184

Biochimica et Biophysica Acta, 561 (1979) 184--193 © Elsevier/North-Holland Biomedical Press

BBA 99350

ISOLATION OF THE T R A N S F E R R N A GENES OF BACTERIOPHAGE T4 AND T R A N S F E R R N A SYNTHESIS IN V I T R O

DONALD A. KAPLAN and DONALD P. NIERLICH

Department of Microbiology, College of Letters and Science, and Molecular Biology Institute, University of California, Los Angeles, CA 90024 (U.S.A.) (Received July 3rd, 1978)

Key words: tRNA gene; Bacteriophage T4; tRNA synthesis

Summary Non-glucosylated T4 DNA was restricted with the endonuclease EcoRI and the mixture of DNA fragments separated by gel electrophoresis and transcribed with purified Escherichia coli RNA polymerase. Three purified fragments were shown to act as templates for t R N A synthesis. A smaller fragment, shown to be hybridizable to 32P-labeled T4 t R N A was n o t transcribable. It was concluded that the promoter for T4 t R N A synthesis had been separated from the structural genes in the smaller fragment by EcoRI and that the distal portion of the t R N A gene cluster lacks internal promoters which display in vitro activity. Preparations of non-glucosylated T4 DNA were never fully restricted with EcoRI and when the larger purified fragments carrying the t R N A were restricted with excess enzyme only a slight cleavage to yield the smaller fragments was obtained. The property of the DNA-limiting complete restriction is n o t known. Introduction

The bacteriophage T4 encodes for a set of 8 tRNAs and t w o additional stable RNAs of u n k n o w n function designated bands 1 and 2 [1,2]. A study of the synthesis of these RNAs would-seem to hold promise for understanding R N A transcription and processing, and a great deal has in fact been learned in the 6 years since their discovery (review see ref. 1). Transfer R N A mutants have been isolated and mapped, demonstrating the clustering of the t R N A genes on the T4 chromosome in a single region near the lysozyme gene [3]. The genes are encoded by the 1-strand of the DNA, as with other 'early' T4 phage functions [4]. The nucleotide fingerprint maps of all 10 species have been pubAbbreviation:

SSC, 0.15 M NaCL 0.015 M trisodium

citrate.

185 lished, and the complete sequences of five species and two dimeric, apparent precursors molecules have been determined [1,2,5,6]. Finally, we have described the in vitro synthesis of these RNAs [7--9]. This synthesis involves the transcription of T4 DNA with purified RNA polymerase, followed by a processing reaction in which the primary transcript is cleaved to individual R N A species b y nuclease present in an extract of Escherichia coli. Such in vitro experiments suggested that the 10 species are grouped into a single transcriptional unit [8]. We have described the cleavage of T4 DNA into specific fragments using the restriction endonuclease EcoRI [10]. The DNA of the T-even phages is unique in that cytosine is replaced by hydroxymethylcytosine, and the latter is glucosylated. In T4 the glucose substitution is virtually complete. Such glucosylated DNA is resistant to restriction by EcoRI; however, DNA isolated from nonglucosylating m u t a n t strains is cleaved. Treatment of non-glucosylated T4 DNA yields, when the reaction is complete, in excess of 50 specific fragments, of average size of a b o u t 3 - 106 daltons. Here we report on the identification of several DNA fragments carrying the t R N A gene cluster. The larger of these fragments retains the template activity in the in vitro formation of t R N A of the native chromosomal DNA. The fragments of approx. 6.5 • 106, 4 . 0 . 1 0 6 , and 3.6 • 106 daltons serve as templates for the synthesis of the tRNAs. By contrast a smaller fragment ( 1 . 5 0 . 1 0 6 daltons) is n o t active in directing t R N A synthesis. Methods

