JOURNAL OF BACTERIOLOGY, May 1975, p. 492-501Copyright 0 1975 American Society for Microbiology

Vol. 122, No. 2Printed in U.S.A.

Multiple Gene Loci for a Single Species of Glycine TransferRibonucleic Acid

EARL W. FLECK' AND JOHN CARBON*

Department of Biological Sciences, University of California, Santa Barbara, California 93106

Received for publication 4 February 1975

The study of suppressors of tryptophan synthase A protein missense mutationsin Escherichia coli has led to the establishment of two nonadjacent genetic loci(glyV and glyW) specifying identical nucleotide sequences for a single isoaccept-ing species of glycine transfer ribonucleic acid (tRNA "YU IIn one case,suppression of the missense mutation trpA78 was due to a mutation in astructural gene (glyW) for tRNA6'y "

c. This mutation resulted in a base change inthe anticodon and modification of an A residue adjacent to the 3 side of theanticodon, leading to the production of a tRNAG'yu3/c species. The resultingglyW5l (SuuGt/c) allele was mapped by interrupted mating and was located atapproximately 37 min on the E. coli genetic map. Other suppressor mutationsaffecting the primary sequence of tRNAc%y3, c and giving rise to the Ins and SuA68phenotypes were positioned at 86 min (glyV). Several independently arisingmissense suppressor mutations resulting in the SUA+58 and SuA78 phenotypes wereisolated and mapped at these two genetic loci (glyV and glyW). The ratio ofappearance of suppressor mutations at glyV and glyW suggests that there arethree or four tRNA"y 3

c structural gene copies at the glyV locus to one copy atthe glyW locus. Structural genes for tRNA ly isoacceptors are now known atfour distinct locations on the E. coli chromosome: glyT (77 min), tRNA"¶/G;glyU (55 min), tRNAGIYl; and glyV (86 min) and glyW (37 min), tRNAGG,LI.

Detailed genetic and biochemical analyses ofmutationally altered transfer ribonucleic acids(tRNA's) have been useful in correlating tRNAstructure and function (16). In many cases, theexamination of Escherichia coli strains bearingsuppressors of nonsense, missense, or frameshiftmutations reveals tRNA's with altered struc-ture. Work in this laboratory has centeredaround the genetic and biochemical analysis ofwild-type and mutant glycine tRNA. Threedistinct species of tRNAG'Y occur in wild-typestrains of E. coli: tRNA Gly , tRNAkly2 andGGG G AI',tRNAG'yu,c. The total nucleotide sequencesof these tRNA's are known (9, 13, 17). Suppres-sor mutations affecting each of these isoac-ceptor tRNA's have been characterized (2, 3,9, 13, 17). These studies have permitted thelocalization of genes that specify the primarynucleotide sequences of the three isoacceptingspecies of tRNAGlY. For example, the geneglyT (77 min) specifies tRNAG'GA/G, glyU (55min) specifies tRNAGly 1 and glyV (86 min)specifies tRNAGGu/c (3, 9, 17).

' Present address: Department of Biology, Whitman Col-lege, Walla Walla, Wash. 99362.

In this paper we present evidence establishingtwo nonadjacent loci for the structural genes fortRNA G'ly,c. One of these loci, glyV, has beenpreviously studied (17) and its location has beenconfirmed at 86 min on the E. coli genetic map.The second, glyW, occurs at approximately37 min and specifies a tRNAGGU'/C of identicalsequence to glyV tRNAG'U/C (2). Mutationsresulting in suppression of trpA58 and trpA78and occurring at these loci indicate a ratio ofthree or four structural gene copies fortRNAGGut,c at glyV to one at glyW.

MATERIALS AND METHODSBacterial strains and bacteriophage. All bacte-

rial strains used in this study are derivatives of E. coliK-12. The characteristics and origin of these strainsare listed in Table 1.The trp allele in strain Ymel A58 (trpA58) involves

a glycine (GGU/C) to aspartic acid (GAU/C) changeat position 234 in the tryptophan synthase (EC4.2.1.20) A protein (6). A suppressed trpA58 strain,Ymel A58su+ (7), presumably contains a tRNA thatinserts glycine into polypeptides in response to thecodon GAU/C (17). The suppressor mutation in strainYmel A58 su+ has been given the conforming genetic

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TABLE 1. Strains of Escherichia coli K-12 used

Strain Genotype Obtaineddesignation (or other pertinent characteristics) from:

Ymel A58 trpA58 C. YanofskyYmel A58su+ trpA58 glyV50 (SUGA OC) C. YanofskyYmel A78 trpA78 C. YanofskyYmel A78su+ trpA78 glyW51 (SuuGu/c) C. YanofskyKL16 HfrKL16 thi B. BachmannAB2072 F- (A-, )r) thr proA2 lacYI B. Bachmann

galK2 trp his ara strmaLAI mtl metE46 ilv thileu tsx C. Squires

CS509 F- AB2072 (multi-auxo-troph) trpA58 C. Squires

CS508 F- AB2072 (multi-auxo-troph) trpA78 C. Squires

CS519 HfrC metB trpA58 C. SquiresCS520 HfrC metB trpA58 glyV50

(SUGAU/C) C. SquiresCS523 HfrC metB trpA78CS524 HrfC metB trpA78 glyW51 C. Squires

