WO2007019603A1

WO2007019603A1 - A method for detecting the interaction of a polypeptide of interest with a target nucleotide sequence

Info

Publication number: WO2007019603A1
Application number: PCT/AU2006/001130
Authority: WO
Inventors: Sergiy Lopato
Original assignee: Australian Centre for Plant Functional Genomics Pty Ltd
Current assignee: Australian Centre for Plant Functional Genomics Pty Ltd
Priority date: 2005-08-12
Filing date: 2006-08-10
Publication date: 2007-02-22
Anticipated expiration: 2008-02-12

Abstract

The present invention relates generally to a method for detecting the interaction of a polypeptide of interest with a target nucleotide sequence. More particularly, the present invention relates to a one-hybrid screening method. The present invention is predicated, in part, on a modification to a standard one-hybrid screening protocol, which involves utilising a mating step to introduce the library construct and reporter construct into the same cell. This modification improves the efficiency of the screen and eliminates the labour intensive step of isolating the library plasmids from the library host strain and subsequently introducing the isolated plasmids into the reporter strain.

Description

A method for detecting the interaction of a polypeptide of interest with a target nucleotide sequence

FIELD OF THE INVENTION

The present invention relates generally to a method for detecting the interaction of a polypeptide of interest with a target nucleotide sequence. More particularly, the present invention relates to a one-hybrid screening method.

BACKGROUND OF THE INVENTION

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

The yeast one-hybrid system is a genetic assay used for, inter alia, isolating novel genes, which encode proteins that bind to a DNA target such as a cis- regulatory element or any other short DNA binding sequence. The one-hybrid assay is highly sensitive because detection of the interactions between the protein and DNA target occur while proteins are in their native configurations in vivo. Furthermore, the one-hybrid system offers the particular advantage of making the gene encoding the DNA binding protein of interest immediately available after screening a library.

Yeast one-hybrid assays, typically first involve the generation of a "reporter strain". The yeast strain used for the generation of the reporter strain is typically auxotrophic for histidine, or another nutritional requirement. The reporter strain is then constructed by inserting a sequence consisting of tandem copies of a DNA target sequence upstream of the HIS3 reporter gene, which are operably connected to minimal promoters. Expression of the HIS3 reporter gene is assayed by looking for rescue of the histidine auxotrophic phenotype. Subsequently, the reporter constructs comprising the DNA target, minimal promoter and reporter gene are integrated site-specifically into the yeast genome to create yeast "reporter strains".

After construction of a reporter strain, cDNA candidates of interest, which putatively encode proteins that bind to the DNA target sequence, are ligated to a nucleotide sequence encoding a GAL4 activator and the cDNA:GAL4 AD sequences are maintained in a plasmid vector in a "library host strain". The plasmids comprising the nucleic acid sequences which encode the cDNA:GAL4 AD fusion proteins are then isolated from the library host cell before the isolated library plasmids are transformed into the yeast reporter strain.

After transformation of the library construct into the reporter strain, the transformed reporter strain is plated onto a selection medium that lacks histidine. If a fusion protein encoded by a particular cDNA:GAL4-AD library construct interacts with the target DNA sequence incorporated into the reporter construct, the activating domain in the fusion protein activates expression of the HIS3 reporter gene in the reporter construct. When expressed, this gene allows the reporter strain, carrying this particular library construct, to grow on minimal medium lacking histidine. If no interaction between the fusion protein and the DNA target occurs, then the HIS3 gene is not expressed and the normally histidine-auxotrophic yeast reporter strain is unable to grow on the selection medium. If a reporter strain comprising both the HIS3 and LacZ reporter genes is used, an "overlay" β-galactosidase assay can be subsequently performed to verify the DNA-protein interaction and help eliminate false positives.

However, the yeast one-hybrid systems in the prior art are typically labour intensive, as they rely on the isolation of the library plasmids from the library host strain followed by subsequent transformation of the isolated library plasmids into the reporter strain to allow the protein-DNA interaction. It would also be desirable, for example, to be able to use a single library for both one- hybrid and two-hybrid screens. Furthermore, it would also be advantageous to improve the confirmation assay, which is reliant on a 3-galactosidase overlay, which is labour intensive and cannot be performed directly on the primary selection medium.

SUMMARY OF THE INVENTION

The present invention is predicated, in part, on a modification to a standard one- hybrid screening protocol, which involves utilising a mating step to introduce the library construct and reporter construct into the same cell. This modification improves the efficiency of the screen and eliminates the labour intensive step of isolating the library plasmids from the library host strain and subsequently introducing the isolated plasmids into the reporter strain.

Therefore, in one aspect, the present invention provides a method for detecting the interaction of a polypeptide of interest with a nucleic acid binding site, the method comprising the steps of: providing a library-host cell comprising one or more library constructs, each library construct comprising a nucleotide sequence encoding a polypeptide; providing a reporter cell, which is a mating partner of the library-host cell, the reporter cell comprising at least one primary reporter construct comprising a nucleotide sequence defining a known or putative nucleic acid binding site for at least part of the polypeptide encoded by the library construct, and at least one downstream reporter gene, wherein binding of the polypeptide encoded by the library construct to the nucleic acid binding site effects modulation of expression of the reporter gene; mating the library-host cell and the reporter cell so as to result in inter-cell transfer of the library construct and/or reporter construct, such that the library construct and reporter construct are both present in the same cell; allowing the cell comprising both the library construct and reporter construct to express the polypeptide encoded by the library construct; and - A -

determining whether the polypeptide encoded by the library construct binds to the nucleic acid binding site by assaying the expression of the reporter gene, wherein the level of expression of the reporter gene is indicative of the binding of the polypeptide to the nucleic acid binding site.

Preferably, the fusion polypeptide encoded by the library construct encodes a polypeptide of interest, which is a known or putative binding partner of the nucleic acid binding site in the reporter construct, fused to an amino acid sequence defining a transcription activating domain which is capable of upregulating expression of a reporter gene in a reporter construct.

