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Poster Full Abstracts - Cell Cycle and Checkpoints
Poster board number is above title. The first author is the presenter
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developmental abnormalities etc. Therefore, analyzing the role of Sld5 in checkpoint regulation is essential for understanding its contribution in the
maintenance of genomic stability.
292A
Regulation of Replication Initiation and Fork Progression during
Drosophila
Follicle Cell Gene Amplification.
Brian Hua, Jessica L Alexander*,
Terry Orr-Weaver. Whitehead Institute, Cambridge, MA.
During follicle cell differentiation six genomic regions undergo repeated origin firing and bidirectional replication fork movement to increase gene copy
number. These
Drosophila Amplicons in Follicle Cells
(
DAFCs
) have specific replication origins that utilize the same machinery as normal DNA replication,
including the Origin Recognition Complex (ORC), DUP/Cdt1, and the DNA helicase MCM2-7. The six
DAFCs
amplify to differing extents because they
undergo distinct numbers of rounds of initiation, but the mechanisms controlling firing of amplification origins are unknown. We found that cis-acting
position effects acting over at least five kb influence whether ORC is bound and amplification occurs at
DAFC-22B
. Transposons with
DAFC
origins
inserted at ectopic sites are subject to position effects influencing the level of amplification. We will present experiments testing whether these position
effects also impact replication fork progression and the extent of the amplified domain. We have generated
DAFC
transposon insertions that depend on
insulator function for amplification to occur. These are being exploited to define the effects of insulators on ORC binding and origin activation.
*First two authors are co-presenters.
293B
Mutation of the lethal(2)denticless gene results in larval lethality and sterility.
S. Catherine S. Key
1
, Roketa Sloan
1
, Christina Swanson
2
, Maryonne
Snow-Smith
1
, Kristen Smith
1
. 1) Department of Biology, North Carolina Central University, Durham, NC; 2) Department of Biology, University of North
Carolina-Chapel Hill, Chapel Hill, NC.
The lethal(2)denticleless (l(2)dtl) gene was originally reported as essential for embryogenesis and formation of the tiny rows of hairs known as the denticle
belt in Drosophila. It is now well-established that l(2)dtl/cdt2 produces an E3 ubiquitin ligase protein which is a key regulator of the cell cycle targeting a
number of essential cell cycle factors including p21, Cdt1, E2F1 and Set8. To investigate the role of l(2)dtl/cdt2 during development, we characterized
existing disruption mutants and generated new deletion strains. We found that heterozygous disruption of the l(2)dtl/cdt2 gene results in a male sterility
phenotype that is corrected by restoration of the gene. All homozygous mutant embryos, selected by negative GFP fluorescence, had intact denticle bands.
The mutant embryos progressed through embryogenesis and died during larval development. New mutant strains generated by P-element mobilization
resulted in deletion strains that are also larval lethal with intact denticle bands. Although mutation of cdt2 in yeast and mice embryos results in replication
defects, based on BrdU assays, there is no detectable replication phenotype during embryogenesis in mutant embryos. However, indirect immunofluorscence
with anti-Cdt2 antibody suggests that L2DTL/Cdt2 is maternally deposited. We conclude that the name l(2)dtl is a misnomer, that lethality occurs during
larval rather than embryonic development, that maternally deposited Cdt2/L2DTL allows progression through embryogenesis, and that l(2)dtl/cdt2 is
important for male fertility.
294C
Integrins are required for proper cell cycle progression and differentiation.
Maria J. Gomez-Lamarca, Laura Cobreros, Maria D. Martin-Bermudo.
Centro Andaluz de Biologia del Desarrollo (CABD), Univ. Pablo Olavide-CSIC, SEVILLA, Spain.
Coordinating differentiation with exit from the cell cycle is critical for proper organogenesis, yet how this is achieved remains largely unknown. The
development of the follicular epithelium of the
Drosophila
ovary represents an ideal system to study the mechanisms controlling the transition from cell
cycle exit to differentiation. The ovary of the adult
Drosophila
female is composed of various tubular structures called ovarioles that contains a line of egg
chambers at different developmental stages. Each egg chamber begins as a 16-cell germline cyst surrounded by a monolayer of somatic follicle cells (FCs)
precursors. During the early stages (up to stage 6), FCs undergo a mitotic division program giving rise to approximately 1000 FCs, which will form a
monolayer known as the follicular epithelium. After stage 6, FCs differentiate and switch from normal mitotic cycle to undergo three rounds of
endoreplication. Later in oogenesis, four different loci synchronously initiate a gene amplification event. By clonal and FACS analysis, we show that
integrins are required for proper proliferation-to-differentiation switch. Interestingly, although integrin mutant cells exit mitosis they remain in an
undifferentiated state and do not enter endocycle. In addition, integrin mutant follicle cells do not initiate the amplification event. At present we are
investigating the molecular mechanisms by which integrins regulate the cell cycle exit to differentiation switch. Our results suggest that integrin mediated
signalling controls this transition by regulating key cell cycle regulator proteins, such as Cyclin B and Dacapo.
295A
Tissue Growth Coordination in the Drosophila Brian via Glia Polyploidization.
Yingdee Unhavaithaya, Terry Orr-Weaver. Whitehead Institute and
Dept. of Biology, Massachusetts Institute of Technology, Cambridge MA 02142.
Proper development requires coordination in growth of the tissue layers comprising an organ. Although there are many examples of large polyploid cells,
little is known about how these polyploid tissues contribute to organ growth. Through examination of nuclear DNA content in situ, we found the Drosophila
subperineurial glia (SPG) to be polyploid in the brain, ventral nerve cord and peripheral nervous system, with ploidy levels ranging up to 22C.
Subperineurial glia cells polyploidize either through endoreplication or endomitosis, producing cells with a single polyploidy or multiple nuclei,
respectively. Inhibition of SPG polyploidy resulted in blood-brain barrier defects, revealing that the increased DNA content and resultant cell size is required
to accomodate the growing brain to maintain the blood-brain barrier. We could rescue these blood-brain barrier defects with dmyc overexpression in the
SPG or by attenuating neuroblast proliferation, indicating that polyploidy is essential for the maintenance of the blood-brain barrier. The increased ploidy of
the SPG defines a new mechanism to coordinate growth of the glial and neuronal tissue layers during brain development; failure of this coordination ruptures
the septate junctions of the SPG envelope around the brain. This mechanism is likely conserved, with potential vertebrates examples in megakaryocytes and
giant trophoblasts.