(Suu G U/c)CS533 F- AB2072 (multiauxo- C. Squires

troph) trpA78 thyACS534 F- AB2072 (multi-auxo- C. Squires

troph) trpA58 thyAJA298 HfrKL16 thi trpA78 This workJA299 HfrKL16 thi trpA78 glyW51 This work

(SuuGU/C)JA303 HfrKL16 thi trpA58 This workJA306 HfrKL16 thi trpA58 glyW53 This work

(SUGAU/C)JA310 HfrKL16 thi trpA58 glyW52 This work

(Su AU/C)JA316 HfrC metB trpA78 glyV54 This work

(Suu U/C)JA320 HfrC metB trpA58 glyW53 This work

(SuG AU/C)JA321 HfrKL16 thi trpA78 glyV54 This work

(SuUGu/C)

symbol glyV50 (SuG,AI,C), the symbol glyV+ beingapplied to the wild-type allele (3, 17). The trpA78mutation results in a change at the same amino acidposition (234) as trpA58 and involves a glycine(GGU/C) to cysteine (UGU/C) substitution (6). Asuppressed revertant has been isolated, Ymel A78 su+(7), which results in a tRNAGI,Y that recognizesUGU/C (2). Since the suppressor mutation in YmelA78su+ occurred in a structural gene for a tRNAG(AT/Cidentical in sequence to the mature gene product ofthe glyV locus but at a different genetic location (2;this work), the genetic symbol glyW51 (SuuG,T/c) hasbeen applied. The wild-type allele of the glyW locusis designated glyW+. Both glyV and glyW specify thesequence of tRNAGGU8C (2, 17).

Plkc and T6 bacteriophage were kindly supplied byC. Squires. The methods for preparing lysates of thesetwo bacteriophage have been described (4, 14).

Media. The following media were used: minimalcitrate medium E of Vogel and Bonner with 0.2%glucose added as a carbon source, with 1.5% agar forplating and supplemented with amino acids 'andvitamins as necessary, or 0.5% Casamino Acids; andL-broth (14).

Genetic procedures. Growth of bacteria was at 37

C. Plkc-mediated transductions were performed,with slight modifications, according to the methods ofRoth (14). Transductants were tested for P1 lysogeny(12). Interrupted matings to determine, as preciselyas possible, the time of entry of various markers weredone exactly as described by Miller (11), except thatequal volumes of exponentially growing F- cells and1:10 diluted exponentially growing Hfr cells weremixed. An apparatus to interrupt mating pairswas constructed by using a minor modification of theplans outlined by Low and Wood (10). Interruptedmatings to obtain a crude estimate of the locations ofrelevant markers were performed essentially as out-lined by Clowes and Hayes (4) except that T6 phageat a multiplicity of infection of 100 to 1 was used tostop chromosome transfer. Ultraviolet light-inducedmutagenesis was performed by spreading the appro-priate strain on a selective medium and irradiatingwith ultraviolet light from a Chromato-Vue apparatus(model CC-20, Ultra-Violet Products, Inc.) for 20 ata distance of 25 cm, the optimal dose necessary formaximum Trp+ revertant frequency. The plates were

incubated for 3 days, at which time revertants were

selected for purification. Ethyl methane sulfonatemutagenesis was performed by placing a sterile filterpaper pad impregnated with ethyl methane sulfonateon a lawn of cells on a selective medium. Revertantswere selected for purification after 72 to 96 h ofincubation. N-methyl-N'-nitro-N-nitrosoguanidinemutagenesis was performed as previously outlined(15). Isolation of auxotrophic mutants was by penicil-lin selection (4).

During the isolation of suppressors of the trpA58and trpA78 alleles, it was necessary to differentiatebetween suppressed revertants and site or near-siterevertants. The presence of suppressors in Trp+revertant strains was indicated by the inability toco-transduce the revertant Trp+ phenotype with cysB(19). The cysB locus is linked with the tryptophanoperon as indicated by transduction by Plkc; therefore, site or near-site revertants should be co-transdu-cible with cysB, whereas most suppressed revertantswould not be linked with cysB.

RESULTS

Mutations affecting the structure oftRNAg'jG,C. In wild-type strains of E. coli K-12, the tRNAGIGu/c population is homogeneousand consists of tRNA molecules of a singleunique sequence (17). Three distinct suppres-sor mutations are known that affect the struc-ture of a portion of the tRNAG'Jy,c pool (seeFig. 1). The mutation giving rise to the Insphenotype (3) results in an anticodon base sub-stitution (G U) in about 25 to 30% of the

tRNA'Gy ,c molecules (17), thus changing thecodon recognition pattern from GGU/C toGGA/G. The original Yanofsky trpA58 sup-pressor mutation (Fig. 1 appears to alter a por-tion of the tRNAg ,c pool to form tRNA'X1/,but in this case the molecular nature of thechange is unknown (17). Both Ins and Su+58

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tRNAGly 3GGU/C

< | ~~~SU+ SU+

Gly 3 Gly 3 tRAGIV 3

GGA/G GAU/C tRNAUGU/C

FIG. 1. Suppressor mutations affecting the codonspecificity of tRNAg ?'/C. The Ins and Su+7 strainsbear mutations resulting in base substitutions in theanticodon of tRNAGGUIC (2,17).

phenotypes result from mutations occurring at86 min on the E. coli chromosome at a locustermed gly V. Possibly several identical copies ofthe tRNA 'G,l gene occur at this location (17).

