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DNA packaging in the lambdoid phages: Identification of the products of φ80 genes 1 and 2
VIROLOGY
111,629-641
(1981)
DNA Packaging in the Lambdoid Phages: Identification of the Products of $80 Genes 7 and 2 M. SUMNER-SMITH’ Department
of Medical
Genetics, Toronto,
AND A. BECKER
Medical Ontario
Accepted
Sciences Building, M&S lA8, Canada
January
University
of Toronto,
14, 1981
We have identified the products of genes 1 and 2 of the lambdoid coliphage 480. These genes code for the subunits of the terminase enzyme that promotes DNA packaging during phage particle assembly. These data serve by analogy to identify the subunits of h terminase as the products of Nul and A, respectively. Identification of the gene 2 product was aided by the isolation of a mutant which causes the overproduction of that polypeptide. Mutants which affect the mobility of the product of gene 1 on 12.5% SDS-polyacrylamide gels were isolated as pseudorevertants of two amber mutations in the gene. Certain of these gene 1 pseudorevertants affect the mobility of the product of gene 2. The hypothesis that these pseudorevertants arise by recombination with a cryptic prophage carried by the host is proposed. The possibility that genes 1 and 2 overlap out of phase for a portion of their coding sequences is discussed. Surprisingly, wild-type 480 produces approximately 280-fold more of the gene 1 than gene 2 product. INTRODUCTION
During the final stages of assembly of a lambdoid phage particle, a monomer length of phage genome is cut at specific cos sites from replicated, concatemeric DNA and packaged into an empty prohead (for reviews see: Hohn and Katsura, 1977; Murialdo and Becker, 1978). An enzyme, called terminase, has been identified and purified from h-infected Escherichia coli cells, which is capable of cleaving DNA endonucleolytically at cos sites and also of promoting packaging in vitro. This terminase has recently been shown to consist of two subunits (M. Cold and A. Becker, in preparation), one of which has previously been identified as the product of A gene A @A) (Becker and Cold, 1975; Becker et al., 1977). The other subunit has not been identified. Active terminase has not been detected in extracts of cells infected with UVulor AAam phage (Becker et al., 1977), but is present in extracts infected with gal trans1 Author dressed.
to whom reprint
requests should be ad629
ducing phage from which all of the morphogenetic genes except Nul and A have been deleted (Becker and Cold, 1975). Recently we have shown that active terminase is produced when XNul - and L4am phage-infected extracts are mixed in vitro (SumnerSmith et al., 1981). Thus it appears likely that the second subunit of terminase is the product of Nul (gpNu1). The work reported here was aimed at confirming this possibility. Unfortunately, the only mutation available in the gene Nul (i.e., Nuldef,,,) is neither a nonsense nor a missense mutant and we have been unable to identify gpNu1 in radiolabeled A-infected extracts by SDS-polyacrylamide gel electrophoresis using this mutant. Weisberg et al. (1979) have shown that genes 1 and 2 of the lambdoid phage 480 are homologous in function to genes Nul and A, respectively. Furthermore, h and 480 are homologous by heteroduplexing in the region of their genomes which code for these genes (Fiandt et al., 1971). Since several nonsense mutations are available in each of genes 1 and 2 (Sat0 et al., 1968; Youderian, 1979) we decided to see if we 0042-6822/81/080629-13$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
630
SUMNER-SMITHANDBECKER
would be more successful in identifying the product of gene 1 (gpl) than we had been in identifying gpNu1. We report here the identification of the products of 480 genes 1 and 2, and show that these correspond to the two subunits of A terminase. Our ability to identify gpl, but not gpNu1, seems to be due to the fortuitous lack of other @O-induced proteins of a similar electrophoretic mobility to gpl, and also possibly to the unexpectedly high level of synthesis of gpl. MATERIALSANDMETHODS
with phage at an m.o.i. of 0.01-0.1. Cultures were grown with shaking at 37” until extensive lysis was observed, a few drops of chloroform were added, and the debris was sedimented by centrifugation and the supernatant retained. Lysates were dialyzed extensively against A diluent before being used for labeling purposes (see below). When required, phage lysates were concentrated as described by Murialdo et aE. (1980). Phage were plated on the appropriate indicator (0.2 ml of a late log culture) strain in 2.5 ml melted top agar over 35 ml solid bottom agar.
Media and buffers. L broth is 10 g Bacto Preparation of labeled extracts of inTryptone (Difco), 5 g yeast extract, and 5 g fected cells Cells were grown in RM meNaCl per liter of distilled H,O which is neu- dium at 37” to a density of 2.5 x lO*/ml, tralized with NaOH. RM medium is 18.7 centrifuged, and resuspended at a concenmM NH&l, 1 mM M&SO,, 20 mJ4 KCl, 0.1 tration of 2.0 x log/ml in fresh RM. A thin 3 pM FeCl,, 49.4 n-&f layer of cell suspension was irradiated with ml4 CaCl,, Na2HP04, 22 mM KHzP04, 22 mM glycbetween 5000 and 6000 ergs/mm2 of ultravierol, and 0.4% maltose. Lambda diluent (A- olet light. The cells- were then added to an dil) is 10 n-&f MgS04, 10 n-&f Tris-HCl, pH equal volume of infecting phage diluted in 7.4. The solid medium used for bacterial A-dil to 1.0 x 10lO/ml and held on ice for 15 colony formation and phage plaque as- to 30 min for adsorption. Four volumes of says has been described (Campbell, 1961; prewarmed RM were added and the mixture incubated for 35 min at 37” with shakMurialdo and Siminovitch, 1971). of L-[35S]methionine Buffer A is 20 mM Tris-HCl (pH 8.0), 1 ing. About 42 &i/ml mM EDTA, 3 mJ4 MgCl, ,5 n-&f 2-mercap(New England Nuclear) or 15 &i/ml of Ltoethanol. Buffer B is 10% sucrose in 0.05 [2, 3-3H]arginine (New England Nuclear) M Tris-HCl (pH 7.4). SM 1 solution is 60 or 5 &i/ml of L-[‘4C]amino acid mixture n&f spermidine, 18 m&f MgC&, 15 mil4 (Amersham) were added and incubation ATP, 30 mM 2-mercaptoethanol in 5 n-&f continued for a further 10 min. Incubation Tris-HCl (pH 7.4). was terminated by cooling the mixture in Strains. The E. coli K12 derivative an ice bath. The cells were harvested by strains used are shown in Table 1. 159supF centrifugation and resuspended in 0.4-vol sample buffer (Laemmli, 1970), placed at was made by selecting for trp+ cells after Pl transduction from QD5003 trp+ supF 100” for 1 min, vortexed, and kept at -20” into a 159A(tonB-trip) derivative. The pres- until thawed for application to a gel. ence of the supF marker was confirmed by Gel electrophoresis. The discontinuous the ability of the strain to suppress the SDS-polyacrylamide gel system Laemmli Sam, mutation in A. 480 strains carrying (1970) was used with a stacking gel of 5% and a separating gel of 12.5%. The reserlam, (Sato et al., 1968>, 1am468, lam,,,, and lam,,,, and Zam,,, (Youderian, 1979) voir buffer was supplemented to 4 mM were the kind gift of Dr. P. Youderian. mercaptoethanol. The gels were run overAimm434TcI,00 (Murialdo et al., 1980) was night at 95 V. After electrophoresis gels used to prepare DNA by phenol extraction were impregnated with 2,5-diphenyloxa(Thomas and Abelson, 1966). zole for fluorography (Bonner and Laskey, 1974), dried under vacuum, and used to exPreparation of phages. Phage lysates with a titer > 101O/ml were routinely pre- pose X-ray film (Kodak, SB-5). Scanning pared by infecting a culture of T5-2 (for was performed in a Gilford spectrophotometer provided with a linear transport and amber phage) or 594 (for nonamber phage) at a concentration of 107-log/ml in L broth the area under the appropriate peaks was
&80 TERMINASE TABLE BACTERIAL
strain T52 594 TC600 XAlOC QD5003 159 WC5043 WC5041 159 supF W3350 (LVuldef,,l&&‘am7
631
GENES 1 AND 2 1 STRAINS
Relevant genotype supE SUP0 supE supc supF gal-, str’, str’, gal-,
tonA
(lacks @30 receptor)
str’, uwrA-, WV-A-, leu-, UWA-, leu-, str’, uvrA-,
sup’ supD supE supF
)
Reference Fuerst (1966) Weigle (1966) Harris et al. (1967) Miller et al. (1977) Yanofsky and Ito (1966) Ptashne (196’7) From M. Howe From M. Howe This work Murialdo and Siminovitch (1972); Weisberg et al. (1979)
594 (AAam,,~am,,cI~~am,)
NS428
sup”, recA
(hAam,,bzredJcIs5$amr)
measured by weighing after they had been cut from the plot. Care was taken to ensure that the optical density of the bands was within the linear response range of the film. Molecular weights of 480 gene products were determined by comparison with a parallel X-infected extract using the published values of A gene products (reviewed by Szybalski and Szybalski, 1979). In vitro packaging of DNA. The twostage packaging system of Becker and Gold (1975) was used. Preparation of stage 1 extract of A lysogens: 400 ml of L broth was inoculated with the indicated strain and the cells were grown at 32” in a shaker to a cell concentration of about 7 x lO’/ml. For thermal induction of the prophage, the cells were incubated with shaking at 70” until the temperature of the cell suspension reached 45”. Subsequently, the flasks were shaken at 45” for 13 min followed by cooling in an ice/salt/water mixture until the temperature dropped to 37”. The cells were incubated at 37” for another 40 min; they were then chilled and spun down at 13,000 g for 10 min. The pellets were resuspended in 0.5 ml of buffer A. Before sonication the volume was adjusted to about 4.0 ml with buffer A. Sonication of the concentrated suspension was done by two pulses of 10 set each in an ice/salt bath. The sonicates were cleared by centrifugation at 4000 g for 10 min.
Becker et al. (1977) Sternberg, Tiemeier, and Enquist (1977)
Preparation of stage 1 extract of cells infected with 480: 300 ml of L broth were inoculated with strain 594 and the cells were grown at 37” in a shaker to a cell concentration of 1.4 x W/ml, pelleted by centrifugation at 13,000 g for 10 min, and resuspended in 42 ml of fresh L broth at O-4”. Purified $80 phage (as indicated) were added to a multiplicity of 5 and the mixture held on ice for 15 min for phage adsorption. Five hundred milliliters of L broth prewarmed to 37” was added and the mixture shaken at 37“ for 40 min. The cells were then pelleted and treated as for an induced A lysogen as described above. Preparation of stage 2 extracts: these extracts were prepared by the lysozymefreeze-thaw method first described by Kaiser and Masuda (1973). The lysogen NS428 was grown in 600 ml L broth, induced, and the cells collected by centrifugation as described above for the preparation of the sonicates. The pellets were subsequently resuspended in 0.8 ml of buffer B. The final volume of this mixture was about 1.5 ml. To 1 ml of this suspension 0.05 ml of a lysozyme solution (2 mg/ml in 0.25 M Tris -HCl, pH 7.4) was added, and this was followed by freezing in liquid N, and shaking in a water bath at 37” just to the point of complete thawing. The viscous extract was incubated on ice for 45 min, then 100 ~1 SMA solution was added.
