Proc. Nati. Acad. Sci.
USA
Vol. 86, pp. 2761-2765, April 1989
Genetics
Null mutation of copper/zinc superoxide dismutase in Drosophila
confers hypersensitivity to paraquat and reduced longevity
JOHN P. PHILLIPS*, SHELAGH D. CAMPBELLt, DENISE MICHAUD, MARC CHARBONNEAUt,
AND ARTHUR J. HILLIKER
Department of Molecular Biology and Genetics, University of Guelph, Guelph, ON, N1G2W1, Canada
Communicated by Bruce N. Ames, December 28, 1988 (received for review November 2, 1988)
ABSTRACT The role of copper/zinc-containing superox-
ide dismutase (cSOD; superoxide:superoxide oxidoreductase,
EC 1.15.1.1) in metabolic defense against 02 toxicity in
Drosophila is examined through the properties of a mutant
strain carrying a cSOD-null mutation, cSOD1'08. Homozygotes
are viable as larvae, which indicates that cSOD is not essential
for cell viability per se. cSOD"'0 confers recessive sensitivity to
the superoxide anion (O )-generator paraquat and to the
transition metal compound CuS04, which indicates that the
cSOD-nufl condition in fact leads to impaired °2 metabolism.
The primary biological consequences of the reduced °2 dis-
mutation capacity of cSOD"IY Drosophila are realized in the
adult as infertility and reduction in life-span. We conclude that
the infertility and reduced life-span of cSODR"'O adults arise as
a consequence of the reduced capacity of embryos, larvae, and
pupae to adequately protect developing preimaginal cells from
02-jnitiated cytotoxic damage.
Reduction of dioxygen (02) in aerobic organisms proceeds
either tetravalently to H20 via the respiratory chain or
univalently to the superoxide anion (O-), hydrogen peroxide
(H202), and the hydroxyl radical (OH-). The products of
univalent reduction, particularly the OHS radical, are highly
reactive and initiate damaging peroxidation of lipids, pro-
teins, carbohydrates, and nucleic acids (1), which in turn can
contribute to a wide variety of acute and chronic pathological
phenomena, such as mutagenesis, carcinogenesis, xenobiotic
toxicity, postischemic tissue injury, diabetes, and inflamma-
tory disease, and to the overall processes of aging (for a
review, see refs. 1-8).
An enzymatic defense system is deployed by aerobic
organisms to catalytically scavenge active oxygen species
and to repair oxidative damage. In general, O2 is scavenged
by the superoxide dismutases (SODs), which catalyze its
dismutation to H202, which in turn is converted by catalase
and peroxidases to H20. The efficient interception of 0° and
H202 by this system prevents the formation of the highly
reactive OHS radical, thought to be responsible for most
02-derived cytotoxicity (1).
The cellular and molecular mechanisms by which these
enzymes, both individually and collectively, carry out the
vital defense functions for which they have been implicated
are poorly understood. Identifying the genes that specify and
regulate the oxygen-defense enzymes will ultimately be
necessary to elucidate these important mechanisms. With
this goal in mind, we recently reported the isolation of a
Drosophila melanogaster strain with a cSOD-null mutation
(cSOD'l0; cSOD is the copper/zinc-containing SOD; super-
oxide:superoxide oxidoreductase, EC 1.15.1.1) (9). Here, we
describe several properties conferred by this mutation that
confirm and clarify the essential function of cSOD in the
metabolism of 0- in this higher eukaryote. We demonstrate
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2761
that cSOD'08 Drosophila have a reduced capacity to suc-
cessfully metabolize the 02 radical, which leads to a striking
reduction in fertility and adult life-span.
MATERIALS AND METHODS
Drosophila Strains. The cSODn108 allele was originally
recovered as an ethyl methane sulfonate-induced recessive
lethal mutation on a chromosome carrying the recessive
markers sr, es, and ca (9). The cSOD108 allele was subse-
quently recombined onto a third chromosome carrying the
larval marker red; in the process, the parental markers, sr, es,
and ca, were eliminated. The resulting chromosome
(cSOD 108, red, sr', es', ca'), hereafter referred to as the
cSOD~n', red chromosome, is maintained as a balanced
heterozygote over TM3 [In(3LR) TM3 Sb Ser y' ri pP sep
bX34e es]. Dft3L)h-76 and Dft3L)h-9 are hybrid dysgenesis-
induced deficiencies respectively encompassing and adjacent
to the cSOD locus (9). A description of the various markers
and chromosomes used is given in Lindsley and Grell (10).
