Ž .
Aquaculture 194 2001 63–74 www.elsevier.nlrlocateraqua-online
DNA cloning and characterization of an allograft inflammatory factor-1 homologue in red sea bream
ž Chrysophrys major
Masato Miyata , Kunio Iinuma, Teruo Miyazaki
Laboratory of Aquaculture Management, Faculty of Bioresources, Fish Pathology, Mie UniÕersity, Tsu, Mie 514-8507, Japan
Received 6 January 2000; received in revised form 11 August 2000; accepted 17 September 2000
Abstract
Ž .
We isolated allograft inflammatory factor-1 AIF-1 homologue cDNA from lipopolysaccha- Ž
. ride LPS -stimulated red sea bream leukocytes. We then determined that this cDNA encodes 444
bp in length and has an open reading frame that encodes 142 amino acids. The deduced amino Ž
. acid sequence was homologous to the AIF-1s in carp and mammals 65.5 sequence identity .
The nucleotide sequence of the protein-coding region in the AIF-1 gene was determined to have six exons and five introns. The exon–intron structure resembles those of the mouse and the
human. Genomic Southern blot analysis revealed the presence of a single AIF-1 gene per haploid in red sea bream. A time course evaluation indicated that the expression level of AIF-1 gene in
LPS-stimulated leukocytes was static in the first hour, but gradually increased from 3 to 24 h. The similarities between AIF-1s in mammals and red sea bream suggest that the AIF-1 in red sea
bream may have a similar function in activated leukocytes as AIF-1s do in mammals. q 2001 Elsevier Science B.V. All rights reserved.
Keywords: Lipopolysaccharide; AIF-1; Chrysophrys major
1. Introduction
Infectious diseases in fish cause significant losses in the commercial aquaculture industry. Investigations into the immunological mechanisms of fish are required for the
Corresponding author. Ž
. E-mail address: miyatabio.mie-u.ac.jp M. Miyata .
0044-8486r01r - see front matter q 2001 Elsevier Science B.V. All rights reserved. Ž
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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
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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
