A comparison of induced mutation at homologous alleles of the tk locus in human cells. II. Molecular analysis of mutants

Amundson, Sally A.; Liber, Howard L.

doi:10.1016/0027-5107(92)90113-g

Cited by 39 publications

(18 citation statements)

References 22 publications

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“…However, the relative difference in mutability between these lines at hprt (about 3-fold) is significantly smaller than at tk (20-to 50-fold [4] (3). However, the finding that LOH by recombination also can lead to slow growth, observed in two cases, is not consistent with this hypothesis.…”

mentioning

confidence: 81%

“…2). Besides being less frequent, LOH was also less extensive in TK6 mutants than in WTK1 and WTKB; 16 to 21% of mutants showed loss of only TK1 (3). Interestingly, the distal long-arm marker D17S24, which was never lost from mutants derived from the tk-7' heterozygotes derived from TK6, was lost in approximately 80% of the mutants from WTKB, the corresponding tk-7' line derived from WIL2-NS.…”

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confidence: 96%

“…LOH was determined by using probes for tk and four other RFLPs on the long arm of chromosome 17, as well as one marker on the short arm. The more mutable tk'l-lines exhibited LOH at an RFLP marker closely linked to tk about twice as often as tk-l' lines, and LOH of the most distal long arm marker occurred in 39% of mutants from tk'l-cells but was never observed in tk-'+ cells (3). This suggested that loss of a region near the latter marker might be survivable if it occurred in one chromosome, but not in the other.…”

mentioning

confidence: 98%

“…However, among these lines there was no difference in X-ray mutability at the hemizygous hprt locus (2). Physical mapping of X-ray-induced mutants further indicated that the differential mutability of tk alleles resulted from a reduced yield of large-scale changes associated with the less mutable allele (3). LOH was determined by using probes for tk and four other RFLPs on the long arm of chromosome 17, as well as one marker on the short arm.…”

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confidence: 99%

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Different capacities for recombination in closely related human lymphoblastoid cell lines with different mutational responses to X-irradiation.

Xia¹,

Amundson²,

Nickoloff³

et al. 1994

Mol. Cell. Biol.

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WIL2-NS and TK6 are two distinct human lymphoblast cell lines derived from a single male donor. WIL2-NS cells are significantly more resistant to the cytotoxic effects of X-irradiation but considerably more sensitive to induced mutation. In an effort to determine the mechanistic basis for these differences, we analyzed the physical structures of thymidine kinase (tk)-deficient mutants isolated after X-ray treatment of tk heterozygotes derived from TK6 and the more mutable WIL2-NS. Southern analysis showed that while 84% of TK6-derived mutants had arisen by loss of heterozygosity (LOH), all 106 mutants from WIL2-NS derivatives arose with LOH at tk and all but one showed LOH at other linked loci on chromosome 17. We adapted a fluorescence in situ hybridization technique to distinguish between LOH due to deletion, which results in retention of only one tk allele, and LOH due to a mechanism involving the homologous chromosome (e.g., recombination), which results in the retention of two alleles. Among the LOH mutants derived that were analyzed in this way, 9 of 26 from WIL2-NS and 11 of 17 from TK6 cell lines arose by deletion. The remaining mutants retained two copies of the tk gene and thus arose by a mechanism involving the homologous allele. Since many of these mutants arising by a homologous mechanism retained partial heterozygosity of chromosome 17, they must have arisen by recombination or gene conversion, and not chromosome loss and reduplication. Finally, the recombinational capacities of WIL2-NS and TK6 were compared in transfection assays with plasmid recombination substrates. Intermolecular recombination frequencies were greater in WIL2-NS than in TK6. These data are consistent with a model suggesting that a recombinational repair system is functioning at a higher level in WIL2-NS than in TK6; the greater mutability of the tk locus in WIL2-NS results from more frequent inter-and intramolecular recombination events.Mutations that activate oncogenes or inactivate tumor suppressors are intimately involved in carcinogenic pathways. Therefore, it is important to study factors that affect mutation rates in human cells, because this will improve our understanding of cancer risk from environmental exposures and spontaneous processes. A number of studies suggest that the frequencies at which mutations are observed at various gene targets vary tremendously, depending on their location in the genome (reviewed in reference 56). Single gene mutations that inactivate a genetic locus are theoretically equally likely to occur at hemizygous loci (+/0: one active allele, no homologous allele) or heterozygous loci (+/-: one active and one inactive allele). Very large deletions may have a higher probability of being lethal if they span hemizygous regions of the genome and include an essential locus, while loss of one copy of such loci from chromosomal regions that exist in duplicate may not be lethal. Homologous recombination or gene conversion, processes that can convert a heterozygous locus to a homozygous state (-/-, with...

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confidence: 81%

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confidence: 96%

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confidence: 98%

mentioning

confidence: 99%

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Different capacities for recombination in closely related human lymphoblastoid cell lines with different mutational responses to X-irradiation.

Xia¹,

Amundson²,

Nickoloff³

et al. 1994

Mol. Cell. Biol.

