Transcription in bacteriophage f1-infected Escherichia coli

.J. Nol.

Hid.

(1983) 164, 377-393

Transcription

in Bacteriophage fl-Infected

Escherichia

cofi

Messenger Populations in the Infected Cell

Transcription of bacteriophage fl 11X.A ire c.iao occurs in two independent regions. They are separated from one another by a strong terminator just downstream from gene \‘I11 on one side. and by the filamentous phage intergenic space on the other. One of these regions contains genes II. V, VII. IX and VIII. and is actively transcribed. In this region there are a number of promoters but only one vffective terminator. Thus. most of the RX& that corn? from t#his region overlap and share secluenres close to the termination site. ‘l’hv other region, which contains genes III. VI. I and IV. is t’ranscrihed much less activt4y. This region gives rise to a long ( -4 x 103 bases) RKA that covers the entire region. and several R?iAs that overlap in the region closest to their 5’ termini. Several other RX& appear to overlap only with the 4x lo3 base transcript. Thus. not only the frequency but the organization of transcript,ion differs in the two portions of the genome.

1. Introduction The male-specific filamentous coliphages. such as fl and the closely related Ml3 and fd. are unique among bacteriophages since they establish a productive infection without killing the host. The nine or ten genes (Pratt et al.. 1966.1969: Beck et al.. 1978) that constitute the entire genome of fl are expressed at verb different levels. The major coat (gene Y’III) prot’ein is made in hundreds of thousands of copies (Pratt et ~1.. 1966). which are continuonsly export,ed as virions. X single-stranded DSA-binding (gene V) 1)rotein is also abundant (Pratt et ~1.. 1966: Alberts it ~1.. 1972: Oey & Knippers. 1972). Other products such as the minor coat prot’eins are rare (Henry & Pratt. 1969: Model B Zinder, 1971). The prope’ties of these phage have been extensively reviewed (Denhardt’ et rrl.. 1978). The limited genetic: complexity of this bacterioljhage makes it an attractive syst,ern with which t’o study differential gene expression. However. due t)o tire continuation of host growth duriug infection. a detailed analysis of the phagr transcriptional activity in GPO has proved somewhat difficult. Early iv vitr,f) st’udies suggested that the phage transcripts were initia,ted at several promoters but terminated at a single termination point. thus generating a series of st,aggered

RSAs that overlap near a, single termina.t.or (Okamoto PI II/.. l!ifiS : (than CA/I(I/. 1975: Edens ut 01.. 1976). This was postulated t,o account f’or differential gene expression. Recent reports by (‘ashman & Webster (1979). (lashman et nl. (1980). and Smits et al. (1978,198O) indicated that the region of the genomr t,hat contains genes II, V. VII. IX and VIII is. in fact, transcribed in this way in c+r-o. ,211 (or almost all) RNA molecules derived from this third of thr genome appear to shary gene VIII sequences at their 3’ end ((‘ashman et nl.. 1980) and thus end at what has been termed the central terminator (Edens P! ~1.. 1975). The remaining two-thirds of the genome cont.ains genes III. t-1. I a.nd l\‘ ant1 is bounded bp the ‘.central terminator” on t,he left and by an intergenic: spacar on thca right (Fig. 1). The rate of transcription of this large region is relatively low (Smits et nl.. 1978.1980: and this paper). No detailed informa.tion has yet, been obtain4 with regard to the RSA sprkes t,hat derive from t,hese gems. Here. we caompare the rate of transcrilkion of the region that contains gent’s II I \I. I and I\7 (region 1) with t)hat, on t,hr l)SA segment that contains genes Il. \VII. IX and VIII (region F). K’e then ident,ify and ana.ly~ the gcnc content of’thy RX.4 molecules that come from thr different part,s of fl 1)K.A. In addition. wf’ compare the relative abundance of the major fl R’K’=\ speck at early and late times of infection. Finally. we study t,he st,ructural decay of the transcript, coding for thfb gene II protein. which seems to be synt,hesized quit)r c>fficiently but does not accumulate in the cell.

fl MESSAGES RNA extraction Modrl, 1978).

IN IKFEPTEI)

(d) RXL4 ertraction was as previously described (Blumberg (e) il~Vas~ digestiotr

E. (‘(/I,1

37!1

8: Malamy.

