Restricted diet delays accelerated ageing and genomic stress in DNA-repair-deficient mice.

Mice deficient in the DNA excision-repair gene Ercc1 (Ercc1∆/-) show numerous accelerated ageing features that limit their lifespan to 4-6 months. They also exhibit a 'survival response', which suppresses growth and enhances cellular maintenance. Such a response resembles the anti-ageing response induced by dietary restriction (also known as caloric restriction). Here we report that a dietary restriction of 30% tripled the median and maximal remaining lifespans of these progeroid mice, strongly retarding numerous aspects of accelerated ageing. Mice undergoing dietary restriction retained 50% more neurons and maintained full motor function far beyond the lifespan of mice fed ad libitum. Other DNA-repair-deficient, progeroid Xpg-/- (also known as Ercc5-/-) mice, a model of Cockayne syndrome, responded similarly. The dietary restriction response in Ercc1∆/- mice closely resembled the effects of dietary restriction in wild-type animals. Notably, liver tissue from Ercc1∆/- mice fed ad libitum showed preferential extinction of the expression of long genes, a phenomenon we also observed in several tissues ageing normally. This is consistent with the accumulation of stochastic, transcription-blocking lesions that affect long genes more than short ones. Dietary restriction largely prevented this declining transcriptional output and reduced the number of γH2AX DNA damage foci, indicating that dietary restriction preserves genome function by alleviating DNA damage. Our findings establish the Ercc1∆/- mouse as a powerful model organism for health-sustaining interventions, reveal potential for reducing endogenous DNA damage, facilitate a better understanding of the molecular mechanism of dietary restriction and suggest a role for counterintuitive dietary-restriction-like therapy for human progeroid genome instability syndromes and possibly neurodegeneration in general.

Effects of bariatric surgery on telomere length and T-cell aging.

BACKGROUND: Obesity adversely affects health and is associated with subclinical systemic inflammation and features of accelerated aging, including the T-cell immune system. The presence of metabolic syndrome (MetS) may accelerate, while bariatric surgery might reverse these phenomena. To examine the effects of MetS and bariatric surgery on T-cell aging, we measured relative telomere length (RTL) and T-cell differentiation status in obese patients before and after bariatric surgery. METHODS: WHO II/III classified obese patients scheduled for bariatric surgery were included: 41 without MetS and 67 with MetS. RTL and T-cell differentiation status were measured in circulating CD4+ and CD8+ T cells via flow cytometry. T-cell characteristics were compared between patients with and without MetS prior to and at 3, 6, and 12 months after surgery considering effects of age, cytomegalovirus-serostatus, and weight loss. RESULTS: Thymic output, represented by numbers of CD31-expressing naive T cells, showed an age-related decline in patients with MetS. MetS significantly enhanced CD8+ T-cell differentiation. Patients with MetS had significant lower CD4+ RTL than patients without MetS. Within the first 6 months after bariatric surgery, RTL increased in CD4+ T cells after which it decreased at month 12. A decline in both thymic output and more differentiated T cells was seen following bariatric surgery, more pronounced in the MetS group and showing an association with percentage of body weight loss. CONCLUSIONS: In obese patients, MetS results in attrition of RTL and accelerated T-cell differentiation. Bariatric surgery temporarily reverses these effects. These data suggest that MetS is a risk factor for accelerated aging of T cells and that MetS should be a more prominent factor in the decision making for eligibility for bariatric surgery.

Programming of metabolic effects in C57BL/6JxFVB mice by in utero and lactational exposure to perfluorooctanoic acid.

Perfluorooctanoic acid (PFOA) is known to cause developmental toxicity and is a suggested endocrine disrupting compound (EDC). Early life exposure to EDCs has been implicated in programming of the developing organism for chronic diseases later in life. Here we study perinatal metabolic programming by PFOA using an experimental design relevant for human exposure. C57BL/6JxFVB hybrid mice were exposed during gestation and lactation via maternal feed to seven low doses of PFOA at and below the NOAEL used for current risk assessment (3-3000 µg/kg body weight/day). After weaning, offspring were followed for 23-25 weeks without further exposure. Offspring showed a dose-dependent decrease in body weight from postnatal day 4 to adulthood. Growth under high fat diet in the last 4-6 weeks of follow-up was increased in male and decreased in female offspring. Both sexes showed increased liver weights, hepatic foci of cellular alterations and nuclear dysmorphology. In females, reductions in perigonadal and perirenal fat pad weights, serum triglycerides and cholesterol were also observed. Endocrine parameters, such as glucose tolerance, serum insulin and leptin, were not affected. In conclusion, our study with perinatal exposure to PFOA in mice produced metabolic effects in adult offspring. This is most likely due to disrupted programming of metabolic homeostasis, but the assayed endpoints did not provide a mechanistic explanation. The BMDL of the programming effects in our study is below the current point of departure used for calculation of the tolerable daily intake.

