Identification and comparison of protein—protein interactions that occur in populations and identification of inhibitors of these interactors

• US 6,410,239 B1
• Filed: 12/14/1999
• Issued: 06/25/2002
• Est. Priority Date: 06/14/1996
• Status: Expired due to Fees

• Current Assignee: CuraGen Corporation (Celldex Therapeutics Incorporated)
• Original Assignee: CuraGen Corporation (Celldex Therapeutics Incorporated)
• Inventors: Rothberg, Jonathan Marc, Kalbfleisch, Theodore Samuel, Knight, James Robert, Yang, Meijia, Nandabalan, Krishnan
• Primary Examiner(s): Campbell, Eggerton A.
• Assistant Examiner(s): Chunduru, Suryaprabha
• Application Number:
  US09/461,125
• Time in Patent Office: 924 Days
• Field of Search: 435/6, 435/69.1, 536/22.1
• US Class Current: 435/6.13
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00695 Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

Abstract

Methods are described for detecting protein-protein interactions, among two populations of proteins, each having a complexity of at least 100. Encoded proteins are fused either to the DNA-binding domain of a transcriptional activator or to the activation domain of a transcriptional activator. Two yeast strains, of the opposite mating type and carrying one type each of the fusion proteins are mated together. Productive interactions between the two halves due to protein-protein interactions lead to the reconstitution of the transcriptional activator, which in turn leads to the activation of a reporter gene containing a binding site for the DNA-binding domain. This analysis can be carried out for two or more populations of proteins. The differences in the genes encoding the proteins involved in the protein-protein interactions are characterized, thus leading to the identification of specific protein-protein interactions, and the genes encoding the interacting proteins, relevant to a particular tissue, stage or disease. Furthermore, inhibitors that interfere with these protein-protein interactions are identified by their ability to inactivate a reporter gene. The screening for such inhibitors can be in a multiplexed format where a set of inhibitors will be screened against a library of interactors. Further, information-processing methods and systems are described. These methods and systems pro vide for identification of the genes coding for detected interacting proteins, for assembling a unified database of protein-protein interaction data, and for processing this unified database to obtain protein interaction domain and protein pathway information.

Claims

1. A method of detecting one or more protein-protein interactions comprising

(a) recombinantly expressing within a population of host cells

(i) a first population of first fusion proteins, each said first fusion protein comprising a first protein sequence and a DNA binding domain in which the DNA binding domain is the same in each said first fusion protein, and in which said first population of first fusion proteins has a complexity of at least 100;

and

(ii) a second population of second fusion proteins, each said second fusion protein comprising a second protein sequence and a transcriptional regulatory domain of a transcriptional regulator, in which the transcriptional regulatory domain is the same in each said second fusion protein, such that a first fusion protein is co-expressed with a second fusion protein in host cells, and wherein said host cells contain at least one nucleotide sequence operably linked to a promoter driven by one or more DNA binding sites recognized by said DNA binding domain such that interaction of a first fusion protein with a second fusion protein results in regulation of transcription of said at least one nucleotide sequence by said regulatory domain, and in which said second population of second fusion proteins has a complexity of at least 100;

wherein a host cell comprises a first fusion protein and a second fusion protein, and the number of host cells in the population is sufficiently large to provide confidence at the level of 50% or greater that every pair wise combination of a first fusion protein and a second fusion protein is represented in the population; and

(b) detecting said regulation of transcription of said at least one nucleotide sequence, thereby detecting cells in which an interaction between a first fusion protein and a second fusion protein has occurred.

17. The method described in any one of claims 1, 2, 7, 12, 13 or 14 wherein the confidence provided is at the level of 95% or greater.

