Kathleen A. Hill

Spontaneous mutations in the Big Blue® transgenic system are primarily mouse derived

The Big Blue transgenic mouse mutation detection system provides a powerful approach for measuring spontaneous and induced mutations in vivo. The observed mutations may contain a fraction of ex vivo or prokaryotic mutational events. Indeed, a modified, selectable form of the Big Blue assay seem to generate artifactual mutants under certain circumstances. Herein we review the evidence that circular mutants (i.e., the plaque circumference is at least 50% blue) collected in the standard Big Blue assay are derived primarily from the mouse. The most direct evidence is the similarity in the types of mutations found in jackpot and nonjackpot mutations. In addition, about half of the spontaneous mutations in the lacI transgene are transitions and transversions at CpG dinucleotides, a mammalian-specific feature. The mutation pattern observed at lacI is consistent with AT mutation pressure operating in a GC rich DNA and approaches that reported for observed germline human factor IX mutations. Furthermore, the spontaneous mutation pattern of circular Big Blue mutants differs significantly from that of an endogenous lacI gene in E. coli. Pinpoint mutants (a dot of blue color peripherally located in a wild type plaque), which a priori were not expected to be mouse-derived, have a mutation pattern consistent with the mutation pattern of an endogenous E. coli lacI gene. Analysis of induced mutagenesis studies reveals mutation frequencies and patterns for the Big Blue circular mutants which are comparable to endogenous genes. In reconstruction experiments, blue plaques derived from a superinfection with wild type and mutant phage produced approximately 50% blue and 50% clear plaques on replating. This phenomenon has not been seen when plaques derived from mouse were replated in the Big Blue assay. Collectively, the evidence strongly supports a murine origin for circular mutants recovered in the standard Big Blue assay. Validation of current assays is an essential step in determining the frequency and pattern of spontaneous murine-specific mutations. Defining this benchmark will be helpful in evaluating the next generation of transgenic mutation detection systems.

Toward efficient analysis of mutations in single cells from ethanol-fixed, paraffin-embedded, and immunohistochemically stained tissues.

Only a few studies have demonstrated successful molecular analysis after whole genome amplification using single cells dissected from paraffin-embedded tissues. The results in these studies were limited by low-amplification efficiency and high rates of allele dropout. In the present study, the amplification rate using a thoroughly modified primer extension and preamplification-PCR protocol was improved significantly for single cells microdissected from paraffin-embedded and immunohistochemically stained tissues. Tissue fixation with ethanol (85%) and the addition of 0.2 mmol/L EDTA helped to achieve an amplification rate between 67% (segments 200 to 400 bp) and 72% (segments <200 bp). Normal tissue sections were immunohistochemically double stained for overabundance of p53 protein and proliferating cell nuclear antigen. Microdissection of single cells was performed with a manual micromanipulator equipped with a Tungsten needle. Sequence analysis of the TP53 gene was performed after improved primer extension preamplification-PCR and multiplex PCR from single microdissected cells. The rate of allele dropout was at least 68%. These technical advances facilitate routine mutation analysis using a single cell or a few cells microdissected from routinely processed paraffin-embedded normal and tumor tissues. Allele dropout still represents a serious problem in single-cell mutation analysis, especially in samples with limited template DNA and prone to DNA damage.

Human germline mutation in the factor IX gene

The molecular epidemiology of factor IX germline mutations in patients with hemophilia B has been studied in detail because it is an advantageous model for analyzing recent germline mutations in humans. It is estimated that mutations have been defined in the majority of nucleotides that are the target for mutation. The likelihood that a factor IX missense mutation will cause disease correlates with the degree of evolutionary conservation of the amino acid. Mutation rates per base-pair have been estimated after careful consideration and correction for biases, predicting about 76 de novo mutations per generation per individual resulting in 0.3 deleterious changes. The male-to-female sex ratio of mutation varies with the type of mutation. There is evidence for a maternal age effect and an excess of non-CpG G:C to A:T transitions. The factor IX mutation pattern is similar among geographically, racially and ethnically diverse human populations. The data support primarily endogenous mechanisms of germline mutation in the factor IX gene. Mutations at splice junctions are compatible with simple rules for predicting disease causing mutations.

Spontaneous mutation frequency and pattern in Big Blue® mice fed a vitamin E-supplemented diet

Endogenous oxidative DNA damage caused by normal cellular processes may play a vital role in carcinogenesis. To directly test the hypothesis that antioxidants will protect DNA from oxidative damage in vivo, Big Blue® mice were fed either a control diet (66 IU vitamin E/kg diet) or a high‐dose vitamin E diet containing 1000 IU vitamin E/kg diet of racemic d,l‐α‐tocopherol acetate from conception until 3 months of age. Using the standard Big Blue® protocol, 15.5 million plaque forming units (pfu) were examined from five tissues (heart, liver, adipose tissue, thymus, and testis) of three control and three high‐dose vitamin E supplemented male mice generating 433 mutants, which represented 373 independent mutations upon sequencing the lacI transgene. The α‐tocopherol tissue concentration increased with high‐dose vitamin E supplementation. In four of the tissues, individually or combined, mutation frequency changed little if any with vitamin E supplementation. In adipose tissue, which accumulated the highest levels of vitamin E, mutation frequency was significantly reduced with high‐dose vitamin E supplementation (P = 0.047). Within the constraints of sample size, the pattern of mutation in adipose tissue was not altered significantly (P = 0.40). When data from all tissues were combined, a reduction in G:C → T:A transversions was observed (P = 0.044). These results may have implications for cancer chemoprevention and provide insight into the efficacy of vitamin E supplementation in reducing spontaneous oxidative DNA damage in vivo. More dramatic alterations of mutation frequency and pattern may be observed with higher doses of vitamin E and substitution of the racemic supplement with d‐α‐tocopherol acetate. Environ. Mol. Mutagen. 34:195–200, 1999

High plating density improves Big Blue® system efficiency without loss of sensitivity

To increase efficiency in the Big Blue system, the plating density was increased from 15000 to 30000 or 45000 plaque forming units (pfus) per plate by increasing the density of the E. coli lawn and decreasing individual plaque size. Small plaque size ensured minimal overlap of the plaques. Liver from one 3- and one 25-month-old mouse (low and high mutation frequencies, respectively) was analyzed and neither plating density nor plaque size affected mutant/mutation frequency and pattern. The color intensity of particular mutant plaques was not affected by plaque size or plating density. Optimal sensitivity is achieved by sequencing mutants to calculate the mutation frequency from the mutant frequency and to identify altered patterns of mutation. Detailed effort and cost accounting of the Big Blue system (including mouse handling, DNA extraction, plaque screening, plaque purification, and DNA sequencing) reveals that one-quarter of the total effort is devoted to plating and screening of plates. This effort is reduced two fold with high plating density. The total cost of the Big Blue system is reduced by 17%. The total cost of the High Plating Density Big Blue system is now only 12% more costly than a selectable assay and offers an extensively validated system with a large mutation database representing a decade of effort.