ProFAT: a web-based tool for the functional annotation of protein sequences

BACKGROUND: The functional annotation of proteins relies on published information concerning their close and remote homologues in sequence databases. Evidence for remote sequence similarity can be further strengthened by a similar biological background of the query sequence and identified database sequences. However, few tools exist so far, that provide a means to include functional information in sequence database searches. RESULTS: We present ProFAT, a web-based tool for the functional annotation of protein sequences based on remote sequence similarity. ProFAT combines sensitive sequence database search methods and a fold recognition algorithm with a simple text-mining approach. ProFAT extracts identified hits based on their biological background by keyword-mining of annotations, features and most importantly, literature associated with a sequence entry. A user-provided keyword list enables the user to specifically search for weak, but biologically relevant homologues of an input query. The ProFAT server has been evaluated using the complete set of proteins from three different domain families, including their weak relatives and could correctly identify between 90% and 100% of all domain family members studied in this context. ProFAT has furthermore been applied to a variety of proteins from different cellular contexts and we provide evidence on how ProFAT can help in functional prediction of proteins based on remotely conserved proteins. CONCLUSION: By employing sensitive database search programs as well as exploiting the functional information associated with database sequences, ProFAT can detect remote, but biologically relevant relationships between proteins and will assist researchers in the prediction of protein function based on remote homologies

Identification of the C. elegans anaphase promoting complex subunit Cdc26 by phenotypic profiling and functional rescue in yeast.

BACKGROUND: RNA interference coupled with videorecording of C. elegans embryos is a powerful method for identifying genes involved in cell division processes. Here we present a functional analysis of the gene B0511.9, previously identified as a candidate cell polarity gene in an RNAi videorecording screen of chromosome I embryonic lethal genes. RESULTS: Whereas weak RNAi inhibition of B0511.9 causes embryonic cell polarity defects, strong inhibition causes embryos to arrest in metaphase of meiosis I. The range of defects induced by RNAi of B0511.9 is strikingly similar to those displayed by mutants of anaphase-promoting complex/cyclosome (APC/C) components. Although similarity searches did not reveal any obvious homologue of B0511.9 in the non-redundant protein database, we found that the N-terminus shares a conserved sequence pattern with the N-terminus of the small budding yeast APC/C subunit Cdc26 and its orthologues from a variety of other organisms. Furthermore, we show that B0511.9 robustly complements the temperature-sensitive growth defect of a yeast cdc26Delta mutant. CONCLUSION: These data demonstrate that B0511.9 encodes the C. elegans APC/C subunit CDC-26.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

SeLOX—a locus of recombination site search tool for the detection and directed evolution of site-specific recombination systems

Site-specific recombinases have become a resourceful tool for genome engineering, allowing sophisticated in vivo DNA modifications and rearrangements, including the precise removal of integrated retroviruses from host genomes. In a recent study, a mutant form of Cre recombinase has been used to excise the provirus of a specific HIV-1 strain from the human genome. To achieve provirus excision, the Cre recombinase had to be evolved to recombine an asymmetric locus of recombination (lox)-like sequence present in the long terminal repeat (LTR) regions of a HIV-1 strain. One pre-requisite for this type of work is the identification of degenerate lox-like sites in genomic sequences. Given their nature—two inverted repeats flanking a spacer of variable length—existing search tools like BLAST or RepeatMasker perform poorly. To address this lack of available algorithms, we have developed the web-server SeLOX, which can identify degenerate lox-like sites within genomic sequences. SeLOX calculates a position weight matrix based on lox-like sequences, which is used to search genomic sequences. For computational efficiency, we transform sequences into binary space, which allows us to use a bit-wise AND Boolean operator for comparisons. Next to finding lox-like sites for Cre type recombinases in HIV LTR sequences, we have used SeLOX to identify lox-like sites in HIV LTRs for six yeast recombinases. We finally demonstrate the general usefulness of SeLOX in identifying lox-like sequences in large genomes by searching Cre type recombination sites in the entire human genome. SeLOX is freely available at http://selox.mpi-cbg.de/cgi-bin/selox/index

Centriole Assembly Requires Both Centriolar and Pericentriolar Material Proteins

AbstractCentrioles organize pericentriolar material to form centrosomes and also template the formation of cilia. Despite the importance of centrioles in dividing and differentiated cells, their assembly remains poorly understood at a molecular level. Here, we develop a fluorescence microscopy-based assay for centriole assembly in the 1-cell stage C. elegans embryo. We use this assay to characterize SAS-6, a centriolar protein that we identified based on its requirement for centrosome duplication. We show that SAS-6, a member of a conserved metazoan protein family, is specifically required for new centriole assembly, a result we confirm by electron microscopy. We further use the centriole assembly assay to examine the roles of three pericentriolar material proteins: SPD-5, the kinase aurora-A, and γ-tubulin. Our results suggest that the pericentriolar material promotes daughter centriole formation by concentrating γ-tubulin around the parent centriole. Thus, both centriolar and pericentriolar material proteins contribute to centriole assembly

