Heat-Inducible degron and the generation of conditional mutants:
A method was developed that allows for a rapid design-based generation of temperature sensitive mutants in Saccharomyces cerevisiae. The method employs
a temperature-inducible degron (td),
which, when linked to the N terminus of a protein to be studied,
targets it for rapid degradation via the ubiquitin-dependent N-end
rule pathway. The degron, however, is only active at elevted
("restrictive") temperatures whereas it is inactive at lower
("permissive") temperatures. The principle of the method as it was originally described in Dohmen, Wu and Varshavsky 1994, and reviewed in Dohmen and Varshavsky 2005, and Dohmen 2006 is illustrated in the following figure.
The method was used to generate a td allele of CDC28 using the
integrative plasmid pPW66R. This pRS306-based integrative plasmid (Sikorski and
Hieter 1989) contains an insert encoding PCUP1-Ub-R-DHFR-td-ha-CDC28(1-) (Sequence of pPW66R). The strategy to generate a genomic td allele by plasmid integration is outlined below. Another available plasmid is pPW58, a pUC19 based plasmid containing PCUP1-Ub-R-DHFR-td-ha (Sequence of pPW58).
A second PCR-based strategy described in Dohmen and Varshavsky 2005 employs pJDtd1 (map), a pUC19 based plasmid (Sequence of pJDtd1 insert) as a template. This template allows for an attachment of targeting sequences to a Ub--URA3--PCUP1-Ub-R-DHFR-td-ha
cassette. Homologous recombination via these target sequences
will direkt this cassett to a target gene of interest. As a result,
a PCUP1 driven td
allele of the target gene is generated. Recombination between the
two Ub modules will result in FOA resistant colonies, in which the td allele of the target gene is controlled by its native promoter as outlined in the figure below.