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Genes to Cells (2009) 14, 457-468. doi:10.1111/j.1365-2443.2009.01282.x
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[FeFe]-hydrogenase-like gene is involved in the regulation of sensitivity to oxygen in yeast and nematode

 Michihiko Fujii*, Noritaka Adachi, Kazuki Shikatani and Dai Ayusawa

International graduate school of Arts and Sciences, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Oxygen is essential for the life of aerobic organisms, but reactive oxygen species (ROS) derived from oxygen can be a threat for it. Many genes are involved in generation of ROS, but not much attention has been focused on the reactions from which ROS are generated. We therefore screened for mutants that showed an increased sensitivity to oxidative stress in the nematode Caenorhabditis elegans, and isolated a novel mutant, oxy-4(qa5001). This mutant showed an increased sensitivity to a high concentration of oxygen, and decreased longevity at 20 °C but not at 26 °C. The genetic analysis has revealed that oxy-4 had a causative mutation in an [FeFe]-hydrogenase-like gene (Y54H5A.4). In the yeast Saccharomyces cerevisiae, a deletion of NAR1, a possible homologue of oxy-4, also caused a similar increased sensitivity to oxygen. [FeFe]-hydrogenases are enzymes that catalyze both the formation and the splitting of molecular hydrogen, and function in anaerobic respiration in anaerobes. In contrast, [FeFe]-hydrogenase-like genes identified in aerobic eukaryotes do not generate hydrogen, and its functional roles are less understood. Our results suggested that [FeFe]-hydrogenase-like genes were involved in the regulation of sensitivity to oxygen in S. cerevisiae and C. elegans.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Oxygen is essential for the life of aerobic organisms. However, reactive oxygen species (ROS), which are by-products in various metabolic pathways, are generated from oxygen. ROS have the potential to damage cellular components such as proteins, DNA, and lipids, and this can result in cellular dysfunction (Finkel & Holbrook 2000). Animals have evolved various defense mechanisms against ROS, but they do not seem to be perfect. Escaped ROS damage cells, and are considered to be a major endogenous factor that accelerates aging (Harman 1957). Considerable efforts have been directed at understanding how cells cope with ROS once they have been generated. Conversely, not much attention has been focused on the reactions from which ROS are generated. Superoxide dismutase (SOD), catalase, and glutathion peroxidase are well-known to eradicate ROS, but there are many other factors or reactions involved in generation or quenching of ROS. In spite of the importance of the genes that affect sensitivity to oxidative stress, they are largely unexplored except for instances, such as well-known anti-oxidant enzymes. We therefore aimed to identify genes that regulate sensitivity to oxidative stress in cells with genetic methods.

The model organisms such as the yeast Saccharomyces cerevisiae, the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and laboratory mice are frequently used because powerful genetic analysis is available in them. However, the genes that can be mutated to confer oxidative stress are far from being saturated by mutations even in these well-characterized model organisms. We then screened for mutants which showed an increased sensitivity to oxidative stress in C. elegans, and isolated a novel mutant, oxy-4(qa5001). At present, several mutants are shown to be hypersensitive to oxidative stress in C. elegans, for example mev-1, mev-3, gas-1, rad-8, skn-1 and nnt-1 (Ishii et al. 1990, 1993, 1994; Yamamoto et al. 1996; Kayser et al. 1999; Hartman et al. 2001; An & Blackwell 2003; Arkblad et al. 2005). mev-1, gas-1, nnt-1, and skn-1 encode the succinate dehydrogenase cytochrome b gene, NADH:ubiquinone-oxidoreductase gene, nicotinamide nucleotide transhydrogenase gene, and a transcription factor, respectively (Ishii et al. 1998; Kayser et al. 1999, 2001; An & Blackwell 2003; Arkblad et al. 2005). The former two are involved in energy production in mitochondria, and the latter two in anti-oxidant defense.

