A quantitative view of the transcriptome of Schistosoma mansoni adult-worms using SAGE

<p>Abstract</p> <p>Background</p> <p>Five species of the genus Schistosoma, a parasitic trematode flatworm, are causative agents of Schistosomiasis, a disease that is endemic in a large number of developing countries, affecting millions of patients around the world. By using SAGE (Serial Analysis of Gene Expression) we describe here the first large-scale quantitative analysis of the Schistosoma mansoni transcriptome, one of the most epidemiologically relevant species of this genus.</p> <p>Results</p> <p>After extracting mRNA from pooled male and female adult-worms, a SAGE library was constructed and sequenced, generating 68,238 tags that covered more than 6,000 genes expressed in this developmental stage. An analysis of the ordered tag-list shows the genes of F10 eggshell protein, pol-polyprotein, HSP86, 14-3-3 and a transcript yet to be identified to be the five top most abundant genes in pooled adult worms. Whereas only 8% of the 100 most abundant tags found in adult worms of S. mansoni could not be assigned to transcripts of this parasite, 46.9% of the total ditags could not be mapped, demonstrating that the 3 sequence of most of the rarest transcripts are still to be identified. Mapping of our SAGE tags to S. mansoni genes suggested the occurrence of alternative-polyadenylation in at least 13 gene transcripts. Most of these events seem to shorten the 3 UTR of the mRNAs, which may have consequences over their stability and regulation.</p> <p>Conclusion</p> <p>SAGE revealed the frequency of expression of the majority of the S. mansoni genes. Transcriptome data suggests that alternative polyadenylation is likely to be used in the control of mRNA stability in this organism. When transcriptome was compared with the proteomic data available, we observed a correlation of about 50%, suggesting that both transcriptional and post-transcriptional regulation are important for determining protein abundance in S. mansoni. The generation of SAGE tags from other life-cycle stages should contribute to reveal the dynamics of gene expression in this important parasite.</p

Developing of a biophysical model to describe DNA double-strand repair process

Esta tese consiste em três estudos relacionados com genomas procarió- ticos. O primeiro estudo trata do desenvolvimento e da aplicação de um método baseado em buscas de similaridade de sequências para a identificação em larga es- cala de genes potencialmente envolvidos em transferência gênica horizontal (TGH). O método usa a distância entre os rRNA 16S dos organismos em estudo como esti- mativa da distância filogenética entre eles. Genes com alvos de alta pontuação em organismos atipicamente distantes são considerados candidatos a TGH. A aplicação deste método a 408 genomas procarióticos anotados nos permite concluir que: (i) genes com funções operacionais estão significativamente enriquecidos, enquanto que genes com funções informacionais estão significativamente empobrecidos em candida- tos a TGH; (ii) há uma forte correlação entre o tamanho do genoma e a proporção de genes candidatos a TGH, que indica que a proporção de TGH é menor em genomas pequenos e maior em genomas grandes; (iii) a hipótese da complexidade, que diz que genes cujos produtos participam de poucas interações proteicas são mais suscetíveis a TGH que aqueles que participam de muitas interações, está de acordo com nossas predições de TGH em dois estudos de interação proteína-proteína em Escherichia coli. O segundo estudo consiste na identificação de genes pertencentes ao regulon SOS de Caulobacter crescentus usando uma estratégia iterativa de predições in si- lico de sítios de ligação da proteína LexA no DNA e experimentos de laboratório para medir a expressão diferencial, entre a cepa selvagem e uma cepa deficiente em lexA, dos genes próximos aos sítios preditos de LexA. Esta estratégia nos permitiu identificar 37 genes pertencentes a este regulon. O terceiro estudo trata de um teste estatístico para a colocalização de posições genômicas. Dados dois conjuntos X e Y de posições genômicas, o método verifica se um número significativo de elementos de Y está posicionado próximo a elementos de X. Este método, aplicado às predições de sítios de ligação de LexA no genoma de C. crescentus, resultou na identificação de uma quantidade significativa de sítios preditos de LexA próximos aos sítios preditos de início de tradução deste genoma. Alguns destes sítios não haviam sido identifi- xii cados no segundo estudo por terem pontuação inferior ao limiar efetivamente usado naquele estudo. O posicionamento destes sítios sugere que os genes correspondentes possam ter regulação dependente de LexA.This thesis comprises three studies related to prokaryotic genomes. The first study consists of the development and application of a sequence similarity-based method for large-scale identification of genes potentially involved in horizontal gene transfer (HGT). The method uses 16S rRNA sequence distances as estimates of phy- logenetic distances between organisms. Genes having high-scoring similarity-search hits in atypically distant organisms are taken as HGT candidates. The application of this method to 408 annotated prokaryotic genomes allowed us to conclude that (i) operational categories (transport, metabolism, mobile elements and DNA restric- tion/modification) are significantly enriched in HGT candidates whereas informati- onal categories (protein synthesis, protein fate, transcription, DNA metabolism) are significantly depleted in HGT candidates; (ii) there is a strong correlation between genome size and HGT proportion, indicating that the proportion of HGT candidates tend to be lower in smaller genomes and higher in bigger ones; (iii) the complexity hypothesis, which states that genes whose products have few interaction partners are more prone to HGT than those with many partners, is supported by our HGT predictions for data from two protein-protein interaction studies in Escherichia coli. The second study is about the identification of genes belonging to the SOS regulon in Caulobacter crescentus. We employed an iterative strategy of in silico predictions of LexA binding sites and differential gene expression measurements, between wild- type and lexA-deficient strains, for genes carrying predicted LexA binding sites in their promoter regions. This strategy allowed us to identify 37 genes belonging to this regulon. In the third study, we have developed a statistical test for the colocalization of genomic positions. Given two sets X and Y of genomic positions, the method verifies if a significant number of Y elements is positioned in close proxi- mity to X elements. This method, applied to the LexA binding-site predictions from the second study, resulted in the identification of a significant number of predicted LexA binding sites near translation start sites in the C. crescentus genome. Some of these sites were not identified in the second study because their scores according to xiv the LexA binding site model were lower than the threshold effectively used in that study. The positioning of these sites suggests that the corresponding genes may be regulated in a LexA-dependent manner

