Divalent cations and cytochalasin B-sensitive processes in activation,synthesis, and release of mixed leukocyte reaction suppressor factor

We have examined the possible rotes of divalent cations and of cytochalasin B-sensitive processes in the production of mixed leukocyte reaction suppressor factor (MLR-TsF). Ca but not Mg was strictly required for MLR-TsF production. A requirement for Mg could be demonstrated only under conditions of Ca depletion, suggesting an intimate linkage in divalent cation requirements. Ca was required for synthesis as well as release of the suppressor factor. Cytochalasin B inhibited MLR-TsF production only when present during the first 4 hr of culture; such inhibition was reversible with incubations of up to 16 hr. This indicated that cytochalasin B inhibited early events of activation, rather than synthesis or release of MLR-TsF. It was found that both Ca and Mg were required either before or during the cytochalasin-sensitive steps in the activation sequence. The relationship of cytochalasin B-sensitive events in early activation to the antigenic stimulus per se was established by limited exposure of suppressor cells to allogeneic stimulators fixed to poly-l-lysine (PLL)-coated plates. Maximal stimulation was observed with a 4-hr exposure, and this antigen-dependent commitment was cytochalasin B sensitive.     

Mechanism of lymphocyte activation. II. Requirements for macromolecular synthesis in the production of lymphokines

Reversible inhibitors of protein synthesis, cycloheximide and puromycin, and an irreversible inhibitor of RNA synthesis, actinomycin D, were employed to study the kinetics and types of macromolecular synthetic events required for the production of migration inhibitory factor (MIF) and macrophage activating factor (MAF) by Con A-stimulated lymphocytes. Reversible inhibition of protein synthesis during the first 2 hr of stimulation completely inhibited MIF and MAF production. The same treatment, performed 4 hr after the beginning of the stimulation, had no effect. When the inhibitors of protein synthesis were left in the cultures, a block of lymphokine production was observed when the drugs were added at 6 hr as well as at time 0. In contrast, irreversible inhibition of RNA synthesis at 6 hr was ineffective and only treatment at the beginning of culture blocked lymphokine production. These data suggest that a critical protein is synthesized during the first few hours of stimulation, which is required for subsequent production of lymphokines. After this special early requirement, however, continued protein synthesis is needed for lymphokine production. In contrast, the RNA required for MIF and MAF production seemed to be completely synthesized within 4 to 6 hr of stimulation. The possibility that suppressor macrophages inhibit lymphokine production via modulation of macromolecular synthesis is discussed.     

Functional analysis of antigen-nonspecific T-cell suppression. I. Effect of mitogen-induced T suppressor cells on helper-T-cell clones

The effect of mitogen-induced nonspecific suppressor T cells (Ts)2 on T-helper-cell activity was investigated using isolated clones of murine T-helper cells as targets. TNP-self-reactive Thy1+, Ly1+ T-cell clones were isolated after continuous culture of T cells derived from picryl chloride-sensitized mice and were characterized by their ability to proliferate in an antigen-specific and MHC-restricted manner. In addition, selected T-cell clones were found to produce both interleukin-2 (Il-2) and T-cell replacing factor (TRF), lymphokines characteristic of helper T cells. Concanavalin A (Con A)-induced Ts cells inhibited the antigen-specific proliferation of these helper-T cell clones in a noncytotoxic manner even in the presence of exogenous Il-2. This implied that failure to proliferate was not merely due to an inability of these clones to produce Il-2. The kinetics of suppression also suggested that early T-cell activation signals were not affected. Furthermore, coculture experiments indicated that while proliferation could be severely inhibited, the actual secretion of lymphokines such as Il-2 and TRF by the T-helper clones was not. Our data suggest that nonspecific Ts modulation of proliferation versus helper factor production are under separate control in cloned T-cell populations, with lymphokine secretion remaining intact in the presence of Con A-induced Ts cells.     

