Control of bacteriophage T4 DNA polymerase synthesis

J. 1Vol. Biol. (1973) 79, 83-94

Control of Bacteriophage T4 DNA Polymerase Synthesis MARJORIE RVSSEL t

D@artement de Biologic Mo&daire Universitt? de GenLve, Geneva, Switzerland (Received 12 February 1973, and in revised form 12 May 1973) Analysis of sodium dodecyl sulphate/acrylamide gels of 14C-labelled proteins from phage-infected bacteria suggests the existence of a self-regulatory control mechanism in bacteriophage T4. Infection of Escherichia coli with phage T4 carrying a mutation in gene 43 (which codes for the phage DNA polymerase) results in a greatly increased rate of synthesis of the gene 43 protein. Such overproduction of defective polymerase occurs in restrictive infections with all gene 43 amber and most gene 43 temperature-sensitive mutants tested. Gene 43 protein synthesis in gene 43+ infections or increased synthesis in gene 43- infections appears to require no additional function of other phage proteins essential for DNA synthesis. Functional gene 43 protein is needed continuously to keep its own levels down to normal. 1.

Introduction

The regulation of gene expression in bacteriophage T4 is known to involve a number of mechanisms which affect the timing of expression (Travers, 1970; Black & Gold, 1971). In addition, it is apparent that phage proteins which appear at about the same times are synthesized at greatly different rates (Hosoda & Levinthal, 1968 ; Laemmli, 1970), indicating that mechanisms affecting the rates of synthesis also exist. It seems likely that many of such rate differences can be explained on the basis of differences in promoter strengths (Brody & Geiduschek, 1970; Miller, 1970) or features of mRNA which determine translation rates. However, data presented in this paper suggest that phage T4 DNA polymerase, the product of gene 43, participates in regulating its own level. In infections with gene 43 mutants, the rate of accumulation of the defective product achieves 10 to 20 times the maximum rate for the wild-type protein 2. Materials and Methods (a) Bacterial strains collection of Dr R. II. Epstein was used as host bacterium in all experiments and as indicator for amber + phage. The permissive strain CR63 (SUP+ 1) was used as indicator for amber mutant phage. Escherichicz

co&i BE (SUP’) from the laboratory

(b) Bacteriophage The following T4D mutant phage from this laboratory were used alone and in various 43amB263, 43amB277, 43amA504, 43amS1252, 43tsP36, combinations : 43amB22, 41amN81, 4ZamN122, 44amN82, 45amE10, 33amN134, 55amBL292, 62amEA1140, and 32amA453. t Present address : Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Cola. 80302, U.S.A. 83

