A search for glucose transporters in trypanosoma brucei leads to identification of collagen like DNA sequences

Abstract

The search for glucose transporter in T.brucei was based upon the fact that the bloodstream forms (BF)of the parasite are highly dependent onglucose uptake for their energy metabolism. Previous work has shown that the BF of T.brucei take up glucose across the plasma membrane by a facilitated diffusion mechanism. The hypothesis put forth in this project, was that the putative gene(s) could be cloned using the Polymerase Chain Reaction (PCR), by taking advantage of the high degree of sequence homology within the conserved stretches of amino acid regions of glucose transporters from prokaryotes and eukaryotes, including a closely related trypanosomatid Leishmania. Oligonucleotide primers constructed from the evolutionary conserved regions of such genes, were used in a PCR using either the genomic DNA or cDNA templates of BF and procyclic (PC) forms. The PCR products as well as the oligonucleot4des from the conserved regions were used as probes to screen T. brucei BF and PC lambda gtlO cDNA librries. Several clones were isolated following the screening exercise. The PCRproduotsand the positive clones were subcloned into M13 phage and partially-sequenced using Sanger's dideoxy chain termination method. None had glucose transporter orglucose transporter related sequences after computer homology searches. However some cDNA clones isolated after screening with the PCR . products generated from genomic DNA templates, had open reading frames having homology to co.lLaqeri of other organisms. The homology is highest in Herpesvirus.collagen-like protein which has 62.4% nucleotide identity. The results of the study showed that the primers constructed were unable to isolate glucose transporter(s) In the trypanosoma brucei, but may be suitable to isolate genomic sequences having collagen like sequences.