In another aspect, the present invention also contemplates the use of one or more secondary reporter constructs that can be used in conjunction with a primary reporter construct to, inter alia, detect false positive and false negative results. Preferably, the expression of the reporter gene in the secondary reporter construct can be assayed on the same medium as the reporter gene in the primary reporter construct.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows vector maps of the plNT-HIS3NB vector (panel A) and a modified plNT-HIS3NB vector, plNT-HIS3NBGate (panel B), which incorporates lambda phage attR sites to facilitate recombination-mediated integration of a test binding site sequence into the reporter construct. DESCRIPTION OF PREFERRED EMBODIMENTS

It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the above description.

As set out above, in one aspect, the present invention provides a method for detecting the interaction of a polypeptide of interest with a nucleic acid binding site, the method comprising the steps of: providing a library-host cell comprising one or more library constructs, each library construct comprising a nucleotide sequence encoding a polypeptide; providing a reporter cell, which is a mating partner of the library-host cell, the reporter cell comprising at least one primary reporter construct comprising a nucleotide sequence defining a known or putative nucleic acid binding site for at least part of the polypeptide encoded by the library construct, and at least one downstream reporter gene, wherein binding of the polypeptide encoded by the library construct to the nucleic acid binding site effects modulation of expression of the reporter gene; mating the library-host cell and the reporter cell so as to result in inter-cell transfer of the library construct and/or reporter construct, such that the library construct and reporter construct are both present in the same cell; allowing the cell comprising both the library construct and reporter construct to express the polypeptide encoded by the library construct; and determining whether the polypeptide encoded by the library construct binds to the nucleic acid binding site by assaying the expression of the reporter gene, wherein the level of expression of the reporter gene is indicative of the binding of the polypeptide to the nucleic acid binding site.

The present invention is predicated, in part, on the ability of the library-host strain and the reporter strain being able to transfer nucleic acids, eg. DNA. As such, the present invention contemplates any library host cell and reporter cell that are able to transfer nucleic acids, such as at least the library construct and/or reporter construct.

Preferably, the library-host cell and the reporter cell are yeast cells. More preferably, the yeast cell is a yeast cell of the genus Saccharomyces, more preferably the yeast cell is from the species Saccharomyces cerevisiae. In another preferred embodiment, the yeast cell may be from the species Schizosaccharomyces pombe. Therefore, in a preferred aspect, the present invention provides a yeast one-hybrid screening method.

Budding yeast, such as Saccharomyces spp. can live with either two genomes (diploid) or one genome (haploid). In either case, reproduction may occur via mitotically generated "buds".

Haploid yeast cells can exist in one of two mating types, either "a" or "alpha (α)". The mating type is determined by the particular allele present at the MAT locus in the yeast. Haploid cells can live, and reproduce via budding, in the haploid state. However, if two haploid cells of opposite mating types meet, they can fuse and enter the diploid phase of the cell cycle. Cells in the diploid phase are more resistant to harsh environmental conditions. However, when diploid cells begin to run out of nutrient, they undergo meiosis, forming four haploid spores in an ascus. When more amenable environmental conditions return, the spores germinate producing four haploid yeast cells, two of each mating type.

Preferably, the yeast cells used in accordance with the present invention are haploid yeast cells. In one preferred embodiment of the invention, the library host cell comprises an "a" allele at the MAT locus and the reporter cell comprises an "α" allele at the MAT locus. In an alternative preferred embodiment, the library host cell comprises an "α" allele at the MAT locus and the reporter cell comprises an "a" allele at the MA T locus. Exemplary MATa and MATα yeast strains that may be used in accordance with the present invention, are known in the art. For example, suitable MATa and MA Ta yeast strains may be identified by searching for the MATa or MATα genotype in the ATCC Yeast Genetic Stock Special Collection (http://www.atcc.org/common/catalog/yeastGeneticStock/yeastGeneticStocklnd ex.cfm).

In accordance with the present invention, it has been identified that Saccharomyces cerevisiae AH 109 (genotype: MATa, trp-901, leu2-3, 112, ura3- 52, his3-200, gal4Δ, galβOA, LYS2::GAL1 UA_S-GAL 1 _TATA-HIS3, GAL2_UAS- GAL2_TATA-ADE2, URA3::GAL1 _UAS-GAL 1_TATA-lacZ, MEL1) and Saccharomyces cerevisiae Y187 (genotype: MATα, ura3-52, his3-Δ200, ade2-101, trp1-901, Ieu2-3, 112) are particularly useful as library host strains and reporter strains. These strains are known in the art, and would be readily obtained by one of skill in the art. For example, AH109 and Y187 yeast strains are commercially available from Clontech.

Therefore, in one embodiment, the library-host cell is an AH 109 (MATa) yeast cell or a derivative thereof and the reporter cell is a MATα mating partner of the library host cell. In another embodiment, the library host cell is a Y187 (MATα) yeast cell or a derivative thereof and the reporter cell is a MATa mating partner of the library host cell.

As referred to herein, a "derivative" of a yeast strain includes any yeast strain that is derived from a parent strain and includes a modified yeast strain, such as a yeast strain generated by mutation, genetic modification and the like of the parent strain. Preferably, the "derivative" is at least of the same mating type, ie. MATa or MATα as the parent strain.

In an alternate embodiment, the reporter cell is an AH 109 (MATa) yeast cell or a derivative thereof and the library-host cell is a MATα mating partner of the reporter cell. In yet another embodiment, the reporter cell is a Y187 (MATα) yeast cell or a derivative thereof and the library-host cell is a MATa mating partner of the reporter cell.

Exemplary MATa mating partners of the Y187 strain include AH109, HF7c, CG- 1945, Y190 and SFY526. Exemplary MATα mating partners of the AH109 strain include Y187 and Y184.

