Relationship between the N function of bacteriophage λ and host RNA polymerase

J. Mol. Biol. (1972) 65, 259-272

Relationship between the N Function of Bacteriophage 5 and Host RNA Polymerase ~~GHYSEN-~

AND MARINAPIRONIO~

Laboratoire de Gkndtique, Ddpartement de Biologic MoELculaire, Faculttk des Sciences Universitt Like de Bruxelles, Bruxelles (Received 13 September 1971) A bacterial mutation (ron) has been isolated which prevents growth of some h lines. These lines were found to carry a mutation in the N gene, mar, which remains undetected in ron + strains. A mar phage in a ron strain behaves as an N mutant both as concerns oomplementation properties and viral messenger RNA synthesis. The ron mutation maps between two mutations which affect the RNA polymerase: rif-r and s&r. Complementation experiments confirm that all three mutations (ram, rif-r and s&r) lie in the same cistron, namely, the structural gene for the fl subunit of RNA polymerase. The ran-mar relationship has been analysed ; results suggest a direct interaction

between the viral N product and the bacterial

RNA polymerase.

1. Introduction The functioning of some viral proteins involves an interaction with a bacterial component (Travers, 1969,197O; Travers, Kamen & Schleif, 1970; Dube & Rudland, 1970). The identification of this bacterial component would undoubtedly increase our understanding of the mechanism of the viral function. One way to obtain this knowledge lies in the isolation of bacterial mutations which interfere with a particular phage function. This approach has been used in the case of the N function of phage X. The N product has a critical role in the positive regulation system of h, since it is indispensible for the expression of most other phage functions (Thomas, 1966; Protass & Korn, 1966). Activation by N takes place at the level of transcription (Skalka, Butler & Echols, 1967 ; Taylor, Hradecna & Szybalski, 1967 ; Kourilsky, Marcaud, Sheldrick, Luzzati & Gras, 1968). Its mechanism is unknown. Bacterial mutations which suppress the N function have been recently isolated (Pironio C%Ghysen, 1970; Georgopoulos & Herskowitz, 1971; Friedman, 1971). In our previous paper (Pironio & Ghysen, 1970) we reported the isolation and preliminary characterization of a bacterial mutation (ron) which prevents growth of some lines of bacteriophage ;\. The affected lines were found to carry a mutation in the N gene, mar, which remains undetected in ron + strains. Here we report the mapping and complementation analysis of five mcGrmutations. We also report mapping and complementation data which assign the ran mutation t Present address: Departamento de Bioquimioa, Facultad ‘de Ciencias, Universitad Casilla 6671, Santiago 4, Chile. f Reprint requests should be sent to: Marina Pironio, Labor&ok de G&&tique, libre de Bruxelles, rue des Chevaux 67, 1640 Rhode-St-Genese, Belgium.

259

de Chile, UniversitFj

260

A.

to the /3 subunit

of RNA

GHYSEN

polymerase.

AND We

M.

show

PIRONIO that

the

effect

of the

ron

mutation

does not take place at the level of transcription of gene N, and that the functioning of N probably involves a direct interaction between the N product and the /3 subunit of RNA polymerase.

2. Materials and Methods (a) Phage strains Phages used for the detection of mar mutations are SU.S mutants of Campbell (1961) and of our laboratory (Thomas et al., 1967). X irnrnal was kindly provided by F. Jacob ; X imm434 by A. D. Kaiser; h Go256 by P. Kourilsky; X Nsus? &us53 r14 ~I857 by P. Brachet. h ~113031 is a clear-plaque mutant obtained in our laboratory. (b) Bacterial (i) Indicator

strains

strains

from a cross All our strains derive from RH1127 (argH Zac,, tsz), which was obtained between SB145 (HfrP4X metB argH (X), N. Glansdoti) and a Xs F- derivative of CA244 (HfrH trya,,, lac,, (X), S. Brenner). Suppressor derivatives of RH1127 were obtained by transducing RH1127, which is lac,,, with a Pl lysate grown on suppressor-carrying strains and selecting for the Lac + phenotype. The ron mutation was introduced in RN1 127 and its suppressor derivatives by crossing them with the original ran. mutant, a derivative of RH1154 (HfrPdX metB argE,, sup.3). (ii) Strain8

used in the mapping

of the ron mutation

RH1306 (argH purD &f-r) was obtained by transducing K12S17 (metA, F. Jacob via R. Lava%) with a Pl lysate grown on a spontaneous r$-r derivative of PA3731 (thr lezl thi hk argH pzlrD xyl mtl lac gal ma1 Xr strA sup2, R. LavallB). RH1582 is a v-if-r d-r derivative of 514 (thr leu thi his argE proA try thy xyl mtl lac gal ara strA ants, J. Davies tia A. Bollen). E&e&h&z coli is normally insensitive to streptolydigin and rifamyoin because these drugs (in contrast with rifampicin) fail to penetrate into the cells. Some strains, including S14, harbour a mutation (ant”) which makes them permeable to these antibiotics. Only from such strains one can isolate stl-r mutants, which can be distinguished from antP (impermeable) revertants because they remain sensitive to rifamycin. A stl-r derivative of 514 was obtained after mutagenesis with nitrosoguanidine. A spontaneous rif-r derivative (RH1582) of this mutant was selected by exposure to rifampicin. (c) Phage crosses

