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- Molin, Magnus, et al.
(författare)
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Two Novel Adenovirus Vector Systems Permitting Regulated Protein Expression in Gene Transfer Experiments
- 1998
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Ingår i: Journal of Virology. - 0022-538X .- 1098-5514. ; 72:10, s. 8358-8861
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Tidskriftsartikel (refereegranskat)abstract
- Two new adenovirus vector systems based on the tetracycline-regulated Tet-ON- (Gossen, M., et al., Science 268:1766-1769, 1995) and the RU 486-regulated progesterone antagonist (Wang, Y., et al., Proc. Natl. Acad. Sci. USA 91:8180-8184, 1994)-induced gene expression systems are described. We show that both systems permit a tight control of chloramphenicol acetyltransferase reporter gene expression in a variety of cell types, with induction levels of approximately 1,800-fold (Tet-ON system) and 600-fold (RU 486-regulated system), respectively. A significant advantage of our vector systems is that reporter protein expression can be adjusted over a wide range by varying the amount of inducer. The Tet-ON system is also shown to permit an efficient control of reporter gene expression in mice.
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| 2. |
- Öhman, Karin
(författare)
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Function of adenovirus early region 4 proteins in RNA processing and cellular transformation
- 1995
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- For almost 30 years, adenovirus has been used as a model system for mechanistic studies of eucaryote gene expression. In this thesis, we have concentrated our efforts to investigate the function of adenovirus early region 4 (E4) gene products in cellular transformation and the post-transcriptional regulation of gene expression. During a Iytic infection, proteins expressed from the E4 region are necessary for efficient mRNA expression from the adenovirus major late transcription unit. By using a transient transfection assay, we could show that E4 post-transcriptionally stimulates the accumulation of major late tripartite leader containing transcripts. The tripartite leader consists of three short leader exons and one long exon, the so called i-leader exon, which is spliced between the second and the third leader exon. Two E4 proteins, E4-ORF3 and E4-ORF6, were shown to have opposing effects on the accumulation of alternatively spliced tripartite leader exons. The E4-ORF3 protein facilitated i-leader exon inclusion, while E4-ORF6 preferentially favoured i-leader exon skipping. The E4-ORF6 protein was also shown to facilitate i-leader exon skipping during a lIytic virus infection. In addition, E4-ORF3 and E4-ORF6 had the same effect on the alternative splicing of chimeric B-globin transcripts, and also similar effects on the accumulation of alternatively spliced E1BmRNAs. The effects of E4-ORF3 and E4-ORF6 on the alternative splicing of tripartite leader mRNAs resembles the activities of the cellular splicing factors ASF /SF2 and hnRNP A1. However, in our DNA transfection assay, we could also show that E4-ORF3 and ASF/SF2 have different effects on the accumulation of alternatively-spliced E1A and E1B mRNAs, whereas E4-ORF6 and hnRNP A1also have different effects on E1A alternative splicing but show the same effect on the accumulation of alternatively-spliced E1B transcripts. The cellular localization of E4-ORF3 and E4-ORF6 were determined by immunofluorescence experiments: E4-ORF3 is localized to the nuclear matrix, while E4-ORF6 is dispersed throughout the nucleus and cytoplasm. Adenoviruses are DNA tumour viruses, and the minimal function required for cellular transformation has been shown to reside within early region 1 (E1). Several studies have shown that adenovirusgenes outside the E1 region can also modulate its transforming properties. We show that transformation of rat CREF fibroblasts by adenovirus-2 El is stimulated by expression of the E4region. Co-transfection of CREF cells with El and E4 did not alter the frequency of focus formation, but resulted in larger, more dense foci than E1 transfection alone. The E1 and E4 co-transfected cells were also capable of anchorage-independent growth. To assay for the activity of individual E4 proteins on El transformation, we used CMV expression vectors encoding single E4 proteins in the CREF cell transformation assay. We could show that two E4 proteins, E4-ORFI and E4-ORF6, had a significant capacity in the stimulation of El-mediated transformation, although none of them were individually capable of enhancing soft-agar growth of E1-transformed cells.
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