632
SUMNER-SMITH
AND BECKER
FIG. 1. Physical map of the head region of h and 480. Regions of homology between A and 480 (Fiandt et al., 1971) are shown by stippling. The sizes and positions of the A genes A, W, B, C, Nu3, D, E, FI, and FZZ are taken from the review of Szybalski and Szybalski (19’79). The order of the 480 genes 1,2,S, &20, and 5 and the sizes of S,4, and 20 are taken from Youderian (1979); their positions are assumed to be the same as the corresponding A genes. The size of genes 1 and 2 are as determined here. The size of the Nuf gene is taken as that of the smaller subunit of A terminase (Cold, M. and Becker, A., in preparation).
The mixture was then centrifuged at 35,000 rpm in the 50 Ti rotor of the Spinco ultracentrifuge for 30 min. The supernatant was used as the stage 2 extract, providing gpD, pgW, gpFI1, and tail function to the assay (Becker et al., 19’7’7). The assay: 4 ~1 of a solution (185 kg/ml) of &nm434TcZ~oo DNA, 4 /.J of SMA solution, and where indicated, 4 ~1 of partially purified terminase (AsI; approximately 1 unit/ml) (Becker and Gold, 1975), 20 ~1 of stage 1 extract, and 30 ~1 of buffer A were incubated at 22” for 15 min. One hundred and fifty microliters of stage 2 extract was added and incubation continued for 60 min at 22”. The phage yield was titered on strain TC600 (supE, tonA). None of the phage used to produce the packaging extracts will plate under these conditions since the h phage carry the Sam, mutation, which is not suppressed by supE and the 480 phage are not adsorbed since TC600 carries the tonA mutation, and is therefore resistant to hmsobut not h* particles. Thus, only particles assembled in vivo from the exogenously added imm434Tc1700 DNA, X or 480 proheads supplied by the stage 1 extract and A tails supplied by the stage 2 extract (Becker et al., 1977) will score by producing a plaque. The results reported here, therefore, confirm the observation of Inokuchi and Ozeki (1970) that 480 DNA-filled
heads will
efficiently
accept X tails
in
vitro. RESULTS
Failure to Identify Nul
the Product
of X Gene
We attempted to identify the Nul gene product by comparing pulse-labeled extracts of cells infected with A phage carrying the absolutely defective Nul mutation t16 to wild-type and t16 revertant phage, by both one-dimensional SDS-polyacrylamide gel electrophoresis and by isoelectric focusing followed by SDS-polyacrylamide gel electrophoresis. We could find no consistent difference between these extracts, however. The $80 gene 1 is homologous to the A gene Nul by heteroduplexing (Fig. 1, Fiandt et al., 1971) and its product can efficiently substitute for gpNu1 in vivo. Since several amber mutations are available in gene 1 (Sato et al., 1968; Youderian, 1979) we decided to see if we could identify the product of that gene. Preliminary experiments comparing 4801 am to l+ infected cell extracts were encouraging. We thought it necessary, however, to first establish that genes 1 and 2 were essential for terminase activity in $80 and also to iden-
&30 TERMINASE
tify gp2, whose A analog (Fig. l), gpA, has already been identified (Murialdo and Siminovitch, 1972a). Lambda
Terminase
GENES 1 AND 2
633 TABLE
&30lam
2
AND%~-INFECTED EXTRACTS DEFICIENT IN TERMINASE
Can Substitute for +?O
ARE
Yield (PFU/ml)
Genes 1 and 2 in vitro Stage 1 extracts prepared from induced A lysogens lacking either Nul or A have been shown to be unable to package exogenous X DNA efficiently in an in vitro packaging system (Becker et al., 1977). This deficit can be overcome by adding partially purified terminase from an induced lysogen lacking all of the morphogenetic genes except Nul and A. We have repeated the above observations (Table 2, lines 3 and 4) and have extended them to show that extracts prepared from cells infected with +SOuir phage carrying either lam or 2am mutations have a similar defect which can be corrected by adding h terminase (Table 2, lines 1 and 2). Thus we conclude that the &30 genes 1 and 2 are essential for endogenous terminase activity and that X terminase is sufficiently similar to substitute for it. Zdentijication
of the Product
of @O Gene 2
A band gpA (i.e., tracts of (Fig. 2,
of the same molecular weight as A ‘79 kdalton) was observed in excells infected with r#doUir phage track c), but was reduced in @&?am,,,vir infected cells (Fig. 2, track b). A host-encoded polypeptide has the same mobility as gp2 (Fig. 2, track a) so that it is not surprising that there is evidence of a faint band remaining after gp2 has been eliminated by a nonsense mutation. Since the amount of putative gp2 made is apparently so small and identification therefore difficult, we attempted to isolate a mutant which would increase the synthesis of the gene 2 product. Selection of an Overproducer Gene 2 and Conjkrnation jkation of gp2
Mutation in, of the Identi-
Recently Murialdo et al. (1980) have isolated a mutation in X which increases the rate of synthesis of the A gene product
Stage 1 extract lam,
2amrss
Nuhf,,,s Aamal,Dam16
-terminase 2 3.6 1.3 4
x x x x
10’ 102 109 10’
+ terminase 3.1 1.6 5.4 3.7
x x x x
10’ 10’ 10’ 10’
Note. Stage 1 extracts were prepared by infecting 594 (sup”) with ~8Olam& of &302amulsvir or by inducing W3350 (ANuld.eftl~I~$am,) or 594 (hAam.,r Dam,&,,Sam,) and used as described under Methods.
(Mop,).
It was found that although a phage is able to plate on a host carrying supE, it is unable to plate on a host carrying supE in which the efficiency of suppression is reduced by a streptomycin mutation. Presumably the yield of phage is limited by the amount of A polypeptide synthesized, and this amount is insufficient in a host carrying a weak suppressor to give a plaque. The op, mutation was selected by picking a phage that still carried the amber mutation, but was able to plate on the “weak suppressor” host. It is thought that the op, mutation increases the efficiency of translation of the A gene product. We reasoned that it should be possible to isolate a similar mutation in $30 gene 2, which would then aid in identifying the gene 2 polypeptide. Although cells carrying either supF or supC suppress amber mutations by inserting tyrosine, their efficiencies of suppression differ: 50 vs 16% (Gorini, 1970). We found that @302am,,,vir was able to plate on QD5003 (supF), but not on XAlOC (supC)(Table 3). A spontaneous mutant was picked which was able to plate on XAlOC, but not on 594 (sup) since it still carried an amber mutation which was unable to complement the parental amber phage. The new mutant phage was designated as +802am,,90p,,,vir. Three spontaneous amber revertants (designated as @02op~~uir, revs. 1,2, and 3) to the mutant XAam,,,
634
SUMNER-SMITH TABLE
AND
3
PLATING OF F’I-IAGE CARRYING AMBER MUTATIONS IN GENES I AND 2 QD5003 (supF)
+8oUir $801 amUlsvir 4801 am,,Bvir Q802amlasvir W@amwvwir
1.0 1.0 1.0 1.0 1.0
XAlOC (supc
)
1.0 0.7 0.9 1.3 x 10-a
0.7
594 (SUP0 )
0.9 9.7 x 3.6 x 7.1 x 3.6 x
10-1 lo+ lo-’ lo-’
Note. The efficiency of plating (e.o.p.) of the designated phage strains on the indicated hosts relative to the permissive host QD5003 is shown.
were isolated. These amber revertants were shown to overproduce the putative gene 2 polypeptide (Fig. 2, tracks d, e, and f, and Table 4). The op10 mutation when combined with the Zam,,, mutation increased the synthesis of an induced polypeptide of lower molecular weight than gp2, which is presumably the “amber fragment,” following infection of a sup0 cell (Fig. 2, track g). This is confirmed by the observation that both the putative amber and wild-type polypeptides are induced when +302am,,,op,,vir infects a cell carrying a suppressor (Fig. 2, track h and Fig. 3, track k). Identifiation
of the Product
of @O Gene 1
A polypeptide with an apparent molecular weight of 20.5 kdalton which was present in +SoVir and @Mam,,,vir-infected cells was absent in cells infected with phage carrying four different gene 1 amber mutations (ambers 3, 468, 516, and 576) when labeled with [35S]methionine. Several hostencoded bands, however, made positive identification difficult. Preliminary attempts to separate the putative gpl from the host polypeptides by isoelectric focusing suggested that it was very basic; in agreement with this observation, labeling with [3H]arginine instead of [35S]methionine was found to greatly enhance the putative gpl band relative to the other bands of similar mobility. Figure 3 shows that the putative gpl polypeptide is present in l’-
BECKER
infected cell extracts (Fig. 3, tracks a, j, and k), but absent in lam,,, (Fig. 3, track b) and lam,,,-infected sup0 cell extracts (Fig. 3, track f). The gpl polypeptide is partially restored when the lam,, and 1 am576 phage were used to infect supD (Fig. 3, tracks c and g), supE (Fig. 3, tracks d and h), and supF (Fig. 3, tracks e and i) cells. Overproduction
of gpl Relative
to gp2
There appeared to be considerably more gpl present than gp2 after 10 min of labeling of a +80uir infected cell extract (see for example, Fig. 3). This result is somewhat surprising since gpl and gp2 are thought to be subunits of terminase. We noticed, however, that while a phage carrying a 2am mutation cannot plate on a host carrying only an ocher suppressor unless it is combined with the overproducer mutation (op,,), two different amber mutations in gene 1 are suppressed sufficiently by the same ocher suppressor to yield a visible plaque (Table 3). These data suggested that gpl is made in excess relative to gp2 by wild-type phage. In addition, we found that mutants which produced considerably less gpl than wild type were still fully viable (see below). In order to assess the relative production of gpl and gp2, 48&r-infected cells were labeled with an Q4C-amino acid mixture (as described under Methods) so as to minimize bias of the results according to the relative amino acid compositions of the two polypeptides. The overproduction of gpl relative to gp2 proved to be so great that we could not reliably measure both polypeptides and remain within the linear range of film sensitivity. We therefore measured the relative density of the gpl and gp2 bands in a $802op,,wir-infected extract (Table 4B) as described under Methods. The overproduction of gp2 caused by the oplo mutation was found to be about 12-fold (Table 4A). Combining these figures and correcting for the relative molecular weights of the two polypeptides we found that gpl is produced in about 280fold relative molar excess to gp2 in @Ovir (Table 4C). While there may be consider-
480 TERMINASE
able error in such a measure, the excess of gpl relative to gp2 is obviously great.
