Just how does the cII selection system work in Muta Mouse?

The lambda CII protein is an essential component in the lytic vs. lysogeny decision a bacteriophage makes upon infection of a host at low temperatures. The protein interacts with numerous phage promoters modulating the expression of the CI repressor, thus providing the mechanism for lysogenization soon after infection. The Big Blue and Muta Mouse are two widely used in vivo mutational model systems. The assays rely on retrievable lambda-based transgenes housing mutational targets (lacI or lacZ, respectively). The transgenes provide an elegant vehicle for the quantification of mutations sustained in virtually any tissue of the rodent. The use of the bacteriophage cII locus as an alternative, or additional mutational target for use with the Big Blue rodent system was first reported by Jakubczak et al. ([1996]: Proc Natl Acad Sci USA 93:9073-9078). More recently, this selection assay has been applied successfully to the Muta Mouse (Swiger et al. [1999]: Environ Mol Mutagen 33:201-207). The use of an Hfl bacterial strain and low temperature allows the determination of mutations sustained at the cII locus in either system, with high fidelity. The cII selection assay in the Big Blue relies on the presence of the lambda repressor protein CI. In contrast, the recombinant construct used to make the Muta Mouse transgene lacks functional CI protein. Nevertheless, we report an excellent system for quantifying mutations at the cII locus in Muta Mouse. Just how does cII selection work in the Muta Mouse? Written in the context of lambda recombinant genetics, this paper explores the question further.

Further characterization and validation of gpt delta transgenic mice for quantifying somatic mutations in vivo.

The utility of any mutation assay depends on its characteristics, which are best discovered using model mutagens. To this end, we report further on the characteristics of the lambda-based gpt delta transgenic assay first described by Nohmi et al. ([1996]: Environ Mol Mutagen 28:465-470). Our studies show that the gpt transgene responds similarly to other transgenic loci, specifically lacZ and cII, after treatment with acute doses of N-ethyl-N-nitrosourea (ENU). Because genetic neutrality is an important factor in the design of treatment protocols for mutagenicity testing, as well as for valid comparisons between different tissues and treatments, a time-course study was conducted. The results indicate that the gpt transgene, like cII and lacZ, is genetically neutral in vivo. The sensitivities of the loci are also equivalent, as evidenced by spontaneous mutant frequency data and dose- response curves after acute treatment with 50, 150, or 250 mg/kg ENU. The results are interesting in light of transgenic target size and location and of host genetic background differences. Based on these studies, protocols developed for other transgenic assays should be suitable for the gpt delta. Additionally, a comparison of the gpt and an endogenous locus, Dlb-1, within the small intestine of chronically treated animals (94 microg/mL ENU in drinking water daily) shows differential accumulation of mutations at the loci during chronic exposure. The results further support the existence of preferential repair at endogenous, expressed genes relative to transgenes.

Dietary restriction during murine development provides protection against MNU-induced mutations.

The developmental stage is the most rapid period for the accumulation of somatic mutations. Epidemiological studies have also suggested a significant role of early life for cancer susceptibility, showing a protective effect of modest dietary restriction early in life. To determine if mutation rate, diet, and cancer risk are related, we have investigated the effect of dietary restriction on somatic mutations early in life. The diet of mouse dams was restricted during pregnancy and lactation by 10% from ad libitum control. F(1) pups (SWRxMutaMouse) were weaned at 3 weeks of age. Pups from dams that were on a restricted diet were kept under dietary restriction (40% until 5 weeks of age and then 20% until sacrifice). Only females from litters of seven or eight were used in this study. A portion of pups from both groups were treated with N-methyl-N-nitrosourea (MNU, 50mg/kg, i.p.) at 5 weeks of age and all mice were sacrificed at 10 weeks of age. The frequency of induced mutations was reduced by about 30% at the three loci studied, lacZ (P=0.028) and cII (P=0.042) and Dlb-1 (P=0.032) in the small intestine in the restricted group. A similar decrease in the lacZ mutant frequency was observed in the bone marrow, but the results did not reach statistical significance (P=0.074). Few differences in the lacZ mutant frequency were observed in the colon and the mammary epithelium, but variability of the mutant frequencies was such that an effect of similar magnitude could not be excluded statistically. Analysis of 47 cII mutants revealed that the majority of MNU-induced mutations were G:C to A:T transition at non-CpG sites, with no difference in the mutation spectrum between the two dietary groups.

