Conditionally lethal mutants of bacteriophage T4 defective in production of a transfer RNA

J. Mol. Biol. (1973) 81, 137-155

Conditionally

Lethal Mutants of Bacteriophage T4 Defective in Production of a Transfer RNA CHRISTINE GUTHRIE~ AND WILLIAM

H. MCCLAIN

Department of Bacteriology The University of Wisconsin Madison, Wis. 53706, U.X.A. (Received 27 March 1973) Mutants of bacteriophage T4 have been isolated that grow normally on Eschwichia coli B/5 but are unable to grow on a strain of E. coli (CT439) isolated from nature and previously shown to restrict growth of transfer RNA-deficient strains of T4. These mutants define five complementation groups, two of which affect the production of bacteriophage-coded tRNAs. One gene appears to affect the appearance of several tRNAs. The second gene (de&red by the mutant HAl) is apparently the structural gene for a single tRNA species (8). Mutant HA1 produces no mature 6 tRNA while revertants of HA1 that have regained the ability to grow on strain CT439 now make this tRNA in amounts correlated with the ability of the various revertants to grow on the restrictive host. The 6 tRNAs made by several revertants of HA1 have been compared to T4 wild-type 6 by two-dimensional fingerprint analyses of pancreatic and T, ribonuolease products. These comparisons have allowed tentative identification of the primary mutational alteration. On the basis of these results we conclude that the inability of mutant HA1 to grow on strain CT439 is due to a mutation iu the structural gene of 6 which interferes with its appearance.

1. Introduction T4 codes for the production of eight transfer RNAs, including acceptor activities for arginine, glycine, isoleucine, leucine, proline and serine (Daniel et al., 1970; Scherberg & Weiss, 1970; Pinkerton et al., 1972; M&lain et al., 1972). A considerable amount of information is accumulating describing various aspects of the structure and genetic control of these tRNAs (Wilson & Kells, 1972 ; Wilson & Abelson, 1972; McClain et al., 1972; Wilson et al., 1972), but little is known about their physiological function. It is clear that at least some of these tRNAs can function in protein synthesis in viva since nonsense suppressor derivatives of several tRNAs have been isolated and characterized (McClain, 1970; Wilson & Kells, 1972). Recently Scherberg & Weiss (1972) have demonstrated the in vitro protein synthetic activity of a number of these tRNAs. On the other hand, none of these species appears to be essential for growth under standard laboratory conditions. The genes coding for these tRNAs appear to be clustered in a small region of the T4 genome; deletions encompassing this entire region are viable (McQain et al., 1972; Wilson et al., 1972; Bacteriophage

t Present address: Department of Biochemistry San Francisco, Calif. 94122, U.S.A. 137

and Biophysics, University

of California,

138

C. GUTHRIE

AND W. MoCLAIN

Wilson, 1973). Nevertheless, the continued presence of the genetic information required for the production of these tRNAs, in phages T2 and T6 as well as T4 (W. H. M&lain, unpublished observations), suggests the existence of a selective pressure for the maintenance of these genes. It was reasoned that the function of one or more of these tRNAs might confer a distinct selective advantage under natural conditions. Strains of E. coli isolated from hospital patients have been screened for their ability to support the growth of T4 strains carrying deletions in the tRNA region (Wilson, 1973). One of these strains, CT439, could not support the growth of a strain bearing a deletion for the entire tRNA region or a point mutant deficient in several tRNAs. We have isolated a large number of point mutants unable to grow on strain CT439 in an attempt to identify a specific tRNA requirement. Five complementation groups have been identified, two of which appear to be involved with tRNA production. One of these groups appears to function in the production of a number of tRNAs. The second group deilnes the structural gene for a single tRNA (6) whose presence is required for growth of phage T4 on strain CT439. The specific nature of this requirement is currently under investigation. 2. Materials and Methods Wild-type T4D and the rI strain r48, from which the mutant strains described here were isolated, were obtained from the Edgar-Wood collection and given to us by Dr J. King. The deletion strain eG192, derived from T4B, was provided by Dr Joyce (Emrich) Owen. The tRNA deletion strains psu;d33 and peucd105 were isolated as described elsewhere(Wilson et al., 1972) and obtained from Dr John H. Wilson. (b) Bacterial strains E. coli strain B/5, used as the permissive host, was obtained from Dr Millard Susman. The restrictive host, CT439, is an E. coli strain isolated from a patient at Los Angeles County Hospital and characterized as described by Wilson (1973). We thank Dr John H. Wilson for making this strain available to us. Unless otherwise noted, saturated overnight cultures (forced aeration) were used; strain B/5 was grown and assayed at 37°C and strain CT439 at 3O’C. (0) Me&a H broth, used for phage and bacterial growth, and EHA top and bottom agar, used for plating assays, were prepared as described previously (Steinberg & Edgar, 1962). Low-phosphate medium used for preparation of high-titer phage stocks and s2P-labeling of phage-infected cells and M9 medium used for U.V. crosses, are described elsewhere (Landy et aZ., 1967). (d) Preparation of phage stocb

High-titer plate stocks were prepared by plating about IO6 phage, obtained from a single 6-h plaque, on EHA agar plates with 2 drops of B/5. The plates were incubated 6 to 8 h at 37’C and washed with 05 ml CHQ,. The phage were eluted by adding 3 ml low-PO, medium without glucose to the surface of each plate and letting the plates stand at room temperature for 1 to 3 h with occasional swirling. The eluants were then centrifuged 1 h at 27,000 g. After decanting the supernatant, the phage pellets were allowed to resuspendin 1 to 2 ml low-PO4 medium overnight at 4°C and finally centrifugedat low speed to remove insoluble aggregates and debris. Titers of 1Ol2plaque-forming units/ml were obtained by this method.

T4 MUTANTS

DEFECTIVE

IN A tRNA

139

(0) ilfzltage~eeis alzd selection of spontaneous na&dd (i) Mutagenesis with hydroxylamine (Allied Chemicals) was performed as described by Tessman (1968). Mutagenesis with 2aminopurine (Sigma), 5bromo-2’-deoxyuride (Sigma), and nitrous acid was performed according to Benzer (1961). Mutagenesis with 9-aminoacridine (Mann Research Laboratories) W&Bperformed as described by Mattson (1968). Mutagenesis with N-methyl-N’-nitro-N-mtrosoguanidine (Sigma) was carried out as follows. B/5 cells growing exponentially in H broth (2 x IO8 cells/ml) at 30°C were infected with T4D at an input multiplicity of 0.1 and nitrosoguanidine was then added to a final concentration of 5 pg/ml. The culture was incubated 4 h under forced aeration and CHCl, was then added to complete the lysis. (ii) Mutants of spontaneous origin were isolated by the turbid plaque method (Barnett et al., 1967) as follows. One drop of CT439 was plated in 25 ml EHA top agar and allowed to set. Suitable aliquots of a suspended r48 plaque were then mixed with 1 drop of B/5 in 1.25 ml EHA top agar and poured on top of the CT439-containing plates. Turbid plaques appearing after overnight incubation at 30% were picked and stabbed on B/5and CT439-seeded plates to identify authentic CT439- phage. (f) Complement&on analysis Plate complementation analysis was performed as follows. Suitable dilutions of tester phage in 0.1 ml (for non-leaky mutants about lo8 phage per plate, for leaky mutants 10 to bO-fold less) were adsorbed to 0.5 ml CT439 cells in exponential growth at 3 x lo* cells/ml for 15 min at room temperature, mixed with 2.5 ml EHA top agar, and plated. Suitable dilutions (as above) of desired mutants were then spotted on each test plate and the plates incubated overnight at 30°C. Because of the difficulty of obtaining clear-cut results with leaky mutants, all tests were performed independently a minimum of three times. (g) Recombination analysis (i) Qualitative recombination analyses were performed by spot crosses as follows. Equal amounts of the two desired phage types, each at about lo9 phage/ml, were mixed with one drop of the permissive host B/5 for 15 mm at room temperature and the mixture was streaked on CT439-seeded EHA agar plates. Results were scored after overnight incubation at 30°C. Clearing of the cell lawn over and above that observed for control crosses was scored as a positive response. (ii) Quantitative recombination analyses were performed as follows (Barnett et al., 1967). For U.V. crosses, the two parental phage types were added to a culture of host B/5 at 2.5 x lOa cells/ml in H broth to give an input multiplicity of 7 each. The oultures were incubated at 37°C with forced aeration for 8 min after phage addition, at which time samples were diluted IO-fold in M9 medium and irradiated with an unfiltered U.V. lamp at a dose equivalent to 20 phage T4 lethal hits. The irradiated cultures were then diluted IO-fold into H broth, aerated at 37% for an additional 60 min, and then lysed with CHCI,. For standard crosses, B/5 cultures in H broth at 1.5 x IO* to 2 x lo8 cells/ml were concentrated to 1 x 10’ cells/ml by centrifugation and mixed with phage as above. Ten min after phage addition, the cultures were diluted 104-fold in fresh H broth, aerated an additional 80 min at 37”C, and lysed with CHCI,. (h) 32P-labeled extracts The standard procedure for obtaining analytical amounts of labeled extracts was as follows. Five-ml cultures of B/5 growing in low-PO, medium at 3 x lo* cells/ml were infected with phage to give an input multiplicity of 10 to 12 phage/cell. Cultures were aerated at 37°C. Cell survival, assayed as colony formers at 2 min after infection, was 0.1% of the input titer. Four min after infection, 1.5 mCi of carrier-free [“2P]orthophosphate (New England Nuclear) were added. Thirteen mm after infection the cultures were extracted by shaking vigorously with 3 ml phenol (saturated with 0.01 M-Tris*Ha, pH 75) for 1 mm at room temperature. Five 0.D.“’ units of tRNA were added as carrier during the extraction procedure. The aqueous phase obtained after centrifugation was

