Johan G. de Boer

Bursts and horizontal evolution of DNA transposons in the speciation of pseudotetraploid salmonids

BackgroundSeveral genome duplications have occurred in the evolutionary history of teleost fish. In returning to a stable diploid state, the polyploid genome reorganized, and large portions are lost, while the fish lines evolved to numerous species. Large scale transposon movement has been postulated to play an important role in the genome reorganization process. We analyzed the DNA sequence of several large loci in Salmo salar and other species for the presence of DNA transposon families.ResultsWe have identified bursts of activity of 14 families of DNA transposons (12 Tc1-like and 2 piggyBac-like families, including 11 novel ones) in genome sequences of Salmo salar. Several of these families have similar sequences in a number of closely and distantly related fish, lamprey, and frog species as well as in the parasite Schistosoma japonicum. Analysis of sequence similarities between copies within the families of these bursts demonstrates several waves of transposition activities coinciding with salmonid species divergence. Tc1-like families show a master gene-like copying process, illustrated by extensive but short burst of copying activity, while the piggyBac-like families show a more random copying pattern. Recent families may include copies with an open reading frame for an active transposase enzyme.ConclusionWe have identified defined bursts of transposon activity that make use of master-slave and random mechanisms. The bursts occur well after hypothesized polyploidy events and coincide with speciation events. Parasite-mediated lateral transfer of transposons are implicated.

Polymorphisms in DNA repair and environmental interactions.

The repair of damage to DNA is critical to the survival of a cell. However, not all organisms nor all individuals express a similar response to challenges to their genetic material. Numerous polymorphisms in genes involved in DNA repair have been found in individuals with DNA repair-related disease as well as in the general population. Studies of these variants are critical in understanding the response of the cell to DNA damage. In some cases, these changes predispose the carrier to a greatly increased risk of cancer. In other cases, the effects are subtler and depend on interactions between the alleles of several genes, or with environmental factors. Consequently, the health effects of exposure to genotoxic or carcinogenic compounds or agents can depend on the variations in these genes. This review will highlight some of the effects that variants, found in many of the genes involved in human DNA repair pathways, have on the response to damage, and their role in susceptibility of the cell and organism to environmental genotoxins. This review will concentrate on the mismatch repair, nucleotide repair, base excision repair, strand break repair, and direct alkyl repair pathways.

Spectrum of spontaneous mutations in liver tissue of lacI transgenic mice

The advent of transgenic technology has greatly facilitated the study of mutation in animals in vivo. The Big Blue® mouse system, transgenic for the lacI gene, permits not only the quantification of mutations in different tissues but also provides for the generation of in vivo‐derived mutational spectra. This report details the sequence alterations of 348 spontaneous mutations recovered from the liver of 6–8‐week‐old male Big Blue® mice. The spectra recovered from two strains of mice, C57Bl/6 and B6C3F1, were compared and found to be very similar. The predominant mutations are G:C → A:T transitions, with 75% of these occurring at 5′‐CpG‐3′ sequences. This mutational bias is consistent with deamination‐directed mutation at methylated cytosine bases. The second most common class of mutations is G:C → T:A transversions. A significant clonal expansion of mutants was found in several animals, and this was used to make an approximate correction of the mutant frequency such that the most conservative estimate of mutation frequency is presented. The establishment of this substantial database of spontaneous mutations in the liver of Big Blue® mice is intended to serve as a reference against which mutations recovered after treatment can be compared. Environ. Mol. Mutagen. 30:273–286, 1997

Diet and Dietary Supplement Intervention Trials for the Prevention of Prostate Cancer Recurrence: A Review of the Randomized Controlled Trial Evidence

