STUDIES OF MUTATIONS IN T4 CONTROL GENES 33 AND 55 H. ROBERT HORVITZl

The Biological Laboratories, Haruard Uniuersity, Cambridge, Massachusetts 02138 Manuscript received October 13, 1974

ABSTRACT

Available mutations in transcriptional control genes 33 and 55 of coliphage T4 have been examined. By complementation analysis and map position, 15 mutants (13 in T4D, 2 i n T4B) have been shown to lie in gene 33 and 6 ( 5 in T4D, 1 i n T4B) in gene 55. According to patterns of suppression and recombination, these mutants define three distinct amber sites in gene 33 and also three distinct amber sites in gene 55. All of these mutations are true amber mutations, in apparent contrast to some traditional T4 “amber” mutants which grow in su+ E. coli CR63 but not in su- E. coli B because of a strain difference other than the suf determinant. Evidence is presented that, contrary and GEI~USCHEK 1970), to previous suggestions (BOLLEet al. 1968; PULITZER the gene 33 product is absolutely essential for T4 development.

HE primary developmental switch from early to late protein synthesis in T4Tinfected Escherichia coli is mediated at the level of transcription by three catalytic phage functions: the products of “maturation” genes 33 and 55 (BOLLE et al. 1968; SNUSTAD 1968; GUHAet al. 1971) and, according to recent studies, 45 (Wu and GEIDUSCHEK 1974; Wu, CASCINOand GEIDUSCHEK 1974). The protein products of genes 33 and 55 co-purify with the host core RNA polymerase (STEVENS 1972; HORVITZ 1973; RATNER 1974b) and may be new, phage-specified subunits of this enzyme. The product of gene 45 also appears to interact with RNA polymerase both in uitro (RATNER 1974a) and in uivo (SNYDER and MONTGOMERY 1974). The products of genes 33, 55, and 45 are the first control proteins believed to interact directly with RNA polymerase which have been identified both genetically and biochemically. They provide strong support for a recently proposed mechanism of general transcriptional control: distinct regulatory proteins interact with a basic “core” RNA polymerase to specify different classes of RNA (BURGESSet al. 1969; TRAVERS, KAMENand SCHLEIF1970). An elucidation of their precise mechanism(s) of action might well prove relevant to an understanding of genetic regulation in many biological systems. As an approach t o the detailed examination of the functions of the products of genes 33 and 55, a general characterization of the properties of available T4 Present address Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England. Genetics 79: 349-360 March, 1975.

350

H. R. HORVITZ

strains mutant in these genes has been performed. These experiments had the following goals: 1) to verify that strains unexamined since their original isolation are indeed defective in genes 33 and 55, using as criteria both the functional test of complementation and the positional test of map location (BENZER1955) ; 2) to determine which of the “amber” mutants available are “true ambers” and which may be “pseudo-ambers” (the latter class includes mutants classified as ambers on the basis of their growth in E. coli CR63 and non-growth in E. coli B but apparently dependent on strain differences other than the presence of the sul+ suppressor in CR63 (KRIEGand STENT1968; YEGIANet al. 1971)); true amber mutants are required for certain types of experiments, such as those which depend upon a missing protein (O’FARRELL, GOLDand HUANG1973; STUDIER 1972),a fragment polypeptide chain (SARABHAI et al. 1964; PUTT et al. 1972; HUANGand LEHMAN1972; FRIEDBERG and LEHMAN1974), or insertion of a specific new amino acid into the protein product at the mutated site ( STRETTON and BRENNER1965; HORVITZ 1973; FILES, WEBER and MILLER 1974) ; 3) to establish which of the mutant strains are genetically distinct. A number of T4 genes have shown multiple isolates of the same mutation (e.g., BENZER m d FREESE 1957; ALLEN,ALBRECHT and DRAKE 1970) and the naive use of such strains can lead to both needless experimental repetition and unfounded generalization. As criteria, both patterns of growth in different bacterial hosts and ability to recombine with other mutations in the same gene have been used. MATERIALS -4ND METHODS

