Eur. J. Biochem.

267, 3885±3890 (2000) q FEBS 2000

Structure and location of a ferritin gene of the yellow fever mosquito

Aedes aegypti
Daphne Q.-D. Pham1, Susan E. Brown2, Dennis L. Knudson2, Joy J. Winzerling3,4, Mark S. Dodson5 and
James J. Shaffer1
1
Department of Biological Sciences and Biomedical Research Institute, University of Wisconsin-Parkside, Kenosha, USA;
2
Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, USA;
3
Department of Nutritional Sciences, 4Center for Insect Science, and 5Department of Biochemistry, University of Arizona, Tucson, USA

We have isolated and sequenced a genomic clone encoding the 24- and 26-kDa ferritin subunits in the mosquito
Aedes aegypti (Rockefeller strain). The A. aegypti gene differs from other known ferritin genes in that it
possesses an additional intron and an unusually large second intron. The additional intron is located within the 5 0
untranslated region, between the CAP site and the start codon. The second intron contains numerous putative
transposable elements. In addition, unlike the human and rat ferritin genes, the A. aegypti ferritin gene is a single
copy gene, located at 88.3% FLpter on the q-arm of chromosome 1. Primer extension analysis indicates that the
A. aegypti ferritin gene has multiple transcriptional start sites. A differential usage of these sites is observed with
varied cellular iron concentrations.
Keywords: Aedes aegypti; ferritin; FISH; gene; mosquito.

Ferritins (Fers) isolated from several insect species show
substantial differences from their vertebrate counterparts [1±7].
Vertebrate Fers are large hollow spheres (450 kDa) that are
found in cytoplasm and serve as stores for ferric ions
maintained in a nontoxic and bioavailable form [8]. They are
composed of 24 nonglycosylated subunits of mass ,20 kDa
[9,10]. These subunits are divided into two groups: heavy chain
(H) and light chain (L) [9,10]. The H and L chains are derived
from different genes [8]. There is a second vertebrate Fer
that is secreted into the blood and has glycosylated subunits
[8]. Little is known of the structure of this vertebrate serum
Fer, but it seems to be very different from the cytoplasmic
protein [8,11±13].
Most insect Fers are larger (. 600 kDa) than vertebrate Fers,
and have larger subunits (24±32 kDa) [1,3,6,14,15]. They are
glycosylated and found in the endoplasmic reticulum of insect
cells and in the blood (hemolymph) [2,16]. Insect Fers are
synthesized with a signal peptide, which presumably serves to
direct the mature subunit to the secretion pathway [6,17]. There
is evidence suggesting that insect Fers move in and out of cells,
and may participate in iron transport [1,2,16,17]. The amino
acid sequences of four insect Fers diverge notably from Fers of
both plants and vertebrates, suggesting that they may have a
distinct role to play in insect metabolism [7].
Might these differences be reflected in the structure of the
insect ferritin gene? Fer gene structures are known for several
vertebrates and one insect, Drosophila melanogaster [18±24].
Correspondence to D. Q.-D. Pham, Department of Biological Sciences,
Biomedical Research Institute, University of Wisconsin-Parkside, Kenosha,
Wisconsin 53141±2000, USA. Fax: 1 1 262 595 2056,
Tel.: 1 1 262 595 2172, E-mail: daphne.pham@uwp.edu
Abbreviations: DAPI, 4 0 ,6-diamidin-2-phenylindole; DIG, digoxigenin;
Fer, ferritin chain; fer, ferritin gene; FISH, fluorescence in situ
hybridization; TBE, Tris/boric acid/EDTA.
(Received 17 February 2000, accepted 25 April 2000)
Most vertebrate genomes have multiple intronless fer pseudo-
genes in addition to the single functional genes for the H and L
subunits [18±20,22,23]. In D. melanogaster, genes for both the
heavy-chain and light-chain reside at adjacent loci, and their
structures show some differences from those of vertebrates
[7,24]. Adams et al. suggest that there is a third ferritin in
D. melanogaster that encodes a cytoplasmic ferritin subunit
[25]. The number of functional fer genes in Drosophila has yet
to be determined.
In the yellow fever mosquito Aedes aegypti (Rockfeller
strain), a cDNA encoding a heavy-chain homologue has been
isolated and sequenced [6]. We used that cDNA clone to isolate
the corresponding gene and determined its sequence. We then
used the genomic clone to map the chromosomal location of the
gene. The data from the chromosomal map and quantitative
Southern analyses suggest that there is a single copy of this
gene. The gene structure differs from known vertebrate as well
as Drosophila fer gene structures. In addition, unlike its
mammalian counterparts, the A. aegypti ferritin gene has
multiple transcriptional start sites, and, interestingly, cellular
iron concentration dictates which of these sites is used.