1974; La Farina &

of the fLV.4 sample

To the RNA sample made 10 ma-sodium acetate (pH 5.5) and 7 mM-M&l,. DNase I. purified according to Wang & Moore (1978). was added to a final concentration of 20 pg/ml. After incubation at 37°C for 30 min, the sample was extracted once at room temperature acetate with 1 vol. water-saturated phenol. the aqueous phase was made 0.1 M-pOtaSSiUm and precipitated by 3 vol. ethanol at -20°C overnight. After centrifugation. the RN.4 pellet was washed once at 4°C with @l M-NaCl. 7576 ethanol, and then resuspended in distilled water. (f) PwiJication of RSA from pulse-l&led cells (‘ells grown in GC.4 medium (Ray & Schekman. 1969) and infected at a multiplicity of infertion of 100 were pulse-labeled bv exposure to [ 3H]uracil (10 &i/ml : 23 Ci/mmol) at’ IO min after infection : 2 min after addition of the label. cells were poured quickly onto frozen medium and RNA was extracted as described (La Farina 8: Model. 1978). Under these caonditions, the label in the medium was not exhausted. To study the structural half-life of the RNA, rifampicin (final concn 200 pg/ml) was added to portions of cells pulse-labeled as described above. Synthesis of new RNA stops within 15 s after addition of the drug (La Farina & Model, 1978). To remove DNA, the RNA samples were fractionated on CsCl step gradients (Brunk & Leick. 1969). Shearing of the RX.4 was then performed by heating it at lOO”(’ for 10 min according to the method of Hayashi rt nl. (1976). (g) Isolation

This

was

performed

of fl as

covnlently

closed. circular

double-stranded

D,V.4 (RFI)

described by Model B Zinder (1974). (h) Filter

hybridizations

Binding of DNA to nitrocellulose filters and filter hybridization of pulse-labeled RSA to the DNA were performed according to the method of Gillespie Xr Spiegelman (1965). fl DSrl fragments bound to the filters were labeled with 32P at low specific activity in order to monitor the DNA fragment retention on each individual filter after the hybridization. Only filters where DNA retention was complete were used in the experiments described here. Before exposure to the pulse-labeled RNA. the DNA filters were pre-incubated for 2 h at 65°C’ in IO ml 2 x SSC (8% is @15 M-N&(:1, 0.015 M-sodium citrate). Then l-ml portions of pulse-labeled RNA 2 x SSC containing Ol”:0 sodium dodecgl sulfate were added to scintillation vials in the presence of a filter containing no DNA and a filter containing the desired DNA, which had been previously incubated as described above. The vials were incubated at 65°C for various lengths of time. The input DNA in each hybridization sample was 6 or 12 pg of RNA. as explained in the Figure legends. The hybridizations were stopped by washing each filter twice with 2 x SSC at room temperature, incubating them at, 37°C in 10 PI of 2 x SSC containing 20 pg of pancreatic RSase/ml (Sigma) for 30 min, and washing them twice again with 2 x SK’. After drying, the filters were counted in the presence of POPOP/PPO/toluene in an Intertechnique scintillation counter. Two different portions were analyzed for each time point. The plateau of hybridization was reached at about 24 to 30 h. The values shown are those of the net cts/min, where the value of the blank (0.030/, of the input counts) has been subtracted from that obtained on the filters that contained the DNA. (i) Preparation of probes Total fl RF1 DNA or specific fragments were nick-translated according to the methods of Rigby d nl. (1977). 20 &‘i of each of the four [ ~-32P]deoxyribonucleoside triphosphates (Sew

380

JI.

I..1

F.4ILIS.4

ASI)

1’

.\lOl,KI,

England Nuclear: 300 (‘i/mmol) were dried and resusprndetl in IO0 PI of 50 rtt\t-‘l’ris (pH 7.8) 5 mM-Mg(‘1,. 10 tni\I-8-mcrcaptoethanol. 04)I pg hovim, serutrt all~rrntin~ntl. which c,ctnt,atttvtl I pg of the desired DNA. After addition of DNA polymerasca I (Miles I,altoratorirs) and I /AI of 0.1 ,qq’rnl DBase I (DPFF: LVorthingt,on). the sample \vas inc.11bated for I h at I.i ( ’ Between 25 and 4(V),, of the inltut counts NX’W ittcvtrporatrvl itrtl, tt,ic~trl,)r.ctit,,c,~tI.ik~,ttl precipitable material. Several different probcbs wrrv sometimes Itrt~~ta~~t~l sitnultattc,ottsl?, II! nick-translating the fragments just after the restric~tiott rrac*tiotr. The fragments u en- theta fractionated on a low melt,ing point agarose (Sea I’laqttc~ Agarose: Marittr (‘olloids I)i\+sion j The fragments were identified by staining or autoradio~t,alth~ atttl tltv albproltriate Ibor‘tiotas of the gel were cut out,. melt4 at, 65°C‘_ and analyzed on agarosrl gels to toast their prtt,it I

