User:Jerome Bonnet/Notebook/day by day notes/2008/11/27
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PCR or digestion. in vitro: Plasterk et al, PNAS 1984
Gin invertase quite slow check indetails
J Mol Biol. 1983 Oct 15;170(1):1-18.Links Knotting of DNA caused by a genetic rearrangement. Evidence for a nucleosome-like structure in site-specific recombination of bacteriophage lambda. Pollock TJ, Nash HA.
Intramolecular recombination between two attachment sites on a circular substrate can invert one segment of the circle with respect to the other. We have studied the topological form of the products of such site-specific inversion as a function of two parameters of the substrate circle: the degree of supercoiling and the distance between the recombining sites. For both integrative and excisive recombination, supercoiled substrates produced knotted recombinants; the complexity of the knots reflects the distance separating the sites. This confirms and extends earlier observations and supports the hypothesis that random interwrapping of segments of the double-helical substrate persists during recombination. For integrative recombination, we find that even at conditions that should limit random interwrapping, absence of supercoiling and very short separation between attachment sites, only about one-half of the recombinant products are simple circles and the rest are knotted. Under the same conditions, excisive recombination yields only simple circular inverted recombinants. We propose that the excess knotting that characterizes integrative recombination reflects the requirement for wrapping of one attachment site, presumably attP, into a nucleosome-like structure. This hypothesis accounts for both the frequency of knots and the observation that the extra knots are trefoils rather than more complex forms.
Modifying yeast artificial chromosomes to generate Cre/LoxP and FLP/FRT site-specific deletions and inversions. Loots GG.
Genome Biology Division, Lawrence Livermore National Laboratory, Livermore, CA, USA.
The ability to efficiently and accurately modify genomic DNA through targeted and tissue-specific mutations is an important goal in animal transgenesis. Here we describe how to exploit two systems of homologous recombination, from yeast and bacteria, to engineer yeast artificial chromosomes (YACs) to generate targeted deletions and inversions in vivo, in transgenic animals, and in the presence of DNA-modifying enzymes known as recombinases. Through homologous recombination in yeast, specific recombinogenic sequences are inserted upstream and downstream of a region in the YAC. The sites of integration of these short sequence elements are chosen carefully, such that the YAC is left functionally intact, and this modified transgene represents the wild-type allele. This YAC is subsequently used to generate transgenic animals, which when bred to animals expressing recombinase proteins result in genetic modifications. By expressing recombinase proteins from different tissue-specific promoters, one can mediate site-specific recombination to generate either ubiquitous or tissue-specific deletions or inversion. These modifications can then be carried through the germline or can be studied somatically. A great advantage of this system is the ability to evaluate subtle genetic effects independent of position-effect variegation, and transgene copy number, eliminating the need to examine several independently generated lines of transgenic animals for each genetic variant.
Site-specific recombination systems for the genetic manipulation of eukaryotic genomes. Thomson JG, Ow DW. Plant Gene Expression Center, USDA, Albany, California 94710, USA.
Site-specific recombination systems, such as the bacteriophage Cre-lox and yeast FLP-FRT systems, have become valuable tools for the rearrangement of DNA in higher eukaryotes. As a first step to expanding the repertoire of recombination tools, we screened recombination systems derived from the resolvase/invertase family for site-specific recombinase activity in the fission yeast Schizosaccharomyces pombe. Here, we report that seven recombination systems, four from the small serine resolvase subfamily (CinH, ParA, Tn1721, and Tn5053) and three from the large serine resolvase subfamily (Bxb1, TP901-1, and U153), can catalyze site-specific deletion in S. pombe. Those from the large serine resolvase subfamily were also capable of site-specific integration and inversion. In all cases, the recombination events were precise. Functional operation of these recombination systems in the fission yeast holds promise that they may be further developed as recombination tools for the site-specific rearrangement of plant and animal genomes. Published 2006 Wiley-Liss, Inc.