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Abstract Background The high degree of sequence conservation between coding regions in fish and mammals can be exploited to identify genes in mammalian genomes by comparison with the sequence of similar genes in fish. Conversely, experimentally characterized mammalian genes may be used to annotate fish genomes. However, gene families that escape this principle include the rapidly diverging cytokines that regulate the immune system, and their receptors. A classic example is the class II helical cytokines (HCII) including type I, type II and lambda interferons, IL10 related cytokines (IL10, IL19, IL20, IL22, IL24 and IL26) and their receptors (HCRII). Despite the report of a near complete pufferfish (Takifugu rubripes) genome sequence, these genes remain undescribed in fish. Results We have used an original strategy based both on conserved amino acid sequence and gene structure to identify HCII and HCRII in the genome of another pufferfish, Tetraodon nigroviridis that is amenable to laboratory experiments. The 15 genes that were identified are highly divergent and include a single interferon molecule, three IL10 related cytokines and their potential receptors together with two Tissue Factor (TF). Some of these genes form tandem clusters on the Tetraodon genome. Their expression pattern was determined in different tissues. Most importantly, Tetraodon interferon was identified and we show that the recombinant protein can induce antiviral MX gene expression in Tetraodon primary kidney cells. Similar results were obtained in Zebrafish which has 7 MX genes. Conclusion We propose a scheme for the evolution of HCII and their receptors during the radiation of bony vertebrates and suggest that the diversification that played an important role in the fine-tuning of the ancestral mechanism for host defense against infections probably followed different pathways in amniotes and fish.

A single species of human interferon α (IFNα) was labelled with 125I to high incorporation for binding studies on tile B-lymphoblastoid cell line, Daudi, whose growth is inhibited by low doses of IFN, the effect being saturated at about 100 U/ml(25 pM). The radiolabelled 1FN was shown to be fully active and the binding affinity to cellular sites was shown to be unchanged by iodination. Experimental conditions were standardized such that binding mid cell growth experiments could be performed on the sam initial culture of cells. 125I-labelled IFNα2 (IFNα prepared from Escherichia coli carrying human α2 gene) was added to exponentially growing cultures (mean specific growth rate 0.77 ±0.07 days-1 at a mean concentration of 235000 ±2000 cells ml-1. Two types of binding could be discerned on growing cultures: the first with a transient peak followed by a loss or discharge of available sites, the second reaching equilibrium some 3 h after the addition of IFN. Large differences in the apparent dissociation constants were evident. The affinity of binding at the ‘steady-state’, appeared to be much higher. An analysis of the displacement rates for bound IFN suggested that the two reactions were occuring consecutively over the whore of tile dose range studied (1–100 U/ml:0.25–25 pM IFN). In this dose range we found that Daudi cells would eventually stop growing at all doses that the rates of deceleration of cellular growth were linearly proportional to the dose of IFN in a double-reciprocal plot (i.e. in analogy to Michaelis-Menten kinetics). A good congruence was found between the equilibrium constants for binding and for growth inhibition (2.65 pM and 2.39 pM, respectively). The amount of IFN bound at steady state thus determines the rate at which growth is inhibited. We propose that the first reaction represents binding of IFN to surface receptors, and the second transfer of IFN to an activation complex on the cell membrane. Appropriate models and their general applicability to IFN action are discussed. [ABSTRACT FROM AUTHOR]

Using membrane preparations of the interferon receptor, prepared from cells of the Burkitt line, Daudi, we have examined the binding of three human recombinant α-interferons. 1. We discovered a binding titration of the interferons IFN-αA and IFN-αD in the pH range 6-9. Receptor binding, negligible at pH 6, rises to a maximum close to pH 9. We have shown that binding of IFN-αA at basic pH is to the same receptors as at neutrality and that IFN-receptor com-plexes extracted with digitonin are more stable at basic pH than they are at neu-trality. 2. The recombinant interferon, IFN-αB, shows little change of binding in the pH range 6-9. At its basic optimum the binding of IFN-αA approaches that of IFN-αB, while at neutral pH the binding of IFN-αA is 3-4 times less. This difference at neutral pH is seen on intact cells as well as on membrane preparations. The specific activity of IFN-αB is close to that of IFN-αA, both of which are 10-20 times more active than IFN-αD; and the binding titration is, therefore, independent of the initial binding affinities. 3. Using hybrid IFNs constructed from the DNA sequences of αD and αB, we have isolated the sequence responsible for the binding titration to the segment com-prising amino acids 61-92. Examination of these sequences reveals that Lys-84 is present in alt the IFN-α except IFN-αB where it is replaced by Glu; and Tyr-90, present in most of the common IFN-α including α A and αD, is replaced by Asp in IFN-αB. Lys and Tyr would normally titrate in the pH range 6-9. We conclude that the binding titration is due to an electrostatic interaction and we propose that the interaction is between IFN-receptor comple-xes. The role of the interaction in the binding losses that accompany the anti-proliferative effects of IFN is discussed. [ABSTRACT FROM AUTHOR]

The Burkitt-derived line, Daudi, whose proliferation is inhibited by human α-interferon (IFN-α), was treated with 125I-labelled recombinant human IFN-αA. After separation from unbound ligand, cell-bound IFN was extracted with the detergent digitonin yielding soluble and insoluble complexes of IFN and receptor, together with a certain amount of uncomplexed IFN. 1. Soluble complexes were stable enough to be separated from uncomplexed IFN by permeation chromatography. Treatment of soluble complexes with the bifunctional reagent, disuccinimidyl suberate, yielded a radioactive product separating with an Mr of 130 000 on electrophoresis in sodium dodecyl sulphate. Similar complexes could be recovered with sodium dodecyl sulphate from the digitonin-insoluble residue, treated with the bi-functional reagent. 2. The total (soluble and insoluble) of complexed IFN obtained after digitonin extraction was a constant fraction (0.62) of the total cell-bound radioactivity, being independent of the concentration of IFN added to the cells (< pM to > nM), and of the time of incubation (1 min to 20 h). However, between 30 min and 3 h of incubation, the insoluble complex increased, at the expense of the soluble complex, and there appeared a cellular pool of degraded ligand. From 3 h to 20 h the distribution of ligand-derived radioactivity remained constant while the total amount decreased to less than 10% of its value at 30 min. This decrease in binding was matched by the appearance of an equivalent quantity of radiolabelled fragments in the culture medium. 3. The inhibition of cellular division due to IFN was shown to be coincident with the disappearance of cellular binding and with the cell-mediated degradation of receptor-complexed IFN. We propose that IFN removes its own receptor and, in doing so, blocks a linked function necessary for the stimulated growth of Daudi cells. [ABSTRACT FROM AUTHOR]