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63
Mitotic crossovers between diverged sequences are regulated by mismatch repair proteins
- in Saccharomyces
, 1996
"... Mismatch repair systems correct replication- and recombination-associated mispaired bases and influence the stability of simple repeats. These systems thus serve multiple roles in maintaining genetic stability in eukaryotes, and human mismatch repair defects have been associated with hereditary pred ..."
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Cited by 43 (5 self)
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Mismatch repair systems correct replication- and recombination-associated mispaired bases and influence the stability of simple repeats. These systems thus serve multiple roles in maintaining genetic stability in eukaryotes, and human mismatch repair defects have been associated with hereditary predisposition to cancer. In prokaryotes, mismatch repair systems also have been shown to limit recombination between diverged (homeologous) sequences. We have developed a unique intron-based assay system to examine the effects of yeast mismatch repair genes (PMS1, MSH2, and MSH3) on crossovers between homeologous sequences. We find that the apparent antirecombination effects of mismatch repair proteins in mitosis are related to the degree of substrate divergence. Defects in mismatch repair can elevate homeologous recombination between 91 % homologous substrates as much as 100-fold while having only modest effects on recombination between 77 % homologous substrates. These observations have implications for genome stability and general mecha-nisms of recombination in eukaryotes. Much of our knowledge concerning the mechanism of ho-mologous recombination in eukaryotes has come from studies done with the yeast Saccharomyces cerevisiae (for a review, see reference 40), in which two basic types of recombination occur:
Targeted mutagenesis by homologous recombination in D. melanogaster. Genes Dev
, 2002
"... We used a recently developed method to produce mutant alleles of five endogenous Drosophila genes, including the homolog of the p53 tumor suppressor. Transgenic expression of the FLP site-specific recombinase and the I-SceI endonuclease generates extrachromosomal linear DNA molecules in vivo. These ..."
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Cited by 31 (2 self)
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We used a recently developed method to produce mutant alleles of five endogenous Drosophila genes, including the homolog of the p53 tumor suppressor. Transgenic expression of the FLP site-specific recombinase and the I-SceI endonuclease generates extrachromosomal linear DNA molecules in vivo. These molecules undergo homologous recombination with the corresponding chromosomal locus to generate targeted alterations of the host genome. The results address several questions about the general utility of this technique. We show that genes not near telomeres can be efficiently targeted; that no knowledge of the mutant phenotype is needed for targeting; and that insertional mutations and allelic substitutions can be easily produced. [Key Words: Gene targeting; Drosophila; recombination; FLP; I-SceI]
Lessons from loricrin-deficient mice: compensatory mechanisms maintaining skin barrier function in the absence of a major cornified envelope protein
- J. Cell
, 2000
"... Abstract. The epidermal cornified cell envelope (CE) is a complex protein–lipid composite that replaces the plasma membrane of terminally differentiated keratinocytes. This lamellar structure is essential for the barrier function of the skin and has the ability to prevent the loss of water and ions ..."
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Cited by 22 (0 self)
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Abstract. The epidermal cornified cell envelope (CE) is a complex protein–lipid composite that replaces the plasma membrane of terminally differentiated keratinocytes. This lamellar structure is essential for the barrier function of the skin and has the ability to prevent the loss of water and ions and to protect from environmental hazards. The major protein of the epidermal CE is loricrin, contributing �70 % by mass. We have generated mice that are deficient for this protein. These mice showed a delay in the formation of the skin barrier in embryonic development. At birth, homozygous mutant mice weighed less than control littermates and showed skin abnormalities, such as congenital erythroderma with a shiny, translucent skin. Tape stripping experiments suggested that the stratum corneum stability was
Design and packaging of adenoassociated virus gene targeting vectors
- J
, 2000
"... Adeno-associated virus (AAV) vectors can transduce cells by several mechanisms, including (i) gene addition by chromosomal integration or episomal transgene expression or (ii) gene targeting by modification of homologous chromosomal sequences. The latter process can be used to correct a variety of m ..."
