Using shotgun sequence data to find active restriction enzyme genes

Whole genome shotgun sequence analysis has become the standard method for beginning to determine a genome sequence. The preparation of the shotgun sequence clones is, in fact, a biological experiment. It determines which segments of the genome can be cloned into Escherichia coli and which cannot. By analyzing the complete set of sequences from such an experiment, it is possible to identify genes lethal to E. coli. Among this set are genes encoding restriction enzymes which, when active in E. coli, lead to cell death by cleaving the E. coli genome at the restriction enzyme recognition sites. By analyzing shotgun sequence data sets we show that this is a reliable method to detect active restriction enzyme genes in newly sequenced genomes, thereby facilitating functional annotation. Active restriction enzyme genes have been identified, and their activity demonstrated biochemically, in the sequenced genomes of Methanocaldococcus jannaschii, Bacillus cereus ATCC 10987 and Methylococcus capsulatus

Detection of Alpha-Rod Protein Repeats Using a Neural Network and Application to Huntingtin

A growing number of solved protein structures display an elongated structural domain, denoted here as alpha-rod, composed of stacked pairs of anti-parallel alpha-helices. Alpha-rods are flexible and expose a large surface, which makes them suitable for protein interaction. Although most likely originating by tandem duplication of a two-helix unit, their detection using sequence similarity between repeats is poor. Here, we show that alpha-rod repeats can be detected using a neural network. The network detects more repeats than are identified by domain databases using multiple profiles, with a low level of false positives (<10%). We identify alpha-rod repeats in approximately 0.4% of proteins in eukaryotic genomes. We then investigate the results for all human proteins, identifying alpha-rod repeats for the first time in six protein families, including proteins STAG1-3, SERAC1, and PSMD1-2 & 5. We also characterize a short version of these repeats in eight protein families of Archaeal, Bacterial, and Fungal species. Finally, we demonstrate the utility of these predictions in directing experimental work to demarcate three alpha-rods in huntingtin, a protein mutated in Huntington's disease. Using yeast two hybrid analysis and an immunoprecipitation technique, we show that the huntingtin fragments containing alpha-rods associate with each other. This is the first definition of domains in huntingtin and the first validation of predicted interactions between fragments of huntingtin, which sets up directions toward functional characterization of this protein. An implementation of the repeat detection algorithm is available as a Web server with a simple graphical output: http://www.ogic.ca/projects/ard. This can be further visualized using BiasViz, a graphic tool for representation of multiple sequence alignments

Corticotropin-Releasing Hormone Receptor Type 1 (CRHR1) Clustering with MAGUKs Is Mediated via Its C-Terminal PDZ Binding Motif.

The corticotropin-releasing hormone receptor type 1 (CRHR1) plays an important role in orchestrating neuroendocrine, behavioral, and autonomic responses to stress. To identify molecules capable of directly modulating CRHR1 signaling, we performed a yeast-two-hybrid screen using the C-terminal intracellular tail of the receptor as bait. We identified several members of the membrane-associated guanylate kinase (MAGUK) family: postsynaptic density protein 95 (PSD95), synapse-associated protein 97 (SAP97), SAP102 and membrane associated guanylate kinase, WW and PDZ domain containing 2 (MAGI2). CRHR1 is co-expressed with the identified MAGUKs and with the additionally investigated PSD93 in neurons of the adult mouse brain and in primary hippocampal neurons, supporting the probability of a physiological interaction in vivo. The C-terminal PDZ (PSD-95, discs large, zona occludens 1) binding motif of CRHR1 is essential for its physical interaction with MAGUKs, as revealed by the CRHR1-STAVA mutant, which harbors a functionally impaired PDZ binding motif. The imitation of a phosphorylation at Thr413 within the PDZ binding motif also disrupted the interaction with MAGUKs. In contrast, distinct PDZ domains within the identified MAGUKs are involved in the interactions. Expression of CRHR1 in primary neurons demonstrated its localization throughout the neuronal plasma membrane, including the excitatory post synapse, where the receptor co-localized with PSD95 and SAP97. The co-expression of CRHR1 and respective interacting MAGUKs in HEK293 cells resulted in a clustered subcellular co-localization which required an intact PDZ binding motif. In conclusion, our study characterized the PDZ binding motif-mediated interaction of CRHR1 with multiple MAGUKs, which directly affects receptor function

Динамическое концентрирование ионов Mn(II), Co(II), Ni(II), Cu(II) на сильносшитых карбоксильных катионитах и создание тест-систем для анализа питьевых вод: автореферат диссертации на соискание учёной степени кандидата химических наук: спец. 02.00.02