GENES
635
2 TABLE
LEVELS 0~gp1
Revertants to Gene 1 Amber Mutations Do Not Always Induce Wild-Type gpl
In order to confirm the identification of gpl, we isolated a spontaneous revertant to each of the amber mutants lam,,, and lamsT6. Our expectation was that the putative gpl product would be induced by these revertants following infection of sup0 cells. However, we found that only the revertants to lam,, appeared to induce the wild-type gpl polypeptide. We therefore
1 AND
A”
gp2+h
480
4.8
2awh
-
2op1, rev 1 2opi0 rev 2 2oplo rev 3
31.4 32.1 26.6
B* 2opi0 rev
1
4
AND gp2 IN PHAGE-INFECTED CELLS h
gp2
2.5 -
2.3 0.0 28.9 29.6 24.1
Increase relative to wild type
1.0 12.6 12.9 I Mean 10.5
= 12.0
gPl
@
gPlkP2
71.5
11.7
6.1
C’ [gpll
kPz1
gp2am
gp20
a
bcdefgh
FIG. 2. Identification of gp2. Ultraviolet-irradiated 159 (sup9, or 159 supF were infected with the phage indicated at the head of the figure (including the three amber revertant isolates described in the text) and labeled with [35S$nethionine as described under Methods. The major capsid protein gp20 is indicated as a reference according to Youderian (1979). Track h is from a separate gel than the other tracks.
= 6.1
x 12.0
79 kdalton x 2. 5 kdalton
= 282
a The levels of either gp2 and the comigrating hostencoded polypeptide called h (i.e., gp2+h), or h alone, in the indicated strains relative to the unidentified phage-encoded band immediately below gp2 were determined by scanning the gel of [35S]methioninelabeled phage-infected extracts shown in Fig. 2 and measuring the weight (mg) of the appropriate plotted peaks. The level of gp2 alone was determined by subtracting h from the determined gp2+h values. b The relative densities of the gpl and gp2 bands in a gel (not shown) of a @3Mopvir-infected cell extract labeled with a W-amino acid mixture were determined as in footnote a above. The contribution to the determined values by other proteins of similar mobility were estimated from 4801amW&rand Zam,opiO-infected extracts and subtracted. c The relative molar amounts of gpl and gp2 in a @8ovir extract were estimated by multiplying the relative density of the gpl to gp2 band in a Zap,,infected extract, by the overproduction of gp2 by 2op,, relative to 2+ and correcting for the molecular weights of the two polypeptides.
isolated a total of seven independent revertants to lamam and six to larnsT6 and examined the induced proteins of each of these phage after labeling with [3H]arginine. Several general results were observed and are summarized in Table 5. Two of the 1 am46s revertants and one of the lam,,, revertants expressed a polypeptide of the same or very similar mobility to wild-type gpl (representatives are shown in Fig. 4, b
636
SUMNER-SMITH
gP2
AND
BECKER
wild-type and one of the “reduction” variants of gpl were quite stable (results not shown). Surprisingly, in those pseudorevertants in which the mobility of gpl was decreased, the mobility of gp2 was found to be increased (gp2* in Fig. 4, c and f, and Fig. 5) although the level of gp2 expression was unchanged even when the level of the mobility variant of gpl was much reduced (Fig. 4, f). DISCUSSION
gPl ~IloM
a
b
E
d
0
fghijkl
FIG. 3. Identification of gpl. Ultraviolet-irradiated 159 (sup”), WC5043 (supD), WC5041 (supE), or 159 supF were infected with the phage indicated at the head of the figure and labeled with [3H]arginine as described under Methods.
and e). These might be true revertants which replaced the amber codon with the original wild-type codon, or pseudorevertants carrying a new codon for a similar amino acid to that of wild type. Two of the revertants to 1am,68 (see for example Fig. 4, c), but none of the revertants to lamsT6 expressed a polypeptide of reduced mobility. The degree of expression of this polypeptide was at least as great and possibly greater than that of wild type. Three of the 1awes revertants and five of the lam,,G revertants at first appeared to have neither the wild-type nor the altered mobility forms of gpl. Careful examination on several different gels of one of these (lam,,,revF, Fig. 4, f) showed that in this case the altered mobility form of gpl was expressed at a level considerably less than that of wild type. The reduced expression of the new gpl seems to be due to a reduced rate of synthesis since pulse-chase experiments showed that both the peptide of the
We have identified the products of $80 genes 1 and 2. Since A and 480 show strong homology by heteroduplex mapping in the Nul-A and l-2 regions (Fiandt et al., 1971) and the products of these genes are functionally homologous (Weisberg et al., 1979; Table 2), it is likely that these products have similar molecular weights. In fact, the molecular weights of gpA and gp2 were found to be identical. The smaller subunit of A terminase, which we believe to be gpNu1 (see Introduction), has an apparent molecular weight of 21.5 kdalton (Cold, M. and Becker, A., in preparation). However, the gpNu1 equivalent of 480, gpl, was found here to have a molecular weight of 20.5 kdalton. Recently, it has been shown that single amino acid changes in a polypeptide can cause substantial mobility changes (equivalent to at least 1000 daltons on SDS -gels (de Jong et al., 1978; Shaw and Murialdo, 1980). Furthermore, we have observed that while the genomes of the two phage are also homologous in the region coding for the major head proteins (gpE in A and gp20 in @O), the apparent molecular weight of these two differ by approximately 3 kdalton when run in parallel on the same gel (unpublished observations). Finally, certain revertants to lam mutations were found to have an altered apparent molecular weight closer to that of the putative gpNu1. Thus we conclude that the smaller subunit of A terminase is indeed gpNu1 and that minor amino acid differences are responsible for the apparent mobility difference between it and gpl on the SDS-gels used. Nul is the left-most gene on the A map.
&?O TERMINASE
GENES TABLE
EXPRESSION
OF ALTERED
FORMS
OF gpl
AND
1 AND
2
637
5
gp2 BY REVERTANTS GENE 1
TO Two
AMBER
MUTATIONS
IN
Phenotype
Mutation Wild type lamlss
lam576
Number of revertant isolates
Level of gpl expression
ml Migration
!2P2 Migration
+ +
+ +
+ +”
+ Low +
Slow NDb +
Fast” ND +
Low
Slow”
Fast”
Crossover interval
True revertant or2+3 2+5 l+? True revertant or2+3 1+5
Note. Independent revertants to gene 1 amber mutants 463 and 576 were isolated and the products of genes 1 and 2 induced by the isolates following infection of 159 were analyzed on 12.5% SDS-polyacrylamide gels after being labeled with [3H]arginine as described under Methods. The level of expression and mobility of the induced gpl compared to wild type were used as criteria to classify each of the isolates. Representatives of four of these classes are shown in Fig. 4. The mobility of the gp2 induced by each of these representatives were examined (Figs. 4 and 5) and are summarized here. The intervals in which crossover events must have occurred to generate these classes by recombination with a cryptic prophage, as described in the Discussion and Fig. 6, are indicated in the last column. ” Determined for a sinale- reoresentative of the class. b Not determined.
The most recent physical maps have placed the start of the next gene: A at 0.5 (Echols and Murialdo, 1978) or 0.9 map unit (Szybalski and Szybalski, 1979) from the left terminus. The first AUG codon from the left end of A is at position 25, although there is no evidence that this is the start of the Nul coding sequence (Nichols and Donelson, 1978). These figures allow either 230 or 416 bp to code for gpNu1. However, if the size of gpNu1 is taken as 21 kdalton, it would require approximately 570-630 bp to code for it. Thus, either the current estimates of the start of gene A are wrong or Nul at least partially overlaps A out of phase. Since the sizes of the morphogenic genes have all been determined from information obtained from SDS-gels, it is clear that such estimates must be made with caution. We note, however, that recently Murialdo et al. (1980) have found that a putative mutation in the ribosome binding site of A gene A (fincslos) seems to map within the coding region of gene Nul. Since it is thought that the stoichiometry
of the two terminase subunits of A is 2: 1 (gpNu1 :gpA) (Gold, M., and Becker, A., in preparation) it is surprising that 480 produces such a large excess of gpl relative to the gp2 subunit. Since we have been unable to identify gpNu1 in radiolabeled extracts of h-infected cells, we do not know if it is produced in excess relative to gpA. However, preliminary results using an in vitro complementation assay suggest that it is not (Sumner-Smith et al., 1981). Furthermore, a mutant A which overproduces the gpA polypeptide (XAop,, Murialdo et al., 1980) does not show more terminase activity in an in vitro packaging assay unless addition.al gpNu1 from a gpNu1 containing extract (i.e., hAam) is also added (M. Sumner-Smith, unpublished observations). One explanation may be that the laboratory strain of $180 carries a mutation resulting in the overproduction of gpl. It is also possible that evolutionarily $80 is a “recent” hybrid between two lambdoid phages and has not yet adjusted the synthesis of gpl and gp2 optimally. Finally, production
SUMNER-SMITH
638
AND
BECKER
replication of the infecting phage is inhibited. An uncharacterized mutation (Xsti) in the Nul -A region of A will overcome this inhibition by the phage 21 gene products (C. Rudolph and M. Feiss, personal communication). It is possible that the gpNu1 (gpl) subunit is important in determining packaging specificity and that a hybrid terminase, containing the gpNu1 subunit from
a
bed
e
f
g
w*
FIG. 4. The level of expression and mobilities of gpl and gp2 as expressed by representatives of four of the classes of revertants to 1 amber mutations described in Table 5. lam468 rwA
of an excess of gpl may confer selective advantage to $80 in a mixed infection with another lambdoid phage. There is already precedent for the concept that overproduction of one of the terminase subunits can be advantageous to the phage, as Murialdo et al. (1980) have shown that overproduction of gpA makes gpF1 partially dispensible. Although the cos site is the same in both A and the lambdoid phage 21, each phage is unable to package the other’s DNA (Hohn, 1975; Feiss et al., 1979). This difference has been attributed to an evolutionary divergence in both a site to the right of cos which is recognized by the packaging mechanism and in other components of that mechanism (Feiss et al., 1979). Recently it has been shown that if a plasmid carrying several phage 21 genes, including the Nul and A analogs, is transactivated by an infecting A,
lams76 revF
lam576 rwA
lame.76
2am489
FIG. 5. The mobility of gp2 is altered in pseudorevertants to two 1 amber mutants. In order to more clearly show the altered mobility form of gp2 (i.e., gp2*) induced by lam,,revA and lam,,,revF tracks c, d, e, f, and g from Fig. 4 were scanned as described under Methods. The plots presented show the region of the tracks immediately around and including gp2 and are oriented with the upper portion of the gel to the left. The host-encoded band (h) of similar mobility to gp2 described in the text and Table 4 is clearly illustrated in the scan of track g (i.e., Zam,,,).
