Altered transfer RNA methylase patterns induced by an avian oncogenic virus

38

BIOCHIMICAET BIOPHYSICAACTA

BBA 96273

A L T E R E D T R A N S F E R RNA M E T H Y L A S E P A T T E R N S INDUCED BY AN AVIAN ONCOGENIC V I R U S B. HACKER* AND L. R. MANDEL Merck Institute ]or Therapeutic Research, Rahway, N.J. o7o65 (U.S.A.) (Received April i4th , 1969)

SUMMARY

The in vitro t R N A methylase activities of livers and spleens from chicks inoculated with the oncogenic viral agent of Marek's disease has been studied. The results demonstrate the following: I. Elevated levels of methylases are associated with the development of discrete tumor nodules in livers during the course of viral transformation. In contrast, spleens from inoculated animals did not possess visible tumor foci nor did they contain increased methylase activities. 2. Extracts from normal as well as tumor-bearing livers can methylate unfractionated methyl-poor t R N A from Escherichia coli KI~ W-6 and form the nucleosides Nl-methyladenosine, N~-methyladenosine, 2-methylguanosine, 2,2,-dimethylguanosine, Nl-methylguanosine, 02'-methylguanosine and 5-methylcytidine in varying proportions. Only extracts from tumorous liver, however, possess methylases for the synthesis of NT-methylguanosine, 5-methyluridine and 3-methylcytidine. Allied studies, using homologous normal chick liver t R N A as the substrate and tumorous liver enzyme, reveal essentially the same type of enzyme pattern but at a lower level of methyl-acceptance capacity. 3- Ancillary studies demonstrate the existence of a completely different methylase pattern for spleen extracts. Moreover, the three new methylases found in tumorous livers are absent from enlarged spleens which were also isolated from infected animals.

INTRODUCTION t R N A ' s have been shown to contain a high proportion of various modified nucleosides 1-~ distributed primarily in single-stranded loops 7. Until the development of adequate resolution techniques, such as reversed-phase column chromatography s, methods were unavailable for the separation of t R N A mixtures or methyl-deficient species. Prior to that time, the precise biological function of modified nucleosides was unclear and remained the subject of speculation 9,1°. It is now evident that methyl substituents do play an important role during amino acid acceptance n and in the codon-recognition process 12. * Present address where inquiries should be directed: Division of Oncology, University of Rochester School of Medicine, Rochester, N.Y. 14620, U.S.A. Abbreviation: MDV, Marek's disease virus. Biochim. Biophys. Acta, 19o (1969) 38-51

tRNA

METHYLASES INDUCED BY AN AVIAN VIRUS

39

Distinctly different enzymes, referred to collectively as methylases, have been shown to be capable of transferring the methyl group from S-adenosyl-L-methionine to accepting sites on tRNA is, rRNA 14,~5 and DNA xn, respectively. That this methyl group transfer occurs at the polynucleotide level has been demonstrated by FLEISSNER AND BOREK 17. Furthermore, there have been several reports which describe methylases from various biological sources x8 which exhibit affinities for specific bases in tRNA x9-24. It has been suggested by SRINIVASAN AND BOREK25 that oncogenic viruses may produce neoplastic transformation as a result of altered tRNA methylation. Recent reports by MITTLEMAN et al. 2e and MACFARLANE AND SHAW 27 concerning elevated levels of t R N A methylase activities in the SV-40 and adenovirus-I2 hamster tumors, respectively, appear to be in accord with this contention. Increased tRNA methylase activity has also been demonstrated in spleens from mice infected with a murine leukemic RNA virus 28 and in rats transplanted with Dunning (R-3323) leukemic cells 29. It is the intention of the present report to record the detailed results of experiments designed to reveal the pattern of tRNA methylases induced in chicks infected with the viral agent associated with Marek's disease (MDV). We have previously described certain quantitative changes in the levels of liver tRNA methylase activities in chicks infected with MDV 3°. Marek's disease is an infectious, lymphoproliferative disease of domestic fowl characterized by lymphoid-cell infiltration of the nervous system and by visceral tumor formationZL Like many human oncogenic diseases 32, studies on the etiology of Marek's disease have strongly implicated a herpestype virus 33-z5 capable of transforming host cells and endowing them with proporties associated with tumor cells.

MATERIALS AND METHODS

Bovine pancreatic deoxyribonuclease (EC 3.1.4.5) (electrophoretically-purified free of ribonuclease), snake venom phosphodiesterase (EC 3.1.4.1 ) and bacterial alkaline phosphatase (EC 3.1.3.1) were obtained from the Worthington Biochemical Corp. Tris was purchased from Sigma Chemical Co. Phenol (Merck) was redistilled before using. All other reagents, unless otherwise indicated, were analytical grade products of the Mallinckrodt Chemical Co. M D V in/ection and preparation o/the GA isolate The GA isolate zn associated with Marek's disease originated with and was kindly provided by Dr. S. C. Schmittle (Poultry Disease Research Center, University of Georgia). Randomly-bred Athens-Canadian chickens were inoculated intraabdominally at 2 days of age with variable quantities of the original isolate. Pooled livers from tumor-beating animals (passage seven) were homogenized (IO %, w/v) in phosphate-buffered saline medium containing penicillin G (ioo units/ml), streptomycin (Ioo#g/ml) and fungazone (0.25 #g/ml) (Buffer A) (Grand Island Biological Co.), using 6 passes in a Tenbroeck tissue grinder. After adding dimethyl sulfoxide to a final concentration of 7 % (v/v) the resulting homogenate (GA-P7) was frozen in 2-ml aliquots at a controlled rate (I ° per min to --5 o°) and stored over Biochim. Biophys. Acta, 19o (1969) 38-51

4°

B. HACKER, L . R . MANDEL

liquid nitrogen in the vapor phase. Samples were thawed in a 37 ° water bath when required. For purposes of obtaining livers and spleens from infected chickens, animals of the Athens-Canadian strain were each inoculated intra-abdominally at 2 days of age with a I : IO dilution of preparation GA-P 7 (o.I ml) which had been diluted with Buffer A after thawing. This quantity of inoculum produces a 50 % mortality 42 days after injection. Animals infected with MDV as well as control chicks were maintained in separate facilities, each possessing independent ventillation systems.

