Nuclear Localization of Schizosaccharomyces pombe Mcm2/Cdc19p Requires MCM Complex Assembly

PubMed Central

Pasion, Sally G.; Forsburg, Susan L.

1999-01-01

The minichromosome maintenance (MCM) proteins MCM2–MCM7 are conserved eukaryotic replication factors that assemble in a heterohexameric complex. In fission yeast, these proteins are nuclear throughout the cell cycle. In studying the mechanism that regulates assembly of the MCM complex, we analyzed the cis and trans elements required for nuclear localization of a single subunit, Mcm2p. Mutation of any single mcm gene leads to redistribution of wild-type MCM subunits to the cytoplasm, and this redistribution depends on an active nuclear export system. We identified the nuclear localization signal sequences of Mcm2p and showed that these are required for nuclear targeting of other MCM subunits. In turn, Mcm2p must associate with other MCM proteins for its proper localization; nuclear localization of MCM proteins thus requires assembly of MCM proteins in a complex. We suggest that coupling complex assembly to nuclear targeting and retention ensures that only intact heterohexameric MCM complexes remain nuclear. PMID:10588642

The Selenocysteine-Specific Elongation Factor Contains Unique Sequences That Are Required for Both Nuclear Export and Selenocysteine Incorporation.

PubMed

Dubey, Aditi; Copeland, Paul R

2016-01-01

Selenocysteine (Sec) is a critical residue in at least 25 human proteins that are essential for antioxidant defense and redox signaling in cells. Sec is inserted into proteins cotranslationally by the recoding of an in-frame UGA termination codon to a Sec codon. In eukaryotes, this recoding event requires several specialized factors, including a dedicated, Sec-specific elongation factor called eEFSec, which binds Sec-tRNASec with high specificity and delivers it to the ribosome for selenoprotein production. Unlike most translation factors, including the canonical elongation factor eEF1A, eEFSec readily localizes to the nucleus of mammalian cells and shuttles between the cytoplasmic and nuclear compartments. The functional significance of eEFSec's nuclear localization has remained unclear. In this study, we have examined the subcellular localization of eEFSec in the context of altered Sec incorporation to demonstrate that reduced selenoprotein production does not correlate with changes in the nuclear localization of eEFSec. In addition, we identify several novel sequences of the protein that are essential for localization as well as Sec insertion activity, and show that eEFSec utilizes CRM1-mediated nuclear export pathway. Our findings argue for two distinct pools of eEFSec in the cell, where the cytoplasmic pool participates in Sec incorporation and the nuclear pool may be involved in an as yet unknown function.

Functional analysis of the C-terminal region of human adenovirus E1A reveals a misidentified nuclear localization signal

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cohen, Michael J.; King, Cason R.; Dikeakos, Jimmy D.

The immortalizing function of the human adenovirus 5 E1A oncoprotein requires efficient localization to the nucleus. In 1987, a consensus monopartite nuclear localization sequence (NLS) was identified at the C-terminus of E1A. Since that time, various experiments have suggested that other regions of E1A influence nuclear import. In addition, a novel bipartite NLS was recently predicted at the C-terminal region of E1A in silico. In this study, we used immunofluorescence microscopy and co-immunoprecipitation analysis with importin-α to verify that full nuclear localization of E1A requires the well characterized NLS spanning residues 285–289, as well as a second basic patch situatedmore » between residues 258 and 263 ({sup 258}RVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP{sup 289}). Thus, the originally described NLS located at the C-terminus of E1A is actually a bipartite signal, which had been misidentified in the existing literature as a monopartite signal, altering our understanding of one of the oldest documented NLSs. - Highlights: • Human adenovirus E1A is localized to the nucleus. • The C-terminus of E1A contains a bipartite nuclear localization signal (NLS). • This signal was previously misidentified to be a monopartite NLS. • Key basic amino acid residues within this sequence are highly conserved.« less

Hidden genetic history of the Japanese sand dollar Peronella (Echinoidea: Laganidae) revealed by nuclear intron sequences.

PubMed

Endo, Megumi; Hirose, Mamiko; Honda, Masanao; Koga, Hiroyuki; Morino, Yoshiaki; Kiyomoto, Masato; Wada, Hiroshi

2018-06-15

The marine environment around Japan experienced significant changes during the Cenozoic Era. In this study, we report findings suggesting that this dynamic history left behind traces in the genome of the Japanese sand dollar species Peronella japonica and P. rubra. Although mitochondrial Cytochrome C Oxidase I sequences did not indicate fragmentation of the current local populations of P. japonica around Japan, two different types of intron sequence were found in the Alx1 locus. We inferred that past fragmentation of the populations account for the presence of two types of nuclear sequences as alleles in the Alx1 intron of P. japonica. It is likely that the split populations have intermixed in recent times; hence, we did not detect polymorphisms in the sequences reflecting the current localization of the species. In addition, we found two allelic sequences of theAlx1 intron in the sister species P. rubra. The divergence times of the two types of Alx1 intron sequences were estimated at approximately 14.9 and 4.0 million years ago for P. japonica and P. rubra, respectively. Our study indicates that information from the intron sequences of nuclear genes can enhance our understanding of past genetic events in organisms. Copyright © 2018 Elsevier B.V. All rights reserved.

Analysis of plastid and mitochondrial DNA insertions in the nucleus (NUPTs and NUMTs) of six plant species: size, relative age and chromosomal localization.

PubMed

Michalovova, M; Vyskot, B; Kejnovsky, E

2013-10-01

We analysed the size, relative age and chromosomal localization of nuclear sequences of plastid and mitochondrial origin (NUPTs-nuclear plastid DNA and NUMTs-nuclear mitochondrial DNA) in six completely sequenced plant species. We found that the largest insertions showed lower divergence from organelle DNA than shorter insertions in all species, indicating their recent origin. The largest NUPT and NUMT insertions were localized in the vicinity of the centromeres in the small genomes of Arabidopsis and rice. They were also present in other chromosomal regions in the large genomes of soybean and maize. Localization of NUPTs and NUMTs correlated positively with distribution of transposable elements (TEs) in Arabidopsis and sorghum, negatively in grapevine and soybean, and did not correlate in rice or maize. We propose a model where new plastid and mitochondrial DNA sequences are inserted close to centromeres and are later fragmented by TE insertions and reshuffled away from the centromere or removed by ectopic recombination. The mode and tempo of TE dynamism determines the turnover of NUPTs and NUMTs resulting in their species-specific chromosomal distributions.

Characterization of karyopherins in androgen receptor intracellular trafficking in the yeast model

PubMed Central

Nguyen, Minh M; Harmon, Robert M; Wang, Zhou

2014-01-01

Background: Mechanisms regulating androgen receptor (AR) subcellular localization represent an essential component of AR signaling. Karyopherins are a family of nucleocytoplasmic trafficking factors. In this paper, we used the yeast model to study the effects of karyopherins on the subcellular localization of the AR. Methods: Yeast mutants deficient in different nuclear transport factors were transformed with various AR based, GFP tagged constructs and their localization was monitored using microscopy. Results: We showed that yeast can mediate androgen-induced AR nuclear localization and that in addition to the import factor, Importinα/β, this process required the import karyopherin Sxm1. We also showed that a previously identified nuclear export sequence (NESAR) in the ligand binding domain of AR does not appear to rely on karyopherins for cytoplasmic localization. Conclusions: These results suggest that while AR nuclear import relies on karyopherin activity, AR nuclear export and/or cytoplasmic localization may require other undefined mechanisms. PMID:25031696

Influence of structural variation on nuclear localization of DNA-binding polyamide-fluorophore conjugates.

PubMed

Edelson, Benjamin S; Best, Timothy P; Olenyuk, Bogdan; Nickols, Nicholas G; Doss, Raymond M; Foister, Shane; Heckel, Alexander; Dervan, Peter B

2004-01-01

A pivotal step forward in chemical approaches to controlling gene expression is the development of sequence-specific DNA-binding molecules that can enter live cells and traffic to nuclei unaided. DNA-binding polyamides are a class of programmable, sequence-specific small molecules that have been shown to influence a wide variety of protein-DNA interactions. We have synthesized over 100 polyamide-fluorophore conjugates and assayed their nuclear uptake profiles in 13 mammalian cell lines. The compiled dataset, comprising 1300 entries, establishes a benchmark for the nuclear localization of polyamide-dye conjugates. Compounds in this series were chosen to provide systematic variation in several structural variables, including dye composition and placement, molecular weight, charge, ordering of the aromatic and aliphatic amino-acid building blocks and overall shape. Nuclear uptake does not appear to be correlated with polyamide molecular weight or with the number of imidazole residues, although the positions of imidazole residues affect nuclear access properties significantly. Generally negative determinants for nuclear access include the presence of a beta-Ala-tail residue and the lack of a cationic alkyl amine moiety, whereas the presence of an acetylated 2,4-diaminobutyric acid-turn is a positive factor for nuclear localization. We discuss implications of these data on the design of polyamide-dye conjugates for use in biological systems.

An N-terminal nuclear localization sequence but not the calmodulin-binding domain mediates nuclear localization of nucleomorphin, a protein that regulates nuclear number in Dictyostelium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Myre, Michael A.; O'Day, Danton H.

2005-06-24

Nucleomorphin is a novel nuclear calmodulin (CaM)-binding protein (CaMBP) containing an extensive DEED (glu/asp repeat) domain that regulates nuclear number. GFP-constructs of the 38 kDa NumA1 isoform localize as intranuclear patches adjacent to the inner nuclear membrane. The translocation of CaMBPs into nuclei has previously been shown by others to be mediated by both classic nuclear localization sequences (NLSs) and CaM-binding domains (CaMBDs). Here we show that NumA1 possesses a CaMBD ({sup 171}EDVSRFIKGKLLQKQQKIYKDLERF{sup 195}) containing both calcium-dependent-binding motifs and an IQ-like motif for calcium-independent binding. GFP-constructs containing only NumA1 residues 1-129, lacking the DEED and CaMBDs, still localized as patchesmore » at the internal periphery of nuclei thus ruling out a direct role for the CaMBD in nuclear import. These constructs contained the amino acid residues {sup 48}KKSYQDPEIIAHSRPRK{sup 64} that include both a putative bipartite and classical NLS. GFP-bipartite NLS constructs localized uniformly within nuclei but not as patches. As with previous work, removal of the DEED domain resulted in highly multinucleate cells. However as shown here, multinuclearity only occurred when the NLS was present allowing the protein to enter nuclei. Site-directed mutation analysis in which the NLS was changed to {sup 48}EF{sup 49} abolished the stability of the GFP fusion at the protein but not RNA level preventing subcellular analyses. Cells transfected with the {sup 48}EF{sup 49} construct exhibited slowed growth when compared to parental AX3 cells and other GFP-NumA1 deletion mutants. In addition to identifying an NLS that is sufficient for nuclear translocation of nucleomorphin and ruling out CaM-binding in this event, this work shows that the nuclear localization of NumA1 is crucial to its ability to regulate nuclear number in Dictyostelium.« less

Dissection of a nuclear localization signal.

PubMed

Hodel, M R; Corbett, A H; Hodel, A E

2001-01-12

The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein import receptors. This study seeks to decipher the energetic details of NLS recognition by the receptor importin alpha through quantitative analysis of variant NLSs. The relative importance of each residue in two monopartite NLS sequences was determined using an alanine scanning approach. These measurements yield an energetic definition of a monopartite NLS sequence where a required lysine residue is followed by two other basic residues in the sequence K(K/R)X(K/R). In addition, the energetic contributions of the second basic cluster in a bipartite NLS ( approximately 3 kcal/mol) as well as the energy of inhibition of the importin alpha importin beta-binding domain ( approximately 3 kcal/mol) were also measured. These data allow the generation of an energetic scale of nuclear localization sequences based on a peptide's affinity for the importin alpha-importin beta complex. On this scale, a functional NLS has a binding constant of approximately 10 nm, whereas a nonfunctional NLS has a 100-fold weaker affinity of 1 microm. Further correlation between the current in vitro data and in vivo function will provide the foundation for a comprehensive quantitative model of protein import.

Unique nuclear localization of Nile tilapia (Oreochromis niloticus) Neu4 sialidase is regulated by nuclear transport receptor importin α/β.

PubMed

Honda, Akinobu; Chigwechokha, Petros Kingstone; Kamada-Futagami, Yuko; Komatsu, Masaharu; Shiozaki, Kazuhiro

2018-06-01

Sialidase catalyzes the removal of sialic acids from glycoconjugates. Different from Neu1 and Neu3 sialidases, Neu4 enzymatic properties such as substrate specificity and subcellular localization are not well-conserved among vertebrates. In fish only zebrafish and medaka neu4 genes have been cloned and their polypeptides have been characterized so far. Thus, characterization of Neu4 from other fish species is necessary to evaluate Neu4 physiological functions. Here, Nile tilapia was chosen for the characterization of Neu4 polypeptide considering that it is one of the major cultured fish all over the world and that its genomic sequences are now available. Coding DNA sequence of tilapia Neu4 was identified as 1,497 bp and its recombinant protein showed broad substrate specificity and optimal sialidase enzyme activity pH at 4.0. Neu4 activity was sustained even in neutral and alkali pH. Interestingly, immunofluorescence analysis revealed that major subcellular localization of tilapia Neu4 was nuclear, quite distinct from zebrafish (ER) and medaka Neu4 (lysosome). Bioinformatic analysis showed the existence of putative nuclear localization signal (NLS) in tilapia Neu4. In general, it is known that importin families bind to several proteins via NLS and transfer them into nucleus. Therefore, to determine the involvement of putative NLS in Neu4 nuclear localization, Neu4 mutant deleting NLS was constructed and expressed in cultured cells. As a result, NLS deletion significantly diminished the nuclear localization. Furthermore, treatment of importazole, interrupter of binding importin β and RanGTP, significantly suppressed Neu4 nuclear localization. In summary, tilapia Neu4 is a unique sialidase localized at nucleus and its transport system into nucleus is regulated by importin. Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Featured Article: Nuclear export of opioid growth factor receptor is CRM1 dependent.

PubMed

Kren, Nancy P; Zagon, Ian S; McLaughlin, Patricia J

2016-02-01

Opioid growth factor receptor (OGFr) facilitates growth inhibition in the presence of its specific ligand opioid growth factor (OGF), chemically termed [Met(5)]-enkephalin. The function of the OGF-OGFr axis requires the receptor to translocate to the nucleus. However, the mechanism of nuclear export of OGFr is unknown. In this study, endogenous OGFr, as well as exogenously expressed OGFr-EGFP, demonstrated significant nuclear accumulation in response to leptomycin B (LMB), an inhibitor of CRM1-dependent nuclear export, suggesting that OGFr is exported in a CRM1-dependent manner. One consensus sequence for a nuclear export signal (NES) was identified. Mutation of the associated leucines, L217 L220 L223 and L225, to alanine resulted in decreased nuclear accumulation. NES-EGFP responded to LMB, indicating that this sequence is capable of functioning as an export signal in isolation. To determine why the sequence functions differently in isolation than as a full length protein, the localization of subNES was evaluated in the presence and absence of MG132, a potent inhibitor of proteosomal degradation. MG132 had no effect of subNES localization. The role of tandem repeats located at the C-terminus of OGFr was examined for their role in nuclear trafficking. Six of seven tandem repeats were removed to form deltaTR. DeltaTR localized exclusively to the nucleus indicating that the tandem repeats may contribute to the localization of the receptor. Similar to the loss of cellular proliferation activity (i.e. inhibition) recorded with subNES, deltaTR also demonstrated a significant loss of inhibitory activity indicating that the repeats may be integral to receptor function. These experiments reveal that OGFr contains one functional NES, L217 L220 L223 and L225 and can be exported from the nucleus in a CRM1-dependent manner. © 2015 by the Society for Experimental Biology and Medicine.

Subcellular localization of full-length human myeloid leukemia factor 1 (MLF1) is independent of 14-3-3 proteins.

PubMed

Molzan, Manuela; Ottmann, Christian

2013-03-01

Myeloid leukemia factor 1 (MLF1) is associated with the development of leukemic diseases such as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, information on the physiological function of MLF1 is limited and mostly derived from studies identifying MLF1 interaction partners like CSN3, MLF1IP, MADM, Manp and the 14-3-3 proteins. The 14-3-3-binding site surrounding S34 is one of the only known functional features of the MLF1 sequence, along with one nuclear export sequence (NES) and two nuclear localization sequences (NLS). It was recently shown that the subcellular localization of mouse MLF1 is dependent on 14-3-3 proteins. Based on these findings, we investigated whether the subcellular localization of human MLF1 was also directly 14-3-3-dependent. Live cell imaging with GFP-fused human MLF1 was used to study the effects of mutations and deletions on its subcellular localization. Surprisingly, we found that the subcellular localization of full-length human MLF1 is 14-3-3-independent, and is probably regulated by other as-yet-unknown proteins.

Plastid, nuclear and reverse transcriptase sequences in the mitochondrial genome of Oenothera: is genetic information transferred between organelles via RNA?

PubMed Central

Schuster, W; Brennicke, A

1987-01-01

We describe an open reading frame (ORF) with high homology to reverse transcriptase in the mitochondrial genome of Oenothera. This ORF displays all the characteristics of an active plant mitochondrial gene with a possible ribosome binding site and 39% T in the third codon position. It is located between a sequence fragment from the plastid genome and one of nuclear origin downstream from the gene encoding subunit 5 of the NADH dehydrogenase. The nuclear derived sequence consists of 528 nucleotides from the small ribosomal RNA and contains an expansion segment unique to nuclear rRNAs. The plastid sequence contains part of the ribosomal protein S4 and the complete tRNA(Ser). The observation that only transcribed sequences have been found i more than one subcellular compartment in higher plants suggests that interorganellar transfer of genetic information may occur via RNA and subsequent local reverse transcription and genomic integration. PMID:14650433

Nuclear localization signal-dependent and -independent movements of Drosophila melanogaster dUTPase isoforms during nuclear cleavage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muha, Villo; Zagyva, Imre; Venkei, Zsolt

2009-04-03

Two dUTPase isoforms (23 kDa and 21 kDa) are present in the fruitfly with the sole difference of an N-terminal extension. In Drosophila embryo, both isoforms are detected inside the nucleus. Here, we investigated the function of the N-terminal segment using eYFP-dUTPase constructs. In Schneider 2 cells, only the 23 kDa construct showed nuclear localization arguing that it may contain a nuclear localization signal (NLS). Sequence comparisons identified a lysine-rich nonapeptide with similarity to the human c-myc NLS. In Drosophila embryos during nuclear cleavages, the 23 kDa isoform showed the expected localization shifts. Contrariwise, although the 21 kDa isoform wasmore » excluded from the nuclei during interphase, it was shifted to the nucleus during prophase and forthcoming mitotic steps. The observed dynamic localization character showed strict timing to the nuclear cleavage phases and explained how both isoforms can be present within the nuclear microenvironment, although at different stages of cell cycle.« less

Prm3p is a pheromone-induced peripheral nuclear envelope protein required for yeast nuclear fusion.

PubMed

Shen, Shu; Tobery, Cynthia E; Rose, Mark D

2009-05-01

Nuclear membrane fusion is the last step in the mating pathway of the yeast Saccharomyces cerevisiae. We adapted a bioinformatics approach to identify putative pheromone-induced membrane proteins potentially required for nuclear membrane fusion. One protein, Prm3p, was found to be required for nuclear membrane fusion; disruption of PRM3 caused a strong bilateral defect, in which nuclear congression was completed but fusion did not occur. Prm3p was localized to the nuclear envelope in pheromone-responding cells, with significant colocalization with the spindle pole body in zygotes. A previous report, using a truncated protein, claimed that Prm3p is localized to the inner nuclear envelope. Based on biochemistry, immunoelectron microscopy and live cell microscopy, we find that functional Prm3p is a peripheral membrane protein exposed on the cytoplasmic face of the outer nuclear envelope. In support of this, mutations in a putative nuclear localization sequence had no effect on full-length protein function or localization. In contrast, point mutations and deletions in the highly conserved hydrophobic carboxy-terminal domain disrupted both protein function and localization. Genetic analysis, colocalization, and biochemical experiments indicate that Prm3p interacts directly with Kar5p, suggesting that nuclear membrane fusion is mediated by a protein complex.

The R35 residue of the influenza A virus NS1 protein has minimal effects on nuclear localization but alters virus replication through disrupting protein dimerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lalime, Erin N.; Pekosz, Andrew, E-mail: apekosz@jhsph.edu

The influenza A virus NS1 protein has a nuclear localization sequence (NLS) in the amino terminal region. This NLS overlaps sequences that are important for RNA binding as well as protein dimerization. To assess the significance of the NS1 NLS on influenza virus replication, the NLS amino acids were individually mutated to alanines and recombinant viruses encoding these mutations were rescued. Viruses containing NS1 proteins with mutations at R37, R38 and K41 displayed minimal changes in replication or NS1 protein nuclear localization. Recombinant viruses encoding NS1 R35A were not recovered but viruses containing second site mutations at position D39 inmore » addition to the R35A mutation were isolated. The mutations at position 39 were shown to partially restore NS1 protein dimerization but had minimal effects on nuclear localization. These data indicate that the amino acids in the NS1 NLS region play a more important role in protein dimerization compared to nuclear localization. - Highlights: • Mutations were introduced into influenza NS1 NLS1. • NS1 R37A, R38A, K41A viruses had minimal changes in replication and NS1 localization. • Viruses from NS1 R35A rescue all contained additional mutations at D39. • NS1 R35A D39X mutations recover dimerization lost in NS1 R35A mutations. • These results reaffirm the importance of dimerization for NS1 protein function.« less

Chitosan Nanoparticles for Nuclear Targeting: The Effect of Nanoparticle Size and Nuclear Localization Sequence Density.

PubMed

Tammam, Salma N; Azzazy, Hassan M E; Breitinger, Hans G; Lamprecht, Alf

2015-12-07

Many recently discovered therapeutic proteins exert their main function in the nucleus, thus requiring both efficient uptake and correct intracellular targeting. Chitosan nanoparticles (NPs) have attracted interest as protein delivery vehicles due to their biocompatibility and ability to escape the endosomes offering high potential for nuclear delivery. Molecular entry into the nucleus occurs through the nuclear pore complexes, the efficiency of which is dependent on NP size and the presence of nuclear localization sequence (NLS). Chitosan nanoparticles of different sizes (S-NPs ≈ 25 nm; L-NP ≈ 150 nm) were formulated, and they were modified with different densities of the octapeptide NLS CPKKKRKV (S-NPs, 0.25, 0.5, 2.0 NLS/nm(2); L-NPs, 0.6, 0.9, 2 NLS/nm(2)). Unmodified and NLS-tagged NPs were evaluated for their protein loading capacity, extent of cell association, cell uptake, cell surface binding, and finally nuclear delivery efficiency in L929 fibroblasts. To avoid errors generated with cell fractionation and nuclear isolation protocols, nuclear delivery was assessed in intact cells utilizing Förster resonance energy transfer (FRET) fluorometry and microscopy. Although L-NPs showed ≈10-fold increase in protein loading per NP when compared to S-NPs, due to higher cell association and uptake S-NPs showed superior protein delivery. NLS exerts a size and density dependent effect on nanoparticle uptake and surface binding, with a general reduction in NP cell surface binding and an increase in cell uptake with the increase in NLS density (up to 8.4-fold increase in uptake of High-NLS-L-NPs (2 NLS/nm(2)) compared to unmodified L-NPs). However, for nuclear delivery, unmodified S-NPs show higher nuclear localization rates when compared to NLS modified NPs (up to 5-fold by FRET microscopy). For L-NPs an intermediate NLS density (0.9 NLS/nm(2)) seems to provide highest nuclear localization (3.7-fold increase in nuclear delivery compared to High-NLS-L-NPs). Results indicate that a higher NLS density does not result in maximum protein nuclear localization and that a universal optimal density for NPs of different sizes does not exist.

Nuclear targeting of the maize R protein requires two nuclear localization sequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shieh, M.W.; Raikhel, N.V.; Wessler, S.R.

1993-02-01

Previous genetic and structural evidence indicates that the maize R gene encodes a nuclear transcriptional activating factor. In-frame carboxyl- and amino-terminal fusions of the R gene to the reporter gene encoding [beta]-glucuronidase (GUS) were sufficient to direct GUS to the nucleus of the transiently transformed onion (Allium cepa) epidermal cells. Further analysis of chimeric constructs containing regions of the R gene fused to the GUS cDNA revealed three specific nuclear localization sequences (NLSs) that were capable of redirecting the GUS protein to the nucleus. Amino-terminal NLS-A (amino acids 100-109, GDRRAAPARP) contained several arginine residues; a similar localization signal is foundmore » in only a few viral proteins. The medial NLS-M (amino acids 419-428, MSERKRREKL) is a simian virus 40 large T antigen-type NLS, and the carboxyl-terminal NLS-C (amino acids 598-610, MISESLRKAIGKR) is a mating type [alpha]2 type. NLSs M and C are independently sufficient to direct the GUS protein to the nucleus when it is fused at the amino terminus of GUS, whereas NLS-A fused to GUS partitioned between the nucleus and cytoplasm. Similar partitioning was observed when localization signals NLS-A and NLS-C were independently fused to the carboxy-terminal portion of GUS. A sequential deletion of the localization signals indicated that the amino-terminal and carboxyl-terminal fusions of R and GUS were redirected to the nucleus only when both NLS-A and -M, or NLS-C and -M, were present. These results indicate that multiple localization signals are necessary for nuclear targeting of this protein. The conservation of the localization signals within the alleles of R and similar proteins from other organisms is also discussed. 45 refs., 6 figs.« less

Identification of amino acid sequences in the polyomavirus capsid proteins that serve as nuclear localization signals

NASA Technical Reports Server (NTRS)

Chang, D.; Haynes, J. I. Jr; Brady, J. N.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1993-01-01

The molecular mechanism participating in the transport of newly synthesized proteins from the cytoplasm to the nucleus in mammalian cells is poorly understood. Recently, the nuclear localization signal sequences (NLS) of many nuclear proteins have been identified, and most have been found to be composed of a highly basic amino acid stretch. A genetic "subtractive" and a biochemical "additive" approach were used in our studies to identify the NLS's of the polyomavirus structural capsid proteins. An NLS was identified at the N-terminus (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) of the major capsid protein VP1 and at the C-terminus (Glu307 -Glu-Asp-Gly-Pro-Glu-Lys-Lys-Lys-Arg-Arg-Leu318) of the VP2/VP3 minor capsid proteins.

Molecular simulations of polycation-DNA binding exploring the effect of peptide chemistry and sequence in nuclear localization sequence based polycations.

PubMed

Elder, Robert M; Jayaraman, Arthi

2013-10-10

Gene therapy relies on the delivery of DNA into cells, and polycations are one class of vectors enabling efficient DNA delivery. Nuclear localization sequences (NLS), cationic oligopeptides that target molecules for nuclear entry, can be incorporated into polycations to improve their gene delivery efficiency. We use simulations to study the effect of peptide chemistry and sequence on the DNA-binding behavior of NLS-grafted polycations by systematically mutating the residues in the grafts, which are based on the SV40 NLS (peptide sequence PKKKRKV). Replacing arginine (R) with lysine (K) reduces binding strength by eliminating arginine-DNA interactions, but placing R in a less hindered location (e.g., farther from the grafting point to the polycation backbone) has surprisingly little effect on polycation-DNA binding strength. Changing the positions of the hydrophobic proline (P) and valine (V) residues relative to the polycation backbone changes hydrophobic aggregation within the polycation and, consequently, changes the conformational entropy loss that occurs upon polycation-DNA binding. Since conformational entropy loss affects the free energy of binding, the positions of P and V in the grafts affect DNA binding affinity. The insight from this work guides synthesis of polycations with tailored DNA binding affinity and, in turn, efficient DNA delivery.

Baculovirus LEF-11 nuclear localization signal is important for viral DNA replication.

PubMed

Chen, Tingting; Dong, Zhanqi; Hu, Nan; Hu, Zhigang; Dong, Feifan; Jiang, Yaming; Li, Jun; Chen, Peng; Lu, Cheng; Pan, Minhui

2017-06-15

Baculovirus LEF-11 is a small nuclear protein that is involved in viral late gene transcription and DNA replication. However, the characteristics of its nuclear localization signal and its impact on viral DNA replication are unknown. In the present study, systemic bioinformatics analysis showed that the baculovirus LEF-11 contains monopartite and bipartite classical nuclear localization signal sequences (cNLSs), which were also detected in a few alphabaculovirus species. Localization of representative LEF-11 proteins of four baculovirus genera indicated that the nuclear localization characteristics of baculovirus LEF-11 coincided with the predicted results. Moreover, Bombyx mori nucleopolyhedrovirus (BmNPV) LEF-11 could be transported into the nucleus during viral infection in the absence of a cNLSs. Further investigations demonstrated that the NLS of BmNPV LEF-11 is important for viral DNA replication. The findings of the present study indicate that the characteristics of the baculovirus LEF-11 protein and the NLS is essential to virus DNA replication and nuclear transport mechanisms. Copyright © 2017 Elsevier B.V. All rights reserved.

Analysis of a developmentally regulated nuclear localization signal in Xenopus

PubMed Central

1992-01-01

The 289 residue nuclear oncoprotein encoded by the adenovirus 5 Ela gene contains two peptide sequences that behave as nuclear localization signals (NLS). One signal, located at the carboxy terminus, is like many other known NLSs in that it consists of a short stretch of basic residues (KRPRP) and is constitutively active in cells. The second signal resides within an internal 45 residue region of E1a that contains few basic residues or sequences that resemble other known NLSs. Moreover, this internal signal functions in injected Xenopus oocytes, but not in transfected Xenopus A6 cells, suggesting that it could be regulated developmentally (Slavicek et al. 1989. J. Virol. 63:4047). In this study, we show that the activity of this signal is sensitive to ATP depletion in vivo, efficiently directs the import of a 50 kD fusion protein and can compete with the E1a carboxy-terminal NLS for nuclear import. In addition, we have delineated the precise amino acid residues that comprise the second E1a NLS, and have assessed its utilization during Xenopus embryogenesis. Using amino acid deletion and substitution analyses, we show that the signal consists of the sequence FV(X)7-20MXSLXYM(X)4MF. By expressing in Xenopus embryos a truncated E1a protein that contains only the second NLS and by monitoring its cytoplasmic/nuclear distribution during development with indirect immunofluorescence, we find that the second NLS is utilized up to the early neurula stage. In addition, there appears to be a hierarchy among the embryonic germ layers as to when the second NLS becomes nonfunctional. For this reason, we refer to this NLS as the developmentally regulated nuclear localization signal (drNLS). The implications of these findings for early development are discussed. PMID:1387407

mRNA localization to the mitochondrial surface allows the efficient translocation inside the organelle of a nuclear recoded ATP6 protein

PubMed Central

Kaltimbacher, Valérie; Bonnet, Crystel; Lecoeuvre, Gaëlle; Forster, Valérie; Sahel, José-Alain; Corral-Debrinski, Marisol

2006-01-01

As previously established in yeast, two sequences within mRNAs are responsible for their specific localization to the mitochondrial surface—the region coding for the mitochondrial targeting sequence and the 3′UTR. This phenomenon is conserved in human cells. Therefore, we decided to use mRNA localization as a tool to address to mitochondria, a protein that is not normally imported. For this purpose, we associated a nuclear recoded ATP6 gene with the mitochondrial targeting sequence and the 3′UTR of the nuclear SOD2 gene, which mRNA exclusively localizes to the mitochondrial surface in HeLa cells. The ATP6 gene is naturally located into the organelle and encodes a highly hydrophobic protein of the respiratory chain complex V. In this study, we demonstrated that hybrid ATP6 mRNAs, as the endogenous SOD2 mRNA, localize to the mitochondrial surface in human cells. Remarkably, fusion proteins localize to mitochondria in vivo. Indeed, ATP6 precursors synthesized in the cytoplasm were imported into mitochondria in a highly efficient way, especially when both the MTS and the 3′UTR of the SOD2 gene were associated with the re-engineered ATP6 gene. Hence, these data indicate that mRNA targeting to the mitochondrial surface represents an attractive strategy for allowing the mitochondrial import of proteins originally encoded by the mitochondrial genome without any amino acid change in the protein that could interfere with its biologic activity. PMID:16751614

Isolation and characterization of a carrot nucleolar protein with structural and sequence similarity to the vertebrate PESCADILLO protein.

PubMed

Ueda, Kenji; Xu, Zheng-Jun; Miyagi, Nobuaki; Ono, Michiyuki; Wabiko, Hiroetsu; Masuda, Kiyoshi; Inoue, Masayasu

2013-07-01

The nuclear matrix is involved in many nuclear events, but its protein architecture in plants is still not fully understood. A cDNA clone was isolated by immunoscreening with a monoclonal antibody raised against nuclear matrix proteins of Daucus carota L. Its deduced amino acid sequence showed about 40% identity with the PESCADILLO protein of zebrafish and humans. Primary structure analysis of the protein revealed a Pescadillo N-terminus domain, a single breast cancer C-terminal domain, two nuclear localization signals, and a potential coiled-coil region as also found in animal PESCADILLO proteins. Therefore, we designated this gene DcPES1. Although DcPES1 mRNA was detected in all tissues examined, its levels were highest in tissues with proliferating cells. Immunofluorescence using specific antiserum against the recombinant protein revealed that DcPES1 localized exclusively in the nucleolus. Examination of fusion proteins with green fluorescent protein revealed that the N-terminal portion was important for localization to the nucleoli of tobacco and onion cells. Moreover, when the nuclear matrix of carrot cells was immunostained with an anti-DcPES1 serum, the signal was detected in the nucleolus. Therefore, the DcPES1 protein appears to be a component of or tightly bound to components of the nuclear matrix. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Comprehensive analysis of the dynamic structure of nuclear localization signals.

PubMed

Yamagishi, Ryosuke; Okuyama, Takahide; Oba, Shuntaro; Shimada, Jiro; Chaen, Shigeru; Kaneko, Hiroki

2015-12-01

Most transcription and epigenetic factors in eukaryotic cells have nuclear localization signals (NLSs) and are transported to the nucleus by nuclear transport proteins. Understanding the features of NLSs and the mechanisms of nuclear transport might help understand gene expression regulation, somatic cell reprogramming, thus leading to the treatment of diseases associated with abnormal gene expression. Although many studies analyzed the amino acid sequence of NLSs, few studies investigated their three-dimensional structure. Therefore, we conducted a statistical investigation of the dynamic structure of NLSs by extracting the conformation of these sequences from proteins examined by X-ray crystallography and using a quantity defined as conformational determination rate (a ratio between the number of amino acids determining the conformation and the number of all amino acids included in a certain region). We found that determining the conformation of NLSs is more difficult than determining the conformation of other regions and that NLSs may tend to form more heteropolymers than monomers. Therefore, these findings strongly suggest that NLSs are intrinsically disordered regions.

A genomic landscape of mitochondrial DNA insertions in the pig nuclear genome provides evolutionary signatures of interspecies admixture.

PubMed

Schiavo, Giuseppina; Hoffmann, Orsolya Ivett; Ribani, Anisa; Utzeri, Valerio Joe; Ghionda, Marco Ciro; Bertolini, Francesca; Geraci, Claudia; Bovo, Samuele; Fontanesi, Luca

2017-10-01

Nuclear DNA sequences of mitochondrial origin (numts) are derived by insertion of mitochondrial DNA (mtDNA), into the nuclear genome. In this study, we provide, for the first time, a genome picture of numts inserted in the pig nuclear genome. The Sus scrofa reference nuclear genome (Sscrofa10.2) was aligned with circularized and consensus mtDNA sequences using LAST software. A total of 430 numt sequences that may represent 246 different numt integration events (57 numt regions determined by at least two numt sequences and 189 singletons) were identified, covering about 0.0078% of the nuclear genome. Numt integration events were correlated (0.99) to the chromosome length. The longest numt sequence (about 11 kbp) was located on SSC2. Six numts were sequenced and PCR amplified in pigs of European commercial and local pig breeds, of the Chinese Meishan breed and in European wild boars. Three of them were polymorphic for the presence or absence of the insertion. Surprisingly, the estimated age of insertion of two of the three polymorphic numts was more ancient than that of the speciation time of the Sus scrofa, supporting that these polymorphic sites were originated from interspecies admixture that contributed to shape the pig genome. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Variations in Nuclear Localization Strategies Among Pol X Family Enzymes.

PubMed

Kirby, Thomas W; Pedersen, Lars C; Gabel, Scott A; Gassman, Natalie R; London, Robert E

2018-06-22

Despite the essential roles of pol X family enzymes in DNA repair, information about the structural basis of their nuclear import is limited. Recent studies revealed the unexpected presence of a functional NLS in DNA polymerase β, indicating the importance of active nuclear targeting, even for enzymes likely to leak into and out of the nucleus. The current studies further explore the active nuclear transport of these enzymes by identifying and structurally characterizing the functional NLS sequences in the three remaining human pol X enzymes: terminal deoxynucleotidyl transferase (TdT), DNA polymerase μ (pol μ), and DNA polymerase λ (pol λ). NLS identifications are based on Importin α (Impα) binding affinity determined by fluorescence polarization of fluorescein-labeled NLS peptides, X-ray crystallographic analysis of the Impα∆IBB•NLS complexes, and fluorescence-based subcellular localization studies. All three polymerases use NLS sequences located near their N-terminus; TdT and pol μ utilize monopartite NLS sequences, while pol λ utilizes a bipartite sequence, unique among the pol X family members. The pol μ NLS has relatively weak measured affinity for Impα, due in part to its proximity to the N-terminus that limits non-specific interactions of flanking residues preceding the NLS. However, this effect is partially mitigated by an N-terminal sequence unsupportive of Met1 removal by methionine aminopeptidase, leading to a 3-fold increase in affinity when the N-terminal methionine is present. Nuclear targeting is unique to each pol X family enzyme with variations dependent on the structure and unique functional role of each polymerase. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Serotype-specific differences in dengue virus non-structural protein 5 nuclear localization.

PubMed

Hannemann, Holger; Sung, Po-Yu; Chiu, Han-Chen; Yousuf, Amjad; Bird, Jim; Lim, Siew Pheng; Davidson, Andrew D

2013-08-02

The four serotypes of dengue virus (DENV-1 to -4) cause the most important arthropod-borne viral disease of humans. DENV non-structural protein 5 (NS5) contains enzymatic activities required for capping and replication of the viral RNA genome that occurs in the host cytoplasm. However, previous studies have shown that DENV-2 NS5 accumulates in the nucleus during infection. In this study, we examined the nuclear localization of NS5 for all four DENV serotypes. We demonstrate for the first time that there are serotypic differences in NS5 nuclear localization. Whereas the DENV-2 and -3 proteins accumulate in the nucleus, DENV-1 and -4 NS5 are predominantly if not exclusively localized to the cytoplasm. Comparative studies on the DENV-2 and -4 NS5 proteins revealed that the difference in DENV-4 NS5 nuclear localization was not due to rapid nuclear export but rather the lack of a functional nuclear localization sequence. Interaction studies using DENV-2 and -4 NS5 and human importin-α isoforms failed to identify an interaction that supported the differential nuclear localization of NS5. siRNA knockdown of the human importin-α isoform KPNA2, corresponding to the murine importin-α isoform previously shown to bind to DENV-2 NS5, did not substantially affect DENV-2 NS5 nuclear localization, whereas knockdown of importin-β did. The serotypic differences in NS5 nuclear localization did not correlate with differences in IL-8 gene expression. The results show that NS5 nuclear localization is not strictly required for virus replication but is more likely to have an auxiliary function in the life cycle of specific DENV serotypes.

Serotype-specific Differences in Dengue Virus Non-structural Protein 5 Nuclear Localization*

PubMed Central

Hannemann, Holger; Sung, Po-Yu; Chiu, Han-Chen; Yousuf, Amjad; Bird, Jim; Lim, Siew Pheng; Davidson, Andrew D.

2013-01-01

The four serotypes of dengue virus (DENV-1 to -4) cause the most important arthropod-borne viral disease of humans. DENV non-structural protein 5 (NS5) contains enzymatic activities required for capping and replication of the viral RNA genome that occurs in the host cytoplasm. However, previous studies have shown that DENV-2 NS5 accumulates in the nucleus during infection. In this study, we examined the nuclear localization of NS5 for all four DENV serotypes. We demonstrate for the first time that there are serotypic differences in NS5 nuclear localization. Whereas the DENV-2 and -3 proteins accumulate in the nucleus, DENV-1 and -4 NS5 are predominantly if not exclusively localized to the cytoplasm. Comparative studies on the DENV-2 and -4 NS5 proteins revealed that the difference in DENV-4 NS5 nuclear localization was not due to rapid nuclear export but rather the lack of a functional nuclear localization sequence. Interaction studies using DENV-2 and -4 NS5 and human importin-α isoforms failed to identify an interaction that supported the differential nuclear localization of NS5. siRNA knockdown of the human importin-α isoform KPNA2, corresponding to the murine importin-α isoform previously shown to bind to DENV-2 NS5, did not substantially affect DENV-2 NS5 nuclear localization, whereas knockdown of importin-β did. The serotypic differences in NS5 nuclear localization did not correlate with differences in IL-8 gene expression. The results show that NS5 nuclear localization is not strictly required for virus replication but is more likely to have an auxiliary function in the life cycle of specific DENV serotypes. PMID:23770669

Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals.

PubMed

Zlopasa, Livija; Brachner, Andreas; Foisner, Roland

2016-06-01

Ankyrin repeats and LEM domain containing protein 1 (Ankle1) belongs to the LEM protein family, whose members share a chromatin-interacting LEM motif. Unlike most other LEM proteins, Ankle1 is not an integral protein of the inner nuclear membrane but shuttles between the nucleus and the cytoplasm. It contains a GIY-YIG-type nuclease domain, but its function is unknown. The mammalian genome encodes only one other GIY-YIG domain protein, termed Slx1. Slx1 has been described as a resolvase that processes Holliday junctions during homologous recombination-mediated DNA double strand break repair. Resolvase activity is regulated in a spatial and temporal manner during the cell cycle. We hypothesized that Ankle1 may have a similar function and its nucleo-cytoplasmic shuttling may contribute to the regulation of Ankle1 activity. Hence, we aimed at identifying the domains mediating Ankle1 shuttling and investigating whether cellular localization is affected during DNA damage response. Sequence analysis predicts the presence of two canonical nuclear import and export signals in Ankle1. Immunofluorescence microscopy of cells expressing wild-type and various mutated Ankle1-fusion proteins revealed a C-terminally located classical monopartite nuclear localization signal and a centrally located CRM1-dependent nuclear export signal that mediate nucleo-cytoplasmic shuttling of Ankle1. These sequences are also functional in heterologous proteins. The predominant localization of Ankle1 in the cytoplasm, however, does not change upon induction of several DNA damage response pathways throughout the cell cycle. We identified the domains mediating nuclear import and export of Ankle1. Ankle1's cellular localization was not affected following DNA damage.

Mutation of the rice XA21 predicted nuclear localization sequence does not affect resistance to Xanthomonas oryzae pv. oryzae.

PubMed

Wei, Tong; Chen, Tsung-Chi; Ho, Yuen Ting; Ronald, Pamela C

2016-01-01

The rice receptor kinase XA21 confers robust resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae ( Xoo ). We previously reported that XA21 is cleaved in transgenic plants overexpressing XA21 with a GFP tag ( Ubi -XA21-GFP) and that the released C-terminal domain is localized to the nucleus. XA21 carries a predicted nuclear localization sequence (NLS) that directs the C-terminal domain to the nucleus in transient assays, whereas alanine substitutions in the NLS disrupt the nuclear localization. To determine if the predicted NLS is required for XA21-mediated immunity in planta , we generated transgenic plants overexpressing an XA21 variant carrying the NLS with the same alanine substitutions ( Ubi -XA21nls-GFP). Ubi- XA21nls-GFP plants displayed slightly longer lesion lengths, higher Xoo bacterial populations after inoculation and lower levels of reactive oxygen species production compared with the Ubi- XA21-GFP control plants. However, the Ubi- XA21nls-GFP plants express lower levels of protein than that observed in Ubi- XA21-GFP. These results demonstrate that the predicted NLS is not required for XA21-mediated immunity.

Assessment of phylogenetic relationship of rare plant species collected from Saudi Arabia using internal transcribed spacer sequences of nuclear ribosomal DNA.

PubMed

Al-Qurainy, F; Khan, S; Nadeem, M; Tarroum, M; Alaklabi, A

2013-03-11

The rare and endangered plants of any country are important genetic resources that often require urgent conservation measures. Assessment of phylogenetic relationships and evaluation of genetic diversity is very important prior to implementation of conservation strategies for saving rare and endangered plant species. We used internal transcribed spacer sequences of nuclear ribosomal DNA for the evaluation of sequence identity from the available taxa in the GenBank database by using the Basic Local Alignment Search Tool (BLAST). Two rare plant species viz, Heliotropium strigosum claded with H. pilosum (98% branch support) and Pancratium tortuosum claded with P. tenuifolium (61% branch support) clearly. However, some species, viz Scadoxus multiflorus, Commiphora myrrha and Senecio hadiensis showed close relationships with more than one species. We conclude that nuclear ribosomal internal transcribed spacer sequences are useful markers for phylogenetic study of these rare plant species in Saudi Arabia.

Protein Sub-Nuclear Localization Based on Effective Fusion Representations and Dimension Reduction Algorithm LDA.

PubMed

Wang, Shunfang; Liu, Shuhui

2015-12-19

An effective representation of a protein sequence plays a crucial role in protein sub-nuclear localization. The existing representations, such as dipeptide composition (DipC), pseudo-amino acid composition (PseAAC) and position specific scoring matrix (PSSM), are insufficient to represent protein sequence due to their single perspectives. Thus, this paper proposes two fusion feature representations of DipPSSM and PseAAPSSM to integrate PSSM with DipC and PseAAC, respectively. When constructing each fusion representation, we introduce the balance factors to value the importance of its components. The optimal values of the balance factors are sought by genetic algorithm. Due to the high dimensionality of the proposed representations, linear discriminant analysis (LDA) is used to find its important low dimensional structure, which is essential for classification and location prediction. The numerical experiments on two public datasets with KNN classifier and cross-validation tests showed that in terms of the common indexes of sensitivity, specificity, accuracy and MCC, the proposed fusing representations outperform the traditional representations in protein sub-nuclear localization, and the representation treated by LDA outperforms the untreated one.

Protein Sub-Nuclear Localization Based on Effective Fusion Representations and Dimension Reduction Algorithm LDA

PubMed Central

Wang, Shunfang; Liu, Shuhui

2015-01-01

An effective representation of a protein sequence plays a crucial role in protein sub-nuclear localization. The existing representations, such as dipeptide composition (DipC), pseudo-amino acid composition (PseAAC) and position specific scoring matrix (PSSM), are insufficient to represent protein sequence due to their single perspectives. Thus, this paper proposes two fusion feature representations of DipPSSM and PseAAPSSM to integrate PSSM with DipC and PseAAC, respectively. When constructing each fusion representation, we introduce the balance factors to value the importance of its components. The optimal values of the balance factors are sought by genetic algorithm. Due to the high dimensionality of the proposed representations, linear discriminant analysis (LDA) is used to find its important low dimensional structure, which is essential for classification and location prediction. The numerical experiments on two public datasets with KNN classifier and cross-validation tests showed that in terms of the common indexes of sensitivity, specificity, accuracy and MCC, the proposed fusing representations outperform the traditional representations in protein sub-nuclear localization, and the representation treated by LDA outperforms the untreated one. PMID:26703574

Characterization of sequences in human TWIST required for nuclear localization

PubMed Central

Singh, Shalini; Gramolini, Anthony O

2009-01-01

Background Twist is a transcription factor that plays an important role in proliferation and tumorigenesis. Twist is a nuclear protein that regulates a variety of cellular functions controlled by protein-protein interactions and gene transcription events. The focus of this study was to characterize putative nuclear localization signals (NLSs) 37RKRR40 and 73KRGKK77 in the human TWIST (H-TWIST) protein. Results Using site-specific mutagenesis and immunofluorescences, we observed that altered TWISTNLS1 K38R, TWISTNLS2 K73R and K77R constructs inhibit nuclear accumulation of H-TWIST in mammalian cells, while TWISTNLS2 K76R expression was un-affected and retained to the nucleus. Subsequently, co-transfection of TWIST mutants K38R, K73R and K77R with E12 formed heterodimers and restored nuclear localization despite the NLSs mutations. Using a yeast-two-hybrid assay, we identified a novel TWIST-interacting candidate TCF-4, a basic helix-loop-helix transcription factor. The interaction of TWIST with TCF-4 confirmed using NLS rescue assays, where nuclear expression of mutant TWISTNLS1 with co-transfixed TCF-4 was observed. The interaction of TWIST with TCF-4 was also seen using standard immunoprecipitation assays. Conclusion Our study demonstrates the presence of two putative NLS motifs in H-TWIST and suggests that these NLS sequences are functional. Furthermore, we identified and confirmed the interaction of TWIST with a novel protein candidate TCF-4. PMID:19534813

Nuclear import of human MLH1, PMS2, and MutLalpha: redundancy is the key.

PubMed

Leong, Vivian; Lorenowicz, Jessica; Kozij, Natalie; Guarné, Alba

2009-08-01

DNA mismatch repair maintains genomic stability by correcting errors that have escaped polymerase proofreading. Defects on mismatch repair genes lead to an increased mutation rate, microsatellite instability and predisposition to human non-polyposis colorectal cancer (HNPCC). Human MutLalpha is a heterodimer formed by the interaction of MLH1 and PMS2 that coordinates a series of key events in mismatch repair. It has been proposed that nuclear import of MutLalpha may be the first regulatory step on the activation of the mismatch repair pathway. Using confocal microscopy and mismatch repair deficient cells, we have identified the sequence determinants that drive nuclear import of human MLH1, PMS2, and MutLalpha. Transient transfection of the individual proteins reveals that MLH1 has a bipartite and PMS2 has a single monopartite nuclear localization signal. Although dimerization is not required for nuclear localization, the MutLalpha heterodimer is imported more efficiently than the MLH1 or PMS2 monomers. Interestingly, the bipartite localization signal of MLH1 can direct import of MutLalpha even when PMS2 encompasses a mutated localization signal. Hence we conclude that the presence of redundant nuclear localization signals guarantees nuclear transport of MutLalpha and, consequently, efficient mismatch repair.

Discrimination between NL1- and NL2-Mediated Nuclear Localization of the Glucocorticoid Receptor

PubMed Central

Savory, Joanne G. A.; Hsu, Brian; Laquian, Ian R.; Giffin, Ward; Reich, Terry; Haché, Robert J. G.; Lefebvre, Yvonne A.

1999-01-01

Glucocorticoid receptor (GR) cycles between a free liganded form that is localized to the nucleus and a heat shock protein (hsp)-immunophilin-complexed, unliganded form that is usually localized to the cytoplasm but that can also be nuclear. In addition, rapid nucleocytoplasmic exchange or shuttling of the receptor underlies its localization. Nuclear import of liganded GR is mediated through a well-characterized sequence, NL1, adjacent to the receptor DNA binding domain and a second, uncharacterized motif, NL2, that overlaps with the ligand binding domain. In this study we report that rapid nuclear import (half-life [t1/2] of 4 to 6 min) of agonist- and antagonist-treated GR and the localization of unliganded, hsp-associated GRs to the nucleus in G0 are mediated through NL1 and correlate with the binding of GR to pendulin/importin α. By contrast, NL2-mediated nuclear transfer of GR occurred more slowly (t1/2 = 45 min to 1 h), was agonist specific, and appeared to be independent of binding to importin α. Together, these results suggest that NL2 mediates the nuclear import of GR through an alternative nuclear import pathway. Nuclear export of GR was inhibited by leptomycin B, suggesting that the transfer of GR to the cytoplasm is mediated through the CRM1-dependent pathway. Inhibition of GR nuclear export by leptomycin B enhanced the nuclear localization of both unliganded, wild-type GR and hormone-treated NL1− GR. These results highlight that the subcellular localization of both liganded and unliganded GRs is determined, at least in part, by a flexible equilibrium between the rates of nuclear import and export. PMID:9891038

FBI-1 enhances transcription of the nuclear factor-kappaB (NF-kappaB)-responsive E-selectin gene by nuclear localization of the p65 subunit of NF-kappaB.

PubMed

Lee, Dong-Kee; Kang, Jae-Eun; Park, Hye-Jin; Kim, Myung-Hwa; Yim, Tae-Hee; Kim, Jung-Min; Heo, Min-Kyu; Kim, Kyu-Yeun; Kwon, Ho Jeong; Hur, Man-Wook

2005-07-29

The POZ domain is a highly conserved protein-protein interaction motif found in many regulatory proteins. Nuclear factor-kappaB (NF-kappaB) plays a key role in the expression of a variety of genes in response to infection, inflammation, and stressful conditions. We found that the POZ domain of FBI-1 (factor that binds to the inducer of short transcripts of human immunodeficiency virus-1) interacted with the Rel homology domain of the p65 subunit of NF-kappaB in both in vivo and in vitro protein-protein interaction assays. FBI-1 enhanced NF-kappaB-mediated transcription of E-selectin genes in HeLa cells upon phorbol 12-myristate 13-acetate stimulation and overcame gene repression by IkappaB alpha or IkappaB beta. In contrast, the POZ domain of FBI-1, which is a dominant-negative form of FBI-1, repressed NF-kappaB-mediated transcription, and the repression was cooperative with IkappaB alpha or IkappaB beta. In contrast, the POZ domain tagged with a nuclear localization sequence polypeptide of FBI-1 enhanced NF-kappaB-responsive gene transcription, suggesting that the molecular interaction between the POZ domain and the Rel homology domain of p65 and the nuclear localization by the nuclear localization sequence are important in the transcription enhancement mediated by FBI-1. Confocal microscopy showed that FBI-1 increased NF-kappaB movement into the nucleus and increased the stability of NF-kappaB in the nucleus, which enhanced NF-kappaB-mediated transcription of the E-selectin gene. FBI-1 also interacted with IkappaB alpha and IkappaB beta.

Sequence and characterization of cytoplasmic nuclear protein import factor p97

PubMed Central

1995-01-01

Nuclear location sequence-mediated binding of karyophilic proteins to the nuclear pore complexes is one of the earliest steps in nuclear protein import. We previously identified two cytosolic proteins that reconstitute this step in a permeabilized cell assay: the 54/56-kD NLS receptor and p97. A monoclonal antibody to p97 localizes the protein to the cytoplasm and the nuclear envelope. p97 is extracted from nuclear envelopes under the same conditions as the O-glycosylated nucleoporins indicating a tight association with the pore complex. The antibody inhibits import in a permeabilized cell assay but does not affect binding of karyophiles to the nuclear pore complex. Immunodepletion of p97 renders the cytosol inactive for import and identifies at least three other cytosolic proteins that interact with p97. cDNA cloning of p97 shows that it is a unique protein containing 23 cysteine residues. Recombinant p97 binds zinc and a bound metal ion is required for the nuclear envelope binding activity of the protein. PMID:7615630

Normal Telomere Length Maintenance in Saccharomyces cerevisiae Requires Nuclear Import of the Ever Shorter Telomeres 1 (Est1) Protein via the Importin Alpha Pathway

PubMed Central

Hawkins, Charlene

2014-01-01

The Est1 (ever shorter telomeres 1) protein is an essential component of yeast telomerase, a ribonucleoprotein complex that restores the repetitive sequences at chromosome ends (telomeres) that would otherwise be lost during DNA replication. Previous work has shown that the telomerase RNA component (TLC1) transits through the cytoplasm during telomerase biogenesis, but mechanisms of protein import have not been addressed. Here we identify three nuclear localization sequences (NLSs) in Est1p. Mutation of the most N-terminal NLS in the context of full-length Est1p reduces Est1p nuclear localization and causes telomere shortening—phenotypes that are rescued by fusion with the NLS from the simian virus 40 (SV40) large-T antigen. In contrast to that of the TLC1 RNA, Est1p nuclear import is facilitated by Srp1p, the yeast homolog of importin α. The reduction in telomere length observed at the semipermissive temperature in a srp1 mutant strain is rescued by increased Est1p expression, consistent with a defect in Est1p nuclear import. These studies suggest that at least two nuclear import pathways are required to achieve normal telomere length homeostasis in yeast. PMID:24906415

Nuclear location of a chromatin insulator in Drosophila melanogaster.

PubMed

Xu, Qinghao; Li, Mo; Adams, Jessica; Cai, Haini N

2004-03-01

Chromatin-related functions are associated with spatial organization in the nucleus. We have investigated the relationship between the enhancer-blocking activity and subnuclear localization of the Drosophila melanogaster suHw insulator. Using fluorescent in situ hybridization, we observed that genomic loci containing the gypsy retrotransposon were distributed closer to the nuclear periphery than regions without the gypsy retrotransposon. However, transgenes containing a functional 340 bp suHw insulator did not exhibit such biased distribution towards the nuclear periphery, which suggests that the suHw insulator sequence is not responsible for the peripheral localization of the gypsy retrotransposon. Antibody stains showed that the two proteins essential for the suHw insulator activity, SUHW and MOD(MDG4), are not restricted to the nuclear periphery. The enhancer-blocking activity of suHw remained intact under the heat shock conditions, which was shown to disrupt the association of gypsy, SUHW and MOD(MDG4) with the nuclear periphery. Our results indicate that the suHw insulator can function in the nuclear interior, possibly through local interactions with chromatin components or other nuclear structures.

Iron-Inducible Nuclear Translocation of a Myb3 Transcription Factor in the Protozoan Parasite Trichomonas vaginalis

PubMed Central

Hsu, Hong-Ming; Lee, Yu; Indra, Dharmu; Wei, Shu-Yi; Liu, Hsing-Wei; Chang, Lung-Chun; Chen, Chinpan; Ong, Shiou-Jeng

2012-01-01

In Trichomonas vaginalis, a novel nuclear localization signal spanning the folded R2R3 DNA-binding domain of a Myb2 protein was previously identified. To study whether a similar signal is used for nuclear translocation by other Myb proteins, nuclear translocation of Myb3 was examined in this report. When overexpressed, hemagglutinin-tagged Myb3 was localized to nuclei of transfected cells, with a cellular distribution similar to that of endogenous Myb3. Fusion to a bacterial tetracycline repressor, R2R3, of Myb3 that spans amino acids (aa) 48 to 156 was insufficient for nuclear translocation of the fusion protein, unless its C terminus was extended to aa 167. The conserved isoleucine in helix 2 of R2R3, which is important for Myb2's structural integrity in maintaining DNA-binding activity and nuclear translocation, was also vital for the former activity of Myb3, but less crucial for the latter. Sequential nuclear influx and efflux of Myb3, which require further extension of the nuclear localization signal to aa 180, were immediately induced after iron repletion. Sequence elements that regulate nuclear translocation with cytoplasmic retention, nuclear influx, and nuclear efflux were identified within the C-terminal tail. These results suggest that the R2R3 DNA-binding domain also serves as a common module for the nuclear translocation of both Myb2 and Myb3, but there are intrinsic differences between the two nuclear localization signals. PMID:23042127

Identification of a nuclear localization signal in the retinitis pigmentosa-mutated RP26 protein, ceramide kinase-like protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Inagaki, Yuichi; Mitsutake, Susumu; Igarashi, Yasuyuki

2006-05-12

Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. A mutation in a new ceramide kinase (CERK) homologous gene, named CERK-like protein (CERKL), was found to cause autosomal recessive retinitis pigmentosa (RP26). Here, we show a point mutation of one of two putative nuclear localization signal (NLS) sequences inhibited the nuclear localization of the protein. Furthermore, the tetra-GFP-tagged NLS, which cannot passively enter the nucleus, was observed not only in the nucleus but also in the nucleolus. Our results provide First evidence of the active nuclear import of CERKL and suggest that the identified NLSmore » might be responsible for nucleolar retention of the protein. As recent studies have shown other RP-related proteins are localized in the nucleus or the nucleolus, our identification of NLS in CERKL suggests that CERKL likely plays important roles for retinal functions in the nucleus and the nucleolus.« less

Mutation of the rice XA21 predicted nuclear localization sequence does not affect resistance to Xanthomonas oryzae pv. oryzae

DOE PAGES

Wei, Tong; Chen, Tsung-Chi; Ho, Yuen Ting; ...

2016-10-05

Background: The rice receptor kinase XA21 confers robust resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae( Xoo). We previously reported that XA21 is cleaved in transgenic plants overexpressing XA21 with a GFP tag ( Ubi-XA21-GFP) and that the released C-terminal domain is localized to the nucleus. XA21 carries a predicted nuclear localization sequence (NLS) that directs the C-terminal domain to the nucleus in transient assays, whereas alanine substitutions in the NLS disrupt the nuclear localization. Methods: To determine if the predicted NLS is required for XA21-mediated immunity in planta, we generated transgenic plants overexpressing an XA21 variant carrying themore » NLS with the same alanine substitutions ( Ubi-XA21nls-GFP). Results: Ubi- XA21nls-GFP plants displayed slightly longer lesion lengths, higher Xoo bacterial populations after inoculation and lower levels of reactive oxygen species production compared with the Ubi- XA21-GFP control plants. However, the Ubi- XA21nls-GFP plants express lower levels of protein than that observed in Ubi- XA21-GFP. Discussion: These results demonstrate that the predicted NLS is not required for XA21-mediated immunity.« less

Phosphorylation and nuclear localization of the varicella-zoster virus gene 63 protein.

PubMed Central

Stevenson, D; Xue, M; Hay, J; Ruyechan, W T

1996-01-01

The protein encoded by varicella-zoster virus open reading frame 63 and carboxy-terminal deletions of the same were expressed either as fusion proteins at the carboxy terminus of the maltose-binding protein in Escherichia coli or independently in transfected mammalian cells. The truncations contained amino acids 1 to 142 (63 delta N) or 1 to 210 (63 delta K) of the complete 278-amino-acid primary sequence. Recombinant casein kinase II phosphorylated the 63F and 63 delta KF fusion proteins in vitro but did not phosphorylate the 63 delta NF fusion protein, implying that phosphorylation occurred between amino acids 142 and 210. Immunoprecipitation of 35S- or 32P-labelled extracts of cells transfected with plasmids expressing 63, 63 delta N, or 63 delta K also indicated that in situ phosphorylation most likely occurred between amino acids 142 and 210. These combined results suggest that casein kinase II plays a significant role in the phosphorylation of the varicella-zoster virus 63 protein. Indirect immunofluorescence of transfected cells indicated nuclear localization of the 63 protein and cytoplasmic localization of 63 delta K and 63 delta N, implying a requirement for sequences between amino acids 210 and 278 for efficient nuclear localization. PMID:8523589

Mutation of the rice XA21 predicted nuclear localization sequence does not affect resistance to Xanthomonas oryzae pv. oryzae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wei, Tong; Chen, Tsung-Chi; Ho, Yuen Ting

Background: The rice receptor kinase XA21 confers robust resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae( Xoo). We previously reported that XA21 is cleaved in transgenic plants overexpressing XA21 with a GFP tag ( Ubi-XA21-GFP) and that the released C-terminal domain is localized to the nucleus. XA21 carries a predicted nuclear localization sequence (NLS) that directs the C-terminal domain to the nucleus in transient assays, whereas alanine substitutions in the NLS disrupt the nuclear localization. Methods: To determine if the predicted NLS is required for XA21-mediated immunity in planta, we generated transgenic plants overexpressing an XA21 variant carrying themore » NLS with the same alanine substitutions ( Ubi-XA21nls-GFP). Results: Ubi- XA21nls-GFP plants displayed slightly longer lesion lengths, higher Xoo bacterial populations after inoculation and lower levels of reactive oxygen species production compared with the Ubi- XA21-GFP control plants. However, the Ubi- XA21nls-GFP plants express lower levels of protein than that observed in Ubi- XA21-GFP. Discussion: These results demonstrate that the predicted NLS is not required for XA21-mediated immunity.« less

The estrogen receptor alpha nuclear localization sequence is critical for fulvestrant-induced degradation of the receptor.

PubMed

Casa, Angelo J; Hochbaum, Daniel; Sreekumar, Sreeja; Oesterreich, Steffi; Lee, Adrian V

2015-11-05

Fulvestrant, a selective estrogen receptor down-regulator (SERD) is a pure competitive antagonist of estrogen receptor alpha (ERα). Fulvestrant binds ERα and reduces the receptor's half-life by increasing protein turnover, however, its mechanism of action is not fully understood. In this study, we show that removal of the ERα nuclear localization sequence (ERΔNLS) resulted in a predominantly cytoplasmic ERα that was degraded in response to 17-β-estradiol (E2) but was resistant to degradation by fulvestrant. ERΔNLS bound the ligands and exhibited receptor interaction similar to ERα, indicating that the lack of degradation was not due to disruption of these processes. Forcing ERΔNLS into the nucleus with a heterologous SV40-NLS did not restore degradation, suggesting that the NLS domain itself, and not merely receptor localization, is critical for fulvestrant-induced ERα degradation. Indeed, cloning of the endogenous ERα NLS onto the N-terminus of ERΔNLS significantly restored both its nuclear localization and turnover in response to fulvestrant. Moreover, mutation of the sumoylation targets K266 and K268 within the NLS impaired fulvestrant-induced ERα degradation. In conclusion, our study provides evidence for the unique role of the ERα NLS in fulvestrant-induced degradation of the receptor. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schwab, Ryan S.; Ihnatovych, Ivanna; Yunus, Sharifah Z.S.A.

Myosin IC is a single headed member of the myosin superfamily that localizes to the cytoplasm and the nucleus, where it is involved in transcription by RNA polymerases I and II, intranuclear transport, and nuclear export. In mammalian cells, three isoforms of myosin IC are expressed that differ only in the addition of short isoform-specific N-terminal peptides. Despite the high sequence homology, the isoforms show differences in cellular distribution, in localization to nuclear substructures, and in their interaction with nuclear proteins through yet unknown mechanisms. In this study, we used EGFP-fusion constructs that express truncated or mutated versions of myosinmore » IC isoforms to detect regions that are involved in isoform-specific localization. We identified two nucleolar localization signals (NoLS). One NoLS is located in the myosin IC isoform B specific N-terminal peptide, the second NoLS is located upstream of the neck region within the head domain. We demonstrate that both NoLS are functional and necessary for nucleolar localization of specifically myosin IC isoform B. Our data provide a first mechanistic explanation for the observed functional differences between the myosin IC isoforms and are an important step toward our understanding of the underlying mechanisms that regulate the various and distinct functions of myosin IC isoforms. - Highlights: ► Two NoLS have been identified in the myosin IC isoform B sequence. ► Both NoLS are necessary for myosin IC isoform B specific nucleolar localization. ► First mechanistic explanation of functional differences between the isoforms.« less

A R/K-rich motif in the C-terminal of the homeodomain is required for complete translocating of NKX2.5 protein into nucleus.

PubMed

Ouyang, Ping; Zhang, He; Fan, Zhaolan; Wei, Pei; Huang, Zhigang; Wang, Sen; Li, Tao

2016-11-05

NKX2.5 plays important roles in heart development. Being a transcription factor, NKX2.5 exerts its biological functions in nucleus. However, the sequence motif that localize NKX2.5 into nucleus is still not clear. Here, we found a R/K-rich sequence motif from Q187 to R197 (QNRRYKCKRQR) was required for exclusive nuclear localization of NKX2.5. Eight truncated plasmids (E109X, Q149X, Q170X, Q187X, Q198X, Y256X, Y259X, and C264X) which were associated with congenital heart disease (CHD) were constructed. Compared with the wild type NKX2.5, the proteins E109X, Q149X, Q170X, Q187X without intact homeodomain (HD) showed no transcriptional activity while Q198X, Y256X, Y259X and C264X with intact HD showed 50 to 66% transcriptional activity. E109X, Q149X, Q170X, Q187X without intact HD localized in the cytoplasm and nucleus simultaneously and Q198X, Y256X, Y259X and C264X with intact HD localized completely in nucleus. These results inferred the indispensability of 187QNRRYKCKRQR197 in exclusive nucleus localization. Additionally, this sequence motif was very conservative among human, mouse and rat, indicating this motif was important for NKX2.5 function. Thus, we concluded that R/K-rich sequence motif 187QNRRYKCKRQR197 played a central role for NKX2.5 nuclear localization. Our findings provided a clue to understand the mechanisms between the truncated NKX2.5 mutants and CHD. Copyright © 2016 Elsevier B.V. All rights reserved.

Transcript variations, phylogenetic tree and chromosomal localization of porcine aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) genes.

PubMed

Sadowska, Agnieszka; Paukszto, Lukasz; Nynca, Anna; Szczerbal, Izabela; Orlowska, Karina; Swigonska, Sylwia; Ruszkowska, Monika; Molcan, Tomasz; Jastrzebski, Jan P; Panasiewicz, Grzegorz; Ciereszko, Renata E

2017-03-01

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor best known for mediating xenobiotic-induced toxicity. AhR requires aryl hydrocarbon receptor nuclear translocator (ARNT) to form an active transcription complex and promote the activation of genes which have dioxin responsive element in their regulatory regions. The present study was performed to determine the complete cDNA sequences of porcine AhR and ARNT genes and their chromosomal localization. Total RNA from porcine livers were used to obtain the sequence of the entire porcine transcriptome by next-generation sequencing (NGS; lllumina HiSeq2500). In addition, both, in silico analysis and fluorescence in situ hybridization (FISH) were used to determine chromosomal localization of porcine AhR and ARNT genes. In silico analysis of nucleotide sequences showed that there were two transcript variants of AhR and ARNT genes in the pig. In addition, computer analysis revealed that AhR gene in the pig is located on chromosome 9 and ARNT on chromosome 4. The results of FISH experiment confirmed the localization of porcine AhR and ARNT genes. In the present study, for the first time, the full cDNAs of AhR and ARNT were demonstrated in the pig. In future, it would be interesting to determine the tissue distribution of AhR and ARNT transcript variants in the pig and to test whether these variants are associated with different biological functions and/or different activation pathways.

Localization of human T-cell lymphotropic virus type II Tax protein is dependent upon a nuclear localization determinant in the N-terminal region.

PubMed

Turci, Marco; Romanelli, Maria Grazia; Lorenzi, Pamela; Righi, Paola; Bertazzoni, Umberto

2006-01-03

Human T-cell lymphotropic viruses (HTLV) types I and II are closely related oncogenic retroviruses that have been associated with lymphoproliferative and neurological disorders. The proviral genome encodes a trans-regulatory Tax protein that activates viral genes and upregulates various cellular genes involved in both cell growth and transformation. Tax proteins of HTLV-I (Tax-I) and HTLV-II (Tax-II) exhibit more than 77% aa homology and expression of either Tax-I or Tax-II is sufficient for immortalization of cultured T lymphocytes. Tax-I shuttles from the nucleus to the cytoplasm and accumulates within the nucleus, whereas Tax-II is found mainly in the cytoplasm. In the present study we have used recombinant vectors to analyze the size and structure of the nuclear localization domain within the Tax-II protein sequence. The Tax-II protein was expressed in HeLa cells either as the complete protein, or regions thereof, that were individually fused to the green fluorescent protein (GFP). Immunoblot analysis of the fused Tax-II products confirmed their expression and size. Fluorescence microscopy studies indicated that the complete Tax-II as well as N-truncated forms presented a punctuate cytoplasmic distribution and that a nuclear localization determinant is confined to within the first 60 aa of Tax-II. Accordingly, site directed mutagenesis and deletion of specific sequences within the first 60 aa showed that the nuclear determinant lies within the first 41 residues of Tax-II. These results point to a direct involvement of the amino-terminal residues of Tax-II protein in determining its nuclear functionality.

Localization of 14-3-3 proteins in the nuclei of arabidopsis and maize.

PubMed

Bihn, E A; Paul, A L; Wang, S W; Erdos, G W; Ferl, R J

1997-12-01

It has been demonstrated that 14-3-3 proteins are present in the nuclei of Arabidopsis thaliana and Zea mays cells using laser scanning confocal microscopy and immunocytochemistry with monoclonal antibodies against plant 14-3-3 proteins. Confirmation of nuclear localization provides insight into the range of functions normally attributed to 14-3-3 proteins, especially since the association of 14-3-3s with transcription factors is (thus far) a phenomenon unique to plants, and since 14-3-3 proteins do not possess a recognizable nuclear targeting sequence.

Autosomal recessive mutations in nuclear transport factor KPNA7 are associated with infantile spasms and cerebellar malformation.

PubMed

Paciorkowski, Alex R; Weisenberg, Judy; Kelley, Joshua B; Spencer, Adam; Tuttle, Emily; Ghoneim, Dalia; Thio, Liu Lin; Christian, Susan L; Dobyns, William B; Paschal, Bryce M

2014-05-01

Nuclear import receptors of the KPNA family recognize the nuclear localization signal in proteins and together with importin-β mediate translocation into the nucleus. Accordingly, KPNA family members have a highly conserved architecture with domains that contact the nuclear localization signal and bind to importin-β. Here, we describe autosomal recessive mutations in KPNA7 found by whole exome sequencing in a sibling pair with severe developmental disability, infantile spasms, subsequent intractable epilepsy consistent with Lennox-Gastaut syndrome, partial agenesis of the corpus callosum, and cerebellar vermis hypoplasia. The mutations mapped to exon 7 in KPNA7 result in two amino-acid substitutions, Pro339Ala and Glu344Gln. On the basis of the crystal structure of the paralog KPNA2 bound to a bipartite nuclear localization signal from the retinoblastoma protein, the amino-acid substitutions in the affected subjects were predicted to occur within the seventh armadillo repeat that forms one of the two nuclear localization signal-binding sites in KPNA family members. Glu344 is conserved in all seven KPNA proteins, and we found that the Glu354Gln mutation in KPNA2 is sufficient to reduce binding to the retinoblastoma nuclear localization signal to approximately one-half that of wild-type protein. Our data show that compound heterozygous mutations in KPNA7 are associated with a human neurodevelopmental disease, and provide the first example of a human disease associated with mutation of a nuclear transport receptor.

Autosomal recessive mutations in nuclear transport factor KPNA7 are associated with infantile spasms and cerebellar malformation

PubMed Central

Paciorkowski, Alex R; Weisenberg, Judy; Kelley, Joshua B; Spencer, Adam; Tuttle, Emily; Ghoneim, Dalia; Thio, Liu Lin; Christian, Susan L; Dobyns, William B; Paschal, Bryce M

2014-01-01

Nuclear import receptors of the KPNA family recognize the nuclear localization signal in proteins and together with importin-β mediate translocation into the nucleus. Accordingly, KPNA family members have a highly conserved architecture with domains that contact the nuclear localization signal and bind to importin-β. Here, we describe autosomal recessive mutations in KPNA7 found by whole exome sequencing in a sibling pair with severe developmental disability, infantile spasms, subsequent intractable epilepsy consistent with Lennox–Gastaut syndrome, partial agenesis of the corpus callosum, and cerebellar vermis hypoplasia. The mutations mapped to exon 7 in KPNA7 result in two amino-acid substitutions, Pro339Ala and Glu344Gln. On the basis of the crystal structure of the paralog KPNA2 bound to a bipartite nuclear localization signal from the retinoblastoma protein, the amino-acid substitutions in the affected subjects were predicted to occur within the seventh armadillo repeat that forms one of the two nuclear localization signal-binding sites in KPNA family members. Glu344 is conserved in all seven KPNA proteins, and we found that the Glu354Gln mutation in KPNA2 is sufficient to reduce binding to the retinoblastoma nuclear localization signal to approximately one-half that of wild-type protein. Our data show that compound heterozygous mutations in KPNA7 are associated with a human neurodevelopmental disease, and provide the first example of a human disease associated with mutation of a nuclear transport receptor. PMID:24045845

A Functional Nuclear Localization Sequence in the C. elegans TRPV Channel OCR-2

PubMed Central

Ezak, Meredith J.; Ferkey, Denise M.

2011-01-01

The ability to modulate gene expression in response to sensory experience is critical to the normal development and function of the nervous system. Calcium is a key activator of the signal transduction cascades that mediate the process of translating a cellular stimulus into transcriptional changes. With the recent discovery that the mammalian Cav1.2 calcium channel can be cleaved, enter the nucleus and act as a transcription factor to control neuronal gene expression, a more direct role for the calcium channels themselves in regulating transcription has begun to be appreciated. Here we report the identification of a nuclear localization sequence (NLS) in the C. elegans transient receptor potential vanilloid (TRPV) cation channel OCR-2. TRPV channels have previously been implicated in transcriptional regulation of neuronal genes in the nematode, although the precise mechanism remains unclear. We show that the NLS in OCR-2 is functional, being able to direct nuclear accumulation of a synthetic cargo protein as well as the carboxy-terminal cytosolic tail of OCR-2 where it is endogenously found. Furthermore, we discovered that a carboxy-terminal portion of the full-length channel can localize to the nucleus of neuronal cells. These results suggest that the OCR-2 TRPV cation channel may have a direct nuclear function in neuronal cells that was not previously appreciated. PMID:21957475

Two short protein domains are responsible for the nuclear localization of the mouse spermine oxidase mu isoform.

PubMed

Bianchi, Marzia; Amendola, Roberto; Federico, Rodolfo; Polticelli, Fabio; Mariottini, Paolo

2005-06-01

In mouse, at least two catalytically active splice variants (mSMOalpha and mSMOmicro) of the flavin-containing spermine oxidase enzyme are present. We have demonstrated previously that the cytosolic mSMOalpha is the major isoform, while the mSMOmicro enzyme is present in both nuclear and cytoplasmic compartments and has an extra protein domain corresponding to the additional exon VIa. By amino acid sequence comparison and molecular modeling of mSMO proteins, we identified a second domain that is necessary for nuclear localization of the mSMOmicro splice variant. A deletion mutant enzyme of this region was constructed to demonstrate its role in protein nuclear targeting by means of transient expression in the murine neuroblastoma cell line, N18TG2.

A chromatin insulator determines the nuclear localization of DNA.

PubMed

Gerasimova, T I; Byrd, K; Corces, V G

2000-11-01

Chromatin insulators might regulate gene expression by controlling the subnuclear organization of DNA. We found that a DNA sequence normally located inside of the nucleus moved to the periphery when the gypsy insulator was placed within the sequence. The presence of the gypsy insulator also caused two sequences, normally found in different regions of the nucleus, to come together at a single location. Alterations in this subnuclear organization imposed by the gypsy insulator correlated with changes in gene expression that took place during the heat-shock response. These global changes in transcription were accompanied by dramatic alterations in the distribution of insulator proteins and DNA. The results suggest that the nuclear organization imposed by the gypsy insulator on the chromatin fiber is important for gene expression.

Identification of essential sequences for cellular localization in the muscle-specific ubiquitin E3 ligase MAFbx/Atrogin 1.

PubMed

Julie, Lagirand-Cantaloube; Sabrina, Batonnet-Pichon; Marie-Pierre, Leibovitch; Leibovitch, Serge A

2012-02-17

In skeletal muscle atrophy, upregulation and nuclear accumulation of the Ubiquitin E3 ligase MAFbx is essential for accelerated muscle protein loss, but the nuclear/cytoplasmic shuttling of MAFbx is undefined. Here we found that MAFbx contains two functional nuclear localization signals (NLS). Mutation or deletion of only one NLS induced cytoplasmic localization of MAFbx. We identified a non-classical NES located in the leucine charged domain (LCD) of MAFbx, which is leptomycin B insensitive. We demonstrated that mutation (L169Q) in LLXXL motif of LCD suppressed cytoplasmic retention of MAFbx. Nucleocytoplasmic shuttling of MAFbx represents a novel mechanism for targeting its substrates and its cytosolic partners in muscle atrophy. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Cyclophilin B enhances HIV-1 Infection

PubMed Central

DeBoer, Jason; Madson, Christian J.; Belshan, Michael

2016-01-01

Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. PMID:26774171

Cyclophilin B enhances HIV-1 infection.

PubMed

DeBoer, Jason; Madson, Christian J; Belshan, Michael

2016-02-01

Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. Copyright © 2015 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kiriyama, Takao; Hirano, Makito; Asai, Hirohide

Triple A syndrome is an autosomal recessive neurological disease, mimicking motor neuron disease, and is caused by mutant ALADIN, a nuclear-pore complex component. We recently discovered that the pathogenesis involved impaired nuclear import of DNA repair proteins, including DNA ligase I and the cerebellar ataxia causative protein aprataxin. Such impairment was overcome by fusing classical nuclear localization signal (NLS) and 137-aa downstream sequence of XRCC1, designated stretched NLS (stNLS). We report here that the minimum essential sequence of stNLS (mstNLS) is residues 239-276, downsized by more than 100 aa. mstNLS enabled efficient nuclear import of DNA repair proteins in patientmore » fibroblasts, functioned under oxidative stress, and reduced oxidative-stress-induced cell death, more effectively than stNLS. The stress-tolerability of mstNLS was also exerted in control fibroblasts and neuroblastoma cells. These findings may help develop treatments for currently intractable triple A syndrome and other oxidative-stress-related neurological diseases, and contribute to nuclear compartmentalization study.« less

Negative transcriptional regulation of mitochondrial transcription factor A (TFAM) by nuclear TFAM

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Eun Jin; Kang, Young Cheol; Park, Wook-Ha

2014-07-18

Highlights: • TFAM localizes in nuclei and mitochondria of neuronal cells. • Nuclear TFAM does not bind the Tfam promoter. • Nuclear TFAM reduced the Tfam promoter activity via suppressing NRF-1 activity. • A novel self-negative feedback regulation of Tfam gene expression is explored. • FAM may play different roles depending on its subcellular localizations. - Abstract: The nuclear DNA-encoded mitochondrial transcription factor A (TFAM) is synthesized in cytoplasm and transported into mitochondria. TFAM enhances both transcription and replication of mitochondrial DNA. It is unclear, however, whether TFAM plays a role in regulating nuclear gene expression. Here, we demonstrated thatmore » TFAM was localized to the nucleus and mitochondria by immunostaining, subcellular fractionation, and TFAM-green fluorescent protein hybrid protein studies. In HT22 hippocampal neuronal cells, human TFAM (hTFAM) overexpression suppressed human Tfam promoter-mediated luciferase activity in a dose-dependent manner. The mitochondria targeting sequence-deficient hTFAM also repressed Tfam promoter activity to the same degree as hTFAM. It indicated that nuclear hTFAM suppressed Tfam expression without modulating mitochondrial activity. The repression required for nuclear respiratory factor-1 (NRF-1), but hTFAM did not bind to the NRF-1 binding site of its promoter. TFAM was co-immunoprecipitated with NRF-1. Taken together, we suggest that nuclear TFAM down-regulate its own gene expression as a NRF-1 repressor, showing that TFAM may play different roles depending on its subcellular localizations.« less

Serine phosphorylation by SYK is critical for nuclear localization and transcription factor function of Ikaros

PubMed Central

Uckun, Fatih M.; Ma, Hong; Zhang, Jian; Ozer, Zahide; Dovat, Sinisa; Mao, Cheney; Ishkhanian, Rita; Goodman, Patricia; Qazi, Sanjive

2012-01-01

Ikaros is a zinc finger-containing DNA-binding protein that plays a pivotal role in immune homeostasis through transcriptional regulation of the earliest stages of lymphocyte ontogeny and differentiation. Functional deficiency of Ikaros has been implicated in the pathogenesis of acute lymphoblastic leukemia, the most common form of childhood cancer. Therefore, a stringent regulation of Ikaros activity is considered of paramount importance, but the operative molecular mechanisms responsible for its regulation remain largely unknown. Here we provide multifaceted genetic and biochemical evidence for a previously unknown function of spleen tyrosine kinase (SYK) as a partner and posttranslational regulator of Ikaros. We demonstrate that SYK phoshorylates Ikaros at unique C-terminal serine phosphorylation sites S358 and S361, thereby augmenting its nuclear localization and sequence-specific DNA binding activity. Mechanistically, we establish that SYK-induced Ikaros activation is essential for its nuclear localization and optimal transcription factor function. PMID:23071339

Expanding the Definition of the Classical Bipartite Nuclear Localization Signal

PubMed Central

Lange, Allison; McLane, Laura M.; Mills, Ryan E.; Devine, Scott E.; Corbett, Anita H.

2010-01-01

Nuclear localization signals (NLSs) are amino acid sequences that target cargo proteins into the nucleus. Rigorous characterization of NLS motifs is essential to understanding and predicting pathways for nuclear import. The best-characterized NLS is the classical NLS (cNLS), which is recognized by the cNLS receptor, importin-α. cNLSs are conventionally defined as having one (monopartite) or two clusters of basic amino acids separated by a 9-12 amino acid linker (bipartite). Motivated by the finding that Ty1 integrase, which contains an unconventional putative bipartite cNLS with a 29 amino acid linker, exploits the classical nuclear import machinery, we assessed the functional boundaries for linker length within a bipartite cNLS. We confirmed that the integrase cNLS is a bona fide bipartite cNLS, then carried out a systematic analysis of linker length in an obligate bipartite cNLS cargo, which revealed that some linkers longer than conventionally defined can function in nuclear import. Linker function is dependent on the sequence and likely the inherent flexibility of the linker. Subsequently, we interrogated the Saccharomyces cerevisiae proteome to identify cellular proteins containing putative long bipartite cNLSs. We experimentally confirmed that Rrp4 contains a bipartite cNLS with a 25 amino acid linker. Our studies reveal that the traditional definition of bipartite cNLSs is too restrictive and linker length can vary depending on amino acid composition PMID:20028483

Identification of the WBSCR9 gene, encoding a novel transcriptional regulator, in the Williams-Beuren syndrome deletion at 7q11.23.

