Volume 6 Number 2 February 1979 Nucleic Acids Research

The nucleotide sequence of the initiator tRNA from Drosophila melanogaster

S.Silverman*, J.Heckmant, G.J.Cowling+, A.D.Delaney+, R.J.Dunn+, I.C.Gillam+, G.M.Tener+,D.S6ll* and U.L.RajBhandaryt

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520,tDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA, and+Department of Biochemistry, University of British Columbia, Vancouver, B.C., V6T IW5, Canada

Received 8 January 1979

ABSTRACT

The nucleotide sequence of DrosqphiAa meZanogaster methionine tRNAi wasdetermined to be: pA-G-C-A-G-A-G-U-m G-m G-C-G-C-A-G-U-G-G-A-A-G-C-G-y-m2G-C-U-G-G-G-C-C-C-A-U-t6A-A-C-C-C-A-G-A-G-m7G-D-m5C-C-G-A-G-G'-A-U-C-G-m A-A-A-C-C-U-U-G-C-U-C-U-G-C-U-A-C-C-AOH. It differs from vertebrate initiator tRNAsin only 6 out of 75 positions.

INTRODUCTION

The nucleotide sequences of several prokaryotic and eukaryotic cyto-plasmic initiator tRNAs have been elucidated (1,2). An interesting result isthe finding that prokaryotic and eukaryotic cytoplasmic initiator tRNAspossess unique structural features which distinguish them as a class fromeach other and from most non-initiator tRNAs (3). The structural featurecommon to all prokaryotic initiator tRNAs is the lack of a Watson-Crick basepair in the acceptor stem between the 5'-terminal nucleotide and the fifthnucleotide from the 3'-end (4). Eukaryotic cytoplasmic initiator tRNAs donot share this feature. Instead, they possess the sequence -AU(or T)CG- inplace of -TTCG(or A) found at the beginning of loop IV of all tRNAs whichfunction in protein synthesis (5).

Another interesting feature of initiator tRNA sequences is the ratherhigh degree of sequence homology among the various initiator tRNAs. Amongthe eukaryotic cytoplasmic initiator tRNAs sequenced to date (2), includingthose from yeast, N. crassa, wheat germ (H.P. Ghosh, K. Ghosh, M. Simsek andU.L. RajBhandary, unpublished), salmon liver, salmon testes, Xenopus Zaevis,rabbit liver, sheep mammary gland, mouse myeloma and human placenta, the ex-

tent of homology varies from 73-100%. Remarkably, the sequence of initiatortRNAs have been conserved among all vertebrates examined to date (6).

In view of the above findings and as a part of our studies on character-ization of precursor tRNAs and tRNA gene organization (7), we have under-

C Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England

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taken the purification and sequence analysis of the cytoplasmic initiator

tRNA from an arthropod. Here we describe the sequence of initiator tRNA

from Drosophila melanogaster (var. Samarkand).

MATERIALS AND METHODS

General. Most of the materials, including enzymes, and methods used in

this work were described previously (1,8). RPC-5 columns were prepared and

packed as described (9).Purification of DrosophiZa tRNAiet. D. melanogaster (Samarkand strain)

was grown as described (9). Adult flies were collected and stored at -700Cuntil used. tRNA was extracted following the procedure of Roe (10). Afterpooling of chromatographic fractions tRNA was recovered by precipitation withtwo volumes of ethanol in the presence of 10 mM Mg2+ at -200C. Small quan-tities (up to 20 A260 units) were then recovered by filtration onto 2.4 cmdiameter membrane filters of 0.45 pm pore size. Pooled fractions from RPC-5columns were extracted twice with small volumes of chloroform before pre-cipitation. Aminoacylation of tRNA was performed in solution as described(9) except that the incubation mixtures contained added 0.16 M KC1 and 50 mMMOPS, pH 7.5 (KOH) in place of Tris buffer.

Analysis of OligonucZeotides Present in Complete Ti RNase and Pancreatic

RNA Digests by in vitro Labeling with Polynucleotide Kinase. Conditionsused for the complete digestion of Drosophila tRNAMet with Tl RNase or pan-

creatic RNase, for the labeling of oligonucleotides present in these digestswith (32P)-phosphate at their 5'-terminal ends, and for the separation ofthe (32P)-labeled oligonucleotides were as described recently (1,8). Thesequence of the 5'-(32P)-oligonucleotides was then established by partialdigestion with snake venom phosphodiesterase and or nuclease P1 and the(32P)-labeled partial degradation products were analyzed by electrophoresison DEAE paper or by two-dimensional homochromatography (1,6,8).