2. A method of detecting one or more protein-protein interactions comprising

(a) recombinantly expressing in a first population of yeast cells of a first mating type, a first population of first fusion proteins, each first fusion protein comprising a first protein sequence and a DNA binding domain, in which the DNA binding domain is the same in-each said first fusion protein;

wherein said first population of yeast cells contains a first nucleotide sequence operably linked to a promoter driven by one or more DNA binding sites recognized by said DNA binding domain such that an interaction of a first fusion protein with a second fusion protein, said second fusion protein comprising a transcriptional activation domain, results in increased transcription of said first nucleotide sequence, and in which said first population of first fusion proteins has a complexity of at least 100;

(b) negatively selecting to reduce the number of those yeast cells expressing said first population of first fusion proteins in which said increased transcription of said first nucleotide sequence occurs in the absence of said second fusion protein;

(c) recombinantly expressing in a second population of yeast cells of a second mating type different from said first mating type, a second population of said second fusion proteins, each second fusion protein comprising a second protein sequence and an activation domain of a transcriptional activator, in which the activation domain is the same in each said second fusion protein, and in which said second population of second fusion proteins has a complexity of at least 100;

(d) mating said first population of yeast cells with said second population of yeast cells to form a third population of diploid yeast cells, wherein a diploid cell comprises a first fusion protein and a second fusion protein, wherein said population of diploid yeast cells contains a second nucleotide sequence operably linked to a promoter driven by a DNA binding site recognized by said DNA binding domain such that an interaction of a first fusion protein with a second fusion protein results in increased transcription of said second nucleotide sequence, in which the first and second nucleotide sequences can be the same or different, and wherein the number of diploid cells in the third population is sufficiently large to provide confidence at the level of 50% or greater that every pair wise combination of a first fusion protein and a second fusion protein is represented in the population; and

(e) detecting said increased transcription of said first and/or second nucleotide sequence, thereby detecting cells in which an interaction between a first fusion protein and a second fusion protein has occurred.

3. The method according to claim 2 in which said negatively selecting is carried out by a method comprising incubating said first population of yeast cells expressing said first population of first fusion proteins in an environment in which substantial death of said first population of host cells occurs if said increased transcription occurs.

4. The method according to claim 2 in which said first nucleotide sequence comprises a functional URA3 coding sequence, and said environment contains 5-fluoroorotic acid.

5. The method according to claim 2 in which the first and second populations of fusion proteins are each expressed from chimeric genes comprising cDNA sequences of mammalian RNA.

6. The method according to claim 2 in which the first population of first fusion proteins is expressed from a first plasmid expression vector that expresses a first selectable marker, and the second population of second fusion proteins is expressed from a second plasmid expression vector that expresses a second selectable marker, and in which the first population of yeast cells is incubated in a first environment in which substantial death of yeast cells occurs in the absence of expression of the first selectable marker, and the second population of yeast cells is incubated in a second environment in which substantial death of yeast cells occurs in the absence of expression of the second selectable marker.

8. A method of determining one or more characteristics of or the identities of nucleic acids encoding an interacting pair of proteins from among a plurality of yeast cell colonies, each colony containing nucleic acids encoding a different pair of interacting proteins, said method comprising carrying out the method of claim 2 in which an interaction between a first fusion protein and a second fusion protein is detected in a plurality of colonies of diploid yeast cells, and which method further comprises:

(f) designating each colony in which an interaction between a first fusion protein and a second fusion protein is detected as one point of a multidimensional array in which the intersection of axes in each dimension uniquely identifies a single said colony;

(g) pooling all colonies along a simple axis to form a plurality of pooled colonies;

(h) amplifying from a first aliquot of each pooled colony a plurality of first nucleic acids, each first nucleic acid comprising a sequence encoding said first fusion protein or a portion thereof comprising said first protein sequence;

(i) amplifying from a second aliquot of each pooled colony a plurality of second nucleic acids, each second nucleic acid comprising a sequence encoding said second fusion protein or a portion thereof comprising said second protein sequence;

(j) subjecting said first nucleic acids from each pooled colony to size separation;

(k) subjecting said second nucleic acids from each pooled colony to size separation;

(l) identifying which at least one of said first nucleic acids are present in samples of first nucleic acids from a pooled colony from axes in each dimension, thereby indicating that said at least one first nucleic acid is present in said array in the colony designated at the intersection of said axes in each dimension;

(m) identifying which at least one of said second nucleic acids are present in samples of a second nucleic acid from a pooled colony from axes in each dimension, thereby indicating that the said at least one second nucleic acid is present in said array in the colony designated at the intersection of said axes in each dimension;

in which the first and second nucleic acids that are indicated to be present in said array in a colony designated at the same intersection are indicated to encode interacting protein sequences.

