The gol site: A Cis-acting bacteriophage T4 regulatory region that can affect expression of all the T4 late genes

J. Mol.

Hiol.

(19X2)

155, 395-407

The gal Site: A C&acting Bacteriophage T4 Regulatory Region that can affect Expression of all the T4 Late Genes WENDY Co01,Ey Michigan (Reruiwd

CHAMPKESS

ASD LARRY S?u’Yl)Elt

Departm,ent of Microbiology and Public State University, East Lansing, Mich. dl March

1981~ and in revised form

Health 48824.

21 September

I’.S.=l 1981)

We have shown that mutations in the Escherichia coli lit gene can prevent the expression of the late genes of bacteriophage T4 at temperatures below 34°C. The defect in late gene expression occurs, at least partially. at the level of transcription, and neither DNA replication nor DNA encapsidation into phage heads is significantly affected. Rare T4 “gal” mutations overcome the defect in late transcription. Refined mapping experiments place gal mutations within gene 23. but an altered gene 23 protein is not responsible for the phenotype. Rather, gol mutations seem to alter a &s-acting site in T4 DNA, the wild-type form of which interferes with late transcription in lit- hosts.

1. Introduction The intracellula,r structure of the DNA of even simple procaryotes and viruses has proved relatively intractable to biochemical a’nalysis. The DNAs are highly folded and often supercoiled, but beyond this little is known. It seems reasonable to suppose that there exist periodic sites on DXA that are involved in the formation and/or maintenance of DNA structures. However, it’ is difficult to predict how one might ident@ such sit’es. since it is not clear what effect, mutations in the sites would have on phenomena such as replication, recombination and tjranscription. It has been proposed that a DKA structure created during replicatjion is required for the t,ranscript’ion of the true-late genes of bacteriophage T4 (Riva et nl.. 1970). The genes are called the “true-late” genes to dist’inguish them from t,hose earl? genes that are transcribed early as well as late after infection, but whose> transcription, at least at early times. is not coupled to the replication of the DNX. The DNA structure is probably fragile because infected cells must be lysed and treated in particular ways to achieve true-late transcription in vitro (Rabussay & (ieiduschek. 192’7). There may be alternative ways of creating the DNA structures other than replication. since some transcription of these genes occurs in the absence of replication. but it is delayed and dependent on the multiplicity of infection (Wu B Geiduschek, 1975). While most) studies of T4 true-late gene expression have focused on the role of T1 395 (~22-2836/82/080395-13 19

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‘C I982

Academic

Press Inc.

(lxmdorr)

Ltd

396

W

gene

products,

would

it seems

likely

he of particular

regulation

in

Escherichin

co/i

1979).

The

uninfected gene. cells

wit)h

expression

at temperatures

replication

are

affected.

Rat-r T1 mutants or “gal”

“p&it”

D’r

showed

T4

19iS).

IIN=\

doesn‘t,

In

rather happen)

have

this

involved.

identified

a

on t’he

in

candidate

genetic

is specific

we

linked gene

site

rates

co-dominant,

present

host

of late for

evidence

that

for

genes

such

for

map

((‘o&y

((‘ooley

7‘4

late

ef 01.. 1979).

expression

gene

gene

the

lat’e

hosts.

\vit,h

wiltf-

in gette 83 (C’ooley

gal mutations host

These

in lit-

exprrssion

mutation

In a lit-

prrvrnts

normal

an

uf ~1..

expression. gene

late

t.o an amber

product.

that

to gene

in a Iit-

normal are

is required

host

transcriptional

mutant, allele (Lit-) fail to support late genv 34°C. Only lat’e gene expression and not T4 T)S,4

effect,

gene

Such

involved

of this

a diffusible at

he

at 25 minutes

exhibit

paper.

also

the below

they are closely

than

are could

allele

go2 muta.tions this

I,. SNYDER

genes they

We

gene.

so the

mutants

t,hat

host.

exist that cat1 multiply

t.ype T4 and that et trl..

lit

.4X1)

because

cells. the (lit’)

and

that.

interest

wild-type

expression.

(‘. (‘HAMPSESS

define

something transcription

a site

happens

on (or

of

T-l

t)act,eriophage.

