Applications of interaction traps/two-hybrid systems to biotechnology research

Mendelsohn, Andrew R; Brent, Roger

doi:10.1016/0958-1669(94)90061-2

Cited by 56 publications

(33 citation statements)

References 32 publications

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“…Our results obtained from the yeast two-hybrid system contrast with those of Quilliam et al (1999), who failed to see any interaction in the yeast two-hybrid system between constitutively active M-Ras Q71L and RalGDS, A-Raf and particularly Rin1. This could be related to dierences in the two-hybrid systems, as we used a Gal4-based two-hybrid system whereas Quilliam et al (1999) used a LexA-based system (Mendelsohn and Brent, 1994). Co-immunoprecipitation studies with fulllength proteins should resolve these discrepancies.…”

Section: Discussionmentioning

confidence: 99%

A novel potential effector of M-Ras and p21 Ras negatively regulates p21 Ras-mediated gene induction and cell growth

et al. 2001

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Here, we report the identi®cation and characterization of a new member of the RalGDS-family, which is widely expressed and interacts strongly and selectively with the GTP-bound forms of M-Ras and p21 Ras. This Ras pathway modulator (RPM), also termed RGL3, exhibited Ras-binding and catalytic domains typical of the RalGDS-family of guanine nucleotide exchange factors, and was most similar to Rlf (RalGDS-like factor), but was distinguished by a unique proline-rich region with multiple candidate SH3-domain binding sites. RPM/ RGL3 resembled AF-6 and Nore1 in interacting strongly with constitutively active M-Ras and p21 Ras. In contrast to Rlf, transiently expressed RPM/RGL3 did not activate an Elk-1-inducible reporter gene alone or in combination with activated p21 Ras, but strongly inhibited induction of this reporter gene by co-expression of activated H-Ras or MEKK-1. This inhibitory eect was independent of the Ras binding domain and required a second signal provided by p21 Ras or MEKK-1, but not Raf-1 or M-Ras. Expression of RPM/RGL3 also strongly inhibited cell growth of ®broblasts transformed by an activated Src Y527F. Thus, RPM/RGL3 is a novel potential eector of both p21 Ras and M-Ras with the novel function of negatively regulating Elk-1-dependent gene induction downstream of p21 Ras or MEKK-1. Oncogene (2001) 20, 188 ± 197.

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Section: Discussionmentioning

confidence: 99%

A novel potential effector of M-Ras and p21 Ras negatively regulates p21 Ras-mediated gene induction and cell growth

et al. 2001

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“…Two approaches were used to identify NS5A-interacting proteins. The first approach involved the yeast two-hybrid system; details are provided in Materials and Methods (18). A LexA DNA-binding domain-NS5A (1a H77) fusion protein was used to a HeLa cDNA library whose translatable products are fused with an acidic transcriptional activation domain.…”