E X P E R I M E N TA L P R O C E D U R E S
Isolation of A. aegypti fer genomic clone and DNA sequence
determination
The ferritin genomic clone Aggeno-7 was isolated from a
lEMBLE3 library [26] using the Non-Radioactive Genius
System (Roche Molecular Biochemicals, Indianapolis, IN,
USA) following the manufacturer's recommendations. Positive
plaques were identified using dideoxygenin-labeled clone 3
generated from previous work [6]. Approximately 300 000
plaques (representing < 9 genome equivalents assuming a
haploid genome of 0.83 pg [27]) were screened. Seven positive
clones were identified and rescreened to homogeneity. Since all
seven clones gave the same restriction enzyme patterns with
3886 D. Q.-D. Pham et al. (Eur. J. Biochem. 267)
EcoRI and BamHI, suggesting that they are identical clones,
subsequent experiments were performed using only one clone.
Phage DNA was isolated using l Quick kit (Bio 101, La Jolla,
CA) according to the manufacturer's instructions. Southern blot
experiments (as described below) using the dideoxygenin-
labeled clone 3 and BamHI-digested phage DNA indicated two
fragments of interest, 5.5 and 1.6 kb. These fragments were
subcloned into a pBluescript KS1 vector (Stratagene, La Jolla,
CA, USA), previously digested with BamHI and dephos-
phorylated with calf intestine phosphatase. The resultant
clones were designated p5AFG and p2AFG, respectively.
Deletion clones were generated from the p5AFG and
p2AFG clones using the Exo III/Mung Bean Nuclease kit
(Stratagene) following the manufacturer's recommendations.
DNA sequencing was accomplished using the dideoxy chain
termination method [28]. Both DNA strands were indepen-
dently sequenced.
Once the nucleic acid sequences of both the p5AFG and
p2AFG clones were determined, two oligonucleotide primers,
located at positions 3791±3811 (sense) and 8036±8058
(antisense), were designed and used for PCR. From this
reaction, a 4.3-kb product was generated and cloned into a
PCR2.1 vector using the TA cloning kit (Invitrogen, San Diego,
CA, USA). The resultant vector was designated p4AFG. The
nucleic acid sequence of this 4.3-kb DNA fragment was
determined, and shown to contain an additional 2.0-kb fragment
of intron 2. A digoxigenin (DIG) probe of the 4-kb DNA
fragment was generated and used to rescreen BamHI-digested
phage DNA for the remainder of intron 2. This probe
hybridized with three fragments (0.5, 0.7, and 0.8 kb) not
seen in the previous screen. These DNA fragments were
subcloned individually into a pBluescript KS 1 vector, and
their nucleic acid sequences were determined. The probe
production and Southern blot analysis were performed as
described below. Subcloning and nucleic acid sequence
analyses were performed as described above.
Primer extension
The oligomer used in the primer extension reaction contains a
sequence antisense to positions 527±561: 5 0 -TCGATGG-
CTGCGATGGAATAATAGGGAAGTTAAAC-3 0 . The standard
ladder was generated using this 35mer and Sequenase II kit
(USB, Cleveland, OH, USA) according to the manufacturer's
recommendations. The 35mer was end-labeled by standard
procedures [29], and the primer extension reaction was
performed using Superscript II Reverse Transcriptase (Life
Technology, Long Island, New York, USA) as recommended by
the manufacturer, except the extension was performed at 50 8C.
Cells were grown and iron-treated (100 mm ferrous ammonium
sulfate) as described previously [30]. For iron-treated cells,
50 mg of total RNA was used. For nontreated cells, 50 and
100 mg of total RNA were used. The higher amount of total
RNA (100 mg) was used for nontreated cells to compensate for
the fact that the ferritin message exists at a < twofold to
threefold lower level in nontreated cells than in iron-treated
cells.
Southern blot analysis
Southern blot analyses were performed using standard
procedures [29] for 16 h at 65 8C. In Southern blot analyses
using DIG-labeled probe, 10 mg of phage DNA and
20 ng´mL21 of probe were used. In Southern blot analyses
using 32P-labeled probe, 20 mg of BamHI and SmaI digested,
q FEBS 2000
genomic DNA and 106 c.p.m.´mL21 of p2AFG probe were
used. Radioactive disintegrations and autoradiographs were
collected using the Phosphoimager System (Molecular
Dynamics, Milpitas, CA, USA). Genomic DNA from A.
aegypti (Rockfeller strain) was prepared using a Quiagen
Genomic column (Qiagen, Valencia, CA, USA) according to
the manufacturer's recommendations.