Our genetic analysis has revealed that oxy-4 had a causative mutation in Y54H5A.4, which encodes an [FeFe]-hydrogenase-like gene. [FeFe]-hydrogenases are enzymes that catalyze the following reversible reaction: H2{leftrightarrow} 2H+ + 2e–, which has been identified in anaerobic respiration of anaerobes, and is well-characterized (Peters 1999; Vignais et al. 2001; Horner et al. 2002; Nicolet et al. 2002; Hackstein 2005; Vignais & Billoud 2007). However, the functional roles of [FeFe]-hydrogenase-like genes identified in aerobic eukaryotes are less understood. They do not produce hydrogen, and are proposed to have acquired novel functions in the evolution of aerobic eukaryotes. Our results indicated that the [FeFe]-hydrogenase-like genes played an important role in regulation of sensitivity to oxygen, and its defect influenced many biological processes that included stress resistance and longevity in C. elegans.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Isolation of oxy-4

To isolate a mutant with an increased sensitivity to oxidative stress, we used paraquat and a high concentration of oxygen gas as selective agents. Both are shown to confer oxidative stress in C. elegans (Ishii et al. 1990). We first selected worms unable to grow on NGM plates supplemented with paraquat. Then, these worms were individually examined for growth in the presence of 90% oxygen in air. We screened a total of approximately 1200 F2 worms born from mutagenized N2 worms, and isolated one mutant. This mutant was designated as oxy-4(qa5001), as oxy-1, oxy-2 and oxy-3 had already been isolated (Honda et al. 1991). oxy-4 was back-crossed six times to N2 for subsequent analyses.

Sensitivity to oxidative stress

Sensitivity to oxygen gas in eggs or L1 larvae was determined by culturing eggs on NGM plates in the presence of various concentrations of oxygen in air for 6 days. As shown in Fig. 1(A), oxy-4 showed an increased sensitivity to a high concentration of oxygen (Fig. 1A). In the presence of 90% oxygen, hatched L1 larvae of oxy-4 were arrested at the L1–L2 stages, and some of these larvae were dead during prolonged culture in oxygen. When adult worms were cultured in the presence of 90% oxygen, oxy-4 died earlier than did N2 (Fig. 1B). However, the difference in survival between N2 adults and oxy-4 adults in 90% oxygen (Fig. 1B) seemed to be comparable to that in normal air, that is, in 21% oxygen (Fig. 4A and Table 1). This indicated that the oxy-4 mutant showed the increased sensitivity to oxygen specifically in the larval stages. Sensitivity to oxygen in eggs or L1 larvae of oxy-4 at 15 and 26 °C (25 °C) was essentially the same as that at 20 °C (data not shown).


Figure 1
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  		Figure 1  Sensitivity to oxidative stress in N2 and oxy-4. (A) Sensitivity to oxygen gas in eggs or L1 larvae. Eggs were incubated on NGM plates in the presence of various concentrations of oxygen for 5 days, and the percentage of worms that reached adulthood was determined. (B) Sensitivity to oxygen gas in adult worms. Adult worms of N2 and oxy-4 were incubated in the presence of 90% oxygen, and their survival was monitored at intervals. (C) Sensitivity to paraquat. Eggs were incubated on NGM plates containing various concentrations of paraquat for 5 days, and the percentage of worms that reached adulthood was determined.

 

Figure 4
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  		Figure 4  Determination of longevity in N2 and oxy-4. Synchronized L1 larvae of N2 and oxy-4 were cultured on NGM plates and their survival was examined at intervals. Graph (A) and (B) show the typical survival curves measured at 20 and 26 °C, respectively.

 

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  		Table 1  Life span of oxy-4 at 20 and 26 °C
 
Next, sensitivity to paraquat was determined by culturing eggs on NGM plates supplemented with various concentrations of paraquat. There was a difference in survival between N2 and oxy-4 in the presence of 0.3–0.4 mM paraquat (Fig. 1C).

