Cloning of the promoter regions of Trypanosoma brucei and Trypanosoma congolense cysteine protease genes.

Abstract

Trypanosoma brucei and T. congolense are protozoan parasites that infect humans, domestic livestock and wildlife in Africa. These parasites undergo complex morphological and biochemical changes, during the various stages of their life cycle. These changes correlate with alterations in the levels of trypanosomal lysosomal cysteine proteases, suggesting a role for transcriptional regulation of the cysteine protease in these parasites. The mechanism of this regulation is not yet understood nor have the promoter regions of the cloned trypanosome cysteine protease genes been investigated. This study involved an attempt to clone the T. brucei and T. congolense DNA fragments containing the promoter regions as the initial step in the investigation of the control elements of the cysteine protease gene. Trypanosomes were isolated from infected rat blood employing a combination of the methods of isopicnic isolation on Percoll gradients and DEAE-cellulose anion exchange resin chromatography. Approximately 5 x 10⁹ viable trypanosome cells were isolated from the infected rat blood and chromosomal DNA (approximately 500 μg) was extracted by alkaline-lysis method. Trypanosome genomic libraries were initially constructed in Eschericia coli HB101 employing the positive selection vector pEcoR251. The Trypanosoma brucei pEcoR251 library contained 6 000 recombinants and the Trypanosoma congolense library contained 15 000 recombinants. Plasmid DNA was then extracted from pools of recombinants, employing the alkaline-lysis method, digested with EcoRl restriction endonuclease and resolved by agarose gel electrophoresis. After Southern hybridisation, the pEcoR251 libraries did not reveal any putative clones containing the fragment of interest when probed with both an oligonucleotide probe and the PCR generated dsDNA probe. Genomic libraries were then constructed in the phagemid pUC119. The T. brucei and T. congolense genomic libraries contained 33 000 and 27 000 recombinants respectively. Recombinants from the T. brucei and T. congolense libraries were pooled in lots of 400 and 300 respectively. Of the 80 T. brucei plasmid pools that were screened 30 pools contained fragments that hybridised with the probe whilst 12 pools from the 90 T. congolense library pools that were screened contained fragments that hybridised with the probe. Putative clones identified appeared to contain inserts, ranging between two and seven kb in size. A partial T. congolense library consisting of approximately 12 pools was screened by colony hybridisation for identification of individual clones and 76 putative clones were identified. After confirmation of these putative clones on a dot blot using a DIG-labelled dsDNA probe, a selection of 30 putative clones were subjected to Southern hybridisation using a DIG-labelled DNA probe. Following Southern hybridisation 23 putative clones were identified to contain DNA inserts of interest in the range of two to seven kb. Five clones, designated pCPC1, pCPC2, pCPC3, pCPC4 and pCPC5 were then selected for further restriction mapping. Clone pCPC4 contains a seven kb fragment of T. congolense genomic DNA. A partial T. brucei library consisting of approximately 30 pools was screened by colony hybridisation for the identification of individual putative clones. Although plasmid pools containing putative clones were identified repeatedly by Southern blotting and DNA/DNA hybridisation, it was not possible to identify individual putative clones following transformation into E. coli MV1184 and colony hybridisation.