Promoter region is the key regulatory region, which enables the gene to be transcribed or repressed by anchoring RNA polymerase and other transcription factors, but it is difficult to determine experimentally. Hence an in silico identification of promoters is crucial in order to guide experimental work and to pin point the key region that controls the transcription initiation of a gene. Analysis of various genome sequences in the vicinity of experimentally identified transcription start sites (TSSs) in prokaryotic as well as eukaryotic genomes had earlier indicated that they have several structural features in common, such as lower stability, higher curvature and less bendability, when compared with their neighboring regions. In this thesis work, the variation observed for these DNA sequence dependent structural properties have been used to identify and delineate promoter regions from other genomic regions. Since the number of bacterial genomes being sequenced is increasing very rapidly, it is crucial to have procedures for rapid and reliable annotation of their functional elements such as promoter regions, which control the expression of each gene or each transcription unit of the genome. The thesis work addresses this requirement and presents step by step protocols followed to get a generic method for promoter prediction that can be applicable across organisms. The each paragraph below gives an overall idea about the thesis organization into chapters. An overview of prokaryotic transcriptional regulation, structural polymorphism adapted by DNA molecule and its impact on transcriptional regulation has been discussed in introduction chapter of this thesis (chapter 1). Standardization of promoter prediction methodology - Part I Based on the difference in stability between neighboring upstream and downstream regions in the vicinity of experimentally determined transcription start sites, a promoter prediction algorithm has been developed to identify prokaryotic promoter sequences in whole genomes. The average free energy (E) over known promoter sequences and the difference (D) between E and the average free energy over the random sequence generated using the downstream region of known TSS (REav) are used to search for promoters in the genomic sequences. Using these cutoff values to predict promoter regions across entire E. coli genome, a reliability of 70% has been achieved, when the predicted promoters were cross verified against the 960 transcription start sites (TSSs) listed in the Ecocyc database. Reliable promoter prediction is obtained when these genome specific threshold values were used to search for promoters in the whole E. coli genome sequence. Annotation of the whole E. coli genome for promoter region has been carried out with 49% accuracy. Reference Rangannan, V. and Bansal, M. (2007) Identification and annotation of promoter regions inmicrobial genome sequences on the basis of DNA stability. J Biosci, 32, 851-862. Standardization of promoter prediction methodology - Part II In this chapter, it has been demonstrated that while the promoter regions are in general less stable than the flanking regions, their average free energy varies depending on the GC composition of the flanking genomic sequence. Therefore, a set of free energy threshold values (TSS based threshold values), from the genomic DNA with varying GC content in the vicinity of experimentally identified TSSs have been obtained. These threshold values have been used as generic criteria for predicting promoter regions in E. coli and B. subtilis and M. tuberculosis genomes, using an in-house developed tool ‘PromPredict’. On applying it to predict promoter regions corresponding to the 1144 and 612 experimentally validated TSSs in E. coli (genome %GC : 50.8) and B. subtilis (genome %GC : 43.5) sensitivity of 99% and 95% and precision values of 58% and 60%, respectively, were achieved. For the limited data set of 81 TSSs available for M. tuberculosis (65.6% GC) a sensitivity of 100% and precision of 49% was obtained. Reference Rangannan, V. and Bansal, M. (2009) Relative stability of DNA as a generic criterion for promoter prediction: whole genome annotation of microbial genomes with varying nucleotide base composition. Mol Biosyst, 5, 1758 - 1769. Standardization of promoter prediction methodology - Part III In this chapter, the promoter prediction algorithm and the threshold values have been improved to predict promoter regions on a large scale over 913 microbial genome sequences. The average free energy (AFE) values for the promoter regions as well as their downstream regions are found to differ, depending on their GC content even with respect to translation start sites (TLSs) from 913 microbial genomes. The TSS based cut-off values derived in chapter 3 do not have cut-off values for both extremes of GC-bins at 5% interval. Hence, threshold values have been derived from a subset of translation start sites (TLSs) from all microbial genomes which were categorized based on their GC-content. Interestingly the cut-off values derived with respect to TSS data set (chapter 3) and TLS data set are very similar for the in-between GC-bins. Therefore, TSS based cut-off values derived in chapter 2 with the TLS based cut-off values have been combined (denoted as TSS-TLS based cutoff values) to predict promoters over the complete genome sequences. An average recall value of 72% (which indicates the percentage of protein and RNA coding genes with predicted promoter regions assigned to