Stimulated rat T cell-derived inhibitory factor for cellular DNA synthesis (STIF). III. Effect on cell proliferation and immune responses

Stimulated T cell-derived inhibitory factor for cellular DNA synthesis (STIF), a lymphokine produced from concanavalin A (Con A)-stimulated rat suppressor T cells, was examined for its inhibitory effect on various cultured cells and on in vitro immune reactions. STIF could inhibit the DNA synthesis of a variety of normal and neoplastic cells from rats, mice, and humans in a dose-dependent fashion. Kinetics studies revealed that STIF selectively inhibited cellular DNA synthesis after incubation for 12 hr, but after 36 hr, it also inhibited RNA and protein syntheses. The inhibited cellular DNA synthesis by 12-hr incubation with STIF was recovered after culturing the cells in STIF-free medium. The inhibitory effect of STIF on the DNA synthesis was not blocked by addition of a sugar (alpha-methyl-D-mannoside, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, L-fucose, or L-rhamnose) in culture, as determined by using rat bone marrow cells. STIF inhibited proliferative responses of rat lymphocytes to T cell mitogens, Con A and phytohemagglutinin, and a B cell mitogen, lipopolysaccharide, as well as IL 2-dependent growth of cloned T572 cells. It could also inhibit both blastogenesis and cytotoxic T cell generation in allogeneic mixed lymphocyte reaction. The release of IL 2 from Con A-stimulated T cells was also inhibited by the added STIF in culture, as demonstrated from the finding that IL 2 activity was not detected in the supernatants even after an anion-exchange column chromatography. These results indicate that STIF could inhibit cellular DNA synthesis in a species-unrestricted manner and thus inhibits the proliferation of various normal and neoplastic cells, and that it could also inhibit lectin- or IL 2-dependent T cell proliferation as well as IL 2 production from T cells in in vitro immune reactions.     

Calcium-antagonists inhibit secretion of very-low-density lipoprotein from cultured rat hepatocytes.

The effects of different calcium-antagonists on secretion of very-low-density lipoprotein (VLDL) from cultured rat hepatocytes were examined. Verapamil (an inhibitor of voltage-dependent calcium channels) and EGTA (a calcium chelator) decreased VLDL-triacylglycerol secretion in a concentration-dependent manner, with maximum inhibition (about 90%) at 0.2 mM-verapamil and 5 mM-EGTA. Inorganic calcium-antagonists such as lanthanum, nickel, cobalt and manganese decreased secretion of VLDL-triacylglycerol by 55-95%, whereas the calcium-agonist barium did not affect secretion. Inhibition of VLDL-triacylglycerol secretion appeared within 30 min, without inhibition of triacylglycerol synthesis. Pulse-chase experiments revealed that verapamil and cobalt inhibited the secretory pathway itself. Cobalt showed a concentration-dependent inhibition of VLDL-triacylglycerol secretion, with maximal effect at 8 mM. Although inhibition by cobalt was not completely reversible, Trypan Blue exclusion and lactate dehydrogenase leakage indicated that the hepatocytes were not injured by cobalt or any of the other calcium-antagonists tested. Inhibition of protein synthesis by cycloheximide did not affect triacylglycerol secretion (up to 2 h), and the observed effects were therefore probably not due to impaired production of apolipoproteins. Taken together, these results suggest that calcium is important for secretion of VLDL particles.     

Quantitative Analysis of Protein Synthesis Altered by Estrogen in Cultured Xenopus Liver Parenchymal Cell

Using the primary culture system of male Xenopus laevis hepatocytes consisting of more than 95% parenchymal cells, the effect of estradiol-17 β (10⁻⁶M) on protein synthesis was quantitatively analyzed by ³H-leucine incorporation kinetics and the estimation of specific radioactivity of newly synthesized secretory protein. The cells in a well defined culture revealed high plating efficiency and very low DNA synthetic activity. The cultured cells could synthesize several secretory proteins containing serum albumin. The pattern of secreted proteins in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) did not alter with culture time but secretion rate of protein increased for 7 days, starting on the third day following inoculation. Estradiol added to the culture media extensively induced the synthesis of yolk precursor protein vitellogenin which accounted for 40–50% of the overall secretory protein synthesis and 20–30% of the total protein synthesis on day 7 of estradiol treatment. Ultimately, the total protein synthesis and secretory protein synthesis were stimulated 1.2–1.3 fold and 2.0–2.2 fold, respectively, over those of the control cells cultured in the absence of estradiol. These results indicated that the stimulation of protein synthesis was largely due to vitellogenin production. Such an estradiol-dependent stimulation of protein synthesis was also detected in the low molecular weight protein(s). On the other hand, albumin synthesis was evidently reduced by estradiol. Thus, estradiol had two different effects on protein synthesis.
The results obtained in this study will be discussed in relation to the findings o in vivo experiments.     