SuA+78 strains (7) bear a mutation that re-sults in the alteration of a small fraction (10% orless) of the tRNA"Y' -,c population to formtRNAGly,3c (Fig. 1). This suppressor mutationhad been shown to produce a C - A change atthe 3' end of the anticodon, along with amodification of the adjacent A residue (2). Theexact map position of the suppressor mutationin strain Ymel A78su+ has not previously beendetermined, however.Map location of the missense suppressor

allele in strain Ymel A78su+. The originalsuppressor of trpA78 was isolated in C. Ya-nofsky's laboratory as a spontaneously arisingnonlinked suppressor revertant of the Ymelstrain carrying the trpA 78 mutation (7). Prelim-inary studies in this laboratory indicated thatthis suppressor allele [termed glyW51(Su G C) I might be located near his (38 min)rather than at glyV (86 min). To get a more ac-curate estimate of the position of the suppres-sor allele, an Hfr strain (KL16) was chosen thattransferred the his+ locus as a relatively earlymarker. From this male strain, an Hfr KL16derivative (JA299) carrying the trpA78 andglyW51 (SUUGu/c) alleles was prepared and usedin an interrupted mating experiment with amulti-auxotrophic female strain (CS533) carry-ing the trpA78 allele. Strain JA299 was pre-pared as follows.An indole-negative derivative of strain KL16

was isolated by nitrosoguanidine mutagenesisand penicillin selection. This strain, KL16 Ind-Trp-, presumably a trpB mutant, was trans-duced to an Ind+ Trp- phenotype with a phagePlkc lysate prepared on the original trpA78strain (trpB strains are unable to grow on mediasupplemented with indole; trpA strains are ableto grow on minimal media supplemented withindole). The KL16 trpA78 (JA298) isolate wastransduced to KL16 trpA78 glyW51 (SuuGu/c)(JA299) with a Plkc lysate prepared on the

original Ymel A78su+ strain. The presence ofthe A78 suppressor in strain JA299 was verifiedby the ability of a Plkc lysate of this strain totransduce trpA78 strains, in high frequency, toa Trp+ phenotype, and the inability to trans-duce trpA58 strains to tryptophan prototrophy.The trpA78 mutation could be recovered fromstrain JA299 by transduction of a,trpAB dele-tion strain to an Ind+ Trp- phenotype with aPlkc lysate of strain JA299.

Strain JA299 was mated with strain CS533 (atrpA 78 derivative of a multi-auxotrophic fe-male). Samples of the mating mixture wereremoved at various times, chromosome transferwas interrupted, and the samples were platedon selective media. Figure 2 gives the time ofappearance of Thy+ Str' His+, Strr, and Trp+Strr recombinants in the mating mixture. It isestimated, based on the time difference be-tween the extrapolation of the times of entry ofThy+, His+, and Trp+, that the glyW51 allele isat 37 min on the E. coli chromosome, approxi-

22

20

18

16EC 14C0c

E 12E0

0: 10x

O 8

6

4

5 10 15 20 25 30 35Time (minutes)

FIG. 2. Mapping of glyW51 (SUUGU,c) by inter-rupted mating. The male, KL16 trpA78 glyW51(Su,Gu,c) (JA299), was mated with a F- thy histrpA78 str multi-auxotroph (CS533). The number ofThy+ Stre, His+ Strr, and Trp+ Strr recombinants thatformed colonies on selective media is plotted versusthe time at which mating was interrupted by agita-tion.

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mately 1.5 min distal to the his allele from thepoint of origin in strain KL16.As a distance of around 1.5 min is possibly

within the co-transduction range of phagePlkc(20), it was of interest to determine if his (38.5min) and glyW51 (SUUGU/C) are co-transduciblewith Plkc. A Plkc lysate of strain JA299 wasused to transduce a trpA78 his strain to a His+or Trp+ phenotype. No Plkc-mediated co-transduction of his+ and glyW51 was observed,although more than 1,000 transductants of eachof the His+ or Trp+ phenotypes were examined.Map location of the missense suppressor

allele in strain Ymel A58su+. Squires andCarbon (17) have suggested that the majority ofthe structural genes for tRNA"'C,c are locatedat glyV near 86 min. They used the suppres-sor allele from strain Ymel A58su+ [glyV50(SUGAU/C)], which is thought to result in atRNASU A 58 species, in an interrupted matingexperiment to determine the approximate lo-cation of the structural genes for tRNAG3GU/C.We thought it desirable to verify the map posi-tion of the original glyV50 allele by using aslightly more accurate mating technique. StrainCS520, an Hfr C metB trpA58 glyV50(SU G AU/C) derivative that transferred themarkers proA, thr, and ilv, in that order, wasmated to strain CS534, a trpA58 multi-auxo-troph. The glyV locus was located at approxi-mately 85 min (Fig. 3), experimental error fromthe location at 86 min previously determined(17).Suppressors of trpA58 occur at both glyV

and glyW. RNA sequence studies of tRNA Y 3,which is specified by a locus at or very near theglyV locus at 86 min, and tRNAGly83, which isspecified by a locus at or near the glyW locus at37 min, indicate that both of these suppressortRNA's are derived by base substitutions intRNAGly species of identical sequence (2, 17).This genetic and biochemical evidence stronglysuggests that there are at least two locations onthe E. coli chromosome that have identicaldeoxyribonucleic acid sequences coding fortRNAGly3/c. The observation of two identicalcistrons occurring in nonadjacent positions hasnot been observed before in E. coli. Glansdorffet al. (5) have shown that wild-type E. coli K-12strains contain two ornithine transcarbamylaseactivities that map at very different locations(argF at 7 min and argI at 85 min); however, noevidence was presented showing that the aminoacid sequences of the proteins specified by theseloci are identical. It was, therefore, of interest topresent further genetic evidence that glyV andglyW specify the same tRNA, by demonstrating

c .0 r Pro+ Strr

1.0 _| l l r t

Eo 2.0

5f)