140

C. GUTHRIE

AND

W.

McCLAIN

then made O-2 M in potassium acetate (pH 5) and precipitated overnight with 3 vol. ethanol at -2O’C. The precipitate was resuspended in 0.5 ml of 10e2 M-Mgcl,, low2 M-NaCl, 10-z M-Tris*HCl (pH 7.5) and 20 pg RNase-free DNase/ml (Worthington, code DPFF) and incubated 15 to 30 min at 37°C. Depending on the solubility of the material from a particular preparation, the DNase-treated extract was either directly reprecipitated before analysis by gel electrophoresis or further purified by stepwise elution from DEAE. cellulose in the following manner. The extract was applied to a column of DEAE-cellulose O-6 cm x 25 cm. The column was washed first with several ml of low2 M-Tris.HCI (pH 7.5) and low2 M-NaCl until the radioactivity in the eluate peaked and plateaued, and then with several ml of the same buffer containing 0.25 M-NaCl, again until the radioactivity peaked and dropped. The tRNA was then eluted in the same buffer containing 1 M-Nacl in 6.5 M-urea in a total volume of about O-5 to 1 ml. This fraction was then ethanolprecipitated 2 to 3 times before gel analysis. To obtain preparative amounts of material, the above procedures were scaled up 10 to 20-fold. (i) Polyacrylamide gel electrophoresis Low molecular weight RNA prepared as in section (h) was analyzed on 20 cm x 40 cm x O-3 cm slabs of 10% polyacrylamide (Smith et al., 1970) of the following composition (per liter) : 10.8 g Tris base, 1.04 g EDTA (tetrasodium salt), 5.5 g boric acid, 95.2 g acrylamide (Eastman, practical grade), 4 g N,N’-methylene-bisacrylamide (Eastman, 8383), 3.84 ml 2-dimethyl-aminoethyl cyanide (British Drug Houses), and 0.96 g ammonium persulfate (Baker). For further analysis of the tRNAs in component 3 (see Plate I), this region was cut out, crushed by extruding through a 5-ml disposable syringe, and repeatedly eluted at 37°C with buffer containing 10m2 M-Tris.HCl (pH 7.5), 0.5 M-NaCl and 2 x 10e2 M-MgCl,. The combined eluates were passed through a sterile Millipore filter (0.45 nm) and ethanol-precipitated. The eluted component 3 material was then applied to a 20 cm x 20 cm x 0.3 om slab of 20% polyacrylamide (Ikemura & Dahlberg, 1973; as above except the concentrations of Tris/borate/EDTA buffer and ammonium persulfate were halved and electrophoretically pure grades (Eastman) of acrylamide (190 g/l) and bisaorylamide (10 g/l) were used) ; electrophoresis was for 50 h at room temperature (11 mA ; 300 V). When further analysis of the resolved species was desired, components were eluted as described above.

(j) RNA composition. and sequence analysis The techniques, materials and methods used were those described in detail by Barrel1 (1971) except that partial digestion with spleen phosphodiesterase was performed with an enzyme concentration of 5 mg/ml for incubation times of 0, 1, and 2 min (Dr R. Roberts, personal communication) and electrophoresed on DEAE paper in 7% formic acid. Conditions for complete enzymatic digestion of tRNA were as follows. Samples of 0.1 to 0.5 0.D.260 units of RNA containing lo4 to lo5 cts/min of 32P were mixed with 10 ~1 of a solution containing (per ml): 50 units RNase T, (Calbiochem), 0.25 mg RNase T,, and 0.25 mg pancreatic RNase A, in 0.05 M-ammonium acetate, pH 4.5. The mixture was incubated for 4 h at 37°C. (k) Thin-layer chromatography Samples were spotted on 20 cm x 20 cm pre-coated thin-layer cellulose plates and subjected to ascending chromatography (Brinkmann Instruments, CELPLATE-22) (6.8: l-76: 1.44, with one of the following solvent systems: (a) isopropanol/HCl/water by vol.), or (b) isopropanol/NH, (7.0: 1.3, v/v). Two-dimensional chromatography was performed as follows (Saneyoshi et al., 1972). A sample of RNase T2 hydrolysate containing lo4 to lo5 cts/min of 3zP material was spotted on a thin-layer cellulose plate and subjected to ascending chromatography for 9 h in isobutyric acid-O.5 M-NH, (5:3, v/v). The plate was air-dried and then chromatographed for 12 h at right angles to the first dimension in isopropanol/HCl/water (70: 15 : 15, by vol.). The products were located on the thin-layer cellulose plates by autoradiography.

T4 MUTANTS

DEFECTIVE

IN

A tRNA

141

3. Results (a) Mutant isolation Mutants of spontaneous and induced origin were isolated from T4D by picking and stabbing plaques on E. co.&strains B/5 and CT439, selected. as the permissive and restrictive hosts, respectively. Six chemical mutagens were used to ensure the production of a broad spectrum of mutant types. As seen in Table 1, all the mutagens employed gave rise to potential mutants of the desired phenotype (a;t an overall frequency of approximately 3%). Because the majority of mutants obtained in the search had only a partially reduced ability to grow on the restrictive host, only a small subset of the total mutant population was selected for further analysis (Table 1, last column). Taking this into account, hydroxylamine appears to be the most effective mutagen and nitrous acid the least.

TAESLE~ Origin of CT439- mutants

(Abbrev.)

Specifioityt

Hydroxylrtmine Nitrous eoid 2-Aminoput!in~ 5-Bromouridine 9-Aminoacridine

(HA) (NA) (AP) (BU)

G-C +A*T A.T .+ G*C A-T +G.C A*T +G.C Insertions/ deletions

Nitrosoguanidine

(NG)

(Spontaneous)

(SP)

Mutagen

(AA)

scE;ed

1000 500 1000 1000 1260 1000 5750 60,000§

T(%)

-

Suitable for further analysis$

9

s

1 1 8

21 61

1 6

23 20

-11

142(2+5%) 6 -6(0+01%)

60,000 Mutagenesis was B/5 and tested for incubated at 3O’C. formed on & mixed

CT439 mutants

1 2 Is 6 ?

carried out as desoribed in Materials and Methods. Survivors were isolated on their ability to grow on CT439 by picking and stabbing onto seeded plates, Spontaneous mutants were selected by picking and stabbing turbid plaques indicator of B/5 and CT439.

t Drake (1970). $ See text. $ Number of plaques screened; 885 turbid plaques were picked and stabbed. I! The starting phage was an rl mutant of T4D.

