Telomere–associated chromosome fragmentation: applications in genome manipulation and analysis

Farr, Carol L.; Stevanović, Milena; Thomson, Eric J.; Goodfellow, Peter N.; Cooke, Howard J.

doi:10.1038/ng1292-275

Cited by 107 publications

(72 citation statements)

References 38 publications

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“…Endonuclease cleavage may assist chromosome fragmentation for the purpose of genome mapping or genetic analysis in vivo (2,12,18), as well as studies of chromosome stability (48) and cell cycle control following DNA damage (for example, see reference 26).…”

Section: Resultsmentioning

confidence: 99%

Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease.

1994

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To maintain genomic integrity, double-strand breaks (DSBs) in chromosomal DNA must be repaired. In mammalian systems, the analysis of the repair of chromosomal DSBs has been limited by the inability to introduce well-defined DSBs in genomic DNA. In this study, we created specific DSBs in mouse chromosomes for the first time, using an expression system for a rare-cutting endonuclease, I-Scel. A genetic assay has been devised to monitor the repair of DSBs, whereby cleavage sites for I-Scel have been integrated into the mouse genome in two tandem neomycin phosphotransferase genes. We find that cleavage of the I-SceI sites is very efficient, with at least 12% of stably transfected cells having at least one cleavage event and, of these, more than 70% have undergone cleavage at both I-Scel sites. Cleavage of both sites in a fraction of clones deletes 3.8 kb of intervening chromosomal sequences. We find that the DSBs are repaired by both homologous and nonhomologous mechanisms. Nonhomologous repair events frequently result in small deletions after rejoining of the two DNA ends. Some of these appear to occur by simple blunt-ended ligation, whereas several others may occur through annealing of short regions of terminal homology. The DSBs are apparently recombinogenic, stimulating gene targeting of a homologous fragment by more than 2 orders of magnitude. Whereas gene-targeted clones are nearly undetectable without endonuclease expression, they represent approximately 10% of cells transfected with the I-Scel expression vector. Gene targeted clones are of two major types, those that occur by two-sided homologous recombination with the homologous fragment and those that occur by one-sided homologous recombination. Our results are expected to impact a number of areas in the study of

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Section: Resultsmentioning

confidence: 99%

Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease.

1994

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“…Random integration of plasmids carrying telomere repeats adjacent to a selectable marker generated selected colonies with a newly seeded telomere next to the marker at a frequency of 20% in Chinese hamster ovary (CHO) cells (23) and 70% in HeLa cells (29). Surprisingly, the majority of such clones carried duplications or other rearrangements at the site of chromosome truncation (14,24,32). The role of telomere sequence in chromosome truncation and terminal rearrangement-beyond its capacity to seed new telomeres-is unclear.…”

mentioning

confidence: 99%

Insertion of a Telomere Repeat Sequence into a Mammalian Gene Causes Chromosome Instability

Kilburn

Shea

Sargent³

et al. 2001

Molecular and Cellular Biology

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Telomere repeat sequences cap the ends of eucaryotic chromosomes and help stabilize them. At interstitial sites, however, they may destabilize chromosomes, as suggested by cytogenetic studies in mammalian cells that correlate interstitial telomere sequence with sites of spontaneous and radiation-induced chromosome rearrangements. In no instance is the length, purity, or orientation of the telomere repeats at these potentially destabilizing interstitial sites known. To determine the effects of a defined interstitial telomere sequence on chromosome instability, as well as other aspects of DNA metabolism, we deposited 800 bp of the functional vertebrate telomere repeat, TTAGGG, in two orientations in the second intron of the adenosine phosphoribosyltransferase (APRT) gene in Chinese hamster ovary cells. In one orientation, the deposited telomere sequence did not interfere with expression of the APRT gene, whereas in the other it reduced mRNA levels slightly. The telomere sequence did not induce chromosome truncation and the seeding of a new telomere at a frequency above the limits of detection. Similarly, the telomere sequence did not alter the rate or distribution of homologous recombination events. The interstitial telomere repeat sequence in both orientations, however, dramatically increased gene rearrangements some 30-fold. Analysis of individual rearrangements confirmed the involvement of the telomere sequence. These studies define the telomere repeat sequence as a destabilizing element in the interior of chromosomes in mammalian cells.

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“…artificial chromosomes ͉ FISH ͉ genetic engineering ͉ telomere truncation A rtificial chromosomes involving de novo centromere formation on an independently assembled unit and engineered minichromosomes produced by telomere truncation provide striking advantages over traditional methods of gene transformation in yeast and mammalian cells (1)(2)(3)(4)(5). The development of such chromosomes in plants would provide these advantages for many applications in basic studies, biotechnology, and agriculture.…”

mentioning

confidence: 99%

Construction and behavior of engineered minichromosomes in maize

Han

Gao

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

142

138

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Engineered minichromosomes were constructed in maize by modifying natural A and supernumerary B chromosomes. By using telomere-mediated chromosomal truncation, it was demonstrated that such an approach is feasible for the generation of minichromosomes of normal A chromosomes by selection of spontaneous polyploid events that compensate for the deficiencies produced. B chromosomes are readily fractionated by biolistic transformation of truncating plasmids. Foreign genes were faithfully expressed from integrations into normal B chromosomes and from truncated miniB chromosomes. Site-specific recombination between the terminal transgene on a miniA chromosome and a terminal site on a normal chromosome was demonstrated. It was also found that the miniA chromosome did not pair with its progenitor chromosomes during meiosis, indicating a useful property for such constructs. The miniB chromosomes are faithfully transmitted from one generation to the next but can be changed in dosage in the presence of normal B chromosomes. This approach for construction of engineered chromosomes can be easily extended to other plant species because it does not rely on cloned centromere sequences, which are species-specific. These platforms will provide avenues for studies on plant chromosome structure and function and for future developments in biotechnology and agriculture.artificial chromosomes ͉ FISH ͉ genetic engineering ͉ telomere truncation A rtificial chromosomes involving de novo centromere formation on an independently assembled unit and engineered minichromosomes produced by telomere truncation provide striking advantages over traditional methods of gene transformation in yeast and mammalian cells (1-5). The development of such chromosomes in plants would provide these advantages for many applications in basic studies, biotechnology, and agriculture. These chromosomes could be used as independent platforms for foreign gene expression without random integration into the normal chromosomes. Further additions of unlimited amounts of DNA could be added to these platforms in a sequential manner via different site-specific recombination cassettes. Genes introduced in this way would be present in a defined context and thus could be expressed at a more predictable level than through random integration (6). Hence, additional genes, multigene complexes, or even whole metabolic pathways could potentially be added to a genotype. Moreover, engineered or artificial chromosomes could be easily introduced or removed from a genotype by genetic crosses and would facilitate introgression of transgenes to different genetic backgrounds.To extend engineered chromosome technology to plants, we developed a method of telomere-mediated chromosomal truncation in maize by Agrobacterium-mediated transformation of constructs with multiple copies of the telomere sequence (7). Here, we report the use of this technology to produce minichromosome platforms by truncating both normal A and supernumerary B maize chromosomes and at the same time introducing site-spe...

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Telomere–associated chromosome fragmentation: applications in genome manipulation and analysis

Cited by 107 publications

References 38 publications

Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease.

Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease.

Insertion of a Telomere Repeat Sequence into a Mammalian Gene Causes Chromosome Instability

Construction and behavior of engineered minichromosomes in maize

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