Transcription in vitro of the bacteriophage P22 antirepressor gene

J. Mol. Biol. (1982) 154, 427-447

Transcription in vitro of the Bacteriophage P22 Antirepressor Gene MIRIAM M. SUSSKINDAND PHILIP ~OUDERIAN

Department of Molecular Genetics and Microbiology University of Massachusetts Medical School Worcester, Mass. 01605, U.S.A.

(Received 31 March 1981, and in revised form 6 August 1981)

By transcribing bacteriophage P22 DNA in vitro with Escherichia coli RNA polymerase, we identify the transcript synthesized from P~t, the promoter for expression of the antirepressor gene (ant). The P~nt in vitro transcript is about 1-2 × 103 bases in size and is one of the most abundant large RNA species synthesized from phage DNA under these conditions. The results of experiments using templates modified by insertion of the translocatable ampicillin resistance element Tnl or by cleavage with restriction endonucleases show that the Pant transcript is synthesized rightward, contains the entire ant coding sequence, and terminates before gene 9, which encodes the P22 tail protein. This finding is consistent with the observation that gene 9 is not expressed from the P~,t promoter in vivo. Six independent spontaneous mutations that prevent synthesis of antirepressor protein in vivo also prevent synthesis of the Pant transcript in vitro; these six mutations most probably inactivate the ])ant promoter, l~eversion of one of these 'promoter-down" (P~n~) mutations by selection for the Ant + phenotype simultaneously restores synthesis of antirepressor protein in vivo and synthesis of the Pant transcript in vitro. Phage that carry a P~,t~ point mutation synthesize no detectable antirepressor protein, although they produce gene 9 protein late in infection. Thus, although the entire ant coding sequence is presumably transcribed in the sense direction from the phage P22 promoter for late gene expression (PI.~), antirepressor protein does not appear to be synthesized from the P~t~ transcript.

1. Introduction Although the genome of temperate Salmonella bacteriophage P22 is organized in much the same manner as that of the related temperate coliphage •, P22 has a novel second cluster of genes (the i m m I region) involved in regulation of gene expression and maintenance of lysogeny. As shown in Figure 1, the primary feature of this regulatory gene cluster is the P22 ant gene, which codes for an antirepressor protein that inhibits the activity of P22 c2 represser and/~ cI represser (Levine et al., 1975; Botstein. et al., 1975: Susskind & Botstein, 1975; R . T . Saner, personal 427 0022-2836/82/030427-21 802.00/0 © 1982 AcademicPress Inc. (London) Ltd.

428

M. M. SUSSKIND AND P. YOUDERIAN

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FIG. 1. Antirepressor model for the bipartite repression/immunity system of phage P22. The prophage genetic map is shown. The probable pattern of t~nseription is shown by thick arrows; broken arrows indicate transcription dependent on gene 23 function. Wavy arrows indicate negative control during lysogeny by repressors acting at operator/promoter sites, shown in boxes. communication). P 2 2 c2 rcpressor (analogous to h cI repressor) is directly responsible for repressing vegetative phage development in the lysogenic state by preventing transcription from the early promoters PL and PR (Poteete et al., 1980; A. R. Poteete & M. Ptashne, unpublished results). The a n t gene is expressed during •lytic infection of sensitive cells (Susskind. 1980), but is repressed in the lysogenic state by the product of the rant gene, which also is located in i m m I . Thus, both c2 and rant repressors are required for maintenance of stable lysogeny by P22 a n t + prophage and for immunity oflysogens to superinfecting P22 a n t + phage (Levine et al., 1975; Botstein et al., 1975). The mechanism of action of Mnt product is not known, but it apparently involves a site between the m n t and a n t genes that is altered by mutations called v i r A or V y . These c i s - d o m i n a n t mutations allow the a n t gene to be expressed in the presence o f M n t repressor, so t h a t V y a n t + phage are virulent (i.e. able to grow in an immune P22 c2 + rant + lysogen) (Levine et al., 1975; Botstein et al., 1975). These findings led Levine et al. (1975) and Botstein et al. (1975) to propose that the a n t gene is transcribed rightward from a promoter between rant and a n t (Pant) that is repressed by Mnt repressor but not by c2 rcpressor. The Vy mutations, by this model, would alter the Pant operator/promoter site so t h a t Mnt repressor no longer exerts its effect. The i m m I region contains another regulatory gene, arc (for antirepressor control), whose product negatively regulates antirepressor synthesis during lytic infection (Susskind, 1980). On infection of s u p ° cells, P22 a r c - a m b e r mutants vastly overproduce antirepressor protein, underproduce phage late proteins, and fail to produce progeny phage. Many revertants of a r c - a m phage selected for ability to grow in s u p ° cells are pseudo-revcrtants which retain the a r c - a m allele and acquire new mutations t h a t prevent synthesis of antirepressor. The isolation of these a r c a n t - pseudo-revertants indicates that Arc function is required for P22 growth only to repress synthesis of antireprcssor. T h a t is, the primary phenotype of the a r c -

PHAGE P22 ant GENE TRANSCRIPT

429

mutants is overproduction of antirepressor, and the lethal phenotype (underproduction of late proteins) is a secondary result of this effect. Most of the a n t - mutations carried by these arc- a n t - pseudo-revertants (18 out of 24 examined) were shown to be mutations in the ant structural gene, which lies to the right of the arc gene (Susskind, 1980). However, six spontaneous arca n t - revertants were found to carry a n t - mutations located to the left of arc. These six a n t - mutations map in the same interval (between rant and arc) as the Vy2 mutation. Therefore, one simple explanation for the a n t - mutations to the left of arc is that these mutations inactivate the Pant promoter. The iramI region is located on the P22 genome between the genes required for head morphogenesis and gene 9, which encodes the phage tail protein. The head and tail genes are expressed only late during infection and are thought to be part of a single operon t h a t is transcribed rightward from a promoter called PI~ (Fig. 1). The late operon is under positive control by the gene 23 product, which is thought to activate transcription from P~a~ by anti-terminating a small leader transcript (transcript a) (Roberts et al., 1976). Since gene 9 appears to be part of the late operon (Weinstock et al., 1980; Adams et al., unpublished results), the Plat+ transcript presumably traverses the entire i m m I region in the same direction as the Pa.t transcript. However, gene 9 does not appear to be expressed from the Pa,t transcript, since the tail protein (p9) is not synthesized early in infection with P22 wild type, when antirepressor is synthesized (Susskind, 1980). To explain why gene 9 is expressed from the rightward Plat~ promoter but not from the rightward P~nt promoter, Susskind & Botstein (1978) proposed that a transcription germination signal is located between ant and gene 9. Early in infection, when Pant transcription occurs, transcription terminates at this site: late in infection, when P~at~ transcription occurs, gene 23 product would allow transcription to proceed through this terminator into gene 9. In the experiments described below, we show t h a t one of the transcripts synthesized by Escherichia coli RNA polymerase in vitro using P22 phage DNA as template has all of the characteristics expected for the in vivo Pa,t transcript. Thus, this in vitro transcript is derived from the region of the P22 genome known to contain the ant gene, is synthesized in the rightward direction from the region expected to contain the Pant promoter, and terminates before gene 9. Furthermore, this transcript is not produced from templates carrying the mutations predicted to inactivate the Pant promoter. In addition, we show that although the ant gene is presumably transcribed in the sense direction from the Plat, promoter, antirepressor protein is not synthesized from the P,~t~ transcript.

