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US20030044866A1 - Yeast arrays, methods of making such arrays, and methods of analyzing such arrays - Google Patents

Yeast arrays, methods of making such arrays, and methods of analyzing such arrays Download PDF

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US20030044866A1
US20030044866A1 US09/930,593 US93059301A US2003044866A1 US 20030044866 A1 US20030044866 A1 US 20030044866A1 US 93059301 A US93059301 A US 93059301A US 2003044866 A1 US2003044866 A1 US 2003044866A1
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yeast
starting
strains
protein
gene
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Charles Boone
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Priority to US09/930,593 priority Critical patent/US20030044866A1/en
Priority to PCT/IB2002/004152 priority patent/WO2003016568A2/fr
Priority to CA002459450A priority patent/CA2459450A1/fr
Priority to EP02794826A priority patent/EP1436625A2/fr
Priority to US10/219,682 priority patent/US7074584B2/en
Priority to AU2002356001A priority patent/AU2002356001A1/en
Publication of US20030044866A1 publication Critical patent/US20030044866A1/en
Priority to US11/484,201 priority patent/US20080287317A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor

Definitions

  • the present invention relates generally to genomics and proteomics. More specifically, it relates to high density output arrays of multiple yeast strains, methods of making the high density output arrays, and methods of using the high density output arrays for functional analysis of genetic and protein-protein interactions.
  • proteomics One of the major goals of the emerging field of proteomics is the establishment of relationships between protein function and particular diseases. Proteomic technologies are used to try to identify important genes and their related proteins implicated in diseases and their treatments and to understand the role these genes and their related proteins play in the onset and progression of disease. A major proteomics challenge is to determine the set of proteins expressed in the cell and the interactions between such proteins, which in turn define the functional pathways of the cell. If a given pathway is linked to a disease, then the proteins within the pathway or a functionally related pathway may represent drug targets for treatment of the disease.
  • yeast arrays of the present invention provide crucial insights into the gene function problem in all eukaryotes.
  • Such synthetic lethal analysis can be performed on the yeast arrays of the present invention.
  • the high density yeast output arrays and the methods of analyzing such arrays of the present invention therefore fulfill a need in the art by providing simple and efficient methods for large-scale, high throughput analysis of genetic and protein-protein interactions.
  • the invention is directed to compositions and methods for performing large-scale analysis of genetic and protein interactions.
  • a high-density output array of multiple resulting yeast strains is constructed.
  • Each resulting yeast strain in the output array contains at least one resulting genetic alteration different from the genetic alterations in the other resulting yeast strains in the output array.
  • the resulting yeast strains in the output array are mating products of at least two input arrays. At least one of the input arrays comprises multiple starting strains of yeast, each carrying at least one genetic alteration, with the genetic alteration being different in each starting yeast strain.
  • the starting and resulting yeast strains are selected from any yeast strain that has two mating types and is capable of mating and meiotic and mitotic reproduction. Examples of species that have such strains are Saccharomyces cerevesiae and Schizosaccharomyces pombe.
  • the input and output arrays are arranged on plates, with between about 96 and about 6144 yeast colonies on one plate, and much higher densities, over 10-fold higher, can be achieved if individual colonies are pooled.
  • the resulting strains in the output array are double mutants.
  • Two different types of output arrays are created, one in which the phenotypes associated with mutations (genetic alterations) are examined within a diploid cell formed by mating the strains on the input arrays and another in which mutations are examined within the context of a haploid cell following sporulation of the diploids.
  • the two mutations can involve a mutation of two different endogenous yeast genes. These yeast genes can be non-essential yeast genes.
  • the entire output arrays can comprise between about 1,000 and about 25 million resulting strains of yeast, or between about 1 million and about 25 million resulting yeast strains.
  • the input array contains starting yeast strains with starting genetic alterations in at least one starting yeast strain.
  • the genetic alterations can be of any of the following type: (i) an alteration in the DNA encoding the gene such as a deletion or mutation of an endogenous essential or non-essential yeast gene; (ii) trans-dominant genetic agents such as genes coding for nucleic acid or peptide aptamers, dominant-negative proteins, antibodies, small molecules, natural products, ribozymes, enzymes, RNAi, and antisense RNA or DNA; (iii) protein and RNA expression vectors of a heterologous gene from a viral, prokaryotic, or eukaryotic genome, wherein the genes can either be wild type, mutated or fragmented (e.g.
  • a protein-protein interaction detection system including expression plasmids coding for a two-hybrid interaction and reporter that registers the interaction, the Ras recruitment system, the split-ubiquitin system, and various other protein fragment complementation systems (e.g. DHR); and (v) a reporter whose expression reflects a change in cellular state such as the activation or the inhibition of a pathway(s).
  • the genetic alterations can be integrated into the yeast genome or propagated on autonomously replicating plasmids.
  • the aptamer can be either a peptide aptamer or a nucleic acid aptamer. It can either inhibit or enhance expression of genes, protein interactions, or the activity of a protein or any other cellular component.
  • the heterologous gene can be a human gene that can be a single nucleotide polymorphism of another human gene.
  • a high-density output array of resulting multiple yeast strains where each resulting yeast strain carries at least one resulting genetic alteration, and the resulting genetic alterations are different in each yeast strain, is constructed through the method disclosed below.
  • Multiple starting yeast strains are generated, each strain carrying a starting genetic alterations.
  • Sets of two starting yeast strains, each of the two sets containing a different starting genetic alteration; are then mated.
  • the mated strains are then made to undergo sporulation, resulting in haploid spore progeny.
  • a single mating type is then germinated and the haploid spore progeny is cultured using selective growth criteria.
  • Multiple haploid yeast strains which carry a resulting genetic alteration which is a combination of at least two starting genetic alterations are selected for through this process.
  • the genetically altered yeast strains are then arrayed in a high-density format on an output array.
  • strains, plates, and genetic alterations used in this embodiment are similar to the previous embodiment.
  • the output array described in this embodiment could also be used to perform synthetic lethal analysis as described in the previous embodiment.
  • Yet another embodiment of the invention is a method for conducting small molecule screening of yeast colonies using a high-density input array of multiple starting yeast strains.
  • the method is carried out by generating an input array containing multiple starting yeast strains as described above. Then exposing this array to at least one biological effector, and detecting change in phenotype in the starting strains in response to the effector.
  • the effector can be a small molecule or any other biological effector.
  • the input array and genetic alterations can be the same array described in previous embodiments.
  • Yet another embodiment of the invention is a method for conducting synthetic lethal analysis of yeast colonies by producing an input array of starting strains, and crossing the starting strains in that array with other starting strains or another input array. Then the diploid resultant strains are studied for changes in phenotype due to the combination of different genetic or chemical alterations.
  • the input arrays are constructed as detailed in the embodiment above.
  • the genetic alterations can be the same as the ones in the embodiments above.
  • Chemical alterations can include biological effectors such as the ones described in the previous embodiment.
  • Yet another embodiment of the invention is a method to perform synthetic lethal analysis of yeast colonies of multiple yeast strains using DNA bar coding.
  • starting strains are constructed as described above, but not placed into arrays.
  • Each of the genetic alterations has a distinct DNA tag associated with it. This tag can be a 20 nucleotide long DNA sequence associated with a certain genetic alteration.
  • These starting strains are mated with other starting strains of a different mating type, which generates the first output strain set, and then stimulated to undergo sporulation, which allows for selected growth of haploid spore progeny that possess of both genetic alterations and generates the second output strain set.
  • the resulting output strains are then studied and isolated through their genetic tags, dispensing with the need to array each strain.
  • the genetic alterations used in this embodiment can be the same alterations used in the previous embodiments.
  • FIG. 1 illustrates the series of replica pinning procedures in which mating and meiotic recombination are used to generate haploid double mutants.
  • FIG. 2A illustrates the array of bni1 ⁇ double mutants resulting from the final pinning and the corresponding wild-type control.