Partial Digestion of 5'-(32P)-ZabeZed OZigonucleotides or Intact tRNA

with NucZease P1. The 5'-(32P)-labeled oligonucleotide (usually 5-10 x 104Cerenkov cpm) was incubated at room temperature with 7.5 ng of nuclease P1per 50 pg carrier yeast tRNA in 20 pl of 50 mM ammonium acetate (pH 5.3).Aliquots were removed at various times (usually 2-30 min), boiled for 3-4 minin 5 mM EDTA to inactivate the enzyme, and analyzed for the extent of diges-tion by homochromatography. Appropriate aliquots were then pooled and ana-

lyzed by two-dimensional homochromatography. Since both snake venom phos-phodiesterase and nuclease P1 cleave phosphodiester bonds to leave 3'-

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hydroxyl and 5'-phosphate ends, timed aliquots of partial digests produced

by each of these enzymes could, if necessary (11), be combined to give an

optimal representation of all the ( 32P)-partial degradation products of an

oligonucleotide; the combined aliquots were analyzed by two-dimensional homo-

chromatography.Partial nuclease Pl digestion, followed by two-dimensional homochroma-

tography, was also used to obtain the sequence of the 5' and 3'-termini of5'- and 3'-( 32P)-labeled tRNA respectively (11). 5'- and 3 _( 32p)_labelingof the tRNA was carried out as described (11).

Conversion of mlA into m6A in Oligonucleotides. Since m A as a posi-

tively charged base gives rise to anomalous mobility shifts during analysisof partial digests of oligonucleotides, mIA in the 5'-(32P)-labeled oligo-nucleotide was converted to m6A prior to analysis (8). About 200,000

Cerenkov cpm of oligonucleotides containing 0.25 A260 units of carrier tRNAwas lyophilized and dissolved in 50 mM NH4HCO3 (0.03 ml) and boiled in a

sealed capillary for 70 min. The reaction mixture was lyophilized and spot-

ted on a polyethylene imine cellulose plate and developed with 2 M pyridiniumformate, pH 3.4, until the solvent front was approximately 2 cm from the top.

After autoradiography, the major spot was eluted and subjected to sequence

analysis.Cleavage of Phosphodiester Bond Adjacent to m7G. The tRNAMet (0.5 A260

units) was subjected to the cleavage procedure as described (8). The pro-

ducts were separated by polyacrylamide gel electrophoresis, and the 5' and3'-terminal fragments were recovered from the gel. The sequence of the 3'-

terminal, 30 nucleotide-long fragment was established by complete digestionwith Tl RNase and pancreatic RNase, followed by 5'-end labeling of the oligo-nucleotides with 32p and analysis of the radioactive spots present therein.

Analysis of Partial Alkali and Ti, U2, B. cereus and Phy I Nuclease32 MetDigests on 5'-( P) tRNA. by PoZyacryZamide GeZ EZectrophoresis. This

was carried as described elsewhere (12).

RESULTSMet Met1. Purification of tRNA .. The procedures used to obtain tRNAM for

determination of its sequence are summarized in Figure 1. tRNA (8700 A260units) was applied to a column of BD-cellulose (1.2 x 90 cm) in 0.50 M NaCl -

10 mM MgC12 and eluted (Figure la) at 200C with an increasing gradient ofNaCl from 600 g of 0.50 M to 300 g of 1.0 M, both containing 10 mM MgC12.Fractions of 4.5 ml were collected every 5 min. At the end of the gradient

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16

12

8

4

b

50 100

IIIi'I

B

I-

150 200

1-2

0-8 F

0-4

0

250

AB

IIIIIII

ML.I50 100 150

FRACTION NUMBER

Figure 1 .

Met clIuChromatographic purification of Drosophila initiator tRNA . a. BD-cellu-lose; b. Sepharose 6B/ammonium sulfate; c. RPC-5 at pH 8.0; d. RPC-5 at pH3.5. Details are qiven in the text.

