THE PERIODICITY OF RNA POLYMERASE INITIATIONS:A NEW REGULATORY FEATURE OF TRANSCRIPTION*

BY ROBERT F. BAKERt AND CHARLES YANOFSKYDEPARTMENT OF BIOLOGICAL SCIENCES, STANFORD UNIVERSITY,

STANFORD, CALIFORNIA

Communicated March 11, 1968

Although repressor regulation of operon expression is reasonably well under-stood in bacteria,'-3 relatively little is known concerning regulation of the maxi-mal rate of transcription initiation. Presumably some factors must determinethe rate of attachment of RNA polymerase molecules to the binding region of aderepressed operon, whether this binding occurs randomly or nonrandomly withtime. If each initiation of transcription of an operon is a random event, thenthe maximal rate of initiation need be governed simply by the availability ofpolymerase molecules in the vicinity of the initiation site and the affinity ofpolymerase for this site. However, if each round of transcription of an operonoccurs with a distinct periodicity, then it seems probable that initiation is gov-erned by a timing mechanism which is an integral feature of operon structure andits transcription and/or translation.

In this paper we describe experiments which demonstrate a definite periodicityin the initiation of transcription of the tryptophan operon of Escherichia coli.

Materials and Methods.-The bacterial strains employed include a prototrophic K12strain, W3110, and a strong polar trpD ochre mutant of W3110, D9778.4. 5 The phagesused as DNA sources for hybridization are: 080, the parental phage which does notcarry the trp operon (supplied by A. Matsushiro), and nondefective transducing phages080ptE-A, 080ptC-A (from S. Deeb and B. Hall), 080ptE-C (from W. F. Doolittle), and080ptBA (from N. C. Franklin). The segments of the trp operon carried by these phages,indicated by letter designations, were determined by genetic mapping and are shown inFigure 3B.

In all transcription experiments, bacteria were grown at 37° with vigorous shaking in aminimal medium6 containing 0.2% glucose or in a rich medium, L-broth. The cultureswere chilled when the cell density reached about 7 X 108 cells/ml. The cells were sedi-mented, washed, and resuspended in fresh minimal medium containing 1% glucose,0.05 mM L-tryptophan, and the other 19 amino acids (0.5 mM each), unless otherwiseindicated, and shaken at 37°. Aliquots of 5 ml were then dispensed into 125-ml Erlen-meyer flasks in a water-bath shaker. After 10 min, the tryptophan analogue, indole-3-propionic acid,7 was added to each flask to a final concentration of 30 ug/ml in order toinitiate trp messenger RNA (mRNA) production. At various times thereafter, repressionwas effected by adding L-tryptophan to a final concentration of 0.5 mM. RNA waslabeled for any desired period by adding H3-uridine (4 c/mmole) at a final level of 35-50Mc/ml to each 5-ml culture. In certain experiments a chase of unlabeled uridine wasemployed at a final concentration of 0.9 mg/ml. The incubation of all cultures wasterminated by rapidly pouring the cells over 4 ml of frozen-crushed medium containing20% sucrose, 400 /Ag/ml of chloramphenicol (CAP),8 0.02 M sodium azide, and 0.005 MMgCl2, in 0.02 M Tris buffer at pH 7.3. The cells were sedimented by centrifugation at12,000 X g for 10 min and resuspended in 3 ml of the same medium with sucrose omitted.RNA was prepared from these cells by a modifications of the procedure of Okamoto et al.9

Lysate preparation, DNA isolation, hybridization, and counting were performed as de-scribed elsewhere.5 10, 11 The results are reported as the percentage of the total labeledRNA that is hybridized with an excess (5 Mg) of 080pt DNA.5 The average of duplicatevalues is presented. The counts trapped by 480 DNA are subtracted in each case.

