Genomic sequences encoding two types of medaka hemopexin-like protein Wap65, and their gene expression profiles in embryos
1 Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School
of Agricultural and Life Sciences, The University of Tokyo, Tokyo113-8657,
Japan
2 Department of Molecular Biology, Keio University School of Medicine, 35
Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
* Author for correspondence (e-mail: awatabe{at}mail.ecc.u-tokyo.ac.jp)
Accepted 7 March 1005
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: medaka, Oryzias latipes, mWap65-1, mWap65-2, hemopexin, genomic sequences, embryo, gene expression, transgenic fish
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Medaka is suitable for developmental and genetic studies as well as for
transgenic experiments, because of the transparency of the embryonic chorion,
high fecundity and short generation times, in addition to its small genome
size (Ishikawa, 2000;
Gong et al., 2001
). With such
advantages, in this study we screened bacterial artificial chromosome (BAC)
genomic clones from medaka containing the full-length sequences of
mWap65-1 and mWap65-2, determined their nucleotide
sequences, including 5'- and 3'-flanking regions, and compared
their genomic organizations with those of the human hemopexin gene. Then, we
examined the expression profiles of mWap65s in medaka embryos using
quantitative real-time PCR. Furthermore, we generated transgenic medaka
expressing green fluorescent protein (GFP) driven by mWap65-1 and
mWap65-2 promoters and observed their expression patterns during
ontogeny.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Approximately 80 embryos incubated at 25°C were collected at each of
the developmental stages: stage 9 [late morula stage, 7 h post-fertilization
(hpf)], 13 (early gastrula stage, 14 hpf), 15 (mid gastrula stage, 19 hpf), 16
(late gastrula stage, 24 hpf), 18 (late neurula stage, 29 hpf), 20 (4 somite
stage, 34 hpf), 22 (9 somite stage, 42 hpf), 24 (16 somite stage, 48 hpf), 28
(30 somite stage, 70 hpf), 36 (heart developmental stage, 156 hpf) and 39
(hatching stage, 240 hpf). Developmental stages were ascertained in a manner
described by Iwamatsu (1994).
Samples were frozen immediately in a liquid nitrogen bath and stored at
-80°C until use for first strand cDNA synthesis.
Embryos at stage 32 (somite completion stage, 120 hpf) were collected and used for whole-mount in situ hybridization. For DNA microinjection, fertilized eggs were collected within 30 min after spawning and kept at 4°C to arrest development until microinjection.
Screening and sequencing of BAC clone
A BAC library of sperm genomic DNA from medaka of HNI strain and its
high-density replica (HDR) filters has been constructed at Keio University
School of Medicine, Japan (Kondo et al.,
2002). The BAC library was screened by colony hybridization with
both mWap65s probes. mWap65-1 and mWap65-2 probes
were composed of 1096-1534 nucleotides (nt) and 838-1376 nt cDNAs
(Hirayama et al., 2004
),
respectively, and labeled with digoxigenin (DIG)-11-dUTP using DIG-High Prime
DNA Labeling and Detection Starter Kit II (Roche Diagnostics, Mannheim,
Germany), according to the manufacturer's instruction. 1 µg of purified BAC
DNAs from positive clones were digested with HindIII and separated on
0.7% agarose gels with a TBE buffer (0.089 mol l-1 Trisborate, 0.5
mol l-1 EDTA). The digested DNAs were transferred to Biodyne B
membranes (Pall BioSupport Division, Washington, NY, USA) and hybridized with
the mWap65-1 and mWap65-2 probes by the method adopted for
screening of the BAC library as described above.
Shotgun strategies were employed for sequencing selected clones, 182O24 including mWap65-1 and 107E17 including mWap65-2. About 1150 and 1550 shotgun clones from BAC clones 182O24 and 107E17, respectively, were sequenced using ABI PRISM BigDye Cycle Terminator Ready Reaction Mix diluted with 5x sequencing buffer (Applied Biosystems, Foster City, CA, USA). Excess dye-terminators were removed by gel filtration and then PCR products were automatically loaded onto an ABI PRISM 377 or 3700 DNA analyzer (Applied Biosystems). To assemble the individual shotgun sequences into contigs, computer programs, Phred, Phrap and Consed were used for base calling, assembly of sequences, and viewing and editing analysis, respectively.