Preparation and restriction of DNA. Phages were grown, banded in CsCl, and the DNA extracted as described before [9]. Non-glucosylated (Glc-) T4 DNA was prepared from T4 ~ gtl~ gt27 and non-giucosylated DNA lacking the modification of certain adenines to 6-methylaminopurine (MeAde-) was prepared from T4 ~ gt57 fl g t l 4 grown on E. coli rglA rglB (formerly r6r2,4-; [10,12]). Restriction with endonuclease EcoRI (a gift from H. Boyer) was carried o u t in 0.1 M Tris-HC1, pH 7.5, 0.05 M NaC1, 5 mM MgC12 and 0.1 mg/ml gelatin at 37°C using the DNA and enzyme concentrations indicated in the text. Gel electrophoresis. Analytical gels were run in 1.2% agarose, stained with ethidium bromide and photographed as described previously [10]. For preparative gels, amounts of DNA of up to 1.5 mg were restricted, heated at 60°C for 3 min and dialyzed against 0.01 M Tris-HC1, pH 7.9, and 0.2 mM EDTA. The DNA was then evaporated to 1 ml and to this was added 0.25 ml dye solution (50% glycerol, 10% Sarkosyl and 0.05% bromphenol blue). The mixtures were run on 1.0 or 1.2% agarose slabs [10,11] at 22°C and 50--80 V for 26--42 h. Both horizontal and vertical gels were used of size 20 × 40 × 0.6 cm. Buffers were recirculated. At the end of the runs the gels were stained with 1 pg/ml ethidium bromide for 1 h and then washed extensively with distilled water. Dye streaking of the gels was reduced by wrapping the gels in plastic wrap and storing them for 18 h at 5--10°C before photography [10]. For visualization of the gels under ultraviolet light, contrast control goggles (UVC-303; Ultra-violet Products, Inc., San Gabriel, Calif.) were found exceptionally good for enhanc-

186

ing the image. The cut bands from the gels were eluted by forcing the cut bands through a No. 14 gauge hypodermic needle and washing the syringe with approx. 2 volumes of 0.25 M NaC1, 2 mM EDTA and 0.01 M Tris-HC1, pH 7.9. This suspension was allowed to stand for 24 h at 3°C and then the aqueous material was extracted by squeezing through 200 mesh bolting silk. The extract was centrifuged for 15 min at 9000 rev./min, the DNA precipitated with 2 volumes of ethanol and, after standing overnight, the pellet was collected and dissolved in 0.01 M Tris-HC1, pH 7.9. This then received a final centrifugation at 9000 rev./min for 15 min and the supernatant stored. Transcription. The transcription, processing and electrophoresis of products were carried o u t as before with a few modifications [9]. The volume of the reaction mixture was increased to 0.75 ml and the concentration of the 32p_ labeled (0.75 Ci/mM) nucleotide (UTP or CTP) was lowered to 0.04 mM. Incubations were carried out at 0.05 or 0.17 M KC1; in the former case KC1 was added to give 0.17 M final concentration for processing. DNA and enzyme, at the concentrations indicated in the text, were preincubated without substrate for 15 min at either 33 or 37°C, substrate was added and the incubation continued as indicated. Processing was carried out for 2 h at 37°C. DNA-RNA hybridization. Transfer RNA, labeled with [32P]phosphate, was isolated from phage-infected cells and purified by 10% polyacrylamide gel electrophoresis [9]. Prior to hybridization [9], the DNA and RNA in 2 × SSC (contained in 0.4 dram polyethylene vials) was heated at 95°C for 15 min and then cooled to 65°C. After hybridization for 18 h, the samples were ribonuclease treated and counted as described before [9]. Results

We have begun a study of the transcription of the T4 tRNA genes because of our interest in understanding the mechanism controlling gene expression [8]. In this framework we previously established that t R N A synthesis in vitro begins at one or a few closely spaced specific sites and transcribes the tRNA genes into a large polycistronic precursor [9]. Fig. 1 shows the products of in vitro transcription and processing after separation by 10% polyacrylamide gel electrophoresis. A comparison of the synthesis of the tRNAs in vivo and in vitro is shown in Fig. 4A. The identity of the various species has been previously shown for b o t h those species expressed in vivo and in vitro by direct fingerprinting [1,7]. In vitro several of the species (in this case, bands 1, 2 and 3) are transcribed, b u t not properly processed and thus a complete set does not appear. The possibility that a chromosomal fragment still active as a template in t R N A synthesis might be isolated stems from finding that non-glucosylated T4 DNA, treated with EcoRI, retains essentially total activity in the in vitro system. This was not a foregone conclusion, in that previous work suggested that the promoter might be several hundred base pairs from the first t R N A gene transcribed. Fig. 1 shows the extent of in vitro tRNA synthesis in reaction mixtures containing T4 DNA, non-glucosylated T4 DNA or EcoRI-treated nonglycosylated T4 DNA. For this experiment RNA polymerase was first incubated with the indicated DNA, and then transcription begun with the