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Results.-The trp operon of E. coli consists of five contiguous genes (trpE-D-C-B-A)4 with an operator region at the E-gene end.'2 Transcription of this operonnormally results in the production of a single polycistronic mRNA carrying in-formation specifying all five polypeptide products of the operon.5 13 Transcrip-tion is initiated at the E-gene end5' 13 and proceeds to the A-gene end when thecorepressor tryptophan is absent or when it is added after initiation.'3 Trypto-phan added to a bacterial culture inhibits initiation of transcription.'3

Synchronization of transcription of the trp operon: In the experiments to bedescribed, conditions were employed such that transcription of the trp operoncould be initiated synchronously in a culture of bacteria and that further initia-tions could be instantaneously prevented. In the procedure developed, cul-tures were preincubated with a low concentration of tryptophan (0.05 mM\) fora ten-minute period in order to repress transcription of the trp operon and to al-low completion of any trp mRNA molecules initiated prior to or at the start ofthis period. (In control experiments it was shown that during the last threeminutes of the ten-minute preincubation period, inhibition of trp mRNA syn-thesis is 95%c complete in cells pregrown in minimal medium and 93% completein cells pregrown in L-broth.) At the end of the ten-minute preincubation pe-riod, the tryptophan analogue indole-3-propionic acid7 was added to synchronizeinitiation of transcription of the operon in all the cells of the culture.That initiation of transcription of the trp operon is rapid and synchronous is

indicated by the data in Table 1. In this experiment, performed as describedabove, a repressing level of tryptophan was added one minute after the indole-propionic acid addition in order to overcome the effect of the analogue and toinhibit subsequent transcription initiations. H3-uridine was present in parallelcultures from either zero to one minute or five to six minutes after the addition ofindolepropionic acid. It can be seen that only about 10 per cent of the trpmRNA labeled during the zero- to one-minute period is hybridizable to the oper-ator-distal genes of the operon (carried by ptBA), and only about 15 per cent ofthe mRNA labeled during the five- to six-minute period is hybridizable with theinitial portion of the operon (carried by ptE-C). These findings indicate thatessentially a single round of transcription was in progress (it takes 6.5 minutes tomake a complete trp mRNA; see Fig. 1) and that transcription was synchronized.From the fact that similar amounts of labeled mRNA were made during the twolabeling periods [compare the two ptE-A values; also ptE-C (0-1) with ptC-A

TABLE 1. Constant rate of trp mRNA synthesis during one round of transcription.Period of labeling of Specific activity ofRNA after initiation total RNA Percentage of H3-RNA Hybridized to DNA of:of derepression (min) (cpm/OD260 X 10-') ptE-A ptE-C ptC-A ptBA

0-1 98 0.110 0.098 0.023 0.0135-6 89 0.102 0.015 0.092 0.080

W3110 pregrown in minimal medium was treated as described in Materials and Methods. Duplicateportions of cells were repressed (tryptophan added) at 1 min after the addition of indole-3-pro-pionic acid and pulsed with H3-uridine (18 Me/ml, 20 c/mmole) during the 1-min periods indicated.The pulse was followed by a cold uridine chase. Six minutes after the addition of indole-3-propionicacid, RNA synthesis was stopped as described, and the RNA was extracted and hybridized to theDNA of various .80pt's and 480. The 480 background values subtracted are 0.031% for the 0- to 1-min period and 0.029 per cent for the 5- to 6-min period.

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FIG. 1.-Time required to synthesizea full-length trp mRNA molecule.Cells pregrown in minimal medium(0-0) or in an enriched medium(0-0) were synchronously derepress- 0.6ed by the addition of indole-3-propionicacid followed 1 min later by a repress- 3H RNA 0.ing amount of tryptophan. H3-uridine HYBRIDIZED 05 -

was added at the moment of derepres-sion (time zero), and parallel cultures pt C-A DA 0.4 -

were pulsed until the time shown in the pt E-A DNAfigure. At the end of the pulse period,RNA was extracted and hybridized 03with ptE-A, ptC-A, and 080 DNA's.(See Fig. 3B for the operon lengths car- 0.2 Iried by the pt's.) The specific activ- 4 S 6 7ities of the bulk RNA samples increased TIME AFTER ADDITION OF 1-3-P (MINUTES)almost linearly with increasing H3-uri-dine pulse length.

(5-6) ], it can also be argued that initiation of transcription is immediate follow-ing the addition of indolepropionic acid and that the rate of transcription is thesame at either end of the operon. This experiment depends in part on the im-mediacy and effectiveness of the cold uridine chase (see legend, Table 1) added atthe end of the first minute to one portion of the cells. That the chase is effectivecan be seen from the similarity of the specific activities of the total RNA ex-tracted from the cells pulsed during the two periods.

