In vivo epitope tagging of Trypanosoma brucei genes using a one step PCR-based strategy

Shen, Suqin; Arhin, George K.; Ullu, Elisabetta; Tschudi, Christian

doi:10.1016/s0166-6851(00)00383-2

Cited by 92 publications

(110 citation statements)

References 9 publications

Supporting

Mentioning

104

Contrasting

Order By: Relevance

“…Our MEPS estimate is somewhat shorter than that made, by very different means, by Papadopoulou and Dumas (45) in Leishmania. Transformation experiments have shown that T. brucei can recombine shorter substrates than the MEPS length determined here, and this can be quite efficient (70,71), but our previous work has suggested that these reactions may be catalyzed by a distinct recombination pathway (see below) from the RAD51-dependent reactions in this present study. Why T. brucei RAD51-dependent recombination requires relatively long substrates awaits analysis.…”

Section: Discussionmentioning

confidence: 43%

Mismatch Repair Regulates Homologous Recombination, but Has Little Influence on Antigenic Variation, in Trypanosoma brucei

Bell

McCulloch

2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

Antigenic variation is critical in the life of the African trypanosome, as it allows the parasite to survive in the face of host immunity and enhance its transmission to other hosts. Much of trypanosome antigenic variation uses homologous recombination of variant surface glycoprotein (VSG)-encoding genes into specialized transcription sites, but little is known about the processes that regulate it. Here we describe the effects on VSG switching when two central mismatch repair genes, MSH2 and MLH1, are mutated. We show that disruption of the parasite mismatch repair system causes an increased frequency of homologous recombination, both between perfectly matched DNA molecules and between DNA molecules with divergent sequences. Mismatch repair therefore provides an important regulatory role in homologous recombination in this ancient eukaryote. Despite this, the mismatch repair system has no detectable role in regulating antigenic variation, meaning that VSG switching is either immune to mismatch selection or that mismatch repair acts in a subtle manner, undetectable by current assays.African trypanosomes are protistan parasites that infect mammals and are considered to have diverged early in eukaryotic evolution (1), prior to the radiation of animals, plants, and fungi. In the mammal Trypanosoma brucei resides in the bloodstream and tissue fluids, where it is subject to immune attack. To evade immune killing, it undergoes antigenic variation, a strategy for changing surface coats found in a diverse range of microbes (2, 3). The surface coat of T. brucei is composed of variant surface glycoprotein (VSG) 1 (4). VSG genes are expressed from telomeric transcription units, called expression sites (ES), of which ϳ20 can be used while the parasite is present in the mammalian host. Only one ES is expressed at a given time from a specific sub-nuclear domain (5), but coordinated transcriptional switches (termed in situ switches) can activate a silent ES and inactivate the transcribed site (6 -12). T. brucei also contains hundreds of silent VSG genes, both in multigene arrays in the megabase chromosomes and at the telomeres of minichromosomes. This silent reservoir is activated by recombination reactions that move the genes into the ES, normally by a gene conversion process (13-15). The available evidence suggests that recombinational VSG switching occurs by homologous recombination. No specific sequence has been shown to be essential in activating VSG gene conversion, but instead the reactions use variable amounts of flanking sequence homologies (2). In addition, disruption of a gene encoding a major enzyme of eukaryotic homologous recombination, RAD51, impairs VSG switching (16). Consistent with this genetic analysis, inactivation of KU70 or KU80, which catalyze a non-homologous end-joining pathway of DNA repair in other organisms (17, 18), does not affect VSG switching (19). Surprisingly, mutation of MRE11 also does not affect VSG switching (20), despite this gene encoding an enzyme that has been proposed to have many rol...

show abstract

Section: Discussionmentioning

confidence: 43%

Mismatch Repair Regulates Homologous Recombination, but Has Little Influence on Antigenic Variation, in Trypanosoma brucei

