NotI Sites Research Articles

Pil1 Co-tethering Assay to Detect Protein-Protein Interactions in the Fission Yeast Schizosaccharomyces pombe.

Protein-protein interactions play critical roles in biological processes. We previously developed the Pil1 co-tethering assay, an imaging-based method to detect protein-protein interactions in living Schizosaccharomyces pombe cells. This assay leverages the distinct localization pattern of the Pil1 protein by fusing a bait protein to Pil1 and examining whether a prey protein co-localize with the Pil1-fused bait. Here, we present an improved protocol of the Pil1 co-tethering assay. In this protocol, modified stable integration vectors (SIVs) with a NotI site as the linearization site are used to express bait and prey proteins. We expect that this protocol will enhance the application of the Pil1 co-tethering assay for studying protein-protein interactions.

P13. Transduction of the rat spinal cord using adenoassociated virus serotype 2.5

<h3>BACKGROUND CONTEXT</h3> Gene therapy is a method by which exogenous genes are introduced to treat disease or deliver drugs of interest. This is commonly done through either direct gene delivery in vivo or ex vivo through transduction of cells that are to be transplanted. There are several vectors for delivering genes, including viral vectors, naked plasmids, electroporation and lipid nanoparticles. Adeno-associated viruses (AAVs) are commonly used and considered to be well tolerated by the brain, muscles, nerves and multiple cell types. Rat Schwann cells are transduced efficiently by AAV1 whereas human Schwann cells are better transduced with AAV2 and 6. Rat nerves segments have demonstrated to be efficiently transduced by AAV1, 5, 7 and 9 whereas human nerves prefer AAV2. Many studies also agree that AAV1, 2, 4, 8 and 9 are optimal serotypes for CNS. We have utilized a novel viral vector, AAV serotype 2.5, that is a chimera of AAV2 vector modified with 5 amino acids from AAV1. AAV2.5 has shared characteristics of AAV1 and AAV2 with enhanced transduction for multiple cell types, such as in the muscle and articular tissue. Since AAV2.5 is not a naturally occurring virus, there is lower cross-reactivity to AAV2 antibodies. However, the ability of AAV2.5 to transduce tissues of the central (CNS) and peripheral nervous system (PNS) is not well understood. <h3>PURPOSE</h3> The purpose of this study is to determine the trophism and transduction efficiency of AAV2.5 for CNS and PNS tissue through in vitro transduction of dorsal root ganglion derived neurons, Schwann cells, and in vivo transduction of spinal cord. We predict that since AAV1 and AAV2 have known tropism for CNS and PNS cells to different extents depending on cell type, that the chimera AAV2.5 would be broadly effective. <h3>STUDY DESIGN/SETTING</h3> Cell culture and animal study. <h3>METHODS</h3> The cDNA encoding green fluorescent protein (GFP) was cloned into the conventional AAV packaging vector pTRUF2. For generation of self-complementing AAV vector plasmids, the cDNAs for GFP were directionally inserted into the SacII, NotI sites of pHpa-trs-SK plasmid. Transcription was driven by the cytomegalovirus promoter. Primary Schwann cells were isolated from the sciatic nerves of postnatal day 5 rat pups and dorsal root ganglion (DRG) neurons from embryonic day 15 rat pups. The cells were plated at a density of 125,000 cells per well and infected with 104 viral genomes per cell. For in vivo transduction of spinal cord tissue, 4 µL of 5 × 106– 5 × 1010vg/mL of AAV2.5-GFP was injected into 4 positions at T9. Immunostaining for markers of different spinal cord cell types colabeled with anti-GFP was done. <h3>RESULTS</h3> AAV2.5 was able to successfully infect DRG neurons and Schwann cells. AAV-2.5 vector was much more efficient than the commonly used AAV2 vector at transducing the Schwann cells. Cell survival and gene product production by AAV2.5 was greater than electroporation. When AAV2.5 -GFP was injected into the spinal cord there was a dose dependent increase in GFP expression by neurons and astrocytes up to 4 mm away from the injection site. <h3>CONCLUSIONS</h3> The study presents a novel vector for broad transduction in vivo or ex vivo of cells in the CNS and PNS. AAV2.5 was more efficient than the more commonly used AAV2 vector or electroporation. Since AAV2.5 is presently in phase 1 trials for osteoarthritis (NCT02790723), this vector type presents an interesting clinical translation opportunity for spinal cord and peripheral nerve injuries/diseases. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.