Length of time for transcription of the trp operon: Under conditions wherethere is but a single round of transcription, a more precise measure of the timenecessary to complete the synthesis of a full-length trp mRNA molecule can bedetermined experimentally by ascertaining the exact time when the proportionof labeled trp mRNA that corresponds to the most distal segment of the operonreaches its highest value, i.e., when the ratio of RNA hybridizable with ptC-ADNA relative to ptE-A DNA reaches a maximum. This measurement is basedon the assumption that there is no degradation of trp mRNA during its synthesis.The data in Figure 1 indicate that it takes about 6.5 minutes to transcribe theentire trp operon in cells pregrown in minimal medium and only 5.5 minutes incells pregrown in a rich medium. Since one trp mRNA molecule should contain6700 nucleotides,5 these times correspond to synthetic rates of ca. 1000 and 1200nucleotides per minute14 for cells pregrown under the two conditions.

Transcription initiation in cells pregrown in minimal medium: In order to de-termine the number of RNA polymerase molecules which are simultaneouslytranscribing each trp operon, the procedure illustrated in Figure 2 and describedin Materials and Methods was adopted. Figure 3A presents the results of an ex-periment in which the time of addition of repressing levels of tryptophan wasvaried during a six-minute labeling period, utilizing cells pregrown in minimalmedium. From the hybridization data it can be seen that when tryptophanis added within 2.5 minutes after the start of derepression, the trp mRNA seg-ments that are detected correspond to portions of a single molecule of messenger,i.e., the relative hybridization values with DNA from the four pt's suggest thatthe addition of tryptophan at any time before the 2.5-minute point permits only

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DEREPRESSIONWITH 1-3-P3

4N (+ H-URIDINE)

REPRESSING AMOUNT OFAT VARIOUS TIMES TO Pr

I 44 41 1 -u a

TR'IRA

PROC. N. A. S.

FIG. 2.-Protocol for tran-STOP WITH CAP, scription initiation experiments.AZIDE, AND LOW Indole-3-propionic acid (I-S-P)TEMPERATURE at a derepressing concentration

and H3-uridine are added atzero time and are present con-

YPTOPHAN ADDED tinuously. Derepression is ter-LLEL CULTURES minated by the addition of tryp-

4+ , tophan at various times in paral-L.. .I' I' lel cultures, prior to stopping4 5 6 RNA synthesis in all cultures at

the end of the sixth minute.

one round of transcription of each operon. In contrast, the ptE-A hybridizationvalues obtained when tryptophan is added at four minutes or after indicate thatadditional trp mRNA was synthesized. That this increase is due to transcrip-tion of the E-C end of the operon can be seen from the higher trp mRNA levelsdetected with ptE-A DNA and ptE-C DNA, and the absence of increased levelswith ptC-A DNA or ptBA DNA. It would appear that between three and fourminutes after the addition of indole-3-propionic acid a second round of transcrip-tion was initiated in those cultures which were not subjected to repressing levelsof tryptophan before the 2.5-minute point. The extent of synchronization ofinitiation of the first round of transcription can be estimated from the slopes ofthe initial portions of the ptE-A and ptE-C hybridization curves. The additionof tryptophan at one minute decreases initiations by only 10-15 per cent com-

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I 0.4

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0 1 2 3 4 5 6TIME OF ADDITION OF TRYPTOPHAN (MINUTES)

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E l)0 C. A OF-NE SEQUENCE0 1 2 3 4 5 6 MINUTES

pt E-A.ptE-CI

U.E

t C-AiCPHASESpt BA

P J-first round

first round, second round

first round I

second round ,...

FIG. 3.-(A) Transcription initiation experiment with cells pregrown in minimal medium.W3110 cells pregrown in minimal medium were concentrated and resuspended at 7 X 109/ml

in fresh minimal medium supplemented with all amino acids (a low level of tryptophan).The protocol outlined in Fig. 2 was followed, with repressing levels of tryptophan added toparallel cultures at the times indicated in (A). The RNA of each culture was hybridized withDNA of the various 480pt's and 480. The specific activities of the bulk RNA samples varied4±6%. -0- ptE-A, -X- ptE-C, --- ptC-A, -V-ptBA.

