THE JOURNAL OF BIOLOQICAL CHEMISTRY Vol.245, No. 9,Issue of May 10, pp. 2259-2267, 1970

Printed in U.S.A.

Regulation of Lac Messenger Ribonucleic Acid Synthesis by Cyclic Adenosine 3’, 5’-Monophosphate and Glucose

(Received for publication, September 11, 1969)

HAROLD E. VARMUS, ROBERT L. PERLMAN, AND IRA PASTAN

From the Clinical Ilinclocrinology Branch, National Institute of Arthritis and Metabolic Diseases, and Molecular Biology Section, lklocrinology Branch, National Cancer Institute, Bethesda, Marylancl SOOld

SUMMARY

Cyclic adenosine 3’,5’-monophosphate reverses the glu- cose repression of fi-galactosidase synthesis in Escherichia coli induced for enzyme synthesis. In this report, we de- scribe hybridization assays for the measurement of synthesis rates of lac messenger RNA and demonstrate that cyclic adenosine 3’,5’-monophosphate and glucose alter rates of mRNA production in proportion to their effects on P-galactosi- dase synthesis. Thus we confirm earlier indirect measure- ments which indicate that control of P-galactosidase synthesis by cyclic adenosine 3’,5’-monophosphate and glucose occurs at the level of transcription. In addition, we show that these effects on lac mRNA synthesis are observed in cultures in- capable of enzyme synthesis and that glucose does not af- feet the rate of degradation of lac mRNA.

Recent studies in this laboratory aud others have demon- strated that cyclic adenosine 3’,5’-monophosphate is capable of reversing both the transient and permanent repression of p- galactosidase synthesis observed following the addition of glucose to a bacterial culture induced for enzyme production (l-4). Be- cause glucose is known to lower cyclic AMP’ levels in starved and growing2 Escherichia coli, these studies suggested that glucose repression may be mediated through reduction in cyclic AMP levels (5). Although much effort has been devoted to an under- standing of the mechanism by which cyclic AMP and glucose alter the rate of enzyme synthesis, scant definitive information has been forthcoming. In 1964, Nakada and Magasanik em- ployed indirect techniques to estimate Zac messenger RNA ac- cumulation and reported that accumulation was decreased in the presence of glucose (6). In those experiments, the half-life of &galactosidase-synthesizing capacity, after removal of inducer, appeared unaffected by glucose. hlore recently Tyler and Magasanik reported that P-galactosidase-synthesizing capacity decreased similarly in cells transiently repressed by glucose and

1 The abbreviations used are: cyclic AMP, cyclic adenosine 3’,5’-monophosphate; IPTG, isopropyl-p-n-thiogalactopyrano- side; SSC, standard saline citrate (0.15 M NaCI, 0.015 M sodium citrate).

2 G. D. Aurbnch, 11. 1~. Perlmsn, and I. Pasta.n, unpublished observations.

in cells from which inducer had been removed (7). Therefore, it was concluded that glucose repression was mediated through a reduction in the rate of formation of Zac mRNA. However, no direct measurements of Zuc messenger levels or synthesis rates have been reported under conditions of glucose repression, and the precise manner in which glucose exerts its effects has remained unclarified.

Likewise, the experiments of Perlman and Pastan were de- signed to measure Zac mRNA accumulation indirectly by separa- tion of the phases of transcription and translation (1). Their results also suggested that Zuc mRNA levels were decreased in the presence of glucose, and, further, that those levels were raised by the treatment of glucose-repressed cells with cyclic AMP. Further indirect verification of these findings has been provided by the work of Jacquet and Kepes, who reported the effect of cyclic AMP and glucose to be exerted upon initiation of Zac mRNA synthesis (8).

The present report confirms these indirect observations by di- rect measurement of the rate of synthesis of Zuc mRNA, under conditions of glucose repression and cyclic AMP treatment, with two different RNA-DNA hybridization assay systems. Several years ago Hayashi et al. (9) and Attardi et al. (10) performed hybridization assays to demonstrate that a rapidly synthesized, unstable, DNA-like RNA species was produced upon the addi- tion of an inducer of P-galactosidase to growing cultures. These assays were, however, crude or cumbersome, and they permitted neither the processing of many samples nor the definition of the limits of each assay system. More recently, Kumar and Szybal- ski have developed a sophisticated hybridization assay for the measurement of RNA transcribed from the i gene (11). Al- though they were able to measure the synthesis of Zac mRNA, this was not their primary concern and they report little data relevant to this study. Brief reports of a hybridization method used to measure Zuc mRNA in polar and promoter mutants have recently appeared, but the characteristics of the assay are not clearly defined (12, 13). In the absence of established methods for the estimation of Zuc mRNA levels, we were obliged to devise and calibrate new ones; part of the present report is devoted to a description of their development.

MATERIALS AND METHODS

Bacteria-Proteus mirabilis 1, P. mirabilis 1 F’lac, and E. coli W4032 Hfr (Zac&l, met-, pro-, Sm*, developed by E. Lederberg) were kindly supplied by Dr. S. Falkow. E. coZi K-12 C600 (Zac i+z+y-, thr-, Zeu-) was obtained from Dr. I. Leder, and E. coZi

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2260 Cyclic AMP Regulation of Lac mRNA Vol. 245, No. 9

RV (XCi8~~hsOk8; XC&&&&c+), a double lysogen for Xh8O and Xh8Odlac, developed by Dr. E. Signer, was acquired through Dr. M. Yarmolinsky. These phages are derivatives of recom- binants containing a X immunity region and 480 host range and attachment site as originally described by Signer (14). E. co& 1103 and E. coli MO X19 (F- and deficient in Enzyme I of the P-enolpyruvate-phosphotransferase system) derived from E. coli K-12 3000) were gifts from Dr. F. Fox. The wild type strain, E. coli K-12 3000 Hfr (lac ifp+o+z+y+a+) was provided by Dr. E. Steers.

