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Interactions between 125I-labelled RNA from RNA tumor viruses and RNA of other sources
316
Biochimica et Biophysica Acta, 442 (1976) 316--323 © Elsevier Scientific Puklishing Company, Amsterdam -- Printed in The Netherlands
BBA 98679 INTERACTIONS BETWEEN 12SI-LABELLED R N A F R O M R N A T U M O R VIRUSES AND R N A OF O T H E R SOURCES
F. WONG-STAAL, S. GILLESPIE and D,H. GILLESPIE Laboratory of Tumor Cell Biology, National Cancer Institute,Bethesda, Md. 20014 and Bionetics Research Laboratories, 7300 Pearl Street, Bethesda, Md. 20014 (U.S.A.)
(Received January 20th, 1976) Summary Fragmented l~SI-labelled R N A from RNA t u m o r viruses was hybridized to unlabelled RNA from cells, viruses, and homoribopolymers. The viral R N A interacted with all R N A tested, except for certain homoribopolymers. Complex formation with unlabelled RNA was verified by nuclease resistance, b u o y a n t density measurements, and thermal stability in solutions of different ionic strength. The RNAase-resistant complex involved 20--30% of the sequences in the 12SI-labelled viral R N A and formed preferentially with nuclear R N A of cells. 12SI-labelled hemoglobin m R N A , 12SI-labelled immunoglobin light chain (k2) m R N A , or 1251-1abe!led viral R N A from encephalomyocarditis virus (EMC) dit not form RNAase-resistant complexes with unlabelled cellurlar R N A . Introduction Since the discovery of reverse transcriptase [1,2] little has been published concerning the presence or absence in virus-infected cells of R N A complementary in nucleotide sequence to the R N A genomes of R N A t u m o r viruses. Durmg the course of such a study using R N A labelled in vitro to high specific activity with ~2sI [3], it was found that the ~2SI-labelled R N A formed complexes with cell R N A under conditions c o m m o n l y used for molecular hybridization (60°C, 0.4 M sodium phosphate, pH 6.8). It is the purpose of this communication to present the characteristics of these complexes, especially those features that distinguish them from complexes formed between random ribocopolymers and from perfectly base-paired R N A • R N A duplexes. Results
Complexes were formed at 60°C in 0.4 M phosphate buffer between ~2sIlabelled R N A of R N A t u m o r viruses and RNA of cells. The complexes were
317 R N A . RNA duplxes stabilized by complementary hydrogen bonds, since: (1) their b u o y a n t density in Cs2SO4 was characteristic of RNA, n o t of R N A • DNA or R N A • protein (Fig. 1A); (2) they were resistant to RNAase under conditions where single-stranded R N A was destroyed (Table I); (3) they were rendered fully RNAase sensitive by heat (Table I) or by reducing the ionic strength (Table I) and (4) the thermal stability (tin) of the RNAase-resistant structure was increased by increasing the ionic strength (Fig. 1B). The R N A • RNA interaction did not appear to be species specific, for 12SI-labelled R N A from Rauscher mouse leukemia virus (MuLVR) and woolly m o n k e y (simian) sarcoma-leukemia virus (SiSV) complexed equally with RNA from mouse, rat, or human cells (Table II). The interaction of 12SI-labelled RNA from RNA t u m o r viruses with unlabelled R N A did not appear to be a random interaction for the following reasons. First, all the ribohomopolymers tested interacted with the 12SI-labelled R N A minimally. Second, little or no hybridization was obtained between 12sIlabelled t u m o r virus R N A and an excess of t R N A from several sources or R N A from R N A t u m o r viruses (Table II). Third, it has been theorized [4] and documented [5] that populations of R N A molecules containing many different nucleotide sequences will contain self-complementary sequences capable of forming partially double-stranded hybrids. The interactions between ~2SI-labelled t u m o r virus R N A and unlabelled cell R N A differ from interactions among random ribonucleotide polymers in that the complexes formed have a tm (Fig. 1B) some 15°C higher and 40°C sharper than that obtained with complexes formed with the synthetic, random, ribonucleotide polymers [5], corrected to similar ionic conditions for tm determinations. Fourth, the concentration of complementary R N A was higher in the nuclear fraction of disrupted cells than in the cytoplasmic fraction (Fig. 1C). Fifth, ~2SI-labelled RNA from encephalomyocarditis (EMC) virus, ~2SI-labelled human hemoglobin mRNA, and ~2sIlabelled mouse immunoglobin "mRNA failed to form complexes with cell R N A (Table III). However, '2SI-labelled human ribosomal R N A did interact with cell RNA, b u t specifically with nuclear RNA. Though the complexes formed with cell RNA involve 20--30% of the ~2sIlabelled t u m o r virus R N A (1000--5000 nucleotides), the complexes contain many internal RNAase-sensitive points. After treatment of the R N A . R N A complexes with RNAase A and subsequent heat denaturation, the largest, single-stranded ~2SI-labelled fragments recovered were some 30 nucleotides long (Fig. 2), compared to an average of 200--300 nucleotides for the original 12sIlabelled R N A (not shown). ~2SI-labelled t u m o r virus R N A fragments prepared in this way failed to rehybridize to cell RNA. The small size of the singlestranded R N A fragments recovered after ribonuclease treatment and the low tm of the R N A • R N A complexes (Fig. 1B) indicate that the hybrids are not perfectly base paired. It should be emphasized that the a m o u n t of complex formation is markedly influenced b y the detection conditions used, e.g. temperature and ionic strength during ribonuclease treatment (Table I). The RNAase conditions chosen for most of the work reported here were selected for maximizing complex yield while maintaining enough specificity so that ~2SI-labelled t u m o r virus R N A would n o t form RNAase-resistant complexes with itself (Table II).
