Biosynthetic labeling of RNA with uracil phosphoribosyltransferase allows cell-specific microarray analysis of mRNA synthesis and decay.
Standard microarrays measure mRNA abundance, not mRNA synthesis, and therefore cannot identify the mechanisms that regulate gene expression. We have developed a method to overcome this limitation by using the salvage enzyme uracil phosphoribosyltransferase (UPRT) from the protozoan Toxoplasma gondii. T. gondii UPRT has been well characterized because of its application in monitoring parasite growth: mammals lack this enzyme activity and thus only the parasite incorporates (3)H-uracil into its nucleic acids. In this study we used RNA labeling by UPRT to determine the roles of mRNA synthesis and decay in the control of gene expression during T. gondii asexual development. We also used this approach to specifically label parasite RNA during a mouse infection and to incorporate thio-substituted uridines into the RNA of human cells engineered to express T. gondii UPRT, indicating that engineered UPRT expression will allow cell-specific analysis of gene expression in organisms other than T. gondii.
Cleary
Michael D
MD
Meiering
Christopher D
CD
Jan
Eric
E
Guymon
Rebecca
R
Boothroyd
John C
JC
D000818:Animals; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D008657:Metabolic Clearance Rate; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010430:Pentosyltransferases / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013194:Staining and Labeling / Q000379:methods; D014122:Toxoplasma / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation / Q000502:physiology*
D012333:RNA, Messenger; D014157:Transcription Factors; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase