This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pan, W. J. Articles by Blackburn, E. H. Search for Related Content PubMed PubMed Citation Articles by Pan, W. J. Articles by Blackburn, E. H.

 Previous Article  |  Next Article 

Mol. Cell. Biol., 06 1995, 3372-3381, Vol 15, No. 6
Copyright © 1995, American Society for Microbiology

Replication of an rRNA gene origin plasmid in the Tetrahymena thermophila macronucleus is prevented by transcription through the origin from an RNA polymerase I promoter

WJ Pan, RC Gallagher and EH Blackburn
Department of Microbiology and Immunology, University of California, San Francisco 94143-0414, USA.

In the somatic macronucleus of the ciliate Tetrahymena thermophila, the palindromic rRNA gene (rDNA) minichromosome is replicated from an origin near the center of the molecule in the 5' nontranscribed spacer. The replication of this rDNA minichromosome is under both cell cycle and copy number control. We addressed the effect on origin function of transcription through this origin region. A construct containing a pair of 1.9-kb tandem direct repeats of the rDNA origin region, containing the origin plus a mutated (+G), but not a wild type, rRNA promoter, is initially maintained in macronuclei as an episome. Late, linear and circular replicons with long arrays of tandem repeats accumulate (W.-J. Pan and E. H. Blackburn, Nucleic Acids Res, in press). We present direct evidence that the +G mutation inactivates this rRNA promoter. It lacks the footprint seen on the wild-type promoter and produces no detectable in vivo transcript. Independent evidence that the failure to maintain wild-type 1.9-kb repeats was caused by transcription through the origin came from placing a short DNA segment containing the rRNA gene transcriptional termination region immediately downstream of the wild-type rRNA promoter. Insertion of this terminator sequence in the correct, but not the inverted, orientation restored plasmid maintenance. Hence, origin function was restored by inactivating the rRNA promoter through the +G mutation or causing termination before transcripts from a wild-type promoter reached the origin region. We propose that transcription by RNA polymerase I through the rDNA origin inhibits replication by preventing replication factors from assembling at the origin.

This article has been cited by other articles:

• Mohammad, M., York, R. D., Hommel, J., Kapler, G. M. (2003). Characterization of a Novel Origin Recognition Complex-Like Complex: Implications for DNA Recognition, Cell Cycle Control, and Locus-Specific Gene Amplification. Mol. Cell. Biol. 23: 5005-5017 [Abstract] [Full Text]  
• Lunyak, V. V., Ezrokhi, M., Smith, H. S., Gerbi, S. A. (2002). Developmental Changes in the Sciara II/9A Initiation Zone for DNA Replication. Mol. Cell. Biol. 22: 8426-8437 [Abstract] [Full Text]  
• Chen, P.-H., Tseng, W.-B., Chu, Y., Hsu, M.-T. (2000). Interference of the Simian Virus 40 Origin of Replication by the Cytomegalovirus Immediate Early Gene Enhancer: Evidence for Competition of Active Regulatory Chromatin Conformation in a Single Domain. Mol. Cell. Biol. 20: 4062-4074 [Abstract] [Full Text]  
• Mohammad, M., Saha, S., Kapler, G. M. (2000). Three different proteins recognize a multifunctional determinant that controls replication initiation, fork arrest and transcription in Tetrahymena. Nucleic Acids Res 28: 843-851 [Abstract] [Full Text]  
• Gallagher, R. C., Blackburn, E. H. (1998). A Promoter Region Mutation Affecting Replication of the Tetrahymena Ribosomal DNA Minichromosome. Mol. Cell. Biol. 18: 3021-3033 [Abstract] [Full Text]  
• Hyrien, O., Maric, C., Méchali, M. (1995). Transition in Specification of Embryonic Metazoan DNA Replication Origins. Science 270: 994-997 [Abstract]  
• Saha, S., Nicholson, A., Kapler, G. M. (2001). Cloning and Biochemical Analysis of the Tetrahymena Origin Binding Protein TIF1. COMPETITIVE DNA BINDING IN VITRO AND IN VIVO TO CRITICAL rDNA REPLICATION DETERMINANTS. J. Biol. Chem. 276: 45417-45426 [Abstract] [Full Text]