Search

Glycerol added to a transcription system containing λgal DNA and Escherichia coli RNA polymerase holoenzyme stimulates total RNA synthesis and replaces the requirement for cyclic adenosine 3' : 5'-monophosphate (cAMP) and cAMP receptor (CRP) for promotion of gal RNA synthesis. The stimulatory effect which is proportional to the concentration of glycerol up to 20% (v/v) occurs at the level of preinitiation complex formation. In stimulating gal transcription, glycerol appears to act at or close to the same site as cAMP and CRP because: (a) glycerol has little effect on gal transcription at saturating levels of cAMP and CRP; (b) although glycerol stimulates transcription from λgal DNA bearing revertible gal promoter mutations, transcription from DNA containing a gal promoter deletion is not stimulated; (c) glycerol stimulation of gal promoter mutants is inhibited by cAMP and CRP; and (d) with either glycerol or cAMP and CRP, transcription of the promoter-proximal galE region precedes transcription of the galKT region. The effect of glycerol on λ early transcription is to stimulate early r-strand RNA synthesis but not early l-strand RNA synthesis. However, if the DNA template contains a defective λsex promoter, the decreased levels of early l-strand RNA are restored to normal by glycerol. Ethylene glycol, dimethylsulfoxide, sucrose, and 1,3-propanediol, all of which lower the Tm of DNA, also stimulate both total RNA and gal RNA synthesis. Furthermore, the glycerol-promoted formation of the gal preinitiation complex is strongly dependent on the preincubation temperature. At lower temperatures, complex formation requires increased glycerol or decreased KCl concentrations. These findings suggest that glycerol may act by melting or changing the conformation of the DNA promoter regions.

The concentration of cyclic AMP has been studied in the NRK line of rat cells transformed by wild-type and a temperature-sensitive derivative of the Kirsten strain of Murine sarcoma virus. The concentration of cyclic AMP is low in cells recently transformed by the Kirsten virus and in two clones derived from a recent infection. The differences in cyclic AMP concentrations are most evident in confluent cultures. Cyclic AMP rises markedly in the contact inhibited cells at confluency but remains low in the transformed cells. Cells transformed with a mutant of Kirsten sarcoma virus, temperature sensitive for the maintenance of transformation, show low levels of cyclic AMP at the permissive temperature and high levels at the nonpermissive temperature. When shifted from 39°C to 32°C it takes 24–48 hr for the cyclic AMP levels to fall in cells transformed by the mutant virus. Exogenous addition of N 6 2′-0-dibutyryl cyclic AMP to Kirsten transformed cells changes their morphology towards that of normal cells and decreases their growth rate, but contact inhibition of growth is not restored. Dibutyryl cyclic AMP and 8-Br-cyclic AMP both decrease the growth rate of NRK cells, but dibutyryl cyclic GMP and 8-Br-cyclic GMP have no effect on the growth rate, saturation density, or morphology of these cells.

To elucidate further how cyclic adenosine 3' : 5'-monophosphate (cyclic AMP) and the cyclic AMP receptor are able to stimulate gal mRNA synthesis we have utilized an antiserum specific for cyclic AMP receptor and also cyclic GMP as inhibitors of cyclic AMP and cyclic AMP receptor action. When cyclic AMP, cyclic AMP receptor, DNA, and RNA polymerase are incubated together a rifampicin-resistant preinitiation complex is formed and, upon the addition of missing ribonucleoside triphosphates or Mg2+, gal mRNA synthesis occurs. If the preinitiation complex is formed in the absence of nucleoside triphosphates the antiserum is able to dissociate the complex. If the complex is formed in the presence of nucleoside triphosphates (no Mg2+), it is converted to a more stable form which is not reversed by the addition of antiserum. Cyclic GMP can prevent the formation of the preinitiation complex. However, we find that once the preinitiation complex has been formed cyclic GMP does not inhibit cyclic AMP receptor-cyclic AMP-dependent transcription, suggesting that RNA polymerase stabilizes cyclic AMP receptor-cyclic AMP binding to the gal promoter region.

The 5′-terminal sequence of mRNA from the galactose operon of E. coli has been determined. gal RNA is synthesized in vitro by means of a kinetically controlled purified transcription system and is isolated by a two-step RNA·DNA hybridization scheme. The following sequence of the first 77 nucleotides has been deduced by analysis of oligonucleotides produced by digestion with T 1 , pancreatic, and carboxymethylated pancreatic ribonucleases: ppA-U-A-C-C-A-U-A-A-G-C-C-U-A-A-U-G-G-A-G-C-G-A-A-U-U-A-U-G-A-G-A-G-U-U-C-U-G-G-U-U-A-C-C-G-G-U-G-G-U-A-G-C-G-G-U-U-A-C-A-U-U-G-G-A-A-G-U-C-A-U-A-C-C-U-G-U. Residues 27-77 correspond to the amino terminal 17 amino acids of UDPgalactose 4-epimerase (EC 5.1.3.2), the protein specified by the promoter-proximal structural gene of the operon. A self-complementary sequence occurs near the 5′ terminus; 12 of 15 nucleotides between residues 4 and 18 are symmetrically located about position 11. The sequence of residues 22-33 closely resembles part of the lactose operator sequence reported previously [Gilbert, W. & Maxam, A. (1973) Proc. Nat. Acad. Sci. USA 70, 3581-3584].