2. Materials a n d M e t h o d s

(a) Bacteria and plasmids Salmonella typhimurium strains are derivatives of strain LT2. DB7000 (leuA-am414 8up °) (Susskind et al., 1974) and its supE derivative MS1363 (Susskind, 1980) have been described. MS1367 (Susskind, 1980) is a 8upE lysogen carrying a P22 prophage deleted for the rant-9 ~region. E. coli strain HB101 (Bolivar et al., 1977) and plasmid vector pMB9 (Rodriguez et al., 1976) were generous gifts from Lydia Villa-Komaroff.

M. M. SUSSKIND AND P. YOUDERIAN

430 Ap515

Ap69

ApSO Ap55

Ap55

R201 amR222 amRHl02 R204 am RH 108 R209 R220 R221 R224

FIG. 2. Genetic map of mutations. The segment of the genetic map shown is bounded by gene 16 on the left and gene 9 on the right. Allele numbers above the line refer to particular Tnl insertion mutations. The filled symbol indicates that the Ap50 mutation is in the orientation that is polar on expression of gene 9; the open symbols indicate Tnl insertions that are in the non-polar orientation. Allele numbers below the line designate the missense mutations sieA44 and mnt-tsl, the amber mutation arc-atoll1605, and the ant- mutations in pseudo-revertants of arc-am phage, either spontaneous (R) or hydroxylamine-induced (RH). (b) Phage P22 strains are derived from the wild-type strain of Levine (1957). Many of the mutations used in this study are shown in the map in Figure 2. Ap50, Ap53, Ap35 and Ap69 are insertions of the translocatable ampicillin resistance element Tnl. The isolation and characterization of these mutations are described by Weinstock et al. (1979). P22 Ap513, which carries a T n l insertion in gene 16, was isolated using the method of Weinstock et al. (1979), by transducing DB7000 to ampicillin resistance with a lysate obtained by ultravioletinduction of DB7189, a P22 wild-type lysogen carrying plasmid RP4. With the exception of P22 Ap513, all of the phages used in this study carry sieA44 (Susskind et al., 1971), a mutation that prevents superinfection exclusion of P22. The .multiple m u t a n t P22 sieA44 mnt-tsl arc-atoll1605 is the parent of the a n t - pseudorevertants R201. R204, R207, R209, R220, R221, R222, R224, RH102 and RH1O8 (Susskind, 1980). As shown in Figure 2, R222, RH102 and RH108 carry amber mutations in ant, R207 carries a mutation in ant that is not amber-suppressible, and the other 6 revertants carry a n t - mutations that presumably inactivate the Pant promoter (see Introduction). I n order to facilitate the preparation of high-titer phage stocks, the mutations 13-amHlO1 (Botstein et al., 1972), which prevents cell lysis, and c2-am08 (Dopatka & Prell, 1973), which prevents lysogeny, were introduced into some phage strains, as indicated in the Figure and Table legends. (c) Isolation of A n t + revertants of P22 R204 Three single-plaque stocks of a c2-am08 13-amH 101 derivative of the mnt-tsl arc-atoll 1605 a n t - pseudo-revertant R204 were grown in MS1363 (supE), which is permissive for both 13amH101 and arc-atoll1605. Spontaneous Ant + revertants were selected by plating on MS1367 at 30°C. One revertant from each stock (designated R204-R1, R204-R2 and R204R3) was purified and grown in MS1363. (d) Preparation of phage D N A P22 Ap phage particles were prepared by induction of lysogens with u.v. (150 ergs/mm 2) or mitomycin C (Sigma; 2 t~g/ml). Other phage strains were grown by lytic infection. Phage were concentrated from cleared lysates by centrifugation at 21,000g for 2 h or by

PHAGE P22 ant GENE T R A N S C R I P T

431

polyethylene glycol/dextran sulfate phase separation (Kourilsky et al., 1968) followed by centrifngation at 30,000g for 70min. Phage were purified twice on discontinuous CsC1 gradients and extracted twice with Tris-buffered phenol (pH 8-0). The DNA was precipitated with 0-25M-NaC1, 700/0 ethanol at - 2 0 ° C and dissolved in 10mm-Tris'HC1 (pH7"5), 0'1 mM-EDTA. (e) Construction of plasmid p M S 1

