15.9. 1975
t h e acid a t d i f f e r e n t t e m p e r a t u r e s , also show some s i m i l a r i t y w i t h d a t a o b t a i n e d a f t e r h y d r o l y s i s in 1 N HC1 a t 60~ for v a r y i n g periods of time. I n f a c t t h e r e s u l t in t h e p r e s e n t s t u d y w i t h 12 N HC1 a t 20~ is i n t e r m e d i a t e b e t w e e n t h a t of 5 N HC1 a t 26~ a n d 1 N HC1 a t 60 ~ Therefore, t h e p r e s e n t a u t h o r feels t h a t t h e process of b r e a k d o w n of p u r i n e in m a t e r i a l s h y d r o l y z e d in 12 N HC1 a t 20 ~ s t a r t s quickly, a n d e v e n m o r e q u i c k l y in 12 N HC1 a t 35~ j u s t like a f t e r 1 N HC1 a t 60~ but w h e r e a s in t h e l a t t e r case f u r t h e r d e g r a d a t i o n of t h e D N A c o m p l e x a n d loss of a p u r i n i c acid are b r o u g h t a b o u t b y
h e a t , in t h e f o r m e r case w i t h 12 N t-IC1 t h e s e are c a u s e d b y t h e o p t i m u m n o r m a l i t y of t h e acid a n d t e m p e r a t u r e .
Rdsurad. O n a 6tudi6 l'effet des c o n d i t i o n s d ' h y d r o l y s e de tissus a m m a l i e n s p a r l ' a c i d e c h l o r h y d r i q u e c o n c e n t r 6 sur la c o l o r a t i o n de F e u l g e n e n lumi&re UV. M. K. DUTT
Depar/ment o~ Zoology, University o/ Delhi, Delhi - 110007 (India), 73 February 1975.
A Rapid and Simple Method for the~.Detection of~.ycoplasma andOther T h e r e are m a n y p u b l i s h e d m e t h o d s for t h e d e t e c t i o n of m y c o p l a s m a s in c u l t u r e d e u k a r y o t i c cells (for reviews see 1, e a n d references w i t h i n ) , m o s t of w h i c h are complex, t i m e c o n s u m i n g a n d o f t e n i n a c c u r a t e giving f a l s e - n e g a t i v e results. R e c e n t l y a m e t h o d h a s b e e n p u b l i s h e d a t h a t i n v o l v e s d e t e c t i o n of c y t o p l a s m i c D N A - c o n t a i n i n g cell contaminants by staining their DNA with either Hoechst 33258 or 4 ' - 6 - d i a m i d i n o - 2 - p h e n y l i n d o l e a n d v i e w i n g b y fluorescence microscopy. T h i s m e t h o d , as are several others, is b a s e d o n t h e f a c t t h a t m y c o p l a s m a s a n d some D N A a n i m a l viruses r e p l i c a t e in t h e c y t o p l a s m . H o w e v e r , one d r a w b a c k of t h i s p r o c e d u r e is t h a t cells m u s t b e g r o w n o n coverslips. I n t h e m e t h o d I describe, a s m a l l c u l t u r e vessel (e.g. a 6 oz m e d i c a l flat) r o u t i n e l y used for s u b - c u l t u r e is a d e q u a t e for t h e assay. A n a n s w e r c a n be o b t a i n e d w i t h i n 2 h, t h u s m a k i n g i t feasible t o c h e c k for t h e p r e s e n c e of m y c o p l a s m a in sister c u l t u r e s before e a c h e x p e r i m e n t , as s u g g e s t e d b y LEVlNE 2. T h e m e t h o d I describe is a simpler a n d m o r e s e n s i t i v e d o u b l e - l a b e l m o d i f i c a t i o n of t h e one described b y SCHNEIDER e t al. ~ a n d m a k e s use of t h e fact t h a t m y c o p l a s m a s synthesize the enzyme pyrimidine phosphorylase 5 which b o t h h y d r o l y s e s u r i d i n e (and t h y m i d i n e ) to uracil (and t h y m i n e ) , a n d c a n ' s a l v a g e ' p y r i m i d i n e s to nucleosides b y t h e reverse r e a c t i o n ; t h i s m e a n s t h a t e x o g e n o u s uracil c a n be i n c o r p o r a t e d i n t o R N A in m y c o p l a s m a - i n f e c t e d cells b u t n o t in u n i n f e c t e d cells ~, 6 w h e r e t h i s e n z y m e is in t h e m a i n a b s e n t ( b u t see ref.2). Methods. A 50 t i m e s c o n c e n t r a t e d m i x t u r e of aHu r i d i n e a n d 14C-uracil was a d d e d to a r a p i d l y growing, s u b c 0 n f l u e n t b o t t l e of ceils to final levels of 1 txCi/ml a n d 0.1 ~xCi/ml respectively, w i t h o u t c h a n g i n g t h e m e d i u m . T h i s b o t t l e was allowed to i n c o r p o r a t e label for 1 h, a f t e r w h i c h t i m e t h e m e d i u m was p o u r e d off a n d t h e a t t a c h e d cells w a s h e d twice w i t h a b a l a n c e d salt solution (e.g. Earle's, H a n k ' s , p h o s p h a t e b u f f e r e d saline).
