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Artykuły w czasopismach na temat "HUMAN TRANSCRIPTOME"
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Brenner, Eric, Gayatri R. Tiwari, Manav Kapoor, Yunlong Liu, Amy Brock i R. Dayne Mayfield. "Single cell transcriptome profiling of the human alcohol-dependent brain". Human Molecular Genetics 29, nr 7 (6.03.2020): 1144–53. http://dx.doi.org/10.1093/hmg/ddaa038.
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Abstract Alcoholism remains a prevalent health concern throughout the world. Previous studies have identified transcriptomic patterns in the brain associated with alcohol dependence in both humans and animal models. But none of these studies have systematically investigated expression within the unique cell types present in the brain. We utilized single nucleus RNA sequencing (snRNA-seq) to examine the transcriptomes of over 16 000 nuclei isolated from the prefrontal cortex of alcoholic and control individuals. Each nucleus was assigned to one of seven major cell types by unsupervised clustering. Cell type enrichment patterns varied greatly among neuroinflammatory-related genes, which are known to play roles in alcohol dependence and neurodegeneration. Differential expression analysis identified cell type-specific genes with altered expression in alcoholics. The largest number of differentially expressed genes (DEGs), including both protein-coding and non-coding, were detected in astrocytes, oligodendrocytes and microglia. To our knowledge, this is the first single cell transcriptome analysis of alcohol-associated gene expression in any species and the first such analysis in humans for any addictive substance. These findings greatly advance the understanding of transcriptomic changes in the brain of alcohol-dependent individuals.
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Londin, Eric R., Eleftheria Hatzimichael, Phillipe Loher, Leonard C. Edelstein, Chad Shaw, Kathleen Delgrosso, Paolo M. Fortina, Paul F. Bray, Steven E. McKenzie i Isidore Rigoutsos. "Towards a Reference Human Platelet Transcriptome: Evaluation Of Inter-Individual Correlations and Its Relationship With a Platelet Proteome". Blood 122, nr 21 (15.11.2013): 2297. http://dx.doi.org/10.1182/blood.v122.21.2297.2297.
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Abstract Next generation sequencing of RNA (RNA-seq) is an emerging technology that has so far been used successfully to profile the transcriptomes of several cell types and cell states. For the platelet transcriptome, RNA-seq descriptions exist for only a few subjects. Additionally, there have been no studies of the same individual’s transcriptome using two different technologies. As such, it has been unclear how well platelet transcriptomes correlate among different donors or across different RNA platforms, and what the transcriptomes’ relationship is with the platelet proteome. We generated RNA-seq profiles of the long RNA transcriptomes from the platelets of 10 healthy young males (5 white and 5 black). In addition to RNA-seq, we profiled the platelet messenger RNAs of the same 10 individuals using the Affymetrix GeneChip System. We observed that the abundance of platelet mRNA transcripts was highly correlated across the 10 individuals, a finding that was independent of race and of the employed technology. Additionally, our RNA-seq data showed that these high inter-individual correlations extend beyond mRNAs to several categories of non-coding RNAs. However, there was a notable exception: the category of pseudogenes exhibited a clear difference in expression by race. Comparison of our mRNA signatures with the only publicly available quantitative platelet proteome data showed that most (87.5%) identified platelet proteins had a detectable corresponding mRNA. Interestingly, there was also a high number of mRNAs that were present in the transcriptomes of all 10 individuals but had no representation in the proteome. Spearman correlation of the relative abundances for those platelet genes that were represented by both an mRNA and a protein, revealed an unexpectedly weak correlation between the transcriptome and the proteome. Further analysis of the overlapping and non-overlapping platelet mRNAs and proteins identified groups of genes with very distinct characteristics. Gene Ontology analysis of the respective gene identifiers revealed that the gene groups corresponded to distinct cellular processes, an interesting finding that provides novel insights for platelet biology. The very high inter-individual correlations of the transcriptome signatures across 10 different subjects representing two races together with the results of our analyses indicate that it is feasible to assemble a platelet mRNA-ome that can serve as a reference for future platelet transcriptomic studies of human health and disease. Disclosures: No relevant conflicts of interest to declare.
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Hu, S., Y. Li, J. Wang, Y. Xie, K. Tjon, L. Wolinsky, R. R. O. Loo, J. A. Loo i D. T. Wong. "Human Saliva Proteome and Transcriptome". Journal of Dental Research 85, nr 12 (grudzień 2006): 1129–33. http://dx.doi.org/10.1177/154405910608501212.
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This paper tests the hypothesis that salivary proteins and their counterpart mRNAs co-exist in human whole saliva. Global profiling of human saliva proteomes and transcriptomes by mass spectrometry (MS) and expression microarray technologies, respectively, revealed many similarities between saliva proteins and mRNAs. Of the function-known proteins identified in saliva, from 61 to 70% were also found present as mRNA transcripts. For genes not detected at both protein and mRNA levels, we made further efforts to determine if the counterpart is present. Of 19 selected genes detected only at the protein level, the mRNAs of 13 (68%) genes were found in saliva by RT-PCR. In contrast, of many mRNAs detected only by microarrays, their protein products were found in saliva, as reported previously by other investigators. The saliva transcriptome may provide preliminary insights into the boundary of the saliva proteome.
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An, Xiuli, Vincent P. Schulz, Jie Li, Kunlu Wu, Jing Liu, Fumin Xue, Jingping Hu, Narla Mohandas i Patrick G. Gallagher. "Global transcriptome analyses of human and murine terminal erythroid differentiation". Blood 123, nr 22 (29.05.2014): 3466–77. http://dx.doi.org/10.1182/blood-2014-01-548305.
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Key Points Transcriptome analyses of human and murine reveal significant stage and species-specific differences across stages of terminal erythroid differentiation. These transcriptomes provide a significant resource for understanding mechanisms of normal and perturbed erythropoiesis.
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Strausberg, R. L., i G. J. Riggins. "Navigating the human transcriptome". Proceedings of the National Academy of Sciences 98, nr 21 (9.10.2001): 11837–38. http://dx.doi.org/10.1073/pnas.221463598.
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Rusk, Nicole. "The human transient transcriptome". Nature Methods 13, nr 8 (28.07.2016): 612. http://dx.doi.org/10.1038/nmeth.3952.
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Đerke, Filip, i Niko Njirić. "Human transcriptome - molecular neurobiology". Gyrus 3, nr 3 (wrzesień 2015): 148–51. http://dx.doi.org/10.17486/gyr.3.1031.
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Goh, Sung-Ho, Matthew Josleyn, Y. Terry Lee, Robert L. Danner, Robert B. Gherman, Maggie C. Cam i Jeffery L. Miller. "The human reticulocyte transcriptome". Physiological Genomics 30, nr 2 (lipiec 2007): 172–78. http://dx.doi.org/10.1152/physiolgenomics.00247.2006.