We made use of the properties of the transloeatable element T n l to select for insertion of a desired region of the P22 genome into plasmid vector pMB9. T n l has a single B a m H I restriction endonuelease cleavage site located 1"4 kb t from one end of the element. Located within this 1"4 kb segment is the ampieillin resistance (amp R) determinant. P22 Ap513 carries T n l inserted in gene 16 in the orientation that places the amp R determinant between the B a m H I cleavage site in T n l and the B a m H I cleavage site in gene 9 (Weinstock & Botstein, 1980). Digestion of P22 Ap513 DNA with B a m H I yields 4 fragments, 1 of which carries the amp '~ determinant and the region of the P22 genome from genes 16 to 9. Insertion of this fragment into the B a m H I cleavage site of pMB9 (which does not itself confer ampicillin resistance) creates a hybrid amp R plasmid. P22 Ap513 DNA (0"6 t~g) and plasmid pMB9 DNA (0"6 t~g) were digested with B a m H I (Boehringer Mannheim GmbH) according to conditions specified by the supplier, and incubated with T4 polynucleotide ligase (New England Biolabs) in 20 ~/1total volume at 15°C for 12 h using specified conditions. The resulting mixture was used to transform E. coli HB101 (Mandel & Higa, 1970; Wensink et al., 1974). Ampicillin resistant transformants were selected on plates containing 100 t~g ampicillin/ml. Only hybrid plasmids containing the P22 Ap513 fragment inserted in either orientation relative to pMB9 DNA were obtained from this selection. The structure of one of these plasmids, designated pMS1, was confirmed by detailed restriction analysis (data not shown). Purified plasmid pMS1 DNA was prepared by the method of Clewell (1972). (f) Preparation and endonuclease cleavage of restriction fragment Msp 2100 The Msp 2100 restriction fragment is the largest fragment generated by complete digestion of plasmid pMSI with MspI. This fragment was purified by electrophoresis in and elution from a 5-0c}~ (w/v) aerylamide/0"2°/o bis-aerylamide gel by the method of Maniatis et al. (1973) as modified by Humayun et al. (1977). The Msp 2100 fragment was digested further with endonueleases EcoRI, HpaI, or HindIII. To remove restriction endonueleases after digestion, the reaction mixtures were extracted twice with phenol and 3 times with ethyl ether, and incubated at 65°C for 15min. The digestion of DNA with restriction endonueleases MspI, Hpal (New England Biolabs), EcoRI (Boehringer Mannheim GmbH), and HindIII (Bethesda Research Laboratories) was carried out in buffer containing 6"0 mMTris- HCI (pH 8"0), 6"0 mM-MgCI 2, i0 mM-dithiothreitol, and 66 mM-NaCI. (g) Transcription in vitro The procedure for RNA synthesis in vitro using E. coli RNA polymerase (Sigma or Miles) was as described by Roberts (1975) except t h a t the concentration of all 4 nucleoside triphosphates was 0-1 mM. [~-32p]ATP (New England Nuclear) or [~-aEp]UTP (Radioehemieal Centre, Amersham) was added to a final concentration of 40 to 250 t~Ci/ml. For the experiment shown in Fig. 4, the concentrations of template DNAs are given in the legend. In all other experiments, the concentration of phage DNA was 50 t~g/ml. Incorporation of labeled nueleotide into acid-insoluble material was measured at the end of each reaction, and ranged from 20~$ to 30% of the input radioactivity. The reaction mixtures were extracted with phenol and RNA was precipitated with ethanol as described by Roberts (1975). t Abbreviations used : kb, 103 bases or base-pairs, as appropriate ; amp, Ap, ampicillin ; SDS, sodium dodecyl sulfate. 15

432

M. M. SUSSKIND AND P. YOUDERIAN (h) Gel eleetroph.oresis of R N A

Labeled RNA was subjected to elcctrophoresis in 2 types of slab gels. To resolve high molecular weight RNA species, 0-5o./0 agarose/2'0°.o acrylamide/6 M-urea gels were prepared as follows. A mixture containing l'0 ml 50~o acrylamide (Eastman), 1"2 ml 2°.~ bisacrylamide (Eastman), and 2-5 ml l0 x Lenning's buffer (0'36 M-Tris base, 0"3 M-NaH2P04, 10 mM-EDTA, pH 7"8)was added to 15 m110 M-urea (Schwartz-Mann, ultrapure) preheated to 48°C. The mixture was degassed and equilibrated to 48°C. Agarose (0'15 g; Sea-Kem. HTP grade) was dissolved by boiling in 5 ml water, cooled to 48°C, and added to the acrylamide mixture. After 5 rain at 48°C, 0'25 ml 10°.~) ammonium persulfate and 5 t~] N,N,N',IV'tetraethyl methylenediamfim were added and the mixture was immediately poured into a slab gel apparatus ( 1 6 c m x l 4 c m × 1 mm) having a 1 cm pre-polymerized 5% polyacrylamide/6 M-urea plug at the bottom. Immediately prior to electrophoresis, RNA was diluted in sample buffer (1 x Lenning's buffer, 70°/() deionized formamide, 10% glycerol), heated at 85°C for 0"5rain. and quick-chilled. Samples (12~l) containing 10,000 cts/min of acid-insoluble material were electrophoresed at 75 V for 5 to 6 h. 32p_ labeled yeast ribosomal RNA used for markers on these gels was a generous gift from Anita K. Hopper. In the experiment shown in Fig. 4, samples were prepared and loaded on a 3-0% to 14% polyacrylamide linear gradient gel containing 7 M-urea and 0"l°/o SDS (Spradling et al., 1977). Samples (30,000 to 175,000 cts/min of acid-insoluble material in 1-5 to 4 ~1) were electrophoresed at 25 mA for 4 h. Autoradiograms of dried gels were prepared on Kodak XR5 X-ray film. (i) Analysis of phage proteins in infected cell lysates Procedures for infection and labeling of unirradiated DB7000 cells with 14C_labeled amino acid mix (New England Nuclear), preparation of concentrated lysates, SDS/polyacrylamide gel electrophoresis, and gel autoradiography were as described by Susskind {1980).

3. Results (a) A n in vitro transcript corresponding to the P 2 2 ant gene I n the experiment shown in Figure 3, various P22 DNA templates were transcribed in vitrd by E. coli R N A polymerase and the resulting transcripts were analyzed by gel electrophoresis and autoradiography. Under these electrophoresis conditions, the high molecular weight transcripts are resolved, while the low molecular weight transcripts (including transcripts a and b characterized b y Roberts et al. (1976)) migrate off the bottom of the gel. As shown in Figure 3 (lane (a)), several discrete high molecular weight transcripts are synthesized in vitro from wild-type P22 DNA. To identify the transcript derived from the ant gene, we made use of P22 derivatives that carry the 5"0 kb translocatable ampicillin resistance element T n l inserted at genetically defined sites in or near the ant gene. The rationale for this experiment, first outlined to us by David Botstein, is that a large insertion in the template D N A will alter the size (and therefore the mobility) of the transcript corresponding to the gene carrying the insertion. P22 Ap50 and Ap53 have the T n l element inserted in opposite orientations at the same site within the ant gene (Weinstock et al., 1979 ; Fig. 2). As shown in lanes (c) and (d) of Figure 3, DNAs from both P22 Ap50 and P22 Ap53 specifically fail to direct the synthesis of the