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M9S medium (Champe & Benzer, 1962) which contains 0.2% Casamino acids was used in the preparation of phage stocks and for the growth of host bacteria. Host cells, grown to no more than lO*/ml were centrifuged and resuspended in M9S medium containing only 10% of the normal concentration of Casamino acids. All experiments were carried out in this medium. Indicator bacteria were grown in Hershey broth (Steinberg & Edgar, 1962), and phage and bacteria were plated on Hershey bottom agar using EHA top layer agar (Steinberg & Edgar, 1962). (d) Experimental procedure In all but one experiment, host bacteria at 2 x lO*/ml were infected with phage at a multiplicity of infection of 5 to 15. At the times indicated in the text and Figure legends, 5 ml of infected cells were added to 0.5 ml of a mixture of 15 14C-labelled n-amino acids at 10 &/ml (from New England Nuclear). Four to five min later, 0.1 ml of 20% Casamino acids was added to terminate the labelling, and after a 4 to 5 min chase, the samples were put on ice. In the temperature-shift experiment, bacteria at 2 x log/ml were infected at 30°C at a m.0.i.t of about 5, superinfected at 4 min with the same phage of ensure lysis inhibition, and subsequently diluted tenfold into medium at 30°C or 42~5°C. (e) Radioactive &ate preparation The labelled cultures were centrifuged at low speed and resuspended in 0.26 ml of 0.01 M-Tris (adjusted with HCl to pH 7.5) and 1.0 ml sample buffer which contained: 10% glycerol, 5% mercaptoethanol and 3% sodium dodecyl sulphate in 0.0625 ivr-Tris (pH 6.8). Sample tubes were immersed in boiling water for 2 min and stored at -20°C. Just before electrophoresis, 20 ~1 of 100% mercaptoethanol were usually added to each sample, and the tubes were again immersed in boiling water for 2 min. Samples were kept frozen to avoid possible hydrolysis of the proteins and were used in general no more than twice. (f ) Gel electrophowsis Disk acrylamide gels containing 7.5% or 10% sodium dodecyl sulphate were prepared and run by the procedure of Laemmli (1970), except that different upper and lower electrode buffers were used. The upper buffer contained 3.0 g Tris*HCl, 14.4 g glyoine, 1.0 g sodium dodecyl sulphate, 2.4 mg sodium thioglycolate and 1.0 ml of O.l”/o bromophenol blue per litre. The lower buffer had the same composition except that the sodium thioglycolate and the bromophenol blue were omitted. Both buffers were at pH 3.5. Fractions of the lysate (0.1 ml) were layered onto each stacking gel, and electrophoresis was carried out with a current of 1 to 2 mA/gel until 30 to 60 mm after the dye marker had reached the bottom of the gel. The gels were treated overnight with 50% trichloroacetic acid to fix the proteins, stained for 1 h at 37°C with a fresh 0.1% soln of Coomassie brilliant blue in 50% trichloroacetic acid, and destained by repeated washings in 7% acetic acid containing 7.5% methanol. Gels were sliced longitudinally, and inner slices containing about one-third of the total volume of the gel were aligned on Mylar sheets so that the Coomassie blue banding patterns corresponded. The slices were then transferred to Whatman 3MM chromatography paper, dried and autoradiographed. Some samples were run on slab gels (Studier, 1973). Procedures were as described above, except that the upper electrode buffer was used in both wells, and only 20-9 samples were applied to each slot in the stacking gel. The electrophoresis was carried out at 20 to 30 mA/slab until about 15 min after the dye marker had reached the bottom of the gel, and gels were dried immediately, without prior fixation or staining. Stacking gels from disk gels were digested with Nuclear Chicago Solubilizer and counted in toluene/POP/POPOP scintillation fluid. They usually contained no more than 2% of the total trichloroacetic acid-precipitable counts added to the gel. Stacking gels from slab gels were dried and exposed with the entire slab. The amount of labelled material remaining in the stacking gel was always small with respect to the proteins of interest if DNA synthesis was not occurring during the labelling. Stacking gels contained more material when lysates, in which DNA synthesis was concurrent t Abbreviations

used: m.o.i., multiplicity

of infection; P43, product of gene 43, etc.

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with the labelhng, were run. In any event, the amount of material remaining in the stack ing gels is never enough to affect the conclusions of the various experiments.

-

(g) Determination of relative protein yields Medical, no-screen X-ray film (Kodak) was exposed to dried gel slices or slabs for 2 to 10 days and was traced with a Joyce-Loebl recording microdensitometer. The areas of the peaks corresponding to proteins of interest were determined vvith a planimeter. Although the specific activities of different lysates from a single experiment were usually about the same, data were standardized by normalizing peak areas to the number of trichloroacetio acid-preoipitable counts layered on to the gel. The peak area/counts per min has been taken as the average rate of synthesis during a given labelling period. The measurements of peak areas are subject to many possible sources of error including non-linearity of the film’s response to radioactivity, local variations in film background, low ratios of peak area to background area and lack of precision in planimeter determinations. An attempt was made to assess these measurement errors by plotting peak area versus time of exposure for many peaks over a large range of exposure times. Error was calculated as the percentage deviation of individual points from the straight lines generated by these data. It was determined from this analysis that the film response to the level of radioactivity was linear over the range of experimental conditions used and that other measurement errors are negligible. The average error in peak size determination is usually less than 3%. To determine the relative molar guantities of whole P43 and of the protein fragment produced in gene 43 amber mutant infections, it was necessary to correct the band intensity measurements described above for the difference in molecular weight of two products. The molecular weights of the amber fragments were determined by comparing their migration on the gel to the migration of the following gene products of known molecular weight: P37, 120,000 (King & Laemmli, 1971); P43, 112,000 (Goulian et al., 1968); P18, 69,000; P20, 63,000; P23, 56,000 and P23, 46,500 (Laemmli, 1970). For proteins of molecular weights above 50,000, the distance migrated varied linearly with the log of the molecular weight. The average deviation from a straight line generated by these proteins of known molecular weight is about 5%. Proteins of molecular weight less than 50,000 did not migrate as far as published values predicted, thus the molecular weight of the amS1252 fragment, which migrated in this non-linear area of the gel, is only approximate.

