Acid-soluble Pool Research Articles

Uptake of nucleosides in density-inhibited cultures of 3T3 cells.

AN increase in the rate of nucleoside incorporation into acid-soluble pools is one of the earliest events seen when fibroblasts arrested in the G1/G0 or A state1 are stimulated to grow by the addition of serum2–5. The stimulation of uridine uptake is preceeded by a lag phase of several minutes6 and is not prevented by inhibitors of protein synthesis7. The uptake of nucleosides by mammalian cells seems to proceed in two steps: (1) the nucleosides are transferred rapidly across the plasma membrane by a facilitated diffusion mechanism8 and (2) the intracellular nucleosides are phosphorylated by specific nucleoside kinases and the nucleosides formed are trapped intracellularly9. Although the regulation of nucleoside uptake has been studied in some detail3–7, it is not known which of these two steps is activated by growth-promoting agents. We have investigated which step of nucleoside uptake is stimulated by serum in density-inhibited cultures of 3T3 cells. The results indicate that phosphorylation is the rate-limiting step in nucleoside uptake by quiescent cells. Growth stimulation by serum enhances the rate of phosphorylation, but apparently not that of transport.

Nerve-dependent regulation of absolute rates of protein synthesis in newt limb regenerates: Measurement of methionine specific activity in peptidyl-tRNA of the growing polypeptide chain

Limb regeneration in the newt requires the presence of nerve fibers at the amputation surface. Regeneration does not occur upon denervation. Incorporation of [ 35S]methionine into hot TCA-precipitable material (cpm/μg of protein) increased sharply in the first few hours after denervation, then declined rapidly and plateaued at about 60% of the innervated control side at 48 hr. The specific activity of methionine was determined from hydrolyzed peptides of peptidyl-tRNA of the growing peptide chain. The hydrolyzed peptides were reacted with 3H-labeled 1-fluoro-2,4-dinitrobenzene (FDNB), and the resulting DNP-methionine was isolated by two-dimensional thin-layer chromatography. Since the specific activity of [ 3H]DNP is given, the ratio of 35S to 3H in DNP-methionine is a measure of methionine specific activity. Our results show that nervous control does not affect the specific activity of methionine in the growing polypeptide chain; the absolute rate of protein synthesis is affected. In both denervated and innervated regenerates, methionine specific activity in nascent peptides of membrane-bound polysomes was approximately half that of free polysomes, suggesting the existence of two distinct intracellular pools which are utilized for protein synthesis. Moreover, our results indicate that the specific activity of methionine in the acid soluble pool is 2–5 times lower than that found in the nascent peptides. Therefore, the acid-soluble pool cannot serve as a precursor pool for protein synthesis. We also estimated the half translation time of the nascent peptide chains. At any given moment, the nascent chain on the polysomes could be assumed to be half synthesized. Half translation time was measured as the time needed to release the labeled nascent peptides into the supernatant fluid. Denervation did not significantly affect the half translation time of average mRNA.

Decreased utilization of [2- 14C]orotic acid for the synthesis of cytidine nucleotides in rat liver after administration of α-hexachlorocyclohexane

Administration of α-1,2,3,4,5,6-hexachlorocyclohexane (α-HCH) to rats decreased the utilization of [2- 14C]orotic acid for the synthesis of liver cytidine nucleotides. The specific radioactivities of uridine components of the acid-soluble pool and rRNA increased during the first hours of treatment with the drug. Later on the specific radioactivities of uridine nucleotides remained unchanged, while those of cytidine components decreased gradually. Administration of hydrocortisone increased the incorporation of labelled orotic acid into rRNA cytidylic acid.

Essential factors in the kinetic analysis of RNA synthesis in HeLa cells.

Further evidence of mRNA in HeLa cells with a half-life two hours or less is given. A kinetic model of RNA synthesis in HeLa cells is described in which equilibration of label occurs first into the acid soluble pool (evidence is given that this pool feeds RNA synthesis) and thence in nuclear and cytoplasmic molecules. The measured accumulation of label in nuclear and cytoplasmic poly (A) is examined with the model and parameters were found which are consistent with the quantitative transfer of nuclear poly(A) to the cytoplasm. The strengths and limitations of the model are discussed.

Mutants of mouse fibroblasts altered in the synthesis of cell surface glycoproteins. Preliminary evidence for a defect in the acetylation of glucosamine 6-phosphate.