PURPOSE We review the effect of diet and dietary supplement interventions on prostate cancer progression, recurrence and survival. MATERIALS AND METHODS A literature search was conducted in MEDLINE, EMBASE and CINAHL to identify diet and dietary supplement intervention studies in men with prostate cancer using prostate specific antigen or prostate specific antigen doubling time as a surrogate serum biomarker of prostate cancer recurrence and/or survival. RESULTS Of the 32 studies identified 9 (28%) were randomized controlled trials and the focus of this review. In these studies men had confirmed prostate cancer and elevated or increasing prostate specific antigen. Only 1 trial included men with metastatic disease. When body mass index was reported, men were overweight or obese. A significant decrease in prostate specific antigen was observed in some studies using a low fat vegan diet, soy beverage or lycopene supplement. While not often reported as an end point, a significant increase in prostate specific antigen doubling time was observed in a study on lycopene supplementation. In only 1 randomized controlled trial in men undergoing orchiectomy was a survival end point of fewer deaths with lycopene supplementation reported. CONCLUSIONS A limited number of randomized controlled trials were identified in which diet and dietary supplement interventions appeared to slow disease progression in men with prostate cancer, although results vary. Studies were limited by reliance on the surrogate biomarker prostate specific antigen, sample size and study duration. Well designed trials are warranted to expand knowledge, replicate findings and further assess the impact of diet and dietary supplement interventions on recurrence and treatment associated morbidities.

Evolution of duplicated IgH loci in Atlantic salmon, Salmo salar.

BackgroundThe Atlantic salmon (Salmo salar) immunoglobulin heavy chain (IgH) locus possesses two parallel IgH isoloci (IGH-A and IGH-B), that are related to the genomic duplication event in the family Salmonidae. These duplicated IgH loci in Atlantic salmon provide a unique opportunity to examine the mechanisms of genome diversity and genome evolution of the IgH loci in vertebrates. In this study, we defined the structure of these loci in Atlantic salmon, and sequenced 24 bacterial artificial chromosome (BAC) clones that were assembled into the IGH-A (1.1 Mb) and IGH-B (0.9 Mb) loci. In addition, over 7,000 cDNA clones from the IgH variable (VH) region have been sequenced and analyzed.ResultsThe present study shows that the genomic organization of the duplicated IgH loci in Atlantic salmon differs from that in other teleosts and other vertebrates. The loci possess multiple Cτ genes upstream of the Cμ region, with three of the Cτ genes being functional. Moreover, the duplicated loci possess over 300 VH segments which could be classified into 18 families. This is the largest number of VH families currently defined in any vertebrate. There were significant structural differences between the two loci, indicating that both IGH-A and -B loci have evolved independently in the short time after the recent genome duplication approximately 60 mya.ConclusionsOur results indicate that the duplication of the IgH loci in Atlantic salmon significantly contributes to the increased diversity of the antibody repertoire, as compared with the single IgH locus in other vertebrates.

Mutational specificity: An efficient laboratory protocol for the sequencing of large numbers of lacl mutants recovered from Big Blue® transgenic animals

Mutational spectra provide a powerful approach to investigate both the mutagenic potential and the mechanism of action of suspected mutagens and carcinogens. Recently, transgenic techniques have made it possible to generate mutational spectra in animals. Such a spectrum may consist of 50 to 200 mutants depending on the nature of the mutations, and many spectra can be generated depending on the design of the experiment. This report describes a practical approach for the processing and sequencing of large numbers of lacl mutants recovered from Big Blue® animals.

Software package for the management of sequencing projects using lacl transgenic animals

The bacterial lacl gene has been used for many years as a mutational target for the study of the mechanisms of mutation. A wealth of information has been collected for many mutagenic treatments and in strains with diverse DNA repair backgrounds. Recently this gene has been used in the construction of a transgenic mouse, named Big Blue®, and a transgenic rat, as well as a rat cell line. The lacl gene in these animals and cells can conveniently be recovered and analyzed in bacteria. This makes it possible to study mutagenic potential of chemical compounds in vivo using a mammal. Tissue, strain, and gender specificity can be addressed. In addition, mutations recovered from tumour tissues or from animals with specific genetic backgrounds can be analyzed conveniently. The mammalian systems can produce large numbers of mutants that require computer assistance to manage the samples and the resulting DNA sequence data. Accordingly, a computer software system was develped. The system maintains an inventory of bacteriophage lambda lacl mutants and allows entry of mutant sequences while performing accuracy checks on the data. The software features several options for displaying lists of mutants. The system can perform several analyses, including mutant class compilations, mutational spectra comparisons, and clonal expansions analysis. An extensive database obtained from the bacterial lacl system is included with the software and can be analyzed along with mutants derived from transgenic animals.

