Hybridization screening

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... 

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. 

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). 

Van Gelder PIAS3 is ubiquitously expressed and is famous for being pulled out of multiple two-hybrid screens. We have not yet immunostained, but I would be fairly confident that PIAS3 is going to be expressed all over the retina. 

Eromont-Racine, M., J.C. Rain, and P. Legrain, Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens. Nat Genet, 1997.16(3) 277-82. 

Uetz, R, and R.E. Hughes, Systematic and large-scale two-hybrid screens. Curr Opin Microbiol, 2000.3(3) 303-8. 

Hybridization analysis using immobilized DNA includes dot blots and slot blots. Dot and slot blots are named for the circular and slotted well shapes in the templates used to present test samples to a membrane surface. They eliminate the need for restriction digestion and electrophoretic resolution steps by depositing samples of DNA to be tested directly onto a hybridizable membrane surface. Dot blot methodology is faster and easier for hybridization screening than Southern blotting, especially when many samples are to be screened... 

Kato-Stankiewicz, J., Hakimi, I., Zhi, G., et al. (2002) Inhibitors of Ras-Raf-1 interaction identified by two-hybrid screening revert Ras-dependent transformation phenotypes in human cancer cells. Proc. Nat. Acad. Sci. USA 99, 14,398-14,403. 

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