SuperSAGE analysis of the Nicotiana attenuata transcriptome after fatty acid-amino acid elicitation (FAC): identification of early mediators of insect responses

<p>Abstract</p> <p>Background</p> <p>Plants trigger and tailor defense responses after perception of the oral secretions (OS) of attacking specialist lepidopteran larvae. Fatty acid-amino acid conjugates (FACs) in the OS of the <it>Manduca sexta </it>larvae are necessary and sufficient to elicit the herbivory-specific responses in <it>Nicotiana attenuata</it>, an annual wild tobacco species. How FACs are perceived and activate signal transduction mechanisms is unknown.</p> <p>Results</p> <p>We used SuperSAGE combined with 454 sequencing to quantify the early transcriptional changes elicited by the FAC <it>N</it>-linolenoyl-glutamic acid (18:3-Glu) and virus induced gene silencing (VIGS) to examine the function of candidate genes in the <it>M. sexta</it>-<it>N. attenuata </it>interaction. The analysis targeted mRNAs encoding regulatory components: rare transcripts with very rapid FAC-elicited kinetics (increases within 60 and declines within 120 min). From 12,744 unique Tag sequences identified (UniTags), 430 and 117 were significantly up- and down-regulated ≥ 2.5-fold, respectively, after 18:3-Glu elicitation compared to wounding. Based on gene ontology classification, more than 25% of the annotated UniTags corresponded to putative regulatory components, including 30 transcriptional regulators and 22 protein kinases. Quantitative PCR analysis was used to analyze the FAC-dependent regulation of a subset of 27 of these UniTags and for most of them a rapid and transient induction was confirmed. Six FAC-regulated genes were functionally characterized by VIGS and two, a putative lipid phosphate phosphatase (LPP) and a protein of unknown function, were identified as important mediators of the <it>M. sexta</it>-<it>N. attenuata </it>interaction.</p> <p>Conclusions</p> <p>The analysis of the early changes in the transcriptome of <it>N. attenuata </it>after FAC elicitation using SuperSAGE/454 has identified regulatory genes involved in insect-specific mediated responses in plants. Moreover, it has provided a foundation for the identification of additional novel regulators associated with this process.</p

Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics

The cellular abundance of transcription factors (TFs) is an important determinant of their regulatory activities. Deriving TF copy numbers is therefore crucial to understanding how these proteins control gene expression. We describe a sensitive selected reaction monitoring–based mass spectrometry assay that allowed us to determine the copy numbers of up to ten proteins simultaneously. We applied this approach to profile the absolute levels of key TFs, including PPARɣ and RXRα, during terminal differentiation of mouse 3T3-L1 pre-adipocytes. Our analyses revealed that individual TF abundance differs dramatically (from ~250 to >300,000 copies per nucleus) and that their dynamic range during differentiation can vary up to fivefold. We also formulated a DNnA binding model for PPARɣ based on TF copy number, binding energetics and local chromatin state. This model explains the increase in PPARɣ binding sites during the final differentiation stage that occurs despite a concurrent saturation in PPARɣ copy number

Relations between Local Authorities and Public Utilities Companies

The development of public utilities (supply) has led to a change in needs, thereby influencing the functions that the local public administration is asked to perform. Globalisation has caused profound socio-political changes (just think of the formation of the European Union, the introduction of the single currency, the fall of some regimes) and has strongly influenced national and local goals. The development of the socio-political context and the development of new technologies have changed the meaning of public service, and local authorities (i.e. elected administrations) have been able to benefit from this discretional power, demonstrating their sensitivity to the matter as well as their vision