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Fig. 1. A n a l y s i s o f t h e c o m p l e x e s f o r m e d w i t h 12 $ l - l a b e l l e d viral R N A a n d cell R N A . H y b r i d i z a t i o n m i x t u r e s c o n t a i n e d 125 I-labelled R N A f r o m M u L V R o r SiSV v i r u s a n d u n l a b e l l e d cell R N A in 0.4 M s o d i u m p h o s p h a t e b u f f e r , p H 6.8. M i x t u r e s w e r e b o i l e d f o r 5 rain, t h e n i n c u b a t e d a t 6 0 ° C . (a) 0.1 ng 125 I-lab e l l e d viral R N A ( 1 0 4 c p m ) w a s i n c u b a t e d w i t h 10 # g o f R N A i s o l a t e d f r o m n u c l e i o f f r e s h h u m a n b l o o d l e u k o e y t e s (final v o l u m e = 0.3 m l ) f o r 6 0 h. T h e m i x t u r e w a s t h e n d i l u t e d w i t h 2 m l o f 0 . 4 5 M NaCl, a n d d i g e s t e d w i t h 2 0 # g / m l R N A a s e A a t 22VC f o r 1 h. S a t u r a t e d Cs2SO 4 s o l u t i o n w a s t h e n a d d e d t o a final d e n s i t y o f 1 . 5 0 g / m l , a n d t h e s a m p l e s w e r e c e n t r i f u g e d t o e q u i l i b r i u m a t 3 5 0 0 0 r e v . / m i n in a SW 50.1 r o t o r , o, 125i.labelled SiSV R N A ; o, 1 2 5 I - l a b e l l e d M u L V R R N A ; ~, 3 H - l a b e l l e d ~ X 1 7 4 R N A . D N A d u p l e x circles g e n e r o u s l y p r o v i d e d b y M, S a r n g a d h a r a n w a s c e n t r i f u g e d in a p a r a l l e l g r a d i e n t as a R N A • D N A h y b r i d m a r k e r . (b) T h e r m a l s t a b i l i t y o f t h e R N A - R N A c o m p l e x e s . H y b r i d s w e r e f o r m e d w i t h 125I.labelled viral R N A a n d l e u k o c y t e n u c l e a r R N A as d e s c r i b e d in a. T h e m i x t u r e w a s t h e n d i l u t e d 300fold i n t o 0 . 0 3 M NaCI, 0 . 1 5 M NaCI a n d 1.0 M NaCI, r e s p e c t i v e l y . A l i q u o t s w e r e h e l d a t t h e i n d i c a t e d t e m p e r a t u r e s f o r 5 rain, t h e n i n c u b a t e d w i t h R N A a s e as d e s c r i b e d in a. @, 1 2 5 i . l a b e l l e d SiSV R N A , 0 . 1 5 M NaCI; o, 1251-1abelled M u L V R R N A , 0 . 1 5 M NaCI; m, 1 2 $ I - l a b e l l e d M u L V R R N A , 0 . 0 3 M NaCI; &, 12 $ I - l a b e l l e d M u L V R R N A , 1.0 M NaCI. (c) C o r r e l a t i o n o f h y b r i d yield a n d i n p u t o f u n l a b e l l e d n u c l e a r or c y t o p l a s m i c R N A . H y b r i d i z a t i o n m i x t u r e s c o n t a i n i n g d i f f e r e n t a m o u n t s o f u n l a b e l l e d cell R N A a n d 1 0 p g o f 125 I-labelled SiSV R N A in 2 pl w e r e i n c u b a t e d f o r 16 h a t 6 0 ° C . @, l e u k e m i c m y e l o b l a s t nuc l e a r R N A ; o, n o r m a l h u m a n l e u k o c y t e n u c l e a r R N A ; A, l e u k e m i c m y e l o b l a s t c y t o p l a s m i c R N A ; ~, normal human leukocyte cytoplasmic RNA. Fig. 2. P o l y a c r y l a m i d e r e / e l e c t r o p h o r e s i s o f t h e h y b r i d i z e d 1251-1abelled viral R N A f r a g m e n t s . C o m p l e x e s o f 12 $ I - l a b e l l e d SiSV R N A a n d n u c l e a r R N A f r o m l e u k e m i c m y e l o b l a s t s o r n o r m a l h u m a n l e u k o c y t e s w e r e f o r m e d as d e s c r i b e d in l e g e n d t o Fig. 1. T h e c o m p l e x e s w e r e t h e n t r e a t e d w i t h R N A a s e A ( 2 0 # g / m ] , 2 2 ° C , 1 h, 0 . 5 M NaCI) f o l l o w e d b y p r o n a s e d i g e s t i o n a n d t h e n p h e n o l e x t r a c t i o n . R i b o n u clease-resistant viral R N A f r a g m e n t s w e r e t h e n o b t a i n e d as e x c l u d e d m a t e r i a l o n Biogel P 6 0 c o l u m n s , d e n a t u r e d w i t h h e a t , a n d a n a l y z e d o n 15% p o l y a c r y l a m i d e gels p r e p a r e d as d e s c r i b e d b y Burd a n d Wells [ 1 5 ] . B r o m o p h e n o l b l u e (BPB) w a s u s e d as a d y e m a r k e r , a, l e u k e m i c m y e l o b l a s t n u c l e a r R N A ; o, n o r m a l
319 TABLE I C O N D I T I O N A L R I B O N U C L E A S E R E S I S T A N C E OF R N A . R N A COMPLEXES F O R M E D BETWEEN 1 2 5 I . L A B E L L E D SiSV R N A A N D N U C L E A R R N A F R O M H U M A N C E L L S A p p r o x . 1 0 0 0 c p m o f 125 I-labelled R N A (108 e p m / p g ) f r o m w o o l l y m o n k e y ( s i m i a n ) s a r c o m a - l e u k e m i a v i r u s (SiSV) was h y b r i d i z e d t o 2 lag o f R N A i s o l a t e d f r o m t h e n u c l e i o f f r e s h , p e r i p h e r a l b l o o d m y e l o blasts. T h e n u c l e a r R N A w a s f r e e d o f D N A b y b r i e f e x p o s u r e t o d e o x y r i b o n u c l e a s e f o l l o w e d b y c e n t r i f u g a t i o n t h r o u g h CsC1 [ 1 3 ] . H y b r i d i z a t i o n w a s c a r r i e d o u t at 6 0 ° C in 0.4 M s o d i u m p h o s p h a t e b u f f e r , p H 6.8, f o r 2 0 b. T h e h y b r i d i z a t i o n s o l u t i o n w a s e x p e l l e d i n t o a s o l u t i o n c o n t a i n i n g t h e i n d i c a t e d c o n c e n t r a t i o n o f NaCl, t h e n i n c u b a t e d w i t h 20 p g / m l of R N A a s e A u n d e r t h e specified c o n d i t i o n s . Acid-insoluble r a d i o a c t i v i t y w a s m e a s u r e d . Salt
0.5M
T e m p e r a t u r e of nuclease incubation (°C)
T i m e of n u c l e a s e incubation (11)
R e s i s t a n t (%) RNA • RNA
Without annealing