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fastest migrating R N A species seen on this gel, designated transcrip~ c. This indicates t h a t transcript c is derived (at least in part) from the a n t gene. Although it is clear t h a t transcript c is not produced from P22 Ap50 and Ap53 templates, we do not know exactly how the insertion alters the form of this transcript. The Ap50 insertion m u t a t i o n exerts a strong polar effect on the

434

M. M. SUSSKIND AND P. YOUDERIAN

expression of gene 9 in vivo, whereas Ap53 is only weakly polar (Weinstock et al., 1980). As shown previously for other T n l insertions in the P22 ant gene (Weinstock et al., 1980), the degree of polarity c l a p 5 0 and Ap53 correlates with the orientation of the T n l element with respect to P22 DNA (unpublished results). The polar effect of T n l insertions m a y be due to a transcription termination site within the T n l element, as is the case with the insertion sequence IS2 (deCrombrugghe et al., 1973), which also is strongly polar in only one orientation (Saedler et al., 1974; Starlinger & Saedler, 1976). Such a termination site may not be observed in vitro, particularly in the absence of E. coli Rho termination factor (see Roberts, 1976), since transcription of Ap50 DNA does not appear to result in any prominent unique RNA species. In the absence of a strong (Rho-independent) termination site within the T n l element, the RNAs corresponding to transcript, c from Ap50 and Ap53 templates may be too variable in size to form discrete bands, or too large to be resolved by our electrophoresis conditions. Transcript, c is synthesized normally with DNA from P22 Ap69 (lane (e)), which carries a T n l insertion immediately to the left of the rant gene (see Fig. 2), and with DNA from P22 Ap35 (lane (b)), which has a T n l insertion in the extreme left end of gene 9 (P. Berget, unpublished results; Fig. 2). These results show that the failure of P22 ant : : T n l templates to synthesize transcript c is not simply due to the presence of the T n l element, but rather depends on the specific location of the insertion. Furthermore, the fact t h a t the Ap69 and Ap35 mutations do not interfere with the synthesis of transcript c indicates that transcript c is derived entirely from the region between the sites of these two insertions. Since the Pant transcript is expected to initiate to the right ofrnnt and to terminate between ant and gene 9 (see Introduction; Fig. 1), the results in Figure 3 are consistent with the idea th/~t transcript c is the Pane transcript. (b) Physical localization and direction of synthesis of the Pa~t transcript In order to demonstrate that transcript c is synthesized rightward from the region of the P22 chromosome known to contain the ant gene, we analyzed the transcripts produced in ,~itro from various restriction fragments within this region. The source of template DNA for this experiment is plasmid pMS1, which carries a 6 kb fragment of the P22 genome from a site in gene 16 to a site in gene 9 (see Materials.and Methods). The largest fragment (2"1 kb) produced by complete digestion of pMS1 with the restriction endonuclease MspI (Map 2100) is derived entirely from P22 DNA, and includes the region from rant to gene 9 (unpublished results). Figure 4, lane (a), shows that purified, supercoiled plasmid pMS1 DNA directs the synthesis of transcript c0 as does the Map 2100 fragment purified from this plasmid (lane (b)). Purified Map 2100 fragment was then cleaved in three separate reactions with restriction endonucleases EcoRI, HpaI and HindIII. The known cleavage sites for these enzymes within the Map 2100 fragment are diagrammed in Figure 5, line (a). The total products of these digestions were then transcribed in vitro and the resulting RNA species are shown in lanes (c) to (e) of the gel in Figure 4. Transcript c is not synthesized from any of the three cleaved templates,

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FIG. 4. Localization and direction of synthesis of the P,.t transcript. Transcripts synthesized in vitro by E. coli RNA polymerase from various DNA templates were analyzed as described in Materials and Methods. Template DNAs are from: (a) supercoiled plasmid pMS1 (15 ~g/ml): (b) restriction fragment Msp 2100 (2"5t,g/ml); (e) Msp 2100 fragment cleaved with EcoRI (2"5t~g/ml): (d) Msp 2100 fragment cleaved with HpaI (2"5~g/ml); (e) Msp 2100 fragment cleaved with HindIII (2.5/xg/ml). i n d i c a t i n g t h a t all t h r e e r e s t r i c t i o n e n d o n u c l e a s e s cleave P22 D N A a t sites w i t h i n the r e g i o n c o r r e s p o n d i n g to t r a n s c r i p t c. F u r t h e r m o r e , t h e m o s t a b u n d a n t t r a n s c r i p t p r o d u c e d f r o m each t e m p l a t e is s h o r t e r t h a n t r a n s c r i p t c, a n d shows a decrease in size c o m m e n s u r a t e w i t h t h e d e c r e a s e in size o f t h e l e f t m o s t D N A f r a g m e n t . As d i a g r a m m e d in F i g u r e 5, t h e s i m p l e s t i n t e r p r e t a t i o n o f t h e s e r e s u l t s is

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FIG. 5. Mapping of the P~,t transcript. (a) Restriction cleavage site map of the Msp 2100 fragment: numbers represent distance in kb from the left end, The arrows in (b) to (e) provide a rationale for the observed transcripts from the Msp 2100 template (b) and from the Msp 2100 fragment cleaved with restriction endonucleases EcoRI (c), HpaI (d), or HindIII (e). The inferred positions of the promoters and terminator are shown above the line in (b).

t h a t the p r o m o t e r for t r a n s c r i p t c is located a b o u t 150 base-pairs to the left of the leftmost HindIII site and t h a t the t e r m i n a t o r for t r a n s c r i p t c is located a b o u t 200 base-pairs to the right 0~fthe EcoRI site. As discussed below, the entire ant gene is contained within this region. I n addition to t r a n s c r i p t c, a minor discrete t r a n s c r i p t (designated x) is p r o d u c e d from the 3Isp 2100 fragment. T r a n s c r i p t x is p r o d u c e d from the HpaLcleaved template, indicating t h a t it is transcribed from a region of D N A internal to one of the two HpaI-MspI fragments. Since a smaller m i n o r transcript is p r o d u c e d from the EcoRI-cleaved Msp 2100 f r a g m e n t , t r a n s c r i p t x is derived from the right HpaIMspI fragment. T h e m i n o r t r a n s c r i p t p r o d u c e d from the HindIII-eleaved Msp 2100 f r a g m e n t is smaller still, indicating t h a t t r a n s c r i p t x is a r i g h t w a r d transcript which initiates f r o m a heretofore u n k n o w n p r o m o t e r (Px) located between the two r i g h t m o s t HindIII cleavage sites in the Msp 2100 f r a g m e n t (Fig. 5). These two HindIII sites are k n o w n to be within the ant s t r u c t u r a l gene (Deans & J a c k s o n , 1979; R . T . Sauer, W. K r o v a t i n , P. Y o u d e r i a n & M . M . Susskind, unpublished results). Since t r a n s c r i p t x is p r o d u c e d from the plasmid template, t r a n s c r i p t x m u s t t e r m i n a t e at a site between the EcoRI site and the right end of the Msp 2100 fragment, p r e s u m a b l y at the same site where t r a n s c r i p t c terminates. The significance of t r a n s c r i p t x in vivo, if any, is u n k n o w n .

