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Duplicate genes for tyrosine transfer RNA in Escherichia coli
J. Mol.
Biol.
(1970) 47, 1-13
Duplicate Genes for Tyrosine Transfer RNA in Escherichia coli R.
L.
RUSSELL,
J. N.
ABELSON?,
S. BRENNER
AND
A. LANDY~, J. D. SMITH
M. L. GEFICER$,
Medical Research Council Laboratory of Molecular Biology Hills Road, Cambridge, England (Received 27 June, 1969) Genetic and biochemical studies of Eschetichia co.5 and the new transducing phage 1$8Opsu& have been used to characterize the tRNA genes of E. coli. The transducing phage stimulates the production of both su& and RU& tyrosine tRNA upon infection, and in hybridization experiments its DNA is saturated with 1.4 tyrosine tRNA molecules per genome. One of its derivatives, selected for its genetic properties, stimulates only SW& tyrosine tRNA, and its DNA is saturated by 0.6 tyrosine tRNA molecule per genome. We conclude that the original phage carries two tyrosine tRNA genes, one su& and one SZ&, while the derivative carries a single su& gene. The single-gene derivative apparently arises by unequal recombination involving the two genes of the original phage; the reciprocal recombination product, carrying three tyrosine tRNA genes, is also detected. Entirely analogous single-gene and three-gene derivatives of E. co&i are found, and we conclude that E. co&i normally carries a pair of closely-linked genes specifying its minor, or 8um tyrosine tRNA.
1. Introduction tyrosine tRNA genes of Escherichia coli have already been partially characterized. Two separableforms of tyrosine tRNA, a major and a minor one, have been identified (Nishimura, Harada, Narushima & Seno, 1967). Since these differ in primary nucleotide sequence(Goodman, Abelson, Landy, Brenner & Smith, 1968), they must be the products of separate genes,and hence E. coli must contain at least two tyrosine tRNA genes.At least one of these genes,specifying the minor tRNA, can mutate to give the suppressorgene su&; this gene maps near the attachment site of phage $80, and has been incorporated into $80-derived transducing phages (Smith, Abelson, Clark, Goodman & Brenner, 1966). Previous studies have suggestedthat this gene may be only one member of a set of identical genes specifying the minor tyrosine tRN14 only a part of the minor tRNA becomesSU&; Goodman et al., 1968). (in sU& strains, The gene or genes specifying the major tyrosine tRNA have not been mapped ; however, they probably do not lie closeto the $80 attachment site, since they are not incorporated into @O-derived transducing phages (Goodman et al., 1968). In this paper we show that the minor tyrosine tRNA of E. coli is the product of two The
identical t Present Calif. 92037, 1 Present R.I. 02912, 8 Present 1
genes
occupying
adjacent
or
nearly
adjacent
positions
on
the
bacterial
address: Department of Chemistry, University of California (San Diego), La Jolla, U.S.A. address : Division of Biological and Medical Sciences, Brown University, Providence, U.S.A. address: Department of Biology, Columbia University, New York, N.Y. 10023, U.S.A. 1
2
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ET
RUSSELL
AL.
chromosome, close to the attachment site of phage 480. This conclusion is based on genetic studies of su& strains, which show that these two-gene strains segregatethe expected one-geneand three-gene derivatives by unequal recombination; on similar studies of the new transducing phage, 48Opsu&, which segregatessimilar derivatives ; and on biochemical experiments which establish that #3Opsu& and its derivatives are as expected from the genetic studies.
2. Materials
Bacterial
(a) CA274t (HfrC derived by curing A. MB0 (araiLer was constructed be isogenic with
la&, the
and Methods
amber trp,,,, SU-) and canonical strains CA244
.&VT@ in an Fthese.
wL strain
strains CA276 (HfrC la& and CA265 (Brenner
kinase amber g4pimerase originally derived (b)
amber trp&ber & Beckwith,
sum) were 1965) of
twAber eC#‘8 @&, kinase amber wJepimeraae) from the CA IIfr strains and is likely to
Bacteriophage
strains
480, #Odsu&, and T4 rI1 mutants were from thecambridge collection. @Oh was the generous gift of Dr B. D. Hall. @Oh am1 and @Oh am.5 were amber mutants derived by N-methyl-N-nitro-N-nitrosoguanidine mutagenesis of @Oh. 48Opsu& was isolated as follows. The double mutant 480h amlam was constructed and used to lysogenize CA275. Among the progeny phages from the induced lysate, 1 in 1011 formed phages on the SILstrain CA274. One of these was picked and shown to have acquired the sum gene of CA275, while retaining all essential phage genes. It retained the host range, immunity and two amber mutations of the original phage, and had nearly the same density (1.490 g/cm3 versus 1.487 for the original). In cells infected with this phage, bacterial amber mutants wore suppressed at high efficiency while non-amber mutations in the same genes were not suppressed. Its lysogens had the suppression pattern of sum, judged by testing with T4 rI1 mutants. Besides su&, this phage had acquired no other identified gene from the vicinity of the 480 attachment site, and it appeared to have lost some non-essential phage genes, as it was integration-defective. This phage differs in important respects from the unstable, non-defective sum transducing phage described by Andoh & Ozeki (1968). It was not isolated from a @Odsu& lysogen, and it has very nearly the same density as its 480 parent. It segregates non-suppressing derivatives only infrequently (see text), and these retain the density of the original phage. It does not behave as though its acquired MJ$, gene were carried between two homologous, recombining regions, as appears to be the case for the phage of Andoh & Ozeki (1968).
(c) Media
and
chewaicals
B broth and low phosphate medium have been described before (Landy, Abelson, Goodman & Smith, 1967). Tris maleate contained, in g/l.; Tris (hydroxymethyl) aminomethane, 6.05; and maleic acid, 5.8; and was adjusted to pH 6.0 with NaOH. Solid (plate) media were of two types; H, which contained, in g/l. ; Difco Bacto Agar, 10; Difco Bacto Tryptone, 10; and NaCl, 8 ; and minimal, which contained in g/l. ; Difco Bacto Agar, 15 ; Na2HP04, 5.8; KH2POI, 3.0; NaCl, 0.5; NH&l, 1.0; MgSO,, 0.12; one or more carbon sources, 2 each; thiamin, 0.002 ; and, on occasion, various amino acids, 0.02 each. All chemicals used were reagent grade, except ICR 364-OH (see Ames & Whitfield, 1966), which was the generous gift of Dr H.J. Creech. Radiochemicals were obtained from the Radiochemical Centre, Amersham. Pancreatic ribonuclease was from Worthington Biochemical Corporation, and Ribonuclease T, from the Sankyo Co., Tokyo. (d)
Mutagenesis
For nitrosoguanidine mutagenesis, cells were grown to 2 x lo8 cells/ml. in B broth, washed once with Tris maleate, and exposed to 100 pg/ml. nitrosoguanidine in Tris maleate for 30 min at 37’C. After mutagenesis the cells were washed once with B broth and grown for t Abbreviations
used:
lac, lactose;
trp,
tryptophan;
ara,
arabinose;
gal, galactose.
DUPLICATE 3 to 4 generations in B broth For ICR’364-OH mutagenesis, posed to 50 pg ICR 364-OH/ml.
tRNA
GENES
3
(to permit expression of mutations) before selective plating. the procedure was the s8me, except that the cells were exin B broth for 1 hr at 37”C, instead of nitrosoguanidine. (e) Mutant
isolation
All bacterial mutants were isolated in MBO, 8 strain specially constructed to allow selection of either sw,+‘s or su-‘s. Su- mutants were selected by pl8ting MB0 on minimal plates with both galactose and glycerol w carbon sources, and with added tryptophan. Suppression of the galactokinase amber mutant U42 by the a&i gene of MB0 allows conversion of galactose to UDP-galectose, which accumulates because of the galactose epimerase mutation and kills the cells, Rare BU- mutants survive, using glycerol as carbon source, because they do not make function81 gal8ctokinase ; tryptophan is required to overcome their unsuppressed trpamber mutation. The only other mutants surviving this plating were the rare gaZeplmerase revertants, which were easily distinguished from su-‘s by their ability to grow without tryptophan. Su+ mutants were selected by plating on minimal glucose plates; sue’s do not grow mutation, and su+ ‘s could be distinguished from because of their unsuppressed trp,,,,, trp+‘s either by their sbility to support the growth of T4 amber mutants or by their inability to grow on minimal galactose-glycerol plates. Sum mutants were distinguished from other su+‘s by lysogenizing with h and testing with T4 rII amber mutants (Brenner & Beckwith, 1965). Frequencies of conversion of sum to sum and vice versa were measured by these selective plating techniques, taking care to svoid large strttistical fluotu8tions by growing cultures from 1 cell to 101r cells and using only a small sample for the me&surement. Su+ Ltnd 8u- deriv8tives of @Opsun, were isol8ted as follows. In the original phage, which carries two amber mutations, .su+ and .su- derivatives are distinguished simply by plating on .YU- bacteria, where the former give pleques and the latter do not. In 8 derived phage used for some experiments, the two amber mutations were removed, one by outcross and the other by reversion. In this phege, su+ and su”- derivatives were distinguished by plating on dye indicator plates containing 0.8 mg 6-bromo-4-chloro-3-indolyl-j3-n-galactoside, 0.24 mg isopropyl-/?-n-thiogalactoside 8nd CA274 bacterie. Su + derivatives suppress of CA274, and the resulting, isopropyl-6-D-thiogalactoside-induced the W& smber mutcttion fl-galactosidase converts the colourless 6-bromo-4-chloro-3-indolyl-6-n-galactoside to an insoluble blue residue which colours the phage plaque. Suplaques remain colourless. (f)
Purification
of tyrosine tRNA from bacteriophage-infected
cells
The procedures of Gefter & Russell (1969) were followed, with the following modifications. For oligonucleotide finger printing, unfractionated, 3aP-labelled tRNA (freed of DNA and ribosomal RNA) ~8s fractionated first on a column of DEAE-Sephadex, as described for peak I tRNA by Gefter & Russell (1969). The partially purified tyrosine tRNA was then acylated with [3H]tyrosine 8nd further purified on 8 column of benzoylated DEAE-cellulose, as described for peak II tRNA by Gefter & Russell (1969). For ribosome binding studies unfrsctionated, unlabelled tRNA (also freed of DNA and ribosomal RNA) was fractionated on 8 reverse phase column eccording to Kelmers et al. (1965). The peak II tRNA described by Gefter & Russell (1969) wa8 selected, to avoid contamination of @Opsum-stimulated tRNA by pre-existing host tyrosine tRNA, and was further purified on a column of benzoylated DEAE-cellulose 8s described before (Gefter & Russell, 1969).
