The Saccharomyces cerevisiae Mod5 protein catalyzes isopentenylation of A to i 6 A on tRNAs in the nucleus, cytosol, and mitochondria. The substrate for Mod5p, dimethylallyl pyrophosphate, is also a substrate for Erg20p that catalyzes an essential step in sterol biosynthesis. Changing the distribution of Mod5p so that less Mod5p is present in the cytosol decreases i 6 A on cytosolic tRNAs and alters tRNA-mediated nonsense suppression. We devised a colony color͞growth assay to assess tRNA-mediated nonsense suppression and used it to search for genes, which, when overexpressed, affect nonsense suppression. We identified SAL6, TEF4, and YDL219w, all of which likely affect nonsense suppression via alteration of the protein synthesis machinery. We also identified ARC1, whose product interacts with aminoacyl synthetases. Interestingly, we identified ERG20. Midwestern analysis showed that yeast cells overproducing Erg20p have reduced levels of i 6 A on tRNAs. Thus, Erg20p appears to affect nonsense suppression by competing with Mod5p for substrate. Identification of ERG20 reveals that yeast have a limited pool of dimethylallyl pyrophosphate. It also demonstrates that disrupting the balance between enzymes that use dimethylallyl pyrophosphate as substrate affects translation.N 6 -(⌬ 2 -isopentenyl)adenosine ͉ sterol biosynthesis ͉ assay for flux in sterol pathway T he Saccharomyces cerevisiae protein Mod5p catalyzes the addition of an isopentenyl group to adenosine (i 6 A) at position 37 of the anticodon loop of some tRNAs (1-5). There are two isoforms of Mod5p, Mod5p-I and Mod5p-II, that differ in the site of their translation initiation codon and in their distribution in the cell. Mod5p-I is translated starting at codon 1 of the MOD5 ORF and is localized to mitochondria and the cytoplasm. Mod5p-II, translated from codon 12 of the MOD5 ORF, is located in the nucleus and the cytoplasm (6, 7).The i 6 A modification promotes the efficiency of SUP7 tRNA in cytosolic suppression of UAA nonsense mutations by the insertion of tyrosine (8). Cells possessing only Mod5p-I have limiting cytosolic amounts of isozyme, and changes in the subcellular distribution and͞or the activity of this isozyme alter nonsense suppression. Hence, genetic screens͞selections based on nonsense suppression can identify cells with altered cytosolic Mod5p-I activity (9).Here we employed the genetic strategy of using overexpression to perturb a pathway (10) and developed a protocol for the selection of cells with lower than normal levels of cytosolic Mod5p-I activity. Using this strategy we were able to sample the entire yeast genome and identify genes that, when overexpressed, lead to lower than normal levels of cytosolic Mod5p-I activity.As a result of that screen, we identified two categories of genes. The first category includes genes that affect nonsense suppression via alteration of the protein synthetic machinery. Our studies suggest that the yeast gene product encoded by YDL219w may function in protein synthesis and that the translation elongatio...

IntroductionBoth clinical experience and experimental evidence have suggested that Adrenocorticotropic hormone (ACTH) might directly exert immunomodulatory effects not dependent on adrenal steroidogenesis.MethodsThe direct effects of H.P. Acthar Gel® (Acthar), a repository preparation containing a porcine ACTH analogue, on human B lymphocyte function were studied in vitro using peripheral blood B cells isolated using anti-CD19 coated magnetic beads and activated by interleukin 4 (IL-4) and CD40 ligand (CD40L). Analysis of expression of messenger RNA (mRNA) encoding activation-induced cytidine deaminase (AICDA) was carried out by quantitative real-time polymerase chain reaction (PCR). Cellular proliferation was assessed by a flow cytometric technique using intracellular staining with carboxyfluorescein succinimidyl ester (CFSE). Immunoglobulin G (IgG) production was measured in cell supernatants using an immunoassay.ResultsActhar was found to exert acute, dose-dependent inhibitory effects on IL-4/CD40L–mediated induction of the expression of activation-induced cytidine deaminase (AICDA) after 24 hours, as well as sustained inhibition of B cell proliferation and IgG production during five more days of culture, without deleterious effects on B cell viability.ConclusionsThese experiments demonstrate that Acthar can exert direct effects on the humoral immune system independent of any role in the regulation of adrenal steroidogenesis. Although the impact of these findings on clinical disease was not evaluated in this study, these data support the therapeutic potential of Acthar for the management of autoimmune diseases characterized by B cell activation and aberrant humoral immune function.

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