.0

5 10 15 20 25 30 35Time (minutes)

FIG. 3. Mapping of glyV50 (SUGAU/C) by interruptedmating. An HfrC trpA58 glyV50 (SUGAUIC) strain(CS520) was mated with a F- proA thr trpA58 strmulti-auxotroph (CS534). The number of Pro+ Strr,Thr+ Strr, and Trp+ Strr recombinants that formedcolonies on selective media is plotted versus the timeat which mating was interrupted by agitation.

that both loci are capable of giving rise tosuppressors of the SuA+, and Su+,, varieties.With this goal in mind, a large number ofSu+,8 isolates were collected and characterizedfor close suppressor gene linkage to thr (glyV) orto his (glyW).To obtain suppressors of the SuA58 variety,

strain CS519, a trpA58 HfrC derivative, wasmutagenized with ultraviolet light. From sev-eral hundred Trp+ revertants of strain CS519,36 isolates were selected that had slow growthrates similar to strain CS520, the isogenictrpA58 glyV50 (Su GAU/c) suppressor strain.(The vast majority of the original revertants hadnormal growth rates similar to the prototrophictrp+ strain). Thirty-five of the 36 slow-growingCS519 Trp+ isolates were suppressed revertantsas determined by the procedure outlined inMaterials and Methods (data not presented).Each of the CS519 Su+68 isolates was mated for60 min with an F- trpA58 multi-auxotroph(CS509), transfer was interrupted with T6phage as outlined in Materials and Methods,and samples of the mating mix were plated toselect for Thr+ Strr or Trp+ Strr recombinants.It has previously been shown that HfrC strainstransfer the glyV locus at high frequency within60 min of mating, after the entry of the thr locusbut before the transfer of ilv (17). Twenty-seven

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of the 35 suppressed CS519 Sul,8 revertantstransferred the Trp+ phenotype at significantlevels within 60 min of mating (Table 2). In nocase was the frequency of Trp+ greater thanThr+, suggesting that no suppressor allelesentered before the thr locus. It was assumedthat the 27 isolates transferring the Trp+phenotype at high frequency represented sup-pressors of trpA58 arising at the glyV locus.From the eight CS519 Su+68 isolates not

transferring the suppressor allele at high fre-quency, Plkc lysates were prepared and thesuppressor alleles were transferred to strainJA303, a KL16 trpA58 derivative. The resultingstrains were mated to strain CS509 and the

mating was interrupted at 60 min, selecting forHis+ Strr and Trp+ Strr recombinants. Prelimi-nary experiments showed that the glyW andhis+ loci, in the male KL16, both enteredrecipient females at high frequency within 60min when mating interruption was performedby the T6 method as outlined in Materials andMethods. Under the same conditions, the trplocus entered the recipient female with very lowfrequency. The suppressor gene from all eight ofthe SuA58 isolates entered at high frequencywithin 60 min of mating (Table 3). Further-more, the gradient of transmission indicatesthat, in all cases, the suppressor alleles enteredafter the his+ locus. This evidence suggests, but

TABLE 2. One-hour interrupted matings of HfrC trpA58 SU+A58 isolates with F- trpA58 thr

10' Recombinants/ml Ratio SuppressorStrains mated Thr+ Strr/ locatioa

Thr+ Strt Trp+ Str' Trp+ Str' location

CS520 [HfrC glyV50 (SUGAU/C)I x CS509 (F- trpA58 thr)CS524 [HfrC glyW51 (Suu(;,l/c) ] x CS508 (F- trpA 78 thr)CS519 (HfrC trpA58 SU+A58) #224 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+A58) #225 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+A58) #227 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+Al.) #228 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+AS8) #229 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58Su+ A5S), #230 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+A58) #50 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+A58) #1 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+A58) #65 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+ASS) #86 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+A58) #102 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+A58) #131 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+A58) #133 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+A58) #24 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+A58) #25 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+A56) #27 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+A58) #28 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+A58) #31 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+Al.) #136 x CS509 (F- trpA58 thr)CS519 (HfrCtrpA58 Su+ A58) #139 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+A58) #140 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+A51) #141 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+A58) #142 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+A58) #52 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+Al.) #53 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 Su+ Al.) #54 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+Al.) #55 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+A,58) #104 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+A58) #220 x CS509 (F- trpA58 thr)C0519 (HfrC trpA58 SU+AIS) #26 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+A,8) #29 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+A58) #30 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+Al.) #137 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+A,l) #138 x CS509 (F- trpA58 thr)CS519 (HfrC trpA58 SU+AIS) #51 x CS509 (F- trpA58 thr)

10.22.32.53.80.83.55.11.23.73.44.33.16.44.47.51.11.08.73.93.24.51.251.21.14.04.37.23.62.42.13.02.04.51.43.12.53.1

3.00.011.120.80.21.71.10.43.01.62.30.61.31.13.90.60.52.31.52.02.00.850.40.21.12.05.11.00.450.040.050.030.040.020.040.010.03

