Vitellin-binding proteins in the nematode Oscheius tipulae (Nematoda, Rhabditida)

We describe the first application of a non-radioactive ligand-blotting technique to the characterization of proteins interacting with nematode vitellins. Chromatographically purified vitellins from the free-living nematode Oscheius tipulae were labeled with fluorescein in vitro. Ligand-blotting assays with horseradish peroxidase-conjugated anti-fluorescein antibodies showed that labeled vitellins reacted specifically with a polypeptide of approximately 100 kDa, which we named P100. This polypeptide is a specific worm`s vitellin-binding protein that is present only in adult worms. Blots containing purified O. tipulae vitellin preparations showed no detectable signal in the 100 kDa region, ruling out any possibility of yolk polypeptides self-assembling under the conditions used in our assay. Experiments done in the presence of alpha-methyl mannoside ruled out the possibility of vitellins binding to P100 through mannose residues. Triton X-114 fractionation of whole worm extracts showed that P100 is either a membrane protein or has highly hydrophobic regions. (C) 2008 Elsevier Inc. All rights reserved.

Oscheius tipulae as an example of eEF1A gene diversity in nematodes

We characterized four eEF1A genes in the alternative rhabditid nematode model organism Oscheius tipulae. This is twice the copy number of eEF1A genes in C. elegans, C. briggsae, and, probably, many other free-living and parasitic nematodes. The introns show features remarkably different from those of other metazoan eEF1A genes. Most of the introns in the eEF1A genes are specific to O. tipulae and are not shared with any of the other genes described in metazoans. Most of the introns are phase 0 (inserted between two codons), and few are inserted in protosplice sites (introns inserted between the nucleotide sequence A/CAG and G/A). Two of these phase 0 introns are conserved in sequence in two or more of the four eEF1A gene copies, and are inserted in the same position in the genes. Neither of these characteristics has been detected in any of the nematode eEF1A genes characterized to date. The coding sequences were also compared with other eEF1A cDNAs from 11 different nematodes to determine the variability of these genes within the phylum Nematoda. Parsimony and distance trees yielded similar topologies, which were similar to those created using other molecular markers. The presence of more than one copy of the eEF1A gene with nearly identical coding regions makes it difficult to define the orthologous cDNAs. As shown by our data on O. tipulae, careful and extensive examination of intron positions in the eEF1A gene across the phylum is necessary to define their potential for use as valid phylogenetic markers.