and complementations

Crosses were performed by mixed infection of a permissive strain. Cells grown in Tryptone broth (Kaiser, 1955) are resuspended in 10 mM-MgS04, infected at a multiplicity of 3 for each phage, adsorbed 15 min at 37”C, diluted loo-fold in Tryptone and grown at 37°C for 90 min. For complementations we used the same technique except that the host cell was non-permissive. (d) Construction

of rif-0

and domimnt

rif-r

e@somes

The technique of Austin & Scaife (1970) was used to construct and characterize rif-0 episomes. Starting strain is AS5 (argG metB his leu lac gal malA malB recA strA hp rif-r/ F’llO m&B+ rif-s, kindly provided by S. Austin), the rif-r mutation of which is recessive. We selected for phenotypically Rif-r derivatives of this strain. tif-0 were discriminated from rif-r episomes by transfer into RH1127 and study of the frequency of appearance of Rif-r phenotypes. A rif-s ret+ /F’rif-0 diploid yields spontaneous rif -r mutants with a frequency lower than 10b7; a rif-s rec+/F’rif-r diploid yields 10e2 to 10m3 r$r/F’ rif-r homogenotes. The technique of Babinet (1971) was used to construct and characterize episomes carrying a dominant rif-r mutation. Starting strain is RH1327 (argG metB his leu recA strA/ F’IIO me@+ ron) obtained by crossing a ron/F’ron homogenote with JC1569 (argG metB h& Zeu recA str, J. Clark). Spontaneous r
N

FUNCTION

AND

RNA

POLYMERASE

simultaneously the ArgH+ and Rif-r phenotypes when (F- argg pro recA &A ma23 Xr, kindly provided by C. rifampicin resistance when the diploid is cured from its was achieved by the sodium dodecyl sulphate technique Tomoeda, 1969). (e) h Messenger RNA

261

crossed with PA505MBdlOlV3 Babmet), and by the loss of any episome. Curing of the episome (Inuzuka, Nakamura, Inuzuka &

hybridization

Cells grown in medium 70 supplemented with glucose (O-4%}, vitamin-free, charcoaltreated Difco Casamino acids (0.2%) and medium 63 (0.5%) were harvested at a concentration of 2 x lo* cells/ml., resuspended in 0.1 M-Tris-Mg buffer, 10 mivr-KCN, and infected at a multiplicity of 5. After adsorption (15 min at 3X?), cells were centrifuged, resuspended in the same vol. of cold 70 medium and diluted IO-fold in warmed supplemented medium 70. After different times, portions were pulse-labelled for 90 set with [3H]uracil, 20 Ci/m-mole. Extraction of RNA was as described by Kourilsky & Luzzati (1967). Filter preparation and hybridization were performed as described by Kourilsky et al. (1968) except for the following modifications: filters were loaded with 4 pg of X or h imma’ DNA and hybridization was allowed to proceed for 48 hr at 65% in 2 x SSC (SSC is 0.15 M-Nacl; 0.015 M-sodium citrate).

3. Results (a) Detection of $ve mar mutations As described previously (Pironio & Ghysen, 1970) the ron mutant was isolated following infection of an sup3 strain with phage h Nsus7cH, as a survivor in which the amber suppressor is still present and fully functional. Among several amber mutants of X tested, two behave like Nsus7, namely, h Psus207 and Ximm434 CsusS. All three lines are defective on a ron strain, whatever the suppressor present (supl, sup2, sup3 or sup6). In fact, the amber mutations are irrelevant; with the exception of hNsus7 itself, which will be discussed below, even SW+ revertants fail to grow on ran derivatives and, conversely, the derivatives of the affected sus mutants selected for growth on ron strains are not SW+. Clearly, the affected phages carry a genetic alteration (mar) which is responsible for their failure to grow on ron strains. Subsequently, the Mar phenotype was found in lines which carried no sus mutations, namely Abio256 and XcII3031. The genetic alterations present in Ximm434, hPsus207, XNsus7, Xbio256 and Xc113031 were provisionally called marl, 2, 3, 4 and 5. Furthermore, it was observed that all Ximm 434derivatives are marl, except Aimm434Nsus213 and all the red int derivatives obtained respectively by crosses with ~~~~213 and Ared int. The marl mutation is also present in 434 wild type, which was used to construct himm434 (Kaiser & Jacob, 1957). A summary of these observations is represented in Table 1. (b) Mappirzg of the mar mutations marl, mar2 and mar5 were first located roughly by three-factor crosses and deletion mapping. The sites, mutations and deletions used for this study are represented in Figure 1. Results are presented in Table 2. marl. himm434 Osus8 marl was crossed with hNsu.s213 c1 (Table 2, cross 1). Most of the sus + recombinants are mar, which locates marl next to or at the left of Ns~.s213. We studied whether or not the marl + allele could be provided by the heteroimmune X and Ximmzl phages. The results (crosses 3 and 4) show that marl lies outside the region of non-homology between Ximm434and /\, but inside the region of non-homology between Ximm434 and himmzl, therefore probably in the N gene. This conclusion is