Functional Hypermethylation of ALDH1L1, PLCL2, and PPP2R3A in Colon Cancer

DNA hypermethylation and mutations are key mechanisms for the downregulation of tumor suppressor genes. NotI-microarrays allowed us to detect hypermethylation and/or deletions in 180 NotI sites associated with 188 genes of human chromosome 3, in 24 paired (tumor/normal) colon samples. The most frequent aberrations (in more than 20% of tumor samples) were detected in the promoter regions of 20 genes. Expression and promoter methylation of these genes were analyzed using the data for paired colon samples from The Cancer Genome Atlas project. Three genes - ALDH1L1, PLCL2, and PPP2R3A - revealed a more than two-fold average decrease in expression and a negative correlation between mRNA level and promoter hypermethylation. The expression of these three genes was then evaluated in 30 paired colon samples by quantitative PCR. Frequent (in more than 60% of cases) and significant (5-9-fold on average) mRNA level decrease was found for each of the genes in the tumor samples. The results indicate a suppressor role of the ALDH1L1, PLCL2, and PPP2R3A genes in colon cancer, as well as functional significance of hypermethylation in the downregulation of these genes.

Assignment and ordering of twenty-three unique NotI-linking clones containing expressed genes including the guanosine 5'-monophosphate synthetase gene to human chromosome 3.

Twenty-three unique NotI-linking clones, mainly isolated from the NRL1 library, were mapped and ordered by fluorescence in situ hybridization to human chromosome 3. All these clones were partially sequenced around the NotI sites and thus represent sequence-tagged sites. The EMBL nucleotide database was then searched with sequences from the NotI-linking clones using the FASTA program. This search revealed that the NRL-090 clone (at 3q24) contains the gene encoding human guanosine 5'-monophosphate synthetase (GMPS-PEN). To our knowledge, this is the first localization of this gene. Clone NL1-320 (at 3p21.3) contains a gene encoding arginine tRNA (97.3% identity in 73 bp), while clones NRL-063, NRL-097 and NRL-143 contain expressed sequences with unknown functions. Other clones displayed 60-85% similarities to cDNAs, CpG islands and other genes.

Mycobacterium tuberculosis Ag85b:hfcγ1 recombinant fusion protein as a selective receptor-dependent delivery system for antigen presentation

Introducing an effective vaccine for tuberculosis (TB) is an urgent need. Mycobacterium tuberculosis (Mtb) Ag85 complex is suggested for making protective immunodominant antigens for design and development of novel TB vaccine. In the present study, a pDR2EF1-Fcγ1 vector has been used to make Ag85B:hFcγ1 recombinant fusion protein. Briefly, specific XbaI and NotI site incorporated primers were used to amplify Mtb-fbpB gene by PCR, TA-cloned and amplified in E.coli DH5α. The resulting vector then subcloned into the pDR2EF1.Fcγ1 vector and transferred to Chinese hamster ovary (CHO) cell line. DNA sequencing was performed to confirm that Ag85B:hFcγ1 construct is precise and in-frame. Then, Ag85B:hFcγ1 protein was produced by CHO expression system and recombinant protein was purified using HiTrap rProtein A Sepharose Fast Flow column. The presence of recombinant fusion protein confirmed by immunofluorescence (IFA) and Western blotting (WB). This fusion protein containing Fc fragment of human IgG1, apart from stability and adjuvanticity potential, could bind to FcRγI (CD64) on the surface of antigen-presenting cells (APCs) and induce cross-presentation in favour of host immune response and can be used as a potential candidate along with other subunit vaccines against Mtb.

