hybridized to different amounts of "1" or "r" strands from. Xplac orX DNA under the conditions described in Table 1. TABLE 1. Effect of H1 on lac transcription.
Proc. Nat. Acad. Sci. USA Vol. 72, No. 1, pp. 333-337, January 1975
Effect of a Low-Molecular-Weight DNA-Binding Protein, H1 Factor, on the In Vitro Transcription of the Lactose Operon in Escherichia coli (lac operon DNA/cAMP binding protein/glycerol)
MICHEL CREPIN*, R]GINE CUKIER-KAHNt, AND Laboratoires de * Biochimie Cellulaire et de t Physico-Chimie, Rue du Dr. Roux, 75015 Paris, France
FRANQOIS GROS*
D6partement de Biologie Mol6culaire, Institut Pasteur, 25,
Communicated by Jacques Monod, September 16, 1974 ABSTRACT Hi protein, a heat-stable low-molecularweight DNA-binding factor previously described by CukierKahn et al. [Proc. Nat. Acad. Sci. USA (1972) 69, 3643-;36471 markedly stimulates in vitro synthesis of lac-specific RNA directed by bacteriophage Xh80 dlac or 480 dlac DNA templates in the presence of purified E. coli RNA polymerase holoenzyme. The extent of stimulation obtained by addition of H1 alone is usually greater than that observed with the cAMP receptor protein-cAMP combination. H1 effect varies quite appreciably (from 4- to 16-fold) with the functional state of the promoter, being much larger with Xh8O dlac p', a transducing DNA carrying a superpromoter mutation, than with Xh8O dlac p+. H1 end cAMP receptor protein effects are nearly additive, although interpretation of the data obtained at high Hi concentration is complicated by the appearance of sqme inhibitory property. While the cAMP-receptor-protein-mediated synthesis is asymmetrical ("I" strand almost exclusively copied), the degree of asymmetry observed with H1 is less pronounced, suggesting asymmetrical copying from the lac promoter and symmetric transcription from other regions of the DNA. Synthesis of lac-specific'RNA from Xh8O dtrp/lac or q80 dlac pr UV5 templates, in which lac promoters are insensitive to cAMP receptor protein, either as a result of lac fusion to the trp operon or mutation in the lac promoter, is totally Hi-insensitive. Glycerol (10-15% w/w) can fully substitute for H1 in stimulating lac RNA synthesis in a fashion analogous to that reported for the cAMP receptor protein-cAMP system. The possibility that H1 acts by causing conformational modifications at the promoter level in a way that increases its functional state, and that this effect ismore pronounced with operons sensitive to cAMP receptor protein, is discussed.
In preceding reports (1, 2), the properties of low-molecularweight Escherichia coli protein factors capable of stimulating DNA transcription have been described. Two such factors, termed H1 and H2, stimulate in vitro total RNA synthesis from bacteriophage Xplac or 480 diac DNA templates. H factors strongly bind native DNA on nitrocellulose Millipore filters. Moreover, the amounts required for maximal stimulation of RNA synthesis vary in proportion to the amount of DNA present, suggesting that these polypeptides may act at the level of the template, possibly by modifying the promoter conformation. Accordingly, preliminary experiments involving the use of rifampicin were supportive of an effect at the initiation step (1). In order to obtain further insight on the mode of action of these DNA binding proteins, it' was necessary to explore
whether they show any kind of specificity with respect to defined phage or bacterial transcription units. Attempts have therefore been made to study H1 effects on lac RNA synthesis from various transducing bacteriophages. H1 effects on the lac operon have therefore been compared with those exerted by the cAMP receptor protein (CRP), since it is known that transcription of catabolite-sensitive genes is entirely dependent upon the CRP-cAMP system (3-a). The results obtained provide the suggestion that H1 is capable of inducing or of stabilizing an active conformational state on the sac promoter even in the absence of CRP and cAMP. MATERIALS AND METHODS