A. GHYSEN

262

AND M. PIRONIO TABLE 1

Origin of the mar mutations mar mutation

x line

mar4

434, all &mm434 lines except Aimm434Nsus213 and red in,t derivatives XPsus207 All lines carrying the XNsus7 mutation Abio256

mar5

Xc113031

mCW1 mar2 mar3

supported by the results of a cross between Ximm 434Osus8 marl and X%0256. As can be seen in cross 5, the Go256 substitution does not delete the marl + allele. mar2. hPsus207 mar2 was crossed with hNsus219 (cross 2). The result indicates that mar2 is next to or at the left of Nsus219. As in the case of marl, the mar2+ allele can be provided by Ximm 434but not by himmzl (crosses 6 and 7). mar5. We first tested whether or not the Mar phenotype of Xc113031 was caused by the ~11 mutation, by crossing h Osu.4 mar5 ~113031 with Ximm434 Nsus213 ~1. 103 SUS+C+ recombinants were tested; none of them grew on a ron strain. The mar mutation is thus unrelated to the cI1 mutation, and maps to the left of the region of non-homology between X and Aimm434. It follows from the preceding results that marl, mar2 and mar5 map to the left of immunity, within or close to the N gene. We then mapped all five mar mutations with respect to three Nsus mutations (Nsus7, Nsus213 and Nsus219) by threefactor crosses. Results are presented in Table 3 and summarized in Figure 2. It can be observed that at least two mar mutations affect the N gene, one of them (mar3) being indistinguishable from Nsus7, the other one (man) mapping between two Nsus mutations. The three remaining mar mutations map to the left of Nsus7. However, these three mutations are very closely linked to Nsus7 as revealed both by the frequencies of recombination and by the high negative interference. (c) Cbmplementation studies of mar mutants The behaviour of m.ar mutants in ran strains was investigated by complementation experiments. Four mar mutants have been analysed; three of them map outside the fed --

N

cm I

-

7

CI I!,# I

x

Y

CII Ic-1 -/

-

213 219

3031

0

8

P --

Q I-I-

--

207

c b ---

c

FIG. 1. Mutations, deletions and regions of non-homology used for the mapping of the mczr mutations. between h and a: region of non-homolo,T between h and Aimm4a4; b: region of non-homology Ximmal; c: bio256 deletion.

ni FUNCTION

AND

RNA

263

POLYMERASE

2

TABLE

Mapping of marl and mar2 mutations Cross Infecting

sus+ ST.L.S+ mar+ Production

Total production

phages

SW+

sus+ mar+ Revertants

I

Ximm434 Osus8 marlc+ + Ximm434 Nsus213 c1

3 x 1Ol0 c+ 1.1 x lO%I

2.3 x lo%+ 2 x IO%1

3.2 x 10% + 8 x lO%I

3 x lo%+ 3.5 x lO%I


2

hPsus207 mar2c+ + XNsus219 CII

1.4 x lo%+ 3.3 x 10%11

7.8 x lo%+ 1.1 x 10%11

5.4 x loso+ 1.6 x 107cII

+ 5.6 x 10% 1.7 x lO%II


3

hirnw-~~~~0.~~8 marl + hNsus219

himm4s4 mar+ production

4

Ximm4s4 0.~~8 marl +

+

Ximm434 mar+ revertants

2.1 x 101s

l-5 x 107

4x105

3.9 x 101s

6.7 x lo4

9 x 104

4.2 x lOlo

2-s x 107

5x105

himma himmez4 5

05~38 +

marl

hbio256 hmar +SUS+ production 6

XPsus207 mar2 +

7.8 x 1O1”

8.5 x 10s

1.8 x 1011

<1os

hmar + 8us + reverts&s <5x102

himm434

7

hPsus207 mar2 + Aimmzl

<5x

10s

known Nsus mutations but very close to one of them (marl, mar4 and mar5), and one which must affect the N gene (mar2). None of the mar mutants is able to complement a W- phage (Table 4, crosses a,c,d,h), whereas in the cases tested they complement normally a hP- (crosses b and f), h0 - or XQ- phage (crosses e and g). Furthermore, we confirmed the defectiveness of the N function of hPsus207 mar2 by complementing this phage with 480. This experiment is based on the following facts (Szpirer & Brachet, 1970) : (1) the P product is interchangeable for $80 and h; but this is not the case for the other early functions ; (2) h DNA cannot be encapsulated in a $80 coat unless there is no synthesis of X late functions. The results of this complementation (Table 4, i) indicate a suboptima1 h replication. Furthermore, all the copies of h produced are found in $80 coats, as shown by the fact that they do not adsorb to a strain resistant to #SO. It follows that a mar2 phage is indeed defective for the N function when in a ron host. N ---

clu

I

I mar I .2

II .s”S 7 mot- 3

I i-nor 2

FIG. 2. Ordering of five mar and three Nsus mutations. mutations were ordered according to the data, of Table was already known (Thomas et al., 1967).

