The stage-regulated expression of Leishmania mexicana CPB cysteine proteases is mediated by an intercistronic sequence element.
The tandemly arranged CPB genes of Leishmania mexicana are polycistronically transcribed and encode cysteine proteases that are differentially stage-specific; CPB1 and CPB2 are expressed predominantly in metacyclics, whereas CPB3-CPB18 are expressed mainly in amastigotes. The mechanisms responsible for this differential expression have been studied via gene analysis and re-integration of individual CPB genes, and variants thereof, into a CPB-deficient parasite mutant. Comparison of the nucleotide sequences of the repeat units of CPB1 and CPB2 with CPB2.8 (typical of CPB3-CPB18) revealed two major regions of divergence as follows: one of 258 base pairs (bp) corresponding to the C-terminal extension of CPB2.8; another, designated InS, of 120 bp, with insertions totaling 57 bp, localized to the intercistronic region downstream of CPB1 and CPB2. Cell lines expressing CPB2.8 or CPB2 with the 3'-untranslated region and intercistronic sequence of CPB2.8 showed up-regulation in amastigotes. Conversely, metacyclic-specific expression occurred with CPB2 or CPB2.8 with the 3'-untranslated region and intercistronic sequence of CPB2. Moreover, the InS down-regulated expression in amastigotes of a reporter gene integrated into the CPB locus. It is proposed that the InS mediates metacyclic-specific stage-regulated expression of CPB by affecting the maturation of polycistronic pre-mRNA. This is the first well defined cis-regulatory element implicated in post-transcriptional stage-specific gene expression in Leishmania.
Brooks
D R
DR
Wellcome Centre for Molecular Parasitology, University of Glasgow, the Anderson College, 56 Dumbarton Road, Glasgow G11 6NU, Scotland, United Kingdom.
Denise
H
H
Westrop
G D
GD
Coombs
G H
GH
Mottram
J C
JC
D020413:3' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D015536:Down-Regulation; D005780:Gelatin / Q000737:chemistry; D005786:Gene Expression Regulation; D005796:Genes; D017930:Genes, Reporter; D007894:Leishmania mexicana / Q000201:enzymology*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011061:Poly A / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection
D020413:3' Untranslated Regions; D012333:RNA, Messenger; D011061:Poly A; D005780:Gelatin; D015500:Chloramphenicol O-Acetyltransferase; D003546:Cysteine Endopeptidases