Procyclic Acidic Repetitive Protein (PARP) Genes Located in an Unusually Small α-Amanitin-Resistant Transcription Unit: PARP Promoter Activity Assayed by Transient DNA Transfection of Trypanosoma brucei

Rudenko, Gloria; Blancq, S Le; Smith, Janet L.; Lee, M G; Rattray, Alison J.; Ploeg, L H Van der

doi:10.1128/mcb.10.7.3492-3504.1990

Cited by 10 publications

(2 citation statements)

References 49 publications

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“…Bellofatto and Cross (3) were the first to develop a transformation system for a kinetoplastid protozoan, identifying the sequences flanking the miniexon gene of Leptomonas seymouri necessary for CAT expression. Recently the applicability of the CAT assay to the analysis of trypanosome promoter regions has been confirmed by the functional characterization of the T. brucei procyclin gene promoter (7,34). Here, as well as identifying the VSG gene promoter, we have shown that this technique can be used to identify 3' sequences which may be important in RNA processing in trypanosomes.…”

Section: Discussionmentioning

confidence: 78%

“…This region seems to contain all of the sequence elements necessary for initiation of transcription, as it was sufficient for CAT expression. It does not appear to share any extensive homology with known promoters, notably those of the T. brucei ribosomal DNA (41) and procyclin genes (7,28,34), which, like the VSG gene, are transcribed by an RNA polymerase resistant to a-amanitin. The only noteworthy feature within this sequence is the presence of three TAT TAC direct repeats 23 to 61 bp upstream of the putative transcription start site.…”

Section: Discussionmentioning

confidence: 99%

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Transient Activity Assays of the Trypanosoma brucei Variant Surface Glycoprotein Gene Promoter: Control of Gene Expression at the Posttranscriptional Level

Jefferies¹,

Tebabi²,

Pays³

1991

Molecular and Cellular Biology

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The putative promoter of the variant surface glycoprotein (VSG) gene of Trypanosoma brucei was cloned into a plasmid containing the chloramphenicol acetyltransferase (CAT) gene. After electroporation into trypanosomes, this construct directed the expression of the CAT reporter gene. The essential region for promoter activity was found to reside within 88 bp upstream of the putative transcription start site. Transcription of the CAT construct occurred at approximately the same level in both bloodstream and procyclic forms and was resistant to alpha-amanitin. However, CAT expression appeared to be modulated in the two forms of the parasite. Sequences 3' to the gene seemed to be important in this respect, as CAT activity in bloodstream forms was readily detectable only when the 3' region of a VSG cDNA was placed downstream of the CAT gene. Two separate VSG gene promoter sequences, both cloned from T. brucei AnTat 1.3A, were equally able to direct CAT expression, which suggests that there are a number of potential VSG gene promoters in the genome, although usually only one expression site is fully active at any one time.

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Section: Discussionmentioning

confidence: 78%

Section: Discussionmentioning

confidence: 99%

Transient Activity Assays of the Trypanosoma brucei Variant Surface Glycoprotein Gene Promoter: Control of Gene Expression at the Posttranscriptional Level

Jefferies¹,

Tebabi²,

Pays³

1991

Molecular and Cellular Biology

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Sorting Signals Required for Trafficking of the Cysteine-Rich Acidic Repetitive Transmembrane Protein in Trypanosoma brucei