Methylation-Sensitive Amplification Length Polymorphism (MS-AFLP) Microarrays for Epigenetic Analysis of Human Genomes.

Somatic, and in a minor scale also germ line, epigenetic aberrations are fundamental to carcinogenesis, cancer progression, and tumor phenotype. DNA methylation is the most extensively studied and arguably the best understood epigenetic mechanisms that become altered in cancer. Both somatic loss of methylation (hypomethylation) and gain of methylation (hypermethylation) are found in the genome of malignant cells. In general, the cancer cell epigenome is globally hypomethylated, while some regions-typically gene-associated CpG islands-become hypermethylated. Given the profound impact that DNA methylation exerts on the transcriptional profile and genomic stability of cancer cells, its characterization is essential to fully understand the complexity of cancer biology, improve tumor classification, and ultimately advance cancer patient management and treatment. A plethora of methods have been devised to analyze and quantify DNA methylation alterations. Several of the early-developed methods relied on the use of methylation-sensitive restriction enzymes, whose activity depends on the methylation status of their recognition sequences. Among these techniques, methylation-sensitive amplification length polymorphism (MS-AFLP) was developed in the early 2000s, and successfully adapted from its original gel electrophoresis fingerprinting format to a microarray format that notably increased its throughput and allowed the quantification of the methylation changes. This array-based platform interrogates over 9500 independent loci putatively amplified by the MS-AFLP technique, corresponding to the NotI sites mapped throughout the human genome.

Comparative evaluation of recombinant LigB protein and heat-killed antigen-based latex agglutination test with microscopic agglutination test for diagnosis of bovine leptospirosis

This study aimed to develop latex agglutination test (LAT) using recombinant leptospiral immunoglobulin-like protein (LigB) (rLigB) antigen and compare its diagnostic efficacy with LAT using conventional heat-killed leptospiral antigen and microscopic agglutination test (MAT) in diagnosing bovine leptospirosis. The PCR-amplified 1053-bp ligB gene sequences from Leptospira borgpetersenii Hardjo serovar were cloned in pET 32 (a) vector at EcoRI and NotI sites and expressed in BL21 E. coli cells as fusion protein with thioredoxin (-57 kDa) and characterized by SDS-PAGE and immunoblot. Out of 390 serum samples [cattle (n = 214), buffaloes (n = 176)] subjected to MAT, 115 samples showed reciprocal titre≥100 up to 1600 against one or more serovars. For recombinant LigB protein/antigen-based LAT, agglutination was observed in the positive sample, while no agglutination was observed in the negative sample. Similarly, heat-killed leptospiral antigen was prepared from and used in LAT for comparison with MAT. A two-sided contingency table was used for analysis of LAT using both the antigens separately against MAT for 390 serum samples. The sensitivity, specificity and positive and negative predictive values of recombinant LigB LAT were found to be 75.65, 91.27, 78.38 and 89.96 %, respectively, and that of heat-killed antigen-based LAT were 72.17, 89.82, 74.77 and 88.53 %, respectively, in comparison with MAT. This developed test will be an alternative/complementary to the existing battery of diagnostic assays/tests for specific detection of pathogenic Leptospira infection in bovine population.

A new vector for efficient gene targeting to the pyrG locus in Aspergillus niger.