480 TERMINASE
1
I
639
GENES 1 AND 2 2
cos
#SO
-: cnms-a Interval:
.,
. ...
,; ~*
i’s ..‘..
2 i
i.I
i . I .. ..i
j4
L”.i
;s
‘it...i
qsr*
I
Nul
A
‘ :
FIG. 6. A mechanism for the pseudoreversion of 1 amber mutations by recombination with the qsr’ cryptic prophage. The qsr’ prophage has been shown to be homologous with h between cos and approximately 2.2 map units (%A) to the leff (Fisher and Feiss, 1980, and personal communication; Kaiser, 1980) and is therefore homologous to #30 in this region (see Fig. 1). The region of qsr’ immediately to the left of cos is homologous to A and is therefore not homologous to @30 (Fiandt et al., 1971). We hypothesize that the determinants for the level of synthesis of gpl and gpNu1 (i.e., + and low, respectively) are at the left end of the genes, i.e., probably the ribosome binding site. We arbitrarily place the determinant for the mobility of these products to the right of the amber mutations. The determinants for the mobility of gp2 and gpA are placed within the coding regions common to these two genes. In order to “cross-out” the amber mutation from d&30,a crossover event must take place in regions 1 or 2, and a second, reciprocal event in regions 3, 4, or 5. Thus, the pseudorevertants described in Table 5 are a subset of all possible permutations of the possible double recombination events. The figure is not drawn to scale. No attempt has been made to show a possible overlap in the coding sequences of Nul and A as this would not necessarily affect the predicted outcomes.
one phage and the gpA from the other, might either be inactive or have the wrong packaging specificity. According to this view, an increased synthesis of one or both terminase subunits by the infecting phage might overcome the competitive inhibition by the phage 21 products. Interestingly, $80, which apparently overproduces gpl, is able to replicate in the presence of the phage 21 products and therefore has the sti phenotype (C. Rudolph and M. Feiss, personal communication), even though it has the same packaging specificity as A. Our inability to identify gpNu1 may be due to two factors. First, gpNu1 may not be produced in excess relative to gpA as gpl is to gp2. Second, A induces the synthesis of several major proteins including one form of gplom (Reeve and Shaw, 1978) and gpI (Ray and Pearson, 1976), which have very similar mobilities to the putative gpNu1 and would therefore obscure it. These proteins are not induced by 480 and thus gpl is relatively easy to detect by the techniques used here. In agreement with this, we were unable to detect gpl in several 480-X hybrids carrying genes 1 and 2 from 480, but all of the other genes from A,
despite its assumed overproduction (unpublished observations). We have not observed any cleavage or degradation of either gpl or g-p2 which would account for the apparent difference in molecular weight of these polypepticles in 1 amber pseudorevertants. Two models can be suggested to explain these clifferences. First, replacement of the amber codon by a codon for a different amino acid than wild type could result in a mobility change in gpl as an artifact of amino acid composition characteristic of SDS-gels (see discussion above). We have found, however, that suppression of both lam,,, and lam,,, by three different amber suppressors did not change the mobility of gpl (Fig. 4). In order to explain the concurrent and opposite mobility changes of gpl and gp2, it is necessary to hypothesize that genes 1 and 2 overlap out of phase and that both of the 1 amber mutations examined fall in this area of overlap. Thus, a base change eliminating the amber codon in gene 1 might cause an amino acid substitution in gene 2, thus altering its mobility. A second explanation which we consider to be more likely is that the 1 amber muta-
640
SUMNER-SMITH
tions can be rescued by recombination with a cryptic lambdoid prophage. Most laboratory strains of E. coli K12 carry at least two and probably three cryptic lambdoid prophage (Kaiser and Murray, 1979; Kaiser, 1980). Recently, Fisher and Feiss (1980) have shown that a A (cosl) which is deleted for cos and at least part of Nul can be rescued by recombination with the cryptic qsr’ prophage carried by the host. It is possible that some or all of the “revertants” of the two gene 1 amber mutants selected here are actually recombinanti carrying all or at least part of the gene N&(l) and possibly gene A (2) equivalents of a cryptic prophage. Figure 6 shows the deduced region of homology between the qsr’ prophage and 480. Any two crossover events between an infecting @30 lam particle and the qsr’ prophage which bracket the amber mutation would lead to a recombinant, hybrid phage which would score as an amber revertant. We hypothesize that the amino acid composition of the Nul (1) and A (2) analogs carried by the cryptic prophage might differ slightly from those of their counterparts in 480. Thus, depending on the position of the crossover points, the resulting recombinant phage might carry part or all of the Nul (1) analog and possibly part of the A (2) gene analog from the cryptic prophage. The “gpl” and 732” expressed by this hybrid phage might then differ in mobility on SDS-gels (because of their “altered” amino acid compositions) as is in fact observed. Furthermore, if we hypothesize that the Nul (1) gene from the cryptic prophage is translated with an efficiency closer to that of the A Nul gene, then recombinants which carry the entire gene would then apparently express much less of “gpl,” which would also have an “altered” mobility on SDS-gels. All of the pseudorevertant classes observed (Table 5) can then be explained as recombinants with qsr’ in which the crossover points differ (compare Fig. 6 with the last column of Table 5). ACKNOWLEDGMENTS
We thank Dale Hawkins for her excellent technical assistance in isolating the op,, mutation; Sam Benchi-
AND
BECKER
mol, Jocelyn Shaw, Helios Murialdo, and Michael Feiss for their many helpful suggestions; Richard Fisher, Michael Feiss, and C. Rudolph for generously sharing their unpublished data; and Phillip Youderian and Martha Howe for strains. Supported by a research grant from the Medical Research Council of Canada (MT-3325) and an MRC Studentship to Martin Sumner-Smith. REFERENCES
BECKER, A., and GOLD, M. (1975). Isolation of the bacteriophage lambda A-gene protein. Proc. Nat. Acad. Sci. USA 72, 581-585. BECKER, A., MURIALDO, H., and GOLD, M. (1977). Studies on an in vitro system for the packaging and maturation of phage h DNA. Virology 78,277-290. BONNER, W. M., and LASKEY, R. A. (1974). A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83-88. CAMPBELL, A. (1961). Sensitive mutants of bacteriophage A. Virology 14, 22-32. DE JONG, W. W., JWEERS, A., and COHEN, L. H. (1978). Influence of single amino acid substitutions on electrophoretic mobility of sodium dodecyl sulfate-protein complexes. Biochem. Biophys. Res. Commun. 82, 532-539. ECHOLS, H., and MURIALDO, H. (1978). Genetic map of bacteriophage lambda. Virology 42, 577-591. FEISS, M., FISHER, R. A., SIEGELE, D. A., NICHOLS, B. P., and DONELSON, J. E. (1979). Packaging of the bacteriophage lambda chromosome: A role for base sequences outside cos. Virology 92, 56-67. FUERST, C. (1966). Defective biotin-transducing mutants of bacteriophage lambda. Virology 30, 581583. FIANDT, M., HRADECNA, Z., LOZERON, H. A., and SZYBALSKI, W. (1971). Electron micrographic mapping of deletions, insertions, inversions and homologies in the DNAs of coliphages lambda and Phi 80. In “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 329-354. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. FISHER, R., and FEISS, M. (1980). Reversion of a cohesive end site mutant of bacteriophage lambda by recombination with a defective prophage. Virology 107, 160-173. GORINI, L. (1970). Informational suppression. Annu. Rev. Gent. 4, 107-134. HARRIS, A. W., MOUNT, D. W. A., FUERST, C. R., and SIMINOVITCH, L. (1967). Mutations in bacteriophage lambda affecting host cell lysis. Virology 32, 553 -569.
HOHN, B. (1975). DNA as substrate for packaging into bacteriophage lambdain vitro. J. Mol. Biol. 98,93106.
. d&O TERMINASE HOHN, T., and KATSURA, I. (197’7). Structure and assembly of bacteriophage lambda. Curr. Top. Microbiol. Immud. 78, 69-110. INOKUCHI, H., and OZEKI,H. (1970). Phenotypic mixing between bacteriophage 480 and lambda in vitro and in vivo. Virology 41, 701-710. KAISER, K., and MURRAY, N. E. (1979). Physical characterisation of the “Rat prophage” in E. coli K12. hfol. Gen. Genet. 175, 154-174. KAISER, K. (1980). The origin of Q-independent derivatives of phage A. Mol. Gen. Genet. 179, 547-554. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. MILLER, J. H., GANEM, D., Lu, P., and SCHITZ, A. (1977). Genetic studies of the lot repressor. I. Correlation of mutational sites with specific amino acid residues: Construction of a colinear gene-protein map. J. Mol. Biol. 109, 275-301. MURIALDO, H., and SIMINOVITCH, L. (1971). The morphogenesis of bacteriophage lambda. III. Identification of genes specifying morphogenetic proteins. In “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 711-723. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. MURIALDO, H., and SIMINOVITCH, L. (1972a). The morphogenesis of bacteriophage lambda. IV. Identification of gene products and control of the expression of the morphogenetic information. Virology 48, 785-823.
MURIALDO, H., and SIMINOVITCH, L. (1972b). The morphogenesis of bacteriophage lambda. V. Formdetermining function of the genes required for assembly of the head. Virology 48, 824-835. MURIALDO, H., and BECKER, A. (1978). Head morphogenesis of complex double-stranded deoxyribonucleic acid bacteriophages. Microb. Rev. 42, 529576.
MURIALDO, H., FIFE, W., BECKER, A., FEISS, M., and YOCHEM, J. (1981). Bacteriophage lambda
GENES 1 AND 2
641
DNA maturation. The functional relationships among the products of genes Nul, A and FI. J. Mol.
Biol.
145, 375-404.
NICHOLS, B. P., and DONELSON, J. E. (1978). 1%nucleotide sequence surrounding the cos site of bacteriophage lambda DNA. J. Viral. 26, 429-434. PTASHNE, M. (1967). Isolation of the X phage repressor. Proc. Nat. Acad. Sci. USA 57, 306313. RAY, P. N., and PEARSON, M. L. (1976). Synthesis of morphogenetic proteins by mutants of bacteriophage lambda carrying tandem genetic duplications. Virology 73, 381-388. SATO, K., NISHIMUNE, Y., SATO, M., NUMICH, R., MATSUSHIRO, A., INOKUCHI, H., and OZEKI, H. (1968). Suppressor-sensitive mutants of coliphage 480.
Virology
34, 637-649.
SHAW, J. E., and MURIALW, H. (1980). Morphogenetic genes C and Nti overlap in bacteriophage A. Nature
(London)
283,
30-35.