(tube 200) elution was continued with 1.0 M NaCl - 10 mM MgCl2 and after tube

424

10 IEC

0'0(N

aLUuzco

0Cl)co

5 -

0

1 2

0-8

0 4I

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220 the eluent also contained 9.5% (v/v) ethanol. This initial fractionationwas designed to give a series of pools to serve in further purification ofspecific tRNAs. It was found that methionine acceptor activity was presentin both pools A and B (Fig. la). Pools B from this and a similar column werecombined and fractionated on the Sepharose 6B/ammonium sulfate system (13).The RNA (1854 A260 units) was dissolved in 1.5 M ammonium sulfate and appliedto a column (1.6 x 95 cm) of Sepharose 6B in the same salt. It was elutedat 210C with a linear gradient from 500 g of 1.5 M ammonium sulfate to 500 gof water. Fractions of 3.4 ml were collected every 12 minutes. Excellentresolution of a number of peaks was obtained (Fig. lb), only the second ofwhich (pool B) contained the methionine acceptor activity. Chromatography ofpool A from the BD-cellulose column (Fig. la) gave a generally similarpattern (data not presented) though with less favorable resolution of tRNAMet.This material was not used in the further purification to be described.

Part of pool B of Fig. lb (90 A260 units) was dissolved in 0.45 M NaCl -

10 mM MgC12 - 10 mM Tris-HCl, pH 8.0 and heated for 10 minutes at 650C. Thesolution was allowed to cool and applied to a column (2.5 x 36 cm) of RPC-5equilibrated with the same buffer. The column was eluted at 370C with a

linear gradient of NaCl in 10 mM MgC12 - 10 mM Tris-HCl, pH 8.0 from 0.45 to1.0 M (total volume 1 Q). Fractions of 10 ml were collected every 3.8 mins.Fig. lc shows the two peaks of optical density eluted. The smaller, secondpeak contained tRNAMet approximately 70-80% pure. Final purification of the1tRNA was achieved by chromatography on RPC-5 at pH 3.5.

A total of 12 A260 units was applied to a column (0.9 x 22 cm) of RPC-5in 0.45 M NaCl - 10 mM MgCl2 - 50 mM formic acid, pH 3.5 (NaOH). The columnwas maintained at 370C and eluted with a linear gradient of NaCl in the samebuffer from 0.45 to 0.65 M (a total of 150 ml). Fractions of 0.75 ml werecollected every 4.5 mins. The major peak (pool B.of Fig. ld) contained themethionine acceptor (9.5 A260 units). After recovery of the tRNA itsmethionine acceptance was found to be 1510 to 1720 pmoles/A260 unit) measuredin water), depending on the concentration of tRNA used. This is within therange expected for a pure tRNA. Using crude E. coli aminoacyl-tRNA syn-thetase preparation the tRNA also accepted methionine to the same level, aproperty of initiator tRNAs of eukaryotes (14). Co-chromatography of thistRNA on RPC-5 at pH 4.5 with total Met-tRNAMet of Drosophila showed that theinitiator comigrated with Met-tRNAMet of White et aZ. (9).3

Only Met-tRNAMet was found to be labeled when a s-imilar co-chromatog-3raphy was performed on Drosophila tRNA labeled with methionine using E. coli

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aminoacyl-tRNA synthetase (data not shown). Thus, the tRNAMet purified in

here is the initiator species and is the same as tRNAMet of White et aZ. (9).Met3

2. Sequence AnaZysis of tRNA. . The procedures used for sequenceMet 11 32analysis of tRNA. involving in vitro ( P) labeling have been described

elsewhere (for a review see 8). A first step in this is the 5 _(32P) label-

ing of all the oligonucleotides present in complete Tl RNase and pancreaticRNase digests of the tRNA using T4 polynucleotide kinase and y-(32P)ATP.The labeled oligonucleotides are separated by two-dimensional fingerprinting,eluted and characterized. Figures 2A and 2B show autoradiograms of the Tl

RNase and pancreatic RNase fingerprints respectively and Tables I and II list

the sequences of all the oligonucleotides therein. Sequence analysis of most

of the oligonucleotides was relatively straightforward, those that required

the use of special procedures are briefly described below.Fragment t8. m7G was found to be at the 5'-end of this oligonucleotide.

