens prepared by s i l i c a g e l chromatography were spotted on a c e l l u l o s e l a y e r , sprayed with a 0 . 5 $ s o l u t i o n of n i n h y d r i n i n acetone, and heated at 105°C f o r 10 minutes. A p l a t e b e a r i n g another 25 p i of erogen p r e p a r a t i o n was placed f o r 5 minutes i n a chromatography tank c o n t a i n i n g c h l o r i n e gas generated by mixing 5 ml of concentrated h y d r o c h l o r i c a c i d with 5 ml of a 10$ s o l u t i o n of potassium permanganate. A f t e r removal from, the tank, the p l a t e was heated at 105°C f o r 15 minutes to d r i v e o f f c h l o r i n e , and sprayed with a s o l u t i o n c o n t a i n i n g 0.2$ potassium i o d i d e and 0 . 1 $ s o l u b l e s t a r c h (Rydon- and/Smith I952). Two TLC p l a t e s were spotted with 50 u l of erogen p r e p a r a t i o n i n one corner, 2 cm. from, each edge. The p l a t e s were developed f i r s t i n chloroform-methanol - 0 . 1 N ammonia (4o/25/2 .5)> and then 128 at r i g h t angles i n e t h y l acetate-ethanol-0.1 K h y d r o c h l o r i c a c i d (40/4o/l5). One p l a t e was sprayed with 0.5$ n i n h y d r i n and heated f o r 15 minutes. The other p l a t e was exposed to c h l o r i n e , heated and sprayed with s t a r c h - i o d i d e s o l u t i o n . 129 R e s u l t s I. I n s t a b i l i t y The erogen samples which had been autoclaved i n 1 N potassium hydroxide and 1 N h y d r o c h l o r i c a c i d showed no conjugation hormone a c t i v i t y . The sample autoclaved i n 10$ ammonia had 0 . 0 2 3 u n i t s per ml (95$ confidence l i m i t s .002 and . 0 5 8 ) . The sample auto-claved i n 1 N KC1 had 0 .15 u n i t s / m l (95$ confidence l i m i t s .06 and . 2 5 ) - I f no erogen a c t i v i t y had been destroyed, the potency expected f o r each sample was about 1 u n i t / m l . I I . F i r s t attempt at p u r i f i c a t i o n When the hormone concentrate s o l u t i o n was a p p l i e d to the anion exchange r e s i n , some of the brown colour was washed s t r a i g h t through the column. More pigment was e l u t e d by the 1 N a c e t i c a c i d , and when the r e s i n was regenerated with potassium hydroxide, more brown colour came o f f . The weight of the mater-i a l e l u t e d from the anion exchange column was I.O92 grams. The b u t a n o l e x t r a c t a f t e r f r e e z e - d r y i n g was a f l u f f y yellow m a t e r i a l weighing 105 mg. The m a t e r i a l e l u t e d from the anion exchange r e s i n , the r e d i s s o l v e d p r e c i p i t a t e and the b u t a n o l e x t r a c t a l l showed ero-genic a c t i v i t y of about 1 u n i t / m l at the c o n c e n t r a t i o n s assayed. The r a f f i n a t e from, the b u t a n o l e x t r a c t i o n contained a t o t a l of 1250 u n i t s . The e t h y l acetate e x t r a c t had very low hormone act i v i t y . The combined b u t a n o l e x t r a c t s had a s p e c i f i c a c t i v i t y of 125 units/mg, and a t o t a l a c t i v i t y of 3 0 , 5 0 0 u n i t s . The y i e l d and p u r i f i c a t i o n at each step of the procedure are summarized i n Table XV. The f r a c t i o n of c e l l s with conjugation tubes i n the assay of each f r a c t i o n from the s i l i c a g e l column has been converted to r e l a t i v e erogen c o n c e n t r a t i o n , and p l o t t e d a gainst f r a c t i o n number i n F i g . 25-130 CD a a a ("lN/SlINTI) NDIlVaiN3DfvDD N C J I - C F i g . 25. Chromatography of f i r s t erogen p r e p a r a t i o n on s i l i c a g e l with a g r a d i e n t of water i n e t h a n o l . Bars re p r e s e n t confidence i n t e r v a l s . 131 Table XV. Progress of the erogen p u r i f i c a t i o n Stage O r i g i n a l c u l t u r e supernatant C h a r c o a l e l u a t e Anion exchange elua t e Butanol e x t r a c t T o t a l erogen S p e c i f i c P u r i f i -a c t i v i t y a c t i v i t y c a t i o n u n i t s / ml u n i t s /mg  124,000 100,000 59,000 3 0 , 5 0 0 0 . 0 3 7 5 - 0 54 125 X 1 x 135 x 1470 x 34oo Y i e l d 100 81 48 25 I I I . I n t e r a c t i o n of the erogens with ion exchange r e s i n s The .05 M KHgPO^ supernatant from the c a t i o n exchange r e s i n i n the K + form contained . 0 3 (95$ confidence l i m i t s .01 and . 0 5 ) times as much erogen a c t i v i t y as the c o n t r o l . The .05 M KHgPO^-50$ acetone supernatant had I . 3 6 (95$ confidence l i m i t s 1.0 and I . 9 ) times as much erogen a c t i v i t y as the c o n t r o l . The 50$ acetone supernatant from the H + r e s i n had no erogen a c t i v i t y . These data i n d i c a t e that a d s o r p t i o n of the erogens from 0 . 0 5 M KHgPOi,. onto the r e s i n was almost complete, but was n e g l i g i b l e from .05 M KH2P02j. c o n t a i n i n g 50$ acetone. R e p e t i t i o n of the experiment gave s i m i l a r r e s u l t s . The .1 M KgHPOij. supernatant from the D u o l i t e A - 4 anion exchange r e s i n contained no d e t e c t a b l e erogen a c t i v i t y . The .1 M K 2HP0^-50$ acetone supernatant, however, had .86 (95$ confidence l i m i t s . 6 1 and l . l ) times as much erogen a c t i v i t y as the c o n t r o l . In the second experiment, the two d i s t i l l e d water super-natants from the D u o l i t e A - 4 r e s i n had very low erogen a c t i v i t y . The 50$ acetone supernatant from the D u o l i t e A - 4 r e s i n which had been e q u i l i b r a t e d with .1 M KgHPOij. contained 0. 14 (95$ confidence l i m i t s 0.11 and 0 . 1 6 ) times as much erogen a c t i v i t y as the c o n t r o l . The 50$ acetone supernatant from the r e s i n i n the f r e e base form had 0.15 (95$ confidence l i m i t s .11 and . 1 8 ) times as much erogen a c t i v i t y as the c o n t r o l . Thus the erogens are almost completely adsorbed on the anion exchange r e s i n from 132 d i s t i l l e d w a t e r o r . 1 M KgHPO^. F r o m 50$ a c e t o n e , 85$ o f t h e e r o g e n s a r e a d s o r b e d on t h e r e s i n a t pH 7 or h i g h e r , b u t f r o m 0 . 1 M-K 2 H P 0 4 - 5 0 $ a c e t o n e , o n l y 15$ o r l e s s o f t h e e r o g e n s a r e a d s o r b e d . I V . F u r t h e r a t t e m p t s a t p u r i f i c a t i o n The s p e c i f i c a c t i v i t y o f t h e e r o g e n s p r e p a r e d . b y P o r a p a k Q a d s o r p t i o n f r o m s h a k e c u l t u r e s u p e r n a t a n t s was 48 u n i t s / m g (95$ c o n f i d e n c e l i m i t s 43 and 5 4 ) . The r a f f i n a t e f r o m t h e n - b u t a n o l e x t r a c t i o n , and t h e e t h y l a c e t a t e e x t r a c t showed l o w e r o g e n i c a c t i v i t y . The n - b u t a n o l e x t r a c t s o l u t i o n c o n t a i n e d 1375 u n i t s o f e r o g e n s . The s e c o n d b u t a n o l e x t r a c t c o n t a i n e d 2 5 , 0 0 0 u n i t s o f e r o g e n s . The d i s t r i b u t i o n o f e r o g e n i c a c t i v i t y 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 g e l c o l u m n i s shown i n F i g . 