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Poster Full Abstracts - Techniques and Functional Genomics
Poster board number is above title. The first author is the presenter
359
Epitope labeling of histidine decarboxylase in
Drosophila melanogaster
.
Benjamin Fair
1
, Marc Vander Vliet
2
, Stephanie Payne
3,4
, Martin Burg
2,3
. 1)
Biology, Grand Valley State Univ., Allendale, MI; 2) Biomedical Sciences, Grand Valley State Univ., Allendale, MI; 3) Cell & Molecular Biology, Grand
Valley State Univ., Allendale, MI; 4) Biology, Johns Hopkins Univ., Baltimore, MD.
Histidine decarboxylase (HDC) plays a critical role in the synthesis of histamine, a central and peripheral nervous system neurotransmitter used by
invertebrates. Past attempts to create antisera that recognize HDC
in vivo
have not produced satisfactory results. While some HDC antisera have been made
in other organisms, they appear not to be useful across species, including
Drosophila melanogaster
. As a result, little is known about the localization or
biochemistry of HDC in the fly. It has been suggested that HDC undergoes a complex maturation process, undergoing cleavage at both the N- and C- termini
of the protein. We report an approach that allows a functional HDC protein to be examined
in vivo
using internal epitope tagging. A genomic fragment that
had been previously shown to contain a completely functional
Hdc
gene was modified by a PCR-mediated insertion of an epitope tag, 6x-HIS, into the
protein coding region of the
Hdc
gene at specific sites. The location of these tags in the protein structure was selected to be in regions of the mature HDC
protein which likely would not affect its function, based on comparisons of the structure of DDC from other species with the HDC protein sequence. Each
Hdc
transgene containing a 6X-HIS tagged
Hdc
gene was transformed into
Hdc
JK910
mutant flies that normally have little to no histamine or HDC activity.
Results indicate that while one of the epitope tags appears to disrupt
Hdc
function (indicated by a lack of histamine staining in the CNS), a 6X-HIS tag in a
different location of the HDC protein structure appears to have no disruptive effect on
Hdc
function (indicated by normal histamine staining in the CNS).
Assuming other epitopes can be used that may be easier to detect in tissue; this approach should enable further studies into the biochemistry and cell biology
of HDC
in vivo
.
853A
Fluorescent fusion protein knockout mediated by anti-GFP nanobody.
Oguz Kanca, Emmanuel Caussinus, Markus Affolter. Cell Biology, Biozentrum
of University of Basel, Basel, Basel-Stadt, Switzerland.
Disruption of protein function has been a central approach in modern biology. Diverse methods such as RNA interference and morpholinos are routinely
used in order to knock down protein function. Nevertheless, those approaches target the RNA encoding for the protein, thus the removal of protein function
depends on the turnover rate of existing target protein. Here we present a new genetic method, named deGradFP, for direct and fast depletion of target Green
Fluorescent Protein (GFP) fusions. deGradFP uses the highly conserved ubiquitination pathway to deplete the target proteins, making it applicable on any
eukaryotic model system. Moreover since it targets GFP fusion protein the knock down can be monitored during live imaging through disappearance of GFP
signal. For many targets it is a ready to use technique as GFP protein trap stocks are being generated through community efforts in Drosophila
(http://flytrap.med.yale.edu/index.html, http://www.flyprot.org/) and in zebrafish (http://kawakami.lab.nig.ac.jp/ztrap/).
854B
Construction of Drosophila strains expressing affinity-tagged Ubiquitins: Investigating the regulation of Epsin by ubiquitination in Notch signaling
cells.
Kristin D. Patterson, Janice A. Fischer. Molecular, Cell and Developmental Biology, The University of Texas at Austin, Austin, TX.
Ubiquitin (Ub) is a 76 amino acid polypeptide that may be attached covalently to other proteins and regulate their activities. Proteins may be mono-
ubiquitinated, or they may be attached to a Ub chain in which several Ub moieties are linked together through one of the seven lysine residues found in Ub
itself. Mono-ubiquitination or polyubiquitination with a particular type of chain linkage affects the fate of the modified protein in different ways. The
Drosophila genome has four genes that encode Ub. We are engineering the fly genome so that most, if not all, of the endogenous Ub is affinity tagged. The
tags do not affect the Ub function, and they will enable purification of any ubiquitinated substrate. We will demonstrate the utility of the tagged Ub fly lines
by purifying and analyzing Ub-Epsin. Extracts from fly stocks that express tagged substrate and tagged Ub will be the starting material for tandem affinity
purification (TAP). The purified Ub-substrate will be analyzed by mass spectrometry to identify the site and type of Ub linkage. The primary role of Epsin in
the developing fly is to activate Notch cell signaling by facilitating ligand endocytosis into the signaling cell. Fly eyes with too much or too little active
Epsin have developmental defects that recapitulate Notch pathway gene mutant phenotypes. Ubiquitination of Epsin, and its deubiquitination by the enzyme
Fat facets, regulates Epsin activity. Two models have been proposed to explain the inactivity of Ub-Epsin: Ub-Epsin may be targeted to the proteasome for
degradation or ubiquitination may prevent the association of Epsin with ligand at the plasma membrane. We will begin to distinguish between these
mechanisms and others by determining the sites and types of Ub linkages on purified Ub-Epsin.
855C
Microarray-based Capture of Novel Expressed Cell type-specific Transcripts (CoNECT) to annotate tissue-specific transcript isoforms.
Xiaojing
Hong
1
, Harshavardhan Doddapaneni
2
, Matthew Rodesch
3
, Heather Halvensleben
3
, Raghu Metpally
1
, Todd Richmond
3
, Bolei Fu
1
, Thomas Albert
3
, J Robert
Manak
1,2,4
. 1) Dept of Biology, Univ of Iowa, Iowa City, IA; 2) Carver Center for Genomics, Univ of Iowa, Iowa City, IA; 3) Roche NimbleGen, Madison,
WI; 4) Dept of Pediatrics, Univ of Iowa, Iowa City, IA.
Faithful annotation of tissue-specific transcript isoforms is important not only to understand how genes are organized and regulated, but also to identify
potential novel, unannotated exons of genes which may be additional targets of mutation in disease states or while performing mutagenic screens. We have
developed a microarray enrichment methodology followed by long-read next generation sequencing for identification of transcript isoforms expressed in
Drosophila
ovaries and the testes. These studies have identified a large number of novel transcript isoforms, including over 2,300 novel 5’ exons/extensions,
over 900 novel 3’ exons/extensions, 282 novel internal exons, 1011 internal exon extensions and 23 gene fusions. Additionally, we identified both germline-
specific transcription start sites and splicing events. As has been suggested in other studies, we find that the testis transcriptome harbors a significantly
higher number of novel transcript isoforms than the ovary, indicating that a more diverse transcriptome is required for male germ cell development. Finally,
comparing our enrichment data set with tiling array analysis, we demonstrate that microarray enrichment 1) is able to capture both highly expressed as well
as low-expressed genes, 2) is quantitative in terms of gene expression levels, and, 3) is able to capture a large number of transcripts which cannot be
identified by microarray analysis. These studies introduce an efficient methodology for cataloguing transcriptomes in which specific classes of genes or
transcripts can be targeted for capture and sequence, thus reducing the significant sequencing depth normally required for accurate annotation.
856A