DR. KATHLEEN HILL
Mutation Scene Investigation
Tracking down mutagens and mutational mechanisms
Research in my laboratory is focused on tracking down the mutagens and mutational mechanisms responsible for the spontaneous mutations observed in all tissues over a lifespan. Spontaneous mutations are implicated in aging and aging-associated diseases such as cancer and neurodegeneration but the mutagens and mechanisms for these mutations are frequently unknown. Understanding the origins of spontaneous mutations is predicted to greatly assist in the design of effective strategies for antimutagenesis and ultimately disease prevention.
I focused my research efforts over the last 12 years on the use of a transgenic mouse mutation detection system to study spontaneous mutations in the soma and germline over the lifespan of the mouse. The work began as my postdoctoral training in the laboratory of Dr. Steve Sommer at the Mayo Clinic and City of Hope and more recently (2003) with the establishment of my research program back home in Canada at The University of Western Ontario.
Transgenic mice permit measurement of the frequency and types of mutations in individual tissues in vivo. Transgenic rodents containing a bacterial gene as a retrievable mutational target are useful in assessing the DNA landscape in the aftermath of exposures to endogenous and exogenous mutagens. These transgenic rodent systems permit quantitative measurements regarding mutation frequency and qualitative analysis regarding the types of mutation. It is possible to characterize the predominant types of endogenous mutations and distinguish novel mutagen signatures. A well-validated system is the Big Blue® transgenic mouse mutation detection assay (Stratagene). The E. coli lacI gene within the lambda phage genome was integrated in the Big Blue mouse genome as a multicopy concatemer on chromosome 4. The lacI sequences can be retrieved from high molecular weight DNA, packaged into virulent phage and used individually to infect E. coli. Those lambda phage genomes containing a wild type lacI sequence produce a clear plaque in the bacterial lawn and those lambda phage genomes harbouring a mutation in the lacI sequence due to a mutational event in the mouse produce a blue plaque in the E. coli lawn. In this genetic screening assay, the blue “mutant” plaque phenotype is the result of the dysfunctional lacI repressor protein that fails to repress the expression of lacZ leading to production of B-galactosidase and cleavage of the chromogenic substrate X-gal in the growth media. Mutations that occur in the mouse are harvested as blue mutant plaques and their frequency compared to the total number of viral genomes harvested is the measure of mutant frequency. The individual mutations are identified following DNA sequencing of the lacI gene and the frequency of individual mutational events is estimated. Transgenic mouse mutation detection systems are currently a national and international standard assay for assessment of in vivo mutation frequency and pattern in individual tissues. A recent review by a team of Canadian researchers is an excellent source of information regarding transgenic rodent mutation detection systems 1
The Big Blue system contributes novel observations that contradict conventional thought on spontaneous mutagenesis. We collaborate with Steve Sommer’s group at The City of Hope and analyze a large database of somatic and germline spontaneous mutations collec ted using the Big Blue® mutation detection assay. We have the ability to compare these mouse mutations with a database of germline mutations in human factor VIII and IX genes.
Here are some of our “Mutation Scene Investigations” that change our thinking in the field of mutagenesis.
The case of paternal age: Spontaneous mutation frequency in male germ cells over the lifespan of the mouse shows no evidence of a paternal age effect 2,3. The frequency of base substitutions and small deletions and insertions in the Big Blue mutation target in the male germ cells is constant from young adulthood into old age. This is consistent with the lack of evidence for a paternal age effect in a detailed analysis of paternal origins of mutation in the human factor IX gene in cases of hemophilia B. These observations are particularly intriguing given the ongoing controversy over whether or not increasing germ cell mutation frequency with paternal age underlies the reports associating older males with a higher incidence of some types of genetic disease.
The case of cell division: The frequency of spontaneous mutations in numerous murine somatic tissues is constant from young to middle adulthood and similar across eight somatic tissues2,3. Our original hypothesis was that a more than three-fold change in age and a broad spectrum of tissue types with different rates of cell proliferation would reveal age and tissue specific differences in mutation frequency. We observed no evidence of an association between mutation frequency and rates of cell division.
The case of a known mutation hotspot: At last year’s Genetics Society of Canada Annual Meeting students in my laboratory reported a mutation pattern in epithelial tissues not typical of other somatic tissues examined to date. Tissues primarily composed of epithelial cells showed a significant elevation in transitions at CpG dinucleotides, a known hotspot of spontaneous mutation in vertebrate species. Students in the Hill laboratory remain in pursuit of the mutagen or mutational process preferentially affecting epithelial tissues.
