Recombination, Phase Variation and Antigenic Variation

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Lecture 6

• Recombination, Phase Variation and Antigenic
  Variation

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Homologous Recombination

• Occurs randomly between homologous sequences.
• Frequency of recombination between two sites is
  proportional to the distance between the sites.
• Provides a means to generate new combinations of
  genes.

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Holliday structures (part I)
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Holliday structures (part II)
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Holliday Structures in Plasmid DNA
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Double-strand breaks in DNA initiate recombination
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Double-stranded breaks are not uncommon
Conjugation (prokaryotes) contact mediated
transfer of chromosomal DNA. Phage/virus
transduction. Meiosis. Mutagens -- X-rays
certain chemicals.
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Initiation of recombination by the RecBCD enzyme
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RecA protein action
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Kowalczykowski, 2000
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Targeted gene disruption by homologous
recombination
Lodish et al. Molecular Cell Biology
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Antigenic and phase variation
Directed adaptive change in specific surface
molecules. Phase variation Often reversible
(ON/OFF). Antigenic variation Usually
uni-directional. Frequencies (10-2 -- 10-6) are
higher than normal mutation rates. Mechanism
often involves homologous recombination.
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Examples of phase/antigenic variation
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General mechanisms to generate diversity
Non-recombinational High mutation rate -- HIV,
flu, adaptive mutation not gene
specific. Slipped strand mispairing -- alters
reading frame. Methylation -- alters level of
transcript. Transcriptional activation.
Recombinational Gene conversion --
non-reciprocal (cassette mechanism). Genomic
rearrangement -- intra- or inter-genomic
(non)reciprocal.
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Deitsch et al. 1997
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Antigenic variation in T. brucei
The African trypanosome -- sleeping sickness
(waves of fever). Many unusual features --
antigenic variation trans-splicing
polycistronic RNA (eukaryote) RNA
editing glycosomes J-DNA DNA -- 20
chromosomes (100 - 1000 kb) gt100 minichromosomes
(50 - 150 kb)
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Trypanosome life-cycle
Can infect many different mammals (cattle). Two
main stages insect (procyclic). PARP/procycli
n mammalian (bloodstream). Variant surface
glycoprotein (VSG).
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(No Transcript)
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African trypanosomes Waves of fever
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Trypanosome antigen switching
At each wave, a different VSG is expressed. 10 -2
to 10-6 per cell in blood. gt100 VSGs expressed
sequentially in one rabbit. Switch not induced by
the immune system. Semi-programmed -- early VSGs
are always early.
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Semi-programmed antigenic variation
Tsetse fly
Rabbit 1 A B C
D E etc.
passage
Rabbit 2 A B D E
etc. What controls this process is unknown..
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Variant surface glycoproteins
Completely cover the blood-stage tryp (107
/cell). except the flagellar pocket. Glycolipid
anchor (released by phospholipase C). VSG protein
-- 450 amino acids C-term is more conserved
(not exposed). N-term highly variable
sequence. 3-D structures are very
similar. 1000s of VSG genes all over the
genome. Only one expressed at a time.
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VSG locus types in T. brucei
Horn and Barry. 2005. Chrom. Res. 13525
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VSG Expression Sites
About 20 expression sites per genome. All are
telomeric (often at telomere ends). Only one
expression site is active at a time.
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Mechanisms of VSG Gene Switching
1. Duplicative transposition -- from internal or
telomeric VSGs displace old gene (cassette
mechanism) 5 side of transposition - 70 bp
repeats few copies at donor site 100s of
copies at expression site 3 side of
transposition - VSGs similar at C-terminus 3
untranslated regions telomeric repeats
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Mechanisms of VSG Gene Switching (II)
2. Telomere conversion -- whole chromosome end
is converted doesnt need 70 bp repeats 3.
Reciprocal telomere exchange (must occur between
VSG and promoter). 4. In situ switch - old
expression site switched off. new ES switched on.
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Expression Sites and ESAGs
20 different expression sites. Different alleles
at different expression sites. All transcribed at
the same rate BUT mRNAs differ greatly (VSG 10
of all mRNA). 2 ESAGs - transferrin receptor
(flagellar pocket) 1 ESAG - adenylate
cyclase other ESAGS -- also expressed in the
flagellar pocket?
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Why 20 Expression Sites?
Increase antigenic diversity? Only one ES is
needed for antigenic switching. And control
becomes a problem All pathogens need to
acquire iron. Mammalian transferrin (Tf, iron
transport) evolved rapidly. Human and bovine Tfs
differ by 30. Different ESAG Tf-receptors bind
Tfs from different species, increasing
trypanosome host range. Do other ESAGs also
encode host-specific functions?
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How is only one ES active at a time?
Cross talk between sites - can two VSG be
expressed simultaneously? Independent
activation/inactivation of sites. Promoter
rearrangement. none near the promoter far
away? Nucleolar location (site of rRNA
synthesis) ES transcribed by pol I (like rRNAs)
but not in the nucleolus DNA modification --
b-glucosyl hydroxymethyluracil (J) J is only
found at silent loci telomeric repeats and 50
bp repeats. Lost from telomere on
activation. Is J the result or the cause of
inactivation? Silencing -- binding of telomeric
proteins (but why not at one?)
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Antigenic variation in Plasmodium
PfEMP1 (var) expressed on the infected
erythrocyte surface, promotes cytoadherence. 50
var genes per genome. Most are located at
telomeres. Only one var expressed per
parasite. Switching rates as high as 2
reported. Recombination at telomeres promotes
gene diversity BUT Switching expression is NOT
associated with recombination. Other gene
families (rif, stevor, clag) may also vary.
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Epigentic factors and var genes

• Perinuclear heterochromatin - role not yet
  demonstrated
• Chromatin remodeling at var promoters
• PfSIR2 silencing of var genes
• Histone acetylation- activation
• Perinuclear movement of var locus
• var intron silencing

Ralph and Scherf. 2005. Curr. Opin. Microbiol.
8434
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Antigenic variation in Neisseria pilin genes
Pilin -- long filaments required for attachment.
Target of neutralizing antibodies.
Encoded by the pilin gene family.
Different pilins mediate attachment to different
cells. Silent genes copied to an expression
site, BUT All silent genes are
incomplete. Silent copy can come from exogenous
DNA intergenic recombination. Intragenic
switches are reciprocal. Nonfunctional gene can
enter expression site loss of pili.
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Phase variation in Neisseria opa genes
Small set of outer membrane protein (Opacity
protein) genes. All copies are transcribed,
control is at translation Signal sequences have
variable of coding repeats (CTCTT). Strand
slippage changes reading frame. Very high
frequency (gt10-2 per division).