A comparison of aH-uridine and 1*C-uracil incorporation in healthy, mycoplasma-infected, and kanamycin-treated infected mouse L ceils Condition of cells
Healthy cells Infected ceils Kanamycin (200 ~xg/ml) treatinent for 7 days
1111
Speeialia
Corrected TCA-insoluble radioactivity (cpm) aH-uridine
1*C-uracil
81,540 3,050
7 5,285
7,488
1,522
Intracellular)Contaminants]
5 m l of 0.1% s o d i u m d o d e c y l s u l p h a t e ( S D S ; w/v) was t h e n a d d e d and, a f t e r l e a v i n g for 5 m i n to allow c o m p l e t e lysis, t h e viscous s o l u t i o n was p o u r e d i n t o 10 m l of 2 0 % t r i c h l o r o a c e t i c acid (TCA; w/v) a t 0~ A further 5 m l of 0.1% S D S was used t o r i n s e t h e c u l t u r e vessel, a n d t h i s was also a d d e d to t h e 2 0 % TCA. T h e m i x t u r e was left for 20 m i n a t 0 ~ before f i l t r a t i o n o n t o a W h a t m a n G F / A or G F / C filter; t h i s filter was w a s h e d s e q u e n t i a l l y w i t h 20 m l of ice-cold 1 0 % TCA, 10 m l of e t h a n o l e t h e r (1 : 1 b y vol.) a n d 10 m l of ether. F i n a l l y t h e filter was dried in a n o v e n a t 100~ for 5 min, a d d e d to a v i a l containing PPO-POPOP toluene scintillant and counted in a P a c k a r d liquid s c i n t i l l a t i o n s p e c t r o m e t e r ; t h e s e t t i n g s were s u c h t h a t *H a n d 14C could b e d i f f e r e n t i a t e d e.g. 3H : w i n d o w 50 400, gain 9 0 % ; 14C: w i n d o w 350 1000, gain 10%. W i t h t h e s e s e t t i n g s aH is c o u n t e d a t 4 7 % r e l a t i v e efficiency of counts, a n d I*C a t 3 5 % ; spillover f r o m 14C c h a n n e l i n t o 3H c h a n n e l is 15% of t h e c o r r e c t e d 14C cpm, while t h e r e is no spillover of aH c p m into t h e 14C c h a n n e l . B a c k g r o u n d values were o b t a i n e d b y s t o p p i n g t h e incorp o r a t i o n a t t i m e zero (i.e. i m m e d i a t e l y a f t e r a d d i t i o n of label), a n d processing e x a c t l y as above. T o t a l cellular D N A was isolated essentially as described b y MARMUR 7. Results and discussion. T h e i m p o r t a n c e of using radioa c t i v e l y - l a b e l l e d nucleic acid p r e c u r s o r c o m p o u n d s in a wide v a r i e t y of t y p e s of e x p e r i m e n t s w i t h tissue c u l t u r e cells o v e r t h e l a s t 20 y e a r s or so is obvious. If cell lines are c o n t a m i n a t e d w i t h I n y c o p l a s m a , t h e n t h e h o s t ' s m e t a b o l i s m is r a d i c a l l y a l t e r e d 1 6. F o r e x a m p l e , m y c o plasmas synthesize pyrimidine phosphorylase, an enzyme t h a t h y d r o l y s e s p y r i m i d i n e nucleosides to ribose a n d t h e free base. A n i m p o r t a n t c o n s e q u e n c e of t h i s is t h a t t h e use of t h y m i d i n e or u r i d i n e e i t h e r to label cellular nucleic acids, or in t h e case of t h y m i d i n e , t o s y n c h r o n i z e ceils, b e c o m e s i m p r a c t i c a l , since t h e s e c o u p o u n d s are d e g r a d e d b y t h e m y c o p l a s m a c o n t a m i n a n t s . T h e p r e s e n c e of p y r i m i dine p h o s p h o r y l a s e , h o w e v e r , c a n be e x p l o i t e d in a c o n v e n i e n t a s s a y for t h e p r e s e n c e of m y c o p l a s m a . SCHNEIDER et al. 4 labelled s e p a r a t e c u l t u r e s w i t h e i t h e r 3H-uridine or all-uracil, p u r i f i e d t o t a l cellular R N A f r o m b o t h , a n d d e t e r m i n e d t h e i r specific r a d i o a c t i v i t i e s . T h u s t h e r a t i o of t h e specific r a d i o a c t i v i t i e s of t h e R N A labelled w i t h a H - u r i d i n e to t h e R N A labelled w i t h 3Huracil g a v e a n i n d i c a t i o n of e l e v a t e d levels of u r i d i n e p h o s p h o r y l a s e a n d h e n c e of m y c o p l a s m a c o n t a m i n a t i o n . T h i s m e t h o d h a s n o w b o t h b e e n simplified a n d m a d e m o r e sensitive b y u s i n g u r i d i n e a n d uracil labelled w i t h d i f f e r e n t r a d i o i s o t o p e s in t h e s a m e c u l t u r e vessel, a n d also s p e e d e d u p b y labelling for m u c h s h o r t e r periods. U s i n g 2 r a d i o i s o t o p e s o b v i a t e s t h e n e c e s s i t y for d e t e r m i n ing t h e precise a m o u n t s of R N A p r e s e n t .