A limited search for spontaneous mutants was conducted by screening turbid plaques formed on a mixed indicator of B/5 and CT439. This method was rather unsatisfactory, largely because of the poor adsorption of T4 to CT439 which results in the

formation

of many

spurious

turbid

plaques.

Only

six mutants

(see Table

1)

by this method, whioh was not pursued because all of these mutants were extremely leaky on CT439 (see Table 3) and, although of independent origin, appeared to define only a single complementation group (see Table 2 and below). These mutants arose at an overall frequency of O*Ol%. The frequency of induced mutants thus represents about a 300-fold increase over the spontaneous level. were

10

isolated

142

C. GUTHRIE

AND

W.

McCLAIN

(b) Mutant characterization (i) Complementation andysis We attempted to perform plate complementation tests with all 148 mutants isolated, but the leakiness of the majority of the mutants allowed only a subset of 20 of them (Table 1, last column) to be classified. Even with this subset the responses of the leakiest members (see Table 3) were sometimes ambiguous. Nevertheless, the 20 mutants tested defined five complementation groups (Table 2). It is interesting to note that all the mutants of spontaneous origin fall within a single complementation group (group V). Only a single induced mutant, BU57, shared this assignment. The spontaneous mutants were derived from an PI mutant of T4D (to facilitate visibility of turbid plaques) and the significance of this factor has not been explored; e.g. a parallel search for spontaneous mutants from an r+ strain was not cbnducted. Curiously, BU57 is also an r mutant, but it should also be noted (see Table 1, column 5) that the frequency of Y induction by 5-bromouridine was unusually high (8%).

TABLE 2

Complementation analysis Groups

I

Members

HA1 HA2 HA6 NC4 BU48 BU53 BU56

II ‘HA4

III

IV

V

HA7 AP4

NG6 CAP21

BUS7 SPl SP2 SP4 SP6 (SP3)

Complementation tests were performed as described in Materisls and Methods. The mutants in parentheses could not be unequivocally assigned to a given group since their leakiness often results in ambiguous responses in the analysis.

Group I is the largest of the five, one possible explanation being the non-leakiness of most of its members (see Table 3), although the members of group III are also non-leaky. Groups II and V are characterized by the extreme leakiness of all their members (Table 3). Only limited analysis of group IV has been carried out because members of this group form poor plaques even on B/5 and are thus difficult to work with. The rationale of using a spectrum of mutagens is supported by the distribution of mutants of different origin among the five groups. Again it can be seen that hydroxylamine was the most effective mutagen employed, giving rise to members in three of the five groups identified. (ii) Time of block Selected members of oomplementation groups I, II, III and V were analyzed for their ability to induce cell lysis of strain CT439 as an indication of whether the genetic defects involve “early ” or “late” functions (Epstein et al., 1963). Although interpretation of the results was complicated by the leakiness of some of the mutants, it appears that groups I, II and V are blocked in a “late” function since significant

T4

MUTANTS

DEFECTIVE

IN

A tRNA

143

TABLE 3 Some properties

Complementation

of the mutants Recombination

Mutant

e.0.p.t on CT439

HA1 HA2 HA6 NC4 BU48 BU53 BU56

3x 10-T 0.02 4x 10-T 4x 10-7 0.02 0.02 3x 10-T

HA4 AA2

0.2 0.2

HA7 AP4

1x10-4 1 x 10-k

+

IV

NG6

3x10-4

-I-

V

BU57 SPl SP2 SP4 SP6

0.1 0.1 0.1 0.1 0.1

+ +

group I

II

III

eG192

p&A33

psu,AlOb

Cell lysis

0

+

0 0 0 f 0 i

+

+

+

+

+

0

+

+

t

I

::

The titer of the mutants on CT439 relative to that on B/5 was adjusted such that T4 has a relative efficiency of plating of 1.0; these values represent the average of 2 to 5 independent measurements. Recombination of the mutants with the indicated deletion strains (see Fig. 1) was analyzed by spot crosses a.s described in Materials and Methods; f, recombination; 0, no reoombination; i, very weak recombination. Cell lysis of CT439 cultures infected with the indicated mutants was performed as described by Epstein et al. (1963). The o.D.*~" of a. oontrol culture infected with T4D showed approximately a twofold decrease 30 min after infeotion; +, comparable decrease; 0, no decrease. Because these mutants have not been cheracterized with respect to their leakiness in liquid culture, a 0 response is considered more significant than a + response. t Abbreviation used: e.o.p., efficiency of plating.

cell lysis occurred (Table 3, last column). In contrast, group III is apparently defective in an “early” function. (iii) Recombination analysis Recombination between groups I to V was examined by spot crosses of representative members. The production of wild-type recombinants was observed in all inter-group crosses. Intra-group recombination was also examined by spot crossing. All possible pairwise combinations were tested with the exception of NGQ x AP2, SP6 x BU57 and SPl x SP2. Since positive results were obtained in each cross, all of the mutants tested appear to define unique sites. The structural genes coding for tRNAs appear to be clustered in a small region of the T4 genetic map about eight map units from gene e (Wilson et al., 1972; McClain et al., 1972). We took advantage of the availability of several deletion mutants in this region as a quick method of screening for mutants having tRNA-related defects. As indicated in Figure 1, deletion eG192 encompasses the entire tRNA region, and

C. GUTHRIE

144

AND

W.

McCLAIN

gene rlc

-

e

gene57

tRNA""'

a

eGl92 pw-A33 psu,-Al05

FIG. 1. Genetic map of T4 in the region of the tRNA genes. The positions of several deletion mutations are indicated.

extends through and beyond the lysozyme gene, e ((Emrich) Owen, personal communication; M&lain & Guthrie, unpublished observations; Wilson et al., 1972). Strain psurA33 deletes the entire tRNA region but stops to the right ofe; psu;A105 falls within the tRNA cluster and includes the gene for serine tRNA as well as the genes for three to four other tRNAs (Wilson et al., 1972). Since none of these deletions will grow on strain CT439 (Wilson, 1973), it seems reasonable that mutants giving no recombination with these deletions should be likely candidates of the desired type. Spot crosses of each of the 20 mutants by eG192 revealed that only members of group I failed to yield wild-type reoombinants (see Table 3). Similarly, members of groups II to V showed recombination with psqA33 while members of group I did not; this observation suggests that group I falls within the tRNA cluster. Confirmation was obtained by spot crosses of group I members by ps$AlO5. Five members gave no recombination, while two (BU48 and BU56) showed very weak recombination. These genetic results indicate that group I affects a region within the tRNA cluster defined by the deletion psu~A105 and probably at one extremity of it. We attempted to obtain a rough estimate of the genetic size of group I by performing quantitative recombination analysis between several members. Mutants were selected which consistently showed strongest recombination in spot crosses, i.e. HA1 and BU56. Mutant BU48 also gave a strong response with HA1 and weak response with BU56. In a standard cross, HAI and BU56 gave O*24o/orecombination (see Table 4). Using the mapping function of Stahl et al. (1964), we can estimate that HA1 and BU56 are separated by 25 to 50 nucleotides. u.v.-stimulated crosses between HAl, BU53 and BU48 gave values consistent with a distance of 25 to 50 nucleotides (Table 4). These findings suggest that the distance separating HA1 and BU48 or BU56 is small and probably within that expected for a tRNA. TABLE 4

Recombination Expt 1 2

cross HA1 HA1 BU48 HA1

x BU56 x BU63 x BU53 x BU48

analysis of HAI Condition Standard U.Y. U.V. U.V.

Recombination

(%)

0.24 o-12 0.40 0.68

Recombination analyses were carried out as described in Materials and Methods. In all cases, the recombination frequency was at least loo-fold above the measured reversion rate (see Table 1). In the case of crosses with BU53, plaques from recombination plates were picked and stabbed on B/6 and CT439 to confirm identtication as wild-type recombinants.

