INRA UMR SPO Montpellier/ Centre de Recherches de Biochimie Macromoléculaire (CRBM) - Centre National de la Recherche Scientifique (CNRS)
Stageonderwerp 1 2005-2006
Analyse van verschillende transcriptiefactoren in de aptabiliteit van de wijnstok onder verschillende soorten stress uit de omgeving, met behulp van de Real Time PCR techniek. Gebruikte technieken: RNA extractie – cDNA synthese – RT PCR – quantitatie van RNA – agarosegel elektroforese – Real Time Quantitatieve PCR
Stageonderwerp 2 2005-2006
Studie van de translatiecontrole op proteïnen waarvan de synthese noodzakelijk is voor de meiotische maturatie. Onderzoek naar de afhankelijkheid van een proteïne van cytoplasmatische polyadenylatie van zijn mRNA. Hierbij wordt gebruik gemaakt van ovocyten van Xenopus laevis.
Gebruikte technieken: Cloning – Sequenering door middel van PCR – Polyadenylatietest – Immunoprecipitatie – Western Blotting – Dubbel hybride
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Samenvatting eindwerk 1 2005-2006: Characterisation of XlRBM9 in relation with the poly(A) polymerase XlGld2
In this study, the oocytes of the Xenopus laevis are used to investigate the process of cytoplasmic polyadenylation and factors that have relations with this process. The Xenopus oocyte offers the perfect biological model for this study, since there is no RNA polymerase-dependent transcription at this stage. More precisely, it is the RNA binding protein XRBM9 that attracts the most attention. The aim is to explore the expression and function of this protein and its interaction with other members of the cytoplasmic polyadenylation complex.
After introducing the laboratory and explaining the general situation and objective of the study, the Xenopus laevis gets presented in the Study of literature. Further on, the importance of Xenopus oocytes as a biological model gets pursued in greater depth, followed by an extensive explanation of translational regulation. Finally, the polyadenylation mechanisms are discussed.
In Materials and methods, every practical operation gets explained in detail, first with a theoretical approach, then with a protocol. The results of these actions, provided with a little explanation and some conclusions, can be found in the chapter Results.
The most important conclusions of this study are that of the four XRBM9 isoforms that are cloned and sequenced, only XRBM9B gets expressed during (every stage) oogenesis. This protein interacts with the poly(A) polymerase XGld2 in the cytoplasmic polyadenylation complex, but does not seem to have an influence on cytoplasmic polyadenylation.
Samenvatting eindwerk 2 2005-2006: The transcription factor DREBP A, induced by environmental constraints, could be important in the grapevine adaptability to the environment
Improving the tolerance of Vitis vinifera to abiotic stresses may be achieved through breeding programs or the definition of specific cultural practices. In both cases, efficient and rational strategies require a better understanding of how grape responds when these stresses occur, what genes are upregulated and how these responsive genes can mitigate the effect of stresses and lead to adjustment of cellular milieu and grape tolerance.
This period of practical training focusses on one grape transcription factor which is involved in the expression of dehydration-, salt- and low-temperature-responsive genes: the Dehydration Responsive Element Binding Protein A (DREBP A). The proposal is aimed at investigating expression patterns of DREBP A by Real Time PCR procedure, using RNAs extracted from leaves, and berries at three different stages of development under or not water, cold and salt stress conditions.
Real-time Polymerase Chain Reaction (PCR) is the ability to monitor the progress of the PCR as it occurs. Data is therefore collected throughout the PCR process, rather than at the end of the PCR. This completely revolutionizes the way one approaches PCR-based quantitation of DNA and RNA. In real-time PCR, reactions are characterized by the point in time during cycling when amplification of a target is first detected rather than the amount of target accumulated after a fixed number of cycles. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed.
Real-time quantitative PCR was first developed to meet specific technical requirements, such as a high sensitivity and specificity, which are not easily achieved with other classical techniques. It is now becoming a routine tool, and it is believed that, thanks to its experienced reliability, its applications will proliferate in the forthcoming years. Thanks to its rapidity, it should even replace some widely used protocols. Most evidently, real-time PCR development is still limited by the high costs of the machine and reagents, but hopefully, future will make this technology economically more widely accessible.
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