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We have cloned the Tac analog of the bovine IL-2 receptor (IL-2R) cDNA. Using mouse and human cDNA probes, we isolated five bovine IL-2R clones from a lambda gtll bovine long-term lymphocyte cDNA library. Three of the clones had inserts of 2600 base pairs (bp), the same size as the bovine IL-2R mRNA visualized on Northern blots. The full-length cDNA contain a 190-bp 5′ untranslated region, followed by a 825-bp coding region, and a 3′ untranslated region that contain t 600 bp. Comparison of the bovine and human IL-2R-coding sequences revealed 71% homology at the nucleotide level. The 3′ and 5′ non-coding regions were not as homologous, apart from a specific site in the 5′-untranslated region that contained a 5′-upstream start codon. In this region, 24 of 26 nucleotides were identical for the human and. bovine cDNAs. Further analysis of the bovine IL-2R sequence also revealed the following: (i) the hydropbobic domains of the IL-2R protein were more conserved between species than the hydrophilic domains, (ii) the predominant site of intracellular IL-2R phosphorylatlon in mouse and human was a conserved Ser which was not conserved in the bovine sequence, and (iii) there exists a statistically significant amino acid homology with the AIDS gag protein. [ABSTRACT FROM AUTHOR]

Background: Circadian rhythms with varying components exist in organisms ranging from humans to cyanobacteria. A simple evolutionarily plausible mechanism for the origin of such a variety of circadian oscillators, proposed in earlier work, involves the non-disruptive coupling of pre-existing ultradian transcriptional-translational oscillators (TTOs), producing "beats," in individual cells. However, like other TTO models of circadian rhythms, it is important to establish that the inherent stochasticity of the protein binding and unbinding does not invalidate the finding of clear oscillations with circadian period., Results: The TTOs of our model are described in two versions: 1) a version in which the activation or inhibition of genes is regulated stochastically, where the 'unoccupied" (or "free") time of the site under consideration depends on the concentration of a protein complex produced by another site, and 2) a deterministic, "time-averaged" version in which the switching between the "free" and "occupied" states of the sites occurs so rapidly that the stochastic effects average out. The second case is proved to emerge from the first in a mathematically rigorous way. Numerical results for both scenarios are presented and compared., Conclusion: Our model proves to be robust to the stochasticity of protein binding/unbinding at experimentally determined rates and even at rates several orders of magnitude slower. We have not only confirmed this by numerical simulation, but have shown in a mathematically rigorous way that the time-averaged deterministic system is indeed the fast-binding-rate limit of the full stochastic model.

Background: Organisms ranging from humans to cyanobacteria undergo circadian rhythm, that is, variations in behavior that cycle over a period about 24 hours in length. A fundamental property of circadian rhythm is that it is free-running, and continues with a period close to 24 hours in the absence of light cycles or other external cues. Regulatory networks involving feedback inhibition and feedforward stimulation of mRNA transcription and translation are thought to be critical for many circadian mechanisms, and genes coding for essential components of circadian rhythm have been identified in several organisms. However, it is not clear how such components are organized to generate a circadian oscillation., Results: We propose a model in which two independent transcriptional-translational oscillators with periods much shorter than 24 hours are coupled to drive a forced oscillator that has a circadian period, using mechanisms and parameters of conventional molecular biology. Furthermore, the resulting circadian oscillator can be entrained by an external light-dark cycle through known mechanisms. We rationalize the mathematical basis for the observed behavior of the model, and show that the behavior is not dependent on the details of the component ultradian oscillators but occurs even if quite generalized basic oscillators are used., Conclusion: We conclude that coupled, independent, transcriptional-translational oscillators with relatively short periods can be the basis for circadian oscillators. The resulting circadian oscillator can be entrained by 24-hour light-dark cycles, and the model suggests a mechanism for its evolution.

