A COMPARATIVE STUDY OF THE EFFECTS OF SEVERAL ANTIMITOTICS By C harles C lark Bowen A DISSERTATION Subm itted to the School o f Graduate S tu d ie s o f M ichigan S ta te C o lle g e o f A g r ic u ltu r e and A p p lied S c ie n c e in p a r t i a l f u lf i llm e n t o f the req uirem en ts f o r the degree o f DOCTOR OF PHILOSOPHY Department o f Botany and P la n t P a th o lo g y 1953 ProQuest Number: 10008231 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10008231 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 ACIOTOVJLEPC3BMBNTS The au th or w ish e s to e x p r ess h is s in c e r e thanks fo r the h e lp and encouragem ent o f the members o f the c y to lo g y group a t M ichigan S t a te C o lle g e o f whose o v e r a ll p r o j e c t t h i s i n v e s t ig a t i o n i s hu t a sm a ll p h a se. In p a r t ic u la r the a u th o r i s in d eb te d to the g r o u p 's le a d e r , Dr. G. B. W ilson, whose guidance and i n t e r e s t have meant so much. Thanks are a ls o due to Dr. W. D. B aten fo r h is a d v ic e on the s t a t i s t i c s in v o lv e d and to Mr. P h i lip Coleman f o r h is in v a lu a b le p h o to g r a p h ic work. A gran t by th e A ll C o lle g e R esearch Committee d efra y ed th e b u lk o f th e expense in v o lv e d in t h i s in v e s t ig a t i o n . The au th or h e ld an Advanced P r e d o c to r a l F e llo w sh ip o f the R a tio n a l S c ie n c e Foundation d u rin g the 1952 - 53 academ ic y e a r and w ish e s to e x p r e ss h is g r e a t e s t a p p r e c ia tio n o f the o p p o r tu n ity a ffo r d e d th ereb y . 328763 A COMPARATIVE STUDY OP THE EFFECTS OF SEVERAL AHTIMITOTICS / By C harles C lark Bowen AIT ABSTRACT Subm itted to th e School o f Graduate S tu d ie s o f M ichigan S ta te C o lle g e o f A g r ic u ltu r e and A p p lied S cien ce in p a r t i a l f u l f il lm e n t o f the requirem ents f o r th e d egree o f DOCTOR OF PHILOSOPHY Department o f Botany and P la n t P a th o lo g y Year 1953 r Approved --- C h a rles C lark Bowen P la n t meristerns a re r e a d ily made p o ly p lo id by exposure to c o lc h i­ c in e . The c h a r a c t e r is t lc p a tte r n o f m it o s is under th e in flu e n c e o f t h i s drug has been termed " C -m ito sis" . Many drugs w hich do not r e a d ily produce p o ly p lo id y , in c lu d in g c e r t a in a n t i b i o t i c s , cau se c y t o lo g ic a l e f f e c t s w hich a t l e a s t s u p e r f i c i a l l y resem ble th o se produced by c o l­ c h ic in e . Such drugs have u s u a lly been c a lle d M C -m ito tic " in the l i t ­ e r a tu r e . The purpose o f t h is in v e s tig a .tio n was to stu d y the c y t o lo g i­ c a l e f f e c t s o f th ree a n t i b i o t i c s — A c t i- d io n e , str ep to m y c in and Chloro­ m ycetin — on a q u a lit a t i v e and q u a n t it a t iv e b a s is , and to conpare th e r e s u l t s w ith s im ila r d ata o b ta in e d from a stu d y o f th e e f f e c t s o f c o lc h ic in e . The r o o t t i p s o f pea s e e d lin g s were immersed in known co n cen tra ­ t io n s o f th e s e drugs d is s o lv e d in a weak b a la n ced m in eral s a l t s o lu ­ t io n and m a te r ia l c o l l e c t e d fo r exam in ation a t known in t e r v a ls up to e ig h t h ou rs. R ecovery was checked by r in s in g s e e d lin g s th orou gh ly a f t e r e ig h t hours o f treatm en t and le a v in g them in m o ist paper to w e lin g fo r f o r t y - e i g h t h ou rs. I t was found th a t th e e f f e c t s o f c o lc h ic in e and A c ti-d io n e d if f e r e d in a lm o st e v er y r e s p e c t. A c ti-d io n e m arkedly d ecrea sed d iv i s io n s by c a u sin g preprophase in h i b i t i o n o f d i v i s i o n s , w h ile c o lc h ic in e caused an in c r e a s e in d iv i s io n s w hich appeared In p a r t to be due to a tru e s t im u la t io n of m it o s is . A c ti-d io n e p r im a r ily a ffe c te d pro­ p h a se s , b lo c k in g t h e i r t r a n s i t io n to raetaphases and th u s r e s u lt in g in an accu m u la tio n o f h ig h ly o v erco n tra .cted prom etaphases. in te r p h a se occu rred in abundance a t a l l prophase s t a g e s . had l i t t l e e f f e c t on p ostp ro p h a se s t a g e s . R ev ersio n to A c ti-d io n e C o lc h ic in e p r im a r ily C h a rles Clark Bowen a f f e c t e d p o stp ro p h a se s ta g e s by s p e c i f i c and com plete impairment o f s p in d le c o n tr o l and showed no e f f e c t on p ro p h a ses. Streptom ycin e f f e c t s were q u a l i t a t i v e l y s im ila r to th o se o f A c ti-d io n e }but s t r e p t o ­ mycin was shown to have a v e r y narrow margin betw een i t s th r e s h o ld o f c y t o l o g i c a l e f f e c t i v e n e s s and i t s l e t h a l th r e s h o ld . C hlorom ycetin c a r r ie d t h i s tendency o f strep to m y cin to th e extrem e, and e x c e p t f o r r a th e r e r r a t i c a l l y e x p r e sse d preprophase in h i b it io n o f d i v i s i o n s , i t s l e v e l o f c y t o lo g ic a l e f f e c t i v e n e s s was n ot sep a ra b le from i t s n e c r o t ic dosage l e v e l . With th e e x c e p tio n o f c o lc h ic in e , the c y t o lo g ic a l a c t i v i t y o f the drugs t e s t e d was a n ta g o n iz e d by d is s o lv e d m ineral s a l t s . T his " s a lt e f f e c t " was v e r y marked w ith strep to m y cin and o n ly moderate w ith A c t i d ion e and C hlorom ycetin. E vidence o f s p in d le impairment was n ot se e n i n s i g n i f i c a n t p r o p o r tio n s i n m a te r ia l tr e a te d w ith the a n t i b i o t i c s e x c e p t a t d osages h ig h enough to p r e v e n t reco v ery . These and o th e r f a c t s a r e u se d to support the h y p o th e s is th a t the s p in d le derangem ents caused by th e th re e a n t i b i o t i c s have a v e r y d if f e r e n t b a s is than th e sp in d le d is tu r b a n c e s cau sed by c o lc h ic in e . f o r th e n o tio n th a t s p in d le d istu rb a n c e A b a s is i s shown to e x i s t caused by treatm en t w ith A c t i- d io n e , strep to m y cin and C hlorom ycetin are a c t u a lly e a r ly s ta g e s in p y k n o tic d e g e n e r a tio n . The term " a k in e tic m i t o s i s 11 i s proposed as a s u b s t it u t e fo r "C— m it o s is " to d e s c r ib e any m it o t ic p r o c e s s where s p in d le c o n tr o l o f p o s t­ prophase chromosomes i s im p aired . "C -m itosis" would be r ese r v e d to d e s c r ib e the c o lc h ic in e type o f " a k in e tic " m ito se s where such s p in d le im­ pairm ent i s a ) a h ig h ly s p e c i f i c r e a c t io n , b) accom panied by l i t t l e or no prophase d is tu r b a n c e , and c) p r o d u c tiv e o f s u b s t a n t ia l amounts o f p o ly p lo TABLE OF CONTENTS PAGE INTRODUCTION...................................................................................................... 1 MATERIALS ARE METHODS.............................................................................. 13 A. E xp erim en tal P r o c e d u r e ........................................................... 13 B. D r u g s ................................................................................. 15 C. C y to lo g ic a l E x a m in a tio n ........................................................... 18 D. Scope o f I n v e s t ig a t io n ......................................... 20 OBSERVATIONS..................................................................................................... 22 A. Frequency o f D i v i s i o n s ........................................................... 22 B. C l a s s i f i c a t i o n o f A b e r r a t i o n s ............................................ 24 C. Range o f T o x i c i t y .......................... 30 D. Changes in E f f e c t s w ith V a r ia tio n in E. Changes i n E f f e c t s w ith D u ra tio n o f Treatm ent F. E ffe c ts o f D ih y d r o s t r e p t o m y c in .......................... 67 G. E f f e c t s o f C h lo r o m y c e t in ........................................ 67 DISCUSSION SUMMARY Dosage . . . . 33 . . . ................................ 47 69 ........................................................................................... . . 80 .............................................................................................................. 83 P la t e I , Normal M i t o s e s ..................................................................... 83 PLATES P l a t e I I , I I I , C o lc h ic in e E f f e c t s ................................................... 8 4 -8 5 P la t e IV, V, A c ti-d io n e E f f e c t s ........................................................8 6 -8 7 P l a t e VI, S trep tom ycin E f f e c t s ................................................... 88 P l a t e V II, C hlorom ycetin E f f e c t s ................................. 89 BIBLIOGRAPHY..................................................................................................... 90 APPENDIX.............................................................................................................. 93 INTRODUCTION The im portance o f the c e l l a s a b a s ic u n it o f o r g a n iz a tio n and c o n tr o l in l i v i n g e n t i t i e s s c a r c e ly needs em phasis to d a y . A c c o r d in g ly , e v e r y scrap o f in fo rm a tio n th a t can be g a in ed con­ c e r n in g the n atu re and m echanics o f th e n u cle u s r e p r e s e n ts an advance i n b i o l o g i c a l knowledge a t the most fundam ental - l e v e l . The stu d y o f c h e m ic a lly ind uced m it o t ic a b e r r a tio n s a s a t o o l in t h is q u est g a in e d i t s f i r s t b ig im petus w ith the d is ­ c o v er y o f the p o ly p lo id iz in g a c t io n o f c o lc h ic in e (B la k e s le e and A very, 1937; D u stin , Havas and L i t s , 1937; N ebel and R u t t le , 1 9 3 8 ). A fte r th e war when s e c u r it y bars were l i f t e d on c e r t a in p h a se s o f b i o l o g i c a l r e se a r c h , i t became known th a t workers on b o th s id e s had su cceed ed in in d u c in g m u tation s by chem ical means (A uerbach and Robson, 1946; O eh lk ers, 1 9 4 3 ). S in c e th a t tim e l i t e r a l l y hundreds o f ch em ica ls have been u sed to t r e a t a w ide v a r i e t y o f l i v i n g t i s s u e s , and r e s u l t s have b een checked c y t o l o g i c a l l y a s w e ll a s g e n e t i c a l l y . Comparison o f r e s u l t s i s com­ p l i c a t e d by th e v a r ie t y o f ex p erim en ta l c o n d itio n s and, in con­ t r a s t to th e s ta n d a r d iz e d t e s t s u se d to d e te c t m u ta tio n s, c y to — l o g i c a l a n a ly s is has b een la r g e l y s u b j e c tiv e and n o n -q u a n tit a tiv e . D e sp ite the problem s o f com parison and la c k o f sta n d a rd i­ z a t io n o f term in o lo g y , c e r t a in g e n e r a l te n d e n c ie s have been ob­ se r v e d and some g e n e r a l c o n c lu s io n s made. Levan in a r e c e n t rev ie w (1952) has d iv id e d th e s e r e a c tio n s In to th re e main h ea d in g s -2 and p o in t s ou t th a t some ch em ica ls may produce o n e, 'two or ev en a l l th r e e o f th e s e r e a c tio n s depending on the c o n d itio n s o f t r e a t ­ ment. Most o f Levan® s c o n c lu s io n s were "based on th e r e a c tio n s o f A lliu m r o o t m eristem s, sp ro u ted i n w a ter, to imm ersion in a s o lu t io n o f the drug f o r a d e f i n i t e p e r io d o f tim e— th e "A lliu m cena t e s t " . The f i r s t c a te g o r y i s th a t o f "mutagenic" r e a c tio n s i . e . r e a c t io n s where the p r in c ip le r e s u lt i s th a t o f g e n e t ic change. Levan fu r th e r d iv id e s th ese in to th e subheadings " g e n e tic a l muta­ g e n s ’® i . e . su b sta n c e s th a t produce gene m u ta tio n s and " c y to lo g i­ c a l m utagens” w hich cause g e n e t ic changes by p ro d u cin g s t r u c t u r a l a l t e r a t i o n s o f th e chromosomes. G en era lly sp ea k in g g e n e t ic a l m utagens a ls o cau se chromosome breakage and v ic e v e r s a , but th e two e f f e c t s do n ot appear to be p a r a l l e l in a l l ch e m ic a ls. The l a s t two c a te g o r ie s a r e th o se w hich p r im a r ily produce c y t o l o g i c a l e f f e c t s o f one s o r t o r an o th er and Levan d e s ig n a te s th e s e a s 1) r e v e r s ib le p h y s io lo g ic a l r e a c tio n s and 2) and t o x i c r e a c t io n s . le th a l Under the f i r s t headin g th e e f f e c t s o f c o l c h i c i n e , so c a l l e d C -m ito s is , i s th e main typ e o f r e a c tio n . T his Levan d e s c r ib e s a s the " r e v e r s ib le in a c t i v a t io n o f th e mechanism o f movement o f the chromosomes”. He r e c o g n iz e s C- tumour form ation and e x c e s s iv e chromosome c o n tr a c tio n a s rea