A novel genetic system to detect protein-protein interactions.
TL;DR: A novel genetic system to study protein-protein interactions between two proteins by taking advantage of the properties of the GAL4 protein of the yeast Saccharomyces cerevisiae, which may be applicable as a general method to identify proteins that interact with a known protein by the use of a simple galactose selection.
Abstract: Protein-protein interactions between two proteins have generally been studied using biochemical techniques such as crosslinking, co-immunoprecipitation and co-fractionation by chromatography. We have generated a novel genetic system to study these interactions by taking advantage of the properties of the GAL4 protein of the yeast Saccharomyces cerevisiae. This protein is a transcriptional activator required for the expression of genes encoding enzymes of galactose utilization. It consists of two separable and functionally essential domains: an N-terminal domain which binds to specific DNA sequences (UASG); and a C-terminal domain containing acidic regions, which is necessary to activate transcription. We have generated a system of two hybrid proteins containing parts of GAL4: the GAL4 DNA-binding domain fused to a protein 'X' and a GAL4 activating region fused to a protein 'Y'. If X and Y can form a protein-protein complex and reconstitute proximity of the GAL4 domains, transcription of a gene regulated by UASG occurs. We have tested this system using two yeast proteins that are known to interact--SNF1 and SNF4. High transcriptional activity is obtained only when both hybrids are present in a cell. This system may be applicable as a general method to identify proteins that interact with a known protein by the use of a simple galactose selection.
Citations
More filters
TL;DR: In this article, an improved two-hybrid system was employed to isolate human genes encoding Cdk-interacting proteins (Cips) and found that CIP1 is a potent, tight-binding inhibitor of Cdks and can inhibit the phosphorylation of Rb by cyclin A-Cdk2.
5,726 citations
TL;DR: Examination of large-scale yeast two-hybrid screens reveals interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes.
Abstract: Two large-scale yeast two-hybrid screens were undertaken to identify protein-protein interactions between full-length open reading frames predicted from the Saccharomyces cerevisiae genome sequence. In one approach, we constructed a protein array of about 6,000 yeast transformants, with each transformant expressing one of the open reading frames as a fusion to an activation domain. This array was screened by a simple and automated procedure for 192 yeast proteins, with positive responses identified by their positions in the array. In a second approach, we pooled cells expressing one of about 6,000 activation domain fusions to generate a library. We used a high-throughput screening procedure to screen nearly all of the 6,000 predicted yeast proteins, expressed as Gal4 DNA-binding domain fusion proteins, against the library, and characterized positives by sequence analysis. These approaches resulted in the detection of 957 putative interactions involving 1,004 S. cerevisiae proteins. These data reveal interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes. The results of these screens are shown here.
4,877 citations
Cites methods from "A novel genetic system to detect pr..."
...We chose to use the yeast two-hybrid system because it can identify pairs of proteins that physically associate with one anothe...
[...]
TL;DR: Limiting discussion to stress-induced phenylpropanoids eliminates few of the structural classes, because many compounds that are constitutive in one plant species or tissue can be induced by various stresses in another species or in another tissue of the same plant.
Abstract: Phenylpropanoid compounds encompass a wide range of structural classes and biological functions. Limiting discussion to stress-induced phenylpropanoids eliminates few of the structural classes, because many compounds thst are constitutive in one plant species or tissue can be induced by various stresses in another species or in another tissue of the same plant (Beggs et al., 1987; Christie et al., 1994).
4,046 citations
TL;DR: P16 seems to act in a regulatory feedback circuit with CDK4, D-type cyclins and retinoblastoma protein, and inhibits the catalytic activity of theCDK4/cyclin D enzymes.
Abstract: The division cycle of eukaryotic cells is regulated by a family of protein kinases known as the cyclin-dependent kinases (CDKs). The sequential activation of individual members of this family and their consequent phosphorylation of critical substrates promotes orderly progression through the cell cycle. The complexes formed by CDK4 and the D-type cyclins have been strongly implicated in the control of cell proliferation during the G1 phase. CDK4 exists, in part, as a multi-protein complex with a D-type cyclin, proliferating cell nuclear antigen and a protein, p21 (refs 7-9). CDK4 associates separately with a protein of M(r) 16K, particularly in cells lacking a functional retinoblastoma protein. Here we report the isolation of a human p16 complementary DNA and demonstrate that p16 binds to CDK4 and inhibits the catalytic activity of the CDK4/cyclin D enzymes. p16 seems to act in a regulatory feedback circuit with CDK4, D-type cyclins and retinoblastoma protein.
3,716 citations
TL;DR: The comprehensive analysis using a system to examine two-hybrid interactions in all possible combinations between the budding yeast Saccharomyces cerevisiae is completed and would significantly expand and improve the protein interaction map for the exploration of genome functions that eventually leads to thorough understanding of the cell as a molecular system.
Abstract: Protein-protein interactions play crucial roles in the execution of various biological functions. Accordingly, their comprehensive description would contribute considerably to the functional interpretation of fully sequenced genomes, which are flooded with novel genes of unpredictable functions. We previously developed a system to examine two-hybrid interactions in all possible combinations between the approximately 6,000 proteins of the budding yeast Saccharomyces cerevisiae. Here we have completed the comprehensive analysis using this system to identify 4,549 two-hybrid interactions among 3,278 proteins. Unexpectedly, these data do not largely overlap with those obtained by the other project [Uetz, P., et al. (2000) Nature (London) 403, 623-627] and hence have substantially expanded our knowledge on the protein interaction space or interactome of the yeast. Cumulative connection of these binary interactions generates a single huge network linking the vast majority of the proteins. Bioinformatics-aided selection of biologically relevant interactions highlights various intriguing subnetworks. They include, for instance, the one that had successfully foreseen the involvement of a novel protein in spindle pole body function as well as the one that may uncover a hitherto unidentified multiprotein complex potentially participating in the process of vesicular transport. Our data would thus significantly expand and improve the protein interaction map for the exploration of genome functions that eventually leads to thorough understanding of the cell as a molecular system.
3,697 citations
References
More filters
TL;DR: New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors and mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences.
14,954 citations
TL;DR: This work has used recently developed hybridization and restriction endonuclease mapping techniques to demonstrate directly the presence of the transforming DNA in the yeast genome and also to determine the arrangement of the sequences that were introduced.
Abstract: A stable leu2- yeast strain has been transformed to LEU2+ by using a chimeric ColE1 plasmid carrying the yeast leu2 gene. We have used recently developed hybridization and restriction endonuclease mapping techniques to demonstrate directly the presence of the transforming DNA in the yeast genome and also to determine the arrangement of the sequences that were introduced. These studies show that ColE1 DNA together with the yeast sequences can integrate into the yeast chromosomes. This integration may be additive or substitutive. The bacterial plasmid sequences, once integrated, behave as a simple Mendelian element. In addition, we have determined the genetic linkage relationships for each newly introduced LEU2+ allele with the original leu2- allele. These studies show that the transforming squences integrate not only in the leu2 region but also in several other chromosomal locations.
2,179 citations
1,491 citations
TL;DR: The utility of this cloning system is demonstrated by isolating the yeast gene encoding the arginine permease, CAN1, from a pool of random yeast DNA fragments inserted into YEp13.
950 citations
TL;DR: Two short regions of GAL4 are identified, each of which activates transcription when fused to the DNA-binding region of the molecule, which is not required for gene activation.
834 citations