Media-P. mirabilis 1 was grown in a peptone-glucose medium (20 g of Difco peptone and 1 g of glucose per liter), and P. mirabi- Lis 1 F’lac in a peptone-lactose medium (10 g of peptone and 20 g of lactose per liter). E. coli RV was grown in the mixture of tryptone (1.6 %), yeast extract (1 yO), and NaCl (0.5%). For other E. coli species, Medium A, containing 14.0 g of K~HPOI, 6.0 g of KH2POI, 2.0 g of (NH&SO+ and 0.2 g of MgS04 per liter, was employed, generally supplemented with 0.5% glycerol or succinate, thiamine, and required amino acids at a concentra- tion of 50 hg per ml.

Chemicals-IPTG, o-nitrophenyl-/?+galactopyranoside, and crystalline lysozyme were purchased from Mann, o-nitrophenyl- O-D-fucoside from Cycle Chemical Company, cyclic AMP from Calhiochem, optical grade cesium chloride and uridine from Schwarz TsioResearch, electrophoretically pure deo‘xyribonu- clease from Worthington, pancreatic ribonuclease from Schwarz BioResearch and Worthington, 14C thymidine from Nuclear Chicago, and 3H-uridine (TRK 178, 27 to 31 Ci per mmole) from Xmersham-Searle and Schwarz BioResearch.

P-Gulactosidase Assay-The presence of @-galactosidase in E. coli or Proteus species was determined by the ability of toluene- treated cells to hydrolyze o-nitrophenyl-B-D-galactopyranoside, at 28”, according to the method ol Pardee, Jacob, and Monod (15). Retention of the F’lac episome by P. mirabilis 1 was also monitored by use of MacConkcy’s and eosin-methylene blue lactose indicator plates.

Preparation of Bacterial DNA-Proteus cultures were harvested in log phase, in some cases after labeling with 0.5 to 1.0 &i of 14C-thymidine for several generations, and the DNA was ex- tracted according to the procedure of Marmur (16). The final suspension of DNA was eluted from a methylated albumin Kieselguhr column with a linear NaCl solution gradient. Be- cause the F’lac DNA (of E. coli origin, with guanine-cytosine content 47 to 48%) is eluted more rapidly than Proteus DNA (with guanine-cytosine content of 39%,), the early fractions of the P. mirubilis 1 F’lac peak were collected and pooled, and the remainder was discarded (17, 18). The major portion of the P. mirabilis 1 DNA peak was collected and both species of DNA were dialyzed in the cold against SSC 0.1 concentration. The P. mirabilis 1 Y’Zuc DNA was shown to contain up to 20% epi- somal DNA by density gradient centrifugation in cesium chlo- ride, by use of the method of Flamm, Bond, and Burr (19).

Preparation of Phase DN/-Log phase cultures of E. coli RV lysogenic for Xh80 and Xh8Odlac were heat-induced at 42” for 15 min, shaken at 30” for 4 to 5 hours (with or without 14C-thymi- dine), and treated with chloroform. The phages were collected by centrifugation and then banded in cesium chloride (4.5 g of CsC12 dissolved in 5.8 ml of a solution containing the phage sus- pended in 0.01 M Tris, pH 7.4, and 0.015 M MgC12) by density gradient centrifugation at 23,500 rpm in a Spinco model 40 rotor for 16 hours at 1”. The two phage bands (the lower containing

X@Odlac, the upper Xh80) were separated by drop collection and rebanded in cesium chloride. Each type of phage DNA was then extracted with redistilled, buffer-saturated, neutralized phenol according to the method of Thomas and Abelson (20). The isolated DNA was dialyzed extensively against 0.01 M Tris buffer (pH 7.8) and stored under chloroform at 2”.

Preparation of RNA-Radioactive RNA was made by expos- ing cultures for 30 to 180 set to 1.0 to 2.0 mCi of 3H-uridine per lOlo cells at a cell density of 5 x lo* per ml. Incubat,ion was stopped by pouring the cells over an equal volume of ice con- taining 0.02 M sodium azide. RNA extraction was then carried out following a modification of the protocol of Okamoto, Sugino, and Nomura (21). Three phenol extractions were done at room temperature for 7 min each; the RNA was twice precipitated in ethanol at -15” and dialyzed overnight in the cold against sev- eral changes of 4-fold concentrated SSC. The same extraction procedure was used for the preparation of unlabeled RNA from E. coli W4032. Optical density at 260 rnp was employed to determine the concentration of RNA solutions. To measure the specific activity, small samples were treated with cold 5% trichloracetic acid for 15 min and precipitates were collected on 25-mm Schleicher and Schuell Bat-T-Flex filters. The filters were then dried and counted in Liquifluor in a Packard Tri-Carb liquid scintillation counter, and activities expressed as counts per min per pg of RNA.

Preparation of DNA Filters-Nitrocellulose filters (Schleicher and Schuell, Type B6), previously cut to 14 mm in diameter, were loaded with 6 fig of Proteus DNA or 1.0 fig of phage DNA as described by Gillespie and Spiegelman (22). Filters 8 mm in diameter, containing 0.3 Mg of phage DNA, were punched out of 25-mm filters loaded with 3 pg of DNA. Alkali-denatured DNA was diluted to 5 ml in 4-fold concentrated SSC, neutralized, and passed dropwise through previously soaked, previously washed filters under moderate suction. The filters were again washed with 4-fold concentrated SSC, dried at room temperature for at least 4 hours, and dried overnight in a vacuum dessicator at 67”. They were then stored at room temperature in screw top glass vials. Blank filters were prepared in the same fashion, but DNA was absent from t,he starting solution.