Cationic liposomal compounds are widely used to introduce DNA and siRNA into viable cells, but none of these compounds are also capable of introducing proteins. Here we describe the use of a cationic amphiphilic lipid SAINT-2:DOPE for the efficient delivery of proteins into cells (profection). Labeling studies demonstrated equal delivery efficiency for protein as for DNA and siRNA. Moreover, proteins complexed with SAINT-2:DOPE were successfully delivered, irrespective of the presence of serum, and the profection efficiency was not influenced by the size or the charge of the protein:cationic liposomal complex. Using beta-galactosidase as a reporter protein, enzymatic activity was detected in up to 98% of the adherent cells, up to 83% of the suspension cells and up to 70% of the primary cells after profection. A delivered antibody was detected in the cytoplasm for up to 7 days after profection. Delivery of the methyltransferase M.SssI resulted in DNA methylation, leading to a decrease in E-cadherin expression. The lipid-mediated multipurpose transport system reported here can introduce proteins into the cell with an equal delivery efficiency as for nucleotides. Delivery is irrespective of the presence of serum, and the protein can exert its function both in the cytoplasm and in the nucleus. Furthermore, DNA methylation by M.SssI delivery as a novel tool for gene silencing has potential applications in basic research and therapy. (c) 2007 Elsevier B.V. All rights reserved

CRHR1-WT and CRHR1-STAVA localize throughout the neuronal plasma membrane including the excitatory post synapse.

<p>Primary hippocampal neurons at DIV 14–20 were transduced with CRHR1-WT and CRHR1-STAVA using AAV8. (A) The spatial pattern of CRHR1-WT and CRHR1-STAVA expression visualized by immunostaining of the GFP tag was confirmed using an anti-CRHR1 antibody. (B) Microtubule-associated protein 2 (MAP2) staining revealed the dendritic presence of the receptor. (C) Co-staining with the axon initial segment marker ankyrin G demonstrated CRHR1-WT and CRHR1-STAVA localization within axons. (D) The presynaptic marker synapsin indicated that CRHR1-WT and CRHR1-STAVA are present in the adjacent post synapse but not in the presynaptic axon terminal. (E) CRHR1-WT and CRHR1-STAVA did not co-localize with the inhibitory postsynaptic marker gephyrin but (F) they co-localized with the MAGUK and excitatory postsynaptic marker PSD95 in spines. (G) CRHR1-WT and mutant co-localized with the candidate interaction partner SAP97.</p

CRHR1 PDZ binding motif is required for clustering with interacting MAGUKs.

<p>CRHR1-WT (A), CRHR1-STAVA (G), or the interacting proteins (first row) were transiently transfected alone. (B–F) Wild-type CRHR1 or (H–L) CRHR1-STAVA were transiently transfected in HEK293 together with (B, H) PSD95-flag, (C, I) HA-SAP97, (D, J) flag-SAP102, (E, K) PSD93-GFP and (F, L) myc-MAGI2. CRHR1-WT co-transfection with the respective interacting partner resulted in a co-clustering of both proteins (B–F). (H–L) Co-transfection of CRHR1-STAVA with the respective interacting partner did not result in any clustering, but both proteins appeared with a subcellular distribution similar to the individual transfections. Immunostaining was performed against the HA tag of HA-CRHR1 or HA-CRHR1-STAVA when co-transfected with PSD95-flag or flag-SAP102. Immunostaining was performed against the flag tag of flag-CRHR1 or flag-CRHR1-STAVA when co-transfected with PSD93-GFP, HA-SAP97, or myc-MAGI2 respectively.</p

CRHR1 interaction with SAP97, SAP102 and PSD93 depends on the PDZ binding motif.

<p>Co-IPs were performed using lysates of HEK293 cells transiently transfected as indicated. (A) myc-GFP-CRHR1 but not myc-GFP-CRHR1STAVA was co-immunoprecipitated with HA-SAP97, and similarly, HA-SAP97 was co-immunoprecipitated with myc-GFP-CRHR1 but not with myc-GFP-CRHR1-STAVA. (B) HA-SAP97 PDZ1-3 and HA-SAP97 PDZ1-2 were co-immunoprecipitated with myc-GFP-CRHR1 and accordingly, these SAP97 variants were co-immunoprecipitated with myc-GFP-CRHR1 using an anti-myc antibody in the Co-IP. No interaction was observed for HA-SAP97 PDZ1. HA-SAP97 PDZ1-2 detected by the anti-HA antibody following the anti-myc Co-IP has the same molecular weight and thus is indistinguishable from the heavy chain of the anti-myc antibody (→). (C) myc-GFP-CRHR1 but not myc-GFP-CRHR1-STAVA was co-immunoprecipitated with flag-SAP102. Similarly, flag-SAP102 was co-immunoprecipitated with myc-GFP-CRHR1 but not with myc-GFP-CRHR1-STAVA. Moreover, flag-SAP102 PDZ3 was not detected following an immunoprecipitation (IP) of myc-GFP-CRHR1. (D) flag-CRHR1 but not flag-CRHR1-STAVA was co-immunoprecipitated with GFP-PSD93 and accordingly, GFP-PSD93 was co-immunoprecipitated with flag-CRHR1 but not with flag-CRHR1-STAVA. CRHR1 always showed high molecular weight complexes (*) together with the monomeric form (>). Therefore, high molecular weight complexes are also shown when necessary. Dashed lines indicate that the samples were run on the same immunoblot (IB), however, not in adjacent lanes. Continuous lines separate different IBs from the same experiment. (B) The ~55 kDa band represents the heavy chain of the primary antibody.</p