Identification of an S-locus glycoprotein allele introgressed from B. napus ssp. rapifera to B. napus ssp. oleifera.

We have previously described a developmentally regulated mRNA in maize that accumulates in mature embryos and is involved in a variety of stress responses in the plant. The sequence of the encoded 16 kDa protein (MA16) predicts that it is an RNA-binding protein, since it possesses a ribonucleoprotein consensus sequence-type RNA-binding domain (CS-RBD). To assess the predicted RNA binding property of the protein and as a starting point to characterize its function we have used ribohomopolymer-binding assays. Here we show that the MA16-encoded protein binds preferentially to uridine- and guanosine-rich RNAs. In light of these results a likely role for this protein in RNA metabolism during late embryogenesis and in the stress response is discussed.     

Evidence of independent gene duplications during the evolution of archaeal and eukaryotic family B DNA polymerases

Eukaryotes and archaea both possess multiple genes coding for family B DNA polymerases. In animals and fungi, three family B DNA polymerases, alpha, delta, and epsilon, are responsible for replication of nuclear DNA. We used a PCR-based approach to amplify and sequence phylogenetically conserved regions of these three DNA polymerases from Giardia intestinalis and Trichomonas vaginalis, representatives of early-diverging eukaryotic lineages. Phylogenetic analysis of eukaryotic and archaeal paralogs suggests that the gene duplications that gave rise to the three replicative paralogs occurred before the divergence of the earliest eukaryotic lineages, and that all eukaryotes are likely to possess these paralogs. One eukaryotic paralog, epsilon, consistently branches within archaeal sequences to the exclusion of other eukaryotic paralogs, suggesting that an epsilon-like family B DNA polymerase was ancestral to both archaea and eukaryotes. Because crenarchaeote and euryarchaeote paralogs do not form monophyletic groups in phylogenetic analysis, it is possible that archaeal family B paralogs themselves evolved by a series of gene duplications independent of the gene duplications that gave rise to eukaryotic paralogs.      

Partial reconstruction of the microvillus core bundle: characterization of villin as a Ca(++)-dependent, actin-bundling/depolymerizing protein

The brush border, isolated from chicken intestine epithelial cells, contains the 95,000 relative molecular mass (M(r)) polypeptide, villin. This report describes the purification and characterization of villin as a Ca(++)-dependent, actin bundling/depolymerizing protein. Then 100,000 g supernatant from a Ca(++) extract of isolated brush borders is composed of three polypeptides of 95,000 (villin), 68,000 (fimbrin), and 42,000 M(r) (actin). Villin, following purification from this extract by differential ammonium sulfate precipitation and ion-exchange chromatography, was mixed with skeletal muscle F-actin. Electron microscopy of negatively stained preparations of these villin-actin mixtures showed that filament bundles were present. This viscosity, sedimentability, and ultrastructural morphology of filament bundles are dependent on the villin:actin molar ratio, the pH, and the free Ca(++) concentration in solution. At low free Ca(++) (less than 10(-6) M), the amount of protein in bundles, when measured by sedimentation, increased as the villin: actin molar ratio increased and reached a plateau at approximately a 4:10 ratio. This behavior correlates with the conversion of single actin filaments into filament bundles as detected in the electron microscope. At high free Ca(++) (more than 10(-6) M), there was a decrease in the apparent viscosity in the villin-actin mixtures to a level measured for the buffer. Furthermore, these Ca(++) effects were correlated with the loss of protein sedimented, the disappearance of filament bundles, and the appearance of short fragments of filaments. Bundle formation is also pH-sensitive, being favored at mildly acidic pH. A decrease in the pH from 7.6 to 6.6 results in an increase in sedimentable protein and also a transformation of loosly associated actin filaments into compact actin bundles. These results are consistent with the suggestions that villin is a bundling protein in the microvillus and is responsible for the Ca(++)-sensitive disassembly of the microvillar cytoskeleton. Thus villin may function in the cytoplasm as a major cytoskeletal element regulating microvillar shape.     

