Selenium metabolism in Drosophila: selenoproteins, selenoprotein mRNA expression, fertility, and mortality.

Abstract

Selenocysteine is a rare amino acid in protein that is encoded by UGA with the requirement of a downstream mRNA stem-loop structure, the selenocysteine insertion sequence element. To detect selenoproteins in Drosophila, the entire genome was analyzed with a novel program that searches for selenocysteine insertion sequence elements, followed by selenoprotein gene signature analyses. This computational screen and subsequent metabolic labeling with (75)Se and characterization of selenoprotein mRNA expression resulted in identification of three selenoproteins: selenophosphate synthetase 2 and novel G-rich and BthD selenoproteins that had no homology to known proteins. To assess a biological role for these proteins, a simple chemically defined medium that supports growth of adult Drosophila and requires selenium supplementation for optimal survival was devised. Flies survived on this medium supplemented with 10(-8) to 10(-6) m selenium or on the commonly used yeast-based complete medium at about twice the rate as those on a medium without selenium or with >10(-6) m selenium. This effect correlated with changes in selenoprotein mRNA expression. The number of eggs laid by Drosophila was reduced approximately in half in the chemically defined medium compared with the same medium supplemented with selenium. The data provide evidence that dietary selenium deficiency shortens, while supplementation of the diet with selenium normalizes the Drosophila life span by a process that may involve the newly identified selenoproteins.

Full text

Page 1

Selenium Metabolism in Drosophila
SELENOPROTEINS, SELENOPROTEIN mRNA EXPRESSION, FERTILITY, AND MORTALITY*
Received for publication, January 17, 2001, and in revised form, May 29, 2001
Published, JBC Papers in Press, June 1, 2001, DOI 10.1074/jbc.M100422200
F. Javier Martin-Romero‡, Gregory V. Kryukov§, Alexey V. Lobanov§, Bradley A. Carlson‡,
Byeong Jae Lee¶, Vadim N. Gladyshev§, and Dolph L. Hatfield‡?
From the ‡Section on the Molecular Biology of Selenium, Basic Research Laboratory, NCI, National Institutes of Health,
Bethesda, Maryland 20892, the §Department of Biochemistry, The Beadle Center, University of Nebraska, Lincoln,
Nebraska 68588, and the ¶Laboratory of Molecular Genetics, School of Biological Sciences, Seoul National University,
Seoul 151-742, Korea
Selenocysteine is a rare amino acid in protein that is
encoded by UGA with the requirement of a downstream
mRNA stem-loop structure, the selenocysteine insertion
sequence element. To detect selenoproteins in Drosoph-
ila, the entire genome was analyzed with a novel pro-
gram that searches for selenocysteine insertion se-
quenceelements,followed
signature analyses. This computational screen and sub-
sequent metabolic labeling with75Se and characteriza-
tion of selenoprotein mRNA expression resulted in iden-
tificationofthree selenoproteins:
synthetase 2 and novel G-rich and BthD selenoproteins
that had no homology to known proteins. To assess a
biological role for these proteins, a simple chemically
defined medium that supports growth of adult Drosoph-
ila and requires selenium supplementation for optimal
survival was devised. Flies survived on this medium
supplemented with 10?8to 10?6M selenium or on the
commonly used yeast-based complete medium at about
twice the rate as those on a medium without selenium or
with >10?6
changes in selenoprotein mRNA expression. The num-
ber of eggs laid by Drosophila was reduced approxi-
mately in half in the chemically defined medium com-
pared with the same medium supplemented with
selenium. The data provide evidence that dietary sele-
nium deficiency shortens, while supplementation of the
diet with selenium normalizes the Drosophila life span
by a process that may involve the newly identified
selenoproteins.
byselenoprotein gene
selenophosphate
M selenium. This effect correlated with
Selenium is recognized as an important dietary micronutri-
ent in mammals. It has anticarcinogenic and antiviral proper-
ties, a role in reproductive function, a role in preventing heart
disease, and a role in development (reviewed in Ref. 1). This
element has been also implicated in enhancing immune func-
tion and in slowing the progression of AIDS in human immu-
nodeficiency virus-positive patients (1). At optimal and subop-
timal levels in the diet, selenium occurs in animals primarily as
selenocysteine (Sec)1in Sec-containing proteins. A deficiency in
dietary selenium results in decreased levels of selenoproteins,
thus compromising biological processes that are maintained by
these proteins.
Sec is the 21st naturally occurring amino acid in protein
(reviewed in Refs. 2–4). It has its own tRNA, which is first
aminoacylated with serine. Serine serves as the backbone for
Sec synthesis on its tRNA, and the tRNA is therefore desig-
nated tRNA[Ser]Sec. Sec also has its own code word, UGA, which
serves a dual function of dictating Sec or the cessation of
protein synthesis. The feature that distinguishes UGA as a Sec
codon is the presence of a stem-loop structure downstream of
UGA called a Sec insertion sequence (SECIS) element (2). In
eukaryotes, the SECIS element is located in the 3?-untrans-
lated region of selenoprotein mRNAs (2), and a specific factor,
the SECIS-binding protein (SBP2), recognizes the SECIS ele-
ment for insertion of Sec into protein (5). Furthermore, a sec-
ond factor, the specific Sec elongation protein (EFsec), recog-
nizes Sec-tRNA[Ser]Sec, and the resulting complex between
EFsec-Sec-tRNA-SBP2 governs the donation of Sec to the nas-
cent selenopeptide in response to UGA (6, 7). In eubacteria, a
single factor, SELB, recognizes both the SECIS element, which is
located immediately downstream of UGA, and Sec-tRNA[Ser]Sec
for Sec insertion (3), while in archaea, the factors required for
Sec insertion resemble those found in eukaryotes (8).
Seventeen selenoproteins are currently known in mammals
(9). The function of most of these selenoproteins is not known,
and thus many of the beneficial effects of selenium remain
elusive. However, the number of selenoproteins appears to be
fewer in lower eukaryotes, and their identification will help
elucidate functions of specific eukaryotic selenoproteins.
Among lower eukaryotes, Drosophila emerges as a very use-
ful organism to characterize roles of selenium in biology and
medicine. Fruit flies have been used as a model organism for
studying a variety of maladies in humans, including cancer
(10), neurological disorders (11), and the aging process (12).
Drosophila has also provided a powerful tool in the investiga-
tion of genetics (13) and development (14).
Several studies support the idea that Drosophila may also
serve as a model system for studying many parameters of
selenium metabolism. For example, two groups have detected a
gene encoding a homolog of selenophosphate synthetase (15,
16). The gene has a role in imaginal disc morphogenesis, brain
* This work was supported in part by National Institutes of Health
Grant GM61603 (to V. N. G.). The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBankTM/EBI Data Bank with accession number(s) AF396454
(G-rich) and AF396455 (BthD).
? To whom correspondence should be addressed. Tel.: 301-496-2797;
Fax: 301-435-4957; E-mail: hatfield@dc37a.nci.nih.gov.
1The abbreviations used are: Sec, selenocysteine; SECIS, Sec inser-
tion sequence; SPS2, selenophosphate synthetase; bp, base pair(s);
ORF, open reading frame; PCR, polymerase chain reaction; RT, reverse
transcription; EST, expressed sequence tag; MSGS, mammalian seleno-
protein gene structure.
THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 276, No. 32, Issue of August 10, pp. 29798–29804, 2001
Printed in U.S.A.
This paper is available on line at http://www.jbc.org
29798

