Abstract
Heterologous reporter genes are widely used for the characterization of gene expression in many organisms. Particularly, constructs bearing reporter genes have greatly contributed to our understanding of gene regulation in kinetoplastids. In some specific circumstances, however, such heterologous reporter has a risk of resulting in irrelevant observations and conclusions, which are primarily due to the introduction of foreign sequence elements. This communication describes our recent experience using the chloramphenicol acetyltransferase (CAT) gene as a reporter for analysis of the translational regulation of HSP70 genes in Leishmania infantum. We show that chimeric mRNAs consisting of the CAT open reading frame (ORF) and the untranslated regions (UTRs) from HSP70-II genes behave differently as endogenous HSP70-II mRNAs and that this difference is due to the presence of CAT sequences. Thus, the main purpose of this communication is to alert researchers working in gene regulation to be cautious when interpreting results based on heterologous reporter genes.
References
Bellofatto V, Cross GA (1989) Expression of a bacterial gene in a trypanosomatid protozoan. Science 244:1167–1169
Boucher N, Wu Y, Dumas C, Dube M, Sereno D, Breton M, Papadopoulou B (2002) A common mechanism of stage-regulated gene expression in Leishmania mediated by a conserved 3′-untranslated region element. J Biol Chem 277:19511–19520
Campbell DA, Thomas S, Sturm NR (2003) Transcription in kinetoplastid protozoa: why be normal? Microbes Infect 5:1231–1240
Charest H, Zhang WW, Matlashewski G (1996) The developmental expression of Leishmania donovani A2 amastigote-specific genes is post-transcriptionally mediated and involves elements located in the 3′-untranslated region. J Biol Chem 271:17081–17090
Clayton CE (2002) Life without transcriptional control? From fly to man and back again. EMBO J 21:1881–1888
D’Orso I, Frasch AC (2001) Functionally different AU- and G-rich cis-elements confer developmentally regulated mRNA stability in Trypanosoma cruzi by interaction with specific RNA-binding proteins. J Biol Chem 276:15783–15793
Folgueira C, Quijada L, Soto M, Abanades DR, Alonso C, Requena JM (2005) The translational efficiencies of the two Leishmania infantum HSP70 mRNAs, differing in their 3′-untranslated regions, are affected by shifts in the temperature of growth through different mechanisms. J Biol Chem 280:35172–35183
Gorman CM, Moffat LF, Howard BH (1982) Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol 2:1044–1051
Laban A, Wirth DF (1989) Transfection of Leishmania enriettii and expression of chloramphenicol acetyltransferase gene. Proc Natl Acad Sci USA 86:9119–9123
Larreta R, Soto M, Quijada L, Folgueira C, Abanades DR, Alonso C, Requena JM (2004) The expression of HSP83 genes in Leishmania infantum is affected by temperature and by stage-differentiation and is regulated at the levels of mRNA stability and translation. BMC Mol Biol 5:3
Lukes J, Paris Z, Regmi S, Breitling R, Mureev S, Kushnir S, Pyatkov K, Jirku M, Alexandrov KA (2006) Translational initiation in Leishmania tarentolae and Phytomonas serpens (Kinetoplastida) is strongly influenced by pre-ATG triplet and its 5′ sequence context. Mol Biochem Parasitol 148:125–132
Mayho M, Fenn K, Craddy P, Crosthwaite S, Matthews K (2006) Post-transcriptional control of nuclear-encoded cytochrome oxidase subunits in Trypanosoma brucei: evidence for genome-wide conservation of life-cycle stage-specific regulatory elements. Nucleic Acids Res 34:5312–5324
McNicoll F, Muller M, Cloutier S, Boilard N, Rochette A, Dube M, Papadopoulou B (2005) Distinct 3′-untranslated region elements regulate stage-specific mRNA accumulation and translation in Leishmania. J Biol Chem 280:35238–35246
Quijada L, Soto M, Alonso C, Requena JM (1997) Analysis of post-transcriptional regulation operating on transcription products of the tandemly linked Leishmania infantum hsp70 genes. J Biol Chem 272:4493–4499
Schurch N, Furger A, Kurath U, Roditi I (1997) Contributions of the procyclin 3′ untranslated region and coding region to the regulation of expression in bloodstream forms of Trypanosoma brucei. Mol Biochem Parasitol 89:109–121
Stanton JD, Mensa-Wilmot K (2006) AUG-proximal nucleotides regulate protein synthesis in Leishmania tropica. Mol Microbiol 61:691–703
Weston D, La Flamme AC, Van Voorhis WC (1999) Expression of Trypanosoma cruzi surface antigen FL-160 is controlled by elements in the 3′ untranslated, the 3′ intergenic, and the coding regions. Mol Biochem Parasitol 102:53–66
Wu Y, El Fakhry Y, Sereno D, Tamar S, Papadopoulou B (2000) A new developmentally regulated gene family in Leishmania amastigotes encoding a homolog of amastin surface proteins. Mol Biochem Parasitol 110:345–357
Zhang K, Kurachi S, Kurachi K (2003) Limitation in use of heterologous reporter genes for gene promoter analysis. Silencer activity associated with the cloramphenicol acetyltransferase reporter gene. J Biol Chem 278:4826–4830
Zilka A, Garlapati S, Dahan E, Yaolsky V, Shapira M (2001) Developmental Regulation of Heat Shock Protein 83 in Leishmania. J Biol Chem 276:47922–47929
Acknowledgments
This work was supported by grants from ISCIII-RETIC RD06/0021/0008-FEDER and Ministerio de Educación y Ciencia (BFU2006-08346). An institutional grant from Fundación Ramón Areces is also acknowledged.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Folgueira, C., Requena, J.M. Pitfalls of the CAT reporter gene for analyzing translational regulation in Leishmania . Parasitol Res 101, 1449–1452 (2007). https://doi.org/10.1007/s00436-007-0640-6
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00436-007-0640-6