(g) DNA-RNA
hybridization
DNA was extrctcted from phsges with phenol and applied to membrane filters (Sohleicher & Schuell, type 13-6, 24 mm diameter) at 2 ~g/filter, 8s described previously (Landy et al., 1967). 32P-18belled tyrosine tRNA for hybridizations was prepared s,s follows. Low phosphate medium was supplemented with [3aP]orthophosph8te (0.15 me/ml.) and unlabelled orthophosphate (to achieve 8 6nal phosph8te concentration of 9.1 M). CA274 was diluted threefold into this medium from an overnight low phosphate medium culture and grown for 1 hr. Unfractionated tRNA from these cells ~8s partially purified on a column of benzoylated DEAE-cellulose 88 described above. Hybridizations were carried out as described by Landy et al. (1967).
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RUSSELL
ET
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3. Results (a) Bacterial experiments Let us consider the properties predicted for E. coli if it contains not one, but two genes for minor tyrosine tRNA, in close linkage. These properties are indicated diagrammatically in Figure 1, where A is the structure of a normal su& strain and B is that of an sum strain derived from it. The important predictions of this two-gene model stem from the possibility of unequal recombination between the adjacent genes of the doubled structure. From an sum strain, this process should generate a novel type of sum derivative (labelled C) which contains only a single sum gene in place of the original two. When this derivative reverts to sum, it should give the single-gene structure E, with properties quite different from those of the original sum strain. Furthermore, the original sum strain should also yield other types of su& derivatives, labelled D, and D,, by mutation in the sum member of the gene pair. Since these retain two genes, they can revert to su& by unequal recombination, as shown in the Figure. The sum revertants generated in this fashion from D, and D, should be quite different. Those from D,, should have the single-gene structure E because the original mutation lies between the anticodon regions of the genes, while those from D, should have the three-gene structure F. These unusual predictions can be tested. If unequal recombination is considerably more frequent than spontaneous mutation, as is likely, most spontaneously arising su;n’s should be C’s. These should revert to sum at the low frequency characteristic of single nucleotide mutation. A smaller fraction of spontaneously arising sun,‘s should
n E
-d-m-
1SUG
DI
i E
+
f F
FIQ. 1. The model. Each structure outside boxes represents the minor tyrosine tRNA gene structure of a class of bacteria. Gene boundaries are shown as heavy vertical lines. The anticodon region of each gene is indicated as either + (specifying tRNA anticodon CUA, which recognizes codon UAG) or (specifying tRNA anticodon G*UA, which recognizes codons UAU and UAC). The sites of mutations in the other regions are indicated by i’s. Structures inside boxes represent out-of-register mispairings between identical chromosomes which can, by the indicated unequal recombination events (W‘NV), generate new structures. Phenotypes are indicated to the right.
DUPLICATE
tRNA
GENES
5
be D1’s and DZ’s, and should revert to M& at the higher frequency characteristic of unequal recombination. Furthermore, since chemical mutagens enhance mutation but not unequal recombination, most induced su&‘s should be the high reverting D1’s and D2’s. As a first check of these predictions, we isolated 20 spontaneous and 22 chemicallyinduced su& derivatives from an su& strain and measured their frequencies of reversion to su& (see Fig. 2(a)). As expected, most (IS/SO) of the spontaneous su&‘s revert at low frequency (< lo-*) and appear to be C’s. The remaining spontaneous sul&‘s and most of the chemically induced su&’ s revert much more frequently ( N 10m6) and appear to be D1’s and D,‘s. If the low reverting su&‘s are really C’s, their su& revertants should have the single-gene structure E. Accordingly, these revertants should differ from the original su& strain in an important respect; chemically-induced su&,‘s derived from them, since they contain only a single gene, should revert to su& at the low frequency characteristic of single nucleotide mutation and not at the higher frequency of unequal recombination. To test this prediction, we isolated two presumptive type E su& revertants from separate low-reverting (type C) su *&‘s, and obtained ten spontaneous and ten chemically induced su&’ s from each. As expected, nearly all of the
Cd)
10-9
10-6
10-7
10-6
10-s
10-J
FIG. 2. Frequencies of reversion to su+ or of su Ijr generation for various bacterial strains. (a) Frequencies of reversion to su + for spontaneous ( 0) and chemically-induced (a) su&‘s from the original au& strain. Of the chemically-induced su&‘s, 10 were induced by N-methylN-nitro-N-nitrosoguanidine and the remaining 13 by ICR 364.OH. Each dot represents the reversion frequency, measured as described in Materials and Methods, for a particular sunI strain. The indicated frequencies are for reversion to su + , including SU:, 8~; and a&. For the highly-reverting class (reversion frequency > 10e8), more than 90% of the su + revertants were 8& ; for the remainder, only about 20% were &,. (b) Frequencies of reversion to 8~ + for spontaneous ( C) and chemically induced (0) sulJ,‘s from two au& strains of presumed structure E. Ten 8% &‘s of each type were isolated from each .YU&; all of the chemically-induced su 1QI’s were induced by N-methyl-N-nitro-N-nitrosoguanidine. (c) Frequency of auIyI generation for the original SUMIII strain. Each dot represents one independent determination of this frequency. (d) Frequencies of ml& generation for some s& strains of presumed structures E (0) and F ( l ), derived as described in the text. (e) Frequency of suIyr generation for an su& strain of presumed structure E, derived as an w,:, revertant from a low-reverting spontaneous su& characterized in (a) above. Each dot represents one independent determination.
0
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RUSSELL
AL.
chemically-induced su&‘s, like the spontaneous ones, revert at low frequencies (
w&C)
I
w&D,)
su,(D,)
cl
I
130
&(E)
su&(E)
74
i
su&
4 1
s&(E)
3000 -
50 I
SU,
30 I s&F) su$(Dz)
I
-=l
=&c(E)
32 I su&iF)
I
3000 -
su$(D,)
FIG. 3. Summary of results for the bacterial tests of the model. Presumed structures for classes of bacteria are indicated in parentheses. Frequencies of events are written, in units of lo-*, beside arrowsrepresentingthoseevents. Frequencies of events presumed to involve mispairing and recombination are underlined, once if a two-gene structure is presumed to be involved, twice if a three-gene structure is presumed to be involved. Compare with Fig. 1.