Page 2

development, and cell proliferation (16, 17), although studies
thus far have failed to detect selenophosphate synthetase ac-
tivity (15). It is of interest to note that two forms of selenophos-
phate synthetase, designated SPS1 and SPS2, have been de-
scribed in mammals (18–20). SPS2 is a selenoprotein (20).
Recently, the occurrence of a SPS2 homolog was described in
Drosophila (21), marking the first identification of a selenopro-
tein in this organism. Thus, Drosophila also has two seleno-
phosphate synthetase forms, a homolog of SPS1 (18–20) and a
homolog of SPS2 (21). In addition, Sec tRNA[Ser]Sechas been
isolated and sequenced from Drosophila, and the tRNA[Ser]Sec
population has been analyzed (22).
Computer algorithms were recently developed that are capa-
ble of identifying selenoprotein genes by searching for SECIS
elements in nucleotide sequence data bases (9, 23). In this
study, we employed a more highly sophisticated computer ap-
proach to scan the recently sequenced Drosophila genome for
the presence of selenoprotein genes. Only three genes were
found. One of these encoded a homolog of SPS2 (20), which was
recently reported by Hirosawa-Takamori et al. (21), and two
genes encoded novel selenoproteins. To analyze the role of
selenium in the expression of these selenoproteins and in fly
mortality, a chemically defined diet lacking selenium was de-
vised. The addition of 10?8to 10?6M selenium to the medium
resulted in a longer life span than that on the same medium
without selenium, indicating a role of dietary selenium in nor-
malizing the life span of Drosophila.
EXPERIMENTAL PROCEDURES
Materials—[75Se]Selenite (1000 Ci/mmol) was purchased from the
University of Missouri Research Reactor; the RNAeasy purification kit
was from Qiagen (Valencia, CA); Monloney murine leukemia virus
reverse transcriptase was from Ambion (Austin, TX); RNase-free
DNase was from Promega (Madison, WI); Pwo DNA polymerase and
CompleteTMinhibitor mixture were from Roche Molecular Biochemi-
cals; UltraPure low melting point agarose was from Life Technologies,
Inc.; and Instant Drosophila Medium (127.95 g/liter corn meal, 22.38
g/liter brewers yeast, 73.89 g/liter glucose, 36.88 g/liter sucrose, 182.69
g/liter soybean fiber, and 1.21 g/liter p-hydroxybenzoic acid methyl
ester), Grace’s insect medium (G-8142), sodium selenite, and p-hydroxy-
benzoic acid methyl ester were from Sigma. The Canton S. strain of
Drosophila was obtained from Dr. Florence Davidson and used through-
out this study. Total poly(A) RNA from adult, larvae, and embryo
Drosophila (Canton S. strain) was purchased from CLONTECH.
Diets—Chemically defined medium contained Grace’s insect medium
(15 ml/vial) and p-hydroxybenzoic acid methyl ester (100 ?l/ml of a 200
mg/ml solution). Low melting point agarose (0.45%) was added as the
support matrix. Either no selenium or selenium at a final concentration
of 10?8, 10?7, 10?6, 10?5, or 10?4M was also included. Newborn flies
(70–75 flies/vial, and 8–10 vials for every experimental condition) were
collected from complete medium (instant Drosophila medium) and
placed on the chemically defined medium with or without selenium
supplementation or on complete medium. Flies were changed to fresh
chemically defined medium every 3 days.
tRNA Analysis—Total tRNA was isolated from adult Drosophila,
0.005 A260units were loaded into each lane of a 15% TBE-urea poly-
acrylamide gel, the tRNA was electroblotted from developed gels onto
nylon membranes, and filters were hybridized with an 188-bp DraIII–
NdeI fragment encoding the Drosophila tRNA[Ser]Secgene (22). Filters
were stripped and reprobed with Drosophila serine tRNASerprobe
(5?-CGCAGCCGGTAGGATTCGAAC-3?) (22). Amounts of probe at-
tached to filters in response to tRNA were determined using a Phos-
phorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
Genomic Search—The entire Drosophila melanogaster genomic se-
quence determined by Celera (24) was analyzed for the presence of
SECIS elements using an advanced version of SECISearch (9).2Candi-
date sequences that resembled eukaryotic SECIS elements (i.e. satis-
fied primary sequence and secondary structure consensus) were sub-
jected to the free energy criterion, which allowed filtering predicted
unstable structures. The sequence, structure, and energy parameters of
the program that were used in the searches allowed identification of
?90% (53 out of 59) selenoproteins from our collection of known eu-
karyotic selenoproteins and SECIS elements. This collection of natural
SECIS elements from nine eukaryotes (human, mouse, rat, bovine,
zebrafish, macaque, Ovis, Caenorhabditis elegans, and Dictyostelium)
was used to tune the SECISearch program. The annotated version of