3. Results Soon after infection

by bacteriophage T4, a number of phage enzymes appear. Wiberg et al. (1962) have studied the synthesis of several of these early proteins by measuring the accumulation of enzyme activity. They have observed that synthesis normally continues for 10 to 20 minutes and then stops, and that this cessation requires phage DNA synthesis. These results are confirmed for one, and extended to another, early protein in the experiment shown in Figure 1. The rate of enzyme synthesis rather than the accumulation of activity wits measured in this experiment. Cells were pulse-labelled at various times during infection, and the relative amounts of protein synthesized during each pulse were determined from autoradiograms of sodium dodecyl sulphate/acrylamide gels (see Materials and Methods). As shown in Figure 1, the rate of synthesis of P46, a protein involved in host DNA degradation (Wiberg, 1966), and of P43, the phage T4 DNA polymerase (De Waard et al., 1965; Warner & Barnes, 1966), begins to decline after 7 minutes (at 42°C) in cells infected with wild-type phage. When DNA synthesis is blocked by infecting cells with a gene 44 mutant (amN82), synthesis of P46 and P43 fails to turn off. Similarly, as shown in Rgure 2(a), if DNA synthesis is blocked with mutants in gene 43 (tsP36 or amB263), P46 synthesis continues at about the maximum wild-type rate until at least 29 minutes at 42°C. In contrast, synthesis of defective gene 43 product of

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FIG. 1. Rates of synthesis of P43 and P46 in wild-type and gene 44 mutant-infected cells. E. wli BE at 1.9 x 108/ml were infected at 42°C with wild type at a m.o.i. of 15 or 44amN82 at a m.o.i. of 11 and labelled at various times with 14C-labelled amino acids. Each labelling was followed by a 4-min chase with unlabelled amino acids. Each point in the Figure represents the average rate of synthesis of a given gene product during a C-min pulse labelling. The rates are plotted at the mid-point of each pulse interval. (Additional experimental details and rate determinations are given in Materials and Methods.) --_n.-_n-, Wild-type infection; -A--A-, amN82 (44) infection.

5

IO 15 20 25 30 Minutes after Infection

FIG. 2. Rates of synthesis of P43 and P46 in gene 43 and gene 44 mutant-infected oells. E. coli BE at 2.5 x lO*/ml were infected at 42’C with 43amB263 (@), 43tsP36 (O), or 44amN82 (A) at a m.o.i. of about 10. Infected cells were labelled with 14C-labelled amino acids for 4 min, followed by a 4-min chase with m&belled amino acids. Rates (see Fig. 1 legend) are plotted at the midpoint of each pulse interval.

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occurs at continually increasing rates in these gene 43- infections (Fig. 2(b)). By 25 to 29 minutes at 42”C, the rate of synthesis of defective P43 is four to five times that of the wild-type product in the gene 44amN82 control infection. The accumulated molar yields of P43 were approximated by determining the area under each curve in Figure 2(b). By 29 minutes, gene 43--infected cells have accumulated about three to four times as much P43 as the DNA-defective control. These rate measurements may reflect the difference between rates of synthesis and degradation of P43. Goldschmidt (1970) has shown that nonsense fragments produced by amber and ochre mutants of the x gene of E. wli are rapidly degraded under conditions in which the wild-type protein, /3-galactosidase, is stable. Similarly, altered lac repressor, produced in E. coli strains carrying an i gene deletion, is less stable than wild-type repressor (Platt et al., 1970). If defective P43 is unstable relative to the wild-type protein, then the actual rate of its synthesis is higher than the rate measured. This possibility would not affect any conclusions presented here; on the contrary, instability of defective P43 would make the phenomenon reported here even more striking. The marked increase in the rate of P43 accumulation is henceforth referred to as “overproduction”. For infections in which the rate of P43 synthesis is similar to the maximum wild-type rate (regardless of whether turn-off occurs), synthesis will be called “normally regulated “. Since overproduction and normal regulation are easily distinguishable by 15 to 20 minutes, infected cells were labelled in a single pulse in most subsequent experiments. Plate I presents an autoradiogram of gels from cells infected with several gene 43 amber mutants. The high intensity of the band corresponding to the P43 fragment from each of the five gene 43 mutant infections is strikingly different from the low intensity of the P43 band in the gene 43+ DNA-negative control. This demonstrates that overproduction is not an allele-specific phenomenon. It can also be seen from this Plate that mutations in gene 43 have no effect on the intensity of any other bands.