We hve reported the isolation and preliminary biochemical characterization of two mutants of mouse fibroblasts selected for a decrease incell-to-substrate adhesion (Pouysségur, J., and Pastan, I. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 544-548). We attributed the adhesive defect of these mutants (AD6 and AD8) to the absence of iodinatable cell surface proteins. This study demonstrates that a defect in glycoprotein synthesis is the biochemical basis for the reduction in proteins exposed at the outer surface of the mutant cells. When D-glucosamine or L-fucose was used as radioactive precursor, analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a decrease in the labeling of all the glycoproteins of both clones. This decrease in glycoprotein labeling was not due to a defect in D-glucosamine uptake, since this precursor was taken up at a higher rate in the mutants than in the wild type. In spite of this high uptake, the rate of D-glucosamine incorporation into macromolecules was decreased by 60% and the carbohydrate content of membranes (mannose, galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid) from clone AD6 was diminished by 40 to 60%. When the various cell lines were labeled for 1 to 3 h with glucosamine and the acid-soluble pool analyzed, the wild type cells were found to accumulate UDP-N-acetylhexosamine as the major component. In contrast, clones AD6 and AD8 accumulated glucosamine 6-phosphate as the major component. This last finding suggests that there is a block in the N-acetylation of glucosamine 6-phosphate in both instants. This suggestion is supported by the finding that feeding the mutant cells 10 mM N-acetylglucosamine reverts them to the wild type phenotype (Pouysségur, J., Willingham, M. and Pastan, I. (1977) Proc. Natl. Acad. Sci. U.S.A., in pressy. In wild type cells all of the iodinatable proteins of the cell surface have the same mobility as the glycoproteins when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that the early block in glycoprotein synthesis accounts for the reduction in iodinatable surface proteins in the mutant cells.

Relationship between thymidine transport and phosphorylation in Novikoff rat hepatoma cells as analyzed by a rapid sampling technique

Incorporation of thymidine into Novikoff rat hepatoma cells was analyzed with a rapid sampling technique which allowed collection of 12 time points in 20 sec. Transport was a rapid, saturable, nonconcentrative process with a Km of about 85 micrometer. The intracellular thymidine pool was also rapidly labeled in cells which phosphorylated thymidine, that a group translocation process involving thymidine kinase can be ruled out. Under all conditions examined, phosphorylation, not the transport, of thymidine was the rate-determining step in its incorporation into the acid-soluble pool. Estimation of transport rates from total incorporation into cells which phosphorylate the substrate is invalid in this cell system and must be questioned in all instances.

Transport, Phosphorylation, and Toxicity of a Tricyclic Nucleoside in Cultured Novikoff Rat Hepatoma Cells and Other Cell Lines and Release of Its Monophosphate by the Cells 2

1,4,5,6,8-Pentaazaacenaphthylene-3-amino-1,5-dihydro-5-methyl-(5-14C)-1-beta-D-ribofuranysly (NSC-154020), a tricyclic 7-deazapurine nucleoside (TCN), was rapidly incorporated into the acid-soluble pool by cultured Novikoff rat hepatoma cells, mouse L-cells, HeLa cells, and HEp-2 cells, but little incorporation into nucleic acids occurred. More than 90% of the intracellular radioactivity was associated with the monophosphate (MP) of the substrate concentration followed normal Michaelis-Menten kinetics. Comparison of the kinetic constants for the uptake of adenosine and the TCN and of the inhibition of their uptake by each other suggests that both were transported by the same system (Michaelis constant approximately 6-10 muM) and with about the same efficiency. The TCN was also phosphorylated in a cellfree extract containing adenosine kinase activity at about the same rate as was adenosine, but not further phosphorylation of the analogue MP occurred. No significant deamination or degradation of the adenosine analogue to its base and ribose-1-phosphate was observed. TCN inhibited the replication of all four types of cells propagated in suspension culture; however, Novikoff cells were several times more sensitive than were the other three cell types, despite the finding that the TCN-MP, probably the main toxic principle, accumulated to about the same concentration in cells of all four lines. Complete inhibition of replication of Novikoff cells were several times more sensitive than were the other three cell types, despite the finding that the TCN-MP, probably the main toxic principle, accumulated to about the same concentration in cells of all four lines. Complete inhibition of replication of Novikoff cells and cell death occurred at concentration as low as 15 muM TCN. At these concentrations, TCN, within 2 hours of its addition, completely inhibited the incorporation of [14C]formate int0 nucleotides and nucleic acids of Novikoff cells. An inhibition of the denovo synthesis of purine and pyrimidines, however, was not the only toxic effect of the TCN since high concentrations of uridine, adenine, guanine, and hypoxanthine, either alone or combined, failed to prevent the inhibition of cell replication by TCN. Also, between 1 and 3 hours of treatment, 70-80% of the Novikoff cells fragmented into four to eight vesicles per cell. These fragments were impermeable to trypan blue, still exhibited some metabolic activity such as the phosphorylation of AMP and TCN, but failed to replicate when the drug was removed. No similar fragmentation was observed with the other cell lines. Novikoff and L-cells rapidly released TCN-MP into the culture fluid. After 4 hours of incubation, 70-100% of the total radioactivity in the medium was associated with the MP. Only a little TCN-MP was released from HeLa and HEp-2 cells. A TCN-resistant mutant of Novikoff cells failed to phosphorylate the analogue and was deficient in adenosine kinase.

DIATOM MINERALIZATION OK SILICIC ACID. I Si(OH)4 TRANSPORT CHARACTERISTICS IN NAVICULA PELLICULOSA

SUMMARYSilicic acid transport was studied in the photosynthetic diatom Navicula pelliculosa (Bréb.) Hilse using [68Ge] germanic acid (68Ge(OH)4) as a tracer of silicic acid (Si(OH)4). The initial uptake rate of Si(OH)4 was dependent on cell number, pH, temperature, light and was promoted by certain monovalent cations in the medium. Na+ was more effective than K+, whereas Li+ and NH+4 were ineffective at promoting uptake.Uncouplers and inhibitors of oxidative phosphorylation and of photophosphorylation reduced uptake by 40–99% of control values. Uptake was also especially sensitive to the sulfhydryl blocking agents at 10−5 M and to the ionophorous compound valinomycin (10−7 M) which inhibited uptake by 82%.The Si(OH)4 transport system displayed Michaelis‐Menten‐type saturation kinetics with kinetic parameters of KS= 4.4 p. mol Si(OH)4· 1−1, Vmax= 334 pmol Si(OH)4· 106 cells−1· min−1. Calculations of the acid soluble silicic acid pool size based on 60 s uptake at 20 μM Si(OH)4 suggested that intracellular levels of Si could reach 20 mM and as much as 5 mM could exist as free silicic acid, representing maintenance of a 250‐fold concentration gradient compared with the medium.Efflux from preloaded cells was dependent on temperature and the Si(OH)4 concentration of the external medium. In the presence of 100 μMM “cold” Si(OH)4, approximately 30% of the Si(OH)4 in preloaded cells was exchanged in 20 min.The initial uptake rate of Si(OH)4 in logarithmic phase cells was constant, but the uptake rate increased in a linear fashion for 6 h in stationary phase cells.These results suggest that the first step in silica mineralization by diatoms is the active transmembrane transport of Si(OH)4 by an energy dependent, saturable, membrane‐carrier mechanism which requires the monovalent cations Na+ and K+ and is sensitive to sulfhydryl blocking agents. Silicic acid transport activity also appears to be regulated during different growth stages of the diatom.

Effects of PHA on rapidly synthesized RNA of murine splenic lymphocytes

The treatment of mouse splenic lymphocytes with PHA resulted in a stimulation of uptake and incorporation of 3H-uridine into rapidly synthesized acid-insoluble RNA. Sucrose gradient centrifugation analyses showed these RNA molecules to be heterodisperse and non-rRNA in nature. When the increase in measured RNA synthesis mediated by PHA ∗, was analyzed according to the amount of radioactivity in the acid-soluble pools, there was little, if any, difference in the measured synthetic activity of treated and non-treated cells. This observation was supported by a comparable increase in the specific activity of both nuclear and cytoplasmic RNA of PHA-treated lymphocytes. Increases in 3H-uridine incorporation under these experimental conditions, appeared to be primarily due to an enhanced permeability of the cell to the radioactive precursor after the binding of the PHA. This alteration in permeability was not limited to the plasma membrane since proportional increases in the amounts of radioactivity were also measured in the intranuclear pools. Analysis of 3H-RNA by means of poly(U) Sepharose chromatography showed little change in the amounts of RNA-poly(A) present in the nucleus and the cytoplasm following PHA treatment.