Preparation o / t R N A methylase enzymes Both normal chicks and those infected with MDV (see previous section for details) were sacrificed at various intervals b y cervical dislocation. For each preparation, livers and spleens from 4-5 animals were pooled, and homogenates (4° °/o, w/v) prepared in ice-cold Buffer B (0.25 M sucrose, 5 mM 2-mercaptoethanol, IO mM MgC12, p H 8.0) using a motor-driven P o t t e r - E l v e h j e m tissue grinder fitted with a Teflon pestle. After filtration through two layers of gauze to remove debris, the homogenate was initially centrifuged at IO ooo × g for 15 rain, followed by centrifugation of the resulting supernatant at lO5 ooo × g for 60 rain. The final supernatant fraction ($40), after suitable dilution with Buffer B when required, constitutes the enzyme fraction prepared for each tissue source. All procedures were conducted at 0-4 °. Protein content was determined according to LOWRY et alY using crystalline serum albumin as the reference standard.

tRNA preparations Unfractionated chick liver t R N A was prepared according to BRUNNGRABER 3s, from 3o-day-old uninfected animals, in yields of 1.6 mg/g of tissue. Methyl-poor t R N A 11 was isolated from methionine-starved cultures of Escherichia coli K12 W-6 using the procedure of lq'LEISSNER AND BOREK39. All starting t R N A preparations possessed A2, 0 mjA2s 0 m~ ratios of 1.9o-1.95. Spectral analyses were performed with a recording Cary-i 5 spectrophotorneter.

Large-scale RNA methylase assays For the qualitative characterization of radioactive t R N A minor nucleosides, each large-scale incubation mixture (5.0 ml) contained: 50 A~60 m~ units of t R N A from either E. coli K12 VV-6 or uninfected chick liver, o.12/*moles (6.0/*C) S-adenosyl-L-EMe-14C]methionine (New England Nuclear Corp., 55.0 mC/mmole), IO/,moles 2-mercaptoethanol, 50/*moles MgC12, 15 nag spleen or 35 mg liver $40 protein in Buffer B and 200/*moles Tris-HC1 buffer, at a final p H of 8.0. After incubating at 37 ° in a Dubnoff metabolic shaker for 45 rain the reaction mixtures were chilled on ice, and o.25-ml aliquots removed and worked up immediately according to the assay procedure described in Fig. I in order to confirm methylase activity. The prim a r y incubates were then terminated by the addition of Sarkosyl 97 (ref, 40) to a final concentration of 0.25 ~o (w/v), and the contents transferred to flasks with a 5o-ml portion of ice-cold Buffer C (o.14 M NaCl-o.I M Tris-HC1, pH 7.4) containing IO mg of carrier E. coli K12 t R N A (General Biochemicals), plus IOO ml phenol saturated with water. After shaking vigorously for 1.5 h at 5 ° the resulting emulsions were centrifuged in 5o-ml plastic tubes at 8200 × g for 30 rain in a Lourdes centrifuge Biochim. Biophys. Acta, 19o (1969) 38-51

t R N A METHYLASESINDUCED BY AN AVIAN VIRUS

41

at o-2 °. The phenol layer was re-extracted with a o.5 vol. of Buffer C and the aqueous layers pooled, o.I vol. potassium acetate (20 °/o, w/v; p H 5.5) and 2 vol. of cold IOO % ethanol were added to the pooled aqueous layers and the precipitate allowed to sediment in the cold (0-5 ° ) overnight. Finally, each preparation was washed sequentially with three portions (15 ml) each of cold IOO % ethanol, ether-ethanol (3 : I, b y vol.) and ether, and then air-dried. Preliminary, small-scale methylase assays were performed in order to assess the time course of MDV infection. The details and results of this study are shown in Fig. I, and were utilized to establish the conditions employed in the large-scale assays described herein.

Deoxyribonuelease treatment o] tRNA preparations In order to prevent interference b y contaminating DNA during the identification of methylated ribonucleosides, all radioactive t R N A preparations were pretreated according to the following procedure: each preparation was dissolved in 4.0 nil of o.i M Tris-HC1 (pH 9.2) and incubated at 37 ° for 30 rain in order to remove aminoacyl amino acids. After adjusting the p H to 7.4 with o.I M acetic acid the following additions were made: 6.0 ml of o.I M Tris-HC1 (pH 7.4), 24 mg MgC12.6 H20, 60/~g bovine pancreatic deoxyribonuclease, and the reaction mixture incubated at 37 ° for 20 rain. Each chilled incubate was next dialyzed against two 3-1 changes of glass-distilled water (0-5 °) for a total of 36 h, and the radioactive t R N A phenoltreated, alcohol precipitated, washed and dried in the manner described in the largescale methylase assay. Purification o] radioactive tRNA on DEA E-cellulose Preparations of radioactive t R N A were each purified using the batch elution procedure devised b y SCHLEICH AND GOLDSTEIN41. Suspensions containing 2 g of DEAE-cellulose (Eastman K o d a k Co.), equilibrated in IO mM Tris-HC1 (pH 7.5), were used to purify up to IOO mg of tRNA, and yielded preparations possessing A260 m~/A2s0 m# 1.95- The r R N A contaminants removed b y this process constituted only 3-4 % of the total input material as estimated b y radioactivity and spectral measurements. =

Enzymic hydrolysis o] tRNA preparations Samples of each radioactive t R N A product containing 2oo A260 ms units, with variable radioactivity, were each incubated together with 35 mg snake venom phosphodiesterase, 3 mg E. coli bacterial alkaline phosphatase, 2.o/~moles MgC12 and 9 ° #moles of Tris-HC1 (pH 8.6) in a final volume of 2.0 ml, for a total of 18 h at 37 °. Use of this protocol results in complete hydrolysis of each t R N A preparation to the nucleoside level, as indicated b y the absence of any radioactivity or ultravioletabsorbing material directly at the origin of paper chromatograms streaked with aliquots of each t R N A hydrolysate and developed in Solvent system ]3. Partition-column chromatography [14ClMethyl nucleoside mixtures were resolved into six major groups (Fig. 2) using small-scale celite partition columns designed after the procedures developed b y HALL42. Each lyophilized t R N A hydrolysate was dissolved in 2.5 ml of Solvent F lower-phase (ethyl acetate-2-ethoxyethanol-water (4 : I :2, b y vol.), and taken up Biochim. Biophys Acta, 19o (1969) 38-51

