ABSTRACT
The coding part of a murine interferon alpha (MuIFN-alpha) gene was cloned into an expression plasmid containing the simian virus 40 early promoter and the rabbit beta-globin polyadenylation signal. This construct was transfected into Chinese hamster ovary cells, together with a plasmid containing the Ecogpt gene as a selection marker. Resulting colonies were assayed for constitutive interferon production and analysed for integration of MuIFN-alpha genes. There was no obvious correlation between the number of genes integrated and the amount of interferon produced. The highest producer, designated CHO-pSV10EF-3, contained four copies of the mouse gene and constitutively secreted up to 100 000 International Units of interferon per ml per day. The MuIFN-alpha subspecies produced by this clone was characterized by analysis of its antiviral activity on heterologous cells, heparin-Sepharose affinity chromatography and chromatofocusing. The results obtained indicate that it is identical or closely related to a minor component present in conventional MuIFN-alpha preparations.
Subject(s)
Interferon Type I/genetics , Animals , Cells, Cultured , Chromatography, Affinity , Cricetinae , Cricetulus , Female , Interferon Type I/analysis , Interferon Type I/pharmacology , Isoelectric Focusing , Ovary , PlasmidsABSTRACT
A number of cloned viral preparations isolated from Rauscher virus-producing JLS-V5 cells were compared in their competence to induce different types of leukaemias. All preparations were able to induce myeloid leukaemias, but the induction of lymphatic or erythroid leukaemias was also observed. Serial infection of newborn mice with either cell-free extracts or serum from animals suffering from a myeloid leukaemia did not result in the occurrence of relatively more myeloid leukaemias nor did the infection with virus harvested from ascites fluid of permanent myeloid cell lines. It appears that the mechanism by which myeloid leukaemias are induced is not virus-specific.
Subject(s)
Leukemia, Experimental/etiology , Leukemia, Myeloid/etiology , Animals , Leukemia, Erythroblastic, Acute/etiology , Leukemia, Lymphoid/etiology , Mice , Mice, Inbred BALB C , Mice, Inbred DBA , Rauscher VirusABSTRACT
Interferon produced by mouse L-929 cells by incubation with poly(rI).poly(rC) is known to be composed of a mixture of MuIFN-alpha and MuIFN-beta. The alpha component was separated from the bete species by affinity chromatography over a monoclonal anti-MuIFN-beta agarose column and partially purified by gel filtration. MuIFN-alpha, prepared by this method was separated into at least five subspecies by chromatofocusing. The approximate pI values of these components are greater than or equal to 7.5, 6.5, 6.2, 5.9 and 5.6, respectively. Component 3 (pI 6.2) was the most prominent subspecies present in our MuIFN-alpha preparations, representing 40 to 50% of the total antiviral activity. Component 1 (pI greater than or equal to 7.5) which accounted for about 5% of the antiviral activity on mouse cells, differed in some properties from the other interferon subspecies. It showed a relatively high antiviral activity on heterologous cells and it was eluted from a Sephadex column after the other alpha subspecies. Furthermore, it showed a diminished binding to heparin as compared to the other MuIFN-alpha subspecies, indicating a lower affinity for polynucleotides.
Subject(s)
Interferon Type I/isolation & purification , Animals , Chromatography, Affinity , Chromatography, Gel , Interferon Type I/classification , Interferon Type I/immunology , Isoelectric Point , L Cells , Mice , Viral InterferenceABSTRACT
A monoclonal antibody against murine interferon-beta (MuIFN-beta) was prepared using standard methods. Antibodies were immobilized by coupling to Sepharose and used for large-scale purification of poly(I) . poly(C)-induced mouse L cell IFN. Antibodies isolated from the serum of one nude mouse which was transplanted with the anti-MuIFN-beta antibodies producing hybridoma were able to bind at least 7 X 10(7)U MuIFN-beta. In one single antibody affinity chromatography step MuIFN-alpha was separated from MuIFN-beta and a 1000-fold purification of MuIFN-beta was obtained. The purified material had a specific activity of 5 X 10(8)U/mg protein. The recovery from the antibody column was 100%. SDS-PAGE analysis of the purified material revealed the presence of one single protein band with a molecular weight of 33 kD, representing MuIFN-beta.
Subject(s)
Interferon Type I/isolation & purification , Animals , Antibodies, Monoclonal/immunology , Electrophoresis, Polyacrylamide Gel , In Vitro Techniques , Interferon Type I/immunology , L Cells/immunology , MiceSubject(s)
Interferons/immunology , Poly I-C/pharmacology , Animals , Cells, Cultured , Cricetinae , L Cells/immunology , Mice , RatsABSTRACT
We have studied the relationship between Friend spleen focus-forming virus (SFFV) and its helper lymphoid leukaemia virus (LLV) by comparing RNase T1 fingerprints of genomic RNAs. Our data indicate that about 70% of the SFFV sequence is a perfect copy of parts of the helper genome. We conclude that our SFFV and LLV isolates have co-evolved very closely and that SFFV-specific sequences are not identical in different Friend virus isolates.
