Use of nonreducing SDS-PAGE for monitoring renaturation of recombinant protein synthesis initiation factor, eIF-4 alpha.

The purification of biologically active human protein synthesis initiation factor 4 alpha, eIF-4 alpha, overexpressed in Escherichia coli, is complicated by its localization in insoluble inclusion bodies, as well as its possession of four cysteines. Two of these cysteines have been reported to be reduced in the native molecule and two form a disulfide bond. A method is described, using nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis, for monitoring renaturation of the polypeptide during staged dialyses in decreasing urea concentrations. The production of biologically active eIF-4 alpha only occurs when the polypeptide is totally reduced during solubilization of the inclusion bodies in 8 M urea. This requires a minimum dithiothreitol concentration of 50 mM. Conversely, reformation of the disulfide bonds only occurs when the staged dialyses are performed at lower concentrations of sulfhydryl reagents. Once renatured, as described, eIF-4 alpha can be purified by affinity chromatography on m7GTP-Sepharose. Approximately 20 micrograms of biologically active eIF-4 alpha per milliliter of bacterial culture can be obtained. The affinity-purified eIF-4 alpha has activity equivalent to that reported for purified native human eIF-4 alpha, as measured by its activity in a rabbit reticulocyte translation system. The method described is applicable to the purification of other cysteine-containing polypeptides that accumulate to high levels in inclusion bodies.

Mechanism of action of developmentally regulated sea urchin inhibitor of eIF-4.

The developmentally regulated inhibitor of eIF-4 function found in unfertilized sea urchin eggs has been partially purified and its mechanism of action studied in vitro using purified recombinant eIF-4 alpha and cell-free translation systems. The results demonstrate that although the phosphorylation of eIF-4 alpha is necessary to promote protein synthesis, it is not sufficient to maintain all aspects of eIF-4 function. The egg inhibitor does not change eIF-4 alpha phosphorylation state. During the blockage of initiation caused by the egg inhibitor, eIF-4 alpha remains phosphorylated but accumulates in a 48S initiation intermediate. This suggests that the egg inhibitor functions by preventing the release of eIF-4 alpha from the small ribosomal subunit. The characteristics of the inhibitor in a reticulocyte translation system demonstrate that eIF-4 activity is inhibited within 3-6 min. However, the inhibitor's characteristics in a mRNA-dependent translation system contrast with this. Preincubation with the inhibitor for 5-25 min prior to the addition of mRNA does not prevent endogenous eIF-4 from participating in translation but diminishes its ability to be reutilized, consistent with the accumulation of eIF-4 alpha on the small ribosomal subunit. The ribosomal localization of the inhibitor suggests that it could prevent eIF-4 alpha release by direct binding. The gradual inactivation of the inhibitor following fertilization indicates that it represents a component of a novel regulatory cascade that modulates eIF-4 activity.

UNC-6, a laminin-related protein, guides cell and pioneer axon migrations in C. elegans.

The unc-6 gene is required for the guidance of pioneer axons and migrating cells along the body wall in C. elegans. In mutants, dorsal and ventral migrations are disrupted, but longitudinal movements are largely unaffected. The gene was tagged for molecular cloning by two independent transposon insertions. Based on genomic and cDNA sequencing, the gene encodes a novel laminin-related protein, UNC-6 (591 amino acids). The N-terminus is homologous to the N-termini (i.e., domains V1, V-1, V-2, and V-3) of laminin subunits, while the C-terminus is a unique domain. We propose that UNC-6 is a component of an extracellular matrix cue that guides dorsoventral migrations on the epidermis.

UNC-5, a transmembrane protein with immunoglobulin and thrombospondin type 1 domains, guides cell and pioneer axon migrations in C. elegans.

The unc-5 gene is required for guiding pioneering axons and migrating cells along the body wall in C. elegans. In mutants, dorsal migrations are disrupted, but ventral and longitudinal movements are largely unaffected. The gene was tagged for molecular cloning by transposon insertions. Based on genomic and cDNA sequencing, the gene encodes UNC-5, a transmembrane protein of 919 aa. The predicted extracellular N-terminus comprises two immunoglobulin and two thrombospondin type 1 domains. Except for an SH3-like motif, the large intracellular C-terminus is novel. Mosaic analysis shows that unc-5 acts in migrating cells and pioneering neurons. We propose that UNC-5 is a transmembrane receptor expressed on the surface of motile cells and growth cones to guide dorsal movements.

The acetylcholinesterase genes of C. elegans: identification of a third gene (ace-3) and mosaic mapping of a synthetic lethal phenotype.

In C. elegans, the newly identified ace-3 is the third gene affecting acetylcholinesterase (AChE) activity. ace-3 II specifically affects class C AChE and is unlinked to ace-1 X or ace-2 I, which affect the other two AChE classes (A and B, respectively). Strains homozygous for an ace-3 mutation have no apparent behavioral or developmental defect; ace-1 ace-3 and ace-2 ace-3 double mutants are also nearly wild type. In contrast, ace-1 ace-2 ace-3 triple mutant animals are paralyzed and developmentally arrested; their embryonic development is relatively unimpaired, but they are unable to grow beyond the hatching stage. Based on the analysis of genetic mosaics, we conclude that in the absence of ace-2 and ace-3 function, the expression of ace-1(+) in muscle cells, but not in neurons, is essential for postembryonic viability.

Genetics of cell and axon migrations in Caenorhabditis elegans.

The Caenorhabditis elegans epidermis comprises 78 cells which cover the external surface of the embryo as a single cell layer. These cells secrete the cuticle from their exterior faces and support the body wall muscles and most of the nervous system on their interior faces. The epidermal cells arise by autonomous embryonic cell lineages but show regulative interactions after their assembly into an epithelium. It is believed that the various epidermal cells express different kinds or amounts of surface molecules that govern their mutual assembly and also guide the attachments and migrations of the underlying body muscles and neurones. The first muscles and neurones may in turn express new surface molecules that refine later cell movements. Mutations in some 30 known genes disrupt the movements of cells or axons along the body wall.