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Orientation of transcription of the lac operon and its represser gene in Escherichia coli
.T. ~~~MoZ. Bid.
(1969) 40, 116-151
Orientation of Transcription of the Zac Operon and its Repressor Gene in Escherichia coli The five genetic elements of the lac operon (p, o, z, y, a) and the structural its repressor (i) lie closely linked on the circular chromosome of Escherichia properties of pIeiotropic mutations have suggested that the kzc operon is counterclockwise (Fig. l), such that transcription is initiated in the promoter
gene for coli. The oriented element
FIG. 1. Genetic map of lysogenic Eschetichia coli. Selected segments of the map of Taylor & Trotter (1967) were updated and modified to include prophage maps of X and 480. The placement of the Zac promoter (p), operator (0) and repressor (i) genes with reference to each other is after Ippen et al. (1968) and Davies & Jacob (1968). A new marker chED, mapping between the gal operon and a.%! (Adhya, Cleary & Campbell, 1968), has been added. The 3’- to 5’- orientation of the complementary DNA &ran& is based on the known order of A and $80 genes in relation to the E. coli genes and to the 3’ and 5’ ends of the mature phage DNA (Wu & Kaiser, 1967). The arrows indicste the direction of transcription for the two transcribing units described in this report (solid -we) and for other units studied in this laboratory by analogous techniques (open arrows). For further details see Fig. 2.
(p), which is near the i gene, and proceeds in the direction of @A (Jacob & Monod, 1961; Newton, Beckwith, Zipser 62Brenner, 1965; Ippen, Miller, Scaife & Beckwith, 1968). The orientation of transcription for the i gene has not been described. We report in this letter the results of studies with i and luc messenger RNA’s, which show that the orientation of transcription of both the i gene and the elements of the luc operon is counterclockwise. The method consisted of annealing 3H-labelled i and luc mRNA’s extracted from appropriate h- and @O-free E. c&i strtbins to the separated DNA strands of #3Ohdkzc phage (Fig. 2), in which in the prophage state the i gene and the elements of the lac operon are oriented with the same polarity as at the normal location on the E. co.5 145
14G
S. KUMAR
AND
W.
SZYBALSKl
3000 -=-I x8606;-i1 ! X*50*( i* X7026i ..._......,._............... i i....--i
N
+
qi8O---h-Structural
E.coli--+blac
cr
OP
3’
2
5’A
r
Q SR
___- --- _--- -
$80 +7zc integration
Lysis immunity
Fm. 2. Map of the vegetative genome of defective, Zccc-transducing, hybrid coliphage 48Ohdlac (abbreviated symbol for 48Ohi”q, ,,,dZac). This phage was derived from an E. wli lysogen in which an P’Eac episome is translocated, with& Signer, 1960). On a out change in polarity, to a site near the at@’ and tdk loci (Beckwith proximal segment of its left arm it carries E. wli genes, including the Zoo repressor (i) and elements of the Zuc operon, p (promoter), o (operator), z (g-galactosidase), y (permease) and a (acetylaee). Its two DNA strands, I and r, are congruent with those of the 980 and X phages, ae shown in Table 2 and explained in the text. The Z strand has been defined (Szybalski, 1969) as that one which is transcribed Zeftward on the conventionally drawn vegetative map of X or 480 and has 6’-guanylate aa its left terminus. In poly(U, G)-containing CsCl gradient (Hwdecne & Seybalski, 1967), the Z strands of ~8OhdZuc, 480h, 480 and X have lower density than the complementary r strands. The 5’-termini of the two strands were positioned by Wu & Kaiser (1967). The orientation of transcription for the phage gene sequences (Taylor et aZ., 1967; Kumar, Bevre, Guha, Hradecna, Maher $ Szybalaki, manuscript in preparation; Loaeron & Szybalski, personal communication; for a review see Szybalaki, 1969) is indicated by open arrows and that for the inserted bacterial Zac and i genes, as determined in the present study, by solid arrows. Hybridization studies with h and 480 mRNA’s (see Table 2) and the genetic data indicate that in 48OAdZac the phage gene sequences on the left arm (genes 1, 2, 3, etc.; sat0 et al., 1968) and the integration region on the right arm are of $80, and a part on the right arm, including the immunity region iA, is of h aa shown on the map; the source of part on the right arm marked by the dashed line is uncertain. Above the bacterial segment of the ph8ge genome are delineated the deletions (dotted lines) in the i-&c region in the bacterial strains used: X7026 (Epstein, 1967), X8606 (Miller et al., 1968) and X8508 (Ippen et al., 1968).