2 6 . The e r o g e n s p o o l e d f r o m f r a c t i o n s 42 t o 174 had a t o t a l a c t i v i t y o f 2 3 , 0 0 0 u n i t s , and a s p e c i f i c a c t i v i t y o f 250 u n i t s p e r mg (95$ c o n f i d e n c e l i m i t s 228 and 2 7 2 ) . The c o m b i n e d P o r a p a k e l u a t e s f r o m t h e foam f r o m 15 l i t r e s o f 2259-7 c u l t u r e i n Gluc-AmS c o n t a i n e d 1 6 2 , 0 0 0 u n i t s o f a c t i v i t y . The b u t a n o l e x t r a c t had a t o t a l a c t i v i t y o f 5 4 , 0 0 0 u n i t s , and a s p e c i f i c a c t i v i t y o f 1500 u n i t s / m g (95$ c o n f i d e n c e l i m i t s 1355 and I670). The r a f f i n a t e f r o m 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 o n l y 1260 u n i t s . The s p e c i f i c a c t i v i t y o f t h e e r o g e n s p r e p a r e d b y P o r a p a k a d s o r p t i o n f r o m c o n d e n s e d f oam was 660 u n i t s / m g (95$ c o n f i d e n c e l i m i t s 596 and 7 2 4 ) . V. T h i n l a y e r c h r o m a t o g r a p h y The d i s t r i b u t i o n o f e r o g e n a c t i v i t y on t h e TLC p l a t e s a f t e r d e v e l o p m e n t i n t h e t h r e e s o l v e n t s y s t e m s i s shown i n F i g . 27 f o r t h e hormone I p r e p a r a t i o n o f s e c t i o n I I , ' and i n F i g . 28 f o r t h e e r o g e n p r e p a r a t i o n o f s e c t i o n I V . Of t h e o r i g i n a l 4600 u n i t s o f e r o g e n a c t i v i t y , l e s s t h a n 1 0 0 u n i t s o f H - l were r e c o v e r e d a f t e r p r e p a r a t i v e TLC i n t h e f o u r s o l v e n t s y s t e m s . The a c t i v i t y o f H-2 a f t e r TLC i n n - b u t a n o l -a c e t i c a c i d - w a t e r was v e r y l o w , and a p p e a r e d a t R f 0 . 4 t o 0 . 6 . 133 INTO f^IlVc^N33vtD N o F i g . 26. Chromatography of second erogen p r e p a r a t i o n on s i l i c a g e l with a g r a d i e n t of water i n e t h a n o l . Bars represent confidence i n t e r v a l s . tn 1.Q4. g o . , M r— 0-4i ^ 0-i O.Q w 0.0 » o-al cn - o-si E 0*4i O.E H 0-0 L O U . O'B O'E 0>4 0-S O.Q 134 0.1 N ammonia 0.2 0.4 0*6 O'B 1.0 e t h y l acetate-ethanol-0.1 N h y d r o c h l o r i c a c i d (40/4o/20) o-s 0.4 0.6 O.B 1-0 c h l o r of or m-me than ol-OraioN ammonia ( 4 0 / 2 5 / 2 . 5 ) " J + + + + 0*0 0*2 0*4 0*6 O'B 1*0 RF 2 7 . Thin l a y e r chromatography of hormone I p r e p a r a t i o n . jL g o . . M 3 o.oJ 135 O.l N ammonia M tn - 0-( & o.a S O-Q o*s 0*4 0-6 0*B 1*0 e t h y l 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*0 1'Qj. 0-B O.E 0-4 0 .£ 0*Q 0-2 0*4 0-6 O.B 1.0 chloroform -methanol -1@m3lo W1 ammo n i a (40/25/2.5) ^ + + + 1 0*0 0*S 0-4 0*6 0*B 1*0 RF 28. Th i n l a y e r chromatography of second erogen p r e p a r a t i o n . 136 The erogen p r e p a r a t i o n gave a pink colour with n i n h y d r i n , and a "bluish purple colour i n the Rydon-Smith t e s t . A f t e r two dimensional TLC, n e i t h e r n i n h y d r i n nor the Rydon-Smith t e s t gave a colour r e a c t i o n i n the area where the erogen a c t i v i t y was expected to be. 137 Dis c u s s i o n The complete l o s s of erogen a c t i v i t y on a u t o c l a v i n g i n e i t h e r 1 N h y d r o c h l o r i c a c i d or 1 N potassium hydroxide i n d i -cates t h a t high temperatures and extremes of pH should be avoided i n the h a n d l i n g of the erogens. A u t o c l a v i n g i n 10$ ammonia destroyed 98$ of the a c t i v i t y and a u t o c l a v i n g at n e u t r a l pH i n 1 N KG1 destroyed 85$. The hi g h s a l t c o n c e n t r a t i o n may have in c r e a s e d the d e s t r u c t i o n . The l o s s of a c t i v i t y during auto-c l a v i n g under the c o n d i t i o n s of the b i o a s s a y has not been measured. Although the erogens are unstable at extreme pH and high temperature, the standard p r e p a r a t i o n has been kept i n the dry s t a t e at f r e e z e r temperatures f o r s e v e r a l months without n o t i c e a b l e l o s s of a c t i v i t y . In the f i r s t p u r i f i c a t i o n attempt, the s p e c i f i c a c t i v i t y was i n c r e a s e d 11 f o l d by a d s o r p t i o n on and e l u t i o n from D u o l i t e A-k anion exchange r e s i n , but the recove r y of a c t i v i t y was low. The f i v e gram p o r t i o n s of concentrate used could have exceeded the c a p a c i t y of the r e s i n , and some of the erogens may have been l o s t i n the column e f f l u e n t . The a d s o r p t i o n and e l u t i o n steps were c a r r i e d out at k°C, and the a c e t i c a c i d eluant was removed by f r e e z e - d r y i n g , t o minimize d e s t r u c t i o n of the erogens by the a c i d . However, some of the a c t i v i t y could have been l o s t be-cause of exposure to the a c i d . A l s o , the 1 N a c e t i c a c i d may not have e l u t e d a l l of the adsorbed erogens from the r e s i n . F u r t h e r p u r i f i c a t i o n of the hormone I I p r e p a r a t i o n a f t e r s i l i c a g e l chromatography by c a t i o n exchange chromatography was attempted, using ammonium acetate and p y r i d i n e acetate b u f f e r s . Under a l l the i n i t i a l c o n d i t i o n s t r i e d the erogen a c t i v i t y was only s l i g h t l y r e t a r d e d by the r e s i n , and l i t t l e s e p a r a t i o n was achieved. The e f f e c t s of pH and organic s o l v e n t s on a d s o r p t i o n of the erogens on ion exchange r e s i n s were s t u d i e d to allow more r a t i o n a l design of ion exchange p u r i f i c a t i o n schemes. The a b i l i t y of 50$ acetone to e l u t e the erogens from Porapak r e s i n s suggested t h a t t h i s s o l v e n t might e l i m i n a t e the non-polar b i n d i n g of the erogens to the p o l y s t y r e n e backbone of the ion exchange r e s i n s . 133 , Comparison of the a d s o r p t i o n of the erogens from 0.05 M KH2P0i,. and 0.05 M KR"2P0^-50$ acetone onto Amberlite IR-120, and from. 0.1 M K2HP0]^ and 0.1 M K2HP0iJ.-50$ acetone onto D u o l i t e A-4 support t h i s view. A d s o r p t i o n from aqueous s o l u t i o n can be i n t e r p r e t e d as the sum of non-polar and i o n i c b i n d i n g , and ad s o r p t i o n from s o l u t i o n s c o n t a i n i n g 50$ acetone can be i n t e r -preted as i o n i c a t t r a c t i o n alone. The complete adsorp-t i o n from 50$ acetone onto the H + form of the r e s i n i n d i c a t e s t h at the erogens can acquire a p o s i t i v e charge at s u f f i c i e n t l y -low pH. The erogen molecules may have an a c i d i c i s o e l e c t r i c p o i n t . On D u o l i t e A-4 r e s i n , 50$ acetone reduces but does not e l i m i n a t e erogen a d s o r p t i o n . Apparently the erogens are neg-a t i v e l y charged at pH 7 and above. The e q u a l i t y of a d s o r p t i o n on r e s i n at pH 7 and i n the f r e e base form suggests that the i s o e l e c t r i c p o i n t of the erogens i s w e l l below 7- The decreased a d s o r p t i o n i n 0.1 M K2HP02j.