Cloning and sequencing of a cuticular collagen gene from the parasitic nematode Ascaris suum

Lulio Ruiz, Purdue University

Abstract

The cuticle of a nematode is a physiologically active structure which is involved in a variety of functions: absorption, excretion, transport, locomotion, and as a protective barrier. The vast majority of proteins in the nematode cuticle are collagens. Cuticular collagen-encoding genes have been described for animal and plant parasites and for free-living nematodes. While examining Ascaris suum for neuropeptide genes, using PCR technology, we amplified one DNA fragment that when cloned and sequenced exhibited high percent identity with collagen genes from parasitic and free-living nematodes. We then tested the hypothesis that this was an Ascaris collagen gene fragment which could be used to identify and characterize collagen gene structure in A. suum. The DNA fragment was used as a probe to screen an A. suum cDNA library. One of the positive recombinant clones was sequenced (1,106 bp) and found to have high percent similarity and identity with other cuticular collagen genes from parasitic nematodes and Caenorhabditis elegans. Analysis of the gene structure indicated an open reading frame of 822 bp with a start codon in frame with a stop codon, and a consensus polyadenylation signal followed by a poly(A) tail. The structure of the gene (interrupted Gly-X-Y blocks, conserved cysteines and other amino acids) matched very closely what is known for collagen genes from C. elegans. Southern blot analysis indicated that this collagen gene is unique in the A. suum genome. Finally, Northern blot analysis of different A. suum larval stages and tissues indicated that this collagen gene is developmentally regulated. To our knowledge, this is the third A. suum cuticular collagen gene that has been cloned and the first one for which a cDNA sequence has been obtained.

Degree

Ph.D.

Advisors

Kazacos, Purdue University.

Subject Area

Molecular biology|Biochemistry|Veterinary services

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