them) and precision of 56% is achieved over the 913 microbial genome dataset. These predicted promoter regions have been given a reliability level (low, medium, high, very high and highest) based on the difference in its relative average free energy, which can help the users design their experiments with more confidence by using the predictions with higher reliability levels. Reference Rangannan, V. and Bansal, M. (2010) High Quality Annotation of Promoter Regions for 913 Bacterial Genomes. Bioinformatics, 26, 3043-3050. Web applications PromBase : The predicted promoter regions for 913 microbial genomes were deposited into a public domain database called, PromBase which can serve as a valuable resource for comparative genomics study for their general genomic features and also help the experimentalist to rapidly access the annotation of the promoter regions in any given genome. This database is freely accessible for the users via the World Wide Web http://nucleix.mbu.iisc.ernet.in/prombase/. EcoProm : EcoProm is a database that can identify and display the potential promoter regions corresponding to EcoCyc annotated TSS and genes. Also displays predictions for whole genomic sequence of E. coli and EcoProm is available at http://nucleix.mbu.iisc.ernet.in/ecoprom/index.htm. PromPredict : The generic promoter prediction methodology described in previous chapters has been implemented in to an algorithm ‘PromPredict’ and available at http://nucleix.mbu.iisc.ernet.in/prompredict/prompredict.html. Analysing the DNA structural characteristic of prokaryotic promoter sequences for their predominance Sequence dependent structural properties and their variation in genomic DNA are important in controlling several crucial processes such as transcription, replication, recombination and chromatin compaction. In this chapter 6, quantitative analysis of sequences motifs as well as sequence dependent structural properties, such as curvature, bendability and stability in the upstream region of TSS and TLS from E. coli, B. subtilis and M. tuberculosis has been carried out in order to assess their predictive power for promoter regions. Also the correlation between these structural properties and GC-content has been investigated. Our results have shown that AFE values (stability) gives finer discrimination rather than %GC in identifying promoter regions and stability have shown to be the better structural property in delineating promoter regions from non-promoter regions. Analysis of these DNA structural properties has been carried out in human promoter sequences and observed to be correlating with the inactivation status of the X-linked genes in human genome. Since, it is deviating from the theme of main thesis; this chapter has been included as appendix A to the main thesis. General conclusion Stability is the ubiquitous DNA structural property seen in promoter regions. Stability shows finer discrimination for promoter prediction rather than directly using %GC-content. Based on relative stability of DNA, a generic promoter prediction algorithm has been developed and implemented to predict promoter regions on a large scale over 913 microbial genome sequences. The analysis of the predicted regions across organisms showed highly reliable predictive performance of the algorithm.

M. Tech. (Department of Biosciences, Faculty of Applied and Computer Sciences) Vaal University of Technology / Most cultivated bananas and plantains (Musa spp. sect. Eumusa), originated from two wild diploid species, Musa acuminata Colla (AA) and Musa balbisiana Colla (BB), which contributed the A and B genomes, respectively. The two genomes confer different traits to a banana plant. Intra- and interspecific hybridization between the wild diploid species, somatic mutations and selection over many thousands of years has given rise to considerable genetic variability in cultivated bananas. Bananas are classified according to its genome composition and a number of morphological traits are used to identify the genomes of a plant. Morphological classification can be misleading since the morphology of plants can be affected by environmental factors. Molecular techniques to identify the genomes of banana have many advantages. The objective of this study was to develop a SCAR (sequence characterized amplified region) marker from a previously reported A genome-specific RAPD fragment that distinguish the A genome of banana from the B genome. This fragment designated OPA17600 was cloned, sequenced and used to design longer 20-mer SCAR primers. Verification of the SCAR primers for its fidelity to the A genome was carried out on a sample of 22 homo-and heterogenomic accessions representing landraces and hybrids of different ploidy and genome combinations. Out of six primers sets that were tested one set (SC3) produced a unique 600 bp in all the A genome containing banana accessions. However, these primers also amplified an 800 bp fragment in all the BB genotypes and some accessions containing the A and B genomes. While previous reports suggested that there was considerable differentiation between the A and B genomes, recent evidence points to the contrary. The presence of the A genome fragment in the B genome genotypes and accessions may be due to recombination between the two genomes, translocations and substitutions. The study concluded that the 600-bp SCAR sequence is conserved across the A genome in Musa and can be used to identify the A genome in banana classification and Musa breeding programmes.