chromosome (Beckwith & Signer, 1966), and determining which DNA strand hybridized with the particular mRNA (details in Fig. 3 and Table 1). The assignment of direction was based on knowledge of the orientation of the 6’-termini of the #Oxdlac DNA strands (Wu & Kaiser, 1967). Pulse-labelled lac [3H]mRNA was extracted from i+ luc+ cells induced to different extents by an inducer of the lac operon and i- lac+ cells grown under derepressed conditions. Figure 3 shows that nearly all the ZUGmRNA present in cells with the derepressed lac operon hybridized with the l-strand of 9%8Oxdlacphage DNA. The [3H]RNA which hybridized to the r-strand remained essentially constant over & wide range of effective concentrations of the inducer. An identical result was obtained when the luc mRNA was 6rst purified by hybridizcttion with denatured @Oxdlac DNA followed by elution, and then annealed with separated strands of 48OXdlac DNA. The data in Figure 3 also permit us to conclude that the level of lac mRNAin induced cells is at least 60 to 200 times higher than in repressed cells. It may be noted here that the specific activity of the t9-galaotosidase in such fully induced cells is about 1000 times higher than in lac repressed cells (see e.g. Gilbert & Miiller-Hill, 1966). We estimate
LETTERS
TO
THE
EDITOR
147
TABLE 1 Prqferentinl
hybridization
of i [3H]mRNA
of 33. coli with the l-strand
of
+SOhdlac DNA
Source of DNA
iq[3H]mRNA annealed
Strain
hch2
i gene
Zuc operon
absent
absent
DNA form
(cts/min)
I-strand
0
r-strand
3.6
l-strand
93.4
r-strand
8.9
Phage +SOxdlac
E. coli
present
present
X7026
deleted
deleted
denatured
5.8
X8508
presentt
deleted
denatured
92.7
X8606
present1
present
denatured
74.2
3000
present
present
denatured
90.6
E. c&i strain G113 (F’i~Zac+/i~km+) was grown with aeration (3’7’C) in M9 minimal medium acids (Difco) and 5 pg thiamine/ (Adams, 1969) supplemented with 0.2% glucose, 1% Casamino ml., to 2 x log cells/ml. The RNA in a 5-ml. portion of the culture was labelled with a 3-mm pulse of [3H]uridine (100 PC/ml.; 26 o/m-mole), terminated by pouring it over 15 ml. of crushed ice containing 10-z M-T& 5 x 10-3 rd-MgCl, and 10-z M-sodium azide. RNA was extracted according to the procedure of Taylor et al. (1967). The P:QmRNA was isolated in two prehybridization elution steps. The total extracted [3H]RNA (7.7 x lo6 acid-precipitable cts/min) was hybridized with 200 pg of denatured 48Ohdkw DNA immobilized on a nitrocellulose filter, treated with pancreatic and T1 RNases, which were subsequently inactivated by iodoacetate, and eluted, as described by Taylor et al. (1967). These Zac and i mRNA’s were then rehybridized with 400 pg of E. ccli X8508 DNA, in which the Zac operon is deleted but the i gene is present, and again eluted (total eluted cts/min = 1510). The last column represents the amount of this eluted iQ[3H]mRNA (input of 110 cts/min per annealing) which hybridized with 3 pg of separated phage DNA strands or 100 fig of denatured bacterial DNA. Hybridization experiments with the strands of +SOhdZuc DNA and with denatured DNA of E. ccli strains were done twice with essentially the same results. The stock of 48Oxdlac (abbreviated symbol for 980M1~r,867 dhc) phage was prepared by thermal induction of the E. c&i lysogen CA5004(480hiaq, 85,) (~8Ohi”cr. a37dZac), followed by three purificationa by banding iu the &Cl gradient. The buoyant densities in C&l gradient of phages 480h( = dSOhi”c,. 86’1) and 48Ohdlac were 1.494, and 1.501, g/cm3, respectively. The 1 and r strands were separated according to Hradecne & Szybalski (1967), using poly (U,G). In armlogy with hdg( = xdgal) phage, in which part of the left arm of the DNA is replaced by bacterial gene sequences (Hradecna & Szybalski, 1967; Murat & Szybalaki, 1968), the distance between the bands containing the 1 and r strands of 48OhdZac DNA (8 mg/om3) is about half of that separating the corresponding bands of 480h DNA strands (17 mg/cm5) in the presence of 5 pg poly(U,G)/ml. Bacterial DNA was prepared by the procedure of Marmur as modified by Szybaleki & Mennigmann (1962). 7 A small segment of the i gene, proximal
to the z gene, is deleted.
$ A small segment of the i gene and the adjoining genes between prcC and i are deleted (Miller et al., 1968). The extents of deletions in the i-Zac region in strains X7026, X8508 and X8606 are indicated in Fig. 2.
148
S. KUMAR
AND
W.
SZYBALSKI
i-Z -Strand IOOO’f-
Isopropyl
-/3 -D.-th~ogalactoside
(M)
FIG. 3. Effect of isopropyl-fi-D-thiogalectoside induction or of the i- mutation in the Zoc repressor gene on the amount of Zuc mRNA transcribed from the individual DNA strands. E. wli strains 3OOoi+ Za.c+ (circles) and 33OOi-lac+ (triangles) were grown with aeration (37OC) iu MQ minimal medium (Adams, 1959) with 0.2% glycerol as carbon source to 2 x lo8 cells/ml. To S-ml. portions of a culture of strain 3000 various concentrations of thiogelaotoside were added, followed 7 mm later by a 3-mm pulse of [3H]uridine (100 @/ml., 26 c/m-mole). An identical sH pulse was given to &ml. portions of & culture of strain 3300. Termination of the pulse and [sH]RNA extraction are described in Table 1. The separated, self-annealed DNA strands I (open symbols) and r (filled symbols) (1 pg) and the [sH]RNA (IO6 acid-precipitable cts/min) were annealed for 6 hr at 66°C in 0.5 ml. 2 x SSC, collected on nitrocellulose filters (B6, Schleicher & Schuell), treated with RNese, washed and the 3H radioactivity couuted (Nygeard & Hall, 1964). The amount of hybridized r3H]RNA is expressed as cts/min (per IO6 cts/min input) and 8s y. of total [3H]RNA. The background of 15 to 20 cts/min, obtained wit11 the I or c strands of hcb2 DNA, has not been subtracted.