To investigate how cell population density influences the intracellular concentration of cyclic AMP we have measured adenylate cyclase and cyclic AMP phosphodiesterase activities and cyclic AMP levels at various stages of cell density in normal rat-kidney (NRK) cells, which exhibit contact-inhibition of growth, and in normal chick-embryo fibroblasts (CEF), which do not show contact inhibition of growth under our conditions. Until NRK cells reach confluency, both activities increase with increasing cell population and cyclic AMP levels are low. As NRK cells reach confluency, cyclic AMP phosphodiesterase activity decreases somewhat whereas adenylate cyclase activity continues to rise. This increase in synthetic ability is accompanied by the increase in cyclic AMP levels which occurs in these cells at confluency. In CEF grown in 5% serum where density-dependent inhibition of growth is not observed, both adenylate cyclase and cyclic AMP phosphodiesterase activities increase proportionately with increasing cell population density. No significant alteration occurs in the ratio between these two enzyme activities and no change is observed in cyclic AMP levels. The NaF-stimulated activity in NRK cells increases with increasing cell density until the cells reach confluency; thereafter the NaF-stimulated activity remains constant. In contrast, the NaF-stimulated activity observed in CEF does not vary appreciably between light and heavy density. The observed changes in the enzymes of cyclic AMP metabolism accurately reflect the changes in cyclic AMP concentration as a function of cell population density. The data indicate that these two enzyme activities respond to increasing cell density to elicit a rise in intracellular cyclic AMP levels. The elevated cyclic AMP levels are thought to be involved in the regulation of cellular growth rate and the mediation of contact inhibition of growth.

Escherichia coli grown in a medium containing 10% (v/v) dimethylsulfoxide (DMSO) show constitutive synthesis of the enzymes of the gal operon, partial induction of λ prophage and extensive filament formation. The effect of DMSO is relatively specific for gal and λ because there is less than a 2 fold effect on the synthesis of several other enzymes studied: UDP-glucose pyrophospholylase, phosphomannose isomerase, phosphoglucomutase, alkaline phosphatase, and β-galactosidse. DMSO also alleviates the functional defects caused by certain promoter mutations in gal and λ. These results are compared with our observations that DMSO and other DNA-denaturing reagents stimulate the synthesis of specific gal and λ RNA transcripts from wild-type and promoter-defective DNA templates in vitro.

Myosin has been identified in three independently derived cell lines of fibroblasts: normal rat kidney cells, mouse 3T3 cells, and mouse L cells. A quantitative two-step procedure for its extraction from as little as 200 mg of cultured fibroblasts (wet weight) has been developed. Myosin constitutes from 0.5 % to over 3 % of the total fibroblast protein. Cell fractionation suggests that the myosin principally resides in the cytoplasm. One virus-transformed, normal rat kidney cell line contained one-half of the myosin of its NRK parent. Treatment of L cells during culture with N6,O2'-dibutyryl cyclic adenosine 3',5'-monophosphate and theophylline had no effect on their myosin content. Actin and tubulin were identified in L cells.

The activity of the enzyme adenylate cyclase has been determined in normal rat kidney (NRK) cells and NRK cells transformed by either the Kirsten sarcoma virus (Ki-SV) or the Moloney sarcoma virus (Mo-SV). The specific activity of the NRK enzyme increases with increasing cell population density. Transformation apparently decreases this response, for the cyclase activity from KNRK cells (NRK cells transformed by Ki-SV) is low and rises only slightly with increasing cell density. The activity in transformed cells is at most 50% that noted in confluent NRK cells. Further, when assayed in the presence of 10 mm NaF, the KNRK and MNRK (NRK cells transformed by Mo-SV) cyclase activity is appreciably lower than that from NRK cells. These changes in adenylate cyclase activity are not due to an altered affinity for ATP; the enzymes from NRK, KNRK, and MNRK cells all exhibit an apparent Km (ATP) ∼0.24 mm. The activity of the NRK enzyme increases with increasing Mg2+ concentration through 15 mm; the enzyme from KNRK and MNRK cells is saturated at 1 mm Mg2+. When assayed in the presence of Mn2+, the cyclase activity of KNRK and MNRK cells is decreased relative to NRK cells. The effects of GTP and prostaglandin E1 were also studied. The adenylate cyclase of NRK homogenates is stimulated 2- to 3-fold by prostaglandin E1; GTP has little effect (∼20% stimulation) on either basal or protaglandin E1-stimulated activity. However, the activity of crude, washed NRK membranes is enhanced 2- to 3-fold by GTP at 10-6 to 10-4 m. Stimulation by prostaglandin E1 is dependent upon the presence of GTP in this membrane preparation. Of particular interest is the observation that the cyclase from KNRK and MNRK cells, either homogenates or membrane preparations, is unresponsive to prostaglandin E1 in either the presence or absence of GTP. Our studies indicate that transformation of fibroblasts with RNA tumor viruses often results in decreased adenylate cyclase activity; the mechanism by which this reduction in enzyme activity is accomplished appears to differ with different viruses.