Analysis on genomic sequences
The exon-intron organizations of mWap65-1 and mWap65-2
were determined with genomic and cDNA nucleotide sequences (the
DDBJ/EMBL/GenBank databases, accession numbers AB075198 for mWap65-1
cDNA and AB075199 mWap65-2 cDNA). The sequence of a 56 kb region
containing 5'- and 3'-flanking sequences of mWap65-1 with
the coding sequence in the middle was compared with those of mWap65-2
and the human hemopexin gene using the PipMaker
(http://bio.cse.psu.edu/pipmaker/)
(Schwartz et al., 2000) and
RepeatMasker (Open-3.0 1996-2004;
http://www.repeatmaster.org/;
A. F. A. Smit, R. Hubley and P. Green, unpublished) (Smit and Green, 1999)
programs. The human hemopexin gene had been mapped to chromosome 11p15.5-p15.4
(Law et al., 1988
) and its
sequence was obtained from NCBI's LocusLink
(http://www.ncbi.nlm.nih.gov/genome/guide/human/).
Putative cis-elements were searched by computer program TFSEARCH
version 1.3
(http://www.cbrc.jp/research/db/TFSEARCH.html)
that has been constructed for highly correlated sequence fragments in the
TFMATRIX transcription factor binding site profile database in the TRANSFAC
databases by GBF-Braunschweig (Heinemeyer
et al., 1998
).
Reverse transcription
Total RNA was extracted from embryos at various developmental stages using
an ISOGEN system (Nippon Gene, Tokyo, Japan). To remove endogenous DNA
contamination, the preparation containing total RNA was digested with DNase.
An aliquot of 5 µg of total RNA was dissolved in 8.5 µl water and added
with 5 U of DNase I (TaKaRa, Otsu, Japan) and 40 U of ribonuclease inhibitor
(TaKaRa), then the RNA solution was incubated at 37°C for 1 h. The enzyme
was inactivated at 98°C for 3 min, and the RNA solution was chilled on
ice. First strand cDNA was synthesized as follows. One microlitre of 10
µmol l-1 adapter primer
(5'-GGCCACGCGTCGACTAGTAC-3'), 2 µl of 10 mmol l-1
dNTP and 1 µl of water were added to the RNA solution treated with DNase.
The reaction mixture was heated at 65°C for 5 min, quickly chilled on ice,
added with 4 µl of 5x first-strand buffer containing 250 mmol
l-1 Tris-HCl (pH 8.3), 375 mmol l-1 KCl and 15 mmol
l-1 MgCl2, 0.1 mol l-1 dithiothreitol (DTT)
and 200 U SuperScriptTM III reverse transcriptase (Invitrogen, Carlsbad,
CA, USA), and incubated at 42°C for 50 min. The enzyme was
heat-inactivated at 70°C for 15 min and first strand cDNA synthesis was
completed by treatment with 1.0 µl of RNase H (Invitrogen).
Real-time polymerase chain reaction (PCR) analysis
Primer pairs m1-F-1250 and m1-R-1319 for mWap65-1, m2-F-111 and
m2-R-173 for mWap65-2, and m-bactin-F-7 and m-bactin-R-70 for medaka
ß-actin (Takagi et al.,
1994) were designed using the Primer Express Software (Applied
Biosystems) (Table 1). Each of
20 µl reaction mixtures contained 1x SYBR Green Master mix (Applied
Biosystems),
25 ng of first strand cDNA and 8 pmol of each primer.