BackgroundThe possibility for efficient gene targeting for the controlled integration of DNA constructs is an important tool in fungal genetics.FindingsIn this study, we report a new targeting vector based on the pyrG marker in Aspergillus niger. The DNA sequence to be targeted is surrounded by two fragments of the pyrG gene to allow homologous recombination of the recombinant DNA at the pyrG locus. The 5’ end of the targeting cassette contains a non-functional truncated pyrG open reading frame (first 112 bases deleted) and the 3’ untranslated region (3’ UTR). At the 3’ end, the targeting cassette consists of the 3’ flanking region of the pyrG gene. A unique NotI site between the flanks allows the insertion of a gene of interest. The linearized targeting cassette is transformed to the A. niger pyrG mutant strain AB4.1 or a derivative thereof. By using a constitutively expressed luciferase reporter gene (mluc) as an example, it is shown that the targeting system is efficient as 4 out of 6 (67%) AB4.1 transformants and 51 out of 66 (77%) MA169.4 (ku70 −) transformants contained the reporter gene at the pyrG locus. A luciferase (lux) activity assay, performed with independently obtained transformants in which the mluc reporter was integrated at the pyrG locus, showed comparable and reproducible lux activities.ConclusionThe new pyrG targeting vector is an important improvement to the existing method for gene targeting in A. niger. Although the vector is specific for A. niger, the presented design and approach is easily applicable for constructing integration vectors for other fungi.

Epigenetic silencing of BTB and CNC homology 2 and concerted promoter CpG methylation in gastric cancer

BTB and CNC homology 2 (BACH2) is a lymphoid-specific transcription factor with a prominent role in B-cell development. Genetic polymorphisms within a single locus encoding BACH2 are associated with various autoimmune diseases and allergies. In this study, restriction landmark genomic scanning revealed methylation at a NotI site in a CpG island covering the BACH2 promoter in gastric cancer cell lines and primary gastric tumors. Increased methylation of the BACH2 promoter was observed in 52% (43/83) of primary gastric tumors, and BACH2 hypermethylation was significantly associated with decreased gene expression. Treatment with 5-aza-2′-deoxycytidine and/or trichostatin. A restored BACH2 expression in BACH2-silenced gastric cancer cell lines, and knockdown of BACH2 using short hairpin RNA (i.e. RNA interference) increased cell proliferation in gastric cancer cells. Clinicopathologic data showed that decreased BACH2 expression occurred significantly more frequently in intestinal-type (27/44, 61%) compared with diffuse-type (13/50, 26%) gastric cancers (P<0.001). Furthermore, BACH2 promoter methylation paralleled that of previously identified targets, such as LRRC3B, LIMS2, PRKD1 and POPDC3, in a given set of gastric tumors. We propose that concerted methylation in many promoters plays a role in accelerating gastric tumor formation and that methylated promoter loci may be targets for therapeutic treatment, such as the recently introduced technique of epigenetic editing.

Construction of an adenoviral expression vector carrying FLAG and hrGFP-1 genes and its expression in bone marrow mesenchymal stem cells

The aim of this study was to construct an adenoviral expression vector for vascular endothelium growth factor 121 (VEGF121)-FLAG and humanized Renilla reniformis green fluorescent protein (hrGFP-1) genes, and to observe their expressions in bone marrow mesenchymal stem cells. Using pTG19T-VEGF121 as a template, polymerase chain reaction technology was adopted to mutate the VEGF121 gene by removing the stop codon and inserting NotI and XhoI restriction sites both before and after the gene sequences. The resultant gene was then subcloned into a pMD19-T plasmid, the pMD19-T-VEGF121 and pShuttle-CMV-IRES-hrGFP-1 plasmids were double-digested, and small and large fragments were linked after gel recovery to complete the construction of recombinant adenovirus vectors. After titer determination, the recombinant adenovirus vectors were used to affect rabbit bone marrow mesenchymal stem cells, and fluorescence intensity was observed under fluorescence microscopy. Enzyme digestion identification and sequencing confirmed that the recombinant plasmids were successfully constructed, and observations under fluorescence microscopy showed significant expression of green fluorescent protein in recombinant adenovirus-infected bone marrow mesenchymal stem cells. The constructed adenoviral gene expression vectors carrying VEGF121-FLAG and hrGFP-1 can be expressed in eukaryotic cells, which may be used for gene therapy of ischemic disorders.

25-Hydroxycholesterol Activates the Integrated Stress Response to Reprogram Transcription and Translation in Macrophages

25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/eIF2α/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity.