10. The method according to claim 8 which further comprises subjecting said pooled colonies of first nucleic acids to a method for identifying, classifying, or quantifying one or more nucleic acids in a sample, said method comprising:

(a) probing said sample with one or more recognition means, each recognition means causing recognition of a target nucleotide subsequence or a set of target nucleotide subsequences;

(b) generating one or more signals from said sample probed by said recognition means, each generated signal arising from a nucleic acid in said sample and comprising a representation of (i) the identities of effective subsequences, each said effective subsequence being a subsequence comprising a target subsequence, or the identities of sets of effective subsequences, each said set having member effective subsequences each of which comprises a different target subsequence from one of said sets of target sequences, and (ii) the length between occurrences of effective subsequences in said nucleic acid or between one occurrence of one effective subsequence and the end of said nucleic acid; and

(c) searching a nucleotide sequence database to determine sequences that match or the absence of any sequences that match said one or more generated signals, said database comprising a plurality of known nucleotide sequences of nucleic acids that may be present in the sample, a sequence from said database matching a generated signal when the sequence from said database has both (i) the same length between occurrences of effective subsequences or the same length between one occurrence of one effective target subsequence and the end of the sequence as is represented by the generated signal, and (ii) the same effective subsequences as are represented by the generated signal, or effective subsequences that are members of the same sets of effective subsequences as are represented by the generated signal,

whereby said one or more nucleic acids in said sample are identified, classified, or quantified.

7. A method of detecting one or more protein-protein interactions comprising

(a) introducing into a first population of cells of Saccharomyces cerevisiae a first population of first plasmids, each said first plasmid encoding and capable of expressing in the first population of cells (i) TRP1, and (ii) a first population of first fusion proteins, each said first fusion protein comprising a GAL4 DNA binding domain and a first protein sequence, in which said first population of first fusion proteins has a complexity of at least 100, and in which said first population of cells (i) is of a first mating type selected from the group consisting of a and α

, (ii) is mutant in endogenous URA3 and IHS3, (iii) contains functional URA3 coding sequences under the control of a promoter containing GAL4 binding sites, and (iv) contains functional lacZ coding sequences under the control of a promoter containing GAL4 binding sites;

(b) introducing into a second population of cells of Saccharomyces cerevisiae a second population of second plasmids, each said second plasmids encoding and capable of expressing in the second population of cells (i) LEU2, and (ii) a second population of second fusion proteins, each said second fusion protein comprising a GAL4 transcriptional activation domain and a second protein sequence, in which said second population of second fusion proteins has a complexity of at least 100, and in which said second population of cells (i) is of a second mating type different from said first mating type and selected from the group consisting of a and α

, (ii) is mutant in endogenous URA3 and HIS3, (iii) contains functional HIS3 coding sequences under the control of a promoter containing GAL4 binding sites, and (iv) contains functional lacZ coding sequences under the control of a promoter containing GAL4 binding sites;

(c) after step (a), incubating said first population of cells in an environment lacking tryptophan and containing 5-fluoroorotic acid;

(d) pooling surviving cells from said first population after step (c);

(e) after step (b), incubating said second population of cells in an environment lacking leucine;

(f) pooling surviving cells from said second population after step (e);

(g) mating the pooled cells from said first population and the pooled cells from said second population by mixing the cells together, applying the cells to a solid medium and incubating the cells, to form diploid cells, wherein a diploid cell comprises a first fusion protein and a second fusion protein and wherein the number of diploid cells is sufficiently large to provide confidence at the level of 50% or greater that every pair wise combination of a first fusion protein and a second fusion protein is represented in the population; and

(h) incubating the diploid cells in an environment lacking uracil, histidine, tryptophan and leucine, to select diploid cells containing a said first plasmid and a said second plasmid and in which transcription of the URA3 and HIS3 coding sequences has been activated, thereby indicating that a first fusion protein has interacted with a second fusion protein within the diploid cell, thereby detecting one or more protein-protein interactions.