  • FIG. 2B illustrates that both the bni1 ⁇ bnr1 ⁇ and bni1 ⁇ cla4 ⁇ double-mutants were inviable and that the bni1 ⁇ bud6 ⁇ double-mutant was associated with a slower growth rate or “synthetic sick” phenotype, reflecting reduced fitness of the double-mutant relative to the respective single mutants.
  • FIG. 3 illustrates a genetic network constructed from the interactions listed in Table 1.
  • a genetic interaction network representing the synthetic lethal/sick interactions is determined by the synthetic genetic analysis of the present invention. Genes are represented as nodes and interactions are represented as edges that connect the nodes, 292 interactions and 205 genes are shown. The genes are colored according to their YPD cellular roles, with the most abundant cellular roles shown.
  • FIG. 4 illustrates two dimensional clustering analysis of synthetic lethal interactions.
  • the set of synthetic lethal interactions associated with mutations in 8 query genes, BIM1, BNI1, ARC40, ARP2, NBP2, BBC1, RAD27 and SGS1, were plotted on the horizontal axis, with the query gene cluster tree above.
  • the 201 genes that showed synthetic lethal interactions with the query genes were plotted on the vertical axis with the cluster tree on the leftmost side of the plot. Synthetic lethal and synthetic sick (slow growth) interactions are represented as shades of red. An expansion of the plot is shown to allow visualization of specific genes.
  • the present invention is directed to high-density output arrays of multiple yeast strains, methods for constructing such arrays, and methods of using such arrays to conduct large-scale high throughput analysis of genetic and protein-protein interactions.
  • the present invention provides a systematic and efficient method for constructing arrays of yeast strains carrying multiple genetic alterations. This invention enables a large-scale analysis of genetic and protein-protein interactions, provides a method for validating potential drug targets and for generating whole cell screens for compounds that perturb the function of these targets.
  • the present invention provides compositions and methods that will enable the systematic and automated construction of arrays of yeast strains containing multiple mutations in a high-throughput manner.
  • the present invention could be used to generate a high density output array of approximately 25 million double mutant yeast strains, through crosses of input arrays involving approximately 5,000 viable haploid deletion mutants.
  • the resulting output arrays can be used for large-scale analysis of genetic and protein interaction networks by identifying the complete set of synthetic lethal double mutant combinations for a model eukaryotic cell.
  • the invention is directed to a variety of high-density yeast arrays, methods of making such arrays, and methods of using such arrays.
  • the arrays include both input and output arrays.
  • Input arrays are those arrays used to generate output arrays.
  • Input arrays are arrays of multiple yeast strains, with each yeast strain containing at least one genetic alteration. These input arrays are crossed with either other starting yeast strains that contain at least one genetic alteration, or other input arrays to produce output arrays.
  • Output arrays are therefore generated by crossing starting yeast strains from input arrays.
  • the starting yeast strains each have at least one genetic alteration. Crossing of the starting yeast strains containing at least one genetic alteration results in strains of the output arrays containing at least two genetic alterations.
  • These genetic alterations present in both the input and output arrays can include, but are not limited to, members of the group consisting of an aptamer, a system for detecting protein-protein interactions, including a yeast two-hybrid system, expression of a heterologous gene from a viral, prokaryotic, or eukaryotic genome with the heterologous gene either having or not having a yeast homolog, transformation with a promoter operably linked to a reference gene, wherein the reference gene can be a reporter gene, mutation or deletion of an endogenous essential or non-essential yeast gene, or the addition of other dominant agents which can perturb any cellular function, including genes coding for dominant negative proteins, antibodies, small molecules, natural products, ribozymes, RNAi, and antisense RNA or DNA.
  • an input array is a grouping of a multitude of starting yeast strains located together on a solid support.
  • the solid support is a plate.
  • the starting strains in the input array are selected from any yeast species that has two mating types and is capable of meiotic and mitotic reproduction.
  • the starting yeast strains can be from either the Saccharomyces cerevesiae species or the Schizosaccharomyces pombe species.
  • the genome of S. cerevisiae has been completely sequenced, and approximately 6,200 genes have been located.
  • the international S. cerevisiae deletion consortium has constructed mutants corresponding to all of the approximately 6,200 suspected genes within the S. cerevisiae genome, with each mutant being a strain of yeast containing a deletion of a different endogenous yeast gene. Tetrad analysis has revealed that approximately 15% of these deletion mutations define genes essential for the viability of haploid cells, also referred to as “essential genes”.
  • essential genes also referred to as “essential genes”.
  • the deletion consortium identified approximately 1,200 essential genes and approximately 5,000 nonessential genes.
  • the set of approximately 1,200 essential genes does not define the minimal set of genes required for life because many genes that are individually dispensable are not simultaneously dispensable. Synthetic genetic interactions are usually identified when a specific mutant is screened for second-site mutations that either suppress or enhance the original phenotype. In particular, two genes show a “synthetically lethal” interaction if they are associated with viability as single mutations but combine to cause a lethal double-mutant phenotype. This phenotype in which a particular gene is only required for cell viability when another gene is also deleted is called a “synthetic lethality defect”.
  • synthetic lethal interactions can also be identified through systematic construction and analysis of double-mutants.
  • the identification of synthetic lethal/fitness double-mutant combinations often indicates significant in vivo interactions between gene products and serves as a key starting point for targeted biochemical or cell biological experiments. Indeed, a synthetic lethal/fitness mutant combination is often observed for genes that impinge on the same essential cellular function.
  • yeast arrays crossing strains with deletions or mutations of non-essential genes can be generated in order to study the effects of double mutations. Accordingly, S. cerevesiae is an organism which works well in the high-density output yeast arrays of the present invention.
  • the same methods described herein for the S. cerevisiae genome wide deletion set can be applied to other yeasts once analogous deletion sets are constructed.
  • the fission yeast Schizosaccharomyces pombe has proven an invaluable complementary organism to budding yeast for cell and molecular genetic analysis. The completion of the fission yeast genome presents an enormous opportunity for comparative biology between the two distantly related yeasts.
  • a complete gene deletion set will certainly be constructed by the S. pombe community, and would be easily amenable to the systematic synthetic lethal analysis using the large-scale genetic and protein interaction analysis methods of the present invention. Any other fungal species that has two mating types can also be used with the present invention.
  • the input array of the present invention contains a multitude of starting yeast strains.
  • the array could contain, for example, about 5000 different yeast strains, each of which contains a different gene deletion.
  • Such an input array could be crossed with a second input array, which also contains about 5000 different yeast strains, each of which also contains a gene deletion.
  • This type of output array containing approximately 25 million (5,000 ⁇ 5,000) different yeast strains is described in detail in Example 5.
  • an input array of about 5000 different yeast strains could be crossed with only one starting strain. Examples of these types of crosses are described in detail in Example 6.
  • more specific input arrays can be crossed with specific deletion mutants, as illustrated in Examples 7-10. The possibilities of different crosses between a first input array and a second input array or between a first input array and a starting strain are quite numerous, and will be discussed in more detail below.
  • a first input array contains yeast strains, which are modified so that they contain at least one non-lethal genetic alteration.
  • the first input array is then crossed with either another yeast starting strain or another input array, which also contains at least one genetic alteration, to form an output array containing double mutants.
  • Double mutants as used herein means yeast strains which contain two genetic alterations, derived from single mutants each of which contains at least one genetic alteration.
  • An output array is the product of a cross between either two input arrays or an input array and a starting strain.
  • the input arrays and/or starting strain to be crossed are of different mating type, to allow for selection of the genetic alterations formed after the cross.
  • the present invention provides a series of replica pinning procedures in which mating and meiotic recombination are used to generate two output arrays, one composed of diploid cells carrying genetic alterations derived from the input arrays and another output array containing haploid meiotic progeny carrying genetic alterations derived from the input arrays (FIG. 1).
  • a query mutation is first introduced into a haploid starting strain, of one mating type, and then crossed to the array of gene deletion mutants of the opposite mating type.
  • a set of diploids that are heterozygous for both mutations represents the first output array that can be analyzed for a phenotype. Sporulation of the diploid cells leads to the formation of double-mutant meiotic progeny and the second output array.