II sus 213

immunity I --sus 219 I

Except for &us213 and sus219, all the 3. The order sus7-sus213-sus219-imm

3 x 1050” 5.1 x 1050 5.1 x 1050 5.1 x 1050

1.4x 1070’ 4.9 x 1060 2.7 x 10Bc+ 1 x 1070 5 x loSo+ 1.8 x 1070

5.6 x lOloo + 1.4 x 10% 3.2 x 10r”c + 2.6 x 10% 1 x 1o11c+ 2.8 x loll0 8.2 x loloo + 6.3 x 101oc 7 x 10IOc + 1.8 x lo~lc

+ XNsus213cI

+ xNsus2l9cII

+ Xmar3su87c+

+ ANsus213c+

+ xNsus219c+

+ hmar5cII3031

7 x lo~lc

1.2 x loec+

8.7 x loec+ 9 x 10%

1.5 x lo$+

7.6 x 101”c+ 9.7 x 101%

+ hmar5cII3031

2 x 10°C

t1.3 x 1070+ 4.3 x 1060

2 x lo%+

2-2 x lore+ 4.5 x 10%

1.4 x loso+ 1 x 10%

2.9 x 10% + 5.3 x loloo

1.6 x 10”~

However,

the ratio

9.4 x 1040 *

2.2 x loBc+

3.9 x 1050 +

2.8 x 1050

7 x 1050

-imm

among the recombinants

mar5

mar4

mar2-su8219-imm

mar2-sus213-immf

mar3 -mar2-imm sus7

marl-sus219-imm

marl-sus213-imm$

-imm

marl

7.5 x loec+ 5.7 x 10%

mm-5

marl -imm mar4

mar3 -imm su.s7

1.5 x loSo+ 1 x 1060

marl-

3.1 x 1050

order

mar2-imm

Inferred

8x106c+ 3.4 x 10%

+ Xmar4bio256cIII

9.3 x 10~0 + 1.2 x 1060

t9.5 x lo$+

6.4 x 101Oc+ 3.6 x 101Oo

sus +

marl

+ hmar3suslcI

Revertants 7.5 x 105Cf 5.1 x 1050

mar+

1.3x107c+ 3.5 x 1060

mar + sus +

7.5 x lo%+ 2.6 x 101lc

+ Amar

Production

t All mar+ recombinants tested (180 out of 180) are SW+. 3 The inference may seem not justified in view of the nearly equal o + /c ratio in the total production. is remarkably insensitive to the parental ratio (Cordone & Radman, 1970).

Xmar4bio256cIII

hmar2cI

himm434ma~lc+

cross

mar+

of Jive mar and three sus mutations

3

Total Production

Ordering

TABLE

N

FUNCTION

AND

RNA

TABLE

Punctional

cross

analysis

Infecting

,&mm434 Osus8 marl hmar4 + Xmar5 + + +

i

+ h N+ AP-

Ximm434 NXimm434 NhoXimm434 P-

hPsus207 mar2

4

of four mar mutations:

marl,

mar *us Production

phages

1.3 83 0.48 2.8 70 160 64 0.16

4.4 166 1.7 3 17.6 82 57 0.4

h

5480

+ 480

265

POLYMERASE

13.3

700

mar4 and mar5

mar2,

mar + sus +

mar + Revertant’s

0,036 0.0075 0.05 0.023 0.023 0.023 0.023 0.00027 X mar+

*us +

0.018 0.34 0.86 0.03 0.24 0.024 0*0028 0.052 mar phages in a h coat? 0,056

ma+

revertants 0.00027

Results are expressed as phage produotion per bacterium. Complementations were performed in a rolz suppressorless strain. Phage lines were: Xmar4: Xmar4 bio256; hmar5: Xmar5 ~113031; himm434 N- : himm434 Nsus213 ~1; kimm 434 P- : himm434 Psus3 Ted int ~1; Ximm434 Q- : Ximm434 Qsus203 Rsus216 red int ~1; X N- : XNsw219 ~11; h O- : hOsus125; XP- : hPsus3. t The phages are titrated on CBOO, a strain resistant to $80.

