Polymerase III (pol III)-dependent genes, like the adenoviral VA1 gene, are of particular interest for expressing small therapeutic RNAs into cells. A new VA1 RNA carrier molecule was generated through the deletion of the VA1 RNA central domain to give rise to the VAdeltaIV RNA vector that was devoid of undesirable physiologic activity (i.e., inhibition of the interferon-induced protein kinase, PKR). This vector was used to express in human cells hammerhead ribozymes targeted against the human immunodeficiency virus (HIV). Eight anti-HIV ribozymes were inserted at the 3'-end of this vector immediately before the four T-residues that serve as a transcription termination signal. Although the constructs were active in vitro, they failed to inhibit HIV replication in transient assays. Analysis of the intracellular ribozyme expression in cells revealed several anomalies. First, using mutant derivatives, we showed that the presence of two or three consecutive T-residues in the ribozyme portion was sufficient to promote the release of anomalous short transcripts. Second, when the ribozyme did not contain T-rich sequence, full-length transcripts were produced, but these transcripts were very unstable and were retained in the cell nucleus. In contrast, insertion of the ribozyme in place of the central domain of VA1 RNA led to production of full-length transcripts that were stable and located in the cytoplasm but that were not found to be active in vitro. Taken together, these results have important consequences for the future design of active intracellular ribozymes based on the use of pol III-transcribed genes.
The differentiation of adipose precursor cells can be divided into early and late events. Growth arrest at the G1/S boundary triggers the activation of early genes, i.e., pOb24 and lipoprotein lipase; the expression of both genes is primarily regulated at a transcriptional level. The expression of late markers, which lead to terminal differentiation and accumulation of neutral lipids, takes place after a limited number of mitoses of early-marker-expressing cells. Only terminal differentiation requires the presence of growth hormone and triiodothyronine as obligatory hormones and insulin as a modulating hormone, and results in the formation of triacylglycerol-filled, non-dividing cells. It appears that terminal differentiation involves the cyclic AMP pathway, the diacylglycerol pathway, and a third pathway triggered by insulinlike growth factor-I and insulin. It is thus proposed that a combination of mitogenic-adipogenic signals is required to trigger terminal differentiation of preadipose cells.
GH has been previously shown in Ob1771 adipose cells to activate transiently the expression of c-fos gene by a protein kinase-C-dependent pathway and to modulate, at last in part by a protein kinase-C-dependent pathway, the expression of the lipoprotein lipase (LPL) gene. In Ob1771 cells exposed to GH, under conditions where protein synthesis is inhibited by cycloheximide, the modulation of LPL gene expression is prevented, suggesting that synthesis of trans-acting factor(s) is required to modulate LPL gene expression. The present results indicate the involvement of c-Fos protein in this modulation; this involvement is supported by various lines of evidence: 1) upon GH stimulation, the increase in c-fos mRNA content is followed by the emergence of c-Fos protein within the nucleus, and this emergence precedes the increase in LPL mRNA content; 2) in GH-treated Ob1771 cells, exposure to antisense sof oligonucleotides abolishes the synthesis of c-Fos protein; and 3) at the same time, the increase in LPL mRNA content and LPL activity does not occur, whereas sense fos oligonucleotides show no effect. It is concluded that c-Fos protein plays an intermediary role in the modulation of LPL gene expression by GH.
GH has been previously shown in Ob1771 adipose cells to activate transiently the expression of c-fos gene by a protein kinase-C-dependent pathway and to modulate, at last in part by a protein kinase-C-dependent pathway, the expression of the lipoprotein lipase (LPL) gene. In Ob1771 cells exposed to GH, under conditions where protein synthesis is inhibited by cycloheximide, the modulation of LPL gene expression is prevented, suggesting that synthesis of trans-acting factor(s) is required to modulate LPL gene expression. The present results indicate the involvement of c-Fos protein in this modulation; this involvement is supported by various lines of evidence: 1) upon GH stimulation, the increase in c-fos mRNA content is followed by the emergence of c-Fos protein within the nucleus, and this emergence precedes the increase in LPL mRNA content; 2) in GH-treated Ob1771 cells, exposure to antisense sof oligonucleotides abolishes the synthesis of c-Fos protein; and 3) at the same time, the increase in LPL mRNA content and LPL activity does not occur, whereas sense fos oligonucleotides show no effect. It is concluded that c-Fos protein plays an intermediary role in the modulation of LPL gene expression by GH.
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