Structure and expression of human IFN-alpha genes.

Abstract

Copy DNA (cDNA) was prepared from induced leucocyte poly(A) RNA and cloned in Escherichia coli. IFN-alpha 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-alpha cDNAs, with characteristic target cell specificities, were identified. The cloned cDNAs typically encode a mature polypeptide of 166 (or, for IFN-alpha 2, 165) amino acids and a signal sequence of 23 amino acids. A human chromosomal library was screened with IFN cDNA and 17 distinct IFN-alpha-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-alpha 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-alpha gene appears to have resulted by a recombinational event between members of the subfamily I and II. IFN-beta is distantly related to IFN-alpha's and may have diverged from a common ancestor at least 500 Ma ago. Both IFN-alpha and IFN-beta genes differ from most other genes of higher organisms by being devoid of introns. The mouse was found to possess an IFN-alpha gene family of a size similar to that of man; the murine genes also do not have introns. IFN-alpha genes devoid of their signal sequence were joined to prokaryotic promoters to produce the mature interferons in E. coli in high yield. IFN-alpha 2, purified to homogeneity, has been crystallized by T. Unge and B. Strandberg (Uppsala). Hybrid genes consisting of IFN-alpha 1 and IFN-alpha 2 segments were constructed and expressed in E. 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-alpha 1 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-beta and IFN-gamma are glycosylated. Because E. coli cells 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-1 and could be propagated for at least several months.