c­ t io n s b e lo n g in g to t h i s g e n e ra l com plex. S u b stan ces o f w id e ly v a r y in g chem ical c o n s t it u t i o n have b een c la s s e d a s C -m ito tic a g e n ts . In the ca se o f a number o f l i p o i d s o lu b le su b sta n ce s o f the n a r c o t ic ty p e , i t has been shown th a t -> in c r e a s e d C -m ito tic a c t i v i t y i s c o r r e la te d w ith in c r e a s e d f a t s o l u b i l i t y and d e c re a sed w ater s o l u b i l i t y . The m a te r ia ls w ith th e lo w e s t known C -m ito tic th r e s h o ld s are th e in o r g a n ic s a l t s o f h eavy m e ta ls , p a r t ic u l a r l y m ercury, the a c t i v i t y o f w hich can be in c r e a s e d even more by a lk y la t io n (Levan, 19^5) • C o lc h ic in e i s u n iq u e among th e s e compounds i n th a t any change w ith in the c o lc h i­ c in e m olecu le d e s tr o y s i t s a c t i v i t y (S te in e g g e r and Levan, 19^5)* Levan (1952) s u g g e s ts th a t the so c a l l e d so m a tic r a eio sis o f H uskins (19^8) c o n s i s t s m ainly o f d e v ia tin g C -m ito ses due to in ­ com p lete impairment o f the s p in d le mechanism. L eth a l and t o x i c r e a c tio n s form the f i n a l c a te g o r y a c c o r d in g to Levan. E f f e c t s in t h i s c a te g o r y ranged from r e v e r s io n o f pro­ p h a se s w ith d is s o l u t i o n o f m atrix and r e la x a t io n o f s p ir a l s to p y k n o s is . Levan d e s c r ib e s t h i s p r o c e s s a s in d u c tio n o f an " a r ti­ f i c i a l i n t e r p h a s e S u b s t a n c e s c a u sin g such e f f e c t s may a ls o cau se C -m ito s is , but t h i s w i l l seldom be n o ted a s the t o x ic th re sh ­ o ld s a r e c lo s e to o r below th e C -m ito tic th r e s h o ld . D*Amato (19^8, 19^9) d e s c r ib e s a la r g e c la s s o f m a te r ia ls he c a l l s " ty p ic a l c e l l ' p o iso n s" (a s opposed to " C -m ito tic p o is o n s " ). He d e s c r ib e s two p r in c ip le ty p es o f e f f e c t s a s t y p ic a l o f the c e l l p o is o n s . a) **Prophase p o iso n in g " in th a t p roph ases a r e reach m etaphase. "unable" to These a r r e s te d prop h ases may r e v e r t to th e r e s t in g c o n d itio n . b) "Preprophase i n h ib it i o n o f m ito sis " in th a t no new d iv i s io n s a r e i n i t i a t e d once trea tm en t has s t a r t e d to take e f f e c t . — Ijw m O nly o c c a s io n a lly c o u ld m a te r ia l w ith in t e n s e r e v e r s io n be re­ c o v er e d and th en o n ly normal d i v i s i o n s c o u ld be found, no p o ly ­ p lo i d y o r diplo-chrom osom es were n o te d . D*Amato fu r th e r p o in t s o u t th a t th e o c c a s io n a l C—m it o t ic e f f e c t s produced a t some con­ c e n t r a t io n s o f th e s e p o i s o n s ” a r e th e r e s u lt o f a “shock a c t i o n ” o r “m a ssiv e t o x ic e f f e c t ” and do n o t r e p r e se n t th e t y p ic a l c y to ­ l o g i c a l e f f e c t o f th e s e d rugs. D uring th e p a s t few y e a rs th e c y to lo g y group a t M ichigan S t a t e C o lle g e has screen ed a la r g e number o f compounds fo r c y to ­ l o g i c a l e f f e c t s (W ilson , 1950? W ilson and Bowen, 1951? P o w e ll, 1951? H uston, 1952; and o th e r s u n p u b lish e d ). These in c lu d e d , among many o th e r m a t e r ia ls , a lo n g l i s t o f a n t i b i o t i c s and in ­ s e c tic id e s . While much o f the work has been on the q u a lit a t i v e l e v e l , i t appears to be d e f i n i t e t h a t , o f the lo n g l i s t o f drugs t e s t e d , o n ly two— c o lc h ic in e and c y clo c h lo ro h ex a n e (L indane)— b e lo n g to the group o f tru e “C -m ito tic ” a g e n ts . A ll o th e r m a teri­ a l s f a l l in to one o r an oth er o f th e fo llo w in g c a t e g o r ie s : A) W ithout e f f e c t , a t l e a s t u n t i l f a n t a s t i c c o n c e n tr a tio n s were a t t a in e d . B) An example i s p e n i c i l l i n (W ilson, 19 5 0 ). Caused p y k n o sis a n d /o r s t i c k i n e s s a t a l e v e l o f con­ c e n tr a tio n low enough to mask any o th e r c y t o lo g ic a l e ffe c ts. An example in t h i s c a te g o r y was th e a n t i - fu n g a l a n t i b i o t i c , R im ocidin (H uston, 1 9 5 2 ). C) S u b s t a n t ia lly answered the requirem ents o f a t y p ic a l “c e l l p o is o n ” in th e D*Amato sen se w ith apparent f a i l u r e o f p rop h ases to reach m etsphase and -5 preprophase in h ib it io n o f d i v i s i o n s . R ev ersio n o f p rop h ases and m etaphases to in te r p h a se commonly hut n ot in v a r ia b ly accom panied th e o th e r symptoms. m ito s e s were o c c a s io n a lly p r e s e n t . C- The h u lk o f ma­ t e r i a l s t e s t e d f e l l in t o t h i s c l a s s and A c t i- d io n e , an a n t ifu n g a l a n t i b i o t i c , may he c i t e d a s r ep re sen ta ­ t iv e o f t h i s type o f compound (W ilson, 1950? Hawthorne and W ilson , 1 9 5 2 ). D uring th e cou rse o f t h is groupf s work a number o f compounds w ith i n t e r e s t i n g p r o p e r t ie s have turned up. I t was the purpose o f t h i s stu d y to in v e s t i g a t e th e e f f e c t s o f s e v e r a l o f th ese drugs on a com parative b a s i s , namely c o lc h i c i n e , A c t i- d io n e , str ep to m y c in , and C hlorom ycetin. A second purpose was to d e v is e a c y t o lo g i c a l t e s t method th a t would e lim in a te th e p r in c ip a l problem s en co u n tered i n th e A lliu m cena t e s t i . e . th e d i f f i c u l t y o f o b ta in in g o n io n b u lb s th a t can be sp ro u ted a t w i l l throughout th e y e a r , th e f r e ­ quent v a r i a b i l i t y in d iv i s io n freq u en cy and f i n a l l y the o c c a s io n a l appearance o f t o x i c i t y symptoms w ith o u t apparent ca u se. C o lc h ic in e The a lk a lo id c o lc h ic in e i s o b ta in e d from th e corm o f Col chi cum autum nale and h as th e fo llo w in g p rob ab le s t r u c t u r a l form ula (o o r tn er and G ortner, 19^9) • ^ II = C H -O C H j C o lc h ic in e ( 022^25^6^) -6 As p r e v io u s ly mentioned^ c o lc h ic in e i s the type su b sta n ce f o r th e c y t o l o g i c a l r e a c t io n la b le d MC -m ito s is w by Levan (1 9 3 8 )- In. th e o r i g i n a l sen se t h i s term was u se d to d e s c r ib e the r e a c t io n a s cau sed by c o lc h ic in e , and thus im p lie d a p r o c e s s w hich, under c o r ­ r e c t c o n d it io n s , w i l l r e s u lt in p o ly p lo id y . Today, however, th e term i s u sed to d e s c r ib e any c y t o l o g i c a l r e a c t io n where p o stp ro — ph ase chromosomes a r e s u b je c t to d e f ic ie n c y o f s p in d le c o n t r o l, even though t h i s may be a symptom o f a c o m p le te ly d if f e r e n t s o r t o f c e l l u l a r d istu r b a n c e than th a t caused by c o lc h ic in e . In t h i s r e s p e c t s e v e r a l w orkers have p o in te d out th a t the C -m ito sis cau sed by the m a jo r ity o f a n t im it o t ic s i s p ro b a b ly a v ery d i f f e r e n t r e a c t io n than th a t produced by c o lc h ic in e and a few o th e r tr u e p o ly p lo id i z in g a g e n ts (D*Amato, 1 9 ^ 1 A lle n , W ilson and P o w e ll, 1 9 5 0 ). Guttman (1952) u s in g A lliu m made a s t a t i s t i c a l a n a ly s is o f th e numbers o f d i v i s i o n f ig u r e s i n ea ch sta g e o f d i v i s i o n p e r ran­ dom f i e l d o f v iew a s ob served under h ig h dry pow er. A pp roxim ately two hundred c e l l s were in c lu d e d i n the average such f i e l d . She combined the d ata from th ree d i f f e r e n t tr e a tm e n ts! a) 200 ppm c o lc h ic in e f o r tw e n ty -fo u r h o u rs, b) 500 ppm c o lc h ic in e f o r tw e n ty -fo u r h o u rs, c) S a tu ra ted s o lu t io n o f acenaphthene f o r tw e n ty -fo u r hou rs. U sin g a P o is s o n d is t r ib u t io n she rep o rted th a t c o lc h ic in e and a c e ­ naphthene l ) 2) Did n o t p rev en t any c e l l s from e n te r in g prophase Doubled the mean le n g t h o f the t o t a l d i v i s i o n c y c le w h ile qu adrupling the mean d u ra tio n o f metaphase -7 3) Caused 8 .5 $ o f m etaphases to r e v e r t to in te r p h a se A ffo rd ed e v er y c e l l r e v e r tin g the same chance a s any o th e r c e l l o f g o in g through a new c y c le o f m it o t ic d iv i s io n . In ord er to dem onstrate th e n atu re o f such d if f e r e n c e s , c o l­ c h ic in e was s e l e c t e d a s one o f th e drugs, th e c y t o l o g i c a l e f f e c t s o f w hich were to he s tu d ie d in t h i s in v e s t ig a t i o n . A c t i- d io n e In 19^7* W h iffen , Bohones and Emerson r ep o rted th e p r e se n c e o f an a n t ifu n g a l a n t i b i o t i c in media ferm ented by str ep to m y c in p ro d u cin g s t r a in s o f Strentom vces g r i s e u s . I s o la t e d and c r y s t a l ­ l i z e d by Eord and Leach (I9h 8) i t has the fo llo w in g s tr u c tu r e : CHa H> H / c -c H3C— H \ c —c ^C=0 / Hx H \ / HC—C—C \ Hv n \ oh C ycloh eslm id e (A c ti-d io n e ) , o Hv || c —c \ / NH c—e 0 A c ti-d io n e i s the r e g is t e r e d trademark o f th e Upjohn Company f o r i t s brand o f t h i s a n t i b i o t i c . P o s s e s s in g no marked a n t i­ b a c t e r i a l p r o p e r t ie s i t h a s, how ever, proved u s e f u l in p la n t p a th o lo g y i n c o n t r o llin g c e r t a in fungus i n f e c t io n s (Vaughn e t a l, 1 9 * 9 ). The c y to lo g y group a t M ichigan S ta te C o lle g e has i n v e s t i ­ g a te d th e c y t o lo g ic a l e f f e c t s o f A c ti-d io n e on A lliu m r o o t t i p s (W ilso n , 1950; Hawthorne and W ilson , 1 9 5 2 ), G en era lly sp ea k in g -8 th e e f f e c t s o f t h i s drag seemed q u ite u n lik e c o lc h ic in e , and in g e n e r a l conformed w ith th o se s e t f o r t h by D*Amato a s "belonging to h i s group o f " c e ll p o is o n s " . Ho r eco v ery o f A llia m r o o t t i p s was o b ta in e d a f t e r treatm en t a t d o sa g es and tim e s s u f f i c i e n t to p rodu ce ob v io u s c y t o lo g ic a l e f f e c t s . B ecause o f th e r e l i a b i l i t y o f th e A c ti-d io n e r e a c tio n , and th e f a c t th a t i t s e f f e c t s a re a s s t r i k i n g a s c o lc h ic in e e f f e c t s , b u t a p p a r e n tly o f a v e ry d i f f e r e n t n a tu r e , i t was s e le c t e d f o r t h i s stu d y . S trep tom ycin S trep tom ycin , th e f i r s t a n t i b i o t i c o f c l i n i c a l im portance to be i s o l a t e d from an a ctin o m y cete was found by Waksman and co­ w orkers (S c h a tz , B ugie and Waksman, 1 9 ^ ) in c u lt u r e s o f S trep t o m vces g r is e u s . The s tr u c tu r e o f strep to m y cin i s a s fo llo w s ( P r a t t and B ufrenoy, 19^ 9): S t r e p t id in e u ,OH NH HjH-? H / C\ H HN-C c — nh \ S trep tom ycin ✓ x S tr e p to s e S Hfc -9 An im portant m o d ific a tio n o f th e drug i s d ih y d ro strep to m y cin w hich i s produced hy r e d u c tio n o f the carb on yl group o f the s t r e p t o s e m o iety to an a lc o h o l. B oth str ep to m y c in and d ih y d ro - s tr e p tom ycin a r e b a s e s and a re u se d c l i n i c a l l y , a s w e ll a s i n t h i s study, a s th e s u l f a t e s . C o n sid era b ly lo w er t o x i c i t y has b een shown f o r the reduced form than fo r strep to m y cin in c l i n i c a l u s e , w ith no change in a n t i b i o t i c a c t i v i t y r e p o r te d (Waksman, 19^9 )• A phenomenon o f in t e r e s t i s th a t th e p r e se n c e o f c e r t a in a n io n s and c a t io n s a l t e r the e f f e c t i v e n e s s o f str ep to m y c in . h as b een c a lle d 11s a l t e f f e c t ” (P r a t t and D ufrenoy, 19^9) • T h is There have b een c o n f l i c t i n g r e p o r ts in th e l i t e r a t u r e , bu t g e n e r a lly n i t r a t e , c h lo r id e , l a c t a t e , p h o sp h a te, t a r t r a t e , c i t r a t e and s u l f a t e among the a n io n s; and b iv a le n t magnesium, calcium and barium among th e c a t io n s have been found to cau se the g r e a t e s t in t e r f e r e n c e w ith a n t ib a c t e r ia l a c t i v i t y . Streptom ycin has s e v e r a l s p e c ia l p r o p e r tie s o f in t e r e s t to the g e n e t i c i s t . P r o v a s o li, Hutner and P in tn e r (1 9 5 2 ) have re­ v iew ed the work th a t has been done on the in d u c tio n o f permanent c h lo r o s i s in see d p la n t s and some a lg a l f l a g e l l a t e s by s tr e p to ­ m ycin. To d ate d e te r m in a tio n s a s to w hether o r n o t such p la ,s tid m u ta tio n s may be in h e r it e d has been h e ld up by th e numerous te c h n i­ c a l problem s in v o lv e d . A second p r o p e r ty worthy o f m ention i s th e u se o f str ep to m y c in dependent s t r a in s o f b a c te r ia (d ev elo p ed by lo n g exposure to low con­ c e n t r a t io n s o f th e drug) in g e n e t ic s t u d ie s . The l o s s o f dependence upon str ep to m y c in i s e a s i l y determ ined q u a n t it a t iv e ly in such a -1 0 c u l t-u re a f t e r exp osu re to chem ical o r p h y s ic a l mutagens (B e r ta n i, 1950). The c y t o lo g ic a l e f f e c t s o f strep to m y cin in A lliu m have heen in ­ v e s t i g a t e d hy s e v e r a l workers (W ilso n , 1950; W ilso n and Bowen, 1 9 5 1 ). I n g e n e r a l th e s e e f f e c t s have in c lu d e d o v e r c o n tr a c tio n and some s p in d le impairment accom panied hy r e v e r s io n o f l a t e r s ta g e s to th e e n e r g ic c o n d it io n . P y k n o sis i s rep o rted to he p r e se n t in v a r y in g d e g r e e s a t d osages o n ly s l i g h t l y o v er th o se n e c e s s a r y to produce c y to lo g ic a l e f f e c t s . No r e p o r ts have heen made o f reco v ery a f t e r dosage a t l e v e l s c y t o l o g i c a l l y e f f e c t i v e . These o b s e r v a tio n s were con firm ed in T ra d esc a n tia and chromosome fra g m en ta tio n in th a t s p e c ie s hy str ep to m y c in was rep o rted hy Tanaka and Sato (1 9 5 2 ). nhlnmTn v c e tin The i s o l a t i o n o f C hlorom ycetin from Strentom vces v e n e z u e la e ( E r lic h e t a l , 19^7) gave man a most u s e f u l a n t i b i o t i c d e s p it e some r e c e n t a d v erse p u b l i c i t y con cern in g s e v e r a l f a t a l c a s e s o f a p l a s t i c anemia fo llo w in g ex ten d ed treatm ent w ith the drug. s t r u c t u r a l form ula i s a s f o llo w s : O V\ . N —c // O B H C -C / \ \-c ' H C —c I HO H H H H — CH r OH h o ? s cf \ C hlorom ycetin (C hloroam phenicol) Cl 2^12^2® 5 Its -1 1 C hlorom ycetin was su b je c te d to the A lliu m t e s t in our la b o r a to r y (W ilson and Bowen, 1951) and was found to cause a lm o st com p lete c e s s a t io n o f d iv i s io n s a t c o n c e n tr a tio n s o f 1000 ppm. The o c c a s io n a l f ig u r e s seen a f t e r tw elv e hours o f treatm en t were s t r o n g ly a ffe c te d ., o v e r c o n tr a c tio n and C -m ito sis b oth b e in g p r e se n t. Root t i p s a f t e r such treatm en t were reco v ered and no a b n o r m a litie s n o te d . B ecause o f th e marked su p p r e ssio n o f m it o s is and e a se o f r e c o v e r y shown by C hlorom ycetin in th e s e p r e lim in a r y A lliu m runs, i t was s e le c t e d f o r t e s t i n g in t h i s in v e s t ig a t i o n . -1 2 MATEEXALS AM) METHODS A. E xp erlm en tal P rocedure The r o o t t i p s o f young s e e d lin g s o f P i sum sativum v a r . A la sk a p r o v id e d th e m e r iste m a tic m a te r ia l f o r th e s e s t u d ie s . F u rn ish ed through th e c o u r te sy o f Ferry-M orse Seed Company, th e s e e d s were r ep o rted to "be f r e s h s to c k from a d is e a s e - f r e e s t r a i n o f r e l a t i v e g e n e t ic u n ifo r m ity . They had n o t “been su b je c te d to any form o f a n t i fu n g a l trea tm en t. Seeds were germ inated in m o ist paper to w e lin g and 72 hours a f t e r s t a r t i n g produced a ro o t a p p ro x im a tely th r e e to fo u r c e n t i ­ m eters lo n g . G erm ination and a l l subseoxu e n t o p e r a tio n s were c a r r ie d o u t a t room tem perature (ab ou t 2^° C ). At t h is tim e the s e e d lin g s were removed from th e p a p er, and t h e i r r o o ts were sub­ m ersed in a m in eral s a l t s o lu t io n . This was accom p lish ed by s t r e t c h i n g wide mesh c h e e s e c lo t h o v er the to p s o f one l i t e r b ea k ers f i l l e d to w ith in one c e n tim e te r o f the brim w ith th e s o lu ­ t io n . The r o o ts were in s e r t e d in t o th e s o lu t io n through the mesh o f th e c h e e s e c lo t h , and the p lu m u le, seed c o a t and c o ty le d o n s were thus suspended ou t o f c o n te n t w ith th e s o lu t io n . The m in eral s a l t s o lu t io n u se d was h a lf - s t r e n g t h o f a modi­ f i e d Hoagland s o lu t io n a s d ev elo p ed by Huskins and S t e i n i t z (19^+8) w ith m inor elem en ts o m itted and pH a d ju s te d to $ . 6 . The compo­ s i t i o n o f one l i t e r o f f u l l s tr e n g th s o lu t io n was a s f o llo w s : .0 9 5 d111, Ca(H0^)2*^HgO .1 2 9 gjn. 180 dm. MgSOlj,* 7H2O .1 3 ^ S®* KH2POI}, .0 0 7 gffl- KgHPOij,* 3H2O -1 3 C onstant a e r a t io n and a g i t a t i o n was o b ta in e d by b u b b lin g a i r a t a r a te o f a p p ro x im a tely 10 cc/m in u te through c e p i l l a r y o p en in g s i n th e ends o f g la s s tu b es in s e r t e d in to the s o lu t io n through the c h e e s e c lo t h . The a i r was o b ta in e d from the la b o r a to r y s e r v ic e o u t l e t s but was f i r s t rendered f r e e o f du st and o i l p a r t i c l e s by b e in g p a s s e d through a f i l t e r c o n s i s t in g e s s e n t i a l l y o f one l i t e r o f e ig h t y mesh c h a r c o a l. By u se o f t h i s tech n iq u e i t was a r e l a t i v e l y sim ple p roced u re to o b ta in any d e s ir e d q u a n tity o f v ig o r o u s ly grow ing s e e d lin g s w ith r o o ts w e ll a c c lim a te d to an aqueous environm ent. A fte r tw e lv e hours in h a l f s tr e n g th Hoagland s o lu tio n , c y t o lo g ic a l exami­ n a t io n dem onstrated th a t r o o t t i p s r a th e r u n ifo r m ly p o s s e s s e d a h ig h r a te o f m e r iste m a tic a c t i v i t y w ith o n ly rare a b n o r m a litie s o f d i v i s i o n and r e l a t i v e l y c o n sta n t p r o p o r tio n s o f c e l l s i n th e s e v e r a l s ta g e s o f m it o s is (Column A, Table X). T his p roced ure was u n ifo r m ly c a r r ie d out to t h i s p o in t , and such ro o t t ip s w i l l h e n c e fo r th be r e f e r r e d to a s nzero hour controls**. Subsequent trea tm en t v a r ie d w ith th e ch em ical to be t e s t e d , b u t i n a l l c a s e s th e s e e d lin g s were removed a f t e r tw elve hours from th e la r g e b e a k e r s, the r o o t t i p s r in s e d in s e v e r a l changes o f d is ­ t i l l e d w a ter and th en in s e r t e d through c h e e s e c lo t h s tr e tc h e d o v e r 250 ml b eak ers w hich were f i l l e d w ith a s o lu t io n o f the drug th e c y t o l o g i c a l e f f e c t s o f which were to be t e s t e d . A e ra tio n and a g i t a t i o n were m a in ta in ed d u rin g treatm ent by b u b b lin g a i r a s b e fo r e . Zero hour c o n tr o ls were alw ays c o l l e c t e d and f ix e d a t t h i s tim e. -1*1Up to f o r t y r o o t t i p s c o u ld "be tr e a te d in one v e s s e l a t one tim e and each c o l l e c t i o n in c lu d e d a minimum o f fo u r ro o t t i p s from one v e s s e l . F ix a t io n was f o r a t l e a s t t h i r t y m inutes in a th r e e to one a b s o lu t e a lc o h o l - a c e t i c a c id m ix tu r e. H y d r o ly sis in one normal h y d r o c h lo r ic a c id f o r tw elv e m in utes a t 60° C was fo llo w e d by s t a in in g by means o f th e F eu lg en r e a c t io n . Squash p r e p a r a tio n s were th en dehyd rated o v e r n ig h t in 95% e th a n o l con­ t a in in g a sm all amount o f f a s t geen a s a c o u n t e r s ta in , and made perm anent w ith D iaphane. C on trol runs were c a r r ie d o u t s im u lta n e o u sly w ith th e t e s t i n g o f a p a r t ic u l a r drug. The c o n tr o l v e s s e l s co n ta in ed o n ly th e p a r t ic u l a r c o n c e n tr a tio n o f Hoagland s o lu t io n or d i s t i l l e d w a ter b e in g u sed a s the s o lv e n t in th e run in q u e s tio n . C o lle c t io n s were made a t in t e r v a ls up to e ig h t hou rs, and a t th e end o f th a t tim e a minimum o f s i x s e e d lin g s were r in s e d i n s e v e r a l changes o f d i s t i l l e d w a ter and p la c e d in m o ist p ap er to w e lin g in ord er to check r e c o v e ry from th e e f f e c t s o f the drug. These s e e d lin g s were in s p e c te d v i s u a l l y fo r g r o ss changes and th e r o o t t i p s c o l l e c t e d and f ix e d f o r subsequent c y t o lo g ic a l examina­ t io n f o r t y - e i g h t hou rs a f t e r rem oval from the treatm en t v e s s e l . £. £rug& S ix d if f e r e n t drug p r e p a r a tio n s were checked f o r c y t o l o g ic a l a c tiv ity . A c ti-d io n e (C on trol Ho. 24?B-JH F-5), dihydro s t r e p t o ­ m ycin s u lf a t e (C o n tro l Ho. T 4520), and str ep to m y c in s u lf a t e (Con­ t r o l Ho. 11>-JA -12) were m anufactured by th e Upjohn Company and fu r n is h e d through the c o u r te sy o f th a t company* s r e se a r c h d i v i s i o n . -1 5 S im ila r ly , p r e p a r a tio n s o f s y n t h e t ic c h lo ro m y cetin (C on trol Ho. 16*4-562) and fe r m e n ta tio n c h lo r o m y c etin (C o n tro l Ho. 168220) were fu r n is h e d through th e c o u r te s y o f P a rk e-D a v is Company r e s e a r c h d iv is io n . The c o lc h ic in e u sed was a p r e p a r a tio n o f the M a llin k ro d t Chem ical Works. S tock s o lu t io n s o f r e l a t i v e l y h ig h c o n c e n tr a tio n o f ea ch o f th e drugs were made up and s to r e d under r e f r ig e r a t io n u n t i l u s e . A l l c o n c e n tr a tio n s were e x p r e sse d i n p a r ts p e r m il lio n a s a con­ v e n ie n t measure o f c o n c e n tr a tio n . The problem o f s o lv e n t f o r th e drugs was in v e s t ig a t e d a t some le n g t h in a s e r i e s o f p r e lim in a ry ex p erim en ts. I t was found th a t a s i g n i f i c a n t drop in freq u en cy o f d iv i s io n s o v e r zero hour c o n tr o ls o c cu rr e d in r o o t t i p s exposed to d i s t i l l e d w ater f o r e ig h t hours (Column C in Table I ) . On the o th e r hand, G alin sk y (19*^8, 19*^9) r e p o r te d c y t o lo g ic a l a b e r r a tio n s caused by v e ry sm a ll amounts o f th e p h osp hate io n , w hich r u le d out a s su sp e c t the u se o f f u l l s tr e n g th b a la n ced m in eral s a l t s o lu t i o n s . I n v e s t ig a t io n showed th a t e ig h t hour treatm en t in a q u a r te r -s tr e n g th Hoagland s o lu t io n , i d e n t i c a l in co m p o sitio n w ith th a t u sed a t h a lf - s t r e n g t h in the p r e lim in a r y p r e p a r a tio n o f s e e d li n g s , gave no s i g n i f i c a n t d e c r e a s e s in d i v i s i o n fr e q u e n c y , changes i n r e la t iv e freq u en cy o f s t a g e s , o r in c r e a s e s in in c id e n c e o f a b n o r m a litie s o ver in itia l c o n t r o ls (Column B in Table I; Text f i g . l ) . In g e n e ra l q u a r te r -s tr e n g th m o d ified Hoagland s o lu t io n was u se d a s s o lv e n t , and a l l Q u a n tita tiv e data were taken from such ru n s. Each drug was a ls o t e s t e d u t i l i z i n g d i s t i l l e d w ater from - 16 TABLE I CYTOLOGICAL ANALYSIS OF CONTROL RUNS (A) Zero Hour C o n tro l Number o f ro o t t i p s Number o f D iv is io n F ig u r e s /1 0 ,0 0 0 c e l l s (B) 8 Hours in ^ Hoag. (C) 8 Hours in D ie t . HgO 30 *+93 ±18 475 ± 5 0 210 ±39 $ o f T o ta l D iv is io n s in E a r ly Prophase 31*1 ± $ o f T o ta l D iv is io n s in Midprophase 2 9 .4 ± - 0 . 7 $ o f T o ta l D iv is io n s in Prom etaphase 5*9 ± 0 .4 $ o f T o ta l D iv is io n s in M etanhase 1 6 .3 d l 0 .7 l6 .1 i2 ,l 19*7 ± 1 * 0 $ o f T o ta l D iv is io n s i n P ostm etap hase 17*3 1 6 .6 ± 0 .9 13*8 ± .1 .2 $ o f D iv is io n s Abnormal 1 .2 i 0 .7 0 .7 ± .0 .2 31*7 ± 2 .0 37*5 ± 1 - 6 i2 ,7 2 0 .3 ^-1*1 6 .4 ± . 0 .6 8 .7 ± 1 . 3 2 9 -3 1 .3 ^ 0 .4 4 .0 ± 0 . 