Dans le cadre de cette thèse nous nous sommes intéressés à l'étude de l'évolution et de la variabilité génétique de la famille des butyrophilines (BTN), des récepteurs de la superfamille des immunoglobulines impliqués dans la régulation de la réponse immunitaire. Par une étude phylogénétique approfondie nous avons caractérisé chez les mammifères 14 groupes phylogénétiques résultant d'une série de duplications à partir de huit gènes ancestraux à la base des thériens. Par la suite, nous avons étudié l'évolution des BTN de la région CMH chez les primates et leur variabilité génétique dans les populations humaines par une analyse minutieuse des données de séquençage générées du projet 1000 Genomes pour plus de 1600 individus à travers le monde. Nous avons montré que l'évolution du gène BTNL2 est marquée par une pression de sélection positive diversifiante chez les mammifères qui est accompagnée chez les hominoïdes d'un niveau de polymorphisme élevé induisant la formation de variants tronqués de BTNL2. Chez l'homme, quatre lignages d'allèles ont été identifiés. Ils ont été maintenus à des fréquences intermédiaires par une forte sélection balancée. D'autre part, l'analyse phylogénétique détaillée du groupe BTN3 (BTN3A1, 3A2 et 3A3) a montré la présence d'une évolution concertée, caractérisée par une homogénéisation forte et récurrente de la région codant pour le peptide signale et le domaine IgV chez les hominoïdes, au cours de laquelle les séquences de 3A1 et 3A3 sont remplacées par la séquence de 3A2. Chez l'homme, ces gènes sont polymorphismes important avec plus de 46 allèles chacun, mais avec la présence d'une homogénéisation extrême des séquences du domaine IgV / In this thesis we were interested in studying the evolution and the genetic variability of the butyrophilin family (BTN), a family of immune receptors belonging to the immunoglobulin superfamily implicated in the regulation of immune response. Through a thorough phylogenetic study of the family we characterized 14 phylogenetic groups in mammals resulting from a series of duplications from eight ancestral genes at the base of therian. Thereafter, we studied the evolution of the BTN of the MHC region and their genetic variability in human populations by a careful analysis of sequencing data generated by the consortium 1000 Genomes for more than 1,600 individuals representing 26 populations worldwide. We have shown that the evolution of BTNL2 gene is marked by a positive diversifying selection in placental mammals. This selection pressure is accompanied in hominoids of a high level of polymorphism inducing the formation of truncated BTNL2 variants. In humans this high level of polymorphism results in the presence of four ancient allele lineages that are maintained at intermediate frequencies by a strong balancing selection. On the other hand, a detailed phylogenetic analysis of BTN3 group (BTN3A1, 3A2 and 3A3) showed that these genes evolve in hominoids in a concerted manner characterized by a strong and recurrent homogenization of the regions encoding for the peptide signal and the IgV domain in which the 3A1 and 3A3 sequences are replaced by the 3A2 sequence. In humans these genes are polymorphic with over 46 alleles each, but with the presence of extreme homogenization of IgV domain sequences

Orientador: Laymert Garcia dos Santos / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Filosofia e Ciencias Humanas / Made available in DSpace on 2018-08-04T18:51:14Z (GMT). No. of bitstreams: 1 Monteiro_MarkoSynesioAlves_D.pdf: 14010644 bytes, checksum: 84f93a9c31977e89c8b6246cdca253a9 (MD5) Previous issue date: 2005 / Resumo: A tese tem por objetivo debater as mudanças atualmente em curso nas concepções modernas do corpo, a partir do impacto das novas tecnologias associadas à genética. Tais concepções, calcadas fortemente na dualidade cartesiana, estariam sendo modificadas pela possibilidade, cada vez mais real, de manipulação da natureza em seu nível molecular. As conseqüências teóricas de transformar o corpo, junto com a própria prática científica, em objetos de analise social, são debatidos. Um estudo de