Vectors for transcription factor cloning and target site identification by means of genetic selection in yeast.
We describe the construction of a number of vectors that can be used in yeast genetic selection systems for cloning of transcription factors or other DNA-binding proteins and for identification of the target sites recognized by transcription factors. For transcription factor cloning we have designed an integration vector with two HIS3 reporter gene cassettes to stably integrate reporter gene constructs at the non-essential yeast PDC6 locus. This set of plasmids was tested in a one-hybrid assay with the rice transcription factor Oshox1, a member of the class of homeodomain leucine zipper proteins. A hybrid protein of Oshox1 and the Gal4 transcriptional activation domain was shown to specifically activate a reporter gene construct with upstream Oshox1 binding sites, which had been integrated at the PDC6 locus using the described vector system. Target site identification by genetic selection in yeast employs a transcriptional activator construct and a library of genomic or random DNA fragments upstream of a reporter gene. We have constructed two variants of a bacteriophage lambda vector which facilitates the construction of the required reporter gene library because of high cloning efficiency and easy conversion into a yeast/Escherichia coli shuttle vector library by Cre-loxP-mediated automatic subcloning. Tests with Oxhox1 as transcriptional activator demonstrated the usefulness of the deprived reporter gene vector.
Meijer
A H
AH
Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, The Netherlands. meijer@rulbim.leidenuniv.nl
Ouwerkerk
P B
PB
Hoge
J H
JH
D010582:Bacteriophage lambda / Q000235:genetics; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics; D015723:Gene Library; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D018398:Homeodomain Proteins / Q000235:genetics; D010940:Plant Proteins; D010957:Plasmids; D029701:Saccharomyces cerevisiae Proteins; D012641:Selection, Genetic; D014157:Transcription Factors / Q000235:genetics*; D015533:Transcriptional Activation; D015003:Yeasts / Q000235:genetics*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D018398:Homeodomain Proteins; C106325:Oshox1 protein, Oryza sativa; D010940:Plant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors