We describe a method for generating gene replacements and deletions in Escherichia coli. The technique is simple and rapid and can be applied to most genes, even those that are essential. What makes this method unique and particularly effective is the use of a temperature-sensitive pSC101 replicon to facilitate the gene replacement. The method proceeds by homologous recombination between a gene on the chromosome and homologous sequences carried on a plasmid temperature sensitive for DNA replication. Thus, after transformation of the plasmid into an appropriate host, it is possible to select for integration of the plasmid into the chromosome at 44°C. Subsequent growth of these cointegrates at 30°C leads to a second recombination event, resulting in their resolution. Depending on where the second recombination event takes place, the chromosome will either have undergone a gene replacement or retain the original copy of the gene. The procedure can also be used to effect the transfer of an allele from a plasmid to the chromosome or to rescue a chromosomal allele onto a plasmid. Since the resolved plasmid can be maintained by selection, this technique can be used to generate deletions of essential genes.
The potential for genome-wide association studies to relate phenotypes to specific genetic variation is greatly increased when data can be combined or compared across multiple studies. To facilitate replication and validation across studies, RTI International (Research Triangle Park, North Carolina) and the National Human Genome Research Institute (Bethesda, Maryland) are collaborating on the consensus measures for Phenotypes and eXposures (PhenX) project. The goal of PhenX is to identify 15 high-priority, well-established, and broadly applicable measures for each of 21 research domains. PhenX measures are selected by working groups of domain experts using a consensus process that includes input from the scientific community. The selected measures are then made freely available to the scientific community via the PhenX Toolkit. Thus, the PhenX Toolkit provides the research community with a core set of high-quality, well-established, low-burden measures intended for use in large-scale genomic studies. PhenX measures will have the most impact when included at the experimental design stage. The PhenX Toolkit also includes links to standards and resources in an effort to facilitate data harmonization to legacy data. Broad acceptance and use of PhenX measures will promote cross-study comparisons to increase statistical power for identifying and replicating variants associated with complex diseases and with gene-gene and gene-environment interactions.
In conjunction with an enhanced system for Agrobacterium-mediated plant transformation, a new binary bacterial artificial chromosome (BIBAC) vector has been developed that is capable of transferring at least 150 kb of foreign DNA into a plant nuclear genome. The transferred DNA appears to be intact in the majority of transformed tobacco plants analyzed and is faithfully inherited in the progeny. The ability to introduce high molecular weight DNA into plant chromosomes should accelerate gene identification and genetic engineering of plants and may lead to new approaches in studies of genome organization.The ability to stably transfer foreign DNA into plant chromosomes is the foundation of plant genetic engineering. DNA transfer to plants has been accomplished by many methods, including Agrobacterium-mediated transformation, biolistic transformation, and microinjection (1, 2). However, no method for routinely introducing DNA fragments larger than about 25 kb into the plant nuclear genome has yet been demonstrated. Genes with related functions, such as disease resistance genes in plants, have been found in clusters (3). A reliable system for transforming large segments (>100 kb) of DNA into plants would make it feasible to introduce a natural gene cluster or a series of previously unlinked foreign genes into a single locus. Such a group of genes could provide resistance to several different pests or pathogens, or it could constitute an entirely new metabolic pathway for production of a novel biomolecule. The integrated "megalocus" would be inherited as a single Mendelian unit and could easily be incorporated into conventional plant breeding programs. Large insert transformation would make it feasible to study the expression of plant genes or gene clusters in their native genomic context and might eliminate site-dependent gene expression, which can be a serious problem in plant transformation experiments. Finally, such a system may make positional cloning applicable to the isolation of genes that encode complex quantitative traits and allow for the transfer of one or more of these genes to various plant species (4).The construction of large insert libraries in bacterial artificial chromosome (BAC) vectors has been reported for several plants (5-7) and animals (8,9
Background Vast sample sizes are often essential in the quest to disentangle the complex interplay of the genetic, lifestyle, environmental and social factors that determine the aetiology and progression of chronic diseases. The pooling of information between studies is therefore of central importance to contemporary bioscience. However, there are many technical, ethico-legal and scientific challenges to be overcome if an effective, valid, pooled analysis is to be achieved. Perhaps most critically, any data that are to be analysed in this way must be adequately ‘harmonized’. This implies that the collection and recording of information and data must be done in a manner that is sufficiently similar in the different studies to allow valid synthesis to take place.Methods This conceptual article describes the origins, purpose and scientific foundations of the DataSHaPER (DataSchema and Harmonization Platform for Epidemiological Research; http://www.datashaper.org), which has been created by a multidisciplinary consortium of experts that was pulled together and coordinated by three international organizations: P3G (Public Population Project in Genomics), PHOEBE (Promoting Harmonization of Epidemiological Biobanks in Europe) and CPT (Canadian Partnership for Tomorrow Project).Results The DataSHaPER provides a flexible, structured approach to the harmonization and pooling of information between studies. Its two primary components, the ‘DataSchema’ and ‘Harmonization Platforms’, together support the preparation of effective data-collection protocols and provide a central reference to facilitate harmonization. The DataSHaPER supports both ‘prospective’ and ‘retrospective’ harmonization.Conclusion It is hoped that this article will encourage readers to investigate the project further: the more the research groups and studies are actively involved, the more effective the DataSHaPER programme will ultimately be.
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