DUPLICATE
tRNA
GENES
7
sum at the frequencies characteristic of the D,‘s examined above, and (2) their sum revertants yielded su&‘s at the very high frequencies (3 x 10m5) found above. Since F’s should yield primarily D, SU~;~‘s by unequal recombination, these results support the assignment of structure F to these four revertants. To summarize, we have provided support for the scheme in Figure 1 by identifying three classes of SU& derivatives with the properties expected for type-C, -D,, and -D, su&‘s; we have also shown that su& revertants from these, as expected, fall into two classes with the properties of type-E and-F SU&‘s. The general scheme of Figure 1 derives further support from quantitative consideration of the data presented above. According to the scheme, two independent measurements of the frequency of unequal recombination have been made; first, the frequency with which type-C suI;,‘s arise from the original su& strain, and second, the frequency with which the D, and D, 8~~;~‘s revert to su.&. Since about three-fourths of spontaneous su&‘s were C’s, the former frequency is 314 x 2.2 x 10V6 = 1.65 x 10-6. The latter frequency (which should be slightly lower, since the interval over which measured recombination occurs is somewhat smaller) is estimated from Figure 2(a) as about 1 x 10V6. The agreement is quite good. Similar agreement is obtained for two independent measurements of the frequency with which mutation inactivates the su& gene. The first is the frequency with which D, and D, su&‘s arise from the original strain, or l/4 x 2.2 x 10-s = 5.5 x 10-7. The second is the frequency with which type-E SU&‘s yield suIn’s, estimated from Figure 2(e) as 7.4 x lo-‘. A summary of this evidence is provided in Figure 3, for comparison with the scheme in Figure 1. We have been unable to generate a reasonable alternative explanation for these results. (b) Bacteriophage experiments Further support for the scheme comes from experiments with the newly isolated transducing phage &3Opsu& (see Materials and Methods). This phage is not defective, I
I
I
(0)
(b) ctr
I
.
tr
&
I
(cl
0
IAll,, I o-5
10-4
10-3
10-2
10-I
100
FIG. 4. Frequencies of reversion to 8u+ or of BU- generation for various bacteriophage strains. (a) Frequency of au- generation for 48Op.9u+ in measured by the dye techniques described in Materials and Methods. The two dots represent independent determinations. (b) Frequencies of reversion to su + for 8u- phages from induced lysates of highly-reverting (type D, and D,) su;Ir lysogens. Two to five phages from each of 25 lysstes were chamcterized, by the selective technique described in Materials and Methods. (0) Frequencies of reversion to 8U+ for 8U- phages from induced lysates of low-reverting (type C) sum lysogens. Two phages from each of 12 lysates were characterized, by the selective technique. (d) Frequencies of 8u- generation for 8u+ phages of presumed structures E (0) and F (a), derived as described in the text. Measurements by the dye technique.
8
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ET
AL.
and consequently the sum genes which it carries can be analysed genetically, as described for bacterial sunI genes above. Furthermore, bacterial sum genes can be readily transferred to this phage; when the phage integrates into the bacterial chromesome, as it doespoorly, it appears to use the sun, region as an attachment site, and upon induction, a considerable fraction of the progeny phages acquire the s%II gene structures of the host (seebelow). The bacterial strain from which $BOpsu.&arose has the two-gene structure B. If +~OPSU.&has this same structure, it should yield type-C $8Opsun, derivatives by unequal recombination. Since recombination is generally enhanced in phages, the frequency with which these arise should be high. Figure 4(a) showsthat the observed frequency of ~80psuI~,‘s is 3 to 6 x 10m3,or 1500 to 3000 times higher than in the bacterial case. Virtually all of these $8OpsuiJ,‘sshould be type C’s, and biochemical studies of someof them (seebelow) show that this is the case. The already high frequency of r$8Opsun,‘sis not raised significantly by chemical mutagens, so that chemically-induced +80psu1;,‘s of type D, and D, could not be obtained in this way. However, when type -D, and -D, sui;r bacteria were lysogenized by $8Opsu& and induced, the lysates contained both L-XL& and ST& phagesin a ratio of about 3 :l. The surerphagesin these lysates, sincethey occurred so frequently, seemed likely to have acquired the D, or D, gene structure of their host. If so, we anticipated that they should revert to sum by unequal recombination, at a frequency (see Fig. 4(a) and above) of approx. 3 to 6 x 10e3. From each lysate we isolated several sum phages. As shown in Figure 4(b), about 70% of them had reversion frequencies compatible with D, or D, structures. The remainder, with lower reversion frequencies, we assumedto be type-C segregantsof +8Opsu&, enhanced in frequency by the processes of integration and excision and by the ultraviolet light-irradiation used to induce the lysogens. In confirmation, when type-C bacteria are lysogenized and induced, the resulting sui& phages (again about one-fourth of the progeny) are all of this same low-reverting type (seeFig. 4(c)). To test further whether the high-reverting sur;r phages were D1’s and D2’s, we examined a number of their sum revertants. Figure 4(d) shows that those from the presumedD1’s yielded $8Opsu,;,‘ssomewhat lessfrequently than the original 5bSOpsu& ( N 5 x 10W4)and appearedto be E’s. Those from the presumedDs’syielded $8Opsun,‘s at very high frequencies (N 2 x 10-l) and appeared to be F’s. As in the bacterial c&se,we have identified ~8Opsui;, derivatives with the properties expected of C’s, Di’s and D,‘s; Their revertants fall into two classeswith the properties expected for E’s and F’s. Furthermore, we have shown that a given sum gene structure retains its properties upon transfer to +8Opsu,,,, subject only to the increase in recombination which characterizes phage growth. Direct support for the schemeof Figure 1 comesfrom biochemical characterization of +8Opsu&, and some of its derivatives. According to the scheme, +8Opsu& itself should stimulate synthesis of both sz& and sur;r tRNA upon infection, whereas a type-E derivative should stimulate only SU& tRNA. Furthermore, +8Opsu& should contain twice as much DNA hybridizing with tyrosine tRNA as does the type-E derivative. Upon infection, $8Opsu& does indeed stimulate the production of large amounts tyrosine tRNA, and the same is true of several of its type-C, -D,, -D, of @%I1 and -E derivatives. The tyrosine tRNA’s stimulated by +8Opsu& and one of its type-E derivatives have been purified and digested with ribonuclease T, to give the
.fwrlrif
,‘,
.\
DUPLICATE
tRNA
E.co/i
9
GENES
Tyr [I(su+)
w&J]
;OH C A pG-C G-C U-A G-C G-C G-C G-C
2’ omG
CGA
G
cuucCUAA
4tU * 4tU
III
‘CAA~
G
II
GAAGG
GCCC III GGG
cu
Tc U
A Cc G-C G” A C-G U’ CAG A-U C-A G-C AU A-@ 2, A’ C c (II) U 2rnt.61A G;
A
f C (S”& G’is
unknown
5. Nucleotide sequences of E. coli tyrosine tRNA’s taken from Goodman et al., (1968). The anticodon region is at the bottom of the Figure; the anticodon itself is CUA in a& tRNA, end G*UA in both wz& tRNA and the major tyrosina tRNA. The minor tyrosine tRNA’s (s& and au&) differ from the major tyrosine tRNA in a dinucleotide sequence at the end of the “finger” adjacent to the anticodon loop; the minor tRNA’s contain a UC sequence in place of the major tRNA’s CA. FIG.
fingerprints presented in Plate I(a) and (b), respectively. (For reference, the entire nucleotide sequenceof E. coli tyrosine tRNA is presented in Fig. 5.) The four oligonucleotide spots labelled 1, 2, 3 and 4 in Plate I( a) are derived from the anticodon region. Their pancreatic ribonuclease digestion products and their inferred sequences are shown in Plate I(c). Spot 3 is derived from sum tRNA and contains the anticodon sequence CUA. Spots 1, 2 and 4 are derived from sum tRNA (anticodon sequence G*UA or GUA) as described in the legend. Since all four spots are present, @Opsu& stimulates the production of both su& and sum tyrosine tRNA. In Plate I(b), these four are replaced by a single spot, labelled 5, whose mobility and composition are identical to those of spot 3; we conclude that the type-E derivative of @Opsu& stimulates the production of only su& tyrosine tRNA. This conclusion is confirmed by the trinucleotide-dependent binding experiments of Table 1. The su& tRNA produced by the type-E derivative recognizes almost exclusively UAG, whereas the tRNA produced by $80psur& binds about equally well in responseto UAG or UAU, as if it were composedof nearly equal amounts of sum and SW& tRNA. (Note also that a type-C derivative produces exclusively sum tRNA.)
10
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TABLE 1 Trinucleotide-dependent
Bacteriophage stimulate tRNA
g8OP&r Type-E Type-C
derivative derivative
used to synthesis
bin&g
to ribosomes of tRNA derivatives
stimulated by 48Opsu& and
Cta/min of 3H-1abelled tryosine tRNA bound to ribosomes with: no triplet UAU UAG 3420 1666 3216
8817 3058 13,496
9512 16,246 3106
Percentage of input tRNA bound specifically by : UAU UAG 17 6.2 42
20 54 0
Binding assays were done by the method of Nirenber & Leder (1964) as previously described (Gefter t Russell, 1969). Phage-stimulated tRNA was prepared as described in Materials and Methods, and then acylated with high specific activity [3H]tyrosine. Input radioactivity for the three experiments was ~BOpsu&; 30,800 cts/min; type-E derivative, 27,000 cts/min; type-C derivative, 24,000 cts/min.
Quantitative hybridization experiments between tyrosine tRNA and the DNA’s of (Fig. 6) show, despite some variation, that +8OP4u and a type-E derivative contains about twice as much DNA hybridizing with tyrosine tRNA as #~OP%L does the type-E derivative, Furthermore, single-step mutations in the SZL& gene of a type-E derivative convert all of the stimulated su& tRNA to an altered form (Abelson et al., 1969), so the type-E derivative does not carry more than one sum gene. We conclude from all these results that @Opsu& contains two tyrosine tRNA genes, one su& and one sum, whereas its type-E derivative contains only a single sum gene. From the genetic studies of E. coli described above, we conclude that the doubled sunI genes of @Opsu& were almost certainly acquired intact from its bacterial host of origin.