In an attempt to unambiguously identify the nucleoside adjacent to m7G and

also to determine the sequence of fragment t8, the intact tRNA was cleaved

at the phosphodiester bond adjacent to m7G (8) and the 3'-terminal 30 nucleo-tide fragment was labeled with (32P)-phosphate at its 5'-end. The 5'-terminal

sequence of this 30 nucleotide long fragment was shown to be D-C-m 5C(c)-C-G-on the basis of the following evidence: (1) It contains D at its 5'-end. (2)Partial digestion with nuclease P1 followed by two-dimensional homochromato-

graphic analysis revealed the sequence pD-C*-C-G and a small percentage of

pD-C-C-G. (3) The dinucleotide pD-C* had identical mobility on DEAE-celluloseelectrophoresis at pH 3.5 to a marker of pD-mi5C. On the basis of all this

we conclude that the complete sequence of fragment t8 is pm7G-D-m5C-C-G.Fragments t13 and p7. Following the conversion of m1A to m6A in these

oligonucleotides, partial digestions followed by two-dimensional homochroma-

tography yielded easily interpretable mobility shifts. Interpretation of the

two-dimensional homochromatogram of fragment p7 (pG-mlA-A-A-C) was aided by

comparison with that of an identical oligonucleotide obtained from a pan-

creatic digest of rabbit liver tRNAMet (6).Fragment t14. Since the length of this fragment and the presence of a

modified nucleoside within it made it difficult to obtain a uniform partialdigest with snake venom phosphodiesterase alone, intermediates from snake

venom and nuclease P1 partial digests were combined to obtain an even dis-tribution for two-dimensional analysis (11).

Fragments pl3a and pl3b. Fragment p13 was resolved into two fragments

by PEI cellulose chromatography. Interpretation of the homochromatographic

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Fi_gure 2.

Fingerprint analysis of Drosophila initiator tRNAMet. a.

digest. b. Complete RNase A digest.Complete RNase Tl

analysis of fragment pl3a was aided by comparison to that of an identical

fragment obtained from rabbit liver tRNAMet (6). Fragment pl3b gave an

easily interpretable homochromatogram.Final Sequence. The oligonucleotides listed in Tables I and II were

aligned into a unique sequence on the basis of the following lines of evi-

dence: (1) partial nuclease P1 digest on 5'-(32P)-labeled tRNA which pro-

vided the sequence of 14 nucleotides from the 5'-end (Fig. 3); (2) a similar

digest on 3'-(32P)-labeled tRNA, which yielded the sequence of 14 nucleotides

from the 3'-end (Fig. 4); (3) identification of oligonucleotides present in

complete Tl RNase and pancreatic RNase digests of a 30 nucleotide long 3'-

terminal fragment obtained by cleavage of phosphodiester bond adjacent to m7G

in the tRNA (6). This analysis together with the data in Figure 4 providedthe sequence of the 3'-terminal 30 nucleotides. Overlap of this sequence

with the fragment pl3a allows us to extend this to 35 nucleotides from the

427

12,4,3 15

i. 14 14 *1240 t1 14r

It~~~~~~~~~~~~Id

*W 10 1041

4: 9 B*7 ~~8 B. 9*8~~~

5*

5s6*:*~~~~~~~~~~4k6..*44

2 3

1~~~~~~~~~~~~~1

S

I

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TABLE 1

Analysis of Ti RNase Digestion Products

Fragment 5-end Partial snake venom or P1 nuclease digest analysed by Conclusion Molar yieldNumber DEAE-electrophoresis 2l- D homo-fnYa

pH 3.5 pH 1.9 chromatography measured sequence

tl pC C-G C-G C-G 2.2 2

t2 pA A-G A-G A-G 3.8 4

t3 pC C-A-G C-A-G C-A-G 2.2 2

t4 pA A-A-G A-A-G A-A-G 1.0 1

t5 pC C-U-A-C-C-.. C-U-A-C-C-A C-U-A-C-C-A C-U-A-C-C-A 1.1 1

t6 pU U-G* U-G* U-m2G 1.0 I

t7 pU U-G U-G U-G 1.0 1

t8 pm7G m7G-D-C*-C-.. m7G- D-C*-C-.. G*-U*-C*-C-G m7G-D-m5C-C-G 0.6 1

t9 pC C-U-G C-U-G C-U-G 1.1 1

tlO pA A-U-C-G A-U-C-G A-U-C-G A-U-C-G 1.1 1

til pU U-mn G-m2G U-mn G-m2G U-G*-G* U-mn G-m2G 0. 7 1

tl2 pC C-U-C-U-G C-U-C-U-G C-U-C-U-G C-U-C-U-G 1.3 1

tl3 pm1A m1A-A-.. m1A-A-.. m1A-A-A-C-C-U-U-G- m1A-A-A-C-C-U-U- 1.3 1

t14 pC C-C-C-A-U-A*-A-C-C- C-C-C-A-U-t6A-A-C-A-G C-C-C-A-G 0.7 1

TABLE 2Analysis of Pancreatic RNase Digestion Products

Fragment 5'-End Partial snake venom or P1 nuclease digest analysed by Conclusion Molar yieldNumber DEAE electrophoresis Z-D homo- final