In one particularly preferred embodiment, when the library-host cell is an AH109 (MATa) yeast cell or a derivative thereof, the reporter cell is a Y187 (MATα) yeast cell or a derivative thereof. In an alternate particularly preferred embodiment, when the library-host cell is a Y187 (MATα) yeast cell or a derivative thereof, the reporter cell is an AH109 (MATa) yeast cell or a derivative thereof.

Controlled crosses of MATa and MATα haploid strains may be carried out using any convenient method known in the art. For example, matings may be simply carried out by mixing approximately equal amounts of each strain on a complete medium and incubating the mixture at 30O for at least 6 hr. Prototrophic diploid colonies can then be selected on appropriate synthetic media if the haploid strains contain complementing auxotrophic markers.

Although haploid MA Ta and MA Ta yeast cells, including AH109 and Y187 yeast cells, are preferred library host cells and reporter cells, the method of the present invention may use any library host cell and reporter cell that can transfer DNA via mating. For example, the methods of the present invention may also be applied to bacterial one-hybrid systems, such as those described by Obrist and Naberhaus (J Bacterid 187(1 1 ): 3807-3813, 2005).

As set out above, the method of the present invention contemplates a library construct that incorporates a nucleotide sequence encoding a polypeptide. Preferably, the polypeptide encoded by the library construct encodes a fusion polypeptide comprising a polypeptide of interest, which is a known or putative binding partner of the nucleic acid binding site in the reporter construct, fused to an amino acid sequence defining a transcription activating domain.

The polypeptide of interest may be any length peptide sequence. As such the "polypeptide" of interest may be a short peptide, a polypeptide, a protein motif, a protein domain, a protein fragment, a truncated protein or a complete protein.

In preferred embodiments of the invention, one or more members of a cDNA library encode the polypeptide of interest. Methods for generating cDNA libraries are well known in the art. Exemplary methods for the generation of cDNA libraries include methods disclosed in: Zhu et al. (Biotechniques 30(4): 892-897, 2001 ); Patanjali et al. (Proc. Natl. Acad. Sci. USA 88: 1943-1947, 1991 ); McCarry and Williams (Curr Opin Biotechnol., 5(1 ): 34-39, 1994); and Suzuki and Sagano (Methods MoI Biol. 175: 143-153, 2001 ).

The library construct preferably comprises a nucleotide sequence encoding a polypeptide of interest fused to a transcription activating domain. The term "transcription activating domain", also referred to herein as "AD", should be understood to encompass any polypeptide domain that is capable of interacting with a transcriptional initiation complex in a manner that increases the rate of transcription of a nucleotide sequence. These interactions often result in bending the DNA, which is believed to help open the double helix and facilitate the initiation of transcription. Transcriptional activation domains typically interact with the preinitiation complex to increase the frequency of transcriptional initiation. As such, the present invention contemplates a library construct, which includes a nucleotide sequence encoding a polypeptide of interest fused to any transcription activating domain. Exemplary transcription activating domains which may be used in accordance with the present invention include: acidic domains such as the GAL4 activating domain (GAL4-AD); glutamine-rich domains including the Sp1 activating domains; proline-rich domains including the CTF activating domain; type I activation domains such as the GAL4, Sp1 , CTF and SW6 activation domains; type HA activation domains such as the Tat activation domain; type HB activation domains such as the VP16, p53 and E2F1 activation domains; the GCN4 activating domain; the ADR1 activating domain; the B42 transactivation domain; and the like.

The library construct may be constructed using standard molecular biology methods that would be readily ascertained by one of ordinary skill in the art (for example see Sambrook and Russell, Molecular Cloning - A Laboratory Manual, 3^rd Ed., Cold Spring Harbor Laboratory Press, 2000). However, in one preferred embodiment, a cDNA insert is incorporated into the library construct via homologous recombination occurring between the library construct and the cDNA insert. In one particularly preferred embodiment, the cDNA insert is prepared and inserted into the library construct using the method described by Zhu et al. (Biotechniques 30(4): 892-897, 2001 ).

The library construct may further comprise other genetic elements, which facilitate its construction and/or maintenance in one or more different cloning hosts. Examples of such elements include origins or replication, origins or transfer, selectable marker genes and the like. Furthermore, the library construct may also include one or more control sequences adapted to effect expression of the library construct in the library host strain, reporter strain and/or mating progeny of the library host and reporter strains.

As used herein, the term "control sequences" should be understood to include all components known in the art, which are necessary or advantageous for the transcription, translation and or post-translational modification of the controlled nucleotide sequence or the transcript or polypeptide encoded thereby. Each control sequence may be native or foreign to the controlled nucleotide sequence. The control sequences may include a leader, polyadenylation sequence, propeptide sequence, promoter, enhancer or upstream activating sequence, signal peptide sequence, and transcription terminator. Typically, a control sequence at least includes a promoter.

The term "promoter" as used herein, describes any nucleic acid that confers, activates or enhances expression of a nucleotide sequence in a cell. Promoters are generally positioned 5' (upstream) to the nucleotide sequences that they control. In the construction of heterologous promoter/structural gene combinations, it is generally preferred to position the promoter at a distance from a gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, ie. the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, ie. the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.

The promoter may regulate the expression of the transgene constitutively or differentially with respect to the developmental stage at which expression occurs, or in response to external stimuli such as chemical inducers, physiological stresses, pathogens, metal ions, and the like. As such, the promoter used in accordance with the methods of the present invention may include a constitutive promoters such as the GAL4, ADH 1 and GAL1 promoters, an inducible promoter such as the Met25 promoter, or a developmentally regulated promoter.

