Nucleotide variations in the lxd region of Drosophila melanogaster: characterization of a candidate modifier of lifespan
Introduction
It is hypothesized that molecular oxidative damage inflicted by reactive oxygen species (ROS) is a main cause of aging (Harman, 1956, Allen, 1998). Aerobic organisms have developed an array of defense mechanisms against the toxic effects of ROS. These mechanisms are believed to include enzymatic and non-enzymatic defenses (Phillips and Hilliker, 1990). With regards to the enzymatic defenses, it has been shown that over-expressing Cu/Zn superoxide dismutase (Cu/Zn SOD) and catalase (CAT) in transgenic Drosophila melanogaster have extended adult mean lifespan (Sohal et al., 1995).
In addition to the enzymatic defense, several metabolites such as uric acid have been suggested to have important antioxidant functions (Hilliker et al., 1992). Uric acid is the product of the two-oxidation steps, hypoxanthine to xanthine to uric acid, that involves the enzyme xanthine dehydrogenase (XDH). In some organisms, further oxidation of uric acid to allantoin is carried out by urate oxidase. This enzyme is not present in humans and some other primates, permitting the accumulation of uric acid in mammals. Based on these findings, and the observations that primates with longer lifespans have higher levels of plasma uric acid, Ames et al. (1981) proposed an in vivo antioxidant role of uric acid in mammals. In D. melanogaster, xanthine dehydrogenase-null mutants, rosy (ry) and maroon-like (ma-l), which are unable to synthesize uric acid (Hilliker et al., 1992, Humphreys et al., 1993), and Cu/Zn SOD and CAT null mutants (Staveley et al., 1991) were found hypersensitive to superoxide (O2−) generating agents. However, compound mutants doubly deficient for uric acid and Cu/Zn SOD are lethals, which are unable to complete metamorphosis under normal growth conditions (Hilliker et al., 1992). These findings reflect the important role of uric acid and SOD in oxygen defense.
Xanthine dehydrogenase and other molybdoenzymes (Mo) have been extensively studied in many organisms (Bogaart and Bernini, 1981). In Drosophila, the molybdoenzyme system consists of xanthine dehydrogenase (ry), pyridoxal oxidase (po), aldehyde oxidase (aldox) and sulfite oxidase (Phillips and Hilliker, 1990). These enzymes require molybdenum for their assembly and function. The structural genes of these enzymes appear to be under the control of three distinct non-structural loci: low xanthine dehydrogenase (lxd), maroon-like (mal), and cinnamon (cin). It has been shown that mutations in any of the three loci affects the activity levels of the four enzymes (Bentley and Williamson, 1979, Finnerty et al., 1979). This complex control system of genetic interaction between lxd, cin and ma-l suggests that the molybdoenzyme system in Drosophila may serve as a multifunctional oxygen defense.
The goal of the present study is to identify the gene(s) that specify and regulate the oxygen defense mechanisms, and consequently affect lifespan. This is accomplished in part by comparing DNA sequences from two highly inbred lines of D. melanogaster: 1L6, a long-lived line, is derived from an outbred (LA) population selected for increased longevity; 1S9 is a control line derived from an un-selected (LD1) population in the same experiment (Lukinbill et al., 1984, Lukinbill and Clare, 1985). These two lines have been used to construct recombinant inbred lines (RIs) for further research on the genetics of lifespan extension (Curtsinger and Khazaeli, 2002). Quantitative trait loci (QTL's) that influence lifespan have been mapped to the left arms of chromosomes II and III, and the centromeric region of chromosome II (Khazaeli et al., 2002). Tanksley (1993) and Falconer and Mackay (1996) have reviewed QTL mapping in a variety of traits in maize, tomato, mice, nematodes and flies. Lifespan QTLs have been identified in Caenorhabditis elegans (Shook et al., 1996) and D. melanogaster (Nuzhdin et al., 1997), but so far there has not been a successful identification of the single gene or genes that produce lifespan QTL peaks. lxd and Sod are candidate longevity genes and, most importantly, lie within the chromosomal region on the left arm of chromosome III that we have previously identified by QTL mapping, in regions 68A3-5 and 68A8-9, respectively.
Here we compare DNA sequences in the lxd region of the long-lived line 1L6 and the control line 1S9. We also characterize lxd, as an integral first step in analysing the candidate loci. Specifically, we explore the hypothesis that in long-lived lines, selection for increased lifespan favored nucleotide substitutions that affect the expression and/or activity of the lxd-encoded protein.
Section snippets
Fly stocks
We used two highly inbred lines of D. melanogaster, 1L6 and 1S9, which are derived respectively from two large random-mating populations: LA, selected for longer life, and LD1, an unselected control (Lukinbill et al., 1984, Lukinbill and Clare, 1985). Details of stock construction are given by Khazaeli et al. (2002). The inbred lines have undergone over 30 generations of full-sib or half-sib mating, giving an inbreeding coefficient greater than 0.99. Stocks were maintained using standard
PCR amplification and sequencing of the lxd gene
The strategies used to amplify and sequence D. melanogaster lxd genomic DNA is diagrammed in Fig. 1. Primers, LXDN and LXDB, derived from the genomic DNA sequence of D. melanogaster BAC clone BACR48O03 were used in the PCR amplification. Two overlapping fragments, approximately 1500 bp (LXDN) and 2300 bp (LXDB) containing the entire lxd gene, and 1099 bp upstream and 341 bp downstream of the transcribed region, respectively, were amplified from 1L6 and 1S9. The lxd region generated by both sets
Discussion
The approach that we are using to find genes that extend lifespan in Drosophila involves two steps. First, QTL mapping is used to find chromosomal regions responsible for lifespan differences between recombinant inbred lines (Khazaeli et al., 2002). Several chromosomal regions have been identified, consisting of 5–8% of the total genome, with large effects mapping to the left arm of chromosome III at polytene chromosome bands 66–68. The second step is to investigate candidate genes located in
Acknowledgements
We thank Amy Steffenhagen for technical assistance. This work is supported by Grants AG 09711 and AG 11722 from the National Institute of Aging at the National Institutes of Health.
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