the Drosophila melanogaster genome was then used to determine the
location of each candidate SECIS element. The sequences that fell to
the 5?-untranslated region, introns, or coding regions of previously
predicted proteins on either DNA strand were discarded. Finally, ORFs
closest to the predicted candidate SECIS elements were manually an-
alyzed with MSGS criteria (9). The principal component of these criteria
was BLAST-based searches for homologs in Drosophila or other eu-
karyotes that contained Cys codons (TGC or TGT) in place of Sec-
encoding TGA codons in Drosophila selenoproteins. Two possibilities
were considered for each candidate sequence. 1) If the ORF was termi-
nated with TGA, we tested whether this codon could encode a Sec
residue. Such analysis resulted in identification of a 12-kDa selenopro-
tein (G-rich), in which Sec was the C-terminal penultimate residue.
2) All candidate ORFs were also analyzed for the possible extension of
their N-terminal sequences that may include in-frame TGA codons.
These analyses identified SPS2 and a 28-kDa selenoprotein (BthD).
mRNA Expression and Northern Hybridization—Total RNA was ex-
tracted from adult flies, purified with an RNAeasy kit, and treated with
RNase-free DNase, and the reverse transcription (RT) reaction was
carried out with Moloney murine leukemia virus reverse transcriptase
and an oligo(dT)18primer. A portion of the RT product (typically 1 ?l)
was used for PCR amplification with Pwo polymerase using specific
primers for each gene. Forward primers contained an EcoRI digestion
site, and reverse primers contained a BamHI site for cloning PCR
products in pUC19. Primers for each cDNA were: SPS2, forward (5?-C-
GGAATTCCGTAGAAATTCGCGCACAATG-3?) and reverse (5?-CGGG-
ATCCTCGTTAATCATCAAAATTATC-3?); 28 kDa, forward (5?-CGGA-
ATTCCGATTCTGATTACTTTAGGATCATGC-3?) and reverse (5?-CGG-
GATCCCGATATATTTTGTAATAACAATGCTC-3?); 12 kDa, forward
(5?-CGGAATTCCGCCTGAGGATAGCGTGGCGCCATGG-3?) and re-
verse (5?-CGGGATCCCGGAAGCAGCGACCCAGGAATCAGTG-3?);
SPS1, forward (5?-CGGAATTCGCAAACCTATAAAGCAAGATG-3?)
and reverse (5?-CGGGATCCCTGAATGTAGCAGCCGTTACG-3?); and
SBP2, forward (5?-CGGAATTCCACTAAGAATCCACAATAAATG-3?)
and reverse (5?-CGGGATCCTTACTCACTTTCGCCGGGGC-3?).
Quantification of mRNA expression was accomplished by RT-PCR
using a competitive strategy. The exogenous standard (competitor) was
constructed by creating a small deletion in the cloned cDNA. Cloned
SPS2 cDNA was digested with EcoRV and BsgI and religated in the
same vector, BthD cDNA was digested with BstXI and BstEII, G-rich
cDNA was digested with EcoRV and XhoI, SPS1 cDNA was digested
with NcoI, and SBP2 cDNA was digested with EcoRV and BsgI. All
cloned cDNAs were religated in the same vector. Using 1 ?l of the RT
product as template, different amounts of competitors were added to the
PCR mix to calculate the relative amount of each gene after
amplification.
PCR products were separated in agarose gels, stained with ethidium
bromide, and scanned using a Molecular Imager FX System (Bio-Rad).
Signals were quantified by volumetric integration of raw data using the
Quantity One Software following the instructions of the manufacturer.
The amount of each PCR product was also compared with the amount
of RT-amplified cDNA for the constitutive gene rp49 (forward primer,
5?-ATGACCATCCGCCCAGCATAC-3?; reverse primer, 5?-CTTGGCGC-
GCTCGACAATCTC-3?) in the same sample under the same experimen-
tal conditions.
Total poly(A) RNA from adult Drosophila was electrophoresed on
formaldehyde-agarose gels, and the RNA was transferred onto nylon
membranes and hybridized with a 550-bp EcoRI-BamHI fragment en-
coding a portion of the G-rich gene, a 900-bp EcoRI-XhoI fragment
encoding a portion of the BthD gene, or a 450-bp BamHI-EcoRV frag-
ment encoding a portion of the SPS2 gene. Filters were stripped and
reprobed with a 550-bp EcoRI-HindIII fragment encoding a portion of
the ribosomal protein gene (rp49) gene as a control, since RP49 is
constitutively expressed at all stages of development.
75Se Labeling—Flies maintained on complete and chemically defined
medium with and without selenium supplementation were transferred
to defined medium without selenium and labeled with 100 ?Ci of75Se
for 24 h. Protein extracts were prepared from 30–35 flies using radio-
immune precipitation buffer (0.3 M NaCl, 1% deoxycholic acid, 1%
Triton X-100, 0.1% SDS, 0.1 M Tris-HCl, pH 7.4); 40 ?g of total protein
from each sample were loaded onto a 10% polyacrylamide gel; and the
gels were run, dried, and exposed to PhosphorImager screens for 48 h.
2G. V. Kryukov, A. V. Lobanov, and V. N. Gladyshev, unpublished
data.
Selenium and Drosophila Mortality
29799