Amplification, Sequencing and Cloning of Iranian Native Bacillus subtilis Alpha-amylase Gene in Saccharomyces cerevisiae

Background: Alpha-amylases are digestive enzymes which hydrolyze starch glycosidic bonds to glucose, maltose, maltotriose and dextrin which have diverse applications in a wide range of industries such as food, textile, paper, detergents representing approximately 30% of the world enzyme production. Objectives: In this study, the gene encoding the alpha-amylase enzyme of native isolated Bacillus subtilis was amplified with specific primers containing of NotI and AscI restriction sites by PCR and then sequenced. Purified PCR product and shuttle episomal vector p316TDH3 were cut by restriction enzymes and cloned into Escherichia coli and yeast hosts. Materials and Methods: The haploid auxotroph (ura3-) strain of S. cerevisiae and p316TDH3 were used as the host and vector for cloning and expression of the alpha amylase gene, respectively. In native Bacillus sp. the amyE gene without signal sequence was amplified with specific primers that introduced AscI and NotI restriction sites. After constructing the recombinant plasmid, it was transformed into E. coli competent cells. Then, colonies selection and confirmation were performed and the extracted plasmid was introduced to competent yeast cells using carrier sperm DNA. Recombinant yeast cells could grow on minimal media and produce extracellular enzyme. Results: The presence of alpha-amylase gene in recombinant bacteria was certificated by colony-PCR method. After extraction of recombinant vector from E. coli, the competent S. cerevisiae cells were transformed using polyethylene glycol and carrier sperm DNA. The recombinant yeast strains were screened by URA3 auxotrophic marker and analyzed for alpha-amylase gene existence. In the other hand, the amylase gene length of native B. subtilis was1887base pairs (bp) with an approximately93.65% similarity with standard bacterial strain. Conclusions: Based on this similarity and our bioinformatics evaluations, this mentioned alpha-amylase gene can be expressed in S. cerevisiae as extracellular enzyme.

Cloning and expression of human granulocyte-macrophage colony stimulating factor (hGM-CSF) in Pichia pastoris

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a glycoprotein, belongs to the family of colony stimulating factors (CSFs) that regulate proliferation and differentiation of hematopoietic cells. Recombinant hGM-CSF (rhGM-CSF) is one of FDA-appoved, therapeutic recombinant proteins that have been successfully used to treat many diseases like neutropenia, leukemia, or in combination with chemical therapy, etc. In this study, we reported the production of rhGM-CSF in methylotrophic yeast Pichia pastoris through secretory expression using the inducible AOX1 promoter. The gene hgm-csf encoding for hGM-CSF comprising of 415 bps was isolated using PCR reaction with two primers hGM-F and hGM-R bearing XhoI and NotI restriction sites, respectively. After double digesting with these enzymes, the hgm-csf fragment was cloned into pPICZαA shuttle vector, between the XhoI and NotI sites. Recombinant vector containing the gene hgm-csf, termed pPICZαA/hGMCSF, under the control of promoter AOX has the ability to express hGM-CSF in P. pastoris. The accuracy of cloning strategy was confirmed by digestion with REs, PCR and sequencing. Sequencing alignment showed that the cloned gene completely homologous to those published on Genebank. SacI linearized pPICZαA/hGM-CSF was transformed into P. pastoris X33 strain to establish the recombinant P. pastoris X33::hgm-csf. In methanol-contained, inducing BMMY medium, the recombinant X33 cells expressed and secreted hGM-CSF into the supernatant. The secreted hGM-CSF migrated as a band at about 20 kDa, a diffuse band at the range of 29 to 35 kDa, indicating differentially glycosylated forms, and a band at 14,7kDa which is a nonglycosylated form on SDS-PAGE gel. This result was confirmed by Western blot with specific antibodies.

