Escherichia coli MW005: lambda Red-mediated recombineering and copy-number induction of oriV-equipped constructs in a single host

<p>Abstract</p> <p>Background</p> <p><it>Escherichia coli </it>strain EL350 contains chromosomally integrated phage lambda Red recombinase genes enabling this strain to be used for modifying the sequence of resident clones <it>via </it>recombineering. BAC and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2) copies per cell, the DNA is difficult to isolate in high yield and purity. To overcome this limitation vectors, e.g. pCC1FOS, have been constructed that contain the additional replication origin, <it>oriV</it>, which permits copy-number to be induced transiently when propagated in a suitable host strain, e.g. EPI300, that supplies the cognate <it>trans</it>-replication protein TrfA. Previously, we used EL350 and EPI300 sequentially to recombineer <it>oriV</it>-equipped fosmid genomic clones and, subsequently, to induce copy-number of the resulting recombinant clone. To eliminate these intervening DNA isolation and transformation steps we retrofitted EL350 with a <it>P</it>_BAD-driven <it>trfA </it>gene generating strain MW005 that supports, independently, both recombineering and copy-number induction.</p> <p>Results</p> <p>The <it>P</it>_BAD-driven copy of <it>cre </it>in EL350 was replaced seamlessly with a copy of <it>trfA</it>, PCR-amplified from EPI300 chromosomal DNA, to generate MW005. This new strain has been used to both generate, via recombineering, a number of reporter gene fusions directly from pCC1FOS-based <it>Caenorhabditis elegans </it>genomic clones and to transiently induce copy-number of fosmid and BAC clones prior to DNA preparation.</p> <p>Conclusions</p> <p>By retrofitting EL350, an established 'recombineering' <it>E. coli </it>strain, with a tightly regulated copy of <it>trfA </it>we have produced a new strain, MW005, which combines recombineering capacity with the useful ability to transiently induce copy-number of <it>oriV</it>-equipped clones. By coupling these two steps in a single strain, use of MW005 will enable the more rapid recombineering-mediated production of recombinant clones in the yield and quality necessary for many downstream purposes.</p

A genome-wide study of PDZ-domain interactions in C. elegans reveals a high frequency of non-canonical binding

<p>Abstract</p> <p>Background</p> <p>Proteins may evolve through the recruitment and modification of discrete domains, and in many cases, protein action can be dissected at the domain level. PDZ domains are found in many important structural and signaling complexes, and are generally thought to interact with their protein partners through a C-terminal consensus sequence. We undertook a comprehensive search for protein partners of all individual PDZ domains in <it>C. elegans </it>to characterize their function and mode of interaction.</p> <p>Results</p> <p>Coupling high-throughput yeast two-hybrid screens with extensive validation by co-affinity purification, we defined a domain-orientated interactome map. This integrates PDZ domain proteins in numerous cell-signaling pathways and shows that PDZ domain proteins are implicated in an unexpectedly wide range of cellular processes. Importantly, we uncovered a high frequency of non-canonical interactions, not involving the C-terminus of the protein partner, which were directly confirmed in most cases. We completed our study with the generation of a yeast array representing the entire set of PDZ domains from <it>C. elegans </it>and provide a proof-of-principle for its application to the discovery of PDZ domain targets for any protein or peptide of interest.</p> <p>Conclusions</p> <p>We provide an extensive domain-centered dataset, together with a clone resource, that will help future functional study of PDZ domains. Through this unbiased approach, we revealed frequent non-canonical interactions between PDZ domains and their protein partners that will require a re-evaluation of this domain's molecular function.</p> <p>[The protein interactions from this publication have been submitted to the IMEx (<url>http://www.imexconsortium.org</url>) consortium through IntAct (PMID: 19850723) and assigned the identifier IM-14654]</p

Pof14, une protéine à F-box impliquée dans un SCF ?

