This proposal describes experiments to initiate molecular analysis of the adhesion disc of Giardia lamblia, a protozoan parasite and significant human pathogen. Specifically I propose to study a protein called giardin, a major structural component of the sucking disc with which G. lamblia adheres to intestinal microvilli. The work will have three main thrust: to characterize giardin at the protein level, to isolate the giardin genes and characterize them at the nucleotide level, and to study functional mutants of the giardin genes after inducing and isolating G. lamblia adhesion mutants. Giardin has been purified and used to riase polyclonal antibodies that cross-react specifically with the ventral adhesion disc of the organism. I propose ways to analyze isomorphs of giardin and to distinguish them from non-giardin proteins of the same molecular weight. I also propose procedures to utilize the anti-giardin antibodies or a partial protein sequence of giardin itself to isolate the G. lamblia giardin genes. These genes will be used to determine the entire derived protein sequence of giardin, the genomic organization of the giardin genes, and elements of transcriptional control and gene processing within G. lamblia. I also describe experiments to initiate chemical and x-ray mutagenesis studies to obtain organisms with a defective adhesion disc, precluding attachment in vitro. I will analyze the giardin genes of such mutants and also will search for ultastructural aberrations in the adhesion disc to localize the site of lesion. The work proposed here will characterize a novel protein and its genes in Giardia. It should provide a means to explore gene regulation during the G. lamblia life cycle and could offer inroads into G. lamblia genetics. In addition, because the adhesion disc is unique to Giardia among parasitic protozoa, these studies also could offer a new approach to devising therapeutics to combat giardiasis. Finally, the early phylogenetic divergence of G. lamblia from the prokaryotic kingdoms, earlier then any known eukaryote, implies that this work could reveal unusual mechanisms of gene processing hereto fore unseen in other organisms.
| Nohria, A; Alonso, R A; Peattie, D A (1992) Identification and characterization of gamma-giardin and the gamma-giardin gene from Giardia lamblia. Mol Biochem Parasitol 56:27-37 |
| Alonso, R A; Peattie, D A (1992) Nucleotide sequence of a second alpha giardin gene and molecular analysis of the alpha giardin genes and transcripts in Giardia lamblia. Mol Biochem Parasitol 50:95-104 |
| Kabnick, K S; Peattie, D A (1990) In situ analyses reveal that the two nuclei of Giardia lamblia are equivalent. J Cell Sci 95 ( Pt 3):353-60 |
| Peattie, D A; Alonso, R A; Hein, A et al. (1989) Ultrastructural localization of giardins to the edges of disk microribbons of Giarida lamblia and the nucleotide and deduced protein sequence of alpha giardin. J Cell Biol 109:2323-35 |
