Expression of active iron regulatory factor from a full length human cDNA by in vitro transcription/translation (original) (raw)

Journal Article

,

Swiss Institute for Experimental Cancer Research, Genetics Unit

CH-1066 Epalinges, Switzerland

Search for other works by this author on:

,

Swiss Institute for Experimental Cancer Research, Genetics Unit

CH-1066 Epalinges, Switzerland

Search for other works by this author on:

,

Swiss Institute for Experimental Cancer Research, Genetics Unit

CH-1066 Epalinges, Switzerland

Search for other works by this author on:

,

Swiss Institute for Experimental Cancer Research, Genetics Unit

CH-1066 Epalinges, Switzerland

Search for other works by this author on:

,

Swiss Institute for Experimental Cancer Research, Genetics Unit

CH-1066 Epalinges, Switzerland

Search for other works by this author on:

Swiss Institute for Experimental Cancer Research, Genetics Unit

CH-1066 Epalinges, Switzerland

* To whom correspondence should be addressed

Search for other works by this author on:

Received:

11 November 1991

Accepted:

06 December 1991

Published:

11 January 1992

• PDF
• Split View
• • Article contents
  • Figures & tables
  • Video
  • Audio
  • Supplementary Data
• Cite

Cite

Harald Hirling, Alice Emery-Goodman, Nancy Thompson, Barbara Neupert, Christian Seiser, Lukas C. Kühn, Expression of active iron regulatory factor from a full length human cDNA by in vitro transcription/translation , Nucleic Acids Research, Volume 20, Issue 1, 11 January 1992, Pages 33–39, https://doi.org/10.1093/nar/20.1.33
Close

Navbar Search Filter Mobile Enter search term Search

Abstract

Iron regulatory factor (IRF), also called iron responsive element-binding protein (IRE-BP), is a cytoplasmic RNA-binding protein which regulates post-transcriptionally transferrin receptor mRNA stability and ferritin mRNA translation. By using the polymerase chain reaction (PCR) and the sequence published by Rouault et al. (1990) a probe was derived which permitted the isolation of three human IRF cDNA clones. Hybridization to genomic DNA and mRNA, as well as sequencing data indicated a single copy gene of about 40 kb specifying a 4.0 kb mRNA that translates into a protein of 98, 400 dalton. By in vitro transcription of a assembled DRF cDNA coupled to in vitro translation in a wheat germ extract, we obtained full sized DRF that bound specifically to a human ferritin IRE. in vitro translated DRF retained sensitivity to sulfhydryl oxidation by diamide and could be reactivated by β-mercaptoethanol in the same way as native placental IRF. An IRF deletion mutant shortened by 132 amino acids at the COOH-terminus was no longer able to bind to an IRE, indicating that this region of the protein plays a role in RNA recognition. Placental DRF has previously been shown to migrate as a doublet on SDS-polyacrylamide gels. After V8 protease digestion the heterogeneity was located in a 65/70 kDa NH 2 -terminal doublet. The liberated 31 kDa COOH-terminal polypeptide was found to be homogeneous by amino acid sequencing supporting the conclusion of a single IRF gene.

This content is only available as a PDF.

© 1992 Oxford University Press

I agree to the terms and conditions. You must accept the terms and conditions.

Submit a comment

Name

Affiliations

Comment title

Comment

You have entered an invalid code

Thank you for submitting a comment on this article. Your comment will be reviewed and published at the journal's discretion. Please check for further notifications by email.

Citations

Views

Altmetric

Metrics

Total Views 145

13 Pageviews

132 PDF Downloads

Since 1/1/2017

Citations

64 Web of Science

×

Email alerts

Citing articles via

More from Oxford Academic