Recombinase-mediated cassette exchange (RMCE) system for functional genomics studies in Mycoplasma mycoides
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We have previously established technologies enabling us to engineer the Mycoplasma mycoides genome while cloned in the yeast Saccharomyces cerevisiae, followed by genome transplantation into Mycoplasma capricolum recipient cells to produce M. mycoides with an altered genome. To expand the toolbox for genomic modifications, we designed a strategy based on the Cre/loxP-based Recombinase-Mediated Cassette Exchange (RMCE) system for functional genomics analyses.In this paper, we demonstrated replacement of an approximately 100 kb DNA segment of the M. mycoides genome with a synthetic DNA counterpart in two orientations. The function of the altered genomes was then validated by genome transplantation and phenotypic characterization of the transplanted cells.This method offers an easy and efficient way to manipulate the M. mycoides genome and will be a valuable tool for functional genomic studies, such as genome organization and minimization.Keywords:
Mycoplasma mycoides
genomic DNA
Functional Genomics
Genome biology aims at using the complete genome sequences to reconstruct all metabolic and signaling pathways that could operate in the target organisms and identify the likely regulatory hubs and potential drug targets. Such analysis requires comprehensive functional annotation of all proteins encoded in each sequenced genome. Standard sequence analysis typically fails to provide (confident) functional assignment for at least a third of the genes even in the relatively small prokaryotic genomes. As a result, comparative genomics has to deal with the constantly growing numbers of proteins whose functions remain unknown. This talk will discuss using comparative genomics to improve our understanding of microbial metabolic and signaling pathways, including some recent examples of identification of missing enzymes and prediction of alternative enzyme variants. It will show that the number of truly enigmatic conserved hypothetical proteins is relatively small, particularly in the reduced genomes of pathogenic bacteria, which suggests that most of their cellular functions are already accounted for. In contrast, the number of uncharacterized genes in free-living organisms remains quite large and their functions remain obscure. Our current hypothesis is that many of these genes have “house-cleaning” function, which is almost as important as house-keeping, particularly for aerobic bacteria and for eukaryotic cells. We shall also briefly discuss how comparative genomics could be used for identification of priority targets for future research and the challenges in characterization of their functions
Comparative Genomics
Functional Genomics
Identification
Genome Biology
Computational genomics
Bacterial genome size
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Abstract Background Large-scale genome reduction has been performed to significantly improve the performance of microbial chassis. Identification of the essential or dispensable genes is pivotal for genome reduction to avoid synthetic lethality. Here, taking Streptomyces as an example, we developed a combinatorial strategy for systematic identification of large and dispensable genomic regions in Streptomyces based on multi-omics approaches. Results Phylogenetic tree analysis revealed that the model strains including S. coelicolor A3(2), S. albus J1074 and S. avermitilis MA-4680 were preferred reference for comparative analysis of candidate genomes. Multiple genome alignment suggested that the Streptomyces genomes embodied highly conserved core region and variable sub-telomeric regions, and may present symmetric or asymmetric structure. Pan-genome and functional genome analyses showed that most conserved genes responsible for the fundamental functions of cell viability were concentrated in the core region and the vast majority of abundant genes were dispersed in the sub-telomeric regions. These results suggested that large-scale deletion can be performed in sub-telomeric regions to greatly streamline the Streptomyces genomes for developing versatile chassis. Conclusions The integrative approach of comparative genomics, functional genomics and pan-genomics can not only be applied to perform a multi-tiered dissection for Streptomyces genomes, but also work as a universal method for systematic analysis of removable regions in other microbial hosts in order to generate more miscellaneous and versatile chassis with minimized genome for drug discovery.
Comparative Genomics
Functional Genomics
Streptomyces avermitilis
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We have previously established technologies enabling us to engineer the Mycoplasma mycoides genome while cloned in the yeast Saccharomyces cerevisiae, followed by genome transplantation into Mycoplasma capricolum recipient cells to produce M. mycoides with an altered genome. To expand the toolbox for genomic modifications, we designed a strategy based on the Cre/loxP-based Recombinase-Mediated Cassette Exchange (RMCE) system for functional genomics analyses.In this paper, we demonstrated replacement of an approximately 100 kb DNA segment of the M. mycoides genome with a synthetic DNA counterpart in two orientations. The function of the altered genomes was then validated by genome transplantation and phenotypic characterization of the transplanted cells.This method offers an easy and efficient way to manipulate the M. mycoides genome and will be a valuable tool for functional genomic studies, such as genome organization and minimization.