-50$ acetone could be caused by com-p e t i t i o n f o r the ion exchange s i t e s by phosphate. In a l l cases where only part of the erogen a c t i v i t y i s adsorbed, each of the a c t i v e components may be bound to a d i f f e r e n t degree. Measure-ment of the erogen d i s t r i b u t i o n between the r e s i n phase and 50$ acetone at more pH's could be used t o determine the i s o e l e c t r i c pH of the erogens, and to check t h a t the r e s u l t s obtained with anion and c a t i o n exchangers are c o n s i s t e n t . E l e c t r o p h o r e s i s could provide an independent estimate of the s i g n and magnitude of the charge on the erogens at va r i o u s pH's. E x t r a c t i o n of the erogens from aqueous s o l u t i o n s with n-butanol i n c r e a s e s the s p e c i f i c a c t i v i t y , but gives low recove r y . The m i s s i n g a c t i v i t y i s not l e f t i n the r a f f i n a t e . P o s s i b l y t h i s a c t i v i t y i s destroyed during evaporation of the but a n o l , or i s h e l d i n the part of the re s i d u e which cannot be r e - d i s -s olved a f t e r e v a poration of the b u t a n o l . An abb r e v i a t e d form of the b i o a s s a y has been used f o r the l a r g e number of samples a r i s i n g from chromatographic s e p a r a t i o n . Only one dose of each sample i s assayed, and the f r a c t i o n of c e l l s producing conjugation tubes i s converted to erogen concen-' 1 3 9 t r a t i o n u sing values f o r the r e g r e s s i o n l i n e slope and standard ED50 from a standard s e r i e s assayed simul t a n e o u s l y . T h i s method i s l e s s accurate than the f u l l b i o a s s a y procedure, but allows the assay of higher numbers of samples. Chromatography on s i l i c a g e l as d e s c r i b e d i n s e c t i o n II c l e a r l y separated two peaks of a c t i v i t y , with a suggestion of a t h i r d peak at the end of the chromatogram. The m a t e r i a l i n the f i r s t peak was unretarded by the column, and emerged with the void volume. There may be a small peak centered on f r a c t i o n 3 3 -The main peak appears to co n t a i n four sub-peaks. However, the response i n the assays of a l l these f r a c t i o n s was near maximum, and the e r r o r s of e s t i m a t i o n are l a r g e . When the hormone p r e p a r a t i o n of s e c t i o n IV was chromato-graphed on s i l i c a g e l , no hormone I peak appeared. A l l of the erogen a c t i v i t y was found i n one broad peak, at approximately the e l u t i o n volume expected f o r hormone I I . I t i s p o s s i b l e that hormone I i s an a r t i f a c t , caused by some chemical change duri n g the p r e p a r a t i o n procedures d e s c r i b e d i n s e c t i o n I I . The r e l a t i o n between hormone I and the erogen peak from the s i l i c a g e l chromatography of s e c t i o n IV was i n v e s t i g a t e d by TLC. A f t e r development with 0 . 1 ET ammonia, both erogen p r e p a r a t i o n s were separated i n t o a component with Rf about 0 . 1 , and a second component with Rf 0 -75 to 1.0. On TLC i n e t h y l 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 / 4 o/20), two components, with approx-imate Rf's 0 . 7 5 a n d O .95 were i n d i c a t e d i n each p r e p a r a t i o n . TLC i n c h l o r of orm-met hanol-0. 1 EF ammonia (40/25/2.5) demonstrated a c t i v e components with Rf's 0 - 3 and O . 9 5 , and p o s s i b l y a t h i r d with Rf 0 . 0 5 , i n both p r e p a r a t i o n s . The hormone I p r e p a r a t i o n seems to c o n t a i n more of the l e s s p o l a r component, and the other erogen p r e p a r a t i o n has more of the more p o l a r component. When the more p o l a r (H-l) and l e s s p o l a r (H-2) components of the second erogen p r e p a r a t i o n were separated by p r e p a r a t i v e TLC, they showed d i s t i n c t Rf's i n the sol v e n t systems e t h y l 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 / 4 o/lO) and n-butanol-a c e t i c acid-water ( 6 0 / 1 5 / 2 5 ) - There was no s e p a r a t i o n of e i t h e r 140 H-l or H-2 i n t o more components during chromatography i n these s o l v e n t s . The y i e l d of erogen a c t i v i t y a f t e r s u c c e s s i v e chroma-tography i n four s o l v e n t systems was very low. The erogens may have "been destroyed dur i n g chromatography, or they may have "been i n c o m p l e t e l y e l u t e d from the powdered c e l l u l o s e . The colour r e a c t i o n s with n i n h y d r i n and the Rydon-Smith t e s t i n d i c a t e t h a t the erogen p r e p a r a t i o n a f t e r s i l i c a g e l chromatography contains amino acids and/or p e p t i d e s . The erogens were separated from the r e a c t i n g m a t e r i a l s "by two dimensional chromatography. The l a c k of d e t e c t a b l e colour formation i n the area where the erogens were expected to be could have been caused by i n s u f f i c i e n t m a t e r i a l . A d s o r p t i o n on Porapak Q, i s a r a p i d and e f f e c t i v e method f o r primary p u r i f i c a t i o n of the erogens from crude c u l t u r e supernatants. The erogens can be e l u t e d i n high y i e l d under gentle c o n d i t i o n s . Chromatography of the erogens on columns of Porapak, using an a p p r o p r i a t e c o n c e n t r a t i o n of acetone i n water as s o l v e n t , might provide good p u r i f i c a t i o n . The p u r i f i c a t i o n of the erogens achieved by foaming can be seen from comparison of the s p e c i f i c a c t i v i t y of erogens prepared by Porapak a d s o r p t i o n from c u l t u r e supernatants (48 units/mg) and from condensed foam (660 units/mg). With present i n f o r m a t i o n , the best p r e l i m i n a r y p u r i f i c a t i o n procedure f o r the erogens appears to i n c l u d e c o n c e n t r a t i o n of the erogens i n the foam from the c u l t u r e , c e n t r i f u g i n g the condensed foam to remove c e l l s , p assing the supernatant through Porapak Q, e l u t i n g the erogens with 50$ acetone, d i s t i l l i n g o f f the acetone, and e x t r a c t i n g the aqueous s o l u t i o n with n-butanol. SUMMARY AND GENERAL DISCUSSION Ikl T r e m e l l a mesenterica 2259-7 c a n u s e ammonium but not n i t r a t e as the so l e n i t r o g e n source f o r growth. A high c o n c e n t r a t i o n of microelements s e l e c t i v e l y slows growth i n media c o n t a i n i n g amino a c i d s . L-asparagine, alone or i n combination with other amino a c i d s , causes slow and abnormal growth of the c e l l s . Thiamine i s the only v i t a m i n f o r which t h i s fungus shows a requirement. Sodium acetate counteracts the pH drop i n c u l t u r e s using ammonium as n i t r o g e n source. A d e f i n e d medium c o n t a i n i n g glucose, ammonium, sulphate, thiamine, s a l t s and microelements supports good growth of T. mesenterica 2 2 5 9 - 7 -The erogens produced by 2 2 5 9 - 7 can be detected by t h e i r a b i l i t y to induce the growth of conjugation tubes from the c e l l s of s t r a i n 2 2 5 9 - 6 . The erogens have a p a r t i t i o n c o e f f i c i e n t of 2-3 from aqueous s o l u t i o n i n t o n-butanol, but are not e x t r a c t e d i n d e t e c t a b l e amounts by l e s s p o l a r organic s o l v e n t s . The erogens are s t r o n g l y adsorbed 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 