  • the starting strain carries a reporter that allows for selected germination of spores, which ensures that conjugation of meiotic progeny does not complicate the final analysis.
  • Both the query mutation and the gene deletion mutations can be linked to dominant selectable markers, which enables selected growth of double-mutants specifically. The final pinning results in an ordered array of double-mutant strains, whose growth rate is monitored by visual inspection or image analysis of colony size.
  • the output arrays are generated by performing the following steps.
  • the first step is to generate multiple starting yeasts strains, with each of these starting yeast strains carrying a genetic alteration. These starting yeast strains are then grouped into either two input arrays, or one input array and a particular starting strain. The input arrays are then crossed (mated), and diploid strains result, forming the first output array. The mated diploid strains then undergo sporulation, resulting in haploid strains. A single mating type is germinated. The haploid spore progeny that result from this sporulation are then grown using selective growth criteria. Multiple haploid yeast strains, which grow on the selective media, can then be selected for the presence of genetic alterations that were in the starting strains. These recovered haploid yeast strains can then be arrayed in a high-density format forming the second output array.
  • output array can contain hundreds, thousands, or millions of resulting yeast strains with genetic alterations.
  • the output array contains between about 1,000 and 25 million resulting yeast strains, and more preferably between about one and about 25 million resulting yeast strains. Because the output array can be produced in such a high-density format, containing millions of yeast strains, the output array can be used to assign gene function to multiple genes simultaneously.
  • the high-density output array also allows for large-scale analysis of genetic and protein interactions, by analyzing the phenotypes of the resulting strains within the output array.
  • Synthetic lethality defect and synthetic fitness defect are phenotypes wherein either cell death or retarded cell growth occurs only when two different genes are deleted at the same time. When a specific gene is required for cell viability under conditions when a different gene is deleted or mutated, this resulting phenotype is termed a “synthetic lethality defect”. This phenotype is so named because the two genes deleted together synthetically lead to cell death.
  • the genetic alterations can be of any of the following types: (i) an alteration in the DNA encoding the gene such as a deletion or mutation of an endogenous essential or non-essential yeast gene; (ii) introduction of trans-dominant genetic agents such as genes coding for peptide or nucleic acid aptamers, dominant-negative proteins, antibodies, small molecules, natural products, nucleic acid aptamers, ribozymes, enzymes, RNAi, and antisense RNA or DNA; (iii) protein and RNA expression vectors of a heterologous gene from a viral, prokaryotic, or eukaryotic genome, wherein the genes can be either wild type, mutated, or fragmented (e.g.
  • a protein-protein interaction detection system including expression plasmids coding for a two-hybrid interaction and reporter that registers the interaction, the Ras recruitment system, the split-ubiquitin system, and various other protein fragment complementation systems (e.g. DHR); and (v) a reporter whose expression reflects a change in cellular state such as the activation or the inhibition of a pathway(s).
  • the genetic alterations can be integrated into the yeast genome or propagated on autonomously replicating plasmids.
  • the input arrays and starting yeast strains which are crossed to produce an output array can contain within them a variety of genetic alterations, which allow for analysis of a variety of different modifications.
  • the yeast starting strains in the input arrays have a gene deletion introduced into them as their genetic alteration. This gene deletion could be of an essential, or non-essential gene.
  • Non-essential gene deletions are deletions or mutations of genes that do not produce a lethal phenotype. In Saccharomyces cerevesiae, there are approximately 5,000 non-essential genes which, when either deleted or mutated, do not produce a lethal phenotype.
  • Combinations of non-essential genes deletions can produce synthetic lethal or synthetic fitness phenotypes that will reveal how these genes interact, what their function is in yeast i, and what the function of their homologs are in humans or other organisms. Combinations of these genes could also lead to other discernible phenotypes, which could suggest the function of the deleted genes.
  • Essential genes are genes that when deleted or mutated cause a lethal phenotype.
  • the function of essential genes can be studied by modulating their expression using inducible promoters.
  • conditional mutations can be introduced into essential genes allowing the mutant phenotype to be analyzed under defined conditions. For example, temperature sensitive mutations are viable at a permissive temperature and inviable at a restrictive temperature.
  • an input array with a non-essential or essential gene deletion can then be crossed with other starting strains that contain other gene deletions, or any other genetic alteration.
  • Genetic alterations mentioned in regards to this invention may include: non-essential gene deletions; essential gene deletions; aptamers; exogenous genes, either wild type, mutated, or fragmented (e.g. coding for a protein domain); genes coding for ribozymes; enzymes; RNAi, and antisense RNA or DNA; systems for detecting protein-protein interactions such as the yeast two-hybrid system; and reporters whose expression reflects changes in cellular state.
  • yeast-starting strains in the input arrays have aptamers either integrated into their genome or introduced as expression plasmids as their genetic alteration.
  • Aptamers are peptide or nucleic acids that are produced through at least partially randomized pools of nucleic acid or amino acid sequences, that are selected for their ability to bind certain epitopes.
  • Peptide aptamers are defined as affinity agents that consist of constrained combinatorial peptide libraries displayed on the surface of scaffold proteins. Peptide aptamers are trans-dominant agents that interact with gene products.
  • Ordered arrays of yeast strains expressing peptide or nucleic acid aptamers can substitute for arrays of yeast deletion strains.
  • starting strains containing gene deletions are crossed to an array of strains expressing peptide or nucleic acid aptamers and haploid meiotic progeny expressing the peptide aptamer and carrying the gene deletion can be selected.
  • the resulting strains that show aptamer-dependent synthetic lethality identify aptamers that inhibit a gene product whose activity is required for viability of the starting gene deletion strain.
  • the array of strains expressing peptide aptamers could also be used to identify dominant inhibitors.
  • a strain carrying a query mutation would be crossed to an array of strains expressing peptide aptamers and the resultant diploid cells examined directly for a phenotype. Those that show aptamer-dependent synthetic lethality as diploids would identify an aptamer that inhibits a gene which shows a genetic interaction with the heterozygous query mutation.
  • Aptamers can inhibit the function of gene products by any one of, but not limited to only, the following mechanisms: (i) modulating the affinity of a protein-protein interaction; (ii) modulating the expression of a protein on a transcriptional level; (iii) modulating the expression of a protein on a post-transcriptional level; (iv) modulating the activity of a protein; and (v) modulating the location of a protein.
  • the precise mechanism of action of peptide aptamers can be determined by biochemical and genetic means to ascertain their specific function in the context of their interaction with other genes, and gene products.
  • Strains carrying characterized aptamers can then be crossed with other starting strains that contain peptide aptamers or any other genetic alteration as described above. The phenotypes of these crosses can then be studied to determine if the resulting strains in the output array develop a synthetic fitness, or synthetic lethal phenotype, or any other discernible phenotype.
  • the starting strains would carry a heterologous gene or gene combination with a readout of gene product activity.
  • the starting strain may contain a heterologous gene encoding an enzyme for which there is a biochemical assay for its activity.
  • the starting strain may carry a yeast two-hybrid protein-protein interaction system or some other protein-protein interaction detection system such as the Ras recruitment system, the split-ubiquitin system and various other protein fragment complementation systems (e.g. DHR), which can be crossed to yeast within the input array that contain other yeast two-hybrid interaction systems, or any other genetic alteration.
  • the genetic alterations in the other starting strain could be any of the ones defined above or any other genetic alterations.
  • the phenotypes of these crosses can be studied to determine if any of the resulting strains within the output array perturb the two-hybrid interaction. For example, if an input starting strain carries a set of genes that allow for the formation and detection of a two-hybrid interaction and the strains within the input array carry yeast gene deletions, then the output array would allow for the identification of deletions that perturb the two-hybrid interaction; or if the strains within the input array carry peptides aptamers, then this system can be used to identify dominant inhibitory peptide aptamers that perturb the two-hybrid interaction.
  • the starting strains in the input arrays can express a heterologous gene(s) from either a prokaryotic, viral, or eukaryotic genome.