25
1 2 1 2
45 26 20 12
5 3 2 2
1 2 1 2
19 8 5 4
1 0 0 0
37
0.3M
25 37
0.15 M
25 25 37
1 2 1 2
4 3 2 2
0 0 0 0
0.03 M
25
1 2 1 2
0 0 0 0
0 0 0 0
37
However, it is known that RNA of RNA tumor viruses contains a high degree of self-complementarity or partial complementarity [6,7] and by altering the conditions of complex detection such interactions can be visualized (Table IV). For this experiment, unlabelled RNA of several kinds was immobilized on phosphocellulose discs [8]. These RNA-containing discs were incubated with dissolved 12SI-labelled viral RNA and complex formation was monitored after simply washing the filters with 0.015 M NaC1. The fragmented, 12SI-labelled viral RNA complexed with unlabelled tumor virus RNA with no specificity concerning the phylogenetic origin of the virus and with cell nuclear and tRNA. Considerable complex formation was also obtained with poly(G), but it should be recalled that '2sI preferentially labels cytosine residues [9]. Also it has been reported recently that RNA tumor virus RNA contain C-rich regions that interact with poly(G) [10]. The tm of complexes formed between 12SI-labelled MULVR RNA and immobilized cytoplasmic RNA, nuclear RNA, tRNA, or poly(G) was 63°C in 0.15 M NaC1 and varied directly with ionic strength (not shown). '2SI-labelled hemoglobin mRNA complexed with immobilized poly(G) and with natural RNAs (Table IV). 12SI-labelled rRNA complexed with immobilized poly(G), nuclear RNA, and tumor virus RNA, but hybridized poorly
320 T A B L E II INTERACTION SAY
OF 125I-LABELLED
VIRAL RNA WITH OTHER RNAs: RNAase RESISTANCE
AS-
C o n d i t i o n s f o r h y b r i d f o r m a t i o n are g i v e n in t h e l e g e n d t o T a b l e I. C o n d i t i o n s f o r h y b r i d d e t e c t i o n in t h i s e x p e r i m e n t w e r e r i b o n u c l e a s e t r e a t m e n t in 3 × SSC f o r 2 h a t 2 5 ~ C . Rot values ~ 5 0 f o r all h y b r i d s ( a p p r o x . 2 /~g u n l a b e l l e d R N A / 4 /~1, 6 0 ° C , l h ) . M u L V R , R a u s c h e r m o u s e l e u k e m i a virus; A 3 1 , c u l t u r e d n o r m a l m o u s e f i b r o b l a s t s ; J L S V 1 0 ( M u L V R ) ~ c u l t u r e d m o u s e cells i n f e c t e d b y a n d p r o d u c i n g M u L V R ; N R K , c u l t u r e d n o r m a l r a t k i d n e y cells; N R K (SiSV), c u l t u r e d n o r m a l r a t k i d n e y cells i n f e c t e d b y a n d p r o d u c t i n g SiSV; N C 3 7 , c u l t u r e d h u m a n l y m p h o i d cells; N C 3 7 ( S i S V ) , c u l t u r e d h u m a n l y m p h o i d cells infected by and producing SiSV; PHA, normal human lymphocytes, obtained fresh then cultured for 72 h in t h e p r e s e n c e o f p h y t o h e m a g g l u t i n i n H L , f r e s h h u m a n m y e l o b l a s t s f r o m a p a t i e n t w i t h a c u t e m y e l o g e n o u s l e u k e m i a . Cells ~vere f r a c t i o n a t e d i n t o n u c l e a r a n d c y t o p l a s m i c c o m p o n e n t s as p r e v i o u s l y d e s c r i b e d [ 1 4 ] . T h e n u c l e a r f r a c t i o n w a s w a s h e d r e p e a t e d l y w i t h 1% T r i t o n in 1 5 % g l y c e r o l a n d b u f f e r c o n t a i n i n g 2 . 5 m M Tris, p H 7 . 4 , 2 . 5 m M N a C l , a n d 0 . 4 m M MgCI 2. R N A w a s p u r i f i e d f r o m t h e c y t o p l a s m i c f r a c t i o n b y s e d i m e n t a t i o n t h r o u g h CsC1 [ 1 3 ] . T h e f i n a l n u c l e a r p e l l e t w a s s u s p e n d e d in 1 0 m M Tris, p H 7 . 4 , 5 0 m M NaC1, 1 0 m M MgC12 a n d 5 0 p g / m l D N A a s e , w a r m e d t o 3 7 ° C f o r 5 r a i n t h e n R N A w a s p u r i f i e d b y sed i m e n t a t i o n t h r o u g h CsC1 [ 1 3 ] . RNAase resistant Poly(A) Poly(C) Poly(G) Poly(U • G) tRNA (mouse)
12$I-labelled M u L V R R N A
125I-labelled SiSV R N A
0 0 2 6 5
3 0 3 7 7
Nuclear RNA A31 JLSV10 (MuLV R) NRK NRK(SiSV) NC37 NC37(SiSV) PHA HL
19 19 17 18 14 15 20 24
22 19 15 16 20 12 23 28
Cytoplasmic RNA A31 JLSV10(MuLVR) NRK NRK(SiSV) NC37 NC37(SiSV) PHA HL
15 -10 12 11 10 17 14
17 14 10 11 12 9 18 19
3 4
4 2
Viral R N A SiSV MuLV R
to tRNA. 12SI-labelled rRNA also complexed with immobilized poly(C), a result expected since oligo(G) regions were detected in 28 S tRNA by hybridization to 3H-labelled poly(C) (Marshall, S. and Gillespie, D., unpublished observations). Experiments were done to see whether different sequences in ~2sI-labelled MuLVR RNA hybridized to each unlabelled RNA. I2SI-labelled RNA was hybridized to immobilized RNA, then the unhybridized RNA was annealed with
321 T A B L E III H Y B R I D I Z A T I O N OF N O N - V I R A L 1 2 $ I - L A B E L L E D RNA TO C E L L U L A R RNAs C o n d i t i o n s o f h y b r i d i z a t i o n are d e s c r i b e d in t h e l e g e n d to T a b l e I. R N A f r o m H e L a cells w a s a gift f r o m T. B o r h n , R N A f r o m e n c e p h a l o m y o c a r d i t i s (EMC) virus w a s d o n a t e d b y A. Burness, h u m a n h e m o g l o b i n m R N A w a s g i v e n t o u s b y D. H o u s m a n , m o u s e i m m u n o g l o b i n light c h a i n (k) m R N A was a gift f r o m S. P e s t k a . R N A w a s l a b e l l e d w i t h 1 2 5 I b y W. P r e n s k y . T h e specific a c t i v i t y o f t h e 1 2 5 I - l a b e l l e d R N A s was 5 • 1 0 7 - - 1 0 - 107 c p m / ~ g .
