Cell-type-specific visualisation and biochemical isolation of endogenous synaptic proteins in mice.

In recent years, the remarkable molecular complexity of synapses has been revealed, with over 1,000 proteins identified in the synapse proteome. Although it is known that different receptors and other synaptic proteins are present in different types of neurons, the extent of synapse diversity across the brain is largely unknown. This is mainly due to the limitations of current techniques. Here, we report an efficient method for the purification of synaptic protein complexes, fusing a high-affinity tag to endogenous PSD95 in specific cell types. We also developed a strategy, which enables the visualisation of endogenous PSD95 with fluorescent-protein tag in Cre-recombinase-expressing cells. We demonstrate the feasibility of proteomic analysis of synaptic protein complexes and visualisation of these in specific cell types. We find that the composition of PSD95 complexes purified from specific cell types differs from those extracted from tissues with diverse cellular composition. The results suggest that there might be differential interactions in the PSD95 complexes in different brain regions. We have detected differentially interacting proteins by comparing data sets from the whole hippocampus and the CA3 subfield of the hippocampus. Therefore, these novel conditional PSD95 tagging lines will not only serve as powerful tools for precisely dissecting synapse diversity in specific brain regions and subsets of neuronal cells, but also provide an opportunity to better understand brain region- and cell-type-specific alterations associated with various psychiatric/neurological diseases. These newly developed conditional gene tagging methods can be applied to many different synaptic proteins and will facilitate research on the molecular complexity of synapses.

Construction of mismatched inverted repeat (IR) silencing vectors for maximizing IR stability and effective gene silencing in plants.

Inverted repeat (IR) RNA silencing vectors containing homologous fragments of target endogenous plant genes, or pathogen genes, are the most widely used vectors to either study the function of genes involved in biotic stress or silence pathogens to induce plant resistance, respectively. RNA silencing has been exploited to produce transgenic plants with resistance to viral pathogens via posttranscriptional gene silencing (PTGS). In some cases, this technology is difficult to apply due to the instability of IR constructs during cloning and plant transformation. We have therefore developed a robust method for the production of long IR vector constructs by introducing base pair mismatches in the form of cytosine to thymine mutations on the sense arm by exposure to sodium bisulfite prior to assembly of the IR.

Construction of effective inverted repeat silencing constructs using sodium bisulfite treatment coupled with strand-specific PCR.

RNA silencing has been exploited to produce transgenic plants with resistance to viral pathogens via posttranscriptional gene silencing (PTGS). In some cases, this technology is difficult to apply due to the instability of inverted repeat (IR) constructs during cloning and plant transformation. Although such constructs have been shown to be stabilized with introns and efficiently induce RNA silencing, we found that the Pdk intron did not stabilize South African cassava mosaic virus (SACMV) silencing constructs. Therefore, we developed a method for producing long SACMV IR constructs through bisulfite-induced base pair mismatches on the sense arm prior to IR assembly. Expression of SACMV BC1 mismatched IR constructs in the model test plant Nicotiana benthamiana resulted in a reduction in viral BC1 transcript levels, hence viral replication, upon SACMV infection. Mismatched SACMV AC1 IR constructs induced PTGS more efficiently in a N. benthamiana callus system than nonmismatched IR constructs. Our novel method for IR construct generation should be applicable to many sequences where the generation of these constructs has proven difficult in the past.

Structure, diversity, and mobility of the Salmonella pathogenicity island 7 family of integrative and conjugative elements within Enterobacteriaceae.

Integrative and conjugative elements (ICEs) are self-mobile genetic elements found in the genomes of some bacteria. These elements may confer a fitness advantage upon their host bacteria through the cargo genes that they carry. Salmonella pathogenicity island 7 (SPI-7), found within some pathogenic strains of Salmonella enterica, possesses features indicative of an ICE and carries genes implicated in virulence. We aimed to identify and fully analyze ICEs related to SPI-7 within the genus Salmonella and other Enterobacteriaceae. We report the sequence of two novel SPI-7-like elements, found within strains of Salmonella bongori, which share 97% nucleotide identity over conserved regions with SPI-7 and with each other. Although SPI-7 within Salmonella enterica serovar Typhi appears to be fixed within the chromosome, we present evidence that these novel elements are capable of excision and self-mobility. Phylogenetic analyses show that these Salmonella mobile elements share an ancestor which existed approximately 3.6 to 15.8 million years ago. Additionally, we identified more distantly related ICEs, with distinct cargo regions, within other strains of Salmonella as well as within Citrobacter, Erwinia, Escherichia, Photorhabdus, and Yersinia species. In total, we report on a collection of 17 SPI-7 related ICEs within enterobacterial species, of which six are novel. Using comparative and mutational studies, we have defined a core of 27 genes essential for conjugation. We present a growing family of SPI-7-related ICEs whose mobility, abundance, and cargo variability indicate that these elements may have had a large impact on the evolution of the Enterobacteriaceae.