Bell

McCulloch

2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Both transfectants were selected with 40 g/ml G418 in addition to 1 g/ml puromycin and then cloned by limiting dilution as described above. For endogenous tagging of TbGntB and TbGolgin63, the PCR-based epitope tagging method was used according to previous publication (46).…”

Section: Methodsmentioning

confidence: 99%

The G1 Cyclin-dependent Kinase CRK1 in Trypanosoma brucei Regulates Anterograde Protein Transport by Phosphorylating the COPII Subunit Sec31

Gourguechon

Wang

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

Transport of secretory proteins from the endoplasmic reticulum to the Golgi is mediated by the coat protein II (COPII) complex comprising a Sec23-Sec24 heterodimer and a Sec13-Sec31 heterotetramer. The mechanisms underlying COPII-mediated protein trafficking have been well defined, but the extent of regulation of this secretory machinery by cellular signaling pathways remains poorly understood. Here, we report that CRK1, a G 1 cyclin-dependent kinase in Trypanosoma brucei, regulates anterograde protein trafficking by phosphorylating Sec31. Depletion of CRK1 abolished anterograde transport of the secretory protein and disrupted the localization of multiple Golgi proteins, reminiscent of Sec31 depletion. CRK1 phosphorylates Sec31 at multiple serine/threonine sites, and mutation of these phosphosites to alanine recapitulates the protein trafficking defects caused by Sec31 depletion. Mutation of these CRK1 phosphosites to aspartate restored Sec31 function. Taken together, these results uncover a novel function of CRK1 in anterograde protein trafficking and elucidate the mechanistic role of CRK1 in protein trafficking through regulation of the COPII subunit Sec31.

show abstract

“…For in situ epitope tagging of the XRNB open reading frame, we used the plasmid containing the V5 tag and Blasticidin selectable marker sequence that is described in Shen et al (2001). We cloned 300 bp of the target open reading frame in frame with, and downstream of, the tag sequence, and inserted a 300-bp fragment from directly 59 to the target gene at the 59 end of the construct, upstream of the selectable marker.…”

Section: Plasmid Constructionmentioning

confidence: 99%

Roles of aTrypanosoma brucei5′→3′ exoribonuclease homolog in mRNA degradation

Irmer²,

Gudjonsdottir-Planck³

et al. 2006

RNA

100

View full text Add to dashboard Cite

The genome of the kinetoplastid parasite Trypanosoma brucei encodes four homologs of the Saccharomyces cerevisiae 59/39 exoribonucleases Xrn1p and Xrn2p/Rat1p, XRNA, XRNB, XRNC, and XRND. In S. cerevisiae, Xrn1p is a cytosolic enzyme involved in degradation of mRNA, whereas Xrn2p is involved in RNA processing in the nucleus. Trypanosome XRND was found in the nucleus, XRNB and XRNC were found in the cytoplasm, and XRNA appeared to be in both compartments. XRND and XRNA were essential for parasite growth. Depletion of XRNA increased the abundances of highly unstable developmentally regulated mRNAs, perhaps by delaying a deadenylation-independent decay pathway. Degradation of more stable or unregulated mRNAs was not affected by XRNA depletion although a slight decrease in average poly(A) tail length was observed. We conclude that in trypanosomes 59/39 exonuclease activity is important in degradation of highly unstable, regulated mRNAs, but that for other mRNAs another step is more important in determining the decay rate.

show abstract

In vivo epitope tagging of Trypanosoma brucei genes using a one step PCR-based strategy

Cited by 92 publications

References 9 publications

Mismatch Repair Regulates Homologous Recombination, but Has Little Influence on Antigenic Variation, in Trypanosoma brucei

Mismatch Repair Regulates Homologous Recombination, but Has Little Influence on Antigenic Variation, in Trypanosoma brucei

The G1 Cyclin-dependent Kinase CRK1 in Trypanosoma brucei Regulates Anterograde Protein Transport by Phosphorylating the COPII Subunit Sec31

Roles of aTrypanosoma brucei5′→3′ exoribonuclease homolog in mRNA degradation

Contact Info

Product

Resources

About