Françoise Mathieu-Daudé

Bio: Françoise Mathieu-Daudé is an academic researcher from Institut de recherche pour le développement. The author has contributed to research in topics: Differential display & Trypanosoma cruzi. The author has an hindex of 16, co-authored 37 publications receiving 1173 citations. Previous affiliations of Françoise Mathieu-Daudé include Torrey Pines Institute for Molecular Studies.

DNA Rehybridization During PCR: The ‘Cot Effect’ and Its Consequences

Abstract: The rate of amplification of abundant PCR products generally declines faster than that of less abundant products in the same tube in the later cycles of PCR. As a consequence, differences in product abundance diminish as the number of PCR cycles increases. Rehybridization of PCR products which may interfere with primer binding or extension can explain this significant feature in late cycles. Rehybridization occurs with a half-time dependent on the reciprocal of the DNA concentration. Thus, if multiple PCR products are amplified in the same tube, reannealing occurs faster for the more abundant PCR products. In RT-PCR using an internal control, this results in a systematic bias against the more abundant of the two PCR products. In RNA fingerprinting by arbitrarily primed PCR (or differentially display of cDNAs), very large or absolute differences in the expression of a transcript between samples are preserved but smaller real differences may be gradually erased as the PCR reaction proceeds. Thus, this 'Cot effect' may systematically cause an underestimate of the true difference in starting template concentrations. However, differences in starting template concentrations will be better preserved in the less abundant PCR products. Furthermore, the slow down in amplification of abundant products will allow these rarer products to become more visible in the fingerprint which may, in turn, allow rarer cDNAs to be sampled more efficiently. In some applications, where the object is to stochiometrically amplify a mixture of nucleic acids, the bias against abundant PCR products can be partly overcome by limiting the number of PCR cycles and, thus, the concentration of the products. In other cases, abundance normalization at later cycles may be useful, such as in the production of normalized libraries.

Socio-economic and Climate Factors Associated with Dengue Fever Spatial Heterogeneity: A Worked Example in New Caledonia.

Abstract: Understanding the factors underlying the spatio-temporal distribution of infectious diseases provides useful information regarding their prevention and control. Dengue fever spatio-temporal patterns result from complex interactions between the virus, the host, and the vector. These interactions can be influenced by environmental conditions. Our objectives were to analyse dengue fever spatial distribution over New Caledonia during epidemic years, to identify some of the main underlying factors, and to predict the spatial evolution of dengue fever under changing climatic conditions, at the 2100 horizon. We used principal component analysis and support vector machines to analyse and model the influence of climate and socio-economic variables on the mean spatial distribution of 24,272 dengue cases reported from 1995 to 2012 in thirty-three communes of New Caledonia. We then modelled and estimated the future evolution of dengue incidence rates using a regional downscaling of future climate projections. The spatial distribution of dengue fever cases is highly heterogeneous. The variables most associated with this observed heterogeneity are the mean temperature, the mean number of people per premise, and the mean percentage of unemployed people, a variable highly correlated with people's way of life. Rainfall does not seem to play an important role in the spatial distribution of dengue cases during epidemics. By the end of the 21st century, if temperature increases by approximately 3°C, mean incidence rates during epidemics could double. In New Caledonia, a subtropical insular environment, both temperature and socio-economic conditions are influencing the spatial spread of dengue fever. Extension of this study to other countries worldwide should improve the knowledge about climate influence on dengue burden and about the complex interplay between different factors. This study presents a methodology that can be used as a step by step guide to model dengue spatial heterogeneity in other countries.

Screening of Differentially Amplified cDNA Products from RNA Arbitrarily Primed PCR Fingerprints Using Single Strand Conformation Polymorphism (SSCP) Gels

Abstract: Arbitrarily primed PCR fingerprinting of RNA and differential display resolved on an acrylamide gel has been extensively used to detect differentially expressed RNAs. However, after a differentially amplified product is detected the next steps are labor-intensive: a small portion of the fingerprinting gel that contains the differentially amplified product is cut out, reamplified and the correct product is determined, typically by cloning and sequencing what is often a mixture of products of similar size. Here we use a native acrylamide gel to separate DNAs in the reamplified mixture based on single-stranded conformation polymorphisms. Reamplifications are performed for the region carrying the differentially amplified product and a corresponding region from an adjacent lane where the product is less prominent or not visible. Denaturation of the reamplified DNA followed by side-by-side comparison on an SSCP gel allows the classification of reamplified material into (i) those that can be directly cloned because the differentially amplified product is relatively pure, (ii) those that need to be reamplified from the SSCP gel before cloning and (iii) those that are too complex for further study. This screen should save considerable effort now wasted on directly cloning unsuitable products from RNA fingerprinting experiments. An example is presented of cloning a gene differentially expressed in Trypanosoma brucei life cycle.

Genome-wide location and function of dna binding proteins

Abstract: The present invention relates to a method of identifying a region (one or more) of a genome of a cell to which a protein of interest binds. In the methods described herein, DNA binding protein of a cell is linked (e.g., covalently crosslinked) to genomic DNA of a cell. The genomic DNA to which the DNA binding protein is linked is removed and combined or contacted with DNA comprising a sequence complementary to genomic DNA of the cell under conditions in which hybridization between the identified genomic DNA and the sequence complementary to genomic DNA occurs. Region(s) of hybridization are region(s) of the genome of the cell to which the protein of binds. A method of identifying a set of genes where cell cycle regulator binding correlates with gene expression and of identifying genomic targets of cell cycle transcription activators in living cells is also encompassed.

