title: Cloning and characterisation of genes encoding two transforming growth factor-beta-like ligands from the hookworm, Ancylostoma caninum.
abstract: To elucidate the role of transforming growth factor beta (TGF-beta) signalling in the arrest/reactivation pathway of the Ancylostoma caninum hookworm, two parasite-encoded TGF-beta-like ligands were cloned and characterised. Ac-dbl-1 showed 60% amino acid identity to the Caenorhabditis elegansdbl-1 gene, which regulates growth while Ac-daf-7 showed 46% amino acid identity to Ce-daf-7 which regulates arrested development. Exon/intron organisation of the genes for Ac-dbl-1 and Ac-daf-7 were different from that of the corresponding C. elegans genes with nine and 10 exons, respectively, and introns ranging in size from 56 to 2,556 bp. Based on real-time reverse transcriptase (RT)-PCR, Ac-dbl-1 and Ac-daf-7 were expressed in all stages tested, i.e. egg, first/second stage larvae (L1/L2), infective third stage larvae (iL3), serum-stimulated third stage larvae (ssL3), and male and female adult worms. Expression of Ac-dbl-1 peaked in the adult male stage suggesting a similar role to Ce-dbl-1 in regulating male tail patterning. Ac-daf-7 expression was at a maximum in the arrested iL3 and reactivated ssL3 stages, which differs from that of Ce-daf-7 expression and may be unique to parasitic nematodes that have an obligate requirement to undergo developmental arrest. In support of the PCR results, antibodies to the A. caninum TGF-beta-like ligands detected proteins in iL3, ssL3, and adult worm extracts. Immunofluorescent studies showed that Ac-daf-7 is expressed in the anterior region of the iL3 similar to Ce-daf-7, which is localised to the ASI chemosensory neurons.
authors.affiliation: Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, 27606, USA.
authors.forename: Tori C
authors.initials: TC
authors.lastname: Freitas
authors.forename: Prema
authors.initials: P
authors.lastname: Arasu
mesh_terms: D000595:Amino Acid Sequence; D000722:Ancylostoma / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D029742:Caenorhabditis elegans Proteins / Q000235:genetics; D003001:Cloning, Molecular; D005455:Fluorescent Antibody Technique; D018507:Gene Expression Regulation, Developmental; D007814:Larva; D008969:Molecular Sequence Data; D018125:Receptors, Transforming Growth Factor beta / Q000032:analysis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017385:Sequence Homology
chemical_list: D029742:Caenorhabditis elegans Proteins; D018125:Receptors, Transforming Growth Factor beta