RNA polymerase I promoter and splice acceptor site recognition affect gene expression in non-pathogenic Leishmania species

• Published in: Memórias do Instituto Oswaldo Cruz Vol. 102; no. 7
• Main Authors: Orlando, Tereza Cristina, Mayer, Mário Gustavo, Campbell, David A, Sturm, Nancy R, Floeter-Winter, Lucile Maria
• Format: Journal Article
• Language: English
• Published: Fundação Oswaldo Cruz, Fiocruz 05-03-2008
• Subjects: intergenic spacer - heterologous protein system - polypyrimidine tract - trans-splicing acceptor site
• ISSN: 1678-8060

Leishmania ( Sauroleishmania) tarentolae has biotechnological
potential for use as live vaccine against visceral leishmaniasis and as
a system for the over expression of eukaryotic proteins that possess
accurate post-translational modifications. For both purposes, new
systems for protein expression in this non-pathogenic protozoan are
necessary. The ribosomal RNA promoter proved to be a stronger
transcription driver since its use yielded increased levels of
recombinant protein in organisms of both genera Trypanosoma or
Leishmania. We have evaluated heterologous expression systems using
vectors with two different polypyrimidine tracts in the splice acceptor
site by measuring a reporter gene transcribed from L. tarentolae RNA
polymerase I promoter. Our data indicate that the efficiency of
chloramphenicol acetyl transferase expression changed drastically with
homologous or heterologous sequences, depending on the polypyrimidine
tract used in the construct and differences in size and/or distance
from the AG dinucleotide. In relation to the promoter sequence the
reporter expression was higher in heterologous lizard-infecting species
than in the homologous L. tarentolae or in the mammalian-infecting L.
(Leishmania) amazonensis.