caso, a partir de pesquisa de campo feita em laboratórios de bioinformática na Universidade de São Paulo e no Instituto Ludwig de Pesquisa com Câncer/Hospital do Câncer, fundamenta empiricamente a discussão. Os microarrays, utilizados em pesquisas que buscam estabelecer biomarcadores para câncer de próstata, são analisados como objetos que encarnam as novas visões do corpo, num contexto marcado pelo avanço da biotecnologia. A tese debate também alguns aspectos políticos que se mostram relevantes na compreensão desse avanço tecnológico, como os temores de um ressurgimento da eugenia. Ao final, debatem-se alternativas analíticas e políticas para a compreensão do novo esatuto do corpo e da matéria viva, a partir de pistas dadas por artistas, engajados com a expressão artística através do corpo e da tecnologia, e através de teóricos que buscam repensar as bases nas quais assenta-se a biologia e a pesquisa científica contemporâneas / Abstract: This thesis analyzes transformations in contemporary conceptions of the body under the light of changes brought on by new technologies associated with genetics. These conceptions, strongly marked by a cartesian opposition between mind/matter, are being shifted in the context of the growing possibilities of the manipulation nature in its molecular leveI through technology. The theoretical consequences of tuming the body, as well as the scientific practice of manipulating it, as an object for the Social Sciences, are also debated. A case study, originated ITom field research with done in bioinformatics laboratories at the Universidade de São Paulo (USP) and the Ludwig Center for Cancer Research/Cancer Hospital, provides the empirical basis of the argument. Microarrays, used in the research being developed in the laboratories cited above in research towards the development of molecular markers for prostate cancer, are analyzed as objects that embody the new conceptions of the body that arise in a context of rapid advance in biotechnology. The thesis debates also some political aspects that are relevant for a deeper understanding of these technological developments, such as the fears surrounding a retum of eugenic practices. ln the conclusion some analytical and political altematives are discussed, as a means of pursuing a richer criticism of the new statute of the body and of living matter, using the example of artists that use the body and technology as medium of artistic expression; also through a debate of theorists that are busy rethinking the bases of contemporary biology and of current scientific practices / Doutorado / Doutor em Ciências Sociais

Submitted in complete fulfillment of the requirements for the Degree of Doctor of Technology: Biotechnology, Durban University of Technology, Durban, South Africa. 2014. / The global demand for green technology has created a need to search for microbes that can play an active role in advancing a greener and cleaner future. Microbial enzymes are nature’s keys to life and their efficiency, specificity and environmental-friendliness has lead to their increased use in industrial processes. Thermomyces lanuginosus is a thermophilic fungus that can degrade plant biomass and produces a variety of enzymes that have industrial application. The fungus T. lanuginosus SSBP has been reported in literature to produce the highest level of xylanase among other Thermomyces strains and some of its enzyme s viz., amylase and lipase are already being used. Because of this ability, it has been identified as one of the organisms that can have various industrial applications. Although a few proteins from this fungus have been cloned and used commercially, the vast majority are still unknown. In order to identify new protein candidates and understand their biochemical interactions, the T. lanuginosus genome (DNA) and the transcriptome (mRNA) were sequenced using 454 Roche and Solexa sequencing platforms. Genome and transcriptome data was assembled using Newbler software forming a genome size of 23.3 Mb contained 30 scaffolds. Protein prediction identified 5105 candidates as protein-coding genes and these gene