Phorbol esters specifically inhibit induction of immunoglobulin secretion in a murine B cell leukemia

We have examined the effect of tumor-promoting phorbol esters such as phorbol myristate acetate (PMA) on the murine B cell leukemia BCL-1 and its in vitro adapted derivative CW.13.20. Phorbol esters, including PMA and phorbol dibutyrate (PDBu), were potent inhibitors of BCL-1 IgM secretion induced by either LPS or lymphokines; half-maximal inhibition was obtained with 0.1 nM PMA and 0.8 nm PDBu. The inhibitory action of PDBu on BCL-1 cells was reversible for over 1 hr, but after 5 hr 70% of the inhibition was irreversible. Irreversible inhibition could be blocked by cycloheximide, suggesting a requirement for protein synthesis. The specificity of PDBu inhibition was examined by comparing the patterns of protein synthesis in PDBu-treated and control BCL-1 cells. Total incorporation of [35S]methionine into protein by BCL-1 cells cultured in the presence of PDBu was similar to that of untreated cells. Analysis of radiolabeled proteins by SDS-PAGE and autoradiography revealed no consistent changes in the pattern of protein synthesis except at those positions corresponding to the heavy and light chains of IgM. Immunoprecipitation with an affinity-purified anti-IgM indicated that PDBu inhibited the increased synthesis of heavy and light chain that follows stimulation by lymphokine but did not diminish control IgM synthesis. Induced IgM secretion from CW.13.20 cells was also inhibited by phorbol esters, indicating a direct action on B cells. Delaying the addition of phorbol ester relative to lymphokine or LPS by 24 hr significantly reduced inhibition of induced IgM secretion from both BCL-1 and CW.13.20 cells. This suggests that phorbol esters specifically interfere with the signal for induction of IgM secretion by both lymphokine and LPS.     

Dibasic cleavage site is required for sorting to the regulated secretory pathway for both pro- and neuropeptide Y

To investigate the signals governing routing of biologically active peptides to the regulated secretory pathway, we have expressed mutated and non-mutated proneuropeptide Y (ProNPY) in pituitary-derived AtT20 cells. The mutations were carried out on dibasic cleavage site and or ProNPY C-terminal sequence. Targeting to the regulated secretory pathway was studied using protein kinase A (8-BrcAMP), protein kinase C (phorbol myristate acetate) specific activators and protein synthesis inhibitor cycloheximide, and by pulse chase. The analysis of expressed peptides in cells and culture media indicated that: neuropeptide Y (NPY) and ProNPY were differently secreted, whilst NPY was exclusively secreted via regulatory pathway; ProNPY was secreted via regulated and constitutive-like secretory pathways. ProNPY secretion behaviour was not Proteolytic cleavage efficiency-dependent. The dibasic cleavage was essential for ProNPY and NPY cAMP-dependent regulated secretion and may have function as a retention signal.     

Suppressor T cell growth and differentiation: production of suppressor T cell differentiation factor by the murine thymoma BW5147

Previous studies have identified a lymphokine, termed Ts differentiation factor (TsDF), in primary MLR supernatants that induces effector function of alloantigen-primed MLR-Ts. The present report describes constitutive production of TsDF by the murine thymoma BW5147, and its use to analyze alloantigen and TsDF requirements for MLR-Ts activation to TsF production. Serum-free supernatants of BW5147 restored the capacity of MLR-TsF production to alloantigen-primed MLR-Ts cultured with glutaraldehyde-fixed allogeneic stimulator cells, and were not themselves directly suppressive in the MLR assay. BW5147 supernatant induced MLR-TsF production from primed L3T4-Ly2+ MLR-Ts in the absence of concomitant proliferation, suggesting that the function of BW5147 supernatant, like that of MLR-derived TsDF, is a differentiative rather than a proliferative one, and is required for the synthesis or release of TsF. The differentiative activity of BW5147 supernatant was associated with a molecular species of approximately 14,500 m.w. by HPLC fractionation and was expressed independently of detectable IL 2, IL 3, IFN-gamma, and IL 1. The functional activity of BW5147 supernatant has therefore been provisionally designated BW5147-derived Ts differentiative factor, or BW-TsDF. By using BW-TsDF, it was demonstrated that MLR-Ts fail to respond to TsDF in the absence of, or preceding, reexposure to priming alloantigen. Instead, alloantigen binding by primed MLR-Ts appears to create a transient state of TsDF responsiveness. Primed MLR-Ts were fully sensitive to delayed addition of TsDF for approximately 12 hr after reexposure to alloantigen, but became TsDF-unresponsive within 24 to 36 hr. MLR-Ts cultured alone for 36 hr were fully responsive to the combined addition of TsDF and alloantigen. Thus, MLR-Ts activation to TsF release requires the sequential events of specific alloantigen binding, which induces a TsDF-responsive state, followed by interaction with TsDF. The transience of induced TsDF responsiveness suggests a precise mechanism for control of antigen-initiated Ts activation to effector function.     