(d) Mapping

of the ron mutation

The genetic map of the bacterial mutations used in these experiments as well as the representation of our results is shown in Figure 3. Preliminary Hfr crosses have shown the ron mutation to be closely linked to the argECBH cluster. We attempted to cotransduce ron with metB. The percentage of cotransduction (10%) localizes ron between the arg cluster and purl). This small region of the E. coli chromosome contains (Tocchini-Valentini, Marino & Colvill, 1968; rifampicin resistance (rif-r) mutations Babinet 8t Condamine, 1968) which have been shown to alter the b subunit of RNA polymerase (Rabussay & Zillig, 1969). We looked for the proximity and relative positions of ron and rif-r by transducing an argH purD rif-r recipient strain with a Pl lysate grown on an arg+ pur+ rif-s ron strain, and selecting either for arg+ or for pur+ recombinants. Results are given in Table 5. Clearly the order is argE-rif-r-ronpurD. Furthermore, rif and ron are closely linked: only 2% of the transduced bacteria are recombined between the two markers. This result led us to check the possibility that ran also affects a subunit of the RNA polymerase. Thus, ron was mapped with respect to another mutation affecting the enzyme: stl-r (streptolydigin resistance, which is also cotransducible with the arg cluster; Schleif, 1969). The recipient strain was RH1582 (argE rif-r d-r; see Material and Methods). The arg+ character was transduced from a ron rif-s stl-s strain. Results are given in Table 6. The order of the mutations is argE-rif-r-ran-d-r. The distance between rif-T and ron is the same as that between ron and &l-r, all three mutations

I

met B

I I

erg ECBH

FIQ. 3. Map of the metB-metA

I

I

rif ran stl

1 I

pur D

I

met A

segment of the E. cold chromosome.

266

A.

GHYSEN

AND

M.

PIRONIO

being extremely closely linked (a’mong the arg+ recombinants, 2% are recombined between rif and ron, and 2% are recombined between ron and stl). This result is consistent with the data of the previous transduction (Table 5). TABLE

Transduction Selected marker

Unselected

rif

w arg +

pur +

5

ordering of the argH, rif-r, ron and purD mutations

I 0 0 0 I

markers

Ton I I I 0 0

I I 0 0 0

I I 0 0 0

I I I 0 0

Number

Pur

I 0 0 0 I

of recombinants

21 50 3 58 1 30 28 1 34 1

The recipient cell is an argH pwD rif-r Ton+ derivative of K12S17. The donor cell is an arg + pur + r-$-s Ton strain. Genotypic description of the recombinants is made as follows: symbol 0 means that the allele of the recipient cell has been kept, whereas I means the presence of the donor cell allele.

(e) Complementation

studies of the ron mutation

Streptolydigin resistance was recently reported to affect the ,B subunit of RNA polymerase, as does rifampioin resistance (Heil & Zillig, 1970). This observation together with our mapping data should indicate that rif-r, ron and stl-r lie in the same cistron. We confirmed this conclusion by a complementation analysis involving all three mutations. The first step of this analysis was the determination of the dominance or recessivity of ran, rif-r and d-r. We crossed a recA proA male containing the FllO episome (which covers the metB-purD segment) with an argE ron female. The resulting arg+pro+ derivatives display a Ron+ phenotype. The episome is not integrated in these derivatives, as judged by their ability to transfer with high frequency the met’ character to a metB recA female. Furthermore, curing the presumed arg ronlIT’ arg+ ran+ diploids made the Ron phenotype reappear. Clearly ron is a recessive mutation. The same set of experiments was performed with the two stl-r mutations used in this work; both proved to be recessive at the streptolydigin concentrations used (100 pglml.). As concerns the rif-r mutations, we used both recessive and dominant mutations. Recessivity was tested as indicated for ron and d-r. Dominant rif-r mutations were used only on an episome. They were characterized as explained in Materials and Methods. A first set of experiments was based on the recessivity of ron, stl-r and some rif-r mutations and on the existence of rif-0 episomes (Austin & Scaife, 1970), where the rif cistron is mutated so as to produce inactive subunits. Such an episome will not prevent the phenotypic expression of recessive rif-r mutations, nor of any other recessive mutation which affects the same cistron as rif-r.

N

FUNCTION

AND

RNA

TABLE

267

POLYMERASE

6

Pl transduction orderilzg of rif-r, stl-r and ran mutations Unselected

marker

rif

mm

stl

I I I 0

I I 0 0

I 0 0 0

Number

of recombinants

129 8 7 215

Donor cell is an arg* rif+ stl-s ~‘0%strain; recipient cell is an argE a strain sensitive to antibiotics. Selected marker is argE +. Genotypic nants is as in Table 5.

