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Based on amino-acid sequence data from Aspergillus niger alpha-L-arabinofuranosidase B (ABF B), and cyanogen bromide fragments derived thereof, deoxyoligonucleotide mixtures were designed to be employed as primers in a polymerase chain reaction (PCR) on A. niger genomic DNA. This resulted in amplification of three related PCR products. The abfB gene encoding ABF B was isolated from a genomic library using such an amplification product as a probe. A 5.1-kb BamHI fragment was subcloned to result in plasmid pIM991. Upon introduction by co-transformation into both A. niger and A. nidulans uridine auxotrophic strains, pIM991 was shown to contain the functional gene since prototrophic transformants overproduced ABF B upon growth on the inducing carbon source sugar beet pulp. A plate assay was developed enabling quick selection of ABF B-overproducing transformants. The sequence of a 4122-bp long BamHI/SstI fragment was determined. The abfB gene does not contain introns and codes for a protein of 499 amino acids. The mature ABF B, 481 amino acids in length, has a deduced molecular weight of 50.7 kDa. A. niger abfB is the first eukaryotic gene encoding an ABF to be characterized.

SUMMARYFour natural murine interferon-~ genes (MuIFN-cd, -~2, -~4 and -cc6) and four hybrid genes (crick4, ~2cM, ~4cd and cc4~2) were transiently expressed in monkey COS cells under the transcriptional control of the simian virus 40 early promoter. The proteins were labelled with [35S]methionine during a 16 h incubation and proteins secreted by the cells during this period were separated by polyacrylamide gel electrophoresis and subsequently visualized by fluorography. Under the conditions used, the IFNs represented 5 to 10~ of the total amount of secreted proteins. All genes were found to encode biologically active IFN subspecies, including ~4 which has a deletion of five amino acids. When the specific activities of the proteins were compared, it appeared that the specific antiviral activity of c~4 on mouse cells was three-to sixfold higher than the activities of the other natural IFN subspecies. The specific activities of the hybrid proteins were similar to those of the natural proteins, except for the ~2c~4 hybrid which had a higher specific activity than the original proteins. The ability of the natural and hybrid subspecies to protect hamster cells against viral infection was determined using MuIFN-~I as a standard. Large differences in activity were found, with ~6 as the most and ~4 as the least active subspecies.

It has been drawn to our attention that the DNA sequence of the MulFN-~10 gene published in this paper is almost identical to one previously published for the MulFN-~7 gene [Dion, M., Vodjdani, G. & Doly, J. (1986). Sequence and expression of a novel murine interferon alpha gene -homology with enhancer elements in the regulatory region of the gene. Biochemical and Biophysical Research Communications 138,[826][827][828][829][830][831][832][833][834]. There is one sequence difference in the 5' non-coding region and one in the 3' non-coding region, but the protein-coding region is identical in both analyses. It is proposed that MulFN-~10 be renamed .

As described earlier the protein products of the murine interferon (IFN) genes MuIFN-alpha 1, -alpha 2, and -alpha 4 differ in their antiviral activity on hamster (CHO) and mouse (L929) cells. For structure-function analysis, hybrids were prepared between the three genes using common restriction enzyme sites. Natural and hybrid genes were transiently expressed in monkey COS cells. Under the conditions used IFN constituted 20-30% of the total amount of secreted proteins. Using a panel of hybrids either between alpha 1 and alpha 2 or between alpha 1, alpha 2, and alpha 4, the amino-terminal region of the protein, from amino acids 10 to 58, was found to determine its antiviral activity on hamster cells. On mouse cells, the antiviral activities of hybrids between alpha 4 and either alpha 1 or alpha 2 were compared. The high activity of alpha 4 (five to ten times that of alpha 1 or alpha 2) was not transmitted to hybrids having the amino-terminal part of alpha 4, but coincided with the presence of the alpha 4 carboxy-terminal region in all but one hybrid construct. The deletion of five amino acids (positions 103-107) located in this region of alpha 4 did not affect antiviral activity when introduced into MuIFN-alpha 2 and a MuIFN-alpha 42 hybrid by site-directed mutagenesis.