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16 PARASITE GENOMICS
clustering by identity, and some BLAST-based
analysis. More complete analyses require an
underlying genomic database, often customized
to deal with the ‘fragmented’ genome
data resulting from ESTs.
Analysis of genome sequence
Genome annotation is an art that is evolving
into a science. The basis of genome annotation
is a reference sequence housed in a database.
This database can then be filled with annotation
‘objects’ that refer to coordinates of the
sequence (and to each other). Thus each parasite
genome project, as it has matured, has
developed a genome database. These range
from simple, hypertext mark-up language
(html) websites to full relational databases with
interactive graphical viewers.
Annotations can be thought of as ‘hard’
and ‘soft’. Hard annotation, given a completed
sequence, refers to objects that rely only on
the primary DNA sequence such as possible
open reading frames, splice sites, and local
repeats. Soft annotation refers to comments
on the sequence and its encoded proteins
based on their relative similarity to other
objects annotated in other genomes. These
soft objects include such things as functional
identification of peptides based on BLAST
similarity to a protein of known function, or
decoration of DNA sequence with promoter
motifs derived from probabilistic searches.
With complete genomes to hand, whole
genome analyses can be carried out to infer the
presence of repetitive DNA segments of various
types, and to perform within-genome
classification of encoded peptides into protein
families. These sorts of analyses are important
in defining the overall structure of the genome,
and in identifying novel features of its encoded
genes.
The annotation snowball
The annotation of a sequence as having a particular
property should be qualified by the relative
goodness-of-fit of that sequence to the
features of the property. For assigning function,
sequence similarity to a protein of known function
is often used. There is a hidden, but known,
danger in this process: what if the functional
assignment of the protein of ‘known’ function
was also based on similarity to another protein?
And what if the chain of functional assignment
is long (many steps before the original biologically
verified functional assignment is
accessed), or one of the assignations is wrong?
For example, many proteins have been functionally
characterized through their roles in
model organisms defined by genetic studies.
If the parasite lacks the structures or biochemistry
displayed by the model organism, the
‘function’ may not exist in the parasite, even
though its proteins are annotated with the
function. One classic example, of relevance to
parasites, is that of the DoxA2 ‘phenoloxidase’
of Drosophila melanogaster. This gene was
identified by mutation as controlling tanning of
the insect cuticle, and knockouts lacked active
phenoloxidase. Such phenoloxidase activity
could underlie the crosslinking and tanning of
nematode cuticles and eggshells, and of platyhelminth
eggshells. The DoxA2 phenoloxidase
has homologues in many other genomes,
including nematodes. However, there are also
homologues in protozoan parasites (such as
P. falciparum and T. brucei) not expected to
encode such activity. In addition, plant homologues
appear to locate to a perinuclear site.
DoxA2 is in fact a regulatory component of the
26S proteasome, which in D. melanogaster is
involved in targeting the pre-pro-phenoloxidase
protein for processing: mutants in DoxA2 are
indeed negative for phenoloxidase, but not
because the phenoloxidase gene is disrupted.
MOLECULAR BIOLOGY