David I. Ratner

A Second UDP-glucose Pyrophosphorylase Is Required for Differentiation and Development in Dictyostelium discoideum

Uridine diphosphoglucose pyrophosphorylase (UDPGP) is a developmentally regulated enzyme in Dictyostelium discoideum, which is involved in trehalose, cellulose, and glycogen synthesis. Two independent UDPGP proteins are believed to be responsible for this activity. To determine the relative contributions of each protein, the genes encoding them were disrupted individually. Cells lacking the udpgp1 gene exhibit normal growth and development and make normal levels of cellulose. In agreement with these phenotypes, udpgp1 − cells still have UDPGP activity, although at a reduced level. This supports the importance of the second UDPGP gene. This newly identified gene,ugpB, encodes an active UDPGP as determined by complementation in Escherichia coli. When this gene is disrupted, cells undergo aberrant differentiation and development ending with small, gnarled fruiting bodies. These cells also have decreased spore viability and decreased levels of glycogen, whose production requires UDPGP activity. These phenotypes suggest that UgpB constitutes the major UDPGP activity produced during development. Sequence analysis of the two UDPGP genes shows that UgpB has higher homology to other eukaryotic UDPGPs than does UDPGP1. This includes the presence of 5 conserved lysine residues. Udpgp1 only has 1 of these lysines.

Potent Neutralization of Staphylococcal Enterotoxin B by Synergistic Action of Chimeric Antibodies

ABSTRACT Staphylococcal enterotoxin B (SEB), a shock-inducing exotoxin synthesized by Staphylococcus aureus, is an important cause of food poisoning and is a class B bioterrorism agent. SEB mediates antigen-independent activation of a major subset of the T-cell population by cross-linking T-cell receptors (TCRs) with class II major histocompatibility complex (MHC-II) molecules of antigen-presenting cells, resulting in the induction of antigen independent proliferation and cytokine secretion by a significant fraction of the T-cell population. Neutralizing antibodies inhibit SEB-mediated T-cell activation by blocking the toxins interaction with the TCR or MHC-II and provide protection against the debilitating effects of this superantigen. We derived and searched a set of monoclonal mouse anti-SEB antibodies to identify neutralizing anti-SEB antibodies that bind to different sites on the toxin. A pair of non-cross-reactive, neutralizing anti-SEB monoclonal antibodies (MAbs) was found, and a combination of these antibodies inhibited SEB-induced T-cell proliferation in a synergistic rather than merely additive manner. In order to engineer antibodies more suitable than mouse MAbs for use in humans, the genes encoding the VL and VH gene segments of a synergistically acting pair of mouse MAbs were grafted, respectively, onto genes encoding the constant regions of human Igκ and human IgG1, transfected into mammalian cells, and used to generate chimeric versions of these antibodies that had affinity and neutralization profiles essentially identical to their mouse counterparts. When tested in cultures of human peripheral blood mononuclear cells or splenocytes derived from HLA-DR3 transgenic mice, the chimeric human-mouse antibodies synergistically neutralized SEB-induced T-cell activation and cytokine production.

Genetic Interactions of the E3 Ubiquitin Ligase Component FbxA with Cyclic AMP Metabolism and a Histidine Kinase Signaling Pathway during Dictyostelium discoideum Development