PHAGE P22 ant GENE TRANSCRIPT

437

(c) P o i n t mutations that affect the synthesis of transcript c As discussed above (see Introduction), six of the a n t - mutations obtained in spontaneous pseudo-revertants of P22 mnt-ts arc-atoll1605 phage were shown to map to the left of all other ant mutations and even to the left of two arc mutations (Fig. 2). One simple explanation for these results is t h a t these six a n t - mutations are lesions in the P~n~ promoter. To test this idea, DNA from these mutants was used as template for transcription i n vitro. Figure 6, lanes (c) to (f), shows that four of these m u t a n t DNAs specifically fail to direct the synthesis of transcript c, while DNA from their immediate parent (lane (b)) directs the synthesis of transcript c normally. Similar results were obtained with the other two a n t - mutations mapping to the left o f arc (R201 and R221 ; data not shown). In contrast, transcript c is produced normally with DNA from four a n t - pseudo-revertants t h a t are derived from the same parental phage by the same ,selection, but which carry mutations in the ant structural gene (Fig. 6, lanes (g) to (j)). These results strongly support the idea t h a t transcript c is the ant transcript, since six mutations that prevent the synthesis of antirepressor protein and confer the A n t - phenotype in vivo (Susskind, 1980; see below) have been shown to prevent the synthesis of transcript c i n vitro. Furthermore~ the finding t h a t these a n t - mutations directly interfere with transcription from Pant provides a rationale for the fact t h a t they map to the left of both the arc and ant structural genes. These six mutations most probably inactivate the P~,t promoter; hereafter we will refer to these putative "promoterdown" mutations as " P ~ t ~ " (e.g. the a n t - mutation in P22 R204 is Pam~R204). (d) A n t + revertants of P 2 2 R204 restore synthesis of transcript c The six P~nt~ mutants were derived in a single step from P22 mnt-ts arc-amH1605 phage by selection for loss of the conditional-lethal phenotype of the arc-am mutation. Each of these pseudo-revertants has acquired a new mutation t h a t interferes with transcription from Pant in vitro and prevents synthesis of antirepressor in vivo. This results in the A n t - phenotype and in suppression of the lethal phenotype of the arc-am mutation, which is known to be retained by these phages (Susskind, 1980). Precise reversion of these Pant~ mutations should regenerate P22 mnt-ts arc-amH1605 ; such true revertants not only shouId regain the ability to produce antirepressor i n vivo and transcript c i n vitro, but also should regain the A r c - conditional-lethal phenotype. Ant + revertants of R204, one of these six arc-am P~nt~ pseudo-revertants, were obtained by selecting for growth on a s u p E ( P 2 2 i m m I ~ ) lysogen. This lysogen is immune to superinfecting a n t - phage, but is sensitive to superinfecting ant + phage ; the superinfecting phage must synthesize antirepressor and inactivate c2 repressor produced by the prophage in order to grow (Botstein etal., 1975; see Fig. 1). A s u p E lysogen was used because this amber suppressor is known to be permissive for P22 arc-atoll1605, and thus should be permissive for precise Ant + revertants of 1~204. Three spontaneous Ant + revertants derived from different single-plaque stocks (and therefore due to independent reversion events) were selected for further study.

438

M. M. SUSSKIND AND P. ¥ O U D E R I A N (a)

b)

{c)

(d)

(e)

(f)

(cJ)

(h]

(i)

(i)

(k)

255~

18S - -

Fro. 6. Mutations that affect the synthesis of the Pa,t transcript. Transcripts synthesized in vitro from various phage DNA templates were analyzed as described in Materials and Methods. All phage DNA templates carry the mnt-tsl and arc-atoll1605 mutations: (b) ant + (parent); (c) ant-R204; (d) ant-R209: (e) ant-R220; (f) ant-R224; (g) ant-amR222: (h) ant-R207; (i) ant-amRHl02; (j) antamRH108. Samples (a) and (k) contained yeast ribosomal RNA.

T h e s e A n t + r e v e r t a n t s o f R204 were first e x a m i n e d for t h e i r a b i l i t y to p r o d u c e a n t i r e p r e s s o r p r o t e i n i n vivo. DB7000 ( s u p °) cells were i n f e c t e d w i t h p h a g e a n d p u l s e - l a b e l e d w i t h r a d i o a c t i v e a m i n o acids b o t h e a r l y a n d l a t e a f t e r infection. T h e cells were h a r v e s t e d i m m e d i a t e l y a f t e r l a b e l i n g a n d e x t r a c t s were a n a l y z e d b y S D S / p o l y a c r y l a m i d e gel e l e e t r o p h o r e s i s . T h e a u t o r a d i o g r a m in F i g u r e 7 shows t h a t P22 arc + p h a g e p r o d u c e a n t i r e p r e s s o r a t a low b u t d e t e c t a b l e r a t e e a r l y a f t e r

PHAGE P22 a n t GENE T R A N S C R I P T (o) {b) (c) .

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pI p9 p16

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. .~ : ,. ::/,... .: >~:):..- . : .... :. : .,";,%. :'~,,:...:'..'.?. ' : ,. ...'; : ' x.

