Linked Life Data

11086160

Source:http://linkedlifedata.com/resource/pubmed/id/11086160

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2000-12-13
pubmed:abstractText
A novel rapid polymerase chain reaction (PCR)-based technique for N-terminal attachment of green fluorescent protein (GFP) to a yeast protein is described. Genomic integration of a PCR-generated loxPkanMX4loxP-yEGFP fusion cassette immediately upstream of the open reading frame (ORF) allows for selection of G418 resistant transformants carrying GFP fused N-terminally to the protein of interest. In a subsequent step, the loxPkanMX4loxP selection marker that is inserted between the tagged ORF and the endogenous promoter is excised upon site-specific recombination between the loxP sites by Cre recombinase, leaving behind in the promoter one loxP site, immediately upstream of the GFP start codon. The essential protein Ydl193wp of unknown function and the oleate-inducible fatty acid activation protein, encoded by FAA2, were N-terminally tagged using the novel technique. Both experiments yielded viable haploid strains with growth phenotypes indistinguishable from the wild type strain. The subcellular localization pattern for the chromosomally expressed GFP-Ydl193wp to the endoplasmic reticulum and lipid particles was identical to the pattern observed for a plasmid-borne GFP construct expressed under control of the MET25(p) promoter, albeit at a lower level and with a more homogeneous distribution among the cell population. GFP-FAA2 was inducible by oleate, as is the wild type gene, demonstrating that specific expression patterns are not grossly affected by the promoter manipulation. In agreement with previous reports, GFP-Faa2p was found to localize to peroxisomes.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
0014-5793
pubmed:author
pubmed:issnType
Print
pubmed:day
17
pubmed:volume
485
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
29-34
pubmed:dateRevised
2008-11-21
pubmed:meshHeading
pubmed:year
2000
pubmed:articleTitle
A novel strategy for constructing N-terminal chromosomal fusions to green fluorescent protein in the yeast Saccharomyces cerevisiae.
pubmed:affiliation
SFB Biomembrane Research Center, Department of Biochemistry, Technical University Graz, Petersgasse 12/2, A-8010 Graz, Austria.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't