(k) fi’rnctir~tlcrtiotl of’ tf.Y.4 frac,tionated on vf.rt ic,al slalt I .1”,, (\I \.) agat’c,sc. pc,is The RSA spcbc*ies \vf~w (IO cm x 10 cm x 0.14 ctn) in the prfwncf of .‘,-rnc~tl-t~lrnrrc.ttt,ic. hytlroxidr (Bailey
Since the mobility of RSA and 1)S.A is t,he satrtt’ in fttlly tlrttatut~ing gels (Kailt~>, ,V Davidson. 1976: McMastrr & (‘armichael. lR77), specific> DSA fragmcants can he r~srcl to estimate the size of the R,N.A species. WP used digests of fl DNA4 as tnarkrlrs. To ha\~t~att equal mass of nucleic acid in each slot. t,hr digest, was ttrixr~tl \\.ith an e(ittaI arttctrtnt of RX.4 from the uninfected cells and fractionated on slots itt parallel with those in which RN.4 frtrrtr infected cells was analyzed. After t,ransfer to I)BWpapt~tr thr different fragment,s every’ visualized by hybridization to fl RF1 [ 32PlI)NLA (Fig. 3(a)). It was found that, the mottilit! of each fragment vorrelated with the natutal logarithm of the- respective ttrttnbet~ 01 nucleotides (data not shown). (iii)

‘Trun.sfPr

of KS.4

,frwn

t//r !/CT/ crd

coutilit/fq

trj /)Il,W-pupct‘

Transfer of RNA fractionated on methylmercttric~ hytlroxitlv gtlls Mas as desc~rilted II,I Alwine it 01. (1977). For transfer of RKA denatured with glyoxal and fractionated ott agarose gels. the procedure was essentially the same except t)hat I-rnrt’c~afttoethanol \~a:, omitted from the 50 rn>f-SaOH treatment and iodoavcltic. xcaiti \vitx omit~tr~d from the 200 m#-sodium phosphate treatment.

Hybridization of nick-t,ranslat,ed fl DSA probes to RNA c:ouplrd t,o DHhl-paptsr \vah performed in the presenre or absence of formamide. (I ) Hybridizatiotls in the prpsenve of formamide were performed ac:c*ording t,o Alwine ut u/. (1977). (2) Hybridizations in thtx absence of formamide were performed as follows. The DBN-ftaprr was nrtttralizrd a,t,6.5 (’ in 5 x SW, 50 m&l-sodium phosphate (pH C.5). 100 pg yeast, tRSX~ml. I”,, (U./V) glycillr. atld 20 pg each of bovine serum albumin. pol~vinvlp?lrolitforlr. and Fic.ol per ml. f(tr 11 t,o 2-C)t The hybridization mixture was the same as the neutralization mixturf, rxcarpt that glyc+nt* was omitted. The samples of Sea Plaque agarosc that contained the various DNA probes

fl

MESSA(:ES

IS

fSFE(‘TEU

3X1

!C. f’O1, I

ww melted at 65°C. diluted with water and denatured by making them 0.1 31 in SaOH. Aft’er neutralization with HCl the? were added to the hybridizat’ion mixture. The strips were hybridized at 65°C for 24 t,o 30 h, after which each strip was rinsed twice for 20 min in 1 x SW, @I”, sodium dodecyl sulfate at room temperature. then twice with 0.1 x SW. O,l”,, sodium dodecyl sulfate at WC for 30 min as described by Shank P( rrl. (1978).

3. Results (a) Rate of transcription

on different

of the fl

regions

genome

\\‘e measured the in rain rat’e of transcription from various regions of the fl genome by hybridizing pulse-labeled RSA to fl restriction fragments. At ten minutes after infection, cells were pulse-labeled with [ 3H]urac4 for two minutes. and the RXA was extracted and purified as described in Materials and Methods. Table 1 shows that the experiments described below, which were carried out with an input of 6 pg of infected cell RP\‘A and 3.8 pmole of DSA on the filters. were at I)NA excess.

of DNA

Demonstration

excess

in jilter

hybridizations

12 pg KS.4 hyl)ritlized to l-9 pm01 fl RF111 f)SA 19 pg RX.4 hyhridizrd to 3.8 pmol fl RF111 f)SA 6 pg RSX hybridized t,o 3.X pmol ff RF111 I)SA

Kl.000 7P.000 :~o.ooo

[‘H IRSA extracted from cells Imlse-labelrd fov 2 min (see Jfat,eCals and Methods) was hybritlizrd to s&s of filters t,o which the indicated amount of denatured linear doubk-stranded RF111 ff 1)S.i hat1 heen bound. The \-slurs shown are net cts/min (cts/min found on the tilts that contained the f)N;\ minus cts!min bound on the blank filter) at the plateau of hybridization (21 to 30 h). ‘I’\vo samples wc~w avrrqrd for rac,h hybridization. ‘I%(~ cwnditicms of hvbridization ww tiex~rihrd m Materials ar111 rnf~thods. 141l’“O1 = 75 Kg ff RF1 Il.