Compound- and sex-specific effects on programming of energy and immune homeostasis in adult C57BL/6JxFVB mice after perinatal TCDD and PCB 153.

Early life exposure to endocrine disrupting compounds has been linked to chronic diseases later in life, like obesity and related metabolic disorders. We exposed C57BL/6JxFVB hybrid mice to the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the constitutive androstane receptor/pregnane X receptor agonist polychlorinated biphenyl 153 (PCB 153) in an experimental design relevant for human exposure. Exposure occurred during gestation and lactation via maternal feed to a wide dose range (TCDD: 10-10,000 pg/kg body weight/day; PCB 153: 0.09-1406 µg/kg body weight/d). Then exposure was ceased and offspring were followed up to 1 year of age. Metabolic parameters like body weight, fat pad weights, glucose tolerance, endocrine serum profile, and neurobehavioral and immunological parameters were determined. Body weight was transiently affected by both compounds throughout the follow-up. TCDD-exposed males showed decreased fat pad and spleen weights and an increase in IL-4 production of splenic immune cells. In contrast, females showed increased fat pad weights and production of IFNγ. PCB 153-exposed males showed an increase in glucose, whereas females showed an increase in glucagon, a decrease in pancreas weight, and an increase in thymus weight. In conclusion, early life exposure to TCDD appears to affect programming of energy and immune homeostasis in offspring, whereas the effects of perinatal PCB 153 were mainly on programming of glucose homeostasis. Both compounds act sex-specifically. Lowest derived BMDLs (lower bounds of the (two sided) 90%-confidence interval for the benchmark dose) for both compounds are not lower than current tolerable daily intakes.

Lifestyle factors and metabolomic aging biomarkers: Meta-analysis of cross-sectional and longitudinal associations in three prospective cohorts.

Biological age uses biophysiological information to capture a person's age-related risk of adverse outcomes. MetaboAge and MetaboHealth are metabolomics-based biomarkers of biological age trained on chronological age and mortality risk, respectively. Lifestyle factors contribute to the extent chronological and biological age differ. The association of lifestyle factors with MetaboAge and MetaboHealth, potential sex differences in these associations, and MetaboAge's and MetaboHealth's sensitivity to lifestyle changes have not been studied yet. Linear regression analyses and mixed-effect models were used to examine the cross-sectional and longitudinal associations of scaled lifestyle factors with scaled MetaboAge and MetaboHealth in 24,332 middle-aged participants from the Doetinchem Cohort Study, Rotterdam Study, and UK Biobank. Random-effect meta-analyses were performed across cohorts. Repeated metabolomics measurements had a ten-year interval in the Doetinchem Cohort Study and a five-year interval in the UK Biobank. In the first study incorporating longitudinal information on MetaboAge and MetaboHealth, we demonstrate associations between current smoking, sleeping ≥8 hours/day, higher BMI, and larger waist circumference were associated with higher MetaboHealth, the latter two also with higher MetaboAge. Furthermore, adhering to the dietary and physical activity guidelines were inversely associated with MetaboHealth. Lastly, we observed sex differences in the associations between alcohol use and MetaboHealth.

Rapid accumulation of genome rearrangements in liver but not in brain of old mice.

Somatic mutations have long been considered a possible cause of ageing. To directly study mutational events in organs and tissues of ageing mammals, a transgenic mouse model has been generated that harbours lacZ reporter genes as part of chromosomally integrated plasmids. Using this model, we determined spontaneous mutant frequencies and spectra in mouse liver and brain as a function of age. In the liver, mutant frequencies increased with age from birth to 34 months; in the brain, an increase was observed only between birth and 4-6 months. Molecular characterization of the mutations showed that a substantial portion involved genome rearrangement events, with one breakpoint in a reporter gene and the other in the mouse flanking sequence. In the liver, these genome rearrangements did not increase with age until after 27 months, when they increased rapidly. In brain, the frequency of genome rearrangements was lower than in liver and did not increase with age.