The kinetically dominant assembly pathway for centrosomal asters in Caenorhabditis elegans is γ-tubulin dependent

γ-Tubulin–containing complexes are thought to nucleate and anchor centrosomal microtubules (MTs). Surprisingly, a recent study (Strome, S., J. Powers, M. Dunn, K. Reese, C.J. Malone, J. White, G. Seydoux, and W. Saxton. Mol. Biol. Cell. 12:1751–1764) showed that centrosomal asters form in Caenorhabditis elegans embryos depleted of γ-tubulin by RNA-mediated interference (RNAi). Here, we investigate the nucleation and organization of centrosomal MT asters in C. elegans embryos severely compromised for γ-tubulin function. We characterize embryos depleted of ∼98% centrosomal γ-tubulin by RNAi, embryos expressing a mutant form of γ-tubulin, and embryos depleted of a γ-tubulin–associated protein, CeGrip-1. In all cases, centrosomal asters fail to form during interphase but assemble as embryos enter mitosis. The formation of these mitotic asters does not require ZYG-9, a centrosomal MT-associated protein, or cytoplasmic dynein, a minus end–directed motor that contributes to self-organization of mitotic asters in other organisms. By kinetically monitoring MT regrowth from cold-treated mitotic centrosomes in vivo, we show that centrosomal nucleating activity is severely compromised by γ-tubulin depletion. Thus, although unknown mechanisms can support partial assembly of mitotic centrosomal asters, γ-tubulin is the kinetically dominant centrosomal MT nucleator

Dietary Restriction Induced Longevity is Mediated by Nuclear Receptor NHR-62 in Caenorhabditis elegans

Dietary restriction (DR) extends lifespan in a wide variety of species, yet the underlying mechanisms are not well understood. Here we show that the C. elegans HNF4a- related nuclear hormone receptor NHR-62 is required for metabolic and physiologic responses associated with DR-induced longevity. nhr-62 mediates the longevity of eat- 2 mutants, a genetic mimetic of dietary restriction, and blunts the longevity response of DR induced by bacterial food dilution at low nutrient levels. Metabolic changes associated with DR, including decreased Oil Red O staining, increased autophagy, and changes in fatty acid composition are partly reversed by mutation of nhr-62. Expression profiles reveal that several hundred genes induced by DR depend on the activity of NHR-62, including a putative lipase required for the DR response. This study provides critical evidence that nuclear hormone receptors regulate the DR response, suggesting hormonal and metabolic control of life span

Whole‐genome comparison between the type strain of Halobacterium salinarum (DSM 3754T) and the laboratory strains R1 and NRC‐1

Halobacterium salinarum is an extremely halophilic archaeon that is widely distributed in hypersaline environments and was originally isolated as a spoilage organism of salted fish and hides. The type strain 91‐R6 (DSM 3754T) has seldom been studied and its genome sequence has only recently been determined by our group. The exact relationship between the type strain and two widely used model strains, NRC‐1 and R1, has not been described before. The genome of Hbt. salinarum strain 91‐R6 consists of a chromosome (2.17 Mb) and two large plasmids (148 and 102 kb, with 39,230 bp being duplicated). Cytosine residues are methylated (m4C) within CTAG motifs. The genomes of type and laboratory strains are closely related, their chromosomes sharing average nucleotide identity (ANIb) values of 98% and in silico DNA–DNA hybridization (DDH) values of 95%. The chromosomes are completely colinear, do not show genome rearrangement, and matching segments show 10 kb). The well‐studied AT‐rich island (61 kb) of the laboratory strains is replaced by a distinct AT‐rich sequence (47 kb) in 91‐R6. Another large replacement (91‐R6: 78 kb, R1: 44 kb) codes for distinct homologs of proteins involved in motility and N‐glycosylation. Most (107 kb) of plasmid pHSAL1 (91‐R6) is very closely related to part of plasmid pHS3 (R1) and codes for essential genes (e.g. arginine‐tRNA ligase and the pyrimidine biosynthesis enzyme aspartate carbamoyltransferase). Part of pHS3 (42.5 kb total) is closely related to the largest strain‐specific sequence (164 kb) in the type strain chromosome. Genome sequencing unraveled the close relationship between the Hbt. salinarum type strain and two well‐studied laboratory strains at the DNA and protein levels. Although an independent isolate, the type strain shows a remarkably low evolutionary difference to the laboratory strains

Chromatin Central: towards the comparative proteome by accurate mapping of the yeast proteomic environment

High resolution mapping of the proteomic environment and proteomic hyperlinks in fission and budding yeast reveals that divergent hyperlinks are due to gene duplications