SUMNER-SMITH, M., BECKER, A., and GOLD, M. (1981). DNA packaging in the lambdoid phages: the role of h genes Nul and A. Virology 111, 593-597. SZYBALSKI, E. H., and SZYBALSKI, W. (1979). A comprehensive molecular map of bacteriophage lambda. Gene
7, 217-270.
THOMAS, C. A., and ABELSON, J. (1966). The isolation and characterization of DNA from bacteriophage. In “Procedures in Nucleic Acid Research” (G. L. Cantoni and D. R. Davies, eds.), p. 553. Harper and Row, New York. WEIGLE, J. (1966). Assembly of phage lambda in vitro. hoc. Nat. Acad. Sci. USA 55, 1462-1466. WEISBERG, R. A., STERNBERG, N., and GALLAY, E. (1979). The Nul gene of coliphage A. Virology 95, 99-106. YANOFSKY, C., and ITO, J. (1966). Nonsense codons and polarity in the tryptophan operon. J. Mol. Biol. 21, 313-334. YOUDERIAN, P. (1979). Ph.D. thesis, Massachusetts Institute of Technology, Cambridge.
111,629-641
(1981)
DNA Packaging in the Lambdoid Phages: Identification of the Products of $80 Genes 7 and 2 M. SUMNER-SMITH’ Department
of Medical
Genetics, Toronto,
AND A. BECKER
Medical Ontario
Accepted
Sciences Building, M&S lA8, Canada
January
University
of Toronto,
14, 1981
We have identified the products of genes 1 and 2 of the lambdoid coliphage 480. These genes code for the subunits of the terminase enzyme that promotes DNA packaging during phage particle assembly. These data serve by analogy to identify the subunits of h terminase as the products of Nul and A, respectively. Identification of the gene 2 product was aided by the isolation of a mutant which causes the overproduction of that polypeptide. Mutants which affect the mobility of the product of gene 1 on 12.5% SDS-polyacrylamide gels were isolated as pseudorevertants of two amber mutations in the gene. Certain of these gene 1 pseudorevertants affect the mobility of the product of gene 2. The hypothesis that these pseudorevertants arise by recombination with a cryptic prophage carried by the host is proposed. The possibility that genes 1 and 2 overlap out of phase for a portion of their coding sequences is discussed. Surprisingly, wild-type 480 produces approximately 280-fold more of the gene 1 than gene 2 product. INTRODUCTION
During the final stages of assembly of a lambdoid phage particle, a monomer length of phage genome is cut at specific cos sites from replicated, concatemeric DNA and packaged into an empty prohead (for reviews see: Hohn and Katsura, 1977; Murialdo and Becker, 1978). An enzyme, called terminase, has been identified and purified from h-infected Escherichia coli cells, which is capable of cleaving DNA endonucleolytically at cos sites and also of promoting packaging in vitro. This terminase has recently been shown to consist of two subunits (M. Cold and A. Becker, in preparation), one of which has previously been identified as the product of A gene A @A) (Becker and Cold, 1975; Becker et al., 1977). The other subunit has not been identified. Active terminase has not been detected in extracts of cells infected with UVulor AAam phage (Becker et al., 1977), but is present in extracts infected with gal trans1 Author dressed.
to whom reprint
requests should be ad629
ducing phage from which all of the morphogenetic genes except Nul and A have been deleted (Becker and Cold, 1975). Recently we have shown that active terminase is produced when XNul - and L4am phage-infected extracts are mixed in vitro (SumnerSmith et al., 1981). Thus it appears likely that the second subunit of terminase is the product of Nul (gpNu1). The work reported here was aimed at confirming this possibility. Unfortunately, the only mutation available in the gene Nul (i.e., Nuldef,,,) is neither a nonsense nor a missense mutant and we have been unable to identify gpNu1 in radiolabeled A-infected extracts by SDS-polyacrylamide gel electrophoresis using this mutant. Weisberg et al. (1979) have shown that genes 1 and 2 of the lambdoid phage 480 are homologous in function to genes Nul and A, respectively. Furthermore, h and 480 are homologous by heteroduplexing in the region of their genomes which code for these genes (Fiandt et al., 1971). Since several nonsense mutations are available in each of genes 1 and 2 (Sat0 et al., 1968; Youderian, 1979) we decided to see if we 0042-6822/81/080629-13$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
630
SUMNER-SMITHANDBECKER
would be more successful in identifying the product of gene 1 (gpl) than we had been in identifying gpNu1. We report here the identification of the products of 480 genes 1 and 2, and show that these correspond to the two subunits of A terminase. Our ability to identify gpl, but not gpNu1, seems to be due to the fortuitous lack of other @O-induced proteins of a similar electrophoretic mobility to gpl, and also possibly to the unexpectedly high level of synthesis of gpl. MATERIALSANDMETHODS
with phage at an m.o.i. of 0.01-0.1. Cultures were grown with shaking at 37” until extensive lysis was observed, a few drops of chloroform were added, and the debris was sedimented by centrifugation and the supernatant retained. Lysates were dialyzed extensively against A diluent before being used for labeling purposes (see below). When required, phage lysates were concentrated as described by Murialdo et aE. (1980). Phage were plated on the appropriate indicator (0.2 ml of a late log culture) strain in 2.5 ml melted top agar over 35 ml solid bottom agar.
Media and buffers. L broth is 10 g Bacto Preparation of labeled extracts of inTryptone (Difco), 5 g yeast extract, and 5 g fected cells Cells were grown in RM meNaCl per liter of distilled H,O which is neu- dium at 37” to a density of 2.5 x lO*/ml, tralized with NaOH. RM medium is 18.7 centrifuged, and resuspended at a concenmM NH&l, 1 mM M&SO,, 20 mJ4 KCl, 0.1 tration of 2.0 x log/ml in fresh RM. A thin 3 pM FeCl,, 49.4 n-&f layer of cell suspension was irradiated with ml4 CaCl,, Na2HP04, 22 mM KHzP04, 22 mM glycbetween 5000 and 6000 ergs/mm2 of ultravierol, and 0.4% maltose. Lambda diluent (A- olet light. The cells- were then added to an dil) is 10 n-&f MgS04, 10 n-&f Tris-HCl, pH equal volume of infecting phage diluted in 7.4. The solid medium used for bacterial A-dil to 1.0 x 10lO/ml and held on ice for 15 colony formation and phage plaque as- to 30 min for adsorption. Four volumes of says has been described (Campbell, 1961; prewarmed RM were added and the mixture incubated for 35 min at 37” with shakMurialdo and Siminovitch, 1971). of L-[35S]methionine Buffer A is 20 mM Tris-HCl (pH 8.0), 1 ing. About 42 &i/ml mM EDTA, 3 mJ4 MgCl, ,5 n-&f 2-mercap(New England Nuclear) or 15 &i/ml of Ltoethanol. Buffer B is 10% sucrose in 0.05 [2, 3-3H]arginine (New England Nuclear) M Tris-HCl (pH 7.4). SM 1 solution is 60 or 5 &i/ml of L-[‘4C]amino acid mixture n&f spermidine, 18 m&f MgC&, 15 mil4 (Amersham) were added and incubation ATP, 30 mM 2-mercaptoethanol in 5 n-&f continued for a further 10 min. Incubation Tris-HCl (pH 7.4). was terminated by cooling the mixture in Strains. The E. coli K12 derivative an ice bath. The cells were harvested by strains used are shown in Table 1. 159supF centrifugation and resuspended in 0.4-vol sample buffer (Laemmli, 1970), placed at was made by selecting for trp+ cells after Pl transduction from QD5003 trp+ supF 100” for 1 min, vortexed, and kept at -20” into a 159A(tonB-trip) derivative. The pres- until thawed for application to a gel. ence of the supF marker was confirmed by Gel electrophoresis. The discontinuous the ability of the strain to suppress the SDS-polyacrylamide gel system Laemmli Sam, mutation in A. 480 strains carrying (1970) was used with a stacking gel of 5% and a separating gel of 12.5%. The reserlam, (Sato et al., 1968>, 1am468, lam,,,, and lam,,,, and Zam,,, (Youderian, 1979) voir buffer was supplemented to 4 mM were the kind gift of Dr. P. Youderian. mercaptoethanol. The gels were run overAimm434TcI,00 (Murialdo et al., 1980) was night at 95 V. After electrophoresis gels used to prepare DNA by phenol extraction were impregnated with 2,5-diphenyloxa(Thomas and Abelson, 1966). zole for fluorography (Bonner and Laskey, 1974), dried under vacuum, and used to exPreparation of phages. Phage lysates with a titer > 101O/ml were routinely pre- pose X-ray film (Kodak, SB-5). Scanning pared by infecting a culture of T5-2 (for was performed in a Gilford spectrophotometer provided with a linear transport and amber phage) or 594 (for nonamber phage) at a concentration of 107-log/ml in L broth the area under the appropriate peaks was
&80 TERMINASE TABLE BACTERIAL
strain T52 594 TC600 XAlOC QD5003 159 WC5043 WC5041 159 supF W3350 (LVuldef,,l&&‘am7
631
GENES 1 AND 2 1 STRAINS
Relevant genotype supE SUP0 supE supc supF gal-, str’, str’, gal-,
tonA
(lacks @30 receptor)
str’, uwrA-, WV-A-, leu-, UWA-, leu-, str’, uvrA-,
sup’ supD supE supF
)
Reference Fuerst (1966) Weigle (1966) Harris et al. (1967) Miller et al. (1977) Yanofsky and Ito (1966) Ptashne (196’7) From M. Howe From M. Howe This work Murialdo and Siminovitch (1972); Weisberg et al. (1979)
594 (AAam,,~am,,cI~~am,)
NS428
sup”, recA
(hAam,,bzredJcIs5$amr)
measured by weighing after they had been cut from the plot. Care was taken to ensure that the optical density of the bands was within the linear response range of the film. Molecular weights of 480 gene products were determined by comparison with a parallel X-infected extract using the published values of A gene products (reviewed by Szybalski and Szybalski, 1979). In vitro packaging of DNA. The twostage packaging system of Becker and Gold (1975) was used. Preparation of stage 1 extract of A lysogens: 400 ml of L broth was inoculated with the indicated strain and the cells were grown at 32” in a shaker to a cell concentration of about 7 x lO’/ml. For thermal induction of the prophage, the cells were incubated with shaking at 70” until the temperature of the cell suspension reached 45”. Subsequently, the flasks were shaken at 45” for 13 min followed by cooling in an ice/salt/water mixture until the temperature dropped to 37”. The cells were incubated at 37” for another 40 min; they were then chilled and spun down at 13,000 g for 10 min. The pellets were resuspended in 0.5 ml of buffer A. Before sonication the volume was adjusted to about 4.0 ml with buffer A. Sonication of the concentrated suspension was done by two pulses of 10 set each in an ice/salt bath. The sonicates were cleared by centrifugation at 4000 g for 10 min.