Chemicals. Cyclic AMP, UTP, ATP, GTP, and CTP were purchased from Sigma. ['H]UTP (10 Ci/mol) was obtained from C.E.A. (Saclay, France). Bacteriophage DNA. DNA was extracted as described by Nissley et al. (6) from bacteriophages Xplac, Xh80 dlac and 480 dlac; strains lysogenic for Xplac, 180 lac pr UV5 were obtained from J. R. Beckwith; 480 dlac E8 and Xh8O dlac p' lysogens were gifts from E. Signer and Weissbach, respectively. We are also indebted to W. J. Schrenk for a strain carrying the prophage Xh8O dlac trp-fus X 7713. In some instances, DNAs from mutants of lac transducing bacteriophages have been used. These mutant strains were Xh8O dtrp X 7713 lac, in which the lactose genes are fused to the tryptophan genes (7); Xh8O dlac p5, a CRP-sensitive lac superpromoter (8, 9), and 080 dlac pr UV6, a CRP-insensitive revertant from the lac-negative LW. CRP, H1 Protein, and RNA Polymerase. Highly purified CRP was a kind gift from B. de Crombrugghe. H1 was obtained as previously described (2). Fig. 1 illustrates a sodium dodecyl sulfate-acrylamide gel pattern of this preparation. E. coli RNA polymerase holoenzyme was purified according to Burgess' technique (10). It was essentially free of "Co"-rlike subfractions
FIG. 1. Polyacrylamide gel electrophoresis of H1 factor. Sodium dodecyl sulfate gel electrophoresis was performed as described by Weber and Osborn (16). A sample containing 25 ug of protein was heated to 1000 for 5 min in the presence of 1% Na dodecyl S04 and 0.1 M 2-mercaptoethanol. Electrophoresis was at 8 mA per tube for 3-4 hr. The gel was stained overnight with Coomassie blue.
Abbreviations: CRP, cAMP receptor protein; SSC, standard saline-citrate solution (0.15 M sodium chloride-0.015 M sodium citrate, pH 7); 4 X SSC means that the concentration of the solution used is 4 times that of the standard saline-citrate solution. 333
334
Biochemistry-: Crepin et al. -~ ~~
0 ~~
~
Proc. Nat. Acad. Sci. USA 72 (1975)
~ ~~~~~~s' ~~~~~0
Aplac, 0~~~~~~
a
20-
P+,2
25
12
9C
4
. ./..
WM
E
i DNA (ug/ml) joKt FIG. 2. Hybridization of a Xh8O dlac ps transcription product with excess Xplac, and Xi DNA. RNA transcribed from Xh8O dlac p' in the presence' of HI or HI + CRP was 'hybridized with various amounts of Xi and Xplaci strands. Standard deviations in experimental values did' not' exceed 5% of the lac-specific radioactive material hybridized.
Transcription Assays and Detection of Specific RNA Products. The transcription reaction mixture (final volume, 0.1 mPI) contained the following components: Tris HCl, pH 7.9, 20 mM; KCl, 130 mM; MgCl, 10 mM; dithiothreitol, 0.1 mM; EDTA, 1 mM; ATP, CTP, and GTP, 0.2 mM each; [8H]UTP (100 Ci/mmol) 0.05 mM; DNA 6t g; 3':5'-cyclic adenosine mononucleotide (cAMP), 0.15 mM; CRP and H1 factor (1-5 Ag) were also added where indicated (see experiments from Tables 1, 2, 3, and 4). Usually, the mixture was preincubated at 31P for 5 min, the reaction being initiated by the addition of RNA polymerase and incubation being continued at the same temperature for 20 min. The amount of lac-specific RNA synthesized from 080 dlac or Oh8O dlac DNA templates was measured by one step DNA RNA hybridization technique. The labeled RNA was hybridized to different amounts of "1" or "r" strands from Xplac or X DNA under the conditions described in Table 1. TABLE 1. Effect of H1 on lac transcription cpm hybridized to
Template t,80 dlac
Additions
None H1 CRP* HI + CRP*
Xplaci
XpkaCr
strand 240 (450) 1130 (470) 840 (460) 1380 (470)
strand
130 760 125 550
O
Total
lacspecific 370 (440) 1890 (L50) 965 (460) (±50) 1930
The reaction was for 20 min at 37°. It was stopped by rapid cooling to zero°C and'sodium dodecyl sulfate (0.5% final concentration) was added. The mixture was further incubated at 370 for 10 min and KCl was added to a final concentration of 0.5 M. The potassiurm-sodium dodecyl sulfate precipitate was discarded by low-speed contrifugation at 40 and the supernatant fraction containing labeled RNA was hybridized to separate DNA strands. Hybridization in liquidphasewas for 6 hr in 4 X SSC and 0.1i% sodium dodecyl sulfate. After cooling and dilution in 2 X SSC, the material was filtered on Millipores, washed, and treated with RNase (10 ug) for 45 min at 250. Millipores were then washed, dried and radioactivity was measured. Input radioactivity:' 30,000 cpm. * cAMP (0.15 mM) was also present.
