Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-02-18
2001-02-13
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S029000, C435S254200, C435S320100, C435S170000, C435S069100, C536S023100, C536S023400, C536S023500, C530S350000
Reexamination Certificate
active
06187535
ABSTRACT:
The present invention is directed to a method for selecting a prey polypeptide that is able to interact with a bait polypeptide of interest, to a prey polynucleotide encoding the prey polypeptide as well as to the prey polypeptide itself. The invention also concerns plasmids used for performing the method of the invention as well as prokaryotic or eukaryotic recombinant host organisms containing such plasmids and also a collection of said recombinant host organisms consisting in a DNA library, such as a collection of recombinant haploid
Saccharomyces cerevisiae.
Finally, the invention is also directed to a technical medium containing the whole information concerning the interactions between metabolically related bait and prey polypeptides and/or polynucleotides coding for bait and prey polypeptides.
BACKGROUND OF THE INVENTION
Most biological processes involve specific protein-protein interactions. General methodologies to identify interacting proteins or to study these interactions have been extensively developed. Among them, the yeast two-hybrid system currently represents the most powerful in vivo approach to screen for polypeptides that could bind to a given target protein. Originally developed by Fields and coworkers [(Fields et al., 1989; Chien et al., 1991). Two U.S. Pat. No. 5,283,173 granted on Feb. 1, 1994 (Fields, S. & Song, O.) and U.S. Pat. No. 5,468,614 granted on Nov. 21, 1995 (Fields, S. & Song, O.) herein incorporated by reference], the two-hybrid system utilizes hybrid genes to detect protein-protein interactions by means of direct activation of a reporter-gene expression (Allen et al., 1995; Transy et al., 1995). In essence, the two putative protein partners are genetically fused to the DNA-binding domain of a transcription factor and to a transcriptional activation domain, respectively. A productive interaction between the two proteins of interest will bring the transcriptional activation domain in the proximity of the DNA-binding domain and will trigger directly the transcription of an adjacent reporter gene (usually lacZ or a nutritional marker) giving a screenable phenotype. The transcription can be activated through the use of two functional domains of a transcription factor: a domain that recognizes and binds to a specific site on the DNA and a domain that is necessary for activation, as reported by Keegan et al. (1986) and Ma et al. (1987).
Recently, Rossi et al. (1997) described a different approach, a mammalian “two-hybrid” system, which uses &bgr;-galactosidase complementation (Ullmann et al., 1968) to monitor protein—protein interactions in intact eukaryotic cells.
The number of genome sequences of prokaryotic as well as eukaryotic host organisms available is increasing exponentially and there is a great need for new tools directed to the functional and global study of these newly characterized complete or partial genomes. As an illustrative example, the genome of the yeast Saccharomyces cerevisiae is now completely sequenced (Goffeau et al., 1996). Despite the tremendous and successful genetic work in past years, 60% of yeast genes have no assigned function and half of those encode putative proteins without any homology with known proteins (Dujon, 1996). In yeast, genetic analyses, such as suppressor or synthetic lethal screens, have suggested many functional links between gene products, some of which have later been confirmed by biochemical means. All together, these approaches have led to a rather extensive knowledge of defined biochemical pathways. However, the integration of these pathways in the complexity of a living cell remains to be accomplished. To explore the integrative functions and find the molecular factors sustaining them, some authors have attempted to design new screens. However, these screens are usually very specific and cannot apply directly to many different cellular functions. In addition, few yeast genes are essential, leading to an additional difficulty for genetic screens. Other approaches developed by cellular biologists seek to precisely localize proteins within the cell. The assumption is that colocalization of factors is indicative of functional interactions. This approach has been very successful, despite the fact that it is usually very elaborate and is rarely considered practicable for a systematic approach (Burns et al., 1994).
Bartel et al. (1996) extended the approach of the typical two-hybrid system consisting in a known protein that forms a part of a DNA-binding domain hybrid, assayed against a library of all possible proteins present as transcriptional activation domain hybrids, using the genome of bacteriophage T7, such that a second library of all possible proteins fused to the DNA-binding domain to be analyzed. This genome-wide approach to the two-hybrid searches has identified 25 interactions among the proteins of T7.
However, the currently available two-hybrid methodology is not suitable for a large scale project without specific methodological improvements. Although the two-hybrid strategy has been a major tool in proving protein:protein interactions between factors known to be functionally related (Fields and Song, 1989), its use for exhaustive and reliable search for unknown partners of a given protein is more problematic. Thus, in most cases, the two-hybrid screen constitutes an initial screen in which many different interactions are found. Among the identified candidates, only some of them are favored due to their appealing sequence. Subsequent functional assays are required for establishing their possible biological significance. For these reasons, the two-hybrid methodology has been considered a difficult, if not misleading experimental approach for screening.
Finley et al. (1994) or Bendixen et al. (1994) have described two-hybrid systems including a step of mating yeast cell colonies by replica-plating diploids, that is to say by mating colonies of yeast cells. Finley et al. (1994) have, in a first step, selected specific inserts of a DNA library using, as selection criteria, the probability for a specific insert contained in the library to comprise a large coding region of an ORF or to contain a coding region associated with a specific biological function. For example, Finley et al. (1994) have made a collection of strains, each of which expressed a different bait (in fact two cyclin dependent kinases [Cdks], namely DmCdc2 and DmCdc2c), and mated them, by replica-plating, with test strains that contained different activation-tagged Cdis (Cyclin-dependent kinase interactors). Then, each of the selected baits was used as a bait in order to screen the prey Cdis of the DNA library. Examination of the resulting interaction matrices showed that each Cdi (preys) associates specifically with a distinct spectrum of Cdks (baits).
Despite the fact that these authors state that their results suggest a number of applications of their method to genetic characterization of larger sets of proteins, it must be pointed out that these screening experiments of prior art lead to the constitution of interactor polypeptide matrices restricted, as the numerous two-hybrid systems of prior art, by the initial choice of the potentially interesting polynucleotide inserts initially identified and/or initially selected in the DNA library.
Moreover, the replica-plating step that makes use of yeast cell colonies does not allow the mating of numerous different recombinant yeast cell colonies in a single culture dish, thus rendering very fastidious, or even materially impossible the study of potential interactions between a given bait polypeptide and a wide collection of prey polypeptides, such as a collection of prey polypeptides encoded by polynucleotides originating from the whole genome of an organism such as a bacterial, viral or yeast organism.
SUMMARY OF THE INVENTION
The aim of the present invention is to provide a new method for selecting a polynucleotide encoding a prey polypeptide in a two-hybrid screening system, said method making use of mating recombinant haploid yeast cells instead of reco
Fromont Micheline
LeGrain Pierre
Rain Jean-Christophe
Finnegan Henderson Farabow Garrett & Dunner
Horlick Kenneth R.
Institut Pasteur
Siew Jeffrey
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