A high throughput screen to identify secreted and transmembrane proteins involved in Drosophila embryogenesis

Abstract

Secreted and transmembrane proteins play an essential role in intercellular communication during the development of multicellular organisms. Because only a small number of these genes have been characterized, we developed a screen for genes encoding extracellular proteins that are differentially expressed during Drosophila embryogenesis. Our approach utilizes a new method for screening large numbers of cDNAs by whole-embryo in situ hybridization. The cDNA library for the screen was prepared from rough endoplasmic reticulum-bound mRNA and is therefore enriched in clones encoding membrane and secreted proteins. To increase the prevalence of rare cDNAs in the library, the library was normalized using a method based on cDNA hybridization to genomic DNA-coated beads. In total, 2,518 individual cDNAs from the normalized library were screened by in situ hybridization, and 917 of these cDNAs represent genes differentially expressed during embryonic development. Sequence analysis of 1,001 cDNAs indicated that 811 represent genes not previously described in Drosophila. Expression pattern photographs and partial DNA sequences have been assembled in a database publicly available at the Berkeley Drosophila Genome Project website (http://fruitf ly.berkeley.edu). The identification of a large number of genes encoding proteins involved in cell-cell contact and signaling will advance our knowledge of the mechanisms by which multicellular organisms and their specialized organs develop.

Signal sequence- and translation-independent mRNA localization to the endoplasmic reticulum

“…As expected, the cytosol fraction was highly enriched in mRNAs encoding cytosolic proteins (EASE value > 8e À8 ). Consistent with previous studies on mRNA partitioning between cytosol and membrane compartments, we observed a partial, but substantial, overlap of cytosolic mRNAs in the nuclear envelope pool (Kopczynski et al 1998;Diehn et al 2000Diehn et al , 2006de Jong et al 2006). Of the 11,386 genes identified in the cytosol fractions, 9263 of these messages were also detected at significant (more than twofold over background) levels in the nuclear envelope fraction.…”

“…Analyses of mRNA partitioning between the cytosol and ER membrane compartments of mammalian cells, fly, and yeast have identified cohorts of cytosolic/nucleoplasmic protein-encoding mRNAs that are highly enriched in the ER, suggesting that ER-directed mRNA localization can occur by a mechanism(s) other than the SRP pathway (Mueckler and Pitot 1981;Kopczynski et al 1998;Diehn et al 2000;Nicchitta et al 2005). In support of this proposal, it has been demonstrated that mRNAs retain their association with the ER in the absence of ribosome engagement and thus participate in ribosome-independent interactions with components of the ER Lerner and Nicchitta 2006).…”

Characterization of differentially expressed genes in purified Drosophila follicle cells: Toward a general strategy for cell type-specific developmental analysis

“…Future screens could also combine the strategies of genetic mutation and spatial selection of follicle cells, for example by crossing GAL4 lines and UAS-GFP into a grkϪ background and analyzing the effect of grk on the expression profiles of specific subregions of the follicular epithelium. This type of targeted mutant analysis will likely be the most useful future application of these methods-screens for purely spatial selection may be supplanted by large-scale in situ screening (37), but it is unlikely to become feasible to perform many thousands of in situ hybridizations for every mutant situation a researcher wishes to analyze. These methods can also be readily applied to other systems; Drosophila imaginal discs are an obvious candidate, and as Krasnow et al (5) have previously pointed out, FACS purification of cells marked with cell type-specific reporter constructs can in principle be applied to any organism receptive to germ-line transformation.…”

The Drosophila synaptotagmin-like protein bitesize is required for growth and has mRNA localization sequences within its open reading frame

“…We first identified btsz in a reverse genetic screen for genes expressed during Drosophila development (18). The btsz transcription unit covers Ϸ50 kb and is predicted to have at least 15 exons (Fig.…”

“…All cDNAs were isolated by screening Drosophila embryonic (LD) and adult head (GH) cDNA libraries (17) with probes derived from clones from the CK cDNA library (18) and by 5Ј RACE, which was accomplished by using the Clontech Marathon cDNA amplification kit. Potential full-length cDNAs were sequenced, analyzed with SEQUENCHER software, and searched against nucleotide and protein databases by using BLAST programs (19).…”