Replication origin location might contribute to genetic variability in Trypanosoma cruzi

Background: DNA replication in trypanosomatids operates in a uniquely challenging environment, since most of their genomes are constitutively transcribed. Trypanosoma cruzi, the etiological agent of Chagas disease, presents high variability in both chromosomes size and copy number among strains, though the underlying mechanisms are unknown. Results: Here we have mapped sites of DNA replication initiation across the T. cruzi genome using Marker Frequency Analysis, which has previously only been deployed in two related trypanosomatids. The putative origins identified in T. cruzi show a notable enrichment of GC content, a preferential position at subtelomeric regions, coinciding with genes transcribed towards the telomeres, and a pronounced enrichment within coding DNA sequences, most notably in genes from the Dispersed Gene Family 1 (DGF-1). Conclusions: These findings suggest a scenario where collisions between DNA replication and transcription are frequent, leading to increased genetic variability, as seen by the increase SNP levels at chromosome subtelomeres and in DGF-1 genes containing putative origins

Development of CaneRegNet platform for functional annotation and analysis of sugarcane transcriptome

A identificação de genes alvos, vias de sinalização e vias metabólicas para melhoramento de cana-de-açúcar associados a características de interesse, ainda são pouco conhecidos e estudados. Alguns estudos do transcriptoma através de plataformas de microarranjo têm buscado identificar listas de genes, para experimentos tecido- específico ou submetidos a condições de estresse bióticos e abióticos. Estudos pontuais destes dados tem sido associados a vias metabólicas ou vias de sinalização já descritas na literatura, de forma a identificar alterações relacionadas a padrões de expressão gênica. Porém, estas relações em cana-de-açúcar são pouco conhecidas e estudadas. O estudo e entendimento de cana-de-açúcar por meio da diversidade genética e de sua adaptação ao ambiente é um grande desafio, principalmente pela ausência de um genoma sequenciado e por possuir um genoma complexo. Apresentamos nossos resultados para tentar superar tais limitações e desafios para estudos de expressão gênica. Foram desenvolvidas metodologias para anotação funcional do transcriptoma, centradas na transferência de anotação, identificação de vias metabólicas e enzimas pelo método de similaridade bi-direcional, predição de genes full-length, análises de ortologia e desenho de oligonucleotídeos para microarranjos customizados, resultando no ORFeoma de cana-de-açúcar, na identificação e classificação de famílias de fatores de transcrição e identificação de genes ortólogos entre gramíneas. Além disso, desenvolvemos uma plataforma para processamento e análise automatizada de experimentos por microarranjo, para armazenamento, recuperação e integração com a anotação funcional. Adicionalmente desenvolvemos e implementamos métodos para seleção de genes diferencialmente e significativamente expressos, e abordagens para análise de enriquecimento de categorias, e escores de atividade de vias metabólicas. De forma a integrar a anotação funcional do transcriptoma aos estudos por expressão gênica, desenvolvemos a plataforma CaneRegNet e uma interface para integração desta rede de dados biológicos e conhecimentos, composta por aplicativos para consulta e prospecção de dados por análises de agrupamento e correlação entre experimentos de microarranjo, possibilitando a geração de novas hipóteses e predições dentro da organização da regulação celular.The identification of target genes, metabolic and signaling pathways associated with characteristics of interest to the sugarcane improvement are still poorly known and studied. Some transcritptome studies through microarray platforms has tried to identify lists of genes, for tissue-specific experiments or subjected to conditions of biotic and abiotic stress. In the literature specific studies of these data has already been associated with metabolic or signaling pathway, in order to identify changes in these tracks related to patterns of gene expression. However, these relations are still little know and generally defined slightly. The study and understanding of sugarcane by means of genetic diversity and its adaptation to the environment is a major challenge, mainly due to the absence of a sequenced genome and by your complex genome. We present our results to surpass this barrier e challenges for the study of gene expression. Methodologies were developed for the transcriptome functional annotation, focused on the annotation transfer, identification of metabolic pathways and enzymes by the bi- directional method; prediction of full-length genes; ortology analysis and probe design for customized microarrays, resulting in the sugarcane ORFeome, the identification and classification of transcription factor families and identification of ortholog genes between grasses. Besides that, we have developed a plataform for automated processing and analysis for microarray experiments, to store, retrieve and integration with the functional annotation. Additionally, we have developed and implemented methods for identification of differentially and significantly expressed genes, and approaches for over-represented analysis and functional class scoring (FCS). To integrate the functional annotation and the studies by gene expression profile, we have developed the CaneRegNet platform and an interface to integrate this network of biological data and knowledge, composed by searching and data mining tools for clustering and correlations between microarray experiments, enabling the generation of new hypothesis and predictions around the organization of cellular regulation

Circadian rhythms of sense and antisense transcription in sugarcane, a highly polyploid crop.