et al. 2006

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The African trypanosome Trypanosoma brucei is a unicellular eukaryotic parasite, causing sleeping sickness in humans and related diseases in livestock. T. brucei has an intricate life cycle, alternating between the bloodstream form, which is free living in the bloodstream of a mammalian host, and the procyclic (or insect) form, which lives in the transmitting vector, the tsetse fly. By undergoing antigenic variation of the variant surface glycoprotein (VSG) coat, which covers the entire cell surface of the bloodstream form, the parasite survives the host immune attack (10, 16). During differentiation into the procyclic form in the midgut of the tsetse fly, the parasite sheds its VSG coat and replaces it with the procyclin (or the procyclic acidic repetitive protein) coat (42).T. brucei, an extracellular organism, is dependent on hostderived nutrients for its growth and development. Underneath a large part of the plasma membrane is the subpellicular microtubule sheath. This distinct surface structure network prohibits the active pinocytosis of plasma membrane as a means for nutrient uptake. The only surface domain where the microtubule network is absent is the flagellar pocket (FP); the FP is an invagination of the plasma membrane, where the flagellum extends from the cell (4,49,50,53). In trypanosomatids, endocytosis and exocytosis are restricted to the FP. Receptors for the uptake of macromolecules are confined to the FP (39, 53). All vesicular trafficking in this elongated and highly polarized parasite takes place between the nucleus and the FP (11,19,31,39,40,53). Membrane-bound proteins, once synthesized, travel from the endoplasmic reticulum (ER) to the Golgi/trans-Golgi network (TGN) and then to the FP membrane. From the pocket, surface coat proteins and some other invariant surface proteins rapidly spread over the entire cell surface, while receptors for the uptake of macromolecules are retained in the FP. We have been investigating mechanisms involved in the sorting of membrane proteins to and from the FP in trypanosomes, a phenomenon that is poorly understood. In the past, most studies on protein trafficking in trypanosomes have focused on the bloodstream form, due to its unique high rate of endocytosis (2,14,17,33,34,51,52). Endocytosis at the FP of the bloodstream form is mediated by large clathrincoated vesicles (2,(18)(19)(20)32). These vesicles are undetectable in the procyclic form, where endocytosis occurs in a much less efficient manner than in the bloodstream form (26).Little is known about the molecular details involved in protein sorting during endocytosis and along the secretory pathway in trypanosomes, though trafficking machinery similar to that of higher eukaryotes may operate (33,34,40). Based on studies in mammalian systems, a general principle underlying directional transfer is that the sorting information (sorting signals or determinants), usually distinct amino acid sequences or structural domains, resides within the sorted protein. The interaction of sorting determinants with specif...

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Active VSG Expression Sites in Trypanosoma brucei Are Depleted of Nucleosomes

2010

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In eukaryotes nuclear DNA is packaged into linear arrays of nucleosomes, which provides one of the main determinants of accessibility to DNA binding proteins (37,60,68,82). Nucleosomes consist of ϳ146 bp of DNA wrapped around a histone octamer composed of two copies each of histones H2A, H2B, H3, and H4 (44). The ability to change how DNA is packaged within nucleosomes allows variation in the accessibility of different DNA binding sites and permits fine modulation of promoter activity (33). Recently, there have been major advances in our understanding of how chromatin structure impacts the regulation of RNA polymerase II (Pol II) transcription units. New developments (72) have enabled the determination of the global organization of nucleosomes in organisms including budding yeast, Drosophila, and humans (3,35,36,48,71,83). These studies have shown evidence for nucleosome depletion at transcriptionally active regulatory regions, with the level of nucleosome occupancy inversely proportional to the rate of transcription initiation at the promoter (3, 36, 83). However, nucleosome remodeling at promoters does not always appear to be simply a consequence of transcriptional activity but is also thought to mechanistically regulate transcription through modulating the access of trans-acting factors (74).Our understanding of the role that chromatin structure plays in the regulation of RNA Pol I transcription has relatively lagged behind that of Pol II (reviewed in references 6, 21, and

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Procyclic Acidic Repetitive Protein (PARP) Genes Located in an Unusually Small α-Amanitin-Resistant Transcription Unit: PARP Promoter Activity Assayed by Transient DNA Transfection of Trypanosoma brucei

Cited by 10 publications

References 49 publications

Transient Activity Assays of the Trypanosoma brucei Variant Surface Glycoprotein Gene Promoter: Control of Gene Expression at the Posttranscriptional Level

Transient Activity Assays of the Trypanosoma brucei Variant Surface Glycoprotein Gene Promoter: Control of Gene Expression at the Posttranscriptional Level

Sorting Signals Required for Trafficking of the Cysteine-Rich Acidic Repetitive Transmembrane Protein in Trypanosoma brucei

Active VSG Expression Sites in Trypanosoma brucei Are Depleted of Nucleosomes

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