Autofluorescence in intestinal cells

Caenorhabditis elegans shows increasing fluorescence signals in intestinal cells with advancing age. These fluorescent materials contain lipofuscin consisting of oxidized and cross-linked macromolecules such as lipids and proteins (Klass 1977; Davis et al. 1982; Hosokawa et al. 1994). In the oxygen-sensitive mutant mev-1, the fluorescent materials accumulate at a greater rate than in N2, and the accumulation rate depends on the concentration of oxygen (Hosokawa et al. 1994). We then examined the fluorescence signals in N2 and oxy-4 at day 10 with fluorescence microscopy, and found that a significant portion of oxy-4 worms (ca. 30%–50%) showed increased fluorescence signals, compared with N2 worms. (Fig. 2)


Figure 2
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  		Figure 2  Autofluorescence in N2 and oxy-4. Fluorescence signals in intestinal cells of worms at day 10 were photographed with fluorescence microscopy.

 
Brood size and life span

N2 and oxy-4 produced 276 ± 15 (n = 19) and 203 ± 15 (n = 16) eggs, respectively (Fig. 3). This result also indicated that oxy-4 showed delayed larval development, as well as reduced fecundity. The mean and maximum life spans in oxy-4 at 20 °C were 23% and 28% shorter, respectively, than those in N2 (Fig. 4A and Table 1). In contrast, the life span of oxy-4 at 26 °C was not shorter than that of N2 (Fig. 4B and Table 1). The recovery of life span at 26 °C was not due to the extended larval development in oxy-4, as oxy-4 reached adulthood 1–1.5 days later than N2 at 26 °C, as at 20 °C (data not shown).


Figure 3
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  		Figure 3  Determination of brood size in N2 and oxy-4. Adult worms were transferred to fresh NGM plates every 24 h and allowed to lay eggs. The number of eggs was scored until they ceased to lay eggs. Vertical bars indicate SE.

 
Sensitivities to heat and UV

We examined sensitivities to heat and UV irradiation. When L1 larvae of N2 and oxy-4 were incubated at 36 °C or irradiated with UV light (40 J/m2), they showed similar sensitivities to these stresses (data not shown). However, when adult worms of N2 and oxy-4 were incubated at 36 °C or irradiated with UV light, oxy-4 survived longer than N2 (Table 2). Thus, adult oxy-4 worms were found to be more resistant to both heat and UV than N2. It is worth noting that oxy-4 was not a mutant with increased sensitivities to general stresses.


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  		Table 2  Survival of worms after exposure to heat or UV in N2 and oxy-4
 
Expression of anti-oxidant enzymes

The expression levels of several anti-oxidant enzymes, cytosolic SOD (sod-1), two mitochondrial SODs (sod-2 and sod-3), extracellular and membrane bound SOD (sod-4), and cytosolic and peroxisomal catalases (ctl-1 and ctl-2), were examined by Northern blot analysis. RNA samples were prepared from mixed-stage cultures of N2 and oxy-4 and probed with each cDNA. When signals were normalized with g3pdh, the expression levels of the two catalase genes were found to be up-regulated (P < 0.05, Fig. 5). The difference in expression level of sod-3 was not statistically significant. These results indicated that the higher sensitivity to oxidative stress in oxy-4 was not caused by down-regulation of these anti-oxidant enzymes.


Figure 5
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  		Figure 5  Expression levels of anti-oxidant enzymes in N2 and oxy-4. RNA was isolated from a mixed-stage culture of worms and subjected to Northern blot analysis. The filter was hybridized with the indicated probes, and the signals were quantified with an image analyzer BAS2000 (Fuji film). Relative expression levels of anti-oxidant enzymes in three independent experiments are shown after normalization with g3pdh. Vertical bars indicate SE.

 
Cloning of oxy-4

The hypersensitivity to oxygen in oxy-4 was found to be a recessive trait and inherited in a Mendelian fashion. Linkage analysis revealed that the oxygen-sensitive trait was concordant with the genetic marker dpy-1 located on LG III. To locate the mutation site more precisely, three-factor-crosses were carried out with the following results: oxy-4 (17/17) (dpy-17 unc-32), (unc-93 dpy-17) (15/15) oxy-4, (unc-79 dpy-17) (7/7) oxy-4, oxy-4 (3/3) (lon-1 unc-36), dpy-17 (2/7) oxy-4 (5/7) lon-1. From these results, oxy-4 was mapped to the region between dpy-17 and lon-1 (Fig. 6A).