Hybridization Procedure-Radioactive RNA of known weight and specific activity was added to filters in their storage vials. Generally, unlabeled RNA from the Zac deletion strain was also added in a fixed ratio to the radioactive material, and a final volume of approximately 300 ~1 for 14-mm filters or 100 ~1 for g-mm filters was obtained by the addition of 4-fold concentrated SSC. The vials were then incubated in a gla.ss jar submerged in a water bath at 74-75” for 16 to 20 hours. Incubation was terminated by chilling the vials on ice. Filters were then washed with 50 ml of 4-fold concentrated SSC on each side, incubated with 20 pg of ribonuclease in 1 ml of 2-fold concentrated SSC at room temperature for 1 hour, and rewashed with 50 ml of 4-fold concentrated SSC on each side. After dlying under a heating lamp for 10 min, the filters were counted in Liquifluor in the Packard Tri-Carb scintillation counter. Counting conditions were adjusted when necessary to count 14C and 3H simultane- ously.

Calculations-The ratio of hybridized counts to acid-precipita- ble input counts was expressed as “percentage hybridized.” For each RNA preparation tested, the percentage hybridized to the contro1 DNA (X&70 or P. mirabilis 1) was subtracted from the percentage hybridized to the Zac-containing DNA (Xh80-

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1500 r Ah80 dfac

Ah80 __------- .

0 _------ , I I

0 5 8 IO 15 20

HOURS

FIG. 1. Hybridization kinetics. 3H-RNA, 1.0 pg, (specific activity = 150,000 cpm per fig) from a culture of E. coli 1103, induced for 6+ min with 5 X 10s4 M IPTG and labeled for 1 min with 3H-uridine, was hybridized at 75” with Xh8Odlac filters con- taining 1.0 pg of DNA in the presence of 50 pg of unlabeled RNA from the Zac deletion strain W4032. Samples were removed from the incubator and processed at 1, 5, 8, and 20 hours. Counts hybridized were plotted as a function of time (-). - - -, the hypothetical curve for counts hybridized to Xh80 DNA filters based on 20.hour incubation of 3H-RNA from the same induced culture of E. coli 1103 with Xh80 DNA filters.

dlac or P. mirabilis 1 F’lac) to give a “percentage difference.” This was considered to be a relative measure of the amount of Zac messenger accumulated during the period of labeling. Be- cause 3H-RNA from the Zac deletion strain W4032 hybridized to a greater extent to the P. miralrilis 1 F’lac than to P. mirabilis 1 filters, percentage differences for Zac-containing strains were corrected for the percentage difference for W4032 RNA. At the levels of 3H-RNA input used, this percentage difference was in the range of 0.12 to 0.27%. No such correction was required for the assay with phage DNA.

FIG. 2. Reconstruction experiment with Xh8OdZac filters. Mix- tures of 3H-RNA, 1.0 pg each, were prepared, containing differing amounts of %-RNA from induced and uninduced cultures of E. coli 1103. These mixtures were hybridized to Xh80dlac filters (containing 1.0 jog of DNA) in the presence of 50 fig of unlabeled RNA from the l& deletion strain, E. coli W4032.- The amount of 3H-RNA from the induced culture added to each mixture is plotted on the abscissa; the ordinate records counts per min, after subtraction of counts remaining bound to blank filters. (Although not shown in the graph, less than 80 cpm per pg of either prepara- tion hybridized with the Ah80 DNA filters.)

Blank filters were tested with each RNA preparation. Counts remaining on these filters were uniformly low (5 to 20 cpm above background). These blank values were subtracted from the experimental results with each preparation.

by cesium chloride centrifugation, and high purity was assured by rebanding the phage particles in cesium chloride. The heavier band was identified as the Zac-carrying species, XhSOdlac, by the capacity of its DNA to hybridize avidly with RNA from an E. coli culture induced for ,&galactosidase.

DNA labeled with 14C was used to monitor the amount of DNA remaining on the filters at the conclusion of each experiment with bacterial DNA and some experiments with phage DNA, but it was not employed to correct the percentage difference. (In general, the remaining amounts of DNA varied from filter to filter by less than lo%.)

RESULTS

The question we hoped to answer with the following experi- ments was a simple one. What is the rate of synthesis of Zac mRNA under conditions of glucose repression and treatment with cyclic AMP? The development of acceptable methods of measurement, however, posed several difficult problems which are discussed in the succeeding sections, along with data answer- ing our initial question.

Filters containing 1.0 pg of Xh80 or Xh80dZac DNA were pre- pared in the described manner. XRNA from a culture of E. co& 1103 induced for &galactosidase production was incubated with both DNA species at 75” in the presence of a 50-fold excess of unlabeled RNA from a lac deletion strain. The kinetics of this reaction are illustrated in Fig. 1. It is apparent that a linear increase in the amount of 3H-RNA hybridized occurred during the first 8 hours of incubation, after which no additional hybrids were formed. The large number of counts hybridized to the Xh80dZac filters, coupled with the low level of background hybridization to Xh80 DNA, indicated a high specificity for lac mRNA under conditions of the assay.

Preliminary Experiments with Phage DNA Filters-Although we employed two hybridization systems in this work, the assay utilizing Xh80 and XhaOdZac DNA was clearly the more success- ful. The two phages were induced from the temperature- sensitive, double lysogen E. co& RV as described under “Ma- terials and Methods.” The phage types were cleanly separated

The linearity of the assay was then inspected by hybridizing l.O+g mixtures of varying ratios of 3H-RNA from induced and uninduced cultures with Xh80dZac DNA filters. As shown in Fig. 2 the assay results were linear over this range of input. Moreover, if the amount of input 3H-RNA was increased to 6.0 pg, assay results remained linear, suggesting a large excess of Zac DNA on the filters (Fig. 3).

