establishment of new methods for the prevention of fish diseases. Leukocytes that can be activated by various mediators are an important link in these self-defense mechanisms.
Since we were interested in the effectors of leukocyte activation, we stimulated the Ž
. Ž
. leukocytes in red sea bream Chrysophrys major with lipopolysaccharide LPS . Total
RNA was subjected to cDNA cloning, and a homology search on database revealed that the mRNAs from the stimulated leukocyte include several copies of a sequence that is
Ž .
homologous to the allograft inflammatory factor-1 AIF-1 . AIF-1 has been recognized as a Ca
2q
-binding peptide expressed predominantly by activated monocytes. It was originally identified in rat cardiac allografts associated with
Ž .
chronic cardiac rejection Autieri et al., 1995 . Rat AIF-1 has been described as a Ž
molecule that is expressed in macrophages, and is responsible to an IFN-g Utans et al., .
1995 . In addition, rat AIF-1 has been found in monocytes located in inflammatory Ž
. lesions resulting from autoimmune diseases Schluesener et al., 1999 . The AIF-1s
Ž encode the EF-hand motief that plays an essential role in calcium binding Negrutskii
. Ž
and El’skaya, 1998 . The deduced amino acid sequences of AIF-1s in human Autieri, .
Ž .
Ž .
Ž 1996 , pig Chen et al., 1997 , mouse Watano and Kimura, 1998 and rat Utans et al.,
.
X
1995 are highly conserved in not only the EF-hand region but also in both the N - and C
X
-ends. In this study, we investigated the full-length sequence of AIF-1 homologous cDNA in
red sea bream leukocytes. We performed Southern blot hybridization of red sea bream genomic DNA to estimate the copy number of AIF-1 genes per haploid. From red sea
Ž bream genomic DNA, we isolated a coding region of an AIF-1 gene from initiation to
. termination codons and then we tried to identify and characterize its exon–intron
structure. Also, the in vitro time course of AIF-1 gene expression was analyzed in LPS-stimulated red sea bream leukocytes.
2. Materials and methods
2.1. Isolation of leukocytes, LPS treatment, and RNA isolation Ž
. Ž .
An adult male red sea bream C. major 40 cm long was exsanguinated using a
Ž .
heperinized syringe NOVO, Denmark . Approximately 40 ml of whole blood was centrifuged at 1000 = g for 5 min at room temperature. The resulting plasma was then
transferred into a new tube, incubated at 558C for 30 min, and sterilized with a 0.22-mm filter. The resulting blood cells were suspended in PBS
Žy.
and poured onto Ficoll 400 Ž
. Sigma, USA . After centrifugation at 1000 = g for 5 min at room temperature, the
leukocytes remaining on the surface of the Ficoll 400 were harvested and suspended in Ž
. RPMI 1640 media without serum
Nissui, Japan . The cells were centrifuged at 1000 = g for 5 min at room temperature, and then resuspended in RPMI 1640 media.
After two washings, the cells were suspended in 20 ml of fresh RPMI 1640 media Ž
. containing 10 FBS Gibco, USA , 5 filtered red sea bream serum, and antibiotics
Ž .
Ž
7
. 200 mg streptomycinrml and 50 mg kanamycinrml . The leukocytes 5 = 10
were
2
Ž .
transferred into a 25-cm flask Corning, USA , incubated at 208C for 24 h, and then Ž
. stimulated with 1 mgrml LPS Escherichia coli, 026:B6rGibco—final concentration
Ž .
for 20 h, when morphological change became rough was observed on cell surfaces. Ž
. Total RNA was harvested with ISOGEN Nippon Gene, Japan following a standard
Ž .
technique developed by Chomczynski and Sacchi 1987 . 2.2. cDNA library
A cDNA library of LPS-stimulated leukocytes was established with TimeSaver
w
Ž .
cDNA synthesis kit Pharmacia, Sweden . Total RNA was reverse-transcribed with Ž
. oligo-dT 12–16 primer. The synthesized cDNA was introduced into l-ZapII cloning
Ž .
vector Stratagene, USA . The vector-insert was packaged with Gigapack III plus
Ž .
w
Ž .
