Identification of gene expression elements in Histomonas meleagridis using splinkerette PCR, a variation of ligated adaptor PCR

Abstract

Histomonas meleagridis is the causative agent of blackhead disease in gallinaceous birds, but little genetic information exists for this organism. The complete genome for this protozoan is unsequenced. The only available sequence information is for coding portions of genes. No information is available for expression elements. In this study, we demonstrate that splinkerette PCR procedure, a variation of ligated adaptor PCR, can be used to identify regions upstream and downstream of known coding sequences. Using this technique, we isolated the upstream sequence of 2 beta-tubulin genes. With sequence analysis of their upstream regions, we identified their upstream intergenic regions and 2 different open reading frames. The intergenic region contained putative polyadenylation and cleavage signals and initiator elements. Our research demonstrates that the use of splinkerette PCR is a valuable tool to identify regions of unknown DNA that are 5’ or 3’ to known sequences in parasites whose genomes remain unsequenced. The identification of the expression elements of H. meleagridis will provide tools for future studies on its gene expression.