rif-r &l-r ran+ derivative description

of of the recombi-

Seven independently obtained rif-0 episomes were characterized as explained in Material and Methods. They were transferred into argE stl-r and argE ron female strains. Five arg+ derivatives of each cross were tested for the presence of the episome by crossing with an appropriate recA female, and for the Stl-r or Ron phenotype. All s&r/F’ rif-0 diploids proved to be phenotypically Stl-r and all ran/F’ rif-0 diploids were phenotypically Ron. This result was found with each of the seven rif-0 episomes used; the rif-8 episome used as a control made both Stl-r and Ron phenotypes disappear. A second set of experiments was based on the in vivo dominance of some rif-r mutations (Babinet, 1971), which will be called rif-R. In a rif-if-slF’rif-R diploid, the addition of rifampicin inactivates the rif-s subunit without affecting the rif-R subunit. Any recessive mutation which affects the same cistron as does the rif-R mutation will then be expressed in the presence of the drug but not in its absence. An argH recA rif-s ron+lF’rif-R ron diploid was thus constructed, the phenotype of which was studied in the absence and in the presence of rifampicin. This diploid displays a Ron + phenotype in the absence of the drug, as one would expect from the recessivity of ran. However, addition of rifampicin makes the phenotype Ron, thus indicating that rif and ron affect the same cistron. The validity of this interpretation is confirmed by the fact that the ran+ allele is inactivated in the presence of rifampicin only if it is located cis Do rif-s; a rif-,f-sron,/F’ rif-R ron + diploid is Ron + even in the presence of rifampicin . (f) Does the ron mutation afleecttranscription of N ati Nmar genes? A trivial explanation for the Ron phenotype could be that there is a decrease in the affinity of the ron RNA polymerase for the N promoter and/or some transcriptionblocking effect at the level of the mar mutation. We investigated this hypothesis by looking at the mRNA synthesized by hN- and /Wmar in ron and ran+ strains. h mRNA synthesized in vivo was extracted and hybridized as explained in Material and Methods. The results are presented in Table 7. It appears that the transcription of a XN- phage is identical whether in a ron or in a ran+ strain. Transcription of an N- phage is limited to the pre-early genes (N and xy) (Kourilsky et al., 1968; Kumar et al., 1969). Thus, any significant difference in the level of transcription of the N gene could have been seen had it existed (as in the case of hsex; Roberts, 1969; Nijkamp, Bsvre t Szybalski, 1970). Furthermore, the level of transcription of a mar

268

A.

GHYSEN

AND

M.

PIRONIO

phage in a ron strain is identical to that of an N- phage in either ron or ron + strains, as concerns both the level of hybridization and the ratio of hybridization with X and himrnzl DNA. Those results make very unlikely the interpretation of the rolzmar interaction as being at the level of the N gene transcription. Furthermore, they give biochemical support to the conclusion that a mar phage in a ron strain behaves as defective for the N function. (g) Functional analysis

of the ron-mar interaction

Most h lines grow in a perfectly normal way on ron strains. However, in addition to the mar mutants, which fail to grow on any ron-carrying strain, some h mutants are affected by the ron mutation in particular conditions. For example, all Nsus mutants fail to plate on ron strains when weak (ochre) suppressors are present; Psus mutants are slightly affected in the same conditions. Efficiencies of plating of QSTLS mutants decrease in the presence of the weakest amber suppressor (sup2) if the strain is ron. Furthermore, Qsus mutants are less “leaky” on suppressorless strains if these are also ron (Dambly & Couturier, personal communication). It has been observed that in SLsystem where N is given in trans to induce the Q gene of a hri”, the induction is less effective in a ran host (Dambly & Couturier, personal communication). Finally, it was found that the three known Nts mutants (Brown & Arber, 1964) fail to grow on ron strains even at low temperature. All these observations would be accounted for by the idea that even in an N+ phage the ron mutation causes a partial inhibition of the N function. We have tried to check this hypothesis, and conversely have studied whether the Nmar product is by itself less active than the Nmar+ product in a ran+ strain. The effects we were looking for cannot be pronounced, since in normal conditions TABLE

7

Synthesis of hNsus and hNmar mRNA in ran+ and ron suppressorless

Host strain

Infecting @age

ran + TO?%* Ton Tim

NNwmr NNmar

Ton

NNmar NN??UT NNWKbT NNWKW

+

ran+ ran Tcm Ton

+

Ton + v-on Ton

Pulse (min after infection)

strains

o/o of RNA input hybridized with Xrnmz= DNA hDNA no DNA

0.03 0.03 0.02 0.03

0.32 1.4 0.31 o-31

0.14 0.72 0.14 0.13

10-11.5

0.02 0.04 0.03 0.03

0,21 2.1 o-29 0.30

0.12 1.66 0.12 0.13

20-21.5

0.04 0.02 0.03 0.03

0.33 4.3 0.32 -

0.14 3.5 0.14 0.15

5-6.5

In viva [3H]RNA samples were prepared as described in Materials and Methods. Hybridization reactions were carried out in 2 x SSC at 65°C for 48 hr in a total volume of 1 ml. The last two columns give results after subtraction of the hybridization background measured with a filter lacking DNA. Phages were: hN- = XNsusBlYcI; hNmur = hNmar2c+.

