Early Protein Synthesis Research Articles

Myeloid growth factor(s) regulation of ornithine decarboxylase: effects of antiproliferative signals interferon-gamma and cAMP

Colony-stimulating factors (CSFs) stimulate the activation and steady- state mRNA accumulation of an important regulatory enzyme for macromolecular synthesis, ornithine decarboxylase (ODC). Cloned murine CSF-dependent cell lines exhibited a rapid activation of ODC enzyme activity, detectable within ten minutes of stimulations with either interleukin-3 (IL-3), GM-CSF, or G-CSF. This early phase of enzyme activation did not require early protein or mRNA synthesis. The subsequent protracted rise in ODC activity occurring four to six hours after CSF treatment was dependent on increases in steady-state ODC mRNA accumulation and de novo protein synthesis. CSF, therefore, modulates both posttranslational activation of preexisting ODC and stabilization and accumulation of ODC mRNA. Antiproliferative signals, such as cAMP or interferon-gamma (IFN-gamma), effectively inhibited the CSF-directed increase in steady-state ODC mRNA. Cotreatment of the murine NSF 60.8 cell line with IFN-gamma and GM-CSF decreased steady-state ODC mRNA greater than 80% as compared with GM-CSF-treated cells alone. IFN treatment did not cause any appreciable destabilization of mature ODC mRNA, suggesting that its major effect may be at the level of ODC mRNA transcription or posttranscriptional processing. These data indicate that the ODC gene-protein system is an important molecular locus of the effects of myeloid proliferative and antiproliferation signals.

Myeloid Growth Factor(s) Regulation of Ornithine Decarboxylase: Effects of Antiproliferative Signals Interferon-γ and cAMP

Colony-stimulating factors (CSFs) stimulate the activation and steady-state mRNA accumulation of an important regulatory enzyme for macromolecular synthesis, ornithine decarboxylase (ODC). Cloned murine CSF-dependent cell lines exhibited a rapid activation of ODC enzyme activity, detectable within ten minutes of stimulations with either interleukin-3 (IL-3), GM-CSF, or G-CSF. This early phase of enzyme activation did not require early protein or mRNA synthesis. The subsequent protracted rise in ODC activity occurring four to six hours after CSF treatment was dependent on increases in steady-state ODC mRNA accumulation and de novo protein synthesis. CSF, therefore, modulates both posttranslational activation of preexisting ODC and stabilization and accumulation of ODC mRNA. Antiproliferative signals, such as cAMP or interferon-gamma (IFN-gamma), effectively inhibited the CSF-directed increase in steady-state ODC mRNA. Cotreatment of the murine NSF 60.8 cell line with IFN-gamma and GM-CSF decreased steady-state ODC mRNA greater than 80% as compared with GM-CSF-treated cells alone. IFN treatment did not cause any appreciable destabilization of mature ODC mRNA, suggesting that its major effect may be at the level of ODC mRNA transcription or posttranscriptional processing. These data indicate that the ODC gene-protein system is an important molecular locus of the effects of myeloid proliferative and antiproliferation signals.

Saturable binding sites mediate the entry of African swine fever virus into VERO cells

Binding experiments of 3H-labeled African swine fever virus to susceptible VERO cells have shown the presence of saturable binding sites for African swine fever virus on the plasma membrane. The Scatchard analysis of the binding data at equilibrium indicates the existence of about 10 4 cellular receptor sites per cell with a dissociation constant ( K d ) of 70 p M. Virus entry into VERO cells is mediated by a saturable component, since tritiated African swine fever virus saturable binding and uptake were competed by the same amounts of unlabeled virus. Similarly, early viral protein synthesis and virus production were inhibited by concentrations of uv-inactivated virus that competed virus attachment to saturable binding sites, suggesting that specific receptors mediate the entry of African swine fever virus particles that initiate a productive infection in VERO cells. African swine fever virus binding to virus-resistant L cells was not mediated by saturable binding sites. As a result of the nonsaturable interaction the virus was not able to enter L cells and neither early viral protein synthesis nor viral DNA synthesis was detected, indicating that the absence of specific receptors for African swine fever virus is a factor that determines the resistance of L cells to the infection.

Molecular events in the activation of murine macrophages.