p y r i d i n e acetate s o l u t i o n f o r d e s o r p t i o n , and on the po l y s t y r e n e r e s i n Porapak, from which they can be e l u t e d with 50$ acetone. The erogens are a l s o adsorbed on both c a t i o n and anion exchange r e s i n s . Part of the b i n d i n g i s due to non-polar f o r c e s , and part to i o n i c a t t r a c t i o n . The i s o e l e c t r i c pH of the erogens appears to be below k.5. The erogens are a l s o adsorbed on Sephadex G -10, so t h a t t h e i r molecular weight cannot be estimated by g e l chroma-tography. However, u l t r a f i l t r a t i o n suggests a molecular weight below 7 5 0 . Chromatography on columns of s i l i c a g e l with a gra d i e n t of water i n eth a n o l separated three a c t i v e 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 gave only one a c t i v e peak. Th i n l a y e r chromatography demonstrated at l e a s t two a c t i v e components i n each erogen p r e p a r a t i o n . The erogens may be amino acids or short peptides with non-polar side chains. The c o n c e n t r a t i o n of erogens has been shown to a f f e c t the f r a c t i o n of 2259-6 c e l l s which produce conjugation tubes, the le n g t h of the conjugation tubes, and the number of conjugation 142" tubes per c e l l . A s t a t i s t i c a l l y v a l i d and p r a c t i c a l q u a n t i t a t i v e b i o a s s a y f o r the erogens has been developed, u s i n g the f r a c t i o n of c e l l s which produce conjugation tubes as the response. The u n i t of erogen a c t i v i t y i s d e f i n e d as the a c t i v i t y of 0.2 mg of a standard p r e p a r a t i o n . Optimum c o n d i t i o n s of i n c u b a t i o n time, temperature, pH and c e l l d e n s i t y f o r the b i o a s s a y have been e s t a b l i s h e d . Complex n i t r o g e n sources such as soytone are best f o r c o n j u g a t i o n tube p r o d u c t i o n , and ammonium i s poor. Maximum, erogen a c t i v i t y i n the supernatant of 2259-7 c u l t u r e s i s reached at the same time as peak t u r b i d i t y . Ammonium i s the best n i t r o g e n source t e s t e d f o r erogen p r o d u c t i o n . The concen-t r a t i o n of v a r i o u s components of the d e f i n e d medium have been adjusted f o r h i g h e s t erogen p r o d u c t i o n . The erogens are removed from 2259-7 c u l t u r e s by the foam which forms spontaneously when a i r i s bubbled through the c u l t u r e s . Foaming i s a u s e f u l method f o r p r e l i m i n a r y c o n c e n t r a t i o n and p u r i f i c a t i o n of the erogens i n l a r g e s c a l e p r o d u c t i o n . Attempts to p u r i f y the erogens have given low y i e l d s of the hormone a c t i v i t y . Future r e s e a r c h w i l l need to develop p u r i f i -c a t i o n methods with high r e c o v e r i e s of the erogens. Then using the methods f o r erogen p r o d u c t i o n developed i n t h i s t h e s i s , s u f f i c i e n t m a t e r i a l can be p u r i f i e d f o r chemical c h a r a c t e r i z a t i o n . When p u r i f i e d erogens become a v a i l a b l e , s t u d i e s on the mechanism by which they r e d i r e c t the growth of the c e l l s from budding to c o n j u g a t i o n tube p r o d u c t i o n can begin. The number of d i s t i n c t erogens produced by T_. mesenterica 2259-7 i s s t i l l u n c l e a r . Chromatography on s i l i c a g e l suggests at l e a s t t h r e e , although some of these may be a r t i f a c t s of the p r e p a r a t i v e process. I n d i v i d u a l peaks of erogen a c t i v i t y from the s i l i c a g e l column are separated i n t o at l e a s t two a c t i v e components by t h i n l a y e r chromatography. The components from, d i f f e r e n t peaks show s i m i l a r Rf v a l u e s . M o n i t o r i n g the s e p a r a t i o n on a s i l i c a g e l column by TLC may c l a r i f y the r e l a t i o n between the peaks of erogen a c t i v i t y and the components r e s o l v e d by TLC. P a r a l l e l s t u d i e s on the conjugation hormones from the other mating type of T. mesenterica, and from other species of T r e m e l l a 143 w o u l d b e o f I n t e r e s t f o r t h e s i m i l a r i t i e s t o a n d d i f f e r e n c e s f r o m t h e e r o g e n s o f 2259-7 t h a t t h e y m i g h t s h o w . 1 BIBLIOGRAPHY 144 Abraham, E. P., and G. G. E. Newton. I967. P e n i c i l l i n s and cep 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, Ed. 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. Hyphal f u s i o n s i n the wood-rotting fungus Schizophyllum commune. Genet. Res. Camb. 15_: I9 - 2 8 . Aiba, S., A. E. Humphrey, and N. F. M i l l i s . 1965 . B i o c h e m i c a l E n g i n e e r i n g . Academic Press, New York. 333 pp. A r i e n s , E. J . , A. M. Simonis, and J. M. van Rossum. I964 . The mode of 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 Molecular Pharmacology, E. J . A r i e n s , Ed. Academic Press, New York. 503 PP-A r s e n a u l t , G. P., K. Biemann, A. W. Barksdale, and T. C. McMorris. I968. The s t r u c t u r e of a n t h e r i d i o l , a sex hormone i n Achlya b i s e x u a l i s . J . Am. Chem. Soc. _9_0: 5 6 3 5 - 5 6 3 6 . Asatoor, A., and C. E. D a l g l i e s h . 1956. The use of d e a c t i v a t e d cha r c o a l s f o r the i s o l a t i o n of aromatic substances. J . Chem. Soc. I 9 5 6 : 2 2 9 1 - 2 2 9 9 -A u s t i n , D. J . , J . D. Bu'Lock, and G. ¥. Gooday. 1969. T r i s p o r i c a c i d s : Sexual hormones from Mucor mu cedo and B l a k e s l e a  t r i s p o r a . Nature 2 2 3 : II78-II79. A u s t i n , D. J . , J . D. Bu'Lock, and D. Drake. 1970. The b i o s y n -t h e s i s of t r i s p o r i c a c i d s f rom/3-carotene v i a r e t i n a l and t r i s p o r o l . E x p e r i e n t i a 15.:. 3 4 8 - 3 4 9 . Banbury, G. H. 1954. Processes c o n t r o l l i n g zygophore formation and zygotropism i n Mucor mu cedo. Nature 173'• ^99 -Banbury, G. H. 1955- P h y s i o l o g i c a l s t u d i e s i n the Mucorales. I I I . The zygotropism of zygophores of Mucor mucedo. J. E x p t l . Botany 6_: 2 3 5 - 2 4 4 . Bandoni, R. J . 1963- Conjugation i n T r e m e l l a mesenterica• Can. J . Bot . 4-1: 4 6 7 - 4 7 4 . Bandoni, R. J . 1965- Secondary c o n t r o l of conjugation i n Tr e m e l l a mesenterica. Can. J . Bot. _43: 6 2 7 - 6 3 0 . Barkley, D. S. 1969. Adenosine - 3 ' > 5'-phosphate: i d e n t i f i c a t i o n as a c r a s i n i n a species of c e l l u l a r slime mold. Science I65 : 1133-1134. Barksdale, A. W. 1963a- The uptake of exogenous hormone A by c e r t a i n s t r a i n s of Achlya. Mycologia 55_: 164-171-145 Barksdale, A. W. 1 9 6 3 b - The r o l e of hormone A during sexual c o n j u g a t i o n i n A c h l y a ambisexualis • Mycologia _5_5_: 6 2 7 - 6 3 2 . . Barksdale, A. W. I969. Sexual hormones of Achlya and other f u n g i . Science 66_: 83I-837 . Barksdale, A. W. 1970- N 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 branching i n Achlya ambisexualis . Mycologia _6_2: 411-420. Bauch, R. 1925- Untersuchungen ttber die 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 bromivora und U s t i l a g o  grandis . Z. Botan. 