Regulated and constitutive secretion. Differential effects of protein synthesis arrest on transport of glycosaminoglycan chains to the two secretory pathways.

Many neural and endocrine cells possess two pathways of secretion: a regulated pathway and a constitutive pathway. Peptide hormones are stored in granules which undergo regulated release whereas other surface-bound proteins are externalized constitutively via a distinct set of vesicles. An important issue is whether proper function of these pathways requires continuous protein synthesis. Wieland et al. (Wieland, F.T., Gleason, M.L., Serafini, T.A., and Rothman, J.E. (1987) Cell 50, 289-300) have shown that a tripeptide containing the sequence Asn-Tyr-Thr can be glycosylated in intracellular compartments and secreted efficiently from Chinese hamster ovary and HepG2 cells, presumably via the constitutive secretory pathway. Secretion is not affected by cycloheximide, suggesting that operation of this pathway does not require components supplied by new protein synthesis. In this report we determined the effects of protein synthesis inhibitor on membrane traffic to the regulated secretory pathway in the mouse pituitary AtT-20 cells. We examined transport of glycosaminoglycan chains since previous studies have shown that these chains enter the regulated secretory pathways and are packaged along with the hormone adrenocorticotropin (ACTH). We found that cycloheximide treatment severely impairs the cell's ability to store and secrete glycosaminoglycan chains by the regulated secretory pathway. In marked contrast, constitutive secretion of glycosaminoglycan chains remains unhindered in the absence of protein synthesis. The differential requirements for protein synthesis indicate differences in the mechanisms for sorting and/or transport of molecules through the constitutive and the regulated secretory pathways. We discuss the possible mechanisms by which protein synthesis may influence trafficking of glycosaminoglycan chains to the regulated secretory pathway.     

Control of mitogen-induced transformation: characterization of a splenic suppressor cell and its mode of action.

The present study demonstrates that mouse spleen cells contain a population of glass wool adherent T lymphocytes which exhibit the capacity to suppress non-glass adherent lymphocyte responses to mitogens. These suppressor cells are stimulated by both low and high doses of PHA¹ and high doses of con A. The suppressor cell effect is observed when UNA, but not RNA or protein synthesis, is studied. This glass-adherent suppressor cell population is characterized as being the primary DNA synthesizing cells during the early (0–8 hr) stages of culture. Suppression still occurs when the suppressor cells are treated with mitomycin C, actinomycin D or cycloheximide. This implies that new macromolecular synthesis may not be required for suppression to occur. Suppression is blocked by inhibiting synthesis of prostaglandin and is mimicked by Bu₂cAMP. This suggests that mitogen activated suppressor cells regulate T cell responses via production of prostaglandin which modulates the concentration of intracellular cyclic nucleotide levels.     

