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We have isolated and characterized DNA segments containing IFN-alpha-related sequences from human lambda and cosmid clone banks. We describe six linkage groups comprising 18 distinct IFN-alpha-related loci, and report the nucleotide sequences of nine chromosomal IFN-alpha-genes with intact reading frames, as well as of five pseudogenes. Taking into account as yet unsequenced genes as well as clones described by others, there are now seven linkage groups and 23 loci, of which 15 correspond to potentially functional genes and six to non-functional genes; two loci remain unsequenced. Eighteen additional sequences are likely to be allelic to the above. The finding that at least two IFN-alpha genes appear to be natural hybrids of other IFN-alpha genes, and that two distinct IFN-alpha loci have completely identical coding sequences, although their flanking regions are different, is evidence for information exchange between the individual genes.

Copy DNA (cDNA) was prepared from induced leucocyte poly (A) RNA and cloned inEscherichia coli. IFN-α cDNA clones were isolated by subculture cloning with the use of a translation hybridization assay. Definitive identification of the clones was based on the production of an interferon-like protein by the transformed bacteria. Different IFN-α cDNAs, with characteristic target cell specificities, were identified. The cloned cDNAs typically encode a mature polypeptide of 166 (or, for IFN-α2, 165) amino acids and a signal sequence of 23 amino acids. A human chromosomal library was screened with IFN cDNA and 17 distinct IFN-α-related sequences were isolated and identified, of which 7 proved to be nonallelic authentic genes and 4 pseudogenes; 6 sequences remain to be elucidated. Taking into account the work of Goeddel and his colleagues, 13 non-allelic authentic genes and 6 pseudogenes can be distinguished. In addition, 9 genes believed to be allelic to the 13 authentic genes have been sequenced. The IFN-α genes may be classified into two major subfamilies, which diverged at least 33 Ma ago, but perhaps much earlier, if sequence rectification occurred. At least one IFN-α gene appears to have resulted by a recombinational event between members of the subfamily I and II. IFN-β is distantly related to IFN-α’s and may have diverged from a common ancestor at least 500 Ma ago. Both IFN-α and IFN-β genes differ from most other genes of higher organisms by being devoid of introns. The mouse was found to possess an IFN-α gene family of a size similar to that of man; the murine genes also do not have introns. IFN-α genes devoid of their signal sequence were joined to prokaryotic promoters to produce the mature interferons inE. coliin high yield. IFN-α2, purified to homogeneity, has been crystallized by T. Unge and B. Strandberg (Uppsala). Hybrid genes consisting of IFN-α1 and IFN-α2 segments were constructed and expressed inE. coli; the target cell specificities of such hybrids were dependent on the arrangement of the segments and were different from those of either parent. The chromosomal gene for HuIFN-α1 was introduced into mouse L cells to study the mechanism of its expression. Correct transcription was only detected after induction (with Newcastle disease virus); expression was transient, with the same kinetics as those of the endogenous mouse IFN mRNA. Natural murine IFNs and human IFN-β and IFN-γ are glycosylated. BecauseE. colicells transformed with the genes of eukaryotic glycoproteins are not expected to yield correctly glycosylated polypeptides, we prepared lines of hamster cells permanently transformed with hybrid plasmids, which contained an IFN gene linked to the SV40 early promoter, as well as dihydrofolate reductase as a selective marker. After intracellular amplification of the introduced genes, cell lines were obtained which constitutively produced IFN at about 40 000 units ml-1and could be propagated for at least several months.

cDNA was prepared from induced leukocyte poly(A) RNA and cloned in Escherichia coli. Interferon (IFN)-alpha cDNA clones were isolated by subculture cloning using a translation hybridization assay. Definitive identification of the clones was based on the production of an interferon-like protein by the transformed bacteria. Different IFN-alpha cDNAs, with characteristic target cell specificities, were identified. The cloned cDNAs typically encode a mature polypeptide of 166 (or, in the case of IFN-alpha 2, 165) amino acids and a signal sequence of 23 amino acids.