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Cited by 21 (4 self)
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Adeno-associated virus (AAV) vectors can transduce cells by several mechanisms, including (i) gene addition by chromosomal integration or episomal transgene expression or (ii) gene targeting by modification of homologous chromosomal sequences. The latter process can be used to correct a variety of mutations in chromosomal genes with high fidelity and specificity. In this study, we used retroviral vectors to introduce mutant alkaline phosphatase reporter genes into normal human cells and subsequently corrected these mutations with AAV gene targeting vectors. We find that increasing the length of homology between the AAV vector and the target locus improves gene correction rates, as does positioning the mutation to be corrected in the center of the AAV vector genome. AAV-mediated gene targeting increases with time and multiplicity of infection, similar to AAV-mediated gene addition. However, in contrast to gene addition, genotoxic stress did not affect gene targeting rates, suggesting that different cellular factors are involved. In the course of these studies, we found that (i) vector genomes less than half of wild-type size could be packaged as monomers or dimers and (ii) packaged dimers consist of inverted repeats with covalently closed hairpins at either end. These studies should prove helpful in designing AAV gene targeting vectors for basic research or gene therapy. Adeno-associated virus (AAV) is a dependent parvovirus with a 4.7-kb single-stranded linear DNA genome. Vectors
Location of crossovers during gene targeting with insertion and replacement
, 1993
"... Gene targeting was used to introduce nonselectable genetic changes into chromosomal loci in mouse embryo-derived stem cells. The nonselectable markers were linked to a selectable marker in both insertion- and replacement-type vectors, and the transfer of the two elements to the Hprt locus was assaye ..."
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Cited by 18 (9 self)
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Gene targeting was used to introduce nonselectable genetic changes into chromosomal loci in mouse embryo-derived stem cells. The nonselectable markers were linked to a selectable marker in both insertion- and replacement-type vectors, and the transfer of the two elements to the Hprt locus was assayed. When insertion vectors were used as substrates, the frequency of transfer was highly dependent upon the distance between the nonselectable marker and the double-strand break in the vector. A marker located close to the vector ends was frequently lost, suggesting that a double-strand gap repair activity is involved in vector integration. When replacement vectors were used, cotransfer of a selectable marker and a nonselectable marker 3 kb apart was over 50%, suggesting that recombination between vector and target often occurs near the ends of the vector. To illustrate the use of replacement vectors to transfer specific mutations to the genome, we describe targeting of the AF508 mutation to the CFTR gene in mouse embryo-derived stem cells. Gene targeting in mouse embryo-derived stem (ES) cells provides the means for generating mice of virtually any desired genotype (3, 4). Most applications of this technology have been directed at creating mice with mutations causing loss of function in the gene of interest. The loss of function
Discriminatory suppression of homologous recombination by p53
- Nucleic Acids Res
, 2004
"... Homologous recombination (HR) is used in vertebrate somatic cells for essential, RAD51-dependent, repair of DNA double-strand-breaks (DSBs), but inappro-priate HR can cause genome instability. A transcrip-tional transactivation-independent role for p53 in suppressing HR has been established, but is ..."
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Cited by 15 (1 self)
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Homologous recombination (HR) is used in vertebrate somatic cells for essential, RAD51-dependent, repair of DNA double-strand-breaks (DSBs), but inappro-priate HR can cause genome instability. A transcrip-tional transactivation-independent role for p53 in suppressing HR has been established, but is not detected in all HR assays. To address the basis of such exceptions, and the possibility that suppression by p53 may be discriminatory, we have conducted a controlled comparison of the effects of p53 depletion onthreedifferentkindsofHR.Weshowthat,within the same cells, p53 depletion promotes both intra-chromosomal HR (ICHR) and extra-chromosomal HR (ECHR), but not homologous DNA integration (gene targeting; GT). This conclusion holds true for both spontaneous and DSB-induced ICHR and GT. We show further that non-conservative ICHR is more sus-ceptible than conservative ICHR to inhibition by p53. These results provide strong evidence that p53 can discriminate between different forms of HR and, des-pite the fact that GT is used experimentally for gene disruption, is consistent with the possibility that p53 preferentially suppresses genome-destabilizing forms of HR. While the mechanism of suppression by p53 remains unclear, our data suggest that it is independent of mismatch repair and of changes in RAD51 protein levels.
Site-directed point mutations in embryonic stem cells: a gene-targeting tag-and-exchange strategy
, 1993
"... Site-directed point mutations in embryonic stem cells: a gene-targeting tag-and-exchange strategy. ..."
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Cited by 14 (0 self)
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Site-directed point mutations in embryonic stem cells: a gene-targeting tag-and-exchange strategy.