0~~~~~~~~1
H, factor (sg) FIG. 3. Effect of H1 factor on lac transcription. Xh8O dlac p DNA (6 Ag) was transcribed with various concentrations of H1 for 20 min at 37'. The amounts of X- and kw-specific RNAs synthesized from the "1" strand were determined by hybridization with Xs and Xplaci strands, respectively. When DNA from Xplac bacteriophage was transcribed the product was directly annealed to separate Xh8O dlac and Xh8O strands. The amount of lac-specific material was measured by taking the difference between the values obtained at DNA saturation with the lac-containing and wild-type detector DNAs corresponding to a given strand (Fig. 2).
RESULTS Effect of H1 factor on lac RNA synthesis
That H1 can stimulate lac-specific RNA synthesis, even in the absence of the CRP plus cAMP system, is evidenced by the results in Table 1. In these experiments, a lac transducing DNA template, 080 dlac, carrying a wild-type lac promoter, has been transcribed in the presence of purified RNA polymerase prepared according to Burgess (10), and the amount of lac-specific RNA synthesized was determined by hybridization. The level of lac RNA synthesis achieved in the presence of RNA polymerase alone was small. Addition of cAMP + CRP (in excess) caused appreciable stimulation of total lac RNA synthesis (sum of the material differentially annealed to each strand). Addition of H1 alone (at optimal concentration) also greatly enhanced lac RNA synthesis (from 2.8- to 5-fold), the extent of stimulation being in all cases somewhat greater than in the presence of CRP + cAMP. In the presence of both HI + CRP (cAMP) the stimulation was somewhat less than additive (see also Table 2) but this should be considered with the fact that at high Hi concentrations the degree of stimulation of lac transcription begins to decrease (Fig. 3). While the CRP effect was exclusively directed towards RNA synthesis from the "correct", or "1" strand (transcription from the "r" strand being generally depressed), H1mediated stimulation concerned primarily the "1" strand, but also to some extent the "r" strand. In this particular experiment the strand'selectivity ratio (lac-specific RNA from "1"/ lac-specific RNA from "r") was close to 1.5, but could reach higher values (see, for instance, experiment from Table 2). Studies with a lac superpromoter mutant It was thought to be of interest to investigate whether the magnitude of HI effect on lac transcription is dependent upon
Proc. Nat. Acad. Sci. USA 72
Effect of H1
(1975)
on
Transcription of the lac Operon
335
TABLE 2. Effect of H1 on lac transcription from a super-promoter mutant Total lao-
cpm hybridized to
specific r lao1 laoi lpk Tplaotal
Additions None
Template
Xh80 dlac p+
CRP* H1 + CRP* None H1 CRP* HI + CRP*
Xh80 dlac pi
2841 1730 2500
870 2300 1220 1760
111 540 510 740
2960 4950 6760 7750
2830 3340 5750 5270
130 1610 1010 2480
980
HI
(±50) (470) (±60) (±80) (±60) (480) (±60) (4100)
3000 3600 3100 2900
2800 3250 3000 2700
200 350 100 200
3850 5810 6550 8240
3790 4980
60 830 120 490
6430 7750
(±60) (±50) (±40) (450) (470) (490) (±60) (±100)
310 890 610 940 190 2440 1130 2970
Same conditions as those described in the legend of Table 1. Input radioactivity: 20,000 cpm for Xh80 dkso p', 10,000 cpm for Xh80. dlac p +. * cAMP (0.15 mM) was also present.