As set out above, the method of the present invention also contemplates the use of at least one primary reporter construct comprising a nucleotide sequence defining a known or putative nucleic acid binding site for at least part of the polypeptide encoded by the library construct, and at least one downstream reporter gene, wherein binding of the polypeptide encoded by the library construct to the nucleic acid binding site effects modulation of expression of the reporter gene.

"Modulation of expression of the reporter gene" includes upregulation and downregulation of transcription of the reporter gene itself and/or translation of the mRNA transcript of the reporter gene. However, in a preferred embodiment, modulation of expression of the reporter gene at least includes modulation of the transcription of the reporter gene.

Preferably, the primary reporter construct comprises a nucleotide sequence defining a putative nucleic acid binding site for the polypeptide of interest, which has a minimal promoter operably linked thereto that drives the transcription of a downstream reporter gene when the polypeptide encoded by the library construct is bound to the putative nucleic acid binding site.

As used herein, the term "minimal promoter" should be understood to include any promoter that does not substantially drive the expression of an operably connected gene itself, but rather requires the binding of a transcription activator to a c/s-acting control sequence, eg. a transcriptional activator binding site. However, as would be understood by one of skill in the art, a minimal promoter may be "leaky" and effect a low basal level of transcription even in the absence of the transcription activator.

In one preferred embodiment, the minimal promoter is a eukaryotic minimal promoter, which incorporates at least a TATA box and transcription initiation site and optionally one or more CAAT boxes.

The primary reporter construct may incorporate any suitable reporter gene and, therefore, the term "reporter gene" as used herein contemplates any nucleic acid sequence which, when transcribed and optionally translated, generates a detectable and/or selectable signal or phenotype. For example, the reporter gene may encode: an auxotrophic rescue gene (eg. HIS3) which, when θxpressed, enables an auxotrophic reporter strain (such as S. cerevisiae AH 109 or S. cerevisiae Y187) to grow on nutritionally selective media; an enzyme which acts on a particular substrate to generate a detectable (eg. coloured) product, such as β-galactosidases, α-galactosidases, glucoamylases and the like; a protein which can be directly detected such as a fluorescent protein including GFP, EGFP, RFP, YFP, CFP and the like; a polypeptide comprising one or more particular epitopes that may be detected with a labelled antibody; and the like. Specific examples of reporter genes that are particularly useful in the modified one hybrid method of the present invention include HIS3, ADE2, /acZ and MEL 1.

Typically, positive clones in a one-hybrid screen are selected by their ability to activate the transcription of a reporter gene that enables an auxotrophic reporter strain to grow on nutritionally selective media, ie. the reporter gene comprises an auxotroph rescue gene. Therefore, in one preferred embodiment of the invention, at least one of the reporter constructs in the reporter strain comprises an auxotroph rescue gene.

In one particularly preferred embodiment, at least one primary reporter construct comprises a histidine auxotroph rescue gene, more preferably the histidine auxotroph rescue gene comprises a HIS3 gene or a functional homolog thereof.

As referred to herein, a "HIS3 gene or functional homolog thereof includes any nucleic acid molecule, which encodes the enzyme imidazoleglycerol-phosphate dehydratase, that catalyses the sixth step in histidine biosynthesis, and which comprises at least 50% nucleotide sequence identity to GenBank Accession

No. NC_001 147 REGION: 721946..722608. More preferably, the HIS3 gene or functional homolog thereof comprises at least 65% sequence identity, yet more preferably at least 80% sequence identity, even more preferably at least 90% sequence identity, and even more preferably at least 95% sequence identity and most preferably at least 100% sequence identity to GenBank Accession No. NC_001 147 REGION: 721946..722608. When comparing sequence identity, the sequences should be compared over a comparison window of at least 100 nucleotides, more preferably at least 200 nucleotides, yet more preferably at least 300 nucleotides, even more preferably at least 500 nucleotides and most preferably over the full length of the compared sequences. In a particularly preferred embodiment, sequences are compared using the BLAST algorithm described by Altschul et al. (J MoI Biol 215: 403- 410, 1990).

The present invention also contemplates the use of two or more reporter constructs in the reporter strain, wherein each reporter construct incorporates a different reporter gene. The subsequent or secondary reporter constructs may be used, for example, to verify a DNA-polypeptide interaction and help eliminate false negative or false positive results. Therefore, in another preferred embodiment, the reporter strain also comprises one or more secondary reporter constructs.

The secondary reporter constructs contemplated by the present invention may be used as either a positive reporter or a negative reporter.

By "positive reporter" is meant that the secondary reporter gene is linked to the putative nucleic acid binding site of the polypeptide of interest in a similar manner to the primary reporter. In this arrangement, specific binding of the AD fusion polypeptide (incorporating the polypeptide of interest) to the putative nucleic acid binding site would activate at least one or both of the primary reporter gene and the secondary reporter gene. In this way, a false negative caused by a failure to activate the primary reporter gene may be detected by activation of the secondary reporter gene.

Therefore, in one embodiment, the reporter strain further comprises at least one secondary reporter construct comprising a nucleotide sequence defining a known or putative nucleic acid binding site for the polypeptide of interest, which has a minimal promoter operably linked thereto that drives the expression of at least one downstream reporter gene when the fusion polypeptide is bound to the putative nucleic acid binding site.

By "negative reporter" is meant that the secondary reporter gene is linked to a nucleic acid sequence that is not a known binding site of the polypeptide of interest. In this way, specific association of the AD fusion polypeptide with the putative nucleic acid binding site should activate the primary reporter gene, but not the secondary reporter gene. However, promiscuous or non-specific binding of the AD fusion polypeptide to non-target nucleotide sequences may be indicated by activation of both the primary and the secondary reporter gene. In this way, a negative secondary reporter gene may be used to detect false positive results caused by non-specific binding of the AD fusion.

Therefore, in another embodiment, the reporter strain further comprises at least one secondary reporter construct comprising a nucleotide sequence, other than the known or putative nucleic acid binding site for the polypeptide of interest, which has a minimal promoter operably linked thereto that drives the expression of at least one downstream reporter gene when the fusion polypeptide is bound to the putative nucleic acid binding site.