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RNA from circulating blood reticulocytes was utilized to provide a robust description of genes transcribed at the final stages of erythroblast maturation. After depletion of leukocytes and platelets, Affymetrix HG-U133 arrays were hybridized with probe generated from the reticulocyte total RNA (blood obtained from 14 umbilical cords and 14 healthy adult humans). Among the cord and adult reticulocyte profiles, 698 probe sets (488 named genes) were detected in each of the 28 samples. Among the highly expressed genes, promoter analyses revealed a subset of transcription factor binding motifs encoded at higher than expected frequencies including the hypoxia-related arylhydrocarbon receptor repressor family. Over 100 probe sets demonstrated differential expression between the cord and adult reticulocyte samples. For verification, the array expression patterns for 21 genes were confirmed by real-time PCR (correlation coefficient 0.98). Only four transcripts (MAP17, FLJ32009 , ARRB2, and FLJ27365 ) were identified as being upregulated in the adult blood transcriptome. Further analysis revealed that the lipid-regulating protein MAP17 was present in the membrane fraction of adult erythrocytes, but not detected in cord blood erythrocytes. Combined with other clinical and experimental data, these reticulocyte transcriptome profiles should be useful to better understand the molecular bases of terminal erythroid differentiation, hemoglobin switching, iron metabolism and malarial pathogenesis.
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Mercer, Tim R., Shane Neph, Marcel E. Dinger, Joanna Crawford, Martin A. Smith, Anne-Marie J. Shearwood, Eric Haugen i in. "The Human Mitochondrial Transcriptome". Cell 146, nr 4 (sierpień 2011): 645–58. http://dx.doi.org/10.1016/j.cell.2011.06.051.
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Cheng, Xuanjin, Junran Yan, Yongxing Liu, Jiahe Wang i Stefan Taubert. "eVITTA: a web-based visualization and inference toolbox for transcriptome analysis". Nucleic Acids Research 49, W1 (21.05.2021): W207—W215. http://dx.doi.org/10.1093/nar/gkab366.
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Abstract Transcriptome profiling is essential for gene regulation studies in development and disease. Current web-based tools enable functional characterization of transcriptome data, but most are restricted to applying gene-list-based methods to single datasets, inefficient in leveraging up-to-date and species-specific information, and limited in their visualization options. Additionally, there is no systematic way to explore data stored in the largest transcriptome repository, NCBI GEO. To fill these gaps, we have developed eVITTA (easy Visualization and Inference Toolbox for Transcriptome Analysis; https://tau.cmmt.ubc.ca/eVITTA/). eVITTA provides modules for analysis and exploration of studies published in NCBI GEO (easyGEO), detailed molecular- and systems-level functional profiling (easyGSEA), and customizable comparisons among experimental groups (easyVizR). We tested eVITTA on transcriptomes of SARS-CoV-2 infected human nasopharyngeal swab samples, and identified a downregulation of olfactory signal transducers, in line with the clinical presentation of anosmia in COVID-19 patients. We also analyzed transcriptomes of Caenorhabditis elegans worms with disrupted S-adenosylmethionine metabolism, confirming activation of innate immune responses and feedback induction of one-carbon cycle genes. Collectively, eVITTA streamlines complex computational workflows into an accessible interface, thus filling the gap of an end-to-end platform capable of capturing both broad and granular changes in human and model organism transcriptomes.
Rozprawy doktorskie na temat "HUMAN TRANSCRIPTOME"
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Werne, Solnestam Beata. "Interpreting the human transcriptome". Doctoral thesis, KTH, Genteknologi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-158320.
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The human body is made of billions of cells and nearly all have the same genome. However, there is a high diversity of cells, resulted from what part of the genome the cells use, i.e. which RNA molecules are expressed. Rapid advances within the field of sequencing allow us to determine the RNA molecules expressed in a specific cell at a certain time. The use of the new technologies has expanded our view of the human transcriptome and increased our understanding of when, where, and how each RNA molecule is expressed. The work presented in this thesis focuses on analysis of the human transcriptome. In Paper I, we describe an automated approach for sample preparation. This protocol was compared with the standard manual protocol, and we demonstrated that the automated version outperformed the manual process in terms of sample throughput while maintaining high reproducibility. Paper II addresses the impact of nuclear transcripts on gene expression. We compared total RNA from whole cells and from cytoplasm, showing that transcripts with long, structured 3’- and 5’-untranslated regions and transcripts with long protein coding sequences tended to be retained in the nucleus. This resulted in increased complexity of the total RNA fraction and fewer reads per unique transcript. Papers III and IV describe dynamics of the human muscle transcriptome. For Paper III, we systematically investigated the transcriptome and found remarkably high tissue homogeneity, however a large number of genes and isoforms were differentially expressed between genders. Paper IV describes transcriptome differences in response to repeated training. No transcriptome-based memory was observed, however a large number of isoforms and genes were affected by training. Paper V describes a transcript profiling protocol based on the method Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification. We designed the method for a few selected transcripts whose expression patterns are important for detecting breast cancer cells, and optimized the method for single cell analysis. We successfully detected cells in human blood samples and applied the method to single cells, confirming the heterogeneity of a cell population.Människokroppen är uppbyggd av miljarder celler och nästan alla innehåller samma arvsmassa. Trots detta finns det många olika celler med olika funktioner vilket är en följd av vilken del av arvsmassan som cellerna använder, dvs vilka RNA-molekyler som finns i varje cell. Den snabba utvecklingen av sekvenseringstekniker har gjort det möjligt att studera när, var och hur varje RNA-molekyl är uttryckt och att få en djupare förståelse för hur människans celler fungerar. Arbetet som presenteras i denna avhandling fokuserar på analys av RNA-molekyler i människans celler. I artikel I beskriver vi en automatiserad metod för att förbereda cellprov för RNA-sekvensering. Det automatiserade protokollet jämfördes med det manuella protokollet, och vi visade att det automatiserade protokollet överträffade det manuella när det gällde provkapacitet samtidigt som en höga reproducerbarheten behölls. I artikel II undersökte vi effekterna som RNA-molekyler från en del av cellen (cellkärnan) har på den totala mängden uttryckta RNA-molekyler. Vi jämförde RNA från hela cellen och från en del av cellen (cytoplasman) och visade att RNA-molekyler med långa och strukturerade 3'- och 5'-otranslaterade regioner och RNA-molekyler med långa proteinkodande sekvenser tenderade att hållas kvar i cellkärnan till en högre grad. Detta resulterade i en ökad komplexitet av RNA-molekylerna i hela cellen, medan vi i cytoplasma-fraktionen lättare kunde hitta de korta och svagt uttryckta RNA-molekyler. I Artikel III och IV studerar vi RNA-molekyler i människans skelettmuskler. I artikel III visar vi att andelen RNA-molekyler uttryckta i skelettmuskler är väldigt lika mellan muskler och mellan olika personer, men att ett stort antal RNA-molekyler var uttryckta i olika nivåer hos kvinnor och män. Artikel IV beskriver RNA-nivåer som svar på upprepade perioder av uthållighetsträning. Artikel V beskriver en metod för att studera ett fåtal utvalda RNA-molekyler. Vi valde RNA-molekyler vars uttryck är viktigt vid analys av bröstcancerceller, och optimerade metoden för analys av enskilda celler. Vi analyserade cancerceller från blodprov och använde metoden för att titta på RNA-nivåer i enskilda celler från en grupp av celler och visade på skillnader i RNA-nivåer inom gruppen.