Recent studies have identified some of the early molecular transductional events, which occur during the activation of murine macrophages. Our current evidence indicate a central role for protein kinase C for the priming effect of interferon-gamma (IFN gamma). IFN gamma also initiates Na+/H+ exchange and 45Ca efflux from murine macrophages (cascade I). Our data further indicate the involvement of multiple transductional pathways in the actions of bacterial lipopolysaccharide (LPS). Specifically, molecular events involved in the action of LPS include production of inositol phosphates and calcium mobilization as well as IFN gamma-regulated alterations in intracellular pH (cascade II). Our data further indicate that additional transductional events (e.g., synthesis of early or competence proteins) in response to LPS (cascade III) are also necessary for macrophage activation. Finally, regulation of important surface (e.g., Ia) and secreted molecules (TNF or IL-1) is exerted at the levels of both transcription and stabilization of specific mRNA in response to transductional cascades I, II and III. Taken together, the data indicate macrophage activation is complexly regulated at multiple levels.

Regulation of the temporal synthesis of proteins in bacteriophage BF23-infected cells.

Regulation of temporal synthesis of pre-early, early, and late proteins in bacteriophage BF23-infected cells has been studied by using five amber mutants defective in genes 1, 2, 10, 14, and 19. The synthesis of pre-early proteins is negatively regulated by the actions of gene 1, a pre-early gene. The switch from pre-early to early protein synthesis is mainly regulated by the second-step DNA transfer reaction, which is controlled by at least genes 1 and 2. Early proteins can be kinetically and genetically divided into two regulatory classes, designated Ea and Eb. The shutoff of Eb-early protein synthesis is associated with the turn-on of late protein synthesis. This step is controlled by genes 10, 14, and 19. Gene 10 also regulates negatively the synthesis of Ea-early proteins, indicating that this gene has a dual function in the regulation of early protein synthesis. The temporal synthesis of phage-encoded proteins is regulated mainly at the transcriptional level. Evidence is presented indicating that the host RNA polymerase is modified by the interaction with the gene products of genes 2, 10, and 14 (gp2, gp10, and gp14, respectively). gp2 interacts with the enzyme in the earlier stage of infection but is replaced by gp10 in the later stage. This exchange reaction depends on the presence of gp14 and gp19 and is related to the switch from Eb to late protein synthesis. Thus, the regulation of BF23 gene expression occurs in a coordinated manner throughout the development of this phage.

Relationships among genes and gene products of bacteriophage BF23

Twenty-five gene products of bacteriophage BF23 were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their functions were studied in relation to type I and II genes classified by means of genetic complementation tests. All the type I mutants were defective in the synthesis of a tail protein, L3. In addition, 4 type I gene products, L5 (gp21), L7 (gp20), L8 (gp29), and L9 (gp25), were identified as constituents of tails (gp21 denotes that a protein is a product of gene 21). Three type IIb mutants in genes 10, 14, and 19 diminished substantially the production of late proteins, including tail and head proteins, and the two other type IIb mutants in genes 1 and 2 were defective in the synthesis of both early and late proteins. Of 14 type IIa mutants, at least 6 were defective in phage DNA synthesis and 2 were defective in the synthesis of head proteins. The defect in the head donor activities of type IIa mutants in extract complementation tests was due to the failure of the formation of mature heads containing DNA. The above results support directly the results of the genetic characterization of BF23 genes.

Effects of Different Interferons on the Replication of Herpes Simplex Virus in Human T Lymphocytes

Human T cells were activated with PHA and after 48 h they were treated with preparations of different human interferons (IFNs). After a further incubation period of 24 h, the cells were washed and infected with herpes simplex virus type 1 (HSV-1). Reduced virus titers were observed in T cells pretreated with IFN-alpha or IFN-beta, whereas IFN-gamma showed no antiviral effects. These findings indicate that IFN-gamma does not exert protection against viral infection in this system. We investigated the synthesis of HSV-coded early and late proteins in T cells pretreated with IFN-alpha and IFN-beta. Immunofluorescence studies revealed inhibition of expression of the immediate early alpha-protein ICP 4. Induction of DNA-polymerase, a viral beta-protein, was inhibited both by IFN-alpha and IFN-beta in a dose-dependent manner. As suggested by SDS-polyacrylamide gel electrophoresis, other viral beta- and gamma-proteins of HSV were inhibited by IFN-alpha and IFN-beta as well.

