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specified. One may have easily missed this phenotype, and one wonders
whether an analogous vertebrate phenotype, say, having a few extra whis-
kers on the face of a mouse, might be noticeable. Finally, it is salient to
mention that derepression of different targets of a miRNA may have distinct
consequences in different locations of miRNA expression. We will return
to this point later in
Section 4.3
.
2.5. On the importance of rescuing mutant phenotypes
It cannot be overemphasized that the careful practice of genetics involves a
certain amount of legwork to prove that a given mutation is causal to an
observed phenotype. Beyond the notes written on a stock label, there is
always the possibility of additional unanticipated mutations that could
underlie a phenotype of interest. First, many
Drosophila
miRNA alleles
were generated by transposon-mediated aberrations.
P
elements are
known to incur a reasonably high frequency of hit-and-run events, such
that the gene tagged by a transposon is not necessarily the same locus
responsible for an associated phenotype. There may be other genes that
were mutated before the transposon landed in its final spot, or transposon
mobilization may have left damaged copies in the genome that are not easily
detected by routine PCR checks. Second, while it is more effort to induce
alleles by HR, and these aberrations are generated more precisely than by
random transposon-induced deletions, it is well documented that the pro-
cess of HR has a high frequency of inducing second-site mutations
(
O'Keefe
et al
., 2007
). If one is unlucky, these might occur in some biased
fashion leading to their presence on independent HR events. Worse yet, the
homology arms themselves might affect neighboring genes in a systematic
fashion. Third, the practice of keeping
Drosophila
stocks in a balanced state
against chromosomes that suppress recombination permits unlinked muta-
tions to accumulate over time.
Therefore, while one may be fortunate to be able to order miRNA loci
tagged with
P
elements from public stock centers, or to have generated
one's own miRNA alleles using HR, one should be cautious in relating
observed phenotypes to the mutated locus. A few tests can be performed. In
the case of transposon alleles, one can ask if a precise excision reverts the
phenotype; if the phenotype persists, it may be a sign that it is due to some
unlinked mutation. For example, in the original
mir-14 P
lethal stock
identified as a suppressor of
GMR-reaper
, lethality was due to a background
mutation and not by loss of
mir-14 per se
(
Xu
et al
., 2003
); of course, the
apoptosis-modifying activity proved
mir-14
dependent.
One may also seek to obtain independent alleles and show that the
phenotype is recapitulated in all of them, as was done with
mir-279
(
Cayirlioglu
et al
., 2008
). This is not a foolproof test, and in unlucky
cases, independent
Drosophila
stocks have been found to contain the same,