Page 3

RESULTS
Analysis of the Drosophila Genome—The entire Drosophila
genome was analyzed with an advanced version of SECISearch,
a computer program that identifies SECIS elements in large
nucleotide sequence data bases, including completely se-
quenced eukaryotic genomes (9). This program entails search-
ing genomic DNA for the primary SECIS element consensus
sequence, followed by analyses of their secondary structure and
then an evaluation of the free energy of identified candidate
SECIS structures (9). Parameters of the program were set such
that the absolute majority of known eukaryotic selenoprotein
genes could be recognized. These automatic analyses of the
Drosophila genome were followed by manual analyses of se-
lected sequences using MSGS criteria (9)3and BLAST searches
to identify possible EST or non-redundant sequences as well as
ORFs within these sequences. In particular, we searched for
homologs that contained Cys residues in place of putative Sec
residues in Drosophila ORFs. Three ORFs encoding proteins of
12, 28, and 43 kDa were detected that contained in frame TGA
codons predicted to encode Sec (Table I). Genes for these pro-
teins (Fig. 1A) have three or four exons separated by small
introns with each exon containing a coding sequence. SECIS
elements in the three selenoprotein genes (Fig. 1B) contained
all features characteristic of eukaryotic SECIS elements, in-
cluding the quartet of non-Watson-Crick-interacting nucleo-
tides UGAN . . . NGAN preceded by an unpaired A as well as
an unpaired AA motif in a bulge separated from the Quartet by
an 11–12 nucleotide stem (Fig. 1B).
The 43-kDa selenoprotein was homologous to SPS2 (see also
Ref. 21), a protein that was previously discovered in mammals
(20). Like mammalian SPS2, this Drosophila protein has a
conserved Sec residue in the N-terminal portion of the protein.
Two other proteins have no homology to known proteins. The
28-kDa protein, designated BthD, has a CXXU motif (i.e. Cys
and Sec are separated by two residues) in the N-terminal
portion, similar to that found in the mammalian selenoproteins
SelT and SelW. Interestingly, the 12-kDa protein, designated
G-rich, contained a C-terminal penultimate Sec residue. This
was similar to mammalian thioredoxin reductases, which also
contain the C-terminal penultimate Sec.
Homology searches revealed additional Cys-containing Dro-
sophila homologs of 12- and 28-kDa selenoproteins (Fig. 2),
consistent with MSGS criteria (9) for selenoprotein genes. Fur-
thermore, Cys-containing and Sec-containing homologs of 12-
and 28-kDa selenoproteins were found in other eukaryotes
including higher plants (Cys) and vertebrates (Sec) (data not
shown). Expression of the three selenoprotein mRNAs was
initially assessed by the presence of multiple Drosophila ESTs
and subsequently by Northern blot and RT-PCR analyses (see
below).
The Drosophila genome was also screened by homology
search analyses for the presence of fruit fly selenoproteins
homologous to known eukaryotic selenoproteins. However, no
additional selenoproteins were detected by this approach.
Chemically Defined Medium—Dietary selenium is known to
regulate expression levels of selenium-containing proteins. To
assess the possible significance of the newly discovered seleno-
proteins and, in particular, their role in the life span of Dro-
sophila, we devised a chemically defined diet that supported
the growth of adult flies. In developing the diet, the following
considerations were taken into account. 1) We reasoned that
the yeast-based medium may not be sufficient to obtain low
selenium levels in the diet, especially if the requirement for
selenium is low in Drosophila. This is because selenium can
enter sulfur pathways and is present as a contaminant in a
number of compounds used in the medium. It should be noted
3G. V. Kryukov and V. N. Gladyshev, unpublished data.
TABLE I
In silico analysis of the Drosophila genome for the presence of
selenoprotein genes
Pattern criteria
applieda
Energy criteria
appliedb
Location criteria
appliedc
MSGS criteria
appliedd
4908179 143
aNumber of candidate sequences after the initial analysis of the
entire Drosophila genome for primary sequences and secondary struc-
tures.
bNumber of candidate sequences after application of the free energy
criterion.
cNumber of candidate SECIS elements that satisfied location criteria
(not present in 5?-untranslated region, introns, or coding regions of
known Drosophila proteins).
dNumber of selenoproteins that satisfied MSGS criteria (9).
FIG. 1. Organization of the Drosophila selenoprotein genes
and their SECIS element structures. A, selenoprotein genes are
depicted as boxes, which represent exons, and lines connecting boxes,
which represent introns. Black boxes represent coding regions, and open
boxes represent untranslated regions within exons. The locations of
initiator ATG codons, Sec-encoding TGA codons, and terminator signals
in the three selenoprotein genes are indicated. Gray boxes indicate the
locations of SECIS elements (also indicated by arrows) within the
3?-untranslated region. Numbers correspond to nucleotides within Dro-
sophila genomic scaffolds containing selenoprotein genes (G-rich, acces-
sion number AE003487; BthD, accession number AE003493; and SPS2,
accession number AE003628). B, SECIS elements in the Drosophila
selenoprotein genes are shown. The quartet of non-Watson-Crick inter-
acting nucleotides, an unpaired A preceding the quartet, and an un-
paired AA motif in the bulge are shown in boldface type. These SECIS
elements are further discussed under “Results.”
Selenium and Drosophila Mortality
29800