that in induced cells about 1.2% of the total pulse-labelled RNA is lac mRNA, which is higher than the value reported previously (Attardi, Naono, Rouviere, Jacob & Gros, 1963; Contesse, Naono & Gros, 1966). Pulse-labelled i [3H]mRNA was extracted from Zuc repressed cells of a homozygous diploid strain with an iq mutation, since the transcribing activity of the iq gene is suspected to be several-fold that of the parent i+ strain (Miiller-Hill, Crapo & Gilbert, 1968). The i mRNA was isolated in two steps (details in Table 1). In the first step, a hybridization with denatured DNA of @OX&c phage selected both i and lac mRNA’s. In the second step, the luc mRNA was eliminated by rehybridization of the RNA eluted in the first step with denatured DNA of E. coli X8508, in which the i gene is retained but the entire lac operon is deleted (Ippen et al., 1968). The resulting RNA, eluted in the second step, hybridized equally well with the denatured DNA of all E. c&i strains carrying the complete or almost complete i gene, whether or not the linked genes on either side of the i gene are deleted, but not with that of a strain in which the i gene is absent (Table 1; Fig. 2), indicating that it was indeed i mRNA. Table 1 also shows that the i mRNA hybridized predominantly with the l-strand of #30X&c DNA. We estimate that about 0.02% of the total pulse-labelled RNA in F’iqkzc+/iqZac + repressed cells represents i mRNA.
LETTERS
TO
THE
TABLE
Hybridization
EDITOR
I49
2
of $80 and A mRNA’s with the I and r strands of 480hdlac, $80 a& h DNA’s
Source of [3H]RNA
Phage
P-0
3H-pulse (min after infection) 3-5
[3H]RSA Input
(cts/min) 37,000
annealed to 1 pg of DKA strands (cts/min)
#40hdZae I
$480
P
1
A r
I
76
4
153
‘7 2635
$80
58-60
33,000
9
638
301
x
28-30
52,000
“7.5
122
-
-
394
1 “921
Host infection was performed &S by Taylor et al. (1967). Procedures for RNA labelliug, RNA extraction, and RNA-DNA annealing (excess of DNA) were similar to those described in the legends for Fig. 3 and Table 1. Background count (11 cts/min) has been subtracted. The 480 and A 13H]RNA’s and ~$80 DNA strands were kindly given by Dr H. A. Lozeron.
The following experiments verified that the 1 and r strands of $8Ohdlac phage DNA (see Fig. 2 and Table 2) are congruent with the corresponding strands of X and $80 DNA. Pulse-labelled RNA’s made early and late during lytic development of 480 and late during lytic development of h were annealed to the separated strands of #OhdZac, $80 and X DNA’s. As evident from Table 2, $80 early [3H]RNA hybridizes preferentially with the 1 strands of both the #SO and ~$80hdlacDNA’s, whereas 480 late [3H]RNA shows a nearly 10: 1 preference for the r-strands of both phages. It is also shown that, of the X late RNA radioactivity which was capable of annealing to the l-strand of h, nearly 70% also hybridized with the l-strand of $80/\dluc DNA. Only 4(1/oof the h late RNA complementary to the r-strand of X hybridized with the r-strand of $8OXdluc DNA. This low figure indicates that h sequences must be absent not only throughout the left arm of ~j8Ohdlac, which consists of $80 and E. wli DNA, but probably also in a large part of the x- to R- region of the right arm (Fig. 2), since over 20% of the late transcription from the r-strand of h DNA originates on the right arm (Taylor, Hradecna & Szybalski, 1967; B4vre & Szybalski, manuscript in preparation; Szybalski, 1969). The results reported here show that strand 1 is the template for the i gene and luc operon in ~80Xdlac DNA, and that strand 1in $80hdlac is congruent with that in h and ~$80.Since transcription proceeds from the 5’-end to the 3’-end of the growing RNA molecule by addition of nucleotide subunits to the free 3’-hydroxyl group antiparallel to the 5’- to 3’- direction of the template DNA strand (Bremer, Konrad, Gaines & Stent, 1965; Maitra & Hurwitz, 1965), and 5’- to 3’- direction of the 1strand on the conventional vegetative map of $8OAdluc DNA is from left to right (Wu & Kaiser, 1967), transcription of the i gene and lac operon in $8OXdluc DNA must proceed from right to left (Fig. 2). The 5’. to 3’- direction of that polynucleotide chain of lysogenic E. coli DNA into which the 1 strands of h, $180or $8OXdlac integrate is clockwise on the conventionally drawn circular recombination map, as in Figure 1. Hence, transcription of the i gene and lac operon in ~8OXdEacprophage must be counterclockwise. Since the i gene and lac operon at their normal location in the
S. KUMAR
160
AND
W.