T4 MUTANTS

DEFECTIVE

IN A tRNA

145

(c) Polyacrylamide gel analysis (i) 10% gel analysis In order to examine the low molecular weight RNAs made by these mutants, cultures of B/5 were infected with representative members of groups I to V and labeled with [32P]orthophosphate from 4 to 13 minutes after infection. The cultures were extracted with phenol and analyzed on 10% polyacrylamide gels to display low molecular weight RNAs. As can be seen in Plate I, eight components are resolved by this method. Components A to D are larger than tRNAs but several lines of evidence indicate either a direct or indirect relation to tRNAs. Components A and B are believed to be tRNA precursor molecules (McClain et al., 1972; (;iuthrie et al., 1973). Component C has been sequenced (Paddock & Abelson, 1973) and appears to be tRNArelated; however, the nature of this link is not clear. Although component D shows no obvious tRNA characteristics (e.g. presence of minor bases (M&lain et al., 1972)), it maps in the tRNA region of the T4 genome (Wilson et al., 1972). Components 1, 2 and 4 are pure species of tRNA that correspond to the amino acids serine (McClain & Barrell, unpublished results), leucine (Pinkerton et al., 1972) and glycine (Barrel1 et al., 1973; Stahl, Paddock & Abelson, personal communication), respectively. Component 3 is a mixture of five tRNAs (McClain et al., 1972). In looking at the pattern of tRNA production by the mutants, we were guided by the observation that mutational alterations that result in functional inactivation of a tRNA generally lead to a dramatic decrease or disappearance of that tRNA, presumably via degradation of the mature species or incorrect processing of the precursor molecule (Smith et al., 1970; Anderson & Smith, 1972). The only known exceptions to this empirical observation to date are cases where the alteration is in the anticodon itself (Altman, 1971; M&lain et al., 1973). We thus reasoned that mutants of the desired type, those which affect tRNA, would be missing or have reduced amounts of one or more tRNA components. As can be seen in Plate I, the 10% gel patterns of mutants from groups I, III, IV and V are essentially the same as the wild-type T4 profile while group II members lack components A and D and the component corresponding to serine tRNA, and show a significant reduction in component 3 label. Although in this particular preparation the group I member HA1 has a greatly reduced amount of component A, it should be emphasized at this time that the amounts of components A and B vary greatly among different preparations (McClain et al., 1972). Independent preparations of HA1 as well as of all other members of group I show normal amounts of component A. In other experiments, for which no data are presented, similar analyses were performed on all members of groups I to IV and several members of group V. With only a siugle exception, the 10% gel patterns of these mutants were equivalent to the comparable profiles shown in Plate I. The group I mutant HA2, however, showed an altered mobility of component C. The significance of this observation is not known. If our hypothesis is correct, that mutational alterations result in the virtual elimination of a tRNA, we are forced to conclude that the alteration in group I mutants is not detected because it resides in a GRNA of component 3. Since this component contains a number of tRNAs, the absence of a single species might result in only partial reduction in intensity of label and thus be virtually undetected. At the time these experiments were initiated, no satisfactory system for fractionation of component 3 was available. We thus decided to analyze this component by examination of RNase T, digestion products in the two-dimensional fingerprinting system of Sanger

146

C. GUTHRIE

AND

W.

MaCLAIN

and his colleagues. We reasoned that we might be able to detect the effects of a tRNA mutation by the specific loss or reduction of a subset of T, oligonuoleotides in the fingerprint of a mutant. To facilitate the comparative analysis between T4 and HA1 we included component 3 from HR4; if only a single tRNA were required for growth on CT439, as would be the simplest case, then HA4, which appears to be defective in the biosynthesis of several tRNAs, should share with HA1 an overlapping set of missing oligonucleotides. The results of this analysis are shown in Plate II. Even though the pattern of T4 is quite complex, it is possible to distinguish several oligonucleotides that are absent in both HA1 and HA4 (Plate II(b) and (c); note arrows (1)). It can be noted that HA4 is missing a set of oligonucleotides in addition to those reduced or absent in HA1 (note arrows (2)). This is consistent with the noticeable decrease in the amount of label in component 3 of HA4. Similar analyses of several other mutants of group I (HA2, BU56) showed the same specific loss of oligonucleotides as seen for HAl. These results are consistent with our predictions and allow us confidence in distinguishing the group I phenotype. (ii) 20% gel analysis In order to extend these preliminary findings we sought a system whereby we could fractionate component 3 into its individual species. When the material in component 3 from a T4 sample is eluted and run on a 20% polyacrylamide gel, five components are resolved (Plate III). Components a, /3, y, 8 and e are homogeneous, distinct species of tRNA (McClain et al., 1972). As seen in Plate III, HA7 (group III) gives a 20% gel pattern indistinguishable from that of T4. Similarly, NG6 (group IV) and SP1 (group V) give essentially wildtype profiles. In contrast, HA1 (group I) has TBlike amounts of a, /I, y and E but is completely missing the component corresponding to 6 in T4. I-IA4 (group II) shows several differences from wild type; the amount of label in every component is reduced, although E appears to be made in the highest yield. The most significant decreases relative to T4 are seen in components j3 and 6. The latter, although barely visible in the Plate, can be identified as present in suitably over-exposed autoradiograms. This marked and preferential decrease in S is consistent with the fingerprint analysis of the total component 3 species discussed above. Specifically, we can now account for the oligonucleotides that are absent in both HA1 and HA4 as being represented in 6 (see below). Those oligonuoleotides that are missing in HA4 uniquely are present in purified preparations of /3 (see M&lain et d., 1972). (d)

HA1 revertant arudy&

(i) Genetic analysis We have presented data which suggest a correlation between the presence of 6 and the ability of phage T4 to grow on strain CT439. In order to test this hypothesis, we proceeded to analyze revertants of the group I member HA1 which had regained the ability to grow on the restrictive host. We were particularly interested in obtaining second-site revertants (i.e. rather than “true” revertants arising from backmutation at the original site) in order to allow the eventual elucidation of both primary and secondary mutational alterations. Since second-site revertants characteristically show less than the wild-type amount of gene product activity (Anderson & Smith, 1972), a set of revertants of both spontaneous (HAlOl, HA102) and induced (HA103, HA104, HA105) origin isolated from CT439-seeded plates were analyzed for

T4 MUTANTS

DEFECTIVE

147

IN A tRNA

the efhciency of plating on this host. At 3O”C, the optimum tem.perature for wildtype phage growth on CT439, the five independent revertants selected plated with about the same efficiency as T4 (Table 5). Because the ability of CT439 to support the growth of T4 falls off significantly with increasing temperature, we also examined the plating properties of the revertants at 37, 40 and 42”C, hoping to distinguish between these revertant classes by differences in efficiency of plating at higher temperatures. The results of this analysis are shown in Table 5. Inspection of the data suggests that more than one class can be distinguished. HA102 plates with the same eEeiency as T4 at all temperatures tested and HA101 has a similar or perhaps slightly reduced efhciency. Revertants HA103-105, however, show significantly reduced plating efficiencies even at 37”C, and these differences become dramatic at 40°C. TABLET

Growth of HA1 revertants at several temperatures

Phage

Origin

1T4

-

‘HA1 HA101 HA102 HA103 HA104 HA105

HA SP SP HA HA HA

Relative efficiency of plating on CT439 at

Relative e.0.p.t on CT439/B/5

30~C/30~C

37”C/3O”C

(1.0) 10-s 1.0 1.0 0.8 0.9 0.8

(1.0) (1.0)

OS

(1.0) (1.0) (l-0) (1.0) (1.0)

0.6 0.7 0.2 0.2 0.2

*

4O”C/3O”C

03 0.3 0.4 0.006
42”C/3O”C

0” 0.3 0.4 0.002
Wild-type T4 and revertant stocks of spontaneous (SP) or hydroxylamine-induced (HA) origin were titrated on B/5 and CT439. The ratios of these titers were then normalized to the wild-type T4 ratio (O-8) set at a value of 1-O (column CT439/B/5). The stocks were then titrated on CT439 at 37”C, 40°C and 42°C. The e.o.p.‘s at these temperatures are reported relative to the 30°C value arbitrarily set at 1-O. Differences of a factor of 2 may not be significant at the higher temperatures, where the quality of plaques on CT439 is poor. The values reported are the average of 2 independent experiments. t Abbreviation used: e.o.p., efficiency of plating.

