ZMBH J.Bericht 2000 - Zentrum für Molekulare Biologie der ...

poral requirement for trx in order to achieve normal
expression patterns. We have shown with X-CHIP a
dynamic association of TRX with CMMs and promoters
of the BX-C and found a close cooperation of TRX
with PcG members at CMMs.
Several members of the Drosophila trxG have counterparts
in humans and mice. The mixed-lineage leukaemia
(MLL) gene (also known as ALL-1, HRX), a
TRX homologue, was found through its implication in
the majority of infantile acute lymphocytic and mixed
lineage leukemias. Disruption of the murine Mll gene
by gene targeting causes homeotic transformations of
the vertebrae in heterozygotes and loss of homeotic
gene expression, supporting the notion that Mll is a
functional equivalent of trx.
To date, not much is known at the molecular level
how translocation of MLL with various other chromosomal
locations generate leukemia. At least 23 fusion
MLL proteins involved in leukemia have been identified.
As part of our collaborative efforts with
the Canaani lab, we have generated transgenic flies
expressing full length MLL, and fusion proteins
MLL-AF9, MLL-AF4, and MLL-AF17 under GAL4
control. Expression of these proteins in Drosophila
may reveal their effects by determining with which
pathway in Drosophila development they influence.
Crosses between our transgenic flies and various
GAL4 drivers revealed that only MLL-AF9 and
MLL-AF17 constructs had a slight effect on Drosophila
development. When we took a closer look at the
molecular level, we could not detect the proteins on
Western, but at the RNA level, we see transcripts from
all four constructs. Interestingly, this same effect is
also observed with MLL-AF9 in mouse. We are currently
testing whether overexpression of the murine
proteins results in cell death as recent published observations
link MLL with apoptosis.
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IV. Drosophila as a model system to identify
components involved in the processing
and the function of the human Amyloid Precursor
Protein
G. Merdes, B. Brückner, DFG (in collaboration
with K. Beyreuther (ZMBH))
The human amyloid precursor protein (APP) is an
integral membrane protein with two homologues in
invertebrates: the amyloid protein-like protein 1 (C.
elegans, APL-1) and the amyloid precursor proteinlike
protein (D. melanogaster, APPL). Flies deficient
for expression of APPL show a phototaxis impairment
that can be rescued by the expression of human APP
suggesting an evolutionary conservation of APP function.
The importance of APP in the pathogenesis of
Alzheimer’s disease (AD) became apparent through
the identification of distinct mutations in the gene
causing early onset familial AD. The major component
of the plaques and amyloid found in senile Alzheimer
patients is a proteolytic product of APP, the
βA4 peptide. APP is cleaved in vivo by several proteases
termed α−, β−, and γ−secretase. Cleavage of
APP by the α−secretase prevents the production of
βA4. Despite the availability of APP-null mutants and
transgenic mice expressing human APP, the physiological
role of APP remains unknown and many factors
involved in the processing of APP have not yet
been clearly identified. Therefore we have established
Drosophila melanogaster as a model system to study
the function and processing of APP.
Various forms of human APP were expressed in
Drosophila tissue culture cells as well as in transgenic
fly lines. Studies in both systems revealed that also in
flies APP is processed in a similar way as in mammalian
cells in regard to the α- and γ-cleavages. In addition,
transgenic flies expressing full-length forms of
APP in the wing imaginal discs are characterized by a
blistered-wing phenotype, suggesting that the expression
of human APP interferes with the cell adhesion
between the dorsal and ventral epithelial cell monolayers
of the wing (Figure 3). To define the involvement
of APP in the blistered wing phenotype in more
detail as well as to identify factors involved in the processing
of APP, we perform biochemical and genetic
studies. Since the observed wing phenotype depends
on full-length APP, an increased processing of APP,
e.g. by the simultaneous overexpression of an APPspecific
protease, results in a suppression of the phenotype.
This observation enables us to identify proteins
involved in the processing of APP by the use of
genetic screens and to subsequently identify the corresponding
mammalian homologues. In a similar way,
gene products required for the effect of APP on cell
adhesion and therefore for APP function, can also be
identified by genetic screens. Mutations in these genes
result in suppression or an enhancement of the blis-
tered wing phenotype, dependent on their function.
Drosophila can be mutagenized by the use of chemicals
or by the use of the transposable P-element.
The advantage of P-element mutagenesis is the easier
identification of the mutagenized genes by isolating
and sequencing the flanking genomic DNA. Therefore
we have recently performed a P-element based genetic
screen as well as screened collections of already existing
Drosophila mutants. The analysis of 150 000 flies
with potential new P-element insertion sites resulted
in the isolation of several enhancer- and suppressormutations,
together with several genes interfering with
wing patterning. The corresponding genes, which we
are currently characterizing further, encode factors
involved in signal transduction, maintenance of cell
shape and cell adhesion, and protein processing.
We utilize the fruit fly Drosophila melanogaster as
a genetic model system. Since Thomas Hunt Morgan
introduced Drosophila as a model organism into genetics
at the beginning of this century and isolated the
Figure 3. Expression of different
isoforms of human APP in transgenic
Drosophila. Only full-length
APP result in a blistered wing
phenotype.
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