Becker et al. (1977) Sternberg, Tiemeier, and Enquist (1977)
Preparation of stage 1 extract of cells infected with 480: 300 ml of L broth were inoculated with strain 594 and the cells were grown at 37” in a shaker to a cell concentration of 1.4 x W/ml, pelleted by centrifugation at 13,000 g for 10 min, and resuspended in 42 ml of fresh L broth at O-4”. Purified $80 phage (as indicated) were added to a multiplicity of 5 and the mixture held on ice for 15 min for phage adsorption. Five hundred milliliters of L broth prewarmed to 37” was added and the mixture shaken at 37“ for 40 min. The cells were then pelleted and treated as for an induced A lysogen as described above. Preparation of stage 2 extracts: these extracts were prepared by the lysozymefreeze-thaw method first described by Kaiser and Masuda (1973). The lysogen NS428 was grown in 600 ml L broth, induced, and the cells collected by centrifugation as described above for the preparation of the sonicates. The pellets were subsequently resuspended in 0.8 ml of buffer B. The final volume of this mixture was about 1.5 ml. To 1 ml of this suspension 0.05 ml of a lysozyme solution (2 mg/ml in 0.25 M Tris -HCl, pH 7.4) was added, and this was followed by freezing in liquid N, and shaking in a water bath at 37” just to the point of complete thawing. The viscous extract was incubated on ice for 45 min, then 100 ~1 SMA solution was added.
632
SUMNER-SMITH
AND BECKER
FIG. 1. Physical map of the head region of h and 480. Regions of homology between A and 480 (Fiandt et al., 1971) are shown by stippling. The sizes and positions of the A genes A, W, B, C, Nu3, D, E, FI, and FZZ are taken from the review of Szybalski and Szybalski (19’79). The order of the 480 genes 1,2,S, &20, and 5 and the sizes of S,4, and 20 are taken from Youderian (1979); their positions are assumed to be the same as the corresponding A genes. The size of genes 1 and 2 are as determined here. The size of the Nuf gene is taken as that of the smaller subunit of A terminase (Cold, M. and Becker, A., in preparation).
The mixture was then centrifuged at 35,000 rpm in the 50 Ti rotor of the Spinco ultracentrifuge for 30 min. The supernatant was used as the stage 2 extract, providing gpD, pgW, gpFI1, and tail function to the assay (Becker et al., 19’7’7). The assay: 4 ~1 of a solution (185 kg/ml) of &nm434TcZ~oo DNA, 4 /.J of SMA solution, and where indicated, 4 ~1 of partially purified terminase (AsI; approximately 1 unit/ml) (Becker and Gold, 1975), 20 ~1 of stage 1 extract, and 30 ~1 of buffer A were incubated at 22” for 15 min. One hundred and fifty microliters of stage 2 extract was added and incubation continued for 60 min at 22”. The phage yield was titered on strain TC600 (supE, tonA). None of the phage used to produce the packaging extracts will plate under these conditions since the h phage carry the Sam, mutation, which is not suppressed by supE and the 480 phage are not adsorbed since TC600 carries the tonA mutation, and is therefore resistant to hmsobut not h* particles. Thus, only particles assembled in vivo from the exogenously added imm434Tc1700 DNA, X or 480 proheads supplied by the stage 1 extract and A tails supplied by the stage 2 extract (Becker et al., 1977) will score by producing a plaque. The results reported here, therefore, confirm the observation of Inokuchi and Ozeki (1970) that 480 DNA-filled
heads will
efficiently
accept X tails
in
vitro. RESULTS
Failure to Identify Nul
the Product
of X Gene
We attempted to identify the Nul gene product by comparing pulse-labeled extracts of cells infected with A phage carrying the absolutely defective Nul mutation t16 to wild-type and t16 revertant phage, by both one-dimensional SDS-polyacrylamide gel electrophoresis and by isoelectric focusing followed by SDS-polyacrylamide gel electrophoresis. We could find no consistent difference between these extracts, however. The $80 gene 1 is homologous to the A gene Nul by heteroduplexing (Fig. 1, Fiandt et al., 1971) and its product can efficiently substitute for gpNu1 in vivo. Since several amber mutations are available in gene 1 (Sato et al., 1968; Youderian, 1979) we decided to see if we could identify the product of that gene. Preliminary experiments comparing 4801 am to l+ infected cell extracts were encouraging. We thought it necessary, however, to first establish that genes 1 and 2 were essential for terminase activity in $80 and also to iden-
&30 TERMINASE
tify gp2, whose A analog (Fig. l), gpA, has already been identified (Murialdo and Siminovitch, 1972a). Lambda
Terminase
GENES 1 AND 2
633 TABLE
&30lam
2
AND%~-INFECTED EXTRACTS DEFICIENT IN TERMINASE
Can Substitute for +?O
ARE
Yield (PFU/ml)
Genes 1 and 2 in vitro Stage 1 extracts prepared from induced A lysogens lacking either Nul or A have been shown to be unable to package exogenous X DNA efficiently in an in vitro packaging system (Becker et al., 1977). This deficit can be overcome by adding partially purified terminase from an induced lysogen lacking all of the morphogenetic genes except Nul and A. We have repeated the above observations (Table 2, lines 3 and 4) and have extended them to show that extracts prepared from cells infected with +SOuir phage carrying either lam or 2am mutations have a similar defect which can be corrected by adding h terminase (Table 2, lines 1 and 2). Thus we conclude that the &30 genes 1 and 2 are essential for endogenous terminase activity and that X terminase is sufficiently similar to substitute for it. Zdentijication
of the Product
of @O Gene 2
A band gpA (i.e., tracts of (Fig. 2,
of the same molecular weight as A ‘79 kdalton) was observed in excells infected with r#doUir phage track c), but was reduced in @&?am,,,vir infected cells (Fig. 2, track b). A host-encoded polypeptide has the same mobility as gp2 (Fig. 2, track a) so that it is not surprising that there is evidence of a faint band remaining after gp2 has been eliminated by a nonsense mutation. Since the amount of putative gp2 made is apparently so small and identification therefore difficult, we attempted to isolate a mutant which would increase the synthesis of the gene 2 product. Selection of an Overproducer Gene 2 and Conjkrnation jkation of gp2
Mutation in, of the Identi-
Recently Murialdo et al. (1980) have isolated a mutation in X which increases the rate of synthesis of the A gene product
Stage 1 extract lam,
2amrss
Nuhf,,,s Aamal,Dam16
-terminase 2 3.6 1.3 4
x x x x
10’ 102 109 10’
+ terminase 3.1 1.6 5.4 3.7
x x x x
10’ 10’ 10’ 10’
Note. Stage 1 extracts were prepared by infecting 594 (sup”) with ~8Olam& of &302amulsvir or by inducing W3350 (ANuld.eftl~I~$am,) or 594 (hAam.,r Dam,&,,Sam,) and used as described under Methods.
(Mop,).
It was found that although a phage is able to plate on a host carrying supE, it is unable to plate on a host carrying supE in which the efficiency of suppression is reduced by a streptomycin mutation. Presumably the yield of phage is limited by the amount of A polypeptide synthesized, and this amount is insufficient in a host carrying a weak suppressor to give a plaque. The op, mutation was selected by picking a phage that still carried the amber mutation, but was able to plate on the “weak suppressor” host. It is thought that the op, mutation increases the efficiency of translation of the A gene product. We reasoned that it should be possible to isolate a similar mutation in $30 gene 2, which would then aid in identifying the gene 2 polypeptide. Although cells carrying either supF or supC suppress amber mutations by inserting tyrosine, their efficiencies of suppression differ: 50 vs 16% (Gorini, 1970). We found that @302am,,,vir was able to plate on QD5003 (supF), but not on XAlOC (supC)(Table 3). A spontaneous mutant was picked which was able to plate on XAlOC, but not on 594 (sup) since it still carried an amber mutation which was unable to complement the parental amber phage. The new mutant phage was designated as +802am,,90p,,,vir. Three spontaneous amber revertants (designated as @02op~~uir, revs. 1,2, and 3) to the mutant XAam,,,
634
SUMNER-SMITH TABLE
AND
3
PLATING OF F’I-IAGE CARRYING AMBER MUTATIONS IN GENES I AND 2 QD5003 (supF)
+8oUir $801 amUlsvir 4801 am,,Bvir Q802amlasvir W@amwvwir
1.0 1.0 1.0 1.0 1.0
XAlOC (supc
)
1.0 0.7 0.9 1.3 x 10-a
0.7
594 (SUP0 )
0.9 9.7 x 3.6 x 7.1 x 3.6 x
10-1 lo+ lo-’ lo-’
Note. The efficiency of plating (e.o.p.) of the designated phage strains on the indicated hosts relative to the permissive host QD5003 is shown.
were isolated. These amber revertants were shown to overproduce the putative gene 2 polypeptide (Fig. 2, tracks d, e, and f, and Table 4). The op10 mutation when combined with the Zam,,, mutation increased the synthesis of an induced polypeptide of lower molecular weight than gp2, which is presumably the “amber fragment,” following infection of a sup0 cell (Fig. 2, track g). This is confirmed by the observation that both the putative amber and wild-type polypeptides are induced when +302am,,,op,,vir infects a cell carrying a suppressor (Fig. 2, track h and Fig. 3, track k). Identifiation
of the Product
of @O Gene 1
A polypeptide with an apparent molecular weight of 20.5 kdalton which was present in +SoVir and @Mam,,,vir-infected cells was absent in cells infected with phage carrying four different gene 1 amber mutations (ambers 3, 468, 516, and 576) when labeled with [35S]methionine. Several hostencoded bands, however, made positive identification difficult. Preliminary attempts to separate the putative gpl from the host polypeptides by isoelectric focusing suggested that it was very basic; in agreement with this observation, labeling with [3H]arginine instead of [35S]methionine was found to greatly enhance the putative gpl band relative to the other bands of similar mobility. Figure 3 shows that the putative gpl polypeptide is present in l’-
BECKER
infected cell extracts (Fig. 3, tracks a, j, and k), but absent in lam,,, (Fig. 3, track b) and lam,,,-infected sup0 cell extracts (Fig. 3, track f). The gpl polypeptide is partially restored when the lam,, and 1 am576 phage were used to infect supD (Fig. 3, tracks c and g), supE (Fig. 3, tracks d and h), and supF (Fig. 3, tracks e and i) cells. Overproduction
of gpl Relative
to gp2
There appeared to be considerably more gpl present than gp2 after 10 min of labeling of a +80uir infected cell extract (see for example, Fig. 3). This result is somewhat surprising since gpl and gp2 are thought to be subunits of terminase. We noticed, however, that while a phage carrying a 2am mutation cannot plate on a host carrying only an ocher suppressor unless it is combined with the overproducer mutation (op,,), two different amber mutations in gene 1 are suppressed sufficiently by the same ocher suppressor to yield a visible plaque (Table 3). These data suggested that gpl is made in excess relative to gp2 by wild-type phage. In addition, we found that mutants which produced considerably less gpl than wild type were still fully viable (see below). In order to assess the relative production of gpl and gp2, 48&r-infected cells were labeled with an Q4C-amino acid mixture (as described under Methods) so as to minimize bias of the results according to the relative amino acid compositions of the two polypeptides. The overproduction of gpl relative to gp2 proved to be so great that we could not reliably measure both polypeptides and remain within the linear range of film sensitivity. We therefore measured the relative density of the gpl and gp2 bands in a $802op,,wir-infected extract (Table 4B) as described under Methods. The overproduction of gp2 caused by the oplo mutation was found to be about 12-fold (Table 4A). Combining these figures and correcting for the relative molecular weights of the two polypeptides we found that gpl is produced in about 280fold relative molar excess to gp2 in @Ovir (Table 4C). While there may be consider-
480 TERMINASE
able error in such a measure, the excess of gpl relative to gp2 is obviously great.