125I-labelledR N A
Human rRNA EMC RNA Hemoglobin R N A IgG m R N A
12$1-1abelledR N A
h y b r i d i z e d (%)
H u m a n nuclear R N A
H u m a n cytoplasmic R N A
17 3 2 4
8 2 4 2
several RNA or DNA preparations, either immobilized or dissolved. In most cases, it appeared t h a t the same sequences in the 12SI-labelled RNA hybridized to different immobilized RNAs. Thus, 12SI-labelled MuLVR RNA sequences t h a t did not hybridize to immobilized poly(G), leukemic myeloblast nuclear TABLE IV COMPLEX FORMATION WITH IMMOBILIZED RNA Urflabelled R N A (1 t~g) w a s i m m o b i l i z e d o n 7 - r a m p h o s p h o c e l l u l o s e discs b y a m o d i f i c a t i o n o f t h e p r o c e d u r e o f S a x i n g e r e t al. [ 8 ] . T h e discs w e r e c o n v e r t e d t o t h e ( b u t y l ) 3 N* f o r m as d e s c r i b e d , i n c u b a t e d w i t h 50 m g / m l c a r b o n y l d i m i d a z o l e in d r y d i m e t h y l f o r m a m i d e at 2 5 ° C f o r 3 h, t h e n w a s h e d t h r e e t i m e s w i t h d r y d i m e t h y l f o r m a m i d e , t h r e e t i m e s w i t h a c e t o n e , a n d d r i e d w i t h N 2 . 5/~1 o f R N A s o l u t i o n in w a t e r w e r e s p o t t e d o n t h e a c t i v a t e d filters a n d d r i e d o v e r n i g h t u n d e r v a c u u m o v e r P 2 0 $ . T h e filters w e r e c o v e r e d w i t h d r y p y r i d i n e a n d i n c u b a t e d 4 h a t 55VC. T h e filters w e r e t h e n w a s h e d w i t h w a t e r , i n c u b a t e d 1 0 m i n a t 2 5 ° C w i t h 10% Tris, p H 9, w a s h e d t h r e e t i m e s w i t h w a t e r , i n c u b a t e d 3 h a t 3 7 ° C w i t h 10% K H 2 P O 4, w a s h e d six t i m e s w i t h 0 . 5 % s o d i u m d o d e c y l s u l f a t e , t w i c e w i t h w a t e r , t w i c e w i t h a c e t o n e , t h e n d r i e d w i t h N 2 a n d s t o r e d a t - - 2 0 ° C . H y b r i d i z a t i o n w a s c a r r i e d o u t in 50 ~1 o f a s o l u t i o n c o n t a i n i n g 1 0 0 0 c p m o f 12 $ I - l a b e l l e d R N A 50% f o r m a m i d e , 0 . 4 5 M NaCI, 4 5 m M s o d i u m c i t r a t e a t 3 7 ° C f o r 4 days. H y b r i d s w e r e s c o r e d as f i l t e r - b o u n d 1 2 s I r e m a i n i n g a f t e r e x t e n s i v e w a s h i n g w i t h 15 m M NaC1 a n d 1.5 m M s o d i u m citrate containing 0.5% sodium d o d e c y l sulfate. Immobilized RNA
12$i.labeHe d R N A ( p e r c e n t c o m p l e x e d ) MuLV R
rRNA
Globin mRNA
Poly(A) Poly(U) Poly(C) Poly(G)
0 0 3 46
0 0 22 35
0 3 2 61
tRNA Adult, chicken, rabbit, or mouse Embryo, mouse
15--20 28--30
Cell n u c l e a r R N A Normal human lymphocyte Leukemic human myeloblast
29 26
23 26
46 49
T u m o r virus R N A SiSV MuLVR AvLVAMV
25 28 25
25 28 25
59 68 77
1--3 7--12
11--25 34--36
322 TABLE V HYBRIDIZATION OF RECYCLED RNA R N A was " r e c y c l e d " b y h y b r i d i z a t i o n to i m m o b i l i z e d R N A as d e s c r i b e d in the t e x t a n d l e g e n d to T a b l e IV. H y b r i d i z a t i o n of r e c y c l e d R N A t o R N A ( p a r t 4) was d o n e w i t h i m m o b i l i z e d n u c l e a r R N A ; h y b r i d i z a t i o n o f r e c y c l e d R N A t o D N A ( p a r t B) was d o n e in s o l u t i o n a n d m o n i t o r e d b y r e s i s t a n c e to R N A a s e . (A) 12$I-labelled R N A u n h y b r i d i z e d to n u c l e a r R N A f r o m n o r m a l h u m a n l e u k o c y t e s I m m o b i l i z e d R N A (2nd cycle)
125I-labelled R N A ( p e r c e n t h y b r i d i z e d ) MuLV R
Normal human leukocyte nuclear RNA Leukemic myeloblast nuclear RNA
Globin mRNA
9
26
16
28
(B) 12$I-labelled R N A u n h y b r i d i z e d to p o l y ( G ) DNA (2nd cycle)
Normal mouse fibroblast MuLV-infected mouse fibroblast
12$I-laballed R N A ( p e r c e n t h y b r i d i z e d )
MuLV R (recycled)
MuLV R (unfractionated)
42 42
25 60
RNA, or mouse e m b r y o t R N A were generally depleted for sequences capable of hybridizing to any of these RNAs or to R N A from the nucleus of human leukocytes or from R N A t u m o r viruses. However, two cases of possible specificity were noted (Table V). First, 12SI-labelled R N A from MULVR selected as not hybridizing t o R N A from the nucleus of normal leukocytes hybridized a b o u t t w o times better to R N A from the nucleus of human leukemic myeloblasts than to nuclear R N A from the normal cells (Table 5A). ~2SI-labelled globin m R N A recycled in the same manner did not show this preference. This suggests the presence of viral-related sequences in the nucleus of leukemic myeloblasts that are missing or at low concentration in the nucleus of normal human leukocytes. Second, ~2SI-labelled R N A from MuLVR selected as not hybridizing to poly(G) hybridized equally to DNA from normal or MuLVa-infected cells, suggesting that viral-specific sequences were selectively removed during the first cycle of hybridization preferentially leaving host-related sequences behind [ 1 1 ] . Discussion The formation of RNAase-resistant, R N A • R N A structures under conditions o f molecular hybridization indicate a partial complementarity between R N A of R N A t u m o r viruses and R N A of cells. The thermal stability of the complexes indicates a non-randomness in the interaction; nervertheless no phylogenetic specificity was observed. RNAase-resistant hybrids were not formed between 12sIlabelled globin, IgG m R N A or EMC virus R N A and cell RNA, though RNAase-
323 sensitive complexes were detected with hemoglobin mRNA. ~2SI-labelled t u m o r virus RNA did n o t form a detectable level of RNAase-resistant structures with itself. ~2SI-labelled t u m o r virus RNA hybridized preferentially to cell nuclear RNA. Measured by RNA titration (Fig. 1C), the complementary RNA sequences appeared to be at least 10-fold more concentrated in the nuclear fraction of disrupted cells than in the cytoplasmic fraction. Half-maximal hybridization was obtained at an Rot value of 5 (50 ng RNA/pl, 60°C, l h [12]; uncorrected for Na + concentration, rate of RNA • RNA vs. DNA • DNA annealing, or influence of mismatching on RNA • RNA annealing), indicating t h a t about 0.3% of the nuclear RNA was complementary to MuLVR RNA. The possibility that the partially complementary RNA in the cytoplasmic fraction of disrupted cells came from nuclear leakage was not excluded. While the biological importance, if any, of the partially complementary sequences in nuclear RNA is not known, their existence presents technical complications in certain molecular hybridization experiments. Experiments designed to detect minus strands of t u m o r virus RNA in cells will be complicated because while stringent conditions of RNAase treatment can be used to eliminate the partially complementary complexes during hybrid detection, their formation might prevent the labelled RNA from hybridizing to homologous RNA. Therefore conditions should be chosen that prevent their formation. Experiments designed to detect plus viral RNA molecules in cells by competitive hybridization can also be complicated, for interaction between the comlSetitor and labelled RNA may give the same results obtained if both RNAs compete for the same DNA sites. Finally, the effect of the partially complementary sequences on hybridization of [3H]DNA complementary to t u m o r virus RNA, synthesized in vitro, to RNA of uninfected or virus-infected cells cannot be predicted. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Temin, H.M. and Mitzutani, S. (1970) Nature 226, 1211--1213 Baltimore, D. (1970) Nature 226, 1209--1211 Prensky, W., Steffensen, D.M. and Hughes, W.L. (1973) Proc. Natl. Acad. Sei. U.S. 70, 1860--1864 Gralla, J. and DeLisi, C. (1974) Nature 248, 330--332 Ricard, B. and Salser, W. (1974) Biochem. Biophys. Res. Commun. 63, 548--554 Bader, J.P. and Steck, T.L. (1969) J. Virol, 4. 454---459 Ginespie, D., Saxinger, W.C. and Gallo, R.C. (1975) Progress in Nucleic Acid Research, Vol. 15, pp. 1--108, Academic Press, New York Saxinger, W.C., P o n n a m p e r u m a . C. and Gillespie, D. (1972) Proc. Natl. Acad. Sci. U.S. 69, 2975-2978 Co mmerford , S.L. (1971) Biochemistry 10, 1 9 3 3 - - 2 0 0 0 Pang, R.H.L. and Phillips, L.A. (1975) Bioehem. Biophys. Res. Commun. 2, 508--517 Gillespie, D., Gillespie, S. and Wong-Staal, F. (1975) M e t h o d s in Cancer Research (Busch, H., ed.), Vol. 11. pp. 205--245, A c a d e m i c Press, N e w York Britten, R. and Kohne, D.E. (1968) Science 161, 529--540 Glisin, V., Crkvenjakov, R. and Byus, C. (1974) Biochemistry 13, 2633--2637 Gallo, R.C., Miller, N.R.. Saxinger, W.C. and Gillespie, D. (1975) Proc. Natl. Acad. Sci. U.S. 70, 3219--3224 Burd, J.F. and Wells, R.D. (1974) J. Biol. Chem. 249, 7094--7103
Biochimica et Biophysica Acta, 442 (1976) 316--323 © Elsevier Scientific Puklishing Company, Amsterdam -- Printed in The Netherlands
BBA 98679 INTERACTIONS BETWEEN 12SI-LABELLED R N A F R O M R N A T U M O R VIRUSES AND R N A OF O T H E R SOURCES
F. WONG-STAAL, S. GILLESPIE and D,H. GILLESPIE Laboratory of Tumor Cell Biology, National Cancer Institute,Bethesda, Md. 20014 and Bionetics Research Laboratories, 7300 Pearl Street, Bethesda, Md. 20014 (U.S.A.)
