In vitro and in vivo analysis of the control of dihydrofolate reductase gene transcription in serum-stimulated mouse fibroblasts

✍ Scribed by Carlos Santiago; Mark Collins; Lee F. Johnson

• Publisher: John Wiley and Sons
• Year: 1984
• Tongue: English
• Weight: 849 KB
• Volume: 118
• Category: Article
• ISSN: 0021-9541
• DOI: 10.1002/jcp.1041180114

✦ Synopsis

We have studied the rate of transcription of t h e gene for dihydrofolate reductase (DHFR) in mouse 3T6 fibroblasts during serum-induced transitions between the resting (Go) and growing states. As a model system, we have used a methotrexate-resistant 3T6 cell line that overproduces DHFR and its mRNA about 300-foId, yet regulates the expression of t h e DHFR gene in t h e same manner as normal 3T6 cells. In previous studies, we showed that the rate of production of cytoplasmic DHFR mRNA relative to total mRNA is about 4 times lower in resting than in exponentially growing cells. The rate increases to the growing value by about 15 h r following serum stimulation of the resting cells. This increase appeared to be controlled by regulating the rate of synthesis of DHFR hnRNA. In this study, we analyze the transcription of the DHFR gene in more detail. We use a variety of labeling times and RNA extraction procedures to measure the rate of synthesis of DHFR hnRNA relative to total hnRNA in pulse-labeled cells or in nuclei isolated from cells at various times following serum stimulation. The amount of labeled DHFR RNA is determined by DNA-excess filter hybridization. In all cases, the relative rate of synthesis of DHFR hnRNA increases at t h e same time, and to the same extent, as t h e rate of production of DHFR mRNA, suggesting that the increase in DHFR mRNA production is due to a corresponding increase in t h e rate of transcription of the DHFR gene. The increase in DHFR gene transcription is not blocked by cytosine arabinoside, showing that the increase does not depend on gene duplication. In isolated nuclei, DHFR RNA synthesis is inhibited by a-amanitin (1 pg/mI), indicating that the DHFR gene is transcribed by RNA polymerase II. Others have shown that when stationary phase cells are stimulated to proliferate, the increase in DHFR mRNA content is controlled primarily at the post-transcriptional level. Therefore, it appears that the rate of production of DHFR mRNA is controlled by different biochemical mechanisms when cells are in different physiological states.