PubMed

Peoples, R J; Cisco, M J; Kaplan, P; Francke, U

1998-01-01

We have identified a novel gene (WBSCR9) within the common Williams-Beuren syndrome (WBS) deletion by interspecies sequence conservation. The WBSCR9 gene encodes a roughly 7-kb transcript with an open reading frame of 1483 amino acids and a predicted protein product size of 170.8 kDa. WBSCR9 is comprised of at least 20 exons extending over 60 kb. The transcript is expressed ubiquitously throughout development and is subject to alternative splicing. Functional motifs identified by sequence homology searches include a bromodomain; a PHD, or C4HC3, finger; several putative nuclear localization signals; four nuclear receptor binding motifs; a polyglutamate stretch and two PEST sequences. Bromodomains, PHD motifs and nuclear receptor binding motifs are cardinal features of proteins that are involved in chromatin remodeling and modulation of transcription. Haploinsufficiency for WBSCR9 gene products may contribute to the complex phenotype of WBS by interacting with tissue-specific regulatory factors during development.

Molecular sled sequences are common in mammalian proteins.

PubMed

Xiong, Kan; Blainey, Paul C

2016-03-18

Recent work revealed a new class of molecular machines called molecular sleds, which are small basic molecules that bind and slide along DNA with the ability to carry cargo along DNA. Here, we performed biochemical and single-molecule flow stretching assays to investigate the basis of sliding activity in molecular sleds. In particular, we identified the functional core of pVIc, the first molecular sled characterized; peptide functional groups that control sliding activity; and propose a model for the sliding activity of molecular sleds. We also observed widespread DNA binding and sliding activity among basic polypeptide sequences that implicate mammalian nuclear localization sequences and many cell penetrating peptides as molecular sleds. These basic protein motifs exhibit weak but physiologically relevant sequence-nonspecific DNA affinity. Our findings indicate that many mammalian proteins contain molecular sled sequences and suggest the possibility that substantial undiscovered sliding activity exists among nuclear mammalian proteins. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

The early UL31 gene of equine herpesvirus 1 encodes a single-stranded DNA-binding protein that has a nuclear localization signal sequence at the C-terminus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Seongman; Chul Ahn, Byung; O'Callaghan, Dennis J.

2012-10-25

The amino acid sequence of the UL31 protein (UL31P) of equine herpesvirus 1 (EHV-1) has homology to that of the ICP8 of herpes simplex virus type 1 (HSV-1). Here we show that the UL31 gene is synergistically trans-activated by the IEP and the UL5P (EICP27). Detection of the UL31 RNA transcript and the UL31P in EHV-1-infected cells at 6 h post-infection (hpi) as well as metabolic inhibition assays indicated that UL31 is an early gene. The UL31P preferentially bound to single-stranded DNA over double-stranded DNA in gel shift assays. Subcellular localization of the green fluorescent protein (GFP)-UL31 fusion proteins revealedmore » that the C-terminal 32 amino acid residues of the UL31P are responsible for the nuclear localization. These findings may contribute to defining the role of the UL31P single-stranded DNA-binding protein in EHV-1 DNA replication.« less

Radial chromatin positioning is shaped by local gene density, not by gene expression

PubMed Central

2009-01-01

G- and R-bands of metaphase chromosomes are characterized by profound differences in gene density, CG content, replication timing, and chromatin compaction. The preferential localization of gene-dense, transcriptionally active, and early replicating chromatin in the nuclear interior and of gene-poor, later replicating chromatin at the nuclear envelope has been demonstrated to be evolutionary-conserved in various cell types. Yet, the impact of different local chromatin features on the radial nuclear arrangement of chromatin is still not well understood. In particular, it is not known whether radial chromatin positioning is preferentially shaped by local gene density per se or by other related parameters such as replication timing or transcriptional activity. The interdependence of these distinct chromatin features on the linear deoxyribonucleic acid (DNA) sequence precludes a simple dissection of these parameters with respect to their importance for the reorganization of the linear DNA organization into the distinct radial chromatin arrangements observed in the nuclear space. To analyze this problem, we generated probe sets of pooled bacterial artificial chromosome (BAC) clones from HSA 11, 12, 18, and 19 representing R/G-band-assigned chromatin, segments with different gene density and gene loci with different expression levels. Using multicolor 3D flourescent in situ hybridization (FISH) and 3D image analysis, we determined their localization in the nucleus and their positions within or outside the corresponding chromosome territory (CT). For each BAC data on local gene density within 2- and 10-Mb windows, as well as GC (guanine and cytosine) content, replication timing and expression levels were determined. A correlation analysis of these parameters with nuclear positioning revealed regional gene density as the decisive parameter determining the radial positioning of chromatin in the nucleus in contrast to band assignment, replication timing, and transcriptional activity. We demonstrate a polarized distribution of gene-dense vs gene-poor chromatin within CTs with respect to the nuclear border. Whereas we confirm previous reports that a particular gene-dense and transcriptionally highly active region of about 2 Mb on 11p15.5 often loops out from the territory surface, gene-dense and highly expressed sequences were not generally found preferentially at the CT surface as previously suggested. PMID:17333233

The Budding Yeast Nucleus

PubMed Central

Taddei, Angela; Schober, Heiko; Gasser, Susan M.

2010-01-01

The budding yeast nucleus, like those of other eukaryotic species, is highly organized with respect to both chromosomal sequences and enzymatic activities. At the nuclear periphery interactions of nuclear pores with chromatin, mRNA, and transport factors promote efficient gene expression, whereas centromeres, telomeres, and silent chromatin are clustered and anchored away from pores. Internal nuclear organization appears to be function-dependent, reflecting localized sites for tRNA transcription, rDNA transcription, ribosome assembly, and DNA repair. Recent advances have identified new proteins involved in the positioning of chromatin and have allowed testing of the functional role of higher-order chromatin organization. The unequal distribution of silent information regulatory factors and histone modifying enzymes, which arises in part from the juxtaposition of telomeric repeats, has been shown to influence chromatin-mediated transcriptional repression. Other localization events suppress unwanted recombination. These findings highlight the contribution budding yeast genetics and cytology have made to dissecting the functional role of nuclear structure. PMID:20554704

Karyopherin α 3 and karyopherin α 4 proteins mediate the nuclear import of methyl-CpG binding protein 2.

PubMed

Baker, Steven Andrew; Lombardi, Laura Marie; Zoghbi, Huda Yahya

2015-09-11

Methyl-CpG binding protein 2 (MeCP2) is a nuclear protein with important roles in regulating chromatin structure and gene expression, and mutations in MECP2 cause Rett syndrome (RTT). Within the MeCP2 protein sequence, the nuclear localization signal (NLS) is reported to reside between amino acids 255-271, and certain RTT-causing mutations overlap with the MeCP2 NLS, suggesting that they may alter nuclear localization. One such mutation, R270X, is predicted to interfere with the localization of MeCP2, but recent in vivo studies have demonstrated that this mutant remains entirely nuclear. To clarify the mechanism of MeCP2 nuclear import, we isolated proteins that interact with the NLS and identified karyopherin α 3 (KPNA3 or Kap-α3) and karyopherin α 4 (KPNA4 or Kap-α4) as key binding partners of MeCP2. MeCP2-R270X did not interact with KPNA4, consistent with a requirement for an intact NLS in this interaction. However, this mutant retains binding to KPNA3, accounting for the normal localization of MeCP2-R270X to the nucleus. These data provide a mechanism for MeCP2 nuclear import and have implications for the design of therapeutics aimed at modulating the function of MeCP2 in RTT patients. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Nuclear Import of the Retrotransposon Tf1 Is Governed by a Nuclear Localization Signal That Possesses a Unique Requirement for the FXFG Nuclear Pore Factor Nup124p

PubMed Central

Dang, Van-Dinh; Levin, Henry L.

2000-01-01

Retroviruses, such as human immunodeficiency virus, that infect nondividing cells generate integration precursors that must cross the nuclear envelope to reach the host genome. As a model for retroviruses, we investigated the nuclear entry of Tf1, a long-terminal-repeat-containing retrotransposon of the fission yeast Schizosaccharomyces pombe. Because the nuclear envelope of yeasts remains intact throughout the cell cycle, components of Tf1 must be transported through the envelope before integration can occur. The nuclear localization of the Gag protein of Tf1 is different from that of other proteins tested in that it has a specific requirement for the FXFG nuclear pore factor, Nup124p. Using extensive mutagenesis, we found that Gag contained three nuclear localization signals (NLSs) which, when included individually in a heterologous protein, were sufficient to direct nuclear import. In the context of the intact transposon, mutations in the NLS that mapped to the first 10 amino acid residues of Gag significantly impaired Tf1 retrotransposition and abolished nuclear localization of Gag. Interestingly, this NLS activity in the heterologous protein was specifically dependent upon the presence of Nup124p. Deletion analysis of heterologous proteins revealed the surprising result that the residues in Gag with the NLS activity were independent from the residues that conveyed the requirement for Nup124p. In fact, a fragment of Gag that lacked NLS activity, residues 10 to 30, when fused to a heterologous protein, was sufficient to cause the classical NLS of simian virus 40 to require Nup124p for nuclear import. Within the context of the current understanding of nuclear import, these results represent the novel case of a short amino acid sequence that specifies the need for a particular nuclear pore complex protein. PMID:11003674

Nuclear import of the retrotransposon Tf1 is governed by a nuclear localization signal that possesses a unique requirement for the FXFG nuclear pore factor Nup124p.

PubMed

Dang, V D; Levin, H L

2000-10-01

Retroviruses, such as human immunodeficiency virus, that infect nondividing cells generate integration precursors that must cross the nuclear envelope to reach the host genome. As a model for retroviruses, we investigated the nuclear entry of Tf1, a long-terminal-repeat-containing retrotransposon of the fission yeast Schizosaccharomyces pombe. Because the nuclear envelope of yeasts remains intact throughout the cell cycle, components of Tf1 must be transported through the envelope before integration can occur. The nuclear localization of the Gag protein of Tf1 is different from that of other proteins tested in that it has a specific requirement for the FXFG nuclear pore factor, Nup124p. Using extensive mutagenesis, we found that Gag contained three nuclear localization signals (NLSs) which, when included individually in a heterologous protein, were sufficient to direct nuclear import. In the context of the intact transposon, mutations in the NLS that mapped to the first 10 amino acid residues of Gag significantly impaired Tf1 retrotransposition and abolished nuclear localization of Gag. Interestingly, this NLS activity in the heterologous protein was specifically dependent upon the presence of Nup124p. Deletion analysis of heterologous proteins revealed the surprising result that the residues in Gag with the NLS activity were independent from the residues that conveyed the requirement for Nup124p. In fact, a fragment of Gag that lacked NLS activity, residues 10 to 30, when fused to a heterologous protein, was sufficient to cause the classical NLS of simian virus 40 to require Nup124p for nuclear import. Within the context of the current understanding of nuclear import, these results represent the novel case of a short amino acid sequence that specifies the need for a particular nuclear pore complex protein.

Nuclear localization and transactivation by Vitis CBF transcription factors are regulated by combinations of conserved amino acid domains.

PubMed

Carlow, Chevonne E; Faultless, J Trent; Lee, Christine; Siddiqua, Mahbuba; Edge, Alison; Nassuth, Annette

2017-09-01

The highly conserved CBF pathway is crucial in the regulation of plant responses to low temperatures. Extensive analysis of Arabidopsis CBF proteins revealed that their functions rely on several conserved amino acid domains although the exact function of each domain is disputed. The question was what functions similar domains have in CBFs from other, overwintering woody plants such as Vitis, which likely have a more involved regulation than the model plant Arabidopsis. A total of seven CBF genes were cloned and sequenced from V. riparia and the less frost tolerant V. vinifera. The deduced species-specific amino acid sequences differ in only a few amino acids, mostly in non-conserved regions. Amino acid sequence comparison and phylogenetic analysis showed two distinct groups of Vitis CBFs. One group contains CBF1, CBF2, CBF3 and CBF8 and the other group contains CBF4, CBF5 and CBF6. Transient transactivation assays showed that all Vitis CBFs except CBF5 activate via a CRT or DRE promoter element, whereby Vitis CBF3 and 4 prefer a CRT element. The hydrophobic domains in the C-terminal end of VrCBF6 were shown to be important for how well it activates. The putative nuclear localization domain of Vitis CBF1 was shown to be sufficient for nuclear localization, in contrast to previous reports for AtCBF1, and also important for transactivation. The latter highlights the value of careful analysis of domain functions instead of reliance on computer predictions and published data for other related proteins. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Controlling Androgen receptor nuclear localization by dendrimer conjugates

NASA Astrophysics Data System (ADS)

Wang, Haoyu

Androgen Receptor (AR) antagonists, such as bicalutamide and flutamide have been used widely in the treatment of prostate cancer. Although initial treatment is effective, prostate cancer cells often acquire antiandrogen resistance with prolonged treatment. AR over-expression and AR mutations contribute to the development of antiandrogen resistant cancer. Second generation antiandrogens such as enzalutamide are more effective and show reduced AR nuclear localization. In this study, derivatives of PAN52, a small molecule antiandrogen previously developed in our lab, were conjugated to the surface of generation 4 and generation 6 PAMAM dendrimers to obtain antiandrogen PAMAM dendrimer conjugates (APDC). APDCs readily enter cells and associate with AR in the cytoplasm. Due to their large size and positive charge, they can not enter the nucleus, thus retaining AR in the cytoplasm. In addition, APDCs are effective in decreasing AR mediated transcription and cell proliferation. APDC is the first AR antagonists that inhibit DHT-induced nuclear localization of AR. By inhibiting AR nuclear localization, APDC represents a new class of antiandrogens that offer an alternative approach to addressing antiandrogen-resistant prostate cancer. Lysine post-translational modification of AR Nuclear Localization Sequence (NLS) has great impact on AR cellular localization. It is of interest to understand which modifications modulate AR translocation into the nucleus. In this study, we prepared dendrimer-based acetyltransferase mimetic (DATM), DATM is able to catalytically acetylate AR in CWR22Rv1 cells, which will be a useful tool for studying AR modification effect on AR cellular localization. Derivatives of DATM, which transfer other chemical groups to AR, can be prepared similarly, and with more dendrimer based AR modification tools prepared in future, we will be able to understand and control AR cellular localization through AR modification.

Molecular characterization of baculovirus Bombyx mori nucleopolyhedrovirus polyhedron mutants.

PubMed

Katsuma, S; Noguchi, Y; Shimada, T; Nagata, M; Kobayashi, M; Maeda, S

1999-01-01

Four newly isolated and two previously isolated polyhedron mutants of Bombyx mori nucleopolyhedrovirus (BmNPV) were studied. Two polyhedron deficient mutants, #126 and #136, produced small uncrystallized particles of polyhedrin in the nuclei and cytoplasm of infected cells. Mutant #211 produced a large number of variably sized polyhedra in the nucleus and #220 produced a few large cuboidal polyhedra in the nucleus. Mutant #24 and #128 were previously isolated BmNPV mutants. Mutant #24 could not produce polyhedrin mRNA and polyhedra produced by mutant #128 lacked oral infectivity. Nucleotide sequence analysis indicated that five mutants (#126, #136, #211, #220 and #128) had amino acid substitutions in polyhedrin and mutant #24 had a point mutation only in the promoter region of the polyhedrin gene. Cotransfection experiments showed that the altered phenotypes were due to the mutations found in the polyhedrin gene regions. In mutants #126 and #136, amino acid sequences of the nuclear localization signal of polyhedrin were identical to those of wild-type BmNPV, suggesting that this sequence was necessary but not sufficient for nuclear localization of polyhedrin. Electron microscopic observation revealed that fewer occluded virions were contained in polyhedra of #128 and #220.

DNA sequence-dependent compartmentalization and silencing of chromatin at the nuclear lamina.

PubMed

Zullo, Joseph M; Demarco, Ignacio A; Piqué-Regi, Roger; Gaffney, Daniel J; Epstein, Charles B; Spooner, Chauncey J; Luperchio, Teresa R; Bernstein, Bradley E; Pritchard, Jonathan K; Reddy, Karen L; Singh, Harinder

2012-06-22

A large fraction of the mammalian genome is organized into inactive chromosomal domains along the nuclear lamina. The mechanism by which these lamina associated domains (LADs) are established remains to be elucidated. Using genomic repositioning assays, we show that LADs, spanning the developmentally regulated IgH and Cyp3a loci contain discrete DNA regions that associate chromatin with the nuclear lamina and repress gene activity in fibroblasts. Lamina interaction is established during mitosis and likely involves the localized recruitment of Lamin B during late anaphase. Fine-scale mapping of LADs reveals numerous lamina-associating sequences (LASs), which are enriched for a GAGA motif. This repeated motif directs lamina association and is bound by the transcriptional repressor cKrox, in a complex with HDAC3 and Lap2β. Knockdown of cKrox or HDAC3 results in dissociation of LASs/LADs from the nuclear lamina. These results reveal a mechanism that couples nuclear compartmentalization of chromatin domains with the control of gene activity. Copyright © 2012 Elsevier Inc. All rights reserved.

Modulation of integrin-linked kinase nucleo-cytoplasmic shuttling by ILKAP and CRM1.

PubMed

Nakrieko, Kerry-Ann; Vespa, Alisa; Mason, David; Irvine, Timothy S; D'Souza, Sudhir J A; Dagnino, Lina

2008-07-15

Integrin-linked kinase (ILK) plays key roles in a variety of cell functions, including cell proliferation, adhesion and migration. Within the cell, ILK localizes to multiple sites, including the cytoplasm, focal adhesion complexes that mediate cell adhesion to extracellular substrates, as well as cell-cell junctions in epidermal keratinocytes. Central to understanding ILK function is the elucidation of the mechanisms that regulate its subcellular localization. We now demonstrate that ILK is imported into the nucleus through sequences in its N-terminus, via active transport mechanisms that involve nuclear pore complexes. In addition, nuclear ILK can be rapidly exported into the cytoplasm through a CRM1-dependent pathway, and its export is enhanced by the type 2C protein phosphatase ILKAP. Nuclear localization of ILK in epidermal keratinocytes is associated with increased DNA synthesis, which is sensitive to inhibition by ILKAP. Our studies demonstrate the importance for keratinocyte proliferation of ILK regulation through changes in its subcellular localization, and establish ILKAP and CRM1 as pivotal modulators of ILK subcellular distribution and activity in these cells.

The Treacher Collins syndrome (TCOF1) gene product, treacle, is targeted to the nucleolus by signals in its C-terminus.

PubMed

Winokur, S T; Shiang, R

1998-11-01

The TCOF1 gene product, treacle, responsible for the craniofacial disorder Treacher Collins syndrome, has been predicted to be a member of a class of nucleolar phosphoproteins based on its primary amino acid sequence. Treacle is a low complexity protein with ten repeating units of acidic and basic residues, each of which contains a large number of putative casein kinase 2 and protein kinase C phosphorylation sites. In addition, the C-terminus of treacle contains multiple putative nuclear localization signals. The overall structure of treacle, as well as sequence similarity to several nucleolar phosphoproteins, predicts that treacle is a member of this class of proteins. Using green fluorescent protein fusion constructs with the full-length and deleted domains of the murine homolog of treacle, we demonstrate that the cellular localization of treacle is nucleolar. This localization is mediated by the last 41 residues of the C-terminus (residues 1262-1302). At least two functional nuclear localization signals have been identified in the protein, one between residues 1176 and 1270 and the second within the last 32 residues of the protein (1271-1302). The nucleolar localization signal is disrupted by two constructs that split the C-terminal region between residues 1270 and 1271. This study provides the first direct analysis of treacle and demonstrates that the protein involved in TCOF1 is a nucleolar protein.

Acetylation of the RhoA GEF Net1A controls its subcellular localization and activity

PubMed Central

Song, Eun Hyeon; Oh, Wonkyung; Ulu, Arzu; Carr, Heather S.; Zuo, Yan; Frost, Jeffrey A.

2015-01-01

ABSTRACT Net1 isoform A (Net1A) is a RhoA GEF that is required for cell motility and invasion in multiple cancers. Nuclear localization of Net1A negatively regulates its activity, and we have recently shown that Rac1 stimulates Net1A relocalization to the plasma membrane to promote RhoA activation and cytoskeletal reorganization. However, mechanisms controlling the subcellular localization of Net1A are not well understood. Here, we show that Net1A contains two nuclear localization signal (NLS) sequences within its N-terminus and that residues surrounding the second NLS sequence are acetylated. Treatment of cells with deacetylase inhibitors or expression of active Rac1 promotes Net1A acetylation. Deacetylase inhibition is sufficient for Net1A relocalization outside the nucleus, and replacement of the N-terminal acetylation sites with arginine residues prevents cytoplasmic accumulation of Net1A caused by deacetylase inhibition or EGF stimulation. By contrast, replacement of these sites with glutamine residues is sufficient for Net1A relocalization, RhoA activation and downstream signaling. Moreover, the N-terminal acetylation sites are required for rescue of F-actin accumulation and focal adhesion maturation in Net1 knockout MEFs. These data indicate that Net1A acetylation regulates its subcellular localization to impact on RhoA activity and actin cytoskeletal organization. PMID:25588829

Nuclear Localization of Suppressor of Cytokine Signaling-1 Regulates Local Immunity in the Lung

PubMed Central

Zimmer, Jana; Weitnauer, Michael; Boutin, Sébastien; Küblbeck, Günter; Thiele, Sabrina; Walker, Patrick; Lasitschka, Felix; Lunding, Lars; Orinska, Zane; Vock, Christina; Arnold, Bernd; Wegmann, Michael; Dalpke, Alexander

2016-01-01

Suppressor of cytokine signaling 1 (SOCS1) is a negative feedback inhibitor of cytoplasmic Janus kinase and signal transducer and activator of transcription (STAT) signaling. SOCS1 also contains a nuclear localization sequence (NLS), yet, the in vivo importance of nuclear translocation is unknown. We generated transgenic mice containing mutated Socs1ΔNLS that fails to translocate in the cell nucleus (MGLtg mice). Whereas mice fully deficient for SOCS1 die within the first 3 weeks due to excessive interferon signaling and multiorgan inflammation, mice expressing only non-nuclear Socs1ΔNLS (Socs1−/−MGLtg mice) were rescued from early lethality. Canonical interferon gamma signaling was still functional in Socs1−/−MGLtg mice as shown by unaltered tyrosine phosphorylation of STAT1 and whole genome expression analysis. However, a subset of NFκB inducible genes was dysregulated. Socs1−/−MGLtg mice spontaneously developed low-grade inflammation in the lung and had elevated Th2-type cytokines. Upon ovalbumin sensitization and challenge, airway eosinophilia was increased in Socs1−/−MGLtg mice. Decreased transepithelial electrical resistance in trachea epithelial cells from Socs1−/−MGLtg mice suggests disrupted epithelial cell barrier. The results indicate that nuclear SOCS1 is a regulator of local immunity in the lung and unravel a so far unrecognized function for SOCS1 in the cell nucleus. PMID:27917175

NMR studies on the structure and dynamics of lac operator DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, S.C.

Nuclear Magnetic Resonance spectroscopy was used to elucidate the relationships between structure, dynamics and function of the gene regulatory sequence corresponding to the lactose operon operator of Escherichia coli. The length of the DNA fragments examined varied from 13 to 36 base pair, containing all or part of the operator sequence. These DNA fragments are either derived genetically or synthesized chemically. Resonances of the imino protons were assigned by one dimensional inter-base pair nuclear Overhauser enhancement (NOE) measurements. Imino proton exchange rates were measured by saturation recovery methods. Results from the kinetic measurements show an interesting dynamic heterogeneity with amore » maximum opening rate centered about a GTG/CAC sequence which correlates with the biological function of the operator DNA. This particular three base pair sequence occurs frequently and often symmetrically in prokaryotic nd eukaryotic DNA sites where one anticipates specific protein interaction for gene regulation. The observed sequence dependent imino proton exchange rate may be a reflection of variation of the local structure of regulatory DNA. The results also indicate that the observed imino proton exchange rates are length dependent.« less

Predicting protein subnuclear location with optimized evidence-theoretic K-nearest classifier and pseudo amino acid composition.

PubMed

Shen, Hong-Bin; Chou, Kuo-Chen

2005-11-25

The nucleus is the brain of eukaryotic cells that guides the life processes of the cell by issuing key instructions. For in-depth understanding of the biochemical process of the nucleus, the knowledge of localization of nuclear proteins is very important. With the avalanche of protein sequences generated in the post-genomic era, it is highly desired to develop an automated method for fast annotating the subnuclear locations for numerous newly found nuclear protein sequences so as to be able to timely utilize them for basic research and drug discovery. In view of this, a novel approach is developed for predicting the protein subnuclear location. It is featured by introducing a powerful classifier, the optimized evidence-theoretic K-nearest classifier, and using the pseudo amino acid composition [K.C. Chou, PROTEINS: Structure, Function, and Genetics, 43 (2001) 246], which can incorporate a considerable amount of sequence-order effects, to represent protein samples. As a demonstration, identifications were performed for 370 nuclear proteins among the following 9 subnuclear locations: (1) Cajal body, (2) chromatin, (3) heterochromatin, (4) nuclear diffuse, (5) nuclear pore, (6) nuclear speckle, (7) nucleolus, (8) PcG body, and (9) PML body. The overall success rates thus obtained by both the re-substitution test and jackknife cross-validation test are significantly higher than those by existing classifiers on the same working dataset. It is anticipated that the powerful approach may also become a useful high throughput vehicle to bridge the huge gap occurring in the post-genomic era between the number of gene sequences in databases and the number of gene products that have been functionally characterized. The OET-KNN classifier will be available at www.pami.sjtu.edu.cn/people/hbshen.

Functional relationship between CABIT, SAM and 14-3-3 binding domains of GAREM1 that play a role in its subcellular localization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishino, Tasuku; Matsunaga, Ryota; Konishi, Hiroaki, E-mail: hkonishi@pu-hiroshima.ac.jp

2015-08-21

GAREM1 (Grb2-associated regulator of Erk/MAPK1) is an adaptor protein that is involved in the epidermal growth factor (EGF) pathway. The nuclear localization of GAREM1 depends on the nuclear localization sequence (NLS), which is located at the N-terminal CABIT (cysteine-containing, all in Themis) domain. Here, we identified 14-3-3ε as a GAREM-binding protein, and its binding site is closely located to the NLS. This 14-3-3 binding site was of the atypical type and independent of GAREM phosphorylation. Moreover, the binding of 14-3-3 had an effect on the nuclear localization of GAREM1. Unexpectedly, we observed that the CABIT domain had intramolecular association withmore » the C-terminal SAM (sterile alpha motif) domain. This association might be inhibited by binding of 14-3-3 at the CABIT domain. Our results demonstrate that the mechanism underlying the nuclear localization of GAREM1 depends on its NLS in the CABIT domain, which is controlled by the binding of 14-3-3 and the C-terminal SAM domain. We suggest that the interplay between 14-3-3, SAM domain and CABIT domain might be responsible for the distribution of GAREM1 in mammalian cells. - Highlights: • 14-3-3ε regulated the nuclear localization of GAREM1 as its binding partner. • The atypical 14-3-3 binding site of GAREM1 is located near the NLS in CABIT domain. • The CABIT domain had intramolecular association with the SAM domain in GAREM1. • Subcellular localization of GAREM1 is affected with its CABIT-SAM interaction.« less

Nuclear localization of foamy virus Gag precursor protein.

PubMed Central

Schliephake, A W; Rethwilm, A

1994-01-01

All foamy viruses give rise to a strong nuclear staining when infected cells are reacted with sera from infected hosts. This nuclear fluorescence distinguishes foamy viruses from all other retroviruses. The experiments reported here indicate that the foamy virus Gag precursor protein is transiently located in the nuclei of infected cells and this is the likely reason for the typical foamy virus nuclear fluorescence. By using the vaccinia virus expression system, a conserved basic sequence motif in the nucleocapsid domain of foamy virus Gag proteins was identified to be responsible for the nuclear transport of the gag precursor molecule. This motif was also found to be able to direct a heterologous protein, the Gag protein of human immunodeficiency virus, into the nucleus. Images PMID:8035493

FOXP2 promotes the nuclear translocation of POT1, but FOXP2(R553H), mutation related to speech-language disorder, partially prevents it.

PubMed

Tanabe, Yuko; Fujita, Eriko; Momoi, Takashi

2011-07-08

FOXP2 is a forkhead box-containing transcription factor with several recognizable sequence motifs. However, little is known about the FOXP2-associated proteins except for C-terminal binding protein (CtBP). In the present study, we attempted to isolate the FOXP2-associated protein with a yeast two-hybrid system using the C-terminal region, including the forkhead domain, as a bait probe, and identified protection of telomeres 1 (POT1) as a FOXP2-associated protein. Immunoprecipitation assay confirmed the association with FOXP2 and POT1. POT1 alone localized in the cytoplasm but co-localized with FOXP2 and the forkhead domain of FOXP2 in nuclei. However, both FOXP2 with mutated nuclear localization signals and (R553H) mutated forkhead, which is associated with speech-language disorder, prevented the nuclear translocation of POT1. These results suggest that FOXP2 is a binding partner for the nuclear translocation of POT1. As loss of POT1 function induces the cell arrest, the impaired nuclear translocation of POT1 in the developing neuronal cells may be associated with the pathogenesis of speech-language disorder with FOXP2(R553H) mutation. Copyright © 2011 Elsevier Inc. All rights reserved.

Cyclophilin B enhances HIV-1 infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

DeBoer, Jason; Madson, Christian J.; Belshan, Michael, E-mail: michaelbelshan@creighton.edu

Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence,more » putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. - Highlights: • CypB has been identified in several proteomic studies of HIV-1 infection. • CypB expression is upregulated in activated and infected T-cells. • Over-expression of CypB enhances HIV nuclear import and infection. • The N-terminus of CypB is necessary for these effects.« less

Import of a major mitochondrial enzyme depends on synergy between two distinct helices of its presequence

USDA-ARS?s Scientific Manuscript database

The human mitochondrial glutamate dehydrogenase isozymes (hGDH1 and 2) are abundant matrix-localized proteins encoded by nuclear genes. The proteins are synthesized in the cytoplasm, with an atypically long N-terminal mitochondrial targeting sequence (MTS). The results of secondary structure predi...

Regulation of the heat shock response under anoxia in the turtle, Trachemys scripta elegans.

PubMed

Krivoruchko, Anastasia; Storey, Kenneth B

2010-03-01

The effects of 20 h of anoxic submergence in cold water and 5 h of aerobic recovery on the heat shock response were analyzed in four organs of the anoxia-tolerant turtle Trachemys scripta elegans. Immunoblotting was used to analyze levels of active and inactive forms of the heat shock transcription factor 1 (HSF1), nuclear translocation of HSF1, and the levels of six heat shock proteins (HSPs). PCR was also used to retrieve the turtle HSF1 nucleotide sequence; its deduced amino acid sequence showed 97% identity with chicken HSF1. White skeletal muscle showed a strong fivefold increase in the amount of active HSF1 under anoxic conditions as well as an 80% increase in nuclear localization. This was accompanied by upregulation of five HSPs by 1.8- to 2.9-fold: Hsp25, Hsp40, Hsp70, Hsc70, and Hsp90, the latter two remained elevated after 5 h of aerobic recovery. Kidney and liver showed little change in active HSF1 content during anoxia and recovery, but a significant increase in the nuclear localization of HSF1 during anoxia. This supported enhanced expression of three HSPs in kidney (Hsp40, Hsc70, and Hsp90) and four in liver (Hsp40, Hsp60, Hsp70, Hsc70). Heart displayed a strong increase in active HSF1 during anoxia and recovery (6.6- to 6.8-fold higher than control) and increased nuclear localization but heart HSP levels did not rise. The data demonstrate organ-specific regulation of HSPs during anoxia exposure and aerobic recovery in T. s. elegans and suggest that the heat shock response is an important aspect of cytoprotection during facultative anaerobiosis, particularly with regard to underwater hibernation of turtles in cold water.

FOXP2 promotes the nuclear translocation of POT1, but FOXP2(R553H), mutation related to speech-language disorder, partially prevents it

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tanabe, Yuko; Fujita, Eriko; Department of Pediatrics, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498

Highlights: {yields} We isolated protection of telomeres 1 (POT1) as a FOXP2-associated protein by a yeast two-hybrid. {yields} FOXP2 associated and co-localized with POT1 in the nuclei. {yields} FOXP2(R553H) also co-localized with POT1 in both the cytoplasm and nuclei. {yields} FOXP2(R553H) partially prevented the nuclear translocation of POT1. {yields} FOXP2(R553H) mutation may be associated with the pathogenesis of speech-language disorder. -- Abstract: FOXP2 is a forkhead box-containing transcription factor with several recognizable sequence motifs. However, little is known about the FOXP2-associated proteins except for C-terminal binding protein (CtBP). In the present study, we attempted to isolate the FOXP2-associated protein withmore » a yeast two-hybrid system using the C-terminal region, including the forkhead domain, as a bait probe, and identified protection of telomeres 1 (POT1) as a FOXP2-associated protein. Immunoprecipitation assay confirmed the association with FOXP2 and POT1. POT1 alone localized in the cytoplasm but co-localized with FOXP2 and the forkhead domain of FOXP2 in nuclei. However, both FOXP2 with mutated nuclear localization signals and (R553H) mutated forkhead, which is associated with speech-language disorder, prevented the nuclear translocation of POT1. These results suggest that FOXP2 is a binding partner for the nuclear translocation of POT1. As loss of POT1 function induces the cell arrest, the impaired nuclear translocation of POT1 in the developing neuronal cells may be associated with the pathogenesis of speech-language disorder with FOXP2(R553H) mutation.« less

Nuclear transport of the Neurospora crassa NIT-2 transcription factor is mediated by importin-α.

PubMed

Bernardes, Natália E; Takeda, Agnes A S; Dreyer, Thiago R; Cupertino, Fernanda B; Virgilio, Stela; Pante, Nelly; Bertolini, Maria Célia; Fontes, Marcos R M

2017-12-06

The Neurospora crassa NIT-2 transcription factor belongs to the GATA transcription factor family and plays a fundamental role in the regulation of nitrogen metabolism. Because NIT-2 acts by accessing DNA inside the nucleus, understanding the nuclear import process of NIT-2 is necessary to characterize its function. Thus, in the present study, NIT-2 nuclear transport was investigated using a combination of biochemical, cellular, and biophysical methods. A complemented strain that produced an sfGFP-NIT-2 fusion protein was constructed, and nuclear localization assessments were made under conditions that favored protein translocation to the nucleus. Nuclear translocation was also investigated using HeLa cells, which showed that the putative NIT-2 nuclear localization sequence (NLS; 915 TISSKRQRRHSKS 927 ) was recognized by importin-α and that subsequent transport occurred via the classical import pathway. The interaction between the N. crassa importin-α (NcImpα) and the NIT-2 NLS was quantified with calorimetric assays, leading to the observation that the peptide bound to two sites with different affinities, which is typical of a monopartite NLS sequence. The crystal structure of the NcImpα/NIT-2 NLS complex was solved and revealed that the NIT-2 peptide binds to NcImpα with the major NLS-binding site playing a primary role. This result contrasts other recent studies that suggested a major role for the minor NLS-binding site in importin-α from the α2 family, indicating that both sites can be used for different cargo proteins according to specific metabolic requirements. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Mapping a nucleolar targeting sequence of an RNA binding nucleolar protein, Nop25

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujiwara, Takashi; Suzuki, Shunji; Kanno, Motoko

2006-06-10

Nop25 is a putative RNA binding nucleolar protein associated with rRNA transcription. The present study was undertaken to determine the mechanism of Nop25 localization in the nucleolus. Deletion experiments of Nop25 amino acid sequence showed Nop25 to contain a nuclear targeting sequence in the N-terminal and a nucleolar targeting sequence in the C-terminal. By expressing derivative peptides from the C-terminal as GFP-fusion proteins in the cells, a lysine and arginine residue-enriched peptide (KRKHPRRAQDSTKKPPSATRTSKTQRRRR) allowed a GFP-fusion protein to be transported and fully retained in the nucleolus. When the peptide was fused with cMyc epitope and expressed in the cells, amore » cMyc epitope was then detected in the nucleolus. Nop25 did not localize in the nucleolus by deletion of the peptide from Nop25. Furthermore, deletion of a subdomain (KRKHPRRAQ) in the peptide or amino acid substitution of lysine and arginine residues in the subdomain resulted in the loss of Nop25 nucleolar localization. These results suggest that the lysine and arginine residue-enriched peptide is the most prominent nucleolar targeting sequence of Nop25 and that the long stretch of basic residues might play an important role in the nucleolar localization of Nop25. Although Nop25 contained putative SUMOylation, phosphorylation and glycosylation sites, the amino acid substitution in these sites had no effect on the nucleolar localization, thus suggesting that these post-translational modifications did not contribute to the localization of Nop25 in the nucleolus. The treatment of the cells, which expressed a GFP-fusion protein with a nucleolar targeting sequence of Nop25, with RNase A resulted in a complete dislocation of the protein from the nucleolus. These data suggested that the nucleolar targeting sequence might therefore play an important role in the binding of Nop25 to RNA molecules and that the RNA binding of Nop25 might be essential for the nucleolar localization of Nop25.« less

Interleukin 33 as a Mechanically Responsive Cytokine Secreted by Living Cells*

PubMed Central

Kakkar, Rahul; Hei, Hillary; Dobner, Stephan; Lee, Richard T.

2012-01-01

Interleukin 33 (IL-33), a member of the Interleukin 1 cytokine family, is implicated in numerous human inflammatory diseases such as asthma, atherosclerosis, and rheumatoid arthritis. Despite its pathophysiologic importance, fundamental questions regarding the basic biology of IL-33 remain. Nuclear localization and lack of an export signal sequence are consistent with the view of IL-33 as a nuclear factor with the ability to repress RNA transcription. However, signaling via the transmembrane receptor ST2 and documented caspase-dependent inactivation have suggested IL-33 is liberated during cellular necrosis to effect paracrine signaling. We determined the subcellular localization of IL-33 and tracked its intracellular mobility and extracellular release. In contrast to published data, IL-33 localized simultaneously to nuclear euchromatin and membrane-bound cytoplasmic vesicles. Fluorescent pulse-chase fate-tracking documented dynamic nucleo-cytoplasmic flux, which was dependent on nuclear pore complex function. In murine fibroblasts in vitro and in vivo, mechanical strain induced IL-33 secretion in the absence of cellular necrosis. These data document IL-33 dynamic inter-organelle trafficking and release during biomechanical overload. As such we recharacterize IL-33 as both an inflammatory as well as mechanically responsive cytokine secreted by living cells. PMID:22215666

The identification of FANCD2 DNA binding domains reveals nuclear localization sequences.

PubMed

Niraj, Joshi; Caron, Marie-Christine; Drapeau, Karine; Bérubé, Stéphanie; Guitton-Sert, Laure; Coulombe, Yan; Couturier, Anthony M; Masson, Jean-Yves

2017-08-21

Fanconi anemia (FA) is a recessive genetic disorder characterized by congenital abnormalities, progressive bone-marrow failure, and cancer susceptibility. The FA pathway consists of at least 21 FANC genes (FANCA-FANCV), and the encoded protein products interact in a common cellular pathway to gain resistance against DNA interstrand crosslinks. After DNA damage, FANCD2 is monoubiquitinated and accumulates on chromatin. FANCD2 plays a central role in the FA pathway, using yet unidentified DNA binding regions. By using synthetic peptide mapping and DNA binding screen by electromobility shift assays, we found that FANCD2 bears two major DNA binding domains predominantly consisting of evolutionary conserved lysine residues. Furthermore, one domain at the N-terminus of FANCD2 bears also nuclear localization sequences for the protein. Mutations in the bifunctional DNA binding/NLS domain lead to a reduction in FANCD2 monoubiquitination and increase in mitomycin C sensitivity. Such phenotypes are not fully rescued by fusion with an heterologous NLS, which enable separation of DNA binding and nuclear import functions within this domain that are necessary for FANCD2 functions. Collectively, our results enlighten the importance of DNA binding and NLS residues in FANCD2 to activate an efficient FA pathway. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Prolonged exposure to particulate chromate inhibits RAD51 nuclear import mediator proteins.

PubMed

Browning, Cynthia L; Wise, John Pierce

2017-09-15

Particulate hexavalent chromium (Cr(VI)) is a human lung carcinogen and a human health concern. The induction of structural chromosome instability is considered to be a driving mechanism of Cr(VI)-induced carcinogenesis. Homologous recombination repair protects against Cr(VI)-induced chromosome damage, due to its highly accurate repair of Cr(VI)-induced DNA double strand breaks. However, recent studies demonstrate Cr(VI) inhibits homologous recombination repair through the misregulation of RAD51. RAD51 is an essential protein in HR repair that facilitates the search for a homologous sequence. Recent studies show prolonged Cr(VI) exposure prevents proper RAD51 subcellular localization, causing it to accumulate in the cytoplasm. Since nuclear import of RAD51 is crucial to its function, this study investigated the effect of Cr(VI) on the RAD51 nuclear import mediators, RAD51C and BRCA2. We show acute (24h) Cr(VI) exposure induces the proper localization of RAD51C and BRCA2. In contrast, prolonged (120h) exposure increased the cytoplasmic localization of both proteins, although RAD51C localization was more severely impaired. These results correlate temporally with the previously reported Cr(VI)-induced RAD51 cytoplasmic accumulation. In addition, we found Cr(VI) does not inhibit interaction between RAD51 and its nuclear import mediators. Altogether, our results suggest prolonged Cr(VI) exposure inhibits the nuclear import of RAD51C, and to a lesser extent, BRCA2, which results in the cytoplasmic accumulation of RAD51. Cr(VI)-induced inhibition of nuclear import may play a key role in its carcinogenic mechanism since the nuclear import of many tumor suppressor proteins and DNA repair proteins is crucial to their function. Copyright © 2017 Elsevier Inc. All rights reserved.

Nesprin 4 is an outer nuclear membrane protein that can induce kinesin-mediated cell polarization

PubMed Central

Roux, Kyle J.; Crisp, Melissa L.; Liu, Qian; Kim, Daein; Kozlov, Serguei; Stewart, Colin L.; Burke, Brian

2009-01-01

Nucleocytoplasmic coupling is mediated by outer nuclear membrane (ONM) nesprin proteins and inner nuclear membrane Sun proteins. Interactions spanning the perinuclear space create nesprin–Sun complexes connecting the cytoskeleton to nuclear components. A search for proteins displaying a conserved C-terminal sequence present in nesprins 1–3 identified nesprin 4 (Nesp4), a new member of this family. Nesp4 is a kinesin-1-binding protein that displays Sun-dependent localization to the ONM. Expression of Nesp4 is associated with dramatic changes in cellular organization involving relocation of the centrosome and Golgi apparatus relative to the nucleus. These effects can be accounted for entirely by Nesp4's kinesin-binding function. The implication is that Nesp4 may contribute to microtubule-dependent nuclear positioning. PMID:19164528

Nesprin 4 is an outer nuclear membrane protein that can induce kinesin-mediated cell polarization.

PubMed

Roux, Kyle J; Crisp, Melissa L; Liu, Qian; Kim, Daein; Kozlov, Serguei; Stewart, Colin L; Burke, Brian

2009-02-17

Nucleocytoplasmic coupling is mediated by outer nuclear membrane (ONM) nesprin proteins and inner nuclear membrane Sun proteins. Interactions spanning the perinuclear space create nesprin-Sun complexes connecting the cytoskeleton to nuclear components. A search for proteins displaying a conserved C-terminal sequence present in nesprins 1-3 identified nesprin 4 (Nesp4), a new member of this family. Nesp4 is a kinesin-1-binding protein that displays Sun-dependent localization to the ONM. Expression of Nesp4 is associated with dramatic changes in cellular organization involving relocation of the centrosome and Golgi apparatus relative to the nucleus. These effects can be accounted for entirely by Nesp4's kinesin-binding function. The implication is that Nesp4 may contribute to microtubule-dependent nuclear positioning.

Delivering Single-Walled Carbon Nanotubes to the Nucleus Using Engineered Nuclear Protein Domains.

PubMed

Boyer, Patrick D; Ganesh, Sairaam; Qin, Zhao; Holt, Brian D; Buehler, Markus J; Islam, Mohammad F; Dahl, Kris Noel

2016-02-10

Single-walled carbon nanotubes (SWCNTs) have great potential for cell-based therapies due to their unique intrinsic optical and physical characteristics. Consequently, broad classes of dispersants have been identified that individually suspend SWCNTs in water and cell media in addition to reducing nanotube toxicity to cells. Unambiguous control and verification of the localization and distribution of SWCNTs within cells, particularly to the nucleus, is needed to advance subcellular technologies utilizing nanotubes. Here we report delivery of SWCNTs to the nucleus by noncovalently attaching the tail domain of the nuclear protein lamin B1 (LB1), which we engineer from the full-length LMNB1 cDNA. More than half of this low molecular weight globular protein is intrinsically disordered but has an immunoglobulin-fold composed of a central hydrophobic core, which is highly suitable for associating with SWCNTs, stably suspending SWCNTs in water and cell media. In addition, LB1 has an exposed nuclear localization sequence to promote active nuclear import of SWCNTs. These SWCNTs-LB1 dispersions in water and cell media display near-infrared (NIR) absorption spectra with sharp van Hove peaks and an NIR fluorescence spectra, suggesting that LB1 individually disperses nanotubes. The dispersing capability of SWCNTs by LB1 is similar to that by albumin proteins. The SWCNTs-LB1 dispersions with concentrations ≥150 μg/mL (≥30 μg/mL) in water (cell media) remain stable for ≥75 days (≥3 days) at 4 °C (37 °C). Further, molecular dynamics modeling of association of LB1 with SWCNTs reveal that the exposure of the nuclear localization sequence is independent of LB1 binding conformation. Measurements from confocal Raman spectroscopy and microscopy, NIR fluorescence imaging of SWCNTs, and fluorescence lifetime imaging microscopy show that millions of these SWCNTs-LB1 complexes enter HeLa cells, localize to the nucleus of cells, and interact with DNA. We postulate that the modification of native cellular proteins as noncovalent dispersing agents to provide specific transport will open new possibilities to utilize both SWCNT and protein properties for multifunctional subcellular targeting applications. Specifically, nuclear targeting could allow delivery of anticancer therapies, genetic treatments, or DNA to the nucleus.

Nuclear Localizing Peptide-Conjugated, Redox-Sensitive Polymersomes for Delivering Curcumin and Doxorubicin to Pancreatic Cancer Microtumors.

PubMed

Anajafi, Tayebeh; Yu, Junru; Sedigh, Abbas; Haldar, Manas K; Muhonen, Wallace W; Oberlander, Seth; Wasness, Heather; Froberg, Jamie; Molla, Md Shahjahan; Katti, Kalpana S; Choi, Yongki; Shabb, John B; Srivastava, D K; Mallik, Sanku

2017-06-05

Improving the therapeutic index of anticancer agents is an enormous challenge. Targeting decreases the side effects of the therapeutic agents by delivering the drugs to the intended destination. Nanocarriers containing the nuclear localizing peptide sequences (NLS) translocate to the cell nuclei. However, the nuclear localization peptides are nonselective and cannot distinguish the malignant cells from the healthy counterparts. In this study, we designed a "masked" NLS peptide which is activated only in the presence of overexpressed matrix metalloproteinase-7 (MMP-7) enzyme in the pancreatic cancer microenvironment. This peptide is conjugated to the surface of redox responsive polymersomes to deliver doxorubicin and curcumin to the pancreatic cancer cell nucleus. We have tested the formulation in both two- and three-dimensional cultures of pancreatic cancer and normal cells. Our studies revealed that the drug-encapsulated polymeric vesicles are significantly more toxic toward the cancer cells (shrinking the spheroids up to 49%) compared to the normal cells (shrinking the spheroids up to 24%). This study can lead to the development of other organelle targeted drug delivery systems for various human malignancies.

Sedimentological indicators of paleoenvironments and siliciclastic stratigraphic sequences in some Miocene deposits of the Calvert Cliffs, southern Maryland

USGS Publications Warehouse

Shideler, G.L.

1994-01-01

Middle Miocene siliciclastic deposits comprising the Calvert Cliffs section at the Baltimore Gas and Electric Company's (BG&E) nuclear power plant site in southern Maryland were analyzed in terms of lithostratigraphy, sedimentary structures, and granulometric parameters, to interprete paleo-environments within a sequence-stratigraphic framework. In terms of sequence-stratigraphic models, the BG&E section can be interpreted as consisting of two genetic stratigraphic sequences (Galloway model), namely, a shelf sequence and an overlying deltaic sequence. Using the Exxon model, the section consists of two third-order (1-5 m.y. duration) depositional sequences. The stratigraphic sequences of the BG&E section reflect both relatively short-term eustatic transgressive events, as well as a long-term regressive trend with associated local deltation and coastal progradation. The regression probably signified a regional basinward shift of depocenters within the Salisbury embayment during Miocene time. -from Author

The Role of Ect2 Nuclear RhoGEF Activity in Ovarian Cancer Cell Transformation

PubMed Central

Huff, Lauren P.; DeCristo, Molly J.; Trembath, Dimitri; Kuan, Pei Fen; Yim, Margaret; Liu, Jinsong; Cook, Danielle R.; Miller, C. Ryan; Der, Channing J.

2013-01-01

Ect2, a Rho guanine nucleotide exchange factor (RhoGEF), is atypical among RhoGEFs in its predominantly nuclear localization in interphase cells. One current model suggests that Ect2 mislocalization drives cellular transformation by promoting aberrant activation of cytoplasmic Rho family GTPase substrates. However, in ovarian cancers, where Ect2 is both amplified and overexpressed at the mRNA level, we observed that the protein is highly expressed and predominantly nuclear and that nuclear but not cytoplasmic Ect2 increases with advanced disease. Knockdown of Ect2 in ovarian cancer cell lines impaired their anchorage-independent growth without affecting their growth on plastic. Restoration of Ect2 expression rescued the anchorage-independent growth defect, but not if either the DH catalytic domain or the nuclear localization sequences of Ect2 were mutated. These results suggested a novel mechanism whereby Ect2 could drive transformation in ovarian cancer cells by acting as a RhoGEF specifically within the nucleus. Interestingly, Ect2 had an intrinsically distinct GTPase specificity profile in the nucleus versus the cytoplasm. Nuclear Ect2 bound preferentially to Rac1, while cytoplasmic Ect2 bound to RhoA but not Rac. Consistent with nuclear activation of endogenous Rac, Ect2 overexpression was sufficient to recruit Rac effectors to the nucleus, a process that required a functional Ect2 catalytic domain. Furthermore, expression of active nuclearly targeted Rac1 rescued the defect in transformed growth caused by Ect2 knockdown. Our work suggests a novel mechanism of Ect2-driven transformation, identifies subcellular localization as a regulator of GEF specificity, and implicates activation of nuclear Rac1 in cellular transformation. PMID:24386507

Distinct Sequence Elements of Cyclin B1 Promote Localization to Chromatin, Centrosomes, and Kinetochores during Mitosis

PubMed Central

Bentley, Anna M.; Normand, Guillaume; Hoyt, Jonathan

2007-01-01

The mitotic cyclins promote cell division by binding and activating cyclin-dependent kinases (CDKs). Each cyclin has a unique pattern of subcellular localization that plays a vital role in regulating cell division. During mitosis, cyclin B1 is known to localize to centrosomes, microtubules, and chromatin. To determine the mechanisms of cyclin B1 localization in M phase, we imaged full-length and mutant versions of human cyclin B1-enhanced green fluorescent protein in live cells by using spinning disk confocal microscopy. In addition to centrosome, microtubule, and chromatin localization, we found that cyclin B1 also localizes to unattached kinetochores after nuclear envelope breakdown. Kinetochore recruitment of cyclin B1 required the kinetochore proteins Hec1 and Mad2, and it was stimulated by microtubule destabilization. Mutagenesis studies revealed that cyclin B1 is recruited to kinetochores through both CDK1-dependent and -independent mechanisms. In contrast, localization of cyclin B1 to chromatin and centrosomes is independent of CDK1 binding. The N-terminal domain of cyclin B1 is necessary and sufficient for chromatin association, whereas centrosome recruitment relies on sequences within the cyclin box. Our data support a role for cyclin B1 function at unattached kinetochores, and they demonstrate that separable and distinct sequence elements target cyclin B1 to kinetochores, chromatin, and centrosomes during mitosis. PMID:17881737

Triangulating the provenance of African elephants using mitochondrial DNA

PubMed Central

Ishida, Yasuko; Georgiadis, Nicholas J; Hondo, Tomoko; Roca, Alfred L

2013-01-01

African elephant mitochondrial (mt) DNA follows a distinctive evolutionary trajectory. As females do not migrate between elephant herds, mtDNA exhibits low geographic dispersal. We therefore examined the effectiveness of mtDNA for assigning the provenance of African elephants (or their ivory). For 653 savanna and forest elephants from 22 localities in 13 countries, 4258 bp of mtDNA was sequenced. We detected eight mtDNA subclades, of which seven had regionally restricted distributions. Among 108 unique haplotypes identified, 72% were found at only one locality and 84% were country specific, while 44% of individuals carried a haplotype detected only at their sampling locality. We combined 316 bp of our control region sequences with those generated by previous trans-national surveys of African elephants. Among 101 unique control region haplotypes detected in African elephants across 81 locations in 22 countries, 62% were present in only a single country. Applying our mtDNA results to a previous microsatellite-based assignment study would improve estimates of the provenance of elephants in 115 of 122 mis-assigned cases. Nuclear partitioning followed species boundaries and not mtDNA subclade boundaries. For taxa such as elephants in which nuclear and mtDNA markers differ in phylogeography, combining the two markers can triangulate the origins of confiscated wildlife products. PMID:23798975

Vaccinia-related kinase 3 (VRK3) sets the circadian period and amplitude by affecting the subcellular localization of clock proteins in mammalian cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Nayoung; Department of Brain Science, Ajou University School of Medicine, 164 Worldcup-ro, Yeongtong-gu, Suwon, Kyunggi-do, 16499; Song, Jieun

In the eukaryotic circadian clock machinery, negative feedback repression of CLOCK (CLK) and BMAL1 transcriptional activity by PERIOD (PER) and CRYPTOCHROME (CRY) underlies the basis for 24 h rhythmic gene expression. Thus, precise regulation of the time-dependent nuclear entry of circadian repressors is crucial to generating normal circadian rhythms. Here, we sought to identify novel kinase(s) that regulate nuclear entry of mammalian CRY1 (mCRY1) with an unbiased screening using red fluorescent protein (RFP)-tagged human kinome expression plasmids in mammalian cells. Transient expression of human vaccinia-related kinase 3 (hVRK3) reduced the nuclear presence of mCRY1. hVRK3 expression also induced alterations in themore » subcellular localization of other core clock proteins, including mCRY2, mPER2, and BMAL1. In contrast, the subcellular localization of mCLK was not changed. Given that singly expressed mCLK mostly resides in the cytoplasm and that nuclear localization sequence (NLS) mutation of hVRK3 attenuated the effect of hVRK3 co-expression on subcellular localization, ectopically expressed hVRK3 presumably reduces the retention of proteins in the nucleus. Finally, downregulation of hvrk3 using siRNA reduced the amplitude and lengthened the period of the cellular bioluminescence rhythm. Taken together, these data suggest that VRK3 plays a role in setting the amplitude and period length of circadian rhythms in mammalian cells. - Highlights: • Screening was performed to identify kinases that regulate CRY1 subcellular localization. • VRK3 alters the subcellular localization of CRY1, CRY2, PER2, and BMAL1. • VRK3 knock-down alters the circadian bioluminescence rhythm in mammalian cells.« less

Mitochondrial and nuclear DNA analysis revealed a cryptic species and genetic introgression in Littorina sitkana (Mollusca, Gastropoda).

PubMed

Azuma, Noriko; Yamazaki, Tomoyasu; Chiba, Susumu

2011-12-01

We investigated mitochondrial and nuclear DNA genotypes in nominal Littorina sitkana samples from 2 localities in Eastern Hokkaido, northern Japan. Our results indicated the existence of cryptic species. In the analysis of partial mitochondrial Cytchrome b gene sequences, haplotypes of L. sitkana samples were monophyletic in a phylogenetic tree with orthologous sequences from other Littorina species, but were apparently separated in 2 clades. One included typical L. sitkana (CBa clade) samples, which formed a clade with an allopatric species, L. horikawai. The other, CBb, was independent from CBa and L. horikawai. Haplotypes of the mitochondrial 16S rRNA gene also separated into 2 clades. We additionally examined intron sequence of the heat shock cognate 70 (HSC70) nuclear gene and identified 17 haplotypes. These were also separated into 2 clades, HSCa and HSCb. Among the examined Hokkaido samples, 60% of individuals were heterozygotes. However, each heterozygote consisted of haplotypes from the same clade, HSCa or HSCb, and no admixture of HSCa and HSCb haplotypes was observed. These results indicate reproductive isolation between the 2 clades. Among the genotyped Hokkaido samples, 93% of individuals had CBa + HSCa or CBb + HSCb genotypes, and 7% had CBb + HSCa genotypes. The discrepancy between the mtDNA and nuclear DNA haplotypes in a few individuals may have been caused by genetic introgression due to past hybridization.

Jaw1/LRMP has a role in maintaining nuclear shape via interaction with SUN proteins.

PubMed

Kozono, Takuma; Tadahira, Kazuko; Okumura, Wataru; Itai, Nao; Tamura-Nakano, Miwa; Dohi, Taeko; Tonozuka, Takashi; Nishikawa, Atsushi

2018-06-06

Jaw1/LRMP is characterized as a type II integral membrane protein that is localized to endoplasmic reticulum (ER), however, its physiological functions have been poorly understood. An alignment of amino acid sequence of Jaw1 with KASH proteins, outer nuclear membrane proteins, revealed that Jaw1 has a partial homology to the KASH domain. Here, we show that the function of Jaw1 is to maintain nuclear shape in mouse melanoma cell line. The siRNA-mediated knockdown of Jaw1 caused a severe defect in nuclear shape, and the defect was rescued by ectopic expression of siRNA-resistant Jaw1. Since co-immunoprecipitation assay indicates that Jaw1 interacts with SUN proteins that are inner nuclear proteins and microtubules, this study suggests that Jaw1 has a role in maintaining nuclear shape via interactions with SUN proteins and microtubules.

The 21.5-kDa isoform of myelin basic protein has a non-traditional PY-nuclear-localization signal

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Graham S.T.; Seymour, Lauren V.; Boggs, Joan M.

2012-06-15

Highlights: Black-Right-Pointing-Pointer Full-length 21.5-kDa MBP isoform is translocated to the nucleus. Black-Right-Pointing-Pointer We hypothesized that the exon-II-encoded sequence contained the NLS. Black-Right-Pointing-Pointer We mutated this sequence in RFP-tagged constructs and transfected N19-cells. Black-Right-Pointing-Pointer Abolition of two key positively-charged residues resulted in loss of nuclear-trafficking. Black-Right-Pointing-Pointer The 21.5-kDa isoform of classic MBP contains a non-traditional PY-NLS. -- Abstract: The predominant 18.5-kDa classic myelin basic protein (MBP) is mainly responsible for compaction of the myelin sheath in the central nervous system, but is multifunctional, having numerous interactions with Ca{sup 2+}-calmodulin, actin, tubulin, and SH3-domains, and can tether these proteins to a lipidmore » membrane in vitro. The full-length 21.5-kDa MBP isoform has an additional 26 residues encoded by exon-II of the classic gene, which causes it to be trafficked to the nucleus of oligodendrocytes (OLGs). We have performed site-directed mutagenesis of selected residues within this segment in red fluorescent protein (RFP)-tagged constructs, which were then transfected into the immortalized N19-OLG cell line to view protein localization using epifluorescence microscopy. We found that 21.5-kDa MBP contains two non-traditional PY-nuclear-localization signals, and that arginine and lysine residues within these motifs were involved in subcellular trafficking of this protein to the nucleus, where it may have functional roles during myelinogenesis.« less

Nuclear localization signal targeting to macronucleus and micronucleus in binucleated ciliate Tetrahymena thermophila.

PubMed

Iwamoto, Masaaki; Mori, Chie; Osakada, Hiroko; Koujin, Takako; Hiraoka, Yasushi; Haraguchi, Tokuko

2018-06-08

Ciliated protozoa possess two morphologically and functionally distinct nuclei: a macronucleus (MAC) and a micronucleus (MIC). The MAC is transcriptionally active and functions in all cellular events. The MIC is transcriptionally inactive during cell growth, but functions in meiotic events to produce progeny nuclei. Thus, these two nuclei must be distinguished by the nuclear proteins required for their distinct functions during cellular events such as cell proliferation and meiosis. To understand the mechanism of the nuclear transport specific to either MAC or MIC, we identified specific nuclear localization signals (NLSs) in two MAC- and MIC-specific nuclear proteins, macronuclear histone H1 and micronuclear linker histone-like protein (Mlh1), respectively. By expressing GFP-fused fragments of these proteins in Tetrahymena thermophila cells, two distinct regions in macronuclear histone H1 protein were assigned as independent MAC-specific NLSs and two distinct regions in Mlh1 protein were assigned as independent MIC-specific NLSs. These NLSs contain several essential lysine residues responsible for the MAC- and MIC-specific nuclear transport, but neither contains any consensus sequence with known monopartite or bipartite NLSs in other model organisms. Our findings contribute to understanding how specific nuclear targeting is achieved to perform distinct nuclear functions in binucleated ciliates. © 2018 The Authors. Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Synthesis and characterization of Her2-NLP peptide conjugates targeting circulating breast cancer cells: cellular uptake and localization by fluorescent microscopic imaging.

PubMed

Cai, Huawei; Singh, Ajay N; Sun, Xiankai; Peng, Fangyu

2015-01-01

To synthesize a fluorescent Her2-NLP peptide conjugate consisting of Her2/neu targeting peptide and nuclear localization sequence peptide (NLP) and assess its cellular uptake and intracellular localization for radionuclide cancer therapy targeting Her2/neu-positive circulating breast cancer cells (CBCC). Fluorescent Cy5.5 Her2-NLP peptide conjugate was synthesized by coupling a bivalent peptide sequence, which consisted of a Her2-binding peptide (NH2-GSGKCCYSL) and an NLP peptide (CGYGPKKKRKVGG) linked by a polyethylene glycol (PEG) chain with 6 repeating units, with an activated Cy5.5 ester. The conjugate was separated and purified by HPLC and then characterized by Maldi-MS. The intracellular localization of fluorescent Cy5.5 Her2-NLP peptide conjugate was assessed by fluorescent microscopic imaging using a confocal microscope after incubation of Cy5.5-Her2-NLP with Her2/neu positive breast cancer cells and Her2/neu negative control breast cancer cells, respectively. Fluorescent signals were detected in cytoplasm of Her2/neu positive breast cancer cells (SKBR-3 and BT474 cell lines), but not or little in cytoplasm of Her2/neu negative breast cancer cells (MDA-MB-231), after incubation of the breast cancer cells with Cy5.5-Her2-NLP conjugates in vitro. No fluorescent signals were detected within the nuclei of Her2/neu positive SKBR-3 and BT474 breast cancer cells, neither Her2/neu negative MDA-MB-231 cells, incubated with the Cy5.5-Her2-NLP peptide conjugates, suggesting poor nuclear localization of the Cy5.5-Her2-NLP conjugates localized within the cytoplasm after their cellular uptake and internalization by the Her2/neu positive breast cancer cells. Her2-binding peptide (KCCYSL) is a promising agent for radionuclide therapy of Her2/neu positive breast cancer using a β(-) or α emitting radionuclide, but poor nuclear localization of the Her2-NLP peptide conjugates may limit its use for eradication of Her2/neu-positive CBCC using I-125 or other Auger electron emitting radionuclide.

Thysanophora penicillioides includes multiple genetically diverged groups that coexist respectively in Abies mariesii forests in Japan.

PubMed

Iwamoto, Susumu; Tokumasu, Seiji; Suyama, Yoshihisa; Kakishima, Makoto

2005-01-01

We investigated intraspecific diversity and genetic structures of a saprotrophic fungus--Thysanophora penicillioides--based on sequences of nuclear ribosomal internal transcribed spacer (ITS) in 15 discontinuous Abies mariesii forests of Japan. In such a well-defined morphological species, numerous unexpected ITS variations were revealed: 12 ITS sequence types detected in 254 isolates collected from 15 local populations were classified into five ITS sequence groups. Maximally, four ITS groups consisted of seven ITS types coexisting in one population. However, group 1 was dominant with approximately 65%; in particular, one haplotype, 1a, was most dominant with approximately 60% in respective populations. Therefore, few differences were recognized in genetic structure among local populations, implying that the gene flow of each lineage of the fungus occurs among local populations without geographic limitations. However, minor haplotypes in some ITS groups were found only in restricted areas, suggesting that they might expand steadily from their places of origin to neighboring A. mariesii forests. Aggregating sequence data of seven European strains and four North American strains from various substrates to those of Japanese strains, 18 ITS sequence types and 28 variable sites were recognized. They were clustered into nine lineages by phylogenetic analyses of the beta-tubulin and combined ITS and beta-tubulin datasets. According to phylogenetic species recognition by the concordance of genealogies, respective lineages correspond to phylogenetic species. Plural phylogenetic species coexist in a local population in an A. mariesii forest in Japan.

The 87-kD A gamma-globin enhancer-binding protein is a product of the HOXB2(HOX2H) locus.

PubMed

Sengupta, P K; Lavelle, D E; DeSimone, J

1994-03-01

Developmental regulation of globin gene expression may be controlled by developmental stage-specific nuclear proteins that influence interactions between the locus control region and local regulatory sequences near individual globin genes. We previously isolated an 87-kD nuclear protein from K562 cells that bound to DNA sequences in the beta-globin locus control region, gamma-globin promoter, and A gamma-globin enhancer. The presence of this protein in fetal globin-expressing cells and its absence in adult globin-expressing cells suggested that it may be a developmental stage-specific factor. A lambda gt11 K562 cDNA clone encoding a portion of the HOXB2 (formerly HOX2H) homeobox gene was isolated on the basis of the ability of its beta-galactosidase fusion protein to bind to the same DNA sequences as the 87-kD K562 protein. Because no other relationship had been established between the 87-kD K562 protein and the HOXB2 protein other than their ability to bind ot the same DNA sequences, we have investigated whether the two proteins are related antigenically. Our data show that antisera produced against the HOXB2-beta-gal fusion protein and a synthetic HOXB2 decapeptide react specifically with an 87-kD protein from K562 nuclear extract, showing that the 87-kD K562 nuclear protein is a product of the HOXB2 locus, and is the first demonstration of cellular HOXB2 protein.

The carboxyl-terminus directs TAF(I)48 to the nucleus and nucleolus and associates with multiple nuclear import receptors.

PubMed

Dynes, Joseph L; Xu, Shuping; Bothner, Sarah; Lahti, Jill M; Hori, Roderick T

2004-03-01

The protein complex Selectivity Factor 1, composed of TBP, TAF(I)48, TAF(I)63 and TAF(I)110, is required for rRNA transcription by RNA polymerase I in the nucleolus. The steps involved in targeting Selectivity Factor 1 will be dependent on the transport pathways that are used and the localization signals that direct this trafficking. In order to investigate these issues, we characterized human TAF(I)48, a subunit of Selectivity Factor 1. By domain analysis of TAF(I)48, the carboxyl-terminal 51 residues were found to be required for the localization of TAF(I)48, as well as sufficient to direct Green Fluorescent Protein to the nucleus and nucleolus. The carboxyl-terminus of TAF(I)48 also has the ability to associate with multiple members of the beta-karyopherin family of nuclear import receptors, including importin beta (karyopherin beta1), transportin (karyopherin beta2) and RanBP5 (karyopherin beta3), in a Ran-dependent manner. This property of interacting with multiple beta-karyopherins has been previously reported for the nuclear localization signals of some ribosomal proteins that are likewise directed to the nucleolus. This study identifies the first nuclear import sequence identified within the TBP-Associated Factor subunits of Selectivity Factor 1.

Insulin-induced translocation of IR to the nucleus in insulin responsive cells requires a nuclear translocation sequence.

PubMed

Kesten, Dov; Horovitz-Fried, Miriam; Brutman-Barazani, Tamar; Sampson, Sanford R

2018-04-01

Insulin binding to its cell surface receptor (IR) activates a cascade of events leading to its biological effects. The Insulin-IR complex is rapidly internalized and then is either recycled back to the plasma membrane or sent to lysosomes for degradation. Although most of the receptor is recycled or degraded, a small amount may escape this pathway and migrate to the nucleus of the cell where it might be important in promulgation of receptor signals. In this study we explored the mechanism by which insulin induces IR translocation to the cell nucleus. Experiments were performed cultured L6 myoblasts, AML liver cells and 3T3-L1 adipocytes. Insulin treatment induced a rapid increase in nuclear IR protein levels within 2 to 5 min. Treatment with WGA, an inhibitor of nuclear import, reduced insulin-induced increases nuclear IR protein; IR was, however, translocated to a perinuclear location. Bioinformatics tools predicted a potential nuclear localization sequence (NLS) on IR. Immunofluorescence staining showed that a point mutation on the predicted NLS blocked insulin-induced IR nuclear translocation. In addition, blockade of nuclear IR activation in isolated nuclei by an IR blocking antibody abrogated insulin-induced increases in IR tyrosine phosphorylation and nuclear PKCδ levels. Furthermore, over expression of mutated IR reduced insulin-induced glucose uptake and PKB phosphorylation. When added to isolated nuclei, insulin induced IR phosphorylation but had no effect on nuclear IR protein levels. These results raise questions regarding the possible role of nuclear IR in IR signaling and insulin resistance. Copyright © 2018 Elsevier B.V. All rights reserved.

Constitutive nuclear lamina-genome interactions are highly conserved and associated with A/T-rich sequence.

PubMed

Meuleman, Wouter; Peric-Hupkes, Daan; Kind, Jop; Beaudry, Jean-Bernard; Pagie, Ludo; Kellis, Manolis; Reinders, Marcel; Wessels, Lodewyk; van Steensel, Bas

2013-02-01

In metazoans, the nuclear lamina is thought to play an important role in the spatial organization of interphase chromosomes, by providing anchoring sites for large genomic segments named lamina-associated domains (LADs). Some of these LADs are cell-type specific, while many others appear constitutively associated with the lamina. Constitutive LADs (cLADs) may contribute to a basal chromosome architecture. By comparison of mouse and human lamina interaction maps, we find that the sizes and genomic positions of cLADs are strongly conserved. Moreover, cLADs are depleted of synteny breakpoints, pointing to evolutionary selective pressure to keep cLADs intact. Paradoxically, the overall sequence conservation is low for cLADs. Instead, cLADs are universally characterized by long stretches of DNA of high A/T content. Cell-type specific LADs also tend to adhere to this "A/T rule" in embryonic stem cells, but not in differentiated cells. This suggests that the A/T rule represents a default positioning mechanism that is locally overruled during lineage commitment. Analysis of paralogs suggests that during evolution changes in A/T content have driven the relocation of genes to and from the nuclear lamina, in tight association with changes in expression level. Taken together, these results reveal that the spatial organization of mammalian genomes is highly conserved and tightly linked to local nucleotide composition.

Initial Genomics of the Human Nucleolus

PubMed Central

Németh, Attila; Conesa, Ana; Santoyo-Lopez, Javier; Medina, Ignacio; Montaner, David; Péterfia, Bálint; Solovei, Irina; Cremer, Thomas; Dopazo, Joaquin; Längst, Gernot

2010-01-01

We report for the first time the genomics of a nuclear compartment of the eukaryotic cell. 454 sequencing and microarray analysis revealed the pattern of nucleolus-associated chromatin domains (NADs) in the linear human genome and identified different gene families and certain satellite repeats as the major building blocks of NADs, which constitute about 4% of the genome. Bioinformatic evaluation showed that NAD–localized genes take part in specific biological processes, like the response to other organisms, odor perception, and tissue development. 3D FISH and immunofluorescence experiments illustrated the spatial distribution of NAD–specific chromatin within interphase nuclei and its alteration upon transcriptional changes. Altogether, our findings describe the nature of DNA sequences associated with the human nucleolus and provide insights into the function of the nucleolus in genome organization and establishment of nuclear architecture. PMID:20361057

Host and viral determinants for MxB restriction of HIV-1 infection.

PubMed

Matreyek, Kenneth A; Wang, Weifeng; Serrao, Erik; Singh, Parmit Kumar; Levin, Henry L; Engelman, Alan

2014-10-25

Interferon-induced cellular proteins play important roles in the host response against viral infection. The Mx family of dynamin-like GTPases, which include MxA and MxB, target a wide variety of viruses. Despite considerable evidence demonstrating the breadth of antiviral activity of MxA, human MxB was only recently discovered to specifically inhibit lentiviruses. Here we assess both host and viral determinants that underlie MxB restriction of HIV-1 infection. Heterologous expression of MxB in human osteosarcoma cells potently inhibited HIV-1 infection (~12-fold), yet had little to no effect on divergent retroviruses. The anti-HIV effect manifested as a partial block in the formation of 2-long terminal repeat circle DNA and hence nuclear import, and we accordingly found evidence for an additional post-nuclear entry block. A large number of previously characterized capsid mutations, as well as mutations that abrogated integrase activity, counteracted MxB restriction. MxB expression suppressed integration into gene-enriched regions of chromosomes, similar to affects observed previously when cells were depleted for nuclear transport factors such as transportin 3. MxB activity did not require predicted GTPase active site residues or a series of unstructured loops within the stalk domain that confer functional oligomerization to related dynamin family proteins. In contrast, we observed an N-terminal stretch of residues in MxB to harbor key determinants. Protein localization conferred by a nuclear localization signal (NLS) within the N-terminal 25 residues, which was critical, was fully rescuable by a heterologous NLS. Consistent with this observation, a heterologous nuclear export sequence (NES) abolished full-length MxB activity. We additionally mapped sub-regions within amino acids 26-90 that contribute to MxB activity, finding sequences present within residues 27-50 particularly important. MxB inhibits HIV-1 by interfering with minimally two steps of infection, nuclear entry and post-nuclear trafficking and/or integration, without destabilizing the inherent catalytic activity of viral preintegration complexes. Putative MxB GTPase active site residues and stalk domain Loop 4 -- both previously shown to be necessary for MxA function -- were dispensable for MxB antiviral activity. Instead, we highlight subcellular localization and a yet-determined function(s) present in the unique MxB N-terminal region to be required for HIV-1 restriction.

Molecular characterization of Fasciola gigantica from Mauritania based on mitochondrial and nuclear ribosomal DNA sequences.

PubMed

Amor, Nabil; Farjallah, Sarra; Salem, Mohamed; Lamine, Dia Mamadou; Merella, Paolo; Said, Khaled; Ben Slimane, Badreddine

2011-10-01

Fasciolosis caused by Fasciola hepatica and Fasciola gigantica (Platyhelminthes: Trematoda: Digenea) is considered the most important helminth infection of ruminants in tropical countries, causing considerable socioeconomic problems. From Africa, F. gigantica has been previously characterized from Burkina Faso, Senegal, Kenya, Zambia and Mali, while F. hepatica has been reported from Morocco and Tunisia, and both species have been observed from Ethiopia and Egypt on the basis of morphometric differences, while the use of molecular markers is necessary to distinguish exactly between species. Samples identified morphologically as F. gigantica (n=60) from sheep and cattle from different geographical localities of Mauritania were genetically characterized by sequences of the first (ITS-1), the 5.8S, and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) genes and the mitochondrial Cytochrome c Oxidase I (COI) gene. Comparison of the sequences of the Mauritanian samples with sequences of Fasciola spp. from GenBank confirmed that all samples belong to the species F. gigantica. The nucleotide sequencing of ITS rDNA of F. gigantica showed no nucleotide variation in the ITS-1, 5.8S, and ITS-2 rDNA sequences among all samples examined and those from Burkina Faso, Kenya, Egypt and Iran. The phylogenetic trees based on the ITS-1 and ITS-2 sequences showed a close relationship of the Mauritanian samples with isolates of F. gigantica from different localities of Africa and Asia. The COI genotypes of the Mauritanian specimens of F. gigantica had a high level of diversity, and they belonged to the F. gigantica phylogenically distinguishable clade. The present study is the first molecular characterization of F. gigantica in sheep and cattle from Mauritania, allowing a reliable approach for the genetic differentiation of Fasciola spp. and providing basis for further studies on liver flukes in the African countries. Copyright © 2011 Elsevier Inc. All rights reserved.

Prevalence and Identity of Taenia multiceps cysts "Coenurus cerebralis" in Sheep in Egypt.

PubMed

Amer, Said; ElKhatam, Ahmed; Fukuda, Yasuhiro; Bakr, Lamia I; Zidan, Shereif; Elsify, Ahmed; Mohamed, Mostafa A; Tada, Chika; Nakai, Yutaka

2017-12-01

Coenurosis is a parasitic disease caused by the larval stage (Coenurus cerebralis) of the canids cestode Taenia multiceps. C. cerebralis particularly infects sheep and goats, and pose a public health concerns. The present study aimed to determine the occurrence and molecular identity of C. cerebralis infecting sheep in Egypt. Infection rate was determined by postmortem inspection of heads of the cases that showed neurological manifestations. Species identification and genetic diversity were analyzed based on PCR-sequence analysis of nuclear ITS1 and mitochondrial cytochrome oxidase (COI) and nicotinamide adenine dinucleotide dehydrogenase (ND1) gene markers. Out of 3668 animals distributed in 50 herds at localities of Ashmoun and El Sadat cities, El Menoufia Province, Egypt, 420 (11.45%) sheep showed neurological disorders. Postmortem examination of these animals after slaughter at local abattoirs indicated to occurrence of C. cerebralis cysts in the brain of 111 out of 420 (26.4%), with overall infection rate 3.03% of the involved sheep population. Molecular analysis of representative samples of coenuri at ITS1 gene marker showed extensive intra- and inter-sequence diversity due to deletions/insertions in the microsatellite regions. On contrast to the nuclear gene marker, considerably low genetic diversity was seen in the analyzed mitochondrial gene markers. Phylogenetic analysis based on COI and ND1 gene sequences indicated that the generated sequences in the present study and the reference sequences in the database clustered in 4 haplogroups, with more or less similar topologies. Clustering pattern of the phylogenetic tree showed no effect for the geographic location or the host species. Copyright © 2017 Elsevier B.V. All rights reserved.

DNA mismatch repair proteins MLH1 and PMS2 can be imported to the nucleus by a classical nuclear import pathway.

PubMed

de Barros, Andrea C; Takeda, Agnes A S; Dreyer, Thiago R; Velazquez-Campoy, Adrian; Kobe, Boštjan; Fontes, Marcos R M

2018-03-01

MLH1 and PMS2 proteins form the MutLα heterodimer, which plays a major role in DNA mismatch repair (MMR) in humans. Mutations in MMR-related proteins are associated with cancer, especially with colon cancer. The N-terminal region of MutLα comprises the N-termini of PMS2 and MLH1 and, similarly, the C-terminal region of MutLα is composed by the C-termini of PMS2 and MLH1, and the two are connected by linker region. The nuclear localization sequences (NLSs) necessary for the nuclear transport of the two proteins are found in this linker region. However, the exact NLS sequences have been controversial, with different sequences reported, particularly for MLH1. The individual components are not imported efficiently, presumably due to their C-termini masking their NLSs. In order to gain insights into the nuclear transport of these proteins, we solved the crystal structures of importin-α bound to peptides corresponding to the supposed NLSs of MLH1 and PMS2 and performed isothermal titration calorimetry to study their binding affinities. Both putative MLH1 and PMS2 NLSs can bind to importin-α as monopartite NLSs, which is in agreement with some previous studies. However, MLH1-NLS has the highest affinity measured by a natural NLS peptide, suggesting a major role of MLH1 protein in nuclear import compared to PMS2. Finally, the role of MLH1 and PMS2 in the nuclear transport of the MutLα heterodimer is discussed. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Identification of candidates for interacting partners of the tail domain of DcNMCP1, a major component of the Daucus carota nuclear lamina-like structure.

PubMed

Mochizuki, Ryota; Tsugama, Daisuke; Yamazaki, Michihiro; Fujino, Kaien; Masuda, Kiyoshi

2017-05-04

NMCP/CRWN (NUCLEAR MATRIX CONSTITUENT PROTEIN/CROWDED NUCLEI) is a major component of a protein fibrous meshwork (lamina-like structure) on the plant inner nuclear membrane. NMCP/CRWN contributes to regulating nuclear shape and nuclear functions. An NMCP/CRWN protein in Daucus carota (DcNMCP1) is localized to the nuclear periphery in interphase cells, and surrounds chromosomes in cells in metaphase and anaphase. The N-terminal region and the C-terminal region of DcNMCP1 are both necessary for localizing DcNMCP1 to the nuclear periphery. Here candidate interacting partners of the amino acid position 975-1053 of DcNMCP1 (T975-1053), which is present in the C-terminal region and contains a conserved sequence that plays a role in localizing DcNMCP1 to the nuclear periphery, are screened for. Arabidopsis thaliana nuclear proteins were subjected to far-Western blotting with GST-fused T975-1053 as a probe, and signals were detected at the positions corresponding to ∼70, ∼40, and ∼18 kDa. These ∼70, ∼40, and ∼18 kDa nuclear proteins were identified by mass spectrometry, and subjected to a yeast 2-hybrid (Y2H) analysis with T975-1053 as bait. In this analysis, the ∼40 kDa protein ARP7, which is a nuclear actin-related protein possibly involved in regulating chromatin structures, was confirmed to interact with T975-1053. Independently of the far-Western blotting, a Y2H screen was performed using T975-1053 as bait. Targeted Y2H assays confirmed that 3 proteins identified in the screen, MYB3, SINAT1, and BIM1, interact with T975-1053. These proteins might have roles in NMCP/CRWN protein-mediated biologic processes.

The evolutionary fate of the chloroplast and nuclear rps16 genes as revealed through the sequencing and comparative analyses of four novel legume chloroplast genomes from Lupinus

PubMed Central

Keller, J.; Rousseau-Gueutin, M.; Martin, G.E.; Morice, J.; Boutte, J.; Coissac, E.; Ourari, M.; Aïnouche, M.; Salmon, A.; Cabello-Hurtado, F.

2017-01-01

Abstract The Fabaceae family is considered as a model system for understanding chloroplast genome evolution due to the presence of extensive structural rearrangements, gene losses and localized hypermutable regions. Here, we provide sequences of four chloroplast genomes from the Lupinus genus, belonging to the underinvestigated Genistoid clade. Notably, we found in Lupinus species the functional loss of the essential rps16 gene, which was most likely replaced by the nuclear rps16 gene that encodes chloroplast and mitochondrion targeted RPS16 proteins. To study the evolutionary fate of the rps16 gene, we explored all available plant chloroplast, mitochondrial and nuclear genomes. Whereas no plant mitochondrial genomes carry an rps16 gene, many plants still have a functional nuclear and chloroplast rps16 gene. Ka/Ks ratios revealed that both chloroplast and nuclear rps16 copies were under purifying selection. However, due to the dual targeting of the nuclear rps16 gene product and the absence of a mitochondrial copy, the chloroplast gene may be lost. We also performed comparative analyses of lupine plastomes (SNPs, indels and repeat elements), identified the most variable regions and examined their phylogenetic utility. The markers identified here will help to reveal the evolutionary history of lupines, Genistoids and closely related clades. PMID:28338826

A Novel Fission Yeast Gene, tht1 +, Is Required for the Fusion of Nuclear Envelopes during Karyogamy

PubMed Central

Tange, Yoshie; Horio, Tetsuya; Shimanuki, Mizuki; Ding, Da-Qiao; Hiraoka, Yasushi; Niwa, Osami

1998-01-01

We have isolated a fission yeast karyogamy mutant, tht1, in which nuclear congression and the association of two spindle pole bodies occurs but the subsequent fusion of nuclear envelopes is blocked. The tht1 mutation does not prevent meiosis, so cells execute meiosis with two unfused nuclei, leading to the production of aberrant asci. The tht1 + gene was cloned and sequenced. Predicted amino acid sequence has no significant homology to previously known proteins but strongly suggests that it is a type I membrane protein. The tht1 + gene is dispensable for vegetative growth and expressed only in conjugating cells. Tht1p is a glycoprotein susceptible to endoglycosilase H digestion. Site- directed mutagenesis showed that the N-glycosylation site, as well as the COOH-terminal region of Tht1p, is essential for its function. A protease protection assay indicated that the COOH terminus is cytoplasmic. Immunocytological analysis using a HA-tagged Tht1p suggested that the protein is localized in nuclear envelopes and in the ER during karyogamy and that its levels are reduced in cells containing fused nuclei. PMID:9442101

Comparative study of photothermolysis of cancer cells with nuclear-targeted or cytoplasm-targeted gold nanospheres: continuous wave or pulsed lasers

NASA Astrophysics Data System (ADS)

Huang, Xiaohua; Kang, Bin; Qian, Wei; Mackey, Megan A.; Chen, Po C.; Oyelere, Adegboyega K.; El-Sayed, Ivan H.; El-Sayed, Mostafa A.

2010-09-01

We conduct a comparative study on the efficiency and cell death pathways of continuous wave (cw) and nanosecond pulsed laser photothermal cancer therapy using gold nanospheres delivered to either the cytoplasm or nucleus of cancer cells. Cytoplasm localization is achieved using arginine-glycine-aspartate peptide modified gold nanospheres, which target integrin receptors on the cell surface and are subsequently internalized by the cells. Nuclear delivery is achieved by conjugating the gold nanospheres with nuclear localization sequence peptides originating from the simian virus. Photothermal experiments show that cell death can be induced with a single pulse of a nanosecond laser more efficiently than with a cw laser. When the cw laser is applied, gold nanospheres localized in the cytoplasm are more effective in inducing cell destruction than gold nanospheres localized at the nucleus. The opposite effect is observed when the nanosecond pulsed laser is used, suggesting that plasmonic field enhancement of the nonlinear absorption processes occurs at high localization of gold nanospheres at the nucleus. Cell death pathways are further investigated via a standard apoptosis kit to show that the cell death mechanisms depend on the type of laser used. While the cw laser induces cell death via apoptosis, the nanosecond pulsed laser leads to cell necrosis. These studies add mechanistic insight to gold nanoparticle-based photothermal therapy of cancer.