The case of a clear mutagen signature: Spontaneous tandem-base mutations (TBM) in mice occur at a frequency of 10 -7 and show age, tissue, pattern and spectrum specificity 4. TBM have a frequency significantly higher than that expected for multiple independent mutational events (10 -10). TBM are rare in germ cells, increase with age in liver and adipose tissue and, in mice, show strong preference for GG to TT transversions. The endogenous mutagen capable of this distinct mutation signature remains unknown.
The case of misleading semantics: The term “indel” has held several definitions but one definition has strongly influenced our thinking about the origins of what many researchers call “complex” mutations. A DNA sequence 5’CCGTGCTT3’ with the following mutation 5’CATGCTT3’ was thought to be a combination of a single-base insertion (A) and a two-base deletion (CG). However, our analysis of a collection of these types of mutations revealed several lines of evidence that a simple deletion and insertion combination mechanism was not at all likely 5-7. Single base insertions almost always (97% of the time) extend or create a monobasic run but this is rarely the case for insertions in indels. Moreover, the small deletions in indels have a significantly broader size distribution than the size of pure deletions. The distinct features of insertions and deletions in indels implicate a diversity of mutational mechanisms not observed in pure insertions and pure deletions.
The case of the larger scene: In the Hill laboratory we extended the “Scene of Investigation” to include the entire genome.Our team collaborates with researchers in computer science and bioinformatics to analyze mutagenesis in the greater context of global DNA sequence structure. We applied Chaos Game Representation to the analysis of DNA sequence organization and revealed genome specific signatures in the organization of the DNA sequence. Genomes are not composed of a random or equal frequency of all possible subsequences but instead show dramatic biases in short sequence composition. We started this study in 1990 prior to the era of genome sequencing and bioinformatics but resumed an in depth Chaos Game Representation of genome sequences in the now “post genomic” era. We now have the ability to quantitate all genomic signatures 10. We now investigate mutations in the context of global genome structure. We hypothesize that patterns of spontaneous mutation are restricted by constraints of global genome structure.
The case of a clustered scene: An interesting feature of non-tandem multiple mutations is proximal spacing 8,9. Double and multiple mutations that are not in tandem show clustering with a median mutation spacing of 120 base pairs. These multiple mutations occur at a frequency (10 -7) that is consistent with a single mutational event occurring in a single cell cycle. We observe that spontaneous multiple mutations do not show a mutator phenotype characterized by high frequency of randomly spaced multiple mutations. Instead, spontaneous multiple mutations show proximal spacing and likely occur in rapid succession in a single cell cycle. In our most recent report, we tested a hypothesis that multiple mutations observed in the lacI mutation target sometimes occur in the context of multiple mutations spanning multiple kilobases. This phenomenon we termed “mutation showers”. Indeed we found evidence for mutation showers in the form of multiple clusters of multiple mutations, with approximately 30 kilobases of the DNA landscape affected by a single “mutation shower”. The most interesting corollaries of the evidence for mutation showers are the potential for “cancer in an instant” and an evolutionary function for "introns as sponges to reduce the deleterious impact of mutation showers."
The Big Blue transgenic mouse mutation detection system has been instrumental in allowing our researchers access to the in vivo mutation scene. The data clearly contradicted conventional thinking in some cases and led to refined hypothesis in others. We look forward to each new assignment and the challenge of a cold case. We are eager to sift through the new clues that mutations have yet to reveal. We continue to interrogate the DNA landscape in pursuit of what we are unable to directly observe; the origins and mechanisms of mutation. Our refined hypotheses and solved cases will directly impact on our understanding of the origins of cancer, the process of aging and evolution of genomes.
1 Lambert, I.B., Singer, T.M., Boucher, S.E. and Douglas, G.R. (2005) Mutat Res 590, 1-280
2 Hill, K.A. et al. (2004) Environ Mol Mutagen 43, 110-20
3 Hill, K.A. et al. (2005) Environ Mol Mutagen 45, 442-54
4 Hill, K.A., Wang, J., Farwell, K.D. and Sommer, S.S. (2003) Mutat Res 534, 173-86
5 Halangoda, A., Still, J.G., Hill, K.A. and Sommer, S.S. (2001) Environ Mol Mutagen 37, 311-23
6 Hill, K.A. et al. (2006) Hum Mutat 27, 55-61
7 Gonzalez, K.D. et al. (2007) Hum Mutat 28, 69-80
8 Buettner, V.L., Hill, K.A., Scaringe, W.A. and Sommer, S.S. (2000) Mutat Res 452, 219-29
9 Hill, K.A. et al. (2004) Mutat Res 554, 223-40
10 Wang, Y., Hill, K., Singh, S. and Kari, L. (2005) Gene 346, 173-85
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April 8, 2010
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