(Received January 20th, 1976) Summary Fragmented l~SI-labelled R N A from RNA t u m o r viruses was hybridized to unlabelled RNA from cells, viruses, and homoribopolymers. The viral R N A interacted with all R N A tested, except for certain homoribopolymers. Complex formation with unlabelled RNA was verified by nuclease resistance, b u o y a n t density measurements, and thermal stability in solutions of different ionic strength. The RNAase-resistant complex involved 20--30% of the sequences in the 12SI-labelled viral R N A and formed preferentially with nuclear R N A of cells. 12SI-labelled hemoglobin m R N A , 12SI-labelled immunoglobin light chain (k2) m R N A , or 1251-1abe!led viral R N A from encephalomyocarditis virus (EMC) dit not form RNAase-resistant complexes with unlabelled cellurlar R N A . Introduction Since the discovery of reverse transcriptase [1,2] little has been published concerning the presence or absence in virus-infected cells of R N A complementary in nucleotide sequence to the R N A genomes of R N A t u m o r viruses. Durmg the course of such a study using R N A labelled in vitro to high specific activity with ~2sI [3], it was found that the ~2SI-labelled R N A formed complexes with cell R N A under conditions c o m m o n l y used for molecular hybridization (60°C, 0.4 M sodium phosphate, pH 6.8). It is the purpose of this communication to present the characteristics of these complexes, especially those features that distinguish them from complexes formed between random ribocopolymers and from perfectly base-paired R N A • R N A duplexes. Results
Complexes were formed at 60°C in 0.4 M phosphate buffer between ~2sIlabelled R N A of R N A t u m o r viruses and RNA of cells. The complexes were
317 R N A . RNA duplxes stabilized by complementary hydrogen bonds, since: (1) their b u o y a n t density in Cs2SO4 was characteristic of RNA, n o t of R N A • DNA or R N A • protein (Fig. 1A); (2) they were resistant to RNAase under conditions where single-stranded R N A was destroyed (Table I); (3) they were rendered fully RNAase sensitive by heat (Table I) or by reducing the ionic strength (Table I) and (4) the thermal stability (tin) of the RNAase-resistant structure was increased by increasing the ionic strength (Fig. 1B). The R N A • RNA interaction did not appear to be species specific, for 12SI-labelled R N A from Rauscher mouse leukemia virus (MuLVR) and woolly m o n k e y (simian) sarcoma-leukemia virus (SiSV) complexed equally with RNA from mouse, rat, or human cells (Table II). The interaction of 12SI-labelled RNA from RNA t u m o r viruses with unlabelled R N A did not appear to be a random interaction for the following reasons. First, all the ribohomopolymers tested interacted with the 12SI-labelled R N A minimally. Second, little or no hybridization was obtained between 12sIlabelled t u m o r virus R N A and an excess of t R N A from several sources or R N A from R N A t u m o r viruses (Table II). Third, it has been theorized [4] and documented [5] that populations of R N A molecules containing many different nucleotide sequences will contain self-complementary sequences capable of forming partially double-stranded hybrids. The interactions between ~2SI-labelled t u m o r virus R N A and unlabelled cell R N A differ from interactions among random ribonucleotide polymers in that the complexes formed have a tm (Fig. 1B) some 15°C higher and 40°C sharper than that obtained with complexes formed with the synthetic, random, ribonucleotide polymers [5], corrected to similar ionic conditions for tm determinations. Fourth, the concentration of complementary R N A was higher in the nuclear fraction of disrupted cells than in the cytoplasmic fraction (Fig. 1C). Fifth, ~2SI-labelled RNA from encephalomyocarditis (EMC) virus, ~2SI-labelled human hemoglobin mRNA, and ~2sIlabelled mouse immunoglobin "mRNA failed to form complexes with cell R N A (Table III). However, '2SI-labelled human ribosomal R N A did interact with cell RNA, b u t specifically with nuclear RNA. Though the complexes formed with cell RNA involve 20--30% of the ~2sIlabelled t u m o r virus R N A (1000--5000 nucleotides), the complexes contain many internal RNAase-sensitive points. After treatment of the R N A . R N A complexes with RNAase A and subsequent heat denaturation, the largest, single-stranded ~2SI-labelled fragments recovered were some 30 nucleotides long (Fig. 2), compared to an average of 200--300 nucleotides for the original 12sIlabelled R N A (not shown). ~2SI-labelled t u m o r virus R N A fragments prepared in this way failed to rehybridize to cell RNA. The small size of the singlestranded R N A fragments recovered after ribonuclease treatment and the low tm of the R N A • R N A complexes (Fig. 1B) indicate that the hybrids are not perfectly base paired. It should be emphasized that the a m o u n t of complex formation is markedly influenced b y the detection conditions used, e.g. temperature and ionic strength during ribonuclease treatment (Table I). The RNAase conditions chosen for most of the work reported here were selected for maximizing complex yield while maintaining enough specificity so that ~2SI-labelled t u m o r virus R N A would n o t form RNAase-resistant complexes with itself (Table II).