The action of recombinant bovine interferons on influenza virus replication correlates with the induction of two Mx-related proteins in bovine cells

Recombinant bovine interferon-α and -γ differ in their action against influenza virus on bovine cells. Bovine IFN-α severely impairs early protein synthesis and replication of influenza virus in bovine cells in contrast to bovine IFN-γ which fails to induce an antiviral state against influenza virus. Otherwise the IFN system seems to function normally in bovine cells since both bovine IFN-α and -γ induce an antiviral state against vesicular stomatitis virus. The establishment of the specific antiviral state against influenza virus correlates with the induction by bovine IFN- a, but not γ, of two cytoplasmic proteins related to the IFN-induced mouse protein Mx involved in the mechanism of resistance of mice to influenza virus infection. This study suggests that bovines possess a system for resistance to influenza virus similar to the mouse Mx system.

Phenotypic characterization of temperature-sensitive mutants of vaccinia virus with mutations in a 135,000-Mr subunit of the virion-associated DNA-dependent RNA polymerase.

The phenotypic defects of three temperature-sensitive (ts) mutants of vaccinia virus, the ts mutations of which were mapped to the gene for one of the high-molecular-weight subunits of the virion-associated DNA-dependent RNA polymerase, were characterized. Because the virion RNA polymerase is required for the initiation of the viral replication cycle, it has been predicted that this type of mutant is defective in viral DNA replication and the synthesis of early viral proteins at the nonpermissive temperature. However, all three mutants synthesized both DNA and early proteins, and two of the three synthesized late proteins as well. RNA synthesis in vitro by permeabilized mutant virions was not more ts than that by the wild type. Furthermore, only one of three RNA polymerase activities that was partially purified from virions assembled at the permissive temperature displayed altered biochemical properties in vitro that could be correlated with its ts mutation: the ts13 activity had reduced specific activity, increased temperature sensitivity, and increased thermolability under a variety of preincubation conditions. Although the partially purified polymerase activity of a second mutant, ts72, was also more thermolabile than the wild-type activity, the thermolability was shown to be the result of a second mutation within the RNA polymerase gene. These results suggest that the defects in these mutants affect the assembly of newly synthesized polymerase subunits into active enzyme or the incorporation of RNA polymerase into maturing virions; once synthesized at the permissive temperature, the mutant polymerases are able to function in the initiation of subsequent rounds of infection at the nonpermissive temperature.

Reduced steady-state levels of vaccinia virus-specific early mRNAs in interferon-treated chick embryo fibroblasts

The molecular mechanism of interferon action on vaccinia virus-specific immediate early protein synthesis was studied in interferon-treated chick cells. In line with previous observations, the synthesis of total vaccinia WR virus-specific mRNA, thymidine kinase (TK) mRNA, and several other early mRNAs was detectable by short [ 3H]uridine pulses. Under conditions of over 90% inhibition of poxvirus-specific TK induction, accumulation of TK mRNA was strongly inhibited. Northern blot analysis revealed strong degradation of residual TK mRNA prepared from interferon-treated chick embryo fibroblasts (CEF). Blot hybridization analysis using total vaccinia DNA and restriction fragment N as probes demonstrated a generally reduced steady-state amount of vaccinia virus-specific early mRNAs in interferon-treated CEF. When CEF were infected with a recombinant vaccinia virus strain into the TK gene of which the chloramphenicol acetyltransferase gene had been inserted, CAT activity was far lower in interferon-treated than in untreated CEF. We conclude that signals that specify rapid breakdown of viral TK mRNA in interferon-treated CEF are located in the regions flanking the coding sequences of the viral TK gene.

Inhibition of T-lymphocyte activation by amiloride.

The T-lymphocyte activation process involves a series of coordinately coupled biochemical events occurring in response to antigen or mitogen. These events have not been completely characterized. The present studies investigate the mechanism of protein synthesis during the initial phase of T-cell activation. Among the early biochemical changes, induction of protein synthesis was observed as early as 10 minutes after mitogen stimulation of T-lymphocytes. This early protein synthesis was inhibited by cycloheximide but was insensitive to actinomycin-D, indicating the presence of preformed mRNA in resting lymphocytes. Since early protein synthesis parallels the increase in protein kinase C activity in activated T-lymphocytes, these two biochemical events may be related. In the present report, amiloride, an inhibitor of Na+/H+ antiport and protein kinase C, significantly inhibited [3H]leucine and [3H]thymidine incorporation in a dose-dependent manner into phytohemagglutinin (PHA)-stimulated T-lymphocytes. Furthermore, when T-lymphocytes were stimulated by phorbol myristate acetate, a known activator of protein kinase C, a similar inhibition of protein and DNA synthesis by amiloride was observed. The partially purified cytosol fraction isolated from PHA-activated T-lymphocytes showed a 75% decrease in protein kinase C-mediated [32P] incorporation from ATP in the presence of 100 microM amiloride. These results suggest that the T-cell activation process following exposure to mitogens involves early protein synthesis, which may be mediated by protein kinase C.

