Yeast two-hybrid

Biological raw data are stored in public databanks (such as Genbank or EMBL for primary DNA sequences). The data can be submitted and accessed via the World Wide Web. Protein sequence databanks like trEMBL provide the most likely translation of all coding sequences in the EMBL databank. Sequence data are prominent, but also other data are stored, e.g.yeast two-hybrid screens, expression arrays, systematic gene-knock-out experiments, and metabolic pathways. 

PIAS (protein inhibitors of activated STATs) proteins were first discovered in yeast-two-hybrid screens as interacting molecules with STAT transcription factors. The mammalian family consists ofthe founding member PIAS3, which was described as a repressor of STAT3, and three additional members, PIAS1, PIASy (also known as PIAS4), and PIASx (also known as... 

Figure 5.1. Yeast two-hybrid system. Interaction of proteins X and Y upstream of a reporter gene leads to transcriptional activation. Protein X is part of a fusion protein that binds to a site on DNA upstream of the reporter gene by means of a DNA binding domain. Protein Y is part of a fusion protein that contains a transcriptional activation domain. Interaction of proteins X and Y places the activation domain in the vicinity of the reporter gene and stimulates its transcription.

Genome-wide yeast two-hybrid analysis of other organisms... 

The bacterial one and two-hybrid systems have potential advantages over the yeast two-hybrid system due to the higher transformation efficiency and faster growth rate of coli. To date, however, the bacterial two-hybrid system has not been used for genome-scale analysis of protein-protein interactions. 

The experiments described above indicate that technology is available to couple SPR with mass spectrometry. These methods should be useful for protein-protein interaction mapping. For example, immobilized proteins can be used as hooks for fishing binding partners from complex protein mixtures under native conditions. The coupling of techniques can lead not only to the rapid identification of interacting proteins but will also provide information on the kinetic parameters of the interaction. This approach should serve as an excellent complement to the use of in vivo techniques such as the yeast two-hybrid system. 

Bai, C., and Elledge, S. J. (1997). Gene identification using the yeast two-hybrid system. Methods Enzymol. 283, 141-156. 

We have previously shown that a 209 amino acid region (aa288-497, asymmetric localization domain) of Insc is necessary and sufficient for apical cortical localization and for mitotic spindle orientation along the apical-basal axis (Tio et al 1999). In a yeast two-hybrid screen we identified Partner of Inscuteable (Pins), a novel 658aa protein with multiple repeats of the Tetratricopeptide (TPR) motif. Affinity purification experiments using embryonic extracts demonstrate that Pins complexes with Insc in vivo. In vitro protein interaction assays demonstrates that Pins interacts with the Insc asymmetric localization domain (see Yu et al 2000). 

Bryant This has not been studied in Drosophila. Several Dig-like MAGUKs have been picked up in a yeast two-hybrid screen using as bait the Erb-B4 receptor (Garcia et al 2000), which is the only one of the mammalian EGF receptor family that has a C-terminus predicted to bind to PDZ domains. This interaction could be involved in controlling receptor localization. Potentially it is the same kind of interaction. 

It should be emphasized that the nature of all presented protocols is very general and, thus, their application for a comprehensive characterization of your favorite multiprotein complex (YFMPC) in yeast might require only minor modifications. The logical sequence of all required steps is schematically shown in Fig. 2.1. The initial large-scale Ni affinity isolation of eIF3 followed by mass spectrometry (MS) of its subunit composition has already been described (Asano et al, 2002), and methods for identification of protein-protein interactions such as yeast two-hybrid (Y2H) and in vitro glutathione-S-transferase (GST) pull-down analysis are presented in volume 429. This chapter focuses on a description of the small-scale one-step in vivo affinity purification techniques that were used to determine the effects of deletions and... 

Criekinge, W.V., and Beyaert, R. (1999) Yeast two-hybrid State of the art. Biological Procedures Online 2(1), October 4, www.biologicalprocedures.com. 

Staudinger, J., Zhou, J., Burgess, R., Elledge, S. J., and Olson, E. N. (1995) PICK1 a perinuclear binding protein and substrate for protein kinase C isolated by the yeast two-hybrid... 

M. C., Yu, C. H., Lin, P. I., and Wu, W. F. Subunit oligomerization and substrate recognition of the Escherichia coli ClpYQ (HslUV) protease implicated by in vivo protein-protein interactions in the yeast two-hybrid system. /. Bacteriol. 2003, 185, 2393-2401. 

The CSN is composed of eight subunits called CSNl to CSN8, which are highly conserved in eukaryotes, although only six of them occur in fission yeast. Two hybrid screens and biochemical methods such as far westerns, pull downs and coprecipitation defined a number of CSN subunit-subunit interactions. Figure 13.1 illustrates known subunit-subunit interactions. Initial insight into the architecture of CSN came from the first 2D electron microscopic analysis of purified CSN from human red blood cells [19] (see also Figure 13.2 below). 

Su(var)3-9 in the yeast two-hybrid interaction assay and increase the Su(var)3-9-mediated H3K9 methylation on autosomes and the male X chromosome. Su(var)3-7, contains seven widely spaced zinc-finger motifs, is involved in the recruitment of Su(var)3-9 (Schotta et al, 2002 Delattre et al, 2004). 

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