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First published online April 22, 2004
doi: 10.1242/10.1242/dev.01064
Molecular and Computational Biology Program, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089-1340, USA
* Author for correspondence (e-mail: jtower{at}USC.edu)
Accepted 8 January 2004
The developmentally regulated amplification of the Drosophila
third chromosome chorion gene locus requires multiple chromosomal elements.
Amplification control element third chromosome (ACE3) appears to function as a
replicator, in that it is required in cis for the activity of nearby DNA
replication origin(s). Ori-ß is the major origin in the locus, and is a
sequence-specific element that is sufficient for high-level amplification in
combination with ACE3. Sequence requirements for amplification were examined
using a transgenic construct that was buffered from chromosomal position
effects by flanking insulator elements. The parent construct supported 18- to
20-fold amplification, and contained the 320 bp ACE3, the 
1.2 kb
S18 chorion gene and the 840 bp ori-ß. Deletion mapping of ACE3
revealed that an evolutionarily conserved 142 bp core sequence functions in
amplification in this context. Several deletions had quantitative effects,
suggesting that multiple, partially redundant elements comprise ACE3. S.
cerevisiae ARS1 origin sequences could not substitute for ori-ß,
thereby confirming the sequence specificity of ori-ß. Deletion mapping of
ori-ß identified two required components: a 140 bp 5' element and a
226 bp A/T-rich 3' element called the ß-region that has significant
homology to ACE3. Antibody to the origin recognition complex subunit 2 (ORC2)
recognizes large foci that localize to the endogenous chorion gene loci and to
active transgenic constructs at the beginning of amplification. Mutations in
Orc2 itself, or the amplification trans regulator satin
eliminated the ORC2 foci. By contrast, with a null mutation of
chiffon (dbf4-like) that eliminates amplification, diffuse
ORC2 staining was still present, but failed to localize into foci. The data
suggest a novel function for the Dbf4-like chiffon protein in ORC
localization. Chromosomal position effects that eliminated amplification of
transgenic constructs also eliminated foci formation. However, use of the
buffered vector allowed amplification of transgenic constructs to occur in the
absence of detectable foci formation. Taken together, the data suggest a model
in which ACE3 and ori-ß nucleate the formation of a ORC2-containing
chromatin structure that spreads along the chromosome in a mechanism dependent
upon chiffon.
Key words: Dbf4, DNA replication, Chiffon, ORC, Amplification, Drosophila
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