9. A method of determining one or more characteristics of or the identities of DNA molecules encoding an interacting pair of proteins from among a plurality of yeast cell colonies, each colony containing DNA molecules encoding a different pair of interacting proteins, comprising carrying out the method of claim 7 in which an interaction between a first fusion protein and a second fusion protein is detected in a plurality of colonies of diploid yeast cells, and which method further comprises:

(f) designating each colony in which an interaction between a first fusion protein and a second fusion protein is detected as one point of a multidimensional array in which the intersection of axes in each dimension uniquely identifies a single said colony;

(g) pooling all colonies along a simple axis to form a plurality of pooled colonies;

(h) amplifying from a first aliquot of each pooled colony a plurality of first DNA molecules, each first DNA molecule comprising a sequence encoding said first fusion protein or a portion thereof comprising said first protein sequence;

(i) amplifying from a second aliquot of each pooled colony a plurality of second DNA molecules, each second DNA molecule comprising a sequence encoding said second fusion protein or a portion thereof comprising said second protein sequence;

(j) subjecting said first DNA molecules from each pooled colony to size separation;

(k) subjecting said second DNA molecules from each pooled colony to size separation;

(l) identifying which at least one of said first DNA molecules are present in samples of first DNA molecules from a pooled colony from axes in each dimension, thereby indicating that said at least one first DNA molecule is present in said array in the colony designated at the intersection of said axes in each dimension;

(m) identifying which at least one of said second DNA molecules are present in samples of a second DNA molecule from a pooled colony from axes in each dimension, thereby indicating that the said at least one second DNA molecule is present in said array in the colony designated at the intersection of said axes in each dimension;

in which the first and second DNA molecules that are indicated to be present in said array in a colony designated at the same intersection are indicated to encode interacting protein sequences.

11. The method according to claim 9 which further comprises subjecting said pooled colonies of first DNA molecules to a method for identifying, classifying, or quantifying one or more DNA molecules in a sample, said method comprising:

(a) probing said sample with one or more recognition means, each recognition means causing recognition of a target nucleotide subsequence or a set of target nucleotide subsequences;

(b) generating one or more signals from said sample probed by said recognition means, each generated signal arising from a nucleic acid in said sample and comprising a representation of (i) the identities of effective subsequences, each said effective subsequence being a subsequence comprising a target subsequence, or the identities of sets of effective subsequences, each said set having member effective subsequences each of which comprises a different target subsequence from one of said sets of target sequences, and (ii) the length between occurrences of effective subsequences in said nucleic acid or between one occurrence of one effective subsequence and the end of said nucleic acid; and

(c) searching a nucleotide sequence database to determine sequences that match or the absence of any sequences that match said one or more generated signals, said database comprising a plurality of known nucleotide sequences of nucleic acids that may be present in the sample, a sequence from said database matching a generated signal when the sequence from said database has both (i) the same length between occurrences of effective subsequences or the same length between one occurrence of one effective target subsequence and the end of the sequence as is represented by the generated signal, and (ii) the same effective subsequences as are represented by the generated signal, or effective subsequences that are members of the same sets of effective subsequences as are represented by the generated signal,

whereby said one or more nucleic acids in said sample are identified, classified, or quantified.

12. A method of detecting one or more protein-protein interactions comprising

(a) recombinantly expressing within a population of host cells

(i) a first population of first fusion proteins, each said first fusion protein comprising a first protein sequence and a DNA binding domain in which the DNA binding domain is the same in each said first fusion protein, and in which said first population of first fusion proteins has a complexity of at least 500;

and

(ii) a second population of second fusion proteins, each said second fusion protein comprising a second protein sequence and a transcriptional regulatory domain of a transcriptional regulator, in which the transcriptional regulatory domain is the same in each said second fusion protein, such that a first fusion protein is co-expressed with a second fusion protein in host cells, and wherein said host cells contain at least one nucleotide sequence operably linked to a promoter driven by one or more DNA binding sites recognized by said DNA binding domain such that interaction of a first fusion protein with a second fusion protein results in regulation of transcription of said at least one nucleotide sequence by said regulatory domain, and in which said second population of second fusion proteins has a complexity of at least 500;

wherein a host cell comprises a first fusion protein and a second fusion protein, and the number of host cells in the population is sufficiently large to provide confidence at the level of 50% or greater that every pair wise combination of a first fusion protein and a second fusion protein is represented in the population; and

(b) detecting said regulation of transcription of said at least one nucleotide sequence, thereby detecting cells in which an interaction between a first fusion protein and a second fusion protein has occurred.