1 ] 12
Specialia
If cells are labelled for 1 h w i t h a m i x t u r e of aH-uridine a n d ~4C-uracil to final c o n c e n t r a t i o n s of 1 ~zCi/ml a n d 0.1 ~Ci/ml respectively, t h e i n c o r p o r a t i o n i n t o R N A c a n b e m e a s u r e d as d e s c r i b e d in M e t h o d s . T h e T a b l e shows results f r o m a t y p i c a l e x p e r i m e n t u s i n g m o u s e L ceils. I s o l a t i o n of t o t a l cellular D N A followed b y a n a l y t i c a l isopycnic CsC1 g r a d i e n t c e n t r i f u g a t i o n ( d a t a n o t shown) c o n f i r m e d t h e p r e s e n c e of m y c o p l a s m a D N A ( d e n s i t y a p p r o x . 1.68 g/ml). F u r t h e r m o r e , t r e a t m e n t of m y c o p l a s m a - i n f e c t e d ceils w i t h k a n a m y c i n (200 ~zg/ml) for 7 d a y s s u p p r e s s e d t h e i n f e c t i o n b u t did n o t e l i m i n a t e it; t h i s suggests t h a t i n c o r p o r a t i o n of ~C-uracil i n d i c a t e s t h e p r e s e n c e of some cell c u l t u r e c o n t a m i n a t i o n . Since t h e m e d i u m used (modified 199) c o n t a i n s uracil (2.68 ~M) b u t n o r uridine, its was n e c e s s a r y to c h e c k if t h e a d d i t i o n of cold u r i d i n e (to 3 v M a n d 30 ~zM) h a d a n y effect o n e i t h e r aH-uridine or 14C-uracil i n c o r p o r a t i o n . As expected, ~H-uridine i n c o r p o r a t i o n was d e c r e a s e d b y a d d i t i o n of cold uridine, b u t 14C-uracil i n c o r p o r a t i o n w a s u n a f f e c t e d . Also u s i n g a m e d u i m (e.g. F10) l a c k i n g uracil s l i g h t l y i n c r e a s e d 14C-uracil i n c o r p o r a t i o n in m y c o p l a s m a i n f e c t e d cells. So far t h i s m e t h o d h a s b e e n successfully a p p l i e d to t h e following cell t y p e s : to h u m a n l y m p h o c y t e s u s p e n s i o n c u l t u r e s ; to m o n o l a y e r c u l t u r e s - h u m a n H E P , D 9 8 / A H 2 a n d F L A ceils, m o u s e L a n d 3T3 cells, k a n g a r o o r a t (Dipodomys ordii) cells, m a r s u p i a l (Sminthopsis crassicaudata) cells a n d Xenopus k i d n e y cells; a n d t o several h u m a n - m o u s e s o m a t i c h y b r i d cells. O b v i o u s l y t h i s m e t h o d will also d e t e c t o t h e r cell c u l t u r e c o n t a m i n a n t s
EXPERIENTIA 31/9
(e.g. viruses) if t h e y p r o d u c e e n z y m e s t h a t utilize uracil. M y c o p l a s m a s , h o w e v e r , are t h e m o s t c o m m o n a n d t r o u b l e some cell c u l t u r e c o n t a m i n a n t s .