Thymidine metabolism and DNA synthesis in Newcastle disease virus-infected cells

The inhibition of thymidine incorporation into DNA in Newcastle disease virus-infected cells has been studied. At 6 h after infection of L-929 cells at high multiplicity, transport of exogenous thymidine across the cell membrane was inhibited. The kinetics of this inhibition, decreased Vmax with no change in Km, suggest that there are fewer sites available for transport in infected cells. The conversion of thymidine to dTTP was not inhibited. Equilibrium of exogenous thymidine with the acid-soluble pool occurred more slowly and at a lower level of radioactivity than in uninfected cells, and there was a reduction in the rate of incorporation of exogenous thymidine into DNA. The reduction of incorporation into the pool and into DNA was proportionate. The size of total cellular dTTP pools was changed very little in infected cells. DNA synthesized in infected cells in the presence of [3H]BrdUrd had reduced incorporation of tritium but similar buoyant density to that from uninfected cells. The results show that Newcastle disease virus inhibits DNA synthesis directly and, in addition, decreases thymidine transport. Together these account for the overall decrease in thymidine incorporation into DNA of infected cells.

Stability of RNA synthesized by the mouse oocyte during its major growth phase

The RNA of growing mouse oocytes and ovulated ova was labeled by injection of a solution containing tritiated uridine into the ovarian bursa. The time course of incorporation into RNA by oocytes was followed by analysis of alkali-labile acid insoluble radioactivity and by autoradiography. The results show that most of the incorporation into RNA by growing oocytes takes place within one day of bursal injection of the precursor, reflecting the rapid fall of label in the acid soluble precursor pool. The RNA of growing oocytes of all sizes is unusually stable, at least 80% of the labeled RNA present two days after bursal injection being retained until ovulation 10 to 20 days later. The fraction of heterogeneous RNA in labeled RNA of ova was estimated as 20% by sucrose gradient analysis. It is likely that egg RNA is synthesized primarily during the period of oocyte growth one to three weeks before ovulation.

Utilization of exogenous pyrimidines as a source of nitrogen by cells of the yeast Rhodotorula glutinis

Uptake and intracellular transformation of pyrimidines supplying cells of the yeast Rhodotorula glutinis with nitrogen have been studied. The amine nitrogen of cytosine was found to be the easiest to utilize. The presence in the medium of inorganic ammonia along with cytosine had a slight effect on cytosine deaminase (EC 3.5.4.1) activity. The uracil produced entered into the nutrient medium with no fission break of the pyridmidine ring. In the absence of any other source of nitrogen, the cells of the yeast R. glutinis utilized nitrogen of the pyrimidine ring of oxypyrimidines. Catabolism of uracil followed the reductive pattern, with release of carbon dioxide; this was accompanied by synthesis of the key enzyme of pyrimidine catabolism, dihydrouracil dehydrogenase (EC 1.3.1.1), whose activity rose 10-fold. With thymidne as the sole source of nitrogen, the lag-phase growth of the yeast cells was maximum. Catabolism of the pyrimidine ring of thymine was possibly preceded by its transformation into uracil. With no source of nitrogen easily utilized, the uridine 5'-monophosphate content in the generally acid-soluble pool rose. Our discussion of the regulation of catabolism of exogenous pyrimidine bases by the yeast R. glutinis takes into account the fact that transformations of pyrimidine bases are determined by how easily the cells can use a particular base as a source of nitrogen.

Action of the Peptide Antibiotic Leucinostatin

Leucinostatin, an antimicrobial and antitumor antibiotic, was found to be cytotoxic to the murine leukemic cell line, L 1210. A complete inhibition of cell growth was achieved with 0.5 mug of the drug per ml. Leucinostatin showed membrane damage against L 1210 cells as well as mouse erythrocytes. L 1210 cells were partially protected from membrane damage by the addition of glucose to the medium. In contrast, no protective effect of glucose was observed with drug-induced hemolysis. The possible involvement of membrane lipid in the mechanism of action of leucinostatin is suggested by its action on liposomes. Liposomes prepared with lecithin, cholesterol, and an amphiphatic molecule, either dicetyl phosphate or stearylamine, were sensitive to leucinostatin and response to the drug was enhanced as the cholesterol level of liposomes was decreased. Changes in the net charge of the membrane did not show significant effects on leucinostatin action. In the presence of the antibiotic, on the other hand, protein synthesis in cells was markedly inhibited under conditions where the uptake of amino acids into an acid-soluble pool was not affected. However, leucinostatin did not exhibit any inhibitory effect on protein synthesis in a cell-free system from L 1210 cells. It is suggested that the inhibition of protein synthesis results from the initial interaction of the drug with membrane phospholipid.