QC 20150115
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Natarajan, Sripriya 1978. "Defining the human endothelial transcriptome". Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/33082.
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Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2005.Includes bibliographical references (leaves 91-100).
Advances in microarray technology facilitate the study of biological systems at a genome-wide level. Meaningful analysis of these transcriptional profiling studies, however, demands the concomitant development of novel computational techniques that take into account the size and complexity of the data. We have devised statistical algorithms that use replicate microarrays to define a genome-wide expression profile of a given cell type and to determine a list of genes that are significantly differentially expressed between experimental conditions. Applying these algorithms to the study of cultured human umbilical vein endothelial cells (HUVEC), we have found approximately 54% of all genes to be expressed at a detectable level in HUVEC under basal conditions. The set of highest expressed genes is enriched in nucleic acid binding proteins, cytoskeletal proteins and isomerases as well as certain known markers of endothelium, and the complete list of genes can be found at ... We have also studied the effect of a 4-hour exposure of HUVEC to 10 U/mL of IL-1, and detected 491 upregulated and 259 downregulated statistically significant genes, including several chemokines and cytokines, as well as members of the TNFAIP3 family, the KLFfamily and the Notch pathway. Applying these rigorous statistical techniques to genome-wide expression datasets underscores known patterns of endothelial inflammatory gene regulation and unveils new pathways as well.
(cont.) Finally, we performed a direct comparison of direct-labeled microarrays with amplified RNA microarrays for an initial assessment of the effect of the additional noise of amplification on the outputs of the statistical algorithms. These techniques can be applied to additional genome-wide profiling studies of endothelium and other cell types to refine our understanding of transcriptomes and the gene regulatory network governing cellular function and pathophysiology.
by Sripriya Natarajan.
S.M.
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Oldham, Michael Clark. "Transcriptome organization in human and chimpanzee brains". Diss., Restricted to subscribing institutions, 2009. http://proquest.umi.com/pqdweb?did=1872073991&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.
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Wetterbom, Anna. "Genome and Transcriptome Comparisons between Human and Chimpanzee". Doctoral thesis, Uppsala universitet, Genomik, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-112893.
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The chimpanzee is humankind’s closest living relative and the two species diverged ~6 million years ago. Comparative studies of the human and chimpanzee genomes and transcriptomes are of great interest to understand the molecular mechanisms of speciation and the development of species-specific traits. The aim of this thesis is to characterize differences between the two species with regard to their genome sequences and the resulting transcript profiles. The first two papers focus on indel divergence and in particular, indels causing premature termination codons (PTCs) in 8% of the chimpanzee genes. The density of PTC genes is correlated with both the distance to the telomere and the indel divergence. Many PTC genes have several associated transcripts and since not all are affected by the PTC we propose that PTCs may affect the pattern of expressed isoforms. In the third paper, we investigate the transcriptome divergence in cerebellum, heart and liver, using high-density exon arrays. The results show that gene expression differs more between tissues than between species. Approximately 15% of the genes are differentially expressed between species, and half of the genes show different splicing patterns. We identify 28 cassette exons which are only included in one of the species, often in a tissue-specific manner. In the fourth paper, we use massive parallel sequencing to study the chimpanzee transcriptome in frontal cortex and liver. We estimate gene expression and search for novel transcribed regions (TRs). The majority of TRs are located close to genes and possibly extend the annotations. A subset of TRs are not found in the human genome. The brain transcriptome differs substantially from that of the liver and we identify a subset of genes enriched with TRs in frontal cortex. In conclusion, this thesis provides evidence of extensive genomic and transcriptomic variability between human and chimpanzee. The findings provide a basis for further studies of the underlying differences affecting phenotypic divergence between human and chimpanzee.
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Symes, A. J. "Epithelial specific transcriptome map of the human prostate". Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1302555/.
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The prostate has a zonal anatomy, with differing susceptibilities to disease (benign prostatic hyperplasia originates from the transition zone, prostate cancer largely arises in the peripheral zone). The molecular reasons for this are not understood. Previous prostate cancer microarray studies have used whole benign, diseased or tissue adjacent to the carcinoma as normal controls, for what is an epithelial disease. This study provides a gene expression profile of normal, non-diseased prostate, or a ‘reference prostate gene expression profile’. This has been compared to prostate cancer to identify novel biomarkers of disease. This study also investigates zonal differences in gene expression between different anatomical zones of the prostate. I used normal, human donor prostate tissue, laser capture microdissection (LCM), and Affymetrix gene expression arrays to achieve these aims. Eight LCM prostate epithelial samples from 3 donor prostates were used. The gene expression data was validated by low density real-time PCR and immunohistochemistry on a prostate tissue microarray. Major differences in gene expression were discovered between whole tissue and LCM epithelium only prostate using homology tables. Novel prostate adenocarcinoma genes were identified using a publicly available LCM prostate cancer gene expression array dataset. 9318 genes showed significant differential expression in normal vs. cancer datasets. Three targets, MCM2, NR1D1 and ABCA1 were validated at the protein level. Expression of NR1D1 and ABCA1 were increased in cancer, suggesting they are novel epithelial biomarkers of prostate cancer. An analysis of zonal differences in gene expression found significant differences between zones. Zonal specific markers included TGM4 (central zone), LPL (peripheral zone), and COL9A1 (transition zone). This study provides: (i) a gene expression profile of the normal prostate epithelium (ii) novel, prostate adenocarcinoma specific gene and protein markers and (iii) the first gene expression profile of normal epithelium on the basis of zonal anatomy of the prostate.
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Corral, Vázquez Celia. "Human sperm transcriptome: characterization, biological relevance, and biomarker functionality". Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/669365.
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Se ha demostrado que la contribución del espermatozoide al embrión va más allá de la transmisión del genoma paterno. Diversos estudios han mostrado que el espermatozoide humano contiene una compleja población de RNAs implicados en funciones relacionadas con la fertilidad. Por tanto, la visión de estas moléculas como meros restos de eventos previos ha quedado atrás. Este nuevo paradigma abre las puertas a nuevas aplicaciones del RNA en el ámbito de los biomarcadores de fertilidad. Sin embargo, los análisis transcriptómicos en espermatozoides presentan diversas limitaciones debidas a la heterogeneidad y delicada naturaleza de estas moléculas, además de la poca cantidad de RNA contenida en dichas células.
En este contexto, el objetivo de esta Tesis Doctoral es caracterizar el transcriptoma del espermatozoide humano y establecer las bases para desarrollar nuevos biomarcadores de fertilidad masculina. Dentro de este objetivo, se plantearon las siguientes metas: 1) optimizar metodologías específicas para analizar el RNA espermático mediante qRT-PCR y RNA-seq; 2) proporcionar un perfil integrado y una caracterización funcional de los mRNAs y lncRNAs espermáticos mediante RNA-seq; y 3) establecer nuevos biomarcadores de fertilidad a partir de la carga transcriptómica del espermatozoide.
Con este propósito, se adaptaron tanto el protocolo experimental como el análisis de datos a las limitaciones propias del RNA espermático y a la tecnología transcriptómica usada. Por tanto, se implementaron métodos de eliminación de células no espermáticas de las muestras seminales, así como controles de calidad para asegurar la ausencia de DNA y RNA no espermático. Además, se usó un método basado en solventes orgánicos para los estudios qRT-PCR, y kits de solventes no orgánicos para RNA-seq.
