On the mechanism of amino acid control of ribonucleic acid biosynthesis

n the Mechanism of Amino Acid Control of Ribonucleic Acid Biosynthesis JONATHAN

GALLANT

AND I~I~ICEAEL CAMEL

Department of Genetics, University of Washington Seattle, Washington, U.S.A. (Received 25 August 1966, and in revisedforna 26 January 1967) Using whole cells which have been rendered permeable to phosphorylated substrates, we have examined the amino acid dependence of the incorporation of uracil, UMP and UTP into RNA. In the presence of the four nuc:leoside triphosphates, the incorporation of labeled UTP into RNA showed only a weak amino acid dependence, which was observed both in polymerase reaction mixtures and in growth med.ium; a weak amino acid dependence of UTP incorporation was seen furthermore in both stringent and relaxed strains. In contrast, the incorporation of uracil or UMP showed a strong amino acid dependence in identically treated stringent cells, and none in relaxed cells. These results suggest that the major effect of amino acid control of RNA synthesis is exerted on the formation of UTP, rather than on the RNA polymerase reaction itself.

1. Introduction Although the synthesis of RNA is clearly dependent upon the presence of a complement of required amino acids (Sands & Roberts, 1952; Pardee & Prestidge? 1956; Gros & Gras, 1958; Stent & Brenner, 1961; Neidhardt, 1964):,the mechanismof amino acid control of RNA synthesis is far from clear. Stent Bt Brenner (1961) and Kurland & Maalrae(1962) postulated that any of 20 amino acid-specific transfer RNA moleculesmight repressRNA synthesis when not neutralized by combination with its cognate amino acid. Tests of this hypothesis, basedon measurement of in vitro RN.bk synthesis catalyzed by RNA polymerase, have yielded equivocal results. Tissi&res, Bourgeois & Gros (1963) observed that free transfer RNA did inhibit RNA polymerase about twioe as effectively as charged transfer RNA. Bremer, Yegian & Konrd (1966) and Qlenick (1966) have reported somewhat smaller differences between the inhibitory action of charged. and uncharged transfer RNA. But even a twofold difference is inconsistent with the lo- or 20-fold reduction in the rate of RNA synthesis oocasionetl by amino acid starvation in V&JO.It is also noteworthy that amino acid-starved celh show little or no decrease in the RNA polymerase activity of crude extracts (Nierlich & Gras, personal

commnnication).

The stringency of the amino acid requirement for RNA synthesis is great,ly relaxed in BMaerichia coli mutants carrying a “relaxed” allele of the .EC gene (Stent $ Brenner, 1961). The aforementioned hypothesis, in its strictest form, predicts that ‘“stringent”

and “relaxed”

bacteria

should

differ

either

in the state

of charging

of

their transfer RNA, or in the sensitivity of their respective RNA polymerases to inhibition by transfer RNA. But experiments with purified comiponents have not 645

546

J. GALLANT

AND

M. CASHEL

sustained either of these predictions (Martin, Yegian & Stent, 1963; Bremer, Yegian & Konrad, 1966; Olenick, 1966). Taken all together, these experiments lend little support to the original hypothesis. However, the possibility remains that purification of RNA polymerase or of transfer RNA frees it of precisely those properties that regulate RNA synthesis in situ. It was accordingly of interest to examine the amino acid dependence of RNA polymerase activity in whole cells which had been rendered permeable to phosphorylated substrates. In the presence of all four nucleoside triphosphate substrates, RNA polymeraseactivity in whole cells showedonly a slight amino acid dependence;in contrast, the incorporation of uracil or of UMP was strongly dependent on the presence of amino acids. This leads us to believe that amino acid control of RNA synthesis operates primarily at the level of UTP production rather than RNA polymerase activity.