Effect of lymphokines on the production of cytostatic protein factors from human monocytes

The effect of activating human monocytes in vitro with lymphokines on the production of cytostatic protein factor(s) (CF) was investigated. Upon exposing the monocytes to either lymphokines or lipopolysaccharide (LPS) the amount of CF released was increased approximately fivefold compared to the amount released from unexposed monocytes. With sequential lymphokine and LPS treatment CF release increased nearly 10-fold. Even 10 min lymphokine activation before LPS exposure enhanced CF production significantly. The enhanced CF production was detected between 5 and 10 hr after lymphokine activation. The RNA synthesis inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide reduced the lymphokine-induced CF production in a dose-dependent manner, indicating that lymphokines augment both CF mRNA and CF protein synthesis. When monocytes were exposed to LPS on both Day 2 and Day 4 of culture, the amount of CF obtained on Day 4 was reduced compared to that obtained on Day 2. A significant increase in CF production, however, was observed when the monocytes were activated with lymphokines before the second exposure to LPS on Day 4, supporting the view that lymphokines initiate synthesis of CF in monocytes. Upon ion exchange chromatography, chromatofocusing, and gel filtration the same elution profiles of CF were obtained irrespectively of whether the monocytes had been activated with lymphokines or not. This indicates that lymphokines induce an increased production of the same factor(s) which was obtained in the absence of lymphokines.     

Alterations in albumin secretion and total protein synthesis in livers of thyroidectomized rats.

Perfused rat livers and isolated rat hepatocytes exhibited a 50% decrease in the secretion of both albumin and total secretory proteins after thyroidectomy. In contrast, synthesis of non-secretory proteins was decreased by only 20% from the rates observed in liver preparations from euthyroid rats. These observations suggested a disproportionate effect of thyroidectomy on the synthesis of secretory proteins compared with non-secretory proteins. Disproportionate decreases in the synthesis of albumin in other endocrine-deficient states such as hypophysectomy and diabetes had previously been shown to be associated with decreases of similar magnitude in the relative abundance of albumin-mRNA sequences. In contrast, thyroidectomy did not affect the activity or amount of albumin mRNA in total liver poly(A)-containing RNA when assayed by cell-free translation and by hybridization with complementary DNA, respectively. Furthermore, labelling experiments in vivo demonstrated that albumin synthesis represented 12.9 +/- 0.5% and 12.4 +/- 0.4% of total protein synthesis in livers of thyroidectomized and euthyroid rats respectively. Therefore the fall in secretion of albumin and total secretory protein after thyroidectomy did not appear to be a reflection of disproportionate decreases in the synthesis of these proteins. Instead, defects in steps involved in the post-synthetic processing and secretion of albumin are suggested. A number of comparisons, including ribosome half-transit times, the size distributions of total and albumin-synthesizing polyribosomes, and the fraction of RNA present as inactive ribosomes, provided evidence that the overall decrease in protein synthesis after thyroidectomy was not due to generalized alterations in translational processes. Instead, the decrease in total protein synthesis appeared to reflect the RNA content of the liver, which fell in proportion to th decrease in protein synthesis.     

Protein synthesis involvement in regulating pituitary-induced progesterone levels in ovarian follicles of Rana pipiens

Involvement of protein synthesis in frog pituitary homogenate (FPH)-induced progesterone production and/or accumulation in ovarian follicles was investigated. In amphibians, cycloheximide (C), an inhibitor of protein synthesis, inhibits progesterone and FPH-induced germinal vesicle breakdown (GVBD). However, the site and mechanisms of action of cycloheximide within ovarian follicles have not been elucidated. Intrafollicular progesterone produced by FPH is considered to mediate oocyte maturation; thus, cycloheximide may interfere with production and/or action of progesterone. Simultaneous treatment of FPH-stimulated follicles with cycloheximide inhibited FPH-induced progesterone accumulation (measured by RIA) and the accompanying-GVBD in a dose-dependent fashion. Inhibitory effects of cycloheximide on either FPH-induced progesterone production or GVBD were not reversed when follicles were washed and returned to fresh medium devoid of FPH and cycloheximide. However, subsequent restimulation of washed follicles with FPH resulted in increased progesterone levels and oocyte maturation. The extent of reversibility, in terms of GVBD and progesterone production, after FPH restimulation varied between animals. Pretreatment of follicles with cycloheximide for 6 hours, without FPH, had little or no effect on progesterone production when follicles were washed and treated with FPH. Delayed addition of cycloheximide to follicles following FPH stimulation blocked further progesterone accumulation as indicated by measurement of intrafollicular progesterone at the time of cycloheximide addition and at the end of the incubation period. The results indicate that cycloheximide rapidly inhibits progesterone production and that continuous protein synthesis is required for progesterone accumulation. Furthermore, protein synthesis does not appear to be required for progesterone metabolism since intrafollicular progesterone declined with prolonged culture even in the presence of cycloheximide. The nature of protein(s) involved in follicular progesterone production remains to be elucidated. FPH mediation of oocyte maturation within ovarian follicles appears to depend upon protein synthesis in somatic follicle cells, which is required for progesterone production, and in the oocyte, to mediate the response to the steroid trigger.     