(B) Probable trp mRNA lengths present in cultures in which repressing levels of tryptophanwere added at 6 min (based on A). The regions of the operon carried by the pt's and themRNA lengths trapped by hybridization are indicated (genes are drawn to scale). The timerequired to transcribe the operon is taken as 6.5 min (see Fig. 1).

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INCUBATION AT37- IN THEPRESENCEOF TRYPTOPHA

-10 0 1 2 3MINUTES

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pared to addition at 2.5 minutes, suggesting that transcription was initiated in80-90 per cent of the cells before the addition of tryptophan at one minute. Thetime period between the second plateau and the first plateau (ca. 1 min) is ameasure of the synchrony of the second round of initiations.

It is possible to estimate the lengths of the mRNA chains synthesized in thefirst and second rounds of transcription from the plateau hybridization values inFigure 3A, from the relative hybridization values with ptE-A versus ptE-C andptC-A DNA, or from the periodicity of initiations. The ptE-A plateau values ofFigure 3A are 0.46 for the first round and 0.68 for the second round. If thedifference represents second-round trp mRNA, its length would be 22/46 or 48 percent of the length of the first-round mRNA. The first-round mRNA should be 92per cent of maximum length (6-min transcription of the 6.5 min required to makea full-length molecule); therefore, the second-round mRNA would be 44 per centof maximum length. Since it takes 6.5 minutes to transcribe the entire operon,the second round of initiation should be in progress by 3.25 minutes (see Fig. 3A).According to this interpretation, ptE-C DNA, which contains ca. 62 per cent ofthe DNA of the operon (Fig. 3B), should trap 62/92 or 68 per cent of the first-round mRNA trapped by ptE-A DNA, and if the second round proceededfor an average of three minutes, 110/140 or 79 per cent of the mRNA trappedby ptE-A DNA after the second round was in progress. The observed values,67 per cent (0.3/0.45) and 85 per cent (0.58/0.68), are in very good agreementwith expectations. Furthermore, ptC-A DNA, containing about 55 per cent

0.6 - Aa 0 0.-0 '

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.1D I ,C I . | AI gene sequence1 2 3 4 5 minutes_______________________ ptEA1A

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_ _,_secondround E-AmRNAO- *i third round LENGTHS

0 I I l I l lfirst round pC-Aj TRAPPEDTIME Of ADDITION OFTRYPTOP(MAN(MINUTES) secondcrounddi--

FIG. 4.-(A) Transcription initiation experiment with cells pregrown in enriched medium.W3110 cells pregrown in L-broth were concentrated and resuspended at 2 X 109/ml in

minimal medium supplemented with all amino acids (a low level of tryptophan). The pro-tocol outlined in Fig. 2 was followed, with repressing levels of tryptophan added to parallelcultures at the times indicated in (A). The RNA of each culture was hybridized with DNA ofthe 4,80pt's and q080. The specific activities of the bulk RNA samples varied ±2%. -0-pt E-A, * ptC-A.

(B) Probable trp mRNA lengths present in cultures in which repressing levels of tryptophanwere added at 6 min (based on A). The regions of the operon carried by the pt's and the mRNAlengths trapped are indicated. The time required to transcribe the operon is taken as 5.5 min(see Fig. 1).

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0. FIG. 5.-Transcription initiation in a° polar mutant. D9778 cells pregrown ina

a: minimal medium were concentratedand resuspended at 7 X 109/ml in fresh

I'0.2_Z! - minimal medium supplemented with allamino acids (a low level of tryptophan).The protocol outlined in Fig. 2 was fol-

,. _i _ lowed with repressing levels of trypto-0 Aphan added to parallel cultures at the

0. times indicated. The RNA of each

;K_10/ culture was hybridized with DNA from

W _ xz < _ the various 080pt's and 080. The spe-cific activities of the bulk RNA samplesvaried 4-3%. -0- ptE-A, -X-