Non-Zac interactions between labeled RNA and Ah80 DNA oc- curred with low frequency and were minimized by the addition of large amounts of unlabeled RNA from the Zac deletion strain, E. coli W4032, to each reaction mixture. Results of a competi-

01 I 1 I I 0 .25 .50 .75 IO

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Cyclic AMP Regulation of Lac mRNA Vol. 245, No: 9

X h80 / dfoc 0 Ir co % 4000-

z "

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Ah80 ____ -__ -- --...-------.

0 ___ I / 0 I 2 3 4 5 6

pg ‘H-RNA

FIG. 3. Saturation experiment with 3H-RNA from induced E. coli 1103 and Ah80 and XhdOdZac DNA filters. Increasing amounts of 3H-RNA (specific activity = 150,000 cpm per pg) prepared from E. coli 1103 induced for p-galactosidase production for Gg min with IPTG were incubated at 75’ for 20 hours with 1.0 pg of Xh80 and XhBOdlac DNA filters, in the presence of a 50-fold excess of unlabeled RNA from E. coli W4032. Hybridized counts per min (ordinate) are plotted against micrograms of 3H-RNA (abscissa). ---, results with Xk80dZac DNA filters; - - -, results with Xh80 filters.

/-I I I 5 25 50 100

pg UNLABELED RNA

FIG. 4. Competition hybridization of 3H-RNA from induced E. coli 1103 and unlabeled RNA from E. coli W4032 with Xh80 DNA filters. 3H-RNA, 1 .O pg, (specific activity = 150,000 cpm per pg) prepared from E. coZi 1103 induced with IPTG was hybridized with 1.0 rg of Xh80 DNA filters in the presence of increasing amounts of unlabeled RNA extracted from the Zac deletion strain E. coli W4032. Micrograms of added unlabeled RNA are recorded on the abscissa; counts per min, after subtraction of counts bound to blank filters, are plotted on the ordinafe.

tion hybridization experiment illustrating this point are graphed in Fig. 4. A similar number of counts could be eliminated in

hybridizations with Ah80 or Xh80dlac DNA. Hybridization Results with Phage Filters--Labeled RhTL4 was

.e

!s Y E .4

a

NONE IPTG 0

I PTG I PTG GLUCOSE GLUCOSE

CAMP

FIG. 5. Lac messenger assay for E. coli 1103 RNA with X/&O and XhBOdZac DNA. The Enzyme I-deficient strain E. coli 1103 F- was grown to log phase in Medium A, supplemented with 0.50/, sodium succinate, 0.1% Casamino acids, and thiamine. Cultures were then incubated alone; with 5 X lo-4 M IPTG; with IPTG and lO+ glucose; and with IPTG, glucose, and 1O-2 M cyclic AMP. After 5 min, specimens were removed for P-galactosidase assay, and the cultures were labeled for 1 min with “II-uridine. RNA (specific activity 154,000 to 194,000 cpm per kg) was extracted and 1.0 fig from each culture was hybridized at 75” for 20 hours with filters containing 1.0 pg of Ah80 or XhBOdZac DNA in the presence of a 50-fold excess of unlabeled RNA from E. coli W4032. Filters were tested in triplicate; deviations from the mean were less than 10%. Stippled bars, relative enzyme levels in each culture; hatched bars, synthetic rates for Zac mRNA expressed as per- centage difference hybridized. Standard errors of the mean are illustrated for the messenger assay.

prepared from cultures of E. coli 1103 induced with 5 x lo-* 1w

IPTG, alone, in the presence of 10m4 1% glucose, and in the pres- ence of glucose and 1O-2 M cyclic AMP. In addition, an unin- duced cult,ure was prepared as a control.

As illustrated by Fig. 5, synthesis rates for Zuc messenger RNA, measured by percentage difference hybridized, correspond well with relative levels of ,&galactosidase. Although not shown in the figure, addition of cyclic AMP to uninduced cultures does not affect the control value. These result,s suggest that glucose, cyclic AMP, and IPTG control enzyme production by altering the rate of transcription of Zac DNA, rather than by affect,ing the translation of mRNA into protein.

Studies of Accumulalion of Labeled Lac mRNA-To examine

the kinetics of accumulation of labeled Zuc messenger, an experi- ment was performed with 3H-RNS from cells labeled for 30, 60, or 180 set during exposure to inducer alone, inducer plus glucose, or inducer plus glucose and cyclic AMP (Fig. 6). The specific activity for each RNA preparation was determined and found to be similar at each time point for the induced and glucose-re- pressed cultures; slightly reduced values for the cyclic AMP- treated culture at 60 and 180 set may reflect the mild inhibitory effect cyclic AMP is known to exert on growth and ribosomal RNA synthesis (23). The decreasing rate of over-all 3H in- corporation into RNA seen by 180 set in all cultures is not readily explained, but may result in part from depletion or degradation of 3H-uridine.

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0 30 60 120 180 0 30 60 120 160 SECONDS

FIG. 6. Kinetics of accumulation of labeled Zac mRNA. E. coli 1103 was grown to log phase in Medium A, succinate, thiamine, and Casamino acids. Thirty milliliters of this culture were placed in each of three flasks. IPTG (5 X 10-d M), IPTG and glucose (lOMa M), and IPTG, glucose, and cyclic AMP (10m2 M) were added simultaneously to the cultures 5) min before labeling. Specimens were removed after 5 min for fl-galactosidase assay, and 30 see later (time zero) 1.5 mCi of 3H-uridine (Schwarz Bio- Research) was added simultaneously ta each flask. Samples of 10 ml each were then removed at 30,60, and 180 set for RNA extrac- tion as described under “Materials and Methods.” Following measurement of optical density at 260 nm, acid precipitable counts in 0.5 pg of RNA were determined for each preparation; the specific activities were computed and plotted in A. RNA species, 0.5 pg each, were then hybridized for 20 hours at 75’ with filters contain- ing 0.3 pg of X&W or Xh8OdZac ‘“C-DNA, in the presence of 22 pg of unlabeled RNA from a Zac deletion strain. Counts bound to the Ah80 filters were subtracted from counts bound to the Xh8Odlac filters, and the difference was assumed to measure Zac specific counts in each preparation. These values are plotted in B as a function of time. The enzyme values were similar to other prepa- rations (Figs. 5 and 8) and are not shown. Induced culture, A- - -A; glucose-repressed culture, ; cyclic AMP-treated culture, W-----W.