6 - 17 - 6 *0} &w oo el •H I a* S A© Time. $Qi a 8 A S' J © 0 o Pm O vr> o vo Numbers □□ with 0 .8 +§* > 0 of Mitoses S' CO to 4 Figure 1A. Control <\! ^ O of Absolute J* 0 0 < D Buns - Variations © k o o VO o on, v i o o -3- _ 1 o o 0^ . ■^ J . , o o _J._ o o s r a s ooo'oi / sasaim £0 huhhqn Text Early Prophase OD O -1 8 an o r d in a r y la b o r a to r y m etal s t i l l a s s o lv e n t in o rd er to d eterm in e i f e f f e c t i v e dosage th r e s h o ld s v a r ie d from th o se found u s in g q u a r te r s t r e n g t h Hoagland s o lu t io n . In th e ca se o f str e p to m y c in , where a w ide d isc r e p a n c y was found to e x i s t , a run was made w ith h a lf - s t r e n g t h H oagland to fu r t h e r e x p lo r e t h is d isc re p a n cy . Runs were p lan n ed w ith th e g o a l o f t e s t i n g each drug o v e r a range o f c o n c e n tr a tio n s wide enough th a t, a f t e r e ig h t hours o f t r e a t ­ m ent, the e f f e c t s would range from com plete n e c r o s is a t one extrem e to i n s i g n i f i c a n t d e v ia t io n from e ig h t hour c o n tr o l s l i d e s a t the o th e r extrem e. T h is proved im p r a c tic a l i n s e v e r a l c a s e s . The l e t h a l th r e s h o ld o f c o lc h ic in e was n ev er a s c e r t a in e d , a s i t was d e c id e d th a t th e in fo rm a tio n to be g a in ed was n o t worth th e ex­ p e n d itu r e o f r e l a t i v e l y la r g e q u a n t it ie s o f t h i s ex p e n siv e drug. In th e c a se o f A c ti-d io n e the p r e c is e low er l i m i t o f c y t o lo g ic a l e f f e c t i v e n e s s was n ot determ ined. Runs made a t c o n c e n tr a tio n s a s low a s 0 .2 ppm showed w e ll d e fin e d r e a c t io n s . A fu r th e r problem was en co u n tered w ith the l a t t e r drug in th a t, even a t the lo w e s t d o sa g es t e s t e d , i t s a c t io n in red u cin g freq u en cy o f d iv i s io n s was so e f f e c t i v e th a t by e ig h t hours c y t o lo g ic a l a n a ly s is was e x tr em e ly d i f f i c u l t due to th e s c a r c it y o f d iv i s io n f i g u r e s . For t h is r ea so n th e e f f e c t s o f d if f e r e n t c o n c e n tr a tio n s o f A c ti-d io n e were s tu d ie d a f t e r fo u r hours o f treatm en t r a th e r than a f t e r e ig h t hou rs. A fte r th e e f f e c t s o f v a r y in g the c o n c e n tr a tio n o f a drug had b een s tu d ie d , runs were made to in v e s t i g a t e changes o c c u r r in g w ith tim e when a t y p i c a l l y e f f e c t i v e dose o f the drug was a p p lie d . For t h i s purpose a dose was s e le c t e d w hich e x h ib ite d a h ig h degree o f -1 9 c y t o l o g i c a l e f f e c t i v e n e s s , "but w hich was "below th e l e v e l o f com­ p l e t e l e t h a l i t y a f t e r e ig h t hours o f trea tm en t. In such runs c o l l e c t i o n s were made a t the end o f 1, 2, 3» 8 hours o f tr e a tm e n t. C. C y to lo g ic a l E xam ination A ll ex am in ation o f s l i d e s was done w ith a 90X o i l im m ersion o b j e c t iv e and 1 2 .5X o c u la r s . C r i t i c a l illu m in a t io n o f th e s l i d e was p r o v id e d "by a rib b on fila m e n t lamp w ith a typ e B green f i l t e r b etw een th e lamp and the m icroscope m irror. Where o n ly . q u a lit a t i v e in fo r m a tio n co n cern in g e f f e c t s was de­ s ir e d s e v e r a l w id e ly sep a ra te s t r i p s o f the s l i d e were c a r e f u l l y exam ined by tr a c k in g a c r o s s the e n t ir e s l i d e u s in g the h o r iz o n ta l ad ju stm en t o f th e m echanical s ta g e o n ly . C a refu l n o te s were tak en on ap p aren t freq u en cy o f d iv i s i o n f ig u r e s , ty p e s and p r e v a le n c e o f a b n o r m a litie s and f i n a l l y e v id e n c e s o f n e c r o t ic changes in b o th d i v i s i o n f ig u r e s and r e s t in g n u c l e i . Where q u a n t it a t iv e data were d e s ir e d , t h i s procedure was r i g i d l y sta n d a r d iz e d . The s l i d e was exam ined s y s t e m a t ic a lly in h o r iz o n t a l s t r i p s a s b e fo r e , bu t e x tr a care was u sed to in su r e th a t an u n b ia sed sample was exam ined, d e s p ite the f a c t th a t w ith good s l i d e prepa­ r a t io n one can e x p e c t a r e l a t i v e l y random d i s t r ib u t i o n o f th e d i f ­ f e r e n t p o r tio n s o f th e ro o t t ip o v er the e n t ir e s l i d e . Hever l e s s than th r e e com plete tr a c k s a c r o s s th e s l i d e were exam ined, d i s ­ t r ib u t e d a p p ro x im a tely one q u a r te r , one h a l f , and th re e q u a rter s o f th e way down the 22 x 22 ram c o v e r g la s s . -2 0 T h is e x a m in a tio n c o n s is t e d o f k eep in g a c c u r a te count o f a l l d i v i s i o n f ig u r e s s e e n . D iv is io n f ig u r e s were s c o r e d by f i r s t d e c id in g to w hich o f th e f i v e g e n e r a l s ta g e s o f m it o s is th e y corresp on d ed . These were somewhat a r b i t r a r i l y d e fin e d a s p ro p h a se, m idprophase, prom etaphase, m etaphase and p o stm eta p h a se. 3Polloxd.ng t h i s d e c is i o n , i t was then determ ined w hether o r n o t the f ig u r e in q u e s tio n was norm al. I f abnorm al, a fu r th e r d e c is i o n had to be made a s to w hich o f s e v e r a l p red eterm in ed ty p es o f a b n o r m a litie s th e f ig u r e corresp on d ed . T his procedure was co n tin u ed u n t i l a minimum o f one hundred d iv i s io n f ig u r e s had been examined p e r s l i d e , e x ce p t in th e c a se o f 6 and 8 hour A c ti-d io n e s l i d e s , where f i f t y and t h ir t y - t h r e e f ig u r e s r e s p e c t iv e ly were examined a s a minimum due to th e extrem e s c a r c i t y o f d i v i s i o n s . At th e same tim e a s the exam in ation o f d i v i s i o n f ig u r e s was b e in g c a r r ie d o u t, a count was k ep t o f r e s t in g n u c le i u n t i l ap­ p r o x im a te ly 1500 had been cou n ted . At t h i s tim e th e r a t io o f d i v i ­ s io n f ig u r e s to t o t a l c e l l s exam ined was c a lc u la t e d . A count o f a l l a b n o r m a litie s se e n in th e r e s t in g n u c le i such a s r a ic r o n u c le i, u n u s u a lly shaped o r o b v io u s ly p o ly p lo id c e l l s was a ls o k e p t. D ata taken in th e s e s t u d ie s are summarized in Appendix T ab les I to VII and form th e b a s is f o r a l l q u a n tit a tiv e a n a ly s is o f th e c y to lo g ic a l e f f e c t s . D. Scone o f I n v e s t ig a t io n The e f f e c t s o f c o lc h i c i n e , A c ti-d io n e and strep to m y cin s u l­ f a t e were s tu d ie d q u a n t it a t iv e ly on b o th a c o n c e n tr a tio n and a tim e b a s i s . The s e v e r a l runs w ith dihydro strep to m y cin s u l f a t e "boro so s t r ik in g a resem blance to th e strep to m y cin s u lf a t e runs th a t o n ly q u a l i t a t i v e s t u d ie s were c a r r ie d o u t. R e s u lts w ith b o th s y n t h e t i c and fe r m e n ta tio n C hlorom ycetin, w h ile in t e r e s t i n g , p ro v ed , a s w i l l be su b se o u e n tly d is c u s s e d , im p r a c tic a l to sc o r e on th e b a s is u sed w ith the o th e r drugs and o n ly q u a li t a t i v e d a ta were o b ta in e d . -2 2 OBSERVATIONS The f i r s t p o r tio n o f the fo llo v d n g o b s e r v a tio n s c o n s i s t s o f a com parative stu d y o f th e q u a n t it a t iv e d ata o b ta in e d from c o lc h ic in e , str ep to m y c in s u l f a t e and A c t id io n e tr e a tm e n ts. F o llo w in g t h i s the q u a lit a t i v e in ­ fo rm a tio n o b ta in e d from th e ch lo ro m y cetin and dihydro­ str ep to m y c in trea tm en ts w i l l be p r e se n te d s e p a r a t e ly . A. Frequency o f D iv is io n s C o lc h ic in e C o lc h ic in e d o sa g es o f 30 ppm and above f o r e ig h t hours ca u sed s i g n i f i c a n t in c r e a s e s in freq u en cy o f d i v i s io n f ig u r e s o v er zero hour c o n t r o ls , and i t i s l i k e l y th a t t h i s e f f e c t o ccu rred a t dosages a s low a s 15 ppm (T ex t f i g s . 2A, 3A ). The e f f e c t o f in c r e a s in g d osage on in c r e a s e in d iv i s io n f ig u r e s appeared to be a g eo m etric one w ith in the range o f 10 ppm to a t l e a s t 50 ppm, the data in ­ d ic a t i n g th a t d o u b lin g the dose in c r e a s e d d i v i s i o n s by a r e l a t i v e l y c o n s ta n t amount (r o u g h ly 120 d iv i s io n s p e r 1 0 ,0 0 0 c e l l s ) . By th ree hours a dose o f 50 ppm r e s u lt e d in a s i g n i f i c a n t i n ­ c r e a s e o f d iv i s io n f ig u r e s (T ext f i g s . 5A, 6A ), While th e b u lk o f t h i s in c r e a s e i s accou n ted f o r by an in c r e a s e i n the number o f metapha.ses , th e r e i s good reason to su sp ect th a t a l l s ta g e s sh are i n th e in c r e a s e . In t h i s r e s p e c t i t was found th a t prop h ases in some e ig h te e n s l i d e s r e p r e s e n tin g treatm ent w ith 50 ppm f o r th r e e to e i g h t hours showed an in c r e a s e in frequency s i g n i f i c a n t a t the th r e e p e r c e n t l e v e l o v er zero hour c o n tr o ls (T able I I ) . -2 > TABLE I I FREQUENCY OF DIVISIONS IN COLCHICINE TREATED ROOT TIPS COMPARED WITH CONTROLS D iv is io n s in Prophase p e r 1 0 ,0 0 0 C e lls D iv is io n s L a te r than Prophase p e r 1 0 ,0 0 0 C e lls 30 3 3 1 .5 1 6 1 .5 18 h 0 7 .3 No. o f S li d e s Zero Hour C o n tr o ls 50 ppm C o lc h ic in e 3 to 8 h r s . 1 3 .4 m 3*1 338. h (1 ) D iffe r e n c e from c o n tr o ls p rob ab ly s i g n i f i c a n t (2 ) D iffe r e n c e from c o n tr o ls h ig h ly s i g n i f i c a n t 7 -5 1 9 . 3 '^ ' A c ti-d io n e A c t i- d io n e , in c o n tr a s t to c o lc h ic in e r e s u lt e d in v e r y s i g ­ n i f i c a n t d e c r e a s e s in d iv i s io n freq u en cy “by fo u r hours a t a l l c o n c e n tr a tio n s t e s t e d (T ext f i g s . 233, 3®)- Maximum e f f e c t i v e n e s s a t fo u r hours was e x h ib it e d by c o n c e n tr a tio n s o f 1 and A ppm. It fu r t h e r appears th a t 16 ppm was s i g n i f i c a n t l y l e s s e f f e c t i v e in t h i s r e s p e c t than a l l low er c o n c e n tr a tio n s t e s t e d . By one hour a dosage o f 6 ppm r e s u lt e d in a s t r ik in g d e c r e a se in fr e q u e n c y and by s i x hours the number o f d i v i s i o n f ig u r e s p e r s l i d e dropped to such a low v a lu e th a t c y t o lo g ic a l a n a ly s is became d i f f i c u l t (T ext f i g s . 5®, 633). S trep to m y cin Strep tom ycin s u lf a t e in g e n e r a l e f f e c t e d a r e d u c tio n in number -2 b - o f d i v i s i o n s , "but n o t n e a r ly a s s e v e r e ly a s d id A c t i- d io n e . Con­ c e n t r a t io n s o f 100 ppm and above showed s i g n i f i c a n t d e c r e a s e s by e ig h t hours (T ext f i g s . 20, 3C ). I t was n o t u n t i l s i x hours o f tr e a tm e n t w ith 75 ppm th a t a s i g n i f i c a n t d e c re a se was o b serv ed (T ext f i g s . 5 0 , 6 0 ). C l a s s i f i c a t i o n o f A b erra tio n s P ron h ase G e n e ra lly sp ea k in g , prophase f ig u r e s in c o lc h ic in e t r e a te d m a te r ia l showed no more d e v ia tio n s from a normal appearance than d id c o n t r o l m a t e r ia l. P rophases in A c ti-d io n e m a te r ia l on th e o th e r hand showed v e r y marked d e v ia tio n s from norm al; in f a c t , a t m oderate d osages the b u lk o f A c ti-d io n e a b e r r a tio n s o ccu rred in t h is sta g e . S trep tom ycin e f f e c t s i n prophase in g e n e ra l resem bled th o s e produced by A c t i- d io n e , but a s a r u le d id n ot approach the same i n t e n s i t y o f e x p r e s s io n o r h ig h freq u en cy. Prophase a b e r r a tio n s o b serv ed i n th ese trea tm en ts can be p la c e d in th re e g e n e r a l c a t e g o r ie s . i s o v e r c o n tr a c tio n . The most s t r ik in g a b n o rm a lity T y p ic a lly chromosomes in such a c e l l a re o r i­ e n te d to form a h o llo w sphere a s i f co n ta in ed i n an in t a c t n u c le a r membrane in normal midprophase arrangem ent. In th e type o f o v e r - c o n tr a c tio n d e s ig n a te d a s ”b a l l prom etaphase1' (P la t e IV, P ig s . 7 , 8 ) , chromosomes are c o n tr a c te d to normal prom etaphase o r metaphase le n g t h and in the ty p e d e sig n a te d a s " o v erco n tra cted prom etaphase11 ( P la t e IV, P ig s . 9 , 10) chromosomes are d e f i n i t e l y s h o r te r than th o se o f a normal m etaphase. Such f ig u r e s show no e v id en ce o f the d isa p p ea ra n ce o f n u c le a r membrane and the clum ping o f chromosomes -2 5 in th e c e n te r o f th e c e l l c h a r a c t e r i s t ic o f th e normal prom etap h ase (P la t e I , P ig s . 7 , 8 , 9 ) . In c o n tr o l and c o lc h ic in e a f f e c t e d m a t e r ia l, such overcon­ t r a c t io n was n o ted but r a r e ly and th en o n ly in the m ild e s t d eg ree, b u t b o th ty p e s were abundant i n A c ti-d io n e tr e a tm e n ts. B a ll p r o - m etap hases were m o d era tely fr e q u e n t in strep to m y cin a f f e c t e d s lid e s ^ b u t c o n tr a c tio n a p p a r e n tly r a r e ly p ro ceed ed to th e p o in t o f p ro d u cin g s i g n i f i c a n t numbers o f o v e r c o n tr a c te d pro me ta p b a se s. The second im portant c a te g o r y o f prophase a b n o rm a lity w i l l be d e s ig n a te d a s " r e v e r tin g prophase" (P la te IV, P ig s , b, 5» 11. 1 2 ). W hile by d e f i n i t i o n o v e r c o n tr a c te d proph ases o f e i t h e r typ e can o n ly o ccu r in prom etaphase, r e v e r s io n appears to occu r a t any tim e from e a r ly prophase to prom etaphase. T y p ic a lly , chromosomes in such f ig u r e s , w h ile r e t a in in g good prophase o r ie n t a t io n , become d i f f u s e and tend to lo s e p r e c is e o u t lin e . R e la x in g o f c o i l i n g o c c u r s and r e l a t i o n a l l y c o ile d double chromonemata fr e q u e n tly be­ come c l e a r l y v i s i b l e . When s e v e r e r e v e r s io n o c c u r s , i t i s se e n in a com plete s e r ie s o f e x p r e s s io n from near normal prophases to c e l l s w hich have what appear to be r e s t in g n u c le i w ith j u s t a s u g g e s tio n o f proph ase chromosome arrangem ent (P la te IV, P ig . 