Biomedical subjects
Publications and source records attributed to J M Short.
Enzymatic synthesis of galactobiosides using a versatile CLONEZYME thermostable glycosidase library was studied. One-pot transglycosylation reactions were demonstrated to synthesize beta(1-->4), beta(1-->6), and alpha(1-->6) disaccharide sequences with high regioselectivity and moderate to high yields.
Aquifex aeolicus was one of the earliest diverging, and is one of the most thermophilic, bacteria known. It can grow on hydrogen, oxygen, carbon dioxide, and mineral salts. The complex metabolic machinery needed for A. aeolicus to function as a chemolithoautotroph (an organism which uses an inorganic carbon source for biosynthesis and an inorganic chemical energy source) is encoded within a genome that is only one-third the size of the E. coli genome. Metabolic flexibility seems to be reduced as a result of the limited genome size. The use of oxygen (albeit at very low concentrations) as an electron acceptor is allowed by the presence of a complex respiratory apparatus. Although this organism grows at 95 degrees C, the extreme thermal limit of the Bacteria, only a few specific indications of thermophily are apparent from the genome. Here we describe the complete genome sequence of 1,551,335 base pairs of this evolutionarily and physiologically interesting organism.
Previously, we developed a model for high incidence, endogenously generated hepatocellular carcinoma (HCC), the human alpha-1-antitrypsin (alpha1AT) Z gene transgenic mouse (Z#2). We now examine the potential utility of a model for endogenous carcinogenesis utilizing the Z#2 mouse also transgenic for the lacI gene, a convenient target for in vivo mutagenesis studies. We crossed the Z#2 line and mice transgenic for lambda/lacI shuttle vector (Big Blue), for determination of lacI mutant frequency during initiation of endogenous carcinogenesis. Five month old double transgenic mice (Z#2+/lacI+) successfully displayed: (1) the expected post-inflammatory stage of Z#2 carcinogenesis; and (2) hepatic lacI mutants measured at frequencies (10(-5)-10(-4)) useful to mutagenesis studies. In this study, hepatic lacI mutation frequencies in Z#2 transgenic mice appeared to be only slightly increased (< 2x) when compared to age matched negative controls. In the future, it may be important to reconcile possibly limited lacI mutagenesis at the time of initiation and demonstrated high incidence of hepatocarcinogenesis.
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Transgenic animals offer a powerful tool to study the mechanisms of spontaneous and induced mutagenesis in vivo. Herein we used a test version of a growth selectable assay to obtain spontaneous mutants in a lacI target transgene recovered from lacI transgenic B6C3F1 mice (Big Blue). This selection system may have certain advantages relative to the more established plaque screening system for mutation detection because: (1) the plating density of the phage is up to 60 times higher in the selectable assay, reducing the number of plates needed to be screened for a comparable amount of mutants; and (2) the mutant frequency obtained from the selectable assay is higher compared to the plaque assay, possibly due to a higher sensitivity for weaker mutants. However, the longer incubation time of the growth selectable assay might allow E. coli host derived mutants to appear. To address this issue, we investigated the sequence changes in the amino-terminal domain of the lacI gene of 405 mutants derived from the liver, spleen, brain, germ cells and skin of five untreated 6-week-old mice. The mutant colonies were isolated after 60, 84, 108 and 150 h of incubation under growth selectable conditions. Tissue-specific differences in the mutational pattern obtained after 60 and 84 h disappear after a longer time of incubation, possibly due to an increasing contribution of E. coli derived mutants. The evolving selectable systems offer the potential to increase screening efficiency, but the results suggest caution in interpreting data from this system because repair by E. coli of DNA lesions or mismatched heteroduplexes either originating in mouse in vivo or produced by ex vivo manipulation as well as de novo mutations in E. coli might contribute significantly to the observed mutational spectra at each timepoint.
The genotoxic response of benzene and tris(2,3-dibromopropyl)-phosphate (TDBP) have been evaluated in several tissues using the standardized lambda/lacI (Big Blue) transgenic mouse mutation assay. Separate groups of four to five male B6C3F1 transgenic lambda/lacI mice were given oral administrations of benzene or TDBP at varying concentrations. Tissues evaluated include lung, bone marrow, and spleen in benzene-treated animals, and liver, kidney, and stomach in TDBP-treated animals. Significant increases in lacI mutations were observed in the spleen and bone marrow of benzene treated mice, and the kidneys of TDBP-treated mice. Where applicable, mutagenesis patterns of tissue sensitivity were consistent with what has been observed previously in other assays. In addition, mutagenicity in tissues not traditionally evaluated for mutations correlated to sites of carcinogenicity for the chemicals tested.
