טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentMaoz-Katz Michal
SubjectCloning of eIF2alpha from Leishmania donovani and Partial
Assessment of its Role in Differentiation
DepartmentDepartment of Biology
Supervisor Professor Emeritus Dan Zilberstein


Abstract

Protozoan parasites of the genus Leishmania are the causative agents of a wide range of visceral and cutaneous diseases in humans. Previous studies indicated that ethanol and azetidine-2-carboxylic acid (AZC), at sub lethal concentrations, induced promastigote to amastigote differentiation at acidic pH without raising the medium temperature. AZC and ethanol are well-established modulators of heat shock response by increasing cellular protein misfolding. We found that the initial steps of differentiation involved attenuation of protein synthesis. Ethanol and geldanamycin that inhibits hsp90 replaced heat in L. donovani differentiation signal, and at the same time cause down regulation of translation. The effects of heat shock modulators on L. donovani differentiation suggested that protein misfolding plays a role in mediating the differentiation signal. We hypothesized that like in other eukaryotics, attenuation of translation is achieved by the phosphorylation of the translation initiation factor eIF2α.  The aim of this work was to clone eIF2α and identify its role in translation during L. donovani differentiation. Using information in the L. major genome project, we cloned the eIF2α from L. donovani genome. Northern analysis indicated a single transcript of 1.5 nt that is constitutively expressed throughout differentiation. Southern analysis indicated a single copy of this gene. 2D gel analysis indicated a 37kDa band with pI ~5. The LdeIF2α shares ~60% of homology and ~30% identity with eIF2α of most organisms. Sequence analysis of LdeIF2α indicated that the highly conserved loop (amino acid residues 51-65) that contained the serine 51, which is the phosphorylation site, moved 20aa downstream. Moreover, the phosphorylating serine changed to threonine. The conserved box contained additional point mutations. This unusual box found to be conserved among all organisms of the family Trypanosomatidae.