187

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nz

Fig. 1. I n v i t r o t r a n s c r i p t i o n o f T 4 D N A r e s t r i c t e d w i t h e n d o n u c l e a s e E c o R I . G l c - M e A d e - D N A w a s r e s t r i c t e d w i t h E c o R I a n d a p o r t i o n o f t h e p r o d u c t s r u n o n a 1.2% a g a r o s c gel to c o n f i r m t h a t t h e restrict i o n h a d g o n e t o c o m p l e t i o n . T o 50 /~g of r e s t r i c t e d or n o n - r e s t r i c t e d G l c - M e A d e - T 4 D N A , 10 ~g R N A p o l y m e r a s e w a s a d d e d . T h e m i x t u r e was i n c u b a t e d at 3 7 ° C f o r 15 rain f o l l o w e d b y t h e s i m u l t a n e o u s addit i o n of t h e f o u r n u c l e o s i d e of t r i p h o s p h a t e s ( w i t h [ 3 2 p ] U T P ) a n d r i f a m p i c i n a t a final c o n c e n t r a t i o n o f 10 ~ug/mi. T h e final KCI c o n c e n t r a t i o n for t h e t r a n s c r i p t i o n w a s 0 . 0 5 M a n d t h e t i m e of t r a n s c r i p t i o n w a s 20 rain. A n i d e n t i c a l e x p e r i m e n t was d o n e using T 4 D N A i n s t e a d o f G l c - M e A d e - T 4 D N A , e v e n t h o u g h n o r e s t r i c t i o n o f g l u c o s y l a t e d D N A is e v e r o b s e r v e d w i t h E c o R I . T h e t r a n s c r i p t s f r o m b o t h e x p e r i m e n t s w e r e p r o c e s s e d w i t h a n E. coli Q 1 3 e n z y m e f r a c t i o n as d e s c r i b e d in M e t h o d s . T h e p r o c e s s e d R N A w a s r u n o n a 10% a c r y l a m i d e gel a n d v i s u a l i z e d b y a u t o r a d i o g r a p h y . T h e b a n d s i n d i c a t e d s.re: 1 , a 1 4 0 n u c l e o t i d e R N A o f u n k n o w n f u n c t i o n ; 4, T 4 t R N A L e u ; 5, a m i x t u r e of five t R N A species; 6, T 4 t R N A G l y [ 1 , 2 ] . F o r f u r t h e r i d e n t i f i c a t i o n of b a n d s see Fig. 4A. I, p r o c e s s e d t r a n s c r i p t s of u n t r e a t e d T 4 D D N A ; II, processed t r a n s c r i p t s of E c o R I - t r e a t e d T 4 D D N A ; I I I , p r o c e s s e d t r a n s c r i p t s of u n t r e a t e d G l c - M e A d e - T 4 D N A ; IV, p r o c e s s e d t r a n s c r i p t s of E c o R I - t r e a t e d G l c - M e A d e - T 4 D N A .

188

simultaneous addition of the nucleoside triphosphates, and rifampicin at 10 pg/ml. Under these conditions only RNA polymerase which can rapidly initiate transcription functions, while polymerase molecules presumed to be bound at sites other than normal promoters are inactivated. Since this experiment was performed at an enzyme to DNA ratio (20 : 1) that just saturates the 'rapid start' sites (data not shown; ref. 15), the data (Fig. 1) indicate that approximately the same fraction of polymerase molecules ia associated with the tRNA promoter in EcoRI-fragmented DNA as with the intact normal templates. Additionally this experiment has been carried out with the salt concentration for transcription adjusted to 0.17 M KC1 and without rifampicin addition. This condition stimulates transcription and allows reinitiation, but recognition of