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Fig. 1. A n a l y s i s o f t h e c o m p l e x e s f o r m e d w i t h 12 $ l - l a b e l l e d viral R N A a n d cell R N A . H y b r i d i z a t i o n m i x t u r e s c o n t a i n e d 125 I-labelled R N A f r o m M u L V R o r SiSV v i r u s a n d u n l a b e l l e d cell R N A in 0.4 M s o d i u m p h o s p h a t e b u f f e r , p H 6.8. M i x t u r e s w e r e b o i l e d f o r 5 rain, t h e n i n c u b a t e d a t 6 0 ° C . (a) 0.1 ng 125 I-lab e l l e d viral R N A ( 1 0 4 c p m ) w a s i n c u b a t e d w i t h 10 # g o f R N A i s o l a t e d f r o m n u c l e i o f f r e s h h u m a n b l o o d l e u k o e y t e s (final v o l u m e = 0.3 m l ) f o r 6 0 h. T h e m i x t u r e w a s t h e n d i l u t e d w i t h 2 m l o f 0 . 4 5 M NaCl, a n d d i g e s t e d w i t h 2 0 # g / m l R N A a s e A a t 22VC f o r 1 h. S a t u r a t e d Cs2SO 4 s o l u t i o n w a s t h e n a d d e d t o a final d e n s i t y o f 1 . 5 0 g / m l , a n d t h e s a m p l e s w e r e c e n t r i f u g e d t o e q u i l i b r i u m a t 3 5 0 0 0 r e v . / m i n in a SW 50.1 r o t o r , o, 125i.labelled SiSV R N A ; o, 1 2 5 I - l a b e l l e d M u L V R R N A ; ~, 3 H - l a b e l l e d ~ X 1 7 4 R N A . D N A d u p l e x circles g e n e r o u s l y p r o v i d e d b y M, S a r n g a d h a r a n w a s c e n t r i f u g e d in a p a r a l l e l g r a d i e n t as a R N A • D N A h y b r i d m a r k e r . (b) T h e r m a l s t a b i l i t y o f t h e R N A - R N A c o m p l e x e s . H y b r i d s w e r e f o r m e d w i t h 125I.labelled viral R N A a n d l e u k o c y t e n u c l e a r R N A as d e s c r i b e d in a. T h e m i x t u r e w a s t h e n d i l u t e d 300fold i n t o 0 . 0 3 M NaCI, 0 . 1 5 M NaCI a n d 1.0 M NaCI, r e s p e c t i v e l y . A l i q u o t s w e r e h e l d a t t h e i n d i c a t e d t e m p e r a t u r e s f o r 5 rain, t h e n i n c u b a t e d w i t h R N A a s e as d e s c r i b e d in a. @, 1 2 5 i . l a b e l l e d SiSV R N A , 0 . 1 5 M NaCI; o, 1251-1abelled M u L V R R N A , 0 . 1 5 M NaCI; m, 1 2 $ I - l a b e l l e d M u L V R R N A , 0 . 0 3 M NaCI; &, 12 $ I - l a b e l l e d M u L V R R N A , 1.0 M NaCI. (c) C o r r e l a t i o n o f h y b r i d yield a n d i n p u t o f u n l a b e l l e d n u c l e a r or c y t o p l a s m i c R N A . H y b r i d i z a t i o n m i x t u r e s c o n t a i n i n g d i f f e r e n t a m o u n t s o f u n l a b e l l e d cell R N A a n d 1 0 p g o f 125 I-labelled SiSV R N A in 2 pl w e r e i n c u b a t e d f o r 16 h a t 6 0 ° C . @, l e u k e m i c m y e l o b l a s t nuc l e a r R N A ; o, n o r m a l h u m a n l e u k o c y t e n u c l e a r R N A ; A, l e u k e m i c m y e l o b l a s t c y t o p l a s m i c R N A ; ~, normal human leukocyte cytoplasmic RNA. Fig. 2. P o l y a c r y l a m i d e r e / e l e c t r o p h o r e s i s o f t h e h y b r i d i z e d 1251-1abelled viral R N A f r a g m e n t s . C o m p l e x e s o f 12 $ I - l a b e l l e d SiSV R N A a n d n u c l e a r R N A f r o m l e u k e m i c m y e l o b l a s t s o r n o r m a l h u m a n l e u k o c y t e s w e r e f o r m e d as d e s c r i b e d in l e g e n d t o Fig. 1. T h e c o m p l e x e s w e r e t h e n t r e a t e d w i t h R N A a s e A ( 2 0 # g / m ] , 2 2 ° C , 1 h, 0 . 5 M NaCI) f o l l o w e d b y p r o n a s e d i g e s t i o n a n d t h e n p h e n o l e x t r a c t i o n . R i b o n u clease-resistant viral R N A f r a g m e n t s w e r e t h e n o b t a i n e d as e x c l u d e d m a t e r i a l o n Biogel P 6 0 c o l u m n s , d e n a t u r e d w i t h h e a t , a n d a n a l y z e d o n 15% p o l y a c r y l a m i d e gels p r e p a r e d as d e s c r i b e d b y Burd a n d Wells [ 1 5 ] . B r o m o p h e n o l b l u e (BPB) w a s u s e d as a d y e m a r k e r , a, l e u k e m i c m y e l o b l a s t n u c l e a r R N A ; o, n o r m a l
319 TABLE I C O N D I T I O N A L R I B O N U C L E A S E R E S I S T A N C E OF R N A . R N A COMPLEXES F O R M E D BETWEEN 1 2 5 I . L A B E L L E D SiSV R N A A N D N U C L E A R R N A F R O M H U M A N C E L L S A p p r o x . 1 0 0 0 c p m o f 125 I-labelled R N A (108 e p m / p g ) f r o m w o o l l y m o n k e y ( s i m i a n ) s a r c o m a - l e u k e m i a v i r u s (SiSV) was h y b r i d i z e d t o 2 lag o f R N A i s o l a t e d f r o m t h e n u c l e i o f f r e s h , p e r i p h e r a l b l o o d m y e l o blasts. T h e n u c l e a r R N A w a s f r e e d o f D N A b y b r i e f e x p o s u r e t o d e o x y r i b o n u c l e a s e f o l l o w e d b y c e n t r i f u g a t i o n t h r o u g h CsC1 [ 1 3 ] . H y b r i d i z a t i o n w a s c a r r i e d o u t at 6 0 ° C in 0.4 M s o d i u m p h o s p h a t e b u f f e r , p H 6.8, f o r 2 0 b. T h e h y b r i d i z a t i o n s o l u t i o n w a s e x p e l l e d i n t o a s o l u t i o n c o n t a i n i n g t h e i n d i c a t e d c o n c e n t r a t i o n o f NaCl, t h e n i n c u b a t e d w i t h 20 p g / m l of R N A a s e A u n d e r t h e specified c o n d i t i o n s . Acid-insoluble r a d i o a c t i v i t y w a s m e a s u r e d . Salt
0.5M
T e m p e r a t u r e of nuclease incubation (°C)
T i m e of n u c l e a s e incubation (11)
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Without annealing