42

B. HACKER, L. R. MANDEL

in io g of a celite mixture comprised of Celite-545-Micro-Cel E (9:1, w/w). This was packed on top of a bed comprised of the same celite mixture" (60 g) predampened with 24 ml of Solvent F lower-phase and then partition-packed in increments of 5 g into a precision-bore glass column (2.0 cm internal diameter). Elution of the column was begun immediately, using Solvent F upper-phase, at a flow rate of 50 ml/ h, until the removal of uridine (Fraction III, Fig. I) was completed. At this time, Solvent G upper-phase (ethyl acetate-i-butanol-ligroine (b.p., 65-9 °° )-water (I: 2: I :I, by vol.), was introduced and continued until elution of the column was terminated. The column effluent was continually monitored for ultraviolet-absorbing material (A~60m#) with the aid of a Uvicord Absorptiometer (LKB Instrument Co., Model 83Ol A). The distribution of radioactive nucleosides was assessed by taking a o.I-ml aliquot from each column fraction (IO ml) and spotting each on a square of Whatman 3 MM paper. The dried papers were immersed in a liquici scintillator (11.3 g 2,5-diphenyloxazole and 135 mg 1,4-bis-I2-(4-methyl-5-phenyloxazolyl)l-benzene per 1 of toluene) and counted in a Packard Tri-carb liquid-scintillation spectrometer having a counting efficiency of 68 %. The appropriate column fractions containing radioactive components were pooled and evaporated to dryness in vacuo. Residues were then extracted with small aliquots of aqueous methanol ( I : I , by vol.) and stored at --20 ° until characterized by paper chromatography. All solvents employed for the partition-column chromatography were of nanograde quality obtained from tile Mallinckrodt Chemical Co. and possesssed absorbance values which were less than 0.04 at 260 mt~.

Paper chromatography o! nucleosides and bases Samples of radioactive nucleosides, isolated by partition-column chromatography of each tRNA hydrolysate, were each co-chromatographed on Whatman 3 MM paper in the descending manner together with synthetic reference markers (1-2 A~60m~ units each) in one or more of the following solvent systems: A, I-butanol-water-I 5 M NH4OH (86:14:5, by vol.); B, 2-propanol-water-I 5 M NH4OH (7:2:1, by vol.); C, ethyl acetate-I-propanol-water (4:1:2, by vol.); D, 2-propan o l - 1 % aq. (NH4)2SO 4 (2 :i, by vol.); E, 2-propanol-I2 M HC1 -water (68o:176:143 , by vol.). After locating the position of the internal reference markers on the dried chromatograms by use of an ultraviolet lamp, each lane was cut into strips perpendicular to the direction of flow. The radioactivity of each section was then estimated in 20 ml of standard scintillation solvent in the usual manner. When feasible, confirmation of the identity of each radioactive nucleoside was sought in as many solvent systems as possible. In those instances where differences in RF values were not substantially different, radioactive components were initially located with the aid of a Packard strip scanner prior to dividing the ehromatogram into sections. Reference nucleosides and bases were obtained from the Cyclo Chemical Co. Nucleosides containing 0~'-methyl substituents were generously supplied by Dr. B. G. Lane (University of Alberta, Canada). The authenticity of each reference compound * E a c h of t h e t w o c e l i t e c o m p o n e n t s ( J o h n s - M a n v i l l e Co.) were w a s h e d s e p a r a t e l y w i t h 6 M HCI u n t i l t h e e f f l u e n t w a s colorless, a n d t h e n e x t e n s i v e l y w i t h g l a s s - d i s t i l l e d w a t e r u n t i l acid-free. A f t e r d r y i n g i n a o v e n (~oo °) t h e m a t e r i a l s w e r e e a c h t r e a t e d w i t h i o vol. of e t h y l a c e t a t e a n d t h e n d r i e d a t 6o °.

t~iochim. Biophys. Mcta, 19o (1969) 38-51

43

t R N A METHYLASES INDUCED BY AN AVIAN VIRUS

was ascertained by its spectral properties at three different pH values using a Cary15 recording spectrophotometer.

Acid hydrolysis o/methylated guanosine nucleosides Aliquots of radioactive material corresponding to Peak IV (Fig. I) were each added to an equal volume of 2 M HE1 (0.2-0.3 ml), together with 3-4 A~e0 m# units each, of Nl-methylguanosine, NT-methylguanosine, 2,2-dimethylguanosine and 2-methylguanosine, and heated in a boiling water bath for I h. This procedure results in the complete conversion to the corresponding methylated guanosine base. The acid hydrolysates were evaporated to dryness in vacuo after repeated additions of small portions of water, then taken up in small volumes of Solvent G upper-phase and streaked on 1.5-cm bands using Whatman 3 MM paper. Synthetic reference markers of each methylated guanine base were run in parallel lanes during development in Solvent E. Radioactive regions were evaluated in the usual manner and the data employed to confirm the qualitative and quantitative compositions of each radioactive tRNA preparation (Figs. 4, 5 and Table II). RESULTS

Quantitative changes in IRNA methylase activities in chicks during the course o/in/ection by MDV Data presented in a previous publication 3° show in vitro tRNA methylase activities to be highest in chick spleen and liver of those organs tested. This was true of both control animals and those infected with MDV. 7 b

6 ,7 x5

x

S

~4 <

<,

~3

< o

o

~2 I

RAYS AFTER INOCULATION

DAYS A~TER INOCULATfON

Fig. I. Q u a n t i t a t i v e c h a n g e s in t R N A m e t h y l a s e a c t i v i t i e s in c h i c k s i n f e c t e d w i t h MDV. Each r e a c t i o n m i x t u r e c o n t a i n e d in o.25 ml: 9 . 6 / * m o l e s Tris-HC1 b u f f e r (pH 8.o), o.48 /tmole 2-merc a p t o e t h a n o l , 2. 5 / z m o l e s MgC12, 6.o m/tmoles (0. 3/~C) of S-adenosyl-L-[Me-14C]methionine, 3 ° 35 Itg E. coli Klz W-6 t R N A plus 15o-6oo/zg Sa0 p r o t e i n in 13uffer 13. (a) P o o l e d livers or excised t u m o r o u s liver n o d u l e s o n D a y s 27-30. (b) P o o l e d spleens, e a c h excised f r o m 5 a n i m a l s . 0 - 0 , u n i n f e c t e d controls; I1-11, c h i c k s i n f e c t e d w i t h t h e GA-Pv i s o l a t e (MATERIALS AND METHODS). I n c u b a t i o n s w e r e c o n d u c t e d at 37 ° for 45 rain. [14C]Methyl i n c o r p o r a t i o n i n t o t r i c h l o r o a c t i c acidp r e c i p i t a b l e m a t e r i a l w a s d e t e r m i n e d a c c o r d i n g to a m o d i f i c a t i o n s° of t h e m e t h o d of SRINIVASAN AND BOREK~tl. Specific r a d i o a c t i v i t y is d e f i n e d as c o u n t s / r a i n p e r / , g of t R N A per m g of $4o enz y m e protein. E a c h v a l u e g i v e n for D a y s 7 - 1 5 r e p r e s e n t s a single e x p e r i m e n t . F o r D a y s 20-38 at least 3 - 4 i n d i v i d u a l a s s a y s w e r e c o n d u c t e d for e a c h tissue p r e p a r a t i o n . In all e x p e r i m e n t s , d a t a are t a k e n f r o m t h e linear p o r t i o n of t h e m e t h y l a s e a c t i v i t y curves.