ACTH and Brain RNA: Changes in Content and Labelling of RNA in Rat Brain Stem

The influence of synthetic ACTH1-24 and ACTH1-10 on rat brain RNA was studied. ACTH1-24 (5 IU/100 g s.c.) treatment resulted in a small decrease in labelling of brain stem RNA (-12%), measured 30 min after the injection of [5-3H] uridine (100 muCi; s.c.), without affecting the distribution of radioactivity of the acid-soluble precursor pool. Furthermore, a small and transitional reduction of total brain stem RNA was found. Similar treatment of adrenalectomized rats (36 h after surgery) resulted in a marked increase (+ 40%) in labelling of brain stem RNA, without affecting the distribution of radioactivity in the acid-soluble pool. In both intact and adrenalectomized rats, treatment with ACTH1-24 did not affect RNA of cortex cerebrum and cerebellum. ACTH1-10, a fragment of ACTH without corticotropic activity in vivo, exhibited no effect on brain stem RNA in either intact or adrenalectomized rats, nor did corticosterone (1 mg/100 g) affect adrenalectomized rats.

Cytidine nucleotide biosynthesis and the level of cytochrome P-450 in rat liver microsomes after administration of colchicine

Colchicine displays a biphasic effect on the biosynthesis of cytidine nucleotides in rat liver; an initial depression is followed by activation of synthesis. Depending on the duration of exposure to colchicine, the changes of the values of the ratio of specific radioactivity of cytosine to uracil in the acid-soluble pool and 3′-CMP to 3′-UMP of cytoplasmic ribosomal RNA are inversely proportional to changes of the levels of cytochrome P-450 in liver microsomes. The utilization of [2- 14C]orotic acid for the biosynthesis of DNA cytosine as affected by the exposure to colchicine reflects the changes in the specific radioactivity of the cytidine components of the acid-soluble pool. However, the maximal radioactivity in DNA thymine is reached under these conditions only at longer time intervals.

Differing effects of inducers of differentiation on the ribonucleotide pool sizes of friend leukemia cells

Friend leukemia cells differentiate when any one of a variety of cryoprotective agents are added to their suspension culture media. While dimethyl sulfoxide (DMSO), dimethylacetamide (DMA) and tetramethylurea (TMU) induce a comparable degree of erythroid maturation their effects on the acid soluble ribonucleotide pools differ. DMSO induced differentiation is accompanied by a 70–80% reduction in pool size while DMA and TMA cause only a 15–20% reduction. Furthermore, inhibition of DMSO-induced differentiation by BUdR does not prevent the DMSO-induced reduction in pool size. These data suggest that the decline in pool size is a toxic effect unrelated to differentiation and these effects should be considered when the biological effects of DMSO-induced differentiation are considered.

N6-(delta2-isopentenyl)adenosine, an inhibitor of cellular transport of uridine and cytidine.

N6-(delta2-Isopentenyl)adenosine (IPAR) inhibited severely the incorporation of uridine and cytidine into S-180 cells in culture. When IPAR and the nucleosides were simultaneously present in the medium the inhibition was competitive (Ki 3.4 muM) and indicated inhibition of transport. However, the inhibition occurred even in the absence of extracellular IPAR if the cells had been preincubated with IPAR. Since 5'-IPAMP was the product which accumulated in large quantities in S-180 cells when incubated with IPAR, the effects of this AMP analog of the intracellular metabolism of uridine had to be considered. No direct correlation between the amount of intracellular IPAMP and the degree of inhibition of uridine utilization was observed and the relative distribution of uridine nucleotides in the acid soluble pool of the cells was unaltered in cells treated with IPAR. Also, IPAMP was not an inhibitor of uridine kinase in a cell free system nor was the activity of this enzyme affected by treatment of cells with IPAR. In addition, a profound inhibition of uridine utilization was also observed in a resistant subline of S-180 cells, which is unable to form IPAMP. These data suggest that IPAMP was not the inhibitory agent. Furthermore, the observation that the inhibition in both sensitive and resistant cells was caused even by a 15-second exposure to 100 muM IPAR, followed by rinsing, suggests that IPAR itself is the effective agent. It is concluded that IPAR exerts its inhibitory effect on uridine and cytidine utilization by becoming lodged in the cell membrane and thereby preventing the passage of these nucleosides into the cells. It is also shown that the inhibition of uridine and cytidine utilization by IPAR and by other potent nucleoside uptake inhibitors is unrelated to inhibition of growth or RNA-synthesis when the cells do not depend on an extracellular source of a nucleoside for growth.