Summary. A simple, r a p i d a n d s e n s i t i v e d o u b l e radioisotopic m e t h o d is described for t h e d e t e c t i o n of m y c o p l a s m a i n f e c t i o n in tissue c u l t u r e cell lines. K. W . C. PEDENS
M R C Mammalian Genome Unit, University o/ Edinburgh, West Mains Road, Edinburgh E H 9 3 J T (Scotland), 2I April 7975. 1 p. p. LUDOVICI and N. B. HOLMGREN,Methods in Cell Biology Ed. D. M. PRESCOTT; Acadenfic Press, Inc., New York 1973), vol. 6, p. 143. 2 E. M. LEVlNE, Methods in Cell Biology (Ed. D. M. PRESCOTT; Academic Press, Inc., New York 1974), vol. 8, p. 229. 3 W. C. RUSSELL, C. NEWMAN a n d D. H . WILLIAMSON, Nature, Lond. 253, 461 (1975). 4 J~. g . SCHNEIDER, E. J . STANBRIDGE a n d C. J. EFSTEIN, Expl. Cell Res. 8d, 311 (1974). E. M. LEVlNE, Expl Cell Res. 7d, 99 (1972). 6 A. G. PEREZ, J . H . I~IM, A. S. GELBARD a n d B. DIORDJEVIC,
Expl. Cell Res. 70, 301 (1972). J. MARMUR,J. molec. Biol. 3, 208 (1961). * The author thanks P. MOUNTS,V. VAN HEYNINGEN, P. R. MUSICtI, J.M.R. HATFIELD and P. M. B. WALKER for discussions and criticism of the manuscript, and gratefully acknowledges receipt of a IV[RC Scholarship for Training in Research Methods.
PRAEMIA
Prize 'Biochemical A n a l y s i s ' T h e prize of D M 1 0 0 0 0 . - is d o n a t e d f r o m B o e h r i n g e r , M a n n h e i m , a n d is a w a r d e d e v e r y 2 y e a r s a t t h e conference ' B i o c h e m i c a l A n a l y t i c ' in Munich, G e r m a n y , for outs t a n d i n g w o r k in t h e field of b i o c h e m i c a l i n s t r u m e n t a t i o n a n d analysis. T h e d o n a t i o n will t a k e place d u r i n g t h e 1976 c o n f e r e n c e b e t w e e n t h e 9 t h a n d 13th of April. One or several p a p e r s c o n c e r n i n g one t h e m e , e i t h e r p u b l i s h e d
or a c c e p t e d for p u b l i c a t i o n b e t w e e n O c t o b e r 1st 1973 a n d S e p t e m b e r 3 0 t h 1975, m a y b e s e n t i n t r i p l i c a t e before N o v e m b e r 1 5 t h 1975 t o : Prof. Dr. I v a r T r a u t s c h o l d , s e c r e t a r y of t h e prize ' B i o c h e m i c a l Analysis', Medizinische H o c h s c h u l e H a n n o v e r , K a r l - W i e c h e r t - A l l e e 9, I)-3000 Hannover-Kleefeld, Germany.
CONGRESSUS
Canada International S y m p o s i u m o n Flammability and Fire Retardants
Italy International S y m p o s i u m on T h r o m b o s i s and Urokinase in Roma, 30 October-7 November 1975
in Toronto, 5-7 May 7976 P a p e r s s h o u l d deal w i t h f l a m m a b i l i t y a n d fire r e t a r d a n c y of p o l y u r e t h a n e s , plastics, t e x t i l e s a n d fabrics, p a i n t s a n d coatings, t e s t i n g p r o c e d u r e s a n d m a r k e t i n g . P a p e r s are n o w b e i n g solicited a n d t e n t a t i v e titles s h o u l d b e s e n t b y O c t o b e r 15, 1975 t o : V i j a y M o h a n B h a t n a g a r , E d i t o r , A d v a n c e s in Fire R e t a r d a n d s , 209 D o v e r R o a d , Cornwall, O n t a r i o , C a n a d a K 6 J 1TT.
T h e S y m p o s i u m is o r g a n i z e d b y t h e I s t i t u t o Superiore di S a n i t s a n d t h e c h a i r m e n are: Prof. Sol S h e r r y of P h i l a d e l p h i a , U S A , a n d Prof. R. P a o l e t t i of Milano, I t a l y . M a i n t o p i c s : P h y s i o p a t h o l o g y of t h r o m b o s i s . Chemical, b i o c h e m i c a l a n d p h a r m a c o l o g i c a l a s p e c t s of urokinase. E f f e c t s of u r o k i n a s e o n t h r o m b o s i s . Clinical a p p l i c a t i o n s of urokinase. R e g i s t r a t i o n fee will be U S Dollars 30.00. I n f o r m a t i o n a n d r e g i s t r a t i o n b y Prof. R o d o l f o P a o l e t t i , V i a A. Del S a r t o 21, 1-20129 Milano, I t a l y .