Cellular and antigenic requirements for production of mixed leukocyte reaction suppressor factor

When murine spleen cells, alloantigen-sensitized previously in vivo, are incubated with spleen cells bearing the sensitizing alloantigens, a supernatant factor is produced that inhibits 3H-thymidine incorporation by responding lymphocytes in the mixed leukocyte reaction. This study evaluates the cellular and antigenic requirements during restimulation for elaboration of this suppressor factor, MLR-TsF. BALB/c spleen cells, sensitized to C57BL/6 (B6) alloantigens in vivo, produced MLR-TsF when cultured with B6 spleen cells in vitro, despite depletion of Sephadex G-10-adherent cells from factor-producing cells, stimulator cells, or from both populations. T cells were not required within the stimulating population, but a requirement for viable stimulator cells was demonstrated when heat-killed or glutaraldehyde-fixed stimulator cells failed to induce MLR-TsF production. The alloantigenic requirements for MLR-TsF production were addressed by 2 approaches. Treatment of stimulator cells with appropriate anti-I region antisera and complement did not affect MLR-TsF production, demonstrating that an absolute requirement for cells expressing I region determinants did not exist. However, spleen cells primed against entire H-2 haplotype differences produced significant quantities of MLR-TsF when they were restimulated with spleen cells homologous to the priming cells in only the I region, in the K and D regions, or in the D region alone. The additive nature of subregion-specific restimulation suggests that distinct subpopulations of K, I, and D region-specific MLR-Ts comprise the MLR-Ts population primed to entire H-2 haplotype differences.     

Interleukin-1 beta stimulates decidual stromal cell cyclo-oxygenase enzyme and prostaglandin production.

The cytokine interleukin-1 (IL-1 beta) increased prostaglandin production by decidual stromal cells in culture in a time and dose dependent manner. Optimum conditions for stimulation were found to be for 24 hours at a concentration of 100 pg IL-1 beta/ml. An apparent increase in cyclo-oxygenase enzyme synthesis accompanied the increase in prostaglandin production, and both changes were inhibited by the protein synthesis inhibitor cycloheximide. This implicates protein synthesis in the stimulatory effects of IL-1 beta, which may be mediated through the increase in cyclo-oxygenase enzyme. A pre-incubation period of 72 hours was found to be necessary to observe the stimulatory effect of IL-1 beta on prostaglandin production, but this did not seem to be due to any change in the sensitivity of the cells to IL-1 beta; the increase in the number of cyclo-oxygenase positive cells was the same if IL-1 beta was added on day 1, day 2 or day 3 of culture, even though prostaglandin production was not stimulated on day 1 or day 2. Cycloheximide increased prostaglandin production on the first two days of culture and had no effect on the third day of culture. This was interpreted as indicating that a factor inhibiting cyclo-oxygenase activity was synthesised during the initial period of culture, which prevented any increase in prostaglandin production following the increase in enzyme synthesis.     

DNA synthesis in polyoma virus infection. V. Kinetic evidence for two requirements for protein synthesis during viral DNA replication.

Protein synthesis in polyoma virus-infected cells was inhibited by 99% within 4 min after exposure to 10 mug of cycloheximide per ml. Subsequent to the block in protein synthesis, the rate of viral DNA synthesis declined via inhibition of the rate of initiation of new rounds of genome replication (Yu and Cheevers, 1976). This process was inhibited with complex kinetics: within 15 min after the addition of cycloheximide, the rate of formation of closed-circular viral DNA was reduced by about one-half. Thereafter, DNA synthesis in cycloheximide-treated cells declined more slowly, reaching a level of 10% of untreated cells only after approximately 2 h. Protein synthesis was also required for normal closure of progeny form I DNA: in the presence of cycloheximide, DNA synthesis was diverted from the production of form I to form Ic, a monomeric closed-circular DNA component deficient in superhelical turns (Yu and Cheevers, 1976). Form I is replaced by Ic with first-order exponential kinetics. It is concluded that at least two proteins are involved in the control of polyoma DNA replication. One is apparently a stoichiometric requirement involved in the initiation step of viral DNA synthesis, since this process cannot be maintained at a normal rate for more than a few minutes in the absence of protein synthesis. The second protein requirement, governing the closure of newly synthesized progeny DNA, is considered distinct from the "initiation" protein on the basis of the kinetic data.     

