Control of recA gene RNA in E. coli: Regulatory and signal genes

Cell, Vol. 20, 731-737.

July

1980.

Copyright

0 1980

by MIT

Control of recA Gene RNA in E. coli: Regulatory and Signal Genes

Ann McPartlancl,* Linda Green and Harrison Echo18 Department of Molecular Biology University of California Berkeley, California 94720

We have studied transcription of the recA gene of E. coli as a probe for regulatory events involved in the coupled control of DNA repair, mutagenesis, cell division, and prophage induction (‘SOS functions”). RecA mRNA production is greatly stimulated by two agents that block normal chromosomal DNA replication, ultraviolet light and nalidixic acid. This specific RNA induction is blocked by recA and dominant IexA mutations that confer ultraviolet sensitivity on the cell. A constitutive high level of RecA mRNA production results from mutations believed to inactivate IexA or from the unique recA mutation fir1 . These results suggest that the LexA protein is a negative regulator of the recA gene at the level of RNA synthesis, whereas RecA is a positive (“autoregulatory”) protein. The earliest (“signal”) events leading to recA gene induction appear to involve different pathways for the two types of replication blocks studied. Mutations affecting the RecBC protein block induction of RecA mRNA by nalidixic acid, but not ultraviolet light. In contrast, a loss of RecF function markedly decreases induction by ultraviolet light, but not nalidixic acid. Thus the signal system for SOS repair seems to have alternative pathways to provide a general response to a variety of DNA-damaging agents. Introduction The recA gene of E. coli has an essential role in a variety of processes that enhance cellular survival following exposure to DNA-damaging agents (Witkin, 1976). In addition to its probable direct participation in DNA repair by recombination, the RecA protein is required for a coupled, induced regulatory response to DNA damage involving mutagenesis, inhibition of cell division, increased DNA repair capacity, and prophage induction (Radman, 1975; Witkin, 1976). This set of inducible responses has been termed the “SOS functions” (Radman, 1975; Devoret, 1978). The SOS functions are induced by a wide variety of agents that block chromosomal DNA replication. The inducing treatment may introduce lesions into DNA (for example, ultraviolet light) (Beukers and Berends, 19601, or may directly inhibit some component of the * Present address: Department nia, Davis, California 95616.

of Biochemistry,

University

of Califor-

replication complex (for example, nalidixic acid) (Sugino et al., 1977). Thus the coupled regulatory response probably involves “signal events” derived from the inhibition of replication, providing in turn for “regulatory events” that turn on the SOS pathways subject to activation. An important advance toward a molecular understanding of the SOS response has been the observation that the level of the RecA protein is greatly increased after treatment of cells with ultraviolet light or nalidixic acid (Gudas and Pardee, 1975. 1976; McEntee, 1977; Emmerson and West, 1977; Gudas and Mount, 1977; Little and Hanawalt, 1977; Little and Kleid, 1977). This RecA induction is blocked by recA and /exA mutations that confer ultraviolet sensitivity, but occurs without DNA damage in cells carrying a unique recA mutation termed tifl that provides for constitutive expression of the SOS functions (Gudas and Pardee, 1976; McEntee, 1977; Gudas and Mount, 1977). These observations suggest that RecA and LexA are regulatory proteins for the SOS response, and that one of the regulated elements of the system is the recA gene. The regulatory role of RecA protein in prophage induction has been shown to involve direct inactivation of the prophage repressor through proteolytic cleavage (Roberts, Roberts and Craig, 1978). Thus one possible mechanism for the induced SOS response is activation of the proteolytic capacity of RecA by a signal of DNA damage, followed by a derepression of several SOS operons through cleavage of the LexA repressor (and perhaps other repressors) by RecA (McEntee, 1977; Gudas and Mount, 1977; Roberts et al., 1978). To begin an analysis of SOS regulation at the DNA level, we have studied the control of mRNA production from the recA gene. In a previous report, we demonstrated ultraviolet induction of mRNA from the recA gene (McPartland, Green and Echols, 1978); these experiments showed that SOS regulation for recA does occur at the gene level, not solely through cleavage reactions involving preexisting proteins. In this paper, we present a detailed characterization of RecA mRNA regulation, including direction of transciption and the effect of mutations in presumptive regulatory and signal genes. Our results show that RecA is a positive regulator and LexA a negative regulator for the recA gene, and that the RecBC and RecF proteins probably provide alternative routes for signal generation for different types of DNA damage. Results Induction of RecA mRNA and Orientation of Transcription We measured the inducibility of recA gene transcription by RecA mRNA production before and after treatment of cells with ultraviolet light or nalidixic acid. For