models were supported by expressed sequence tag and transcriptomic data. The annotated data was assembled into metabolic pathways in order to identify functional pathways and validate the accuracy of the annotation process. T. lanuginosus is usually found in composting plant material thus protein related to plant hydrolysis were analysed. The total number of plant biomass-degrading and related proteins that fall into the carbohydrate-active enzyme (CAZy) family was 224. Most of these proteins were similar to proteins found in other filamentous fungi. Surprisingly, T. lanuginosus contained a single gene coding for xylanase which hydrolyses xylan although this organism is well known for being among the highest producers of this enzyme. An important subset of the above group of proteins is the cellulose degrading-proteins as this can be used in biofuel production. Eight candidates belonging to this group were identified, making this fungus significant in the biofuels. Among the eight cellulase candidates, phylogenetic analysis revealed that three of them were closely related to Trichoderma reesei, a well known industrial cellulase-producer. Utilization of cellulase-related compounds was validated by phenotypic microarray experiments, with cellobiose having inducing biomass in T. lanuginosus. Proteins that are involved in high temperature survival are vital for the survival. of this thermophilic fungus. Interestingly, T. lanuginosus contains 19 heat shocking proteins which are responsible for thermostability. Another adaptation identified in this fungus is the accumulation of trehalose to combat heat stress. Furthermore, T. lanuginosus contains the highest reported number methyltransferases, which have been linked to producing thermostable proteins and higher energy production. Also because of this organism’s ability to grow on composting environments, the assimilation and ability to produce biomass on different carbon sources were analysed using phenotypic microarray technique. The results showed that xylose was the best compound to induce biomass followed by trehalose, maltose and maltotriose. The genomic sequencing of this fungus has provided valuable information that can be used for various biotechnological applications, as well as providing greater insights into its thermostability. Understanding the metabolic pathways involved may allow for manipulation to increase production of these enzymes or cloning into other hosts. This can have an impact in the field of biofuel production and other plant biomass-related processes.

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Microbiology / Master / Master of Philosophy

Homologous Recombination (HR) can promote genome stability through its capacity to faithfully repair DNA gouble 2trand !;!reak2 (DSBs) and preventing the demise of stalled replication forks in part by catalysing template switching to enable DNA polymerase to bypass lesions. Despite these beneficial roles, inappropriate or untimely HR events can have deleterious consequences. HR can cause genome instability by recombining "inappropriate" homologous sequences, especially if the recombination intermediates are resolved to form crossovers. Over the past few years, study of the rare inherited chromosome instability disorder, Eanconi Anaemia (FA), has uncovered a novel DNA damage response pathway. Although the FA pathway is required primarily for interstrand DNA cross link repair, its precise role in DNA repair reactions is still unclear. FA.Qomplementation group M (FANCM) is the sole component within the FA core complex which possesses a DNA helicase/ATPase domain and an endonuclease domain (albeit non-functional), suggesting that FANCM could translocate along DNA and target the FA core complex to blocked replication forks. To further elucidate the role of FANCM in HR, I have purified Fm11, the FANCM orthologue in the fission yeast Schizosaccharomyces pombe and tested its activity on a range of synthetic replication and recombination intermediates in vitro. Fml1 binds both replication forks and Holliday Junctions (HJs) which are key intermediates of HR.