Elements of the lactose operon were used to study parameters affecting gene expression in cultured cells and transgenic animals. A Lac repressor protein containing a nuclear transport signal was shown to inhibit expression of a reporter gene by interacting with lac operator sequences. In cultured cells, operator sequence, operator placement and induction parameters were all shown to be important for obtaining tight repression of a reporter gene followed by high level expression upon induction. Induction levels were also dependent on the reporter gene, with the luciferase gene yielding higher induction levels than the chloramphenicol acetyltransferase gene. In transgenic animals, the lacI mRNA was not detected in the C57BL/6 mouse strain until the animal was exposed to a demethylating agent. After 5-azacytidine treatment, expression of lacI mRNA was detected in the brain, heart, kidney, lung and ovary. In the FVB transgenic mouse strain, expression of lacI mRNA was detected without 5-azacytidine treatment in the kidney, liver, lung, and testes. Preliminary experiments with double transgenic animals containing both lacI and operator/luciferase transgenes showed a decrease in luciferase expression compared to the luciferase-only animals in both tissue extracts and transgenic fetal primary cultures, although IPTG induction was not achieved in these animals or primary cultures. The applicability and challenges of the system for regulation of gene expression are discussed.
Spontaneous mutant frequency in livers of two transgenic mouse strains, each carrying identical lambda shuttle vectors with a lacI target gene, was evaluated by two laboratories. These studies investigated variability in spontaneous mutant frequency between animals and as a function of the number of phage screened. Liver DNA was independently isolated from 7-11 week old C57BL/6 and B6C3F1 Big Blue transgenic mice. At least 500,000 phage were screened for mutation at lacI for each animal using standardized assay procedures. In the two labs, the C57BL/6 liver spontaneous mutant frequency was 45 +/- 9 x 10(-6) and 41 +/- 7 x 10(-6). The B6C3F1 liver spontaneous mutant frequency was 42 +/- 10 x 10(-6) at one lab and 43 +/- 12 x 10(-6) and 41 +/- 8 x 10(-6) in two trials at the second lab. Mean mutant frequency data from both labs, calculated in increments of 100,000 plaque forming units (pfu) scored for each mouse strain, show stabilized mean mutant frequency and standard deviation after approximately 200,000-300,000 pfu screened. The frequency of spontaneous lacI mutants was reproducible both within and between labs and was comparable between the two transgenic mouse strains.
A lambda/lacI shuttle vector transgenic mouse mutagenesis assay has been optimized and standardized for reproducible mutant detection. The mutagenic endpoints are blue lacI- phage plaques on a bacterial lawn resulting from the de-repression of beta-galactosidase activity acting on the chromogenic substrate X-gal. Non-mutant lacI phage plaques remain colorless. Factors demonstrated to affect mutant detection include X-gal concentration per assay tray, plaque density per assay tray, pH of plating agar, incubation time at 37 degrees C and the use of a red translucent screening filter over a light source to enhance mutant plaque visibility. In vivo mutant frequencies for liver in untreated animals using standard protocols and internal controls were repeatable in separate experiments using lambda/lacI B6C3F1 mice (4.3 +/- 1.2 x 10(-5) and 4.1 +/- 0.8 x 10(-5)). These studies analyze the use of internal controls to monitor the level of mutant phage plaque detection in a given experiment and evaluate the repeatability of observed mutant frequencies obtained when using standardized procedures.
Statistical analysis of the lambda/lacI transgenic mutagenicity assay was used to determine optimal sample size and resource allocation in terms of number of animals and number of recovered target genes (recovered phage) required to demonstrate a statistically significant induction in mutant frequency. Statistical assumptions as applied to mutagenicity data are discussed for a number of frequently used statistical analyses. Log transformations are suggested as a means of meeting statistical assumptions and examples are given on interpreting results of analyses of log transformed data. The data analyzed in this study indicate that 300,000 lambda plaques from each of five animals should be analyzed per treatment group in order to detect a doubling of mutant frequencies. Additional sensitivity is gained primarily through increase of animal number and not the number of phage rescued, due to inherent animal-to-animal variability.
A rat embryo cultured cell line was generated that carries stably integrated copies of a lambda/lacI shuttle vector, containing the lacI gene as a mutational target. After the desired treatment of the cells, this vector can be rapidly and efficiently recovered from the cell DNA by in vitro packaging and then screened for mutations in the lacI gene, using bacterial detection systems. The vector is identical to that integrated into the Big Blue transgenic mouse, which was developed for in vivo mutation analysis. Characterization of the cell line by fluorescence in situ hybridization showed that the phage DNA is integrated at two distinct sites on separate chromosomes at approximately 50-70 copies per cell and the cell line is polyploid. The rescue efficiency is approximately 100,000 pfu/micrograms of genomic DNA. To examine the ability of the cell line to detect mutations in the lacI gene, the cells were treated with 100 micrograms/ml of the direct-acting alkylating agent N-methyl-N-nitrosourea (MNU) for 30 min at 37 degrees C and grown to confluence. The shuttle vector was rescued from untreated and mutagen treated cells, and spontaneous and induced mutant frequencies were determined to be 4.0 x 10(-5) and 92.7 x 10(-5), respectively. The cell line can be used to detect mutations in the lacI gene, followed by recovery of mutants for sequence analysis. The cell line may be valuable for short-term in vitro mutagenesis studies, oncogene and tumor suppressor studies, and DNA repair studies.