Maleyl-BSA and fucoidan induce expression of a set of early proteins in murine mononuclear phagocytes.

We examined the effect of maleyl-BSA on specific protein expression in murine peritoneal macrophages by radiolabeling treated macrophages with [35S]methionine followed by SDS-polyacrylamide gel electrophoresis. Such treatment induces the expression of a set of at least seven proteins (38, 42, 57, 65, 75, 80, and 85 kD). A similar set of proteins is also induced by treatment of macrophages with the algal polysaccharide fucoidan. The proteins resemble those induced in response to treatment of this same cell population with bacterial lipopolysaccharide (LPS), as judged by co-migration in both one- and two-dimensional electrophoresis. Two proteins induced by either LPS or maleyl-BSA (e.g., p57 and p85) show similar primary structure, as assessed by partial proteolytic peptide mapping confirming their identity. The induction of these proteins by maleyl-BSA is a transient phenomenon, being expressed as early as 1 hr after treatment and declining after 8 hr even in the continuous presence of the stimulus. The dose of maleyl-BSA required to induce the response varies to some extent with the protein in question, but agrees with the Kd for ligand-receptor binding. Chloroquine, which blocks the degradation of ligand, does not inhibit the induction of early protein synthesis. Whereas the induction of these proteins is blocked by inhibition of RNA synthesis with actinomycin D, the reversible inhibition of protein synthesis with cycloheximide during the induction phase does not prevent their expression. LPS, maleyl-BSA, and fucoidan previously have been shown to stimulate protease secretion and tumoricidal function in appropriately primed macrophages. The present findings now demonstrate that all three agents can also mediate the expression of early genes which may participate in the acquisition of functional competence.

Regulation of adenovirus gene expression in human WI38 cells by an E1B-encoded tumor antigen.

Adenovirus mutants carrying alterations in the gene encoding the E1B 19-kilodalton tumor antigen (19K protein) cause enhanced cytopathic effect (cyt phenotype) and the degradation of host-cell chromosomal DNA (deg phenotype) upon infection of human HeLa or KB cells. Furthermore, E1B 19K gene mutant viruses are defective for cellular transformation. We report that these mutant viruses possess a host-range phenotype for growth in human cells. In human HeLa cells the mutant viruses grew to the same levels as the wild-type virus, but they were severely defective for growth in KB cells. In human WI38 cells, the E1B 19K gene mutant viruses had a substantial growth advantage over the wild-type virus, yielding 500-fold-higher titers. Viral DNA synthesis was reduced 10- to 20-fold in WI38 cells infected with the wild-type virus relative to that synthesized by the E1B mutant viruses. Viral early and late protein synthesis was similarly reduced in wild type- relative to mutant-infected cells. These reduced levels of early gene expression in wild-type virus-infected cells were paralleled by comparably reduced levels of early cytoplasmic mRNA. The primary cause of this host-range phenotype appeared at the level of early gene transcription, since transcription of viral early genes in the mutant-infected cells was substantially greater than levels found in cells infected with the wild-type virus. These results implicate the E1B 19K tumor antigen in the regulation of adenovirus early gene expression. Specifically, the E1B 19K protein directly or indirectly exerts a negative effect on early gene transcription accounting for efficient gene expression from the E1B mutant viruses in WI38 cells. Based on these findings it is probable that the cyt and deg phenotypes observed in mutant-infected HeLa and KB cells are the result of the pleiotropic effect of this altered gene regulation.

Regulation of adenovirus gene expression in human WI38 cells by an E1B-encoded tumor antigen.