Submitted in complete fulfillment for the Degree of Master of Technology (Biotechnology), Durban University of Technology, Durban, South Africa, 2014. / Chitin, a highly insoluble 1,4- -linked polymer of N-acetyl- -D-glucosamine, is the second-most abundant bio-polysaccharide in nature after cellulose. Most chitinolytic fungi are known to produce more than one kind of chitinase. The recent sequencing of the Thermomyces lanuginosus SSBP genome by our group has revealed four putative family 18 chitinases. In this study, three novel chitinase genes (chitl, chit2 and chit3) and the previously reported chit4 gene were cloned from Thermomyces lanuginosus SSBP and their gene structures were analysed. chit3, encoding a 36.6 kDa protein, and chit4, encoding a 44.1 kDa protein, were successfully expressed in Pichia pastoris. The recombinant Chit3 and Chit4 enzymes exhibited optimum activity at pH 4.0 and 5.0 and at 40oC and 50°C, respectively. Chit3 was stable at 40oC and retained 71% of its activity at 50°C after 60 min, while Chit4 was stable at 50°C and retained 56% of its activity at 60°C after 30 min. Both enzymes produced chitobiose as the major product using colloidal chitin, chitooligosaccharides and shrimp shell powder as substrates. Of the fungal strains tested, Chit3 displayed antifungal activity against Penicillium sp. and Aspergillus sp. This is the first report on the multi-chitinolytic system of T. lanuginosus and enzyme characterization has shown the potential of the enzymes to be used in degradation of the under-utilized bio-resource chitin. / PDF Full-text unavailable. Please refer to hard copy for Full-text / M

Orientador: Ricardo da Fonseca / Coorientador: Marcos Vinícius Gualberto Barbosa da Silva / Coorientador: José Fernando Garcia / Banca: Henrique Nunes de Oliveira / Banca: Roberto Carvalheiro / Banca: João Cláudio do Carmo Panetto / Banca: Adriana Santana do Carmo / Resumo: A base genética que rege os processos fisiológicos para expressão dos fenótipos de produção de leite ainda não está completamente compreendida, pois poucos genes causais ou marcadores associados com a variação na expressão desses fenótipos foram relatados e espera-se que mais genes estejam envolvidos. Com o surgimento da era genômica, os esforços para identificar polimorfismos de sítio único (Single Nucleotide Polymorphisms - SNPs) foram expressivos. Os SNPs permitem estabelecer uma forte relação entre a expressão de características economicamente importantes e regiões específicas do genoma de um indivíduo. Tal relação é confirmada por estudos de associação ampla do genoma (GWAS), gerando conhecimento a cerca dos genes e fragmentos cromossômicos ligados a características importantes, os quais são posteriormente explorados na biologia dos sistemas. Qualquer inferência acerca de segmentos cromossômicos que possam estar associados a fenótipos de interesse utiliza uma montagem de um genoma de referência, onde todos os genes estão ancorados. Porém, o processo de montagem de um genoma é complexo e erros quanto ao posicionamento de sequências são esperados. Desta forma, este trabalho propõe avaliar a montagem de referência do genoma bovino produzido pelo grupo de pesquisa da universidade de Maryland e a aplicação do GWAS na raça Gir (Bos indicus) aos fenótipos de produção de leite, proteína e gordura, porcentagem de proteína e gordura e idade ao primeito parto, com o intuito de identificar regiões cromossômicas que possam estar relacionadas com aspectos importantes da produção de leite e fertilidade, contribuindo para a melhor compreensão dos fenômenos que regem tais aspectos / Abstract: The genetic basis of physiological processes underlying milk production traits are not completely understood, and few causal genes and markers associated with these traits have been reported to date. The emergence of the genomics era, efforts for the discovery of single nucleotide polymorphisms (SNPs) are numerous. These markers allow for establishing relationships between differences in economically important traits and specific genomic coordinates. These relationships are confirmed in genome-wide association studies (GWAS), which provide knowledge about genes and chromosomal segments affecting traits of interest that can be further explored in systems biology. Inferences about genomic localtions that are potentially implicated in phenotypic differences rely on a reference genome assembly where genes are annotated. However, genome assembly is a complex task that is prone to errors, and cases of wrong positioning of nucleotide sequences are not rare. Therefore, this thesis aimed at assessing candidate misassembled regions in the reference bovine genome assembly and performing a GWAS for milk traits in Gir cattle (Bos indicus), including milk, protein and fat yield, percentage of protein and fat, and age at first calving, targeting the identication of genomic regions that are potentially related to important aspects of fertility and milk production / Doutor