Construction of an expression vector for the fission yeast Schizosaccharomyces pombe.
نویسندگان
چکیده
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.
منابع مشابه
A transcriptionally regulated expression vector for the fission yeast Schizosaccharomyces pombe.
An expression vector for the fission yeast Schizosaccharomyces pombe is described. The vector is designed to facilitate the construction of transcriptional fusions to the promoter of the S. pombe fructose bisphosphatase gene. Transcription from this promoter is regulated by glucose repression over a range of greater than 100-fold. The tight regulation by this promoter should allow for the maint...
متن کاملCassette for the generation of sequential gene disruptions in the yeast Schizosaccharomyces pombe.
The ability to conveniently construct gene disruptions is an important methodology for genetic analysis of the fission yeast Schizosaccharomyces pombe. Because of the limited number of selectable markers available for generating gene disruptions in fission yeast, the construction of strains that contain multiple gene disruptions can be quite difficult. This becomes a particular problem when epi...
متن کاملEffect of human immunodeficiency virus type 1 protein R (vpr) gene expression on basic cellular function of fission yeast Schizosaccharomyces pombe.
The human immunodeficiency virus type 1 (HIV-1) Vpr protein affects cell morphology and prevents proliferation of human cells by induction of cell cycle G2 arrest. In this study, we used the fission yeast Schizosaccharomyces pombe as a model system to investigate the cellular effects of HIV-1 vpr gene expression. The vpr gene was cloned into an inducible fission yeast gene expression vector and...
متن کاملComparative proteomic and transcriptomic profiling of the fission yeast Schizosaccharomyces pombe
The fission yeast Schizosaccharomyces pombe is a widely used model organism to study basic mechanisms of eukaryotic biology, but unlike other model organisms, its proteome remains largely uncharacterized. Using a shotgun proteomics approach based on multidimensional prefractionation and tandem mass spectrometry, we have detected approximately 30% of the theoretical fission yeast proteome. Apply...
متن کاملPlasmid Construction Using Recombination Activity in the Fission Yeast Schizosaccharomyces pombe
BACKGROUND Construction of plasmids is crucial in modern genetic manipulation. As of now, the common method for constructing plasmids is to digest specific DNA sequences with restriction enzymes and to ligate the resulting DNA fragments with DNA ligase. Another potent method to construct plasmids, known as gap-repair cloning (GRC), is commonly used in the budding yeast Saccharomyces cerevisiae....
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
- Nucleic acids research
دوره 16 17 شماره
صفحات -
تاریخ انتشار 1988