The cII locus in the MutaMouse system.

Here, we report the first application and characterization of the cII locus as a mutational target for use with the Muta(trade mark)Mouse system for quantifying somatic mutations in vivo. This locus can be analyzed for mutations using positive selection and is identical in sequence to the cII in the Big Blue((R)) Mouse. The cII displays similar spontaneous (5.5 x 10(-5)) and induced mutation frequencies when compared to the lacZ gene in the small intestine of MutaMice treated with ENU (N-ethyl-N-nitrosourea). After acute treatment with 250 mg/kg ENU (ip) the mutant frequencies were 127 x 10(-5) at the cII and 147 x 10(-5) at the lacZ loci, reaching a maximal mutant frequency 10 days posttreatment and remaining constant thereafter. These data prove that this transgene is genetically neutral, conferring neither selective advantage nor disadvantage on the host cells. The cII dose response curve was linear (R(2) = 0.93) comparable to the lacZ after treatments with 0, 50, 150, or 250 mg/kg ENU. Use of the cII locus (0.3 kb) addresses the single most significant drawback associated with the MutaMouse system, namely the inability to obtain sequence spectra efficiently, due to the large size of the lacZ gene (3.0 kb). Moreover, a less obvious application, but nevertheless of considerable importance, is the easy identification of jackpot mutations, without sequencing. The cII, identical in both sequence and origin on the transgenic constructs used in producing the Big Blue and MutaMouse systems, provides the first transgenic locus common to the two widely used in vivo mutagenesis assays.

The persistence of aberrations in mice induced by gamma radiation as measured by chromosome painting.

Fluorescence in situ hybridization, or chromosome painting, has become an invaluable tool in the cytogenetic evaluation of historical or chronic exposure because it can be used to detect stable genetic damage, such as translocations, which persist through cell division, quickly and easily. The recent development of chromosome-specific composite DNA probes for the mouse has allowed the use of chromosome painting in this commonly used animal model. In order to measure the persistence of radiation-induced translocations, C57BL/6 female mice were given a whole body acute dose of 0, 1, 2, 3 or 4 Gy 137Cs gamma rays at 8 weeks of age. Metaphase chromosomes from both peripheral blood and bone marrow cells were obtained from four mice in each dose group at 1, 8, 15 and 30 days post-irradiation. Chromosomes 2 and 8 were painted, while the remaining chromosomes were counterstained with propidium iodide. DAPI counterstain was used to differentiate between translocations and dicentrics because it brightly labels the centromeric heterochromatin. The equivalent of 100 cells from each tissue was scored from each mouse. The results show that the percentage of reciprocal translocations, at least at doses of 3 Gy or lower, did not decrease with time in either tissue. In contrast, the frequency of non-reciprocal translocations induced by doses of 3 Gy or lower, remained unchanged in the peripheral blood, but decreased after a week in the bone marrow, then remained constant. An increase in these two types of aberration was observed between 15 and 30 days in the bone marrow and may have been due to clonal expansion. Dicentrics decreased with time in both tissues, almost none remained in the bone marrow after 8 days. These data suggest that reciprocal translocations are persistent and will serve as an effective biodosimeter for radiation exposure.

Risk estimation from somatic mutation assays.

The ability to quantify somatic mutations in vivo provides a new source of toxicological information that is relevant to the assessment of cancer risk. The major experimental factors that influence the mutant frequency are age, time after treatment, treatment protocol, and tissue analyzed. In untreated mice, the mutant frequency increases very rapidly with age from conception to birth, more slowly from birth to adulthood, and very slowly thereafter. All somatic tissues studied so far in adults have similar mutant frequencies. The time after treatment (expression time) is the most important experimental variable. The minimum time for expression varies from one tissue to another. To be valid, comparisons between tissues and treatments must be made after complete expression of the mutations. Unfortunately, the minimum expression time has not been characterized in most tissues. Since carcinogens are tissue specific, and many chemicals are distributed in the body in complex patterns, it is to be expected that there will be differences in the frequency of mutation induced in different tissues. As yet this has not been extensively studied. Since the mutations detected by the transgenic assays are neutral, the mutants should accumulate as the integral of the mutation rate. Hence chronic treatment protocols should be more effective than acute and subacute protocols whenever they permit substantially larger doses to be delivered. Such protocols are more relevant to human exposure and are preferable for dose extrapolations. The importance of transcription in determining mutation rates is not yet known, but it is noteworthy that the transgenes are not transcribed whereas the Ioci involved in carcinogenesis are. The mutation spectrum is important for quantitative risk estimation. Risk estimation must also take into account the spectrum of mutations that are involved in the carcinogenic process in the tissue and the spectrum of mutations that are detectable by the assay. New assays are being used to quantify mutations in vivo in order to understand the carcinogenic process, to search for the environmental factors involved in human cancer, and to evaluate the carcinogenic hazard qualitatively.