Corrigendum MARIAN DORR, I-IANNAH STEINBERG a n d M. SHAPERO : Stimulation o/ Sexual Behaviour in Rats by a Benzo-
dioxane Derivative, E x p e r i e n t i a 37, 91 (1975). I n p a r a g r a p h 1, line 6, a m i n o e t h y l s h o u l d r e a d c o r r e c t l y a m i n o m e t h y l .
t h e acid a t d i f f e r e n t t e m p e r a t u r e s , also show some s i m i l a r i t y w i t h d a t a o b t a i n e d a f t e r h y d r o l y s i s in 1 N HC1 a t 60~ for v a r y i n g periods of time. I n f a c t t h e r e s u l t in t h e p r e s e n t s t u d y w i t h 12 N HC1 a t 20~ is i n t e r m e d i a t e b e t w e e n t h a t of 5 N HC1 a t 26~ a n d 1 N HC1 a t 60 ~ Therefore, t h e p r e s e n t a u t h o r feels t h a t t h e process of b r e a k d o w n of p u r i n e in m a t e r i a l s h y d r o l y z e d in 12 N HC1 a t 20 ~ s t a r t s quickly, a n d e v e n m o r e q u i c k l y in 12 N HC1 a t 35~ j u s t like a f t e r 1 N HC1 a t 60~ but w h e r e a s in t h e l a t t e r case f u r t h e r d e g r a d a t i o n of t h e D N A c o m p l e x a n d loss of a p u r i n i c acid are b r o u g h t a b o u t b y
h e a t , in t h e f o r m e r case w i t h 12 N t-IC1 t h e s e are c a u s e d b y t h e o p t i m u m n o r m a l i t y of t h e acid a n d t e m p e r a t u r e .
Rdsurad. O n a 6tudi6 l'effet des c o n d i t i o n s d ' h y d r o l y s e de tissus a m m a l i e n s p a r l ' a c i d e c h l o r h y d r i q u e c o n c e n t r 6 sur la c o l o r a t i o n de F e u l g e n e n lumi&re UV. M. K. DUTT
Depar/ment o~ Zoology, University o/ Delhi, Delhi - 110007 (India), 73 February 1975.
A Rapid and Simple Method for the~.Detection of~.ycoplasma andOther T h e r e are m a n y p u b l i s h e d m e t h o d s for t h e d e t e c t i o n of m y c o p l a s m a s in c u l t u r e d e u k a r y o t i c cells (for reviews see 1, e a n d references w i t h i n ) , m o s t of w h i c h are complex, t i m e c o n s u m i n g a n d o f t e n i n a c c u r a t e giving f a l s e - n e g a t i v e results. R e c e n t l y a m e t h o d h a s b e e n p u b l i s h e d a t h a t i n v o l v e s d e t e c t i o n of c y t o p l a s m i c D N A - c o n t a i n i n g cell contaminants by staining their DNA with either Hoechst 33258 or 4 ' - 6 - d i a m i d i n o - 2 - p h e n y l i n d o l e a n d v i e w i n g b y fluorescence microscopy. T h i s m e t h o d , as are several others, is b a s e d o n t h e f a c t t h a t m y c o p l a s m a s a n d some D N A a n i m a l viruses r e p l i c a t e in t h e c y t o p l a s m . H o w e v e r , one d r a w b a c k of t h i s p r o c e d u r e is t h a t cells m u s t b e g r o w n o n coverslips. I n t h e m e t h o d I describe, a s m a l l c u l t u r e vessel (e.g. a 6 oz m e d i c a l flat) r o u t i n e l y used for s u b - c u l t u r e is a d e q u a t e for t h e assay. A n a n s w e r c a n be o b t a i n e d w i t h i n 2 h, t h u s m a k i n g i t feasible t o c h e c k for t h e p r e s e n c e of m y c o p l a s m a in sister c u l t u r e s before e a c h e x p e r i m e n t , as s u g g e s t e d b y LEVlNE 2. T h e m e t h o d I describe is a simpler a n d m o r e s e n s i t i v e d o u b l e - l a b e l m o d i f i c a t i o n of t h e one described b y SCHNEIDER e t al. ~ a n d m a k e s use of t h e fact t h a t m y c o p l a s m a s synthesize the enzyme pyrimidine phosphorylase 5 which b o t h h y d r o l y s e s u r i d i n e (and t h y m i d i n e ) to uracil (and t h y m i n e ) , a n d c a n ' s a l v a g e ' p y r i m i d i n e s to nucleosides b y t h e reverse r e a c t i o n ; t h i s m e a n s t h a t e x o g e n o u s uracil c a n be i n c o r p o r a t e d i n t o R N A in m y c o p l a s m a - i n f e c t e d cells b u t n o t in u n i n f e c t e d cells ~, 6 w h e r e t h i s e n z y m e is in t h e m a i n a b s e n t ( b u t see ref.2). Methods. A 50 t i m e s c o n c e n t r a t e d m i x t u r e of aHu r i d i n e a n d 14C-uracil was a d d e d to a r a p i d l y growing, s u b c 0 n f l u e n t b o t t l e of ceils to final levels of 1 txCi/ml a n d 0.1 ~xCi/ml respectively, w i t h o u t c h a n g i n g t h e m e d i u m . T h i s b o t t l e was allowed to i n c o r p o r a t e label for 1 h, a f t e r w h i c h t i m e t h e m e d i u m was p o u r e d off a n d t h e a t t a c h e d cells w a s h e d twice w i t h a b a l a n c e d salt solution (e.g. Earle's, H a n k ' s , p h o s p h a t e b u f f e r e d saline).