Cell 732

specific detection of RNA from the recA region, we used two transducing derivatives of phage X, whose genomic structures are depicted in Figure 1 (McEntee and Epstein, 1977). Both phages were derived from lysogens in which h was integrated into the srlA gene. The XsrlC recA phage carries part of srlA and the intact bacterial genes srlC and recA, whereas XsrlC presumably has the same srlAsrlC segment but does not appear to have acquired any recA gene DNA. Although the srl distal terminus of the bacterial substitution of XsrlC recA might be as far as 1.5 kb downstream from the recA gene, as judged by restriction enzyme analysis (McEntee and Epstein, 1977; Sancar and Rupp, 1979). the only specific bacterial proteins synthesized after infection by this phage are the srlC and recA gene products (McEntee, Hesse and Epstein, 1976). A quantitative assay for RecA RNA synthesis should therefore be derived from a comparison of hybridization reactions between total pulse-labeled cellular RNA and DNA from either hsrlC recA or hsrlC; that this is the case is shown in Table 1. The results of a typical ultraviolet induction experiment are shown in Table 1. Two logarithmic cultures of wild-type E. coli were treated identically, except that one was exposed to ultraviolet light (600 ergs/ mm*), then incubated for 20 min at 37°C before pulselabeling and RNA extraction. Radioactive RNA from each culture was tested for ability to hybridize to the isolated I and r strands of XsrlC recA and XsrlC DNA. Very little of the RNA from unirradiated cells hybridized to either hsrlC recA or XsrlC DNA (line 1). However, a small fraction of the RNA did hybridize preferentially to the I strand of hsrlC recA DNA. These data indicate that the normal rate of recA gene tranBacterial .... DNA ....

recll

*r/c

SrlA

.. . .. .

b’b’.

scription is rather low, but that a basal level is detectable. Following ultraviolet irradiation, the fraction of RNA which hybridized to the I strand of XsrlC recA DNA increased markedly (line 2). In contrast, ultraviolet treatment had no effect on RNA complementary to either hsrlC recA r strand DNA or AsrlC DNA. RecA RNA synthesis is therefore ultraviolet-inducible, and the coding strand for the RecA transcript is the I strand of hsrlC recA DNA. We can estimate the ultraviolet inducibility of RecA RNA production from the difference between the hsrlC recA and XsrlC I strand values. The observed increase is about 10 fold (column 5). Since the RecA transcript hybridizes specifically to the I strand of XsrlC recA DNA, the direction of transcription in vivo can be deduced from the orientation of the srlC and recA genes in the phage genome. This information will be needed for a biochemical analysis of the system in vitro. The arrangement of the bacterial genes within XsrlC recA DNA shown in Figure 1 is inferred from the position of the substitution in the X genome and the probable mechanism for its formation (McEntee and Epstein, 1977). The designated I and r strands indicate the coding strands for X transcripts synthesized in a “leftward” or “rightward” direction with respect to the standard genetic map. The singlestranded hsrlC recA DNA strands used in our hybridization experiments were matched to the corresponding I or r strands of X by relative ability to hybridize to purified X 6s transcript. Since the RecA transcript hybridizes to the I strand of hsrlC recA DNA, the direction of recA transcription is from right to left on the linear A map. Because sr/C is positioned to the right of the recA gene in the phage genome, the recA promoter lies on the srlC side of the recA gene. This location of the recA promotor agrees with that inferred by Sancar and Rupp (1979) based on a deletion Table 1. Ultraviolet

