3. Results
3.1. Sequence of AIF-1 cDNA and AIF-1 gene open reading frames A total of 150 randomly chosen clones were sequenced with forward primer. Aside
from the numerous unknown sequences that could not be identified in the homology search, the most frequent gene encountered was 12 clones of cDNA with homology to
mammalian AIF-1s.
Fig. 2. Nucleotide sequence of red sea bream AIF-1 cDNA. Deduced amino acid sequence is aligned. Three asterisks represent a termination codon. The sense strand is displayed from the 5
X
to 3
X
direction.
The AIF-1 homologous cDNA was 444 bp in length with an open reading frame that Ž
. encodes 147 amino acids Fig. 2 . All of the cDNA clones encoded exactly the same
sequence. The amplified PCR product from genomic DNA was single banded. The sequence of ORFs in the genomic DNA was exactly the same as the cDNA encoding
sequence. The red sea bream AIF-1 gene included six exons and five introns. The ORFs were 19, 62, 67, 42, 166, and 88 bp in length from ORF1 to ORF6, respectively. All the
Ž .
intervening sequences IVS follow the GT-AG rule, and there were 93, 150, 90, 160, Ž
. and 408 bp, from IVS1 to IVS6, respectively Fig. 3 .
Ž .
The predicted amino acid sequence of red sea bream AIF-1 DDBJrAB019540 was Ž
. Ž
homologous to that of the AIF-1 in human GenBankrHSU49392 , pig
Swiss .
Ž .
Ž .
protrAIF1 PIG , mouse GenBankrAB013745 , rat GenBankrRNU17919 , and carp –
Ž . Fig. 3. Nucleotide sequence and structure of protein coding region in red sea bream AIF-1 gene. A The six
open reading frames are underlined, and the deduced amino acid sequence is aligned. The three asterisks
X X
Ž . represent a termination codon. The sense strand is displayed from the 5 to 3 direction. B Shaded boxes
represent open reading frames, and open boxes show introns.
Table 1 Similarities of the AIF-1 chains in red sea bream, carp, and mammals
RS Carp
Mouse Rat
Pig Carp
59.9 Mouse
65.5 59.5
Rat 66.9
62.2 93.9
Pig 65.5
62.2 85.7
87.1 Human
65.5 63.5
89.1 89.8
91.2 Ž
. Ž .
Red sea bream RS identical matches .
Ž .
GenBankrAB012309 . The amino acid sequence from red sea bream AIF-1 were found to be 65.5 homologous to those from human AIF-1, 65.5 homologous to those from
pig AIF-1, 66.9 homologous to those from rat AIF-1, 65.5 homologous to those Ž
. from mouse AIF-1 and 59.9 homologous to those from carp Table 1 .
3.2. Southern blot analysis of red sea bream genomic DNA Southern blot analysis of genomic DNA was performed using an AIF-1 gene
Ž .
fragment 1343 bp of PCR product; see Fig. 3 as a hybridization probe. As shown in
Fig. 4. Comparison of red sea bream AIF-1 chain with those of carp and mammals. Dashes indicate the amino acid gaps that are necessary to align these sequences. The conserved residues in all sequences are indicated by
Ž .
asterisk .
Ž .
Fig. 4, a 4.4-kb long positive band was observed in the EcoRI lane Fig. 5 , and an additional faint band was observed just above the 4.4-kb band. In the lanes of BamHI
and Pst I digestion, a 3.1- and 1.8-kb long single positive band was observed, respec- Ž
. tively Fig. 5 . However, there were no faint bands in the lanes of BamHI and Pst I
Ž .
digestion Fig. 5 . 3.3. Time course of AIF-1 expression in LPS-stimulated leukocytes
From the calibration assay, both the density of PCR products of b-actin or AIF-1 Ž
. were found to be proportional to the amount of template cDNA Fig. 1 . The assays
Ž .
were normalized with b-actin. The ratio of density values AIF-1rb-actin were static at 0, 5, 15, 30 min, and 1 h of LPS stimulation, but gradually increased from 3 to 24 h. The
Fig. 5. Southern blot analysis of the red sea bream AIF-1 gene. Red sea bream genomic DNA was digested Ž
. Ž
. Ž
. with EcoRI lanes E , BamHI lanes B , and Pst I lanes B . The digests were separated on 0.6 agarose gel
Ž .
Ž .
left panel , blotted onto a nylon membrane and hybridized with radiolabeled probes right panel . Marker Ž
. Ž
. DNA HindIII-digested l-DNA was electrophoresed in parallel lane M . The filters were hybridized with a
Ž .
radiolabeled fragment of AIF-1 gene that was amplified by PCR see Section 2 .
Fig. 6. Time course of AIF-1 expression level in LPS-stimulated red sea bream leukocytes. Red sea bream Ž
6
. Ž
leukocytes 5=10
cells were placed into a 12-well plate, and subjected to LPS stimulation
final .
concentration: 0.1 mgrml . The RNAs were reverse transcribed with oligo dT, and amplified with primers for b-actin and AIF-1, respectively. The PCR conditions were calibrated to be consistent with template quantity.
Ž .
Ž .
Ž .
The bars represent density values DPI of PCR products by AIF-1 white and b-actin gray primers. The line indicates estimated expression level of AIF-1 based on the expression level of b-actin.
Ž maximum ratio of expression values in AIF-1rb-actin was 24 h of LPS stimulation Fig.
. 6 .
4. Discussion
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