The structure and transcription of an element interspersed between tandem arrays of mini-exon donor RNA genes inTrypanosoma brucei

Abstract

Messenger RNAs in Trypanosoma brucei brucei have the same 35 bases at the 5' end. These 35 bases are not encoded contiguously in the genome, but are donated from a 140 base RNA (the mini-exon donor RNA). The mini-exon donor RNA (medRNA) is encoded by 1.35 kbp genes that occur in tandem repeats. A DNA element that is associated with mini-exon donor RNA medRNA genes has been identified and characterised by restriction enzyme mapping and partial sequencing. This element (the medRNA gene associated element: MAE) varies in length between 5.5 and 7 kbp. There are between 20 and 40 copies of the element per haploid genome. In clones of genomic DNA MAEs occurred between two medRNA gene arrays and on both sides of a medRNA gene array. The MAEs were always in the same orientation with respect to the medRNA genes. It is proposed that in the genome MAEs are interspersed between tandem arrays of medRNA genes. The transcription of the element has been investigated. Low levels of a 70-80 base transcript derived from a small part of MAE were detected in steady state RNA. Nuclear run off transcript studies indicated that MAEs were transcribed at high levels and that they possibly contain at least one start and stop of transcription.

Duplication and transcription of procyclin genes inTrypanosoma brucei

“…Hybridisation was carried out as described previously (19), except that oligonucleotides were hybridised at 370C. Washing conditions are described in the figure legends.…”

“…Plasmid probes were labelled with 32p by nick translation (20); oligonucleotides were end-labelled with 32p using polynucleotide kinase (21). Isolation of genomic clones and construction of mapS The constuction of a genomic library from ILTat 1.21 DNA in XEMBL 3 has been described (19). Genomic clones of procyclin were selected by hybridisation with the cDNA clone pPRO2001 (4).…”

“…DNA (17) and RNA (18) were isolated as described previously. Nuclear run-ons were performed as described by Carrington et al (19).…”

A novel strategy to identify the location of necessary and sufficient cis-acting regulatory mRNA elements in trypanosomes

“…Northern blotting was as described (Carrington et al 1987); all blots were washed in 15 mM sodium chloride, 1.5 mM tri-sodium citrate (0.13 SSC), and 0.1% sodium dodecyl sulphate at 60 C.…”

A new member of a family of site-specific retrotransposons is present in the spliced leader RNA genes of Trypanosoma cruzi.

“…In all trypanosomatid species examined, multiple copies of SL-RNA genes exist in tandem arrays in discrete genomic loci (2,16,22,39,40). It has previously been reported that in the African trypanosome Trypanosoma brucei gambiense, several copies of these SL-RNA genes are interrupted by a 5.5-to 7.0-kb retrotransposon, SLACS (spliced leader-associated conserved sequence) (2,3) or MAE (miniexon donor RNA gene-associated element) (11). A similar 4.0-kb element, CRE1, has been described in the distantly related mosquito trypanosomatid Crithidia fasciculata (23).…”