GENES
635
2 TABLE
LEVELS 0~gp1
Revertants to Gene 1 Amber Mutations Do Not Always Induce Wild-Type gpl
In order to confirm the identification of gpl, we isolated a spontaneous revertant to each of the amber mutants lam,,, and lamsT6. Our expectation was that the putative gpl product would be induced by these revertants following infection of sup0 cells. However, we found that only the revertants to lam,, appeared to induce the wild-type gpl polypeptide. We therefore
1 AND
A”
gp2+h
480
4.8
2awh
-
2op1, rev 1 2opi0 rev 2 2oplo rev 3
31.4 32.1 26.6
B* 2opi0 rev
1
4
AND gp2 IN PHAGE-INFECTED CELLS h
gp2
2.5 -
2.3 0.0 28.9 29.6 24.1
Increase relative to wild type
1.0 12.6 12.9 I Mean 10.5
= 12.0
gPl
@
gPlkP2
71.5
11.7
6.1
C’ [gpll
kPz1
gp2am
gp20
a
bcdefgh
FIG. 2. Identification of gp2. Ultraviolet-irradiated 159 (sup9, or 159 supF were infected with the phage indicated at the head of the figure (including the three amber revertant isolates described in the text) and labeled with [35S$nethionine as described under Methods. The major capsid protein gp20 is indicated as a reference according to Youderian (1979). Track h is from a separate gel than the other tracks.
= 6.1
x 12.0
79 kdalton x 2. 5 kdalton
= 282
a The levels of either gp2 and the comigrating hostencoded polypeptide called h (i.e., gp2+h), or h alone, in the indicated strains relative to the unidentified phage-encoded band immediately below gp2 were determined by scanning the gel of [35S]methioninelabeled phage-infected extracts shown in Fig. 2 and measuring the weight (mg) of the appropriate plotted peaks. The level of gp2 alone was determined by subtracting h from the determined gp2+h values. b The relative densities of the gpl and gp2 bands in a gel (not shown) of a @3Mopvir-infected cell extract labeled with a W-amino acid mixture were determined as in footnote a above. The contribution to the determined values by other proteins of similar mobility were estimated from 4801amW&rand Zam,opiO-infected extracts and subtracted. c The relative molar amounts of gpl and gp2 in a @8ovir extract were estimated by multiplying the relative density of the gpl to gp2 band in a Zap,,infected extract, by the overproduction of gp2 by 2op,, relative to 2+ and correcting for the molecular weights of the two polypeptides.
isolated a total of seven independent revertants to lamam and six to larnsT6 and examined the induced proteins of each of these phage after labeling with [3H]arginine. Several general results were observed and are summarized in Table 5. Two of the 1 am46s revertants and one of the lam,,, revertants expressed a polypeptide of the same or very similar mobility to wild-type gpl (representatives are shown in Fig. 4, b
636
SUMNER-SMITH
gP2
AND
BECKER
wild-type and one of the “reduction” variants of gpl were quite stable (results not shown). Surprisingly, in those pseudorevertants in which the mobility of gpl was decreased, the mobility of gp2 was found to be increased (gp2* in Fig. 4, c and f, and Fig. 5) although the level of gp2 expression was unchanged even when the level of the mobility variant of gpl was much reduced (Fig. 4, f). DISCUSSION
gPl ~IloM
a
b
E
d
0
fghijkl
FIG. 3. Identification of gpl. Ultraviolet-irradiated 159 (sup”), WC5043 (supD), WC5041 (supE), or 159 supF were infected with the phage indicated at the head of the figure and labeled with [3H]arginine as described under Methods.
and e). These might be true revertants which replaced the amber codon with the original wild-type codon, or pseudorevertants carrying a new codon for a similar amino acid to that of wild type. Two of the revertants to 1am,68 (see for example Fig. 4, c), but none of the revertants to lamsT6 expressed a polypeptide of reduced mobility. The degree of expression of this polypeptide was at least as great and possibly greater than that of wild type. Three of the 1awes revertants and five of the lam,,G revertants at first appeared to have neither the wild-type nor the altered mobility forms of gpl. Careful examination on several different gels of one of these (lam,,,revF, Fig. 4, f) showed that in this case the altered mobility form of gpl was expressed at a level considerably less than that of wild type. The reduced expression of the new gpl seems to be due to a reduced rate of synthesis since pulse-chase experiments showed that both the peptide of the
We have identified the products of $80 genes 1 and 2. Since A and 480 show strong homology by heteroduplex mapping in the Nul-A and l-2 regions (Fiandt et al., 1971) and the products of these genes are functionally homologous (Weisberg et al., 1979; Table 2), it is likely that these products have similar molecular weights. In fact, the molecular weights of gpA and gp2 were found to be identical. The smaller subunit of A terminase, which we believe to be gpNu1 (see Introduction), has an apparent molecular weight of 21.5 kdalton (Cold, M. and Becker, A., in preparation). However, the gpNu1 equivalent of 480, gpl, was found here to have a molecular weight of 20.5 kdalton. Recently, it has been shown that single amino acid changes in a polypeptide can cause substantial mobility changes (equivalent to at least 1000 daltons on SDS -gels (de Jong et al., 1978; Shaw and Murialdo, 1980). Furthermore, we have observed that while the genomes of the two phage are also homologous in the region coding for the major head proteins (gpE in A and gp20 in @O), the apparent molecular weight of these two differ by approximately 3 kdalton when run in parallel on the same gel (unpublished observations). Finally, certain revertants to lam mutations were found to have an altered apparent molecular weight closer to that of the putative gpNu1. Thus we conclude that the smaller subunit of A terminase is indeed gpNu1 and that minor amino acid differences are responsible for the apparent mobility difference between it and gpl on the SDS-gels used. Nul is the left-most gene on the A map.
&?O TERMINASE
GENES TABLE
EXPRESSION
OF ALTERED
FORMS
OF gpl
AND
1 AND
2
637
5
gp2 BY REVERTANTS GENE 1
TO Two
AMBER
MUTATIONS
IN
Phenotype
Mutation Wild type lamlss
lam576
Number of revertant isolates
Level of gpl expression
ml Migration
!2P2 Migration
+ +
+ +
+ +”
+ Low +
Slow NDb +
Fast” ND +
Low
Slow”
Fast”
Crossover interval
True revertant or2+3 2+5 l+? True revertant or2+3 1+5
Note. Independent revertants to gene 1 amber mutants 463 and 576 were isolated and the products of genes 1 and 2 induced by the isolates following infection of 159 were analyzed on 12.5% SDS-polyacrylamide gels after being labeled with [3H]arginine as described under Methods. The level of expression and mobility of the induced gpl compared to wild type were used as criteria to classify each of the isolates. Representatives of four of these classes are shown in Fig. 4. The mobility of the gp2 induced by each of these representatives were examined (Figs. 4 and 5) and are summarized here. The intervals in which crossover events must have occurred to generate these classes by recombination with a cryptic prophage, as described in the Discussion and Fig. 6, are indicated in the last column. ” Determined for a sinale- reoresentative of the class. b Not determined.
The most recent physical maps have placed the start of the next gene: A at 0.5 (Echols and Murialdo, 1978) or 0.9 map unit (Szybalski and Szybalski, 1979) from the left terminus. The first AUG codon from the left end of A is at position 25, although there is no evidence that this is the start of the Nul coding sequence (Nichols and Donelson, 1978). These figures allow either 230 or 416 bp to code for gpNu1. However, if the size of gpNu1 is taken as 21 kdalton, it would require approximately 570-630 bp to code for it. Thus, either the current estimates of the start of gene A are wrong or Nul at least partially overlaps A out of phase. Since the sizes of the morphogenic genes have all been determined from information obtained from SDS-gels, it is clear that such estimates must be made with caution. We note, however, that recently Murialdo et al. (1980) have found that a putative mutation in the ribosome binding site of A gene A (fincslos) seems to map within the coding region of gene Nul. Since it is thought that the stoichiometry
of the two terminase subunits of A is 2: 1 (gpNu1 :gpA) (Gold, M., and Becker, A., in preparation) it is surprising that 480 produces such a large excess of gpl relative to the gp2 subunit. Since we have been unable to identify gpNu1 in radiolabeled extracts of h-infected cells, we do not know if it is produced in excess relative to gpA. However, preliminary results using an in vitro complementation assay suggest that it is not (Sumner-Smith et al., 1981). Furthermore, a mutant A which overproduces the gpA polypeptide (XAop,, Murialdo et al., 1980) does not show more terminase activity in an in vitro packaging assay unless addition.al gpNu1 from a gpNu1 containing extract (i.e., hAam) is also added (M. Sumner-Smith, unpublished observations). One explanation may be that the laboratory strain of $180 carries a mutation resulting in the overproduction of gpl. It is also possible that evolutionarily $80 is a “recent” hybrid between two lambdoid phages and has not yet adjusted the synthesis of gpl and gp2 optimally. Finally, production
SUMNER-SMITH
638
AND
BECKER
replication of the infecting phage is inhibited. An uncharacterized mutation (Xsti) in the Nul -A region of A will overcome this inhibition by the phage 21 gene products (C. Rudolph and M. Feiss, personal communication). It is possible that the gpNu1 (gpl) subunit is important in determining packaging specificity and that a hybrid terminase, containing the gpNu1 subunit from
a
bed
e
f
g
w*
FIG. 4. The level of expression and mobilities of gpl and gp2 as expressed by representatives of four of the classes of revertants to 1 amber mutations described in Table 5. lam468 rwA
of an excess of gpl may confer selective advantage to $80 in a mixed infection with another lambdoid phage. There is already precedent for the concept that overproduction of one of the terminase subunits can be advantageous to the phage, as Murialdo et al. (1980) have shown that overproduction of gpA makes gpF1 partially dispensible. Although the cos site is the same in both A and the lambdoid phage 21, each phage is unable to package the other’s DNA (Hohn, 1975; Feiss et al., 1979). This difference has been attributed to an evolutionary divergence in both a site to the right of cos which is recognized by the packaging mechanism and in other components of that mechanism (Feiss et al., 1979). Recently it has been shown that if a plasmid carrying several phage 21 genes, including the Nul and A analogs, is transactivated by an infecting A,
lams76 revF
lam576 rwA
lame.76
2am489
FIG. 5. The mobility of gp2 is altered in pseudorevertants to two 1 amber mutants. In order to more clearly show the altered mobility form of gp2 (i.e., gp2*) induced by lam,,revA and lam,,,revF tracks c, d, e, f, and g from Fig. 4 were scanned as described under Methods. The plots presented show the region of the tracks immediately around and including gp2 and are oriented with the upper portion of the gel to the left. The host-encoded band (h) of similar mobility to gp2 described in the text and Table 4 is clearly illustrated in the scan of track g (i.e., Zam,,,).