Proteome Modulation in H9c2 Cardiac Cells by microRNAs miR-378 and miR-378

MicroRNAs are a novel class of powerful endogenous regulators of gene expression. MiR-378 and miR-378* are localized in the first intron of the Ppargc1b gene that codes the transcriptional co-activator PGC-1β. The latter regulates energy expenditure as well as mitochondrial biogenesis. The miR-378:miR-378* hairpin is highly expressed in cardiac cells. To better assess their role in cardiomyocytes, we identified miR-378 and miR-378* targets via a proteomic screen. We established H9c2 cellular models of overexpression of miR-378 and miR-378* and identified a total of 86 down-regulated proteins in the presence of either one of these miRs. Functional annotation clustering showed that miR-378 and miR-378* regulate related pathways in cardiomyocytes, including energy metabolism, notably glycolysis, cytoskeleton, notably actin filaments and muscle contraction. Using bioinformatics algorithms we found that 20 proteins were predicted as direct targets of the miRs. We validated eight of these targets by quantitative RT-PCR and luciferase reporter assay. We found that miR-378 targets lactate dehydrogenase A and impacts on cell proliferation and survival whereas miR-378* targets cytoskeleton proteins actin and vimentin. Proteins involved in endoplasmic reticulum stress response such as chaperone and/or calcium buffering proteins GRP78, PPIA (cyclophilin A), calumenin, and GMMPA involved in glycosylation are repressed by these miRs. Our results show that the miR-378/378* hairpin establishes a connection among energy metabolism, cytoskeleton remodeling, and endoplasmic reticulum function through post-transcriptional regulation of key proteins involved in theses pathways.

Agonist-biased Trafficking of Somatostatin Receptor 2A in Enteric Neurons

Somatostatin (SST) 14 and SST 28 activate somatostatin 2A receptors (SSTR2A) on enteric neurons to control gut functions. SST analogs are treatments of neuroendocrine and bleeding disorders, cancer, and diarrhea, with gastrointestinal side effects of constipation, abdominal pain, and nausea. How endogenous agonists and drugs differentially regulate neuronal SSTR2A is unexplored. We evaluated SSTR2A trafficking in murine myenteric neurons and neuroendocrine AtT-20 cells by microscopy and determined whether agonist degradation by endosomal endothelin-converting enzyme 1 (ECE-1) controls SSTR2A trafficking and association with β-arrestins, key regulators of receptors. SST-14, SST-28, and peptide analogs (octreotide, lanreotide, and vapreotide) stimulated clathrin- and dynamin-mediated internalization of SSTR2A, which colocalized with ECE-1 in endosomes and the Golgi. After incubation with SST-14, SSTR2A recycled to the plasma membrane, which required active ECE-1 and an intact Golgi. SSTR2A activated by SST-28, octreotide, lanreotide, or vapreotide was retained within the Golgi and did not recycle. Although ECE-1 rapidly degraded SST-14, SST-28 was resistant to degradation, and ECE-1 did not degrade SST analogs. SST-14 and SST-28 induced transient interactions between SSTR2A and β-arrestins that were stabilized by an ECE-1 inhibitor. Octreotide induced sustained SSTR2A/β-arrestin interactions that were not regulated by ECE-1. Thus, when activated by SST-14, SSTR2A internalizes and recycles via the Golgi, which requires ECE-1 degradation of SST-14 and receptor dissociation from β-arrestins. After activation by ECE-1-resistant SST-28 and analogs, SSTR2A remains in endosomes because of sustained β-arrestin interactions. Therapeutic SST analogs are ECE-1-resistant and retain SSTR2A in endosomes, which may explain their long-lasting actions.