Adenovirus mutants carrying alterations in the gene encoding the E1B 19-kilodalton tumor antigen (19K protein) cause enhanced cytopathic effect (cyt phenotype) and the degradation of host-cell chromosomal DNA (deg phenotype) upon infection of human HeLa or KB cells. Furthermore, E1B 19K gene mutant viruses are defective for cellular transformation. We report that these mutant viruses possess a host-range phenotype for growth in human cells. In human HeLa cells the mutant viruses grew to the same levels as the wild-type virus, but they were severely defective for growth in KB cells. In human WI38 cells, the E1B 19K gene mutant viruses had a substantial growth advantage over the wild-type virus, yielding 500-fold-higher titers. Viral DNA synthesis was reduced 10- to 20-fold in WI38 cells infected with the wild-type virus relative to that synthesized by the E1B mutant viruses. Viral early and late protein synthesis was similarly reduced in wild type- relative to mutant-infected cells. These reduced levels of early gene expression in wild-type virus-infected cells were paralleled by comparably reduced levels of early cytoplasmic mRNA. The primary cause of this host-range phenotype appeared at the level of early gene transcription, since transcription of viral early genes in the mutant-infected cells was substantially greater than levels found in cells infected with the wild-type virus. These results implicate the E1B 19K tumor antigen in the regulation of adenovirus early gene expression. Specifically, the E1B 19K protein directly or indirectly exerts a negative effect on early gene transcription accounting for efficient gene expression from the E1B mutant viruses in WI38 cells. Based on these findings it is probable that the cyt and deg phenotypes observed in mutant-infected HeLa and KB cells are the result of the pleiotropic effect of this altered gene regulation.

Synthesis of heat shock proteins in rat brain cortex after transient ischemia.

Cell-free protein synthesis and two-dimensional gel autoradiography were used to characterize early postischemic protein synthesis in rat neocortex. Severe forebrain ischemia was induced for 30 min (four-vessel occlusion model) and followed by 3 h of recirculation. Polysomes were isolated from the cerebral cortex, translated in vitro in a reticulocyte system, and analyzed by two-dimensional gel electrophoresis. The translation products of postischemic polysomes included a major new protein family (70 kDa) with multiple isoelectric variants that was found to comigrate with the 68- to 70-kDa "heat shock" protein synthesized from polysomes of hyperthermic rats. Two other stress proteins (93 and 110 kDa) also appeared to be synthesized in increased amounts after ischemia. A complement of proteins that was indistinguishable from that of controls was also synthesized after ischemia, indicating that messenger ribonucleic acid coding for most brain proteins is preserved after ischemia and is bound to polysomes.

Two-dimensional analysis of african swine fever virus proteins and proteins induced in infected cells

Two-dimensional (2D) analysis of African swine fever (ASF) virus purified by Percoll gradient centrifugation resolves 54 structural proteins, 30 in conventional IEF gels and 24 in NEPHGE gels, while only 26 structural proteins are separated by SDS-PAGE. The two main bands separated by SDS-PAGE, with mol wt 150K and 72K, correspond to single spots in 2D gels. Other bands, including major bands of 38K, 35K, 24K, 17K, and 15.5K mol wt, correspond to multiple proteins of the same molecular weight but different p I. One hundred six virus-specific proteins were resolved by 2D analysis, 59 in conventional IEF gels and 47 in NEPHGE gels. Thirty-five of the virus-specific proteins are early proteins, synthesized before DNA replication, and the remaining 71 proteins are late proteins. Early proteins belong to two groups: 11 transient early proteins are synthesized only early in infection and the other 24 are persistent early proteins, synthesized at both early and late phases. Treatment with cytosine arabinoside prevents the synthesis of late proteins and blocks the shut-off of the synthesis of transient early proteins. Eleven structural proteins are major early proteins and 28 are late proteins. The remaining 15 structural proteins migrate in 2D gels like cellular proteins. Three of these cellular proteins, with mol wt 58K, 56K, and 45K were identified by immunoblotting as α-tubulin, β-tubulin, and actin, respectively.

Bacteriophage T4 regA protein. Purification of a translational repressor.

The bacteriophage T4 regA protein translationally regulates its own synthesis and the synthesis of several other T4 early proteins. In order to study the mechanism of translational regulation, we have purified the regA protein. Initially a mutant protein, incapable of autogenous repression, was placed under lambda PL transcriptional control and amplified to approximately 10% of total cell protein. The membrane-associated mutant protein was extracted with organic solvent mixtures and purified by reverse phase-high performance liquid chromatography. Polyclonal antibodies prepared against the mutant protein were used in Western blot assays to monitor purification of the wild-type protein from T4-infected cells. Phosphocellulose and poly(U)-agarose chromatography were important steps in its purification. The binding properties of regA protein to polyribonucleotides are discussed in relation to the mechanism by which the protein recognizes its mRNA targets.