A uniform amount of RNA from each culture was then hy- bridized to Xh80 and Xh80dZuc DNA filters and the number of Zac specific counts hybridized from each preparation was calcu- lated. As indicated by Fig. 64 there is an initial period of rapid incorporation of label into Zac mRNA. This incorpora- tion is approximately linear for at least 60 see, following which there is a slowing of the accumulation of labeled Zuc mRNA. The initial linearity indicates that experiments performed with *H-RNA from cultures labeled for 1 min provide approximate measurements of the rates of Zac mRNA synthesis. The subse- quent decrease in accumulation rates mainly reflects the decline in over-all RNA incorporation of 3H but appears more marked, perhaps because of degradation of labeled messenger proceeding concurrently with its further synthesis.

In the glucose-repressed culture, a small amount of labeled Zac mRNA is consistently found. The addition of cyclic AMP to the glucose-repressed culture restores the accumulation of Zuc mRNA to unrepressed levels. Because the E. coli strain 1103 is particularly sensitive to transient repression, it is difficult to examine the kinetics of accumulation of the small amount of labeled Zuc mRNA present during exposure to glucose. We therefore performed similar experiments employing strain MO- X19, which is less sensitive to transient repression but, like

i=-

v -.07 5

E - .06 m

2 -.05 A

a

-.04 z

.03 Fl 8

-.02 2 o g

-.Ol g j

5 IO 150 m 60 120 160 MINUTES SECONDS

FIG. 7. Lac mRNA accumulation and p-galactosidase synthesis in induced and glucose-repressed cultures of E. coli MO-X19. E. co& strain MO-X19 was grown to log phase at 35” in Medium A, supplemented with 0.5% succinate and 5 rg per ml of thiamine. Cultures exposed to 5 X lo-’ M IPTG, with or without 1O-4 M glu- cose, were assayed at 0,5, 10, and 15 min for p-galactosidase pro- duction; these results are shown in the left-hand panel. A similar pair of cultures, after 6 min of exposure to inducer with or without glucose, were labeled with 2.2 pg per ml of 3H-uridine. The spe- cific activity of material added to the glucose-treated culture was 28 Ci per mM; material added to the culture induced in the absence of glucose had a specific activity of 5.6 Ci per mu. Samples were removed from each culture for RNA extraction 30,60,120, and 180 set after labeling. At 120 set, unlabeled uridine (I rng- per ml) and o-nitronhenvl-&n-fucoside (5 X 10m3 M) were added to each

- 1.

culture (see text and Fig. 8). RNA preparations, 4.2~g each. were hybridized with filters containing 0.3 ig of Xh8d or %80dZ& 14C- DNA. in the nresence of 320 UP of unlabeled RNA from the Zac de- letion strain.- Lac mRNA speiific counts were determined by sub- tracting the counts hybridized to the X/z80 DNA from those hybridized to the Xh80dluc DNA. On the basis of specific activi- ties and calculated quenching under the employed counting condi- ditions, these results were converted to picomoles of added 3H- uridine incorporated into hybridized luc mRNA per 4.2 pg of RNA. These values are expressed on the ordinate in the right-hand panel and allow representation of relative amounts of Zuc mRNA accu- mulated in the two cultures. Glucose-treated cells, 0 ; induced control culture, 0.

strain 1103, fully responsive to cyclic AMP, unable to utilize glu- cose, and F- (24). As described in detail in the legend to Fig. 7, pairs of cultures of thii strain were induced with IPTG, in the presence and absence of glucose and assayed for accumulation of Zuc mRNA and &galactosidase. Enzyme production is only 20 to 25% of control levels in the glucose-repressed culture, and preliminary experiments showed that corresponding amounts of Zuc mRNA are synthesized during a 60-set labeling period. In order to detect changes caused by the synthesis or degradation of this small amount of measurable Zuc mRNA, the glucose-re- pressed culture was labeled with 5-fold more radioactivity than the control culture. Consequently, the number of Zuc mRNA specific counts is approximately equal for RNA preparations from each culture at each time point. Moreover, with the use of higher concentrations of labeled uridine, incorporation of label into total RNA proceeds linearly throughout this phase of the experiment. In order to show the relative rate of accumulation of Zuc niRNA in the glucose-repressed culture in comparison with enzyme synthesis in that culture, results are expressed as pico- moles of aH-uridine incorporated into hybridizable Zuc mRNA. It is apparent that lac mRNA accumulates in the glucose-re- pressed culture at a rate approximately one-fifth that seen in the control culture and thus comparable to the rate of enzyme syn-

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.001p MINUTES

FIG. 8. Degradation of lac mRNA in induced and glucose- repressed cultures of MO-X19. Following the addition of unla- beled uridine and the competitive inhibitor o-nitrophenyl-p-n- fucoside to the induced and glucose-repressed cultures described in the legend to Fig. 7, specimens were removed every 60 see for RNA extraction. Hybridizations and calculations were per- formed in the manner noted for Fig. 7, but the data for the degradative phase are plotted logarithmically versus time. Re- sults are again expressed as picomoles of 3H-uridine present in hybridized Zac mRNA for 4.2 pg of RNA. Glucose-treated cul- ture, 0 ; induced control culture, 0.

thesis. Most importantly, the kinetics of accumulation appear identical in the control and glucose-repressed cultures, suggest- ing that the rates of synthesis, rather than rates of degradation, are being altered by glucose.