Biochim. Biophys. Acta, 19o (1969) 38-51

44

B. HACKER, L. R. MANDEL

Time-course studies, shown in Figs. I a and Ib, were conducted in an a t t e m p t to establish whether quantitative changes had occurred with regard to methylase activities in chicks following inoculation with the viral agent of Marek's disease. Examination of Fig. i a reveals little variation in liver t R N A methylase levels until about 15 days after inoculation at which time small but visible tumor foci are discernable. 5 days later, when discrete nodular liver tumors are first obvious, liver t R N A methylase activity is almost 2-fold greater than that found in control animals. After an initial period of 2o days, enzyme activity is seen to diminish somewhat in control animals, while that from infected chicks continues to increase, attaining a m a x i m u m value at 27 days when tumor nodules are visibly enlarged. The most noteworthy feature is the continuously increasing difference that occurs between control and inoculated animals with regard to liver methylase activities during the course of viral infection. The transformation is reflected b y the degree of tumor involvement leading ultimately to animal death. Although spleen methylase levels were consistently higher than those found in liver, it is obvious from Fig. I b that spleens from MDVinoculated animals possessed somewhat diminished enzyme capacity compared to spleens from control birds. From a histopathological viewpoint, spleens from MDV-inoculated animals were generally enlarged (splenomegaly) and contained some immature lymphoid cells. They did not, however contain the large compact tumor foci present in liver tumor nodules. This information, when correlated with the data presented in Fig. I, suggests the possibility that overall enhanced t R N A methylase activity m a y be a prelude to or associated with tumor formation.

The characterization o! tRNA E14C]methyl nucleosides Fig. 2 illustrates a typical partition-column elution profile obtained when mixtures of non-radioactive reference nucleosides were resolved alone, or together with E14C]methyl nucleosides obtained from enzymic hydrolysates of radioactive tRNA.

O0 m'~. A

03 2

m~A

o

=TG ~2G =~o

m'G

b

O2 I

150 300 450 600 750 900 I050 1200 1350 1500 1650 1800 1950 EFFLUENT VOLUME (ml)

5

10

15

20 25 t5 20 DISTANCE FROM ORIGIN(cm)

25

30

35

Fig. 2. Partition column chromatography of t R N A methylated nucieosides. Nuc]eoside components, obtained from enzymic hydrolysis of [14C]methyl-tRNA reaction products, were resolved into six major fractions using the small-scale celite partition columns described in MATERIALS AND METHODS.

Fig. 3. Paper c h r o m a t o g r a p h y of radioactive m e t h y l a t e d adenosine and guanosine nucleosides. (a) An aliquot of Fraction I (Fig. 4, I ) was e h r o m a t o g r a p h e d together with appropriate reference nucleosides in Solvent s y s t e m A. (b) C h r o m a t o g r a p h y of Fraction IV (Fig. 4, I ) in Solvent s y s t e m B. OV-methylguanosine, whose RF is intermediary between 2-methylguanosine and 2,2d i m e t h y l g u a n o s i n e in this system, is resolved best in S y s t e m A (Table I). See MATERIALS AND METHODS for further details, mlA, N l - m e t h y l a d e n o s i n e ; A, adenosine; m"A, N6-methyladenosine; mTG, N~-methylguanosine; m2G, 2-methylguanosine; m2~G, 2,2-dimethylguanosine; m~G, N l - m e thylguanosine.

Biochim. Biophys. Acla, 19o (1969) 38-5 t

tRNA

METHYLASES INDUCED BY AN AVIAN VIRUS

45

When separation of nucleoside mixtures into six major peaks was completed, each partition column was routinely eluted with about 200 ml of water. This particular fraction, which contained somewhat less than 1 % of the total input radioactivity, was a heterogenous mixture comprised of 2-4 components, all of which eluded characterization by paper chromatography. The relatively high RF values ( ~ 0.80) obtained for each unidentified compound in Solvent systems A, B and C suggest, however, that these may either be corresponding bases of parent nucleosides or degradation products. The small quantity of actual chemical compound associated with radioactivity values, obviously, precluded identification of these compounds by physicochemical techniques. TABLE

I

PAPER CHROMATOGRAPHIC IDENTIFICATION OF METHYLATED NUCLEOSIDES AND BASES OBTAINED FROM t R N A HYDROLYSATES T h e d e v e l o p m e n t t i m e r e q u i r e d for t h e r e s o l u t i o n of n u c l e o s i d e s w a s as follows: S o l v e n t s y s t e m s A, B a n d D, 18-2o h; S y s t e m C, 5 h; S y s t e m E, 36 h. A d e q u a t e s e p a r a t i o n of ttle m e t h y l a t e d g u a n o sine n u c l e o s i d e s in S y s t e m B w a s a c h i e v e d in 3 ° h. Compound

Adenosine N1-Methyladenosine Ne-Methyladenosine Uridine 5-Methyluridine Guanosine 2-Methylguanosine 2, z - D i m e t h y l g u a n o s i n e N 1-Methylguanosine N T - M e t h y l g u a n o s i n e *" O~'-Methylguanosine 2-Methylguanine 2, z - D i m e t h y l g u a n i n e N1-Methylguanine N~-Methylguanine Inosine N~-Methylinosine Pseudouridine 5-Methylcytidine 3-Methylcytidine Cytidine

R F in solvent system: A

B

C

D

E*

0.27 0.22 o.47 o.io o.27 o.05 o. IO o. 15 o. 17 0.04 o.21 . . . . 0.o 5 o. 02 o.o 4 o.2o 0.34 o. 15

0.54 0.53 o.72 o.38 o.54 o.26 o.39 0.49 o.67 o.28 0.44 . . . . 0.37 o. 34 o.29 o.44 o.62 o.44

0.35 0.03 o.57 0.27 o.44 o.13 o.21 0.26 o. 18 0.o2 0.30

o.61 0. 51 o.76 o.61 o.68 0.47 o.6o 0.62 o.57 0.36 0.67

o. 14 o. 02 o. I I o.o7 0.04 o. i o

0.55 --o.56 0.59 0.56

0.34 0.29 o.5 I 0.64 o.74 o.30 o.45 0.40 o.34 o.31 0.44 i. o o. 87 o. 65 o. 76 0.33 o. 20 o.52 o.48 0.54 o.47

. . . .