Induction

RNA in Hybrid

uv Figure 1. Genetic RecA mRNA

Structure

of A Transducing

Phages

used to Identify

The designations I and r indicate the coding strands for transcripts synthesized in a leftward or rightward direction with respect to the linear h map. The dark lines identify bacterial DNA. pes is the promoter site for initiation of the 6s h transcript: P,-,, is the promoter site for the recA gene. The aberrant host attachment site for A integration in srlA is designated b-b’*; X insertion occurs by a b l b” (host) x aa’ (phage) recombination to generate a b l a’Aab’* prophage. Thus the s&-transducing derivatives of h excision from the W/A gene have a hybrid attachment site b *a’ and the bacterial segments shown in the figure. The h genes A. J. int. N. Q and R are also shown, as are the single-stranded cohesive sites m and m’.

ASTIC recA DNA (1 Strand)

of RecA mRNA

(cpm per 2 X lo5 Input CPm)

Asrlc DNA (I Strand)

XsrlC recA DNA (r Strand)

hsrlC recA (I Strand) Minus XsrlC (I Strand)

RecA RNA (% Input RNA)

-

156

75

72

63

0.04

+

1036

94

82

942

0.47

150 ml of a log-phase culture of strain AS1 157 grown at 37’C were transferred to a Pyrex baking dish (9 x 9 inches) and ultravioletirradiated to a dose of 600 ergs/mm’ under a GE germicidal lamp. Incubation at 37’C was continued 20 min before pulse labeling with ‘H-uridine and RNA extraction. Liquid hybridization reactions were performed in DNA excess with 5-20 pg of RNA representing 0.5-3 x 1 O5 cpm. Each number is the average of several determinations at different RNA concentrations. RecA RNA (column 5) is the difference in percent total labeled RNA retained as hybrid with XsrlC recA (I strand) and Asr/C (1 strand) DNA.

Regulation 733

of RecA

RNA

analysis of RecA protein synthesis in vivo and RNA synthesis in vitro. The kinetics of ultraviolet induction of RecA mRNA are shown in Figure 2a. Ultraviolet irradiation causes a rapid increase in recA gene transcription, which reaches a maximum in approximately 10 min and remains constant for at least 20 additional min. Nalidixic acid, an inhibitor of DNA gyrase, also produces an induction of RecA mRNA (Figure 2b). RecA RNA begins to increase in a linear fashion shortly after addition of nalidixic acid; in this case the maximum production is reached after approximately 20 min. Thus rapid induction of recA gene transcription is one common cellular response elicited by both ultraviolet light and nalidixic acid, two agents having different primary modes of action on DNA synthesis. Regulation of RecA mRNA by the LexA and RecA Proteins Mutations in the recA and IexA genes affect inducibility of SOS functions and production of RecA protein (Radman, 1975; Witkin, 1976; Devoret, 1978-see Introduction). To relate these observations to regulation of the recA gene at the level of transcription, we have measured RecA mRNA production in strains

carrying various recA and IexA mutations. Mutations that prevent SOS induction completely abolish induction of RecA mRNA by either ultraviolet light or nalidixic acid (Table 2). From these results, we infer that induction of RecA mRNA by ultraviolet light and nalidixic acid proceeds through a common regulatory step involving both the RecA and LexA proteins. Because recA1 has been shown to confer a missense alteration in RecA protein (Gudas and Mount, 1977) the effect of the recAl mutation on recA gene transcription cannot result from nonsense polarity. Thus the RecA protein regulates transcription of its own gene. We have also studied the effects of mutations that confer constitutive expression on SOS functions: the tif (recA) and spr (/exA) lesions (Table 3). For the temperature-sensitive mutation tifl , a low basal rate of RecA mRNA is observed during growth at 30X, but constitutive production occurs at 41°C (line 2). Table 2. Dependence RecA Proteins

of RecA

mRNA

Induction

RecA RNA (Percentage

on the LexA and

RNA in Hybrid)

