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P. Dimitri - Drosophila melanogaster as a model organism for functional genomic analyses of essential genes
300 heterochromatin genes. Among the genes studied,
interesting results were obtained by inactivation
of CG17528 which encodes a putative microtubule
associated protein. After inactivation of CG17528,
several defects occurred: 1) aberrant anaphases, 2)
binucleate cells and 3) abnormal shape cells. The frequency
of these defects was significantly higher
than that found in the controls. These data are compatible
with a role of CG17528 in spindle and
cytoskeleton organization. RNAi analyses also
revealed 5 genes (CG17168, CG40461, CG41281
and concertina) whose protein product may be
required for cytokinesis or proper cell shape.
The GC40218 protein
The CG40218 protein belongs to the conserved family
of BCNT-like proteins found in animals and plants,
whose functions is still unknown. Immuno-staining
and western blotting experiments were carried out
using antibody against the mouse CP27 protein which
share homology with the CG40218 protein. In western
blotting, the antibody recognizes a band of about
27 Kda absent in CG40218 mutants. On polytene
chromosomes of wild-type larvae, the immuno-staining
is distributed all over the euchromatic arms and
the chromocenter, but disappears in CG40218
mutants. We also found that on polytene chromosomes
deposition of H2Av, HP1 and H3trimetk9
chromatin markers is affect in CG40218 mutants.
RNAi mediated inactivation of CFDP1, the
human ortholog of CG40218, in HeLa cells
CFDP1 (craniofacial development protein 1), the
human ortholog of CG40218, encodes a 299 a. a.
belonging to the BCNT family, whose function is still
unknown. RNAi mediated inactivation of CFDP1 in
HeLa cells using a specific siRNA-expressing vector
called psiUx (Denti et al. 2004). Expression of
CFDP1 after RNAi was monitored by RT PCR and
immunoblotting using policlonal antibodies against
CFDP1 protein. The cytological analysis of treated
cells revealed the presence of metaphase chromosomes
with aberrant morphology and condensation
defects similar to those observed after inactivation of
CG40218 in Drosophila.
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In vivo RNAi in S2 Drosophila cells
We tested the RNAi-transgene in vivo silencing of
endogenous heterochromatin gene targets by monitoring
the presence of visible phenotypes and verifying
transcript reduction of the endogenous gene by
Northern blots.
Ory gene: Inactivation of Ory, one of the two Ylinked
genes (Carvalho et al., 2001) of the collection,
induces a complete male sterile phenotype with
testes of mutant males that lack mature spermatids
and show empty seminal vesicles. Since mutant
testes do not show any apparent defects in cells of
the earlier stage of spermatogenesis we speculated
that in Ory mutants a potential disruption of the
individualization process occurs.
CG17528 gene: inactivation of CG17528 generates
in adult flies clear phenotypes characterized by
defects in wing, notum and abdomen development.
Since most of the observed defects perfectly matches
the phenotype caused by disruption of wingless
pathway in the wing disc (Morata and Lawrence,
1977), this may represent a good starting point to
study the role of CG17528 and the opportunity to
use wing disc development as model.
Selected publications
Corradini N, Rossi F, Giordano E, Caizzi R,
Verní F, Dimitri P. Drosophila melanogaster as a
model for studying protein-encoding genes that
are resident in constitutive heterochromatin.
Heredity 2007, 98:3-12.
Hoskins RA, Carlson JW, Kennedy C, Acevedo D,
Evans-Holm M, Frise E, Wan KH, Park S, Mendez-
Lago M, Rossi F, Villasante A, Dimitri P, Karpen
GH, Celniker SE. Sequence finishing and mapping of
Drosophila melanogaster heterochromatin. Science
2007, 316:1625-8.
Rossi F, Moschetti R, Caizzi R, Corradini N,
Dimitri P. Cytogenetics and molecular characterization
of heterochromatin gene models in Drosophila
melanogaster. Genetics 2007, 175:595-607.