The pattern of somatic mutation observed in tumor suppressor genes, such as the p53 gene, vary dramatically with tumor type. Some of the observed differences are due to tissue specific effects of mutagens, but it is also possible that some differences may reflect the tissue/cell type specificity of spontaneous mutation. Transgenic mouse models with recombinant shuttle vectors containing the lacI or lacZ target genes may shed light on the extent to which spontaneous mutation displays tissue specificity. Herein we utilize a recently described selectable system to obtain spontaneous mutants for analysis of the molecular lesions. Spontaneous mutations were isolated in the lacI gene recovered from five transgenic mice carrying a lambda shuttle vector. Seventy-three and 67 independent mutations derived from liver and spleen DNA, respectively, were defined in the amino terminal region of lacI. Although technical barriers preclude a direct assessment of the E. coli derived pattern of mutation in this system, five pieces of circumstantial evidence suggest that many of the mutations arose in mouse rather than in E. coli. In DNA from both liver and spleen, mutations at CpG dinucleotides predominate (58% and 51%, respectively). In spleen, most of the mutations at CpG are transitions, while in liver most are transversions. In addition, liver has a higher frequency of GC-->TA transversions at non-CpG dinucleotides while spleen had a higher frequency of deletions and insertions. The data provide evidence that the spontaneous pattern of mutation is tissue specific. In addition, the high frequency of transversions at CpG suggests the need to reevaluate the mechanisms by which mutations occur at this methylated dinucleotide.
Transgenic B6C3F1 mice carrying a lacI target gene were exposed to acute and multiple doses of ethylnitrosourea (ENU), and germ cells from the seminiferous tubules were assayed for mutation 3 and 90 days after treatment. Relative to untreated controls, the mutation frequency increased 3.2- and 19.9-fold at 3 and 90 days after treatment, respectively. Mutant lacI genes recovered from untreated and treated groups were sequenced, and the spectra of mutations were determined. Eighty-five percent (11/13) of the spontaneous mutations resulted in G.C-->A.T transitions, all of which occurred at CpG dinucleotides. Fifteen of 22 sites (68%) found mutated 3 days after ENU treatment occurred at G.C base pairs, although some of these are expected to be spontaneous mutations. Ninety days after treatment, 13 of 19 sites (68%) found mutated occurred at A.T base pairs. The mutation spectra seen are consistent with proposed mechanisms of ENU mutagenesis and correlate with the in vivo spectra seen in ENU studies by using transmissibility assays and the hprt gene. These findings represent significant progress toward defining the in vivo spectra of ENU mutagenesis in mammalian germ cells.
The establishment in recent years of transgenic shuttle vector-based mutagenicity assays has provided improved systems for analysis of mutagenic and carcinogenic processes. Results in the mouse have stimulated the development of an alternate species suitable for mutation analysis and have increased our understanding of the existing models. A previously described shuttle vector (lambda LIZ), based on a lacI target gene, was constructed in this laboratory for the study of mutagenesis in transgenic mice and in cultured cell lines. The shuttle vector allows for several options in its recovery from the host genome and in mutant identification. Of the 9 transgenic lineages that were generated with the lambda LIZ vector, one was chosen for use in a standardized mutagenicity assay (Big Blue, mouse lineage A1). Characterization of this lineage included copy-number determination, chromosomal localization of transgene integration and analysis of copy-number stability. As part of the validation process, the standardized color-screening assay has been tested in the mouse, both for spontaneous mutant frequencies and with a variety of model mutagenic compounds, and has been shown to identify most major classes of mutations as evidenced by mutant spectra data. A discussion of the relative sensitivity of the shuttle vector to each of these classes of mutations is included. These studies have now been extended to the generation of transgenic rats containing the same shuttle vector for cross-species analysis. Spontaneous mutant frequencies in two transgenic rat lineages were measured in liver and in germ cells. Preliminary data suggest that spontaneous mutant frequencies in somatic tissue are lower in rats than in mice, a result consistent with historical observations of DNA damage and repair in these two species. Also under evaluation are alternative selectable systems for mutant identification, and hybrid animals obtained from mating lambda LIZ transgenics with genetically engineered mice possessing an inactivated tumor suppressor gene. It is expected that each of these widely varying endeavors will contribute, not only in furthering our understanding of the role transgenic systems should play in human risk assessment, but in illuminating the mechanisms of mutation in general.
Experimental features of a transgenic mouse mutation assay based on a lacI target transgene from Escherichia coli are considered in detail. Sources of variability in the experimental protocol that can affect the statistical nature of the observations are examined with the goal of identifying sources of excess variation in the observed mutant fractions. The sources include plate-to-plate (within packages), package-to-package (within animals), and animal-to-animal (within study) variability. Data from two laboratories are evaluated, using various statistical methods to identify excess variability. Results suggest only scattered patterns of excess variability, except possibly in those cases where genomic DNA from test animals is stored for extended periods (e.g., > 90 days) after isolation from tissues. Further study is encouraged to examine the validity and implications of this time/storage-related effect.