Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2000-10-06
2004-09-14
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S069100, C435S069700, C435S071100, C435S006120
Reexamination Certificate
active
06790607
ABSTRACT:
BACKGROUND OF THE PRESENT INVENTION
Genetically-based interaction systems are commonly used in scientific research and in commercial and therapeutic applications derived from that research. Current genetically-based interaction systems are severely limited by a fixed level of interaction sensitivity which is either completely “on” or completely “off” (Fields and Song, 1989; Bartel et al., 1993; Gyuris et al., 1993; Mendelsohn and Brent, 1994; Phizicky and Fields, 1995; Bai and Elledge, 1997; Brachmann and Boeke, 1997; Finley and Brent, 1997; Young, 1998). This creates problems related to both the detection of numerous biologically irrelevant interactions, as well as a failure to detect relevant biological interactions. The consequences of this problem may be either a complete inability or prolonged time required to elucidate important biologically relevant interactions, cellular pathways, and potentially related modulatory agents and drugs.
Historically, the first description of a genetic system to detect molecular interactions is the two-hybrid system (Fields and Song, 1989; FIG.
1
). This set forth the original concept and practice of detecting protein-protein interactions in
Saccharomyces cerevisiae.
This original system features detection of an in vivo protein-protein interaction within the nucleus of the yeast cells. These cells were engineered to express the visually detectable bacterial gene lacZ in the presence of an interaction. Basically, the host cells were transformed with an expressible gene coding for a first hybrid protein composed of a DNA binding domain and a first polypeptide. The host cells were additionally transformed with a second hybrid protein consisting of a transcriptional activation domain and a second polypeptide of stable interaction with the first protein fragment. Finally, the cells were also transformed with a lacZ reporter gene containing at least one DNA binding sequence for the DNA binding domain of the first hybrid protein and capable of being transcribed at increased and detectable levels when the transcriptional activation domain of the second hybrid protein was in close proximity. Field and Song demonstrated that when the two hybrid proteins were expressed, levels of the LacZ reporter protein dramatically increased in the host cell. This indicated that the DNA binding domain in the first hybrid protein was binding to the DNA binding sequence of the reporter gene and that the first polypeptide of the first hybrid protein was interacting with the second polypeptide of the second hybrid protein in such a manner as to bring the transcriptional activation domain of the second hybrid protein into proximity of the lacZ gene and thus increase its transcription and subsequent expression.
This basic approach has been employed in all later two-hybrid and three-hybrid systems. Extensions of this work describe such detection capability in nuclear, cytoplasmic, or membrane locations of eukaryotes (Aronheim et al., 1997; Gyuris et al., 1997), as well as in prokaryotes (Bustos and Schleif, 1993; Bunker and Kingston, 1995; Hays et al., 2000). The initial art has also been subsequently extended to include multiple prokaryotic (Bustos and Schleif, 1993; Bunker and Kingston, 1995; Hays et al., 2000) and eukaryotic organisms (other fungal strains, arthropod, plant, and mammalian cells) (e.g. Vasavada et al., 1991; Fearon et al., 1992; Luo et al., 1997; Shoda et al., 2000).
Parallel approaches to genetic molecular interaction detection have been described for detecting protein interactions with RNA and DNA, as well as with small ligands, including peptides and drugs (Li and Herskowitz, 1993; Yang et al., 1995; SenGupta et al., 1996; Brachmann and Boeke, 1997; Young, 1998). All of these systems work on the same basic concept of using the living cell as a means of detecting the interaction between two intracellular molecules.
Genetic molecular detection systems following the original Fields two-hybrid system also usually include the additional feature of genetic selection (Fields and Song, 1989). Selection allows the detection of an interaction by choosing the phenotype of survival; cells containing proteins that do not interact strongly enough or at all are unable to grow, and are no longer considered. The current methods of selection are limited to an “all or nothing” auxotrophic nutrient, antibiotic selection or other means of affecting survival (Fields and Song, 1989; Gyuris et al., 1993; Bai and Elledge, 1997). Selection yields a great advantage for the various detection systems, since cells containing potentially irrelevant pairs of candidate interacting molecules are eliminated without intervention from the scientist or other automated analysis.
However, the introduction of genetic selection introduced a new and severely limiting aspect to the in vivo genetic molecular detection systems. All current methods of selecting for molecular interactions in vivo must make a priori assumptions about the strength of the interactions that they detect. The system must be constructed such that there is a threshold above which an interaction will be detected, and below which it will not. That is, there is an implicit assumption that very weak or transient interactions are probably less likely to be real or important. Systems are designed to exclude these interactions because, if systems are too sensitive, they will detect too much background. However, if the system is not sensitive at all, important interactions will be missed. Those constructing these systems built them and tested them, and then used the systems with the most reasonable compromise of detection sensitivity. In short, they chose the compositions that yielded, on average, a tolerable background while missing a tolerable number of biologically relevant interactions.
Early crude attempts to overcome this “all or nothing” threshold of reporting output have included: (a) exposure of yeast to toxic nutrient analogues at sub-lethal concentrations, for example. 3-AT as a histidine synthesis inhibitor (Mangus et al., 1998); and (b) the creation of complicated genetic modifications of the reporter, which gives several different fixed (nonadjustable) levels of detection (James et al., 1996; Finley and Brent, 1997; Serebriiskii et al., 1999). Such complicated modifications include the use of (b.1.) variable numbers of reporter binding sites, (exemplified by the use of multiple LexA binding sites (by, e.g. multiple LexA binding sites for a Leucine reporter as described in Finley and Brent, 1997), for a Leucine reporter as described in Finely and Brent, 1997), and (b.2) variable distance between reporter building site and the transcriptional start site (West et al., 1984).
A feature of current detection systems is the capacity to turn the detection of protein interactions on or off completely by providing for the expression or lack of expression of the two-hybrid library fusion under standard nutrient conditions. Gyuris et al. (1993) found that by being able to express one of the two hybrid proteins at high levels or by being able to limit expression of one such protein completely, it was possible to show in vivo that the presence of both of the hybrid proteins were necessary for activation of the reporter gene; in other words, they added a switch enabling on or off control of one of the interacting components. This control is useful and exerts its effects by modulating reporter activity, but it does not provide for the continuous adjustability of the sensitivity of a two-hybrid protein interaction system. Thus, the Gyuris system further demonstrates the limitation of the prior art; it is either on or off, above or below the same detection threshold set by the reporters chosen when the system was constructed.
The level of reporter gene expression that will result from any given molecule-molecule interaction in a two-hybrid system is uniform for those molecules used in combination with that reporter. The Brent lab first demonstrated this in experiments using a traditional two-hybrid protein-protein interaction sys
Edwards David N.
Leon Arlene
Ranney David F.
Baker & Botts L.L.P.
Hybrigen, Inc.
Ketter James
Lambertson David
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