2. Materials (a)

Ractprinl

and Methods and

phacgp

stmins

The amber mutants were obtained from K. \Yantlcrslice stock collection. The mutations will be referred t,o by gene mutation Hl 1 in gene Z? is 23amHll. T4 got6B was forming mutant on E. coli MPH6. a lithost. (Poole?; et

and II;. B. Wood or w-uerr from our and by name. For example, amber isolated as a spont,aneous plaqueal.. 1979).

(ii) E. coli stmins CR63 (supD) was used for crosses

and B834 (su -) (Wood. 1966) was used to detect ant+ recombinants and as the lit+ host for physiological experiments. In the original experiment,s. t)he effect of host lit- mutations on T4 development was studied using the-K-12 strains from which lif - mutant,s were first isolated (Cooley it al.. 1979). It is difficult to achieve synchronous infections of K-12 strains, so we transferred a lit- mutation into the B strain, clockwise from E. coli B834, as follows. An Hfr strain. ltH041, which is purB- and transfers 97 min. was obtained from B. Bachmann and the Yale Stock Collection. A lit- mutation, lit-c. was transduced with bacteriophage PI int,o this strain from MPH6 using PUT+ as the select,ed marker. .An Hfr litrecombinant was then mated with E. coli B834 made hl:s- by mut.agenesis with nitrosoguanidine (Adelberg et al., 1965). Two out of 80 his+ recombinants of the mating were IX. One of these, E. fnfi B834-l&03, was the lithost used for all the experiments to be described. Since only one lithost was used it will be referred to simply as “the lithost”.

Crosses and infections were performed in M9 medium as described previously (Snyder at al., 1976). To measure phage yields the cells were grown in tryptone broth at 37°C and infected at a total multiplicity of infection (m.o.i.) of 10. After 5 min, T4 antiserum (Cappel) was added and at, 13 min the cells were diluted lo3 times. At 90 min chloroform was added, and the phage were diluted further and plated. In some experiments it was necessary to determine accurately the ratio of gal- and gal+ phages in mixed infections. To accomplish this. the mixture of phage to be used or the phage released were plated on lit+ E. coli. A sector of the plate that contained about 100 plaques was chosen and the percentage of the plaques due t,o gal- mutants was determined by t.oothpicking all of the plaques in this sector onto a plate with litindicator.

A ~~.Y-ACTING

SITE

IS

of’

T4 protein

(c) Analysis

T1

QE:ICE and

E:XPRESSIOS

307

RLVAsynthP.sis

To label proteins, [3sS]methionine (1000 Ci/mmol, 10 pC’i/ml) was added to cells infected at 30°C in M9 medium without Casamino acids. The procedures and buffers of Studier (1973) were used for the processing of the samples and slab gel electrophoresis. The gels were 11 (‘Cl (w/v) acrylamide. with the exception of the gel shown in Fig. 3. which was 95’?4 acrylamidr. (:els were stained (to ensure that total protein per column was uniform), dried, and used to expose XRP-1 Kodak X-rap film for varying lengths of time. To label RNA, [5-3HJuridine (26 C’i/mmol, 10 &i/ml) was added to cells infected at 30°C as above. The RNA was extracted wit,h phenol as described by Belle et al. (1968). except that the Ri’CA was and treated with 1Opg RNAasr-free precipit)ated with alcohol after 1 extractions DSAase/ml (Worthington) for 200 min at 37°C before the final :! extractions with phenol. The purified “I” and “r” separated complement,ary strands of T1 RSA were a very generous gift from Dietmar Rabussay, who also helped with the hybridization assays. Hybridization was hy the liquid method in 2 x SSC (SSC is 0.15 M-NaCl, 0015 M-sodium citrate) for 8 h at 60°C’. The hybrids were collected on nitrocellulose filters and washed with 05 M-KU, 041 MTris. HCI (pH 7.9).