0 2 3 4 5 6 ptE-C, ---o--ptC-A.TIME OF ADDITION OF TRYPTOPHAN (MINUTES)

of the operon (Fig. 3B), should trap 51 per cent (47/92) of the first-roundmRNA trapped by ptE-A DNA. With both first and second rounds in progress,

ptC-A DNA should trap 34 per cent (47/139) of the amount trapped by ptE-ADNA. The observed values are 0.22/0.45 (or 49%) and 0.22/0.68 (or 32%,)again agreeing well with expectations. Thus, the trp mRNA lengths hybridizedto ptE-A DNA, as shown in Figure 3B, probably are reasonably representativeof in vivo mRNA lengths at the particular time that cell activities were stopped.The data in Figure 3A and the interpretation in Figure 3B can be used to argue

that the addition of tryptophan represses second-round initiations instantane-ously. Thus, tryptophan added prior to 2.5 minutes prevents initiation andsynthesis of second-round mRNA, when it is known that second-round mRNAsynthesis is well under way at the three-minute point under the conditions ofthese experiments. The relative plateau values suggest that the same number ofinitiations occur in the first and second rounds.

In other experiments the same periodicity of initiations and hybridizationplateau ratios were obtained with cells growing logarithmically in minimalmedium.

Transcription initiation in cells pregrown in a rich medium: Figure 4A presentshybridization data from a transcription initiation experiment in which the cellsemployed were pregrown in L-broth. With such cells it takes about 5.5 minutesto transcribe the entire operon (Fig. 1). From the data in Figure 4A (and inter-pretation in Fig. 4B), it can be seen that there are three initiations per operon

during the six-minute period. The periodicity of initiation is about two minutes,and the second initiation occurs in all the cells between 2 and 2.5 minutes.The relative lengths of trp mRNA made in the second and third rounds, cal-culated from the plateau values, are (0.56-0.34)/0.34 (or 65%) of full length,and (0.67-0.56)/0.34 (or 32%) of full length, respectively. In this case we con-

sider the first plateau value, 0.34, to represent full-length molecules, since only5.5 minutes of the 6-minute labeling period are required to transcribe the entireoperon. If the rates of transcription are the same for all three rounds, then theobserved portion of the second-round period should be of 3.5-minute duration,and the third round should represent the last 1.5 minutes of the 6-minute

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period. These times correspond to 64 and 28 per cent of a full-length mRNA,for the second and third rounds, respectively. These values are in excellentagreement with the values calculated from the plateaus. These data precludethe possibility that third-round messenger results from an initiation after thefinish of the first round (5.5 minutes). This would result in a very short third-round piece, about 9 per cent of full-length rather than the observed 32 per cent.The ptC-A DNA hybridization data also indicate that the first-round mRNA isapproximately full length (0.18/0.34, or 53%7) and that there is no additionalptC-A hybridizable RNA made during the second round. However, it can beseen that the five- and six-minute ptC-A points are elevated (see also the 6-minpoint in Figs. 3A and 5). Since there is no comparable increase in the ptE-Ahybridization values, we do not believe that this increase is due to the forma-tion of RNA complementary to any part of the tryptophan operon. We shallconsider this RNA irrelevant to our arguments and calculations.

The periodicity of initiations and the lengths of mRNA molecules detected in apolar trpD nonsense mutant: A transcription initiation experiment was performedwith a polar D-gene nonsense mutant in which the introduced nonsense codon isapproximately at the two-minute point in the operon map pictured in Figure3B.4 5 It can be seen in Figure 5 that the second round of transcription isinitiated between 2.5 and 3 minutes, about the same time as in wild-type cells(compare Fig. 3A). It is also evident that the ptE-C values are slightly lowerthan the ptE-A values and that there is little mRiNA detectable with ptC-A DNA(cf. Fig. 3A). These findings agree with previous observations,5 and indicatethat trp mRNA regions corresponding to the D-A region of the operon are poorlyrepresented in the hybridizable RNA extracted from this strain.The relative plateau values in Figure a are somewhat surprising because they

indicate that there is about 1.3 times as much second-round trp mRNA as first-round mRNA. This could result from degradation of a portion of the first-roundmRNA during the six-minute period.Discussion.-The observations described in this paper indicate that, at most,