TABLE 1

Relative yield of P43 fragments after infection with gene 43 amber mutants from Plate I

Mutation

Molecular weight?

amNS2 (43+ control) amB22 amB263 amB277 amA amS1252

112,000 92,000

74,000 66,000 49,000 30,000-40,000

Relative molecular weight

P43 (intensity)t

1.0 0.82 0.66 0.59 0.44 0.27-0.36

P43 (relative molar yield)

18.1 13.6 11.3 9.0

1-o

28 23 26 33-25

Infected cells at 30°C were labelled with 14C-labelled amino acids from 15 to 20 min. Labelling was terminated by the addition of unlabelled amino acids, and 5 min later the infected cultures were put on ice. t See Materials and Methods. $ The amB22 fragment was not sufficiently resolved from a neighbouring band to permit determination of the yield.

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The relative quantities of P43 labelled during these 15 to 20-minute pulses at 30°C are presented in Table 1. In the last column the relative molar yield (see Materials and Methods) for each P43 species is known; it is clear that the same number of fragments are labelled in each gene 43- infection. The fragments labelled during this pulse period are synthesized in 25fold molar excess over P43 in the gene 43+ control. It should be emphasized that these values indicate rate differences rather than differences in total accumulation. Seven of eight gene 43 temperature-sensitive mutants tested at the non-permissive temperature made P43 at an elevated rate (data not shown except for tsP36 in Figure 2(b)). Thus, overproduction is not limited to infections with amber mutants. Overproduction at 42°C is less than at 30°C. Furthermore, it is consistently less in temperature-sensitive than in amber mutant infections. The latter difference is probably not due to greater leakiness of the temperature-sensitive mutant for DNA synthesis because tsP36-infected cells stop DNA synthesis immediately upon transfer to the restrictive temperature (Riva et al., 1970; M. Russell, unpublished results). However, it may be that at high temperature the inactive P43 retains some regulatory function, suggesting that the role of functional P43 in DNA synthesis and in selfregulation may be separable. One temperature-sensitive mutant did not overproduce P43. In fact, this mutant, tsA58 (Allen et al., 1970), made no detectable P43 at the restrictive temperature. The nature of this mutant is being investigated. TABLET Relative yield of P43 after infection with DNA-defective mutants Mutation amA amN81 amN122 amB263 rtmN82 amEl a.mN81-itmN122-amN82-amEl

Gene 32 41 42 43 44 45 41-42-44-45

P43 (relative molar yield) 0.97 1.0 0.97 14.0 1.0 0.94 l.lT

Infected cells at 30°C were labelled with 14C-labelled amino acids from 15 to 20 min. L&belling was terminated by the addition of m&belled amino aoids, and 5 min later the infected cultures were put on ice. 5 This represents about 2% of total protein as determined from the densitometer tracing of an entire gel.

