View - ResearchGate

314 Serebriiskii et al.results obtained with nondigested PCR, and load the digestion products on a 1.5%agarose gel. Run out the DNA fragments a sufficient distance to get good resolutionof DNA products in the 200–1000-bp size range. This will generally yield distinctiveand unambiguous groups of inserts, confirming whether multiple isolates of a smallnumber of cDNAs have been obtained.20. Primer FPP1 provides enough specificity to sequence library plasmid even fromthis crude mixture.21. It is possible to use 5 µL of unpurified plasmid mixture (see Subheading 3.2.1.,step 2) to transform the original reporter strain (and, in parallel, pBaitC-zip and/orpBaitC-Ras control bait strains) used in Subheading 3.1.1. (see also Note 11).22. A further option is to test a few additional bait proteins that are related to one’sprotein of interest and a few that are unrelated. Interaction with related baitproteins and not with unrelated bait proteins might indicate that the isolated preyspecifically interacts with a family of proteins, whereas interaction with anynonspecific baits tested can indicate a widespread or a nonspecific interaction.References1. Fields, S. and Song, O. (1989) A novel genetic system to detect protein-proteininteraction. Nature 340, 245–246.2. Fashena, S. J., Serebriiskii, I. G., and Golemis, E. A. (2000) The continued evolutionof hybrid screening approaches in yeast: how to outwit different baits with differentpreys. Gene 250, 1–14.3. Dove, S. L., Joung, J. K., and Hochschild, A. (1997) Activation of prokaryotictranscription through arbitrary protein-protein contacts. Nature 386, 627–630.4. Dove, S. L. and Hochschild, A. (1998) Conversion of the omega subunit of Escherichiacoli RNA polymerase into a transcriptional activator or an activation target. GenesDev. 12, 745–754.5. Joung, J. K., Ramm, E. I., and Pabo, C. O. (2000) A bacterial two-hybrid selectionsystem for studying protein-DNA and protein-protein interactions. Proc. Natl.Acad. Sci. USA 97, 7382–7387.6. Vallet-Gely, I., Donovan, K. E., Fang, R., Joung, J. K., and Dove, S. L. (2005)Repression of phase-variable cup gene expression by H-NS-like proteins inPseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 102, 11,082–11,087.7. Dove, S. L. and Hochschild, A. (2004) A bacterial two-hybrid system based ontranscription activation. Methods Mol. Biol. 261, 231–246.8. Serebriiskii, I. G., Fang, R., Latypova, E., et al. (2005) A combined yeast/bacteriatwo-hybrid system: development and evaluation. Mol. Cell Proteomics 4, 819–826.9. Dove, S. L., Huang, F. W., and Hochschild, A. (2000) Mechanism for a transcriptionalactivator that works at the isomerization step. Proc. Natl. Acad. Sci. USA97, 13,215–13,220.10. Shaywitz, A. J., Dove, S. L., Kornhauser, J. M., Hochschild, A., and Greenberg, M. E.(2000) Magnitude of the CREB-dependent transcriptional response is determinedby the strength of the interaction between the kinase-inducible domain of CREB andthe KIX domain of CREB-binding protein. Mol. Cell Biol. 20, 9409–9422.