Advertisement

RNA Analysis by Biosynthetic Tagging Using 4-Thiouracil and Uracil Phosphoribosyltransferase

  • Gusti M. Zeiner
  • Michael D. Cleary
  • Ashley E. Fouts
  • Christopher D. Meiring
  • Edward S. Mocarski
  • John C. Boothroyd
Part of the Methods In Molecular Biology™ book series (MIMB, volume 419)

Summary

RNA analysis by biosynthetic tagging (RABT) enables sensitive and specific queries of (a) how gene expression is regulated on a genome-wide scale and (b) transcriptional profiling of a single cell or tissue type in vivo. RABT can be achieved by exploiting unique properties of Toxoplasma gondii uracil phosphoribosyltransferase (TgUPRT), a pyrimidine salvage enzyme that couples ribose-5-phosphate to the N1 nitrogen of uracil to yield uridine monophosphate (UMP). When 4-thiouracil is provided as a TgUPRT substrate, the resultant product is 4-thiouridine monophosphate which can, ultimately, be incorporated into RNA. Thio-substituted nucleotides are not a natural component of nucleic acids and are readily tagged, detected, and purified with commercially available reagents. Thus, one can do pulse/chase experiments to measure synthesis and decay rates and/or use cell-specific expression of the TgUPRT to tag only RNA synthesized in a given cell type. This chapter updates the original RABT protocol (1) and addresses methodological details associated with RABT that were beyond the scope or space allotment of the initial report.

Key Words

RABT 4-thiouracil 4-thiouridine monophosphate microarray gene regulation cell-specific profiling RNA synthesis decay RNA purification Toxoplasma gondii cre lox 

Notes

Acknowledgments

We thank Sean Curran and Tammy Doukas for helpful suggestions and comments during the course of this work. G.M.Z. was supported by the NIH (5F32AI066538). M.D.C. was supported by the NIH (CMB GM07276) and the University of California Universitywide AIDS Research Program (D02-ST-405). C.D.M. was supported by the NIH (5T32AI07328 and 1F32AI056959). A.E.F. was supported by a Stanford Graduate Fellowship and Cell and Molecular Training Grant fellowship (GM07276). E.S.M. was supported by the NIH (AI30363 and AI20211). J.C.B. was supported by the NIH (AI41014 and AI21423).

References

  1. 1.
    Cleary, M. D., Meiering, C. D., Jan, E., Guymon, R., and Boothroyd, J. C. (2005) Biosynthetic labeling of RNA with uracil phosphoribosyltransferase allows cell-specific microarray analysis of mRNA synthesis and decay. Nat Biotechnol 23, 232–7.CrossRefPubMedGoogle Scholar
  2. 2.
    Iltzsch, M. H. (1993) Pyrimidine salvage pathways in Toxoplasma gondii. J Eukaryot Microbiol 40, 24–8.CrossRefPubMedGoogle Scholar
  3. 3.
    Pfefferkorn, E. R., and Pfefferkorn, L. C. (1977) Specific labeling of intracellular Toxoplasma gondii with uracil. J Protozool 24, 449–53.PubMedGoogle Scholar
  4. 4.
    Iltzsch, M. H., and Tankersley, K. O. (1994) Structure-activity relationship of ligands of uracil phosphoribosyltransferase from Toxoplasma gondii. Biochem Pharmacol 48, 781–92.CrossRefPubMedGoogle Scholar
  5. 5.
    Donald, R. G., and Roos, D. S. (1995) Insertional mutagenesis and marker rescue in a protozoan parasite: cloning of the uracil phosphoribosyltransferase locus from Toxoplasma gondii. Proc Natl Acad Sci USA 92, 5749–53.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Gusti M. Zeiner
    • 1
  • Michael D. Cleary
    • 1
  • Ashley E. Fouts
    • 1
  • Christopher D. Meiring
    • 1
  • Edward S. Mocarski
    • 1
  • John C. Boothroyd
    • 1
  1. 1.Department of Microbiology and ImmunologyStanford University School of MedicineStanfordUSA

Personalised recommendations