Biology Reference
In-Depth Information
resulted in the identification of Drosophila proteins important for centrosome
biogenesis and function (Table
1.1
). This includes the use of anti-centrosomal
antibodies, the use of reverse genetic approaches, and the use of forward genetic
approaches such as the cloning of genes from sterile, mechanosensory, or PCL
mutants.
1.6.1 Anti-Centrosomal Antibodies
Analysis of the centrosomal gene CP190 (Whitfield et al.
1988
) was facilitated by
the use of an antibody raised against isolated centrosomes and that were later
found to bind CP190. CP60 was identified as a protein that interacts with CP190
(Kellogg and Alberts
1992
). CP190 and CP60 localized to the centrosome in a cell
cycle-dependent manner and their amount at the centrosome was shown to be
maximal during mitosis and was barely detected during interphase. However,
CP190 and CP60 are not centrosome-specific and are also found in the nucleus
during interphase. A mutant for the CP190 gene was identified using standard
genetic approaches and it was found that CP190 is not essential for centrosome
biogenesis or function, but its function in the nucleus is essential for fly viability
(Butcher et al.
2004
). RNAi study of CP60 suggests it is also not essential for
centrosome biogenesis or function (Butcher et al.
2004
). Orthologs of CP190 and
CP60 have not been identified in vertebrates.
1.6.2 Reverse Genetics
Another way to obtain mutants in centrosomal proteins is to study the Drosophila
ortholog of known centriolar proteins in other organisms. This approach benefits
from the availability of large collections of mutant flies in identified genes. For
example, Sas-4 (Kirkham et al.
2003
), Sas-6 (Dammermann et al.
2004
; Leidel
et al.
2005
), Spd-2 (Kemp et al.
2004
; Pelletier et al.
2004
), Bld10 (Matsuura et al.
2004
) and PLK4 (Habedanck et al.
2005
) are all conserved centriolar proteins first
identified in other model organisms.
1.6.3 Bioinformatic subtractive screen for ciliary
and centrosomal proteins
Several centrosomal proteins were identified in a bioinformatic subtractive screen
for ciliary and centrosomal proteins (Avidor-Reiss et al.
2004
; Li et al.
2004
). Such
a screen is based on the idea that only organisms that have cilia should have ciliary
genes and is used to identify genes that are conserved in organisms with cilia but