Mapping the genes in the terminal redundancy of bacteriophage SPO1 with restriction endonucleases.

Although most early transcription from SPO1, a lytic DNA bacteriophage of Bacillus subtilis, is specified by the 12.6-kilobase region of the terminal redundancy, early genes from this region have not been identified by standard genetic means. We mapped genes to DNA regions of the SPO1 terminal redundancy by analyzing in vitro protein synthesis from isolated SPO1 restriction fragments in an Escherichia coli-coupled transcription-translation cell-free system. DNA from the terminal redundancy directs the synthesis in vitro of eleven proteins, e3, e4, e6, e7, e9, e12, e15, e16, e18, e20, and e21, which correspond in mobility on sodium dodecyl sulfate-polyacrylamide gels with authentic SPO1 early proteins. From their mapped positions on the DNA, genes were positioned downstream from most, but not all, of the twelve early promoter regions identified in vitro in the terminal redundancy. The temporal patterns of early protein synthesis in vivo suggest a differential turning on and off of early promoters in the terminal redundancy. Both in vivo and in vitro evidence suggests the existence of previously unidentified early promoter regions upstream from the genes for e6 and e4 as well as a middle promoter region upstream from the gene for e16.

Early protein synthesis in activated human peripheral blood mononuclear cells

Though B-cell division and Ig synthesis in response to pokeweed mitogen (PWM) require interaction with T-cells and monocytes, it is not clear which earlier events in B-cell activation share this requirement, and which are the result of direct interaction of mitogen with the B-cell. Having previously shown that the acceleration of lecithin synthesis in human B-cells at 16–20 hr requires both T-cells and monocytes, we now examine whether B-cells require similar interactions to increase their protein synthetic rate, another important activation event. At 21–24hr of PWM stimulation, the stimulation index (SI) for incorporation of [ 35S]methionine into protein was 2.1 ± 0.4 for unfractionated cells, 1.7 ± 0.1 for B-cells, 2.5 ± 0.1 for T-cells, and 3.4 ± 0.5 for monocytes. Thus monocytes contributed substantially to early mitogen-induced protein synthesis by human peripheral blood mononuclear cells. When the monocyte/B-cell fraction (MB) and T-cell fraction (T) were mixed at various ratios in PWM-stimulated cultures, synergy was apparent at MB:T ratios of 1:1 and 1:2, indicating cell interactions augmented early mitogen-driven protein synthesis in at least one of these cell types. However, much or all of this synergy could be attributed to T-cells, whose protein synthetic response was augmented by B-cells and monocytes. In contrast, the early increase in B-cell protein synthesis appeared to be independent of cell interactions, since their SI of 1.7 was not influenced by varying the proportion of M- or T-cells over a 50-fold range. These contrasting results between two contemporary events fits the hypothesis that one (accelerated phospholipid synthesis) requires a first signal plus one or more cell interaction signals, whereas the other (accelerated protein synthesis) requires only the first signal.

Modulation of the expression of interleukin 2 receptors of human thymocytes by recombinant interleukin 2

The effect of purified recombinant interleukin 2 on the expression of the receptors for interleukin 2 by human thymocytes was examined. Interleukin 2 augmented the expression of interleukin 2 receptors and interferon-γ synthesis by thymocytes activated with concanavalin A, and it was required to maintain the growth of thymocytes in vitro and the expression of interleukin 2 receptors. The increase observed in the number of receptor bearing thymocytes and in the density of receptors due to interleukin 2 occurred within the first 2 days of culture. Dexamethasone inhibited the expression of interleukin 2 receptors, the synthesis of interferon-γ, and the early proliferation and protein synthesis of lectin-activated thymocytes during the first 2 days of culture. The inhibitory effect of dexamethasone on the expression of interleukin 2 receptors and on the synthesis of interferon-γ was reversed by interleukin 2, whereas its effect on proliferation and on protein synthesis during the first two days of culture was not reversed by interleukin 2. Interleukin 2 induced the proliferation of thymocytes in vitro, even in the absence of activation by lectin; however, the number of cells displaying receptors which could be detected with anti-Tac remained low throughout the first week of culture and interferon-γ synthesis was not observed. Nonetheless, interleukin 2-induced proliferation was inhibited by anti-Tac on a dose dependent manner. The results of the study document that recombinant interleukin 2, like purified natural interleukin 2, is required for the expression of interleukin 2 receptors, for interferon-γ synthesis, and for the growth of thymocytes in Vitro.