-Oral presentation- Au sein du laboratoire, nous travaillons sur l’homologue de Skp1 chez Schizosaccharomyces pombe. Cette protéine, conservée chez tous les eucaryotes, forme avec la culline le corps d’une ubiquitine ligase de type SCF (composée de Skp1, d’une Culline et d’une protéine à F-box). La protéine à F-box est l’adaptateur qui recrute spécifiquement la protéine à ubiquitiner en vue de sa dégradation par le protéasome 26S. Dans un premier temps, nous avons montré par coimmunoprécipitation que Shp1 forme in vivo un complexe SCF chez S. pombe ;elle interagit avec la culline Pcu1 et des protéines à F-box Pop1 et Pop2. Ensuite, un crible double-hybride avec Skp1 comme appât, ainsi qu’une analyse in silico du protéome de S. pombe ont permis d’identifier 16 protéines possédant un motif à F-box. Parmi celles-ci, nous nous intéressons particulièrement à Pof14.info:eu-repo/semantics/nonPublishe

Regulation of the Mcs2 C-type cyclin in fission yeast

CDK activation requires activating phosphorylation of a conserved residue in the T-loop by the CAK (CDK-activating kinase). In both fission yeast and higher eukaryotes, CAK is a trimeric complex composed of Mcs6-Mcs2-Pmh1 or its homologue [1–3]. Two hybrid assays revealed interaction of both Mcs2 and Pmh1 with Shp1, a subunit of the SCF ubiquitin ligase. This prompted us to test the stability of these CAK regulators. Although the steady-state level of Mcs2 does not seem to change during the cell cycle [4], we show that it is strongly correlated to Mcs6 kinase activity and that Mcs2 was nearly undetectable when Mcs6 was strongly overexpressed. Interestingly, this effect is reversed by a mutation in shp1. Taken together, these results suggest a putative regulation of Mcs2 by Mcs6 phosphorylation and SCF ubiquitin ligase complex. Thus, the hypothesis is under investigation. (1) Hermand D, Westerling T. Pihlak A, et al. 2001. EMBO J 20: 82–90. (2) Hermand D, Pihlak A, Westerling T, et al. 1998. EMBO J 17: 7230–7238. (3) Kaldis P. 1999. Cell Mol Life Sci 55: 284–296. (4) Molz L, Beach D. 1993. EMBO J 12: 1723–1732.S.B. is a FNRS Research Fellow. D.H. is a FNRS Scientific Research Worker. L.T. is recipient of a FRIA Fellowship.info:eu-repo/semantics/publishe

Skp1 and the F-box protein Pof6 are essential for cell separation in fission yeast

Here we report functional characterization of the essential fission yeast Skp1 homologue. We have created a conditional allele of skp1 (skp1-3f) mimicking the mutation in the budding yeast skp1-3 allele. Although budding yeast skp1-3 arrests at the G(1)/S transition, skp1-3f cells progress through S phase and instead display two distinct phenotypes. A fraction of the skp1-3f cells arrest in mitosis with high Cdc2 activity. Other skp1-3f cells as well as the skp1-deleted cells accumulate abnormal thick septa leading to defects in cell separation. Subsequent identification of 16 fission yeast F-box proteins led to identification of the product of pof6 (for pombe F-box) as a Skp1-associated protein. Interestingly, cells deleted for the essential pof6 gene display a similar cell separation defect noted in skp1 mutants, and Pof6 localizes to septa and cell tips. Purification of Pof6 demonstrates association of Skp1, whereas the Pcu1 cullin was absent from the complex. These findings reveal an essential non-Skp1-Cdc53/Cullin-F-box protein function for the fission yeast Skp1 homologue and the F-box protein Pof6 in cell separation.info:eu-repo/semantics/publishe

Repression of ergosterol level during oxidative stress by fission yeast F-box protein Pof14 independently of SCF

We describe a new member of the F-box family, Pof14, which forms a canonical, F-box dependent SCF (Skp1, Cullin, F-box protein) ubiquitin ligase complex. The Pof14 protein has intrinsic instability that is abolished by inactivation of its Skp1 interaction motif (the F-box), Skp1 or the proteasome, indicating that Pof14 stability is controlled by an autocatalytic mechanism. Pof14 interacts with the squalene synthase Erg9, a key enzyme in ergosterol metabolism, in a membrane-bound complex that does not contain the core SCF components. pof14 transcription is induced by hydrogen peroxide and requires the Pap1 transcription factor and the Sty1 MAP kinase. Pof14 binds to and decreases Erg9 activity in vitro and a pof14 deletion strain quickly loses viability in the presence of hydrogen peroxide due to its inability to repress ergosterol synthesis. A pof14 mutant lacking the F-box and an skp1-3 ts mutant behave as wild type in the presence of oxidant showing that Pof14 function is independent of SCF. This indicates that modulation of ergosterol level plays a key role in adaptation to oxidative stress