Mutant isolation

About 15,000 F2 progeny of EMS-mutagenized hermaphrodites were examined for hypersensitivity to methyl viologen. During the initial screening, about 360 candidates were isolated, which presumably included many individuals that died of reasons related to the drug treatment. In the rescreening 2 strains proved to be highly sensitive to methyl viologen. One strain, mev-1(kn1), was back-crossed 5 times with wild type males using methyl viologen sensitivity as a marker to identify mutant progeny (Ishii et al., 1990).

Sensitivity to methyl viologen and oxygen

L1 larvae were cultured on plates containing various concentrations of methyl viologen. At the highest concentrations of methyl viologen examined (0.2mM), most wild-type animals developed into L4 larvae or adults within 4 days. However, mev-1 mutants usually arrested as L1 or L2 larvae (Ishii et a., 1990).

The mutant is even more hypersensitive to oxygen gas than to methyl viologen. Whereas wild-type animals can develop nearly normally under 90% oxygen, few mev-1 larvae survive a 3-day exposure. The growth and movement of mev-1 larvae were nearly normal for the first day of exposure, but arrested thereafter (Ishii et al., 1990).

Fecundity

The mev-1 mutants have normal morphology. However, they have low vital activity, i.e., slow growth and low fecundity. Egg-laying in the wild type begins on the third day after hatching, reaches a maximum of about 130 eggs per animals per day on the fifth day and continues for another week. The average number of eggs laid per individual was 287. Egg production was delayed for about half a day in mev-1 mutant and the average number of eggs laid per animals was only 77 (Ishii et al., 1990).

Life span

The mean and maximum life spans of both the wild type and mev-1 increased and decreased under low and high concentrations of oxygen, respectively. The mean and maximum life spans of the wild type under 21% oxygen were 26 days and 33 days, respectively. Their life spans under 1% oxygen were extended significantly (mean, 30 days; maximum, 41 days), while those under 60% oxygen were shortened considerably (mean, 23 days; maximum, 28 days). Those under oxygen concentrations within a range between 2 and 40% remained unchanged. On the other hand, the mean and maximum life spans of the mutant under 21% oxygen were 21 and 26 days, respectively. Their life spans under 1% oxygen were also longer (mean, 26 days; maximum, 35 days), while those under 60% oxygen were much shorter (mean, 8 days; maximum, 10 days). The life spans were observed to vary over a wide range of oxygen concentration (Ishii et al., 1990, Honda et al., 1993).

The Gompertz component, a parameter of aging rate, of the wild type was smaller under 1% oxygen than under 2% or more oxygen. Further, the Gompertz component of the mutant increased with an increase in oxygen concentration. These effects of oxygen on the perturbation of life span and aging rate were more pronounced in the mev-1 mutant than in wild type. A 1% oxygen exposure at the early phase of life span was ineffective for life span extension in the mutant, suggesting that the effect of oxygen concentrations on life span is not secondary to the effects of development and maturation (Honda et al., 1993).

Aging marker

Fluorescent materials (repofuscin) and protein carbonyl derivatives are formed in vitro as a result of metal-catalyzed oxidation and accumulate during aging in disparate model systems(Epstein et al., 1972, Spoerri et al., 1974, Strehler et al., 1959, Stadman E.R and Oliver, 1991, Stadman, 1992). These results indicate that Fluorescent materials and protein carbonyl modifications can be a specific indicator of oxidized lipid and protein.

a)fluorescent materials

As observed using fluorescence microscopy, C. elegans contains blue autofluorescent granules and materials in the intestinal cells. These granules and materials accumulated in wild type in an age-dependent fashion. On day 5 after hatching, the amount of fluorescence seemed to be approximately the same in wild type and mev-1 mutants. Conversely, on day 10, the substances in the mev-1 mutant had accumulated to a much higher level than in wild type.

Also, fluorescent material in methanol/water extracts of both wild type and mev-1 accumulated with increasing age. The fluorescent material in mev-1 accumulated more than in the wild type. The amount of the extracts of mev-1 on day 10 was approximately two times that observed in the wild type on the same days.

When incubated under 90% oxygen, the fluorescent materials in the mev-1 but not wild-type accumulated more rapidly compared with incubation under atmospheric conditions. Conversely, the materials did not accumulate in either wild type or mev-1 under 2% oxygen. (Hosokawa et. al., 1994)

b)protein carbonyl

The protein carbonyl contents in young wild-type and mev-1 adults at the age of 4-8 days were similar. Afterwards, different accumulations of carbonyl were observed with the genotype and increasing age. In wild type, an age-dependent accumulation in carbonyl content was observed until the end of life span around 20 days to reach 4.7 nmol/mg protein, whereas in mev-1 it occurred at a faster rate to reach 5.7 nmol/mg protein at the end of life span (age 15-16 days).