Los datos obtenidos se normalizaron usando métodos específicos de la técnica empleada. En concreto, para la normalización de los datos de expresión de miRNAs espermáticos en estudios singleplex qRT-PCR era necesario establecer miRNAs normalizadores. Esto se consiguió comparando los resultados derivados de unos datos que se normalizaron mediante: i) el método Mean-Centering Restricted (MCR); y ii) el nivel de expresión de diferentes miRNAs. Los miRNAs hsa-miR-100-5p y hsa-miR-30a-5p mostraron una expresión estable y ubicua, y su uso derivó en resultados con una calidad semejante a los conseguidos mediante la normalización por MCR. Por tanto, se sugirió esta combinación de miRNAs como la mejor opción para la normalización de futuros estudios singleplex qRT-PCR de miRNAs espermáticos.
Por otro lado, se empleó RNA-seq, para caracterizar el transcriptoma espermático de individuos fértiles. Los resultados revelaron una red de mRNAs y lncRNAs en alto estado de fragmentación, pero que contenían un grupo de transcritos ubicuos. Los análisis de Ontología Génica de todos los mRNAs expresados mostraron una implicación en procesos de espermatogénesis y reproducción, la cual era más significativa en los análisis de los mRNAs altamente expresados, ubicuos y altamente estables. Aparte, los potenciales genes dianas en cis de los lncRNAs mostraron relación con procesos de desarrollo embrionario y adhesión celular, la cual prevalecía en los genes dianas que no estaban expresados en espermatozoides.
Finalmente, el hecho de hallar transcritos ubicuos y de expresión correlacionada indicó un posible uso de estas moléculas como biomarcadores de fertilidad. Por tanto, se evaluó y se validó la presencia de pares de miRNAs espermáticos con una expresión correlacionada en individuos fértiles y no correlacionada en pacientes infértiles de diferentes etiologías (astenozoospermia, teratozoospermia, oligozoospermia e infertilidad inexplicable [UMI]). El par hsa-miR-942-5p/hsa-miR-1208 permitió clasificar correctamente el 85.71% de los casos de infertilidad, alcanzando el mayor potencial de diagnóstico de pacientes con alteraciones seminales. El par hsa-miR-34b-3p/hsa-miR-93-3p destacó por su potencial para discernir pacientes UMI. Aparte, varios pares de mRNAs y lncRNAs también mostraron expresiones correlacionadas en individuos fértiles, constituyendo unos candidatos potenciales para futuros estudios.The biological relevance of sperm contribution to the embryo has been shown to go beyond a mere transmission of the paternal genome. Several findings revealed that human spermatozoa carry a complex population of coding and non-coding RNAs with potential implications in multiple fertility-related pathways. Accordingly, the consideration of these molecules as simple residual pools of earlier processes has been left behind. This new paradigm also opens the possibility for potential applications in the field of male fertility biomarkers. However, sperm transcriptomic analysis has several limitations due to the heterogeneity and delicate nature of these molecules, besides the small amount of RNA contained in spermatozoa. In this context, the objective of this Doctoral Thesis is to characterize the human sperm transcriptome to set up the basis for developing new biomarkers of male fertility. Within this goal, the following aims were undertaken: 1) To optimize specific methodologies of sperm RNA analysis using qRT-PCR and RNA-seq strategies; 2) To provide an integrative profiling and functional characterization of sperm mRNAs and lncRNAs by RNA-seq technologies; and 3) To establish new fertility biomarkers among the transcriptomic cargo of the human spermatozoa. For this purpose, the experimental protocols and data analysis were adapted to the inherent limitations of sperm RNA and to the used transcriptomic technology. Therefore, methods for the elimination of non-sperm cells from semen samples were implemented, together with strict quality controls for ensuring the absence of DNA and non-sperm RNA. Besides, an organic solvent-based method was used for qRT-PCR studies, and non-organic solvent kits were employed for RNA-seq. The obtained data were normalized by specific methods depending on the used technique. In particular, the normalization of sperm miRNA qRT-PCR singleplex studies required the determination of a suitable set of normalizing miRNAs molecules. This was achieved by comparing the results derived from a sperm miRNA expression dataset normalized by: i) the reference Mean Centering Restricted (MCR) method; and ii) the expression level of different miRNAs. The miRNAs hsa-miR-100-5p and hsa-miR-30a-5p showed ubiquitous and stable expressions, and data normalized by their mean expression led to results with an appropriate quality when compared to MCR. Therefore, this miRNA combination was suggested as the most suitable choice for data normalization in further sperm singleplex studies. RNA-seq analysis was used to characterize the sperm transcriptome cargo of fertile individuals. Results revealed a complex network of mRNAs and lncRNAs with a high fragmentation status, but containing a host of ubiquitous transcripts. Gene ontology analyses of the whole set of expressed mRNAs showed an enrichment of spermatogenesis and reproduction processes, which was more significant in the sets of highly expressed, ubiquitous, and highly stable mRNAs. Additionally, the functional profiling of potential cis-target genes of the observed lncRNAs showed a significant involvement in embryo development and cell adhesion. This implication became more evident in those cis-target genes that were not present among the sperm mRNA cargo. Finally, the detection of ubiquitous transcripts and pairs of RNAs with correlated expressions suggested a potential use of these molecules as fertility biomarkers. Accordingly, the presence of sperm miRNA pairs with a correlated expression in fertile individuals that was disrupted in infertile patients of different ethiologies (asthenozoospermia, teratozoospermia, oligozoospermia, and Unexplained Male Infertility or UMI) was evaluated and validated by qRT-PCR. The hsa-miR-942-5p/hsa-miR-1208 pair allowed correctly classifying the 85.71% of infertile individuals, thus achieving the highest potential for discerning infertility cases with seminal alterations. Additionally, the pair hsa-miR-34b-3p/hsa-miR-93-3p was highlighted due to its high potential for discerning UMI patients. Besides, several pairs of ubiquitous lncRNAs and mRNAs were also observed to display a correlated expression in fertile individuals, becoming potential candidates for further biomarker studies.
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Khuder, Basil. "Human Genome and Transcriptome Analysis with Next-Generation Sequencing". University of Toledo Health Science Campus / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=mco1501886695490104.
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Chen, Jenny (Jennifer). "Evolutionary signatures for unearthing functional elements in the human transcriptome". Thesis, Massachusetts Institute of Technology, 2018. http://hdl.handle.net/1721.1/117792.
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Thesis: Ph. D. in Bioinformatics and Integrative Genomics, Harvard-MIT Program in Health Sciences and Technology, 2018.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged student-submitted from PDF version of thesis.
Includes bibliographical references (pages 141-156).