Self-rated health in individuals with and without disease is associated with multiple biomarkers representing multiple biological domains.

Self-rated health (SRH) is one of the most frequently used indicators in health and social research. Its robust association with mortality in very different populations implies that it is a comprehensive measure of health status and may even reflect the condition of the human organism beyond clinical diagnoses. Yet the biological basis of SRH is poorly understood. We used data from three independent European population samples (N approx. 15,000) to investigate the associations of SRH with 150 biomolecules in blood or urine (biomarkers). Altogether 57 biomarkers representing different organ systems were associated with SRH. In almost half of the cases the association was independent of disease and physical functioning. Biomarkers weakened but did not remove the association between SRH and mortality. We propose three potential pathways through which biomarkers may be incorporated into an individual's subjective health assessment, including (1) their role in clinical diseases; (2) their association with health-related lifestyles; and (3) their potential to stimulate physical sensations through interoceptive mechanisms. Our findings indicate that SRH has a solid biological basis and it is a valid but non-specific indicator of the biological condition of the human organism.

Glucose levels and genetic variants across transcriptional pathways: interaction effects with BMI.

OBJECTIVE: Much of the genetic variation in glucose levels remains to be discovered. Especially, research on gene-environment interactions is scarce. Overweight is one of the main risk factors for hyperglycemia. As transcriptional regulation is important for both weight maintenance and glucose control, we analyzed 353 single nucleotide polymorphisms (SNPs), occurring in transcriptional pathways of glucose and lipid metabolism in interaction with body mass index (BMI) on glucose levels. RESEARCH DESIGN AND METHODS: SNPs were measured in 3244 participants of the Doetichem cohort. Non-fasting glucose levels and BMI were measured twice in 6 years. SNP x BMI interactions were analyzed by mixed models and adjusted for age, sex, time since last meal, and follow-up time. False discovery rate (FDR) <0.2 was used to adjust for multiple testing. RESULTS: Two SNPs in the PPARGC1A gene (rs8192678, FDR=0.07; rs3755863, FDR=0.17) showed a significant interaction with BMI. The rare allele of both SNPs was associated with significantly lower glucose levels in subjects with a BMI28 kg m(-2). A small intervention study (n=120) showed similar, though non-significant, results. CONCLUSIONS: Using a pathway-based approach, we found that BMI significantly modified the association between two SNPs in the PPARGC1A gene and glucose levels. The association between glucose and PPARGC1A was only present in lean subjects. This suggests that the effect of the PPARGC1A gene, which is involved both in fatty acid oxidation and glucose metabolism, is modified by BMI.

Genetic variations in regulatory pathways of fatty acid and glucose metabolism are associated with obesity phenotypes: a population-based cohort study.

BACKGROUND: As nuclear receptors and transcription factors have an important regulatory function in adipocyte differentiation and fat storage, genetic variation in these key regulators and downstream pathways may be involved in the onset of obesity. OBJECTIVE: To explore associations between single nucleotide polymorphisms (SNPs) in candidate genes from regulatory pathways that control fatty acid and glucose metabolism, and repeated measurements of body mass index (BMI) and waist circumference in a large Dutch study population. METHODS: Data of 327 SNPs across 239 genes were analyzed for 3575 participants of the Doetinchem cohort, who were examined three times during 11 years, using the Illumina Golden Gate assay. Adjusted random coefficient models were used to analyze the relationship between SNPS and obesity phenotypes. False discovery rate q-values were calculated to account for multiple testing. Significance of the associations was defined as a q-value < or = 0.20. RESULTS: Two SNPs (in NR1H4 and SMARCA2 in women only) were significantly associated with both BMI and waist circumference. In addition, two SNPs (in SIRT1 and SCAP in women only) were associated with BMI alone. A functional SNP, in IL6, was strongly associated with waist. CONCLUSION: In this explorative study among participants of a large population-based cohort, five SNPs, mainly located in transcription mediator genes, were strongly associated with obesity phenotypes. The results from whole genome and candidate gene studies support the potential role of NR1H4, SIRT1, SMARCA2 and IL6 in obesity. Although replication of our findings and further research on the functionality of these SNPs and underlying mechanism is necessary, our data indirectly suggest a role of GATA transcription factors in weight control.