6 ). R e v e r tin g p ro p h a ses were seldom encountered i n c o n tr o l and c o lc h ic in e m a t e r ia l, but were m od erately fr e q u e n t in strep to m y cin s l i d e s and, e x c e p t fo r o v e r c o n tr a c te d p ro p h a ses, were the most fr e q u e n t a b e r r a tio n found in A c ti-d io n e a f f e c t e d c e l l s . The f i n a l prophase anomaly o f s ig n if ic a n c e i s d e sig n a te d a s " p yk n osis" . P y k n o tic p roph ases (P la t e VI, P ig s . 1 1 , 12) assume a -2 6 v a r i e t y o f form s, some o f w hich when m ild ly e x p r e s s e d , are d i f f i ­ c u l t to se p a r a te from r e v e r s io n p ro p h a ses. In g e n e r a l, th e p ic t u r e i s one o f n u c le a r d is in t e g r a t i o n and chromosomal d efo rm a tio n , s t i c k i n e s s , m e ltin g o r fu s in g . b le ( P la t e VI, P ig . 1 1 ) . O ften c o i l i n g "becomes c le a .r ly v i s i ­ P y k n o sis in prophase i s in v a r ia b ly ac­ companied by s im ila r phenomena in a l l o th e r s ta g e s a s w e ll a s i n a d ja c e n t r e s t in g n u c le i. A t th e d o sa g es u se d f o r the runs from w hich q u a n t it a t iv e data were drawn p y k n o sis in prophase was a lm o st n o n -e x is t e n t e x c e p t f o r str e p to m y c in tr e a tm e n ts where i t was m od erately fr e q u e n t a t th e h ig h e s t d o sa g e s. Meta-phase In c o n tr a s t to i t s e f f e c t on prophase A c ti-d io n e produced r e l a t i v e l y few m etaphase f ig u r e s o f extrem e a b n o rm a lity . On th e o th e r hand, m etaphases a f f e c t e d by c o lc h ic in e bore l i t t l e or no resem blan ce to c o n tr o l m etaphases. S trep tom ycin , l i k e A c t i- d io n e , produ ced a much lo w er p r o p o r tio n o f ab erran t m etaphases than d id c o lc h ic in e . A berrant m etaphase f ig u r e s see n in th e s e s t u d ie s f a l l in to th re e g e n e r a l c a t e g o r ie s . The f i r s t are d e s ig n a te d " o v erco n tra cted m etaphases" (P la t e IV, P ig s . 1 3 , 1 4 ) . In such fig u r e s the chromo­ somes have normal o r ie n t a t io n on an e q u a to r ia l p l a t e , but are m arkedly o v e r c o n tr a c te d in com parison to normal metaphase chromo­ somes (P la te I , P ig s . 10, 1 1 ). The second and most s t r ik in g s o r t o f abnormal metaphase i s d e s ig n a te d a s " a k in e tic m etaphase". In such f ig u r e s chromosome -2 7 le n g t h may be the same or s h o r te r than th a t o f normal m etaphase chromosomes. No arrangem ent on an e q u a to r ia l p l a t e has taken p la c e d e s p it e the f a c t th a t chrom atid arms show some s e p a r a tio n w ith o n ly the k in e to chore h o ld in g th e chrom atids to g e th e r . A c tu a l l o c a t i o n o f th e chromosomes in the c e l l may v a ry from s c a t t e r e d ( P la t e V, P ig . 2) to clumped (P la te I I , P ig s . 6 , 7» &)• The f i n a l c a te g o r y o f m etaphases f a l l s under the h ead in g o f p k y n o s is . '^Pyknotic m etaphases" l i k e p y k n o tic p rop h ases are r a r e ly se e n e x c e p t in company w ith p y k n o tic r e s t in g n u c le i and o th e r sta g e s. These f ig u r e s r e p r e se n t n u c le a r d is s o lu t i o n and d e g e n e r a tio n and th e p ic t u r e i s one o f fu s in g , amorphous chromosomes u s u a lly clumped in a h ig h ly a lv e o la r mass (P la te V II, P ig . 6 ). G e n e ra lly sp e a k in g , m etaphases s tr o n g ly a f f e c t e d by c o l c h i ­ c in e were c lo s e to 100$ a k in e t ic w ith the o th er anom alies a p p ea rin g r a r e ly i f a t a l l . Streptom ycin a f f e c t e d t i s s u e showed o c c a s io n a l a k i n e t i c m etaphases and o c c a s io n a l o v e r c o n tr a c tio n . A c ti-d io n e a f ­ f e c t e d m etaphases were r a r e ly a k in e t ic but o v e r c o n tr a c tio n was common. P y k n o sis was r a r e ly caused by any o f the drugs a t th e con­ c e n t r a t io n s u sed f o r the q u a n t it a t iv e runs e x c e p t o c c a s io n a l pyk­ n o t ic m etaphases in th e strep to m y cin t e s t e d m a te r ia l. P ostm etan hase As w ith m etap h ases, the g r e a t m a jo rity o f p ostm etap h ases in c o lc h ic in e a f f e c t e d m a te r ia l were v e ry d if f e r e n t in appearance from th o s e in c o n t r o ls . A c ti-d io n e and strep to m y cin tr e a te d m a te r ia l p r e s e n te d few er and f a r l e s s s t r ik i n g a b e r r a tio n s a t t h i s s ta g e than were produced by c o lc h ic in e tr e a tm e n ts. -2 8 At t h i s s ta g e a b e r r a tio n s were d iv id e d in to fo u r more o r l e s s d i s t i n c t groups. On o c c a s io n th e f i r s t two groups w i l l be r e fe r r e d to a s a s in g le broad c l a s s — " a k in e tic p o stm eta p h a ses" . In u n ip o la r f ig u r e s the e n t ir e 41T complement o f chromosomes forms a s in g le more o r l e s s clumped group r a th e r c e n t r a l l y lo c a te d in th e c e l l . In such c e l l s chromosomes range from n e a r ly normal in le n g t h to c o n s id e r a b ly o v e r c o n tr a c te d (P la t e I I , P i g s . 1 1 , 1 2 , 15, l 6 ) . I t was o f t e n d i f f i ­ c u l t on a n a ly s is to d is t in g u is h such a c e l l from a clumped a k in e t ic m etap hase. The second c l a s s , d e sig n a te d " d iso rg a n ized p o stm eta p h a ses" , in c lu d e s a l l d e v ia t io n s from normal anaphase or te lo p h a se sep ara­ t io n and grouping e x c e p t th o se f ig u r e s w hich can be s t r i c t l y c l a s s i f i e d a s u n ip o la r . Thus unequal or m u ltip o la r g ro u p in g s, b r id g e s , la g g in g chromosomes and random s c a t t e r in g f a l l in to t h i s c l a s s (P la t e I I , P ig s . 9> 10, 13» 1 4 ). The th ir d c l a s s " o v erco n tra cted p o s t m e t a p h a s e s in c lu d e s th ose f ig u r e s in w hich chromosomes have sep a ra ted in to two eq u al w e ll o r i­ e n te d groups, bu t where chromosomes a re s tr o n g ly o v e r c o n tr a c te d com­ p ared to normal postm etap hase chromosomes (P la te IV, P ig s . 1 5 , 1 6 ). The f i n a l c a te g o r y , a lm o st n o n -e x is t e n t a t th e dosages em­ p lo y e d f o r the q u a n t it a t iv e s t u d ie s , i s th at o f "pyknotic p ostm eta­ p h a se s" . Such p o stm eta p h a ses, l i k e p y k n o tic f ig u r e s in prophase and m etaphase, c o n s is t o f a v a r ie t y o f d e t e r io r a t in g n e c r o t ic form s. ( P la t e V, P ig s . 15, 1 6 ). In g e n e ra l c o lc h ic in e treatm en t r e s u lt e d in an abundance o f u n ip o la r and to a l e s s e x te n t d is o r g a n iz e d p o stm eta p h a ses. On th e -2 9 o th e r hand, o v e r c o n tr a c te d p o stm eta p h a ses w ith t h e ir normal grouping and o r ie n t a t io n o f chromosomes were n ever found in c o lc h ic in e a f ­ f e c t e d m a t e r ia l, bu t formed th e p r in c ip le a b e r r a tio n in A c ti-d io n e t r e a t e d m a t e r ia l. E xcept a t th e h ig h e s t c o n c e n tr a tio n s , u n ip o la r and d is o r g a n iz e d p ostm etap h a ses r e s u lt e d o n ly in f r e q u e n tly from A c t i- d io n e tr e a tm e n t. Strep tom ycin showed o n ly in fr e q u e n t abno3>- m a l i t i e s i n t h i s s ta g e a t th e d o sa g es u se d , o v e r c o n tr a c te d , u n i­ p o la r and d is o r g a n iz e d f ig u r e s b e in g seen a t th e lo n g e r tim es and h ig h e r d o sa g e s. R e s tin g N u c le i D e v ia tio n s o f r e s t in g n u c le i can be se p a ra ted in to two p r in c i p l e c l a s s e s . The most i n t e r e s t i n g o f th e se are th o se where th e chrom atin has become d is t r ib u t e d in m ic r o n u c le i. E xcep t in r ec o v e r e d m a te r ia l c y t o k in e s is r a r e ly o ccu rs to form m ic r o c y te s . B in u c le a te c e l l s w ith or w ith o u t e q u a lit y in the s i z e o f n u c le i and c e l l s w ith g r o te s q u e ly shaped, b ilo b e d or m u ltilo b e d n u c le i were fr e q u e n t ly ob served a f t e r some trea tm en ts and appear to be­ lo n g to t h i s same c a te g o r y (P la t e I I I , P ig s . 1, 2 ) . A ll th e p r e - c e e d in g ty p es were seen in c o lc h ic in e tr e a te d m a te r ia l, but were n o t found a f t e r any o f the o th e r trea tm en ts i n any s i g n i f i c a n t freq u en cy . The second c l a s s o f a b erra n t r e s t in g n u c le i are d e s ig n a te d a s p y k n o tic . Here th e appearance ranges from r e s t in g n u c le i w hich are s m a lle r than any found in c o n tr o ls and are v e r y h e a v ily s ta in e d ( P la t e V II, P ig . 3 ) , to n u c le i in w hich th e chrom atic m a te r ia l has con d en sed in t o a v a s t number o f t i n y d r o p le ts w hich may be arran ged -3 0 more o r l e s s h is c o n tin u o u s ly in t o str a n d s l i k e s t r in g s o f beads ( P la t e VI, P ig . 1 2 ) . At d eg rees o f n e c r o s is betw een th e se extrem es th e c o i l i n g o f th e chromonemata i s o f t e n c l e a r l y v i s i b l e in p o r tio n s o f th e d e g e n e r a tin g n u c le i. P y k n o sis was n o t en cou n tered in r e s t in g n u c le i o f c o lc h ic in e t r e a t e d m a t e r ia l, and o n ly r a r e ly d id A c ti-d io n e cause i t a t d o se s b elow 16 ppm. I t was en cou n tered f a i r l y fr e q u e n t ly in most s t r e p t o ­ m ycin t r e a t e d m a t e r ia l. P o lv n lo id v E xcep t f o r some o b v io u s ly p o ly p lo id r e s t i n g n u c le i seen a f t e r c o lc h ic in e tr e a tm e n ts, none o f the drugs a t th e end o f e ig h t hou rs had produced any s i g n i f i c a n t v i s i b l e e v id e n c e o f p o ly p lo id y . In r e c o v e r e d m a te r ia l o n ly c o lc h ic in e trea tm en ts produced s i g ­ n i f i c a n t amounts o f p o ly p lo id d i v i s i o n (P la te I I I , F ig s . 3* 4 , 5> 6 ). However, inasmuch a s q u a n tit a tiv e a n a ly s is was n ot made o f re­ co v ered m a te r ia l, no c l a s s i f i c a t i o n o f the e x p r e s s io n o f t h i s phenomenon was a ttem p ted . C. Range o f T o x ic it y R e s u lts o f the- in v e s t ig a t i o n o f the dosage l e v e l s req u ir ed to produce c y t o lo g ic a l d e v ia tio n s from normal and to perm anently k i l l th e t i s s u e s are summarized in Table I I I . S e v e r a l item s o f in t e r ­ e s t were brought out b y t h is phase o f the in v e s t ig a t i o n . C on sid era b ly h ig h e r d oses o f a l l drugs e x c e p t c o lc h ic in e were r e q u ir e d to produce th e same l e v e l o f e f f e c t s when Hoagland s o lu ­ t io n in s t e a d o f d i s t i l l e d w ater was u sed a s the drug s o lv e n t . T h is e f f e c t was most marked by f a r in the strep to m y cin tr e a tm e n ts, Jo2i * £5 -H H EH eh r o e5 a E o s § o CYTOLOGICAL O Eh rt|* oJ »H R s pi &o M ft -P o £h O m CM *H ft O R O o A cti o A {V cti O O O o rH o O •A O A O- O- O o A o A CM O p o o O O A A rH o A C O cti o o o o 1—1 A IV CA oCti o A IV O P O A o tv A o O P o p O O iH o o A iH p R CD i—1 £ O o O O (PARTS PER MILLION) NECESSARY TO PRODUCE SUMMARY OF DOSAGE LEVEL *-4*cti t§ o 'A tN- CM rH A tv cti o o CM o A oCti O h(c\j cti O S 3 S§ W) o A £V *-<<* Cti o o VO o o 00 o o o P A CM A O P o o O CO A O p o O ij- A A P O 01 CM •H f t A R O P o o tV A CM A rtfa oCd O ft CO Eh p W CM «H cti O A —-j o *h trj R O CO CM O Cti VO iH CO rH O P o CM VO *H O O P O CM o W) CD P rH cti ft iH R co R O >s a o p ft CD R P CO O P W A CM o CM p R W) £ CD t> rH O CO o P A CM iH cti . O »d •H rH t*D O O ft rH O OQ <1> P U >S ft O EH o A O P o A A O •H ft R e O ft ft O CQ P R R O a Cti P R cti o •rH ft •rH P R rd CD P F-t CD •H CQ 5> R o o R •H O S o p ft CD O R o R *d ft o p P cti CQ O CD SH P CO 'd *d •rH o R g R £ R •rH £ rd CM fi O iH ft •H Cti •H o' •H o o CM A rH I— I £ CD I— 1 A +»o ca CM *H ft R R • J AS BP R -rH a £ a & S cti o A r ’s u CD > o o O CD f t in ft o CD £ a tfti f t 10 o g CD tti) Cti w O R & •H P CD CD > R 'H ft o R CM rH CM >>»✓ ' —-1 CD ' d y j © S +& > © a © o 2A. Bight W A fa ou Figure □ □□□Q o O o c^ o o VO o o V A O 5 o oCA O O CM O o O r -i s m s o 000*01 /SH50IIM £0 S3HK021 B pJ d *H O O S Wo ■p Text © Hour Colchicine } Treatment. Variation CM Dose - 39 - o with VO o u p c6 m © $ S +a © e 2 JS CQ n cm 5 Pi W of Mitoses L Variation □0 □□ Treatment. o PH p? 2B. Four Hoar A cti-dione PH CM °• S C-* CM a aoi—i uv «=ii O * Eh O Pi o JZI o e-t Text Figure SI s o *o o o - o o VO o o V\ o § o o o oo o o Cvi ev STISO 0 0 0 ‘ 01 / SUOXSIAia 10 HEHMftfl 05 tP P P o r* p p O O -p P i W P o o - *K> « o o 0> i oP< & •H © w J5 £ •A § u of Absolute t—i © Numbers o 'A © © j3 w +1 .