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TABLE 8

Activity

of Nmar+ product in a ron strain and of Nmar product in a ran+ strain Infecting

ran+ host

phages

N-r14 N-r32 N+ONmar ON-r14 + N+ON-r32 + N+ON-r14 + Nmar ON-r32 + Nmar O-

0.02 0.016 0,028 0.029 310 210 210 220

ron host 0.016 0.017 0.027 0.013 91 13 0.023 0~028

Results are expressed as phage production per bacterium. Complement&ions are performed in suppressorless strains isogenic except for the rolt character. Phages used are: N-r14 = hNsus7 Nsus53 mar3 ~14 ~1857; N-r32 = ANsus7 Nszcs53 mar3 r32 ~1857; N+O- = Ximm434 05~~8 red OsusS marl c+. int ~13068; Nmar O- = Ximm434

Nmar + phages grow well in the presence of the ran mutation, and Nmar phages grow as well as Nmar + phages in the absence of ran. Therefore we used a complementation system in which the requirement for the N function would be stringent. The principle of this complementation was to look at the gene 0 production of an N- phage while supplying the N product in trans. Pull expression of the 0 gene requires the action of the N product (Thomas, 1966, 1970). Furthermore, the N- phage we used carries an r14 or r32 insertion in the ~11 region, which makes gene 0 expression even more dependent upon N than it normally is (Brachet, Eisen & Rambach, 1970). The phage which gives the N product is itself O-, so that its growth requires the supply of 0, which in tram is necessary in huge quantities (Brachet 82;Green, 1970). Ultimately, growth of both phages relies on the functioning of the N product of the O- phage. This sytem was used to emphasize any functional difference between N+ and Nmar in a ran+ host, or any effect of ron on the normal N product. Results of this experiment are presented in Table 8. As expected, the complementation is complete when a Nmar+ product acts in a ran.+ strain; and there is no complementation at all when the N product is mar, and the host is ran. The ron mutation clearly affects the functioning of the Nmur + product, which confirms our initial idea. However, this inhibition is far from complete. This is expected, for if this inhibition were complete, even N + phages would not grow in ron strains. On the other hand, there is no detectable difference between N + and Nmar in a ron + host, which suggests that, the two products are functionally indistinguishable in a ran+ strain.

4. Discussion moor mutations are characterized by their failure to grow on any ron-carrying A survey of all h lines available in our laboratory resulted in the identification of four such mutations (marl, 2, 4 and 5; the case of mar3 will be discussed below). None of those mutations is distinguishable on any ran+ strain; all of them map in gene N and behave as N- mutations in ron hosts. In addition, as mentioned in section (g) above, there is a heterogeneous set of h mutants which are affected by the ron mutation to variable extents and in particular strain.

18

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conditions only. Most of those lines are clearly not mar: they grow at least on some roll strains, moreover, the mutation responsible for the occasional Mar phenotype can be detected in ran+ strains and maps outside of the N gene. The situation of mar3 and the Nts is less obvious. In fact, mar3 and Nsus7 could not be separated by recombination, and part of the SW+ derivatives are mar+ (Thomas, personal communication), suggesting that the same genetic alteration is responsible for both abnormalities. As for the Nts, it is not clear in what category they should be classified. The mar mutations were not selected for; they were present, but undetected, in several X lines. Such an abundancy of unselected mutations may seem surprising. However, the existence of most of them can be explained, at least tentatively. marl is not a true mutation. It is found in wild-type phage 434 and in most of its derivatives. One is presumably dealing here with some pecularity of the N gene of phage 434. marl is much closer to the immunity region than the marker (~11144) which was used in the construction of Ximm4a4; it is thus not surprising that it was conserved through all the crosses with h. On the other hand, marl is closely linked to the Nsus213+ allele, which explains why introducing Nsus213 in himm434 makes this phage (and consequently all its derivatives) mar +. The marl mutation has some strange characteristics; for example 20% of the mar+ revertants have a clear phenotype, and marl mutants do not complement AimmzlP- in a ran host (Pironio, unpublished results), whereas usual N- phages do (Herskowitz & Signer, 1970). Among the other mar mutations, only mar5 shares the characteristics of marl (Pironio, unpublished results), which led us to suppose that mar5 might be identical to marl. The finding that marl and mar5 do not yield recombinants when crossed is consistent with this view. Furthermore, Amar ~113031 results from a cross between X and Ximm434mar5cI13031. This genealogy supports the idea that mar5 might be the marl mutation acquired by X through this cross. The origin of the mar2 mutation, which was found in hPsus207, is not obvious. The case of mar3, associated with Nsus7, has been discussed above. The mar4 mutation affects hbio256, the deletion of which extends close to the N gene. A trivial possibility is that the bio substitution exerts a position effect on the N gene since Inokuchi, Franklin & Dove (personal communication) have found bio256 to be deficient in N activity. However, one finds mar+ revertants of bio256. The particular case of marl, which is in fact a determinant of the N gene of phage 434, led us to study whether other lambdoid phages could display a Mar phenotype. Of course if this were the case it would suggest the existence of an N-like function in the affected phage. Roth 21 and #30 grow normally on ron strains. This negative result does not allow any conclusion regarding the positive regulation mechanism of these phages. If we look now at the interaction which makes mar phages defective on ron strains, we may ask whether this interaction is direct or indirect. The finding that ron slightly affects the N function of mar+ phages does not by itself permit one to decide. Any N inhibiting effect can be explained by an indirect interaction; for example, by supposing that the RNA polymerase mutation prevents the transcription of some other bacterial product which is the one directly interacting with the N product. Furthermore, the slight inhibition of N function we observed in ron strains might suggest a “cumulative ” interpretation of the ronmar interaction : if the mar mutation makes N protein less active in a ran+ strain, then the additional inhibition by ran might account for complete defectivity. However, this seems not to be the case. SO