3. Results (a) T4 genr

Pxprmsion

in the lit

h,ost

\Ve reported previously that host lit- mutations prevent T4 late gene expression (Cooley et al.. 1979). Figure 1 (columns A and a) shows t,he synthesis of T4 proteins late in infection of the lit+ and lit- host. respectively. At’ this labeling time, some T1 late prot,ein synthesis occurs in the lit - host, hut’ it is reduced in rate compared t>o the lit’ contjrol. At later labeling times, T4 protein synthesis has almost totally c>easd in a lit- host (Cooley et al., 1979). Host lit- mutations only prevent T4 late gene expression at temperatures belo\% 31’C’ (Cooley et nl.. 1979). Furthermore, we have found that the temperature at which the cells were grown prior to infection is important,. More lat)e protein synthesis occurs in lit - cells grown at 37°C’ and infected at 30°C’ t.han in cells grown and infected at 30°C (data not shown). This indicates that the temperature dependence of late gene expression in a lit- host is not due to the temperature dependence of a particular reaction, but rather to differences in cells grown at different temperatures. It is interesting to contrast the block imposed on T4 late gene expression by host lit mutat,ions with t,hat imposed by most T1 mutations. which prevent the synthesis of true-late proteins: for example, DSA-negative or gene .55 mutants. In the latter cases. early protrin synthesis contjinurs and t)he product of T4 gene 32 is sometimes grossly overproduced (cf. Gold et 01. 1 1976). After infection of the lit - host by wildtype T1, true-late proteins are made in reduced amounts, but early protein synthesis also ceases. probably somewhat’ earlier than usual, and the gene ,‘J2 product is not overproduced. This is demonstrat’ed in Figure 1, where we show the results of infecting with T4 having amber mutations in genes 44 and 42. the products of which are required for DNA replication. Now. as mentioned above, the synthesis of the products of many early genes, including 42 and 4.3, continued late into infection (Fig. 1. lanes b and c). In fact, the program of gene expression in the lit- mutant was not very different from that in the lit+ host (compare with Fig. 1. lanes H and C). A similar result was obtained with a T4 gene ,55 mutant. except, that

.0 p37-

_ _I

-

p43

-

p42

II

f

pIEi-

p23p23*-

I

III att earlier

papw

((‘ool~~y rt trl.. I %!I). NY’ ~m~posed that

T4 late grtte c~xptwsiott rx~tcrin~rtit Ii/+

itlld

T-I I)SA.

lit-

at the Ir\-rl of tri~ttsc~ril)tiott.

showti in T;t\)lc

1 \\‘P t~;~dioac~ti~rly lalwled

hosts atltl hybridized If t,hta tlrfwt

tflr

host lit rntttatiotts

I)lock

To f)rove this \v(’ prrfortttrti KS.\

Iatrb in itifwtiott

KS.4 to the c~ornf)lrn7~titwt~~ )’ atttl I

o~~~urs at tttr, level of trartwriptiott.

WP wortltf

thrl of tlw

Stl~~IltlS

not cq)wt

Of

A cis-.4(‘TIS(:

fhP lit

SITE

IS

T4

GESE

EXPRESSIOS

3!)9

Hyhridizntion of KS.4 labeled ufter infection of host to thP sepnrrrtrd con~plmentnry strands of T4 DAL4

much hybridization to the r strand wit,h the RNA made in the Zif- host, since T-t late RX=\ hybridizes mostly to t’he r strand of T4 DR’A. With RNA prepared aft,et ittfectiott of the lit+ host. 520~ of the input RSA h,vbridized to the r &t-and of T-l DN.4. while in the lit- case only IO”,, hybridized to the r strand. Sot only did the percentaye of KS.4 hybridizing to the r strand drop, but the total hybridization tjo the wparatd strands also dropped. The latter result is reproducible and suggests that the distribution of t)he RSA may be grossly altered. or that the RR’As may be much smaller. or that some host RSA may be synthesized. In any case. it seems calear that the host lit- mutation affects T4 lat,e transcription and this is probabl> suffic*irttt to explain the defect in late gene expression and phage production.