4. Discussion The results described above strongly support the following general picture for tyrosine tRNA genes in E. coli. The major portion (60%) of tyrosine is produced by a gene or genes of unknown location, probably not close to the $80 attachment site (since $30 derivatives do not stimulate synthesis of this tRNA). The remaining minor tyrosine tRNA is the product of two closely linked and normally identical genes, located near the 480 attachment site. Su& mutants arise by mutation in the anticodon region of one of these two, leaving the other intact. When $30 transducing phages acquire the resulting sum gene, they are very likely to acquire its sur;r neighbour as well. In both phage and bacteria, unequal recombination between the neighbouring genes occurs, sometimes eliminating one gene, sometimes inserting an additional gene. This picture is consistent with most previous evidence, and it enables us to explain two previous paradoxical observations. The production of both sum and sunI tRNA by the defective transducing phage 480dsum (Goodman et al., 1968) is almost certainly due to incorporation into this phage of the tightly linked su& and sz& gene pair from its host of origin, Second, the finding that most su& mutants derived from the original strain appeared to be anticodon sum+ -to-su,;, changes (Abelson, J. N., Barnett,
DUPLICATE
'2P-lobelled
tRNA
GENES
tyrosine
tRNA
11
added
(pg)
FIQ. 6. Saturation curves for hybridization of tyrosine tRNA with DNA from ~SOpsu& and a derivative. Hybridizations were performed as described in Materials and Methods. Each filter contained 2 pg of added DNA, and the specific activity of the s2P-labelled tyrosine tRNA used was 582,500 cts/min/pg. The filled symbols are for DNA from @Opal & (three separate DNA preparations), the open symbols are for DNA from a type E derivative phage (three separate DNA preparations). Each value in the Figure has been oorrected by subtraction of the relatively small background which bound to 480 DNA at the same added-tRNA concentration. The arrows at the right denote the levels of bound tRNA expected for one and two tyrosine tRNA genes per genome, respectively, based on the following assumptions; (1) a molecular weight of 30.4 x 10s for the sodium salt of @Opaz& DNA, computed from the densities of @Opsu& and $80 (1.490 and 1.487 g/ems, respectively), and the molecular weight of 29.5 x 10s for the sodium salt of 480 DNA (Yamagashi, Nakamura & Ozeki, 1965; Burgi & Hershey, 1963), by the method of Weigle, Meselson & Paigen (1959), and (2) a molecular weight of 29.2 x lo3 for the sodium salt of tyrosine tRNA, computed from bhe su& tyrosine tRNA sequence of Fig. 5.
L., Brenner, S., Goodman, H. M., Landy, A. & Smith, J. D., unpublished results) can now be ascribed to the generation of type-C SU~;~‘s by unequal recombination. However, the picture is contradicted by one important piece of evidence; previous DNA-RNA hybridization experiments (Landy et al., 1967) seemed to show that +80dsu:,, contains only a single s%II gene, judged by the amount of tRNA hybridized to +8Odsu& DNA under saturating conditions. A possible resolution of this disagreement, can be seen by close examination of Figure 6. By genetic criteria, more than 99.9% of the genomes of a type-E $3Opsu,,, derivative contain an su& gene. But according to Figure 6, DNA from this derivative is saturated with tyrosine tRNA at a level of only about 0.6 molecule per genome. This may result from some systematic error in the experiments, but it may also imply that about 40% of the tRNA-complementary sequences in the DNA are not available for hybridization. Similarly, the DNA of $3Opsu:,,, which almost certainly carries two s%II genes, is saturated at a level of about 1.4 molecules per genome. These low-saturation levels, if they are real, are not difficult to explain; we suspect that they may result either from a particular tendency of tRNA-complementary sequences in the DNA to fold up on themselves, or, more generally, from interactions between DNA and the filter which render significant stretches of DNA inaccessible. Either of these difficulties, but particularly the latter, might well affect other hybridization studies, rendering their estimates of complementary DNA low by as much as 40%. If these are the only factors affecting hybridization studies with +8Odsu&, its DNA should also saturate at about 1.2 to 1.4 molecules per genome. We have repeated
12
R.
L.
RUSSELL
1T
AL.
hybridization experiments with #30dsu&, in parallel with those reported in Figure 6. Although we observed considerable variation (somewhat greater than that of Fig. 6), the average saturation level for these experiments was 0.96 molecule per genome, in agreement with the published value of 1.0. This low value may well reflect the difficulty of working with the defective &Odsu&. In particular, this phage must be prepared by induction of a doubly lysogenic strain, and the process of excision from the bacterial chromosome may well enhance the frequency with which unequal recombination reduces an original doubled structure to a single one. (This apparently happens during induction of $80psum lysogens-see Fig. 4(b) and above.) Preliminary experiments suggest that the frequency of single-gene-carrying phages in a +80dsu& preparation may be as high as 30%. Moreover, the 48Odsum particles must be separated from the ~$80particles in the same lysate ; failure to achieve proper separation would dilute the sunI genes carried by @Odsu& DNA. We suspect that both of these factors, as well as the general effects observed with $8Opsu& have operated to reduce the saturation level for @Odsu & DNA from a theoretical 2.0 molecules per genome to the observed 1.0 molecule per genome. The role of the duplicate sum genes in E. coli is not clear. They are apparently the only genes which specify the minor tyrosine tRNA (preliminary experiments by J. N. Abelson and J. D. Smith show that all of the minor tyrosine tRNA in type-E bacteria is sum and none SU~;~). Nonetheless, they do not appear to be essential, since viable mutants with apparent deletions of both genes have been obtained (Russell, R. L., unpublished results). Moreover, if one gene is eliminated by unequal recombination and the other is subverted from its normal coding function by conversion to su& (as in type-E bacteria), there is no observable effect on growth rate. Nevertheless, there must be active maintenance of these duplicate genes, particularly when unequal recombination could easily eliminate one of them, and one must assume that they confer upon E. coli some slight selective advantage, not apparent under normal laboratory conditions. Although other minor tRNA’s may have similar functions, at least one, the minor serine tRNA specified by the 8% gene, does not appear to be specified by a similar pair of closely linked genes. flu: strains of E. coli behave like type-E su& derivatives in the sense that both chemically-induced and spontaneous 8%‘~ derived from them revert to su: at the low frequency characteristic of single nucleotide mutation. By inference, the su: gene does not have a dispensable su; neighbour. Similar experiments with su; strains have proved inconclusive. While the role of the duplicate su& genes is not clear, they do provide information on the properties of gene duplications in general. Of particular interest is the observation that an increase in gene number from two to three (as from type-D, to type-F bacteria) produces a 20- to 30-fold increase in the frequency of unequal recombination (from 1 to 1.5 x 1O-6 to 3 x 10m5). Since this increased recombination tends to eliminate the three-gene structure, it is clear that gene amplification in this fashion, at least in E. coli, rather rapidly encounters problems in the stability of the amplified product; only sufficiently strong selective pressures will maintain gene multiples created in this way. We thank Miss E. Higgins and Mr T. V. Smith for their expert technical assistance. by N.A.T.O. Postdoctoral Fellowships,
Two of us (R.L.R. and A.L.) were supported another (J.N.A.) by a U.S. Public Health Service (M.L.G.) by a Jane Coffin Childs Memorial Fund
Postdoctoral for Medical
Fellowship, and Research Fellowship.
another
DUPLICATE
tRNA
GENES
13
REFERENCES Abelson, J. N., Barn&t, L., Brenner, S., Gefter, M. L., Landy, A., Russell, R. L. & Smith, J. D. (1969). FEBS Letters, 3, 1. Ames, B. N. & Whitfield, H. J., Jr. (1966). Cold Spr. Had. Symp. Quant. Biol. 31, 221. Andoh, T. & Ozeki, H. (1968). Proc. Nat. Acd Sci., Wash. 59, 792. Brenner, S. & Beckwith, J. R. (1965). J. Mol. Biol. 13, 629. Burgi, E. & Hershey, A. D. (1963). Biophys. J. 3, 309. Gefter, M. L. & Russell, R. L. (1969). J. Mol. Biol. 39, 145. Goodman, H. M., Abelson, J. N., Landy, A., Brenner, S. & Smith, J. D. (1968). Nature, 217, 1019. Kelmers, A. D., Novelli, G. D. & Stulberg, M. P. (1965). J. BioZ. Chem. 240, 3979. Landy, A., Abelson, J. N., Goodman, H. M. & Smith, .J. D. (1967). J. Mol. BioZ. 29, 457, Nirenherg, M. W. & Leder, P. (1964). Science, 145, 1399. Nishimura, S., Harada, J., Narushima, U. & Seno, T. (1967). Biochim. biophys. Actu, 142, 133. Sanger, F., Brownlee, G. 0. & Barrell, B. G. (1965). J. Mol. BioZ. 13, 373. Smith, J. D. Abelson, J. N., Clark, B. F. C., Goodman, H. M. & Brenner, S. (1966). Cold Spr. Harb. Symp. Quant. BioZ. 31, 479. Weiglo, J-. J., Meselson, M. & Paigen, K. (1959). J. Mol. BioZ. 1, 379. Yamagashi, H., Nakamura, K. & Ozeki, H. (1965) Biochem. Biophys. Res. Comm. 20, 727.
Biol.