Figure 6
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  		Figure 6  Cloning of oxy-4. (A) A genetic map of LG III. Approximate genetic positions of the mutants used are shown. (B) A physical map. The cosmids between dpy-17 and lon-1, which were used in this study, are shown. (C) The genes and plasmids in Y54H5A. DNA regions cloned in plasmids are shown as black bars. Triangles indicate frame-shift mutations in Y54H5A.1 and Y54H5A.4 at the position of BamHI and SpeI, respectively. The rescuing abilities of these plasmids are shown as + or – at right. A mutation found in oxy-4 is shown at the bottom.

 
To identify the oxy-4 gene, oxy-4 was microinjected with the cosmids that resided between dpy-17 and lon-1 (Fig. 6B). Transgenic worms carrying these cosmids were examined for their sensitivity to 90% oxygen, but no cosmids rescued the increased sensitivity to oxygen in oxy-4. As a small region (Y54H5A) that was not covered with these cosmids was left unexplored, we then amplified the parts of the sequence in this region by PCR and cloned them into a plasmid vector to make pYBT20 and pYBT21 (Fig. 6C). Microinjection of these plasmids has revealed that the oxygen sensitivity in oxy-4 was complemented by pYBT20, which contained two genes, Y54H5A.1 and Y54H5A.4. To determine which gene was responsible for the rescuing ability, a frame-shift mutation was inserted in the first exon of Y54H5A.1 or in the second exon of Y54H5A.4 (shown as triangles in Fig. 6C). These mutations were created by digestion of the genes with restriction enzymes, followed by end-filling with klenow enzyme and self-ligation. From the result of microinjection of these plasmids (pYBT20S and pYBT21B), oxy-4 was revealed to be Y54H5A.4.

This result was further confirmed by the finding of a mutation in the fourth exon of Y54H5A.4 in oxy-4: a conversion of G to A at the position 1117 was identified, which indicated a change in an amino acid from aspartic acid(D)-373 (GAT) to asparagine(N)-373 (AAT) (Fig. 6C). Importantly, no mutation was found in Y54H5A.1, Y54H5A.2, and Y54H5A.3. Therefore, we have concluded that the causal gene for oxy-4 was Y54H5A.4, which encodes an [FeFe]-hydrogenase-like protein.

RNAi

To further confirm the above results, we down-regulated the oxy-4 expression by RNAi (Fraser et al. 2000). N2 worms fed with bacteria in which double-stranded RNA for Y54H5A.4 cDNA was expressed, were examined for growth in normal air and 90% oxygen. They grew normally in normal air, but were arrested at L2–L3 stages in 90% oxygen (data not shown). This result indicated that knock-down of oxy-4 (Y54H5A.4) led to an increased sensitivity to oxygen in C. elegans. Other phenotypic changes were also observed in these worms (oxy-4(RNAi)), though the extent of changes in oxy-4(RNAi) was less marked than that in oxy-4(qa5001).

nar1 in yeast

To examine the functional conservation of [FeFe]-hydrogenase-like genes in other organisms, we characterized the possible yeast homologue, NAR1. As NAR1 is an essential gene, it was deleted in the yeast strain that expressed NAR1 driven from the GAL1 promoter, as previously described (Balk et al. 2004). The resulting strain, Gal-nar1, showed galactose-dependent growth, coincident with the previous study (Balk et al. 2004) (Fig. 7A). We then examined the effect of a decrease in the expression of NAR1 on sensitivity to oxygen in Gal-nar1. The low expression levels of NAR1 were achieved by addition of low concentrations of galactose in medium that contained 2% raffinose as a carbon source, because raffinose neither suppresses nor induces the GAL1 promoter.


Figure 7
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  		Figure 7  Increased sensitivity to oxygen in nar1{Delta}. (A) Galactose-dependent growth in Gal-nar1. The wild-type and Gal-nar1 strains were cultured in medium containing 2% Glucose or 2% Galactose, under normal air. (B) Growth of Gal-nar1 under various concentrations of oxygen. The wild-type and Gal-nar1 strains were cultured in medium containing 2% raffinose and the indicated concentrations of galactose, under 21% (normal air), 90%, and 2% oxygen.