13. A method of detecting one or more protein-protein interactions comprising

(a) recombinantly expressing in a first population of yeast cells of a first mating type, a first population of first fusion proteins, each first fusion protein comprising a first protein sequence and a DNA binding domain, in which the DNA binding domain is the same in each said first fusion protein;

wherein said first population of yeast cells contains a first nucleotide sequence operably linked to a promoter driven by one or more DNA binding sites recognized by said DNA binding domain such that an interaction of a first fusion protein with a second fusion protein, said second fusion protein comprising a transcriptional activation domain, results in increased transcription of said first nucleotide sequence, and in which said first population of first fusion proteins has a complexity of at least 500;

(b) negatively selecting to reduce the number of those yeast cells expressing said first population of first fusion proteins in which said increased transcription of said first nucleotide sequence occurs in the absence of said second fusion protein;

(c) recombinantly expressing in a second population of yeast cells of a second mating type different from said first mating type, a second population of said second fusion proteins, each second fusion protein comprising a second protein sequence and an activation domain of a transcriptional activator, in which the activation domain is the same in each said second fusion protein, and in which said second population of second fusion proteins has a complexity of at least 500;

(d) mating said first population of yeast cells with said second population of yeast cells to form a third population of diploid yeast cells wherein a diploid cell comprises a first fusion protein and a second fusion protein, wherein said population of diploid yeast cells contains a second nucleotide sequence operably linked to a promoter driven by a DNA binding site recognized by said DNA binding domain such that an interaction of a first fusion protein with a second fusion protein results in increased transcription of said second nucleotide sequence, in which the first and second nucleotide sequences can be the same or different, and wherein the number of diploid cells in the third population is sufficiently large to provide confidence at the level of 50% or greater than every pair wise combination of a first fusion protein and a second fusion protein is represented in the population; and

(e) detecting said increased transcription of said first and/or second nucleotide sequence, thereby detecting cells in which an interaction between a first fusion protein and a second fusion protein has occurred.

15. A method of determining one or more characteristics of or the identities of nucleic acids encoding an interacting pair of proteins from among a plurality of yeast cell colonies, each colony containing nucleic acids encoding a different pair of interacting proteins, said method comprising carrying out the method of claim 13 which an interaction between a first fusion protein and a second fusion protein is detected in a plurality of colonies of diploid yeast cells, and which method further comprises:

(f) designating each colony in which an interaction between a first fusion protein and a second fusion protein is detected as one point of a multidimensional array in which the intersection of axes in each dimension uniquely identifies a single said colony;

(g) pooling all colonies along a simple axis to form a plurality of pooled colonies;

(h) amplifying from a first aliquot of each pooled colony a plurality of first nucleic acids, each first nucleic acid comprising a sequence encoding said first fusion protein or a portion thereof comprising said first protein sequence;

(i) amplifying from a second aliquot of each pooled colony a plurality of second nucleic acids, each second nucleic acid comprising a sequence encoding said second fusion protein or a portion thereof comprising said second protein sequence;

(j) subjecting said first nucleic acids from each pooled colony to size separation;

(k) subjecting said second nucleic acids from each pooled colony to size separation;

(l) identifying which at least one of said first nucleic acids are present in samples of first nucleic acids from a pooled colony from axes in each dimension, thereby indicating that said at least one first nucleic acid is present in said array in the colony designated at the intersection of said axes in each dimension;

(m) identifying which at least one of said second nucleic acids are present in samples of a second nucleic acid from a pooled colony from axes in each dimension, thereby indicating that the said at least one second nucleic acid is present in said array in the colony designated at the intersection of said axes in each dimension;

in which the first and second nucleic acids that are indicated to be present in said array in a colony designated at the same intersection are indicated to encode interacting protein sequences.