the efficiency of the promoter region. For this purpose the DNA from Xh8O dlac p5, a transducing phage carrying a lac region with a superpromoter mutation, was used (Table 2). Here, total RNA synthesis was stimulated up to 16-fold in the presence of H1 alone, as opposed to 3- to 4-fold when DNA from the same transducing phage, but with a normal lac p+, is used. It should be noted that the CRP-cAMP effect was also greater with this superpromoter mutant than when a normal lac promoter was involved. Fig. 3 shows the comparative stimulatory effect on Xspecific and lac-specific RNA synthesis of different HI concentrations with a Xh8O dlac pS template. Hi-dependent stimulation goes through an optimum, maximal stimulation corresponding to 0.16 ug/gg of DNA or roughly 150 molecules per DNA chain if one assumes that HI exists in the form of a dimer, as is suggested by recent investigations (H. Buc, personal communication). For higher HI/DNA ratios the extent of stimulation begins to decrease. It is unlikely that a contaminant might be responsible for the stimulation effect inasmuch as examination of gel electrophoretic patterns reveals there is a maximum of 5% contamination. Much effort has been devoted to testing the possibility that stimulation of lac RNA synthesis is due to the effect of contaminating deoxyribonuclease. No conversion of replicative form RF1 supercoiled simian virus 40 DNA into relaxed doubled-stranded circles could be obtained after prolonged incubation with H1. After 25-min incubation of X DNA with
TABLE 3. Comparative H1 effects on lac transcription with Xh80 dlac, Xplac, and Oh8O trp-lac DNA templates
H1 under the conditions of the DNA transcription test, no loss in transfecting ability could be detected, until the H1/DNA ratio was close to 1.0, at which stage a 40% reduction was observed. Whether this latter effect can be accounted for by some contaminating "nickase" or DNA coating is difficult to assess, but it is clear that at the lower H1/DNA ratios that are compatible with maximal stimulation of RNA synthesis, the templa te DNA does not suffer any permanent nucleolytic cleavage. Relationship between H1 and CRP sensitivity Although we have not yet analyzed how H1 and CRP mutually influence each other for binding to DNA, results from the preceding section are indicative that the effects of both factors might require a similar DNA conformation if not a strictly common target. Furthermore, examination of results from Table 2 indicates that although H1 exerts an enhancing effect on X-specific RNA synthesis, in agreement with previous reports (2), the overall transcription from a wild-type X DNA template is seldom stimulated more than 2-fold. More generally speaking, since it is admitted that transcription from X P1 or X Pr promoters is not affected by the presence of the CRP + cAMP system, the possibility did exist that transcription units whose expression is strictly dependent upon the cAMP binding protein are more susceptible to H1 stimulation than are CRP-insensitive operons. The following lines of evidence lend support to this hypothesis: (a) When Xplac DNA, in which expression of the lac region is under the command of X promoters rather than of the lac promoter (such as in Xh8O dlac p +), is used as template, H1
cpm hybridized to
Templates
Additions
xh80 dlac
None
Xplac
None
Xh8O dtrp-lac
None H1
H1 H1
TABLE 4. H1 insensitivity of lac transcription in a pr Uv5 mutant
Xh8O Xplaci
Xi
dlacj Xh801
laci cpm hybridized to laci strand
1120 970 3380 2620 - 2310 1960 4920 4230
150 (±50) 760 (±60) 350 (=60) 690 (480)
Template
3160 2960
-
260 (460)
080 dlac pr UV5
5650 5600
-
-
50 (±50)
Same conditions as in Table 1, except that the amount of H1 factor was 0.8 jyg for Xplac transcription. Input radioactivity: 28,000 cpm for Xplac transcription; 20,000 cpm for Xh8O dtrp-lac and 15,000 cpm for Xh8O dlac.
Additions None
H1 CRP* H1 + CRP*
(xplaci - XI) 1520 (±50) 1490 (470) 1960 (440) 1780 (450)
Same conditions as for Table 1. Input radioactivity: 16,000
cpm. *
cAMP (0.15 mM) was also present.
Proc. Nat. Acad. Sci. USA 72 (1975)
Biochemistry: Crepin et al.
336
0
i CD
a
.0
'La
,.
0LQ 0
0
5
10
s
20
25 0
5
10
15
20
25
Glycerol (%)
FIG. 4. Effect of glycerol on lac RNA synthesis in the presence and absence of H1 factor. Total trichloroacetic-acid-precipitable radioactivity (a) aid lac-specific RNA (b) were determined after transcription of Xh80 dlac pi DNA in the presence of various glycerol concentrations. (a) Total RNA made in the presence (2 Mxg) (0) and absence (@) of H1 factor; (b) lac RNA made in the presence (0), and absence (-) of H1 factor.
