Expression of GARP, a major surface glycoprotein of Trypanosoma congolense, on the surface of Trypanosoma brucei: characterization and use as a selectable marker.
Procyclic and epimastigote forms of Trypanosoma congolense express an immunodominant glutamic acid/alanine-rich protein (GARP) that covers the parasite surface. Although GARP shows no sequence similarity to procyclins from T. brucei, the general characteristics of the two sets of surface glycoproteins suggest that they have analogous functions, in much the same way that variant surface glycoproteins with unrelated primary sequences fulfil the same function in bloodstream form trypanosomes. Since T. brucei and T. congolense do not follow the same pathway through the tsetse fly, one possible function of procyclins might be to direct parasites to the correct compartments. As a first step towards testing this hypothesis, we have produced stably transformed procyclic forms of T. brucei in which the GARP coding region has been integrated into a procyclin expression site. GARP can be detected on the surface of these transgenic trypanosomes, uniformly distributed within the endogenous procyclin coat, but there are differences in post-translational modification when it is expressed in T. brucei rather than in T. congolense. The fact that GARP is readily accessible to antibodies which were raised against a bacterial fusion protein led us to examine its potential as a selectable surface marker for transfection. We have established a rapid and simple procedure for isolating stable transformants that provides an alternative to conventional methods of selection for antibiotic resistance.
Hehl
A
A
Institut für Allgemeine Mikrobiologie, Universität Bern, Switzerland.
Pearson
T W
TW
Barry
J D
JD
Braun
R
R
Roditi
I
I
D000818:Animals; D030801:Animals, Genetically Modified; D000953:Antigens, Protozoan / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D000954:Antigens, Surface / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D006790:Host-Parasite Interactions; D016056:Immunodominant Epitopes / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D018189:Immunomagnetic Separation; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008863:Microspheres; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012641:Selection, Genetic; D014162:Transfection; D014348:Trypanosoma congolense / Q000737:chemistry* / Q000254:growth & development / Q000276:immunology; D014370:Tsetse Flies / Q000378:metabolism / Q000469:parasitology
D000953:Antigens, Protozoan; D000954:Antigens, Surface; C406724:GARP protein, Trypanosoma; D016056:Immunodominant Epitopes; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C053277:procyclic acidic repetitive protein, Trypanosoma