Cloning and analysis of expression of a gilthead sea bream (Sparus aurata) Mx cDNA.
In the current work, we have cloned and sequenced the full cDNA for a Mx protein in the gilthead sea bream (Sparus aurata) by RACE PCR. The Mx cDNA of 2182 bp contained an open reading frame of 1857 bp that codes for a protein of 618 aa. Within the coding sequence, characteristic features of Mx proteins were found, such as a tripartite guanosine-5'-triphosphate (GTP)-binding motif (GXXXSGKS/T, DXXG and T/NKXD), the signature of the dynamin family, LPRG(S/K)GIVTR, and a sequence that codes for a leucine zipper at the C-terminal region of the protein. An RT-PCR was optimised to estimate the level of expression of Mx protein in sea bream. Through this method we determined that Mx is constitutively expressed in head kidney, liver, spleen, heart, gills, muscle and brain of healthy sea bream. Intramuscular challenge of sea bream with polyinosinic:polycytidylic acid (Poly I:C) up-regulated Mx expression in liver, head kidney, spleen and muscle. Constitutive expression was also found in isolated head kidney macrophages and blood leukocytes. This expression was significantly up-regulated by addition of Poly I:C. Mx was not constitutively expressed in the sea bream established cell line, SAF-1, but Poly I:C and nodavirus were also capable of inducing Mx expression in this cell line.
Tafalla
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C
Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Cientificas, Eduardo Cabello 6, Vigo 36208, Spain.
Aranguren
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R
Secombes
C J
CJ
Figueras
A
A
Novoa
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D034281:Dynamins / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics*; D015870:Gene Expression; D016350:Leucine Zippers / Q000235:genetics; D008969:Molecular Sequence Data; D064547:Myxovirus Resistance Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D021541:Sea Bream / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D018076:DNA, Complementary; D064547:Myxovirus Resistance Proteins; D019204:GTP-Binding Proteins; D034281:Dynamins