Hybridimtion

of pulse-labeled

diffe,rent

parts

Svt cts,!min hybritfizrd HUc’fff

R I A HfwIIf (‘ f’lobr r, f’rotw 2 Hrtrfl

RXA to prohresfrom

of the fl

genorrre

Su&otitfw

19.200 5100

1(i”3 I. 2HXB

4300

x-1-9

.‘,Mu ) !MH)

x4 1 3 13

f)errsity of hybridization$ 11.x 1.!) 5.1 6% 2.9

B-pg portions of [ 3HjRS.A (we lrgentf to Table I ) wew hybridized to rritrocetlulose filters to whl1.h 3.8 x IO-” mol of a specific fl 1)S.i fragment had hren bound. All conditions were as dexribed in thr Ieg~nd to Table 1. t See Fig. 1. 1 (‘ts/min per nucleotidr~.

transcription rates in the two regions. F and I (Fig. I). Table 2 shows t,hc cbc)unth hybridized as well as the density of hybridization. which is the number, of’ coounts hybridized divided by the number of nucleotides in the releva,nt fiagn~rnt. Transcription is about six times as high in the region c~o~~rred 1,~. fftrll I-R as t hit1 in HneIII-A. As expected. the spanning fraymcbnt. ffnrlll-C’. shoav:, ittl intermedia& level. Two additional fragments (sty Fig, I ) wart’ inc4utletl. t&h shows a transcription rate c*onsist,ent with t)he region from which it wah tifJri\-c+ I 211,il low r:rtjc. a result c.orrsisttarrt Thus. genes III, VI. I and IV are transcribed with data obtained by Smits it (IL. (1980) for phag,rr S3IX

We fractionated RSA extracted from inf’rcted ~11s on denaturing gfbls. transferred it to DKM-paper. and hybridizc>tl i~r)l)~‘~)l)riat~~ strips to Hu~III fragments of fl Similar input, counts were used for t hcht,hrrca li~br~ic~izat.ioris: thus. t,he intensity of the bands should be relat,etl to thca (~otlc~c~ntrwt.iotr of the relr\.;~nt RX=\. Figure:! shows t,hat t,he bands present iu t,llc) strip that h>.l)ridizrd to the* H&II-R fragment are much darker t,han those in the other, two st’riljs. In gent~~,;tl. bands that’ hybridize with HczPIII-A do not, hybridize: with Hoc1 II-H (in l)articulat, a lighter exposure of lane (h) shows that, there is no f)antl at 16 S) while t,hr Hc/rjII I (’ probe reacts wit’11 all of the bands. These observat,ions suggest, that there are two sets of’ transcripts. One. cirtrc.ted by HneIII-A. is composed of RNAs derived from the rrgion in which genes III. \-I, I and IV are located. and ranges from 1.1 to 4 kbt. The other set comes from thr, region containing genes IT, \‘. \‘IT. I?i and \‘IIT. is detec+tf by the HwIII-K probt,. ranges from 04 t,o 2 kb. and is relatively abundant.

(c) .A ttulysis (

. .

of lhr gettt

wtttettt

ctJ’ tttitrctr ,f’l fr’,V:l

sftpcic~s

hpeclhc probes for md~v~dual fl gcncs wflt’e l)rt~l)arc4. Thr l)osition of’cac*h f)robcL on the phage DNA and its relat,ionship t,o t,he genetic. map arr S~IOM.~I in Figure I ‘I’hc~ mobilitv of various fl I)SA fragments t,ha,t were run on tJhcbsame ltlrth?llner,c.lcl,\gel on which the MNAs were fractionated and whic*h scr\~rtl as size markers is show11 in Figure 3(a). An autoradiograph of I)BM-palmer stril)s h>.hridizetl to thr diffcArc>nt probes is shown in Figure R(b). The strips are orderrd wcwding to the Iwsition of each DSA segment on the genetic and physical mal). brginniny with a fragment t,hat comes from the c-entral part of gent> III and mo\cs to~artl gene \‘I I1 acc*ortling to the dire&ion of t,ransc*ription (Model R: Zinder. I!W: Konings rt (I/.. 107~). Probes 1 through 4 are derived from the infrequently transc~ribrd region. :!I1 four probes hybridized to a large ItSA1 (band -A). the largest. fl RNA found so fat. in /-i~o. This RNA moves slightly slower than the tf(reITI-A fmgment of fl OSA (9.?%~ nucleotides): the estimate of a,bout. 1000 nur-leotides. based on it,s mobilit~y. is in agreement with the c:apac:it.y of this molecule t.ct hybridizrtl to probes 1 through -C

fl

MESSAGES

IS

(al

ISFE(TEI)

(b)

