skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Exploring strategies for protein trapping in Drosophila

Abstract

The use of fluorescent protein tags has had a huge impact oncell biological studies in virtually every experimental system.Incorporation of coding sequence for fluorescent proteins such as greenfluorescent protein (GFP) into genes at their endogenous chromosomalposition is especially useful for generating GFP-fusion proteins thatprovide accurate cellular and subcellular expression data. We testedmodifications of a transposon-based protein trap screening procedure inDrosophila to optimize the rate of recovering useful protein traps andtheir analysis. Transposons carrying the GFP-coding sequence flanked bysplice acceptor and donor sequences were mobilized, and new insertionsthat resulted in production of GFP were captured using an automatedembryo sorter. Individual stocks were established, GFP expression wasanalyzed during oogenesis, and insertion sites were determined bysequencing genomic DNA flanking the insertions. The resulting collectionincludes lines with protein traps in which GFP was spliced into mRNAs andembedded within endogenous proteins or enhancer traps in which GFPexpression depended on splicing into transposon-derived RNA. We report atotal of 335 genes associated with protein or enhancer traps and aweb-accessible database for viewing molecular information and expressiondata for these genes.

Authors:
; ; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
COLLABORATION - Yale U. School ofMedicine
OSTI Identifier:
923654
Report Number(s):
LBNL-62587
Journal ID: ISSN 0016-6731; GENTAE; R&D Project: L0111; BnR: 600305000; TRN: US200804%%1379
DOE Contract Number:
DE-AC02-05CH11231
Resource Type:
Journal Article
Resource Relation:
Journal Name: Genetics; Journal Volume: 175; Journal Issue: 3; Related Information: Journal Publication Date: March 2007
Country of Publication:
United States
Language:
English
Subject:
59; DNA; DROSOPHILA; EMBRYOS; GENES; MODIFICATIONS; OOGENESIS; PRODUCTION; PROTEINS; RNA; SPLICING; TRANSPOSONS; TRAPPING