Nuclear counterparts of the cytoplasmic mitochondrial 12S rRNA gene: a problem of ancient DNA and molecular phylogenies.

PubMed

van der Kuyl, A C; Kuiken, C L; Dekker, J T; Perizonius, W R; Goudsmit, J

1995-06-01

Monkey mummy bones and teeth originating from the North Saqqara Baboon Galleries (Egypt), soft tissue from a mummified baboon in a museum collection, and nineteenth/twentieth-century skin fragments from mangabeys were used for DNA extraction and PCR amplification of part of the mitochondrial 12S rRNA gene. Sequences aligning with the 12S rRNA gene were recovered but were only distantly related to contemporary monkey mitochondrial 12S rRNA sequences. However, many of these sequences were identical or closely related to human nuclear DNA sequences resembling mitochondrial 12S rRNA (isolated from a cell line depleted in mitochondria) and therefore have to be considered contamination. Subsequently in a separate study we were able to recover genuine mitochondrial 12S rRNA sequences from many extant species of nonhuman Old World primates and sequences closely resembling the human nuclear integrations. Analysis of all sequences by the neighbor-joining (NJ) method indicated that mitochondrial DNA sequences and their nuclear counterparts can be divided into two distinct clusters. One cluster contained all temporary cytoplasmic mitochondrial DNA sequences and approximately half of the monkey nuclear mitochondriallike sequences. A second cluster contained most human nuclear sequences and the other half of monkey nuclear sequences with a separate branch leading to human and gorilla mitochondrial and nuclear sequences. Sequences recovered from ancient materials were equally divided between the two clusters. These results constitute a warning for when working with ancient DNA or performing phylogenetic analysis using mitochondrial DNA as a target sequence: Nuclear counterparts of mitochondrial genes may lead to faulty interpretation of results.

The role of nuclear localization signal in parvovirus life cycle.

PubMed

Liu, Peng; Chen, Shun; Wang, Mingshu; Cheng, Anchun

2017-04-14

Parvoviruses are small, non-enveloped viruses with an approximately 5.0 kb, single-stranded DNA genome. Usually, the parvovirus capsid gene contains one or more nuclear localization signals (NLSs), which are required for guiding the virus particle into the nucleus through the nuclear pore. However, several classical NLSs (cNLSs) and non-classical NLSs (ncNLSs) have been identified in non-structural genes, and the ncNLSs can also target non-structural proteins into the nucleus. In this review, we have summarized recent research findings on parvovirus NLSs. The capsid protein of the adeno-associated virus has four potential nuclear localization sequences, named basic region 1 (BR), BR2, BR3 and BR4. BR3 was identified as an NLS by fusing it with green fluorescent protein. Moreover, BR3 and BR4 are required for infectivity and virion assembly. In Protoparvovirus, the canine parvovirus has a common cNLS located in the VP1 unique region, similar to parvovirus minute virus of mice (MVM) and porcine parvovirus. Moreover, an ncNLS is found in the C-terminal region of MVM VP1/2. Parvovirus B19 also contains an ncNLS in the C-terminal region of VP1/2, which is essential for the nuclear transport of VP1/VP2. Approximately 1 or 2 cNLSs and 1 ncNLS have been reported in the non-structural protein of bocaviruses. Understanding the role of the NLS in the process of parvovirus infection and its mechanism of nuclear transport will contribute to the development of therapeutic vaccines and novel antiviral medicines.

Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins.

PubMed

Jong, A Y; Clark, M W; Gilbert, M; Oehm, A; Campbell, J L

1987-08-01

To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly homologous to a peptide in calf thymus UP1 and UP2 and a human heterogeneous nuclear ribonucleoprotein. Residues 125 to 162 constitute a fivefold tandem repeat of the sequence RGGFRG, the composition of which suggests a nucleic acid-binding site. Near the C terminus, residues 233 to 245 are homologous to several RNA-binding proteins. Of 18 C-terminal residues, 10 are acidic, a characteristic of the procaryotic single-stranded DNA-binding proteins and eucaryotic DNA- and RNA-binding proteins. In addition, examination of the subcellular distribution of SSB1 by immunofluorescence microscopy indicated that SSB1 is a nuclear protein, predominantly located in the nucleolus. Sequence homologies and the nucleolar localization make it likely that SSB1 functions in RNA metabolism in vivo, although an additional role in DNA metabolism cannot be excluded.

Microsporidia, amitochondrial protists, possess a 70-kDa heat shock protein gene of mitochondrial evolutionary origin.

PubMed

Peyretaillade, E; Broussolle, V; Peyret, P; Méténier, G; Gouy, M; Vivarès, C P

1998-06-01

An intronless gene encoding a protein of 592 amino acid residues with similarity to 70-kDa heat shock proteins (HSP70s) has been cloned and sequenced from the amitochondrial protist Encephalitozoon cuniculi (phylum Microsporidia). Southern blot analyses show the presence of a single gene copy located on chromosome XI. The encoded protein exhibits an N-terminal hydrophobic leader sequence and two motifs shared by proteobacterial and mitochondrially expressed HSP70 homologs. Phylogenetic analysis using maximum likelihood and evolutionary distances place the E. cuniculi sequence in the cluster of mitochondrially expressed HSP70s, with a higher evolutionary rate than those of homologous sequences. Similar results were obtained after cloning a fragment of the homologous gene in the closely related species E. hellem. The presence of a nuclear targeting signal-like sequence supports a role of the Encephalitozoon HSP70 as a molecular chaperone of nuclear proteins. No evidence for cytosolic or endoplasmic reticulum forms of HSP70 was obtained through PCR amplification. These data suggest that Encephalitozoon species have evolved from an ancestor bearing mitochondria, which is in disagreement with the postulated presymbiotic origin of Microsporidia. The specific role and intracellular localization of the mitochondrial HSP70-like protein remain to be elucidated.

Detection of PIWI and piRNAs in the mitochondria of mammalian cancer cells.

PubMed

Kwon, ChangHyuk; Tak, Hyosun; Rho, Mina; Chang, Hae Ryung; Kim, Yon Hui; Kim, Kyung Tae; Balch, Curt; Lee, Eun Kyung; Nam, Seungyoon

2014-03-28

Piwi-interacting RNAs (piRNAs) are 26-31 nt small noncoding RNAs that are processed from their longer precursor transcripts by Piwi proteins. Localization of Piwi and piRNA has been reported mostly in nucleus and cytoplasm of higher eukaryotes germ-line cells, where it is believed that known piRNA sequences are located in repeat regions of nuclear genome in germ-line cells. However, localization of PIWI and piRNA in mammalian somatic cell mitochondria yet remains largely unknown. We identified 29 piRNA sequence alignments from various regions of the human mitochondrial genome. Twelve out 29 piRNA sequences matched stem-loop fragment sequences of seven distinct tRNAs. We observed their actual expression in mitochondria subcellular fractions by inspecting mitochondrial-specific small RNA-Seq datasets. Of interest, the majority of the 29 piRNAs overlapped with multiple longer transcripts (expressed sequence tags) that are unique to the human mitochondrial genome. The presence of mature piRNAs in mitochondria was detected by qRT-PCR of mitochondrial subcellular RNAs. Further validation showed detection of Piwi by colocalization using anti-Piwil1 and mitochondria organelle-specific protein antibodies. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Isolation with differentiation followed by expansion with admixture in the tunicate Pyura chilensis

PubMed Central

2013-01-01

Background Pyura chilensis, a tunicate commercially exploited as food resource in Chile, is subject to management strategies, including restocking. The goal of this study was to examine the genetic structure of P. chilensis using information from a mitochondrial gene (Cytochrome Oxidase I, COI) and a nuclear gene (Elongation 1 alpha, EF1a), to characterize the geographic distribution of genetic diversity and differentiation, and to identify the main processes that have shaped it. We analyzed 268 and 208 sequences of COI and EF1a, respectively, from samples of eight local populations covering ca. 1800 km. Results For Pyura chilensis, partial sequences of the gene COI revealed three highly supported haplogroups that diverged 260000 to 470000 years ago. Two haplogroups currently are widely distributed and sympatric, while one is dominant only in Los Molinos (LM, 39°50′S). The two widespread COI haplogroups underwent a geographic expansion during an interglacial period of the Late Pleistocene ca. 100000 years ago. The nuclear gene was less divergent and did not resolve the COI haplogroups. Bayesian clustering of the nuclear gene’s SNPs revealed that individuals from the two widespread COI haplogroups were mostly assigned to two of the three detected clusters and had a marked degree of admixture. The third cluster predominated in LM and showed low admixture. Haplotypic diversity of both genes was very high, there was no isolation by distance, and most localities were genetically undifferentiated; only LM was consistently differentiated with both genes analyzed. Conclusions Pyura chilensis has less genetic structure than expected given its life history, which could be a consequence of dispersal on ship hulls. The only differentiated local population analyzed was LM. Coincidentally, it is the one furthest away from main maritime routes along the coast of Chile. The use of mitochondrial and nuclear markers allowed detection of divergent mitochondrial haplogroups in P. chilensis, two of which revealed nuclear admixture. The genetic structure of P. chilensis has likely been shaped by Pleistocene’s climatic effect on sea level leading to population contraction with isolation, followed by geographic range expansions with concomitant secondary contact and admixture. PMID:24238017

Asparagine 326 in the extremely C-terminal region of XRCC4 is essential for the cell survival after irradiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wanotayan, Rujira; Fukuchi, Mikoto; Imamichi, Shoji

2015-02-20

XRCC4 is one of the crucial proteins in the repair of DNA double-strand break (DSB) through non-homologous end-joining (NHEJ). As XRCC4 consists of 336 amino acids, N-terminal 200 amino acids include domains for dimerization and for association with DNA ligase IV and XLF and shown to be essential for XRCC4 function in DSB repair and V(D)J recombination. On the other hand, the role of the remaining C-terminal region of XRCC4 is not well understood. In the present study, we noticed that a stretch of ∼20 amino acids located at the extreme C-terminus of XRCC4 is highly conserved among vertebrate species.more » To explore its possible importance, series of mutants in this region were constructed and assessed for the functionality in terms of ability to rescue radiosensitivity of M10 cells lacking XRCC4. Among 13 mutants, M10 transfectant with N326L mutant (M10-XRCC4{sup N326L}) showed elevated radiosensitivity. N326L protein showed defective nuclear localization. N326L sequence matched the consensus sequence of nuclear export signal. Leptomycin B treatment accumulated XRCC4{sup N326L} in the nucleus but only partially rescued radiosensitivity of M10-XRCC4{sup N326L}. These results collectively indicated that the functional defects of XRCC4{sup N326L} might be partially, but not solely, due to its exclusion from nucleus by synthetic nuclear export signal. Further mutation of XRCC4 Asn326 to other amino acids, i.e., alanine, aspartic acid or glutamine did not affect the nuclear localization but still exhibited radiosensitivity. The present results indicated the importance of the extremely C-terminal region of XRCC4 and, especially, Asn326 therein. - Highlights: • Extremely C-terminal region of XRCC4 is highly conserved among vertebrate species. • XRCC4 C-terminal point mutants, R325F and N326L, are functionally deficient in terms of survival after irradiation. • N326L localizes to the cytoplasm because of synthetic nuclear export signal. • Leptomycin B restores the nuclear localization of N326L but only partially reverses radiosensitivity. • Other N326 mutants (N326A, N326D and N326Q) are functionally deficient in terms of survival after irradiation.« less

Tunneling induced electron transfer between separated protons

NASA Astrophysics Data System (ADS)

Vindel-Zandbergen, Patricia; Meier, Christoph; Sola, Ignacio R.

2018-04-01

We study electron transfer between two separated protons using local control theory. In this symmetric system one can favour a slow transfer by biasing the algorithm, achieving high efficiencies for fixed nuclei. The solution can be parametrized using a sequence of a pump followed by a dump pulse that lead to tunneling-induced electron transfer. Finally, we study the effect of the nuclear kinetic energy on the efficiency. Even in the absence of relative motion between the protons, the spreading of the nuclear wave function is enough to reduce the yield of electronic transfer to less than one half.

AtLAZY1 is a signaling component required for gravitropism of the Arabidopsis thaliana inflorescence.

PubMed

Yoshihara, Takeshi; Spalding, Edgar P; Iino, Moritoshi

2013-04-01

The present study identified a family of six A. thaliana genes that share five limited regions of sequence similarity with LAZY1, a gene in Oryza sativa (rice) shown to participate in the early gravity signaling for shoot gravitropism. A T-DNA insertion into the Arabidopsis gene (At5g14090) most similar to LAZY1 increased the inflorescence branch angle to 81° from the wild type value of 42°. RNA interference lines and molecular rescue experiments confirmed the linkage between the branch-angle phenotype and the gene consequently named AtLAZY1. Time-resolved gravitropism measurements of atlazy1 hypocotyls and primary inflorescence stems showed a significantly reduced bending rate during the first hour of response. The subcellular localization of AtLAZY1 protein was investigated to determine if the nuclear localization predicted from the gene sequence was observable and important to its function in shoot gravity responses. AtLAZY1 fused to green fluorescent protein largely rescued the branch-angle phenotype of atlazy1, and was observed by confocal microscopy at the cell periphery and within the nucleus. Mutation of the nuclear localization signal prevented detectable levels of AtLAZY1 in the nucleus without affecting the ability of the gene to rescue the atlazy1 branch-angle phenotype. These results indicate that AtLAZY1 functions in gravity signaling during shoot gravitropism, being a functional ortholog of rice LAZY1. The nuclear pool of the protein appears to be unnecessary for this function, which instead relies on a pool that appears to reside at the cell periphery. © 2013 The Authors The Plant Journal © 2013 Blackwell Publishing Ltd.

Importin α-importin β complex mediated nuclear translocation of insulin-like growth factor binding protein-5.

PubMed

Sun, Min; Long, Juan; Yi, Yuxin; Xia, Wei

2017-10-28

Insulin-like growth factor-binding protein (IGFBP)-5 is a secreted protein that binds to IGFs and modulates IGF actions, as well as regulates cell proliferation, migration, and apoptosis independent of IGF. Proper cellular localization is critical for the effective function of most signaling molecules. In previous studies, we have shown that the nuclear IGFBP-5 comes from ER-cytosol retro-translocation. In this study, we further investigated the pathway mediating IGFBP-5 nuclear import after it retro-translocation. Importin-α5 was identified as an IGFBP-5-interacting protein with a yeast two-hybrid system, and its interaction with IGFBP-5 was further confirmed by GST pull down and co-immunoprecipitation. Binding affinity of IGFBP-5 and importins were determined by surface plasmon resonance (IGFBP-5/importin-β: K D =2.44e-7, IGFBP-5/importin-α5: K D =3.4e-7). Blocking the importin-α5/importin-β nuclear import pathway using SiRNA or dominant negative impotin-β dramatically inhibited IGFBP-5-EGFP nuclear import, though importin-α5 overexpress does not affect IGFBP-5 nuclear import. Furthermore, nuclear IGFBP-5 was quantified using luciferase report assay. When deleted the IGFBP-5 nuclear localization sequence (NLS), IGFBP-5 ΔNLS loss the ability to translocate into the nucleus and accumulation of IGFBP-5 ΔNLS was visualized in the cytosol. Altogether, our findings provide a substantially evidence showed that the IGFBP-5 nuclear import is mediated by importin-α/importin-β complex, and NLS is critical domain in IGFBP-5 nuclear translocation.

A Method of Trajectory Design for Manned Asteroids Exploration

NASA Astrophysics Data System (ADS)

Gan, Q. B.; Zhang, Y.; Zhu, Z. F.; Han, W. H.; Dong, X.

2014-11-01

A trajectory optimization method of the nuclear propulsion manned asteroids exploration is presented. In the case of launching between 2035 and 2065, based on the Lambert transfer orbit, the phases of departure from and return to the Earth are searched at first. Then the optimal flight trajectory in the feasible regions is selected by pruning the flight sequences. Setting the nuclear propulsion flight plan as propel-coast-propel, and taking the minimal mass of aircraft departure as the index, the nuclear propulsion flight trajectory is separately optimized using a hybrid method. With the initial value of the optimized local parameters of each three phases, the global parameters are jointedly optimized. At last, the minimal departure mass trajectory design result is given.

Genetic Structure and Potential Environmental Determinants of Local Genetic Diversity in Japanese Honeybees (Apis cerana japonica)

PubMed Central

Nagamitsu, Teruyoshi; Yasuda, Mika; Saito-Morooka, Fuki; Inoue, Maki N.; Nishiyama, Mio; Goka, Koichi; Sugiura, Shinji; Maeto, Kaoru; Okabe, Kimiko; Taki, Hisatomo

2016-01-01

Declines in honeybee populations have been a recent concern. Although causes of the declines remain unclear, environmental factors may be responsible. We focused on the potential environmental determinants of local populations of wild honeybees, Apis cerana japonica, in Japan. This subspecies has little genetic variation in terms of its mitochondrial DNA sequences, and genetic variations at nuclear loci are as yet unknown. We estimated the genetic structure and environmental determinants of local genetic diversity in nuclear microsatellite genotypes of fathers and mothers, inferred from workers collected at 139 sites. The genotypes of fathers and mothers showed weak isolation by distance and negligible genetic structure. The local genetic diversity was high in central Japan, decreasing toward the peripheries, and depended on the climate and land use characteristics of the sites. The local genetic diversity decreased as the annual precipitation increased, and increased as the proportion of urban and paddy field areas increased. Positive effects of natural forest area, which have also been observed in terms of forager abundance in farms, were not detected with respect to the local genetic diversity. The findings suggest that A. cerana japonica forms a single population connected by gene flow in its main distributional range, and that climate and landscape properties potentially affect its local genetic diversity. PMID:27898704

Facile rhenium-peptide conjugate synthesis using a one-pot derived Re(CO)3 reagent.

PubMed

Chanawanno, Kullapa; Kondeti, Vinay; Caporoso, Joel; Paruchuri, Sailaja; Leeper, Thomas C; Herrick, Richard S; Ziegler, Christopher J

2016-03-21

We have synthesized two Re(CO)3-modified lysine complexes (1 and 2), where the metal is attached to the amino acid at the Nε position, via a one-pot Schiff base formation reaction. These compounds can be used in the solid phase synthesis of peptides, and to date we have produced four conjugate systems incorporating neurotensin, bombesin, leutenizing hormone releasing hormone, and a nuclear localization sequence. We observed uptake into human umbilical vascular endothelial cells as well as differential uptake depending on peptide sequence identity, as characterized by fluorescence and rhenium elemental analysis.

Molecular characterization of Fasciola spp. from the endemic area of northern Iran based on nuclear ribosomal DNA sequences.

PubMed

Amor, Nabil; Halajian, Ali; Farjallah, Sarra; Merella, Paolo; Said, Khaled; Ben Slimane, Badreddine

2011-07-01

Fasciolosis caused by Fasciola spp. (Platyhelminthes: Trematoda: Digenea) is considered as the most important helminth infection of ruminants in tropical countries, causing considerable socioeconomic problems. In the endemic regions of the North of Iran, Fasciola hepatica and Fasciola gigantica have been previously characterized on the basis of morphometric differences, but the use of molecular markers is necessary to distinguish exactly between species and intermediate forms. Samples from buffaloes and goats from different localities of northern Iran were identified morphologically and then genetically characterized by sequences of the first (ITS-1) and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA). Comparison of the ITS of the northern Iranian samples with sequences of Fasciola spp. from GenBank showed that the examined specimens had sequences identical to those of the most frequent haplotypes of F. hepatica (n=25, 48.1%) and F. gigantica (n=20, 38.45%), which differed from each other in different variable nucleotide positions of ITS region sequences, and their intermediate forms (n=7, 13.45%), which had nucleotides overlapped between the two Fasciola species in all the positions. The ITS sequences from populations of Fasciola isolates in buffaloes and goats had experienced introgression/hybridization as previously reported in isolates from other ruminants and humans. Based on ITS-1 and ITS-2 sequences, flukes are scattered in pure F. hepatica, F. gigantica and intermediate Fasciola clades, revealing that multiple genotypes of Fasciola are able to infect goats and buffaloes in North of Iran. Furthermore, the phylogenetic trees based upon the ITS-1 and ITS-2 sequences showed a close relationship of the Iranian samples with isolates of F. hepatica and F. gigantica from different localities of Africa and Asia. In the present study, the intergenic transcribed spacers ITS-1 and ITS-2 showed to be reliable approaches for the genetic differentiation of Fasciola spp., providing bases for further studies on F. hepatica, F. gigantica and their intermediate forms in the endemic areas in Asia. Copyright © 2011 Elsevier Inc. All rights reserved.

A dual role of p53 in the control of autophagy.

PubMed

Tasdemir, Ezgi; Chiara Maiuri, M; Morselli, Eugenia; Criollo, Alfredo; D'Amelio, Marcello; Djavaheri-Mergny, Mojgan; Cecconi, Francesco; Tavernarakis, Nektarios; Kroemer, Guido

2008-08-01

Genotoxic stress can induce autophagy in a p53-dependent fashion and p53 can transactivate autophagy-inducing genes. We have observed recently that inactivation of p53 by deletion, depletion or inhibition can trigger autophagy. Thus, human and mouse cells subjected to knockout, knockdown or pharmacological inhibition of p53 manifest signs of autophagy such as depletion of p62/SQSTM1, LC3 lipidation, redistribution of GFP-LC3 in cytoplasmic puncta, and accumulation of autophagosomes and autolysosomes, both in vitro and in vivo. Inhibition of p53 causes autophagy in enucleated cells, indicating that the cytoplasmic, non-nuclear pool of p53 can regulate autophagy. Accordingly, retransfection of p53(-/-) cells with wild-type p53 as well as a p53 mutant that is excluded from the nucleus (due to the deletion of the nuclear localization sequence) can inhibit autophagy, whereas retransfection with a nucleus-restricted p53 mutant (in which the nuclear localization sequence has been deleted) does not inhibit autophagy. Several distinct autophagy inducers (e.g., starvation, rapamycin, lithium, tunicamycin and thapsigargin) stimulate the rapid degradation of p53. In these conditions, inhibition of the p53-specific E3 ubiquitin ligase HDM2 can avoid p53 depletion and simultaneously prevent the activation of autophagy. Moreover, a p53 mutant that lacks the HDM2 ubiquitinylation site and hence is more stable than wild-type p53 is particularly efficient in suppressing autophagy. In conclusion, p53 plays a dual role in the control of autophagy. On the one hand, nuclear p53 can induce autophagy through transcriptional effects. On the other hand, cytoplasmic p53 may act as a master repressor of autophagy.

The Enamovirus P0 protein is a silencing suppressor which inhibits local and systemic RNA silencing through AGO1 degradation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fusaro, Adriana F.; CSIRO Plant Industry, Canberra, P.O. Box 1600, ACT 2601; Correa, Regis L.

The P0 protein of poleroviruses and P1 protein of sobemoviruses suppress the plant's RNA silencing machinery. Here we identified a silencing suppressor protein (SSP), P0{sup PE}, in the Enamovirus Pea enation mosaic virus-1 (PEMV-1) and showed that it and the P0s of poleroviruses Potato leaf roll virus and Cereal yellow dwarf virus have strong local and systemic SSP activity, while the P1 of Sobemovirus Southern bean mosaic virus supresses systemic silencing. The nuclear localized P0{sup PE} has no discernable sequence conservation with known SSPs, but proved to be a strong suppressor of local silencing and a moderate suppressor of systemicmore » silencing. Like the P0s from poleroviruses, P0{sup PE} destabilizes AGO1 and this action is mediated by an F-box-like domain. Therefore, despite the lack of any sequence similarity, the poleroviral and enamoviral SSPs have a conserved mode of action upon the RNA silencing machinery.« less

The evolutionary fate of the chloroplast and nuclear rps16 genes as revealed through the sequencing and comparative analyses of four novel legume chloroplast genomes from Lupinus.

PubMed

Keller, J; Rousseau-Gueutin, M; Martin, G E; Morice, J; Boutte, J; Coissac, E; Ourari, M; Aïnouche, M; Salmon, A; Cabello-Hurtado, F; Aïnouche, A

2017-08-01

The Fabaceae family is considered as a model system for understanding chloroplast genome evolution due to the presence of extensive structural rearrangements, gene losses and localized hypermutable regions. Here, we provide sequences of four chloroplast genomes from the Lupinus genus, belonging to the underinvestigated Genistoid clade. Notably, we found in Lupinus species the functional loss of the essential rps16 gene, which was most likely replaced by the nuclear rps16 gene that encodes chloroplast and mitochondrion targeted RPS16 proteins. To study the evolutionary fate of the rps16 gene, we explored all available plant chloroplast, mitochondrial and nuclear genomes. Whereas no plant mitochondrial genomes carry an rps16 gene, many plants still have a functional nuclear and chloroplast rps16 gene. Ka/Ks ratios revealed that both chloroplast and nuclear rps16 copies were under purifying selection. However, due to the dual targeting of the nuclear rps16 gene product and the absence of a mitochondrial copy, the chloroplast gene may be lost. We also performed comparative analyses of lupine plastomes (SNPs, indels and repeat elements), identified the most variable regions and examined their phylogenetic utility. The markers identified here will help to reveal the evolutionary history of lupines, Genistoids and closely related clades. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

The fanconi anemia proteins FANCA and FANCG stabilize each other and promote the nuclear accumulation of the Fanconi anemia complex.

PubMed

Garcia-Higuera, I; Kuang, Y; Denham, J; D'Andrea, A D

2000-11-01

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with 8 complementation groups. Four of the FA genes have been cloned, and at least 3 of the encoded proteins, FANCA, FANCC, and FANCG/XRCC9, interact in a multisubunit protein complex. The FANCG protein binds directly to the amino terminal nuclear localization sequence (NLS) of FANCA, suggesting that FANCG plays a role in regulating FANCA nuclear accumulation. In the current study the functional consequences of FANCG/FANCA binding were examined. Correction of an FA-G cell line with the FANCG complementary DNA (cDNA) resulted in FANCA/FANCG binding, prolongation of the cellular half-life of FANCA, and an increase in the nuclear accumulation of the FA protein complex. Similar results were obtained upon correction of an FA-A cell line, with a reciprocal increase in the half-life of FANCG. Patient-derived mutant forms of FANCA, containing an intact NLS sequence but point mutations in the carboxy-terminal leucine zipper region, bound FANCG in the cytoplasm. The mutant forms failed to translocate to the nucleus of transduced cells, thereby suggesting a model of coordinated binding and nuclear translocation. These results demonstrate that the FANCA/FANCG interaction is required to maintain the cellular levels of both proteins. Moreover, at least one function of FANCG and FANCA is to regulate the nuclear accumulation of the FA protein complex. Failure to accumulate the nuclear FA protein complex results in the characteristic spectrum of clinical and cellular abnormalities observed in FA.

A protein that interacts with members of the nuclear hormone receptor family: identification and cDNA cloning.

PubMed Central

Zeiner, M; Gehring, U

1995-01-01

In search of proteins which interact with activated steroid hormone receptors, we screened a human liver lambda gt11 expression library with the glucocorticoid receptor. We identified and cloned a cDNA sequence of 1322 bp that encodes a protein of 274 aa. This protein consists predominantly of hydrophilic amino acids and contains a putative bipartite nuclear localization signal. The in vitro translated receptor-associating protein runs in SDS/polyacrylamide gels with an apparent molecular mass of 46 kDa. By use of the bacterially expressed fusion protein with glutathione S-transferase we have found that interaction is not limited to the glucocorticoid receptor but included other nuclear receptors--most notably, the estrogen and thyroid receptors. Binding also occurs with the glucocorticoid receptor complexed with the antiglucocorticoid RU 38486, with the estrogen receptor complexed with the antiestrogen 4-hydroxytamoxifen or ICI 164,384, and even with receptors not complexed with ligand. Association with steroid hormone receptors depends on prior receptor activation--i.e., release from heat shock proteins. The sequence identified here appears to be a general partner protein for nuclear hormone receptors, with the gene being expressed in a variety of mammalian tissues. Images Fig. 2 Fig. 3 Fig. 4 PMID:8524784

A phylogenetic study of Laeliinae (Orchidaceae) based on combined nuclear and plastid DNA sequences

PubMed Central

van den Berg, Cássio; Higgins, Wesley E.; Dressler, Robert L.; Whitten, W. Mark; Soto-Arenas, Miguel A.; Chase, Mark W.

2009-01-01

Background and Aims Laeliinae are a neotropical orchid subtribe with approx. 1500 species in 50 genera. In this study, an attempt is made to assess generic alliances based on molecular phylogenetic analysis of DNA sequence data. Methods Six DNA datasets were gathered: plastid trnL intron, trnL-F spacer, matK gene and trnK introns upstream and dowstream from matK and nuclear ITS rDNA. Data were analysed with maximum parsimony (MP) and Bayesian analysis with mixed models (BA). Key Results Although relationships between Laeliinae and outgroups are well supported, within the subtribe sequence variation is low considering the broad taxonomic range covered. Localized incongruence between the ITS and plastid trees was found. A combined tree followed the ITS trees more closely, but the levels of support obtained with MP were low. The Bayesian analysis recovered more well-supported nodes. The trees from combined MP and BA allowed eight generic alliances to be recognized within Laeliinae, all of which show trends in morphological characters but lack unambiguous synapomorphies. Conclusions By using combined plastid and nuclear DNA data in conjunction with mixed-models Bayesian inference, it is possible to delimit smaller groups within Laeliinae and discuss general patterns of pollination and hybridization compatibility. Furthermore, these small groups can now be used for further detailed studies to explain morphological evolution and diversification patterns within the subtribe. PMID:19423551

Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins.

PubMed Central

Jong, A Y; Clark, M W; Gilbert, M; Oehm, A; Campbell, J L

1987-01-01

To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly homologous to a peptide in calf thymus UP1 and UP2 and a human heterogeneous nuclear ribonucleoprotein. Residues 125 to 162 constitute a fivefold tandem repeat of the sequence RGGFRG, the composition of which suggests a nucleic acid-binding site. Near the C terminus, residues 233 to 245 are homologous to several RNA-binding proteins. Of 18 C-terminal residues, 10 are acidic, a characteristic of the procaryotic single-stranded DNA-binding proteins and eucaryotic DNA- and RNA-binding proteins. In addition, examination of the subcellular distribution of SSB1 by immunofluorescence microscopy indicated that SSB1 is a nuclear protein, predominantly located in the nucleolus. Sequence homologies and the nucleolar localization make it likely that SSB1 functions in RNA metabolism in vivo, although an additional role in DNA metabolism cannot be excluded. Images PMID:2823109

Regulation of NT-PGC-1alpha subcellular localization and function by protein kinase A-dependent modulation of nuclear export by CRM1.

PubMed

Chang, Ji Suk; Huypens, Peter; Zhang, Yubin; Black, Chelsea; Kralli, Anastasia; Gettys, Thomas W

2010-06-04

Peroxisome proliferator-activated receptor gamma co-activator-1alpha (PGC-1alpha) plays a central role in the regulation of cellular energy metabolism and metabolic adaptation to environmental and nutritional stimuli. We recently described a novel, biologically active splice variant of PGC-1alpha (NT-PGC-1alpha, amino acids 1-270) that retains the ability to interact with and transactivate nuclear hormone receptors through its N-terminal transactivation domain. Whereas PGC-1alpha is an unstable nuclear protein sensitive to ubiquitin-mediated targeting to the proteasome, NT-PGC-1alpha is relatively stable and predominantly cytoplasmic, suggesting that its ability to interact with and activate nuclear receptors and transcription factors is dependent upon regulated access to the nucleus. We provide evidence that NT-PGC-1alpha interacts with the nuclear exportin, CRM1, through a specific leucine-rich domain (nuclear export sequence) that regulates its export to the cytoplasm. The nuclear export of NT-PGC-1alpha is inhibited by protein kinase A-dependent phosphorylation of Ser-194, Ser-241, and Thr-256 on NT-PGC-1alpha, which effectively increases its nuclear concentration. Using site-directed mutagenesis to prevent or mimic phosphorylation at these sites, we show that the transcriptional activity of NT-PGC-1alpha is regulated in part through regulation of its subcellular localization. These findings suggest that the function of NT-PGC-1alpha as a transcriptional co-activator is regulated by protein kinase A-dependent inhibition of CRM1-mediated export from the nucleus.

Localization of the putative precursor of Alzheimer's disease-specific amyloid at nuclear envelopes of adult human muscle.

PubMed Central

Zimmermann, K; Herget, T; Salbaum, J M; Schubert, W; Hilbich, C; Cramer, M; Masters, C L; Multhaup, G; Kang, J; Lemaire, H G

1988-01-01

Cloning and sequence analysis revealed the putative amyloid A4 precursor (pre-A4) of Alzheimer's disease to have characteristics of a membrane-spanning glycoprotein. In addition to brain, pre-A4 mRNA was found in adult human muscle and other tissues. We demonstrate by in situ hybridization that pre-A4 mRNA is present in adult human muscle, in cultured human myoblasts and myotubes. Immunofluorescence with antipeptide antibodies shows the putative pre-A4 protein to be expressed in adult human muscle and associated with some but not all nuclear envelopes. Despite high levels of a single 3.5-kb pre-A4 mRNA species in cultured myoblasts and myotubes, the presence of putative pre-A4 protein could not be detected by immunofluorescence. This suggests that putative pre-A4 protein is stabilized and therefore functioning in the innervated muscle tissue but not in developing, i.e. non-innervated cultured muscle cells. The selective localization of the protein on distinct nuclear envelopes could reflect an interaction with motor endplates. Images PMID:2896589

Nup93, a Vertebrate Homologue of Yeast Nic96p, Forms a Complex with a Novel 205-kDa Protein and Is Required for Correct Nuclear Pore Assembly

PubMed Central

Grandi, Paola; Dang, Tam; Pané, Nelly; Shevchenko, Andrej; Mann, Matthias; Forbes, Douglass; Hurt, Ed

1997-01-01

Yeast and vertebrate nuclear pores display significant morphological similarity by electron microscopy, but sequence similarity between the respective proteins has been more difficult to observe. Herein we have identified a vertebrate nucleoporin, Nup93, in both human and Xenopus that has proved to be an evolutionarily related homologue of the yeast nucleoporin Nic96p. Polyclonal antiserum to human Nup93 detects corresponding proteins in human, rat, and Xenopus cells. Immunofluorescence and immunoelectron microscopy localize vertebrate Nup93 at the nuclear basket and at or near the nuclear entry to the gated channel of the pore. Immunoprecipitation from both mammalian and Xenopus cell extracts indicates that a small fraction of Nup93 physically interacts with the nucleoporin p62, just as yeast Nic96p interacts with the yeast p62 homologue. However, a large fraction of vertebrate Nup93 is extracted from pores and is also present in Xenopus egg extracts in complex with a newly discovered 205-kDa protein. Mass spectrometric sequencing of the human 205-kDa protein reveals that this protein is encoded by an open reading frame, KIAAO225, present in the human database. The putative human nucleoporin of 205 kDa has related sequence homologues in Caenorhabditis elegans and Saccharomyces cerevisiae. To analyze the role of the Nup93 complex in the pore, nuclei were assembled that lack the Nup93 complex after immunodepletion of a Xenopus nuclear reconstitution extract. The Nup93-complex–depleted nuclei are clearly defective for correct nuclear pore assembly. From these experiments, we conclude that the vertebrate and yeast pore have significant homology in their functionally important cores and that, with the identification of Nup93 and the 205-kDa protein, we have extended the knowledge of the nearest-neighbor interactions of this core in both yeast and vertebrates. PMID:9348540

Entire plastid phylogeny of the carrot genus (Daucus, Apiaceae):Concordance with nuclear data and mitochondrial and nuclear DNA insertions to the plastid

USDA-ARS?s Scientific Manuscript database

We explored the phylogenetic utility of entire plastid DNA sequences in Daucus and compared the results to prior phylogenetic results using plastid, nuclear, and mitochondrial DNA sequences. We obtained, using Illumina sequencing, full plastid sequences of 37 accessions of 20 Daucus taxa and outgrou...

Nuclear accumulation of the Arabidopsis immune receptor RPS4 is necessary for triggering EDS1-dependent defense.

PubMed

Wirthmueller, Lennart; Zhang, Yan; Jones, Jonathan D G; Parker, Jane E

2007-12-04

Recognition of specific pathogen molecules inside the cell by nucleotide-binding domain and leucine-rich repeat (NB-LRR) receptors constitutes an important layer of innate immunity in plants. Receptor activation triggers host cellular reprogramming involving transcriptional potentiation of basal defenses and localized programmed cell death. The sites and modes of action of NB-LRR receptors are, however, poorly understood. Arabidopsis Toll/Interleukin-1 (TIR) type NB-LRR receptor RPS4 recognizes the bacterial type III effector AvrRps4. We show that epitope-tagged RPS4 expressed under its native regulatory sequences distributes between endomembranes and nuclei in healthy and AvrRps4-triggered tissues. RPS4 accumulation in the nucleus, mediated by a bipartite nuclear localization sequence (NLS) at its C terminus, is necessary for triggering immunity through authentic activation by AvrRps4 in Arabidopsis or as an effector-independent "deregulated" receptor in tobacco. A strikingly conserved feature of TIR-NB-LRR receptors is their recruitment of the nucleocytoplasmic basal-defense regulator EDS1 in resistance to diverse pathogens. We find that EDS1 is an indispensable component of RPS4 signaling and that it functions downstream of RPS4 activation but upstream of RPS4-mediated transcriptional reprogramming in the nucleus.

Identification of the methylation preference region in heterogeneous nuclear ribonucleoprotein K by protein arginine methyltransferase 1 and its implication in regulating nuclear/cytoplasmic distribution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Yuan-I; Hsu, Sheng-Chieh; Chau, Gar-Yang

2011-01-21

Research highlights: {yields} Verifying by direct methylation assay the substrate sites of PRMT1 in the hnRNP K protein. {yields} Identifying the preferred PMRT1 methylation regions in hnRNP K by kinetic analysis. {yields} Linking methylation in regulating nuclear localization of hnRNP K. -- Abstract: Protein arginine methylation plays crucial roles in numerous cellular processes. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multi-functional protein participating in a variety of cellular functions including transcription and RNA processing. HnRNP K is methylated at multiple sites in the glycine- and arginine-rich (RGG) motif. Using various RGG domain deletion mutants of hnRNP K as substrates,more » here we show by direct methylation assay that protein arginine methyltransferase 1 (PRMT1) methylated preferentially in a.a. 280-307 of the RGG motif. Kinetic analysis revealed that deletion of a.a. 280-307, but not a.a. 308-327, significantly inhibited rate of methylation. Importantly, nuclear localization of hnRNP K was significantly impaired in mutant hnRNP K lacking the PRMT1 methylation region or upon pharmacological inhibition of methylation. Together our results identify preferred PRMT1 methylation sequences of hnRNP K by direct methylation assay and implicate a role of arginine methylation in regulating intracellular distribution of hnRNP K.« less

Transportin acts to regulate mitotic assembly events by target binding rather than Ran sequestration

PubMed Central

Bernis, Cyril; Swift-Taylor, Beth; Nord, Matthew; Carmona, Sarah; Chook, Yuh Min; Forbes, Douglass J.

2014-01-01

The nuclear import receptors importin β and transportin play a different role in mitosis: both act phenotypically as spatial regulators to ensure that mitotic spindle, nuclear membrane, and nuclear pore assembly occur exclusively around chromatin. Importin β is known to act by repressing assembly factors in regions distant from chromatin, whereas RanGTP produced on chromatin frees factors from importin β for localized assembly. The mechanism of transportin regulation was unknown. Diametrically opposed models for transportin action are as follows: 1) indirect action by RanGTP sequestration, thus down-regulating release of assembly factors from importin β, and 2) direct action by transportin binding and inhibiting assembly factors. Experiments in Xenopus assembly extracts with M9M, a superaffinity nuclear localization sequence that displaces cargoes bound by transportin, or TLB, a mutant transportin that can bind cargo and RanGTP simultaneously, support direct inhibition. Consistently, simple addition of M9M to mitotic cytosol induces microtubule aster assembly. ELYS and the nucleoporin 107–160 complex, components of mitotic kinetochores and nuclear pores, are blocked from binding to kinetochores in vitro by transportin, a block reversible by M9M. In vivo, 30% of M9M-transfected cells have spindle/cytokinesis defects. We conclude that the cell contains importin β and transportin “global positioning system”or “GPS” pathways that are mechanistically parallel. PMID:24478460

Modular structural elements in the replication origin region of Tetrahymena rDNA.

PubMed Central

Du, C; Sanzgiri, R P; Shaiu, W L; Choi, J K; Hou, Z; Benbow, R M; Dobbs, D L

1995-01-01

Computer analyses of the DNA replication origin region in the amplified rRNA genes of Tetrahymena thermophila identified a potential initiation zone in the 5'NTS [Dobbs, Shaiu and Benbow (1994), Nucleic Acids Res. 22, 2479-2489]. This region consists of a putative DNA unwinding element (DUE) aligned with predicted bent DNA segments, nuclear matrix or scaffold associated region (MAR/SAR) consensus sequences, and other common modular sequence elements previously shown to be clustered in eukaryotic chromosomal origin regions. In this study, two mung bean nuclease-hypersensitive sites in super-coiled plasmid DNA were localized within the major DUE-like element predicted by thermodynamic analyses. Three restriction fragments of the 5'NTS region predicted to contain bent DNA segments exhibited anomalous migration characteristic of bent DNA during electrophoresis on polyacrylamide gels. Restriction fragments containing the 5'NTS region bound Tetrahymena nuclear matrices in an in vitro binding assay, consistent with an association of the replication origin region with the nuclear matrix in vivo. The direct demonstration in a protozoan origin region of elements previously identified in Drosophila, chick and mammalian origin regions suggests that clusters of modular structural elements may be a conserved feature of eukaryotic chromosomal origins of replication. Images PMID:7784181

Plant DNA sequences from feces: potential means for assessing diets of wild primates.

PubMed

Bradley, Brenda J; Stiller, Mathias; Doran-Sheehy, Diane M; Harris, Tara; Chapman, Colin A; Vigilant, Linda; Poinar, Hendrik

2007-06-01

Analyses of plant DNA in feces provides a promising, yet largely unexplored, means of documenting the diets of elusive primates. Here we demonstrate the promise and pitfalls of this approach using DNA extracted from fecal samples of wild western gorillas (Gorilla gorilla) and black and white colobus monkeys (Colobus guereza). From these DNA extracts we amplified, cloned, and sequenced small segments of chloroplast DNA (part of the rbcL gene) and plant nuclear DNA (ITS-2). The obtained sequences were compared to sequences generated from known plant samples and to those in GenBank to identify plant taxa in the feces. With further optimization, this method could provide a basic evaluation of minimum primate dietary diversity even when knowledge of local flora is limited. This approach may find application in studies characterizing the diets of poorly-known, unhabituated primate species or assaying consumer-resource relationships in an ecosystem. (c) 2007 Wiley-Liss, Inc.

A TALE-inspired computational screen for proteins that contain approximate tandem repeats.

PubMed

Perycz, Malgorzata; Krwawicz, Joanna; Bochtler, Matthias

2017-01-01

TAL (transcription activator-like) effectors (TALEs) are bacterial proteins that are secreted from bacteria to plant cells to act as transcriptional activators. TALEs and related proteins (RipTALs, BurrH, MOrTL1 and MOrTL2) contain approximate tandem repeats that differ in conserved positions that define specificity. Using PERL, we screened ~47 million protein sequences for TALE-like architecture characterized by approximate tandem repeats (between 30 and 43 amino acids in length) and sequence variability in conserved positions, without requiring sequence similarity to TALEs. Candidate proteins were scored according to their propensity for nuclear localization, secondary structure, repeat sequence complexity, as well as covariation and predicted structural proximity of variable residues. Biological context was tentatively inferred from co-occurrence of other domains and interactome predictions. Approximate repeats with TALE-like features that merit experimental characterization were found in a protein of chestnut blight fungus, a eukaryotic plant pathogen.

A TALE-inspired computational screen for proteins that contain approximate tandem repeats

PubMed Central

Krwawicz, Joanna

2017-01-01

TAL (transcription activator-like) effectors (TALEs) are bacterial proteins that are secreted from bacteria to plant cells to act as transcriptional activators. TALEs and related proteins (RipTALs, BurrH, MOrTL1 and MOrTL2) contain approximate tandem repeats that differ in conserved positions that define specificity. Using PERL, we screened ~47 million protein sequences for TALE-like architecture characterized by approximate tandem repeats (between 30 and 43 amino acids in length) and sequence variability in conserved positions, without requiring sequence similarity to TALEs. Candidate proteins were scored according to their propensity for nuclear localization, secondary structure, repeat sequence complexity, as well as covariation and predicted structural proximity of variable residues. Biological context was tentatively inferred from co-occurrence of other domains and interactome predictions. Approximate repeats with TALE-like features that merit experimental characterization were found in a protein of chestnut blight fungus, a eukaryotic plant pathogen. PMID:28617832

Exportin Crm1 is repurposed as a docking protein to generate microtubule organizing centers at the nuclear pore.

PubMed

Bao, Xun X; Spanos, Christos; Kojidani, Tomoko; Lynch, Eric M; Rappsilber, Juri; Hiraoka, Yasushi; Haraguchi, Tokuko; Sawin, Kenneth E

2018-05-29

Non-centrosomal microtubule organizing centers (MTOCs) are important for microtubule organization in many cell types. In fission yeast Schizosaccharomyces pombe , the protein Mto1, together with partner protein Mto2 (Mto1/2 complex), recruits the g-tubulin complex to multiple non-centrosomal MTOCs, including the nuclear envelope (NE). Here, we develop a comparative-interactome mass spectrometry approach to determine how Mto1 localizes to the NE. Surprisingly, we find that Mto1, a constitutively cytoplasmic protein, docks at nuclear pore complexes (NPCs), via interaction with exportin Crm1 and cytoplasmic FG-nucleoporin Nup146. Although Mto1 is not a nuclear export cargo, it binds Crm1 via a nuclear export signal-like sequence, and docking requires both Ran in the GTP-bound state and Nup146 FG repeats. In addition to determining the mechanism of MTOC formation at the NE, our results reveal a novel role for Crm1 and the nuclear export machinery in the stable docking of a cytoplasmic protein complex at NPCs. © 2018, Bao et al.

Exportin Crm1 is repurposed as a docking protein to generate microtubule organizing centers at the nuclear pore

PubMed Central

Bao, Xun X; Spanos, Christos; Kojidani, Tomoko; Lynch, Eric M; Rappsilber, Juri; Hiraoka, Yasushi; Haraguchi, Tokuko

2018-01-01

Non-centrosomal microtubule organizing centers (MTOCs) are important for microtubule organization in many cell types. In fission yeast Schizosaccharomyces pombe, the protein Mto1, together with partner protein Mto2 (Mto1/2 complex), recruits the γ-tubulin complex to multiple non-centrosomal MTOCs, including the nuclear envelope (NE). Here, we develop a comparative-interactome mass spectrometry approach to determine how Mto1 localizes to the NE. Surprisingly, we find that Mto1, a constitutively cytoplasmic protein, docks at nuclear pore complexes (NPCs), via interaction with exportin Crm1 and cytoplasmic FG-nucleoporin Nup146. Although Mto1 is not a nuclear export cargo, it binds Crm1 via a nuclear export signal-like sequence, and docking requires both Ran in the GTP-bound state and Nup146 FG repeats. In addition to determining the mechanism of MTOC formation at the NE, our results reveal a novel role for Crm1 and the nuclear export machinery in the stable docking of a cytoplasmic protein complex at NPCs. PMID:29809148

Nuclear and cytoplasmic delivery of lactoferrin in glioma using chitosan nanoparticles: Cellular location dependent-action of lactoferrin.

PubMed

Tammam, Salma N; Azzazy, Hassan M E; Lamprecht, Alf

2018-08-01

Lactoferrin (Lf) exerts anti-cancer effects on glioma, however, the exact mechanism remains unclear. Despite possessing a nuclear localization sequence (NLS), Lf was found to allocate only in the cytoplasm of glioma 261. Lf was therefore loaded into nuclear and cytoplasmic targeted nanoparticles (NPs) to determine whether nuclear delivery of Lf would enhance its anti-cancer effect. Upon treatment with 300 and 800 µg/mL Lf loaded chitosan NPs, nuclear targeted Lf-NPs showed 1.3 and 2.7 folds increase in cell viability, whereas cytoplasmic targeted Lf-NPs at 300 µg/mL decreased cell viability by 0.8 folds in comparison to free Lf and controls. Results suggest that the cytotoxicity of Lf on glioma is attributable to its cytoplasmic allocation. Nuclear delivery of Lf induced cell proliferation rather than cytotoxicity, indicating that the mode of action of Lf in glioma is cell location dependent. This calls for caution about the general use of Lf as an anti-cancer protein. Copyright © 2018. Published by Elsevier B.V.

[Analysis of nuclear localization and signal function of MITF protein predisposing to Warrdenburg syndrome].

PubMed

Zhang, Hua; Feng, Juan; Chen, Hongsheng; Li, Jiada; Luo, Hunjin; Feng, Yong

2015-12-01

To study the role of dysfunction of nuclear localization signals (NLS) of MITF protein in the pathogenesis of Waardenburg syndrome. Eukaryotic expression plasmid pCMV-MITF-Flag was used as a template to generate mutant plasmid pCMV-MITF△NLS-Flag by molecular cloning technique in order to design the mutagenic primers. The UACC903 cells were transfected transiently with MITF and MITF△NLS plasmids, and the luciferase activity assays were performed to determine their impact on the transcriptional activities of target gene tyrosinase (TYR). The oligonucleotide 5'-GAACGAAGAAGAAGATTT-3' was subcloned into pEGFP-N1 to generate recombinant eukaryotic expression plasmid pEGFP-N1-MITF-NLS. The NIH3T3 cells were transfected separately with MITF, MITF△NLS, pEGFP-N1 and pEGFP-N1-NLS plasmids, and their subcellular distribution was observed by immunoflorescence assays. Expression plasmids for the mutant MITF△NLS with loss of core NLS sequence and pEGFP-N1-NLS coupled with MITF△NLS were successfully generated. Compared with the wild-type MITF, MITF△NLS was not able to transactivate the transcriptional activities of promoter TYR and did not affect the normal function of MITF. MITF△NLS was only localized in the cytoplasm and pEGFP-N1 was found in both the cytoplasm and nucleus, whereas pEGFP-N1-NLS was mainly located in the nucleus. This study has confirmed the localization function of NLS sequence 213ERRRRF218 within the MITF protein. Mutant MITF with loss of NLS has failed to transactivate the transcriptional activities of target gene TYR, which can result in melanocyte defects and cause WS.

A Novel Nuclear Trafficking Module Regulates the Nucleocytoplasmic Localization of the Rabies Virus Interferon Antagonist, P Protein*

PubMed Central

Oksayan, Sibil; Wiltzer, Linda; Rowe, Caitlin L.; Blondel, Danielle; Jans, David A.; Moseley, Gregory W.

2012-01-01

Regulated nucleocytoplasmic transport of proteins is central to cellular function and dysfunction during processes such as viral infection. Active protein trafficking into and out of the nucleus is dependent on the presence within cargo proteins of intrinsic specific modular signals for nuclear import (nuclear localization signals, NLSs) and export (nuclear export signals, NESs). Rabies virus (RabV) phospho (P) protein, which is largely responsible for antagonising the host anti-viral response, is expressed as five isoforms (P1–P5). The subcellular trafficking of these isoforms is thought to depend on a balance between the activities of a dominant N-terminal NES (N-NES) and a distinct C-terminal NLS (C-NLS). Specifically, the N-NES-containing isoforms P1 and P2 are cytoplasmic, whereas the shorter P3–P5 isoforms, which lack the N-NES, are believed to be nuclear through the activity of the C-NLS. Here, we show for the first time that RabV P contains an additional strong NLS in the N-terminal region (N-NLS), which, intriguingly, overlaps with the N-NES. This arrangement represents a novel nuclear trafficking module where the N-NLS is inactive in P1 but becomes activated in P3, concomitant with truncation of the N-NES, to become the principal targeting signal conferring nuclear accumulation. Understanding this unique switch arrangement of overlapping, co-regulated NES/NLS sequences is vital to delineating the critical role of RabV P protein in viral infection. PMID:22700958

The autoantigen Ro52 is an E3 ligase resident in the cytoplasm but enters the nucleus upon cellular exposure to nitric oxide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Espinosa, Alexander; Oke, Vilija; Elfving, Ase

2008-12-10

Patients with the systemic autoimmune diseases Sjoegrens's syndrome and systemic lupus erythematosus often have autoantibodies against the intracellular protein Ro52. Ro52 is an E3 ligase dependent on the ubiquitin conjugation enzymes UBE2D1 and UBE2E1. While Ro52 and UBE2D1 are cytoplasmic proteins, UBE2E1 is localized to the nucleus. Here, we investigate how domains of human Ro52 regulate its intracellular localization. By expressing fluorescently labeled Ro52 and Ro52 mutants in HeLa cells, an intact coiled-coil domain was found to be necessary for the cytoplasmic localization of Ro52. The amino acids 381-470 of the B30.2 region were essential for translocation into the nucleus.more » Furthermore, after exposure of HeLa cells to the inflammatory mediator nitric oxide (NO), Ro52 translocated to the nucleus. A nuclear localization of Ro52 in inflamed tissue expressing inducible NO synthetase (iNOS) from cutaneous lupus patients was observed by immunohistochemistry and verified in NO-treated cultures of patient-derived primary keratinocytes. Our results show that the localization of Ro52 is regulated by endogenous sequences, and that nuclear translocation is induced by an inflammatory mediator. This suggests that Ro52 has both cytoplasmic and nuclear substrates, and that Ro52 mediates ubiquitination through UBE2D1 in the cytoplasm and through UBE2E1 in the nucleus.« less

Regulation of mIκBNS stability through PEST-mediated degradation by proteasome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Koog Chan; Jeong, Jiyeong; Kim, Keun Il, E-mail: kikim@sookmyung.ac.kr

2014-01-24

Highlights: • mIκBNS is degraded rapidly by proteasome without ubiquitylation. • N-terminal PEST sequence is responsible for the unstable nature of mIκBNS. • PEST sequence is not critical for nuclear localization of mIκBNS. • There is single bona fide NLS at the C-terminus of mIκBNS. - Abstract: Negative regulatory proteins in a cytokine signaling play a critical role in restricting unwanted excess activation of the signaling pathway. At the same time, negative regulatory proteins need to be removed rapidly from cells to respond properly to the next incoming signal. A nuclear IκB protein called IκBNS is known to inhibit amore » subset of NF-κB target genes upon its expression by NF-κB activation. Here, we show a mechanism to control the stability of mIκBNS which might be important for cells to prepare the next round signaling. We found that mIκBNS is a short-lived protein of which the stability is controlled by proteasome, independent of ubiquitylation process. We identified that the N-terminal PEST sequence in mIκBNS was critical for the regulation of stability.« less

Problem-solving test: Southwestern blotting.

PubMed

Szeberényi, József

2014-01-01

Terms to be familiar with before you start to solve the test: Southern blotting, Western blotting, restriction endonucleases, agarose gel electrophoresis, nitrocellulose filter, molecular hybridization, polyacrylamide gel electrophoresis, proto-oncogene, c-abl, Src-homology domains, tyrosine protein kinase, nuclear localization signal, cDNA, deletion mutants, expression plasmid, transfection, RNA polymerase II, promoter, Shine-Dalgarno sequence, polyadenylation element, affinity chromatography, Northern blotting, immunoprecipitation, sodium dodecylsulfate, autoradiography, tandem repeats. Copyright © 2014 The International Union of Biochemistry and Molecular Biology.

Cloning, expression and nuclear localization of human NPM3, a member of the nucleophosmin/nucleoplasmin family of nuclear chaperones

PubMed Central

Shackleford, Gregory M; Ganguly, Amit; MacArthur, Craig A

2001-01-01

Background Studies suggest that the related proteins nucleoplasmin and nucleophosmin (also called B23, NO38 or numatrin) are nuclear chaperones that mediate the assembly of nucleosomes and ribosomes, respectively, and that these activities are accomplished through the binding of basic proteins via their acidic domains. Recently discovered and less well characterized members of this family of acidic phosphoproteins include mouse nucleophosmin/nucleoplasmin 3 (Npm3) and Xenopus NO29. Here we report the cloning and initial characterization of the human ortholog of Npm3. Results Human genomic and cDNA clones of NPM3 were isolated and sequenced. NPM3 lies 5.5 kb upstream of FGF8 and thus maps to chromosome 10q24-26. In addition to amino acid similarities, NPM3 shares many physical characteristics with the nucleophosmin/nucleoplasmin family, including an acidic domain, multiple potential phosphorylation sites and a putative nuclear localization signal. Comparative analyses of 14 members of this family from various metazoans suggest that Xenopus NO29 is a candidate ortholog of human and mouse NPM3, and they further group both proteins closer with the nucleoplasmins than with the nucleophosmins. Northern blot analysis revealed that NPM3 was strongly expressed in all 16 human tissues examined, with especially robust expression in pancreas and testis; lung displayed the lowest level of expression. An analysis of subcellular fractions of NIH3T3 cells expressing epitope-tagged NPM3 revealed that NPM3 protein was localized solely in the nucleus. Conclusions Human NPM3 is an abundant and widely expressed protein with primarily nuclear localization. These biological activities, together with its physical relationship to the chaparones nucleoplasmin and nucleophosmin, are consistent with the proposed function of NPM3 as a molecular chaperone functioning in the nucleus. PMID:11722795

Phosphorylation and subcellular redistribution of high mobility group proteins 14 and 17, analyzed by mass spectrometry.

PubMed Central

Louie, D. F.; Gloor, K. K.; Galasinski, S. C.; Resing, K. A.; Ahn, N. G.

2000-01-01

High mobility group (HMG) proteins 14 and 17 are nonhistone nuclear proteins that have been implicated in control of transcription and chromatin structure. To examine the posttranslational modifications of HMG-14 and -17 in vivo, HMG proteins were prepared from nuclear vs. cytosolic fractions of human K562 cells treated with 12-O-tetradecanoylphorbol 13-acetate (TPA) or okadaic acid (OA) and examined by electrospray mass spectrometry. Analysis of full-length masses demonstrated mono-, di-, and triphosphorylation of HMG-14 and mono- and diphosphorylation of HMG-17 from OA treated cells, whereas HMG-14 and -17 from TPA treated cells were monophosphorylated. Peptide mass and sequence analysis showed major and minor phosphorylation sites, respectively, at Ser24 and Ser28 in HMG-17, and Ser20 and Ser24 in HMG-14. These sites were found in the consensus sequence RRSARLSAK, within the nucleosomal binding domain of each protein. A third phosphorylation site in HMG-14 was located at either Ser6 or Ser7. Interestingly, the proportion of HMG-14 and -17 found in cytosolic pools increased significantly after 1 h of treatment compared to control cells and showed preferential phosphorylation compared with proteins from nuclear fractions. These results suggest that phosphorylation of HMG-14 and -7 interferes with nuclear localization mechanisms in a manner favoring release from nuclei. PMID:10739259

Phosphorylation and subcellular redistribution of high mobility group proteins 14 and 17, analyzed by mass spectrometry.

PubMed

Louie, D F; Gloor, K K; Galasinski, S C; Resing, K A; Ahn, N G

2000-01-01

High mobility group (HMG) proteins 14 and 17 are nonhistone nuclear proteins that have been implicated in control of transcription and chromatin structure. To examine the posttranslational modifications of HMG-14 and -17 in vivo, HMG proteins were prepared from nuclear vs. cytosolic fractions of human K562 cells treated with 12-O-tetradecanoylphorbol 13-acetate (TPA) or okadaic acid (OA) and examined by electrospray mass spectrometry. Analysis of full-length masses demonstrated mono-, di-, and triphosphorylation of HMG-14 and mono- and diphosphorylation of HMG-17 from OA treated cells, whereas HMG-14 and -17 from TPA treated cells were monophosphorylated. Peptide mass and sequence analysis showed major and minor phosphorylation sites, respectively, at Ser24 and Ser28 in HMG-17, and Ser20 and Ser24 in HMG-14. These sites were found in the consensus sequence RRSARLSAK, within the nucleosomal binding domain of each protein. A third phosphorylation site in HMG-14 was located at either Ser6 or Ser7. Interestingly, the proportion of HMG-14 and -17 found in cytosolic pools increased significantly after 1 h of treatment compared to control cells and showed preferential phosphorylation compared with proteins from nuclear fractions. These results suggest that phosphorylation of HMG-14 and -7 interferes with nuclear localization mechanisms in a manner favoring release from nuclei.

Genomic organization of the human mi-er1 gene and characterization of alternatively spliced isoforms: regulated use of a facultative intron determines subcellular localization.

PubMed

Paterno, Gary D; Ding, Zhihu; Lew, Yuan-Y; Nash, Gord W; Mercer, F Corinne; Gillespie, Laura L

2002-07-24

mi-er1 (previously called er1) is a fibroblast growth factor-inducible early response gene activated during mesoderm induction in Xenopus embryos and encoding a nuclear protein that functions as a transcriptional activator. The human orthologue of mi-er1 was shown to be upregulated in breast carcinoma cell lines and breast tumours when compared to normal breast cells. In this report, we investigate the structure of the human mi-er1 (hmi-er1) gene and characterize the alternatively spliced transcripts and protein isoforms. hmi-er1 is a single copy gene located at 1p31.2 and spanning 63 kb. It contains 17 exons and includes one skipped exon, a facultative intron and three polyadenylation signals to produce 12 transcripts encoding six distinct proteins. hmi-er1 transcripts were expressed at very low levels in most human adult tissues and the mRNA isoform pattern varied with the tissue. The 12 transcripts encode proteins containing a common internal sequence with variable N- and C-termini. Three distinct N- and two distinct C-termini were identified, giving rise to six protein isoforms. The two C-termini differ significantly in size and sequence and arise from alternate use of a facultative intron to produce hMI-ER1alpha and hMI-ER1beta. In all tissues except testis, transcripts encoding the beta isoform were predominant. hMI-ER1alpha lacks the predicted nuclear localization signal and transfection assays revealed that, unlike hMI-ER1beta, it is not a nuclear protein, but remains in the cytoplasm. Our results demonstrate that alternate use of a facultative intron regulates the subcellular localization of hMI-ER1 proteins and this may have important implications for hMI-ER1 function.

Broadband excitation in nuclear magnetic resonance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tycko, Robert

1984-10-01

Theoretical methods for designing sequences of radio frequency (rf) radiation pulses for broadband excitation of spin systems in nuclear magnetic resonance (NMR) are described. The sequences excite spins uniformly over large ranges of resonant frequencies arising from static magnetic field inhomogeneity, chemical shift differences, or spin couplings, or over large ranges of rf field amplitudes. Specific sequences for creating a population inversion or transverse magnetization are derived and demonstrated experimentally in liquid and solid state NMR. One approach to broadband excitation is based on principles of coherent averaging theory. A general formalism for deriving pulse sequences is given, along withmore » computational methods for specific cases. This approach leads to sequences that produce strictly constant transformations of a spin system. The importance of this feature in NMR applications is discussed. A second approach to broadband excitation makes use of iterative schemes, i.e. sets of operations that are applied repetitively to a given initial pulse sequences, generating a series of increasingly complex sequences with increasingly desirable properties. A general mathematical framework for analyzing iterative schemes is developed. An iterative scheme is treated as a function that acts on a space of operators corresponding to the transformations produced by all possible pulse sequences. The fixed points of the function and the stability of the fixed points are shown to determine the essential behavior of the scheme. Iterative schemes for broadband population inversion are treated in detail. Algebraic and numerical methods for performing the mathematical analysis are presented. Two additional topics are treated. The first is the construction of sequences for uniform excitation of double-quantum coherence and for uniform polarization transfer over a range of spin couplings. Double-quantum excitation sequences are demonstrated in a liquid crystal system. The second additional topic is the construction of iterative schemes for narrowband population inversion. The use of sequences that invert spin populations only over a narrow range of rf field amplitudes to spatially localize NMR signals in an rf field gradient is discussed.« less

Chromosomal localization and partial genomic structure of the human peroxisome proliferator activated receptor-gamma (hPPAR gamma) gene.

PubMed

Beamer, B A; Negri, C; Yen, C J; Gavrilova, O; Rumberger, J M; Durcan, M J; Yarnall, D P; Hawkins, A L; Griffin, C A; Burns, D K; Roth, J; Reitman, M; Shuldiner, A R

1997-04-28

We determined the chromosomal localization and partial genomic structure of the coding region of the human PPAR gamma gene (hPPAR gamma), a nuclear receptor important for adipocyte differentiation and function. Sequence analysis and long PCR of human genomic DNA with primers that span putative introns revealed that intron positions and sizes of hPPAR gamma are similar to those previously determined for the mouse PPAR gamma gene[13]. Fluorescent in situ hybridization localized hPPAR gamma to chromosome 3, band 3p25. Radiation hybrid mapping with two independent primer pairs was consistent with hPPAR gamma being within 1.5 Mb of marker D3S1263 on 3p25-p24.2. These sequences of the intron/exon junctions of the 6 coding exons shared by hPPAR gamma 1 and hPPAR gamma 2 will facilitate screening for possible mutations. Furthermore, D3S1263 is a suitable polymorphic marker for linkage analysis to evaluate PPAR gamma's potential contribution to genetic susceptibility to obesity, lipoatrophy, insulin resistance, and diabetes.

Cell cycle dependent intracellular distribution of two spliced isoforms of TCP/ILF3 proteins.

PubMed

Xu, You Hai; Leonova, Tatyana; Grabowski, Gregory A

2003-12-01

TCP80 is an approximately 80kDa mammalian cytoplasmic protein that binds to a set of mRNAs and inhibits their translation in vitro and ex vivo. This protein has high sequence similarity to interleukin-2 enhancer-binding factors (NF90/ILF3) and the M-phase phosphoprotein (MPP4)/DRBP76. A 110kDa immunologic isoform of TCP80/NF90/MPP4/DRBP76, termed TCP110, also is present in cytoplasm and nuclei of many types of cells. A cDNA sequence coding for TCP110 was derived by 5(')RACE. The TCP110 sequence is identical to ILF3. The gene coding for TCP110/ILF3 mapped to human chromosome 19 and the gene organization was analyzed using TCP80 and TCP110/ILF3 cDNA sequences. The TCP/ILF3 gene spans >34.8kb and contains 21 exons. At least one alternatively spliced product involving exons 19-21 exists and predicts the formation of either TCP80 or TCP110/ILF3. However, the functional relationships of TCP80 and TCP110/ILF3 required elucidation. The metabolic turnover rates and subcellular distribution of TCP80 and TCP110/ILF3 during the cell cycle showed TCP80 to be relatively stable (t(1/2)=5 days) in the cytoplasmic compartment. In comparison, TCP110/ILF3 migrated between the cytoplasmic and nuclear compartments during the cell cycle. The TCP110 C-terminal segment contains an additional nuclear localizing signal that plays a role in its nuclear translocation. This study indicates that the multiple cellular functions, i.e., translation control, interleukin-2 enhancer binding, or cell division, of TCP/ILF3 are fulfilled by alternatively spliced isoforms.

Kicking Triturus arntzeni when it's down: large-scale nuclear genetic data confirm that newts from the type locality are genetically admixed.

PubMed

Wielstra, B; Arntzen, J W

2014-05-27

We collected nuclear DNA data (52 markers) with next-generation sequencing for nine Triturus newt specimens, including the holotype and two of the paratypes of T. arntzeni, from the type locality at Vrtovać in eastern Serbia. We compare these data to a reference set composed of the four crested newt species distributed in eastern Serbia namely T. cristatus, T. dobrogicus, T. ivanbureschi and T. macedonicus to determine to which of these species the newts from the type locality of T. arntzeni should be attributed. The majority of alleles in individuals from Vrtovać is derived from T. macedonicus, but a considerable number of T. ivanbureschi alleles is also present; alleles typical for T. cristatus and T. dobrogicus are found at low frequency. Accordingly, we interpret Vrtovać as a T. macedonicus - T. ivanbureschi hybrid population, albeit not composed of F1 hybrids but of genetically admixed individuals derived through multiple generations of backcrossing. The data support the notion that the name T. arntzeni should not be applied to a species newly distinguished in T. karelinii sensu lato (to which the name T. ivanbureschi has been given). We conclude that because of the hybrid nature of the individuals from Vrtovać, the name T. arntzeni should be placed not only in the synonymy of T. macedonicus but also in the synonymy of T. ivanbureschi. In this study we demonstrate that next-generation sequencing can provide high quality data for type material with degraded DNA and therefore can play an important role in taxonomy.

NMRDSP: an accurate prediction of protein shape strings from NMR chemical shifts and sequence data.

PubMed

Mao, Wusong; Cong, Peisheng; Wang, Zhiheng; Lu, Longjian; Zhu, Zhongliang; Li, Tonghua

2013-01-01

Shape string is structural sequence and is an extremely important structure representation of protein backbone conformations. Nuclear magnetic resonance chemical shifts give a strong correlation with the local protein structure, and are exploited to predict protein structures in conjunction with computational approaches. Here we demonstrate a novel approach, NMRDSP, which can accurately predict the protein shape string based on nuclear magnetic resonance chemical shifts and structural profiles obtained from sequence data. The NMRDSP uses six chemical shifts (HA, H, N, CA, CB and C) and eight elements of structure profiles as features, a non-redundant set (1,003 entries) as the training set, and a conditional random field as a classification algorithm. For an independent testing set (203 entries), we achieved an accuracy of 75.8% for S8 (the eight states accuracy) and 87.8% for S3 (the three states accuracy). This is higher than only using chemical shifts or sequence data, and confirms that the chemical shift and the structure profile are significant features for shape string prediction and their combination prominently improves the accuracy of the predictor. We have constructed the NMRDSP web server and believe it could be employed to provide a solid platform to predict other protein structures and functions. The NMRDSP web server is freely available at http://cal.tongji.edu.cn/NMRDSP/index.jsp.

Detection of PIWI and piRNAs in the mitochondria of mammalian cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwon, ChangHyuk, E-mail: netbuyer@hanmail.net; Tak, Hyosun, E-mail: chuberry@naver.com; Rho, Mina, E-mail: minarho@hanyang.ac.kr

2014-03-28

Highlights: • piRNA sequences were mapped to human mitochondrial (mt) genome. • We inspected small RNA-Seq datasets from somatic cell mt subcellular fractions. • Piwi and piRNA transcripts are present in mammalian somatic cancer cell mt fractions. - Abstract: Piwi-interacting RNAs (piRNAs) are 26–31 nt small noncoding RNAs that are processed from their longer precursor transcripts by Piwi proteins. Localization of Piwi and piRNA has been reported mostly in nucleus and cytoplasm of higher eukaryotes germ-line cells, where it is believed that known piRNA sequences are located in repeat regions of nuclear genome in germ-line cells. However, localization of PIWImore » and piRNA in mammalian somatic cell mitochondria yet remains largely unknown. We identified 29 piRNA sequence alignments from various regions of the human mitochondrial genome. Twelve out 29 piRNA sequences matched stem-loop fragment sequences of seven distinct tRNAs. We observed their actual expression in mitochondria subcellular fractions by inspecting mitochondrial-specific small RNA-Seq datasets. Of interest, the majority of the 29 piRNAs overlapped with multiple longer transcripts (expressed sequence tags) that are unique to the human mitochondrial genome. The presence of mature piRNAs in mitochondria was detected by qRT-PCR of mitochondrial subcellular RNAs. Further validation showed detection of Piwi by colocalization using anti-Piwil1 and mitochondria organelle-specific protein antibodies.« less

A preliminary assessment of genetic divergence and distribution of Malagasy cave fish in the genus Typhleotris (Teleostei: Milyeringidae).

PubMed

Vences, Miguel; Rasoloariniaina, Jean R; Riemann, Jana C

2018-02-08

The genus Typhleotris contains three poorly known blind fish species, inhabiting aquifers in the limestone plateau of south-western Madagascar. Until recently these species were known from only few localities, and their pattern of genetic differentiation remains poorly studied. In this study we analyse 122 Typhleotris tissue samples collected from 12 localities, spanning the entire known range of the genus, and use DNA sequences to assign these samples to the three species known. The phylogeny based on the mitochondrial marker cox1 revealed three main clades corresponding to the three species: Typhleotris madagascariensis, T. mararybe and T. pauliani, differing by uncorrected pairwise sequence divergences of 6.3-9.8%. The distribution ranges of the three species overlapped widely: T. mararybe was collected only in a southern group of localities, T. madagascariensis was found in both the southern and the central group of localities, and T. pauliani occurred from the northernmost site to the southern group of localities; yet the three species did not share haplotypes in two nuclear genes, except for three individuals that we hypothesize are hybrids of T. pauliani with T. madagascariensis and T. mararybe. This pattern of concordant mitochondrial and nuclear divergence despite sympatry strongly supports the status of all three taxa as separate species. Phylogeographic structure was obvious in T. madagascariensis, with two separate shallow mitochondrial clades occupying (1) the central vs. (2) the southern group of populations, and in T. pauliani, with separate mitochondrial clades for (1) the northern vs. (2) the central/southern populations. The widespread occurrence of these three cave fish species suggests that the aquifers in south-western Madagascar have at least in the past allowed episodic dispersal and gene flow of subterraneous organisms, whereas the phylogeographic pattern of T. madagascariensis and T. pauliani provides evidence for isolation and loss of connectivity in the more recent past.

RNA localization in Xenopus oocytes uses a core group of trans-acting factors irrespective of destination.

PubMed

Snedden, Donald D; Bertke, Michelle M; Vernon, Dominic; Huber, Paul W

2013-07-01

The 3' untranslated region of mRNA encoding PHAX, a phosphoprotein required for nuclear export of U-type snRNAs, contains cis-acting sequence motifs E2 and VM1 that are required for localization of RNAs to the vegetal hemisphere of Xenopus oocytes. However, we have found that PHAX mRNA is transported to the opposite, animal, hemisphere. A set of proteins that cross-link to the localization elements of vegetally localized RNAs are also cross-linked to PHAX and An1 mRNAs, demonstrating that the composition of RNP complexes that form on these localization elements is highly conserved irrespective of the final destination of the RNA. The ability of RNAs to bind this core group of proteins is correlated with localization activity. Staufen1, which binds to Vg1 and VegT mRNAs, is not associated with RNAs localized to the animal hemisphere and may determine, at least in part, the direction of RNA movement in Xenopus oocytes.

Identification of a subunit of NADH-dehydrogenase as a p49/STRAP-binding protein.

PubMed

Zhang, Xiaomin; Azhar, Gohar; Helms, Scott; Zhong, Ying; Wei, Jeanne Y

2008-01-29

The p49/STRAP (or SRFBP1) protein was recently identified in our laboratory as a cofactor of serum response factor that contributes to the regulation of SRF target genes in the heart. In the present study, we report that NDUFAB1, a nuclear encoded subunit of NADH dehydrogenase, represented the majority of the cDNA clones that interacted with p49/STRAP in multiple screenings using the yeast two-hybrid system. The p49/STRAP and NDUFAB1 proteins interacted and co-localized with each other in the cell. The p49/STRAP protein contains four classic nuclear localization sequence motifs, and it was observed to be present predominantly in the nucleus. Overexpression of p49/STRAP altered the intracellular level of NAD, and reduced the NAD/NADH ratio. Overexpression of p49/STRAP also induced the deacetylation of serum response factor. These data suggest that p49/STRAP plays a role in the regulation of intracellular processes such as cardiac cellular metabolism, gene expression, and possibly aging.

Selection and sex-biased dispersal in a coastal shark: the influence of philopatry on adaptive variation.

PubMed

Portnoy, D S; Puritz, J B; Hollenbeck, C M; Gelsleichter, J; Chapman, D; Gold, J R

2015-12-01

Sex-biased dispersal is expected to homogenize nuclear genetic variation relative to variation in genetic material inherited through the philopatric sex. When site fidelity occurs across a heterogeneous environment, local selective regimes may alter this pattern. We assessed spatial patterns of variation in nuclear-encoded, single nucleotide polymorphisms (SNPs) and sequences of the mitochondrial control region in bonnethead sharks (Sphyrna tiburo), a species thought to exhibit female philopatry, collected from summer habitats used for gestation. Geographic patterns of mtDNA haplotypes and putatively neutral SNPs confirmed female philopatry and male-mediated gene flow along the northeastern coast of the Gulf of Mexico. A total of 30 outlier SNP loci were identified; alleles at over half of these loci exhibited signatures of latitude-associated selection. Our results indicate that in species with sex-biased dispersal, philopatry can facilitate sorting of locally adaptive variation, with the dispersing sex facilitating movement of potentially adaptive variation among locations and environments. © 2015 John Wiley & Sons Ltd.

Xp11.2 translocation renal cell carcinoma with PSF-TFE3 rearrangement.

PubMed

Zhong, Minghao; Weisman, Paul; Zhu, Bing; Brassesco, Maria; Yang, Youfeng; Linehan, W Marston; Merino, Maria J; Zhang, David; Rohan, Stephen; Cai, Dongming; Yang, Ximing

2013-06-01

Xp11.2 translocation renal cell carcinoma (Xp11.2 RCC) is a subtype of RCC characterized by translocations involving a breakpoint at the TFE3 gene (Xp11.2). Moderate to strong nuclear TFE3 immunoreactivity has been recognized as a specific diagnostic marker for this type of tumor. However, exclusive cytoplasmic localization of a TFE3 fusion protein was reported in UOK 145 cells, a cell line derived from an Xp11.2 RCC harboring the PSF-TFE3 translocation. If reproducible using immunohistochemistry (IHC), this finding would have important implications for pathologists in the diagnosis of Xp11.2 RCC, calling into question the specificity of nuclear immunoreactivity for TFE3 in these tumors. The purpose of this study was to determine whether the above-noted cytoplasmic localization of the TFE3 fusion protein could be reproduced using IHC. UOK 145 cells and fresh frozen tissue from 2 clinical cases of Xp11.2 RCC found to harbor the PSF-TFE3 gene rearrangement (by cytogenetic testing) were collected. All samples were subjected to histopathologic evaluation by board-certified pathologists, TFE3 IHC, reverse transcription polymerase chain reaction, and Sanger sequencing analysis. A strong nuclear TFE3 immunoreactivity was demonstrated in all samples including the UOK 145 cell line. No cytoplasmic immunoreactivity was seen. Reverse transcription polymerase chain reaction and Sanger sequencing confirmed the previously reported PSF-TFE3 gene fusion between exon 9 of PSF and exon 6 of TFE3 in the UOK 145 cell line and in one of 2 clinical cases of Xp11.2 RCC. A novel PSF-TFE3 gene fusion between exon 9 of PSF and exon 5 of TFE3 was detected in the second clinical case of Xp11.2 RCC.

Target of Rapamycin Regulates Development and Ribosomal RNA Expression through Kinase Domain in Arabidopsis1[W][OA

PubMed Central

Ren, Maozhi; Qiu, Shuqing; Venglat, Prakash; Xiang, Daoquan; Feng, Li; Selvaraj, Gopalan; Datla, Raju

2011-01-01

Target of rapamycin (TOR) is a central regulator of cell growth, cell death, nutrition, starvation, hormone, and stress responses in diverse eukaryotes. However, very little is known about TOR signaling and the associated functional domains in plants. We have taken a genetic approach to dissect TOR functions in Arabidopsis (Arabidopsis thaliana) and report here that the kinase domain is essential for the role of TOR in embryogenesis and 45S rRNA expression. Twelve new T-DNA insertion mutants, spanning 14.2 kb of TOR-encoding genomic region, have been characterized. Nine of these share expression of defective kinase domain and embryo arrest at 16 to 32 cell stage. However, three T-DNA insertion lines affecting FATC domain displayed normal embryo development, indicating that FATC domain was dispensable in Arabidopsis. Genetic complementation showed that the TOR kinase domain alone in tor-10/tor-10 mutant background can rescue early embryo lethality and restore normal development. Overexpression of full-length TOR or kinase domain in Arabidopsis displayed developmental abnormalities in meristem, leaf, root, stem, flowering time, and senescence. We further show that TOR, especially the kinase domain, plays a role in ribosome biogenesis by activating 45S rRNA production. Of the six putative nuclear localization sequences in the kinase domain, nuclear localization sequence 6 was identified to confer TOR nuclear targeting in transient expression assays. Chromatin immunoprecipitation studies revealed that the HEAT repeat domain binds to 45S rRNA promoter and the 5′ external transcribed spacer elements motif. Together, these results show that TOR controls the embryogenesis, postembryonic development, and 45S rRNA production through its kinase domain in Arabidopsis. PMID:21266656

Identification of a Functional, CRM-1-Dependent Nuclear Export Signal in Hepatitis C Virus Core Protein

PubMed Central

Cerutti, Andrea; Maillard, Patrick; Minisini, Rosalba; Vidalain, Pierre-Olivier; Roohvand, Farzin; Pecheur, Eve-Isabelle; Pirisi, Mario; Budkowska, Agata

2011-01-01

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified. We show here that the aa(109–133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1–173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication. Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection. PMID:22039426

Evolutionary rates of mitochondrial genomes correspond to diversification rates and to contemporary species richness in birds and reptiles

PubMed Central

Eo, Soo Hyung; DeWoody, J. Andrew

2010-01-01

Rates of biological diversification should ultimately correspond to rates of genome evolution. Recent studies have compared diversification rates with phylogenetic branch lengths, but incomplete phylogenies hamper such analyses for many taxa. Herein, we use pairwise comparisons of confamilial sauropsid (bird and reptile) mitochondrial DNA (mtDNA) genome sequences to estimate substitution rates. These molecular evolutionary rates are considered in light of the age and species richness of each taxonomic family, using a random-walk speciation–extinction process to estimate rates of diversification. We find the molecular clock ticks at disparate rates in different families and at different genes. For example, evolutionary rates are relatively fast in snakes and lizards, intermediate in crocodilians and slow in turtles and birds. There was also rate variation across genes, where non-synonymous substitution rates were fastest at ATP8 and slowest at CO3. Family-by-gene interactions were significant, indicating that local clocks vary substantially among sauropsids. Most importantly, we find evidence that mitochondrial genome evolutionary rates are positively correlated with speciation rates and with contemporary species richness. Nuclear sequences are poorly represented among reptiles, but the correlation between rates of molecular evolution and species diversification also extends to 18 avian nuclear genes we tested. Thus, the nuclear data buttress our mtDNA findings. PMID:20610427

The GCKIII Kinase Sps1 and the 14-3-3 Isoforms, Bmh1 and Bmh2, Cooperate to Ensure Proper Sporulation in Saccharomyces cerevisiae

PubMed Central

Slubowski, Christian J.; Paulissen, Scott M.; Huang, Linda S.

2014-01-01

Sporulation in the budding yeast Saccharomyces cerevisiae is a developmental program initiated in response to nutritional deprivation. Sps1, a serine/threonine kinase, is required for sporulation, but relatively little is known about the molecular mechanisms through which it regulates this process. Here we show that SPS1 encodes a bona-fide member of the GCKIII subfamily of STE20 kinases, both through phylogenetic analysis of the kinase domain and examination of its C-terminal regulatory domain. Within the regulatory domain, we find Sps1 contains an invariant ExxxPG region conserved from plant to human GCKIIIs that we call the EPG motif; we show this EPG motif is important for SPS1 function. We also find that Sps1 is phosphorylated near its N-terminus on Threonine 12, and that this phosphorylation is required for the efficient production of spores. In Sps1, Threonine 12 lies within a 14-3-3 consensus binding sequence, and we show that the S. cerevisiae 14-3-3 proteins Bmh1 and Bmh2 bind Sps1 in a Threonine 12-dependent fashion. This interaction is significant, as BMH1 and BMH2 are required during sporulation and genetically interact with SPS1 in sporulating cells. Finally, we observe that Sps1, Bmh1 and Bmh2 are present in both the nucleus and cytoplasm during sporulation. We identify a nuclear localization sequence in Sps1 at amino acids 411–415, and show that this sequence is necessary and sufficient for nuclear localization. Taken together, these data identify regions within Sps1 critical for its function and indicate that SPS1 and 14-3-3s act together to promote proper sporulation in S. cerevisiae. PMID:25409301

Localization and regulation of glucagon receptors in the chick eye and preproglucagon and glucagon receptor expression in the mouse eye.

PubMed

Feldkaemper, Marita P; Burkhardt, Eva; Schaeffel, Frank

2004-09-01

Myopia is a condition in which the eye is too long for the focal length of cornea and lens. Analysis of the messengers that are released by the retina to control axial eye growth in the animal model of the chicken revealed that glucagon-immunoreactive amacrine cells are involved in the retinal image processing that controls the growth of the sclera. It was found that the amount of retinal glucagon mRNA increased during treatment with positive lenses and pharmacological studies supported the idea that glucagon may act as a stop signal for eye growth. Glucagon exerts its regulatory effects by binding to a single type of glucagon receptor. In this study, we have sequenced the chicken glucagon receptor and compared its DNA and amino acid sequence with the human and mouse homologues. After sequencing about 80% of the receptor, we found a homology between 79.4 and 75.6% on cDNA level. At the protein level, about 73% of the amino acids were identical. Moreover, the cellular localization and regulation of the glucagon receptor in the chick retina was studied. In situ hybridization studies showed that many cells in the ganglion cell layer and inner nuclear layer, and some cells in the outer nuclear layer, express the receptor mRNA. Injection of the glucagon agonist Lys17,18,Glu21-glucagon induced a down-regulation of glucagon receptor mRNA content. Since the mouse would be an attractive mammalian model to study the biochemical and genetic basis of myopia, and because recent studies have demonstrated that form deprivation myopia can be induced, the expression of preproglucagon and glucagon receptor genes were also studied in the mouse retina and were found to be expressed.

Arabidopsis ASYMMETRIC LEAVES2 protein required for leaf morphogenesis consistently forms speckles during mitosis of tobacco BY-2 cells via signals in its specific sequence.

PubMed

Luo, Lilan; Ando, Sayuri; Sasabe, Michiko; Machida, Chiyoko; Kurihara, Daisuke; Higashiyama, Tetsuya; Machida, Yasunori

2012-09-01

Leaf primordia with high division and developmental competencies are generated around the periphery of stem cells at the shoot apex. Arabidopsis ASYMMETRIC-LEAVES2 (AS2) protein plays a key role in the regulation of many genes responsible for flat symmetric leaf formation. The AS2 gene, expressed in leaf primordia, encodes a plant-specific nuclear protein containing an AS2/LOB domain with cysteine repeats (C-motif). AS2 proteins are present in speckles in and around the nucleoli, and in the nucleoplasm of some leaf epidermal cells. We used the tobacco cultured cell line BY-2 expressing the AS2-fused yellow fluorescent protein to examine subnuclear localization of AS2 in dividing cells. AS2 mainly localized to speckles (designated AS2 bodies) in cells undergoing mitosis and distributed in a pairwise manner during the separation of sets of daughter chromosomes. Few interphase cells contained AS2 bodies. Deletion analyses showed that a short stretch of the AS2 amino-terminal sequence and the C-motif play negative and positive roles, respectively, in localizing AS2 to the bodies. These results suggest that AS2 bodies function to properly distribute AS2 to daughter cells during cell division in leaf primordia; and this process is controlled at least partially by signals encoded by the AS2 sequence itself.

The unusually large Plasmodium telomerase reverse-transcriptase localizes in a discrete compartment associated with the nucleolus

PubMed Central

Figueiredo, Luisa M.; Rocha, Eduardo P. C.; Mancio-Silva, Liliana; Prevost, Christine; Hernandez-Verdun, Danièle; Scherf, Artur

2005-01-01

Telomerase replicates chromosome ends, a function necessary for maintaining genome integrity. We have identified the gene that encodes the catalytic reverse transcriptase (RT) component of this enzyme in the malaria parasite Plasmodium falciparum (PfTERT) as well as the orthologous genes from two rodent and one simian malaria species. PfTERT is predicted to encode a basic protein that contains the major sequence motifs previously identified in known telomerase RTs (TERTs). At ∼2500 amino acids, PfTERT is three times larger than other characterized TERTs. We observed remarkable sequence diversity between TERT proteins of different Plasmodial species, with conserved domains alternating with hypervariable regions. Immunofluorescence analysis revealed that PfTERT is expressed in asexual blood stage parasites that have begun DNA synthesis. Surprisingly, rather than at telomere clusters, PfTERT typically localizes into a discrete nuclear compartment. We further demonstrate that this compartment is associated with the nucleolus, hereby defined for the first time in P.falciparum. PMID:15722485

A comprehensive characterization of simple sequence repeats in pepper genomes provides valuable resources for marker development in Capsicum.

PubMed

Cheng, Jiaowen; Zhao, Zicheng; Li, Bo; Qin, Cheng; Wu, Zhiming; Trejo-Saavedra, Diana L; Luo, Xirong; Cui, Junjie; Rivera-Bustamante, Rafael F; Li, Shuaicheng; Hu, Kailin

2016-01-07

The sequences of the full set of pepper genomes including nuclear, mitochondrial and chloroplast are now available for use. However, the overall of simple sequence repeats (SSR) distribution in these genomes and their practical implications for molecular marker development in Capsicum have not yet been described. Here, an average of 868,047.50, 45.50 and 30.00 SSR loci were identified in the nuclear, mitochondrial and chloroplast genomes of pepper, respectively. Subsequently, systematic comparisons of various species, genome types, motif lengths, repeat numbers and classified types were executed and discussed. In addition, a local database composed of 113,500 in silico unique SSR primer pairs was built using a homemade bioinformatics workflow. As a pilot study, 65 polymorphic markers were validated among a wide collection of 21 Capsicum genotypes with allele number and polymorphic information content value per marker raging from 2 to 6 and 0.05 to 0.64, respectively. Finally, a comparison of the clustering results with those of a previous study indicated the usability of the newly developed SSR markers. In summary, this first report on the comprehensive characterization of SSR motifs in pepper genomes and the very large set of SSR primer pairs will benefit various genetic studies in Capsicum.

A comprehensive characterization of simple sequence repeats in pepper genomes provides valuable resources for marker development in Capsicum

PubMed Central

Cheng, Jiaowen; Zhao, Zicheng; Li, Bo; Qin, Cheng; Wu, Zhiming; Trejo-Saavedra, Diana L.; Luo, Xirong; Cui, Junjie; Rivera-Bustamante, Rafael F.; Li, Shuaicheng; Hu, Kailin

2016-01-01

The sequences of the full set of pepper genomes including nuclear, mitochondrial and chloroplast are now available for use. However, the overall of simple sequence repeats (SSR) distribution in these genomes and their practical implications for molecular marker development in Capsicum have not yet been described. Here, an average of 868,047.50, 45.50 and 30.00 SSR loci were identified in the nuclear, mitochondrial and chloroplast genomes of pepper, respectively. Subsequently, systematic comparisons of various species, genome types, motif lengths, repeat numbers and classified types were executed and discussed. In addition, a local database composed of 113,500 in silico unique SSR primer pairs was built using a homemade bioinformatics workflow. As a pilot study, 65 polymorphic markers were validated among a wide collection of 21 Capsicum genotypes with allele number and polymorphic information content value per marker raging from 2 to 6 and 0.05 to 0.64, respectively. Finally, a comparison of the clustering results with those of a previous study indicated the usability of the newly developed SSR markers. In summary, this first report on the comprehensive characterization of SSR motifs in pepper genomes and the very large set of SSR primer pairs will benefit various genetic studies in Capsicum. PMID:26739748

Design, synthesis, nuclear localization, and biological activity of a fluorescent duocarmycin analog, HxTfA.

PubMed

Kiakos, Konstantinos; Englinger, Bernhard; Yanow, Stephanie K; Wernitznig, Debora; Jakupec, Michael A; Berger, Walter; Keppler, Bernhard K; Hartley, John A; Lee, Moses; Patil, Pravin C

2018-05-01

HxTfA 4 is a fluorescent analog of a potent cytotoxic and antimalarial agent, TfA 3, which is currently being investigated for the development of an antimalarial vaccine, PlasProtect®. HxTfA contains a p-anisylbenzimidazole or Hx moiety, which is endowed with a blue emission upon excitation at 318 nm; thus enabling it to be used as a surrogate for probing the cellular fate of TfA using confocal microscopy, and addressing the question of nuclear localization. HxTfA exhibits similar selectivity to TfA for A-tract sequences of DNA, alkylating adenine-N3, albeit at 10-fold higher concentrations. It also possesses in vitro cytotoxicity against A549 human lung carcinoma cells and Plasmodium falciparum. Confocal microscopy studies showed for the first time that HxTfA, and by inference TfA, entered A549 cells and localized in the nucleus to exert its biological activity. At biologically relevant concentrations, HxTfA elicits DNA damage response as evidenced by a marked increase in the levels of γH2AX observed by confocal microscopy and immunoblotting studies, and ultimately induces apoptosis. Copyright © 2018 Elsevier Ltd. All rights reserved.

Echinoderm phosphorylated matrix proteins UTMP16 and UTMP19 have different functions in sea urchin tooth mineralization.

PubMed

Alvares, Keith; Dixit, Saryu N; Lux, Elizabeth; Veis, Arthur

2009-09-18

Studies of mineralization of embryonic spicules and of the sea urchin genome have identified several putative mineralization-related proteins. These predicted proteins have not been isolated or confirmed in mature mineralized tissues. Mature Lytechinus variegatus teeth were demineralized with 0.6 N HCl after prior removal of non-mineralized constituents with 4.0 M guanidinium HCl. The HCl-extracted proteins were fractionated on ceramic hydroxyapatite and separated into bound and unbound pools. Gel electrophoresis compared the protein distributions. The differentially present bands were purified and digested with trypsin, and the tryptic peptides were separated by high pressure liquid chromatography. NH2-terminal sequences were determined by Edman degradation and compared with the genomic sequence bank data. Two of the putative mineralization-related proteins were found. Their complete amino acid sequences were cloned from our L. variegatus cDNA library. Apatite-binding UTMP16 was found to be present in two isoforms; both isoforms had a signal sequence, a Ser-Asp-rich extracellular matrix domain, and a transmembrane and cytosolic insertion sequence. UTMP19, although rich in Glu and Thr did not bind to apatite. It had neither signal peptide nor transmembrane domain but did have typical nuclear localization and nuclear exit signal sequences. Both proteins were phosphorylated and good substrates for phosphatase. Immunolocalization studies with anti-UTMP16 show it to concentrate at the syncytial membranes in contact with the mineral. On the basis of our TOF-SIMS analyses of magnesium ion and Asp mapping of the mineral phase composition, we speculate that UTMP16 may be important in establishing the high magnesium columns that fuse the calcite plates together to enhance the mechanical strength of the mineralized tooth.

Light-modulated abundance of an mRNA encoding a calmodulin-regulated, chromatin-associated NTPase in pea

NASA Technical Reports Server (NTRS)

Hsieh, H. L.; Tong, C. G.; Thomas, C.; Roux, S. J.

1996-01-01

A CDNA encoding a 47 kDa nucleoside triphosphatase (NTPase) that is associated with the chromatin of pea nuclei has been cloned and sequenced. The translated sequence of the cDNA includes several domains predicted by known biochemical properties of the enzyme, including five motifs characteristic of the ATP-binding domain of many proteins, several potential casein kinase II phosphorylation sites, a helix-turn-helix region characteristic of DNA-binding proteins, and a potential calmodulin-binding domain. The deduced primary structure also includes an N-terminal sequence that is a predicted signal peptide and an internal sequence that could serve as a bipartite-type nuclear localization signal. Both in situ immunocytochemistry of pea plumules and immunoblots of purified cell fractions indicate that most of the immunodetectable NTPase is within the nucleus, a compartment proteins typically reach through nuclear pores rather than through the endoplasmic reticulum pathway. The translated sequence has some similarity to that of human lamin C, but not high enough to account for the earlier observation that IgG against human lamin C binds to the NTPase in immunoblots. Northern blot analysis shows that the NTPase MRNA is strongly expressed in etiolated plumules, but only poorly or not at all in the leaf and stem tissues of light-grown plants. Accumulation of NTPase mRNA in etiolated seedlings is stimulated by brief treatments with both red and far-red light, as is characteristic of very low-fluence phytochrome responses. Southern blotting with pea genomic DNA indicates the NTPase is likely to be encoded by a single gene.

Design of Conditionally Active STATs: Insights into STAT Activation and Gene Regulatory Function

PubMed Central

Milocco, Lawrence H.; Haslam, Jennifer A.; Rosen, Jonathan; Seidel, H. Martin

1999-01-01

The STAT (signal transducer and activator of transcription) signaling pathway is activated by a large number of cytokines and growth factors. We sought to design a conditionally active STAT that could not only provide insight into basic questions about STAT function but also serve as a powerful tool to determine the precise biological role of STATs. To this end, we have developed a conditionally active STAT by fusing STATs with the ligand-binding domain of the estrogen receptor (ER). We have demonstrated that the resulting STAT-ER chimeras are estrogen-inducible transcription factors that retain the functional and biochemical characteristics of the cognate wild-type STATs. In addition, these tools have allowed us to evaluate separately the contribution of tyrosine phosphorylation and dimerization to STAT function. We have for the first time provided experimental data supporting the model that the only apparent role of STAT tyrosine phosphorylation is to drive dimerization, as dimerization alone is sufficient to unmask a latent STAT nuclear localization sequence and induce nuclear translocation, sequence-specific DNA binding, and transcriptional activity. PMID:10082558

Genomic Resources of Three Pulsatilla Species Reveal Evolutionary Hotspots, Species-Specific Sites and Variable Plastid Structure in the Family Ranunculaceae.

PubMed

Szczecińska, Monika; Sawicki, Jakub

2015-09-15

The European continent is presently colonized by nine species of the genus Pulsatilla, five of which are encountered only in mountainous regions of southwest and south-central Europe. The remaining four species inhabit lowlands in the north-central and eastern parts of the continent. Most plants of the genus Pulsatilla are rare and endangered, which is why most research efforts focused on their biology, ecology and hybridization. The objective of this study was to develop genomic resources, including complete plastid genomes and nuclear rRNA clusters, for three sympatric Pulsatilla species that are most commonly found in Central Europe. The results will supply valuable information about genetic variation, which can be used in the process of designing primers for population studies and conservation genetics research. The complete plastid genomes together with the nuclear rRNA cluster can serve as a useful tool in hybridization studies. Six complete plastid genomes and nuclear rRNA clusters were sequenced from three species of Pulsatilla using the Illumina sequencing technology. Four junctions between single copy regions and inverted repeats and junctions between the identified locally-collinear blocks (LCB) were confirmed by Sanger sequencing. Pulsatilla genomes of 120 unique genes had a total length of approximately 161-162 kb, and 21 were duplicated in the inverted repeats (IR) region. Comparative plastid genomes of newly-sequenced Pulsatilla and the previously-identified plastomes of Aconitum and Ranunculus species belonging to the family Ranunculaceae revealed several variations in the structure of the genome, but the gene content remained constant. The nuclear rRNA cluster (18S-ITS1-5.8S-ITS2-26S) of studied Pulsatilla species is 5795 bp long. Among five analyzed regions of the rRNA cluster, only Internal Transcribed Spacer 2 (ITS2) enabled the molecular delimitation of closely-related Pulsatilla patens and Pulsatilla vernalis. The determination of complete plastid genome and nuclear rRNA cluster sequences in three species of the genus Pulsatilla is an important contribution to our knowledge of the evolution and phylogeography of those endangered taxa. The resulting data can be used to identify regions that are particularly useful for barcoding, phylogenetic and phylogeographic studies. The investigated taxa can be identified at each stage of development based on their species-specific SNPs. The nuclear and plastid genomic resources enable advanced studies on hybridization, including identification of parent species, including their roles in that process. The identified nonsynonymous mutations could play an important role in adaptations to changing environments. The results of the study will also provide valuable information about the evolution of the plastome structure in the family Ranunculaceae.

Genomic Resources of Three Pulsatilla Species Reveal Evolutionary Hotspots, Species-Specific Sites and Variable Plastid Structure in the Family Ranunculaceae

PubMed Central

Szczecińska, Monika; Sawicki, Jakub

2015-01-01

Background: The European continent is presently colonized by nine species of the genus Pulsatilla, five of which are encountered only in mountainous regions of southwest and south-central Europe. The remaining four species inhabit lowlands in the north-central and eastern parts of the continent. Most plants of the genus Pulsatilla are rare and endangered, which is why most research efforts focused on their biology, ecology and hybridization. The objective of this study was to develop genomic resources, including complete plastid genomes and nuclear rRNA clusters, for three sympatric Pulsatilla species that are most commonly found in Central Europe. The results will supply valuable information about genetic variation, which can be used in the process of designing primers for population studies and conservation genetics research. The complete plastid genomes together with the nuclear rRNA cluster can serve as a useful tool in hybridization studies. Methodology/principal findings: Six complete plastid genomes and nuclear rRNA clusters were sequenced from three species of Pulsatilla using the Illumina sequencing technology. Four junctions between single copy regions and inverted repeats and junctions between the identified locally-collinear blocks (LCB) were confirmed by Sanger sequencing. Pulsatilla genomes of 120 unique genes had a total length of approximately 161–162 kb, and 21 were duplicated in the inverted repeats (IR) region. Comparative plastid genomes of newly-sequenced Pulsatilla and the previously-identified plastomes of Aconitum and Ranunculus species belonging to the family Ranunculaceae revealed several variations in the structure of the genome, but the gene content remained constant. The nuclear rRNA cluster (18S-ITS1-5.8S-ITS2-26S) of studied Pulsatilla species is 5795 bp long. Among five analyzed regions of the rRNA cluster, only Internal Transcribed Spacer 2 (ITS2) enabled the molecular delimitation of closely-related Pulsatilla patens and Pulsatilla vernalis. Conclusions/significance: The determination of complete plastid genome and nuclear rRNA cluster sequences in three species of the genus Pulsatilla is an important contribution to our knowledge of the evolution and phylogeography of those endangered taxa. The resulting data can be used to identify regions that are particularly useful for barcoding, phylogenetic and phylogeographic studies. The investigated taxa can be identified at each stage of development based on their species-specific SNPs. The nuclear and plastid genomic resources enable advanced studies on hybridization, including identification of parent species, including their roles in that process. The identified nonsynonymous mutations could play an important role in adaptations to changing environments. The results of the study will also provide valuable information about the evolution of the plastome structure in the family Ranunculaceae. PMID:26389887

Histone H3 Variants in Trichomonas vaginalis

PubMed Central

Zubáčová, Zuzana; Hostomská, Jitka

2012-01-01

The parabasalid protist Trichomonas vaginalis is a widespread parasite that affects humans, frequently causing vaginitis in infected women. Trichomonad mitosis is marked by the persistence of the nuclear membrane and the presence of an asymmetric extranuclear spindle with no obvious direct connection to the chromosomes. No centromeric markers have been described in T. vaginalis, which has prevented a detailed analysis of mitotic events in this organism. In other eukaryotes, nucleosomes of centromeric chromatin contain the histone H3 variant CenH3. The principal aim of this work was to identify a CenH3 homolog in T. vaginalis. We performed a screen of the T. vaginalis genome to retrieve sequences of canonical and variant H3 histones. Three variant histone H3 proteins were identified, and the subcellular localization of their epitope-tagged variants was determined. The localization of the variant TVAG_185390 could not be distinguished from that of the canonical H3 histone. The sequence of the variant TVAG_087830 closely resembled that of histone H3. The tagged protein colocalized with sites of active transcription, indicating that the variant TVAG_087830 represented H3.3 in T. vaginalis. The third H3 variant (TVAG_224460) was localized to 6 or 12 distinct spots at the periphery of the nucleus, corresponding to the number of chromosomes in G1 phase and G2 phase, respectively. We propose that this variant represents the centromeric marker CenH3 and thus can be employed as a tool to study mitosis in T. vaginalis. Furthermore, we suggest that the peripheral distribution of CenH3 within the nucleus results from the association of centromeres with the nuclear envelope throughout the cell cycle. PMID:22408228

Interaction of dengue virus nonstructural protein 5 with Daxx modulates RANTES production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khunchai, Sasiprapa; Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok; Junking, Mutita

Highlights: Black-Right-Pointing-Pointer For the first time how DENV NS5 increases RANTES production. Black-Right-Pointing-Pointer DENV NS5 physically interacts with human Daxx. Black-Right-Pointing-Pointer Nuclear localization of NS5 is required for Daxx interaction and RANTES production. -- Abstract: Dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), caused by dengue virus (DENV) infection, are important public health problems in the tropical and subtropical regions. Abnormal hemostasis and plasma leakage are the main patho-physiological changes in DHF/DSS. A remarkably increased production of cytokines, the so called 'cytokine storm', is observed in the patients with DHF/DSS. A complex interaction between DENV proteinsmore » and the host immune response contributes to cytokine production. However, the molecular mechanism(s) by which DENV nonstructural protein 5 (NS5) mediates these responses has not been fully elucidated. In the present study, yeast two-hybrid assay was performed to identify host proteins interacting with DENV NS5 and a death-domain-associate protein (Daxx) was identified. The in vivo relevance of this interaction was suggested by co-immunoprecipitation and nuclear co-localization of these two proteins in HEK293 cells expressing DENV NS5. HEK293 cells expressing DENV NS5-K/A, which were mutated at the nuclear localization sequences (NLS), were created to assess its functional roles in nuclear translocation, Daxx interaction, and cytokine production. In the absence of NLS, DENV NS5 could neither translocate into the nucleus nor interact with Daxx to increase the DHF-associated cytokine, RANTES (CCL5) production. This work demonstrates the interaction between DENV NS5 and Daxx and the role of the interaction on the modulation of RANTES production.« less

An atypical topoisomerase II sequence from the slime mold Physarum polycephalum.

PubMed

Hugodot, Yannick; Dutertre, Murielle; Duguet, Michel

2004-01-21

We have determined the complete nucleotide sequence of the cDNA encoding DNA topoisomerase II from Physarum polycephalum. Using degenerate primers, based on the conserved amino acid sequences of other eukaryotic enzymes, a 250-bp fragment was polymerase chain reaction (PCR) amplified. This fragment was used as a probe to screen a Physarum cDNA library. A partial cDNA clone was isolated that was truncated at the 3' end. Rapid amplification of cDNA ends (RACE)-PCR was employed to isolate the remaining portion of the gene. The complete sequence of 4613 bp contains an open reading frame of 4494 bp that codes for 1498 amino acid residues with a theoretical molecular weight of 167 kDa. The predicted amino acid sequence shares similarity with those of other eukaryotes and shows the highest degree of identity with the enzyme of Dictyostelium discoideum. However, the enzyme of P. polycephalum contains an atypical amino-terminal domain very rich in serine and proline, whose function is unknown. Remarkably, both a mitochondrial targeting sequence and a nuclear localization signal were predicted respectively in the amino and carboxy-terminus of the protein, as in the case of human topoisomerase III alpha. At the Physarum genomic level, the topoisomerase II gene encompasses a region of about 16 kbp suggesting a large proportion of intronic sequences, an unusual situation for a gene of a lower eukaryote, often free of introns. Finally, expression of topoisomerase II mRNA does not appear significantly dependent on the plasmodium cycle stage, possibly due to the lack of G1 phase or (and) to a mitochondrial localization of the enzyme.

Mutation in TOR1AIP1 encoding LAP1B in a form of muscular dystrophy: a novel gene related to nuclear envelopathies.

PubMed

Kayman-Kurekci, Gulsum; Talim, Beril; Korkusuz, Petek; Sayar, Nilufer; Sarioglu, Turkan; Oncel, Ibrahim; Sharafi, Parisa; Gundesli, Hulya; Balci-Hayta, Burcu; Purali, Nuhan; Serdaroglu-Oflazer, Piraye; Topaloglu, Haluk; Dincer, Pervin

2014-07-01

We performed genome-wide homozygosity mapping and mapped a novel myopathic phenotype to chromosomal region 1q25 in a consanguineous family with three affected individuals manifesting proximal and distal weakness and atrophy, rigid spine and contractures of the proximal and distal interphalangeal hand joints. Additionally, cardiomyopathy and respiratory involvement were noted. DNA sequencing of torsinA-interacting protein 1 (TOR1AIP1) gene encoding lamina-associated polypeptide 1B (LAP1B), showed a homozygous c.186delG mutation that causes a frameshift resulting in a premature stop codon (p.E62fsTer25). We observed that expression of LAP1B was absent in the patient skeletal muscle fibres. Ultrastructural examination showed intact sarcomeric organization but alterations of the nuclear envelope including nuclear fragmentation, chromatin bleb formation and naked chromatin. LAP1B is a type-2 integral membrane protein localized in the inner nuclear membrane that binds to both A- and B-type lamins, and is involved in the regulation of torsinA ATPase. Interestingly, luminal domain-like LAP1 (LULL1)-an endoplasmic reticulum-localized partner of torsinA-was overexpressed in the patient's muscle in the absence of LAP1B. Therefore, the findings suggest that LAP1 and LULL1 might have a compensatory effect on each other. This study expands the spectrum of genes associated with nuclear envelopathies and highlights the critical function for LAP1B in striated muscle. Copyright © 2014 Elsevier B.V. All rights reserved.

Successful Recovery of Nuclear Protein-Coding Genes from Small Insects in Museums Using Illumina Sequencing.

PubMed

Kanda, Kojun; Pflug, James M; Sproul, John S; Dasenko, Mark A; Maddison, David R

2015-01-01

In this paper we explore high-throughput Illumina sequencing of nuclear protein-coding, ribosomal, and mitochondrial genes in small, dried insects stored in natural history collections. We sequenced one tenebrionid beetle and 12 carabid beetles ranging in size from 3.7 to 9.7 mm in length that have been stored in various museums for 4 to 84 years. Although we chose a number of old, small specimens for which we expected low sequence recovery, we successfully recovered at least some low-copy nuclear protein-coding genes from all specimens. For example, in one 56-year-old beetle, 4.4 mm in length, our de novo assembly recovered about 63% of approximately 41,900 nucleotides in a target suite of 67 nuclear protein-coding gene fragments, and 70% using a reference-based assembly. Even in the least successfully sequenced carabid specimen, reference-based assembly yielded fragments that were at least 50% of the target length for 34 of 67 nuclear protein-coding gene fragments. Exploration of alternative references for reference-based assembly revealed few signs of bias created by the reference. For all specimens we recovered almost complete copies of ribosomal and mitochondrial genes. We verified the general accuracy of the sequences through comparisons with sequences obtained from PCR and Sanger sequencing, including of conspecific, fresh specimens, and through phylogenetic analysis that tested the placement of sequences in predicted regions. A few possible inaccuracies in the sequences were detected, but these rarely affected the phylogenetic placement of the samples. Although our sample sizes are low, an exploratory regression study suggests that the dominant factor in predicting success at recovering nuclear protein-coding genes is a high number of Illumina reads, with success at PCR of COI and killing by immersion in ethanol being secondary factors; in analyses of only high-read samples, the primary significant explanatory variable was body length, with small beetles being more successfully sequenced.

Successful Recovery of Nuclear Protein-Coding Genes from Small Insects in Museums Using Illumina Sequencing

PubMed Central

Dasenko, Mark A.

2015-01-01

In this paper we explore high-throughput Illumina sequencing of nuclear protein-coding, ribosomal, and mitochondrial genes in small, dried insects stored in natural history collections. We sequenced one tenebrionid beetle and 12 carabid beetles ranging in size from 3.7 to 9.7 mm in length that have been stored in various museums for 4 to 84 years. Although we chose a number of old, small specimens for which we expected low sequence recovery, we successfully recovered at least some low-copy nuclear protein-coding genes from all specimens. For example, in one 56-year-old beetle, 4.4 mm in length, our de novo assembly recovered about 63% of approximately 41,900 nucleotides in a target suite of 67 nuclear protein-coding gene fragments, and 70% using a reference-based assembly. Even in the least successfully sequenced carabid specimen, reference-based assembly yielded fragments that were at least 50% of the target length for 34 of 67 nuclear protein-coding gene fragments. Exploration of alternative references for reference-based assembly revealed few signs of bias created by the reference. For all specimens we recovered almost complete copies of ribosomal and mitochondrial genes. We verified the general accuracy of the sequences through comparisons with sequences obtained from PCR and Sanger sequencing, including of conspecific, fresh specimens, and through phylogenetic analysis that tested the placement of sequences in predicted regions. A few possible inaccuracies in the sequences were detected, but these rarely affected the phylogenetic placement of the samples. Although our sample sizes are low, an exploratory regression study suggests that the dominant factor in predicting success at recovering nuclear protein-coding genes is a high number of Illumina reads, with success at PCR of COI and killing by immersion in ethanol being secondary factors; in analyses of only high-read samples, the primary significant explanatory variable was body length, with small beetles being more successfully sequenced. PMID:26716693

Reconciling Apparent Conflicts between Mitochondrial and Nuclear Phylogenies in African Elephants

PubMed Central

Georgiadis, Nicholas J.; David, Victor A.; Zhao, Kai; Stephens, Robert M.; Kolokotronis, Sergios-Orestis; Roca, Alfred L.

2011-01-01

Conservation strategies for African elephants would be advanced by resolution of conflicting claims that they comprise one, two, three or four taxonomic groups, and by development of genetic markers that establish more incisively the provenance of confiscated ivory. We addressed these related issues by genotyping 555 elephants from across Africa with microsatellite markers, developing a method to identify those loci most effective at geographic assignment of elephants (or their ivory), and conducting novel analyses of continent-wide datasets of mitochondrial DNA. Results showed that nuclear genetic diversity was partitioned into two clusters, corresponding to African forest elephants (99.5% Cluster-1) and African savanna elephants (99.4% Cluster-2). Hybrid individuals were rare. In a comparison of basal forest “F” and savanna “S” mtDNA clade distributions to nuclear DNA partitions, forest elephant nuclear genotypes occurred only in populations in which S clade mtDNA was absent, suggesting that nuclear partitioning corresponds to the presence or absence of S clade mtDNA. We reanalyzed African elephant mtDNA sequences from 81 locales spanning the continent and discovered that S clade mtDNA was completely absent among elephants at all 30 sampled tropical forest locales. The distribution of savanna nuclear DNA and S clade mtDNA corresponded closely to range boundaries traditionally ascribed to the savanna elephant species based on habitat and morphology. Further, a reanalysis of nuclear genetic assignment results suggested that West African elephants do not comprise a distinct third species. Finally, we show that some DNA markers will be more useful than others for determining the geographic origins of illegal ivory. These findings resolve the apparent incongruence between mtDNA and nuclear genetic patterns that has confounded the taxonomy of African elephants, affirm the limitations of using mtDNA patterns to infer elephant systematics or population structure, and strongly support the existence of two elephant species in Africa. PMID:21701575

Crystal structure of importin-α3 bound to the nuclear localization signal of Ran-binding protein 3.

PubMed

Koyama, Masako; Matsuura, Yoshiyuki

2017-09-23

Ran-binding protein 3 (RanBP3) is a primarily nuclear Ran-binding protein that functions as an accessory factor in the Ran GTPase system. RanBP3 associates with Ran-specific nucleotide exchange factor RCC1 and enhances its catalytic activity towards Ran. RanBP3 also promotes CRM1-mediated nuclear export as well as CRM1-independent nuclear export of β-catenin, Smad2, and Smad3. Nuclear import of RanBP3 is dependent on the nuclear import adaptor protein importin-α and, RanBP3 is imported more efficiently by importin-α3 than by other members of the importin-α family. Protein kinase signaling pathways control nucleocytoplasmic transport through phosphorylation of RanBP3 at Ser58, immediately C-terminal to the nuclear localization signal (NLS) in the N-terminal region of RanBP3. Here we report the crystal structure of human importin-α3 bound to an N-terminal fragment of human RanBP3 containing the NLS sequence that is necessary and sufficient for nuclear import. The structure reveals that RanBP3 binds to importin-α3 residues that are strictly conserved in all seven isoforms of human importin-α at the major NLS-binding site, indicating that the region of importin-α outside the NLS-binding site, possibly the autoinhibotory importin-β1-binding domain, may be the key determinant for the preferential binding of RanBP3 to importin-α3. Computational docking simulation indicates that phosphorylation of RanBP3 at Ser58 could potentially stabilize the association of RanBP3 with importin-α through interactions between the phosphate moiety of phospho-Ser58 of RanBP3 and a cluster of basic residues (Arg96 and Lys97 in importin-α3) on armadillo repeat 1 of importin-α. Copyright © 2017 Elsevier Inc. All rights reserved.

Coordination of tRNA transcription with export at nuclear pore complexes in budding yeast.

PubMed

Chen, Miao; Gartenberg, Marc R

2014-05-01

tRNAs are encoded by RNA polymerase III-transcribed genes that reside at seemingly random intervals along the chromosomes of budding yeast. Existing evidence suggests that the genes congregate together at the nucleolus and/or centromeres. In this study, we re-examined spatial and temporal aspects of tRNA gene (tDNA) expression. We show that tDNA transcription fluctuates during cell cycle progression. In M phase, when tRNA synthesis peaks, tDNAs localize at nuclear pore complexes (NPCs). Docking of a tDNA requires the DNA sequence of the contacted gene, nucleoporins Nup60 and Nup2, and cohesin. Characterization of mutants that block NPC localization revealed that docking is a consequence of elevated tDNA transcription. NPC-tDNA contact falters in the absence of the principal exportin of nascent tRNA, Los1, and genetic assays indicate that gating of tDNAs at NPCs favors cytoplasmic accumulation of functional tRNA. Collectively, the data suggest that tDNAs associate with NPCs to coordinate RNA polymerase III transcription with the nuclear export of pre-tRNA. The M-phase specificity of NPC contact reflects a regulatory mechanism that may have evolved, in part, to avoid collisions between DNA replication forks and transcribing RNA polymerase III machinery at NPCs.

Coordination of tRNA transcription with export at nuclear pore complexes in budding yeast

PubMed Central

Chen, Miao; Gartenberg, Marc R.

2014-01-01

tRNAs are encoded by RNA polymerase III-transcribed genes that reside at seemingly random intervals along the chromosomes of budding yeast. Existing evidence suggests that the genes congregate together at the nucleolus and/or centromeres. In this study, we re-examined spatial and temporal aspects of tRNA gene (tDNA) expression. We show that tDNA transcription fluctuates during cell cycle progression. In M phase, when tRNA synthesis peaks, tDNAs localize at nuclear pore complexes (NPCs). Docking of a tDNA requires the DNA sequence of the contacted gene, nucleoporins Nup60 and Nup2, and cohesin. Characterization of mutants that block NPC localization revealed that docking is a consequence of elevated tDNA transcription. NPC–tDNA contact falters in the absence of the principal exportin of nascent tRNA, Los1, and genetic assays indicate that gating of tDNAs at NPCs favors cytoplasmic accumulation of functional tRNA. Collectively, the data suggest that tDNAs associate with NPCs to coordinate RNA polymerase III transcription with the nuclear export of pre-tRNA. The M-phase specificity of NPC contact reflects a regulatory mechanism that may have evolved, in part, to avoid collisions between DNA replication forks and transcribing RNA polymerase III machinery at NPCs. PMID:24788517

Gadd45a Is an RNA Binding Protein and Is Localized in Nuclear Speckles

PubMed Central

Sytnikova, Yuliya A.; Kubarenko, Andriy V.; Schäfer, Andrea; Weber, Alexander N. R.; Niehrs, Christof

2011-01-01

Background The Gadd45 proteins play important roles in growth control, maintenance of genomic stability, DNA repair, and apoptosis. Recently, Gadd45 proteins have also been implicated in epigenetic gene regulation by promoting active DNA demethylation. Gadd45 proteins have sequence homology with the L7Ae/L30e/S12e RNA binding superfamily of ribosomal proteins, which raises the question if they may interact directly with nucleic acids. Principal Findings Here we show that Gadd45a binds RNA but not single- or double stranded DNA or methylated DNA in vitro. Sucrose density gradient centrifugation experiments demonstrate that Gadd45a is present in high molecular weight particles, which are RNase sensitive. Gadd45a displays RNase-sensitive colocalization in nuclear speckles with the RNA helicase p68 and the RNA binding protein SC35. A K45A point mutation defective in RNA binding was still active in DNA demethylation. This suggests that RNA binding is not absolutely essential for demethylation of an artificial substrate. A point mutation at G39 impared RNA binding, nuclear speckle localization and DNA demethylation, emphasizing its relevance for Gadd45a function. Significance The results implicate RNA in Gadd45a function and suggest that Gadd45a is associated with a ribonucleoprotein particle. PMID:21249130

On the value of nuclear and mitochondrial gene sequences for reconstructing the phylogeny of vanilloid orchids (Vanilloideae, Orchidaceae)

PubMed Central

Cameron, Kenneth M.

2009-01-01

Background and Aims Most molecular phylogenetic studies of Orchidaceae have relied heavily on DNA sequences from the plastid genome. Nuclear and mitochondrial loci have only been superficially examined for their systematic value. Since 40% of the genera within Vanilloideae are achlorophyllous mycoheterotrophs, this is an ideal group of orchids in which to evaluate non-plastid gene sequences. Methods Phylogenetic reconstructions for Vanilloideae were produced using independent and combined data from the nuclear 18S, 5·8S and 26S rDNA genes and the mitochondrial atpA gene and nad1b-c intron. Key Results These new data indicate placements for genera such as Lecanorchis and Galeola, for which plastid gene sequences have been mostly unavailable. Nuclear and mitochondrial parsimony jackknife trees are congruent with each other and previously published trees based solely on plastid data. Because of high rates of sequence divergence among vanilloid orchids, even the short 5·8S rDNA gene provides impressive levels of resolution and support. Conclusions Orchid systematists are encouraged to sequence nuclear and mitochondrial gene regions along with the growing number of plastid loci available. PMID:19251715

Organization and evolution of highly repeated satellite DNA sequences in plant chromosomes.

PubMed

Sharma, S; Raina, S N

2005-01-01

A major component of the plant nuclear genome is constituted by different classes of repetitive DNA sequences. The structural, functional and evolutionary aspects of the satellite repetitive DNA families, and their organization in the chromosomes is reviewed. The tandem satellite DNA sequences exhibit characteristic chromosomal locations, usually at subtelomeric and centromeric regions. The repetitive DNA family(ies) may be widely distributed in a taxonomic family or a genus, or may be specific for a species, genome or even a chromosome. They may acquire large-scale variations in their sequence and copy number over an evolutionary time-scale. These features have formed the basis of extensive utilization of repetitive sequences for taxonomic and phylogenetic studies. Hybrid polyploids have especially proven to be excellent models for studying the evolution of repetitive DNA sequences. Recent studies explicitly show that some repetitive DNA families localized at the telomeres and centromeres have acquired important structural and functional significance. The repetitive elements are under different evolutionary constraints as compared to the genes. Satellite DNA families are thought to arise de novo as a consequence of molecular mechanisms such as unequal crossing over, rolling circle amplification, replication slippage and mutation that constitute "molecular drive". Copyright 2005 S. Karger AG, Basel.

Conditionally controlling nuclear trafficking in yeast by chemical-induced protein dimerization

PubMed Central

Xu, Tao; Johnson, Cole A; Gestwicki, Jason E; Kumar, Anuj

2016-01-01

We present here a protocol to conditionally control the nuclear trafficking of target proteins in yeast. In this system, rapamycin is used to heterodimerize two chimeric proteins. one chimera consists of a FK506-binding protein (FKBp12) fused to a cellular ‘address’ (nuclear localization signal or nuclear export sequence). the second chimera consists of a target protein fused to a fluorescent protein and the FKBp12-rapamycin-binding (FrB) domain from FKBp-12-rapamycin associated protein 1 (Frap1, also known as mtor). rapamycin induces dimerization of the FKBp12- and FrB-containing chimeras; these interactions selectively place the target protein under control of the cell address, thereby directing the protein into or out of the nucleus. By chemical-induced dimerization, protein mislocalization is reversible and enables the identification of conditional loss-of-function and gain-of-function phenotypes, in contrast to other systems that require permanent modification of the targeted protein. Yeast strains for this analysis can be constructed in 1 week, and the technique allows protein mislocalization within 15 min after drug treatment. PMID:21030958

Conditionally controlling nuclear trafficking in yeast by chemical-induced protein dimerization.

PubMed

Xu, Tao; Johnson, Cole A; Gestwicki, Jason E; Kumar, Anuj

2010-11-01

We present here a protocol to conditionally control the nuclear trafficking of target proteins in yeast. In this system, rapamycin is used to heterodimerize two chimeric proteins. One chimera consists of a FK506-binding protein (FKBP12) fused to a cellular 'address' (nuclear localization signal or nuclear export sequence). The second chimera consists of a target protein fused to a fluorescent protein and the FKBP12-rapamycin-binding (FRB) domain from FKBP-12-rapamycin associated protein 1 (FRAP1, also known as mTor). Rapamycin induces dimerization of the FKBP12- and FRB-containing chimeras; these interactions selectively place the target protein under control of the cell address, thereby directing the protein into or out of the nucleus. By chemical-induced dimerization, protein mislocalization is reversible and enables the identification of conditional loss-of-function and gain-of-function phenotypes, in contrast to other systems that require permanent modification of the targeted protein. Yeast strains for this analysis can be constructed in 1 week, and the technique allows protein mislocalization within 15 min after drug treatment.

Promoter- and RNA polymerase II–dependent hsp-16 gene association with nuclear pores in Caenorhabditis elegans

PubMed Central

Rohner, Sabine; Kalck, Veronique; Wang, Xuefei; Ikegami, Kohta; Lieb, Jason D.; Meister, Peter

2013-01-01

Some inducible yeast genes relocate to nuclear pores upon activation, but the general relevance of this phenomenon has remained largely unexplored. Here we show that the bidirectional hsp-16.2/41 promoter interacts with the nuclear pore complex upon activation by heat shock in the nematode Caenorhabditis elegans. Direct pore association was confirmed by both super-resolution microscopy and chromatin immunoprecipitation. The hsp-16.2 promoter was sufficient to mediate perinuclear positioning under basal level conditions of expression, both in integrated transgenes carrying from 1 to 74 copies of the promoter and in a single-copy genomic insertion. Perinuclear localization of the uninduced gene depended on promoter elements essential for induction and required the heat-shock transcription factor HSF-1, RNA polymerase II, and ENY-2, a factor that binds both SAGA and the THO/TREX mRNA export complex. After induction, colocalization with nuclear pores increased significantly at the promoter and along the coding sequence, dependent on the same promoter-associated factors, including active RNA polymerase II, and correlated with nascent transcripts. PMID:23460676

Quantitative analyses of the 3D nuclear landscape recorded with super-resolved fluorescence microscopy.

PubMed

Schmid, Volker J; Cremer, Marion; Cremer, Thomas

2017-07-01

Recent advancements of super-resolved fluorescence microscopy have revolutionized microscopic studies of cells, including the exceedingly complex structural organization of cell nuclei in space and time. In this paper we describe and discuss tools for (semi-) automated, quantitative 3D analyses of the spatial nuclear organization. These tools allow the quantitative assessment of highly resolved different chromatin compaction levels in individual cell nuclei, which reflect functionally different regions or sub-compartments of the 3D nuclear landscape, and measurements of absolute distances between sites of different chromatin compaction. In addition, these tools allow 3D mapping of specific DNA/RNA sequences and nuclear proteins relative to the 3D chromatin compaction maps and comparisons of multiple cell nuclei. The tools are available in the free and open source R packages nucim and bioimagetools. We discuss the use of masks for the segmentation of nuclei and the use of DNA stains, such as DAPI, as a proxy for local differences in chromatin compaction. We further discuss the limitations of 3D maps of the nuclear landscape as well as problems of the biological interpretation of such data. Copyright © 2017 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Manisha; Jamieson, Cara; Lui, Christina

β-catenin is a key mediator of Wnt signaling and its deregulated nuclear accumulation can drive cancer progression. While the central armadillo (Arm) repeats of β-catenin stimulate nuclear entry, the N- and C-terminal “tail” sequences are thought to regulate turnover and transactivation. We show here that the N- and C-tails are also potent transport sequences. The unstructured tails of β-catenin, when individually fused to a GFP-reporter, could enter and exit the nucleus rapidly in live cells. Proximity ligation assays and pull-down assays identified a weak interaction between the tail sequences and the FG-repeats of nucleoporins, consistent with a possible direct translocationmore » of β-catenin through the nuclear pore complex. Extensive alanine mutagenesis of the tail sequences revealed that nuclear translocation of β-catenin was dependent on specific uniformly distributed patches of hydrophobic residues, whereas the mutagenesis of acidic amino acids had no effect. Moreover, the mutation of hydrophobic patches within the N-tail and C-tail of full length β-catenin reduced nuclear transport rate and diminished its ability to activate transcription. We propose that the tail sequences can contribute to β-catenin transport and suggest a possible similar role for hydrophobic unstructured regions in other proteins. - Highlights: • We show that the N- and C-tails of beta-catenin possess nuclear transport activity. • Nuclear transport of the N- or C-tails requires specific hydrophobic amino acids. • Mutagenesis of the N-terminus diminished nuclear entry of full-length beta-catenin. • We propose the N-tail contributes to beta-catenin nuclear entry and transactivation.« less

Oncoprotein p28 GANK binds to RelA and retains NF-kappaB in the cytoplasm through nuclear export.

PubMed

Chen, Yao; Li, Hong Hai; Fu, Jing; Wang, Xue Feng; Ren, Yi Bin; Dong, Li Wei; Tang, Shan Hua; Liu, Shu Qing; Wu, Meng Chao; Wang, Hong Yang

2007-12-01

p28(GANK) (also known as PSMD10, p28 and gankyrin) is an ankyrin repeat anti-apoptotic oncoprotein that is commonly overexpressed in hepatocellular carcinomas and increases the degradation of p53 and Rb. NF-kappaB (nuclear factor-kappaB) is known to be sequestered in the cytoplasm by I kappaB (inhibitor of NF-kappaB) proteins, but much less is known about the cytoplasmic retention of NF-kappaB by other cellular proteins. Here we show that p28(GANK) inhibits NF-kappaB activity. As a nuclear-cytoplasmic shuttling protein, p28(GANK) directly binds to NF-kappaB/RelA and exports RelA from nucleus through a chromosomal region maintenance-1 (CRM-1) dependent pathway, which results in the cytoplasmic retention of NF-kappaB/RelA. We demonstrate that all the ankyrin repeats of p28(GANK) are required for the interaction with RelA and that the N terminus of p28(GANK), which contains the nuclear export sequence (NES), is responsible for suppressing NF-kappaB/RelA nuclear translocation. These results suggest that overexpression of p28(GANK) prevents the nuclear localization and inhibits the activity of NF-kappaB/RelA.

The nuclear matrix protein NMP-1 is the transcription factor YY1.

PubMed Central

Guo, B; Odgren, P R; van Wijnen, A J; Last, T J; Nickerson, J; Penman, S; Lian, J B; Stein, J L; Stein, G S

1995-01-01

NMP-1 was initially identified as a nuclear matrix-associated DNA-binding factor that exhibits sequence-specific recognition for the site IV regulatory element of a histone H4 gene. This distal promoter domain is a nuclear matrix interaction site. In the present study, we show that NMP-1 is the multifunctional transcription factor YY1. Gel-shift and Western blot analyses demonstrate that NMP-1 is immunoreactive with YY1 antibody. Furthermore, purified YY1 protein specifically recognizes site IV and reconstitutes the NMP-1 complex. Western blot and gel-shift analyses indicate that YY1 is present within the nuclear matrix. In situ immunofluorescence studies show that a significant fraction of YY1 is localized in the nuclear matrix, principally but not exclusively associated with residual nucleoli. Our results confirm that NMP-1/YY1 is a ubiquitous protein that is present in both human cells and in rat osteosarcoma ROS 17/2.8 cells. The finding that NMP-1 is identical to YY1 suggests that this transcriptional regulator may mediate gene-matrix interactions. Our results are consistent with the concept that the nuclear matrix may functionally compartmentalize the eukaryotic nucleus to support regulation of gene expression. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7479833

A masked NES in INI1/hSNF5 mediates hCRM1-dependent nuclear export: implications for tumorigenesis

PubMed Central

Craig, Errol; Zhang, Zhi-Kai; Davies, Kelvin P.; Kalpana, Ganjam V.

2002-01-01

INI1 (integrase interactor 1)/hSNF5 is a component of the mammalian SWI/SNF complex and a tumor suppressor mutated in malignant rhabdoid tumors (MRT). We have identified a nuclear export signal (NES) in the highly conserved repeat 2 domain of INI1 that is unmasked upon deletion of a downstream sequence. Mutation of conserved hydrophobic residues within the NES, as well as leptomycin B treatment abrogated the nuclear export. Full-length INI1 specifically associated with hCRM1/exportin1 in vivo and in vitro. A mutant INI1 [INI1(1–319) delG950] found in MRT lacking the 66 C-terminal amino acids mislocalized to the cytoplasm. Full-length INI1 but not the INI1(1–319 delG950) mutant caused flat cell formation and cell cycle arrest in cell lines derived from MRT. Disruption of the NES in the delG950 mutant caused nuclear localization of the protein and restored its ability to cause cell cycle arrest. These observations demonstrate that INI1 has a masked NES that mediates regulated hCRM1/exportin1-dependent nuclear export and we propose that mutations that cause deregulated nuclear export of the protein could lead to tumorigenesis. PMID:11782423

Identification of a subunit of NADH-dehydrogenase as a p49/STRAP-binding protein

PubMed Central

Zhang, Xiaomin; Azhar, Gohar; Helms, Scott; Zhong, Ying; Wei, Jeanne Y

2008-01-01

Background The p49/STRAP (or SRFBP1) protein was recently identified in our laboratory as a cofactor of serum response factor that contributes to the regulation of SRF target genes in the heart. Results In the present study, we report that NDUFAB1, a nuclear encoded subunit of NADH dehydrogenase, represented the majority of the cDNA clones that interacted with p49/STRAP in multiple screenings using the yeast two-hybrid system. The p49/STRAP and NDUFAB1 proteins interacted and co-localized with each other in the cell. The p49/STRAP protein contains four classic nuclear localization sequence motifs, and it was observed to be present predominantly in the nucleus. Overexpression of p49/STRAP altered the intracellular level of NAD, and reduced the NAD/NADH ratio. Overexpression of p49/STRAP also induced the deacetylation of serum response factor. Conclusion These data suggest that p49/STRAP plays a role in the regulation of intracellular processes such as cardiac cellular metabolism, gene expression, and possibly aging. PMID:18230186

Evolutionary Origin of OwlRep, a Megasatellite DNA Associated with Adaptation of Owl Monkeys to Nocturnal Lifestyle

PubMed Central

Nishihara, Hidenori; Stanyon, Roscoe; Kusumi, Junko; Hirai, Hirohisa

2018-01-01

Abstract Rod cells of many nocturnal mammals have a “non-standard” nuclear architecture, which is called the inverted nuclear architecture. Heterochromatin localizes to the central region of the nucleus. This leads to an efficient light transmission to the outer segments of photoreceptors. Rod cells of diurnal mammals have the conventional nuclear architecture. Owl monkeys (genus Aotus) are the only taxon of simian primates that has a nocturnal or cathemeral lifestyle, and this adaptation is widely thought to be secondary. Their rod cells were shown to exhibit an intermediate chromatin distribution: a spherical heterochromatin block was found in the central region of the nucleus although it was less complete than that of typical nocturnal mammals. We recently demonstrated that the primary DNA component of this heterochromatin block was OwlRep, a megasatellite DNA consisting of 187-bp-long repeat units. However, the origin of OwlRep was not known. Here we show that OwlRep was derived from HSAT6, a simple repeat sequence found in the centromere regions of human chromosomes. HSAT6 occurs widely in primates, suggesting that it was already present in the last common ancestor of extant primates. Notably, Strepsirrhini and Tarsiformes apparently carry a single HSAT6 copy, whereas many species of Simiiformes contain multiple copies. Comparison of nucleotide sequences of these copies revealed the entire process of the OwlRep formation. HSAT6, with or without flanking sequences, was segmentally duplicated in New World monkeys. Then, in the owl monkey linage after its divergence from other New World monkeys, a copy of HSAT6 was tandemly amplified, eventually forming a megasatellite DNA. PMID:29294004

The coding region of the UFGT gene is a source of diagnostic SNP markers that allow single-locus DNA genotyping for the assessment of cultivar identity and ancestry in grapevine (Vitis vinifera L.)

PubMed Central

2013-01-01

Background Vitis vinifera L. is one of society’s most important agricultural crops with a broad genetic variability. The difficulty in recognizing grapevine genotypes based on ampelographic traits and secondary metabolites prompted the development of molecular markers suitable for achieving variety genetic identification. Findings Here, we propose a comparison between a multi-locus barcoding approach based on six chloroplast markers and a single-copy nuclear gene sequencing method using five coding regions combined with a character-based system with the aim of reconstructing cultivar-specific haplotypes and genotypes to be exploited for the molecular characterization of 157 V. vinifera accessions. The analysis of the chloroplast target regions proved the inadequacy of the DNA barcoding approach at the subspecies level, and hence further DNA genotyping analyses were targeted on the sequences of five nuclear single-copy genes amplified across all of the accessions. The sequencing of the coding region of the UFGT nuclear gene (UDP-glucose: flavonoid 3-0-glucosyltransferase, the key enzyme for the accumulation of anthocyanins in berry skins) enabled the discovery of discriminant SNPs (1/34 bp) and the reconstruction of 130 V. vinifera distinct genotypes. Most of the genotypes proved to be cultivar-specific, and only few genotypes were shared by more, although strictly related, cultivars. Conclusion On the whole, this technique was successful for inferring SNP-based genotypes of grapevine accessions suitable for assessing the genetic identity and ancestry of international cultivars and also useful for corroborating some hypotheses regarding the origin of local varieties, suggesting several issues of misidentification (synonymy/homonymy). PMID:24298902

MARs and MARBPs: key modulators of gene regulation and disease manifestation.

PubMed

Chattopadhyay, Samit; Pavithra, Lakshminarasimhan

2007-01-01

The DNA in eukaryotic genome is compartmentalized into various domains by a series of loops tethered onto the base of nuclear matrix. Scaffold/Matrix attachment regions (S/MAR) punctuate these attachment sites and govern the nuclear architecture by establishing chromatin boundaries. In this context, specific proteins that interact with and bind to MAR sequences called MAR binding proteins (MARBPs), are of paramount importance, as these sequences spool the proteins that regulate transcription, replication, repair and recombination. Recent evidences also suggest a role for these cis-acting elements in viral integration, replication and transcription, thereby affecting host immune system. Owing to the complex nature of these nucleotide sequences, less is known about the MARBPs that bind to and bring about diverse effects on chromatin architecture and gene function. Several MARBPs have been identified and characterized so far and the list is growing. The fact that most the MARBPs exist in a co-repressor/co-activator complex and bring about gene regulation makes them quintessential for cellular processes. This participation in gene regulation means that any perturbation in the regulation and levels of MARBPs could lead to disease conditions, particularly those caused by abnormal cell proliferation, like cancer. In the present chapter, we discuss the role of MARs and MARBPs in eukaryotic gene regulation, recombination, transcription and viral integration by altering the local chromatin structure and their dysregulation in disease manifestation

Specific interaction of capsid protein and importin-{alpha}/{beta} influences West Nile virus production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhuvanakantham, Raghavan; Chong, Mun-Keat; Ng, Mah-Lee, E-mail: micngml@nus.edu.sg

2009-11-06

West Nile virus (WNV) capsid (C) protein has been shown to enter the nucleus of infected cells. However, the mechanism by which C protein enters the nucleus is unknown. In this study, we have unveiled for the first time that nuclear transport of WNV and Dengue virus C protein is mediated by their direct association with importin-{alpha}. This interplay is mediated by the consensus sequences of bipartite nuclear localization signal located between amino acid residues 85-101 together with amino acid residues 42 and 43 of C protein. Elucidation of biological significance of importin-{alpha}/C protein interaction demonstrated that the binding efficiencymore » of this association influenced the nuclear entry of C protein and virus production. Collectively, this study illustrated the molecular mechanism by which the C protein of arthropod-borne flavivirus enters the nucleus and showed the importance of importin-{alpha}/C protein interaction in the context of flavivirus life-cycle.« less

Ultra-barcoding in cacao (Theobroma spp.; malvaceae) using whole chloroplast genomes and nuclear ribosomal DNA

USDA-ARS?s Scientific Manuscript database

High-throughput next-generation sequencing was used to scan the genome and generate reliable sequence of high copy number regions. Using this method, we examined whole plastid genomes as well as nearly 6000 bases of nuclear ribosomal DNA sequences for nine genotypes of Theobroma cacao and an indivi...

Weighted low-rank sparse model via nuclear norm minimization for bearing fault detection

NASA Astrophysics Data System (ADS)

Du, Zhaohui; Chen, Xuefeng; Zhang, Han; Yang, Boyuan; Zhai, Zhi; Yan, Ruqiang

2017-07-01

It is a fundamental task in the machine fault diagnosis community to detect impulsive signatures generated by the localized faults of bearings. The main goal of this paper is to exploit the low-rank physical structure of periodic impulsive features and further establish a weighted low-rank sparse model for bearing fault detection. The proposed model mainly consists of three basic components: an adaptive partition window, a nuclear norm regularization and a weighted sequence. Firstly, due to the periodic repetition mechanism of impulsive feature, an adaptive partition window could be designed to transform the impulsive feature into a data matrix. The highlight of partition window is to accumulate all local feature information and align them. Then, all columns of the data matrix share similar waveforms and a core physical phenomenon arises, i.e., these singular values of the data matrix demonstrates a sparse distribution pattern. Therefore, a nuclear norm regularization is enforced to capture that sparse prior. However, the nuclear norm regularization treats all singular values equally and thus ignores one basic fact that larger singular values have more information volume of impulsive features and should be preserved as much as possible. Therefore, a weighted sequence with adaptively tuning weights inversely proportional to singular amplitude is adopted to guarantee the distribution consistence of large singular values. On the other hand, the proposed model is difficult to solve due to its non-convexity and thus a new algorithm is developed to search one satisfying stationary solution through alternatively implementing one proximal operator operation and least-square fitting. Moreover, the sensitivity analysis and selection principles of algorithmic parameters are comprehensively investigated through a set of numerical experiments, which shows that the proposed method is robust and only has a few adjustable parameters. Lastly, the proposed model is applied to the wind turbine (WT) bearing fault detection and its effectiveness is sufficiently verified. Compared with the current popular bearing fault diagnosis techniques, wavelet analysis and spectral kurtosis, our model achieves a higher diagnostic accuracy.

Serine 192 in the tiny RS repeat of the adenoviral L4-33K splicing enhancer protein is essential for function and reorganization of the protein to the periphery of viral replication centers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oestberg, Sara, E-mail: sara.ostberg@imbim.uu.se; Toermaenen Persson, Heidi, E-mail: heidi.tormanen.persson@imbim.uu.se; Akusjaervi, Goeran, E-mail: goran.akusjarvi@imbim.uu.se

2012-11-25

The adenovirus L4-33K protein is a key regulator involved in the temporal shift from early to late pattern of mRNA expression from the adenovirus major late transcription unit. L4-33K is a virus-encoded alternative splicing factor, which enhances processing of 3 Prime splice sites with a weak sequence context. Here we show that L4-33K expressed from a plasmid is localized at the nuclear margin of uninfected cells. During an infection L4-33K is relocalized to the periphery of E2A-72K containing viral replication centers. We also show that serine 192 in the tiny RS repeat of the conserved carboxy-terminus of L4-33K, which ismore » critical for the splicing enhancer function of L4-33K, is necessary for the nuclear localization and redistribution of the protein to viral replication sites. Collectively, our results show a good correlation between the activity of L4-33K as a splicing enhancer protein and its localization to the periphery of viral replication centers.« less

Navigating the tip of the genomic iceberg: Next-generation sequencing for plant systematics.

PubMed

Straub, Shannon C K; Parks, Matthew; Weitemier, Kevin; Fishbein, Mark; Cronn, Richard C; Liston, Aaron

2012-02-01

Just as Sanger sequencing did more than 20 years ago, next-generation sequencing (NGS) is poised to revolutionize plant systematics. By combining multiplexing approaches with NGS throughput, systematists may no longer need to choose between more taxa or more characters. Here we describe a genome skimming (shallow sequencing) approach for plant systematics. Through simulations, we evaluated optimal sequencing depth and performance of single-end and paired-end short read sequences for assembly of nuclear ribosomal DNA (rDNA) and plastomes and addressed the effect of divergence on reference-guided plastome assembly. We also used simulations to identify potential phylogenetic markers from low-copy nuclear loci at different sequencing depths. We demonstrated the utility of genome skimming through phylogenetic analysis of the Sonoran Desert clade (SDC) of Asclepias (Apocynaceae). Paired-end reads performed better than single-end reads. Minimum sequencing depths for high quality rDNA and plastome assemblies were 40× and 30×, respectively. Divergence from the reference significantly affected plastome assembly, but relatively similar references are available for most seed plants. Deeper rDNA sequencing is necessary to characterize intragenomic polymorphism. The low-copy fraction of the nuclear genome was readily surveyed, even at low sequencing depths. Nearly 160000 bp of sequence from three organelles provided evidence of phylogenetic incongruence in the SDC. Adoption of NGS will facilitate progress in plant systematics, as whole plastome and rDNA cistrons, partial mitochondrial genomes, and low-copy nuclear markers can now be efficiently obtained for molecular phylogenetics studies.

II. Inhibited Diffusion Driven Surface Transmutations

NASA Astrophysics Data System (ADS)

Chubb, Talbot A.

2006-02-01

This paper is the second of a set of three papers dealing with the role of coherent partitioning as a common element in Low Energy Nuclear Reactions (LENR), by which is meant cold-fusion related processes. This paper discusses the first step in a sequence of four steps that seem to be necessary to explain Iwamura 2-α-addition surface transmutations. Three concepts are examined: salt-metal interface states, sequential tunneling that transitions D+ ions from localized interstitial to Bloch form, and the general applicability of 2-dimensional vs. 3-dimensional symmetry hosting networks.

A COMPARATIVE ANALYSIS BETWEEN FRANCE AND JAPAN ON LOCAL GOVERNMENTS' INVOLVEMENT IN NUCLEAR SAFETY GOVERNANCE

NASA Astrophysics Data System (ADS)

Sugawara, Shin-Etsu; Shiroyama, Hideaki

This paper shows a comparative analysis between France and Japan on the way of the local governments' involvement in nuclear safety governance through some interviews. In France, a law came into force that requires related local governments to establish "Commision Locale d'Information" (CLI), which means the local governments officially involve in nuclear regulatory activity. Meanwhile, in Japan, related local governments substantially involve in the operation of nuclear facilities through the "safety agreements" in spite of the lack of legal authority. As a result of comparative analysis, we can point out some institutional input from French cases as follows: to clarify the local governments' roles in the nuclear regulation system, to establish the official channels of communication among nuclear utilities, national regulatory authorities and local governments, and to stipulate explicitly the transparency as a purpose of safety regulation.

A smart polymeric platform for multistage nucleus-targeted anticancer drug delivery.

PubMed

Zhong, Jiaju; Li, Lian; Zhu, Xi; Guan, Shan; Yang, Qingqing; Zhou, Zhou; Zhang, Zhirong; Huang, Yuan

2015-10-01

Tumor cell nucleus-targeted delivery of antitumor agents is of great interest in cancer therapy, since the nucleus is one of the most frequent targets of drug action. Here we report a smart polymeric conjugate platform, which utilizes stimulus-responsive strategies to achieve multistage nuclear drug delivery upon systemic administration. The conjugates composed of a backbone based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer and detachable nucleus transport sub-units that sensitive to lysosomal enzyme. The sub-units possess a biforked structure with one end conjugated with the model drug, H1 peptide, and the other end conjugated with a novel pH-responsive targeting peptide (R8NLS) that combining the strength of cell penetrating peptide and nuclear localization sequence. The conjugates exhibited prolonged circulation time and excellent tumor homing ability. And the activation of R8NLS in acidic tumor microenvironment facilitated tissue penetration and cellular internalization. Once internalized into the cell, the sub-units were unleashed for nuclear transport through nuclear pore complex. The unique features resulted in 50-fold increase of nuclear drug accumulation relative to the original polymer-drug conjugates in vitro, and excellent in vivo nuclear drug delivery efficiency. Our report provides a strategy in systemic nuclear drug delivery by combining the microenvironment-responsive structure and detachable sub-units. Copyright © 2015 Elsevier Ltd. All rights reserved.

Nuclear Mitochondrial DNA Activates Replication in Saccharomyces cerevisiae

PubMed Central

Chatre, Laurent; Ricchetti, Miria

2011-01-01

The nuclear genome of eukaryotes is colonized by DNA fragments of mitochondrial origin, called NUMTs. These insertions have been associated with a variety of germ-line diseases in humans. The significance of this uptake of potentially dangerous sequences into the nuclear genome is unclear. Here we provide functional evidence that sequences of mitochondrial origin promote nuclear DNA replication in Saccharomyces cerevisiae. We show that NUMTs are rich in key autonomously replicating sequence (ARS) consensus motifs, whose mutation results in the reduction or loss of DNA replication activity. Furthermore, 2D-gel analysis of the mrc1 mutant exposed to hydroxyurea shows that several NUMTs function as late chromosomal origins. We also show that NUMTs located close to or within ARS provide key sequence elements for replication. Thus NUMTs can act as independent origins, when inserted in an appropriate genomic context or affect the efficiency of pre-existing origins. These findings show that migratory mitochondrial DNAs can impact on the replication of the nuclear region they are inserted in. PMID:21408151

Nuclear mitochondrial DNA activates replication in Saccharomyces cerevisiae.

PubMed

Chatre, Laurent; Ricchetti, Miria

2011-03-08

The nuclear genome of eukaryotes is colonized by DNA fragments of mitochondrial origin, called NUMTs. These insertions have been associated with a variety of germ-line diseases in humans. The significance of this uptake of potentially dangerous sequences into the nuclear genome is unclear. Here we provide functional evidence that sequences of mitochondrial origin promote nuclear DNA replication in Saccharomyces cerevisiae. We show that NUMTs are rich in key autonomously replicating sequence (ARS) consensus motifs, whose mutation results in the reduction or loss of DNA replication activity. Furthermore, 2D-gel analysis of the mrc1 mutant exposed to hydroxyurea shows that several NUMTs function as late chromosomal origins. We also show that NUMTs located close to or within ARS provide key sequence elements for replication. Thus NUMTs can act as independent origins, when inserted in an appropriate genomic context or affect the efficiency of pre-existing origins. These findings show that migratory mitochondrial DNAs can impact on the replication of the nuclear region they are inserted in.

Purification and partial sequencing of the nuclear autoantigen RA33 shows that it is indistinguishable from the A2 protein of the heterogeneous nuclear ribonucleoprotein complex.

PubMed Central

Steiner, G; Hartmuth, K; Skriner, K; Maurer-Fogy, I; Sinski, A; Thalmann, E; Hassfeld, W; Barta, A; Smolen, J S

1992-01-01

RA33 is a nuclear autoantigen with an apparent molecular mass of 33 kD. Autoantibodies against RA33 are found in about 30% of sera from RA patients, but only occasionally in sera from patients with other connective tissue diseases. To characterize RA33, the antigen was purified from HeLa cell nuclear extracts to more than 90% homogeneity by affinity chromatography on heparin-Sepharose and by chromatofocusing. Sequence analysis of five tryptic peptides revealed that their sequences matched corresponding sequences of the A2 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex. Furthermore, RA33 was shown to be present in the 40S hnRNP complex and to behave indistinguishably from A2 in binding to single stranded DNA. In summary, these data strongly indicate that RA33 and A2 are the same protein, and thus identify on a molecular level a new autoantigen. Images PMID:1522214

From famine to feast? Selecting nuclear DNA sequence loci for plant species-level phylogeny reconstruction

PubMed Central

Hughes, Colin E; Eastwood, Ruth J; Donovan Bailey, C

2005-01-01

Phylogenetic analyses of DNA sequences have prompted spectacular progress in assembling the Tree of Life. However, progress in constructing phylogenies among closely related species, at least for plants, has been less encouraging. We show that for plants, the rapid accumulation of DNA characters at higher taxonomic levels has not been matched by conventional sequence loci at the species level, leaving a lack of well-resolved gene trees that is hindering investigations of many fundamental questions in plant evolutionary biology. The most popular approach to address this problem has been to use low-copy nuclear genes as a source of DNA sequence data. However, this has had limited success because levels of variation among nuclear intron sequences across groups of closely related species are extremely variable and generally lower than conventionally used loci, and because no universally useful low-copy nuclear DNA sequence loci have been developed. This suggests that solutions will, for the most part, be lineage-specific, prompting a move away from ‘universal’ gene thinking for species-level phylogenetics. The benefits and limitations of alternative approaches to locate more variable nuclear loci are discussed and the potential of anonymous non-genic nuclear loci is highlighted. Given the virtually unlimited number of loci that can be generated using these new approaches, it is clear that effective screening will be critical for efficient selection of the most informative loci. Strategies for screening are outlined. PMID:16553318

A population study of the minicircles in Trypanosoma cruzi: predicting guide RNAs in the absence of empirical RNA editing.

PubMed

Thomas, Sean; Martinez, L L Isadora Trejo; Westenberger, Scott J; Sturm, Nancy R

2007-05-24

The structurally complex network of minicircles and maxicircles comprising the mitochondrial DNA of kinetoplastids mirrors the complexity of the RNA editing process that is required for faithful expression of encrypted maxicircle genes. Although a few of the guide RNAs that direct this editing process have been discovered on maxicircles, guide RNAs are mostly found on the minicircles. The nuclear and maxicircle genomes have been sequenced and assembled for Trypanosoma cruzi, the causative agent of Chagas disease, however the complement of 1.4-kb minicircles, carrying four guide RNA genes per molecule in this parasite, has been less thoroughly characterised. Fifty-four CL Brener and 53 Esmeraldo strain minicircle sequence reads were extracted from T. cruzi whole genome shotgun sequencing data. With these sequences and all published T. cruzi minicircle sequences, 108 unique guide RNAs from all known T. cruzi minicircle sequences and two guide RNAs from the CL Brener maxicircle were predicted using a local alignment algorithm and mapped onto predicted or experimentally determined sequences of edited maxicircle open reading frames. For half of the sequences no statistically significant guide RNA could be assigned. Likely positions of these unidentified gRNAs in T. cruzi minicircle sequences are estimated using a simple Hidden Markov Model. With the local alignment predictions as a standard, the HMM had an ~85% chance of correctly identifying at least 20 nucleotides of guide RNA from a given minicircle sequence. Inter-minicircle recombination was documented. Variable regions contain species-specific areas of distinct nucleotide preference. Two maxicircle guide RNA genes were found. The identification of new minicircle sequences and the further characterization of all published minicircles are presented, including the first observation of recombination between minicircles. Extrapolation suggests a level of 4% recombinants in the population, supporting a relatively high recombination rate that may serve to minimize the persistence of gRNA pseudogenes. Characteristic nucleotide preferences observed within variable regions provide potential clues regarding the transcription and maturation of T. cruzi guide RNAs. Based on these preferences, a method of predicting T. cruzi guide RNAs using only primary minicircle sequence data was created.

Analysis of Sir2E in the cellular slime mold Dictyostelium discoideum: cellular localization, spatial expression and overexpression.

PubMed

Katayama, Takahiro; Yasukawa, Hiro

2008-10-01

It has been reported that Dictyostelium discoideum encodes four silent information regulator 2 (Sir2) proteins (Sir2A-D) showing sequence similarity to human homologues of Sir2 (SIRT1-3). Further screening in a database revealed that D. discoideum encodes an additional Sir2 homologue (Sir2E). The amino acid sequence of Sir2E is not similar to those of SIRTs but is similar to those of proteins encoded by Giardia lamblia, Cryptosporidium hominis and Cryptosporidium parvum. Fluorescence of Sir2E-green fluorescent protein fusion protein was detected in the D. discoideum nucleus, indicating that Sir2E is a nuclear localizing protein. Reverse transcription-polymerase chain reaction and whole-mount in situ hybridization analyses showed that D. discoideum expressed sir2E in amoebae in the growth phase and in prestalk cells in the developmental phase. D. discoideum overexpressing sir2E grew faster than the wild type. These results indicate that Sir2E plays important roles both in the growth phase and developmental phase of D. discoideum.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Griffith, K.D.; Read, E.J.; Carrasquillo, J.A.

Patients with metastatic melanoma undergoing therapy with cyclophosphamide (CPM), tumor-infiltrating lymphocytes (TIL), and interleukin-2 (IL-2) were studied for the ability of their 111In-labeled TIL or peripheral blood lymphocytes (PBL) to localize in sites of tumor using gamma camera imaging and biopsies. Nineteen infusions of radiolabeled TIL were given to 18 patients, while five patients received radiolabeled autologous PBL during TIL therapy. Clear tumor localization was seen on 13 of 18 nuclear scan series performed on 111In-TIL recipients, while tumor was imaged in only one of four scan sequences on patients given 111In-PBL. Nineteen paired biopsies of tumor and normal skinmore » were completed on 10 patients receiving 111In-TIL, while eight biopsies were done on three PBL patients receiving 111In-PBL. The mean percentage of total injectate activity localizing per gram of tumor tissue was 0.0049% in the TIL group and 0.0010% in the PBL group (P2 = .0004). The mean of the tumor to normal skin ratios of the 111In-TIL group was three times that for 111In-PBL (P2 = .0072). One patient was studied by nuclear scanning on three consecutive treatment courses of CPM, TIL, and IL-2. He initially demonstrated clear tumor localization by 111In-TIL at several sites, then faint localization with 111In-PBL at a single site, and subsequently positive tumor imaging on repeat 111In-TIL infusion at multiple sites. These results confirm and expand our initial data demonstrating that human TIL transferred with CPM pretreatment and followed by IL-2 preferentially localize to tumor sites and indicate that this localization is greater for TIL than PBL.« less

Molecular characterization of a nuclear topoisomerase II from Nicotiana tabacum that functionally complements a temperature-sensitive topoisomerase II yeast mutant.

PubMed

Singh, B N; Mudgil, Yashwanti; Sopory, S K; Reddy, M K

2003-07-01

We have successfully expressed enzymatically active plant topoisomerase II in Escherichia coli for the first time, which has enabled its biochemical characterization. Using a PCR-based strategy, we obtained a full-length cDNA and the corresponding genomic clone of tobacco topoisomerase II. The genomic clone has 18 exons interrupted by 17 introns. Most of the 5' and 3' splice junctions follow the typical canonical consensus dinucleotide sequence GU-AG present in other plant introns. The position of introns and phasing with respect to primary amino acid sequence in tobacco TopII and Arabidopsis TopII are highly conserved, suggesting that the two genes are evolved from the common ancestral type II topoisomerase gene. The cDNA encodes a polypeptide of 1482 amino acids. The primary amino acid sequence shows a striking sequence similarity, preserving all the structural domains that are conserved among eukaryotic type II topoisomerases in an identical spatial order. We have expressed the full-length polypeptide in E. coli and purified the recombinant protein to homogeneity. The full-length polypeptide relaxed supercoiled DNA and decatenated the catenated DNA in a Mg(2+)- and ATP-dependent manner, and this activity was inhibited by 4'-(9-acridinylamino)-3'-methoxymethanesulfonanilide (m-AMSA). The immunofluorescence and confocal microscopic studies, with antibodies developed against the N-terminal region of tobacco recombinant topoisomerase II, established the nuclear localization of topoisomerase II in tobacco BY2 cells. The regulated expression of tobacco topoisomerase II gene under the GAL1 promoter functionally complemented a temperature-sensitive TopII(ts) yeast mutant.

Entire plastid phylogeny of the carrot genus (Daucus, Apiaceae): Concordance with nuclear data and mitochondrial and nuclear DNA insertions to the plastid.

PubMed

Spooner, David M; Ruess, Holly; Iorizzo, Massimo; Senalik, Douglas; Simon, Philipp

2017-02-01

We explored the phylogenetic utility of entire plastid DNA sequences in Daucus and compared the results with prior phylogenetic results using plastid and nuclear DNA sequences. We used Illumina sequencing to obtain full plastid sequences of 37 accessions of 20 Daucus taxa and outgroups, analyzed the data with phylogenetic methods, and examined evidence for mitochondrial DNA transfer to the plastid ( Dc MP). Our phylogenetic trees of the entire data set were highly resolved, with 100% bootstrap support for most of the external and many of the internal clades, except for the clade of D. carota and its most closely related species D. syrticus . Subsets of the data, including regions traditionally used as phylogenetically informative regions, provide various degrees of soft congruence with the entire data set. There are areas of hard incongruence, however, with phylogenies using nuclear data. We extended knowledge of a mitochondrial to plastid DNA insertion sequence previously named Dc MP and identified the first instance in flowering plants of a sequence of potential nuclear genome origin inserted into the plastid genome. There is a relationship of inverted repeat junction classes and repeat DNA to phylogeny, but no such relationship with nonsynonymous mutations. Our data have allowed us to (1) produce a well-resolved plastid phylogeny of Daucus , (2) evaluate subsets of the entire plastid data for phylogeny, (3) examine evidence for plastid and nuclear DNA phylogenetic incongruence, and (4) examine mitochondrial and nuclear DNA insertion into the plastid. © 2017 Spooner et al. Published by the Botanical Society of America. This work is licensed under a Creative Commons public domain license (CC0 1.0).

The nuclear proteome of Trypanosoma brucei

PubMed Central

Goos, Carina; Dejung, Mario; Janzen, Christian J.; Butter, Falk

2017-01-01

Trypanosoma brucei is a protozoan flagellate that is transmitted by tsetse flies into the mammalian bloodstream. The parasite has a huge impact on human health both directly by causing African sleeping sickness and indirectly, by infecting domestic cattle. The biology of trypanosomes involves some highly unusual, nuclear-localised processes. These include polycistronic transcription without classical promoters initiated from regions defined by histone variants, trans-splicing of all transcripts to the exon of a spliced leader RNA, transcription of some very abundant proteins by RNA polymerase I and antigenic variation, a switch in expression of the cell surface protein variants that allows the parasite to resist the immune system of its mammalian host. Here, we provide the nuclear proteome of procyclic Trypanosoma brucei, the stage that resides within the tsetse fly midgut. We have performed quantitative label-free mass spectrometry to score 764 significantly nuclear enriched proteins in comparison to whole cell lysates. A comparison with proteomes of several experimentally characterised nuclear and non-nuclear structures and pathways confirmed the high quality of the dataset: the proteome contains about 80% of all nuclear proteins and less than 2% false positives. Using motif enrichment, we found the amino acid sequence KRxR present in a large number of nuclear proteins. KRxR is a sub-motif of a classical eukaryotic monopartite nuclear localisation signal and could be responsible for nuclear localization of proteins in Kinetoplastida species. As a proof of principle, we have confirmed the nuclear localisation of six proteins with previously unknown localisation by expressing eYFP fusion proteins. While proteome data of several T. brucei organelles have been published, our nuclear proteome closes an important gap in knowledge to study trypanosome biology, in particular nuclear-related processes. PMID:28727848

Morphological and molecular differentiation of two new species of Pseudoacanthocephalus Petrochenko, 1958 (Acanthocephala: Echinorhynchidae) from amphibians and reptiles in the Philippines, with identification key for the genus.

PubMed

Tkach, Vasyl V; Lisitsyna, Olga I; Crossley, Janna L; Binh, Tran Thi; Bush, Sarah E

2013-05-01

The genus Pseudoacanthocephalus Petrochenko, 1958 currently includes 14 species of acanthocephalans parasitic in amphibians and reptiles worldwide. This work describes two new species of Pseudoacanthocephalus from amphibians and reptiles collected in several localities on Luzon Island, Philippines. Pseudoacanthocephalus nickoli n. sp. was found in two species of frogs, Rana luzonensis Boulenger and Rana similis (Günther), and Pseudoacanthocephalus smalesi n. sp. was found in a scincid lizard, Sphenomorphus abdictus Brown & Alcala. Differential diagnoses of the two new species of Pseudoacanthocephalus from their congeners are provided. Comparative analysis of nuclear ribosomal rRNA sequences encompassing the 3' end of 18S nuclear rDNA gene, internal transcribed spacer region (ITS1+5.8S+ITS2), and 5' end of the 28S gene strongly corroborated the morphological evidence and demonstrated significant differences between the two new species as well as between these species and closely related species from continental China and Vietnam. No intraspecific sequence variability was detected among different individuals representing each of the examined species. This is the first report of Pseudoacanthocephalus in the Philippines. A key to known species of Pseudoacanthocephalus is provided.

Genetic variability in Melipona quinquefasciata (Hymenoptera, Apidae, Meliponini) from northeastern Brazil determined using the first internal transcribed spacer (ITS1).

PubMed

Pereira, J O P; Freitas, B M; Jorge, D M M; Torres, D C; Soares, C E A; Grangeiro, T B

2009-01-01

Melipona quinquefasciata is a ground-nesting South American stingless bee whose geographic distribution was believed to comprise only the central and southern states of Brazil. We obtained partial sequences (about 500-570 bp) of first internal transcribed spacer (ITS1) nuclear ribosomal DNA from Melipona specimens putatively identified as M. quinquefasciata collected from different localities in northeastern Brazil. To confirm the taxonomic identity of the northeastern samples, specimens from the state of Goiás (Central region of Brazil) were included for comparison. All sequences were deposited in GenBank (accession numbers EU073751-EU073759). The mean nucleotide divergence (excluding sites with insertions/deletions) in the ITS1 sequences was only 1.4%, ranging from 0 to 4.1%. When the sites with insertions/deletions were also taken into account, sequence divergences varied from 0 to 5.3%. In all pairwise comparisons, the ITS1 sequence from the specimens collected in Goiás was most divergent compared to the ITS1 sequences of the bees from the other locations. However, neighbor-joining phylogenetic analysis showed that all ITS1 sequences from northeastern specimens along with the sample of Goiás were resolved in a single clade with a bootstrap support of 100%. The ITS1 sequencing data thus support the occurrence of M. quinquefasciata in northeast Brazil.

Nuclear medicine. Bibliography from Nuclear Science Abstracts, Volumes 31--33

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1976-12-01

References to 4362 publications related to nuclear medicine announced in Nuclear Science Abstracts (NSA) volumes 31(Jan.--June 1975), 32(July--Dec. 1975), and 33(Jan.--June 1976) are contained in this bibliography. References are arranged in order by the original NSA abstract number which approximately places them in chronological order. Sequence numbers appear beside each reference, and the indexes refer to these sequence numbers. Indexes included are: Corporate, Personal Author, Subject, and Report Number.

Genetic Diversity of Sheep Breeds from Albania, Greece, and Italy Assessed by Mitochondrial DNA and Nuclear Polymorphisms (SNPs)

PubMed Central

Pariset, Lorraine; Mariotti, Marco; Gargani, Maria; Joost, Stephane; Negrini, Riccardo; Perez, Trinidad; Bruford, Michael; Ajmone Marsan, Paolo; Valentini, Alessio

2011-01-01

We employed mtDNA and nuclear SNPs to investigate the genetic diversity of sheep breeds of three countries of the Mediterranean basin: Albania, Greece, and Italy. In total, 154 unique mtDNA haplotypes were detected by means of D-loop sequence analysis. The major nucleotide diversity was observed in Albania. We identified haplogroups, A, B, and C in Albanian and Greek samples, while Italian individuals clustered in groups A and B. In general, the data show a pattern reflecting old migrations that occurred in postneolithic and historical times. PCA analysis on SNP data differentiated breeds with good correspondence to geographical locations. This could reflect geographical isolation, selection operated by local sheep farmers, and different flock management and breed admixture that occurred in the last centuries. PMID:22125424

Myosin-1C uses a novel phosphoinositide-dependent pathway for nuclear localization.

PubMed

Nevzorov, Ilja; Sidorenko, Ekaterina; Wang, Weihuan; Zhao, Hongxia; Vartiainen, Maria K

2018-02-01

Accurate control of macromolecule transport between nucleus and cytoplasm underlines several essential biological processes, including gene expression. According to the canonical model, nuclear import of soluble proteins is based on nuclear localization signals and transport factors. We challenge this view by showing that nuclear localization of the actin-dependent motor protein Myosin-1C (Myo1C) resembles the diffusion-retention mechanism utilized by inner nuclear membrane proteins. We show that Myo1C constantly shuttles in and out of the nucleus and that its nuclear localization does not require soluble factors, but is dependent on phosphoinositide binding. Nuclear import of Myo1C is preceded by its interaction with the endoplasmic reticulum, and phosphoinositide binding is specifically required for nuclear import, but not nuclear retention, of Myo1C. Our results therefore demonstrate, for the first time, that membrane association and binding to nuclear partners is sufficient to drive nuclear localization of also soluble proteins, opening new perspectives to evolution of cellular protein sorting mechanisms. © 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

Searching for nuclear export elements in hepatitis D virus RNA.

PubMed

Freitas, Natália; Cunha, Celso

2013-08-12

To search for the presence of cis elements in hepatitis D virus (HDV) genomic and antigenomic RNA capable of promoting nuclear export. We made use of a well characterized chloramphenicol acetyl-transferase reporter system based on plasmid pDM138. Twenty cDNA fragments corresponding to different HDV genomic and antigenomic RNA sequences were inserted in plasmid pDM138, and used in transfection experiments in Huh7 cells. The relative amounts of HDV RNA in nuclear and cytoplasmic fractions were then determined by real-time polymerase chain reaction and Northern blotting. The secondary structure of the RNA sequences that displayed nuclear export ability was further predicted using a web interface. Finally, the sensitivity to leptomycin B was assessed in order to investigate possible cellular pathways involved in HDV RNA nuclear export. Analysis of genomic RNA sequences did not allow identifying an unequivocal nuclear export element. However, two regions were found to promote the export of reporter mRNAs with efficiency higher than the negative controls albeit lower than the positive control. These regions correspond to nucleotides 266-489 and 584-920, respectively. In addition, when analyzing antigenomic RNA sequences a nuclear export element was found in positions 214-417. Export mediated by the nuclear export element of HDV antigenomic RNA is sensitive to leptomycin B suggesting a possible role of CRM1 in this transport pathway. A cis-acting nuclear export element is present in nucleotides 214-417 of HDV antigenomic RNA.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hiraishi, Nobuhiro; Ishida, Yo-ichi; Nagahama, Masami, E-mail: nagahama@my-pharm.ac.jp

Nuclear VCP-like 2 (NVL2) is a chaperone-like nucleolar ATPase of the AAA (ATPase associated with diverse cellular activities) family, which exhibits a high level of amino acid sequence similarity with the cytosolic AAA-ATPase VCP/p97. These proteins generally act on macromolecular complexes to stimulate energy-dependent release of their constituents. We previously showed that NVL2 interacts with RNA processing/degradation machinery containing an RNA helicase MTR4/DOB1 and an exonuclease complex, nuclear exosome, and involved in the biogenesis of 60S ribosomal subunits. These observations implicate NVL2 as a remodeling factor for the MTR4-exosome complex during the maturation of pre-ribosomal particles. Here, we used amore » proteomic screen and identified a WD repeat-containing protein 74 (WDR74) as a factor that specifically dissociates from this complex depending on the ATPase activity of NVL2. WDR74 shows weak amino acid sequence similarity with the yeast ribosome biogenesis protein Nsa1 and is co-localized with NVL2 in the nucleolus. Knockdown of WDR74 decreases 60S ribosome levels. Taken together, our results suggest that WDR74 is a novel regulatory protein of the MTR4-exsosome complex whose interaction is regulated by NVL2 and is involved in ribosome biogenesis. - Highlights: • WDR74 accumulates in MTR4-exosome complex upon expression of dominant-negative NVL2. • WDR74 is co-localized with NVL2 in the nucleolus. • WDR74, along with NVL2, is involved in the synthesis of 60S ribosomal subunits.« less

SPK1 is an essential S-phase-specific gene of Saccharomyces cerevisiae that encodes a nuclear serine/threonine/tyrosine kinase.

PubMed

Zheng, P; Fay, D S; Burton, J; Xiao, H; Pinkham, J L; Stern, D F

1993-09-01

SPK1 was originally discovered in an immunoscreen for tyrosine-protein kinases in Saccharomyces cerevisiae. We have used biochemical and genetic techniques to investigate the function of this gene and its encoded protein. Hybridization of an SPK1 probe to an ordered genomic library showed that SPK1 is adjacent to PEP4 (chromosome XVI L). Sporulation of spk1/+ heterozygotes gave rise to spk1 spores that grew into microcolonies but could not be further propagated. These colonies were greatly enriched for budded cells, especially those with large buds. Similarly, eviction of CEN plasmids bearing SPK1 from cells with a chromosomal SPK1 disruption yielded viable cells with only low frequency. Spk1 protein was identified by immunoprecipitation and immunoblotting. It was associated with protein-Ser, Thr, and Tyr kinase activity in immune complex kinase assays. Spk1 was localized to the nucleus by immunofluorescence. The nucleotide sequence of the SPK1 5' noncoding region revealed that SPK1 contains two MluI cell cycle box elements. These elements confer S-phase-specific transcription to many genes involved in DNA synthesis. Northern (RNA) blotting of synchronized cells verified that the SPK1 transcript is coregulated with other MluI box-regulated genes. The SPK1 upstream region also includes a domain highly homologous to sequences involved in induction of RAD2 and other excision repair genes by agents that induce DNA damage. spk1 strains were hypersensitive to UV irradiation. Taken together, these findings indicate that SPK1 is a dual-specificity (Ser/Thr and Tyr) protein kinase that is essential for viability. The cell cycle-dependent transcription, presence of DNA damage-related sequences, requirement for UV resistance, and nuclear localization of Spk1 all link this gene to a crucial S-phase-specific role, probably as a positive regulator of DNA synthesis.

Comparative analysis of seven viral nuclear export signals (NESs) reveals the crucial role of nuclear export mediated by the third NES consensus sequence of nucleoprotein (NP) in influenza A virus replication.

PubMed

Chutiwitoonchai, Nopporn; Kakisaka, Michinori; Yamada, Kazunori; Aida, Yoko

2014-01-01

The assembly of influenza virus progeny virions requires machinery that exports viral genomic ribonucleoproteins from the cell nucleus. Currently, seven nuclear export signal (NES) consensus sequences have been identified in different viral proteins, including NS1, NS2, M1, and NP. The present study examined the roles of viral NES consensus sequences and their significance in terms of viral replication and nuclear export. Mutation of the NP-NES3 consensus sequence resulted in a failure to rescue viruses using a reverse genetics approach, whereas mutation of the NS2-NES1 and NS2-NES2 sequences led to a strong reduction in viral replication kinetics compared with the wild-type sequence. While the viral replication kinetics for other NES mutant viruses were also lower than those of the wild-type, the difference was not so marked. Immunofluorescence analysis after transient expression of NP-NES3, NS2-NES1, or NS2-NES2 proteins in host cells showed that they accumulated in the cell nucleus. These results suggest that the NP-NES3 consensus sequence is mostly required for viral replication. Therefore, each of the hydrophobic (Φ) residues within this NES consensus sequence (Φ1, Φ2, Φ3, or Φ4) was mutated, and its viral replication and nuclear export function were analyzed. No viruses harboring NP-NES3 Φ2 or Φ3 mutants could be rescued. Consistent with this, the NP-NES3 Φ2 and Φ3 mutants showed reduced binding affinity with CRM1 in a pull-down assay, and both accumulated in the cell nucleus. Indeed, a nuclear export assay revealed that these mutant proteins showed lower nuclear export activity than the wild-type protein. Moreover, the Φ2 and Φ3 residues (along with other Φ residues) within the NP-NES3 consensus were highly conserved among different influenza A viruses, including human, avian, and swine. Taken together, these results suggest that the Φ2 and Φ3 residues within the NP-NES3 protein are important for its nuclear export function during viral replication.

Nuclear import of glucokinase in pancreatic beta-cells is mediated by a nuclear localization signal and modulated by SUMOylation.

PubMed

Johansson, Bente Berg; Fjeld, Karianne; Solheim, Marie Holm; Shirakawa, Jun; Zhang, Enming; Keindl, Magdalena; Hu, Jiang; Lindqvist, Andreas; Døskeland, Anne; Mellgren, Gunnar; Flatmark, Torgeir; Njølstad, Pål Rasmus; Kulkarni, Rohit N; Wierup, Nils; Aukrust, Ingvild; Bjørkhaug, Lise

2017-10-15

The localization of glucokinase in pancreatic beta-cell nuclei is a controversial issue. Although previous reports suggest such a localization, the mechanism for its import has so far not been identified. Using immunofluorescence, subcellular fractionation and mass spectrometry, we present evidence in support of glucokinase localization in beta-cell nuclei of human and mouse pancreatic sections, as well as in human and mouse isolated islets, and murine MIN6 cells. We have identified a conserved, seven-residue nuclear localization signal ( 30 LKKVMRR 36 ) in the human enzyme. Substituting the residues KK 31,32 and RR 35,36 with AA led to a loss of its nuclear localization in transfected cells. Furthermore, our data indicates that SUMOylation of glucokinase modulates its nuclear import, while high glucose concentrations do not significantly alter the enzyme nuclear/cytosolic ratio. Thus, for the first time, we provide data in support of a nuclear import of glucokinase mediated by a redundant mechanism, involving a nuclear localization signal, and which is modulated by its SUMOylation. These findings add new knowledge to the functional role of glucokinase in the pancreatic beta-cell. Copyright © 2017 Elsevier B.V. All rights reserved.

The Hsp90 Inhibitor, 17-AAG, Prevents the Ligand-Independent Nuclear Localization of Androgen Receptor in Refractory Prostate Cancer Cells

PubMed Central

Saporita, Anthony J.; Ai, Junkui; Wang, Zhou

2010-01-01

BACKGROUND Androgen receptor (AR) is the key molecule in androgen-refractory prostate cancer. Despite androgen ablative conditions, AR remains active and is necessary for the growth of androgen-refractory prostate cancer cells. Nuclear localization of AR is a prerequisite for its transcriptional activation. We examined AR localization in androgen-dependent and androgen-refractory prostate cancer cells. METHODS AND RESULTS We demonstrate increased nuclear localization of a GFP-tagged AR in the absence of hormone in androgen-refractory C4-2 cells compared to parental androgen-sensitive human prostate cancer LNCaP cells. Analysis of AR mutants impaired in ligand-binding indicates that the nuclear localization of AR in C4-2 cells is truly androgen-independent. The hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), inhibits basal PSA expression and disrupts the ligand-independent nuclear localization of AR at doses much lower than required to inhibit androgen-induced nuclear import. CONCLUSIONS Hsp90 is a key regulator of ligand-independent nuclear localization and activation of AR in androgen-refractory prostate cancer cells. PMID:17221841

Poly(A) polymerase contains multiple functional domains.

PubMed Central

Raabe, T; Murthy, K G; Manley, J L

1994-01-01

Poly(A) polymerase (PAP) contains regions of similarity with several known protein domains. Through site-directed mutagenesis, we provide evidence that PAP contains a functional ribonucleoprotein-type RNA binding domain (RBD) that is responsible for primer binding, making it the only known polymerase to contain such a domain. The RBD is adjacent to, and probably overlaps with, an apparent catalytic region responsible for polymerization. Despite the presence of sequence similarities, this catalytic domain appears to be distinct from the conserved polymerase module found in a large number of RNA-dependent polymerases. PAP contains two nuclear localization signals (NLSs) in its C terminus, each by itself similar to the consensus bipartite NLS found in many nuclear proteins. Mutagenesis experiments indicate that both signals, which are separated by nearly 140 residues, play important roles in directing PAP exclusively to the nucleus. Surprisingly, basic amino acids in the N-terminal-most NLS are also essential for AAUAAA-dependent polyadenylation but not for nonspecific poly(A) synthesis, suggesting that this region of PAP is involved in interactions both with nuclear targeting proteins and with nuclear polyadenylation factors. The serine/threonine-rich C terminus is multiply phosphorylated, including at sites affected by mutations in either NLS. Images PMID:8164653

The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lawrence, Paul; Schafer, Elizabeth A.; Rieder, Elizabeth, E-mail: elizabeth.rieder@ars.usda.gov

2012-03-30

Picornavirus infection can lead to disruption of nuclear pore traffic, shut-off of cell translation machinery, and cleavage of proteins involved in cellular signal transduction and the innate response to infection. Here, we demonstrated that the FMDV 3C{sup pro} induced the cleavage of nuclear RNA-binding protein Sam68 C-terminus containing the nuclear localization sequence (NLS). Consequently, it stimulated the redistribution of Sam68 to the cytoplasm. The siRNA knockdown of Sam68 resulted in a 1000-fold reduction in viral titers, which prompted us to study the effect of Sam68 on FMDV post-entry events. Interestingly, Sam68 interacts with the internal ribosomal entry site within themore » 5 Prime non-translated region of the FMDV genome, and Sam68 knockdown decreased FMDV IRES-driven activity in vitro suggesting that it could modulate translation of the viral genome. The results uncover a novel role for Sam68 in the context of picornaviruses and the proteolysis of a new cellular target of the FMDV 3C{sup pro}.« less

Loss of a Trans-Splicing nad1 Intron from Geraniaceae and Transfer of the Maturase Gene matR to the Nucleus in Pelargonium

PubMed Central

Grewe, Felix; Zhu, Andan; Mower, Jeffrey P.

2016-01-01

The mitochondrial nad1 gene of seed plants has a complex structure, including four introns in cis or trans configurations and a maturase gene (matR) hosted within the final intron. In the geranium family (Geraniaceae), however, sequencing of representative species revealed that three of the four introns, including one in a trans configuration and another that hosts matR, were lost from the nad1 gene in their common ancestor. Despite the loss of the host intron, matR has been retained as a freestanding gene in most genera of the family, indicating that this maturase has additional functions beyond the splicing of its host intron. In the common ancestor of Pelargonium, matR was transferred to the nuclear genome, where it was split into two unlinked genes that encode either its reverse transcriptase or maturase domain. Both nuclear genes are transcribed and contain predicted mitochondrial targeting signals, suggesting that they express functional proteins that are imported into mitochondria. The nuclear localization and split domain structure of matR in the Pelargonium nuclear genome offers a unique opportunity to assess the function of these two domains using transgenic approaches. PMID:27664178

The viral protein A238L inhibits cyclooxygenase-2 expression through a nuclear factor of activated T cell-dependent transactivation pathway.

PubMed

Granja, Aitor G; Nogal, Maria L; Hurtado, Carolina; Vila, Virginia; Carrascosa, Angel L; Salas, María L; Fresno, Manuel; Revilla, Yolanda

2004-12-17

Cyclooxygenase-2 is transiently induced upon cell activation or viral infections, resulting in inflammation and modulation of the immune response. Here we report that A238L, an African swine fever virus protein, efficiently inhibits cyclooxygenase-2 gene expression in Jurkat T cells and in virus-infected Vero cells. Transfection of Jurkat cells stably expressing A238L with cyclooxygenase-2 promoter-luciferase constructs containing 5'-terminal deletions or mutations in distal or proximal nuclear factor of activated T cell (NFAT) response elements revealed that these sequences are involved in the inhibition induced by A238L. Overexpression of a constitutively active version of the calcium-dependent phosphatase calcineurin or NFAT reversed the inhibition mediated by A238L on cyclooxygenase-2 promoter activation, whereas overexpression of p65 NFkappaB had no effect. A238L does not modify the nuclear localization of NFAT after phorbol 12-myristate 13-acetate/calcium ionophore stimulation. Moreover, we show that the mechanism by which the viral protein down-regulates cyclooxygenase-2 activity does not involve inhibition of the binding between NFAT and its specific DNA sequences into the cyclooxygenase-2 promoter. Strikingly, A238L dramatically inhibited the transactivation mediated by a GAL4-NFAT fusion protein containing the N-terminal transactivation domain of NFAT1. Taken together, these data indicate that A238L down-regulates cyclooxygenase-2 transcription through the NFAT response elements, being NFAT-dependent transactivation implicated in this down-regulation.

Identification of a Signal-Responsive Nuclear Export Sequence in Class II Histone Deacetylases

PubMed Central

McKinsey, Timothy A.; Zhang, Chun Li; Olson, Eric N.

2001-01-01

Activation of muscle-specific genes by the MEF2 transcription factor is inhibited by class II histone deacetylases (HDACs) 4 and 5, which contain carboxy-terminal deacetylase domains and amino-terminal extensions required for association with MEF2. The inhibitory action of HDACs is overcome by myogenic signals which disrupt MEF2-HDAC interactions and stimulate nuclear export of these transcriptional repressors. Nucleocytoplasmic trafficking of HDAC5 is mediated by binding of the chaperone protein 14-3-3 to two phosphoserine residues (Ser-259 and Ser-498) in its amino-terminal extension. Here we show that HDAC4 and -5 each contain a signal-responsive nuclear export sequence (NES) at their extreme carboxy termini. The NES is conserved in another class II HDAC, HDAC7, but is absent in class I HDACs and the HDAC-related corepressor, MEF2-interacting transcription repressor. Our results suggest that this conserved NES is inactive in unphosphorylated HDAC5, which is localized to the nucleus, and that calcium-calmodulin-dependent protein kinase (CaMK)-dependent binding of 14-3-3 to phosphoserines 259 and 498 activates the NES, with consequent export of the transcriptional repressor to the cytoplasm. A single amino acid substitution in this NES is sufficient to retain HDAC5 in the nucleus in the face of CaMK signaling. These findings provide molecular insight into the mechanism by which extracellular cues alter chromatin structure to promote muscle differentiation and other MEF2-regulated processes. PMID:11509672

A 5.8S nuclear ribosomal RNA gene sequence database: applications to ecology and evolution

NASA Technical Reports Server (NTRS)

Cullings, K. W.; Vogler, D. R.

1998-01-01

We complied a 5.8S nuclear ribosomal gene sequence database for animals, plants, and fungi using both newly generated and GenBank sequences. We demonstrate the utility of this database as an internal check to determine whether the target organism and not a contaminant has been sequenced, as a diagnostic tool for ecologists and evolutionary biologists to determine the placement of asexual fungi within larger taxonomic groups, and as a tool to help identify fungi that form ectomycorrhizae.

Elucidation of Motifs in Ribosomal Protein S9 That Mediate Its Nucleolar Localization and Binding to NPM1/Nucleophosmin

PubMed Central

Lindström, Mikael S.

2012-01-01

Biogenesis of eukaryotic ribosomes occurs mainly in a specific subnuclear compartment, the nucleolus, and involves the coordinated assembly of ribosomal RNA and ribosomal proteins. Identification of amino acid sequences mediating nucleolar localization of ribosomal proteins may provide important clues to understand the early steps in ribosome biogenesis. Human ribosomal protein S9 (RPS9), known in prokaryotes as RPS4, plays a critical role in ribosome biogenesis and directly binds to ribosomal RNA. RPS9 is targeted to the nucleolus but the regions in the protein that determine its localization remains unknown. Cellular expression of RPS9 deletion mutants revealed that it has three regions capable of driving nuclear localization of a fused enhanced green fluorescent protein (EGFP). The first region was mapped to the RPS9 N-terminus while the second one was located in the proteins C-terminus. The central and third region in RPS9 also behaved as a strong nucleolar localization signal and was hence sufficient to cause accumulation of EGFP in the nucleolus. RPS9 was previously shown to interact with the abundant nucleolar chaperone NPM1 (nucleophosmin). Evaluating different RPS9 fragments for their ability to bind NPM1 indicated that there are two binding sites for NPM1 on RPS9. Enforced expression of NPM1 resulted in nucleolar accumulation of a predominantly nucleoplasmic RPS9 mutant. Moreover, it was found that expression of a subset of RPS9 deletion mutants resulted in altered nucleolar morphology as evidenced by changes in the localization patterns of NPM1, fibrillarin and the silver stained nucleolar organizer regions. In conclusion, RPS9 has three regions that each are competent for nuclear localization, but only the central region acted as a potent nucleolar localization signal. Interestingly, the RPS9 nucleolar localization signal is residing in a highly conserved domain corresponding to a ribosomal RNA binding site. PMID:23285058

Transforming growth factor beta-independent shuttling of Smad4 between the cytoplasm and nucleus.

PubMed

Pierreux, C E; Nicolás, F J; Hill, C S

2000-12-01

Smad4 plays a pivotal role in all transforming growth factor beta (TGF-beta) signaling pathways. Here we describe six widely expressed alternatively spliced variants of human Smad4 with deletions of different exons in the linker, the region of Smad4 that separates the two well-conserved MH1 and MH2 domains. All these Smad4 variants form complexes with activated Smad2 and Smad3 and are incorporated into DNA-binding complexes with the transcription factor Fast-1, regardless of the amount of linker they contain. However, sequences encoded by exons 5 to 7 in the linker are essential for transcriptional activation. Most importantly, our observation that different Smad4 isoforms have different subcellular localizations has led us to the identification of a functional CRM1-dependent nuclear export signal in the Smad4 linker and a constitutively active nuclear localization signal in the N-terminal MH1 domain. In the absence of TGF-beta signaling, we conclude that Smad4 is rapidly and continuously shuttling between the nucleus and the cytoplasm, the distribution of Smad4 between the nucleus and the cytoplasm being dictated by the relative strengths of the nuclear import and export signals. We demonstrate that inhibition of CRM1-mediated nuclear export by treatment of cells with leptomycin B results in endogenous Smad4 accumulating very rapidly in the nucleus. Endogenous Smad2 and Smad3 are completely unaffected by leptomycin B treatment, indicating that the nucleocytoplasmic shuttling is specific for Smad4. We propose that, upon TGF-beta signaling, complex formation between Smad4 and activated Smad2 or -3 leads to nuclear accumulation of Smad4 through inhibition of its nuclear export. We demonstrate that after prolonged TGF-beta signaling Smad2 becomes dephosphorylated and Smad2 and Smad4 accumulate back in the cytoplasm.

Transforming Growth Factor β-Independent Shuttling of Smad4 between the Cytoplasm and Nucleus

PubMed Central

Pierreux, Christophe E.; Nicolás, Francisco J.; Hill, Caroline S.

2000-01-01

Smad4 plays a pivotal role in all transforming growth factor β (TGF-β) signaling pathways. Here we describe six widely expressed alternatively spliced variants of human Smad4 with deletions of different exons in the linker, the region of Smad4 that separates the two well-conserved MH1 and MH2 domains. All these Smad4 variants form complexes with activated Smad2 and Smad3 and are incorporated into DNA-binding complexes with the transcription factor Fast-1, regardless of the amount of linker they contain. However, sequences encoded by exons 5 to 7 in the linker are essential for transcriptional activation. Most importantly, our observation that different Smad4 isoforms have different subcellular localizations has led us to the identification of a functional CRM1-dependent nuclear export signal in the Smad4 linker and a constitutively active nuclear localization signal in the N-terminal MH1 domain. In the absence of TGF-β signaling, we conclude that Smad4 is rapidly and continuously shuttling between the nucleus and the cytoplasm, the distribution of Smad4 between the nucleus and the cytoplasm being dictated by the relative strengths of the nuclear import and export signals. We demonstrate that inhibition of CRM1-mediated nuclear export by treatment of cells with leptomycin B results in endogenous Smad4 accumulating very rapidly in the nucleus. Endogenous Smad2 and Smad3 are completely unaffected by leptomycin B treatment, indicating that the nucleocytoplasmic shuttling is specific for Smad4. We propose that, upon TGF-β signaling, complex formation between Smad4 and activated Smad2 or -3 leads to nuclear accumulation of Smad4 through inhibition of its nuclear export. We demonstrate that after prolonged TGF-β signaling Smad2 becomes dephosphorylated and Smad2 and Smad4 accumulate back in the cytoplasm. PMID:11074002

Localized population divergence of vervet monkeys (Chlorocebus spp.) in South Africa: evidence from mtDNA

PubMed Central

Turner, Trudy R.; Coetzer, Willem G.; Schmitt, Christopher A.; Lorenz, Joseph G.; Freimer, Nelson B.; Grobler, J. Paul

2015-01-01

Objectives Vervet monkeys are common in most tree-rich areas of South Africa, but their absence from grassland and semi-desert areas of the country suggest potentially restricted and mosaic local population patterns that may have relevance to local phenotype patterns and selection. A portion of the mtDNA control region was sequenced to study patterns of genetic differentiation. Materials and Methods DNA was extracted and mtDNA sequences were obtained from 101 vervet monkeys at 15 localities which represent both an extensive (widely across the distribution range) and intensive (more than one troop at most of the localities) sampling strategy. Analyses utilized Arlequin 3.1, MEGA 6, BEAST v1.5.2 and Network V3.6.1 Results The dataset contained 26 distinct haplotypes, with six populations fixed for single haplotypes. Pairwise P-distance among population pairs showed significant differentiation among most population pairs, but with non-significant differences among populations within some regions. Populations were grouped into three broad clusters in a maximum likelihood phylogenetic tree and a haplotype network. These clusters correspond to (i) north-western, northern and north-eastern parts of the distribution range as well as the northern coastal belt; (ii) central areas of the country; and (iii) southern part of the Indian Ocean coastal belt, and adjacent inland areas. Discussion Apparent patterns of genetic structure correspond to current and past distribution of suitable habitat, geographic barriers to gene flow, geographic distance and female philopatry. However, further work on nuclear markers and other genomic data is necessary to confirm these results. PMID:26265297

Adipogenesis stimulates the nuclear localization of EWS with an increase in its O-GlcNAc glycosylation in 3T3-L1 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Qiang; Kamemura, Kazuo, E-mail: k_kamemura@nagahama-i-bio.ac.jp

2014-07-18

Highlights: • The majority of EWS localizes stably in the cytosol in 3T3-L1 preadipocytes. • Adipogenic stimuli induce the nuclear localization of EWS. • Adipogenesis promotes O-GlcNAcylation of EWS. • O-GlcNAcylation stimulates the recruitment of EWS to the nuclear periphery. - Abstract: Although the Ewing sarcoma (EWS) proto-oncoprotein is found in the nucleus and cytosol and is associated with the cell membrane, the regulatory mechanisms of its subcellular localization are still unclear. Here we found that adipogenic stimuli induce the nuclear localization of EWS in 3T3-L1 cells. Tyrosine phosphorylation in the C-terminal PY-nuclear localization signal of EWS was negative throughoutmore » adipogenesis. Instead, an adipogenesis-dependent increase in O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation of EWS was observed. Pharmacological inactivation of O-GlcNAcase in preadipocytes promoted perinuclear localization of EWS. Our findings suggest that the nuclear localization of EWS is partly regulated by the glycosylation.« less

An Out-of-frame Overlapping Reading Frame in the Ataxin-1 Coding Sequence Encodes a Novel Ataxin-1 Interacting Protein*

PubMed Central

Bergeron, Danny; Lapointe, Catherine; Bissonnette, Cyntia; Tremblay, Guillaume; Motard, Julie; Roucou, Xavier

2013-01-01

Spinocerebellar ataxia type 1 is an autosomal dominant cerebellar ataxia associated with the expansion of a polyglutamine tract within the ataxin-1 (ATXN1) protein. Recent studies suggest that understanding the normal function of ATXN1 in cellular processes is essential to decipher the pathogenesis mechanisms in spinocerebellar ataxia type 1. We found an alternative translation initiation ATG codon in the +3 reading frame of human ATXN1 starting 30 nucleotides downstream of the initiation codon for ATXN1 and ending at nucleotide 587. This novel overlapping open reading frame (ORF) encodes a 21-kDa polypeptide termed Alt-ATXN1 (Alternative ATXN1) with a completely different amino acid sequence from ATXN1. We introduced a hemagglutinin tag in-frame with Alt-ATXN1 in ATXN1 cDNA and showed in cell culture the co-expression of both ATXN1 and Alt-ATXN1. Remarkably, Alt-ATXN1 colocalized and interacted with ATXN1 in nuclear inclusions. In contrast, in the absence of ATXN1 expression, Alt-ATXN1 displays a homogenous nucleoplasmic distribution. Alt-ATXN1 interacts with poly(A)+ RNA, and its nuclear localization is dependent on RNA transcription. Polyclonal antibodies raised against Alt-ATXN1 confirmed the expression of Alt-ATXN1 in human cerebellum expressing ATXN1. These results demonstrate that human ATXN1 gene is a dual coding sequence and that ATXN1 interacts with and controls the subcellular distribution of Alt-ATXN1. PMID:23760502

Few mitochondrial DNA sequences are inserted into the turkey (Meleagris gallopavo) nuclear genome: evolutionary analyses and informativity in the domestic lineage.

PubMed

Schiavo, G; Strillacci, M G; Ribani, A; Bovo, S; Roman-Ponce, S I; Cerolini, S; Bertolini, F; Bagnato, A; Fontanesi, L

2018-06-01

Mitochondrial DNA (mtDNA) insertions have been detected in the nuclear genome of many eukaryotes. These sequences are pseudogenes originated by horizontal transfer of mtDNA fragments into the nuclear genome, producing nuclear DNA sequences of mitochondrial origin (numt). In this study we determined the frequency and distribution of mtDNA-originated pseudogenes in the turkey (Meleagris gallopavo) nuclear genome. The turkey reference genome (Turkey_2.01) was aligned with the reference linearized mtDNA sequence using last. A total of 32 numt sequences (corresponding to 18 numt regions derived by unique insertional events) were identified in the turkey nuclear genome (size ranging from 66 to 1415 bp; identity against the modern turkey mtDNA corresponding region ranging from 62% to 100%). Numts were distributed in nine chromosomes and in one scaffold. They derived from parts of 10 mtDNA protein-coding genes, ribosomal genes, the control region and 10 tRNA genes. Seven numt regions reported in the turkey genome were identified in orthologues positions in the Gallus gallus genome and therefore were present in the ancestral genome that in the Cretaceous originated the lineages of the modern crown Galliformes. Five recently integrated turkey numts were validated by PCR in 168 turkeys of six different domestic populations. None of the analysed numts were polymorphic (i.e. absence of the inserted sequence, as reported in numts of recent integration in other species), suggesting that the reticulate speciation model is not useful for explaining the origin of the domesticated turkey lineage. © 2018 Stichting International Foundation for Animal Genetics.

Psr1, a nuclear localized protein that regulates phosphorus metabolism in Chlamydomonas.

PubMed

Wykoff, D D; Grossman, A R; Weeks, D P; Usuda, H; Shimogawara, K

1999-12-21

Understanding the ways in which phosphorus metabolism is regulated in photosynthetic eukaryotes is critical for optimizing crop productivity and managing aquatic ecosystems in which phosphorus can be a major source of pollution. Here we describe a gene encoding a regulator of phosphorus metabolism, designated Psr1 (phosphorus starvation response), from a photosynthetic eukaryote. The Psr1 protein is critical for acclimation of the unicellular green alga Chlamydomonas reinhardtii to phosphorus starvation. The N-terminal half of Psr1 contains a region similar to myb DNA-binding domains and the C-terminal half possesses glutamine-rich sequences characteristic of transcriptional activators. The level of Psr1 increases at least 10-fold upon phosphate starvation, and immunocytochemical studies demonstrate that this protein is nuclear-localized under both nutrient-replete and phosphorus-starvation conditions. Finally, Psr1 and angiosperm proteins have domains that are similar, suggesting a possible role for Psr1 homologs in the control of phosphorus metabolism in vascular plants. With the identification of regulators such as Psr1 it may become possible to engineer photosynthetic organisms for more efficient utilization of phosphorus and to establish better practices for the management of agricultural lands and natural ecosystems.

Highly conserved D-loop-like nuclear mitochondrial sequences (Numts) in tiger (Panthera tigris).

PubMed

Zhang, Wenping; Zhang, Zhihe; Shen, Fujun; Hou, Rong; Lv, Xiaoping; Yue, Bisong

2006-08-01

Using oligonucleotide primers designed to match hypervariable segments I (HVS-1) of Panthera tigris mitochondrial DNA (mtDNA), we amplified two different PCR products (500 bp and 287 bp) in the tiger (Panthera tigris), but got only one PCR product (287 bp) in the leopard (Panthera pardus). Sequence analyses indicated that the sequence of 287 bp was a D-loop-like nuclear mitochondrial sequence (Numts), indicating a nuclear transfer that occurred approximately 4.8-17 million years ago in the tiger and 4.6-16 million years ago in the leopard. Although the mtDNA D-loop sequence has a rapid rate of evolution, the 287-bp Numts are highly conserved; they are nearly identical in tiger subspecies and only 1.742% different between tiger and leopard. Thus, such sequences represent molecular 'fossils' that can shed light on evolution of the mitochondrial genome and may be the most appropriate outgroup for phylogenetic analysis. This is also proved by comparing the phylogenetic trees reconstructed using the D-loop sequence of snow leopard and the 287-bp Numts as outgroup.

A calmodulin-like protein (LCALA) is a new Leishmania amazonensis candidate for telomere end-binding protein.

PubMed

Morea, Edna G O; Viviescas, Maria Alejandra; Fernandes, Carlos A H; Matioli, Fabio F; Lira, Cristina B B; Fernandez, Maribel F; Moraes, Barbara S; da Silva, Marcelo S; Storti, Camila B; Fontes, Marcos R M; Cano, Maria Isabel N

2017-11-01

Leishmania spp. telomeres are composed of 5'-TTAGGG-3' repeats associated with proteins. We have previously identified LaRbp38 and LaRPA-1 as proteins that bind the G-rich telomeric strand. At that time, we had also partially characterized a protein: DNA complex, named LaGT1, but we could not identify its protein component. Using protein-DNA interaction and competition assays, we confirmed that LaGT1 is highly specific to the G-rich telomeric single-stranded DNA. Three protein bands, with LaGT1 activity, were isolated from affinity-purified protein extracts in-gel digested, and sequenced de novo using mass spectrometry analysis. In silico analysis of the digested peptide identified them as a putative calmodulin with sequences identical to the T. cruzi calmodulin. In the Leishmania genome, the calmodulin ortholog is present in three identical copies. We cloned and sequenced one of the gene copies, named it LCalA, and obtained the recombinant protein. Multiple sequence alignment and molecular modeling showed that LCalA shares homology to most eukaryotes calmodulin. In addition, we demonstrated that LCalA is nuclear, partially co-localizes with telomeres and binds in vivo the G-rich telomeric strand. Recombinant LCalA can bind specifically and with relative affinity to the G-rich telomeric single-strand and to a 3'G-overhang, and DNA binding is calcium dependent. We have described a novel candidate component of Leishmania telomeres, LCalA, a nuclear calmodulin that binds the G-rich telomeric strand with high specificity and relative affinity, in a calcium-dependent manner. LCalA is the first reported calmodulin that binds in vivo telomeric DNA. Copyright © 2017 Elsevier B.V. All rights reserved.

A genetic polymorphism repurposes the G-protein coupled and membrane-associated estrogen receptor GPER to a transcription factor-like molecule promoting paracrine signaling between stroma and breast carcinoma cells

PubMed Central

Pupo, Marco; Bodmer, Alexandre; Berto, Melissa; Maggiolini, Marcello; Dietrich, Pierre-Yves; Picard, Didier

2017-01-01

GPER is a membrane-associated estrogen receptor of the family of G-protein coupled receptors. For breast cancer, the contribution of GPER to promoting the proliferation and migration of both carcinoma cells and cancer-associated fibroblasts (CAFs) in response to estrogen and other agonists has extensively been investigated. Intriguingly, GPER was previously found to be localized to the nucleus in one isolate of breast CAFs. Moreover, this nuclear GPER was shown to bind regulatory sequences of cancer-relevant target genes and to induce their expression. We decided to find out what induces the nuclear localization of GPER, how general this phenomenon is, and what its functional significance is. We discovered that interfering with N-linked glycosylation of GPER, either by mutation of the predicted glycosylation sites or pharmacologically with tunicamycin, drives GPER into the nucleus. Surveying a small set of CAFs from breast cancer biopsies, we found that a relatively common single nucleotide polymorphism, which results in the expression of a GPER variant with the amino acid substitution P16L, is associated with the nuclear localization of GPER. GPER with P16L fails to be glycosylated, presumably because of a conformational effect on the nearby glycosylation sites. GPER P16L is defective for membrane-associated signaling, but instead acts like an estrogen-stimulated transcription factor. In CAFs, it induces the secretion of paracrine factors that promote the migration of carcinoma cells. This raises the possibility that the GPER P16L polymorphism could be a risk factor for breast cancer. PMID:28596490

Toward a DNA Taxonomy of Alpine Rhithrogena (Ephemeroptera: Heptageniidae) Using a Mixed Yule-Coalescent Analysis of Mitochondrial and Nuclear DNA

PubMed Central

Vuataz, Laurent; Sartori, Michel; Wagner, André; Monaghan, Michael T.

2011-01-01

Aquatic larvae of many Rhithrogena mayflies (Ephemeroptera) inhabit sensitive Alpine environments. A number of species are on the IUCN Red List and many recognized species have restricted distributions and are of conservation interest. Despite their ecological and conservation importance, ambiguous morphological differences among closely related species suggest that the current taxonomy may not accurately reflect the evolutionary diversity of the group. Here we examined the species status of nearly 50% of European Rhithrogena diversity using a widespread sampling scheme of Alpine species that included 22 type localities, general mixed Yule-coalescent (GMYC) model analysis of one standard mtDNA marker and one newly developed nDNA marker, and morphological identification where possible. Using sequences from 533 individuals from 144 sampling localities, we observed significant clustering of the mitochondrial (cox1) marker into 31 GMYC species. Twenty-one of these could be identified based on the presence of topotypes (expertly identified specimens from the species' type locality) or unambiguous morphology. These results strongly suggest the presence of both cryptic diversity and taxonomic oversplitting in Rhithrogena. Significant clustering was not detected with protein-coding nuclear PEPCK, although nine GMYC species were congruent with well supported terminal clusters of nDNA. Lack of greater congruence in the two data sets may be the result of incomplete sorting of ancestral polymorphism. Bayesian phylogenetic analyses of both gene regions recovered four of the six recognized Rhithrogena species groups in our samples as monophyletic. Future development of more nuclear markers would facilitate multi-locus analysis of unresolved, closely related species pairs. The DNA taxonomy developed here lays the groundwork for a future revision of the important but cryptic Rhithrogena genus in Europe. PMID:21611178

Activation of Beta-Catenin Signaling in Androgen Receptor–Negative Prostate Cancer Cells

PubMed Central

Wan, Xinhai; Liu, Jie; Lu, Jing-Fang; Tzelepi, Vassiliki; Yang, Jun; Starbuck, Michael W.; Diao, Lixia; Wang, Jing; Efstathiou, Eleni; Vazquez, Elba S.; Troncoso, Patricia; Maity, Sankar N.; Navone, Nora M.

2012-01-01

Purpose To study Wnt/beta-catenin in castrate-resistant prostate cancer (CRPC) and understand its function independently of the beta-catenin–androgen receptor (AR) interaction. Experimental Design We performed beta-catenin immunocytochemical analysis, evaluated TOP-flash reporter activity (a reporter of beta-catenin–mediated transcription), and sequenced the beta-catenin gene in MDA PCa 118a, MDA PCa 118b, MDA PCa 2b, and PC-3 prostate cancer (PCa) cells. We knocked down beta-catenin in AR-negative MDA PCa 118b cells and performed comparative gene-array analysis. We also immunohistochemically analyzed beta-catenin and AR in 27 bone metastases of human CRPCs. Results Beta-catenin nuclear accumulation and TOP-flash reporter activity were high in MDA PCa 118b but not in MDA PCa 2b or PC-3 cells. MDA PCa 118a and 118b cells carry a mutated beta-catenin at codon 32 (D32G). Ten genes were expressed differently (false discovery rate, 0.05) in MDA PCa 118b cells with downregulated beta-catenin. One such gene, hyaluronan synthase 2 (HAS2), synthesizes hyaluronan, a core component of the extracellular matrix. We confirmed HAS2 upregulation in PC-3 cells transfected with D32G-mutant beta-catenin. Finally, we found nuclear localization of beta-catenin in 10 of 27 human tissue specimens; this localization was inversely associated with AR expression (P = 0.056, Fisher’s exact test), suggesting that reduced AR expression enables Wnt/beta-catenin signaling. Conclusion We identified a previously unknown downstream target of beta-catenin, HAS2, in PCa, and found that high beta-catenin nuclear localization and low or no AR expression may define a subpopulation of men with bone-metastatic PCa. These findings may guide physicians in managing these patients. PMID:22298898

Frequent Nuclear/Cytoplasmic Localization of β-Catenin without Exon 3 Mutations in Malignant Melanoma

PubMed Central

Rimm, David L.; Caca, Karel; Hu, Gang; Harrison, Frank B.; Fearon, Eric R.

1999-01-01

β-Catenin has a critical role in E-cadherin-mediated cell-cell adhesion, and it also functions as a downstream signaling molecule in the wnt pathway. Mutations in the putative glycogen synthase kinase 3β phosphorylation sites near the β-catenin amino terminus have been found in some cancers and cancer cell lines. The mutations render β-catenin resistant to regulation by a complex containing the glycogen synthase kinase 3β, adenomatous polyposis coli, and axin proteins. As a result, β-catenin accumulates in the cytosol and nucleus and activates T-cell factor/lymphoid enhancing factor transcription factors. Previously, 6 of 27 melanoma cell lines were found to have β-catenin exon 3 mutations affecting the N-terminal phosphorylation sites (Rubinfeld B, Robbins P, Elgamil M, Albert I, Porfiri E, Polakis P: Stabilization of beta-catenin by genetic defects in melanoma cell lines. Science 1997, 275:1790–1792). To assess the role of β-catenin defects in primary melanomas, we undertook immunohistochemical and DNA sequencing studies in 65 melanoma specimens. Nuclear and/or cytoplasmic localization of β-catenin, a potential indicator of wnt pathway activation, was seen focally within roughly one third of the tumors, though a clonal somatic mutation in β-catenin was found in only one case (codon 45 Ser→Pro). Our findings demonstrate that β-catenin mutations are rare in primary melanoma, in contrast to the situation in melanoma cell lines. Nonetheless, activation of β-catenin, as indicated by its nuclear and/or cytoplasmic localization, appears to be frequent in melanoma, and in some cases, it may reflect focal and transient activation of the wnt pathway within the tumor. PMID:10027390

A genetic polymorphism repurposes the G-protein coupled and membrane-associated estrogen receptor GPER to a transcription factor-like molecule promoting paracrine signaling between stroma and breast carcinoma cells.

PubMed

Pupo, Marco; Bodmer, Alexandre; Berto, Melissa; Maggiolini, Marcello; Dietrich, Pierre-Yves; Picard, Didier

2017-07-18

GPER is a membrane-associated estrogen receptor of the family of G-protein coupled receptors. For breast cancer, the contribution of GPER to promoting the proliferation and migration of both carcinoma cells and cancer-associated fibroblasts (CAFs) in response to estrogen and other agonists has extensively been investigated. Intriguingly, GPER was previously found to be localized to the nucleus in one isolate of breast CAFs. Moreover, this nuclear GPER was shown to bind regulatory sequences of cancer-relevant target genes and to induce their expression. We decided to find out what induces the nuclear localization of GPER, how general this phenomenon is, and what its functional significance is. We discovered that interfering with N-linked glycosylation of GPER, either by mutation of the predicted glycosylation sites or pharmacologically with tunicamycin, drives GPER into the nucleus. Surveying a small set of CAFs from breast cancer biopsies, we found that a relatively common single nucleotide polymorphism, which results in the expression of a GPER variant with the amino acid substitution P16L, is associated with the nuclear localization of GPER. GPER with P16L fails to be glycosylated, presumably because of a conformational effect on the nearby glycosylation sites. GPER P16L is defective for membrane-associated signaling, but instead acts like an estrogen-stimulated transcription factor. In CAFs, it induces the secretion of paracrine factors that promote the migration of carcinoma cells. This raises the possibility that the GPER P16L polymorphism could be a risk factor for breast cancer.

Introduced Scotch broom (Cytisus scoparius) invades the genome of native populations in vulnerable heathland habitats.

PubMed

Rostgaard Nielsen, Lene; Brandes, Ursula; Dahl Kjaer, Erik; Fjellheim, Siri

2016-06-01

Cytisus scoparius is a global invasive species that affects local flora and fauna at the intercontinental level. Its natural distribution spans across Europe, but seeds have also been moved among countries, mixing plants of native and non-native genetic origins. Hybridization between the introduced and native gene pool is likely to threaten both the native gene pool and the local flora. In this study, we address the potential threat of invasive C. scoparius to local gene pools in vulnerable heathlands. We used nuclear single nucleotide polymorphic (SNP) and simple sequence repeat (SSR) markers together with plastid SSR and indel markers to investigate the level and direction of gene flow between invasive and native heathland C. scoparius. Analyses of population structures confirmed the presence of two gene pools: one native and the other invasive. The nuclear genome of the native types was highly introgressed with the invasive genome, and we observed advanced-generation hybrids, suggesting that hybridization has been occurring for several generations. There is asymmetrical gene flow from the invasive to the native gene pool, which can be attributed to higher fecundity in the invasive individuals, measured by the number of flowers and seed pods. Strong spatial genetic structure in plastid markers and weaker structure in nuclear markers suggest that seeds spread over relatively short distances and that gene flow over longer distances is mainly facilitated by pollen dispersal. We further show that the growth habits of heathland plants become more vigorous with increased introgression from the invaders. Implications of the findings are discussed in relation to future management of invading C. scoparius. © 2016 John Wiley & Sons Ltd.

Low-Field Nuclear Polarization Using Nitrogen Vacancy Centers in Diamonds

NASA Astrophysics Data System (ADS)

Hovav, Y.; Naydenov, B.; Jelezko, F.; Bar-Gill, N.

2018-02-01

It was recently demonstrated that bulk nuclear polarization can be obtained using nitrogen vacancy (NV) color centers in diamonds, even at ambient conditions. This is based on the optical polarization of the NV electron spin, and using several polarization transfer methods. One such method is the nuclear orientation via electron spin locking (NOVEL) sequence, where a spin-locked sequence is applied on the NV spin, with a microwave power equal to the nuclear precession frequency. This was performed at relatively high fields, to allow for both polarization transfer and noise decoupling. As a result, this scheme requires accurate magnetic field alignment in order preserve the NV properties. Such a requirement may be undesired or impractical in many practical scenarios. Here we present a new sequence, termed the refocused NOVEL, which can be used for polarization transfer (and detection) even at low fields. Numerical simulations are performed, taking into account both the spin Hamiltonian and spin decoherence, and we show that, under realistic parameters, it can outperform the NOVEL sequence.

Budding yeast chromatin is dispersed in a crowded nucleoplasm in vivo

PubMed Central

Chen, Chen; Lim, Hong Hwa; Shi, Jian; Tamura, Sachiko; Maeshima, Kazuhiro; Surana, Uttam; Gan, Lu

2016-01-01

Chromatin organization has an important role in the regulation of eukaryotic systems. Although recent studies have refined the three-dimensional models of chromatin organization with high resolution at the genome sequence level, little is known about how the most fundamental units of chromatin—nucleosomes—are positioned in three dimensions in vivo. Here we use electron cryotomography to study chromatin organization in the budding yeast Saccharomyces cerevisiae. Direct visualization of yeast nuclear densities shows no evidence of 30-nm fibers. Aside from preribosomes and spindle microtubules, few nuclear structures are larger than a tetranucleosome. Yeast chromatin does not form compact structures in interphase or mitosis and is consistent with being in an “open” configuration that is conducive to high levels of transcription. From our study and those of others, we propose that yeast can regulate its transcription using local nucleosome–nucleosome associations. PMID:27605704

Local dynamic nuclear polarization using quantum point contacts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wald, K.R.; Kouwenhoven, L.P.; McEuen, P.L.

1994-08-15

We have used quantum point contacts (QPCs) to locally create and probe dynamic nuclear polarization (DNP) in GaAs heterostructures in the quantum Hall regime. DNP is created via scattering between spin-polarized Landau level electrons and the Ga and As nuclear spins, and it leads to hysteresis in the dc transport characteristics. The nuclear origin of this hysteresis is demonstrated by nuclear magnetic resonance (NMR). Our results show that QPCs can be used to create and probe local nuclear spin populations, opening up new possibilities for mesoscopic NMR experiments.

High-Resolution Whole-Genome Sequencing Reveals That Specific Chromatin Domains from Most Human Chromosomes Associate with Nucleoli

PubMed Central

van Koningsbruggen, Silvana; Gierliński, Marek; Schofield, Pietá; Martin, David; Barton, Geoffey J.; Ariyurek, Yavuz; den Dunnen, Johan T.

2010-01-01

The nuclear space is mostly occupied by chromosome territories and nuclear bodies. Although this organization of chromosomes affects gene function, relatively little is known about the role of nuclear bodies in the organization of chromosomal regions. The nucleolus is the best-studied subnuclear structure and forms around the rRNA repeat gene clusters on the acrocentric chromosomes. In addition to rDNA, other chromatin sequences also surround the nucleolar surface and may even loop into the nucleolus. These additional nucleolar-associated domains (NADs) have not been well characterized. We present here a whole-genome, high-resolution analysis of chromatin endogenously associated with nucleoli. We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy. The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner. NADs have in common a high density of AT-rich sequence elements, low gene density and a statistically significant enrichment in transcriptionally repressed genes. Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope. PMID:20826608

High-resolution whole-genome sequencing reveals that specific chromatin domains from most human chromosomes associate with nucleoli.

PubMed

van Koningsbruggen, Silvana; Gierlinski, Marek; Schofield, Pietá; Martin, David; Barton, Geoffey J; Ariyurek, Yavuz; den Dunnen, Johan T; Lamond, Angus I

2010-11-01

The nuclear space is mostly occupied by chromosome territories and nuclear bodies. Although this organization of chromosomes affects gene function, relatively little is known about the role of nuclear bodies in the organization of chromosomal regions. The nucleolus is the best-studied subnuclear structure and forms around the rRNA repeat gene clusters on the acrocentric chromosomes. In addition to rDNA, other chromatin sequences also surround the nucleolar surface and may even loop into the nucleolus. These additional nucleolar-associated domains (NADs) have not been well characterized. We present here a whole-genome, high-resolution analysis of chromatin endogenously associated with nucleoli. We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy. The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner. NADs have in common a high density of AT-rich sequence elements, low gene density and a statistically significant enrichment in transcriptionally repressed genes. Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope.

Cdc6 localizes to S- and G2-phase centrosomes in a cell cycle-dependent manner

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Gwang Su; Kang, Jeeheon; Bang, Sung Woong

2015-01-16

Highlights: • Cdc6 protein is a component of the pre-replicative complex required for chromosomal replication initiation. • Cdc6 localized to centrosomes of S and G2 phases in a cell cycle-dependent manner. • The centrosomal localization was governed by centrosomal localization signal sequences of Cdc6. • Deletions or substitution mutations on the centrosomal localization signal interfered with centrosomal localization of the Cdc6 proteins. - Abstract: The Cdc6 protein has been primarily investigated as a component of the pre-replicative complex for the initiation of chromosome replication, which contributes to maintenance of chromosomal integrity. Here, we show that Cdc6 localized to the centrosomesmore » during S and G2 phases of the cell cycle. The centrosomal localization was mediated by Cdc6 amino acid residues 311–366, which are conserved within other Cdc6 homologues and contains a putative nuclear export signal. Deletions or substitutions of the amino acid residues did not allow the proteins to localize to centrosomes. In contrast, DsRed tag fused to the amino acid residues localized to centrosomes. These results indicated that a centrosome localization signal is contained within amino acid residues 311–366. The cell cycle-dependent centrosomal localization of Cdc6 in S and G2 phases suggest a novel function of Cdc6 in centrosomes.« less

The nuclear 18S ribosomal RNA gene as a source of phylogenetic information in the genus Taenia.

PubMed

Yan, Hongbin; Lou, Zhongzi; Li, Li; Ni, Xingwei; Guo, Aijiang; Li, Hongmin; Zheng, Yadong; Dyachenko, Viktor; Jia, Wanzhong

2013-03-01

Most species of the genus Taenia are of considerable medical and veterinary significance. In this study, complete nuclear 18S rRNA gene sequences were obtained from seven members of genus Taenia [Taenia multiceps, Taenia saginata, Taenia asiatica, Taenia solium, Taenia pisiformis, Taenia hydatigena, and Taenia taeniaeformis] and a phylogeny inferred using these sequences. Most of the variable sites fall within the variable regions, V1-V5. We show that sequences from the nuclear 18S ribosomal RNA gene have considerable promise as sources of phylogenetic information within the genus Taenia. Furthermore, given that almost all the variable sites lie within defined variable portions of that gene, it will be appropriate and economical to sequence only those regions for additional species of Taenia.

Growth hormone-specific induction of the nuclear localization of porcine growth hormone receptor in porcine hepatocytes.

PubMed

Lan, H N; Hong, P; Li, R N; Shan, A S; Zheng, X

2017-10-01

The phenomenon of nuclear translocation of growth hormone receptor (GHR) in human, rat, and fish has been reported. To date, this phenomenon has not been described in a domestic animal (such as pig). In addition, the molecular mechanisms of GHR nuclear translocation have not been thoroughly elucidated. To this end, porcine hepatocytes were isolated and used as a cell model. We observed that porcine growth hormone (pGH) can induce porcine GHR's nuclear localization in porcine hepatocytes. Subsequently, the dynamics of pGH-induced pGHR's nuclear localization were analyzed and demonstrated that pGHR's nuclear localization occurs in a time-dependent manner. Next, we explored the mechanism of pGHR nuclear localization using different pGHR ligands, and we demonstrated that pGHR's nuclear translocation is GH(s)-dependent. We also observed that pGHR translocates into cell nuclei in a pGH dimerization-dependent fashion, whereas further experiments indicated that IMPα/β is involved in the nuclear translocation of the pGH-pGHR dimer. The pGH-pGHR dimer may form a pGH-GHR-JAK2 multiple complex in cell nuclei, which would suggest that similar to its function in the cell membrane, the nuclear-localized pGH-pGHR dimer might still have the ability to signal. Copyright © 2017 Elsevier Inc. All rights reserved.

Mitochondrial Genome Sequence of the Legume Vicia faba

PubMed Central

Negruk, Valentine

2013-01-01

The number of plant mitochondrial genomes sequenced exceeds two dozen. However, for a detailed comparative study of different phylogenetic branches more plant mitochondrial genomes should be sequenced. This article presents sequencing data and comparative analysis of mitochondrial DNA (mtDNA) of the legume Vicia faba. The size of the V. faba circular mitochondrial master chromosome of cultivar Broad Windsor was estimated as 588,000 bp with a genome complexity of 387,745 bp and 52 conservative mitochondrial genes; 32 of them encoding proteins, 3 rRNA, and 17 tRNA genes. Six tRNA genes were highly homologous to chloroplast genome sequences. In addition to the 52 conservative genes, 114 unique open reading frames (ORFs) were found, 36 without significant homology to any known proteins and 29 with homology to the Medicago truncatula nuclear genome and to other plant mitochondrial ORFs, 49 ORFs were not homologous to M. truncatula but possessed sequences with significant homology to other plant mitochondrial or nuclear ORFs. In general, the unique ORFs revealed very low homology to known closely related legumes, but several sequence homologies were found between V. faba, Beta vulgaris, Nicotiana tabacum, Vitis vinifera, and even the monocots Oryza sativa and Zea mays. Most likely these ORFs arose independently during angiosperm evolution (Kubo and Mikami, 2007; Kubo and Newton, 2008). Computational analysis revealed in total about 45% of V. faba mtDNA sequence being homologous to the Medicago truncatula nuclear genome (more than to any sequenced plant mitochondrial genome), and 35% of this homology ranging from a few dozen to 12,806 bp are located on chromosome 1. Apparently, mitochondrial rrn5, rrn18, rps10, ATP synthase subunit alpha, cox2, and tRNA sequences are part of transcribed nuclear mosaic ORFs. PMID:23675376

Functional Conservation of the Transportin Nuclear Import Pathway in Divergent Organisms

PubMed Central

Siomi, Mikiko C.; Fromont, Micheline; Rain, Jean-Christophe; Wan, Lili; Wang, Fan; Legrain, Pierre; Dreyfuss, Gideon

1998-01-01

Human transportin1 (hTRN1) is the nuclear import receptor for a group of pre-mRNA/mRNA-binding proteins (heterogeneous nuclear ribonucleoproteins [hnRNP]) represented by hnRNP A1, which shuttle continuously between the nucleus and the cytoplasm. hTRN1 interacts with the M9 region of hnRNP A1, a 38-amino-acid domain rich in Gly, Ser, and Asn, and mediates the nuclear import of M9-bearing proteins in vitro. Saccharomyces cerevisiae transportin (yTRN; also known as YBR017c or Kap104p) has been identified and cloned. To understanding the nuclear import mediated by yTRN, we searched with a yeast two-hybrid system for proteins that interact with it. In an exhaustive screen of the S. cerevisiae genome, the most frequently selected open reading frame was the nuclear mRNA-binding protein, Nab2p. We delineated a ca.-50-amino-acid region in Nab2p, termed NAB35, which specifically binds yTRN and is similar to the M9 motif. NAB35 also interacts with hTRN1 and functions as a nuclear localization signal in mammalian cells. Interestingly, yTRN can also mediate the import of NAB35-bearing proteins into mammalian nuclei in vitro. We also report on additional substrates for TRN as well as sequences of Drosophila melanogaster, Xenopus laevis, and Schizosaccharomyces pombe TRNs. Together, these findings demonstrate that both the M9 signal and the nuclear import machinery utilized by the transportin pathway are conserved in evolution. PMID:9632798

Regulation of Nuclear Import and Export of Negative Cofactor 2*S⃞

PubMed Central

Kahle, Joerg; Piaia, Elisa; Neimanis, Sonja; Meisterernst, Michael; Doenecke, Detlef

2009-01-01

The negative cofactor 2 (NC2) is a protein complex composed of two subunits, NC2α and NC2β, and plays a key role in transcription regulation. Here we investigate whether each subunit contains a nuclear localization signal (NLS) that permits individual crossing of the nuclear membrane or whether nuclear import of NC2α and NC2β depends on heterodimerization. Our results from in vitro binding studies and transfection experiments in cultured cells show that each subunit contains a classical NLS (cNLS) that is recognized by the importin α/β heterodimer. Regardless of the individual cNLSs the two NC2 subunits are translocated as a preassembled complex as co-transfection experiments with wild-type and cNLS-deficient NC2 subunits demonstrate. Ran-dependent binding of the nuclear export receptor Crm1/exportin 1 confirmed the presence of a leucine-rich nuclear export signal (NES) in NC2β. In contrast, NC2α does not exhibit a NES. Our results from interspecies heterokaryon assays suggest that heterodimerization with NC2α masks the NES in NC2β, which prevents nuclear export of the NC2 complex. A mutation in either one of the two cNLSs decreases the extent of importin α/β-mediated nuclear import of the NC2 complex. In addition, the NC2 complex can enter the nucleus via a second pathway, facilitated by importin 13. Because importin 13 binds exclusively to the NC2 complex but not to the individual subunits this alternative import pathway depends on sequence elements distributed among the two subunits. PMID:19204005

Internal Associations of the Acidic Region of Upstream Binding Factor Control Its Nucleolar Localization.

PubMed

Ueshima, Shuhei; Nagata, Kyosuke; Okuwaki, Mitsuru

2017-11-15

Upstream binding factor (UBF) is a member of the high-mobility group (HMG) box protein family, characterized by multiple HMG boxes and a C-terminal acidic region (AR). UBF is an essential transcription factor for rRNA genes and mediates the formation of transcriptionally active chromatin in the nucleolus. However, it remains unknown how UBF is specifically localized to the nucleolus. Here, we examined the molecular mechanisms that localize UBF to the nucleolus. We found that the first HMG box (HMG box 1), the linker region (LR), and the AR cooperatively regulate the nucleolar localization of UBF1. We demonstrated that the AR intramolecularly associates with and attenuates the DNA binding activity of HMG boxes and confers the structured DNA preference to HMG box 1. In contrast, the LR was found to serve as a nuclear localization signal and compete with HMG boxes to bind the AR, permitting nucleolar localization of UBF1. The LR sequence binds DNA and assists the stable chromatin binding of UBF. We also showed that the phosphorylation status of the AR does not clearly affect the localization of UBF1. Our results strongly suggest that associations of the AR with HMG boxes and the LR regulate UBF nucleolar localization. Copyright © 2017 American Society for Microbiology.

Ecological niche modelling and nDNA sequencing support a new, morphologically cryptic beetle species unveiled by DNA barcoding.

PubMed

Hawlitschek, Oliver; Porch, Nick; Hendrich, Lars; Balke, Michael

2011-02-09

DNA sequencing techniques used to estimate biodiversity, such as DNA barcoding, may reveal cryptic species. However, disagreements between barcoding and morphological data have already led to controversy. Species delimitation should therefore not be based on mtDNA alone. Here, we explore the use of nDNA and bioclimatic modelling in a new species of aquatic beetle revealed by mtDNA sequence data. The aquatic beetle fauna of Australia is characterised by high degrees of endemism, including local radiations such as the genus Antiporus. Antiporus femoralis was previously considered to exist in two disjunct, but morphologically indistinguishable populations in south-western and south-eastern Australia. We constructed a phylogeny of Antiporus and detected a deep split between these populations. Diagnostic characters from the highly variable nuclear protein encoding arginine kinase gene confirmed the presence of two isolated populations. We then used ecological niche modelling to examine the climatic niche characteristics of the two populations. All results support the status of the two populations as distinct species. We describe the south-western species as Antiporus occidentalis sp.n. In addition to nDNA sequence data and extended use of mitochondrial sequences, ecological niche modelling has great potential for delineating morphologically cryptic species.

Ecological Niche Modelling and nDNA Sequencing Support a New, Morphologically Cryptic Beetle Species Unveiled by DNA Barcoding

PubMed Central

Hawlitschek, Oliver; Porch, Nick; Hendrich, Lars; Balke, Michael

2011-01-01

Background DNA sequencing techniques used to estimate biodiversity, such as DNA barcoding, may reveal cryptic species. However, disagreements between barcoding and morphological data have already led to controversy. Species delimitation should therefore not be based on mtDNA alone. Here, we explore the use of nDNA and bioclimatic modelling in a new species of aquatic beetle revealed by mtDNA sequence data. Methodology/Principal Findings The aquatic beetle fauna of Australia is characterised by high degrees of endemism, including local radiations such as the genus Antiporus. Antiporus femoralis was previously considered to exist in two disjunct, but morphologically indistinguishable populations in south-western and south-eastern Australia. We constructed a phylogeny of Antiporus and detected a deep split between these populations. Diagnostic characters from the highly variable nuclear protein encoding arginine kinase gene confirmed the presence of two isolated populations. We then used ecological niche modelling to examine the climatic niche characteristics of the two populations. All results support the status of the two populations as distinct species. We describe the south-western species as Antiporus occidentalis sp.n. Conclusion/Significance In addition to nDNA sequence data and extended use of mitochondrial sequences, ecological niche modelling has great potential for delineating morphologically cryptic species. PMID:21347370

Compartmentalization of the yeast meiotic nucleus revealed by analysis of ectopic recombination.

PubMed

Schlecht, Hélène B; Lichten, Michael; Goldman, Alastair S H

2004-11-01

As yeast cells enter meiosis, chromosomes move from a centromere-clustered (Rabl) to a telomere-clustered (bouquet) configuration and then to states of progressive homolog pairing where telomeres are more dispersed. It is uncertain at which stage of this process sequences commit to recombine with each other. Previous analyses using recombination between dispersed homologous sequences (ectopic recombination) support the view that, on average, homologs are aligned end to end by the time of commitment to recombination. We have undertaken further analyses incorporating new inserts, chromosome rearrangements, an alternate mode of recombination initiation, and mutants that disrupt nuclear structure or telomere metabolism. Our findings support previous conclusions and reveal that distance from the nearest telomere is an important parameter influencing recombination between dispersed sequences. In general, the farther dispersed sequences are from their nearest telomere, the less likely they are to engage in ectopic recombination. Neither the mode of initiating recombination nor the formation of the bouquet appears to affect this relationship. We suggest that aspects of telomere localization and behavior influence the organization and mobility of chromosomes along their entire length, during a critical period of meiosis I prophase that encompasses the homology search.

Complete Sequence and Analysis of Coconut Palm (Cocos nucifera) Mitochondrial Genome.

PubMed

Aljohi, Hasan Awad; Liu, Wanfei; Lin, Qiang; Zhao, Yuhui; Zeng, Jingyao; Alamer, Ali; Alanazi, Ibrahim O; Alawad, Abdullah O; Al-Sadi, Abdullah M; Hu, Songnian; Yu, Jun

2016-01-01

Coconut (Cocos nucifera L.), a member of the palm family (Arecaceae), is one of the most economically important crops in tropics, serving as an important source of food, drink, fuel, medicine, and construction material. Here we report an assembly of the coconut (C. nucifera, Oman local Tall cultivar) mitochondrial (mt) genome based on next-generation sequencing data. This genome, 678,653bp in length and 45.5% in GC content, encodes 72 proteins, 9 pseudogenes, 23 tRNAs, and 3 ribosomal RNAs. Within the assembly, we find that the chloroplast (cp) derived regions account for 5.07% of the total assembly length, including 13 proteins, 2 pseudogenes, and 11 tRNAs. The mt genome has a relatively large fraction of repeat content (17.26%), including both forward (tandem) and inverted (palindromic) repeats. Sequence variation analysis shows that the Ti/Tv ratio of the mt genome is lower as compared to that of the nuclear genome and neutral expectation. By combining public RNA-Seq data for coconut, we identify 734 RNA editing sites supported by at least two datasets. In summary, our data provides the second complete mt genome sequence in the family Arecaceae, essential for further investigations on mitochondrial biology of seed plants.

Arachidonate 15-Lipoxygenase 2 as an Endogenous Inhibitor of Prostate Cancer Development

DTIC Science & Technology

2006-03-01

dehydrogenase; NHP, normal human prostate epithelial cells; PCa, prostate cancer; NLS, nuclear localization signal; PPAR -, peroxisome proliferator...cloned, i.e., 15-LOX2sv-a/b/c, are mostly excluded from the nucleus. A potential bi-partite nuclear localization signal (NLS...only partially involved in the nuclear import of 15-LOX2. To elucidate the relationship between nuclear localization , enzymatic activity, and tumor

Exaptation of Bornavirus-Like Nucleoprotein Elements in Afrotherians

PubMed Central

Kobayashi, Yuki; Horie, Masayuki; Nakano, Ayumi; Murata, Koichi; Itou, Takuya; Suzuki, Yoshiyuki

2016-01-01

Endogenous bornavirus-like nucleoprotein elements (EBLNs), the nucleotide sequence elements derived from the nucleoprotein gene of ancient bornavirus-like viruses, have been identified in many animal genomes. Here we show evidence that EBLNs encode functional proteins in their host. Some afrotherian EBLNs were observed to have been maintained for more than 83.3 million years under negative selection. Splice variants were expressed from the genomic loci of EBLNs in elephant, and some were translated into proteins. The EBLN proteins appeared to be localized to the rough endoplasmic reticulum in African elephant cells, in contrast to the nuclear localization of bornavirus N. These observations suggest that afrotherian EBLNs have acquired a novel function in their host. Interestingly, genomic sequences of the first exon and its flanking regions in these EBLN loci were homologous to those of transmembrane protein 106B (TMEM106B). The upstream region of the first exon in the EBLN loci exhibited a promoter activity, suggesting that the ability of these EBLNs to be transcribed in the host cell was gained through capturing a partial duplicate of TMEM106B. In conclusion, our results strongly support for exaptation of EBLNs to encode host proteins in afrotherians. PMID:27518265

Using Partial Genomic Fosmid Libraries for Sequencing CompleteOrganellar Genomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

McNeal, Joel R.; Leebens-Mack, James H.; Arumuganathan, K.

2005-08-26

Organellar genome sequences provide numerous phylogenetic markers and yield insight into organellar function and molecular evolution. These genomes are much smaller in size than their nuclear counterparts; thus, their complete sequencing is much less expensive than total nuclear genome sequencing, making broader phylogenetic sampling feasible. However, for some organisms it is challenging to isolate plastid DNA for sequencing using standard methods. To overcome these difficulties, we constructed partial genomic libraries from total DNA preparations of two heterotrophic and two autotrophic angiosperm species using fosmid vectors. We then used macroarray screening to isolate clones containing large fragments of plastid DNA. Amore » minimum tiling path of clones comprising the entire genome sequence of each plastid was selected, and these clones were shotgun-sequenced and assembled into complete genomes. Although this method worked well for both heterotrophic and autotrophic plants, nuclear genome size had a dramatic effect on the proportion of screened clones containing plastid DNA and, consequently, the overall number of clones that must be screened to ensure full plastid genome coverage. This technique makes it possible to determine complete plastid genome sequences for organisms that defy other available organellar genome sequencing methods, especially those for which limited amounts of tissue are available.« less

The MB2 gene family of Plasmodium species has a unique combination of S1 and GTP-binding domains

PubMed Central

Romero, Lisa C; Nguyen, Thanh V; Deville, Benoit; Ogunjumo, Oluwasanmi; James, Anthony A

2004-01-01

Background Identification and characterization of novel Plasmodium gene families is necessary for developing new anti-malarial therapeutics. The products of the Plasmodium falciparum gene, MB2, were shown previously to have a stage-specific pattern of subcellular localization and proteolytic processing. Results Genes homologous to MB2 were identified in five additional parasite species, P. knowlesi, P. gallinaceum, P. berghei, P. yoelii, and P. chabaudi. Sequence comparisons among the MB2 gene products reveal amino acid conservation of structural features, including putative S1 and GTP-binding domains, and putative signal peptides and nuclear localization signals. Conclusions The combination of domains is unique to this gene family and indicates that MB2 genes comprise a novel family and therefore may be a good target for drug development. PMID:15222903

Characterization of the N-terminal segment used by the barley yellow dwarf virus movement protein to promote interaction with the nuclear membrane of host plant cells.

PubMed

Dennison, Sarah Rachel; Harris, Frederick; Brandenburg, Klaus; Phoenix, David Andrew

2007-11-01

The barley yellow dwarf virus movement protein (BYDV-MP) requires its N-terminal sequence to promote the transport of viral RNA into the nuclear compartment of host plant cells. Here, graphical analysis predicts that this sequence would form a membrane interactive amphiphilic alpha-helix. Confirming this prediction, NT1, a peptide homologue of the BYDV-MP N-terminal sequence, was found to be alpha-helical (65%) in the presence of vesicles mimics of the nuclear membrane. The peptide increased the fluidity of these nuclear membrane mimics (rise in wavenumber of circa 0.5-1.0 cm(-1)) and induced surface pressure changes of 2 mN m(-1) in lipid monolayers with corresponding compositions. Taken with isotherm analysis these results suggest that BYDV-MP forms an N-terminal amphiphilic alpha-helix, which partitions into the nuclear membrane primarily through thermodynamically stable associations with the membrane lipid headgroup region. We speculate that these associations may play a role in targeting of the nuclear membrane by BYDM-MP.

Simulation of concomitant magnetic fields on fast switched gradient coils used in advanced application of MRI

NASA Astrophysics Data System (ADS)

Salinas-Muciño, G.; Torres-García, E.; Hidalgo-Tobon, S.

2012-10-01

The process to produce an MR image includes nuclear alignment, RF excitation, spatial encoding, and image formation. To form an image, it is necessary to perform spatial localization of the MR signals, which is achieved using gradient coils. MRI requires the use of gradient coils that generate magnetic fields, which vary linearly with position over the imaging volume. Safety issues have been a motivation to study deeply the relation between the interaction of gradient magnetic field and the peripheral nerve stimulation. In this work is presented a numerical modeling between the concomitant magnetic fields produced by the gradient coils and the electric field induced in a cube with σ conductivity by the gradient field switching in pulse sequences as Eco planar Imaging (EPI), due to this kind of sequence is the most used in advance applications of magnetic resonance imaging as functional MRI, cardiac imaging or diffusion.

[Polymorphic loci and polymorphism analysis of short tandem repeats within XNP gene].

PubMed

Liu, Qi-Ji; Gong, Yao-Qin; Guo, Chen-Hong; Chen, Bing-Xi; Li, Jiang-Xia; Guo, Yi-Shou

2002-01-01

To select polymorphic short tandem repeat markers within X-linked nuclear protein (XNP) gene, genomic clones which contain XNP gene were recognized by homologous analysis with XNP cDNA. By comparing the cDNA with genomic DNA, non-exonic sequences were identified, and short tandem repeats were selected from non-exonic sequences by using BCM search Launcher. Polymorphisms of the short tandem repeats in Chinese population were evaluated by PCR amplification and PAGE. Five short tandem repeats were identified from XNP gene, two of which were polymorphic. Four and 11 alleles were observed in Chinese population for XNPSTR1 and XNPSTR4, respectively. Heterozygosities were 47% for XNPSTR1 and 70% for XNPSTR4. XNPSTR1 and XNPSTR4 localized within 3' end and intron 10, respectively. Two polymorphic short tandem repeats have been identified within XNP gene and will be useful for linkage analysis and gene diagnosis of XNP gene.

Promoter mapping of the mouse Tcp-10bt gene in transgenic mice identifies essential male germ cell regulatory sequences.

PubMed

Ewulonu, U K; Snyder, L; Silver, L M; Schimenti, J C

1996-03-01

Transgenic mice were generated to localize essential promoter elements in the mouse testis-expressed Tcp-10 genes. These genes are expressed exclusively in male germ cells, and exhibit a diffuse range of transcriptional start sites, possibly due to the absence of a TATA box. A series of transgene constructs containing different amounts of 5' flanking DNA revealed that all sequences necessary for appropriate temporal and tissue-specific transcription of Tcp-10 reside between positions -1 to -973. All transgenic animals containing these sequences expressed a chimeric transgene at high levels, in a pattern that paralleled the endogenous genes. These experiments further defined a 227 bp fragment from -746 to -973 that was absolutely essential for expression. In a gel-shift assay, this 227-bp fragment bound nuclear protein from testis, but not other tissues, to yield two retarded bands. Sequence analysis of this fragment revealed a half-site for the AP-2 transcription factor recognition sequence. Gel shift assays using native or mutant oligonucleotides demonstrated that the putative AP-2 recognition sequence was essential for generating the retarded bands. Since the binding activity is testis-specific, but AP-2 expression is not exclusive to male germ cells, it is possible that transcription of Tcp-10 requires interaction between AP-2 and a germ cell-specific transcription factor.

Identification of the maize gravitropism gene lazy plant1 by a transposon-tagging genome resequencing strategy.

PubMed

Howard, Thomas P; Hayward, Andrew P; Tordillos, Anthony; Fragoso, Christopher; Moreno, Maria A; Tohme, Joe; Kausch, Albert P; Mottinger, John P; Dellaporta, Stephen L

2014-01-01

Since their initial discovery, transposons have been widely used as mutagens for forward and reverse genetic screens in a range of organisms. The problems of high copy number and sequence divergence among related transposons have often limited the efficiency at which tagged genes can be identified. A method was developed to identity the locations of Mutator (Mu) transposons in the Zea mays genome using a simple enrichment method combined with genome resequencing to identify transposon junction fragments. The sequencing library was prepared from genomic DNA by digesting with a restriction enzyme that cuts within a perfectly conserved motif of the Mu terminal inverted repeats (TIR). Paired-end reads containing Mu TIR sequences were computationally identified and chromosomal sequences flanking the transposon were mapped to the maize reference genome. This method has been used to identify Mu insertions in a number of alleles and to isolate the previously unidentified lazy plant1 (la1) gene. The la1 gene is required for the negatively gravitropic response of shoots and mutant plants lack the ability to sense gravity. Using bioinformatic and fluorescence microscopy approaches, we show that the la1 gene encodes a cell membrane and nuclear localized protein. Our Mu-Taq method is readily adaptable to identify the genomic locations of any insertion of a known sequence in any organism using any sequencing platform.

Identification of the Maize Gravitropism Gene lazy plant1 by a Transposon-Tagging Genome Resequencing Strategy

PubMed Central

Howard, Thomas P.; Hayward, Andrew P.; Tordillos, Anthony; Fragoso, Christopher; Moreno, Maria A.; Tohme, Joe; Kausch, Albert P.; Mottinger, John P.; Dellaporta, Stephen L.

2014-01-01

Since their initial discovery, transposons have been widely used as mutagens for forward and reverse genetic screens in a range of organisms. The problems of high copy number and sequence divergence among related transposons have often limited the efficiency at which tagged genes can be identified. A method was developed to identity the locations of Mutator (Mu) transposons in the Zea mays genome using a simple enrichment method combined with genome resequencing to identify transposon junction fragments. The sequencing library was prepared from genomic DNA by digesting with a restriction enzyme that cuts within a perfectly conserved motif of the Mu terminal inverted repeats (TIR). Paired-end reads containing Mu TIR sequences were computationally identified and chromosomal sequences flanking the transposon were mapped to the maize reference genome. This method has been used to identify Mu insertions in a number of alleles and to isolate the previously unidentified lazy plant1 (la1) gene. The la1 gene is required for the negatively gravitropic response of shoots and mutant plants lack the ability to sense gravity. Using bioinformatic and fluorescence microscopy approaches, we show that the la1 gene encodes a cell membrane and nuclear localized protein. Our Mu-Taq method is readily adaptable to identify the genomic locations of any insertion of a known sequence in any organism using any sequencing platform. PMID:24498020

Evolutionarily Conserved Sequence Features Regulate the Formation of the FG Network at the Center of the Nuclear Pore Complex

PubMed Central

Peyro, M.; Soheilypour, M.; Lee, B.L.; Mofrad, M.R.K.

2015-01-01

The nuclear pore complex (NPC) is the portal for bidirectional transportation of cargos between the nucleus and the cytoplasm. While most of the structural elements of the NPC, i.e. nucleoporins (Nups), are well characterized, the exact transport mechanism is still under much debate. Many of the functional Nups are rich in phenylalanine-glycine (FG) repeats and are believed to play the key role in nucleocytoplasmic transport. We present a bioinformatics study conducted on more than a thousand FG Nups across 252 species. Our results reveal the regulatory role of polar residues and specific sequences of charged residues, named ‘like charge regions’ (LCRs), in the formation of the FG network at the center of the NPC. Positively charged LCRs prepare the environment for negatively charged cargo complexes and regulate the size of the FG network. The low number density of charged residues in these regions prevents FG domains from forming a relaxed coil structure. Our results highlight the significant role of polar interactions in FG network formation at the center of the NPC and demonstrate that the specific localization of LCRs, FG motifs, charged, and polar residues regulate the formation of the FG network at the center of the NPC. PMID:26541386

Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A.

PubMed

Wiley, J C; Wailes, L A; Idzerda, R L; McKnight, G S

1999-03-05

Regulation of protein kinase A by subcellular localization may be critical to target catalytic subunits to specific substrates. We employed epitope-tagged catalytic subunit to correlate subcellular localization and gene-inducing activity in the presence of regulatory subunit or protein kinase inhibitor (PKI). Transiently expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking the nuclear export signal blocked gene induction but not nuclear accumulation, demonstrating that nuclear export is not essential to inhibit gene induction. When the catalytic subunit was targeted to the nucleus with a nuclear localization signal, it was not sequestered in the cytoplasm by regulatory subunit, although its activity was completely inhibited. PKI redistributed the nuclear catalytic subunit to the cytoplasm and blocked gene induction, demonstrating that the nuclear export signal of PKI can override a strong nuclear localization signal. With increasing PKI, the export process appeared to saturate, resulting in the return of catalytic subunit to the nucleus. These results demonstrate that both the regulatory subunit and PKI are able to completely inhibit the gene-inducing activity of the catalytic subunit even when the catalytic subunit is forced to concentrate in the nuclear compartment.

A New Member of the Sin3 Family of Corepressors Is Essential for Cell Viability and Required for Retroelement Propagation in Fission Yeast

PubMed Central

Dang, Van Dinh; Benedik, Michael J.; Ekwall, Karl; Choi, Jeannie; Allshire, Robin C.; Levin, Henry L.

1999-01-01

Tf1 is a long terminal repeat (LTR)-containing retrotransposon that propagates within the fission yeast Schizosaccharomyces pombe. LTR-retrotransposons possess significant similarity to retroviruses and therefore serve as retrovirus models. To determine what features of the host cell are important for the proliferation of this class of retroelements, we screened for mutations in host genes that reduced the transposition activity of Tf1. We report here the isolation and characterization of pst1+, a gene required for Tf1 transposition. The predicted amino acid sequence of Pst1p possessed high sequence homology with the Sin3 family of proteins, known for their interaction with histone deacetylases. However, unlike the SIN3 gene of Saccharomyces cerevisiae, pst1+ is essential for cell viability. Immunofluorescence microscopy indicated that Pst1p was localized in the nucleus. Consistent with the critical role previously reported for Sin3 proteins in the histone acetylation process, we found that the growth of the strain with the pst1-1 allele was supersensitive to the specific histone deacetylase inhibitor trichostatin A. However, our analysis of strains with the pst1-1 mutation was unable to detect any changes in the acetylation of specific lysines of histones H3 and H4 as measured in bulk chromatin. Interestingly, the pst1-1 mutant strain produced wild-type levels of Tf1-encoded proteins and cDNA, indicating that the defect in transposition occurred after reverse transcription. The results of immunofluorescence microscopy showed that the nuclear localization of the Tf1 capsid protein was disrupted in the strain with the pst1-1 mutation, indicating an important role of pst1+ in modulating the nuclear import of Tf1 virus-like particles. PMID:10022921

Analysis and Quantitation of NF-[kappa]B Nuclear Translocation in Tumor Necrosis Factor Alpha (TNF-[alpha]) Activated Vascular Endothelial Cells

NASA Astrophysics Data System (ADS)

Fuseler, John W.; Merrill, Dana M.; Rogers, Jennifer A.; Grisham, Matthew B.; Wolf, Robert E.

2006-07-01

Nuclear factor kappa B (NF-[kappa]B) is a heterodimeric transcription factor typically composed of p50 and p65 subunits and is a pleiotropic regulator of various inflammatory and immune responses. In quiescent cells, p50/p65 dimers are sequestered in the cytoplasm bound to its inhibitors, the I-[kappa]Bs, which prevent entry into the nucleus. Following cellular stimulation, the I-[kappa]Bs are rapidly degraded, activating NF-[kappa]B. The active form of NF-[kappa]B rapidly translocates into the nucleus, binding to consensus sequences in the promoter/enhancer region of various genes, promoting their transcription. In human vascular endothelial cells activated with tumor necrosis factor-alpha, the activation and translocation of NF-[kappa]B is rapid, reaching maximal nuclear localization by 30 min. In this study, the appearance of NF-[kappa]B (p65 subunit, p65-NF-[kappa]B) in the nucleus visualized by immunofluorescence and quantified by morphometric image analysis (integrated optical density, IOD) is compared to the appearance of activated p65-NF-[kappa]B protein in the nucleus determined biochemically. The appearance of p65-NF-[kappa]B in the nucleus measured by fluorescence image analysis and biochemically express a linear correlation (R2 = 0.9477). These data suggest that localization and relative protein concentrations of NF-[kappa]B can be reliably determined from IOD measurements of the immunofluorescent labeled protein.

Cytoplasmically Retargeted HSV1-tk/GFP Reporter Gene Mutants for Optimization of Noninvasive Molecular-Genetic Imaging

PubMed Central

Ponomarev, Vladimir; Doubrovin, Michael; Serganova, Inna; Beresten, Tatiana; Vider, Jelena; Shavrin, Aleksander; Ageyeva, Ludmila; Balatoni, Julius; Blasberg, Ronald; Tjuvajev, Juri Gelovani

2003-01-01

Abstract To optimize the sensitivity of imaging HSV1-tk/GFP reporter gene expression, a series of HSV1-tk/GFP mutants was developed with altered nuclear localization and better cellular enzymatic activity, compared to that of the native HSV1-tk/GFP fusion protein (HSV1-tk/GFP). Several modifications of HSV1-tk/GFP reporter gene were performed, including targeted inactivating mutations in the nuclear localization signal (NLS), the addition of a nuclear export signal (NES), a combination of both mutation types, and a truncation of the first 135 bp of the native hsv1-tk coding sequence containing a “cryptic” testicular promoter and the NLS. A recombinant HSV1-tk/GFP protein and a highly sensitive sandwich enzyme-linked immunosorbent assay for HSV1-tk/GFP were developed to quantitate the amount of reporter gene product in different assays to allow normalization of the data. These different mutations resulted in various degrees of nuclear clearance, predominant cytoplasmic distribution, and increased total cellular enzymatic activity of the HSV1-tk/GFP mutants, compared to native HSV1-tk/GFP when expressed at the same levels. This appears to be the result of improvedmetabolic bioavailability of cytoplasmically retargeted mutant HSV1-tk/GFP enzymes for reaction with the radiolabeled probe (e.g., FIAU). The analysis of enzymatic properties of different HSV1-tk/GFP mutants using FIAU as a substrate revealed no significant differences from that of the native HSV1-tk/GFP. Improved total cellular enzymatic activity of cytoplasmically retargeted HSV1-tk/GFP mutants observed in vitro was confirmed by noninvasive imaging of transduced subcutaneous tumor xenografts bearing these reporters using [131I]FIAU and a γ-camera. PMID:12869307

An Essential Role for COPI in mRNA Localization to Mitochondria and Mitochondrial Function.

PubMed

Zabezhinsky, Dmitry; Slobodin, Boris; Rapaport, Doron; Gerst, Jeffrey E

2016-04-19

Nuclear-encoded mRNAs encoding mitochondrial proteins (mMPs) can localize directly to the mitochondrial surface, yet how mMPs target mitochondria and whether RNA targeting contributes to protein import into mitochondria and cellular metabolism are unknown. Here, we show that the COPI vesicle coat complex is necessary for mMP localization to mitochondria and mitochondrial function. COPI inactivation leads to reduced mMP binding to COPI itself, resulting in the dissociation of mMPs from mitochondria, a reduction in mitochondrial membrane potential, a decrease in protein import in vivo and in vitro, and severe deficiencies in mitochondrial respiration. Using a model mMP (OXA1), we observed that COPI inactivation (or mutation of the potential COPI-interaction site) led to altered mRNA localization and impaired cellular respiration. Overall, COPI-mediated mMP targeting is critical for mitochondrial protein import and function, and transcript delivery to the mitochondria or endoplasmic reticulum is regulated by cis-acting RNA sequences and trans-acting proteins. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Kar5p is required for multiple functions in both inner and outer nuclear envelope fusion in Saccharomyces cerevisiae.

PubMed

Rogers, Jason V; Rose, Mark D

2014-12-02

During mating in the budding yeast Saccharomyces cerevisiae, two haploid nuclei fuse via two sequential membrane fusion steps. SNAREs (i.e., soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and Prm3p mediate outer nuclear membrane fusion, but the inner membrane fusogen remains unknown. Kar5p is a highly conserved transmembrane protein that localizes adjacent to the spindle pole body (SPB), mediates nuclear envelope fusion, and recruits Prm3p adjacent to the SPB. To separate Kar5p's functions, we tested localization, Prm3p recruitment, and nuclear fusion efficiency in various kar5 mutants. All domains and the conserved cysteine residues were essential for nuclear fusion. Several kar5 mutant proteins localized properly but did not mediate Prm3p recruitment; other kar5 mutant proteins localized and recruited Prm3p but were nevertheless defective for nuclear fusion, demonstrating additional functions beyond Prm3p recruitment. We identified one Kar5p domain required for SPB localization, which is dependent on the half-bridge protein Mps3p. Electron microscopy revealed a kar5 mutant that arrests with expanded nuclear envelope bridges, suggesting that Kar5p is required after outer nuclear envelope fusion. Finally, a split-GFP assay demonstrated that Kar5p localizes to both the inner and outer nuclear envelope. These insights suggest a mechanism by which Kar5p mediates inner nuclear membrane fusion. Copyright © 2015 Rogers and Rose.

Kar5p Is Required for Multiple Functions in Both Inner and Outer Nuclear Envelope Fusion in Saccharomyces cerevisiae

PubMed Central

Rogers, Jason V.; Rose, Mark D.

2014-01-01

During mating in the budding yeast Saccharomyces cerevisiae, two haploid nuclei fuse via two sequential membrane fusion steps. SNAREs (i.e., soluble N-ethylmaleimide–sensitive factor attachment protein receptors) and Prm3p mediate outer nuclear membrane fusion, but the inner membrane fusogen remains unknown. Kar5p is a highly conserved transmembrane protein that localizes adjacent to the spindle pole body (SPB), mediates nuclear envelope fusion, and recruits Prm3p adjacent to the SPB. To separate Kar5p’s functions, we tested localization, Prm3p recruitment, and nuclear fusion efficiency in various kar5 mutants. All domains and the conserved cysteine residues were essential for nuclear fusion. Several kar5 mutant proteins localized properly but did not mediate Prm3p recruitment; other kar5 mutant proteins localized and recruited Prm3p but were nevertheless defective for nuclear fusion, demonstrating additional functions beyond Prm3p recruitment. We identified one Kar5p domain required for SPB localization, which is dependent on the half-bridge protein Mps3p. Electron microscopy revealed a kar5 mutant that arrests with expanded nuclear envelope bridges, suggesting that Kar5p is required after outer nuclear envelope fusion. Finally, a split-GFP assay demonstrated that Kar5p localizes to both the inner and outer nuclear envelope. These insights suggest a mechanism by which Kar5p mediates inner nuclear membrane fusion. PMID:25467943

Type 1 IGF Receptor Localization in Paediatric Gliomas: Significant Association with WHO Grading and Clinical Outcome.

PubMed

Clément, Florencia; Martin, Ayelen; Venara, Marcela; de Luján Calcagno, Maria; Mathó, Cecilia; Maglio, Silvana; Lombardi, Mercedes García; Bergadá, Ignacio; Pennisi, Patricia A

2018-06-01

Nuclear localization of insulin-like growth factor receptor type 1 (IGF-1R) has been described as adverse prognostic factor in some cancers. We studied the expression and localization of IGF-1R in paediatric patients with gliomas, as well as its association with World Health Organization (WHO) grading and survival. We conducted a single cohort, prospective study of paediatric patients with gliomas. Samples were taken at the time of the initial surgery; IGF-1R expression and localization were characterized by immunohistochemistry (IHC), subcellular fractionation and western blotting. Tumours (47/53) showed positive staining for IGF-1R by IHC. IGF-1R nuclear labelling was observed in 10/47 cases. IGF-1R staining was mostly non-nuclear in low-grade tumours, while IGF-1R nuclear labelling was predominant in high-grade gliomas (p = 0.0001). Survival was significantly longer in patients with gliomas having non-nuclear IGF-1R localization than in patients with nuclear IGF-1R tumours (p = 0.016). In gliomas, IGF-1R nuclear localization was significantly associated with both high-grade tumours and increased risk of death. Based on a prospective design, we provide evidence of a potential usefulness of intracellular localization of IGF-1R as prognostic factor in paediatric patients with gliomas.

Suppressor of cytokine signaling 1 (SOCS1) limits NFkappaB signaling by decreasing p65 stability within the cell nucleus.

PubMed

Strebovsky, Julia; Walker, Patrick; Lang, Roland; Dalpke, Alexander H

2011-03-01

Suppressor of cytokine signaling (SOCS) proteins are inhibitors of cytoplasmic Janus kinases (Jak) and signal transducer and activator of transcription (STAT) signaling pathways. Previously the authors surprisingly observed that SOCS1 translocated into the nucleus, which was because of the presence of a nuclear localization sequence. This report now hypothesizes that SOCS1 mediates specific functions within the nuclear compartment because it is instantly transported into the nucleus, as shown by photoactivation and live cell imaging in human HEK293 cells. The NFκB component p65 is identified as an interaction partner for SOCS1 but not for other members of the SOCS family. SOCS1 bound to p65 only within the nucleus. By means of its SOCS box domain, SOCS1 operated as a ubiquitin ligase, leading to polyubiquitination and proteasomal degradation of nuclear p65. Thus, SOCS1 limited prolonged p65 signaling and terminated expression of NFκB inducible genes. Using mutants that lack either nuclear translocation or a functional SOCS box, this report identifies genes that are regulated in a manner dependent on the nuclear availability of SOCS1. Data show that beyond its receptor-proximal function in Jak/STAT signaling, SOCS1 also regulates the duration of NFκB signaling within the cell nucleus, thus exerting a heretofore unrecognized function.

Effect of the linkers between the zinc fingers in zinc finger protein 809 on gene silencing and nuclear localization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ichida, Yu, E-mail: ichida-y@ncchd.go.jp; Utsunomiya, Yuko; Onodera, Masafumi

2016-03-18

Zinc finger protein 809 (ZFP809) belongs to the Kruppel-associated box-containing zinc finger protein (KRAB-ZFP) family and functions in repressing the expression of Moloney murine leukemia virus (MoMLV). ZFP809 binds to the primer-binding site (PBS)located downstream of the MoMLV-long terminal repeat (LTR) and induces epigenetic modifications at integration sites, such as repressive histone modifications and de novo DNA methylation. KRAB-ZFPs contain consensus TGEKP linkers between C2H2 zinc fingers. The phosphorylation of threonine residues within linkers leads to the inactivation of zinc finger binding to target sequences. ZFP809 also contains consensus linkers between zinc fingers. However, the function of ZFP809 linkers remainsmore » unknown. In the present study, we constructed ZFP809 proteins containing mutated linkers and examined their ability to silence transgene expression driven by MLV, binding ability to MLV PBS, and cellular localization. The results of the present study revealed that the linkers affected the ability of ZFP809 to silence transgene expression. Furthermore, this effect could be partly attributed to changes in the localization of ZFP809 proteins containing mutated linkers. Further characterization of ZFP809 linkers is required for understanding the functions and features of KRAB-ZFP-containing linkers. - Highlights: • ZFP809 has three consensus linkers between the zinc fingers. • Linkers are required for ZFP809 to silence transgene expression driven by MLV-LTR. • Linkers affect the precise nuclear localization of ZFP809.« less

Preliminary study on the inhibition of nuclear internalization of Tat peptides by conjugation with a receptor-specific peptide and fluorescent dyes

NASA Astrophysics Data System (ADS)

Shen, Duanwen; Liang, Kexiang; Ye, Yunpeng; Tetteh, Elizabeth; Achilefu, Samuel

2006-02-01

Numerous studies have shown that basic Tat peptide (48-57) internalized non-specifically in cells and localized in the nucleus. However, localization of imaging agents in cellular nucleus is not desirable because of the potential mutagenesis. When conjugated to the peptides that undergo receptor-mediated endocytosis, Tat peptide could target specific cells or pathologic tissue. We tested this hypothesis by incorporating a somatostatin receptor-avid peptide (octreotate, Oct) and two different fluorescent dyes, Cypate 2 (Cy2) and fluorescein 5'-carboxlic acid (5-FAM), into the Tat-peptide sequence. In addition to the Cy2 or 5-FAM-labeled Oct conjugated to Tat peptide (Tat) to produce Tat-Oct-Cypate2 or Tat-Oct-5-FAM, we also labeled the Tat the Tat peptide with these dyes (Tat-Cy2 and Tat-5-FAM) to serve as positive control. A somatostatin receptor-positive pancreatic tumor cell line, AR42J, was used to assess cell internalization. The results show that Tat-5-FAM and Tat-Cypate2 localized in both nucleus and cytoplasm of the cells. In contrast to Tat-Oct-Cypate2, which localized in both the cytoplasm and nucleus, Tat-Oct-5-FAM internalized in the cytoplasm but not in the nucleus of AR42J cells. The internalizations were inhibited by adding non-labeled corresponding peptides, suggesting that the endocytoses of each group of labeled and the corresponding unlabeled compounds occurred through a common pathway. Thus, fluorescent probes and endocytosis complex between octreotate and somatostatin receptors in cytoplasm could control nuclear internalization of Tat peptides.

The human haptoglobin gene promoter: interleukin-6-responsive elements interact with a DNA-binding protein induced by interleukin-6.

PubMed Central

Oliviero, S; Cortese, R

1989-01-01

Transcription of the human haptoglobin (Hp) gene is induced by interleukin-6 (IL-6) in the human hepatoma cell line Hep3B. Cis-acting elements responsible for this response are localized within the first 186 bp of the 5'-flanking region. Site-specific mutants of the Hp promoter fused to the chloramphenicol acetyl transferase (CAT) gene were analysed by transient transfection into uninduced and IL-6-treated Hep3B cells. We identified three regions, A, B and C, defined by mutation, which are important for the IL-6 response. Band shift experiments using nuclear extracts from untreated or IL-6-treated cells revealed the presence of IL-6-inducible DNA binding activities when DNA fragments containing the A or the C sequences were used. Competition experiments showed that both sequences bind to the same nuclear factors. Polymers of oligonucleotides containing either the A or the C regions confer IL-6 responsiveness to a truncated SV40 promoter. The B region forms several complexes with specific DNA-binding proteins different from those which bind to the A and C region. The B region complexes are identical in nuclear extracts from IL-6-treated and untreated cells. While important for IL-6 induction in the context of the haptoglobin promoter, the B site does not confer IL-6 inducibility to the SV40 promoter. Our results indicate that the IL-6 response of the haptoglobin promoter is dependent on the presence of multiple, partly redundant, cis-acting elements. Images PMID:2787245

Molecular Characterization of the Calvin Cycle Enzyme Phosphoribulokinase in the Stramenopile Alga Vaucheria litorea and the Plastid Hosting Mollusc Elysia chlorotica

PubMed Central

Rumpho, Mary E.; Pochareddy, Sirisha; Worful, Jared M.; Summer, Elizabeth J.; Bhattacharya, Debashish; Pelletreau, Karen N.; Tyler, Mary S.; Lee, Jungho; Manhart, James R.; Soule, Kara M.

2009-01-01

Phosphoribulokinase (PRK), a nuclear-encoded plastid-localized enzyme unique to the photosynthetic carbon reduction (Calvin) cycle, was cloned and characterized from the stramenopile alga Vaucheria litorea. This alga is the source of plastids for the mollusc (sea slug) Elysia chlorotica which enable the animal to survive for months solely by photoautotrophic CO2 fixation. The 1633-bp V. litorea prk gene was cloned and the coding region, found to be interrupted by four introns, encodes a 405-amino acid protein. This protein contains the typical bipartite target sequence expected of nuclear-encoded proteins that are directed to complex (i.e. four membrane-bound) algal plastids. De novo synthesis of PRK and enzyme activity were detected in E. chlorotica in spite of having been starved of V. litorea for several months. Unlike the algal enzyme, PRK in the sea slug did not exhibit redox regulation. Two copies of partial PRK-encoding genes were isolated from both sea slug and aposymbiotic sea slug egg DNA using PCR. Each copy contains the nucleotide region spanning exon 1 and part of exon 2 of V. litorea prk, including the bipartite targeting peptide. However, the larger prk fragment also includes intron 1. The exon and intron sequences of prk in E. chlorotica and V. litorea are nearly identical. These data suggest that PRK is differentially regulated in V. litorea and E. chlorotica and at least a portion of the V. litorea nuclear PRK gene is present in sea slugs that have been starved for several months. PMID:19995736

Identification of Novel Saccharomyces cerevisiae Proteins with Nuclear Export Activity: Cell Cycle-Regulated Transcription Factor Ace2p Shows Cell Cycle-Independent Nucleocytoplasmic Shuttling

PubMed Central

Jensen, Torben Heick; Neville, Megan; Rain, Jean Christophe; McCarthy, Terri; Legrain, Pierre; Rosbash, Michael

2000-01-01

Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the NES receptor CRM1/Crm1p. We have carried out a yeast two-hybrid screen with Crm1p as a bait. The Crm1p-interacting clones were subscreened for nuclear export activity in a visual assay utilizing the Crm1p-inhibitor leptomycin B (LMB). This approach identified three Saccharomyces cerevisiae proteins not previously known to have nuclear export activity. These proteins are the 5′ RNA triphosphatase Ctl1p, the cell cycle-regulated transcription factor Ace2p, and a protein encoded by the previously uncharacterized open reading frame YDR499W. Mutagenesis analysis show that YDR499Wp contains an NES that conforms to the consensus sequence for leucine-rich NESs. Mutagenesis of Ctl1p and Ace2p were unable to identify specific NES residues. However, a 29-amino-acid region of Ace2p, rich in hydrophobic residues, contains nuclear export activity. Ace2p accumulates in the nucleus at the end of mitosis and activates early-G1-specific genes. We now provide evidence that Ace2p is nuclear not only in late M-early G1 but also during other stages of the cell cycle. This feature of Ace2p localization explains its ability to activate genes such as CUP1, which are not expressed in a cell cycle-dependent manner. PMID:11027275

Diversity of chloroplast genome among local clones of cocoa (Theobroma cacao, L.) from Central Sulawesi

NASA Astrophysics Data System (ADS)

Suwastika, I. Nengah; Pakawaru, Nurul Aisyah; Rifka, Rahmansyah, Muslimin, Ishizaki, Yoko; Cruz, André Freire; Basri, Zainuddin; Shiina, Takashi

2017-02-01

Chloroplast genomes typically range in size from 120 to 170 kilo base pairs (kb), which relatively conserved among plant species. Recent evaluation on several species, certain unique regions showed high variability which can be utilized in the phylogenetic analysis. Many fragments of coding regions, introns, and intergenic spacers, such as atpB-rbcL, ndhF, rbcL, rpl16, trnH-psbA, trnL-F, trnS-G, etc., have been used for phylogenetic reconstructions at various taxonomic levels. Based on that status, we would like to analysis the diversity of chloroplast genome within species of local cacao (Theobroma cacao L.) from Central Sulawesi. Our recent data showed, there were more than 20 clones from local farming in Central Sulawesi, and it can be detected based on phenotypic and nuclear-genome-based characterization (RAPD- Random Amplified Polymorphic DNA and SSR- Simple Sequences Repeat) markers. In developing DNA marker for this local cacao, here we also included analysis based on the variation of chloroplast genome. At least several regions such as rpl32-TurnL, it can be considered as chloroplast markers on our local clone of cocoa. Furthermore, we could develop phylogenetic analysis in between clones of cocoa.

Contrasting genetic structure between mitochondrial and nuclear markers in the dengue fever mosquito from Rio de Janeiro: implications for vector control

PubMed Central

Rašić, Gordana; Schama, Renata; Powell, Rosanna; Maciel-de Freitas, Rafael; Endersby-Harshman, Nancy M; Filipović, Igor; Sylvestre, Gabriel; Máspero, Renato C; Hoffmann, Ary A

2015-01-01

Dengue is the most prevalent global arboviral disease that affects over 300 million people every year. Brazil has the highest number of dengue cases in the world, with the most severe epidemics in the city of Rio de Janeiro (Rio). The effective control of dengue is critically dependent on the knowledge of population genetic structuring in the primary dengue vector, the mosquito Aedes aegypti. We analyzed mitochondrial and nuclear genomewide single nucleotide polymorphism markers generated via Restriction-site Associated DNA sequencing, as well as traditional microsatellite markers in Ae. aegypti from Rio. We found four divergent mitochondrial lineages and a strong spatial structuring of mitochondrial variation, in contrast to the overall nuclear homogeneity across Rio. Despite a low overall differentiation in the nuclear genome, we detected strong spatial structure for variation in over 20 genes that have a significantly altered expression in response to insecticides, xenobiotics, and pathogens, including the novel biocontrol agent Wolbachia. Our results indicate that high genetic diversity, spatially unconstrained admixing likely mediated by male dispersal, along with locally heterogeneous genetic variation that could affect insecticide resistance and mosquito vectorial capacity, set limits to the effectiveness of measures to control dengue fever in Rio. PMID:26495042

Phylogeography of the Lutzomyia gomezi (Diptera: Phlebotominae) on the Panama Isthmus

PubMed Central

2014-01-01

Background Lutzomyia gomezi (Nitzulescu, 1931) is one of the main Leishmania (Vianna) panamensis vectors in Panama, and despite its medical significance, there are no population genetic studies regarding this species. In this study, we used the sequences of the mitochondrial gene cytochrome b/start of NADH1 and the nuclear elongation gene α-1 in order to analyze genetic variation and phylogeographic structure of the Lu. gomezi populations. Methods A total of 86 Lu. gomezi individuals were captured in 38 locations where cutaneous leishmaniasis occurred. DNA was extracted with phenol/chloroform methods and amplification of genes was performed using PCR primers for mitochondrial and nuclear markers. Results We found a total of 37 and 26 haplotypes of mitochondrial and nuclear genes, high haplotype diversity (h) for all three populations were detected with both molecular markers. Nucleotide diversity (π) was estimated to be high for all three populations with the mitochondrial marker, which was opposite to the estimate with the nuclear marker. In the AMOVA Φst recorded moderate (mitochondrial) and small (nuclear) population structure with statistical significance among populations. The analysis of the fixation index (Fst) used to measure the differentiation of populations showed that with the exception of the population located in the region of Bocas del Toro, the other populations presented with minor genetic differentiation. The median-Joining network of the mitochondrial marker reveled three clusters and recorded four haplotypes exclusively of localities sampled from Western Panama, demonstrating strong divergence. We found demographic population expansion with Fu´s Fs neutrality test. In the analysis mismatch distribution was observed as a bimodal curve. Conclusion Lu. gomezi is a species with higher genetic pool or variability and mild population structure, due to possible capacity migration and local adaptation to environmental changes or colonization potential. Thus, knowledge of the genetic population and evolutionary history is useful to understand the implications of different population genetic structures for cutaneous leishmaniasis epidemiology. PMID:24398187

Development of single-copy nuclear intron markers for species-level phylogenetics: Case study with Paullinieae (Sapindaceae).

PubMed

Chery, Joyce G; Sass, Chodon; Specht, Chelsea D

2017-09-01

We developed a bioinformatic pipeline that leverages a publicly available genome and published transcriptomes to design primers in conserved coding sequences flanking targeted introns of single-copy nuclear loci. Paullinieae (Sapindaceae) is used to demonstrate the pipeline. Transcriptome reads phylogenetically closer to the lineage of interest are aligned to the closest genome. Single-nucleotide polymorphisms are called, generating a "pseudoreference" closer to the lineage of interest. Several filters are applied to meet the criteria of single-copy nuclear loci with introns of a desired size. Primers are designed in conserved coding sequences flanking introns. Using this pipeline, we developed nine single-copy nuclear intron markers for Paullinieae. This pipeline is highly flexible and can be used for any group with available genomic and transcriptomic resources. This pipeline led to the development of nine variable markers for phylogenetic study without generating sequence data de novo.

Relationships in subtribe Diocleinae (Leguminosae; Papilionoideae) inferred from internal transcribed spacer sequences from nuclear ribosomal DNA.

PubMed

Varela, Eduardo S; Lima, João P M S; Galdino, Alexsandro S; Pinto, Luciano da S; Bezerra, Walderly M; Nunes, Edson P; Alves, Maria A O; Grangeiro, Thalles B

2004-01-01

The complete sequences of nuclear ribosomal DNA (nrDNA) internal transcribed spacer regions (ITS/5.8S) were determined for species belonging to six genera from the subtribe Diocleinae as well as for the anomalous genera Calopogonium and Pachyrhizus. Phylogenetic trees constructed by distance matrix, maximum parsimony and maximum likelihood methods showed that Calopogonium and Pachyrhizus were outside the clade Diocleinae (Canavalia, Camptosema, Cratylia, Dioclea, Cymbosema, and Galactia). This finding supports previous morphological, phytochemical, and molecular evidence that Calopogonium and Pachyrhizus do not belong to the subtribe Diocleinae. Within the true Diocleinae clade, the clustering of genera and species were congruent with morphology-based classifications, suggesting that ITS/5.8S sequences can provide enough informative sites to allow resolution below the genus level. This is the first evidence of the phylogeny of subtribe Diocleinae based on nuclear DNA sequences.

Further studies on Boreonectes Angus, 2010, with a molecular phylogeny of the Palaearctic species of the genus

PubMed Central

Angus, Robert B.; Ribera, Ignacio; Jia, Fenglong

2017-01-01

Abstract Karyotypes are given for Boreonectes emmerichi (Falkenström, 1936) from its type locality at Kangding, China, and for B. alpestris (Dutton & Angus, 2007) from the St Gotthard and San Bernardino passes in the Swiss Alps. A phylogeny based on sequence data from a combination of mitochondrial and nuclear genes recovered western Palaearctic species of Boreonectes as monophyletic with strong support. Boreonectes emmerichi was placed as sister to the north American forms of B. griseostriatus (De Geer, 1774), although with low support. The diversity of Palaearctic species of the B. griseostriatus species group is discussed. PMID:28919958

Further studies on Boreonectes Angus, 2010, with a molecular phylogeny of the Palaearctic species of the genus.

PubMed

Angus, Robert B; Ribera, Ignacio; Jia, Fenglong

2017-01-01

Karyotypes are given for Boreonectes emmerichi (Falkenström, 1936) from its type locality at Kangding, China, and for B. alpestris (Dutton & Angus, 2007) from the St Gotthard and San Bernardino passes in the Swiss Alps. A phylogeny based on sequence data from a combination of mitochondrial and nuclear genes recovered western Palaearctic species of Boreonectes as monophyletic with strong support. Boreonectes emmerichi was placed as sister to the north American forms of B. griseostriatus (De Geer, 1774), although with low support. The diversity of Palaearctic species of the B. griseostriatus species group is discussed.

EphB4 localises to the nucleus of prostate cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mertens-Walker, Inga, E-mail: inga.mertenswalker@qut.edu.au; Australian Prostate Cancer Research Centre—Queensland, Translational Research Institute, 37 Kent Street, Woolloongabba 4102, QLD; Lisle, Jessica E.

2015-04-10

The EphB4 receptor tyrosine kinase is over-expressed in a variety of different epithelial cancers including prostate where it has been shown to be involved in survival, migration and angiogenesis. We report here that EphB4 also resides in the nucleus of prostate cancer cell lines. We used in silico methods to identify a bipartite nuclear localisation signal (NLS) in the extracellular domain and a monopartite NLS sequence in the intracellular kinase domain of EphB4. To determine whether both putative NLS sequences were functional, fragments of the EphB4 sequence containing each NLS were cloned to create EphB4NLS-GFP fusion proteins. Localisation of bothmore » NLS-GFP proteins to the nuclei of transfected cells was observed, demonstrating that EphB4 contains two functional NLS sequences. Mutation of the key amino residues in both NLS sequences resulted in diminished nuclear accumulation. As nuclear translocation is often dependent on importins we confirmed that EphB4 and importin-α can interact. To assess if nuclear EphB4 could be implicated in gene regulatory functions potential EphB4-binding genomic loci were identified using chromatin immunoprecipitation and Lef1 was confirmed as a potential target of EphB4-mediated gene regulation. These novel findings add further complexity to the biology of this important cancer-associated receptor. - Highlights: • The EphB4 protein can be found in the nucleus of prostate cancer cell lines. • EphB4 contains two functional nuclear localisation signals. • Chromatin immunoprecipitation has identified potential genome sequences to which EphB4 binds. • Lef1 is a confirmed target for EphB4-mediated gene regulation.« less

Crystal structure of the Xpo1p nuclear export complex bound to the SxFG/PxFG repeats of the nucleoporin Nup42p.

PubMed

Koyama, Masako; Hirano, Hidemi; Shirai, Natsuki; Matsuura, Yoshiyuki

2017-10-01

Xpo1p (yeast CRM1) is the major nuclear export receptor that carries a plethora of proteins and ribonucleoproteins from the nucleus to cytoplasm. The passage of the Xpo1p nuclear export complex through nuclear pore complexes (NPCs) is facilitated by interactions with nucleoporins (Nups) containing extensive repeats of phenylalanine-glycine (so-called FG repeats), although the precise role of each Nup in the nuclear export reaction remains incompletely understood. Here we report structural and biochemical characterization of the interactions between the Xpo1p nuclear export complex and the FG repeats of Nup42p, a nucleoporin localized at the cytoplasmic face of yeast NPCs and has characteristic SxFG/PxFG sequence repeat motif. The crystal structure of Xpo1p-PKI-Nup42p-Gsp1p-GTP complex identified three binding sites for the SxFG/PxFG repeats on HEAT repeats 14-20 of Xpo1p. Mutational analyses of Nup42p showed that the conserved serines and prolines in the SxFG/PxFG repeats contribute to Xpo1p-Nup42p binding. Our structural and biochemical data suggest that SxFG/PxFG-Nups such as Nup42p and Nup159p at the cytoplasmic face of NPCs provide high-affinity docking sites for the Xpo1p nuclear export complex in the terminal stage of NPC passage and that subsequent disassembly of the nuclear export complex facilitates recycling of free Xpo1p back to the nucleus. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Genetic Rearrangements Can Modify Chromatin Features at Epialleles

PubMed Central

Foerster, Andrea M.; Dinh, Huy Q.; Sedman, Laura; Wohlrab, Bonnie; Mittelsten Scheid, Ortrun

2011-01-01

Analogous to genetically distinct alleles, epialleles represent heritable states of different gene expression from sequence-identical genes. Alleles and epialleles both contribute to phenotypic heterogeneity. While alleles originate from mutation and recombination, the source of epialleles is less well understood. We analyze active and inactive epialleles that were found at a transgenic insert with a selectable marker gene in Arabidopsis. Both converse expression states are stably transmitted to progeny. The silent epiallele was previously shown to change its state upon loss-of-function of trans-acting regulators and drug treatments. We analyzed the composition of the epialleles, their chromatin features, their nuclear localization, transcripts, and homologous small RNA. After mutagenesis by T-DNA transformation of plants carrying the silent epiallele, we found new active alleles. These switches were associated with different, larger or smaller, and non-overlapping deletions or rearrangements in the 3′ regions of the epiallele. These cis-mutations caused different degrees of gene expression stability depending on the nature of the sequence alteration, the consequences for transcription and transcripts, and the resulting chromatin organization upstream. This illustrates a tight dependence of epigenetic regulation on local structures and indicates that sequence alterations can cause epigenetic changes at some distance in regions not directly affected by the mutation. Similar effects may also be involved in gene expression and chromatin changes in the vicinity of transposon insertions or excisions, recombination events, or DNA repair processes and could contribute to the origin of new epialleles. PMID:22028669

Breaking the DNA-binding code of Ralstonia solanacearum TAL effectors provides new possibilities to generate plant resistance genes against bacterial wilt disease.

PubMed

de Lange, Orlando; Schreiber, Tom; Schandry, Niklas; Radeck, Jara; Braun, Karl Heinz; Koszinowski, Julia; Heuer, Holger; Strauß, Annett; Lahaye, Thomas

2013-08-01

Ralstonia solanacearum is a devastating bacterial phytopathogen with a broad host range. Ralstonia solanacearum injected effector proteins (Rips) are key to the successful invasion of host plants. We have characterized Brg11(hrpB-regulated 11), the first identified member of a class of Rips with high sequence similarity to the transcription activator-like (TAL) effectors of Xanthomonas spp., collectively termed RipTALs. Fluorescence microscopy of in planta expressed RipTALs showed nuclear localization. Domain swaps between Brg11 and Xanthomonas TAL effector (TALE) AvrBs3 (avirulence protein triggering Bs3 resistance) showed the functional interchangeability of DNA-binding and transcriptional activation domains. PCR was used to determine the sequence of brg11 homologs from strains infecting phylogenetically diverse host plants. Brg11 localizes to the nucleus and activates promoters containing a matching effector-binding element (EBE). Brg11 and homologs preferentially activate promoters containing EBEs with a 5' terminal guanine, contrasting with the TALE preference for a 5' thymine. Brg11 and other RipTALs probably promote disease through the transcriptional activation of host genes. Brg11 and the majority of homologs identified in this study were shown to activate similar or identical target sequences, in contrast to TALEs, which generally show highly diverse target preferences. This information provides new options for the engineering of plants resistant to R. solanacearum. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

The N-terminal leucine-zipper motif in PTRF/cavin-1 is essential and sufficient for its caveolae-association

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wei, Zhuang; Laboratory of System Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031; Zou, Xinle

2015-01-16

Highlight: • The N-terminal leucine-zipper motif in PTRF/cavin-1 determines caveolar association. • Different cellular localization of PTRF/cavin-1 influences its serine 389 and 391 phosphorylation state. • PTRF/cavin-1 regulates cell motility via its caveolar association. - Abstract: PTRF/cavin-1 is a protein of two lives. Its reported functions in ribosomal RNA synthesis and in caveolae formation happen in two different cellular locations: nucleus vs. plasma membrane. Here, we identified that the N-terminal leucine-zipper motif in PTRF/cavin-1 was essential for the protein to be associated with caveolae in plasma membrane. It could counteract the effect of nuclear localization sequence in the molecule (AAmore » 235–251). Deletion of this leucine-zipper motif from PTRF/cavin-1 caused the mutant to be exclusively localized in nuclei. The fusion of this leucine-zipper motif with histone 2A, which is a nuclear protein, could induce the fusion protein to be exported from nucleus. Cell migration was greatly inhibited in PTRF/cavin-1{sup −/−} mouse embryonic fibroblasts (MEFs). The inhibited cell motility could only be rescued by exogenous cavin-1 but not the leucine-zipper motif deleted cavin-1 mutant. Plasma membrane dynamics is an important factor in cell motility control. Our results suggested that the membrane dynamics in cell migration is affected by caveolae associated PTRF/cavin-1.« less

Properties of Localized Protons in Neutron Star Matter at Finite Temperatures

NASA Astrophysics Data System (ADS)

Szmaglinski, A.; Kubis, S.; Wójcik, W.

2014-02-01

We study properties of the proton component of neutron star matter for realistic nuclear models. Vanishing of the nuclear symmetry energy implies proton-neutron separation in dense nuclear matter. Protons which form admixture tend to be localized in potential wells. Here, we extend the description of proton localization to finite temperatures. It appears that the protons are still localized at temperatures typical for hot neutron stars. That fact has important astrophysical consequences. Moreover, the temperature inclusion leads to unexpected results for the behavior of the proton localized state.

Cloning, structure, and chromosome localization of the mouse glutaryl-CoA dehydrogenase gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koeller, D.M.; DiGiulio, A.; Frerman, F.E.

Glutaryl-CoA dehydrogenase (GCDH) is a nuclear-encoded, mitochondrial matrix enzyme. In humans, deficiency of GCDH leads to glutaric acidemia type I, and inherited disorder of amino acid metabolism characterized by a progressive neurodegenerative disease. In this report we describe the cloning and structure of the mouse GCDH (Gcdh) gene and cDNA and its chromosomal localization. The mouse Gcdh cDNA is 1.75 kb long and contains and open reading frame of 438 amino acids. The amino acid sequences of mouse, human, and pig GCDH are highly conserved. The mouse Gcdh gene contains 11 exons and spans 7 kb of genomic DNA. Gcdhmore » was mapped by backcross analysis to mouse chromosome 8 within a region that is homologous to a region of human chromosome 19, where the human gene was previously mapped. 14 refs., 3 figs.« less

A new species of Alsodes (Anura: Alsodidae) from Altos de Cantillana, central Chile.

PubMed

Charrier, Andrés; Correa, Claudio; Castro, Camila; Méndez, Marco A

2015-02-05

Based on morphological and molecular evidence (mitochondrial and nuclear sequences) we describe a new species of spiny-chest frog, Alsodes cantillanensis, from central Chile (around 34°S). The type locality, Quebrada Infiernillo, is located in the Coastal Range at approximately 65 km from Santiago (Metropolitan Region), the capital of Chile. The distribution of the new species is included entirely in that of A. nodosus (32-36°S approximately), which was identified as the sister taxon according to molecular phylogenetic analyses. Moreover, both species are sympatric in the type locality. The new species was found in a Nothofagus macrocarpa relict forest potentially threatened by gold mining activities. We identify other threats for its conservation and some biological data needed for understanding the evolution of this species. This discovery reveals the scarce knowledge about biogeography, evolution and ecology of spiny-chest frogs from central Chile. 

A Fungal Effector With Host Nuclear Localization and DNA-Binding Properties Is Required for Maize Anthracnose Development.

PubMed

Vargas, Walter A; Sanz-Martín, José M; Rech, Gabriel E; Armijos-Jaramillo, Vinicio D; Rivera, Lina P; Echeverria, María Mercedes; Díaz-Mínguez, José M; Thon, Michael R; Sukno, Serenella A

2016-02-01

Plant pathogens have the capacity to manipulate the host immune system through the secretion of effectors. We identified 27 putative effector proteins encoded in the genome of the maize anthracnose pathogen Colletotrichum graminicola that are likely to target the host's nucleus, as they simultaneously contain sequence signatures for secretion and nuclear localization. We functionally characterized one protein, identified as CgEP1. This protein is synthesized during the early stages of disease development and is necessary for anthracnose development in maize leaves, stems, and roots. Genetic, molecular, and biochemical studies confirmed that this effector targets the host's nucleus and defines a novel class of double-stranded DNA-binding protein. We show that CgEP1 arose from a gene duplication in an ancestor of a lineage of monocot-infecting Colletotrichum spp. and has undergone an intense evolution process, with evidence for episodes of positive selection. We detected CgEP1 homologs in several species of a grass-infecting lineage of Colletotrichum spp., suggesting that its function may be conserved across a large number of anthracnose pathogens. Our results demonstrate that effectors targeted to the host nucleus may be key elements for disease development and aid in the understanding of the genetic basis of anthracnose development in maize plants.

Eukaryotic DING Proteins Are Endogenous: An Immunohistological Study in Mouse Tissues

PubMed Central

Collombet, Jean-Marc; Elias, Mikael; Gotthard, Guillaume; Four, Elise; Renault, Frédérique; Joffre, Aurélie; Baubichon, Dominique; Rochu, Daniel; Chabrière, Eric

2010-01-01

Background DING proteins encompass an intriguing protein family first characterized by their conserved N-terminal sequences. Some of these proteins seem to have key roles in various human diseases, e.g., rheumatoid arthritis, atherosclerosis, HIV suppression. Although this protein family seems to be ubiquitous in eukaryotes, their genes are consistently lacking from genomic databases. Such a lack has considerably hampered functional studies and has fostered therefore the hypothesis that DING proteins isolated from eukaryotes were in fact prokaryotic contaminants. Principal Findings In the framework of our study, we have performed a comprehensive immunological detection of DING proteins in mice. We demonstrate that DING proteins are present in all tissues tested as isoforms of various molecular weights (MWs). Their intracellular localization is tissue-dependant, being exclusively nuclear in neurons, but cytoplasmic and nuclear in other tissues. We also provide evidence that germ-free mouse plasma contains as much DING protein as wild-type. Significance Hence, data herein provide a valuable basis for future investigations aimed at eukaryotic DING proteins, revealing that these proteins seem ubiquitous in mouse tissue. Our results strongly suggest that mouse DING proteins are endogenous. Moreover, the determination in this study of the precise cellular localization of DING proteins constitute a precious evidence to understand their molecular involvements in their related human diseases. PMID:20161715

Local structures of mesoporous bioactive glasses and their surface alterations in vitro: inferences from solid-state nuclear magnetic resonance

PubMed Central

Gunawidjaja, Philips N.; Mathew, Renny; Lo, Andy Y. H.; Izquierdo-Barba, Isabel; García, Ana; Arcos, Daniel; Mattias Edén, María Vallet-Regí

2012-01-01

We review the benefits of using 29Si and 1H magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy for probing the local structures of both bulk and surface portions of mesoporous bioactive glasses (MBGs) of the CaO–SiO2−(P2O5) system. These mesoporous materials exhibit an ordered pore arrangement, and are promising candidates for improved bone and tooth implants. We discuss experimental MAS NMR results from three MBGs displaying different Ca, Si and P contents: the 29Si NMR spectra were recorded either directly by employing radio-frequency pulses to 29Si, or by magnetization transfers from neighbouring protons using cross polarization, thereby providing quantitative information about the silicate speciation present in the pore wall and at the MBG surface, respectively. The surface modifications were monitored for the three MBGs during their immersion in a simulated body fluid (SBF) for intervals between 30 min and one week. The results were formulated as a reaction sequence describing the interconversions between the distinct silicate species. We generally observed a depletion of Ca2+ ions at the MBG surface, and a minor condensation of the silicate-surface network over one week of SBF soaking. PMID:22349247

Diverged composition and regulation of the Trypanosoma brucei origin recognition complex that mediates DNA replication initiation

PubMed Central

Marques, Catarina A.; Tiengwe, Calvin; Lemgruber, Leandro; Damasceno, Jeziel D.; Scott, Alan; Paape, Daniel; Marcello, Lucio; McCulloch, Richard

2016-01-01

Abstract Initiation of DNA replication depends upon recognition of genomic sites, termed origins, by AAA+ ATPases. In prokaryotes a single factor binds each origin, whereas in eukaryotes this role is played by a six-protein origin recognition complex (ORC). Why eukaryotes evolved a multisubunit initiator, and the roles of each component, remains unclear. In Trypanosoma brucei, an ancient unicellular eukaryote, only one ORC-related initiator, TbORC1/CDC6, has been identified by sequence homology. Here we show that three TbORC1/CDC6-interacting factors also act in T. brucei nuclear DNA replication and demonstrate that TbORC1/CDC6 interacts in a high molecular complex in which a diverged Orc4 homologue and one replicative helicase subunit can also be found. Analysing the subcellular localization of four TbORC1/CDC6-interacting factors during the cell cycle reveals that one factor, TbORC1B, is not a static constituent of ORC but displays S-phase restricted nuclear localization and expression, suggesting it positively regulates replication. This work shows that ORC architecture and regulation are diverged features of DNA replication initiation in T. brucei, providing new insight into this key stage of eukaryotic genome copying. PMID:26951375

Plant U13 orthologues and orphan snoRNAs identified by RNomics of RNA from Arabidopsis nucleoli

PubMed Central

Kim, Sang Hyon; Spensley, Mark; Choi, Seung Kook; Calixto, Cristiane P. G.; Pendle, Ali F.; Koroleva, Olga; Shaw, Peter J.; Brown, John W. S.

2010-01-01

Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs whose main function in eukaryotes is to guide the modification of nucleotides in ribosomal and spliceosomal small nuclear RNAs, respectively. Full-length sequences of Arabidopsis snoRNAs and scaRNAs have been obtained from cDNA libraries of capped and uncapped small RNAs using RNA from isolated nucleoli from Arabidopsis cell cultures. We have identified 31 novel snoRNA genes (9 box C/D and 22 box H/ACA) and 15 new variants of previously described snoRNAs. Three related capped snoRNAs with a distinct gene organization and structure were identified as orthologues of animal U13snoRNAs. In addition, eight of the novel genes had no complementarity to rRNAs or snRNAs and are therefore putative orphan snoRNAs potentially reflecting wider functions for these RNAs. The nucleolar localization of a number of the snoRNAs and the localization to nuclear bodies of two putative scaRNAs was confirmed by in situ hybridization. The majority of the novel snoRNA genes were found in new gene clusters or as part of previously described clusters. These results expand the repertoire of Arabidopsis snoRNAs to 188 snoRNA genes with 294 gene variants. PMID:20081206

Nuclear localization signal regulates porcine circovirus type 2 capsid protein nuclear export through phosphorylation.

PubMed

Hou, Qiang; Hou, Shaohua; Chen, Qing; Jia, Hong; Xin, Ting; Jiang, Yitong; Guo, Xiaoyu; Zhu, Hongfei

2018-02-15

The open reading frame 2 (ORF2) of Porcine circovirus type 2 (PCV2) encodes the major Capsid (Cap) protein, which self-assembles into virus-like particle (VLP) of similar morphology to the PCV2 virion and accumulates in the nucleus through the N-terminal arginine-rich nuclear localization signal (NLS). In this study, PCV2 Cap protein and its derivates were expressed via the baculovirus expression system, and the cellular localization of the recombinant proteins were investigated using anti-Cap mAb by imaging flow cytometry. Analysis of subcellular localization of Cap protein and its variants demonstrated that NLS mediated Cap protein nuclear export as well as nuclear import, and a phosphorylation site (S17) was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the NLS domain to regulate Cap protein nuclear export. Phosphorylation of NLS regulating the PCV2 Cap protein nuclear export was also demonstrated in PK15 cells by fluorescence microscopy. Moreover, the influence of Rep and Rep' protein on Cap protein subcellular localization was investigated in PK15 cells. Phosphorylation of NLS regulating Cap protein nuclear export provides more detailed knowledge of the PCV2 viral life cycle. Copyright © 2018 Elsevier B.V. All rights reserved.

Protein Sub-Nuclear Localization Prediction Using SVM and Pfam Domain Information

PubMed Central

Kumar, Ravindra; Jain, Sohni; Kumari, Bandana; Kumar, Manish

2014-01-01

The nucleus is the largest and the highly organized organelle of eukaryotic cells. Within nucleus exist a number of pseudo-compartments, which are not separated by any membrane, yet each of them contains only a specific set of proteins. Understanding protein sub-nuclear localization can hence be an important step towards understanding biological functions of the nucleus. Here we have described a method, SubNucPred developed by us for predicting the sub-nuclear localization of proteins. This method predicts protein localization for 10 different sub-nuclear locations sequentially by combining presence or absence of unique Pfam domain and amino acid composition based SVM model. The prediction accuracy during leave-one-out cross-validation for centromeric proteins was 85.05%, for chromosomal proteins 76.85%, for nuclear speckle proteins 81.27%, for nucleolar proteins 81.79%, for nuclear envelope proteins 79.37%, for nuclear matrix proteins 77.78%, for nucleoplasm proteins 76.98%, for nuclear pore complex proteins 88.89%, for PML body proteins 75.40% and for telomeric proteins it was 83.33%. Comparison with other reported methods showed that SubNucPred performs better than existing methods. A web-server for predicting protein sub-nuclear localization named SubNucPred has been established at http://14.139.227.92/mkumar/subnucpred/. Standalone version of SubNucPred can also be downloaded from the web-server. PMID:24897370

The low information content of Neurospora splicing signals: implications for RNA splicing and intron origin.

PubMed

Collins, Richard A; Stajich, Jason E; Field, Deborah J; Olive, Joan E; DeAbreu, Diane M

2015-05-01

When we expressed a small (0.9 kb) nonprotein-coding transcript derived from the mitochondrial VS plasmid in the nucleus of Neurospora we found that it was efficiently spliced at one or more of eight 5' splice sites and ten 3' splice sites, which are present apparently by chance in the sequence. Further experimental and bioinformatic analyses of other mitochondrial plasmids, random sequences, and natural nuclear genes in Neurospora and other fungi indicate that fungal spliceosomes recognize a wide range of 5' splice site and branchpoint sequences and predict introns to be present at high frequency in random sequence. In contrast, analysis of intronless fungal nuclear genes indicates that branchpoint, 5' splice site and 3' splice site consensus sequences are underrepresented compared with random sequences. This underrepresentation of splicing signals is sufficient to deplete the nuclear genome of splice sites at locations that do not comprise biologically relevant introns. Thus, the splicing machinery can recognize a wide range of splicing signal sequences, but splicing still occurs with great accuracy, not because the splicing machinery distinguishes correct from incorrect introns, but because incorrect introns are substantially depleted from the genome. © 2015 Collins et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

High-Content Positional Biosensor Screening Assay for Compounds to Prevent or Disrupt Androgen Receptor and Transcriptional Intermediary Factor 2 Protein–Protein Interactions

PubMed Central

Hua, Yun; Shun, Tong Ying; Strock, Christopher J.

2014-01-01

Abstract The androgen receptor–transcriptional intermediary factor 2 (AR-TIF2) positional protein–protein interaction (PPI) biosensor assay described herein combines physiologically relevant cell-based assays with the specificity of binding assays by incorporating structural information of AR and TIF2 functional domains along with intracellular targeting sequences and fluorescent reporters. Expression of the AR-red fluorescent protein (RFP) “prey” and TIF2-green fluorescent protein (GFP) “bait” components of the biosensor was directed by recombinant adenovirus constructs that expressed the ligand binding and activation function 2 surface domains of AR fused to RFP with nuclear localization and nuclear export sequences, and three α-helical LXXLL motifs from TIF2 fused to GFP and an HIV Rev nucleolar targeting sequence. In unstimulated cells, AR-RFP was localized predominantly to the cytoplasm and TIF2-GFP was localized to nucleoli. Dihydrotestosterone (DHT) treatment induced AR-RFP translocation into the nucleus where the PPIs between AR and TIF2 resulted in the colocalization of both biosensors within the nucleolus. We adapted the translocation enhanced image analysis module to quantify the colocalization of the AR-RFP and TIF2-GFP biosensors in images acquired on the ImageXpress platform. DHT induced a concentration-dependent AR-TIF2 colocalization and produced a characteristic condensed punctate AR-RFP PPI nucleolar distribution pattern. The heat-shock protein 90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) and antiandrogens flutamide and bicalutamide inhibited DHT-induced AR-TIF2 PPI formation with 50% inhibition concentrations (IC50s) of 88.5±12.5 nM, 7.6±2.4 μM, and 1.6±0.4 μM, respectively. Images of the AR-RFP distribution phenotype allowed us to distinguish between 17-AAG and flutamide, which prevented AR translocation, and bicalutamide, which blocked AR-TIF2 PPIs. We screened the Library of Pharmacologically Active Compounds (LOPAC) set for compounds that inhibited AR-TIF2 PPI formation or disrupted preexisting complexes. Eleven modulators of steroid family nuclear receptors (NRs) and 6 non-NR ligands inhibited AR-TIF2 PPI formation, and 10 disrupted preexisting complexes. The hits appear to be either AR antagonists or nonspecific inhibitors of NR activation and trafficking. Given that the LOPAC set represents such a small and restricted biological and chemical diversity, it is anticipated that screening a much larger and more diverse compound library will be required to find AR-TIF2 PPI inhibitors/disruptors. The AR-TIF2 protein–protein interaction biosensor (PPIB) approach offers significant promise for identifying molecules with potential to modulate AR transcriptional activity in a cell-specific manner that is distinct from the existing antiandrogen drugs that target AR binding or production. Small molecules that disrupt AR signaling at the level of AR-TIF2 PPIs may also overcome the development of resistance and progression to castration-resistant prostate cancer. PMID:25181412

Inter-individual and intragenomic variations in the ITS region of Clonorchis sinensis (Trematoda: Opisthorchiidae) from Russia and Vietnam.

PubMed

Tatonova, Yulia V; Chelomina, Galina N; Nguyen, Hung Manh

2017-11-01

Here we examined the intraspecific genetic variability of Clonorchis sinensis from Russia and Vietnam using nuclear DNA sequences (the 5.8S gene and two internal transcribed spacers of the ribosomal cluster). Despite the low level of variability in the ITS1 region, this marker has revealed some features of C. sinensis across multiple geographic regions. The genetic diversity levels for the Russian and Vietnamese populations were similar (0.1 and 0.09%, respectively) but were significantly lower than the C. sinensis from China (0.31%). About half of the sequences of the Chinese (53%) and Korean (47%) populations and about a tenth of the Vietnamese (12%) and Russian (8%) sequences included a 5bp insertion. No sequences with nucleotide substitutions both upstream and downstream of the 5bp insertion were found within the whole data set. The population of northern China had both sequence variants (with substitutions either upstream or downstream of the insertion), while only one of these variants was presented at the other localities. The Vietnamese population had a higher frequency of intragenomic polymorphism than the Russian population (69% vs. 46% and 23% vs. 3% at the 114bp and 339bp positions, respectively). These data are discussed in connection with parasite origin and adaptation, and also its invasive capacity and drug-resistance. Copyright © 2017 Elsevier B.V. All rights reserved.

Transformation of Chloroplast Ribosomal RNA Genes in Chlamydomonas: Molecular and Genetic Characterization of Integration Events

PubMed Central

Newman, S. M.; Boynton, J. E.; Gillham, N. W.; Randolph-Anderson, B. L.; Johnson, A. M.; Harris, E. H.

1990-01-01

Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas. PMID:1981764

Multifunctionality of a picornavirus polymerase domain: nuclear localization signal and nucleotide recognition.

PubMed

Ferrer-Orta, Cristina; de la Higuera, Ignacio; Caridi, Flavia; Sánchez-Aparicio, María Teresa; Moreno, Elena; Perales, Celia; Singh, Kamalendra; Sarafianos, Stefan G; Sobrino, Francisco; Domingo, Esteban; Verdaguer, Nuria

2015-07-01

The N-terminal region of the foot-and-mouth disease virus (FMDV) 3D polymerase contains the sequence MRKTKLAPT (residues 16 to 24) that acts as a nuclear localization signal. A previous study showed that substitutions K18E and K20E diminished the transport to the nucleus of 3D and 3CD and severely impaired virus infectivity. These residues have also been implicated in template binding, as seen in the crystal structures of different 3D-RNA elongation complexes. Here, we report the biochemical and structural characterization of different mutant polymerases harboring substitutions at residues 18 and 20, in particular, K18E, K18A, K20E, K20A, and the double mutant K18A K20A (KAKA). All mutant enzymes exhibit low RNA binding activity, low processivity, and alterations in nucleotide recognition, including increased incorporation of ribavirin monophosphate (RMP) relative to the incorporation of cognate nucleotides compared with the wild-type enzyme. The structural analysis shows an unprecedented flexibility of the 3D mutant polymerases, including both global rearrangements of the closed-hand architecture and local conformational changes at loop β9-α11 (within the polymerase motif B) and at the template-binding channel. Specifically, in 3D bound to RNA, both K18E and K20E induced the opening of new pockets in the template channel where the downstream templating nucleotide at position +2 binds. The comparisons of free and RNA-bound enzymes suggest that the structural rearrangements may occur in a concerted mode to regulate RNA replication, processivity, and fidelity. Thus, the N-terminal region of FMDV 3D that acts as a nuclear localization signal (NLS) and in template binding is also involved in nucleotide recognition and can affect the incorporation of nucleotide analogues. The study documents multifunctionality of a nuclear localization signal (NLS) located at the N-terminal region of the foot-and-mouth disease viral polymerase (3D). Amino acid substitutions at this polymerase region can impair the transport of 3D to the nucleus, reduce 3D binding to RNA, and alter the relative incorporation of standard nucleoside monophosphate versus ribavirin monophosphate. Structural data reveal that the conformational changes in this region, forming part of the template channel entry, would be involved in nucleotide discrimination. The results have implications for the understanding of viral polymerase function and for lethal mutagenesis mechanisms. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Multifunctionality of a Picornavirus Polymerase Domain: Nuclear Localization Signal and Nucleotide Recognition

PubMed Central

Ferrer-Orta, Cristina; de la Higuera, Ignacio; Caridi, Flavia; Sánchez-Aparicio, María Teresa; Moreno, Elena; Perales, Celia; Singh, Kamalendra; Sarafianos, Stefan G.; Sobrino, Francisco; Domingo, Esteban

2015-01-01

ABSTRACT The N-terminal region of the foot-and-mouth disease virus (FMDV) 3D polymerase contains the sequence MRKTKLAPT (residues 16 to 24) that acts as a nuclear localization signal. A previous study showed that substitutions K18E and K20E diminished the transport to the nucleus of 3D and 3CD and severely impaired virus infectivity. These residues have also been implicated in template binding, as seen in the crystal structures of different 3D-RNA elongation complexes. Here, we report the biochemical and structural characterization of different mutant polymerases harboring substitutions at residues 18 and 20, in particular, K18E, K18A, K20E, K20A, and the double mutant K18A K20A (KAKA). All mutant enzymes exhibit low RNA binding activity, low processivity, and alterations in nucleotide recognition, including increased incorporation of ribavirin monophosphate (RMP) relative to the incorporation of cognate nucleotides compared with the wild-type enzyme. The structural analysis shows an unprecedented flexibility of the 3D mutant polymerases, including both global rearrangements of the closed-hand architecture and local conformational changes at loop β9-α11 (within the polymerase motif B) and at the template-binding channel. Specifically, in 3D bound to RNA, both K18E and K20E induced the opening of new pockets in the template channel where the downstream templating nucleotide at position +2 binds. The comparisons of free and RNA-bound enzymes suggest that the structural rearrangements may occur in a concerted mode to regulate RNA replication, processivity, and fidelity. Thus, the N-terminal region of FMDV 3D that acts as a nuclear localization signal (NLS) and in template binding is also involved in nucleotide recognition and can affect the incorporation of nucleotide analogues. IMPORTANCE The study documents multifunctionality of a nuclear localization signal (NLS) located at the N-terminal region of the foot-and-mouth disease viral polymerase (3D). Amino acid substitutions at this polymerase region can impair the transport of 3D to the nucleus, reduce 3D binding to RNA, and alter the relative incorporation of standard nucleoside monophosphate versus ribavirin monophosphate. Structural data reveal that the conformational changes in this region, forming part of the template channel entry, would be involved in nucleotide discrimination. The results have implications for the understanding of viral polymerase function and for lethal mutagenesis mechanisms. PMID:25903341

Classic Nuclear Localization Signals and a Novel Nuclear Localization Motif Are Required for Nuclear Transport of Porcine Parvovirus Capsid Proteins

PubMed Central

Boisvert, Maude; Bouchard-Lévesque, Véronique; Fernandes, Sandra

2014-01-01

ABSTRACT Nuclear targeting of capsid proteins (VPs) is important for genome delivery and precedes assembly in the replication cycle of porcine parvovirus (PPV). Clusters of basic amino acids, corresponding to potential nuclear localization signals (NLS), were found only in the unique region of VP1 (VP1up, for VP1 unique part). Of the five identified basic regions (BR), three were important for nuclear localization of VP1up: BR1 was a classic Pat7 NLS, and the combination of BR4 and BR5 was a classic bipartite NLS. These NLS were essential for viral replication. VP2, the major capsid protein, lacked these NLS and contained no region with more than two basic amino acids in proximity. However, three regions of basic clusters were identified in the folded protein, assembled into a trimeric structure. Mutagenesis experiments showed that only one of these three regions was involved in VP2 transport to the nucleus. This structural NLS, termed the nuclear localization motif (NLM), is located inside the assembled capsid and thus can be used to transport trimers to the nucleus in late steps of infection but not for virions in initial infection steps. The two NLS of VP1up are located in the N-terminal part of the protein, externalized from the capsid during endosomal transit, exposing them for nuclear targeting during early steps of infection. Globally, the determinants of nuclear transport of structural proteins of PPV were different from those of closely related parvoviruses. IMPORTANCE Most DNA viruses use the nucleus for their replication cycle. Thus, structural proteins need to be targeted to this cellular compartment at two distinct steps of the infection: in early steps to deliver viral genomes to the nucleus and in late steps to assemble new viruses. Nuclear targeting of proteins depends on the recognition of a stretch of basic amino acids by cellular transport proteins. This study reports the identification of two classic nuclear localization signals in the minor capsid protein (VP1) of porcine parvovirus. The major protein (VP2) nuclear localization was shown to depend on a complex structural motif. This motif can be used as a strategy by the virus to avoid transport of incorrectly folded proteins and to selectively import assembled trimers into the nucleus. Structural nuclear localization motifs can also be important for nuclear proteins without a classic basic amino acid stretch, including multimeric cellular proteins. PMID:25078698

Predicting nuclear gene coalescence from mitochondrial data: the three-times rule.

PubMed

Palumbi, S R; Cipriano, F; Hare, M P

2001-05-01

Coalescence theory predicts when genetic drift at nuclear loci will result in fixation of sequence differences to produce monophyletic gene trees. However, the theory is difficult to apply to particular taxa because it hinges on genetically effective population size, which is generally unknown. Neutral theory also predicts that evolution of monophyly will be four times slower in nuclear than in mitochondrial genes primarily because genetic drift is slower at nuclear loci. Variation in mitochondrial DNA (mtDNA) within and between species has been studied extensively, but can these mtDNA data be used to predict coalescence in nuclear loci? Comparison of neutral theories of coalescence of mitochondrial and nuclear loci suggests a simple rule of thumb. The "three-times rule" states that, on average, most nuclear loci will be monophyletic when the branch length leading to the mtDNA sequences of a species is three times longer than the average mtDNA sequence diversity observed within that species. A test using mitochondrial and nuclear intron data from seven species of whales and dolphins suggests general agreement with predictions of the three-times rule. We define the coalescence ratio as the mitochondrial branch length for a species divided by intraspecific mtDNA diversity. We show that species with high coalescence ratios show nuclear monophyly, whereas species with low ratios have polyphyletic nuclear gene trees. As expected, species with intermediate coalescence ratios show a variety of patterns. Especially at very high or low coalescence ratios, the three-times rule predicts nuclear gene patterns that can help detect the action of selection. The three-times rule may be useful as an empirical benchmark for evaluating evolutionary processes occurring at multiple loci.

Transcriptional Activity and Nuclear Localization of Cabut, the Drosophila Ortholog of Vertebrate TGF-β-Inducible Early-Response Gene (TIEG) Proteins

PubMed Central

Belacortu, Yaiza; Weiss, Ron; Kadener, Sebastian; Paricio, Nuria

2012-01-01

Background Cabut (Cbt) is a C2H2-class zinc finger transcription factor involved in embryonic dorsal closure, epithelial regeneration and other developmental processes in Drosophila melanogaster. Cbt orthologs have been identified in other Drosophila species and insects as well as in vertebrates. Indeed, Cbt is the Drosophila ortholog of the group of vertebrate proteins encoded by the TGF-ß-inducible early-response genes (TIEGs), which belong to Sp1-like/Krüppel-like family of transcription factors. Several functional domains involved in transcriptional control and subcellular localization have been identified in the vertebrate TIEGs. However, little is known of whether these domains and functions are also conserved in the Cbt protein. Methodology/Principal Findings To determine the transcriptional regulatory activity of the Drosophila Cbt protein, we performed Gal4-based luciferase assays in S2 cells and showed that Cbt is a transcriptional repressor and able to regulate its own expression. Truncated forms of Cbt were then generated to identify its functional domains. This analysis revealed a sequence similar to the mSin3A-interacting repressor domain found in vertebrate TIEGs, although located in a different part of the Cbt protein. Using β-Galactosidase and eGFP fusion proteins, we also showed that Cbt contains the bipartite nuclear localization signal (NLS) previously identified in TIEG proteins, although it is non-functional in insect cells. Instead, a monopartite NLS, located at the amino terminus of the protein and conserved across insects, is functional in Drosophila S2 and Spodoptera exigua Sec301 cells. Last but not least, genetic interaction and immunohistochemical assays suggested that Cbt nuclear import is mediated by Importin-α2. Conclusions/Significance Our results constitute the first characterization of the molecular mechanisms of Cbt-mediated transcriptional control as well as of Cbt nuclear import, and demonstrate the existence of similarities and differences in both aspects of Cbt function between the insect and the vertebrate TIEG proteins. PMID:22359651

Occurrence of mitochondrial CO1 pseudogenes in Neocalanus plumchrus (Crustacea: Copepoda): Hybridization indicated by recombined nuclear mitochondrial pseudogenes

PubMed Central

Lin, Ya-Ying

2017-01-01

A portion of the mitochondrial cytochrome c oxidase I gene was sequenced using both genomic DNA and complement DNA from three planktonic copepod Neocalanus species (N. cristatus, N. plumchrus, and N. flemingeri). Small but critical sequence differences in CO1 were observed between gDNA and cDNA from N. plumchrus. Furthermore, careful observation revealed the presence of recombination between sequences in gDNA from N. plumchrus. Moreover, a chimera of the N. cristatus and N. plumchrus sequences was obtained from N. plumchrus gDNA. The observed phenomena can be best explained by the preferential amplification of the nuclear mitochondrial pseudogenes from gDNA of N. plumchrus. Two conclusions can be drawn from the observations. First, nuclear mitochondrial pseudogenes are pervasive in N. plumchrus. Second, a mating between a female N. cristatus and a male N. plumchrus produced viable offspring, which further backcrossed to a N. plumchrus individual. These observations not only demonstrate intriguing mating behavior in these species, but also emphasize the importance of careful interpretation of species marker sequences amplified from gDNA. PMID:28231343

Complete Sequence and Analysis of Coconut Palm (Cocos nucifera) Mitochondrial Genome

PubMed Central

Zhao, Yuhui; Zeng, Jingyao; Alamer, Ali; Alanazi, Ibrahim O.; Alawad, Abdullah O.; Al-Sadi, Abdullah M.; Hu, Songnian; Yu, Jun

2016-01-01

Coconut (Cocos nucifera L.), a member of the palm family (Arecaceae), is one of the most economically important crops in tropics, serving as an important source of food, drink, fuel, medicine, and construction material. Here we report an assembly of the coconut (C. nucifera, Oman local Tall cultivar) mitochondrial (mt) genome based on next-generation sequencing data. This genome, 678,653bp in length and 45.5% in GC content, encodes 72 proteins, 9 pseudogenes, 23 tRNAs, and 3 ribosomal RNAs. Within the assembly, we find that the chloroplast (cp) derived regions account for 5.07% of the total assembly length, including 13 proteins, 2 pseudogenes, and 11 tRNAs. The mt genome has a relatively large fraction of repeat content (17.26%), including both forward (tandem) and inverted (palindromic) repeats. Sequence variation analysis shows that the Ti/Tv ratio of the mt genome is lower as compared to that of the nuclear genome and neutral expectation. By combining public RNA-Seq data for coconut, we identify 734 RNA editing sites supported by at least two datasets. In summary, our data provides the second complete mt genome sequence in the family Arecaceae, essential for further investigations on mitochondrial biology of seed plants. PMID:27736909

JAK/STAT1 signaling promotes HMGB1 hyperacetylation and nuclear translocation

PubMed Central

Lu, Ben; Antoine, Daniel J.; Kwan, Kevin; Lundbäck, Peter; Wähämaa, Heidi; Schierbeck, Hanna; Robinson, Melissa; Van Zoelen, Marieke A. D.; Yang, Huan; Li, Jianhua; Erlandsson-Harris, Helena; Chavan, Sangeeta S.; Wang, Haichao; Andersson, Ulf; Tracey, Kevin J.

2014-01-01

Extracellular high-mobility group box (HMGB)1 mediates inflammation during sterile and infectious injury and contributes importantly to disease pathogenesis. The first critical step in the release of HMGB1 from activated immune cells is mobilization from the nucleus to the cytoplasm, a process dependent upon hyperacetylation within two HMGB1 nuclear localization sequence (NLS) sites. The inflammasomes mediate the release of cytoplasmic HMGB1 in activated immune cells, but the mechanism of HMGB1 translocation from nucleus to cytoplasm was previously unknown. Here, we show that pharmacological inhibition of JAK/STAT1 inhibits LPS-induced HMGB1 nuclear translocation. Conversely, activation of JAK/STAT1 by type 1 interferon (IFN) stimulation induces HMGB1 translocation from nucleus to cytoplasm. Mass spectrometric analysis unequivocally revealed that pharmacological inhibition of the JAK/STAT1 pathway or genetic deletion of STAT1 abrogated LPS- or type 1 IFN-induced HMGB1 acetylation within the NLS sites. Together, these results identify a critical role of the JAK/STAT1 pathway in mediating HMGB1 cytoplasmic accumulation for subsequent release, suggesting that the JAK/STAT1 pathway is a potential drug target for inhibiting HMGB1 release. PMID:24469805

The absence of p53 during Human Cytomegalovirus infection leads to decreased UL53 expression, disrupting UL50 localization to the inner nuclear membrane, and thereby inhibiting capsid nuclear egress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuan, Man I; O’Dowd, John M.; Fortunato, Elizabeth

Our electron microscopy study (Kuan et al., 2016) found HCMV nuclear capsid egress was significantly reduced in p53 knockout cells (p53KOs), correlating with inhibited formation of infoldings of the inner nuclear membrane (IINMs). Molecular examination of these phenomena has found p53KOs expressed UL97 and phosphorylated lamins, however the lamina failed to remodel. The nuclear egress complex (NEC) protein UL50 was expressed in almost all cells. UL50 re-localized to the inner nuclear membrane (INM) in ~90% of wt cells, but only ~35% of p53KOs. UL53 expression was significantly reduced in p53KOs, and cells lacking UL50 nuclear staining, expressed no UL53. Re-introductionmore » of p53 into p53KOs largely recovered UL53 positivity and UL50 nuclear re-localization. Nuclear rim located UL50/53 puncta, which co-localized with the major capsid protein, were largely absent in p53KOs. We believe these puncta were IINMs. In the absence of p53, UL53 expression was inhibited, disrupting formation of the NEC/IINMs, and reducing functional virion secretion. -- Highlights: •Phosphorylated nuclear lamins were inefficiently remodeled in p53KO cells. •p53KO cells expressed UL50, but it was not efficiently targeted to the nuclear rim. •UL53 was not expressed in the large majority of p53KO cells. •Cells failing to express UL53 did not localize UL50 to the nucleus. •NEC puncta/infoldings of the inner nuclear membrane were scarce in p53KO cells.« less

Characterization of Coffee ringspot virus-Lavras: A model for an emerging threat to coffee production and quality

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramalho, T.O.; Figueira, A.R.; Sotero, A.J.

2014-09-15

The emergence of viruses in Coffee (Coffea arabica and Coffea canephora), the most widely traded agricultural commodity in the world, is of critical concern. The RNA1 (6552 nt) of Coffee ringspot virus is organized into five open reading frames (ORFs) capable of encoding the viral nucleocapsid (ORF1p), phosphoprotein (ORF2p), putative cell-to-cell movement protein (ORF3p), matrix protein (ORF4p) and glycoprotein (ORF5p). Each ORF is separated by a conserved intergenic junction. RNA2 (5945 nt), which completes the bipartite genome, encodes a single protein (ORF6p) with homology to RNA-dependent RNA polymerases. Phylogenetic analysis of L protein sequences firmly establishes CoRSV as a membermore » of the recently proposed Dichorhavirus genus. Predictive algorithms, in planta protein expression, and a yeast-based nuclear import assay were used to determine the nucleophillic character of five CoRSV proteins. Finally, the temperature-dependent ability of CoRSV to establish systemic infections in an initially local lesion host was quantified. - Highlights: • We report genome sequence determination for Coffee ringspot virus (CoRSV). • CoRSV should be considered a member of the proposed Dichorhavirus genus. • We report temperature-dependent systemic infection of an initially local lesion host. • We report in planta protein and localization data for five CoRSV proteins. • In silico predictions of the CoRSV proteins were validated using in vivo assays.« less

Intergradation between discrete lineages of Tevnia jerichonana, a deep-sea hydrothermal vent tubeworm

NASA Astrophysics Data System (ADS)

Zhang, Haibin; Johnson, Shannon B.; Flores, Vanessa R.; Vrijenhoek, Robert C.

2015-11-01

We describe a broad zone of intergradation between genetically differentiated, northern and southern lineages of the hydrothermal vent tubeworm, Tevnia jerichonana. DNA sequences from four genes, nuclear HSP and ATPsα and mitochondrial COI and Cytb were examined in samples from eastern Pacific vent localities between 13°N and 38°S latitude. Allelic frequencies at these loci exhibited concordant latitudinal clines, and genetic differentiation (pairwise ΦST's) increased with geographical distances between sample localities. Though this pattern of differentiation suggested isolation-by-distance (IBD), it appeared to result from hierarchical population structure. Genotypic assignment tests identified two population clusters comprised of samples from the northern East Pacific Rise (NEPR: 9-13°N) and an extension of the Pacific-Antarctic Ridge (PAR: 31-32°S) with a zone of intergradation along the southern East Pacific Rise (SEPR: 7-17°S). The overall degrees of DNA sequence divergence between the NEPR and PAR populations were slight and not indicative of lengthy isolation. Bayesian assignment methods suggested that the SEPR populations constitute intergrades that connect the NEPR and PAR populations. Though it typically is difficult to distinguish between primary and secondary intergradation, our results were consistent with parallel studies of vent-restricted species that suggest a high degree of demographic instability along the superfast-spreading SEPR axis. Frequent local extinctions and immigration from NEPR and PAR refugia probably shaped the observed pattern of intergradation.

Evolutionary relationships among Pinus (Pinaceae) subsections inferred from multiple low-copy nuclear loci.

Treesearch

John Syring; Ann Willyard; Richard Cronn; Aaron Liston

2005-01-01

Sequence data from nrITS and cpDNA have failed to fully resolve phylogenetic relationships among Pinus species. Four low-copy nuclear genes, developed from the screening of 73 mapped conifer anchor loci, were sequenced from 12 species representing all subsections. Individual loci do not uniformly support either the nrITS or cpDNA hypotheses and in...

Aberrant localization of lamin B receptor (LBR) in cellular senescence in human cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arai, Rumi; En, Atsuki; Ukekawa, Ryo

2016-05-13

5-Bromodeoxyuridine (BrdU), a thymidine analogue, induces cellular senescence in mammalian cells. BrdU induces cellular senescence probably through the regulation of chromatin because BrdU destabilizes or disrupts nucleosome positioning and decondenses heterochromatin. Since heterochromatin is tethered to the nuclear periphery through the interaction with the nuclear envelope proteins, we examined the localization of the several nuclear envelope proteins such as lamins, lamin-interacting proteins, nuclear pore complex proteins, and nuclear transport proteins in senescent cells. We have shown here that lamin B receptor (LBR) showed a change in localization in both BrdU-induced and replicative senescent cells.

Immunological and biochemical evidence for nuclear localization of annexin in peas

NASA Technical Reports Server (NTRS)

Clark, G. B.; Dauwalder, M.; Roux, S. J.

1998-01-01

Immunofluorescent localization of annexins using an anti-pea annexin polyclonal antibody (anti-p35) in pea (Pisum sativum) leaf and stem epidermal peels showed staining of the nuclei and the cell periphery. Nuclear staining was also seen in cell teases prepared from pea plumules. The amount of nuclear stain was reduced both by fixation time and by dehydration and organic solvent treatment. Observation with confocal microscopy demonstrated that the anti-p35 stain was diffusely distributed throughout the nuclear structure. Immunoblots of purified nuclei, nuclear envelope matrix, nucleolar, and chromatin fractions showed a cross-reactive protein band of 35 kDa. These data are the first to show annexins localized in plant cell nuclei where they may play a role in nuclear function.

Amplicon Sequencing Reveals Microbiological Signatures in Spent Nuclear Fuel Storage Basins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bagwell, Christopher E.; Noble, Peter A.; Milliken, Charles E.

Water quality is an important determinant for the structural integrity of alloy cladded fuels and assemblies during long-term wet storage. Detailed characterization of a water filled storage basin for spent nuclear reactor fuel was performed following the formation and proliferation of an amorphous white flocculent. White precipitant was sampled throughout the storage basin for chemical and spectroscopic characterization, and eDNA was extracted for pyrosequencing of bacterial rRNA gene diversity. Accordingly, spectroscopic analyses indicated the precipitant to be primarily amorphous to crystalline aluminum (oxy) hydroxides with minor associated elemental components including Fe, Si, Ti, and U. High levels of dissolved carbonmore » were co-localized with the precipitant relative to bulk water. Bacterial densities were highly variable between sampling locations and with depth; cell numbers (log scale) ranged from 5.6 to 4.89 cells / mL. Bacterial diversity that was physically associated with the aluminum (oxy) hydroxide complexes exceeded an estimated 4,000 OTUs / amplicon library (3% cutoff) and the greatest percent majority of sequences were aligned to the families Burkholderiales (23%), Nitrospiraceae (23%), Hyphomicrobiaceae (17%), and Comamonadaceae (6%). We surmise that episodic changes in the physical and chemical properties of the basin contribute to the polymerization of aluminum (oxy) hydroxides, which in turn can chemisorb nutrients, carbon ligands and bacterial cells from the surrounding bulk aqueous phase. As such, these precipitants should establish favorable microhabitats for bacterial colonization and growth. Comparative analyses of 16S rRNA gene amplicon libraries across diverse environmental landscapes were performed and microbiological signatures unique to the spent nuclear fuel storage basin environment were revealed. These insights could spur the development of tractable bioindicators that are specific of and diagnostic for water quality at discrete locations and finer scales of resolution, marking an important contribution for improved water quality and management of spent nuclear fuel storage facilities.« less

Lysine 271 but not lysine 210 of XRCC4 is required for the nuclear localization of XRCC4 and DNA ligase IV

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fukuchi, Mikoto; Wanotayan, Rujira; Liu, Sicheng

2015-06-12

XRCC4 and DNA Ligase IV (LIG4) cooperate to join two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). However, it is not fully understood how these proteins are localized to the nucleus. Here we created XRCC4{sup K271R} mutant, as Lys271 lies within the putative nuclear localization signal (NLS), and XRCC4{sup K210R} mutant, as Lys210 was reported to undergo SUMOylation, implicated in the nuclear localization of XRCC4. Wild-type and mutated XRCC4 with EGFP tag were introduced into HeLa cell, in which endogenous XRCC4 had been knocked down using siRNA directed to 3′-untranslated region,more » and tested for the nuclear localization function by fluorescence microscopy. XRCC4{sup K271R} was defective in the nuclear localization of itself and LIG4, whereas XRCC4{sup K210R} was competent for the nuclear localization with LIG4. To examine DSB repair function, wild-type and mutated XRCC4 were introduced into XRCC4-deficient M10. M10-XRCC4{sup K271R}, but not M10-XRCC4{sup K210R}, showed significantly reduced surviving fraction after 2 Gy γ-ray irradiation as compared to M10-XRCC4{sup WT}. The number of γ-H2AX foci remaining 2 h after 2 Gy γ-ray irradiation was significantly greater in M10-XRCC4{sup K271R} than in M10-XRCC4{sup WT}, whereas it was only marginally increased in M10-XRCC4{sup K210R} as compared to M10-XRCC4{sup WT}. The present results collectively indicated that Lys271, but not Lys210, of XRCC4 is required for the nuclear localization of XRCC4 and LIG4 and that the nuclear localizing ability is essential for DSB repair function of XRCC4. - Highlights: • XRCC4{sup K271R} is defective in the nuclear localization of itself and LIG4. • XRCC4{sup K210R} is competent for the nuclear localization of itself and LIG4. • XRCC4{sup K271R} is deficient in DSB repair function. • XRCC4{sup K210R} is mostly normal in DSB repair function.« less

The fate of the Antennae galaxies

NASA Astrophysics Data System (ADS)

Lahén, Natalia; Johansson, Peter H.; Rantala, Antti; Naab, Thorsten; Frigo, Matteo

2018-04-01

We present a high-resolution smoothed particle hydrodynamic simulation of the Antennae galaxies (NGC 4038/4039) and follow the evolution 3 Gyr beyond the final coalescence. The simulation includes metallicity-dependent cooling, star formation, and both stellar feedback and chemical enrichment. The simulated best-match Antennae reproduce well both the observed morphology and the off-nuclear starburst. We also produce for the first time a simulated two-dimensional (2D) metallicity map of the Antennae and find good agreement with the observed metallicity of off-nuclear stellar clusters; however, the nuclear metallicities are overproduced by ˜0.5 dex. Using the radiative transfer code SKIRT, we produce multiwavelength observations of both the Antennae and the merger remnant. The 1-Gyr-old remnant is well fitted with a Sérsic profile of n = 7.07, and with an r-band effective radius of re = 1.6 kpc and velocity dispersion of σe = 180 km s-1 the remnant is located on the Fundamental Plane of early-type galaxies (ETGs). The initially blue Antennae remnant evolves on to the red sequence after ˜2.5 Gyr of secular evolution. The remnant would be classified as a fast rotator, as the specific angular momentum evolves from λRe ≈ 0.11 to 0.14 during its evolution. The remnant shows ordered rotation and a double peaked maximum in the mean 2D line-of-sight velocity. These kinematical features are relatively common amongst local ETGs and we specifically identify three local ETGs (NGC 3226, NGC 3379, and NGC 4494) in the atlas3D sample, whose photometric and kinematic properties most resemble the Antennae remnant.

Small nuclear RNA U2 is base-paired to heterogeneous nuclear RNA.

PubMed

Calvet, J P; Meyer, L M; Pederson, T

1982-07-30

Eukaryotic cells contain a set of low molecular weight nuclear RNA's. One of the more abundant of these is termed U2 RNA. The possibility that U2 RNA is hydrogen-bonded to complementary sequences in other nuclear RNA's was investigated. Cultured human (HeLa) cells were treated with a psoralen derivative that cross-links RNA chains that are base-paired with one another. High molecular weight heterogeneous nuclear RNA was isolated under denaturing conditions, and the psoralen cross-links were reversed. Electrophoresis of the released RNA and hybridization with a human cloned U2 DNA probe revealed that U2 is hydrogen-bonded to complementary sequences in heterogeneous nuclear RNA in vivo. In contrast, U2 RNA is not base-paired with nucleolar RNA, which contains the precursors of ribosomal RNA. The results suggest that U2 RNA participates in messenger RNA processing in the nucleus.

The 2017 North Korea M6 seismic sequence: moment tensor, source time function, and aftershocks

NASA Astrophysics Data System (ADS)

Ni, S.; Zhan, Z.; Chu, R.; He, X.

2017-12-01

On September 3rd, 2017, an M6 seismic event occurred in North Korea, with location near previous nuclear test sites. The event features strong P waves and short period Rayleigh waves are observed in contrast to weak S waves, suggesting mostly explosion mechanism. We performed joint inversion for moment tensor and depth with both local and teleseismic waveforms, and find that the event is shallow with mostly isotropic yet substantial non-isotropic components. Deconvolution of seismic waveforms of this event with respect to previous nuclear test events shows clues of complexity in source time function. The event is followed by smaller earthquakes, as early as 8.5 minutes and lasted at least to October. The later events occurred in a compact region, and show clear S waves, suggesting double couple focal mechanism. Via analyzing Rayleigh wave spectrum, these smaller events are found to be shallow. Relative locations, difference in waveforms of the events are used to infer their possible links and generation mechanism.

Characterization of a nuclear export signal within the human T cell leukemia virus type I transactivator protein Tax.

PubMed

Alefantis, Timothy; Barmak, Kate; Harhaj, Edward W; Grant, Christian; Wigdahl, Brian

2003-06-13

Human T cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T cell leukemia and HTLV-I-associated myelopathy/tropical spastic paraparesis. The HTLV-I transactivator protein Tax plays an integral role in the etiology of adult T cell leukemia, as expression of Tax in T lymphocytes has been shown to result in immortalization. In addition, Tax is known to interface with numerous transcription factor families, including activating transcription factor/cAMP response element-binding protein and nuclear factor-kappaB, requiring Tax to localize to both the nucleus and cytoplasm. In this report, the nucleocytoplasmic localization of Tax was examined in Jurkat, HeLa, and U-87 MG cells. The results reported herein indicate that Tax contains a leucine-rich nuclear export signal (NES) that, when fused to green fluorescent protein (GFP), can direct nuclear export via the CRM-1 pathway, as determined by leptomycin B inhibition of nuclear export. However, cytoplasmic localization of full-length Tax was not altered by treatment with leptomycin B, suggesting that native Tax utilizes another nuclear export pathway. Additional support for the presence of a functional NES has also been shown because the NES mutant Tax(L200A)-GFP localized to the nuclear membrane in the majority of U-87 MG cells. Evidence has also been provided suggesting that the Tax NES likely exists as a conditionally masked signal because the truncation mutant TaxDelta214-GFP localized constitutively to the cytoplasm. These results suggest that Tax localization may be directed by specific changes in Tax conformation or by specific interactions with cellular proteins leading to changes in the availability of the Tax NES and nuclear localization signal.

Nuclear accumulation of epidermal growth factor receptor and acceleration of G1/S stage by Epstein-Barr-encoded oncoprotein latent membrane protein 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tao Yongguang; Song Xing; Deng Xiyun

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is considered to be the major oncogenic protein of EBV-encoded proteins and has always been the core of the oncogenic mechanism of EBV. Advanced studies on nuclear translocation of the epidermal growth factor receptor (EGFR) family have greatly improved our knowledge of the biological function of cell surface receptors. In this study, we used the Tet-on LMP1 HNE2 cell line as a cell model, which is a dual-stable LMP1-integrated nasopharyngeal carcinoma (NPC) cell line and the expression of LMP1 which could be regulated by the Tet system. We found that LMP1 couldmore » regulate the nuclear accumulation of EGFR in a dose-dependent manner quantitatively and qualitatively. We also demonstrated that the nuclear localization sequence of EGFR played some roles in the location of the protein within the nucleus under LMP1 regulation and EGFR in the nucleus could bind to the promoters of cyclinD1 and cyclinE, respectively. We further demonstrated that EGFR is involved in the acceleration of the G1/S phase transition by LMP1 through binding to cyclinD1 and cyclinE directly. These findings provided a novel view that the acceleration of LMP1 on the G1/S transition via the nuclear accumulation of EGFR was critical in the process of nasopharyngeal carcinoma.« less

Fukushima Accident: Sequence of Events and Lessons Learned

NASA Astrophysics Data System (ADS)

Morse, Edward C.

2011-10-01

The Fukushima Dai-Ichi nuclear power station suffered a devastating Richter 9.0 earthquake followed by a 14.0 m tsunami on 11 March 2011. The subsequent loss of power for emergency core cooling systems resulted in damage to the fuel in the cores of three reactors. The relief of pressure from the containment in these three reactors led to sufficient hydrogen gas release to cause explosions in the buildings housing the reactors. There was probably subsequent damage to a spent fuel pool of a fourth reactor caused by debris from one of these explosions. Resultant releases of fission product isotopes in air were significant and have been estimated to be in the 3 . 7 --> 6 . 3 ×1017 Bq range (~10 MCi) for 131I and 137Cs combined, or approximately one tenth that of the Chernobyl accident. A synopsis of the sequence of events leading up to this large release of radioactivity will be presented, along with likely scenarios for stabilization and site cleanup in the future. Some aspects of the isotope monitoring programs, both locally and at large, will also be discussed. An assessment of radiological health risk for the plant workers as well as the general public will also be presented. Finally, the impact of this accident on design and deployment of nuclear generating stations in the future will be discussed.

The 193-Kd Vault Protein, Vparp, Is a Novel Poly(Adp-Ribose) Polymerase

PubMed Central

Kickhoefer, Valerie A.; Siva, Amara C.; Kedersha, Nancy L.; Inman, Elisabeth M.; Ruland, Cristina; Streuli, Michel; Rome, Leonard H.

1999-01-01

Mammalian vaults are ribonucleoprotein (RNP) complexes, composed of a small ribonucleic acid and three proteins of 100, 193, and 240 kD in size. The 100-kD major vault protein (MVP) accounts for >70% of the particle mass. We have identified the 193-kD vault protein by its interaction with the MVP in a yeast two-hybrid screen and confirmed its identity by peptide sequence analysis. Analysis of the protein sequence revealed a region of ∼350 amino acids that shares 28% identity with the catalytic domain of poly(ADP-ribose) polymerase (PARP). PARP is a nuclear protein that catalyzes the formation of ADP-ribose polymers in response to DNA damage. The catalytic domain of p193 was expressed and purified from bacterial extracts. Like PARP, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction; thus, the 193-kD protein is a new PARP. Purified vaults also contain the poly(ADP-ribosyl)ation activity, indicating that the assembled particle retains enzymatic activity. Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP. Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s). A portion of p193 is nuclear and localizes to the mitotic spindle. PMID:10477748

An ethylene-responsive enhancer element is involved in the senescence-related expression of the carnation glutathione-S-transferase (GST1) gene.

PubMed

Itzhaki, H; Maxson, J M; Woodson, W R

1994-09-13

The increased production of ethylene during carnation petal senescence regulates the transcription of the GST1 gene encoding a subunit of glutathione-S-transferase. We have investigated the molecular basis for this ethylene-responsive transcription by examining the cis elements and trans-acting factors involved in the expression of the GST1 gene. Transient expression assays following delivery of GST1 5' flanking DNA fused to a beta-glucuronidase receptor gene were used to functionally define sequences responsible for ethylene-responsive expression. Deletion analysis of the 5' flanking sequences of GST1 identified a single positive regulatory element of 197 bp between -667 and -470 necessary for ethylene-responsive expression. The sequences within this ethylene-responsive region were further localized to 126 bp between -596 and -470. The ethylene-responsive element (ERE) within this region conferred ethylene-regulated expression upon a minimal cauliflower mosaic virus-35S TATA-box promoter in an orientation-independent manner. Gel electrophoresis mobility-shift assays and DNase I footprinting were used to identify proteins that bind to sequences within the ERE. Nuclear proteins from carnation petals were shown to specifically interact with the 126-bp ERE and the presence and binding of these proteins were independent of ethylene or petal senescence. DNase I footprinting defined DNA sequences between -510 and -488 within the ERE specifically protected by bound protein. An 8-bp sequence (ATTTCAAA) within the protected region shares significant homology with promoter sequences required for ethylene responsiveness from the tomato fruit-ripening E4 gene.

Reversible Oxidation of a Conserved Methionine in the Nuclear Export Sequence Determines Subcellular Distribution and Activity of the Fungal Nitrate Regulator NirA.

PubMed

Gallmetzer, Andreas; Silvestrini, Lucia; Schinko, Thorsten; Gesslbauer, Bernd; Hortschansky, Peter; Dattenböck, Christoph; Muro-Pastor, María Isabel; Kungl, Andreas; Brakhage, Axel A; Scazzocchio, Claudio; Strauss, Joseph

2015-07-01

The assimilation of nitrate, a most important soil nitrogen source, is tightly regulated in microorganisms and plants. In Aspergillus nidulans, during the transcriptional activation process of nitrate assimilatory genes, the interaction between the pathway-specific transcription factor NirA and the exportin KapK/CRM1 is disrupted, and this leads to rapid nuclear accumulation and transcriptional activity of NirA. In this work by mass spectrometry, we found that in the absence of nitrate, when NirA is inactive and predominantly cytosolic, methionine 169 in the nuclear export sequence (NES) is oxidized to methionine sulfoxide (Metox169). This oxidation depends on FmoB, a flavin-containing monooxygenase which in vitro uses methionine and cysteine, but not glutathione, as oxidation substrates. The function of FmoB cannot be replaced by alternative Fmo proteins present in A. nidulans. Exposure of A. nidulans cells to nitrate led to rapid reduction of NirA-Metox169 to Met169; this reduction being independent from thioredoxin and classical methionine sulfoxide reductases. Replacement of Met169 by isoleucine, a sterically similar but not oxidizable residue, led to partial loss of NirA activity and insensitivity to FmoB-mediated nuclear export. In contrast, replacement of Met169 by alanine transformed the protein into a permanently nuclear and active transcription factor. Co-immunoprecipitation analysis of NirA-KapK interactions and subcellular localization studies of NirA mutants lacking different parts of the protein provided evidence that Met169 oxidation leads to a change in NirA conformation. Based on these results we propose that in the presence of nitrate the activation domain is exposed, but the NES is masked by a central portion of the protein (termed nitrate responsive domain, NiRD), thus restricting active NirA molecules to the nucleus. In the absence of nitrate, Met169 in the NES is oxidized by an FmoB-dependent process leading to loss of protection by the NiRD, NES exposure, and relocation of the inactive NirA to the cytosol.