In order to examine directly the rates of degradation of mRNA made in the presence and absence of glucose, unlabeled uridine and o-nitrophenyl-P-D-fucoside, a competitive inhibitor of induc- tion by IPTG, were added after 2 min of exposure of induced cells to labeled uridine. These additives decrease incorporation of label into total RNA by 99% and into Zac mRNA completely, as judged by comparison of BO-set pulse labeling of control and glucose-repressed cultures 1 min before and 1 min after addition of o-nitrophenyl-fi-D-fucoside and uridine. Under conditions of the reported experiment, incorporation of 3H-uridine into total RNA continues for 1 min after addition and then ceases; accumulation of Zac mRNA specific counts slows, as may be seen in Fig. 7, for the 1st min after addition. This decline is likely because of degradation of labeled Zac mRNA as well as slowed synthesis of newly labeled messenger. During the next 2 min, as shown in Fig. 8, there is rapid disappearance of labeled Zac mRNA at very similar rates for the glucose-repressed and control cultures. Half-lives determined from the semilogarithmic plot are 54 set for the glucose-repressed culture and 60 set for the culture induced in the absence of glucose.

This result is supported by a simpler experiment, depicted in Fig. 9, in which an induced culture of E. coli 1103 is labeled for 3 min with Wuridine, treated with o-nitrophenyl-P-D-fucoside

‘OO I I I

180 330 480 SECONDS

FIG. 9. Degradation rates for Zac mRNA. E. coli 1103 was grown to log phase at 35” in Medium A, succinate, thiamine, and 0.1% Casamino acids. The culture was induced from -13 min with 5 X 10-’ M IPTG and, during the last 3 min of induction la- beled with 3H-uridine. At zero time, induction was inhibited by the addition of 5 X 1O-3 M o-nitrophe&-n-fucoside; 1 mg per ml of uridine was also added. The culture was divided. and at 60 set one flask was exposed to lo+ M glucose while the other served as a control. Samples were removed simultaneously from each flask at 180, 330, and 480 set, and 3H-RNA was extracted. 3H- RNA preparations, 4.0 pg each (specific activities = 73,000 cpm per pg), were then hybridized to filters containing 0.3 pg of Ah80 or Xh80dZac ‘%-DNA in the presence of 50 rg of unlabeled RNA from the Zac deletion strain. Counts specific for Zac mRNA were de- termined for each preparation and plotted logarithmically versus time. The zero time value is an approximation based on several similar experiments. Glucose-treated cells, 0 ; control cells, l .

and unlabeled uridine, then divided 1 min later into flasks with and without glucose. The half-life of Zac mRNA under these conditions is about 90 set and is unaffected by the presence of glucose.

These findings confirm the earlier conclusion that glucose pri- marily affects the rate of Zac mRNA synthesis. These experi- ments do not, however, specify whether glucose and cyclic AMP act upon frequency of initiation of mRNA synthesis or upon rate of chain elongation.

Control of Lac Messenger Synthesis in Amino Acid-deprived Culture-In order to examine the possibility that the observed alterations in transcription rates were mediated through a direct effect of glucose or cyclic AMP on translational events, rates of messenger synthesis were examined in the absence of enzyme production. E. coli C600, a derivative of E. coli K-12 auxo- trophic for leucine and threonine, has been shown in this labora- tory and others to produce ,f%galactosidase at a slow rate and only after a lag period of 4 to 6 min when induced during starva- tion for threonine and a carbon source (1, 6). This slow rate of enzyme synthesis virtually ceases in the presence of lop3 M glu-

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Issue of May 10, 1970 H. E. Varmus, R. L. Perlman, and I. Pastan 2265

TABLE I Hybridization results with 3H-RNA from E. Coli C600

As described in the text, 3H-RNA extracted from the threonine- leucine auxotroph, E. coli C600, incubated under various condi- tions, was hybridized with filters containing 1.0 ~g of Ah80 and Xh8Odlac DNA in the presence of a IO-fold excess of unlabeled RNA from E. coli W4032. “Additions” were compounds supple- menting Medium A, leucine, and thiamine (concentrations noted in the text). Specific activities were determined as described under “Materials and Methods.” Enzyme levels were deter- mined for cells incubated under the indicated conditions for 30 min.

Additions

None.................. IPTG . IPTG and glucose. IPTG, glucose, and

cyclic AMP.. . IPTG, glycerol, and

threonine

Specific Hybrid- activity ized

CP~d~~ ia 8,000 10.0 6,000 12.5

22 ) 000 5.0

22,000 5.0

1.0 10.0

150,000

Percentage difference

hybridized

0.04 0.69 f 0.07 0.10 f 0.01

r/ml 0.03 1.76 0.14

0.52 f 0.07 0.42

0.65 f 0.11 0.71 f 0.11

30.8

L

case; approximately one-fourth of the initial rate is restored by the addition of cyclic AMP (1).

This strain was grown to log phase in Medium A supplemented with 0.5% glycerol, 50 pg per ml of threonine and leucine, and 5 pg per ml of thiamine. The cells were centrifuged at room tem- perature and resuspended in Medium A containing only leucine and thiamine. After 15 min of starvation at 37”, cells were in- cubated alone; with 5 x 10h4 M IPTG; with IPTG and 10m3 M glucose; and with IPTG, glucose, and 10m2 M cyclic AMP. After 3 min of incubation, they were labeled for 1 min with 3H-uridine and RNA was extracted from each. Because RNA synthesis or uridine uptake (or both) appeared to be under stringent control as well as partially dependent upon a carbon source, the specific activities of the RNA preparations varied in relation to the cul- ture media (25). The properties of these RNA preparations and of a control preparation from a culture grown on complete medium in the presence of inducer are summarized in Table I.