pH 3.5 pH 1.9 chromatography measured sequence

p2 pA A-C A-C A-C 1.1 1

p3 pm2G m2G-C m2G-C m2G-C 0.9 1

p4 pG G-C G-C G-C 3.0 3

p5 pA A-U A-U A-U 1.0 1

p6 pA A-G-C A-G-C A-G-C 1.2 1

p7 pG G-m1A-A-A-C G-m1A-A-A-C 0.8 1

p8 pm G m1G-m2G-C m G-m2G-C mlG-G*-C m1G-m2G-C 0.9 1

P9 pt6A t6A-A-C t6A-A-C t6A-A-C t6A-A-C 1.1 1

plO pG G-U G-U G-U 1.3 1

pl1 pA A-G-U A-G-U A-G-U 1.0 1

p12 pG G-G-G-C G-G-G-... G-G-G-C G-G-G-C 0.8 1

pl3a pA A-G-... A-G-A-G-... A-G-A-G-m7G-D A-G-A-G-m7G-D1.3 1

pl3b pG G-G-... G-G-A-G-... G-G-A-A-G-C G-G-A-A-G-C

p14 pA A-G-A-... A-G-A-G-... A-G-A-G-U A-G-A-G-U 0.8 1

p15 pG G-A-G-G-... G-A-G-G-A-U G-A-G-G-A-U 0.8 1

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A

G

A

G

2~~~~~~~~~~m

#,~~~~~~~~~ G

Figure 3.

Autoradiogram of a partial P1 nuclease digest of 5'-(32P)-labeled DrosophiZainitiator tRNAMet.

3'-end. (4) The remainder of the overlap necessary for obtaining the finalsequence was provided by polyacrylamide gel electrophoretic analysis ofpartial alkali and nuclease digests of 5'-(32P)-labeled tRNA (Fig. 5).

The sequence thus derived is shown in Figure 6 in cloverleaf form.

DISCUSSIONThe nucleotide sequence of D. meZanogaster tRNAMet fits into the general

pattern observed with other eukaryotic cytoplasmic initiator tRNAs in thatit also contains the sequence -AUCG- in loop IV instead of -TTCG(or A)- foundin other tRNAs. In fact, except for differences due to post-transcriptionalmodification (U versus TP for instance), the entire sequence of this loop,

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Figure 4.

Autoradiogram of a partial P1 nuclease digest ofDrosophiZa initiator tRNAMet.

3'-( P)-AMP labeled

-AUCGm1AAA-, is identical to the sequence occurring in other eukaryotic cyto-plasmic initiator tRNAs. Thus our finding that the unique feature in thesequence of loop IV first observed in yeast initiator tRNA (5) is present ininitiator tRNA from other fungal, higher plant, vertebrate and now an arthro-pod suggests that this feature is indeed a common one among all eukaryoticcytoplasmic initiator tRNAs.

As described in the Introduction, there is a rather strong conservation

430

b~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~JA4*

4: 2L, /,5

nix - a

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qm-.mI.uIIImhhhmmmEhmEIp~~~~~~~~~~~~~~~~~~~~~~~~-

..-__s

a

-4tto

4Ui_MW...

S

.:

Figure 5.

Autoradiogram of partial digests on 5'-( 32P)-labeled Drosophila initiatortRNA. From left to right, - enzyme, incubated in the absence of enzyme; TI,T2, U2, B.c and P.p, incubation of RNA at two different levels of Tl-RNase,T2-RNase, U2-RNase, B. cereus RNase and RNA from Physarwn poZycephalwn, re-spectively. BB, XC and SBB indicate location of bromophenol blue, xylenecyanole blue and Sigma brilliant blue tracing dyes respectively. Because ofthe presence of background bands in the - enzyme track, Physarum RNase couldnot be used to unambiguously distinguish between the pyrimidine residues, inthe sequence shown here these residues are, therefore, designated as N. How-ever, the information provided by partial digestion with Tl, T2, U2 and B.cereus RNase along with the data discussed in the text was sufficient to de-rive the complete sequence of the tRNA.