Phage strains: Most of the T4 phage amber mutants used in these experiments were isolated and preliminarily characterized by R. EEGAR,R. EPSTEIN,and their collaborators (EPSTEIN et al. 1963). These strains and the direct sources from which they were obtained are described in Table 1. Wild-type T4D was from M. MESELSON. The multiple mutants amN134-amCi8 (gene 33-33) and amN134-amC18-amBL292 (genes 33-33-55) were from G. NOTANI.“am” NG130 (gene 17) and “am”N56 (gene 17) were from W. WOOD,“am”HM26 (gene 60) from and E. FLOOR. J. LEVY,and copies of “am”N57 (gene 4 1 ) were obtained from both R. JAYARAMAN Bacterial strains; These strains are described in Table 2. CR63 was generally used as the permissive host for amber mutants; B/r was the non-permissive host. Media: All plates and media were prepared according t o JAYARAMAN (1970). Plate top agar: 6 gm/l bactoagar; 10 gm/l bactotryptone; 8 gm/l NaC1; 2 gm/l sodium citrate; 1 gm/l glucose; 1.5 ml/l I N NaOH. Plate bottom agar: 12 gm/l bactoagar; 10 gm/l bactotryptone; 8 gm/l NaC1; 2 gm/l sodium citrate; 1 gm/l glucose; 1.5 ml/l I N NaOH. Broth: 10 gm/l bactopeptone; 5 gm/l NaC1; 3 gm/l beef extract; 1 gm/l glucose. Phage wash fluid: 10 m M TrisHCI, pH 7.4; 0.01% gelatin; 1 mM MgC1,; 0.1% NaC1. Phage stocks: Phage stocks were prepared from single plaques. Generally, 5 x IO4 plaqueforming units were incubated for about 12 hours at 34” on each of two petri dishes seeded with CR63. After nearly confluent lysis, top agar containing phage and cell debris was transferred to a tube containing 3 ml of phage wash fluid and 2 drops of chloroform and vortexed vigorously. After sitting 15 minutes at room temperature, agar and debris were removed by low speed centrifugation (10 minutes at 4” at 8000 rpm in a Sorvall SS-34 rotor). Titers vaned from 1 x 1O10/ml to 3 x 1011/ml. Complementation spot tests: Complementation tests were performed essentially as described (1968). A lawn of the non-permissive host, B/r, (0.1 ml of cells grown in broth to by SYMONDS

MUTATIONS I N T4 GENES

351

33 AND 55

TABLE 1 Phage strains Phage

T4D + ad134 amNG46 adG405 amBUi3 amE306 ad18 amEl120 ad033 amEl259 amEl266 amC2l ad490 ad666 amE583 amEl136 amBL292 ad64 amBU27 amE552 amNG561 amNG372

Type*

D D D D B D D D B D D D D D D D D D B D D D

Isolated by*

Gene

33 33 33 33 33 33 33 33 33 33 33 33 33 33 33 55 55 55 55

55 55

Mutagen*+

-

A. DOERMANN NA R. EPSTEIN NG R. EDGAR NG R. EDGAR BU G. EDLIN AP R. EDGAR AP+BU S. BRENNER AP R. EDGAR NA R. &STEIN AP R. EDGAR AP R. EDGAR AP BU S. BRENNER AP R. EDGAR AP R. EDGAR AP R. EDGAR AP R. EDGAR BU R. EPSTEXN AP BU S . BRENNER BU G. EWN AP R. EDGAR NG R. EDGAR NG R. EDGAR

+

+

Source

M. MESELSON R. JAYARAMAN

w. WOOD w. WOOD w. WOOD

R. HASELKORN R. HASELKORN

w. WOOD w. WOOD w. WOOD w. WOOD

R. HASELKORN R. HASELKORN R. HASELKORN

w. WOOD w. WOOD

R. JAYARAMAN

w. WOOD w. WOOD

A. STEVENS

w. WOOD w. WOOD

* (W. WOOD, I. LI~ELAUSIS and S. BRENNER, personal communications.)

tAP, 2-aminapunne

BU, 5-bromouracil or 5-bromdemyuridine NA, nitrous acid NG, nitrosoguanidine

a n O.D.,,, of 1.0-1.5) containing 5 x IO7 to 108 of one phage strain was poured and allowed to set. Drops (about 0.02 ml) of each test phage at 106 to lO7/ml were then applied and allowed to absorb. Plates were incubated overnight. A region of clearing indicated complementation. Growth spot tests: Phage were spotted (one drop of phage at lO4/ml) onto a freshly poured lawn of the appropriate host. Spots were allowed to absorb and the plates incubated overnight. At this dilution, single plaques are visible and can be easily checked for distinct morphological properties. Cross-streaking growth tests: Phage at about 4 x IOg/ml were streaked from a capillary tube (1.61.8 mm diameter; Kimax No. 34500) alosng the diameter of a plate, and the liquid allowed to absorb into the agar. Cells picked from a single colony with the broad end of a flat toothpick were streaked across the line of the phage. About twelve such cross streaks of different hosts were applied per plate. Plates were then incubated overnight. Phage growth is indicated by a break in the streak of cells that sometimes extends beyond the line of the phage streak. Recombination spot tests: A rapid method of identifying distinct mutations within a single gene was developed from a protocol described by DUERMANN and BOEHNER (1970). This technique differs from that previously described in that it did not require prior knowledge concerning the specific markers being tested and allowed screening of phage stored in buffer instead of from plaques o n plates. The protocol calls for mixing permissive and non-permissive cells (the latter in substantial excess) with a given T4 mutant in top agar and plating this seeded lawn. After the agar sets, liquid spots of other phage mutants are dropped onto this lawn and allowed

