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

Abstract

This patent describes a novel method of detecting genetic interactions in yeast. This method can also be used to screen for function of biological effectors on yeast. The method encompasses crossing yeast strains with genetic alterations to acquire double mutants. The phenotypes of these double mutants are then checked to detect genetic interactions between the double mutants. This method can be used to assign function to yeast genes and their viral, prokaryotic, and eukaryotic homologs, and aptamers. It can also be used to study yeast two hybrid interactions and to find genes that regulate certain yeast promoters.

1 . A high density output array of multiple yeast strains, wherein each resulting yeast strain in the output array contains at least two resulting genetic alterations, and wherein the resulting genetic alterations are different in each resulting yeast strain, the output array being the mating product of at least two input arrays, wherein at least one of the input arrays comprises multiple starting strains of yeast, wherein each starting yeast strain carries at least one genetic alteration, with the genetic alteration being different in each starting yeast strain. 
     
     
         2 . The output array of  claim 1 , wherein the resulting yeast strains are in the diploid state. 
     
     
         3 . The output array of  claim 1 , wherein the resulting yeast strains are in the haploid state. 
     
     
         4 . The input or output array of  claim 1 , wherein the starting and resulting yeast strains are selected from any yeast strain that has two mating types and is capable of meiotic and mitotic reproduction. 
     
     
         5 . The input or output array of  claim 4 , wherein the starting and resulting yeast strains are from either the  Saccharomyces cerevesiae  or the  Schizosaccharomyces pombe  species. 
     
     
         6 . The input or output array of  claim 1 , wherein the yeast strains are located on plates, with between about 9 and about 6200 yeast colonies on one plate. 
     
     
         7 . The output array of  claim 1 , wherein the resulting genetic alteration is a double mutant, the double mutant involving a mutation of two different endogenous yeast genes. 
     
     
         8 . The output array of  claim 7 , wherein the double mutant carries the deletion of two different non-essential yeast genes. 
     
     
         9 . The output array of  claim 8 , wherein the double mutant is either a synthetic lethal double mutant or a synthetic fitness double mutant. 
     
     
         10 . The output array of  claim 1 , which comprises between about 1,000 and about 25 million resulting strains of yeast. 
     
     
         11 . The input array of  claim 1 , wherein the starting genetic alteration in at least one starting yeast strain is selected from the group consisting of introduction of genes coding for an aptamer, introduction of a protein-protein interaction detection 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, transfection with a promoter operably linked to a reporter gene, and mutation or deletion of an endogenous yeast gene. 
     
     
         12 . The input array of  claim 11 , wherein the aptamer is either a peptide aptamer or a nucleic acid aptamer. 
     
     
         13 . The input array of  claim 11 , wherein the aptamer performs a function selected from the group consisting of inhibiting expression of a gene, increasing expression of a gene, inhibiting protein-protein interactions, enhancing protein-protein interactions, inhibiting the activity of a protein, and enhancing the activity of a protein. 
     
     
         14 . The input array of  claim 11 , wherein the protein-protein interaction detection system is selected from the group consisting of a yeast two-hybrid system, the Ras recruitment system, the split ubiquitin system, and protein fragment complementation systems. 
     
     
         15 . The input array of  claim 11 , wherein the heterologous gene is a human gene. 
     
     
         16 . The input array of  claim 15 , wherein the human gene comprises a set of alleles, each differing by one or more SNPs. 
     
     
         17 . A method for generating a high-density output array of resulting multiple yeast strains, wherein each resulting yeast strain carries at least two resulting genetic alterations, and wherein the resulting genetic alterations are different in each yeast strain, the method comprising:
 a) generating multiple starting yeast strains, each strain carrying a starting genetic alteration;   b) mating sets of two starting yeast strains, wherein each of the two starting yeast strains contains a starting genetic alteration; and   c) recovering multiple diploid yeast strains which carry a resulting genetic alteration, wherein the resulting genetic alteration comprises the starting genetic alterations from each of the two mated starting yeast strains; and   d) arraying the genetically altered yeast strains in a high-density diploid output array.