Except where otherwise noted, all cultures were maintained
on standard cornmeal/dextrose/agar medium at 250C.
Polyacrylamide Gel Electrophoresis. Adult Drosophila
were homogenized in 10 mM dithiothreitol/0.1% Triton
X-100 (5 ILI per fly) in 1.5-ml Microfuge tubes on ice. After
centrifugation at 10,000 x g for 10 min at 40C, aliquots were
electrophoresed on 7.5-cm discontinuous polyacrylamide
gels [stacking gel, 4% acrylamide (pH 6.8); separating gel,
10% acrylamide (pH 8.8)]. Gels were stained for SOD activity
(11) by incubation with gentle agitation in 2.45 mM nitroblue
tetrazolium/28 mM N,N,N',N'-tetramethylethylenediamine/
0.028 mM riboflavin/36 mM K2HPO4 for 20 min in the dark,
followed by illumination on a fluorescent lightbox for ='30 min.
As determined by dilution analysis, the minimum level of
cSOD activity detectable under these conditions is -1/200th
of that found in cSOD' strains such as Canton S.
Xenobiotic Administration. Adults (0-2 day) were exposed
for 48 hr at 250C in vials containing filter pads saturated with
aqueous solutions of the compound in 1% sucrose and were
scored immediately for response. Larvae were allowed to
develop from eggs through to eclosion in vials containing
instant medium (Carolina Biological Supply) hydrated with
aqueous solutions of the compound.
Longevity Determinations. Newly eclosed adults (0-24 hr)
were maintained at 250C and serially transferred to fresh corn
meal in vials at 2-3 day intervals. Percent survival is
calculated at the end of the experiment after correcting for
total loss incurred from handling.
Abbreviations: SOD, superoxide dismutase; cSOD, copper/zinc-
containing SOD.
*To whom reprint requests should be addressed.
tPresent address: Genetics and Cell Biology Section, Biological
Sciences Group, University of Connecticut, Storrs, CT 06268.
tPresent address: Department of Botany, University of Guelph,
Guelph, ON, NMG2W1, Canada.
2762 Genetics: Phillips et al.
RESULTS
The original cSODn108 chromosome (cSOD"08, sr, es, ca)
produces a recessive lethality pattern in which homozygotes
die during the process of eclosion; rare eclosing adults are
completely sterile, are devoid of detectable cSOD activity,
and die within 2-3 days (9). The cSODn'08, red chromosome
(cSODn"08, red, sr', es+, ca+) gives a recessive premature
adult lethality pattern in which development proceeds to
adulthood. Preadult development of cSOD~108, red homozy-
gotes is somewhat retarded; otherwise the chromosome has
no obvious effect on larval viability. cSOD"n08, red homozy-
gotes are devoid of detectable cSOD activity in both larvae
and adults as shown on histochemically stained polyacryl.
amide gels (Fig. 1). These results are confirmed by spectro-
photometric assay of SOD activity in extracts (B. Staveley
and J.P.P., unpublished observations). By these criteria, we
now consider cSOD~108 to be a cSOD-null activity allele.
cSOD""0 Confers Sensitivity to Paraquat. The O2 scaveng-
ing activity characteristic of cSOD suggests that cSODn'08Y,
red Drosophila should have a reduced metabolic capacity to
dismutate O° and, because of the cytotoxicity of O2 and its
active derivatives, should be hypersensitive to agents that
generate O- radicals. Paraquat (1,1'-dimethyl-4,4'-bi-
pyridinium dichloride; Pq2+) generates O2 radicals in vivo
(12-15) by undergoing an NADH-dependent reduction to
form a relatively stable Pq2' radical (Pq+) that reacts very
rapidly (k2 = 7.7 x 108 M-l so1) with 02 to generate O2-
radical and pq2+ (13).
We investigated the sensitivity of cSOD1'08, red Drosoph-
ila to enhanced production of O2 radical by exposure of
adults to aqueous Pq2+ (Fig. 2). LC50 values for exposure
under these conditions were 0.05 mM for cSOD'08, red
homozygotes and 10 mM for cSODn08 red heterozygotes
, sensitive to pq2+, while heterozygotes carrying Dft3L)h-9
and controls. These results show that, under the conditions
of acute exposure used here, the absence of cSOD activity in
cSODnI08, red homozygous adults confers hypersensitivity to
Pq2+-generated 0° radicals. Furthermore, the Pq2+ sensitiv-
ity of cSODnI'8, red heterozygotes is normal despite their
ostensibly reduced level of cSOD activity. That is, the SOD
activity of heterozygotes is still sufficient to afford normal
levels of protection against Pq2+ toxicity. Overall, the hyper-
sensitivity of cSODn108, red adults to Pq2+ shows that their
lack of cSOD leads to a reduced metabolic capacity to
detoxify the °- radical.