SZYBALSKI
chromosome and in 48OXdlac prophage are oriented with the same polarity (Beckwith $ Signer, 1966), the direction of their transcription at the normal location must also be counterclockwise. This conclusion means that transcription of the i gene starts at the end linked to proC and proceeds in the direction of the promoter element of the luc operon. The direction of transcription of the luc operon previously deduced from genetic experiments (see Ippen et al., 1968) is confirmed. In summary, we conclude that the orientation of transcription is counterclockwise for a number of genes distributed throughout a segment spanning one-sixth of the E. coli chromosome, including the lac operon, i gene (this report), gal operon (Guha, Tabaczynski & Szybalski, 1968) CUFF (= e,,) gene (Lozeron, Szybalski, Landy, Abelson & Smith, 1969), trp operon (Lozeron, Kumar & Szybalski, unpublished results) and h repressor gene (Taylor et al., 1967), since the corresponding mRNA’s hybridize only with the l-strands of transducing lambdoid phages carrying the respective genes. Drs W. Gilbert, W. Epstein and J. Beckwith generously supplied E. coli strains G113, X7026 and X8606. Dr J. Davies kindly gave the +8Ohdlac lysogen and all other bacterial strains. We wish to thank Drs J. Davies, K. Bevre, A. Guha and H. A. Lozeron for their frequent advice and Drs J. Beckwith and E. Signer for critical reading of this manuscript. The work was supported in part by grants from the National Cancer Institute (CA-07175), National Science Foundation (B-14976), and The Alexander and Margaret Stewart Trust Fund. McArdle Laboratory University of Wisconsin Madison, Wis. 53706, U.S.A. Received 21 October 1968, and in revised form 30 November
SUSEIL KUMAR WACLAW
SZYBALSKI
1968
REFERENCES Adams, M. H. (1969). In Bacteriophagea, p. 446. New York: Interscience. Adhya, S., Clear-y, P. t Campbell, A. (1968). Proc. Nat. Acad. Sci., Wad. 61, 966. Attardi, G., Naono, S., Rouviere, J., Jacob, F. & Gros, F. (1963). Cold Spr. Harb. Symp. &want.
Biol.
23, 363.
Beckwith, J. R. & Signer, E. R. (1966). J. Mol. Biol. 19, 264. Bremer, H., Konrad, M. W., Gaines, K. t Stent, G. S. (1966). J. Mol. Biol. 13, 540. Contesse, G., Naono, S. & Gros, F. (1966). C.R. Acad. Sci. Pad, 263, 1007. Davies, J. & Jacob, F. (1968). J. Mol. Bid. 86. 413. Epstein, W. (1967). J. Mol. Bid. 30, 629. Gilbert, W. BEMu&r-Hill, B. (1966). Proc. Nat. Acad. Sci., Wash. 56, 1891. Guha. A., Tabaczyriski, M. & Szybalski, W. (1968). J. Mol. Biol. 35, 207. Hradecna, Z. & Szybalski, W. (1967). Virology, 92, 633. Ippen, K., Miller, J. H., Scaife, J. t Beckwith, J. (1968). Nature, 21’7, 826. Jacob, F. & Monod, J. (1961). J. Mol. Bid. 3, 318. Lozeron, H. A., Szybalski, W., Landy, A., Abelson, J. & Smith, J. D. (1969). J. Mol. Biol. 39, 239. Maitra, U. t Hurwitz, J. (1966). Proc. Nat. Acad. Sci., Vu&. 54, 816. Mtiller-Hill, B., Crapo, L. & Gilbert, W. (1968). Proc. Nat. Acad. Sci., Wud. 59, 1269. Murata, N. & Szybalski, W. (1968). J. B en. Appl. Microbial., Tokyo, 14, 67. Newton, W. A., Be&with, J. R., Zipser, D. & Brenner, S. (1966). J. Mol. B&d. 14, 290. Nygaard, A. P. & Hall, B. D. (1964). J. Mol. Bid. 2, 125.
LETTERS
TO
THE
15 I
EDITOR
&to,
K., Nishimune, Y., Sato, M., Numich, It., Matsushiro, A., Inokuchi, (1968). Virology, 34, 637. Szybalski, W. (1969). Canadian Cancer Conference, 8, in the press. Szybalski, W. & Mennigmann, H.-D. (1962). Anal@ Biochenz. 3, 267. Taylor, K., Hradecna, Z. t Szybalski, W. (1967). Proc. Nat. Acad. Sci., Taylor, A. L. & Trotter, C. D. (1967). Bact. Rev. 31, 332. Wu, R. & Kaiser, A. D. (1967). PTOC. Nat. Acud. Sci., Wash. 57, 170.
H. & Ozeki,
H.
Wash. 57, 1618.
Note added ir, proof. mr this paper was accepted for publication we received a manuscript kindly sent by Dr J. Beckwith, which supplies independent genetic evidence supporting our conclusion on the orientation of the i gene (Miller, Beckwith t MiillerHill, 1968).