Page 4

that even when selenium-deficient Torula yeast was used in
studies on rats and mice, many selenoproteins, especially those
expressed in the endocrine organs, were apparently expressed
in sufficient levels to maintain essential processes. Thus, a
simple diet for Drosophila was required that would eliminate
as many selenium-containing, contaminant compounds as pos-
sible. 2) In addition to designing a medium that is chemically
defined (to be able to regulate the amount of selenium intake by
Drosophila), it was important to obtain a different support
matrix for the medium, since the agar routinely used for this
purpose contained selenium. We therefore used a highly puri-
fied agarose as matrix (see “Experimental Procedures”). 3) We
considered and/or tested a number of supplement compounds
and growth conditions that could be sufficient to maintain the
flies for a period long enough to assess their life span charac-
teristics. For example, we tested several concentrations of a
regular agarose (0.4–2%) as a matrix for the media. However,
the only kind of agarose that could be used without affecting
normal life in flies was low melting point agarose. We also
tested the survival of flies using a selenium-deficient Torula
yeast extract. This extract failed to support life of adult flies
even after supplementation with Grace’s vitamin solution and
a lipid mixture. As a result of numerous attempts to find a diet
that was dependent on selenium, we found that Grace’s me-
dium supplemented with p-hydroxybenzoic acid methyl ester
provided a simple, chemically defined medium that supported
growth of adult flies and manifested a growth response to
selenium. Flies could be maintained on this medium without
any other supplementation for more than 5 weeks.
Drosophila Mortality—The life span of flies maintained on
the chemically defined medium with varying levels of selenium
was compared with that of flies maintained on the yeast-based
complete medium routinely used for growing Drosophila (Fig.
3). Selenium levels varied from none to 10?8, 10?7, 10?6, 10?5,
and 10?4M. Flies maintained on defined medium without se-
lenium or with 10?5M selenium died at more than twice the
rate of flies maintained on complete medium or on defined
medium with 10?8, 10?7, or 10?6
survival in the medium with the latter levels of selenium was
similar to those in the complete medium. More than 90% of the
flies maintained on chemically defined medium in the presence
of 10?4M selenium died at the end of the 35-day period (data
not shown). These data suggested that selenium in the 10?8to
10?6M range was beneficial to the life span of flies under these
growth conditions, whereas higher levels of selenium (10?5and
10?4M) were toxic. It also appears that the requirement for
selenium in Drosophila was less than 10 nM.
Drosophila Fertility—To assess the relationship between se-
lenium and fertility in Drosophila, we determined the number
and the hatching of laid eggs in flies maintained on defined and
M selenium. The rate of
FIG. 2. Alignments between Dro-
sophila selenoproteins and their Dro-
sophila homologs. A, alignment be-
tween G-rich and
(accession number AAF48112). B, align-
ment between BthD and its Cys homolog
(accession number AAF58585). C, align-
ment between SPS2 and Drosophila SPS1
(accession number AAF58266). Identical
residues in each alignment are high-
lighted. U, which has been used previ-
ously to designate Sec (1–9), indicates
Sec. Numbers correspond to amino acids
in proteins. Only the N-terminal Sec-con-
taining domain of BthD is shown in the
alignment shown in B.
its Cys homolog
Selenium and Drosophila Mortality
29801