A factor in bovine colostrum (colostrum inhibitory factor, CIF) inhibits interleukin 2 (IL2) production in activated T helper cells by blocking the accumulation of IL2 mRNA. To determine whether CIF blocks at the level of IL2 transcription, we introduced reporter plasmids into the human T leukemia cell line Jurkat by transient transfection. These contained the luciferase gene under the control of either the human IL2 upstream enhancer region (segments -326 to +45) or three repeats of the NFAT element contained within it (segments -255 to -285). Expression of luciferase in these cells was induced by phorbol myristate acetate plus a calcium ionophore. CIF inhibited induction of either construct as did cyclosporine, which is known to block activation of the NFAT element. CIF failed to inhibit several other enhancer elements. The NFAT-controlled luciferase gene system distinguishes CIF from other T cell inhibitory activities present in colostrum, in particular, TGF beta 1 and TGF beta 2 and the glucocorticoids. Stably transfected Jurkat cells behaved similarly to the transiently transfected ones with respect to inhibition by CIF and cyclosporine. The NFAT-luc assay is a useful technique for the rapid, sensitive measurement of CIF or other immunosuppressants with a similar mode of action.

Human colostrum contains a factor that inhibits the induction of interleukin 2 (IL2) in T lymphocyte cell lines (colostrum inhibitory factor, CIF). In PMA-stimulated EL4.E1 cells, inhibition is the result of blocking the accumulation of IL2 mRNA. Human colostrum contains on the order of 100 U/ml of CIF activity, where 1 U/ml inhibits 50% of the IL2 response. Transient acidification to pH 3.0 increased CIF activity severalfold. Although it resembles TGF beta in some respects, antisera against TGF beta neutralized 10% or less of the CIF activity in human colostrum. Furthermore, whereas authentic TGF beta blocked the induction of IL2 by PMA-plus-calcium-stimulated cells by only about 50%, CIF inhibited it completely. A similar CIF activity was also detected in bovine colostrum, but not in normal bovine milk.

The mouse T lymphocyte cell line EL4.E1 synthesizes a proteoglycan core protein (PGCP) mRNA which is identical to serglycin mRNA found in mouse bone marrow-derived mast cells and a mouse mastocytoma cell line. PGCP mRNA was strongly induced in EL4.E1 cells by phorbol myristate acetate, which also induces mRNAs for several cytokines in these cells. In contrast to the induction of cytokine mRNAs, however, the induction of PGCP mRNA was not inhibited by Cyclosporine. PGCP mRNA was also inducible by allogeneic stimulation of normal mouse spleen cells, and by Con A stimulation of an Interleukin 2-producing T hybridoma cell line. A number of other cell lines expressed an identical or similar, mRNA, including two cytotoxic T cell lines, and three tumor cell lines related to bone marrow-derived cells. The levels of several proteoglycans have previously been reported to increase in cells of bone marrow origin under activating conditions, but this appears to be the first report of an induction of the corresponding PGCP mRNA by immune stimulation of T lymphocytes.

Interleukin 2 (IL2) mRNA has a short half-life in the cytoplasm of T lymphocytes, relative to most mRNA. We have discovered a candidate ribonuclease to account for the rapid turnover of IL2 mRNA in the cytosol of the human T lymphocyte cell line Jurkat. In partially purified form, this RNase is about 7 times as active on IL2 as on beta-globin mRNA. Pancreatic RNase, by contrast, does not show a significant preference for IL2 mRNA. Neither 5' capping, nor polyadenylation of the substrate mRNAs affects their degradation by the IL2-selective mRNase, whose activity is optimal in 0.5 mM Mg++ and 100 mM potassium acetate. The mRNase behaves like a protein of molecular weight 60-70,000 on gel chromatography, and is unusual in that it is insensitive to placental RNase inhibitor (RNasin). The mRNase cleaves IL2 mRNA at a small number of sites in the coding region, and IL2 mRNA containing only the coding region and 36 nucleotides of the 3'-noncoding region competes efficiently with full-length IL2 mRNA for the mRNase, whereas beta-globin mRNA does not.