2. Materials

and Methods

(a) Bacterial strains and growth conditions E. coli B333 is a derivative of B3 which requires leucine, proline and methionme, as well as thymine. It shows “stringent” control of RNA synthesis: cultures grown in minimal medium supplemented with 0.2% casein hydrolysate (Casamino acids) show a &O-fold reduction in the rate of RNA synthesis shortly after downshift into amino acid-free medium. EAl and EA2 are a closely related pair of K12 strains carrying, respectively, the stringent and relaxed alleles of the RC gene. EAl is the 58-161 “archetype” which requires methionine and biotin and shows stringent control of RNA synthesis (Alfoldi, Stent & Clowes, 1962); EA2 is one of the biotin-independent, RC relaxed descendents of 58-161, discovered in the laboratory of Dr W. Hayes. Although not strictly isogenic, the two strains are not known to differ at loci other than RC and biotin (Alfoldi et al., 1962). Both strains were obtained from Dr F. Gros, who in turn obtained them from Dr S. Brenner. The minimal medium employed was a Tris-glucose medium (Gallant & Suskind, 1962) supplemented with 2 x 1O-6 M-Fecla and (in cultures of B333) 20 pg thymine/ml. In all experiments, cultures were grown in this medium plus 0.2% Casamino acids to a density of 5 to 10 x lo8 cells/ml. They were then centrifuged, washed twice and resuspended in amino acid-free medium. Following 20 min of amino acid starvation, the cells were centrifuged again, washed once in 0.01 br-Tris (pH 8.0) and resuspended at a density of 1 to 2 x lOlo cells/ml. in 2 M-sucrose in 0.01 M-T&s (pH 8-O). (b) Decryptification and assay of RNA polymerase RNA polymerase activity was decryptitled through plasmolysis in hypertonic sucrose, as described by Gros, Gallant, Weisberg & Cashel (1967). In the experiment recorded in Fig. 1, the plasmolyzed cells were centrifuged, washed and resuspended in minimal medium. UTP incorporation is optimal under these conditions, but the incorporation of uracil and of UMP is very poor. However, when the cells were diluted directly out of sucrose into growth medium, substantial uptake of these precursors was obtained. Substantial UTP incorporation could be obtained in growth medium by fortifying it with O-004 m-Mgcl, and 0.05 M-KCl. For experiments in which the incorporation of uracil, UMP and UTP were to be compared directly, the cells were plasmolyzed for 5 to 15 min then diluted immediately 20-fold into fortified growth medium plus the appropriate labeled precursor. Under these conditions, the rates of incorporation of the 3 different precursors are roughly comparable. The reaction mixtures used are described in the legends to the Figures. All reactions were carried out at 3O’C. Incorporation of the labeled substrate into RNA was assayed by transfer of 0*05- or 0.1 ml. portions into about 1-O ml. of 10% trichloroacetic acid plus 1% Casamino acids. The samples were then filtered, washed with trichloroacetic acid and then with ethanol, dispersed in Bray’s counting fluid (Bray, 1960), and counted in a

CONTROL

OF RNA

SYNTHESIS

cd47

Packard liquid-scintillation counter. In each experiment, a measured amount of labeled substrate was added to a filter containing the trichloroacetic acid precipitate of the 2ppropriate volume of unlabeled cells and counted to standardize absolute incorporation. Tritium-labeled uracil, UMP and UTP were obtained from Schwartz BiochemicaEs, Counting eflicienoy was about 10%. Aqueous solutions of nucleotides, adjusted to pH 7.0, were stored at -20°C. We occasionally checked the purity of our stock solutions of tritiated UMP aad UTP, using the thin-layer chromatographio procedure of Randerath & Randerath (1964). 1.5 to 28% of the radioactive material in our UMP migrated with unlabeled uridine, and 1.7 to 4.4”‘/;, with unlabeled uracil. Less (generally much less) than 25% of the radioactive material In our UTP migrated with unlabeled uridine, and 1 to 4% with unlabeled uracil. Thus, non-phosphorylated breakdown products represented of the order of 5% of each precursor. The UTP was found to contain about 5% UMP and UDP (mainly UDP) when fresh+ prepared; on prolonged storage, this value increased to 10 to 20%.

3. Results The starting point of the present investigation is the fact that the RNA polymera~e activity of whole, plasmolyzed cells is only slightly reduced in the absenceof a,mino acids (Fig, I). This could mean either that amino acid control doesnot operat,eupon RNA polymerase after all, or that the mechanism of ammo acid control is deranged in plasmolyzed cells.

0.6

a

2

4 Time

6 (min)

Fro. 1. Effect of amino acids on UTP incorporation. Cells of E. cc& B333 were treated and plasmolyzed as described in Materials and Methods. The reaction mixtures contained Tris minimal medium plus 0.05 n-potassium maleate (pH 7~5), 0.054 M-MnC1, and 0.004 n-mercaptoethanolamine; ATP, CTP and GTP at 0.5 pmola/ml. each; IOn cells/ml.; and 0.1 pmole/ml. tritiated UTP (100 pc/pmole). A (@), plus 0.2% Casamtio acida; B ( 0 ), no Casamino acids.