  • heterologous genes are from the human genome. Different alleles of these genes can be tested in yeast to see how they interact with mutated yeast genes that are homologous to human genes. This method can be used to assign function to different exogenous genes. Single nucleotide polymorphisms (SNPs) of human genes could also be characterized to identify SNP-dependent interactions. The genes of any organism could be similarly manipulated with this system. These heterologous genes can replace their deleted yeast homolog, or they can be genes that are not homologous to any yeast gene. These exogenous genes could be crossed with strains that contain either any of the genetic alterations described above, or any other genetic alteration.
  • yeast genes are conserved from yeast to humans and thus it is possible to functionally replace a yeast gene with its human homolog. There are many different alleles of a given human gene and some of these may be associated with a diseased state.
  • the replacement of a yeast gene by set of alleles of its human homologue, each differing by one or more SNP (single nucleotide polymorphism), in the context of the described genetic arrays offers a means to assess the functional interactions of a given allele within a model eukaryotic cell.
  • SNP single nucleotide polymorphism
  • the analysis of different alleles of a human gene may reveal that one allele in particular is associated with a greater number of synthetic lethal interactions, which suggests it is compromised for function relative to other alleles and, therefore, may be associated with a diseased state. If more than one conserved human gene is implicated in a diseased state, then in theory all combinations of different alleles can be tested for function within the context of a genetic array.
  • Yeast genetic arrays also permit the functional analysis of heterologous genes that do not have yeast counterparts.
  • hXXX a human gene, designated hXXX, whose product is involved in reorganization of the actin cytoskeleton and for which there is no yeast counterpart.
  • yeast cells do not contain a homolog hXXX
  • yeast cells have a highly conserved actin cytoskeleton and therefore will likely contain gene products, such as actin, that the hXXX gene product may interact with.
  • expression of the human gene within the context of a yeast genetic array will likely result in synthetic lethal/fitness defects that link the function of hXXX to actin reorganization.
  • the heterologous gene could be taken from any viral, prokaryotic, or other eukaryotic genome.
  • Starting strains carrying heterologous genes can be crossed with starting strains containing another exogenous gene, or any other genetic alteration.
  • the genetic alterations in the other starting strain could be any of the ones defined above or any other genetic alterations.
  • the phenotypes of these crosses can then be studied to determine if the resulting strains in the output array develop a synthetic fitness, or synthetic lethal phenotype, or any other discernible phenotype.
  • the starting strains in the input array contain a promoter from either a prokaryotic, viral, or eukaryotic genome operably linked to a reference gene.
  • Starting strains that carry a promoter operably linked to a reporter gene can be crossed with starting strains containing another promoter and reporter gene, or any other genetic alteration.
  • the genetic alterations in the other starting strain can be any of the ones defined above or any other genetic alterations.
  • the phenotypes of these crosses can then be studied to determine if the resulting strains in the output array develop a synthetic fitness, or synthetic lethal phenotype, synthetic dosage lethality, expression of the reporter gene, or lack thereof, or any other discernible phenotype.
  • Synthetic dosage lethality is a specialized version of a classical synthetic lethality screen.
  • a reference gene is overexpressed in set of mutant strains carrying potential target mutations.
  • This reference gene can be an exogenous gene from a viral, prokaryotic, or eukaryotic genome. More specifically, the gene could be a human gene. Increasing the amount of the reference gene product may not produce a phenotype in a wild-type strain. However, a lethal phenotype may result when overexpression of a gene product is combined with decreased activity of another gene product that impinges on the same essential function.
  • synthetic dosage lethality has been used to identify genetic interactions between CTF3, which encodes a centromere binding protein, and a set of conditional kinetichore mutants.
  • the synthetic dosage lethality gene could also contain single nucleotide polymorphisms.
  • Genes can be overexpressed by cloning the open reading frame (ORF) behind a strong promoter, such as the galactose-induced GAL1 promoter.
  • ORF open reading frame
  • Input arrays containing starting strains with the approximately 5,000 different yeast deletions can be crossed to a starting strain carrying a plasmid that contains a GAL1-regulated reference gene.
  • Haploid meiotic progeny carrying GAL1-regulated reference gene and carrying the gene deletion can be selected.
  • the output array containing yeast deletions combined with the GAL1-regulated reference gene can be pinned from glucose medium, where the reference gene is not expressed, to galactose medium, where it is overexpressed, to score for synthetic dosage lethality.
  • the gene-encoding reporter such as green fluorescence protein (GFP) may be placed on a plasmid under the control of a regulated promoter.
  • a regulated promoter is the pheromone-induced FUS1 promoter, FUS1pr, which is massively induced by stimulation of the MAP-kinase pathway that mediates the pheromone response in yeast.
  • FUS1pr-GFP gene could be constructed and introduced into the set of yeast deletion mutants for expression analysis. Mutations that lead to increased basal levels of FUS1pr-GFP expression, i.e. levels above that displayed by wild-type cells, would identify genes that encode potential negative regulators of the pheromone response signal transduction pathway.
  • GFP genome reporter matrix
  • each of the yeast deletion mutants is constructed such that it is tagged with two unique 20 mer oligonucleotide sequences.
  • bar codes allow for identification and analysis of specific deletion mutants within large populations.
  • a microarray printed with probes for the bar codes that correspond to the approximately 5,000 viable deletion mutants can be used to follow synthetic lethality of particular strains following batch mating and sporulation experiments.
  • Microarray-based synthetic lethal analysis with bar-coded mutants follows the same series of steps outlined in the pinning procedure for double-mutant construction. However, in this case, the yeast mutants are manipulated as a pooled population of cells and the growth of the cells is monitored as an array of bar codes.
  • a bar code is included into the components of the genetic array, manipulations of the cells can be carried out in batch format for array analysis via the bar codes. Details of the bar-code methodology are described in Example 12.
  • microarray approach to synthetic lethal analysis stems from its experimental simplicity.
  • the approximately 5,000 viable deletion mutants are manipulated as a single pooled population, which eliminates the need for high-density arrays of mutants and the volumes of media associated with manipulations of these arrays.
  • individual labs should be able to carry out synthetic lethal screens rapidly.
  • the parallel development of both the systematic and microarray-based approaches to synthetic lethal screening will allow for exploitation of the strengths of each strategy.
  • mutations and plasmids can be introduced into an input array via standard transformation procedures, e.g. lithium acetate or electroporation for the transformation of yeast cells. In this case, the resultant transformants would form the output array.
  • nucleic alterations can be combined using genetic manipulations or strain transformation protocols.
  • large-scale double-mutant combinations are constructed using a specialized starting strain and an automated pinning method for manipulation of high density input arrays of defined starting yeast mutants.
  • the manipulations to be performed can be performed by a robot.
  • robotic manipulations are described in detail in the examples, including Example 2.
  • the development of robotic methods for manipulation of the budding yeast genome-wide deletion set will set the stage for exciting uses of the present invention.
  • the high-density output arrays of multiple yeast strains of the present invention can be used in a variety of ways to analyze genetic interactions on a large-scale, high throughput basis. A description of some of these uses follows.
  • Mutations within many different yeast genes will lead to multiple synthetic lethal/fitness defects, generating a synthetic lethal profile for a given mutant.
  • Cluster analysis of a set of synthetic lethal profiles should identify mutant alleles that result in similar compromised states and therefore perturb similar functions within the cell (FIG. 3 and FIG. 4).
  • large-scale synthetic lethal/fitness analysis with yeast genetic arrays will provide a method of determining gene function. Mutations in nonessential genes are most easily analyzed; however, conditional alleles of essential genes, i.e. those genes required for cell growth, e.g. temperature sensitive alleles or those placed under the control of a regulated promoter (e.g.
  • the Tet-regulated promoter can be analyzed for synthetic effects following introduction into a genetic array.
  • the essential genes would be tested for synthetic lethal/fitness defects at a temperature below the nonpermissive/lethal temperature.
  • synthetic lethal/fitness defects would be tested at an intermediate gene expression level, i.e. at a level that compromises fitness but does not prevent cell growth.