3.4230

2.24.84.02.14.63.01.22.11.95.24.94.01.91.82.03.82.61.62.21.53.05.53.62.151.43.65.3

52.560.066.8112.070.051.6

250.0103.00

glyVglywglyVglyVglyVglyVglyVglyVglyVglyVglyVglyVglyVglyVglyVglyVglyVglyVglyVglyVglyVglyVglyVglyVglyVglyVglyVglyVglyVglyWglyWglywglywglyWglyWglywglyw

a Those strains not transferring the suppressor allele with the thr+ locus (that is, a ratio of Thr+ Strr/Trp+Strr recombinants of from 50 to 250) were tentatively designated as glyW- (SUGAU/C) isolates. Those suppressorstrains that transferred the suppressor locus with a Thr+ Strr/Trp+ Strr ratio of approximately 1 to 5 wereassumed to be closely linked to thr+ and to be located at the glyV locus.

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TABLE 3. One-hour interrupted matings of KL16 trpA58 SU+AIS isolates with F- trpA58104 Recombinants/ml

Strains mated RHas Stio/ Suppressor

Str'

KL16 trpA78 glyW51 (Suu6,,/c) x CS508 (F- trpA78) 7.2 3.35 2.1 glyWKL16 trpA58 glyV50 (SuGAu/c) x CS509 (F- trpA58) 5.1 0.02 250.0 glyVKL16 trpA58 SU+ Al. #26 x CS509 (F- trpA58) 1.5 0.8 1.9 glyWKL16 trpA58 SU+ A.. #29 x CS509 (F- trpA58) 4.3 2.1 2.0 glyWKL16 trpA58 Su+ Ai, #30 x CS509 (F- trpA58) 4.4 2.5 1.8 glyWKL16 trpA58 Su+ Al. #51 x CS509 (F- trpA58) 5.9 2.1 2.8 glyWKL16 trpA58 SU+ AlS #104 x CS509 (F- trpA58) 4.3 3.3 1.3 glyWKL16 trpA58 SU+A,. #220 x CS509 (F- trpA58) 4.2 3.0 1.4 glyWKL16 trpA58 SU+ Al. #137 x CS509 (F- trpA58) 7.1 3.7 1.9 glyWKL16 trpA58 Su+ A.. #138 x CS509 (F- trpA58) 5.1 2.6 2.0 glyW

a Any Su+ All strain that transferred His+ and Trp+ with an His+ Strrp+ Strr ratio of approximately 2 wasassumed to have the suppressor allele located at the glyW locus.

does not prove, that the suppressor mutation inthese eight Su+A58 isolates arose at the glyWlocus.Two of the KL16 trpA58 Su',58 isolates were

selected for further study-KL16 trpA58 SuA+58no. 29 and KL16 trpA58 Su+S8 no. 104 (desig-nated strains JA306 and JA310, respectively).Interrupted matings of these strains indicatedthat the suppressor alleles in both strains[glyW52 (SUGAU,C) and glyW53 (SUGAIT/C),respectively ] were located about 2 min counter-clockwise from his on the standard E. coligenetic map. The time of entry data for thesuppressor allele in strain JA310 is presented inFig. 4. It is estimated that this suppressor alleleis located at 37 min. Since this position is verynear to the glyW51 (SUucU/C) allele, it seemslikely that the trpA58 suppressor mutationsmapping at 37 min arise at the glyW locus andare derived from tRNAGG'U/C.Suppressors of trpA78 occur at both glyV

and glyW. In the previous section we showedthat it is possible to obtain suppressors oftrpA58 arising at both the glyV and glyW loci.To be consistent with the hypothesis that thereare two gene loci coding for tRNAGG1Us, (glyVand glyW), it should also be possible to selectsuppressors of trpA78 arising at both glyV andglyW.

In a manner analogous to the experimentaldesign indicated above, suppressors of trpA78arising near the thr locus and probably locatedat glyV were sought. Revertants of strainCS523, a trpA78 HfrC derivative, were obtainedby mutagenesis with ethyl methane sulfonate asoutlined in Materials and Methods. From sev-eral hundred such revertants, 38 isolates wereselected that grew at approximately the sameslow rate on minimal agar plates as strainCS524 [trpA78glyW51 (SuUGU/c) ]. Thirty-six ofthe 38 CS523 Trp+ revertant isolates were

10

9

a

-.- 7

U ; ~~~Hie Strr

E0 05

to4

0

2 _ y-[>Thy+ Strr > S

5 10 15 20 25 30 35Time (minutes)

FIG. 4. Mapping ofglyW53 (SUCAU/C) by interruptedmating. Strain JA310 [KL16 trpA58 gly W53(SuO,AU,c)] was mated with strain CS534 (F- thyA histrpA58 str). The number of Thy+ Strr, His+ Strr, andTrp+ Str' recombinants that formed colonies onselective media is plotted versus the time at whichmating was interrupted by agitation.

suppressed revertants, as determined by themethod outlined in Materials and Methods(data not presented).Ten of the CS523 SuA7,, isolates were mated

for 60 min with an F- trpA78 multi-auxotroph.Mating was interrupted with T6 phage, andsamples were plated to select for Thr+ Strr or