Genotype

uv

Nalidixic

recA’

0.46

0.49

0.01

0.04

recA1

0.03

0.03

recA3

0.01

/exA+

lexA3

Acid

Each strain was grown at 37°C to log-phase. One set of cultures received nalidixic acid to a final concentration of 40 pgg/ml and was subsequently incubated at 37°C for 30 min; another group of cultures was treated with ultraviolet light (600 ergs/mm’) as described in the legend to Table 1. Pulse labeling, RNA extractions and DNA-RNA hybridizations were performed as described in Experimental Procedures. The data in column 2 were obtained by the filter hybridization method; the data in column 3 were derived from the liquid hybridization method. RecA RNA is defined in Table 1. 0 Minutes

10 after

Figure 2. Kinetics and Nalidixic Acid

20 30 UV irradiation

of recA

Gene

0

10 Minutes after

Induction

20 30 NAL oddllion

by Ultraviolet

Irradiation

Two 150 ml cultures of strain AD1 157 were grown to log-phase at 37% One culture was ultraviolet-irradiated with a dose of 600 ergs/ mm2 as described in the legend to Table 1, These cells were immediately returned to a prewarmed flask and incubation at 37’C continued. 25 ml aliquots were subsequently removed to small flasks and pulse-labeled at various times. Nalidixic acid treatment was accomplished by adding aliquots of a second culture to prewarmed flasks containing enough nalidixic acid to achieve a final concentration of 40 gg/ml: incubation at 37°C was continued for the times indicated before pulse labeling. RNA extractions and DNA-RNA hybridization reactions were performed as described in Experimental Procedures. The data in Figure 2a were obtained by the liquid hybridization method, using isolated DNA strands. RecA RNA is defined in the legend to Table 1. The data in Figure 2b were derived by the filter hybridization method with immobilized denatured DNA in which the strands were not separately isolated. For this experiment, RecA RNA is defined as the difference between cpm retained in hybrid with AsrlC recA and hsr/C DNA. The data are expressed as percentage of total cpm added to the hybridization reaction found in RecA-specific hybrid.

Table 3. Constitutive

RecA mRNA

by tif and spr Mutants RecA RNA (Percentage brid)

Genotype” tif+ spr+

tifl (recA spr51

30°C (recA’

flexA

tifl spr51

rexA+)

mutation) mutation)

RNA in Hy-

41 “C

0.02

0.05

0.03

0.36

0.44

0.39

0.30

0.42

‘All strains carry an sfiA mutation (suppressor of filamentation) (George et al., 1975); the spr mutants probably have a second mutation in /exA (/exA3), which was present in the parent from which spr51 was selected (Mount, 1977). The tifl mutation has been shown to affect RecA protein (McEntee, 1977; Gudas and Mount, 1977). Logarithmic cultures of each strain prepared by growth at 30°C or 41°C were pulse-labeled with 3H-uridine. RNA extraction and liquid DNA-RNA hybridizations were as described in Experimental Procedures and the legend to Table 1.