17_: 129-177. Bhalerao, U. T., J . J . P l a t t n e r , and H. Rapoport. 1 9 7 0 - Syn-t 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 : 3 4 2 9 - 3 4 3 3 -Bishop, H. 1 9 4 0 . A study of s e x u a l i t y i n Sapromyces r e i n s c h i i . Mycologia _3_2: 5 0 5 - 5 2 9 -B i s t i s , G. N. I956. S e x u a l i t y i n Ascobolus s t e r c o r a r i u s I. Morphology of the ascogonium; plasmogamy; evidence f o r a sexual hormone mechanism. Am. J . Bot. _4_3_: 3 8 9 - 3 9 4 . B i s t i s , G. N. 1 9 5 7 - S 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 experiments on v a r i o u s aspects of the sexual process. Am. J . Bot. 4-4: 4 3 6 - 4 4 3 -B i s t i s , G. N., and J . R. Raper. 1 9 6 3 - H e t e r o t h a l l i s m and s e x u a l i t y i n As cobolus s t e r c o r a r i u s . Am. J . Bot. _5_0: 88O-89I. Brough, S. G. 1 9 7 0 - The B i o l o g y of T r e m e l l a bambusina Sacc. 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 Columbia. B u l l e r , A. H. R. 1933- A 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 hyphal 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 Researches on Fungi V o l . 5: 6 8 - 7 2 . Bu'Lock, J . D., D. Drake, and D. J . Winstanley. 1 9 7 2 . Spec-i f i c i t y and t r a n s f o r m a t i o n s of the 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 1 1 : 2 0 1 1 - 2 0 1 8 . Burgeff, H. 1924. Untersuchungen Uber S e x u a l i t a t und Para-s i t i s m u s b e i Mucorineen I. Botan. Abhandl.. _4: 5-135-( c i t e d i n M a c h l i s , I 9 6 6 ) . Burnett, J . H. I 9 6 8 . Fundamentals of Mycology. Edward Arn o l d ( P u b l i s h e r s ) L t d . , London. 546 pp. C a g l i o t i , L., G. C a i n e l l i , B. Camerino, 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 . 1 9 6 7 - The 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 ' acido t r i s p o r l c o B. Chim. Ind. (Milan) 49: 628-629. C a r l i l e , M. J . , and L. Maehlis. 1965 . The response of male gametes of Allomyces t o the sexual hormone s i r e n i n . Am. J . Bot. _5_2~ 4 7 8 - 4 8 3 . Corey, E. J . , K. Achiwa, and J . A. Katzenellenbogen. 1 9 6 9 . 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. Soc. 91: 4 3 I 8 - 4 3 2 O . Corey, E. J . , and K. Achiwa. I97O. A simple s y n t h e t i c route to d l - s i r e n i n . Tetrahedron L e t t . 2_6: 2245-2246. D r i v e r , C. H., and H. E. Wheeler. 1 9 5 5 - A se x u a l hormone i n Gl o m e r e l l a . Mycologia hj_: 3II - 3 1 6 . Duntze, 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 . Science 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 . I969. The .synthesis of the f u n g a l sex hormone a n t h e r i d i o l . J . Am. Chem. Soc. £ l : 1248 - 1 2 4 9 . Edwards, J . A.,-V. Schwarz, J . Fajkos, M. L. Maddox, and J . H. F r i e d . 1 9 7 1 - Fungal sex hormones. The s y n t h e s i s of ( i ) -7 ( t ) , 9 ( t ) - t r i s p o r i c a c i d B methyl e s t e r . The stereochem-i s t r y at C-9 of the 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. F r i e d . 1 9 7 2 . A new s y n t h e t i c route to the f u n g a l sex hormone a n t h e r i d i o l , and the deter m i n a t i o n of i t s absolute s t e r e o c h e m i s t r y . Tetrahedron L e t t . 1 9 7 2 : 7 9 1 - 7 9 4 . Ende, H. van den. 1 9 6 7 . Sexual f a c t o r of the Mucorales. Nature 215 : 2 1 1 - 2 1 2 . Ende, H. van den. I968. R e l a t i o n s h i p between s e x u a l i t y and carotene 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 . J . Bact. 96: 1298-1303. 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. van den, and D. Stegwee. 1 9 7 1 - P h y s i o l o g y of sex i n Mucorales. Bot. Rev. _3J_: 22-36. Ende, H. van den, B. A. Werkman, and M. L. van 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 of the fungus B l a k e s l e a t r i s p o r a . Arch. M i k r o b i o l . 86: 1 7 5 - 1 8 4 . ihi F e o f i l o v a , E. P. 1970. C o n d i t i o n s of formation 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 plus 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 3 9 : 2 6 6 - 2 7 0 . Finney, D. J . 1964. S t a t i s t i c a l Method i n B i o l o g i c a l Assay. Second e d i t i o n . C l a r k s G r i f f i n & Company L t d . , London. 668 pp. Finney, D. J . 1971- P r o b i t A n a l y s i s . T h i r d e d i t i o n . Cambridge U n i v e r s i t y Press. 333 PP-F l e g e l , T. W. I968. Some aspects of conjugation i n the 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 sexual 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. ¥. Barksdale, and T. C. McMorris. 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 the s y n t h e s i s of i t s C-22 epimer. Tetrahedron 2 7 : H 9 9 -1203-G r i e c o , P. A. I969. 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 . J . Am. Chem. Soc. 91: 566O-566I. Kehl, H. .1937- E i n B e i t r a g zur Morphologie und P h y s i o l o g i e der Zygophoren 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. 1967- Die chemischen Grundlagen der Befruchtung (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, ¥. Ruhland, Ed. S p r i n g e r - V e r l a g , B e r l i n . pp 2 8 2 - 3 2 0 . Krafczyk, H. I93I. Die Zygosporen 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 . Deut. Botan. Bes . 4_9: 141-146 . ( c i t e d i n M a c h l i s , I966). Krafczyk, H. 1935- Die B i l d u n g und Keimung der Zygosporen 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 Myzel. 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) Kubota, T., T. Tokoroyama, Y. Tsukuda, H. Koyama, and A. Miyake. 1973- I 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 conjugation i n i t i a t i n g gamone i n the c i l i a t e Blepharisma. Science 179: 400 - 4 0 2 . Lemlich, 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 Progress 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. Perry, Ed. 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 John Wiley & Sons), New York. pp I - 5 6 . 148 L e v i , J . D. 1956. Mating r e a c t i o n i n yeast. Nature 1 7 7 ' 7 5 3 -7 5 4 . McGahen, J . W., and H. E. Wheeler. 1951- Genetics of G l o m e r e l l a IX. P e r i t h e c i a l development and plasmogamy. Am. J . Bot. 3 8 : 6 1 0 - 6 1 7 . McMorris, T. C., and A. W. Barksdale. I967. I s o l a t i o n of a sex hormone from the water mold Achlya b i s e x u a l i s . Nature 2 1 5 : 3 2 0 - 3 2 1 . McMorris, T. C., and R. S e s h a d r i . 1971 . S y n t h e t i c s t u d i e s on a n t h e r i d i o l . Chem. Comm. I971: 1646. McMorris, T. C , T. Arunachalam, 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 L e t t . 