Activity banding of human chromosomes as shown by histone acetylation.

The expression of genes in mammalian cells depends on many factors including position in the cell cycle, stage of differentiation, age, and environmental influences. As different groups of genes are expressed, their packaging within chromatin changes and may be detected at the chromosomal level. The organization of DNA within a chromosome is determined to a large extent by the positively charged, highly conserved histones. Histone subtypes and the reversible chemical modifications of histones have been associated with gene activity. Active or potentially active genes have been associated with hyperacetylated histones and inactive genes with nonacetylated histones. Sodium butyrate increases the acetylation levels of histones in cell cultures and acts as both an inducer of gene activity and as a cell-cycle block. We describe a method to label the interphase distribution of DNA associated with various histone acetylation stages on chromosomes. Nucleosomes from untreated and butyrate-treated HeLa cells were fractionated by their acetylation level and the associated DNA labeled, and hybridized to normal human chromosomes. In the sodium butyrate-treated cells the resulting banding patterns of the high- and low-acetylated fractions were strikingly different. DNA from low-acetylated chromatin labeled several pericentric regions, whereas hybridization with DNA from highly acetylated chromatin resulted in a pattern similar to inverse G-bands on many chromosomes. The results from noninduced cells at both high and low acetylation levels were noticeably different from their induced counterparts. The capture and hybridization of DNA from interphase chromatin at different acetylation states provides a "snapshot" of the distribution of gene activity on chromosomes at the time of cell harvest.

Cytogenetic analysis of mice chronically fed the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine.

The cytogenetic effects in mice chronically fed the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP) were evaluated by chromosome painting, micronucleated normochromatic erythrocytes (MN NCEs) and sister chromatid exchanges (SCEs). PhIP and numerous other heterocyclic amines have been isolated from cooked foods, and many have been found to be carcinogenic in laboratory rodents. Female C57BL/6N mice were chronically fed a diet containing 0, 100, 250 or 400 ppm of PhIP beginning at 8 weeks of age. Peripheral blood and bone marrow were taken from 5 mice per treatment group at 1, 4 and 6 months from the start of exposure. PhIP was removed from the diet for a final month of the experiment, at which time blood was taken from the remaining animals. Chromosome-specific composite DNA probes for mouse chromosomes 2 and 8 were hybridized to metaphase cells from each tissue. The 1- and 4-month time points showed no statistically significant difference between the control and exposed mice for either tissue in chromosome aberration frequencies. Both MN NCEs and SCEs were analyzed at a single time point during exposure (4 months for MN NCEs and 6 months for SCEs) and again 1 month after removing PhIP from the diet. MN NCEs in the peripheral blood showed a statistically significant dose response, with all values decreasing significantly 1 month after removing PhIP from the diet. SCE frequencies in the peripheral blood showed an approximate doubling compared to control mice, and decreased to control levels 1 month after removing PhIP from the diet. SCE frequencies in the bone marrow of exposed mice showed no difference from the control animals. These results show that chronic ingestion of PhIP by female C57BL/6 mice does not produce persistent cytogenetic damage as visualized by chromosome aberrations, MN NCEs or SCEs.

Fluorescence in situ hybridization: a brief review.

Fluorescence in situ hybridization (FISH) is used for many purposes, including analysis of chromosomal damage, gene mapping, clinical diagnostics, molecular toxicology and cross-species chromosome homology. FISH allows an investigator to identify the presence and location of a region of cellular DNA or RNA within morphologically preserved chromosome preparations, fixed cells or tissue sections. This report describes in situ hybridization, and discusses the past, present and future applications of this method for genetic analysis and molecular toxicology.

