1,720,974 research outputs found
The Drosophila minifly (mfl) gene is essential for the formation of intestinal stem cells
No full textLaser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc
No full textHeterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context
Human dyskerin: beyond telomeres
No full textHuman dyskerin is an evolutively conserved protein
that participates in diverse nuclear complexes: the
H/ACA snoRNPs, that control ribosome biogenesis, RNA
pseudouridylation, and stability of H/ACA snoRNAs; the
scaRNPs, that control pseudouridylation of snRNAs; and the
telomerase active holoenzyme, which safeguards telomere
integrity. The biological importance of dyskerin is further
outlined by the fact that its deficiency causes the X-linked
dyskeratosis congenita disease, while its over-expression
characterizes several types of cancers and has been proposed
as prognostic marker. The role of dyskerin in telomere
maintenance has widely been discussed, while its functions
as H/ACA sno/scaRNP component has been so far mostly
overlooked and represent the main goal of this review. Here
we summarize how increasing evidence indicates that the
snoRNA/microRNA pathways can be interlaced, and that
dyskerin-dependent RNA pseudouridylation represents a
flexible mechanism able to modulate RNA function in different
ways, including modulation of splicing, change of
mRNA coding properties, and selective regulation of IRESdependent translation. We also propose a speculative model that suggests that the dynamics of pre-assembly and nuclear import of H/ACA RNPs are crucial regulatory steps that can be finely controlled in the cytoplasm in response to developmental, differentiative and stress stimuli
Drosophila midgut as a model for stem cell dysfunction caused by Dyskeratosis congenita X-linked
No full textDomesticazione e feralizzazione in Sus scrofa, adattamenti funzionali della mucosa olfattiva
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Silencing of Drosophila H/ACA snoRNP pseudouridine synthase dysregulates key developmental pathways
No full textThe snoRNP pseudouridine synthase is required for self-renewal of larval Adult Midgut Precursor (AMP) cells
No full textThe role of Drosophila pseudouridine synthase component of H/ACA snoRNPs in compensatory proliferation and tissue remodeling
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