REFERENCE Miller,
J. H., Beckwith,
J. & Miiller-Hill,
B. (1968).
Nature
220, 1287.
(1969) 40, 116-151
Orientation of Transcription of the Zac Operon and its Repressor Gene in Escherichia coli The five genetic elements of the lac operon (p, o, z, y, a) and the structural its repressor (i) lie closely linked on the circular chromosome of Escherichia properties of pIeiotropic mutations have suggested that the kzc operon is counterclockwise (Fig. l), such that transcription is initiated in the promoter
gene for coli. The oriented element
FIG. 1. Genetic map of lysogenic Eschetichia coli. Selected segments of the map of Taylor & Trotter (1967) were updated and modified to include prophage maps of X and 480. The placement of the Zac promoter (p), operator (0) and repressor (i) genes with reference to each other is after Ippen et al. (1968) and Davies & Jacob (1968). A new marker chED, mapping between the gal operon and a.%! (Adhya, Cleary & Campbell, 1968), has been added. The 3’- to 5’- orientation of the complementary DNA &ran& is based on the known order of A and $80 genes in relation to the E. coli genes and to the 3’ and 5’ ends of the mature phage DNA (Wu & Kaiser, 1967). The arrows indicste the direction of transcription for the two transcribing units described in this report (solid -we) and for other units studied in this laboratory by analogous techniques (open arrows). For further details see Fig. 2.
(p), which is near the i gene, and proceeds in the direction of @A (Jacob & Monod, 1961; Newton, Beckwith, Zipser 62Brenner, 1965; Ippen, Miller, Scaife & Beckwith, 1968). The orientation of transcription for the i gene has not been described. We report in this letter the results of studies with i and luc messenger RNA’s, which show that the orientation of transcription of both the i gene and the elements of the luc operon is counterclockwise. The method consisted of annealing 3H-labelled i and luc mRNA’s extracted from appropriate h- and @O-free E. c&i strtbins to the separated DNA strands of #3Ohdkzc phage (Fig. 2), in which in the prophage state the i gene and the elements of the lac operon are oriented with the same polarity as at the normal location on the E. co.5 145
14G
S. KUMAR
AND
W.
SZYBALSKl
3000 -=-I x8606;-i1 ! X*50*( i* X7026i ..._......,._............... i i....--i
N
+
qi8O---h-Structural
E.coli--+blac
cr
OP
3’
2
5’A
r
Q SR
___- --- _--- -
$80 +7zc integration
Lysis immunity
Fm. 2. Map of the vegetative genome of defective, Zccc-transducing, hybrid coliphage 48Ohdlac (abbreviated symbol for 48Ohi”q, ,,,dZac). This phage was derived from an E. wli lysogen in which an P’Eac episome is translocated, with& Signer, 1960). On a out change in polarity, to a site near the at@’ and tdk loci (Beckwith proximal segment of its left arm it carries E. wli genes, including the Zoo repressor (i) and elements of the Zuc operon, p (promoter), o (operator), z (g-galactosidase), y (permease) and a (acetylaee). Its two DNA strands, I and r, are congruent with those of the 980 and X phages, ae shown in Table 2 and explained in the text. The Z strand has been defined (Szybalski, 1969) as that one which is transcribed Zeftward on the conventionally drawn vegetative map of X or 480 and has 6’-guanylate aa its left terminus. In poly(U, G)-containing CsCl gradient (Hwdecne & Seybalski, 1967), the Z strands of ~8OhdZuc, 480h, 480 and X have lower density than the complementary r strands. The 5’-termini of the two strands were positioned by Wu & Kaiser (1967). The orientation of transcription for the phage gene sequences (Taylor et aZ., 1967; Kumar, Bevre, Guha, Hradecna, Maher $ Szybalaki, manuscript in preparation; Loaeron & Szybalski, personal communication; for a review see Szybalaki, 1969) is indicated by open arrows and that for the inserted bacterial Zac and i genes, as determined in the present study, by solid arrows. Hybridization studies with h and 480 mRNA’s (see Table 2) and the genetic data indicate that in 48OAdZac the phage gene sequences on the left arm (genes 1, 2, 3, etc.; sat0 et al., 1968) and the integration region on the right arm are of $80, and a part on the right arm, including the immunity region iA, is of h aa shown on the map; the source of part on the right arm marked by the dashed line is uncertain. Above the bacterial segment of the ph8ge genome are delineated the deletions (dotted lines) in the i-&c region in the bacterial strains used: X7026 (Epstein, 1967), X8606 (Miller et al., 1968) and X8508 (Ippen et al., 1968).