In order t,o distinguish critically between these alternatives, we devised reaction under which uracil, UMP or UTP could be incorporated at roughly comparable rates in otherwise identical reaction mixtures. These conditions are described in Materials and Methods. Representa,tive results for the incorporation of uracil and UTP are shown in Fig. 2. in plasmollyzed cells of E. coli B333, a stringent strain, uracil uptake is almost cornplateiy dependent upon the presence of amino acids (circles); ura.cii uptake is indetjendent of ammo acids in identically treated cells of E92, a relaxed strain (triangles). 37 conditions

648

J.

GALLANT

AND I

M.

I

I

CSSHEL I

I

Time

FIO.

2. Incorporation

(min)

of uracil

and UTP.

Cells of E. coli B333 and EA2 were cultured, starved of ammo acids and plasmolyzed for 15 min es described in Materials and Methods. They were then diluted directly to-fold (to lo9 cells/ml.) into reaction mixtures containing Tris-glucose medium plus 2 x 10eS M-potassium phosphate (pH 7.4), 0.004 M-MgCl,, and 0.05 M-KCI. For uracil uptake, tritiated uracil (50 rc/pmole) was present at 0.1 pmole/ml.; for UTP uptake, tritiated UTP was present at 0.1 pmole/ml. ( 0, q , A, +,) Uracil or UTP uptake in the presence of 0.2 % Casamino acids; ( 0, q , A, 0) uracil or UTP uptake without Casamino acids. (0, 0) Uracil uptake by B333; (A., A) uracil uptake by EA2; (a, 0) uracil uptake by B333 in the presence of ATP, CTP and GTP (0.5 qole/ml. each); (+, 0) UTP uptake by B333 in the presence of ATP, CTP and GTP (0.5 pmoIe/ml. each). The squares and diamonds compare uracil and UTP uptake by B333 in otherwise identical reaction mixtures.

The addition of ATP, CTP and GTP doesnot alter the amino acid requirement of the stringent strain (squares). (Since the medium employed is a growth medium containing glucoseas carbon and energy source, it is not surprising that the plasmolyzed cells are able to synthesize the other three triphosphates endogenously. In all subsequent experiments, we included the other three triphosphates in order to isolate the formation of UTP as the sole experimental variable.) If, however, UTP is substituted for uracil in precisely the same reaction mixture, then omission of amino acids produces only a modest decrease-less than a factor of two-in the rate of incorporation (diamonds). Therefore, the mechanism of amino acid control of RNA synthesis is intact in plasmolyzed cells, but it applies to uracil incorporation rather than to UTP incorporation. It is difficult to escapethe conclusion that the a,mino acid-dependent step is not the RN-4 polymerase reaction, but rather a biosynthetic step between uracil and UTP. It should be noted that, in the absence of amino acids, the incorporation of UTP is much greater than that of uracil; therefore it is unlikely that the relatively amino acid-independent incorporation of UTP occurs in a fraction of the cell population which has been rendered insensitive to amino acid control by someunknown consequenceof plasmolysis. Conversely, the uracil is a,pparently taken up by plasmolyzed

CONTROL

OF

RNA

1

TABLE

Actinomycin

inhibition

of urac2:l incorporution exp 1 1%min incubation

Uracil incorporation (mpmoles/ml.) in the presence of

No actinomycin

0.81

50 pg actinomycin/ml.

0.07

100 pg actinomycin/ml.

0.03

Cells of E. coli B333, 0.2% casein hydrolysate).

649

SYNTHESIS

exp 2 20-min incubation

exp 3 20-mm incubation

0.31

0.67

0.01

0.06

treated as in Fig. 2, were assayed in the same reaction Tritiated uracil was at 0.1 pmole/ml.

mixture

(containing

cells, inasmuch as uracil incorporation, like UTP incorporation, can be inhibite by actinomycin D (Table 1). Finally, competition experiments suggest that uracil and UTP are taken up by the same cells, since cold UTP reduces the incorporation of labeled uracil, and cold uracil reduces the incorporation of labeled UTP (Fig. 3). (We have no way of knowing whether this competition is at the Level of uptake or intracellular isotope dilution). Uracil is converted to UMP by UMP pyrophosphorylase (Crawford, Kornberg C% Simms, 1957; Brockman, Davis & Stutts, 1960) and UMP is converted to UTP by one or two kinases (Lieberman, Kornberg & Simms, 1955). In order to find out, whether the amino acid-dependent step comesbefore or after the formation of UMP we examined the incorporation of UMP into RNA in plasmolyzed cells. Figure 4 showsthat UMP incorporation is strongly dependent on the presenceof amino acids in the stringent strain, but not in the relaxed strain. The dependence of UMP incorporation on amino acid is an observation of past;-