1 2 3 4
FIG. 1. Effect of cSODn'08 on SOD activity. Nondenaturing polyacrylamide gels of extracts from adults (lanes 1-4) and larvae (lanes 5 and
6) were histochemically stained for SOD activity. red and sr, es, ca specify a fast electromorph of cSOD; cn, bw specifies a slow electromorph.
Lanes: 1, red; 2, cn, bw; 3, cSODn'08, red/sr, el, ca; 4-6, red. mSOD, manganese-containing SOD.
Proc. Natl. Acad. Sci. USA 86 (1989)
100 -
80 -
g 60-
.2
CO 40-
20
0-
0 5 10 15 20 25 30 35 40
paraquat, mM
FIG. 2. cSODn'08 confers sensitivity to Pq2+ in adults. a,
cSODn'08, red; *, cSOD"n08, red/sr, es, ca; m, sr, es, ca. Two
hundred adults of each genotype were tested.
We also investigated the relative sensitivity of cSOD-null
larvae to Pq2' by raising the larval progeny from inter se
crosses of cSODn,08, red/TM3 parents on Pq2+-containing
medium (Fig. 3). The ratio (k) of the two progeny genotypes
eclosing as adults provides an accurate measure of their
relative sensitivities of Pq2+. The survival of cSOD'08, red
homozygotes relative to their heterozygous siblings is seen to
decrease with increasing Pq2+ concentration from the Men-
delian ratio of 0.33 for controls to 0.00 at 2.0 mM pq2+. Thus,
the hypersensitivity to Pq2+ of cSODn'08, red larvae, as in
adults, parallels their lack of detectable cSOD activity. Fig.
3 also shows that cSODn]08, red heterozygotes carrying
Df(3L)h-76 (which encompasses the cSOD locus) are hyper-
(which adjoins but does not encompass the cSOD locus) are
not. These results strengthen the conclusion that larval and
adult hypersensitivity to Pq2+ is due to the cSODn'0 allele.
cSOD1'08 Confers Sensitivity to Copper(I). The hypersen-
sitivity of cSODn""0, red to Pq2+ is evidence that the cSOD-
null condition leads to a decreased capacity to metabolize O2-
generated by a xenobiotic agent. We also would like to know
whether O2 generated from normal metabolism has a lower
turnover rate in cSODnI08, red Drosophila. To investigate
this, we used a bioassay based on the O2 activation of
transition metals, which then act as Fenton catalysts to
5 6
mSOD
cSOD
 Genetics: Phillips et al.
0.0 0.5 1.0 1.5 2.0
paraquat, mM
FIG. 3. ccSOD"08 confers sensitivity to Pq2 in larvae. Progeny
from the foallowing four crosses were examined: a , cSOD"n08,
red/TM3 x (cSODn'08, red/TM3; *, cSODn8, red/TM3 x Df(3L)h-
76/TM3; *, cSODen, , rediTM3 x Df(3L)h-9/TM3;o , cSODn'08,
red/TM3 x s
to total prog eeny from each cross that develop to adulthood. The
number of a lult progeny scored from each cross ranged between 318
and 1052.
generate thLe highly reactive and cytotoxic OHS radical from
endogenou s H202 (16):
M(n + 1)+ +
0- Mn- + 02
Mn + H202 M(n + ) + OH + OH-
Net: 0- + H202 OH + OH + 02.
Accordingly, free transition metals should be especially
toxic to cSOD1'08, red Drosophila, in which enzymatic
dismutation of O- is impaired but in which H202 from other
metabolic sources is still present, in a manner analogous to
0- -initiated iron-overload toxicity in mammals (for a review,
see ref. 17). We examined the relative toxicity of copper(I) to
the two classes of larval progeny from cSODn'08, red/TM3
parents as described above. Copper(I) (derived from CuSO4)
has an obvious differential toxic effect on cSOD'08, red
homozygotes and heterozygotes (Fig. 4). As before, controls
with Df(3L)h-9 and Df(3L)h-76 (Fig. 4) implicate the
cSODn'08 allele as the origin of copper(I) hypersensitivity.