RNA and Protein Synthesis during Progesterone-Induced Germinal Vesicle Breakdown in R. pipiens Ovarian Tissue

Rana pipiens oocytes with intact follicle cells were induced to undergo meiotic maturation in vitro under the influence of progesterone or related steroids or with pituitary extract. Uptake and incorporation of 3H-uridine and 14C-leucine were measured using pulse-labelling techniques. A transient increase in uridine incorporation (expressed as percent intracellular acid soluble pool) occurred 1–4 h after exposure to inducing levels of progesterone with both amino acid-containing and amino acid-free incubation media; but total incorporation was greatest in amino acid-containing medium. Nanomolar concentrations of a single amino acid such as leucine, tryptophan, or arginine (but not glycine) stimulated uridine incorporation into TCA precipitable material 8–10-fold in non-induced follicles, but had little effect on total uridine uptake. Hydrocortisone or testosterone also induced germinal vesicle breakdown, but did not stimulate uridine incorporation above the control level. A comparison of pulse-labelled RNA recovered in ovulated eggs with that in follicular tissue indicated that progesterone-induced RNA synthesis occurred primarily in the oocyte. Induction in Cal+-free Ringer's solution inhibited uridine incorporation more than 95%, with no effect on total uridine uptake and only 30% inhibition of germinal vesicle breakdown. Twenty μg/ml ethidium bromide inhibited uridine incorporation about 70% in non-induced follicles and only about 30% in progesterone-induced follicles, with no effect on germinal vesicle breakdown. 10–40 μg/ml actinomycin D did not significantly effect either uridine incorporation or germinal vesicle breakdown in non-induced or progesterone-induced follicles. These findings suggest that progesterone (or its metabolites) may act at a transcriptional as well as a post transcriptional level and confirm that the induction of germinal vesicle breakdown is post-transcriptional.

Diguanosine nucleotides of fungi that regulate RNA polymerases isolated and partially characterised

Three unusual phosphorylated diguanosine compounds called ‘hot spots’ HS-1, HS-2 and HS-3 (ref. LéJohn, H.B. Proc. Can. Fed. Biol. Soc. 18, 159, 1975) have been isolated as acid-soluble materials from several fungi, Achlya, Blastocladiella emersonii, Aspergillus niger and Rhizopus stolonifer in their vegetative phase. The nucleotides were purified from acid extracts of Achlya and Blastocladiella . The tentative structures of HS-3 and HS-2 determined are GppppG and GppppGp. HS-1 structure is still in doubt but it is related to HS-2. The structures were deduced from enzymatic digestion and UV analyses of the products, molar ratios of guanosine and phosphate, and chromatographic behaviour on PEI-cellulose. All three compounds accumulated in an inverse manner with rates of RNA synthesis and directly with rates of protein synthesis. The acid-soluble pools of the three compounds fluctuated during the life cycle of Achlya , and just prior to sporulation, were excreted into the medium. HS-2 was convertible to HS-3 by acid hydrolysis.

Macromolecular synthesis in T7 infected F′ cells

The abortive development of bacteriophage T7 in Escherichia coli cells carrying F′ factors has previously been attributed to lack of virus-directed modification of ribosomes in such cells. However, we find that 6–8 min after T7 infection of male bacteria there is an overall cessation of macromolecular synthesis, resulting in reduced production of DNA, late messenger RNA, and both early and late T7 proteins. Processing of an early viral protein has been detected in F − hosts; this processing is incomplete in F′ infection. The overall paralysis of macromolecular metabolism is correlated in time with a permeability change resulting in loss of the entire acid-soluble pool of phosphorus-containing compounds from the cells ( Britton and Haselkorn, 1975). Thus T7 development in F′ cells appears to be arrested at a particular time without regard to the developmental stage reached by T7 at that time.