A comparison of aH-uridine and 1*C-uracil incorporation in healthy, mycoplasma-infected, and kanamycin-treated infected mouse L ceils Condition of cells
Healthy cells Infected ceils Kanamycin (200 ~xg/ml) treatinent for 7 days
1111
Speeialia
Corrected TCA-insoluble radioactivity (cpm) aH-uridine
1*C-uracil
81,540 3,050
7 5,285
7,488
1,522
Intracellular)Contaminants]
5 m l of 0.1% s o d i u m d o d e c y l s u l p h a t e ( S D S ; w/v) was t h e n a d d e d and, a f t e r l e a v i n g for 5 m i n to allow c o m p l e t e lysis, t h e viscous s o l u t i o n was p o u r e d i n t o 10 m l of 2 0 % t r i c h l o r o a c e t i c acid (TCA; w/v) a t 0~ A further 5 m l of 0.1% S D S was used t o r i n s e t h e c u l t u r e vessel, a n d t h i s was also a d d e d to t h e 2 0 % TCA. T h e m i x t u r e was left for 20 m i n a t 0 ~ before f i l t r a t i o n o n t o a W h a t m a n G F / A or G F / C filter; t h i s filter was w a s h e d s e q u e n t i a l l y w i t h 20 m l of ice-cold 1 0 % TCA, 10 m l of e t h a n o l e t h e r (1 : 1 b y vol.) a n d 10 m l of ether. F i n a l l y t h e filter was dried in a n o v e n a t 100~ for 5 min, a d d e d to a v i a l containing PPO-POPOP toluene scintillant and counted in a P a c k a r d liquid s c i n t i l l a t i o n s p e c t r o m e t e r ; t h e s e t t i n g s were s u c h t h a t *H a n d 14C could b e d i f f e r e n t i a t e d e.g. 3H : w i n d o w 50 400, gain 9 0 % ; 14C: w i n d o w 350 1000, gain 10%. W i t h t h e s e s e t t i n g s aH is c o u n t e d a t 4 7 % r e l a t i v e efficiency of counts, a n d I*C a t 3 5 % ; spillover f r o m 14C c h a n n e l i n t o 3H c h a n n e l is 15% of t h e c o r r e c t e d 14C cpm, while t h e r e is no spillover of aH c p m into t h e 14C c h a n n e l . B a c k g r o u n d values were o b t a i n e d b y s t o p p i n g t h e incorp o r a t i o n a t t i m e zero (i.e. i m m e d i a t e l y a f t e r a d d i t i o n of label), a n d processing e x a c t l y as above. T o t a l cellular D N A was isolated essentially as described b y MARMUR 7. Results and discussion. T h e i m p o r t a n c e of using radioa c t i v e l y - l a b e l l e d nucleic acid p r e c u r s o r c o m p o u n d s in a wide v a r i e t y of t y p e s of e x p e r i m e n t s w i t h tissue c u l t u r e cells o v e r t h e l a s t 20 y e a r s or so is obvious. If cell lines are c o n t a m i n a t e d w i t h I n y c o p l a s m a , t h e n t h e h o s t ' s m e t a b o l i s m is r a d i c a l l y a l t e r e d 1 6. F o r e x a m p l e , m y c o plasmas synthesize pyrimidine phosphorylase, an enzyme t h a t h y d r o l y s e s p y r i m i d i n e nucleosides to ribose a n d t h e free base. A n i m p o r t a n t c o n s e q u e n c e of t h i s is t h a t t h e use of t h y m i d i n e or u r i d i n e e i t h e r to label cellular nucleic acids, or in t h e case of t h y m i d i n e , t o s y n c h r o n i z e ceils, b e c o m e s i m p r a c t i c a l , since t h e s e c o u p o u n d s are d e g r a d e d b y t h e m y c o p l a s m a c o n t a m i n a n t s . T h e p r e s e n c e of p y r i m i dine p h o s p h o r y l a s e , h o w e v e r , c a n be e x p l o i t e d in a c o n v e n i e n t a s s a y for t h e p r e s e n c e of m y c o p l a s m a . SCHNEIDER et al. 4 labelled s e p a r a t e c u l t u r e s w i t h e i t h e r 3H-uridine or all-uracil, p u r i f i e d t o t a l cellular R N A f r o m b o t h , a n d d e t e r m i n e d t h e i r specific r a d i o a c t i v i t i e s . T h u s t h e r a t i o of t h e specific r a d i o a c t i v i t i e s of t h e R N A labelled w i t h a H - u r i d i n e to t h e R N A labelled w i t h 3Huracil g a v e a n i n d i c a t i o n of e l e v a t e d levels of u r i d i n e p h o s p h o r y l a s e a n d h e n c e of m y c o p l a s m a c o n t a m i n a t i o n . T h i s m e t h o d h a s n o w b o t h b e e n simplified a n d m a d e m o r e sensitive b y u s i n g u r i d i n e a n d uracil labelled w i t h d i f f e r e n t r a d i o i s o t o p e s in t h e s a m e c u l t u r e vessel, a n d also s p e e d e d u p b y labelling for m u c h s h o r t e r periods. U s i n g 2 r a d i o i s o t o p e s o b v i a t e s t h e n e c e s s i t y for d e t e r m i n ing t h e precise a m o u n t s of R N A p r e s e n t .