F i g . 2. ( A ) A p r e p a r a t i v e a g a r o s e g e l o f G l c - T 4 D N A r e s t r i c t e d w i t h E c o R I . G l c - D N A ( 1 . 2 m g ) w a s restricted with 100 units of EcoRI for 7 h at 37°C. The restricted DNA was run on a 1.0% agarose slab (40 X 20 X 0.6 cm) for 42 h at 50 V. The time and voltage were chosen to provide increased resolution o f t h e l a r g e r f r a g m e n t s . ( B ) A n a n a l y t i c a l a g a r o s e gel o f p a r t i a l l y p u r i f i e d E c o R I r e s t r i c t i o n f r a g m e n t s o f G l c - T 4 D N A . G l c - T 4 D N A ( 2 4 6 /~g) w a s r e s t r i c t e d w i t h 2 0 0 u n i t s o f E c o R I f o r 7 h a t 3 7 ° C a n d t h e n r u n a t 8 0 V f o r 2 8 h o n a 1 . 0 % p r e p a r a t i v e a g a r o s e g e l ( 4 0 X 2 0 X 0 . 6 c m ) as d e s c r i b e d i n M e t h o d s . T h e g e l w a s s l i c e d i n t o 2 8 f r a c t i o n s a n d t h e D N A w a s e l u t e d f r o m e a c h . P o r t i o n s o f D N A ( 0 . 5 /zg) f r o m adjacent fractions were combined to reduce the number of fractions from 28 to 14 and the samples dried w i t h a g e n t l e a i r s t r e a m . T h e s e w e r e t a k e n u p i n 3 0 #1 o f d y e s o l u t i o n a n d w e r e r u n o n a 1 . 2 % a g a r o s e s l a b ( 4 0 X 2 0 X 0 . 2 5 c m ) a t 9 0 V f o r 2 2 h, w i t h r e c y c l i n g o f t h e b u f f e r . L a n e s 1 a n d 2 2 , t h e u n f r a c t i o n a t e d m a t e r i a l ~ l a n e s 2 - - 8 , 0 . 5 /~g o f D N A f r o m f r a c t i o n s 3 - - 9 o f t h e o r i g i n a l 2 8 f r a c t i o n s : l a n e s 9 - - 2 1 , t h e 1 4 combined fractions.

189 promoter sites may n o t be as selective as in low-salt [15]. Under these conditions, as with the low-salt, the EcoRI-cleaved template functions a b o u t as well as intact DNA. We have used 1% agarose gels for t h e fractionation of the restriction fragments of T4 DNA. Such gels require exceptional care in their use, as they have little mechanical strength, b u t at the scale used, 20 × 40 × 0.6 cm, a very satisfactory fractionation can be obtained on substantial quantities of DNA. Fig. 2A shows the fractionation of 1.2 mg of T4 DNA treated with EcoRI. For these experiments T4 DNA was prepared from phage T4 ~ gtl fl gt27, grown in E. coli K12 rglA rglB [10,12]. The DNA obtained in this way contains h y d r o x y m e t h y l c y t o s i n e , b u t these residues are n o t substituted (less than 3%) with glucose as is normally the case with T4 [16]. The gels have been stained with ethidium bromide, and the bands cut from the gel on the stage of a long wavelength ultraviolet light transilluminator. The bands are cut, eluted, and the DNA precipitated with ethanol. Fig. 2B shows a 1.2% analytical agarose gel (0.25 cm thick} and the fractionation typically obtained.

Fig. 3. R e r e s t r i c t i o n o f D N A f r a g m e n t s 10, 18 a n d 19. E c o R I ( 1 5 u n i t s ) w a s a d d e d t o 0 . 1 2 p m o l o f e a c h of b a n d s 10, 18 a n d 19 in a v o l u m e of 1 0 0 ~1. T h e r e a c t i o n w a s r u n f o r 8 h at 3 7 ° C a n d s t o p p e d b y h e a t i n g t h e s a m p l e s t o 6 0 ° C . T h e s a m p l e s w e r e c o o l e d t o 2 0 ° C a n d 2 0 ~1 o f d y e s o l u t i o n a d d e d . A p o r t i o n ( 5 0 /~l) o f e a c h w a s r u n o n a 1.2% a g a r o s e a n a l y t i c a l gel f o r 18 h at 1 2 0 V, s t a i n e d w i t h e t h i d i u m b r o m i d e a n d p h o t o g r a p h e d . L a n e s 1, 7, a n d 9, G l c - D N A r e s t r i c t e d w i t h E c o R I ; 2, b a n d 19; 3, E c o R I - r e s t r i c t e d b a n d 19; 4, b a n d 18; 5, E c o R I - r e s t r i c t e d b a n d 18; 6, b a n d 10; 7, E c o R I - r e s t r i c t e d b a n d 10.