Effects of cycloheximide on chromatin biosynthesis.

In the presence of sufficient cycloheximide, puromycin or NaCl to quantitatively inhibit protein synthesis in HeLa cells, thymidine incorporation continues at 20% of control rates for 60 to 90 minutes, after which incorporation gradually ceases. Both DNA and protein synthesis revert to control rates in about five minutes after removal of cycloheximide.DNA synthesis in the presence of cycloheximide appears to be a continuation of the replicative process by several criteria. The persistent DNA synthesis in the presence of cycloheximide is abolished by hydroxyurea, which does not inhibit repair synthesis, while ethidium bromide, an inhibitor of mitochondrial DNA synthesis, is without effect. Nuclear DNA is not nicked during incubation in cycloheximide. Low molecular weight Okazaki fragments (4 to 5 S) are both synthesized and processed to high molecular weight DNA in cells treated with cycloheximide. Replication forks, identified in alkaline CsCl gradients by incorporation of bromodeoxyuridine as a density marker just before the addition of cycloheximide, are selectively labeled with radioactive thymidine during DNA synthesis.In the presence of cycloheximide the maturation of DNA intermediates into high molecular weight DNA is defective. All size classes of DNA fragments, normally present during progression of low to high molecular weight DNA, are demonstrable in cells preincubated in cycloheximide for prolonged periods. However, 21 S fragments, intermediate in size between Okazaki pieces and mature, high molecular weight DNA, accumulate in cells treated with cycloheximide, demonstrating a defect in maturation of the 21 S intermediates into high molecular weight DNA. After removal of the cycloheximide, the 21 S DNA fragments are processed to high molecular weight DNA at a significantly impaired rate, requiring about three hours for completion of chain growth as compared to 40 to 60 minutes in controls. The slowed growth of DNA fragments synthesized in the presence of cycloheximide following drug removal is not due to persisting effects of cyeloheximide since DNA synthesis immediately following removal of the drug has chain growth rates similar to that of controls.Pools of chromatin proteins exist in HeLa cells, as demonstrated by a brief, labeled amino acid pulse followed by a chase with cycloheximide. The specific activity of chromatin proteins increases significantly during 60 minutes of cycloheximide inhibition. Histone f2a1 accumulates preferentially during this chase period, suggesting that a supply of this highly conserved histone might be requisite to continued replication.Comparison of chromatin synthesized during cycloheximide treatment with pulse-labeled control chromatin has provided insight into the mechanism of assembly of proteins and DNA into the nucleoprotein complex. The DNA of ch-chromatin² is more susceptible to nuclease digestion than control chromatin, suggesting that it is deficient in protein content. Upon reversal of cycloheximide inhibition, the recovery of nuclease digestibility of ch-chromatin to control values takes two to three hours, a time similar to that required for conversion of the corresponding 21 S chDNA fragments to high molecular weight DNA. Briefly pulse-labeled (30 to 60 s) DNA in control chromatin also has an enhanced susceptibility to nuclease digestion of the same degree as found in ch-ehromatin. The time of recovery of increased nuclease susceptibility of newly made chromatin DNA (via protein addition) to control levels is about 10 to 15 minutes and corresponds to the time required for synthesis of replicon-sized units of DNA.In addition to being nuclease-sensitive, both cycloheximide and newly synthesized (30 to 60 s) chromatin have lighter buoyant densities in CsCl gradients than bulk chromatin. This property exists for only one to two minutes in controls and is probably due to structural properties distinct from those rendering nuclease sensitivity.Limit digests of chromatin by micrococcal nuclease yield a characteristic pattern of polynucleotides when resolved in polyacrylamide gels. The radioactivity profiles of limit digest polynucleotides from control and ch-chromatin are identical, indicating that pre-existing chromatin proteins remain in place on newly replicated DNA in the same fashion as in mature chromatin.