Real-time PCR was performed with ABI PRISM 7300 (Applied Biosystems). Thermal
cycling conditions consisted of the initial steps for 2 min at 50°C then
10 min at 95°C followed by 45 cycles of denaturation at 95°C for 15 s,
annealing at 60°C for 1 min. Relative quantification was carried out by
normalization the values relative to those of ß-actin.
Amplification specificity was examined using the melting curve following the
manufacturer's instructions. Analysis and quantification using the comparative
Ct method were carried out with the ABI Prism 7300 Sequence Detection Software
(SDS) version 1.2 (Applied Biosystems).
|
Whole-mount in situ hybridization
Sense and antisense DIG-labeled RNA probes were synthesized from the DNA
fragments of nt 1-1610 of mWap65-1 cDNA and nt 96-1510 of
mWap65-2 cDNA with a DIG RNA labeling kit (Roche Diagnostics).
DIG-labeling of RNA probes was carried out according to the manufacturer's
instruction (Promega, Madison, WI, USA). Whole-mount in situ
hybridization with the DIG-labeled RNA probes was performed according to the
method of Westerfield (2000)
with some modifications, as follows: after fixation at 4°C for 4 h in 4%
paraformaldehyde in phosphate-buffered saline (PBS) containing 0.1% Tween 20
(PBSTw), the chorion was removed and embryos then refixed overnight at 4°C
with the same solution. Treatment with proteinase K (TaKaRa; 5 µg ml
l-1 in PBSTw) was carried out for 10 min at 37°C and the
DIG-labeled RNA probes were used for hybridization at an approximate
concentration of 1 µg ml l-1.
Construction of recombinant GFP plasmids
mWap65-1 and mWap65-2 upstream regions of 5 kb were
amplified from BAC clones 182O24 and 107E17, respectively, by PCR using
primers mWap65-1Fgfp5 and mWap65-1Rgfp5 for mWap65-1 and
mWap65-2Fgfp5 and mWap65-2Rgfp5 for mWap65-2
(Table 2,
Fig. 1). The amplified products
of mWap65-1 and mWap65-2 were double-digested with
BamHI/HindIII and NotI/HindIII,
respectively. The digested mWap65-1 fragment was inserted into
BamHI and HindIII sites of the phrGFP vector (Stratagene, La
Jolla, CA, USA), whereas that of mWap65-2 was inserted into
NotI and HindIII sites
(Fig. 1). Plasmids of
8.5
kb, containing mWap65-1 and mWap65-2 upstream regions, were
designated as mWap65-1-hrGFP and mWap65-2-hrGFP,
respectively.
|
|
Microinjection
Microinjection was carried out according to the method of Kinoshita et al.
(1996). A DNA solution of
20 ng µl-1 in distilled water was injected into the
cytoplasm of blastomeres at the single cell stage. Eggs injected with
recombinant GFP plasmids were incubated at 25°C and GFP fluorescence was
observed by fluorescence microscope.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Exon-intron organizations were predicted using the genomic nucleotide sequences of mWap65-1 and mWap65-2, and compared with that of the human hemopexin gene (Fig. 2). Both mWap65s consisted of 10 exons and 9 introns, as is the case for the human hemopexin gene. Although mWap65s were smaller than the human hemopexin gene (9.9 kb), their exon-intron organizations were almost identical.
|
The sequence of a 56 kb region containing 5'- and 3'-flanking sequences of mWap65-1 with the coding sequence in the middle was compared with those of the corresponding regions of mWap65-2 and the human hemopexin gene using the PipMaker program. As shown in Fig. 3, a high homology between mWap65-1 and mWap65-2 was found in the fifth, sixth and eighth exons, and in the seventh and ninth introns. In the 5'- and 3'-flanking regions, homology was found 15.3 and 11.6 kb upstream and 2.1 and 22.5 kb downstream of the coding region of mWap65-1. mWap65-1 showed an apparent homology with the human hemopexin gene only in the fifth and sixth exons (Fig. 3). mWap65-1 also showed homology with mWap65-2 and the human hemopexin gene in a very short sequence, considered to be a repetitive element.
|
Previously, Hirayama et al.