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Trp+ Strr recombinants. Eight of the ten CS523SuA78 isolates transferred their Trp+ phenotypeat high frequency, indicating that the suppres-sor alleles were located just distal to the thrlocus from the point of origin in HfrC, and weretentatively classified as glyV- (SuU,G u/c) strains(Table 4). The remaining two CS523 Su'78isolates were presumed to be located at theglyW locus and were not examined further.One of the CS523 SuA78 revertants that trans-

ferred the Trp+ phenotype at high frequencywithin 60 min (designated strain JA316) wasselected for mapping of the suppressor allele bycareful interrupted mating. Analysis indicatedthat the suppressor mutation in strain JA316was located at approximately 85 min on thestandard genetic map (Fig. 5). This location isvery close to, or is the same as, the location ofthe glyV locus. Therefore, this Su+78 strainprobably contains the glyV54 (Suu G I/c) allele.The fact that two different suppressors de-

rived from tRNAGGU/C could be obtained ateither the glyV or glyW locus is in agreementwith the concept that glyV and glyWboth codefor the same tRNAGI', ,c species. Furthermore,the ratio of appearance of the various suppres-sors at the glyV and glyW loci, regardless ofwhich suppressor was sought or which mutagenwas used, was approximately three or four atglyV to one at glyW. Since it is known thatsuppressor mutations affecting the nucleotidesequence of tRNAGlGy 3c alter only a fraction ofthe tRNAGly¶,c pool (3, 17), it seems clear thatmultiple copies of this gene exist, with three orfour copies at glyV and one at gly W.

Allelic specificity of A58 and A78 sup-pressors arising at the glyV and glyW loci.

The allelic specificity of suppressors of manytrpA mutants has been demonstrated. For ex-ample, the extragenic suppressor of the trpAllmutation does not suppress the trpA3 mutationor any other trp mutant; likewise, the extra-genic suppressor of trpA3 does not suppress thetrpAl mutation or any other trp mutation (22).Preliminary transduction analysis showed thatthe original suppressors in strains Ymel A58su+and Ymel A78su+ do not cross-suppress; that is,they are allele specific. Presumably, an A78suppressor mutation arising at either the glyVor glyW locus would result in the suppression oftrpA 78 but not trpA58; conversely, an A58suppressor mutation occurring at either theglyV or glyW locus should suppress trpA58 butnot trpA78. It was, therefore, of interest to askwhether the new trpA58 and trpA78 suppressorsmapping at glyV and glyW are allele specific.Plkc lysates, prepared on suppressor strains

of the Su 78 variety arising at either glyV orglyW, were capable of transducing a trpA78derivative to Trp+ but were unable to transducetrpA58 to a Trp+ phenotype. Similarly, Plkclysates, prepared on suppressor strains of theSu+58 variety arising at either of the two loci,transduced trpA58 derivatives to tryptophanprototrophy but were unable to transducetrpA78 to Trp+ (Table 5). It thus geems unlikelythat the newly derived suppressor strains con-tain generalized suppressors causing a relaxa-tion in the fidelity of translation.

DISCUSSIONStructural genes for tRNAGlY isoacceptors are

now known at four distinct locations on the E.coli chromosome (Fig. 6). A single gene, glyT, at

TABLE 4. One-hour interrupted matings of HfrC trpA78 Su'78 isolates with F- trpA78 thr

104 Recombinants/mlStrainsmated - ~~~~~~~~~~~RatioSup-Strains mated Thr+ SWR/ pressorThr+ S-trr Tr Trp+ Strr locationaSte'

CS520 [HfrC trpA58 glyV50 (Su6 AU/C)] x CS509 (F- trpA58 thr) 7.5 1.7 4.4 glyVCS524 [HfrC trpA 78 glyW51 (Su u c; I/c)l x CS508 (F- trpA 78 thr) 20.0 0.04 500.0 glYWCS523 (HfrC trpA58 SU+ A,S) #7-1 x CS508 (F- trpA78 thr) 0.6 0.13 4.6 glyVCS523 (HfrC trpA58 SU+ A58) #7-3 x CS508 (F- trpA78 thr) 1.7 1.1 1.5 glyVCS523 (HfrC trpA58 SU+ A,.) #5-4 x CS508 (F- trpA78 thr) 1.8 0.7 2.6 glyVCS523 (HfrC trpA58 SU+ A58) #5-6 x CS508 (F- trpA78 thr) 1.8 0.52 3.5 glyVCS523 (HfrC trpA58 SU+A,S) #5-7 x CS508 (F- trpA78 thr) 1.25 0.6 2.1 glyVCS523 (HfrC trpA58 SU+A58) #15-7 x CS508 (F- trpA78 thr) 0.83 0.26 3.2 glyVCS523 (HfrC trpA58 SU+ A,.) #6-3 x CS508 (F- trpA78 thr) 1.1 0.34 3.2 glyVCS523 (HfrC trpA58 SU+ A5S) #8-1 x CS508 (F- trpA78 thr) 5.4 2.6 2.1 glyVCS523 (HfrC trpA58 Su+ A,.) #24-4 x CS508 (F- trpA78 thr) 0.97 0.01 97.0 glyWCS523 (HfrC trpA58 SU+A,.) #15-3 x CS508 (F- trpA78 thr) 5.0 0.05 100.0 glyW

aAny strain that transferred the suppressor allele at high frequency (Thr+ StrrtTrp+ Strr ratio equal to1.5 to 5.0) within 1 h of mating was assumed to have the suppressor mutation located at the glyV locus. Thosenot transferring the Trp+ phenotype within 1 h had a much larger Thr+ Str"/Trp+ Strr ratio and were tentativelypositioned at the glyW locus.

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77 min, specifies tRNAG"G'AIG. This gene occursin a closely spaced cluster with two other tRNAgenes: thrT (tRNAThru,c) and tyrT (tRNATuy, C)(1, 18. 21). A tRNA precursor, containing thetRNA.7A". and tRNAAhcU,C sequences linked intandem through a short six-nucleotide "spacer,"has been isolated and sequenced (1). Similarly,a single gene, glyU, at 55 min, specifies thesynthesis of tRNAGly (9). Several suppressor

Thr+ Strr7

E TPro+TStrtr

5 10 15 20 25 30 35

Time (minutes)

FIG. 5. Mapping of glyV54 (SUuGu/,c) by interrupted

mating. An HfrC trpA78 glyV54 (SucuGuc) isolate

(JA316) was mated with a F- proA thr trpA78 str

multi-auxotroph (CS533). The number of Pro+ Str',

Thr+ Sti', and Trp+ Str' recombinants that formed

colonies on selective media is plotted versus the time

at which mating was interrupted by agitation.

mutations are known to occur at both glyT andglyU (Fig. 6).

Genetic and biochemical evidence has nowestablished two non-adjacent genetic loci thatspecify the synthesis oftRNAY'I/c. The nucleo-tide sequence of wild-type tRNAGly/,c has beenreported (17). In addition, two suppressor

tRNA's, tRNAGly and tRNAe,'YA'., are knownto be derived from tRNAGC]u/c by single basesubstitutions (2, 17). Since the original Ins andSUA78 strains contain suppressor mutations thatmap at different locations on the E. colichromosome, it seems clear that identicaltRNAGGI;u/c genes occur at these nonadjacent

tiyr Tl

gtrTlyTorgH 9

7supT5SUA36 suA8)A78p

g~ ~ ~~~~iglyW0

FIG. 6. Relative locations of structural genes speci-

fying tRNAJ'ly (glyU), tRNA7?JJA4/G (glyT), and

tRNAC64f,c(glyV and glyW) on the E. coli chromo-

some (not to scale). Various suppressor mutations

occurring at these loci are indicated.

TABLE 5. Allelic specificity of suppressors of trpA58 and trpA78 arising at the glyV and glyW loci

No. of Trp+Recipient cell Donor strain for Plkc lysate colonies/0.1 ml of

transduction mixturea

CS519 (trpA58) None 12CS523 (trpA78) None 10None CS520 [trpA58 glyV50 (SUG AU/C)] 0None JA310 [trpA58 glyW53 (SUGAU/C)] 0None JA316 [trpA78 glyV54 (SuuGu/C)] 0None JA299 [trpA 78 glyW51 (SuuGu,c)] 0CS519 (trpA58) CS520 [trpA58 glyV50 (SUG AU/C)] 190CS519 (trpA58) JA310 [trpA58 glyW53 (SU c A U/C)] 145CS519 (trpA58) JA316 [trpA78 glyV54 (SuuGu,c)J 9CS519 (trpA58) JA299 [trpA 78 glyW51 (SuuGu,c) ] 8CS523 (trpA 78) CS520 [trpA58 glyV50 (SU c A tl / C)] 9CS523 (trpA78) JA310 [trpA58 glyW53 (SUGAU/C)] 5CS523 (trpA 78) JA316 [trpA78 glyV54 (SuuuGu/c) ] 340CS523 (trpA 78) JA299 [trpA 78 glyW51 (Su uG u/c)] 279

a Transductions were performed as outlined in the text. The Plkc lysates used were 10- dilutions of theoriginal lysates prepared on the donor strains.

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loci, glyVat 86 min and glyWat 37 min (Fig. 6).Three distinct suppressor classes derived by

genetic alteration of tRNAGGYUC structure areknown, Ins, SUA,,,, and SUA78 (Fig. 1). If identi-cal tRNAGII,c genes occur at glyV and glyW,than it should be possible to isolate eachof these suppressor classes in at least twogenetic forms, e.g., glyV- (SUGAU/c) and glyW-(SUGA,/c), and glyV- (SUUG,T/c) and glyW-(SuUGu/c). A genetic analysis of several inde-pendently arising Su',, and SU'78 strains in-dicates that, in each, the suppressor mutationcan indeed occur at either glyV or glyW (seeResults). Similar studies on the Ins strains havenot been carried out owing to difficulties inselecting and mapping this mutation.Our analysis of the frequency of independ-

ently arising missense suppressor mutations atglyV and glyW suggests that, regardless ofwhich suppressor mutation is selected or whichmutagen is used, the ratio of appearance isthree to four at glyV to one at glyW. Onereasonable interpretation of these data is thatthere are three to four identical tRNAGI,Ic genecopies at glyV for every one occurring at glyW.This model is supported by studies on tRNAGYisolated from strains carrying the episomeKLF17, which probably covers the region from84 to 89 min and thus renders the entire glyVregion diploid (17). The presence of this epi-some causes over a twofold increase in theamount of tRNAG'GU,c (relative to tRNAG'YGI), aswould be expected if the majority of tRNAG'GU7Cgenes occurred in the glyV region (assumingequal chromosomal and episomal gene dosage).