FIG. 7. Synthesis of antirepressor protein by Ant + revertants of P22 R204. Proteins synthesized at 37°C in unirradiated DB7000 (sup °) cells infected with P22 mutants (multiplicity of infection, 10 phage/cell) were analyzed on a 12"5~o SDS/polyacrylamide gel. All infecting phages carry the c2am08, 13-amHlO1, and mnt-tsl alleles: (b) and (c) arc + P+nt; (d) and (e) arc-atoll1605 P+nt; (f) and (g) arcamH1605 P~.t~R204; (h) and (i) arc-atoll1605 Pa.t~R204 Pa, t~R1 ; (j) and (k) arc-atoll1605 Pa,t~R204 Pa,tTR2 ; (1) and (m) arc-atoll1605 Pan,$R204 Pa,tTR3. For each infection, cells were labeled for 5 min beginning at (i~om lei~ to right) 15 and 35 rain after infection. Samples (a) and (n) contained purified P22 )articles. At the right, several P22 proteins are labeled according to the genes that specify them Botstein et al.. 1973; Lew & Casjens, 1975; Youderian & Susskind~ 1980). infection (lane (b)); l a t e in infection, a n t i r e p r e s s o r s y n t h e s i s is n o t d e t e c t a b l e (lane (c)). I n t h i s case, a n t i r e p r e s s o r s y n t h e s i s is r e p r e s s e d b y t h e a c t i o n o f t h e a r c gene p r o d u c t , since P22 a r c - a t o l l 1 6 0 5 (P~+nt) p h a g e p r o d u c e a n t i r e p r e s s o r a t high r a t e s b o t h e a r l y a n d l a t e a f t e r infection (lanes (d) a n d (e)). P22 R204 ( a r c - a r n H 1 6 0 5 P,nt~R204) p h a g e p r o d u c e no d e t e c t a b l e a n t i r e p r e s s o r (lanes (f) a n d (g)). I n c o n t r a s t , t w o o f t h e A n t + r e v e r t a n t s , R 2 0 4 - R 2 (lanes (j) a n d (k)) a n d R 2 0 4 - R 3 (lanes (1) a n d (m)) p r o d u c e a n t i r e p r e s s o r a t a p p r o x i m a t e l y t h e s a m e high r a t e s as P22 a r c - a t o l l 1 6 0 5 P~+.t p h a g e . T h e o t h e r A n t + r e v e r t a n t , R 2 0 4 - R 1 , p r o d u c e s significant levels o f a n t i r e p r e s s o r p r o t e i n b o t h e a r l y a n d l a t e a f t e r infection (lanes

440

M. M. SUSSKIND AND P. YOUDERIAN TABLE 1 Growth o f A n t + revertants of P 2 2 R 2 0 4

Infecting phage arc + P2~, arc-atoll1605 arc-atoll1605 arc-atoll1605 arc-atoll1605 arc-atoll1605

P£e

P~.t~R204P~.t~R204 P~.t~'R1 P~t~l~204 P..t]'R2 P~,tSR204 Pant~'R3

Burst size 130 0'4 140 50 0.1 0.2

DB7000 (sup °) cells were grown in M9CAAat 37°C to 2 x l0 s cells/ml,infected with the indicated phages (multiplicity of infection, 0"2 phage/cell), and incubated at 40°C. 15 rain after infection, the cultures were diluted 10¢-fold in isothermal M9CAA and incubation was continued. Samples were removed and treated with chloroform at 100 rain after infection, and burst sizes (progeny phage/input phage) were determined by plating on MS1363. All of the phages in this experiment also carry the c2-am08~ 13-amHlO1, and mnt-tsl mutations. (h) and (i)); at both times, this phage produces more antirepressor t h a n its R204 parent, but less t h a n P22 a r c - a m H l 6 0 5 Pant+ As described previously (Susskind, 1980), the overproduction of antirepressor by P 2 2 a r c - a t o l l 1605 (P~+~t)phage leads secondarily to underproduction of late proteins and failure to produce progeny phage. This is illustrated by the patterns of late protein synthesis shown in the autoradiogram in,Figure 7 (compare lanes (e) and (e)) and b y the burst size measurements presented in Table 1 (compare lines 1 and 2). Since the P~nt~R.204 m u t a t i o n prevents antirepressor synthesis, it suppresses these secondary effects; R204 phage produce late proteins (Fig. 7, lane (g)) and progeny phage (Table 1, line 3) normally. The Ant + revertants R204-R2 and R204R3 regain the A r e - lethal phenotype, since they both fail to synthesize late proteins (Fig. 7, lanes-(k) and (m)) and produce few progeny (Table 1, lines 5 and 6). In contrast, the Ant + r e v e r t a n t R204-R1 produces normal levels of late proteins (Fig. 7, lane (i)) and a substantial burst of progeny phage (Table 1, line 4). DNA from these three Ant + revertants of R204 was transcribed in vitro and the products were analyzed by gel electrophoresis. The autoradiogram in Figure 8 shows t h a t DNA from R204-R2 and R204-R3 (lanes (d) and (e)) directs the synthesis of a p p r o x i m a t e l y the same a m o u n t of transcript c as the P+nt control (lane (a)), whereas D N A from their P,nt~R204 parent directs the synthesis of far less transcript c (lane (b)). Thus, reversion of the A n t - phenotype of R204 phage can simultaneously restore the ability to direct the synthesis of transcript c in vitro. This finding demonstrates t h a t the m u t a t i o n in P22 R204 t h a t prevents antirepressor synthesis in vivo is also responsible for the failure to synthesize transcript c in vitro. The relative amounts of transcript c synthesized b y R204 and R204-Rl templates are difficult to estimate in this experiment, since an R N A species t h a t normally migrates slightly slower than transcript c (see Figs 3 and 6) co-migrates with transcript c in the gel shown in Figure 8. However, it appears t h a t R204-Rl directs the synthesis of more transcript c than R204, but less t h a n R204R2, R204-R3 and the Pa+t control.

P H A G E P22 a n t G E N E T R A N S C R I P T (o

(b)

(¢)

(d)

(e)

(f)

~25 S

--18 S

• /

, •[

Fro. 8. Reversion of P~ntlR204 restores synthesis of the Pant transcript• Transcripts synthesized in vitro from various phage DNA templates were analyzed as described in Materials and Methods. All phage DNA templates carrv~ the c2-am08,13-amHlO1, mnt-tsl, and arc-atoll1605 mutations: (a) Pant+ ,' (b) P~t~R204; (c)P~t~R204 P~ntTR1 : (d) P~.tSR204 PantTR2; (e) P,,t~R204 P~tSR3. Sample (f) contained yeast ribosomal RNA.