 
In normal air (21% oxygen), Gal-nar1 grew on a plate containing 0.02% galactose, whereas it did not grow on a plate containing 0.002% galactose (Fig. 7B, left). As these plates contained raffinose as a carbon source, the growth defect on a plate containing 0.002% galactose was probably due to an insufficient expression level of NAR1. Then, we cultured Gal-nar1 on a plate containing 0.02% galactose in the presence of 90% oxygen, and found that Gal-nar1 was unable to grow on this plate (Fig. 7B, middle). In contrast, when Gal-nar1 was cultured in the presence of 2% oxygen, it was able to grow even on a plate containing 0.002% galactose (Fig. 7B, right). The growth of the wild-type strain was not essentially influenced by concentrations of oxygen nor galactose.

These observations clearly indicated that repression of NAR1 led to an increased sensitivity to oxygen, and also that lethality in nar1{Delta} under normal air was, at least in part, caused by oxidative stress. These results in C. elegans and S. cerevisiae suggested that the function of [FeFe]-hydrogenase-like proteins might be conserved across phylogeny.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Although oxygen is essential for the life of aerobic organisms, genes that affect oxygen sensitivity in cells are not well-studied. We therefore screened for such mutants, and isolated a mutant, oxy-4, that showed an increased sensitivity to a high concentration of oxygen. oxy-4 exhibited a decrease in longevity at 20 °C as expected, but showed some unexpected phenotypes. First, oxy-4 showed increased resistance to heat and UV. As oxy-4 showed growth retardation as well as hypersensitivity to oxygen, this mutant seemed to be suffering from oxidative damage even under normal culture conditions. If so, genes involved in defense against oxidative stress and other stresses are considered to be up-regulated in this mutant, and the increased resistance to UV and heat may be explained along with this context. In agreement with this, the expression levels for catalases were up-regulated in oxy-4.

Second, oxy-4 showed almost normal life span at 26 °C. As the sensitivity to oxygen in oxy-4 was essentially the same at 20 and 26 °C (data not shown), it follows that a decrease in longevity was suppressed at 26 °C by other genetic mechanisms. When worms are grown at 26 °C, they show decreased longevity probably due to chronic heat stress (Van Voorhies & Ward 1999). We therefore speculate that the enhanced resistance to heat stress in oxy-4 compensated the decrease in the longevity at 26 °C, leading to an apparently normal level of life span. Regulation of life span that depends on ambient temperature is also observed in rad-8, a mutant of C. elegans with increased sensitivities to both UV and oxidative stresses (Hartman & Herman 1982). Interestingly, rad-8 shows normal life span at 25 °C, but extended life span at 16 °C (Ishii et al. 1994). Longevity is considered to be affected by many parameters that include sensitivities to environmental stresses, and to be specified through combined effects of these parameters. Then, it would be useful to characterize various mutants for a better understanding of complex processes in aging.

Our genetic analysis has indicated that a mutation in an [FeFe]-hydrogenase-like gene led to hypersensitivity to oxygen in C. elegans and S. cerevisiae. In addition, it was recently shown that knock-down of an [FeFe]-hydrogenase-like gene in the plant Arabidopsis thaliana causes a dwarf phenotype, which is repressed when cultured under hypoxic conditions (Cavazza et al. 2008). These findings suggested that an [FeFe]-hydrogenase-like gene was involved in the regulation of sensitivity to oxygen in a variety of organisms.