190

To identify the bands which contain the T4 tRNA genes, fractions (see Fig. 2B) covering the entire gel from the largest to the smallest visible band, were prepared for an initial screening by DNA-RNA hybridization. The RNA used was a reconstituted mixture of 32P-labeled tRNAs, made in vivo, and purified through 10% acrylamide gel elctrophoresis. DNA bands hybridizing to the tRNA mixture were further purified by additional electrophoresis as necessary.

Fig. 4. ( A ) I, T 4 t R N A s y n t h e s i z e d in v i v o . II, t R N A s y n t h e s i s in v i t r o f r o m u n r e s t r i c t e d T 4 D N A t e m p l a t e . B a n d i is n o r m a l l y f o r m e d in t h e s e r e a c t i o n s b u t h a s b e e n d e g r a d e d h e r e d u e t o t h e p r e s e n c e o f R N A a s e I I I in the cell-free e x t r a c t [ 1 ] . B a n d s 2 a n d 3 are n o t n o r m a l l y f o r m e d in t h e in v i t r o s y s t e m : t h e y a p p e a r t r a n s c r i b e d b u t n o t p r o c e s s e d [ 7 , 9 ] . T h e i d e n t i t y o f t h e s p e c i e s c o n t a i n e d in b a n d s 4, 5 a n d 6 h a s b e e n f u r t h e r e s t a b l i s h e d b y e l e c t r o p h o r e s i s o n 20% a c r y l a m i d e gels a n d b y R N A f i n g e r p r i n t i n g . (B) I n v i t r o t r a n s c r i p t i o n o f D N A r e s t r i c t i o n f r a g m e n t s . T h e i n d i c a t e d D N A s w e r e t r a n s c r i b e d in M e t h o d s w i t h [ 3 2 p ] U T P , t h e p r o d u c t p r o c e s s e d w i t h an E. coli Q 1 3 e x t r a c t e d a n d t h e R N A s r u n o n a 10% p o l y a c r y l a m i d e gel. L a n e s I - - I I I , t r a n s c r i p t i o n o f p u r i f i e d D N A b a n d s 10, 18 a n d 19, r e s p e c t i v e l y . R e a c t i o n m i x t u r e s c o n t a i n i n g 0 . 1 2 p m o l D N A a n d 3 ~zg R N A p o l y m e r a s e w e r e i n c u b a t e d 4 5 rain. L a n e I V , t r a n s c r i p t i o n o f p o o l e d b a n d s 10, 18 a n d 19. L a n e V, a t r a n s c r i p t o f p a r t i a l l y p u r i f i e d D N A b a n d 36. T h e r e a c t i o n m i x t u r e c o n t a i n e d 0 . 1 0 p m o l D N A b a n d 36 a n d 3 ~ g R N A p o l y m e r a s e . L a n e V I , p r o c e s s e d t r a n s c r i p t o f p o o l e d l o w m o l e c u l a r w e i g h t f r a g m e n t s , e x c l u s i v e o f b a n d 36.

