Evaluation of methods for the estimation of mutation rates in cultured mammalian cell populations

Li, I-Chian; Chu, Ernest H. Y.

doi:10.1016/0165-7992(87)90010-8

Cited by 16 publications

(9 citation statements)

References 7 publications

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“…Furthermore, the observed rate of phenotypic reversion tends to vary tremendously as a function of the specific site of mutation within a gene due to "DNA context effects" (5). In the present study, use of direct selection facilitated large-scale fluctuation tests and their analysis by the maximum-likelihood method, which represents the most accurate and statistically efficient strategy widely used to measure mutation rates in microorganisms (15,17). The growth capabilities of S. acidocaldarius also facilitated efforts to directly estimate the possible impact of differential growth rates and phenotypic lag on mutation rate determinations (5).…”

Section: Discussionmentioning

confidence: 99%

Rates of spontaneous mutation in an archaeon from geothermal environments

Jacobs

Grogan

1997

J Bacteriol

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To estimate the efficacy of mechanisms which may prevent or repair thermal damage to DNA in thermophilic archaea, a quantitative assay of forward mutation at extremely high temperature was developed for Sulfolobus acidocaldarius, based on the selection of pyrimidine-requiring mutants resistant to 5-fluoro-orotic acid. Maximum-likelihood analysis of spontaneous mutant distributions in wild-type cultures yielded maximal estimates of (2.8 ؎ 0.7) ؋ 10 ؊7 and (1.5 ؎ 0.6) ؋ 10 ؊7 mutational events per cell per division cycle for the pyrE and pyrF loci, respectively. To our knowledge, these results provide the first accurate measurement of the genetic fidelity maintained by archaea that populate geothermal environments. The measured rates of forward mutation at the pyrE and pyrF loci in S. acidocaldarius are close to corresponding rates reported for protein-encoding genes of Escherichia coli. The normal rate of spontaneous mutation in E. coli at 37°C is known to require the functioning of several enzyme systems that repair spontaneous damage in DNA. Our results provide indirect evidence that S. acidocaldarius has cellular mechanisms, as yet unidentified, which effectively compensate for the higher chemical instability of DNA at the temperatures and pHs that prevail within growing Sulfolobus cells.In the past 25 years, diverse microorganisms have been isolated from geothermal environments and have been shown to require temperatures of 75 to 105°C (depending on the species) for optimal growth; these organisms have been variously termed extreme thermophiles or hyperthermophiles (30). The fact that the preferred growth temperatures of these organisms can effectively denature the RNA, DNA, and protein of mesophilic microorganisms raises basic questions regarding the potential stability of biological macromolecules and the means by which extreme thermophiles and hyperthermophiles realize this potential stability in vivo. Considerable effort has been invested in understanding, in molecular terms, how the tertiary and secondary structures of enzymes and nucleic acids are preserved in these microorganisms. The fact that growth temperatures of 75 to 105°C should also disrupt the primary structure of biological macromolecules has received much less attention, despite indications that spontaneous processes of this type affect the growth and survival of bacteria growing at 37°C (16,29).The chemical instability of DNA is a major source of spontaneous mutation in mesophiles and has been studied extensively. Hydrolytic depurination represents the most frequent spontaneous decomposition of duplex DNA, followed by hydrolytic deamination of cytosine and adenine residues (19)(20)(21)(22)29). In aerobes, additional oxidative reactions form a variety of damaged bases (21). Biochemical and genetic studies using bacteria have shown that these various spontaneous reactions (i) occur in vivo (indeed, some metabolic processes actually contribute to them [21,29]); (ii) are potentially mutagenic, i.e., they cause mutation if the lesions escape rep...

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Section: Discussionmentioning

confidence: 99%

Rates of spontaneous mutation in an archaeon from geothermal environments

Jacobs

Grogan

1997

J Bacteriol

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“…The FA-resistant (FA r ) or 6TG-resistant colonies were fixed with Carnoy's solution, stained with crystal violet, and counted. Mutation rate was calculated by the P 0 method as described (12,13) and corrected for colony forming efficiency. Rates are presented per cell per generation.…”

Section: Methodsmentioning

confidence: 99%

“…The asterisk indicates that no spontaneously arising Hprt mutants were detected (mutation frequency Ͻ10 Ϫ8 ). (b) Preexisting APRT and HPRT mutants were eliminated in ES cell and MEF cultures, which were then treated with EMS or left untreated as described in a. Mutation rate was calculated as previously described, but adapted for mammalian cells (13) and corrected for colony-forming efficiency. Solid bars indicate Aprt mutation rate and hatched bars represent Hprt mutation rate.…”