(ii) Gel analysis To correlate these results with the production of 6 in these phage, component 3 tRNAs made by revertants HAlOl-105 were analyzed on 20% polyacrylamide gels together with HA1 and T4. As can be seen in Plate IV, label in the 6 position can be seen for T4, HA101 and HA102 only. Revertants HA.103-105, which show the poorest plating efllciency on CT439, show no detectable amounts of 6. The quality of resolution on 20% gels is somewhat variable; we have observed the co-migration of j3 and y on several occasions. For this reason, all bands from T4, HAI, HA101 and HA103 were eluted and analyzed in a one-dimensional system after RNase T, digestion. Analysis of these results showed no detectable differences between the CI,,B, y and E components of HAI, HA101 and HA103 and the analogous counterparts of T4 (data not shown; the sensitivity of this method is sufficient to distinguish among the various tRNA species but would probably not allow the detection of a single base change). The faint band moving slightly ahead of y (y’; Plate IV) has been analyzed

148

C. GUTHRIE

AND

W.

McCLAIN

by two-dimensional RNase T, fingerprinting and shown to be indistinguishable from y. This fractionation occurs occasionally and is specially pronounced when heating of the gel occurs during the electrophoresis; the significance of it is not clear. The faint band moving ahead of S in HA101 (6’; Plate IV) has been shown by two-dimensional T, fingerprint analysis to be S (see below). Again, the basis for this fractionation is not understood but one likely possibility is inaccurate processing of S from its precursor molecule. Although it is only faintly visible in the preparation shown in Plate IV, HA103 has a lightly labeled band moving slightly ahead of 6, in the 6’ position. Two-dimensional fingerprinting of a RNase T, digest of this band identified it as S (see below). A thorough search for S in revertants HA104 and HA105 has not been made. We have estimated the approximate amounts of S made in these revertants with respect to T4 as 25 to 50%, 100% and 5 to 15% for HAIOl, HA102 and HA103, respectively. These values were obtained by visual inspection of the autoradiograms and quantitation of the eluted bands. More accurate calculations are difficult because elution is not quantitative, and because these bands are of variable purity as determined by fingerprint analysis (see below). Nevertheless, a comparison of these values with the plating efficiencies of the different revertants (Table 5) clearly shows a correlation between efliciency of growth on CT439 and amount of S produced. (e) Sequence analysis

of

StRNAs

The purpose of this work is the identification of a phage-coded tRNA requirement for growth on a bacterial host. Among the mutants isolated we have been able to define two complementation groups (I, II) whose function is somehow involved with the production of tRNA; the disappearance of the RNA species S is the only discernible alteration shared by these groups. Furthermore, revertants of HA1 (group I) that have regained the ability to grow on the restrictive host have been shown to reacquire S in amounts proportional to their efficiency of growth on CT439. We have thus clearly demonstrated a correlation between the presence of S and the ability of T4 to grow on CT439. Although the data are most simply interpreted by postulating that this relation is direct (i.e. causal), the possibility exists that the mutational alteration responsible for the HA1 phenotype is in a gene unrelated to S tRNA, one which is only indirectly responsible for the absence of S (e.g. by causing S to “stick” to cell wall or membrane and thus be resistant to phenol extraction). If, however, the HA1 gene is directly responsible for S production, it should be possible to identify the presence of sequence differences between wild-type and revertant S‘s compatible with this hypothesis. Specifically, S from second-site revertants of HA1 should differ from T4 S by at least two nucleotides. (1) The first change should correspond to the HAl-induced change. This alteration should be consistent with the specificity of the mutagen used (hydroxylamine: G-C -+ A.T) and should be present in all second-site revertants but not in true revertants. (2) The second change should correspond to the compensating mutation. This alteration should be unique to each distinct revertant analyzed and should be absent for authentic revertants. To characterize S with respect to its purity, composition and size, 32P-labeled material in the S position from wild-type T4 was eluted from a 20% gel and analyzed by two-dimensional fingerprinting of the RNase T, digestion products (Plate V(i)). Since RNase T, splits after Gp residues only, one would expect 80~4, or about 20

Origin

-A

-B

n?

Q

T4

H

I

m

PLATE I. Autoradiograph of 32P-labeled RNA from cells infected with wild-type T4 and members of each complementation group. The entire amount of 3ZP-labeled low molecular weight RNA derived from a 5-ml infected culture was prepared and analyzed on a slab of 10% polyacrylamide (without prior passage over DEAE-cellulose) as described in Materials and Methods. Specific mutants from each group were: I, HAl; II, HA4; III, HA7; IV, NG6; V, SPl. Component 4 had migrated 25 cm from the origin. Transfer RNAs migrate in components 1, 2, 3 and 4 (see text). [faeingp.118

_

.-.

PLATE II. Autoradiograph of ribonuclease T, products of component 3 RNA from (a) wild-type T4, (b) HA4 and (0) HAl. Extracts were fractionated on 10% polyacrylamide gels and the material from component 3 was eluted as described in Materials and Methods. Samples containing approx. 1 x 106 cts/min were digested with RNase T,. 0 Marks the origin. Electrophoresis on cellulose acetate in pyridine acetate, 7 M-urea (pH 3.5), from right to left (first dimension); and on DEAE-paper in 7% formic acid (v/v) from top to bottom (second dimension). Arrows (1) indicate the position of oligonuoleotides which are reduced or absent in both mutant d&e&s; arrows (2) mark the position of oligonucleotides t,hat are reduced or absent in HA4 only.

-Origin

1-

H

I

I

r.

LI

PLX~E 111. ATutoradiograph of 32P-labeled RXA from component 3 (Plate I), fractionated on a slab of 2076 polyacrylamide. The entire amount of mat*erial from &ted component 3 of HA4, atid half that from component 3 of the other strains (Plate I), was analyzed on a 20% gel as described in Materials and Methods. Component E had migrated 13 om from the origin.

---

Origin

I-

6’

-I--E

101

102

103

104

105

HAI

T4

PLATE IV. Autoradiograph of 32P-labeled RNA from component 3 of T4, HA1 and 5 revertants (HAlOl-HA105) of HA1 fractionated on a slab of 20% polyacrylamide. Five-ml extracts were first fractionated on 10% gels and the entire amount of material in component 3 was then eluted and analyzed on a 20% gel as described in Materials and Methods. Components y’ and 6’ were shown to be y and 8, respectively, by two-dimensional RNase T, fingerprint analysis (see text). Component E had migrated 13 cm from the origin.

PLATE V(ii) Autoradiograph of ribonuclease T, products of 6 tRNA from (i)(a) T4, (ii)(a) HAlOl, (ii)(b) HA102 and (ii)(c) HA103. Extracts were fractionated on 10% and 20% gels of polgacrylamidc as described in Materials and Methods. RNA moving in the 8 (T4, HA101 and HA102) or 6’ (HA103) position (see Plate IV) was fingerprint&l. Uncqwl amount s of labeled material were digested; fingerprints were autoradiographed to obtain exposures of comparable intensities. The sequences of oligx- Luzleotidcs di%ring among the tRNAs are given and positions are indicated by arrows when missing. The schematic diagram is a composite showing oli~~:~~xkIide.~ found in all 4 digests. Several of the differences among the patterns are due to contamination of 6 with component 4 in the HA103 preparat,ion. Sepnwtion and origin as in Plate II. tRNA, which co-migrates wi:h 5-3’ in 20% c:rIs:; this is most, pronounced

PLATE VI. Autoradiograph of two-dimensional thin-layer chromatography of an RNase ‘I’, digest of 6. 32P-labeled material of T4 from the 8 position in a 20% gel was eluted and digested with RTu’ase T,, and subjected to thin-layer chromatography as described in Materials and Methods. The tentative identification of standard and minor nucleo&des found in E. coli tRNAs (Saneyoshi et al., 1972) are indicated. Np is an unknown product; it has not been found in the ot,her 7 t)RK_r\s of T4 (McClain & Guthrie, unpublished observations).