Page 5

complete media. Both sexes were kept separately for 14 days on
either complete or chemically defined medium with 10?6M or
without selenium. Subsequently, populations of flies were
mixed in different combinations, and the number of laid eggs
was counted in each case (Fig. 4). The number of laid eggs was
similar if either males or females were maintained on complete
diet or diet supplemented with selenium, but this number
dropped to less than 50% when both sexes were maintained on
chemically defined medium without selenium. The viability of
eggs (hatching) was higher than 90% under all conditions ex-
amined. The data suggest a role of dietary selenium in egg
production and/or fertilization.
Sec tRNA[Ser]SecLevels—The Sec tRNA[Ser]Secpopulation in
Drosophila migrates as two bands on gel electrophoresis (18).
The primary sequences of both bands are identical, suggesting
that Drosophila tRNA[Ser]Secexists in two stable conforma-
tions. Furthermore, the levels of these two isoforms did not
change throughout development (22). We investigated whether
the amounts of the two tRNA[Ser]Secisoforms from flies main-
tained on complete medium or on defined medium with and
without different levels of selenium varied. As an internal
control, we used serine tRNASer. The total amount of tRNA[Ser]Sec
for each sample relative to the amount of tRNASerwas deter-
mined. The amounts of two tRNA[Ser]Secisoforms did not
change in response to selenium status after 7, 21, or 35 days of
growth on the various media (data not shown). It should also be
noted that a sufficient number of flies that were grown on
defined media containing 10?5or 10?4
survive for 35 days to perform tRNA analysis.
mRNA Expression—Total RNA was isolated from flies main-
tained on the various diets for 21 days. Expression levels of the
three selenoprotein mRNAs (SPS2, G-rich, and BthD) as well
as mRNAs for proteins involved in selenocysteine biosynthesis
(SPS1 and SBP2) were examined by competitive RT-PCR in
flies grown on media supplemented with 0–10?4M selenium
(Fig. 5). The amounts of each mRNA expressed in complete
medium relative to SPS1, which was assigned a value of 1.0,
were as follows: SPS2, 3.0; SBP2, 2.0; G-rich, 14.0; and BthD,
6.0. Thus, the selenoprotein mRNAs were expressed in higher
levels than mRNAs of the two nonselenium containing proteins
with G-rich and BthD mRNAs being expressed at highest lev-
els. The levels of SPS1 mRNA manifested only a slight varia-
tion in relation to selenium status. SBP2 showed slightly
M selenium did not
greater changes but not nearly as much as those found with the
three selenoprotein mRNAs. The amounts of SPS2 and BthD
mRNAs increased 2-fold in the presence of selenium, and that
of G-rich mRNA increased 4-fold. Comparison of mRNA levels
in the chemically defined medium with those in the complete
medium revealed that selenium deficiency resulted in the de-
crease in selenoprotein mRNA levels, whereas supplementa-
tion of the diet with selenium normalized selenoprotein mRNA
expression.
Selenoprotein mRNAs were analyzed by Northern blotting
from adults, larva, and embryos (Fig. 6) to assess the expres-
sion of these mRNAs during development and to better assess
their relative sizes. The constitutively expressed rp49 gene was
used as a control. G-rich protein and SPS2 mRNAs were ex-
pressed throughout development, although SPS2 appeared to
be more weakly expressed at the embryonic and larval stages.
BthD was expressed in embryos and adults, but very weakly in
larvae. The relative sizes of each mRNA are given in the legend
of Fig. 6, and they agree with the predicted sizes of each mRNA
FIG. 3. Survival rates of adult Drosophila in response to sele-
nium. Newborn flies were transferred from complete medium to de-
fined medium supplemented with or without selenium or to complete
medium. A total of 600–700 flies (70–75 flies/vial and a total of 8–10
vials containing each medium condition) were used to assess survival of
flies in each medium, and the number of surviving flies was counted at
the time intervals shown. Error bars correspond to S.D. for the 8–10
vials in every experimental condition. Statistical analyses were done
using an analysis of variance test. p values of significance were repre-
sented as follows: *, p ? 0.05; **, p ? 0.001; ***, p ? 0.0001.
FIG. 4. Effect of selenium on fertility. Fresh newborn flies were
collected, and males and females were maintained separately on chem-
ically defined media with 10?6M selenium or without selenium during
the first postnatal 14 days. Subsequently, 40 males and 40 females from
each group were combined in different combinations and kept on sele-
nium-free medium for 36 h. The total number of eggs laid in each
combination was counted. Analysis of variance was performed in order
to describe significant differences between groups. (Se0/Se0, p ? 0.05,
significant difference between chemically defined medium without se-
lenium for both males and females and any combination).
FIG. 5. Expression of SPS2, the 12- and 28-kDa selenoprotein
genes, and SPS1 and SBP2 in response to selenium. Total RNA
was isolated from flies maintained on complete and chemically defined
medium at the selenium concentrations shown for 21 days, and the
amounts of each cDNA were determined by RT-PCR as given under
“Experimental Procedures.” Values obtained for each cDNA on com-
plete medium were assumed to represent optimal or 100% expression,
