Download Rapid communication: Nucleotide sequence of red seabream

Rapid communication: Nucleotide sequence of red seabream,
Pagrus major, β-actin cDNA
C. Y. Choi1 and F. Takashima
Department of Aquatic Biosciences, Tokyo University of Fisheries, Konan, Minato-ku, Tokyo 108-8477, Japan
Name of the Sequence. Red seabream β-actin cDNA.
Genus and Species. Pagrus major.
Origin of Clone. Total RNA was isolated from ovary
of red seabream. Poly(A)+ RNA was purified using an
Oligotex-dT30 (Super) mRNA purification kit (Takara,
Japan). Double-stranded complementary DNA synthesis was carried out using a TimeSaver cDNA synthesis
kit (Pharmacia Biotech, Uppsala, Sweden). The cDNA
library was constructed with a Lamda gt10/EcoRI/
CIAP-treated vector (Stratagene, La Jolla, CA). To target β-actin cDNA from red seabream, primers for PCR
were synthesized referring to DNA nucleotide sequences reported for medaka β-actin (Takagiri et al.,
1997). The PCR primers used to screen the red seabream ovary cDNA library were ACTACCTCATGAAG
ATCCTG for the forward primer and TTGCTGATCCA
CATCTGCTG for the reverse primer. Reverse transcriptase-PCR amplified a 517-bp band from red seabream cDNA synthesized from the ovary RNA. The PCR
product as a probe was labeled with [α-32P]dCTP (Oligolabelling kit, Pharmacia Biotech) to screen the cDNA.
The positive clones containing the insert were digested
with Sau3AI, SacI, and ScaI and then subcloned into
pBlue Script SK+. Both strands were sequenced with
an ALFexpress DNA Sequencer System (Pharmacia
Biotech).
Comparison with Related Sequences. The deduced
amino acid sequence of red seabream β-actin showed
96.3% identity to both human (Ponte et al., 1984) and
medaka (Takagiri et al., 1997) and 95.7% identity to
gilthead seabream β-actin (Santos et al., 1997).
1
Present address for correspondence: Dr. Cheol Young Choi, Sesoko
Station, Tropical Biosphere Research Center, University of the Ryukyus, 3422 Sesoko, Motobu, Okinawa 905-0227, Japan (phone: +81
980 47 6215; fax: +81 980 47 4919; E-mail: [email protected]).
Received January 20, 2000.
Accepted July 20, 2000.
2000 American Society of Animal Science. All rights reserved.
Sequence Data. The red seabream β-actin cDNA clone
contained 1,521 nucleotides (nt), including the open
reading frame that encoded 375 amino acids. The stop
codon was followed by a 3′ untranslated region of 342
nt, including the polyadenylation signal, AATAAA.
DDBJ/EMBL/GenBank
Accession
Number.
AB036756.
Comments. From the total of 375 amino acids, the
human β-actin amino acid sequences differed by 7
amino acids and 14 amino acids from gilthead seabream
and red seabream β-actin amino acid sequences, respectively (Figure 1). The amino acid sequences of red seabream β-actin differed from those of β-actin of gilthead
seabream, which belongs to the same family as red
seabream, by 16 amino acids out of 375. Comparison
of the deduced amino acid sequence of the red seabream
β-actin gene was found to be highly conserved in relation to other known actins. However, the red seabream
β-actin isoform has a higher homology to human β-actin
than β-actin of gilthead seabream, which belongs to the
same family as red seabream. Therefore, the possibility
that red seabream β-actin and gilthead seabream βactin have different isoforms must be considered.
Literature Cited
Katagiri, T., I. Hirono, and T. Aoki. 1997. Identification of a cDNA
for Medaka cytoskeletal β-actin and construction for the reverse
transcriptase-polymerase chain reaction (RT-PCR) primers.
Fisheries Sci. (Tokyo) 63:73–76.
Ponte, P., S.-Y. Ng, J. Engel, P. Gunning, and L. Kedes. 1984. Evolutionary conservation in the untranslated regions of actin
mRNAs: DNA sequence of human beta-actin cDNA. Nucleic
Acids Res. 12:1687–1696.
Santos, C. R. A., D. M. Power, P. Kille, L. Llewellyn, V. Ramsurn,
T. Wigham, and G. E. Sweeney. 1997. Cloning and sequencing
of full-length seabream (Sparus aurata) β-actin cDNA. Comp.
Biochem. Physiol. 117B:185–189.
Key Words: Actin, Complementary DNA, DNA Cloning,
Red Seabream
2990
J. Anim. Sci. 2000. 78:2990–2991
Rapid Communication
2991
Figure 1. The amino acid sequences were extracted from the DDBJ/EMBL/GenBank sequence databases. Comparison
of the amino acid sequences encoded by the red seabream β-actin (RsACT) (this paper, AB036756) with gilthead
seabream (GsACT) (X89920), medaka (MeACT) (D89627), and human β-actin (HuACT) (84144061). The amino acid
sequences were optimally aligned so as to match identical residues, which are indicated by asterisks.