[FeFe]-Hydrogenases are enzymes that contain [Fe-S] clusters and catalyze the formation and the splitting of hydrogen: H2{leftrightarrow}2H+ + 2e–. [FeFe]-hydrogenases were originally identified as a key enzyme involved in anaerobic respiration in anaerobic bacteria, and were later found in a few anaerobic eukaryotes such as anaerobic fungi, ciliates, trichomonads, and green algae (Meyer 2007; Vignais & Billoud 2007). Aerobic eukaryotes produce energy in mitochondria with use of oxygen as an electron acceptor, although some anaerobic eukaryotes do it in different organelles called "hydrogenosomes", with use of a proton as an electron acceptor (Rotte et al. 2000). However, recent genome analysis of a number of organisms has indicated that aerobic eukaryotes that do not produce hydrogen also possess genes homologous to [FeFe]-hydrogenases, that is, [FeFe]-hydrogenase-like genes (Horner et al. 2002; Hackstein 2005). It is suggested that, in mitochondriate eukaryotes, craniates have two homologues, whereas others have one homologue in their genomes (Meyer 2007). For instance, S. cerevisiae and C. elegans have single homologues, NAR1 and oxy-4(Y54H5A.4), respectively. Human has two homologues, Narf/IOP2 and Narf-like/IOP1 (Barton & Worman 1999; Balk et al. 2004; Huang et al. 2007; Meyer 2007). These [FeFe]-hydrogenase-like proteins conserve essential amino acids in their [Fe-S] domains, but they do not show hydrogenase activity because of a lack of the catalytic moiety (Vignais & Billoud 2007). Hence, the OXY-4 protein was presumed not to produce hydrogen. Although [FeFe]-hydrogenase-like proteins in aerobic eukaryotes would be probably derived from [FeFe]-hydrogenases in anaerobes, the exact roles of [FeFe]-hydrogenase-like proteins are not well-studied. They are proposed to have acquired novel functions under aerobic environments, other than hydrogen production (Horner et al. 2002). At present, Narf/IOP2 is shown to interact with prelamin A/C in human cells (Barton & Worman 1999). NAR1 and Narf-like/IOP1 are shown to be involved in the formation of [Fe-S] proteins in yeast and human cells, respectively (Balk et al. 2004; Song & Lee 2008). Narf-like/IOP1 is also shown to regulate the expression of HIF-1 in human cells, which functions as a key transcription factor under hypoxic conditions (Huang et al. 2007).

Why would a defect in oxy-4 lead to an increased sensitivity to oxygen? One key to understanding of the function of oxy-4 may lie in the possibility that oxy-4 encodes a protein that contains [Fe-S] clusters. [Fe-S] proteins are known to be involved in diverse biological functions, such as electron transport, oxygen level sensing, and formation of other [Fe-S] proteins (Johnson et al. 2005). Then, if oxy-4 is involved in the above biological functions, it is likely that its mutation causes a change in sensitivity to oxygen. Another possibility is suggested by the finding that knock-down of Narf-like/IOP1 up-regulates the expression of HIF-1 in human cells (Huang et al. 2007). The basic functions of HIF-1 are well-conserved in a variety of organisms that include C. elegans (Jiang et al. 2001; Shen & Powell-Coffman 2003). We then examined the expression of hif-1 in N2 and oxy-4, but did not find the evidence that the mutation of oxy-4(qa5001) influenced the expression of hif-1 (data not shown). This result suggested that hif-1 was not involved in an increase in sensitivity to oxygen in oxy-4, though it still remains possible that oxy-4 functions as a transcriptional regulator of genes other than hif-1, whose mis-regulation lead to a change in sensitivity to oxygen.

Several mutants are shown to be hypersensitive to oxidative stress in C. elegans, e.g. mev-1, mev-3, gas-1, rad-8, skn-1 and nnt-1 (Ishii et al. 1990, 1993, 1994; Yamamoto et al. 1996; Hartman et al. 2001; An & Blackwell 2003; Arkblad et al. 2005). mev-1, gas-1, nnt-1 and skn-1 encode the succinate dehydrogenase cytochrome b gene, NADH:ubiquinone-oxidoreductase gene, nicotinamide nucleotide transhydrogenase gene, and a transcription factor, respectively (Ishii et al. 1998; Kayser et al. 1999, 2001; An & Blackwell 2003; Arkblad et al. 2005). The former two are involved in energy production in mitochondria, and the latter two in anti-oxidant defense. At present, it is not clear whether oxy-4 is involved in these pathways or not. oxy-4 may be classified in a novel class of genes that affect oxygen sensitivity.

