Curr Genet (1990)17:473-479 Current Genetics ?9 Springer-Verlag 1990 Evolutionary conservation of transcriptional machinery between yeast and plants as shown by the efficient expression from the CaMV 35S promoter and 35S terminator H. Hirt, M. Kiigl, T. Murbacher, and E. Heberle-Bors Institute of Microbiology and Genetics, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria Received February 16, 1990 Summary. Complementation of fission yeast mutants by plant genomic libraries could be a promising method for the isolation of novel plant genes. One important prereq- uisite is the functioning of plant promoters and termina- tors in Schizosaccharomyces pombe and Saccharomyces cerevisiae. Therefore, we studied the expression of the bacterial fl-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 termina- tor is appropriately recognized leading to a polyadeny- lated 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. Interest- ingly, 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 dehy- drogenase) 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 tran- scription rates. Key words: Schizosaccharomyces pombe - Saccharomyces cerevisiae - CaMV 35S promoter - CaMV 35S termina- tor - Heterologous expression Introduction At present several approaches can be used to isolate plant genes with regulatory functions in plant growth or devel- Offprint requests to: H. Hirt opment. One method requires the construction of cDNA libraries from different developmental stages of a plant organ. Differential screening of these cDNA banks al- lows the isolation of stage-specific genes which are, how- ever, usually not the regulatory genes themselves. An- other method makes use of knowledge obtained from studies on other organisms, such as animals or lower eukaryotes. Specifically, heterologous probes, consisting of either gene fragments or synthetic oligonucleotides, are hybridized to plant gene banks. A third approach employs an isolation procedure that selects for func- tional, rather than for sequence, homology. Since the function of a protein appears to be much more conserved during evolution than its sequence, this method may be particularly attractive for the isolation of genes which cannot be obtained by cross-hybridization methods. Specific temperature-sensitive yeast mutants are comple- mented with either genomic or cDNA expression librar- ies. Yeast is ideally suited for this purpose, since many mutants are available and the respective genes have, in many cases, been biochemically defined. Furthermore, transformation and screening of large quantities of cells is experimentally easy. This method has been successfully employed for the isolation of the human CDC2 homo- logue (Lee and Nurse 1987). For this purpose, a human cDNA bank was cloned into an expression vector which contained the SV40 promoter. As the SV40 promoter is strongly recognized in S. pombe (Jones et al. 1985) the human cDNA clones could be expressed and comple- mented the mutant fission yeast cdc2 gene. For successful complementation with genomic plant libraries, several requirements must be fulfilled. These are proper tran- scription (i.e. selection of start site, processing of introns, termination and polyadenylation) and translation (in- cluding post-translational modifications) of the plant genes. Although the budding yeast S. cerevisiae has been shown to recognize some, but not all, of several plant promoters (Langridge etal. 1984; Cramer etal. 1985; Coraggio et al. 1986), this yeast seemed unsuitable for heterologous complementation, because of the unusual branch point requirement of a TACTAAC box in the introns.