Probe production
Probes for clone 3 and p4AFG were generated as follows.
Ten nanograms of the desired DNA template were used in
each PCR reaction. Both PCRs were performed with M13
reverse and forward primers using standard conditions [29]
for 30 cycles (denaturation: 95 8C, 1 min; annealing: for
clone 3, 55 8C, 1 min, and for clone p4AFG, 45 8C, 1 min;
extension: 72 8C, 2 min). At the end of 30 cycles, the reaction
was left at 72 8C for 10 min so that all unfinished extensions
could be completed. The DIG-labeled UTP was purchased from
Roche Molecular Biochemicals and used at a final concentra-
tion of 35 mm. The p2AFG probe was generated using the
Radprime Labeling kit (Life Tech., Gaithersburg, MD, USA).
FISH mapping
Fluorescence in situ hybridization (FISH), microscopy, and
digital imaging methods were used to map probes p2AFG and
p5AFG to A. aegypti metaphase chromosome preparations as
described [31,32]. Probes p2AFG and p5AFG were labeled
with biotin, and the A. aegypti FISH landmark plasmid
p2392 was labeled with DIG following standard procedures
[31]. A Cot1 suppression±hybridization was carried out
where repetitive elements in p2AFG and p5AFG were
suppressed by an excess of an A. aegypti DNA fraction
prepared where Cot ˆ 1. The biotinylated probe was
detected with FITC-conjugated avidin and DIG was detected
using rhodamine-conjugated anti-DIG. The slides were
counterstained with 4 0 ,6 0 -diamidin-2-phenylindole (DAPI)
(0.2 mg´mL21) and stored at 4 8C until examined optically.
Digital images were captured of the DAPI, FITC, and
rhodamine stained images and they were processed as described
[31,32]. The chromosome-specific landmark probe p2392 FISH
signals allowed chromosome identification and orientation,
permitting measurements from the p-terminus of the metaphase
chromosome to be made. The distance of the p2AFG and
p5AFG signals was recorded as a percent fractional length from
the p-terminus (%FLpter).
R E S U LT S A N D D I S C U S S I O N
Isolation, nucleotide sequence analysis, and primer
extension analysis
The genomic clone Aggeno-7 encoding the A. aegypti
24/26-kDa ferritin subunit was isolated from an A. aegypti
chromosomal DNA library. The clone carried a 9-kb DNA
fragment containing five exons that encode the entire mosquito
ferritin 24/26 subunit (Fig. 1). The present nucleic acid
sequence showed differences from that reported previously
(Fig. 1A, nucleotides in bold). Comparative analyses (gap,
Genetics Computer Group, Madison, WI, USA) revealed a 2%
difference in the open reading frame and a 5% difference in the
noncoding region. The nucleic acid sequence reported pre-
viously was derived from the Bahamas strain, whereas here the
Rockefeller strain was used. The nucleic acid sequence
q FEBS 2000 Aedes aegypti ferritin gene: structure and location (Eur. J. Biochem. 267) 3887

indicated that all introns of this gene conform to the GT-AG
rule (Fig. 1A). Primer extension analysis showed the ferritin
gene has several transcriptional start sites, at positions 469 and
477 (Figs 1A and 2), and the primary site for transcriptional
initiation is at position 477. When the mosquito cells were
treated with 100 mm ferrous ammonium sulfate, two transcrip-
tional start-sites (position 469, nucleotide C; and position 477,
nucleotide A) were used (Fig. 1A; Fig. 2, lane 3). When the
cells were not treated with iron, only position 477 was used
(Fig. 1A; Fig. 2, lanes 1 and 2). These results further confirm
our previous observation that transcriptional regulation plays a
role in the modulation of the mosquito ferritin gene [30].
In both D. melanogaster and A. aegypti, iron availability
induces the expression of fer message. In D. melanogaster, iron
availability dramatically alters the distribution of fer message,
and this re-distribution of fer mRNA has been attributed to an
alternative splicing event. A splicing event, however, could not
explain the increased level in fer message [33]. In A. aegypti,
the increase in fer message level in the presence of excess iron
has been attributed to increased expression [30]. Current data
suggested that mosquito cells rely on the multiple transcrip-
tional start sites to provide a more efficient transcriptional
machinery, which in turn supplies more fer message, and thus
more Fer protein for iron storage.

