Preparation of DNA and RNA from Trypanosoma brucei

The protozoan Trypanosoma brucei is the most destructive parasite of domestic livestock in sub-Saharan Africa. The parasite lives extracellularly in its two hosts; alternately in the bloodstream of a mammal, and in the midgut and subsequently the salivary glands of the tsetse fly (Glossina spp.) vector. Most laboratory work has concentrated on two of these life cycle stages; rodent-adapted blood- stream forms propagated by syringe passage, and cultured procyclic forms representing the form found in the tsetse fly midgut. The blood- stream form has been intensively studied in order to describe and elucidate the control of antigenic variation that occurs through successive use of a series of genes, each encoding an antigenically distinct variant specific glycoprotein (VSG) (1 ,2 ). In this context VSGs were the first parasite antigens to be characterized at the level of cDNA and genomic clones (3 –5 ). Such a characterization is, of course, dependent on the ability to obtain undegraded RNA of high quality. The use of VSG cDNAs to probe Southern blots of genomic DNA provided the demonstration that in most cases antigenic variation is the result of the duplicative transposition of a VSG gene to an expression site (6 –8 ). Subsequently Northern blotting (9 ), primer extension (9 –11 ), and S1 mapping (11 ) of RNA were used to demonstrate the presence of a mini-exon at the 5′ end of all mRNAs, leading to the discovery of trans-splicing.