T-l go/ mutations are single point mutations that overcome the defect in late protein syttt hesis in lit - hosts ((‘ooley et ~1.. 1979). If the defect in late transcription is c~ausitt~ the defect in protein synbhesis. we would also expect gal mutations t)o o\-et-come the effect on late tratiscriptioti. In Table 1 we have included hybridizatiott results from experiments with RlVA labeled in the lit- host, after ittfect~iott with the go! mutant. This lat,e RNA hybridized with an efficiency of 77:, t,o the separated strands of T1 DNA and 71 O/b of the input RNA hybridized to the I strand. Thus the RNA labeled after infection by the go1 mutant was more like normal T-C late RSA in that most of it hybridized to the r strand of T1 DNA. \/E’e c~ttc~ludr that ‘I’4 go1 mutations over~me the effect on late RKA synthesis caused hy host lit mutations. (c) (ktletic

trrrnlysis

of Td go1 rn ~rtrrtions

In art earlier paper. we reported that go/ mutations are closely linked t,o T1 gene 23 ((‘o&y rt ~1.. 1979). The 200 go/ mutant’s we have tested so far all had mutations in this region. If mutations to the (;ol phenotype can occur elsewhere. they mnst hr less frequent by at least two orders of maqtitude. To localize further a particular go1 mutation, we performed three-factor crosses. The results are shown in Table 2. This particular go1 mutation. go/G/j. ;t/)1)i~t~(~tttl~ lies within gene 33. (4 or .k wise (or carhoxy-terminal) to t)he ret-y a ttri~~~~~tc~t~ttlitlal

400

w.

(“.

CHAMPNESS

ASI) TARLE

I,. SSYDEK

%

1. ornH11,

qoKRxrrmB270

2. crmH11.

goKHxnmBli

3. 0mB17.

g&R

4. omBl7.

yolGHxrrrnB26

17il4X

1%

5. nmB17.

gol6H x nmE3XU

1:w,a

I4

6. nmE389,

gol6B x nrr,Bli

138/146

9-l

‘7. wmE389.

g&R

105/143

73

xa.mHll

13jus

I4

llT/l%ti

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s/w 1

x nmB270

B270-HI

1 -yoZ

YOl HI1 (-R17 go1 HII I-Bl’i p’I mi7gol ( go1 pm-

x

H27O--gol

H26 E389 E389 E389

T)ouhle mutants consisting of the go&H mutation and amber mutations in genes 22. 23 and 24 were crossed against other single wry mutants and awl+ recombinants were tested for growth on the lit- host. The crosses unambiguously place the gal mutation between the gene 23 mutations nv/,Hll and nnaE389, very close to amB17. The crosses do not clearly show on which side of nmB17 the gal mutation lies. We have indicated this by brackets.

83amHll and close to t,he 23amB17 mutation. A. H. Doermann (personal communication) has confirmed this map position using “marker rescue” mapping techniques and has further concluded that this gal mutation lies clockwise (or carboxy-terminal) to 23amBl7. XII expanded map of this region is shown in Figure 2. The product of gene 23 is the major capsid protein. During head formation, the X-terminal portion of the polypept,ide is removed by proteolytic cleavage (Laemmli. 1970) and t’he site of the cleavage lies between 23amHll and 23amB17. (d) Evidence

that

go1 mutations

&fine

n cis-acting

site ~a T4 11X.4

III spite of the map position of go2 mutations, the Go1 phenotype is not due to an altered gene 23 product. In the experiment shown in Figure 3(b) (lane 3) a double mutant, having both the go1 mutation and 23amE389. was used 6 infect t,he lithost. The gal mutation stimulated the synthesis of all the T4 late proteins, including the amber fragment of gene 23. Thus t,he Col phenotype is unchanged

63

even in the ahser~e of a functional gene 23 polypeptide. \Ve repeated t’his experimerlt with the mutation 23arnHll. which, as mentioned above, is K-terminal to the yol mutation and in the part of gene 23 coding for the fragment removed during head formation. Xow the amber fragment was too small to be detected, but the gol. 2da~nHI 1 double mutant synthesized all the late proteins except the gene 33 product (data not shown). We conclude that. since the yol mutation could stimulate the expression of the other late genes in the absence of the gene 23 product or even the N-terminal fragment, the Go1 phenotype is not due to an alteration of either the gene 23 protein or the X-terminal fragment of the gene 2;~ protein. The possibilitirs remain that the Go1 phenotype is due t’o altering the gene 23 mRNA rat,her than protein : or to altering a gene whose seyuence overlaps that of prrw 23 or is on the opposite DSA strand. Alternatively. go1 mutations may define a site. The evidence presented immediately below suggests that gol mutations define a site on the DIVA rather than a diffusible gene product. If qo/ mut,ations define a site on the 1)K.A. they may be more closely linked than if their target is a diffusible gene product. Of the five spontaneous go/ mutations we have tested. four seemed to be in the same base-pair, since they gave recombination frequencies of less than O~Ol(~,,, the frequenry of recombination between mutations in adjacent) base-pairs. The other mutation gave a frequency of about 0+4?~,, wit’h the other go/ mutations, which is consistent with its being about, four base-p&s away. Therefore, the “target size” for go/ mutations seems to be very small.