352

H . R. HORVITZ

TABLE 2

Bacterial strains Name

B/r/Tl B4Q CR63 B5 XAlOOic

XAlOlc XA102c XA103c XAIMc XAlOBc XAlOCc XAlMc

Strain

B B

K K K K K K K K K K

Responds su'

to*+

Inserts'

Insertion efficiency*

1 1 5

-

-

-

am am am,och -

ser ser lys

3040% 3040% 5%

1 2 3 6

am ser gln am tyr am am leu am,ach gln$ am,och tyr am,och lys

-

B C

5

-

-

3040%

1560% 3&55% 55% ? 15% 5%

Source

Genotype

R. S a " UVRTIRsuR. JAYARAMAN su,+

E.FLOOR D. GANEM D. GANEM

D.GANEM D.GANEM D.GANEM D. GANEM D. GANEM D. GANEM D. GANEM

+

SUI

HfrC su,+ F-B,-del(luc-pro) metB- argEamrifRsrc su,+, from XAlOOc sus+, from XAlOOc su,+, fromXA100c sus+, from XAlOQic .vu,+, from XAlOOc su,+, from XAlOOc su,+, from XAlOOc

* According to KAPLAN,S T R E ~and N BRENNER(1965); GAREN(1968); GORINI (1970). -f am: amber; och: ochre. $ SIGNER, BECKWITH and BRENNER (1965). to absorb. The plates are then incubated. Mixed infection in a permissive host allows phage growth. If the two phage mutants are distinct, wild-type recombinants will be produced during this growth cycle, and these wild-type progeny will then multiply on the lawn of predominantly non-permissive cells; a cleared spot (generally somewhat turbid) will result. If the two phage are mutated at the same site, no recombination and thus no spot production will occur. If the phage are mutated in two different genes, complementation occurs and a clear spot will be produced. Three parameters are relevant to this process: (a) the ratio of permissive to non-permissive cells i n the lawn, (b) the number of phage spread with these cells in the lawn, and (c) the number of phage spotted onto the lawn. Optimal parameters have been determined in preliminary experiments (not shown), resulting in the following protocol. Cells are grown in broth to O.D.,,, = 1.5 and mixed in a 1:25 ratio of permissive to nonpermissive (0.01 ml of CR63 and 0.25 ml of B/r per plate). Then 5 x IO7 phage and 2.5 m l of top agar are added and the lawn poured. After it sets, one drop of phage to be tested is applied from a 0.1 ml pipette. Phage from solutions at both lO7/ml and 108/ml are used; these t w o dilutions allow readings of all mutant pairs. Not counting controls, two strains can be checked against that in the lawn per plate. After phage are spotted and the liquid allowed to absorb, plates are incubated overnight. Phage crosses: Two-factor crosses were performed i n a manner similar to that of SYMONDS (1968). The permissive host, CR63, was grown at 30" in broth to about 2 x lO7/ml, chilled, and concentrated by centrifugation to 2 x lO*/ml. The two parental phage (at a multiplicity of infection of about 7 each) were added and allowed to adsorb at 30" in the presence of 4mM KCN ( t = 0 min). At t = 8 min, antiserum to phage T 4 was added (final inactivation constant K = 2 ) . At t = 14 min, infected cells were diluted 1:IO5 into broth at 30". At t = 104 min, infected cells were lysed with chloroform. Total progeny were titered on CR63; wild-type recombinants, on B/r. The recombination frequency was calculated as twice the titer on B/r divided by the titer o n CR63. Burst sizes were generally between 200 and 300.