Fig. 1. The nucleic acid sequence and deduced

amino acid sequence of the A. aegypti ferritin
genomic clone encoding the 24/26-kDa subunit
(A) and schematic representation of the
A. aegypti fer. CAP (B). (A) Carat,
transcriptional start sites. Nucleotides in the
introns are in lower case. Nucleotides in the
exons are capitalized. Amino acids are
capitalized and italicized. Nucleotides in the
iron-responsive element are capitalized and
underlined. Nucleotides in bold are nucleotides
that are different from those previously reported
[6]. The nucleotide sequence described here has
been deposited with the GenBank/EMBL
sequence data banks and is available under
accession number AF126431. (B) CAP,
CAP-site; ATG, initiation codon; TAA, stop
codon; AATAA poly adenylation site. The
boxes represents the exons, and the lines
represent the introns. The exons and introns are
not drawn to scale. The numbers of amino
acids in the exons are listed above the boxes.
The numbers of nucleotides in the exons are
listed under the black boxes, and the numbers
of nucleotides in the introns are listed between
the boxes.
3888 D. Q.-D. Pham et al. (Eur. J. Biochem. 267)
Fig. 2. Primer extension. G, A, T and C refer to standard sequencing
reactions using the 35mer oligonucleotide with termination mix ddG, ddA,
ddT or ddC, respectively; 1 I, primer extension for iron-treated cells using
50 mg of RNA; -I, primer extension for non iron-treated cells using 50 mg
or 100 mg of RNA. Briefly, the oligomer used in the primer extension
reaction contained a sequence antisense to positions 527±561. For iron-
treated cells (100 mm ferrous ammonium sulfate), 50 mg of total RNA was
used. For nontreated cells, 50 and 100 mg of total RNA were used. The
higher amount of total RNA (100 mg) was used for nontreated cells to
compensate for the fact that the ferritin message exists at <twofold to
threefold lower in nontreated cells than in iron-treated cells.
Structural analysis of genomic clone
The organization of the A. aegypti fer gene is notably different
from other known fer genes. The first difference is an additional
intron in the 5 0 upstream region in the mosquito gene, between
the CAP site and the translational start site (Fig. 1B). This
intron seems also to occur in D. melanogaster, but not in the
vertebrates [24].
The second difference is a much larger intron corresponding
to the first vertebrate intron. This larger second intron in the
mosquito gene contains numerous sequences similar to
q FEBS 2000
transposable elements, a feature not seen in other fer genes.
The putative transposable elements are located in positions:
(1) 1612±1736 (MosquI-family) [34]; (2) 1759±1951
(Wujin-family) [35]; (3) 2372±2519 (Zebeedee-family)
[36]; (4) 2752±3167 (Dufu-aa2) [35]; (5) 3179±3659
(Pony-aa2) (Z. Tu, personal communication); (6) 3826±
3920 (incomplete Wujin-Aa6) [35]; (7) 4151±4284 (heT-A)
[37]; (8) 4352±4258 (MosquI-Aa1) [34]; (9) 4753±4467
(mgd1) [38]; (10) 4500±4762 (Z. Tu, personal communi-
cation); (11) 5510±5390 (MosquI-Aa1) [34]; and (12)
6278±6609 (Z. Tu, personal communication) (Fig. 1). The
insertion of repetitive elements seems to be a common feature
of the A. aegypti genome [35] (D. L. Knudson, unpublished
data). Although there are numerous nucleotide differences
between this fer gene and the cDNA isolated earlier [6], the
deduced amino acid sequence differs by only a single amino
acid, suggesting that the differences may simply be the
consequence of differences in the alleles or strains of
A. aegypti used.
Computer analyses (gene finder, Bayor College of
Medicine, Houston, TX, USA) suggested that there are other
putative codon sequences that can be derived from the current
nucleic acid sequence. However, four cDNA clones that we
have isolated showed only one coding sequence, that presented
in Fig. 1A.