402

W.

(‘. t”H;\MPNKSS

ANI)

I,. SNYDER

p23 p23*

-F

(b)

~23 p23*

-F

I

2

3

4

5

6

Synthesis ofT4 gene 23 product (~23) in mixed infections with a gene 23 amber mutation and i* of T4 proteins labeled from 2R to .71 min after infection yol metal tion on lit E. co/i. An electropherogram is shown (a) lit+ li:. CC&: (h) tit- E. eoli. Lanes 1. T4 g&B: 2. 23nmE389: :<. the doublr mutant NC:.

3.

A cis-AC’TING

SlTE

Ix

T4

GESE

EXPRESSION

403

If go/ mutations define a site 011 T4 DXA they should be &s-acting for the of T-l late genes in mixed infections. To test this, we performed the experiment shown in Figure 3. lanes 5 and 6. We coinfected cells with the T4 go/ mutant and gal+ T4 with the 23amE389 mutation (lane 5) and, conversely, with goZ+ T4 and a double mutant) having the go1 mutation and 83amE389 (lane 6). In Iit+ E. coli. gene 23 was expressed from both t,he gal mutant and gal+ DNA since the NIL fragment, and complete polypeptide were synthesized at approximateI) equal rates (Fig. 3(a), lanes 5 and 6). In contrast. in the Zit- E. coli gene 23 was only expressed from the DNA with the go1 mut)ation. In other words, if the goZ+ DIVA had the amber mutant, allele, only the complete gene 23 polypeptide was synthesized (Fig. 3(b). lane 5), but if the go1 mutarrt DNA had the amber allele, only the arnber fragment was synthesized (Fig. 3(b), lane 6). Thus. the gal mutation stimulat,ed the synthesis of t’he gene 23 product only in cis. \Ve atternpted a semi-quantit,ative determination of the stimulation of the c-omplete polypeptide and the UM fragment in each experiment by determining the area under each band, using a scanning and integrating densitometer. r\ background was subtracted that u-as taken to be the area in the same region of thr gel when the am mutant or wild-type ‘1’4, respectively, had infected the lit- host’ without t’he coinfecting go1 mutant’. Subject to the limitations of this method. it appears t’hat the presence of a go1 rnutation on the same DNA stimulated t,hta synthesis of eit,her the complete polypeptide or the amber fragrnent at least, 300fold without stimulating gene expression frorn the goZ+ DNA in the same cell. The synthesis of the am fragment appears t,o the eye t,o be st,imulated more by the go/ mutation t,han the complete polypeptidr. hut, the densitometer reveals that this is an illusion based on the width of the bands in different parts of the gel. To determine if the cis effect of go/ mutations extends to late genes other than 23. we repeated the experiment’ shown in Figure 3. but with an amber mutation in gene 18. The results are shown in Figure 1. Like the gene 23 product, the gene 28 product \vas only synthesized from the DNA with the gal mutation. We tried the same experiment with gene 34. The results were less clear but there did seem to be some cis effect on this gene as well (data not shown). Apparently. the cis effect of go/ mutations extends some distance. and in both directions, from the site of the go/ mutation. 01re c~)uld argue. frorn the experitnrnts shown in Figures 3 and 4, that go/ mutations are totally rbcessive. but that a subset of the cells received gal rnut,ant phage exc~lusively. Since these were the only cells in which late proteins were synthesized. only one allele of gene 2.3 or gene IX was expressed, because this was tlrcs only allele in those cells that were making any late proteins. This seems unlikeI\.. The rate of late protein q-nthesis in the mixed infections was about 30(‘,, that of’the /it’ caontrol. so 30”,, of the cells could not have been infected by goZ+ T4. But to eliminate t,his trivial explanation for the cis effect. we designed the c~xpcrirnrnt shown in Table 3. The experiment is based on the observation that host