In summary, we have shown that oxy-4 encodes an [FeFe]-hydrogenase-like protein, whose defect caused an increase in oxygen sensitivity in C. elegans. It is an interesting finding that [FeFe]-hydrogenases play an essential role in the absence of oxygen in some anaerobic organisms, and its possible offspring, [FeFe]-hydrogenase-like proteins, play an essential role in the presence of oxygen in some aerobic organisms.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Strains and culture conditions

The C. elegans strains used were obtained from the Caenorhabditis Genetics Center, USA. Worms were grown and maintained at 20 °C on NGM plates seeded with E. coli OP50 as a food source as described by Brenner (1974) unless otherwise stated.

Mutant isolation

L4 larvae of the wild-type N2 were treated with 50 mM ethylmethane sulfonate for 4 h, and cultured to bear F2 progeny. F2 worms were singly cultured overnight in 96 well-titer plates that contained E. coli OP50 in liquid medium to lay eggs. Aliquots of the resulting mixture of the eggs and hatched L1 larvae were transferred to NGM plates supplemented with 0.3 mM paraquat. After incubation for 5–7 days, the plates were examined under a microscope to select worms that showed retarded growth. Then, eggs from the selected worms were individually prepared by treatment with alkaline hypochorine as previously described (Emmons et al. 1979), and were incubated for 4–6 days on NGM plates placed in a container filled with 90% oxygen gas. The worm unable to grow to adulthood in the high concentration of oxygen gas was retained as an oxidative stress-sensitive mutant.

Genetic analysis

Genetic crosses were performed as previously described (Brenner 1974). The following markers were used to assign the linkage of oxy-4: dpy-5(e61) for LG (Linkage group) I, rol-5(sc13) for LG II, dpy-1(e1) for LG III, unc-22(e66) for LG IV, dpy-11(e224) for LG V, and lon-2(e678) for LG X. To regionally map the oxy-4 locus, three factor-crosses were carried out with the following double mutants: dpy-17(e164) unc-32(e189), unc-93(e1500) dpy-17(e164), unc-79(e1068) dpy-17(e164), lon-1(e185) unc-36(e251) and dpy-17(e164) lon-1(e1820).

Photography of autofluorescence

Fluorescent materials in intestinal cells were monitored with a fluorescent microscope (MZFLIII, Leica). Fluorescence signals were captured with a CCD camera (DC350F, Leica) and a filter set for DAPI fluorescence (excitation 359 nm).

Measurement of brood size

Synchronized L1 larvae were obtained by overnight incubation of eggs in S-basal buffer, and were allowed to develop to L3–L4 larvae on a seeded NGM plate. Then they were singly transferred to fresh NGM plates every 24 h, and the number of eggs on the plates was scored until they ceased to lay eggs.

Determination of life span

Life span was determined as described previously (Fujii et al. 2004). Synchronized L1 larvae were cultured on a NGM plate to develop to L4 larvae. A hundred of the L4 larvae were transferred to NGM plates supplemented with 40 µM 5-fluoro-2'-deoxyuridine that suppresses the production of their progeny. The worms were examined daily and dead worms, which did not respond to multiple touches of their heads with a platinum wire, were removed and scored. Dead worms because of desiccation by crawling up the wall of the plate were excluded from the analysis. Worms on the plates contaminated by other microorganisms were also excluded from the analysis because the life span of C. elegans is affected by concentration of dietary bacteria (Hosono et al. 1989).

Assay of sensitivities to stresses

Eggs were prepared as described above. Adult worms were obtained by culturing synchronized L1 larvae of N2 and oxy-4 for 4 and 5 days, respectively. Sensitivity to oxygen in eggs or L1 larvae was determined as described by Ishii et al. with a slight modification (Ishii et al. 1990; Fujii et al. 2005). Eggs were plated on NGM plates placed in a container filled with various concentrations of oxygen gas. After incubation for 5–6 days, the percentage of worms that reached adulthood was determined. Sensitivity to paraquat in eggs or L1 larvae was similarly determined by culturing eggs on NGM plates supplemented with various concentrations of paraquat. After incubation for 5–6 days, the percentage of worms that reached adulthood was determined. Sensitivity to oxygen in adult worms was determined by monitoring the survival of adult N2 and oxy-4 worms in 90% oxygen.