Typically, the E. coli lacZ gene is used as a secondary reporter gene in one- hybrid screening assays. When this reporter gene is used, the colonies growing on the selection plates are assayed for the activation of the reporter gene lacZ in a second screen by a filter-lift assay. Accordingly, in another embodiment of the invention, at least one reporter construct used in the reporter strain comprises a lacZ reporter gene.

However, in another aspect, the present invention contemplates a modification to the standard one-hybrid protocol, wherein the modification improves the efficiency of the method and eliminates the labour intensive step of the lacZ reporter gene overlay assay, the modification comprising the use of one or more secondary reporter constructs, which may be used in conjunction with a primary auxotrophic rescue reporter gene, wherein the expression of the reporter gene in the secondary reporter construct can be assayed on the same medium as the reporter gene in the primary reporter construct. In one preferred embodiment, the secondary reporter construct comprises a reporter gene which can be assayed on an auxotroph selection medium.

In a more preferred embodiment of the present invention, an α-galactosidase encoding reporter gene is used as a secondary reporter gene. The use of α- galactosidase as a secondary reporter gene allows the activity of the reporter gene to be assessed directly on nutritional selection plates by supplementing these plates with 5-Bromo-4-Chloro-3-indolyl α-D-galactopyranoside (X-α-gal) as described by Aho et al. (Anal. Biochem. 253: 270-272, 1997).

X-α-Gal is a chromogenic substrate for α-galactosidase (also known as melibiase or alpha-D-galactoside galactohydrolase, EC 3.2.1 .22), an enzyme that enables yeast to use the disaccharide melibiose as a carbon source during growth or fermentation. In the yeast Saccharomyces cerevisiae, the MEL1 gene encodes this enzyme. Secretion of this enzyme leads to hydrolysis of X-α-Gal in the medium causing yeast colonies to develop a blue colour.

Accordingly, in one particularly preferred embodiment, the reporter gene that encodes α-galactosidase comprises a MEL 1 gene or functional homolog thereof.

As referred to herein, a "MEL 1 gene or functional homolog thereof" includes any nucleic acid molecule which encodes α-galactosidase and which comprises at least 50% nucleotide sequence identity to GenBank Accession No. X03102. More preferably, the MEL1 gene or functional homolog thereof comprises at least 65% sequence identity, yet more preferably at least 80% sequence identity, even more preferably at least 90% sequence identity, and even more preferably at least 95% sequence identity and most preferably at least 100% sequence identity to GenBank Accession No. X03102. When comparing sequence identity, the sequences should be compared over a comparison window of at least 100 nucleotides, more preferably at least 200 nucleotides, yet more preferably at least 500 nucleotides, even more preferably at least 1000 nucleotides and most preferably over the full length of the compared sequences. In a particularly preferred embodiment, sequences are compared using the BLAST algorithm described by Altschul et al. (J MoI Biol 215: 403- 410, 1990).

The one or more reporter constructs used in the reporter strain each may be either integrated into a chromosome of the reporter strain or may be maintained as an autonomous genetic element, eg. an autonomously replicating plasmid. In one preferred embodiment, one or more of the reporter constructs is integrated into the a chromosome of the reporter cell. In this preferred embodiment, the reporter construct is present as a single copy, there is no need to maintain selection for the reporter construct in the cell, and the DNA-protein interaction takes place in the nucleus of the reporter cell.

One particularly preferred reporter construct, into which a DNA target sequence may be incorporated for use in accordance with the present invention, is the plNT-HIS3NB plasmid (Figure 1 ).

The reporter constructs of the present invention may be constructed using standard molecular biology methods that would be readily ascertained by one of ordinary skill in the art (for example see Sambrook and Russell, Molecular Cloning - A Laboratory Manual, 3^rd Ed., Cold Spring Harbor Laboratory Press, 2000). Also, site specific recombination methods, including site-specific lambda phage recombination may be used to assemble all or part of the reporter construct. Exemplary site-specific lambda phage recombination protocols are described in the Clontech Gateway Technology brochure (Version E, dated 22 September 2003). In one embodiment, lambda phage aff-site mediated recombination is used to insert the nucleotide sequence defining a known or putative nucleic acid binding site into the reporter construct.

As would be evident to one of skill in the art, the present invention is in no way limited to the particular sequence of the nucleic acid target sequences or the amino acid sequences of the polypeptides encoded in the library.

In light of the foregoing, one of skill in the art would appreciate that the methods of the present invention may be used to identify polypeptides that interact with a known nucleic acid binding site sequence. As such, in another aspect, the present invention provides polypeptides that interact with a known nucleic acid binding site, wherein the polypeptide is identified according to the method of the present invention.

Furthermore, the one-hybrid systems, such as the method of the present invention, make the gene encoding a polypeptide of interest immediately available after screening a library. For example, once a positive clone is identified, the library construct of the positive clone may be isolated and sequenced. Accordingly, in another aspect, the present invention also extends to an isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide of interest that has been identified according to the method of the present invention.

Alternatively, when a known polypeptide of interest is used, the present invention may also be used to identify a nucleic acid binding site for the protein of interest. Therefore, the present invention should also be understood to extend to an isolated nucleic acid molecule comprising a nucleotide sequence, which defines a nucleic acid binding site for a known polypeptide of interest, wherein the nucleic acid binding site is identified in accordance with the method of the present invention. As would be evident to one of skill in the art, the methods of the present invention are suitable for detecting any protein-nucleic acid interactions, such as the interaction of transcription factors, DNA replication factors, DNA repair factors and the like with their nucleic acid binding targets. Again, as would be evident to one of skill the art, the present invention allows the identification of proteins that bind to a nucleic acid binding target as monomers or multimers (either homo- or hetero-).