© Variation OD © O o o v~. O © VT\ 4 O o VA © © © CA O o CM — o © r~i STEED 000*01 I SSSOLIW 10 hkhmin I *p i: Text Figure 20. Eight Hour Streptomycin with D ose. H Treatment. [ | Early Prophase {>i— t of M ito ses CM «*° ^Kl_ — IQ H -P d o o •H e o a* n iH O U Pi •P o o «H o V i* » O -P •H a V \ o /v \ \ O g B 4 \ 8 \ \ \ \ \ o I Eh -3- \ CM n —- i - - r — r O o O 00 o VO o CM SSVMKOSKd - ^3 ** o o •H aJ -P •H £ © -p a o o © c®tf o p ■p O o -p n oJ M TO O cn o s a 3 jJ--Jn PRO­ METAPHASE METAPHASE o EH p R © 8 POST­ METAPHASE "ZZZ - EIGHT hour 5 10 15 CONCENTRATION OP COLCHICINE IN 20 30 PARTS PER MILLION 50 CONTROL T ext P ig u r e 4A, C o lc h ic in e ( e ig h t h ou rs) E f f e c t s w ith Dosage V a r ia tio n . □ ■ ■ - k5 NORMAL PI REVERTING - EARLY PROPHASB n OVERCONTRACTED PROPHASES II BALL PROPHASES *h 0VERCONTRACTEL POSTPROPHASES □ SCATTERED AKINETIC METAP-------^KINETIC >ORGANI >the tap: PYKNOSIS hifc'f'.',:?; POSTMETAPHASE POUR HOUR CONTROL 0 .^* 0.5 l.o 4-.0 8.0 16.0 CONCENTRATION OF ACTI-DIONE IN PARTS PER MILLION T ext F igu re 4B, A c ti-d io n e (f o u r hours) E f f e c t s w ith Dosage V a r ia tio n - 46 . “ MU>PFJQPHASE = $ v '' J'H I 'i 02 4-> Ci . ^ © TO © S' fa o •H to •H > E l O vercon tr. P o stp ro p h a ses □ R e v e r tin g □ B a ll P roph ases I 1 S c a tte r e d A k in e tic M etaphases and B is o r g . A k in e tic P ostm etap h ases yij P y k n o sis © rH G5 O □ | I Normal i I O vercon tr. P rophases TO .3 O cS PRO­ METAPHASE © m 8 METAPHASE 8 8 s' Vi POST­ METAPHASE 1 s § (X , SIGHT HOUR CONTROL 50 100 200 150 175 CONCENTRATION OP STREPTOMYCIN SULPHATE IN PARTS PER MILLION T ext F ig u re 4C. S trep tom ycin ( e ig h t hour) E f f e c t s w ith D osage V a r ia tio n -4 7 “ E. Changes jja E f f e c t s w ith D u ration o f Treatm ent C o lc h ic in e a t 50 PP®, A c ti-d io n e a t 6 ppm and strep to m y cin s u l f a t e a t 175 PP^ were u sed in t h i s phase o f th e i n v e s t ig a t i o n in tr e a tm e n ts ran gin g in le n g th from one to e ig h t hou rs. Appendix T ab les V, VI and VII s e t fo r t h th e c o n s o lid a te d data o b ta in e d from th e s e runs. T ext f i g s . to 50 g iv e the a b s o lu te number o f f ig u r e s f o r each sta g e a t the s e v e r a l tim es fo r ea ch drug. T ext f i g s . 7A to 7® show th e v a r ia t io n w ith time o f th e r e l a t i v e p r o p o r tio n s o f each s ta g e and o f the r e l a t i v e p r o p o r tio n s o f ea ch typ e o f ab n o rm a lity on a com parative b a s is fo r ea ch drug. T ext f i g s . 8A to 8E show the change w ith time o f the r e l a t i v e pro­ p o r t io n s o f ea ch s ta g e fo r each treatm en t compared w ith th e 95 p e r c e n t c o n fid e n c e l i m i t s o f the v a r ia t io n o f each s ta g e in c o n tr o l ms. te r i a l . C o lc h ic in e (P la te IX, I I I ) A fte r one hour treatm ent w ith 50 PPm c o lc h ic in e a la r g e p r o p o r tio n o f th e m etaphases were se e n to be a k in e t ic and more than o n e -t h ir d o f p ostm etap h ases e x h ib ite d a d egree o f s p in d le d istu r b a n c e a s e v id e n c ed by m u ltip o la r an ap h ases, unequal g ro u p in g s, la g g a r d s and g e n e r a l d is o r g a n iz a tio n . By two hours the propor­ t io n o f m etaphases had in c r e a se d s u b s t a n t ia lly a t the expense o f the o th e r s t a g e s , however, no g e n e r a l in c r e a s e in d iv i s i o n f r e ­ quency was n o te d . S u b s t a n t ia lly a l l o f the m etaphases were a k in e t ic and tw o -th ir d s o f th e p ostm etap h ases were u n ip o la r , the b a la n ce b e in g more o r l e s s d is o r g a n iz e d . At the end o f fo u r hours t o t a l d i v i s i o n f ig u r e s had in c r e a s e d a lm o st f i f t y p er c e n t and th e -4 8 p r o p o r tio n o f m etaphases was double th a t in c o n t r o ls , a c c o u n tin g f o r m ost bu t p ro b a b ly n ot a l l o f th e in c r e a s e in number o f d i v i s i o n s . A l l m etap hases were a k in e t ic , m o stly o f the clumped ty p e , w hereas a l l p o stm eta p h a ses were seen to be u n ip o la r . At t h i s p o in t an e q u i­ lib r iu m was a p p a r e n tly e s t a b lis h e d and an a d d it io n a l fo u r hours tr e a tm e n t brought about l i t t l e change in the o v e r a ll p ic t u r e . No s i g n i f i c a n t numbers o f a b n o r m a litie s or im portant s h i f t s in r e l a t i v e p r o p o r tio n s were ob served in th e prophase s ta g e s a t any tim e d u rin g t h i s treatm en t e x c e p t fo r a m oderate o v e r a ll drop in p r o p o r tio n s a s m etaphase in c r e a s e d i t s r e l a t i v e share o f d i v i s i o n f i g u r e s . A c t i- d io n e (P la te IV) The e f f e c t o f 6 ppm o f A c ti-d io n e was imm ediate and d r a s t ic . T o ta l d iv i s io n freq u en cy dropped to l e s s than tw o -th ir d s o f c o n tr o l v a lu e s by one hour, t h is drop c o n tin u in g s t e a d i l y w ith time u n t i l by e ig h t hours th er e were about o n e -te n th a s many d iv is io n s a s in c o n t r o ls . E a r ly p rop h ases dropped s im ila r ly u n t i l by fo u r hours a l l normal e a r ly p rop h ases had d isa p p ea red . B a ll prom etaphases were much in e v id e n c e a f t e r one hour, and by two hours prometa­ p h a se s had s t a r t e d to in c r e a s e in p r o p o r tio n to a l l o th e r s ta g e s w hich co n tin u ed u n t i l , by e ig h t h o u rs, ab erra n t prom etaphases o f one s o r t or a n o th e r accou n ted f o r more than s e v e n ty p e r c en t o f a l l d iv is io n fig u r e s . The im p re ssio n was one o f c o n s ta n tly in ­ c r e a s in g numbers o f prophases show ing in c r e a s in g d eg rees o f over­ c o n t r a c t io n and d isap p earan ce o f a l l o th e r s t a g e s . A c tu a lly by th e end o f e ig h t h o u rs, in s p it e o f the t e n f o ld d ecrea se in t o t a l numbers o f d iv i s io n s seen , more f ig u r e s were c la s s e d a s prom etaphases -4 9 th an in c o n t r o ls . R eversio n o f p ro p h a ses was s i g n i f i c a n t by two hou rs and r e v e r tin g f ig u r e s in c r e a s e d r a p id ly , becom ing second o n ly to v a r io u s d e g r ee s o f o v e r c o n tr a c tio n a s a major a b n o rm a lity . p h a se s and p o stm eta p h a ses, showing l i t t l e Meta­ i f any s p in d le d is tu r b a n c e , became o v e r c o n tr a c te d and decrea.sed i n p r o p o r tio n s even more than was n e c e s s a r y to accou n t fo r th e g r e a t in c r e a s e in p r o p o r tio n o f pro— m etap h ases. I t w i l l be n o ted th a t a f t e r e ig h t hours trea tm en t w ith t h i s dosage no d i v i s i o n f ig u r e s were sco r e d a s norm al, d e s p it e the f a c t th a t such r o o t t i p s were n o ted to be r ec o v e re d f o r t y - e i g h t h ou rs a f t e r removal from tr e a tm e n t. S trep tom ycin As p r e v io u s ly n o ted , strep to m y cin s u lf a t e a f f e c t e d m a te r ia l shows th e same o v e r a ll freq u en cy ch an ges, k in d s o f a b e r r a tio n s and changes in r e l a t i v e p r o p o r tio n s o f a ta g e s a s does m a te r ia l tr e a t e d w ith A c t i- d io n e , b u t g e n e r a lly w ith c o n sid e r a b ly l e s s i n t e n s i t y o f e x p r e s s io n . v e s tig a tio n . T h is was s u b s ta n tia te d in t h i s phase o f the in ­ L i t t l e o f consequence was ob serv ed in m a te r ia l tr e a t e d w ith 175 PP^ strep to m y cin u n t i l fo u r hours o f trea tm en t. D uring the l a s t fo u r hours o f trea tm en t t o t a l d i v i s io n freq u en cy dropped, e a r ly prophase d ecrea sed and prometaphase in c r e a s e d in r e l a t i v e p r o p o r tio n s . In proph ases r e v e r sio n s became v e r y fr e ­ quent a s d id s l i g h t o v e r c o n tr a c tio n a s ev id en ced by b a l l prom eta­ p h a se s w hich in c r e a s e d w ith tim e. M etaphases and p ostm etap h ases were l i t t l e a lt e r e d from c o n tr o ls ex cep t f o r m oderate overcon­ t r a c t i o n , o c c a s io n a l sp in d le f a i l u r e s and a few p y k n o tic meta­ p h a se s by e ig h t hours o f trea tm en t. - U eneral 50- finrm ng rrr T ext f i g s . 6a , 6b and 6C summarize in a g e n e r a l way th e d i f f e r e n c e s betw een c o lc h ic in e , A c ti-d io n e and str ep to m y c in w ith r e s p e c t to th e e f f e c t o f d u ra tio n o f treatm en t on (a ) th e fr e q u e n c y o f d i v i s i o n f i g u r e s , (b ) th e p r o p o r tio n o f a b n o r m a litie s in t o t a l prophase and (c ) the p r o p o r tio n o f a b n o r m a litie s in t o t a l p o stp ro p h a se. L i t t l e can be s a id a t t h i s p o in t to supplem ent th e s e graphs and th e in fo rm a tio n g iv e n i n the p r e e e e d in g s e c t i o n s , e x c e p t to re-em p h a size some fundam ental d if f e r e n c e s betw een th e a c t io n o f th e s e drugs a t d osages below th e l e t h a l l e v e l . The e f f e c t s o f c o lc h ic in e were found to be v e r y d if f e r e n t from th o se o f A c t i- d io n e , the form er m od erately in c r e a s in g numbers o f d iv i s io n s and p r im a r ily a f f e c t i n g p ostp rop h ase s ta g e s by c a u s in g d e v ia n ts w hich can b e n e r a lly be c l a s s i f i e d a s s p in d le derangem ents. A c t i- d io n e , on the o th e r hand, c a u ses extrem e re­ d u c tio n in number o f d iv is io n s and p r im a r ily a f f e c t s prophase by c a u s in g e f f e c t s th a t can g e n e r a lly be c la s s e d a s r e v e r s io n s and v a r io u s d e g r ee s o f o v e r c o n tr a c tio n . Streptom ycin can be s a id to ca u se e f f e c t s s im ila r , b u t l e s s in d eg ree, to th o se cau sed by A c t i- d io n e . - 51 - f 1^'' © © loo V2 $ & © CO a 5 6 p ft S P (1) o ?-* ' a & p © a p CO o ft I © co JS ft © © & ft 8 ft 8 C D ft <£• .» r^v\ - . - , :V•--■r v ' CO © P! «H O O ao s© O a ■ S S I rH ,© o a ft- 3 !z CM v- 1 ft © ft -p Pi O rH \r * o ©6 0 . . - g 5 5 a © *H ft © p W .H O cd P H^ © rt E+ > o o 00 _ ” O l ip o o 0 - . - P - O O VO o o VO o o CO O O CM O o 3TIS0 0 0 0 401 / SHSOIIW JO HSDaWOK O F TREATMENT .... . -------------------------------- 52 P4 JSA. Su & w o u » © «2 < D W JS +§* > © 6 © m J5 +& > Time m o p s* s P< d •H fl «3 Q} ’3 * O d o> & *m o 8 Pi a CO 6 ppm A c ti-d io n e . X \ X\/ \\ \\ D eviations \ 63. \ \ from nonnal with treatm ent \ \ \ *H Figure ej-P *r4 DURATION OF TREATIfflUT I3ST HOURS ©P Pi O d u ra tio n , PJr— 1 Pin 02 ©o 0 © o® r f P( PJ 02 d 02 C^p rQOPO .O / / 1 o V\ *--------------------------------i ^ v n CO o o - ,1 —, VP\ £>- ........ >1 — o vy .............. . * V O CM (KMMM n E4 - 56 ~ oo D u ra tio n \ / Treatment / with ,vO from Normal / / / / / / \ \ *H 0 Vi. M 0$ 01 < D m o •p \ \ •a O !>* 0 a 1 s I 03 ai 01 o 00)1 •P Qi 8 £ w o p< a © 03 •H 10 a> •H JSP4 S P< a 01 © •rl •H ■P •H rH «H § 'S S *3 Vi. I -P V i. I D eviations u a o o -p Sulphate# / , M 1■ “f;* > :• m l # I .n p i ■I 0 40 175ppm STREPTOMYCIN **••- ' 30 1 I 20 ^ m ; I : . i 1 10 f s';1: TO;r| OF TOTAL MI TO: 40 PERCENTAGE oa o ® 50 20 f 6 ppm ACTL-DIONE 30 | | Normal f ) R e v er tin g ■ fe ; 10i 0 7T DURATION OF TREATMENT IN HOURS T ext F igu re 7 B. V a r ia tio n s in Mi&prophase w ith D u ration o f Treatm ent. CONTROL 5 0 p m eOLCHICI^ ___ — — _ ; v ___ 175ppm STREPTOMYCIN SULPHATE _____ 6ppm ACTI-DIONE ----■ ■ ___ | Normal ] B a ll ] O v erco n tra cted R e v e r tin g m B >' ! '$vf b": 1 0 DURATION OF TREATMENT IN HOURS 8 T ex t F ig u re 70. V a r ia tio n s in Prom etaphase w ith D u ration o f Treatm ent. - 60 CONTROL 10 50ppm COLCHICINE 30 20 10 0 20 17^Di>m STREPTOMYCIN SUO’HATE -----b = PERCENTAGE OF TOTAL MITOSES 10 ih ■. .. 20 . 6px>m ACTX—DXQNB [ Normal 1 O v erco n tra cted [ ] A k in e tic 10 0 1 2 3 ^ DURATION OF TREATMENT IN HOURS T ext F ig u re 7D. V a r ia tio n s in Metaphase w ith D u ration o f Treatm ent. - 6l - CONTROL 20 50FPM OOLCHXCXNB 10 0 175PPM STREPTOMYCIN SmPHAlS m ito se s 20 r 10 - percentage or total ____ 6PPM ACTI-DIONE 20 □ [ Normal 1 O v erco n tra cted D is o r g . A k in e tic U n ip o la r A k in e tic 10 0 1 2 3 DURATION OF TREATMENT IN HOURS T ext F ig u r e 7E. 8 V a r ia tio n in Postm etaphase w ith D uration o f Treatm ent. - 62 © © a ft 8 & ft ft t’s * © N © C3 © WO * *H 5 s i>» . fH W Tl ©o cl 4^> o •H *. o / _U _ o VT\ I ' o Duration s4 *P © Wo $ „ 00 © © with 6© 3O © 03 Prophase <3 rH Cl Gj of Early 8-1 in Proportion ft o CM sasodiH m o n £o ssviiiaoHS^ 8A. V ariations if t Text Figure H P s in Proportion © O P -P •H O «H H a gj OO Cti r— 1 ■tS tti p © O pj +3 V ariations AP P »H 83. $ e H Gj -P • *H a a o o O O 0) p 02• f-t a a o (b © Pi • s . 