(2004) determined the
full-length of mWap65-1 and mWap65-2 cDNAs. In this study,
we found that the sequences at the 5' ends of the cDNA clones containing
mWap65-1 and mWap65-2 were all identical (data not shown),
and so the putative transcription start points of mWap65s were
confirmed. Thus, putative transcription start points of mWap65-1 and
mWap65-2 were located at 29 and 89 bases, respectively, upstream of
the translation start site (Fig.
4). Putative cis-elements were searched using prediction
programs. Although a 1 kb 5'-flanking region showed no obvious sequence
homology among mWap65-1, mWap65-2 and the human hemopexin gene (data
not shown), various transcription factor binding sites known in vertebrates
were found in mWap65s (Fig.
4). The binding sites for HNF-3ß, which is a liver-enriched
transcription factor, were found at -344 for mWap65-1 and -963 for
mWap65-2. A Cdx1 binding site, which is an important element for
developmental regulation (Subramanian et
al., 1995
), was abundant in the 5'-flanking regions of both
mWap65-1 (-790, -741, -672, -567, -471, -370, -307, -246) and
mWap65-2 (-940, -874, -812, -801, -793, -765, -707, -556, -539, -509,
-494, -446, -339, -166). In addition, the 5'-flanking region of
mWap65-1 contained putative binding sites for SRY (-994, -532, -463,
-343, -230, -155), USF (-854), Nkx-2.5 (-838, -687), MZF1 (-654, -201), Oct-1
(-516, -499), XFD-1 (-514), C/EBP
(-496), HFH-2 (-344), Evi-1 (-279),
GATA-1 (-257) and AML-1a (-87), whereas that of mWap65-2 consisted of
SRY (-946, -500, -463, -419, -180, -89, -48), Pbx-1 (-837), Prx-2 (-861,
-819), HFH-2 (-854), Nkx-2.5 (-815, -637, +58), XFD-1 (-778),
EF1
(-718), Brn-2 (-539), Oct-1 (-514), Sox-5 (-499), HNF-1 (-246) and c-Ets-1
(-72).
|
Real-time PCR analysis on expressional changes of mWap65-1 and mWap65-2 during development
Quantitative real-time RCR was performed to examine changes in the
expression levels of mWap65s in embryos at stages 9-39, using those
of ß-actin as the internal standard. As shown in
Fig. 5, the mWap65-1
transcripts were detected in embryos at the beginning of the experiment, at
stage 9, the late morula stage. The expression level at stage 13 (early
gastrula) was about 30-fold higher than that at stage 9, and the highest
expression level of mWap65-1 was observed at stage 16 (late
gastrula). Thereafter, the amount of transcripts was reduced at stages 18
(late neurula) to 24 (16 somite), but the expression level was increased again
at stage 28, when 30 somites are formed in the trunk muscle, and almost
constant levels of transcripts were maintained until hatching. In contrast,
mWap65-2 transcripts were not detected until stage 15 (mid gastrula)
(Fig. 5). The mWap65-2
transcripts were first observed at stage 16. Then the expression levels were
rapidly increased until hatching, although the levels were slightly decreased
at stage 24 (16 somite) compared with those at stage 22 (9 somite). Thus,
expression patterns of mWap65-1 and mWap65-2 were markedly
different from each other.
|
Spatial expression patterns of mWap65-1 and mWap65-2 in medaka embryos
To examine spatial expression patterns of mWap65s, whole-mount
in situ hybridization was performed on embryos at stage 32, at which
point somite formation is complete. mWap65-1 hybridization signals
were observed along the edge of pectoral fin bud and median fin fold of the
tail bud, but not in the liver (Fig.