Although the present invention is described with particular reference to one- hybrid screening methods, the utilisation of a mating step as described in the present invention, would also be applicable to a range of other protein-nucleic acid interaction screening methodologies that involve an inter-cell transfer of a

DNA construct. For example, the utilization of a mating step, as described in the methods of the present invention, may also be adapted to translational repression assays which identify interactions between a protein and an RNA target, such as the method of Paraskeva ef a/. (Proc Natl Acad Sci USA 95:

951 -956, 1998).

Finally, reference is made to standard textbooks of molecular biology that contain methods for carrying out basic techniques encompassed by the present invention, including DNA restriction and ligation for the generation of the various genetic constructs described herein. See, for example, Maniatis ef a/.,

Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press,

New York, 1982) and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^nd Ed. (Cold Spring Harbor Laboratory Press, New York, 1989).

The present invention is further exemplified by the following non-limiting examples: EXAMPLE 1 Generation of a yeast reporter strain

DNA fragments (e.g. a mapped part of a promoter) were generated using PCR amplification with primers having introduced restriction sites. These fragments were then cloned into the Λ/ofl-Spel unique restriction sites of the plNT-HIS3NB plasmid (Figure 1 ). This plasmid provides the advantage of being able to perform the cloning in one step and allows the use of the same vector in both bacteria and yeast.

Alternatively, the fragments may be cloned into a modified plNT-HIS3NB vector which incorporates lambda phage att sites. For example, Figure 1 B shows a modified plNT-HIS3NB vector, plNT-HIS3NBGate, which incorporates two attR sites. These sites may be used to recombine with a donor vector having the fragment of interest flanked by attL sites. The attL on the donor vector and attR sites in the plNT-HIS3NB vector would recombine to insert the fragment of interest between the attR sites in the plNT-HIS3NB vector.

In the case of well-characterized short specific DNA sequences, such as cis- elements, the oligonucleotides are designed such that after annealing they will generate 2-4 tandem repeats of the c/s-element with sticky ends for Noti and Spe\ sites. These tandem repeated sequences may then be cloned into Λ/ofl- Spe\ restriction sites of the plasmid, with successful insertions being selected by PCR and confirmed by sequencing.

Transformation of the reporter construct into yeast was performed by growing yeast strain Y187 (MATα ura3-52 his3-Δ200 ade2-101 trp1-901 Ieu2-3, 112; Harper et al., Cell 75: 805-816, 1993) before introducing the plasmid into the Y187 reporter strain using a lithium acetate transformation procedure as described below. For yeast transformation using lithium acetate (LiAc), overnight cultures were set up by transferring a large colony of the strain of interest (eg. y187) into 100 ml YPDA in 250 ml conical flask, followed by incubation with shaking overnight at 30⁹C. After incubation, cells were pelleted by centrifugation at 2000 rpm for 5min in 50 ml tubes. The cell pellets were then resuspended in 50 ml of fresh YPDA by vortexing before being pooled into a 250 ml flask and incubated at 30 °C shaking for a further 2-3 hours. The cells were then washed by centrifugation at 2000 rpm for 5 min and resuspension in autoclaved distilled water. The cells were then again centrifuged and resuspended in 2 ml TE/LJAc.

Herring Testes Carrier DNA (ClonTech) was boiled for 10 minutes, to denature, before being cooled on ice. 1 ug of DNA for transformation (in 1 -2 ul) and 5 ul Herring DNA was then added to 100 ul competent yeast cells in TE/LiAc and the mixture was vortexed. 60OuI PEG/TE/LiAc was added before vortexing again followed by incubation at 30O for 30 min with shaking. 7OuI DMSO was then added before vortexing again.

The cells were then subjected to a heat shock at 42 °C for 15 min before placing into ice for 2 min. The cells were then centrifuged for 30 sec at full speed. The pellet was then washed with 780 ul TE, before being resuspended in 50OuI TE.

2OuI and 20OuI of the potentially transformed cells were then plated onto SD plates, which were grown inverted at 30O. For co-transformation with multiple two plasmids, 200 ul or more may be used per plate as it is less efficient. Visible colonies were seen after 2-3 days.

Colonies strongly growing on the YAPD-G418 plates after 3 days of incubation at 30⁹C were selected. These colonies represent transformants, which contain the reporter construct, incorporating the c/s-element of interest, stably integrated into their genomic DNA. Glycerol cultures of the transformed strains were stored at -80⁹C until needed. EXAMPLE 2 Construction of the cDNA library using SMART cDNA synthesis technology and recombination-mediated cloning in yeast

cDNA synthesis from total or poly A+ RNA was performed using either the oligo dT (CDSIII primer, Clontech) or a random oligo (CDSIII/6 primer, Clontech). The SMARTIII oligonucleotide, which has an oligo(G) sequence at its 3' end was used to extend a template. The resulting ss cDNA contained the complementary 5' end of the mRNA as well as the sequence complementary to the SMART III oligo, which then served as universal priming site in the subsequent amplification by long-distance PCR (SMART technology, Clontech). This methodology provides the advantages of: (i) the generated library is enriched with full length sequences; (ii) the library can be prepared from a very small amount of tissue; (iii) the oligo dT and SMART primers do not recognize ribosomal RNA, therefore, no amplification from ribosomal RNA occurs and the library prepared from total RNA contains no cDNA clones complementary to rRNA; and (iv) the library can be stored in aliquots as a yeast glycerol cultures for at least several years at -80O.