The effect of mutations in several early genes required for DNA synthesis on the rate of P43 synthesis is demonstrated by the data shown in Table 2. Bacteria were infected at 30°C with phage carrying amber mutations in genes 32, 41, 42, 43, 44 or 45 and labelled from 15 to 20 minutes. P43 is not overproduced in the presence of DNA-negative mutants other than those in gene 43. Thus overproduction is due to the specific absence of functional P43, and none of the products of the genes tested is required to maintain synthesis at a normal rate. The multiple mutant, 41-42-4445-, was also tested. It was imagined that the proposed DNA replication complex,

am s 1252

am A504

am B217

am B263

am

822

controt (gene43+)

PLATE I. Autoradiograms of 10% sodium dodecyl sulphate/polyacrylamide gels of phage ‘r4 early proteins. Arrows indicate the position of wild-type P43 from an amN82 (gene 44) control infection and of P43 fragments from infections with several gene 43 amber mutants. Infeoted cells at 30°C were pulse-labelled with 14C-labelled amino acids from 15 to 20 min after infect,ion. For additional d&ails, see Materials and Methods.

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which is thought to include the products of these genes (Barry eital., 1973; Chiu & Greenberg, 1968), might play a role in the regulation of P43 synthesis. It seemed possible that a complex missing only one of the small component proteins might continue to regulate normally, but that the elimination of several of these proteins might lead to overproduction of the wild-type gene 43 product. This was not the case. Thus regulation of P43 is not mediated by the proposed complex. It is, of course, still possible that the product of a gene which has not been tested is required for the regulation of P43. The data in Table 2 show that P43 synthesis occurs at wild-type rates in infections with phage carrying mutations in genes other than gene 43. Similarly, one can ask if overproduction of the P43 fragment in gene 43 amber mutant infections requires these functions. Bacteria were infected at 30°C either with an am‘ber double mutant (43-X-) or a single mutant (43+X-) and labelled from 15 to 20 minutes. The data in Table 3 show that none of the mutants (in genes 32, 33, 41, 42, 44, 45, 55 or 62)

TABLE 3

Relative yield of P43 fragments after infection with multiple mutants

Genes

Mutations

amA453-amB263 amA amN13PamB263 amN134 amN8l-amN122-amB263-amN82 rtmNSl-rtmN122-amN82-amEl amBL292-amB263 amBL292 amlZA1140-amB263 amEAl amN82

-amElO

32-43 32 33-43 33 41-42-43-44-45 41-42-4&45 55-43 55 62-43 62 44

P43 (relative molar yield) 21.0 1.4 21.0 0.9 19.0 1.1 19.0 l-5 20.0 1.2 1.0

Infected cells at 30°C were labelled with 14C-labelledamino acids from 15 to 20 min. Labelling was terminated by the addition of m&belled amino acids, and 5 min later the infected cultures were put on ice.

eliminate P43 overproduction when coupled with the gene 43 amber mutant. Since none of the gene 43+X- controls overproduce P43, the products of genes 33, 55 and 62, in addition to those listed in Table 2, are not required for P43 regulation. The capacity of infected cells to overproduce P43 is dependent only on the absence of functional P43 and not on the function of the other genes tested. The temperature-shift experiment presented in Figure 3 was done to determine whether functional P43 is required continuously to maintain synthesis at the wildtype rate. Bacteria were infected at 30°C with a gene 43 temperature-sensitive mutant, tsP36, or with wild-type phage. Each culture was shifted to the restrictive temperature at 18 minutes after infection. Portions of each culture were pulse-labelled at two different times, both before and after the temperature shift. At’ 3O”C, both tsP36 and wild type synthesized P43 at nearly the same rate (Fig. 3(b)). In both infections,

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3 12,z z3 10.3

0’

;

6-

30°C

Mmutes

E’IG.

offer

infec!lon

3. Rates of synthesis of P43 and P46 at 30°C and after shift to 42.5°C.

E. coli BE at 2 x log/ml were infected with wild type at & m.o.i. of 8 and 43 tsP36 phage at a m.o.i. of 5 at 30°C. Each culture w&s super-infectedwith the same phage at the same m.o.i.‘s at 4 min. Portions of the infected cultures were diluted tenfold into medium at 30°C and labelled at 30°C in two different 4-min pulses. Additional portions of the infected cultures were diluted tenfold into medium at 42.5T at 18 min and labelled in two different 4-min pulses. Rates of synthesis of P43 and P46 are plotted at the beginning of each pulse interval. Each 4-min pulse was terminated by the addition of excess m&belled amino acids. (e), tsP36 (43); (0) wild type.

