Multipurpose vectors designed for the fast generation of N‐ or C‐terminal epitope‐tagged proteins
- 29 January 1994
- Vol. 10 (1) , 105-112
- https://doi.org/10.1002/yea.320100110
Abstract
In this paper are described a set of new high‐copy‐number yeast vectors, which are specially designed for the conditional expression of epitope‐tagged proteins in vivo. One of the major advantages of these plasmids is that they allow polymerase chain reaction‐amplified open reading frames to be automatically fused in frame with the epitope‐coding sequence, avoiding longer procedures such as site‐directed mutagenesis. This heterologous construction can be realized either at the 5′‐end of the coding sequence, in the pYeF1 vector, or at its 3′‐end, in pYeF2, generating N‐ or C‐terminal tagged proteins, respectively. Moreover, to increase the usefulness of the method, derivatives of the two basic URA3‐borne pYeF1 and pYeF2 were constructed, carrying either the HIS3 or TRP1 gene as a marker of selection. These vectors could be of use for the purpose of functional analysis of the newly discovered genes resulting from the systematic sequencing of the yeast genome. Here, we present results showing the functional expression and the efficient immunoprecipitation of the epitope‐tagged Rna15 protein, which is involved in Saccharomyces cerevisiae mRNA stability.Keywords
This publication has 24 references indexed in Scilit:
- [10] 5-Fluoroorotic acid as a selective agent in yeast molecular geneticsPublished by Elsevier ,2004
- The yeast PRP19 protein is not tightly associated with small nuclear RNAs, but appears to associate with the spliceosome after binding of U2 to the pre-mRNA and prior to formation of the functional spliceosome.Molecular and Cellular Biology, 1993
- Two distinct sequences control the targeting and anchoring of the mouse P450 1A1 into the yeast endoplasmic reticulum membraneBiochemical and Biophysical Research Communications, 1992
- A family of low and high copy replicative, integrative and single‐stranded S. cerevisiae/E. coli shuttle vectorsYeast, 1991
- Mutations in the yeast RNA14 and RNA15 genes result in an abnormal mRNA decay rate; sequence analysis reveals an RNA-binding domain in the RNA15 protein.Molecular and Cellular Biology, 1991
- The yeast PRP6 gene encodes a U4/U6 small nuclear ribonucleoprotein particle (snRNP) protein, and the PRP9 gene encodes a protein required for U2 snRNP binding.Molecular and Cellular Biology, 1990
- RNA polymerase II subunit RPB3 is an essential component of the mRNA transcription apparatus.Molecular and Cellular Biology, 1989
- Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method.Molecular and Cellular Biology, 1988
- A mitochondrial RNA maturase gene transferred to the yeast nucleus can control mitochondrial mRNA splicingCell, 1986
- A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistanceMolecular Genetics and Genomics, 1984