Comparative genomics is a powerful method for identifying functional genetic elements by their evolutionary patterns across species. However, current studies largely focus on analysis of genome sequences. The recent development of RNA-sequencing reveals dimensions of regulatory information previously inaccessible to us by sequence alone. The comparison of RNA-sequencing data across mammals has great potential for addressing two open problems in biology: identifying the regulatory mechanisms crucial to mammalian physiology, and deciphering how gene regulation contributes to the diversity of mammalian phenotypes. For my thesis, I developed two methodologies for interrogating comparative transcriptomic data for biological inference. First, I developed a framework for quantifying the evolutionary forces acting on gene expression and inferring evolutionarily optimal expression levels. I demonstrate how to use this framework to identify expression pathways underlying conserved, adaptive, and disease states of mammalian biology. Second, I developed novel metrics of transcriptional evolution to evaluate the conservation of long noncoding RNAs. These metrics further reveal that long noncoding RNAs harbor distinct evolutionary signatures, suggesting that they are not a homogenous class of molecules but rather a mixture of multiple functional classes with distinct biological roles. My thesis work provides fundamental quantitative tools for asking biological questions about transcriptome evolution. These tools provide a pivotal framework for interpreting transcriptional data across species and pave the way for deciphering the regulatory changes that lead to mammalian phenotypic variation.
by Jenny Chen.
Ph. D. in Bioinformatics and Integrative Genomics
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Xu, Guorong. "Computational Pipeline for Human Transcriptome Quantification Using RNA-seq Data". ScholarWorks@UNO, 2011. http://scholarworks.uno.edu/td/343.
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The main theme of this thesis research is concerned with developing a computational pipeline for processing Next-generation RNA sequencing (RNA-seq) data. RNA-seq experiments generate tens of millions of short reads for each DNA/RNA sample. The alignment of a large volume of short reads to a reference genome is a key step in NGS data analysis. Although storing alignment information in the Sequence Alignment/Map (SAM) or Binary SAM (BAM) format is now standard, biomedical researchers still have difficulty accessing useful information. In order to assist biomedical researchers to conveniently access essential information from NGS data files in SAM/BAM format, we have developed a Graphical User Interface (GUI) software tool named SAMMate to pipeline human transcriptome quantification. SAMMate allows researchers to easily process NGS data files in SAM/BAM format and is compatible with both single-end and paired-end sequencing technologies. It also allows researchers to accurately calculate gene expression abundance scores.
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Sherwood, Karen. "Preparation, characterisation and transcriptome analysis of RNA from human vCJD brains". Thesis, University of Edinburgh, 2008. http://hdl.handle.net/1842/4226.
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The pathological mechanisms of variant Creutzfeldt-Jakob disease (vCJD) in the human brain remain poorly understood. Gene expression data may provide insight into the molecular mechanisms involved. This requires analysis of human postmortem brain tissue however; the variability in RNA preparations from human brain material is a concern. A method for the isolation of RNA from vCJD brains which minimized infectivity and reduced Proteinase K resistant prion protein levels to undetectable by biochemical assay was developed. RNA preparations were made from sample of the frontal parasagital cortex, sub-frontal cortex and cerebellum of 78 human autopsy cases; 21 vCJD, 26 other neurological disease (OND) and 31 nonneurological disease (NND). Suitable RNA metrics for these human brain RNA preparations were evaluated and the intra- and inter-case variability of RNA preparations was determined. There was marked intra- and inter-case variability in RNA integrity number (RIN), A260:280 absorbance ratio and RNA yield. In particular, RIN and A260:280 showed little variation intra-patient, although RNA yield was more variable. The effects of postmortem interval, tissue pH, age at death, gender, freeze-thaw cycles (including storage method and temperature) and agonal state were investigated; none of these parameters correlated with the marked variability observed. Parameters for matching vCJD and OND/NND cases were considered and RNA from three age and gender matched comparison groups, each containing one OND, one NND and one vCJD case, were used for gene expression analysis. Data was generated using Superarray GEArray® Focused DNA Microarray and analysed using the GEArray Expression Analysis Suite and Significance Analysis of Microarray software. A comparison between matched vCJD and NND control cases identified 26 up-regulated and 16 down-regulated genes, showing >1.5-fold change with a false discovery rate of 9%. The modulated genes were involved in cell signaling, cell death, cholesterol and lipid metabolism. Involvement of these pathways is consistent with findings in other transmissible spongiform encephalopathy studies.
Książki na temat "HUMAN TRANSCRIPTOME"
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Einson, Jonah. Common and rare genetic effects on the transcriptome and their contribution to human traits. [New York, N.Y.?]: [publisher not identified], 2022.
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LeGrice, Stuart, i Matthias Gotte, red. Human Immunodeficiency Virus Reverse Transcriptase. New York, NY: Springer New York, 2013. http://dx.doi.org/10.1007/978-1-4614-7291-9.
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Genome transcriptome and proteome analysis. Chichester: Wiley, 2004.
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Bernot, Alain. Genome Transcriptome and Proteome Analysis. New York: John Wiley & Sons, Ltd., 2005.
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Ho, Charmaine. Development of human immunodeficiency virus type-1 reverse transcriptase activity assays. Ottawa: National Library of Canada, 1993.
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Schadt, Eric E. Network Methods for Elucidating the Complexity of Common Human Diseases. Redaktorzy Dennis S. Charney, Eric J. Nestler, Pamela Sklar i Joseph D. Buxbaum. Oxford University Press, 2017. http://dx.doi.org/10.1093/med/9780190681425.003.0002.
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The life sciences are now a significant contributor to the ever expanding digital universe of data, and stand poised to lead in both the generation of big data and the realization of dramatic benefit from it. We can now score variations in DNA across whole genomes; RNA levels and alternative isoforms, metabolite levels, protein levels, and protein state information across the transcriptome, metabolome and proteome; methylation status across the methylome; and construct extensive protein–protein and protein–DNA interaction maps, all in a comprehensive fashion and at the scale of populations of individuals. This chapter describes a number of analytical approaches aimed at inferring causal relationships among variables in very large-scale datasets by leveraging DNA variation as a systematic perturbation source. The causal inference procedures are also demonstrated to enhance the ability to reconstruct truly predictive, probabilistic causal gene networks that reflect the biological processes underlying complex phenotypes like disease.
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Bittner, Edward A., i Shawn P. Fagan. The host response to trauma and burns in the critically ill. Oxford University Press, 2016. http://dx.doi.org/10.1093/med/9780199600830.003.0304.
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Following severe traumatic injury, patients enter a state of immune dysregulation consisting of both exaggerated inflammation and immune suppression. Traditionally, the host response has been viewed as an early systemic inflammatory response syndrome (SIRS) followed temporally by a compensatory anti-inflammatory or immune-suppressive response syndrome (CARS). While this paradigm has been widely accepted across both medical and scientific fields, recent advances have challenged this concept. The Glue grant investigators recently characterized both the initial inflammatory response to injury and the dynamic evolving recovery process. They found: (1) severe injury produces a rapid (< 12 hours) genomic reprioritization in which 80% of the leukocyte transcriptome is altered; (2) similarities in gene expression patterns between different injuries reveal an apparently fundamental response to severe inflammatory stress, which is far more common than different; (3) alterations in the expression of classical inflammatory and anti-inflammatory as well as adaptive immunity genes occur simultaneously, not sequentially after severe injury; (4) the temporal nature of the current SIRS/CARS paradigm is not supported at the level of the leukocyte transcriptome. Complications are not associated with genomic evidence of a ‘second hit’ and differ only in the magnitude and duration of this genomic reprioritization. Furthermore, the delayed clinical recovery with organ injury is not associated with dramatic qualitative differences in the leukocyte transcriptome. Finally, poor correlation between human and rodent inflammatory genomic responses will alter how the host response is studied in the future.