25
1 2 1 2
45 26 20 12
5 3 2 2
1 2 1 2
19 8 5 4
1 0 0 0
37
0.3M
25 37
0.15 M
25 25 37
1 2 1 2
4 3 2 2
0 0 0 0
0.03 M
25
1 2 1 2
0 0 0 0
0 0 0 0
37
However, it is known that RNA of RNA tumor viruses contains a high degree of self-complementarity or partial complementarity [6,7] and by altering the conditions of complex detection such interactions can be visualized (Table IV). For this experiment, unlabelled RNA of several kinds was immobilized on phosphocellulose discs [8]. These RNA-containing discs were incubated with dissolved 12SI-labelled viral RNA and complex formation was monitored after simply washing the filters with 0.015 M NaC1. The fragmented, 12SI-labelled viral RNA complexed with unlabelled tumor virus RNA with no specificity concerning the phylogenetic origin of the virus and with cell nuclear and tRNA. Considerable complex formation was also obtained with poly(G), but it should be recalled that '2sI preferentially labels cytosine residues [9]. Also it has been reported recently that RNA tumor virus RNA contain C-rich regions that interact with poly(G) [10]. The tm of complexes formed between 12SI-labelled MULVR RNA and immobilized cytoplasmic RNA, nuclear RNA, tRNA, or poly(G) was 63°C in 0.15 M NaC1 and varied directly with ionic strength (not shown). '2SI-labelled hemoglobin mRNA complexed with immobilized poly(G) and with natural RNAs (Table IV). 12SI-labelled rRNA complexed with immobilized poly(G), nuclear RNA, and tumor virus RNA, but hybridized poorly
320 T A B L E II INTERACTION SAY
OF 125I-LABELLED
VIRAL RNA WITH OTHER RNAs: RNAase RESISTANCE
AS-
C o n d i t i o n s f o r h y b r i d f o r m a t i o n are g i v e n in t h e l e g e n d t o T a b l e I. C o n d i t i o n s f o r h y b r i d d e t e c t i o n in t h i s e x p e r i m e n t w e r e r i b o n u c l e a s e t r e a t m e n t in 3 × SSC f o r 2 h a t 2 5 ~ C . Rot values ~ 5 0 f o r all h y b r i d s ( a p p r o x . 2 /~g u n l a b e l l e d R N A / 4 /~1, 6 0 ° C , l h ) . M u L V R , R a u s c h e r m o u s e l e u k e m i a virus; A 3 1 , c u l t u r e d n o r m a l m o u s e f i b r o b l a s t s ; J L S V 1 0 ( M u L V R ) ~ c u l t u r e d m o u s e cells i n f e c t e d b y a n d p r o d u c i n g M u L V R ; N R K , c u l t u r e d n o r m a l r a t k i d n e y cells; N R K (SiSV), c u l t u r e d n o r m a l r a t k i d n e y cells i n f e c t e d b y a n d p r o d u c t i n g SiSV; N C 3 7 , c u l t u r e d h u m a n l y m p h o i d cells; N C 3 7 ( S i S V ) , c u l t u r e d h u m a n l y m p h o i d cells infected by and producing SiSV; PHA, normal human lymphocytes, obtained fresh then cultured for 72 h in t h e p r e s e n c e o f p h y t o h e m a g g l u t i n i n H L , f r e s h h u m a n m y e l o b l a s t s f r o m a p a t i e n t w i t h a c u t e m y e l o g e n o u s l e u k e m i a . Cells ~vere f r a c t i o n a t e d i n t o n u c l e a r a n d c y t o p l a s m i c c o m p o n e n t s as p r e v i o u s l y d e s c r i b e d [ 1 4 ] . T h e n u c l e a r f r a c t i o n w a s w a s h e d r e p e a t e d l y w i t h 1% T r i t o n in 1 5 % g l y c e r o l a n d b u f f e r c o n t a i n i n g 2 . 5 m M Tris, p H 7 . 4 , 2 . 5 m M N a C l , a n d 0 . 4 m M MgCI 2. R N A w a s p u r i f i e d f r o m t h e c y t o p l a s m i c f r a c t i o n b y s e d i m e n t a t i o n t h r o u g h CsC1 [ 1 3 ] . T h e f i n a l n u c l e a r p e l l e t w a s s u s p e n d e d in 1 0 m M Tris, p H 7 . 4 , 5 0 m M NaC1, 1 0 m M MgC12 a n d 5 0 p g / m l D N A a s e , w a r m e d t o 3 7 ° C f o r 5 r a i n t h e n R N A w a s p u r i f i e d b y sed i m e n t a t i o n t h r o u g h CsC1 [ 1 3 ] . RNAase resistant Poly(A) Poly(C) Poly(G) Poly(U • G) tRNA (mouse)
12$I-labelled M u L V R R N A
125I-labelled SiSV R N A
0 0 2 6 5
3 0 3 7 7
Nuclear RNA A31 JLSV10 (MuLV R) NRK NRK(SiSV) NC37 NC37(SiSV) PHA HL
19 19 17 18 14 15 20 24
22 19 15 16 20 12 23 28
Cytoplasmic RNA A31 JLSV10(MuLVR) NRK NRK(SiSV) NC37 NC37(SiSV) PHA HL
15 -10 12 11 10 17 14
17 14 10 11 12 9 18 19
3 4
4 2
Viral R N A SiSV MuLV R
to tRNA. 12SI-labelled rRNA also complexed with immobilized poly(C), a result expected since oligo(G) regions were detected in 28 S tRNA by hybridization to 3H-labelled poly(C) (Marshall, S. and Gillespie, D., unpublished observations). Experiments were done to see whether different sequences in ~2sI-labelled MuLVR RNA hybridized to each unlabelled RNA. I2SI-labelled RNA was hybridized to immobilized RNA, then the unhybridized RNA was annealed with
321 T A B L E III H Y B R I D I Z A T I O N OF N O N - V I R A L 1 2 $ I - L A B E L L E D RNA TO C E L L U L A R RNAs C o n d i t i o n s o f h y b r i d i z a t i o n are d e s c r i b e d in t h e l e g e n d to T a b l e I. R N A f r o m H e L a cells w a s a gift f r o m T. B o r h n , R N A f r o m e n c e p h a l o m y o c a r d i t i s (EMC) virus w a s d o n a t e d b y A. Burness, h u m a n h e m o g l o b i n m R N A w a s g i v e n t o u s b y D. H o u s m a n , m o u s e i m m u n o g l o b i n light c h a i n (k) m R N A was a gift f r o m S. P e s t k a . R N A w a s l a b e l l e d w i t h 1 2 5 I b y W. P r e n s k y . T h e specific a c t i v i t y o f t h e 1 2 5 I - l a b e l l e d R N A s was 5 • 1 0 7 - - 1 0 - 107 c p m / ~ g .