Analysis of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments.

Abstract: Denaturing gradient gel electrophoresis (DGGE) is a powerful and convenient tool for analyzing the sequence diversity of complex natural microbial populations. DGGE was evaluated for the identification of ammonia oxidizers of the beta subdivision of the Proteobacteria based on the mobility of PCR-amplified 16S rDNA fragments and for the analysis of mixtures of PCR products from this group generated by selective PCR of DNA extracted from coastal sand dunes. Degenerate PCR primers, CTO189f-GC and CTO654r, incorporating a 59 GC clamp, were designed to amplify a 465-bp 16S rDNA region spanning the V-2 and V-3 variable domains. The primers were tested against a representative selection of clones and cultures encompassing the currently recognized beta-subdivision ammonia oxidizer 16S rDNA sequence diversity. Analysis of these products by DGGE revealed that while many of the sequences could be separated, some which were known to be different migrated similarly in the denaturant system used. The CTO primer pair was used to amplify 16S rDNA sequences from DNA extracted from soil sampled from Dutch coastal dune locations of differing in pH and distance from the beach. The derived DGGE patterns were reproducible across multiple DNA isolations and PCRs. Ammonia oxidizer-like sequences from different phylogenetic groupings isolated from gene libraries made from the same sand dune DNA samples but prepared with different primers gave DGGE bands which comigrated with most of the bands detected from the sand dune samples. Bands from the DGGE gels of environmental samples were excised, reamplified, and directly sequenced, revealing strong similarity or identity of the recovered products to the corresponding regions of library clones. Six of the seven sequenced clusters of beta-subdivision ammonia oxidizers were detected in the dune systems, and differences in community structure between some sample sites were demonstrated. The most seaward dune site contained sequences showing affinity with sequence clusters previously isolated only from marine environments and was the only site where sequences relate to Nitrosomonas genes could be detected. Nitrosospira-like sequences were present in all sites, and there was some evidence of differences between Nitrosospira populations in acid and alkaline dune soils. Such differences in community structure may affect physiological differences within beta-subdivision ammonia oxidizers, with consequent effects on nitrification rates in response to key environmental factors.

High-fidelity mRNA amplification for gene profiling.

Abstract: The completion of the Human Genome Project has made possible the comprehensive analysis of gene expression, and cDNA microarrays are now being employed for expression analysis in cancer cell lines or excised surgical specimens. However, broader application of cDNA microarrays is limited by the amount of RNA required: 50-200 microg of total RNA (T-RNA) and 2-5 microg poly(A) RNA. To broaden the use of cDNA microarrays, some methods aiming at intensifying fluorescence signal have resulted in modest improvement. Methods devoted to amplifying starting poly(A) RNA or cDNA show promise, in that detection can be increased by orders of magnitude. However, despite the common use of these amplification procedures, no systematic assessment of their limits and biases has been documented. We devised a procedure that optimizes amplification of low-abundance RNA samples by combining antisense RNA (aRNA) amplification with a template-switching effect (Clonetech, Palo Alto, CA). The fidelity of aRNA amplified from 1:10,000 to 1:100,000 of commonly used input RNA was comparable to expression profiles observed with conventional poly(A) RNA- or T-RNA-based arrays.

ENSO representation in climate models: from CMIP3 to CMIP5

Abstract: We analyse the ability of CMIP3 and CMIP5 coupled ocean–atmosphere general circulation models (CGCMs) to simulate the tropical Pacific mean state and El Nino-Southern Oscillation (ENSO). The CMIP5 multi-model ensemble displays an encouraging 30 % reduction of the pervasive cold bias in the western Pacific, but no quantum leap in ENSO performance compared to CMIP3. CMIP3 and CMIP5 can thus be considered as one large ensemble (CMIP3 + CMIP5) for multi-model ENSO analysis. The too large diversity in CMIP3 ENSO amplitude is however reduced by a factor of two in CMIP5 and the ENSO life cycle (location of surface temperature anomalies, seasonal phase locking) is modestly improved. Other fundamental ENSO characteristics such as central Pacific precipitation anomalies however remain poorly represented. The sea surface temperature (SST)-latent heat flux feedback is slightly improved in the CMIP5 ensemble but the wind-SST feedback is still underestimated by 20–50 % and the shortwave-SST feedbacks remain underestimated by a factor of two. The improvement in ENSO amplitudes might therefore result from error compensations. The ability of CMIP models to simulate the SST-shortwave feedback, a major source of erroneous ENSO in CGCMs, is further detailed. In observations, this feedback is strongly nonlinear because the real atmosphere switches from subsident (positive feedback) to convective (negative feedback) regimes under the effect of seasonal and interannual variations. Only one-third of CMIP3 + CMIP5 models reproduce this regime shift, with the other models remaining locked in one of the two regimes. The modelled shortwave feedback nonlinearity increases with ENSO amplitude and the amplitude of this feedback in the spring strongly relates with the models ability to simulate ENSO phase locking. In a final stage, a subset of metrics is proposed in order to synthesize the ability of each CMIP3 and CMIP5 models to simulate ENSO main characteristics and key atmospheric feedbacks.