E. (‘(/I>1

383

(cl

1”1(:. 2. Hybridization of fl RSA to HwIII-A, H and (’ UN;\ fragments. RNA was rxtracted 13 mm infection and fractionated on a 1.4’$;, (w/r) vertical agarose gel containing 5 mwmethylmercuri~~ hydroxide as described in Materials and Methods. POpg of RNA were loaded onto a 3% cm slot. After transfer to DBM-paper and neutralization (see Materials and Methods for details), the DBM-paper was cut into 3 strips and these were hybridized to: (a) HcreIII-.A fl [“P]I)NA (3x IO6 cts/min: spec. act. 2.6 x 10’ cts/min per pg DNA); (b) HaeIII-B fl [32P]DNA (29 x 106 cts/min: 2.4 x 10’ cts/min per pg DSA); (c) HaeIII-C fl [32P]DNA (1% x 10’ cts/min; 2.0 x 10’ cts/min per pg DSA). Hybridizations were performed at fWC for 24 h as described in Materials and Methods. The time of exposure was 12 h on a DuPont (konex film with Hi-Plus intensifying scwens at -7OY’. after

(genes III, VI, I and IV). Probes 1 and 2, both of which contain gene III sequences. give a major band (E) at about 1.7 to 1.8 kb: t’he probe for gene IV (4) gives a major band at about 1.4 kb (F). Two other fainter bands (B and C) are present in the samples hybridized to thr, gene III-containing probes (1 and 2): for these two samples. a band (F’) also appears in the same position of the major gene IV-specific band (F). Since the segment of D1JA located between probes 1 and 4 is longer than the 1.4 kb sizca estimated for RNA migrating in the band F position, we assume that bands F and

3x1 cm

kb

l-

E- 4.0

3-

4 --

2.0

5-

I.0

6-

?- 0.5

8-

F’ are due to at least t,wo different m KSAs (if’ sl~licAng does not occ*uI’ in bacterial cells). into N Vvtay long RSA. The four fl genes present in this qion a~‘(’ a II t t’i~llS(~titWd Gene III seyuences. which are fIresent at the 5’ cxtltl of’ this RSA. appear to be present in several shorter species as well. (itme 11. secluencrs. whkh are transcribed into t,he 3’ end of t,hat same long RSA. are also found in one shorter R?;A. Two different models can explain the pattern of KSA molecules actually observed (see Discussion). (d) ilnu1;y.si.r of

t?re

QWWc’or/tent

c/f thr tttct,jor. ,fl hLV.4 .vpeck,v

Lanes 5 through 9 in Figure 3(b) show t,he bands visualized upon hybridization fractionated fl R,K\‘X to several probes that (Y)VPI the wgion where penes II.

of \-.

cm

I

kb

No. 3

4

5 6 (b)

7

9

9’

IO

D

16s

-

G H

I

6

7

a

FIG:. 4. Hybridization of fl RX;\ to lwobrs that itr~lud~~ genres II atttl \’ RN.1 \~as vxtractul. fractionated. and transferred to DHM-palwr as described ill the le~c~nd to Fig. 2. 2Opg of KSA wew loaded on a 35 cm slot and, after transfer. 3 DKKpaper. strips w’ew hybrid&i to probes 6. 7 and X as shown at the bottom of the Figure. Each strip was hybridized to about IoO.oM cts/min of probe (spee act. about 1 x 10’ cts/min p+x pg DNA) at (ifiT (see legend to Fig. 2) for 30 h. The time of exposure was 10 h on Dupont (‘ronex film with Hi-Plus intensifying screens.

VII, IX and VIII are located. ‘I’he last strip (probe 10) shows the bands after hybridization to a probe derived from HaeIII-C’ that contains sequences from both genes VIII and III. and this probe hybridizes t,o ItSAs from both the freyuentl~~ and the infrequently transcribed region. The position of the bands shows that as we move from the 5’ end of gene 11 towards gene VIII (lanes 5 to 9). the contiguous. non-overlapping probes hybridize to more numerous bands. Seyuences coding for t,hc S-t)erminal region of the gene II protein are present only in band I), which is the largest, KSA from this region of the genome (about 2.0 kb long) and inc*ludes genes II through \‘TII. Sequences that code for the central and distal regions of gene II (probes 6 and 5) are found in t)hree other bands in the lower region (bands G. H and I) as well as in band 1). Gene \7

fl

MESSAGES

IN

ISFE(‘TEU

E. COLl

387

(probe 8) sequences hybridize to all but the smallest R?;A bands. Quite a high background is present in the lower region of the strip hybridized to probes 6 through 8. In particular, probe 8 shows some hpbridizat’ion to the region of band K. In replicate experiments, however, probe 8 did not hybridize to that region. This can be seen more clearly in Figure 4, in which probes 6 to 8 are hybridized to a different RNA preparation. Gene VIII sequences (probe 9) hybridize to all the bands. The fastest moving band, to which only probe 9 hybridizes. is quite broad. Lane 9’ shows a shorter exposure of strip 9, which indicates t,hat this band has two components, J and K. A comparable strip from a different experiment (9”) in which RSA was extracted later in infection resolves the two species more clearly. It also shows that late in infection. species K is more abundant than ,J. U’e note that band I hybridizes to probes 6 to 10. The length of the DNA segment covered by these probes is 1.7 kb and the distance between the distal part of probe 6 and the proximal part of probe 10 is 1.1 kb. The RXA contained in band I is about 0.8 to 0.9 kb. Thus it seems likely that band I actually contains several RSA molecules, and that one of them must lack gene VIII sequences. From their size. it seems likely that the I species are composed of RSAs D, E and F described by (‘ashman & Webster (1979) and the “11 S” of Smits et trl. (1980). With this exception, our results agree with the general conclusion of Cashman 8; Webster (1979). (‘ashman et ul. (1980). and of Smits et al. (1980) : most of the RXA molecules from the gene II-gene VIII region share gene VIII seyuences at their 3’ end.