125I-labelledR N A
Human rRNA EMC RNA Hemoglobin R N A IgG m R N A
12$1-1abelledR N A
h y b r i d i z e d (%)
H u m a n nuclear R N A
H u m a n cytoplasmic R N A
17 3 2 4
8 2 4 2
several RNA or DNA preparations, either immobilized or dissolved. In most cases, it appeared t h a t the same sequences in the 12SI-labelled RNA hybridized to different immobilized RNAs. Thus, 12SI-labelled MuLVR RNA sequences t h a t did not hybridize to immobilized poly(G), leukemic myeloblast nuclear TABLE IV COMPLEX FORMATION WITH IMMOBILIZED RNA Urflabelled R N A (1 t~g) w a s i m m o b i l i z e d o n 7 - r a m p h o s p h o c e l l u l o s e discs b y a m o d i f i c a t i o n o f t h e p r o c e d u r e o f S a x i n g e r e t al. [ 8 ] . T h e discs w e r e c o n v e r t e d t o t h e ( b u t y l ) 3 N* f o r m as d e s c r i b e d , i n c u b a t e d w i t h 50 m g / m l c a r b o n y l d i m i d a z o l e in d r y d i m e t h y l f o r m a m i d e at 2 5 ° C f o r 3 h, t h e n w a s h e d t h r e e t i m e s w i t h d r y d i m e t h y l f o r m a m i d e , t h r e e t i m e s w i t h a c e t o n e , a n d d r i e d w i t h N 2 . 5/~1 o f R N A s o l u t i o n in w a t e r w e r e s p o t t e d o n t h e a c t i v a t e d filters a n d d r i e d o v e r n i g h t u n d e r v a c u u m o v e r P 2 0 $ . T h e filters w e r e c o v e r e d w i t h d r y p y r i d i n e a n d i n c u b a t e d 4 h a t 55VC. T h e filters w e r e t h e n w a s h e d w i t h w a t e r , i n c u b a t e d 1 0 m i n a t 2 5 ° C w i t h 10% Tris, p H 9, w a s h e d t h r e e t i m e s w i t h w a t e r , i n c u b a t e d 3 h a t 3 7 ° C w i t h 10% K H 2 P O 4, w a s h e d six t i m e s w i t h 0 . 5 % s o d i u m d o d e c y l s u l f a t e , t w i c e w i t h w a t e r , t w i c e w i t h a c e t o n e , t h e n d r i e d w i t h N 2 a n d s t o r e d a t - - 2 0 ° C . H y b r i d i z a t i o n w a s c a r r i e d o u t in 50 ~1 o f a s o l u t i o n c o n t a i n i n g 1 0 0 0 c p m o f 12 $ I - l a b e l l e d R N A 50% f o r m a m i d e , 0 . 4 5 M NaCI, 4 5 m M s o d i u m c i t r a t e a t 3 7 ° C f o r 4 days. H y b r i d s w e r e s c o r e d as f i l t e r - b o u n d 1 2 s I r e m a i n i n g a f t e r e x t e n s i v e w a s h i n g w i t h 15 m M NaC1 a n d 1.5 m M s o d i u m citrate containing 0.5% sodium d o d e c y l sulfate. Immobilized RNA
12$i.labeHe d R N A ( p e r c e n t c o m p l e x e d ) MuLV R
rRNA
Globin mRNA
Poly(A) Poly(U) Poly(C) Poly(G)
0 0 3 46
0 0 22 35
0 3 2 61
tRNA Adult, chicken, rabbit, or mouse Embryo, mouse
15--20 28--30
Cell n u c l e a r R N A Normal human lymphocyte Leukemic human myeloblast
29 26
23 26
46 49
T u m o r virus R N A SiSV MuLVR AvLVAMV
25 28 25
25 28 25
59 68 77
1--3 7--12
11--25 34--36
322 TABLE V HYBRIDIZATION OF RECYCLED RNA R N A was " r e c y c l e d " b y h y b r i d i z a t i o n to i m m o b i l i z e d R N A as d e s c r i b e d in the t e x t a n d l e g e n d to T a b l e IV. H y b r i d i z a t i o n of r e c y c l e d R N A t o R N A ( p a r t 4) was d o n e w i t h i m m o b i l i z e d n u c l e a r R N A ; h y b r i d i z a t i o n o f r e c y c l e d R N A t o D N A ( p a r t B) was d o n e in s o l u t i o n a n d m o n i t o r e d b y r e s i s t a n c e to R N A a s e . (A) 12$I-labelled R N A u n h y b r i d i z e d to n u c l e a r R N A f r o m n o r m a l h u m a n l e u k o c y t e s I m m o b i l i z e d R N A (2nd cycle)
125I-labelled R N A ( p e r c e n t h y b r i d i z e d ) MuLV R
Normal human leukocyte nuclear RNA Leukemic myeloblast nuclear RNA
Globin mRNA
9
26
16
28
(B) 12$I-labelled R N A u n h y b r i d i z e d to p o l y ( G ) DNA (2nd cycle)
Normal mouse fibroblast MuLV-infected mouse fibroblast
12$I-laballed R N A ( p e r c e n t h y b r i d i z e d )
MuLV R (recycled)
MuLV R (unfractionated)
42 42
25 60
RNA, or mouse e m b r y o t R N A were generally depleted for sequences capable of hybridizing to any of these RNAs or to R N A from the nucleus of human leukocytes or from R N A t u m o r viruses. However, two cases of possible specificity were noted (Table V). First, 12SI-labelled R N A from MULVR selected as not hybridizing t o R N A from the nucleus of normal leukocytes hybridized a b o u t t w o times better to R N A from the nucleus of human leukemic myeloblasts than to nuclear R N A from the normal cells (Table 5A). ~2SI-labelled globin m R N A recycled in the same manner did not show this preference. This suggests the presence of viral-related sequences in the nucleus of leukemic myeloblasts that are missing or at low concentration in the nucleus of normal human leukocytes. Second, ~2SI-labelled R N A from MuLVR selected as not hybridizing to poly(G) hybridized equally to DNA from normal or MuLVa-infected cells, suggesting that viral-specific sequences were selectively removed during the first cycle of hybridization preferentially leaving host-related sequences behind [ 1 1 ] . Discussion The formation of RNAase-resistant, R N A • R N A structures under conditions o f molecular hybridization indicate a partial complementarity between R N A of R N A t u m o r viruses and R N A of cells. The thermal stability of the complexes indicates a non-randomness in the interaction; nervertheless no phylogenetic specificity was observed. RNAase-resistant hybrids were not formed between 12sIlabelled globin, IgG m R N A or EMC virus R N A and cell RNA, though RNAase-
323 sensitive complexes were detected with hemoglobin mRNA. ~2SI-labelled t u m o r virus RNA did n o t form a detectable level of RNAase-resistant structures with itself. ~2SI-labelled t u m o r virus RNA hybridized preferentially to cell nuclear RNA. Measured by RNA titration (Fig. 1C), the complementary RNA sequences appeared to be at least 10-fold more concentrated in the nuclear fraction of disrupted cells than in the cytoplasmic fraction. Half-maximal hybridization was obtained at an Rot value of 5 (50 ng RNA/pl, 60°C, l h [12]; uncorrected for Na + concentration, rate of RNA • RNA vs. DNA • DNA annealing, or influence of mismatching on RNA • RNA annealing), indicating t h a t about 0.3% of the nuclear RNA was complementary to MuLVR RNA. The possibility that the partially complementary RNA in the cytoplasmic fraction of disrupted cells came from nuclear leakage was not excluded. While the biological importance, if any, of the partially complementary sequences in nuclear RNA is not known, their existence presents technical complications in certain molecular hybridization experiments. Experiments designed to detect minus strands of t u m o r virus RNA in cells will be complicated because while stringent conditions of RNAase treatment can be used to eliminate the partially complementary complexes during hybrid detection, their formation might prevent the labelled RNA from hybridizing to homologous RNA. Therefore conditions should be chosen that prevent their formation. Experiments designed to detect plus viral RNA molecules in cells by competitive hybridization can also be complicated, for interaction between the comlSetitor and labelled RNA may give the same results obtained if both RNAs compete for the same DNA sites. Finally, the effect of the partially complementary sequences on hybridization of [3H]DNA complementary to t u m o r virus RNA, synthesized in vitro, to RNA of uninfected or virus-infected cells cannot be predicted. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
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