Southern blot analysis and chromosomal localization
Data from our quantitative Southern blot analysis indicated that
A. aegypti has only one copy of fer (data not shown). Initially,
Southern blot analyses were performed using both genomic
clones p5AFG (position 1 to position 5421) and p2AFG
(position 7430 to position 9043); the analyses resulted in
smears throughout the gel lanes. Since clone p5AFG contains
repetitive elements and these elements are abundant in the
A. aegypti genome [35], the smearing in these analyses was due
probably to the cross-hybridization between the repetitive
elements within probe p5AFG and their sister elements
Fig. 3. Chromosomal mapping of the fer gene
by FISH. FISH of fer gene related probes p2AFG
and p5AFG and the A. aegypti FISH landmark
plasmid p2392 is represented. (A,C,D) The
depicted digital images result from image capture
using specific band-pass filter sets and these are
represented as grayscale images. (B) A composite
color image, made from the grayscale images,
which are pseudo-colored and merged or overlaid
in software, is represented. A DAPI stained,
metaphase chromosome spread is represented in
(A). Probes p2AFG and p5AFG produced
detectable, paired FISH signals on one arm of the
smallest chromosome (C). In (B), FISH signals
resulting from the specific hybridization of
probes p2AFG and p5AFG are depicted (green
color) and overlaid on the FISH image of the
FISH landmark probe p2392 (red color). The
specific FISH landmark signals resulting from
probe p2392 may be seen in (D) and also as red
in (B). Use of the landmark probe identifies and
orients the paired chromosomes [left to right is 3,
1, and 2 as indicated in (B)] and the p2AFG and
p5AFG signals were located on the q-arm of
chromosome 1 at 88.3% FLpter.
q FEBS 2000 Aedes aegypti ferritin gene: structure and location (Eur. J. Biochem. 267) 3889
elsewhere in the A. aegypti genome. When Southern blot
analyses were performed with a fragment of fer [position 7832
(SmaI) to position 9038 (BamHI site)] that has no recognizable
repetitive element, a weak but clear signal was observed.
Genomic clones p0.8AFG (position 5421 to position 6207),
p0.5AFG (position 6207 to position 6730) and p0.7AFG
(position 6730 to position 7430) were not used in Southern
blot analysis because these clones are either too AT-rich or also
contain repetitive elements.
In spite of the fact that p5AFG contained repetitive elements,
p2AFG and p5AFG were used together as FISH probes to
metaphase chromosomes. These two probes were used in
concert to increase the FISH DNA target size because small
probes that are represented as single copy sequences in the
genome are often difficult to detect using standard FISH
procedures. The Cot1 suppression±hybridization was able to
suppress any potentially confounding signal due to the
repetitive elements in p5AFG because paired signals were
seen on the q-arm of chromosome 1 (Fig. 3). Even though the
FISH signal background was higher than that normally seen in
FITC-detected images, the paired signals were consistently
seen in metaphase spreads (Fig. 3C). Use of the landmark probe
p2392, which was detected with rhodamine, allowed us to
visually identify that the specific signals were located clearly
on the q-arm of chromosome 1 (Fig. 3D). The location of the
specific p2AFG and p5AFG paired signals was confirmed
through measurements of final images (Fig. 3B), which were
derived from the grayscale images (Fig. 3A,C,D) that were
pseudo-colored and merged in software. The paired signals
were located at 88.3 ^ 2.5% FLpter (n ˆ 15) on chromosome
1. When p2AFG was used as a single FISH probe, paired
signals were seen on chromosome 1 on the q-arm, but they were
not seen consistently in all metaphase chromosome spreads.
This latter finding is also consistent with the notion that the
sequence is represented in the haploid genome as a low copy
and possibly a single copy sequence.
FISH data indicate that the mosquito fer gene is localized on
the q-arm of chromosome 1 (Fig. 3). The homologous gene in
D. melanogaster is located on the right arm of the third
chromosome [7]. The single locations indicate that, unlike
vertebrate genomes, the dipteran genomes are not littered
with fer pseudogenes. Previous work has suggested that
D. melanogaster has virtually no pseudogenes because of
rampant deletions of DNA in unconstrained regions [39,40].
While FISH data are not conclusive as to whether there is only
a single fer gene in the Rockefeller A. aegypti haploid genome,
they clearly indicate that there are at most a few. However,
when considering the quantitative Southern analysis and the
FISH data together, the presence of a single functional gene is
further substantiated. In this respect, A. aegypti fer is similar to
its vertebrate counterparts, in that only one functional gene
exists [18,21,23,41].
ACKNOWLEDGEMENTS
The authors thank Z. Tu for the analysis of the putative transposable
elements, E. M. Siegel for her assistance with the identification of clone
Aggeno7, J. C. Villegas for his assistance with the primer extension, J. L.
Stephens for her assistance in the FISH mapping, and J. H. Law and G. C.
Mayer for their comments on the manuscript. This work was supported by
funds from the Public Health Services awarded to DQDP (DK 09121 and
GM 55866). The FISH work was supported by the National Institutes of
Health, National Institute of Allergy and Infectious Disease grant AI 34337
awarded to D. L. K.
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