Cell 734

The spr51 mutation also confers constitutive production of RecA mRNA (at both 30” and 41 “C) (line 3). In combination, the fill and spr51 mutations lead to RecA mRNA levels approximately the same as for either mutation alone when constitutively expressed (Table 3, column 2). This corresponds to the maximum induced RecA mRNA levels found for wild-type cells with either ultraviolet light or nalidixic acid. Thus the RecA and LexA proteins presumably have coordinate rather than completely independent roles in controlling recA gene transcription. To clarify the regulatory interaction between the RecA and LexA proteins, we studied RecA mRNA production in strains carrying combinations of mutations that individually confer opposite effects on induction (Table 4). The derepressed level of RecA mRNA is given in line 1 by the “double constitutive” mutant carrying tif and spr lesions. If a spr lesion in IexA is present in combination with recA1 (which by itself prevents induction), a high constitutive level of recA mRNA ensues (line 3). On the other hand, if the constitutive recA mutation tifl is in combination with the pleiotropic-negative lexA3, only a low basal rate of RecA mRNA formation occurs (line 2). Thus in each case a /exA mutation is epistatic with respect to the recA mutation. Because the lexA3 mutation is dominant to wildtype (Mount, Low and Edmiston, 1972) and the spr mutations are recessive (Mount, 1977; Pacelli, Edmiston and Mount, 1979), the spr mutations presumably result in loss of LexA function and the lexA3 mutation a functionally altered LexA. Taken together, the results of Tables 2-4 and the dominance data suggest strongly that LexA is a negative regulator of recA gene transcription. Since ultraviolet-sensitive recA mutations such as recA1 and recA3 presumably result in loss of RecA function (Clark, 19731, the data in Tables 2 and 3 suggest that RecA is a positive regulator of the recA gene. The results in Table 4 indicate that LexA protein has the more direct effect on recA gene transcription and that positive regulation by RecA occurs through an inactivation of LexA. Alternate Routes for Signal Generation: RecBC and RecF Pathways As noted above, RecA mRNA is low in wild-type cells. High-level transcription occurs only after treatment

with inhibitors of chromosomal DNA replication. The inhibitory treatment presumably leads to an “inducing signal,” which interacts with the RecA/LexA control system to release negative regulation of the recA gene. To identify potential genetic elements governing formation of the presumptive signal, we screened a number of known DNA repair mutations for effects on induction of recA gene transcription following exposure to ultraviolet irradiation or nalidixic acid. We expected that some DNA repair mutations would owe their ultraviolet sensitivity, at least in part, to effects on recA gene expression. A strong indication for a signal gene is a differential response to different inhibitors of DNA replication, suggesting an action prior to the general regulatory response mediated by RecA and LexA. We observed no drastic effects of mutations in uvrA, recL, recBC or various combinations thereof on ultraviolet-induced RecA mRNA. Mutations in recBC prevent nalidixic acid induction, however, and a recF lesion markedly reduces ultraviolet-inducibility. Data on recBC and recF are given in Table 5. Typical wildtype levels of RecA mRNA occur in a recBC strain following ultraviolet irradiation, but nalidixic acid induction is completely blocked (line 2). A recF lesion sharply reduces ultraviolet induction but does not impair the nalidixic acid response (line 3). In contrast to the absolute block on nalidixic acid induction by recBC mutations, we have found in every experiment with recF mutants a residual (and somewhat variable) ultraviolet-induced increase of RecA mRNA. This residual ultraviolet inducibility could not be eliminated by additional lesions in recBC or uvrA (Table 5, lines 4 and 5). From these results, we conclude that active RecBC protein is essential for induction of RecA mRNA if nalidixic acid is the inducing agent, but has no major Table 5. Differential Effects of Mutations in recB Induction of recA gene Transcription by Nalidixic Ultraviolet Irradiation

RecA RNA (Percentage brid)

Genotype’

Effect

of /exA Mutations

on RecA

Nalidixic

UV

Wild-type

0.49

0.42

0.05

0.41

0.75

0.11

RecA RNA (Percentage

recC22

recFl43

mRNA

RNA in Hybrid)

RNA in Hy-

Genotype

recB21 Table 4. Epistatic

and recF on Acid versus

recB21

recC22

recFl43

uvfA6

0.10

recFl43

0.12

fifl spr55

0.16

recA1

fifl /exA3

0.02

.spr5 1 recA 1

0.18

Cell growth, ultraviolet irradiation (400 ergs/mm*) and nalidixic acid treatment, pulse labeling, and RNA extractions were as described in the legend to Table 2. Liquid DNA-RNA hybridization was as described in the legend to Table 1. The values obtained with a strain carrying recF143 varied somewhat in different experiments. The numbers given are from an experiment which is representative of the average effect observed.

a All strains carry an sfiA mutation; the spr mutants probably carry /exAS. spr5.5 is an amber mutation. The growth temperature was 41 “C. Experimental procedures otherwise as described for Table 3.

