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

a technology of arrays and yeast, applied in the field of yeast arrays and high density output arrays, can solve the problems of phenotypic analysis of the set of viable deletion strains within certain species of yeast, the absence of obvious functions for a large fraction of encoded proteins will quickly become an enormous problem in biology, and the scope of the challenge is immens

Inactive Publication Date: 2008-11-20
BOONE CHARLES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention is about a method for studying genetic interactions and protein interactions in yeast cells. The method involves creating multiple strains of yeast cells with different genetic alterations and mating them with each other. The resulting strains are then analyzed to determine the effects of the genetic alterations on their phenotype. The method can be used to study the interactions of genes, proteins, or other biological factors, and can also be used for small molecule screening. The resulting strains can be arrayed in a high-density format for further analysis.

Problems solved by technology

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.
With genome sequence in hand, the monumental challenge is to understand the roles of the approximately 6,200 predicted yeast gene products.
The scope of the challenge is immense.
Simple extrapolation to more complex genomes suggests that the absence of obvious functions for a large fraction of encoded proteins will quickly become an enormous problem in biology.
The phenotypic analysis of the set of viable deletion strains within certain species of yeast represents a major challenge because the role of many genes will only be manifest under very specialized growth conditions.

Method used

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

Examples

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example 1

Creation of Double Mutant Output Array

[0086]An example of a starting strain that could be used in this embodiment is the 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.

[0087]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 ...

example 2

Use of Robotics to Generate Output Array

[0096]Following construction of a natR-marked mutant for synthetic lethal analysis, simple replica plating or pinning manipulations will enable us to complete steps 2-6 of Example 1. These steps can be carried out through the use of a robotic colony arrayer, as described below. The Colony Arrayer used in this Example, the Virtek Vision CPCA was designed by our labs in conjunction with Virtek Vision. The CPCA is based upon a system used for genome-wide two-hybrid arrays, since the manipulations required for automated two-hybrid screens are very similar to those required for automated double-mutant construction and synthetic lethal screens.

[0097]For a rapid genome-wide two-hybrid screening procedure, we have created a robotic colony array which includes replica-plate pinhead that transfers 768 individual colonies from one standard microtiter-sized agar-slab plate to another in a single move. This cell density allows efficient mating, on the orde...

example 3

Recovery of Haploid Spore Progeny

[0104]The recovery of haploid spore progeny is mentioned in step 5 in Example 1, and is described in greater detail below. The MATα starting strain described in Example 1, Y2454, carries two selectable markers, can1Δ0 and MFA1pr-HIS3, both of which permit efficient recovery of haploid spore progeny.

[0105]i) MFA1pr-HIS3

[0106]MFA1 encodes the a-factor precursor, which is expressed constitutively in MATa cells. The MFA1 promoter, MFA1pr, is repressed in MATa and MATa / α 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 MATα starting strain, Y2454, fails to grow on synthetic medium lacking histidine because the MFA1pr-HIS3 reporter is repressed in MATα 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 MATa / α cells. Following sporulation of...

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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.

Description

RELATED APPLICATION[0001]This application claims priority to U.S. Ser. No. 09 / 930,593, filed Aug. 15, 2001, pending, which is incorporated by reference in its entirety.FIELD OF THE INVENTION[0002]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, methods of using the high density output arrays for functional analysis of genetic and protein-protein interactions, and methods of mapping genetic mutations using the arrays.BACKGROUND OF THE INVENTION[0003]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 ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C40B40/02C40B50/06C12N15/81C12Q1/04
CPCC12N15/81C12Q1/04
Inventor BOONE, CHARLES
Owner BOONE CHARLES
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