less P43 was labelled during the second 30°C pulse than the first because of the normal DNA synthesis-dependent turn-off that occurs after 15 minutes in wild-type infections. After the shift to the restrictive temperature at 18 minutes, the rate of P43 synthesis in wild type remains very low. In contrast, P43 is overproduced following transfer of tsP36-infected cells to the restrictive temperature. Similar results were obtained when amN82-tsP36, which does not synthesize DNA at either temperature, was shifted to 42°C at a late time (data not shown). Figure 3(a) presents data of the synthesis of another early protein, P46, from this experiment. The arrest of DNA synthesis following shift of tsP36-infected cells to the restrictive temperature has no effect on P46 turn-off. Other bands identified as early proteins show qualitatively similar behaviour. After the temperature shift, a few bands become more intense in the tsP36 infection than in the wild-type control. The identities of these bands have not been rigorously established. However, their behaviour suggests that they may be unprocessed precursor proteins (Laemmli, 1970) which accumulate in the absence of continued DNA synthesis. In any event, these observations indicate that for most, if not all early proteins, normal turn-off is not reversed when DNA synthesis is blocked at a late time. The mechanism leading to overproduction of defective P43 therefore appears to be independent of the normal turn-off of early proteins. The observation that P43 is overproduced following denaturation of the enzyme (and consequent arrest of DNA synthesis) indicates that functional P43 is required continuously to prevent the high rate of synthesis characteristic of gene 43 mutant infections.

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4. Discussion Expression of gene 43 involves a number of separate mechanisms. The first appearance of gene 43 product, like that of many early genes (H’osoda & Levinthal, 1968), may depend on the distance of gene 43 from a promoter (Brody et al., 1971; Jayaraman, 1972), on modification of the host RNA polymerase (Goff & Weber, 1970; Schachner 8t Zillig, 1971), and on the appearance of a phage T4 transcription factor (Travers, 1970). Subsequently, shortly after the onset of DNA replication and at about the time when late genes begin to be expressed, synthesis of P43 and other early proteins diminishes (Hosoda & Levinthal, 1968). This early turn-off is dependent in some way on DNA synthesis (Wiberg et al., 1962; Young & van Houwe, 1970), but otherwise little is known about it. In addition to these general controls on temporal expression, it now appears possible that the gene 43 product is specifically involved in determining the rate of its own synthesis. The experiments reported here demonstrate that restrictive infections with amber or temperature-sensitive mutations in gene 43 synthesize the defective product of this gene at unusually high rates. Rough estimates from a variety of pulse-labellings suggest that by 30 minutes at 30°C gene 43- infections accumulate as much as 10 to 20-fold more P43 than a DNA-negative gene 43+ control. These observations may have some bearing on some unusual results obtained in other experiments. Karam (personal communication) has observed that the transmission coefficient of gene 43 amber mutants on sup’ strains exhibiting ribosomal ambiguity (ram) suppression is very high, while on &f derivatives, apparently lacking this suppression ability, it is very low (low transmissions on both sup0 hosts were observed for amber mutants in many other genes). This high transmission frequency of gene 43 mutants has been interpreted to indicate that extremely small amounts of active P43 are sufficient for function. However, overproduction undoubtedly contributes to these high transmission values: P43 fragments can accumulate in large excess during infection of a sup0 host with gene 43 ambex mutants, and the number of complete chains probably increases by a similar factor. If this is the case, it is not clear that the substantial function that occurs does so with unusually small amounts of active P43. Karam (personal communication) obtained similarly high transmission frequencies on ram hosts with amber mutants in gene 62, and preliminary evidence (Russel & Kirsch, unpublished observations) suggests that defective P62 may be overproduced during a gene 62 amber mutant infection. Neither P41, P42 nor P45 are overproduced during infection with phage carrying mutations in those genes (Krisch, unpublished observations). This is consistent with Karam’s observation (personal communication) that gene 42, and probably 41 and 45, amber mutants have low transmission coeillcients on both types of sup” host. Gene 32 mutant infections do overproduce P32 (Krisch, unpublished results) but do not have the high transmission coefticients of gene 43 mutants on the ram hosts. However, independent evidence indicates that P32 is required in much larger quantities than P43 (Alberts & Frey, 1970; Sinha & Snustad, 1971). Thus it is unlikely that an increase in the number of complete chains of P32 would lead to striking increases in the transmission frequency of gene 32 amber mutants. It is not yet known whether protein overproduction reflects events originating at the level of transcription or translation (although the question is currently being investigated). If gene 43 mRNA is synthesized at increased rates, one would expect