A GAL4 AD fusion library was then produced by co-transforming AH 109 yeast (MATa, trp-901, leu2-3, 112, ura3-52, his3-200, gal4A, galδOΛ, LYS2::GAL1_UAs- GAL 1 _TATA-HIS3, GAL2_UAS-GAL2_TATA-ADE2, URA3::GAL 1UA_S-GAL 1 _TATA-lacZ, MEL1; James et ai, Genetics 144: 1425-1436, 1996) with the SMART ds cDNA described above and a Sma\ linearised form of pGADT7-Rec (Clontech). The linearised plasmid and cDNA recombine in vivo to form a closed circular expression vector which incorporates the cDNA fused to the GAL4 AD. Transformants carrying a circular form of the vector are selected on leucine deficient media such that colonies containing plasmids with cDNA inserts, but not the empty plasmids, grow. This methodology allows more efficient cloning of the library into the library vector as there is no ligation of adaptors or other operations. Furthermore, as described below, the AH109 strain (MATa) can be used for mating with Y187 strain, or any other (MATα) reporter strain.

EXAMPLE 3 Yeast one-hybrid screen using yeast mating

Before mating of the library and reporter strains, the concentration of 3-AT required to reduce possible leaky expression of the HIS3 reporter gene was determined. Specifically, G418-resistant colonies were streaked out onto selective media (SD) and on SD-His plates containing different concentrations of 3-amino-1 ,2,4-triazole (3-AT). After incubation of plates at 3OO for one week, the concentration that is required to reduce growth was determined to be 5-10 mM 3-AT.

The library host strain (AH 109) and the reporter strain (Y187) were then mated. Specifically, glycerol cultures of the library strain and reporter strain were thawed before rich YPDA media with Kan (50μg/ml) was added. The cultures were then incubated together with rotation (30-50 rpm) for 12-24 hours at 30⁹C.

After incubation, the cells were then washed with water or TE buffer, re- suspended in a small volume of TE buffer and spread on SD (-Leu, -His, + 5- 10mM 3-AT) plates. Colonies that appeared after 3-5 days of incubation at 3OO were streaked out on a new plate with the same media containing X-α-GAL. However, this step could be excluded if X-α-GAL was included in the original nutritional selection medium.

In this protocol, a MEL1 gene operably connected to a non-target sequence was used as a negative reporter and, accordingly, white colonies (which do not express MEL1 ) were selected and used for further analysis. The mating method described herein has significant advantages over the isolation of the library plasmids from the library host and subsequent transformation of the reporter strain with the isolated library plasmids, including: (i) the mating is less time and labour consuming than yeast transformation with the cDNA library as a plasmid mix; (ii) the mating provides higher screen efficiency (>6 000 000 clones can be screened in one experiment); and (iii) the MEL 1 gene can be used instead of, or in parallel with, lacZ for particularly efficient primary selection of true positives; (iv) The same cDNA library can be used to identify transcription factors/ DNA binding polypeptides using the yeast one-hybrid screen and to identify interacting partners of the transcription factor/DNA binding polypeptide using the yeast two-hybrid screen, that is the same library strain can be used for both one-hybrid and two-hybrid screens.

Once putative positive clones are identified using the one-hybrid method described above, further analysis may be performed on the clones, including: (i) isolation of the library plasmids from positive clones to identify the nucleic acids encoding the putative DNA-binding polypeptides; (ii) elimination of colonies bearing the same library construct by PCR and restriction analysis; (iii) transformation of the plasmids from positive clones into E. coli and purification of the plasmid DNA; (iv) confirmation of the interaction of a polypeptide with a particular DNA c/s-element by direct transformation of a specific plasmid into a reporter strain; (v) sequencing the cDNA inserts of positive clones; (vi) if the DNA c/s-element was not previously analysed earlier (eg. a novel c/s-element), confirmation of the one-hybrid result in vitro, using a gel-shift assay.

EXAMPLE 4

Yeast one-hybrid screening for HD-Zip proteins using well-characterized cis- elements

The one-hybrid method described herein was used to screen for HD-Zip proteins in a cDNA library from wheat embryos at 0-6 DAP using an Arabidopsis-όeύveό cis-element as a bait sequence.

The DNA binding site cis-elements of HD-Zip proteins are highly conserved in different plant species and the DNA-protein binding is very strong and specific. The HD-Zip proteins have diverse functions. For example, HD-Zip IV (HD-GB2, GLABRA2-like factors) are involved in the regulation of epidermal cell fate, while some HD-Zip III have a role in vascular differentiation. Furthermore, HD- Zip I and Il might be specifically related to the regulation of developmental adaptations to environmental changes. However, the cis-elements to which these proteins bind are relatively conserved between HD-Zip families, as shown in Table 1 .

Table 1 - DNA binding site cis-elements for different HD-Zip protein families.

Transcriptional DNA cis-acting Organism Reference factor/family elements

HD-Zip family I -CAAT(A/T)ATTG- Arabidopsis Sessa et al. EMBO J.

12(9): 3507-17, 1993 HD-Zip family Il -CAAT(G/C)ATTG- Arabidopsis Sessa et al. J MoI Biol.

274(3): 303-9, 1997

Based on the sequences above, a "4x HD-Zip recognition element" was developed for use as a bait sequence in the reporter construct. This 4x HD-Zip recognition element had the sequence 5'-

(CAATAATTG)(CAATTATTG)(CAATCATTG)(CAATGATTG)-S'

Using the methods described in the previous examples, about 200 white positive colonies, carrying library constructs derived from a cDNA library prepared from developing wheat grain, were generated. The majority of these positive colonies contained the full length coding region for one of several HD- Zip proteins. Three different proteins (two HD-Zip I and one HD-Zip II) and several homeologues of these proteins were identified, one of which is not represented in EST-databases.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