I f we assume t h a t the a n t - m u t a t i o n in R204 inactivates the P~,t promoter, these results suggest t h a t the r e v e r t a n t R204-R1 has only partially restored the activity of the Pant promoter, whereas the Pant p r o m o t e r in the revertants R204-R2 and R204-R3 is as active as the wild-type promoter. T h a t is, R204-R2 and R204-R3

442

M. M. SUSSKIND AND P. YOUDERIAN

m a y be true revertants in which the wild-type Pa~ltsequence has been regenerated, so that these phages are indistinguishable in phenotype from arc-am P+m phage. In contrast, R204-R1 appears to be a pseudo-revertant whose Pant promoter is more active than t h a t of R204, but less active than the wild-type promoter. Consequently, the level of antirepressor produced by this phage in the absence of Arc function (in s u p ° cells) is reduced sufficiently to relieve the A r c - lethal phenotype ; on the other hand, the level of antirepressor is high enough, even under Arc + conditions (in s u p E cells, which suppress the arc-am mutation), to allow the phage to grow in a s u p E ( i m m I ~ ) lysogen. Such a pseudo-revertant could result if the nucleotide base-pair affected by the P~nt~R204 mutation were replaced by a third, different base-pair; alternatively, R204-RI may retain the Pant~R204 mutation and acquire a suppressing mutation at a second site. 4. D i s c u s s i o n

We have examined the products of in vitro transcription of bacteriophage P22 wild-type and m u t a n t DNA templates in order to investigate the control of expression of the P22 ant gene. We have found that P22 DNA directs the synthesis of a transcript about 1"2 kb in length that is derived in part from the ant gene. This is demonstrated by the observation t h a t this transcript is not produced from templates carrying insertions of the translocatable element T n l within ant. Furthermore, by transcribing restriction fragments of P22 DNA, we have localized this transcript to the region of the P22 genome known to contain the ant gene, and have shown that this transcript is synthesized rightward: The Pant transcript is one of the most abundant large transcripts synthesized by E, coli RNA polymerase from wild-type P22 DNA in vitro. This indicates that Pant is a strong promoter whose activity does not depend upon accessory positive activators. Furthermore, when relieved of negative regulation by the Arc and Mnt repressors in vivo, Pant directs the synthesis of extremely high levels of antirepressor protein. The Pant transcript is not produced from templates carrying any of six mutations that confer the A n t - phenotype in vivo yet map to the left of the arc gene. Reversion of one of these mutations to Ant + simultaneously restores synthesis of the ant transcript i n vitro (Fig. 8). As discussed below, these six A n t - mutations probably inactivate the Pant promoter. (a) M a p p i n g of the P.,~t in vitro transcript The results of experiments using templates modified by T n l insertion (Fig. 3) or by cleavage with restriction endonucleases (Figs 4 and 5) allow us to align the P~.t in vitro transcript with respect to the genetic and physical map of the P22 genome. The Pa, t transcript initiates approximately 150 nucleotides to the left of P22 H i n d I I I site 1 and extends rightward to terminate approximately 200 nucleotides to the right of P22 E c o R I site 3 (Figs 4 and 5). The ant structural gene is entirely contained within the transcribed region. Various T n l insertion mutations that inactivate the ant gene h a v e been shown to lie in the region between H i n d I I I site 1

PHAGE

P22 ant GENE

TRANSCRIPT

443

and E c o R I site 3 (Deans & Jackson, 1979). Furthermore, correlation of the DNA sequence of this region with the amino acid sequence of antirepressor protein (R. T. Sauer, W. Krovatin, P. ¥ o u d e r i a n & M.M. Susskind, unpublished results) demonstrates that the ant coding sequence starts between H i n d I I I sites 1 and 2 and ends about 100 base-pairs to the right of E c o R I site 3. Thus, the ant gene corresponds to about 800 nucleotides of the 1"2 kb Pant transcript. The arc gene maps to the right of the mutations t h a t affect Pant transcription in vitro (Pant~ mutations) and the Vy2 mutation (a putative operator-constitutive mutation) (Susskind, 1980). Therefore, the arc gene should be transcribed from the Pant promoter. In the accompanying paper (Youderian et al., 1981) we present evidence t h a t the arc gene is expressed from the P~nt promoter in vivo. Since we estimate the are gene product to be about 8000 M~, we expect the arc gene to be about 200 base-pairs long ; the arc and ant genes would therefore account for about l'0 kb of the 1-2 kb P~nt transcript. The Pant transcript terminates approximately 200 nucleotides to the right of P22 E c o R I site 3. Since gene 9 begins approximately 300 base-pairs to the right of E c o R I site3 (R. T. Sauer, W. Krovatin, P. Youderian & M.M. Susskind, unpublished results), the terminator observed in vitro is located to the left of all of gene 9. (b) Mutations directly affecting transcription f r o m

Pa~t

Transcript c is not synthesized from template DNAs carrying any of six independent, spontaneous mutations that confer the A n t - phenotype in vivo yet map to ~the left of the arc gene. This finding lends further support to the idea t h a t transcript c is the Pant transcript. At the same time, it shows that these six mutations interfere with transcription from P~t, probably by inactivating the P~nt promoter. I t should be noted that these mutations, all of which revert to Ant + at frequencies typical for single point mutations (Susskind, 1980), are markedly severe in their effects ; for example, the P~,tSR204 mutation results in a more than 50-fold decrease in synthesis of antirepressor in vivo (Fig. 7). Thus, the peculiarities of the A r c - lethal phenotype afford a selection t h a t yields mutants defective in transcription from Pant ("promoter-down" mutants) at high frequency. We are currently using this selection to isolate large numbers of different P~t~ mutations in order to investigate the relationship between promoter primary structure and activity. (c) Differential expression of ant and gene 9 To provide a rationale for the observations that indicate that gene 9 is not expressed from the Pant promoter in vivo, Susskind & Botstein (1978) proposed that transcription from Pant terminates in vivo at a terminator, T~nt, between ant and gene 9 (see Introduction) ; the observation t h a t transcription from Pant terminates in this region in vitro, even in the absence of added Rho termination factor, provides direct evidence for this idea. Although gene 9 is separated from the rest of the P22 late genes by the i m m I region, gene 9 appears to be part of the late operon, since Weinstock et al. (1980)