chromosome (Beckwith & Signer, 1966), and determining which DNA strand hybridized with the particular mRNA (details in Fig. 3 and Table 1). The assignment of direction was based on knowledge of the orientation of the 6’-termini of the #Oxdlac DNA strands (Wu & Kaiser, 1967). Pulse-labelled lac [3H]mRNA was extracted from i+ luc+ cells induced to different extents by an inducer of the lac operon and i- lac+ cells grown under derepressed conditions. Figure 3 shows that nearly all the ZUGmRNA present in cells with the derepressed lac operon hybridized with the l-strand of 9%8Oxdlacphage DNA. The [3H]RNA which hybridized to the r-strand remained essentially constant over & wide range of effective concentrations of the inducer. An identical result was obtained when the luc mRNA was 6rst purified by hybridizcttion with denatured @Oxdlac DNA followed by elution, and then annealed with separated strands of 48OXdlac DNA. The data in Figure 3 also permit us to conclude that the level of lac mRNAin induced cells is at least 60 to 200 times higher than in repressed cells. It may be noted here that the specific activity of the t9-galaotosidase in such fully induced cells is about 1000 times higher than in lac repressed cells (see e.g. Gilbert & Miiller-Hill, 1966). We estimate
LETTERS
TO
THE
EDITOR
147
TABLE 1 Prqferentinl
hybridization
of i [3H]mRNA
of 33. coli with the l-strand
of
+SOhdlac DNA
Source of DNA
iq[3H]mRNA annealed
Strain
hch2
i gene
Zuc operon
absent
absent
DNA form
(cts/min)
I-strand
0
r-strand
3.6
l-strand
93.4
r-strand
8.9
Phage +SOxdlac
E. coli
present
present
X7026
deleted
deleted
denatured
5.8
X8508
presentt
deleted
denatured
92.7
X8606
present1
present
denatured
74.2
3000
present
present
denatured
90.6
E. c&i strain G113 (F’i~Zac+/i~km+) was grown with aeration (3’7’C) in M9 minimal medium acids (Difco) and 5 pg thiamine/ (Adams, 1969) supplemented with 0.2% glucose, 1% Casamino ml., to 2 x log cells/ml. The RNA in a 5-ml. portion of the culture was labelled with a 3-mm pulse of [3H]uridine (100 PC/ml.; 26 o/m-mole), terminated by pouring it over 15 ml. of crushed ice containing 10-z M-T& 5 x 10-3 rd-MgCl, and 10-z M-sodium azide. RNA was extracted according to the procedure of Taylor et al. (1967). The P:QmRNA was isolated in two prehybridization elution steps. The total extracted [3H]RNA (7.7 x lo6 acid-precipitable cts/min) was hybridized with 200 pg of denatured 48Ohdkw DNA immobilized on a nitrocellulose filter, treated with pancreatic and T1 RNases, which were subsequently inactivated by iodoacetate, and eluted, as described by Taylor et al. (1967). These Zac and i mRNA’s were then rehybridized with 400 pg of E. ccli X8508 DNA, in which the Zac operon is deleted but the i gene is present, and again eluted (total eluted cts/min = 1510). The last column represents the amount of this eluted iQ[3H]mRNA (input of 110 cts/min per annealing) which hybridized with 3 pg of separated phage DNA strands or 100 fig of denatured bacterial DNA. Hybridization experiments with the strands of +SOhdZuc DNA and with denatured DNA of E. ccli strains were done twice with essentially the same results. The stock of 48Oxdlac (abbreviated symbol for 980M1~r,867 dhc) phage was prepared by thermal induction of the E. c&i lysogen CA5004(480hiaq, 85,) (~8Ohi”cr. a37dZac), followed by three purificationa by banding iu the &Cl gradient. The buoyant densities in C&l gradient of phages 480h( = dSOhi”c,. 86’1) and 48Ohdlac were 1.494, and 1.501, g/cm3, respectively. The 1 and r strands were separated according to Hradecne & Szybalski (1967), using poly (U,G). In armlogy with hdg( = xdgal) phage, in which part of the left arm of the DNA is replaced by bacterial gene sequences (Hradecna & Szybalski, 1967; Murat & Szybalaki, 1968), the distance between the bands containing the 1 and r strands of 48OhdZac DNA (8 mg/om3) is about half of that separating the corresponding bands of 480h DNA strands (17 mg/cm5) in the presence of 5 pg poly(U,G)/ml. Bacterial DNA was prepared by the procedure of Marmur as modified by Szybaleki & Mennigmann (1962). 7 A small segment of the i gene, proximal
to the z gene, is deleted.
$ A small segment of the i gene and the adjoining genes between prcC and i are deleted (Miller et al., 1968). The extents of deletions in the i-Zac region in strains X7026, X8508 and X8606 are indicated in Fig. 2.
148
S. KUMAR
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SZYBALSKI
i-Z -Strand IOOO’f-
Isopropyl
-/3 -D.-th~ogalactoside
(M)
FIG. 3. Effect of isopropyl-fi-D-thiogalectoside induction or of the i- mutation in the Zoc repressor gene on the amount of Zuc mRNA transcribed from the individual DNA strands. E. wli strains 3OOoi+ Za.c+ (circles) and 33OOi-lac+ (triangles) were grown with aeration (37OC) iu MQ minimal medium (Adams, 1959) with 0.2% glycerol as carbon source to 2 x lo8 cells/ml. To S-ml. portions of a culture of strain 3000 various concentrations of thiogelaotoside were added, followed 7 mm later by a 3-mm pulse of [3H]uridine (100 @/ml., 26 c/m-mole). An identical sH pulse was given to &ml. portions of & culture of strain 3300. Termination of the pulse and [sH]RNA extraction are described in Table 1. The separated, self-annealed DNA strands I (open symbols) and r (filled symbols) (1 pg) and the [sH]RNA (IO6 acid-precipitable cts/min) were annealed for 6 hr at 66°C in 0.5 ml. 2 x SSC, collected on nitrocellulose filters (B6, Schleicher & Schuell), treated with RNese, washed and the 3H radioactivity couuted (Nygeard & Hall, 1964). The amount of hybridized r3H]RNA is expressed as cts/min (per IO6 cts/min input) and 8s y. of total [3H]RNA. The background of 15 to 20 cts/min, obtained wit11 the I or c strands of hcb2 DNA, has not been subtracted.
