Lecture B5 - Gene Switching and Antigenic Variation in Trypanosoma Brucei Flashcards
Describe the antigenic variation of Trypanosomes.
Infection characterised by periodic waves of parasitemia.
Each wave represents a single antigenically distinct clone or serotype.
Antibodies produced in the first week against clone A will not react with clone B.
This changing display of antigens is called antigenic variation.
Describe the surface coat of trypanosomes.
Covered with a. dense surface coat.
Variant specific antisera strongly react with the surface coat.
Surface coats from different clones are antigenic ally distinct.
What suggests that the antigenic determinant on the surface us a protein?
Trypsin treatment completely removes the surface coat from trypanosomes and also abolishes antibody binding.
Describe the genome organisation of VSG genes.
1000 genes or basic copies.
Exclusively 1 VSG gene expressed at a given time.
VSGs only expressed at telomeres - telomeric expression sites.
Describe VSG.
Mature VSG is 60kDa
500 amino acids
N-term is 20-40 amino acids = signal sequence
360 amino acid = variable region
C-term is 110-120 amino acids = more conserved
Final 17-23 amino acids cleaved and replaced with GPI anchor.
Describe VSG structure.
Despite highly divergent sequences in the variable region, the tertiary structure of the VSGs remains highly conserved.
VSG molecules pack on the cell surface to make a uniform coat.
Variable domain has many antigenecitys but has a confined structure.
Packed together as dimers (2 molecules).
Describe VSG expression sites.
Telomeres specifically recruited for expression.
VSG located 5-10kb from chromosome end.
Surrounded by 2 barren regions devoid of restriction enzyme sites - 3’ barren region containing telomeric repeats TTAGGG, 5’ barren region contains 76 bp repeats = recombinational hotspots.
VSG expression sites - bloodstream form in mammalian hosts, metacyclic form in Tsetse fly salivary gland.
What are bloodstream-form expression sites?
20 in total
Polycistronic transcription units of 40-60kb.
Encode VSG and expression site associated genes.
Upstream 50bp repeat arrays can extend - >40kb stretch
Where is a parking area for VSG genes that need to be silent?
Mini chromosomes.
What are the 3 mechanisms of switching VSG coats?
Silent VSG gene can be copied into active VSG site
Silent VSG gene can be inserted into active VSG site
Expression site activation/inactivation
How is gene replacement achieved in VSG?
Recombinational events
Gene conversion end points frequency - 76bp repeats of 5’ barren region and conserved 3’ end of VSG gene.
Reciprocal recombination between telomeres also possible.
Describe gene conversion - duplicate transposition.
Gene is being transcribed and VSG coat protein D is being made, A B and C there internally.
A combination events can take place between the repeat regions allowing for the C gene to be copied at the active site and the D gene is now deleted and 2 copies of the C gene so C gene is expressed.
Name the 4 gene switching mechanisms.
Duplicative transposition of a non-telomeric gene
Telomere conversion
Reciprocal telomere exchange
In situ switch
What cellular factor has been identified in VSG switching?
RAD51 - highly conserved euk enzyme responsible for transfer of ssDNA ends into intact duplex DNA, searches for homology and creates DNA recombination intermediates.
RAD51 gene knockout in trypanosome impairs but does not prevent switching suggesting multiple recombination pathways.
What is epigenetic switching?
Identical DNA sequence but different phenotypic feature.
Chromatin and DNA modifications.
Stable over several rounds of cell division.
Inherited.