Also, it must be noted that, as used herein, the singular forms "a", "an" and "the" include plural aspects unless the context already dictates otherwise. Thus, for example, reference to "a library host cell" or "a reporter cell" includes a single library host cell or reporter cell as well as two or more of such cells; "a "reporter construct" or "library construct" includes a single construct as well as two or more constructs; while "a nucleotide sequence defining a known or putative nucleic acid binding site" may include a single binding site or multiple binding sites; and so forth.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1 . A method for detecting the interaction of a polypeptide of interest with a nucleic acid binding site, the method comprising the steps of: providing a library-host cell comprising one or more library constructs, each library construct comprising a nucleotide sequence encoding a polypeptide; providing a reporter cell, which is a mating partner of the library-host cell, the reporter cell comprising at least one primary reporter construct comprising a nucleotide sequence defining a known or putative nucleic acid binding site for at least part of the polypeptide encoded by the library construct, and at least one downstream reporter gene, wherein binding of the polypeptide encoded by the library construct to the nucleic acid binding site effects modulation of expression of the reporter gene; mating the library-host cell and the reporter cell so as to result in inter-cell transfer of the library construct and/or reporter construct, such that the library construct and reporter construct are both present in the same cell; allowing the cell comprising both the library construct and reporter construct to express the polypeptide encoded by the library construct; and determining whether the polypeptide encoded by the library construct binds to the nucleic acid binding site by assaying the expression of the reporter gene, wherein the level of expression of the reporter gene is indicative of the binding of the polypeptide to the nucleic acid binding site.

2. The method of claim 1 wherein the polypeptide encoded by the library construct comprises a polypeptide of interest fused to an amino acid sequence defining a transcription activating domain.

3. The method of claim 1 or 2 wherein at least one primary reporter construct comprises a nucleotide sequence defining a putative nucleic acid binding site for at least part of the polypeptide encoded by the library construct, which has a minimal promoter operably linked thereto that drives the expression of a downstream reporter gene when the polypeptide encoded by the library construct is bound to the putative nucleic acid binding site.

4. The method of any one of claims 1 to 3 wherein the library-host cell is a yeast cell.

5. The method of claim 4 wherein the library host cell is a MATa yeast cell.

6. The method of claim 5 wherein the MATa yeast cell is an AH109 yeast cell or a derivative thereof.

7. The method of claim 4 wherein the library host cell is a MA Ta yeast cell.

8. The method of claim 7 wherein the MATa yeast cell is a Y187 yeast cell.

9. The method of any one of claims 1 to 3 wherein the reporter cell is a yeast cell.

10. The method of claim 9 wherein the reporter cell is a MATa yeast cell.

1 1 . The method of claim 10 wherein the MATa yeast cell is an AH109 yeast cell or a derivative thereof.

12. The method of claim 9 wherein the reporter cell is a MATα yeast cell.

13. The method of claim 12 wherein the MATα yeast cell is a Y187 yeast cell.

14. The method of any one of claims 1 to 13 wherein the library host cell is a MATa yeast cell and the reporter cell is a MATα yeast cell.

15. The method of claim 14 wherein the library-host cell is an AH 109 yeast cell or a derivative thereof and the reporter cell is a Y187 yeast cell or a derivative thereof.

16. The method of any one of claims 1 to 13 wherein the library-host cell is a MATayeasl cell and the reporter cell is a MATa yeast cell.

17. The method of claim 16 wherein the library-host cell is a Y187 yeast cell or a derivative thereof and the reporter cell is a AH 109 yeast cell or a derivative thereof.

18. The method of any one of claims 1 to 17 wherein at least one primary reporter construct comprises a reporter gene selected from an auxotrophic rescue gene; a gene encoding an enzyme which acts on a particular substrate to generate a detectable product; a gene encoding a fluorescent protein; gene encoding a polypeptide comprising one or more particular epitopes that may be detected with a labelled antibody.

19. The method of claim 18 wherein at least one primary reporter construct comprises an auxotroph rescue gene as a reporter gene.

20. The method of claim 19 wherein the auxotroph rescue gene comprises a histidine auxotroph rescue gene.

21 . The method of claim 20 wherein the histidine auxotroph rescue gene comprises a HIS3 gene or a functional homolog thereof.

22. The method of any one of claims 1 to 21 wherein the reporter strain further comprises at least one secondary reporter construct comprising a nucleotide sequence defining a known or putative nucleic acid binding site for the polypeptide of interest, which has a minimal promoter operably linked thereto that drives the expression of at least one downstream reporter gene when the fusion polypeptide is bound to the putative nucleic acid binding site.

23. The method of claim 22 wherein expression of the reporter gene in the secondary reporter construct is indicative of specific binding of the fusion polypeptide to the putative nucleic acid binding site.

24. The method of any one of claims 1 to 21 wherein the reporter strain further comprises at least one secondary reporter construct comprising a nucleotide sequence, other than the known or putative nucleic acid binding site for the polypeptide of interest, which has a minimal promoter operably linked thereto that drives the expression of at least one downstream reporter gene when the fusion polypeptide is bound to the putative nucleic acid binding site.

25. The method of claim 24 wherein expression of the reporter gene in the secondary reporter construct is indicative of non-specific binding of the fusion polypeptide to nucleotide sequences.

26. The method of any one of claims 22 to 25 wherein the expression of the reporter gene in the secondary reporter construct may be assayed on the same medium as the reporter gene in a primary reporter construct.

27. The method of any one of claims 22 to 26 wherein the secondary reporter construct comprises a reporter gene encoding an enzyme that acts on a particular substrate to generate a detectable product.

28. The method of claim 27 wherein the reporter gene encodes α- galactosidase.

29. The method of claim 28 wherein the reporter gene comprises a MEL 1 gene or functional homolog thereof.

30. A polypeptide that interacts with a known nucleic acid binding site, wherein the polypeptide is identified according to the method of any one of claims 1 to 29.

31 . An isolated nucleic acid molecule comprising a nucleotide sequence that encodes the polypeptide of claim 30.

32. An isolated nucleic acid molecule comprising a nucleotide sequence, which defines a nucleic acid binding site for a known polypeptide of interest, wherein the nucleic acid binding site is identified in accordance with the method of any one of claims 1 to 29.