(1970) 47, 1-13
Duplicate Genes for Tyrosine Transfer RNA in Escherichia coli R.
L.
RUSSELL,
J. N.
ABELSON?,
S. BRENNER
AND
A. LANDY~, J. D. SMITH
M. L. GEFICER$,
Medical Research Council Laboratory of Molecular Biology Hills Road, Cambridge, England (Received 27 June, 1969) Genetic and biochemical studies of Eschetichia co.5 and the new transducing phage 1$8Opsu& have been used to characterize the tRNA genes of E. coli. The transducing phage stimulates the production of both su& and RU& tyrosine tRNA upon infection, and in hybridization experiments its DNA is saturated with 1.4 tyrosine tRNA molecules per genome. One of its derivatives, selected for its genetic properties, stimulates only SW& tyrosine tRNA, and its DNA is saturated by 0.6 tyrosine tRNA molecule per genome. We conclude that the original phage carries two tyrosine tRNA genes, one su& and one SZ&, while the derivative carries a single su& gene. The single-gene derivative apparently arises by unequal recombination involving the two genes of the original phage; the reciprocal recombination product, carrying three tyrosine tRNA genes, is also detected. Entirely analogous single-gene and three-gene derivatives of E. co&i are found, and we conclude that E. co&i normally carries a pair of closely-linked genes specifying its minor, or 8um tyrosine tRNA.
1. Introduction tyrosine tRNA genes of Escherichia coli have already been partially characterized. Two separableforms of tyrosine tRNA, a major and a minor one, have been identified (Nishimura, Harada, Narushima & Seno, 1967). Since these differ in primary nucleotide sequence(Goodman, Abelson, Landy, Brenner & Smith, 1968), they must be the products of separate genes,and hence E. coli must contain at least two tyrosine tRNA genes.At least one of these genes,specifying the minor tRNA, can mutate to give the suppressorgene su&; this gene maps near the attachment site of phage $80, and has been incorporated into $80-derived transducing phages (Smith, Abelson, Clark, Goodman & Brenner, 1966). Previous studies have suggestedthat this gene may be only one member of a set of identical genes specifying the minor tyrosine tRN14 only a part of the minor tRNA becomesSU&; Goodman et al., 1968). (in sU& strains, The gene or genes specifying the major tyrosine tRNA have not been mapped ; however, they probably do not lie closeto the $80 attachment site, since they are not incorporated into @O-derived transducing phages (Goodman et al., 1968). In this paper we show that the minor tyrosine tRNA of E. coli is the product of two The
identical t Present Calif. 92037, 1 Present R.I. 02912, 8 Present 1
genes
occupying
adjacent
or
nearly
adjacent
positions
on
the
bacterial
address: Department of Chemistry, University of California (San Diego), La Jolla, U.S.A. address : Division of Biological and Medical Sciences, Brown University, Providence, U.S.A. address: Department of Biology, Columbia University, New York, N.Y. 10023, U.S.A. 1
2
R.
L.
ET
RUSSELL
AL.
chromosome, close to the attachment site of phage 480. This conclusion is based on genetic studies of su& strains, which show that these two-gene strains segregatethe expected one-geneand three-gene derivatives by unequal recombination; on similar studies of the new transducing phage, 48Opsu&, which segregatessimilar derivatives ; and on biochemical experiments which establish that #3Opsu& and its derivatives are as expected from the genetic studies.
2. Materials
Bacterial
(a) CA274t (HfrC derived by curing A. MB0 (araiLer was constructed be isogenic with
la&, the
and Methods
amber trp,,,, SU-) and canonical strains CA244
.&VT@ in an Fthese.
wL strain
strains CA276 (HfrC la& and CA265 (Brenner
kinase amber g4pimerase originally derived (b)
amber trp&ber & Beckwith,
sum) were 1965) of
twAber eC#‘8 @&, kinase amber wJepimeraae) from the CA IIfr strains and is likely to
Bacteriophage
strains
480, #Odsu&, and T4 rI1 mutants were from thecambridge collection. @Oh was the generous gift of Dr B. D. Hall. @Oh am1 and @Oh am.5 were amber mutants derived by N-methyl-N-nitro-N-nitrosoguanidine mutagenesis of @Oh. 48Opsu& was isolated as follows. The double mutant 480h amlam was constructed and used to lysogenize CA275. Among the progeny phages from the induced lysate, 1 in 1011 formed phages on the SILstrain CA274. One of these was picked and shown to have acquired the sum gene of CA275, while retaining all essential phage genes. It retained the host range, immunity and two amber mutations of the original phage, and had nearly the same density (1.490 g/cm3 versus 1.487 for the original). In cells infected with this phage, bacterial amber mutants wore suppressed at high efficiency while non-amber mutations in the same genes were not suppressed. Its lysogens had the suppression pattern of sum, judged by testing with T4 rI1 mutants. Besides su&, this phage had acquired no other identified gene from the vicinity of the 480 attachment site, and it appeared to have lost some non-essential phage genes, as it was integration-defective. This phage differs in important respects from the unstable, non-defective sum transducing phage described by Andoh & Ozeki (1968). It was not isolated from a @Odsu& lysogen, and it has very nearly the same density as its 480 parent. It segregates non-suppressing derivatives only infrequently (see text), and these retain the density of the original phage. It does not behave as though its acquired MJ$, gene were carried between two homologous, recombining regions, as appears to be the case for the phage of Andoh & Ozeki (1968).
(c) Media
and
chewaicals
B broth and low phosphate medium have been described before (Landy, Abelson, Goodman & Smith, 1967). Tris maleate contained, in g/l.; Tris (hydroxymethyl) aminomethane, 6.05; and maleic acid, 5.8; and was adjusted to pH 6.0 with NaOH. Solid (plate) media were of two types; H, which contained, in g/l. ; Difco Bacto Agar, 10; Difco Bacto Tryptone, 10; and NaCl, 8 ; and minimal, which contained in g/l. ; Difco Bacto Agar, 15 ; Na2HP04, 5.8; KH2POI, 3.0; NaCl, 0.5; NH&l, 1.0; MgSO,, 0.12; one or more carbon sources, 2 each; thiamin, 0.002 ; and, on occasion, various amino acids, 0.02 each. All chemicals used were reagent grade, except ICR 364-OH (see Ames & Whitfield, 1966), which was the generous gift of Dr H.J. Creech. Radiochemicals were obtained from the Radiochemical Centre, Amersham. Pancreatic ribonuclease was from Worthington Biochemical Corporation, and Ribonuclease T, from the Sankyo Co., Tokyo. (d)
Mutagenesis
For nitrosoguanidine mutagenesis, cells were grown to 2 x lo8 cells/ml. in B broth, washed once with Tris maleate, and exposed to 100 pg/ml. nitrosoguanidine in Tris maleate for 30 min at 37’C. After mutagenesis the cells were washed once with B broth and grown for t Abbreviations
used:
lac, lactose;
trp,
tryptophan;
ara,
arabinose;
gal, galactose.
DUPLICATE 3 to 4 generations in B broth For ICR’364-OH mutagenesis, posed to 50 pg ICR 364-OH/ml.
tRNA
GENES
3
(to permit expression of mutations) before selective plating. the procedure was the s8me, except that the cells were exin B broth for 1 hr at 37”C, instead of nitrosoguanidine. (e) Mutant
isolation
All bacterial mutants were isolated in MBO, 8 strain specially constructed to allow selection of either sw,+‘s or su-‘s. Su- mutants were selected by pl8ting MB0 on minimal plates with both galactose and glycerol w carbon sources, and with added tryptophan. Suppression of the galactokinase amber mutant U42 by the a&i gene of MB0 allows conversion of galactose to UDP-galectose, which accumulates because of the galactose epimerase mutation and kills the cells, Rare BU- mutants survive, using glycerol as carbon source, because they do not make function81 gal8ctokinase ; tryptophan is required to overcome their unsuppressed trpamber mutation. The only other mutants surviving this plating were the rare gaZeplmerase revertants, which were easily distinguished from su-‘s by their ability to grow without tryptophan. Su+ mutants were selected by plating on minimal glucose plates; sue’s do not grow mutation, and su+ ‘s could be distinguished from because of their unsuppressed trp,,,,, trp+‘s either by their sbility to support the growth of T4 amber mutants or by their inability to grow on minimal galactose-glycerol plates. Sum mutants were distinguished from other su+‘s by lysogenizing with h and testing with T4 rII amber mutants (Brenner & Beckwith, 1965). Frequencies of conversion of sum to sum and vice versa were measured by these selective plating techniques, taking care to svoid large strttistical fluotu8tions by growing cultures from 1 cell to 101r cells and using only a small sample for the me&surement. Su+ Ltnd 8u- deriv8tives of @Opsun, were isol8ted as follows. In the original phage, which carries two amber mutations, .su+ and .su- derivatives are distinguished simply by plating on .YU- bacteria, where the former give pleques and the latter do not. In 8 derived phage used for some experiments, the two amber mutations were removed, one by outcross and the other by reversion. In this phege, su+ and su”- derivatives were distinguished by plating on dye indicator plates containing 0.8 mg 6-bromo-4-chloro-3-indolyl-j3-n-galactoside, 0.24 mg isopropyl-/?-n-thiogalactoside 8nd CA274 bacterie. Su + derivatives suppress of CA274, and the resulting, isopropyl-6-D-thiogalactoside-induced the W& smber mutcttion fl-galactosidase converts the colourless 6-bromo-4-chloro-3-indolyl-6-n-galactoside to an insoluble blue residue which colours the phage plaque. Suplaques remain colourless. (f)
Purification
of tyrosine tRNA from bacteriophage-infected
cells
The procedures of Gefter & Russell (1969) were followed, with the following modifications. For oligonucleotide finger printing, unfractionated, 3aP-labelled tRNA (freed of DNA and ribosomal RNA) ~8s fractionated first on a column of DEAE-Sephadex, as described for peak I tRNA by Gefter & Russell (1969). The partially purified tyrosine tRNA was then acylated with [3H]tyrosine 8nd further purified on 8 column of benzoylated DEAE-cellulose, as described for peak II tRNA by Gefter & Russell (1969). For ribosome binding studies unfrsctionated, unlabelled tRNA (also freed of DNA and ribosomal RNA) was fractionated on 8 reverse phase column eccording to Kelmers et al. (1965). The peak II tRNA described by Gefter & Russell (1969) wa8 selected, to avoid contamination of @Opsum-stimulated tRNA by pre-existing host tyrosine tRNA, and was further purified on a column of benzoylated DEAE-cellulose 8s described before (Gefter & Russell, 1969).