that in induced cells about 1.2% of the total pulse-labelled RNA is lac mRNA, which is higher than the value reported previously (Attardi, Naono, Rouviere, Jacob & Gros, 1963; Contesse, Naono & Gros, 1966). Pulse-labelled i [3H]mRNA was extracted from Zuc repressed cells of a homozygous diploid strain with an iq mutation, since the transcribing activity of the iq gene is suspected to be several-fold that of the parent i+ strain (Miiller-Hill, Crapo & Gilbert, 1968). The i mRNA was isolated in two steps (details in Table 1). In the first step, a hybridization with denatured DNA of @OX&c phage selected both i and lac mRNA’s. In the second step, the luc mRNA was eliminated by rehybridization of the RNA eluted in the first step with denatured DNA of E. coli X8508, in which the i gene is retained but the entire lac operon is deleted (Ippen et al., 1968). The resulting RNA, eluted in the second step, hybridized equally well with the denatured DNA of all E. c&i strains carrying the complete or almost complete i gene, whether or not the linked genes on either side of the i gene are deleted, but not with that of a strain in which the i gene is absent (Table 1; Fig. 2), indicating that it was indeed i mRNA. Table 1 also shows that the i mRNA hybridized predominantly with the l-strand of #30X&c DNA. We estimate that about 0.02% of the total pulse-labelled RNA in F’iqkzc+/iqZac + repressed cells represents i mRNA.
LETTERS
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TABLE
Hybridization
EDITOR
I49
2
of $80 and A mRNA’s with the I and r strands of 480hdlac, $80 a& h DNA’s
Source of [3H]RNA
Phage
P-0
3H-pulse (min after infection) 3-5
[3H]RSA Input
(cts/min) 37,000
annealed to 1 pg of DKA strands (cts/min)
#40hdZae I
$480
P
1
A r
I
76
4
153
‘7 2635
$80
58-60
33,000
9
638
301
x
28-30
52,000
“7.5
122
-
-
394
1 “921
Host infection was performed &S by Taylor et al. (1967). Procedures for RNA labelliug, RNA extraction, and RNA-DNA annealing (excess of DNA) were similar to those described in the legends for Fig. 3 and Table 1. Background count (11 cts/min) has been subtracted. The 480 and A 13H]RNA’s and ~$80 DNA strands were kindly given by Dr H. A. Lozeron.
The following experiments verified that the 1 and r strands of $8Ohdlac phage DNA (see Fig. 2 and Table 2) are congruent with the corresponding strands of X and $80 DNA. Pulse-labelled RNA’s made early and late during lytic development of 480 and late during lytic development of h were annealed to the separated strands of #OhdZac, $80 and X DNA’s. As evident from Table 2, $80 early [3H]RNA hybridizes preferentially with the 1 strands of both the #SO and ~$80hdlacDNA’s, whereas 480 late [3H]RNA shows a nearly 10: 1 preference for the r-strands of both phages. It is also shown that, of the X late RNA radioactivity which was capable of annealing to the l-strand of h, nearly 70% also hybridized with the l-strand of $80/\dluc DNA. Only 4(1/oof the h late RNA complementary to the r-strand of X hybridized with the r-strand of $8OXdluc DNA. This low figure indicates that h sequences must be absent not only throughout the left arm of ~j8Ohdlac, which consists of $80 and E. wli DNA, but probably also in a large part of the x- to R- region of the right arm (Fig. 2), since over 20% of the late transcription from the r-strand of h DNA originates on the right arm (Taylor, Hradecna & Szybalski, 1967; B4vre & Szybalski, manuscript in preparation; Szybalski, 1969). The results reported here show that strand 1 is the template for the i gene and luc operon in ~80Xdlac DNA, and that strand 1in $80hdlac is congruent with that in h and ~$80.Since transcription proceeds from the 5’-end to the 3’-end of the growing RNA molecule by addition of nucleotide subunits to the free 3’-hydroxyl group antiparallel to the 5’- to 3’- direction of the template DNA strand (Bremer, Konrad, Gaines & Stent, 1965; Maitra & Hurwitz, 1965), and 5’- to 3’- direction of the 1strand on the conventional vegetative map of $8OAdluc DNA is from left to right (Wu & Kaiser, 1967), transcription of the i gene and lac operon in $8OXdluc DNA must proceed from right to left (Fig. 2). The 5’. to 3’- direction of that polynucleotide chain of lysogenic E. coli DNA into which the 1 strands of h, $180or $8OXdlac integrate is clockwise on the conventionally drawn circular recombination map, as in Figure 1. Hence, transcription of the i gene and lac operon in ~8OXdEacprophage must be counterclockwise. Since the i gene and lac operon at their normal location in the