ABSTRACT Dictyostelium discoideum amoebae with an altered fbxA gene, which is thought to encode a component of an SCF E3 ubiquitin ligase, have defective regulation of cell type proportionality. In chimeras with wild-type cells, the mutant amoebae form mainly spores, leaving the construction of stalks to wild-type cells. To examine the role of fbxA and regulated proteolysis, we have recovered the promoter of fbxA and shown that it is expressed in a pattern resembling that of a prestalk-specific gene until late in development, when it is also expressed in developing spore cells. Because fbxA cells are developmentally deficient in pure culture, we were able to select suppressor mutations that promote sporulation of the original mutant. One suppressor mutation resides within the gene regA, which encodes a cyclic AMP (cAMP) phosphodiesterase linked to an activating response regulator domain. In another suppressor, there has been a disruption of dhkA, a gene encoding a two-component histidine kinase known to influence Dictyostelium development. RegA appears precociously and in greater amounts in the fbxA mutant than in the wild type, but in an fbxA/dhkA double mutant, RegA is restored to wild-type levels. Because the basis of regA suppression might involve alterations in cAMP levels during development, the concentrations of cAMP in all strains were determined. The levels of cAMP are relatively constant during multicellular development in all strains except the dhkA mutant, in which it is reduced at least sixfold. The level of cAMP in the double mutant dhkA/fbxA is relatively normal. The levels of cAMP in the various mutants do not correlate with spore formation, as would be expected on the basis of our present understanding of the signaling pathway leading to the induction of spores. Altered amounts of RegA and cAMP early in the development of the mutants suggest that both fbxA and dhkA genes act earlier than previously thought.

Prespore gene expression in Dictyostelium requires concomitant protein synthesis.

It has been established previously that the maintenance of expression of prespore-specific genes of Dictyostelium discoideum is prevented by the translational inhibitor cycloheximide. The drug had no effect upon the level of transcripts of the other genes examined, prestalk-specific or cell type non-specific (Mehdy, M., Ratner, D. and Firtel, R., (1983) Cell 32, 763-771). We have now characterized the cellular specificity and temporal profiles of mRNA accumulation of additional Dictyostelium cDNA clones. Other inhibitors of in vivo protein synthesis have been examined, with emetine shown to be a particularly effective but reversible agent. Four structurally and mechanistically distinct translational inhibitors each prevented the reaccumulation of prespore transcripts in cyclic AMP-primed disaggregated amoebae. These results establish a role for protein synthesis in the transcription or transcript stability of prespore genes.

Use of a Probabilistic Motif Search to Identify Histidine Phosphotransfer Domain-Containing Proteins.

The wealth of newly obtained proteomic information affords researchers the possibility of searching for proteins of a given structure or function. Here we describe a general method for the detection of a protein domain of interest in any species for which a complete proteome exists. In particular, we apply this approach to identify histidine phosphotransfer (HPt) domain-containing proteins across a range of eukaryotic species. From the sequences of known HPt domains, we created an amino acid occurrence matrix which we then used to define a conserved, probabilistic motif. Examination of various organisms either known to contain (plant and fungal species) or believed to lack (mammals) HPt domains established criteria by which new HPt candidates were identified and ranked. Search results using a probabilistic motif matrix compare favorably with data to be found in several commonly used protein structure/function databases: our method identified all known HPt proteins in the Arabidopsis thaliana proteome, confirmed the absence of such motifs in mice and humans, and suggests new candidate HPts in several organisms. Moreover, probabilistic motif searching can be applied more generally, in a manner both readily customized and computationally compact, to other protein domains; this utility is demonstrated by our identification of histones in a range of eukaryotic organisms.

Effects of protein synthesis inhibition on the transcription and transcript stability of Dictyostelium prespore genes

The in vivo accumulation of several prespore transcripts of Dictyostelium discoideum has previously been shown to depend upon concomitant protein synthesis (Ratner, D.I., Pentz, W.H. and Pelletier, D.A. (1989) Biochim. Biophys. Acta 1008, 71-78). Measurements of in vivo mRNA decay and nuclear run-on transcription assays have now been used to learn whether protein synthesis is required primarily for mRNA synthesis or transcript stability. The translational inhibitors cycloheximide and pactamycin stabilized existing prespore transcripts, despite their effect upon mRNA accumulation. Transcriptional assays, performed at intervals throughout the developmental cycle, demonstrated that temporal changes in the abundance of several cell-specific transcripts correlated closely with changes in their rates of synthesis. Finally, blocking protein synthesis strongly inhibited the transcription of the prespore genes examined. These results imply that one or more developmentally regulated, labile proteins are needed for the activation of prespore gene transcription.