expression

L’kcuE::C-S!). goKH: 4. u ild-tvpe T4 plus T4 gol6R: 5. ZlorrrE3HR plus T4 g&H: 6. the double mutant 2.h I/l Iw3’). go/6 H p I us wild-&w T4. In the single infections (lanes I to 3) the m.o.i. was 10. In the mixed infections (lanes 4 to 8) the m.o.i. wits 10 of each. The grne 29 product and the amber fragment left b! 2.?rcw E::SX!) (F) arc identified.

(a)

Lb)

pi8

I FIG:. 4. Synthesis mutation in litinfection. (a) lit‘ wild-type T4: 3. 18nmE18. g&B mixed infections fragment left by

2

-

3

of T4 gene IX product in rnixrd infections with it gene IX amber mutation and a gul E:. coli. shown in an elrctropherogram of the ‘I’4 proteins labeled from 28 to 31 min afti E. eoli: (b) lit&F:. coli. Lane 1. IXowtClX: 2. the double mutant IXunrElX. go1611 plw T4wild-type: 4. ZXnntElX: 5. thedouble mutant 18rrmElS. gol6R: 6. thedouble mutant plus wild-type T4. For single infections (lanes 1. 3. 4 and 5) the m.o.i. was 10. For the (lanes 2 and 6), the m.o.i. was 10 of each. The T4 gene 18 product (~18) and the amber 18omKZR (F) are identified.

Pro&&ion

of goI+

Phage T4+ T4 gol6B T4 gol6H + 2.‘;cr~~/H 11 rcn/H 11 T-4+ T4 g&H 1’1 g&H + 2.‘In ,II H 11 23tr ,tt H 1 1

and

Bacterium

go1

mutant

phuye Phage infected

8. cozi lit-

5.5

E.

roli

135

E. E. E. E. E.

coli co/i

coli coli coli

l3. eoli

At least 100 plaques were tested produced. In this experiment. the This is nut generalI>the case.

litlitlit+

lit-~ /it-

litlit

in per cell

mixed

(I; gol6H

input

0 loo 51 0 0 l(H) 51 0

15 1 x 1or4 w;;i 135 15 1.5 x 1oP

for the &I phenotype to determine yield of T-t g&H WBY higher in the

infwtions

the lit-

percentages mutant than

“/o yol6H

output 0 IO0 53 0 0 100 67 0

ofgol mutants in lit+ K’. w/i.

do not substanbially affect 7’4 DEA replication : so. in a lit host. both goZ+ and go/ mutant DKA is replicated. Thus. even if go1 mutat,ions are cis-acting for gene expression, those late proteins synthesized from the go1 mutant DXA could package go/+ USA in the same cell. Therefore. in a mixed infection of the lit- host. such as that shown in Figures 3 and 4. both gal’ and gal mutant phage should be produced if go1 mutations are G--acting. In contrast, if the explanation for the apparent cis effect is that gal mutations are recessive and the only cells synthesizing lat,e prot,eins are those infected exclusively with go1 mutants, then only go1 mutant phage will be produced in mixed infections. Even if go1 mutations are cis-actSing we expect some bias toward the gal mutant phage because those cells that are infected mostly with go2 mutants will produce more phage, since they will synthesize more late proteins. III the experiment shown in Table 3. t’he goZ+ phage had an amber mut’ation. d,llamHl 1, so any goZ+ phage produced must obtain their major coat protein from the gal mutant genomes in the same cell. The results were that in the mixed infection about nine phage per cell or 334; of the total phage released were of’ t,he gol+ genotype. This represents a stimulation in the production of golf phage of about, 20.000 times more than that produced after infect’ion by the goZ+ phagr alone. It is also many more phage than are produced after gal’ phage without an amber mutation infect the IX host (see Table 3). We conclude that late proteins are synthesized in cells infected by both gal’ and gal mutant phage. and that go/ mutations are not simply recessive. However. t’here is some interference from gal’ genomes on t,hc expression of go/ mutant genomes in the same cell. In mixed infections of gal+ and gal mutant phage, such as those shown in Figures 3 and 4. the rate of late protAn synthesis was less than half t)hat of normal even if half of the infktirlp phagr had the go1 mutation. lit mutations

