Genomic organisation of rainbow trout, Oncorhynchus mykiss TGF-beta.

The genomic organisation of Oncorhnchus mykiss TGF-beta has been determined through the generation of contiguous clones by PCR. The O. mykiss TGF-beta gene is approximately 3.4 Kb in length and consists of 7 coding exons with no introns in the 5'-UTR. Whilst this is the same number of exons found in TGF-beta genes of amphibians, birds and mammals, in the O. mykiss gene intron 2 of other vertebrates is absent and an additional intron is present at the 3' end of the molecule, splitting exon 7 of the other known TGF-beta genes into two exons (trout exons 6 and 7). Comparison of exon sizes in the coding region support the suggestion that the Xenopus TGF-beta5 and trout TGF-beta sequences are the forerunners of TGF-beta1. Conservation of exons coding for the mature TGF-beta peptide is relatively high (63-73% identity) but other exons show lower identities (37-58%). Comparison of the TGF-beta intron sequences reveals that in general the O. mykiss introns are considerably shorter than the avian homologs. The impact of the teleost TGF-beta gene organisation on theories of the gene evolution of this cytokine family are discussed.

Cloning of two chemokine receptor homologs (CXC-R4 and CC-R7) in rainbow trout Oncorhynchus mykiss.

Two rainbow trout chemokine receptors have been sequenced, with homology to CXC-R4 and CC-R7 molecules. The CXC-R4 sequence consisted of 1681 nucleotides, which translated into a mature protein of 357 amino acids, with 80.7% similarity to human CXC-R4. The CC-R7 sequence consisted of 2287 nucleotides, which translated into a 368-amino acid mature protein with 64.5% similarity to human CC-R7. Both sequences contained seven hydrophobic regions, representing the seven transmembrane domains (TM) typical of G-protein-coupled receptors. Extracellular cysteines, transmembrane prolines, and the DRY motif immediately following TM3 were conserved. Phylogenetic tree analysis revealed a tight clustering of trout CXC-R4 with CXC-R3-5 genes. Trout CC-R7 clustered with CC-R6-7 and CXC-R1-2. Reverse transcriptase-polymerase chain reaction analysis demonstrated a wide tissue distribution of CXC-R4 and CC-R7 message in trout, being present in head-kidney leukocytes, blood, gill, brain, spleen, and liver.

Isolation of the first piscine transforming growth factor beta gene: analysis reveals tissue specific expression and a potential regulatory sequence in rainbow trout (Oncorhynchus mykiss).

The nucleotide sequence of a rainbow trout transforming growth factor beta (TGF-beta) peptide is presented, which translates into a 382 amino acid precursor molecule containing a 20 amino acid leader and a mature peptide of 112 amino acids. The mature peptide has nine conserved cysteines and a conserved proline (position 36) and glycine (position 46), all characteristics of TGF-beta superfamily molecules. Within the precursor region are three glycosylation sites, two in common with known TGF-beta s, an integrin binding site (RGD) and the tetrabasic peptide cleavage site (RKKR). The full 3' untranslated region (UTR) consists of 542 nucleotides with a polyadenylation signal 16 nucleotides upstream of the poly(A) tail. The 5' UTR contains an open reading frame with the potential to encode an eleven amino acid peptide, which may have significance for regulation of TGF-beta translation. A wide tissue distribution of TGF-beta message was detected by RT-PCR; in blood leukocytes, kidney macrophages, brain, gill, and spleen tissue but not liver. A phylogenetic tree reveals the trout TGF-beta sequence is most related to xenopus TGF-beta 5, with these sequences and that of chicken TGF-beta 4 grouping with mammalian TGF-beta 1 s. The impact of the trout sequence on current theories of TGF-beta isotype evolution is discussed.