A Naturally Variable Residue in the S1 Subsite of M1 Family Aminopeptidases Modulates Catalytic Properties and Promotes Functional Specialization

M1 family metallo-aminopeptidases fulfill a wide range of critical and in some cases medically relevant roles in humans and human pathogens. The specificity of M1-aminopeptidases is dominated by the interaction of the well defined S1 subsite with the side chain of the first (P1) residue of the substrate and can vary widely. Extensive natural variation occurs at one of the residues that contributes to formation of the cylindrical S1 subsite. We investigated whether this natural variation contributes to diversity in S1 subsite specificity. Effects of 11 substitutions of the S1 subsite residue valine 459 in the Plasmodium falciparum aminopeptidase PfA-M1 and of three substitutions of the homologous residue methionine 260 in Escherichia coli aminopeptidase N were characterized. Many of these substitutions altered steady-state kinetic parameters for dipeptide hydrolysis and remodeled S1 subsite specificity. The most dramatic change in specificity resulted from substitution with proline, which collapsed S1 subsite specificity such that only substrates with P1-Arg, -Lys, or -Met were appreciably hydrolyzed. The structure of PfA-M1 V459P revealed that the proline substitution induced a local conformational change in the polypeptide backbone that resulted in a narrowed S1 subsite. The restricted specificity and active site backbone conformation of PfA-M1 V459P mirrored those of endoplasmic reticulum aminopeptidase 2, a human enzyme with proline in the variable S1 subsite position. Our results provide compelling evidence that changes in the variable residue in the S1 subsite of M1-aminopeptidases have facilitated the evolution of new specificities and ultimately novel functions for this important class of enzymes.

Dbf4 and Cdc7 Proteins Promote DNA Replication through Interactions with Distinct Mcm2–7 Protein Subunits

The essential cell cycle target of the Dbf4/Cdc7 kinase (DDK) is the Mcm2-7 helicase complex. Although Mcm4 has been identified as the critical DDK phosphorylation target for DNA replication, it is not well understood which of the six Mcm2-7 subunits actually mediate(s) docking of this kinase complex. We systematically examined the interaction between each Mcm2-7 subunit with Dbf4 and Cdc7 through two-hybrid and co-immunoprecipitation analyses. Strikingly different binding patterns were observed, as Dbf4 interacted most strongly with Mcm2, whereas Cdc7 displayed association with both Mcm4 and Mcm5. We identified an N-terminal Mcm2 region required for interaction with Dbf4. Cells expressing either an Mcm2 mutant lacking this docking domain (Mcm2ΔDDD) or an Mcm4 mutant lacking a previously identified DDK docking domain (Mcm4ΔDDD) displayed modest DNA replication and growth defects. In contrast, combining these two mutations resulted in synthetic lethality, suggesting that Mcm2 and Mcm4 play overlapping roles in the association of DDK with MCM rings at replication origins. Consistent with this model, growth inhibition could be induced in Mcm4ΔDDD cells through Mcm2 overexpression as a means of titrating the Dbf4-MCM ring interaction. This growth inhibition was exacerbated by exposing the cells to either hydroxyurea or methyl methanesulfonate, lending support for a DDK role in stabilizing or restarting replication forks under S phase checkpoint conditions. Finally, constitutive overexpression of each individual MCM subunit was examined, and genotoxic sensitivity was found to be specific to Mcm2 or Mcm4 overexpression, further pointing to the importance of the DDK-MCM ring interaction.

Alteration of Plasma Membrane Organization by an Anticancer Lysophosphatidylcholine Analogue Induces Intracellular Acidification and Internalization of Plasma Membrane Transporters in Yeast

The lysophosphatidylcholine analogue edelfosine is a potent antitumor lipid that targets cellular membranes. The underlying mechanisms leading to cell death remain controversial, although two cellular membranes have emerged as primary targets of edelfosine, the plasma membrane (PM) and the endoplasmic reticulum. In an effort to identify conditions that enhance or prevent the cytotoxic effect of edelfosine, we have conducted genome-wide surveys of edelfosine sensitivity and resistance in Saccharomyces cerevisiae presented in this work and the accompanying paper (Cuesta-Marbán, Á., Botet, J., Czyz, O., Cacharro, L. M., Gajate, C., Hornillos, V., Delgado, J., Zhang, H., Amat-Guerri, F., Acuña, A. U., McMaster, C. R., Revuelta, J. L., Zaremberg, V., and Mollinedo, F. (January 23, 2013) J. Biol. Chem. 288,), respectively. Our results point to maintenance of pH homeostasis as a major player in modulating susceptibility to edelfosine with the PM proton pump Pma1p playing a main role. We demonstrate that edelfosine alters PM organization and induces intracellular acidification. Significantly, we show that edelfosine selectively reduces lateral segregation of PM proteins like Pma1p and nutrient H(+)-symporters inducing their ubiquitination and internalization. The biology associated to the mode of action of edelfosine we have unveiled includes selective modification of lipid raft integrity altering pH homeostasis, which in turn regulates cell growth.