  • the identified genes may represent a new target for a drug that will enhance the effectiveness of the known drug. For example, if we identify gene deletion mutants that are hypersensitive to the growth inhibitory effects associated with Cisplatin, a cancer therapeutic agent, then inhibitors of the identified genes would represent potential targets for drugs that would be used in combination with Cisplatin to kill cancer cells. In another example, if we identify gene deletion mutants that were hypersensitive to an antifungal drug, then the inhibitors of the identified genes would represent potential targets for drugs that would used in combination with the antifungal drug to kill a fungal pathogen. This procedure could also be used with any genetic manipulation either defined above or elsewhere.
  • results of the comprehensive synthetic lethal analysis in yeast provide a whole cell screen for inhibitors of specific target molecules.
  • the specificity of the screen will depend upon the specificity of the synthetic lethal profile associated with the ste20 ⁇ mutation. Given that each mutation will be tested for synthetic lethality in approximately 5,000 different contexts, it is anticipated that even genes within the same pathway may show a distinct synthetic lethal profile.
  • Genetic arrays can be used to identify mutations that function as suppressors of lethality. For example, consider a mutation in a gene whose product functions within a DNA damage check point signal transduction pathway. The combination of a DNA damage check point mutant and a DNA damaging agent leads to lethality and we can use a genetic array to screen for gene deletion mutations that suppress the conditional lethal situation. As another example, consider a gene that leads to lethality when overexpressed; in this case, we can use a genetic array to screen for mutations that are either hypersensitive or resistant to overexpression of the detrimental gene.
  • Use of the synthetic genetic arrays of the present invention should also allow for backcrossing the entire set of deletion mutations into another genetic background to analyze traits specific to that background.
  • genetic backgrounds which can be analyzed include the ⁇ 1278 background that is competent for filamentous growth and the SK1 background that is hyperactive for sporulation.
  • haploid yeast double-mutants within the output array are formed by meiotic recombination, the analysis of strains within the output array can be used to map gain of function phenotypes, even those that are multigenic traits. In this case, the gene(s) associated with the gain of function phenotype would fail to form double mutants efficiently with genetically linked gene deletions, resulting in a series of output strains that fail to inherit the gain of function phenotype.
  • the synthetic genetic array analysis of the present invention can be extended from yeast cells to mammalian cells by using an array of transfection constructs that lead to the expression of peptide or nucleic acid aptamers or other genetic alterations.
  • the peptide aptamer expression plasmids are first suspended in gelatin solution and arrayed on glass slides using a robotic microarrayer. Mammalian cells are then cultured on the glass slides containing the peptide aptamer expression plasmids. Cells growing in the vicinity of the gelatin spots uptake the peptide aptamer expression plasmids creating spots of localized transfection within a lawn of nontransfected cells.
  • the peptide aptamer expression plasmids Once the peptide aptamer expression plasmids are incorporated into cells they can function as dominant agents, dominant agents being agents which perturb the function of the cell in any way.
  • an input starting mammalian cell line might carry a set of genes that allow for the formation and detection of a two-hybrid interaction and the input array might carry a set of peptide aptamer expression plasmids.
  • the output array would consist of mammalian cells transfected with the aptamers that may perturb the two-hybrid interaction.
  • the cell microarrays can be designed such that the positions of individual aptamers in the yeast array are cross-correlated to the positions of the same aptamers in the cell microarray. This correlation will allow aptamers that have observable phenotypes in the yeast array, much as the disruption of a mammalian protein interaction, to be directly assessed in the cell microarray.
  • high density output arrays of multiple yeast strains of the present invention including but not limited to synthetic lethal analysis of high density output arrays to assign gene function, synthetic lethal analysis of high density output arrays for drug discovery and potential cancer therapeutics, synthetic lethal analysis of high density output arrays to generate cocktail therapies, synthetic lethal analysis of high density input arrays screened against small molecules to analyze small molecule-target interaction, and suppressed analysis of a conditional lethal silation.
  • Y2454 Saccharomyces cerevesiae strain termed Y2454.
  • the Y2454 strain is characterized by being a MAT ⁇ mating type with ura3, leu2, his3, and lys2 mutations, and a HIS3 gene linked to an MFA1 promoter.
  • the ura3, leu2, his3, and lys2 mutations require the strain to be grown in supplemented media to survive. They also carry a can1 null allele which confers canavinine resistance to the cells.
  • a mutant gene for example, one of the approximately 5,000 non-lethal mutations found in Saccharomyces cerevesiae, is introduced into this strain. The deleted gene is being replaced by a NAT gene which confers noureseothricin resistance to these cells.
  • This strain can be crossed with a starting array of yeast strains of the MATa mating type.
  • the strains in this starting array contain ura3, leu2, his3, and met15 knockouts, so that they can only survive on supplemented media.
  • These cells can also contain a mutation of one of the approximately 5,000 non-lethal gene deletions known in Saccharomyces cerevesiae.
  • the deleted gene is replaced with an operably linked KAN gene, which gives the yeast cells resistance to kanamycin derivatives like Geneticin.
  • These starting array strains carry a wild-type CAN1 locus, which makes them sensitive to canavinine.
  • a double mutant haploid cell could then be developed by the following steps:
  • Step 1 Construction of a Y2454-derivative that carries a nat-marked mutant allele, e.g. bni1 ⁇ ::nat where the given gene is deleted and replaced with nat, which results in nourseothricin-resistance, for genome-wide synthetic lethal analysis.
  • a nat-marked mutant allele e.g. bni1 ⁇ ::nat
  • Step 2 Mating of the MAT ⁇ Y2454-derivative to the array of MATa xxx ⁇ ::kan deletion mutants on rich medium to facilitate zygote formation.
  • Step 3 Transfer zygotes to medium containing geneticin and nourseothricin to select for growth of MATa/ ⁇ diploid cells.
  • Step 4 Transfer MATa/ ⁇ diploid cells to sporulation medium to induce spore formation.
  • Step 5 Transfer spores to synthetic medium lacking histidine and containing canavanine to select for growth MATa haploid spore-progeny.
  • Step 6 Transfer MATa haploid spore-progeny to medium containing geneticin and nourseothricin to score for growth and viability of MATa bni1 ⁇ ::nat xxx ⁇ ::kan double-mutants.
  • step 2 yeast cells are arrayed on rich medium to allow efficient mating.
  • step 3 the mating reactions are arrayed onto medium containing geneticin and nourseothricin, which allows for selected growth of diploid cells.
  • step 4 the diploid cells are transferred to medium that is low in nitrogen and carbon and induces sporulation.
  • step 5 the spores are transferred to germination medium that selects for growth of the haploid MATa cells (this step is described in detail below).
  • step 6 double mutant strains are selected for growth the two mutations are scored as synthetic lethal/fitness defect if the MATa haploid double-mutants form a colony that is smaller than that associated with either of the single mutants.
  • the mutations predicted to be synthetically lethal can be analyzed in more detail through tetrad analysis of the heterozygous diploid cells created in Step 2.
  • Wash/Dry station for cleaning the pins between runs and includes the 5 stages of water, ethanol or bleach, sonicator (water), ethanol, and air-drying;
  • Example 1 The recovery of haploid spore progeny is mentioned in step 5 in Example 1, and is described in greater detail below.
  • MFA1 encodes the a-factor precursor, which is expressed constitutively in MATa cells.
  • the MFA1 promoter, MFA1pr is repressed in MAT ⁇ and MATa/ ⁇ cells.
  • the MFA1pr-HIS3 reporter was constructed by replacing the MFA1 ORF (open reading frame) with the HIS3 ORF such that MFA1pr drives HIS3 expression.
  • the MAT ⁇ starting strain, Y2454 fails to grow on synthetic medium lacking histidine because the MFA1pr-HIS3 reporter is repressed in MAT ⁇ cells.
  • the cells constructed in step 2 of Example 1 will also fail to grow on synthetic medium lacking histidine because the MFA1pr-HIS3 reporter is repressed in MAT a/ ⁇ cells.
