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Concept

Double hybride

La technique de double hybride est une technique de biologie moléculaire permettant de détecter une interaction physique entre deux protéines. La technique est la suivante : on utilise une protéine test (telle Gal4) dont une des extrémités peut se fixer à une séquence activatrice, et l'autre activer la transcription du gène rapporteur (comme lacZ) mis sous contrôle de cette séquence. Par génie génétique, on synthétise ensuite deux protéines hybrides : une constituée de l'une des extrémités de la protéine test fusionnée avec la première protéine d'intérêt, l'autre formée de l'autre extrémité fusionnée à l'autre protéine d'intérêt. Si les deux protéines interagissent physiquement, ces deux hybrides vont se rapprocher et permettre la transcription du gène rapporteur. Cette technique peut cependant échouer, à cause des manipulations faites sur les protéines, et nécessite de nombreux contrôles. En outre, on teste systématiquement la réciprocité, c'est-à-dire que l'on inverse les fusions

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thumb|upright=1.2|L'inhibiteur de la ribonucléase en forme de fer à cheval (en représentation « fil de fer ») forme une interaction protéine–protéine avec la protéine de la ribonucléase. Les contacts

redresse=1.36|vignette|Représentation d'une protéine, ici deux sous-unités d'une molécule d'hémoglobine. On observe les représentées en couleur, ainsi que deux des quatre molécules d'hème, qui sont l

Saccharomyces cerevisiae est une espèce de levures employée notamment dans la fermentation de la bière. Elle occupe une place particulière parmi les ferments, levains et levures utilisés depuis la Hau

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Quantitative analysis of gene regulation in Drosophila

Antonina Iagovitina

In this project, characterization of the MAPK signaling cascade was done using the terminal patterning of Drosophila embryo as an experimental system. MAPK acts downstream of the Torso receptor tyrosine kinase (RTK) whose activation is limited to the poles of the embryo. This results in a localized activation of MAPK which is essential for the specification of the embryonic termini. Active MAPK controls the expression of the tailless (tll) and huckebein (hkb) gap genes by downregulating a transcriptional repressor, Capicua (Cic). In this work we are interested in understanding the mechanisms that allow differential specification and precise positioning of gene expression boundaries. We quantitatively analyzed information flow from MAPK to Cic and the two target genes, tll and hkb, by constructing input-output maps in wild type embryos. Our results show that the sharp MAPK activation is translated into a graded gradient of Cic concentration and switch-like responses of tll and hkb genes. This indicates that the ultrasensitive response occurs at the level of the Cic repression which led us to test the hypothesis that Cic alone could be sufficient for tll and hkb boundary specification. However, data from mutants containing lower levels of Cic showed that boundaries of two genes become indistinguishable in terms of Cic concentration while still clearly different in terms of position. This suggests that MAPK downregulation of Cic alone is not sufficient to explain the observed localization and that, other factors are required for the robust patterning of the termini of the Drosophila embryo. To find the missing components of this regulatory system we have started an in vitro Yeast One Hybrid screen. This technique, allowing detection of protein-DNA interaction, was optimized for high-throughput screening. Together with a library Drosophila transcription factors we hope to identify the missing regulators of tll and hkb genes.

2010

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Potential Direct Regulators of the Drosophila yellow Gene Identified by Yeast One-Hybrid and RNAi Screens

Bart Deplancke

The regulation of gene expression controls development, and changes in this regulation often contribute to phenotypic evolution. Drosophila pigmentation is a model system for studying evolutionary changes in gene regulation, with differences in expression of pigmentation genes such as yellow that correlate with divergent pigment patterns among species shown to be caused by changes in cis- and trans-regulation. Currently, much more is known about the cis-regulatory component of divergent yellow expression than the trans-regulatory component, in part because very few trans-acting regulators of yellow expression have been identified. This study aims to improve our understanding of the trans-acting control of yellow expression by combining yeast-one-hybrid and RNAi screens for transcription factors binding to yellow cis-regulatory sequences and affecting abdominal pigmentation in adults, respectively. Of the 670 transcription factors included in the yeast-one-hybrid screen, 45 showed evidence of binding to one or more sequence fragments tested from the 5 intergenic and intronic yellow sequences from D. melanogaster, D. pseudoobscura, and D. willistoni, suggesting that they might be direct regulators of yellow expression. Of the 670 transcription factors included in the yeast-one-hybrid screen, plus another TF previously shown to be genetically upstream of yellow, 125 were also tested using RNAi, and 32 showed altered abdominal pigmentation. Nine transcription factors were identified in both screens, including four nuclear receptors related to ecdysone signaling (Hr78, Hr38, Hr46, and Eip78C). This finding suggests that yellow expression might be directly controlled by nuclear receptors influenced by ecdysone during early pupal development when adult pigmentation is forming.

Genetics Society of America2016

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Analysis of expression of PDCP and MAL13P1.308 of Plasmodium falciparum employing a quantitative proteomics approach based on SILAC

Céline Freymond

Plasmodium falciparum is a deadly parasite that causes malaria in humans. This disease causes the death of one million people every year. To find new means to fight this parasite, it is important to learn more about its biology. As the pathways of protein expression are better understood, it becomes easier to find out how to block these mechanisms. The completion of the genome sequencing has opened new perspective of genome wide analysis. One of these studies was done by LaCount et al. who used the yeast1two1hybrid system to map the complete interaction network between proteins of P. falciparum. ¨ From this network of interaction an interesting protein was studied further by Daubenberger et al. which is PDCP. This protein is closely related to MSP11 in this protein network, a protein involved in erythrocyte invasion. PDCP is a CCCH1type zinc finger protein, a family of proteins that are involved in protein1protein interaction, nucleic acid binding and binding in small ligands. It was shown that its expression was dependant on the density of the parasites. In this study we used the SILAC technology to confirm these previous results. A culture is grown with isotopically heavy isoleucine in the medium as a control sample. After a few cell cycles, almost all the natural isoleucines are replaced with the labeled ones. Three cultures with parasitemia of 2, 5 and 10% are grown and mixed with an equal amount of the control culture. When the proteins are analyzed by mass spectrometry, the peptides that contained isoleucine will be detected as two separate peaks. After normalization of each peptide area with this internal control, the quantity of PDCP can be compared between cultures of different parasitemias. 11 peptides of PDCP have been detected by mass spectrometry, proving its existence for the first time. These peptides were labeled to more than 95%, allowing the comparison of PDCP expression between the cultures. But because of difficulty of reproducibility in in vitro cultures, the regulation of PDCP by the parasitemia has not been observed by SILAC. We wanted to study further the protein interaction network in which PDCP is involved. MAL13P1.308, a protein directly interacting with PDCP and MSP11 in the protein network map, was analyzed by IFA. It was found to be expressed during all stages of the asexual blood stage cycle and it was not imported in the host cell. The next step will be to see if it interacts with PDCP by co1localization studies

2008

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BIO-212: Biological chemistry I

Biochemistry is a key discipline for the Life Sciences. Biological Chemistry I and II are two tightly interconnected courses that aim to describe and understand in molecular terms the processes that make life possible.

BIO-471: Cancer biology I

The course covers in detail molecular mechanisms of cancer development with emphasis on cell cycle control, genome stability, oncogenes and tumor suppressor genes.

CH-412: Frontiers in chemical biology

Chemical biology is a key discipline in biomedical research for drug discovery, synthetic biology and protein functional annotation. We will give a broad perspective of the field ranging from seminal classical experiments to state-of-the-art approaches to dissect and perturb biological systems.