. . . .

* R F = RRelative for t h e four m e t h y l a t e d g u a n i n e b a s e s r e l a t i v e t o t h e m o b i l i t y of z - m e t h y l g u a n i n e . T h e d e v e l o p m e n t t i m e w a s 72~)6 h. ** I n d i s t i n g u i s h a b l e , in S o l v e n t s y s t e m s A - D , f r o m t h e d e g r a d a t i o n p r o d u c t 2 - a m i n o - 4 - h y droxy-5-methylformamido-6-ribosylaminopurine.

The paper chromatographic mobilities for tRNA components, isolated according to the procedures described in MATERIALSAND METHODS, are listed in Table I. In those instances where total radioactivity was _< 500 counts/rain samples were recovered by elution of paper chromatogram bands, concentrated, and rechromatographed in another suitable solvent system. Schematic representations of typical cbromatograms and corresponding radioactivity recordings are depicted in Fig. 3. Biochi m . B i o p h y s . Acta, 19o (I969) 38-51

40

B. HACKER, L. R. MANDEL

In order to clearly distinguish N:-methylguanosine from other related methylated guanosines, acid hydrolysis to the corresponding bases was performed as detailed in MATERIALSAND METHODS. Under the conditions employed, all methylated guanosine nucleosides were converted to their corresponding bases. IWANAMIAND BI~OWN43 have recently reported that more rigorous hydrolysis will convert N7-me thylguanosine to sarcosine (N-methylglycine). The methylated adenosines were not amenable to such treatment, and were degraded to the extent of 3o-4 o °/o under the mild hydrolytic conditions employed. The radioactive nucleosides, which reflect the activities of specific t R N A methylases, are summarized in Figs. 4, 5 and Table II.

40[ 70

w

35

60

30

I

/ Orllll

i P

ITION COLUMN FRACTION

T

N COLUMN F R A C T I O N

Fig. 4- N a t u r e of r a d i o a c t i v e t R N A m i n o r nucleosides. L a r g e - s c a l e m e t h y l a s e a s s a y s c o n t a i n e d m e t h y l - p o o r t R N A f r o m E. cull K12 W - 6 a n d c h i c k Sa0 e n z y m e s . UI, livers f r o m c o n t r o l a n i m a l s (32 d a y s old); I , t u m o r - b e a r i n g livers f r o m 3 2 - d a y -old chicks, i n o c u l a t e d w i t h M D V (GA-P7) a t 2 d a y s of age. See MATERIALS AND METHODS for details. Fig. 5. N a t u r e of r a d i o a c t i v e t R N A m i n o r nucleosides. L a r g e - s c a l e m e t h y l a s e a s s a y s c o n t a i n e d m e t h y l - p o o r t R N A E. cull KI~ \V-6 a n d c h i c k $4o e n z y m e s . [~, s p l e e n s f r o m c o n t r o l a n i m a l s (32 clays old); I , s p l e e n s f r o m 3 2 - d a y - o l d chicks, i n o c u l a t e d w i t h M D V (GA-PT) at 2 d a y s of age. See MATERIALS AND METHODS for details.

Studies using methyl-poor t R N A /rum E. cull and chick liver enzymes Unfractionated t R N A from methionine-starved cultures of E. cull K12 W-6 was methylated in the presence of S-adenosyl-L-EMe-14Clmethionine using preparations of heterologous liver enzyme from either control or MDV-infected chicks. Each reisolated, radioactive t R N A reaction product was then hydrolyzed enzymatically, and the resulting nueleosides separated into six major fractions on celite partition columns. Subsequent paper chromatography in three to five different solvent systems facilitated the identification of each radioactive nucleoside component, whose existence reflects a distinct liver t R N A methylase enzyme. The details of the foregoing protocols are described in MATERIALSAND METHODS. Biochim. Biophys. Acla, i9o (i969) 38-51

tRNA

METHYLASES INDUCED BY AN AVIAN VIRUS

47

TABLE II NATURE OF METHYLATED NUCLEOSIDIgSFORMED USING UNINFECTED CHICK-LIVER t R N A SUBSTRATE LIVER TUMOR ENZYME DERIVED FROM MDV-INFECTED CHICKS

AND

Large-scale t R N A m e t h y l a s e assays contained t R N A isolated f r o m uninfected control chicks (33 d a y s of age), and S,0 liver e n z y m e f r o m MDV-infected chicks (31 days after inoculation), as described in MATERIALS AND METHODS. All radioactive nucleosides o b t a i n e d from e n z y m e h y d r o lysates (see text) were resolved on p a r t i t i o n c o l u m n s (Figs. 2) and characterized using five p a p e r c h r o m a t o g r a p h y s y s t e m s (MATERIALS AND METHODS, Table I).

Partitioncolumn Fraction

Nucleosides

?4C ]3/Iethylincorporation (counts/rain)

I

N1-Methyladenosine N~-Methy ladenosine

2oo6 3 I98

II

5-Methyluridine

22oo

III

Uridine

IV

2-Methylguanosine 2,2-Dimethylguanosine N~-Methylguanosine NT-Methylguanosine

OV-Metkylguanosine V

Inosine

V[

5-Methylcytidine 3-Methylcytidine Cytidine

42 4309 5998 i996 5213 407 193 2oi 26 t 8 84

Fig. 4 lists the constituent nucleosides according to their chemical nature, radioactivities and column elution profile. Since an overall increase in total liver t R N A methylases had been detected following inoculation with MDV (Fig. Ia), it was expected that the levels of some selected methylated nucleosides would be elevated. Those included in this category are the following: Nl-methyladenosine and NC-methyladenosine, Fraction I; 2-methylguanosine, 2,2-dimethylguanosine and O r - m e thylguanosine, Fraction IV; 5-methylcytidine, Fraction VI (Fig. 4). BROOKES AND LAWLEY44 have demonstrated an alkaline-catalyzed N 1- to NC-methyl rearrangement for adenosine. In view of this property, the relative quantities of Nl-methyladenosine and Ne-methyladenosine reported are considered to be tentative, despite the mild hydrolytic conditions of our experimental workup. N~-Methyladenosine has been found in various tissues by DUNN AND LITTLEFIELD 45, and its corresponding methylase isolated and purified from spleens of leukemic rates b y BAGULEY AND STAEHELIN29. In contrast to an earlier report ~, we have successfully isolated all radioactive methylated guanosines as nucleosides rather than as free bases. Identifications were established b y employing at least three chromatographic systems (A-C) for resolving nucleosides, and the chemical nature confirmed using acid hydrolysis to the corresponding bases (MATERIALSAND METHODS).N1-Guanosine methylases have been reported by HURWlTZ et al. 46 in E . coli and by MITTLEMAN el al. 26 in an SV-4o hamster tumor. Although we have demonstrated the existence of this enzyme in livers and spleens from control chicks, no further increase was apparent during infection with MDV (Figs. 4 and 5, Peak IV). 0V-Methylguanosine has been reported as a component of mammalian and bacterial RNA 47-~9. l~iochim. Biophys. Acta, 19o (1969) 38-51

48

B. HACKER, L. R. MANDEL

Small amounts of radioactivity were also formed in endocyclic carbon atoms of uridine, pseudouridine and inosine, probably because of the entrance of L14C]methyl substituents into one-carbon fragment pools. This result is in agreement with the labeling pattern shown for carbon skeletons of ribonucleates from L cells pulsed with radioactive methioninO 9. The most significant feature conveyed by the data in Fig. 4 is the demonstration of three new chick liver tRNA methylases induced by MDV during the transformation process. The new methylating enzymes, responsible for the tormation of 5-methyluridine (Fraction II), NV-methylguanosine (Fraction IV) and 3methylcytidine (Fraction VI), reside exclusively in tumor-bearing livers. The inclusion of 5-methyluridine and 3-methylcytidine as naturally-occurring constituents of tRNA and other heterogenous RNA preparations from various biological sources has been reported by several laboratories 1,5°-52. It is rather unique that 3-methylcytidine found in tRNA s~r from rat liver 53 is contained in the minor loop at a position occupied by cytidine in tRNA set I (yeast) and by uridine in tRNA set ii (yeast) in the clover-leaf model ~. Of further interest is the fact that NT-methylguanosine is also found in the same minor loop but in tRNA Phe isolated from yeast ~5, as well as in the maior type of N-formylmethionine tRNA from E. coli 56. Perhaps fortuitously, is the fact that N7-methylguanosine is also known to be the major product found during reactions involving RNA and certain of the alkylating carcinogens, particularly dimethylnitrosaminO v. Studies using normal chick-liver t R N A and tumorous liver enzyme The results given in Table II represent an experiment designed to establish a true expression of liver tRNA methylases induced during transformation by the oncogenic viral agent of Marek's disease. Since homologous tRNA from normal chick livers was employed as the substrate, the nucleoside data presented is a more direct indication of new methylase activities. The results of experiments presented in the previous section (Fig. 4), in which heterologous, methyl-poor tRNA from E. coli was used, were significant but gave a "relative" representation of liver enzyme activities before and after viralinfection. As LAZZARINIAND PETERKOFSKY58have pointed out, however, such heterologous systems may be plagued by low methyl-acceptor capability. This does not appear to be of any consequence in the current studies. The total amount of E14C]methyl radioactivity incorporated into normal chick liver tRNA was about 17 °/o of that found using methyl-poor E. coli K12 W-6 as substrate when the methylase enzyme utilized in both instances was from tumorous chick livers (MATERIALSAND METHODS).Although present in smaller quantities, all the modified nucleosides demonstrated in the previous experiment (Fig. 4) were also found when tRNA from normal chick liver was used. Of possible significance, is the higher NT-inethylguanosine/2-methylguanosine+2,2-dimethylguanosine ratio obtained, almost unity, when tRNA from an homologous system was used (Table II). Studies using methyl-poor E. coli t R N A and spleen enzymes The experiments employing spleen methylases (Fig. 5) were prompted by concern that the liver methylase data (Fig. 4 and Table II) should reflect the effects of viral oncogenesis, and should not be an artefact produced by the presence of non-hepatic cellsa~. In this connection, it is interesting to note that the pattern of tRNA methylases characteristic of normal chick spleens is distinctly different from that Biochim. Biophys. Acta, 19o (1969) 38-51

tRNA

METHYLASES INDUCED BY AN AVIAN VIRUS

49

found in livers of normal chicks or those infected with MDV. Of further importance is the fact that, unlike the liver methylases, there were no marked increases in any specific spleen methylases in infected animals. Only slight increases were noted for Nl-methyladenosine and N6-methyladenosine methylases, while those specific for other nucleosides tended to generally decrease after viral infection. This agrees with the overall patterns shown in Fig. I, and is in accord with recent observations by B A G U L E Y AND STAEHELIN 29.