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271

far as we can tell, there is no difference in activity between N+ and Nrnur proteins. The fact that the ron product discriminates N from Nmar suggests that it distinguishes structural features of these products, and this in turn means that there is a direct interaction between the N protein and the ran product, the p subunit of the RNA polymerase. In summary, we propose the following interpretation of our results: the N protein forms a complex with the /3 subunit of the RNA polymerase. ron mutation somehow loosens this interaction or makes the complex less active. mar mutations modify the structure of N in such a way that it completely prevents formation of the Nmar-/3ron complex or makes this complex inactive. We thank Professor R. Thomas and all our colleagues of the laboratory for helpful discussions on X, Drs C. Babinet and S. Austin for strains and advices on the genetics of RNA polymerase, Drs P. Kourilsky and N. Sternberg for their help in the hybridization experiments, Drs C. Allende and R. Thomas for critical reading of this manuscript. This work was carried out under the contract Euratom-ULB 007-61-10 ABIB and with support from the Fonds de la Reoherche Fondamentale Collective and by the Belgian Government. One of us (A. G.) is an aspirant au Fonds National de la Recherche Scientifique; the other (M. P.) holds a Euratom fellowship. REFERENCES Austin, S. & Scaife, J. (1970). J. Mol. Biol. 49, 263. Babinet, C. (1971). Biochimie, 53, 507. Babinet, C. & Condamine, H. (1968). C.R. Acad. Sci. Paris, 267 D, 231. Brachet, P., Eisen, H. & Rambach, A. (1970). Molec. Gen. Genetics, 108, 266. Brachet, P. & Green, B. R. (1970). V&roZogy, 40, 792. Brown, A. & Arber, W. (1964). Virology, 24,237. Campbell, A. (1961). Virology, 14, 22. Cordone, L. & Radman, M. (1970). Virology, 41, 166. Dube, S. K. & Rudland, P. S. (1970). Nature, 226, 820. Friedman, D. I. (1971). The Bacteriophage A, ed. by 9. D. Hershey. New York: Cold Spring Harbor. Georgopoulos, C. P. & Herskowitz, I. (1971). The Bacteriophage X, ed. by A. D. Hershey. New York: Cold Spring Harbor. He& A. & Z&g, W. (1970). FEBS Letters, 11, 165. Herskowitz, I. & Signer, E. R. (1970). Cold Spr. Harb. Symp. Quant. Biol. 35, 355. Inuzuka, N., Nakamura, S., Inuzuka, M. & Tomoeda, M. (1969). J. Bact. 100, 827. Kaiser, A. D. (1955). Vivirology, 1, 424. Kaiser, A. D. & Jacob, F. (1957). Vi’irology, 4, 509. Kourilsky, P. & Luzzati, D. (1967). J. MoZ. BioZ. 25, 357. Kourilsky, P., Marcaud, L., Sheldrick, P., Luzzati, D. & Gras, F. (1968). Proc. Nat. Acad. Sci., Wash. 61, 1013. Kumar, S., Bcvre, K., Guha, A., Hradecna, Z., Maher, Sr. V. M. & Szybalslri, W. (1969). Nature, 221, 823. Nijkamp, II. J. J., Bsvre, M. & Szybalski, W. (1970). J. Mol. BioZ. 54, 599. Pironio, M. & Ghysen, A. (1970). ilfolec. Gen. Genetics, 108: 374. Protass, J. J. & Korn, D. (1966). Proc. Nat. Acad. Sci., Wash, 55, 1089. Rabussay, D. & Zillig, W. (1969). PEBS Letters, 5, 104. Roberts, J. W. (1969). Nature, 224, 1168. Schleif, R. (1969). Nature, 223, 1068. Skalka, A., Butler, B. & Echols, H. (1967). Proc. Nat. Acad. Sci., Wash. 58, 576. Szpirer, J. & Brachet, P. (1970). Molec. Gen. Genetics, 108, 78. Taylor, K.: Hradecna, Z. & Szybalski, W. (1967). Proc. Nat. Acad. Sci., Wash. 57, 1618. Thomas, R. (1966). J. Mol. Biol. 22, 79. Thomas, R. (1970). J. Mol. Biol. 49, 393.

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