4. Discussion Host lit - mutations severely alter the transcription of the T4 genome late in infection and t,hereby prevent’ the expression of the late genes. One site. the go1 site on 1‘4 DSA. is responsible for the defect in late gene expression, and T4 with

406

1%‘. (‘. (‘HAMPSESS

ASI)

I.. SSYI)E:K

mutations in this site multiply normally on /it - hoAs. Our evidence that yol mutations define a site rather than a diffusible gene product is twofold. First. the! are very closely linked. Second, they are cis-acatirlg f’or the expression of the late genes. e.g. when lit- cells are mixedly infected uith yol+ and y0/ mutant l)hage having different alleles of a late gene. onI;\ the allele on the yol mutant lISAA is expressed, There is no cis effect on 1)X=\ packaging. Iro\vt~vtv, and I&r yoI+ and yol mutant IIS,- are packaged in late prot,eins made from the yol mutant 1JN.A. N’e have demonstrated a cis effect on t,he expression of genes 23 and IX. both of which are c*losely linked to the yol mutation. Somewhat unexpec+dl~. ~(1 also observed some cis effect on the expression of genr $1. which is about IOO away on t,he circular map (or about 10pm on 1)Xx) from the site of the yol mutation. although the results were less dramatic bec*ausc of the lo\vrr rate of exijression ot gene 33 than that of either gene 23 or IX. Hecbause ‘I’4 recombination is so ac+ive. one might not’ expect, to be able to demonstrate a cis effect OII t’he expression of a gene so far a\vay. since the yol mutation kvill often he separat’ed from the gene .‘,‘I amber mutat’ion by rec~ornl)irlat,ioll. Severthrless. this result need not vitiate our interpretat,ion. Most recombinat,ion o(~(urs late in ‘I‘4 development (Lrvinthal 6 Visconti, 1953), perhaps later than t,he times at jvhich WY‘ labeled the proteins. Models of the function of the T4 go1 site must alvait informatjion on the nature of yol mutations and of t’he function of the host lit gene product In a lit - host. T4 late gene expression is temperat,ure-dependent. This t,emperat ure dependence is at least partially due to differences in E. coli cells that have been grown at different temperatures and not, t’o the thermal sensitivity of a protein or reaction. Also. the effect of host lit mutations on T4 gene expression is qualit~atirel~ different from that of most types of phage mutat,ions that block late gene expression. %‘hen truelate gene expression is prevented by mutations in replication genes 01’ gene .Sj or 33, the synthesis of many early proteins contjinurs lat,e into infection. Ho\vever. la,tr int,o infect’ion of a lit- host,, \ve see the tsessation of synthesis of even those earl! gene products that are normally synthesized throughout infection. Other situat,ions are known in which true-late gene expression is prevented and the synthesis of the early gene products also ceases. These occur when 7’4 1)S.q replicat’es t)o cont,ain evt,osine and t,he T4 are &+ (Snyder et al.. 1978). and when F)olvlluc,leotide kinase or R,lA ligase-deficient mutants of T4 inf&t 6. di (“l’r.?x (Sirotkin rt 01.. 197X: Runnels rt ~1.. 1982). It is interesting that t,he air gene product also unfolds the bacterial nucleoid after infection (Sirotkin Pt (11.. 1977: Tigges et (11.. 1977) and that mutants restrict,ed polgnucleot,ide kinase-deticient some of the original litmutants of T4 (Cooley rt al.. 1979). Perhaps all these gene products are involved in a common pathway. We can only speculate on how the wild-t?pe yol’ site can int,erfere with lat,e gene expression in a lit- host,. Host, lit mutations might prevent t,he utilization of the site. and yol mutations alt,er the sit,e so it can function in a lithost YOl+ Alternat~ivelg. in a lit - host, something may happen to the yol+ site that prevents the expression of the genome harboring the site. For example, host lit- mutat’ions may activate a restrictiorl-like nuclease. which makes a break specificall?: a,t the yol site. If this is true. then late gene expression must be particularly sensitive to cleavage at this site. since after infection bq’ gene 46 or 17 mutants. for example. double-