05 P« 2 0 o Ui O and 10 w ith r e v e r s io n s a t a l l s ta g e s o f prophase shown by P la t e IV, F i g s . 2 , 5 , 11 and 12. S trep to m y cin E f f e c t s The e f f e c t s o f strep to m y cin a t n o n -le th a l l e v e l s appear to bear l i t t l e resem blance to c o lc h ic in e e f f e c t s . On the o th e r hand, s tr e p to m y c in , l i k e A c ti-d io n e , answ ers D*Amato*s d e s c r ip t io n o f a " c e l l p o iso n " , show ing e f f e c t s q u a l i t a t i v e l y i d e n t ic a l w ith th o se produ ced by A c ti-d io n e but c o n s id e r a b ly weaker from a q u a n t it a t iv e p o in t o f v ie w . T his i s seen most s t r ik in g l y when a com parison i s ma.de o f Text f i g s . 3^, F and C. is little m argin betw een I t i s apparent th a t th ere strep to m y cin d o sa g es th a t cause marked c y t o lo g ic a l e f f e c t s and d o sa g es th a t cau se n e c r o s is as e v id e n c e d by appearance o f p y k n o sis. There i s o n ly one apparent e x c e p t io n to t h i s c o n c lu s io n , and th a t i s th a t preprophase in ­ h i b i t i o n o f m it o s is by strep to m y cin appears s i g n i f i c a n t l y a t d o sa g es lo w er than th o se req u ired to produce c y t o lo g ic a l e f f e c t s . C y t o lo g ic a l e f f e c t s w ith strep to m y cin m ight w e ll appear i f , fo r exam ple, treatm en t w ith 100 and 150 ppm strep to m y cin were a llo w e d to c o n tin u e lo n g e r than the e ig h t hours u sed in t h i s in v e s t ig a t i o n . However, i t seems more rea so n a b le to p o s tu la te th a t preprophase in h i b i t i o n o f d i v i s i o n ( a t l e a s t a s caused by str ep to m y c in f d i f f e r s fu n d a m en ta lly from th e r e a c tio n s p r e v e n tin g p rop h ases rea ch in g m etaphase and c a u s in g r e v e r s io n . The p o s s i b i l i t y th a t t h i s may a ls o b e the ca se w ith A c ti-d io n e i s su g g e ste d when one exam ines th e d ata w ith r e s p e c t to a dosage o f 0 .2 ppm and 0 .5 ppm a s sum­ m arized in Text f i g . 3®* -7 5 W hile th e q u a n t it a t iv e in fo rm a tio n on w hich th e se c o n c lu s io n s a r e "based was o b ta in e d through th e u se o f str ep to m y c in s u l f a t e , the e f f e c t s o f dihydro strep to m y cin s u l f a t e showed no fundam ental de­ v i a t i o n s th erefrom and a s f a r a s com parisons were made, i t seems r ea so n a b le to assume th a t th e two drugs are i d e n t i c a l in e f f e c t . C h lorom ycetin E f f e c t s W hile no q u a n tit a tiv e d ata a re a t hand w ith r e s p e c t to th e de­ t a i l e d e f f e c t s o f C hlorom ycetin, i t seems apparent th a t, e x c e p t f o r r a th e r e r r a t i c a l l y e x p r essed i n h ib it io n o f d i v i s i o n s , c le a r cu t c y t o l o g i c a l e f f e c t s o f th e type produced by the o th e r drugs t e s t e d a r e masked by n e c r o t ic changes a t a l l c o n c e n tr a tio n s o f th e drug a b le to e f f e c t such change. I t can be s t a t e d t h a t , compared to A c t i- d io n e and str ep to m y c in , C hlorom ycetin on a c o n c e n tr a tio n b a s is i s c o n s id e r a b ly l e s s t o x ic . However, the th r e s h o ld o f c y t o lo g ic a l e f f e c t i v e n e s s i s no low er than the th r e sh o ld o f l e t h a l i t y . The n e c r o t ic e f f e c t s o f C hlorom ycetin appear to in v o lv e the p o is o n in g o f l o c a l s e c t o r s o f m eristem s r a th e r than a sy ste m ic p o is o n in g o f th e e n t ir e m eristem , a s seems to occu r w ith A c ti-d io n e and str ep to m y c in . U n til fu r th e r in v e s t ig a t i o n i s made and many more d ata are o b ta in e d , s p e c u la tio n con cern in g t h i s apparent d i f ­ fe r e n c e betw een C hlorom ycetin and th e o th er drugs seems f r u i t l e s s . As f a r a s stu d y was c a r r ie d o u t, a s might be ex p e cted , syn­ t h e t i c and fe r m e n ta tio n p r e p a r a tio n s o f C hlorom ycetin appeared i d e n t i c a l in t h e ir a c t io n . S a lt E f f e c t s One o f th e most s t r ik in g phenomena noted in t h i s stu d y was -7 6 th e trem endous d egree o f antagonism shown by c e r t a in m in eral s a l t s to str ep to m y c in a c t io n . E f f e c t i v e th r e s h o ld s o f the drug were r a is e d by a f a c t o r o f ap p ro x im a tely tw enty in g o in g from d i s t i l l e d w a te r to q u a r te r ^ str e n g th Hoagland s o lu t io n , and by a p p ro x im a tely e ig h t y betw een d i s t i l l e d w ater and h a lf - s t r e n g t h Hoagland. Sim i­ l a r s a l t e f f e c t s have been r ep o r te d a s a n ta g o n iz in g a n t i b a c t e r i a l a c t i v i t y o f str ep to m y c in in v it r o (P r a tt and D ufrenoy, 1 9 ^ ) . This im m ed ia te ly s u g g e s ts th a t a c lo s e r e la t io n s h ip e x i s t s betw een the a n t i b i o t i c p r o p e r t ie s o f t h is drug and the e f f e c t s we have n o ted on p la n t m eristem s. As to th e s ig n if ic a n c e th a t t h i s phenomenon has w ith r e s p e c t to th e mode o f a c t io n o f str ep to m y c in , one can o n ly sp e c u la te th a t e i t h e r th e s a l t e f f e c t i s one o f d e c r e a sin g the p e r m e a b ility o f th e plasm a membrane f o r strep to m y cin o r th a t th e e f f e c t in v o lv e s an a l t e r a t i o n o f th e c o l l o i d a l p r o p e r tie s o f protop lasm ren d erin g i t d i r e c t l y or i n d i r e c t l y more r e s i s t a n t to the a c t io n o f the drug. A s a l t e f f e c t was noted w ith A c ti-d io n e and C hlorom ycetin, b u t was n o t n e a r ly a s marked a s w ith strep to m y cin (a f a c t o r o f be­ tw een two and fo u r f o r A c ti-d io n e and about two w ith C hlorom ycetin betw een d i s t i l l e d w a te r and q u a r te r -s tr e n g th Hoagland s o lu t io n ) . W ithout much more in fo rm a tio n th e r e i s l i t t l e to be gain ed in s p e c u la t in g w hether or not t h i s has the same b a s is a s s a l t an­ tagon ism o f str ep to m y c in . An im portant o b s e r v a tio n , however, was th a t v a r ia t io n s i n s a l t c o n c e n tr a tio n d id n o t a l t e r the e f f e c t i v e th r e s h o ld s o f c o lc h i c i n e . This f a c t p e r m its one to -7 7 p o s t u la t e a fundam ental d if f e r e n c e in mode o f a c t io n o f c o lc h ic in e compared to the o th e r drags te sted * C -m ito s is ' \ The term nC - m it o s is w o r i g i n a l l y was in ten d e d to a p p ly to the a l t e r e d m it o t ic p a t t e r n caused hy c o lc h ic in e (Levan, 1938)* Today, a s a lr e a d y p o in te d o u t, i t i s u se d "by most workers to d e s c r ib e any a l t e r a t i o n o f m it o s is where s p in d le c o n tr o l o f postm etaphase chromosomes i s im p aired . One s u s p e c ts th a t h ig h ly c o n tr a c te d pro­ m etap hases a s produced hy exten d ed treatm ent w ith A c ti-d io n e (P la te IV, F i g s . 9 and 10) may fr e q u e n tly he rep o rted a s wC -m ito tic w hy th e c a su a l o b se r v e r . The c a r e fu l workers in t h i s f i e l d have a l ­ read y p o in te d out (!DlAmato, 19^9; Hawthorne and W ilson, 1952: L evan, 1952 and o th e r s ) th a t, d e s p ite the r e l a t i v e l y s im ila r appearance o f m etaphases in a number o f d i f f e r e n t trea tm en ts, the fundam ental cau ses and mechanisms may he q u ite d i f f e r e n t . An advantage o f the u se o f Pisum root t ip s and the tech n iq u e h e r e in d e sc r ib e d i s th a t reco v ery o f tr e a te d ro o t t i p s i s ob­ ta in e d a t a much h ig h e r l e v e l o f e f f e c t than w ith A llium m a te r ia l. L e th a l c o n c e n tr a tio n th r e sh o ld s can be r a th e r p r e c i s e l y determ ined and, c o n tr a r y to p r e v io u s r e p o r ts (W ilson, 1950? W ilson and Bowen 1951; W ilson and Hawthorne, 1 9 5 2 ), th e se were found to be h ig h e r th an th e th r e s h o ld s o f v i s i b l e c y t o lo g ic a l e f f e c t s in th e c a se o f A c t i- d io n e and str ep to m y c in . T h is p r e c is io n o f d e t e m in a t io n o f n o n -r e c o v e r y l e v e l s o f dosage has le d to two o b s e r v a tio n s th a t may be o f fundam ental i n t e r e s t . F i r s t o f a l l , r e c o v e ry o f t is s u e was n o t o b ta in e d a t any c o n c e n tr a tio n l e v e l produ cin g s i g n i f i c a n t -7 8 q u a n t it ie s o f p y k n o s is. S eco n d ly , r ec o v e ry , e x c e p t w ith c o lc h ic in e , was n o t obtained, a t any c o n c e n tr a tio n l e v e l p ro d u cin g s i g n i f i c a n t q u a n t i t i e s o f p ostp rop h ase f ig u r e s showing s p in d le im pairm ent. I t i s th e r e fo r e su g g e ste d th a t sp in d le impairment cau sed by t y p i c a l " c e ll-p o is o n s " i s the r e s u l t o f l e t h a l p r o c e s s e s , perhaps o f ’’p o o r f i x a t i o n ” , and m ight w e ll be an e a r ly sta g e in p y k n o tic d e g e n e r a tio n . I t may be fu r th e r p o in te d out th a t sp in d le derange­ ment cau sed by n o n -r ec o v e ra b le d o sa g es o f A c ti-d io n e (P la te V, F i g s . 2 , 3 , and h ) , strep to m y cin (P la te VI, F i g s . 8 and 15) and C hlorom ycetin (P la te V II, F ig s . h and 5 ) p r e s e n ts a v ery d i f f e r e n t g r o s s appearance compared to th a t caused by e f f e c t i v e p o ly p lo i­ d iz in g d o sa g es o f c o lc h ic in e (P la te I I , F ig s . 5 through 1 6 ). Exami­ n a t io n o f Pisum r o o t t i p s tr e a te d w ith p o ly p lo id iz in g concen tra­ t io n s o f cy clo ch lo ro h ex a n e sh o u ld prove o f i n t e r e s t in t h i s r e s p e c t. I t i s proposed th a t the u se o f the term ”C -m ito s is ” and th e con seq u en t term "C-metaphase" be r e se r v e d fo r s p in d le d is r u p tio n caused by th e few tru e p o ly p lo id iz in g m a te r ia ls . Thus b e fo r e s p in d le impairment co u ld be l a b e l l e d ”C -m ito s is ” , i t would have to be shown th a t i t was a ) a h ig h ly s p e c i f i c r e a c tio n , b) accom panied by l i t t l e or no propha.se d istu rb a n ce and, c) p r o d u ctiv e o f sub­ s t a n t i a l amounts o f p o ly p lo id y . A g en era l term (w ith o u t any p o ly ­ p l o i d i z i n g im p lic a t io n s ) sh ou ld be u sed to d e s c r ib e s p in d le d is ­ turbance r e g a r d le s s o f ca u se. " A kin etic m ito s is " i s su g g e ste d a s a s u it a b le term to d e sc r ib e a l l s o r t s o f s p in d le d is r u p tio n and " a k in e tic m etaphases" o r " a k in e tic postm etaphases" might be u sed to d e s c r ib e f ig u r e s showing s p in d le d istu rb a n ce r e g a r d le s s o f ca u se* T his ought n o t in any way to in t e r f e r e or be co n fu sed w ith th e p r e se n t term " a k in e tic chromosome*1in lim it e d u se synony­ m ously w ith " a c e n tr ic chromosome" to d e sc r ib e a chromosome w ith ­ o u t a k in e to ch o re. -8 0 .SUMMARY 1. The tech n iq u e o f imm ersing ro o t m eristem s o f Pisum sativum s e e d lin g s in known c o n c e n tr a tio n s o f an a n t im it o t ic d is s o lv e d i n a weak b a la n ced m in eral s o lu t io n , produced c o n s is t e n t ly r e pro d u cab le c y t o lo g ic a l a b e r r a tio n s w hich were r e a d ily a n a ly z a b le on b oth a Q u a n tita tiv e a s w e ll a s a q u a lit a t i v e b a s i s . T h is tech n iq u e p o s s e s s e d c e r t a in ad van tages o v er the stan d ard A lliu m c y t o lo g ic a l t e s t p a r t ic u l a r ly a ) in b e in g r e a d ily a v a ila b le throughout th e y e a r , b) in p e r m ittin g reco v ery from s tr o n g e r trea tm en ts and, c ) in p e r m ittin g d eterm in a tio n o f l e t h a l th r e s h o ld s w ith g r e a te r p r e c is io n . 2. C o lc h ic in e was ob served p r im a r ily to a f f e c t p ostp rop h ase s t a g e s , e lim in a t in g s p in d le c o n tr o l o f chromosome o r ie n t a t io n and move­ m ent, thus c a u sin g form ation o f p o ly p lo id r e s t in g n u c le i. p h a ses g e n e r a lly appeared norm al. th e d u ra tio n o f metaphase Pro­ At c o n c e n tr a tio n s t e s t e d , was in c r e a s e d r e s u lt in g in a moderate in c r e a s e in the p r o p o r tio n o f m etaphases to o th e r s t a g e s . E vid en ce i s p r e se n te d s u g g e s tin g a sh o r te n in g o f the tim e re­ q u ir e d fo r a com plete d iv i s i o n c y c le and a consequent in c r e a s e in freq u en cy o f d iv i s io n f ig u r e s . 