1 ] 12
Specialia
If cells are labelled for 1 h w i t h a m i x t u r e of aH-uridine a n d ~4C-uracil to final c o n c e n t r a t i o n s of 1 ~zCi/ml a n d 0.1 ~Ci/ml respectively, t h e i n c o r p o r a t i o n i n t o R N A c a n b e m e a s u r e d as d e s c r i b e d in M e t h o d s . T h e T a b l e shows results f r o m a t y p i c a l e x p e r i m e n t u s i n g m o u s e L ceils. I s o l a t i o n of t o t a l cellular D N A followed b y a n a l y t i c a l isopycnic CsC1 g r a d i e n t c e n t r i f u g a t i o n ( d a t a n o t shown) c o n f i r m e d t h e p r e s e n c e of m y c o p l a s m a D N A ( d e n s i t y a p p r o x . 1.68 g/ml). F u r t h e r m o r e , t r e a t m e n t of m y c o p l a s m a - i n f e c t e d ceils w i t h k a n a m y c i n (200 ~zg/ml) for 7 d a y s s u p p r e s s e d t h e i n f e c t i o n b u t did n o t e l i m i n a t e it; t h i s suggests t h a t i n c o r p o r a t i o n of ~C-uracil i n d i c a t e s t h e p r e s e n c e of some cell c u l t u r e c o n t a m i n a t i o n . Since t h e m e d i u m used (modified 199) c o n t a i n s uracil (2.68 ~M) b u t n o r uridine, its was n e c e s s a r y to c h e c k if t h e a d d i t i o n of cold u r i d i n e (to 3 v M a n d 30 ~zM) h a d a n y effect o n e i t h e r aH-uridine or 14C-uracil i n c o r p o r a t i o n . As expected, ~H-uridine i n c o r p o r a t i o n was d e c r e a s e d b y a d d i t i o n of cold uridine, b u t 14C-uracil i n c o r p o r a t i o n w a s u n a f f e c t e d . Also u s i n g a m e d u i m (e.g. F10) l a c k i n g uracil s l i g h t l y i n c r e a s e d 14C-uracil i n c o r p o r a t i o n in m y c o p l a s m a i n f e c t e d cells. So far t h i s m e t h o d h a s b e e n successfully a p p l i e d to t h e following cell t y p e s : to h u m a n l y m p h o c y t e s u s p e n s i o n c u l t u r e s ; to m o n o l a y e r c u l t u r e s - h u m a n H E P , D 9 8 / A H 2 a n d F L A ceils, m o u s e L a n d 3T3 cells, k a n g a r o o r a t (Dipodomys ordii) cells, m a r s u p i a l (Sminthopsis crassicaudata) cells a n d Xenopus k i d n e y cells; a n d t o several h u m a n - m o u s e s o m a t i c h y b r i d cells. O b v i o u s l y t h i s m e t h o d will also d e t e c t o t h e r cell c u l t u r e c o n t a m i n a n t s
EXPERIENTIA 31/9
(e.g. viruses) if t h e y p r o d u c e e n z y m e s t h a t utilize uracil. M y c o p l a s m a s , h o w e v e r , are t h e m o s t c o m m o n a n d t r o u b l e some cell c u l t u r e c o n t a m i n a n t s .