S. KUMAR
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chromosome and in 48OXdlac prophage are oriented with the same polarity (Beckwith $ Signer, 1966), the direction of their transcription at the normal location must also be counterclockwise. This conclusion means that transcription of the i gene starts at the end linked to proC and proceeds in the direction of the promoter element of the luc operon. The direction of transcription of the luc operon previously deduced from genetic experiments (see Ippen et al., 1968) is confirmed. In summary, we conclude that the orientation of transcription is counterclockwise for a number of genes distributed throughout a segment spanning one-sixth of the E. coli chromosome, including the lac operon, i gene (this report), gal operon (Guha, Tabaczynski & Szybalski, 1968) CUFF (= e,,) gene (Lozeron, Szybalski, Landy, Abelson & Smith, 1969), trp operon (Lozeron, Kumar & Szybalski, unpublished results) and h repressor gene (Taylor et al., 1967), since the corresponding mRNA’s hybridize only with the l-strands of transducing lambdoid phages carrying the respective genes. Drs W. Gilbert, W. Epstein and J. Beckwith generously supplied E. coli strains G113, X7026 and X8606. Dr J. Davies kindly gave the +8Ohdlac lysogen and all other bacterial strains. We wish to thank Drs J. Davies, K. Bevre, A. Guha and H. A. Lozeron for their frequent advice and Drs J. Beckwith and E. Signer for critical reading of this manuscript. The work was supported in part by grants from the National Cancer Institute (CA-07175), National Science Foundation (B-14976), and The Alexander and Margaret Stewart Trust Fund. McArdle Laboratory University of Wisconsin Madison, Wis. 53706, U.S.A. Received 21 October 1968, and in revised form 30 November
SUSEIL KUMAR WACLAW
SZYBALSKI
1968
REFERENCES Adams, M. H. (1969). In Bacteriophagea, p. 446. New York: Interscience. Adhya, S., Clear-y, P. t Campbell, A. (1968). Proc. Nat. Acad. Sci., Wad. 61, 966. Attardi, G., Naono, S., Rouviere, J., Jacob, F. & Gros, F. (1963). Cold Spr. Harb. Symp. &want.
Biol.
23, 363.
Beckwith, J. R. & Signer, E. R. (1966). J. Mol. Biol. 19, 264. Bremer, H., Konrad, M. W., Gaines, K. t Stent, G. S. (1966). J. Mol. Biol. 13, 540. Contesse, G., Naono, S. & Gros, F. (1966). C.R. Acad. Sci. Pad, 263, 1007. Davies, J. & Jacob, F. (1968). J. Mol. Bid. 86. 413. Epstein, W. (1967). J. Mol. Bid. 30, 629. Gilbert, W. BEMu&r-Hill, B. (1966). Proc. Nat. Acad. Sci., Wash. 56, 1891. Guha. A., Tabaczyriski, M. & Szybalski, W. (1968). J. Mol. Biol. 35, 207. Hradecna, Z. & Szybalski, W. (1967). Virology, 92, 633. Ippen, K., Miller, J. H., Scaife, J. t Beckwith, J. (1968). Nature, 21’7, 826. Jacob, F. & Monod, J. (1961). J. Mol. Bid. 3, 318. Lozeron, H. A., Szybalski, W., Landy, A., Abelson, J. & Smith, J. D. (1969). J. Mol. Biol. 39, 239. Maitra, U. t Hurwitz, J. (1966). Proc. Nat. Acad. Sci., Vu&. 54, 816. Mtiller-Hill, B., Crapo, L. & Gilbert, W. (1968). Proc. Nat. Acad. Sci., Wud. 59, 1269. Murata, N. & Szybalski, W. (1968). J. B en. Appl. Microbial., Tokyo, 14, 67. Newton, W. A., Be&with, J. R., Zipser, D. & Brenner, S. (1966). J. Mol. B&d. 14, 290. Nygaard, A. P. & Hall, B. D. (1964). J. Mol. Bid. 2, 125.
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EDITOR
&to,
K., Nishimune, Y., Sato, M., Numich, It., Matsushiro, A., Inokuchi, (1968). Virology, 34, 637. Szybalski, W. (1969). Canadian Cancer Conference, 8, in the press. Szybalski, W. & Mennigmann, H.-D. (1962). Anal@ Biochenz. 3, 267. Taylor, K., Hradecna, Z. t Szybalski, W. (1967). Proc. Nat. Acad. Sci., Taylor, A. L. & Trotter, C. D. (1967). Bact. Rev. 31, 332. Wu, R. & Kaiser, A. D. (1967). PTOC. Nat. Acud. Sci., Wash. 57, 170.
H. & Ozeki,
H.
Wash. 57, 1618.
Note added ir, proof. mr this paper was accepted for publication we received a manuscript kindly sent by Dr J. Beckwith, which supplies independent genetic evidence supporting our conclusion on the orientation of the i gene (Miller, Beckwith t MiillerHill, 1968).
REFERENCE Miller,
J. H., Beckwith,
J. & Miiller-Hill,
B. (1968).
Nature
220, 1287.