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Gotte, Matthias, i Stuart LeGrice. Human Immunodeficiency Virus Reverse Transcriptase. Springer, 2015.
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Gotte, Matthias, i Stuart LeGrice. Human Immunodeficiency Virus Reverse Transcriptase. Springer, 2013.
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Gotte, Matthias, i Stuart LeGrice. Human Immunodeficiency Virus Reverse Transcriptase. Springer London, Limited, 2013.
Znajdź pełny tekst źródłaCzęści książek na temat "HUMAN TRANSCRIPTOME"
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Peres, Nalu T. A., Gabriela F. Persinoti, Elza A. S. Lang, Antonio Rossi i Nilce M. Martinez-Rossi. "Transcriptome in Human Mycoses". W Transcriptomics in Health and Disease, 227–63. Cham: Springer International Publishing, 2014. http://dx.doi.org/10.1007/978-3-319-11985-4_13.
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Peres, Nalu T. A., Tamires A. Bitencourt, Gabriela F. Persinoti, Elza A. S. Lang, Antonio Rossi i Nilce M. Martinez-Rossi. "Transcriptome in Human Mycoses". W Transcriptomics in Health and Disease, 395–435. Cham: Springer International Publishing, 2022. http://dx.doi.org/10.1007/978-3-030-87821-4_17.
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Moreira-Filho, Carlos Alberto, Silvia Yumi Bando, Fernanda Bernardi Bertonha i Magda Carneiro-Sampaio. "Functional Genomics of the Infant Human Thymus: AIRE and Minipuberty". W Thymus Transcriptome and Cell Biology, 235–45. Cham: Springer International Publishing, 2019. http://dx.doi.org/10.1007/978-3-030-12040-5_10.
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Okubo, Kousaku, i Teruyoshi Hishiki. "Knowledge Discovery from the Human Transcriptome". W Introduction to Bioinformatics, 693–710. Totowa, NJ: Humana Press, 2003. http://dx.doi.org/10.1007/978-1-59259-335-4_36.
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Marinotti, Osvaldo, i Anthony A. James. "The Transcriptome of Human Malaria Vectors". W Molecular Approaches to Malaria, 516–30. Washington, DC, USA: ASM Press, 2014. http://dx.doi.org/10.1128/9781555817558.ch27.
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Russo, Jose, i Irma H. Russo. "Methodological Approach for Studying the Human Breast". W Role of the Transcriptome in Breast Cancer Prevention, 243–68. Boston, MA: Springer US, 2012. http://dx.doi.org/10.1007/978-1-4614-4884-6_5.
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Russo, Jose, i Irma H. Russo. "The Role of Spliceosome in the Human Breast". W Role of the Transcriptome in Breast Cancer Prevention, 337–90. Boston, MA: Springer US, 2012. http://dx.doi.org/10.1007/978-1-4614-4884-6_8.
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Osada, Naoki. "An Overview of Transcriptome Studies in Non-Human Primates". W Post-Genome Biology of Primates, 9–22. Tokyo: Springer Tokyo, 2011. http://dx.doi.org/10.1007/978-4-431-54011-3_2.
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Bombonato-Prado, Karina F., Adalberto L. Rosa, Paulo T. Oliveira, Janaína A. Dernowsek, Vanessa Fontana, Adriane F. Evangelista i Geraldo A. Passos. "Transcriptome Analysis During Normal Human Mesenchymal Stem Cell Differentiation". W Transcriptomics in Health and Disease, 109–19. Cham: Springer International Publishing, 2014. http://dx.doi.org/10.1007/978-3-319-11985-4_6.
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Ouedraogo, Wend Yam Donald Davy, i Aida Ouangraoua. "Inferring Clusters of Orthologous and Paralogous Transcripts". W Comparative Genomics, 19–34. Cham: Springer Nature Switzerland, 2023. http://dx.doi.org/10.1007/978-3-031-36911-7_2.
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AbstractThe alternative processing of eukaryote genes allows producing multiple distinct transcripts from a single gene, thereby contributing to the transcriptome diversity. Recent studies suggest that more than 90% of human genes are concerned, and the transcripts resulting from alternative processing are highly conserved between orthologous genes.In this paper, we first present a model to define orthology and paralogy relationships at the transcriptome level, then we present an algorithm to infer clusters of orthologous and paralogous transcripts. Gene-level homology relationships are used to define different types of homology relationships between transcripts and a Reciprocal Best Hits approach is used to infer clusters of isoorthologous and recent paralogous transcripts.We applied the method to transcripts of gene families from the Ensembl-Compara database. The results are agreeing with those from previous studies comparing orthologous gene transcripts. The results also provide evidence that searching for conserved transcripts beyond orthologous genes will likely yield valuable information. The results obtained on the Ensembl-Compara gene families are available at https://github.com/UdeS-CoBIUS/TranscriptOrthology. Supplementary material can be found at https://doi.org/10.5281/zenodo.7750949.
Streszczenia konferencji na temat "HUMAN TRANSCRIPTOME"
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Combs, Joseph, Tracy Mandichak, Elizabeth Ferree i Aaron E. Hoffman. "Abstract 1798: The human mammary circadian transcriptome." W Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC. American Association for Cancer Research, 2013. http://dx.doi.org/10.1158/1538-7445.am2013-1798.
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Walters, Matthew S., Neil R. Hackett, Renat Shaykhiev, Rui Wang, Rachel K. Zwick, Jacqueline Salit i Ronald G. Crystal. "The Human Airway Epithelial Basal Cell Transcriptome". W American Thoracic Society 2011 International Conference, May 13-18, 2011 • Denver Colorado. American Thoracic Society, 2011. http://dx.doi.org/10.1164/ajrccm-conference.2011.183.1_meetingabstracts.a6019.
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Xu, Xiaoxiao, Arye Nehorai i Joseph Dougherty. "Cell type specific analysis of human transcriptome data". W 2012 IEEE International Workshop on Genomic Signal Processing and Statistics (GENSIPS). IEEE, 2012. http://dx.doi.org/10.1109/gensips.2012.6507737.
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Schamberger, Andrea, Fien Verhamme, Michael Lindner, Jürgen Behr i Oliver Eickelberg. "Transcriptome analysis of the human airway epithelium duringin vitrodifferentiation". W ERS International Congress 2016 abstracts. European Respiratory Society, 2016. http://dx.doi.org/10.1183/13993003.congress-2016.pa3993.
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Galatro, Thais Fernanda, Antonio M. Lerario, Sueli M. Oba-Shinjo, Bart J. Eggen i Suely K. Marie. "Abstract 2958: Transcriptome analysis of astrocytomaversusnon-neoplastic human microglia". W Proceedings: AACR Annual Meeting 2017; April 1-5, 2017; Washington, DC. American Association for Cancer Research, 2017. http://dx.doi.org/10.1158/1538-7445.am2017-2958.
