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Poster Full Abstracts - Regulation of Gene Expression
Poster board number is above title. The first author is the presenter
343
terminals. Mutants of the second gene CG17816, named
bumblebee
(
bbe
), also leads to loss of GluRIIA but no disruption in production of other GluR
protein subunits. Both opr and bbe are novel highly conserved proteins. Optimus prime is the founding member of a novel protein family. SNPs in the
human opr homolog are associated with autism spectrum disorders.
791B
Investigating Duplicated Ribosomal Proteins Reveals Differential Post-Translational Modification of the RpL22e Family in the Male Germline:
Evidence for SUMOylation of RpL22.
Michael Kearse, Jill Ireland, Vassie Ware. Department of Biological Sciences, Lehigh University, Bethlehem, PA.
Duplicated ribosomal protein (Rp) genes are found throughout eukaryotic genomes, at times encoding highly similar or identical proteins. Whether these
paralogues are functionally redundant or provide a unique cellular role, not limited to translation, remains largely unknown. We have focused on the the
eukaryotic-specific RpL22e family members in
Drosophila
, RpL22 and RpL22-like. Using paralogue-specific C-terminal peptide-derived polyclonal
antibodies, Western analysis shows RpL22-like is primarily testis-specific, found at its predicted molecular weight (MW), and is a component of ribosomes
(Kearse
et al
., 2011). Furthermore, RpL22 is detected not only at its expected MW of 33kD, but more predominantly at a higher MW of ~50kD, suggestive
of post-translational modification. Computational probing predicts a SUMOylation consensus motif, localized at different sites within the fly-specific N-
terminal extension of both family members. In this report, we investigate the possible SUMOylation of RpL22. Results from S2 cell-based experiments
replicate the RpL222 higher MW pattern when a FLAG-tagged RpL22, but not a K39R mutation within the proposed SUMOylation site, is co-expressed
with HA-SUMO. These results support the
in silico
prediction and demonstrate that RpL22 can be SUMOylated, likely with two SUMO moieties.
Interestingly, Western analysis shows that SUMOylated RpL22 is more predominant in adult testis than in S2 cells. Western analysis of RpL23a-FLAG-
affinity purified complexes from S2 cells shows that SUMOylated RpL22 is only a minor component of complexes containing RpL23a. Whole-mount testis
immunohistochemistry reveals distinct nucleoplasmic, but not nucleolar localization for RpL22 and cytoplasmic localization for RpL22-like within the germ
cells. Taken together, these data suggest that RpL22 and RpL22-like have distinct roles within the germline.
792C
The evolutionarily canalized expression of
eve
stripe 2 in
Drosophila
and the Sepsidae.
Ah-Ram Kim
1,2
, Carlos Martinez
1
, Bin He
1
, Michael Ludwig
1
,
Martin Kreitman
1
, John Reinitz
1,3
. 1) Department of Ecology and Evolution, Chicago Center for Systems Biology, University of Chicago, IL, U.S.A; 2)
Department of Biochemistry and Cell Biology, Stony Brook University, NY, U.S.A; 3) Departments of Statistics and Molecular Genetics & Cell Biology,
University of Chicago, IL, U.S.A.
We employ a theoretical model that is intermediate between, on one hand, a content-based picture in which only the number of binding sites for each factor
in an enhancer is significant, and, on the other hand, a grammar-based approach in which a precise arrangement of binding sites is required for regulatory
function. The model quantitatively treats 1) proteins binding to DNA; 2) Steric competition for binding sites; 3) Cooperative binding; 4) Short range
repression by quenching; 5) Coactivation; 6) Direct repression; and 7) Transcriptional initiation as a diffusion limited enzymatic process driven by an
Arrhenius rate law. In our model, the physical arrangement of binding sites is quite important, but it is specified by rules that are sufficiently flexible to
permit many solutions, reflecting the observed variability in binding site arrangement. Here we report new progress in understanding the naturally evolved
robustness of transcriptional control at single functional binding site resolution. It is a remarkable fact that
eve
enhancers from 12
Drosophila
species and
from the Sepsidae, which bear almost no homology to those from melanogaster, nevertheless express in that organism in patterns only slightly different from
the native ones. We make use of the fact that our model can be used to untangle the relative roles of many simultaneously acting regulatory mechanisms to
show how the evolutionarily canalized expression of eve stripe 2 in
Drosophila
and the Sepsidae is achieved.
793A
Turning off Bruno-dependent Translational Repression.
Goheun Kim
1
, Keiji Sato
2
, Akira Nakamura
2
, Paul Macdonald
1
. 1) Molecular Cell &
Developmental Biology Dept, University of Texas at Austin, Austin, TX; 2) Laboratory for Germline Development, RIKEN Center for Developmental
Biology, Kobe, Japan.
Oskar (Osk) is a posterior body patterning determinant in Drosophila and is highly concentrated at the posterior pole of the oocyte. Tight spatial and
temporal restriction of the Osk patterning activity is essential for the viability of the embryo. Bruno (Bru) directly binds to the
osk
mRNA and represses
translation during mRNA localization to the posterior pole. In one model for repression, the interaction between Bru, Cup and eIF4E is thought to inhibit
translational initiation. In another model, Bru promotes oligomerization of multiple
osk
mRNAs into large particles that are inaccessible to the translational
machinery. Evidence from our lab suggests that translational activation of
osk
involves at least two spatially distinct mechanisms: one acts throughout the
oocyte and requires
cis
-acting elements; another appears to act only at the posterior pole of the oocyte and may be independent of
cis
elements. Insights into
the second mechanism come from analysis of Bru protein interactions and phosphorylation. We showed that Bru dimerizes, which can explain how Bru can
oligomerize
osk
mRNA. We mapped domains involved in Bru-Bru and Bru-Cup interactions, and found that aa1-146 and aa334-416 contribute to both
interactions, with a stronger requirement for aa1-146. We found that a small fraction of Bru is phosphorylated. Several predicted sites of phosphorylation in
Bru lie within the regions important for Bru-Bru and -Cup interactions, and could be targets for inactivation of repression. Phosphomimetic mutants of some
of the sites that are predicted targets of Protein Kinase A (PKA) are defective in Bru binding to both itself and Cup, while corresponding phosphosilent
mutants have no effect. aa1-146 of Bru that contains these sites can be phosphorylated in vitro by purified PKA. We propose that local phosphorylation of
Bru by PKA at the posterior of the oocyte is a likely mechanism for inactivating Bru-dependent translational repression by either of two models.
794B
Dose dependent buffering effects in Drosophila melanogaster.
Lina E Lundberg, Per Stenberg, Margarida Figueiredo, Jan Larsson. Dept. of molecular
biology, Umeå, Umeå, Sweden.
Chromosomal aneuploidy is a common feature of cancers and involves loss or duplication of chromosomal copies. This normally lethal condition can be
tolerated when small regions are affected, but the transcriptional effects and mechanisms that are triggered by these segmental aneuploidies have been
elusive. However, as better and more accurate techniques have evolved, more is known about transcriptional responses in these region and it has become
clear that the remaining chromosome copy in a haploid (single) region is not unaffected by this state. Rather, the transcriptional output from a single region