Cloning and expression of the turtle (Trachemys scripta) immunoglobulin joining (J)-chain cDNA.

The J-chain protein is a M(r) 15000 polypeptide associated with polymeric IgA and IgM. The complete cDNA sequences of human, mouse, cow, brushtail possum, chicken and frog J chains have been previously reported, but nothing is known about the cDNA and amino acid sequences of reptilian J chain. Here, we determined a turtle J-chain cDNA sequence by RT-PCR and RACE, and examined J-chain mRNA and protein expression by Northern blotting and immunohistochemistry. This turtle J-chain cDNA was 1934 bp and had an open reading frame of 477 nucleotides, encoding 159 amino acids. The mature J-chain protein is composed of 137 amino acids, M(r) approximately 15000. The deduced amino acid sequence of the turtle J chain was highly homologous to that of human (60%), mouse (61%), cow (60%), rabbit (60%), chicken (69%), brushtail possum (65%), Rana catesbeiana (47%) and Xenopus laevis (58%). Eight cysteine residues were located at the same positions as in these other species, with the exception of X. laevis. PROSITE database analysis indicated the presence of two N-glycosylation sites in turtle, one of which was novel. Northern blot analysis revealed that turtle J-chain mRNA was expressed in lung, stomach, spleen and intestine. In addition, immunohistochemistry showed J-chain-positive plasma cells in the intestine and spleen. These results suggest the presence of a mucosal immune system mainly composed of J-chain-containing Ig in the turtle.

Cloning of the chicken immunoglobulin joining (J)-chain gene and characterization of its promoter region.

Three overlapping genomic clones of the chicken immunoglobulin joining (J) chain were isolated and then characterized using restriction enzyme analysis, Southern blot analysis with cDNA probes, and DNA sequencing. The gene consisted of four exons separated by a 2.6-kb intron 1, a 0.9-kb intron 2, and a 0.5-kb intron 3. A transcriptional initiation site was identified by a primer extension method using mRNA and cDNA, indicating that exon 1 was 86 bp encoding 20 amino acid residues. A TATA box was positioned at 29 approximately 25 bp upstream of exon 1. Exons, 2, and 3 consisted of 133 bp and 81 bp, encoding 43 and 26 amino acid residues of the mature protein, respectively. Exon 4 consisted of 202 bp encoding 66 amino acid residues and 1.2 kb of untranslated sequence. Deletion mutants of a 4.1-kb genomic fragment containing exon 1 showed high levels of promoter activities when examined in luciferase reporter assays following transfection into the DT-40 chicken B-cell line. These results suggest that the chicken J-chain gene consists of four exons and three introns and that the transcriptional regulatory elements may be present within 3.8 kb upstream of exon 1.