Concentrations of total NAD+ and NADH have been measured and the NAD+:NADH ratio calculated for 3T3 fibroblasts and SV40 transformed 3T3 cells and for normal rat kidney fibroblasts and murine sarcoma virus (Kirsten strain)-transformed normal rat kidney fibroblasts. The absolute levels were found to decrease 2- to 3-fold with transformation. The NAD+:NADH ratio in the normal cells ranged from 3.1 to 3.6, and increased 3- to 4-fold as they grew from logarithmic to stationary phase. The final ratio was found to be a direct function of the final cell density achieved for the 3T3 cells. In contrast, the NAD+:NADH ratio of transformed cells was low, (1.2 to 3.2) and did not rise when the cells reached a heavy density. Comparable results were obtained for the ratio of free NAD+:NADH. This ratio was determined by analysis of cellular pyruvate and lactate concentrations and calculated using the Keq of lactate dehydrogenase. Attempts to alter the ratio by treatment of the cells with agents capable of elevating intracellular cyclic adenosine 3′ : 5′-monophosphate levels, such as N6, O2′-dibutyryl cyclic AMP or prostaglandin E1, were unsuccessful. Nor did serum deprivation or agents capable of lowering cyclic adenosine 3′ : 5′-monophosphate levels affect the NAD+:NADH ratio. We suggest that the inability of cells to elevate their NAD+: NADH ratio at confluency is a characteristic of transformed cells and related to their defective growth control.

Cyclic 3',5'-AMP stimulates the rate of tryptophanase synthesis in Escherichia coli treated with tris(hydroxymethyl)aminomethane and ethylenediaminetetraacetic acid and induced with tryptophan. A maximal effect occurs at 4 x 10-4 m cyclic 3',5'-AMP and a half-maximal response at 1.5 x 10-4 m. The action of cyclic 3',5'-AMP is specific for tryptophanase since the over-all rate of protein and RNA synthesis and the synthesis of the biosynthetic enzyme tryptophan synthetase are unaffected. One site of cyclic 3',5'-AMP action appears to be after the transcription of DNA into messenger RNA since increased enzyme synthesis is observed in induced cells in which cyclic 3',5'-AMP is added after messenger RNA synthesis is arrested by actinomycin D, proflavine, or inducer removal. The half-life of the messenger RNA for tryptophanase is 3 min and is unaffected by cyclic 3',5'-AMP. Cyclic 3',5'-AMP also fails to stimulate the conversion of tryptophanase precursor into active enzyme. Thus cyclic 3',5'-AMP appears to act at the level of the polysome to increase the rate of polypeptide synthesis.

An enzyme that hydrolyzes sphingomyelin to ceramide and phosphorylcholine has been purified from the growth medium of Clostridium perfringens. The activity of the enzyme is stimulated about 2-fold by magnesium chloride and by diethyl ether. The activity of the enzyme is completely inhibited by 10-3 m ethylenediaminetetraacetic acid or 10-3 m calcium chloride. Lysolecithin and dipalmitoyl lecithin are hydrolyzed at about 10% of the rate of sphingomyelin. No hydrolysis of phosphatidylserine, phosphatidyl-ethanolamine, or phosphatidylinositol is detected. The enzyme does not catalyze the exchange of phosphorylcholine-14C into sphingomyelin.

Cyclic adenosine 3',5'-monophosphate (cyclic AMP) overcomes the repression by glucose of the synthesis of the following inducible enzymes and proteins in Escherichia coli: β-galactosidase, lac permease, galactokinase, glycerokinase, l-α-glycerophosphate permease, Enzyme II for fructose of the phosphoenolpyruvate phosphotransferase system, l-arabinose permease, tryptophanase, d-serine deaminase, and thymidine phosphorylase. Cyclic AMP overcomes the repression by glucose of the synthesis of β-galactosidase in Serratia marcescens F lac, Salmonella typhimurium F lac, Proteus inconstans P lac, and Aerobacter aerogenes; it also overcomes the repression by glucose of galactokinase synthesis in S. typhimurium. A number of analogues of cyclic AMP failed to mimic or inhibit the action of the cyclic nucleotide on β-galactosidase synthesis. Since glucose lowers the intracellular concentration of cyclic AMP in E. coli, we propose that the intracellular level of cyclic AMP regulates the rate of synthesis of many inducible enzymes in E. coli and other microorganisms and that glucose lowers the rate of synthesis of these enzymes by decreasing the intracellular level of cyclic AMP.

