Biology Reference
In-Depth Information
repository, has resulted in mapping and curation of several hundred thou-
sand transposon mobilization events over the years. The 20-year-old
Dro-
sophila
Gene Disruption Project (GDP) is a prominent community resource
project that seeks to isolate insertions in all
Drosophila
genes. In addition to
an immense work from the core project members (
Bellen
et al
., 2011
), the
GDP has also consolidated transposon collections from many other labora-
tories and even companies (e.g., the Exelixis and GenExel collections;
Bellen
et al
., 2004
;
Deak
et al
., 1997
;
Lee
et al
., 2005
;
Spradling
et al
.,
1999
;
Thibault
et al
., 2004
).
Some transposons, most notably the
P
element, prefer to insert in active
promoters (
Fig. 8.1
B). When viewed alongside genome annotation tracks
(e.g.,
http://flybase.org/
), this preference becomes quite obvious, with
many insertions piling up at the 5
0
ends of annotated genes. Of course,
not every insertion represents a promoter, and many insertions are not
associated with annotated genes. However, in the case of hotspots associated
with multiple insertions, one can infer that some type of promoter has been
tagged. A number of these “orphan” insertion hotspots were later recog-
nized to identify alternative 5
0
exons of protein-coding genes (
Manak
et al
.,
2006
), but a number of them proved to identify miRNA genes (
Brennecke
et al
., 2003
;
Cayirlioglu
et al
., 2008
;
Hipfner
et al
., 2002
;
Raisin
et al
., 2003
).
Transposons frequently affect the expression of the inserted loci, thereby
acting as mutant alleles (
Fig. 8.1
B). One of the very first
Drosophila
miRNAs
reported was recovered on the basis of a
P
insertion in the
mir-14
locus,
which is indeed a
P
hotspot (
Xu
et al
., 2003
). In this study, flies were
sensitized by expression of the proapoptotic gene reaper in the developing
eye (
GMR
>
reaper
), which induces small, rough eyes. This stock was crossed
to a collection of lethal
P
insertions to find ones that could modify the rough
eye phenotype, thus representing loci that putatively affect cell death.
An insertion in
mir-14
enhanced
GMR
>
reaper
, yielding smaller eyes and
increased pupal lethality. This proved to be due to loss of
mir-14
, since the
have potential to be mutagenic for miRNA loci. The P element strongly prefers to
insert in promoters and can disrupt transcription. The Minos and piggyBac elements,
whose insertion preferences are less biased, can disrupt miRNA primary transcripts. (C)
P elements (and to a lesser extent Minos elements, but not piggyBacs) can be used to
generate local deletions by imprecise excision. (D) FRT-mediated deletion. Flp recom-
binase can induce deletions between different FRT-containing transposons located in
trans. This can result in the precise deletion of the intervening region. (E) Homologous
recombination (HR) allows the precisely engineered generation of a mutant allele. The
miRNA hairpin can be replaced with a sequence of choice. (F) Recently, the advent of
genomic engineering has made it possible to generate a founder knockout line by HR
that contains a phage attachment site (not shown) which enables relatively easily
modification of the founder line as desired, for example, adding various markers,
Gal4 transgenes or modified hairpins to the endogenous locus.