exerts a smaller stimulatory effect on transcription from the "1" strand (1.9-fold instead of 5-fold; see Table 3). (b) Similarly, when the lac region is fused by an appropriate deletion to the tryptophan (trp) operon, in such a way that the lac promoter is deleted and lac-specific RNA synthesis is initiated at the trp promoter (7), the resulting low level of lac transcription is, if anything, slightly reduced by H1 (Table 3). (c) Finally, and more significantly, when the lac promoter is modified so as to become CRP-insensitive, as is the case in pr uv5, a superpromoter that arises by reversion from a point mutant, L87, lac RNA is transcribed with high efficiency but addition of H1 causes little stimulation (Table 4). This is in contrast to what is observed with the CRP-sensitive highly efficient promoter lac p' (Table 3). H1 effect in the presence of glycerol RNA synthesis from E. coli DNA, rDNA (11), or Xpgal DNA (12) templates is greatly stimulated by glycerol at final concentrations between 10 and 20%. This effect has been ascribed to modifications of the promoter conformation which would render it accessible to RNA polymerase, thereby increasing
the rounds of transcription initiation. We have reinvestigated the effect of glycerol on total and lao-specific RNA synthesis, using Xh8O dlac pm as a template, in the absence of the CRP and cAMP effectors. The overall rate of transcription increases with the concentration of glycerol, maximal stimulation (about 4-fold) being achieved at a concentration of 20%. With respect to lac-specific RNA synthesis, an optimum is observed with 10% glycerol; at higher concentrations, glycerol shows a somewhat smaller effect. As it has already been reported for the CRP and cAMP system (12), concentrations of glycerol causing optimal stimulation eliminate the transcription-enhancing effect of H1 for total or lac-specific RNA synthesis (Fig. 4). DISCUSSION The present work indicates that the effect of H1 resembles, in some respects, that of the cAMP binding protein, the CRP factor. Accordingly, Hi-dependent stimulation of in vitro RNA
0
1
2
S
4
5
H1 factor (Mug) FIG. 5. H1 effect on lac RNA synthesis in the presence of saturating CRP. Xh80 dlac ps (6 Mg) was transcribed in the presence of a saturating amount of CRP (2 j.g) and various concentrations of H1 factor. RNA synthesized for 20 min was hybridized with the "1" (0-O) or "r" strand (0-0) of Xplac and X DNA. Self-annealing of RNA (A---- -) was determined by measuring the amount of RNase-resistant material after incubating the RNA product for 60 min at 220 in 2 X SSC buffer and precipitating the remaining material with 5% cold trichloroacetic acid. The insert illustrates the effect of CRP (0.15 mM cAMP) on the rate of lack RNA synthesis from a Xh80 dkac ps template (6 ag).
synthesis, although significant, is of moderate extent (the average stimulation determined from 13 independent experiments being 1.5- to 2.0-fold) when X-apecific promoters are involved, but amounts to between 4- and 16-fold (depending upon the state of the promoter) when transcription of the lac region is studied. That sensitivity to H1 stimulation more or less parallels sensitivity to the CRP + cAMP effect is clearly shown by a series of observations, the most significant of which are that: (i) When expression of the lac region is placed under the command of the trp promoter as a result of a deletion, no H1-dependent enhancement of lac RNA synthesis is observed. (ii) Transcription of the lac region from Xh8O dlac pr uv5, a transducing phage with a "CRP insensitive" superpromoter, fails to be stimulated by H1. H1 and CRP effects are additive up to a certain limit. When a system containing an optimal concentration of CRP is further supplemented with H1, lac RNA synthesis is stimulated over the CRP-dependent level but full additivity is never reached (Fig. 5). This is to be related with the fact that, at high H1/DNA ratio, the H1-dependent stimulation begins to gradually decrease (Fig. 3), perhaps as a result of the H1 coating effect. H1-dependent stimulation of lac-specific RNA synthesis is not fully asymmetrical, contrary to the effect due to CRP + cAMP addition. This can be interpreted by assuming that H1 activates transcription at the lac promoter site asymmetrically, but could cause symmetrical transcription from other (presumably "internal") regions of the DNA, a situation resembling in some respect the one described by Brody and Leautey (13) when using ethyleneglycol. Independent observations (C. Reiss, R. Cukier-Kahn,
Proc. Nat. Acad. Sci. USA 72
(1975)