(g) DNA-RNA
hybridization
DNA was extrctcted from phsges with phenol and applied to membrane filters (Sohleicher & Schuell, type 13-6, 24 mm diameter) at 2 ~g/filter, 8s described previously (Landy et al., 1967). 32P-18belled tyrosine tRNA for hybridizations was prepared s,s follows. Low phosphate medium was supplemented with [3aP]orthophosph8te (0.15 me/ml.) and unlabelled orthophosphate (to achieve 8 6nal phosph8te concentration of 9.1 M). CA274 was diluted threefold into this medium from an overnight low phosphate medium culture and grown for 1 hr. Unfractionated tRNA from these cells ~8s partially purified on a column of benzoylated DEAE-cellulose 88 described above. Hybridizations were carried out as described by Landy et al. (1967).
4
R.
L.
RUSSELL
ET
AL.
3. Results (a) Bacterial experiments Let us consider the properties predicted for E. coli if it contains not one, but two genes for minor tyrosine tRNA, in close linkage. These properties are indicated diagrammatically in Figure 1, where A is the structure of a normal su& strain and B is that of an sum strain derived from it. The important predictions of this two-gene model stem from the possibility of unequal recombination between the adjacent genes of the doubled structure. From an sum strain, this process should generate a novel type of sum derivative (labelled C) which contains only a single sum gene in place of the original two. When this derivative reverts to sum, it should give the single-gene structure E, with properties quite different from those of the original sum strain. Furthermore, the original sum strain should also yield other types of su& derivatives, labelled D, and D,, by mutation in the sum member of the gene pair. Since these retain two genes, they can revert to su& by unequal recombination, as shown in the Figure. The sum revertants generated in this fashion from D, and D, should be quite different. Those from D,, should have the single-gene structure E because the original mutation lies between the anticodon regions of the genes, while those from D, should have the three-gene structure F. These unusual predictions can be tested. If unequal recombination is considerably more frequent than spontaneous mutation, as is likely, most spontaneously arising su;n’s should be C’s. These should revert to sum at the low frequency characteristic of single nucleotide mutation. A smaller fraction of spontaneously arising sun,‘s should
n E
-d-m-
1SUG
DI
i E
+
f F
FIQ. 1. The model. Each structure outside boxes represents the minor tyrosine tRNA gene structure of a class of bacteria. Gene boundaries are shown as heavy vertical lines. The anticodon region of each gene is indicated as either + (specifying tRNA anticodon CUA, which recognizes codon UAG) or (specifying tRNA anticodon G*UA, which recognizes codons UAU and UAC). The sites of mutations in the other regions are indicated by i’s. Structures inside boxes represent out-of-register mispairings between identical chromosomes which can, by the indicated unequal recombination events (W‘NV), generate new structures. Phenotypes are indicated to the right.
DUPLICATE
tRNA
GENES
5
be D1’s and DZ’s, and should revert to M& at the higher frequency characteristic of unequal recombination. Furthermore, since chemical mutagens enhance mutation but not unequal recombination, most induced su&‘s should be the high reverting D1’s and D2’s. As a first check of these predictions, we isolated 20 spontaneous and 22 chemicallyinduced su& derivatives from an su& strain and measured their frequencies of reversion to su& (see Fig. 2(a)). As expected, most (IS/SO) of the spontaneous su&‘s revert at low frequency (< lo-*) and appear to be C’s. The remaining spontaneous sul&‘s and most of the chemically induced su&’ s revert much more frequently ( N 10m6) and appear to be D1’s and D,‘s. If the low reverting su&‘s are really C’s, their su& revertants should have the single-gene structure E. Accordingly, these revertants should differ from the original su& strain in an important respect; chemically-induced su&,‘s derived from them, since they contain only a single gene, should revert to su& at the low frequency characteristic of single nucleotide mutation and not at the higher frequency of unequal recombination. To test this prediction, we isolated two presumptive type E su& revertants from separate low-reverting (type C) su *&‘s, and obtained ten spontaneous and ten chemically induced su&’ s from each. As expected, nearly all of the
Cd)
10-9
10-6
10-7
10-6
10-s
10-J
FIG. 2. Frequencies of reversion to su+ or of su Ijr generation for various bacterial strains. (a) Frequencies of reversion to su + for spontaneous ( 0) and chemically-induced (a) su&‘s from the original au& strain. Of the chemically-induced su&‘s, 10 were induced by N-methylN-nitro-N-nitrosoguanidine and the remaining 13 by ICR 364.OH. Each dot represents the reversion frequency, measured as described in Materials and Methods, for a particular sunI strain. The indicated frequencies are for reversion to su + , including SU:, 8~; and a&. For the highly-reverting class (reversion frequency > 10e8), more than 90% of the su + revertants were 8& ; for the remainder, only about 20% were &,. (b) Frequencies of reversion to 8~ + for spontaneous ( C) and chemically induced (0) sulJ,‘s from two au& strains of presumed structure E. Ten 8% &‘s of each type were isolated from each .YU&; all of the chemically-induced su 1QI’s were induced by N-methyl-N-nitro-N-nitrosoguanidine. (c) Frequency of auIyI generation for the original SUMIII strain. Each dot represents one independent determination of this frequency. (d) Frequencies of ml& generation for some s& strains of presumed structures E (0) and F ( l ), derived as described in the text. (e) Frequency of suIyr generation for an su& strain of presumed structure E, derived as an w,:, revertant from a low-reverting spontaneous su& characterized in (a) above. Each dot represents one independent determination.
0
R.
L.
ET
RUSSELL
AL.
chemically-induced su&‘s, like the spontaneous ones, revert at low frequencies (
w&C)
I
w&D,)
su,(D,)
cl
I
130
&(E)
su&(E)
74
i
su&
4 1
s&(E)
3000 -
50 I
SU,
30 I s&F) su$(Dz)
I
-=l
=&c(E)
32 I su&iF)
I
3000 -
su$(D,)
FIG. 3. Summary of results for the bacterial tests of the model. Presumed structures for classes of bacteria are indicated in parentheses. Frequencies of events are written, in units of lo-*, beside arrowsrepresentingthoseevents. Frequencies of events presumed to involve mispairing and recombination are underlined, once if a two-gene structure is presumed to be involved, twice if a three-gene structure is presumed to be involved. Compare with Fig. 1.
