Initiation of a Sarcocystis neurona expressed sequence tag (EST) sequencing project: a preliminary report

To accelerate genetic and molecular characterization of Sarcocystis neurona , the primary causative agent of equine protozoal myeloencephalitis (EPM), a sequencing project has been initiated that will generate approximately 70 00-8000 expressed sequence tags (ESTs) from this apicomplexan parasite. Pol y(A)(+) RNA was isolated from culture-derived S. neurona merozoites, and a cDNA library was constructed in a unidirectional lambda phage cloning vecto r. Sixty phage clones were randomly picked from the library, and the cDNA i nserts were amplified from these clones using the T3 and T7 primers that fl ank the multi-cloning site of the lambda vector. This analysis demonstrated that 100% (60/60) of the clones selected from this library contained recom binant cDNA inserts ranging in size from 0.4 to 4.0 kilobases (kb) with an average size of 1.23 kb. Single-pass sequencing from the 5' end of the 60 a mplified cDNAs produced high-quality nucleotide sequence from 53 of the clo nes. Comparison of these ESTs to the current gene databases revealed signif icant matches for 10 of the ESTs, six of which are similar to sequences fro m other Apicomplexa (i.e,, Toxoplasma gondii). Importantly, none of the EST s were of obvious mammalian origin, thus indicating that the cDNAs in this library were derived primarily from parasite mRNA and not from mRNA of the bovine turbinate host cells. Collectively, these data indicate that the des cribed cDNA library will provide an excellent substrate for generating a po rtion of the ESTs that are planned from S. neurona. This sequencing project will greatly hasten gene discovery for this protozoan pathogen thereby enh ancing efforts towards the development of improved diagnostics, treatments, and preventatives for EPM. In addition, the S. neurona ESTs will represent a significant contribution to the extensive database of sequences from the Apicomplexa. Comparative analyses of these apicomplexan sequences will lik ely offer a multitude of important information about the biology and evolut ionary history of this phylogenetic grouping of parasites. (C) 2001 Elsevie r Science B.V. All rights reserved.