The Genetic Risk in Mice from Radiation: An Estimate of the Mutation Induction Rate per Genome

Restriction Landmark Genome Scanning (RLGS) is a method that uses end-labeled (32)P NotI sites that are mostly associated with coding genes to visualizes thousands of DNA fragments as spots in two-dimensional autoradiograms. This approach allows direct detection of autosomal deletions as spots with half normal intensity. The method was applied to mouse offspring derived from spermatogonia exposed to 4 Gy of X rays. A genome-wide assessment of the mutation induction rate was estimated from the detected deletions. Examinations were made of 1,007 progeny (502 derived from control males and 505 from irradiated males) and 1,190 paternal and 1,240 maternal spots for each mouse. The results showed one deletion mutation in the unirradiated paternal genomes of 502 offspring (0.2%) and 5 deletions in the irradiated paternal genomes of 505 offspring (1%). The difference was marginally significant, with the deletion sizes ranged from 2-13 Mb. If the frequencies are taken at face value, the net increase was 0.8% after an exposure of 4 Gy, or 0.2% per Gy per individual if a linear dose response is assumed. Since the present RLGS analysis examined 1,190 NotI sites, while the mouse genome contains ∼25,000 genes, the genomic probability of any gene undergoing a deletion mutation would be 25× 0.2%, or 5% per Gy. Furthermore, since the present RLGS screened about 0.2% of the total genome, the probability of detecting a deletion anywhere in the total genome would be estimated to be 500 times 0.2% or 100% (i.e., 1 deletion per Gy). These results are discussed with reference to copy number variation in the human genome.

Neuronal Per Arnt Sim (PAS) Domain Protein 4 (NPAS4) Regulates Neurite Outgrowth and Phosphorylation of Synapsin I

Neuronal Per Arnt Sim domain protein 4 (NPAS4), a brain-specific basic helix-loop-helix transcription factor, has recently been shown to regulate the development of the GABAergic inhibitory synapses and transcription program for contextual memory formation in the hippocampus. We previously reported that chronic social isolation or restriction stress in mice resulted in an impairment in memory and emotional behavior, which was associated with a decrease in Npas4 mRNA levels. In this study, we investigated the role of NPAS4 in neuronal function in vitro and in vivo. Differentiation medium-induced neurite outgrowth was inhibited in Npas4 knockdown Neuro2a cells, whereas overexpression of NPAS4 accelerated the neurite outgrowth in Neuro2a cells. Furthermore, depolarization-induced neurite outgrowth was abolished in Npas4 KO hippocampal neurons. NPAS4 overexpression increased cyclin-dependent kinase 5 (CDK5)-dependent synapsin I phosphorylation in Neuro2a cells and primary cultured hippocampal neurons. A CDK5 inhibitor, roscovitine, inhibited the neurite outgrowth and the increase in phosphorylated synapsin I (p-SYN I) levels in Npas4-overexpressed Neuro2a cells. Interaction of NPAS4 with promoters of Cdk5 and NeuN genes was demonstrated by a chromatin immunoprecipitation assay. In an in vivo study, pentylenetetrazole-induced convulsions in mice resulted in an increase in NPAS4 and p-SYN I levels in the prefrontal cortex of wild-type mice, although no changes in p-SYN I levels were observed in Npas4 knock-out mice. These results suggest that NPAS4 plays an important role in the structural and functional plasticity of neurons.