Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-12-10
2001-05-15
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S483000, C435S254110, C435S254210
Reexamination Certificate
active
06232074
ABSTRACT:
CROSS-REFERENCES TO RELATED APPLICATIONS
Not Applicable.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
Not Applicable.
FIELD OF THE INVENTION
This invention relates to functional gene arrays of yeast. Novel aspects include the individual yeast cells, methods for making the yeast and the arrays, the arrays and uses for the arrays. A diploid bearing special genetic properties has been constructed to facilitate cloning of heterologous genes capable of providing essential functions. A selection method, using this strain allows the identification haploid yeast strains dependent for life on heterologous essential genes. The arrays of these strains comprise a library of unique members where each member is dependent for survival on the function of a heterologous gene complementing a different essential host gene which has been inactivated by the insertion of a dominant selectable marker. These arrays provide screening platforms for agents that specifically target the activity of these heterologous genes.
BACKGROUND OF THE INVENTION
The understanding of the interaction of extraneous molecules with the machinery of a living cell is a central topic for all science and industrial activities where one desires to effect a change on a biological system by treatment with a chemical. This includes basic cellular research, pharmaceutical discovery, toxicology, agricultural sciences and environmental testing. The interaction can be studied at several levels of intricacy, however one is usually interested in relating the gross effect on a biological system with the molecular interactions that cause the effect. The usual mode of approaching this problem has been to identify a chemical that has an affect on an organism, then find the cellular molecule with which the chemical interacts. Alternatively, one can isolate cellular molecules, find chemicals that interact with them either by measuring binding of the two or by measuring a change in the function of the cellular molecule by the chemical. The chemical is then tested for its affect on the cell or organism as a whole. In an industry where discovery of the biological effect of chemicals at the molecular level is central to the discovery of useful compounds, the process of identifying biologically active compounds and their cellular “targets”, becomes a key economic factor. Systems, materials and techniques that increase the efficiency of this process are useful and valuable.
The development of new techniques of measuring interaction between chemicals and biological molecules such as proteins, nucleic acids and membrane lipids has lead to the application of systems with the capacity for assaying many reactions simultaneously. These systems, referred to as high through put (HTP) systems, although automated and rapid, still demand either choosing the type of assay based on the known characteristics of the cellular target, or isolating the cellular target so that a physical interaction between the target and the chemical compound can be measured. Here we disclose a genetic system for generating ordered arrays of hundreds of functional cellular targets which can be used to assay the biological activities of large numbers of chemical compounds, with no prior understanding of the function of the chemical compound, nor prior choice of type of target to be assayed. The system can be used to both determine if a compound has an activity against one of the targets and identify the target.
The genetic system employs the yeast,
Saccharomyces cerevisiae.
The genetics of the organism are better understood than any other eukaryote and it is relatively easily manipulated genetically. The organism can grow either as a diploid with 16 pairs of homologous chromosomes, or after undergoing sporulation, as a haploid having only one member of each chromosome pair.
Saccharomyces cerevisiae
has long served as a useful model for the analysis of eukaryotic gene expression and as a system to study the function of genes isolated from other organisms (heterologous genes) since yeast can be efficiently transformed by DNA molecules consisting of circular or linear plasmids carrying foreign genes under the control of a yeast transcription promoter. In some cases the protein product of the heterologous gene can functionally replace a missing or mutated yeast protein. This type of functional complementation has been used to identify and study genes from more complex organisms, such as higher plants and humans, since yeast can be much more easily grown than higher organisms or their cells. However, it requires that the yeast be made dependent on the foreign gene to grow. Current methods to do this are tedious and require cloning of the yeast gene to be replaced, as well as a complex selection procedure.
The elucidation of the function of many yeast genes followed the completion of the nucleotide sequence in 1996. A systematic study in which each open reading frame (orf) is deleted and the growth the haploid yeast carrying the deletion is measured has thus far revealed that of the some 6200 genes about 20% appear to be essential for growth.
The present invention provides for construction of yeast strains in which selection for haploid cells containing inactivated essential genes allows the simultaneous selection of strains in which the inactivated essential gene is replaced by a heterologous gene which may support growth or other critical cell functions. This invention also provides for the construction of arrays of such strains in which each member of the array contains a heterologous gene upon which that strain is dependent. These arrays in turn serve as platforms for the identification of chemical compounds that affect the function of heterologous genes expressed by individual members of the array. The array is therefore a system that can be used for both determining if a chemical compound is biologically active and identifying the target of the activity.
SUMMARY OF THE INVENTION
This invention provides for a stable haploid population of yeast cells having at least two selecting markers and a recombinant plasmid where: (a) the first marker is a dominant selectable marker wherein said marker is recombinantly inserted into an essential yeast gene rendering that essential gene inactive; (b) the second marker is a defined haploid-selecting recessive marker; and (c) the recombinant plasmid comprises a functional gene that complements the essential gene inactivated by the dominant selectable marker. A preferred species of yeast is
Saccharomyces cerevisiae.
Preferred dominant selectable markers are selected from the group consisting of: KAN, LEU2, LYS2, URA3, TRP1, HIS3 and ADE2. The preferred defined haploid-selecting recessive (resistant) marker are those selectable markers having a corresponding dominant counter selectable (sensitive) allele in the parental diploid yeast. The recessive markers are preferrably created by insertion, deletion or point mutation which result in recessive selectable phenotypes. The preferred haploid-selecting recessive markers are can1, cyh2-1, lys2, met15 and ura3. The preferred dominant counter selectable (sensitive) allele is a wild type allele selected from the group consisting of CAN1, CYH2, LYS2, MET15 and URA3. Using this method in a preferred manner one can create a haploid yeast population wherein the diploid population is less than one in 1,000,000. It is further preferred that the population carries a functional gene on the plasmid that is a heterologous gene from a non-
Saccharomyces cerevisiae
organism.
In an alternative preferred population of haploid yeast, the population has a multiplicity of different dominant selectable markers inserted into more than one gene. A second and different defined haploid-selecting recessive marker having a corresponding dominant counter selectable (sensitive) allele in the parental diploid yeast may also be used to ensure and maintain high levels of haploidy in the population.
In a preferred embodiment the populations of haploid yeast are arranged as an array
Dawson Dean
Swindle John
CompleGen, Inc.
Ketter James
Townsend and Townsend / and Crew LLP
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