A set of epitope-tagging integration vectors for functional analysis in Saccharomyces cerevisiae.
Functional analysis of genes from Saccharomyces cerevisiae has been the major goal after determination of genome sequences. Even though several tools for molecular-genetic analyses have been developed, only a limited number of reliable genetic tools are available to support functional assay at protein level. Epitope tagging is a powerful tool for detecting, purifying, and functional studying of proteins. But systematic tagging systems developed with integration vectors are not available. Here, we have constructed a set of integration vectors allowing a translational fusion of interested proteins to the four different epitope tags (HA, Myc, Flag, and GFP). To confirm function and expression of C-terminal-tagged proteins, we used Cdc11, a component of the septin filament that encircles the mother bud neck and consists of five major proteins: Cdc3, Cdc10, Cdc11, Cdc12, and Sep7. The tagged version of Cdc11 expressed under its endogenous promoter was found to be physiologically functional, as evidenced by localization at the neck and suppression of the growth defect associated with the temperature-sensitive mutation of cdc11-6. The expressed proteins were efficiently detected with antibodies against Cdc11 or the epitopes. When immunoprecipitated with anti-Myc antibody, each septin protein tagged with Myc was effectively copurified with other septin components, indicating formation of a stable septin complex. Because the modules of the tags were located under the same array of eighteen restriction sites on integration vectors containing four different markers (HIS3, TRP1, LEU2, or URA3), this tagging system provides efficient multiple tagging and stable expression of a gene of interest.
Sung
Hyeran
H
College of Pharmacy, Chungbuk National University, 48 Gaeshindong, Cheongju, Chungbuk, Republic of Korea.
Chul Han
Kyung
K
Chul Kim
Jun
J
Wan Oh
Ki
K
Su Yoo
Hwan
H
Tae Hong
Jin
J
Bok Chung
Youn
Y
Lee
Chong-Kil
CK
Lee
Kyung S
KS
Song
Sukgil
S
D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism; D000939:Epitopes / Q000235:genetics*; D018390:Gene Targeting; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology
C099502:CDC11 protein, S cerevisiae; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D000939:Epitopes; D029701:Saccharomyces cerevisiae Proteins