Multi-locus DNA sequence data reveal a history of deep cryptic vicariance and habitat-driven convergence in the desert night lizard Xantusia vigilis species complex (Squamata: Xantusiidae).

PubMed

Leavitt, Dean H; Bezy, Robert L; Crandall, Keith A; Sites, Jack W

2007-11-01

The lizard genus Xantusia of southwestern North America has received recent attention in relation to delimiting species. Using more than 500 lizards from 156 localities, we further test hypothesized species boundaries and clarify phylogeographical patterns, particularly in regions of potential secondary contact. We sequenced the entire mitochondrial cytochrome b gene for every lizard in the study, plus a second mitochondrial DNA (mtDNA) region and two nuclear introns for subsets of the total sample. Phylogenetic analyses of the mtDNA recover a well-resolved, novel hypothesis for species in the Xantusia vigilis complex. The nuclear DNA (nDNA) data provide independent support for the recognition of X. arizonae, X. bezyi and X. wigginsi. Differences between the respective mtDNA and nDNA topologies result from either the effects of lineage sorting or ancient introgression. Nuclear data confirm the inference that some populations of X. vigilis in northwestern Arizona converged on rock-crevice-dwelling morphology and are not X. arizonae with an introgressed X. vigilis mtDNA genome. The historical independence of ancient cryptic lineages of Xantusia in southern California is also corroborated, though limited introgression is detected. Our proposed biogeographical scenario indicates that diversification of this group was driven by vicariance beginning in the late Miocene. Additionally, Pleistocene climatical changes influenced Xantusia distribution, and the now inhospitable Colorado Desert previously supported night lizard presence. The current taxonomy of the group likely underestimates species diversity within the group, and our results collectively show that while convergence on the rock-crevice-dwelling morphology is one hallmark of Xantusia evolution, morphological stasis is paradoxically another.

Shuttling imbalance of MLF1 results in p53 instability and increases susceptibility to oncogenic transformation.

PubMed

Yoneda-Kato, Noriko; Kato, Jun-Ya

2008-01-01

Myeloid leukemia factor 1 (MLF1) stabilizes the activity of the tumor suppressor p53 by suppressing its E3 ubiquitin ligase, COP1, through a third component of the COP9 signalosome (CSN3). However, little is known about how MLF1 functions upstream of the CSN3-COP1-p53 pathway and how its deregulation by the formation of the fusion protein nucleophosmin (NPM)-MLF1, generated by t(3;5)(q25.1;q34) chromosomal translocation, leads to leukemogenesis. Here we show that MLF1 is a cytoplasmic-nuclear-shuttling protein and that its nucleolar localization on fusing with NPM prevents the full induction of p53 by both genotoxic and oncogenic cellular stress. The majority of MLF1 was located in the cytoplasm, but the treatment of cells with leptomycin B rapidly induced a nuclear accumulation of MLF1. A mutation of the nuclear export signal (NES) motif identified in the MLF1 sequence enhanced the antiproliferative activity of MLF1. The fusion of MLF1 with NPM translocated MLF1 to the nucleolus and abolished the growth-suppressing activity. The introduction of NPM-MLF1 into early-passage murine embryonic fibroblasts allowed the cells to escape from cellular senescence at a markedly earlier stage and induced neoplastic transformation in collaboration with the oncogenic form of Ras. Interestingly, disruption of the MLF1-derived NES sequence completely abolished the growth-promoting activity of NPM-MLF1 in murine fibroblasts and hematopoietic cells. Thus, our results provide important evidence that the shuttling of MLF1 is critical for the regulation of cell proliferation and a disturbance in the shuttling balance increases the cell's susceptibility to oncogenic transformation.

Shuttling Imbalance of MLF1 Results in p53 Instability and Increases Susceptibility to Oncogenic Transformation▿ †

PubMed Central

Yoneda-Kato, Noriko; Kato, Jun-ya

2008-01-01

Myeloid leukemia factor 1 (MLF1) stabilizes the activity of the tumor suppressor p53 by suppressing its E3 ubiquitin ligase, COP1, through a third component of the COP9 signalosome (CSN3). However, little is known about how MLF1 functions upstream of the CSN3-COP1-p53 pathway and how its deregulation by the formation of the fusion protein nucleophosmin (NPM)-MLF1, generated by t(3;5)(q25.1;q34) chromosomal translocation, leads to leukemogenesis. Here we show that MLF1 is a cytoplasmic-nuclear-shuttling protein and that its nucleolar localization on fusing with NPM prevents the full induction of p53 by both genotoxic and oncogenic cellular stress. The majority of MLF1 was located in the cytoplasm, but the treatment of cells with leptomycin B rapidly induced a nuclear accumulation of MLF1. A mutation of the nuclear export signal (NES) motif identified in the MLF1 sequence enhanced the antiproliferative activity of MLF1. The fusion of MLF1 with NPM translocated MLF1 to the nucleolus and abolished the growth-suppressing activity. The introduction of NPM-MLF1 into early-passage murine embryonic fibroblasts allowed the cells to escape from cellular senescence at a markedly earlier stage and induced neoplastic transformation in collaboration with the oncogenic form of Ras. Interestingly, disruption of the MLF1-derived NES sequence completely abolished the growth-promoting activity of NPM-MLF1 in murine fibroblasts and hematopoietic cells. Thus, our results provide important evidence that the shuttling of MLF1 is critical for the regulation of cell proliferation and a disturbance in the shuttling balance increases the cell's susceptibility to oncogenic transformation. PMID:17967869

A Role for Caenorhabditis elegans Importin IMA-2 in Germ Line and Embryonic Mitosis

PubMed Central

Geles, Kenneth G.; Johnson, Jeffrey J.; Jong, Sena; Adam, Stephen A.

2002-01-01

The importin α family of nuclear-cytoplasmic transport factors mediates the nuclear localization of proteins containing classical nuclear localization signals. Metazoan animals express multiple importin α proteins, suggesting their possible roles in cell differentiation and development. Adult Caenorhabditis elegans hermaphrodites express three importin α proteins, IMA-1, IMA-2, and IMA-3, each with a distinct expression and localization pattern. IMA-2 was expressed exclusively in germ line cells from the early embryonic through adult stages. The protein has a dynamic pattern of localization dependent on the stage of the cell cycle. In interphase germ cells and embryonic cells, IMA-2 is cytoplasmic and nuclear envelope associated, whereas in developing oocytes, the protein is cytoplasmic and intranuclear. During mitosis in germ line cells and embryos, IMA-2 surrounded the condensed chromosomes but was not directly associated with the mitotic spindle. The timing of IMA-2 nuclear localization suggested that the protein surrounded the chromosomes after fenestration of the nuclear envelope in prometaphase. Depletion of IMA-2 by RNA-mediated gene interference (RNAi) resulted in embryonic lethality and a terminal aneuploid phenotype. ima-2(RNAi) embryos have severe defects in nuclear envelope formation, accumulating nucleoporins and lamin in the cytoplasm. We conclude that IMA-2 is required for proper chromosome dynamics in germ line and early embryonic mitosis and is involved in nuclear envelope assembly at the conclusion of mitosis. PMID:12221121

Between Two Fern Genomes

PubMed Central

2014-01-01

Ferns are the only major lineage of vascular plants not represented by a sequenced nuclear genome. This lack of genome sequence information significantly impedes our ability to understand and reconstruct genome evolution not only in ferns, but across all land plants. Azolla and Ceratopteris are ideal and complementary candidates to be the first ferns to have their nuclear genomes sequenced. They differ dramatically in genome size, life history, and habit, and thus represent the immense diversity of extant ferns. Together, this pair of genomes will facilitate myriad large-scale comparative analyses across ferns and all land plants. Here we review the unique biological characteristics of ferns and describe a number of outstanding questions in plant biology that will benefit from the addition of ferns to the set of taxa with sequenced nuclear genomes. We explain why the fern clade is pivotal for understanding genome evolution across land plants, and we provide a rationale for how knowledge of fern genomes will enable progress in research beyond the ferns themselves. PMID:25324969

Enlightenment of Yeast Mitochondrial Homoplasmy: Diversified Roles of Gene Conversion

PubMed Central

Ling, Feng; Mikawa, Tsutomu; Shibata, Takehiko

2011-01-01

Mitochondria have their own genomic DNA. Unlike the nuclear genome, each cell contains hundreds to thousands of copies of mitochondrial DNA (mtDNA). The copies of mtDNA tend to have heterogeneous sequences, due to the high frequency of mutagenesis, but are quickly homogenized within a cell (“homoplasmy”) during vegetative cell growth or through a few sexual generations. Heteroplasmy is strongly associated with mitochondrial diseases, diabetes and aging. Recent studies revealed that the yeast cell has the machinery to homogenize mtDNA, using a common DNA processing pathway with gene conversion; i.e., both genetic events are initiated by a double-stranded break, which is processed into 3′ single-stranded tails. One of the tails is base-paired with the complementary sequence of the recipient double-stranded DNA to form a D-loop (homologous pairing), in which repair DNA synthesis is initiated to restore the sequence lost by the breakage. Gene conversion generates sequence diversity, depending on the divergence between the donor and recipient sequences, especially when it occurs among a number of copies of a DNA sequence family with some sequence variations, such as in immunoglobulin diversification in chicken. MtDNA can be regarded as a sequence family, in which the members tend to be diversified by a high frequency of spontaneous mutagenesis. Thus, it would be interesting to determine why and how double-stranded breakage and D-loop formation induce sequence homogenization in mitochondria and sequence diversification in nuclear DNA. We will review the mechanisms and roles of mtDNA homoplasmy, in contrast to nuclear gene conversion, which diversifies gene and genome sequences, to provide clues toward understanding how the common DNA processing pathway results in such divergent outcomes. PMID:24710143

Activation of β-catenin signaling in androgen receptor-negative prostate cancer cells.

PubMed

Wan, Xinhai; Liu, Jie; Lu, Jing-Fang; Tzelepi, Vassiliki; Yang, Jun; Starbuck, Michael W; Diao, Lixia; Wang, Jing; Efstathiou, Eleni; Vazquez, Elba S; Troncoso, Patricia; Maity, Sankar N; Navone, Nora M

2012-02-01

To study Wnt/β-catenin in castrate-resistant prostate cancer (CRPC) and understand its function independently of the β-catenin-androgen receptor (AR) interaction. We carried out β-catenin immunocytochemical analysis, evaluated TOP-flash reporter activity (a reporter of β-catenin-mediated transcription), and sequenced the β-catenin gene in MDA prostate cancer 118a, MDA prostate cancer 118b, MDA prostate cancer 2b, and PC-3 prostate cancer cells. We knocked down β-catenin in AR-negative MDA prostate cancer 118b cells and carried out comparative gene-array analysis. We also immunohistochemically analyzed β-catenin and AR in 27 bone metastases of human CRPCs. β-Catenin nuclear accumulation and TOP-flash reporter activity were high in MDA prostate cancer 118b but not in MDA prostate cancer 2b or PC-3 cells. MDA prostate cancer 118a and MDA prostate cancer 118b cells carry a mutated β-catenin at codon 32 (D32G). Ten genes were expressed differently (false discovery rate, 0.05) in MDA prostate cancer 118b cells with downregulated β-catenin. One such gene, hyaluronan synthase 2 (HAS2), synthesizes hyaluronan, a core component of the extracellular matrix. We confirmed HAS2 upregulation in PC-3 cells transfected with D32G-mutant β-catenin. Finally, we found nuclear localization of β-catenin in 10 of 27 human tissue specimens; this localization was inversely associated with AR expression (P = 0.056, Fisher's exact test), suggesting that reduced AR expression enables Wnt/β-catenin signaling. We identified a previously unknown downstream target of β-catenin, HAS2, in prostate cancer, and found that high β-catenin nuclear localization and low or no AR expression may define a subpopulation of men with bone metastatic prostate cancer. These findings may guide physicians in managing these patients.

Five Conditions Commonly Used to Down-regulate Tor Complex 1 Generate Different Physiological Situations Exhibiting Distinct Requirements and Outcomes*

PubMed Central

Tate, Jennifer J.; Cooper, Terrance G.

2013-01-01

Five different physiological conditions have been used interchangeably to establish the sequence of molecular events needed to achieve nitrogen-responsive down-regulation of TorC1 and its subsequent regulation of downstream reporters: nitrogen starvation, methionine sulfoximine (Msx) addition, nitrogen limitation, rapamycin addition, and leucine starvation. Therefore, we tested a specific underlying assumption upon which the interpretation of data generated by these five experimental perturbations is premised. It is that they generate physiologically equivalent outcomes with respect to TorC1, i.e. its down-regulation as reflected by TorC1 reporter responses. We tested this assumption by performing head-to-head comparisons of the requirements for each condition to achieve a common outcome for a downstream proxy of TorC1 inactivation, nuclear Gln3 localization. We demonstrate that the five conditions for down-regulating TorC1 do not elicit physiologically equivalent outcomes. Four of the methods exhibit hierarchical Sit4 and PP2A phosphatase requirements to elicit nuclear Gln3-Myc13 localization. Rapamycin treatment required Sit4 and PP2A. Nitrogen limitation and short-term nitrogen starvation required only Sit4. G1 arrest-correlated, long-term nitrogen starvation and Msx treatment required neither PP2A nor Sit4. Starving cells of leucine or treating them with leucyl-tRNA synthetase inhibitors did not elicit nuclear Gln3-Myc13 localization. These data indicate that the five commonly used nitrogen-related conditions of down-regulating TorC1 are not physiologically equivalent and minimally involve partially differing regulatory mechanisms. Further, identical requirements for Msx treatment and long-term nitrogen starvation raise the possibility that their effects are achieved through a common regulatory pathway with glutamine, a glutamate or glutamine metabolite level as the sensed metabolic signal. PMID:23935103

Rat Humanin is encoded and translated in mitochondria and is localized to the mitochondrial compartment where it regulates ROS production.

PubMed

Paharkova, Vladislava; Alvarez, Griselda; Nakamura, Hiromi; Cohen, Pinchas; Lee, Kuk-Wha

2015-09-15

Evidence for the putative mitochondrial origin of the Humanin (HN) peptide has been lacking, although its cytoprotective activity has been demonstrated in a variety of organismal and cellular systems. We sought to establish proof-of-principle for a mitochondria-derived peptide (MDP) in a rat-derived cellular system as the rat HN sequence is predicted to lack nuclear insertions of mitochondrial origin (NUMT). We found that the rat HN (Rattin; rHN) homologue is derived from the mitochondrial genome as evidenced by decreased production in Rho-0 cells, and that peptide translation occurs in the mitochondria as it is unaffected by cycloheximide. Rat HN localizes to the mitochondria in cellular subfractionation and immunohistochemical studies. Addition of a HN analogue to isolated mitochondria from rat INS-1 beta cells reduced hydrogen peroxide production by 55%. In summary, a locally bioactive peptide is derived and translated from an open reading frame (ORF) within rat mitochondrial DNA encoding 16S rRNA. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Nup82 functions redundantly with Nup136 in a salicylic acid-dependent defense response of Arabidopsis thaliana.

PubMed

Tamura, Kentaro; Fukao, Yoichiro; Hatsugai, Noriyuki; Katagiri, Fumiaki; Hara-Nishimura, Ikuko

2017-05-04

The nuclear pore complex (NPC) comprises more than 30 nucleoporins (Nups). NPC mediates macromolecular trafficking between the nucleoplasm and the cytoplasm, but specific roles of individual Nups are poorly understood in higher plants. Here, we show that the novel nucleoporin unique to angiosperm plants (designated as Nup82) functions in a salicylic acid-dependent defense in a redundant manner with Nup136, which is a component of the nuclear basket in the NPC. Arabidopsis thaliana Nup82 had a similar amino acid sequence to the N-terminal half of Nup136 and a Nup82-GFP fusion was localized on the nuclear envelope. Immunoprecipitation and bimolecular fluorescence complementation analyses revealed that Nup82 interacts with the NPC components Nup136 and RAE1. The double knockout mutant nup82 nup136 showed severe growth defects, while the single knockout mutant nup82 did not, suggesting that Nup82 functions redundantly with Nup136. nup82 nup136 impaired benzothiadiazole (an analog of salicylic acid)-induced resistance to the virulent bacteria Pseudomonas syringae pv. tomato DC3000. Furthermore, transcriptome analysis of nup82 nup136 indicates that deficiency of Nup82 and Nup136 causes noticeable downregulation of immune-related genes. These results suggest that Nup82 and Nup136 are redundantly involved in transcriptional regulation of salicylic acid-responsive genes through nuclear transport of signaling molecules.

Role of cyclophilins in somatolactogenic action.

PubMed

Rycyzyn, M A; Clevenger, C V

2000-01-01

Prolactin (PRL) and growth hormone (GH) are members of the somatolactogenic hormone family, the pleiotropic actions of which are necessary for vertebrate growth and mammary differentiation. The basis for the specific function of these hormones has remained uncertain; however, their action is associated with internalization and translocation into the nucleus. A yeast two-hybrid screen identified an interaction between PRL and cyclophilin B (CypB), a peptidyl prolyl isomerase (PPI) found in the endoplasmic reticulum (ER), extracellular space, and nucleus. The interaction between CypB and PRL/GH was confirmed in vitro and in vivo through the use of recombinant proteins and coimmunoprecipitation studies. The exogenous addition of CypB potentiated the proliferation of PRL- and GH-dependent cell lines 18- and 40-fold, respectively. The potentiation of PRL action by CypB was accompanied by a dramatic increase in the nuclear retrotranslocation of PRL. Immunogold electron microscopy has revealed this retrotransport to occur via a vesicular pathway. A CypB mutant, termed CypB-NT, was generated that lacked the putative wild-type N-terminal nuclear localization sequence. Although CypB-NT demonstrated levels of PRL binding and PPI activity equivalent to wild-type CypB, it was incapable of mediating the nuclear retrotranslocation of PRL or enhancing PRL-driven proliferation. These studies reveal CypB as an important chaperone facilitating the nuclear retrotransport and action of the somatolactogenic hormone family.

A dated molecular phylogeny of manta and devil rays (Mobulidae) based on mitogenome and nuclear sequences.

PubMed

Poortvliet, Marloes; Olsen, Jeanine L; Croll, Donald A; Bernardi, Giacomo; Newton, Kelly; Kollias, Spyros; O'Sullivan, John; Fernando, Daniel; Stevens, Guy; Galván Magaña, Felipe; Seret, Bernard; Wintner, Sabine; Hoarau, Galice

2015-02-01

Manta and devil rays are an iconic group of globally distributed pelagic filter feeders, yet their evolutionary history remains enigmatic. We employed next generation sequencing of mitogenomes for nine of the 11 recognized species and two outgroups; as well as additional Sanger sequencing of two mitochondrial and two nuclear genes in an extended taxon sampling set. Analysis of the mitogenome coding regions in a Maximum Likelihood and Bayesian framework provided a well-resolved phylogeny. The deepest divergences distinguished three clades with high support, one containing Manta birostris, Manta alfredi, Mobula tarapacana, Mobula japanica and Mobula mobular; one containing Mobula kuhlii, Mobula eregoodootenkee and Mobula thurstoni; and one containing Mobula munkiana, Mobula hypostoma and Mobula rochebrunei. Mobula remains paraphyletic with the inclusion of Manta, a result that is in agreement with previous studies based on molecular and morphological data. A fossil-calibrated Bayesian random local clock analysis suggests that mobulids diverged from Rhinoptera around 30 Mya. Subsequent divergences are characterized by long internodes followed by short bursts of speciation extending from an initial episode of divergence in the Early and Middle Miocene (19-17 Mya) to a second episode during the Pliocene and Pleistocene (3.6 Mya - recent). Estimates of divergence dates overlap significantly with periods of global warming, during which upwelling intensity - and related high primary productivity in upwelling regions - decreased markedly. These periods are hypothesized to have led to fragmentation and isolation of feeding regions leading to possible regional extinctions, as well as the promotion of allopatric speciation. The closely shared evolutionary history of mobulids in combination with ongoing threats from fisheries and climate change effects on upwelling and food supply, reinforces the case for greater protection of this charismatic family of pelagic filter feeders. Copyright © 2014 Elsevier Inc. All rights reserved.

Molecular evidence of hybridization in sympatric populations of the Enantia jethys complex (Lepidoptera: Pieridae).

PubMed

Jasso-Martínez, Jovana M; Machkour-M'Rabet, Salima; Vila, Roger; Rodríguez-Arnaiz, Rosario; Castañeda-Sortibrán, América Nitxin

2018-01-01

Hybridization events are frequently demonstrated in natural butterfly populations. One interesting butterfly complex species is the Enantia jethys complex that has been studied for over a century; many debates exist regarding the species composition of this complex. Currently, three species that live sympatrically in the Gulf slope of Mexico (Enantia jethys, E. mazai, and E. albania) are recognized in this complex (based on morphological and molecular studies). Where these species live in sympatry, some cases of interspecific mating have been observed, suggesting hybridization events. Considering this, we employed a multilocus approach (analyses of mitochondrial and nuclear sequences: COI, RpS5, and Wg; and nuclear dominant markers: inter-simple sequence repeat (ISSRs) to study hybridization in sympatric populations from Veracruz, Mexico. Genetic diversity parameters were determined for all molecular markers, and species identification was assessed by different methods such as analyses of molecular variance (AMOVA), clustering, principal coordinate analysis (PCoA), gene flow, and PhiPT parameters. ISSR molecular markers were used for a more profound study of hybridization process. Although species of the Enantia jethys complex have a low dispersal capacity, we observed high genetic diversity, probably reflecting a high density of individuals locally. ISSR markers provided evidence of a contemporary hybridization process, detecting a high number of hybrids (from 17% to 53%) with significant differences in genetic diversity. Furthermore, a directional pattern of hybridization was observed from E. albania to other species. Phylogenetic study through DNA sequencing confirmed the existence of three clades corresponding to the three species previously recognized by morphological and molecular studies. This study underlines the importance of assessing hybridization in evolutionary studies, by tracing the lineage separation process that leads to the origin of new species. Our research demonstrates that hybridization processes have a high occurrence in natural populations.

Genomic diversity in switchgrass (Panicum virgatum): from the continental scale to a dune landscape

PubMed Central

Morris, Geoffrey P.; Grabowski, Paul; Borevitz, Justin O.

2011-01-01

Connecting broad-scale patterns of genetic variation and population structure to genetic diversity on a landscape is a key step towards understanding historical processes of migration and adaptation. New genomic approaches can be used to increase the resolution of phylogeographic studies while reducing locus sampling effects and circumventing ascertainment bias. Here, we use a novel approach based on high-throughput sequencing to characterize genetic diversity in complete chloroplast genomes and >10,000 nuclear loci in switchgrass, across a continental and landscape scale. Switchgrass is a North American tallgrass species, which is widely used in conservation and perennial biomass production, and shows strong ecotypic adaptation and population structure across the continental range. We sequenced 40.9 billion base pairs from 24 individuals from across the species’ range and 20 individuals from the Indiana Dunes. Analysis of plastome sequence revealed 203 variable SNP sites that define eight haplogroups, which are differentiated by 4 to 127 SNPs and confirmed by patterns of indel variation. These include three deeply divergent haplogroups, which correspond to the previously described lowland-upland ecotypic split and a novel upland haplogroup split that dates to the mid-Pleistoscene. Most of the plastome haplogroup diversity present in the northern switchgrass range, including in the Indiana Dunes, originated in the mid- or upper-Pleistocene prior to the most recent postglacial recolonization. Furthermore, a recently colonized landscape feature (~150 ya) in the Indiana Dunes contains several deeply divergent upland haplogroups. Nuclear markers also support a deep lowland-upland split, followed by limited gene flow, and show extensive gene flow in the local population of the Indiana Dunes. PMID:22060816

The PCBP1 gene encoding poly(rC) binding protein I is recurrently mutated in Burkitt lymphoma.

PubMed

Wagener, Rabea; Aukema, Sietse M; Schlesner, Matthias; Haake, Andrea; Burkhardt, Birgit; Claviez, Alexander; Drexler, Hans G; Hummel, Michael; Kreuz, Markus; Loeffler, Markus; Rosolowski, Maciej; López, Cristina; Möller, Peter; Richter, Julia; Rohde, Marius; Betts, Matthew J; Russell, Robert B; Bernhart, Stephan H; Hoffmann, Steve; Rosenstiel, Philip; Schilhabel, Markus; Szczepanowski, Monika; Trümper, Lorenz; Klapper, Wolfram; Siebert, Reiner

2015-09-01

The genetic hallmark of Burkitt lymphoma is the translocation t(8;14)(q24;q32), or one of its light chain variants, resulting in IG-MYC juxtaposition. However, these translocations alone are insufficient to drive lymphomagenesis, which requires additional genetic changes for malignant transformation. Recent studies of Burkitt lymphoma using next generation sequencing approaches have identified various recurrently mutated genes including ID3, TCF3, CCND3, and TP53. Here, by using similar approaches, we show that PCBP1 is a recurrently mutated gene in Burkitt lymphoma. By whole-genome sequencing, we identified somatic mutations in PCBP1 in 3/17 (18%) Burkitt lymphomas. We confirmed the recurrence of PCBP1 mutations by Sanger sequencing in an independent validation cohort, finding mutations in 3/28 (11%) Burkitt lymphomas and in 6/16 (38%) Burkitt lymphoma cell lines. PCBP1 is an intron-less gene encoding the 356 amino acid poly(rC) binding protein 1, which contains three K-Homology (KH) domains and two nuclear localization signals. The mutations predominantly (10/12, 83%) affect the KH III domain, either by complete domain loss or amino acid changes. Thus, these changes are predicted to alter the various functions of PCBP1, including nuclear trafficking and pre-mRNA splicing. Remarkably, all six primary Burkitt lymphomas with a PCBP1 mutation expressed MUM1/IRF4, which is otherwise detected in around 20-40% of Burkitt lymphomas. We conclude that PCBP1 mutations are recurrent in Burkitt lymphomas and might contribute, in cooperation with other mutations, to its pathogenesis. © 2015 Wiley Periodicals, Inc.

TCOF1 gene encodes a putative nucleolar phosphoprotein that exhibits mutations in Treacher Collins Syndrome throughout its coding region.

PubMed

Wise, C A; Chiang, L C; Paznekas, W A; Sharma, M; Musy, M M; Ashley, J A; Lovett, M; Jabs, E W

1997-04-01

Treacher Collins Syndrome (TCS) is the most common of the human mandibulofacial dysostosis disorders. Recently, a partial TCOF1 cDNA was identified and shown to contain mutations in TCS families. Here we present the entire exon/intron genomic structure and the complete coding sequence of TCOF1. TCOF1 encodes a low complexity protein of 1,411 amino acids, whose predicted protein structure reveals repeated motifs that mirror the organization of its exons. These motifs are shared with nucleolar trafficking proteins in other species and are predicted to be highly phosphorylated by casein kinase. Consistent with this, the full-length TCOF1 protein sequence also contains putative nuclear and nucleolar localization signals. Throughout the open reading frame, we detected an additional eight mutations in TCS families and several polymorphisms. We postulate that TCS results from defects in a nucleolar trafficking protein that is critically required during human craniofacial development.

TCOF1 gene encodes a putative nucleolar phosphoprotein that exhibits mutations in Treacher Collins Syndrome throughout its coding region

PubMed Central

Wise, Carol A.; Chiang, Lydia C.; Paznekas, William A.; Sharma, Mridula; Musy, Maurice M.; Ashley, Jennifer A.; Lovett, Michael; Jabs, Ethylin W.

1997-01-01

Treacher Collins Syndrome (TCS) is the most common of the human mandibulofacial dysostosis disorders. Recently, a partial TCOF1 cDNA was identified and shown to contain mutations in TCS families. Here we present the entire exon/intron genomic structure and the complete coding sequence of TCOF1. TCOF1 encodes a low complexity protein of 1,411 amino acids, whose predicted protein structure reveals repeated motifs that mirror the organization of its exons. These motifs are shared with nucleolar trafficking proteins in other species and are predicted to be highly phosphorylated by casein kinase. Consistent with this, the full-length TCOF1 protein sequence also contains putative nuclear and nucleolar localization signals. Throughout the open reading frame, we detected an additional eight mutations in TCS families and several polymorphisms. We postulate that TCS results from defects in a nucleolar trafficking protein that is critically required during human craniofacial development. PMID:9096354

Biodiversity of the Betta smaragdina (Teleostei: Perciformes) in the northeast region of Thailand as determined by mitochondrial COI and nuclear ITS1 gene sequences☆

PubMed Central

Kowasupat, Chanon; Panijpan, Bhinyo; Laosinchai, Parames; Ruenwongsa, Pintip; Phongdara, Amornrat; Wanna, Warapond; Senapin, Saengchan; Phiwsaiya, Kornsunee

2014-01-01

In Thailand, there are currently five recognized species members of the bubble-nesting Betta genus, namely Betta splendens, B. smaragdina, B. imbellis, B. mahachaiensis and B. siamorientalis. In 2010, we indicated the possibility, based on COI barcoding evidence, that there might be two additional species, albeit cryptic, related to the type-locality B. smaragdina in some provinces in the northeast of Thailand. In the present study, after a more extensive survey of the northeast, and phylogenetic analyses based on COI and ITS1 sequences, the B. smaragdina group may be composed of at least 3 cryptic species members. The phylogenetic positions of these B. smaragdina group members in the bubble-nesting bettas' tree together with those of their congeners have been consolidated by better DNA sequence quality and phylogenetic analyses. With a better supported tree, the species statuses of B. siamorientalis and the Cambodian B. smaragdina-like fish, B. stiktos, are also confirmed. PMID:25606392

Stable CoT-1 repeat RNA is abundant and associated with euchromatic interphase chromosomes

PubMed Central

Hall, Lisa L.; Carone, Dawn M.; Gomez, Alvin; Kolpa, Heather J.; Byron, Meg; Mehta, Nitish; Fackelmayer, Frank O.; Lawrence, Jeanne B.

2014-01-01

SUMMARY Recent studies recognize a vast diversity of non-coding RNAs with largely unknown functions, but few have examined interspersed repeat sequences, which constitute almost half our genome. RNA hybridization in situ using CoT-1 (highly repeated) DNA probes detects surprisingly abundant euchromatin-associated RNA comprised predominantly of repeat sequences (“CoT-1 RNA”), including LINE-1. CoT-1-hybridizing RNA strictly localizes to the interphase chromosome territory in cis, and remains stably associated with the chromosome territory following prolonged transcriptional inhibition. The CoT-1 RNA territory resists mechanical disruption and fractionates with the non-chromatin scaffold, but can be experimentally released. Loss of repeat-rich, stable nuclear RNAs from euchromatin corresponds to aberrant chromatin distribution and condensation. CoT-1 RNA has several properties similar to XIST chromosomal RNA, but is excluded from chromatin condensed by XIST. These findings impact two “black boxes” of genome science: the poorly understood diversity of non-coding RNA and the unexplained abundance of repetitive elements. PMID:24581492

Verification of voltage/frequency requirement for emergency diesel generator in nuclear power plant using dynamic modeling

NASA Astrophysics Data System (ADS)

Hur, Jin-Suk; Roh, Myung-Sub

2014-02-01

One major cause of the plant shutdown is the loss of electrical power. The study is to comprehend the coping action against station blackout including emergency diesel generator, sequential loading of safety system and to ensure that the emergency diesel generator should meet requirements, especially voltage and frequency criteria using modeling tool. This paper also considered the change of the sequencing time and load capacity only for finding electrical design margin. However, the revision of load list must be verified with safety analysis. From this study, it is discovered that new load calculation is a key factor in EDG localization and in-house capability increase.

Genetic diversity and demographic instability in Riftia pachyptila tubeworms from eastern Pacific hydrothermal vents

PubMed Central

2011-01-01

Background Deep-sea hydrothermal vent animals occupy patchy and ephemeral habitats supported by chemosynthetic primary production. Volcanic and tectonic activities controlling the turnover of these habitats contribute to demographic instability that erodes genetic variation within and among colonies of these animals. We examined DNA sequences from one mitochondrial and three nuclear gene loci to assess genetic diversity in the siboglinid tubeworm, Riftia pachyptila, a widely distributed constituent of vents along the East Pacific Rise and Galápagos Rift. Results Genetic differentiation (FST) among populations increased with geographical distances, as expected under a linear stepping-stone model of dispersal. Low levels of DNA sequence diversity occurred at all four loci, allowing us to exclude the hypothesis that an idiosyncratic selective sweep eliminated mitochondrial diversity alone. Total gene diversity declined with tectonic spreading rates. The southernmost populations, which are subjected to superfast spreading rates and high probabilities of extinction, are relatively homogenous genetically. Conclusions Compared to other vent species, DNA sequence diversity is extremely low in R. pachyptila. Though its dispersal abilities appear to be effective, the low diversity, particularly in southern hemisphere populations, is consistent with frequent local extinction and (re)colonization events. PMID:21489281

Genetic diversity and demographic instability in Riftia pachyptila tubeworms from eastern Pacific hydrothermal vents

USGS Publications Warehouse

Coykendall, D.K.; Johnson, S.B.; Karl, S.A.; Lutz, R.A.; Vrijenhoek, R.C.

2011-01-01

Background: Deep-sea hydrothermal vent animals occupy patchy and ephemeral habitats supported by chemosynthetic primary production. Volcanic and tectonic activities controlling the turnover of these habitats contribute to demographic instability that erodes genetic variation within and among colonies of these animals. We examined DNA sequences from one mitochondrial and three nuclear gene loci to assess genetic diversity in the siboglinid tubeworm, Riftia pachyptila, a widely distributed constituent of vents along the East Pacific Rise and Galpagos Rift. Results: Genetic differentiation (FST) among populations increased with geographical distances, as expected under a linear stepping-stone model of dispersal. Low levels of DNA sequence diversity occurred at all four loci, allowing us to exclude the hypothesis that an idiosyncratic selective sweep eliminated mitochondrial diversity alone. Total gene diversity declined with tectonic spreading rates. The southernmost populations, which are subjected to superfast spreading rates and high probabilities of extinction, are relatively homogenous genetically. Conclusions: Compared to other vent species, DNA sequence diversity is extremely low in R. pachyptila. Though its dispersal abilities appear to be effective, the low diversity, particularly in southern hemisphere populations, is consistent with frequent local extinction and (re)colonization events. ?? 2011 Coykendall et al; licensee BioMed Central Ltd.

A hydrodynamic study of a slow nova outburst. [computerized simulation of thermonuclear runaway in white dwarf envelope

NASA Technical Reports Server (NTRS)

Sparks, W. M.; Starrfield, S.; Truran, J. W.

1978-01-01

The paper reports use of a Lagrangian implicit hydrodynamics computer code incorporating a full nuclear-reaction network to follow a thermonuclear runaway in the hydrogen-rich envelope of a 1.25 solar-mass white dwarf. In this evolutionary sequence the envelope was assumed to be of normal (solar) composition and the resulting outburst closely resembles that of the slow nova HR Del. In contrast, previous CNO-enhanced models resemble fast nova outbursts. The slow-nova model ejects material by radiation pressure when the high luminosity of the rekindled hydrogen shell source exceeds the local Eddington luminosity of the outer layers. This is in contrast to the fast nova outburst where ejection is caused by the decay of the beta(+)-unstable nuclei. Nevertheless, radiation pressure probably plays a major role in ejecting material from the fast nova remnants. Therefore, the sequence from slow to fast novae can be interpreted as a sequence of white dwarfs with increasing amounts of enhanced CNO nuclei in their hydrogen envelopes, although other parameters such as the white-dwarf mass and accretion rate probably contribute to the observed variation between novae.

Electrophoretic mobility shift assay reveals a novel recognition sequence for Setaria italica NAC protein.

PubMed

Puranik, Swati; Kumar, Karunesh; Srivastava, Prem S; Prasad, Manoj

2011-10-01

The NAC (NAM/ATAF1,2/CUC2) proteins are among the largest family of plant transcription factors. Its members have been associated with diverse plant processes and intricately regulate the expression of several genes. Inspite of this immense progress, knowledge of their DNA-binding properties are still limited. In our recent publication,1 we reported isolation of a membrane-associated NAC domain protein from Setaria italica (SiNAC). Transactivation analysis revealed that it was a functionally active transcription factor as it could stimulate expression of reporter genes in vivo. Truncations of the transmembrane region of the protein lead to its nuclear localization. Here we describe expression and purification of SiNAC DNA-binding domain. We further report identification of a novel DNA-binding site, [C/G][A/T][T/A][G/C]TC[C/G][A/T][C/G][G/C] for SiNAC by electrophoretic mobility shift assay. The SiNAC-GST protein could bind to the NAC recognition sequence in vitro as well as to sequences where some bases had been reshuffled. The results presented here contribute to our understanding of the DNA-binding specificity of SiNAC protein.

Electrophoretic mobility shift assay reveals a novel recognition sequence for Setaria italica NAC protein

PubMed Central

Puranik, Swati; Kumar, Karunesh; Srivastava, Prem S

2011-01-01

The NAC (NAM/ATAF1,2/CUC2) proteins are among the largest family of plant transcription factors. Its members have been associated with diverse plant processes and intricately regulate the expression of several genes. Inspite of this immense progress, knowledge of their DNA-binding properties are still limited. In our recent publication,1 we reported isolation of a membrane-associated NAC domain protein from Setaria italica (SiNAC). Transactivation analysis revealed that it was a functionally active transcription factor as it could stimulate expression of reporter genes in vivo. Truncation of the transmembrane region of the protein lead to its nuclear localization. Here we describe expression and purification of SiNAC DNA-binding domain. We further report identification of a novel DNA-binding site, [C/G][A/T] [T/A][G/C]TC[C/G][A/T][C/G][G/C] for SiNAC by electrophoretic mobility shift assay. The SiNAC-GST protein could bind to the NAC recognition sequence in vitro as well as to sequences where some bases had been reshuffled. The results presented here contribute to our understanding of the DNA-binding specificity of SiNAC protein. PMID:21918373

Karyopherin-mediated nuclear import of the homing endonuclease VMA1-derived endonuclease is required for self-propagation of the coding region.

PubMed

Nagai, Yuri; Nogami, Satoru; Kumagai-Sano, Fumi; Ohya, Yoshikazu

2003-03-01

VMA1-derived endonuclease (VDE), a site-specific endonuclease in Saccharomyces cerevisiae, enters the nucleus to generate a double-strand break in the VDE-negative allelic locus, mediating the self-propagating gene conversion called homing. Although VDE is excluded from the nucleus in mitotic cells, it relocalizes at premeiosis, becoming localized in both the nucleus and the cytoplasm in meiosis. The nuclear localization of VDE is induced by inactivation of TOR kinases, which constitute central regulators of cell differentiation in S. cerevisiae, and by nutrient depletion. A functional genomic approach revealed that at least two karyopherins, Srp1p and Kap142p, are required for the nuclear localization pattern. Genetic and physical interactions between Srp1p and VDE imply direct involvement of karyopherin-mediated nuclear transport in this process. Inactivation of TOR signaling or acquisition of an extra nuclear localization signal in the VDE coding region leads to artificial nuclear localization of VDE and thereby induces homing even during mitosis. These results serve as evidence that VDE utilizes the host systems of nutrient signal transduction and nucleocytoplasmic transport to ensure the propagation of its coding region.

Karyopherin-Mediated Nuclear Import of the Homing Endonuclease VMA1-Derived Endonuclease Is Required for Self-Propagation of the Coding Region

PubMed Central

Nagai, Yuri; Nogami, Satoru; Kumagai-Sano, Fumi; Ohya, Yoshikazu

2003-01-01

VMA1-derived endonuclease (VDE), a site-specific endonuclease in Saccharomyces cerevisiae, enters the nucleus to generate a double-strand break in the VDE-negative allelic locus, mediating the self-propagating gene conversion called homing. Although VDE is excluded from the nucleus in mitotic cells, it relocalizes at premeiosis, becoming localized in both the nucleus and the cytoplasm in meiosis. The nuclear localization of VDE is induced by inactivation of TOR kinases, which constitute central regulators of cell differentiation in S. cerevisiae, and by nutrient depletion. A functional genomic approach revealed that at least two karyopherins, Srp1p and Kap142p, are required for the nuclear localization pattern. Genetic and physical interactions between Srp1p and VDE imply direct involvement of karyopherin-mediated nuclear transport in this process. Inactivation of TOR signaling or acquisition of an extra nuclear localization signal in the VDE coding region leads to artificial nuclear localization of VDE and thereby induces homing even during mitosis. These results serve as evidence that VDE utilizes the host systems of nutrient signal transduction and nucleocytoplasmic transport to ensure the propagation of its coding region. PMID:12588991

Identification of multiple nuclear localization signals in murine Elf3, an ETS transcription factor.

PubMed

Do, Hyun-Jin; Song, Hyuk; Yang, Heung-Mo; Kim, Dong-Ku; Kim, Nam-Hyung; Kim, Jin-Hoi; Cha, Kwang-Yul; Chung, Hyung-Min; Kim, Jae-Hwan

2006-03-20

We investigated nuclear localization signal (NLS) determinants within the AT-hook and ETS DNA-binding domains of murine Elf3 (mElf3), a member of the subfamily of epithelium-specific ETS transcription factors. Deletion mutants containing the AT-hook, ETS domain or both localized strictly in the nucleus, suggesting that these individual domains contain independent NLS motif(s). Within the AT-hook domain, four basic residues (244KRKR247) were critical for strong NLS activity, and two potent bipartite NLS motifs (236-252 and 249-267) were sufficient for nuclear import of mElf3, although less efficient than the full domain. In addition, one stretch of basic residues (318KKK320) within the ETS domain appears to be essential for mElf3 nuclear localization. Taken together, mElf3 contains multiple NLS motifs, which may function cooperatively to effect efficient nuclear transport.

Mapping the nuclear localization signal in the matrix protein of potato yellow dwarf virus.

PubMed

Anderson, Gavin; Jang, Chanyong; Wang, Renyuan; Goodin, Michael

2018-05-01

The ability of the matrix (M) protein of potato yellow dwarf virus (PYDV) to remodel nuclear membranes is controlled by a di-leucine motif located at residues 223 and 224 of its primary structure. This function can be uncoupled from that of its nuclear localization signal (NLS), which is controlled primarily by lysine and arginine residues immediately downstream of the LL motif. In planta localization of green fluorescent protein fusions, bimolecular fluorescence complementation assays with nuclear import receptor importin-α1 and yeast-based nuclear import assays provided three independent experimental approaches to validate the authenticity of the M-NLS. The carboxy terminus of M is predicted to contain a nuclear export signal, which is belived to be functional, given the ability of M to bind the Arabidopsis nuclear export receptor 1 (XPO1). The nuclear shuttle activity of M has implications for the cell-to-cell movement of PYDV nucleocapsids, based upon its interaction with the N and Y proteins.

Interim reliability-evaluation program: analysis of the Browns Ferry, Unit 1, nuclear plant. Appendix C - sequence quantification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mays, S.E.; Poloski, J.P.; Sullivan, W.H.

1982-07-01

This report describes a risk study of the Browns Ferry, Unit 1, nuclear plant. The study is one of four such studies sponsored by the NRC Office of Research, Division of Risk Assessment, as part of its Interim Reliability Evaluation Program (IREP), Phase II. This report is contained in four volumes: a main report and three appendixes. Appendix C generally describes the methods used to estimate accident sequence frequency values. Information is presented concerning the approach, example collection, failure data, candidate dominant sequences, uncertainty analysis, and sensitivity analysis.

Fission yeast Lem2 and Man1 perform fundamental functions of the animal cell nuclear lamina.

PubMed

Gonzalez, Yanira; Saito, Akira; Sazer, Shelley

2012-01-01

In animal cells the nuclear lamina, which consists of lamins and lamin-associated proteins, serves several functions: it provides a structural scaffold for the nuclear envelope and tethers proteins and heterochromatin to the nuclear periphery. In yeast, proteins and large heterochromatic domains including telomeres are also peripherally localized, but there is no evidence that yeast have lamins or a fibrous nuclear envelope scaffold. Nonetheless, we found that the Lem2 and Man1 proteins of the fission yeast Schizosaccharomyces pombe, evolutionarily distant relatives of the Lap2/Emerin/Man1 (LEM) sub-family of animal cell lamin-associated proteins, perform fundamental functions of the animal cell lamina. These integral inner nuclear membrane localized proteins, with nuclear localized DNA binding Helix-Extension-Helix (HEH) domains, impact nuclear envelope structure and integrity, are essential for the enrichment of telomeres at the nuclear periphery and by means of their HEH domains anchor chromatin, most likely transcriptionally repressed heterochromatin, to the nuclear periphery. These data indicate that the core functions of the nuclear lamina are conserved between fungi and animal cells and can be performed in fission yeast, without lamins or other intermediate filament proteins.

Interim Reliability Evaluation Program: analysis of the Browns Ferry, Unit 1, nuclear plant. Main report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mays, S.E.; Poloski, J.P.; Sullivan, W.H.

1982-07-01

A probabilistic risk assessment (PRA) was made of the Browns Ferry, Unit 1, nuclear plant as part of the Nuclear Regulatory Commission's Interim Reliability Evaluation Program (IREP). Specific goals of the study were to identify the dominant contributors to core melt, develop a foundation for more extensive use of PRA methods, expand the cadre of experienced PRA practitioners, and apply procedures for extension of IREP analyses to other domestic light water reactors. Event tree and fault tree analyses were used to estimate the frequency of accident sequences initiated by transients and loss of coolant accidents. External events such as floods,more » fires, earthquakes, and sabotage were beyond the scope of this study and were, therefore, excluded. From these sequences, the dominant contributors to probable core melt frequency were chosen. Uncertainty and sensitivity analyses were performed on these sequences to better understand the limitations associated with the estimated sequence frequencies. Dominant sequences were grouped according to common containment failure modes and corresponding release categories on the basis of comparison with analyses of similar designs rather than on the basis of detailed plant-specific calculations.« less

Diatom centromeres suggest a mechanism for nuclear DNA acquisition

DOE PAGES

Diner, Rachel E.; Noddings, Chari M.; Lian, Nathan C.; ...

2017-07-18

Centromeres are essential for cell division and growth in all eukaryotes, and knowledge of their sequence and structure guides the development of artificial chromosomes for functional cellular biology studies. Centromeric proteins are conserved among eukaryotes; however, centromeric DNA sequences are highly variable. We combined forward and reverse genetic approaches with chromatin immunoprecipitation to identify centromeres of the model diatom Phaeodactylum tricornutum. We observed 25 unique centromere sequences typically occurring once per chromosome, a finding that helps to resolve nuclear genome organization and indicates monocentric regional centromeres. Diatom centromere sequences contain low-GC content regions but lack repeats or other conserved sequencemore » features. Native and foreign sequences with similar GC content to P. tricornutum centromeres can maintain episomes and recruit the diatom centromeric histone protein CENH3, suggesting nonnative sequences can also function as diatom centromeres. Thus, simple sequence requirements may enable DNA from foreign sources to persist in the nucleus as extrachromosomal episomes, revealing a potential mechanism for organellar and foreign DNA acquisition.« less

Diatom centromeres suggest a mechanism for nuclear DNA acquisition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Diner, Rachel E.; Noddings, Chari M.; Lian, Nathan C.

Centromeres are essential for cell division and growth in all eukaryotes, and knowledge of their sequence and structure guides the development of artificial chromosomes for functional cellular biology studies. Centromeric proteins are conserved among eukaryotes; however, centromeric DNA sequences are highly variable. We combined forward and reverse genetic approaches with chromatin immunoprecipitation to identify centromeres of the model diatom Phaeodactylum tricornutum. We observed 25 unique centromere sequences typically occurring once per chromosome, a finding that helps to resolve nuclear genome organization and indicates monocentric regional centromeres. Diatom centromere sequences contain low-GC content regions but lack repeats or other conserved sequencemore » features. Native and foreign sequences with similar GC content to P. tricornutum centromeres can maintain episomes and recruit the diatom centromeric histone protein CENH3, suggesting nonnative sequences can also function as diatom centromeres. Thus, simple sequence requirements may enable DNA from foreign sources to persist in the nucleus as extrachromosomal episomes, revealing a potential mechanism for organellar and foreign DNA acquisition.« less

Capturing novel mouse genes encoding chromosomal and other nuclear proteins.

PubMed

Tate, P; Lee, M; Tweedie, S; Skarnes, W C; Bickmore, W A

1998-09-01

The burgeoning wealth of gene sequences contrasts with our ignorance of gene function. One route to assigning function is by determining the sub-cellular location of proteins. We describe the identification of mouse genes encoding proteins that are confined to nuclear compartments by splicing endogeneous gene sequences to a promoterless betageo reporter, using a gene trap approach. Mouse ES (embryonic stem) cell lines were identified that express betageo fusions located within sub-nuclear compartments, including chromosomes, the nucleolus and foci containing splicing factors. The sequences of 11 trapped genes were ascertained, and characterisation of endogenous protein distribution in two cases confirmed the validity of the approach. Three novel proteins concentrated within distinct chromosomal domains were identified, one of which appears to be a serine/threonine kinase. The sequence of a gene whose product co-localises with splicesome components suggests that this protein may be an E3 ubiquitin-protein ligase. The majority of the other genes isolated represent novel genes. This approach is shown to be a powerful tool for identifying genes encoding novel proteins with specific sub-nuclear localisations and exposes our ignorance of the protein composition of the nucleus. Motifs in two of the isolated genes suggest new links between cellular regulatory mechanisms (ubiquitination and phosphorylation) and mRNA splicing and chromosome structure/function.

Population genomics and geographical parthenogenesis in Japanese harvestmen (Opiliones, Sclerosomatidae, Leiobunum).

PubMed

Burns, Mercedes; Hedin, Marshal; Tsurusaki, Nobuo

2018-01-01

Naturally occurring population variation in reproductive mode presents an opportunity for researchers to test hypotheses regarding the evolution of sex. Asexual reproduction frequently assumes a geographical pattern, in which parthenogenesis-dominated populations are more broadly dispersed than their sexual conspecifics. We evaluate the geographical distribution of genomic signatures associated with parthenogenesis using nuclear and mitochondrial DNA sequence data from two Japanese harvestman sister taxa, Leiobunum manubriatum and Leiobunum globosum . Asexual reproduction is putatively facultative in these species, and female-biased localities are common in habitat margins. Past karyotypic and current cytometric work indicates L. globosum is entirely tetraploid, while L. manubriatum may be either diploid or tetraploid. We estimated species phylogeny, genetic differentiation, diversity, and mitonuclear discordance in females collected across the species range in order to identify range expansion toward marginal habitat, potential for hybrid origin, and persistence of asexual lineages. Our results point to northward expansion of a tetraploid ancestor of L. manubriatum and L. globosum , coupled with support for greater male gene flow in southern L. manubriatum localities. Specimens from localities in the Tohoku and Hokkaido regions were indistinct, particularly those of L. globosum , potentially due to little mitochondrial differentiation or haplotypic variation. Although L. manubriatum overlaps with L. globosum across its entire range, L. globosum was reconstructed as monophyletic with strong support using mtDNA, and marginal support with nuclear loci. Ultimately, we find evidence for continued sexual reproduction in both species and describe opportunities to clarify the rate and mechanism of parthenogenesis.

Structural Studies of the Tandem Tudor Domains of Fragile X Mental Retardation Related Proteins FXR1 and FXR2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adams-Cioaba, Melanie A.; Guo, Yahong; Bian, ChuanBing

Expansion of the CGG trinucleotide repeat in the 5'-untranslated region of the FMR1, fragile X mental retardation 1, gene results in suppression of protein expression for this gene and is the underlying cause of Fragile X syndrome. In unaffected individuals, the FMRP protein, together with two additional paralogues (Fragile X Mental Retardation Syndrome-related Protein 1 and 2), associates with mRNA to form a ribonucleoprotein complex in the nucleus that is transported to dendrites and spines of neuronal cells. It is thought that the fragile X family of proteins contributes to the regulation of protein synthesis at sites where mRNAs aremore » locally translated in response to stimuli. Here, we report the X-ray crystal structures of the non-canonical nuclear localization signals of the FXR1 and FXR2 autosomal paralogues of FMRP, which were determined at 2.50 and 1.92 {angstrom}, respectively. The nuclear localization signals of the FXR1 and FXR2 comprise tandem Tudor domain architectures, closely resembling that of UHRF1, which is proposed to bind methylated histone H3K9. The FMRP, FXR1 and FXR2 proteins comprise a small family of highly conserved proteins that appear to be important in translational regulation, particularly in neuronal cells. The crystal structures of the N-terminal tandem Tudor domains of FXR1 and FXR2 revealed a conserved architecture with that of FMRP. Biochemical analysis of the tandem Tudor doamins reveals their ability to preferentially recognize trimethylated peptides in a sequence-specific manner.« less

Genome skimming identifies polymorphism in tern populations and species

PubMed Central

2012-01-01

Background Terns (Charadriiformes: Sterninae) are a lineage of cosmopolitan shorebirds with a disputed evolutionary history that comprises several species of conservation concern. As a non-model system in genetics, previous study has left most of the nuclear genome unexplored, and population-level studies are limited to only 15% of the world's species of terns and noddies. Screening of polymorphic nuclear sequence markers is needed to enhance genetic resolution because of supposed low mitochondrial mutation rate, documentation of nuclear insertion of hypervariable mitochondrial regions, and limited success of microsatellite enrichment in terns. Here, we investigated the phylogenetic and population genetic utility for terns and relatives of a variety of nuclear markers previously developed for other birds and spanning the nuclear genome. Markers displaying a variety of mutation rates from both the nuclear and mitochondrial genome were tested and prioritized according to optimal cross-species amplification and extent of genetic polymorphism between (1) the main tern clades and (2) individual Royal Terns (Thalasseus maxima) breeding on the US East Coast. Results Results from this genome skimming effort yielded four new nuclear sequence-based markers for tern phylogenetics and 11 intra-specific polymorphic markers. Further, comparison between the two genomes indicated a phylogenetic conflict at the base of terns, involving the inclusion (mitochondrial) or exclusion (nuclear) of the Angel Tern (Gygis alba). Although limited mitochondrial variation was confirmed, both nuclear markers and a short tandem repeat in the mitochondrial control region indicated the presence of considerable genetic variation in Royal Terns at a regional scale. Conclusions These data document the value of intronic markers to the study of terns and allies. We expect that these and additional markers attained through next-generation sequencing methods will accurately map the genetic origin and species history of this group of birds. PMID:22333071

Hedysarum L. (Fabaceae: Hedysareae) Is Not Monophyletic – Evidence from Phylogenetic Analyses Based on Five Nuclear and Five Plastid Sequences

PubMed Central

Liu, Pei-Liang; Wen, Jun; Duan, Lei; Arslan, Emine; Ertuğrul, Kuddisi; Chang, Zhao-Yang

2017-01-01

The legume family (Fabaceae) exhibits a high level of species diversity and evolutionary success worldwide. Previous phylogenetic studies of the genus Hedysarum L. (Fabaceae: Hedysareae) showed that the nuclear and the plastid topologies might be incongruent, and the systematic position of the Hedysarum sect. Stracheya clade was uncertain. In this study, phylogenetic relationships of Hedysarum were investigated based on the nuclear ITS, ETS, PGDH, SQD1, TRPT and the plastid psbA-trnH, trnC-petN, trnL-trnF, trnS-trnG, petN-psbM sequences. Both nuclear and plastid data support two major lineages in Hedysarum: the Hedysarum s.s. clade and the Sartoria clade. In the nuclear tree, Hedysarum is biphyletic with the Hedysarum s.s. clade sister to the Corethrodendron + Eversmannia + Greuteria + Onobrychis clade (the CEGO clade), whereas the Sartoria clade is sister to the genus Taverniera DC. In the plastid tree, Hedysarum is monophyletic and sister to Taverniera. The incongruent position of the Hedysarum s.s. clade between the nuclear and plastid trees may be best explained by a chloroplast capture hypothesis via introgression. The Hedysarum sect. Stracheya clade is resolved as sister to the H. sect. Hedysarum clade in both nuclear and plastid trees, and our analyses support merging Stracheya into Hedysarum. Based on our new evidence from multiple sequences, Hedysarum is not monophyletic, and its generic delimitation needs to be reconsidered. PMID:28122062

Hedysarum L. (Fabaceae: Hedysareae) Is Not Monophyletic - Evidence from Phylogenetic Analyses Based on Five Nuclear and Five Plastid Sequences.

PubMed

Liu, Pei-Liang; Wen, Jun; Duan, Lei; Arslan, Emine; Ertuğrul, Kuddisi; Chang, Zhao-Yang

2017-01-01

The legume family (Fabaceae) exhibits a high level of species diversity and evolutionary success worldwide. Previous phylogenetic studies of the genus Hedysarum L. (Fabaceae: Hedysareae) showed that the nuclear and the plastid topologies might be incongruent, and the systematic position of the Hedysarum sect. Stracheya clade was uncertain. In this study, phylogenetic relationships of Hedysarum were investigated based on the nuclear ITS, ETS, PGDH, SQD1, TRPT and the plastid psbA-trnH, trnC-petN, trnL-trnF, trnS-trnG, petN-psbM sequences. Both nuclear and plastid data support two major lineages in Hedysarum: the Hedysarum s.s. clade and the Sartoria clade. In the nuclear tree, Hedysarum is biphyletic with the Hedysarum s.s. clade sister to the Corethrodendron + Eversmannia + Greuteria + Onobrychis clade (the CEGO clade), whereas the Sartoria clade is sister to the genus Taverniera DC. In the plastid tree, Hedysarum is monophyletic and sister to Taverniera. The incongruent position of the Hedysarum s.s. clade between the nuclear and plastid trees may be best explained by a chloroplast capture hypothesis via introgression. The Hedysarum sect. Stracheya clade is resolved as sister to the H. sect. Hedysarum clade in both nuclear and plastid trees, and our analyses support merging Stracheya into Hedysarum. Based on our new evidence from multiple sequences, Hedysarum is not monophyletic, and its generic delimitation needs to be reconsidered.

Basic amino acid residues located in the N-terminal region of BEND3 are essential for its nuclear localization.

PubMed

Shiheido, Hirokazu; Shimizu, Jun

2015-02-20

BEN domain-containing protein 3 (BEND3) has recently been reported to function as a heterochromatin-associated protein in transcriptional repression in the nucleus. BEND3 should have nuclear localization signals (NLSs) to localize to the nucleus in light of its molecular weight, which is higher than that allowed to pass through nuclear pore complexes. We here analyzed the subcellular localization of deletion/site-directed mutants of human BEND3 by an immunofluorescence assay in an attempt to identify the amino acids essential for its nuclear localization. We found that three basic amino acid residues located in the N-terminal region of BEND3 (BEND356-58, KRK) are essential, suggesting that these residues play a role as a functional NLS. These results provide valuable information for progressing research on BEND3. Copyright © 2015 Elsevier Inc. All rights reserved.

Subcellular localization of celery mannitol dehydrogenase. A cytosolic metabolic enzyme in nuclei.

PubMed Central

Yamamoto, Y T; Zamski, E; Williamson, J D; Conkling, M A; Pharr, D M

1997-01-01

Mannitol dehydrogenase (MTD) is the first enzyme in mannitol catabolism in celery (Apium graveolens L. var dulce [Mill] Pers. cv Florida 638). Mannitol is an important photoassimilate, as well as providing plants with resistance to salt and osmotic stress. Previous work has shown that expression of the celery Mtd gene is regulated by many factors, such as hexose sugars, salt and osmotic stress, and salicylic acid. Furthermore, MTD is present in cells of sink organs, phloem cells, and mannitol-grown suspension cultures. Immunogold localization and biochemical analyses presented here demonstrate that celery MTD is localized in the cytosol and nuclei. Although the cellular density of MTD varies among different cell types, densities of nuclear and cytosolic MTD in a given cell are approximately equal. Biochemical analyses of nuclear extracts from mannitol-grown cultured cells confirmed that the nuclear-localized MTD is enzymatically active. The function(s) of nuclear-localized MTD is unknown. PMID:9414553

Spatiotemporal dynamics of OCT4 protein localization during preimplantation development in mice.

PubMed

Fukuda, Atsushi; Mitani, Atsushi; Miyashita, Toshiyuki; Kobayashi, Hisato; Umezawa, Akihiro; Akutsu, Hidenori

2016-11-01

Spatiotemporal expression of transcription factors is crucial for genomic reprogramming. Pou5f1 (Oct4) is an essential transcription factor for reprogramming. A recent study reported that OCT4A, which is crucial for establishment and maintenance of pluripotent cells, is expressed in oocytes, but maternal OCT4A is dispensable for totipotency induction. Whereas another study reported that OCT4B, which is not related to pluripotency, is predominantly expressed instead of OCT4A during early preimplantation phases in mice. To determine the expression states of OCT4 in murine preimplantation embryos, we conducted in-depth expression and functional analyses. We found that pluripotency-related OCT4 mainly localizes to the cytoplasm in early preimplantation phases, with no major nuclear localization until the 8-16-cell stage despite high expression in both oocytes and early embryos. RNA-sequencing analysis using oocytes and early preimplantation embryos could not identify the splice variants creating alternative forms of OCT4 protein. Forced expression of OCT4 in zygotes by the injection of polyadenylated mRNA clearly showed nuclear localization of OCT4 protein around 3-5-fold greater than physiological levels and impaired developmental competency in a dose-dependent manner. Embryos with modest overexpression of OCT4 could develop to the 16-cell stage; however, more than 50% of the embryos were arrested at this stage, similar to the results for OCT4 depletion. In contrast, extensive overexpression of OCT4 resulted in complete arrest at the 2-cell stage accompanied by downregulation of zygotically activated genes and repetitive elements related to the totipotent state. These results demonstrated that OCT4 protein localization was spatiotemporally altered during preimplantation development, and strict control of Oct4 protein levels was essential for proper totipotential reprogramming. © 2016 Society for Reproduction and Fertility.

Local alignment of two-base encoded DNA sequence

PubMed Central

Homer, Nils; Merriman, Barry; Nelson, Stanley F

2009-01-01

Background DNA sequence comparison is based on optimal local alignment of two sequences using a similarity score. However, some new DNA sequencing technologies do not directly measure the base sequence, but rather an encoded form, such as the two-base encoding considered here. In order to compare such data to a reference sequence, the data must be decoded into sequence. The decoding is deterministic, but the possibility of measurement errors requires searching among all possible error modes and resulting alignments to achieve an optimal balance of fewer errors versus greater sequence similarity. Results We present an extension of the standard dynamic programming method for local alignment, which simultaneously decodes the data and performs the alignment, maximizing a similarity score based on a weighted combination of errors and edits, and allowing an affine gap penalty. We also present simulations that demonstrate the performance characteristics of our two base encoded alignment method and contrast those with standard DNA sequence alignment under the same conditions. Conclusion The new local alignment algorithm for two-base encoded data has substantial power to properly detect and correct measurement errors while identifying underlying sequence variants, and facilitating genome re-sequencing efforts based on this form of sequence data. PMID:19508732

The Heart of Matter: A Nuclear Chemistry Module. Teacher's Guide.

ERIC Educational Resources Information Center

Viola, Vic; Hearle, Robert

This teacher's guide is designed to provide science teachers with the necessary guidance and suggestions for teaching nuclear chemistry. In this book, the fundamental concepts of nuclear science and the applications of nuclear energy are discussed. The material in this book can be integrated with the other modules in a sequence that helps students…

Nup124p Is a Nuclear Pore Factor of Schizosaccharomyces pombe That Is Important for Nuclear Import and Activity of Retrotransposon Tf1

PubMed Central

Balasundaram, David; Benedik, Michael J.; Morphew, Mary; Dang, Van-Dinh; Levin, Henry L.

1999-01-01

The long terminal repeat (LTR)-containing retrotransposon Tf1 propagates within the fission yeast Schizosaccharomyces pombe as the result of several mechanisms that are typical of both retrotransposons and retroviruses. To identify host factors that contribute to the transposition process, we mutagenized cultures of S. pombe and screened them for strains that were unable to support Tf1 transposition. One such strain contained a mutation in a gene we named nup124. The product of this gene contains 11 FXFG repeats and is a component of the nuclear pore complex. In addition to the reduced levels of Tf1 transposition, the nup124-1 allele caused a significant reduction in the nuclear localization of Tf1 Gag. Surprisingly, the mutation in nup124-1 did not cause any reduction in the growth rate, the nuclear localization of specific nuclear localization signal-containing proteins, or the cytoplasmic localization of poly(A) mRNA. A two-hybrid analysis and an in vitro precipitation assay both identified an interaction between Tf1 Gag and the N terminus of Nup124p. These results provide evidence for an unusual mechanism of nuclear import that relies on a direct interaction between a nuclear pore factor and Tf1 Gag. PMID:10409764

Nup124p is a nuclear pore factor of Schizosaccharomyces pombe that is important for nuclear import and activity of retrotransposon Tf1.

PubMed

Balasundaram, D; Benedik, M J; Morphew, M; Dang, V D; Levin, H L

1999-08-01

The long terminal repeat (LTR)-containing retrotransposon Tf1 propagates within the fission yeast Schizosaccharomyces pombe as the result of several mechanisms that are typical of both retrotransposons and retroviruses. To identify host factors that contribute to the transposition process, we mutagenized cultures of S. pombe and screened them for strains that were unable to support Tf1 transposition. One such strain contained a mutation in a gene we named nup124. The product of this gene contains 11 FXFG repeats and is a component of the nuclear pore complex. In addition to the reduced levels of Tf1 transposition, the nup124-1 allele caused a significant reduction in the nuclear localization of Tf1 Gag. Surprisingly, the mutation in nup124-1 did not cause any reduction in the growth rate, the nuclear localization of specific nuclear localization signal-containing proteins, or the cytoplasmic localization of poly(A) mRNA. A two-hybrid analysis and an in vitro precipitation assay both identified an interaction between Tf1 Gag and the N terminus of Nup124p. These results provide evidence for an unusual mechanism of nuclear import that relies on a direct interaction between a nuclear pore factor and Tf1 Gag.

Emerin and histone deacetylase 3 (HDAC3) cooperatively regulate expression and nuclear positions of MyoD, Myf5, and Pax7 genes during myogenesis

PubMed Central

Demmerle, Justin; Koch, Adam J.; Holaska, James M.

2016-01-01

The spatial organization of chromatin is critical in establishing cell-type dependent gene expression programs. The inner nuclear membrane protein emerin has been implicated in regulating global chromatin architecture. We show emerin associates with genomic loci of muscle differentiation promoting factors in murine myogenic progenitors, including Myf5 and MyoD. Prior to their transcriptional activation Myf5 and MyoD loci localized to the nuclear lamina in proliferating progenitors and moved to the nucleoplasm upon transcriptional activation during differentiation. The Pax7 locus, which is transcribed in proliferating progenitors, localized to the nucleoplasm and Pax7 moved to the nuclear lamina upon repression during differentiation. Localization of Myf5, MyoD, and Pax7 to the nuclear lamina and proper temporal expression of these genes required emerin and HDAC3. Interestingly, activation of HDAC3 catalytic activity rescued both Myf5 localization to the nuclear lamina and its expression. Collectively, these data support a model whereby emerin facilitates repressive chromatin formation at the nuclear lamina by activating the catalytic activity of HDAC3 to regulate the coordinated spatiotemporal expression of myogenic differentiation genes. PMID:24062260

Evolutionary gradient of predicted nuclear localization signals (NLS)-bearing proteins in genomes of family Planctomycetaceae.

PubMed

Guo, Min; Yang, Ruifu; Huang, Chen; Liao, Qiwen; Fan, Guangyi; Sun, Chenghang; Lee, Simon Ming-Yuen

2017-04-04

The nuclear envelope is considered a key classification marker that distinguishes prokaryotes from eukaryotes. However, this marker does not apply to the family Planctomycetaceae, which has intracellular spaces divided by lipidic intracytoplasmic membranes (ICMs). Nuclear localization signal (NLS), a short stretch of amino acid sequence, destines to transport proteins from cytoplasm into nucleus, and is also associated with the development of nuclear envelope. We attempted to investigate the NLS motifs in Planctomycetaceae genomes to demonstrate the potential molecular transition in the development of intracellular membrane system. In this study, we identified NLS-like motifs that have the same amino acid compositions as experimentally identified NLSs in genomes of 11 representative species of family Planctomycetaceae. A total of 15 NLS types and 170 NLS-bearing proteins were detected in the 11 strains. To determine the molecular transformation, we compared NLS-bearing protein abundances in the 11 representative Planctomycetaceae genomes with them in genomes of 16 taxonomically varied microorganisms: nine bacteria, two archaea and five fungi. In the 27 strains, 29 NLS types and 1101 NLS-bearing proteins were identified, principal component analysis showed a significant transitional gradient from bacteria to Planctomycetaceae to fungi on their NLS-bearing protein abundance profiles. Then, we clustered the 993 non-redundant NLS-bearing proteins into 181 families and annotated their involved metabolic pathways. Afterwards, we aligned the ten types of NLS motifs from the 13 families containing NLS-bearing proteins among bacteria, Planctomycetaceae or fungi, considering their diversity, length and origin. A transition towards increased complexity from non-planctomycete bacteria to Planctomycetaceae to archaea and fungi was detected based on the complexity of the 10 types of NLS-like motifs in the 13 NLS-bearing proteins families. The results of this study reveal that Planctomycetaceae separates slightly from the members of non-planctomycete bacteria but still has substantial differences from fungi, based on the NLS-like motifs and NLS-bearing protein analysis.

The SAMI Galaxy Survey: spatially resolving the main sequence of star formation

NASA Astrophysics Data System (ADS)

Medling, Anne M.; Cortese, Luca; Croom, Scott M.; Green, Andrew W.; Groves, Brent; Hampton, Elise; Ho, I.-Ting; Davies, Luke J. M.; Kewley, Lisa J.; Moffett, Amanda J.; Schaefer, Adam L.; Taylor, Edward; Zafar, Tayyaba; Bekki, Kenji; Bland-Hawthorn, Joss; Bloom, Jessica V.; Brough, Sarah; Bryant, Julia J.; Catinella, Barbara; Cecil, Gerald; Colless, Matthew; Couch, Warrick J.; Drinkwater, Michael J.; Driver, Simon P.; Federrath, Christoph; Foster, Caroline; Goldstein, Gregory; Goodwin, Michael; Hopkins, Andrew; Lawrence, J. S.; Leslie, Sarah K.; Lewis, Geraint F.; Lorente, Nuria P. F.; Owers, Matt S.; McDermid, Richard; Richards, Samuel N.; Sharp, Robert; Scott, Nicholas; Sweet, Sarah M.; Taranu, Dan S.; Tescari, Edoardo; Tonini, Chiara; van de Sande, Jesse; Walcher, C. Jakob; Wright, Angus

2018-04-01

We present the ˜800 star formation rate maps for the Sydney-AAO Multi-object Integral field spectrograph (SAMI) Galaxy Survey based on H α emission maps, corrected for dust attenuation via the Balmer decrement, that are included in the SAMI Public Data Release 1. We mask out spaxels contaminated by non-stellar emission using the [O III]/H β, [N II]/H α, [S II]/H α, and [O I]/H α line ratios. Using these maps, we examine the global and resolved star-forming main sequences of SAMI galaxies as a function of morphology, environmental density, and stellar mass. Galaxies further below the star-forming main sequence are more likely to have flatter star formation profiles. Early-type galaxies split into two populations with similar stellar masses and central stellar mass surface densities. The main-sequence population has centrally concentrated star formation similar to late-type galaxies, while galaxies >3σ below the main sequence show significantly reduced star formation most strikingly in the nuclear regions. The split populations support a two-step quenching mechanism, wherein halo mass first cuts off the gas supply and remaining gas continues to form stars until the local stellar mass surface density can stabilize the reduced remaining fuel against further star formation. Across all morphologies, galaxies in denser environments show a decreased specific star formation rate from the outside in, supporting an environmental cause for quenching, such as ram-pressure stripping or galaxy interactions.

Investigation of the mechanism of meiotic DNA cleavage by VMA1-derived endonuclease uncovers a meiotic alteration in chromatin structure around the target site.

PubMed

Fukuda, Tomoyuki; Ohta, Kunihiro; Ohya, Yoshikazu

2006-06-01

VMA1-derived endonuclease (VDE), a homing endonuclease in Saccharomyces cerevisiae, is encoded by the mobile intein-coding sequence within the nuclear VMA1 gene. VDE recognizes and cleaves DNA at the 31-bp VDE recognition sequence (VRS) in the VMA1 gene lacking the intein-coding sequence during meiosis to insert a copy of the intein-coding sequence at the cleaved site. The mechanism underlying the meiosis specificity of VMA1 intein-coding sequence homing remains unclear. We studied various factors that might influence the cleavage activity in vivo and found that VDE binding to the VRS can be detected only when DNA cleavage by VDE takes place, implying that meiosis-specific DNA cleavage is regulated by the accessibility of VDE to its target site. As a possible candidate for the determinant of this accessibility, we analyzed chromatin structure around the VRS and revealed that local chromatin structure near the VRS is altered during meiosis. Although the meiotic chromatin alteration exhibits correlations with DNA binding and cleavage by VDE at the VMA1 locus, such a chromatin alteration is not necessarily observed when the VRS is embedded in ectopic gene loci. This suggests that nucleosome positioning or occupancy around the VRS by itself is not the sole mechanism for the regulation of meiosis-specific DNA cleavage by VDE and that other mechanisms are involved in the regulation.

Investigation of the Mechanism of Meiotic DNA Cleavage by VMA1-Derived Endonuclease Uncovers a Meiotic Alteration in Chromatin Structure around the Target Site

PubMed Central

Fukuda, Tomoyuki; Ohta, Kunihiro; Ohya, Yoshikazu

2006-01-01

VMA1-derived endonuclease (VDE), a homing endonuclease in Saccharomyces cerevisiae, is encoded by the mobile intein-coding sequence within the nuclear VMA1 gene. VDE recognizes and cleaves DNA at the 31-bp VDE recognition sequence (VRS) in the VMA1 gene lacking the intein-coding sequence during meiosis to insert a copy of the intein-coding sequence at the cleaved site. The mechanism underlying the meiosis specificity of VMA1 intein-coding sequence homing remains unclear. We studied various factors that might influence the cleavage activity in vivo and found that VDE binding to the VRS can be detected only when DNA cleavage by VDE takes place, implying that meiosis-specific DNA cleavage is regulated by the accessibility of VDE to its target site. As a possible candidate for the determinant of this accessibility, we analyzed chromatin structure around the VRS and revealed that local chromatin structure near the VRS is altered during meiosis. Although the meiotic chromatin alteration exhibits correlations with DNA binding and cleavage by VDE at the VMA1 locus, such a chromatin alteration is not necessarily observed when the VRS is embedded in ectopic gene loci. This suggests that nucleosome positioning or occupancy around the VRS by itself is not the sole mechanism for the regulation of meiosis-specific DNA cleavage by VDE and that other mechanisms are involved in the regulation. PMID:16757746

Phosphatidic acid interacts with a MYB transcription factor and regulates its nuclear localization and function in Arabidopsis.

PubMed

Yao, Hongyan; Wang, Geliang; Guo, Liang; Wang, Xuemin

2013-12-01

Phosphatidic acid (PA) has emerged as a class of cellular mediators involved in various cellular and physiological processes, but little is known about its mechanism of action. Here we show that PA interacts with werewolf (WER), a R2R3 MYB transcription factor involved in root hair formation. The PA-interacting region is confined to the end of the R2 subdomain. The ablation of the PA binding motif has no effect on WER binding to DNA, but abolishes its nuclear localization and its function in regulating epidermal cell fate. Inhibition of PA production by phospholipase Dζ also suppresses WER's nuclear localization, root hair formation, and elongation. These results suggest a role for PA in promoting protein nuclear localization.

Phosphatidic Acid Interacts with a MYB Transcription Factor and Regulates Its Nuclear Localization and Function in Arabidopsis[C][W

PubMed Central

Yao, Hongyan; Wang, Geliang; Guo, Liang; Wang, Xuemin

2013-01-01

Phosphatidic acid (PA) has emerged as a class of cellular mediators involved in various cellular and physiological processes, but little is known about its mechanism of action. Here we show that PA interacts with WEREWOLF (WER), a R2R3 MYB transcription factor involved in root hair formation. The PA-interacting region is confined to the end of the R2 subdomain. The ablation of the PA binding motif has no effect on WER binding to DNA, but abolishes its nuclear localization and its function in regulating epidermal cell fate. Inhibition of PA production by phospholipase Dζ also suppresses WER’s nuclear localization, root hair formation, and elongation. These results suggest a role for PA in promoting protein nuclear localization. PMID:24368785

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paterson, Carolyn P.; Ayalew, Lisanework E.; Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5E3 S7N 5B4 Canada

The L1 region of bovine adenovirus (BAdV)-3 encodes a non-structural protein designated 52K. Anti-52K serum detected a protein of 40 kDa, which localized to the nucleus but not to the nucleolus in BAdV-3-infected or transfected cells. Analysis of mutant 52K proteins suggested that three basic residues ({sup 105}RKR{sup 107}) of the identified domain (amino acids {sup 102}GMPRKRVLT{sup 110}) are essential for nuclear localization of 52K. The nuclear import of a GST-52K fusion protein utilizes the classical importin {alpha}/{beta}-dependent nuclear transport pathway. The 52K protein is preferentially bound to the cellular nuclear import receptor importin {alpha}3. Although deletion of amino acidmore » 102-110 is sufficient to abrogate the nuclear localization of 52K, amino acid 90-133 are required for interaction with importin-{alpha}3 and localizing a cytoplasmic protein to the nucleus. These results suggest that 52K contains a bipartite NLS, which preferentially utilize an importin {alpha}3 nuclear import receptor-mediated pathway to transport 52K to the nucleus.« less

Regulation of pokemon 1 activity by sumoylation.

PubMed

Roh, Hee-Eun; Lee, Min-Nyung; Jeon, Bu-Nam; Choi, Won-Il; Kim, Yoo-Jin; Yu, Mi-Young; Hur, Man-Wook

2007-01-01

Pokemon 1 is a proto-oncogenic transcriptional regulator that contains a POZ domain at the N-terminus and four Kruppel-like zinc fingers at the C-terminus. Pokemon 1 plays an important role in adipogenesis, osteogenesis, oncogenesis, and transcription of NF-kB responsive genes. Recent reports have shown that biological activities of transcription factors are regulated by sumolylation. We investigated whether Pokemon 1 is post-translationally modified by sumoylation and whether the modification affects Pokemon 1's transcriptional properties. We found that Pokemon 1 is sumoylated in vitro and in vivo. Upon careful analysis of the amino acid sequence of Pokemon 1, we found ten potential sumoylation sites located at lysines 61, 354, 371, 379, 383, 396, 486, 487, 536 and 539. We mutated each of these amino acids into arginine and tested whether the mutation could affect the transcriptional properties of Pokemon 1 on the Pokemon 1 responsive genes, such as ADH5/FDH and pG5-FRE-Luc. Wild-type Pokemon 1 potently represses transcription of ADH5/FDH. Most of the mutants, however, were weaker transcription repressors and repressed transcription 1.3-3.3 fold less effective. Although potential sumoylation sites were located close to the DNA binding domain or the nuclear localization sequence, the mutations did not alter nuclear localization or DNA binding activity. In addition, on the pG5-FRE-Luc test promoter construct, ectopic SUMO-1 repressed transcription in the presence of Pokemon 1. The sumoylation target lysine residue at amino acid 61, which is located in the middle of the POZ-domain, is important because K61R mutation resulted in a much weaker molecular interaction with corepressors. Our data suggest that Pokemon 1's activity as a transcription factor may involve sumoylation, and that sumoylation might be important in the regulation of transcription by Pokemon 1.

Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo.

PubMed

Gandhi, Shashank; Piacentino, Michael L; Vieceli, Felipe M; Bronner, Marianne E

2017-12-01

The advent of CRISPR/Cas9 has made genome editing possible in virtually any organism, including those not previously amenable to genetic manipulations. Here, we present an optimization of CRISPR/Cas9 for application to early avian embryos with improved efficiency via a three-fold strategy. First, we employed Cas9 protein flanked with two nuclear localization signal sequences for improved nuclear localization. Second, we used a modified guide RNA (gRNA) scaffold that obviates premature termination of transcription and unstable Cas9-gRNA interactions. Third, we used a chick-specific U6 promoter that yields 4-fold higher gRNA expression than the previously utilized human U6. For rapid screening of gRNAs for in vivo applications, we also generated a chicken fibroblast cell line that constitutively expresses Cas9. As proof of principle, we performed electroporation-based loss-of-function studies in the early chick embryo to knock out Pax7 and Sox10, key transcription factors with known functions in neural crest development. The results show that CRISPR/Cas9-mediated deletion causes loss of their respective proteins and transcripts, as well as predicted downstream targets. Taken together, the results reveal the utility of this optimized CRISPR/Cas9 method for targeted gene knockout in chicken embryos in a manner that is reproducible, robust and specific. Copyright © 2017 Elsevier Inc. All rights reserved.

Cbp80 is needed for the expression of piRNA components and piRNAs

PubMed Central

Colombo, Martino; Hernandez, Greco; Beuchle, Dirk; Berger, Fabienne; Peischl, Stephan; Bruggmann, Rémy

2017-01-01

Cap binding protein 80 (Cbp80) is the larger subunit of the nuclear cap-binding complex (nCBC), which is known to play important roles in nuclear mRNA processing, export, stability and quality control events. Reducing Cbp80 mRNA levels in the female germline revealed that Cbp80 is also involved in defending the germline against transposable elements. Combining such knockdown experiments with large scale sequencing of small RNAs further showed that Cbp80 is involved in the initial biogenesis of piRNAs as well as in the secondary biogenesis pathway, the ping-pong amplification cycle. We further found that Cbp80 knockdown not only led to the upregulation of transposons, but also to delocalization of Piwi, Aub and Ago3, key factors in the piRNA biosynthesis pathway. Furthermore, compared to controls, levels of Piwi and Aub were also reduced upon knock down of Cbp80. On the other hand, with the same treatment we could not detect significant changes in levels or subcellular distribution (nuage localization) of piRNA precursor transcripts. This shows that Cbp80 plays an important role in the production and localization of the protein components of the piRNA pathway and it seems to be less important for the production and export of the piRNA precursor transcripts. PMID:28746365

The nucleoporin Mlp2 is involved in chromosomal distribution during mitosis in trypanosomatids

PubMed Central

Morelle, Christelle; Sterkers, Yvon; Crobu, Lucien; MBang-Benet, Diane-Ethna; Kuk, Nada; Portalès, Pierre; Bastien, Patrick; Pagès, Michel; Lachaud, Laurence

2015-01-01

Nucleoporins are evolutionary conserved proteins mainly involved in the constitution of the nuclear pores and trafficking between the nucleus and cytoplasm, but are also increasingly viewed as main actors in chromatin dynamics and intra-nuclear mitotic events. Here, we determined the cellular localization of the nucleoporin Mlp2 in the ‘divergent’ eukaryotes Leishmania major and Trypanosoma brucei. In both protozoa, Mlp2 displayed an atypical localization for a nucleoporin, essentially intranuclear, and preferentially in the periphery of the nucleolus during interphase; moreover, it relocated at the mitotic spindle poles during mitosis. In T. brucei, where most centromeres have been identified, TbMlp2 was found adjacent to the centromeric sequences, as well as to a recently described unconventional kinetochore protein, in the periphery of the nucleolus, during interphase and from the end of anaphase onwards. TbMlp2 and the centromeres/kinetochores exhibited a differential migration towards the poles during mitosis. RNAi knockdown of TbMlp2 disrupted the mitotic distribution of chromosomes, leading to a surprisingly well-tolerated aneuploidy. In addition, diploidy was restored in a complementation assay where LmMlp2, the orthologue of TbMlp2 in Leishmania, was expressed in TbMlp2-RNAi-knockdown parasites. Taken together, our results demonstrate that Mlp2 is involved in the distribution of chromosomes during mitosis in trypanosomatids. PMID:25690889

Two host cytoplasmic effectors are required for pathogenesis of Phytophthora sojae by suppression of host defenses.

PubMed

Liu, Tingli; Ye, Wenwu; Ru, Yanyan; Yang, Xinyu; Gu, Biao; Tao, Kai; Lu, Shan; Dong, Suomeng; Zheng, Xiaobo; Shan, Weixing; Wang, Yuanchao; Dou, Daolong

2011-01-01

Phytophthora sojae encodes hundreds of putative host cytoplasmic effectors with conserved FLAK motifs following signal peptides, termed crinkling- and necrosis-inducing proteins (CRN) or Crinkler. Their functions and mechanisms in pathogenesis are mostly unknown. Here, we identify a group of five P. sojae-specific CRN-like genes with high levels of sequence similarity, of which three are putative pseudogenes. Functional analysis shows that the two functional genes encode proteins with predicted nuclear localization signals that induce contrasting responses when expressed in Nicotiana benthamiana and soybean (Glycine max). PsCRN63 induces cell death, while PsCRN115 suppresses cell death elicited by the P. sojae necrosis-inducing protein (PsojNIP) or PsCRN63. Expression of CRN fragments with deleted signal peptides and FLAK motifs demonstrates that the carboxyl-terminal portions of PsCRN63 or PsCRN115 are sufficient for their activities. However, the predicted nuclear localization signal is required for PsCRN63 to induce cell death but not for PsCRN115 to suppress cell death. Furthermore, silencing of the PsCRN63 and PsCRN115 genes in P. sojae stable transformants leads to a reduction of virulence on soybean. Intriguingly, the silenced transformants lose the ability to suppress host cell death and callose deposition on inoculated plants. These results suggest a role for CRN effectors in the suppression of host defense responses.