Mutational specificity: Spectrum of mutations in kidney, stomach, and liver from lacl transgenic mice recovered after treatment with tris(2,3‐dibromopropyl)phosphate

The flame retardant tris(2,3‐dibromopropyl)phosphate (TDBP), once used in cotton sleepware for children, is presently banned from commerce. It produces tumors in rodents in both a sex‐ and tissue‐specific manner. The kidney is the main target for tumor formation in male and female rats, as well as in male mice. In contrast, tumors are formed in the liver of female animals. We have used lacl transgenic male B6C3F1 mice (Big Blue®) to examine the induction of mutation in kidney, liver, and stomach after exposure to 150 mg/kg (2 days), 300 mg/kg (4 days), and 600 mg/kg (4 days of TDBP. At the highest dose, the mutant frequency was approximately 50% above control values in the kidney (P < 0.01). A smaller increase was observed in the liver (P = 0.07), while no increase was seen in the stomach (P = 0.28). Sequence analysis of the recovered mutants showed a TDBP‐specific change in mutation spectrum in kidney, which was not observed in liver and stomach. In kidney, a dose‐dependent decrease in G:C → A:T transitions, including at 5′‐CpG‐3′ sites, was observed. This was accompanied by an increase in the loss of single G:C base pairs from approximately 3% to 15%. These results illustrate both the sensitivity and specificity of the lacl transgenic system in the analysis of tissue‐specific mutation. This study also reinforces the importance of examining mutational spectra when mutant induction levels are low.

Interpretation of mutational spectra from different genes: analyses of PhIP-induced mutational specificity in the lacI and cII transgenes from colon of Big Blue® rats

The cII assay provides an alternative choice to the lacI transgene for mutational studies involving Big Blue(R) transgenic mice and rats, or permits the evaluation of mutational responses in both genes. Here, we compare the mutational response of the cII gene from colon of Big Blue(R) F344 rats treated with a dietary mutagen and animal carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), to those previously determined in the lacI transgene from colon of the same group of animals. A cursory inspection of PhIP-induced mutational spectra (MS) in cII and lacI suggests that the two transgenes respond differently to PhIP-induced mutation. However, a more thorough analysis of the MS in the two transgenes, including consideration of the number of mutational target sequences in each gene and nearest neighbor analyses of mutated nucleotides, indicates that PhIP-induced mutational specificity is similar in both genes. The evaluation of PhIP-induced mutational responses in these two transgenes serves as a model for intergenic mutational analyses.

Protective effect of green tea against benzo[a]pyrene-induced mutations in the liver of Big Blue® transgenic mice

We assessed the ability of green tea to protect against benzo[a]pyrene (B[a]P)-induced mutations in the liver of lacI transgenic male C57BL/6 Big Blue mice. The mice were given a 2% Japanese green tea hot water extract as their sole source of drinking water for 10 weeks. After 7 weeks, they received a total dose of 150 mg/kg B[a]P. Treatment with B[a]P resulted in a two-fold higher lacI mutant frequency than the untreated controls (8.6+/-0.8 x 10(-5) versus 4.0+/-0.7 x 10(-5), P=0.01). B[a]P increased the frequency of its characteristic mutation (GC-->TA transversions) nearly five-fold, from 0.75 x 10(-5) to 3.7 x 10(-5). In mice treated with green tea, the induced B[a]P mutant frequency decreased by 63%, while GC-->TA transversions were reduced by 54%. Thus, we report evidence that green tea extract significantly suppressed B[a]P-induced mutation by lowering its specific transversion mutation in the lacI transgene in vivo. Further studies will address the correlation between the modulation of metabolic enzymes and the protection against induced mutation by green tea.