U-

c-

G-

CU,C)GCClJ,C)GC CCU,C)G-

UCCU.C)G-

HA101

T4

PLAYCEVII. Autoradiograph of products of partial digestion with spleen phosphodiesterase of the (C,,U)G RNase T, oligonucleotide from T46, and the (C,,U,)G RNase T, oligonucleotide from HA101 6. The digestions were performed for the indicated times (0,l and 2 min) and subsequently subjected to electrophoresis on DEAE paper in 7% formic acid as described in Materials and Methods. Since any phosphorylated oligonucleotide moves faster than the corresponding structure but with an extra nucleotide at its 5’.terminus, a spectrum of products arranged in order of their size is generated. The Ioss of a particular nucleotide results in a characteristic change in mobility (e.g. the loss of a Up produces a larger effect than the loss of a Cp). The intermediate products were not analyzed; the indicated structures are therefore tentative.

32P-labeled 6 RNA was prepared as described in Materials PLATE VIII. Autoradiograph of ribonuclease A products of 6 tRNA from (a) T4 and (b) HAlOl. and Methods. The sequences of oligonucleotides differing between T4 and HAIOl are given and indicated by an arrow when absent. The HA101 preparation is contaminated with component 4 tRNA (see legend to Plate V) ; the composite schematic diagram indicates position of oligonucleotides shared by 6 and component 4 (open), unique to component 4 (crosshatched), and unique to S (dark).

G,G! AG CAG AsG PGP,PGP! UAG CC(UC)G (DsA)G (U&)G UC(UC)G (CLA&)GmG (CsA,Usm7G)G (CsAsUsTWG (‘&A,UIN)G UCACCA,,

1,l’ 2 3 4 5,6’ 6 I 8 9 10 11,ll’ 12,12’ 13,13’ 14,14 15 6 0 2 1 1 1 1 1 1 1 1 1 1 1

6.3 0.2 2.0 0.8 0.6 1.0 1.1 1.0 1.3 1.1 1.2 1.1 0.7 1.3

Molar yield Observed Suggested l,l’,l” 2 3 4 4a 5 6 7 8 9,9’ 10,lO’ 11 12,12’ 13,13’ 14 14% 15 16,16’

Pancrestic oligo. no.

U,U!,Y,Y! C AC (NAW CC AU AGC AsU AsU GU (AG)U -I- (AG)T GsC GGD,GGD ! GmGU,GmGU ! pGGC (GsA)U GsU (G,Asm7G)U

Composition

8.2 12.4 2.2 0.6 0.3 0.7 2.9 1.1 1.1 1.3 2.5 1.5 0.4 1.0 0.2 0.9 1.2

1 0 1 1

2

12 2 1 0 1 3 1 1 1 2 -

8

Molar yield Suggested Observed

The oligonucleotides (numbered according to the schematic drawings in Plates V and VIII) obtained after digestion with either RNaae T1 or pancreatic RNase A were cut out from the DEAE paper, quantitated by liquid-scintillation counting, and their oompositions determined from analysis on 540 paper at pH 3.6 of products yielded by alkaline hydrolysis. From these data, molar yields of each oligonuoleotide were calculated and are expressed relative to UAG = 1.0 for RNase T, products and AsU and G,U = I.0 for pancreatic RNase A products. Because RN&se T1 product 7 is in 1 molar yield (relative to UAG) and is completely absent in HA101 and HA103, there is probably 1 mole of UAG in the 6 tRNA sequence. Oligonucleotides with primed numbers are related either by cyclization ( !), or by composition and by minor bases and differ only in the extent or state of modification (e.g. RNase T, products 11 and 11’ are identical in composition except that 11’ lacks the modified sequence GmG) and have been combined for these calculations. Sequences of smaller oligonuoleotides were determined by analysis of pancreatic RNase A digestion products of RN&se T, oligonuoleotides or of RNase T, digestion products of pancreatic RNase A oligonucleotides (e.g. the RNase T1 oligonuoleotide CAG yields the two products C and AG after digestion with pancreatic RN&se A). The minor base designations are described in Table 7. All procedures are as cited in Materials and Methods. Dashes (-) indicate sequences present only in mutant digests (see text).

Composition

T1 oligo. no.

RNase T, and pancreatic RNase A products of T4 6

TABLE 6

160

C. GUTHRIE

AND

W.

McCLAIN

products after complete digestion of a tRNA 80 nucleotides in length. Thus, in contrast to the complexity of the component 3 fingerprint of T4 (Plate II(a)), we find only 15 unique RNase T1 products for T4 S (Plate V(i)). Each T, product of Plate V(i) was out out from the DEAE paper, counted, eluted and its composition (given in Table 6) determined by a&dine hydrolysis. The product that contains the 3’-end group is characterized by its failure to yield a Gp residue after alkaline hydrolysis while 5’-end groups yield a mononucleotide diphosphate with a characteristic electrophoretic mobility on paper at pH 3.5 that is due to the presence of 5’-phosphate. We find only one set of end groups for T4 S and these are present in good yield, indicating a high degree of purity. From the composition data it can be estimated that S is approximately 80 nucleotides in chain length. The presence of several modified nucleotides is a characteristic property of tRNAs. To look for modified nucleotides in 6, a suitable amount of 32P-labeled material (approx. lo4 to lo5 ots/min) was extensively hydrolyzed by RNase T, and the resulting nucleotide products were analyzed by two-dimensional thin-layer chromatography (Plate VI ; Saneyoshi et al., 1972). The positions of the four common nucleotides Cp, Ap, Up and Gp and the 5’-end group pGp have been indicated in Plate VI. In addition to these common nucleotides, at least seven minor products are also seen. Most of these minor nucleotides are in positions in the ohromatograph characteristic of structures previously identified in E. coli tRNAs, including Tp, Yp, Dp, GmGp and m7Gp (Saneyoshi et al., 1972). The indicated identification of these minor nuoleotides in S is only tentative. A minor nucleotide survey of the RNase T, products seen in Plate V(i) provides support for the above structures and identifies each minor nucleotide with its specific T, product (Table 7). We note the presence of an oligonucleotide, in about one molar yield, containing Tp, Up and Cp; the sequence TYC is common to all known tRNAs (Zaohau, 1969). To summarize, we conclude that S is a tRNA, approximately 80 nucleotides in length, and, at least in the case of T4, fairly pure. Finally, we wish to point out that the fingerprint of purified S (Plate II, arrows (l), cf. Plate V (i)) accounts for the shared oligonucleotide differences in the component 3 fingerprints of T4, HA1 and HA4. For comparative studies, RNase T, fingerprints of S from HAlOl, HA102 and HA103 were prepared. Comparison of the T, 6ngerprints of T4 and HA102 (Plate V(i) and (ii)(b)) reveals no apparent differences between the two patterns. In a separate experiment, the RNase T, products were fractionated in one dimension and each of these 12 products eluted and analyzed by one-dimensional separation of pancreatic RNase A (C- and U-specific) products (data not shown). Again, no differences between S from T4 and HA102 could be detected by this method. These results are consistent with the T4-like plating ef%ency of HA102 on CT439 (see Table 5 and above) and the apparent full production of S by this revertant (Plate IV), and suggest that HA102 has arisen by a back-mutation at the HAl-induced site. In contrast, a comparison between S from HA101 and HA103 on the one hand and T4 on the other reveals several differences (Plate V(i), (ii)(a) and (ii)(c)). Perhaps the most striking difference noted is the absence in HA101 and HA103 of the oligonucleotide (C,,U)G, which is present in T4 S in about one molar yield (Plate V; Table 6). Correlated with this absence, we find a new oligonucleotide in the T, digests of both HA101 and HA103 that is not present in T4 and which has the composition (U,,C.JG. Evidence for the connection by mutation betweenthese two oligonucleotides

