Anatomy of the parp gene promoter of Trypanosoma brucei.

Abstract

While growing in the tsetse fly, Trypanosoma brucei expresses a major surface glycoprotein, the procyclic acidic repetitive protein (PARP). The parp genes are transcribed by an alpha‐amanitin‐resistant RNA polymerase. We have determined the sequence requirements for parp promoter activity. Studies of RNA produced from input DNA in transiently transfected trypanosomes indicate that the RNA is correctly processed by trans‐splicing and polyadenylation. Deletion analyses show that 330 bp are sufficient for full promoter and splicing activity and that the promoter structure is complex, involving at least three elements whose mutual spacing is important. Mutagenesis pin‐pointed two sequences vital for promoter activity; neither bears any resemblance to known prokaryotic or eukaryotic promoter elements.

In vivo import of firefly luciferase into the glycosomes of Trypanosoma brucei and mutational analysis of the C-terminal targeting signal.

“…This sequence contains a unique Mlu I restriction endonuclease site that allows linearization of the vector DNA and insertion of the entire plasmid into a tubulin locus of T. brucei by homologous recombination . To construct pTSA-NEO2, the multicloning site of pBluescript (Stratagene, San Diego, CA) was purified as a 90 bp Apa I to Sst I fragment, blunt-ended with T4 polymerase (Sambrook et al, 1989), and inserted into the blunt-ended HindIll site of pJP25 (Sherman et al, 1991) between the splice acceptor site and the CAT gene, resulting in the plasmid pJP25.MCS1. The orientation of the insert is Xho I to Sst II.…”

Function of N‐terminal import signals in trypanosome microbodies

“…To construct the expression vector pAldE a PCR was performed with pAldl7 [18] as template, The primers CZ031 5'TTCAC-AAGCTTCACAATGTCCAAGCGTGTTGAAGTT3' and CZ049 5'ATATCCAGTGATTTTTTTCTCGAGCGCTTCATATGGCGTC-TTCAG3' (restriction sites underlined) were used to amplify a fragment encoding the 24 N-terminal amino acids of aldolase joined to the first six amino acids of CAT starting with glutamic acid (the amino acid immediately following the initiator methionine), pJP44 [19] was used as template in a second PCR to amplify the CAT gene with the primers CZ030 5'CTGAAGACGCCATATGAAGCGCTCGAG-AAAAAAATCACTGGATAT3' and CC12 5'GTTTCGTTCCTCC-GAGGCGC3'. The product is a CAT coding region that can hybridise to the first via a junction segment containing an XhoI site.…”

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

“…1C-1), as possible explanation for the efficiency of that construct. We constructed another vector, pAB3.0tbCAT, replacing the synthetic acceptor site by the splice acceptor site (tb) of Trypanosoma brucei EP1 (or PARP B) gene (Sherman et al 1991), which contains a longer polypyrimidine tract (3 from the ETS plus 28 from tb, see Fig. 1C-3), positioned upstream the CAT gene.…”

“…Moreover, CAT activity in heterologous lizard-infecting species L. hoogstraali was 4.5 fold higher than observed in the homologous species, indicating that expression driven (Orlando et al 2002); B: schematic of plasmids: the two constructions containing the L. tarentolae IGS/ ETS (white box) upstream to the synthetic (st) or to T. brucei derived (tb) splice acceptor site followed by CAT coding sequence (gray box); C: sequence of ETS/acceptor site junction of the plasmid constructions. 1: construct pLa∆14ASCAT: 3'end of L. (Leishmania) amazonensis ETS-synthetic splice acceptor site (Uliana et al, 1996); 2: construct pAB3.0stCAT: 3' end of L. tarentolae ETS-synthetic splice acceptor site; 3: construct pAB3.0tbCAT: 3'end of L. tarentolae ETS-T. brucei derived splice acceptor site (Sherman et al 1991). Normal characters correspond to the ETS sequence; boxes indicate additional run of pyrimidines in the 3'ETS; letters after the dash indicate the polypyrimidine tract and splice acceptor site with AG indicated by underlining.…”