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Functional analysis of yeast essential genes using a promoter‐substitution cassette and the tetracycline‐regulatable dual expression system
Author(s) -
Bellí Gemma,
Garí Eloi,
Aldea Martí,
Herrero Enrique
Publication year - 1998
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/(sici)1097-0061(19980915)14:12<1127::aid-yea300>3.0.co;2-#
Subject(s) - biology , gene , homologous recombination , genetics , transformation (genetics) , homology (biology) , promoter , tetracycline , tata box , microbiology and biotechnology , gene expression , antibiotics
A promoter‐substitution cassette has been constructed that allows one‐step substitution of chromosomal gene promoters for the tetracycline‐regulatable tetO promoter in yeast cells, which uses kanMX4 as selective marker for geneticin resistance. Oligonucleotides for PCR amplification of the cassette are designed to allow homologous recombination through short flanking regions of homology with the upstream sequences of the chromosomal gene, upon transformation of target cells. By testing three essential genes of chromosome XV (YOL135c, YOL142w and YOL144w), the system causes tetracycline‐dependent conditional growth of the cells, being modulatable by intermediate concentrations of the effector. Analysis of terminal phenotypes of the promoter‐substituted cells in the presence of the antibiotic may facilitate functional analysis of essential orphan genes. © 1998 John Wiley & Sons, Ltd.