Stratagene , and transformed into bacteria using the ExAssist system Stratagene .
2.3. Sequencing and characterization Plasmids of randomly chosen cDNA clones were isolated using an alkaline lysis
Ž .
method Sambrook et al., 1989 . The sequencing reaction for cDNA clones was
performed using a dye terminator cycle sequencing kit for a DNA sequencer ABI Ž
. Prisme 377 Perkin Elmer, USA . The sequence data was subjected to a homology
Ž .
search using the GeneBank database. http:rrwww.blast.genome.ad.jpr . 2.4. Genomic DNA isolation
Ž .
Ž Liver 100 mg was homogenized with 2 ml of 1 = proteinase K buffer 500 mM
Ž .
. Tris–HCl, 100 mM EDTA, 20
wrv SDS, 100 mgrml Proteinase K . After
Ž .
incubation at 558C for 1 h, an equal volume of Tris–saturated phenol pH 8.0 was Ž
. added and mixed by slow inversion 1 cycle every 2 s over a period of 30 min. After
centrifugation at 20,000 = g for 5 min at 208C, the supernatant was transferred into a new tube containing 200 ml of 3 M sodium acetate and 5 ml of 99 ethanol. Genomic
DNA was hooked up with a AJB-shaped glass rod. The glass rod was placed into a new
Ž .
tube containing 100 ml of TE buffer 10 mM Tris–HCl, 1 mM EDTA, pH 8.0 to dissolve the adhered genomic DNA.
2.5. Amplification of AIF-1 gene coding region Two primers, AIF-1 initiate: 5
X
-GGGATCCATGGACAGCACAGCTCAAGGA-3
X X
X
Ž and AIF-1 terminate: 5 -GGGATCCTCAGGGCAGGTCTGAAAAGGT-3
underlines .
indicate BamHI site , which include the initiation and termination codons, respectively, were synthesized. PCR was carried out using 100 ng of template DNA, 0.2 mM of each
dNTP, 16 pmol of each primer, 5 ml of 10 = reaction buffer, and 1.25 units of LA-Taq Ž
. DNA polymerase Takara, Japan , in a total volume of 50 ml. Twenty-eight thermal
cycles were performed, each consisting of 30 s at 948C to denature, 30 s at 508C to
anneal, and 10 min at 728C for extension. The samples were post-heated at 728C for 10 min. The obtained DNA fragments were completely digested with BamHI, and con-
nected with plasmid vector pUC118. Recombinant plasmid was transferred into E. coli Ž
. XL1Blue cell Stratagene . The resulting transformants were cultured separately, and the
amplified recombinant plasmids were recovered using an alkaline lysis method Ž
. Sambrook et al., 1989 . Reaction for genomic clones were performed with a Thermo
Ž sequenase, fluorescent-labeled, primer cycle sequencing kit Amersham life science,
. Ž
. USA for a DNA sequencer DNA 4000L Li-Cor, USA .
2.6. Southern blot analysis of red sea bream genomic DNA Ž
. Genomic DNA 20 mg isolated from red sea bream was digested with EcoRI,
BamHI and Pst I, passed through 0.8 agarose gel, transferred to a nitrocellulose Ž
. membrane, and subjected to capillary blotting using immobilon NC Pure Millipore . A
probe was prepared using PCR-amplified AIF-1 open reading frames from red sea Ž
bream genomic DNA by primers AAIF-1 initiateB and AAIF-1 terminateB; see Section .
w
32
x Ž
. 2.5 and labeled with
a- P -dATP, using a random primer labeling kit Takara . Pre-hybridization, hybridization with
32
P-labeled probe, and washing were carried out Ž
. using the method described by Sambrook et al. 1989 . The membrane was then exposed
Ž .
to X-ray film Kodak at y808C using an intensifying screen. 2.7. Time course of AIF-1 expression in LPS-stimulated leukocytes
Ž
6
. Leukocytes 5 = 10 cells in 2 ml of RPMI 1640 media were placed into chambers
of a 12-well plate. Media with LPS was added to chambers to a final concentration of 0.1 mgrml LPS. The cells were kept at 258C for 24 h in the absence of LPS stimulation
and for 24, 12, 6, 3, 1 h, 30, 15, and 5 min in the presence of LPS stimulation. The cells Ž
were harvested at the same time. The total RNA was isolated with ISOGEN Nippon .
Gene . The isolated RNAs were reverse-transcribed, and PCRs were performed using
primers for b-actin and AIF-1. 2.5 mg of total RNA was mixed with 1 ml of 0.5 mgrml Ž
. Ž .
oligo dT 12–16 Pharmacia . The 10-ml mixture was heated at 708C for 10 min, and
then chilled in ice immediately afterward. Reverse transcription was performed in 20 ml Ž
. of total volume including 10 ml of total RNA with oligo dT 12–16 , 2 ml of 0.1 M
DTT, 1 ml of 10 mM dNTPs, 2 ml of DEPC-treated distilled water, 4 ml of 5 = first Ž
. strand buffer, and 200 units of reverse transcriptase, Superscript II Gibco . The mixture
was incubated at 378C for 30 min, and terminated with 30 ml of 10 mM EDTA. PCRs were performed in a 10-ml reaction volume. The reagents were premixed to a total
volume of 100 ml, and then divided into 10 tubes; nine tubes for reaction and one tube for excess. For each 10-ml reaction mixture, 0.2 ml of cDNA template, 0.1 ml of each
100 pmol primers, 0.2 ml of 25 mM dNTPs, 1 ml of 10 = buffer, and 0.05 ml of Taq
Ž .
polymerase BMH, Germany were included. The thermal condition was calibrated using a pilot experiment described in Section 2.8. Twenty-six thermal cycles were
performed, each consisting of 30 s at 948C to denature, 30 s at 508C to anneal, and 1 min
at 728C for extension. The samples were post-heated at 728C for 10 min. The primers for
X X
Ž .
X
the b-actin probe were 5 -TTGCTGATCCACATCTGCTG-3 B-ACrFWD and 5 -
X
Ž . Ž
. ACTACCTCATGAAGATCCTG-3 B-ACrRV
will produce 736 bp . The primers for the AIF-1 probe were derived from the determined cDNA: 5
X
CGCTCAGAAATG-
X
Ž .
X X
Ž GACAGCACAGC-3
AIF-FWD and 5 -CTGCTGGGATGGAGGGCGTCTT-3 AIF-
. Ž .
RV will produce 476 bp .
2.8. Calibration assay Reverse-transcribed cDNA from leukocytes without stimulation was used for calibra-
tion. The cDNA was serially diluted with sterilized water to concentrations of 120, 60, 30, and 15 ngrml. Diluted cDNA, at 2 ml each, were added to each appropriate PCR
Ž .
Ž .
mixture with b-actin primers Fig. 1 or with AIF-1 primers data not shown . A total of 22, 24, 26, and 28 thermal cycles were performed for each dilution step. The results of
the PCR amplifications were visualized by electrophoresis using a 2.0 agarose gel in Ž
. half-strength TBE 45 mM Tris–borate, 2 mM EDTA with serially diluted molecular
Ž weight markers 100, 50 and 25 ng of HindIII-digested l-DNA; density values of
. 560-bp bands were analyzed . The gel was stained with 1 mgrml of ethidium bromide
for 15 min. Electrophoregrams were photographed using a digital camera DC120 Ž
. Kodak , and the density value of each band was estimated and analyzed with software,
Ž .
NIH image and Excel see Fig. 1 .
Fig. 1. Calibration assay. Reverse-transcribed cDNA was serially diluted, and 22, 24, 26, and 28 thermal cycles were performed for each dilution step. The results of the PCR amplifications were visualized by
Ž .
electrophoresis with serially diluted molecular weight markers HindIII digested l-DNA . The density value in each band was estimated with an NIH image.
3. Results