Pure monoiodo ACTH- 125 I was prepared that was biologically active and free of unlabeled ACTH. Extracts of adrenal cortex that contained ACTH-sensitive adenyl cyclase, bound ACTH- 125 I; extracts that lacked the ACTH-sensitive cyclase did not bind ACTH- 125 I. Unlabeled ACTH inhibited the binding of ACTH- 125 I. Five ACTH derivatives which varied widely in biological activity were tested. All inhibited the binding of ACTH- 125 I in direct proportion to their biological activity. Albumin, insulin, and four unrelated iodinated hormones were inert. The addition of excess hormone or acetic acid produced rapid dissociation of bound ACTH- 125 I. This study demonstrates directly the binding of ACTH to its biologically significant site.

A cyclic AMP binding protein has been purified over 100-fold from E. coli extracts. Protein purified from wild-type strains binds cyclic AMP with an apparent dissociation constant of 1-2 × 10 -6 M . Two mutant strains that are unresponsive to exogenous cyclic AMP have altered binding activity; the protein purified from one of these mutants has a decreased affinity for cyclic AMP (apparent dissociation constant = 2 × 10 -5 M ). Extracts of this mutant are deficient in their ability to support β-galactosidase synthesis in vitro . The addition of purified, wild-type binding protein to these extracts restores enzyme synthesis toward normal. Because this binding protein appears to be required for cyclic AMP action, we suggest it be called the cyclic AMP receptor protein (CR protein).

Several analogues of cyclic adenosine 3',5'-monophosphate (cyclic AMP) have been tested for their ability to inhibit the binding of cyclic AMP to cyclic AMP receptor (CRP) and to inhibit the cyclic AMP-CRP-dependent transcription of the gal operon. A number of the cyclic AMP derivatives are able to inhibit cyclic AMP binding to CRP, but of all the analogues studied only tubercidin 3',5'-monophosphate is able to replace cyclic AMP in promoting gal transcription. Those analogues which inhibit cyclic AMP binding also decrease cyclic AMP-CRP-dependent transcription. Derivatives with a modified cyclic phosphate structure are potent inhibitors of gal transcription although they do not influence cyclic AMP binding to CRP as observed in the ammonium sulfate precipitation assay. These results indicate a marked specificity for cyclic AMP in stimulating gal mRNA synthesis and also point to the probability that the purine base, the ribose moiety, and the cyclic phosphate group all are involved in binding to CRP and eliciting the correct conformational change of the protein required to mediate cyclic AMP-dependent gene transcription.

Sphingomyelinase, like thyroid-stimulating hormone, increases the oxidation of [1- 14 C]glucose to 14 CO 2 and the incorporation of 32 P i into phospholipid in dog and bovine thyroid slices. The increase in glucose oxidation is inhibited by 3- O -methyl-glucose, indicating that the glucose oxidized enters the cell via a stereospecific transport system. In sphingomyelinase-treated beef thyroids, there is a 65–90% fall in sphingomyelin content and a 10–30% fall in lecithin. Thus most of the sphingomyelin appears to be in the plasma membrane. Lecithinase C produced a small increase in glucose oxidation and phospholipid synthesis in only 7 of 15 experiments. In lecithinase C-treated tissue, the levels of all phospholipids fell about equally. Sphingomyelinase and lecithinase C, unlike thyroid-stimulating hormone, failed to induce the formation of intracellular colloid droplets in vitro , and Sphingomyelinase failed to produce a fall in the radioiodine content of chick thyroids.

DNA synthesis and mitosis in synchronized Swiss 3T3-4 and Balb 3T3 cells is prevented by the addition of (but) 2cAMP during the early G1 phase of the cell cycle. Removal of (but) 2cAMP at up to eleven hours, or addition of (but) 2cAMP as early as three hours after planting fails to block DNA synthesis. However, in Swiss 3T3-4 cells, but not Balb 3T3 C1A31 cells, the addition or removal of (but) 2cAMP at three or six hours after planting causes DNA synthesis to reach a peak at an earlier time. Adding (but) 2cAMP in early S-phase does not inhibit DNA synthesis, but prevents subsequent mitosis. This indicates the presence of a cyclic AMP-dependent block in G2. Our results suggest that cyclic AMP has at least three regulatory functions during the cell cycle, two negative and one positive.

Cultures of Clostridium perfringens produce a factor that stimulates the thyroid. The stimulating factor has been purified 300-fold by fractionation of the growth medium with ammonium sulfate, filtration on Sephadex G-100, and chromatography on diethylaminoethyl cellulose. The purified factor contains no proteolytic activity, and has been separated from neuraminidase and lecithinase C. When incubated with thyroid slices the purified factor, like thyroid-stimulating hormone stimulates glucose-1-14C oxidation, phospholipid synthesis, and the formation of intracellular colloid droplets. When injected into chicks, the factor causes the depletion of radioiodine. The factor is thought to be a protein since its activity is destroyed by treatment with Pronase. Its behavior on Sephadex G-100 suggests a molecular weight of about 30,000.