191 From these experiments, four fragments were identified as carrying t R N A genes (Fig. 3). The three larger fragments designated were active as templates for the in vitro transcription of the tRNAs (Fig. 4B). The smaller, was n o t (Fig. 4B, lane V). Transcription and processing of the larger fragments indicate that they serve as template for bands 4, 5 and 6. Band 1, the most distal species, does n o t appear, either because it is degraded in the processing reaction (Fig. 4A) or n o t made. Hybridization does show that the band 1 gene is present on the three larger fragments (not shown). Thus the three bands represent different partial digestion products, each carrying the entire set of t R N A genes. Data (Fukada, K. and Abelson, J., personal communication) indicate the presence of an EcoRI site within the t R N A cluster of genes separating band 1 and t w o closely linked species (2 and t R N A Arg) from the others. From their work, it is clear that our smaller fragment, which is relatively inactive in transcription, is a fragment carrying these three species. The DNAs active in transcription have molecular weights of approx. 6.5 • 106 (band 10); 4.0 • 106 (band 18) and 3 . 5 . 1 0 6 (band 19) as measured from their mobilities relative to fragments of bacteriophage k DNA of known size [17]. They are partial digestion fragments still containing the distal fragments carrying the band 1 species.

Relationship o f bands 10, 18 and 19 We have previously shown that the pattern of fragments obtained in gel electrophoresis of EcoRI-restricted DNA was stable over more than a 12-fold range of time and enzyme concentration. This fact and the fact that this enzyme readily yields complete digestion products with other substrates led us to anticipate that only a single fragment carrying the t R N A genes would be found [10]. We have examined the relationship between the fragments by attempting their further restriction. For this, small amounts of fragments 10, 18 and 19 were treated with 150 units/ml EcoRI, approx. 20 times more concentrated than used for the initial digestion. The products were then fractionated on a 1.2% agarose gel (Fig. 3). The results show that a small portion of band 10 can be converted to bands 18 and 19, and likewise a small portion of band 18 converted to band 19 and other smaller fragments one being band 36 (1.5 • 106 daltons) (refer to Fig. 3), which is inactive in transcription. The sum of the molecular weights of band 19 and 36 is 5.1 • 106 which is considerably greater than the molecular weight of band 18 (4 • 106). It appears that band 36 is another partial restriction product. This is consistent with F u k u d a and Abelson's data where they have identified an even smaller EcoRI restriction fragment of 0.39 • 106 daltons carrying genes 1, 2 and t R N A Arg. Discussion

Non-glucosylated T4 DNA is subjected to restriction by EcoRI to yield in excess of 50 fragments. From the fragments produced we have identified several carrying the T4 t R N A genes. The three largest of these are active as templates in in vitro transcription. While it is difficult to compare the template activity of the isolated fragments to the intact template, because equivalent ratios of polymerase to DNA cannot be ascertained [18], the fact that the unfractionated restricted template yields approximately the same a m o u n t of

192 transfer RNA as the intact DNA indicates that the transcriptional activity of the system has n o t been significantly altered. Moreover, these experiments have been carried o u t under conditions defining the 'rapid-start' sites, indicating that the specificity of the system is retained as well. Based on these experiments as well as others to be reported, the fragments retain the same high specificity of transcriptional initiation that characterizes the transcription of the tRNAs from the intact template [8]. A smaller fragment is inactive as template for t R N A synthesis under our standard conditions of assay. The existence of this fragment indicates that there is no p r o m o t e r site active in vitro in the distal portion of the cluster, supporting our argument [9] that the t R N A genes are organized into a single transcriptional unit. We did n o t anticipate obtaining multiple fragments carrying the t R N A region. That the pattern of bands obtained in gel electrophoresis is both stable and reproducible under all conditions in which the reaction is not purposely restricted, and that the average fragment size is that anticipated for this restriction enzyme, suggested that the cleavage of the T4 DNA would yield a set of unique fragments, as it does for the lambdoid phage DNAs we have used as controls [10]. Our experiments show that the larger fragments are n o t simply the result of an incomplete reaction, since the same set of fragments is obtained over a substantial range of e n z y m e / D N A ratios and with experimental protocols including multiple additions of enzyme [10]. In addition, we have shown here that • restriction of the purified larger bands yields the smaller ones, although this reaction, even with great excess enzyme, proceeds only to a very limited extent. Recently Polisky et al. [19] have shown that E c o R I shows substrate site variability depending on the pH and ionic strength of the reaction mixture. Under restriction conditions which they term EcoRI* we have found that nonglucosylated T4 DNA is degraded to smaller fragments than are obtained under the standard conditions. This is anticipated because EcoRI* recognizes the sequence -AATT- wherease the sequence -GAATTC- is cleaved under the standard conditions. Under EcoRI* conditions band 10 is cleaved to bands 18 and 19, b u t at the same time these bands are cleaved to yield smaller products. It does therefore n o t appear that the appearance of the partial digestion products is the consequence of different rates of attack by the enzyme at different sites or to a combined attack by the EcoRI and EcoRI* activities of the enzyme. One obvious possibility is that the DNA itself is heterogeneous, possessing some incomplete modification at various sites, the modified sites then being resistant to EcoRI cleavage. For example, a low level of glucosylation, particularly if n o t randomly distributed, could partially protect the DNA against restriction, and create the observed pattern. In regard to glucosylation, phage mutants with varying glucose content have been tested, and while increasing glucosylation increases the" level of uncleaved DNA and larger fragments (see ref. 10, Fig. 2), the relative amounts of fragments 10, 18 and 19 are n o t changed. A similar result is obtained with mutants lacking the 6-methyl amino purine modification as well [10].