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"Transcriptome of failing human heart reveals atrial myocytes reprogramming". W Bioinformatics of Genome Regulation and Structure/Systems Biology (BGRS/SB-2022) :. Institute of Cytology and Genetics, the Siberian Branch of the Russian Academy of Sciences, 2022. http://dx.doi.org/10.18699/sbb-2022-450.
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Deng, Nan, i Dongxiao Zhu. "RNA-Seq analyses to reveal the human transcriptome landscape". W BCB'13: ACM-BCB2013. New York, NY, USA: ACM, 2013. http://dx.doi.org/10.1145/2506583.2506603.
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Staudt, Michelle R., Jennifer A. Fuller, Rui Wang, Rachel K. Zwick, Jacqueline Salit, Neil R. Hackett i Ronald G. Crystal. "Whole Transcriptome Analysis Of Human Airway Epithelial Basal Cells". W American Thoracic Society 2010 International Conference, May 14-19, 2010 • New Orleans. American Thoracic Society, 2010. http://dx.doi.org/10.1164/ajrccm-conference.2010.181.1_meetingabstracts.a6402.
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Narozna, Beata, Wojciech Langwiński, Zuzanna Stachowiak, Ewelina Bukowska-Olech i Aleksandra Szczepankiewicz. "Changes in human airway cells transcriptome during epithelial wound repair". W ERS International Congress 2021 abstracts. European Respiratory Society, 2021. http://dx.doi.org/10.1183/13993003.congress-2021.pa3685.
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Schreiter, Thomas, RobertK Gieseler, Ramiro Vílchez-Vargas, Ruy Jauregui, Jan-Peter Sowa, Susanne Klein-Scory, Ruth Broering i in. "Age-related analysis of transcriptome-wide sequencing of human liver". W 38. Jahrestagung der Deutsche Arbeitsgemeinschaft zum Studium der Leber. Georg Thieme Verlag, 2022. http://dx.doi.org/10.1055/s-0041-1740685.
Pełny tekst źródłaRaporty organizacyjne na temat "HUMAN TRANSCRIPTOME"
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Libray, Spring. The Booming Field of Epitranscriptomics and its Role in Human Disease. Spring Library, kwiecień 2021. http://dx.doi.org/10.47496/sl.blog.26.
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Currently, the detection techniques used for transcriptome-wide identification of chemical modifications mainly depend on chemical and antibody-based detection methods followed by sequencing analysis.
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Miller, Gad, i Jeffrey F. Harper. Pollen fertility and the role of ROS and Ca signaling in heat stress tolerance. United States Department of Agriculture, styczeń 2013. http://dx.doi.org/10.32747/2013.7598150.bard.
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The long-term goal of this research is to understand how pollen cope with stress, and identify genes that can be manipulated in crop plants to improve reproductive success during heat stress. The specific aims were to: 1) Compare heat stress dependent changes in gene expression between wild type pollen, and mutants in which pollen are heat sensitive (cngc16) or heat tolerant (apx2-1). 2) Compare cngc16 and apx2 mutants for differences in heat-stress triggered changes in ROS, cNMP, and Ca²⁺ transients. 3) Expand a mutant screen for pollen with increased or decreased thermo-tolerance. These aims were designed to provide novel and fundamental advances to our understanding of stress tolerance in pollen reproductive development, and enable research aimed at improving crop plants to be more productive under conditions of heat stress. Background: Each year crop yields are severely impacted by a variety of stress conditions, including heat, cold, drought, hypoxia, and salt. Reproductive development in flowering plants is highly sensitive to hot or cold temperatures, with even a single hot day or cold night sometimes being fatal to reproductive success. In many plants, pollen tube development and fertilization is often the weakest link. Current speculation about global climate change is that most agricultural regions will experience more extreme environmental fluctuations. With the human food supply largely dependent on seeds, it is critical that we consider ways to improve stress tolerance during fertilization. The heat stress response (HSR) has been intensively studied in vegetative tissues, but is poorly understood during reproductive development. A general paradigm is that HS is accompanied by increased production of reactive oxygen species (ROS) and induction of ROS-scavenging enzymes to protect cells from excess oxidative damage. The activation of the HSR has been linked to cytosolic Ca²⁺ signals, and transcriptional and translational responses, including the increased expression of heat shock proteins (HSPs) and antioxidative pathways. The focus of the proposed research was on two mutations, which have been discovered in a collaboration between the Harper and Miller labs, that either increase or decrease reproductive stress tolerance in a model plant, Arabidopsis thaliana (i.e., cngc16--cyclic nucleotide gated channel 16, apx2-1--ascorbate peroxidase 2,). Major conclusions, solutions, achievements. Using RNA-seq technology, the expression profiles of cngc16 and apx2 pollen grains were independently compared to wild type under favourable conditions and following HS. In comparison to a wild type HSR, there were 2,776 differences in the transcriptome response in cngc16 pollen, consistent with a model in which this heat-sensitive mutant fails to enact or maintain a normal wild-type HSR. In a comparison with apx2 pollen, there were 900 differences in the HSR. Some portion of these 900 differences might contribute to an improved HSR in apx2 pollen. Twenty-seven and 42 transcription factor changes, in cngc16 and apx2-1, respectively, were identified that could provide unique contributions to a pollen HSR. While we found that the functional HS-dependent reprogramming of the pollen transcriptome requires specific activity of CNGC16, we identified in apx2 specific activation of flavonol-biosynthesis pathway and auxin signalling that support a role in pollen thermotolerance. Results from this study have identified metabolic pathways and candidate genes of potential use in improving HS tolerance in pollen. Additionally, we developed new FACS-based methodology that can quantify the stress response for individual pollen in a high-throughput fashion. This technology is being adapted for biological screening of crop plant’s pollen to identify novel thermotolerance traits. Implications, both scientific and agricultural. This study has provided a reference data on the pollen HSR from a model plant, and supports a model that the HSR in pollen has many differences compared to vegetative cells. This provides an important foundation for understanding and improving the pollen HSR, and therefor contributes to the long-term goal of improving productivity in crop plants subjected to temperature stress conditions. A specific hypothesis that has emerged from this study is that pollen thermotolerance can be improved by increasing flavonol accumulation before or during a stress response. Efforts to test this hypothesis have been initiated, and if successful have the potential for application with major seed crops such as maize and rice.
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Lers, Amnon, Majid R. Foolad i Haya Friedman. genetic basis for postharvest chilling tolerance in tomato fruit. United States Department of Agriculture, styczeń 2014. http://dx.doi.org/10.32747/2014.7600014.bard.