  • the MFA1pr-HIS3 reporter selects for growth of MATa haploid progeny. Twenty-five per cent of the haploid progeny generated by sporulation of the diploid cells will be MATa MFA1pr-HIS3.
  • Other a-specific reporters can be constructed using promoters from different a-specific genes (e.g. MFA2, ASG7, STE2) or different reporters (e.g. URA3, LEU2, or heterologous genes conferring resistance to antibiotics or other chemicals)
  • the CAN1 gene encodes an arginine permease.
  • the Y2454 starting strain has been engineered to carry a recessive can1 ⁇ 0 null allele, which renders the cells resistant to canavanine, a toxic arginine analog that is transported by the CAN1 gene product.
  • the knock-out strains constructed by the deletion consortium are canavanine-sensitive because they carry a wild-type CAN1 locus.
  • the MAT ⁇ starting strain, Y2454 carries can1 ⁇ 0 rendering it canavanine-resistant.
  • the MATa/ ⁇ can1 ⁇ 0/CAN1 diploids isolated in step 3 of Example 1 will be canavanine-sensitive.
  • the can1 ⁇ 0 allele allows for selection of canavanine-resistant haploid progeny. Fifty percent the haploid spore progeny will be canavanine-resistant. Other recessive drug resistant genes, such as cyh2 mutations which leads to cycloheximide resistance, can also be used.
  • Both the can1 ⁇ 0 and the MFA1pr-HIS3 reporter allow selection for haploid spore progeny in Step 5 of the pinning procedure described in Example 1.
  • the selection provided by the MFA1pr-HIS3 reporter enables the specific isolation of MATa cells. It is important to isolate spore progeny of a single mating type when ultimately scoring for the presence or absence of two marked mutant alleles; otherwise progeny of opposite mating type may conjugate to generate diploid cells heterozygous for each mutation, which would appear, perhaps falsely, as a viable double-mutant.
  • the MATa/ ⁇ can1 ⁇ 0/CAN1 MFA1pr-HIS3/MFA1 diploids constructed in steps 2 and 3, can become canavanine-resistant as diploid cells through mitotic recombination involving the can1 ⁇ 0 locus, creating MATa/ ⁇ can1 ⁇ 0/can1 ⁇ 0 MFA1pr-HIS3/MFA1 cells.
  • sporulation medium we do not observe the formation of cells that are both canavanine-resistant and competent for growth on medium lacking histidine.
  • MFA1pr-HIS3 and the can1 ⁇ alleles are essential for synthetic lethal analysis via a pinning procedure, they present a position-based problem.
  • the xxx ⁇ ::KANR deletion mutations that are linked tightly to these alleles will be paired into the genome of the haploid spores at reduced frequency and may lead to a false synthetic lethal score.
  • the problematic double-mutant combinations are predictable.
  • MFA2pr-HIS3 starting strain For mutations in the vicinity of MFA1pr-HIS3 on chromosome X, we will employ an MFA2pr-HIS3 starting strain.
  • the MFA2 gene encodes a second copy of the a-factor structural gene.
  • MFA2pr leads to a-specific gene expression and will facilitate selection for MATa spores. Because 1 cM in S. cerevisiae is roughly equivalent to 1.5 Kb of DNA sequence and the average S. cerevisiae gene is approximately 2 Kb, we anticipate that 10-30 genes on either side of the MFA1 locus will have to be mated to a starting strain containing MFA2pr-HIS3. Alternatively the MFA1pr-HIS3 could be moved to another position in the genome. The can1 ⁇ 0 allele presents a slightly different problem because it cannot be moved like the MAT ⁇ -specific reporter.
  • Some xxx ⁇ ::kanR deletion mutations may cause synthetic lethality in synthetic lethal starting strains carrying the MFA1pr-HIS3 and can1 ⁇ alleles. Because MFA1 is a specialized gene devoted to conjugation, it is unlikely that MFA1pr-HIS3 will be associated with any synthetic lethal interactions unless localized alterations of the genome affect the function of neighboring genes. Because the CAN1 gene product facilitates arginine uptake, the can1 ⁇ deletion mutation will be synthetically lethal with any gene that prevents arginine biosynthesis. All the genes required for arginine biosynthesis will be defined/confirmed simply by creating all the double mutants with the wild-type version of the starting strain.
  • Some xxx ⁇ ::KANR deletion mutations will be defective for one of the cellular functions required for double mutant construction or spore formation.
  • mating defective mutants e.g. ste4 ⁇
  • a genome-wide synthetic lethal screen will also identify all the genes required for sporulation in Step 4.
  • diploids that are homozygous for certain mutations will lead to a sporulation defect; however, other genes may prove haploinsufficient for sporulation or two mutations may exhibit nonallelic-noncomplementation, giving rise to a sporulation defect.
  • analysis of the growth of MATa MFA1pr-HIS3 can1 ⁇ cells in Step 5 will be important.
  • a large-scale comprehensive synthetic lethal analysis can be performed either by constructing approximately 5,000 gene deletions in the synthetic lethal starting strain, Y2454, or by establishing an automated method to transfer the knockout alleles constructed by the deletion consortium into the Y2454 starting strain.
  • Strain growth and transformation will be carried out using a 96 well format; therefore, approximately 52 rounds of transformation will allow us to switch the panel of approximately 5000 viable MATa ura3 ⁇ 0 leu2 ⁇ 0 his3 ⁇ 1 met15 ⁇ 0 xxx ⁇ ::kan strains to MAT ⁇ ura3 ⁇ 0 leu2 ⁇ 0 his3 ⁇ 1 met15 ⁇ 0 xxx ⁇ ::nat strains.
  • the next challenge is to move the approximately 5,000 xxx ⁇ ::nat alleles from the MATa ura3 ⁇ 0 leu2 ⁇ 0 his3 ⁇ 1 met15 ⁇ 0 xxx ⁇ ::nat cells into the strain that carries the haploid selection alleles, MFA1pr-HIS3 and can1 ⁇ 0.
  • MFA1pr-HIS3 haploid selection alleles
  • lys2 ⁇ 0 and can1 ⁇ 0 are associated with drug-mediated selections; however, we must construct a haploid selection marker that is functionally equivalent to MFA1pr-HIS3 and selects specifically for growth of MAT ⁇ cells.
  • MFA1pr-HIS3 :MF ⁇ 1pr-LEU2
  • MFA1pr-HIS3 reporter provides a selection for haploid MATa cells on medium lacking histidine.
  • MF ⁇ 1pr controls the expression of the ⁇ -factor structural gene and is expressed only in MAT ⁇ cells.
  • MF ⁇ 1pr-LEU2 provides a selection for MAT ⁇ cells on synthetic medium lacking leucine.
  • MFA1pr-HIS3 is tightly linked to MF ⁇ 1pr-LEU2 within the context of the dual reporter, the MAT ⁇ cells recovered on synthetic medium lacking leucine will also carry MFA1pr-HIS3.
  • the MFA1pr-HIS3 could be placed at CAN1 locus, creating can1 ⁇ 0::MFA1pr-HIS3; in this context, growth of MAT ⁇ MF ⁇ 1pr-LEU2 can1 ⁇ 0::MFA1pr-HIS3 cells can be selected on synthetic medium containing canavanine and lacking leucine.
  • Step 1 96-well format transformation will be used to switch a panel of approximately 5000 viable MATa ura3 ⁇ 0 leu2 ⁇ 0 his3 ⁇ 1 met15 ⁇ 0 xxx ⁇ ::kanR strains to MATa ura3 ⁇ 0 leu2 ⁇ 0 his3 ⁇ 1 met15 ⁇ 0 xxx ⁇ ::NAT strains. Transformants that grow on medium containing nourseothricin will be screened for Geneticin sensitivity to confirm the switching event.
