METHODS TO PREPARE RNA AND TO ISOLATE DEVELOPMENTALLY-REGULATED GENESFROM EIMERIA

Coccidians represent a large class of important intracellular parasite s that traverse multiple developmental stages that are distinct and re quired to complete the life cycle. The biochemical details underlying the regulation of transformation Front one developmental form to the n ext are limited and the study of such details presents unique obstacle s. However, the genetic program is critical and may provide a basis fo r understanding the biology of these organisms in addition to the oppo rtunity to suppress development and infection. We provide a basic over view of several strategies, including previously unpublished results, used by this laboratory to isolate stage-specific genes from Eimeria b ovis, Additionally, we have included detailed discussions that summari ze the associated advantages and disadvantages of each as applied to c occidia and potentially to other parasites in the phylum Apicomplexa. Given that the purification of sufficient quantities of high-quality R NA is vital, we have included detailed protocols for the isolation of RNA from various parasite stages. Also included is a detailed protocol to apply mRNA differential display to investigate stage-specific deve lopmental regulation. (C) 1997 Academic Press.