Cyclic 3′,5′-AMP increases the synthesis of lac mRNA in a cell-free extract of E. coli using λh80dlac DNA as template. Lac mRNA was measured by DNA-RNA hybridization to F'lac DNA and shown to be specific by competition of the 3 H-lac mRNA with unlabeled lac mRNA prepared from whole cells.

The repressor of the galactose operon of Escherichia coli has been partially purified and identified as a protein. Induction of a lysogen in which λ was linked to the bacterial gal R and lysine genes resulted in a large increase in the production of the gal repressor. Single-step purification by affinity chromatography, using the ligand p -aminophenyl-β-D-thiogalactoside linked to beaded agarose, provided a convenient method of separating the gal repressor from other DNA-binding proteins. Binding of gal repressor to λp gal [ 32 P]DNA was studied by assay of binding to a nitrocellulose filter. Interaction between gal repressor and λp gal DNA showed a high degree of specificity; the dissociation constant of the complex was estimated to be 1.0 × 10 -12 M. Unlabeled λp gal DNA competed for binding to gal repressor, but λDNA and λh80d lac DNA did not. Fucose and galactose, which function as inducers of the galactose operon in vivo , produced one-half maximal inhibition of gal repressor-λp gal DNA binding at concentrations of 5 × 10 -5 M.

THE study of the absorption of iodide by the intestinal tract was initiated by Cohn (1), who showed that iodide was well absorbed by the ileum, jejunum and colon of the dog, and poorly by the stomach. Albert et al. (2) found that the rat also absorbed iodide well from the small and large bowel and only slightly from the stomach. Pearlman, Morton and Chaikoff (3) demonstrated in the rat that the tissue of the entire small intestine accumulated at the end of one hour 0.6% of the I131 administered into the stomach. The studies of Johnson and Albert (4) revealed I131 in the contents of the small intestine of the rat in significant concentrations, but they did not establish whether it was gastric iodide being passed down the intestinal tract or actively secreted at that site. Furthermore, Honour et al. (5) concluded that while the iodide concentration of stomach secretions was thirty times that of plasma, the iodide concentration of duodenal secretions was approximately equal to that of plasma.

Cyclic AMP-phosphodiesterase is present in various mouse fibroblasts. Contact-inhibited 3T3 cells contain two forms of the enzyme, one with a K m of 2.5 μM and the second with a K m of 71 μM. As 3T3 cells grow to confluency and cAMP concentrations rise, the activity of the first enzyme increases, whereas that of the second is unchanged. A line of SV40-transformed 3T3 cells with low cAMP concentration also has low levels of the cAMP-phosphodiesterase with a K m of 2.5 μM. Treatment of 3T3 and SV40-transformed 3T3 cells with dibutyryl cAMP and theophylline increases cAMP-phosphodiesterase accumulation. This accumulation is blocked by cycloheximide and actinomycin D. The newly formed enzyme resembles the higher affinity enzyme present in unstimulated cells, since it has a K m of 1.2-2.0 μM, and is stimulated by snake venom. In L cells in which cAMP concentrations are elevated by treatment with prostaglandin E 1 , cAMP phosphodiesterase also accumulates. We conclude that intracellular concentrations of cAMP regulate the synthesis of cAMP-phosphodiesterase, and that cAMP functions as an inducer of the enzyme.

Treatment of intact chicken embryo fibroblasts in tissue culture with trypsin leads to an alteration of the kinetic properties of a particulate cyclic adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterase. Distribution studies show that this phosphodiesterase is principally located in the plasma membrane. Little activity is found in nuclear, mitochondrial, and microsomal fractions. The trypsin-sensitive phosphodiesterase activity does not function on the exterior of the cell membrane. The Km and Vmax of the enzyme are altered by trypsin treatment of intact cells and this effect is quantitatively reproduced by trypsin treatment of the isolated plasma membrane fraction. The supernatant fraction of chicken embryo fibroblasts contains phosphodiesterases capable of hydrolyzing both cyclic AMP and cyclic GMP. DEAE-cellulose chromatography of the supernatant fraction resolves phosphodiesterase activity into two peaks. One fraction is specific for cyclic AMP and is kinetically similar to the plasma membrane enzyme, whereas the other fraction hydrolyzes both cyclic nucleotides in a Michaelis-Menten fashion. The cyclic AMP-specific phosphodiesterase appears to be under negative cooperative regulation. These studies show that chicken embryo fibroblasts contain a cyclic AMP phosphodiesterase in the plasma membrane, the kinetic properties of which can be altered by an enzyme that acts on the outer cell membrane.

6-Hydroxynicotinic acid was demonstrated to be the first intermediate in the degradation of nicotinic acid by a clostridium. In the presence of pyruvate, other intermediates accumulated; two of these were identified, one as 1,4,5,6-tetrahydro-6-oxonicotinic acid, and the other as α-methyleneglutaric acid. The pathways for nicotinic acid degradation may be considered as involving successive oxidative and reductive steps to 1,4,5,6-tetrahydro-6-oxonicotinic acid, then through some unidentified steps to α-methyleneglutaric acid, which is subsequently converted to propionic and acetic acids and carbon dioxide. Pyruvate and lactate were shown not to be obligatory intermediates in the production of propionate. This microorganism has a high content of B12 coenzyme, but its function in nicotinic acid fermentation is not yet understood.

We have studied the in vitro repression of gal mRNA synthesis by the gal repressor from Escherichia coli . By use of a four-step purification procedure involving chromatography on phosphocellulose, DEAE-cellulose, and an affinity resin, the gal repressor has been purified about 1600-fold from a crude cell extract. The purification was aided by use of a cell extract made after prophage induction of cells lysogenic for bacteriophage λ that carries the gal repressor gene ( galR ). The highly purified gal repressor is an effective and specific repressor of in vitro synthesis of gal mRNA with λ gal DNA as template. Both D-fucose and D-galactose overcome the action of gal repressor; the half-maximal concentrations of D-fucose and D-galactose for overcoming the action of repressor are 1 mM and 0.5 mM, respectively. The repressor fails to repress gal -specific transcription when the gal DNA contains a cis-dominant operator constitutive ( O c ) mutation. We conclude that the gal repressor recognizes the gal operator site and acts by preventing gal transcription.

A procedure is described for the purification of cyclic adenosine 3',5'-monophosphate receptor protein (CRP) from Escherichia coli involving chromatography on DEAE-cellulose and phosphocellulose, (NH4)2SO4 precipitation, and filtration on Sephadex G-100. The preparation appears to be homogeneous as determined by sedimentation equilibrium studies, isoelectric focusing, and amino-terminal amino acid sequence analysis. The ability of CRP to promote selected transcription is lost upon elution of the protein from phosphocellulose but activity is usually regained within 20 to 48 hours. The active CRP preparation is stable for several months stored either frozen or at 4°. The weight average molecular weight of the native protein is ∼45,000, based on sedimentation equilibrium data; it is composed of two apparently identical subunits of molecular weight ∼22,500. A sedimentation constant s20,w = 3.5 was determined by sedimentation velocity. Isoelectric focusing data show the protein to have an isoelectric point of 9.12. Titration of free sulfhydryl groups by either 5,5'-dithiobis(2-nitrobenzoic acid) or 4,4'-dithiodipyridine in the presence of 6 m guanidine-HCl shows the presence of four free sulfhydryl groups per native dimer; these account for the four half-cystines observed by amino acid analysis.

An adrenocorticotropic hormone (ACTH)-sensitive, steroid-producing mouse adrenal tumor was homogenized and fractionated by differential centrifugation. The adenyl cyclase activity that was stimulated by ACTH and by fluoride was purified 10- to 20-fold and localized to particulate fractions. The enzyme activity was not affected by several other polypeptide hormones, by epinephrine, or by prostaglandin PGE1. The ACTH-induced increase in adenyl cyclase activity was maximal at pH 7.4 to pH 7.7. The optimal Mg2+:ATP ratio was 1 to 1.5. Enzyme activity in the presence of ACTH was decreased by Ca2+, Na+, K+, and propranolol. Other nucleotides at equimolar concentrations to ATP also decreased the ACTH effect to a variable extent; dATP was the most effective inhibitor.

Pathways of glucose oxidation using glucose-1-C14 and glucose-6-C14 have been studied in vitro in adrenal, ovary, testis, pituitary and parathyroid slices. The results indicate the existence of an active hexose monophosphate pathway in all endocrine glands studied. Although in similar experiments TSH in vitro was capable of stimulating glucose oxidation in thyroid (1), no effects of FSH, LH, chorionic gonadotrophin or anterior pituitary powder were observed in ovary or testis. Neither ACTH nor 3′5′ cyclic adenosine monophosphate inhibited glucose oxidation in slices of adrenal cortex. Possible causes for this absence of inhibition are discussed in light of the recent work by Haynes on the mechanism of action of ACTH (6). The hexose monophosphatc pathway for glucose metabolism in endocrine tissue is a source of TPNH, and this cofactor may be of importance in the regulation of hormone production.

Incubation of thyroid slices with added thyroid stimulating hormone results in an increase in the concentration of oxidized triphosphopyridine nucleotide. Since thyroid stimulating hormone also stimulates acetate and pyruvate oxidation, it is suggested that the previously reported increase in glucose oxidation is not a primary effect of thyroid stimulating hormone but results secondarily from the ability of this hormone to increase the TPN concentration in the thyroid. Attempts to demonstrate an effect of thyroid stimulating hormone on TPNH oxidase were unsuccessful, and it was not possible to stimulate glucose oxidation in thyroid homogenates fortified with TPN or TPNH. The amount of glucose oxidized by thyroid homogenates appeared to be a function of the concentration of TPN added. An effect of 3·4·1o−11M TSH was obtained when dog thyroid slices were used. This concentration is considered to be in the physiological range. Monoiodotyrosine and diiodotyrosine also stimulated glucose oxidation in thyroid slices. Acetylated thyroid stimulating hormone neither stimulated glucose oxidation in thyroid slices nor did it inhibit the action of one-tenth as much thyroid stimulating hormone. Thyroid glands obtained from guinea pigs injected with thyroid stimulating hormone for two days prior to sacrifice oxidized more glucose than did glands from control animals.