M U T A T I O N S IN

+ + + + + + + + + + + i

T4

#

z + + + + + I

33

GENES

++ ++ ++ + I

++ ++ ++

55

AND

+ #

+ a

++ ++ ++ ++

++

I

I

I

I

# #

I

I

+ +

++ ++

4-

I

I

I

1

++ ++ I cu

In

y"

6

Ac\l

s3 5

:+

:Q ib

353

I

3544

H. R. HORVITZ RESULTS

Complementation tests: All mutants in genes 33 and 55 listed in Table 1 were confirmed to be in the expected complementation group (data not shown). Patterns of growth: All amber mutants in genes 33 and 55 were tested for growth at 34” in twelve different strains of E. coli; this set of bacterial strains includes both su+ and su- B and K strains and an isogenic series of sui+, su2+, su3+, sus+, sue+,suc+ and sus+ hosts. Both cross-streaking and growth spot tests were used. Patterns of growth are presented in Table 3, with mutants grouped appropriately. As shown, the 15 amber mutants in gene 33 fall into three distinct classes; the six amber mutants in gene 55 fall into two classes. Since different amber mutations could have equivalent suppression patterns, these results suggest minimal estimates for the number of distinct mutations. All of the mutants in genes 33 and 55 appear to be true ambers; i.e., they fail to grow on both K and B strain su- hosts (XAlOOc and B/r) and grow on both types of su+ hosts (e.g., CR63 and B40). As a control, it was shown that previously described (KRIEGand STENT 1968; YEGIANet al. 1971) “pseudo-ambers” “am”N56 (gene 1 7 ) and “am” HL626 (gene 60) grew on all ten K strain hosts but not on either of the B strain hosts. During the course of these experiments with mutations in genes 33 and 55, “pseudo-amber’’ growth patterm were discovered for “am”N57 (gene 4 1 ) and “am”NG130 (gene 1 7 ) . Recombination spot tests: Each amber mutant was tested by the recombination spot test against other mutants in the same gene. Patterns of recombination are given in Table 4. According to these tests, the gene 33 ambers fall into two classes and the gene 55 ambers into three classes. These classes correspond to those observed in the patterns of growth, except (a) two classes (“c” and “d” of gene 55 mutants with indistinguishable growth patterns readily recombine, and (b) two classes (“2”and “3” of gene 33 mutants with distinct growth patterns recombine very rarely, if at all. Combining these two sets of observations, there appear TABLE 4 Recombination spot tests a. Gene 33

spots

a. amN134, amNG46, amNG405, amBU13, amE306 b. amC18, amEl120, amN033, amE1259, amE1266, amC21, amE490, amE666, amE583, amE1136

amCf8

-*

+t

+

+

-

-

b. Gene 55

amBL292

amBL292, amC64, amBU27 d. amE552 e. amNG372, amNG561 c.

* -, no spot.

t +,turbid spost caused by recombination.

Lawn

amNi34

-

++

Lawn amE552

+ +

-

amE583

-~~

amNG561

++ -

MUTATIONS I N

T4 GENES 33 AND 55

355

TABLE 5 Classificationof available mutants in genes 33 and 55 Class

Gene 33

Gene 55

Mutants

I I1 I11

amN134, amNG46, amNG405, amBUI3, amE306 amCl8, am.Ell20, amN033, amE1259, amEl266, amC21, mnE490, amE666 amE583, amE1136

I I1 I11

amBL292, amC64, amBU27 amE552 amNG372, amNG561

to be three distinguishable amber mutations in each gene (Table 5 ) . Attempts to resolve classes I1 and I11 of gene 33 by varying parameters of the recombinatioc spot test failed, suggesting that these mutations are very closely linked. Direct mapping data from phage crosses (see below) confirmed this hypothesis. Phage crosses: Mapping data for the six apparently distinct amber mutations in genes 33 and 55 have been obtained using two-factor phage crosses (Table 6). These data are consistent with the observations described above, directly verifying that the mutations studied map in the regions of genes 33 and 55, respectively, and confirming the results of the spot recombination tests. Representatives of both other classes of presumed gene 33 mutants map very close to the traditional gene 33 mutation amN134 (EPSTEINet al. 1963); similarly, both other classes of 55 amber mutants map very close to the traditional gene 55 mutation amBL292. I n gene 5 5 , amNG372 appears to map between amBL292 and amE552, closer to the latter site. In gene 33, amNI34 and amCl8 are clearly distinct, and amE583 is extremely close to amCl8. Temperature-dependence of suppression patterns: After three apparently distinct amber mutations were identified in each of these genes, more complete suppression patterns were obtained using growth spot tests at three different temperatures: 23", 34", and 42". This screening was done primarily to determine if any other phage-host combinations had a temperature-sensitive phenotypz like that shown by amE552 (gene 55) when grown on CR63 (PULITZER 1970). Conditions effecting a temperature-sensitive gene 33 phenotype would be TABLE 6 Recombination frequencies for distinct amber mutations

Gene 33: amNl34 amCl8 amN134

cross x ad18

x

x x x

amE583 amE583

Gene 55: amBL292 amE552 amBL292 amNG372 amE552 X amNG372

% recombination

1.1, 1.2 Less than 0.1 1.2 0.9, 1.3 0.7, 0.6 0.3, 0.2

356

H.