480 TERMINASE
1
I
639
GENES 1 AND 2 2
cos
#SO
-: cnms-a Interval:
.,
. ...
,; ~*
i’s ..‘..
2 i
i.I
i . I .. ..i
j4
L”.i
;s
‘it...i
qsr*
I
Nul
A
‘ :
FIG. 6. A mechanism for the pseudoreversion of 1 amber mutations by recombination with the qsr’ cryptic prophage. The qsr’ prophage has been shown to be homologous with h between cos and approximately 2.2 map units (%A) to the leff (Fisher and Feiss, 1980, and personal communication; Kaiser, 1980) and is therefore homologous to #30 in this region (see Fig. 1). The region of qsr’ immediately to the left of cos is homologous to A and is therefore not homologous to @30 (Fiandt et al., 1971). We hypothesize that the determinants for the level of synthesis of gpl and gpNu1 (i.e., + and low, respectively) are at the left end of the genes, i.e., probably the ribosome binding site. We arbitrarily place the determinant for the mobility of these products to the right of the amber mutations. The determinants for the mobility of gp2 and gpA are placed within the coding regions common to these two genes. In order to “cross-out” the amber mutation from d&30,a crossover event must take place in regions 1 or 2, and a second, reciprocal event in regions 3, 4, or 5. Thus, the pseudorevertants described in Table 5 are a subset of all possible permutations of the possible double recombination events. The figure is not drawn to scale. No attempt has been made to show a possible overlap in the coding sequences of Nul and A as this would not necessarily affect the predicted outcomes.
one phage and the gpA from the other, might either be inactive or have the wrong packaging specificity. According to this view, an increased synthesis of one or both terminase subunits by the infecting phage might overcome the competitive inhibition by the phage 21 products. Interestingly, $80, which apparently overproduces gpl, is able to replicate in the presence of the phage 21 products and therefore has the sti phenotype (C. Rudolph and M. Feiss, personal communication), even though it has the same packaging specificity as A. Our inability to identify gpNu1 may be due to two factors. First, gpNu1 may not be produced in excess relative to gpA as gpl is to gp2. Second, A induces the synthesis of several major proteins including one form of gplom (Reeve and Shaw, 1978) and gpI (Ray and Pearson, 1976), which have very similar mobilities to the putative gpNu1 and would therefore obscure it. These proteins are not induced by 480 and thus gpl is relatively easy to detect by the techniques used here. In agreement with this, we were unable to detect gpl in several 480-X hybrids carrying genes 1 and 2 from 480, but all of the other genes from A,
despite its assumed overproduction (unpublished observations). We have not observed any cleavage or degradation of either gpl or g-p2 which would account for the apparent difference in molecular weight of these polypepticles in 1 amber pseudorevertants. Two models can be suggested to explain these clifferences. First, replacement of the amber codon by a codon for a different amino acid than wild type could result in a mobility change in gpl as an artifact of amino acid composition characteristic of SDS-gels (see discussion above). We have found, however, that suppression of both lam,,, and lam,,, by three different amber suppressors did not change the mobility of gpl (Fig. 4). In order to explain the concurrent and opposite mobility changes of gpl and gp2, it is necessary to hypothesize that genes 1 and 2 overlap out of phase and that both of the 1 amber mutations examined fall in this area of overlap. Thus, a base change eliminating the amber codon in gene 1 might cause an amino acid substitution in gene 2, thus altering its mobility. A second explanation which we consider to be more likely is that the 1 amber muta-
640
SUMNER-SMITH
tions can be rescued by recombination with a cryptic lambdoid prophage. Most laboratory strains of E. coli K12 carry at least two and probably three cryptic lambdoid prophage (Kaiser and Murray, 1979; Kaiser, 1980). Recently, Fisher and Feiss (1980) have shown that a A (cosl) which is deleted for cos and at least part of Nul can be rescued by recombination with the cryptic qsr’ prophage carried by the host. It is possible that some or all of the “revertants” of the two gene 1 amber mutants selected here are actually recombinanti carrying all or at least part of the gene N&(l) and possibly gene A (2) equivalents of a cryptic prophage. Figure 6 shows the deduced region of homology between the qsr’ prophage and 480. Any two crossover events between an infecting @30 lam particle and the qsr’ prophage which bracket the amber mutation would lead to a recombinant, hybrid phage which would score as an amber revertant. We hypothesize that the amino acid composition of the Nul (1) and A (2) analogs carried by the cryptic prophage might differ slightly from those of their counterparts in 480. Thus, depending on the position of the crossover points, the resulting recombinant phage might carry part or all of the Nul (1) analog and possibly part of the A (2) gene analog from the cryptic prophage. The “gpl” and 732” expressed by this hybrid phage might then differ in mobility on SDS-gels (because of their “altered” amino acid compositions) as is in fact observed. Furthermore, if we hypothesize that the Nul (1) gene from the cryptic prophage is translated with an efficiency closer to that of the A Nul gene, then recombinants which carry the entire gene would then apparently express much less of “gpl,” which would also have an “altered” mobility on SDS-gels. All of the pseudorevertant classes observed (Table 5) can then be explained as recombinants with qsr’ in which the crossover points differ (compare Fig. 6 with the last column of Table 5). ACKNOWLEDGMENTS
We thank Dale Hawkins for her excellent technical assistance in isolating the op,, mutation; Sam Benchi-
AND
BECKER
mol, Jocelyn Shaw, Helios Murialdo, and Michael Feiss for their many helpful suggestions; Richard Fisher, Michael Feiss, and C. Rudolph for generously sharing their unpublished data; and Phillip Youderian and Martha Howe for strains. Supported by a research grant from the Medical Research Council of Canada (MT-3325) and an MRC Studentship to Martin Sumner-Smith. REFERENCES
BECKER, A., and GOLD, M. (1975). Isolation of the bacteriophage lambda A-gene protein. Proc. Nat. Acad. Sci. USA 72, 581-585. BECKER, A., MURIALDO, H., and GOLD, M. (1977). Studies on an in vitro system for the packaging and maturation of phage h DNA. Virology 78,277-290. BONNER, W. M., and LASKEY, R. A. (1974). A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83-88. CAMPBELL, A. (1961). Sensitive mutants of bacteriophage A. Virology 14, 22-32. DE JONG, W. W., JWEERS, A., and COHEN, L. H. (1978). Influence of single amino acid substitutions on electrophoretic mobility of sodium dodecyl sulfate-protein complexes. Biochem. Biophys. Res. Commun. 82, 532-539. ECHOLS, H., and MURIALDO, H. (1978). Genetic map of bacteriophage lambda. Virology 42, 577-591. FEISS, M., FISHER, R. A., SIEGELE, D. A., NICHOLS, B. P., and DONELSON, J. E. (1979). Packaging of the bacteriophage lambda chromosome: A role for base sequences outside cos. Virology 92, 56-67. FUERST, C. (1966). Defective biotin-transducing mutants of bacteriophage lambda. Virology 30, 581583. FIANDT, M., HRADECNA, Z., LOZERON, H. A., and SZYBALSKI, W. (1971). Electron micrographic mapping of deletions, insertions, inversions and homologies in the DNAs of coliphages lambda and Phi 80. In “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 329-354. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. FISHER, R., and FEISS, M. (1980). Reversion of a cohesive end site mutant of bacteriophage lambda by recombination with a defective prophage. Virology 107, 160-173. GORINI, L. (1970). Informational suppression. Annu. Rev. Gent. 4, 107-134. HARRIS, A. W., MOUNT, D. W. A., FUERST, C. R., and SIMINOVITCH, L. (1967). Mutations in bacteriophage lambda affecting host cell lysis. Virology 32, 553 -569.
HOHN, B. (1975). DNA as substrate for packaging into bacteriophage lambdain vitro. J. Mol. Biol. 98,93106.
. d&O TERMINASE HOHN, T., and KATSURA, I. (197’7). Structure and assembly of bacteriophage lambda. Curr. Top. Microbiol. Immud. 78, 69-110. INOKUCHI, H., and OZEKI,H. (1970). Phenotypic mixing between bacteriophage 480 and lambda in vitro and in vivo. Virology 41, 701-710. KAISER, K., and MURRAY, N. E. (1979). Physical characterisation of the “Rat prophage” in E. coli K12. hfol. Gen. Genet. 175, 154-174. KAISER, K. (1980). The origin of Q-independent derivatives of phage A. Mol. Gen. Genet. 179, 547-554. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. MILLER, J. H., GANEM, D., Lu, P., and SCHITZ, A. (1977). Genetic studies of the lot repressor. I. Correlation of mutational sites with specific amino acid residues: Construction of a colinear gene-protein map. J. Mol. Biol. 109, 275-301. MURIALDO, H., and SIMINOVITCH, L. (1971). The morphogenesis of bacteriophage lambda. III. Identification of genes specifying morphogenetic proteins. In “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 711-723. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. MURIALDO, H., and SIMINOVITCH, L. (1972a). The morphogenesis of bacteriophage lambda. IV. Identification of gene products and control of the expression of the morphogenetic information. Virology 48, 785-823.
MURIALDO, H., and SIMINOVITCH, L. (1972b). The morphogenesis of bacteriophage lambda. V. Formdetermining function of the genes required for assembly of the head. Virology 48, 824-835. MURIALDO, H., and BECKER, A. (1978). Head morphogenesis of complex double-stranded deoxyribonucleic acid bacteriophages. Microb. Rev. 42, 529576.
MURIALDO, H., FIFE, W., BECKER, A., FEISS, M., and YOCHEM, J. (1981). Bacteriophage lambda
GENES 1 AND 2
641
DNA maturation. The functional relationships among the products of genes Nul, A and FI. J. Mol.
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