also are

0.03

0.02

Regulation 735

of RecA RNA

role in ultraviolet induction (at least under the conditions used in our work). The RecF protein, on the other hand, has a major role for ultraviolet induction, but not for nalidixic acid. We suggest that the differential requirements of RecBC and RecF for recA gene induction indicate alternative routes from the replication block to an early step in the SOS response, “signal generation.” Discussion The coupled regulatory response of the SOS functions can be considered in terms of a signal of DNA damage and a regulatory reply that provides for enhanced DNA repair and mutagenesis, inhibited cell division, and prophage induction. In an effort to define more clearly the genetic elements involved, we have focused on recA gene transcription as a direct assay for a crucial early event in SOS control. To analyze the generality of the response, we have used two inhibitors of DNA replication with widely different biochemical mechanisms, ultraviolet light (pyrimidine dimer) and nalidixic acid (DNA gyrase). We find that mutations inactivating RecA protein block induction of RecA mRNA for either nalidixic or ultraviolet light, whereas mutations inactivating LexA produce a high constitutive level of RecA mRNA. Some mutations that probably generate RecA and LexA proteins with altered activity confer the opposite phenotypes: constitutive transcription of the recA gene for the tif form of RecA, and blocked induction by the mutant form of LexA associated with a dominant, ultraviolet-sensitive phenotype. From the genetic properties and general effect of these mutations, we conclude that RecA and LexA are the principal regulatory elements controlling the recA gene, and that RecA exerts positive regulation and LexA negative regulation. From experiments with double mutants, we argue that LexA is the more direct regulator, and that RecA probably acts by inactivation of LexA (Figure 3). A similar regulatory model has been derived from measurements of RecA protein production (McEntee, 1977; Gudas and Mount, 1977). In contrast to the general effect of recA and IexA mutations, we find a differential effect on RecA mRNA induction exerted by mutations in genes for two other proteins, RecBC and RecF. An inactive RecBC blocks nalidixic acid induction, but not ultraviolet induction; an inactive RecF inhibits ultraviolet induction, but not nalidixic acid induction. We interpret these results as indicative of alternative signal pathways from DNA damage to the general regulatory response mediated by RecA and LexA (Figure 3). Such alternative pathways presumably allow the cell to generate the coupled SOS response from different inhibitor effects on chromosome replication (for example, direct synthesis block or DNA unwinding block). The signal for the RecA/LexA regulatory system might be a small-mQl-

Figure

3. Postulated

An inhibition tein to carry inactivation employs the presumably

Early Events

in Induction

of SOS Functions

of chromosomal DNA replication signals the RecA proout positive regulation of the recA gene through an of the LexA repressor. The pathway to signal generation RecBC protein for an unwinding block and the RecF (and yet another route) for a pyrimidine dimer.

ecule effector or the DNA structure generated by RecBC or RecF. For nalidixic acid, the RecBC pathway appears to be obligatory, as indicated also by work with RecA protein (Gudas and Pardee, 1976). For ultraviolet light, the RecF pathway seems to be preferred, but still another route (besides RecBC) probably exists, since a significant residual level of ultraviolet-induced RecA RNA is found in a recFrecBC double mutant. The existence of alternative signal pathways probably accounts for some previous complexities concerning the role of RecBC and RecF in the SOS response. RecBC does have an important role for ultraviolet induction in plasmolyzed cells (Oishi and Smith, 1978), and mutations affecting RecF diminish but do not eliminate ultraviolet induction of h prophage and RecA protein (Horii and Clark, 1973; Roberts and Roberts, 1975; Armengod and Blanco, 1978; Clark, Volkert and Margossian, 1978). Experimental Procedures Bacterial and Bacteriophage Strains The wild-type bacterial strain used was AB1157, a derivative of E. coli K12 (Bachmann. 1972). Mutants carrying recA1, recA3. recB21, recC22, recFl43 or lexA3 were isogenic with AB1157: these strains were provided by A. J. Clark. Strain JM-1, a sup+ (nonsuppressing) derivative of AB1157, was the parent of mutants carrying tifl , spr51 and spr55. All strains harboring tif or spr lesions also carry an sfiA mutation (George, Castellazzi and Buttin. 1975) to suppress RecA/ LexA-mediated filamentation. The tif/spr strains used in this work were obtained from D. Mount; their construction and properties have been described (Mount. 1977; Gudas and Mount, 1977; Pacelli et al., 1979). The bacteriophage strains X.sr/C and hsrfC recA are plaque-forming specialized transducing phages isolated by K. McEntee (McEntee et al.. 1976; McEntee and Epstein, 1977). Both phage were derived from E. coli lysogens in which h&357 was integrated into the sr/A gene; in each case the b2 region of X has been replaced by bacterial DNA. The srf-distal terminus of the bacterial substitution of AsrlC recA is estimated by restriction analysis to be somewhere between the end of the recA gene and a site approximately 1.5 kb downstream (McEntee and Epstein, 1977; Sancar and Rupp. 1979; D. Willis, personal communication). However, the only AsrlC recA-specific bacterial proteins synthesized after phage infection are the SrlC and RecA products (McEntee et al.. 1976).

