Cloning, characterization and expression of cDNA containing major histocompatibility complex class I, IIα and IIβ genes of Japanese flounder Paralichthys olivaceus

ABSTRACT:   The nucleotide sequences of Japanese flounder Paralichthys olivaceus , major histocompatibility complex (MHC) cDNA, classical MHC class Iα, non-classical MHC class Iβ, MHC class IIα and IIβ, were determined. The domain structures and antigen binding motifs of vertebrate MHC are conserved in the Japanese flounder MHC. A phylogenetic analysis supports the classification of these genes into class I and class II MHC. Classical MHC class Iα was ubiquitously expressed, whereas the non-classical MHC class Iβ was expressed mainly in lymphoid organs, gills, intestine and stomach. The MHC classes IIα and IIβ were also ubiquitously expressed.     

Cloning and characterization of the haemolysin determinants from Aeromonas hydrophila

Abstract. Two haemolysin genes (AHH4 and AHH-2) of Aeromonas hydrophila ATCC7966 were cloned into a plasmid vector in Escherichia coli K-12. An open reading frame (ORF) of the AHH-1 haemolysin gene was 1734 base pairs (bp). and corresponded to a protein of 577 amino acid residues. Analysis of the deduced amino sequence indicated a highly hydrophobic N-terminal region which had the characteristics of a leader peptide. The sequence also included the -10 region and the -35 region of a promoter, and a ribosome- binding site upstream from the ORF. The termination site was located downstream from the ORF. The haemolysin was a thermolabile protein with the predicted molecular mass of 60 kDa. The AHH-1 gene is distributed in various A. hydrophila and A. salmonicida strains. The nucleotide sequence of a 981 bp ORF of the AHH-2 gene was encoded with the predicted molecular mass of 377 kDa polypeptides. The homology of the nucleotide sequence was very low between the AHH-1 and AHH-2 genes, and also with the aerolysin gene cloned by Howard & Buckley (19S6). No leader peptide was found in the N-terminal region of the ORF of the AHH2 gene. The AHH-2 gene was detected in the original strain ATCC7966, but was not detected in other tested strains of A. hydrophila and A. salmonicida.     

Cloning of estrogen receptor-β cDNA of common Japanese conger Conger myriaster and expression of the gene during oocyte development

ABSTRACT:   Estrogen receptor (ER) cDNA was cloned from the liver of common Japanese conger Conger myriaster . Common Japanese conger ER cDNA contains 2543 nucleotides including an open reading frame that encodes 596 amino acids, with a molecular weight of 66.1 kDa. The deduced amino-acid sequence of the common Japanese conger ER shares 85% identity with that of eel Anguilla japonica ER-β, and 39–46% and 53–59% identity with the deduced sequences of ER-α and ER-β of other species, respectively. In a phylogenetic analysis, the common Japanese conger ER was found to cluster with the ER-β of other species. The common Japanese conger ER-β also has six domains (A–F), which are conserved in the nuclear receptor superfamily. The amino-acid sequences in the C and E domains were highly conserved between common Japanese conger ER-β and other ER. However, the other domains showed low homology. In the female common Japanese conger, ER-β mRNA was highly expressed in the liver and pituitary gland. The expression levels of the ER-β gene increased from the oil droplet stage to the primary yolk globule stage and were maintained until the migratory nucleus stage.     

Cloning of kuruma prawn Marsupenaeus japonicus crustin-like peptide cDNA and analysis of its expression

ABSTRACT:   Antimicrobial peptides serve as an important component of the innate immune system of all species by functioning to provide a rapid first line defense against infection. Arthropod antimicrobial peptides have been well described in insects, whereas only a few molecules have been identified in crustaceans. Five variants (types 1–5) of Marsupenaeus japonicus crustin-like peptide cDNA that were obtained from a hemocyte cDNA library and polymerase chain reaction (PCR) amplification are reported here. Marsupenaeus japonicus crustin-like peptide type 1, the predominant type, has a cDNA consisting of 679 nucleotides and an open reading frame consisting of 573 base pairs coding for 191 amino acid residues. Other types contain varying glycine-rich repeats at the N-terminal amino acid sequences. The deduced amino acid sequences of these variants are highly similar to those of Litopenaeus setiferus (80% identity), Litopenaeus vannamei (80% identity) and Carcinus maenas crustins (44% identity). Expression of Marsupenaeus japonicus crustin-like peptide mRNA was detected in hemocytes, but not in the heart, hepatopancreas, gill, fore-gut, mid-gut, muscle, subcuticular epithelium or ovary. The expression level of crustin-like peptide mRNA increased significantly 1, 3 and 7 days post-peptidoglycan feeding as determined by quantitative real-time PCR. These results suggest that crustin-like peptide could have an important role in shrimp defense mechanisms.