download report - Istituto Pasteur

P a r t i c i p a n t s :
Nicolay Junakovic, CNR researcher; Melina Accardo,
Nicoletta Corradini, Fabrizio Rossi, post-doc fellows.
C o l l a b o r a t i o n s :
Dipartimento di Anatomia Patologica e Genetica, Università di
Bari (Prof. Ruggiero Caizzi); Dipartimento di Genetica, Biologia
Generale e Molecolare, Università di Napoli “Federico II” (Dr.
Ennio Giordano).
Report of activity
The main aim of our project was to study the functions
of heterochromatin genes of D. melanogaster.
We found that the 15 genes tested are expressed
throughout all development, despite their location in
a supposedly silent region of the genome. In addition,
some of them are required for important functions
such has chromosome condensation and cytokinesis.
Future analyses will be carried out to delucidate
the role played by CG40218 protein and its
human hortolog CFDP1 in chromosome organization
and function.
Heterochromatin gene expression
throughout development
Using Northern blotting, we investigated transcription
throughout developmental stages in wild-type
Oregon-R strain of the vital gene Nipped-A and of
15 putative genes: CG12567 and CG40042 of 2Lh,
CG40218, CG41063, CG17691, CG40080, CG40130,
CG40129, CG17665, CG2944, CG2682, CG30440,
CG17514, CG10837 and CG17419. Notably, the protein
products encoded by 13 of these gene models
are conserved in humans. A Nipped-A-homologous
transcript of about 11 kb was detected in all stages
suggesting that the expression of this essential gene
is required throughout all development. A similar
picture emerges from the analysis of the putative
genes tested: transcripts of all 15 genes are present
Principal investigator: Patrizio Dimitri
Professor of Genetics
Dipartimento di Genetica e Biologia Molecolare
Tel/Fax: (+39) 06 49917948
Patrizio.dimitri@uniroma1.it
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Molecular genetics of eukaryotes - AREA 3
Drosophila melanogaster as a model organism for functional
genomic analyses of essential genes resident in heterochromatin
at different stages of development, with the exception
of CG17514 transcript not seen in embryos.
Insertional mutagenesis of vital
heterochromatic genes with P-elements
Insertional mutagenesis (see original program) was
performed with 3 different P{w+} inserts located in
chromosome 2 heterochromatin (Corradini et al.,
2003). We screened 5899 chromosomes and found 23
lethal mutations induced by P-element transposition.
Among the recovered mutations we found insertional
alleles in the following vital genes of 2Rh:
l(2)41Aa (3 alleles), l(2)41Ae (2 alleles), l(2)41Af (2
allele), Nipped-A (2 alleles) and Nipped-B (4 alleles).
Linking l(2)41Aa vital gene and CG40218
putative gene
Using inverse PCR and molecular sequencing of
LP1 insertional lethal allele, we identified the putative
gene CG40218 (971bp) which is candidate to
correspond to l(2)41Aa. Sequencing of CG40218 on
genomic DNA from larvae homozygous for EMS-31,
a lethal allele of l(2)41Aa, revealed that the EMS-31
allele carries a single base pair substitution that gives
rise to a stop codon in the aminoacid sequence. A
GC40218-homologous transcript of the expected
size (0.9 Kb) is present during all stages of development
in the wild-type, but is absent in the LP1
mutants. These results indicate that CG40218 corresponds
to l(2)41Aa.
RNAi mediated inactivation of
heterochromatin genes in S2 cells
Cytological analysis of metaphases after RNAi
mediated inactivation of l(2)41Aa-CG40218 in
Drosophila S2 tissue culture cells revealed that chromosome
condensation is highly defective upon
depletion of the CG40218 gene product. This defect
is consistent with that observed in l(2)41Aa mutants
(Cenci et al., 2003). We also performed doublestranded
RNA-mediated interference of a group of