Cell 736

Buffers Phage buffer is 10 mM Tris-HCI. 10 mM MgSO, (pH 7.5); DNA buffer is 10 mM Tris-HCI. 1 mM EDTA (pH 7.5); hybridization buffer is 10 mM Tris, 10 mM sodium citrate, 1 mM EDTA. 500 mM N&l (pH 7.4); SSC buffer is 15 mM sodium citrate, 150 mM NaCl (pH 7.0). Cell Growth, Pulse Labeling and RNA Extractions Prior to an RNA labeling experiment, the cells were grown from single colony isolates to saturation in minimal M9 salts supplemented with glucose (0.4%). casamino acids (0.2%). and thiamine hydrochloride (2 pg/ml). At this point a sample was taken and used to test for the appropriate phenotypic characteristics of the strain. The saturated cultures were subsequently used to inoculate fresh samples of the same media (1% inoculum) for the labeling experiment. RNA was labeled with a 60 set pulse of tritiated (3H-5) uridine added to logarithmic cultures at a final concentration of 25 @i/ml. Growth was halted by pouring the culture over 0.5 vol of frozen minimal M9 salts containing sodium azide (20 mM), followed by quick chilling in a dry ice-ethanol bath. The cells were concentrated by centrifugation and resuspended in a small volume of 20 mM potassium acetate (pH 5.2). Sodium dodecylsulfate was added to a final concentration of 3.5% and lysis at 4°C was accelerated by agitation with a vortex mixer. RNA was extracted by three successive hot phenol treatments [1.5 vol phenol equilibrated with 50 mM potassium acetate (pH 5.2) at 6O”C]. The radioactive RNA was separated from small molecules in the final aqueous phase by repeated precipitation with 2.5 vol of cold 95% ethanol. The precipitate from the last treatment was resuspended in a small amount of hybridization buffer, passed through a nitrocellulose filter (13 mm). and stored at -20°C. RNA concentrations were determined by AZGO; purity was estimated from the A260/A280 ratios. Specific activity was estimated after determining the radioactivity (3H-cpm) present in a small aliquot of the RNA preparation: samples were loaded onto nitrocellulose filters (13 mm). which were dried and suspended in Toluene-Omnifluor scintillation fluid for counting. Phage Propagation, DNA Isolation and Strand Separation Large quantities of phage were prepared by infection of a sup+ strain of E. coli with lysis-defective (hS7) derivatives of hcl857 sr/C and A~1857 sr/C recA. Log phase cells growing in media containing tryptone (1%). yeast extract (0.02%). MgS04 (1 mM) and maltose (0.2%) were concentrated by centrifugation and resuspended in phage buffer (l/20 vol). and phages were added at a multiplicity of 5. After adsorption at room temperature, the cells were diluted into an 80 fold excess of media composed of tryptone (1%). yeast extract (0.02%). and glycerol (0.25%). and then incubated at 37°C for 3 hr. Cell lysis was achieved by addition of chloroform and the cellular DNA was digested with pancreatic DNAase (0.1 pg/ml). Cell debris was removed by centrifugation. and the phages were concentrated in the presence of polyethylene glycol and sodium chloride and subjected to equilibrium density centrifugation in CsCl (Wu et al.. 1972). Phages recovered from the gradient were rebanded, then dialyzed against phage buffer. Double-stranded DNA was isolated by successive cold phenol extractions of phage preparations, then dialyzed against DNA buffer. The yield and purity of DNA were estimated by AZeO and AZeO measurements. To isolate separated DNA strands, intact phages were subjected to the procedure described by Szybalski et al. (1971). Briefly. the phages were first dialyzed against 1 mM EDTA (pH 8.0). Sarkosyl and poly(U. G) were added to the phage suspension, which was heated to 100°C for 5 min. then chilled rapidly. The resultant extract was adjusted to pH 7.5 and subjected to equilibrium density centrifugation in CsCl (p = 1 .4. 70 hr at 30,000 rpm). Poly(U. G) binds preferentially to the de-rich strand of bacteriophage A. facilitating strand separation in a neutral gradient. After centrifugation. three drop fractions were collected from the gradient and diluted with DNA buffer. The fractions containing DNA were identified by AZBO measurements. Two distinct peaks were distinguished in all gradients, indicating that the DNA strands of hsr/C and AsrlC recA were clearly separated by this technique. Peak fractions were pooled and stored