Also listed in Table I are the results of hybridization incuba- tions of these 3H-RNA species with Xh8O and Xh80dZac DNA filters in the presence of a IO-fold excess of unlabeled RNA from the Zac deletion strain. Because of the relatively low specific activity of the RNA, larger amounts than usual were employed in the experiments. (To show that Zuc DNA sites were not satu- rated at this high level of input, 3H-RNh from the culture grown on complete medium was shown to hybridize with equal efficiency whether 1.0 or 10.0 pg was added to the hybridization mixture.)

Considerable variation in the specific activities of the RNA preparations complicates interpretation of the data. Comparing only RNAs of similar radioactivity, it is evident that 1PTG stimulates messenger synthesis in cells starved for both an es- sential amino acid and a carbon source, and that cyclic AMP stimulates transcription in glucose-repressed cells lacking an es- sential amino acid. Most importantly, this stimulation of ZUC

mRNA production occurs during a lag phase in which no meas- urable ,&galactosidase is formed. Thus it appears unlikely that the studied materials exert their effect on transcription indirectly

-

FIG. 10. P. mirabilis 1 and P. mirabilis 1 F’lac lac mRNA assay. The experiment described in Fig. 5 was performed with the bac- terial DNA assay system. Cultures of E. coli 1103 were induced for 64 min with 5 X 1OV M IPTG; alone, with 1Cr3 M glucose, and with lwz M cyclic AMP and glucose. An uninduced culture was used as a control. After 5 min, specimens were removed for p- galactosidase assays, and the cultures were labeled with %f-uri- dine for 1 min. Extracted 3H-RNA, 1.0 rg, in the presence of a 50.fold excess of unlabeled RNA from E. coli W4032, was hybrid- ized for 20 hours at 75” with P. mirabilis 1 and P. miribilis 1 F’lac filters containing 6.2 fig of 14C-DNA. Stippled bars, P-galactosi- dase levels; hatched bars, percentage difference for hybrid forma- tion for each RNA preparation, with standard errors of the mean.

by some direct effect on translation, although the experiments do not rule out this possibility.

I f stability of the uridine pool size is assumed, it would appear that a larger absolute amount of Zac mRNA was formed in the cyclic AMP-treated cells than in those induced in the absence of a carbon source. However, enzyme production was found to be several-fold greater under the latter conditions when incubation was allowed to proceed for 30 min. One plausible explanation for this discrepancy might be that the amino acid-starved cell has materials with which to carry out only a very small fraction of the protein synthesis for which messages have been made. Under such circumstances, the relative frequency, rather than the absolute amount, of any one species of mRNA will determine the extent to which it is translated.

Results with Bacterial DNA System-Initial experiments designed to measure Zac mRNA under conditions of glucose re- pression and cyclic AMP treatment were performed with a hybridization system employing P. mirabilis 1 and P. mirabilis 1 F’Zac DNA. Although a high degree of background hybridiza- tion of labeled E. coli RNA with P. mira6iZis 1 DNA presented an early obstacle in the development of this assay, we were able to reduce this background by the addition of large amounts of unlabeled RNA from a Zac deletion strain to each hybridization mixture. When wild type strain E. coli K-12 3000 Hfr was

examined during exposure to inducer alone, to inducer and glu- cose, and to inducer with cyclic AMP and glucose, Zac mRNA levels were found to be lowered by glucose and elevated by cyclic AMP. However, differences in mRNA levels did not correlate

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2266 Cyclic AMP Regulation of Lac mRN-4 Vol. 245, Nd. 9

well with differences in enzyme levels; when control cultures, treated with glucose or cyclic AMP in the absence of inducer, were studied, it was found that these compounds apparently stimulated the transcription of non-Zac RNA complementary to genetic material contained in the F’Zac episome. This difficulty was surmounted by use of a derivative strain of E. coli 3000, E. coli 1103, possessing several useful properties. First it was F-.3 Secondly, it was deficient in Enzyme I of the P-enolpyruvate- phosphotransferase system and could not transport or metabolize glucose at a significant rate. In addition, it was more sensit,ive t.o glucose repression than the parent strain, and t.his repression was readily overcome by cyclic AMP (24).

An experiment illustrating results of a lac mRNA assay in uninduced, induced, glucose-repressed, and cyclic AMP-treated cultures of E. coli 1103, with P. mirabilis 1 and P. miralrilis F’lac filters, is shown in Fig. 10. As shown with the phage system, there is good correlation between lac mRNA and enzyme values, indicating that glucose and cyclic AMP exert their effects at the level of transcription. Att’empts to perform additional experi- ments with this assay system were frustrated by considerable variation among replicate samples, as discussed below.

DISCUSSION

In the experiments described above, we have developed hy- bridization assays for the rates of synthesis of Zac mRNA in E. coli grown under a variety of conditions. Our principal findings are that Zac mRNA production is slowed upon the addition of glucose to induced cells and that production of Zac mRNA returns toward normal upon treatment of glucose-repressed cells with cyclic AMP. These results strongly suggest, as predicted by earlier, indirect approaches, that glucose and cyclic AMP exert their effect on enzyme induction at the level of transcription in a manner as yet undefined (1, 6-8).

It should be re-emphasized that these assays measure the rate of Eat mRNA synthesis relative to the rate of non-Zac RNA syn- thesis in the cell and do not directly measure Zac mRNA con- centration. Cells were generally pulse labeled for 60 set with aH-uridine of very high specific activity and the number of counts incorporated into RN,4 complementary to Zac DNA was com- pared to the total number of counts incorporated into all species of RNA.