Parameters determining the efficiency of gene targeting in the moss Physcomitrella patens. Nucleic Acids Res
, 2005
"... In the moss Physcomitrella patens, transforming DNA containing homologous sequences integrates pre-dominantly by homologous recombination with its 10 genomic target. A systematic investigation of the parameters that determine gene targeting efficiency shows a direct relationship between homology len ..."
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Cited by 12 (6 self)
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In the moss Physcomitrella patens, transforming DNA containing homologous sequences integrates pre-dominantly by homologous recombination with its 10 genomic target. A systematic investigation of the parameters that determine gene targeting efficiency shows a direct relationship between homology length and targeting frequency for replacement vectors (a selectable marker flanked by homologous DNA). 15 Overall homology of only 1 kb is sufficient to achieve a 50 % yield of targeted transformants. Targeting may occur through homologous recombination in one arm, accompanied by non-homologous end-joining by the other arm of the vector, or by allele replacement 20 following two homologous recombination events. Allele replacement frequency depends on the sym-metry of the targeting vector, being proportional to the length of the shorter arm. Allele replacement may involve insertion of multiple copies of the trans-25 forming DNA, accompanied by ectopic insertions at non-homologous sites. Single-copy and single inser-tions at targeted loci (targeted gene replacements, ‘TGR’) occur with a frequency of 7–20 % of all trans-formants when the minimum requirements for allele 30 replacement are met. Homologous recombination in Physcomitrella is substantially more efficient than in any multicellular eukaryote, recommending it as the outstanding model for the study of homologous recombination in plants. 35
Use of double-replacement gene targeting to replace the murine alpha-lactalbumin gene with its human counterpart in embryonic stem cells and mice
, 1994
"... The mouse a-lactalbumin gene has been replaced with the human gene by two consecutive rounds of gene targeting in hypoxanthine phosphoribosyltransferase (HPRT)-deficient feeder-independent murine embryonic stem (ES) cells. One mouse a-lactalbumin allele was first replaced by an HPRT minigene which w ..."
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Cited by 8 (3 self)
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The mouse a-lactalbumin gene has been replaced with the human gene by two consecutive rounds of gene targeting in hypoxanthine phosphoribosyltransferase (HPRT)-deficient feeder-independent murine embryonic stem (ES) cells. One mouse a-lactalbumin allele was first replaced by an HPRT minigene which was in turn replaced by human a-lactalbumin. The end result is a clean exchange of defined DNA fragments with no other DNA remaining at the target locus. Targeted ES cells at each stage remained capable of contributing efficiently to the germ line of chimeric animals. Double replacement using HPRT-deficient ES cells and the HPRT selection system is therefore a powerful and flexible method of targeting specific alterations to animal genes. A typical strategy for future use would be to generate a null mutation which could then be used to produce multiple second-step alterations at the same locus. Gene targeting in murine embryonic stem (ES) cells has had an enormous impact on biology over the past 3 to 4 years because of its ability to produce mice with specific genetic alterations. Although the first mice produced by this method contained a corrected hypoxanthine phosphoribosyltrans-ferase (HPRT) gene (32), most subsequent reports describe
The frequency of gene targeting in Trypanosoma brucei is independent of target-site copy number. Nucleic Acids Res 2003;31:3993–4000
"... We have investigated the effect of target copy number on the ef®ciency of stable transformation of the protozoan parasite Trypanosoma brucei. Using a single strain of the organism, we targeted integra-tive vectors to several different genomic sequences, occurring at copy numbers ranging from 1 to ~3 ..."
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Cited by 7 (1 self)
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We have investigated the effect of target copy number on the ef®ciency of stable transformation of the protozoan parasite Trypanosoma brucei. Using a single strain of the organism, we targeted integra-tive vectors to several different genomic sequences, occurring at copy numbers ranging from 1 to ~30 000 per diploid genome, and undertook a systematic assessment of both transformation and integration ef®ciencies. Even over this vast copy number range, frequency of gene targeting was the same for all sites. An independence of targeting frequency and target copy number is characteristic of mammalian homologous recombination and is unlike the situation in budding yeast. It is also not seen in the related parasite Leishmania, a distinc-tion that may be the consequence of the different usage of recombination within the mechanisms of pathogenicity in the two species.
