Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-05-18
2003-03-11
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S006120, C435S069100, C435S483000
Reexamination Certificate
active
06531289
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods and compositions for regulated expression of specific genes in
Saccharomyces cerevisiae
. The invention can be used to identify and clone genes of interest and to identify antifungal agents using high-throughput screening techniques. The invention also relates to the use of the
Saccharomyces cerevisiae
strains of the invention in the isolation and analysis of antifungal agents.
BACKGROUND OF THE INVENTION
The ability to regulate the expression of particular genes of interest is important for many purposes, including, for example, (i) investigation of the biological function of a particular gene product; (ii) design of variants of the gene product that are tailored for different ends; and (iii) identification of agents that influence the activity of the gene product, including, e.g., inhibitors or activators. The ease of performing genetic and molecular manipulations in
S. cerevisiae
has made it an extremely useful experimental organism for regulated expression of recombinant genes. However, many gene expression systems based on
S. cerevisiae
are limited in their applicability by (i) the degree of regulation that can be achieved, i.e., the extent to which genes can be turned on and off, as well as the timing of these events; (ii) the relative stability of certain gene products, which makes it difficult to quickly deplete the cell of a gene product; and (iii) potential metabolic side effects of the procedures used to trigger or initiate changes in gene expression.
Thus, there is a need in the art for
S. cerevisiae
expression systems in which gene expression can be tightly and efficiently regulated, with respect to both transcription of the gene and accumulation of the protein product.
SUMMARY OF THE INVENTION
The present invention encompasses yeast strains in which expression of a particular protein (the “subject” protein) can be tightly regulated. The invention provides
Saccharomyces cerevisiae
cells in which expression of the subject protein can be repressed by exogenous metal. These cells comprise, for example:
(i) a first gene encoding a transcriptional repressor protein, the expression of which has been placed under the control of a metal ion-responsive element, wherein expression of the repressor protein is stimulated by the addition of a metal ion to the growth medium of the cells;
(ii) a second gene encoding a subject protein, wherein expression of the subject protein is controlled by a promoter, the activity of which is inhibited by said repressor protein; and
(iii) a third gene encoding a biomineralization protein, wherein the third gene is inactivated and wherein inactivation of the third gene enhances the transcriptional response of the metal-responsive element to added metal ions.
In a preferred embodiment, the first gene is ROX1; the second gene is controlled by an ANB1 promoter; and the third gene is SLF1.
In another embodiment, the yeast cell comprises a fourth gene encoding a protein that targets ubiquitin-containing polypeptides for degradation, where the fourth gene is placed under the control of a metal ion-responsive element. In a preferred embodiment, the fourth gene is the UBR1 gene.
The invention further comprises yeast cells in which expression of the subject protein is stimulated by exogenous metal ions. These cells comprise:
(i) a first gene encoding a subject protein, wherein expression of the gene encoding the subject protein is under the control of a metal ion-responsive element and is stimulated by the addition of a metal ion to the growth medium of the cells; and
(ii) a second gene encoding a biomineralization protein, wherein the second gene is inactivated and wherein inactivation of the second gene enhances the transcriptional response of the metal-responsive element to added metal ions.
In a preferred embodiment, the metal-responsive element is the Sc3451 promoter and the second gene is SLF1.
In another aspect, the invention relates to a method for the introduction of a subject gene under the control a predetermined promoter DNA sequence into a yeast cell genome, comprising the steps of providing a shuffled gene fragment, where the fragment comprises a restriction enzyme cleavage sequence, ligating the shuffled gene fragment into a vector, where the ligation results in the shuffled gene fragment being operably linked to a predetermined transcriptional control DNA sequence, cutting the vector with a restriction enzyme specific for the restriction enzyme cleavage sequence to yield a linearized vector, and transforming a yeast cell with the linearized vector.
The invention also provides methods for repressing or activating expression of a gene encoding a subject protein in
S. cerevisiae
to a predetermined level, comprising culturing the strains described above in the presence of metal, wherein the metal is present at sufficient concentration to activate the metal-responsive element so as to achieve the predetermined level of repression or activation of the gene.
In a further embodiment, the invention also encompasses methods for generating the yeast strains of the invention comprising:
(a) generating a yeast cell comprising
(i) a first gene encoding a transcriptional repressor protein whose expression is under the control of a metal ion-responsive element, wherein expression of said first gene encoding said repressor protein is stimulated by the addition of a metal ion to growth medium of said yeast cell;
(ii) a second gene encoding a subject protein, wherein expression of said second gene encoding said subject protein is controlled by a transcriptional control sequence whose activity is inhibited by said repressor protein; and
(iii) a third gene encoding a biomineralization protein, wherein said third gene is inactivated and wherein inactivation of said third gene enhances transcriptional response of said metal ion-responsive element to metal ions in said growth medium of said yeast cell;
(b) culturing the yeast cell in a growth medium comprising metal ions, wherein said metal ions are present in sufficient concentration to activate said metal ion-responsive element to a level which will result in said predetermined level of repression of expression of said subject gene;
(c) assessing whether the rapid depletion of the second gene from the yeast cell leads to inhibition of cell growth or cell death.
In a preferred embodiment the identifies the target gene as an essential target gene.
In another embodiment the invention is directed to methods of screening a candidate antifungal compound for interaction with an essential target gene comprising:
(a) generating a regulated yeast strain comprising a regulated essential target gene of the invention;
(b) establishing a concentration of metal ion at which the growth or viability of the regulated yeast strain ceases;
(c) generating a serial dilution of metal ion in yeast growth media;
(d) culturing the regulated yeast strain in the serially diluted growth media, wherein the serial dilution leads to a dose-dependent modulation of expression of the regulated essential target yeast gene product;
(e) screening the serially diluted cultures for altered sensitivity of the strain to the candidate antifungal compound;
(f) determining the metal ion concentration present in a culture demonstrating altered sensitivity to the candidate antifungal compound;
(g) comparing the metal ion concentration of step (b) with the metal ion concentration of the culture determined in step (f), and identifying a candidate antifungal compound for which a lower concentration of metal ion is required to eliminate growth or viability in step (f) as compared to step (b).
The method of screening may screen a single candidate or a plurality of candidate antifungal compounds. When a plurality of candidates screeened, the screen is selected from the group consisting of screening together in a single assay and screening individually using multiple simultaneous individual detecting steps.
The invention also encompasses methods of rapidly cloning a DNA complementary
Bailey David A.
Bradley John D.
Moore Jeffrey B.
Thompson Craig M.
Wobbe C. Richard
Anadys Pharmaceuticals, Inc.
David R Preston & Associates
Ketter James
Loeb Bronwen M.
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