26 : 2 6 7 3 - 2 6 7 6 . M a c h l i s , L. 1958a- Evidence f o r a sexual hormone i n Allomyces. P h y s i o l . P l a n t arum 11.: I8I-I92 . M a c h l i s , L. 1958b. A procedure f o r p u r i f i c a t i o n of s i r e n i n . Nature l 8 l : 1 7 9 0 - 1 7 9 1 . M a c h l i s , L. 1 9 5 8 c . A study of s i r e n i n , the chemotactic sexual hormone from, the watermold Allomyces • P h y s i o l . Plantarum 1 1 : 845 - 8 5 4 . M a c h l i s , L. I966. Sex hormones i n f u n g i , i n The Fungi V o l . I I , G. C. Ainsworth and A. S. Sussman, Ed. Academic Press, New York. pp 4 1 5 - 4 3 3 . M a c h l i s , L. 1969. Zoospore chemotaxis i n the watermold Allomyces. P h y s i o l . Plantarum 22: I26-I39. Ma c h l i s , L. 1972. The coming of age of sex hormones i n p l a n t s . Mycologia 6_4: 2 3 5 - 2 4 7 . M a c h l i s , L., W. H. N u t t i n g , M. W. W i l l i a m s , and H. Rapoport. I966. Production, 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 s i r e n i n . B i o c h e m i s t r y 5: 2 1 4 7 - 2 1 5 2 . M a c h l i s , L., and E. Rawitscher-Kunkel. 1967. Mechanisms of gametic approach 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. B. Metz and A. Monroy, Ed. Academic Press, New York. pp 117-1 6 1 . 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 chemical 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. Ribbons, Ed. Academic Press, New York, pp 5 2 1 - 5 6 6 . 149 Markert, C. L. I949. S e x u a l i t y i n the fungus, G l o m e r e l l a . Am. N a t u r a l i s t _8_3: 227-231. Mori, K., and M. Matsui. I969. S y n t h e s i s of racemic s i r e n i n , a pl a n t sex hormone. Tetrahedron L e t t . 51: 44-35-4438. MUller, D. G., L. Jaenicke, M. Donike, 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 . Science 17_1: 8 l 5 - 8 l 6 . N a i r , 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 of 3**5 ' - c y c l i c n u c l e o t i d e phosphodiesterase from dog he a r t . Biochemistry 5_: 150-157-N i c h o l a s , D. J . D. 1965- U 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 Fungi V o l . I, G. C. Ainsworth and A. S. Sussman, Ed. Academic Press, New York. pp 349-376. Niederwieser, A. 1971- Use of 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 non-polar amino acids and non-polar o l i g o p e p t i d e s . J . Chromatog. _6l: 81-94. Niederwieser, A., and P. G i l i b e r t i . I97I. Simple e x t r a c t i o n of i n d o l e d e r i v a t i v e s from aqueous s o l u t i o n by a d s o r p t i o n on n e u t r a l p o l y s t y r e n e r e s i n . J . Chromatog. 6 l : 95-99-N u t t i n g , W. H., H. Rapoport, 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. Soc. _9_0: 6434-6438. P l a t t n e r , J . J . , U. T. Bhalerao, and H. Rapoport. I969. Synthesis of d l - s i r e n i n . J . Am. Chem. Soc. _9_1: 4933-4934. P l a t t n e r , J . J . , and H. Rapoport. 1971- The s y n t h e s i s of d-and 1 - s i r e n i n and t h e i r absolute c o n f i g u r a t i o n s . J . Am. Chem. Soc. _9_1: 1758-1761. Plempel, M. 1957- Die S e x u a l s t o f f e der Mucoraceae. Arch. M i k r o b i o l . 2_6: 151-174. Plempel, M. i960. Die zygotr opis che Reaktion b e i Mucor in eenrz. I. M i t t e i l u n g . P l a n t a 5_5_: 254-258. Plempel, M. I962. Die zyg o t r o p i s c h e Reaktion b e i Mucorineen I I I . P l a n t a 58.: 5O9-52O. Plempel, M. 1963- Die chemischen Grundlagen der S e x u a l r e a k t i o n b e i Zygomyceten. P l a n t a _59_: 492-508. Plempel, M., and W. Dawid. 1961. Die zygot r o p i s c h e Reaktion b e i Mucorineen 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 . 1970. I n f l u e n c e of g i b b e r e l l i c a c i d on the i n c o r p o r a t i o n of 8--^C adenosine i n t o adenosine 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. Biophys. A c t a 201: 511 -512 . Raper, J . R. 1939- Sexual hormones i n Achlya• I. I n d i c a t i v e evidence f o r a hormonal c o - o r d i n a t i n g mechanism.. Am.. J . Bot. 2_6: 6 3 9 - 6 5 0 . Raper, J . R. I9U0. Sexual hormones i n Achlya. I I . D i s t a n c e 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 . Bot. _2J_: 162-I73. Raper, J . R. 1942a. Sexual hormones i n Ac h l y a . I I I . Hormone A and the i n i t i a l male r e a c t i o n . Am. J . Bot. _29: I59-I66. Raper, J . R. 1942b. Sexual hormones i n Ac 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 of hormone A. Proc. Wat. Acad. S c i . _2§_: 5 0 9 - 5 1 6 . Raper, J . R. 1 9 5 0 a . Sexual hormones i n Achlya. VI. The hormones of the A-complex. Proc. Nat. Acad. S c i . _3_6: 524 -533-Raper, J . R. 1950b. Sexual hormones i n Achlya• V I I . The hormon-a l mechanism i n h o m o t h a l l i c s p e c i e s . Bot. Gaz . 112: 1 - 2 4 . Raper, J . R. 1952. Chemical r e g u l a t i o n of sexual processes i n the T h a l l o p h y t e s . Bot. Rev. _ l 8 : 4 4 7 - 5 4 5 -Raper, J . R. 1967- The r o l e of s p e c i f i c s e c r e t i o n s i n the i n d u c t i o n and development of sexual organs and i n the det e r m i n a t i o n of sexual a f f i n i t y , 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 , W. Ruhland, Ed. S p r i n g e r - V e r l a g , B e r l i n . pp 2 1 4 - 2 3 4 . Raper, J . R., and A. J . Haagen-Smit. 1942. Sexual hormones i n Achlya. IV. P r o p e r t i e s of hormone A of A. b i s e x u a l i s . J . B i o l . Chem. JL4_3: 311-320. Reschke, T. 1969. Die Gamone aus B l a k e s l e a t r i s p o r a . T e t r a -hedron L e t t . 39_: 3 4 3 5 - 3 4 3 9 -Robison, G. A., R. W. Butcher, and E. W. Sut h e r l a n d . 1971 . C y c l i c AMP. Academic Press, New York. 532 pp. Ronsdorf, L. 1931- liber d i e chemischen Bedingungen von Wachstum und Zygotenbi-ldung b e i Phycomyces blakesleeanus . P l a n t a 14: 4 8 2 - 5 1 4 . ( c i t e d i n Ma c h l i s , I 9 6 6 ) . R o t h s t e i n , A. 1965- Uptake and t r a n s l o c a t i o n . I. Uptake, i n The Fungi V o l . I, G. C. Ainsworth and A. S. Sussman, Ed. Academic Press, New York. pp 4 2 9 - 4 5 6 . Rydon and Smith. 1952- see p. 153-1 5 1 Sakai, K., and N. Yanagishima. 1 9 7 2. Mating r e a c t i o n i n Sac-charomyces c e r e v i s i a e . I I . Hormonal r e g u l a t i o n of agglu-t i n a b i l i t y of a type c e l l s . Arch. M i k r o b i o l . 8 4 : 191-I98. Salomon, D., and J . P. Mascarenhas. 1 9 7 1 - Auxin-induced s y n t h e s i s of c y c l i c 3S5 '-adenosine monophosphate i n Avena c o l e o p t i l e s . L i f e Sciences _10: 8 7 9 - 8 8 5 . Schroeder, W. A., R. T. Jones, J . Cormick, and K. M c C a l l a . I962. Chromatographic s e p a r a t i o n of peptides on ion exchange r e s i n s . S e p a r a t i o n of peptides from enzymatic h y d r o l y s a t e s of the , p> , and X chains of human hemoglobins. An a l . Chem. _3_4: 1 5 7 0 - 1 5 7 5 -Sherwood, W. A. 1 9 6 6. Evidence f o r a sexual hormone i n the water mold Dictyuchus . Mycologia . 5_8 : 2 1 5 - 2 2 0 . Shimoda, C., and N. Yanagishima. 1 9 7 2 . 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 a c t i v i t y . Arch. M i k r o b i o l . _8_5_: 3IO -318 . Smith, I. 1 9 5 8 . Chromatographic Techniques. W i l l i a m Heinemann M e d i c a l Books, L t d . , London. 3 0 9 pp. S t e e l , R. G. D., and J . H. T o r r i e . i 9 6 0 . P r i n c i p l e s and Prodedures of S t a t i s t i c s . McGraw-Hill Book Company, Ltd., New York. 4 8 l pp. S u t t e r , R. P. 1 9 7 0. 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 . Science l 6 8 : I59O-I592. S u t t e r , R. P., and M. E. R a f e l s o n , J r . I 9 6 8 . S e p a r a t i o n of beta 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 . B a c t e r i o l . 9 5 : 4 2 6 - 4 3 2 . S u t t e r , R. P., D. Capage, and T. L. H a r r i s o n . 1971- Aspects 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 . A b s t r . F i r s t I n t e r n . M y c o l o g i c a l Congress, p. 92~ ( c i t e d i n Werkman and van den Ende, 1 9 7 3 ) -Thomas, D. des S., and J . T. M u l l i n s . 1 9 6 7 - Role of enzymatic w a l l s o f t e n i n g i n p l a n t morphogenesis: Hormonal i n d u c t i o n i n A c h l y a . Science 1 5 6 : 84-85-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 extension i n the water mold Achlya ambisexualis. P h y s i o l . . P l a n t . 2 2 : 347-353-152 Thomas, D. M., and T. ¥. Goodwin. 1967- S t u d i e s on caroteno-genesis i n B l a k e s l e a 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.: 3 5 5 - 3 6 0 . Thomas, D. M., R. C. H a r r i s , J . T. 0 . K i r k , and T. W. Goodwin. 1967- S t u d i e s on carotenogenesis i n B l a k e s l e a t r i s p o r a . I I . The mode of a c t i o n of t r i s p o r i c a c i d . Phytochemistry 6_: 3 6 1 - 3 6 6 . Ueyama, A. 1972 . Chemical b i o l o g y of sexual f a c t o r s i n the f u n g i . I. Induction of the gamete i n i t i a l s by the 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 glauca Hagem. Trans. Mycol. Soc. Jap. 13_: 6 6 - 7 0 . Verkaik, C. 1930- Uber das Entstehen von Zygophoren von Mucor  mu cedo ( + ) unter B e e i n f l u s s u n g eines 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 i n A chlya ambisexualis. Am. J . Bot. jj6_: 1135-1142. Warren, C. 0 . , and B. H. S e l l s . 1971- C e l l u l a s e i n d u c t i o n during 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 Achlya. J . Gen. M i c r o b i o l . 6j_: 3 6 7 - 3 6 9 . Werkman, T. A., and H. van den Ende. 1973- 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 . I n t e r a c t i o n between plus and minus mating types. Arch. M i k r o b i o l . ( i n p r e s s ) . Wolf, F. J . I969. S e p a r a t i o n Methods i n Organic Chemistry and Bioc h e m i s t r y . Academic Press, New York. 237 PP• Wood, H., M. L i n , and A. Braun. 1972. The i n h i b i t i o n of p l a n t and animal adenosine 3 ' > 5 ' - c y c l i c monophosphate phosphodi-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_: 4 0 3 - 4 0 6 . Yanagishima, N. I969. Sexual hormones i n yeas t . P l a n t a 8 7 : 110-118 . Yanagishima, N. 1971- Induced p r o d u c t i o n of a sexual hormone i n yeast. P h y s i o l . P l a n t . 2_4: 26O-263. Yanagishima, N., C. Shimoda, K. Sakai, and N. Takao. I 9 6 9 . 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 Int. Bot. Congr. A b s t r a c t p. 245-Yanagishima, N., and C. Shimoda. 1970- S e p a r a t i o n of hormone-s e n s i t i v e yeast 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 : 9 7 1 - 9 7 4 . 153 Yanagishima, N. , and C. Shimoda. 1973- Auxin and yeast. Bot Rev. _39: 1-14. Rydon, H. N., and P. ¥. G. Smith. 1952. A new method f o r the t e c t i o n of peptides and s i m i l a r compounds on paper chroma tograms. Nature 169 : 9 2 2 - 9 2 3 . i APPENDICES 15 4 Appendix A. F r e q u e n t l y used media. Malt Yeast Peptone (MYP). Bacto Malt E x t r a c t 7 . 0 gm Bacto Yeast E x t r a c t 0 . 5 gm, Bacto Soytone 1 .0 gm D i s t i l l e d water 1 l i t r e Glu'cps-eeS:oyib:one Medium (GS) D-Glucose 4.0 gm. Bacto Soytone 4.0 gm D i s t i l l e d water 1 l i t r e Growth Medium # 3 (GM3); D-Glucose Bacto Soytone KH 2P0^ MgS0 v7H 20 CaCl 2-2H 20 4.0 gm 4.0 gm 1.0 gm 0-5 gm 0 .1 gm. Microelement Stock S o l u t i o n C i t r i c a c id-H 20 5 -0 gm. ZnSOj^HgO 5-0 gm FeS0^-7H 20 0 . 7 1 gm. CuS0^-5H 20 0 . 2 5 gm MnS0^-H20 0 . 0 5 gm H^BO^ 0 . 0 5 gm Ra 2Mo0^-2H 20 0 . 0 5 gm D i s t i l l e d water 95 ml st o r e d i n r e f r i g e r a t o r Conjugation Medium (CjM) D-Glucose Bacto Soytone D i s t i l l e d water 2.0 gm 2.0 gm. 1 l i t r e 155 Glucose-Ammonium Sulphate Medium (Gluc-AmS). D-Glucose 10.0 gm. (NH4) 2S0^ 1.0 gm KH2POi4. 1.0 gm MgS0j^-7H20 0.5 gm CaCl 2-2H 20 0.1 gm Thiamine-HC1 100 ugm Microelement stock s o l u t i o n .13 ml D i s t i l l e d water 1 l i t r e 156 Appendix B.. Measurements of c o n j u g a t i o n tube l e n g t h at v a r i o u s c o n c e n t r a t i o n s of erogen. Doses are expressed i n micrograms of hormone p r e p a r a t i o n per ml. Lengths are measured i n microns. __2.22.Q_e.0_ U J . Q A P : MSJLQ l l o 14a 8« 21 o 12e l l o X I * 13e 18e 17a 10* 26a 14a 5© 18e 2e 9© 2o 19e - 20. 13e 9e l l o 18-* 4e 4e 4e 6« 19* 18 a 6 a l i e 4 a 16e 11« 3 a 12J> LQ_ _ 1 J L J 19a 13. 13. 14a 6_s 7_o_ 22« 15o 7e 5e 14a 3e 14a 3e 1 1 * 18e 18e 18e 4« l l o 4a 4e 3e 9o 15-« 1 5 * — 4 © — 6 0 — ~4e -1-2 e 1-L* 8-e~ 12* 16e 8e 14e l i e l i e l i e l i t 6e 12e 16e 7e 4a 16a 6e 13& 4e 7o 6-e 14-e 8..e 9-e 3<s -13.e _9.* 5_e __6JL_ l i d 14e 13e 18o 5e 5o 21* 2* 9e 5o 9* 17o l l o 4 e 12e 4s l i e 5e „ l - l - i ! X* , 7.e . . .6.0- _ 1.5.e 2.a 2J» it.«_. 14e 12* 15e 5o 10* 7o 9a 4s 4e 6 a 13* 7o I4e 18*. l i e l i e 9a 10* _L_ As _ll_e 1JL_ l i u 9_c 7 A 10. :5_e_ 1 5 . 19e 14« H e 13a 12e 5« l i e 4e H e 14a 4e 21a 1 4 0 l l o 6e 6 . 5o 9-o 1.8.6 8e 9o l i e 5B l i e 1A 6»__ _ 6 66-«Q 4A_M>-0— '— 2-2-2-e-O 12e 12e 7o 5o 5e 5e 5© 4a 6e 9e 4e 8o 7e 2e 3* 6e> 4e 2s 4 a 6s 15 e 4 a 5© 8« 5 e 3 o 5 « 1 2 a 7a 5 a 7a 8c 6 a 5a 2e 2a . 9.o 3_s 8__ 8_» 8j» 4 e_ . 4 e 4 o 3_»... 13e 2 a 9 o 9e 4o 3o 4o 4a 4e 9e 4» 8e 3e 5a 4e 5e 2a l o 7J» , 5.e ,_9_*. . Ao . 4o 8_» . §.*, . 3J>_ 4 o H e 7o ' 4 a 4 e 4 a 3 a l a 6a 12e 15* 15e - 4e 10a 5 e 4 e 5e l a JL4-6 6-» 3JJ 8_a l.n Ao. Ae A* A_o_ l i e 7 a 5 e 4 a 8 • 4 e 4o 4e 4a 10 a • 9 a 7e 10. 11. 4 a 4a 4a 4a .1.4 .o 13J> JL6.a ; : 5 el 1.1. * l i e 5_e J U 9e 4a 2e 5e 4e 10a 4« 4a 6a 5a H e - 6 a 5 a 4e 4e 5o 4a 5a Aa l i f t . , 4 s _8_e JL2_e 5„a 8 a l a 1 _e_ 6 e 8e ' 5 e 7 a . 8e 7e 4 s l a 7e 13e 7 a 4o 8 c i l 0 e 7a 5a 3c 4 e 4. a. 8_e__ 5_.e_ ; 9 a 5_a 4 e _5 a 4_o 4 a 157 I J J J L Q __£§n5 3 s 3 o 3 s 2 © 2 o 2d 4 * 4 o 3o lb 2e 2 @ 4 « 8a 3e 2o 2« 4e 4 e 4© 5 e 2 s A« 4 e 3 o 3 « 4 e 4 e l o 2s _3_9 7_» 4e 2 c 4.0 4 o 3 e 4 e 4 » 4 o l o 2e 2e l o 2 o l o 4 e -3.0 4_« _ _ _ A o 4,s 2.o___ 3..*_^ 4 o 2 s 2 * 4 o 2 o 3 s 4 e 4 e 4 o l e 3 e 1 e -5_e A a kA IJS 3 J 4_s_._ 3 c 4 s 3 o So 2 o 2 o 3 e 2 a 2 o 3a l o l o A e . - 2_o_ l , e . 3.© 4 o 5 o 3 e 2 e 2 a 2 o 6 o 4 * 3 e 2 e 4o I s l o _2_B_ 3_s_ 2.a 2_e L 3jt 4 s 4 e 3e 2 6 4 e 2 e -'4« 2 o 3 e 2 o 2 o "3 e 158 Appendix G. A computer program to process b i o a s s a y data. T h i s program has been w r i t t e n i n F o r t r a n IV, and run on an IBM 1130 computer at the B i o l o g y Data Center, U n i v e r s i t y of B r i t i s h Columbia. Input: Data cards r e q u i r e d a re: 1. A f i r s t card, s p e c i f y i n g the number ( 1 - 9 9 ) °f assays t o be processed. 2. For each assay, an i n i t i a l card g i v i n g the number of prepar-a t i o n s assayed ( 1 -20) , i n 12 format, and the date (Ah) . 3 . For each p r e p a r a t i o n a card g i v i n g the number of doses ( l - 5 ) : i n I I format, a sequence number ( 1 2 ) , the name of the prepar-a t i o n (Ah), and f o r each dose, the logarithmn to the base two of the d i l u t i o n ( 0 , - 1 , - 2 , e t c . ) i n Fh.l format, the number of c e l l s with c o n j u g a t i o n tubes ( F 4 . 0 ) , and the t o t a l number of c e l l s counted ( F 4 . 0 ) . Output: The program, f i r s t l i s t s the data f o r each p r e p a r a t i o n along with the c a l c u l a t e d f r a c t i o n of c e l l s with conjugation tubes (P), the l o g i t of these f r a c t i o n s (Y), and the expected l o g i t s from the r e g r e s s i o n l i n e s (Y3). The slope of the r e g r e s s i o n l i n e and the chi-squared f o r the d e v i a t i o n s of the data from the r e g r e s -s i o n l i n e are given along with the p r o b a b i l i t y t h a t the d e v i a t i o n s are caused by random sampling. Next comes a l i s t of a c t u a l and expected l o g i t s from the r e g r e s s i o n with a l l l i n e s p a r a l l e l . The value and confidence i n t e r v a l f o r the common slope are p r i n t e d , along with G, a f i g u r e of merit f o r the p r e c i s i o n of the assay. An a n a l y s i s of the d e v i a t i o n s of the data from the r e g r e s s i o n l i n e s f o l l o w s with t e s t s f o r d e v i a t i o n s g r e a t e r than can be explained by random sampling ( 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 of the r e g r e s s i o n l i n e s . I f h e t e r o g e n e i t y i s s i g n i f i c a n t , a h e t e r o g e n e i t y f a c t o r i s c a l c u l a t e d , and used to broaden the confidence i n t e r v a l s f o r 159 the parameter est i m a t e s . F i n a l l y , f o r each p r e p a r a t i o n , the ED50, with 95$ confidence l i m i t s , the logarithmn to the base two of r e l a t i v e potency (M ) , and the potency r e l a t i v e to the standard, with 95$ confidence l i m i t s , are l i s t e d . PAGE 1 I, REID I. REID 1 LOG DRIVE CART SPEC CART AVAIL PHY DRIVE OOOO 0001 0001 0000 ' VZ MIC ACTUAL 8K CONFIG 8K _ _.*EQUATIPRNTZ.PRNTY) _ 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 *»COMPILER M E S S A G E S * * 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 ) . Y3 ( 20 . 5 ) > CH 111 2 0 ) . NF1 ( 2 0 ) . B ( 20 ) . S S N I 20 I . 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 FORMAT!12) DO 100 L=1.NR READ!2.3)NP.DATE 00003 FORMAT ( I 2 .A4 ) • . WRITEI3.601DATE 60 FORMAT(111.1TREMELLA HORMONE BIOASSAY PERFORMED 1 .AA.//>6X1 PREP DO 1SE R T P Y Y 3 ' l DO 101 J=1 .NP SY = 0. s x * 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 = SY+Y( ID.I 1 sx*b>:+D( 11 ID) SX2=SX2+D!I. ID ) * *2 SXY = SXY + D( I.ID)*Y( ID.I) 4 CONTINUE MIO )=ND AND=ND XBAR ( ID )=SX /AND YBAR(ID)=SY/AND S S X=£X2 - S X**2 / A N D SSXY=SXY-SX»SY/AND B1= S S X Y / S S X 27 SN-=0. SX=0. SX2"=0. i o v. // JOB T -PAGE - ! - . - « E-ID-C-ERRS. ..STNO.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 **COMPILER MESSAGES** SY = 0. SY? = 0.._ SXY<=0. C B Y B A R ( I D I - B 1 * X B A R < I D ) DO, 11 I=1.ND Y4(ID.I)=C+B1*D(I.ID) Z = Y 4 ( I D . I ) _UZ=I ../XL . .+.EXE1.2._*ZJJ YW' = Z T ( P { I D . I i +QZ-1. ) / 12. t.' = 4 . * l l . - Q Z ) * Q Z WN-.-;*T ( I . I D ) SN = Si;+v;N SX=SX+WM«D< I . ID) _S.Y_=.SYJ-J«J*X1J »(1.-QZ)*QZI 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 X B A R . L i 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 H CA H Y3I ID.I )=C+fc2*D<I >ID) DIr-ABS(Y3IID.II-Y4IID.I)) 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 1 ) GOTO 101 3 0 CHI I ( ID) = S.SY-.SSXY*»2/SSX !!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( 1 1.10XF5.1.2I2XF4.0).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 ( 1 0 ' . 'NUMBER OF ITERATIONS* '.12/' SLOPE" '.F7.4/ 1FITY CHISO" ' » F8•2 . 1 WITH '.13.' DEGREES OF FREEDOM AND HETEROGEN PROB= 1lF7 1.4) 101 CO.NTK4UE WRITE(3.64) 64 FORMAT(1OPREP DOSE Y Y4'./.' ') ITER"! p t r , F •>, I . R F I D f C-ERRS. R T R A N S O U R C E ••COMPILER MESSAGES** 34 DO 61 1=1iNP ND = f, ( ; ] r DO Cl J=l.ND 61 Y4(:.J)=Y3(I.J) S=,XY = n. ssx = c . SSY=0. DO 104 1=1, Sis =0. <;x =n. NP 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 ' ' ' " " ~ ' DO 20 1=1.NP C = YBAR(I )-B2*XBAR( I ) N D = 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 31 GOTO 20 32 K = 2 20 CONTINUE GOTOI33.63)>K 63 ITER=ITER+1 B1-B2 ! 163 H * oj -i ™ —i _ J <£ . 3_' _ a .-n U ZZ — O J rs: _) 11 -1 LU - 1 U a u 2 r :_ a. o_ - J u II CL 1 s> CL 11 < II x uj X _ _ _ _ - » < _ _> a _ _ _ _ _ _ —j -3" + - O J Q _ O J I— a — a c U ^ - J L H o u_ ; *-4 - \ <-< ^ I oj c a, K i • 3 II a u i— CL. *-* H L o _q x ir i <_> 5J — •< -q oj •di <r 4-i o 4- <. ' i™# r—4 <—I t CO I I • o a : I t— _t .it -• O U. ii. : O <ri _ J U) f - • O _ > J JO_!r-:><___ ) u> _> ^  . -sj * V : <j _  L :q_' i f j cn -- h-j - 1 r- _ - OJ t— OJ O i — • r—4 y— r u u j i— n u' _r> ; i I— O I—,3 I I— tl . t ul \J L O O < ui O i _  O LiJ'a. _t -„__> :_ •_. I. RI-'ID C-ERRS. ..STNO.C. 0 R T R A N S O U R C E 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 1M AND PRCB = ' ,F7.4) ... .. IFIPRP-.C5149.50.50 49 WR1TE13.51) 51 FORMAT(10 PREP ND CHISQ CONTRIBUTION') 0 0_i _i_I_=l J ii£ , DEGREES OF FREEDO 5 3 52 50 WhlTe(3.52)1.ALAB( I ) ,M(I I .DCHI( I) FORMAT I ' ' . 12.2XA4.14.5XF9.4) 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 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 t Ai-l=EOaO 1 1_ir.EJir_0 i l l RELATIVE POTENCY CONFI =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 58 59' 100 RL=2.**CL F.U = 2 .**CU WRITE! 3 .59I_ALAB( I ) .ED50! I U CLL ( I )_. CLU ( i ) .AM. RMj RLj_RU FORMAT! • ' . A4. 1XF6. 2 . 1XF6.2 i 1XF6".~2 . 2XF6.2 > 5XF 7 . 3 • 7XF 7 . 3 . 2XF~7 . 3 f CGNTINUE CALL EXIT  END H CA -f=-FEATURES SUPPORTED _ • ONE WORD INTEGERS' " " • " STANDARD PRECISION _Lp AD GO  ICCS-1132 PRINTER CARD CORE REQUIREMENTS FOR -COMMON- 0. VARIABLES AND TEMPORARIES- 2016. CONSTANTS AND PROGRAM- 2496 COMPILATION // XEQ «• COMMENCE PROGRAM EXECUTION •• TREMELLA HOBt-'ONE ttl-OASSAY-BEKFOft-iEO fe 5 PREP DOSE R 1 STD 0.0 237. _!.._ 12-6.. -2.0 97. -3.0 98. NUMBER OF ITERATIONS* SLOPE* 0.324'. -H_I_RO_U.E-IJ-_.XH 14U«_ 274. _IX0.. 142. 214. 0.923 0 .__,__ 0.683 0.384 0.457 -0.084 0.864 0.74-1 Y3 0.92 0 _D-.-5V.5_ 0.271 -0.053 _2_-4.l_wXT.H_ _2_D£ ORE ES_OF_J.liEEDQl__A.__ J ____!_ 0.0. 123.... 263.......0.4S7_ -0 . C64...„-0 .05 3.. _ -i.'J 108. 338. 0.319 -0.377 -0.398 -2.0 41. 230. 0.178 -0.764 -0.742 NUMBER OF ITERATIONS11 SLGPE= 0.3445 . n£T£RCGEi.£lT.Y.CHlSa„_ FB __0 ..0_ -1.0 -2.0 15.6.. 125. 49. ..0.21. WITH l_DEGREES_OP l:RE.ED0»_A/S ___R.0.3« .0_.-64.8J_ OJJJ.6.4. 0...0.3.7 , _2-9.3_. 0...2._2 414. .0.301 261. ,0.187 -0.419 -0.373 -0.732 -0.784 DUMBER OF ITERATIONS* SLGPE= 0.4110 _t_-__KU5 E ;.£lXY...ai !_.(_• H il WITH 1 OFGREES OF FREEDOM AND PR06 = 0.2387 PREP JOSE Y 1 0*0 - i .0 -2..0 0.928 0.526 0.364 0.968 0.616 0.263 "l -3.0 -0.064 -0.038 2 0.0 -0.064 -0.0-4 7 2 . - i • 0 ., -0.3 77. .. -0.399 2 -2.0 -0.764 -0.751 3 ' 0.0 0.064 -0.011 -a -1..Q - _ _ . _ i _ _ _Q...3.6.3 3 -2.0 -C.732 -0.716 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 STD -2.74 -3.04 -2.49 7 0_J._ _0.._0_ (jj_t_ _2_28_ RELATIVE POTENCY CONFIDENCE LIMITS _0__U__ FB 0.03 -0.18 0.28 .78 0. 145 0.114 0.175 

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