Evolutionary conservation of transcriptional machinery between yeast and plants as shown by the efficient expression from the CaMV 35S promoter and 35S terminator.
Complementation of fission yeast mutants by plant genomic libraries could be a promising method for the isolation of novel plant genes. One important prerequisite is the functioning of plant promoters and terminators in Schizosaccharomyces pombe and Saccharomyces cerevisiae. Therefore, we studied the expression of the bacterial beta-glucuronidase (GUS) reporter gene under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter and 35S terminator. We show here that S. pombe initiates transcription at exactly the same start site as was reported for tobacco. The 35S CaMV terminator is appropriately recognized leading to a polyadenylated mRNA of the same size as obtained in plant cells transformed with the same construct. Furthermore, the GUS-mRNA is translated into fully functional GUS protein, as determined by an enzymatic assay. Interestingly, expression of the 35S promoter in the budding yeast S. cerevisiae was found to be only moderate and about hundredfold lower than in S. pombe. To investigate whether different transcript stabilities are responsible for this enormous expression difference in the two yeasts, the 35S promoter was substituted by the ADH (alcohol dehydrogenase) promoter from fission yeast. In contrast to the differential expression pattern of the 35S promoter, the ADH promoter resulted in equally high expression rates in both fission and budding yeast, comparable to the 35S promoter in S. pombe. Since the copy number of the 35S-GUS constructs differs only by a factor of two in the two yeasts, it appears that differential recognition of the 35S promoter is responsible for the different transcription rates.
Institute of Microbiology and Genetics, University of Vienna, Austria.
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Hirt
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Kögl
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Murbacher
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Heberle-Bors
D000426:Alcohol Dehydrogenase / Q000235:genetics; D005075:Biological Evolution; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D015966:Gene Expression Regulation, Fungal; D005966:Glucuronidase / Q000235:genetics; D009029:Mosaic Viruses / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D012260:Ribonucleases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D004718:Saccharomycetales / Q000235:genetics*; D012568:Schizosaccharomyces / Q000235:genetics*; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic
D000426:Alcohol Dehydrogenase; D012260:Ribonucleases; D005966:Glucuronidase