and H. Buc, manuscript in preparation) have shown that H1 can cause drastic changes in the Xplac DNA temperature transition profile, indicating that a large fraction of the DNA has its half-melting point at a temperature 8-10 degrees greater than the normal Tm. Ghosh and Echols (14) reported that "D" factor, a DNA binding protein with properties very similar to Hi, also markedly modifies the DNA melting profile. An attractive hypothesis would be that H, could act by a direct or, what seems more likely, by a "long range" effect on the promoter regions. In support of this proposal is our observation that high concentrations of glycerol, known to lower the Tm of lambdoid phage DNAs and to abolish the requirement for CRP and cAMP during transcription of gal genes (12), also totally compete for H1 effect on lac RNA synthesis. Our work provides a clear suggestion that H1 causes changes in promoter conformation and activity to an extent and in a direction which depend upon the class of transcription unit involved. Accordingly, our data indicate, for instance, that H1 would greatly favor the "opening" of promoters from "catabolite-sensitive" genes, have only a modest effect on X-specific promoters and have virtually no effect on trp promoters. Moreover, independent observations (15) show that, at temperatures below 42°, E. coli ribosomal promoters are made less accessible to RNA polymerase in vitro when H1 is present. These "differential" effects could be imputed to the fact that H1 "stabilizes" certain DNA regions from the template (particularly in the vicinity of the promoter) while inducing local melting of others. But it could also be assumed, as has already been done, (11), that all promoters exist in a state of equilibrium between a "closed" and an "open" form, depending upon the temperature and ionic conditions. H1 could prevent establishment of this equilibrium by "freezing" the promoter in its "closed" or "open" conformation according to the category of transcription unit with which it interacts. In any case, the possibility that E. coli DNA binding proteins, like D (14), H1, and H2 (2), as well as a recently discovered factor termed "Hu" (J. Yaniv, manuscript in preparation), can impose structural constraints on phage or bacterial chromo-
Effect of H1 on Transcription of the lac Operon somes in a manner resembling the effect of nuclear on eukaryotic DNA is a challenging hypothesis.
337
proteins
This work i dedicated to the memory of our coworker Dr. CukierKahn, who died soon after completion of the manuscript. We thank Dr. B. de Crombrugghe and Dr. M. Gottesman for CRP protein, and Dr. J. Beckwith for p' mutants. We thank particularly B. Lescure who has made various RNA polymerase preparations, and other colleagues from the Institute (particularly Dr. Buc) for useful advice. This work was supported by grants from the Fonds de D6veloppement de la Recherche Scientifique, the Centre National de la Recherche Scientifique, the Commissariat A l'Energie Atomique, the Ligue Nationale Frangaise contre le Cancer, and the Fondation pour la Recherche M6dicale Fran-
gaise. 1. Jacquet, M., Cukier-Kahn, R., Pla, J. & Gros, F. (1971) Biochem. Biophys. Res. Commun. 45, 1597-1607. 2. Cukier-Kahn, R., Jacquet, M. & Gros, F. (1972) Proc. Nat.
Acad. Sci. USA 69, 3643-3647. 3. Zubay, G., Schwartz, D. & Beckwith, J. (1970) Proc. Nat. Acad. Sci. USA 66, 104-110. 4. Emmer, M., de Crombrugghe, B., Pastan, I. & Perlman, R. (1970) Proc. Nat. Acad. Sci. USA 66, 480-487. 5. Arditti, R. R., Eron, L., Zubay, G., Tocchini-Valentini, G., Connaway, S. & Beckwith, J. R. (1970) Cold Spring Harbor Symp. Quant. Biol. 35, 437-442. 6. Nissley, S. P., Anderson, W. B., Gottesman, M. E., Perlman, R. & Pastan I. (1971) J. Biol. Chem. 246, 4671-4678. 7. Zubay, G., Morse, D. E., Schrenk, W. J. & Miller, J. H. M. (1972) Proc. Nat. Acad. Sci. USA 69, 1100-1103. 8. de Crombrugghe, B., Chen, B., Anderson, W., Nissley, P., Gottesman, M., Pastan, I. & Perlman, R. (1971) Nature New Biol. 231, 139-142. 9. Eron, L. & Block, R. (1971) Proc. Nat. Acad. Sci. USA 68, 1828-1832. 10. Burgess, R. R. (1969) J. Biol. Chem. 244, 6160-6167. 11. Travers, A., Baillie, D. L. & Pedersen, S. (1973) Nature New Biol. 243, 161-163. 12. Nakanishi, S., Adhya, S., Gottesman, M. E. & Pastan, I. (1974) J. Biol. Chem. 249, 4050-4056. 13. Brody, E. N. & Leautey, J. (1973) Eur. J. Biochem. 36, 347361. 14. Ghosh, S. & Echols, H. (1972) Proc. Nat. Acad. Sci. USA 69, 3660-3664. 15. Travers, A. & Cukier-Kahn, R. (1974) FEBS Lett. 43, 86-88. 16. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 44064412.
