emo-1(oz1) is a member of a class of hermaphrodite sterile mutations in Caenorhabditis elegans that produce endomitotic oocytes in the gonad arm. Oocytes in emo-1(oz1) mutants exhibit multiple defects during oogenesis. After meiotic maturation, ovulation fails, trapping oocytes in the gonad arm where they become endomitotic. emo-1 encodes a homologue of the Sec61p gamma subunit, a protein necessary for translocation of secretory and transmembrane proteins into the endoplasmic reticulum of yeast and mammalian cells. A putative emo-1 null mutation, oz151, displays embryonic lethality. The oz1 sterile mutation is a transposable element insertion into the emo-1 3' untranslated region that almost completely eliminates germline mRNA accumulation. Genetic mosaic analysis using the oz1 allele indicates that emo-1(+) expression in germ cells is required for fertility. The J67 monoclonal antibody, which recognizes an oocyte surface antigen (Strome, S. 1986. In Gametogenesis and the Early Embryo. J.G. Gall, editor. Alan R. Liss, Inc., New York. 77-95.), does not stain oz1 oocytes, a finding consistent with defective protein transport in the mutant. We propose that the emo-1 gene product acts in the transport of secreted and transmembrane proteins in C. elegans oocytes, and is necessary for both oogenesis and the coupling of ovulation with meiotic maturation.
The temporal relationship between tubulin expression and the assembly of the mitotic spindle microtubules has been investigated during the naturally synchronous cell cycle of the Physarum plasmodium. The cell cycle behavior of the tubulin isoforms was examined by two-dimensional gel electrophoresis of proteins labeled in vivo and by translation of RNA in vitro. alpha 1-, alpha 2-, beta 1-, and beta 2-tubulin synthesis increases coordinately until metaphase, and then falls, with beta 2 falling more rapidly than beta 1. Nucleic acid hybridization demonstrated that alpha- and beta-tubulin RNAs accumulate coordinately during G2, peaking at metaphase. Quantitative analysis demonstrated that alpha-tubulin RNA increases with apparent exponential kinetics, peaking with an increase over the basal level of greater than 40-fold. After metaphase, tubulin RNA levels fall exponentially, with a short half-life (19 min). Electron microscopic analysis of the plasmodium showed that the accumulation of tubulin RNA begins long before the polymerization of mitotic spindle microtubules. By contrast, the decay of tubulin RNA after metaphase coincides with the depolymerization of the spindle microtubules.
Three alpha-tubulins and two beta-tubulins have been resolved by two-dimensional gel electrophoresis of whole cell lysates of Physarum myxamoebae or plasmodia. Criteria used to identify the tubulins included migration on two-dimensional gels with myxamoebal tubulins purified by self-assembly into microtubules in vitro, peptide mapping with Staphylococcus V8 protease and with chymotrypsin, immunoprecipitation with a monoclonal antibody specific for beta-tubulin, and, finally, hybrid selection of specific mRNA by cloned tubulin DNA sequences, followed by translation in vitro. Differential expression of the Physarum tubulins was observed. The alpha 1- and beta 1-tubulins were detected in both myxamoebae and plasmodia; alpha 2 and beta 2 were detected only in plasmodia, alpha 3 was detected only in the myxamoebal phase, and may be specific to the flagellate. Observation of more tubulin species in plasmodia than in myxamoebae was remarkable; the only microtubules detected in plasmodia are those of the mitotoic spindle, whereas myxamoebae display cytoplasmic, centriolar, flagellar, and mitotic-spindle microtubules. In vitro translation of myxamoebal and plasmodial RNAs indicated that there are distinct mRNAs, and therefore probably separate genes, for the alpha 1-, alpha 2-, beta 1-, and beta 2-tubulins. Thus, the different patterns of tubulin expression in myxamoebae and plasmodia reflect differential expression of tubulin genes.