  • Step 2 The MATa ura3 ⁇ 0 leu2 ⁇ 0 his3 ⁇ 1 met15 ⁇ 0 xxx ⁇ ::natR strains will be mated to MAT ⁇ ura3 ⁇ 0 leu2 ⁇ 0 his3 ⁇ 1 lys2 ⁇ 0 MFA1pr-HIS3::MF ⁇ 1pr-URA3 can1 ⁇ 0 on rich medium.
  • the resultant diploid cells will be selected for growth on synthetic medium lacking methionine and lysine. As mentioned above, this selection is not ideal because the met15 ⁇ 0 allele does not completely eliminate growth on medium lacking methionine; however, because each strain will only require mating once, we will be able to follow the diploid selection carefully using relatively large patches of cells and double replica plating to selective medium.
  • Step 3 Sporulation of the resultant diploid cells and selection for MAT ⁇ ura3 ⁇ 0 leu2 ⁇ 0 his3 ⁇ 1 lys2 ⁇ 0 MFA1pr-HIS3::MF ⁇ 1pr-URA3 can1 ⁇ xxx ⁇ ::LEU2 on medium that lacks uracil and leucine but contains ⁇ -aminoadipate and canavanine.
  • ⁇ -aminoadipate selects for lys2 ⁇ 0 mutant cells and canavanine selects for can1 ⁇ 0 cells; 50% of the resultant haploids will be met15 ⁇ 0.
  • the presence of the met15 marker can be scored by successive replica-platings to medium lacking methionine.
  • Step 4 Each of the approximately 5000 MAT ⁇ ura3 ⁇ 0 leu2 ⁇ 0 his3 ⁇ 1 lys2 ⁇ 0 MFA1pr-HIS3::MF ⁇ 1pr-URA3 can1 ⁇ xxx ⁇ ::LEU2 strains will each be mated to the array of MATa xxx ⁇ ::KANR deletion mutants and run through steps 2-6 of the pinning procedure outlined in Example 1 for systematic genome-wide synthetic lethal analysis.
  • Pathways critical for the fitness of bni1 ⁇ cells were revealed by multiple interactions with subsets of genes involved in bud emergence (BEM1, BEM2, and BEM4), chitin synthase III activity (CHS3, SKT5, CHS5, CHS7, and BNI4), MAP kinase pathway signaling (BCK1 and SLT2), the cell cycle-dependent transition from apical to isotropic bud growth (CLA4, ELM1, GIN4, and NAP1), and the dynein/dynactin spindle orientation pathway (DYN1, DYN2, PAC1, PAC11, ARP1, JNM1, NIP100).
  • the present example demonstrates that the replica pinning procedure of the present invention can be used for synthetic lethal analysis.
  • a deletion of the BNI1 gene, bni1 ⁇ was selected as the query mutation.
  • a test-array of gene deletion mutants was assembled that included bnr1 ⁇ , which is synthetically lethal with bni1 ⁇ .
  • BNI1 and BNR1 both encode members of the formin family, proteins that appear to control actin polymerization in response to signaling by Rho-type GTPases.
  • Growing yeast cells contain two major filamentous actin structures, cortical actin patches, which polarize to the cortex of the growing bud and act as sites of endocytosis, and actin cables, which align along the mother bud axis and act as tracks for myosin motors that coordinate polarized cell growth and spindle orientation.
  • Bni1 and Bnr1 are required for the formation of actin cables.
  • bni1 ⁇ mutants show defects in polarized cell growth and spindle orientation, whereas bnr1 ⁇ mutants display no obvious phenotype, indicating that Bni1 functions as the predominant formin in yeast cells.
  • the array contained 96 strains, each of which was included in quadruplicate and positioned next to each other in a square pattern, resulting in a matrix with 384 elements.
  • bnr1 ⁇ was included at two positions and enriched the array for mutations in other genes with roles in actin assembly and cell polarity.
  • FIG. 2A The array of bni1 ⁇ double-mutants resulting from the final pinning and the corresponding wild-type control are shown in FIG. 2A.
  • the cells at the bnr1 ⁇ positions failed to grow, forming a residual colony with a reduced size relative to the control.
  • the resultant double-mutants are created by meiotic recombination, gene deletions that are genetically linked to the query mutation form double mutants at a reduced frequency.
  • double mutants can not form.
  • the cells at the bni1 ⁇ position would fail to grow under double-mutant selection.
  • Cla4 is a PAK-like kinase involved in actin patch assembly and the cell cycle-dependent transition from apical to isotropic bud growth;
  • Bud6 forms a complex with Bni1 and actin to control actin cable assembly and cell polarity.
  • Tetrad analysis confirmed that both the bni1 ⁇ bnr1 ⁇ and bni1 ⁇ cla4 ⁇ double-mutants were inviable and that the bni1 ⁇ bud6 ⁇ double-mutant was associated with a slower growth rate or “synthetic sick” phenotype, reflecting reduced fitness of the double-mutant relative to the respective single mutants (FIG. 2B).
  • This example thus demonstrates that the replica pinning procedure can identify genetic interactions corresponding to the spectrum of fitness defects from synthetic sick to synthetic lethal phenotypes.
  • Bbc1 ⁇ showed interactions with several genes whose products control actin polymerization and localize to cortical actin patches (CAP1, CAP2, SAC6, and SLA1), suggesting BBC1 may be involved in the assembly actin patches or their dependent processes.
  • CAP1, CAP2, SAC6, and SLA1 genes whose products control actin polymerization and localize to cortical actin patches
  • BBC1 may be involved in the assembly actin patches or their dependent processes.
  • Bbc1 localized predominantly to cortical actin patches and that its SH3 domain binds directly to Las17 (Bee1), a member of the WASP (Wiskott-Aldrich syndrome protein) family proteins that controls the assembly of cortical actin patches through regulation of the Arp2/3 actin nucleation complex.
  • WASP Wikott-Aldrich syndrome protein
  • SGS1 encodes the yeast homolog of the human Werner's Syndrome protein, WRN, a member of the RecQ family of DNA helicases
  • RAD27 encodes an enzyme that processes Okazaki fragments during DNA synthesis and repair.
  • the phenotype of yeast cells deleted for the SGS1 gene mirrors the chromosomal instabilities and premature aging associated with Werner's syndrome.
  • SGS1 is known to show a synthetic lethal/sick relationship with YNL218W, which encodes the yeast homolog of human Werner helicase interacting protein, and seven other genes (SLX1, MMS4, SLX3, SLX4, HEX3, HRP5, and SLX8), which are thought to mediate the resolution of recombination intermediates generated in the absence of SGS1.
  • SLX1, MMS4, SLX3, SLX4, HEX3, HRP5, and SLX8 seven other genes (SLX1, MMS4, SLX3, SLX4, HEX3, HRP5, and SLX8), which are thought to mediate the resolution of recombination intermediates generated in the absence of SGS1.
  • SLX1, MMS4, SLX3, SLX4, HEX3, HRP5, and SLX8 seven other genes (SLX1, MMS4, SLX3, SLX4, HEX3, HRP5, and SLX8), which are thought to mediate the resolution of
  • RRM3 a gene that encodes a closely related helicase involved in rDNA replication
  • WSS1 a gene identified as a high dosage suppressor of SMT3, which codes a conserved ubiquitin-related protein that interacts with HEX3 in the two-hybrid system
  • YBR094W a highly conserved gene of unknown function with an NH2-terminal SurE domain and COOH-terminal tubulin-tyrosine ligase-like domain
  • ESC4 which codes for an uncharacterized gene with 3 BRCT domains, peptide recognition modules that appear to be found exclusively in proteins involved in DNA synthesis and repair.
  • nbp2 ⁇ a complete deletion of NBP2, gene of uncharacterized function that showed a synthetic lethal/sick interaction with BNI1; the Nbp2 product contains an SH3 domain and shows a two-hybrid interaction with Nap1.
  • arc40-1 a temperature sensitive allele of ARC40, an essential gene that codes for a component of the Arp2/3 complex, a seven-member complex that functions to nucleate actin filaments, controlling the assembly, movement, and localization of cortical actin patches in yeast.
  • arp2-2 a temperature sensitive allele of ARP2, coding for one of the key actin-related proteins of the Arp2/3 complex.