Recently, a tRNA precursor has been studiedthat is processed to mature tRNAGGU,C whenincubated with E. coli extracts (1). This precur-sor RNA is approximately 450 nucleotides longand appears to contain three tandemly repeatedcopies of tRNAGGc U,c. Although it seems likelythat this tRNA"'cY,c precursor is transcribedfrom the glyV region, this has not yet beenestablished. Confirmation of the exact numberof glyV tRNAGG U/C gene copies and their ar-rangement and spacing awaits further biochem-ical analysis of precursors containing a knownglyV suppressor tRNA sequence.

It is not clear why tRNAG ',/c should bespecified by multiple copies of identical genes,whereas tRNA GI~ and tRNA Gly 'G are eachtranscribed from single gene copies (9). Inwild-type strains of E. coli, the tRNAGlY popula-tion consists predominantly of tRNA"''Y c(70 to80%), indicating that a simple gene dosageeffect controls the relative quantities of theseisoacceptors. Perhaps the relatively largeamounts of tRNA Gly;G are needed to satisfy

functions unrelated to protein synthesis. Forexample, Gentner and Berg (Fed. Proc. 30:1218,1967) have shown that tRNAGI,3/c can serve asglycine donor for the synthesis of a glycyllipopolysaccharide in an E. coli membranefraction.

ACKNOWLEDGMENTSThis work was supported by research grants from the

Public Health Service (CA-11034, from the National CancerInstitute) and the National Science Foundation (GB-23429).

LITERATURE CITED1. Carbon, J., S. Chang, and L. L. Kirk. 1974. Clustered

tRNA genes in Escherichia coli: transcription andprocessing. Brookhaven Symp. Biol., RNA Processing,in press.

2. Carbon, J., and E. W. Fleck. 1974. Genetic alteration ofstructure and function in glycine transfer RNA ofEscherichia coli: mechanism of suppression of thetryptophan synthetase A78 mutation. J. Mol. Biol.85:371-391.

3. Carbon, J., C. Squires, and C. W. Hill. 1970. Glycinetransfer RNA of Escherichia coli. H. Impaired GGA-recognition in strains containing a genetically alteredtransfer RNA; reversal by a secondary suppressormutation. J. Mol. Biol. 52:571-584.

4. Clowes, R. C., and W. Hayes. 1968. Experiments inmicrobial genetics. John Wiley and Sons, New York.

5. Glansdorff, N., G. Sand, and C. Verhoef. 1967. The dualgenetic control of ornithine transcarbamylase synthesisin Escherichia coli K12. Mutat. Res. 4:743-751.

6. Guest, J. R., and C. Yanofsky. 1964. Mutationallyinduced amino acid substitutions in a tryptic peptideof the tryptophan synthetase A protein. J. Biol. Chem.240:679-689.

7. Guest, J. R., and C. Yanofsky. 1965. Amino acid replace-ments associated with reversion and recombinationwithin a coding unit. J. Mol. Biol. 12:793-804.

8. Hill, C. W., G. Combriato, W. Steinhart, D. L. Riddle,and J. Carbon. 1973. The nucleotide sequence of theGGG-specific glycine transfer ribonucleic acid of Esch-erichia coli and of Salmonella typhimurium. J. Biol.Chem. 248:4252-4262.

9. Hill, C. W., C. Squires, and J. Carbon. 1970. Glycinetransfer RNA of Escherichia coli. I. Structural gense fortwo glycine tRNA species. J. Mol. Biol. 52:557-569.

10. Low, B., and T. H. Wood. 1965. A quick and efficientmethod for interruption of bacterial conjugation. Ge-net. Res. 6:300-303.

11. Miller, J. H. 1972. Experiments in molecular genetics.Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.

12. Orias, E., T. K. Gartner, J. E. Lannan, and M. Betlach.1972. Close linkage between ochre and missense sup-pressors in Escherichia coli. J. Bacteriol.109:1125-1133.

13. Roberts, J. W., and J. Carbon. 1974. The molecularmechanism for missense suppression in E. coli. Nature(London) 250:412-414.

14. Roth, J. R. 1970. Genetic techniques in studies ofbacterial metabolism. Methods Enzymol. 17A:3-35.

15. Russell, R. L., J. N. Abelson, A. Landy, M. L. Gefter, S.Brenner, and J. D. Smith. 1970. Duplicate genes fortyrosine transfer RNA in Escherichia coli. J. Mol. Biol.47:1-13.

16. Smith, J. D. 1972. Genetics of transfer RNA. Annu. Rev.Genet. 6:235-255.

17. Squires, C., and J. Carbon. 1971. Normal and mutantglycine transfer RNAs. Nature (London) New Biol.

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233:274-277.18. Squires, C., B. Konrad, J. Kirschbaum, and J. Carbon.

1973. Three adjacent transfer RNA genes in Esche-richia coli. Proc. Natl. Acad. Sci. U.S.A. 70:438-441.

19. Stadler, J., and C. Yanofsky. 1959. Studies on a series oftryptophan-independent strains derived from a trypto-phan-requiring mutant of Escherichia coli. Genetics44:105-123.

20. Taylor, A. L., and C. D. Trotter. 1967. Revised linkage

map of Escherichia coli. Bacteriol. Rev. 31:332-353.21. Wu, M., N. Davidson, and J. Carbon. 1973. Physical

mapping of the transfer RNA genes on Xh80dglyTsu+,.J. Mol. Biol. 78:23-34.

22. Yanofsky, C., D. R. Helinski, and B. Maling. 1961. Theeffects of mutation on the composition and propertiesof the A protein of Escherichia coli tryptophan synthe-tase. Cold Spring Harbor Symp. Quant. Biol. 26:11-24.

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