R.

HORVITZ

TABLE 7 Temperaiure-dependence of growth patterns Temp.

Strain

42"

T4D + amCl8(33) amN134(33) amE583 (33) amBL292(55) amE552(55) amNG372(55) amN134-amC18 amN134-amC18amBL292

23"

T4Df amClS(33) amN134(33) amE583(33) amBL292(55) amE552(55) amNG372(55) amN134-amC18 amN134-amC18amBL292

CR63

+++* ++ ++ + +++ ++ ++ + +

B/r

XA

lOOc

XA

lOlc

XA

102c

XA

103c

XA

101;~

XA

106c

+#$: ++ ++ ++ ++ ++ # + ++ ++ ++ -+ # + +++ -++ +++ -+# +++ + + + - + + + + - + ++ ++ ++ ++ ++ +++ +++ +++ -++ -++ ++ ++ ++ ++ ++ + + + - + + + + - +

g m e h like T4D+. +3* -+, , no vlsible growth. #, visible, but deficient, growth.

particularly useful, since no temperature-sensitive mutants in gene 33 now exist and I. LIELAUSIS, personal communication). These patterns are pre(W. WOOD sented in Table 7 for growth at 23" and 42". Patterns at 34" were very similar to those shown here for 23" and can essentially be obtained from Table 3. NO phenotypically heat-sensitive mutants in gene 33 were identified. Gene 33 amN134 was phenotypically cold-sensitive when grown on a sus+ host. DISCUSSION

1. There exist 13 strains of T4D and 2 strains of T4B carrying amber mutations in gene 33; there are 5 strains of T4D and 1 strain of T4B carrying amber mutations in gene 55. These strains probably define 3 distinct sites in gene 33 (represented by amNI34, amCI8, and amE583) and 3 distinct sites in gene 55 (amBL292, amE552, and amNG372). Each of these values is a minimal estimate, as both tests used in defining these classes (suppression patterns and recombination spot tests) could fail to distinguish distinct mutations. It is conceivable that amE583 and amCI8 are at the same site in gene 33 but display different suppression patterns because amE583 carries a secondary amber mutation which cannot be suppressed by an sus+ host (amE583, unlike amCI8, does not grow on

MUTATIONS I N

T4 GENES 33 A N D 55

35 7

an suG+host). Such second-site mutations have explained similar apparent discrepancies between recombination and suppression patterns among T4 strains mutant in gene 43 (KARAM and O'DONNELL1973). However, the occurrence of two such mutants in gene 33 (amE583 and amE2136) makes this possibility unlikely. 2. All six of these apparent distinct amber mutations in genes 33 and 55 are true amber mutations. 3. The amber codon in amE552 (gene 5 5 ) is probably derived from a tryptophan residue; the other amber mutants are most likely a t sites which code for glutamine in the wild-type gene products. These conclusions are based upon the specificities of the mutagens used. All six mutations studied were generated by mutagenesis with 5-bromouracil, 2-aminopurine, nitrosoguanidine, and/or nitrous acid, each of which specifically induces transition mutations (A-T+ G-C) in T 4 (CHAMPEand BENZER1962; BAKERand TESSMAN 1968; DRAKE 1969). Thus, only two codons could have been mutated to the UAG amber genotype: CAG (glutamine) and UGG (tryptopan) (CRICK1966). (The third similarly related codon, UAA, is itself a nonsense codon.) Since glutamine is a far more common amino acid than tryptophan (SPAHR1962; DAYHOFF 1972), most transition-induced amber mutations, like those studied in this paper, have probably arisen from glutamine residues. However, amE552 displays heat-sensitive growth in an sus+ host (Table 7), suggesting that its glutamine-containing suppressed gene 55 product is temperature-sensitive. Thus, the non-temperaturesensitive wild-type gene 55 product probably does not contain glutamine at the site of the amE552 mutation. 4. The data in Table 7 question a previous suggestion (BOLLEet al. 1968; PULITZER and GEIDUSCHEK 1970) that the gene 33 product is not absolutely essential. This claim was based upon an observed leakiness of late transcription for five separately isolated gene 33 amber mutants, amN134, amE306, amE490, amC28, and amC22 (HOSODA and LEVINTHAL 1968; PULITZER and GEIDUSCHEK 1970). It was not realized that these five define but two distinct mutations (see Table 5), and it was assumed that since all were leaky, the function for which they coded was probably itself not absolutely required. However, an alternative explanation seems equally reasonable. Since the gene 33 function is catalytic ( SNUSTAD 1968), either slight misreading through the amber codon (STRIGINI and LEHMAN and BRICKMAN 1973) or an active amber fragment (FRIEDMAN 1974) or restart protein (PLATT et aZ. 1972) could cause the observed leakiness. According to these second possibilities, double mutations in the gene would be expected to eliminate the leaky phenotype; if the first explanation were true, a double amber would have no effect. Table 7 shows that, unlike the three leaky gene 33 single amber mutants, the double mutant amN134-amC28 does not grow at 42". This non-leakiness of the gene 33 double amber has been confirmed by examining its pattern of protein synthesis. Even very late after infection of B/r, its level of T 4 late protein synthesis is f a r below that observed with a single gene 33 amber mutant (T. POTEETE and R. HORVITZ, unpublished observations). Similar confirmation comes from