Association of leukocyte DNA methylation changes with dietary folate and alcohol intake in the EPIC study.

BACKGROUND: There is increasing evidence that folate, an important component of one-carbon metabolism, modulates the epigenome. Alcohol, which can disrupt folate absorption, is also known to affect the epigenome. We investigated the association of dietary folate and alcohol intake on leukocyte DNA methylation levels in the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Leukocyte genome-wide DNA methylation profiles on approximately 450,000 CpG sites were acquired with Illumina HumanMethylation 450K BeadChip measured among 450 women control participants of a case-control study on breast cancer nested within the EPIC cohort. After data preprocessing using surrogate variable analysis to reduce systematic variation, associations of DNA methylation with dietary folate and alcohol intake, assessed with dietary questionnaires, were investigated using CpG site-specific linear models. Specific regions of the methylome were explored using differentially methylated region (DMR) analysis and fused lasso (FL) regressions. The DMR analysis combined results from the feature-specific analysis for a specific chromosome and using distances between features as weights whereas FL regression combined two penalties to encourage sparsity of single features and the difference between two consecutive features. RESULTS: After correction for multiple testing, intake of dietary folate was not associated with methylation level at any DNA methylation site, while weak associations were observed between alcohol intake and methylation level at CpG sites cg03199996 and cg07382687, with qval = 0.029 and qval = 0.048, respectively. Interestingly, the DMR analysis revealed a total of 24 and 90 regions associated with dietary folate and alcohol, respectively. For alcohol intake, 6 of the 15 most significant DMRs were identified through FL. CONCLUSIONS: Alcohol intake was associated with methylation levels at two CpG sites. Evidence from DMR and FL analyses indicated that dietary folate and alcohol intake may be associated with genomic regions with tumor suppressor activity such as the GSDMD and HOXA5 genes. These results were in line with the hypothesis that epigenetic mechanisms play a role in the association between folate and alcohol, although further studies are warranted to clarify the importance of these mechanisms in cancer.

The Association between BMI and Different Frailty Domains: A U-Shaped Curve?

OBJECTIVES: Previous studies showed a U-shaped association between BMI and (physical) frailty. We studied the association between BMI and physical, cognitive, psychological, and social frailty. Furthermore, the overlap between and prevalence of these frailty domains was examined. DESIGN: Cross-sectional study. SETTING: The Doetinchem Cohort Study is a longitudinal population-based study starting in 1987-1991 examining men and women aged 20-59 with follow-up examinations every 5 yrs. PARTICIPANTS: For the current analyses, we used data from round 5 (2008-2012) with 4019 participants aged 41-81 yrs. MEASUREMENTS: Physical frailty was defined as having ≥ 2 of 4 frailty criteria from the Frailty Phenotype (unintentional weight loss, exhaustion, physical activity, handgrip strength). Cognitive frailty was defined as the < 10th percentile on global cognitive functioning (based on memory, speed, flexibility). Psychological frailty was defined as having 2 out of 2 criteria (depression, mental health). Social frailty was defined as having ≥ 2 of 3 criteria (loneliness, social support, social participation). BMI was divided into four classes. Analyses were adjusted for sex, age, level of education, and smoking. RESULTS: A U-shaped association was observed between BMI and physical frailty, a small linear association for BMI and cognitive frailty and no association between BMI and psychological and social frailty. The four frailty domains showed only a small proportion of overlap. The prevalence of physical, cognitive and social frailty increased with age, whereas psychological frailty did not. CONCLUSION: We confirm that not only underweight but also obesity is associated with physical frailty. Obesity also seems to be associated with cognitive frailty. Further, frailty prevention should focus on multiple domains and target individuals at a younger age (<65yrs).

A signature of renal stress resistance induced by short-term dietary restriction, fasting, and protein restriction.

During kidney transplantation, ischemia-reperfusion injury (IRI) induces oxidative stress. Short-term preoperative 30% dietary restriction (DR) and 3-day fasting protect against renal IRI. We investigated the contribution of macronutrients to this protection on both phenotypical and transcriptional levels. Male C57BL/6 mice were fed control food ad libitum, underwent two weeks of 30%DR, 3-day fasting, or received a protein-, carbohydrate- or fat-free diet for various periods of time. After completion of each diet, renal gene expression was investigated using microarrays. After induction of renal IRI by clamping the renal pedicles, animals were monitored seven days postoperatively for signs of IRI. In addition to 3-day fasting and two weeks 30%DR, three days of a protein-free diet protected against renal IRI as well, whereas the other diets did not. Gene expression patterns significantly overlapped between all diets except the fat-free diet. Detailed meta-analysis showed involvement of nuclear receptor signaling via transcription factors, including FOXO3, HNF4A and HMGA1. In conclusion, three days of a protein-free diet is sufficient to induce protection against renal IRI similar to 3-day fasting and two weeks of 30%DR. The elucidated network of common protective pathways and transcription factors further improves our mechanistic insight into the increased stress resistance induced by short-term DR.