14. A method of detecting one or more protein-protein interactions comprising

(a) introducing into a first population of cells of Saccharomyces cerevisiae a first population of first plasmids, each said first plasmid encoding and capable of expressing in the first population of cells (i) TRP1, and (ii) a first population of first fusion proteins, each said first fusion protein comprising a GAL4 DNA binding domain and a first protein sequence, in which said first population of first fusion proteins has a complexity of at least 500, and in which said first population of cells (i) is of a first mating type selected from the group consisting of a and α

, (ii) is mutant in endogenous URA3 and HIS3, (iii) contains functional URA3 coding sequences under the control of a promoter containing GAL4 binding sites, and (iv) contains functional lacZ coding sequences under the control of a promoter containing GAL4 binding sites;

(b) introducing into a second population of cells of Saccharomyces cerevisiae a second population of second plasmids, each said second plasmids encoding and capable of expressing in the second population of cells (i) LEU2, and (ii) a second population of second fusion proteins, each said second fusion protein comprising a GAL4 transcriptional activation domain and a second protein sequence, in which said second population of second fusion proteins has a complexity of at least 500, and in which said second population of cells (i) is of a second mating type different from said first mating type and selected from the group consisting of a and , (ii) is mutant in endogenous URA3 and HIS3, (iii) contains functional HIS3 coding sequences under the control of a promoter containing GAL4 binding sites, and (iv) contains functional lacZ coding sequences under the control of a promoter containing GALA binding sites;

(c) after step (a), incubating said first population of cells in an environment lacking tryptophan and containing 5-fluoroorotic acid;

(d) pooling surviving cells from said first population after step (c);

(e) after step (b), incubating said second population of cells in an environment lacking leucine;

(f) pooling surviving cells from said second population after step (e);

(g) mating the pooled cells from said first population and the pooled cells from said second population by mixing the cells together, applying the cells to a solid medium and incubating the cells, to form diploid cells, wherein a diploid cell comprises a first fusion protein and a second fusion protein and wherein the number of diploid cells is sufficiently large to provide confidence at the level of 50% or greater that every pair wise combination of a first fusion protein and second fusion protein is represented in the population; and

(h) incubating the diploid cells in an environment lacking uracil, histidine, tryptophan and leucine, to select diploid cells containing a said first plasmid and a said second plasmid and in which transcription of the URA3 and HIS3 coding sequences has been activated, thereby indicating that a first fusion protein has interacted with a second fusion protein within the diploid cell, thereby detecting one or more protein-protein interactions.

16. A method of determining one or more characteristics of or the identities of DNA molecules encoding an interacting pair of proteins from among a plurality of yeast cell colonies, each colony containing DNA molecules encoding a different pair of interacting proteins, comprising carrying out the method of claim 14 in which an interaction between a first fusion protein and a second fusion protein is detected in a plurality of colonies of diploid yeast cells, and which method further comprises:

(f) designating each colony in which an interaction between a first fusion protein and a second fusion protein is detected as one point of a multidimensional array in which the intersection of axes in each dimension uniquely identifies a single said colony;

(g) pooling all colonies along a simple axis to form a plurality of pooled colonies;

(h) amplifying from a first aliquot of each pooled colony a plurality of first DNA molecules, each first DNA molecule comprising a sequence encoding said first fusion protein or a portion thereof comprising said first protein sequence;

(i) amplifying from a second aliquot of each pooled colony a plurality of second DNA molecules, each second DNA molecule comprising a sequence encoding said second fusion protein or a portion thereof comprising said second protein sequence;

(j) subjecting said first DNA molecules from each pooled colony to size separation;

(k) subjecting said second DNA molecules from each pooled colony to size separation;

(l) identifying which at least one of said first DNA molecules are present in samples of first DNA molecules from a pooled colony from axes in each dimension, thereby indicating that said at least one first DNA molecule is present in said array in the colony designated at the intersection of said axes in each dimension;

(m) identifying which at least one of said second DNA molecules are present in samples of a second DNA molecule from a pooled colony from axes in each dimension, thereby indicating that the said at least one second DNA molecule is present in said array in the colony designated at the intersection of said axes in each dimension;

in which the first and second DNA molecules that are indicated to be present in said array in a colony designated at the same intersection are indicated to encode interacting protein sequences.