Cloning and expression of the canine interferon-gamma gene.
The canine interferon-gamma (cIFN-gamma) chromosomal gene was isolated from a recombinant library, containing the entire dog genome, by screening with a rat IFN-gamma genomic probe. The structural organization of the cIFN-gamma gene closely resembles that of the human, murine, and rat IFN-gamma genes. It contains three intervening sequences and encodes a signal sequence of 23 amino acids followed by a mature protein of 143 amino acids. Two potential N-glycosylation sites are located at positions 16 and 83 of the mature protein. Comparison of the cIFN-gamma protein sequence with that of the corresponding murine, rat, human, and bovine proteins revealed a homology of 40%, 42%, 65%, and 76%, respectively. The cIFN-gamma gene was expressed under control of the simian virus 40 early promoter in Chinese hamster ovary (CHO) cells, deficient in dihydrofolate reductase (DHFR), after cotransformation with a plasmid containing the murine dhfr gene. Initial transformants with dhfr+ phenotype produced cIFN-gamma titers ranging from 10 to 10,000 laboratory units (LU)/ml of culture medium. A cIFN-gamma cDNA sequence was identified in a cDNA library constructed with partially purified RNA from a cIFN-gamma-producing CHO cell line. In vitro transcription of the cDNA and translation of the mRNA in Xenopus laevis oocytes resulted in secreted IFN-gamma with an antiviral activity specific for canine cells.
Devos
K
K
Laboratory of Molecular Biology, State University of Gent, Belgium.
Duerinck
F
F
Van Audenhove
K
K
Fiers
W
W
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004285:Dogs; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D015698:Genomic Library; D007371:Interferon-gamma / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D015183:Restriction Mapping; D014407:Tumor Cells, Cultured; D010948:Viral Plaque Assay
D011994:Recombinant Proteins; D007371:Interferon-gamma