Kinetic experiments described above indicated that the amount of labeled Zuc mRNA accumulated in 60 set is a fun&ion of the rate of synthesis, since degradative processes do not seem to af- fect this measurement significantly. Although the assay does not directly measure the concentration of Zac mRNA, it does provide an estimate of Zac mRNA levels under steady state con- ditions in which no alteration of degradation rate occurs. Since the kinetic data do not suggest any effect of glucose or cyclic AMP upon rates of degradation and since labeling was performed under relatively stable conditions, it may be assumed that our assay does provide an indirect measure of Zac mRNA concentra- tion. We have recently performed competition hybridization ex- periments which confirm these alterations in mRNA concentra- tions.4 However, the kinetics of the hybridization reaction itself is poorly defined; it is therefore hazardous to assign to either the rate measurement or the concentration estimate more than a relative value.

3 F. Fox, personal communication. 4 H. E. Varmus, R. L. Perlman, and I. Pastan, manuscript in

preparation.

A RNA-DNA hybridization method of this sort depends upon a technique for isolating E. coli Zac DNA from the bulk of the E. coli chromosome and purifying it sufficiently -lo be used in excess amounts in each assay. We have employed two sources of Zac DNA: an F’lac episome previously transferred from E. coli to a Proteus strain and a Xh8O phage known to transduce lac DNA from an E. coli genome (26, 27).

The use of P. mirabilis 1 F’Zac DNA in a hybridization assay for E. coli lac mRNA presents several difficulties. Since RNA preparations vary in their hybridizability, their capacity to hy- bridize with a lac-conta.ining DNd must be measured in relation to their affinity for a control DNA. In these experiments, hhe control DNA, P. mirabilis 1, hybridized to a significant extent wiOh the E. coli RNA, although it was possible to minimize this “background” activity by carrying out the incubnt,ions at, 75” and in Dhe presence of large amounts of unlabeled lac deletion RNA. A more perplexing problem concerns the complex and generally unspecified composition of the F’Zac episome. The mo- lecular weight of the episome is believed to be approximately 74 X lo6 daltons, with F itself weighing about 45 x lo6 daltons (28). However, specific products of the F genes are not known and the apparently large amount of non-F, non-Zac DNA has not been identified. The tendency of uninduced lacf strains (both F- and Hfr) and a Zuc delet.ion Hfr strain to hybridize preferentially to P. mirabilis 1 F’lac, as compared to P. mirabilis 1 DNA, indica- ted that the production of non-Zac RNA was detected by this as- say. Moreover, control experiments with uninduced wild strain cells suggested that glucose and cyclic AMP could alter the tran- scription of some of these noa-Zac elements. We attempted to circumvent these difficulties by correcting our results for the per- centage difference seen with aH-RNA from the Zac deletion strain and by performing experiments with an F- strain incapable of growth on glucose. Despite t,hese adjustments, the P. mirabilis

1 F’lac technique was severely hampered by a paucity of lac DNA available for hybridization. Although we enriched P. mirabilis 1 F’Zac DNA for its episomal fract.ion by passage over methylated albumin Kieselguhr column and estimated the amount of lac DNA on each filter to be at least lo-fold in excess of the Zac mRNA expected to hybridize, results of saturation experiments indicated that we were operating close to t,he saturation point for available Zac DNS. Consequently, small differences in the amount of Zac DNA from filter to filter probably caused the large variation observed among replicate samples in some experiments. Rather than attempt the cumbersome isolation of large amounts of episome by preparative centrifugation in cesium chloride, we decided instead to develop an assay system with the use of DNA from the Zac-transducing phage Xh80.

Ah.80 DNA has a molecular weight of 3 x 10’ daltons (29). When it transduces part of the E. co& genome, it leaves behind some of its own genetic material coding for late functions, and hence the final molecular weight is essentially unchanged. Lac DNA, therefore, comprises about 5 to 10% of the Xh80dZuc DNA. This phage is a hybrid of X and $80; since it may transduce other elements of the E. co& chromosome along with Zac, it appeared theoretically possible that significant non-lac inter- action would occur. However, with this system, labeled RNA from the Zuc deletion &rain W4032 Hfr hybridized equally and to a very small extent with both XhsO and Xh80dlac DNA. Moreover, with use of filters with 1 .O pg of phage DNA (or about 0.05 to 0.10 pg of Zac DNA), “background” hybridization with Xh80 DNA was very low, RN,4 from an induced culture formed

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Issue of May lo,1970 H. E. Varmus, R. L. Perlman, and I. Pastan 2267

less t,han 5% as many hybrids with Xh80 as with Xh80dlac DNA, percentage differences were 3 to 4 times as large as with the P. mirabilis 1-P. mirabilis 1 F’lac assay, and saturation of the DNA was not observed even with inputs as large as 10.0 pg of labeled RNA. Since the estimated amount of Zac DNA on the phage filters was only 2 to 10 times as much as that estimated to be on the bacterial filters, this marked difference in DNA saturability and efficiency of hybridization is not, fully explained.

With the vastly improved phage assay, it was possible to con- firm the less reproducible results obtained with the episomal sys- tem. In addition, experiments were performed to study the synthesis of Zac mRNA in cells unable to carry out net protein synthesis. These experiments, with the reservations set forth in an earlier section, tentatively suggest that glucose and cyclic AMP exert their effects on Zac mRNA synthesis directly through regulation of transcription, and not indirectly through some ef- fect on translation. The precise manner in which these actions occur remains largely unknown. Recent work with lac promoter mutants indicates that cyclic AMP or glucose may influence transcription by some unspecified action at the promoter locus (30, 31). Satisfactory elucidation of these processes, however, will probably require their reproduction in a cell-free system. Investigations are now proceeding in this direction in this laboratory and others.

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Harold E. Varmus, Robert L. Perlman and Ira Pastan3',5'-Monophosphate and Glucose

Messenger Ribonucleic Acid Synthesis by Cyclic AdenosineLacRegulation of

1970, 245:2259-2267.J. Biol. Chem. 

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