444

M. M. SUSSKIND AND P. YOUDERIAN

found that insertions of Tnl in the polar orientation in genes 16 and 20 are polar on expression of gene 9. This would imply that transcripts initiating at PI~, the promoter for late gene expression, continue rightward through the entire i m m I region, and in particular through the Tan t terminator. This can be explained by postulating that at late times during infection, an anti-termination function (presumably the product of gene 23) allows rightward transcription from PJa~ to continue through the T~nt terminator into gene 9 (Susskind & Botstein, 1978; Weinstock et al., 1980). Even in the presence of p23, it appears that little, if any, transcription of gene 9 begins at Pant, since polar insertion mutations in gene 20 are polar to the same degree upon expression of gene 9 as polar insertions in ant (Weinstock et al., 1980). If a substantial proportion of gene 9 transcripts initiated at Pant, insertions upstream from Pant (e.g. in gene 20) would be expected to be less polar than insertions in ant. To explain why P~nt does not contribute to expression of gene 9, Susskind & Botstein (1978) proposed that p23 can prevent termination at Tant for transcripts originating at PI~ but not for transcripts originating at Pant" This would not be surprising, since the anti-termination functions endoded by the 2 N, P22 24, and 2 Q genes are known to work only on transcripts initiated at particular promoters (Franklin, 1974; Adhya et al., 1974; Friedman & Ponce-Campos, 1975; Hilliker & Botstein, 1976; Hilliker et al., 1978; Forbes~ 1978). However, the discovery that ant gene expression is negatively regulated during infection by the arc gene product (Susskind, 1980) obviates the necessity for postulating that p23 is unable to anti-terminate P~,lt transcripts. In wild-type infections, synthesis of antirepressor occurs early but is shut off by the arc gene product before the activity of p23 is apparent. During infection of s u p ° cells with P22 a r c - a m phage, the Pant promoter remains active at late times, but overproduction of antirepressor apparently interferes with the synthesis and/or function of p23 (unpublished results). Thus, under neither of these conditions are both Pant and p23 known to be active simultaneously, and we do not presently know whether p23 is able to antiterminate transcription originating at the Pant promoter. Although the Pl~ transcript should include all of the ant coding sequence, antirepressor does not appear to be produced from the Pl~ transcript. Thus, P22 R204, which carries a point mutation that abolishes transcription from P~t, produces gene 9 product in normal amounts late in infection of s u p ° cells at 37°C, but synthesizes no antirepressor protein (Fig. 8, lanes (e) and (g)). Since this phage carries both an m n t - t s and an a r c - a m mutation, the failure to synthesize antirepressor from the Pla~ transcript is not due to the action of either Mnt or Arc, the only known negative regulators of antirepressor synthesis. If we assume that the P,~ transcript extends through the ant gene, the failure to produce antirepressor may be due to some form of post-transcriptional control. This situation is reminiscent of the regulation of expression of the ~int gene, which is transcribed from two promoters. PL and Pint, but is translated efficiently only from the shorter Pint transcript (Guarneros & Galindo, 1979; Belfort, 1980; Epp & Pearson, 1981). The current model to explain this effect is that the Pint transcript terminates shortly before a recognition site for RNAase III; this site is present in the longer PL transcript. Following endonucleolytic attack by RNAase III at this

PHAGE P22 ant GENE TRANSCRIPT

445

site, presumably the int coding sequences on the PL transcript are rapidly degraded (U. Schmeissner, K. MeKenney, D. Court & M. Rosenberg, personal communication). Possibly a similar mechanism results in rapid degradation of ant sequences in the P22 Plato transcript. (d) Uses of the translocatable ampicillin resistance element T n l We have made novel use of insertions of the translocatable ampicillin resistance element T n l to identify an in vitro transcript and localize it with respect to the genetic and physical map of the P22 genome. Since the T n l element is large (5"0 kb), an insertion of T n l should alter substantially the size of any transcript that normally traverses the site of insertion, but should not affect other transcripts. Thus, the fact t h a t P22 Ap50 and Ap53 templates specifically fail to produce transcript c, whereas P22 Ap69 and Ap35 templates produce this transcript normally (Fig. 3), indicates that transcript c crosses the Ap50/Ap53 site but not the Ap69 and Ap35 sites. Because these mutations have been mapped both genetically and physically, these results localize transcript c with respect to the genetic and physical map. This method is an extension of the well-established technique of using templates with other types of multisite mutations (usually deletions or substitutions) to map in vitro transcripts (see, for example. Roberts et al., 1976). The advantages of using insertions of translocatable drug-resistance elements for this purpose lie principally in the well-documented virtues of these elements as genetic and physical markers (see Kleekner et al.. 1977; Weinstock et al., 1979; Weinstock & Botstein, 1980). We have also used the T n l insertion element in the in vitro construction of a recombinant plasmid containing the P22 i m m I region. As suggested previously by Kleckner et al. (1977), for any particular region of DNA to be cloned, the presence of a linked antibiotic resistance element allows a clone of the region of interest to be selected. (e) Organization of the i m m I region

The genetic organization of the i m m I region of phage P22 bears a striking resemblance to that of the immunity region of phage 2 (and to the i m m C region of P22). In both cases, gene(s) expressed from a rightward promoter (PR in 2; Pant in immI) are negatively regulated by a gene immediately to the right of the promoter during the lytic cycle (cro in 2: arc in immI), and by a gene immediately to the left of the promoter during lysogeny (cI in 2; rant in immI). Since the 2 cro gene is known to be expressed from PR, this analogy predicts that the arc gene is expressed from Pant and t h a t the Arc protein negatively regulates its own synthesis. In the accompanying paper we present evidence to support these predictions. We are indebted to Jeff Roberts for communicating his unpublished results demonstrating that one of the in vitro transcripts from P22 DNA is derived from the imm[ region. We are also grateful to David Botstein for the idea of using Tnl insertion mutations to facilitate the identification of the Pant transcript and to Bob Sauer and Peter Berget for communicating unpublished results. We thank Nancy Kleckner and Jeff Way for helpful suggestions. The

446

M. M. SUSSKIND AND P. Y O U D E R I A N

expert technical assistance of Susan Chadwick was invaluable to this work, which was supported by a grant from the National Institutes of Health (R01-GM22877) to M.M.S.P.Y. was the recipient of an American Cancer Society Postdoctoral Fellowship (PF-1385).

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Edited by M. Gottesman