  • the data set was first imported into the Biomolecular Interaction Network Database (BIND), then formatted with BIND tools and exported to the Pajek package, a program originally designed for the analysis of social networks.
  • BIND Biomolecular Interaction Network Database
  • the genetic interaction network shown in FIG. 3 contains 205 genes, represented as nodes on the graph, 292 synthetic lethal/sick interactions, represented as edges connecting genes. All of these interactions were first identified using the automated methodology and then confirmed by tetrad analysis.
  • To visualize sets of genes with related functions we color-coded the genes according to their YPD cellular roles and aligned the genes based upon both their roles and connectivity.
  • each of the query genes were biased towards interactions with genes of particular cellular roles. Moreover, subsets of interacting genes with the same cellular roles could distinguished from one another by their connectivity.
  • the BIM1 screen identified a large group of genes involved in mitosis (red genes), which include several components of the Bub2p- and Mad2p-dependent spindle assembly checkpoints (BUB1, BUB2, BUB3, BFA1, MAD1, MAD2, and MAD3) and multiple genes involved nuclear migration and spindle orientation during mitosis (BIK1, MCK1, SLK19, KIP3, PAC11, PAC1, NUM1, DYN1, JNM1, ARP1, ASE1), a subset of which also interacted with BNI1 and function specifically as part of the Dyn1 kinesin pathway.
  • red genes include several components of the Bub2p- and Mad2p-dependent spindle assembly checkpoints (BUB1, BUB2, BUB3, BFA1, MAD1, MAD2, and MAD3) and multiple genes involved nuclear migration and spin
  • BIM1 interacted with a group genes that have a chromatin/chromosome structure cellular role (yellow genes), many of which have been implicated kinetichore function (CTF8, CTF19, MCM21, MCM22, and CHL4), and a total of 15 genes of with unknown cellular roles (black genes).
  • CTF8 CTF8, CTF19, MCM21, MCM22, and CHL4
  • black genes a total of 15 genes of with unknown cellular roles
  • NBP2 screen which showed interactions with several genes involved in nuclear migration and spindle function (KAR9, CIN2 and KIP3), actin assembly (CAP1 and CAP2), and de novo folding of actin and tubulin (PAC10 and GIM5), suggestive of a general role in cytoskeletal organization.
  • the RAD27 screen resulted in 35 interactions, the majority of which occurred for genes with DNA synthesis/repair cellular roles (FIG. 3 green genes).
  • the unprocessed Okazaki fragments of rad27 ⁇ cells are probably recognized as nicks or converted into double-strand breaks as evidenced by a large set of previously known synthetic lethal/sick interactions with the genes encoding multiple components of the recombinational repair apparatus (RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, MRE11, XRS2) and the DNA damage checkpoint signaling pathway (RAD9, RAD17, RAD24, DDC1).
  • HST1 and HST2 two genes encoding Sir3-like deacetylases and CAC2, a gene coding for a chaperone that delivers acetylated histones to newly synthesized DNA, which may be indicative of a functional relationship amongst the products of these genes in chromatin assembly or silencing.
  • RAD27 also showed a synthetic lethal/fitness with the SOD1 superoxide dismutase gene, and its copper chaperone, LYS7, suggesting that the antioxidant functions of Sod1 are required to protect the rad27 ⁇ cells from accumulated DNA damage.
  • RAD27 interacted with 4 genes of unknown function, for which we predict a possible role in a DNA synthesis and/or repair.
  • the pinning procedure described for the construction of double mutants provides a simple method to move a plasmid of interest into the set of approximately 5,000 viable xxx ⁇ ::KAN deletion mutants.
  • the Y2454 starting strain is transformed with a URA3- or LEU2-based plasmid and then crossed into the haploid mutants by following steps 1-6 of the pinning procedure as described in Example 1 for genome-wide double-mutant construction.
  • the ability to undertake plasmid-based screens with the complete deletion set greatly extends the number of possible genome-wide screens including synthetic dosage lethality and green-fluorescence protein-based reporter screens.
  • Step 1 The pool of MAT ⁇ xxx ⁇ ::KAN cells, containing the entire set of approximately 5,000 viable deletion mutants, will be mated to a Y2454-derivative for synthetic lethal analysis with a mutant allele marked with NAT (e.g. Y2454 made bni1 ⁇ ::NAT).
  • NAT e.g. Y2454 made bni1 ⁇ ::NAT
  • Step 2 The resultant pool of diploid cells is transferred to sporulation medium and MATa can1 ⁇ MFA1-HIS3 cells are selected on synthetic medium that contains canavanine but lacks histidine.
  • Step 3 The pool of haploid MATa can1 ⁇ MFA1-HIS3 cells are grown on medium containing geneticin to select for MATa can1 ⁇ MFA1-HIS3 xxx ⁇ ::KAN deletion mutants
  • Step 4 The MATa can1 ⁇ MFA1-HIS3 xxx ⁇ ::KAN cells are split into two samples. To determine the set of mutant cells that mated and sporulated efficiently, DNA will be prepared from one sample and used to probe the bar-coded microarray. DNA preparation of the DNA involves isolating genomic DNA and preparing bar code probes via a PCR-based method.
  • Step 5 To determine the set of synthetic lethal double mutants, the other sample of cells will first be grown on medium containing nourseothricin, which selects for double-mutant cells, e.g. MATa bni1 ⁇ ::LEU2 xxx ⁇ ::KANR can1 ⁇ MFA1-HIS3 cells. Then, DNA will prepared for bar-coded microarray analysis. Comparison of the bar-coded mutants that are present in sample 1 but not present in sample 2 identifies potential synthetic-lethal combinations.
  • medium containing nourseothricin which selects for double-mutant cells, e.g. MATa bni1 ⁇ ::LEU2 xxx ⁇ ::KANR can1 ⁇ MFA1-HIS3 cells.
  • DNA will prepared for bar-coded microarray analysis. Comparison of the bar-coded mutants that are present in sample 1 but not present in sample 2 identifies potential synthetic-lethal combinations.

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US20060160169A1 (en) * 2004-12-03 2006-07-20 Board Of Regents, The University Of Texas System Cell microarray for profiling of cellular phenotypes and gene function
US20080287317A1 (en) * 2001-08-15 2008-11-20 Charles Boone Yeast arrays, methods of making such arrays, and methods of analyzing such arrays

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EP1616939A1 (fr) * 2003-03-24 2006-01-18 National Institute for Environmental Studies Milieu de culture cellulaire et preparation solidifiee de proteine ou de peptide d'adhesion cellulaire
US20150368639A1 (en) * 2011-04-14 2015-12-24 Ryan T. Gill Compositions, methods and uses for multiplex protein sequence activity relationship mapping
WO2014059370A1 (fr) * 2012-10-12 2014-04-17 Institute For Systems Biology Système à haut débit amélioré pour les études génétiques
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US10011849B1 (en) 2017-06-23 2018-07-03 Inscripta, Inc. Nucleic acid-guided nucleases
US9982279B1 (en) 2017-06-23 2018-05-29 Inscripta, Inc. Nucleic acid-guided nucleases
CN109376842B (zh) * 2018-08-20 2022-04-05 安徽大学 一种基于属性优化蛋白质网络的功能模块挖掘方法
EP4001417A1 (fr) * 2020-11-13 2022-05-25 serYmun Yeast GmbH Plate-forme de levure pour la production de vaccins

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US20080287317A1 (en) * 2001-08-15 2008-11-20 Charles Boone Yeast arrays, methods of making such arrays, and methods of analyzing such arrays
US20060051770A1 (en) * 2004-09-03 2006-03-09 Affymetrix, Inc. Methods of genetic analysis of yeast
US7312035B2 (en) 2004-09-03 2007-12-25 Affymetrix, Inc. Methods of genetic analysis of yeast
US20060160169A1 (en) * 2004-12-03 2006-07-20 Board Of Regents, The University Of Texas System Cell microarray for profiling of cellular phenotypes and gene function

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