The yeast split-ubiquitin system has previously been shown to be suitable to detect protein interactions of membrane proteins and of transcription factors in vivo. Therefore, this technology complements the classical split-transcription factor based yeast two-hybrid system (Y2H). Success or failure of the Y2H depends primarily on the ability to avoid false-negative and false-positive hits that become a limiting factor for the value of the system, especially in large scale proteomic analyses. We provide here a systematic assessment of parameters to help improving the quality of split-ubiquitin cDNA-library screenings. We experimentally defined the optimal 5-fluoroorotic acid (5-FOA) concentration as a key parameter to increase the reproducibility of interactions and, at the same time, to keep non-specific background growth low. Furthermore, we show that the efficacy of the 5-FOA selection is modulated by the plating density of the yeast clones. Moreover, a reporter-specific class of false-positive hits was identified, and a simple phenotypic assay for efficient de-selection was developed. We demonstrate the application of this improved system to identify novel interacting proteins of the human Frizzled 1 receptor. We identified several novel interactors with components of the Wnt-Frizzled signalling pathways and discuss their potential roles as direct mediators of Frizzled receptor signalling. The present work is the first example of a split-ubiquitin interaction screen using an in-situ expressed receptor of the serpentine class, emphasizing the suitability of the described improvements in the screening protocol.
SUMMARYFour bacteriophages (A 16, CK235, q~ 1.2 and K31) which specifically attack different encapsulated strains of Escherichia coli have been shown to be related to bacteriophage T7 (which is unable to grow on encapsulated hosts). The conclusion that phages A16 and CK235 are related to T7 is based on (i) similarities in the pattern of expression of intracellular phage proteins, (ii) early appearance, in infected host cells, of a phage DNA-specific RNA polymerase and (iii) hybridization (albeit to a low extent) of A16 DNA and of CK235 DNA to T7 DNA. The first two criteria also apply to phages ~bl. 2 and K31 but hybridization of their DNAs with T7 DNA could not be detected. The RNA polymerases of CK235 and A 16 have similar template specificities and the same applies to the RNA polymerases of ~b 1.2 and K3 i. None of the new RNA polymerases can use T7 DNA as template. INTRODUCTIONEnterobacteria with lipopolysaccharide capsules are lysed by phages which possess specific hydrolases as virion constituents. By means of these particle-bound enzymes the phages tunnel through the thick layer of capsule material until they find the outer membrane where specific adsorption and DNA injection occurs (Lindberg, 1977). One group of such capsule-specific phages is characterized by an icosahedral head with a diameter of about 60 nm, and a short tail of about 20 nm, to which spikes are attached with sixfold symmetry. These spikes display the capsule-lysing activity (Bessler et al., 1975;Rieger-Hug & Stirm, 1981). Since this particle morphology is reminiscent of that of the classical coliphage T7 (which, however, does not infect capsulated strains), Korsten et al. (1979) examined two such capsule-specific phages, Klebsiella phage K11, and Citrobacter phage Villi, with regard to their genetic relatedness to T7; they found that indeed the genome structures of T7, K11 and VilII were similar in various respects. The most striking feature was the synthesis, early in infection by K11, and by VilII, of a phagecoded RNA polymerase which recognizes only DNA of the homologous phage as a template. This observation closely corresponds to what is known for phage T7, and thus it was hypothesized that, in spite of the lack of heterologous transcription (i.e. transcription of the DNA of one phage by the RNA polymerase of another), there was a phylogenetic relationship between T7, K11 and Villi. In the meantime Dietz (1985) and Dietz et al. (1985) have confirmed this hypothesis for T7 and K11 by means of base sequence analysis of some segments of K11 DNA and comparison to the corresponding sequences of T7 DNA, as determined by , and to the DNA sequences of T3 (which is related to T7) as determined by Fujisawa & Sugimoto (1983) and by McAllister et al. (1983). However, so far no capsule-specific Escherichia coli phages have been shown to be related to T7. Considering this, we have investigated a series of phages which specifically lyse one of three different capsule-producing E. coli strains. We did this mainly by (i) comparison, by PAGE and auto...
The gene expression of nine phages of the T7 group was compared after infection of Escherichia coli B(P1). With the exception of phage 13a which grew normally, all of them infected E. coli B(P1) abortively. Differences were found in the efficiency of host killing which ranged from 100% for phage 13a to 37% for phage A1122. Infection by T7 prevented colony formation by about 70% of the cells but they showed filamentous growth until about 2 h after infection. It was shown by SDS-polyacrylamide gel electrophoresis and autoradiography of [35S]methionine-labelled phage-coded proteins that all phages except for 13a showed measurable expression only of the early genes. No correlation was observed between killing capacity and the pattern of gene expression, and the ability to hydrolyse S-adenosyl-methionine (SAM, a cofactor for the P1 restriction endonuclease) by means of a phage-coded SAMase. Mixed infection of E. coli B(P1) with 13a and T7 yielded mixed progeny indistinguishable from that observed after mixed infection of the normal host E. coli B. Genetic crosses with amber mutants of 13a and T7 showed that the 13a marker opo+ (overcomes P one), required for growth on B(P1), is located in the early region, to the left of gene 1 (RNA polymerase gene).
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