B5- Antigenic Switch Trypanosoma Flashcards
What are the waves of parasitemia
The immunogenic response 5-7 days mounted then peak again when the 1% of cells changing have outgrown other populations
How does it cover surface
The final 23 aa are cleaved for a gpi anchor
How many gene copies of vsg
1000
What is the one expressed called
Expression linked copy
How many expression sites are there and why
20-44 because max of 44 telomeres in the 11 pairs of chr
Vsg lie in subtelomeric regiosnnonly in expression sites
Explain structure of vsg
About 500 aa
Signal sequence in n terminus
Central region is highly variable
C terminus is conserved
Final 17-23 aa are cleaved and replaced by gpi anchor
If they are highly diverse, are they diverse in tertiary structure
No, they have a highly conserved homodimer strucrure where each monomer held by hinge region at central region
What can cleave the gpi anchor to release coat for turnover
Pl-C
What is on the 5’ and 3’ of the vsg gene in the expression site at Subtelomeric regions
2 barren regions of repeats not targeted by RE
5’ is a 76bp repeat
3’ is the TTAGGG telomeric repeats
What other repeat can be present (only in blood form not metacyclic salivary gland) before promoter
50bp repeat
What is in between the promoter and 76bp repeat on blood form expression sites
Exp site associated genes polycistron (ESAGs)
Exp at the same time as the vsg
Give example of what this can be
Transferrin receptor for grabbing iron
How many Expressjon sites in blood form can be found
20
How does blood form es differ to salivary metacyclic forms of tsetse
They don’t have the 50bp repeats, promoter immediately 5’ of the vsg
No 76 bp repeat
No esags
In metacyclic only 20 vsg genes are ever expressed vs 1000 in bes
Where else can silent vsgs park
On the 100 mini chromosomes
What sort of repeats are here
5’ long 177 bp repeat and also telomerix 3’
What is the most common way of gene switching (all involve recombination)
Duplicative transposition
A gene not in telomeric region ie esag can form a loop (chr loops) and displace the vsg at the expression site telomere to become active
The other vsg discarded
Duplicative because the Solent vsg still kept in silent library
How is telomere conversion different
Where another gene at another telomere site will displace the active vsg-ES
It is copied and transposed into that spot
What other 2 ways can occur
Telomere reciprocal exchange where both genes at a telomeric site switch places and 1 of them becomes in the active ES
In situ switch where promoter can become activated or inactivated
Are the promoter sequences diff between active and inactive Es
No
Give example of a enzyme for homologous recomb which ko can disrupt gene switching (but not fully) and how does it work
Rad51
Can insert ssdna into homologous dna duplexes and causes recombination events
What sort of ptm modification associated with silenced ES enriched at telomere regions
Base J (modified base)
In active vs inactive where are modified j bases (active shows loss in Es, inactive shows gain)
Active- only in 50bp repeats before promoter and at telomere ttaggg repeats
Inactive - along whole orf and the repeats including at the ESAGS and vsg site
Was J base modification actually shown to control expression or not
It was later found to not affect expression despite different patterns in active and inactive vsgs but instead likely involved in switching and recombination
How is telomeric position effect a factor in silencing (suggested by reporter cloning exp)
Closer the gene to the telomere the more silenced it is
Further away the reporter gene becomes expressed much more
independent of promoter (works with a constitutive promoter)
Suggesting active can switch further from telomere
Which initiation factor associated with the active ES and increases occupancy of rna pol 1. (Enriched at active ES)
CIFTA
What type of ptm is seen close to promoter and can increase occupancy in active ES
SUMOylation
What protein displaces h1,2a and 3 in active ES and opens the chromatin structure for txn
Tdp1
What does this show
Tertiary structure also a factor in expression
Which methylation by dot1b associated with silent ES
Trimethylation at h3k76
What was used which blocks rna pol II and Iii but not rna pol I and still found vsg expression confirming pol 1
A-amanitin
Where is activity of rna pol 1 found usually (and only ever found for insect stages but not blood form)
Nucleolus periphery (for rrna txn)
Where apart from nucleolus was active ES expression found by tagging with gfp aswell as rna pol activity - found only in the blood form.
Extranucleolar body
(Inactive vsg did not colocalise with rna pol in the extranucleolar body)
How does this explain exclusion
1 chr / Es needs to find way to this Extranucleolar body and the rest are silenced
Was this found in insect stage
No
How did scientists looking at tertiary structure through hi-c and pacbio come to conclusion that chromatin compactness affects accessibility for switching AND exclusion
Subtelomeric vsg highly packaged chromatin in inactive state
When deleting the h3 and h4 variant histones they found increased exp of more than 1 vsg
Further identified vsgs were able to homologously recombine better in this state and switch vsgs that were active (usually low frequency / low rate switching)
How could this potentially give a therapy
Target genome compact structure ? To increase exp of more vsgs
Which protein part of the nucleoskeleton keeps stability of heterochromatin in silent vsg ES and what happens in knockdown of it
NUP-1
Knockdown shown to increase vsg switchinf and also results in reduced vsg silencing
Showing importance of tertiary structure
What other proteins present in periphery help the heterochromstin stability of inactive ES (epigentic control in addition to dot1b)
Hdac3
What is present in the Extranucleolar body that inactive Es don’t have access to
Sl-array (rna pol II whcih transcribes SLrna to donate sl for maturation of the active vsg)
Why doesn’t telomeric position effect explain full story of gene exp
Because there will be a vsg that is the expression linked copy which will be expressed despite being close to telomeric regions
Suggests other factors of regulating like chromatin/ tertiary dna structures
Why is pacbio particularly important to look at vsg genes in Subtelomeric regions
High amount of repeats = long sequences
Which form of parasite is nuclear compartmentalisation extra nuclear body txn machinery specific to
Blood form
