Cost-Effective Sequencing of Full-Length cDNA Clones Powered by a De Novo-Reference Hybrid Assembly

Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded., to confirm that its ability was competent even for non-human species.The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only ∼US$3 per clone, demonstrating a significant advantage over previous approaches

Transcriptome Profile of Trichoderma harzianum IOC-3844 Induced by Sugarcane Bagasse

Profiling the transcriptome that underlies biomass degradation by the fungus Trichoderma harzianum allows the identification of gene sequences with potential application in enzymatic hydrolysis processing. in the present study, the transcriptome of T. harzianum IOC-3844 was analyzed using RNA-seq technology. the sequencing generated 14.7 Gbp for downstream analyses. de novo assembly resulted in 32,396 contigs, which were submitted for identification and classified according to their identities. This analysis allowed us to define a principal set of T. harzianum genes that are involved in the degradation of cellulose and hemicellulose and the accessory genes that are involved in the depolymerization of biomass. An additional analysis of expression levels identified a set of carbohydrate-active enzymes that are upregulated under different conditions. the present study provides valuable information for future studies on biomass degradation and contributes to a better understanding of the role of the genes that are involved in this process.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Campinas UNICAMP, CBMEG, Campinas, SP, BrazilBrazilian Ctr Res Energy & Mat CNPEM, Brazilian Bioethanol Sci & Technol Lab CTBE, Campinas, SP, BrazilUniv São Paulo, Phys Inst Sao Carlos, Sao Carlos, SP, BrazilFed Univ São Paulo UNIFESP, Inst Sci & Technol, Sao Jose Dos Campos, SP, BrazilUniv Campinas UNICAMP, Dept Plant Biol, Inst Biol, Campinas, SP, BrazilFed Univ São Paulo UNIFESP, Inst Sci & Technol, Sao Jose Dos Campos, SP, BrazilWeb of Scienc

Nonoverlapping Clone Pooling for High-Throughput Sequencing

Simultaneously sequencing multiple clones using second-generation sequencers can speed up many essential clone-based sequencing methods. However, in applications such as fosmid clone sequencing and full-length cDNA sequencing, it is important to create pools of clones that do not overlap on the genome for the identification of structural variations and alternatively spliced transcripts, respectively. We define the nonoverlapping clone pooling problem and provide practical solutions based on optimal graph coloring and bin-packing algorithms with constant absolute worst-case ratios, and further extend them to cope with repetitive mappings. Using theoretical analysis and experiments, we also show that the proposed methods are applicable.Japanese Government Ministry of Education, Culture, Sports, Science, and Technology (MEXT)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, Inst Ciencia & Tecnol, BR-12231280 Sao Jose Dos Campos, SP, BrazilUniversidade Federal de São Paulo, Inst Ciencia & Tecnol, BR-12231280 Sao Jose Dos Campos, SP, BrazilWeb of Scienc

eIF5A and EF-P: two unique translation factors are now traveling the same road

Translational control is extremely important in all organisms, and some of its aspects are highly conserved among all primary kingdoms, such as those related to the translation elongation step. The previously classified translation initiation factor 5A (eIF5A) and its bacterial homologue elongation factor P (EF-P) were discovered in the late 70's and have recently been the object of many studies. eIF5A and EF-P are the only cellular proteins that undergo hypusination and lysinylation, respectively, both of which are unique posttranslational modifications. Herein, we review all the important discoveries related to the biochemical and functional characterization of these factors, highlighting the implication of eIF5A in translation elongation instead of initiation. The findings that eIF5A and EF-P are important for specific cellular processes and play a role in the relief of ribosome stalling caused by specific amino acid sequences, such as those containing prolines reinforce the hypothesis that these factors are involved in specialized translation. Although there are some divergences between these unique factors, recent studies have clarified that they act similarly during protein synthesis. Further studies may reveal their precise mechanism of ribosome activity modulation as well as the mRNA targets that require eIF5A and EF-P for their proper translation. (C) 2014 John Wiley & Sons, Ltd.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Contribution to the taxonomy and ecology of the Azorean benthic marine algae

Evolution and Conservation in the North Atlantic Islands (Madeira, Canaries, Azores). Manchester, Reino Unido, 4-6 de Setembro de 1990

Business Models with Additive Manufacturing : opportunities and Challenges from the Perspective of Economics and Management

Trichoderma harzianum IOC-3844 secretes high levels of cellulolytic-active enzymes and is therefore a promising strain for use in biotechnological applications in second-generation bioethanol production. However, the T. harzianum biomass degradation mechanism has not been well explored at the genetic level. The present work investigates six genomic regions (~150 kbp each) in this fungus that are enriched with genes related to biomass conversion. A BAC library consisting of 5,760 clones was constructed, with an average insert length of 90 kbp. The assembled BAC sequences revealed 232 predicted genes, 31.5% of which were related to catabolic pathways, including those involved in biomass degradation. An expression profile analysis based on RNA-Seq data demonstrated that putative regulatory elements, such as membrane transport proteins and transcription factors, are located in the same genomic regions as genes related to carbohydrate metabolism and exhibit similar expression profiles. Thus, we demonstrate a rapid and efficient tool that focuses on specific genomic regions by combining a BAC library with transcriptomic data. This is the first BAC-based structural genomic study of the cellulolytic fungus T. harzianum, and its findings provide new perspectives regarding the use of this species in biomass degradation processes

Molecular functions, biological process distribution and cellular localization of the transcriptome assembly.

<p>Contigs were assigned putative classifications based on homology and evaluated for their predicted involvement in molecular functions (A), biological processes (B) and cellular localization (C).</p

Identified sequences that catalyze reactions that are potentially involved in biomass degradation.

<p>The results of identification based on homology using the NCBI NR database indicate the presence of genes that are related to the depolymerization of biomass in the transcriptome.</p

Filter paper activity enzymatic profiles (FPU mL⁻¹ h⁻¹).

<p><i>T. harzianum</i> fermentation on a complex substrate (DSB) from extracts grown on preculture media using DSB (▵), cellulose (◊) or lactose (□) as the carbon source. The differences were considered significant at P<0.05 (Student's t-test) and are indicated by (a) for cellulose, (b) for DSB and (c) for lactose samples.</p