We developed a new program called GeneTack for ab initio frameshift detection in intronless protein-coding nucleotide sequences. The GeneTack program uses a hidden Markov model (HMM) of a genomic sequence with possibly frameshifted protein-coding regions. The Viterbi algorithm nds the maximum likelihood path that discriminates between true adjacent genes and a single gene with a frameshift. We tested GeneTack as well as two other earlier developed programs FrameD and FSFind on 17 prokaryotic genomes with frameshifts introduced randomly into known genes. We observed that the average frameshift prediction accuracy of GeneTack, in terms of (Sn+Sp)/2 values, was higher by a signicant margin than the accuracy of the other two programs. GeneTack was used to screen 1,106 complete prokaryotic genomes and 206,991 genes with frameshifts (fs-genes) were identifed. Our goal was to determine if a frameshift transition was due to (i) a sequencing error, (ii) an indel mutation or (iii) a recoding event. We grouped 102,731 genes with frameshifts (fs-genes) into 19,430 clusters based on sequence similarity between their protein products (fs-proteins), conservation of predicted frameshift position, and its direction. While fs-genes in 2,810 clusters were classied as conserved pseudogenes and fs-genes in 1,200 clusters were classied as hypothetical pseudogenes, 5,632 fs-genes from 239 clusters pos- sessing conserved motifs near frameshifts were predicted to be recoding candidates. Experiments were performed for sequences derived from 20 out of the 239 clusters; programmed ribosomal frameshifting with eciency higher than 10% was observed for four clusters. GeneTack was also applied to 1,165,799 mRNAs from 100 eukaryotic species and 45,295 frameshifts were identied. A clustering approach similar to the one used for prokaryotic fs-genes allowed us to group 12,103 fs-genes into 4,087 clusters. Known programmed frameshift genes were among the obtained clusters. Several clusters may correspond to new examples of dual coding genes. We developed a web interface to browse a database containing all the fs-genes predicted by GeneTack in prokaryotic genomes and eukaryotic mRNA sequences. The fs-genes can be retrieved by similarity search to a given query sequence, by fs- gene cluster browsing, etc. Clusters of fs-genes are characterized with respect to their likely origin, such as pseudogenization, phase variation, programmed frameshifts etc. All the tools and the database of fs-genes are available at the GeneTack web site http://topaz.gatech.edu/GeneTack/

Deficiency in Msh2, a component of the mismatch repair (MMR) system, leads to a ~10-fold increase in the mutation frequency in most tissues. By contrast, Msh2-deficiency in germinal center (GC) B cells decreases the mutation frequency at the IgH V-region, as a dU:dG mismatch produced by AID initiates modifications by MMR resulting in mutations at nearby A:T basepairs. This raises the possibility that GC B cells express a factor that converts MMR into a globally mutagenic pathway. To test this notion, we investigated whether MMR corrects mutations in GC B cells at a gene not mutated by AID. We found that GC B cells accumulate 5-times more mutations than follicular B cells. Notably, the mutation frequency was ~10 times higher in Msh2-/- compared to wildtype GC B cells. These results show that in GC B cells MMR functions normally at an AID-insensitive gene.

Deficiency in Msh2, a component of the mismatch repair (MMR) system, leads to a ~10-fold increase in the mutation frequency in most tissues. By contrast, Msh2-deficiency in germinal center (GC) B cells decreases the mutation frequency at the IgH V-region, as a dU:dG mismatch produced by AID initiates modifications by MMR resulting in mutations at nearby A:T basepairs. This raises the possibility that GC B cells express a factor that converts MMR into a globally mutagenic pathway. To test this notion, we investigated whether MMR corrects mutations in GC B cells at a gene not mutated by AID. We found that GC B cells accumulate 5-times more mutations than follicular B cells. Notably, the mutation frequency was ~10 times higher in Msh2-/- compared to wildtype GC B cells. These results show that in GC B cells MMR functions normally at an AID-insensitive gene.

Deficiency of antithrombin caused by mutations affecting the gene encoding this key anticoagulant (SERPINC1) significantly increases the risk of thrombosis. We aim to identify new mechanisms involved in antithrombin deficiency. Using molecular, cellular and biochemical methods, we studied 29 patients with antithrombin deficiency without SERPINC1 mutation, a family study, three case-control studies including 2,980 patients and 3,996 controls, and two patients with congenital disorder of glycosylation (CDG). We identified the first mutation affecting the SERPINC1 promoter causing antithrombin deficiency. We confirmed the low genetic variability of SERPINC1 and its minor role on the heritability of antithrombin. Genome wide association studies and silencing experiments identified the first modulating gene of antithrombin, LARGE. We diagnosed a patient with CDG based on his antithrombin deficiency. Finally, we described a new disorder with identical biochemical features than CDGs, but only thrombosis, which is caused by a single mutation in PMM2 and concomitant alcohol consumption. / La deficiencia de antitrombina causada por mutaciones en el gen SERPINC1 incrementa el riesgo trombótico. Nuestro objetivo fue identificar nuevos mecanismos implicados en la deficiencia de este anticoagulante. Empleando metodología molecular, celular y bioquímica estudiamos 29 pacientes con deficiencia de antitrombina sin mutaciones en SERPINC1, un estudio familiar, tres estudios caso-control (2,980 pacientes/3,996 controles) y dos pacientes con trastornos congénitos de glicosilación (CDG). Identificamos la primera mutación en el promotor de SERPINC1 que causa deficiencia de antitrombina. Confirmamos la baja variabilidad genética en SERPINC1 y su escasa influencia en la heredabilidad de antitrombina. Un GWAS y experimentos de silenciamiento mostraron que LARGE es el primer gen modulador de antitrombina. Diagnosticamos un CDG por la deficiencia de antitrombina de un paciente con trombosis recurrente y descubrimos nuevo desorden con patrón bioquímico similar al CDG pero solo con trombosis que es causado por una sola mutación en PMM2 y consumo de alcohol.

Phosphofructokinase from Escherichia coli (EcPFK) is allosterically regulated by MgADP and phospho(enol)pyruvate (PEP). Both molecules compete for binding to the same allosteric site, however, MgADP activates and PEP inhibits the binding of fructose-6-phosphate (F6P) to the active site. The mode by which this enzyme can differentiate between the two ligands and cause the appropriate response is important for the understanding of the basis of allosteric regulation. We studied the interactions between an active site and an allosteric site (heterotropic interactions) within the protein, and found that each of the four unique heterotropic interactions is unique and the magnitudes of the coupling free energies for MgADP activation sum up to 100% that of wildtype EcPFK without homotropic cooperativity in F6P binding. We took on the kinetic and structural characterization of phosphofructokinase from Lactobacillus bulgaricus (LbPFK) to reveal an enzyme that exhibits allosteric properties in spite of previous kinetic studies performed by Le Bras et al. (1991). We have identified residues in EcPFK (Asp59, Gly184 and Asp273), which are important for the allosteric responses to both MgADP and PEP. Interestingly, Lys214 is only important in PEP inhibition and not MgADP activation. We can also differentially disrupt the MgADP heterotropic interactions with the introduction of G184C within the protein. These results suggest that there are different pathways for allosteric communication within the enzyme: different paths for MgADP activation and PEP inhibition, and different paths for each heterotropic interaction with Gly184 being important for the 33Å MgADP heterotropic interaction.

Cette étude s'est proposée d'examiner les liens entre la croissance démographique et les mutations agraires et vise à déterminer dans quelle mesure et de quelle manière l'évolution de la population du Sahel a influé sur les changements agraires.<br /> L'espace du Sahel des Doukkala fut en effet le lieu de transformations considérables, cependant à vitesses inégales, et à tendances différentes, d'un espace à l'autre, voire d'une exploitation à l'autre.<br />La période coloniale, puis celle de l'indépendance, ont vu s'accroître le poids économique et humain de la région du Sahel des Doukkala, marqué par l'augmentation de la population sur ses pourtours, essentiellement sur les crêtes de la falaise morte, dominant à la fois la dépression de l'Oulja et les terres de parcours du Sahel Intérieur. Cette nouvelle dynamique démographique a été concomitante à une amélioration des conditions de la production agricole et un développement appréciable des productions orientées vers l'exportation.<br />C'est une autre logique qui commença alors à régir l'économie rurale du Sahel, non seulement par l'importance de la part de la production consacrée à la vente, ou par la nature des marchés (national et international) et des techniques, mais aussi parce que la terre (à l'Oulja notamment) est devenue, elle aussi, matière à transactions marchandes après sa «privatisation » et a entraîné une véritable exaltation de défrichements.<br />Ces transformations, n'ont pas touché toute la population, ni tout le territoire du Sahel. Une grande partie de la population et du territoire conserva les traits d'une paysannerie traditionnelle où persistaient encore pratiques et valeurs agro-pastorales traditionnelles et resta à l'écart de ces mutations. Néanmoins, face à ces changements, la population locale n'a pas tardé à mettre en question son système de production agricole traditionnel. Progressivement cette population a commencé à adopter, elle aussi, les cultures et techniques introduites par les colons européens. Le changement qui s'effectuât alors ne fut pas une simple réorientation du système de production, mais une vraie transformation qui a touché les fondements techniques et sociaux du système de production agricole

