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Background 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 C. elegans to characterize their function and mode of interaction. Results 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 C. elegans and provide a proof-of-principle for its application to the discovery of PDZ domain targets for any protein or peptide of interest. Conclusions 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. [The protein interactions from this publication have been submitted to the IMEx ( http://www.imexconsortium.org ) consortium through IntAct (PMID: 19850723) and assigned the identifier IM-14654]

SCF‐type (SCF: Skp1–Cullin–F‐box protein complex) E3 ligases regulate ubiquitin‐dependent degradation of many cell cycle regulators, mainly at the G1/S transition. Here, we show that SCF Grr1 functions during cytokinesis by degrading the PCH protein Hof1. While Hof1 is required early in mitosis to assemble a functional actomyosin ring, it is specifically degraded late in mitosis and remains unstable during the entire G1 phase of the cell cycle. Degradation of Hof1 depends on its PEST motif and a functional 26S proteasome. Interestingly, degradation of Hof1 is independent of APC Cdh1 , but instead requires the SCF Grr1 E3 ligase. Grr1 is recruited to the mother–bud neck region after activation of the mitotic‐exit network, and interacts with Hof1 in a PEST motif‐dependent manner. Our results also show that downregulation of Hof1 at the end of mitosis is necessary to allow efficient contraction of the actomyosin ring and cell separation during cytokinesis. SCF Grr1 ‐mediated degradation of Hof1 may thus represent a novel mechanism to couple exit from mitosis with initiation of cytokinesis.

Background Escherichia coli strain EL350 contains chromosomally integrated phage lambda Red recombinase genes enabling this strain to be used for modifying the sequence of resident clones via 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, oriV , which permits copy-number to be induced transiently when propagated in a suitable host strain, e.g. EPI300, that supplies the cognate trans -replication protein TrfA. Previously, we used EL350 and EPI300 sequentially to recombineer oriV -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 P BAD -driven trfA gene generating strain MW005 that supports, independently, both recombineering and copy-number induction. Results The P BAD -driven copy of cre in EL350 was replaced seamlessly with a copy of trfA , 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 Caenorhabditis elegans genomic clones and to transiently induce copy-number of fosmid and BAC clones prior to DNA preparation. Conclusions By retrofitting EL350, an established 'recombineering' E. coli strain, with a tightly regulated copy of trfA we have produced a new strain, MW005, which combines recombineering capacity with the useful ability to transiently induce copy-number of oriV -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.

SCF-type (SCF: Skp1-Cullin-F-box protein complex) E3 ligases regulate ubiquitin-dependent degradation of many cell cycle regulators, mainly at the G1/S transition. Here, we show that SCF(Grr1) functions during cytokinesis by degrading the PCH protein Hof1. While Hof1 is required early in mitosis to assemble a functional actomyosin ring, it is specifically degraded late in mitosis and remains unstable during the entire G1 phase of the cell cycle. Degradation of Hof1 depends on its PEST motif and a functional 26S proteasome. Interestingly, degradation of Hof1 is independent of APC(Cdh1), but instead requires the SCF(Grr1) E3 ligase. Grr1 is recruited to the mother-bud neck region after activation of the mitotic-exit network, and interacts with Hof1 in a PEST motif-dependent manner. Our results also show that downregulation of Hof1 at the end of mitosis is necessary to allow efficient contraction of the actomyosin ring and cell separation during cytokinesis. SCF(Grr1)-mediated degradation of Hof1 may thus represent a novel mechanism to couple exit from mitosis with initiation of cytokinesis.

Sequence-specific DNA-binding proteins, with their paramount importance in the regulation of expression of the genetic material, are encoded by approximately 5% of the genes in an animal's genome. But it is unclear to what extent alternative transcripts from these genes may further increase the complexity of the transcription factor complement. Of the 938 potential C. elegans transcription factor genes, 197 were annotated in WormBase as encoding at least two distinct isoforms. Evaluation of prior evidence identified, with different levels of confidence, 50 genes with alternative transcript starts, 23 with alternative transcript ends, 35 with alternative splicing and 34 with alternative transcripts generated by a combination of mechanisms, leaving 55 that were discounted. Expression patterns were determined for transcripts for a sample of 29 transcription factor genes, concentrating on those with alternative transcript starts for which the evidence was strongest. Seamless fosmid recombineering was used to generate reporter gene fusions with minimal modification to assay expression of specific transcripts while maintaining the broad genomic DNA context and alternative transcript production. Alternative transcription factor gene transcripts were typically expressed with identical or substantially overlapping distributions rather than in distinct domains. Increasingly sensitive sequencing technologies will reveal rare transcripts but many of these are clearly non-productive. The majority of the transcription factor gene alternative transcripts that are productive may represent tolerable noise rather than encoding functionally distinct isoforms.