0

IO

20

30

0 Time

IO

20

30

(min)

FIQ. 3. Competition between uracil and UTP. Cells of E. coli B333 were treated as in Fig. 2 and assayed in the same reaction mixture in the presence of 0.2% casein hydrolysate. Cold UTP or uracil, in the lower curves of (a) and (b) respec.. tively, were added at 0.4 ~mole/ml., a fourfold excess over the concentration of the labeled sub &rate.

550

J.

GALLANT

AND

M.

CASHEL

cular interest, for two reasons. First of all, UMP is a normal endogenous intermediate in RNA synthesis (which uracil is not), and amino acid control applies equally to endogenous RNA synthesis and uracil uptake (Friesen, 1966). Secondly, UMP is a phosphorylated intermediate, like UTP, and there is no reason to expect the entry of UMP and UTP into plasmolyzed cells to be subject to different rules.

Time

FIG.

4. Amino

acid

depeadence

(mid

of UMP

incorporation.

Cells of E. coli B333 and EA2 were treated as in Fig. 2 and assayed in the same reaction mixture, containing ATP, CTP and GTP (0.5 pmole/ml.) and tritiated UMP (100 pc/pmole) at 0.04 pmole/ ml. (@, a) UMP incorporation plus 0.2% Casamino acids. (0, 0) UMP incorporation without Casamino acids. (a, 0) B333; ( q , 0) EA2.

Our central finding, therefore, is that the incorporation of UTP into RNA in plasmolyzed stringent cells shows only a weak ammo acid dependence; while the incorporation of UMP into RNA under identical conditions shows a very strong amino acid dependence. The two strains compared in Fig. 4 differ in a variety of ways other than their respective RC alleles. We have therefore exa,mined the incorporation of UMP and UTP in EAl and EA2, which are a closely related stringent and relaxed pair. Figure 5 shows that UMP incorporation is strongly amino acid dependent in the stringent EAl, but not in its relaxed descendent EA.2. In contrast, UTP incorporation in both strains shows again the slight amino acid dependence (about 30%) observed in strain B333. EA2 does show a higher specific activity of UTP incorporation than EAI; we presume that this is an indirect consequenceof the RCrel mutation, like the increased transfer RNA content reported by Martin et al. (1963).

4. Discussion Our experiments were designedto identify the biosynthetic step in the production of RNA which is dependent upon a supply of amino acids in whole cells. In normal cells, the rate of uracil incorporation or of net RNA synthesis in the absenceof amino acids is 095 to O-1that observed in the presenceof amino acids (Gras & Gras, 1958; Stent & Brenner, 1961; Friesen, 1966). The biosynthetic step in RNA synthesis which

CONTROL

OF

RNA

RYNTH.ESIS

0.30

0.6

2 0.25 4 2 0.20 Y E 0.15

0.5 0.4

0.10

0.2

0.05

0.1

0

561

0.3

IO

20

0

IO

20

Time (min) FIG.

5. Incorporation

of UMP

and UTP

in isogenic

stringent

and relaxed

strains.

Cells of %. coli EAl (stringent) and EA2 (relaxed) were cultured, starvesd of amino acids, and harvested as described in Materials and Methods. They were then plasmolyzed for 5 mm, and diluted immediately into reaction mixtures identical to those described iu the legend to Fig. 4. The labeled substrates, UMP and UTP, were each present at a concentration of 0.1 ~mole/ml, (a) UMP incorporation; (b) UTP incorporation. (a) EAI plus Casamino acids (CA); (01 EfLI without Casmino acids; ( q ); EA2 plus C&amino acids; (0) EA2 without Casamino acids.