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ABSTRACT Postharvest losses of fresh produce are estimated globally to be around 30%. Reducing these losses is considered a major solution to ensure global food security. Storage at low temperatures is an efficient practice to prolong postharvest performance of crops with minimal negative impact on produce quality or human health and the environment. However, many fresh produce commodities are susceptible to chilling temperatures, and the application of cold storage is limited as it would cause physiological chilling injury (CI) leading to reduced produce quality. Further, the primary CI becomes a preferred site for pathogens leading to decay and massive produce losses. Thus, chilling sensitive crops should be stored at higher minimal temperatures, which curtails their marketing life and in some cases necessitates the use of other storage strategies. Development of new knowledge about the biological basis for chilling tolerance in fruits and vegetables should allow development of both new varieties more tolerant to cold, and more efficient postharvest storage treatments and storage conditions. In order to improve the agricultural performance of modern crop varieties, including tomato, there is great potential in introgression of marker-defined genomic regions from wild species onto the background of elite breeding lines. To exploit this potential for improving tomato fruit chilling tolerance during postharvest storage, we have used in this research a recombinant inbred line (RIL) population derived from a cross between the red-fruited tomato wild species SolanumpimpinellifoliumL. accession LA2093 and an advanced Solanum lycopersicumL. tomato breeding line NCEBR-1, developed in the laboratory of the US co-PI. The original specific objectives were: 1) Screening of RIL population resulting from the cross NCEBR1 X LA2093 for fruit chilling response during postharvest storage and estimation of its heritability; 2) Perform a transcriptopmic and bioinformatics analysis for the two parental lines following exposure to chilling storage. During the course of the project, we learned that we could measure greater differences in chilling responses among specific RILs compared to that observed between the two parental lines, and thus we decided not to perform transcriptomic analysis and instead invest our efforts more on characterization of the RILs. Performing the transcriptomic analysis for several RILs, which significantly differ in their chilling tolerance/sensitivity, at a later stage could result with more significant insights. The RIL population, (172 lines), was used in field experiment in which fruits were examined for chilling sensitivity by determining CI severity. Following the field experiments, including 4 harvest days and CI measurements, two extreme tails of the response distribution, each consisting of 11 RILs exhibiting either high sensitivity or tolerance to chilling stress, were identified and were further examined for chilling response in greenhouse experiments. Across the RILs, we found significant (P < 0.01) correlation between field and greenhouse grown plants in fruit CI. Two groups of 5 RILs, whose fruits exhibited reproducible chilling tolerant/sensitive phenotypes in both field and greenhouse experiments, were selected for further analyses. Numerous genetic, physiological, biochemical and molecular variations were investigated in response to postharvest chilling stress in the selected RILs. We confirmed the differential response of the parental lines of the RIL population to chilling stress, and examined the extent of variation in the RIL population in response to chilling treatment. We determined parameters which would be useful for further characterization of chilling response in the RIL population. These included chlorophyll fluorescence Fv/Fm, water loss, total non-enzymatic potential of antioxidant activity, ascorbate and proline content, and expression of LeCBF1 gene, known to be associated with cold acclimation. These parameters could be used in continuation studies for the identification and genetic mapping of loci contributing to chilling tolerance in this population, and identifying genetic markers associated with chilling tolerance in tomato. Once genetic markers associated with chilling tolerance are identified, the trait could be transferred to different genetic background via marker-assisted selection (MAS) and breeding. The collaborative research established in this program has resulted in new information and insights in this area of research and the collaboration will be continued to obtain further insights into the genetic, molecular biology and physiology of postharvest chilling tolerance in tomato fruit. The US Co-PI, developed the RIL population that was used for screening and measurement of the relevant chilling stress responses and conducted statistical analyses of the data. Because we were not able to grow the RIL population under field conditions in two successive generations, we could not estimate heritability of response to chilling temperatures. However, we plan to continue the research, grow the RIL progeny in the field again, and determine heritability of chilling tolerance in a near future. The IS and US investigators interacted regularly and plan to continue and expand on this study, since combing the expertise of the Co-PI in genetics and breeding with that of the PI in postharvest physiology and molecular biology will have great impact on this line of research, given the significant findings of this one-year feasibility project.
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Fridman, Eyal, i Eran Pichersky. Tomato Natural Insecticides: Elucidation of the Complex Pathway of Methylketone Biosynthesis. United States Department of Agriculture, grudzień 2009. http://dx.doi.org/10.32747/2009.7696543.bard.
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Plant species synthesize a multitude of specialized compounds 10 help ward off pests. and these in turn may well serve as an alternative to synthetic pesticides to reduce environmental damage and health risks to humans. The general goal of this research was to perform a genetic and biochemical dissection of the natural-insecticides methylketone pathway that is specific to the glandular trichomes of the wild species of tomato, Solanumhabrochaites f. glabratum (accession PI126449). Previous study conducted by us have demonstrated that these compounds are synthesized de novo as a derivate pathway of the fatty acid biosynthesis, and that a key enzyme. designated MethylketoneSynthase 1 (MKS 1). catalyzes conversion of the intermediate B-ketoacyl- ACPs to the corresponding Cn-1 methylketones. The approach taken in this proposed project was to use an interspecific F2 population. derived from the cross between the cultivated lV182 and the wild species PIl26449. for three objectives: (i) Analyze the association between allelic status of candidate genes from the fatty acid biosynthesis pathway with the methylketone content in the leaves (ii) Perform bulk segregant analysis of genetic markers along the tomato genome for identifying genomic regions that harbor QTLs for 2TD content (iii) Apply differential gene expression analysis using the isolated glands of bulk segregant for identifying new genes that are involved in the pathway. The genetic mapping in the interspecific F2 population included app. 60 genetic markers, including the candidate genes from the FAS pathway and SSR markers spread evenly across the genome. This initial; screening identified 5 loci associated with MK content including the candidate genes MKS1, ACC and MaCoA:ACP trans. Interesting observation in this genetic analysis was the connection between shape and content of the glands, i.e. the globularity of the four cells, typical to the wild species. was associated with increased MK in the segregating population. In the next step of the research transcriptomic analysis of trichomes from high- and 10w-MK plants was conducted. This analysis identified a new gene, Methy1ketone synthase 2 (MKS2), whose protein product share sequence similarity to the thioesterase super family of hot-dog enzymes. Genetic analysis in the segregating population confirmed its association with MK content, as well as its overexpression in E. coli that led to formation of MK in the media. There are several conclusions drawn from this research project: (i) the genetic control of MK accumulation in the trichomes is composed of biochemical components in the FAS pathway and its vicinity (MKS 1 and MKS2). as well as genetic factors that mediate the morphology of these specialized cells. (ii) the biochemical pathway is now realized different from what was hypothesized before with MKS2 working upstream to I\1KS 1 and serves as the interface between primary (fatty acids) and secondary (MK) metabolism. We are currently testing the possible physical interactions between these two proteins in vitro after the genetic analysis showed clear epistatic interactions. (iii) the regulation of the pathway that lead to specialized metabolism in the wild species is largely mediated by transcription and one of the achievements of this project is that we were able to isolate and verify the specificity of the MKS1 promoter to the trichomes which allows manipulation of the pathways in these cells (currently in progress). The scientific implications of this research project is the advancement in our knowledge of hitherto unknown biochemical pathway in plants and new leads for studying a new family in plants (hot dog thioesterase). The agricultural and biotechnological implication are : (i) generation of new genetic markers that could assist in importing this pathway to cultivated tomato hence enhancing its natural resistance to insecticides, (ii) the discovery of MKS2 adds a new gene for genetic engineering of plants for making new fatty acid derived compounds. This could be assisted with the use of the isolated and verified MKS1 promoter. The results of this research were summarized to a manuscript that was published in Plant Physiology (cover paper). to a chapter in a proceeding book. and one patent was submitted in the US.