(e) Accwr~ulation

of 8 S RSA

la.te in injection

Figure 5 shows the bands obtained after hybridization of Rh’A extracted from cells 20 minutes after infection (a) or of RXA from uninfected cells (b) to total 32Plabeled fl RFI. Lane (c) shows markers that consist of a HaeIII digest of RF1 t,ogether with linear and circular fl single-stranded Dh’A. It is clear that at this time of infection the amount of 0.4 kb RNA is much greater than any other species The bands at the top of the gel are due to DXA. since this sample was not digested with DNase. The O-4 kb RNA hybridizes only to probe 9 (see Fig. 3(b)) and actually contains two species (J and K in Fig. 3(b), lane 9”). RSA of this size codes only for gene VIII in vitro (Chan et rrl.. 1975 : Rirera et al.. 197X : Cashman & Webster, 1979), although it probably contains gene IX coding sequences (Beck & Zink, 1981). and it,s accumulation accounts in large measure for the enormous amount of gene VIII protein made in infected cells.

(f) &ru&mzZ

decay of mRXA

coding for the gegenef f protein

The large (2 kb) RXA present in band D (Figs 3 and 4) appears to be the only fl RNA molecule that can code for the entire gene II protein. The signal detected in band D (as well as in band G) with probes 7 and 8 (see Figs 3(b) and 4) is weaker than the signals obtained with the same probes in bands H and I. Thus, in the cell. the concentration of 2 kb RIYA appears to be lower than that of smaller RXAs that (*ode only for the gene V and gene ‘I’111 products. Transcription of the sequence

-

ss fl DNA

__

ss fl DNA Lopen

--_-----------

Harllt

A

-

tiocm c

-

HoeIll

D,E,F

[closed

form1

form1

fl

MESS.4GES

IS

lSFE(‘TED

.&. (‘OLI

389

that can hybridize to probe 5; which is derived from the intergenic space and the S-terminal region of gene II, is quite active (see Table 2). It seemed likel?,. therefore, that the absence of RSX coding for the S-terminal portion of gene IT is due to its rapid decay. To ask explicitly whether the RNA coding for the amino-terminal part of gene II has a rery short half-life, we pulse-labeled fl-infected cells and extracted RX=\ immediately or two minut,es after the addition of rifampicin. This RSA war; hybridized to HueIII-B (see Fig. 1). which includes gene VIII. and to probe 5 (Fig, 1). which is specific for gene II. Table 3 shows that pulse-labeled RX;\ ext’racted after rifampicin treatment hybridizes to the HaeIII-B fragment at about half the efficiency of the untreated control RSA. In contrast. hybridization to t#hfJ gene II probe (5) is reduced almost to background levels when RrZA from rifarnpic+l-treated cells is used. Measurable hybridization was obtained wit)h t’wic*fJ as much input : these results suggest that the half-life for this RSA is less than one minute. This estimate agrees well with the less than two minutes half-life (‘ashman rt al. (1980) found for their RSX species A. which is probably the same as band I) in Figure 1.

4. Discussion Figure 6 describes the RSA population of fl-infected cells. The phage I>ISX can be separated into two domains : the frequently (F) and infrequently (I) transcribed regions. The frequency of transcription was measured by quantitative filt,er hybridization between pulse-labeled RNA frorn infected cells and specific DSA fragments. and the transcripts were mapped by blot hybridizat)ions between fractionated fl RXA and nick-translat’ed DSA fragment’s. The F region corresponds to that part of the phage genome in which strong promobers (initiating wit’h pppG) have been located by several in vitro studies (Seeburg & Schaller. 1975: Edens et al., 1976: Okamoto et al.. 1969). The I region corresponds to the remainder of the phage DNA on which in vitro studies have been mapped the weak Xpromoters. which initiate with pppA (Seeburg & Schaller. 1975: Edens et ~1.. 1976: Okarnoto rl al.. 1969). The I region contains genes III. YI. I and IV. while F caontains genes II. (X), 1’. VII. IX and VIII. Xo phage RSA spans the two regions of the fl genome, i.e. there appear to be two transcription termination points. One (the central terminator) is located at the gene VlII/gene III border (Edens et nl.. 1975: (‘ban et al.. 1975: Sugimot,o rf (ll., 1977) and the other just in front of or within the intergenic region. which is located between the F and I regions on the phage genome. A number of workers have inserted foreign DNA into the intergenic region (Messing et al.. 1977 : Boeke et al.. 1979) and still obtained viable phage. In particular. Moses et ~1. (1980) inserted an fl-derived fragment that carries the central t’erminator into this region and obtained phage that grew almost as well as the parent phage. These experimenf,s reinforce the idea that t,ranscription oc~rs independently on the two regions of thr genorne. The RSAs that come from the I region accumulatJe at low levels whereas those from the F region are much more abundant. The transcriptional patterns in the t\ro

