Bulk replicative DNA synthesis in eukaryotes is highly accurate and efficient, primarily because of two DNA polymerases (Pols): Pols δ and ε. The high fidelity of these enzymes is due to their intrinsic base selectivity and proofreading exonuclease activity which, when coupled with post-replication mismatch repair, helps to maintain human mutation rates at less than one mutation per genome duplication. Conditions that reduce polymerase fidelity result in increased mutagenesis and can lead to cancer in mice. Whereas yeast Pol ε has been well characterized, human Pol ε remains poorly understood. Here, we present the first report on the fidelity of human Pol ε. We find that human Pol ε carries out DNA synthesis with high fidelity, even in the absence of its 3′→5′ exonucleolytic proofreading and is significantly more accurate than yeast Pol ε. Though its spectrum of errors is similar to that of yeast Pol ε, there are several notable exceptions. These include a preference of the human enzyme for T→A over A→T transversions. As compared with other replicative DNA polymerases, human Pol ε is particularly accurate when copying homonucleotide runs of 4–5 bases. The base pair substitution specificity and high fidelity for frameshift errors observed for human Pol ε are distinct from the errors made by human Pol δ.

During embryogenesis, organisms undergo considerable cellular remodelling requiring the combined action of thousands of proteins. In the case of the well studied model Drosophila melanogaster, transcriptomic studies, most notably from the modENCODE project, have described in detail changes in gene expression at the mRNA level across development. Although such data are clearly very useful for understand how the genome is regulated during embryogenesis, it is important to understand how changes in gene expression are reflected at the level of the proteome. In this study, we describe a combination of two quantitative label free approaches, SWATH and Data Dependent Acquisition, to monitor changes in protein expression across a timecourse of Drosophila embryonic development. We demonstrate that both approaches provide robust and reproducible methods for the analysis of proteome changes. In a preliminary analysis of Drosophila embryogenesis, we identified several pathways, including the heat-shock response, nuclear protein import and energy production, that are regulated during embryo development. In some cases changes in protein expression mirrored transcript levels across development, whereas other proteins showed signatures of post-transcriptional regulation. Taken together, our pilot study provides a good platform for a more detailed exploration of the embryonic proteome.

Quantitative proteomics methods have emerged as powerful tools for measuring protein expression changes at the proteome level. Using mass-spectrometry (MS) based approaches, it is now possible to routinely quantify thousands of proteins. However, pre-fractionation of the samples at the protein or peptide level is usually necessary to go deep into the proteome, increasing both MS analysis time and technical variability. Recently, a new MS acquisition method named SWATH was introduced with the potential to provide good coverage of the proteome as well as a good measurement precision without prior sample fractionation. In contrast to shotgun based MS however, a library containing experimental acquired spectra is necessary for the bioinformatics analysis of SWATH data. In this study, we built spectral libraries for two widely used models to study crop ripening or animal embryogenesis, Solanum lycopersicum (tomato) and Drosophila melanogaster, respectively. The spectral libraries comprise fragments for 5,197 and 6,040 proteins for Solanum lycopersicum and Drosophila melanogaster, respectively, and allow reproducible quantification for thousands of peptides per MS analysis. The spectral libraries and all massspectrometry data are available in the MassIVE repository with the dataset identifiers MSV000081074 and MSV000081075 and the PRIDE repository with the dataset identifiers PXD006493 and PXD006495. For DIA, the spectral library contains experimental acquired spectra of the precursors and can be produced directly from the SWATH data, by reconstituting the MSMS spectra using the retention time of the precursors and the fragments [7], or by DDA analysis of the sample, which can be fractionated to increase the number of spectra in the library [9]. Ideally, the spectral library should be generated on a MS instrument similar to the one used to acquire further SWATH-MS data as the correlation of the fragment intensities for a peptide acquired on different instrument was shown to be rather low [10]. Although Rosenberger et al. recently published a library containing assays for 10,000 human proteins [9] and deep libraries for other applications such as SRM (Selected Reaction Monitoring) have been generated [11][12][13], the number of publicly available spectral libraries for SWATH-MS is still limited. Several other studies have produced libraries for other species [14][15][16]. However, spectral libraries are still missing for many species and, when available, the size of the libraries are limited and a deeper coverage of the proteome would increase the number of potential identifications in SWATH-MS analysis. In the present study, we produced spectral libraries for two well established models to study fruit ripening or animal embryogenesis, respectively Solanum lycopersicum L. and Drosophila melanogaster, for which no or low depth spectral libraries for SWATH-MS on TripleTOF instruments have been produced so far [17,18]. These libraries contain assays for more than 5,000 proteins for both species and are best suited...

The recent development of transposon and CRISPR-Cas9-based tools for manipulating the fly genome in vivo promises tremendous progress in our ability to study developmental processes. Tools for introducing tags into genes at their endogenous genomic loci facilitate imaging or biochemistry approaches at the cellular or subcellular levels. Similarly, the ability to make specific alterations to the genome sequence allows much more precise genetic control to address questions of gene function.

GPR34 translocation and mutation are specifically associated with salivary gland MALT lymphoma (SG-MALT-Lymphoma). Majority of GPR34 mutations are clustered in its C-terminus, resulting in truncated proteins lacking the phosphorylation motif important for receptor desensitization. It is unclear why GPR34 genetic changes associate with SG-MALT-Lymphoma and how these mutations contribute to the lymphoma development. We generated isogenic Flp-InTRex293 cell lines that stably expressed a single copy of GPR34 or its various mutants, and performed a range of in vitro assays. We showed that the GPR34 Q340X truncation, but not R84H and D151A mutants conferred a significantly increased resistance to apoptosis, and greater transforming potential than the GPR34 wild type. The GPR34 truncation mutant had a significantly delayed internalization than the wild type following ligand (lysophosphatidylserine) stimulation. Among 9 signaling pathways examined, the GPR34 Q340X truncation, to a lesser extent the D151A mutant, significantly activated CRE, NFkB and AP1 reporter activities, particularly in the presence of ligand stimulation. We further demonstrated enhanced activities of phospholipase-A1/2 in the culture supernatant of Flp-InTRex293 cells that expressed the GPR34 Q340X mutant, and their potential to catalyze the synthesis of lysophosphatidylserine from phosphatidylserine. Importantly, phospholipase-A1 was abundantly expressed in the duct epithelium of salivary glands and those involved in lymphoepithelial lesions (LELs). Our findings advocate a model of paracrine stimulation of malignant B-cells via GPR34, in which PLA is released by LELs, and hydrolyzes the phosphatidylserine exposed on apoptotic cells, generating lysophosphatidylserine, the ligand for GPR34. Thus, GPR34 activation potentially bridges LELs to genesis of SG-MALT-Lymphoma.