and the amounts of each cDNA relative to those found in complete
medium are shown.
Selenium and Drosophila Mortality
29802

Page 6

determined by sequencing cDNA clones, aligning all EST se-
quences found in the Drosophila EST data base and analyzing
selenoprotein gene structures (see legend to Fig. 6).
Metabolic Labeling of Flies with
tained on the chemically defined medium with and without
selenium or on complete medium for 7 days and were then
labeled with75Se for 24 h. The resulting protein extracts were
analyzed by SDS-polyacrylamide gel electrophoresis and Phos-
phorImager analysis (Fig. 7). In the absence of added selenium
(Fig. 7B, lane 1), three bands were clearly discernible. The
strongest labeled band corresponded in size to the selenopro-
tein that we identified in this study as the 12-kDa (G-rich)
protein. It was present as a double band, as is clearly shown in
lane 5, that contained labeled extract from flies maintained on
complete medium. The reason why the 12-kDa selenoprotein
migrated as a double band is not clear. The slower migrating
portion of this band is far more enriched in flies maintained on
complete medium than on defined medium either with or with-
out supplemented selenium. In addition, a 43-kDa band and an
?80-kDa band were visible in the samples. The 43-kDa band
corresponds to SPS2, one of the three selenoproteins we iden-
tified in this study. The 80-kDa protein was not detected in the
Drosophila genome by SECISearch, and the possibility exists
that it might be of bacterial origin (see “Discussion”).
When selenium was added to the medium, an additional
band appeared that migrated just above SPS2 (Fig. 7, lanes 2
and 3). This band and the one that runs as the higher of the two
12-kDa bands were more enriched in labeled extracts from flies
maintained on diets containing 10?4M selenium, which is a
toxic level. The 28-kDa (BthD) selenoprotein that we described
in this study was not detected in adult flies. It is not clear why
higher levels of BthD mRNA were expressed in response to
selenium in adults (see above), while expression of the corre-
sponding selenoprotein was not detected.
75Se—Flies were main-
DISCUSSION
This report describes the identification of selenoprotein
genes in Drosophila and provides evidence for the role of die-
tary selenium in the life span of the fruit fly. It should be noted
that the presence of Drosophila components of the Sec insertion
machinery, including Sec tRNA[Ser]Sec(22) and SPS1 (15–17),
was previously reported. In addition, the selenoprotein, SPS2,
was identified recently in Drosophila (21). Although the com-
pletely sequenced genome of Drosophila is available, it does not
provide correct annotation to selenoproteins. This is because
selenoprotein genes contain in-frame TGA code words encoding
Sec residues that are recognized as termination codons by
currently available genome analysis programs. Such programs
are therefore prone to incorrect annotation of sequences con-
taining in-frame TGA codons.
To identify selenoproteins in Drosophila, we applied a com-
puter program that identifies selenoproteins through recogni-
tion of SECIS elements. The initial version of this program was
previously used to discover new selenoproteins in the human
dbEST (9). However, analyses of completely sequenced ge-
FIG. 6. Northern blot analysis of selenoprotein mRNAs during development. 1.0 ?g of total poly(A) RNA from embryos, larva, and adults
was electrophoresed on formaldehyde-agarose gels, transferred to nylon membranes, and hybridized with the probe (see “Experimental Proce-
dures”) specific for G-rich mRNA (A), BthD mRNA (B), and SPS2 mRNA (C), and the amount of probe attached to the corresponding mRNA was
determined using a PhosphorImager. RNA markers were also run on gels, and mRNA sizes were estimated to be 0.8 kilobases for G-rich mRNA,
1.1 kilobases for BthD mRNA, and 1.3 kilobases for SPS2 mRNA. These sizes are in agreement with those estimated from sequencing DNA clones,
aligning EST sequences found in the Drosophila data base, and analyzing selenoprotein gene structures, which were 876 bases for G-rich, 994 bases
for BthD, and 1309 bases for SPS2 excluding poly(A) tails.
FIG. 7. Detection of selenoproteins in adult Drosophila. Flies
that had been maintained on complete and chemically defined medium
at the selenium concentrations shown for 21 days were transferred to
medium without selenium supplementation, labeled with75Se for 24 h,
protein-extracted, and electrophoresed. The gels were stained with
Coomassie Blue and dried, and the amount of labeling was determined
in a PhosphorImager. A and C, the Coomassie Blue-stained gels are
shown from two separate labelings with75Se, and the lanes contained
the following. A, lane 1, chemically defined medium without selenium
supplementation; lane 2, chemically defined medium plus 10?8M so-
dium selenite; lane 3, chemically defined medium plus 10?7M sodium
selenite; lane 4, chemically defined medium plus 10?4M sodium sele-
nite; lane 5, complete medium. C, lane 1, chemically defined medium
without selenium supplementation; lane 2, chemically defined medium
plus 10?8M sodium selenite; lane 3, chemically defined medium plus
10?7M sodium selenite; lane 4, chemically defined medium plus 10?4M
sodium selenite. B and D, the labeled bands are shown from two
separate labelings with75Se, and the lanes correspond to those shown
in A and C, respectively. MW, molecular mass.
Selenium and Drosophila Mortality
29803