358

H. R. HORVITZ

experiments by NOTANI(1973), who has shown that this double amber allows a phage burst greatly reduced from that with either mutation alone. Since double mutations in gene 33 eliminate the leaky phenotype of single gene 33 mutants, that leakiness does not reflect dispensibility of the gene 33 function. 5 . The leucine-containing suppressed amE583 gene 33 product may inhibit the normally functional wild-type polypeptide. Gene 33 amE583 does not grow at 42" in the six6+ host XA106c, even though it is capable of some growth in the otherwise isogenic SLC XAlOOc (Table 7). This observation suggests that sus+amE583-gene 33 product may "poison" the otherwise functional wild-type polypeptide, as has been previously suggested for the 42O-tsA81-gene 55 product (PULITZER 1970). For both of these cases, one plausible mechanism is the saturation of RNA polymerase with the non-functional polypeptides, thereby preventing its interaction with the competent gene products available. 6. Increased temperature may decrease the need for functional gene 33 product. Two observations suggest this possibility. Firstly, all three single ambers show partial growth at 42O, but not at 34" or 23" (Tables 3 and 7), reflecting either increased synthesis of or decreased requirement for functional gene 33 product at the elevated temperature. Secondly, the phenotypic cold sensitivity of amNI34 in an sus+ host (Table 7) could result from a decreased need at higher temperatures of the suppressed polypeptide produced at a low level in an sus+ host. 7. The suppression data presented in Table 7 might facilitate genetic analysis of the interactions between the products of genes 33 and 55 and the host RNA polymerase. Three proteins (42"-su6+-amE583-gene33 product; suz+-amE552gene 55 product; and CR63-suI+-amE552-gene 55 product) may be present in high levels (see Table 2 ) and yet still non-functional (Table 7). Thus, these T4 strains may effectively carry missense mutations under these growth conditions and might be used to isolate host mutants which restore their abilities to grow. Since the products of genes 33 and 55 are known to interact with the host RNA polymerase (STEVENS 1972; HORVITZ 1973; RATNER1974b), one class of such host mutants might well be in a structural gene for RNA polymerase, possibly outside the one subunit (beta) for which genetic data currently exist (CHAMBERLIN 1974). I thank many individuals who supplied phage and bacterial strains for this work, particularly D. GANEMfor the complete set d isogenic suppressor host strains. I also thank DR. J. LEVY, T. POTEETE and DR. N.MAIZELS for assistance. This work was supported by a U.S. National Institutes of Health Training Grant to the author and National Institutes of Health Grant No. GM09541-12 to DRS. J. D. WATSON and W. GILBERT. L I T E R A T U R E CITED

ALLEN,E., I. ALBRECHT and J. DRAKE,1970 Properties of bacteriophage T4 mutants defective in DNA polymerase. Genetics 65: 187-200. BAKER,R. and I. TESSMAN, 1968 Different mutagenic specificities in phages SI3 and T4: in uiuo treatment with N-Methyl-N'-nitro-N-nitrosoguanidine. J. Mol. Biol. 35: 439-448.

MUTATIONS I N T 4 GENES

33

AND

55

359

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