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Poster Full Abstracts - Regulation of Gene Expression
Poster board number is above title. The first author is the presenter
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is higher than the expected 50% of wildtype level, meaning that compensatory mechanisms stimulate transcription in these regions in response to
chromosomal loss, acting to push the level of transcripts from an aneuploid region closer to wildtype level. This mechanism has been termed “buffering”. It
should be noted that genome-wide studies inevitably include analyses of non-expressed genes and genes expressed at non-detectable levels; two groups of
genes that thus will be scored as fully compensated. By taking these genes into consideration, we have recently shown that this buffering effect is in fact
lower than previously reported. To further study this buffering effect and to find out which factors influence the level of buffering, 7
Drosophila
deficiency
stocks were analyzed by microarray, both as single replicates and in pairwise combinations. In addition, 6 wildtype replicates were used to create a model of
normal biological variation, an important understanding when examining small effects in genomic expression. We show that buffering is a general
mechanism which targets whole regions and that the buffering effect is larger in short regions compared to long regions. On individual gene level, a long
gene length is the primary determinant for buffering effect. For short genes, the expression level is more important, where genes with lower expression levels
are more buffered. Further, we have discovered that proteolytic genes are upregulated as a general response to aneuploidy.
795C
A Global Transcriptomics Approach Identifies Sex-Specific and Immune-System Changes in Gene Expression and pre-mRNA Splicing Induced by
Doa and fne Mutations.
Leonard Rabinow
1
, Marie-Laure Samson
1
, David Sturgill
2
, Xia Sun
1
, John Malone
2
, Yunpo Zhao
1
, Brian Oliver
2
. 1) University
Paris Sud, UMR 8195, Orsay, France; 2) Laboratory of Developmental Genomics NIDDK, NIH, Bethesda MD USA.
We are using high-throughput sequencing of cDNAs (RNA-Seq) Drosophila melanogaster cDNAs to identify splicing and expression-level targets of the
Doa
and
fne
loci.
Doa
encodes a protein kinase, which among other things phosphorylates SR proteins, influencing alternative splicing.
Doa
mutations affect
somatic sex-determination due to aberrant splicing of doublesex.
fne
is one of the three
elav
-family paralogues present in the Drosophila genome. Members
of this neuronally-expressed gene family, conserved in all metazoans, bind RNAs to influence diverse processes, including neuronal development, memory
and sleep regulation. Flies were raised under controlled conditions of temperature and low-density population for 3 generations, to avoid excessive
competition among larvae; constant lighting, to ablate circadian rhythms; and aging for 7 days post-eclosion in mixed-sex cultures to allow for
developmental stabilization of transcript levels. Flash-freezing was followed by isolation of fly heads on dry-ice. Poly-adenylated RNAs were prepared from
sex-sorted heads of wild-type,
fne
and
Doa
mutants and cDNAs subjected to high-throughput sequencing. Three rounds of sequencing were performed on
two independent samples. Potential target genes for
Doa
and
fne
affected either at the expression level or alternative splicing are being validated via RT-PCR
and genetic crosses. Importantly, the RNA-Seq results reproduce the aberrant alternative splicing of doublesex transcripts previously described for
Doa
mutants. Further results suggest that DOA kinase represses transcription of several anti-microbial peptide genes, since their expression is dramatically
elevated in mutants. Differences in the alternative splicing of several potential
Doa
target loci has also been observed.
796A
Regulation of
twin-of-eyeless
, a
Drosophila Pax6
gene.
John Skottheim Honn, Linn Jacobsson, Karin Ekström, Åsa Rasmuson-Lestander. Dept. of
Molecular Biology, Umeå University, Umeå, Sweden.
There are two
Pax6
genes in
Drosophila
;
eyeless (ey)
and
twin-of-eyeless (toy)
. They encode transcription factors that are important for the correct
development of head structures, a function that is conserved also in humans. Heterozygosity in humans leads to eye abnormalities known as aniridia,
homozygous mice are lacking eyes and in severe mutants in
Drosophila
most of the head is missing. This indicates that
Pax6
is not only involved in eye
formation and development but in formation of the early brain and central nervous system as well.
toy
is considered to be the first eye specification gene
expressed in the regulatory network that governs eye formation and the gene that, in turn, activates
eyeless
. In this study our focus is on how the
toy
gene is
regulated since that is still an open question. Toy is expressed very early during development (stage 5, 2-3 hrs after egg laying) and it is conceivable that
early transcription factors, like maternally deposited proteins or gap gene products are involved. By using a Toy specific antibody we study the expression of
Toy in various mutant backgrounds to find out which genes are involved in the temporal and spatial regulation of Toy. We show, by misexpression and
mutant analysis, that the head-specific gene
empty spiracles
alters the expression pattern of Toy in the head region around the visual primordia.
797B
Effect of matrix and nucleocapsid on multimerization of gypsy structural protein Gag.
Boris V. Syomin
1,2
, Tatjana A. Trendeleva
1,2
, Yurii V. Ilyin
2
,
Vladimir I. Popenko
2
. 1) Russian Institute of Experimental Vet Medicine, Moscow; 2) Institute of Molecular Biology, Russian Academy of Sciences,
Moscow, 119991 Russia.
Gypsy is an endogenous retrovirus that was first revealed in Drosophila. Amino acid sequence of the gypsy Gag does not contain a canonical motif known
for the majority of vertebrate retroviruses. Moreover, protein translation can theoretically begin with two separated initiation codons located within its
unique open reading frame. Therefore there are two theoretically possible variants of Gag gypsy that differ from each other by 41 amino acid residues of the
N-terminal. The Gag contains matrix domain in the case of the “long” variant, “short” variant of the Gag lacks matrix. We designed constructs for
expression of two variants of Gag polypeptide and investigated the ability of each product to form virus-like particles in the bacterial cell, i.e., in the absence
of eukaryotic cell factors. Both variants of structural protein Gag gypsy can form ellipsoid virus like particles with average diameter 27 nm in bacterial cells.
However, “short” variant of Gag is preferably synthesized in a eukaryotic cell because this variant of gypsy Gag was detected by anti-Gag antibodies in the
preparations of virus-like particles isolated from Drosophila. Expression of an alone capsid domain of gypsy Gag was also sufficient for particle assembly in
Escherichia coli. However, particles assembled from the capsid domain were variable in size and displayed much less organization than particles formed by
the whole Gag or its deleted mutant containing capsid fused to N-termini (proximal) part of nucleocapsid. The assembly of examined proteins in vitro can be
driven through interactions with RNA or single strand DNA oligonucleotide as well.
798C
Molecular mechanism of the miRNA machinery responding to serum deprivation in Drosophila.
Pei-Hsuan Wu, Richard Carthew. Northwestern
University, Department of Molecular Biosciences, 2205 Tech Drive, Hogan 2-100, Evanston, IL60208.
miRNAs have the remarkable capacity to modulate their target gene expression in response to environmental challenges ranging from various cellular
stress to nutritional deprivation, which reflects on its crucial role in numerous cellular processes. Serum deprivation has been shown to alter the function of