These results further argue that the cSOD-null condition of
cSOD'08, red leads to a reduced metabolic capacity to
dismutate 0-2
cSOD"08 Confers Reduced Adult Longevity. The sensitivity
to Pq2' and CuSO4 is evidence for impaired active oxygen
Proc. Natl. Acad. Sci. USA 86 (1989) 2763
metabolism in cSOD'10, red Drosophila. Because active
oxygen species have been implicated in processes contrib-
uting to aging, we examined the effects of the cSODW'O allele
on the adult life-span (Fig. 5). Males were used exclusively in
these experiments for the reason that the life-span in females
has been shown to be dependent upon reproductive history
(18). The two parent stocks of cSODI'0, red-namely, sr, es,
ca, and red-and their hybrid, cSOD'08, red/sr, e , ca, show
mean adult life-spans under our conditions of 55.4 days, 57.8
days, and 61.4 days, respectively, while cSOD108, red itself
has a mean adult life-span of 11.8 days. These results
demonstrate that, despite the near normal development of
cSOD'08, red larvae, the absence of cSOD activity leads to
serious life-shortening consequences in the adult.
cSOD"'08 Confers Sterility. In higher eukaryotes, impaired
fertility is a sensitive indicator of cytotoxic or genotoxic
events in cells of the reproductive system. Thus, if cSOD
plays an important protective role in reproductive cells,
cSODn'08, red homozygotes might show reduced fertility.
Indeed, cSOD'08, red males are completely sterile and
cSODn0 , red females are nearly so, yielding only occasional
progeny. These observations are evidence for an important
role for cSOD in the reproductive system and suggest that, if
ineffectively scavenged, oxygen radicals produced under
normal culture conditions can have a profound deleterious
effect on reproduction.
DISCUSSION
The discovery of SOD by McCord and Fridovich (19) has led
to the concept of an enzymatic system to defend aerobic cells
against the noxious products of the univalent reduction of 02.
Concurrently, the catalogue of pathologies related to active
oxygen has been expanding at an impressive rate. Unfortu-
nately, very little direct experimental evidence can be cited
that describes the in vivo relationship between active oxygen
species, the putative oxygen defense system, and specific
consequences of the failure of this system in higher eukary-
otes. The cSOD1108 mutation of Drosophila described here
provides an opportunity to examine these relationships more
closely.
cSOD-null larvae are viable and develop to adulthood.
cSOD is not essential, therefore, for cell viability per se. The
apparent lack of effect of the cSOD-null condition on larval
viability might reflect a lower exposure of larvae to atmo-
spheric 02 through their semiliquid habitat or possibly
through a lower basal metabolic rate. Several other features
of larval biology also may contribute to the apparent invul-
100 -

0.60 -
80
0.504
0.40- co 60
o-
0.
he~e0.30 I 40
0.20-
20
0.10.
0
-0.00 I . .

0 1 2 3 4 5 days
CuS04, mM
FIG. 5. cSOD!Y"1 confers reduced adult longevity. a , red; o, sr,
FIG. 4. cSOD '08 confers sensitivity to CuS04 in larvae. Sym- e , ca; *, cSOD'08, red/sr, es, ca; *, cSOD"108, red. Effective
bols are as described in the legend to Fig. 3. The number of adult population sizes (number of mortality events scored) ranged between
progeny scored from each cross ranged between 133 and 867. 175 and 434.
 2764 Genetics: Phillips et al.
nerability of cSOD'108, red larvae to the lack of cSOD. Larval
life-span is rather short (-5 days, including embryogenesis,
at 25TC) compared to that of adults. Thus, O-initiated
cellular damage may simply not accumulate to toxic levels in
this short time. Also, most cells of the larva proper are
polytene and postmitotic. Such cells may be less vulnerable
to O-initiated cytological damage than are dividing diploid
cells. Finally, oogenesis may provide a maternal dowry of
cSOD sufficient to carry the development of cSODn,08, red
embryos to adulthood. These and other features of larval
biology may conspire to minimize the apparent consequences
of the failure of cSOD'08, red larvae to produce active cSOD.
However, the cSOD-null condition of these larvae may not be
without severe consequences to the diploid, mitotically
active preimaginal cells, which give rise to the adult.
Reduced life-span is one such important consequence of
cSOD"l01 on adults. The counteractive roles of active oxygen
and the putative oxygen defense system in normal species
life-span have been the subject of much discussion (2, 3, 5, 20,
21). The reduced life-span of cSODn'08, red adults supports
the view that normal adult longevity is dependent upon
normal levels of cSOD activity. However, the observation
that cSOD"n08, red heterozygotes have a normal adult life
span clearly indicates that longevity is not simply a function
of cSOD levels. Furthermore, it predicts that cSOD over-
production, as for example in mutants or in cSOD transform-
ants, will not per se extend adult life-span. In fact, cSOD
overproduction in cultured mammalian cells appears to
generate a variety of deleterious membrane effects related to
active oxygen (22, 23).
