PolI-driven integrative expression vectors for yeast

• Published in: Journal of biotechnology Vol. 56; no. 1; pp. 41 - 47
• Main Authors: Blancafort, Pilar, Ferbeyre, Gerardo, Sariol, Carlos, Cedergren, Robert
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 23.07.1997; Amsterdam Elsevier; New York, NY
• Subjects: Biological and medical sciences; Biotechnology; Fundamental and applied biological sciences. Psychology; Gene expression; Genetic engineering; Genetic technics; Genetic Vectors; Methods. Procedures. Technologies; plasmid vectors; Ribosomal RNA; RNA Polymerase I - genetics; RNA, Messenger - metabolism; Vectors; Vectors (cloning, transfer, expression). Insertion sequences and transposons; Yeast; Yeasts - genetics; hh, hammerhead coding RNA domain; ETS, external transcribed spacer; BSA, bovine serum albumin; nt(s), nucleotide(s); Yeast; kb, kilobases; tADH1, alcohol dehydrogenase terminator of transcription; ITS, internal transcribed spacer; Vectors; rRNA, ribosomal RNA; Gene expression; ADH1, alcohol dehydrogenase gene; polIII, RNA polymerase III; E/T, enhancer/termination region of ribosomal DNA transcription unit; Ribosomal RNA; PAGE, polyacrylamide gel electrophoresis; rDNA, ribosomal DNA; polI, RNA polymerase I; polII, RNA polymerase II; Genetic transformation; Transcription; Enzyme; Transferases; DNA-directed RNA polymerase; Fungi; Promoter; Nucleotidyltransferases; Enhancer sequence; Polyadenylation; Ascomycetes; Saccharomyces cerevisiae; Vector; Thallophyta
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/S0168-1656(97)00078-3

A novel expression vector for yeast has been constructed from the regulatory elements present in the polI promoter and the enhancer/termination region (E/T) of rDNA. Under some conditions, this promoter/vector combination produces small RNAs such as the hammerhead RNA sequence at levels comparable to polII- and polIII-dependent systems. No stable transcription product can be demonstrated with this vector when the enhancer/termination sequence is less than 100 nucleotides downstream from the promoter. On the other hand, high expression of a stable, hammerhead RNA molecule can be obtained from this vector by inserting a 400-bp fragment containing the ADH1 transcription termination region upstream of the E/T. RNAs produced by this vector are polyadenylated and multiple copies of this plasmid can be stably integrated into the yeast chromosome.