DUPLICATE
tRNA
GENES
7
sum at the frequencies characteristic of the D,‘s examined above, and (2) their sum revertants yielded su&‘s at the very high frequencies (3 x 10m5) found above. Since F’s should yield primarily D, SU~;~‘s by unequal recombination, these results support the assignment of structure F to these four revertants. To summarize, we have provided support for the scheme in Figure 1 by identifying three classes of SU& derivatives with the properties expected for type-C, -D,, and -D, su&‘s; we have also shown that su& revertants from these, as expected, fall into two classes with the properties of type-E and-F SU&‘s. The general scheme of Figure 1 derives further support from quantitative consideration of the data presented above. According to the scheme, two independent measurements of the frequency of unequal recombination have been made; first, the frequency with which type-C suI;,‘s arise from the original su& strain, and second, the frequency with which the D, and D, 8~~;~‘s revert to su.&. Since about three-fourths of spontaneous su&‘s were C’s, the former frequency is 314 x 2.2 x 10V6 = 1.65 x 10-6. The latter frequency (which should be slightly lower, since the interval over which measured recombination occurs is somewhat smaller) is estimated from Figure 2(a) as about 1 x 10V6. The agreement is quite good. Similar agreement is obtained for two independent measurements of the frequency with which mutation inactivates the su& gene. The first is the frequency with which D, and D, su&‘s arise from the original strain, or l/4 x 2.2 x 10-s = 5.5 x 10-7. The second is the frequency with which type-E SU&‘s yield suIn’s, estimated from Figure 2(e) as 7.4 x lo-‘. A summary of this evidence is provided in Figure 3, for comparison with the scheme in Figure 1. We have been unable to generate a reasonable alternative explanation for these results. (b) Bacteriophage experiments Further support for the scheme comes from experiments with the newly isolated transducing phage &3Opsu& (see Materials and Methods). This phage is not defective, I
I
I
(0)
(b) ctr
I
.
tr
&
I
(cl
0
IAll,, I o-5
10-4
10-3
10-2
10-I
100
FIG. 4. Frequencies of reversion to 8u+ or of BU- generation for various bacteriophage strains. (a) Frequency of au- generation for 48Op.9u+ in measured by the dye techniques described in Materials and Methods. The two dots represent independent determinations. (b) Frequencies of reversion to su + for 8u- phages from induced lysates of highly-reverting (type D, and D,) su;Ir lysogens. Two to five phages from each of 25 lysstes were chamcterized, by the selective technique described in Materials and Methods. (0) Frequencies of reversion to 8U+ for 8U- phages from induced lysates of low-reverting (type C) sum lysogens. Two phages from each of 12 lysates were characterized, by the selective technique. (d) Frequencies of 8u- generation for 8u+ phages of presumed structures E (0) and F (a), derived as described in the text. Measurements by the dye technique.
8
R. L. RUSSELL
ET
AL.
and consequently the sum genes which it carries can be analysed genetically, as described for bacterial sunI genes above. Furthermore, bacterial sum genes can be readily transferred to this phage; when the phage integrates into the bacterial chromesome, as it doespoorly, it appears to use the sun, region as an attachment site, and upon induction, a considerable fraction of the progeny phages acquire the s%II gene structures of the host (seebelow). The bacterial strain from which $BOpsu.&arose has the two-gene structure B. If +~OPSU.&has this same structure, it should yield type-C $8Opsun, derivatives by unequal recombination. Since recombination is generally enhanced in phages, the frequency with which these arise should be high. Figure 4(a) showsthat the observed frequency of ~80psuI~,‘s is 3 to 6 x 10m3,or 1500 to 3000 times higher than in the bacterial case. Virtually all of these $8OpsuiJ,‘sshould be type C’s, and biochemical studies of someof them (seebelow) show that this is the case. The already high frequency of r$8Opsun,‘sis not raised significantly by chemical mutagens, so that chemically-induced +80psu1;,‘s of type D, and D, could not be obtained in this way. However, when type -D, and -D, sui;r bacteria were lysogenized by $8Opsu& and induced, the lysates contained both L-XL& and ST& phagesin a ratio of about 3 :l. The surerphagesin these lysates, sincethey occurred so frequently, seemed likely to have acquired the D, or D, gene structure of their host. If so, we anticipated that they should revert to sum by unequal recombination, at a frequency (see Fig. 4(a) and above) of approx. 3 to 6 x 10e3. From each lysate we isolated several sum phages. As shown in Figure 4(b), about 70% of them had reversion frequencies compatible with D, or D, structures. The remainder, with lower reversion frequencies, we assumedto be type-C segregantsof +8Opsu&, enhanced in frequency by the processes of integration and excision and by the ultraviolet light-irradiation used to induce the lysogens. In confirmation, when type-C bacteria are lysogenized and induced, the resulting sui& phages (again about one-fourth of the progeny) are all of this same low-reverting type (seeFig. 4(c)). To test further whether the high-reverting sur;r phages were D1’s and D2’s, we examined a number of their sum revertants. Figure 4(d) shows that those from the presumedD1’s yielded $8Opsu,;,‘ssomewhat lessfrequently than the original 5bSOpsu& ( N 5 x 10W4)and appearedto be E’s. Those from the presumedDs’syielded $8Opsun,‘s at very high frequencies (N 2 x 10-l) and appeared to be F’s. As in the bacterial c&se,we have identified ~8Opsui;, derivatives with the properties expected of C’s, Di’s and D,‘s; Their revertants fall into two classeswith the properties expected for E’s and F’s. Furthermore, we have shown that a given sum gene structure retains its properties upon transfer to +8Opsu,,,, subject only to the increase in recombination which characterizes phage growth. Direct support for the schemeof Figure 1 comesfrom biochemical characterization of +8Opsu&, and some of its derivatives. According to the scheme, +8Opsu& itself should stimulate synthesis of both sz& and sur;r tRNA upon infection, whereas a type-E derivative should stimulate only SU& tRNA. Furthermore, +8Opsu& should contain twice as much DNA hybridizing with tyrosine tRNA as does the type-E derivative. Upon infection, $8Opsu& does indeed stimulate the production of large amounts tyrosine tRNA, and the same is true of several of its type-C, -D,, -D, of @%I1 and -E derivatives. The tyrosine tRNA’s stimulated by +8Opsu& and one of its type-E derivatives have been purified and digested with ribonuclease T, to give the
.fwrlrif
,‘,
.\
DUPLICATE
tRNA
E.co/i
9
GENES
Tyr [I(su+)
w&J]
;OH C A pG-C G-C U-A G-C G-C G-C G-C
2’ omG
CGA
G
cuucCUAA
4tU * 4tU
III
‘CAA~
G
II
GAAGG
GCCC III GGG
cu
Tc U
A Cc G-C G” A C-G U’ CAG A-U C-A G-C AU A-@ 2, A’ C c (II) U 2rnt.61A G;
A
f C (S”& G’is
unknown
5. Nucleotide sequences of E. coli tyrosine tRNA’s taken from Goodman et al., (1968). The anticodon region is at the bottom of the Figure; the anticodon itself is CUA in a& tRNA, end G*UA in both wz& tRNA and the major tyrosina tRNA. The minor tyrosine tRNA’s (s& and au&) differ from the major tyrosine tRNA in a dinucleotide sequence at the end of the “finger” adjacent to the anticodon loop; the minor tRNA’s contain a UC sequence in place of the major tRNA’s CA. FIG.
fingerprints presented in Plate I(a) and (b), respectively. (For reference, the entire nucleotide sequenceof E. coli tyrosine tRNA is presented in Fig. 5.) The four oligonucleotide spots labelled 1, 2, 3 and 4 in Plate I( a) are derived from the anticodon region. Their pancreatic ribonuclease digestion products and their inferred sequences are shown in Plate I(c). Spot 3 is derived from sum tRNA and contains the anticodon sequence CUA. Spots 1, 2 and 4 are derived from sum tRNA (anticodon sequence G*UA or GUA) as described in the legend. Since all four spots are present, @Opsu& stimulates the production of both su& and sum tyrosine tRNA. In Plate I(b), these four are replaced by a single spot, labelled 5, whose mobility and composition are identical to those of spot 3; we conclude that the type-E derivative of @Opsu& stimulates the production of only su& tyrosine tRNA. This conclusion is confirmed by the trinucleotide-dependent binding experiments of Table 1. The su& tRNA produced by the type-E derivative recognizes almost exclusively UAG, whereas the tRNA produced by $80psur& binds about equally well in responseto UAG or UAU, as if it were composedof nearly equal amounts of sum and SW& tRNA. (Note also that a type-C derivative produces exclusively sum tRNA.)
10
R.
L.
RUSSELL
ET
AL.
TABLE 1 Trinucleotide-dependent
Bacteriophage stimulate tRNA
g8OP&r Type-E Type-C
derivative derivative
used to synthesis
bin&g
to ribosomes of tRNA derivatives
stimulated by 48Opsu& and
Cta/min of 3H-1abelled tryosine tRNA bound to ribosomes with: no triplet UAU UAG 3420 1666 3216
8817 3058 13,496
9512 16,246 3106
Percentage of input tRNA bound specifically by : UAU UAG 17 6.2 42
20 54 0
Binding assays were done by the method of Nirenber & Leder (1964) as previously described (Gefter t Russell, 1969). Phage-stimulated tRNA was prepared as described in Materials and Methods, and then acylated with high specific activity [3H]tyrosine. Input radioactivity for the three experiments was ~BOpsu&; 30,800 cts/min; type-E derivative, 27,000 cts/min; type-C derivative, 24,000 cts/min.
Quantitative hybridization experiments between tyrosine tRNA and the DNA’s of (Fig. 6) show, despite some variation, that +8OP4u and a type-E derivative contains about twice as much DNA hybridizing with tyrosine tRNA as #~OP%L does the type-E derivative, Furthermore, single-step mutations in the SZL& gene of a type-E derivative convert all of the stimulated su& tRNA to an altered form (Abelson et al., 1969), so the type-E derivative does not carry more than one sum gene. We conclude from all these results that @Opsu& contains two tyrosine tRNA genes, one su& and one sum, whereas its type-E derivative contains only a single sum gene. From the genetic studies of E. coli described above, we conclude that the doubled sunI genes of @Opsu& were almost certainly acquired intact from its bacterial host of origin.