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overproduction of the products of any genes co-transcribed with gene 43. P42 and P62, the products of two genes closely linked to gene 43 and 42, are not overproduced. This is consistent with Stahl’s observation (1970) which suggests that genes 43 and 42 are not co-transcribed. Furthermore, it predicts that gene 62, which Stahl did not examine, is not co-transcribed with gene 43 . Although much additional information will be necessary to understand P43 overproduction, it may be helpful to consider two general models. One possibility is that functional P43 directly represses its own synthesis ; that is, in the gene 43 mutant infections examined here, P43 is overproduced because the defective product is unable to act as a repressor. According to this model, in wildtype infections P43 would reach the level required for maximal repression shortly after its synthesis begins. Early turn-off would be a separate mechanism which reduces the level of expression of early genes and which is “superimposed” on the gene 43 self-regulation system. The result of the temperature-shift experiment (Fig. 3), which demonstrates that functional P43 is needed continuously to maintain the wildtype rate of synthesis, would be due to “derepression” as an immediate consequence of P43 inactivation. The constant rate of synthesis seen in the infection with a DNAdefective mutant in genes other than gene 43 would then be the maximally repressed level not influenced by shut-off. Other aspects of these results, however, are somewhat more difficult to explain on this simple repressor model. The maximally repressed rate of P43 synthesis is rather substantial, suggesting that additional factors would be involved in the extent of repressor binding. Likewise, in gene 43- infections (Fig. 2), the long period during which the rate of P43 synthesis is continually increasing is unexpected. Here again, additional factors involved in determining the capacity to synthesize the defective product would have to be invoked. Another possible mechanism is that P43 overproduction arises indirectly from the failure of the defective product to function, i.e. that functional P43 is not under active self-regulatory control, but that in its absence synthesis of P43 is abexrant. The phage T4 DNA polymerase has been shown to be capable of several functions involving DNA synthesis or degradation (Epstein et al., 1963; Baldy, 1968; Nossal, 1969). A defect in any one of these functions might be the basis for P43 overproduction. For example, if P43 functions in initiating new rounds of DNA replication at an origin, located near gene 43 (Mosig, 1970), its absence might open this region to a high rate of transcription. Alternatively, the failure of the enzyme to carry out a repair function might result in the accumulation of nicks in the DNA. If such nicks could be sites for additional transcriptional initiations, the increasing rate of P43 overproduction (Fig. 2) could be due to the accumulation of nicks in the vicinity of gene 43. Nicks might accumulate uniquely near the replicative origin, or they might occur elsewhere as well but ineffeotively. The conclusion that functional P43 is required continuously to avoid overproduction (Fig. 3) would mean that nicks (or whatever might be the actual locus for P43 regulation) must be continually created. Similarly, the continued synthesis of P43 in DNA-defective gene 43’ infections would be due to a steady state between creation and repair of the sites. Enzyme overproduction due to the presence of mutations in the corresponding structural gene has been observed for the histidine repressor in Sal?nonella typhimu&urn (Smith & Magasanik, 1971), di-hydrofolate reductase in Diplococc~ pneumo-

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(Sirotnak, 1971), and for glucose 6-phosphate dehydrogenase (Yoshida, 1970) and pseudo-choline esterase (Yoshida & Motulsky, 1969) in humans. The fact that in phage T4 this phenomenon seems to be restricted to two or perhaps three early proteins (P43, P32, and perhaps P62), and that these proteins are all essential for DNA synthesis, suggests that this overproduction has biological significance. Such overproduction might increase the capacity for phage DNA replication under a variety of conditions limiting either the activity of these proteins or the level of protein synthesis in general.

niae

This research was supported by the Ponds National Suisse de la Recherche Scientifique. no. 3.597.71. I am extremely grateful to H. Krisch, T. Mattson and especially R. H. Epstein for many valuable discussions and for help with the preparation of the manuscript.

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