Complexity and dynamics of kinetoplast DNA in the sleeping sickness parasite Trypanosoma brucei
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Date
07/07/2017Item status
Restricted AccessEmbargo end date
31/12/2100Author
Cooper, Sinclair
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Abstract
The mitochondrial genome (kinetoplast or kDNA) of Trypanosoma brucei is highly
complex in terms of structure, content and function. It is composed of two types of
molecules: 10-50 copies of identical ~23-kb maxicircles and 5,000-10,000 highly
heterogeneous 1-kb minicircles. Maxicircles and minicircles form a concatenated
network that resembles chainmail. Maxicircles are the equivalent of mitochondrial
DNA in other eukaryotes, but 12 out of the 18 protein-coding genes encoded on the
maxicircle require post-transcriptional RNA editing by uridylate insertion and removal
before a functional mRNA can be generated. The 1-kb minicircles make up the bulk of
the kDNA content and facilitate the editing of the maxicircle-encoded mRNAs by
encoding short guide RNAs (gRNAs) that are complementary to blocks of edited
sequence. It is estimated that there are at least hundred classes of minicircle, each class
encoding a different set of gRNAs. At each cycle of cell division the contents of the
kDNA genome must be faithfully copied and segregated into the daughter cells.
Mathematical modelling of kDNA replication has shown that failure to segregate
evenly will eventually result in loss of low copy number minicircle classes from the
population. Depending on the type of minicircle that is lost this can result in immediate
parasite death or, if the loss occurred in the bloodstream stage, render the cells unable
to complete the canonical life-cycle in the tsetse fly vector.
In order to investigate minicircle complexity and replication in T. brucei further
we i) first established the true complexity of the kDNA genome using a Illumina short
read sequencing and a bespoke assembly pipeline, ii) annotated the minicircles to
establish the editing capacity of the cells, iii) analysed expression levels of predicted
gRNA gene cassettes using small RNA data, and iv) carried out a long term time course
to measure how kDNA complexity changes over time and compared this to preliminary
model predictions. The structure of this thesis follows these steps.
Using these approaches, 365 unique and complete minicircle sequences were
assembled and annotated, representing 99% of the minicircle genome of the
differentiation competent (i.e. transmission competent) T. brucei strain AnTat90.13.
These minicircles encode 593 canonical gRNAs, defined as having a match in the
known editing space, and a further 558 non-canonical gRNAs with unknown function.
Transcriptome analysis showed that the non-canonical gRNAs, like the canonical set,
have 3' U-tails and showed the same length distribution. Canonical and non-canonical
sets differ, however, in their sense to anti-sense transcript ratios.
In vitro culturing of bloodstream form T. brucei for ~500 generations resulted in
loss of ~30 minicircle classes. After incorporating parameters for network size and
minicircle diversity determined above, model fitting to longitudinal kDNA complexity
data will provide estimations for the fidelity of kDNA segregation. The refined
mathematical model for kDNA segregation will permit insight into time constraints for
transmissibility during chronic infections due to progressive minicircle loss. It also has
the potential to shed light on the selective pressures that may have led to the apparent
co-evolution of the concatenated kDNA network structure and parasitism in
kinetoplastids.