To determine if the antioxidant defense ability in mev-1 operated under higher levels of oxidative stress, protein carbonyl contents were compared after exposure to 70% oxygen between age 4 and 11 days. At the age of 11 days, such hyperoxia caused 100% and 31 % increases in carbonyl in mev-1 and wild type over the respective basal levels in the ambient atmosphere of 21% oxygen, respectively (Adachi et al., 1998).

Superoxide dismutase (SOD) activity

SOD, one of antioxidant defense systems in eukaryotic cells is composed of 2 types, one containing Zn and Cu in the active center and another containing Mn. The activities of these 2 types can be distinguished by adding 1 mM KCN to the reaction mixtures, which specifically inhibits the Zn/Cu enzyme.

The SOD activity in mev-1 is about 50% that of the wild type. Most wild-type activity is inhibited by KCN, indicating that there is relatively little, if any Mn-SOD. In contrast,14-21% of the residual activity in mev-1 could be attributed to the Mn-SOD. As a caveat, these measurements were made from asynchronous populations and are conceivably affected by unrecognized difference in the stage distributions of the populations (Ishii et al., 1990)

Molecular cloning

Three-factor crosses using visible genetic markers placed mev-1 between unc-50(e306) and unc-49(e382) of chromosome III. We tested cosmids from this region for their abilities to rescue mev-1 mutants from oxygen-hypersensitivity after germline transformation. Only cosmid T07C4 was able to rescue the mev-1 mutant phenotype. By testing various subclones from this cosmid, we identified a 5.6kb fragment that also restored wild-type resistance. This fragment includes a putative gene, named cyt-1, that is homologous to the bovine succinate dehydrogenase (SDH) cytochrome b560 (GenBank accession number L26545). We found the mev-1 strain contained a missense mutation resulting in a glycine to glutamic acid substitution in cyt-1. The mutation created a restriction fragment length polymorphism such that the restriction enzyme Mor-1 should cleave wild-type but not mev-1 DNA at position 323. As predicted by these sequence data, Mor-1 digestion of RT-PCR products yielded two bands with wild type, one band with mev-1 and three bands in transgenic animals. This confirms that the wild-type cyt-1 gene introduced in the mev-1 strain was expressed and that rescue was provided by this gene.(Ishii et al., 1998)

Electron transport is mediated by five multimeric complexes (complex I-V) that are embedded in the inner membrane of the mitochondrion. Mitochondrial succinate-ubiquinone reductase (complex II), which catalyzes electron transport from succinate to ubiquinone, is composed of succinate dehydrogenase (SDH)(flavin protein: Fp and iron-sulfur protein: Ip) and two other subunits containing cytochrome b560. In vivo, SDH is anchored to the inner membrane with the cytochrome b560 and is the catalytic component of complex II. Using separate assays, it is possible to quantify specifically both SDH activity and complex II activity. This we did after wild-type and mev-1 extracts were subjected to differential centrifugation to separate mitochondria and mitochondrial membranes from cytosol. The SDH activity in the mev-1 mitochondrial fraction was experimentally identical to that of wild type. Conversely, complex II activity in the mev-1 membrane fraction was reduced over 80% relative to wild type. As expected of a mitochondrial enzyme, no SDH activity was observed in the cytosol. Thus, the mev-1 mutation affects neither SDH anchoring to the membrane nor SDH activity per se. However, it dramatically compromises the ability of complex II to participate in electron transport.

How then does the mev-1 mutation exert its effects on mitochondria and, ultimately, the nematode? Cytochrome b560 is predicted to have three membrane-spanning domains. The substitution of glutamic acid for glycine is at position 71, only two amino acids removed from a histidine residue (His-73) that is thought to serve as a haem ligand. This could affect the ability of iron to accept and relinquish electrons, thus explaining the complex II deficiency in the mev-1 mutant. As a consequence, the precocious aging and free-radical hypersensitivity of mev-1 could result from two distinct mechanisms. First, the mutation could cause electron transport to be deregulated such that oxygen uptake into mitochondria is higher in wild type. This would lead to increased free radical production. Second, mutational perturbation of electron transport would compromise ATP production and, as a result, lead to precocious aging.

In summary, much attention has focused on the role that oxidative damage plays in cellular and organismal aging. A mev-1(kn1) mutant of Caenorhabditis elegans, isolated on the basis of its methyl viologen (paraquat) hypersensitivity, is also hypersensitive to elevated oxygen levels. Unlike wild type, its life span decreases dramatically as oxygen concentrations are increased from 1% to 60%. Strains bearing this mutation accumulate fluorescent materials and protein carbonyls, markers of aging, at faster rates than wild type. We have cloned mev-1 gene by transformation rescue and found that it is the previously sequenced gene (cyt-1) that encodes succinate dehydrogenase cytochrome b. A missense mutation abolishes complex II activity in the mitochondrial membrane but not succinate dehydrogenase enzyme activity per se. It is suggested that CYT-1 directly participates in electron transport from FADH2 to coenzyme Q. Moreover, mutational inactivation of this process renders animals susceptible to oxidative stress and, as a result, leads to precocious aging.

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