three RNA polymerase molecules can simultaneously transcribe one tryptophanoperon. The initiation of second and third rounds of transcription is not randomin time but exhibits a marked periodicity which itself sets an upper limit on thenumber of messenger molecules that can be transcribed per operon in any timeperiod. This number varies somewhat as a function of the rate of transcription:when transcription is rapid, e.g., in cells pregrown in enriched medium, reinitia-tion occurs every 2 to 2.5 minutes; in cells pregrown in minimal medium,reinitiation has a periodicity of 2.5-3 minutes. These times correspond to a2400- to 3000-nucleotide spacing of polymerase molecules in either case, since,as calculated before, the transcription rates are 1200 nucleotides per minute and1000 nucleotides per minute, respectively. The spacing also can be calculatedfrom the difference in relative lengths of first- and second-round mRNA molecules-the values so obtained are 3000 nucleotides for cells grown in enriched mediumand 2900 nucleotides for cells grown in minimal medium.The explanation of the periodicity of initiation is not yet known. Possible

mechanisms include repressor relaxation, promoter or operator modification,

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RNA polymerase activation, and coupling of initiation with transcription ortranslation. In view of the relationship noted here between initiation periodicityand transcription rate, it can be suggested that either a definite portion of anascent messenger must be completed or a certain number of ribosomes must beattached to the growing mRNA chain before the operon is freed for a subsequentinitiation (see Stent'5). Our findings with the polarity mutant could be con-sidered contradictory to this interpretation, but it remains to be establishedwhether transcription is normal in this case with degradation influencing thedetected lengths of hybridizable mRNA. Alternatively, the effect of the growthconditions may be superimposed on a pacemaking mechanism which could berelatively independent of transcription rate. We do not have any definitivedata to distinguish between the alternatives mentioned. It is undoubtedlypertinent, however, that mutational alteration of the promoter region of the lacoperon can markedly reduce the maximal rate of expression of this operon.16The findings in this study permit us to estimate the number of trp mRNA

molecules which can be synthesized per operon copy per generation. For cellsgrowing in minimal medium with a generation time of about 70 minutes, thisnumber is about 28, if we take the periodicity of initiation as 2.5 minutes. Thisnumber of trp mRNA molecules is in good agreement with an estimate based onother considerations.17The evidence presented here demonstrates the existence of a control mech-

anism, probably not under repressor control, which determines the periodicityof initiation of RNA polymerase transcription of the trp operon.

Essentially similar findings are reported in the accompanying paper by Ima-moto.18

The authors are indebted to Miriam Bonner for technical assistance and to Ethel Jack-son, Naomi Franklin, and our other colleagues for helpful suggestions.

* Supported by grants from the National Science Foundation and the U.S. Public HealthService.

t Postdoctoral trainee of the U.S. Public Health Service.1Jacob, F., and J. Monod, J. Mol. Biol., 3, 318 (1961).2 Beckwith, J. R., Science, 156, 597 (1967).3Gilbert, W., and B. Miller-Hill, these PROCEEDINGS, 56, 1891 (1966).4 Yanofsky, C., and J. Ito, J. Mol. Biol., 21, 313 (1966).5Imamoto, F., and C. Yanofsky, J. Mol. Biol., 28, 1 (1967).6 Vogel, H., and D. M. Bonner, J. Biol. Chem., 218, 97 (1956).7Doolittle, W. F., R. F. Baker, and C. Yanofsky, in preparation.8 Mangiarotti, G., and D. Schlessinger, J. Mol. Biol., 20, 123 (1966).O Okamoto, K., Y. Sugino, and M. Nomura, J. Mot. Biol., 5, 527 (1962).

10 Kaiser, A. D., and D. S. Hogness, J. Mol. Biol., 2, 392 (1960).11 Nygaard, A. P., and B. D. Hall, J. Mol. Biol., 9, 125 (1964).12Matsushiro, A., K. Sato, J. Ito, S. Kida, and F. Imamoto, J. Mol. Biol., 11, 54 (1965).13Imamoto, F., N. Morikawa, and K. Sato, J. Mol. Biol., 13, 169 (1965).14Zimmermann, R. A., and C. Levinthal, J. Mol. Biol., 30, 349 (1967).15Stent, G. S., Proc. Roy. Soc. (London) B, 164, 181 (1966).1Scaife, J., and J. R. Beckwith, in Cold Spring Harbor Symposia on Quantitative Biology,

vol. 31 (1966), p. 403.17Edlin, G., G. S. Stent, R. F. Baker, and C. Yanofsky, to be published.18 Imamoto, F., these PROCEEDINGS, 60, 305 (1968).

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