Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Jun 15;88(12):5287–5291. doi: 10.1073/pnas.88.12.5287

Newly synthesized RNA: simultaneous measurement in intact cells of transcription rates and RNA stability of insulin-like growth factor I, actin, and albumin in growth hormone-stimulated hepatocytes.

T R Johnson 1, S D Rudin 1, B K Blossey 1, J Ilan 1, J Ilan 1
PMCID: PMC51857  PMID: 1711226

Abstract

The levels of several RNA transcripts in cultured hepatocytes are regulated by transcriptional and post-transcriptional mechanisms and are affected by growth hormone and insulin. We assessed the effects of these hormones on transcription rates and the stability of insulin-like growth factor I, actin, and albumin transcripts in intact cells of primary cultures of rat hepatocytes by analyzing thiol-labeled, newly synthesized RNA isolated by mercurated agarose affinity chromatography. The application of this concept to the measurement of transcript stability is presented in detail. The data indicate that growth hormone stimulates the transcription rates of insulin-like growth factor I, actin, and albumin genes. The stability of all three transcripts, particularly albumin, appears to be lower in growth hormone-containing medium than it is in insulin-containing medium. The experiments indicate that the rates of transcription and/or degradation of albumin mRNA are influenced by hormonal treatment. However, the cells maintain roughly constant albumin transcript levels independent of hormone treatment by compensatory changes in the rates of transcription and degradation.

Full text

PDF
5287

Images in this article

5288Image
on p.5288
5288Image
on p.5288
5288Image
on p.5288

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aiello L. P., Shia M. A., Robinson G. S., Pilch P. F., Farmer S. R. Characterization and hepatic expression of rat alpha 1-inhibitor III mRNA. J Biol Chem. 1988 Mar 15;263(8):4013–4022. [PubMed] [Google Scholar]
  2. Bezerra R., Favre A. In vivo incorporation of the intrinsic photolabel 4-thiouridine into Escherichia coli RNAs. Biochem Biophys Res Commun. 1990 Jan 15;166(1):29–37. doi: 10.1016/0006-291x(90)91907-a. [DOI] [PubMed] [Google Scholar]
  3. Cavalieri R. L., Havell E. A., Vilcek J., Pestka S. Induction and decay of human fibroblast interferon mRNA. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4415–4419. doi: 10.1073/pnas.74.10.4415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  5. Guyette W. A., Matusik R. J., Rosen J. M. Prolactin-mediated transcriptional and post-transcriptional control of casein gene expression. Cell. 1979 Aug;17(4):1013–1023. doi: 10.1016/0092-8674(79)90340-4. [DOI] [PubMed] [Google Scholar]
  6. Helms S. R., Rottman F. M. Characterization of an inducible promoter system to investigate decay of stable mRNA molecules. Nucleic Acids Res. 1990 Jan 25;18(2):255–259. doi: 10.1093/nar/18.2.255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Idzerda R. L., Behringer R. R., Theisen M., Huggenvik J. I., McKnight G. S., Brinster R. L. Expression from the transferrin gene promoter in transgenic mice. Mol Cell Biol. 1989 Nov;9(11):5154–5162. doi: 10.1128/mcb.9.11.5154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Isom H. C., Secott T., Georgoff I., Woodworth C., Mummaw J. Maintenance of differentiated rat hepatocytes in primary culture. Proc Natl Acad Sci U S A. 1985 May;82(10):3252–3256. doi: 10.1073/pnas.82.10.3252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Johnson T. R., Blossey B. K., Denko C. W., Ilan J. Expression of insulin-like growth factor I in cultured rat hepatocytes: effects of insulin and growth hormone. Mol Endocrinol. 1989 Mar;3(3):580–587. doi: 10.1210/mend-3-3-580. [DOI] [PubMed] [Google Scholar]
  10. McKnight G. S., Palmiter R. D. Transcriptional regulation of the ovalbumin and conalbumin genes by steroid hormones in chick oviduct. J Biol Chem. 1979 Sep 25;254(18):9050–9058. [PubMed] [Google Scholar]
  11. Nagaoka I., Trapnell B. C., Crystal R. G. Regulation of insulin-like growth factor I gene expression in the human macrophage-like cell line U937. J Clin Invest. 1990 Feb;85(2):448–455. doi: 10.1172/JCI114458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Peavy D. E., Taylor J. M., Jefferson L. S. Time course of changes in albumin synthesis and mRNA in diabetic and insulin-treated diabetic rats. Am J Physiol. 1985 Jun;248(6 Pt 1):E656–E663. doi: 10.1152/ajpendo.1985.248.6.E656. [DOI] [PubMed] [Google Scholar]
  13. Rapid switching of plant gene expression induced by fungal elicitor. Science. 1985 Mar 8;227(4691):1240–1243. doi: 10.1126/science.227.4691.1240. [DOI] [PubMed] [Google Scholar]
  14. Rodgers J. R., Johnson M. L., Rosen J. M. Measurement of mRNA concentration and mRNA half-life as a function of hormonal treatment. Methods Enzymol. 1985;109:572–592. doi: 10.1016/0076-6879(85)09116-9. [DOI] [PubMed] [Google Scholar]
  15. Scott C. D., Baxter R. C. Production of insulin-like growth factor I and its binding protein in rat hepatocytes cultured from diabetic and insulin-treated diabetic rats. Endocrinology. 1986 Nov;119(5):2346–2352. doi: 10.1210/endo-119-5-2346. [DOI] [PubMed] [Google Scholar]
  16. Singer R. H., Penman S. Messenger RNA in HeLa cells: kinetics of formation and decay. J Mol Biol. 1973 Aug 5;78(2):321–334. doi: 10.1016/0022-2836(73)90119-8. [DOI] [PubMed] [Google Scholar]
  17. Tannenbaum G. S. Growth hormone secretory dynamics in streptozotocin diabetes: evidence of a role for endogenous circulating somatostatin. Endocrinology. 1981 Jan;108(1):76–82. doi: 10.1210/endo-108-1-76. [DOI] [PubMed] [Google Scholar]
  18. Woodford T. A., Schlegel R., Pardee A. B. Selective isolation of newly synthesized mammalian mRNA after in vivo labeling with 4-thiouridine or 6-thioguanosine. Anal Biochem. 1988 May 15;171(1):166–172. doi: 10.1016/0003-2697(88)90138-8. [DOI] [PubMed] [Google Scholar]
  19. Zaret K. S., DiPersio C. M., Jackson D. A., Montigny W. J., Weinstat D. L. Conditional enhancement of liver-specific gene transcription. Proc Natl Acad Sci U S A. 1988 Dec;85(23):9076–9080. doi: 10.1073/pnas.85.23.9076. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES