Int J Biochem Mol Biol 2013;4(2):73-82

Original Article
Identification of coding sequences from a freshly prepared Trypanosoma brucei brucei
expression library by polymerase chain reaction

Uthman Okalang, Ann Nanteza, Enock Matovu, George W Lubega

Department of Biology, Faculty of Science and Education, Busitema University, Tororo, Uganda; Department of Biomolecular Resources and
Biolaboratory Sciences, Animal Resources and Biosecurity, College of Veterinary Medicine, Makerere University, Kampala, Uganda

Received May 25, 2013; Accepted July 6, 2013; Epub July 29, 2013; Published August 15, 2013

Abstract: Animal African trypanosomiasis (AAT) also known as Nagana is a devastating disease among domestic animals in large parts of
Sub-Saharan Africa causing loses in milk and meat production as well as traction power. However, there is currently no commercial vaccine
against AAT. The parasites have also developed resistance to some of the drugs in use. Moreover, the use of affordable computer-aided wet
bench methods in the search for vaccine and/or new drug targets against this disease have not yet been fully explored in developing countries.
This study, therefore, explored the use of PCR to screen a freshly prepared bloodstream form Trypanosoma brucei brucei (T. b. brucei)
expression library for coding sequences followed by bioinformatics analyses specifying the functions and importance of these proteins to
parasite survival. Eleven protein coding sequences were identified from twenty nine purified clones. The putative retro transposon hot spot
protein 4 (RHSP 4) was the only protein with a fully annotated DNA sequence. All the others were hypothetical or had partial or unqualified
annotations. RHSP 4 and pyruvate dehydrogenase E1 component, alpha sub-unit (PDE1α) are involved in aerobic respiration whereas
succinyl-Co A-3-ketoacid-coenzyme A transferase mitochondrial precursor (SKTMP) is predicted to be involved in ketone body catabolism.
Cystathionine beta-synthase (CBS) and alpha-1,3-mannosyltransferase (αMT) have been predicted in cysteine biosynthesis and vesicular
transport respectively. The functions of the hypothetical proteins encountered have neither been experimentally determined nor predicted. We
hypothesize that both CBS and PDE1α are good drug targets. Overall, about 300 plates are required to PCR screen the entire Trypanosoma
brucei genome in approximately eight months. This method is therefore, applicable and affordable in the search for new drug targets under
conditions of limited resources among developing countries. (IJBMB1305003).

Keywords: Nagana, drug targets, proteins, expression library, PCR, bioinformatics

Address correspondence to: Mr. Uthman Okalang, Department of Biology, Faculty of Science and Education, Busitema University, P.O Box
236, Tororo, Uganda. Phone: +256 772 968176; Fax: +256 454436517; E-mail: uokalang@sci.busitema.ac.ug
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