ESSENTIAL COMPONENTS OF THE MINI-EXON GENE PROMOTER IN THE TRYPANOSOMATID LEPTOMONAS-SEYMOURI

In members of the Trypanosomatidae family of parasitic protozoa, the m ini-exon (MX) genes encode the mini-exon donor RNA (medRNA) that contr ibutes a small, 39-nt exon to all pre-mRNAs during mRNA maturation. Pr eviously we have shown that a single copy of a MX gene can be expresse d continuously from a stable episome transfected into the monogenetic trypanosomatid Leptomonas seymouri. We now identify components of the MX gene promoter. A series of 10-bp block substitution mutations in a tagged MX gene were transfected into Leptomonas on an episomal vector. Expression of tagged and endogenous medRNA was assessed in stably tra nsformed clonal cell populations. Results show that less than half of the 757-bp MX gene is necessary for medRNA transcription and that the key components of the MX gene promoter lie within the proximal 70-bp s equence upstream from the transcription initiation site. Transcription requires several sequence-specific blocks within this 70-bp region. L eptomonas cell extracts contain protein(s) that appear to interact wit h a subset of these sequences in gel mobility shift assays. All trypan osomatid MX genes contain an AT-rich region at the + 10 to + 20 positi on within the transcribed region of the MX gene. Mutagenesis of this r egion within an episomal copy of the MX gene did not block tagged medR NA synthesis but did cause a 10-fold increase in the steady-state amou nt of endogenous medRNA.