Summary. A simple, r a p i d a n d s e n s i t i v e d o u b l e radioisotopic m e t h o d is described for t h e d e t e c t i o n of m y c o p l a s m a i n f e c t i o n in tissue c u l t u r e cell lines. K. W . C. PEDENS
M R C Mammalian Genome Unit, University o/ Edinburgh, West Mains Road, Edinburgh E H 9 3 J T (Scotland), 2I April 7975. 1 p. p. LUDOVICI and N. B. HOLMGREN,Methods in Cell Biology Ed. D. M. PRESCOTT; Acadenfic Press, Inc., New York 1973), vol. 6, p. 143. 2 E. M. LEVlNE, Methods in Cell Biology (Ed. D. M. PRESCOTT; Academic Press, Inc., New York 1974), vol. 8, p. 229. 3 W. C. RUSSELL, C. NEWMAN a n d D. H . WILLIAMSON, Nature, Lond. 253, 461 (1975). 4 J~. g . SCHNEIDER, E. J . STANBRIDGE a n d C. J. EFSTEIN, Expl. Cell Res. 8d, 311 (1974). E. M. LEVlNE, Expl Cell Res. 7d, 99 (1972). 6 A. G. PEREZ, J . H . I~IM, A. S. GELBARD a n d B. DIORDJEVIC,
Expl. Cell Res. 70, 301 (1972). J. MARMUR,J. molec. Biol. 3, 208 (1961). * The author thanks P. MOUNTS,V. VAN HEYNINGEN, P. R. MUSICtI, J.M.R. HATFIELD and P. M. B. WALKER for discussions and criticism of the manuscript, and gratefully acknowledges receipt of a IV[RC Scholarship for Training in Research Methods.
PRAEMIA
Prize 'Biochemical A n a l y s i s ' T h e prize of D M 1 0 0 0 0 . - is d o n a t e d f r o m B o e h r i n g e r , M a n n h e i m , a n d is a w a r d e d e v e r y 2 y e a r s a t t h e conference ' B i o c h e m i c a l A n a l y t i c ' in Munich, G e r m a n y , for outs t a n d i n g w o r k in t h e field of b i o c h e m i c a l i n s t r u m e n t a t i o n a n d analysis. T h e d o n a t i o n will t a k e place d u r i n g t h e 1976 c o n f e r e n c e b e t w e e n t h e 9 t h a n d 13th of April. One or several p a p e r s c o n c e r n i n g one t h e m e , e i t h e r p u b l i s h e d
or a c c e p t e d for p u b l i c a t i o n b e t w e e n O c t o b e r 1st 1973 a n d S e p t e m b e r 3 0 t h 1975, m a y b e s e n t i n t r i p l i c a t e before N o v e m b e r 1 5 t h 1975 t o : Prof. Dr. I v a r T r a u t s c h o l d , s e c r e t a r y of t h e prize ' B i o c h e m i c a l Analysis', Medizinische H o c h s c h u l e H a n n o v e r , K a r l - W i e c h e r t - A l l e e 9, I)-3000 Hannover-Kleefeld, Germany.
CONGRESSUS
Canada International S y m p o s i u m o n Flammability and Fire Retardants
Italy International S y m p o s i u m on T h r o m b o s i s and Urokinase in Roma, 30 October-7 November 1975
in Toronto, 5-7 May 7976 P a p e r s s h o u l d deal w i t h f l a m m a b i l i t y a n d fire r e t a r d a n c y of p o l y u r e t h a n e s , plastics, t e x t i l e s a n d fabrics, p a i n t s a n d coatings, t e s t i n g p r o c e d u r e s a n d m a r k e t i n g . P a p e r s are n o w b e i n g solicited a n d t e n t a t i v e titles s h o u l d b e s e n t b y O c t o b e r 15, 1975 t o : V i j a y M o h a n B h a t n a g a r , E d i t o r , A d v a n c e s in Fire R e t a r d a n d s , 209 D o v e r R o a d , Cornwall, O n t a r i o , C a n a d a K 6 J 1TT.
T h e S y m p o s i u m is o r g a n i z e d b y t h e I s t i t u t o Superiore di S a n i t s a n d t h e c h a i r m e n are: Prof. Sol S h e r r y of P h i l a d e l p h i a , U S A , a n d Prof. R. P a o l e t t i of Milano, I t a l y . M a i n t o p i c s : P h y s i o p a t h o l o g y of t h r o m b o s i s . Chemical, b i o c h e m i c a l a n d p h a r m a c o l o g i c a l a s p e c t s of urokinase. E f f e c t s of u r o k i n a s e o n t h r o m b o s i s . Clinical a p p l i c a t i o n s of urokinase. R e g i s t r a t i o n fee will be U S Dollars 30.00. I n f o r m a t i o n a n d r e g i s t r a t i o n b y Prof. R o d o l f o P a o l e t t i , V i a A. Del S a r t o 21, 1-20129 Milano, I t a l y .
Corrigendum MARIAN DORR, I-IANNAH STEINBERG a n d M. SHAPERO : Stimulation o/ Sexual Behaviour in Rats by a Benzo-
dioxane Derivative, E x p e r i e n t i a 37, 91 (1975). I n p a r a g r a p h 1, line 6, a m i n o e t h y l s h o u l d r e a d c o r r e c t l y a m i n o m e t h y l .