Section: The Spontaneous Mutation Frequency and Rate In Es Cells Are mentioning

confidence: 99%

Embryonic stem cells and somatic cells differ in mutation frequency and type

Cervantes

Stringer²,

Shao³

et al. 2002

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

288

221

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Pluripotent embryonic stem (ES) cells have been used to produce genetically modified mice as experimental models of human genetic diseases. Increasingly, human ES cells are being considered for their potential in the treatment of injury and disease. Here we have shown that mutation in murine ES cells, heterozygous at the selectable Aprt locus, differs from that in embryonic somatic cells. The mutation frequency in ES cells is significantly lower than that in mouse embryonic fibroblasts, which is similar to that in adult cells in vivo. The distribution of spontaneous mutagenic events is remarkably different between the two cell types. Although loss of the functional allele is the predominant mutation type in both cases, representing about 80% of all events, mitotic recombination accounted for all loss of heterozygosity events detected in somatic cells. In contrast, mitotic recombination in ES cells appeared to be suppressed and chromosome loss͞reduplication, leading to uniparental disomy (UPD), represented more than half of the loss of heterozygosity events. Extended culture of ES cells led to accumulation of cells with adenine phosphoribosyltransferase deficiency and UPD. Because UPD leads to reduction to homozygosity at multiple recessive disease loci, including tumor suppressor loci, in the affected chromosome, the increased risk of tumor formation after stem cell therapy should be viewed with concern.T he frequency and types of spontaneous mutation that occur in pluripotent stem cells may have an impact on their future clinical uses. Embryonic stem (ES) cells are intrinsically different from somatic cells in that they retain the capacity to differentiate into multiple cell types (1, 2). Maintenance of genomic stability in these cells is essential for their potential to serve as donor cells for tissue transplants. Here we have examined spontaneous and induced mutagenic events in ES cells. We have taken advantage of a recently described murine model that utilizes mice heterozygous at the selectable marker encoding the purine salvage enzyme adenine phosphoribosyltransferase (APRT) to examine this issue (3-5).The Aprt gene, resident on mouse chromosome 8, is an endogenous marker that supports selection for cells that are APRT deficient and, conversely, for cells that have APRT activity (6). The model uses cells derived from 129 SvEv ϫ C3H͞HeJ mice or embryos in which the strain 129 SvEv Aprt allele has been interrupted by a targeted neo insertion (7). These cells are therefore heterozygous at Aprt, with the neo insertion allowing for rapid discrimination between the targeted and untargeted alleles. Polymorphic microsatellites that span the length of chromosome 8 can be used to confirm loss of heterozygosity (LOH) resulting from either mitotic recombination, or multilocus deletion. Centromeres in mouse strains 129 SvEv and C3H͞HeJ are different sizes. Hence, the parental origin of copies of chromosome 8 can be determined, providing visual determination of uniparental disomy (UPD), defined as the presence of homolo...

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“…Their first method used the proportion of cultures in which no mutants could be detected (P 0 ). At low mutation rates, the distribution of mutations will be approximately Poisson, with mean m. Accordingly a proportion P 0 =e 2m cultures will have no mutant colonies, and m can be estimated by 2ln(P 0 ), and the mutation rate by m/(N t 2N 0 ), where N t is the population size at time t. Although it is efficient and simple (Li & Chu, 1987), this method has drawbacks for phase variation experiments, in particular that all cells in a culture are plated and all mutants detected. The efficiency declines greatly if investigation of the whole culture is impractical (Kendal & Frost, 1988;Jones et al, 1994).…”

Section: Introductionmentioning

confidence: 99%

“…It is, therefore, more common to use the median. One comparison of the methods indicated that performance decreases from maximum likelihood, the median, the upper quartile, to the mean method (Li & Chu, 1987). Each method was used to derive the parameters of the Lea & Coulson distribution from an experiment, then this distribution was compared to the actual data.…”

Section: Introductionmentioning

confidence: 99%

Mutation rates: estimating phase variation rates when fitness differences are present and their impact on population structure

2003

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Phase variation is a mechanism of ON-OFF switching that is widely utilized by bacterial pathogens. There is currently no standardization to how the rate of phase variation is determined experimentally, and traditional methods of mutation rate estimation may not be appropriate to this process. Here, the history of mutation rate estimation is reviewed, describing the existing methods available. A new mathematical model that can be applied to this problem is also presented. This model specifically includes the confounding factors of back-mutation and the influence of fitness differences between the alternate phenotypes. These are central features of phase variation but are rarely addressed, with the result that some previously estimated phase variation rates may have been significantly overestimated. It is shown that, conversely, the model can also be used to investigate fitness differences if mutation rates are approximately known. In addition, stochastic simulations of the model are used to explore the impact of 'jackpot cultures' on the mutation rate estimation. Using the model, the impact of realistic rates and selection on population structure is investigated. In the absence of fitness differences it is predicted that there will be phenotypic stability over many generations. The rate of phenotypic change within a population is likely, therefore, to be principally determined by selection. A greater insight into the population dynamics of mutation rate processes can be gained if populations are monitored over successive time points. INTRODUCTIONPhase variation describes a process of reversible, highfrequency phenotypic switching that is mediated by DNA mutations, reorganization or modification. Phase variation is used by several bacterial species to generate population diversity that increases bacterial fitness and is important in niche adaptation including immune evasion (Saunders, 2003; Salaün et al., 2003). Being able to determine the rate at which these processes occur and the nature of any factors that influence them is integral to understanding the impact of these processes on the evolution and dynamics of the population as a whole and on the host-bacterium interaction. To do this, tools with which to reliably determine and compare phase variation rates within and between experiments and bacterial populations are needed. The estimation of mutation rates in bacteria has a long history. The methods in use, however, are not general across all systems and it is important that the assumptions behind the methods are recognized. Here, we present a new mathematical model that can be used to estimate phase variation rates in the presence of fitness differences, and explore the impact of proposed rates on population structure over time. It is also timely to compare and contrast some of the many different methods and terminology in this field. To put our approach in context, we begin with a brief review of the previous work in this field.

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Evaluation of methods for the estimation of mutation rates in cultured mammalian cell populations

Cited by 16 publications

References 7 publications

Rates of spontaneous mutation in an archaeon from geothermal environments

Rates of spontaneous mutation in an archaeon from geothermal environments

Embryonic stem cells and somatic cells differ in mutation frequency and type

Mutation rates: estimating phase variation rates when fitness differences are present and their impact on population structure

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