Thyroid-stimulating hormone increased adenyl cyclase activity in canine and bovine thyroid homogenates. Activation of ade-nyl cyclase was impaired by 3 adrenergic blocking agents, dl-propranolol, d-propranolol and phentolamine, over the range 3−7.5 × 10−4m. The concentrations are approximately 100 times greater than those which produce alpha- or beta-adrenergic blockade. dl-Propranolol and phen-tolamine at 1.5−3.0 × 10−4M impaired thyroid-stimulating hormone stimulation of glucose-l-14C oxidation in canine thyroid slices. A similar effect was seen when the slices were preincubated with propranolol, washed, and then incubated with thyroid-stimulating hormone in the absence of propranolol. Propranolol and phentolamine at concentrations which abolished thyroid-stimulating hormone stimulated glucose oxidation in canine thyroid slices had little effect on the stimulation of glucose oxidation by dibutyryl cyclic 3′,5′-AMP. In bovine thyroids concentrations of propranolol which abolished stimulation of adenyl cy...

DNA-RNA hybridization experiments show that transcription of the gal operon is regulated by cyclic AMP as well as inducer. Cyclic AMP stimulates gal mRNA synthesis and does not affect its half-life. Mutants unable to make cyclic AMP synthesize gal mRNA only after the nucleotide is added. Mutants lacking a protein required for cyclic AMP action do not make gal mRNA. We conclude that lac and gal transcription is similarly controlled by cyclic AMP.

A system has been developed to study the regulation of the lactose operon in vitro. Transcription requires cyclic AMP and cyclic AMP binding protein and is repressed by lactose repressor protein.

Cyclic adenosine 3',5'-monophosphate reverses the glucose repression of β-galactosidase synthesis in Escherichia coli induced for enzyme synthesis. In this report, we describe 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 β-galactosidase synthesis. Thus we confirm earlier indirect measurements which indicate that control of β-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 incapable of enzyme synthesis and that glucose does not affect the rate of degradation of lac mRNA.

The fusion of XC cells induced by murine leukemia virus (MuLV)-infected cells is also induced by homogenates prepared from the infected cells and by purified MuLV. The fusion-inducing factor appears to contain a heat-labile lipoprotein. No synthesis of specific macromolecules by the XC cells is necessary to obtain fusion. The results suggest that specific components of the viral particle are the activators for the fusion process and they may also be present in the membranes of infected cells.

In a transcription system containing λpgal8 DNA as template and Escherichia coli RNA polymerase, the addition of cyclic adenosine monophosphate receptor protein (CRP) and 3',5'-cyclic AMP causes a 15-fold increase in gal mRNA synthesis. Gal mRNA was measured by hybridizing the [3H]RNA product to the separated strands of λpgal8 DNA after removal of λ mRNA by preliminary hybridization to λ DNA. Competition experiments indicate that a large fraction of this RNA is identical with in vivo gal mRNA. With λpgal8 DNA as template but not with λ DNA, 3',5'-cyclic AMP and CRP cause a 2-fold increase in λ mRNA. Since the increment in λ mRNA hybridizes to the left-hand half of the ι strand of λ DNA and is abolished by the addition of ρ, it is interpreted as read through from the 3',5'-cyclic AMP-CRP sensitive site in the gal region of λpgal8 DNA. The concentrations of CRP and 3',5'-cyclic AMP necessary for half-maximal stimulation of gal mRNA synthesis are 5 x 10-8 and 5 x 10-6 m, respectively. 2',3'-Cyclic AMP and 5'-AMP do not stimulate gal mRNA synthesis; 3',5'-cyclic GMP is an inhibitor. When 3',5' cyclic AMP, CRP, DNA, and RNA polymerase are incubated together for 10 min, a rifampicin-resistant preinitiation complex is formed, and, upon the addition of nucleoside triphosphates, gal mRNA synthesis occurs. 3',5'-Cyclic GMP prevents the formation of this complex and can dissociate it after it has been formed. These results suggest that 3',5'-cyclic AMP and CRP stimulate gal transcription at a step prior to chain elongation.

