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1.  Analytical biochemistry of DNA–protein assemblies from crude cell extracts 
Nucleic Acids Research  2007;35(13):e92.
Purification of specific DNA–protein complexes is a challenging task, as the involved interactions can be both electrostatic/H-bond and hydrophobic. The chromatographic stringency needed to obtain reasonable purifications uses salts and detergents. However, these components elicit the removal of proteins unspecifically bound to the chromatographic support itself, thus contaminating the purification products. In this work, a photocleavable linker connected the target oligonucleotidic sequence to the chromatographic beads so as to allow the irradiation-based release of the purified DNA–protein complexes off the beads. Our bioanalytical conditions were validated by purifying the tetracycline repressor protein onto a specific oligonucleotide. The purification factor was unprecedented, with a single contaminant. The robustness of our method was challenged by applying it to the purification of multiprotein assemblies forming onto DNA damage-mimicking oligonucleotides. The purified components were identified as well-known DNA repair proteins, and were shown to retain their enzymatic activities, as seen by monitoring DNA ligation products. Remarkably, kinase activities, also monitored, were found to be distinct on the beads and on the purified DNA–protein complexes, showing the benefits to uncouple the DNA–protein assemblies from the beads for a proper understanding of biochemical regulatory mechanisms involved in the DNA–protein assemblies.
PMCID: PMC1935021  PMID: 17617645
2.  Basal Body Positioning Is Controlled by Flagellum Formation in Trypanosoma brucei 
PLoS ONE  2007;2(5):e437.
To perform their multiple functions, cilia and flagella are precisely positioned at the cell surface by mechanisms that remain poorly understood. The protist Trypanosoma brucei possesses a single flagellum that adheres to the cell body where a specific cytoskeletal structure is localised, the flagellum attachment zone (FAZ). Trypanosomes build a new flagellum whose distal tip is connected to the side of the old flagellum by a discrete structure, the flagella connector. During this process, the basal body of the new flagellum migrates towards the posterior end of the cell. We show that separate inhibition of flagellum assembly, base-to-tip motility or flagella connection leads to reduced basal body migration, demonstrating that the flagellum contributes to its own positioning. We propose a model where pressure applied by movements of the growing new flagellum on the flagella connector leads to a reacting force that in turn contributes to migration of the basal body at the proximal end of the flagellum.
PMCID: PMC1857822  PMID: 17487282
3.  GNU polyxmass: a software framework for mass spectrometric simulations of linear (bio-)polymeric analytes 
BMC Bioinformatics  2006;7:226.
Nowadays, a variety of (bio-)polymers can be analyzed by mass spectrometry. The detailed interpretation of the spectra requires a huge number of "hypothesis cycles", comprising the following three actions 1) put forth a structural hypothesis, 2) test it, 3) (in)validate it. This time-consuming and painstaking data scrutiny is alleviated by using specialized software tools. However, all the software tools available to date are polymer chemistry-specific. This imposes a heavy overhead to researchers who do mass spectrometry on a variety of (bio-)polymers, as each polymer type will require a different software tool to perform data simulations and analyses. We developed a software to address the lack of an integrated software framework able to deal with different polymer chemistries.
The GNU polyxmass software framework performs common (bio-)chemical simulations–along with simultaneous mass spectrometric calculations–for any kind of linear bio-polymeric analyte (DNA, RNA, saccharides or proteins). The framework is organized into three modules, all accessible from one single binary program. The modules let the user to 1) define brand new polymer chemistries, 2) perform quick mass calculations using a desktop calculator paradigm, 3) graphically edit polymer sequences and perform (bio-)chemical/mass spectrometric simulations. Any aspect of the mass calculations, polymer chemistry reactions or graphical polymer sequence editing is configurable.
The scientist who uses mass spectrometry to characterize (bio-)polymeric analytes of different chemistries is provided with a single software framework for his data prediction/analysis needs, whatever the polymer chemistry being involved.
PMCID: PMC1524818  PMID: 16643644
4.  Functional complementation of RNA interference mutants in trypanosomes 
BMC Biotechnology  2005;5:6.
In many eukaryotic cells, double-stranded RNA (dsRNA) triggers RNA interference (RNAi), the specific degradation of RNA of homologous sequence. RNAi is now a major tool for reverse-genetics projects, including large-scale high-throughput screens. Recent reports have questioned the specificity of RNAi, raising problems in interpretation of RNAi-based experiments.
Using the protozoan Trypanosoma brucei as a model, we designed a functional complementation assay to ascertain that phenotypic effect(s) observed upon RNAi were due to specific silencing of the targeted gene. This was applied to a cytoskeletal gene encoding the paraflagellar rod protein 2 (TbPFR2), whose product is essential for flagellar motility. We demonstrate the complementation of TbPFR2, silenced via dsRNA targeting its UTRs, through the expression of a tagged RNAi-resistant TbPFR2 encoding a protein that could be immunolocalized in the flagellum. Next, we performed a functional complementation of TbPFR2, silenced via dsRNA targeting its coding sequence, through heterologous expression of the TbPFR2 orthologue gene from Trypanosoma cruzi: the flagellum regained its motility.
This work shows that functional complementation experiments can be readily performed in order to ascertain that phenotypic effects observed upon RNAi experiments are indeed due to the specific silencing of the targetted gene. Further, the results described here are of particular interest when reverse genetics studies cannot be easily achieved in organisms not amenable to RNAi. In addition, our strategy should constitute a firm basis to elaborate functional-dissection studies of genes from other organisms.
PMCID: PMC549545  PMID: 15703078

Results 1-4 (4)