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“…Thymidine kinase gene can be used as a selectable marker for mutations ranging from point to megabase deletions, besides recombination events can be studied using the tk heterozygous cell line TK6 [30,31]. The construction of the cell line, reported here, began with the use of TX528 cells [32] which is derived from TK6 lymphoblasts, an immortalized cell line characterized by Giver et al [33].…”

Section: Introductionmentioning

confidence: 99%

Use of E18 Cell Model to Quantify DNA Strand Break Associated Bystander Effect (DSB-ABE)

Jalal¹

2017

J Carcinog Mutagen

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Citation: Nasir J (2017) Use of E18 Cell Model to Quantify DNA Strand Break Associated Bystander Effect (DSB-ABE). J Carcinogene Mutagene 8: 295. doi: 10.4172/2157-2518.1000295 Copyright: © 2017 Nasir J. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. AbstractThe lymphoblast cell line E18 is a derivative of TK6 and has an I-Sce1 insert in the intron 2 of heterozygous TK1/ tk1 gene. E18 can be targeted with I-Sce1 restriction endonuclease to induce double strand breaks at the I-Sce1 site to measure radiation independent DNA damage and the associated bystander effect. For construction of E18 cell line an Eco47III site was inserted into the pTK-UAS linearized plasmid backbone and annealed with blunt-ended oligonucleotides of ISce1 restriction sequence in the presence of DNA ligase. DH5-α cells were transformed by heat transfecting the cells with pTK-UAS-Eco47III-Sce1 and selecting ampicillin resistant colonies. Plasmid DNA was extracted and analyzed for the presence of Eco47III and I-Sce1 sites. A clone with I-Sce site in intron 2 of the thymidine kinase active allele was selected for further experimentation and named E18. This model was tested to demonstrate the double strand break induced mutations and these strand breaks can also produce DNA strand break associated bystander effect (DSB-ABE) as measured by increased mutation frequency in naïve cells. The plasmid pAdTrackCMV I-Sce1 carrying GFP marker was electroporated in E18 cells to express the I-Sce1 restriction endonuclease which specifically targets the I-Sce1 site at intron 2 of the active TK allele. Mutation fraction assay was employed to measure direct mutation fraction (DMF) or accompanied by conditioned medium transfer to naïve cells for measuring bystander mutation fraction (BMF) as an end-point of targeting E18 with an exogenous I-Sce1 expression via pAdTrackCMV I-Sce1 to induce DNA damage and quantify DMF and BMF.

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Requirement of wild-type p53 protein for maintenance of chromosomal integrity

et al. 2000

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Chromosomal double‐strand breaks (DSBs) occurring in mammalian cells can initiate genomic instability, and their misrepairs result in chromosomal deletion, amplification, and translocation, common findings in human tumors. The tumor‐suppressor protein p53 is involved in maintaining genomic stability. In this study, we demonstrate that the deficiency of wild‐type p53 protein may allow unrepaired DSBs to initiate chromosomal instability. The human lymphoblastoid cell line TK6‐E6 was established by transfection with human papilloma virus 16 (HPV16) E6 cDNA into parental TK6 cells via a retroviral vector. Abrogation of p53 function by E6 resulted in an increase in the spontaneous mutation frequencies at the heterozygous thymidine kinase (TK) locus but not at the hemizygous hypoxanthine phosphoribosyl transferase (HPRT) locus. Almost all TK‐deficient mutants from TK6‐E6 cells exhibited loss of heterozygosity (LOH) with the hemizygous TK allele. LOH analysis with microsatellite loci spanning the long arm of chromosome 17, which harbors the TK locus, showed that LOH extended over half of 17q toward the terminal end. Cytogenetic analysis of LOH mutants by chromosome painting indicated a mosaic of chromosomal aberrations involving chromosome 17, in which partial chromosome deletions, amplifications, and multiple translocations appeared heterogeneously in a single mutant. We speculate that spontaneous DSBs trigger the breakage‐fusion bridge cycle leading to such multiple chromosome aberrations. In contrast, no chromosomal alterations were observed in TK‐deficient mutants from TK6‐20C cells expressing wild‐type p53. In wild‐type p53 cells, spontaneous DSBs appear to be promptly repaired through recombination between homologous chromosomes. These results support a model in which p53 protein contributes to the maintenance of genomic integrity through recombinational repair. Mol. Carcinog. 28:203–214, 2000. © 2000 Wiley‐Liss, Inc.

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A comparison of induced mutation at homologous alleles of the tk locus in human cells. II. Molecular analysis of mutants

Cited by 39 publications

References 22 publications

Different capacities for recombination in closely related human lymphoblastoid cell lines with different mutational responses to X-irradiation.

Different capacities for recombination in closely related human lymphoblastoid cell lines with different mutational responses to X-irradiation.

Use of E18 Cell Model to Quantify DNA Strand Break Associated Bystander Effect (DSB-ABE)

Requirement of wild-type p53 protein for maintenance of chromosomal integrity

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