Characterization of the SOS Regulon of Caulobacter crescentus▿ ‡

The SOS regulon is a paradigm of bacterial responses to DNA damage. A wide variety of bacterial species possess homologs of lexA and recA, the central players in the regulation of the SOS circuit. Nevertheless, the genes actually regulated by the SOS have been determined only experimentally in a few bacterial species. In this work, we describe 37 genes regulated in a LexA-dependent manner in the alphaproteobacterium Caulobacter crescentus. In agreement with previous results, we have found that the direct repeat GTTCN7GTTC is the SOS operator of C. crescentus, which was confirmed by site-directed mutagenesis studies of the imuA promoter. Several potential promoter regions containing the SOS operator were identified in the genome, and the expression of the corresponding genes was analyzed for both the wild type and the lexA strain, demonstrating that the vast majority of these genes are indeed SOS regulated. Interestingly, many of these genes encode proteins with unknown functions, revealing the potential of this approach for the discovery of novel genes involved in cellular responses to DNA damage in prokaryotes, and illustrating the diversity of SOS-regulated genes among different bacterial species

Differential L1 regulation in pluripotent stem cells of humans and apes.

Identifying cellular and molecular differences between human and non-human primates (NHPs) is essential to the basic understanding of the evolution and diversity of our own species. Until now, preserved tissues have been the main source for most comparative studies between humans, chimpanzees (Pan troglodytes) and bonobos (Pan paniscus). However, these tissue samples do not fairly represent the distinctive traits of live cell behaviour and are not amenable to genetic manipulation. We propose that induced pluripotent stem (iPS) cells could be a unique biological resource to determine relevant phenotypical differences between human and NHPs, and that those differences could have potential adaptation and speciation value. Here we describe the generation and initial characterization of iPS cells from chimpanzees and bonobos as new tools to explore factors that may have contributed to great ape evolution. Comparative gene expression analysis of human and NHP iPS cells revealed differences in the regulation of long interspersed element-1 (L1, also known as LINE-1) transposons. A force of change in mammalian evolution, L1 elements are retrotransposons that have remained active during primate evolution. Decreased levels of L1-restricting factors APOBEC3B (also known as A3B) and PIWIL2 (ref. 7) in NHP iPS cells correlated with increased L1 mobility and endogenous L1 messenger RNA levels. Moreover, results from the manipulation of A3B and PIWIL2 levels in iPS cells supported a causal inverse relationship between levels of these proteins and L1 retrotransposition. Finally, we found increased copy numbers of species-specific L1 elements in the genome of chimpanzees compared to humans, supporting the idea that increased L1 mobility in NHPs is not limited to iPS cells in culture and may have also occurred in the germ line or embryonic cells developmentally upstream to germline specification during primate evolution. We propose that differences in L1 mobility may have differentially shaped the genomes of humans and NHPs and could have continuing adaptive significance