6). However, mWap65-2 was observed only in liver, clearly
demonstrating different functions of mWap65-1 and mWap65-2
during ontogeny. No signal was detected with sense probes for mWap65s
(data not shown).
|
Expression of GFP in embryos injected with mWap65-1-hrGFP and mWap65-2-hrGFP constructs
mWap65-1-hrGFP and mWap65-2-hrGFP constructs were
injected into embryos at the single cell stage. One day after injection with
the mWap65-1-hrGFP construct, GFP fluorescence spots were observed
throughout the blastoderm including the embryonic shield, when embryos
attained stage 16 (late gastrula) (Fig.
7A). Following development from stage 16 to stage 29, GFP
fluorescence was observed not only in yolk sac but also various areas of the
embryonic body. Large and many small fluorescent spots were observed in the
yolk sac at stages 23 (12 somite; Fig.
7C) and 26 (22 somite; Fig.
7E) as well as in the embryonic body
(Fig. 7C, right part). However,
the small fluorescent spots tended to disappear at stage 26 and in turn a new
fluorescent spot was found in the area of the liver anlage, a primitive form
of liver in embryos (Fig. 7E).
The fluorescence of liver anlage was more clearly observed in the embryo at
stage 29 (Fig. 7G).
|
Embryos injected with the mWap65-2-hrGFP construct expressed GFP at stage 16 (Fig. 8A), as was the case with mWap65-1. At this stage also fluorescent spots of mWap65-2-hrGFP were again observed in various areas of the blastoderm, including the embryonic shield. Similarly to the expression patterns of mWap65-1-hrGFP, the mWap65-2-hrGFP fluorescent spots mostly disappeared at stage 26. However, mWap65-2-hrGFP was first observed in liver anlage in the embryos at stage 30 (35 somite) (Fig. 8E) in contrast to mWap65-1-hrGFP, which was activated in this tissue as early as at stage 26 (22 somite). These differences in expression patterns between mWap65-1-hrGFP and mWap65-2-hrGFP appeared to reflect those of the accumulated mRNA levels as shown in Fig. 5. Although mWap65-1-hrGFP was examined for its activation profiles until hatching, an intense GFP fluorescence was observed only in liver (Fig. 9).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alignment of nucleotide sequences using the PipMaker program revealed that
not only fWap65-1 (Hirayama et
al., 2003) but also mWap65-1 showed apparent homology
with the human hemopexin gene in the fifth and sixth exons (see
Fig. 3). These encode a
potential receptor binding site, by which rabbit hemopexin is bound to
hepatoma cells (Morgan et al.,
1988
,
1993
). It has been reported
for rabbit hemopexin that eight hydrophobic residues are important in the
structural stability around the heme binding site
(Paoli et al., 1999
). In the
case of Fugu, fWap65-1 contained five, and fWap65-2 seven, out of the
eight hydrophobic residues found in rabbit hemopexin and most of these
residues were located in the sixth exon. These residues were also found in
mWap65s (data not shown), suggesting their importance in structural
integrity.
No apparent sequence homology was found in the 5'-flanking region 1
kb upstream the coding sequence among mWap65-1, mWap65-2 and human
hemopexin gene. However, this region was found to contain various
transcription factor binding sites (see
Fig. 4). Interestingly, several
transcriptional elements, which are thought to regulate development, are
contained in both mWap65s. While Cdx1 is considered to be a regulator
of Hox gene expression (Subramanian et
al., 1995), putative binding sites for Cdx1 are rich in the
5'-flanking regions of both mWap65s. Found also in
mWap65s were the binding sites for Nkx-2.5, a vertebrate
homologue of tinman from Drosophila melanogaster involved in cardiac
mesoderm formation (Bodmer et al.,
1990
; Lints et al.,
1993
), and Prx-2, a homeobox transcription factor possibly playing
roles in development of the heart and forebrain
(Leussink et al., 1995
), as in
the case of fWap65s (Hirayama et
al., 2003
). The presence of these binding sites in the
5'-flanking regions of Wap65s indicates their involvement in
the transcriptional regulation during development.
Rat hemopexin transcripts were first detected in liver on day 24 of
gestation and rapidly increase during the postnatal period
(Nikkila et al., 1991).
