Identification of genes induced by factor deprivation in hematopoietic cells undergoing apoptosis using gene-trap mutagenesis and site-specific recombination

A strategy employing gene-trap mutagenesis and site-specific recombination (Cre/loxP) has been developed to isolate genes that are transcriptionally activated during programmed cell death. Interleukin-3 (IL-3)-dependent hematopoietic precursor cells (FDCP1) expressing a reporter plasmid that codes for herpes simplex virus–thymidine kinase, neomycin phosphotransferase, and murine IL-3 were transduced with a retroviral gene-trap vector carrying coding sequences for Cre-recombinase (Cre) in the U3 region. Activation of Cre expression from integrations into active genes resulted in a permanent switching between the selectable marker genes that converted the FDCP1 cells to factor independence. Selection for autonomous growth yielded recombinants in which Cre sequences in the U3 region were expressed from upstream cellular promoters. Because the expression of the marker genes is independent of the trapped cellular promoter, genes could be identified that were transiently induced by IL-3 withdrawal.

On the failure of de novo-designed peptides as biocatalysts.

While the elegance and efficiency of enzymatic catalysis have long tempted chemists and biochemists with reductionist leanings to try to mimic the functions of natural enzymes in much smaller peptides, such efforts have only rarely produced catalysts with biologically interesting properties. However, the advent of genetic engineering and hybridoma technology and the discovery of catalytic RNA have led to new and very promising alternative means of biocatalyst development. Synthetic chemists have also had some success in creating nonpeptide catalysts with certain enzyme-like characteristics, although their rates and specificities are generally much poorer than those exhibited by the best novel biocatalysts based on natural structures. A comparison of the various approaches from theoretical and practical viewpoints is presented. It is suggested that, given our current level of understanding, the most fruitful methods may incorporate both iterative selection strategies and rationally chosen small perturbations, superimposed on frameworks designed by nature.

Posttranslational modification of the glycosylation inhibiting factor (GIF) gene product generates bioactive GIF

Glycosylation inhibiting factor (GIF) and macrophage migration inhibitory factor (MIF) share an identical structure gene. Here we unravel two steps of posttranslational modifications in GIF/MIF molecules in human suppressor T (Ts) cell hybridomas. Peptide mapping and MS analysis of the affinity-purified GIF from the Ts cells revealed that one modification is cysteinylation at Cys-60, and the other is phosphorylation at Ser-91. Cysteinylated GIF, but not the wild-type GIF/MIF, possessed immunosuppressive effects on the in vitro IgE antibody response and had high affinity for GIF receptors on the T helper hybridoma cells. In vitro treatment of wild-type recombinant human GIF/MIF with cystine resulted in preferential cysteinylation of Cys-60 in the molecules. The cysteinylated recombinant human GIF and the Ts hybridoma-derived cysteinylated GIF were comparable both in the affinity for the receptors and in the immunosuppressive activity. Polyclonal antibodies specific for a stretch of the amino acid sequence in α2-helix of GIF bound bioactive cysteinylated GIF but failed to bind wild-type GIF/MIF. These results strongly suggest that cysteinylation of Cys-60 and consequent conformational changes in the GIF/MIF molecules are responsible for the generation of GIF bioactivity.

Phosphoglycerylethanolamine Posttranslational Modification of Plant Eukaryotic Elongation Factor 1α1

Eukaryotic elongation factor 1α (eEF-1A) is a multifunctional protein. There are three known posttranslational modifications of eEF-1A that could potentially affect its function. Except for phosphorylation, the other posttranslational modifications have not been demonstrated in plants. Using matrix-assisted laser desorption/ionization-mass spectrometry and peptide mass mapping, we show that carrot (Daucus carota L.) eEF-1A contains a phosphoglycerylethanolamine (PGE) posttranslational modification. eEF-1A was the only protein labeled with [14C]ethanolamine in carrot cells and was the predominant ethanolamine-labeled protein in Arabidopsis seedlings and tobacco (Nicotiana tabacum L.) cell cultures. In vivo-labeling studies using [3H]glycerol, [32P]Pi, [14C]myristic acid, and [14C]linoleic acid indicated that the entire phospholipid phosphatidylethanolamine is covalently attached to the protein. The PGE lipid modification did not affect the partitioning of eEF-1A in Triton X-114 or its actin-binding activity in in vitro assays. Our in vitro data indicate that this newly characterized posttranslational modification alone does not affect the function of eEF-1A. Therefore, the PGE lipid modification may work in combination with other posttranslational modifications to affect the distribution and the function of eEF-1A within the cell.