Galactokinase has been synthesized in vitro in a coupled system for transcription and translation containing λpgal DNA and a cell-free extract prepared from a gal deletion strain of E. coli. Galactokinase synthesis requires gal operon DNA, and is stimulated 2- to 12-fold by 10-4 m cyclic adenosine 3',5'-monophosphate (cyclic AMP). The action of cyclic AMP requires cyclic AMP receptor protein and is completely reversed by 10-3 m cyclic guanosine 3',5'-monophosphate. Synthesis of galactokinase is partially inhibited by 10-4 m guanosine 3'-diphosphate, 5'-diphosphate. Galactokinase synthesis begins after a 15-min lag and is linear for 45 to 60 min at 30° and 37°. No enzyme synthesis is detected at 23°, and no enzyme is made in the presence of deoxyribonuclease, ribonuclease, or chloramphenicol. The galactokinase made in vitro resembles the enzyme made in vivo in its molecular weight, inhibition by calcium, and resistance to inactivation by toluene.

The effect of dibutyryl-cyclic adenosine 3',5'-monophosphate and theophylline on the incorporation of [35S]sulfate into acid mucopolysaccharides by 3T3 and SV3T3 fibroblasts has been studied. SV40 transformation of 3T3 fibroblasts produced a marked decrease in the rate of synthesis of acid mucopolysaccharides, in agreement with the data of others. When the transformed cells were grown in medium supplemented with dibutyryl-cyclic AMP and theophylline for 3 days, they secreted, on the basis of cell protein, approximately 250% more labeled acid mucopolysaccharides during a 24-hour labeling period than did the nontreated controls. The secretion of a variety of sulfated acid mucopolysaccharides was increased including chondroitin-4- and -6-sulfate and dermatan sulfate. Comparison of the rate of sulfate incorporation into acid mucopolysaccharides and the rate of loss of previously labeled acid mucopolysaccharides from the cells indicated that the effect was due to a greater rate of synthesis of these compounds at the time of labeling rather than due to a decrease in the rate of their degradation.

The addition of SV40 to contact inhibited Balb 3T3 cells causes a 2-fold decrease in intracellular cAMP levels. The levels reach a minimum 3 hours after virus addition, and after a few hours begin to rise toward normal. No significant changes in cAMP levels are observed after cells are exposed to UV-inactivated virus or are mock-infected. This is the earliest known effect of SV40 infection. We propose that SV40 induces host DNA synthesis by lowering cAMP levels.

Chick embryo fibroblasts (CEF) transformed by the Schmidt-Ruppin strain of Rous sarcoma virus (RSV-SR) have decreased adenylate cyclase activity. In cells infected by a temperature-sensitive mutant of this virus (RSV-SR-T5), enzyme activity is near normal when the cells are grown at the non-permissive temperature (41°C) but decreases at the permissive temperature (36°). Adenylate cyclase activity decreases slowly over a 24 hr period to one half normal levels when CEF-RSV-SR-T5 are shifted from 41° to 36°C. The low enzyme activity in CEF-RSV-SR is not due to an alteration in the K m ATP or a change in the kinetics of Mg ++ activation, and is not observed when the enzyme is assayed in the presence of NaF. We conclude that transformation by RSV-SR reduces adenylate cyclase activity by a different mechanism than the Bryan high-titer strain of RSV.

Glucose and α-methyl glucoside repress β-galactosidase synthesis in wild type Escherichia coli and in mutant strains deficient in Enzyme I or in the heat-stable protein of the phosphoenolpyruvate-phosphotransferase system. Although the mutants do not grow on glucose and accumulate only a small amount of α-methyl glucoside, they are more sensitive to repression by these compounds than are their parent strains. This repression is presumably due to the lowering of the intracellular concentration of cyclic 3',5'-AMP by glucose and α-methyl glucoside, since it can be prevented by addition of the nucleotide. In contrast, a mutant deficient in glucose Enzyme II activity was resistant to repression by glucose and α-methyl glucoside. Evidently, the repression of β-galactosidase synthesis by these sugars does not require phosphorylation by the P-enolpyruvate-phosphotransferase system. It does, however, require the presence of Enzyme II activity for the sugars. An Enzyme I mutant and a heat-stable protein mutant which did not grow on lactose would grow on lactose in the presence of cyclic 3',5'-AMP or of isopropylthio-β-d-galactoside. Therefore, an intact P-enolpyruvate-phosphotransferase system is not required for lactose utilization by E. coli.

Myosin has been isolated from cloned mouse fibroblasts, line L-929. Fibroblast myosin: ( i ) binds to rabbit muscle actin and is dissociated from it by ATP, ( ii ) has an ATPase activity that is suppressed by Mg 2+ in 0.6 M KCl and is activated by rabbit muscle actin in the presence of Mg 2+ in 14 mM KCl, ( iii ) forms thin bipolar aggregates in 0.1 M KCl when viewed in the electron microscope, ( iv ) possesses a heavy chain with the same mobility as muscle myosin (molecular weight 200,000) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In these respects, fibroblast myosin appears to be similar to muscle myosin in structure and function.

2-mercapto-1(β-4-pyridethyl)benzimidazole (MPB) inhibited the development of antiviral activity induced by interferon in chick cells. The MPB effect was reversed by washing the cells. The action of MPB was on a step immediately following interferon binding but before the cellular RNA and protein synthesis that appear to be required for interferon action. The step blocked by MPB possibly involves cellular phospholipid synthesis.