Construction of an expression vector for the fission yeast Schizosaccharomyces pombe

• Published in: Nucleic acids research Vol. 16; no. 17; pp. 8603 - 8617
• Main Authors: KUDLA, B, PERSUY, M.-A, GAILLARDIN, C, HESLOT, H
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 12.09.1988
• Subjects: Amino Acid Sequence; Base Sequence; beta-Galactosidase - genetics; Biochemistry; Biochemistry, Molecular Biology; Biological and medical sciences; Cloning, Molecular; DNA Restriction Enzymes; Escherichia coli; Fundamental and applied biological sciences. Psychology; Genes; Genes, Fungal; Genetic Vectors; Kinetics; Life Sciences; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Plasmids; Promoter Regions, Genetic; Pseudomonas putida; Saccharomycetales - genetics; Schizosaccharomyces - enzymology; Schizosaccharomyces - genetics; Schizosaccharomyces pombe; Transcription. Transcription factor. Splicing. Rna processing; Yeast; Molecular structure; Nucleotide sequence; Enzyme; Transcription promoter; Escherichia coli; Method; β-D-Galactosidase; Gene expression; In vitro; Fungi; Plasmid; Enzymatic activity; Gene; Restriction map; Schizosaccharomyces pombe; Bacteria; Ascomycetes; Escherichieae; Vector; Enterobacteriaceae; Thallophyta
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/16.17.8603

We have isolated and characterized a S. pombe promoter using a functional heterologous gene product assay. Random S. pombe genomic fragments were cloned upstream from the promoterless 'lacZ gene and tested in vivo for their efficiency to promote expression of the beta-galactosidase protein in the fission yeast. An efficient S. pombe promoter called 54/1 was isolated and shown to drive up to 5% of total protein synthesis as beta-galactosidase. The structure and nucleotide sequence of this promoter were determined, precise localization of its mRNA transcriptional start points established. Translational fusion of the Pseudomonas putida XylE gene with the 54/1 gene was shown to allow expression of catechol oxidase activity in S. pombe. An expression vector suitable for transcriptional fusions was then constructed from engineered 54/1 promoter sequences and used to drive expression of the E. coli Tn5 ble gene, thus confering resistance to the fission yeast against bleomycin and phleomycin antibiotics.