Les lentivirus présentent une évolution rapide du gène env, notamment dans la région codant la glycoprotéine de surface (SU). Un fait remarquable est que les mutations de la SU sont localisées dans des zones spécifiques, appelées régions variables (V), séparées par des régions dites constantes (C). Afin de déterminer s'il existe des signatures spécifiques des régions C et V, nous avons développé des modèles de Markov cachés, ou HMM (Hidden Markov Models), basés sur la composition en oligonucléotides de chaque type de région, capables de différencier les régions C et V des lentivirus. Nous avons entraîné des modèles de Markov cachés sur des séquences des SU des lentivirus équins, humains, simiens et des petits ruminants. Nous avons obtenu une délimitation claire des régions C et V de tous ces lentivirus ainsi que des lentivirus bovins et félins qui n'avaient pas été utilisés pour définir les modèles. Nos résultats suggèrent que les régions C et V des lentivirus ont des compositions statistiques en mots de nucléotides et d'acides aminés différentes. Des signatures caractéristiques des régions C et V ont été extraites à partir des modèles définis.

This work is a revised edition of the former article "Evolution and Mutation Physics” by the same author. Some unclear formulations have been eliminated. New ideas and new calculations have been included, especially the important connection between successive entropy - changes and increasing DNA –length at slowly decreasing temperature-decrease of surroundings.

The evolution of antibiotic resistance is shaped by interactions between genes, the chemical environment, and an antibiotic's mechanism of action. This thesis explores these interactions with experiments, theory, and analysis, seeking a mechanistic understanding of how different interactions between genes and drugs can enhance or constrain the evolution of antibiotic resistance. Chapter 1 investigates the effects of the chemical decay of an antibiotic. Tetracycline resistant and sensitive bacteria were grown competitively in the presence of tetracycline and its decay products. Antibiotic decay did not only remove selection for resistance, but long-lived decay products favored tetracycline sensitivity by inducing costly drug efflux pumps in the resistant strain. Selection against resistance by antibiotic-related compounds may contribute to the coexistence of drug-sensitive and resistant bacteria in nature. Chapter 2 investigates how genetic interactions can favor particular combinations of resistance-conferring mutations. All possible combinations of a set of trimethoprim resistance-conferring mutations in the drug's target gene were constructed and phenotyped. Incompatibilities between mutations arose in a high-order, not pairwise, manner. One mutation was found to induce this ruggedness and create a multi-peaked adaptive landscape. Chapters 1 and 2 observed that non-optimal expression of a drug resistance gene or a drug's target could compromise antibiotic resistance. Chapter 3 broadly characterizes non-optimal gene expression under antibiotic treatment, using a functional genetic screen to identify over one hundred pathways to antibiotic resistance through positive and negative changes in gene expression. Genes with the potential to confer antibiotic resistance were found to often go unused during antibiotic stress. The optimization of gene expression for drug-free growth was found to cause non-optimal expression under drug treatment, creating a situation where regulatory mutations can confer resistance by correcting errors in gene expression. Chapter 4 investigates whether it is beneficial to up-regulate the genes encoding antibiotic targets when they are inhibited. Drug target genes were quantitatively over-expressed, and drug resistance was found to not always increase, but alternatively to remain unchanged or even decrease. These diverse effects were explained by simple models that consider toxicity arising from gene over-expression, and mechanisms of drug action in which drugs induce harmful enzymatic reactions.

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