Commercial sugarcane (Saccharum hybrid) is a highly polyploid and aneuploid grass that stores large amounts of sucrose in its stem. We have measured circadian rhythms of sense and antisense transcription in a commercial cultivar (RB855453) using a custom oligoarray with 14,521 probes that hybridize to sense transcripts (SS) and 7,380 probes that hybridize to antisense transcripts (AS).We estimated that 32% of SS probes and 22% AS probes were rhythmic. This is a higher proportion of rhythmic probes than the usually found in similar experiments in other plant species. Orthologs and inparalogs of Arabidopsis thaliana, sugarcane, rice, maize and sorghum were grouped in ortholog clusters. When ortholog clusters were used to compare probes among different datasets, sugarcane also showed a higher proportion of rhythmic elements than the other species. Thus, it is possible that a higher proportion of transcripts are regulated by the sugarcane circadian clock. Thirty-six percent of the identified AS/SS pairs had significant correlated time courses and 64% had uncorrelated expression patterns. The clustering of transcripts with similar function, the anticipation of daily environmental changes and the temporal compartmentation of metabolic processes were some properties identified in the circadian sugarcane transcriptome. During the day, there was a dominance of transcripts associated with photosynthesis and carbohydrate metabolism, including sucrose and starch synthesis. During the night, there was dominance of transcripts associated with genetic processing, such as histone regulation and RNA polymerase, ribosome and protein synthesis. Finally, the circadian clock also regulated hormone signalling pathways: a large proportion of auxin and ABA signalling components were regulated by the circadian clock in an unusual biphasic distribution

Rhythmic probes associated with sugar storage.

<p>Z-score normalized time courses of rhythmic probes for transcripts associated with sugar storage pathways were separated into (<b>A</b>) sucrose synthesis (light green); (<b>B</b>) sucrose degradation (dark green); (<b>C</b>) starch synthesis (dark red); (<b>D</b>) starch degradation (cyan); (<b>E</b>) starch branching (red); and starch debranching (light blue). White boxes represent periods of subjective day and light grey boxes represent periods of subjective night.</p

Proportion of rhythmic probes in different plant species using different algorithms.

1<p>Khan et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071847#pone.0071847-Khan1" target="_blank">[18]</a>;</p>2<p>Filichkin et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071847#pone.0071847-Filichkin2" target="_blank">[17]</a>;</p>3<p>Covington and Harmer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071847#pone.0071847-Covington2" target="_blank">[39]</a>;</p>4<p>Edwards et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071847#pone.0071847-Edwards1" target="_blank">[20]</a>;</p>5<p>estimated.</p

Temporal coordination of probes associated with sucrose and starch metabolism pathways.

<p>The time of peak of rhythms in transcript levels of genes associated with sucrose and starch metabolism was identified in a schematic metabolic pathway. Each circle corresponds to a specific gene model. Metabolic pathways were colored according to the median of the phase of their constitutive genes. The time of peak of probes associated with starch and sucrose synthesis pathways was between ZT20 and ZT4, while the time of the peak of probes associate to sucrose and starch degradation was between ZT8 to ZT16. Genes that were not circadian (n. c.) were in gray. Rhythmic with a time of peak at ZT0 are in yellow, ZT4 in dark orange, ZT8 in red, ZT12 in blue, ZT16 in dark blue and ZT20 in light blue. Enzymes for sucrose synthesis are: (<b>1</b>) sucrose-phosphate synthase; (<b>2</b>) sucrose phosphatase; (<b>3</b>) sucrose synthase; (<b>4</b>) neutral invertase; (<b>5</b>) hexokinase; (<b>6</b>) fructokinase; (<b>7</b>) glucose-6-phosphate isomerase; (<b>8</b>) phosphoglucomutase; (<b>9</b>) UDP-glucose pyrophosphorylase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071847#pone.0071847-Rohwer1" target="_blank">[75]</a>. Abbreviations: S6P – sucrose 6-phosphate; UDP-G - UDP-glucose; G1P – glucose 1-phosphate; G6P – glucose 6-phosphate; F6P – fructose 6-phosphate; ADP-G – ADP-glucose; 3P glycerate – 3-phospho glycerate.</p

Sense and antisense transcripts are modulated differently by the circadian clock.

<p>(<b>A</b>) Overlap between SAS that had their probes for sense transcripts (SS) considered circadian and SAS that had their probes for antisense transcripts (AS) considered circadian. (<b>B</b>) Distribution of Spearman's rank correlation coefficient (ρ) for each of all the 428 pairs of SS/AS (light blue) and only the 207 pairs of SS/AS that had at least one probe considered circadian (orange). If ρ >0.56, correlation is positive and significant. If ρ <−0.56, correlation is negative and significant. (<b>C to F</b>) Z-score normalized expression levels of SS (red) and AS (dark blue) for a gene that have (<b>C</b>) both SS and AS in the same phase (RuBisCo activase; SCBGLR1044D06.g,), (<b>D</b>) SS and AS in opposite phases (urease, SCSGLR1045A02.g), (<b>E</b>) AS peaking before SS (glucose 1-phosphate adenylyltransferase, SCVPCL6061A06.g) and (<b>F</b>) SS peaking before AS (violaxanthin de-epoxidase, SCVPHR1095C07.g). White boxes represent periods of subjective day and light grey boxes represent periods of subjective night.</p