431

;....."WINOWOMm'ok 04mppmpol"

..:.:.:k4:-;.I .M..:000. '.

a"%"a" .ammimm,

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DROSOPHILA METHIONINE

u G A

GC G0@

G AG CA

Figure 6.

Cloverleaf model of

AccA

G * CC * GA * UG * CA * UG * C

mIUmGm2 Ic G

* * nG Um2G j

C * GU * AG * CG * CG * C

OH

aU*0

C Gn5C

ING

u c c M-A

A G GD A u Cm7G

c Ac t6A

c U

A

DrosophiZa initiator tRNAMet.

of nucleotide sequence among initiator tRNAs in general; the sequence ofall vertebrate cytoplasmic initiator tRNAs has been fully conserved (6).Drosophila tRNAMet differs from vertebrate initiator tRNAs in only 6 out of

75 positions, whereas it differs from yeast in 15 and from N. crassa in 19

out of 75 positions. These observations are in agreement with the much

earlier divergence of fungi during evolution prior to the divergence of in-

sects and the vertebrates.

Every tRNA sequenced to date (2) has been found to contain the "invar-

iant" nucleoside U before the anticodon. The only exceptions are vertebrate

initiator tRNAs and the initiator tRNA from wheat germ (H.P. Ghosh, K. Ghosh,M. Simsek and U.L. RajBhandary, unpublished), both of which contain C in

432

A A IA

G

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place of U. In contrast, yeast and N. crassa initiator tRNAs (19) containthe more usual U residue before the anticodon. The finding that DrosophiZainitiator tRNA also contains C before the anticodon may be taken as a furtherindication of its "closer" relationship to the corresponding vertebraterather than fungal initiator tRNAs.

ACKNOWLEDGEMENTSWe wish to thank Drs. D.T. Suzuki and T.A. Grigliatti for raising and

providing the flies used in this study (supported by grants A-1764 from theNational Research Council and 6051 from the National Cancer Institute of

Canada). This work was supported in part by grants from the Medical Re-

search Council of Canada (MT-1279), the National Institutes of Health

(GM 17151 and GM 22854), the National Science Foundation (PCM 78-18541), andthe American Cancer Society (NP 114).

REFERENCES1. Heckman, J.E., Hecker, L., Schwartzbach, S., Barnett, W.E., Baumstark, B.

and RajBhandary, U.L. (1978) CeZl 13, 83-95.2. Sprinzl, M., GrUter, F. and Gauss, D.H. (1979) NucZeic Acids Res. 6 rl-r203. Rich, A. and RajBhandary, U.L. (1976) Ann. Rev. Biochem. 45, 805-860.4. Dube, S.K., Marcker, K.A., Clark, B.F.C. and Cory, S. (1968) Nature

218, 232-233.5. Simsek, M. and RajBhandary, U.L. (1972) Biochem. Biophys. Res. Cormnun.

49, 508-517.6. Gillum, A.M., Urquhart, N., Smith, M. and RajBhandary, U.L. (1975)

CeZZ 6, 395-405.7. Schmidt, O., Mao, J. Silverman, S., Hovemann, B. and Sbll, D. (1978)

Proc. Nat. Acad. Sci. USA 75, 4819-4823.8. Silberklang, M., Gillum, A.M. and RajBhandary, U.L. (1979) Methods in

EnzymoZogy 60, in press.9. White, B.N., Tener, G.M., Holden, J. and Suzuki, D.T. (1973) DeveZop.

BioZ. 33, 185-195.10. Roe, B.N. (1974) NucZeic Acids Res. 2, 21-42.11. Silberklang, M., Gillum, A.M. and RajBhandary, U.L. (1977) NucZeic Acids

Res. 4, 4091-4108.12. Heckman, J.E., Alzner-DeWeerd, B. and RajBhandary, U.L. (1979) Proc.

Nat. Acad. Sci. USA, in press.13. Holmes, W.M., Hurd, R.E., Reid, B.R., Rimerman, R.A. and Hatfield, G.W.

(1975) Proc. Nat. Acad. Sci. USA 72, 1068-1071.14. Gillum, A.M., Hecker, L.I., Silberklang, M., Schwartzbach, S.D.

RajBhandary, U.L. and Barnett, W.E. (1977) NucZeic Acids Res. 4,4109-4131.

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