T4

MUTANTS

DEFECTIVE

IN

A tRNA

161

TAJ~LE 7

Chromutogra~hicprqetiies of minor nucleotidtrs Mobility in Ru in solvent

source Nucleotide PGP “P m7Gp GmGp NP Tp YP

(RN&se T1 oligo. no.) 6 8 12,12’ 11,ll’ 14,14’ 13,13’ 13,13’

pH 3.6 electrophoresis

(a)

“Very fast u” “f&St U” “f&l& c” “slow U”

0.62 0.94,1*16 0.67 0.19 O-26-0*32t 0.73 1.13

“81OW

c”

U “streaky

U”

(b) 0.39 0.86,0.93 0.84 0.64 0.89 1.38

T4 S RNase T, oligonucleotides (see Plate V) were subjected to alkaline hydrolysis and the products analyzed by electrophoresis on no. 640 paper at pH 3.6. Labeled material running in positions other than those of the standard Cp (0*67), Ap (O-63), Gp (0.47) and Up (1-O) nucleotides was eluted and analyzed by thin-layer chromatography in the two solvent systems isopropanol/HCl (a) and isopropanol/NH,(b) es described in Materials and Methods. Their mobilities are reported relative to Up(Ru) . The design&ion of the minor nucleotide struotures is only tentative and, in the case of N, completely unknown. Certain structures are labile to alkaline hydrolysis (e.g. Dp breaks down to give two produots after exposure to alkali). However these designations are consistent with the minor products found in 8 two-dimensional chromatographic system after complete enzymatic hydrolysis (see Plate VI). t This mobility characterized the major product; a minor product had a mobility of 0.65, similar to Cp or Ap.

provided by the related spectrum of products generated by partial digestion of these oligonucleotides with the 5’-exonuclease spleen phosphodiesterase (Plate VII). Since only small amounts of material were available, it was not possible to further extend this analysis. We suggest, however, that these results are consistent with the origin of the UC(U,C)G oligonucleotide in revertants HA101 and HA103 via a C -+ U transition in the wild-type oligonucleotide CC(U,C)G. We furthermore note that this nucleotide change is compatible with the known specificity of hydroxylamine. In theory, of oourse, one would desire to show the presence of this change, and only this change, in mutant HAI. Although 8 is not present in HAl, we investigated the possibility of using component B, the putative precursor, for these studies. Although the 10% gel profile of Plate I shows little distinct banding in the component B position, when this region of the gel was eluted and digested. with RNase T, a fingerprint was obtained which was essentially indistinguishable from T4 component B. Qualitative comparison of the component B fingerprints from T4, HA1 and HA101 revealed the presence of the (C,U)G oligonucleotide in component B from T4 only. HA1 and HA101 component B, on the other hand, were both found to contain the new oligonucleotide (C,U,)G. Finally, we wish to note that the T1 fingerprint of HA102 6, the presumptive full revertant, contains the (C,U)G oligonucleotide of the wild-type sequence and lacks the altered counterpart (C,U,)G of HA101 andHA103. Takentogether with the above results, a C + U transition in the oligonucleotide (G)CC(U,C)G is the likely primary alteration caused by the HA1 mutation: is

152

C. GUTHRIE

AND

W.

McCLAIN

T4 : (G)CC(U,C)G 3 hydroxylamine HA1 : (G)UC(U,C)G The secondary alteration should be unique to each distinct revertant. Turning again to the RNase T, digests, we note the complete absence in HA101 S of the oligonucleotide UAG, which is present in T4 S and HA103 S in molar yield (Plate V; Table 6). In the corresponding pancreatic digests (Plate VIII; Table 6), we find the product G,U in molar yield in T4. This product is completely absent in HAlOl, but instead we find the new sequence G,C. These two differences are presumably related via a single nucleotide change. Due to the low yield and purity of S from HA103, we were unable to draw specific conclusions as to the identity of the secondary mutation We point out, however, that since HA103 S contains a high yield of UAG it has reverted by a different alteration than has HAlOl. In summary, then: we have generated a spectrum of revertants from HA1 which could be distinguished by their relative eEiciencies of growth on the restrictive host CT439. This classification was uniquely correlated with the amount of S found to be present in these revertants and was, in turn, consistent with the preliminary sequence analysis of the respective S tRNAs. 4. Discussion Approximately 150 spontaneous and induced mutants of T4 which are unable to grow on strain CT439 have been isolated and a subset of 20 classified into five complementation groups. Two groups, defined by the mutants HA1 (group I) and HA4 (group II), affect the production of tRNA. Three groups (III, IV and V) do not affect the banding pattern of RNA and control unknown functions. While the function of groups III to V is not defined, we wish to point out that since RNA production was examined in the permissive host, B/S, the possibility exists that groups III to V have tRNA-related defects that are only detectable in the non-permissive host, CT439. Mutants of group II manifest a fairly complex phenotype. Polyacrylamide gel electrophoretic analyses of 32P-labeled low molecular weight RNAs produced on permissive infection reveal that these phage are lacking components A and D and serine tRNA and have reduced amounts of M, p, y and E and almost undetectable amounts of 6. Although the mutants of this group, HA4 and AA2, were obtained after mutagenization, the possibility that this phenotype is due to multiple or multisite mutations seems unlikely since (1) the two mutants are clearly of independent origin and yet have indistinguishable phenotypes, and (2) although both mutants are quite leaky, apparent revertants (which are distinguished by their plaque size) do arise. Furthermore, since these mutants show strong recombination with deletions for the tRNA region, it is difficult to account for this phenotype by simultaneous alterations of several tRNA structural genes. If these mutants do arise by point mutation, one could postulate two types of mechanisms to account for the observed pleiotropy. The affected gene might have only an indirect role in the elimination of these tRNAs, e.g. as mentioned above by somehow interfering with their recovery from the infected cell. Alternatively, the group II gene might play a direct role in the processing of a number of tRNAs from their respective precursor molecules.

T4 MUTANTS

DEFECTIVE

IN

A tRNA

153

The putative tRNA precursors A and B can be cleaved in vitro with uninfected cell extract (Guthrie et al., 1973; cf. Altman & Smith, 1971). Since these in vitro reactions have not yet been characterized with respect to accuracy, efficiency, or role of minor base modification, the possible participation of phage-coded enzymes in the processing of tRNA remains an open question. We suggest that the HA4 gene product might in fact have such a role in tRNA maturation. If this were the case, inactivation of this product might well be expected to interfere with the appearance of a number of tRNAs simultaneously. It is interesting to note that the HA4 gene also interferes with the production of component D. As mentioned earlier, although this component has no apparent tRNA counterpart, its map position within the tRNA cluster (Wilson et al., 1972; McClain et al., 1972) suggests some as yet undetermined link to the phage-coded tRNAs. This observation that the HA4 mutation eliminates the appearance of component D coincident with the simultaneous elimination of several known tRNAs is thus further support for the possible tRNA-related nature of component D. A final comment about the HA4 gene is its likely analogy to the mb gene described by Wilson & Abelson (1972). Suppressor-negative derivatives 01 the T4-coded nonsense suppressor psub+fall into two compl ementation groups, one defining the structural gene of serine tRNA, and the second designated mb, which has been positioned ten map units from TII (Wilson & Abelson, 1972). The 10% gel pattern of mb-infected cells is indistinguishable from that of the HA4 group (our data, not shown). Furthermore, a gene (Ml) with similar properties to mb has been identified by McClain among suppressor-negative derivatives of the psui+ T4 serine suppressor, presumably analogous to Wilson’s psub+gene (McClaiu et al., 1973). The Ml gene fails to complement HA4 and 10% gel analysis as well as fingerprints of component 3 tRNAs of Ml mutants are also indistinguishable from the comparable HA4 results. We thus tentatively conclude that the HA4 gene is equivalent to the mb and Ml genes, mutational alterations in which interfere with production of several tRNAs, including serine and 6. Mutant HA1 of group I, in contrast, has a simple phenotype. It displays a normal profile on 10% polyacrylamide gels and on fractionation of the complex component 3 by 200/ gels can be shown to differ from T4 only in its lack of component 8. Revertants isolated by their ability to form plaques on the restrictive host CT439 show a range in plating efliciency when tested at temperatures above 3O”C, the optimum temperature for T4 growth on this strain. Revertants HA101 and HA102, which plate with close to wild-type efficiency at all temperatures tested, produce amounts of 6 estimated at 25 to 50% and lOOo/oof T4, respectively. Revertant HA103, which shows significantly reduced plating efficiencies at 37°C (0.2 against 0.8 for T4) and dramatic reductions above this temperature (e.g. 0.006 against 0.3 for T4 at 4O”C), produced only about loo/, the amount of 6 made by T4. These data show a strong correlation between production of 6 and ability of T4 to grow on strain CT439. In order to establish a direct causal link as the basis of this correlation, we attempted to demonstrate that it is the gene for component 6 which has undergone the primary mutational alteration, and not some other gene which only indirectly results in the lack of 6 seen in these mutants. We have presented data to support the presence of primary and secondary mutational alterations that are at least consistent with our hypothesis that mutant HA1 is in fact affected in the structural gene coding for 6 tRNA.

