Age-related molecular variability in Caenorhabditis elegans, T. R. Golden et al. 185 appearance of DNA masses containing actively replicating DNA, as well as the loss of some nuclei. Individuals of the long-lived strain daf-2(e1370) are more heterogeneous than N2 at ages less than 14 days. This may reflect differences in nuclear content or size in the mutant strain, and suggests that characterization of the development and nuclear content of this mutant, which is so important to aging research, should be conducted. After 17 days of age, the nuclear content of daf-2(e1370) falls to 4-day-old levels (Fig. 5b), and a similar decline is seen in daf- 2(e1368) (Supplementary Fig. S3b). A decline in nuclear genome copy number with age is not seen in N2 hermaphro- dites, likely because the effect is swamped by the accumulation of large DNA masses in the body cavity of aging worms. How- ever, an age-related decline is observed in N2 hermaphrodites raised on FUDR (Fig. 5d) and N2 males (Fig. 5c). This loss in nuclear genome content may reflect loss of nuclei, such as the loss of tail nuclei we detected via DAPI staining (Figs 2 and 3). It may also reflect the accumulation of DNA damage with age. Alternatively, it may result from animals with the highest genome content dying first. This could be consistent with hypotheses related to the relative fitness of the ‘oldest old’ of a population (Perls, 2002). These theories predict that the most-fit individuals remain at the end of the maximum lifespan of an organism, the weak having succumbed to disease, trauma or stochastic events. In the case of a population of C. elegans in which all individuals are genetically identical, the oldest old may be those who escape detrimental stochastic insults. The decay of functions other than genome maintenance would cause aging in this remaining population; evolutionary theory predicts that multiple interacting factors are responsible for the aging phenotype (Finch & Kirkwood, 2000). As they age, individual C. elegans have recently been observed to develop morphologic differences in a stochastic fashion consistent with the data presented here (Herndon et al., 2002). These data indicate that although single-gene effects may significantly affect lifespan (Friedman & Johnson, 1988a; Guarente & Kenyon, 2000), molecular stochastic processes also have to be taken into account as significant factors that limit lifespan in individual animals. In conclusion, we have described striking age-related changes in nuclear maintenance and morphology, and in nuclear genome maintenance in the nematode C. elegans. Certain nuclei are systematically lost from the tail with age, and this loss is delayed in the long-lived strain daf-2(e1368) and daf-2(e1370). Nuclear genome copy number declines dramatically in N2 males with age, and also in daf-2(e1370), which may reflect this nuclear loss. The fact that the loss is delayed in daf-2 worms suggests that maintenance of nuclear integrity is associated with the daf-2 longevity. Experimental procedures Strains Strains used were wild-type Bristol N2, daf-2(e1370), daf- 2(e1368), and glp-4(bn2ts). The nematode strains and the © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2007 No claim to original US government works Escherichia coli strain OP50 used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health National Center for Research Resources (NCRR). Culture of nematodes Nematodes were cultured at 20 °C on nematode growth media (NGM) plates, and fed E. coli strain OP50 (Wood, 1988). Syn- chronized populations were obtained by conducting a timed lay in which gravid adults were placed on a plate and removed after being allowed to lay eggs for 2–4 h. The resulting eggs were allowed to hatch and develop for 3 days, during which time the progeny were moved to new plates for survival experiments. Worms in survival experiments were moved to fresh plates at least every other day to avoid contamination by young. For culture in the presence of FUDR, worms were transferred to NGM plates containing 0.1 mg mL−1 FUDR at 3 days of age. Production of males To generate a population of males for qPCR, N2 L4 hermaph- rodites were incubated at 30 °C for 4–6 h and then returned to 20 °C. Resulting male progeny were placed onto NGM plates with L4 hermaphrodites at a ratio of 1 : 10 (hermaphrodite : males) for mating overnight at 20 °C. Males were removed, and mated hermaphrodites allowed to lay eggs on NGM plates for 4 h at 20 °C. On day 3, males were removed from lay plates and transferred to fresh NGM plates seeded with OP50 for survival analysis at 20 °C. Worm lysis For the qPCR experiments shown in Supplementary Fig. S2, individual worms were picked from the plate into a PCR tube containing 5 µ L of water, and frozen immediately on dry ice. Two-x lysis buffer was added to result in a final concentration of 10 mM Tris-HCl pH 8.3, 20 mM KCl, 150 µ g mL−1 proteinase K. Lysis was carried out by incubation at 60 °C for 1 h and subsequent inactivation of the proteinase K by incubating at 99 °C for 10 min. Quantitative real-time PCR Primer design and optimization Primers were designed using AcePrimer (http://elegans.bcgsc.bc.ca/ gko/aceprimer.shtml) and the following settings: [primer sets = 5; product size = 100 ± 50; primer size = 20 ± 2; temperature = 60 ± 2; GC clamp OFF; Nested primer interval OFF; Q value = 0.5; Primers in nearby gene ON; e-PCR word size = 7 (default); e-PCR allowed mismatches = 3; poison primers = none]. Initially, primers were designed for all five chromosomes, using the following target gene sequences: Chromosome I = smd-1 (F47G4.7), Chromosome II = cct-1 (T05C12.7), Chromosome III = ced-7 (C48B4.4), Chromosome IV = fat-3 (W08D2.4),