Accelerated accumulation of somatic mutations in mice deficient in the nucleotide excision repair gene XPA.

Inheritable mutations in nucleotide excision repair (NER) genes cause cancer-prone human disorders, such as xeroderma pigmentosum, which are also characterized by symptoms of accelerated ageing. To study the impact of NER deficiency on mutation accumulation in vivo, mutant frequencies have been determined in liver and brain of 2-16 month old NER deficient XPA-/-, lacZ hybrid mice. While mutant frequencies in liver of 2-month old XPA-/-, lacZ mice were comparable to XPA+/-, lacZ and the lacZ parental strain animals, by 4 months of age mutant frequencies in the XPA-deficient mice were significantly increased by a factor of two and increased further until the age of 16 months. In brain, mutant frequencies were not found to increase with age. These results show that a deficiency in the NER gene XPA causes an accelerated accumulation of somatic mutations in liver but not in brain. This is in keeping with a higher incidence of spontaneous liver tumors reported earlier for XPA-/- mice after about 15 months of age.

Liver DNA methylation analysis in adult female C57BL/6JxFVB mice following perinatal exposure to bisphenol A.

Bisphenol A (BPA) is a compound released from plastics and other consumer products used in everyday life. BPA exposure early in fetal development is proposed to contribute to programming of chronic diseases like obesity and diabetes, by affecting DNA methylation levels. Previously, we showed that in utero and lactational exposure of C57BL/6JxFVB hybrid mice via maternal feed using a dose range of 0-3000µg/kg body weight/day resulted in a sex-dependent altered metabolic phenotype in offspring at 23 weeks of age. The most univocal effects were observed in females, with reduced body weights and related metabolic effects associated with perinatal BPA exposure. To identify whether the effects of BPA in females are associated with changes in DNA methylation, this was analyzed in liver, which is important in energy homeostasis. Measurement of global DNA methylation did not show any changes. Genome-wide DNA methylation analysis at specific CpG sites in control and 3000µg/kg body weight/day females with the digital restriction enzyme analysis of methylation (DREAM) assay revealed potential differences, that could, however, not be confirmed by bisulfite pyrosequencing. Overall, we demonstrated that the observed altered metabolic phenotype in female offspring after maternal exposure to BPA was not detectably associated with liver DNA methylation changes. Still, other tissues may be more informative.

Levels of DNA damage are unaltered in mice overexpressing human catalase in nuclei.

Two types of transgenic mice were generated to evaluate the role of hydrogen peroxide in the formation of nuclear DNA damage. One set of lines overexpresses wild-type human catalase cDNA, which is localized to peroxisomes. The other set overexpresses a human catalase construct that is targeted to the nucleus. Expression of the wild-type human catalase transgene was found in liver, kidney, skeletal muscle, heart, spleen, and brain with muscle and heart exhibiting the highest levels. Animals containing the nuclear-targeted construct had a similar pattern of expression with the highest levels in muscle and heart, but with lower levels in liver and spleen. In these animals, immunofluorescence detected catalase present in the nuclei of kidney, muscle, heart, and brain. Both types of transgenic animals had significant increases of catalase activities compared to littermate controls in most tissues examined. Despite enhanced activities of catalase, and its presence in the nucleus, there were no changes in levels of 8OHdG, a marker of oxidative damage to DNA. Nor were there differences in mutant frequencies at a Lac Z reporter transgene. This result suggests that in vivo levels of H(2)O(2) may not generate 8OHdG or other types of DNA damage. Alternatively, antioxidant defenses may be optimized such that additional catalase is unable to further protect nuclear DNA against oxidative damage.

Genotyping the Prop-1 mutation in Ames dwarf mice.

