Biomedical Engineering Reference
In-Depth Information
transgenic fish will be the first genetically modified
animals to enter the food-chain.
by internal deletion events. The elements are char-
acterized by perfect 31-bp inverted terminal repeats,
which are recognized by the transposase. The proto-
type element contains a single gene, comprising four
exons, encoding the transposase (a truncated ver-
sion of the transposase may act as the repressor).
The transposase primary transcript is differentially
spliced in germ cells and somatic cells, such that
functional transposase is produced only in germ
cells. Laski
et al
. (1986) showed this clearly by
making a P-element construct in which the differen-
tially spliced third intron was precisely removed.
This element showed a high level of somatic transposi-
tion activity. Naturally occurring short P elements
are generally defective, because they do not encode
functional transposase. However, they do possess
the inverted terminal repeats and can be activated
in trans
by transposase supplied by a non-defective
P element in the same nucleus.
Spradling and Rubin (1982) devised an approach
for introducing P-element DNA into
Drosophila
chromosomes. Essentially, a recombinant plasmid
comprising a 2.9 kb P element together with some
flanking
Drosophila
DNA sequences, cloned in the
pBR322 vector, was microinjected into the posterior
pole of M-cytotype embryos. The embryos were
injected at the syncytial blastoderm stage, when the
cytoplasm has not yet become partitioned into
individual cells (Fig. 11.9). The posterior pole was
chosen because this is where the germ line ori-
ginates, and P-element DNA in this region was
expected to be incorporated into the genome in a
proportion of the germ cells.
A screen of progeny lines showed that P elements
had indeed integrated at a variety of sites in each of
the five major chromosomal arms, as revealed by
in situ
hybridization to polytene chromosomes. P-
element integration occurred by transposition, not
by random integration. This was proved by probing
Southern blots of restricted DNA and showing that
the integrated P element was not accompanied by
the flanking
Drosophila
or pBR322 DNA sequences
present in the recombinant plasmid (Spradling
& Rubin 1982). The injected plasmid DNA must
therefore have been expressed at some level
before
integration, so as to provide transposase.
These experiments showed that P elements could
transpose with a high efficiency from injected plasmids
DNA transfer to invertebrates
Transgenic flies
Drosophila
P elements
P elements are transposable DNA elements that, under
certain circumstances, can be highly mobile in the
germ line of
D. melanogaster
. The subjugation of these
sequences as specialized vector molecules in
Droso-
phila
was a landmark in
Drosophila
genetics. Through
the use of P-element vectors, any DNA sequence can
be introduced into the genome of the fly.
P elements cause a syndrome of related genetic
phenomena called
P-M hybrid dysgenesis
(Bingham
et al
. 1982, Rubin
et al
. 1982). Dysgenesis occurs
when males of a P (paternally contributing) strain
are mated with females of an M (maternally contrib-
uting) strain, but not when the reciprocal cross is
made. The syndrome predominantly affects the
germ line and induces a high rate of mutation and
frequent chromosomal aberrations, resulting in
abnormal (dysgenic) hybrid offspring. In extreme
cases, there is failure to produce any gametes at all.
Hybrid dysgenesis occurs because P strains
contain transposable genetic elements, P elements,
which are mobilized in the eggs of M-strain females
(eggs that are permissive for P-element transposi-
tion are described as 'M-cytotype'). The P elements
do not cause dysgenesis in crosses within P strains,
because they are not mobilized in P-cytotype eggs.
This is because the P element encodes a repressor of
its own transposase, which prevents transposition.
When a sperm from a P-cytotype male fertilizes the
egg of an M-cytotype female, the absence of repres-
sor in the egg results in temporary derepression of
the transposase, such that P-element transposition
occurs at a high frequency. The high rate of muta-
tion characteristic of the dysgenesis syndrome
reflects the insertion of P elements into multiple
genetic loci.
Several members of the P-element family have
been cloned and characterized (O'Hare & Rubin
1983). The prototype is a 2.9-kb element, while
other members of the family appear to have arisen