Chicken hemopexin is found around hatching and increases in 4-day-old chicken
to more than 1000-fold over embryonic levels as revealed by electroimmunoassay
of serum (Grieninger et al.,
1986
).
Quantitative real-time PCR in this study revealed that mWap65-1
transcripts were detected even at stage 9, the beginning of the experiments
(see Fig. 5). Our previous
study showed that mWap65-1 transcripts were first detected at stage
24 by semi-quantitative RT-PCR (Hirayama et
al., 2004). Interestingly, quantitative real-time PCR analysis
showed that embryos expressed mWap65-1 transcripts even when
embryonic shield was not observed, and their maximum levels were observed in
embryos at stage 16, when the blastoderm covers three-quarters of the yolk
sphere and the embryonic shield becomes evident
(Iwamatsu, 1994
). The
embryonic shield is formed at stage 16, followed by the formation of somite,
notochord and organs until hatching. The high expression levels of
mWap65-1 in early embryonic stages indicate a possible role of this
gene in early embryogenesis.
mWap65-2 transcripts were first detected at stage 16 and thereafter gradually increased during ontogeny, as embryos undergo dynamic changes of their morphology. These results, together with the presence of putative transcriptional elements involving development in the 5'-flanking region of mWap65-2, suggests that mWap65-2 also plays important roles in embryonic development.
Medaka embryos injected with mWap65-1-hrGFP- and mWap65-2-hrGFP expressed GFP fluorescence at stage 16, 1 day after injection, suggesting that mWap65-1 and mWap65-2 promoters were both activated at early developmental stages. Such GFP expression in the early embryonic stages was consistent with the results obtained by real-time RT-PCR (Fig. 5).
mWap65-2 transcripts were expressed in liver as revealed by in
situ hybridization (see Fig.
6). Unexpectedly, those of mWap65-1 were expressed along
the edge of pectoral fin bud and median fin fold of the tail bud in embryos at
stage 32, suggesting a function of mWap65-2 in the development of
these two tissues. GFP fluorescence was restricted to the liver of hatched
medaka injected with mWap65-1-hrGFP (see
Fig. 9), shifting its
localization during hatching. While the msx homeobox genes are known
to be important for limb development in mouse (Muneoka and Sasoon, 1992),
their homologs are expressed in zebrafish embryos along the edge of the
pectoral fin bud and median fin fold of the tail bud during development
(Akimenko et al., 1995).
We observed transient expression profiles of GFP driven by two
mWap65s promoter in this study, but the localization of expressed GFP
varied among individuals (data not shown). It is known that the F0
generation of transgenic fish show the mosaic expression patterns of
transgenes (Ju et al., 1999)
and the uniform expression of transgenes is normally attained in the
F1 or F2 generation
(Chou et al., 2001
). We are now
generating stable transgenic lines to reveal more precisely the spatiotemporal
expression patterns of mWap65s.
In conclusion, we determined the genomic nucleotide sequences of two mWap65s and their flanking sequences. Furthermore, the expression profiles of mWap65s were determined using quantitative real-time PCR and in situ hybridization. We also generated transgenic medaka expressing GFP driven by two mWap65 promoters, and examined their expression patterns. The spatiotemporal patterns in embryos were different between mWap65-1 and mWap65-2, suggesting their distinct roles, at least during ontogeny.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akimenko M. A., Johnson, S. L., Westerfield, M. and Ekker,
M. (1995). Differential induction of four msx
homeobox genes during fin development and regeneration in zebrafish.
Development 121,347
-357.
Altruda, F., Poli, V., Restagno, G., Argos, P., Cortese, R. and Silengo, L. (1985). The primary structure of human hemopexin deduced from cDNA sequence: evidence for internal, repeating homology. Nucleic Acids Res. 13,3841 -3859.[Abstract]
Bodmer, R., Jan, L. Y. and Jan, Y. N. (1990). A new homeobox-containing gene, msh-2, is transiently expressed early during mesoderm formation in Drosophila. Development 110,661 -669.[Abstract]
Chou, C. Y., Horng, L. S. and Tsai, H. J. (2001). Uniform GFP-expression in transgenic medaka (Oryzias latipes) at the F0 generation. Transgenic Res. 10,303 -315.[CrossRef][Medline]
Gong, Z., Ju, B. and Wan, H. (2001). Green fluorescent protein (GFP) transgenic fish and their applications. Genetica 111,213 -225.[CrossRef][Medline]
Grieninger, G., Liang, T. J., Beuving, G., Goldfarb, V.,
Metcalfe, S. A. and Muller-Eberhard, U. (1986). Hemopexin is
a developmentally regulated, acute-phase plasma protein in the chicken.
J. Biol. Chem. 261,15719
-15724.
Heinemeyer, T., Wingender, E., Reuter, I., Hermjakob, H., Kel,
A. E., Kel, O. V., Ignatieva, E. V., Ananko, E. A., Podkolodnaya, O. A.,
Kolpakov, F. A. et al. (1998). Databases on transcriptional
regulation: TRANSFAC, TRRD and COMPEL. Nucleic Acids
Res. 26,362
-367.
Hirayama, M., Nakaniwa, M., Ikeda, D., Hirazawa, N., Otaka, T., Mitsuboshi, T., Shirasu, K. and Watabe, S. (2003). Primary structures and gene organizations of two types of Wap65 from the pufferfish Takifugu rubripes. Fish Physiol. Biochem. 29,211 -224.[CrossRef]
Hirayama, M., Kobiyama, A., Kinoshita, S. and Watabe, S.
(2004). The occurrence of two types of hemopexin-like protein in
medaka and differences in their affinity to heme. J. Exp.
Biol. 207,1387
-1398.
Ishikawa, Y. (2000). Medakafish as a model system for vertebrate developmental genetics. BioEssays 22,487 -495.[CrossRef][Medline]
Iwamatsu, T. (1994). Stages of normal development in the medaka Oryzias latipes. Zool. Sci. 11,825 -839.
Ju, B., Xu, Y., He, J., Liao, J., Yan, T., Hew, C. L., Lam, T. J. and Gong, Z. (1999). Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters. Dev. Genet. 25,158 -167.[CrossRef][Medline]
Kikuchi, K., Watabe, S., Suzuki, Y., Aida, K. and Nakajima, H. (1993). The 65-kDa cytosolic protein associated with warm temperature acclimation in goldfish, Carassius auratus. J. Comp. Physiol. B 163,349 -354.
Kikuchi, K., Yamashita, M., Watabe, S. and Aida, K.
(1995). The warm temperature acclimation-related 65-kDa protein,
Wap65, in goldfish and its gene expression. J. Biol.
Chem. 270,17087
-17092.
Kikuchi, K., Watabe, S. and Aida, K. (1997). The Wap65 gene expression of goldfish (Carassius auratus) in association with warm water temperature as well as bacterial lipopolysaccharide (LPS). Fish Physiol. Biochem. 17,423 -432.[CrossRef]
Kinoshita, M., Toyohara, H., Sakaguchi, M., Inoue, K., Yamashita, S., Satake, M., Wakamatsu, Y. and Ozato, K. (1996). A stable line of transgenic medaka (Oryzias latipes) carrying the CAT gene. Aquaculture 143,267 -276.[CrossRef]
Kinoshita, S., Itoi, S. and Watabe, S. (2001). cDNA cloning and characterization of the warm-temperature-acclimation-associated protein Wap65 from carp, Cyprinus carpio. Fish Physiol. Biochem. 24,125 -134.[CrossRef]
Kondo, M., Froschauer, A., Kitano, A., Nanda, I., Hornung, U., Volff, J. N., Asakawa, S., Mitani, H., Naruse, K., Tanaka, M. et al. (2002). Molecular cloning and characterization of DMRT genes from the medaka Oryzias latipes and the platyfish Xiphophorus maculatus. Gene 295,213 -222.[CrossRef][Medline]
Law, M. L., Cai, G. Y., Hartz, J. A., Jones, C. and Kao, F. T. (1988). The hemopexin gene maps to the same location as the ß-globin gene cluster on human chromosome 11. Genomics 3,48 -52.[CrossRef][Medline]
Leussink, B., Brouwer, A., El Khattabi, M., Poelmann, R. E., Gittenberger-de Groot, A. C. and Meijlink, F. (1995). Expression patterns of the paired-related homeobox genes MHox/Prx1 and S8/Prx2 suggest roles in development of the heart and the forebrain. Mech. Dev. 52, 51-64.[CrossRef][Medline]
Lints, T. J., Parsons, L. M., Hartley, L., Lyons, I. and Harvey,
R. P. (1993). Nkx-2.5: a novel murine homeobox gene
expressed in early heart progenitor cells and their myogenic descendants.
Development 119,419
-431.
Morgan, W. T., Muster, P., Tatum, F. M., McConnell, J., Conway,
T. P., Hensley, P. and Smith, A. (1988). Use of hemopexin
domains and monoclonal antibodies to hemopexin to probe the molecular
determinants of hemopexin-mediated heme transport. J. Biol.
Chem. 263,8220
-8225.
Morgan, W. T., Muster, P., Tatum, F. M., Kao, S. M., Alam, J.
and Smith, A. (1993). Identification of the histidine
residues of hemopexin that coordinate with heme-iron and of a receptor-binding
region. J. Biol. Chem.
268,6256
-6262.
Muneoka, K. and Sassoon, D. (1992). Molecular aspects of regeneration in developing vertebrate limbs. Dev. Biol. 152,37 -49.[CrossRef][Medline]
Nikkila, H., Gitlin, J. D. and Muller-Eberhard, U. (1991). Rat hemopexin. Molecular cloning, primary structural characterization, and analysis of gene expression. Biochemistry 30,823 -829.[CrossRef][Medline]
Paoli, M., Anderson, B. F., Baker, H. M., Morgan, W. T., Smith, A. and Baker, E. N. (1999). Crystal structure of hemopexin reveals a novel high-affinity heme site formed between two ß-propeller domains. Nature Struct. Biol. 6, 926-931.[CrossRef][Medline]
Schwartz, S., Zhang, Z., Frazer, K. A., Smit, A., Riemer, C.,
Bouck, J., Gibbs, R., Hardison, R. and Miller, W. (2000).
PipMaker - a web server for aligning two genomic DNA sequences.
Genome Res. 10,577
-586.
Subramanian, V., Meyer, B. I. and Gruss, P. (1995). Disruption of the murine homeobox gene Cdx1 affects axial skeletal identities by altering the mesodermal expression domains of Hox genes. Cell 83,641 -653.[CrossRef][Medline]
Takagi, S., Sasado, T., Tamiya, G., Ozato, K., Wakamatsu, Y., Takeshita, A. and Kimura, M. (1994). An efficient expression vector for transgenic medaka construction. Mol. Mar. Biol. Biotechnol. 3,192 -199.[Medline]
Takahashi, N., Takahashi, Y. and Putnam, F. W.
(1985). Complete amino acid sequence of human hemopexin, the
heme-binding protein of serum. Proc. Natl. Acad. Sci.
USA 82,73
-77.
Tolosano, E. and Altruda, F. (2002). Hemopexin: structure, function, and regulation. DNA Cell Biol. 21,297 -306.[CrossRef][Medline]
Watabe, S., Kikuchi, K. and Aida, K. (1993). Cold- and warm-temperature acclimation induces specific cytosolic proteins in goldfish and carp. Nippon Suisan Gakkaishi 59,151 -156.
Westerfield, M. (2000). The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio), 4th edn. Eugene, Oregon, USA: University of Oregon Press.