4. Discussion The results described above strongly support the following general picture for tyrosine tRNA genes in E. coli. The major portion (60%) of tyrosine is produced by a gene or genes of unknown location, probably not close to the $80 attachment site (since $30 derivatives do not stimulate synthesis of this tRNA). The remaining minor tyrosine tRNA is the product of two closely linked and normally identical genes, located near the 480 attachment site. Su& mutants arise by mutation in the anticodon region of one of these two, leaving the other intact. When $30 transducing phages acquire the resulting sum gene, they are very likely to acquire its sur;r neighbour as well. In both phage and bacteria, unequal recombination between the neighbouring genes occurs, sometimes eliminating one gene, sometimes inserting an additional gene. This picture is consistent with most previous evidence, and it enables us to explain two previous paradoxical observations. The production of both sum and sunI tRNA by the defective transducing phage 480dsum (Goodman et al., 1968) is almost certainly due to incorporation into this phage of the tightly linked su& and sz& gene pair from its host of origin, Second, the finding that most su& mutants derived from the original strain appeared to be anticodon sum+ -to-su,;, changes (Abelson, J. N., Barnett,
DUPLICATE
'2P-lobelled
tRNA
GENES
tyrosine
tRNA
11
added
(pg)
FIQ. 6. Saturation curves for hybridization of tyrosine tRNA with DNA from ~SOpsu& and a derivative. Hybridizations were performed as described in Materials and Methods. Each filter contained 2 pg of added DNA, and the specific activity of the s2P-labelled tyrosine tRNA used was 582,500 cts/min/pg. The filled symbols are for DNA from @Opal & (three separate DNA preparations), the open symbols are for DNA from a type E derivative phage (three separate DNA preparations). Each value in the Figure has been oorrected by subtraction of the relatively small background which bound to 480 DNA at the same added-tRNA concentration. The arrows at the right denote the levels of bound tRNA expected for one and two tyrosine tRNA genes per genome, respectively, based on the following assumptions; (1) a molecular weight of 30.4 x 10s for the sodium salt of @Opaz& DNA, computed from the densities of @Opsu& and $80 (1.490 and 1.487 g/ems, respectively), and the molecular weight of 29.5 x 10s for the sodium salt of 480 DNA (Yamagashi, Nakamura & Ozeki, 1965; Burgi & Hershey, 1963), by the method of Weigle, Meselson & Paigen (1959), and (2) a molecular weight of 29.2 x lo3 for the sodium salt of tyrosine tRNA, computed from bhe su& tyrosine tRNA sequence of Fig. 5.
L., Brenner, S., Goodman, H. M., Landy, A. & Smith, J. D., unpublished results) can now be ascribed to the generation of type-C SU~;~‘s by unequal recombination. However, the picture is contradicted by one important piece of evidence; previous DNA-RNA hybridization experiments (Landy et al., 1967) seemed to show that +80dsu:,, contains only a single s%II gene, judged by the amount of tRNA hybridized to +8Odsu& DNA under saturating conditions. A possible resolution of this disagreement, can be seen by close examination of Figure 6. By genetic criteria, more than 99.9% of the genomes of a type-E $3Opsu,,, derivative contain an su& gene. But according to Figure 6, DNA from this derivative is saturated with tyrosine tRNA at a level of only about 0.6 molecule per genome. This may result from some systematic error in the experiments, but it may also imply that about 40% of the tRNA-complementary sequences in the DNA are not available for hybridization. Similarly, the DNA of $3Opsu:,,, which almost certainly carries two s%II genes, is saturated at a level of about 1.4 molecules per genome. These low-saturation levels, if they are real, are not difficult to explain; we suspect that they may result either from a particular tendency of tRNA-complementary sequences in the DNA to fold up on themselves, or, more generally, from interactions between DNA and the filter which render significant stretches of DNA inaccessible. Either of these difficulties, but particularly the latter, might well affect other hybridization studies, rendering their estimates of complementary DNA low by as much as 40%. If these are the only factors affecting hybridization studies with +8Odsu&, its DNA should also saturate at about 1.2 to 1.4 molecules per genome. We have repeated
12
R.
L.
RUSSELL
1T
AL.
hybridization experiments with #30dsu&, in parallel with those reported in Figure 6. Although we observed considerable variation (somewhat greater than that of Fig. 6), the average saturation level for these experiments was 0.96 molecule per genome, in agreement with the published value of 1.0. This low value may well reflect the difficulty of working with the defective &Odsu&. In particular, this phage must be prepared by induction of a doubly lysogenic strain, and the process of excision from the bacterial chromosome may well enhance the frequency with which unequal recombination reduces an original doubled structure to a single one. (This apparently happens during induction of $80psum lysogens-see Fig. 4(b) and above.) Preliminary experiments suggest that the frequency of single-gene-carrying phages in a +80dsu& preparation may be as high as 30%. Moreover, the 48Odsum particles must be separated from the ~$80particles in the same lysate ; failure to achieve proper separation would dilute the sunI genes carried by @Odsu& DNA. We suspect that both of these factors, as well as the general effects observed with $8Opsu& have operated to reduce the saturation level for @Odsu & DNA from a theoretical 2.0 molecules per genome to the observed 1.0 molecule per genome. The role of the duplicate sum genes in E. coli is not clear. They are apparently the only genes which specify the minor tyrosine tRNA (preliminary experiments by J. N. Abelson and J. D. Smith show that all of the minor tyrosine tRNA in type-E bacteria is sum and none SU~;~). Nonetheless, they do not appear to be essential, since viable mutants with apparent deletions of both genes have been obtained (Russell, R. L., unpublished results). Moreover, if one gene is eliminated by unequal recombination and the other is subverted from its normal coding function by conversion to su& (as in type-E bacteria), there is no observable effect on growth rate. Nevertheless, there must be active maintenance of these duplicate genes, particularly when unequal recombination could easily eliminate one of them, and one must assume that they confer upon E. coli some slight selective advantage, not apparent under normal laboratory conditions. Although other minor tRNA’s may have similar functions, at least one, the minor serine tRNA specified by the 8% gene, does not appear to be specified by a similar pair of closely linked genes. flu: strains of E. coli behave like type-E su& derivatives in the sense that both chemically-induced and spontaneous 8%‘~ derived from them revert to su: at the low frequency characteristic of single nucleotide mutation. By inference, the su: gene does not have a dispensable su; neighbour. Similar experiments with su; strains have proved inconclusive. While the role of the duplicate su& genes is not clear, they do provide information on the properties of gene duplications in general. Of particular interest is the observation that an increase in gene number from two to three (as from type-D, to type-F bacteria) produces a 20- to 30-fold increase in the frequency of unequal recombination (from 1 to 1.5 x 1O-6 to 3 x 10m5). Since this increased recombination tends to eliminate the three-gene structure, it is clear that gene amplification in this fashion, at least in E. coli, rather rapidly encounters problems in the stability of the amplified product; only sufficiently strong selective pressures will maintain gene multiples created in this way. We thank Miss E. Higgins and Mr T. V. Smith for their expert technical assistance. by N.A.T.O. Postdoctoral Fellowships,
Two of us (R.L.R. and A.L.) were supported another (J.N.A.) by a U.S. Public Health Service (M.L.G.) by a Jane Coffin Childs Memorial Fund
Postdoctoral for Medical
Fellowship, and Research Fellowship.
another
DUPLICATE
tRNA
GENES
13
REFERENCES Abelson, J. N., Barn&t, L., Brenner, S., Gefter, M. L., Landy, A., Russell, R. L. & Smith, J. D. (1969). FEBS Letters, 3, 1. Ames, B. N. & Whitfield, H. J., Jr. (1966). Cold Spr. Had. Symp. Quant. Biol. 31, 221. Andoh, T. & Ozeki, H. (1968). Proc. Nat. Acd Sci., Wash. 59, 792. Brenner, S. & Beckwith, J. R. (1965). J. Mol. Biol. 13, 629. Burgi, E. & Hershey, A. D. (1963). Biophys. J. 3, 309. Gefter, M. L. & Russell, R. L. (1969). J. Mol. Biol. 39, 145. Goodman, H. M., Abelson, J. N., Landy, A., Brenner, S. & Smith, J. D. (1968). Nature, 217, 1019. Kelmers, A. D., Novelli, G. D. & Stulberg, M. P. (1965). J. BioZ. Chem. 240, 3979. Landy, A., Abelson, J. N., Goodman, H. M. & Smith, .J. D. (1967). J. Mol. BioZ. 29, 457, Nirenherg, M. W. & Leder, P. (1964). Science, 145, 1399. Nishimura, S., Harada, J., Narushima, U. & Seno, T. (1967). Biochim. biophys. Actu, 142, 133. Sanger, F., Brownlee, G. 0. & Barrell, B. G. (1965). J. Mol. BioZ. 13, 373. Smith, J. D. Abelson, J. N., Clark, B. F. C., Goodman, H. M. & Brenner, S. (1966). Cold Spr. Harb. Symp. Quant. BioZ. 31, 479. Weiglo, J-. J., Meselson, M. & Paigen, K. (1959). J. Mol. BioZ. 1, 379. Yamagashi, H., Nakamura, K. & Ozeki, H. (1965) Biochem. Biophys. Res. Comm. 20, 727.