Sensitivities to thermal stress and UV irradiation were determined as previously described (Lithgow et al. 1995; Murakami & Johnson 1996). After L1 larvae or adult worms of N2 and oxy-4 were incubated on fresh NGM plates at 36 °C, or were exposed to UV light (40 J/m2) on bacteria-free plates, their survival was monitored at intervals.

Northern blot analysis

Total RNA samples were prepared from mixed-stage cultures of C. elegans as previously described (Fujii et al. 1998). RNAs were resolved by formaldehyde gel electrophoresis, blotted on to a nylon membrane (Bio-dyne), and hybridized with a cDNA probe labeled with [{alpha}-32P]-dCTP as previously described (Fujii et al. 1995). After washing, the signals were quantified by an image-analyzer BAS2000 (Fuji film, Japan).

Microinjection

Cosmids were provided by the Sanger Center (UK), and prepared with the alkaline lysis method, followed by purification with QIAGEN columns (QIAGEN). Two sequences in Y54H5A were amplified with the following two sets of primers, 5'-TGTT GAAGGGTGAGCGTTTCAG-3', 5'-ATTCGAAACCGGTT GCAGCAA-3', and 5'-TCAGCCTGAAACGCTACCC-3', 5'-AAAATGTAGAAAAACGAATCG-3', and were cloned into pBluescript to make pYBT20 and pYBT21, respectively. To introduce frame-shift mutations in Y54H5A.4, a 6kb BamHI-fragment from pYBT20 was sub-cloned into pUC19, digested with SpeI, end-filled by klenow enzyme, and self-ligated. This modified BamHI-fragment was re-cloned at the BamHI site in pYBT20 to make pYBT20S, which carried a frame-shift mutation at SpeI site in Y54H5A.4. Similarly, two BamHI-fragments derived from pYBT20 were treated with klenow enzyme and re-ligated to make pYBT20B, which carried a frame-shift mutation at BamHI site in Y54H5A.1. Cosmids and plasmids were microinjected with pRF4 to oxy-4 as previously described (Fire 1986). The molar ratio of test DNAs to pRF4 was approximately 1 : 100. After microinjection, worms with a Rol phenotype were isolated, and examined for their sensitivity to 90% oxygen.

Yeast

NNY11 (MATa lys2-801 ura3-52 trp1 leu2 his3) was used as the wild-type strain of the yeast S. cerevisiae (Fujii et al. 2002). The NAR1 gene was amplified from the genomic DNA of NNY11 with the following primers, 5'-TGATTAAGAGAGCAGGT GAC-3' and 5'-GGCGAGTTTTCCGTACATTG3-', and then cloned into pGEM-T (Promega) to make pYBT22. The sequence between HindIII (2.5 kb) in pYBT22 was replaced with HIS3 to make a knock-out construct for NAR1, pYBT23. The NAR1-expressing vector, pYBT24, was constructed by inserting the DraI-PvuII sequence (1.6 kb) from pYBT22 into pYES2 that contains the GAL1 promoter and the CYC1 terminator (Invitrogen). NNY11 was first transformed with pYBT24 and selected on SD (synthetic glucose) plates lacking uracil, followed by transformation with linearized pYBT23 and selection on SG (synthetic galactose) plates lacking uracil and histidine. From these transformants, the NAR knock-out strain, designated as Gal-nar1, was isolated. Oxygen concentration was controlled as previously described (Ishii et al. 1990), and the growth of Gal-nar1 was examined with a spot assay (Fujii et al. 2002) in normal air (21% oxygen), 90% oxygen, and 2% oxygen.


   Acknowledgements
 
Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR). This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Yuji Kohara

* Correspondence: mifuji{at}yokohama-cu.ac.jp


   References
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 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
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Accepted: 8 January 2009





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