DISCUSSION

In 1966, TsuTsui et al. 19 presented evidence to show that total methylase activity in several mammalian tumors was higher compared to corresponding normal tissues. This was in agreement with earlier interpretations by this group ~5 and by others 26,27,59 which contend that oncogenic viruses elicit a modified pattern of methylated nucleic acids because of their ability to induce new methylase enzymes within the host system. In this connection, recent reports by TR)[VNI~EK6° and BEAUDREAU et al. 61, provide evidence that the oncogenic virus of avian erythromyeloblastic leukosis possesses viral-associated tRNA whose amino acid acceptance properties are distinctly different from those found in the host cells. Modified chromatographic profiles for tRNA, following various viral infections, have been observed by several groups 62-66. Distinct differences have been reported for tRNA~riSand tRNA Tyr in the Novikoff hepatoma 67. TAYLOR et al. 6s have recently demonstrated different elution patterns, on methylated albumin-kieselguhr columns, for distinct amino acid-accepting tRNA species which were isolated from Ehrlich ascites tumor cells. These same workers also found an additional peak of isoaccepting tRNA Tyr in HeLa cells and adeno-7-virus-transformed hamster cells 69. Review articles by ZAMECNIK70 and WEINSTEIN71suggest that oncogenic viruses may exert their capacity to "transform ''32,72 host cells, and what the possible role of tRNA may be during carcinogenesis. These findings are all consistent with the concept that some discrimination occurs at the level of peptide bond synthesis during the interaction of tRNA and polyribosomes 73. This type of interaction could conceivably be altered because of certain conformational changes 7e initiated by hypermethylation of the tRNA. It has recently been shown that methyl-poor tRNA Leu and tRNA Phe have altered codon recognition patterns 12,~6. On the other hand, SHUGARTet al. n present convincing evidence which demonstrates that methyl groups are vital for recognition of amino acids. In the current study, spleens and livers from normal and MDV-infected chicks have been found to possess enzyme systems which are capable of hypermethylating unfractionated tRNA from methionine-starved cultures of E . coli K ~ W-6. The latter has been shown to contain about 50 % of undermethylated tRNA, which is methyl deficient by an average of 2.2-2.3 methyl groups per macromolecule n. Spleen methylases are generally enriched with adenosine-methylating capacity, while liver extracts favor guanosine sites for this purpose (Figs. 4 and 5). Of the tissues studied, only tumor-bearing livers possess the ability to synthesize NT-methylguanosine, 5-methyluridine (ribosylthymine) and 3-methylcytidine, using either source of tRNA as the substrate. The former nucleoside has been shown to be the primary reaction product found after methylation of RNA by certain alkylating carcinogens 57,74. It has been Biochim. Biophys. Acta, 19o (1969) 38-51

5°

B. H A C K E R , L. R. M A N D E L

suggested that the methyl group of NT-methylguanosine can sterically hinder normal base-pairing, and thereby permit miscoding during the translation step 7'5. Comparison of the data presented in Fig. 4 and Table II show that the combined quantities of radioactive methylated adenosines constitute approx. 19 % and the methylated guanosines about 65 % ot the total material recovered from each partition column, using either type of t R N A as substrate. Yet, it is most interesting to note that the NT-methylguanosine/2-methylguanosine+2,2-dinlethylguanosine ratio is almost 4.5 times greater when homologous normal chick liver tRNA is employed in combination with tumorous liver extracts. A plausible explanation for this "sparing" effect favoring NT-methylguanosine synthesis might reside in the presence of nucleotide sequences in homologous t R N A which are more susceptible than methyl-poor E. coli t R N A to this type of enzymic methylation. Certain conformational changes in the t R N A structures should not, however, be excluded as a possible factor contributing to this phenomenon. BAGULEY AND STAEHELIN 24 have demonstrated two specific sequences in methyl-deficient t R N A from E. coli which are methylated by rat liver extracts. It is apparent from the data presented in Figs. 4, 5 and in Table II that the pattern of liver and spleen t R N A methylases, from normal as well as from MDVinfected chicks, are distinct and different from each other. Moreover, it is eminently clear tttat an altered methylase pattern, both in a quantitative and qualitative sense, is associated with transformation by the avian oncogenic virus of Marek's disease. It remains to be further clarified whether the observed changes are responsible for the neoplastic transformation, or are the result of this particular neoplasic state. Studies are currently in progress to assess which species of t R N A fronl tumorous liver contain the three new modified nucleosides, and whether their presence alters amino acid acceptance or codon-recognition capacity. REFERENCES I D. B. DUNN, J. D. SMITH AND D. F. SPAHR, J. Mol. Biol., 2 (196o) 1i 3. P. L. BERQUIST AND R. E. F. MATHEWS, Biochem. J., 85 (1962) 3o5 . 3 M. SLUYSER AND L. BOSCH, Biochim. Biophys. Acta, 55 (1962) 479. 4 R. H. HALL, Biochemistry, 4 (1965) 661. 5 H. G. ZACHAU, D. DOTTING, H. FELDMAN, Angew. Chem., 78 (1962) 392. 6 W . J. BURROWS, D. J. SKOOG, S. M. HECHT, J. T. A. BOYLE AND N. J. LEONARD, Science, 161 (1968) 691. 7 J- T. MADISON, Ann. Rev. Biochem., 37 (1968) 131. 8 A. D. KELMERS, G. D. NOVELLI AND M. P. STULBERG, J. Biol. Chem., 240 (1965) 3979. 9 J. L. STARR, Biochem. Biophys. Res. Commun., i o (1963) 181. lO J. D. CAPRA AND A. PETERKOFSKY, J . Mol. Biol., 21 ( t 9 6 6 ) 455' 11 L. SHUGART, B. H. CHASTAIN, G. D. NOVELLI AND M. P. STULBERG, Biochem. Biophys. Res. Commun., 31 (1968) 404 . 12 J. D. CAPRA AND A. PETERKOFSKY, J. Mol. Biol., 33 (1968) 591. 13 L. R. MANDEL AND E . BORE, K, Biochem. Biophys. Res. Commun., 8 (1961) 138. 14 J. L. STARR AND R. FEFFERMAN, J . Biol. Chem., 239 (1964) 3457. 15 J. L. NICHOLS AND B. C;. LANE, Can. J. Biochem., 46 (1968) lO9. 16 M. GOLD, J. HURWlTZ AND M. ANDERS, Biochem. Biophys. Res. Comm~t~., I E (1963) lO 7. 1 7 E . FLEISSNER AND E. BOREK, Biochemistry, 2 (1963) lO93. 18 E. FLEISSNER AND E . BOREK, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1199. 19 E. TSUTSUI, P. R . SRINIVASAN AND E. BOREK, Proc. Natl. Acad. Sci. U.S., 58 (1966) lOO 3. 20 R. RODEH, M. FELDMAN AND U. Z. LITTAUER, Biochemistry, 6 (1967) 451. 2 I L. A. CDLP AND G. M. BROWN, Arch. Biochem. Biophys., 124 (1968) 483 . 22 R. L. HANCOCK, Cancer Res., 27 (~967) 646. 23 G. ],. VIALE, &. F. IKESTELLI AND E . VIALE, Tamori, 53 (1967) 533. 24 B. ('. BAGULEY AND M. STAEtIELIN, Biochemistry, 7 (1968) 45-

Biochim. Biophys. Acta, 19o (1969) 38-51

tRNA 25 26 27 28 29 3° 31 32 33 34 35 36 37 38 39 4° 41 42 43 44 45 46 47 48 49 5° 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69