Mutation of a Conserved Nuclear Export Sequence in Chikungunya Virus Capsid Protein Disrupts Host Cell Nuclear Import.

PubMed

Jacobs, Susan C; Taylor, Adam; Herrero, Lara J; Mahalingam, Suresh; Fazakerley, John K

2017-10-20

Transmitted by mosquitoes; chikungunya virus (CHIKV) is responsible for frequent outbreaks of arthritic disease in humans. CHIKV is an arthritogenic alphavirus of the Togaviridae family. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleus. In encephalitic alphaviruses nuclear translocation induces host cell shut off; however, the role of capsid protein nuclear localisation in arthritogenic alphaviruses remains unclear. Using replicon systems, we investigated a nuclear export sequence (NES) in the N-terminal region of capsid protein; analogous to that found in encephalitic alphavirus capsid but uncharacterised in CHIKV. The chromosomal maintenance 1 (CRM1) export adaptor protein mediated CHIKV capsid protein export from the nucleus and a region within the N-terminal part of CHIKV capsid protein was required for active nuclear targeting. In contrast to encephalitic alphaviruses, CHIKV capsid protein did not inhibit host nuclear import; however, mutating the NES of capsid protein (∆NES) blocked host protein access to the nucleus. Interactions between capsid protein and the nucleus warrant further investigation.

The LINC complex contributes to heterochromatin organisation and transcriptional gene silencing in plants.

PubMed

Poulet, Axel; Duc, Céline; Voisin, Maxime; Desset, Sophie; Tutois, Sylvie; Vanrobays, Emmanuel; Benoit, Matthias; Evans, David E; Probst, Aline V; Tatout, Christophe

2017-02-01

The linker of nucleoskeleton and cytoskeleton (LINC) complex is an evolutionarily well-conserved protein bridge connecting the cytoplasmic and nuclear compartments across the nuclear membrane. While recent data support its function in nuclear morphology and meiosis, its involvement in chromatin organisation has not been studied in plants. Here, 3D imaging methods have been used to investigate nuclear morphology and chromatin organisation in interphase nuclei of the model plant Arabidopsis thaliana in which heterochromatin clusters in conspicuous chromatin domains called chromocentres. Chromocentres form a repressive chromatin environment contributing to transcriptional silencing of repeated sequences, a general mechanism needed for genome stability. Quantitative measurements of the 3D position of chromocentres indicate their close proximity to the nuclear periphery but that their position varies with nuclear volume and can be altered in specific mutants affecting the LINC complex. Finally, we propose that the plant LINC complex contributes to proper heterochromatin organisation and positioning at the nuclear periphery, since its alteration is associated with the release of transcriptional silencing as well as decompaction of heterochromatic sequences. © 2017. Published by The Company of Biologists Ltd.

Big and slow: phylogenetic estimates of molecular evolution in baleen whales (suborder mysticeti).

PubMed

Jackson, J A; Baker, C S; Vant, M; Steel, D J; Medrano-González, L; Palumbi, S R

2009-11-01

Baleen whales are the largest animals that have ever lived. To develop an improved estimation of substitution rate for nuclear and mitochondrial DNA for this taxon, we implemented a relaxed-clock phylogenetic approach using three fossil calibration dates: the divergence between odontocetes and mysticetes approximately 34 million years ago (Ma), between the balaenids and balaenopterids approximately 28 Ma, and the time to most recent common ancestor within the Balaenopteridae approximately 12 Ma. We examined seven mitochondrial genomes, a large number of mitochondrial control region sequences (219 haplotypes for 465 bp) and nine nuclear introns representing five species of whales, within which multiple species-specific alleles were sequenced to account for within-species diversity (1-15 for each locus). The total data set represents >1.65 Mbp of mitogenome and nuclear genomic sequence. The estimated substitution rate for the humpback whale control region (3.9%/million years, My) was higher than previous estimates for baleen whales but slow relative to other mammal species with similar generation times (e.g., human-chimp mean rate > 20%/My). The mitogenomic third codon position rate was also slow relative to other mammals (mean estimate 1%/My compared with a mammalian average of 9.8%/My for the cytochrome b gene). The mean nuclear genomic substitution rate (0.05%/My) was substantially slower than average synonymous estimates for other mammals (0.21-0.37%/My across a range of studies). The nuclear and mitogenome rate estimates for baleen whales were thus roughly consistent with an 8- to 10-fold slowing due to a combination of large body size and long generation times. Surprisingly, despite the large data set of nuclear intron sequences, there was only weak and conflicting support for alternate hypotheses about the phylogeny of balaenopterid whales, suggesting that interspecies introgressions or a rapid radiation has obscured species relationships in the nuclear genome.

Phylogenetic position of avian nocturnal and diurnal raptors.

PubMed

Mahmood, Muhammad Tariq; McLenachan, Patricia A; Gibb, Gillian C; Penny, David

2014-02-01

We report three new avian mitochondrial genomes, two from widely separated groups of owls and a falcon relative (the Secretarybird). We then report additional progress in resolving Neoavian relationships in that the two groups of owls do come together (it is not just long-branch attraction), and the Secretarybird is the deepest divergence on the Accipitridae lineage. This is now agreed between mitochondrial and nuclear sequences. There is no evidence for the monophyly of the combined three groups of raptors (owls, eagles, and falcons), and again this is agreed by nuclear and mitochondrial sequences. All three groups (owls, accipitrids [eagles], and falcons) do appear to be members of the "higher land birds," and though there may not yet be full "consilience" between mitochondrial and nuclear sequences for the precise order of divergences of the eagles, falcons, and the owls, there is good progress on their relationships.

Phylogenetic Position of Avian Nocturnal and Diurnal Raptors

PubMed Central

Mahmood, Muhammad Tariq; McLenachan, Patricia A.; Gibb, Gillian C.; Penny, David

2014-01-01

We report three new avian mitochondrial genomes, two from widely separated groups of owls and a falcon relative (the Secretarybird). We then report additional progress in resolving Neoavian relationships in that the two groups of owls do come together (it is not just long-branch attraction), and the Secretarybird is the deepest divergence on the Accipitridae lineage. This is now agreed between mitochondrial and nuclear sequences. There is no evidence for the monophyly of the combined three groups of raptors (owls, eagles, and falcons), and again this is agreed by nuclear and mitochondrial sequences. All three groups (owls, accipitrids [eagles], and falcons) do appear to be members of the “higher land birds,” and though there may not yet be full “consilience” between mitochondrial and nuclear sequences for the precise order of divergences of the eagles, falcons, and the owls, there is good progress on their relationships. PMID:24448983

A protocol for isolating insect mitochondrial genomes: a case study of NUMT in Melipona flavolineata (Hymenoptera: Apidae).

PubMed

Françoso, Elaine; Gomes, Fernando; Arias, Maria Cristina

2016-07-01

Nuclear mitochondrial DNA insertions (NUMTs) are mitochondrial DNA sequences that have been transferred into the nucleus and are recognized by the presence of indels and stop codons. Although NUMTs have been identified in a diverse range of species, their discovery was frequently accidental. Here, our initial goal was to develop and standardize a simple method for isolating NUMTs from the nuclear genome of a single bee. Subsequently, we tested our new protocol by determining whether the indels and stop codons of the cytochrome c oxidase subunit I (COI) sequence of Melipona flavolineata are of nuclear origin. The new protocol successfully demonstrated the presence of a COI NUMT. In addition to NUMT investigations, the protocol described here will also be very useful for studying mitochondrial mutations related to diseases and for sequencing complete mitochondrial genomes with high read coverage by Next-Generation technology.

Structural determinants of nuclear export signal orientation in binding to exportin CRM1

DOE PAGES

Fung, Ho Yee Joyce; Fu, Szu -Chin; Brautigam, Chad A.; ...

2015-09-08

The Chromosome Region of Maintenance 1 (CRM1) protein mediates nuclear export of hundreds of proteins through recognition of their nuclear export signals (NESs), which are highly variable in sequence and structure. The plasticity of the CRM1-NES interaction is not well understood, as there are many NES sequences that seem incompatible with structures of the NES-bound CRM1 groove. Crystal structures of CRM1 bound to two different NESs with unusual sequences showed the NES peptides binding the CRM1 groove in the opposite orientation (minus) to that of previously studied NESs (plus). A comparison of minus and plus NESs identified structural and sequencemore » determinants for NES orientation. The binding of NESs to CRM1 in both orientations results in a large expansion in NES consensus patterns and therefore a corresponding expansion of potential NESs in the proteome.« less

Fascioliasis transmission by Lymnaea neotropica confirmed by nuclear rDNA and mtDNA sequencing in Argentina.

PubMed

Mera y Sierra, Roberto; Artigas, Patricio; Cuervo, Pablo; Deis, Erika; Sidoti, Laura; Mas-Coma, Santiago; Bargues, Maria Dolores

2009-12-03

Fascioliasis is widespread in livestock in Argentina. Among activities included in a long-term initiative to ascertain which are the fascioliasis areas of most concern, studies were performed in a recreational farm, including liver fluke infection in different domestic animal species, classification of the lymnaeid vector and verification of natural transmission of fascioliasis by identification of the intramolluscan trematode larval stages found in naturally infected snails. The high prevalences in the domestic animals appeared related to only one lymnaeid species present. Lymnaeid and trematode classification was verified by means of nuclear ribosomal DNA and mitochondrial DNA marker sequencing. Complete sequences of 18S rRNA gene and rDNA ITS-2 and ITS-1, and a fragment of the mtDNA cox1 gene demonstrate that the Argentinian lymnaeid belongs to the species Lymnaea neotropica. Redial larval stages found in a L. neotropica specimen were ascribed to Fasciola hepatica after analysis of the complete ITS-1 sequence. The finding of L. neotropica is the first of this lymnaeid species not only in Argentina but also in Southern Cone countries. The total absence of nucleotide differences between the sequences of specimens from Argentina and the specimens from the Peruvian type locality at the levels of rDNA 18S, ITS-2 and ITS-1, and the only one mutation at the mtDNA cox1 gene suggest a very recent spread. The ecological characteristics of this lymnaeid, living in small, superficial water collections frequented by livestock, suggest that it may be carried from one place to another by remaining in dried mud stuck to the feet of transported animals. The presence of L. neotropica adds pronounced complexity to the transmission and epidemiology of fascioliasis in Argentina, due to the great difficulties in distinguishing, by traditional malacological methods, between the three similar lymnaeid species of the controversial Galba/Fossaria group present in this country: L. viatrix, Galba truncatula and L. neotropica. It also poses a problem with regard to the use, for lymnaeid vector species discrimination, of several molecular techniques which do not show sufficient accuracy, as those relying on the 18S rRNA gene or parts of it, because both L. neotropica and L. viatrix present identical 18S sequence.

Subcellular targeting of p33ING1b by phosphorylation-dependent 14-3-3 binding regulates p21WAF1 expression.

PubMed

Gong, Wei; Russell, Michael; Suzuki, Keiko; Riabowol, Karl

2006-04-01

ING1 is a type II tumor suppressor that affects cell growth, stress signaling, apoptosis, and DNA repair by altering chromatin structure and regulating transcription. Decreased ING1 expression is seen in several human cancers, and mislocalization has been noted in diverse types of cancer cells. Aberrant targeting may, therefore, functionally inactivate ING1. Bioinformatics analysis identified a sequence between the nuclear localization sequence and plant homeodomain domains of ING1 that closely matched the binding motif of 14-3-3 proteins that target cargo proteins to specific subcellular locales. We find that the widely expressed p33(ING1b) splicing isoform of ING1 interacts with members of the 14-3-3 family of proteins and that this interaction is regulated by the phosphorylation status of ING1. 14-3-3 binding resulted in significant amounts of p33(ING1b) protein being tethered in the cytoplasm. As shown previously, ectopic expression of p33(ING1b) increased levels of the p21(Waf1) cyclin-dependent kinase inhibitor upon UV-induced DNA damage. Overexpression of 14-3-3 inhibited the up-regulation of p21(Waf1) by p33(ING1b), consistent with the idea that mislocalization blocks at least one of ING1's biological activities. These data support the idea that the 14-3-3 proteins play a crucial role in regulating the activity of p33(ING1b) by directing its subcellular localization.

Short Interspersed Nuclear Element (SINE) Sequences in the Genome of the Human Pathogenic Fungus Aspergillus fumigatus Af293

PubMed Central

Kanhayuwa, Lakkhana; Coutts, Robert H. A.

2016-01-01

Novel families of short interspersed nuclear element (SINE) sequences in the human pathogenic fungus Aspergillus fumigatus, clinical isolate Af293, were identified and categorised into tRNA-related and 5S rRNA-related SINEs. Eight predicted tRNA-related SINE families originating from different tRNAs, and nominated as AfuSINE2 sequences, contained target site duplications of short direct repeat sequences (4–14 bp) flanking the elements, an extended tRNA-unrelated region and typical features of RNA polymerase III promoter sequences. The elements ranged in size from 140–493 bp and were present in low copy number in the genome and five out of eight were actively transcribed. One putative tRNAArg-derived sequence, AfuSINE2-1a possessed a unique feature of repeated trinucleotide ACT residues at its 3’-terminus. This element was similar in sequence to the I-4_AO element found in A. oryzae and an I-1_AF long nuclear interspersed element-like sequence identified in A. fumigatus Af293. Families of 5S rRNA-related SINE sequences, nominated as AfuSINE3, were also identified and their 5'-5S rRNA-related regions show 50–65% and 60–75% similarity to respectively A. fumigatus 5S rRNAs and SINE3-1_AO found in A. oryzae. A. fumigatus Af293 contains five copies of AfuSINE3 sequences ranging in size from 259–343 bp and two out of five AfuSINE3 sequences were actively transcribed. Investigations on AfuSINE distribution in the fungal genome revealed that the elements are enriched in pericentromeric and subtelomeric regions and inserted within gene-rich regions. We also demonstrated that some, but not all, AfuSINE sequences are targeted by host RNA silencing mechanisms. Finally, we demonstrated that infection of the fungus with mycoviruses had no apparent effects on SINE activity. PMID:27736869

Short Interspersed Nuclear Element (SINE) Sequences in the Genome of the Human Pathogenic Fungus Aspergillus fumigatus Af293.

PubMed

Kanhayuwa, Lakkhana; Coutts, Robert H A

2016-01-01

Novel families of short interspersed nuclear element (SINE) sequences in the human pathogenic fungus Aspergillus fumigatus, clinical isolate Af293, were identified and categorised into tRNA-related and 5S rRNA-related SINEs. Eight predicted tRNA-related SINE families originating from different tRNAs, and nominated as AfuSINE2 sequences, contained target site duplications of short direct repeat sequences (4-14 bp) flanking the elements, an extended tRNA-unrelated region and typical features of RNA polymerase III promoter sequences. The elements ranged in size from 140-493 bp and were present in low copy number in the genome and five out of eight were actively transcribed. One putative tRNAArg-derived sequence, AfuSINE2-1a possessed a unique feature of repeated trinucleotide ACT residues at its 3'-terminus. This element was similar in sequence to the I-4_AO element found in A. oryzae and an I-1_AF long nuclear interspersed element-like sequence identified in A. fumigatus Af293. Families of 5S rRNA-related SINE sequences, nominated as AfuSINE3, were also identified and their 5'-5S rRNA-related regions show 50-65% and 60-75% similarity to respectively A. fumigatus 5S rRNAs and SINE3-1_AO found in A. oryzae. A. fumigatus Af293 contains five copies of AfuSINE3 sequences ranging in size from 259-343 bp and two out of five AfuSINE3 sequences were actively transcribed. Investigations on AfuSINE distribution in the fungal genome revealed that the elements are enriched in pericentromeric and subtelomeric regions and inserted within gene-rich regions. We also demonstrated that some, but not all, AfuSINE sequences are targeted by host RNA silencing mechanisms. Finally, we demonstrated that infection of the fungus with mycoviruses had no apparent effects on SINE activity.

The Human Polycomb Group Complex Associates with Pericentromeric Heterochromatin to Form a Novel Nuclear Domain

PubMed Central

Saurin, Andrew J.; Shiels, Carol; Williamson, Jill; Satijn, David P.E.; Otte, Arie P.; Sheer, Denise; Freemont, Paul S.

1998-01-01

The Polycomb group (PcG) complex is a chromatin-associated multiprotein complex, involved in the stable repression of homeotic gene activity in Drosophila. Recently, a mammalian PcG complex has been identified with several PcG proteins implicated in the regulation of Hox gene expression. Although the mammalian PcG complex appears analogous to the complex in Drosophila, the molecular mechanisms and functions for the mammalian PcG complex remain unknown. Here we describe a detailed characterization of the human PcG complex in terms of cellular localization and chromosomal association. By using antibodies that specifically recognize three human PcG proteins— RING1, BMI1, and hPc2—we demonstrate in a number of human cell lines that the PcG complex forms a unique discrete nuclear structure that we term PcG bodies. PcG bodies are prominent novel nuclear structures with the larger PcG foci generally localized near the centromeres, as visualized with a kinetochore antibody marker. In both normal fetal and adult fibroblasts, PcG bodies are not randomly dispersed, but appear clustered into defined areas within the nucleus. We show in three different human cell lines that the PcG complex can tightly associate with large pericentromeric heterochromatin regions (1q12) on chromosome 1, and with related pericentromeric sequences on different chromosomes, providing evidence for a mammalian PcG–heterochromatin association. Furthermore, these heterochromatin-bound PcG complexes remain stably associated throughout mitosis, thereby allowing the potential inheritance of the PcG complex through successive cell divisions. We discuss these results in terms of the known function of the PcG complex as a transcriptional repression complex. PMID:9722603

Use of DNA barcodes to identify flowering plants.

PubMed

Kress, W John; Wurdack, Kenneth J; Zimmer, Elizabeth A; Weigt, Lee A; Janzen, Daniel H

2005-06-07

Methods for identifying species by using short orthologous DNA sequences, known as "DNA barcodes," have been proposed and initiated to facilitate biodiversity studies, identify juveniles, associate sexes, and enhance forensic analyses. The cytochrome c oxidase 1 sequence, which has been found to be widely applicable in animal barcoding, is not appropriate for most species of plants because of a much slower rate of cytochrome c oxidase 1 gene evolution in higher plants than in animals. We therefore propose the nuclear internal transcribed spacer region and the plastid trnH-psbA intergenic spacer as potentially usable DNA regions for applying barcoding to flowering plants. The internal transcribed spacer is the most commonly sequenced locus used in plant phylogenetic investigations at the species level and shows high levels of interspecific divergence. The trnH-psbA spacer, although short ( approximately 450-bp), is the most variable plastid region in angiosperms and is easily amplified across a broad range of land plants. Comparison of the total plastid genomes of tobacco and deadly nightshade enhanced with trials on widely divergent angiosperm taxa, including closely related species in seven plant families and a group of species sampled from a local flora encompassing 50 plant families (for a total of 99 species, 80 genera, and 53 families), suggest that the sequences in this pair of loci have the potential to discriminate among the largest number of plant species for barcoding purposes.

Death-domain associated protein-6 (DAXX) mediated apoptosis in hantavirus infection is counter-balanced by activation of interferon-stimulated nuclear transcription factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khaiboullina, Svetlana F., E-mail: sv.khaiboullina@gmail.com; Morzunov, Sergey P.; Boichuk, Sergei V.

2013-09-01

Hantaviruses are negative strand RNA species that replicate predominantly in the cytoplasm. They also activate numerous cellular responses, but their involvement in nuclear processes is yet to be established. Using human umbilical vein endothelial cells (HUVECs), this study investigates the molecular finger-print of nuclear transcription factors during hantavirus infection. The viral-replication-dependent activation of pro-myelocytic leukemia protein (PML) was followed by subsequent localization in nuclear bodies (NBs). PML was also found in close proximity to activated Sp100 nuclear antigen and interferon-stimulated gene 20 kDa protein (ISG-20), but co-localization with death-domain associated protein-6 (DAXX) was not observed. These data demonstrate that hantavirusmore » triggers PML activation and localization in NBs in the absence of DAXX-PLM-NB co-localization. The results suggest that viral infection interferes with DAXX-mediated apoptosis, and expression of interferon-activated Sp100 and ISG-20 proteins may indicate intracellular intrinsic antiviral attempts.« less

Stronger activation of SREBP-1a by nucleus-localized HBx

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Qi; Qiao, Ling; Yang, Jian

2015-05-08

We previously showed that hepatitis B virus (HBV) X protein activates the sterol regulatory element-binding protein-1a (SREBP-1a). Here we examined the role of nuclear localization of HBx in this process. In comparison to the wild-type and cytoplasmic HBx, nuclear HBx had stronger effects on SREBP-1a and fatty acid synthase transcription activation, intracellular lipid accumulation and cell proliferation. Furthermore, nuclear HBx could activate HBV enhancer I/X promoter and was more effective on up-regulating HBV mRNA level in the context of HBV replication than the wild-type HBx, while the cytoplasmic HBx had no effect. Our results demonstrate the functional significance of themore » nucleus-localized HBx in regulating host lipogenic pathway and HBV replication. - Highlights: • Nuclear HBx is more effective on activating SREBP-1a and FASN transcription. • Nuclear HBx is more effective on enhancing intracellular lipid accumulation. • Nuclear HBx is more effective on enhancing cell proliferation. • Nuclear HBx up-regulates HBV enhancer I/X promoter activity. • Nuclear HBx increases HBV mRNA level in the context of HBV replication.« less

The Hepatitis C Virus-Induced Membranous Web and Associated Nuclear Transport Machinery Limit Access of Pattern Recognition Receptors to Viral Replication Sites

PubMed Central

Neufeldt, Christopher J.; Joyce, Michael A.; Van Buuren, Nicholas; Levin, Aviad; Kirkegaard, Karla; Gale Jr., Michael; Tyrrell, D. Lorne J.; Wozniak, Richard W.

2016-01-01

Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family and a major cause of liver disease worldwide. HCV replicates in the cytoplasm, and the synthesis of viral proteins induces extensive rearrangements of host cell membranes producing structures, collectively termed the membranous web (MW). The MW contains the sites of viral replication and assembly, and we have identified distinct membrane fractions derived from HCV-infected cells that contain replication and assembly complexes enriched for viral RNA and infectious virus, respectively. The complex membrane structure of the MW is thought to protect the viral genome limiting its interactions with cytoplasmic pattern recognition receptors (PRRs) and thereby preventing activation of cellular innate immune responses. Here we show that PRRs, including RIG-I and MDA5, and ribosomes are excluded from viral replication and assembly centers within the MW. Furthermore, we present evidence that components of the nuclear transport machinery regulate access of proteins to MW compartments. We show that the restricted assess of RIG-I to the MW can be overcome by the addition of a nuclear localization signal sequence, and that expression of a NLS-RIG-I construct leads to increased immune activation and the inhibition of viral replication. PMID:26863439

Genome-wide profiling of chromosome interactions in Plasmodium falciparum characterizes nuclear architecture and reconfigurations associated with antigenic variation

PubMed Central

Lemieux, Jacob E; Kyes, Sue A; Otto, Thomas D; Feller, Avi I; Eastman, Richard T; Pinches, Robert A; Berriman, Matthew; Su, Xin-zhuan; Newbold, Chris I

2013-01-01

Spatial relationships within the eukaryotic nucleus are essential for proper nuclear function. In Plasmodium falciparum, the repositioning of chromosomes has been implicated in the regulation of the expression of genes responsible for antigenic variation, and the formation of a single, peri-nuclear nucleolus results in the clustering of rDNA. Nevertheless, the precise spatial relationships between chromosomes remain poorly understood, because, until recently, techniques with sufficient resolution have been lacking. Here we have used chromosome conformation capture and second-generation sequencing to study changes in chromosome folding and spatial positioning that occur during switches in var gene expression. We have generated maps of chromosomal spatial affinities within the P. falciparum nucleus at 25 Kb resolution, revealing a structured nucleolus, an absence of chromosome territories, and confirming previously identified clustering of heterochromatin foci. We show that switches in var gene expression do not appear to involve interaction with a distant enhancer, but do result in local changes at the active locus. These maps reveal the folding properties of malaria chromosomes, validate known physical associations, and characterize the global landscape of spatial interactions. Collectively, our data provide critical information for a better understanding of gene expression regulation and antigenic variation in malaria parasites. PMID:23980881

Identification of ZASP, a novel protein associated to Zona occludens-2.

PubMed

Lechuga, Susana; Alarcón, Lourdes; Solano, Jesús; Huerta, Miriam; Lopez-Bayghen, Esther; González-Mariscal, Lorenza

2010-11-15

With the aim of discovering new molecular interactions of the tight junction protein ZO-2, a two-hybrid screen was performed on a human kidney cDNA library using as bait the middle segment of ZO-2. Through this assay we identified a 24-kDa novel protein herein named ZASP for ZO-2 associated speckle protein. ZO-2/ZASP interaction further confirmed by pull down and immunoprecipitation experiments, requires the presence of the intact PDZ binding motif SQV of ZASP and the third PDZ domain of ZO-2. ZASP mRNA and protein are present in the kidney and in several epithelial cell lines. Endogenous ZASP is expressed primarily in nuclear speckles in co-localization with splicing factor SC-35. Nocodazole treatment and wash out reveals that ZASP disappears from the nucleus during mitosis in accordance with speckle disassembly during metaphase. ZASP amino acid sequence exhibits a canonical nuclear exportation signal and in agreement the protein exits the nucleus through a process mediated by exportin/CRM1. ZASP over-expression blocks the inhibitory activity of ZO-2 on cyclin D1 gene transcription and protein expression. The identification of ZASP helps to unfold the complex nuclear molecular arrays that form on ZO-2 scaffolds. Copyright © 2010 Elsevier Inc. All rights reserved.

Rapid isolation of microsatellite DNAs and identification of polymorphic mitochondrial DNA regions in the fish rotan (Perccottus glenii) invading European Russia

USGS Publications Warehouse

King, Timothy L.; Eackles, Michael S.; Reshetnikov, Andrey N.

2015-01-01

Human-mediated translocations and subsequent large-scale colonization by the invasive fish rotan (Perccottus glenii Dybowski, 1877; Perciformes, Odontobutidae), also known as Amur or Chinese sleeper, has resulted in dramatic transformations of small lentic ecosystems. However, no detailed genetic information exists on population structure, levels of effective movement, or relatedness among geographic populations of P. glenii within the European part of the range. We used massively parallel genomic DNA shotgun sequencing on the semiconductor-based Ion Torrent Personal Genome Machine (PGM) sequencing platform to identify nuclear microsatellite and mitochondrial DNA sequences in P. glenii from European Russia. Here we describe the characterization of nine nuclear microsatellite loci, ascertain levels of allelic diversity, heterozygosity, and demographic status of P. glenii collected from Ilev, Russia, one of several initial introduction points in European Russia. In addition, we mapped sequence reads to the complete P. glenii mitochondrial DNA sequence to identify polymorphic regions. Nuclear microsatellite markers developed for P. glenii yielded sufficient genetic diversity to: (1) produce unique multilocus genotypes; (2) elucidate structure among geographic populations; and (3) provide unique perspectives for analysis of population sizes and historical demographics. Among 4.9 million filtered P. glenii Ion Torrent PGM sequence reads, 11,304 mapped to the mitochondrial genome (NC_020350). This resulted in 100 % coverage of this genome to a mean coverage depth of 102X. A total of 130 variable sites were observed between the publicly available genome from China and the studied composite mitochondrial genome. Among these, 82 were diagnostic and monomorphic between the mitochondrial genomes and distributed among 15 genome regions. The polymorphic sites (N = 48) were distributed among 11 mitochondrial genome regions. Our results also indicate that sequence reads generated from two three-hour runs on the Ion Torrent PGM can generate a sufficient number of nuclear and mitochondrial markers to improve understanding of the evolutionary and ecological dynamics of non-model and in particular, invasive species.

Nuclear DNA analyses in genetic studies of populations: practice, problems and prospects.

PubMed

Zhang, De-Xing; Hewitt, Godfrey M

2003-03-01

Population-genetic studies have been remarkably productive and successful in the last decade following the invention of PCR technology and the introduction of mitochondrial and microsatellite DNA markers. While mitochondrial DNA has proven powerful for genealogical and evolutionary studies of animal populations, and microsatellite sequences are the most revealing DNA markers available so far for inferring population structure and dynamics, they both have important and unavoidable limitations. To obtain a fuller picture of the history and evolutionary potential of populations, genealogical data from nuclear loci are essential, and the inclusion of other nuclear markers, i.e. single copy nuclear polymorphic (scnp) sequences, is clearly needed. Four major uncertainties for nuclear DNA analyses of populations have been facing us, i.e. the availability of scnp markers for carrying out such analysis, technical laboratory hurdles for resolving haplotypes, difficulty in data analysis because of recombination, low divergence levels and intraspecific multifurcation evolution, and the utility of scnp markers for addressing population-genetic questions. In this review, we discuss the availability of highly polymorphic single copy DNA in the nuclear genome, describe patterns and rate of evolution of nuclear sequences, summarize past empirical and theoretical efforts to recover and analyse data from scnp markers, and examine the difficulties, challenges and opportunities faced in such studies. We show that although challenges still exist, the above-mentioned obstacles are now being removed. Recent advances in technology and increases in statistical power provide the prospect of nuclear DNA analyses becoming routine practice, allowing allele-discriminating characterization of scnp loci and microsatellite loci. This certainly will increase our ability to address more complex questions, and thereby the sophistication of genetic analyses of populations.

Microgeographic Genetic Variation of the Malaria Vector Anopheles darlingi Root (Diptera: Culicidae) from Córdoba and Antioquia, Colombia

PubMed Central

Gutiérrez, Lina A.; Gómez, Giovan F.; González, John J.; Castro, Martha I.; Luckhart, Shirley; Conn, Jan E.; Correa, Margarita M.

2010-01-01

Anopheles darlingi is an important vector of Plasmodium spp. in several malaria-endemic regions of Colombia. This study was conducted to test genetic variation of An. darlingi at a microgeographic scale (approximately 100 km) from localities in Córdoba and Antioquia states, in western Colombia, to better understand the potential contribution of population genetics to local malaria control programs. Microsatellite loci: nuclear white and cytochrome oxidase subunit I (COI) gene sequences were analyzed. The northern white gene lineage was exclusively distributed in Córdoba and Antioquia and shared COI haplotypes were highly represented in mosquitoes from both states. COI analyses showed these An. darlingi are genetically closer to Central American populations than southern South American populations. Overall microsatellites and COI analysis showed low to moderate genetic differentiation among populations in northwestern Colombia. Given the existence of high gene flow between An. darlingi populations of Córdoba and Antioquia, integrated vector control strategies could be developed in this region of Colombia. PMID:20595475

In vivo binding of hot pepper bZIP transcription factor CabZIP1 to the G-box region of pathogenesis-related protein 1 promoter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Boo-Ja; Park, Chang-Jin; Kim, Sung-Kyu

2006-05-26

We find that salicylic acid and ethephon treatment in hot pepper increases the expression of a putative basic/leucine zipper (bZIP) transcription factor gene, CabZIP1. CabZIP1 mRNA is expressed ubiquitously in various organs. The green fluorescent protein-fused transcription factor, CabZIP1::GFP, can be specifically localized to the nucleus, an action that is consistent with the presence of a nuclear localization signal in its protein sequence. Transient overexpression of the CabZIP1 transcription factor results in an increase in PR-1 transcripts level in Nicotiana benthamiana leaves. Using chromatin immunoprecipitation, we demonstrate that CabZIP1 binds to the G-box elements in native promoter of the hotmore » pepper pathogenesis-related protein 1 (CaPR-1) gene in vivo. Taken together, our results suggest that CabZIP1 plays a role as a transcriptional regulator of the CaPR-1 gene.« less

Cloning of cDNA encoding the nuclear form of chicken sterol response element binding protein-2 (SREBP-2), chromosomal localization, and tissue expression of chicken SREBP-1 and -2 genes.

PubMed

Assaf, S; Hazard, D; Pitel, F; Morisson, M; Alizadeh, M; Gondret, F; Diot, C; Vignal, A; Douaire, M; Lagarrigue, S

2003-01-01

Sterol regulatory element binding protein-1 and -2 (SREBP-1 and -2) are key transcription factors involved in the biosynthesis of cholesterol and fatty adds. The SREBP have mainly been studied in rodents in which lipogenesis is regulated in both liver and adipose tissue. There is, however, a paucity of information on birds, in which lipogenesis occurs essentially in the liver as in humans. As a prelude to the investigation of the role of SREBP in lipid metabolism regulation in chicken, we sequenced the cDNA, encoding the mature nuclear form of chicken SREBP-2 protein, mapped SREBP-1 and -2 genes and studied their tissue expressions. The predicted chicken SREBP-2 amino acid sequence shows a 77 to 79% identity with human, mouse, and hamster homologues, with a nearly perfect conservation in all the important functional motifs, basic, helix-loop-helix, and leucine zipper (bHLH-Zip) region as well as cleavage sites. As in the human genome, SREBP-1 and SREBP-2 chicken genes are located on two separate chromosomes, respectively microchromosome 14 and macrochromosome 1. Tissue expression data show that SREBP-1 and SREBP-2 are expressed in a wide variety of tissues in chicken. However, unlike SREBP-2, SREBP-1 is expressed preferentially in the liver and uropygial gland, suggesting an important role of SREBP-1 in the regulation of lipogenesis in avian species.

Molecular authentication of Radix Puerariae Lobatae and Radix Puerariae Thomsonii by ITS and 5S rRNA spacer sequencing.

PubMed

Sun, Ye; Shaw, Pang-Chui; Fung, Kwok-Pui

2007-01-01

In the present study, we examined nuclear DNA sequences in an attempt to reveal the relationships between Pueraria lobata (Willd). Ohwi, P. thomsonii Benth., and P. montana (Lour.) Merr. We found that internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA are highly divergent in P. lobata and P. thomsonii, and four types of ITS with different length are found in the two species. On the other hand, DNA sequences of 5S rRNA gene spacer are highly conserved across multiple copies in P. lobata and P. thomsonii, they could be used to identify P. lobata, P. thomsonii, and P. montana of this complex, and may serve as a useful tool in medical authentication of Radix Puerariae Lobatae and Radix Puerariae Thomsonii.

Predicting RNA-protein binding sites and motifs through combining local and global deep convolutional neural networks.

PubMed

Pan, Xiaoyong; Shen, Hong-Bin

2018-05-02

RNA-binding proteins (RBPs) take over 5∼10% of the eukaryotic proteome and play key roles in many biological processes, e.g. gene regulation. Experimental detection of RBP binding sites is still time-intensive and high-costly. Instead, computational prediction of the RBP binding sites using pattern learned from existing annotation knowledge is a fast approach. From the biological point of view, the local structure context derived from local sequences will be recognized by specific RBPs. However, in computational modeling using deep learning, to our best knowledge, only global representations of entire RNA sequences are employed. So far, the local sequence information is ignored in the deep model construction process. In this study, we present a computational method iDeepE to predict RNA-protein binding sites from RNA sequences by combining global and local convolutional neural networks (CNNs). For the global CNN, we pad the RNA sequences into the same length. For the local CNN, we split a RNA sequence into multiple overlapping fixed-length subsequences, where each subsequence is a signal channel of the whole sequence. Next, we train deep CNNs for multiple subsequences and the padded sequences to learn high-level features, respectively. Finally, the outputs from local and global CNNs are combined to improve the prediction. iDeepE demonstrates a better performance over state-of-the-art methods on two large-scale datasets derived from CLIP-seq. We also find that the local CNN run 1.8 times faster than the global CNN with comparable performance when using GPUs. Our results show that iDeepE has captured experimentally verified binding motifs. https://github.com/xypan1232/iDeepE. xypan172436@gmail.com or hbshen@sjtu.edu.cn. Supplementary data are available at Bioinformatics online.

Intracellular Localization of Arabidopsis Sulfurtransferases1

PubMed Central

Bauer, Michael; Dietrich, Christof; Nowak, Katharina; Sierralta, Walter D.; Papenbrock, Jutta

2004-01-01

Sulfurtransferases (Str) comprise a group of enzymes widely distributed in archaea, eubacteria, and eukaryota which catalyze the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. In all organisms analyzed to date, small gene families encoding Str proteins have been identified. The gene products were localized to different compartments of the cells. Our interest concerns the localization of Str proteins encoded in the nuclear genome of Arabidopsis. Computer-based prediction methods revealed localization in different compartments of the cell for six putative AtStrs. Several methods were used to determine the localization of the AtStr proteins experimentally. For AtStr1, a mitochondrial localization was demonstrated by immunodetection in the proteome of isolated mitochondria resolved by one- and two-dimensional gel electrophoresis and subsequent blotting. The respective mature AtStr1 protein was identified by mass spectrometry sequencing. The same result was obtained by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts, whereas AtStr2 was exclusively localized to the cytoplasm by this method. Three members of the single-domain AtStr were localized in the chloroplasts as demonstrated by transient expression of green fluorescent protein fusions in protoplasts and stomata, whereas the single-domain AtStr18 was shown to be cytoplasmic. The remarkable subcellular distribution of AtStr15 was additionally analyzed by transmission electron immunomicroscopy using a monospecific antibody against green fluorescent protein, indicating an attachment to the thylakoid membrane. The knowledge of the intracellular localization of the members of this multiprotein family will help elucidate their specific functions in the organism. PMID:15181206

Intracellular localization of Arabidopsis sulfurtransferases.

PubMed

Bauer, Michael; Dietrich, Christof; Nowak, Katharina; Sierralta, Walter D; Papenbrock, Jutta

2004-06-01

Sulfurtransferases (Str) comprise a group of enzymes widely distributed in archaea, eubacteria, and eukaryota which catalyze the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. In all organisms analyzed to date, small gene families encoding Str proteins have been identified. The gene products were localized to different compartments of the cells. Our interest concerns the localization of Str proteins encoded in the nuclear genome of Arabidopsis. Computer-based prediction methods revealed localization in different compartments of the cell for six putative AtStrs. Several methods were used to determine the localization of the AtStr proteins experimentally. For AtStr1, a mitochondrial localization was demonstrated by immunodetection in the proteome of isolated mitochondria resolved by one- and two-dimensional gel electrophoresis and subsequent blotting. The respective mature AtStr1 protein was identified by mass spectrometry sequencing. The same result was obtained by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts, whereas AtStr2 was exclusively localized to the cytoplasm by this method. Three members of the single-domain AtStr were localized in the chloroplasts as demonstrated by transient expression of green fluorescent protein fusions in protoplasts and stomata, whereas the single-domain AtStr18 was shown to be cytoplasmic. The remarkable subcellular distribution of AtStr15 was additionally analyzed by transmission electron immunomicroscopy using a monospecific antibody against green fluorescent protein, indicating an attachment to the thylakoid membrane. The knowledge of the intracellular localization of the members of this multiprotein family will help elucidate their specific functions in the organism.

Cellular stress stimulates nuclear localization signal (NLS) independent nuclear transport of MRJ

PubMed Central

Andrews, Joel F.; Sykora, Landon J.; Barik-Letostak, Tiasha; Menezes, Mitchell E.; Mitra, Aparna; Barik, Sailen; Shevde, Lalita A.; Samant, Rajeev S.

2012-01-01

HSP40 family member MRJ (DNAJB6) has been in the spot light for its relevance to Huntington’s, Parkinson’s diseases, limb-girdle muscular dystrophy, placental development, neural stem cells, cell cycle and malignancies such as breast cancer and melanoma. This gene has two spliced variants coding for 2 distinct proteins with significant homology. However, MRJ(L) (large variant) is predominantly localized to the nucleus whereas MRJ(S) (small variant) is predominantly cytoplasmic. Interestingly MRJ(S) translocates to the nucleus in response to heat shock. The classical heat shock proteins respond to crises (stress) by increasing the number of molecules, usually by transcriptional up-regulation. Our studies imply that a quick increase in the molar concentration of MRJ in the nuclear compartment is a novel method by which MRJ responds to stress. We found that MRJ(S) shows NLS (nuclear localization signal) independent nuclear localization in response to heat shock and hypoxia. The specificity of this response is realized due to lack of such response by MRJ(S) when challenged by other stressors, such as some cytokines or UV light. Deletion analysis has allowed us to narrow down on a 20 amino acid stretch at the C-terminal region of MRJ(S) as a potential stress sensing region. Functional studies indicated that constitutive nuclear localization of MRJ(S) promoted attributes of malignancy such as proliferation and invasiveness overall indicating distinct phenotypic characteristics of nuclear MRJ(S). PMID:22504047

Origins and Widespread Distribution of Co-existing Polyploids in Arnica cordifolia (Asteraceae)

PubMed Central

Kao, Rebecca Hufft

2008-01-01

Background and Aims Polyploidy is a central force structuring genetic diversity in angiosperms, but its ecological significance and modes of origin are not fully understood. This work investigated the patterns of coexistence and molecular relatedness of polyploids in the perennial herb, Arnica cordifolia. Methods The local- and broad-scale distributions of cytotypes were analysed using flow cytometry. Samples were collected from both roadside and understorey habitats to test the hypothesis of niche separation between triploids and tetraploids. The nuclear rDNA internal transcribed spacer (ITS) and plastid rpl16 spacer, trnL intron plus trnL-trnF spacer and trnK 3' intron regions were sequenced. Key Results Broad-scale sampling established that both triploids and tetraploids were common throughout the range of the species, pentaploids were rare, and diploids were not found. Local-scale sampling revealed coexistence of both triploids and tetraploids within the majority of sites. Triploids and tetraploids were equally represented in the understorey and roadside habitat. Triploids were more variable than tetraploids, but both cytotypes shared polymorphisms in ITS. Conclusions Coexistence of cytotypes appears to be the norm in A. cordifolia, but habitat differentiation (roadside vs. understorey) is not supported as a coexistence mechanism. Molecular analyses supported multiple events creating triploids but revealed a lack of variation in the tetraploids. Additionally, sequence polymorphisms in ITS suggested a hybridization event prior to polyploidization. PMID:17993653

Origins and widespread distribution of co-existing Polyploids in Arnica cordifolia (Asteraceae).

PubMed

Kao, Rebecca Hufft

2008-01-01

Polyploidy is a central force structuring genetic diversity in angiosperms, but its ecological significance and modes of origin are not fully understood. This work investigated the patterns of coexistence and molecular relatedness of polyploids in the perennial herb, Arnica cordifolia. The local- and broad-scale distributions of cytotypes were analysed using flow cytometry. Samples were collected from both roadside and understorey habitats to test the hypothesis of niche separation between triploids and tetraploids. The nuclear rDNA internal transcribed spacer (ITS) and plastid rpl16 spacer, trnL intron plus trnL-trnF spacer and trnK 3' intron regions were sequenced. Broad-scale sampling established that both triploids and tetraploids were common throughout the range of the species, pentaploids were rare, and diploids were not found. Local-scale sampling revealed coexistence of both triploids and tetraploids within the majority of sites. Triploids and tetraploids were equally represented in the understorey and roadside habitat. Triploids were more variable than tetraploids, but both cytotypes shared polymorphisms in ITS. Coexistence of cytotypes appears to be the norm in A. cordifolia, but habitat differentiation (roadside vs. understorey) is not supported as a coexistence mechanism. Molecular analyses supported multiple events creating triploids but revealed a lack of variation in the tetraploids. Additionally, sequence polymorphisms in ITS suggested a hybridization event prior to polyploidization.

Secuencias evolutivas e isocronas para estrellas de baja masa e intermedia

NASA Astrophysics Data System (ADS)

Panei, J.; Baume, G.

2016-08-01

We present theoretical evolutionary sequences for low- and intermediate-mass stars. The masses calculated range from 1.7 to 10 M. The initial chemical composition is . In addition, we have taken into account a nuclear network with 17 isotopes and 34 nuclear reactions. With respect to the mix, we considered overshooting with a parameter . The evolutionary calculations were initialized from the region of instability of Hayashi, in order to calculate isochrones of pre-sequence, too.

Resolving the phylogenetic position of Darwin's extinct ground sloth (Mylodon darwinii) using mitogenomic and nuclear exon data.

PubMed

Delsuc, Frédéric; Kuch, Melanie; Gibb, Gillian C; Hughes, Jonathan; Szpak, Paul; Southon, John; Enk, Jacob; Duggan, Ana T; Poinar, Hendrik N

2018-05-16

Mylodon darwinii is the extinct giant ground sloth named after Charles Darwin, who first collected its remains in South America. We have successfully obtained a high-quality mitochondrial genome at 99-fold coverage using an Illumina shotgun sequencing of a 12 880-year-old bone fragment from Mylodon Cave in Chile. Low level of DNA damage showed that this sample was exceptionally well preserved for an ancient subfossil, probably the result of the dry and cold conditions prevailing within the cave. Accordingly, taxonomic assessment of our shotgun metagenomic data showed a very high percentage of endogenous DNA with 22% of the assembled metagenomic contigs assigned to Xenarthra. Additionally, we enriched over 15 kb of sequence data from seven nuclear exons, using target sequence capture designed against a wide xenarthran dataset. Phylogenetic and dating analyses of the mitogenomic dataset including all extant species of xenarthrans and the assembled nuclear supermatrix unambiguously place Mylodon darwinii as the sister-group of modern two-fingered sloths, from which it diverged around 22 million years ago. These congruent results from both the mitochondrial and nuclear data support the diphyly of the two modern sloth lineages, implying the convergent evolution of their unique suspensory behaviour as an adaption to arboreality. Our results offer promising perspectives for whole-genome sequencing of this emblematic extinct taxon. © 2018 The Authors.

Paralogues of nuclear ribosomal genes conceal phylogenetic signals within the invasive Asian fish tapeworm lineage: evidence from next generation sequencing data.

PubMed

Brabec, Jan; Kuchta, Roman; Scholz, Tomáš; Littlewood, D Timothy J

2016-08-01

Complete mitochondrial genomes and nuclear rRNA operons of eight geographically distinct isolates of the Asian fish tapeworm Schyzocotyle acheilognathi (syn. Bothriocephalus acheilognathi), representing the parasite's global diversity spanning four continents, were fully characterised using an Illumina sequencing platform. This cestode species represents an extreme example of a highly invasive, globally distributed pathogen of veterinary importance with exceptionally low host specificity unseen elsewhere within the parasitic flatworms. In addition to eight specimens of S. acheilognathi, we fully characterised its closest known relative and the only congeneric species, Schyzocotyle nayarensis, from cyprinids in the Indian subcontinent. Since previous nucleotide sequence data on the Asian fish tapeworm were restricted to a single molecular locus of questionable phylogenetic utility-the nuclear rRNA genes-separating internal transcribed spacers-the mitogenomic data presented here offer a unique opportunity to gain the first detailed insights into both the intraspecific phylogenetic relationships and population genetic structure of the parasite, providing key baseline information for future research in the field. Additionally, we identify a previously unnoticed source of error and demonstrate the limited utility of the nuclear rRNA sequences, including the internal transcribed spacers that has likely misled most of the previous molecular phylogenetic and population genetic estimates on the Asian fish tapeworm. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Nuclear distribution of the Trypanosoma cruzi RNA Pol I subunit RPA31 during growth and metacyclogenesis, and characterization of its nuclear localization signal.

PubMed

Canela-Pérez, Israel; López-Villaseñor, Imelda; Cevallos, Ana María; Hernández, Roberto

2018-03-01

Trypanosoma cruzi is the aetiologic agent of Chagas disease. Our research group studies ribosomal RNA (rRNA) gene transcription and nucleolus dynamics in this species of trypanosomes. RPA31 is an essential subunit of RNA polymerase I (Pol I) whose presence is apparently restricted to trypanosomes. Using fluorescent-tagged versions of this protein (TcRPA31-EGFP), we describe its nuclear distribution during growth and metacyclogenesis. Our findings indicate that TcRPA31-EGFP alters its nuclear presence from concentrated nucleolar localization in exponentially growing epimastigotes to a dispersed granular distribution in the nucleoplasm of stationary epimastigotes and metacyclic trypomastigotes. These changes likely reflect a structural redistribution of the Pol I transcription machinery in quiescent cellular stages where downregulation of rRNA synthesis is known to occur. In addition, and related to the nuclear internalization of this protein, the presence of a classical bipartite-type nuclear localization signal was identified towards its C-terminal end. The functionality of this motif was demonstrated by its partial or total deletion in recombinant versions of the tagged fluorescent protein. Moreover, ivermectin inhibited the nuclear localization of the labelled chimaera, suggesting the involvement of the importin α/β transport system.

The Defective Nuclear Lamina in Hutchinson-Gilford Progeria Syndrome Disrupts the Nucleocytoplasmic Ran Gradient and Inhibits Nuclear Localization of Ubc9▿

PubMed Central

Kelley, Joshua B.; Datta, Sutirtha; Snow, Chelsi J.; Chatterjee, Mandovi; Ni, Li; Spencer, Adam; Yang, Chun-Song; Cubeñas-Potts, Caelin; Matunis, Michael J.; Paschal, Bryce M.

2011-01-01

The mutant form of lamin A responsible for the premature aging disease Hutchinson-Gilford progeria syndrome (termed progerin) acts as a dominant negative protein that changes the structure of the nuclear lamina. How the perturbation of the nuclear lamina in progeria is transduced into cellular changes is undefined. Using patient fibroblasts and a variety of cell-based assays, we determined that progerin expression in Hutchinson-Gilford progeria syndrome inhibits the nucleocytoplasmic transport of several factors with key roles in nuclear function. We found that progerin reduces the nuclear/cytoplasmic concentration of the Ran GTPase and inhibits the nuclear localization of Ubc9, the sole E2 for SUMOylation, and of TPR, the nucleoporin that forms the basket on the nuclear side of the nuclear pore complex. Forcing the nuclear localization of Ubc9 in progerin-expressing cells rescues the Ran gradient and TPR import, indicating that these pathways are linked. Reducing nuclear SUMOylation decreases the nuclear mobility of the Ran nucleotide exchange factor RCC1 in vivo, and the addition of SUMO E1 and E2 promotes the dissociation of RCC1 and Ran from chromatin in vitro. Our data suggest that the cellular effects of progerin are transduced, at least in part, through reduced function of the Ran GTPase and SUMOylation pathways. PMID:21670151

The defective nuclear lamina in Hutchinson-gilford progeria syndrome disrupts the nucleocytoplasmic Ran gradient and inhibits nuclear localization of Ubc9.

PubMed

Kelley, Joshua B; Datta, Sutirtha; Snow, Chelsi J; Chatterjee, Mandovi; Ni, Li; Spencer, Adam; Yang, Chun-Song; Cubeñas-Potts, Caelin; Matunis, Michael J; Paschal, Bryce M

2011-08-01

The mutant form of lamin A responsible for the premature aging disease Hutchinson-Gilford progeria syndrome (termed progerin) acts as a dominant negative protein that changes the structure of the nuclear lamina. How the perturbation of the nuclear lamina in progeria is transduced into cellular changes is undefined. Using patient fibroblasts and a variety of cell-based assays, we determined that progerin expression in Hutchinson-Gilford progeria syndrome inhibits the nucleocytoplasmic transport of several factors with key roles in nuclear function. We found that progerin reduces the nuclear/cytoplasmic concentration of the Ran GTPase and inhibits the nuclear localization of Ubc9, the sole E2 for SUMOylation, and of TPR, the nucleoporin that forms the basket on the nuclear side of the nuclear pore complex. Forcing the nuclear localization of Ubc9 in progerin-expressing cells rescues the Ran gradient and TPR import, indicating that these pathways are linked. Reducing nuclear SUMOylation decreases the nuclear mobility of the Ran nucleotide exchange factor RCC1 in vivo, and the addition of SUMO E1 and E2 promotes the dissociation of RCC1 and Ran from chromatin in vitro. Our data suggest that the cellular effects of progerin are transduced, at least in part, through reduced function of the Ran GTPase and SUMOylation pathways.

Nuclear localization of the dystrophin-associated protein α-dystrobrevin through importin α2/β1 is critical for interaction with the nuclear lamina/maintenance of nuclear integrity.

PubMed

Aguilar, Areli; Wagstaff, Kylie M; Suárez-Sánchez, Rocío; Zinker, Samuel; Jans, David A; Cisneros, Bulmaro

2015-05-01

Although α-dystrobrevin (DB) is assembled into the dystrophin-associated protein complex, which is central to cytoskeletal organization, it has also been found in the nucleus. Here we delineate the nuclear import pathway responsible for nuclear targeting of α-DB for the first time, together with the importance of nuclear α-DB in determining nuclear morphology. We map key residues of the nuclear localization signal of α-DB within the zinc finger domain (ZZ) using various truncated versions of the protein, and site-directed mutagenesis. Pulldown, immunoprecipitation, and AlphaScreen assays showed that the importin (IMP) α2/β1 heterodimer interacts with high affinity with the ZZ domain of α-DB. In vitro nuclear import assays using antibodies to specific importins, as well as in vivo studies using siRNA or a dominant negative importin construct, confirmed the key role of IMPα2/β1 in α-DB nuclear translocation. Knockdown of α-DB expression perturbed cell cycle progression in C2C12 myoblasts, with decreased accumulation of cells in S phase and, significantly, altered localization of lamins A/C, B1, and B2 with accompanying gross nuclear morphology defects. Because α-DB interacts specifically with lamin B1 in vivo and in vitro, nuclear α-DB would appear to play a key role in nuclear shape maintenance through association with the nuclear lamina. © FASEB.

A nuclear fraction of turnip crinkle virus capsid protein is important for elicitation of the host resistance response.

PubMed

Kang, Sung-Hwan; Qu, Feng; Morris, T Jack

2015-12-02

The N-terminal 25 amino acids (AAs) of turnip crinkle virus (TCV) capsid protein (CP) are recognized by the resistance protein HRT to trigger a hypersensitive response (HR) and systemic resistance to TCV infection. This same region of TCV CP also contains a motif that interacts with the transcription factor TIP, as well as a nuclear localization signal (NLS). However, it is not yet known whether nuclear localization of TCV CP is needed for the induction of HRT-mediated HR and resistance. Here we present new evidence suggesting a tight correlation between nuclear inclusions formed by CP and the manifestation of HR. We show that a fraction of TCV CP localized to cell nuclei to form discrete inclusion-like structures, and a mutated CP (R6A) known to abolish HR failed to form nuclear inclusions. Notably, TIP-CP interaction augments the inclusion-forming activity of CP by tethering inclusions to the nuclear membrane. This TIP-mediated augmentation is also critical for HR resistance, as another CP mutant (R8A) known to elicit a less restrictive HR, though still self-associated into nuclear inclusions, failed to direct inclusions to the nuclear membrane due to its inability to interact with TIP. Finally, exclusion of CP from cell nuclei abolished induction of HR. Together, these results uncovered a strong correlation between nuclear localization and nuclear inclusion formation by TCV CP and induction of HR, and suggest that CP nuclear inclusions could be the key trigger of the HRT-dependent, yet TIP-reinforced, resistance to TCV. Copyright © 2015 Elsevier B.V. All rights reserved.

Piwi Nuclear Localization and Its Regulatory Mechanism in Drosophila Ovarian Somatic Cells.

PubMed

Yashiro, Ryu; Murota, Yukiko; Nishida, Kazumichi M; Yamashiro, Haruna; Fujii, Kaede; Ogai, Asuka; Yamanaka, Soichiro; Negishi, Lumi; Siomi, Haruhiko; Siomi, Mikiko C

2018-06-19

In Drosophila ovarian somatic cells (OSCs), Piwi represses transposons transcriptionally to maintain genome integrity. Piwi nuclear localization requires the N terminus and PIWI-interacting RNA (piRNA) loading of Piwi. However, the underlying mechanism remains unknown. Here, we show that Importinα (Impα) plays a pivotal role in Piwi nuclear localization and that Piwi has a bipartite nuclear localization signal (NLS). Impα2 and Impα3 are highly expressed in OSCs, whereas Impα1 is the least expressed. Loss of Impα2 or Impα3 forces Piwi to be cytoplasmic, which is rectified by overexpression of any Impα members. Extension of Piwi-NLS with an additional Piwi-NLS leads Piwi to be imported to the nucleus in a piRNA-independent manner, whereas replacement of Piwi-NLS with SV40-NLS fails. Limited proteolysis analysis suggests that piRNA loading onto Piwi triggers conformational change, exposing the N terminus to the environment. These results suggest that Piwi autoregulates its nuclear localization by exposing the NLS to Impα upon piRNA loading. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Cloud prediction of protein structure and function with PredictProtein for Debian.

PubMed

Kaján, László; Yachdav, Guy; Vicedo, Esmeralda; Steinegger, Martin; Mirdita, Milot; Angermüller, Christof; Böhm, Ariane; Domke, Simon; Ertl, Julia; Mertes, Christian; Reisinger, Eva; Staniewski, Cedric; Rost, Burkhard

2013-01-01

We report the release of PredictProtein for the Debian operating system and derivatives, such as Ubuntu, Bio-Linux, and Cloud BioLinux. The PredictProtein suite is available as a standard set of open source Debian packages. The release covers the most popular prediction methods from the Rost Lab, including methods for the prediction of secondary structure and solvent accessibility (profphd), nuclear localization signals (predictnls), and intrinsically disordered regions (norsnet). We also present two case studies that successfully utilize PredictProtein packages for high performance computing in the cloud: the first analyzes protein disorder for whole organisms, and the second analyzes the effect of all possible single sequence variants in protein coding regions of the human genome.

Cloud Prediction of Protein Structure and Function with PredictProtein for Debian

PubMed Central

Kaján, László; Yachdav, Guy; Vicedo, Esmeralda; Steinegger, Martin; Mirdita, Milot; Angermüller, Christof; Böhm, Ariane; Domke, Simon; Ertl, Julia; Mertes, Christian; Reisinger, Eva; Rost, Burkhard

2013-01-01

We report the release of PredictProtein for the Debian operating system and derivatives, such as Ubuntu, Bio-Linux, and Cloud BioLinux. The PredictProtein suite is available as a standard set of open source Debian packages. The release covers the most popular prediction methods from the Rost Lab, including methods for the prediction of secondary structure and solvent accessibility (profphd), nuclear localization signals (predictnls), and intrinsically disordered regions (norsnet). We also present two case studies that successfully utilize PredictProtein packages for high performance computing in the cloud: the first analyzes protein disorder for whole organisms, and the second analyzes the effect of all possible single sequence variants in protein coding regions of the human genome. PMID:23971032

Predicting residue-wise contact orders in proteins by support vector regression.

PubMed

Song, Jiangning; Burrage, Kevin

2006-10-03

The residue-wise contact order (RWCO) describes the sequence separations between the residues of interest and its contacting residues in a protein sequence. It is a new kind of one-dimensional protein structure that represents the extent of long-range contacts and is considered as a generalization of contact order. Together with secondary structure, accessible surface area, the B factor, and contact number, RWCO provides comprehensive and indispensable important information to reconstructing the protein three-dimensional structure from a set of one-dimensional structural properties. Accurately predicting RWCO values could have many important applications in protein three-dimensional structure prediction and protein folding rate prediction, and give deep insights into protein sequence-structure relationships. We developed a novel approach to predict residue-wise contact order values in proteins based on support vector regression (SVR), starting from primary amino acid sequences. We explored seven different sequence encoding schemes to examine their effects on the prediction performance, including local sequence in the form of PSI-BLAST profiles, local sequence plus amino acid composition, local sequence plus molecular weight, local sequence plus secondary structure predicted by PSIPRED, local sequence plus molecular weight and amino acid composition, local sequence plus molecular weight and predicted secondary structure, and local sequence plus molecular weight, amino acid composition and predicted secondary structure. When using local sequences with multiple sequence alignments in the form of PSI-BLAST profiles, we could predict the RWCO distribution with a Pearson correlation coefficient (CC) between the predicted and observed RWCO values of 0.55, and root mean square error (RMSE) of 0.82, based on a well-defined dataset with 680 protein sequences. Moreover, by incorporating global features such as molecular weight and amino acid composition we could further improve the prediction performance with the CC to 0.57 and an RMSE of 0.79. In addition, combining the predicted secondary structure by PSIPRED was found to significantly improve the prediction performance and could yield the best prediction accuracy with a CC of 0.60 and RMSE of 0.78, which provided at least comparable performance compared with the other existing methods. The SVR method shows a prediction performance competitive with or at least comparable to the previously developed linear regression-based methods for predicting RWCO values. In contrast to support vector classification (SVC), SVR is very good at estimating the raw value profiles of the samples. The successful application of the SVR approach in this study reinforces the fact that support vector regression is a powerful tool in extracting the protein sequence-structure relationship and in estimating the protein structural profiles from amino acid sequences.

Human beta-globin gene polymorphisms characterized in DNA extracted from ancient bones 12,000 years old.

PubMed

Béraud-Colomb, E; Roubin, R; Martin, J; Maroc, N; Gardeisen, A; Trabuchet, G; Goosséns, M

1995-12-01

Analyzing the nuclear DNA from ancient human bones is an essential step to the understanding of genetic diversity in current populations, provided that such systematic studies are experimentally feasible. This article reports the successful extraction and amplification of nuclear DNA from the beta-globin region from 5 of 10 bone specimens up to 12,000 years old. These have been typed for beta-globin frameworks by sequencing through two variable positions and for a polymorphic (AT) chi (T) gamma microsatellite 500 bp upstream of the beta-globin gene. These specimens of human remains are somewhat older than those analyzed in previous nuclear gene sequencing reports and considerably older than those used to study high-copy-number human mtDNA. These results show that the systematic study of nuclear DNA polymorphisms of ancient populations is feasible.

NUREBASE: database of nuclear hormone receptors.

PubMed

Duarte, Jorge; Perrière, Guy; Laudet, Vincent; Robinson-Rechavi, Marc

2002-01-01

Nuclear hormone receptors are an abundant class of ligand activated transcriptional regulators, found in varying numbers in all animals. Based on our experience of managing the official nomenclature of nuclear receptors, we have developed NUREBASE, a database containing protein and DNA sequences, reviewed protein alignments and phylogenies, taxonomy and annotations for all nuclear receptors. The reviewed NUREBASE is completed by NUREBASE_DAILY, automatically updated every 24 h. Both databases are organized under a client/server architecture, with a client written in Java which runs on any platform. This client, named FamFetch, integrates a graphical interface allowing selection of families, and manipulation of phylogenies and alignments. NUREBASE sequence data is also accessible through a World Wide Web server, allowing complex queries. All information on accessing and installing NUREBASE may be found at http://www.ens-lyon.fr/LBMC/laudet/nurebase.html.

Custos controls β-catenin to regulate head development during vertebrate embryogenesis.

PubMed

Komiya, Yuko; Mandrekar, Noopur; Sato, Akira; Dawid, Igor B; Habas, Raymond

2014-09-09

Precise control of the canonical Wnt pathway is crucial in embryogenesis and all stages of life, and dysregulation of this pathway is implicated in many human diseases including cancers and birth defect disorders. A key aspect of canonical Wnt signaling is the cytoplasmic to nuclear translocation of β-catenin, a process that remains incompletely understood. Here we report the identification of a previously undescribed component of the canonical Wnt signaling pathway termed Custos, originally isolated as a Dishevelled-interacting protein. Custos contains casein kinase phosphorylation sites and nuclear localization sequences. In Xenopus, custos mRNA is expressed maternally and then widely throughout embryogenesis. Depletion or overexpression of Custos produced defective anterior head structures by inhibiting the formation of the Spemann-Mangold organizer. In addition, Custos expression blocked secondary axis induction by positive signaling components of the canonical Wnt pathway and inhibited β-catenin/TCF-dependent transcription. Custos binds to β-catenin in a Wnt responsive manner without affecting its stability, but rather modulates the cytoplasmic to nuclear translocation of β-catenin. This effect on nuclear import appears to be the mechanism by which Custos inhibits canonical Wnt signaling. The function of Custos is conserved as loss-of-function and gain-of-function studies in zebrafish also demonstrate a role for Custos in anterior head development. Our studies suggest a role for Custos in fine-tuning canonical Wnt signal transduction during embryogenesis, adding an additional layer of regulatory control in the Wnt-β-catenin signal transduction cascade.

PGE2/EP3/SRC signaling induces EGFR nuclear translocation and growth through EGFR ligands release in lung adenocarcinoma cells

PubMed Central

Bazzani, Lorenzo; Donnini, Sandra; Finetti, Federica; Christofori, Gerhard; Ziche, Marina

2017-01-01

Prostaglandin E2 (PGE2) interacts with tyrosine kinases receptor signaling in both tumor and stromal cells supporting tumor progression. Here we demonstrate that in non-small cell lung carcinoma (NSCLC) cells, A549 and GLC82, PGE2 promotes nuclear translocation of epidermal growth factor receptor (nEGFR), affects gene expression and induces cell growth. Indeed, cyclin D1, COX-2, iNOS and c-Myc mRNA levels are upregulated following PGE2 treatment. The nuclear localization sequence (NLS) of EGFR as well as its tyrosine kinase activity are required for the effect of PGE2 on nEGFR and downstream signaling activities. PGE2 binds its bona fide receptor EP3 which by activating SRC family kinases, induces ADAMs activation which, in turn, releases EGFR-ligands from the cell membrane and promotes nEGFR. Amphiregulin (AREG) and Epiregulin (EREG) appear to be involved in nEGFR promoted by the PGE2/EP3-SRC axis. Pharmacological inhibition or silencing of the PGE2/EP3/SRC-ADAMs signaling axis or EGFR ligands i.e. AREG and EREG expression abolishes nEGFR induced by PGE2. In conclusion, PGE2 induces NSCLC cell proliferation by EP3 receptor, SRC-ADAMs activation, EGFR ligands shedding and finally, phosphorylation and nEGFR. Since nuclear EGFR is a hallmark of cancer aggressiveness, our findings reveal a novel mechanism for the contribution of PGE2 to tumor progression. PMID:28415726

Dynamics of miRNA biogenesis and nuclear transport.

PubMed

Kotipalli, Aneesh; Gutti, Ravikumar; Mitra, Chanchal K

2016-12-01

MicroRNAs (miRNAs) are short noncoding RNA sequences ~22 nucleotides in length that play an important role in gene regulation-transcription and translation. The processing of these miRNAs takes place in both the nucleus and the cytoplasm while the final maturation occurs in the cytoplasm. Some mature miRNAs with nuclear localisation signals (NLS) are transported back to the nucleus and some remain in the cytoplasm. The functional roles of these miRNAs are seen in both the nucleus and the cytoplasm. In the nucleus, miRNAs regulate gene expression by binding to the targeted promoter sequences and affect either the transcriptional gene silencing (TGS) or transcriptional gene activation (TGA). In the cytoplasm, targeted mRNAs are translationally repressed or cleaved based on the complementarity between the two sequences at the seed region of miRNA and mRNA. The selective transport of mature miRNAs to the nucleus follows the classical nuclear import mechanism. The classical nuclear import mechanism is a highly regulated process, involving exportins and importins. The nuclear pore complex (NPC) regulates all these transport events like a gate keeper. The half-life of miRNAs is rather low, so within a short time miRNAs perform their function. Temporal studies of miRNA biogenesis are, therefore, useful. We have carried out simulation studies for important miRNA biogenesis steps and also classical nuclear import mechanism using ordinary differential equation (ODE) solver in the Octave software.

Dynamics of miRNA biogenesis and nuclear transport.

PubMed

Kotipalli, Aneesh; Gutti, Ravikumar; Mitra, Chanchal K

2016-12-22

MicroRNAs (miRNAs) are short noncoding RNA sequences ~22 nucleotides in length that play an important role in gene regulation-transcription and translation. The processing of these miRNAs takes place in both the nucleus and the cytoplasm while the final maturation occurs in the cytoplasm. Some mature miRNAs with nuclear localisation signals (NLS) are transported back to the nucleus and some remain in the cytoplasm. The functional roles of these miRNAs are seen in both the nucleus and the cytoplasm. In the nucleus, miRNAs regulate gene expression by binding to the targeted promoter sequences and affect either the transcriptional gene silencing (TGS) or transcriptional gene activation (TGA). In the cytoplasm, targeted mRNAs are translationally repressed or cleaved based on the complementarity between the two sequences at the seed region of miRNA and mRNA. The selective transport of mature miRNAs to the nucleus follows the classical nuclear import mechanism. The classical nuclear import mechanism is a highly regulated process, involving exportins and importins. The nuclear pore complex (NPC) regulates all these transport events like a gate keeper. The half-life of miRNAs is rather low, so within a short time miRNAs perform their function. Temporal studies of miRNA biogenesis are, therefore, useful. We have carried out simulation studies for important miRNA biogenesis steps and also classical nuclear import mechanism using ordinary differential equation (ODE) solver in the Octave software.

Large-Scale Paraphrasing for Natural Language Understanding

DTIC Science & Technology

2018-04-01

to manufacture , use, or sell any patented invention that may relate to them. This report is the result of contracted fundamental research deemed...station contaminated local fish populations Atomic power generation in Springfield polluted indigenous seafood stocks Radioactive power generation...from PPDB. Springfield’s nuclear power plant contaminated local fish populations nuclear power station nuclear plant power plant fish stocks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Piccioli, Zachary; McKee, Courtney H.; Leszczynski, Anna

We investigated the nuclear import of low risk HPV11 E7 protein using 1) transfection assays in HeLa cells with EGFP fusion plasmids containing 11E7 and its domains and 2) nuclear import assays in digitonin-permeabilized HeLa cells with GST fusion proteins containing 11E7 and its domains. The EGFP-11E7 and EGFP-11cE7{sub 39-98} localized mostly to the nucleus. The GST-11E7 and GST-11cE7{sub 39-98} were imported into the nuclei in the presence of either Ran-GDP or RanG19V-GTP mutant and in the absence of nuclear import receptors. This suggests that 11E7 enters the nucleus via a Ran-dependent pathway, independent of nuclear import receptors, mediated bymore » a nuclear localization signal located in its C-terminal domain (cNLS). This cNLS contains the zinc binding domain consisting of two copies of Cys-X-X-Cys motif. Mutagenesis of Cys residues in these motifs changed the localization of the EGFP-11cE7/-11E7 mutants to cytoplasmic, suggesting that the zinc binding domain is essential for nuclear localization of 11E7.« less

Tau regulates the subcellular localization of calmodulin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barreda, Elena Gomez de; Avila, Jesus, E-mail: javila@cbm.uam.es; CIBER de Enfermedades Neurodegenerativas, 28031 Madrid

Highlights: {yields} In this work we have tried to explain how a cytoplasmic protein could regulate a cell nuclear function. We have tested the role of a cytoplasmic protein (tau) in regulating the expression of calbindin gene. We found that calmodulin, a tau-binding protein with nuclear and cytoplasmic localization, increases its nuclear localization in the absence of tau. Since nuclear calmodulin regulates calbindin expression, a decrease in nuclear calmodulin, due to the presence of tau that retains it at the cytoplasm, results in a change in calbindin expression. -- Abstract: Lack of tau expression in neuronal cells results in amore » change in the expression of few genes. However, little is known about how tau regulates gene expression. Here we show that the presence of tau could alter the subcellular localization of calmodulin, a protein that could be located at the cytoplasm or in the nucleus. Nuclear calmodulin binds to co-transcription factors, regulating the expression of genes like calbindin. In this work, we have found that in neurons containing tau, a higher proportion of calmodulin is present in the cytoplasm compared with neurons lacking tau and that an increase in cytoplasmic calmodulin correlates with a higher expression of calbindin.« less

Nuclear Lipids in the Nervous System: What they do in Health and Disease.

PubMed

Garcia-Gil, Mercedes; Albi, Elisabetta

2017-02-01

In the last 20 years it has been widely demonstrated that cell nucleus contains neutral and polar lipids localized in nuclear membranes, nucleoli, nuclear matrix and chromatin. Nuclear lipids may show specific organization forming nuclear lipid microdomains and have both structural and functional roles. Depending on their localization, nuclear lipids play different roles such as the regulation of nuclear membrane and nuclear matrix fluidity but they also can act as platforms for vitamin and hormone function, for active chromatin anchoring, and for the regulation of gene expression, DNA duplication and transcription. Crosstalk among different kinds of lipid signalling pathways influence the physiopathology of numerous cell types. In neural cells the nuclear lipids are involved in cell proliferation, differentiation, inflammation, migration and apoptosis. Abnormal metabolism of nuclear lipids might be closely associated with tumorigenesis and neurodegenerative diseases such as Alzheimer disease and Parkinson disease among others.

Microtubule Actin Crosslinking Factor 1 Regulates the Balbiani Body and Animal-Vegetal Polarity of the Zebrafish Oocyte

PubMed Central

Gupta, Tripti; Marlow, Florence L.; Ferriola, Deborah; Mackiewicz, Katarzyna; Dapprich, Johannes; Monos, Dimitri; Mullins, Mary C.

2010-01-01

Although of fundamental importance in developmental biology, the genetic basis for the symmetry breaking events that polarize the vertebrate oocyte and egg are largely unknown. In vertebrates, the first morphological asymmetry in the oocyte is the Balbiani body, a highly conserved, transient structure found in vertebrates and invertebrates including Drosophila, Xenopus, human, and mouse. We report the identification of the zebrafish magellan (mgn) mutant, which exhibits a novel enlarged Balbiani body phenotype and a disruption of oocyte polarity. To determine the molecular identity of the mgn gene, we positionally cloned the gene, employing a novel DNA capture method to target region-specific genomic DNA of 600 kb for massively parallel sequencing. Using this technique, we were able to enrich for the genomic region linked to our mutation within one week and then identify the mutation in mgn using massively parallel sequencing. This is one of the first successful uses of genomic DNA enrichment combined with massively parallel sequencing to determine the molecular identity of a gene associated with a mutant phenotype. We anticipate that the combination of these technologies will have wide applicability for the efficient identification of mutant genes in all organisms. We identified the mutation in mgn as a deletion in the coding sequence of the zebrafish microtubule actin crosslinking factor 1 (macf1) gene. macf1 is a member of the highly conserved spectraplakin family of cytoskeletal linker proteins, which play diverse roles in polarized cells such as neurons, muscle cells, and epithelial cells. In mgn mutants, the oocyte nucleus is mislocalized; and the Balbiani body, localized mRNAs, and organelles are absent from the periphery of the oocyte, consistent with a function for macf1 in nuclear anchoring and cortical localization. These data provide the first evidence for a role for spectraplakins in polarization of the vertebrate oocyte and egg. PMID:20808893

Microtubule actin crosslinking factor 1 regulates the Balbiani body and animal-vegetal polarity of the zebrafish oocyte.

PubMed

Gupta, Tripti; Marlow, Florence L; Ferriola, Deborah; Mackiewicz, Katarzyna; Dapprich, Johannes; Monos, Dimitri; Mullins, Mary C

2010-08-19

Although of fundamental importance in developmental biology, the genetic basis for the symmetry breaking events that polarize the vertebrate oocyte and egg are largely unknown. In vertebrates, the first morphological asymmetry in the oocyte is the Balbiani body, a highly conserved, transient structure found in vertebrates and invertebrates including Drosophila, Xenopus, human, and mouse. We report the identification of the zebrafish magellan (mgn) mutant, which exhibits a novel enlarged Balbiani body phenotype and a disruption of oocyte polarity. To determine the molecular identity of the mgn gene, we positionally cloned the gene, employing a novel DNA capture method to target region-specific genomic DNA of 600 kb for massively parallel sequencing. Using this technique, we were able to enrich for the genomic region linked to our mutation within one week and then identify the mutation in mgn using massively parallel sequencing. This is one of the first successful uses of genomic DNA enrichment combined with massively parallel sequencing to determine the molecular identity of a gene associated with a mutant phenotype. We anticipate that the combination of these technologies will have wide applicability for the efficient identification of mutant genes in all organisms. We identified the mutation in mgn as a deletion in the coding sequence of the zebrafish microtubule actin crosslinking factor 1 (macf1) gene. macf1 is a member of the highly conserved spectraplakin family of cytoskeletal linker proteins, which play diverse roles in polarized cells such as neurons, muscle cells, and epithelial cells. In mgn mutants, the oocyte nucleus is mislocalized; and the Balbiani body, localized mRNAs, and organelles are absent from the periphery of the oocyte, consistent with a function for macf1 in nuclear anchoring and cortical localization. These data provide the first evidence for a role for spectraplakins in polarization of the vertebrate oocyte and egg.

Sequence specificity of the human mRNA N6-adenosine methylase in vitro.

PubMed Central

Harper, J E; Miceli, S M; Roberts, R J; Manley, J L

1990-01-01

N6-adenosine methylation is a frequent modification of mRNAs and their precursors, but little is known about the mechanism of the reaction or the function of the modification. To explore these questions, we developed conditions to examine N6-adenosine methylase activity in HeLa cell nuclear extracts. Transfer of the methyl group from S-[3H methyl]-adenosylmethionine to unlabeled random copolymer RNA substrates of varying ribonucleotide composition revealed a substrate specificity consistent with a previously deduced consensus sequence, Pu[G greater than A]AC[A/C/U]. 32-P labeled RNA substrates of defined sequence were used to examine the minimum sequence requirements for methylation. Each RNA was 20 nucleotides long, and contained either the core consensus sequence GGACU, or some variation of this sequence. RNAs containing GGACU, either in single or multiple copies, were good substrates for methylation, whereas RNAs containing single base substitutions within the GGACU sequence gave dramatically reduced methylation. These results demonstrate that the N6-adenosine methylase has a strict sequence specificity, and that there is no requirement for extended sequences or secondary structures for methylation. Recognition of this sequence does not require an RNA component, as micrococcal nuclease pretreatment of nuclear extracts actually increased methylation efficiency. Images PMID:2216767

Phylogenetic relationships and timing of diversification in gonorynchiform fishes inferred using nuclear gene DNA sequences (Teleostei: Ostariophysi).

PubMed

Near, Thomas J; Dornburg, Alex; Friedman, Matt

2014-11-01

The Gonorynchiformes are the sister lineage of the species-rich Otophysi and provide important insights into the diversification of ostariophysan fishes. Phylogenies of gonorynchiforms inferred using morphological characters and mtDNA gene sequences provide differing resolutions with regard to the sister lineage of all other gonorynchiforms (Chanos vs. Gonorynchus) and support for monophyly of the two miniaturized lineages Cromeria and Grasseichthys. In this study the phylogeny and divergence times of gonorynchiforms are investigated with DNA sequences sampled from nine nuclear genes and a published morphological character matrix. Bayesian phylogenetic analyses reveal substantial congruence among individual gene trees with inferences from eight genes placing Gonorynchus as the sister lineage to all other gonorynchiforms. Seven gene trees resolve Cromeria and Grasseichthys as a clade, supporting previous inferences using morphological characters. Phylogenies resulting from either concatenating the nuclear genes, performing a multispecies coalescent species tree analysis, or combining the morphological and nuclear gene DNA sequences resolve Gonorynchus as the living sister lineage of all other gonorynchiforms, strongly support the monophyly of Cromeria and Grasseichthys, and resolve a clade containing Parakneria, Cromeria, and Grasseichthys. The morphological dataset, which includes 13 gonorynchiform fossil taxa that range in age from Early Cretaceous to Eocene, was analyzed in combination with DNA sequences from the nine nuclear genes and a relaxed molecular clock to estimate times of evolutionary divergence. This "tip dating" strategy accommodates uncertainty in the phylogenetic resolution of fossil taxa that provide calibration information in the relaxed molecular clock analysis. The estimated age of the most recent common ancestor (MRCA) of living gonorynchiforms is slightly older than estimates from previous node dating efforts, but the molecular tip dating estimated ages of Kneriinae (Kneria, Parakneria, Cromeria, and Grasseichthys) and the two paedomorphic lineages, Cromeria and Grasseichthys, are considerably younger. Copyright © 2014 Elsevier Inc. All rights reserved.

Hybridization and massive mtDNA unidirectional introgression between the closely related Neotropical toads Rhinella marina and R. schneideri inferred from mtDNA and nuclear markers

PubMed Central

2011-01-01

Background The classical perspective that interspecific hybridization in animals is rare has been changing due to a growing list of empirical examples showing the occurrence of gene flow between closely related species. Using sequence data from cyt b mitochondrial gene and three intron nuclear genes (RPL9, c-myc, and RPL3) we investigated patterns of nucleotide polymorphism and divergence between two closely related toad species R. marina and R. schneideri. By comparing levels of differentiation at nuclear and mtDNA levels we were able to describe patterns of introgression and infer the history of hybridization between these species. Results All nuclear loci are essentially concordant in revealing two well differentiated groups of haplotypes, corresponding to the morphologically-defined species R. marina and R. schneideri. Mitochondrial DNA analysis also revealed two well-differentiated groups of haplotypes but, in stark contrast with the nuclear genealogies, all R. schneideri sequences are clustered with sequences of R. marina from the right Amazon bank (RAB), while R. marina sequences from the left Amazon bank (LAB) are monophyletic. An Isolation-with-Migration (IM) analysis using nuclear data showed that R. marina and R. schneideri diverged at ≈ 1.69 Myr (early Pleistocene), while R. marina populations from LAB and RAB diverged at ≈ 0.33 Myr (middle Pleistocene). This time of divergence is not consistent with the split between LAB and RAB populations obtained with mtDNA data (≈ 1.59 Myr), which is notably similar to the estimate obtained with nuclear genes between R. marina and R. schneideri. Coalescent simulations of mtDNA phylogeny under the speciation history inferred from nuclear genes rejected the hypothesis of incomplete lineage sorting to explain the conflicting signal between mtDNA and nuclear-based phylogenies. Conclusions The cytonuclear discordance seems to reflect the occurrence of interspecific hybridization between these two closely related toad species. Overall, our results suggest a phenomenon of extensive mtDNA unidirectional introgression from the previously occurring R. schneideri into the invading R. marina. We hypothesize that climatic-induced range shifts during the Pleistocene/Holocene may have played an important role in the observed patterns of introgression. PMID:21939538

Nucleolar Association and Transcriptional Inhibition through 5S rDNA in Mammals

PubMed Central

Fedoriw, Andrew M.; Starmer, Joshua; Yee, Della; Magnuson, Terry

2012-01-01

Changes in the spatial positioning of genes within the mammalian nucleus have been associated with transcriptional differences and thus have been hypothesized as a mode of regulation. In particular, the localization of genes to the nuclear and nucleolar peripheries is associated with transcriptional repression. However, the mechanistic basis, including the pertinent cis- elements, for such associations remains largely unknown. Here, we provide evidence that demonstrates a 119 bp 5S rDNA can influence nucleolar association in mammals. We found that integration of transgenes with 5S rDNA significantly increases the association of the host region with the nucleolus, and their degree of association correlates strongly with repression of a linked reporter gene. We further show that this mechanism may be functional in endogenous contexts: pseudogenes derived from 5S rDNA show biased conservation of their internal transcription factor binding sites and, in some cases, are frequently associated with the nucleolus. These results demonstrate that 5S rDNA sequence can significantly contribute to the positioning of a locus and suggest a novel, endogenous mechanism for nuclear organization in mammals. PMID:22275877

Comparative NMR analysis of the decadeoxynucleotide d-(GCATTAATGC)2 and an analogue containing 2-aminoadenine.

PubMed Central

Chazin, W J; Rance, M; Chollet, A; Leupin, W

1991-01-01

The dodecadeoxynucleotide duplex d-(GCATTAATGC)2 has been prepared with all adenine bases replaced by 2-NH2-adenine. This modified duplex has been characterized by nuclear magnetic resonance (NMR) spectroscopy. Complete sequence-specific 1H resonance assignments have been obtained by using a variety of 2D NMR methods. Multiple quantum-filtered and multiple quantum experiments have been used to completely assign all sugar ring protons, including 5'H and 5'H resonances. The assignments form the basis for a detailed comparative analysis of the 1H NMR parameters of the modified and parent duplex. The structural features of both decamer duplexes in solution are characteristic of the B-DNA family. The spin-spin coupling constants in the sugar rings and the relative spatial proximities of protons in the bases and sugars (as determined from the comparison of corresponding nuclear Overhauser effects) are virtually identical in the parent and modified duplexes. Thus, substitution by this adenine analogue in oligonucleotides appears not to disturb the global or local conformation of the DNA duplex. PMID:1945828

Spatially Resolved Quantification of Chromatin Condensation through Differential Local Rheology in Cell Nuclei Fluorescence Lifetime Imaging

PubMed Central

Spagnol, Stephen T.; Dahl, Kris Noel

2016-01-01

The linear sequence of DNA encodes access to the complete set of proteins that carry out cellular functions. Yet, much of the functionality appropriate for each cell is nested within layers of dynamic regulation and organization, including a hierarchy of chromatin structural states and spatial arrangement within the nucleus. There remain limitations in our understanding of gene expression within the context of nuclear organization from an inability to characterize hierarchical chromatin organization in situ. Here we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to quantify and spatially resolve chromatin condensation state using cell-permeable, DNA-binding dyes (Hoechst 33342 and PicoGreen). Through in vitro and in situ experiments we demonstrate the sensitivity of fluorescence lifetime to condensation state through the mechanical effects that accompany the structural changes and are reflected through altered viscosity. The establishment of FLIM for resolving and quantifying chromatin condensation state opens the door for single-measurement mechanical studies of the nucleus and for characterizing the role of genome structure and organization in nuclear processes that accompany physiological and pathological changes. PMID:26765322