154

C. GUTHRIE

AND

W.

MoCLAIN

These results thus suggest that the failure of HA1 to grow on strain CT439 is due to the specify lack of 6 tRNA. We have not shown that lack of 6 is uniquely responsible for the inability of group I members to grow on CT439. Though the members of group I unambiguously define a single complementation group, one must exercise caution in interpreting the significance of this fact. Since 6 appears to be co-transoribed with cc, it is conceivable that a mutation in one structural gene might interfere with the maturation, and hence production, of a second tRNA. That is, complementation group I might correspond to more than one tRNA. Although our recombination analysis of group I members suggest that this possibility is unlikely, further analysis of all other group I members (e.g. on 20% gels) is needed for clarification on this point. The mechanism by which 6 is prevented from appearing in the mature form is certainly not clear at this time. The first possibility would be that the precursor from which it is derived is altered in such a way as to prevent its proper processing. The presumptive 6 precursor, component B, can be detected in HA1 extracts, but only in very low amounts. This suggests either that B is very unstable in these extracts, or alternatively, that B is derived from a larger, as yet unidentified precursor, which is processed abnormally and/or at a much reduced rate. The final point to be considered about group I mutants is the significance of the variability in plating efficiency on CT439. We have already pointed out the correlation between efficiency of plating and production of 6 observed in the analysis of HA1 revertants. This observation suggests in turn that those members of group I which are leaky on CT439 possess alterations in 6 which allow a small amount of gene product to ‘leak through.’ Although we have not made an exhaustive test of such a possibility, it is relevant to consider the group II mutants in this context. Mutants HA4 and AA2 are quite leaky on CT439 (efficiency of plating about 0.2) and have been shown to produce small but detectable amounts of 6. Taking all these results into consideration, it seems quite likely that there is a single tRNA requirement for growth on strain CT439 and furthermore that there is a direct relation between the amount of this tRNA present in its mature form and the efficiency with which phage growth occurs. Subak-Sharpe (1966) first suggested the possibility of viral-coded tRNAs as a consequence of the tendency of natural selection to promote a tRNA population optimally adapted to the translation of mRNAs which might have codon recognition requirements considerably different from those of the host. Scherberg & Weiss (1972) have in fact obtained triplet binding results that are consistent with such a hypothesis, and Wilson (1973) has invoked a similar model to explain his genetic and biochemical results obtained with T4 strains carrying deletions for a number of tRNAs. Alternatively, speculations have also been put forth that invoke a regulatory role for tRNA in developmental control (Sueoka & Kano-Sueoka, 1972). We are hopeful that our system, in which only a single, well-defined tRNA is absent, will allow us to determine unequivocally the specific mechanism of this requirement.

We are indebted to Dr J. H. Wilson for his generous provision of phage and bacterial strains. The technical assistance of Mrs P. Krie is gratefully acknowledged. This work was supported by a grant from the University of Wisconsin Graduate School Research Committee and by U. S. Public Health Service grant A110257.

T4 MUTANTS

DEFECTIVE

IN A tRNA

155

REFERENCES Altman, 8. (1971). Nature New Biol. 229, 19-21. Altman, 8. & Smith, J. D. (1971). Nature New Biol. 223, 35-39. Anderson, K. W. & Smith, J. D. (1972). J. Mol. Biol. 69, 349-356. Barn&t, L., Brenner, S., Crick, F. H. C., Shulman, R. G. & Watts-Tobin, R. J. (1967). Phil. Trans. Roy. Sot. London, 252, 467-560. Barrell, B. G. (1971). In Procedures in Nucleic Acid Research (Cantoni, G. L. and Davies, D. R., eds), vol. 2, pp. 751-779, Harper and Row, New York. Barrell, B. G., Co&on, A. R. & M&lain, W. H. (1973). l?EBS Letters, in the press. Benzer, S. (1961). Proc. Nat. Acad. Sk., U.S.A. 47, 403-415. Daniel, V., Sarid, S. & Littauer, U. Z. (1970). Scielzce, 167, 1682-1688. Drake, J. W. (1970). In The Molecular B&s of Mutation, pp. l-273, Holden-Day, San Francisco. Epstein, R. H., Belle, A., Steinberg, C. M., Kellenberger, E., Boy de la Tour, E., Chevalley, R., Edgar, R. S., Susman, M., Denhardt, G. H. & Lielausis, A. (1963). Cold Sprirzg Harbor Symp. &want. Biol. 28, 375-394.

Guthrie, C., Seidman, J. G., Altman, S., Barrell, B. G. Smith, J. D. & McClain, W. H., (1973). Nature New Biol. in the press. Ikemura, T. & Dahlberg, J. E. (1973). J. BioE. Chem. 248, 5024-5032. Landy, A., Abelson, J. N., Goodman, H. J. & Smith, J. D. (1967). J. Mol. Biol. 29,457-471. Mattson, T. L. (1968). Ph.D. Thesis, University of Wisconsin, Madison, Wisconsin. McClain, W. H. (1970). .E’EBS Letters, 6, 99-101. M&lain, W. H., Guthrie, C. & Barrell, B. G. (1972). Proc. Nut. Acad. Sci., U.S.A. 69, 3703-3707.

McClain, W. H., Guthrie, C. & Barrel& B. G. (1973). J. Mol. Biol. 81, 157-171. Paddock, G. & Abelson, J. N. (1973). Nature New Biol. in the press. Pinkerton, T. C., Paddock, G. & Abelson, J. (1972). Nature New Biol. 240, 88-90. Saneyoshi, M., Ohashi, Z., Harada, F. & Nishimura, S. (1972). Biochim. Biophys. Acta, 262, l-10. Scherberg, N. H. & Weiss, S. B. (1970). Proc. Nat. Acad.Sci., U.S.A. 67, 1164-1171. Scherberg, N. H. &Weiss, S. B. (1972). Proc. Nat. Acad.Sci., U.S.A. 69, 1114-1118. Smith, J. D., Barnett, L., Brenner, S. & Russell, R. L. (1970). J. Mol. B&Z. 54, 1-14. Stahl, F. W., Edgar, R. S. & Steinberg, J. (1964). Genetics, 50, 539-552. Steinberg, C. M. & Edgar, R. S. (1962). Genetics, 47, 187-208. Subak-Sharps, H. (1966). Cold Spring Harbor Symp. Quant. Biol. 31, 583-594. Sueoka, N. & Kane-Sueoka, T. (1970). Prog. Nucleic Acid Res. & Mol. Biol. 10,23-55. Tessman, H. (1968). virology, 35, 331-333. Wilson, J. H. (1973). J. Mol. Biol. ‘71, 547-556. Wilson, J. H. & Abelson, J. N. (1972). J. Mol. Biol. 69, 57-73. Wilson, J. H. & Kells, S. (1972). J. Mol. Biol. 69, 39-56. Wilson, J. H., Kim, J. S. & Abelson, J. N. (1972). J. Mol. Biol. 71, 547-556. Zachau, H. 0. (1969). Angew. Chem. internat. Edit. 8, 711-727.