Section: Resultsmentioning

confidence: 99%

Cellular cofactors affecting hepatitis C virus infection and replication

Randall

Panis²,

Cooper

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

496

454

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Recently identified hepatitis C virus (HCV) isolates that are infectious in cell culture provide a genetic system to evaluate the significance of virus-host interactions for HCV replication. We have completed a systematic RNAi screen wherein siRNAs were designed that target 62 host genes encoding proteins that physically interact with HCV RNA or proteins or belong to cellular pathways thought to modulate HCV infection. This includes 10 host proteins that we identify in this study to bind HCV NS5A. siRNAs that target 26 of these host genes alter infectious HCV production >3-fold. Included in this set of 26 were siRNAs that target Dicer, a principal component of the RNAi silencing pathway. Contrary to the hypothesis that RNAi is an antiviral pathway in mammals, as has been reported for subgenomic HCV replicons, siRNAs that target Dicer inhibited HCV replication. Furthermore, siRNAs that target several other components of the RNAi pathway also inhibit HCV replication. MicroRNA profiling of human liver, human hepatoma Huh-7.5 cells, and Huh-7.5 cells that harbor replicating HCV demonstrated that miR-122 is the predominant microRNA in each environment. miR-122 has been previously implicated in positively regulating the replication of HCV genotype 1 replicons. We find that 2-O-methyl antisense oligonucleotide depletion of miR-122 also inhibits HCV genotype 2a replication and infectious virus production. Our data define 26 host genes that modulate HCV infection and indicate that the requirement for functional RNAi for HCV replication is dominant over any antiviral activity this pathway may exert against HCV.antivirals ͉ miR-122 ͉ RNAi ͉ HCVcc-siRNA H epatitis C virus (HCV) has a notable ability to establish persistent infections in Ϸ70% of cases, resulting in 130 million chronically infected people throughout the world (1). This prevalence has spurred considerable interest in the study of HCV-host interactions, on both cellular and molecular levels. The inability to grow HCV in cell culture led some groups to focus on the identification of cellular proteins that interact with individual HCV proteins or RNA elements, resulting in the accumulation of a large number of putative HCV-host interactions. Unfortunately, the significance of most of these with respect to the HCV life cycle is currently unknown (reviewed in ref.2). Over the past 6 years, cell culture systems have been developed that enable the characterization of HCV replication and entry (3-6). This effort recently culminated in the development of cell culture systems that reproduce the entire viral life cycle (7-9). A number of virus-host interactions have been characterized by using these experimental systems. For example, CD81 has been demonstrated to play a role in HCV entry (10-12).Sequence-specific gene silencing of RNAi is ideal for assessing the genetic phenotypes associated with virus-host interactions. We have previously shown that siRNAs are highly effective at silencing either host or viral RNAs in cells that contain replicating HCV, demonstrating th...

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“…As a second example, in targeted examination of the interaction of a single activation domain-fused protein with two defined partners (for example, interaction of activation domain-fused cyclin D with LexA-fused CDK4 and cI-fused CDK6), a randomly mutagenized pool of activation domainfused partners could be screened to identify mutations that disrupt interaction with either one or both of the partner proteins. As a third example, one source of interest in two-hybrid systems is their use in drug screening approaches to identify compounds that disrupt interactions between discrete pairs of interacting proteins (8,32,33); dual bait reagents would apply a simultaneous control to the specificity of such interactions.…”

Section: Resultsmentioning

confidence: 99%

A Two-hybrid Dual Bait System to Discriminate Specificity of Protein Interactions

Serebriiskii

Khazak²,

Golemis

1999

Journal of Biological Chemistry

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Biological regulatory systems require the specific organization of proteins into multicomponent complexes. Two hybrid systems have been used to identify novel components of signaling networks based on interactions with defined partner proteins. An important issue in the use of two-hybrid systems has been the degree to which interacting proteins distinguish their biological partner from evolutionarily conserved related proteins and the degree to which observed interactions are specific. We adapted the basic two-hybrid strategy to create a novel dual bait system designed to allow single-step screening of libraries for proteins that interact with protein 1 of interest, fused to DNA binding domain A (LexA), but do not interact with protein 2, fused to DNA binding domain B ( cI). Using the selective interactions of Ras and Krev-1(Rap1A) with Raf, RalGDS, and Krit1 as a model, we systematically compared LexA-and cI-fused baits and reporters. The LexA and cI baitr reporter systems are well matched for level of bait expression and sensitivity range for interaction detection and allow effective isolation of specifically interacting protein pairs against a nonspecific background. These reagents should prove useful to refine the selectivity of library screens, to reduce the isolation of false positives in such screens, and to perform directed analyses of sequence elements governing the interaction of a single protein with multiple partners.

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Applications of interaction traps/two-hybrid systems to biotechnology research

Cited by 56 publications

References 32 publications

A novel potential effector of M-Ras and p21 Ras negatively regulates p21 Ras-mediated gene induction and cell growth

A novel potential effector of M-Ras and p21 Ras negatively regulates p21 Ras-mediated gene induction and cell growth

Cellular cofactors affecting hepatitis C virus infection and replication

A Two-hybrid Dual Bait System to Discriminate Specificity of Protein Interactions

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