at 4°C. The lower band in the gradient of AsrlC recA was identified as the dC-rich “right” ADNA strand by its ability to hybridize with purified A6S RNA transcript (gift from R. Fischer). DNA-RNA Hybridizations Liquid Hybridization The method used was a modification of the procedure of Takeda. Matsubara and Ogata (1975). A typical reaction mixture contained 2-3 fig of single-stranded phage DNA and 2-30 pg of tritium-labeled E. coli RNA (corresponding to l-4 x IO5 cpm). Afler a 2.5 hr incubation at 67°C. the samples were treated with a mixture of RNAase A (30 pg/ml) and RNAase Tl (15 U/ml). Each sample was diluted 10 fold with 2 x SSC buffer and filtered slowly through a Schleicher and SchuellB6 nitrocellulose filter (13 mm), which retains the DNA-RNA hybrids. The filter was rinsed by successive filtration with 2 X SSC buffer under suction, dried, and suspended in TolueneOmnifluor. and the radioactivity retained was determined by scintillation counting. Fitter Hybridization The procedure used was that of Gillespie and Spiegelman (1965) as modified by Bertrand, Squires and Yanofsky (1976). Heat-denatured DNA was loaded onto Schleicher and Schuell B6 13 mm nitrocellulose membrane filters (10 pg per filter). which served as the source of DNA for hybridization. Between 5 and 40 pg of tritiated RNA (corresponding to l-4 x 1 O5 cpm) was used in each hybridization reaction. All DNA-RNA hybridizations (liquid or filter) were performed under conditions of DNA excess. In every experiment the RNA concentration was varied and the DNA concentration held constant. In all cases the radioactivity retained on the filter was linearly proportional to the input 3H-RNA. In most cases the data are expressed as percentage of total input tritium cpm retained as hybrid with DNA. The numbers given represent an average of at least four determinations, all of which were similar. Acknowledgments We thank John Clark, Kevin McEntee and David Mount for strains, Robert Fischer, Linda Margossian. David Mount, Yoshinori Takeda. David Willis and Michael Volkert for advice and unpublished data, and Ruby Anderson for help with the experiments. This research was supported in part by a research grant from the NSF and by a postdoctoral fellowship (to A. M.) from the NIH. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

February

26, 1980;

revised

April 7. 1980

References Armengod. M.-E. and Blanco. M. (1978). Influence of the recF143 mutation of Escherichia coli K12 on prophage A induction. Mutation Res. 52, 37-47. Bachmann. B. J. (1972). Pedigrees of some mutant erichia coli Ki 2. Bacterial. Rev. 36, 525-557.

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