Citation Formats

Quinones-Coello, Ana T., Petrella, Lisa N., Ayers, Kathleen, Melillo, Anthony, Mazzalupo, Stacy, Hudson, Andrew M., Wang, Shu, Castiblanco, Claudia, Buszczak, Michael, Hoskins, Roger A., and Cooley, Lynn. Exploring strategies for protein trapping in Drosophila. United States: N. p., 2006. Web. doi:10.1534/genetics.106.065995.
Quinones-Coello, Ana T., Petrella, Lisa N., Ayers, Kathleen, Melillo, Anthony, Mazzalupo, Stacy, Hudson, Andrew M., Wang, Shu, Castiblanco, Claudia, Buszczak, Michael, Hoskins, Roger A., & Cooley, Lynn. Exploring strategies for protein trapping in Drosophila. United States. doi:10.1534/genetics.106.065995.
Quinones-Coello, Ana T., Petrella, Lisa N., Ayers, Kathleen, Melillo, Anthony, Mazzalupo, Stacy, Hudson, Andrew M., Wang, Shu, Castiblanco, Claudia, Buszczak, Michael, Hoskins, Roger A., and Cooley, Lynn. Mon . "Exploring strategies for protein trapping in Drosophila". United States. doi:10.1534/genetics.106.065995.
@article{osti_923654,
title = {Exploring strategies for protein trapping in Drosophila},
author = {Quinones-Coello, Ana T. and Petrella, Lisa N. and Ayers, Kathleen and Melillo, Anthony and Mazzalupo, Stacy and Hudson, Andrew M. and Wang, Shu and Castiblanco, Claudia and Buszczak, Michael and Hoskins, Roger A. and Cooley, Lynn},
abstractNote = {The use of fluorescent protein tags has had a huge impact oncell biological studies in virtually every experimental system.Incorporation of coding sequence for fluorescent proteins such as greenfluorescent protein (GFP) into genes at their endogenous chromosomalposition is especially useful for generating GFP-fusion proteins thatprovide accurate cellular and subcellular expression data. We testedmodifications of a transposon-based protein trap screening procedure inDrosophila to optimize the rate of recovering useful protein traps andtheir analysis. Transposons carrying the GFP-coding sequence flanked bysplice acceptor and donor sequences were mobilized, and new insertionsthat resulted in production of GFP were captured using an automatedembryo sorter. Individual stocks were established, GFP expression wasanalyzed during oogenesis, and insertion sites were determined bysequencing genomic DNA flanking the insertions. The resulting collectionincludes lines with protein traps in which GFP was spliced into mRNAs andembedded within endogenous proteins or enhancer traps in which GFPexpression depended on splicing into transposon-derived RNA. We report atotal of 335 genes associated with protein or enhancer traps and aweb-accessible database for viewing molecular information and expressiondata for these genes.},
doi = {10.1534/genetics.106.065995},
journal = {Genetics},
number = 3,
volume = 175,
place = {United States},
year = {Mon Dec 18 00:00:00 EST 2006},
month = {Mon Dec 18 00:00:00 EST 2006}
}
  • Drosophila virilis genomic DNA corresponding analysis of a 3.8-kb genomic piece allowed identification of (1) an open reading frame (ORF) with striking homology to the previously identified D. melanogaster ORF and (2) conserved sequence elements of possible regulatory relevance within and flanking the second intron. Conceptual translation of the D. virilis ORF predicts a 519-amino-acid-long ribonucleoprotein consensus sequence-type protein. Similar to D. melanogaster ELAV protein, it contains three tandem RNA-binding domains and an alanine/glutamine-rich amino-terminal region. The sequence throughout the RNA-binding domains, comprising the carboxy-terminal 346 amino acids, shows an extraordinary 100% identify at the amino acid level, indicating amore » strong structural constraint for this functional domain. Thus, the divergence of the amino-terminal region of the ELAV protein reflects lowered functional constraint rather than species-specific functional specification.« less
  • A scheme for a Ramsey-coherent population trapping (CPT) atomic clock that eliminates the acousto-optic modulator (AOM) is proposed and experimentally studied. Driven by a periodically microwave modulated current, the vertical-cavity surface-emitting laser emits a continuous beam that switches between monochromatic and multichromatic modes. Ramsey-CPT interference has been studied with this mode-switching beam. In eliminating the AOM, which is used to generate pulsed laser in conventional Ramsey-CPT atomic clock, the physics package of the proposed scheme is virtually the same as that of a conventional compact CPT atomic clock, although the resource budget for the electronics will slightly increase as amore » microwave switch should be added. By evaluating and comparing experimentally recorded signals from the two Ramsey-CPT schemes, the short-term frequency stability of the proposed scheme was found to be 46% better than the scheme with AOM. The experimental results suggest that the implementation of a compact Ramsey-CPT atomic clock promises better frequency stability.« less
  • A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. In this paper, we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix–loop–helix–loop structural motif. Eighty-three designs with sequences unrelatedmore » to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 °C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 Å. Finally, our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering.« less
  • Embedding nanostructures within a bulk matrix is an important practical approach towards the electronic engineering of high performance thermoelectric systems. For power generation applications, it ideally combines the efficiency benefit offered by low dimensional systems along with the high power output advantage offered by bulk systems. In this work, we uncover a few crucial details about how to embed nanowires and nanoflakes in a bulk matrix so that an overall advantage over pure bulk may be achieved. First and foremost, we point out that a performance degradation with respect to bulk is inevitable as the nanostructure transitions to a multimore » moded one. It is then shown that a nano embedded system of suitable cross-section offers a power density advantage over a wide range of efficiencies at higher packing fractions, and this range gradually narrows down to the high efficiency regime, as the packing fraction is reduced. Finally, we introduce a metric - the advantage factor, to elucidate quantitatively, the enhancement in the power density offered via nano-embedding at a given efficiency. In the end, we explore the maximum effective width of nano-embedding which serves as a reference in designing generators in the efficiency range of interest.« less