A cis-.4(‘TISG

SITE

IS T4 GE:?r’N ~NPRESSION

1fli

stranded breaks accumulate in T-t DNA (Hosoda rt al.. 1971) but late gent expression is not affected. Furthermore. the observation that gal+ genomes can Iw packaged in mixed infections of lit- hosts argues against the “restric,tion-site” hypothesis. sinw packaging requires concatameric genomes. N:e have recently observed that plasmids with T-i USA “inserts” containing the hut not /itE. cf~li. In cont,rast 1 if’ M-ii&type (g01+ ) site can he used to transform lit + the T-4 I)SA insert contains the go1 rnutant site or deletions of this s&e. then either /it+ or /it- E. wli can be transformed. Apparently. something happens at this site eveu in the alwnce of t)he rest of the 1’1 genome. Sate that, bhese observatiotw sul)f)ort the letter of the two possibilities mentioned above: that somcthiny host that is deleterious to the expression of’ ha,ppws at. the gal+ s&e in a litgenomrs harboring t.he site. This raises the possibility t.hat, t,he got site is not trormally required for late gene expression jr/ ?~ico but can act as a “spoiler” undtir some conditions. It may not be required for lat)e transcription because other similar sites can substitute for it or because its real role is tjo co-ordinat,e some other event. such as I)SA packaging. with late transcription. In any case. even if the go/ site is Ilot normally involved in transcript,ional activation. an understanding of how an event at one place in the 1)N.A can affect the expression of genes so far iway should rnhauw out’ understanding of the intracellular struc%ure of T1 DXA and how this structure act,ivates the tjranscription of the late genes. \Vv thank Dietmar Kabussay for help with the hybridization experiments and Peter Wduschr~k for critical discussions. This work was supported 1,~ National Svicnw Foundation grant 3Y%24422 and Sationaf Inst.itut,es of Hra1t.h grant GM28001 -01. \V.(‘. ac*knowlrdgw an XSF predortoral fellowship.

788-795.

Bollr. ;\.. Epsteill. K.. Salser. LV. & (&aiduschek. E. P. (1968). J. Mol. Hiol. 33, 339-362. (‘o&y. IV.. Sirotkin. K., (preen, R. & Snyder. L. R. (1979). J. Ba~cteriol. 140, 83-91. (+old. L.. O’Parrell, I’. Z. B Russcl, M. (1976). d. Hiol. Chem. 251, 7251-7dfX. Hosoda. fJ,. Mathca\vs. E. & .Jansw, B. (1971 ). J. C’irol. 8. 372-387. Laemmli. IT. K. (1970). X&UY (Loudott). 227. Bt(O-~j85. Lrvinthal, (‘. & Yisconti, N. (1953). Grrtetics. 38. ,50051 I. Kabuwsay. I). C;:(iriduschek. E. 1’. (1977). I’ror. ;Vat. =Iccld. ~‘ci.. l~,AS..A. 74. 5304-3309. Kiva, S.. (‘asc4no. A. & Geiduschek. E. P. (1970). J. Mol. Biol. 54, 85-102. Kunnrls. ?J. AI.. Soltis, D.. Hey. T. & Snyder, L. (1982). .I Mol. Biol. 154, 273-286. Sirotkin. K.. \Vei. .I. & Snyder. L. (1977). Sut~rv (London). 265. 28-32. Sirotkin. K.. (‘ooley. W., Runnels. ,J. B Snyder. L. K. (1978). J. MOM. Biol. 123, 22lbd33. Snydrr. L.. (iold. 1,. d Kutter. E. (1976). P rot. AVat. Acad. AX., l’.S-4, 73, 3098-3102. Stud&. F. IV. (1973). J. MO/. Biol. 79. 237-248. ‘l’igpw. ,\I. (‘.. Rursch. ,I. H. 8 Snustad. D. 1’. (1977). J. ITirol. 24. 775-785. \\‘ood, L\‘. IL (196.X). ./. Mol. Biol. 16, I 1X -133. \Vu. K. & (:eiduwhek. E. I’. (1975). J. Mol. Biol. 96. 513-538.