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THE PARASITES AND THEIR GENOMES 21
efforts are underway to examine stage-specific
expression, involvement in virulence and
potential vaccine candidacy through microarray,
genetic and mass-vaccine approaches.
A small but significant EST dataset accompanies
this: trypanosomatid mRNAs all carry the same
5 trans-spliced leader sequence, permitting
facile construction of full length cDNA libraries
which have been sampled to define both stagespecific
transcripts and housekeeping genes.
The genes of Leishmania species revealed by
EST and genomic sequencing display relatively
limited overall similarity to genes of other
organisms, with a relatively high proportion
being classified as ‘unknown’ or ‘novel’, as would
be expected from their phylogenetic distance
from other sequenced eukaryotes.
The general patterns of genome organization
found in Leishmania are also observed in other
trypanosomatids, but with taxon-specific variations.
In T. cruzi, homologous chromosomes
can differ in size by over 50% and there are
suggestions of aneuploidy, and thus genome
sequencing of this species must rely on a cloneby-clone
scaffold informing chromosome shotguns.
The T. cruzi genome is also more replete
with multigene families, and appears to be
more variable (in gene content) between isolates,
again posing problems in analysis and
application. The T. cruzi genome is being
sequenced from the reference strain CL Brener
TC3 by a combination of physical map-based
and whole chromosome strategies. Many
T. cruzi ESTs have been generated.
The genome of T. brucei is also more repetitive
overall than Leishmania, but these repeats
are in general restricted in distribution to particular
segments of chromosomes. One repetitive
gene family, the variable surface glycoprotein
(vsg) family, is distributed between chromosome
ends (where it resides in ‘expression
sites’, only one of which is active in each cell),
chromosome-internal sites, and a population
of very small minichromosomes. Active transposition
and gene conversion serve to move
genes from the silent sites (chromosome internal,
minichromosome, and most telomeric
sites) to the active expression site. This plasticity
in the genome for vsg genes does not
appear to be true for other genes. The genome
sequence of the reference strain of T. brucei,
TREU 927/4, is being determined by a combination
of whole-chromosome and physical
mapping strategies assisted by GSS and EST
sequencing.
Apicomplexan parasite genomes
The apicomplexan parasites (Plasmodium,
Toxoplasma, Eimeria and related genera) have
relatively simple genomes, but genomic analysis
is significantly complicated by the extreme
sequence composition bias of their DNA. For
example, the human malaria parasite P. falciparum
has an AT content of over 80% overall,
with genes (protein-coding regions) having a
slightly lower overall % AT than intergenic and
other non-coding segments. This AT bias makes
sequencing very difficult, as Plasmodium DNA
does not clone well in bacterial systems (and
those clones that are recovered are often
derived from only a subsection of the genome)
and sequencing chemistries do not yield very
long reads from AT-biased DNA . Despite these
problems, the P. falciparum genome is near
completion, and there are significant genomics
datasets available for Toxoplasma, Babesia,
Eimeria and Cryptosporidium.
The first malaria chromosomes to be
sequenced were from the 3D7 strain of P. falciparum,
and reveal the overall composition and
structure of apicomplexan chromosomes. As in
trypanosomatids, relatively repetitive telomeric
and subtelomeric regions flank a gene-rich core.
In the malaria chromosomes, there is an obvious
centromere, central to the core region,
MOLECULAR BIOLOGY