193 Acknowledgements We thank Christina Wong for her superb technical assitance and Steven Friedman and Paul Geshelin for their help with the gel electrophoresis. The gel apparatus was constructed by Ken Meadors of the UCLA Life Sciences Design and Development Facility. We thank J. Abelson and K. Fukuda for communicating to us the results of their restriction mapping of the T4 tRNA region. This work was supported by a grant GM 15381 from the United States Public Health Service. References 1 Abelson, J., Fukada, K., Johnson, P., Lamfrom, H., Nierlich, D.P., Otsuka, P., Paddock, G.V., Pinkerton, T.C., Sarabhai, A., Stahl, S., Wilson, J.H. and Yesian, H. (1974) Brookhaven Symp. Biol. 26, 77--88 2 McClaln, W.H., Guthrie, C. and Barrel], B.G. (1972) Proe. Natl. Aead. Sci. U.S. 63, 3703--3707 3 Wilson, J.H., Kim, J.S. and Abelson, J.N. (1972) J. Mol. BioL 71, 547--556 4 Scherberg, N.H., Guha, A., Hsu, W. and Weiss, S.B. (1970) Biochem. Biophys. Res. Commun. 40, 919--924 5 Barrell, B.G., Seidman, J.G., Guthrie, C. and McClain, W.H. (1974) Proc. Natl. Acad. Sci. U.S. 71, 413--416 6 Guthrie, C. (1975) J. Mol. Biol. 95, 529--547 7 Nierlich, D.P., Lamfrom, H., Sarabhai, A. and Abelson, J. (1973) Proc. Natl. Acad. Sci. 70, 179---182 8 Nierlich, D.P. and Kaplan, D.A. (1976) ICN-UCLA Sy mpos i a on Molecular and Cellular Biol. Vol 5, pp. 121--134 9 Kaplan, D.A. and Nierlich, D.P. (1975) J. Biol. Chem. 250, 934--938 10 Kaplan, D.A. and Nierlich, D.P. (1975) J. Biol. Chem. 250, 2395---2397 11 Kaplan, D.A., Russo, R. and Wilcox, G. (1977) Anal. Biochem. 78, 235--243 12 Revel, H.R. and Georgopoulus, C.P. (1969) Virology 39, 1--17 13 Hattman , S. (1 970) Virology 42, 359--367 14 Reid, M.S. and Bieleski, R.L. (1968) Anal. Biochem. 22, 374--381 15 Chamberlin, M.J. (1974) Annu. Rev. Biochem. 43, 721--775 16 Georgopoulos, C.P. and Revel, H.R. (1971) Virology 44, 271--285 17 Thomas, M. and Davis, R.W. (1975) J. Mol. Biol. 91, 315--328 18 Kaplan, D.A. and Nierlich, D.P. (1976) ICN-UCLA Symposia on Molecular and Cellular Biol. Vol. 5, pp. 611--621 19 Polisky, B., Greene, P., Garfin, D.E., McCarthy, B.J., Goodman, H.M. and Boyer, H.W. (1975) Proc. Natl. Acad. Sci. U.S. 72, 3 3 1 0 - - 3 3 1 4