The Ames dwarf mouse phenotype is based on a homozygous single gene mutation in the Prop-1 gene that markedly extends life span. Since its discovery, interest in breeding these mice as a model to study retardation of aging has increased dramatically. However, the homozygous Prop-1 mutants are infertile, which necessitates breeding heterozygotes. Heterozygotes cannot be distinguished from the wildtype, while the homozygote dwarf phenotype only becomes apparent after about 3 weeks. Hence, there is a need for a simple test to genotype individual animals at an early stage for the absence or presence of one or two copies of the Prop-1 mutant gene. Here we present a Prop-1 genotyping protocol, based on a PCR reaction followed by a PflMI digestion.

Transgenic mouse models for studying mutations in vivo: applications in aging research.

To study mutation accumulation in the DNA of somatic cells and tissues during aging in vivo, a transgenic mouse model has been constructed. The model harbors plasmid vectors, containing the lacZ reporter gene, integrated head to tail at various chromosomal locations. Procedures have been worked out to efficiently recover the plasmids into E. coli host cells. A positive selection system, permitting only E. coli cells with a lacZ mutated plasmid to grow, allows for the accurate determination of mutation frequencies as the ratio of mutant colonies versus the total number of transformants, i.e., the total number of plasmid copies recovered. Results obtained from a life span study of plasmid mice with vector clusters on chromosome 3 and 4 indicated age-related mutation accumulation in the liver, but not in the brain. Comparison of the mutational spectra revealed a significantly larger proportion of large size-change mutations in liver than in brain.

Transgenic mouse models for studying mutations in vivo: applications in aging research.

To study mutation accumulation in the DNA of somatic cells and tissues during aging in vivo, a transgenic mouse model has been constructed. The model harbors plasmid vectors, containing the lacZ reporter gene, integrated head to tail at various chromosomal locations. Procedures have been worked out to efficiently recovery the plasmids into E. coli host cells. A positive selection system, permitting only E. coli cells with a lacZ mutated plasmid to grow, allows for the accurate determination of mutation frequencies as the ratio of mutant colonies versus the total number of transformants, i.e., the total number of plasmid copies recovered. Results obtained from a life span study of plasmid mice with vector clusters on chromosome 3 and 4 indicated age-related mutation accumulation in the liver, but not in the brain. Comparison of the mutational spectra revealed a significantly larger proportion of large size-change mutations in liver than in brain.

Mutation frequency and type during ageing in mouse seminiferous tubules.

Mutations arise in the germline by errors of replication, recombination and repair, and the movement of transposable elements. Transgenic mice bearing reporter genes such as lacZ have proven useful for measurements of spontaneous and induced mutation frequencies, as well as studies of the effects of ageing. In this study, testicular DNA from lacZ transgenic mice was examined for age-related effects on mutation frequency and type. The recovered transgene was tested for simple substitutions and rearrangements including transposition of endogenous mobile elements. There was no evidence for either an age-related accumulation of mutations, or for the insertion of retrotransposons into the lacZ reporter gene in the testis. We conclude that the frequency of retrotransposition of several mouse mobile elements into the lacZ reporter gene is less than 3.73x10(-8). This is significantly less than the known frequency of approximately 7% of all spontaneous mutations in the mouse being due to retrotransposition of these elements.

Background mutations and polymorphisms in lacZ-plasmid transgenic mice.

Transgenic animal models harboring chromosomally integrated shuttle vectors with bacterial reporter genes are now widely used to measure in vivo mutant frequencies. The lacZ-plasmid transgenic mouse model has a unique sensitivity to large rearrangements compared to systems using bacteriophage lambda vectors, which mainly detect point mutations and small deletions or insertions. In this study, the background mutant frequencies and spectra in the lacZ-plasmid transgenic mouse model were investigated. While the majority of the recovered lacZ-mutants appeared to have originated in the mouse, a subset of mutants are likely to represent artifacts, and occur with a frequency of about 1.3 x 10(-5), irrespective of the total mutant frequency. Galactose-insensitive host cells, due to galE back mutations or galK or galT forward mutations, grow through the positive selection system and cause a small subset of the background. When using HindIII to excise the plasmids from genomic DNA, the largest contribution to the background, (1.1 +/- 0.3) x 10(-5), appeared to be caused by star activity, i.e., cleavage at nucleotide sequences other than the HindIII restriction enzyme recognition sequence, during the recovery procedure. Finally, a total of 10 polymorphic sites in different copies of the lacZ-plasmid cluster in founder line 60 were discovered.