Page 7

nomes are more challenging and have not been reported. The
present study constitutes the first analysis of a completely
sequenced eukaryotic genome, in which new selenoprotein
genes were identified by searching for mRNA structures.
Our search of the Drosophila genome for selenoproteins iden-
tified three such genes. Two of the selenoprotein genes that we
identified, G-rich and BthD, do not have sequence homology to
known proteins, and their functions are currently not known.
The third protein, SPS2, was a previously identified mamma-
lian (20) and Drosophila (21) selenoprotein that is involved in
selenocysteine biosynthesis by activating intracellular sele-
nium to form a selenium donor compound, selenophosphate.
Labeling of flies with75Se resulted in more bands on SDS
gels than expected from the computational analysis of the
genome for selenoproteins. It is possible that our computational
analysis may not have identified all selenoprotein genes in
Drosophila. However, since the program was sufficiently spe-
cific to identify the majority of selenoprotein genes from vari-
ous eukaryotes, it is likely that we have identified most, if not
all, of such genes in the Drosophila genome. This raises a
question of the origin of the 80-kDa band and the occurrence of
two bands in the 12- and 43-kDa ranges observed on SDS gels
(see Fig. 7B). One possibility is that since selenium can enter
sulfur pathways, then these additional bands may be proteins
that are particularly rich in cysteine and/or methionine and
arise by nonspecific labeling with selenium. Another possibility
is that the two bands in the 12- and 43-kDa ranges may be the
result of processing of these proteins. Interestingly, an 80-kDa
selenoprotein has been observed in Drosophila extracts that
was identified as bacterial formate dehydrogenase.4Thus, it is
possible that the labeled 80-kDa band we observed may be of
bacterial origin.
To elucidate the possible role of these identified selenopro-
teins, we developed a chemically defined medium. When this
medium was supplemented with 10?6to 10?8M selenium, flies
had the same life span as flies on a complete yeast-based
medium over a 35-day period, indicating the requirement of
this element for supporting the normal life in adult flies. When
selenium was not included in the medium, the survival rate of
flies was about one-half that of flies supplemented with optimal
levels of selenium. We also found that a concentration of 10 nM
selenium was sufficient to support a normal life span for the
35-day period. Concentrations over 10 ?M were highly toxic.
Dietary selenium also had a dramatic effect on Drosophila
fertility. If both male and female flies were maintained on a
selenium deficiency diet, the number of laid eggs decreased
more than twice compared with conditions in which either
males or females were placed on selenium-supplemented or
complete diets.
The data clearly indicate that dietary selenium has a role in
normalizing the life span and fertility of Drosophila. Selenium
was previously implicated in delaying the aging process in
mammals through changes in antioxidant selenoproteins and
oxidative modifications in proteins in aging cells (4). In hu-
mans, selenium deficiency was associated with early aging (25,
26). Mice and rats maintained on selenium-deficient yeast-
based diets do not show any effect of selenium on aging. In
these dietary studies in mammals, the diets contained trace
amounts of selenium that were sufficient to maintain expres-
sion of some selenoproteins at the expense of others. The rela-
tive significance of these selenoproteins in the life span of these
organisms is not known, and such studies do not seem at
present to provide a means of assessing the role of selenopro-
teins in mortality.
Since only three selenoproteins appear to be encoded in the
Drosophila genome, it seems likely that fruit flies will provide
a simple model system to examine the role of selenoproteins in
the life span of this organism. Two of the newly identified
selenoproteins, G-rich and BthD, are possible candidates. The
third selenoprotein, SPS2, appears to be involved in Sec bio-
synthesis and may provide an autoregulatory mechanism for
selenoprotein expression (20). An additional mechanism, which
we found to regulate selenoprotein expression, is selenium-de-
pendent changes in the abundances of selenoprotein mRNAs.
Taken together, these processes should provide dramatic re-
sponses in selenoprotein levels in response to selenium supple-
mentation and should provide a model system for exploring the
role of selenoproteins in the life span of the fruit fly.
Acknowledgments—We express our sincere appreciation to Drs. Flor-
ence Davidson, Mark Mortin, Bruce Paterson, and Carl Wu for advice
during the course of this study.
REFERENCES
1. Gladyshev, V. N., Martin-Romero, F. J., Xu, X.-M., Kumaraswamy, E.,
Carlson, B. A., Hatfield, D. L., and Lee, B. J. (1999) Recent Res. Dev.
Biochem. 1, 145–167
2. Low, S. C., and Berry, M. J. (1996) Trends Biochem. Sci. 21, 203–208
3. Bock, A. (2000) Biofactors 11, 77–78
4. Hatfield, D. L., Gladyshev, V. N., Park, J. M., Park, S. I., Chittum, H. S., Huh,
J. R., Carlson, B. A., Kim, M., Moustafa, M. E., and Lee, B. J. (1999)
Comprehensive Natural Products Chemistry (Kelly, J. W., ed) Vol. 4, pp.
353–380, Elsevier Science Ltd., Oxford
5. Copeland, P. R., Fletcher, J. E., Carlson, B. A., Hatfield, D. L., and Driscoll,
D. M. (2000) EMBO J. 19, 306–314
6. Tujebajeva, R. M., Copeland, P. R., Xu, X.-M., Carlson, B. A., Harney, J. W.,
Driscoll, D. M., Hatfield, D. L., and Berry, M. J. (2000) EMBO Rep. 1,
158–163
7. Fagegaltier, D., Hubert, N., Yamada, K., Mizutani, T., Carbon, P., and Krol, A.
(2000) EMBO J. 19, 4796–4805
8. Rother, M., Wilting, R., and Bock, A. (2000) J. Mol. Biol. 299, 351–358
9. Kryukov, G. V., Kryukov, V. M., and Gladyshev, V. N. (1999) J. Biol. Chem.
274, 33888–33897
10. Bilder, D., Li, M., and Perrimon, N. (2000) Science 289, 113–116
11. Phillips, J. P., Tainer, J. A., Getzoff, E. D., Boulianne, G. L., Kirby, K., and
Hilliker, A. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8574–8578
12. Tower, J. (2000) Mech. Ageing Dev. 118, 1–14
13. Vernooy, S. Y., Copeland, J., Ghaboosi, N., Griffin, E. E., Yoo, S. J., and Hay,
B. A. (2000) J. Cell Biol. 150, F69–F76
14. Wieschaus E., Nusslein-Volhard, C., and Kluding, H. (1984) Dev. Biol. 104,
172–186
15. Persson, B. C., Bock, A., Jackle, H., and Vorbruggen, G. (1997) J. Mol. Biol.
274, 174–180
16. Alsina, B., Serras, F., Bagun ˜a, J., and Corominas, M. (1998) Mol. Gen. Genet.
257, 113–123
17. Alsina, B., Corominas, M., Berry, M. J., Bagun ˜a, J., and Serras, F. (1999)
J. Cell Sci. 112, 2875–2884
18. Low, S. C., Harney, J. W., and Berry, M. J. (1995) J. Biol. Chem. 270,
21659–21664
19. Kim, I. Y., and Stadtman, T. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92,
7710–7713
20. Guimaraes, M. J., Peterson, D., Vicari, A., Cocks, B. G., Copeland, N. G.,
Gilbert, D. J., Jenkins, N. A., Ferrick, D. A., Kastelein, R. A., Bazan, J. F.,
and Zlotnik, A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 15086–15091
21. Hirosawa-Takamori, M., Jackle, H., and Vorbruggen, G. (2000) EMBO Rep. 1,
441–446
22. Zhou, X., Park, S. I., Moustafa, M. E., Carlson, B. A., Crain, P. F., Diamond,
A. M., Hatfield, D. L., and Lee, B. J. (1999) J. Biol. Chem. 274, 18729–18734
23. Lescure, A., Gautheret, D., Carbon, P., and Krol, A. (1999) J. Biol. Chem. 274,
38147–38154
24. Myers, E. W., Sutton, G. G., Delcher, A. L., Dew, I. M., Fasulo, D. P., Flanigan,
M. J., Kravitz, S. A., Mobarry, C. M., Reinert, K. H., Remington, K. A.,
Anson, E. L., Bolanos, R. A., Chou, H. H., Jordan, C. M., Halpern, A. L.,
Lonardi, S., Beasley, E. M., Brandon, R. C., Chen, L., Dunn, P. J., Lai, Z.,
Liang, Y., Nusskern, D. R., Zhan, M., Zhang, Q., Zheng, X., Rubin, G. M.,
Adams, M. D., and Venter, J. C. (2000) Science 287, 2196–2204
25. Li, F. S., Duan, Y. J., Yan, S. J., Guan, J. Y., Zou, L. M., Wei, F. C., Mong, L. Y.,
Li, L., and Li, S. Y. (1990) Mech. Ageing Dev. 54, 103–120
26. Foster, H. D., and Zhang, L. (1995) Sci. Total Environ. 170, 133–139
4R. A. Sunde, personal communication.
Selenium and Drosophila Mortality
29804