Ectopic expression of dentin sialoprotein during amelogenesis hardens bulk enamel

Dentin sialophosphpoprotein (Dspp) is transiently expressed in the early stage of secretory ameloblasts. The secretion of ameloblast-derived Dspp is short-lived, correlates to the establishment of the dentinoenamel junction (DEJ), and is consistent with Dspp having a role in producing the specialized first-formed harder enamel adjacent to the DEJ. Crack diffusion by branching and dissipation within this specialized first-formed enamel close to the DEJ prevents catastrophic interfacial damage and tooth failure. Once Dspp is secreted, it is subjected to proteolytic cleavage that results in two distinct proteins referred to as dentin sialoprotein (Dsp) and dentin phosphoprotein (Dpp). The purpose of this study was to investigate the biological and mechanical contribution of Dsp and Dpp to enamel formation. Transgenic mice were engineered to overexpress either Dsp or Dpp in their enamel organs. The mechanical properties (hardness and toughness) of the mature enamel of transgenic mice were compared with genetically matched and age-matched nontransgenic animals. Dsp and Dpp contributions to enamel formation greatly differed. The inclusion of Dsp in bulk enamel significantly and uniformly increased enamel hardness (20%), whereas the inclusion of Dpp weakened the bulk enamel. Thus, Dsp appears to make a unique contribution to the physical properties of the DEJ. Dsp transgenic animals have been engineered with superior enamel mechanical properties.     

The COOH terminus of the amelogenin, LRAP, is oriented next to the hydroxyapatite surface

The organic matrix in forming enamel consists largely of the amelogenin protein self-assembled into nanospheres that are necessary to guide the formation of the unusually long and highly ordered hydroxyapatite (HAP) crystallites that constitute enamel. Despite its ability to direct crystal growth, the interaction of the amelogenin protein with HAP is unknown. However, the demonstration of growth restricted to the c-axis suggests a specific protein-crystal interaction, and the charged COOH terminus is often implicated in this function. To elucidate whether the COOH terminus is important in the binding and orientation of amelogenin onto HAP, we have used solid state NMR to determine the orientation of the COOH terminus of an amelogenin splice variant, LRAP (leucine-rich amelogenin protein), which contains the charged COOH terminus of the full protein, on the HAP surface. These experiments demonstrate that the methyl 13C-labeled side chain of Ala46 is 8.0 A from the HAP surface under hydrated conditions, for the protein with and without phosphorylation. The experimental results provide direct evidence orienting the charged COOH-terminal region of the amelogenin protein on the HAP surface, optimized to exert control on developing enamel crystals.     

AGGREGATION FORMATION AND ASSORTATIVE MATING IN TWO MELOID BEETLES

Males and females of the meloid beetles Lytta magister and Tegrodera aloga form large aggregations in Sonoran Desert habitat. Males and females of L. magister fly to prominent ridgetop landmarks, where they feed on flowering shrubs, mate (probably just once in any one aggregation), and disperse, with the group forming and disbanding in a few days. Males and females of T. aloga form very large, mobile bands that march across the desert flatland feeding and mating; females probably leave after a single copulation, with the result that the group becomes increasingly male-biased in its sex ratio. The aggregations persist for a minimum of 2–3 weeks. Great variation in body size characterizes both species. Positive assortative mating occurs in L. magister but not in T. aloga, possibly because males and females of the two species incur different costs and benefits for selective mating. It is probable that body size in Lytta correlates both with female fecundity and with male spermatophore size or amount of cantharidin contributed during copulation. This should favor males that mate with large females while also favoring females that mate with large males. If large individuals pair off, this will leave smaller individuals to settle for one another, leading to positive assortative mating. The same argument may not apply to T. aloga, perhaps because its copulations are shorter and its aggregations longer-lived, and therefore any one mating does not eliminate chances to copulate again within the band.