Genomics and Transcriptomics

Techniques PCR (polymerase chain reaction) mass arrays (Sequenom.com) 

Biomolecular MS and in particular MALDI-TOF-MS (see Sections 2.1.22 and 2.2.1) permit the routine analysis of oligonucleotides up to 70-mers, intact nucleic acids, and the direct detection of DNA products with no primer labels with an increase in analysis speed and mass accuracy especially in contrast to traditional DNA separation techniques such as slab gels or capillary electrophoresis. Applications focus on the characterization of single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs). Precise and accurate gene expression measurements show relative and absolute numbers of target molecules determined independently of the number of PCR cycles. DNA methylation can be studied quantitatively. 

Comparative sequence analysis based on MALDI-TOF-MS analysis of nucleic acids cleaved at specific bases and reference sequences used to construct in silico cleavage patterns enable cross-correlation of theoretical and experimental mass signal patterns. Observed signal pattern differences are indicators of sequence variations and 

accurate SNP validation can be carried out using a sample-pooling technique—allele frequency—that rapidly screens and confirms the presence of an SNP and its allelic frequency in patient populations. Conventional technologies typically analyze each SNP in each individual of the population in question, and individual results are then consolidated to yield the overall SNP allele-frequency distribution. MS-based technology is able to determine SNP allele frequencies with high precision in pooled samples, thereby replacing hundreds of individual measurements with one consolidated analysis. To that end, DNA is isolated, purified, and quantitated. A pool of DNA is formed from a high number of different individuals, amplified, and mass measured. 

Study object Proteins, peptides, amino acids 

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Cellular activities are mediated by complex networks of interactions in response to physiological signals, and the nature of the response is dependent on the cell type and states. These aspects cannot be accounted for by investigating at the genomic or transcriptomic levels alone, as genomes and transcriptomes are fairly conserved between cell states and types within a system. 

HUMAN GENOME AND TRANSCRIPTOME FROM GENE LOCI TO GENE PRODUCTS... 

Proteogenomics refers to the integration of the proteomic data with the genomic and transcriptomic data with the goal of improving our understanding of complex living systems (100,101). The main idea behind this approach... 

Similarly, we have described how information related to protein-splicing variants, amino acid peptide variants (polymorphisms), and PTMs is being introduced in data analysis pipelines in order to increase the rate of identifiable peptides. The introduction of genomics and transcriptomics information, often neglected in routine proteomics analyses, will enable the characterization of myriad splicing variants and amino acid polymorphisms, as well as the development of robust proteomics analysis pipelines, which will contribute to elucidate their role in health and disease states. These tools will make it possible to uncover a new layer of the proteome complexity and to expand the information available from cellular systems and the potential implication of these protein variants in different physiological processes. Finally, several bioinformatics tools that perform unbiased PTM analysis have also been reviewed. The tools that are currently being developed and refined tackle... 

This chapter aims to review different combinations of data fusion. Genomics and transcriptomics currently reach total coverage for a sequenced organism. Not all gene function might be known, but at least predicted ORFs can be tested for expression. Currently, metabolomics is far away for complete metabolome coverage. Today numbers between 5% and 30% of known metabolites in a typical MS-based dataset are the normal case. The remaining... 

Lukes J, Hashimi H, Zikova A. Unexplained complexity of the mitochondrial genome and transcriptome in kinetoplastid flagellates. Curr Genet 2005 48(5) 277-99. 

The composition of the genome determines many biological processes in an organism, thereby influencing the susceptibility toward genetic diseases or the response to xenobiotic compounds." The transcriptome is the complete set of RNA products that can be produced from the genome, and transcriptomics is the study of the transcriptome. For example, microarrays and chips represent... 

Flu, S, Zheng, H, Gu, Y, Zhao, J. et al (2011) Comparative genomic and transcriptomic analysis revealed genetic characteristics related to solvent formation and xylose utilization in Clostridium acetobutylicum EA 2018. 

Table 7.1). The analysis of both microbial genome and transcriptome represents a rich molecular toolbox for the study of microbial community in food matrices. 

In contrast to the large quantity of genome and transcriptome information available for model plants on a variety of websites (A. thaliana (http //arabidopsis.org), O. sativa (http //rice. plantbiology.msu.edu http //rapdb.dna.affrc. go.jp) and others), genome information is not yet available for most medicinal plants. Conse-... 

Coupled with metabolite analyses of natural plant mutants and/or ecotypes deficient in particular compoimds as well as engineered plant lines silenced for particular P450s, these genomic and transcriptomic resources are providing details on the exceptionally large number of P450 transcripts expressed in different plant species, those co-regulated in branch pathways and metabolic interactions between primary and specialized compounds in individual species. 

Genomic and transcriptomic technologies have been used to rapidly identify biosynthetic steps. There are currently over 40,000 expressed enzyme tags (ESTs) generated fi om alkaloid-producing plants that have been used to isolate genes involved in the alkaloid pathway [7]. Some alkaloid biosynthetic steps occur as spontaneous chemical reactions without the use of enzymes, for example, conversion of the intermediate neopine into codeinone in the morphine biosynthetic pathway. Also, some enzymes may catalyze two or more separate reactions in the pathway, for example, hyoscyamine 6-hydroxylase, which carries out two consecutive steps in the scopolamine biosynthetic pathway. Alkaloid biosynthesis also involves compartmentalization. Tissue-specific localization studies have shown that sequential biosynthetic enzymes can occur in distinct cell types [8, 9]. During the biosynthesis of the indole alkaloids vinblastine and vincristine in Catharanthus roseus, different enzymatic steps are carried out in different cellular compartments (Fig. 8.5) [10]. Various steps in the pathway are carried out in different types of cell. This requires the intercellular transport of metabolic intermediates. Similarly, scopolamine biosynthesis also involves two different cell types. 

In this chapter, we will snmmarize the current status of plant genome and transcriptome projects, describe strategies and examples that exploit the resulting information in the context of plant natural product research, and give an overview of how proteome and metabolome analyses are being used to promote the field. 

Extensive characterization of an evolved ethanol-tolerant R coli strain through both genome and transcriptome analysis recently identified three mutations that counteract the detrimental effect of ethanol on transcription and translation machinery [144]. Specifically, Haft etal. identified mutations within a ribosomal protein RpsQ, methionine synthesis regulatory MetJ, and the transcription termination factor Rho that improved ethanol tolerance, though at the time of this writing the effect of these mutations on ethanol production has not yet been publically described. 

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