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regions are also quite different from eavh other. In region I2 we tiucl a gradient ot’ RXVA concentrations that increases as we move from gene I I towards gene \.I II This is the consequence of two phenomena. First. gene \‘I11 sequences are ~~r~w~~t in all (or almost all) of t,hese transcripts. but gene II sec4uenc~s are ljresent in their entirety only at the 5’ end of the large, 2 kb R,VA. Thus. gene \‘I1 I sec4uenccv ark present in many (about 7) species of RNA. completr gene I1 sec4uencrs in onl?~ ollt’. Sequences from the ot,her genes are present in an intermediate number of spec,it*s. Second. the smaller species. which conta,in sequences from genes 1. and VIII. a~ more abundant on a molar basis than the larger KS.&. the most abundant being all RNA that contains only gene VIII sec4uences (band K of Pig. 3). It is not by any means clear whether the overlapping transcripts that c~mr f’rorrl the F region have much biological function. The larger amount of gene VIII protein synthesized in infected cells is attributable mainly to the ac+c:umulation of thv smallest RNA. which Kivera et nl. (197X). (‘ashman & LC’rbster (1879). Smits P[ rrl.

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(1980) and (‘ashman et al. (19X0) have shown to be a primary transcript. A similar inference can be drawn from the work of Moses et 01. (1980). who showed that’ if gene \‘I11 (and its promoter) is moved to another part of the genome. phage growth is essentially normal. Thus. it seems that the strength of the promoter for this R?\‘A. together with its stability. accounts for t)he preferential synthesis of gene 1X1 1)rotein. The 2 kb RNA (band D, Fig. 3) appears to be much less abundant than the other RSXs synthesized from the F region. Sequences that code for the N-terminal region of the gene 11 protein present only in the 2 kb band. although transcribed quite efficiently, appear to turn over rapidly. Such a gradient is not present in region 1. Here, in addition to a 4 kb molecule that spans the whole region. several shorter molecules are observed that contain sequences from either end of t’he region. This pattern of RNA molecules is incompatible with a mechanistn of transcription in which there are several promoters but’ only one terminator, such ax applies in the F region. In the 1 region. the upstream (gene Ill) sequenc~es are represented in at least’ as many RNA species as are the distal. Pratt rt al. (1966) found that early amber mutants in gene Ill are polar on t,h
V. Enea for his constant help and suggestions during the course of tfrt> and the actual writing of this I)aper. The work was supported by grants from Science Foundation ad the Sational lnstit’utes of Health.

