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iRNA in yeast
Posted on November 27th, 2009 No commentsiRNA is a tool currently used in molecular biology to define the function of a gene: indeed scientists block mRNA transcription of specific gene and observe the cellular response. This technique is extremely potent and specific and allows to study one protein at time. In yeast, certain strains lack the normal RNA interference machinery and have alternative enzyme variants which can be transplanted into truly deficient species. iRNA is specially useful to study budding yeast, but this model lacks the Dicer enzyme, responsible for processing the double stranded RNAs into small interfering RNAs.
Scientists from the Whitehead Institute identified many proteins that show hallmarks of Dicer- mediated cleavage; they moved these proteins into new strains in order to reconstitute the iRNA machinery and they observed the complete silencing of all gene of interest. Thus, no limitations were found to study genes and proteins involved in yeast biology. Moreover, transposons are efficiently silenced without interfering with other genes. The system is greatly specific. Based on this positive result, scientists want to apply this finding to pathogenic yeast Candida Albicans in order to better understand the behaviour of this organism and identify some pharmacological target. -
New technologies to identify gene function
Posted on October 30th, 2009 No commentsAfter advances in DNA sequencing technology, the major task is to determine the functional role of proteins coded by these sequenced genes. Given the broad range of different functions carried out by proteins, it’s obvious that a multiplicity of techniques will be necessary, while DNA sequencing is achieved by few, easy and simply technique. A series of strategies based on generalization and systemization of genetics are emerging now as important tool to fill the gap between sequence and activity. One of these approaches is the analysis of the effect of perturbations of gene expression, by deletion, mutation or over-expression: after one of these modifications, we expect to observe a phenotypic change.
The challenge is to quantitatively measure phenotypes with enough accuracy and depth to define gene function. Two complementary approaches for determining complex phenotypes are currently used: in the first one many different parameters are simultaneously analysed, this is an high content screen; otherwise a single or limited number of aspects are observed, but the effect of perturbing each gene is followed in combination with a second perturbation, either another mutation or a chemical treatment. This genetic interaction profiling offers a high-resolution view of the function of each gene. Saccaromyces Cerevisiae is a model really useful for this kind of studies: a complete series of deletion strains of nonessential genes has been produced and has allowed to better understand the role of proteins important for yeast biochemistry and biology. Important results have been achieved also by using conditioning mutants that selectively grow in rich media: also in this case precious information has been retrieved. Rather than loss of function studies, methods for systematic gene over-expression have been optimized. Novel approaches in this field are interested in construction of untagged proteins in order to exclude that the presence of tag could interfere with the normal function of protein self.
The main goal of this systematic studies is to maximize the information flow, while minimally compromising the accuracy of phenotype detection. The introduction of large biomolecules into cells, such as DNA, RNA allows to directly analyse the role of one gene in the cellular life, and different kind of cells (mammals, primary cells, stem cells) can be used in this approach. Biomolecule is printed in an array onto glass slides, as done in conventional microarray. A monolayer of cells is deposited on top of the arrayed molecules and cells are transfected by taking up the material from glass. By using 96-well format plate, it’s possible to analyse the effects of a large number of biomolecules in a quantitative way. A plausible example of this method application is the effect of iRNAs on cellular proliferation: iRNA can be printed on bottom plate, cells are transfected (please note that is important to define the efficiency of transfection) and proliferation rate can be measured with normal treatment with MTT. In this way, genes important in proliferation could be identified.
Future efforts will be done to exploit a vast array of data that will emerge from large-scale genomic and proteomic projects to gain a deeper knowledge of the function of biological system.
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