Mycoplasma mycoides
genomic DNA
Functional Genomics
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Comparative Genomics
Schistosoma
Functional Genomics
Sequence assembly
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Identifying functions for all gene products in all sequenced organisms is a central challenge of the post-genomic era. However, at least 30-50% of the proteins encoded by any given genome are of unknown or vaguely known function, and a large number are wrongly annotated. Many of these 'unknown' proteins are common to prokaryotes and plants. We set out to predict and experimentally test the functions of such proteins. Our approach to functional prediction integrates comparative genomics based mainly on microbial genomes with functional genomic data from model microorganisms and post-genomic data from plants. This approach bridges the gap between automated homology-based annotations and the classical gene discovery efforts of experimentalists, and is more powerful than purely computational approaches to identifying gene-function associations.Among Arabidopsis genes, we focused on those (2,325 in total) that (i) are unique or belong to families with no more than three members, (ii) occur in prokaryotes, and (iii) have unknown or poorly known functions. Computer-assisted selection of promising targets for deeper analysis was based on homology-independent characteristics associated in the SEED database with the prokaryotic members of each family. In-depth comparative genomic analysis was performed for 360 top candidate families. From this pool, 78 families were connected to general areas of metabolism and, of these families, specific functional predictions were made for 41. Twenty-one predicted functions have been experimentally tested or are currently under investigation by our group in at least one prokaryotic organism (nine of them have been validated, four invalidated, and eight are in progress). Ten additional predictions have been independently validated by other groups. Discovering the function of very widespread but hitherto enigmatic proteins such as the YrdC or YgfZ families illustrates the power of our approach.Our approach correctly predicted functions for 19 uncharacterized protein families from plants and prokaryotes; none of these functions had previously been correctly predicted by computational methods. The resulting annotations could be propagated with confidence to over six thousand homologous proteins encoded in over 900 bacterial, archaeal, and eukaryotic genomes currently available in public databases.
Prokaryote
Comparative Genomics
Homology
Functional Genomics
Gene prediction
Gene Annotation
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Functional genomics represents a systematic approach to elucidating the function of the novel genes revealed by complete genome sequences. Such an approach should adopt a hierarchical strategy since this will both limit the number of experiments to be performed and permit a closer and closer approximation to the function of any individual gene to be achieved. Moreover, hierarchical analyses have, in their early stages, tremendous integrative power and functional genomics aims at a comprehensive and integrative view of the workings of living cells. The first draft of the human genome sequence has just been produced, and the complete genome sequences of a number of eukaryotic human pathogens (including the parasitic protozoa Plasmodium, Leishmania, and Trypanosoma ) will soon be available. However, the most rapid progress in the elucidation of gene function will initially be made using model organisms. Yeast is an excellent eukaryotic model and at least 40% of single–gene determinants of human heritable diseases find homologues in yeast. We have adopted a systematic approach to the functional analysis of the Saccharomyces cerevisiae genome. A number of the approaches for the functional analysis of novel yeast genes are discussed. The different approaches are grouped into four domains: genome, transcriptome, proteome, and metabolome. The utility of genetic, biochemical, and physico–chemical methods for the analysis of these domains is discussed, and the importance of framing precise biological questions, when using these comprehensive analytical methods, is emphasized. Finally, the prospects for elucidating the function of protozoan genes by using the methods pioneered with yeast, and even exploiting Saccharomyces itself, as a surrogate, are explored.
Functional Genomics
Proteome
Comparative Genomics
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Comparative Genomics
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Genome biology aims at using the complete genome sequences to reconstruct all metabolic and signaling pathways that could operate in the target organisms and identify the likely regulatory hubs and potential drug targets. Such analysis requires comprehensive functional annotation of all proteins encoded in each sequenced genome. Standard sequence analysis typically fails to provide (confident) functional assignment for at least a third of the genes even in the relatively small prokaryotic genomes. As a result, comparative genomics has to deal with the constantly growing numbers of proteins whose functions remain unknown. This talk will discuss using comparative genomics to improve our understanding of microbial metabolic and signaling pathways, including some recent examples of identification of missing enzymes and prediction of alternative enzyme variants. It will show that the number of truly enigmatic conserved hypothetical proteins is relatively small, particularly in the reduced genomes of pathogenic bacteria, which suggests that most of their cellular functions are already accounted for. In contrast, the number of uncharacterized genes in free-living organisms remains quite large and their functions remain obscure. Our current hypothesis is that many of these genes have “house-cleaning” function, which is almost as important as house-keeping, particularly for aerobic bacteria and for eukaryotic cells. We shall also briefly discuss how comparative genomics could be used for identification of priority targets for future research and the challenges in characterization of their functions
Comparative Genomics
Identification
Functional Genomics
Genome Biology
Computational genomics
Cite
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Summary: The Human Genome Project is now almost completed, and we are about to move into the post-genome sequence era of functional genomics. The advent of genome science has markedly changed the way life science research including pharmacological study is conducted; thus, systematic and integrated 'genome-wide' survey is feasible. The stream of 'Genome→Transcriptome→ Proteomics' is logical and, in each aspect, approaches for functional genomics are now pursued at a high pace. We have recently developed a standardized technical platform (in various levels, such as transcription, cell and whole animal levels, etc.), and applied these techniques to the study of functional genomics of G-proteincoupled receptors, particularly α1-adrenoceptors as a model. Combining the genome information and technology, future pharmacological studies would become the genome-based search and research.
Functional Genomics
Genome Biology
Computational genomics
Pace
Personal genomics
Comparative Genomics
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Genomics is the study of the structure and function of the genome: the set of genetic information encoded in the DNA of the nucleus and organelles of an organism. It is a dynamic field that combines traditional paths of inquiry with new approaches that would have been impossible without recent technological developments. Much of the recent focus has been on obtaining the sequence of entire genomes, determining the order and organization of the genes, and developing libraries that provide immediate physical access to any desired DNA fragment. This has enabled functional studies on a genome-wide level, including analysis of the genetic basis of complex traits, quantification of global patterns of gene expression, and systematic gene disruption projects. The successful contribution of genomics to problems in applied entomology requires the cooperation of the private and public sectors to build upon the knowledge derived from the Drosophila genome and effectively develop models for other insect Orders.
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