Studies with Drosophila have contributed substantially to
our overall understanding of the genetic determination of
life-span (18, 24-28). Reduced adult life span is a pleiotropic
characteristic of many Drosophila mutants, and genetic
variation in natural populations of Drosophila can be selected
to produce lines with significantly altered adult life-span.
However, the significance of cSODWI, red lies in the fact
that its reduced adult life-span arises from a single gene
specifying an enzyme that protects a number of cellular
processes that, until now, have been implicated only indi-
rectly as being important for normal longevity. Thus, the
effect of cSODn'08 on adult longevity in Drosophila signifi-
cantly clarifies our understanding of the fundamental biolog-
ical importance of cSOD.
Infertility is another obvious and important biological
consequence of the lack of cSOD activity in cSODY"0, red
Drosophila. Although we do not yet know the basis for this
reduced fertility, several observations (J.P.P., unpublished
data) suggest directions for further investigation: cSOD"08,
red males and females both actively engage in mating;
cSODn'08, red males do not appear to possess normal motile
spermatozoa; and cSOD+ adults can be made transiently
infertile by pulse exposure to Pq2+. These observations
suggest that the reduced fertility of cSOD '08, redDrosophila
arises from cytotoxic damage to cellular components of the
reproductive system. Furthermore, the known reactivity of
active oxygen with DNA in vivo, as measured directly (1, 5,
29, 30) and indirectly by mutagenesis (31), suggests that
genotoxic exposure of cells to unscavenged O- radicals
during larval and early-adult development may also contrib-
ute to infertility. Investigation of somatic and germ-line
mutation frequency in cSODn'08, redDrosophila should more
clearly define the overall biological role of cSOD.
The hypersensitivity of cSODn08, red Drosophila to Pq2+
and to CuS04 is a consequence of the lack of cSOD activity
and supports the interpretation that the phenotypes of re-
duced longevity and infertility characteristic of this mutant
arise from inadequate scavenging of O2 produced under
normal culture conditions. This clearly establishes an impor-
tant fundamental relationship between cSOD activity in vivo,
Proc. Nati. Acad. Sci. USA 86 (1989)
the metabolism of O2, and two biological properties of vital
importance in higher eukaryotes, namely, life-span and
fertility. It will now be of interest to examine cSOD'108, red
Drosophila for direct molecular evidence consistent with the
action of active oxygen species on structural biomolecules
such as lipids and proteins and especially on informational
macromolecules such as DNA (5, 29, 30) and RNA.
The relationship between metabolic rate, antioxidant phys-
iology, and adult-life span has been extensively investigated
in D. melanogaster and in another dipteran insect, Musca
domestica (for reviews, see refs. 26, 27, and 32). Our
interpretation of the cSODn,08, red phenotype is consistent
with known antioxidant physiology in both species, although
there are significant differences between Drosophila and
Musca. For example, D. melanogaster (Fig. 5) has a mean
adult life-span =3 times longer than M. domestica (32); the
level of cSOD remains relatively constant throughout adult
life in D. melanogaster (33, 34) but decays by 30%o in M.
domestica (35); and, contrary to M. domestica (34), the bulk
of SOD activity in D. melanogaster adults is attributable to
cSOD rather than to manganese-containing SOD (B. Staveley
and J.P.P., unpublished observations; see also Fig. 1).
Finally, we wish to emphasize that the results reported
here on cSOD~108, red Drosophila should be interpreted in
terms of the holometabolous development of Drosophila, in
which the adult is assembled during metamorphosis from
imaginal cells formed and propagated during development of
the larvae. Our results could be interpreted to suggest that the
phenotypes of reduced adult life-span and infertility arise
primarily, if not exclusively, from the reduced capacity of
cSOD'08, red embryos, larvae, and pupae to adequately
protect preimaginal cells from O-initiated damage.
We thank Brenda Duyf and Kim Kirby for technical assistance.
This work was supported by grants from the Natural Sciences and
Engineering Research Council of Canada to J.P.P.; S.D.C. and D.M.
were supported by a postgraduate fellowship and an undergraduate
summer research fellowship, respectively, from the Natural Sciences
and Engineering Research Council of Canada.
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