is responsible for this dramatic amino acid effect ought to show amino acid dependence of the same order of magnitude. In plasmolyzed cells, the amino acid dependence of the RNA polymerase reaction, measured as UTP incorporation in the presence of the other three triphosphates, is olearly much too small. Since the RNA polymerase activity of plasmolyzed oellls is only a few per cent of the capacity for RNA synthesis of untreated cells, tbis observation is not, in itself, a very critical one. But with plasmolyzed cells, we can also examine the incorporation of precursors earlier in the metabolic pathway than the triphosphates. This is precisely the rationale of the present experiments. It is evident (Fig. 2) that plasmolyzed cells retain the normal amino acid dependence of uracil incorporation. In the absence of ammo acids, UTP incorporation is much greater than uracil incorporation (Fig. 2). To ascribe the incorporation of uraeil. and of UTP to different cells in the population, therefore, it is necessary to assume that .the cells which incorporate uracil cannot incorporate UTP. This assumption is rendered quite unlikely by the fact that uracil incorporation, like UTP incorporation, is sensitive to actinomyoin inhibition (Table 1); and by the mutual competition displayed by uracil and UTP (Fig. 3). Furthermore, the incorporation of UMP, a phosphorylated precursor like UTP, also shows a normal degree of amino acid dependence (Figs 4 and 5). We have direot1.y compared the amino acid dependence of UMP and UTP incorporation in four different preparations of cells plasmolyzed and assayed as indicated in Figs 2 to 5. In eac:ii experiment, the ammo acid dependence of UMP incorporation, measured in incubation periods of 15 to 30 minutes, was much greater than the amino acid dependence of UTP incorporation. The ratio of UMP uptake in the absence of amino acids to tha,t

552

J.

GALLANT

AND

M.

CASHEL

in the presence of amino acids averaged O-15, with a range of 048 to 0.22; the corresponding value for UTP averaged 0.77, with a range of 0.37 to 1.1~ In several other experiments we have examined the amino acid dependence of the uptake of either UMP or UTP, with almost identical results. In either case, the difference in t.he ratios for UMP and UTP uptake is significant at below the 1% level by the t test. We also note that the ratio for UMP incorporation lies within the range displayed by RCst recombinants in a cross of RW x RCW, whereas the ratio for UTP incorporation lies within the range displayed by RCPel recombinants (Alfoldi, Stent, Hoogs $ Hill, 1963). We believe that the effect of Casamino acids on UMP incorporation is a manifestation of the control mechanism in which the RC gene plays a part for two reasons, First, we find that a synthetic mixture of amino acids mimics the effect of Casamino acids. Second, the amino acid dependence of UMP incorporation is abolished by the presence of the relaxed allele of the RC gene (Figs 4 and 5). On the other hand, a weak amino acid dependence of UTP incorporation is displayed by both a relaxed and a stringent strain which are nearly isogenic (Fig. 5). This small amino acid effect on the RNA polymerase reaction is comparable to the small differential effect on RNA polymerase of charged and uncharged transfer RNA discovered by Tissieres et al. (1963) and may well reflect that phenomenon. Under identical conditions, however, we observe a much stronger amino acid-dependence of UMP incorporation in the stringent strain, and little if any in the relaxed strain (Fig. 5). Therefore, the role of the RC gene product, and thus the action of amino acids in controlling RNA synthesis, must again intervene between UMP and UTP in the formation of RNA. Our observations are completely consistent with the observation of Edlin & Neuhard (1967) that a stringent strain of E. coli is unable to convert uracil and ci3uorouracil to nucleoside triphosphates in the absence of amino acids whereas an isogenic relaxed strain is. Our observations further indicate that: (1) the amino acid-dependent step lies between UMP and UTP; (2) RNA synthesis from exogenously supplied UTP shows only a weak amino acid dependence; (3) therefore, the amino acid dependence of UTP formation is probably the primary site of amino acid control of RNA synthesis, and not an indirect reflection of control of any step after UTP formation. Since our experiments were carried out in the presence of ATP, GTP and CTP, they do not exclude the possibility that amino acid control applies to other phosphorylation steps as well as those between UXP and IJTP. On the other hand, Edlin & Neuhard (accompanying paper) have observed that amino acid starvation decreases ATP synthesis much less than UTP synthesis. NaYvely, our interpretation seems to predict that amino acid starvation would result in rapid depletion of the UTP pool. Edlin $ Neuhard (1967) observe a decline in all four ribonucleoside triphosphates following amino acid starvation, but this decline appears to be rather slow. Measurements of total triphosphate pools may be difficult to i.nt.erpret if physiological substrates are compartmentalized. In the case of uracil metabolites, there is kinetic evidence suggesting the existence of at least two distinct pools (McCarthy & Britten, 1962; Midgley & McCarthy, 1962; Midgley, 1963). In the case of orthophosphate, we find that phosphate starvation produces only a slight decrease in the intracellular concentration of orthophosphate, again implying compartmentalization (unpublished experiments).