Why do stem cells exist?

Self-renewing tissues have a differentiation hierarchy such that the stem cells are the only permanent residents of the tissue, and it is in these cells that most cancerous mutations arise. The progeny of the stem cells either remain stem cells or enter a transient proliferating cell population that differentiates to produce the functional cells of the tissue. The reason that this differentiation hierarchy exists has not been established. We show here that alternative hierarchies, in which there would be no stem cells, are feasible and biologically plausible. We show that current evidence from somatic mutation frequencies at both transgenic and endogenous loci implicates cell division in the origin of most somatic mutations. We suggest, therefore, that the existence of stem cells is an evolutionary consequence of a selective pressure to avoid cancer by reducing the number of somatic mutations. The stem cell hierarchy reduces the number of cell divisions of those cells that reside permanently in the tissue, which reduces the number of somatic mutations and thus minimizes the cancer rate. In the small intestine, the existence of stem cells reduces the mutant frequency in the stem cells by about one order of magnitude. Since two or more mutations are required to transform a cell, the protective effect may be 100-fold or more. Similar factors may be expected in other tissues.

The transmission rate of the lacI transgene from the Big Blue mouse.

Since transgenic mice are being used to analyze somatic and germinal mutation rates in vivo, it is of interest to know to what extent these mice are normal or abnormal in any way. During experiments designed to compare the mutational response of the transgene and an endogenous gene, Big Blue mice hemizygous for the transgene were bred to create a hybrid mouse in which the comparisons could be made. The fraction of these mice that inherited the transgene was 37% rather than the Mendelian expectation of 50%.

The development of painting probes for dual-color and multiple chromosome analysis in the mouse.

The recent development of mouse chromosome painting probes for fluorescence in situ hybridization has extended the use of this common laboratory mammal in cytogenetics. We now report the development of additional painting probes by degenerate-oligonucleotide-primed PCR on chromosomes from mouse lung fibroblast cultures, each homozygous for a single Robertsonian translocation chromosome. These probes are for Rb(1.2), Rb(1.3), Rb(4.6), and Rb(6.7). Probes were also made for the sex chromosomes by isolating shoulders from larger peaks (X) or small, clearly resolved peaks (Y) in the flow karyotype. Combinations of probes were used to paint four chromosomes simultaneously in a single color. Multicolor painting was achieved with a biotinylated Rb(1.2) probe and a digoxigenin-labeled Rb(2.8) probe. Each of the three different homologous pairs was uniquely colored by avidin-Texas Red, anti-digoxigenin-FITC, or both simultaneously. These results extend the usefulness of the mouse as a model for understanding adverse environmental exposures and genetic diseases in humans.

The LacZ transgene in MutaMouse maps to chromosome 3.

Transgenic mouse models are being used with increasing frequency for mutational and toxicological studies. One such system. MutaMouse, contains a stably integrated lambda-gt10LacZ shuttle vector in the mouse genome. We describe the use of dual color fluorescence in situ hybridization (FISH) with Mus musculus whole chromosome paints and lambda DNA to map the integration site of the lambda transgene to band C on mouse chromosome 3.

Isolation and characterization of mouse Xrcc-1, a DNA repair gene affecting ligation.

Human DNA repair gene XRCC1 complements the strand-break rejoining defect in Chinese hamster mutant EM9 and encodes a protein that is apparently required for optimal activity of DNA ligase III. Toward the goal of producing transgenic mice that carry a mutation in the Xrcc-1 locus, the murine homolog of XRCC1 was cloned from both cosmid genomic and cDNA libraries. Upon transfection into EM9 cells, cosmids containing the functional mouse gene efficiently corrected (94-100%) the high sister-chromatid-exchange defect. Mouse Xrcc-1 is 26 kb in length, contains 17 exons, and maps by metaphase in situ hybridization to the 7A3-7B2 region of mouse chromosome 7. Isolated cDNA clones were highly truncated and were extended by anchored polymerase chain reactions. The 1893-bp open reading frame of mouse Xrcc-1 encodes 631 amino acids, compared with 633 for the human homolog. The predicted mouse Xrcc-1 protein of 69.1 kDa and pI of 5.95 is 86% identical and 93% similar to human XRCC1.