7° 7I 72 73 74 75 76

METHYLASES INDUCED BY AN AVIAN VIRUS

51

P. R. SRINIVASAN AND E. BOREK, Science, 145 (1964) 548. A. MITTLEMAN, R. H. HALL, D. S. YOHN AND J. T. GRACE, Cancer Res., 27 (1967) 14o 9. E. S. McFARLANE AND G. J. SHAW', Can. J. Microbiol., 14 (1968) 499. R. SILBER, B. GOLDSTEIN, E. BERMAN J. DECTER AND G. FRIEND, Cancer Res., 27 (1967) 1246. B. C. BAGULEY AND M. STAEHELIN, European J. Biochem., 6 (1968) i. L. R. 1V[ANDEL, ]3. HACKER AND T. MAAG, Canc. Res., 1969, in the press. P. M. BIGGS, Vet. Record, 81 (1967) 583 . W. HENLE, Cancer, 21 (1968) 580. K. NAZERIAN, J. J. SOLOMON, R. L. WITTLER AND B. ][~. BURMESTER, Proo. Soc. Exptl. Biol. 31ed., 127 (1968) 177. U. HEINE, D. BEARD AND J. W. BEARD, Cancer Res., 28 (1968) 585 . K. NAZERIAN AND B. R. BURMESTER, Cancer Res., 28 (1968) 2454. C. S. EIDSON AND S. C. SCHMITTLE, Avian Diseases, 12 (1968) 467 . O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265, E. F. ]3RUNNGRABER, Biochem. Biophys. Res. Commun., 8 (1962) i. E. FLEISSNER AND E. BOREK, in G. L. CANTONI AND D. R. DAVIES, Procedures in Nucleic Acid Research, H a r p e r and Row, N e w York, 1968, p. 461-465. E. F. ZIMMERMAN, Biochemistry, 7 (1968) 3156. T. SCHLEICH AND J. GOLDSTEIN, J . Mol. Biol., 15 (1966) 136. R. H. HALL, in S. P. COLOXVICKAND •. O. I~APLAN, Methods in Enzymology, Vol. 12, P a r t A, Academic Press, New York, 1968, p. 305 . Y. IWANAMI AND G. M. BROWN, Arch. Biochem. Biophys., 124 (1968) 472. p- BROOKES AND P. D. LA~VLEY, J. Chem. Soc., (196o) 539. D. ]3. DUNN AND J. ~,v. LITTLEFIELD, Nature, 181 (1958) 254. J. HURWlTZ, M. GOLD AND M. ANDERS, J. Biol. Chem., 239 (1964) 3462. R. H. HALL, Biochemistry, 3 (1964) 876S. MORISAWA AND E. CHARGAFF, Biochim. Biophys. Acta, 68 (1963) 147. T. TAMAOKI AND B. G. LANE, Biochemistry, 7 (1968) 3431. 1{. H. HALL, Biochem. Biophys. Res. Commun., 12 (1963) 361. T. D. PRlCE, H. A. HINDS AND R. S. BROWN, J. Biol, Chem., 238 (1963) 311. J. VV. LITTLEFIELD AND D. B. DUNN, Bioehem. J., 7° (1958) 642. M. STAEHELIN, H. ROBB, B. C. BAGULEY, T. GINSBERG AND W. WEHRLI, Nature, 219 (1968) 1363. R. W. HOLLEY, J. APGAR, G. A. EVERETT, J. T. MADISON, M. MARQUISEE, S. H. MERRILL, J. R. PENSWlCK AND A. ZAMIR, Science, 147 (1965) 1462. [J" L. RAJBHANDARY, A. STUART, R. D. FAULKNER, S. H. CHANG AND H. G. KHORANA, Proc. Natl. Acad. Sci. U.S., 57 (1967) 751. S. K. DUBE, K. A. :¥~ARCKER, B. F. C. CLARK AND S. CORY, Nature, 218 (1968) 232. p- F. SVCANN AND P. N. MAGEE, Bioehem. J., i i o (1968) 39. R. A. LAZZARINI AND A. PETERKOFSKY, Proc. Natl. Acad. Sci. U.S., 53 (1965) 549. R. SILBER, E. BERMAN, B. GOLDSTEIN, H. STEIN, G. FARNHAM AND J. 1{. BERTINO, Biochim. Biophys. Acta, 123 (1966) 638. I~{. TR~VN~EK, Biochim. Biophys. Acta, 166 (1968) 757. G. S. BEAUDREAU, L. SVERAK, R. ZISCHKA AND J. \¥. BEARD, Natl. Cancerlnst. Monograph, 17 (1964) 791. N. SUEOKA AND T. KANO-SUEOKA, Proc. Natl. Acad. Sci. U,S., 52 (1964) 1535. T. KANO-SUEOKA AND N. SUEOKA, J. Mol. Biol., 20 (1966) 183. L. C. WATERS AND G. D. NOVELLI, Proc. Natl. Acad. Sci. U.S., 57 (1967) 979. H. SUBAK-SHARPE AND J. HAY, J. 3/fol. Biol., 12 (1965) 924H. SUBAK-SHARPE, H. 1~. BURKE, L. CRAWFORD, J. MORRISON, J. HAY AND H. KEIR, Cold Spring Harbor Syrup. Quant. Biol., 31 (1966) 538. t3. ]~ALIGA, P. R. SRIN1VASAN AND E. BOREK, Federation Proc., Abstr. No. 3256 (1968) 794. M. W. TAYLOR, C. A. BUCK, G. A. GRANGER AND J. J. HOLLAND, Proc. Natl. Acad. Sci. U.S., 57 (1967) 1712. M. W. TAYLOR, C. A. BUCK, G. A. GRANGER AND J. J. HOLLAND, J. Mol. Biol., 33 (1968) 800. P. C. ZAMECNIK, Cancer Res., 26 (1966) i. B. WEINSTEIN, Cancer Res., 28 (1968)1871. S. E. LURIA AND J. E. DARNELL, in General Virology, J o h n Wiley, New York, 1967, p. 387 . N. SUEOKA, T. KANO-SUEOKA AND W. J, GARTLAND, Cold Spring Harbor Syrup. Quant. Biol., 31 (1966) 571 . N. Y. Acad. Sci., Monograph Con/. Biol. Effects Alkylating Agents, 1968. R. C. WILHELM AND D. B. LUDLUM, Science, 153 (1966) 14o3. U. Z. LITTAUER, M. REVEL AND t{. STERN, Cold Spring Harbor Syrup. Ouant. Biol., 31 (1966) 5Ol. Biochim. Biophys. Acta, I9o (1969) 38-51