CONTROL

OF RNA

SYNTHESIS

553

0:~ &dings supply a reasonable explanation of the fact tha,t the synthesis of f.2 BNA is under the same amino acid control as host cell RNA syntheels (Friosen, 1965). Since f2 phage infection apparently does not induce the formation of a new UMP kinase (Hiraga & Sugino, 1966) it is entirely reasonablethat the synthesis of f2 RNA by the phage-induced RNA polymerase (August, Cooper, Shapiro & Zinder, 1963) should require the amino acid-dependent formati.on of UTP by bacterial enzymes. FinaPly, we note that control of RNA biosynthesis at the level of precursor produo‘r.ion is consistent with the results of Friesen (1966) and of Gros et al. (19631, w&b indicate that amino acid starvation decreasesthe rate of synthesis of different species of RNA co-ordinately. In principle, a regulat,ory mechanism controlling one or more phosphorylation stepsmight respond either to the levels of free amino acids, or to the degreeof charging of transfer RNA, or to the rate of protein synthesis itself. ILL vitro studies on the phosphorylation of UMP are in progress. It is a pleasureto acknowledgethe encouragementand hospitality of Dr Francois Gras, phage

who initiated us into the mysteries of plasmolysis. We are indebted to Mrs Catherine Walker for capable technical assistance. This work was supported by U. S. Public Meal& Service grant 1 ROl GM 13626. One of us (M. C.) is a U.S. Public Health Service Trainee in Geneties on leave from the Laboratory of Molecular Biology, N.I.N.D.B., National Institutes of Health. REFERENCES AlfGlidi, L., Stent, 6. S. & Clowes, R. C. (1962). J. Mol. Biol. 5, 348. A&k%, L., Stent, 0. S., Hoogs, M. & Hill, R. (1963). 2. Vererbungskhre, 94, 285. August, J. T., Cooper, S., Shapiro, L. & Zinder, N. D. (1963). Gold Xpr. $%rb. Symp. Quczn~. Biol. 28, 95. Bray, G. A. (1960). Analyt. Biochem. 1, 279. Bremer, H., Yegian, C. & Konrad, M. (1966). J. Mol. Biol. 16, 94. .&o&man, R. W., Davis, J. M. & Stutts, P. (1960). Biochim. biophys. Acta, 40, 22. Crawford, I., Kornberg, A. & Simms, E. S. (1957). J. Biot. Them. 226, 1093. Edlin, G. & Neuhard, J. (1967). J. 1UoZ. Biol. 24, 225. Friesen, J. D. (1965). J. Mol. Biol. 13, 220. Friesen, J. D. (1966). J. Mol. Biol. 20, 559. Gal.lant, J. & Suskind, S. R. (1962). Biochim. biophys. Acta, 55, 627. Gras, F., Dub&, J. M., TissiBres, A., Bourgeois, S., Michelson, M., Soffer, R. & Legaul”;, L. (1963). Cold Spr. Harb. Symp. QtLan,t. Biol. 28, 299. Gras, F., Gallant, J., Weisberg, R. & Cashel, M. (1967). J. MOE. Biol. 25, 555. Gros, F. Kr. Gros, F. (1958). Exp. Cell Res. 14, 104. IXiraga,

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Kurland, C. G. & Maalee, 0. (1962). J. Mol. Biol. 4, 193. Lieberman, I., Kornberg, A. & Simms, E. S. (1955). J. Biol. C’hem. 2115, 429. %Xartin, E. M., Yegian, C. 8: Stent, G. S. (1963). 8. Vererbungslehre, 94, 303. McCarthy, B. J. & Britten, R. J. (1962). Biophys. J. 2, 35. Mid&y, J. E. K (1963). Biochivm. biophys. Acta, 68, 354. Xidgley, J. E. M., 85 McCarthy, B. J. (1962). Biochim. biophys. Acta, 61, 696. Neidhardt, F. C. (1964). Progr. Nucleic Acid Res. 3, 145. Olenick, J. G. (1966). Fed. Proc. 25, 520. Pardee, A. B. & Prestidge, L. S. (1956). J. Bact. ‘71, 677. Randcrath, K. & Randerath, E. (1964). J. Chromatog. 16, 111. Sands, M. K. & Roberts, R. B. (1952). J. Bact. 63, 505. Stent, G. X. $ Brenner, S. (1961). Proc. Nat. Acad. Sci., Wash. 47, 2005. ‘T&i&es, A., Bourgeois, S. & Gras, B. (1963). J. Mol. Biol. 7, 100