Background Sequence-specific DNA-binding proteins, with their paramount importance in the regulation of expression of the genetic material, are encoded by approximately 5% of the genes in an animal’s genome. But it is unclear to what extent alternative transcripts from these genes may further increase the complexity of the transcription factor complement. Results Of the 938 potential C. elegans transcription factor genes, 197 were annotated in WormBase as encoding at least two distinct isoforms. Evaluation of prior evidence identified, with different levels of confidence, 50 genes with alternative transcript starts, 23 with alternative transcript ends, 35 with alternative splicing and 34 with alternative transcripts generated by a combination of mechanisms, leaving 55 that were discounted. Expression patterns were determined for transcripts for a sample of 29 transcription factor genes, concentrating on those with alternative transcript starts for which the evidence was strongest. Seamless fosmid recombineering was used to generate reporter gene fusions with minimal modification to assay expression of specific transcripts while maintaining the broad genomic DNA context and alternative transcript production. Alternative transcription factor gene transcripts were typically expressed with identical or substantially overlapping distributions rather than in distinct domains. Conclusions Increasingly sensitive sequencing technologies will reveal rare transcripts but many of these are clearly non-productive. The majority of the transcription factor gene alternative transcripts that are productive may represent tolerable noise rather than encoding functionally distinct isoforms.

We describe a new member of the F‐box family, Pof14, which forms a canonical, F‐box dependent SCF ( S kp1, C ullin, 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.

Regulatory transcription factors operate in networks, conferring biological robustness that makes dissection of such gene control processes difficult. The nematode Caenorhabditis elegans is a powerful molecular genetic system that allows the close scrutiny needed to understand these processes in an animal, in vivo. Strikingly lower levels of gene expression were observed when a gfp reporter was inserted into C. elegans transcription factor genes, in their broader genomic context, in comparison to when the reporter was fused to just the promoter regions. The lower level of expression is more consistent with endogenous levels of the gene products, based on independent protein and transcript assays. Through successive precise manipulations of the reporter fusion genes, elements essential for the lower level of expression were localised to the protein-coding region. With a closer focus on four transcription factor genes, the expression of both genes encoding transcriptional activators was found to be restricted by a post-transcriptional mechanism while expression of both genes encoding transcriptional repressors was delimited by transcriptional repression. An element through which the transcriptional repression acts for unc-4 was localised to a 30 base-pair region of a protein-encoding exon, with potentially wider implications for how homeobox genes operate. The hypothesis that the distinction in mechanisms delimiting expression of the two types of transcription factor genes, as observed here, may apply more widely is raised. This leads to observations concerning the implications of these different mechanisms on stochastic noise in gene expression and the consequent significance for developmental decisions in general.

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 C. elegans to characterize their function and mode of interaction. 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 C. elegans and provide a proof-of-principle for its application to the discovery of PDZ domain targets for any protein or peptide of interest. 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.

Background Potentially inappropriate prescribing (PIP) is usually associated with a higher risk of adverse health outcomes. It is therefore important to identify PIP in older adults. However, there are no clear prioritisation strategies to select patients requiring prescription reviews. Aim The aim of this study was to assess the association between the identification of seniors at risk (ISAR) score and the number of PIPs. Method A 12-month retrospective hospital-based study was conducted. PIPs, including potentially inappropriate medications (PIMs) and potential prescribing omissions (PPOs), were detected using the STOPP/START tool. Multivariate linear regressions were conducted to identify factors associated with the number of PIPs. Sensitivity, specificity, Youden index, and ROC curve were calculated to determine the predictive power of ISAR score. Results This study included 266 records. The analysis led to the detection of 420 PIMs and 210 PPOs, with a prevalence of 80.1% and 54.9%, respectively. Multivariate linear regression revealed that the ISAR score ( p  = 0.041), and the number of medications ( p  < 0.001) were determinants of PIP. The number of medications remained the sole determinant of the number of PIMs ( p  < 0.001), while living in a nursing home was the only determinant of the number of PPOs ( p  = 0.036). Conclusion The study showed that the ISAR score and the number of medications were independently associated with the number of PIPs. Considering the use of the ISAR score and the number of medications may be useful strategies to prioritise patients for whom prescribing appropriateness should be assessed using explicit criteria.