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Nucleic Acids Research, 2000, Vol. 28, No. 6 1428-1438
© 2000 Oxford University Press

Functional {alpha}-fragment of ß-galactosidase can be expressed from the mobile group I intron PpLSU3 embedded in yeast pre-ribosomal RNA derived from the chromosomal rDNA locus

Jue Lin and Volker M. Vogt*

Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853, USA

PpLSU3, a mobile group I intron found in the ribo­somal RNA genes of Physarum polycephalum, encodes the I-PpoI homing endonuclease. This enzyme represents one of the rare cases in nature where a protein is expressed from an RNA polymerase I transcript. Our previous results showed that the full length intron, but not a further processed species, is the messenger for I-PpoI, implying a role of the untranslated region (UTR) in gene expression. To study the function of the 3'-UTR in expression of the endonuclease and in splicing of the intron, we replaced the I-PpoI gene in PpLSU3 with the gene for the {alpha}-fragment of Escherichia coli ß-galactosidase, and then integrated this chimeric intron into all the chromosomal rDNA repeats of yeast. The resulting cells synthesized functional {alpha}-fragment, as evidenced by a complementation assay analogous to that used in E.coli. The ß-galactosidase activity thus provides an unusual and potentially valuable readout for Pol I transcription from chromosomal rDNA. This is the first example in which a eucaryotic homing endonuclease gene has been successfully replaced by a heterologous gene. Using deletion mutagenesis and a novel rando­mization approach with the {alpha}-fragment as a reporter, we found that a small segment of the 3'-UTR dramatically influences both splicing and protein expression.

* To whom correspondence should be addressed. Tel: +1 607 255 2443; Fax: +1 607 255 2428; Email: vmv1@cornell.edu


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