3. D if f e r in g from c o lc h ic in e in alm o st every r e s p e c t, A c ti-d io n e was shown p r im a r ily to a f f e c t p rop h ases, p r e v e n tin g a t l e a s t th e g r e a t e s t p a r t o f them from e n te r in g metapha.se and th u s re­ s u l t i n g in much o v e r c o n tr a c tio n o f chromosomes. R e v er sio n s to an a r t i f i c i a l in te r p h a se were freq u en t a t a l l prophase s t a g e s . P ostp rop h ase s ta g e s were r e l a t i v e l y norm al, but d e c re a sed in p r o p o r tio n s due to f a il u r e A lm ost com plete in h ib i t io n o f prophases to e n te r m etaphase. o f nev; d iv i s io n s was ob served . S trep tom ycin e f f e c t s were s im ila r to th o se produced by A c t i d io n e , but a t reco v e ra b le d osages were not n e a r ly a s s tr o n g ly exp ressed . I t was shown th a t l i t t l e margin e x is t e d betw een th e th e minimum dosage capable o f producing c y t o lo g ic a l e f f e c t s and th e l e t h a l th r e s h o ld o f t h is drug. E xcep t fo r somewhat e r r a t ic prepropha.se in h ib i t io n o f d iv i s io n , C hlorom ycetin had no c y t o lo g ic a l e f f e c t s th a t could be c l e a r l y se p a r a te d from n e c r o t ic p r o c e s s e s . D e sp ite t h i s , on a concen­ t r a t i o n b a s is t h i s drug was the l e a s t t o x ic o f the th ree a n t i­ b io t ic s te ste d . W ith th e e x c e p tio n o f c o lc h ic in e , the c y t o lo g ic a l a c t i v i t y o f th e drugs t e s t e d was a n ta g o n ized by d is s o lv e d m ineral s a l t s . T h is was v e ry marked in the c a se o f str ep to m y c in , and w ith the o th e r two drugs was o n ly m oderate. o f t h i s " s a lt e f f e c t " a re d is c u s s e d . The p o s s ib le im p lic a tio n s Most im portant o f th e s e i s th e s u g g e s tio n th a t c o lc h ic in e has a mode o f a c tio n funda­ m e n ta lly d if f e r e n t than the o th e r drugs. In th e c a se o f the th re e a n t i b i o t i c s ev id en ce o f n e c r o s is and o f s p in d le impairment was not seen in s i g n i f i c a n t p r o p o r tio n s e x c e p t a t d osages h ig h enough to p rev en t r ec o v e r y . T his and o th e r f a c t s are u sed to support the h y p o th e s is th a t th e s p in d le derangem ents cau sed by th e th ree a n t i b i o t i c s have a v e r y d i f ­ f e r e n t b a s is than th e s p in d le d istu rb a n c es caused by c o lc h ic in e . There i s some b a s is fo r th e p o s s i b i l i t y th a t s p in d le im pairm ents ca u sed by treatm en t w ith A c ti-d io n e , strep to m y cin and Chloro­ m y c e tin a r e a c t u a l l y e a r ly s ta g e s in p k y n o tic d e g e n e r a tio n . The term " a k in e tic m ito sis " i s proposed a s a s u b s t it u t e fo r th e term " C -m itosis" to d e s c r ib e any m it o t ic p r o c e s s where s p in d le c o n tr o l o f p ostp rop h ase chromosomes i s im paired. "C- m it o s is " would be r ese rv e d to d e sc r ib e the c o lc h ic in e typ e o f " a k in e tic " m ito se s where such sp in d le impairment i s a ) a h ig h ly s p e c i f i c r e a c t io n , b) accom panied by l i t t l e or no prophs.se d istu r b a n c e and c ) p r o d u ctiv e o f s u b s t a n t ia l amounts o f p o ly ­ p lo id y . PLATE I Normal M ito s e s from U n tr e a te d M a te r ia l F ig s . 1 -3 E a r ly p r o p h a se s F i g s . h- 6 M idprophases F i g s . 7 -9 P rom etap h a ses F i g s . 1 0 -1 1 M etaphases F i g s . 1 2 -1 3 A naphases F i g s . lif—16 T e lo p h a se s E ach d i v i s i o n o f th e s c a l e r e p r e s e n t s t e n m ic r o n s. 83- » f m & r„v r V v i* * * * n ah & i tk « J 10 9 # ^ **+ * 11 x ‘Y'K < •* • a iv *1 %Oa u. /1 ^ &■ 13 "<>*> >L*y 14* m m 15 | 16 PLATE 11 E f f e c t s o f C o lc h ic in e M ito s e s from m a t e r ia l t r e a t e d f o r e i g h t h o u rs w ith 50 ppm d i s s o l v e d i n q u a r t e r - s t r e n g t h H oagland s o l u t i o n u n l e s s o th e r w is e s p e c i f i e d P ig . 1 Normal e a r l y p r o p h a se F ig . 2 Normal m idprophase F i g s . 3~h Normal p ro m eta p h a ses F ig . 5 S c a t t e r e d a k i n e t i c m etaphase F i g s . 6 -8 Clumped a k i n e t i c m etap h ases F ig , 9 S c a t t e r e d a k i n e t i c m etaphase and d is o r g a n iz e d an ap h ase from m a t e r ia l t r e a t e d w ith 30 ppm a k in e tic F ig . 10 D is o r g a n iz e d a k i n e t i c anapha.se F ig . 11 U n ip o la r a k i n e t i c anaphase from m a t e r ia l t r e a t e d w ith 30 ppm F i g . 12 U n ip o la r a k i n e t i c a n a p h a ses F i g . 13 D is o r g a n iz e d a k i n e t i c w it h 30 ppm F ig , l h D is o r g a n iz e d a k i n e t i c t e lo p h a s e t e lo p h a s e s from m a t e r ia l t r e a t e d F i g s . 1 5 -1 6 U n ip o la r a k i n e t i c t e lo p h a s e s E ach d i v i s i o n o f th e s c a l e r e p r e s e n t s t e n m ic r o n s . -8*1r\ ■ M V . • .'v A » • « t V €• s. . ... . V*-». ' , , Tip; , r » ;> ; « • M. £. I H < p p f ’N « 4 **.■ ' * £ # 10 vk % 1 * 12 11 V; . » ■. *1 *1 **4.^ V* > * i AH %'-r 4* 13 V 14 FUTB I I v >*' *?, •/-■ f ; , ')/ « . *15 I 18 I PLATE I I I E f f e c t s o f C o lc h ic in e M ito s e s from m a t e r ia l t r e a t e d w ith c o l c h i c i n e i n q u a r t e r - s t r e n g t h H oagland s o l u t i o n P ig . 1 M ic r o n u c le i from m a t e r ia l t r e a t e d w ith 30 PPm e i g h t hou rs F ig . 2 M ic r o n u c le i from m a t e r ia l t r e a t e d w ith 20 ppm f o r e i g h t hou rs P i g s . 3 -6 P o l y p lo id m eta p h a ses from m a t e r ia l t r e a t e d w ith 30 ppm f o r e i g h t h ou rs and r e c o v e r e d f o r f o r t y - e i g h t h o u rs E ach d i v i s i o n o f th e s c a l e r e p r e s e n t s te n m ic r o n s . m ~85“ « * {« Mtff & & * * 2 * * $ F aa nt/ >^y ... v > * •«^\ C r \ 4 # ' r * ■ i f - A rtian 7l f l < ft * • * It • • •< - r.C • . #**" ■ * O i 9 10 U' . * • * ‘i 12 It ■ ■ ■ ■ * > -rJ >»• *1 13 r*© & < l*V *1 $ 'X I ? f * f ? . -a V. .A K *■ *3 if :4 T Li " g y ■*^ 9 Jk A w A 10 ii 12 ■ ■ tA » $ ^ • r\^ /*> m V.* * JK * f 13 i “M X— ■ -> ft - V ?> « PLAOffi T I IS "4 PLAlE VII Effects of Chloromycetin Mitoses from material treated for four hours with 1000 ppm synthetic Chloromycetin dissolved in quarter-strength Hoagland solution F ig . 1 R e v e r tin g e a r l y p rop h a se F ig . 2 B a l l p rom etap hase F ig . 3 R e v e r tin g m idprophase F ig . k S c a t t e r e d a k in e t ic - m etap h ases F ig . 5 D is o r g a n iz e d a k i n e t i c an ap h ase F ig . 6 P y k n o tic m etap h ases F ig . 7 P y k n o tic p ro p h a se E ach d i v i s i o n o f th e s c a l e r e p r e s e n t s te n m ic r o n s . 8 > r ' f e 2 y»«* * 4 "o> •5~ * $ 6 £1*3® T II * 90 ' BIBLIOGRAPHY A lle n , IT. 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C y to lo g ic a l e f f e c t s o f some a n t i b i o t i c s . Jou r. Hered. 4 1 :2 2 6 -2 3 1 . _______________ and C. C. Bowen. 1951* C y to lo g ic a l e f f e c t s o f some more a n t i b i o t i c s . Jour. Hered. 4 2 :2 5 1 -2 5 6 . co On 00 00 r\ i— i d iv isio n s CM except 93 - PYKNOTIC co H ON ON iH CO 00 VO o VO ON Run Suumarized 00 VO On CM e\ O NUMBER OP SLIDES vo o g g CO H CO o g g CO n H CO Ei o o o O CO Values are p ercen tag es as n o te d . H vo Control VO CO o rffW M o CO CO (IX TABLE I. o- Data. co of to ta l UNIPOLAR as MICRONUCLEI O•s 4- + V\ IN • o tN +- 4~ PYKNOTIC DISORG. | OVERCONTR, ) NORMAL IN rH PYKNOTIC i UN rH c- .. j!— ...... VO NORMAL CM 3 3EH § ON S | Ai w CH T " j j REVERTING > CO on rH UN --------- i 1 i i \ ; VO CO IN IN ON CM W ^ ftl NORMAL CM IN- ! CM ■ -------------------- -----------H — f i i1 1 IN CM £N CM cm H ; ^ rH -3 CM UN CM ! -MCM ON CM ON ON VO W, M O I—l B t— i ON CM VO 1 ON on M O t-H K M ON CM ON rH ON -------------------- ! UN CM r -1 ON NUMBER OP SLIDES eh IN j rH i REVERTING REVERTING CO CM rH VO NORMAL On i—1 CM rH ---------------------------------:-------M J ON rH pH BALL NORMAL to rH ■ "- CO rl OVERCONTR. U3 VO i j OYERCONTR. I CM rH rH I "1 AKINETIC IN rH rH on 1 CM Values noted* 1 m o PH UNIPOLAR ■=3r Data. 1 rH -3* CM i I o o g O M tn o »-3 8 o o £h s c* b8 M W UN 3 O o & O I—1 W o H) o o S §3 UN rH 8 O f-H W o 8o 5 6 o iH K i 1 o o o S & o " O t-H w o 80 1 o UN Summarized 5 * -*• 8 hour ^ Colchicine ! TABLE II. CM ra. APPENDIX $ CELLS DIVIDING are percentages of total d iv isio n s except Qjiarter-strength Hoagland as s o lv e n t. - 9*+ - CM vo VV CO CM cm CM rv o~\ MICRONUCLEI PYKNOTIC CM DISORG. UNIPOLAR m o 0* CM OVEBCONTR. vo NORMAL "vzr C O I—I H rH CM ON PYKNOTIC CM AKINETIC OVEBCONTR. CM Ov CO rH H rH rH VO OVEBCONTR, r -i rH bail * C M CM tNCM U>v 0~\ OV CM CM r\ CM REVERTING C °\ vo REVERTING S S * s p* w p< NORMAL g O w A EH Q EH i— I VO VT\ rH rv CM P"\ rv § o Iw A Ei O ***? *4 EH H EH ft ft s ft ft CM XTv o CM rH -BT- rH NUMBER op sl id e s CM CM REVERTING ims gPh NORMAL CM CO Acti-dione co NORMAL O CM O t O «*5 S3 & O Pi I EH o < S3 P h PM JH O A Eh O O w • ® cj O Q • - 97 - . •d- ON • -d- -d• vo • 7 .0 o 7 .9 # CELLS DIVIDING ON • + + + + DISORG, v r\ -d UNIPOLAR rH CM rH [SrH NORMAL h J- rH JN> rH ON rH CO rH O d 0 0 rH rH INCM -3 “ rH CM CO £NCM VO CO d d CO CM OVEBCONTR. vo NORMAL ft rH BALL rH rH rH rH -d rH § REVERTING ft ft VO NORMAL rH REVERTING ipsj lf t l w ft 00 NORMAL -d rH i—1 VO CM o oo rH CO D- VO CM CO CM ■—1 rH rH H CM rH CM rH i—1 co O CO CM VO* CM rH CM CM CM O CM CO CM o CO co CO, CO CO VO CM O- CM REVERTING ft ^ ft NORMAL NUMB3BI OP SLIDES EH & g ji co i o o e § o m s H o s ift o 0 0 t£ OVEBCONTR. Q A 0 •p d ft rH AKINETIC s «H O ft d a> « d © W) «P d » t0 O rH PYKNOTIC ?S ^d rt .d QVERCONTR. 8 •> >d d ■p 1 ft CO •H a d o o W -4-5 PYKNOTIC ca d rH O t> 4h MICRONUCLEI % pl O •H 8 @ ft 8 H CO : f - ----------- iw EH ft c!> t— I ft rd • © -P •p d o 0 d > rH ©O d oq O . *0 o rH m icro :tTUCLEI PYKNOTIC DISORGANIZE ) 1 UNIPOLAR rH OVEHCONTR. -3- rs EVi-l NORMAL VO rH 00 j SO _________ f—..... - 00 -S’ ... ....L__ PYKNOTIC 0 H A K IN ETIC 1 OVEHCONTR. VO NORMAL 8 j' rH -3- rH rH rH 00 vo BALL 1 ..... h “....... ... INCr \ CM vs 9 rH OS rs -3- CM rH rH -3- rH rH rH O CSJ il REVERTING § VO NORMAL CM REVERTING 8 ^ 8 S p * s NORMAL A O C"S -3- i? REVERTING R R « 3pH * PH PH NORMAL I— l ; c^s Q ^ v\ H rH CM o g .A-irm«tL-a- jtmfS 1 ^ 1 VS CM VO *""* rH CM ! i o 0 s — 1 j j 1 f i ? 00 | CM CM t ----- c!— ;----- ^ ■- -J.~r ■---------j------------! * so ] so 1 VO ! CM I I rl rl j j ________ i-------------- i-------------- ------------J \ j : o~\ c\ ------------ --------------- !-------------- 1------------- o E-* CM TABLE VI. PH 4 1 rl CQ o iM W i H 0 © us i s o o O US 0 g 1M 1 w i ] ................. i . _ Values are p ercen tag es of to tal d iv is io n s e x c e p t as noted. Q u a rte r-s tre n g th Hoagland solution a s s o lv e n t. • CM Data. . Summarized \6 • CM rH I M m APPENDIX OS • -3- CELLS D IV ID IN G 1 .6 % 6 PPM A cti-dione - 98 D IV ID IN G - Ov * 4 00 . VO 2 -V cv• VO iH • CO 4 . rH M IC R O N U C L E I P Y K N O T IC rH 8 D IS O R G . rH §Eh U N IP O L A R c/a o Ph OVERCONTR. rH i—I C^- NORMAL 1—1 rH 00 i—1 ON rH OV «H Ov 00 1—1 VO rH 1—I rH ’ CM rH OVERCONTR. vo i—1 NORM AL VO H 00 rH o rH VO i—1 i—1 rH CM VO O- 00 1—1 rH VO CO VO CM CM 4 o CO Ov CO Ov CO vo CO 4 r- VO co CO rH OVERCONTR. BALL NORMAL P ) d> < 3 g VO VO C"- R E V E R T IN G o CO, Pi NORMAL g < i @ rH R E V E R T IN G Pi NORMAL NUM BER OP S L ID E S rH CO CO CO «—i O CO CO CM co CM CO CM 00 00 1—1 rH o CO CO CO CO CO CO CO rH 711. W vo I Eh {25 &H & CO o & § o w o g a o w o s I HOUR s f t. s g rH R E V E R T IN G i s g § £ c/a E IG H T s Sulphate 1—1 A K IN E T IC Si Streptomycin w CO Summarized Data. CM P Y K N O T IC 175 Si TABLE CELLS APPENDIX i> Values are $ of total d iv isio n s ex cep t as noted. Q uarter-strength Hoagland s o ln . used as s o lv e n t. - 99 - Charles Clark Bowen candidate fo r the degree o f Doctor o f P hilosophy P in a l exam ination: D is s e r t a tio n : May 20, 1953 , 2:00 p .m ., Botany Seminar Boom A Comparative Study o f the E f f e c ts o f S everal A n tim ito tic s O u tlin e o f S tu d ies Major su b je ct: Minor su b jec t: Botany (C ytology and G en etics) B iochem istry B io g ra p h ica l Items . B om : March 18, 1917» D e tr o it Michigan Undergraduate S tu d ies: U n iv e r sity o f Michigan 1935 -3 9 , Michigan S ta te C ollege 1948 - 49 Graduate S tu d ie s: E xp erien ce: Awards: Michigan S ta te C ollege 1949 ~ 53 A ctiv e duty U nited S ta te s Naval Reserve 1941-46 Member o f organized reserve 1946 - 1953 Graduate A s s is ta n t, Michigan S ta te C ollege 1949 - 1952 In str u c to r in n a u tic a l astronomy and c e l e s t i a l n a v ig a tio n a t U.S. Navy Reserve O ffic e r Candidate School Summers o f 1949. $ 0 , 51 and 52 P ellow o f the N ational Science Foundation 1952-53 Awarded U.S. P u b lic Health S ervice p o std o cto ra l fello w sh ip o f the N ational Cancer I n s t it u t e fo r 1953-54 Member o f: G enetic S o c ie ty o f America Sem Bot S o c ie ty o f the Sigma Xi Alpha D elta Phi fr a te r n it y