RESUMO
Singapore's National Flower, Papilionanthe (Ple.) Miss Joaquim 'Agnes' (PMJ) is highly prized as a horticultural flower from the Orchidaceae family. A combination of short-read sequencing, single-molecule long-read sequencing and chromatin contact mapping was used to assemble the PMJ genome, spanning 2.5 Gb and 19 pseudo-chromosomal scaffolds. Genomic resources and chemical profiling provided insights towards identifying, understanding and elucidating various classes of secondary metabolite compounds synthesized by the flower. For example, presence of the anthocyanin pigments detected by chemical profiling coincides with the expression of ANTHOCYANIN SYNTHASE (ANS), an enzyme responsible for the synthesis of the former. Similarly, the presence of vandaterosides (a unique class of glycosylated organic acids with the potential to slow skin aging) discovered using chemical profiling revealed the involvement of glycosyltransferase family enzymes candidates in vandateroside biosynthesis. Interestingly, despite the unnoticeable scent of the flower, genes involved in the biosynthesis of volatile compounds and chemical profiling revealed the combination of oxygenated hydrocarbons, including traces of linalool, beta-ionone and vanillin, forming the scent profile of PMJ. In summary, by combining genomics and biochemistry, the findings expands the known biodiversity repertoire of the Orchidaceae family and insights into the genome and secondary metabolite processes of PMJ.
Assuntos
Antocianinas , Orchidaceae , Cromatina/metabolismo , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Glicosiltransferases/genética , Redes e Vias Metabólicas , Orchidaceae/genética , SingapuraRESUMO
BACKGROUND: Melanoma is the major cause of skin cancer deaths and melanoma incidence doubles every 10 to 20 years. However, little is known about melanoma pathway aberrations. Here we applied the robust Gene Identification Signature Paired End diTag (GIS-PET) approach to investigate the melanoma transcriptome and characterize the global pathway aberrations. METHODS: GIS-PET technology directly links 5' mRNA signatures with their corresponding 3' signatures to generate, and then concatenate, PETs for efficient sequencing. We annotated PETs to pathways of KEGG database and compared the murine B16F1 melanoma transcriptome with three non-melanoma murine transcriptomes (Melan-a2 melanocytes, E14 embryonic stem cells, and E17.5 embryo). Gene expression levels as represented by PET counts were compared across melanoma and melanocyte libraries to identify the most significantly altered pathways and investigate the expression levels of crucial cancer genes. RESULTS: Melanin biosynthesis genes were solely expressed in the cells of melanocytic origin, indicating the feasibility of using the PET approach for transcriptome comparison. The most significantly altered pathways were metabolic pathways, including upregulated pathways: purine metabolism, aminophosphonate metabolism, tyrosine metabolism, selenoamino acid metabolism, galactose utilization, nitrobenzene degradation, and bisphenol A degradation; and downregulated pathways: oxidative phosphorylation, ATPase synthesis, TCA cycle, pyruvate metabolism, and glutathione metabolism. The downregulated pathways concurrently indicated a slowdown of mitochondrial activities. Mitochondrial permeability was also significantly altered, as indicated by transcriptional activation of ATP/ADP, citrate/malate, Mg++, fatty acid and amino acid transporters, and transcriptional repression of zinc and metal ion transporters. Upregulation of cell cycle progression, MAPK, and PI3K/Akt pathways were more limited to certain region(s) of the pathway. Expression levels of c-Myc and Trp53 were also higher in melanoma. Moreover, transcriptional variants resulted from alternative transcription start sites or alternative polyadenylation sites were found in Ras and genes encoding adhesion or cytoskeleton proteins such as integrin, beta-catenin, alpha-catenin, and actin. CONCLUSION: The highly correlated results unmistakably point to a systematic downregulation of mitochondrial activities, which we hypothesize aims to downgrade the mitochondria-mediated apoptosis and the dependency of cancer cells on angiogenesis. Our results also demonstrate the advantage of using the PET approach in conjunction with KEGG database for systematic pathway analysis.
Assuntos
Perfilação da Expressão Gênica/métodos , Melaninas/biossíntese , Melanoma/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Neoplasias Cutâneas/genética , Animais , Apoptose/genética , Vias Biossintéticas/genética , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/genética , Melaninas/genética , Melanoma/patologia , Camundongos , RNA Mensageiro/genética , Elementos Reguladores de Transcrição/genética , Sensibilidade e Especificidade , Neoplasias Cutâneas/patologia , Células Tumorais CultivadasRESUMO
BACKGROUND: We recently developed the Paired End diTag (PET) strategy for efficient characterization of mammalian transcriptomes and genomes. The paired end nature of short PET sequences derived from long DNA fragments raised a new set of bioinformatics challenges, including how to extract PETs from raw sequence reads, and correctly yet efficiently map PETs to reference genome sequences. To accommodate and streamline data analysis of the large volume PET sequences generated from each PET experiment, an automated PET data process pipeline is desirable. RESULTS: We designed an integrated computation program package, PET-Tool, to automatically process PET sequences and map them to the genome sequences. The Tool was implemented as a web-based application composed of four modules: the Extractor module for PET extraction; the Examiner module for analytic evaluation of PET sequence quality; the Mapper module for locating PET sequences in the genome sequences; and the Project Manager module for data organization. The performance of PET-Tool was evaluated through the analyses of 2.7 million PET sequences. It was demonstrated that PET-Tool is accurate and efficient in extracting PET sequences and removing artifacts from large volume dataset. Using optimized mapping criteria, over 70% of quality PET sequences were mapped specifically to the genome sequences. With a 2.4 GHz LINUX machine, it takes approximately six hours to process one million PETs from extraction to mapping. CONCLUSION: The speed, accuracy, and comprehensiveness have proved that PET-Tool is an important and useful component in PET experiments, and can be extended to accommodate other related analyses of paired-end sequences. The Tool also provides user-friendly functions for data quality check and system for multi-layer data management.