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Alberts. H.. Pwy. I,. & 1)elirts. H. (1!)74). ./. Mol. /iid. 68. 139 ISP. ,Ilwinfx. C‘. A.. Kemp. I). ,I. K- Stark, (:. K. (1977). /‘UK. .2irt. AIccrtl. 8%,i.. 1 ‘..<..-I. 74. Xl.i(~5354. IGley. tJ. M. & l)avidsotI. N. (1976). A-J,/rr/. /1I’oc/w/1/. 70. 7,i S.i. Beck. 1-Z.& Zink. 1%.(IHXI). ~:VIIP. 16. ::.? 58. Rwk. E.. Sommrr. E. A.. Auf~t~swalt1. IC. A.. Kurt. C’.. Zittk. PI.. Ostt~thttrg. (:.. Sfhallf~r. H Sngimot,o. K., Sugisaki. H.. Okamoto. 7’. & Taka,na.mi. J1. (l!UX). ,Yuc/. .-lcid.v Ho.s 5. 419.5 4503. Hlumbrtg, I). I). & Malatny. $1. H. (l!W). -1. I-id. 13. :JiX : Kof,kr. .I. I).. \.c)vis. (:. I’. CQZindf~r. S. I). (1!17!1). /‘WC. .\ilt. .Iurd. Ci.. /..,q..l. 76. PC!)!) 2io2. 8runk. (‘. (:. & Lf~ic~k. \.. (ICW!)). Kiochirr~. Biophys. .lrtu. 179. I%-lM 76. II69 117:1. (‘ashman. .J. S. & \Vf+ster. R. E:. (1!17!)). I ‘rot. ;?nt. .Icod. SC;.. I’.S..l. (‘ashman. .I. S., LVf~bster. Ii. b:. 8~ Stergf,. 1). .A. (1980). ./. Wiol. C’//~nr. 255. 2d;iGPT,6Z (‘hall. ‘1’. S.. Modrl, 1’. CVZirdrr. S. I). (IW;,). -1. Mol. Bid. 99. 389 382. /).V.l I’l~yrs. (‘oltl I~f~nhartlt. I). ‘I’.. I)trwlf~~~. T). k Ray. I). S. (197X). ‘P//c ,Sir,y/r~,Str(ltrdcd Spring Harl)c)r 1,ahoratory. (‘old Spring Hathor. Sfa~. York. Edrns. I,.. Konings, 1%. S. H. c!! Sf~hornmakers. .I. (:. (:. (l!Kr,). .V/d. .lrir/s Kv.u. 2. Itill I X20. Edetls. L.. TatI 1Vf:zfhrrl)tlfxk. I’.. Konings. Ii. S. H. & Slrf)f,ltrllakf,t.s. ,I. (:. (;. (1!)7ti). E//t.. .I Ilio~hPlrt 70. *isi 5%. (:illrspif~. I). S: Spiegdmatt. S. (I!G). ./. Mol. Bid. 12. 81”!) ML!. Hayaslti. %I.. I~‘~tjimttra. 13’.K. & Hay&i. Xl. (1976). /‘,,or. .L’u/. .-Irctrl. ,Sri.. I‘.S..i. 73. :%.il!l 3523. Hfwt:\,. .I. 1’. c( I’ratt. I). (I!%!,). 1’1.f~. .\ilt. A/x/d. SC/.. /...\‘..I. 62. X00 X07. I’hu~yrHorirtuhi. K.. \-ovis. (:. F. ci Motif~l. I’. (1!)78). Itr 7’/1r Sir/glr~,Str/r,cdrcli~l~ /).\‘.-I (l)f~nhardt. I). ‘I’., I)twslf~r. I). & Kay. 1). S.. ds). 1,1,. 113 IX. (‘old Sl~rittg Hartwt~ Laboratory. (‘old Spring Harbor. SW York. I).z:-l I’hncJr’.\ Konings. 11. N. H. & Schoenmakrrs. .I. (:. Cl. (197X). III 7’1)(’ Sir/y/c- JSil~/ltdrrl (Ihlllaldt. 1). T.. I~rrsslf~t~. D. & Ray. I). s.. cds). 1’1’. r,o7 530. (‘oltl Spring Harlwr Laboratory. (‘old Spring H;trhor, Se\v 1’ork. La Farina. IV. C? Motlrl. 1’. (197X). I’irolqy. 86. 36X :375. M&aster. G. I(. &z ~‘;trtrric:haf~l. (:. (‘_ (1977). I’roc. .\‘/I/. .lud. Sri.. (-.‘\‘..I 74. -PA3 MIX, Messing. ,I,, (~t~onc~r~l~ot~n,K.. Miillf~t~-Hill. H. & Hofkf~hnriclf~r. 1’. H. (1977). l’wr. Srr/. .-Id ,sc;.. I ..A,‘1 74. 361% :wtti. Morkl, I’. & Zintlcr. N. 1). (1974). ,/. dlrd. /lid. 88. 131 Zl Mows. 1’. K.. I~fwkf~. .J. I)., Horiuchi. K. & Zintlf~r. N. 1). (l!MO). l’i,o/oy~~. 104. 267 ‘7s Only. .J. 1,. & Kttil)lwtx. It. (l!K%). ,/. Md. Bid. 68. 125 IX% Okamotjo. ‘I’.. Sugiura. Xl. k Takanami. 11. (1969). -1. Mol. /
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Serburg. I’. H. cV Sfhallw. H. (1!175). ./. Nol. Bid. 92, P61 KY. S.. (:tttttatkw. 1:. \ Shank. 1’. ft.. Hrghfx S. H., Kuny, H. .I.. ~Vayors. .J. E.. Qttitltwll. Kishop. .J. M. & \Tarmrts. H. E. (197X). (‘t/I, 15. I Xi8 13%~. .I. t;. c:. (I!j’iX). /~iOd/i///. Smits. Al. A.. Simons. (j.. Konings. K. N. H. k Sc)rof~nrnakf,ts. fkyh!/s. .d /Y/r. 521. 27-l-t. Smits. 31. A.. Svhornmakf~rs. S. (:. (:. ti Korrings. I<. S. H. (IOXO). E/O,. ./. Riochert/. 112. :9x--321

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Sugimoto. K.. Sugisaki. H.. Okamoto. T. & Takanami. &I. (1977). J. Mol. Rid. 111. 487 507 Takanami. hf.. Okamoto. T. K: Sugiura. M. (1971 ). ./. LWd. Hid. 62. HlLXX. LVany. I). K- %ore. S. (1978). J. Kid. f’hm. 253. 7216-7219. Edited by M. (htt~sman