Leptomonas seymouri, Trypanosoma brucei:A Method for Isolating Trypanosomatid Nuclear Factors Which BindT. bruceiSingle-Stranded g-Rich Telomere Sequence

Abstract

Sequential expression of variant surface glycoproteins (VSG) inTrypanosoma bruceiis the basis of antigenic variation which is essential for parasite survival. Telomere distal copies of VSG genes, so-called basic copies, provide a repository of VSG sequence information for variability, but actively expressed copies are found only at subtelomeric regions of chromosomes. Of eight or so expression sites (ES) in theT. bruceigenome, only one is active at one time. Movement of a basic copy VSG gene to an ES requires a recombination event of unknown mechanism. The properties of telomeres have been speculated to be important for control of VSG expression or basic copy mobilization, prompting us to begin to investigate telomere-binding proteins in trypanosomatids. TheT. bruceitelomere sequence is known, facilitating design of synthetic telomeric DNAs. Here we describe a method for preparation of active trypanosomatid nuclear extracts. We show that inT. bruceiandLeptomonas seymouri,factors can be detected which bind a g-rich single-strand telomere sequence based on theT. bruceitelomere. TheL. seymouritelomere-binding factor, LST-1, dissociates in the presence of high salt to produce a core factor, LST-2, migrating similarly to theT. bruceitelomere-binding factor TBT-1. The affinity of LST-2 and TBT-1 for DNA under high salt conditions is characteristic of telomere proteins.